Antimalarial Chalcones
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4451
0.2 mmol) in absolute alcohol (10 mL), and the mixture was
stirred for 4 h at room temperature and then diluted with
water (40 mL). The mixture was extracted with ethyl acetate
(50 mL × 3), and the combined organic phases were concen-
trated in vacuo to give the crude hydroxylated chalcone. The
crude product was purified by column chromatography using
silica gel (230-400 mesh ASTM) as the stationary phase and
CHCl3/hexane as the mobile phase. For all compounds, re-
crystallization was done twice and purity was checked by TLC
before characterization by 1H NMR and accurate mass and
elemental analyses. The yields of the synthesized compounds,
their melting points, and spectroscopic and elemental analyses
data are given in the Supporting Information (Table 1).
mobile phase and an aliquot (10 µL) was injected for the
determination of retention time. Triplicate determinations
were done for each concentration of test compound at each
mobile-phase composition. The capacity factor (k′) was deter-
mined from log k′ ) log[(Vs - Vo)/Vo], where Vs and Vo are the
retention volumes of the test compound and acetone, respec-
tively. Linear regression of log k′ of each compound against
mobile-phase composition and extrapolation to 100% aqueous
phase gave log kw of the compound at pH 7.0.
Deter m in a tion of th e Ch em ica l Sh ift of th e Ca r bon yl
Ca r bon . 13C NMR spectroscopy was used to determine the
chemical shift of the carbonyl carbon in the trimethoxy series
of chalcones. The difference in chemical shift is given by ∆δ )
δX - δR, where δX is the chemical shift of the trimethoxychal-
cone with a substituted A ring and δR is that of the reference
compound (2′,3′,4′-trimethoxychalcone with no A ring substitu-
tion, 189.976 ppm). ∆δ is known to be sensitive to the
electronic influence of the alkyl/aryl moieties18,19 and is used
here to give a direct assessment of the electronic effects of the
A ring, which is attached by conjugation to the carbonyl
carbon. The 13C NMR spectra of the chalcones (CDCl3 or
dimethyl-d6 sulfoxide, tetramethylsilane as reference) were
determined on a Bruker ACF 300 instrument.
Molecu la r Mod elin g Meth od s. The following parameters
were determined from the force-field-minimized geometries of
the chalcones using the SYBYL 6.6 force field MMFF94 (Tripos
Associates, St Louis, MO), with calculations continued until
the rms gradient was less than 0.001 kcal mol-1 Å: ClogP,
molecular refractivity (MR), total dipole moment (TDM),
Connolly surfaces (volume and surface area, calculated from
MOLCAD in SYBYL), and negative charge on the carbonyl
oxygen using the Gasteiger-Huckel method. Orbital energies
for HOMO and LUMO were calculated from MOPAC (QCPE
program 455, version 6.0), which is interfaced with SYBYL.
Sta tistica l Meth od s. Multiple linear regression analyses
were carried out using SPSS 10 (SPSS, Inc., Chicago, IL). The
following statistical parameters were determined for each
regression equation: 95% confidence interval variables, mea-
sure of explained variance r2, Fischer significance ratio F at
P ) 0.05, and standard error SE. Cross-validated r2 and SE
were determined using the QSAR module of SYBYL 6.6.
Multivariate data analyses were performed with SIMCA-P
(version 8.0)20 using default settings.
Eva lu a tion of in Vitr o An tim a la r ia l Activity. The in
vitro antimalarial activities of compounds 1-92 were evalu-
ated by the method of Desjardins et al.,16 with modifications.
Briefly the assay measures the incorporation of [3H] hypox-
anthine by the parasites and the inhibition of the incorporation
in the presence of the test compound. A strain of chloroquine
resistant (K1) Plasmodium falciparum was used in the assay.
The test compounds were dissolved in DMSO and serially
diluted 10-fold with complete culture media (RPMI-1640, 5%
sodium bicarbonate, and 10% normal type “O” human serum)
to give a 106-fold concentration range. The diluted drugs (25
µL) were transferred to wells in a 96-well microtiter plate,
together with 200 µL of parasitized erythrocytes (1-2%
parasitemia and 1.5% hematocrit), and the whole was incu-
bated at 37 °C for 24 h in a candle jar. The control well in
each plate contained 25 µL of complete medium instead of the
test compound. Chloroquine was also tested as a positive
control. After 24 h, 25 µL of [3H] hypoxanthine was added,
and the plates were incubated for an additional 24 h, after
which the cells were filtered onto glass fiber filters (Whatman
934-AH) and counted in a scintillation counter. For each test
compound, the concentration-response profile was determined
and analyzed by a nonlinear, logistic dose response program
to give its IC50, which is the concentration of test compound
required to inhibit [3H] hypoxanthine uptake by 50% compared
to the control.
Eva lu a tion of in Vivo An tim a la r ia l Activity. The in vivo
test measures the survivability of mice following administra-
tion of the drug. Swiss albino mice (male, 4 weeks, ap-
proximately 25 g) were inoculated intraperitoneally with 107
parasitized erythrocytes (P. berghei ANKA). The test com-
pound was given intraperitoneally at a daily dose of 100 mg/
kg in DMSO for 3 consecutive days after the day of infection
(day 0). Each compound was tested against three mice. Three
groups of control infected mice were maintained, and they were
given chloroquine (52 mg/kg, ip, 0.5% Tween buffer solution,
pH 7.4) on day 1 and DMSO or 41 (2,4-dimethoxy-4′-butoxy-
chalcone, 100 mg/kg in DMSO, ip) on days 1-3. Thin blood
smears were made from the tail blood of the mice from day 1
to day 14 or until their demise. The blood smears were fixed
with 5% Giemsa, examined microscopically, and graded ac-
cording to WHO protocol for evaluating the degree of para-
sitemia.17 Control infected mice treated with DMSO or chlo-
roquine would normally perish within 8 days and 14-16 days,
respectively. Mice that received 41 would live on the average
for 8-9 days.
Ack n ow led gm en t. Mei Liu gratefully acknowledges
the National University of Singapore for granting her
a research scholarship. This work has been supported
by Grant RP-140-000-016-112 from the National Uni-
versity of Singapore (M.-L.G.) and the Thailand Re-
search Fund (P.W.).
Su p p or tin g In for m a tion Ava ila ble: Tables containing
physical and analytical data of synthesized compounds (Table
1), their physicochemical descriptors (Table 2), correlation
maxtrix of descriptors (Table 3) and a summary of PLS models
(Table 4) and figures showing the score plots of principal
components for alkoxylated chalcones (Figure 1), PLS score
plots for actives (Figure 2a), and a plot of predicted vs observed
activities of actives (Figure 2b). This material is available free
Det er m in a t ion of Lip op h ilicit y b y R ever sed -P h a se
HP LC. Lipophilicity was determined experimentally from
their capacity factors (k′) by a reversed-phase HPLC method.
Separation was achieved on a LiChrosorb RP-18 (10 µM)
stationary phase with a methanol/0.02 M phosphate buffer (pH
7.0) mobile phase. At least four mobile-phase compositions
were investigated for each compound, with the methanol
content ranging from 50% to 85% w/w for each composition.
Determinations were carried out at 30 °C, with the flow rate
adjusted to 1.0-1.5 mL/min depending on the mobile-phase
composition, with UV detection set at 280 and 330 nm. A stock
solution (10 mg/mL) of the compound was prepared in metha-
nol. For each mobile phase composition, equal volumes (20 µL)
of the stock solution and an acetone stock solution (10% v/v
acetone in the mobile phase) were diluted to 200 µL with the
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