Synthesis and biological evaluation of some pyrazole derivatives as anti-malarial agents

Article abstract of DOI:10.1002/ardp.201100078

Novel series of pyrazole derivatives were synthesized and tested for their in vivo anti-malarial activity using mice infected with chloroquine sensitive P. berghei at a dose level of 50μmol/kg. The most active compounds were further tested in vitro agains

Full text of DOI:10.1002/ardp.201100078

Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
147
Full Paper
Synthesis and Biological Evaluation of Some Pyrazole
Derivatives as Anti-Malarial Agents
Adnan A. Bekhit1,2, Ariaya Hymete2, Henok Asfaw2, and Alaa El-Din A. Bekhit3
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2 Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, Addis Ababa,
Ethiopia
3 Food Sciences, University of Otago, Dunedin, New Zealand
Novel series of pyrazole derivatives were synthesized and tested for their in vivo anti-malarial activity
using mice infected with chloroquine sensitive P. berghei at a dose level of 50 mmol/kg. The most active
compounds were further tested in vitro against chloroquine resistant (RKL9) strain of P. falciparum. The
in vivo anti-malarial activity study indicated that compounds 2a, 2b, 8a and 8b had mean percent
suppression of 85%, 83%, 95% and 97%, respectively at equimolar dose level of the standard drug
chloroquine diphosphate. Moreover, compounds 2a, 2b, 8a and 8b showed in vitro IC50 values lower
(p < 0.05) than that of the standard drug chloroquine phosphate (0.188 ꢀ 0.003 mM) using the RKL9
strain. Compound 8b was the most active with IC50 of 0.033 ꢀ 0.014 mM. Generally, among the tested
compounds, those containing a free carboxylic acid functional group on the pyrazole ring were the
most active and this finding was supported by the docking results performed for the active compounds.
The acute toxicity studies of the active compounds revealed that they have a good safety profile.
Keywords: Acute toxicity / Anti-malarial activity / Docking / P. berghei / P. falciparum / Pyrazoles
Received: March 3, 2011; Revised: August 6, 2011; Accepted: August 26, 2011
DOI 10.1002/ardp.201100078
Introduction
the search for new and effective anti-malarial drugs remains
a challenging quest for medicinal chemists.
Malaria continues to be a global serious health problem.
According to the WHO 2009 malaria report, half of the world
population is at risk of malarial infection. An estimated
1 million deaths caused by malaria occurred in the year 2009
among which 89% were in the African region [1]. Despite the
introduction of a large number of chemotherapeutic agents
for the treatment of malaria there is still an unmet medical
need for new effective drugs due to the emergence of resist-
ance for current available anti-malarial drugs. This type of
resistance has been reported for almost all the therapeutic
agents approved for the treatment of malaria which could
lead to the situation where no effective drug for treating this
serious health problem becomes available [2, 3]. Therefore,
The present work was carried out in an attempt to find
new effective anti-malarial drugs among newly synthesized
pyrazole derivatives series. Pyrazole derivatives were chosen
because they were found to have a wide range of pharmaco-
logical activities, including anti-inflammatory [4, 5], anti-
microbial [6, 7], antiviral [8] and antitumor activity [9, 10],
and because they are versatile to be synthesized.
Furthermore, several reports have shown that pyrazoles
have significant antimalarial activities. For instance, chlor-
oquine-pyrazole analogues (Fig. 1) have been reported to
exhibit a significant in vitro anti-malarial activity using an
experimental model against P. falciparum [11, 12]. Ethyl 3-
amino-5-phenylamino-4H-pyrazole-4-carboxylate showed
a
potent antimalarial activity (IC50 ¼ 1.32 mmol/L) when tested
in vitro against P. falciparum [13]. Similarly, some aryl substi-
tuted pyrazole derivatives such as methyl 5-amino-3-anisidi-
nylpyrazole-4-carboxylic acid (IC50 ¼ 0.149 mmol/L) [14].
There are also other reports that demonstrated the antima-
larial activities of pyrazole derivatives [15, 16]. Hence, this
Correspondence: Adnan A. Bekhit, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria
21521, Egypt.
E-mail: adnbekhit@alexpharmacy.edu.eg
Fax: þ20-3-4873273
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A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
1HNMR spectrum of these two compounds revealed two sin-
glets at d 8.55 and 10.1 that were assigned to pyrazole-
C5H and aldehydic proton. Pyrazole aldehyde 2a and 2b were
condensed with the primary amine functional group of
hydrazine derivatives, namely isonicotinic hydrazide, 2,4-
dinitrophenylhydrazine and fluorophenylhydrazine deriva-
tives in the presence of few drops of hydrochloric acid as a
catalyst to give the corresponding hydrazone derivatives 3–7.
Structures of the synthesized hydrazone derivatives were
proved by the absence of the aldehydic characteristic bands
at 2726, 2669 and1670 cmꢁ1 in their IR spectra. In addition,
the absence of the singlet aldehydic peak of the starting
OH
N
N
CF3
CF3
N
N
N
Cl
N
Cl
Figure 1. Reported antimalarial pyrazole derivatives.
1
aldehydes at d 10.1 in their HNMR spectrum was taken as
work aims to synthesize new pyrazole derivatives and to
study the effect of the molecular variation among these
compounds on their anti-malarial activity.
an indication of the formation of these compounds.
Oxidation of aldehydes 2a and 2b by KMnO4 afforded the
corresponding carboxylic acid derivatives 8a and 8b. The
latter showed a characteristic IR broad band at 3170–2520
assigned for COOH stretching vibration and a sharp band at
Results and discussion
–
1700–1702 attributed to C O stretching vibration. Their
–
Chemistry
1HNMR spectra showed broad D2O exchangeable singlet at
The target compounds were synthesized according to the
steps outlined in Scheme 1. Initially the hydrazones 1a
and 1b were subjected to Vilsmeyer Haack reaction, to yield
the corresponding pyrazole aldehydes 2a, and 2b. The struc-
tures of the obtained aldehydes were confirmed by IR spectra
that showed characteristic bands at 2726, 2669 and
d 12.72–12.76 characteristic for COOH.
Biological activity
In vivo anti-malarial activity
Results for the in vivo anti-malarial activity tests of the
synthesized compounds are shown in Table 1. All the tested
compounds, except 3b, 4a, and 6a, showed parasitaemia
suppression in mice above 50% compared to those which
1670 cmꢁ1 that were attributed to the C–H and C O stretch-
–
–
ing vibration of the aldehydic group, respectively. Moreover,
F
H
HN
N
HN
N
O
N
N
N
N
H N
2
H N
2
N
NH
N
N
N
R
NH
F
R
R
2a,b
o-Fluoro: 5a,b
m-Fluoro: 6a,b
p-Fluoro:7a,b
3a,b
H N
2
KMnO4
POCl3 /
DMF
NH
NO
2
O N
2
O
NO
2
HN
NO
2
OH
N
N
N
N
N
N
NH
R = CH3, Cl
R
R
R
8a,b
1a,b
4a,b
Scheme 1. Sythesis of intermediate and target compounds.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
Pyrazole Derivatives as Anti-malarial Agents
149
Table 1. In vivo antimalarial activity of the target compounds 2–8.
may undergo many nucleophilic reactions with vital elecro-
philes present in the body [17, 18]. These side effects can be
avoided by preparing their carboxylic acid analogous, such as
in 8a and 8b.
Comp. No.
% Parasitaemia % Suppression Mean survival
time (days)
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
12 ꢀ 1.7
13 ꢀ 0.3
18 ꢀ 2.3
75 ꢀ 4.5
45 ꢀ 4.1
18 ꢀ 0.4
25 ꢀ 1.6
37 ꢀ 2.3
42 ꢀ 2.8
39 ꢀ 1.8
39 ꢀ 1.8
38 ꢀ 2.6
4 ꢀ 0.4
2 ꢀ 0.3
79 ꢀ 1.6
0.0
85
83
78
4
8.6 ꢀ 0.3
9.8 ꢀ 0.6
13.0 ꢀ 0.4
11.4 ꢀ 0.8
6.6 ꢀ 0.6
9.2 ꢀ 0.5
6.9 ꢀ 0.2
6.1 ꢀ 0.6
6.8 ꢀ 0.2
6.3 ꢀ 0.7
7.3 ꢀ 0.2
6.8 ꢀ 0.3
>14
In vitro antiplasmodial activity
The most active compounds 2a, 2b, 3a, 4b, 8a and 8b, which
showed reasonable in vivo activity, were further examined for
their antiplasmodial activities against chloroquine resistant
(RKL9) strain parasite (Table 2). All the tested compounds
showed better activity than chloroquine diphosphate
(IC50 ¼ 0.188 mM) against chloroquine resistant (RKL9) strain
parasite (Table 2). Compound 8b was found to be the most
potent against RKL9 strain (IC50 ¼ 0.033 mM) and this may be
attributed to the Cl group at the para-position which makes
the compound more lipophilic and increases the tissue
penetration power.
43
78
69
54
46
51
53
52
95
97
0.0
100
>14
5.8 ꢀ 0.4
Control
Chloroquine
>14
In vivo acute toxicity study
were treated with the standard drug chloroquine.
Compounds 8b, 8a, 2a, 2b, 3a, and 4b exhibited the highest
activity with percent suppression >75% (97%, 95%, 85%, 83%,
78% and 78%, respectively).
Compounds 2a, 2b, 3a, 4b, 8a and 8b were further evaluated
for their oral acute toxicity in male mice using a previously
reported method [18, 19]. The results indicated that the tested
compounds were non-toxic and well tolerated by experimen-
tal animals up to 300 mg/kg. Moreover, these compounds
were tested for their toxicity through the parenteral route
and the results revealed that all test compounds were non-
toxic up to 100 mg/kg.
Among the initially tested compounds, those that con-
tained free aldehydic functionality on the pyrazole nucleus
2a and 2b generally showed higher in vivo anti-malarial
activity. This was probably due to stronger binding of these
groups to receptor backbone compared to hydrazone
analogues since such groups serve as better hydrogen donors
and acceptors. This suggestion was supported by the docking
results obtained for these molecules which demonstrated
that while the aldehydic derivatives 2a and 2b exhibited
strong hydrogen bonding with the backbone of dihydrofolate
reductase enzyme, the hydrazone derivatives undergo fewer
and weaker interactions. The activity of compound 2a was
slightly higher than that of compound 2b, which may be due
to the p-tolyl group which makes the aldehydic group suited
to form stronger hydrogen bond by the steric effect of the
methyl group.
Docking
Molecular docking studies further helped in understanding the
various interactions between the ligands and enzyme active
sites. The determination of the three dimensional co-crystal
structure of dihydrofolate reductase (PDB ID: 1J3I), complexed
with a selective inhibitor, WR99210 (Fig. 2) led to the develop-
ment of a model for the topography of the anti-malarial drugs
binding site in dihydrofolate reductase enzyme. This compound
WR99210 displayed hydrogen bond interactions with Ile 14,
Asp 54 and Ile 164, in addition to hydrophobic interactions
with Ile 14, Cys 15, Ala 16, Asp 54, Met 55, Phe 58 and Pro 113.
By realizing the possible in vivo metabolism of the aldehy-
dic group to its corresponding carboxylic acid, we synthes-
ized and tested the biological activity of compounds 8a and
8b and an excellent anti-malarial activity was observed which
was more pronounced than that found with the two aldehy-
dic derivatives 2a and 2b. This finding was also rationalized
by performing molecular docking for these compounds
where compound 8a was shown to form six hydrogen bonds
and some hydrophobic interaction while compound 8b
formed four hydrogen bonds and few hydrophobic inter-
actions with the receptor backbone. This finding is quite
interesting because of the possible long term toxicity of
aldehyde derivatives which have very reactive nature and
Table 2. In vitro antiplasmodial activity against chloroquine
resistant (RKL9) strain of P. falciprum
Compound
IC50, mM W SDꢂ
2a
2b
3a
4b
8a
8b
0.051 ꢀ 0.003
0.041 ꢀ 0.004
0.076 ꢀ 0.011
0.068 ꢀ 0.130
0.036 ꢀ 0.018
0.033 ꢀ 0.014
0.188 ꢀ 0.003
Chloroquine
ꢂ
Results of two separate determinations
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150
A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
Figure 4. 3D view from a molecular modeling study of the mini-
mum-energy structure of the complex of 4a docked in DHFR (PDB
ID: 1J3I). Viewed using Molecular Operating Environment (MOE)
module.
Figure 2. 3D view from a molecular modeling study, of the mini-
mum-energy structure of the complex of WR99210 docked in DHFR
(PDB ID: 1J3I). Viewed using Molecular Operating Environment
(MOE) module.
Also it showed hydrophobic interactions with Phe 116,
Met 55 and Ser 108, Tyr 170, respectively.
The molecular docking studies revealed that compounds
8a and 8b exhibited the strongest interaction with the
target enzyme. Compound 8a undergoes six hydrogen
bonds with Ala 16, Ala 16, Leu 40, Tyr 170, Ala 16 and Tyr
170 and weak hydrophobic interactions with Cys 15, Ala 16,
Leu 40, Leu 46, Met 55, Gly 166 and Ser 167 (Fig. 5). Similarly,
compound 8b binds with backbone of the receptor of
the target enzyme by strong hydrogen bonding with Ile
164, Tyr 170, Ser 111 and Tyr 170 and weak hydrophobic
interactions with Leu 40, Gly 44, Val 45, Leu 46, Ile 112 and
Tyr 170 (Fig. 6).
The result of the docking study for compound 2a showed
that the free aldehydic group of the compound exhibited a
very strong hydrogen bonding interaction with Ser111
(Fig. 3). In addition, the compound showed hydrophobic
interaction with Phe 58, Phe 116, Met 55, and hydrogen
bonding with Ser 108. The binding mode of compound 4b
was not different from compound 2a (Fig. 4). Compound 4b
showed a relatively strong hydrogen bonding with Ser 111.
Figure 3. 3D view from a molecular modeling study of the mini-
mum-energy structure of the complex of 2a docked in DHFR (PDB
ID: 1J3I). Viewed using Molecular Operating Environment (MOE)
module.
Figure 5. 3D view from a molecular modeling study of the mini-
mum-energy structure of the complex of 8a docked in DHFR (PDB
ID: 1J3I). Viewed using Molecular Operating Environment (MOE)
module.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
Pyrazole Derivatives as Anti-malarial Agents
151
on FTIR-8400 Shimadzu infrared spectrophotometer using Nujol
mules or KBr plates. 1HNMR spectra was recorded on BRUKER
Avance DMX400 400-MHz FT-NMR spectrometer using one or two
of the following solvents DMSO-d6, CDCl3 and MeOD and the
chemical shifts are given in d (ppm) downfield from tetrame-
thylsilane (TMS) which served as an internal standard. Splitting
patterns were designated as follows: s: singlet; d: doublet;
m: multiplet. Elemental analyses was performed on Perkin
Elmer 2400 elemental analyzer and values obtained were within
ꢀ0.4% of the theoretical values. Follow up of the reactions and
checking the purity of the compounds was made by thin layer
chromatography (TLC) on silica gel-precoated aluminum sheets
(Type 60 GF254, Merck) and the spots were detected by exposure to
UV-lamp at l ¼ 254nm and/or iodine chamber. Parasites were
counted using BIO-PLUS microscope.
Chemistry
Synthesis of 3-phenyl-1-(p-tolyl)-1H-pyrazole-4-
carboxaldehyde (2a) and 1-(4-chlorophenyl)-3-phenyl-1H-
pyrazole-4-carboxaldehyde (2b)
Figure 6. 3D view from a molecular modeling study of the mini-
mum-energy structure of the complex of 8b docked in DHFR (PDB
ID: 1J3I). Viewed using Molecular Operating Environment (MOE)
module.
Phosphorous oxychloride (2.42 g, 15.8 mmol) was added drop-
wise to an ice cold dimethylformamide (6.54 g, 6.93 mL,
89.5 mmol) with continuous stirring over a period of 45 min.
1-(1-Phenylethylidene)-2-(p-tolyl) hydrazine (1.5 g, 6.88 mmol)
or 2-(4-chlorophenyl)-1-(1-phenylethylidene) hydrazine (1.7 g,
6.88 mmol) was then added and the reaction mixture was
allowed to attain room temperature. The mixture was then
heated at 708C for 2 hours, allowed to cool and poured onto
crushed ice (55 g) and water (100 ml). Finally the mixture was
boiled and the copious white precipitate obtained after cooling
was filtered, dried and recrystallized from methanol as white
needles.
In general the result from molecular docking for the active
compounds supports the findings and the biological activi-
ties obtained from the in vivo and in vitro experimental
models. Both the in vivo and in vitro anti-malarial activity
study and docking results revealed that the most active
compounds were those that contained free carboxylic acid
group attached to the pyrazole functionality 8a and 8b.
Compound 2a, yield % ¼ 76, m.p. ¼ 120–1218C, microanlysis
for C17H14N2O (262.31), calculated C% ¼ 77.84, H% ¼ 5.38,
N% ¼ 10.68, Found C% ¼ 77.47, H% ¼ 5.61, N% ¼ 10.92. IR
Conclusion
(cmꢁ1): 1670 (C O), 1610 (C N). 1H-NMR (CDCl3): d 2.50 (s, 3H,
–
–
–
–
CH3), 7.31–7.63 (m, 5H, phenyl-H), 7.75 (d, J ¼ 8.0 Hz, 2H, tolyl-
C3,5H), 7.85 (d, J ¼ 8.0 Hz,2H, tolyl-C2,6H), 8.55 (s, 1H, pyrazole-
C5H), 10.1 (s, 1H, CHO).
Compounds 2a, 2b, 8a and 8b showed appreciable in vivo anti-
malarial activity against chloroquine sensitive P. berghei in
mice and excellent in vitro antiplasmodial activity
against chloroquine resistant (RKL9) strain of P. falciparum.
Among all the synthesized compounds, the two pyrazole
derivatives which contained free carboxylic acid functional
group 8a and 8b showed the highest anti-malarial activity.
Docking studies for both 8a and 8b with DHFR (PDB ID: 1J3I)
showed good binding profile. The results of acute toxicity
studies for the most active compounds revealed that these
compounds have a good safety margin. Therefore, com-
pounds 8a and 8b would represent a fruitful matrix for
the development of a new class of anti-malarial agents that
would deserve further investigation and derivatization.
Compound 2b, yield % ¼ 71, m.p. ¼ 134–1368C, microanlysis
for C16H11ClN2O (282.72), calculated C% ¼ 67.97, H% ¼ 3.92,
N% ¼ 9.91, Cl% ¼ 12.54, Found C% ¼ 68.22, H% ¼ 3.75,
N% ¼ 10.21, Cl% ¼ 12.32. IR (cmꢁ1): 1670 (C O), 1600 (C N).
–
–
–
–
1H-NMR (CDCl3):
d
7.45–7.55 (m, 5H, phenyl-H), 7.82
(d, J ¼ 7.8 Hz, 2H, chlorophenyl-C3,5H), 7.85 (d, J ¼ 7.8 Hz, 2H,
chlorophenyl-C2,6H), 8.52 (s, 1H, pyrazole-C5H), 10.11 (s, 1H, CHO).
Synthesis of N0-[3-phenyl-1-(4-methylphenyl)-
1H-pyrazol-4-yl)methylene]isonicotinohydrazide (3a) and
N0-[1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)-
methylene]isonicotinohydrazide (3b)
Three drops of HCl were added to a solution of the selected
aldehyde 2a or 2b (2 mmol) and isonicotinic hydrazide (2 mmol,
0.27 g) in 25 mL of ethanol. The mixture was heated under reflux
for 7 h with stirring. The reaction mixture was then cooled and
the yellow precipitate formed was filtered, washed with ethanol,
dried and recrystallized from methanol/water (1:1) for 2a and
from ethanol/water (1:1) for 2b.
Experimental
General procedures
Melting points were determined in open glass capillaries using
electro thermal BUCHI (B-540) hot storage melting-point appar-
atus and are uncorrected. The infrared (IR) spectra were recorded
Compound 3a, yield % ¼ 72, m.p. ¼ 212–2158C, microanlysis
for C23H20N4 (352.43), calculated C% ¼ 78.38, H% ¼ 5.72,
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A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
N% ¼ 15.90, Found C% ¼ 78.51, H% ¼ 4.29, N% ¼ 16.92.
hydrazine (2 mmol, 0.25 g) in 25 mL of ethanol. The mixture was
heated under reflux for 5 h, then cooled and the yellow pre-
cipitate formed was filtered, washed with ethanol, dried and
recrystallized from methanol/water (4:1).
IR (cmꢁ1): 3120 (NH), 1610 (C N). 1H-NMR (CDCl3): d 2.50
–
–
(s, 3H, CH3 ), 7.32–7.53 (m, 5H, phenyl-H), 7.55 (d, J ¼ 7.8 Hz,
ꢃ
2H, tolyl-C3,5H), 7.82 (d, J ¼ 7.8 Hz, 2H, tolyl-C2,6H), 7.85
(d, J ¼ 6.1 Hz, 2H, pyridine-C2,6H), 8.45 (s, 1H, pyrazole-C5H),
8.71 (d, J ¼ 6.1 Hz, 2H, pyridine-C3,5H) 8.85 (s, 1H, CH).
Compound 5a, yield % ¼ 82, m.p. ¼ 252–2538C, microanlysis
for C23H19FN4 (370.42), calculated C% ¼ 74.58, H% ¼ 5.17,
N% ¼ 15.13, Found C% ¼ 74.86, H% ¼ 5.28, N% ¼ 14.97. IR
Compound 3b, yield % ¼ 74, m.p. ¼ 265–2668C, microanlysis
for C22H17ClN4 (372.85), calculated C% ¼ 70.8, H% ¼ 4.60,
N% ¼ 15.03, Cl% ¼ 9.51, Found C% ¼ 70.54, H% ¼ 4.86,
(cmꢁ1): 3155 (NH), 1612 (C N). 1H-NMR (MeOD): d 2.50 (s, 3H,
–
–
CH3), 6.57-7.53 (m, 9H, fluorophenyl-H
& phenyl-H), 7.52
N% ¼ 14.74, Cl% ¼ 9.28. IR (cmꢁ1): 3150 (NH), 1612 (C N).
(d, J ¼ 7.8 Hz, 2H, tolyl-C3,5H), 7.76 (d, J ¼ 7.8 Hz, 2H, tolyl-
–
–
1H-NMR (CDCl3):
d
6.72–6.94 (m, 5H, phenyl-H), 7.05
C2,6H), 8.41 (s, 1H, pyrazole-C5H), 8.78 (s, 1H, CH).
(d, J ¼ 8.1 Hz, 2H, chlorophenyl C3,5H), 7.15 (d, J ¼ 6.1 Hz, 2H,
pyridine-C2,6H), 7.20 (d, J ¼ 8.1 Hz, 2H, chlorophenyl C2,6H), 7.80
(s, 1H, pyrazole -C5H), 8.05 (d, J ¼ 6.1 Hz, 2H, pyridine-C3,5H) 8.25
(s, 1H, CH).
Compound 5b, yield % ¼ 79, m.p. ¼ 244–3458C, microanlysis
for C22H16ClFN4 (390.84), calculated C% ¼ 67.61, H% ¼ 4.13,
N% ¼ 14.33, Cl% ¼ 9.07, Found C% ¼ 67.92, H% ¼ 3.79,
N% ¼ 14.11, Cl% ¼ 9.28. IR (cmꢁ1): 3280 (NH), 3146 (NH), 1607
(C N). 1H-NMR (MeOD): d 6.51–7.58 (m, 13H, fluorophenyl-H,
–
–
chlorophenyl-H & phenyl-H), 7.95 (s, 1H, pyrazole -C5H), 8.28
(s, 1H, CH).
Synthesis of 2-(2,4-dinitrophenyl)-1-{[3-phenyl-1-(4-
methylphenyl)-1H-pyrazol-4-yl]methylene}hydrazine (4a)
and 2-(2,4-dinitrophenyl)-1-{[1-(4-chlorophenyl)-3-phenyl-
Compound 6a, yield % ¼ 86, m.p. ¼ 251–2528C, microanlysis
for C23H19FN4 (370.42), calculated C% ¼ 74.58, H% ¼ 5.17,
N% ¼ 15.13, Found C% ¼ 74.71, H% ¼ 5.44, N% ¼ 15.42. IR
1H-pyrazol-4yl]methylene}-hydrazine (4b)
–
–
Three drops of HCl were added to a solution of the appropriate
aldehyde 2a or 2b (2 mmol) and 2,4-dinitrophenylhydrazine
(2 mmol, 0.40 g) in ethanol (25 mL). The reaction mixture was
heated under reflux for 7 h, cooled and resultant red precipitate
formed was filtered, washed with ethanol and recrystallized
from ethyl acetate.
(cmꢁ1): 3164 (NH), 1614 (C N). 1H-NMR (CD3Cl): d 2.53 (s, 3H,
CH3), 6.26–7.49 (m, 9H, fluorophenyl-H & phenyl-H), 7.53
(d, J ¼ 7.8 Hz, 2H, tolyl-C3,5H), 7.69 (d, J ¼ 7.8 Hz, 2H, tolyl-
C2,6H), 8.42 (s, 1H, pyrazole-C5H), 8.74 (s, 1H, CH).
Compound 6b, yield % ¼ 86, m.p. ¼ 258–2598C, microanlysis
for C22H16ClFN4 (390.84), calculated C% ¼ 67.61, H% ¼ 4.13,
N% ¼ 14.33, Cl% ¼ 9.07, Found C% ¼ 67.88, H% ¼ 4.41,
Compound 4a, yield % ¼ 93, m.p. ¼ 312–3148C, microanlysis
for C23H18N6O4 (442.43), calculated C% ¼ 62.44, H% ¼ 4.10,
N% ¼ 19.00, Found C% ¼ 62.68, H% ¼ 4.53, N% ¼ 18.87.
N% ¼ 14.58, Cl% ¼ 8.78. IR (cmꢁ1): 3157 (NH), 1615 (C N).
–
–
1H-NMR (CD3Cl): d 6.24–7.56 (m, 13H, fluorophenyl-H, chloro-
phenyl-H & phenyl-H), 7.98 (s, 1H, pyrazole -C5H), 8.26 (s, 1H, CH).
Compound 7a, yield % ¼ 85, m.p. ¼ 248–2508C, microanlysis
for C23H19FN4 (370.42), calculated C% ¼ 74.58, H% ¼ 5.17,
N% ¼ 15.13, Found C% ¼ 74.28, H% ¼ 5.36, N% ¼ 15.46.
IR (cmꢁ1): 3225 (NH), 1615 (C N), 1463, 1388 (NO ). 1H-NMR
–
–
2
(DMSO-d6): d 7.41–7.63 (m, 5H, phenyl-H), 7.70 (d, J ¼ 8.1 Hz,
2H, tolyl-C3,5H), 8.01 (d, J ¼ 8.1 Hz, 2H, tolyl-C2,6H), 8.11
(d, J¼ 9.3 Hz, 1H, 2,4-dinitrophenyl-C6H), 8.32 (d, J ¼ 9.3 Hz,
1H, 2,4-dinitrophenyl-C5H), 8.82 (s, 1H, pyrazole-C5H), 8.91
(s, 1H, CH), 9.12 (s, 1H, 2,4-dinitrophenyl-C3H), 11.71 (s, 1H, NH).
Compound 4b, yield % ¼ 86, m.p. ¼ 299–3018C, microanlysis
for C22H15ClN604 (462.85), calculated C% ¼ 57.09, H% ¼ 3.27,
N% ¼ 18.16, Cl% ¼ 7.66, Found C% ¼ 56.87, H% ¼ 3.52,
IR (cmꢁ1): 3170 (NH),1612 (C N). 1H-NMR (CD3Cl): d 2.50 (s, 3H,
–
–
CH3), 6.52 (d, J ¼ 8.8 Hz, 2H, fluorophenyl-C2,6H), 6.78
(d, J ¼ 8.8 Hz, 2H, fluoophenyl-C3,5H), 7.31–7.52 (m, 5H, phenyl-
H), 7.54 (d, J ¼ 7.8 Hz 2H, p-tolyl-C3,5H), 7.58 (d, J ¼ 7.8 Hz, 2H,
tolyl-C2,6H), 8.44 (s, 1H, pyrazole-C5H), 8.72 (s, 1H, CH).
Compound 7b, yield % ¼ 81, m.p. ¼ 264–2658C, microanlysis
for C22H16ClFN4 (390.84), calculated C% ¼ 67.61, H% ¼ 4.13,
N% ¼ 14.33, Cl% ¼ 9.07, Found C% ¼ 67.76, H% ¼ 4.36,
N% ¼ 17.78, Cl% ¼ 7.36. IR (cmꢁ1): 3280 (NH), 1618 (C N),
–
–
1475, 1388 (NO2). 1H-NMR (DMSO-d6): d 7.42–7.61 (m, 5H,
phenyl-H), 7.80 (d, J ¼ 8.1 Hz, 2H, chlorophenyl –C3,5H), 8.00
(d, J ¼ 8.1 Hz, 2H, chlorophenyl–C2,6H), 8.12 (d, J ¼ 9.6 Hz,
1H, 2,4-dinitrophenyl-C6H), 8.32 (d, J ¼ 9.6 Hz, 1H, 2,4-dinitro-
phenyl-C5H), 8.80 (s, 1H, pyrazole-C5H), 8.92 (s, 1H, CH), 9.11–9.21
(s, 1H, 2,4-dinitrophenyl-C3H), 11.71 (s, 1H, NH).
N% ¼ 14.60, Cl% ¼ 9.32. IR (cmꢁ1): 3168 (NH), 1616 (C N).
–
–
1H-NMR (CD3Cl): d 6.49–7.58 (m, 13H, fluorophenyl-H, chloro-
phenyl-H & phenyl-H), 78.01 (s, 1H, pyrazole -C5H), 8.24 (s, 1H, CH).
Synthesis of 3-phenyl-1-(4-methylphenyl)-1H-pyrazole-4-
carboxylic acid (8a) and 1-(4-chlorophenyl)-3-phenyl-1H-
pyrazole-4-carboxylic acid (8b)
Synthesis of 2-(2-fluorophenyl)-1-{[3-phenyl-1-(4-
methylphenyl)-1H-pyrazol-4-yl]methylene}hydrazine (5a);
1-{[1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl]-
methylen}-2-(2-fluorophenyl)-hydrazine (5b); 2-(3-
fluorophenyl)-1-{[3-phenyl-1-(4-methylphenyl)-1H-
pyrazol-4-yl]methylene}hydrazine (6a); 1-{[1-(4-
Potassium permanganate (2 mmol, 316 mg) was added portion-
wise to a cold solution of the appropriate aldehyde 2a or 2b
(2 mmol) in aqueous acetone (20 mL, 3:1 v/v) under continuous
stirring and temperature below 208C. The mixture was stirred
until the violet color disappeared, then an aqueous NaOH
solution (30 mL, 5% w/v) was added and reaction mixture was
stirred for one hour. The reaction mixture was filtered and the
filtrate was acidified with cold dilute hydrochloric acid with
vigorous stirring. The precipitate formed was filtered, washed
with excess of cold water, dried and re-crystallized from ethanol.
Compound 8a, yield % ¼ 78, m.p. ¼ 178–1808C, microanlysis
for C17H14N2O2 (278.31), calculated C% ¼ 73.37, H% ¼ 5.07,
chlorophenyl)-3-phenyl-1H-pyrazol-4-yl]methylene}-2-(3-
fluorophenyl)-hydrazine (6b); 2-(4-fluorophenyl)-1-{[3-
phenyl-1-(4-methylphenyl)-1H-pyrazol-4-yl]methylene}-
hydrazine (7a) and 1-{[1-(4-chlorophenyl)-3-phenyl-1H-
pyrazol-4-yl]methylene}-2-(4-fluorophenyl)-hydrazine (7b)
Three drops of HCl were added to a solution of the selected
aldehyde 2a or 2b (2 mmol) and the appropriate flourophenyl-
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Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
Pyrazole Derivatives as Anti-malarial Agents
153
N% ¼ 10.07, Found C% ¼ 73.50, H% ¼ 5.32, N% ¼ 10.28. IR
(Sigma) [23] and used for testing. Assay was carried out in 96 well
microtitre flat-bottomed tissue culture plates incubated at 378C
for 24 h in presence of two fold serial dilutions of compounds
and chloroquine diphosphate for their effect on schizont matu-
ration. Initial culture was maintained in small vials with 10%
haematocrit, i.e. 10 mL erythrocytes containing 1.0% ring stage
parasite in 100 mL complete media. The assay culture volume
was 100 mL per well. Number of parasites for the assay was
adjusted at 1–1.5% by dilution with fresh human B(þ) RBC.
Compounds were dissolved in ethanol and further diluted with
RPMI 1640 medium (the final ethanol concentration did
not exceed 0.5% which did not affect parasite growth).
Chloroquine diphosphate was dissolved in aqueous medium.
Test was done in duplicate wells for each dose of the drugs.
Solvent control culture containing the same concentrations of
the solvent as present in the test wells was done with RPMI-1640
containing 10% AB(þ) serum.
(cmꢁ1): 3170–2510 (br, OH), 1702 (C O), 1605 (C N). 1H-NMR
–
–
–
–
(CD3Cl): d 2.51 (s, 3H, CH3), 7.32-7.54 (m, 5H, phenyl-H), 7.53
(d, J ¼ 7.8 Hz, 2H, tolyl-C3,5H), 7.56 (d, J ¼ 7.8 Hz, 2H, tolyl-
C2,6H), 8.42 (s, 1H, pyrazole-C5H), 8.71 (s, 1H, CH), 12.76
(br s, D2O exchangeable, 1H, COOH).
Compound 8b, yield % ¼ 74, m.p. ¼ 191–1928C, microanlysis
for C16H11ClN2O2(298.72), calculated C% ¼ 64.33, H% ¼ 3.71,
N% ¼ 9.38, Cl% ¼ 11.87, Found C% ¼ 64.50, H% ¼ 3.32,
N% ¼ 9.28, Cl% ¼ 12.11. IR (cmꢁ1): 3168–2520 (br, OH), 1700
1
–
–
–
(C O), 1602 (C N). H-NMR (CD Cl): d 6.49–7.58 (m, 13H, fluoro-
–
3
phenyl-H, chlorophenyl-H & phenyl-H), 78.01 (s, 1H, pyrazole-
C5H), 8.24 (s, 1H, CH), 12.72 (br s, D2O exchangeable, 1H, COOH).
In vivo anti-malarial activity
The in vivo anti-malarial activity of the synthesized compounds
was evaluated using the standard 4 day suppressive assay [19].
Male Swiss albino mice (weight 20–25 g, obtained from
the Ethiopian Health and Nutrition Institute) were used in
this study. The animals were acclimatized for a period of
7 days to room temperature (23–258C) with relative humidity
of 60–65% before starting the assay. The animals were housed
in standard cages and maintained on standard pelleted diet
and water [20]. Accordingly, the test mice were infected with
0.2 mL of 2 ꢄ 107 parasitized erythrocytes, (P. berghei ANKA
strain) intravenously on day 0. Parasitized erythrocytes were
obtained from the blood of a donor mouse with approximately
20–30% parasitemia which was diluted with normal saline
(1:4).
After 2 h, the infected mice were weighed and randomly
divided into sixteen groups of five mice per cage for each test
compound. Groups 1–14 received the synthesized compounds
orally which was suspended in a vehicle containing 7% Tween80
and 3% ethanol in water, at 50 mmol/kg dose level and served as
treatment group. Group fifteen received the vehicle and served
as a negative control. Group sixteen received the standard
drug chloroquine (50 mmol/Kg) and served as a positive control
[19, 21].
On days 1 to 3, animals in the treated groups were adminis-
trated the same dose of the synthesized compounds as in day 0.
On day 4 (24 h after the last dose or 96 h post-infection)
the weight of the animals was determined and blood smear
from all mice was prepared using Giemsa stain. Level of
parasitemia was determined microscopically by counting 4 fields
of approximately 100 erythrocytes per field. The difference
between the mean parasitemia level of the negative control
group (taken as 100%) and that of the experimental group
was calculated and expressed as percent suppression. Finally
the survival time for each mouse was recorded except for
chloroquine treated ones which were completely cured of the
parasite.
Parasite growth was found unaffected at the solvent concen-
trations used. Growth of the parasites from duplicate wells of
each concentration was monitored in Giemsa stained blood
smears by counting number of schizont per 100 asexual para-
sites. Percent schizont maturation inhibition was calculated
using the formula: (1-Nt/Nc) ꢄ 100 where, Nt and Nc represent
the number of schizonts in the test and control well respectively.
Acute toxicity
The oral acute toxicity of the most active compounds 2a, 2b, 3a,
4b, 8a and 8b was investigated using male mice (average
weight ¼ 20 g obtained from the Medical Research Institute,
Alexandria University) following previously reported method
[24]. The animals were divided into groups of six mice each.
The compounds were given orally, suspended in 1% gum
acacia, in doses of 1, 10, 100, 200, 250, 300 mg/kg. The mortality
percentage in each group was recorded after 24 h. The
test compounds were also investigated for their parenteral
acute toxicity in groups of mice of six animals each. The
compounds or their vehicle, propylene glycol (control), were
given by intraperitoneal injection in doses of 10, 25, 50, 75,
100 mg/kg. The percentage survival was followed up to 7 days
[25].
Modeling study
Docking
The coordinate from the X-ray crystal structure of dehydro-
folate reductase enzyme (DHFR) used in this simulation was
obtained from the Protein Data Bank (PDB ID: 1J3I), where the
selective DHFR inhibitor WR99210 is bound to the active site.
The ligand molecules were constructed using the builder
module and were energy minimized. The active site of
DHFR was generated using the MOE-Alpha Site Finder,
Molecular Operating Environment’s (MOE Dock 2005)
module to rationalize the observed biological results in
the animal experiment [26]. Then ligands were docked within
this active site using the MOE-Dock. The lowest energy con-
formation was selected and the ligand interactions (hydro-
gen bonding and hydrophobic interaction) with DHFR were
determined.
In vitro anti-malarial assay
P. falciparum strain RKL9 (CQ resistant) was maintained in a
continuous culture using the standard method described by
Trager and Jensen [22]. Parasites were cultured in human B(þ)
erythrocytes in RPMI-1640 media (GIBCOBRL, Paisley, Scotland)
supplemented with 25 mM HEPES buffer, 10% human AB(þ)
serum and 0.2% sodium bicarbonate (Sigma) and maintained
under 5% CO2 atmosphere. Cultures containing predominantly
early ring stages were synchronized by addition of 5% D-sorbitol
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

154
A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 147–154
The authors wish to thank the Egyptian Fund for Technical Cooperation
with Africa, Ministry of Foreign Affairs, Egypt for financial support
provided for the availability of the first author in Addis Ababa University.
[12] W. Cunico, C. A. Cechinel, H. G. Bonacorso, A. P. Martins,
et al. Bioorg. Med. Chem. Lett. 2006, 16, 649–653.
[13] M. Verma, V. Gupta, R. K. Nema, U. Misra, JOPHAS, 2007, 4 (1),
390–394.
The authors have declared no conflict of interest.
´
[14] J. N. Domınguez, J. E. Charris, M. Caparelli, F. Riggione,
Arzneimittelforschung. 2002, 52 (6), 482–488.
[15] N. J. Thumar, M. P. Patel, Arch. Pharm. Chem. Life Sci. 2011, 344
(2), 91–101.
References
[16] E. Talaat, J. Chin. Chem. Soc, 2007, 54, 507–518.
[1] WHO. 2009, The World Malaria Report from WHO and UNICEF,
World Health Organization, Geneva
[17] P. J. O’Brien, A. G. Siraki, N. Shangari, Crit Rev Toxicol 2005, 35,
609–662.
[2] S. Martin, Arch. Pharm. Chem. Life Sci. 2008, 341, 149–163.
[3] I. M. Hastings, Trends Parasitol. 2004, 20, 512–518.
[18] J. W. Deneer, W. Seinen, J. L. M. Hermens, Aquat. Toxicol. 1988,
12, 185–192.
[4] A. A. Bekhit, T. A. Aziem, Bioorg. Med. Chem. 2004, 12, 1935–
1945.
[19] W. Peters, B. L. Robinson, Parasitic infection models: Handbook of
animal models of infection, London Academic Press, 1999, 757–
773
[5] J. Milano, M. J. Rossato, S. M. Oliveira, C. Drewes, et al. Life Sci.
2008, 83, 739–746.
[20] D. Dikasso, E. Makonnen, A. Debela, D. Abebe, K. Urga, et al.
Ethiopian J. Heal. Dev, 2006, 20, 112–118.
[6] B. F. Abdel-Wahab, H. A. Abdel-Aziz, E. M. Ahmed, Eur. J. Med.
Chem. 2008, 30, 1–4.
[21] J. N. Dominguez, C. Leon, J. Rodrigues, G. D. Neira, et al. Eur. J.
Med. Chem. 2009, 44, 1457–1462.
[7] B. F. Abdel-Wahab, H. A. Abdel-Aziz, E. M. Ahmed, Arch.
Pharm. Chem. Life Sci, 2008, 341, 734–739.
[22] W. Trager, J. B. Jensen, Science. 1976, 193, 673–675.
[8] R. Aggarwal, V. Kumar, P. Tyagib, S. P. Singh, Bioorg. Med.
Chem. 2006, 14, 1785–1791.
[23] C. Lambros, J. P. Vanderberg, J. Parasitol. 1979, 65, 418–420.
[24] M. Verma, M. Tripathi, A. K. Saxena, K. Shanker, Eur. J. Med.
Chem. 1994, 29, 941–946.
[9] I. A. Zanze, D. H. Albert, P. F. Bousquet, G. A. Cunha, et al.
Bioorg. Med. Chem. Lett. 2007, 17, 3136–3140.
[25] A. A. Bekhit, H. T. Y. Fahmy, Arch. Pharm. Pharm Med Chem.
2003, 336, 111–118.
[10] P. Diana, A. Carbone, P. Barraja, A. Martorana, et al. Bioorg.
Med. Chem. Lett. 2007, 17, 6134–6137.
[26] Molecular Operating Environment (MOE). Version 2005–
06.Chemical Computing Group, Inc Montreal, Canada:
http://www.chemcomp.com.
[11] F. E. Goda, A. R. Maarouf, E. R. El-Bendary, Saudi Pharm. J.
2003, 11, 111–117.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com