Synthesis, in silico experiments and biological evaluation of 1,3,4-trisubstituted pyrazole derivatives as antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.067

New 1,3,4-trisubstituted pyrazole derivatives were synthesized and evaluated for their antiplasmodial activity. Compounds 4b, 4c, 7a and 7d were the most potent antiplasmodial agents against P. berghei with percent of suppression ranging from 90 to 100%.

Full text of DOI:10.1016/j.ejmech.2018.11.067

Accepted Manuscript
Synthesis, in silico experiments and biological evaluation of 1,3,4-trisubstituted
pyrazole derivatives as antimalarial agents
Adnan A. Bekhit, Manal N. Saudi, Ahmed M.M. Hassan, Salwa M. Fahmy, Tamer M.
Ibrahim, Doaa Ghareeb, Aya M. El-Seidy, Sherry N. Nasralla, Alaa El-Din A. Bekhit
PII:
S0223-5234(18)31026-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.067
EJMECH 10925
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 September 2018
Revised Date: 5 November 2018
Accepted Date: 28 November 2018
Please cite this article as: A.A. Bekhit, M.N. Saudi, A.M.M. Hassan, S.M. Fahmy, T.M. Ibrahim, D.
Ghareeb, A.M. El-Seidy, S.N. Nasralla, A.E.-D.A. Bekhit, Synthesis, in silico experiments and biological
evaluation of 1,3,4-trisubstituted pyrazole derivatives as antimalarial agents, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.11.067.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, in silico experiments and biological evaluation of 1,3,4-
trisubstituted pyrazole derivatives as antimalarial agents
a,b*
a
a
a
Adnan A. Bekhit , Manal N. Saudi , Ahmed M. M. Hassan , Salwa M. Fahmy , Tamer M.
c,d
e
a
b
f
Ibrahim , Doaa Ghareeb , Aya M. El-Seidy , Sherry N. Nasralla , Alaa El-Din A. Bekhit
.
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21521, Egypt
Pharmacy Program, Pharmacology Stream, Allied Health Department, College of Health
b
Sciences, University of Bahrain, P.O. Box 32038, Kingdom of Bahrain
c
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafr
El-Sheikh 33516, Egypt.
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, The British University in
Egypt, Al-Sherouk City, Cairo, Egypt
e
Department of Biohemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
f
Food Sciences, University of Otago, Dunedin, New Zealand
Correspondence: Adnan A. Bekhit,
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Alexandria University, Alexandria
2
1521, Egypt.
E-mail: adnbekhit@pharmacy.alexu.edu.eg
Fax: 20-3-4873273
1

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1
Abstract
New 1,3,4-trisubstituted pyrazole derivatives were synthesized and evaluated for their
antiplasmodial activity. Compounds 4b, 4c, 7a and 7d were the most potent antiplasmodial
agents against P. berghei with percent of suppression ranging from 90-100 %. They were also
screened for their in vitro antimalarial activity against the chloroquine resistant strain P.
falciparum, (RKL9). Compound 4c displayed the highest in vitro antimalarial activity; 13-fold
higher than standard chloroquine phosphate. Molecular docking of the most active compounds
against the wildtype and quadruple mutant pf DHFR-TS structures rationalized the in vitro
antimalarial activity. Furthermore, these compounds exhibited reasonable in silico drug-
likeness and pharmacokinetic properties. Toxicity studies of the most active compounds
revealed that all tested compounds were non-toxic and well-tolerated up to 150 mg/kg via oral
route and 75 mg/kg via parentral route. According to RBC hemolysis assay, it was found that
compound 7a was the most potent anti-inflammatory and least toxic derivative with IC value
5
0
7
1-fold higher than IC50 value related to the antimalarial activity. Moreover, cytotoxicity
assessment revealed that compound 4c was the least toxic derivative with IC value 70000-
5
0
fold higher than IC50 value related to the antimalarial activity.
Key words
Pyrazole, hydrazone, oxadiazole, triazole, thiazole, thiazolidinone, thiadiazole, antimalarial, in
silico experiments, cytotoxicity, RBCs hemolysis and acute toxicity.
2

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2
Introduction
Malaria is one of the most significant parasitic diseases endemic to Africa and parts of Asia. It
is caused by four species of Plasmodium genus, but only two species are responsible for the
majority of infections; P. falciparum and P. vivax [1]. According to the WHO report in 2016,
although there is a substantial decline in the number of malaria cases and malaria deaths, the
disease accounted for 420,000 deaths in the year 2015, mainly in African children under five
years old. [2]. In addition, more than 50% of population is exposed to malaria risk in Ethiopia.
The main cause of infection is Plasmodium vivax, followed by P. falciparum and mixed
infection [3]. Despite the many interventions planned and adopted by malaria-inflicted
countries, further strategies should be implemented to target 90% or more reduction in the
global malaria incidence and mortality. One of the most important obstacles to complete
malaria eradication, is the rapidly developing resistance and severe adverse effects of currently
used drugs [2, 3]. In order to decrease the incidence of developing resistance, combinations of
two or more drugs is recommended. These drugs should target multiple stages of parasitic life
cycles as sporozoites, hypnozoites, merozoites and gametocytes. They also should include
transmission blocking compounds and ensure post-treatment prophylaxis [4-6]. Therefore,
there is a crucial need to synthesize new derivatives, with a wider safety margin, capable of
targeting novel pathways and enzymes inside protozoa. Also, there is a need to develop new
innovative strategies to ensure sustainable treatment and eradication of parasitic diseases.
These strategies include generating new formulations with variable combinations and
strengths of existing drugs, drug repurposing or repositioning and piggy-back approaches [7,
8
].
Antifolates, such as pyrimethamine and cycloguanil are inhibitors of DHFR domain of
Plasmodium falciparum dihydrofolate reductase thymidylate synthase (pf DHFR-TS). This
inhibition leads to prevention of DNA synthesis, growth, proliferation and finally parasitic cell
death [9-11]. It is worth mentioning that the resistance to pyrimethamine and its analogues
appeared quickly due to site specific amino acid mutation that led to a steric clash between the
previous compounds and Asn 108 [12, 13]. There is an ongoing need to discover new
antimalarial agents which are effective against the mutant pf DHFR.
It is well known that pyrazole ring, whether free or hybridized with other heterocyclic rings,
displayed a wide array of biological activities, such as antibacterial, antiviral, antitubercular,
anti-inflammatory, antioxidant, anticancer, antimalarial and antileishmanial [14-23]. For
instance, hybridization strategies of pyrazole ring with different scaffolds such as
thiazolidinone A showed promising in vivo and in vitro antimalarial activities especially for
the chloroquine-resistant strain of P. falciparum (RKL9) [24]. Also, other hybridization
approaches with heterylhydrazone B [25], oxadiazole C, [26, 27] were observed to enhance
antiprotozoal activity [24]. Also, heterocyclic moieties containing thiazole D, thiadiazole E,
and triazole F [28, 29] comprised a range of promising antiprotozoal agents, as seen in Figure
3

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1
. Based on such rational and promising results of previously reported analogues of (A), a
series of shiff bases I were designed and obtained via condensation of acid hydrazide
derivatives with different heterocyclic aldehydes. Also, new hybrids of pyrazoles with 4-
thiazolidinones II, 3,4-diarylthiazoles III, thia- / oxadiazoles IV and triazoles V were also
designed, then synthesized and biologically evaluated (Figure 2) as an effort to extend the
chemical space of pyrazole-based chemotypes [30] for antimalarial activity.
NO2
R1
OCH3
N
S
O
N
N
N
N
H C
O
O
NH
3
N
R3
N
N
N
N
N
N
R2
A
F
B
C
Br
IC50 for resistant (RKL9) p. falciparum = 0.0364 - 0.0420 uM
Excellent in vivo activity
Safe parentrally up to 100 mg/kg
Putative target for antimalarial activity is Pf-DHFR [24]
NH
N
O
S
O
HN
O
O
N
N
S
O
N
NH
O N
2
N
O
S
N
N
N
N
N
F
H
D
E
F
Figure 1. Rational and structures of lead antimalarial compounds.
4

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Figure 2. General formulae of the target compounds.
3
Results and discussion
3
.1 Chemistry
Target compounds were synthesized according to the steps outlined in Scheme 1 and Scheme
. The title starting material, acid hydrazide 1, was prepared by heating the methyl ester with
2
excess amounts of hydrazine hydrate in ethanol, taking into consideration the reaction
conditions as reported [31, 32]. In Scheme 1, the N-formyl derivative 2 was synthesized by
heating under reflux acid hydrazide 1 in formic acid [33, 34]. While, the proposed N-acetyl
derivative 3 was obtained, in excellent yield, by the reaction of acid hydrazide 1 with acetyl
chloride in dioxane [35]. Moreover, N-arylidinepyrazole-4-carbohydrazides 4a-f were
synthesized by heating under reflux acid hydrazide 1 with different aryl aldehydes [31]. In
Scheme 2, the key intermediates, substituted thiosemicarbazide 5a,b, were obtained via
heating acid hydrazide 1 with phenyl and allyl isothiocyanate, respectively, in ethanol [35,
3
6]. Furthermore, they were cyclized into the corresponding thiazolidin-4-one 6a,b and 3,4-
diarylthiazoline 7a-d derivatives via reaction with chloroacetyl chloride [37, 38] and
phenacylbromides [24, 39-41], respectively. In addition, intra-molecular cyclization of
substituted thiosemicarbazide 5a,b under basic, acidic and presence of HgO conditions
afforded triazolethione 8a,b, thiadiazole 9a,b and oxadiazole 10a,b derivatives, respectively
[35, 42-46]. In the presence bases, the nucleophilcity of the nitrogen atom of NHR group
5

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would increase enough to intramolecularly attack the carbonyl group with simultaneous
dehydration [42-45]. In acidic conditions, the nucleophilicity of the nitrogen atom of NHR
group would decrease, due to protonation. While, the nucleophilicity of the thiol group would
increase enough to intramolecularly attack the carbonyl group, followed by dehydration [44,
4
7, 48]. In the presence of HgO, the mechanism involved desulfurization, followed by
nucleophilic attack of the hydroxyl group on the carbonyl moiety with concurrent dehydration
44, 48, 49].
[
Scheme 1. Synthetic route of compounds 1-4.
6

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Scheme 2. Synthetic route of compounds 5-10.
3
.1.1 Analytical results
The Results and Discussion of the analytical results is summarized in the Supplementary
Material.
3
.2 In silico experiments
3
.2.1 Molecular docking
Antifolates, such as pyrimethamine and cycloguanil are considered as inhibitors of DHFR
domain of Plasmodium falciparum dihydrofolate reductase thymidylate synthase (pf DHFR-
TS). This inhibition leads to prevention of DNA synthesis, growth, proliferation and finally
parasitic cell death. The resistance to pyrimethamine and its analogues appeared quickly due
to site specific amino acid mutation that led to a steric clash between the previous compounds
and mutated Asn 108 [12, 13]. On the other hand, the co-crystallized ligand WR99210 has
high binding affinity to the mutant pf DHFR-TS due to the presence of flexible side chain that
adopts a conformation can still fit into the active site modified by mutations [13, 50].
7

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To rationalize the observed in vitro antimalarial activity, we explored the binding modes of
4
b, 4c, 7a and 7d in the active site for pf DHFR-TS as a putative antimalarial target to our
compounds. For this we performed molecular docking experiments on both the wildtype and
the quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS structures guided by
previous studies [51, 52]. Challenging the most active compounds by such highly mutant
model would provide clues about the in silico affinity of the most active compounds compared
to the reference pf DHFR-TS binders (e.g., pyrimethamine, cycloguanil and WR99210)
especially in a resistant variant of malaria.
Table 1. AutoDock Vina docking scores (kcal mol-1) of the most active compounds along
with the reference pf DHFR-TS binders (pyrimethamine, cycloguanil and WR99210) bound to
the wildtype and quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS structures.
Docking Score (SD*)
Compounds
Wildtype Pf-DHFR Quadruple mutant Pf-DHFR
4
4
7
7
b
c
a
d
-9.5 (0.06)
-9.9 (0)
-9.5 (0.06)
-10 (0)
-8.1 (0.06)
-9 (0.06)
-8.8 (0.06)
-8.4 (0)
-9.1 (0.06)
-9.8 (0)
-7.5 (0)
Cycloguanil
Pyrimethamine
WR99210
-7.5 (0)
-8.8 (0.06)
-8.8 (0)
*SD is the standard deviation of three consecutive docking runs.
The docking scores of the most active compounds obviously indicated significant in silico
binding affinities towards both wildtype and mutant pf DHFR-TS structures, especially when
compared to the reference pf DHFR-TS binders (e.g., pyrimethamine, cycloguanil and
WR99210) as shown in Table 1. Also, the most active compounds exhibited superior docking
score towards the mutant pf DHFR-TS structure compared to the wildtype, with exception to
only 4b which showed similar scores for both pf DHFR-TS structures. This indicates a
particular affinity of the most active compounds towards the mutant pf DHFR-TS structures
and as a possible resistance mechanism of malaria.
The docking pose of 4b shows favorable interactions in the binding site of wildtype pf DHFR-
TS as seen in Figure 3A. The phenyl pyrazolyl moiety demonstrated favorable hydrophobic
contact with the Leu46, Val45 and Ile112. The trimethoxybenzylidene moiety is involved in
H-bonding interaction as an acceptor with NH backbone and OH sidechain of Ser120. The p-
methoxy phenyl group is packed deeply between the co-factor NDP610, Ala16, Cys15, Leu46,
Ile164 and Phe58 indicating favorable van der Waal interactions. This binding pose is
reproduced upon docking into the quadruple mutant (N51I, C59R, S108N and I164L) pf
8

ACCEPTED MANUSCRIPT
DHFR-TS structure (Figure 3B), however, with the mutant residues appeared to be involved
in the interaction pattern. For instance, the mutant Arg59 is observed to show H-bonding
interaction with trimethoxybenzylidene moiety of 4b. In addition, mutant Asn108 and Leu164
appear to surround the p-methoxy phenyl group of 4b via favorable hydrophobic interactions.
Likewise, 4c shows binding mode similar to its congener 4b for both wildtype and mutant pf
DHFR-TS structures as shown in Figure 4.
Figure 3. The docking poses of 4b (green sticks) in the binding site of wildtype pf DHFR-TS
(
(
PDB code: 1j3i) [53] and quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS
PDB code: 1j3k) [53] for (A) and (B), respectively. Yellow-colored dashed lines indicate H-
bonding interactions. Non-polar hydrogen atoms were omitted for clarity.
9

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Figure 4. The docking poses of 4c (purple sticks) in the binding site of wildtype pf DHFR-TS
(PDB code: 1j3i) and quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS (PDB
code: 1j3k) for (A) and (B), respectively. Yellow-colored dashed lines indicate H-bonding
interactions. Non-polar hydrogen atoms were omitted for clarity.
The postulated binding modes of 7a and 7d did not differ dramatically from the predicted
poses of 4b or 4c, while also some dissimilarity can be observed due to differences of the
topological features between 7 and 4 compounds. Taking compound 7a as an example, the
phenyl pyrazolyl moiety of 7a pose is packed between the side chains of Val45 and Ile46 of
the wildtype pf DHFR-TS structure, as seen in Figure 5A. Like 4b, the p-methoxyphenyl
group of 7a is packed deeply between the co-factor NDP610, Ala16, Cys15, Leu46, Ile164
and Phe58. Also, the diphenylthiazolylidene moiety of 7a appeared to be packed between the
side chains of Met55, Cys59, Phe116, Arg122 and Cys59. Again, this postulated binding pose
did not differ intensely from the docked pose into the quadruple mutant (N51I, C59R, S108N
and I164L) pf DHFR-TS structure, as shown in Figure 5B. The mutant Arg59 is in a close
1
0

ACCEPTED MANUSCRIPT
proximity to the diphenylthiazolylidene indicating a favorable cation- π interaction of the
protonated guanidino group of the residue with the aromatic group N-phenylthiazolyl group of
7
a. In addition, like 4b and 4c, Asn108 and Leu164 are observed to border the p-methoxy
phenyl group of 7a via favorable hydrophobic interactions.
Figure 5. The docking poses of 7a (yellow sticks) in the binding site of wildtype pf DHFR-TS
(PDB code: 1j3i) and quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS (PDB
code: 1j3k) for (A) and (B), respectively. Yellow-colored dashed lines indicate H-bonding
interactions. Non-polar hydrogen atoms were omitted for clarity.
1
1

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3
.2.2 In silico prediction of physicochemical properties, pharmacokinetic, drug likeness
score and toxicity profile
The Results and Discussion of this section is summarized in the Supplementary Material.
.3 Biological screening
.3.1 In vivo antimalarial activity testing against P. berghei
3
3
All the newly synthesized compounds were screened for their in vivo antimalarial activity
using a 4-day suppressive test in P. berghei infected mice at a dose of 48.4 µmol/Kg/day [54],
Table 2.
Table 2: In vivo antimalarial activity of the newly synthesized compounds against P. berghei
at dose 48.4 µM/kg/day.
Compoud
Number
%
Parasitemia*
26±1.6
32±2.8
13±0.4
6±0.6
%
Suppression
70.11
63.21
85.05
93.10
97.70
67.81
86.20
79.31
73.56
70.11
82.75
79.31
97.70
81.60
71.26
90.80
73.56
63.21
72.21
74.71
85.05
68.96
0.00
Mean survival
time (days)*
6.66±0.2
6.66±0.4
9.33±0.2
ND**
2
3
4
a
4
b
4
c
2±0.4
ND**
4
d
14±0.6
12±0.6
18±0.2
23±0.6
26±1.2
15±0.8
18±1.4
2±0.4
10.50±0.4
11.50±0.3
8.83±0.3
6.66±0.5
6.66±0.3
12.83±0.5
11.33±0.4
ND**
4
4
f
5
a
5
b
6
a
6
b
7
a
7
b
16±0.6
25±0.8
8±0.2
11.66±0.2
7.33±0.3
ND**
7
c
7
d
8
a
23±0.6
32±1.8
24±0.2
22±0.4
13±0.8
27±0.6
87±1.2
0.0
7.66±0.6
5.50±0.2
7.16±0.4
8.33±0.6
11.16±0.2
7.33±0.4
4.33±0.7
ND**
8
b
9
a
9
b
1
0a
1
0b
Control
Pyrimethamine
Chloroquine
phosphate
100
100
0.0
12.22±0.2
1
2

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*
Values are M±SD, P< 0.05
**
ND: No death recorded during experimental period
3
.3.1.1 Structure activity relationship
At a certain stage of the design of this work, hydrazone derivatives were prepared by
condensation of acid hydrazide derivative 1 with different aryl/heterylaldehydes. Molecular
modeling of the proposed compounds showed formation of different hydrogen bonds with
amino acid residues in the active site of pfDHFR-TS. The results of in vivo antimalarial
screening of N-arylhydrazone 4b is in coherence with the rational concluded from the docking
experiment since emphasizing the high activity against P. berghei. On the other hand,
introduction of halogens in N-arylhydrazone derivative 4d slightly decreased their activity.
This could be attributed to the lower solubility rate of these compounds. Moreover,
condensation of acid hydrazide 1 with thiophene-2-carboxaldehyde resulted in thiophene
derivative 4e displaying good anti-plasmodial activity against P. berghei. This could be due to
the increased hydrophobicity of these compounds. The highest antimalarial activity of schiff
base 4c could be attributed to its interaction similarity with the backbone of target enzyme
pfDHFR-TS, similar to that of the co-crystalized ligand. Additionally, its solubility increased,
due to the presence of hydroxyl group. In a trial to increase the activity of the synthesized
compounds, hybridization of pyrazole ring with thiazolidinone or thiazole ring through three
atom spacer to produce 6a,b and 7a-d, respectively, was achieved. In general, thiazole
derivatives 7a-d showed higher activity than thiazolidinone derivatives 6a,b. Moreover,
compound 7a displayed the highest percentage of suppression, 97.0%, compared with other
thiazole derivatives. Direct attachment of different heterocyclic rings to the parent pyrazole
moiety resulted in compounds having variable antimalarial activity profile. Therefore,
cyclization of
thiosemicarbazides 5a,b showed different effects on the activity of
corresponding derivatives. For example, higher activity was associated with formation of
oxadiazole derivative 10a and thiadiazole 9b. Besides, no change in the activity was attributed
to triazole derivatives 8a. and a decrease in the activity of 8b, 9a and 10b was clear.
3
.3.2 In vitro antimalarial activity testing against P. falciparum
The compounds most active against P. berghei, compounds 4b, 4c, 7a and 7d, were screened
for their in vitro antimalarial activity against chloroquine resistant strain P. falciparum
(RKL9) using the standard method described by Trager and Jensen [55] (Table 3).
Table 3: In vitro antimalarial activity of the most active compounds against chloroquine-
resistant strain P. falciparum (RKL9).
Comp No.
IC µM±SD*
50
4
b
0.0398±0.004
1
3

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4c
0.0142±0.002
7
a
0.0492±0.014
0.0319±0.002
0.1930±0.003
0.01246±0.002
7
d
Chloroquine phosphate
Pyrimethamine
*
Results of two separate determinations.
3
.3.2.1 Structure activity relationship
Results of in vitro antiplasmodial screening showed that all test compounds displayed higher
antimalarial activity than the standard chloroquine phosphate against chloroquine-resistant P.
falciparum (RKL9). It is worth mentioning that m-hydroxy benzylidenehydrazone derivative
4
c was the most potent with IC50 = 0.0142 µM. This is in agreement with the previously
mentioned results of the in vivo antimalarial screening against P. berghei.
3
.3.3 Toxicity studies
3
.3.3.1 Lysosomal anti-inflammatory activity and hemolytic effect on RBCs
Lysosomal anti-inflammatory activity and hemolytic effect on RBCs of the most active
antimalarial compounds were evaluated according to Chatterjee et al [56] and Evans et al [57]
(Table 4). It was noticeable that compound 7a was the most potent anti-inflammatory with
IC 71-fold higher than IC of the corresponding antimalarial activity. It also displayed the
5
0
50
least hemolytic effect, even at very high concentrations. While, compound 4b was the least
active anti-inflammatory and most toxic derivative, with IC50 1700-fold higher than IC50
corresponding to the antimalarial activity.
Table 4: RBCs hemolysis assay of the most active antimalarial compounds.
Anti-inflammatory%
IC50
IC50
Antihemolytic %
IC50
IC50
µg/ml
(µM)
µg/m (µM)
l
Cpd
No.
5
20
(µg/ml)
40
(µg/ml)
5.34±
0.6
at
82.27
µM
5
20
40
(
2
µg/ml)
9.69±2 17.52±0.
(µg/ml)
41±1.3
at
(µg/ml) (µg/ml)
85 ±3.5 97.8±6.
at
41.13
µM
4
b
.5
35
at
41.13
µM
3
at
82.27
µM
5
2.45±2
120.8±
at
0.28
µM
25.50±1.1
10.28 µM
58.75
5.1
.
3
1
±2.5
4
±
9.03
3.2
at
2.12
54.31±
2.5
at
48.49
µM
59.58±6.
7
at
96.98
µM
32±1.02
at
12.12 µM
40±2.1 44±2.03
4c
1
at
at
96.98
µM
1
32.6±
11.8
16±2.2
54.71
±4.9
6
.60±0.9
1
µM
48.49
µM
5
6.11±1 81.19 at
106.28
±3.8
at
41±1.91
at
9.19 µM
79±3.0 99.8±5.
7
a
.
at
.19
6
±3.7
36.78
µM
3.49±0.
37
5
at
81
at
138.03
±6.6
7
±
5.15
3.6
1.90±0.2
9
73.58
36.78
73.58
1
4

ACCEPTED MANUSCRIPT
µM
µM
µM
µM
3
5.23±0 57.79±2. 80.34±1.
16±0.06
at
8.52 µM
59±4.0 68±5.02
7
d
.
94
at
1
at
3
at
6
at
at
68.20
µM
1
3.74±0
.85
79.52±
9.03
46.64
±5.7
7
.90±0.5
8
µM
.52
34.10
µM
68.20
µM
34.10
µM
3
.3.3.2 White blood cell cytotoxicity assessment
The effect of different concentrations of test compounds on proliferation of normal peripheral
blood mono nuclear cells (PBMC) was determined in order to measure their safety (Table 5).
Neutral red uptake assay was performed as the amount of retained dye is directly proportional
to the number of viable cells with an intact membrane [58]. It was noticeable that compound
4
c was the least toxic derivative, displaying the highest IC value equal to 1000.18 µM. This
50
IC50 was 70000-fold higher than IC50 related to the antimalarial activity. Additionally,
compound 4b was considered the most toxic compound as it showed the least IC50 value.
However, this IC was 783-fold higher than IC of the antimalarial activity.
5
0
50
Table 5: cytotoxicity assessment of test compounds on normal peripheral blood mono nuclear
cells (PBMC).
Comp
25
(µg/
ml)
IC (µM)
50
6
.25
100
(µg/ml)
IC50
(µg/ml)
.
(
µg/ml)
No.
4
3.00±1. 60.00
4
b
77.93±2.9
y = 0.3344x + 45.678
y = 0.0849x + 12.293
15.15±0.02 31.16±0.041
9
±2.3
-
5
4.42 12.78±0.2
420.00±12. 1000.18±30.
4
c
19.18±0.
3
±1.8
1
1
2
-
7
a
-6.20±0.4 1.63± 48.27±1.3
.06
6.05
0.9
y = 0.5989x - 12.178
y = 0.3307x + 7.01
103.34±5.2
130.30±3.2
190.02±9.6
222.16±5.5
0
1
±
7d
8.46±0.9
39.93±1.3
3
.3.3.3 In vivo acute toxicity testing
The most active antiplasmodial compounds 4b, 4c, 7a and 7d were evaluated for their acute
toxicity in mice. The absence of any toxicity signs, difference in the weight of the mice and
death cases were recorded during 3 days of observation post administration of the test
compounds. Thus, it could be concluded that the test compounds were non-toxic and well
tolerated by the experimental animals orally up to 150 mg/kg. In addition, evaluation of their
toxicity through the parenteral route was performed and the results revealed that all the
selected test compounds were non-toxic up to 75 mg/kg.
1
5

ACCEPTED MANUSCRIPT
4
Conclusion
New 1,3,4-trisubstituted pyrazole derivatives were synthesized and biologically evaluated for
their antiplasmodial activity. All synthesized compounds were evaluated for their in vivo
antimalarial activity using a 4-day suppressive test using Swiss Albino mice infected with P.
berghei ANKA strain. It is worth mentioning that compounds 4b, 4c, 7a and 7d displayed the
highest percent of suppression, above 90%, among all test compounds. These compounds
were further subjected to in vitro antiplasmodial testing against chloroquine-resistant P.
falciparum strain RKL9 using chloroquine phosphate as a reference drug. All compounds
showed inhibitory effects higher than chloroquine and compound 4c was found to be the most
potent with IC = 0.0142 µM. 4b, 4c, 7a and 7d were docked into the active site of the
5
0
wildtype and quadruple mutant (N51I, C59R, S108N and I164L) pf DHFR-TS to rationalize
their observed antimalarial activities. In addition, these compounds were subjected to in silico
prediction of physicochemical properties and pharmacokinetic profiles. They showed
reasonable drug-likeness scores, as well as pharmacokinetic properties. Other important
issues, such as compliance with Lipiniskiʹs rule of five, acceptable cell permeability through
Caco-2 model and low toxicity profile advocated these compounds to be drug-like candidates.
In addition, toxicity studies on the most active antimalarial compounds were performed. It was
found that compound 7a was the most potent anti-inflammatory and safe derivative with IC50
7
1-fold higher than IC related to the antimalarial activity. Moreover, cytotoxicity assessment
50
revealed that compound 4c was the least toxic derivative with IC 70000-fold higher than IC
50
5
0
related to the antimalarial activity. At the least, acute toxicity studies showed that all tested
compounds were non-toxic and well-tolerated up to 150 mg/kg via oral route and 75 mg/kg
via parenteral route.
5
Experimental:
5
.1 Chemistry
Melting points were determined in open-glass capillaries using a Griffin melting point
apparatus and all were uncorrected. Infrared spectra (IR) were recorded using KBr discs, on
Perkin-Elmer 1430 infrared spectrophotometer. Proton and carbon nuclear magnetic resonance
1
13
spectra ( H-NMR & C-NMR) were scanned on Joel-500 MHz, Brucker-400 MHz and
mercury-300 MHz NMR-spectrometer (DMSO-d6 & CDCl ). Chemical shifts were given in δ
ppm) using tetramethylsilane (TMS) as internal standard. Microanalyses were performed on
3
(
elemental analyzer at The Regional Center for Mycology and Biotechnology. Following up of
the reactions rates was performed by thin-layer chromatography (TLC) on ready-made silica
sheets and the spots were visualized by exposure to iodine vapors or UV-lamp at λ = 254 nm.
Electron impact mass spectra (EIMS) were run on a gas chromatograph/mass spectrometer at
1
6

ACCEPTED MANUSCRIPT
The Regional Center for Mycology and Biotechnology and relative intensity percentage
corresponding to the most characteristic fragments was recorded.
5
.1.1 General method for preparation of 3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carboxylic acid hydrazide 1[31, 32]
To a suspension of pyrazole-4-carboxylic acid methyl ester (1.54 g, 5 mmol) in absolute
ethanol (10 ml), excess hydrazine hydrate (10 ml) was added. The reaction mixture was stirred
for 24 h at room temperature. Then the solvent was evaporated and the residue washed with
0
water, filtered, dried, and crystallized from ethanol as white solid, mp 158-160 C (Reported,
0
-1
1
1
57-158 C [31]), in yield (97.5%). IR (cm ): 3470, 3312 (NH ); 3206 (NH); 1649 (C=O);
2
617 (C=N).
5
.1.2 General method for preparation of N'-Formyl-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazide 2
A solution of acid hydrazide 1 (0.3 g, 1 mmol) in formic acid (2 ml) was heated under reflux
for 6 h. After cooling, the reaction mixture was concentrated and the residue obtained was
o
filtered, washed, dried and crystallized from methanol as off-white crystals, mp 170-172 C, in
-1
1
yield (82%). IR (cm ): 3310, 3215 (NH); 1715 (CHO); 1643 (amidic C=O); 1611 (C=N). H -
NMR (DMSO-d , δ ppm): 3.79 (s, 3H, OCH ); 6.95, 7.01 (2d, J = 8.7 Hz, 2H, methoxy
6
3
phenyl-C3,5-H); 7.36-7.40 (m, 1H, phenyl-C
4
-H); 7.52-7.57 (m, 1H, phenyl-C3,5-H); 7.4, 7.81
(
2d, J = 8.7 Hz, 2H, methoxyphenyl-C -H); 7.83, 7.98 (2d, J = 8.7 Hz, 2H, phenyl-C -H);
2,6 2,6
9
.16 (s, 1H, NH, D
2
O-exchangeable); 9.24 (s, 1H, pyrazole-C
5
-H); 9.27 (s, 1H, CHO); 11.34
1
3
(s, 1H, NH, D O-exchangeable). C-NMR (DMSO-d , δ ppm): 55.76 (OCH ); 105.79
2 6 3
4
(pyrazole-C ); 114.23 (methoxyphenyl-C3,5); 119.49 (phenyl-C2,6); 124.32 (methoxy phenyl-
C ); 127.89 (phenyl-C ); 130.19 (methoxyphenyl-C ); 130.52 (phenyl-C ); 131.72
1
4
2,6
3,5
(pyrazole-C
5
1 3 4
); 139.27 (phenyl-C ); 151.03 (pyrazole-C ); 154.29 (methoxyphenyl-C ); 159.69
(
amidic C=O), 160.37 (CHO). Elemental analysis Calcd for C H N O : C, 64.28; H, 4.79; N,
18 16 4 3
8
.80. Found: C, 64.57; H, 4.83; N, 16.92.
5
.1.3 General method for preparation of N'-Acetyl-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazide 3
To a solution of acid hydrazide 1 (0.3 g, 1 mmol) in dioxane (10 ml), acetyl chloride (0.07 ml,
mmol) was added. The reaction mixture was heated under reflux for 6 h, then the solvent
was removed and the residue was triturated with an ice-H O mixture. The product formed was
filtered, washed with H O, dried and crystallized from methanol as yellow crystals, mp 130-
1
2
2
o
-1
1
1
32 C, in yield (83%). IR (cm ): 3244, 3128 (NH); 1725 (CH
608 (C=N). H -NMR (CDCl , δ ppm): 2.33 (s, 3H, CH ); 3.78 (s, 3H, OCH ); 6.94 (d, J =
3
CO); 1692 (amidic C=O);
1
3
3
3
1
7

ACCEPTED MANUSCRIPT
8
.8 Hz, 2H, methoxyphenyl-C -H); 7.28 (t, J = 7.4 Hz, 1H, phenyl-C -H); 7.38-7.42 (m, 3H,
3
,5
4
phenyl-C3,5-H and NH); 7.64-7.66 (m, 5H, methoxyphenyl-C2,6-H and phenyl-C2,6-H and NH);
1
3
8
.49 (s, 1H, pyrazole-C -H). C-NMR (CDCl , δ ppm): 25.09 (CH3); 55.41 (OCH ); 114.52
5 3 3
4
(pyrazole-C ); 114.74 (methoxyphenyl-C3,5); 119.60 (phenyl-C2,6); 124.00 (methoxyphenyl-
C ); 127.72 (phenyl-C ); 129.65 (methoxyphenyl-C ); 130.72 (phenyl-C ); 131.99
1
4
2,6
3,5
(pyrazole-C
5
1 3 4
); 139.07 (phenyl-C ); 151.53 (pyrazole-C ); 160.72 (methoxyphenyl-C ); 163.21
(
amidic C=O); 171.67 (CH CO). Elemental analysis Calcd for C H N O : C, 65.13; H, 5.18;
3
19 18
4
3
N, 15.99. Found: C, 65.37; H, 5.16; N, 16.25.
5
.1.4 General method for preparation of N'-arylidene-3-(4-methoxyphenyl)-1-phenyl-
H-pyrazole-4-carbohydrazide 4a-f
1
A mixture of acid hydrazide 1 (0.3 g, 1 mmol) and appropriate aldehyde (1 mmol) in absolute
ethanol (10 ml) was heated under reflux for 1-3 h. The precipitate formed was filtered, washed
with ethanol, dried and crystallized from ethanol as white solid.
5
.1.4.1 N'-benzylidene-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbohydrazide 4a
0
-1
The product was obtained as white solid, mp 242-244 C, in yield (89%). IR (cm ): 3190
1
(NH); 1646 (C=O); 1605 (C=N). H -NMR (DMSO-d
6 3
, δ ppm): 3.80 (s, 3H, OCH ); 7.00 (d, J
=
8.7 Hz, 2H, methoxyphenyl-C -H); 7.36-7.45 (m, 5H, N-phenyl-C3,4,5-H and phenyl-C3,5-
3
,5
H); 7.54-7.71 (m, 3H, N-phenyl-C2,6-H and phenyl-C
4
-H); 7.83 (d, J = 8.7 Hz, 2H,
methoxyphenyl-C -H); 7.92 (d, J = 7.5 Hz, 2H, phenyl-C -H); 8.29 (s, 1H, pyrazole-C -H);
2
,6
2,6
5
8
.99 (s, 1H, -CH=N); 11.68 (s, 1H, NH, D
2
O-exchangeable). Elemental analysis Calcd for
C H N O : C, 72.71; H, 5.08; N, 14.13. Found: C, 72.98; H, 5.16; N, 14.37.
24
20
4
2
5
.1.4.2 3-(4-Methoxyphenyl)-1-phenyl-N'-(3,4,5-trimethoxybenzylidene)-1H-pyrazole-4-
carbohydrazide 4b
0
-1
The product was obtained as white solid, mp 232-234 C, in yield (77%). IR (cm ): 3166
1
6 3
(NH); 1661 (C=O); 1643, 1605 (C=N). H -NMR (DMSO-d , δ ppm): 3.71 (1s, 3H, OCH );
3
.80 (1s, 3H, OCH ); 3.84 (1s, 6H, 2 OCH ); 7.01 (d, J = 8.25 Hz, 2H, methoxyphenyl-C -
3 3 3,5
H); 7.08 (s, 2H, tri methoxyphenyl-C2,6-H); 7.38 (t, J = 7.35 Hz, 1H, phenyl-C
4
-H); 7.54-7.59
(
m, 2H, phenyl-C -H); 7.83 (d, J = 8.25 Hz, 2H, methoxyphenyl-C -H); 7.92 (d, J = 7.8 Hz,
3,5 2,6
2
H, phenyl-C2,6-H); 8.22 (s,1H, pyrazole-C
5
2
-H); 8.99 (s,1H, -CH=N); 11.68 (s, 1H, NH, D O-
exchangeable). Elemental analysis Calcd for C H N O : C, 66.65; H, 5.39; N, 11.52. Found:
2
7
26
4
5
C, 66.91; H, 5.45; N, 11.78.
5
.1.4.3 N'-(3-Hydroxybenzylidene)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carbohydrazide 4c
0
-1
The product was obtained as white solid, mp 219-220 C, in yield (68%). IR (cm ): 3419
OH); 3204 (NH); 1662 (C=O); 1634, 1600 (C=N). H -NMR (DMSO-d , δ ppm): 3.80 (s, 3H,
1
(
6
3 4
OCH ); 6.82 (d, J = 8.7 Hz, 1H, hydroxyphenyl-C -H); 7.00 (d, J = 9 Hz, 2H, methoxyphenyl-
1
8

ACCEPTED MANUSCRIPT
C -H); 7.08-7.25 (m, 3H, hydroxyphenyl-C -H); 7.38 (t, J = 7.35 Hz, 1H, phenyl-C -H);
3
,5
2,5,6
4
7
.54-7.59 (m, 2H, phenyl-C3,5-H); 7.83 (d, J = 9 Hz, 2H, methoxyphenyl-C2,6-H); 7.92 (d, J =
7
.8 Hz, 2H, phenyl-C -H); 8.19 (s, 1H, pyrazole-C -H); 8.99 (s, 1H, -CH=N); 9.6 (s, 1H, OH,
2
,6
5
D
2
O-exchangeable); 11.99 (s, 1H, NH, D
2
O-exchangeable). Elemental analysis Calcd for
C H N O : C, 69.89; H, 4.89; N, 13.58. Found: C, 70.12; H, 4.96; N, 13.79.
24
20
4
3
5
.1.4.4 N'-(4-Fluorobenzylidene)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carbohydrazide 4d
0
-1
The product was obtained as white solid, mp 223-225 C, in yield (66%). IR (cm ): 3185
NH); 1660 (C=O); 1603 (C=N). H -NMR (300 MHz, DMSO-d , δ ppm): 3.80 (s, 3H,
6
1
(
OCH ); 7.00 (d, J = 8.7 Hz, 2H, methoxyphenyl-C -H); 7 (m, 2H, fluorophenyl-C -H); 7.38
3
3,5
3,5
4
(t, J = 7.35 Hz, 1H, phenyl-C -H); 7.54-7.59 (m, 2H, phenyl-C3,5-H); 7.8-7.84 (m, 4H,
methoxyphenyl-C -H and fluorophenyl-C -H); 7.92 (d, J = 7.8 Hz, 2H, phenyl-C -H); 8.29
2
,6
2,6
2,6
(
s, 1H, pyrazole-C
5
-H); 8.99 (s, 1H, -CH=N); 11.77 (s, 1H, NH, D
2
O-exchangeable). EIMS
+
·
+·
m/z (% relative abundance): 415 (4) (M +1), 414 (12) (M ), 293 (16), 278 (20), 277 (100),
2
6
34 (5), 145 (6), 104 (9), 77 (16), 75 (21). Elemental analysis Calcd for C24
9.55; H, 4.62; N, 13.52. Found: C, 69.81; H, 4.67; N, 13.74.
4 2
H19FN O : C,
5
.1.4.5 3-(4-Methoxyphenyl)-1-phenyl-N'-(thiophen-2-ylmethylene)-1H-pyrazole-4-
carbohydrazide 4e
0
-1
The product was obtained as white solid, mp 213-215 C, in yield (62%). IR (cm ): 3173
1
6
(NH); 1654 (C=O); 1600 (C=N); 1058 (C-S-C). H -NMR (DMSO-d , δ ppm): 3.80 (s, 3H,
OCH ); 7.00 (d, J = 8.25 Hz, 2H, methoxyphenyl-C -H); 7.13 (m, 1H, thiophen-C -H); 7.42
3
3,5
4
4
(t, J = 7.35 Hz, 1H, phenyl-C -H); 7.46-7.67 (m, 4H, phenyl-C3,5-H, thiophen-C3,5-H); 7.82 (d,
J = 8.25 Hz, 2H, methoxyphenyl-C -H); 7.92 (d, J = 7.7 Hz, 2H, phenyl-C -H); 8.50 (s, 1H,
2
,6
2,6
pyrazole- C
5
-H); 8.96 (s,1H, -CH=N); 11.64 (s, 1H, NH, D
2
O-exchangeable). Elemental
analysis Calcd for C H N O S: C, 65.65; H, 4.51; N, 13.92; S, 7.97. Found: C, 65.89; H,
2
2
18
4
2
4
.54; N, 14.15; S, 8.04.
5
.1.4.6 3-(4-Methoxyphenyl)-1-phenyl-N'-(pyridin-4-ylmethylene)-1H-pyrazole-4-
carbohydrazide 4f
0
-1
The product was obtained as white solid, mp 198-200 C, in yield (70%). IR (cm ): 3181
NH); 1664 (C=O); 1590 (C=N). H -NMR (DMSO-d , δ ppm): 3.78 (s, 3H, OCH ); 6.98 (d, J
1
(
6
3
=
8.7 Hz, 2H, methoxyphenyl-C3,5-H); 7.37 (m, 1H, phenyl-C
4
-H); 7.53-7.65 (m, 4H, phenyl-
C2,3,5,6-H); 7.79 (d, J = 8.7 Hz, 2H, methoxyphenyl-C -H); 7.90 (d, J = 8.7 Hz, 2H, pyridine-
2,6
C
5
2,6-H); 8.26 (s, 1H, pyrazole-C -H); 8.63 (d, J = 8.7 Hz, pyridine-C2,6-H); 9.02 (s, 1H, -
1
3
CH=N); 11.99 (s, 1H, NH, D O-exchangeable). C-NMR (DMSO-d , δ ppm): 55.70 (OCH );
2
6
3
1
1
4
14.15 (methoxyphenyl-C3,5 and pyrazole-C ); 119.21 (phenyl-C2,6); 121.49 (pyridine-C2,6);
25.32 (methoxyphenyl-C ); 127.60 (phenyl-C ); 130.06 (methoxy phenyl-C ); 130.29
1
4
2,6
(phenyl-C3,5); 131.26 (pyrazole-C
5
1 1
); 139.50 (phenyl-C ); 144.77 (pyridine-C ); 146.80
1
9

ACCEPTED MANUSCRIPT
(
CH=N); 150.82 (pyridine-C3,5 and pyrazole-C ); 160.07 (methoxy phenyl-C ); 161.00 (C=O).
3
4
19 5 2
Elemental analysis Calcd for C23H N O : C, 69.51; H, 4.82; N, 17.62. Found: C, 69.82; H,
4
.88; N, 17.89.
5
.1.5 General method for preparation of 2-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazole-
-carbonyl]-N-(pheny/allyl)hydrazine- carbothioamides 5a,b
4
A suspension of acid hydrazide 1 (0.3 g, 1 mmol) and isothiocyanate (1 mmol) in absolute
ethanol (10 ml) was heated under reflux for 4 h and then cooled to room temperature. The
precipitate formed was filtered, washed with ethanol, dried and crystallized from ethanol.
5
.1.5.1 2-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbonyl]-N-
phenylhydrazinecarbo- thioamide 5a
-1
The product was obtained as white solid, mp 183-185°C, in yield (85%). IR (cm ): 3411,
328, 3132 (NH); 1674 (C=O); 1601 (C=N); 1531, 1250, 1224 and 966 (N-C=S I, II, III and
3
1
IV bands, respectively). H -NMR (DMSO-d , δ ppm): 3.77 (s, 3H, OCH ); 6.95 (d, J = 8.4
6
3
4
Hz, 2H, methoxyphenyl-C3,5-H); 7.15 (t, J = 7.25 Hz, 1H, NH-phenyl-C -H); 7.31-7.43 (m,
5
H, phenyl-C3,4,5-H and NH-phenyl-C -H); 7.54-7.57 (m, 2H, NH-phenyl-C -H); 7.18 (d, J
3,5 2,6
=
8.4 Hz, 2H, methoxyphenyl-C2,6-H); 7.85 (d, J = 7.65 Hz, 2H, phenyl-C2,6-H); 9.00 (s, 1H,
pyrazole-C -H); 9.70 (s, 1H, NH, D O-exchangeable); 9.75 (s, 1H, S=C-NH, D O-
5
2
2
1
3
exchangeable); 10.25 (s, 1H, O=C-NH, D
2
O-exchangeable). C-NMR (DMSO-d
6
, δ ppm):
5
1
5.70 (OCH ); 113.92 (methoxyphenyl-C ); 114.99 (pyrazole-C ); 119.17 (phenyl-C );
3
3,5
4
2,6
24.97 (NH-phenyl-C2,6); 125.82 (methoxy phenyl-C
1
); 126.63 (phenyl-C
4
); 127.69 (NH-
phenyl-C ); 128.63 (NH-phenyl-C ); 130.42 (methoxyphenyl-C and pyrazole-C ); 130.52
4
3,5
2,6
5
(phenyl-C3,5); 139.46 (NH-phenyl-C
1
); 139.67 (phenyl-C
1
3
); 152.21 (pyrazole-C ); 160.05
(
methoxyphenyl-C ); 163.50 (C=O); 170.02 (C=S). Elemental analysis Calcd for
4
24 21 5 2
C H N O S: C, 64.99; H, 4.77; N, 15.79; S, 7.23. Found: C, 65.23; H, 4.81; N, 15.94; S,
7
.41.
5
.1.5.2 N-Allyl-2-{3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carbonyl}hydrazinecarbothioamide 5b
-1
as white solid, mp 132-134°C, in yield (90%). IR (cm ): 3326, 3220, 3129 (NH); 1668 (C=O);
1
1
616 (C=N); 1541, 1249, 1225 and 959 (N-C=S I, II, III and IV bands, respectively). H -NMR
(
DMSO-d , δ ppm): 3.81 (s, 3H, OCH ); 4.15 (d, J = 5.2 Hz, 2H, CH =CH-CH ); 5.07(d, J =
6
3
2
2-
1
2 2 2 2
0.4 Hz, 1H, Hcis-CH =CH-CH -); 5.15 (d, J = 17.2 Hz, 1H, Htrans-CH =CH-CH -); 5.84-5.90
(
m, 1H, CH =CH-CH -); 6.97 (d, J = 8.4 Hz, 2H, methoxyphenyl-C -H); 7.41 (t, J = 7.6 Hz,
2
2
3,5
1
4
H, phenyl-C -H); 7.57-7.61 (m, 2H, phenyl-C3,5-H); 7.83-7.86 (m, 4H, methoxyphenyl-C2,6-
H and phenyl-C -H); 8.24 (s, 1H, NH, D O-exchangeable); 8.97 (s, 1H, pyrazole-C -H);
2
,6
2
5
9
.42 (s, 1H, S=C-NH, D
2
O-exchangeable); 10.07 (s, 1H, O=C-NH, D
2
O-exchangeable).
Elemental analysis Calcd for C H N O S: C, 61.90; H, 5.19; N, 17.19; S, 7.87. Found: C,
21
21
5
2
6
2.21; H, 5.24; N, 17.38; S, 7.94.
2
0

ACCEPTED MANUSCRIPT
5
.1.6 General method for preparation of
3-(4-Methoxyphenyl)-N'-[4-oxo-3-
(phenyl/allyl) thiazolidin-2-ylidene]-1-phenyl-1H-pyrazole-4-carbohydrazides 6a,b
A mixture of substituted thiosemicarbazide 5a or 5b (1 mmol) and chloroacetyl chloride (0.08
ml, 1 mmol) in dry dioxane (10 ml) was heated under reflux for 8 h. After cooling, the
precipitate formed was washed with ethanol, filtered, washed again with H O, dried and
2
crystallized from appropriate solvents.
5
.1.6.1 3-(4-Methoxyphenyl)-N'-(4-oxo-3-phenylthiazolidin-2-ylidene)-1-phenyl-1H-
pyrazole-4-carbohydrazide 6a
The product was crystallized from chloroform as yellow crystals, mp 156-158 °C, in yield
-
1
(
1
3
55%). IR (cm ): 3439 (NH); 1738 (cyclic amidic C=O); 1683 (C=O); 1605 (C=N); 1246,
1
068 (C-S-C). H -NMR (DMSO-d , δ ppm): 3.79 (d, J = 11.2 Hz, 2H, thiazolidinone-C -H);
6 5
.83 (s, 3H, OCH ); 7.01 (t, J = 7.7 Hz, 1H, thiazolidinone-N-phenyl-C -H); 7.06 (d, J = 8.4
3
4
Hz, 2H, methoxyphenyl-C3,5-H); 7.32-7.43 (m, 3H, phenyl-C
4
-H and
thiazolidinone-N-
phenyl-C -H); 7.55-7.59 (m, 4H, phenyl-C -H and thiazolidinone-N-phenyl -C -H); 7.85
3
,5
3,5
2,6
(d, J = 8.4 Hz, 2H, methoxyphenyl-C3,5-H); 7.99 (d, J = 7.7, 2H, phenyl-C2,6-H); 9.10 (s, 1H,
pyrazole-C -H); 10.55 (s, 1H, NH). Elemental analysis Calcd for C H N O S: C, 64.58; H,
5
26 21
5
3
4
.38; N, 14.48; S, 6.63. Found: C, 64.81; H, 4.43; N, 14.63; S, 6.70
5
.1.6.2 N'-(3-Allyl-4-oxothiazolidin-2-ylidene)-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazide 6b
The product was crystallized from DMF: H O (9:1) as white solid, mp 148-150 °C, in yield
2
-1
1
(
93%). IR (cm ): 3130 (NH); 1698, 1653 (C=O); 1607 (C=N); 1068 (C-S-C). H -NMR
(
DMSO-d , δ ppm): 3.77, 3.97 (2s, 2H, thiazolidinone-C -H, E and Z isomers); 3.80 (s, 3H,
6
5
OCH ); 3.81, 4.24 (2d, J = 5.6 Hz, 2H, CH =CH-CH , E and Z isomers); 4.13, 4.37 (2d, J =
3
2
2
1
2 2
0.4 Hz, 1H, Hcis-CH =CH-CH , E and Z isomers); 4.24, 4.75 (2d, J = 17.2 Hz, 1H, Htrans-
CH =CH-CH -); 5.02-5.08 and 5.64-5.69 (2m, 1H, CH =CH-CH , E and Z isomers); 6.98,
2
2
2
2
7
.01 (2d, J = 8.7 Hz, 2H, methoxyphenyl-C3,5–H, E and Z isomers); 7.37-7.39 (m, 2H, phenyl-
C -H, E and Z isomers); 7.49-7.58 (m, 4H, phenyl-C -H, E and Z isomers); 7.78 (d, J = 8.7
4
3,5
Hz, 2H, methoxyphenyl-C2,6-H); 7.84-7.89 (m, 4H, phenyl-C2,6-H and methoxyphenyl-C2,6
-
H); 7.98 (d, J = 7.8 Hz, 2H, phenyl-C -H); 8.79, 8.88 (2s, 1H, pyrazole-C -H, E and Z
2
,6
5
isomers); 9.09, 9.40 (2s, 1H, NH, D
2
O-exchangeable, E and Z isomers). Elemental analysis
Calcd for C H N O S: C, 61.73; H, 4.73; N, 15.65; S, 7.17. Found: C, 62.02; H, 4.80; N,
2
3
21
5
3
1
5.89; S, 7.23.
5
.2.7. General method for preparation of N'-[3-(Phenyl/allyl)-4-(phenyl/4-
thiazol-2(3H)-ylidene]-3-(4-methoxy phenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazides 7a-d
bromophenyl)
2
1

ACCEPTED MANUSCRIPT
To a suspension of substituted thiosemicarbazide 5a or 5b (1 mmol) in absolute ethanol (10
ml), phenacyl bromide or p-Br-phenacyl bromide (1 mmol) and anhydrous Na acetate (0.08 g,
1
mmol) were added. The reaction mixture was heated under reflux for (8-10) h, then cooled
and the precipitate formed was filtered, washed with H O, dried and crystallized from ethanol.
2
5
.1.6.3 N'-(3,4-Diphenylthiazol-2(3H)-ylidene)-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazide 7a
The product was obtained as white solid, mp 230-232 °C, in yield (82%). IR (cm ): 3177
-1
1
(
NH); 1677 (C=O); 1582 (C=N); 1248, 1063 (C-S-C). H -NMR (DMSO-d , δ ppm): 3.82 (s,
6
3
2
H, OCH
3 4 5
); 6.98-7 (m, 2H, thiazole-N-phenyl-C -H and thiazole-C -H); 7.05 (d, J = 8.4 Hz,
H, methoxyphenyl-C -H); 7.31-7.43 (m, 4H, pyrazole-N-phenyl-C -H, phenyl-C -H and
3
,5
4
4
thiazole-N-phenyl-C3,5-H); 7.54-7.59 (m, 6H, pyrazole-N-phenyl-C3,5-H, thiazole-N-phenyl-
C -H and phenyl-C -H); 7.85 (d, J = 8.4 Hz, 2H, methoxyphenyl-C -H); 7.99 (d, J = 7.8
2
,6
3,5
2,6
Hz, 4H, pyrazole-N-phenyl-C2,6-H and phenyl-C2,6-H); 9.08 (s, 1H, pyrazole-C
5
-H); 10.52 (s,
1
H, NH, D O-exchangeable). Elemental analysis Calcd for C H N O S: C, 70.70; H, 4.64;
2
32 25
5
2
N, 12.88; S, 5.90. Found: C, 70.89; H, 4.68; N, 13.17; S, 5.98.
5
.1.6.4 N'-[4-(4-Bromophenyl)-3-phenylthiazol-2(3H)-ylidene]-3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazole-4-carbohydrazide 7b
-1
The product was obtained as white solid, mp 233-235 °C, in yield (79%). IR (cm ): 3179
1
(NH); 1678 (C=O); 1582 (C=N); 1249, 1063 (C-S-C); 742 (C-Br). H -NMR (400 MHz,
DMSO-d , δ ppm): 3.82 (s, 3H, OCH ); 6.99-7.02 (m, 2H, thiazole-N-phenyl-C -H and
6
3
4
thiazole-C -H); 7.06 (d, J = 8.4 Hz, 2H, methoxyphenyl-C -H); 7.32-7.42 (m, 4H,
5
3,5
bromophenyl-C2,6-H and thiazole-N-C
6
H
5
-C3,5-H); 7.55-7.59 (m, 5H, methoxyphenyl-C2,6-H
and pyrazole-N-phenyl-C3,4,5-H); 7.85 (d, J = 8.36 Hz, 2H, bromo phenyl-C -H); 7.99 (d, J =
3,5
7
.8 Hz, 4H, pyrazole-N-phenyl-C2,6-H and thiazole-N-phenyl-C2,6-H); 9.09 (s, 1H, pyrazole-
1
3
C -H); 10.55 (s, 1H, NH, D O-exchangeable). C-NMR (100 MHz, DMSO-d , δ ppm): 55.68
5
2
6
(
OCH
3
); 106.15 (thiazole-C
5
4
); 114.3 (methoxyphenyl-C3,5); 114.28 (pyrazole-C ); 117.5 (Br-
phenyl-C ); 117.53 (thiazole-N-phenyl-C ); 119.36 (phenyl-C ); 119.38 (thiazole-N-phenyl-
4
2,6
2,6
C
4
); 122.28 (methoxyphenyl-C
1
); 124.43 (phenyl-C
4
5
); 127.67 (pyrazole-C ); 129.51 (methoxy
phenyl-C2,6 and Br-Phenyl-C ); 130.16 (phenyl-C , thiazole-N-phenyl-C , Br-phenyl-
2,6
3,5
3,5
C
1,3,5); 130.78 (phenyl-C
1
); 139.17 (thiazole-N-phenyl-C
1
4
); 139.31 (thiazole-C ); 150.58
(
(
(
(
thiazole-C ); 153.42 (pyrazole-C ); 160.07 (methoxyphenyl-C ); 160.23 (C=O). EIMS m/z
2 3 4
+
·
+·
% relative abundance): 623 (5) (M +2), 621 (5) (M ), 335 (7), 323 (6), 319 (5), 271 (6), 242
7), 225 (25), 223 (9), 204 (6), 175 (12), 138 (12), 135 (100), 109 (53), 104 (21), 97 (17), 54
24). Elemental analysis Calcd for C H BrN O S: C, 61.74; H, 3.89; N, 11.25; S, 5.15.
3
2
24
5
2
Found: C, 61.89; H, 3.87; N, 11.48; S, 5.23.
2
2

ACCEPTED MANUSCRIPT
5
.1.6.5 N'-(3-Allyl-4-phenylthiazol-2(3H)-ylidene)-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbohydrazide 7c
-
The product was obtained as yellowish brown solid, mp 119-121 °C, in yield (75%). IR (cm
1
1
)
: 3440 (NH); 1673 (C=O); 1598 (C=N); 1248, 1056 (C-S-C). H -NMR (DMSO-d
6
, δ ppm):
3
.82 (s, 3H, OCH ); 3.85 (d, J = 5.6 Hz, 2H, CH =CH-CH ); 5.13 (d, J = 10.4 Hz, 1H, H -
3
2
2
cis
CH
2
=CH-CH
2
); 5.24 (d, J = 17.2 Hz, 1H, Htrans-CH
2
=CH-CH
2
); 5.85-5.95 (m, 1H, CH
2
=CH-
CH ); 7.03 (d, J = 8.6 Hz, 2H, methoxy phenyl-C -H); 7.39 (t, J = 7.6 Hz, 2H, N-phenyl-C -
2
3,5
4
H and phenyl-C
4
-H); 7.54-7.72 (m, 5H, N-phenyl-C3,5-H, phenyl-C3,5-H and thiazole-C
5
-H);)
7
.84-7.87 (m, 4H, methoxy phenyl-C -H and phenyl-C -H); 7.98 (d, J = 7.6 Hz, 2H, N-
2,6 2,6
1
3
phenyl-C2,6-H); 9.03 (s, 1H, pyrazole-C
5 6 2
-H). C-NMR (DMSO-d , δ ppm): 45.36 (CH =CH-
CH ); 55.69 (OCH ); 106.64 (thiazole-C ); 114.17 (methoxyphenyl-C and pyrazole-C4);
2
3
5
3,5
1
16.39 (CH =CH-CH ); 119.28 (N-phenyl-C ); 124.54 (methoxyphenyl-C ); 124.54 (2
2 2 2,6 1
phenyl-C ); 129.08 (phenyl-C ); 129.91 (pyrazole-C ); 130.12 (methoxyphenyl-C and
4
1
5
2,6
phenyl-C ); 130.38 (2 phenyl-C ); 135.17 (CH =CH-CH and thiazole-C ); 139.34 (N-
2
,6
3,5
2
2
4
phenyl-C ); 150.29 (thiazole-C ); 153.18 (pyrazole-C ); 160.14 (methoxyphenyl-C ); 163.61
1
2
3
4
(
C=O). Elemental analysis Calcd for C H N O S: C, 68.62; H, 4.96; N, 13.80; S, 6.32.
29 25 5 2
Found: C, 68.90; H, 4.99; N, 14.07; S, 6.41.
5
.1.6.6 N'-[3-Allyl-4-(4-bromophenyl)thiazol-2(3H)-ylidene]-3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazole-4-carbohydrazide 7d
-1
The product was obtained as brown solid, mp 128-130 °C, in yield (72%). IR (cm ): 3194
1
(NH); 1673 (C=O); 1595 (C=N); 1247, 1062 (C-S-C); 755 (C-Br). H -NMR (DMSO-d
6
, δ
ppm): 3.82 (s, 3H, OCH ); 3.85 (d, J = 5.6 Hz, 2H, CH =CH-CH ); 5.13 (d, J = 10.4 Hz, 1H,
3
2
2
H -CH =CH-CH ); 5.24 (d, J = 17.2 Hz, 1H, H -CH =CH-CH ); 5.88-5.95 (m, 1H,
cis
2
2
trans
2
2
CH =CH-CH ); 7.03 (d, J = 8.6 Hz, 2H, methoxyphenyl-C -H); 7.39 (t, J =7.6 Hz, 1H,
2
2
3,5
phenyl-C -H); 7.54-7.77 (m, 5H, bromophenyl-C -H, phenyl-C -H and thiazole-C -H);)
4
2,6
3,5
5
7
2
.85 (d, J = 8.6 Hz, 4H, methoxyphenyl-C -H and bromo phenyl-C -H); 7.98 (d, J = 7.7 Hz,
2,6 3,5
H, phenyl-C -H); 9.03 (s,1H, pyrazole-C -H). Elemental analysis Calcd for C H BrN O S:
2
,6
5
29 24
5
2
C, 59.39; H, 4.12; N, 11.94; S, 5.47. Found: C, 59.64; H, 4.18; N, 12.17; S, 5.60.
5
.2.8. General method for preparation of 3-[3-(4-Methoxyphenyl)-1-phenyl-
1
H-pyrazol-4-yl]-4-(phenyl/allyl)-1H-1,2,4-triazole-5(4H)-thiones 8a,b
A suspension of substituted thiosemicarbazide 5a or 5b (1 mmol) in ethanol (10 ml) was
dissolved in aquous KOH (3 ml, 4N). The reaction mixture was gently heated under reflux for
3
h, then it was concentrated and cooled to room temperature. The pH of the reaction mixture
was adjusted to 5-6 with dilute HCl and the solid formed was filtered, washed with H O, dried
2
and crystallized from appropriate solvents.
2
3

ACCEPTED MANUSCRIPT
5
.1.6.7 3-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-4-phenyl-1H-1,2,4-triazole-
5
(4H)-thione 8a
The product was crystallized from DMF:H O (9:1) as white solid, mp 253-255 °C, in yield
2
-1
(
94%). IR (cm ): 3421 (NH); 1598 (C=N); 1501, 1247, 1220 and 957 (N-C=S I, II, III and IV
bands, respectively). H -NMR (DMSO-d , δ ppm): 3.79 (s, 3H, OCH ); 6.95 (d, J = 8.4 Hz,
1
6
3
2
H, methoxyphenyl-C3,5-H); 7.07-7.15 (m, 2H, triazole-N-phenyl-C2,6-H); 7.20-7.38 (m, 6H,
pyrazole-N-phenyl-C3,4,5-H and triazole-N-phenyl-C3,4,5-H); 7.49-7.54 (m, 2H, pyrazole-N-
phenyl-C2,6-H); 7.77 (d, J = 8.4 Hz, 2H, methoxyphenyl-C2,6-H); 8.69 (s,1H, pyrazole-C -H);
5
1
4
4.16 (s,1H, SH, D O-exchangeable). Elemental analysis Calcd for C H N OS: C, 67.74; H,
.50; N, 16.46; S, 7.54. Found: C, 67.98; H, 4.47; N, 16.72; S, 7.69.
2
24 19
5
5
.1.6.8 4-Allyl-3-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-1H-1,2,4-triazole-
(4H)-thione 8b
The product was crystallized from ethanol as off-white solid, mp 186-188 °C, in yield (95%).
5
-1
IR (cm ): 3421 (NH); 1608 (C=N); 1503, 1249, 1087 & 957 (N-C=S I, II, III & IV bands,
respectively). H -NMR (DMSO-d , δ ppm): 3.78 (s, 3H, OCH ); 4.49 (d, J = 5.2 Hz, 2H,
6 3
1
CH =CH-CH ); 4.79 (d, J = 17.2 Hz, 1H, H -CH =CH-CH ); 4.99 (d, J = 10.4 Hz, 1H, H -
2
2
trans
2
2
cis
CH
2
=CH-CH
2
); 5.62-5.69 (m, 1H, CH
2
2
=CH-CH ); 7.01 (d, J = 8.6 Hz, 2H, methoxy phenyl-
C -H); 7.38 (t, J = 7.6 Hz, 1H, phenyl-C -H) 7.49 (d, J = 7.8 Hz, 2H, phenyl-C -H); 7.55-
3
,5
4
3,5
7
.59 (m, 2H, phenyl-C2,6-H) 7.93 (d, J = 8.6 Hz, 2H, methoxyphenyl-C2,6-H); 8.9 (s,1H,
1
3
pyrazole-C -H); 14.02 (s, 1H, SH, D O-exchangeable). C-NMR (DMSO-d , δ ppm): 45.88
5
2
6
(
CH
2
=CH-CH
2
); 55.66 (OCH
3
); 106.21 (pyrazole-C
4
); 114.66 (methoxyphenyl-C3,5); 118.11
(
CH =CH-CH ); 119.23 (phenyl-C ); 124.38 (methoxyphenyl-C ); 127.62 (2phenyl-C );
2 2 2,6 1 4
1
28.90 (methoxyphenyl-C ); 130.17 (phenyl-C ); 131.64 (pyrazole-C ); 131.72 (CH =CH-
2,6 3,5 5 2
CH ); 139.39 (phenyl-C ); 145.69 (triazole-C ); 151.06 (pyrazole-C ); 160.14 (methoxy
2
1
2
3
phenyl-C ); 167.53 (triazole-C ). Elemental analysis Calcd for C H N OS: C, 64.76; H, 4.92;
4
5
21 19
5
N, 17.98; S, 8.23. Found: C, 64.98; H, 4.96; N, 18.23; S, 8.30.
5
.2.9. General method for preparation of 5-[3-(4-Methoxyphenyl)-1-phenyl-
1
H-pyrazol-4-yl]-N-(phenyl/allyl)-1,3,4-thiadiazol-2-amines 9a,b
The substituted thiosemicarbazide 5a or 5b (1 mmol) was added gradually with stirring on an
ice-cold conc H SO (5 ml) and the reaction mixture was further stirred for 4 h in ice bath. The
2
4
2
rection mixture was poured onto ice-H O mixture. The pH of the solution was adjusted to 7-8
with NH OH solution and the resulting solid was filtered, washed with H O, dried and
4
2
crystallized from appropriate solvents.
2
4

ACCEPTED MANUSCRIPT
5
.1.6.9 2-{3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl}-N-phenyl-1,3,4-thiadiazol-5-
amine 9a
The product was crystallized from DMF:H O (9:1) as greenish yellow solid, mp 192-194 °C,
2
-1
1
in yield (95%). IR (cm ): 3178 (NH); 1612 (C=N); 1202, 1072 (C-S-C). H -NMR (DMSO-
d , δ ppm): 3.83 (s, 3H, OCH ); 5.67 (s, 1H, NH, D O-exchangeable); 7.09 (d, J = 8.5 Hz, 2H,
6
3
2
methoxyphenyl-C3,5-H); 7.11-7.24 (m, 1H, NH-phenyl-C
C -H and phenyl-C -H); 7.51-7.70 (m, 6H, phenyl-C
4
-H); 7.31-7.39 (m, 3H, NH-phenyl -
-H and NH-phenyl-C -H); 7.97 (d,
3
,5
4
2,3,5,6
2,6
1
3
5 6
J = 8.5 Hz, 2H, methoxyphenyl-C2,6-H); 9.06 (s,1H, pyrazole-C -H). C-NMR (DMSO-d , δ
ppm): 56.22 (OCH ); 112.25 (pyrazole-C ); 112.66 (NH-phenyl-C ); 116.90 (methoxy
3
4
2,6
phenyl-C3,5); 119.15 (phenyl-C2,6); 123.17 (NH-phenyl-C
4
); 127.01 (methoxyphenyl-C2,6);
1
27.53 (methoxyphenyl-C ); 129.37 (phenyl-C ); 130.14 (phenyl-C ); 132.08 (NH-phenyl-
1 4 3,5
C ); 134.90 (pyrazole-C ); 139.30 (NH-phenyl-C ); 141.16 (phenyl-C ); 150.38 (pyrazole-
3
,5
5
1
1
C ); 150.50 (thiadiazole-C ); 157.17 (methoxy phenyl-C ); 164.06 (thiadiazole-C ). Elemental
3
2
4
5
analysis Calcd for C H N OS: C, 67.74; H, 4.50; N, 16.46; S, 7.54. Found: C, 67.88; H,
2
4
19
5
4
.57; N, 16.80; S, 7.62.
5
.1.6.10 N-Allyl-5-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-1,3,4-thiadiazol-2-
amine 9b
-1
ethanol as pale yellow solid, mp 140-142 °C, in yield (92%). IR (cm ): 3201 (NH); 1600
1
6
(C=N); 1222 & 1056 (C-S-C). H -NMR (DMSO-d , δ ppm): 3.26 (d, J = 10.8 Hz, 1H, Hcis-
CH =CH-CH -); 3.40 (d, J = 16.7 Hz, 1H, H -CH =CH-CH ); 3.82 (s, 3H, OCH ); 3.87 (d,
2
2
trans
2
2
3
J = 5.9 Hz, 2H, CH
2
=CH-CH
2
); 4.59 (m, 1H, CH
2
2
=CH-CH , masked by DMSO); 7.08 (d, J =
8
.4 Hz, 2H, methoxyphenyl-C -H); 7.39 (t, J = 7.6, 1H, phenyl-C -H); 7.52-7.56 (m, 2H,
3,5 4
phenyl-C -H); 7.67 (d, J = 8.4 Hz, 2H, methoxyphenyl-C -H); 7.96 (d, J = 7.8 Hz, 2H,
3
,5
2,6
phenyl-C -H); 9.15 (s, 1H, pyrazole-C -H); 9.46 (s, 1H, NH, D O-exchangeable). Elemental
2
,6
5
2
analysis Calcd for C H N OS: C, 64.76; H, 4.92; N, 17.98; S, 8.23. Found: C, 65.01; H,
2
1
19
5
4
.97; N, 18.19; S, 8.31.
5
.1.6.11 General method for preparation of 5-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-
-yl]-N-(phenyl/allyl)-1,3,4-oxadiazol-2-amines 10a,b
4
A mixture of substituted thiosemicarbazide 5a or 5b (1 mmol) and mercuric oxide (2.16 g, 10
mmol) in dioxane (20 ml) was heated under reflux for 10 h, then filtered while hot and the
precipitate washed with hot dioxane. The filtrate was concentrated, cooled and the precipitate
2
formed was filtered, dried and crystallized from dioxane: H O (9:1).
5
.1.6.12 2-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-N-phenyl-1,3,4-oxadiazol-5-
amine 10a
The product was obtained as brownish yellow solid, mp 160-162 °C, in yield (75%). IR (cm
-
1
1
)
: 3396 (NH); 1675, 1580 (C=N). H -NMR (DMSO-d , δ ppm): 3.82 (s, 3H, OCH ); 7 (t, J =
6
3
2
5

ACCEPTED MANUSCRIPT
7
.36 Hz, 1H, NH-phenyl-C -H); 7.05 (d, J = 8.8 Hz, 2H, methoxyphenyl-C -H); 7.34-7.40
4
3,5
4
(m, 3H, phenyl-C -H and NH-phenyl-C3,5-H); 7.53-7.55 (m, 2H, phenyl-C3,5-H); 7.64 (d, J =
7
7
.6 Hz, 1H, NH-phenyl-C -H); 7.69 (d, J = 8.8 Hz, 2H, methoxyphenyl-C -H); 7.98 (d, J =
2,6 2,6
.8 Hz, 2H, phenyl-C2,6-H); 9.09 (s, 1H, pyrazole-C
5
2
-H); 10.39 (s, 1H, NH, D O-
exchangeable). Elemental analysis Calcd for C H N O : C, 70.40; H, 4.68; N, 17.10. Found:
2
4
19
5
2
C, 70.58; H, 4.76; N, 17.34.
5
.1.6.13 N-Allyl-2-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-1,3,4-oxadiazol-5-
amine 10b
-1
The product was obtained as white solid, mp 138-140 °C, in yield (80%). IR (cm ): 3420
1
(NH); 1673, 1599 (C=N). H -NMR (DMSO-d , δ ppm): 33.82 (s, 3H, OCH ); 3.86 (d, J = 5.6
6
3
Hz, 2H, CH
2
=CH-CH
2
); 5.13 (d, J = 10.4 Hz, 1H, Hcis-CH
2
=CH-CH
2
); 5.24 (d, J = 17.2 Hz,
1
H, Htrans-CH =CH-CH ); 5.88-5.98 (m, 1H, CH =CH-CH ); 7.03 (d, J = 8.6 Hz, 2H, methoxy
2 2 2 2
phenyl-C3,5-H); 7.39 (t, J =7.4 Hz, 1H, phenyl-C
4
-H); 7.53-7.58 (m, 2H, phenyl-C3,5-H); 7.85
(
d, J = 8.6 Hz, 2H, methoxyphenyl-C -H); 7.98 (d, J = 7.7 Hz, 2H, phenyl-C -H); 9.03
3,5 2,6
1
3
(s,1H, pyrazole-C
5
-H). C-NMR (DMSO-d
6
, δ ppm): 45.36 (CH
2
=CH-CH
2
3
); 55.68 (OCH );
1
06.64 (pyrazole-C ); 114.18 (methoxyphenyl-C ); 116.38 (CH =CH-CH ); 119.25 (phenyl-
4
3,5
2
2
C
2,6); 124.54 (methoxyphenyl-C
1
4
); 127.55 (phenyl-C ); 130.12 (methoxyphenyl-C2,6 and
phenyl-C ); 130.37 (pyrazole-C ); 135.17 (CH =CH-CH ); 139.34 (phenyl-C ); 150.28
3
,5
5
2
2
1
(pyrazole-C
3
); 153.18 (oxadiazole-C
5
); 160.15 (methoxy phenyl-C
4
2
); 163.61 (oxadiazole-C ).
Elemental analysis Calcd for C H N O : C, 67.55; H, 5.13; N, 18.76. Found: C, 67.79; H,
2
1
19
5
2
5
.19; N, 18.97.
.2 In silico experiments
.2.1 Molecular docking
5
5
The coordinates for wildtype (PDB code:1j3i) and quadruple mutant (N51I, C59R, S108N and
I164L) pf DHFR-TS (PDB code:1j3k) crystal structures were retrieved from the protein data
bank and handled consequently with MOE [59]. Redundant chains, non-essential ions, water
molecules and ligands were discarded. Only exception was made for one structural water
molecule that mediates salt-bridge for the co-crystal ligand for both crystal structures. The
preparation procedure was conducted by the “Quick Prep” module at default settings. The
PDB files were converted to PDBQT files by employing a python script
(prepare_receptor4.py) provided by the MGLTools package (version 1.5.4) [60] for
AutoDock Vina (version 1.1.2) [61] docking experiments.
The most active compounds (4b, 4c, 7a and 7d) were built and prepared by MOE. ‘Molecule
wash’ function was used to generate meaningful protonation states by deprotonating strong
acids and protonating strong bases (if required). Energy minimization of all molecules was
performed using the Amber: 10EHT force field at a gradient of 0.01 RMSD. Conformational
2
6

ACCEPTED MANUSCRIPT
search was conducted to sample maximum number of minimized conformations including
different stereoisomers and ring conformations (if necessary) using “Stochastic” method, with
option “Allow unconstrained double bond rotation” enabled to sample different geometrical
isomers of the double bond-containing compounds. All other options were set at default levels.
The resulting conformers were saved as SD file for the docking experiments. The SD files
were converted and split into PDB files by MOE, which were further converted into PDBQT
files by a MGLTools (version 1.5.4) python script (prepare_ligand4.py) for AutoDock Vina
docking experiments.
AutoDock Vina (version 1.1.2) was used for docking experiments of the most active
compounds against both wildtype and quadruple mutant (N51I, C59R, S108N and I164L) pf
DHFR-TS structures. We employed default docking parameters and the size of the docking
grid was 20 Å × 20 Å × 20 Å, with a grid spacing of 1 Å. By default, the docking was
terminated when the maximum energy difference between the best scored pose and the worst
one was 3 kcal/ mol. Figures 3-5 were rendered using Pymol v2.2 [62]. This docking setup
was validated by successful pose-retrieval docking experiment for the co-crystal ligand on
both PDB crystal structures.
5
.2.2 In silico prediction of physicochemical properties, pharmacokinetic profiles, drug
likeness scores and toxicity profiles
In the present work, the biologically active compounds were subjected to prediction of the
physical and molecular properties using various tools, such as PreADMET, Molinspiration,
Mol-Soft and Osiris softwares. Molinspiration chemoinformatic server was used to calculate
Lipinski's violation, topological polar surface area (PSA) and percentage of oral absorption
(%ABS) [63]. While, PreADMET calculator was used to predict the pharmacokinetic
properties, such as absorption, distribution, metabolism, excretion and toxicity [64]. Drug
likeness score and solubility parameters were calculated using Mol-Soft software [65], while
toxicity risks like mutagenecity, tumorigenecity, irritation and reproductive side effects were
calculated using Osiris property explorer [66].
5
.3 Biological Screening
5
.3.1 In vivo antimalarial activity testing against P. berghei
A 4-day suppressive test using P. berghei ANKA strain infected mice was used for in vivo
antimalarial screening of the newly synthesized compounds as described by Fidock et al [54].
It is the most widely used preliminary test as the efficacy of a compound is calculated in the
form of percentage suppression in blood parasitemia level. So efficacy is assessed by a
comparison of blood parasitemia levels in treated and untreated mice [67].
7
On day 0, the test mice were injected intravenously with 0.2 ml of 2 x 10 parasitized
2
7

ACCEPTED MANUSCRIPT
erythrocytes infected with P. berghei ANKA strain. These parasitized erythrocytes were
obtained from the blood of a donor mouse with approximately 20-30 % parasite and the blood
was diluted with normal saline. After 2 h injection, the infected mice were weighed and
randomly divided into 47 groups of five mice per cage. Treatment groups were the groups
from 1-45 (45 cages) and they received the synthesized compounds orally, each at a dose of
0
.048 mmol/kg/day [67]. The negative control group (group 46) received the vehicle, which
consisted of 7% Tween and 3% ethanol in distilled water. The positive control group (group
7) received the reference drug, chloroquine phosphate, at a dose of 0.048 mmol/kg/day (25
4
mg/kg/day). On days 1-3, every 24 h interval, the treatment groups received the same single
dose of the synthesized compounds orally as in day 0. On day 4, after 96 h post infection,
blood smears from all test animals was prepared using Giemsa stain and level of parasitemia
was determined microscopically by counting 5 fields of approximately 100 erythrocytes per
field. The difference between the mean value for the negative control group (taken as 100%)
and those of the experimental groups was calculated and expressed as percent suppression or
activity. The survival time for each test mouse was recorded and the mean survival time was
calculated in comparison with that of the negative group. On the other hand, the survival time
for chloroquine treated mice was not recorded since they were completely cured from the
parasite [68]. Percentage parasitemia and percentage suppression were calculated using the
following equations:
Number of infected RBC
%
parasitemia =
x 100
Number of total RBC
Parasitemia in negative control-parasitemia in treatment group
Parasitemia in negative control
%Suppression =
x 100
5
.3.2 In vitro antimalarial activity testing against P. falciparum
In vitro antimalarial activity was evaluated for the most active compounds which showed
reasonable activity in in vivo screening. We evaluated their antiplasmodial activity against
chloroquine resistant P. falciparum strain (RKL9) which was maintained in a continuous
culture using the standard method described by Trager and Jensen [55]. The
assay was carried out in 96-well microtitre flat-bottomed tissue culture plates incubated at
o
3
7 C for 24 h in the presence of two-fold serial dilutions of test compounds and chloroquine
diphosphate, which were examined for their effect on schizont maturation. The initial culture
2
8