Karavilagenin C derivatives as antimalarials

Article abstract of DOI:10.1016/j.bmc.2010.11.015

Karavilagenin C (1), a cucurbitane-type triterpenoid, previously isolated from the aerial parts of Momordica balsamina, was acylated with different alkanoyl, aroyl and cinnamoyl chlorides/anydrides, yielding ten new mono or diesters, karavoates F (7) and

Full text of DOI:10.1016/j.bmc.2010.11.015

Bioorganic & Medicinal Chemistry 19 (2011) 330–338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Karavilagenin C derivatives as antimalarials
Cátia Ramalhete a, Dinora Lopes b, Joseph Molnár c, Silva Mulhovo d
Virgílio E. Rosário b, Maria-José U. Ferreira a,
⇑
a iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. das Forças Armadas, 1600-083 Lisbon, Portugal
b UEI Malaria, CMDT.LA, Institute of Hygiene and Tropical Medicine, UNL, R. da Junqueira 96, 1349-008 Lisbon, Portugal
c Department of Medical Microbiology and Immunobiology, University of Szeged, H-6720 Szeged, Hungary
d Departamento de Ciências Agro-Pecuárias, Universidade Pedagógica, Campus de Lhanguene, Av. de Mozambique, Mozambique
a r t i c l e i n f o
a b s t r a c t
Article history:
Karavilagenin C (1), a cucurbitane-type triterpenoid, previously isolated from the aerial parts of Momor-
dica balsamina, was acylated with different alkanoyl, aroyl and cinnamoyl chlorides/anydrides, yielding
ten new mono or diesters, karavoates F (7) and H-P (8–16). Furthermore, the new compound cucurbal-
saminol C (17) was isolated from the same plant. Their structures were assigned by spectroscopic meth-
ods, including 2D NMR experiments. Compounds 1 and 17 and the acyl derivatives 8–16 along with other
five esters (2–6, karavoates A–E), previously prepared from 1, were evaluated for their in vitro antimalar-
ial activity against the chloroquine-sensitive (3D7) and the chloroquine-resistant (Dd2) strains of
Plasmodium falciparum. Compound 1 exhibited a moderate activity and 17 was inactive. However, a
remarkable antiplasmodial activity was observed for most of karavilagenin C alkanoyl and monoaroyl/
cynamoyl derivatives. Karavoates B, D, E, I, and M were the most active, displaying IC50 values similar
Received 17 June 2010
Revised 1 October 2010
Accepted 8 November 2010
Available online 11 November 2010
Keywords:
Momordica balsamina
Cucurbitaceae
Cucurbitane-type triterpenes
Karavilagenin C
to those found for chloroquine, particularly against the resistant strain (IC50 <0.6 lM). Structure–activity
Plasmodium falciparum
Antimalarial activity
relationships (SAR) are discussed. Moreover, the preliminary toxicity toward human cells of compounds
1–17 was also evaluated in breast cancer cell line (MCF-7). Most of the esters showed no toxicity, display-
ing, in general, much higher selectivity index values than those obtained for the parent compound.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
(Cucurbitaceae), also referred as the balsam apple, or African
pumpkin, is a vegetable extensively cultivated and used as food
In 2008, there were more than 200 million cases of malaria and
nearly one million deaths, mostly among African children.1 In spite
of all efforts in drug development, malaria remains one of the most
devastating tropical diseases. A major contributor to malaria mor-
tality and morbidity is the increasing resistance of malarial para-
sites, particularly Plasmodium falciparum, the most dangerous
species, to front-line drugs, like chloroquine, and antifolates.2,3
Thus, there are a continuing need for new therapeutic agents,
mainly with novel mechanisms of action and structurally unre-
lated to existing antimalarial agents.4 Natural product-derived
compounds have played a major role in drug discovery and devel-
opment. In case of malaria, 11 drugs out of 15 included in the WHO
therapeutic schemes for malaria treatment are natural products or
related with natural products.5 The great significance of plant-
derived drugs for the treatment of the disease is highlighted by
quinine, artemisinin and their derivatives, which are presently
the mainstay of antimalarial therapy. Momordica balsamina L.
in many tropical and subtropical regions of the world.6,7 It has also
been widely used in traditional medicine in Africa to treat various
diseases mostly diabetes, and malaria symptoms.8–10
Cucurbitane triterpenoids are the main constituents of Cucur-
bitaceae family.11 They exhibit a broad range of potent biological
activities namely hepatoprotective, cytotoxic, anti-inflammatory,
cardiovascular, anti-diabetic and antiparasitic effects.11,12
In previous work, bioassay-guided fractionation of the metha-
nol extract of the aerial parts of M. balsamina led to the isolation
of several cucurbitane-type triterpenoids.13–15 Most of the com-
pounds displayed antimalarial activity,13 and were found to be
strong P-glycoprotein modulators in multidrug resistant cancer
cells.14 In order to find out some structure–activity relationships,
the cucurbitane triterpene, karavilagenin C (1) (Fig. 1), isolated in
large amount from the referred extract, was esterified with differ-
ent acylating agents to afford the alkanoyl esters 2–6, previously
reported.14 Further acylation of compound 1 yielded ten new ester
derivatives (7–16). Compounds 1–16 along with a new cucurbitane
(17) isolated from the same plant were evaluated for their antima-
larial activity against the P. falciparum CQ-sensitive (3D7) and the
CQ-resistant (Dd2) strains. Moreover, the cytotoxicity of all
⇑
Corresponding author. Tel.: +351 21 7946475; fax: +351 21 7946470.
E-mail address: mjuferreira@ff.ul.pt (Maria-José U. Ferreira).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.015

C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
331
H3
C
R3O
CH3
H3
CH3
C
R2
H
R1
R2
R3
Karavilagenin C (1)
H
H
H
H
2
Karavoate A ( )
H
H
COCH3
Karavoate B (3)
Karavoate C (4)
COCH3
H
COCH3
CH3
CH3
H
H
COCH2CH3
COCH2CH3
COCH2CH2CH3
H
R1O
5
Karavoate D ( )
COCH2CH3
OCH3
H
H3
C
CH3
Karavoate E (6)
H
H
H
Cucurbalsaminol C (17)
OH
Figure 1. Structures of compounds 2–6, and cucurbalsaminol C (17).
Table 1
NMR data of cucurbalsaminol C (17), (MeOD, 1H 400 MHz, 13C 100.61 MHz; d in ppm,
J in Hz)
compounds was evaluated on the human breast cancer (MCF-7)
cell line, and a selectivity index was determined.
Position
1H
13C
DEPT
2. Results and discussion
2.1. Chemistry
1
2
3
4
5
6
7
8
9
1.63 m; 1.70 m
1.71 m; 1.98 m
3.49 br sa
—
22.5
CH2
CH2
CH
C
30.1
77.4
42.4
149.1
120.3
78.4
48.8
37.4
41.6
44.4
72.0
52.4
51.1
36.2
25.7
52.6
10.7
29.3
31.0
22.4
44.8
67.8
130.6
133.4
18.2
25.9
28.7
26.0
18.6
56.4
—
C
Bioassay-guided fractionation of the methanol extract of the
aerial parts of M. balsamina led to the isolation of several cucurbi-
tane-type triterpenoids.13–15 A large amount of karavilagenin C (1,
7b-methoxycucurbita-5,24-diene-3b,23(R)-diol) was isolated.14
Acylation of its free hydroxyl groups at C-23 or C-3/C-23 with ace-
tic, propionic, and butyric anhydrides yielded five mono or diacy-
lated derivatives (2–6, karavoates A–E, Fig. 1).14
5.78 d (4.8)
3.49 br sa
1.98 br s
—
2.33 br d (10.0)
1.38 m; 1.88 m
3.86 dd (11.2, 4.8)
—
CH
CH
CH
C
CH
CH2
CH
C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7-OMe
Compound 17, named cucurbalsaminol C, was obtained as a
white amorphous powder, with a molecular formula C31H52O4 de-
duced by HR-EIMS, which showed a molecular ion [M]+ at m/z
488.3867 (calcd for C31H52O4: 488.3866). Its IR spectrum exhibited
a characteristic absorption band for the hydroxyl function at
3443 cmꢀ1. The low resolution EIMS showed significant ions at
m/z 470 [MꢀH2O]+, 452 [Mꢀ2 ꢁ H2O]+ and 434 [Mꢀ3 ꢁ H2O]+,
due to the sequential loss of water molecules, suggesting the pres-
ence of three hydroxyl groups. The 1H NMR spectrum showed res-
onances for seven tertiary methyl groups as singlets at dH 0.76,
0.93, 1.01, 1.02, 1.18, 1.67, and 1.70, one secondary methyl as a
doublet at dH 1.07 (J = 6.4 Hz), and a methoxyl group at dH 3.33.
Moreover, four oxygenated methine protons [dH 3.49 (br s, 2H),
3.86 (dd, J = 11.2, 4.8 Hz), 4.41 (td, J = 8.9, 3.6 Hz)], and protons of
two trisubstituted double bonds [dH 5.16 (1H, d, J = 8.4 Hz), and
5.78 (1H, d, J = 4.8 Hz)] were also observed. The 13C NMR spectrum
of 17 revealed 31 carbon signals, which were assigned by a DEPT
experiment as nine methyls, six methylenes, ten methines (includ-
ing four oxygenated and two vinylic), and six quaternary carbons
(two sp2). All the above structural features were corroborated by
two-dimensional NMR data (1H–1H COSY, HMQC and HMBC),
which allowed clearly the assignment of all carbon signals (Ta-
ble 1). In this way, 1H–1H COSY experiments revealed the structure
of four spin-systems (Fig. 2), which were connected on the basis of
—
C
1.30 m; 1.51 m
1.65 m; 1.92 m
1.88 m
0.93 s
1.01 s
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH
CH
C
CH3
CH3
CH3
CH3
CH3
CH3
1.86 m
1.07 d (6.4)
0.98 m; 1.77 m
4.41 td (8.9, 3.6)
5.16 d (8.4)
–
1.67 s
1.70 s
1.02 s
1.18 s
0.76 s
3.33 s
a
Overlapped signals.
parison of the coupling constant pattern with that reported in liter-
ature for similar compounds. Significant NOE effects between H-12/
H-10 (dH 2.33), H-12/Me-30 (dH 0.76), and H12/H-17, together with
coupling constants, indicated an equatorial b-oriented OH group at
C-12 also found in cucurbalsaminols A and B.15 The configuration
at C-23 was assigned as R, mainly by comparison of the 13C NMR data
of the side chain carbons of 17 with those reported for a lanostane-
type triterpene, which stereochemistry was assigned by X-ray dif-
fraction.17 Therefore, the structure of compound 17 was established
as 7b-methoxycucurbita-5,24-diene-3b,12b 23(R)-triol.
Further acylation of 1 with butyric anhydride gave rise to a new
alkanoyl diester, named karavoate F (7). In the same way, acylation
of karavilagenin C with four different aroyl chlorides, namely ben-
zoyl, p-nitrobenzoyl, p-chlorobenzoyl, and p-methoxybenzoyl
chlorides (Scheme 1), yielded seven new ester derivatives: three
monoesters at C-23: karavoates I (9), K (11) and M (13), and four
2
3
key heteronuclear JC–H and JC–H correlations displayed in the
HMBC spectrum that also allowed the location of the functional
groups in the molecule (Fig. 2). Thus, the presence of an unusual
OH group at C-12, indicated by the marked downfield shift of C-
3
12 (
D
dC ffi +40 ppm,
a
-carbon),15 was corroborated by the JC–H
couplings between C-18 and H-12, and between C-10 and H-11,
and the JC–H correlations between C-11, C-13 and H-12.
2
The relative configuration of the tetrahedral stereocenters of
17 was determined by a NOESY experiment ( Fig. 3), taking into
account cucurbitane-type triterpenoids biosynthesis,16 and by com-

332
C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
(IC50) activities, the cytotoxic activity of all the compounds was
HO
determined on breast cancer cells (MCF-7). According to
some authors, a promising lead in drug discovery should have a SI
>10.22 The effects of compounds (1–17) against both strains of
D
23
21
25
OH
12
P. falciparum, expressed as IC50 values in
rized in Table 2, as well as their cytotoxic activity, expressed as IC50
valuesin M, and theselectivityindex. It shouldbe noted thata great
lM and lg/mL, are summa-
18
30
27
l
C
diversity of criteria has been adopted for assessing in vitro antima-
19
larial activity of pure compounds. Some authors consider com-
pounds with IC50 values of 100
others use a stricter endpoint criteria, in which, compounds worthy
of further studies should have IC50 values below 25
M.24 Taking
into account these criteria, in this work the antimalarial activity of
compounds was defined as: IC50 6 1 M, excellent/potent activity;
1 < IC50 6 10 M, good activity; and 10 < IC50 6 30 M, moderate
activity. A compound with an IC50 >30 M, was considered inactive.
l
M and higher as active,23 while
1
4
9
l
A
7
l
HO
OCH3
l
l
B
l
28
COSY
HMBC
29
As can be observed in Table 2, most of the esters derivatives dis-
played antimalarial activity, having the parent compound (1)
exhibited a moderate activity (IC50 = 10.4 and 11.2 lM for 3D7
and Dd2, respectively). Compound 17 was inactive. Interestingly,
a remarkable activity was observed for most of the alkanoyl esters
(2–7). The lowest IC50 values against both P. falciparum strains
were displayed by the diacylated derivatives, karavoates B (3,
(13C → 1H)
Figure 2. Key 1H–1H COSY and HMBC correlations of compound 17.
diesters (at C-3 and C-23), karavoates H (8), J (10), L (12), and N
(14). Two new cinnamoyl derivatives were also obtained, namely
the monoester, karavoate O (15), and the diester, karavoate P (16).
The structure elucidation of these new compounds was based
on a comparison of their spectroscopic data with those of karavi-
lagenin C (1).14,18 In this way, the main differences, between the
NMR data of 1 and the esters 7–16, were observed for carbon
and proton signals of ring A and side chain. In the 1H NMR spectra
of the monoacylated derivatives (9, 11, 13, 15) a significant para-
magnetic effect was observed for the chemical shift of H-23, which
appeared approximately, 1.4 ppm downfield. Similarly, deshielding
IC50 = 0.5 and 0.5
IC50 = 1.5 and 0.4
l
M, for 3D7 and Dd2, respectively) and D (5,
lM, for 3D7 and Dd2, respectively). When com-
pared with karavilagenin C (1), the former ester was approximately
20-fold more active against both strains. Importantly, it is interest-
ing to point that compound 5 was much more active against the
resistant strain (7 and 28-fold against 3D7 and Dd2, respectively).
Similar results were also found for the monobutanoyl derivative,
karavoate E (6) that showed a strong antiplasmodial activity,
mainly against Dd2 strain (IC50 = 3.5 and 0.6 lM, for 3D7 and
Dd2, respectively). According to some authors, the development
of resistance to one agent, in drug-resistant cells (both prokaryotic
and eukaryotic), can confer greater sensitivity to an alternate agent
than observed in the parental (sensitive) cell line.25 The phenom-
enon, named ‘‘collateral sensitivity’’, was first reported for
Escherichia coli,26 however its mechanistic basis is still unclear.
Regarding the set of the aroyl derivatives of karavilagenin C (Table
effects at C-23
dC ffi +1.1, and +2.7 ppm,
effects at C-22 and C-24 (
(
D
dC ffi +5.9 ppm,
a-carbon), C-20, and C-25
-carbons, respectively), and shielding
(D
c
D
dC ffi ꢀ1.7, ꢀ3.9 ppm, b-carbons) were
observed. In the diacyl derivatives (8, 10, 12, 14, 16), besides the
difference in chemical shift of H-23, the signal of H-3 was also
shifted downfield (
NMR spectra, a paramagnetic effect at C-3 (
D
dH ffi +1.24 ppm). In the same way, in the 13C
D
dC ffi +3.3 ppm,
a
-car-
2), karavoates I (9, IC50 = 2.6 and 0.5
tively) and M (13, IC50 = 1.3 and 0.6
l
M, for 3D7 and Dd2, respec-
bon), and diamagnetic effects at C-2 and C-4
ꢀ1.1 ppm, b-carbons) were also observed.
(D
dC ffi ꢀ1.8,
lM, for 3D7 and Dd2, respec-
tively), bearing a p-nitrobenzoyl and a p-methoxybenzoyl moiety
at C-23, respectively, displayed the strongest activity, mainly
against the Dd2 resistant strain. However, karavoate K (11), with
a p-chlorobenzoyl residue at C-23, exhibited an activity lower
2.2. Antimalarial activity in vitro
Compounds (1–17) were evaluated for their in vitro antimalarial
activity against chloroquine-sensitive (3D7) and resistant (Dd2)
P. falciparum strains. A standardized SYBR Green I-based fluores-
cence assay was used in the experiment.19–21 Chloroquine was used
as a positive control. Moreover, in order to calculate the selectivity
index (SI), defined as the ratio between cytotoxic (IC50) and parasitic
(IC50 = 13.3 and 23.3 lM, for 3D7 and Dd2, respectively) than
karavilagenin C, against both strains. A decrease of activity was still
more evident when both positions, C-3 and C-23, bear an aroyl
moiety, as shown in karavoates H (8), J (10), and L (12) (Table 2).
In the same way, a large increase in IC50 values was also found
H3C
OH
18
27
H3C
21
H
CH3
25
CH3
OH
OH
H3C
23
19
24
H
20
9
12
17
1
H
4
8
5
H3C
H
OMe
H
29
7
30CH3
28 CH3
H
H
H
Figure 3. Key NOESY correlations of compound 17.

C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
333
5''
3''
5'
O
3'
7''
1''
O
O
7'
4''
O
2''
1'
O
3''
23
3
1''
O
OCH3
O
4'
2'
8
3'
OCH3
5'
1'
O
OCH3
O
O
3'
O
O
Cl
7'
7
O
N
1'
N
R
O
5'
3'
7'
O
1'
26
O
27
HO
HO
OCH3
25
21
23
18
13
HO
OCH3
N
N
1
19
R = NO2 : 9
R = Cl : 11
O
7
OCH3
5''
3
5
30
OCH3
O
HO
Cl
3''
Cl
H3CO
29
28
7''
O
R
1''
1
O
OCH3
5'
R
3'
5''
3''
7'
1'
7''
O
O
O
1''
O
OCH3
O
N
R
Cl
5'
14
3'
7'
1'
O
7''
O
5''
3''
OCH3
7'
9''
5'
2''
R = NO2 : 10
R = Cl : 12
O
9'
1''
2'
1'
O
3'
O
7'
O
5'
9'
3'
2'
1'
O
OCH3
O
HO
OCH3
16
15
Scheme 1. Preparation of karavilagenin C esters (7–16).
for the dicinnamoyl derivative (16, IC50 = 65.2 and 56.6
3D7 and Dd2, respectively), showing the monocinnamoyl ester a
good antimalarial activity (15, IC50 = 6.6 and 26.7 M, for 3D7
l
M, for
importantly, all derivatives (2–16) showed selectivity index values
higher than those obtained for the original compound, karavilage-
nin C (1, SI = 1.6 and 1.4, for 3D7 and Dd2, respectively).
l
and Dd2, respectively). Regarding cytotoxicity, no significant cyto-
toxic activity was found for karavilagenin C (1) esters. In fact, for
the alkanoyl (5 and 7), aroyl ( 8–14) and cinnamoyl derivatives
Compounds 1–17 represent a set of compounds that may allow
considerations of structure–activity relationship. As illustrated
above, most of the esters showed better activities than the parent
compound, karavilagenin C (1). The lowest IC50 values were found
when both positions C-3 and C-23 bear acetyl or propanoyl groups,
(15 and 16) of karavilagenin C an IC50 >133.3
remaining esters (2–4 and 6) were also inactive or showed a weak
activity, displaying IC50 values ranging from 19.1 to 73.8 M. More
lM was found. The
l
as in karavoates B (3, IC50 = 0.5 and 0.5 lM, for 3D7 and Dd2,

334
C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
Table 2
Antimalarial activity, cytotoxicity, and selectivity index of compounds 1–17
Compounds
IC50 SD
P. falciparum Dd2
g/mL
Selectivity indexa
MCF7/3D7 MCF7/Dd2
P. falciparum 3D7
MCF7 cells
l
M
lg/mL
l
M
l
lM
Karavilagenin C (1)
Karavoate A (2)
Karavoate B (3)
Karavoate C (4)
Karavoate D (5)
Karavoate E (6)
Karavoate F (7)
Karavoate H (8)
Karavoate I (9)
Karavoate J (10)
Karavoate K (11)
Karavoate L (12)
Karavoate M (13)
Karavoate N (14)
Karavoate O (15)
Karavoate P (16)
Cucurbalsaminol C (17)
CQ
10.4 0.7
6.7 1.1
0.5 0.01
5.1 0.02
1.5 0.04
3.5 0.02
6.9 1.9
36.4 7.4
2.6 0.1
18.6 2.5
13.3 0.9
22.5 0.5
1.3 0.01
2.4 0.1
6.6 3.5
65.2 5.3
52.7 2.4
0.016
4.9 0.3
3.5 0.6
11.2 0.7
9.2 0.6
0.5 0.03
22.0 2.3
0.4 0.04
0.6 0.2
8.4 3.2
30.5 3.9
0.5 0.1
20.7 4.1
23.3 2.9
66.0 2.2
0.6 0.2
47.9 12.7
26.7 9.9
56.6 1.2
67.6 7.4
0.2
5.3 0.3
4.7 0.3
0.3 0.01
11.6 1.2
0.2 0.02
0.3 0.1
16.7 2.1
22.9 1.24
68.1 1.62
19.1 0.76
>133.3
73.8 2.13
>133.3
>133.3
>133.3
>133.3
>133.3
>133.3
>133.3
>133.3
>133.3
>133.3
43.3 3.7
b
1.6
3.4
151.2
3.7
1.4
2.5
126.0
0.9
0.3 0.01
2.7 0.01
0.9 0.02
1.9 0.01
4.2 1.2
24.8 5.1
1.6 0.05
14.3 1.9
8.1 0.5
16.8 0.4
0.8 0.01
1.8 0.07
3.9 2.1
47.8 3.9
25.7 1.2
>89.0
20.8
>19.4
>3.7
>51.4
>7.2
>10.0
>5.9
>43.6
>55.4
>20.3
>2.0
0.8
>349.9
116.3
>15.8
>4.4
>296.2
>6.43
>5.7
>2.0
>214.8
>2.8
>5.0
>2.4
0.6
5.1 1.9
20.8 2.7
0.3 0.08
16.0 3.2
14.3 1.8
49.4 1.7
0.4 0.1
35.5 9.4
16.0 6.0
41.5 0.9
33.0 3.6
b
b
a
Selectivity index (SI) = cytotoxicity (IC50)/antiplasmodial activity (IC50).
b
IC50 >100 l
M;28,29 SI (MCF7/3D7) >6250; SI (MCF7/Dd2) >500.
Table 3
respectively) and D (5, IC50 = 1.5 and 0.4 lM, for 3D7 and Dd2,
Physico-chemical properties of compounds 1–17 (topological polar surface area,
number of hydrogen bond acceptors and donors, molecular weight, octanol/water
partition coefficient, and volume)a
respectively). Surprisingly, between butanoyl esters, the most ac-
tive was the monoacylated derivative, karavoate E (6, IC50 = 3.5
and 0.6
played a very strong antimalarial activity against the Dd2 resistant
strain, which was comparable to that found for CQ (IC50 = 0.016
and 0.20 M for 3D7 and Dd2, respectively). In the same way, for
lM, for 3D7 and Dd2, respectively). This compound dis-
Compound
TPSA
No. H
acc. Don.
MW
log P
Volume
lM
l
Karavilagenin C (1)
Karavoate A (2)
Karavoate B (3)
Karavoate C (4)
Karavoate D (5)
Karavoate E (6)
Karavoate F (7)
Karavoate H (8)
Karavoate I (9)
Karavoate J (10)
Karavoate K (11)
Karavoate L (12)
Karavoate M (13)
Karavoate N (14)
Karavoate O (15)
Karavoate P (16)
Cucurbalsaminol C (17)
49.7
55.8
61.8
55.8
61.8
55.8
61.8
61.8
101.6
153.5
55.8
61.8
65.0
80.3
55.8
61.8
69.9
3
4
5
4
5
4
5
5
7
11
4
5
5
7
4
5
4
2
1
0
1
0
1
0
0
0
0
1
0
1
0
1
0
3
472
514
556
528
584
542
612
680
621
770
610
748
606
740
602
732
488
7.3
8.0
8.6
8.3
8.9
8.7
9.3
9.6
9.0
9.6
9.2
9.9
9.0
9.7
9.2
9.8
6.4
499.6
536.1
572.6
552.9
606.2
569.7
639.8
682.3
614.3
729.0
604.5
709.4
616.5
733.4
618.4
737.1
507.6
the aroyl and cinnamoyl derivatives of karavilagenin C, the highest
antimalarial activity was found for the monoesters. This is high-
lighted by the IC50 values obtained for karavoates I (9, IC50 = 2.6
and 0.5
0.6 M, for 3D7 and Dd2, respectively), and O (15, IC50 = 6.6 and
26.7 M, for 3D7 and Dd2, respectively), which showed much
lM, for 3D7 and Dd2, respectively), M (13, IC50 = 1.3 and
l
l
better antiplasmodial activity than the corresponding diesters
(10, 14, and 16, respectively). Moreover, these differences were
more accentuated for the esters without any substituent at the ar-
oyl/cinnamoyl moiety, karavoates H (8, IC50 = 36.4 and 30.5
3D7 and Dd2, respectively) and P (16, IC50 = 65.2 and 56.6
l
M, for
lM, for
3D7 and Dd2, respectively). Therefore, when analyzing some phys-
ico-chemical properties of karavilagenin C and its esters (Table 3),
the results suggest that their antimalarial activity might be influ-
enced by molecular steric effects. In fact, compounds with molec-
ular volumes between 536.1 and 618.4 showed an excellent/good
activity, and those with values between 682.3 and 737.1 exhibited
a weak effect or were inactive. Furthermore, one the highest IC50
values was found for compound 16 with the highest molecular
volume.
a
Physico-chemical parameters were determined by using the JME molecular
editor (version April 2010, http://www.molinspiration.com/).
as solvents. ESIMS were taken on a MicromassÒ Quattro micro™
API. EIMS, HR-EIMS were recorded on a Micromass Autospec spec-
trometer. TLC was performed on precoated SiO2 F254 plates (Merck
5554 and 5744), with visualization under UV light and by spraying
with sulfuric acid/methanol (1:1), followed by heating. Column
chromatography (CC) was carried out on silica gel (Merck 9385).
HPLC was carried out on a Merck–Hitachi instrument, with UV
detection (210 and 220 nm), using a Merck LiChrospher 100 RP-
In conclusion, this study suggests that karavilagenin C, a major
constituent of the African plant medicinal plant M. balsamina, may
be valuable as lead for the development of new antimalarials.
3. Experimental section
3.1. Chemistry
18 (10
l
m, 250 ꢁ 10 mm) column.
3.1.1. General experimental procedures
3.1.2. Extraction and isolation
Optical rotations were obtained using a Perkin Elmer 241 polar-
imeter. IR spectra were determined on a FTIR Nicolet Impact 400,
and NMR spectra recorded on a Bruker ARX-400 NMR spectrome-
ter (1H 400 MHz; 13C 100.61 MHz), using MeOD, CDCl3 and acetone
Dried aerial parts of M. balsamina (1.2 kg) were powdered and
exhaustively extracted with methanol (11 ꢁ 8 L) at room temper-
ature, as previously described.14 Briefly, the MeOH extract was
evaporated to afford a residue (280 g), which was suspended in

C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
335
H2O (1 L) and extracted with EtOAc (9 ꢁ 0.5 L). The EtOAc residue
(85 g) was suspended in MeOH/H2O (9:1; 1 L), and extracted with
n-hexane (5 ꢁ 0.5 L) for removal of waxy material that was not fur-
ther studied. The remaining extract was evaporated under vacuum
(40 °C), yielding a residue (45 g) that was chromatographed over
silica gel (1 kg), using mixtures of n-hexane/EtOAc (1:0–0:1) and
EtOAc/MeOH (19:1–0:1) as eluents to obtain six fractions (Fr
1–6), which were combined according to TLC analysis. Compound
17 (50 mg) was obtained from the crude fraction Fr 3, eluted with
mixtures of n-hexane/EtOAc (9:11–1:19), after successive column
chromatography, and further purification by HPLC (210 nm,
MeOH/H2O, 17:3, 5 mL/min, Rt = 9 min).
(2H, d, J = 7.1 Hz, H-30/H-70), 7.56 (2H, m, H-50/H-500), 7.45 (4H,
m, H-40/H-400/H-60/H-600), 5.89 (1H, d, J = 4.7 Hz, H-6), 5.87 (1H, td,
J = 9.2, 2.8 Hz, H-23), 5.24 (1H, d, J = 8.9 Hz, H-24), 5.03 (1H, br
s, H-3), 3.49 (1H, br d, J = 4.3 Hz, H-7), 3.40 (3H, s, 7-OMe), 2.40
(1H, dd, J = 11.2, 4.4 Hz, H-10), 2.08 (1H, br s, H-8), 1.84 (3H, s,
Me-26), 1.74 (3H, s, Me-27), 1.22 (3H, s, Me-29), 1.17 (3H, s, Me-
28), 1.04 (3H, s, Me-19), 1.03 (3H, d, J = 6.3 Hz, Me-21), 0.92 (3H,
s, Me-18), 0.78 (3H, s, Me-30). 13C NMR (101 MHz, CDCl3): d
166.1 (C-100), 166.0 (C-10), 146.8 (C-5), 135.9 (C-25), 132.8 (C-50),
132.7 (C-500), 130.9 (C-200), 130.6 (C-20), 129.7 (C-30/C-70), 129.5
(C-300/C-700), 128.4 (C-40/C-60), 128.3 (C-400/C-600), 124.6 (C-24),
119.5 (C-6), 79.3 (C-3), 77.4 (C-7), 70.3 (C-23), 56.3 (7-OMe), 50.5
(C-17), 48.2 (C-8), 48.0 (C-14), 46.2 (C-13), 42.1 (C-22), 40.3 (C-
4), 38.6 (C-10), 34.7 (C-15), 34.1 (C-9), 33.0 (C-20), 32.2 (C-11),
30.1 (C-12), 28.5 (C-19), 28.1 (C-28), 28.0 (C-16), 26.5 (C-2), 25.8
(C-27), 25.2 (C-29), 21.7 (C-1), 19.1 (C-21), 18.4 (C-26), 18.0 (C-
30), 15.4 (C-18).
3.1.2.1. Cucurbalsaminol C, 7b-methoxycucurbita-5,24-diene-
3b,12b 23(R)-triol (17). Amorphous white powder; ½a D26
Š
+117 (c
0.11, MeOH); IR (KBR) mmax 3443, 1455, 1382, 1182, 1138, 1024,
978, 939 cmꢀ1 1H NMR and 13C NMR data, see Table 1; EIMS m/
;
z: 488 [M]+ (30), 470 [MꢀH2O]+ (43), 452 [Mꢀ2 ꢁ H2O]+ (16),
438 (8), 434 [Mꢀ3 ꢁ H2O]+ (12), 420 [Mꢀ2 ꢁ H2O–CH3OH]+ (12),
402 (3), 388 (2), 344 (15), 312(13), 270 (7), 223 (23), 189 (19),
182 (18), 172 (17), 164 (25), 109 (100); HR-EIMS m/z: 488.3867
[M]+ (calcd for C31H52O4, 488.3866).
3.1.3.3. Acylation with p-nitrobenzoyl chloride. To compound 1
(43.5 mg) was added pyridine (1 mL) and p-nitrobenzoyl chloride
in excess (150 mg), and the mixture was stirred at room tempera-
ture for 24 hours. The excess of pyridine was removed with N2 and
the residue was purified by silica gel column chromatography
(n-hexane/EtOAc, 0:1–1:1) and preparative TLC (n-hexane/EtOAc,
3:2) to afford 45 mg of compound 9 and 10 mg of 10.
3.1.3. Preparation of karavilagenin C esters
3.1.3.1. Acylation with butyric anhydride. 31 mg of compound 1
were suspended in butyric anhydride (0.5 mL) and pyridine
(0.5 mL), and the reaction mixture was stirred for 72 hours at
80 °C. The mixture was diluted with ethyl acetate and washed suc-
cessively with Na2CO3 (5%) and HCl (1%) solutions, dried over
Na2SO3, and filtered. The crude product was purified twice by pre-
parative TLC, using n-hexane/EtOAc (13:7) and n-hexane/EtOAc
(9:1), as eluents, to afford 26 mg of compound 7.
3.1.3.3.1. Karavoate I, 23(R)-(p-nitrobenzoyloxy)-7b-methoxycu-
curbita-5,24-dien-3b-ol (9). Compound 9 colorless oil; IR (KBr)
mmax 3447, 1716, 1606, 1529, 1457, 1273, 1085, 850, 758 cmꢀ1
.
ESIMS m/z (rel. int.): 644 [M+Na]+ (68), 509 (100), 495 (25), 477
[M+Na–C7H5O4N]+ (21), 423 (45), 316 (29), 288 (83), 249 (22), 241
(13). 1H NMR (400 MHz, CD3COCD3): d 8.34 (2H, d, J = 7.8 Hz, H-40/
H-60), 8.25 (2H, d, J = 7.9 Hz, H-30/H-70), 5.91 (1H, td, J = 8.8, 2.0 Hz,
H-23), 5.73 (1H, d, J = 5.0 Hz, H-6), 5.27 (1H, d, J = 8.9 Hz, H-24),
3.46 (1H, br s, H-3), 3.36 (1H, br d, J = 5.2 Hz, H-7), 3.23 (3H, s, 7-
OMe), 2.30 (1H, br d, J = 10.5 Hz, H-10), 1.98 (1H, br s, H-8), 1.79
(3H, s, Me-26), 1.70 (3H, s, Me-27), 1.15 (3H, s, Me-29), 1.02
(3H, d, J = 6.3 Hz, Me-21), 1.00 (3H, s, Me-28), 0.91 (3H, s, Me-19),
0.86 (3H, s, Me-18), 0.75 (3H, s, Me-30). 13C NMR (101 MHz,
CD3COCD3): d 164.6 (C-10), 151.5 (C-50), 148.0 (C-5), 137.1 (C-20),
137.0 (C-25), 131.4 (C-30/C-70), 125.1 (C-24), 124.5 (C-40/C-60),
120.1 (C-6), 77.8 (C-7), 76.5 (C-3), 71.7 (C-23), 56.2 (7-OMe), 51.2
(C-17), 49.1 (C-8), 48.8 (C-14), 46.9 (C-13), 42.7 (C-22), 42.1 (C-4),
39.6 (C-10), 35.4 (C-15), 34.7 (C-9), 33.8 (C-20), 33.3 (C-11), 31.0
(C-12), 30.0 (C-2), 29.2 (C-19), 28.7 (C-16), 28.5 (C-28), 26.0 (C-
29), 25.8 (C-27), 21.9 (C-1), 19.4 (C-21), 18.6 (C-26), 18.5 (C-30),
15.7 (C-18).
3.1.3.1.1. Karavoate F, 3b,23(R)-dibutanoyloxy-7b-methoxycucur-
bita-5,24-diene (7). Compound 7 colorless oil; (KBr) mmax 1730,
1243 cmꢀ1. ESIMS m/z (rel. int.): 635 [M+Na]+ (62), 547 [M+Na–
CH3CH2CH2COOH]+ (50), 517 (33), 454 (100). 1H NMR (400 MHz,
CD3COCD3): d 5.79 (1H, d, J = 5.0 Hz, H-6), 5.67 (1H, td, J = 10.5,
3.0 Hz, H-23), 5.13 (1H, d, J = 8.9 Hz, H-24), 4.72 (1H, br s, H-3),
3.43 (1H, br d, J = 5.1 Hz, H-7), 3.30 (3H, s, 7-OMe), 2.42 (1H, br
d, J = 10.6 Hz, H-10), 2.26 (2H, t, J = 7.3 Hz, H-20), 2.24 (2H, t,
J = 7.3 Hz, H-200), 2.04 (1H, br s, H-8), 1.72 (3H, s, Me-26), 1.69
(3H, s, Me-27), 1.62 (4H, m, H-30/H-300), 1.12 (3H, s, Me-28), 1.11
(3H, s, Me-29), 0.98 (3H, d, J = 5.3 Hz, Me-21), 0.98 (3H, s, Me-
19), 0.95 (3H, s, Me-18), 0.94 (3H, t, J = 7.3 Hz, Me-40), 0.92 (3H, t,
J = 7.4 Hz, Me-400), 0.78 (3H, s, Me-30). 13C NMR (101 MHz,
CD3COCD3): d 173.0 (C-100), 172.9 (C-10), 147.0 (C-5), 135.7 (C-25),
126.0 (C-24), 120.4 (C-6), 78.9 (C-3), 77.7 (C-7), 69.1 (C-23), 56.3
(7-OMe), 51.3 (C-17), 49.3 (C-8), 48.8 (C-14), 46.9 (C-13), 42.8 (C-
22), 40.7 (C-4), 39.3 (C-10), 36.9 (C-20/C-200), 35.3 (C-15), 34.8 (C-
9), 33.6 (C-11), 33.2 (C-20), 30.9 (C-12), 29.3 (C-19), 28.6 (C-16),
28.3 (C-28), 27.0 (C-2), 25.7 (C-27), 25.4 (C-29), 22.4 (C-1), 19.3
(C-30/C-300), 19.2 (C-21), 18.4 (C-26/C-30), 15.7 (C-18), 13.9 (C-40/
C-400).
3.1.3.3.2. Karavoate J, 3b,23(R)-di-(p-nitrobenzoyloxy)-7b-meth-
oxycucurbita-5,24-diene (10). Compound 10 colorless oil; IR (KBr)
m
max 1716, 1605, 1530, 1455, 1383, 1276, 1102, 850 cmꢀ1. 1H NMR
(400 MHz, CD3COCD3): d 8.39 (4H, dd, J = 8.9, 1.9 Hz, H40/H400/H-60/
H-600), 8.30 (2H, d, J = 8.9 Hz, H-40/H-60), 8.23 (2H, d, J = 8.9 Hz, H-
400/H-600), 5.96 (1H, td, J = 9.0, 3.0 Hz, H-23), 5.90 (1H, d, J = 4.7 Hz,
H-6), 5.32 (1H, d, J = 9.0 Hz, H-24), 5.06 (1H, br s, H-3), 3.49 (1H,
br d, J = 4.7 Hz, H-7), 3.34 (3H, s, 7-OMe), 2.55 (1H, br d,
J = 10.0 Hz, H-10), 2.10 (1H, br s, H-8), 1.84 (3H, s, Me-26), 1.74
(3H, s, Me-27), 1.24 (3H, s, Me-28), 1.21 (3H, s, Me-29), 1.06 (3H,
d, J = 6.4 Hz, Me-21), 1.03 (3H, s, Me-19), 0.93 (3H, s, Me-18),
0.84 (3H, s, Me-30). 13C NMR (101 MHz, CD3COCD3): d 164.7 (C-
100), 164.4 (C-10), 151.6 (C-50/C-500), 146.6 (C-5), 137.1 (C-20/C-200),
137.0 (C-25), 131.4 (C-30/C-300/C-70/C-700), 125.1 (C-24), 124.6 (C-
40/C-400/C-60/C-600), 121.0 (C-6), 81.3 (C-3), 77.6 (C-7), 71.8 (C-23),
56.3 (7-OMe), 51.3 (C-17), 49.1 (C-8), 48.8 (C-14), 47.0 (C-13),
42.7 (C-22), 41.0 (C-4), 39.1 (C-10), 35.4 (C-15), 34.8 (C-9), 33.8
(C-20), 33.1 (C-11), 30.9 (C-12), 29.2 (C-19), 28.7 (C-16), 28.1 (C-
28), 26.9 (C-2), 25.8 (C-27), 25.6 (C-29), 22.6 (C-1), 19.4 (C-21),
18.5 (C-26/C-30), 15.7 (C-18).
3.1.3.2. Acylation with benzoyl chloride. To compound
1
(30.0 mg) was added pyridine (1 mL) and benzoyl chloride
(0.5 mL), and the mixture was stirred at room temperature for 1
hour. The reaction mixture was treated as above. The residue
obtained was purified, sequentially, by column chromatography
(n-hexane/CH2Cl2, 2:3 to n-hexane/CH2Cl2, 0:1) and preparative
TLC (n-hexane/CH2Cl2, 1:9), to afford 15 mg of compound 8.
3.1.3.2.1. Karavoate H, 3b,23(R)-dibenzoyloxy-7b-methoxycucur-
bita-5,24-diene (8). Compound 8 colorless oil; IR (KBr) mmax 1716,
1520, 1455, 1270, 934, 712, 736 cmꢀ1. ESIMS m/z (rel. int.): 681
[M+H]+ (18), 703 [M+Na]+ (19), 581 [M+Na–C6H5COOH]+ (28). 1H
NMR (400 MHz, CDCl3): d 8.04 (2H, d, J = 7.1 Hz, H-300/H-700), 8.02

336
C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
3.1.3.4. Acylation with p-chlorobenzoyl chloride. To a solution
of compound 1 (25 mg) in pyridine, 60 L of p-chlorobenzoyl chlo-
J = 10.3 Hz, H-10), 2.01 (1H, br s, H-8), 1.79 (3H, s, Me-26), 1.69
(3H, s, Me-27), 1.18 (3H, s, Me-29), 1.03 (3H, s, Me-28), 1.02 (3H,
d, J = 7.7 Hz, Me-21), 0.94 (3H, s, Me-19), 0.88 (3H, s, Me-18),
0.77 (3H, s, Me-30). 13C NMR (101 MHz, CD3COCD3): d 165.9 (C-
10), 164.3 (C-50), 148.0 (C-5), 135.9 (C-25), 132.1 (C-30/C-70), 125.9
(C-24), 124.0 (C-20), 120.1 (C-6), 114.5 (C-40/C-60), 77.8 (C-7), 76.5
(C-3), 69.9 (C-23), 56.2 (7-OMe), 55.8 (50-OMe), 51.2 (C-17), 49.1
(C-8), 48.7 (C-14), 46.9 (C-13), 42.8 (C-22), 42.0 (C-4), 39.6 (C-
10), 35.3 (C-15), 34.7 (C-9), 33.7 (C-20), 33.3 (C-11), 30.9 (C-12),
30.1 (C-2), 29.2 (C-19), 28.7 (C-16), 28.5 (C-28), 26.0 (C-29), 25.8
(C-27), 22.0 (C-1), 19.4 (C-21), 18.5 (C-30), 18.4 (C-26), 15.6 (C-18).
3.1.3.5.2. Karavoate N, 3b,23(R)-di-(p -methoxybenzoyloxy)-7b-
methoxycucurbita-5,24-diene (14). Compound 14 colorless oil; IR
(KBr) mmax 1716, 1593, 1455, 1271, 1171, 1093, 934, 850, 759 cm
ꢀ1. ESIMS m/z (rel. int.): 763 [M+Na]+ (67), 611 [M+Na–C8H8O3]+
(100), 589 (28), 561 (38), 517 (57), 501 (42), 473 (43), 457 (18).
1H NMR (400 MHz, CD3COCD3): d 7.96 (2H, d, J = 8.8 Hz, H-300/H-
700), 7.93 (2H, d, J = 8.8 Hz, H-30/H-70), 7.02 (4H, m, H-40/H-400/H-
60/H-600), 5.88 (1H, d, J = 5.0 Hz, H-6), 5.86 (1H, td, J = 10.0,
3.2 Hz, H-23), 5.26 (1H, d, J = 8.2 Hz, H-24), 4.95 (1H, br s, H-3),
3.88 (6H, s, 50-OMe/500-OMe), 3.47 (1H, br d, J = 4.7 Hz, H-7), 3.33
(3H, s, 7-OMe), 2.50 (1H, dd, J = 11.0, 3.0 Hz, H-10), 2.08 (1H, br
s, H-8), 1.80 (3H, s, Me-26), 1.71 (3H, s, Me-27), 1.19 (3H, s, Me-
29), 1.17 (3H, s, Me-28), 1.03 (3H, s, Me-19), 1.02 (3H, d,
J = 5.8 Hz, Me-21), 0.91 (3H, s, Me-18), 0.82 (3H, s, Me-30). 13C
NMR (101 MHz, CD3COCD3): d 165.9 (C-100), 165.6 (C-10), 164.4
(C-500), 164.3 (C-50), 147.0 (C-5), 136.0 (C-25), 132.1 (C-300/C-700),
132.0 (C-30/C-70), 125.9 (C-24), 124.0 (C-200), 123.9 (C-20), 120.6
(C-6), 114.6 (C-40/C-400/C-60/C-600), 79.5 (C-3), 77.7 (C-7), 69.9 (C-
23), 56.3 (7-OMe), 55.9 (50-OMe/500-OMe), 51.2 (C-17), 49.1 (C-8),
48.8 (C-14), 46.9 (C-13), 42.8 (C-22), 41.0 (C-4), 39.1 (C-10), 35.3
(C-15), 34.8 (C-9), 33.7 (C-20), 33.1 (C-11), 30.9 (C-12), 29.1 (C-
19), 28.7 (C-16), 28.1 (C-28), 27.0 (C-2), 25.8 (C-27), 25.6 (C-29),
22.5 (C-1), 19.4 (C-21), 18.5 (C-30), 18.4 (C-26), 15.7 (C-18).
l
ride were added. The mixture was stirred at room temperature, and
after 15 min the excess of pyridine was removed with N2. The crude
product was purified by column chromatography (n-hexane/EtOAc,
1:0–7:3) and preparative TLC (n-hexane/EtOAc, 3:2), giving rise to
8 mg of compound 11 and 24 mg of 12.
3.1.3.4.1. Karavoate K, 23(R)-(p-chlorobenzoyloxy)-7b-methoxycu-
curbita-5,24-dien-3b-ol (11). Compound 11 colorless oil; IR (KBr)
mmax 3431, 1716, 1594, 1455, 1381, 1272, 1088, 935, 851,
759 cmꢀ1. ESIMS m/z (rel. int.): 649 [M+K]+ (29), 633 [M+Na]+
(55.8), 477 [M+Na–C7H5O2Cl]+ (100). 1H NMR (400 MHz, CD3-
COCD3): d 8.00 (2H, d, J = 8.8 Hz, H-30/H-70), 7.54 (2H, d, J = 8.8 Hz,
H-40/H-60), 5.88 (1H, td, J = 9.0, 3.3 Hz, H-23), 5.75 (1H, d,
J = 5.0 Hz, H-6), 5.25 (1H, d, J = 8.8 Hz, H-24), 3.49 (1H, br s, H-3),
3.39 (1H, br d, J = 5.3 Hz, H-7), 3.26 (3H, s, 7-OMe), 2.33 (1H, br d,
J = 10.3 Hz, H-10), 2.00 (1H, br s, H-8), 1.79 (3H, s, Me-26), 1.70
(3H, s, Me-27), 1.17 (3H, s, Me-29), 1.02 (3H, s, Me-28), 1.02 (3H, d,
J = 7.7 Hz, Me-21), 0.93 (3H, s, Me-19), 0.88 (3H, s, Me-18), 0.77
13
(3H, s, Me-30). C NMR (101 MHz, CD3COCD3): d 165.3 (C-10),
148.1 (C-5), 139.4 (C-50), 136.6 (C-25), 131.8 (C-30/C-70), 130.5
(C-20), 129.6 (C-40/C60), 125.4 (C-24), 120.1 (C-6), 77.8 (C-7), 76.5
(C-3), 70.9 (C-23), 56.2 (7-OMe), 51.2 (C-17), 49.2 (C-8), 48.8 (C-
14), 46.9 (C-13), 42.7 (C-22), 42.1 (C-4), 39.6 (C-10), 35.3 (C-15),
34.7 (C-9), 33.8 (C-20), 33.3 (C-11), 30.9 (C-12), 30.1 (C-2), 29.2 (C-
19), 28.7 (C-16), 28.5 (C-28), 26.0 (C-29), 25.7 (C-27), 21.9 (C-1),
19.4 (C-21), 18.5 (C-30), 18.4 (C-26), 15.6 (C-18).
3.1.3.4.2. Karavoate L, 3b,23(R)-di-(p-chlorobenzoyloxy)-7b-meth-
oxycucurbita-5,24-diene (12). Compound 12 colorless oil; IR (KBr)
mmax 1717, 1559, 1455, 1381, 1272, 1088, 935, 850, 758 cmꢀ1
.
ESIMS m/z (rel. int.): 771 [M+Na]+ (2). 1H NMR (400 MHz,
CD3COCD3): d 8.04 (2H, d, J = 8.8 Hz, H-300/H-700), 7.98 (2H, d,
J = 8.8 Hz, H-30/H-70), 7.56 (4H, m, H-40/H-400/H-60/H-600), 5.88 (1H,
dt, J = 9.0, 3.3 Hz, H-23), 5.87 (1H, d, J = 4.7 Hz, H-6), 5.27 (1H,
d, J = 8.8 Hz, H-24), 4.99 (1H, br s, H-3), 3.47 (1H, br d, J = 5.0 Hz,
H-7), 3.31 (3H, s, 7-OMe), 2.52 (1H, br d, J = 10.0 Hz, H-10), 2.12
(1H, br s, H-8), 1.82 (3H, s, Me-26), 1.73 (3H, s, Me-27), 1.19 (3H,
s, Me-29), 1.22 (3H, s, Me-28), 1.04 (3H, d, J = 6.0 Hz, Me-21),
1.03 (3H, s, Me-19), 0.93 (3H, s, Me-18), 0.84 (3H, s, Me-30). 13C
NMR (101 MHz, CD3COCD3): d 165.4 (C-100), 165.1 (C-10), 146.8
(C-5), 139.5 (C-50/C-500), 136.6 (C-25), 131.8 (C-30/C-300/C-70/C-700),
130.5 (C-20/C-200), 129.7 (C-40/C-400/C60/C-600), 125.4 (C-24), 120.8
(C-6), 80.5 (C-3), 77.7 (C-7), 70.9 (C-23), 56.3 (7-OMe), 51.3 (C-
17), 49.1 (C-8), 48.8 (C-14), 46.9 (C-13), 42.7 (C-22), 41.0 (C-4),
39.1 (C-10), 35.4 (C-15), 34.9 (C-9), 33.8 (C-20), 33.2 (C-11), 30.9
(C-12), 29.1 (C-19), 28.8 (C-16), 28.1 (C-28), 27.0 (C-2), 25.8 (C-
27), 25.6 (C-29), 22.6 (C-1), 19.4 (C-21), 18.5 (C-30), 18.4 (C-26),
15.6 (C-18).
3.1.3.6. Acylation with cinnamoyl chloride. Compound
1
(30.6 mg), dissolved in pyridine (1 mL), was added to cinnamoyl
chloride (50 mg), and the reaction mixture was kept at room tem-
perature for 48 hours. After removal of the excess of pyridine, the
mixture was purified by column chromatography (n-hexane/
CH2Cl2, 1:4–0:1, and CH2Cl2/Me2CO, 0:1–9:1), and preparative
TLC (n-hexane/EtOAc, 7:3) to give 12 mg of compound 15 and
8 mg of 16.
3.1.3.6.1. Karavoate O, 23(R)-cinnamoyloxy-7b-methoxycucurbita-
5,24-dien-3b-ol (15). Compound 15 colorless oil; IR (KBr) mmax
3446, 1718, 1450, 1271, 1171, 759, 735 cmꢀ1. ESIMS m/z (rel.
int.): 625 [M+Na]+ (100), 477 [M+Na–C9H8O2]+ (49). 1H NMR
(400 MHz, CD3COCD3): d 7.68–7.63 (2H, m, H-50/H-90), 7.67 (1H,
d, J = 16.0 Hz, H-30), 7.45 (3H, m, H-60/H-80/H-70), 6.56 (1H, d,
J = 16.0 Hz, H-20), 5.80 (1H, td, J = 9.2, 2.8 Hz, H-23), 5.77 (1H, d,
J = 5.2 Hz, H-6), 5.22 (1H, d, J = 8.9 Hz, H-24), 3.50 (1H, br s, H-
3), 3.41 (1H, br d, J = 5.3 Hz, H-7), 3.28 (3H, s, 7-OMe), 2.36 (1H,
br d, J = 11.2 Hz, H-10), 1.80 (3H, s, Me-26), 1.72 (3H, s, Me-27),
1.20 (3H, s, Me-29), 1.05 (3H, s, Me-28), 1.03 (3H, d, J = 6.2 Hz,
Me-21), 0.97 (3H, s, Me-19), 0.94 (3H, s, Me-18), 0.79 (3H, s, Me-
3.1.3.5. Acylation with p-methoxybenzoyl chloride. To a solu-
tion of compound
1 (32.7 mg) in pyridine (1 mL), 50 lL of
p-methoxybenzoyl chloride were added. The mixture was stirred
at room temperature. After 24 hours, the excess of pyridine was
removed with N2. The crude product was purified by column chro-
matography, (n-hexane/EtOAc, 1:0–4:1), and preparative TLC
(n-hexane/EtOAc, 4:1), to afford 20 mg of compound 13 and
23 mg of compound 14.
13
30). C NMR (101 MHz, CD3COCD3): d 166.5 (C-10), 148.0 (C-5),
144.9 (C-30), 136.0 (C-25), 135.4 (C-40), 131.0 (C-60/C-80), 129.7
(C-70), 128.9 (C-50/C90), 125.8 (C-24), 120.0 (C-6), 119.4 (C-20),
77.7 (C-7), 76.4 (C-3), 69.5 (C-23), 56.3 (7-OMe), 51.2 (C-17),
49.1 (C-8), 48.7 (C-14), 46.8 (C-13), 42.7 (C-22), 42.0 (C-4), 39.6
(C-10), 35.2 (C-15), 34.6 (C-9), 33.6 (C-20), 33.2 (C-11), 30.7
(C-12), 29.9 (C-2), 29.1 (C-19), 28.5 (C-16), 28.4 (C-28), 25.9
(C-27), 25.6 (C-29), 21.8 (C-1), 19.3 (C-21), 18.3 (C-26/C-30), 15.6
(C-18).
3.1.3.5.1. Karavoate M, 23(R)-(p-methoxybenzoyloxy)-7b-meth-
oxycucurbita-5,24-dien-3b-ol (13). Compound 13 colorless oil; IR
(KBr) mmax 3446, 1716, 1593, 1455, 1271, 1171, 1093, 934, 851.
759 cmꢀ1. ESIMS m/z (rel. int.): 629 [M+Na]+ (2), 454 [MꢀC8H8O3]+
(100). 1H NMR (400 MHz, CD3COCD3): d 7.97 (2H, d, J = 8.8 Hz,
H-30/H-70), 7.02 (2H, d,
J
= 8.8 Hz, H-40/H-60), 5.86 (1H, td,
J = 10.0, 3.2 Hz, H-23), 5.76 (1H, d, J = 5.0 Hz, H-6), 5.24 (1H, d,
J = 8.8 Hz, H-24), 3.87 (3H, s, 50-OMe), 3.49 (1H, br s, H-3), 3.39
(1H, br d, J = 5.3 Hz, H-7), 3.26 (3H, s, 7-OMe), 2.33 (1H, br d,
3.1.3.6.2. Karavoate P, 3b,23(R)-dicinnamoyloxy-7b-methoxycu-
curbita-5,24-diene (16). Compound 16 colorless oil; IR (KBr) mmax

C. Ramalhete et al. / Bioorg. Med. Chem. 19 (2011) 330–338
337
1718, 1455, 1271, 1171, 759, 735 cmꢀ1. ESIMS m/z (rel. int.): 755
[M+Na]+ (54), 607 [M+Na–C9H8O2]+ (100). 1H NMR (400 MHz,
CD3COCD3): d 7.70–7.62 (4H, m, H-50/H-500/H-90/H-900), 7.68 (1H,
d, J = 16.0 Hz, H-300), 7.66 (1H, d, J = 16.0 Hz, H-30), 7.47–7.40 (6H,
m, H-60/H-600/H-70/H-700/H-80/H-800), 6.55 (1H, d, J = 16.0 Hz, H-200),
6.54 (1H, d, J = 16.0 Hz, H-20), 5.82 (1H, d, J = 5.6 Hz, H-6), 5.80
(1H, td, J = 9.6, 3.2 Hz, H-23), 5.20 (1H, d, J = 9.0 Hz, H-24), 4.85
(1H, br s, H-3), 3.44 (1H, br d, J = 4.6 Hz, H-7), 3.31 (3H, s, 7-
OMe), 2.46 (1H, br d, J = 11.2 Hz, H-10), 1.76 (3H, s, Me-26), 1.70
(3H, s, Me-27), 1.16 (3H, s, Me-28), 1.14 (3H, s, Me-29), 1.01 (3H,
d, J = 6.0 Hz, Me-21), 1.00 (3H, s, Me-19), 0.93 (3H, s, Me-18),
0.80 (3H, s, Me-30). 13C NMR (101 MHz, CD3COCD3): d 166.3
(C-10/C-100), 146.9 (C-5), 145.1 (C-30), 144.9 (C-300), 136.0 (C-25),
135.4 (C-40/C-400), 131.0 (C-60/C-600/C-80/C-800), 129.7 (C-70/C-700),
128.9 (C-50/C-500/C90/C-900), 125.8 (C-24), 120.5 (C-6), 119.4 (C-20/
C-200), 79.2 (C-3), 77.6 (C-7), 69.6 (C-23), 56.2 (7-OMe), 51.2 (C-
17), 49.0 (C-8), 48.7 (C-14), 46.8 (C-13), 42.7 (C-22), 40.8 (C-4),
39.1 (C-10), 35.2 (C-15), 34.7 (C-9), 33.6 (C-20), 33.1 (C-11), 30.8
(C-12), 29.1 (C-19), 28.5 (C-16), 28.1 (C-28), 26.9 (C-2), 25.7 (C-
27), 25.4 (C-29), 22.4 (C-1), 19.3 (C-21), 18.4 (C-26), 18.3 (C-30),
15.6 (C-18).
dium dodecyl sulfate (SDS) (Sigma, St Louis, MO, USA) solution
(10%) was measured into each well and the plates were further
incubated at 37 °C overnight. The cell growth was determined by
measuring the optical density (OD) at 550 nm (ref. 630 nm) with
a Dynatech MRX vertical beam ELISA reader. Inhibition of cell
growth (as
a
percentage) was determined according to the
formula:
ꢀ
ꢁ
ODsample ꢀ ODmedium
control
100 ꢀ
ꢁ 100
ODcell control ꢀ ODmedium
control
where IC50 is defined as the inhibitory dose that reduces the growth
of the compound-exposed cells by 50%. The IC50 values are ex-
pressed as means SD from three experiments.
Acknowledgments
The Science and Technology Foundation, Portugal (FCT, grant
SFRH/BD/22321/2005) supported this work. The authors thank
Mrs. Vigyikán Várady Aniko for technical assistance with the tissue
cultures, and also Dr. Catarina Arruda, from the Portuguese
Embassy in Mozambique, as well as the Portuguese Office of
International Affairs for plant transport.
3.2. Biological assays
Supplementary data
3.2.1. Antimalarial assay
Human malaria parasites were cultured as previously described
by Trager and Jensen (1976), with minor modifications.27 Briefly,
3D7 and Dd2 P. falciparum strains were cultivated in recently col-
lected erythrocytes as host cells in RPMI 1640 medium (Gibco)
containing 25 mM HEPES (Sigma) and 6.8 mM hypoxanthine (Sig-
ma) supplemented with 10% AlbuMAX II (Invitrogen). Cultures
were maintained at 37 °C under an atmosphere of 5% O2, 3–5%
CO2, and N2. The antimalarial activity of the compounds was deter-
mined by a fluorometric method using SYBR Green I.19,21 In brief,
stock solutions of the samples were prepared in DMSO (10 mg/
mL), and were diluted to give a series of concentrations ranging
Supplementary data (1D and 2D NMR spectra of compound 17)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.11.015. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
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