Antimalarial pharmacodynamics of chalcone derivatives in combination with artemisinin against Plasmodium falciparum in vitro

Article abstract of DOI:10.1016/j.ejmech.2009.02.008

Use of artemisinin based combination therapies (ACTs) is increasing in treatment of malaria. Their extensive and indiscriminate deployment will ultimately lead to selection of resistance. Thus, alternate ACTs are needed. We reported in vitro antimalarial

Full text of DOI:10.1016/j.ejmech.2009.02.008

European Journal of Medicinal Chemistry 44 (2009) 3388–3393
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimalarial pharmacodynamics of chalcone derivatives in combination with
artemisinin against Plasmodium falciparum in vitro
,
a,
**
Amit Bhattacharya a, Lokesh C. Mishra a, Manish Sharma a, Satish K. Awasthi b , Virendra K. Bhasin
*
a Department of Zoology, North Campus, University of Delhi, New Delhi 110007, India
b Chemical Biology Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Use of artemisinin based combination therapies (ACTs) is increasing in treatment of malaria. Their
extensive and indiscriminate deployment will ultimately lead to selection of resistance. Thus, alternate
ACTs are needed. We reported in vitro antimalarial potential of chalcone derivatives. A few potent
chalcones were selected for their antimalarial interaction in combination with artemisinin in vitro.
Combinations evaluated show synergistic or additive interactions. Chalcones act on broad range of
asexual stages of the parasite. The synergistic combinations decrease hemozoin formation in parasitized
erythrocytes. These combinations do not affect new permeation pathways induced in the host cells. This
is the first report showing antiplasmodial interactions between artemisinin and synthetic chalcone azole
derivatives. Thus, chalcones and artemisinin combinations open the possibility of novel ACTs.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 6 December 2008
Received in revised form
2 February 2009
Accepted 12 February 2009
Available online 21 February 2009
Keywords:
Artemisinin
Antimalarial chalcone combination
Chalcone derivatives
In vitro interaction
1. Introduction
malaria with artemisinin based combination therapies (ACTs) [3] in
areas of chloroquine resistance, leading to increase in demand of
Many modern medicines developed over the recent past have
been derived from or inspired by particular natural products [1].
Natural products have also been the rich source of antimalarial
drugs. Artemisinin was isolated and identified in 1970s as the
principal antimalarial compound from extracts of a shrub Artemisia
annua. The foliage decoctions of A. annua, though, have been used
in China for over 2000 years for fever resolution [2]. Artemisinins
(artemisinin and its derivatives) now represent the most powerful
antimalarial drugs: resounding the success of quinine in the
treatment of malaria, discovered some 350 years ago. These plant
products have made a lasting impact in malaria treatment for long
periods – spanning centuries – whereas; therapeutic life span of
most synthetic antimalarial drugs has often been short-lived,
lasting at the most for decades. Today artemisinins are the only
drugs that cure chloroquine-resistant Plasmodium falciparum
infections, and clinical resistance to these compounds has not yet
been reported. To avert quick emergence of resistance to this vital
class of compound, the malariologists have agreed not to deploy
artemisinins as monotherapy but in combination with other anti-
malarial(s). WHO now recommends treating even uncomplicated
artemisinin. Artemisinin being a natural product is at times in short
supply [4]. Synthesis of artemisinin is difficult and expensive.
Mounting demand and use of the few available ACTs is likely to
result in selection of the resistant-parasites sooner than expected.
Thus, there is a need for alternate ACTs. Continuous in vitro cultures
of P. falciparum provide a good initial screen system for new ACTs.
Natural products or their synthetic derivatives as partner drugs
which decrease dependence on artemisinin form an attractive
search option for novel ACTs.
Chalcones are aromatic ketones and key biosynthetic interme-
diates for combinatorial assembly of different heterocyclic scaffolds.
They form an important group of natural compounds which are easy
to synthesize, and some of them show wide range of biological
activities. Licochalcone A, an oxygenated chalcone, isolated from the
roots of Chinese licorice was reported to have antiplasmodial
activity [5]. This prompted researchers to design and synthesize
a variety of chalcone derivatives, and to evaluate their antimalarial
potential [6,7]. Likewisewefirst reported the antiplasmodial activity
of chalcones with azoles on acetophenone ring [8]. Three of these
azole derivatives inhibited the parasite multiplication rate to 50%
(IC50) at concentrations of less than 3
mg/ml, indicating the potential
to be developed as an antimalarial [7]. We considered it interesting
to investigate the antiplasmodial interactions of artemisinin in
combination with each of these three synthesized novel chalcone
derivatives (Scheme 1). These chalcone derivatives alone or each in
fixed-ratio combinations with artemisinin were employed to assess
* Corresponding author. Tel.: þ91 11 27662104.
** Corresponding author. Tel.: þ91 11 27667989; fax: þ91 11 27667524.
E-mail addresses: awasthisatish@yahoo.com (S.K. Awasthi), virendrabhasin@
hotmail.com (V.K. Bhasin).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.008

A. Bhattacharya et al. / European Journal of Medicinal Chemistry 44 (2009) 3388–3393
3389
Scheme 1.
their antiplasmodial activity in vitro. An isobologram method [9]
was used to analyze the synergistic, antagonistic or additive inter-
actions of the combinations. We also determined the combination
effect of these compounds on hemozoin formation by the parasites;
sorbitol-induced hemolysis of the parasitized erythrocytes and
compared stage-specific inhibition of parasites by chalcone deriva-
tives, with an aim to obtain clues to the mode of action of these
combinations.
artemisinin (Fig. 1A,B,C). The isobolograms show that antimalarial
interaction of the chalcone derivatives in vitro with artemisinin is
not antagonistic. Compound SK1 in combination with artemisinin
shows synergistic antiplasmodial interaction in three of the four
fixed-ratio combinations evaluated and additive in the other.
Similarly the combination of SK2 and artemisinin displays syner-
gistic interaction in two combinations and additive in rest of the
two combinations. Interaction of artemisinin with SK7 is consis-
tently found to be additive.
2. Results
2.4. Effect of drugs on hemozoin formation
2.1. In vitro sensitivity of parasites
Amount of hemozoin formed reflects the extent of hemoglobin
digestion. Table 6 shows the percentage hemozoin formation by the
parasites in the presence of artemisinin or chalcone derivatives and
some synergistic combinations of these. Hemozoin formation in
pyrimethamine treated parasites serve as a negative control.
Hemozoin formation in parasites treated with IC50 concentration of
artemisinin shows less than expected 50% pigment formation, so
also treatment with the chalcone derivatives and some synergistic
combinations evaluated. The results indicate that chalcone deriv-
atives or artemisinin treatment affected the hemoglobin digestion
in the parasites leading to reduced amount of hemozoin than
expected.
The concentrations required to achieve IC50 with each of the
compound evaluated is given in Table 1. Artemisinin was found to
be the most effective followed by chloroquine, licochalcone A, SK7,
SK2 or SK1 as shown by molar IC50. These concentrations were in
nM range for known antimalarials like artemisinin and chloro-
quine, whereas in mM range for the chalcone derivatives assayed.
2.2. In vitro stage-specific inhibition
Chalcone derivatives were found to be toxic to different asexual
stages of P. falciparum. They are more toxic to trophozoite stages
followed by ring and schizont stages (Table 2).
Table 1
2.3. Antiplasmodial interactions between artemisinin and chalcone
derivatives
In vitro sensitivity of P. falciparum strains 3D7 to artemisinin, licochalcone A, chlo-
roquine and chalcone derivatives.
Compounds
Mean IC50 ꢀ SEa
4 nM ꢀ 0.1
In every combination assay IC50 was determined from two sets
of drug response curves obtained from each replicate. Each set
Artemisinin
Licochalcone A
Chloroquine
SK1
SK2
SK7
4.2
0.03
9.5
7
m
m
m
m
m
M ꢀ 0.2
M ꢀ 0.01
M ꢀ 0.3
M ꢀ 0.8
M ꢀ 0.1
representing four combination curves and
a curve of drug/
compound alone. Mean FIC50 values derived from these curves are
tabulated in Tables 3–5, for each chalcone derivative combination
with artemisinin. Sum of FICs are presented in three isobolograms
for each chalcone derivative SK1, SK2 or SK7 in combination with
4.9
a
Standard error (n ¼ 3).

3390
A. Bhattacharya et al. / European Journal of Medicinal Chemistry 44 (2009) 3388–3393
Table 2
Table 4
Stage-specific effect of artemisinin and chalcone derivatives was determined by
exposing ring, trophozoite and schizont stages to IC50 of each of the compounds for
a period of 18 h for ring and trophozoite stages and 12 h for schizont, their respective
residence time in each of these stages. Percent inhibition of parasitemia in relation
to control was determined at the end of 48 h erythrocytic cycle.
Interaction between artemisinin and SK2 chalcone derivative against P. falciparum
(3D7 strain) at six different preparations.
P
Combination Compound A (Artemisinin) Compound B (SK 2)
FICs,
a
a
preparation
Mean FIC50
Mean FIC50
interactionb
1
2
3
4
5
6
1.0 ꢀ 0.01
0.9 ꢀ 0.008
0.4 ꢀ 0.003
0.3 ꢀ 0.01
0.3 ꢀ 0.002
0
0
1.0, ADD
1.1, ADD
0.7, SYN
0.7, SYN
1.3, ADD
1.0, ADD
Compound
Percentage Inhibition ꢀ S.E
0.2 ꢀ 0.01
0.3 ꢀ 0.01
Ring stage
Trophozoite stage
Schizont stage
0.4 ꢀ 0.004
1.00 ꢀ 0.01
1.0 ꢀ 0.004
SK1
SK2
SK7
51.4 ꢀ 0.4
47.9 ꢀ 1.3
48.7 ꢀ 1.0
61.5 1.1
70.9 0.9
65.8 0.8
48.9 ꢀ 1.5
41 ꢀ 0.7
40.1 ꢀ 0.8
a
Standard error (n ¼ 3).
b
SYN, synergy; ADD, additive.
2.5. Effect of drugs on sorbitol-induced hemolysis
New permeation pathways are produced in the host erythro-
cytes by the developing intracellular parasites. These novel chan-
nels are blocked to the extent of about 50% by 10 mM frusemide
resulting in reduced hemolysis. The results presented in Table 6
show that neither of the drugs used alone or in combination with
chalcone derivatives reduced the hemolysis following sorbitol
treatment, except for SK7 treated parasites. The results indicate
that SK7 blocks some sorbitol-induced hemolysis.
T and S-stages at their respective IC50 values implying broad range
inhibition of asexual stages. Similar observations have been made
for ART [10]. Relatively, however, chalcone derivatives are more
toxic to T-stages. ART is also known to be toxic to sexual stages [15].
We further employed erythrocyte cultures to study interactions
of ART in combination with a chalcone derivative to determine
schizontocidal activity of the combination on P. falciparum in vitro.
Chalcones constitute an important group of natural products with
selective inhibition against P. falciparum, first reported for lico-
chalcone A [5], and low cost of production; thus making them
promising compounds for evaluation in combination with artemi-
sinin. Antiplasmodial activity of chalcone derivatives in combina-
tion with artemisinin has not been previously reported. ART in
combination with SK1 or SK2 showed synergistic or additive
interactions. SK7 being most effective chalcone derivative evalu-
ated in inhibiting the parasite growth showed additive interaction
in combination with ART. We recently reported that licochalcone A
and ART combination shows synergistic interaction in vitro [16].
Interactions between ART and chalcone derivatives evaluated and
analyzed using the fixed-ratio isobolograms demonstrate that none
of the interactions are antagonistic.
Ideally components of the antimalarial combinations should
target different metabolic pathways. Exact mode of antiplasmodial
action of ART as well as chalcone derivatives is not known. There
are varied views regarding ARTs’ mode of action on P. falciparum.
ART is a sesquiterpene lactone, with an endoperoxide bridge, which
is believed to interact with the iron in the parasitized red blood
cells to form free radicals that then destroy proteins in nanodomain
vicinity [17], and exert pleiotropic ultrastructural damage to the
parasite, leading to total disintegration of the parasite [18]. It has
also been demonstrated that ART and its derivatives inhibit an
essential calcium adenosine triphosphatase (PfATPase) enzyme
[19], disrupting calcium homeostasis. Inhibition of this vital
enzyme causes global release of calcium stored in endoplasmic
reticulum into the cytoplasm, causing parasite death. Artemisinin
has also been reported to be an effective inhibitor of heme poly-
merization activity mediated by P. falciparum histidine-rich protein
II [20]. It has also been suggested that artemisinin react covalently
3. Discussion
Artemisinin and quinine continue to retain their therapeutic
efficacy against malaria for centuries [10]. Probably for most part of
the history these two natural products had been used as multi-
component extracts of plant parts for therapeutics. There is
consensus now that appropriate antimalarial combination thera-
pies are capable to postpone the emergence of resistance [11].
Today ACTs are regarded as drugs of choice to cure any malaria,
even chloroquine-resistant infections. Mounting deployment of the
few available ACTs is likely to select resistance to these combina-
tions sooner than expected. Thus, there is a huge unmet need for
novel ACTs. Accordingly, it is an attractive proposition to search for
alternate combinations which could decrease dependence on
artemisinin without compromising on the potency to cure malaria.
Blood stage cultures P. falciparum provide a good initial screen
for identifying schizontocidal action of a compound. Using this in
vitro system we measured the effect of compounds on parasite
multiplication by counting the ‘live’ parasites from a Giemsa
stained thin blood films at the end of 48 h erythrocytic cycle. This
morphological method, though tedious, was found to be more
reproducible compared to the [3H]-hypoxanthine incorporation
method [12], in our lab as well. Antimalarials and chalcone deriv-
atives studied for their inhibitory effect on parasites showed
concentration dependent growth inhibition. IC50 values of lico-
chalcone A and other antimalarials evaluated have been found to be
comparable to those reported by others [13,14]. The stage-specific
toxicity of three chalcone derivatives showed that they all inhibit R,
Table 3
Table 5
Interaction between artemisinin and SK1 chalcone derivative against P. falciparum
Interaction between artemisinin and SK7 chalcone derivative against P. falciparum
(3D7 strain) at six different preparations.
(3D7 strain) at six different preparations.
P
P
Combination Compound A (Artemisinin) Compound B (SK 1)
FICs,
Combination Compound A (Artemisinin) Compound B (SK 7)
FICs,
interactionb
preparation
Mean FIC50
Mean FIC50
interactionb
a
a
a
a
preparation Mean FIC50
Mean FIC50
1
2
3
4
5
6
1.0 ꢀ 0.02
0.5 ꢀ 0.1
0.3 ꢀ 0.05
0.3 ꢀ 0.01
0.2 ꢀ 0.002
0
0
1.0, ADD
0.6, SYN
0.5, SYN
0.7, SYN
1.1, ADD
1.0, ADD
1
2
3
4
5
6
1.0 ꢀ 0.055
1.0 ꢀ 0.1
0.8 ꢀ 0.004
0.6 ꢀ 0.4
0.3 ꢀ 0.01
0
0
1.0, ADD
1.2, ADD
1.3, ADD
1.5, ADD
1.4, ADD
1.0, ADD
0.1 ꢀ 0.02
0.2 ꢀ 0.04
0.4 ꢀ 0.01
0.9 ꢀ 0.01
1.0 ꢀ 0.01
0.2 ꢀ 0.03
0.5 ꢀ 0.01
0.9 ꢀ 0.01
1.1 ꢀ 0.08
1.0 ꢀ 0.03
a
a
Standard error (n ¼ 3).
Standard error (n ¼ 3).
b
b
SYN, synergy; ADD, additive.
ADD, additive.

A. Bhattacharya et al. / European Journal of Medicinal Chemistry 44 (2009) 3388–3393
3391
Table 6
In vitro sensitivity of artemisinin, chalcone derivatives and their synergistic
combinations against P. falciparum (3D7 strain) to determine the percent hemozoin
formation, and percent sorbitol-induced hemolysis.
Drug
Concentration % Hemozoin
Percent sorbitol-
induced hemolysis
used (IC50
)
formationa
Concentration
used
%
Hemolysisa
Artemisinin
SK 1
SK 2
SK 7
ART-SK1
4 nM
48.3 ꢀ 1.4
48.8 ꢀ 0.5
47.7 ꢀ 0.2
47.8 ꢀ 1.4
47.8 ꢀ 0.5
5 nM
90.9 ꢀ 0.2
85.9 ꢀ 0.2
90.0 ꢀ 0.2
36.1 ꢀ 0.2
84.7 ꢀ 1.5
9.5
m
M
10 mM
10 mM
10 mM
7 mM
4.9
19.2 nM:
30.8
mM
3 nM:4 mM
m
M
3:2
ART-SK1
12.8 nM:
46.2
48.6 ꢀ 0.4
47.6 ꢀ 0.8
47.1 ꢀ 1.8
2 nM: 6
m
M
88.8 ꢀ 1.7
91 ꢀ 1.1
m
M
2:3
ART-SK2
19.2 nM:
22.4
3 nM:4
2 nM: 6
10 nM
mM
m
M
3:2
ART-SK2
12.8 nM:
33.6
m
M
88.6 ꢀ 0.6
mM
2:3
Pyrimethamine 3.5 nM
Frusemide ND
56.3 ꢀ 1.0
92.2 ꢀ 0.4
56.3 ꢀ 0.2
ND
10 mM
ND, not determined.
a
Standard error (n ¼ 3).
process proves fatal for the parasite. Structure-based drug design of
antimalarial chalcone derivatives predicts trophozoite cysteine
protease as the most likely target enzyme [6,25]. Chalcones are a, b-
unsaturated ketones [26] that assume linear or nearly planar
structure. These compounds are stable in the presence of falcipain
enzyme and adopt a unique folded conformation to fit into the long
cleft of the active site of the cysteine protease enzyme [8]. We also
assume that the protonation of triazolyl chalcone derivative under
acidic condition may boost their interaction with cysteine protease
His 67 amino acid present at the active site [8].
All the compounds showed inhibition of plasmodial hemozoin
formation in culture and percent hemozoin formed in combination
was comparable with concentration of either compound used
alone, suggesting that both the compounds act on hemozoin
formation pathways but may be at different parasite stages [27],
considering our stage-specific inhibition results. Pyrimethamine,
a known antimalarial did not inhibit hemozoin formation and was
used as negative control.
Of the three novel chalcone derivatives, only SK7, a triazole
substituted chalcone was found to inhibit parasite-induced
permeation pathways to a significant extent. Few alkoxylated and
hydroxylated chalcones have been previously reported to inhibit
new permeation pathways induced by the parasite in the host
erythrocyte membrane [28]. Artemisinin, SK1, SK2 or their
synergistic combination ratios do not retard the parasite growth
through inhibition of parasite-induced channels. Frusemide
(Furosemide) [28] and artemisinin were used as positive and
negative controls at their approximate IC50 values, respectively.
These results suggest that different substituted chalcone analogs
may exert their antimalarial activity through different pathways
other than new permeation pathways in the parasitized
erythrocytes.
In conclusion our results show that antiplasmodial activity of
chalcone derivatives is exerted on all stages of asexual cycle
predominantly acting on trophozoite stages by inhibiting the
hemozoin formation and interaction of ARTand chalcone derivative
combination is not antagonistic thus hold a promise being an
appropriate partner of alternate ACTs.
Fig. 1. Isobologram showing interaction between artemisinin and chalcone derivatives
against P. falciparum 3D7 strain.
with heme of P. falciparum hemozoin both in vitro and in situ [21].
Thus multi-targeted attack on the parasite by ART may also be
responsible for the non-emergence of resistance to this drug,
despite its use for centuries. The available ACTs are safe and well
tolerated [22]. The chalcone derivatives are believed to interact and
inhibit the P. falciparum enzyme cysteine protease (falcipain), one
of the key enzymes involved in the hemoglobin degradation within
the acidic food vacuole of the intra-erythrocytic parasite [23]. The
toxic heme (ferriprotoporphyrin IX) molecules released from
hemoglobin catabolism is converted to non-toxic hemozoin
(b-hematin) within the food vacuole [24] and inhibition of this

3392
A. Bhattacharya et al. / European Journal of Medicinal Chemistry 44 (2009) 3388–3393
4. Experimental protocol
4.1.4. Slide preparation, staining and assessment
Thin blood smear slides were air dried, methanol fixed, and
stained in Giemsa solution for 40 min. After staining slides were
removed from coupling jar, washed in running tap water and air
dried. The Giemsa stained slides were examined for counting the
number of parasites in random adjacent microscopic fields,
equivalent to about 4000 erythrocytes at 1000ꢃ magnification.
Percent parasitemia was calculated. Reproducibility of counts was
checked by two other readers to maintain the quality control.
4.1. Estimation of in vitro antiplasmodial activity
4.1.1. Parasite culture
Stock culture of malaria parasite P. falciparum 3D7 strain
(chloroquine sensitive) was continuously maintained in vitro
using the candle jar method of Trager and Jensen [29]. P. falcipa-
rum parasites for normal growth and multiplication were incu-
bated at low-oxygen, high carbon-di-oxide atmosphere
–
generated by the candle – at 37 ꢁC. The parasites were maintained
on Bþ human red blood cells suspended in a complete culture
medium. Each liter of RPMI-1640 aqueous culture medium was
prepared with 10.4 g of powdered RPMI-1640 (with glutamine but
without bicarbonate), 5.94 g of HEPES buffer, 1 g of dextrose and
40 mg of gentamycin. Complete medium was constituted just
before use by adding sterile 5% sodium bicarbonate at the rate of
4 ml per 96 ml, and supplemented with 10% (v/v) pooled Bþ
human serum. Infected erythrocytes were suspended in these
culture media at a starting hematocrit of 5% and parasitemia kept
between 2 and 4% with sub-culturing done beyond 5%. Medium
was changed once a day and percentage parasitemia was moni-
tored by Giemsa stained slides.
4.2. In vitro stage-specific inhibition assay
Synchronized P. falciparum parasites (0.8–1.5% parasitemia and
2% hematocrit) were challenged with IC50 concentration of each
compound at the ring (R) stage (between 0 and 18 h), trophozoite
(T) stage (between 18 and 36 h) and schizont (S) stage (between 36
and 48 h) in triplicate along with control well (without compound).
For each well 20 ml of parasitized blood and 480 ml of culture
medium (with or without the compound) was used. Each test well
containing R, T or S was loaded with drug (compound) for the
specific time period (starting from 0 h); in a 48 h asexual life-cycle.
The drug pressure was removed after desired time and was main-
tained in drug free-culture medium. Thin blood smears were
prepared and processed for Giemsa staining at the end of 48 h
asexual cycle. Stage (ring, trophozoite or schizont) specific
percentage inhibition for each drug was calculated relative to
control (without drug, 100% growth) by counting 3500 cells for
each stage. This assay allows determining the sensitivity of each
asexual stage to the compound evaluated.
4.1.2. Synthesis of substituted 1,3-diarylpropenone derivatives
The Claisen–Schmidt condensation method was employed for
synthesis of chalcone derivatives (SK1, SK2, SK7 compounds) as
reported earlier [8].
SK 1. 1-(4-(1H-pyrrol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-
1-one: Yield 68%, Rf 0.67, off white crystals, m.p. 132–134 ꢁC, MS m/z
(M þ 1) 309.15, 1H NMR (CDCl3, 300 MHz):
d
7.19–8.0 (m, 14H, Ar).
4.3. Antiplasmodial interaction assay of artemisinin and chalcone
derivative combinations
SK 2. 1-(4-(1H-benzo[d][1,2,3] triazol-1-yl)-phenyl)-3-(4-chloro
phenyl)-prop-2-en-1-one: Yield 60%, Rf 0.68, off white crystals, m.p.
154–155 ꢁC, MS m/z 360 (M þ 1), 1H NMR (CDCl3, 300 MHz):
4.3.1. Preparation of fixed-ratio combinations
d
6.52–8.15(m, 14H, Ar).
In each combination assays; two compounds (Compound A,
SK 7. 1-(4-(1H-1,2,4-triazole-1-lyl)-phenyl)-3-(4-chlorophenyl)-
artemisinin and Compound B, a chalcone derivative) were
prop-2-en-1-one: Yield 79%, Rf 0.55, yellow crystals, m.p.
combined in four fixed ratios (4:1, 3:2, 2:3, and 1:4). Approximately
eight-fold IC50 compound concentration of respective compound A
or B was taken as 100%, so that IC50 of the individual compound falls
in between third and fourth two-fold serial dilution. Six solutions in
160–162 C, MS m/z 309.12 (Mþ ), 1H NMR (CDCl3, 300 MHz):
d
,
ꢁ
ꢂ
7.31–7.90 (m, 12H, Ar).
4.1.2.1. Stock solution of compounds. Artemisinin (Sigma–Aldrich,
USA), pyrimethamine (Sigma–Aldrich, USA) and licochalcone A
(Calbiochem, Merck Bioscience, Pvt. Ltd., USA) were each prepared
separately in DMSO to get the stock solution of 1 mg/ml strength.
Compounds SK1, SK2 and SK7 were synthesized according to the
procedure described by Mishra et al. [8] and each compound was
made to strength of 1 mg/ml stock solution. Chloroquine phosphate
ampoule (Ipca Laboratories, India) containing 40 mg/ml chloro-
quine aqueous solution drug was procured locally. The stock solu-
tion was diluted on the day of experiment to get the desired
concentrations for each drug. The amount of DMSO in diluted
concentrations used had no effect on parasite growth.
combination assay of ART (nM): SK1 (
25.6:15.4, 19.2:30.8, 12.8:46.2, 6.4:61.6, 0:72; respectively, for ART
(nM): SK2 ( M) were 32:0, 25.6:11.2, 19.2:22.4, 12.8:33.6, 6.4:44.8,
mM) used were 32:0,
m
0:56; respectively and for ART (nM): SK7 (mM) were 32:0, 25.6:8,
19.2:16, 12.8:24, 6.4:32, 0:40; respectively.
4.3.2. Plate preparation for antiplasmodial interaction assay
Compound dilutions of each combination solution were made in
sterile flat-bottom 96-well tissue culture plates as described by
Fivelman and others [9]. Six times two-fold serial dilution was done
for each combination in triplicate. Each well contained a total
volume of 200 ml of complete culture medium with or without
compound and pre-synchronized infected RBCs (1% parasitemia at
2.5% hematocrit). Control cultures (without compound) were
maintained on the same plate in triplicate. Two 96-well plates (for
six combinations) were used for each combination experiment. The
plates were stacked in a candle jar and gassed to increase carbon-
di-oxide concentration [29]. The plates were incubated at 37 ꢁC for
48 h with a brief interruption after 24 h to reconstitute gas mixture
by burning the candle and change the medium.
4.1.3. Compound (drug) concentration response assay
The concentration of an individual compound required to
inhibit multiplication of parasites by 50% (IC50) against P. falciparum
was determined using concentration response assay in 24-well
tissue culture plates in triplicates. Synchronous parasite [30]
cultures were subjected to graded concentration of a compound,
prepared in gentamycin-free RPMI culture medium, for 48 h at
37 ꢁC in a candle jar [29]. Medium was changed in each well after
24 h with or without the compound. The results were expressed as
the mean percentage inhibition ꢀ standard error in relation to
control; examined by thin smear Giemsa stained slides. IC50 values
were computed from semi-log plots.
4.3.3. Isobologram preparation and data analysis
For each combination assay, IC50 was calculated from two sets of
concentration response graphs, each containing compound alone
curve and four combination curves. Fractional inhibitory

A. Bhattacharya et al. / European Journal of Medicinal Chemistry 44 (2009) 3388–3393
3393
concentration values of compound A (FICA) and compound B (FICB)
were calculated separately in each combination. FIC values were
calculated by the following equation to plot isobologram:
extent (ꢅ40% of control values) near to the IC50 concentration were
considered as a good channel blocker [28].
Fraction of drug concentration required to produce IC50when used in combination
Fraction of drug concentration required to produce IC50when used alone
FIC ¼
The sum FIC for each combination ratio of two combined
Acknowledgements
compounds shows that the drug–drug schizontocidal interaction
between them [31,32] was determined by the following equation:
Work was supported in part by the Department of Biotechnology,
Ministry of Science and Technology, Government of India and Univer-
sity of Delhi, Delhi. AB and LCM received fellowship from the Council of
Scientific and Industrial Research, New Delhi, India. MS received
fellowship from University Grants Commission (UGC), New Delhi.
ꢀ
ꢁ
X
IC50of A in mixture IC50of B in mixture
SumFIC
FIC
¼
þ
IC50of A alone
IC50of B alone
P
P
FIC < 1 represents synergism,
additive interaction,
nism while
FIC ꢄ 1 and <2 represents
P
FIC ꢄ 2 and <4 represents slight antago-
P
References
FIC ꢄ 4 represents marked antagonism [33–35].
[1] D.J. Newman, G.M. Cragg, J. Nat. Prod. 70 (2007) 461–477.
[2] C. Liu, Y. Zhao, Y. Wang, Appl. Microbiol. Biotechnol. 72 (2006) 11–20.
[3] WHO 1998, ed. World Health Organization, The use of artemisinin and its
derivatives as antimalarial drugs, Report of a joint CTD/DMP/TDR informal
consultation, WHO document WHO/MAL/98.1086.
4.4. Hemozoin inhibition assay
The hemozoin (b-hematin) inhibition by drugs in P. falciparum
cultures was determined using drug concentrations in the vicinity
of IC50 concentrations at the end of 48 h [35]. The test cultures were
centrifuged at 1300 rpm for 5–10 min to remove the culture
medium. Infected erythrocyte pellet (mixture of erythrocyte
[4] V.K. Bhasin, L. Nair, Lancet. Infectious. Dis. 3 (2003) 609.
[5] M. Chen, T.G. Theander, S.B. Christensen, L. Hviid, L. Zhai, A. Kharazmi, Anti-
microb. Agents. Chemother. 38 (1994) 1470–1475.
[6] R. Li, G.L. Kenyon, F.E. Cohen, X. Chen, B. Gong, J.N. Dominguez, E. Davidson,
G. Kurzban, R.E. Miller, E.O. Nuzum, P.J. Rosenthal, J.H. McKerrow, J. Med.
Chem. 38 (1995) 5031–5037.
[7] M. Liu, P. Wilairat, S.L. Croft, A.L. Tan, M.L. Go, Bioorg. Med. Chem. 11 (2003)
2729–2738.
[8] N. Mishra, P. Arora, B. Kumar, L.C. Mishra, A. Bhattacharya, S.K. Awasthi,
V.K. Bhasin, Eur. J. Med. Chem. 43 (2008) 1530–1535.
membrane and b-hematin) was subjected to 0.01% saponin lysis for
10 min at 25 ꢁC to release parasites from erythrocyte. The parasites
were washed three times with PBS, re-suspended in 2.5% sodium-
dodecyl sulfate buffer solution (SDS in PBS) and subjected to spin at
20,000g for 1 h. The supernatant was discarded and the insoluble
hemozoin pellet was washed in 2.5% SDS in PBS and then dissolved
in 20 mM NaOH. The hemozoin content was estimated by deter-
mining the absorbance at 400 nm and using a standard curve
[9] Q.L. Fivelman, I.S. Adagu, D.C. Warhurt, Antimicrob. Agents. Chemother. 48
(2004) 4097–4102.
[10] N.J. White, Science 320 (2008) 330–334.
[11] World Health Organisation, Antimalarial Drug Combination Therapy. Report of
WHO Technical Consultation. (WHO/CDS/RBM/2001.35), WHO, Geneva, 2001.
[12] R.E. Desjardins, C.J. Canfield, J.D. Haynes, J.D. Chulay, Antimicrob. Agents.
Chemother. 16 (1979) 710–718.
[13] H.L. Ziegler, H.S. Hansen, D. Stærk, S.B. Christensen, H. Ha¨gerstrand,
J.W. Jaroszewski, Antimicrob. Agents. Chemother 48 (2004) 4067–4071.
[14] S.F. Nielsen, A. Kharazmi, S.B. Christensen, Bioorg. Med. Chem. 6 (1998) 937–945.
[15] P.Q. Chen, G.Q. Li, X.B. Guo, K.R. He, Y.X. Fu, L.C. Fu, Y.Z. Song, Chin. Med. J.
(Engl.) 107 (1994) 709–711.
prepared from b-hematin (Sigma) [36]. The amount of hemozoin
formed in relation to control was calculated.
4.5. Sorbitol-induced hemolysis assay
[16] L.C. Mishra, A. Bhattacharya, V.K. Bhasin, Acta Trop. 109 (2009) 194–198.
[17] D.L. Klayman, Science 228 (1985) 1049–1054.
Late trophozoite to schizont stage parasites (36–44 h old post
invasion) when suspended in an iso-osmotic sorbitol solution;
there is net uptake of sorbitol and water into the parasitized
erythrocytes, resulting in cell swelling and hemolysis. Hemolysis is
reduced in the presence of channel blockers.
The hemolysis was estimated spectrophotometrically by
measuring absorbance at 540 nm with drug at a concentration near
to the antiplasmodial IC50 [28] estimated earlier. Infected cell
suspension was prepared by harvesting 36–44 h old synchronized
culture (with 15% parasitemia), by 600g spin for 5 min. Pellet was
washed two times with a 7.4 pH solution of NaCl (150 mM)–HEPES
(20 nM) and finally re-suspended in this solution to give 50%
hematocrit of infected cells. Stock solutions of drugs were diluted
using sorbitol (300 mM)–HEPES (20 mM) buffer (pH 7.4) to make
[18] Y. Maeno, T. Toyoshima, H. Fujioka, Y. Ito, S.R. Meshnick, A. Benakis,
W.K. Milhous, M. Aikawa, Am. J. Trop. Med. Hyg. 49 (1993) 485–491.
[19] U.E. Ludwig, R.J. Webb, I.D.A. van Goethem, J.M. East, A.G. Lee, M. Kimura,
P.M. O’Neill, P.G. Bray, S.A. Ward, S. Krishna, Nature 424 (2003) 957–961.
[20] A.V. Pandey, B.L. Tekwani, R.L. Singh, V.S. Chauhan, J. Biol. Chem. 274 (1994)
19383–19388.
[21] Y.L. Hong, Y.Z. Yang, S.R. Meshnick, Mol. Biochem. Parasitol. 63 (1994) 121–128.
[22] I.R. Ribeiro, P. Olliaro, Med. Trop. (Mars) 58 (1998) 50–53.
[23] B.R. Shenai, P.S. Sijwali, A. Singh, P.J. Rosenthal, J. Biol. Chem. 275 (2000)
29000–29010.
[24] A.U. Orjih, Exp. Biol. Med. (Maywood) 226 (2001) 746–752.
[25] R. Li, X. Chen, B. Gong, P.M. Selzer, Z. Li, E. Davidson, G. Kurzban, R.E. Miller,
E.O. Nuzum, J.H. McKerrow, R.J. Fletterick, S.A. Gillmor, C.S. Craik, I.D. Kuntz,
F.E. Cohen, G.L. Kenyon, Bioorg. Med. Chem. 4 (1996) 1421–1422.
[26] Z. Nowakowska, Eur. J. Med. Chem. 42 (2007) 125–137.
[27] A.V. Pandey, B.L. Tekwani, Febs. Lett. 402 (1997) 236–240.
[28] M.L. Go, M. Liu, P. Wilairat, P.J. Rosenthal, K.J. Saliba, K. Kirk, Antimicrob.
Agents. Chemother. 48 (2004) 3241–3245.
0.1 mM buffered drug stock solutions. 100 ml of infected cell prep-
aration (50% hematocrit) was mixed with desired concentration of
buffered drug solution to make a total volume of 1 ml. The cell
suspension was incubated at 37 ꢁC for 15 min, centrifuged and
aliquots of supernatant were used to estimate the amount of
hemoglobin released by determining absorbance at 540 nm. The
degree of hemolysis was compared with that of control, to observe
the channel blocking effect of drugs. Compounds that inhibited
sorbitol-induced lysis of parasitized erythrocytes to a significant
[29] W. Trager, J.B. Jensen, Science 193 (1976) 673–675.
[30] C. Lambros, J.P. Vanderberg, J. Parasitol. 65 (1979) 418–420.
[31] M.C. Berenbaum, J. Infect. Dis. 137 (1978) 122–130.
[32] S. Gupta, M.M. Thapar, W.H. Wernsdorfer, A. Bjorkman, Antimicrob. Agents.
Chemother. 46 (2002) 1510–1515.
[33] F.C. Odds, J. Antimicrob. Chemother. 52 (2003) 1.
[34] L.C. Mishra, A. Bhattacharya, V.K. Bhasin, Am. J. Trop. Med. Hyg. 76 (2007) 497–501.
[35] A. Bhattacharya, L.C. Mishra, V.K. Bhasin, Am. J. Trop. Med. Hyg. 78 (2008) 721–728.
[36] T. Akompong, N. Ghori, K. Haldar, Antimicrob. Agents. Chemother. 44 (2000)
88–96.