Design, synthesis and biological evaluation of several aromatic substituted chalcones as antimalarial agents

Article abstract of DOI:10.1002/ddr.21727

Malaria is a communicable disease which is caused by protozoan's mainly Plasmodium species (P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi). The increasing resistance of Plasmodium to available malarial drugs poses a great responsibility f

Full text of DOI:10.1002/ddr.21727

Received: 15 April 2020
DOI: 10.1002/ddr.21727
Revised: 6 June 2020
Accepted: 10 July 2020
R E S E A R C H A R T I C L E
Design, synthesis and biological evaluation of several aromatic
substituted chalcones as antimalarial agents
1
1
1
Adarsh Gopinathan
| Mahreen Moidu | Minil Mukundan
|
1
1
2
Dr. Siju Ellickal Narayanan | Dr. Hariraj Narayanan | Navin Adhikari
1
Department of Pharmacy, Government
Abstract
Medical College, Kannur, India
2
CSIR, Centre for Cellular and Molecular
Biology, Hyderabad, India
Malaria is a communicable disease which is caused by protozoan's mainly Plasmo-
dium species (P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi). The increas-
ing resistance of Plasmodium to available malarial drugs poses a great responsibility
for the researchers in the field of malaria. To overcome this problem of resistance,
this study aimed to design and synthesize a new class of antimalarial agent with
chalcone as the main moiety. Chalcones, a member of flavanoid family, consist of
two aromatic rings of 1,3-diphenyl-2-propen-1-one linked by a three carbon
α,β-unsaturated carbonyl system. Five derivatives were designed and among them
one was selected. The CC2 was then synthesized by esterification of Para amino
acetophenone followed by treatment with hydrazide to form 2-(4 acetylphenoxy)
acetohydrazide. This was then coupled with 2-Bromo substituted Diazotized esteri-
fied anilines, which was finally linked with substituted benzaldehyde to yield CC2.
These were then structurally verified by Infra Red (IR) and Nuclear Magnetic Reso-
nance (NMR) spectroscopy. The chalcone was then tested for in vitro growth inhibi-
tion assays using SYBR GREEN-1 Based assay and IC50 values were identified. The
compound CC2 showed quite promising antimalarial activity by inhibiting cysteine
protease enzyme. The acute toxicity studies of the compound were carried out as
per OECD guideline 425 and the results showed no toxic signs and symptoms indi-
cating CC2 as a safe and less toxic compound.
Correspondence
Adarsh Gopinathan, M Pharm Student,
Department of Pharmacy, College of
Pharmaceutical Sciences, Government Medical
college, Kannur, Kerala, India.
Email: adarshgopinathan10@gmail.com
K E Y W O R D S
antimalarial drugs, chalcones, malaria, plasmodium, SYBR GREEN-1 based assay
1
|
INTRODUCTION
the highest of acquiring malaria with 93% cases in 2018. Also, almost
85% malarial cases were in 19 countries, which includes India and
Malaria, one among the most disastrous African nightmares often
called as King of diseases (Tangpukdee, Duangdee, Wilairatana, &
Krudsood, 2009), is a communicable disease caused by Plasmodium
species. In humans, five species of plasmodium parasites have been
reported to cause malaria namely Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium
knowlesi. However, P. falciparum infections were recorded the most
across the globe. As per 2019 WHO report, African region still bears
18 African countries. Between 2010 and 2018, deaths due to malaria
declined from 585,000 to 405,000 cases. Also, 67% malaria deaths
(272,000 cases) were found in children aged below 5 years of age
(World Health Organization, 2019). There are mainly 6 identified
malarial vectors in India and they include Anopheles culicifacies, Anoph-
eles stephensi, Anopheles dirus, Anopheles fluviatilis, Anopheles minimus
and Anopheles sundaicus (Dash, Adak, Raghavendra, & Singh, 2007).
India currently contributes for approximately 4% of the global malarial
Drug Dev Res. 2020;1–9.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals LLC
1

2
GOPINATHAN ET AL.
burden and adds to the 87% of the total malarial cases in South East
Asia. Altogether an aggregate of 0.84 million confirmed cases of
malaria and 194 malarial deaths were reported by the National Vector
Borne Disease Control Programme (NVBDCP) in 2017. The states
mainly Odisha, Chhattisgarh, Madhya Pradesh and Jharkhand
reported 74.1% of the total malarial cases in the country. The peak
value of malarial cases (40%) was reported from the Odisha state
Plasmodium. It can be studied by looking into consideration of FP-2
with E-64, which is an epoxide and a potential FP-2 inhibitor. The
crystal structure is also available in Protein data bank as 3BPF. The
E64 proceeds toward FP-2 and binds with it tightly and inhibit it
(Salawu, 2018).
(
Dhiman, Veer, & Dev, 2018).
2
|
MATERIALS AND METHODS
| Molecular docking
Chalcones, a member of flavanoid family, consist of two aromatic
rings of 1,3-diphenyl-2-propen-1-one linked by a three carbon α,β-
unsaturated carbonyl system. Chalcones exposed their importance in
the field of antimalarial drug discovery when licochalcone A, a natural
product isolated from Chinese liquorice roots, was reported to exhibit
2.1
Five novel chalcone analogues (CC1, CC2, CC3, CC4 and CC5) were
designed with pyrazolone functions (as shown in Figure 1) as cyste-
ine protease inhibitors which could be used as a better antimalarial
agent. In order to reveal the possible intermolecular interactions
behind the inhibitory activities of novel chalcone derivatives, a
molecular modeling study was performed using docking program
Argus lab 4.0.1.version. Hence five designed compounds were
docked into binding site of the enzyme cysteine protease and
recorded the possible docking scores. Based on the docking score
one compound was selected from the designed series with better
binding affinity and binding mode.
potent antimalarial activity. After that
a synthetic analogue
0
4
-hydroxy-2-methoxy- 4 -butoxy chalcone was reported to have out-
standing antimalarial activity. Afterward, a succession of natural and
synthesized alkoxylated, hydroxylated, prenylated, oxygenated, quin-
olylated chalcones have been examined as antiplasmodial agents
(
Tomar et al., 2015). A systematic PubMed literature research survey
conducted between January 2002 and December 2018 showed
results of rising artemisinin resistance mutation. Also, the role of
Genetic Algorithm (GA) toward the development of cure against
malaria has been emerged. GAs are in silico models of Darwinian evo-
lution which mimics the principals of genetic variations. GA consist of
class of optimization routines that are mainly population based search
metaheuristics. This method is employed to make in silico technique
more robust as it has the character of mimicking natural selection
2.1.1
|
Preparation of receptor
The cysteine proteases crystal structure (PDB entry: 3BPF) was taken
from RCSB Protein Data Bank (http://www.rcsb.org) which is com-
posed of falcipain 2 and its inhibitor, E64. The ligand from 3BPF was
extracted from the crystal structure. Then the protein was filtered out
by removing the water molecules. The addition of hydrogen atoms
was performed to correct the tautomeric and ionization states of
corresponding amino acid residues. The energy minimization of the
refined protein was also carried out. Then the newly modified protein
structure obtained was saved in PDB format and used for future
docking studies.
(
Hati, Bhattacharya, & Sen, 2015). Based on the mentioned situation,
the need for new antimalarial agents has motivated the research to
find synthetic molecules which are able to answer the problem. This
resistance barricade could be tackled by the emergence of a versatile
molecule having a virtuous antimalarial property and the Chalcones
are apt one to resolve the same. These all information's guided us to
synthesize chalcone to be a potent antimalarial agent. They mainly act
by inhibiting the malarial cysteine protease and thus can be novel
approach in antimalarial therapy. The chalcones are comparatively
simple to synthesize requiring only normal laboratory conditions and
also offering good yield.
The functions of Cysteine proteases in malarial parasites have
been identified from studies conducted over protease inhibitors. The
best function of plasmodium cysteine proteases was the characteristic
hydrolysis of hemoglobin. As hemoglobin is processed, the haem por-
tion is converted into hemozoin pigment, and the globin moiety was
hydrolyzed to its constituent amino acids. Hemoglobin hydrolysis is
essential to provide amino acids for parasite protein synthesis, to bal-
ance the osmotic stability of malaria parasites and to provide space
for the growing intra erythrocytic parasite. Evidence regarding the
cysteine proteases in the hydrolysis of hemoglobin came from studies
of the effects of cysteine protease inhibitors on cultured parasites
2.1.2
|
Determination of the active site
The active site determination (PDB entry: 3BPF) was performed by
using scfbio-iit.res.in: an energy-based method for the prediction of
protein-ligand binding sites. The active sites determined were selected
and saved as the binding site for the docking studies.
2.1.3
|
Preparation of ligands
All the ligand molecules were built by using Marvin Sketch 19.17.0
version software. The structures obtained were saved in mol format
which then can be imported into the work area of Argus lab 4.0.1.ver-
sion. The geometry optimization was done by using Universal Force
Field molecular mechanics method. Hydrogen bonds were added to
(
Ettari, Bova, Zappalà, Grasso, & Micale, 2009; Rosenthal, 2004). So
obstructing the Cysteine Protease can eventually destroys the Plasmo-
dium species and thus Falcipain 2 (FP-2) would be a potential target
for the antimalarial drug action as its inhibition leads to death of the

GOPINATHAN ET AL.
3
Cl
Cl
O
O
N
O
O
N
O
O
N
N
O
O
CH 3
Br
CH 3
N
N
NH
NH
Br
CC1
CC2
H3C
O
CH3
H C
3
N
CH3
O
O
O
H3C
O
O
N
O
O
O
N
N
N
O
CH3
Br
O
CH3
N
N
NH
NH
Br
CC3
CC4
H C
O
O
CH3
O
3
CH3
O
O
N
O
N
O
CH3
N
NH
Br
CC5
FIGURE 1 Structure of designed compounds. The pictures depict the structure of the five designed chalcone derivatives
each molecule and check the hybridization level and valency of atoms
in the ligand. Clean hybridization option was done to make sure of the
exact hybridization pattern of the molecule. Final geometry
optimization was done by using semi empirical quantum mechanics
PM3 method. The energy minimized structures were saved in PDB
format for further docking studies.

4
GOPINATHAN ET AL.
2
.1.4
|
Docking protocol
2.2.2
|
Synthesis of ethyl (4-acetylphenoxy)
acetate
Argus lab 4.0.1.version was used in the coupling between protein and
ligand using Argus dock with a fast and simplified medium strength
potential. Argus lab is a simple available molecular modeling and drug
designing software, with user friendly interface and is free licensed
software. The coupling of a ligand at the binding site was performed
by “Argus Dock” as the coupling engine. “Regular accuracy” was
selected in the coupling accuracy menu, “Dock” was selected as the
type of calculation, “Flexible” for the ligand and “A score” was used
for the scoring function. The junction site box was set to (26.33
P-Hydroxy Acetophenone (5 mmol) and potassium carbonate
(5.5 mmol) were taken and dissolved in Dimethyl Formamide (DMF)
(5 ml) (Razavi et al., 2013). The solution was allowed to stir at room
temperature for several minutes and then ethyl chloroacetate
(5.2 mmol) was added drop wise to the mixture. The solution was then
ꢀ
heated to 90 C for 5 hr. After completion of the reaction, the mixture
was allowed cool down to room temperature and diluted with cold
water. The precipitate was filtered off and washed. The solid was
dried and recrystallized from ethanol.
×
22.66 × 17.24) angstroms to enclose the entire active binding site
of the protein with a grid resolution of 0.4 Å. The coupling process is
repeated until a constant coupling score value is obtained. The
resulting final coupled structures were saved in PDB format and the
coupling snapshots were sent into Molegro Molecular viewer 7 version
2.2.3
acetohydrazide
|
Synthesis of 2-(4-acetylphenoxy)
2
019.7.0.0. This program can generate the amino acid residue around
each segment of the synthesized final derivatives and interpret the
different drug-receptor interactions, such as hydrogen bonds, hydro-
phobic interaction and electrostatic attraction force.
In a 500 ml round bottom flask take required quantity of ethanol
(20 ml), to this add 7 g of Ethyl (4-acetylphenoxy) acetate and 1.3 ml
of hydrazine hydrate (Kardile, Holam, Ankit, & Ramani, 2011). This
was then refluxed for 5–6 hr. After completion of the reaction, the
mixture was added into ice-coldwater, which resulted in separation of
solid, which was filtered, dried and recrystallized from ethanol.
2
.2
|
Synthesis of 3-chloro chalcone
derivative (CC2)
2
.2.1
|
Chemistry
2.2.4
|
Diazotization
All chemicals were purchased from Sigma-Aldrich. Melting Point was
determined by using BUCHI-M-560 apparatus. The NMR spectra
were recorded BRUCKER 500 MHz spectrometer in CDCl3/DMSO–
d6/CD3OH. Chemical shifts are reported in parts per million (ppm)
from Tetramethylsilane. The progress of reaction was examined by
thin layer chromatography using 60F-254 precoated silica gel (Merck,
India).
Substituted aromatic amines (2-bromo aniline) 0.01 mol was taken in
a beaker and then add 40 ml of hydrochloric acid (8 ml) and water
(6 ml) (Organic Chemistry Portal, n.d.). The solution was then cooled
ꢀ
to 0–5 C in ice water. To this add a cold solution of sodium nitrite
(0.03 mol). The diazotization salt solution was filtered directly into the
cold solution of ethyl acetoacetate (0.01 mol) and sodium acetate
(0.122 mol) in 50 ml of ethanol. The resultant solid was filtered off,
O
O
CH3
K CO
3
O
2
O
CH3
Cl
DMF
+
H C
O
O
CH3
3
HO
O
1
-(4-hydroxyphenyl)ethanone
ethyl chloroacetate
ethyl (4-acetylphenoxy)acetate
O
O
CH3
CH
3
H N
NH2
2
O
O
H C
O
NH
3
H
N
2
O
O
ethyl (4-acetylphenoxy)acetate
2-(4-acetylphenoxy)acetohydrazide

GOPINATHAN ET AL.
5
washed with water and then recrystallized from ethanol. The purity of
the compound was established by single spot on TLC.
ohydrazide(0.002 mol)in glacial acetic acid were taken in a round bot-
tom flask and refluxed for 5 hr, cooled and then allowed to stand
overnight (Delos & Detar, 1958). The product was precipitated out by
addition of cold water. The resultant solid separated by filtration. It
was then dried and recrystallized from ethanol. The purity of the
resultant compound was determined by single spot on TLC.
Br
NH2
Br
NH N
O
O
H C
3
CH3
O
2-bromoaniline
ethyl (2Z)-2-[2-(2-bromophenyl)hydrazinylidene]-3-oxobutanoate
2.2.6
|
Chalcone synthesis
2
.2.5
|
Cyclization
The chalcone was synthesized by reacting equimolar amount of
cyclized product (10 mmol) and 3 Chloro substituted benzaldehyde
(10 mmol) in ethanol (25 ml) and stirred (Syahri, Yuanita, Nurohmah,
Armunanto, & Purwono, 2017). To the stirred solution 60% NaOH
The two mixtures namely, diazotized and esterified aromatic
substituted amines (0.002 mol) and 2-(4-acetylphenoxy) acet-
O
CH3
Br
O
NH N
O
O
+
NH
H C
3
H N
2
CH3
O
O
2
-(4-acetylphenoxy)acetohydrazide
ethyl
2Z)-2-[2-(2-bromophenyl)hydrazinylidene]-
(
3
-oxobutanoate
H C
3
O
O
O
N
N
O
CH3
N
NH
Br
(4E)-2-[(4-acetylphenoxy)acetyl]-4-[2-(2-bromophenyl)hydrazinylidene]-5-methyl-2,
4
-dihydro-3H-pyrazol-3-one
H C
3
Cl
O
O
N
O
H
O
N
O
O
O
N
CH3
+
O
N
N
Cl
NH
O
CH3
Br
Br
N
NH
3-chlorobenzaldehyde
(
4E)-2-[(4-acetylphenoxy)acetyl]-4-[2-(2-bromoph
CC2
enyl)hydrazinylidene]-5-methyl-2,4-dihydro-3H-py
razol-3-one

6
GOPINATHAN ET AL.
(
15 ml) was added dropwise and stirred at room temperature over-
these mentioned techniques have limitations, such as radioactive
waste disposal, expensive equipment, as well as a requirement for a
high level of expertise and skill. So, the screening is performed by
SYBR Green-1 based assay.
night. After the completion of the reaction, it was poured onto
crushed ice and neutralized with 2 M HCl and filtered and dried.
2
.2.7
|
Synthesized compound
2
.3.1
|
SYBR GREEN-1 based assay
The synthesized compound was characterized by MP, NMR and
MASS spectrometric analysis, TLC was performed using 10% metha-
nol in chloroform as mobile phase.
Principle
SYBR Green is an asymmetrical cyanine dye which binds directly to dou-
ble strand DNA, preferring G and C base pairs. When incorporated into
DNA, it is highly fluorescent, absorbs light at a wavelength between
Cl
390 and 505 nm, with a peak at 497 nm and a secondary peak near
254 nm. It emits light at 505–615 nm, with a peak at 520 nm.
Method
O
O
N
P. falciparum 3D7 was obtained from the Malaria Research and Refer-
ence Reagent Resource Centre (MR4), cell culture reagents and SYBR
Green 1 were from Thermo Fisher Scientific, Dimethyl Sulfoxide
O
N
O
(
DMSO) was from Sigma, Albumax II was from Thermo Fisher Scientific.
The RPMI 1640 medium (supplemented with 2 g/L sodium bicarbonate,
2 g/L glucose, 25 μg/ml gentamicin, 300 mg/L glutamine, 0.4%
CH3
N
NH
Albumax and human erythrocytes at 2% hematocrit) was used for para-
site culture. Parasites were synchronized by treatment with 5% D-
sorbitol at ring stage. The compound (CC2) was assessed for the
growth inhibition of P. falciparum. The compounds CC2 (100 μg/ml)
was dissolved in DMSO, and serially diluted twofold in 75 μl culture
medium across rows of a 96 well tissue culture plate. DMSO (0.05%) or
chloroquine (500 nM) was added to control wells. 75 μl parasite sus-
pension (2% ring-infected erythrocytes at 4% hematocrit) was added to
Br
CC2
ꢀ
(2E)-3-(3-chlorophenyl)-1-phenylprop-2-enoyl [4-[2-(2-bromophenyl)
each well. The plate was incubated at 37 C for 50 hr as described
hydrazinylidene] 5-methyl-2,4-dihydro-3H-pyrazol-3-one]: Amorphous
above. At the end of incubation, 75 μl of the culture was used for SYBR
Green assay. 75 μl lysis buffer (20 mM Tris-Cl, 5 mM EDTA, 0.008%
saponin, 0.08% Triton X-100, pH 7.5) with SYBR Green 1 (at 2X dilu-
ꢀ
powder (65%); MP- 214 to 216 C; R
f
value = 0.64 (10% Methanol:Chlo-
roform =1:9)
1
ꢀ
H
NMR- (500 MHz,DMSO-d6,δ,ppm) 2.35 (s,3H,CH
3
), 4.24
tion) was added to each well, the plate was incubated at 37 C for
(
(
s,2H,CH
2
), 6.871–6.958 (d,J = 5.5 Hz,2H,ArH),7.133(s,1H,ArH),7.365
30 min, and fluorescence was measured (Ex: 485 nm, Em: 530 nm, gain
setting: 50) using a multimode microplate reader. Background fluores-
cence was corrected by subtracting the fluorescence of standard cul-
tures (chloroquine-treated) from the fluorescence values of test
compounds and DMSO. Fluorescence values of cultures treated with
the compound were normalized as percentage of the fluorescence of
DMSO-treated cultures (positive control). The relative fluorescence unit
(RFU) for each concentration was plotted against logarithmic concen-
trations and analyzed using nonlinear regression analysis to determine
IC50 concentrations.
d,J = 8.0 Hz,1H,ArH),7.491(dd,J = 12.5 Hz,9.0 Hz,1H,ArH), 7.551(d,
J = 9.0 Hz,1H,ArH), 7.569 (dd,J = 9.0 Hz,15.5 Hz,1H,ArH), 7.663 (d,J =
5.5 Hz,1H, =CH), 7.694 (dd,J = 7.0 Hz,15.0 Hz,1H,ArH), 7.78 (d,
J = 7 Hz,1H,=CH), 7.918 (d,J = 8 Hz,1H,ArH), 7.947 (d,J = 15.5 Hz,1H,
1
13
=
CH), 7.978–8.11 (d,J = 7.0 Hz,2H,ArH), 11.777 (s,1H,NH). C NMR-
(500 MHz,DMSO-d6,δ,ppm) 189.7,170.7,163.9,162.1,148.2,145.1,140.1,
1
1
37.6,136.6,134.2,132.8,130.9,130.9,130.2,130.0,128.8,128.4,127.4,
+
26.5,126.1,125.1,121.3,115.3,114.8,114.8,66.4,11.5; ESI-MS: [M + H]
calculated 579.5721 found 578.0511.
2
.3
|
In vitro pharmacological studies
2.4
|
Toxicity studies
A number of antiplasmodial screening techniques have been identi-
fied, which include the [3H] hypoxanthine uptake test and the WHO
micro-test (Dery et al., 2015; Rajesh Prasad, 2013). However, some of
The toxicity studies of the synthesized compound CC2 was carried
according to OECD Guidelines 425 (Up and Down Procedure) (OECD
Guidelines for Testing, 2008).

GOPINATHAN ET AL.
7
TABLE 1 Docking score and molecular properties of designed compounds
Sl. No.
Compounds
CC1
Binding Energy (kcal/Mol)
−10.9819
Log p
6.32
6.29
5.09
5.74
5.29
Molecular Weight
579.84
HA
8
HD
1
Number of Violations
1
2
3
4
5
2
2
3
2
3
CC2
−10.9852
579.84
8
1
CC3
−9.4993
635.47
11
9
1
CC4
−9.3228
588.46
1
CC5
−9.5204
635.47
11
1
Abbreviations: HA, hydrogen bond acceptor; HD, hydrogen bond donor.
2
.4.1
|
Procedure
Food should be withheld overnight prior to dosing. The test substance
1 ml/100 g) is administered a single dose by gavage using stomach
(
tube or suitable intubation cannula. One animal was given the test
dose. If the animal dies, conduct the main test to determine the Lethal
Dose at 50% (LD50). If the animal survives, dose four additional ani-
mals sequentially so that a total of five animals are tested. However, if
three animals die, the limit test is terminated and the main test is per-
formed. The LD50 is greater than 2000 mg/kg if three or more animals
survive.
2
.4.2
|
Animal procurement and observation
FIGURE 2 Interaction of CC2 with Cys 42 with H bonding. The
figure depicts the H-bonding interaction of CC2 with the Cysteine
Female albino mice weighing between 24 and 38 g were obtained
from Kerala Veterinary and Animal Science University, Mannuthy,
Thrissur and housed in polycarbonate cages and was maintained at
Animal House, College of Pharmaceutical Sciences, Govt. Medical Col-
lege, Kannur. Institutional Animal Ethics Committee (IAEC) constituted
as per CPCSEA guidelines with the clearance number CPCSEA/IAEC-
42 amino acid
1
8/19-01.
Animals are observed individually at least once during the first
0 min after dosing, followed by observing during the first 24 hr
3
(
with special attention given during the first 4 hr), and daily there-
after, for 14 days, except where they need to be removed from
the study and humanely killed for animal welfare reasons or are
found dead. Observations should include changes in skin and fur,
eyes and mucous membranes, and also respiratory, circulatory,
autonomic and central nervous systems, somatomotor activity and
behavior pattern. Attention should be directed to observations of
tremors, convulsions, salivation, diarrhea, lethargy, sleep
and coma.
3
3
|
RESULTS AND DISCUSSION
| Docking results
FIGURE 3 Hydrophobic interaction zone around CC2. The figure
depicts the hydrophobic interaction zone around CC2 molecule
.1
unique configurations were considered for further interaction studies.
The binding energy and molecular parameters of all the designed
structures are shown in Table 1.
The docking studies were carried out on Argus lab 4.0.1 version. The
synthesized derivative with good binding energy and showing more

8
GOPINATHAN ET AL.
FIGURE 5 IC50 of CC2. The figure depicts a plot between relative
fluorescence units (RFU) versus log concentration
FIGURE 4 Steric hindrance zone around CC2. The figure depicts
the Steric hindrance across the CC2 molecule
TABLE 2 Antimalarial activity indicated by IC50 value
Sl. No.
Concentration
2.699
Relative Fluorescence Units (RFU)
0
The docking results showed that all derivatives have almost
similar binding energy modes with different docking scores. The
docking study suggested that the compound CC2 showed a bet-
ter binding energy than the other derivatives with a binding
energy of −10.9852 kcal/mol. In the compound CC2, the pyrazole
ring contributed to the H-bonding interaction with the bond
length of Cys 42 (2.68 Å) with the active site of cysteine protease
1
2
3
4
5
6
7
8
9
2.398
0
2.097
0
1.796
16.35 ± 0.4575
24.46 ± 0.6357
65.88 ± 2.770
85.18 ± 6.380
98.11 ± 4.372
99.38 ± 10.24
107.9 ± 0.3309
109.3 ± 5.999
106.9 ± 2.237
1.495
1.194
0.893
(
Figure 2) and other interactions like hydrophobic and Steric hin-
0.592
drance were obtained. (Figures 3 and 4). This shows that the CC2
has comparatively less binding toward 3BPF compared to E64, as
it blocks more catalytic residues like N-38, N-81, H-174, C-42, Q-
0.291
10
11
12
13
−0.010
−0.311
−0.612
IC50 = 21.67 μg/ml
1
71, C-39, G-40 and G-82 (Salawu, 2018). The results also show
the CC2 has got significant binding toward FP-2 through Cys
2 binding.
4
Chloroquine (IC50 range, μg/ml) = (0.005–0.006 μg/ml)
Note: IC50 value is the average of the duplicate readings with SD.
3
3
.2
|
Pharmacological studies
.2.1
|
In vitro antimalarial activity by SYBR
3.2.2
|
In vivo toxicity studies
GREEN-1 based assay
The toxicity study for the designed compound CC2 was done as per
OECD guideline 425 in one swiss albino female mouse weighing
28–34 g by administering an oral dose of 2,000 mg/kg. The animal was
observed for mortality as well as for behavioral changes initially during
the first 30 min, followed by 24 hr, with special care given during the
first 4 hr and observation continued daily for a period of 14 days. All
observations were systematically recorded. After the end of 14th day,
the dose was well tolerated and no morbidity features was found. So
CC2 was found to be safe up to a dose 2,000 mg/kg in mouse.
The compound inhibited parasite growth at low microgram concentra-
tions. The designed compound acts by inhibiting the plasmodium cys-
teine protease which is required for the degradation of hemoglobin
into fragments. The compound was amorphous powder in nature and
sparingly soluble in DMSO and the concentration was calculated as
mass/volume.
The compound CC2 possess significant activity with IC50 concen-
tration of 21.67 μg/ml. (see Figure 5 and Table 2). Also, the effect of
the test compounds on the morphology of the parasite would add
more value to the inhibition property of the compounds. The toxicity
of compounds on a panel of mammalian cells needs to be determined
to determine if they are specific for Plasmodium. The further question
would be to ask the molecular mechanisms of inhibition of the para-
site growth by these compounds.
4
|
CONCLUSION
A group of five novel aromatic chalcone derivatives were designed
and out of which a single entity (CC2) was selected on the basis of

GOPINATHAN ET AL.
9
molecular docking and Lipinski's rule of five and the compound was
synthesized thereafter.
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https://doi.org/10.1007/s11693-015-9170-1
In the present study, the result obtained confirmed that the syn-
thesized derivative was found to possess promising antimalarial prop-
erty, which was proved by the in vitro SYBR GREEN-1 based assay.
The designed compound acts by inhibiting the plasmodium cysteine
protease which is required for the degradation of hemoglobin into
fragments.
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However, the effect of the test compound on the morphology of
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compound. The toxicity of compound on a panel of mammalian cells
needs to be determined to determine if they are specific for Plasmo-
dium. Further, the molecule has to be screened for in vivo
antiplasmodial studies to explore the mechanism of action and to
develop the lead compound.
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via dual inhibition of hemoglobin degradation and the ubiquitin
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0.1016/j.ijpara.2004.10.003
The authors declare no conflicts of interest.
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ORCID
Syahri, J., Yuanita, E., Nurohmah, B. A., Armunanto, R., & Purwono, B.
Adarsh Gopinathan
https://orcid.org/0000-0003-4183-3722
(
2017). Chalcone analogue as potent anti-malarial compounds against
Plasmodium falciparum: Synthesis, biological evaluation, and docking
simulation study. Asian Pacific Journal of Tropical Biomedicine, 7(8),
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