Design, in silico and in?vitro evaluation of curcumin analogues against Plasmodium falciparum

Article abstract of DOI:10.1016/j.exppara.2017.02.006

The polyphenolic compound curcumin has been reported for its antimalarial properties in various scientific studies. Plasmodium falciparum ATP6, the parasite orthologue of mammalian sarcoplasmic Ca²⁺ ATPase (SERCA) has been identified as a key molecular target of both artemisinin and curcumin. The work was thereby undertaken to study the anti-malarial properties of two different series of curcumin analogues based on their docking interactions with PfATP6 and correlating the results with their anti-malarial activity. The compounds were designed retaining similar functional groups as that of the parent curcumin nucleus while incorporating changes in the carbon chain length, unsaturated groups and the number of ketone groups. The compounds (1E, 4E)-1,5-bis(4-methylphenyl)penta-1,4-dien-3-one (CD-9), (1E, 4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (CD-8) and (E)-1,3-bis(4-hydroxylphenyl)prop-2-en-1-one (CD-1) showed IC₅₀ values of 1.642?μM, 1.764?μM and 2.59?μM in 3D7 strain and 3.039?μM, 7.40?μM and 11.3?μM in RKL-2 strain respectively. Detailed structure-activity relationship studies of the compounds showed that CD-9 and CD-8 had a common hydrophobic interaction with the residue Leu268 of the PfATP6 protein and has been postulated through our study to be the reason for their antimalarial activity as seen after corroborating the results with the in?vitro study. The study provided valuable insight about the ligand-protein interaction of the various functional groups of curcumin and its analogues against the PfATP6 protein and their importance in imparting antimalarial action.

Full text of DOI:10.1016/j.exppara.2017.02.006

Accepted Manuscript
Design, in silico and in vitro evaluation of curcumin analogues against Plasmodium
falciparum
Chandrajit Dohutia, Dipak Chetia, Kabita Gogoi, Kishore Sarma
PII:
S0014-4894(17)30070-X
10.1016/j.exppara.2017.02.006
YEXPR 7367
DOI:
Reference:
To appear in: Experimental Parasitology
Received Date: 17 August 2016
Revised Date: 27 December 2016
Accepted Date: 3 February 2017
Please cite this article as: Dohutia, C., Chetia, D., Gogoi, K., Sarma, K., Design, in silico and in vitro
evaluation of curcumin analogues against Plasmodium falciparum, Experimental Parasitology (2017),
doi: 10.1016/j.exppara.2017.02.006.
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ACCEPTED MANUSCRIPT

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Design, in silico and in vitro evaluation of curcumin
analogues against Plasmodium falciparum
Keywords: Curcumin, synthesis, PfATP6, Plasmodium falciparum, SAR.
Name, affiliation and contact address of authors:
Chandrajit Dohutia1*, Dipak Chetia1, Kabita Gogoi2, Kishore Sarma2
1Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh-786004
2Regional Medical Research Centre NE (Indian Council of Medical Research), Dibrugarh-
786001
*corresponding author
Chandrajit Dohutia
Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh-786004
Contact No: +91-8011713589
Email: chnadrajit@gmail.com

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Abstract
The polyphenolic compound curcumin has been reported for its antimalarial
properties in various scientific studies. Plasmodium falciparum ATP6, the parasite orthologue
of mammalian sarcoplasmic Ca2+ ATPase (SERCA) has been identified as a key molecular
target of both artemisinin and curcumin. The work was thereby undertaken to study the anti-
malarial properties of two different series of curcumin analogues based on their docking
interactions with PfATP6 and correlating the results with their anti-malarial activity. The
compounds were designed retaining similar functional groups as that of the parent curcumin
nucleus while incorporating changes in the carbon chain length, unsaturated groups and the
number of ketone groups. The compounds (1E,4E)-1,5-bis(4-methylphenyl)penta-1,4-dien-3-
one (CD-9), (1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (CD-8) and (E)-1,3-
bis(4-hydroxylphenyl)prop-2-en-1-one (CD-1) showed IC50 values of 1.642 µM, 1.764 µM
and 2.59 µM in 3D7 strain and 3.039 µM, 7.40 µM and 11.3 µM in RKL-2 strain
respectively. Detailed structure-activity relationship studies of the compounds showed that
CD-9 and CD-8 had a common hydrophobic interaction with the residue Leu268 of the
PfATP6 protein and has been postulated through our study to be the reason for their
antimalarial activity as seen after corroborating the results with the in vitro study. The study
provided valuable insight about the ligand-protein interaction of the various functional groups
of curcumin and its analogues against the PfATP6 protein and their importance in imparting
antimalarial action.

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1. Introduction
Globally malaria is beginning to show signs of abatement yet it still afflicts millions
across the globe. Its worldwide figures border on two hundred million clinical cases and half
a million deaths 1. Artemisinin has long been the backbone of anti-malarial studies but of late
concerns over its resistance in Southeast Asian countries and Greater Mekong areas have led
2
to newer combinations of anti-malarial agents being employed . Thereby, newer, more
potent and less toxic anti-malarial leads are the need of the hour. Despite the requirement of a
novel anti-malarial agent, drug discovery for malaria is a time consuming and expensive
process 3, 4. The continuous evolution of the drug discovery methods and high-quality lead
generation process is likely to deliver potential compounds with better therapeutic activity 5.
The PfATP6 protein, an orthologue of the mammalian sarcoplasmic endoplasmic reticulum
6
Ca2+ ATPase (SERCA) is considered as a target for artemisinin and its derivatives .
7
Curcumin possesses incredible properties to combat a variety of ailments plaguing people .
Studies have shown that curcumin exhibits a considerable amount of activity against both
human and rodent strains of malaria parasite in in-vitro and in vivo 8-11. The antiparasitic
action of curcumin could be attributed to its binding to a hydrophobic pocket in the
transmembrane region of the PfATP6 (modelled) protein in the parasite and thereby
interfering with its calcium transport 12. A detailed structural elucidation of the curcumin
molecule had earlier revealed that the phenolic groups in the curcumin nucleus play a crucial
role in its anti-malarial activity. Any change in these groups apart from esterification has
been reported to have decreased the anti-malarial efficacy of the compound 13. The ß-
diketone moiety has been associated with the instability of curcumin and has undergone
considerable modifications of late 14. However, some researchers consider the diketo group to
be essential for antimalarial activity 15. Ring closure of the ß-diketone moiety by
condensation reactions involving hydrazines has led to considerable improvement in the
antimalarial properties 16. Aher et al. (2011) synthesized and screened a series of
dibenzalacetone analogues against P. falciparum using different substituents and obtained
promising results 17. Various synthetic and natural chalcones have been reported to have
proficient antimalarial property, some, of which also showed good activity against CQ-
resistant P. falciparum strains 18-22. Encouraging results were earlier obtained through
docking studies of different chalcone derivatives against the cysteine protease (falcipain)
23
enzyme . Studies using the homology models of falcipains 1, 2 and 3 and their screening
against the W2 and D6 strains showed promising results 24-26. Based on these studies we

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designed a library of fifteen dibenzalacetones and chalcones respectively which were
structurally similar to curcumin and docked them against the PfATP6 protein. Based on their
binding scores we synthesized and evaluated a total of ten compounds and tested against the
CQ sensitive 3D-7 and mutant RKL-2 strain of P. falciparum. Though studies based on some
19-23
chalcones and dibenzalacetones as anti-malarial agents have been earlier performed
,
molecular docking studies against PfATP6 protein, impact of the different functional groups,
particularly that of the hydroxyl groups, difference in the carbon chain lengths, unsaturated
carbons, ketone groups and the presence/absence of electron donating/withdrawing groups on
the in vitro anti-malarial efficacy have been reported for the first time. The ideology behind
the study was to the synthesize compounds based on their binding energies to PfATP6,
corroborating the results with their in vitro activity and formulate an SAR to identify the
residues and groups responsible for increase/decrease in antimalarial activity of the
compounds. The work is likely to help in developing potential leads for the development of
newer drugs/hybrid molecules against malaria.
2. Materials and methods
2.1. Target identification
The protein PfATP6, also known as PfSERCA is a 139 kDa protein composed of
1228 amino acids which share 51% identity with mammalian SERCA protein and has been
27
proved to be a major molecular drug target of artemisinin antimalarials . PfATP6 has been
identified as the common binding site of both artemisinin and curcumin. Hong-Fang Ji and
Liang Shen used PfATP6 as the drug target for curcumin binding which indicated its
interactions with PfATP6 through hydrophobic and hydrogen bonds and its subsequent
inhibition 28. As a consequence, in the present study a series of curcumin analogues were
designed and targeted against PfATP6 to check for their binding affinity and pattern
considering curcumin as a secondary control and chloroquine as a primary control. This, in
turn, unveils their anti-malarial potential. In absence of crystallographic structure of PfATP6,
the modelled structure of PfATP6, (PDB ID: 1U5N) designed by Krishna et al. and Salas-
Burgos et al. was used 29, 30
.
2.2. Ligand dataset preparation and optimization
A ligand library of two different series of curcumin analogues each containing 15
compounds was designed using ChemDraw Professional (PerkinElmer Informatics 2016).
Selection of the ligands was based on structural similarity to the parent compound curcumin.

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The carbon chain was decreased and modification was made in the aryl groups using
substituents found in the curcumin molecule in the hope of increasing its anti-malarial
efficacy. Table 1 shows the positions of different substituents on the synthesized analogues.
The “Prepare ligand” protocol of DS 4.5 was used to prepare the ligands which remove
duplicate structures, standardizes the charges of common groups, calculates the ions and
ionization of the ligand’s functional groups, generates isomers and tautomers, 2D-3D
conversion, verifying and optimizing the structures, and other tasks established by user-
defined parameters. Energy minimizations of all the ligands were done by applying
CHARMM force field.
2.3. Docking of receptor with ligand
PfATP6 was used as receptor molecule for the docking study to probe the binding free
energy between the ligand library and receptor using AutoDock 4.2 31. Autodock Tools
(ADT) was used to optimize the receptor and ligand molecules. For the preparation of the
receptor molecule, polar hydrogen’s, Kollman charges and AD4 type of atoms were added,
while Gasteiger charges were added on the ligands and maximum numbers of active torsions
were given. AutoGrid4 was used to prepare a grid map of interaction energies around
Leu263, Phe264, Gln267, Ile977, Ile981, Ala985, Asn1039, Leu1040, Ile1041 and Asn1042
with a grid box of 90 X 90 X 90 Å3 centred on X, Y, Z = 52.27, 16.45, 11.48 with a grid
spacing of 0.375Å. The residues in the current study have been used with reference to earlier
reports of their interactions with artemisinin and curcumin32-34. Molecular docking was
performed using Lamarckian Genetic Algorithm (LGA), keeping the receptor molecule rigid
throughout the docking simulation and rest of the docking parameters was set to default
values. Ten different poses were generated for each ligand and scored using AutoDock 4.2
scoring functions and were ranked according to their docked energy. AutoDock Tools,
35, 36
PyMOL
and Discovery studio 4.5 client (BIOVIA Discovery Studio Client 4.5) were
used for post-docking analysis.
2.4. Chemistry
Melting points were measured with a Buchi B-540 melting point apparatus and are
uncorrected. IR spectra were recorded on Bruker ALPHA FT-IR spectrometer on a thin film
13
1
using chloroform. C and H NMR spectra were recorded on Bruker Avance II 400-NMR
spectrometer using tetramethylsilane (TMS) as an internal standard. Mass spectra were
recorded on Waters, Q-TOF micromass (ESI-MS) spectrometer. All the commercially

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available reagents were used as received. All experiments were monitored by thin layer
chromatography (TLC). TLC was performed on prepared silica glass plates. Column
chromatography was performed on silica gel (60-120 mesh, Merck Chemicals). In
this
study, one step facile synthesis of dibenzalacetone and chalcones based on their structural
homology with curcumin and binding energy scores were carried out via the pathways
illustrated in Scheme 1, 2 (Fig. 2, Fig. 3). Dibenzalacetones (CD 8, 9, 10) were prepared with
substituted benzaldehydes and acetone (2:1) in a base catalyzed reaction via aldol
condensation 37, 38. Synthesis of various benzylideneacetophenones (chalcones) was carried
out using Claisen condensation 39. Hydroxyllated dibenzalacetones (CD 5, 12) were
synthesized under protic conditions using an alternate route 40.
2.4.1 General procedure for synthesis of chalcone derivatives (CD-1, 2, 13, 14, 15):
Equimolar amounts of benzaldehydes and acetophenones were dissolved and stirred
in a beaker to which 50% sodium hydroxide was added dropwise with moderate heating.
Stirring was continued till the mixture solidified. The residue was washed with ice cold water
and recrystallized with hot ethanol.
2.4.2 (E)-1,3-bis(4-hydroxylphenyl)prop-2-en-1-one (CD-1): Yield 71%; Rf = 0.78
(Toluene: Ethyl acetate: Formic acid = 5:4:1); Light brown crystals; m.p. 203-205°C. IR (cm-
1): 3147, 1646, 1654. 1H NMR (DMSO, 400 Mhz): δ 8.1206 (s. 2H), 7.4904-7.5143 (m, 3H),
13
6.8060-6.9429 (m, 2H), 6.6034-6.6336 (m, 2H), 7.2725-7.3155 (m, 3H). C NMR (DMSO,
400 Mhz): δ 191.72, 168.49, 159.70, 120.80, 143.22, 130.22, 123.22, 120.80, 113.94, 135.45.
MS (EI, m/z) = 224 (M-60+).
2.4.3 (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (CD-2): Yield 87%; Rf = 0.2
1
(EtOAc/Hexane = 1:4); Pale yellow crystals; m.p. 71-73°C. IR (cm-1): 3721, 1664, 1651. H
NMR (CDCl3, 400 MHz): δ 3.84 (s, 3H), 6.9210-6.9429 (m, 2H), 7.9949-8.0165 (m, 2H),
7.3919-7.6083 (m, 6H), 15.68 (d, 1H) 13C NMR (CDCl3, 400 MHz): δ 190.57, 161.71,
144.72, 119.76, 138.51, 114.45, 128.44, 128.59, 127.61, 132.60, 130.27 and 55.42. MS (EI,
m/z) = 239 (M-60+).
2.4.4 (2E)-1,3-diphenylprop-2-en-1-one
(CD-13):
Yield
93%;
Rf
=
0.3
(CHCl3/CH3OH/C6H6 = 6:3:1); Lemon yellow coloured crystals; m.p. 57-59°C. IR (cm-1):
1
1680, 1645. H NMR (CdCl3, 400 Mhz): δ 7.7933-7.8326 (m, 2H), 7.5895-7.6549 (m, 2H),
7.4878-7.5051 (m, 2H), 7.4130-7.4816 (m, 2H), 7.5124-7.5686 (m, 2H), 7.5784-7.5838 (m,
2H). 13C NMR (CDCl3): δ 190.61, 145.2, 138.22, 134.89, 132.85, 130.60, 129.00, 128.67,
128.55, 128.50, 122.10. MS (EI, m/z) = 209 (M-60+).
2.4.5 (E)-3-(4-hydroxylphenyl)-1-phenylprop-2-en-1-one (CD-14): Yield 71%; Rf = 0.4
(CHCl3/CH3OH/C6H6 = 5:2:1); Pale brown coloured crystals; m.p. 83-85°C. IR (cm-1): 3640,
1
1670, 1644. H NMR (DMSO, 400 Mhz): δ 9.3494 (s, 1H), 6.336-6.6705 (t, 2H), 7.3919-
7.6036 (m, 3H), 7.6083-7.6152 (m, 2H), 8.3765-8.3783 (m, 2H). 13C NMR (DMSO): δ

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193.45, 173.09, 138.22, 134.03, 133.85, 128.66, 128.43, 114.46, 144.90, 123.99, 118.76. MS
(EI, m/z) = 224 (M-60+).
2.4.6 (E)-1-phenyl-3-(p-tolyl)prop-2-en-1-one (CD-15): Yield 89%; Rf
=
0.7
(CHCl3/CH3OH/C6H6 = 7:2:1); Yellow coloured crystals; m.p. 99-101°C. IR (cm-1): 1680,
1260, 1650. 1H NMR (CdCl3, 400 Mhz): δ 2.3865 (s, 3H), 8 (t, 2H), 7.4686-7.5738 (m, 6H),
15.72 (d, 1H), 8.68 (t, 2H). 13C NMR (CdCl3, 400 Mhz): 190.67, 144.98, 141.13, 138.38,
132.71, 132.17, 129.74, 128.62, 128.52, 128.50, 121.10, 21.58. MS (EI, m/z) = 222 (M-60+).
2.5.1 General procedure for synthesis of dibenzalacetone derivatives (CD-8, 9, 10):
Substituted benzaldehydes and acetone were mixed together (2:1) in 99% ethanol and
10% sodium hydroxide was added drop wise till the contents solidified. The reaction was
monitored by TLC using a solvent mixture of ethyl acetate and hexane (3:7). The product was
washed repeatedly with cold water, dried and recrystallized with ethyl acetate to obtain pure
crystals.
2.5.3 (1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (CD-8): Yield 87%; Rf = 0.4
(EtOAc/Hexane = 1:8); lemon yellow coloured crystals; m.p. 79°C. IR (cm-1): 3280, 1250,
1
1710, 1652. H NMR (CdCl3, 400 Mhz): δ 3.8187 (s, 3H), 6.9176-6.9691 (m, 2H), 7.5730-
7.5800 (d, 1H), 7.4512-7.5011 (m, 1H), 7.5442-7.5683 (m, 2H). 13C NMR (CDCl3): δ 188.81,
161.60, 161.53, 55.37, 130.08, 129.97, 114.46, 127.02, 127.58. MS (EI, m/z) = 294 (M-60+).
2.5.4 (1E,4E)-1,5-di-p-tolylpenta-1,4-dien-3-one (CD-9): Yield 83%; Rf
=
0.3
(EtOAc/Hexane = 2:7); Light Yellow coloured crystals; m.p. 87-89°C. IR (cm-1): 1711, 1260,
1650. 1H NMR (CdCl3, 400 Mhz): δ 2.3836 (s, 3H), 7.0149-7.0548 (d, 1H), 7.2033-7.2532 (t,
2H), 7.4990-7.5193 (d, 2H), 7.6893-7.7721 (d, 1H). 13C NMR (CDCl3): δ 21.56, 189.07,
143.18, 140.98, 132.13, 129.73, 128.43, 124.60. MS (EI, m/z) = 263 (M-60+).
2.5.5 (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (CD-10): Yield 91%; Rf
=
0.5
(EtOAc/Hexane = 3:7); Yellow coloured crystals; m.p. 95-97°C. IR (cm-1): 1650, 1255, 1645.
1H NMR (CdCl3, 400 Mhz): δ 7.0566-7.0964 (d, 2H), 7.3799-7.4145 (m, 3H), 7.5911-7.6512
(m, 2H), 7.7142-7.7541 (d, 1H). 13C NMR (CDCl3): δ 188.95, 143.35, 134.81, 130.55,
129.01, 128.45, 125.45. MS (EI, m/z) = 235 (M-60+).
2.6.1 General procedure for synthesis of dibenzalacetone derivatives (CD-5, 12):
Substituted aldehyde and ketone (2:1) were dissolved in glacial acetic acid saturated
with anhydrous HCl in an ice bath. The mixture was then kept at room temperature for an
hour and subsequently heated in a water bath at 25-35°C for 2 hours. The resultant mixture
was kept at room temperature for two days after which it was treated with cold water and
filtered. The solid obtained was washed, dried and purified using column chromatography.
2.5.2 (1E,4E)-1,5-bis(2-hydroxylphenyl)penta-1,4-dien-3-one (CD-5): Yield 70%; Rf = 0.6
(EtOAc/Hexane = 1:8); cream coloured flaky crystals; m.p. >300°C. IR (cm-1): 3227, 1672,
1644. 1H NMR (CdCl3, 400 Mhz): δ 9.9402 (s, 1H), 8.3773 (m, 1H), 7.3604-7.4039 (m, 2H),

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7.4933-7.5176 (m, 1H), 6.6198-6.7226 (m, 2H). 13C NMR (CdCl3): δ 195.25, 170.53,
137.93, 129.62, 123.57, 121.87, 119.76, 115.83, 113.94. MS (EI, m/z) = 266 (M-60+).
2.6.2 (1E,4E)-1,5-bis(4-hydroxylphenyl)penta-1,4-dien-3-one (CD-12): Yield 77%; Rf = 0.4
(EtOAc/Hexane = 4:6); Green coloured crystals; m.p. 239-241°C. IR (cm-1): 3227, 1672,
1
1644. H NMR (DMSO, 400 Mhz): δ 9.4700 (s, 1H), 8.3773-8.3783 (d, 1H), 7.6848-7.7066
(m, 2H), 7.0149-7.0548 (d, 1H), 6.7183-6.7500 (t, 2H). 13C NMR (DMSO): δ 193.63, 160.40,
133.75, 124. 58, 118. 40, 158.40. MS (EI, m/z) = 266 (M-60+).
3. Antimalarial activity
3.1 Preparation of parasites
The chloroquine sensitive 3D7 (West Africa) and chloroquine resistant RKL-2 strain
(Raurkela, Orissa, India) of P. falciparum were routinely maintained in stock cultures in
medium RPMI-1640 supplemented with 25 mmol HEPES, 1% D-glucose, 0.23% sodium
bicarbonate and 10% heat inactivated human AB +ve serum in a CO2 incubator maintained at
37°C and 5% CO2 level 41. The asynchronous parasites of P. falciparum were synchronized
after 5% D-sorbitol treatment to obtain only the ring stage of the parasite. For carrying out
the assay, the initial ring stage parasitemia was maintained at 1% and 3% hematocrit.
3.2 In vitro anti-malarial screening
The in-vitro antimalarial screening was carried out following the microassay methods
of Reickmann and Desjardins with minor modifications 42 - 44. We used two different dosages
5ug/ml and 50 µg/ml for preliminary screening of the compounds and the compounds with
good antimalarial activity were further tested for determination IC50 values. The compounds
were dissolved in 1:200 dimethyl sulfoxide (DMSO) to get a stock solution of 5 mg/ml
concentration. The further required dilutions of the stocks were prepared with incomplete
media (without serum). To a 96-well flat bottom microtiter plate required amount of the test
compounds of the secondary standard curcumin and different test dosages were charged per
well in triplicates and accordingly the required volume of synchronized parasites in 3%
hematocrit containing 1% parasitemia was added to get the final test dose. Chloroquine
(Sigma chemicals) as a primary standard and curcumin (Sigma chemicals) was used as a
secondary standard for this test to validate the integrity of the assay. The negative control
wells inoculated with 20 µl of CRPMI to which 180 µl of 3% hematocrit with 1%
parasitaemia were added while another set of negative control wells received 20 µl of

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incomplete culture medium and 0.5 % DMSO (Fig. 5). The plates were incubated for 40 hrs
in a water jacketed incubator at 37⁰C and 5% CO2 environment, after which thin blood
smears were prepared from each well, fixed with methanol, stained with 3% Giemsa and
observed under microscope. The level of parasitemia in terms of % dead rings and
trophozoites was determined by counting a total of 100 asexual parasites (both live and dead)
microscopically.
3.3 Determination of IC50 values of the screened compounds
The compounds which showed a minimal antimalarial activity of 20% (at 5 ug/ml) to
80% (at 50ug/ml) of inhibition were selected for determination of their IC50 values. Multiple
doses were taken based on their parasite inhibiting properties as determined microscopically.
The compounds and the parasite concentrations were adjusted in the same way as that of the
screening method. Percentage reductions were used to plot percentage inhibition of growth as
a function of drug concentrations. IC50 values are determined by log-concentration–response
probit analysis program 45
.
4. Results and Discussion
4.1. Molecular docking study
The docking studies of all the synthetized compounds were performed into the
binding pocket of PfATP6 (1U5N.pdb) protein (Leu263, Phe264, Gln267, Ile977, Ile981,
Ala985, Asn1039, Leu1040, Ile1041). Selection of the ten compounds for synthesis was
based on the binding energies obtained by using AutoDock 4.2 against the PfATP6 protein
(Table 1) Compounds CD-1, 8 and 9 had slightly higher binding energies (-5.44, -6.36 and -
6.37 Kcal/mol respectively) then the secondary standard curcumin (-4.95 Kcal/mol) while the
rest of the compounds (CD-2, 5, 10, 12, 13, 14, 15) displayed binding energies closer to that
of curcumin (-4.58, -4.64, -4.79, -4.95, -4.44, -4.86 and -4.99 Kcal/mol respectively). 2D
interactions using discovery studio client 4.5 show that the major binding source of the
designed compounds is through Van der Waals forces, Pi-Sigma and other hydrophobic
interactions (Fig. 4).
4.2. Chemistry

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As stated earlier the compounds were designed based on the structural features of
curcumin. In various synthesis the substituents used consisted of electron donating groups
such as hydroxyl, methoxy and methyl groups. The chalcones were prepared using the facile
one step Claisen-Schmidt condensation reaction while the dibenzalacetone analogues not
containing hydroxyl groups were prepared using Aldol condensation. Dibenzalacetone
analogues containing hydroxyl groups were prepared using an alternative route under
anhydrous conditions using hydrogen chloride gas produced simultaneously using sodium
chloride and sulphuric acid and passing it through the reaction mixture. All the spectroscopic
data have been presented in the experimental section. For compound CD-8 IR stretching
bonds at 3294, 1259, 1625 were observed signalling the formation of aromatic rings, methoxy
groups and ketones. A strong band at 3721 in compound CD-1 expressed the stretching
pattern of the hydroxyl group while a band at 1625 indicated the presence of a ketone group.
The stretching bands from 2945-3295 show the presence of methyl groups while the band at
1625 denotes the keto group in the compound CD-9.
4.2 Antimalarial evaluation and Structure-Activity Relationship
All ten compounds passed the screening conditions and were further evaluated for the
determination of their IC50 values. Compounds (1E,4E)-1,5-bis(4-methylphenyl)penta-1,4-
dien-3-one (CD-9) with methyl substitution and (1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-
dien-3-one (CD-8) with methoxy substitution showed the best activity with an IC50 of 1.64
µM and 1.764 µM against the 3D-7 strain and 3.03 µM, 7.40 µM against the RKL-2 strain
respectively. (E)-3-(2-hydroxylphenyl)-1-phenylprop-2-en-1-one (CD-1) with an hydroxyl
substitution showed an IC50 value of 2.59 µM against the 3D-7 strain and 11.3 µM against
the RKL-2 strain respectively. CD-2, CD-14, CD-15 were chalcone derivatives containing
methoxy, hydroxyl and methyl groups respectively while CD-13 was devoid of any
functional group substituents. CD-14 had a higher binding energy than CD-2 and CD-13 (-
4.86, -4.58, -4.44 Kcal/mol respectively) due to the interaction of its hydroxyl group with
Asn 1042. This can be observed from their IC50 values (14.03, 29.68, 41.372 against 3D-7
and 25.26, 44.55, and more than 100 higher than 100 <mu>g/ml against RKL-2 strains).
Dibenzalacetone analogues CD-5 and CD-12 containing hydroxyl groups showed binding
energies of -4.64 and -4.95 Kcal/mol but higher IC50 values than that of CD-9 and CD-8. This
showed that hydrogen bond interactions between the hydroxyl groups CD-5 and CD-12 with
residues Leu 257 of the PfATP6 protein do not play an active part in altering the antimalarial
properties of these compounds. In view of the hydrophobic nature of dibenzalacetones and

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chalcones, it can be inferred that the hydrophobic interactions play more important roles in
the binding of these compounds with PfATP6. Curcumin has also been reported to have
significant number of hydrophobic interactions with PfATP6 25. Two residues are involved in
the hydrogen bond formation between Asn267 with one of the phenolic groups and Arg 1034
with a ketonic group of curcumin respectively. Arg1048 showed an unfavourable linkage
with the second phenolic group of curcumin. Compounds CD-9, CD-8, CD-1 were observed
to fit in the same binding pocket as that of curcumin and interact with the PfATP6 protein
through a series of hydrophilic and hydrophobic interactions. Compound CD-9 was showed
to exhibit π-alkyl interaction between the methyl groups and Lys260, Leu1040, Ile981 and
Ile272. The aromatic ring of CD-9 was found to interact with Leu268 via π-σ bonds. No
hydrogen bond formation was seen in the CD-9 compound. Compound CD-8 showed π-alkyl
interactions in between the aromatic rings and the residues Ile1041, Ile977, Ile271.
Interaction via π-σ bonds was observed in-between the aromatic ring of CD-8 and Leu 268. A
common site of interaction was seen in case of CD-9 and curcumin with the Leu1040 residue
though its role in antimalarial activity remains to be seen. In-vitro antimalarial activity results
showed that the most active compounds CD-9 and CD-8 showed higher antimalarial activity
against both strains of P. falciparum, which could be confirmed by the interactions with
Leu268. Both the compounds belonged to the class of dibenzalacetones. Presence of electron
donating groups (methyl, methoxy) in CD-9 and CD-8 contributed significantly to their anti-
malarial activity as can been seen from table 1. All chalcone based compounds had different
binding interactions with the PfATP6 molecule, making it difficult to isolate the specific
interaction responsible for their antimalarial properties. CD-1 showed good anti-malarial
activity comparable to that of curcumin. This could be attributed to the hydrogen bonding
interactions between the hydroxyl groups of the acetophenone and benzaldehydic segments
of the chalcones with Arg1048 and Asn1042. Presence of an unfavourable linkage in the
curcumin molecule was observed which might hinder the attachment of the molecule to the
receptor and therefore lead to lesser duration of anti-malarial action (Fig. 4). In earlier studies
it was reported that the antimalarial action of curcumin was mainly due to the interaction of
its phenolic groups with the PfATP6 molecule via hydrogen bonding with Leu1040 and
Ala985 residues respectively 13, 28. However the dibenzalacetone compounds CD-9 and CD-8
did not contain hydroxyl groups as substituents even though they showed higher binding
energies and antimalarial activity then curcumin. This showed that hydrophobic interactions
between the PfATP6 residues and dibenzalacetones is mainly responsible for its antimalarial

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activity and though sharing a close structural resemblance to curcumin, their ways of protein
interaction with PfATP6 are not the same.
5. Conclusion
In the present work we synthesized ten curcumin analogues based on their binding
energies to the modelled structure of the PfATP6 protein. Visualization studies showed
favourable hydrophobic interactions (π-σ, π-alkyl) between the aromatic rings of CD-9 and
CD-8 with the residue Leu268 which we have postulated as being the reason for their better
in vitro antimalarial activity against the 3D7 and RKL-2 strains when compared to curcumin.
Electron donating groups increased the antimalarial activity of the synthesized analogues.
Further in vivo studies would be required to determine the efficacy of the compounds, their
toxicities and the effect of biotransformation on the compounds. The study is likely to
provide valuable insight and in-depth assessment to researchers in the design and
development of curcumin derivatives/analogues as promising drug candidates in the battle
against malaria. The decreased molecular weight of the compounds can be useful in the
formation of synthetic hybrids with piperine which is well known to augment the activity and
bioavailability of curcumin 46, 47
.

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Fig 1. Docked orientation showing the fitting of compounds CD-8, CD-9, CD-1 and
curcumin into the binding pockets of PfATP6 protein.
Fig 2. Reaction scheme: Synthesis of dibenzalacetone derivatives
a) 99% Ethanol, 10% Sodium hydroxide, stir, 24 hours, rt.
b) Glacial acetic acid, Hydrogen chloride gas, stir, 2-3 hrs 25-35°C, kept for two days.

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Fig 3. Reaction scheme: Synthesis of chalcones
a) 95% Ethanol, 50% Sodium hydroxide added dropwise.
Fig 4. CD-9, CD-8, CD-1 and curcumin ligand protein interactions. All interacting amino
acids exhibiting Cation- π and π- π interactions with the ligands are shown as dotted violet

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lines, while hydrogen bonds are shown as dotted green lines. The pink coloured lines indicate
π- Alkyl interactions while the red dotted lines indicate unfavourable bumps. Orange dotted
lines indicated π- cation linkage. The green spheres represent Van der Waals forces.
Fig 5.A) Negative control (3D7 strain) and B) CD-9 treated slide (3D7 strain)

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Table 1: Binding energies, substituents and IC50 values of the synthesized compounds
Compound
code
Substituents
Binding
energy
(Kcal/m
ol)
3D7
*R2=
RKL-2
R2=
R
R1
R’
IC50
(ug/m
l)
#Y=
IC50
(ug/ml)
Y=
H
H
OH
H
CD1
CD2
CD5
-5.44
2.59
0.809 6.741 X
+32.5
11.3
0.851
0.957
0.977X +
38.96
OCH3 OH
-4.58
-4.64
29.68 0.945 0.828X +
25.42
44.55
0.606X +
23
O
H
H
OH
-
21.32 0.860 0.507X +
31.66
40.584 0.908
0.650X +
23.62
OCH3
CH3
H
-
-
CD8
-6.36
-6.37
-4.79
-4.95
-4.44
-4.86
-4.95
-
1.764 0.886 7.182X
+37.33
7.40
0.843
0.935
0.900
0.865
1.116X +
41.74
H
H
H
H
H
-
CD9
1.642 0.924 13.17X +
28.37
3.039
35.6
15.3X +
3.5
-
CD10
18.66 0.876 1.885X +
14.82
0.68X +
25.79
OH
H
-
CD12
12.67 0.844 1.901X +
25.91
21.86
1.293X +
21.73
H
H
-
CD13
41.37 0.900 0.561X +
2
Higher than 100ug/ml
26.79
OH
-
CD14
14.03 0.867 2.073X +
20.91
25.26
5.59
1.44
0.892
0.893
-
1.191X
19.91
14.79X +
4.1
Curcumin
Chloroquine
2.13
0.903 13.11X +
26.33
-
-
-
0.7
-
-
-
*R2= Chi-square value
#Y= mx + c
Acknowledgements
We would like to thank the Department of Pharmaceutical Sciences, Dibrugarh
University for providing the facilities to carry out the synthetic work and Regional Medical
Research Centre, Dibrugarh for providing the Plasmodium falciparum strains for antimalarial
assay. We would also like to thank SAIF, Panjab University for carrying out the NMR and
MS analysis of the compounds. In this regard, we would like to appreciate the efforts of
Partha Pratim Kaishap of CSIR, NEIST in providing assistance while analyzing spectral data.

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Highlights
‹
The work involves design of dibenzalacetones and chalcones based on their structural
similarities to curcumin and their subsequent docking against the Plasmodium
falciparum ATP6 protein (PfATP6). Based on their binding energies ten compounds
were synthesized, screened and evaluated against the sensitive 3D-7 and mutant RKL-
2 strains of Plasmodium falciparum.
‹
The compounds (1E,4E)-1,5-bis(4-methylphenyl)penta-1,4-dien-3-one (CD-9),
(1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (CD-8) and (E)-1,3-bis(4-
hydroxylphenyl)prop-2-en-1-one (CD-1) showed better binding energies (-6.37, -6.36,
-5.44 Kcal/mol respectively) and in vitro antimalarial activity (IC50 1.642 µM, 1.764 µM
and 2.59 µM in 3D7 and 3.039 µM, 7.40 µM and 11.3 µM in RKL-2) than the
secondary standard curcumin (-4.95 Kcal/mol; IC50 2.13 µM in 3D7, 5.59 µM in
RKL-2).
‹
The study is likely to provide valuable insight and in-depth assessment to researchers
in the design and development of newer curcumin analogues and synthetic hybrid
molecules which could serve as promising drug candidates in the battle against
malaria.