Pyrazole schiff base hybrids as anti-malarial agents: Synthesis, in vitro screening and computational study

Article abstract of DOI:10.2174/1386207321666180213092911

Background: Malaria is one of the most vital infectious diseases caused by protozoan parasites of the Plasmodium genus. As P. falciparum, the cause of most of the severe cases of malaria, is increasingly resistant to available drugs such as amodioquine, chloroquine, artemisinin, and antifolates, there is an urgent need to identify new targets for chemotherapy Objective: This study screened novel pyrazole derivatives carrying iminium & benzothiazole group for antimalarial potential against P. falciparum chloroquine sensitive (3D7) strain. Materials & Methods: Several pyrazole schiff base hybrids with a wide range of substitution have been synthesized via condensation of substituted aniline with substituted 4-formylpyrazole and evaluated for their in vitro antimalarial activity against asexual blood stages of human malaria parasite, Plasmodium falciparum. The interaction of these conjugate hybrids was also investigated by molecular docking studies in the binding site of P. falciparum cystein protease falcipain-2. The pharmacokinetic properties were also studied using ADME prediction. Results: Among all compounds, 6bf and 6bd were found to be potential molecules with EC₅₀ 1.95μg/ml and 1.98μg/ml respectively. Docking study results reveal that the pyrazole schiff base derivatives occupy the PfFP binding sites and they show good interactions with significant values of binding energies. Conclusion: We provide evidence which implicates pyrazole Schiff base hybrids as potential prototypes for the development of antimalarial agents.

Full text of DOI:10.2174/1386207321666180213092911

194
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Combinatorial Chemistry & High Throughput Screening, 2018, 21, 194-203
RESEARCH ARTICLE
ISSN: 1386-2073
eISSN: 1875-5402
Pyrazole Schiff Base Hybrids as Anti-Malarial Agents: Synthesis, In Vitro
Impact
Factor:
0.952
Screening and Computational Study
BENTHAM
S
CIENCE
Shilpy Aggarwal1, Deepika Paliwal2, Dhirender Kaushik2, Girish Kumar Gupta3 and Ajay Kumar2,*
1R.K.S.D (PG) College, Kaithal, Haryana, India; 2Institute of Pharmaceutical Sciences, Kurukshetra University,
Kurukshetra 136119, India; 3Department of Pharmaceutical Chemistry, M.M. College of Pharmacy, Maharishi
Markandeshwar (Deemed to be University), Mullana-Ambala 133203, India
Abstract: Background: Malaria is one of the most vital infectious diseases caused by protozoan
parasites of the Plasmodium genus. As P. falciparum, the cause of most of the severe cases of
malaria, is increasingly resistant to available drugs such as amodioquine, chloroquine, artemisinin,
and antifolates, there is an urgent need to identify new targets for chemotherapy
Objective: This study screened novel pyrazole derivatives carrying iminium & benzothiazole group
for antimalarial potential against P. falciparum chloroquine sensitive (3D7) strain.
A R T I C L E H I S T O R Y
Materials & Methods: Several pyrazole schiff base hybrids with a wide range of substitution have
been synthesized via condensation of substituted aniline with substituted 4-formylpyrazole and
Received: October 29, 2017
Revised: January 14, 2018
evaluated for their in vitro antimalarial activity against asexual blood stages of human malaria
Accepted: February 3, 2018
parasite, Plasmodium falciparum. The interaction of these conjugate hybrids was also investigated
by molecular docking studies in the binding site of P. falciparum cystein protease falcipain-2. The
DOI:
10.2174/1386207321666180213092911
pharmacokinetic properties were also studied using ADME prediction.
Results: Among all compounds, 6bf and 6bd were found to be potential molecules with EC50
1.95µg/ml and 1.98µg/ml respectively. Docking study results reveal that the pyrazole schiff base
derivatives occupy the PfFP binding sites and they show good interactions with significant values of
binding energies.
Conclusion: We provide evidence which implicates pyrazole Schiff base hybrids as potential
prototypes for the development of antimalarial agents.
Keyword: Pyrazole, Plasmodium falciparum, antimalarial, Falcipain-2, docking, ADME.
identification to lead optimization and beyond [4-6], and
approaches such as ligand- [4] or structure based virtual
screening [7] techniques are widely used in many discovery
efforts. Along this line, there is also great inclination of
using distinctive heterocyclic skeletons for the development
of dual therapeutic drugs. The rationale of this approach
relies on the hybridization of two drugs, both dynamic
compounds and pharmacophoric units familiar and derived
from known bioactive molecules [8].
1. INTRODUCTION
Malaria is one of the prehistoric parasitic infectious
diseases which are caused by the protozoan parasite of the
Plasmodium genus and is transmitted to human by the
infected female Anopheles mosquito. According to World
Health Organization malaria reports, half of the world
population is at threat to malarial infection [1]. P. falciparum
is becoming resistant to the existing drugs in the market;
therefore, there is an imperative need for the introduction of
new therapeutic agents to act against the disease. Enzyme
cysteine protease falcipain-2 (FP-2) that is involved in P
falciparum development emerges to be a good target.
Mounting facts suggest that cysteine proteases are concerned
with host cell rupture and release of merozoites. In the
presence of such inhibitors, merozoites mature usually but
are unable to run off from host erythrocytes [2, 3]. This
makes FP-2 an attractive target for antimalarial drug designs.
Computational methodologies have emerged as a vital
component of many drug discovery programs, from hit
Schiff bases are the prospective molecules, which can be
affective against the problem of drug resistance [9]. In
addition to the synthetic derivatives, ancistrocladidine having
iminium group moiety, is a natural product formed by plants
belonging
to the family Dioncophyllaceae and
Ancistrocladiceae and is known as an anti-malarial agent
with activity against P. falciparum strains 3D7 and K1 [10].
Schiff bases obtained by the condensation of 2,6-
diarylsubstituted
piperidin-4-ones
with
7-chloro-4-
hydrazinoquinoline have also been tested for antimalarial
activities and reported to show strong antimalarial activity
against the P. falciparum strains [11]. Therefore, in the quest
for new effective drug molecules against malaria, Schiff
bases can be a potential possibility of research. Furthermore,
*Address correspondence to this author at the Institute of Pharmaceutical
Sciences, Kurukshetra University, Kurukshetra 136119, India;
Tel: +91 9416194419; E-mail: ajkumar@kuk.ac.in
1875-5402/18 $58.00+.00
© 2018 Bentham Science Publishers

Pyrazole Schiff Base Hybrid as an Anti-Malarial Agent
Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3 195
numerous reports have shown that pyrazoles have significant
antimalarial activities. For illustration, chloroquine
substituted 4-formylpyrazole analogues have been accounted
for to show a huge in vitro anti-malarial activity using an
experimental model against P. falciparum [12]. In another
research, antiparasitic activities of the pyrazole azomethines
were also reported [13]. There are likewise different reports
that demonstrated the antimalarial activities of pyrazole
derivatives [14]. Further, it has been reported that
compounds containing benzothiazole moiety displayed
intense anti-plasmodial activity against P. falciparum [15,
16]. Hence, based on the literature review [17], the present
work aims to synthesize novel pyrazole derivatives carrying
iminium & benzothiazole groups and evaluated as potential
antimalarial agents against P. falciparum chloroquine
sensitive (3D7) strain. Furthermore, we investigated the
interaction of these hybrids in the binding site of P.
falciparum falcipain-2 (PfFP) protein structures using
molecular docking studies and ADME prediction was carried
out computationally to get an insight of the structural
parameters leading to activity.
2-[(Z)-2-(1-phenylethylidene)hydrazin-1-yl]-1,3-benzothia-
zole (3a)
1
205-207°C, yield 81%. H-NMR (DMSO-d6) δ 9.78 (s,
1H, NH), 7.32-7.71 (m, 9H, ArH), 2.37 (s, 3H, CH3).
2-[(Z)-2-[1-(4-methylphenyl)ethylidene]hydrazin-1-yl]-1,3-
benzothiazole (3b)
1
210-212°C, yield 81%. H-NMR (DMSO-d6) δ 9.57 (s,
1H, NH), 7.28-7.82 (m, 8H, ArH), 2.53 (s, 3H, CH3), 2.32 (s,
3H, CH3).
2-[(Z)-2-[1-(4-fluorophenyl)ethylidene]hydrazin-1-yl]-1,3-
benzothiazole (3c)
1
230-232°C, yield 81%. H-NMR (DMSO-d6) δ 9.73 (s,
1H, NH), 7.32-7.73 (m, 8H, ArH), 2.32 (s, 3H, CH3).
General procedure for the synthesis of 4-formylpyrazoles
(4a-c)
To a cold, stirred solution of dimethylformamide (6 mL)
and phosphorous oxychloride (2.5 mmol) was added
hydrazone 3 (6, 1 mmol) following the literature procedure
[18]. The reaction mixture was stirred at 55-60°C for 5 h,
cooled to room temperature, poured into ice chilled water
and neutralized with saturated aqueous sodium bicarbonate
solution where upon a solid was separated that was filtered,
washed with excess of chilly water, dried and crystallized
from acetic acid to afford aldehydes.
2. MATERIALS & METHODS
2.1. Chemistry
All the reagents were purchased from commercial
sources and were used without purification. Chromatography
was carried on silica gel (60-120 and 100-200 mesh). All the
reactions were monitored by thin layer chromatography
(TLC). Melting points were determined in open glass
capillaries in an electrical melting point apparatus and are
uncorrected.1H NMR and 13C NMR spectra were recorded
either in pure DMSO-d6 or in CDCl3/DMSO-d6 mixture on
Bruker NMR spectrometers at 300/400 MHz and 75.5/100
MHz respectively using tetra methylsilane (TMS) as internal
standard. Chemical shifts are expressed in δ, ppm. The purity
1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazole-4-carbalde-
hyde (4a)
1
175-177°C, yield 81%. H-NMR (DMSO-d6) δ 10.07(s,
1H, CHO), 8.86 (s, 1H, pyrazole C5-H), 7.32-7.82 (m, 9H,
ArH).
1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-pyrazole-4-
carbaldehyde (4b)
1
166-168°C, yield 81%. H-NMR (DMSO-d6) δ 10.15 (s,
1H, CHO), 8.83 (s, 1H, pyrazole C5-H, 7.33-7.78 (m, 8H,
ArH), 2.28 (s, 3H, CH3).
1
of the compounds was checked by H NMR and thin layer
chromatography (TLC) on silica gel plates using a mixture of
petroleum ether and ethyl acetate as eluent. Iodine or UV
lamp was used as a visualizing agent. Abbreviations ‘s’ for
singlet and ‘m’ for multiplet were used for NMR
assignments. The mass spectral data were obtained on Q-
TOF MICROMASS (LC-MS) spectrometer at Punjab
University of Chandigarh India. The antimalarial activity of
synthesized compounds was assessed against chloroquine-
sensitive P. falciparum (3D7) isolate obtained from the
National Institute of Malaria Research (NIMR), New Delhi,
India. P. falciparum were cultivated in human A Rh+ red
blood cells using RPMI 1640 medium (Sigma, India)
supplemented with AB Rh+ serum (10%), 5% sodium
bicarbonate (Sigma, India) and 40 μg/mL of gentamycin
sulfate (Sigma, India).
1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-pyrazole-4-
carbaldehyde (4c)
196-198°C, yield 73%. 1H-NMR (DMSO-d6) δ 10.09 (s,
1H, CHO), 8.82 (s, 1H, pyrazole C5-H), 7.28-7.85 (m, 8H,
ArH).
General procedure for the conversion of 4-formylyrazole
into 4-arylidene pyrazole derivatives (6)
An equimolar mixture of substituted aromatic amines 5
and 4-formylpyrazoles 4 was refluxed in solvent benzene for
10-12 hrs in the presence of a catalytic amount of sulphuric
acid. The resulting solution was cooled to room temperature
and the precipitated solid was filtered under suction and
washed with cooled ethanol, dried and recrystallized from
ethanol [19].
General procedure for the synthesis of hydrazones (3a-c)
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
An equimolar amount of compound 2-hydrazino-
benzothiazole 2 (1.5mmol), substituted with methyl ketone 1
(1.5mmol) and glacial acetic acid (2-3drop) was taken in
absolute ethanol (20ml) and refluxed for 4-5 hrs. On cooling,
a solid was separated which was filtered, dried and
crystallized from ethanol to afford hydrazones 3.
4-yl)methylene)benzenamine (6aa) m.p. 172-174°C, yield
1
81%. H-NMR (DMSO-d6) δ 8.91 (s, 1H, CH=N), 8.86 (s,
1H, CH, pyrazole), 7.39-8.18 (m, 14H, ArH). 13C NMR
(DMSO-d6): δ 160.46, 152.74, 151.63, 150.58, 148.63,
133.40, 131.37, 129.41, 129.10, 128.93, 128.63, 127.68,
127.36, 126.76, 124.94, 124.21, 121.55, 121.06, 119.86. MS

196 Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3
Aggarwal et al.
(ESI) m/z = 381.16 (M +H+). Anal. Calc. for C23H16N4S: C,
72.61; H, 4.24; N, 14.73; S 8.43 Found: C, 72.66; H, 4.27;
N, 14.80; S 8.49
129.38, 128.83, 128.37, 127.86, 127.55, 126.64, 124.64,
121.06, 121.04, 119.74. MS (ESI) m/z = 395.14 (M +H+).
Anal. Calc. for C24H18N4S: C, 73.07; H, 4.60; N, 14.20; S
8.13 Found: C, 73.14; H, 4.52; N, 14.26; S 8.11
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
4-yl)methylene)-4-methylbenzenamine (6ab) m.p.= 178-
180°C, yield 89%. 1H-NMR (DMSO-d6) δ 8.92 (s, 1H,
CH=N), 8.86 (s, 1H, CH, pyrazole), 7.39-8.18 (m, 13H,
ArH), 2.49 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 160.35,
152.37, 151.96, 151.83, 148.07, 133.26, 132.53, 132.46,
130.42, 129.38, 128.83, 128.27, 127.86, 127.55, 126.64,
124.64, 121.06, 121.04, 119.74, 21.28. MS (ESI) m/z =
395.16 (M +H+). Anal. Calc. for C24H18N4S: C, 73.07; H,
4.60; N, 14.20; S 8.13 Found: C, 73.16; H, 4.57; N, 14.18; S
8.19
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazol-4-yl)methylene)-4-methyl-benzenamine (6bb) m.p.=
173-175 °C, yield 73% . 1H-NMR (DMSO-d6) δ 8.83 (s, 1H,
CH=N), 8.77 (s, 1H, CH, pyrazole), 7.24-8.09 (m, 13H,
ArH), 2.29 (s, 3H, CH3), 2.16 (s, 3H, CH3). 13C NMR
(DMSO-d6): 160.74, 152.20, 151.99, 151.78, 148.94, 139.41,
133.53, 132.46, 132.42, 130.38, 129.83, 129.27, 127.86,
126.55, 125.64, 124.64, 121.06, 121.04, 119.77. MS (ESI)
m/z = 409.14 (M +H+). Anal. Calc. for C25H20N4S: C, 73.50;
H, 4.93; N, 13.71; S 7.85 Found: C, 73.54; H, 4.93; N,
13.76; S 7.79
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
4-yl)methylene)-4-methoxybenzenamine (6ac) m.p.=184-185
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
1
°C, yield 79%. H-NMR (DMSO-d6) δ 8.84 (s, 1H, CH=N),
8.75 (s, 1H, CH, pyrazole), 7.24-8.06 (m, 13H, ArH), 3.79
(s, 3H, OCH3). 13C NMR (DMSO-d6): δ 160.36, 156.17,
152.13, 151.83, 151.68, 148.26, 133.46, 132.60, 129.42,
128.83, 128.74, 127.86, 127.55, 126.21, 124.96, 122.64,
121.06, 119.04, 114.74, 55.47. MS (ESI) m/z = 411.49 (M
+H+). Anal. Calc. for C24H18N4OS: C, 70.22; H, 4.42; N,
13.65; S 7.81 Found: C, 70.16; H, 4.47; N, 13.68; S 7.89
pyrazol-4-yl)methylene)-4-methoxy-benzenamine
(6bc)
1
m.p.= 180-182 °C, yield 72% . H-NMR (DMSO-d6) δ 8.86
(s, 1H, CH=N), 8.76 (s, 1H, CH, pyrazole), 7.36-8.06 (m,
12H, ArH), 3.79 (s, 3H, OCH3), 2.43 (s, 3H, CH3). 13C NMR
(DMSO-d6): δ 160.78, 156.53, 152.17, 151.93, 151.83,
148.68, 139.46, 133.60, 132.42, 129.83, 129.74, 127.86,
126.55, 125.21, 124.96, 122.64, 121.06, 119.04, 114.74,
55.26, 21.26. MS (ESI) m/z = 425.16 (M +H+). Anal. Calc.
for C25H20N4OS: C, 70.73; H, 4.75; N, 13.20; S 7.55 Found:
C, 70.78; H, 4.73; N, 13.26; S 7.59
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
4-yl)methylene)-4-chlorobenzenamine (6ad) m.p.= 198-199
°C, yield 73% . 1H-NMR (DMSO-d6) δ 8.86 (s, 1H, CH=N),
8.81 (s, 1H, CH, pyrazole), 7.30-8.09 (m, 13H, ArH). 13C
NMR (DMSO-d6): δ 160.46, 152.86, 151.74, 151.58, 148.20,
133.76, 132.27, 129.60, 129.42, 129.38, 128.83, 128.87,
127.96, 127.56, 126.86, 124.64, 121.06, 121.04. 119.74. MS
(ESI) m/z = 415.09 (M +H+). Anal. Calc. for C23H15ClN4S:
C, 66.58; H, 3.64; N, 13.50; S 7.73 Found: C, 66.56; H, 3.67;
N, 13.58; S 7.81
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazol-4-yl)methylene)-4-chloro-benzenamine (6bd) m.p.=
184-185 °C, yield 79% . 1H-NMR (DMSO-d6) δ 8.87 (s, 1H,
CH=N), 8.75 (s, 1H, CH, pyrazole), 7.23-8.02 (m, 12H,
ArH), 2.13 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 160.75,
152.37, 151.96, 151.87, 148.77, 139.27, 133.73, 132.46,
129.42, 129.38, 129.13, 127.86, 126.55, 125.67, 125.64,
121.27, 121.04, 119.74, 21.27. MS (ESI) m/z = 429.14 (M
+H+). Anal. Calc. for C24H17ClN4S: C, 67.20; H, 3.99; N,
13.06; S 7.48 Found: C, 67.14; H, 3.93; N, 13.14; S 7.49
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
4-yl)methylene)-2-chlorobenzenamine (6ae) m.p.= 205-207
°C, yield 71% . 1H-NMR (DMSO-d6) δ 8.84 (s, 1H, CH=N),
8.75 (s, 1H, CH, pyrazole), 7.24-8.06(m, 13H, ArH), 3.79 (s,
3H, OCH3). 13C NMR (DMSO-d6): δ 160.76, 152.83, 151.74,
151.48, 149.56, 133.20, 132.76, 130.29, 129.60, 129.42,
128.86, 128.64, 127.74, 127.58, 127.22, 126.36, 125.15,
121.38, 119.56, 118.27. MS (ESI) m/z = 415.11 (M +H+).
Anal. Calc. for C23H15ClN4S: C, 66.58; H, 3.64; N, 13.50; S
7.73 Found: C, 66.59; H, 3.69; N, 13.55; S 7.78
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazol-4-yl)methylene)-2-chloro-benzenamine (6be) m.p.=
179-181 °C, yield 69% . 1H-NMR (DMSO-d6) δ 8.85 (s, 1H,
CH=N), 8.73 (s, 1H, CH, pyrazole), 7.29-8.04 (m, 12H,
ArH), 2.13 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 160.86,
152.17, 151.83, 151.41, 149.20, 139.46, 133.27, 132.60,
130.07, 129.96, 129.26, 129.13, 127.36, 127.31, 126.86,
125.64, 125.21, 124.04, 121.74, 119.87, 118.47, 21.27. MS
(ESI) m/z = 429.12 (M +H+). Anal. Calc. for C24H17ClN4S:
C, 67.20; H, 3.99; N, 13.06; S 7.48 Found: C, 67.16; H, 3.91;
N, 13.09; S 7.51
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-phenyl-1H-pyrazol-
4-yl)methylene)-3-chlorobenzenamine (6af) m.p.=202-204
°C, yield 72% . 1H-NMR (DMSO-d6) δ 8.83 (s, 1H, CH=N),
8.75 (s, 1H, CH, pyrazole), 7.23-8.03 (m, 13H, ArH). 13C
NMR (DMSO-d6): δ. 160.76, 152.83, 152.74, 151.58,
151.20, 134.76, 133.27, 132.60, 130.42, 129.38, 128.83,
128.27, 127.86, 127.64, 126.56, 124.74, 123.48, 121.22,
121.07, 121.04, 119.29. MS (ESI) m/z = 415.05 (M +H+).
Anal. Calc. for C23H15ClN4S: C, 66.58; H, 3.64; N, 13.50; S
7.73 Found: C, 66.55; H, 3.67; N, 13.57; S 7.76
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazol-4-yl)methylene)-3-chloro-benzenamine (6bf) m.p.=
185-187 °C, yield 79% . 1H-NMR (DMSO-d6) δ 8.95 (s, 1H,
CH=N), 8.81 (s, 1H, CH, pyrazole), 7.28-8.04 (m, 12H,
ArH), 2.15 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 160.83,
152.86, 152.17, 151.49, 151.20, 139.46, 134.27, 133.60,
132.30, 130.96, 129.41, 129.13, 127.36, 126.21, 125.86,
124.64, 123.21, 121.87, 121.74, 121.27, 119.74. MS (ESI)
m/z = 429.13 (M +H+). Anal. Calc. for C24H17ClN4S: C,
67.20; H, 3.99; N, 13.06; S 7.48 Found: C, 67.24; H, 4.03;
N, 13.11; S 7.41
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazol-4-yl)methylene)benzenamine (6ba) m.p.= 158-160
°C, yield 65% . 1H-NMR (DMSO-d6) δ 8.93 (s, 1H, CH=N),
8.86 (s, 1H, CH, pyrazole), 7.33-8.12 (m, 13H, ArH), 2.46
(s, 3H, CH3). 13C NMR (DMSO-d6): δ 160.35, 152.37,
151.96, 151.83, 148.07, 133.26, 132.53, 132.46, 130.42,
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)benzenamine (6ca) m.p.= 193-195

Pyrazole Schiff Base Hybrid as an Anti-Malarial Agent
Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3 197
°C, yield 73% . 1H-NMR (DMSO-d6) δ 8.95 (s, 1H, CH=N),
8.86 (s, 1H, CH, pyrazole), 7.27-8.07 (m, 13H, ArH). 13C
NMR (DMSO-d6): δ 163.38, 160.07, 152.44, 151.83, 151.22,
148.74, 133.34, 132.35, 129.86, 129.14, 127.96, 127.41,
126.07, 124.36, 124.15, 121.14, 121.06, 119.04, 115.74. MS
(ESI) m/z = 399.07 (M +H+). Anal. Calc. for C23H15FN4S: C,
69.33; H, 3.79; N, 14.06; S 8.05 Found: C, 69.24; H, 3.83;
N, 14.11; S 8.11
2.2. Assay for in vitro Antimalarial Activity
2.2.1. Parasite Cultivation
The antimalarial activity of synthesized compounds was
assessed against chloroquine-sensitive P. falciparum (3D7)
isolated obtained from the National Institute of Malaria
Research (NIMR), New Delhi, India. P. falciparum are
cultivated in human A (+) red blood cells using RPMI 1640
medium supplemented with AB (+) serum (10%), 5%
sodium bicarbonate and 40 μg/mL of gentamycin sulfate
[20].
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-4-methyl-benzenamine (6cb) m.p.=
209-211 °C, yield 67% . 1H-NMR (DMSO-d6) δ 8.89 (s, 1H,
CH=N), 8.75 (s, 1H, CH, pyrazole), 7.24-8.09 (m, 12H,
ArH), 2.16 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 163.35,
160.37, 152.53, 151.83, 151.07, 148.26, 133.46, 132.80,
132.40, 130.48, 129.39, 127.96, 127.55, 126.86, 124.64,
121.64, 121.06, 119.04, 115.74, 21.11. MS (ESI) m/z =
413.15 (M +H+). Anal. Calc. for C24H17FN4S: C, 69.88; H,
4.15; N, 13.58; S 7.77 Found: C, 69.84; H, 4.13; N, 13.61; S
7.74
2.2.2. In vitro Test for Antimalarial Activity
The in vitro activity of P. falciparum intra erythrocytic
stage on test compounds was evaluated by Schizont
Maturation Inhibition method as described previously [21].
Firstly, a stock solution of synthesized compounds was
prepared by dissolving in DMSO and serially diluted with
RPMI 1640 medium to reach 1 mg/mL prior to use. Serial
double dilutions were made in 96-well microliter plates with
a concentration range of 1.98-500 μg/mL against a control
containing incomplete medium with the same concentration
of DMSO. The cultures, before testing, were synchronized
by treatment with 5% D-sorbitol with a parasitemia of 0.6-
0.8%. Each well received 10 μL of parasite-infected
erythrocytes, 5% hematocrit and 90 μL of different drug
dilutions. The plates were incubated at 37°C for 24 hours.
After confirmation of the presence of 10% mature schizonts
in control well (without drug), the blood from each well was
harvested, and a thick blood smear was prepared on a glass
slide. Growth of the parasites from duplicate wells of each
concentration was monitored in Giemsa stained blood smears
by counting the number of schizonts per 200 asexual
parasites. Growth inhibition was expressed as the percentage
of schizonts in each concentration, compared with controls.
(Z)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-4-methoxy-benzenamine (6cc) m.p.
1
181-183 °C, yield 74% . H-NMR (DMSO-d6) δ 8.84 (s,1H,
CH=N), 8.75 (s, 1H, CH, pyrazole), 7.33-8.05 (m, 12H,
ArH), 3.73 (s, 3H, OCH3). 13C NMR (DMSO-d6): δ 163.36,
160.17, 156.53, 152.83, 151.68, 151.26, 148.46, 133.60,
132.42, 129.38, 127.74, 127.47, 126.55, 124.96, 122.86,
121.64, 119.06, 115.04, 114.74, 55.37. MS (ESI) m/z =
429.15 (M +H+). Anal. Calc. for C24H17FN4OS: C, 67.27; H,
4.00; N, 13.08; S 7.48 Found: C, 67.24; H, 4.08; N, 13.11; S
7.44
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-4-chloro-benzenamine (6cd) m.p.
1
229-231 °C, yield 71% . H-NMR (DMSO-d6) δ 8.86 (s,1H,
CH=N), 8.78 (s, 1H, CH, pyrazole), 7.30-8.07 (m, 12H,
ArH). 13C NMR (DMSO-d6): δ 163.20, 160.41, 152.86,
151.64, 151.21, 148.46, 133.47, 132.67, 129.78, 129.39,
129.16, 127.74, 127.41, 126.21, 124.86, 121.64, 121.21,
119.04, 115.74. MS (ESI) m/z = 433.13 (M +H+). Anal.
Calc. for C23H14ClFN4S: C, 63.81; H, 3.26; N, 12.94; S 7.41
Found: C, 63.84; H, 3.28; N, 12.88; S 7.47
2.2.3. Antimalarial Activity Calculation and Analysis
The number of schizonts counted per well was directly
entered into the nonlinear regression software, HN NonLin V
1.1 [22], which was particular for the analysis of in vitro
drug sensitivity assay for malaria. Individual dose response
curves were generated and their EC50 values were
determined.
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-2-chloro-benzenamine (6ce) m.p.
245-247 °C, yield 78% . 1H-NMR (DMSO-d6) δ 8.85 (s, 1H,
CH=N), 8.78 (s, 1H, CH, pyrazole), 7.28-8.05 (m, 12H,
ArH). 13C NMR (DMSO-d6): δ 163.86, 160.17, 152.83,
151.86, 151.20, 149.46, 133.27, 132.69, 130.07, 129.39,
129.26, 127.96, 127.55, 127.21, 126.86, 125.64, 124.21,
121.07, 119.74, 118.87, 115.47. MS (ESI) m/z = 433.09 (M
+H+). Anal. Calc. for C23H14ClFN4S: C, 63.81; H, 3.26; N,
12.94; S 7.41 Found: C, 63.87; H, 3.29; N, 12.98; S 7.46
2.3. Molecular Docking Study
Docking study is used to diminish false positive and
recognize suitable orientation for the ligand in a protein
active site. For the docking of ligands to protein active sites,
an advanced molecular docking program, AutoDock [23]
was used in the present study. The scoring functions and
hydrogen bonds formed with the surrounding amino acids
are used to predict their binding modes, their binding
affinities and orientation of these compounds at the active
site. The ligands were built in Marvin Sketch. All hydrogens
in the structure were added, 2D molecules were cleaned into
3D and conformational energy of the molecules was
minimized using MMFF94 force field and the ligands were
saved as pdb files. MGL Tools 1.5.4 was used to prepare the
ligands’ pdbqt files, setting the number of rotatable bonds to
(E)-N-((1-(1,3-benzothiazol-2-yl)-3-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-3-chloro-benzenamine(6cf)
m.p.
1
223-225 °C, yield 78% . H-NMR (DMSO-d6) δ 8.82 (s,1H,
CH=N), 8.78 (s, 1H, CH, pyrazole), 7.28-8.06 (m, 12H,
ArH). 13C NMR (DMSO-d6): δ. 163.46, 152.97, 152.53,
151.58, 151.20, 134.76, 133.27, 132.60, 130.42, 129.38,
127.26, 127.21, 126.55, 124.96, 123.86, 121.64, 121.06,
121.04, 119.74, 115.47. MS (ESI) m/z = 433.17 (M +H+).
Anal. Calc. for C23H14ClFN4S: C, 63.81; H, 3.26; N, 12.94; S
7.41 Found: C, 63.86; H, 3.31; N, 12.88; S 7.48

198 Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3
Aggarwal et al.
maximum, since most of the ligands used in this study have
fewer than eight rotatable bonds (http://mgltools.
scripps.edu/). The 3D crystal structure of PfFP with good
resolution of 3.1 Å was taken from Protein Data Bank
(www.rcsb.org) with (PDB Id: 2GHU). The crystal
structures of protein were prepared for docking by adding
hydrogen atoms, assigning bond orders, removal of all ions,
water molecules and all the ligands from the structure. The
polypeptide chain was processed in MGL Tools 1.5.4 to
obtain the grid.pdbqt files. A grid-box was generated that
was large enough to cover the entire receptor binding site. In
the present study, the dimensions of the grid were 40×40×40
grid points with spacing of 0.375 A° between grid points and
center on the ligands (51.36, 0.384, -31.12 coordinates).
Docking simulations were performed using Lamarckian
Genetic Algorithm (LGA) [24]. For each ligand, 10 docking
poses were generated out of which best docked confirmation
was considered for further analysis about hydrogen bonding.
Further characterization via MD simulations was conducted
using complexes that were selected according to their
binding energy values and the interactions made with the
surrounding residues. The resulting conformations were
descriptors such as octanol/water partitioning coefficient
(logP), aqueous solubility (QPlogS), brain/blood partition
coefficient (QPlogBB), human serum albumin binding
(QPlogKhsa) and others.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The synthetic approach adopted to obtain the inter-
mediate and targeted compounds is outlined in Scheme 1.
First, 2-hydrazinobenzothiazole was condensed with various
substituted methyl ketones in aqueous ethanol affording
corresponding hydrazone. The subsequent reaction of
hydrazone under Vilsmeiere-Hack condition afforded 4-
formylpyrazole [18]. Further condensation of 4-
formylpyrazole with aromatic amine using concentrated
H2SO4 gave the target product [19]. Spectral data (1H NMR,
13C NMR, mass and elemental analysis) of the newly
synthesized compounds were in complete agreement with the
proposed structures. The 1HNMR spectra in general
displayed a characteristic signal at δ 9.96-10.18 assigned for
aldehydic proton and a singlet at δ 8.66-8.95 for C5-H of
visualized
in
the
PyMol
Viewer
tool
1
(http://www.pymol.org/).
pyrazole ring. The formation of schiff base in HNMR was
ascertained based on a singlet at δ 8.49-8.86.
2.4. In silico ADME Prediction Method
3.2. In vitro Antimalarial Activity
The pharmacokinetic profile of the synthesized
compounds showing good antimalarial activity was predicted
by using programs Qikprop v3.6 (Schrodinger, Inc., New
York, NY, 2012). All the compounds prepared by LigPrep
were used for the calculation of pharmacokinetic properties
by QikProp. The program QikProp, utilizes the method of
Jorgensen [25] to calculate pharmacokinetic properties and
All the eighteen synthesized compounds were evaluated
for their in vitro antimalarial activity against the asexual
blood stage of human malaria parasite, P. falciparum as
described previously [21]. EC50 value of these compounds is
depicted in Table 1. To get structural insights, two variations
were made in the pyrazole hybrid. In the structural motif of
H
S
N
O
N
S
Ar1
EtOH
+
NHNH2
N
Ar1
CH3
N
2
reflux
1
3
N
Ar2-NH2
N
POCl3
5
S
H
C
N
S
Ar2
CHO
N
N
N
DMF, 55O
C
N
EtOH, reflux
Ar1
Ar1
4
6
F
CH3
CH3
Ar1
=
OCH3
Cl
Ar2
=
Cl
Cl
Scheme 1. Synthesis of Pyrazole schiff base hybrid.

Pyrazole Schiff Base Hybrid as an Anti-Malarial Agent
Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3 199
these hybrids, pyrazole and benzothiazole substitution at
position N-1 was kept common and variations were made in
phenyl ring substitution at position 3 and 4. Most of the
compounds showed good to moderate activity. Among the
series, compounds 6ba-6bf having p-CH3 substitution at the
phenyl ring on pyrazole nucleus at position 3 showed
maximum antimalarial activity in the range 1.953-3.518
µg/ml while compounds 6ca-6cf having a p-F substitution
showed moderate antimalarial activity in the range 2.59-5.9
µg/ml. Unsubstituted compounds 6aa-6af showed least
antimalarial activity in the range 2.7-8.3 µg/ml. Among the
synthesized compounds, compound with m-Cl substitution at
position 4 was found to be the most active compound 6bf
with EC50 1.953µg/ml followed by compounds 6bd, 6bb
with EC50 1.98µg/ml and 2.24µg/ml respectively.
Substitution of phenyl ring at position 4 of pyrazole nucleus
with halogen group, showed potential antimalarial activity in
the range 1.95-4.03 µg/ml. Compound 6aa was found to be
the least active compound of the series with EC50 8.34
µg/ml.
phenyl ring at position 4 of pyrazole nucleus with electron
withdrawing group like halogen, showed potential
antimalarial activity. Para and Meta substitution of chloro
group showed excellent activity whereas ortho substitution
led to partially decreased antimalarial activity. Compounds
containing moderate electron donating group like methyl
also showed potential antimalarial activity while methoxy
group and no substitution at phenyl ring decreased the
biological activity. The antimalarial activity among pyrazole
derivatives showed that substituted aromatic ring showed
maximum activity than the unsubstituted aromatic ring. The
SAR studies developed from the screening data are displayed
in Fig. (1).
3.4. Docking Studies
To study the probable mode of action for antimalarial
activity and to predict orientation of the molecules at the
active site, docking simulations were performed using Auto
Dock Vina program.
A
cysteine protease inhibitor
obstructing the development of cultured P. falciparum
parasites was chosen for docking because it is an important
target for antimalarial chemotherapy [26, 27]. All the
eighteen compounds were docked into the crystal structure
of P. falciparum falcipain-2. The scoring functions and
interactions with the surrounding amino acids were used to
predict their binding modes, binding affinities and
orientation of these compounds at the active site. The results
of docking studies for the interaction between Falcipain-2
and the representative schiff base of pyrazole derivatives are
given in Table 2. The result reveals that the pyrazole Schiff
base derivatives occupy the PfFP binding sites and they
show good interactions with significant values of binding
energies. Binding energies for these compounds were in the
range of -7.9 to -8.6 kcal/mol. Important hydrophobic bond
interactions were observed with the energy minimized
structure of ligands and PfFP protein.
Table 1. In vitro antimalarial activity of Pyrazole Schiff base
hybrid against P. falciparum (3D7) strain.
Compounds
Ar1
Ar2
EC50(µg/ml)
6aa
Ph
Ph
Ph
p -CH3Ph
p -OCH3Ph
p –ClPh
o-ClPh
m-ClPh
Ph
8.342
2.760
4.126
2.769
4.038
2.702
3.518
2.241
3.923
1.983
2.621
1.953
5.920
2.726
5.009
2.594
3.753
2.621
1.3a
6ab
6ac
Ph
6ad
Ph
6ae
Ph
6af
Ph
6ba
p-CH3Ph
p -CH3Ph
p -CH3Ph
p -CH3Ph
p -CH3Ph
p -CH3Ph
p -FPh
p-FPh
p -FPh
p -FPh
p -FPh
p -FPh
-
6bb
p -CH3Ph
p -OCH3Ph
p –ClPh
o-ClPh
m-ClPh
Ph
6bc
According to the binding affinity, the most active
compound was 6bf which matched with our results of in
vitro studies on P. falciparum. Fig. (2) and (3) illustrate the
binding pose as well as interactions of compound 6ba and
6bf with docking score -8.5 and -8.6, respectively. All
compounds showed π-π stacking with the phenyl ring of
amino acid TRP206. In compound 6ba, pyrazole ring
showed π-π stacking with phenyl ring of amino acid PHE158
and phenyl ring at position 4 of the pyrazole ring showed π-π
interaction with amino acid TRP206. The fair interactions
with good binding energies suggest that schiff base of
pyrazole derivatives ligands are potential compounds in
inhibiting falcipain-2 protein.
6bd
6be
6bf
6ca
6cb
p-CH3Ph
p-OCH3Ph
p-ClPh
o-ClPh
m-ClPh
-
6cc
6cd
6ce
6cf
Chloroquinine
a Activity reported [21]
3.5. Prediction of Pharmacokinetic Properties
All Pyrazole Schiff base hybrids were screened for their
different pharmacokinetic parameters of compounds using
ADME predictions by QIKProp 3.6. All the synthesized
compounds were primarily screened considering the primary
parameters of Lipinski’s rule of 5. All the synthesized
hybrids had drug-likeness properties per Lipinski’s rules
specified in Table 3 and ADME properties is depicted in
Table 4. An orally active drug should not have more than 1
3.3. Structure Activity Relationship
The SAR study revealed that various substitutions at
position 3 and 4 on pyrazole motif were responsible for a
range of antimalarial activities. Substitutions of phenyl ring
at position 3 of pyrazole nucleus with electron donating
group like methyl, showed good antimalarial activity while
substitution of electron withdrawing group like fluoro
partially decreased antimalarial activity. Substitutions of

200 Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3
Aggarwal et al.
p-Cl,m-Cl
o-Cl
excellent activity
lowers activity
p-CH3 retain activity
p-OCH3 decreased activity
p-H
vanish activity
RING B
Rigid Fragment
N
N
CH
N
Essential template
for activity
S
N
Pharmacophore
RING A
p-CH3 retain activity
p-F,p-H
lowers activity
Fig. (1). Structure-Activity Relationship of Pyrazole Schiff base hybrid.
Table 2. Docking score with the π-π interactions of Pyrazole Schiff base hybrid with amino acids.
Compound
6aa
Docking Score
Interaction Type
Amino Acid Involved
Interaction with Structural Feature
-8.2
π-π
π-π
TRP206
TRP206
Pyrazole ring
Ph at position 3
6ab
-8.1
π-π
π-π
TRP206
TRP206
Ph at position 3
p-CH3Ph at position 4
6ac
6ad
6ae
6af
-7.9
-8.0
-8.1
-8.1
π-π
π-π
π-π
TRP206
TRP206
TRP206
p-OCH3Ph at position 4
p-ClPh at position 4
o-ClPh at position 4
π-π
π-π
TRP206
TRP206
Ph at position 3
m-ClPh at position 4
6ba
-8.5
π-π
π-π
PHE158
TRP206
Pyrazole ring
Ph at position 4
6bb
6bc
6bd
6be
6bf
-8.3
-7.9
-8.2
-8.6
-8.6
π-π
π-π
π-π
π-π
TRP206
TRP206
TRP206
TRP206
p-CH3Ph at position 4
p-OCH3Ph at position 3
p-ClPh at position 4
o-ClPh at position 4
π-π
π-π
TRP206
TRP206
Pyrazole ring
p-CH3Ph at position 4
6ca
-8.4
π-π
π-π
TRP206
TRP206
Pyrazole ring
p-FPh ring at position 3
6cb
6cc
6cd
-8.2
-7.9
-8.4
π-π
π-π
TRP206
TRP206
p-CH3Ph at position 4
p-OCH3Ph at position 4
π-π
π-π
TRP206
TRP206
Pyrazole ring
p-FPh at position 3
6ce
6cf
-8.1
-8.1
π-π
π-π
TRP206
TRP206
Pyrazole ring
p-FPh at position 3
π-π
π-π
TRP206
TRP206
Pyrazole ring
p-FPh at position 3

Pyrazole Schiff Base Hybrid as an Anti-Malarial Agent
Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3 201
Fig. (2). Binding mode of Compound 6ba in pocket ofPfFP (PDB: 2GHU).
Fig. (3). Binding mode of Compound 6bf in pocket ofPfFP (PDB: 2GHU).
violation of these rules [28]. The most favorable values of
the descriptors like polar surface area and rotatable bonds
have profound influence on the oral bioavailability of the
molecules [29]. In the present study, all the synthesized
compounds possessed several rotatable bonds less than 15
and polar surface area fell suitably in the acceptable range
(7-200 Å). Caco-2 cell model and Madin-Darby canine
kidney (MDCK) cell model have been suggested as a reliable
in-vitro model for the calculation of oral drug absorption,
present as a model for the gut-blood barrier [30]. QPPCaco
and QPP MDCK predictions for all the test compounds
showed significant values. The solubility (QPlogS) of
organic molecules in water has a major impact on numerous
ADME-related properties like absorption, distribution,
transport, and ultimately bioavailability. All the synthesized
compounds showed solubility values within the limits (-6.5
to 0.5). Further, the results for the QPlogkhsa descriptor of
Qikprop representing predicted values of human serum
albumin binding indicated that synthesized molecules were
found to fall in the permitted range (1.5 to 1.5). Blood-Brain
Barrier (BBB) penetration is crucial in the pharmaceutical
sphere because CNS-active compounds must cross it and all
the synthesized compounds showed trustworthy prediction
for brain/blood partition coefficient.
CONCLUSION
In the present work, we have developed a series of
molecules comprised of pyrazole schiff base hybrid where
most of the compounds showed significant in vitro
antimalarial activity against CQS strains of P. falciparum.
Among all compounds, 6bf and 6bd were found to be

202 Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3
Aggarwal et al.
Table 3. Prediction of Lipinski's ‘Rule of 5’ for the Pyrazole Schiff base hybrid.
Compound
M.W.
Polar Surface Area
logP
HBD
HBA
‘N’ of violation
6aa
6ab
6ac
6ad
6ae
6af
6ba
6bb
6bc
6bd
6be
6bf
6ca
6cb
6cc
6cd
6ce
6cf
380.10
394.12
410.12
414.07
414.07
414.07
394.12
408.14
424.13
428.08
428.08
428.08
398.10
412.11
428.11
432.06
432.06
432.06
43.08
43.08
52.32
43.08
43.08
43.08
43.08
43.08
52.32
43.08
43.08
43.08
43.08
43.08
52.32
43.08
43.08
43.08
5.78
6.22
5.83
6.45
6.41
6.43
6.22
6.67
6.28
6.90
6.85
6.88
5.94
6.30
6.00
6.62
6.57
6.59
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
4
5
4
4
4
4
4
5
4
4
4
4
4
5
4
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Table 4. Calculated ADME properties.
Comps
QlogBB
(-3.0 to 1.5)
QP
logKHSA
QPP
MDCK
(<25 poor, >500 great)
QPP
caco(<25 poor,
>500 great)
QPlogS
(-6.5 to 5)
No of rotatable
bond
Percent Human
oral absorption
(>80%— high
<15
and <25%—poor)
6aa
6ab
6ac
6ad
6ae
6af
6ba
6bb
6bc
6bd
6be
6bf
6ca
6cb
6cc
6cd
6ce
6cf
0.181
0.172
0.049
0.294
0.295
0.287
0.159
0.174
0.166
0.039
0.287
0.109
0.101
-0.021
0.222
0.553
0.789
0.690
1.179
1.153
1.353
1.307
0.843
1.518
1.493
1.692
1.648
1.429
1.166
1.263
1.237
1.437
1.391
1.237
1.391
1.177
508
508
4057
921
924
721
100
510
510
404
923
506
506
404
916
906
100
100
5297
5297
804
529
531
429
531
527
527
429
527
531
531
430
539
859
859
856
-6.273
-6.505
-6.399
-6.649
-5.360
-5.475
-4.994
-4.607
-4.945
-4.102
-4.217
-4.227
-4.512
-5.012
-5.045
-4.489
-4.639
-4.724
4
4
5
4
4
4
4
4
5
4
4
4
4
4
5
4
4
4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

Pyrazole Schiff Base Hybrid as an Anti-Malarial Agent
Combinatorial Chemistry & High Throughput Screening, 2018, Vol. 21, No. 3 203
dihydro–1H–pyrazoles
as
potential
anticancer,antifungal,
potential molecules with EC50 1.95µg/ml and 1.98µg/ml
respectively. The observed activity was further correlated via
molecular docking study. The molecular modeling studies
revealed that the pyrazole Schiff base analogues completely
occupy the site of PfFP-2 with different modes. The ascer-
tained ADME parameters for the synthesized compounds
showed good pharmacokinetic properties. This outcome
shows that the pyrazole Schiff base hybrids may be
promising leads and provide a significant model for further
structural as well as biological optimization.
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Hout, S.; Azas, N.; Darque, A.; Robin, M.; Di Giorgio, C.; Gasquet,
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CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
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The authors report no conflict of interest. The authors
alone are responsible for the content of the manuscript.
ACKNOWLEDGEMENTS
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We are grateful to National Malaria Research Institutes,
Dwarka New Delhi for providing facility for antimalarial
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