Targeting malaria and leishmaniasis: Synthesis and pharmacological evaluation of novel pyrazole-1,3,4-oxadiazole hybrids. Part II

Article abstract of DOI:10.1016/j.bioorg.2019.102986

In continuance with earlier reported work, an extension has been carried out by the same research group. Mulling over the ongoing condition of resistance to existing antimalarial agents, we had reported synthesis and antimalarial activity of certain pyrazole-1,3,4-oxadiazole hybrid compounds. Bearing previous results in mind, our research group ideated to design and synthesize some more derivatives with varied substitutions of acetophenone and hydrazide. Following this, derivatives 5a–r were synthesized and tested for antimalarial efficacy by schizont maturation inhibition assay. Further, depending on the literature support and results of our previous series, certain potent compounds (5f, 5n and 5r) were subjected to Falcipain-2 inhibitory assay. Results obtained for these particular compounds further strengthened our hypothesis. Here, in this series, compound 5f having unsubstituted acetophenone part and a furan moiety linked to oxadiazole ring emerged as the most potent compound and results were found to be comparable to that of the most potent compound (indole bearing) of previous series. Additionally, depending on the available literature, compounds (5a–r) were tested for their antileishmanial potential. Compounds 5a, 5c and 5r demonstrated dose-dependent killing of the promastigotes. Their IC₅₀ values were found to be 33.3 ± 1.68, 40.1 ± 1.0 and 19.0 ± 1.47 μg/mL respectively. These compounds (5a, 5c and 5r) also had effects on amastigote infectivity with IC₅₀ of 44.2 ± 2.72, 66.8 ± 2.05 and 73.1 ± 1.69 μg/mL respectively. Further target validation was done using molecular docking studies. Acute oral toxicity studies for most active compounds were also performed.

Full text of DOI:10.1016/j.bioorg.2019.102986

Accepted Manuscript
Targeting Malaria and Leishmaniasis: Synthesis and Pharmacological Evalua-
tion of Novel Pyrazole-1,3,4-Oxadiazole Hybrids. Part II
Garima Verma, Mohemmed Faraz Khan, Lalit Mohan Nainwal, Mohd Ishaq,
Mymoona Akhter, Afroz Bakht, Tariq Anwer, Farhat Afrin, Mohammad
Islamuddin, Ibraheem Husain, Mohammad Mumtaz Alam, Mohammad
Shaquiquzzaman
PII:
S0045-2068(18)31531-1
https://doi.org/10.1016/j.bioorg.2019.102986
102986
DOI:
Article Number:
Reference:
YBIOO 102986
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 December 2018
6 April 2019
16 May 2019
Please cite this article as: G. Verma, M. Faraz Khan, L. Mohan Nainwal, M. Ishaq, M. Akhter, A. Bakht, T. Anwer,
F. Afrin, M. Islamuddin, I. Husain, M. Mumtaz Alam, M. Shaquiquzzaman, Targeting Malaria and Leishmaniasis:
Synthesis and Pharmacological Evaluation of Novel Pyrazole-1,3,4-Oxadiazole Hybrids. Part II, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102986
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Targeting Malaria and Leishmaniasis: Synthesis and Pharmacological Evaluation of
Novel Pyrazole-1,3,4-Oxadiazole Hybrids. Part II
Garima Verma1, Mohemmed Faraz Khan1, Lalit Mohan Nainwal1, Mohd Ishaq2, Mymoona
Akhter1, Afroz Bakht3, Tariq Anwer4, Farhat Afrin5, Mohammad Islamuddin5, Ibraheem
Husain5, Mohammad Mumtaz Alam1*, Mohammad Shaquiquzzaman1*
1Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and
Research, Jamia Hamdard, New Delhi-110062
2Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia
Hamdard, New Delhi-110062.
3Department of Chemistry, College of Science and Humanities, Prince Sattam bin Abdulaziz
University, P.O. Box-173, Al-Kharj, Saudi Arabia.
4Department of Pharmacology, College of Pharmacy, Jazan University, P.O. Box 114, Gizan,
Saudi Arabia.
5Molecular Virology Lab, Centre for Interdisciplinary Research in Basic Sciences, Jamia
Millia Islamia University, New Delhi.
*Corresponding Authors
Dr. Mohammad Shaquiquzzaman
Department of Pharmaceutical Chemistry
School of Pharmaceutical Education & Research
Jamia Hamdard
New Delhi-110062
Email: shaqiq@gmail.com
Phone: +91-9990663405
Dr. Mohammad Mumtaz Alam
Department of Pharmaceutical Chemistry
School of Pharmaceutical Education & Research
Jamia Hamdard
New Delhi-110062
Email: drmmalam@gmail.com
Phone: +91-7004101839
Keywords: Pyrazole-4-Acrylic Acid,1,3,4-Oxadiazole, Antimalarial, Antileishmanial,
Falcipain-2.

Abstract
In continuance with earlier reported work, an extension has been carried out by the same
research group. Mulling over the ongoing condition of resistance to existing antimalarial
agents, we had reported synthesis and antimalarial activity of certain pyrazole-1,3,4-
oxadiazole hybrid compounds. Bearing previous results in mind, our research group ideated
to design and synthesize some more derivatives with varied substitutions of acetophenone
and hydrazide. Following this, derivatives 5a-r were synthesized and tested for antimalarial
efficacy by schizont maturation inhibition assay. Further, depending on the literature support
and results of our previous series, certain potent compounds (5f, 5n and 5r) were subjected to
Falcipain-2 inhibitory assay. Results obtained for these particular compounds further
strengthened our hypothesis. Here, in this series, compound 5f having unsubstituted
acetophenone part and a furan moiety linked to oxadiazole ring emerged as the most potent
compound and results were found to be comparable to that of the most potent compound
(indole bearing) of previous series. Additionally, depending on the available literature,
compounds (5a-r) were tested for their antileishmanial potential. Compounds 5a, 5c and 5r
demonstrated dose-dependent killing of the promastigotes. Their IC50 values were found to be
33.3±1.68, 40.1±1.0 and 19.0±1.47μg/mL respectively. These compounds (5a, 5c and 5r)
also had effects on amastigote infectivity with IC50 of 44.2±2.72, 66.8±2.05 and 73.1±1.69
μg/mL respectively. Further target validation was done using molecular docking studies.
Acute oral toxicity studies for most active compounds were also performed.

1. Introduction
Malaria is considered as the most threatening parasitic infection in humans and has been a
matter of concern for centuries [1]. As per the World Malaria Report, 2016 by WHO, around
429000 deaths occurred due to malaria globally. Majority of the deaths were reported in
WHO African region, particularly in the Sub-Saharan Africa. Plasmodium falciparum (P.
falciparum) was found to be responsible for majority of the deaths. 303000 deaths (equivalent
to 70% of the global total are estimated to have occurred in children under five years of age
[2].
Considering such an alarming condition of spread of these menaces and the datum of
emergence of resistance to the available antimalarial agents, our research group had
previously envisioned to develop novel antimalarials. So, a series of pyrazole-1,3,4-
oxadiazole based compounds targeting malaria was previously synthesized and reported [3].
Amongst compounds of the preceding series, compound 6v (Figure 1) was the most active
one (against both chloroquine sensitive and resistant strains of P. falciparum) having
unsubstituted acetophenone part connected to pyrazole and a heteroaromatic ring (indole)
linked to oxadiazole. From results, it was inferred that the presence of unsubstituted
acetophenone part along with a heteroaromatic ring attached to oxadiazole moiety greatly
favored antimalarial effects.
Heteroaromatic Ring
(Indole)
H
N
O
N
N
N
N
Unsubstituted
Acetophenone
Part
Figure 1:
Most Active Compound of Previously reported Series
From results of the previous study, it was found that amide derivatives elicited promising
anticancer effects whereas oxadiazole derivatives emerged as potent antimalarial agents.

On the other hand, leishmaniasis continues to prevail in the list of neglected tropical diseases.
This malevolent infectious disease is known to be caused by intramacrophage protozoa of the
genus Leishmania. Amongst the various known forms of leishmaniasis, visceral, cutaneous
and mucocutaneous, visceral form is the most fatal one. Leishmania donovani (L. donovani)
is the causative organism behind this form of disease while this gets transmitted by the bite of
phlebotomine sand flies [4,5]. Countries like India, Brazil, South Sudan, Ethiopia and
Bangladesh contribute to the 90% global burden of this disease. Despite availability of a
number of treatment options, there is a pressing need for the development of novel, effective,
cost effective and better patient compliant treatment options to curb this menace. Toxicity,
long duration of treatment, high cost of treatment and parasitic resistance are the problems
associated with the current therapeutic regimen [6].
Literature gives strong evidences regarding antileishmanial activity of pyrazole based
compounds (Figure 2). Compound I synthesized by Santos and group demonstrated very
promising effects against Leishmania amazonensis (L. amazonensis) with an IC50 value of
15.5 ± 6.8 µM [7]. Faria et al. determined antileishmanial potential of pyrazole based
carbonitriles and its derivatives and reported compound II as the most potent candidate
against Leishmania braziliensis (L. braziliensis) with an IC50 value of 15±0.14 µM [8].
Certain pyrazole derivatives were developed and screened for antileishmanial activity by
Taha et al. Amongst those synthesized compounds, compound III was highly active with
IC50 value of 0.0112 µg/mL when tested against L. donovani [9]. Amongst a series of
pyrazole based compounds prepared and evaluated by Bekhit and co-workers, compound IV
exhibited maximum activity with IC50 value of 0.13 ± 0.02 µg/mL against Leishmania
aethiopica (L. aethiopica) [10].

O
N+
O-
N
N
N
N
N
Br
NH
NH
N
O
N
S
N
N
N
NH2
N
N
N
N
N
O
N
N
O
Cl
Cl
Cl
IV
I
II
III
Pyrazole based Antileishmanial Compounds
Figure 2:
Antileishmanial activity of oxadiazole based compounds is also well documented in the
literature. Phenyl linked oxadiazole-phenylhydrazone hybrids synthesized by Taha et al. were
assessed for antileishmanial potential. The group reported compound V as the most potential
candidate with an IC50 value of 0.95±0.01 µM [11]. In another series of quinolinyl-
oxadiazole hybrids reported by Taha and group, compound VI emerged as the most potent
agent with IC50 value of 0.10±0.001 µM [12] (Figure 3).
OH
OH
O
H
H
O
N
N
N
N
N
O
H
S
F
F
H
O
N
O
N
F
N
VI
1,3,4-Oxadiazole based Antileishmanial Agents
N
N
V
Figure 3:
Incited by promising results of the previous series, we synthesized another series of
compounds (Scheme I) bearing pyrazole and 1,3,4-oxadiazole with differently substituted
acetophenones and hydrazides (5a-r). These derivatives were tested for evaluated for both
antimalarial and antileishmanial efficacy. Previously, promising antimalarial results were
obtained for 1,3,4-oxadiazole derivatives. Since, leishmaniasis is also one of the protozoal
diseases, it was envisaged to assess antileishmanial efficacy for these compounds. Available
literature in this context also gave substantial support.

2. Results & Discussion
2.1 Chemistry
Desired compounds were obtained via different reaction steps mentioned in Scheme I.
Substituted acetophenone derivatives reacted with phenylhydrazine leading to the formation
of corresponding hydrazone derivatives involving addition reaction. Further, Vilsmeir-Haack
reaction was used involving formylation of the aromatic compounds using N-N-dimethyl
formamide (DMF) as an acylating agent in the presence of activating agent (POCl3) [13].
After this, acrylic acid derivatives were synthesized by treatment of pyrazole carbaldehydes
with malonic acid in the presence of pyridine and piperidine. Mechanism for formation of the
acrylic acid derivatives involved Knoevenagel condensation followed by Doebner
modification [14, 15].
Finally, the prepared acrylic acid derivatives reacted with substituted benzohydrazide
derivatives and lead to the formation of desired 1,3,4-oxadiazole derivatives in the presence
of POCl3. Synthesized derivatives have been listed in Table IV.
2.1.1 Spectral Characterization
Formation of the desired 1,3,4-oxadiazole derivatives was supported by spectral and
analytical data. For α,β-unsaturated carboxylic acid derivatives (2a-c), characteristic acidic
peak was observed in the region of 12.31 to 12.33 ppm in 1HNMR spectra.
Trans conformation of acrylic protons (CH=CH) was observed using 1H NMR spectra (2a-c).
J value was found to be in the range of 15.6-16.4 Hz, thereby confirming existence of trans
conformation. α and β protons were observed in the range of 6.39- 6.94 and 7.40-7.68 ppm
respectively. Pyrazole proton appeared as a singlet (s) in the region of 8.27-9.12 ppm.
Further evidence in the support of formation of 1,3,4-oxadaizole derivatives was obtained
from 13C-NMR, IR and mass spectral data (5a-r). Peaks in the region of 161-164 ppm in 13C
NMR spectra gave a confirmation for oxadiazole formation. IR spectral data also supported
the formation of proposed oxadiazole derivatives. Characteristic peaks for C=N, C=C and C-
O-C were observed at their expected values.
Experimental and calculated values of elemental analysis were in consonance with each
other. Results were found to be well within range of ±0.4 % of theoretical values. Mass
spectral data further confirmed the formation of desired compounds. Isotopic peaks were seen
in case of chloro bearing compounds i.e. 5l, 5m and 5r. In 5l and 5m, owing the presence of

dichloro groups, [M+H]+, [M-2+H]+ and [M-4+H]+ peaks were observed. However, in case of
5r (mono chloro substituted) [M+H]+ and [M-2+H]+ peaks were observed.
2.2 Pharmacological Activity
2.2.1 Antimalarial Activity
2.2.1.1 Schizont Maturation Inhibition Assay
Schizont maturation inhibition assay was employed for evaluating in vitro antimalarial
activity of the synthesized compounds (5a-r). Activity was done using a culture of 3D7 strain
of P. falciparum. Following confirmation of schizont maturation, blood smears were
prepared. These prepared slides were examined under microscope. Total number of schizonts
per 200 parasites was calculated. Following this, IC50 value for all the compounds was
determined (Table-I). On evaluation, compound 5f emerged as the most potent compound
with IC50 value of 0.248 µg/mL against 3D7 strain. Certain other compounds of the series
(5n, 5r, 5o, 5g and 5a) displayed promising antimalarial effects with IC50 value of less than
1.0 µg/mL. Results obtained for this particular set of compounds were quite comparable to
the previously reported compounds [3].
Table I: Antimalarial Effects (IC50 CQ Sensitive: 3D7 strain of P. falciparum) of
Compounds 5a-r
S. No. Compd. IC50 CQ Sensitive
S. No.
Compd.
IC50 CQ Sensitive
(g/mL)
1.708
(g/mL)
1.
3.
5.
7.
5a
5c
5e
5g
5i
0.886
1.180
2.203
0.647
2.736
2.769
4.316
0.582
3.382
2.
4.
6.
8.
10.
12.
14.
16.
18.
5b
5d
5f
5h
5j
5l
5n
5p
2.234
0.248
2.302
1.650
3.073
0.322
1.942
0.494
9.
11.
13.
15.
17.
5k
5m
5o
5q
5r
19. Chloroq
uine
0.405
CQ: Chloroquine
Antimalarial effects of compound 5f (IC50=0.248 µg/mL) can be visualized from Figure 4.
Figure 4a represents the view taken from microscope for control well. In this figure, a

number of schizonts can be seen (bright violet dots/cluster of dots present over the
background of RBCs). In the view taken for compound 5f, schizonts are not visible (Figure
4b). In this microscopic view, a clear background of stained RBCs can be seen. For this
study, chloroquine was taken as the standard agent.

Figure 4: (a) Control Well; (b): Antimalarial Effects of Compound
5f
A few compounds (5a, 5f, 5g, 5n, 5o and 5r) showed promising antimalarial effects with IC50
value of less than 1 µg/mL against chloroquine sensitive strain. These compounds were also
evaluated against the chloroquine resistant strain (RKL 9) of the parasite. Further, in order to
assess toxicity of these compounds, in vitro cytotoxicity evaluation was performed using
Vero cell line. Their Selectivity Index (SI) was also determined (Table II).
2.2.1.2 Falcipain-2 Assay
For target identification, further screening for inhibitory activity against recombinant
Falcipain-2 using Cbz-Phe-Arg-AMC (fluorogenic substrate) was carried out for compounds,
5a, 5f, 5g, 5n, 5o and 5r. An equivalent concentration of DMSO served as the negative
control whereas E64 served the purpose of positive control. This assay was performed at
concentrations of 100, 50 and 25 µM (Table II). Falcipain-2 inhibitory activity was assessed
by cleavage of the fluorogenic substrate, releasing the fluorescent AMC group. A decrease in
the fluorescence intensity of the sample was indicative of inhibition of enzyme activity.
Compounds which showed more than 50% inhibition at a concentration of 25 µM were taken
for IC50 value determination. Compound 5f was the most potent inhibitor of Falcipain-2 with
an IC50 value of 14 µM.
Table II: Selectivity Index and IC50 (CQ Resistant: RKL 9 strain of P. falciparum and
Falcipain-2 Inhibition) of Compounds
S. No. Compd.
SI
IC50 CQ Resistant Resistance Index
IC50
(Falcipain-2)
(µM)
(g/mL)
1.
5a
29.1
2.129
2.40
ND

2.
3.
4.
5.
6.
7.
5f
5g
5n
5o
5r
13.4
27.3
22.5
17.6
19.2
-
0.863
2.025
0.882
1.705
1.659
2.154
3.47
3.12
2.73
2.92
3.35
5.31
14
ND
21
ND
110
-
Chloroq
uine
DMSO
E64
-
-
8.
9.
-
-
-
-
0
0.02
ND: Not Determined; SI: Selectivity Index
2.2.1.3 Molecular Docking Studies
An effort for identification of novel and potential antimalarial agents has been made in the
present study. Further, in order to determine their probable mode of action, docking studies
were done against falcipain-2 enzyme. Glide score for the most active compound was found
to be -5.532. Dock pose and two dimensional ligand interaction diagram for the same is
presented as Figures 5a and 5b respectively.
(a)

(b)
(c)
Figure 5(a): Dock Pose of Compound 5f with 1YVB; (b): Ligand Interaction Diagram of
Compound 5f with Ligand Binding Domain of Falcipain 2 (1YVB); (c) Legends for
Interactions
Dock pose for compound 5f with catalytic domain of falcipain-2 (PDB ID: 1YVB) is
represented in Figure 5a. 2D ligand interaction diagram for the same is given in Figure 5b.
Formation of hydrogen bond between one of the nitrogen atoms of the oxadiazole ring and
side chain of protein (HIP 159) is clearly evident from Figure 5b. This bond is shown in the
figure as purple dotted arrow. For better understanding of the interactions, different
interactions along with their color coding have been shown in Figure 5c. Polar amino acid
residues, represented in cyan color, found responsible for interaction were Ser 29, Asn 64,
Asn 21, Gln 19, Asn 158, Ser 24, Ser 133, Asn 69 and Gln 156. Interactions were also
observed between the compound and certain non polar amino acid residues (green color) like
Cys 63, Ala 160, Cys 22, Trp 177, Val 136, Cys 25, Trp 26, Ile 132, Phe 207, Leu 67, Ile 68,

Leu 157 and Val 134. Positively charged amino acid residues (blue color) having interactions
with 5f were Lys 20 and Hip 159. One negatively charged residue (red color), Asp 205
showed interaction with the compound. Compound 5f also interacted with Gly 65, Gly 23 and
Gly 66, represented in golden color. Apart from all these interactions, significant solvent
exposure was seen in the region of furan, 1,3,4-oxadiazole, biphenyl ring systems attached to
pyrazole. This solvent exposure was thought to promote the interactions between our
compound and the protein.
2.2.2 Antileishmanial Activity
2.2.2.1 Initial Screening of Compounds
Synthesized compounds were tested for antileishmanial activity as well against L. donovani
(MHOM/IN/83/AG83) promastigotes. Parasites were treated with compounds at a
concentration of 100μg/mL. Viability of compounds was determined under phase contrast
microscope (400X). It was observed that compounds 5a, 5c and 5r exhibited significant
inhibitory effects against promastigotes. However, other tested compounds did not show
significant inhibition (Figure 6).

Figure 6: Screening of compounds. Exponential-phase L. donovani promastigotes (2x106
cells/mL) were incubated with 100 μg/mL of synthesized compounds for 48 h and analyzed
by light microscopy (X400 magnification)

2.2.2.2 Determination of IC50 of Compounds against Promastigotes
MTT reduction assay was used for evaluating the antileishmanial activity of compounds
against L. donovani promastigotes. Compounds 5a, 5c and 5r (0–100 μg/mL) demonstrated
dose-dependent killing of the promastigotes. Their IC50 values were found to be 33.3±1.68,
40.1±1.0 and 19.0±1.47 μg/mL respectively (Figure 7). Established antileishmanial drug,
Pentamidine was taken as positive drug control. This drug showed a similar trend in dose
dependent parasite killing with IC50 value of 2.6 ± 0.32 μg/mL. Parasite viability was not
affected by the presence of negative control, DMSO (0.2%).
Figure 7: Log phase Leishmania donovani promastigotes were incubated with compounds
5a, 5c and 5r (0-100 μg/mL) for 48h at 22°C for IC50 determination. Each point or bar
corresponds to the mean of triplicate samples and is representative of one of three
independent experiments.
2.2.2.3 Antileishmanial Effect of Compounds against Intracellular Amastigotes
Activity of compounds 5a, 5c and 5r on internalized amastigotes was determined
microscopically on Giemsa-stained macrophages. Macrophages entry by promastigotes
involves the formation of membrane bound parasitophorous vacuoles, where they get
transformed into nonmotile amastigote form. Survival of parasites within the parasitophorous
vacuoles is an indicator of pathogenesis. Thereby, it was important to determine activity of
5a, 5c and 5r against the macrophage resident amastigotes. Results depicted a dose-
dependent inhibition of 5a, 5c and 5r compounds (0-100 μg/mL) on amastigote infectivity
with IC50 of 44.2±2.72, 66.8±2.05 and 73.1±1.69 μg/mL respectively (Figure 8).

Figure 8: Promastigote-infested RAW264.7 macrophages were incubated at 37°C in CO2
incubator with compounds 5a, 5c and 5r (0-100 μg/mL) for 48h at 37°C. % infectivity
estimated for determination of IC50 on amastigotes (inset). Each point corresponds to the
mean of triplicate samples and is representative of one of three independent experiments.
2.2.2.4 Cytotoxicity of the Test Compounds on RAW264.7 Macrophage
In vitro cytotoxicity assay was carried out with the murine macrophage cell line RAW264.7
to determine the adverse effects of 5a, 5c and 5r, using Pentamidine as a reference drug. The
toxicity assay showed that compounds 5a, 5c and 5r had no significant adverse effects on the
viability and morphology of the macrophages (Figure 9).
Figure 9: Cytotoxicity of compounds 5a, 5c and 5r to RAW 264.7 macrophages ascertained
as % viability 48 h post-incubation with increasing concentrations of 5a, 5c and 5r or

Pentamidine (50, 100 and 200 mg/mL). Each point corresponds to the mean ± SE of triplicate
samples and is representative of one of three independent experiments.
2.5 Acute Oral Toxicity Studies
OECD guidelines were followed for carrying out acute oral toxicity studies. Dose used in
these studies was 300 mg/Kg. For toxicity evaluation, histopathological evaluation along with
biochemical testing were done. General behavior and appearance of the rats were observed
initially during first 30 minutes of dosing, at an interval of 6h, 14 h and thereafter, regularly
for a period of 14 days for compounds 5f and 5r. In both control and test groups, rats
appeared completely normal, both in terms of appearance and behavior. All the parameters
observed during the observation period have been tabulated in Table III.
Table III: Parameters observed
Observation
Control
6h 14h
Compounds 5f and 5r
6h 14h
30 min
Till
14th
Day
30 min
N
Till
14th
Day
Skin
and N
N
N
N
N
N
N
Fur
Eyes
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Mucous
Membrane
Behavioral
Pattern
Salivation
Lethargy
Sleep
N
N
N
N.O.
N
N
N
N
N.O.
N
N
N
N
N.O.
N
N
N
N.O.
N
N
N
N.O.
N
N
N.O.
N
N
N.O.
N
N
N.O.
N
Diarrhea
Coma
Tremors
N: Normal
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.
N.O.: Not Observed
Following an observation period of 14 days, all the animals were sacrificed on 15th day. Liver
tissue of all the animals was excised. Slides were prepared using this tissue to examine
histopathological changes, if any. Liver sections of control and test group are shown in
Figure 10a and 10b for 5f and 10c and 10d for 5r respectively. In the microscopic view of
slides, architecture of both the groups appeared normal, showing no evidences of toxicity.

a
b
c
d
Figure 10: (a) Liver architecture for control group (b) Liver architecture for test group
(Compound 5f) (c) Liver architecture for control group (d) Liver architecture for test group
(Compound 5r)
For carrying out biochemical estimation of both the groups, blood was collected from rats,
plasma portion was separated and sent for evaluation. Three tests, namely liver function test
(LFT), kidney function test (KFT) and lipid profile were performed. Results of all the three
tests suggested no signs of toxicity. Values for all the tested parameters lied well within the
normal prescribed range (Figure 11).

Figure 11: Biochemical Estimation for Compounds 5f and 5r (a) Kidney Function Test (b)
Liver Function Test (c) Lipid Profile

3. Experimental
3.1 Chemistry
All the chemicals required for synthesis of the desired compounds were obtained from
commercial suppliers and were utilized without any further purification. For thin layer
chromatography (TLC), aluminum backed silica plates (Merck, silica gel 60 F254) were
used. Melting points were taken using pen capillaries and are uncorrected. Bruker alpha-T
spectrophotometer was used for recording infra-red (IR) spectra. 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded on Bruker Avance-400 (400 and 100 MHz,
respectively). Tetramethylsilane (TMS) was as an internal standard for recording NMR
spectra. CDCl3 and DMSO were used as NMR solvents. Chemical shift values (δ) and
coupling constants (J) have been reported in parts per million (ppm) and Hertz (Hz)
respectively. Perkin-Elmer model 240 was used for elemental analyses. Yields refer to
products obtained after crystallization.
Desired compounds were synthesized in accordance with the steps mentioned in Scheme I.
Substitutions for all the synthesized derivatives are given in Table IV.
N
O
N
(i, ii)
R
R
HN
H2N
N
R
(iii)
N
CH3
Phenyl
3
6
Subsituted
Acetophenone
4
5
2
Hydrazine
C
1a-1c
C
O
HO
2a-2c
H
O
3'
2'
N
N
a: R=H, b: R= CH3, c: R=F
N
R
COOH
CONHNH2
5' 6'
COOCH2CH3
(v)

(iv)
CH3CH2OH
Ethanol
(viii)

X
O
X
Substituted Benzoic
Acid Derivatives
N
X
4a-h
X
3a-h
O
5a-r
Cl
Cl
(vi)
(vii)
Cl
HN NH2
O
OH
Cinnamic Acid
REAGENTS & CONDITIONS:
S
S
4i
O
(i, ii) Glacial acetic acid, methanol, refulx, dimethyl formamide, phosphorus oxyhloride; (iii)
Malonic acid, pyridine, piperidine, reflux; (iv) Conc. H2SO4, reflux; (v) Hydrazine hydrate, reflux; (vi) SOCl2, DMF, Pyridine;
(vii) Hydrazine hydrate, reflux (viii) Phosphorus oxychloride, stirring, heating (60-70oC)
SCHEME I

Table IV: List of Synthesized Derivatives in Scheme I
Comp. R
X
Comp.
5b
R
4-
CH3
X
Comp.
5c
R
H
X
5a
H
O
O
O
2"
6"
2"
6"
2"
O
O
O
O
6" 5"
O
O
O
O
3"
5d
4-
5e
4-F
H
5f
H
4"
5"
CH3
2"
2"
O
O
6" 5"
3"
6" 5"
2" 3"
2" 3"
5g
5j
4-
CH3
5h
5k
5i
5l
4-
CH3
4"
5"
4"
5"
4"
O
5"
6"
6"
Cl
Cl
2" 3"
2" 3"
H
4-
H
3"
CH3
O
O
4"
5"
5"
6"
6"
2"
5"
Cl
Cl
2"
5m
5p
4-
CH3
5n
5q
H
5o
5r
4-
CH3
3"
N
3"
N
3"
4"
6" 5"
6" 5"
Cl
5"
3"
H
4-
CH3
4-
CH3
2"
6"
2"
6"
3"
5"
OH
OH
S
5"
3.1.1General procedure to synthesize 1,3-Diphenyl-1H-pyrazole-4-carbaldehyde (1a-c)
Dimethyl formamide (DMF) and phosphorus oxychloride (POCl3) were separately cooled
down to a temperature of 0ºC. To previously cooled DMF (0.075 mol) at 0-5ºC, POCl3 was
added drop-wise through dropping funnel. The resulting solution was stirred for 15 min. A
solution of hydrazone (prepared by refluxing desired acetophenone in methanol in the
presence of catalytic amount of glacial acetic acid), made using minimum quantity of DMF
was added to the mixture in drop-wise manner. The resulting mixture was warmed to room
temperature and subsequently heated to a temperature of 75-80ºC for a period of 5 hrs.
Completion of the reaction was judged using TLC. Following completion, reaction mixture

was cooled to room temperature and basified using a saturated solution of NaHCO3.
Precipitate obtained on basification was filtered, washed and dried [16,17].
3.1.2 General procedure to synthesize 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid
derivatives (2a-c)
Pyrazole-4-carbaldehyde derivatives (0.01 mol) were dissolved in pyridine (10 mL). To this
solution, malonic acid (0.05 mol) and piperidine in catalytic amount (0.5 mL) were added.
The resulting mixture was stirred for a period of 8 hrs. After stirring, the mixture was heated
to a temperature of 95-100ºC. Progress of the reaction was determined using TLC. Following
completion, processing for the reaction was done. The reaction mixture was poured on to
crushed ice followed by basification using NaOH solution. Further acidification using dilute
HCl gave the desired acid derivatives.
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid (2a)
Yield (%): 89; Appearance: White powder; m.p. 184-186˚C; IR (cm-1): 1684 (C=O); 1H NMR
(400 MHz, DMSO): 6.41 (d, 1H, H-α, J=15.6), 7.35 (t, 1H, H-4, J=7.2), 7.46-7.62 (m, 8H, H-
β, 2,3,5,6,3’,4’,5’), 7.90 (d, 2H, H-2’,6’, J=8.0), 9.21 (s, 1H, H-Pyrazole), 12.33 (s, 1H, -
COOH); Elemental analysis (%) of C18H14N2O2, calculated/found: C (74.45/74.47),
H (4.89/4.86), N (9.62/9.65).
3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)acrylic acid (2b)
Yield (%): 87; Appearance: White powder; m.p. 194-196˚C; IR (cm-1): 1688 (C=O); 1H NMR
(400 MHz, DMSO): 6.39 (d, 1H, H-α, J=15.6), 7.33-7.37 (m, 3H, H-4,3’,5’), 7.46-7.55 (m,
5H, H-2,3,5,6,β), 7.88 (d, 2H, H-2’,6’, J=8.0), 9.19 (s, 1H, H-Pyrazole), 12.31 (s, 1H, -
COOH); Elemental analysis (%) of C19H16N2O2, calculated/found: C (74.99/74.98),
H (5.32/5.30), N (9.21/9.20).
3-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)acrylic acid (2c)
Yield (%): 84; Appearance: White powder; m.p. 190-192˚C; IR (cm-1): 1692 (C=O); 1H NMR
(400 MHz, DMSO): 6.39 (d, 1H, H-α, J=16.0), 7.34-7.36 (m, 3H, H-4,3’,5’), 7.38 (d, 1H, H-
β, J=16.0), 7.46-7.52 (m, 2H, H-3,5), 7.62-7.66 (m, 2H, H-2’,6’), 7.88-7.90 (d, 2H, H-2,6,
J=8.0), 9.19 (s, 1H, H-Pyrazole), 12.31 (s, 1H, -COOH); Elemental analysis (%) of
C19H16N2O2, calculated/found: C (70.12/70.11), H (4.25/4.24), N (9.09/9.10).
General procedure to synthesize benzohydrazide derivatives (4a-i)

Derivatives 3a-i were synthesized in accordance with the reported methods [18-20].
Derivative 4i was prepared using the method reported by Pakray and Castle [21].
3.1.3 General procedure to synthesize 2-(2-(1,3-diphenyl-1H-pyrazol-4-yl)vinyl)-5-
phenyl-1,3,4-oxadiazole derivatives (5a-v)
Scheme I was followed for synthesizing oxadiazole derivatives. Substitution for these
derivatives is given in Table IV.
1,3,4-oxadiazole derivatives were prepared via direct coupling of the appropriately
substituted hydrazide to the necessary acid derivative. To 10 mL of POCl3, hydrazide
(2mmol) and acid (2mmol) were added. The resulting reaction mixture was stirred at a
temperature of 60-70˚C. Progress of the reaction was monitored using TLC. Following
completion of the reaction, the reaction mixture was poured on crushed ice and neutralized
with 10% sodium bicarbonate solution in water. Neutralized solution was filtered, washed
with water and dried. The dried product was re-crystallized from methanol.
2-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)vinyl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (5a)
Yield (%): 85; Appearance: Pale yellow solid; m.p. 216-220°C; IR (cm-1): 1626 (C=N), 1592
1
(C=C), 1243 (C-O-C); H NMR (400 MHz, CDCl3): 3.92 (s, 3H, OCH3); 3.94 (s, 6H, 2 x
OCH3); 6.89 (d,1H, H-α, J=16.4 Hz); 7.29 (s, 2H, H-2”,6”); 7.34 (t, 1H, H-4, J=16.4 Hz);
7.45-7.53 (m, 5H, H-3,5,3’,4’,5’); 7.62 (d, 1H, H-β, J=16.4 Hz); 7.73 (d, 2H, H-2,6, J=6.8
Hz); 7.79 (d, 2H, H-2’,6’, J=8.0 Hz); 8.30 (s, 1H, H-Pyrazole); Mass (m/z): 481 [M+H]+;
Elemental analysis (%) of C28H24N4O4, calculated/found: C (69.99/69.70), H (5.03/5.01),
N (11.66/11.68)
2-(2-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazole (5b)
Yield (%): 87; Appearance: Pale yellow solid; m.p. 222-224°C; IR (cm-1): 1618 (C=N), 1596
1
(C=C), 1234 (C-O-C); H NMR (400 MHz, CDCl3): 2.44 (s, 3H, CH3); 3.92 (s, 3H, OCH3);
3.95 (s, 6H, 2XOCH3); 6.88 (d, 1H, H-α, J=16.4 Hz); 7.26-7.37 (m, 5H, H-4,3’,5’,2”,6”);
7.48 (t, 2H, H-3,5, J=7.6 Hz); 7.57-7.65 (m, 3H, H-2,6,β); 7.78 (d, 2H, H-2’,6’, J=8.0 Hz);
8.28 (s, 1H, H-Pyrazole); Mass (m/z): 495 [M+H]+; Elemental analysis (%) of C29H26N4O4,
calculated/found: C (70.43/70.41), H (5.30/5.30), N (11.33/11.34)
2-(3,4-Dimethoxyphenyl)-5-(2-(1,3-diphenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5c)

Yield (%): 88; Appearance: Yellow solid; m.p.158°C; IR (cm-1): 1614 (C=N), 1603 (C=C),
1
1248 (C-O-C); H NMR (400 MHz, DMSO): 3.96 (s, 3H, OCH3); 3.97 (s, 3H, OCH3); 6.90
(d, 1H, H-α, J=16.4 Hz); 6.95 (d, 1H, H-5”, J=8.8 Hz); 7.34 (t, 1H, H-4, J=6.8 Hz); 7.47-7.55
(m, 8H, H-2,3,5,6,3’,4’,5’,β); 7.68 (d, 2H, H-2’,6’, J=7.2 Hz); 7.90 (d, 2H, H-2”,6”, J=8.0
Hz); 9.23 (s, 1H, H-Pyrazole); Mass (m/z): 451 [M+H]+; Elemental analysis (%) of
C27H22N4O3, calculated/found: C (71.99/71.98), H (4.92/4.93), N (12.44/12.46)
2-(3,4-Dimethoxyphenyl)-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole
(5d)
Yield (%): 86; Appearance: Pale yellow solid; m.p. 164°C; IR (cm-1): 1618 (C=N), 1601
1
(C=C), 1244 (C-O-C); H NMR (400 MHz, CDCl3): 2.50 (s, 3H, CH3); 3.86 (s, 3H, OCH3);
3.87 (s, 3H, CH3); 7.17 (d, 1H, H-5”, J= 8.8 Hz); 7.19 (d, 1H, H-α, J= 20.0 Hz); 7.39 (t, 1H,
H-4, J= 7.2 Hz); 7.50-7.62 (m, 7H, H-3,5,3’,5’,2”,6”,β); 7.72 (d, 2H, H-2,6, J=7.2 Hz); 7.94
(d, 2H, H-2’6’, J= 8.0 Hz); Mass (m/z): 465 [M++H]; Elemental analysis (%) of C28H24N4O3,
calculated/found: C (72.40/72.41), H (5.21/5.19), N (12.06/12.07)
2-(3,4-Dimethoxyphenyl)-5-(2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-
oxadiazole (5e)
Yield (%): 85; Appearance: Yellow solid; m.p. 212-214°C; IR (cm-1): 1632 (C=N), 1593
(C=C), 1239 (C-O-C); 1H NMR (400 MHz, CDCl3): 3.95 (s, 3H, OCH3); 3.96 (s, 3H, OCH3);
6.87 (d, 1H, H-α, J=16.8 Hz); 6.94 (d, 1H, H-5”, J=8.8 Hz); 7.18 (t, 2H, H-3’,5’, J=8.8 Hz);
7.33 (t, 1H, H-4, J=7.2 Hz); 7.46-7.71 (m, 7H, H-3,5,2’,6’,2”,6”,β); 7.76 (d, 2H, H-2,6, J=8.4
Hz); 8.27 (s, 1H, H-Pyrazole); Mass (m/z): 469 [M+H]+; Elemental analysis (%) of
C27H21FN4O3, calculated/found: C (69.22/69.23), H (4.52/4.50), N (11.96/11.97)
2-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)vinyl)-5-(furan-2-yl)-1,3,4-oxadiazole (5f)
Yield (%): 89; Appearance: Yellow solid; m.p. 190-192°C; IR (cm-1): 1635 (C=N), 1590
1
(C=C), 1243 (C-O-C); H NMR (400 MHz, CDCl3): 66.60 (m, 1H, H-4”); 6.88 (d, 1H, H-α,
J=16.4 Hz); 7.17 (d, 1H, H-3”, J=3.6 Hz); 7.34 (t, 1H, H-4, J=7.2 Hz); 7.45-7.64 (m, 7H, H-
3,5,3’,4’,5’, 5”, ); 7.71 (d, 2H, H-2,6, J=7.2 Hz); 7.79 (d, 2H, H-2’,6’, J=8.0 Hz); 8.30 (s,
1H, H-Pyrazole); Mass (m/z): 381 [M+H]+; Elemental analysis (%) of C23H16N4O2,
calculated/found: C (72.62/72.63), H (4.24/4.22), N (14.73/14.74)
2-(Furan-2-yl)-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5g)
Yield (%): 84; Appearance: Yellow solid; m.p. 182-186°C; IR (cm-1): 1639 (C=N), 1596
1
(C=C), 1240 (C-O-C); H NMR (400 MHz, CDCl3): 2.45 (s, 3H, CH3); 6.61 (m, 1H, H-4”);

6.87 (d, 1H, H-α, J= 16.4 Hz); 7.17 (d, 1H, H-3”, J=3.2 Hz); 7.32-7.37 (m, 3H, H- 4,3’,5’);
7.48 (t, 2H, H-3,5, J=8.0 Hz); 7.59-7.65 (m, 4H, H-2,6,5”, β); 7.78 (d, 2H, H-2’,6’, J=8.0
Hz); 8.28 (s, 1H, H-Pyrazole); Mass (m/z): 395 [M+H]+; Elemental analysis (%) of
C24H18N4O2, calculated/found: C (73.08/73.10), H (4.60/4.58), N (14.20/14.22)
2-Benzyl-5-(2-(1,3-diphenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5h)
Yield (%): 89; Appearance: Yellow solid; m.p. 170-172°C; IR (cm-1): 1646 (C=N), 1591
1
(C=C), 1247 (C-O-C); H NMR (400 MHz, CDCl3): 5.26 (s, 2H, CH2); 6.83 (d, 1H, H-α,
J=16.8 Hz); 7.03 (d, 2H, H-2”,6”, J=6.4 Hz); 7.30-7.52 (m, 9H, H-3,4,5,3’,4’,5’,3”,4”,5”);
7.56 (d, 1H, H-β, J=16.4 Hz); 7.67 (d, 2H, H-2,6, J=7.6 Hz); 7.78 (d, 2H, H-2’,6’, J=8.0 Hz);
8.28 (s, 1H, H-Pyrazole); Mass (m/z): 405 [M+H]+; Elemental analysis (%) of C26H20N4O,
calculated/found: C (77.21/77.23), H (4.98/4.97), N (13.85/13.85)
2-Benzyl-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5i)
Yield (%): 82; Appearance: pale yellow solid; m.p. 188°C; IR (cm-1): 1635 (C=N), 1589
1
(C=C), 1246 (C-O-C); H NMR (400 MHz, CDCl3): 2.43 (s, 3H, CH3); 5.26 (s, 2H, CH2);
6.82 (d, 1H, H-α, J=16.4 Hz); 7.03 (d, 2H, H-2”,6”, J=6.8 Hz); 7.30-7.36 (m, 5H, H-
3,4,5,3’,5’); 7.47-7.60 (m, 6H, H-2,6,3”,5”,4”, ); 7.77 (d, 2H, H-2’,6’, J=7.6 Hz); 8.26 (s,
1H, H-Pyrazole); Mass (m/z): 419 [M+H]+; Elemental analysis (%) of C27H22N4O,
calculated/found: C (77.49/77.52), H (5.30/5.32), N (13.39/13.40)
2-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)vinyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (5j)
Yield (%): 84; Appearance: Pale yellow solid; m.p.220°C; IR (cm-1):1633 (C=N), 1590
1
(C=C), 1244 (C-O-C); H NMR (400 MHz, CDCl3): 3.88 (s, 3H, OCH3); 6.89 (d, 1H, H-α,
J=16.4 Hz); 6.99 (d, 2H, H-3”,5”, J=8.8 Hz); 7.33 (t, 1H, H-4, J=7.6 Hz); 7.45-7.55 (m, 5H,
H-3,5,3’,4’,5’); 7.57 (d, 1H, H-, J=16.4 Hz); 7.72 (d, 2H, H-2,6, J=7.2 Hz); 7.79 (d, 2H, H-
2’,6’, J=8.0 Hz); 7.99 (d, 2H, H-2”,6”, J=8.8 Hz); 8.29 (s, 1H, H-Pyrazole); Mass (m/z): 421
[M+H]+; Elemental analysis (%) of C26H20N4O2, calculated/found: C (74.27/74.28),
H (4.79/4.77), N (13.33/13.32)
2-(4-Methoxyphenyl)-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5k)
Yield (%): 80; Appearance: Yellow solid; m.p. 206-208°C; IR (cm-1):1639 (C=N), 1588
1
(C=C), 1240 (C-O-C); H NMR (400 MHz, CDCl3): 2.45 (s, 3H, CH3); 3.88 (s, 3H, OCH3);
6.88 (d, 1H, H-α, J=16.4 Hz); 7.00 (d, 2H, H-3”,5”, J=8.8 Hz); 7.33 (triplet merged with
doublet, 3H, H-4,3’,5’, J=6.8 Hz); 7.48 (t, 2H, H-3,5, J=7.6 Hz); 7.56-7.63 (m, 3H, H-2,6,β);

7.78 (d, 2H, H-2’,6’, J=8.0 Hz); 8.00 (d, 2H, H-2”,6”, J=8.4 Hz); 8.28 (s, 1H, H-Pyrazole);
Mass (m/z): 435 [M+H]+; Elemental analysis (%) of C27H22N4O2, calculated/found:
C (74.64/74.66), H (5.10/5.13), N (12.89/12.88)
2-(2,6-Dichlorophenyl)-5-(2-(1,3-diphenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole (5l)
1
Yield (%): 87; Appearance: Yellow solid; m.p. 194-196°C; IR (cm-1):; H NMR (400 MHz,
CDCl3): 6.84 (d, 1H, H-α, J=16.4 Hz); 7.34-7.40 (m, 2H, H-4,4”); 7.45-7.57 (m, 8H, H-
3,5,3’,4’,5’,3”,5”,β); 7.69 (d, 2H, H-2,6, J=6.4 Hz); 7.78 (d, 2H, H-2’,6’, J=8.4 Hz); 8.29 (s,
1H, H-Pyrazole); Mass (m/z): 459 [M+H]+, 461 [M-2+H]+, 463 [M-4+H]+ ; Elemental
analysis (%) of C25H16Cl2N4O, calculated/found: C (65.37/65.38), H (3.51/3.49), N
(12.20/12.19)
2-(2,6-Dichlorophenyl)-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazole
(5m)
Yield (%): 89; Appearance: Yellow solid; m.p. 210-212°C; IR (cm-1):1627 (C=N), 1595
1
(C=C), 1238 (C-O-C); H NMR (400 MHz, CDCl3): 2.44 (s, 3H, CH3); 6.83 (d, 1H, H-α,
J=16.4 Hz); 7.29-7.36 (m, 5H, H-3,4,5,3’,5’); 7.48-7.52 (m, 3H, H-3”,4”,5”); 7.53 (d, 1H, H-
β, J=16.4 Hz); 7.59 (d, 2H, H-2,6, J=8.0 Hz); 7.78 (d, 2H, H-2’,6’, J=8.0 Hz); 8.26 (s, 1H, H-
Pyrazole); Mass (m/z):473 [M+H]+; 475 [M-2+H]+, 477 [M-4+H]+; Elemental analysis (%) of
C26H18Cl2N4O, calculated/found: C (65.97/65.98), H (3.83/3.85), N (11.84/11.84)
2-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)vinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (5n)
Yield (%): 85; Appearance: Pale yellow solid; m.p. 200-202°C; IR (cm-1):1639 (C=N), 1593
1
(C=C), 1246 (C-O-C); H NMR (400 MHz, CDCl3): 6.91 (d, 1H, H-α, J=16.4 Hz); 7.35 (t,
1H, H-4, J=6.8 Hz); 7.47-7.56 (m, 5H, H-3,5,3’,4’,5’); 7.66 (d, 1H, H-β, J=16.0Hz); 7.71 (d,
2H, H-2,6, J=7.2 Hz); 7.79 (d, 2H, H-2’,6’, J=8.0 Hz); 7.92 (s, 2H, H-2”,6”); 8.33 (s, 1H, H-
Pyrazole); 8.82 (s, 2H, H-3”,5”); 13C NMR (100 MHz, CDCl3): 108.32, 117.48, 118.93,
119.81, 125.67, 126.89, 128.28, 128.33, 128.41, 129.14, 130.21, 130.50, 131.77, 138.91,
150.35, 152.59, 161.55, 164.70; Mass (m/z): 392 [M+H]+; Elemental analysis (%) of
C24H17N5O, calculated/found: C (73.64/73.66), H (4.38/4.39), N (17.89/17.91)
2-(2-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (5o)
Yield (%): 81; Appearance: Yellow solid; m.p. 172-174°C; IR (cm-1): 1629 (C=N), 1596
1
(C=C), 1240 (C-O-C); H NMR (400 MHz, CDCl3): 2.46 (s, 3H, CH3); 6.90 (d, 1H, H-α,
J=16.4 Hz); 7.30 (m, 3H, H-4,3’,5’); 7.49 (t, 2H, H-3,5, J=8.0 Hz); 7.60 (d, 2H, H-2,6, J=8.0
Hz); 7.67 (d, 1H, H-β, J=16.8 Hz); 7.79 (d, 2H, H-2’,6’, J=7.6 Hz); 7.92 (d, 2H, H-2”,6”);

8.31 (s, 1H, H-Pyrazole); 8.82 (s, 2H, H-3”,5”); Mass (m/z): 406 [M+H]+; Elemental analysis
(%) of C25H19N5O, calculated/found: C (74.06/74.09), H (4.72/4.70), N (17.27/17.29)
4-(5-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazol-2-yl)phenol (5p)
Yield (%): 87; Appearance: Yellow solid; m.p. 172-174°C; IR (cm-1): 1631 (C=N), 1594
(C=C), 1237 (C-O-C), 3315 (OH); 1H NMR (400 MHz, CDCl3): 6.93 (d, 2H, H-3”,5”, J=8.4
Hz); 7.16 (d, 1H, H-α, J=16.4 Hz); 7.36 (t, 1H, H-4, J=7.2 Hz); 7.49-7.58 (m, 6H, H-β,
3,5,3’,4’,5’); 7.69 (d, 2H, H-2,6, J=7.2 Hz); 7.83 (d, 2H, H-2’,6’, J=8.4 Hz); 7.91 (d, 2H, H-
2”,6”, J=7.6 Hz); 10.27 (bs, 1H, OH); Mass (m/z): 407 [M+H]+; Elemental analysis (%) of
C25H18N4O2, calculated/found: C (73.88/70.91), H (4.46/4.44), N (13.78/13.78)
4-(5-(2-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-oxadiazol-2-yl)phenol (5q)
Yield (%): 80; Appearance: Yellow solid; m.p. 190-194°C; IR (cm-1):1635 (C=N), 1591
(C=C), 1247 (C-O-C), 3328 (OH); 1H NMR (400 MHz, CDCl3): 2.49 (s, 3H, CH3); 7.14-7.20
(m, 3H, H-3”,5”,α); 7.31-7.38 (m, 5H, H-3,4,5,3’,5’); 7.49-7.59 (m,3H, H-2,6,β); 7.82 (d, 2H,
H-2’,6’, J=8.4 Hz); 7.90 (d, 2H, H-2”,6”, J=8.0 Hz); 9.25 (s, 1H, H-Pyrazole); 9.27 (bs, 1H,
OH); Mass (m/z): 421 [M+H]+; Elemental analysis (%) of C26H20N4O2, calculated/found:
C (74.27/74.27), H (4.79/4.82), N (13.33/13.35)
2-(3-chlorobenzo[b]thiophen-2-yl)-5-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)vinyl)-1,3,4-
oxadiazole (5r)
Yield (%): 80; Appearance: Yellow solid; m.p. 188°C; IR (cm-1): 1638 (C=N), 1595 (C=C),
1242 (C-O-C); 1H NMR (400 MHz, CDCl3): 2.48 (s, 3H, CH3); 6.94 (d, 1H, H-α, J=16.4 Hz);
7.35 (t, 3H, H-4,3’,5’, J=8.0 Hz); 7.51-7.57 (m, 4H, H-3,5,4”,7”); 7.65 (d, 2H, H-2,6, J=8.0
Hz); 7.75 (d, 1H, H-β, J= 16.0 Hz); 7.82 (d, 2H, H-2’,6’, J= 7.6 Hz); 7.88-7.91 (m, 1H, H-6”);
7.99 (m, 1H, H-5”); 8.34 (s, 1H, H-Pyrazole); Mass (m/z): 495 [M+H]+; 497 [M-2+H]+;
Elemental analysis (%) of C26H20N4O2, calculated/found: C (67.94/67.95), H (3.87/3.85), N
(11.32/11.33)
3.2 Pharmacological Activity
3.2.1 Antimalarial Activity
3.2.1.1 Schizont Maturation Inhibition Assay
Synchronization of culture of chloroquine sensitive 3D7 strain of P. falciparum was done
using 5% aqueous solution of sorbitol. All stages except rings were degenerated. With this, a
collection of parasites of similar stage was obtained. Centrifugation for 5 min at 1500 rpm

was done for removing degenerated stages. Supernatant was discarded and the pellet was
washed twice using incomplete media.
Adjustment of parasitemia to about 1% for assay was done by dilution with fresh washed
RBCs. Synthesized compounds that were to be assessed for antimalarial efficacy were
dissolved in 100 µL of DMSO. Control well also received the same concentration of DMSO.
Dilution of the stock solution was done in RPMI-1640 for obtaining different concentrations.
Chloroquine served as the standard for carrying out this study.
Testing was done in 96 well plate using chloroquine sensitive isolates. Prepared
concentrations of the compounds were dispensed in 96 well plate using chloroquine sensitive
isolates. Drug was not added to the first well of all the rows and was considered as the control
well. Synchronized parasites were inoculated in all the wells, even the control one to get a
final concentration of 5% haematocrit. Same protocol was adopted to determine the efficacy
of compounds against chloroquine resistant strain of P. falciparum (RKL 9).
Depending on the maturation of schizont, these plates were incubated at 37ºC in gas mixture
containing 90% N2, 5% CO2 and 5% O2 for a period of 24-30 h. Following confirmation of
schizont maturation, smears were prepared from all the wells. Staining of these smears was
done using JSB II and JSB I stains. The dried slides were observed under power of the Nikon
Eclipse E200 microscope. Total number of schizonts (3 or more merozoites) per 200 asexual
parasites was counted. IC50 value for each compound was calculated from total number of
schizonts using HN-NonLin Regression analysis (malaria.farch.net). Active compounds of
the series were further tested against chloroquine resistant strain, RKL9 [22]. Cytotoxicity
studies for the synthesized compounds were carried out using Vero cell line. Mossman
method with certain modifications was adopted for performing in vitro cytotoxicity assay
[23]. Cells with different dilutions of compounds were incubated for a period of 72 h using
MTT reagent. Using the formula, CC50/IC50, SI of the compounds was calculated. Here, CC50
refers to cytotoxic concentration, required to bring about death of 50% of the fibroblast cells.
Values for SI of compounds are given in Table II. For these compounds, resistance index
(RI) was determined by dividing IC50:PfRKL9/IC50:Pf3D7.
3.2.1.2 In Vitro Falcipain-2 Assay
Diluted, soluble P. falciparum Falcipain-2 (30 nM) was incubated at room temperature in 100
mM sodium acetate, pH 5.5, 10 mM DTT, along with different concentrations of compounds

to be tested or the standard agent, E64 for a duration of 10 minutes. After incubation for the
stipulated time period, a fluorescent substrate, Cbz-Phe-Arg-AMC was added to a final
concentration of 25 µM. Intensity of fluorescence (excitation: 355 nm, emission: 460 nm)
was determined for 10 minutes at room temperature using SynergyTM 4 Multi-Mode
Microplate Reader (BioTek). Following formula was used for determining the inhibition rate
(%).
% Inhibition = [1-(F test/F control)] X 100
Here, F test refers to the fluorescence intensity of test compound whereas F control is the
fluorescence intensity of standard agent, E64. All the values are mean values of triplicate
independent determinations and deviations are <10% of the mean value. IC50 values for
falcipain-2 were determined for compounds exhibiting 50% or more enzyme inhibition at a
dose of 25 µM.
3.2.1.3 Molecular Docking Studies
Molecular docking studies were performed for antimalarial compounds. Catalytic domain for
falcipain-2 (PDB ID: 1YVB) was obtained from protein data bank. Protein preparation
wizard [24], available in Schrödinger suite 2016-1 was used for preparing protein.
Crystallographic water molecules i.e. without 3H bonds were deleted and hydrogen bonds
corresponding to pH 7 were added considering the appropriate ionization states for both
acidic and basic amino acid residues. For energy minimization of the crystal structure,
OPLS_2005 force field was used. Site map was run to identify the active site of the
Falcipain-2 enzyme. Selection amongst the generated sites was made on the basis of site
score and further, centroid was taken for further grid generation. Active site was defined with
a radius of 16Å around the centroid. For docking, low energy conformations of all the
compounds were docked into the catalytic domain of both enzymes using Grid based Ligand
Docking with Energetics (Glide v7.0, Schrödinger 2016-1) in standard precision (SP) mode
in the absence of any constraint.
3.2.3 Antileishmanial Activity
For carrying out antileishmanial activity, fetal bovine serum (FBS) was obtained from Gibco-
BRL, DMSO from SRL and methanol from Merck. All the other chemicals required in the
study were procured from Sigma-Aldrich, unless mentioned.

L. donovani parasites, strain MHOM/IN/83/AG83, were maintained in vivo in BALB/c mice.
Culturing of promastigotes was carried out in M199 medium supplemented with 10%
hematin activated FBS, penicillin G sodium (100 U/mL) and streptomycin sulfate (100
g/mL) at a temperature of 22oC. Sub-culturing was done every 72 h in the same medium at a
mean density of 2x106 cells/mL [25]. Cell line, RAW264.7 was grown at 37oC in RPMI 1640
medium (pH 7.4) supplemented with 10% heat-inactivated FBS for 48–72 h in 5% CO2 and
sub-cultured in fresh RPMI 1640 medium at a mean density of 2x105 cells/mL.
Screening of compounds was performed against the promastigote stage of L. donovani in
vitro. Incubation of promastigotes of L. donovani in M199 medium was done for a period of
48 h at a temperature of 22◦C with each compound at a concentration of 100μg/mL.
Pentamidine served as the standard drug for the whole study while 0.2% DMSO was taken as
the solvent control. Parasites with media alone were taken as control. Viability of parasites
was determined microscopically after a period of 48 h [26].
For determination of IC50 values, promastigotes (2 x 106 cells/mL) were incubated for 48 h at
a temperature of 22°C without or with serial two fold dilutions of 5a, 5c and 5r starting from
100 μg/mL. MTT assay was adopted for determining mean percent (%) viability. IC50,
inhibitory concentration responsible for 50% reduction in promastigote growth was
graphically extrapolated by plotting percent (%) viability versus drug concentration. DMSO
was taken as the negative control while Pentamidine served as the reference drug [27].
3.4 Acute Oral Toxicity Studies
Acute oral toxicity testing for compounds 5f and 5r was carried out in accordance with
OECD guideline for testing chemicals. Two groups i.e. control and test comprising of three
Wistar rats each (female) were made. Each animal selected for inclusion into each group was
between 8 and 12 weeks old. Animals were initially housed in the Central Animal Housing
Facility of Jamia Hamdard. Rats were kept at a temperature of 22±3°C. Relative humidity
was maintained between 40-50%. They were provided with 12 hours cycle each of light and
dark. They were fed with conventional laboratory diets and unlimited availability of drinking
water.
Five days prior to our experimentation, animals were shifted to the laboratory where
experiments were to be performed. This was done to permit acclimatization to the laboratory
conditions.