Synthesis, mechanistic and synergy studies of diarylidenecyclohexanone derivatives as new antiplasmodial pharmacophores

Article abstract of DOI:10.1007/s00044-018-2237-2

Diarylidenecyclohexanone (DAC) derivatives (Ia-i, IIa-c and IIIa-b) were synthesized, characterized and screened for their invitro antiplasmodial activities against erythrocytic stages of chloroquine (CQ) sensitive and resistant strains of P. falciparum by using SYBR green I fluorescence assay. SAR studies of DAC derivatives showed antiplasmodial activity in the order of 3-NO₂ (Ib, IC₅₀ 0.95 μM) > 3-chloro (Id, IC₅₀ 3 μM) > 4-chloro (Ie, IC₅₀ 8.5 μM) > 2-chloro (Ic, IC₅₀ 13 μM). Further Ib and Id exhibited nearly equal potencies against CQ-resistant strains P. falciparum Dd2, {IC₅₀ 1 μM (Ib) and 2.7 μM (Id)} and PfINDO {IC₅₀ 1.1 μM (Ib) and 2.5 μM (Id)}. Drug exposure followed by drug withdrawal-based stage-specific kill kinetic studies showed that Ib is shizonticidal within 3 h while the earliest killing actions against Trophozoites and Rings were seen at >3 h and >6 h, respectively. Combination studies of the most potent leads viz. Ib and Id showed strong to moderate synergistic effects with Artemisinin (?FIC₅₀: 0.34 to 0.63) whereas no interaction (?FIC₅₀: 0.65 to 2.36) was observed with Chloroquine. The DACs showed significant insilico binding affinity with β-haematin and P. falciparum lactate dehydrogenase (PfLDH) suggesting these to be the targets of their antiplasmodial action. High compliance with Lipinski rule of 5 and high selectivity index of Ib (105.3) and Id (8.3) against HeLa cell line indicated that Diarylidenecyclohexanones could serve as structural templates towards lead optimization of compounds for discovery of novel, potent, safe and affordable drugs against malaria.

Full text of DOI:10.1007/s00044-018-2237-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2237-2
ORIGINAL RESEARCH
Synthesis, mechanistic and synergy studies of
diarylidenecyclohexanone derivatives as new antiplasmodial
pharmacophores
Bishnu P. Joshi1 Dinesh Mohanakrishnan Garima Mittal Swayamsiddha Kar Jeevan Kumar Pola
2 ●
2 ●
1 ●
1 ●
●
Nageswara Rao Golakoti Jagadeesh Babu Nanubolu Rajesh Babu D. Sai Suraj Kumar S. Dinkar Sahal2
1 ●
3 ●
1 ●
1 ●
Received: 22 February 2018 / Accepted: 17 August 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Diarylidenecyclohexanone (DAC) derivatives (Ia-i, IIa-c and IIIa-b) were synthesized, characterized and screened for their
in vitro antiplasmodial activities against erythrocytic stages of chloroquine (CQ) sensitive and resistant strains of P.
falciparum by using SYBR green I fluorescence assay. SAR studies of DAC derivatives showed antiplasmodial activity in
the order of 3-NO (Ib, IC 0.95 µM) > 3-chloro (Id, IC50 3 µM) > 4-chloro (Ie, IC50 8.5 µM) > 2-chloro (Ic, IC50 13 µM).
2
50
Further Ib and Id exhibited nearly equal potencies against CQ-resistant strains P. falciparum Dd2, {IC50 1 µM (Ib) and 2.7
µM (Id)} and PfINDO {IC50 1.1 µM (Ib) and 2.5 µM (Id)}. Drug exposure followed by drug withdrawal-based stage-
specific kill kinetic studies showed that Ib is shizonticidal within 3 h while the earliest killing actions against Trophozoites
and Rings were seen at >3 h and >6 h, respectively. Combination studies of the most potent leads viz. Ib and Id showed
strong to moderate synergistic effects with Artemisinin (ƩFIC : 0.34 to 0.63) whereas no interaction (ƩFIC : 0.65 to 2.36)
5
0
50
was observed with Chloroquine. The DACs showed significant in silico binding affinity with β-haematin and P. falciparum
lactate dehydrogenase (PfLDH) suggesting these to be the targets of their antiplasmodial action. High compliance with
Lipinski rule of 5 and high selectivity index of Ib (105.3) and Id (8.3) against HeLa cell line indicated that
Diarylidenecyclohexanones could serve as structural templates towards lead optimization of compounds for discovery of
novel, potent, safe and affordable drugs against malaria.
●
●
●
●
Keywords Diarylidenecyclohexanone Synthesis Antimalarial activity Synergy studies Molecular docking
Introduction
Although massive efforts at prevention, diagnostic testing
These authors contributed equally: Bishnu P. Joshi, Dinesh
Mohanakrishnan, Garima Mittal.
and cure with antimalarial medicines have shown some
promise, about 3.2 billion people continue to remain at risk
of malaria. In 2014 alone, there were an estimated 214
million new cases of malaria and 438,000 deaths (WHO
World Malaria Report 2015). The fact that malaria con-
tinues to cause economic and emotional trauma in today’s
global world suggests that a concerted effort towards
effective vector control together with safe, potent and
affordable antimalarial drugs is the need of our times. The
tendency of rising resistance (Cui et al. 2015) to clinically
available antimalarial drugs urges the search for new, potent
and affordable alternatives. The α, β-unsaturated ketone
curcumin (Fig. 1A), a popular food additive and constituent
of traditional medicines in Southeast Asia is also well
known for its biological activities (Maheshwari et al. 2006;
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2237-2) contains supplementary
material, which is available to authorized users.
*
*
Nageswara Rao Golakoti
gnageswararao@sssihl.edu.in
Dinkar Sahal
dinkar@icgeb.res.in
1
2
3
Department of Chemistry, Sri Sathya Sai Institute of Higher
Learning, Puttaparthi, AP, India
International Center for Genetic Engineering and Biotechnology
(
ICGEB), New Delhi, India
Indian Institute of Chemical Technology (IICT), Hyderabad, India

Medicinal Chemistry Research
Fig. 1 Transition from
dicarbonyl curcumin (A) to the
two aromatic regions linked
together by either an enone (B)
or dienone (C)
Zorofchian Moghadamtousi et al. 2014) including promis-
ing antimalarial properties (Reddy et al. 2005; Mishra et al.
IC50 16 µM. Thus freezing the conformational flexibility of
the keto group by introduction of cyclohexane moiety was
found to enhance the antiplasmodial potency by >20-fold.
This motivated us to synthesize and evaluate a series of
diarylidenecyclohexanone derivatives leading us to identify
2,6-bis(3-nitro-benzylidene) cyclohexanone Ib (Pf3D7,
IC50 0.95 µM) and 2,6-bis(3-chloro-benzylidene) cyclo-
hexanone Id (Pf3D7, IC50 3 µM) as promising anti-
plasmodial molecules. Here we describe the detailed
antiplasmodial properties including synergistic activities
with artemisinin, promising selectivity of Ib & Id and
stage-specific kill kinetics of Ib.
Since the diarylidenecyclohexanone derivatives showed
potential antiplasmodial activity, we wanted to explore the
potential targets of their action. Towards this, we studied the
in silico binding affinities of DACs with β-haematin and
lactate dehydrogenase (PfLDH) of P. falciparum.
2
008). However, being a dicarbonyl, curcumin, is suscep-
tible to keto-enol tautomerism mediated by light, pH and
heat-induced decomposition (Kumavat et al. 2013). Among
other compounds with structural similarity to Curcumin,
antimalarial activity of chalcones (Fig. 1B) was first noticed
in licochalcone A (a natural product isolated from Chinese
liquorice roots) which was reported to exhibit potent in vitro
antiplasmodial
activity
(IC ,
1.8 µM
against
5
0
Pf3D7 &PfDd2) (Chen et al. 1994). Subsequently, a syn-
thetic chalcone derivative, l-(2,5-dichlorophenyl)-3-(4-qui-
nolinyl)-2-propen-l-one was reported for its potent
antiplasmodial activities (IC , 200 nM) (Li et al. 1995).
50
Antimalarial chalcones are widely thought to act against
́
malarial cysteine proteases(Li et al. 1995; Dominguez et al.
001; Rosenthal 2005; Sriwilaijaroen et al. 2006), which are
2
used by the parasite for the degradation of host haemoglo-
bin for its nutritional purposes. Dibenzylideneacetone, a
dienone bis-chalcone (Fig. 1C), differs from curcumin
Materials and methods
(
Fig. 1A) in having only one keto group, is more symmetric
than the monoenone chalcones (Fig. 1B) and more stable
than curcumin. The nonlinear optical properties of sym-
metrically substituted bis-chalcones with D-π-A-π-D type
organic chromophores have earlier been reported by us
The various aldehydes and ketones used were obtained from
S.D fine and Sigma-Aldrich chemicals. All solvents were
distilled before use. The chloroquine used for the studies
was procured as chloroquine diphosphate salt from Sigma-
Aldrich.
(
Rajashekar et al. 2012; Kiran et al. 2014; Reddy et al.
014). Dibenzylideneacetones have shown promising bio-
2
logical activities including inhibition of the activation of
NF-κB, IN-LEDGF/p75 interaction in HIV-1 and growth
inhibition of colorectal carcinoma HCT116 p53+/+ (Cao
et al. 2012) and mucoepidermoid carcinoma cells (Lee et al.
Measurement of in vitro inhibition of P. falciparum
growth
Chloroquine-sensitive Pf3D7 and chloroquine-resistant
PfDd2 and Pf INDO strains obtained from MR4 were
used to determine the in vitro antiplasmodial activities.
Parasite strains were cultivated by the method of Trager and
Jensen (1976) with minor modifications. Cultures were
2
014). In our previous studies, dibenzylideneacetone deri-
vatives have also shown potent antiplasmodial activities
against CQ-sensitive (3D7) (IC : 1.97 µM) and CQ-
50
resistant (RKL9) (IC : 1.69 µM) strains of P. falciparum
50
+
(
Aher et al. 2011). Towards further investigations of the
maintained in fresh O human erythrocytes at 4% haema-
dibenzylidenealacetone pharmacophore, here we have
explored the antiplasmodial properties of cyclohexanone
analogues (derivatives) of dibenzylideneactone. During the
course of our investigations, we observed that introduction
of cyclohexane moiety into dibenzylideneacetone
tocrit in complete medium (RPMI 1640 with 0.2% sodium
bicarbonate, 0.5% Albumax I, 45 mg/L hypoxanthine, and
50 mg/L gentamicin) at 37 °C under reduced O (gas mix-
2
ture 5% O , 5% CO , and 90% N ). Stock solutions of
2
2
2
chloroquine (CQ) (1 mM) were prepared in water (Milli-Q
grade), and test compounds (25 mM) were dissolved in
DMSO. All stocks were then diluted with complete culture
(
IC Pf3D7 > 308 µM) (Aher et al. 2011), Fig. 1C provided
50
us diarylidenecyclohexanone (Fig. 1D) with anti- Pf3D7

Medicinal Chemistry Research
medium to achieve the required concentrations. (In all cases
the final concentration of DMSO was 0.4% v/v, which was
found to be non-toxic to the parasite.) CQ and test com-
pounds were then placed in triplicate wells of 96-well flat-
bottom tissue-culture grade plates with drug concentrations
96-well plate reader (Versa Max). Triplicate values of each
data set were used to calculate mean and standard devia-
tions and IC50 values were determined by analysis of dose
response curves as described above. The selectivity index
was calculated as IC50 (mammalian cell)/IC (Pf 3D7).
50
0
, 12.5, 25, 50 and 100 μM in a final well volume of 100 μL
for primary screening. Chloroquine (100 nM for Pf3D7; 1
µM for PfDd2 &PfINDO) was used as a positive control in
all experiments. Parasite culture was synchronized at ring
stage with 5% sorbitol (Lambros and Vanderberg 1979).
Synchronized culture was aliquoted to drug containing 96-
well plate at 2% haematocrit and 1% parasitemia. After 48 h
of incubation under standard culture conditions, plates were
harvested and read by the SYBR green I fluorescence-based
method (Smilkstein et al. 2004) using a 96-well fluores-
cence plate reader (Victor, Perkin-Elmer), with excitation
and emission wavelengths of 485 and 530 nm, respectively.
The mean fluorescence readings from triplicate wells (Y
axis) which are indices of growth were plotted against drug
concentration (X axis). A straight line connecting 50%
growth inhibition on the Y axis was extended to intersect
with the graph line. From the point of intersection, a per-
pendicular straight line was drawn to intersect on the X axis
at a point which was taken to be the IC50 (Sharma et al.
Combination studies of antimalarial drugs (CQ,
artemisinin (ART)) with Ib and Id against CQ-
resistant PfINDO
For the combination studies, the 8× IC50 (PfINDO) stock
solutions (CQ: 3.2 μM, ART: 0.12 μM, Ib: 8.8 μM, Id: 20
μM) and dilutions of individual compounds were prepared
in complete RPMI. Stock solutions of CQ/ART were then
combined with stock solutions of Ib or Id in the molar
ratios of 1:0, 1:0.25, 1:0.5, 1:1, 1:2, 1:3, 1:5 and 0:1. The
stock mixtures of each of the two drugs were placed into the
wells of sterile 96-wells plate and serially two-fold diluted.
Finally, the above mentioned stocks and their respective
dilutions were transferred into 96-well flat-bottomed cell
culture plates to give triplicates of each combination dose
for determination of antiplasmodial IC50 values as described
above.
2
011). The compounds, which showed >50% growth inhi-
Data analysis
bition at 12.5 µM in the first screen were examined in tri-
plicate wells at concentrations lower than 12.5 µM to
determine the precise IC50 values in the second screen.
Further the SYBR green assay results have been validated
by microscopic analysis of treated and untreated parasites.
To study the effect of each combination, IC50 values were
determined for individual drugs (ART, CQ, Ib, Id) and
drug combinations (ART + Ib, CQ + Ib, ART + Id, CQ
+ Id). The IC50 values obtained were used to derive 50%
Fractional inhibitory concentration (FIC ) values of each
5
0
Measurement of in vitro cytotoxicity against
drug by using following equation.
mammalian cell lines
FIC50 ¼ IC50 for drug in combination=IC50 for drug alone
Animal cell line (HeLa) was used to determine drug toxicity
by using MTT assay for mammalian cell viability assay as
described by Mosmann (1983). Cells were cultured in
complete RPMI (cRPMI) containing 10% fetal bovine
serum, 0.2% sodium bicarbonate, and 50 μg/mL gentamy-
The values of FIC50 for each of the two drugs in each
combination were used to plot isobologram, a graphical
representation of drug–drug interactions. Each data point on
such a graph connects the FIC50 of drug 1 on the X axis to
FIC50 of drug 2 on the Y axis. Addition of the two FIC50
values for each data point provides ΣFIC50 for each specific
combination. A diagonal connecting FIC = 1 on each axis
then allows one to see the significance of each point:
ΣFIC = ≤ 0.5: Synergy, ΣFIC = > 0.5 to 4: no interac-
4
cin. Briefly, cells (10 cells/200 μL/well) were seeded into
9
6-well flat-bottom tissue-culture plates in complete culture
medium. Drug concentrations were added in triplicate after
overnight seeding and cells were incubated in triplicate
wells for 24 h in a humidified atmosphere at 37 °C and 5%
5
0
50
tion, ΣFIC = > 4 antagonism (Odds 2003; Bhattacharya
5
0
CO . DMSO (final concentration 10%) was added as +ve
et al. 2009).
2
control. An aliquot of a stock solution of 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5
mg/mL in 1× phosphate-buffered saline) was added at 20
μL per well and incubated for another 3 h. After the plate
was spun (1500 rpm, 5 min, 27 °C) the supernatant was
removed and 100 μL of the stop agent DMSO was added to
each well to dissolve the formazan crystals. Formation of
formazan, an index of cell growth, was read at 570 nm by
Microscopic evaluation of Ib-mediated stage-specific
kill kinetics of erythrocytic stages of P. falciparum
Stage specificity in the action of Ib was determined by
taking highly synchronized P. falciparum (3D7) cultures at
ring (R), trophozoite (T) and schizont (S) stages and treating
each in triplicate wells separately with Ib at its IC90 (3.1

Medicinal Chemistry Research
μM). The ring stage P. falciaprum culture was tightly
synchronized for two continuous life cycles using 5% sor-
bitol to maintain the synchronicity of Plasmodium stages
and the synchrony of each stage was confirmed by micro-
scopy. One percent of synchronized early rings (0–6 h of
post invasion), early trophozoites (~24 h post synchroniza-
tion) and early schizonts (~40 h post synchronization) were
used for the stage-specific drug action studies. Drug expo-
sure followed by drug withdrawal-based microscopic
method (Sharma et al. 2011) was used to find the stage most
vulnerable to the action of Ib. Treatment with Ib was for 6,
the wells were processed using the pyridine-ferrochrome
method by adding acetone (26.25 µL) and pyridine solution
{(50% pyridine, 20% acetone and 200 mM HEPES, pH 7.4
in water)} (14 µL). Then the plates were kept under shaking
for 30 min to dissolve the free haem. After incubation, the
plates were read at 405 nm using SpectraMax plate reader.
Percentage of β-haematin formation inhibition by DACs
was assessed by comparing the positive (CQ) and negative
controls (4% DMSO). Finally, the dose response curves
were generated to calculate the IC50 values of DACs.
Molecules IIa, IIb and IIIa were interfering in the data
analysis due to their coloured nature. So these molecules
were not taken for assay.
1
8, 24, 30, 42 and 48 h (Rings); 3, 6, 18, 24 h (Tropho-
zoites) and 3, 6 and 12 h (Schizonts). Following drug
treatment as indicated above, the drugs were withdrawn by
centrifugal removal of conditioned medium followed by
media wash. The washed cells were maintained in drug-free
complete RPMI medium for total of 96, 72 and 60 h for
ring, trophozoites and schizonts, respectively. After incu-
bation, percentage of stage-specific inhibition was calcu-
lated in comparison to drug-free control by counting of total
of a 3000 cells in microscopic images using Plasmodium
auto count 0.1 (Ma et al. 2010) enabling us to determine the
percent parasitemia at each time point and for each stage.
The parasites with pycnotic morphology were considered as
dead.
In silico studies
The structures of the lactate dehydrogenase of Plasmodium
falciparum was obtained from the Protein Data Bank (PDB)
(http://www.rcsb.org/pdb/home/home.do). The PDB ID is:
1CET. GaussView (3.09) (Dennington et al. 2003) was used
to model the β-haematin structure having four haem units.
The DACs used for the docking studies were modelled
using GaussView (3.09). The energy minimization of these
molecules has been carried out using DFT (B3LYP, 6-31G)
theory (Frisch et al. 2004). After carrying out the energy
minimization procedures, the DACs were docked with the
prepared target using Autodock-Vina (Trott and Olson
2010). The docking studies were carried out after identify-
ing the potential active site in the protein as the complex/co-
crystal structure of the protein with ligand are available. For
docking against the β-haematin unit, the general area was
selected. The binding interactions were then reviewed using
PyMol (DeLano 2015) and MOE (Molecular Operating
Environment 2017).
Inhibition of detergent-mediated β-haematin
formation assay
The detergent-mediated β-haematin formation method was
adopted as previously described by Sandlin et al (Sandlin
et al. 2016) with minor modifications. A 25 mM stock
solution of hemin was prepared by dissolving hemin (Fluka,
Switzerland) in DMSO and the solution was filtered through
0
.22 µM membrane syringe filter. This stock solution was
diluted with vortexing to prepare 228 µM of hemin sus-
pension in 2 M acetate buffer (pH 4.9). A 348 µM solution
of NP-40 (Fluka, Switzerland) was prepared in Sterile and
filtered milli Q water. Stock solutions of chloroquine (CQ,
Results and discussion
Synthesis
5
0 mM) and test compounds (25 mM) were prepared in
water and DMSO respectively. The stocks of CQ and test
compounds were diluted in their respective solvents to
achieve the required concentrations. In all cases, the final
concentration of DMSO was 4% v/v, which showed no
effect on β-haematin formation. Test compounds (4 μL)
were then placed in triplicate wells of 96-well flat-bottom
plates followed by water (31.25 µL), NP-40 solution (8.75
µL), acetone (12.25 µL) and haem suspension (43.75 µL) in
a final well volume of 100 μL. The final concentrations of
test compounds/CQ were 0, 125, 250, 500, 750 and 1000
μM while additional concentrations up to 2 mM were tested
for CQ. Further the plates were incubated (6 h) in shaking
incubator (37 °C, 50 rpm) following which the samples in
Diarylidenecyclohexanone derivatives (Ia-i, IIa-c and IIIa-
b) were synthesized by base catalysed Claisen–Schmidt
condensation using substituted aryl aldehydes and cyclo-
hexanone (Furniss 1989), as outlined below (Scheme 1)
Characterization
The compounds were purified by recrystallization and
1
13
characterized by UV, FT-IR, H-NMR,
C-NMR and
Mass. (Supp. Info). In general, the UV spectra for all
compounds showed two bands in the region ~250 and 380
nm as an evidence of extended conjugation. FT-IR spectra
−
1
indicated the presence of C = O (1640-1670 cm ), C = C

Medicinal Chemistry Research
Scheme 1 Synthesis of novel
diarylidenecyclohexanone
derivatives
−
1
(
1600-1635 cm ), aromatic groups (two bands in the
The presence of isopropyl group in Ig is confirmed by
−
1
1
region 1450–1600 cm and aromatic C–H out of plane
bending at 900–650 cm ) and cyclohexanone ring (C–H
δ1.27 ppm (d, 12H) and δ 2.9 ppm (2H, septet) of H-NMR
−
1
13
along with δ23.87 and δ33 ppm in the C NMR. In the IR
−
1
stretch 2850–2950 cm ). PMR spectra also confirmed
cyclohexanone ring by the signal at ~δ 1.88 ppm (2 H,
quintet) and a triplet ~δ 2.9 ppm (4 H). The β proton of the
α–β unsaturated ketone was seen as a singlet at ~δ 7.5 ppm
in all the compounds, and the aromatic proton signals were
in the region δ 6.6–7.5 ppm, C-NMR signals for aromatic
carbons were observed between δ 120 ppm and 160 ppm.
The signal at ~δ 190 ppm in C-NMR indicated C=O
group. The mass spectra of all these compounds showed
expected molecular ions confirming their structures.
In case of Ih, the presence of –OH group was inferred by
spectra of the furanyl derivatives IIa and IIIa C–O–C
−1
asymmetric absorption is observed at ~1280 cm . In the
−
1
pyrrolyl derivative IIb, IR band at 3469 cm corresponds
1
to N–H stretch and in H-NMR, δ10.45 ppm {d(br), 2 H}
corresponds to N–H proton. The thienylidenecyclohex-
13
+
+
anones IIc and IIIb, MS (m/z):287 [M + H] , 288[M + 2]
peak is characteristic of Sulphur compounds. Complete
spectral data along with assignments are given in the sup-
plementary section.
13
X-ray diffraction analysis of single crystals of 2,6-
Bis-(4-isopropyl-benzylidene)-cyclohexanone (Ig)
−
1
the peak at 3356 cm (br-s) due to the -OH and 1257, 1171
C–O stretch). The corresponding H-NMR peak at δ 9.88
1
(
ppm (s, 1H) confirmed the same. In Ib, the IR bands at
Crystal growth
−
1
1
521, 1325 cm are due to asymmetric and symmetric
stretch of –NO . In the chloro derivatives, C–Cl stretch is
characterized by the IR peaks at 1052 and 1066 cm ,
The crystal growth of novel 2, 6-Bis-(4-iso-propylbenzyli-
dene) cyclohexanone (Ig) was done by slow evaporation
growth technique at ambient temperature. The purified
compound was used for growing single crystals using var-
ious solvents like chloroform, ethanol, acetone, cyclohex-
ane, methanol and mixtures of the solvents. We observed
that chloroform/ethanol mixture (1:1, v/v) was the best
solvent composition for yielding single crystals of good
quality.
2
−
1
respectively for 2-Cl (Ic) and 3-Cl (Id). However, in case of
1
4
-Cl (Ie), it is observed at a higher wavenumber 1091 cm−
as it is in conjugation with carbonyl group which seems to
lead to more double bond character to the C–Cl bond. In If,
−
1
the IR band at 1274 cm (Ar C–O stretch), δ3.84 ppm (s,
1
13
6
H) of H-NMR and δ59.93 ppm of C NMR correspond
1
to -OCH groups. In the H-NMR spectrum of the cinnamyl
3
derivative Ii, the six olefinic proton signals are seen at δ
6
.97 ppm (d, 2H, J = 15.6 Hz), δ7.07 ppm (t, 2H) and δ
Crystal structure determination
7
.50 ppm (2H). The alkene carbons gave corresponding
1
3
signals at δ136.34, δ123.68 and δ140.78 ppm in the
C
X-ray data for the compound were collected at room tem-
perature using a Bruker Smart Apex CCD diffractometer
NMR spectrum.

Medicinal Chemistry Research
The disordered atoms were refined isotropically. Refine-
ment was unstable with anisotropic treatment. The C–C
bond distances of the disordered isopropyl groups were
restrained to 1.5 Å using DFIX command. ORTEP diagram
was prepared in PLATON (Spek 2001) and crystal packing
diagram in CCDC Mercury (Macrae et al. 2008).
Crystal data for Ig C H O, M = 358.50, colourless
2
6
30
3
block, 0.43 × 0.32 × 0.26 mm , monoclinic, space group C2
(
=
No. 5), a = 18.380(2), b = 6.1575(6), c = 19.171(2) Å, β
3
3
93.317(3)°, V = 2166.1(4) Å , Z = 4, D = 1.099 g/cm ,
c
F000 = 776, CCD area detector, MoKα radiation, λ =
.71073 Å, T = 293(2)K, 2θmax = 50.0°, 10,471 reflections
0
collected, 2108 unique (Rint = 0.0308), Final GooF = 1.054,
R1 = 0.0692, wR2 = 0.1965, R indices based on 1835
2
reflections with I > 2σ(I) (refinement on F ), 232 para-
meters, 13 restraints, and largest difference peak and hole
are 0.403 and −0.201 e.Å-3. CCDC 1452365 contains the
supplementary crystallographic data for this paper. These
data can be obtained free of charge at www.ccdc.cam.ac.uk/
conts/retrieving.html or from the Cambridge Crystal-
lographic Data Centre (CCDC).
The ORTEP diagram and the packing of the molecules in
the unit cell is shown in Fig. 2B. The cyclohexanone ring is
in a sofa conformation. Atom C4 deviates from rest of the
ring atoms C1/C2/C3/C5/C6, and is inclined at a dihedral
angle of 51.68°. The molecule is predominantly hydro-
phobic containing a single polar carbonyl functional group.
This group is considered to be a good H-bond acceptor and
can normally result in strong hydrogen bonding usually
with polar amine or hydroxyl donor groups. However, in Ig,
the molecule lacks strong hydrogen bond donors, as a
result, the carbonyl C=O is made to involve in two C–H∙∙∙O
interactions as shown in Fig. 2C, and facilitate the
propagation of molecules as an infinite tape along the b-
axis. The three-dimensional molecular packing in the crystal
is stabilized by van der Waals interactions. Displacement
ellipsoids are drawn at the 30% probability level and H
atoms are shown as small spheres of arbitrary radius. Only
major component of the disordered isopropyl groups is
shown for clarity.
The carbonyl involves in two C‒H…O interactions with
methyl groups of cyclohexanone ring and propagates the
molecules as an infinite tape along the b-axis. Bond
distances (H…A and D…A) and bond angle (∠D-H…A)
values are 2.64Å, 3.325(5) Å, 127.7(2)°, respectively for
C5-H5A…O1 and 2.70 Å, 3.383(5)Å, 127.1(2)°, respec-
tively for C13-H13A…O1 interactions.
We have compared the bond angles and bond lengths
obtained from the single crystal XRD experiment and the
Gaussian calculations in order to establish the soundness of
the chosen theory and basis set for the calculations. The two
are summarized in SD Table 2a, b in the supplementary
Fig. 2 2, 6-Bis-(4-isopropyl-benzylidene)-cyclohexanone (Ig):
Chemical Structure, B ORTEP diagram with the atom-numbering
scheme, C crystal packing diagram
A
with graphite monochromated MoKα radiation (λ =
0
.71073Å) with ω-scan method. Preliminary lattice para-
meters and orientation matrices were obtained from four
sets of frames. Unit cell dimensions were determined using
3
755 reflections. Integration and scaling of intensity data
were accomplished using SAINT program (SAINT 2001).
The structures were solved by direct methods using
SHELXS97 (Sheldrick 1997) and refinement was carried
out by full-matrix least-squares technique using SHELXL97
(
Sheldrick 1997). Anisotropic displacement parameters
were included for all non-hydrogen atoms. All H atoms
were positioned geometrically and treated as riding on their
parent C atoms, with C–H distances of 0.93–0.97Å, and
with U (H) = 1.2U or 1.5Ueq for methyl atoms. The
iso
eq
molecule contains two isopropyl groups on two aromatic
rings, and both these isopropyl groups are disordered over
two sites. For the first disordered isopropyl group, the site
occupancy factors were 0.60 for C14/C15/C16 atoms
(
(
major component) and 0.4 for C14D/C15D/C16D atoms
minor component). Similarly, for the second disordered
isopropyl group, the site occupancy factors were 0.75 for
C24/C25/C26 atoms (major component) and 0.25 for
C24D/C25D/C26D atoms (minor component). The disorder
modelling was handled with PART and FVAR commands.

Medicinal Chemistry Research
and Ie showing Pf3D7 IC50 values (μM) of 13, 3 and 8.5,
respectively. It is interesting to note that both nitro and
chloro have an electron withdrawing effect and the greater
inductive effect of nitro than chloro may be responsible for
the greater antiplasmodial potency of Ib over Id. It may be
noted that presence of electron withdrawing groups (nitro or
chloro) specifically at the 3rd position of the benzene ring
seems to enhance antiplasmodial potency. The importance
of electron withdrawing group at position 3 is also evident
from the fact that replacement of nitro/chloro with the
electron donating –OCH (If) led to an enhanced Pf3D7
3
IC50 value of >100 μM. The selectivity index (IC50 HeLa/
IC Pf3D7) for Ib against HeLa cell line was found to be
5
0
105. This reveals that Ib is highly selective in targeting the
Fig. 3 Dose-dependent growth inhibition curves of diarylidenecyclo-
hexanones treated P. falciparum (3D7). The ring stage parasites were
treated with different concentrations of DAC derivatives and the
growth measured by SYBR green I assay
malaria parasite. It is important to point out that even as
nitro compounds have been found to be associated with
toxicity, it is not true that all nitro compounds are toxic.
Indeed, there are many Nitro compounds which gain target
specificity by the fact that they become selectively toxic to
the pathogen since their prodrug forms are selectively
activated only in the parasite by virtue of the presence of a
parasite specific nitroreductase which is absent from the
host. Systematic studies currently on in different labora-
tories have indeed revealed the potential of nitroaromatic
drugs to treat neglected parasitic diseases and the long held
prejudice against them is giving way to a more rational
approach to proceed with caution rather than disregarding
them (Patterson and Wyllie 2014). Nitroaromatic drug like
molecules may have the added advantage that as our data
given below shows, in synergy with other drugs, their
effective concentration is further reduced by three orders of
magnitude to levels which are unlikely to be toxic. Inter-
estingly replacing H with OH in Ih led to 3-fold increase in
potency against PfINDO with no substantial changes in
potency against Pf3D7 and PfDd2. These results may sug-
gest similarities and differences in drug targets across CQ-
resistant and CQ-sensitive strains. Replacing the six mem-
bered benzene rings by five membered furan (IIa & IIIa),
pyrrol (IIb), and thienyl (IIc) rings resulted in loss of
antiplasmodial activity. However, the thienyl containing
section. The regression analysis of the comparison between
the experimental XRD data and the simulated Gaussian data
2
returned a significant R of 0.9134 in case of the bond
2
lengths. Similarly, we recorded an R of 0.8841 in case of
the bond angles. These high coefficients indicate the
minimized conformation of the Gaussian structure to align
with the synthesized XRD structure. This proves the
accuracy of the chosen Gaussian basis set, level of theory
and the soundness of our computed values.
In vitro screening of antiplasmodial activity
The synthesized compounds (Scheme 1) were screened for
in vitro antiplasmodial activity against chloroquine-
sensitive 3D7 and chloroquine-resistant Dd2 and INDO
strains of P. falciparum using SYBR-green-I fluorescence
assay (Odds 2003). The growth inhibition curves (Fig. 3)
enabled the determination of IC50 values (Table 1). With
reference to the unsubstituted compound Ia, it was inter-
esting to observe that the IC50 was identical at 16 μM for
chloroquine-sensitive 3D7 and also CQ-resistant INDO
strains. However, an IC50 of 9 μM was observed against
another CQ-resistant strain viz. Dd2. The significantly
higher potency of dibenzylidinecyclohexanone Ia (IC50 16
μM) over cinnamylidinecyclohexanone Ii (IC > 100 μM)
sulphur at position 3 (IIIb) (Pf3D7 IC : 17 µM) was sig-
50
nificantly more potent than IIc where the sulphur is present
at position 2.
50
may suggest the importance of spacer length in binding the
target proteins. Interestingly, the best activity was displayed
by nitro substituted Ib against Pf3D7 (IC50 0.95 μM),
PfDd2 (IC50 1 μM) and PfINDO (IC50 1.1 μM) strains. It
may be noted that unlike Ia, the IC50 of Ib is close to 1 μM
across all the three strains tested. Similar potentiating effect
was observed also in the case of chloro substituted com-
pounds Ic-Ie. However, the effect of chloro substitution was
position specific with 2-Cl, 3-Cl and 4-Cl compounds Ic, Id
Drug combination studies of Ib/Id with chloroquine and
artemisinin
The first line antimalarial drugs CQ or ART (kept fixed 8X
IC50 values) were combined with changing concentrations
of Ib/Id to obtain differing molar ratios as indicated in SD
Table 1. Drug combination studies against CQ-resistant
PfINDO (Fig. 4) revealed that both Ib and Id exhibit strong
to moderate synergistic effects with artemisinin (∑FIC :
5
0

Medicinal Chemistry Research
Table 1 In vitro antiplasmodial and toxicity studies of DAC derivatives
Compounds
IC50 (μM)
Resistance indicesa
IC50 CQ-resistant
strain/ IC50 CQ-
Sensitive strain)
Selectivity indicesb Inhibition of β-
(
(IC50 mammalian
cells/IC50P.
haematin
formation IC50
(µM)
falciparum)
Pf3D7 PfDd2 PfINDO HeLa IC PfDd2/
IC P-
50
IC50 HeLa/
50
IC50Pf3D7
fINDO/
IC50Pf3D7
IC50Pf3D7
c
Ia (R=H)
16
0.95
13
9
16
35
0.6
1
2.2
Ib (R=NO2)
Ic (R=2-Cl)
Id (R=3-Cl)
Ie (R=4-Cl)
If (R=O-Me)
Ig(R=isopropyl)
Ih(R=OH)
1
1.1
4.8
2.5
9.4
n.d.
13.8
4.8
n.d.
100 1.1
1.2
0.4
0.8
1.1
n.d.
1.3
0.3
n.d.
105.3
5.2
>1000
>1000
>1000
>1000
370
9.2
2.7
6.7
67
25
64
0.7
0.9
0.8
3
8.3
8.5
7.5
>100 n.d.
n.d. n.d.
>100 0.6
>100 0.4
n.d. n.d.
n.d.
>9.5
>5.3
n.d.
10.5
18.8
6.8
8.2
>1000
>1000
520
Ii (2,6-Bis-
>100 n.d.
cinnamylidinecyclohexanone)
c
c
IIa (X=O)
IIb (X=NH)
IIc (X=S)
>100 n.d.
>100 n.d.
>100 n.d.
>100 n.d.
n.d.
n.d.
n.d.
n.d.
22.6
n.d. n.d.
n.d. n.d.
n.d. n.d.
n.d. n.d.
n.d.
n.d.
n.d.
n.d.
1.3
n.d.
n.d.
n.d.
n.d.
3.5
>1000
c
IIIa (X=O)
IIIb (X=S)
Chloroquine
17
14.6
60
0.9
760
40 nM 170 nM 440 nM >100 4.3
11
>2500
1380
n.d. not done for compounds that showed Pf3D7 IC50 > 100 μM
aResistance index (Ri): ratio of IC50 for drug-resistant strain/drug-sensitive strain: 10-100 indicates high-level resistance, > 1 but < 10 may indicate
an intermediate resistance level and < 1 low-level resistance.
bSelectivity Index (SI) ratio of IC50 for mammalian cell line/IC50 for P. falciparum:100 or more is considered to be good for safe potential drugs
cColoured compounds that caused interference in absorbance
combinations of Ib/Id with chloroquine showed little or no
interaction (∑FIC : 0.65 to 1.8 and 1.24 to 2.26, respec-
5
0
tively). Since both partners in each of the two combinations
Ib + Art and Id + Art) exhibit promising antiplasmodial
(
activity, the occurrence of synergy may suggest that the
partner drugs may interact with different molecular targets
across different biochemical pathways which have points of
convergence (Yin et al. 2014).
Note the strong to moderate synergy observed for the
combinations of Ib/Id with ART. Ib and CQ combinations
showed moderate synergy to no interaction while Id and CQ
combinations displayed strong antagonism in Fig. 4.
Drug exposure followed by drug withdrawal-based stage-
Fig. 4 Combinatorial effects (determined by SYBR Green assay) of Ib/
Id with chloroquine and artemisinin against CQ-resistant PfINDO
specific kill kinetic studies
Drugs must be studied for (a) the speed of their action (b)
for their static vs cidal effects on the target organism. Both
kinetics and net effect on the pathogen are crucial to the
larger question of the tendency of drugs to elicit drug
resistance. The faster a drug acts and the more decisive or
cidal is its action, the lesser is the opportunity for the
0
.34 to 0.63). A combined examination of Fig. 4 and SD
Table 1 indicates that in synergy combinations, while the
concentrations of ART are reduced 1.6 to 3-fold the
reduction in concentrations of Ib and Id were 34–478-fold
and 46–926-fold, respectively. On the other hand, the

Medicinal Chemistry Research
Fig. 5 Microscopic study of
antiplasmodial kill kinetics of Ib
against rings, trophozoites and
schizonts
pathogen to proliferate and develop resistance. In order to
find kinetics of action of Ib against ring, trophozoite and
schizont stages, we first incubated the parasite to Ib (IC90
histograms in panel D. Note that while Ib kills all stages, its
kill kinetics is fastest for S, intermediate for T and slowest
for R.
3
.1 μM) for different durations of time and then scored the
parasite for growth in drug-free medium. The results of
these studies (Fig. 5) indicating cidal activity of Ib against
all stages suggest that the target(s) for DACs may be present
in all the three stages. However, as shown in Fig. 5D, it was
of particular interest to see that the cidal action of Ib was
fastest (3 h) against schizonts followed by trophozoites (>3
to 6 h) and rings (>6 to 18 h). This may have to do with the
fact that the permeability and metabolic activity of malaria
parasite is known to increase from the relatively inert rings
to the far more active trophozoites and schizonts (Elford
et al. 1985).
The untreated cultures in each case have been compared
with cultures treated with Ib (D+; at IC100) for the variable
time points indicated, followed by washing the cultures and
letting the parasite grow in Ib free (D−) medium. The
combined total duration of culture in drug+ and drug−
media was 96 h (rings, R), 72 h (trophozoites, T) and 60 h
Docking studies
Microscopic stage-specific results clearly suggested that Ib
is equipotent against all the three erythrocytic stages of P.
falciparum. Further Ib showed fast cidal activity like
chloroquine. So we attempted to perform in silico docking
studies against the known targets of chloroquine viz. -β.
Haematin (Olafson et al. 2015) and P. falciparum lactate
dehydrogenase (PfLDH) (Read et al. 1999). The compounds
that showed significant antiplasmodial activities were taken
for in silico docking studies. Interestingly all these com-
pounds showed high binding affinities against PfLDH
(1CET) (Fig. 6, Table 2).
The compound Ib showed the highest binding affinities
and also has shown the best in vitro IC50 values among the
DACs. The binding affinity trend is mostly similar to the
2
potency of the DACs observed in vitro. R value of 0.8554
(
schizonts, S). The D+ images have been placed immedi-
was obtained when the binding affinities for the lactate
dehydrogenase (PfLDH) of P. falciparum and the IC50
values for the DACs against Pf3D7 were compared (plot
shown in the supplementary section). These significant
ately above the corresponding D− images. Microscopic
counting of ~3000 red blood cells enabled estimation of the
counts of live vs pycnotic R, T and S have been plotted as

Medicinal Chemistry Research
Table 3 Binding affinities for the DACs docked with the β-haematin
fragment
MOLECULE
Binding energy (kcal/mol) for β-haematin
Ia
−7.8
−6.9
−8.1
−7.9
−8.3
−7.2
−7.2
−6.0
−6.7
−6.9
−6.6
−6.5
−6.5
−6.4
Ib
Ic
Id
Ie
If
Ig
Fig. 6 Image of compound Ib bound to 1CET respectively
Ih
Ii
Table 2 Binding affinities for the DACs docked with the lactate
dehydrogenase (PfLDH) of P. falciparum
IIa
IIb
IIc
IIIa
IIIb
Binding energy in kcal/mol
Molecule
1CET
Chloroquine
−9.0
−8.8
−7.8
−7.5
−7.0
−7.5
−7.0
−6.3
−6.6
Ib
Id
Ie
Ig
Ic
Ia
IIIb
Ih
correlations between the in vitro and the in silico studies
suggest that the antimalarial efficacy of the DACs might be
due to their binding to the protein, hence providing a pos-
sible mechanism of action.
All the DACs showed significant binding to the β-hae-
matin fragment (Table 3) which further suggests that these
DACs may also be potent β-haematin growth inhibitors.
One docked pose of β-haematin with Ie is shown in Fig. 7.
The DACs and their interaction with the protein was
reviewed using PyMol and MOE where the active site
amino acid residues interacting with the ligands were
identified. The list includes both hydrophobic and hydro-
philic interactions. The details are shown in Table 4.
In the active site of the lactate dehydrogenase of plas-
modium falciparum (1CET) any common amino acid resi-
due interacting with all the DACs was not observed.
However, a few interesting points can be gleaned through
the results. The compound Ih contains a –OH substituent.
Barring it all the other DACs interact with the Ala 98 and
Gly 99 residues. Compounds Ia and IIIb have unsub-
stituted aromatic rings and they interact with the same set of
amino acid residues. The position of the substituents seems
to have some effect on the amino acid residues interacting
with the respective DAC. For example, the residues Gly 29,
Fig. 7 Image of compound Ie bound to the β-haematin fragment
Met 30, Ile 31, Thr 97 and Pro 250 were observed to
interact only in case of meta or ortho substituted aromatic
rings. Similarly, Ser 170, Arg 171, Tyr 174, Tyr 175, Arg
231, Glu 238 were seen to interact in case of para
substitutions.
These active site interactions help us to identify the
particular amino acid residues these DACs prefer to interact.
In turn, these interactions may help us to understand how
these ligands bind to the target protein and thereby result in
the corresponding antiplasmodial activity.
Lipinski’s parameters for active compounds
The active compounds were analysed for the drug likeness
properties using open-source DruLiTo 1. The results sum-
marized in Table 5 indicate that the in silico molecular

Medicinal Chemistry Research
Table 4 List of amino acid
residues interacting with each
ligand in the active site of 1CET
Molecule Interacting amino acids in 1CET
Ia
Ib
Asp 53, Ile 54, Ala 98, Gly 99, Phe 100, Ile 119, Glu 122
Gly 29, Met 30, Ile 31, Gly 32, Thr 97, Ala 98, Gly 99, Val 138, Leu 167, His 195, Ala 236, Pro
46, Pro 250
(
(
the lactate dehydrogenase
PfLDH) of P. falciparum)
2
Ic
Gly 29, Met 30, Ile 31, Thr 97, Ala 98, Gly 99, Leu 167, Pro 246, Pro 250
Gly 27, Gly 29, Met 30, Ile 31, Asp 53, Ile 54, Thr 97, Ala 98, Gly 99, Pro 246, Tyr 247
Ser 170, Arg 171, Tyr 174, Tyr 175, Arg 231, Glu 238, Leu 242
Id
Ie
Ig
Ser 170, Arg 171, Lys 173, Tyr 174, Tyr 175, Arg 185, Arg 231, Thr 235, Glu 238, Ile 239,
Ser 170, Arg 171, Tyr 174, Ile 239, Ser 245, Pro 246, Val 248, Ala 249, Pro 250
Asp 53, Ile 54, Ala 98, Gly 99, Phe 100, Ile 119, Glu 122
Ih
IIIb
Table 5 Lipinski’s parameters for active compounds across all strains
Lipinski’s parameters
the same assay. No correlation was observed between
antiplasmodial potency and anti-β haematin formation
potency since both If and Ii exhibited poor antiplasmodial
activity Pf3D7 IC > 100 μM. However, 2,6-bis-(3-thieny-
lidene) cyclohexanone (IIIb) with an IC50 of 17 µM against
Pf3D7 also showed a promising anti β-haematin IC50 of
S. no.
Compound
M. wt.
Log P
HBA
HBD
TPSA
5
0
1
2
3
4
5
Ib
347.98
325.93
325.93
325.93
271.94
2.781
3.501
3.501
3.501
3.211
1
1
1
1
1
0
0
0
0
0
103.35
17.07
17.07
17.07
67.67
Ic
Id
760 µM. Hence it is quite likely that with introduction of
Ie
additional drug like features into IIIb, its derivatives may
turn out to provide potent antimalarials in the future.
IIIb
HBA hydrogen bond acceptors, HBD hydrogen bond donors, TPSA
total polar surface area
Conclusions
characteristics for the active compounds are in compliance
with Lipinski’s rule of five for drug like compounds.
Given the established predictions of drug likeness
In conclusion, this work has through the study of several
different derivatives has identified the diarylidenecyclo-
hexanones as new pharmacophores against malaria para-
sites. Two of these derivatives Ib and Id displayed good
antiplasmodial activity against P. falciparum 3D7, Dd2 and
INDO strains compared to unsubstituted diarylidenecyclo-
hexanone (Ia). This investigation allows us to conclude that
the presence of electron withdrawing nitro (Ib)/chloro (Id)
at meta position is highly desirable for antiplasmodial
activity. Though nitro-based compounds are known for
toxicity, not all nitro compounds are toxic to the host. Thus
in the present study, Ib showed high selectivity (>100)
against mammalian cell line. Furthermore, Ib exhibited high
synergy in combination with Artemisinin, which led to
reduction by three orders of magnitude in the effective
concentration of Ib in the combination. In silico studies
returned significant binding affinities of several potent
compounds for Pf Lactate dehydrogenase on the one hand
and beta haematin fragement on the other. This may indi-
cate possible mechanism of action since the former is a
crucial glycolytic enzyme of the malaria parasite and the
latter is key to the haem detoxification strategy of the
parasite. Further the conformity to Lipinski’s parameters
establishes that the DACs can be explored as potential
leads. Coupled with the features of synergy and selectivity,
rapid kill kinetics of action against all erythrocytic stages of
malaria parasites and in silico data suggestive of targeting a
(
Ghose et al. 1999), we compared and contrasted the
obtained predicted values of the above derivatives prepared
to verify their potential as drugs. Firstly, all the log P Par-
tition coefficient values are well within the range of −0.4 to
+
2
forms to the desired range of 180–500. The net number of
atoms in each molecule falls in the lower domain of the
prescribed range of 20–70 (including H-bond donors and H-
bond acceptors). The calculated polar surface area is at max
1
5.6 ranges. The molecular weight is observed to start from
70 with the maximum going up to 348 which again con-
2
04 maintaining the sanctity of no >140 Ǻ .
Inhibition of detergent-mediated β-haematin formation
Since the in silico studies showed high affinity for β-hae-
matin, we tested the in vitro inhibitory activities of DACs
against detergent-mediated β-haematin formation (Table 1).
However the active molecules Ib and Id failed to show
inhibition of β-haematin formation (IC : >1000 μM). It was
interesting that while CQ a potent antimalarial (IC Pf 3D7:
0
tion showed a high IC50 value of 1380 μM in the detergent-
mediated β-haematin formation assay, some of the test
compounds including If (IC50 370 μM), Ii (IC50 520 μM)
and IIIb (IC50 760 μM) showed higher potency than CQ in
50
50
.04 μM) and a well-known inhibitor of hemozoin forma-

Medicinal Chemistry Research
key enzyme of glycolysis or the crucial haem detoxification
pathway, it appears that diarylidenecyclohexanones may be
good antiplasmodial pharmacophores for future design of
drugs against malaria.
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Acknowledgements BPJ, SK, GNR, DRB, SS and JK dedicate the
work to Founder Chancellor Bhagawan Sri Sathya Sai Baba. We thank
Sri Sai Giridhar Sharma (SSSIHL), for his valuable suggestions. We
also thank Sri Parth Gupta (IIT-Chennai) for his help with the MOE
software. Financial assistance from the CSIR (No. 01 (2286)/08/EMR-
II) is gratefully acknowledged. DM thanks to ICMR, New Delhi for
Senior Research fellowship. DM, GM and DS thank MR4 who gen-
erously provided the chloroquine-resistant Dd2 and INDO strains used
in the study.
Macrae CF, Bruno IJ, Chisholm JA et al. (2008) Mercury CSD 2.0--
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biological activities of curcumin: a short review. Life Sci
Conflict of interest The authors declare that they have no conflict of
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