Synthesis and molecular modelling studies of phenyl linked oxadiazole-phenylhydrazone hybrids as potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2016.12.019

Molecular hybridization yielded phenyl linked oxadiazole-benzohydrazones hybrids 6–35 and were evaluated for their antileishmanial potentials. Compound 10, a 3,4-dihydroxy analog with IC₅₀value of 0.95 ± 0.01 μM, was found to be the most potent antileishmanial agent (7 times more active) than the standard drug pentamidine (IC₅₀= 7.02 ± 0.09 μM). The current series 6–35 conceded in the identification of thirteen (13) potent antileishmanial compounds with the IC₅₀values ranging between 0.95 ± 0.01–78.6 ± 1.78 μM. Molecular docking analysis against pteridine reductase (PTR1) were also performed to probe the mode of action. Selectivity index showed that compounds with higher number of hydroxyl groups have low selectivity index. Theoretical stereochemical assignment was also done for certain derivatives by using density functional calculations.

Full text of DOI:10.1016/j.ejmech.2016.12.019

Accepted Manuscript
Synthesis and molecular modelling studies of phenyl linked oxadiazole-
phenylhydrazone hybrids as potent antileishmanial agents
Muhammad Taha, Nor Hadiani Ismail, Syahrul Imran, El Hassane Anouar,
Manikandan Selvaraj, Waqas Jamil, Muhammad Ali, Syed Muhammad Kashif, Fazal
Rahim, Khalid Mohammed Khan, Mohd Ilham Adenan
PII:
S0223-5234(16)31025-X
10.1016/j.ejmech.2016.12.019
EJMECH 9110
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2016
Revised Date: 6 December 2016
Accepted Date: 9 December 2016
Please cite this article as: M. Taha, N.H. Ismail, S. Imran, E.H. Anouar, M. Selvaraj, W. Jamil, M. Ali,
S.M. Kashif, F. Rahim, K.M. Khan, M.I. Adenan, Synthesis and molecular modelling studies of phenyl
linked oxadiazole-phenylhydrazone hybrids as potent antileishmanial agents, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.019.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and Molecular Modelling
Studies of Phenyl Linked
Oxadiazole-Phenylhydrazone Hybrids as Potent
Antileishmanial Agents
∗
a,b
a,b
a,b
c
d,e
Muhammad Taha , Nor Hadiani Ismail , Syahrul Imran , El Hassane Anouar , Manikandan Selvaraj ,
f
f
f
g
h
Waqas Jamil , Muhammad Ali Syed Muhammad Kashif , Fazal Rahim , Khalid Mohammed Khan , Mohd
a,b
Ilham Adenan
a
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor, Malaysia; Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia; Chemistry
Department, College of Sciences and Humanities,Prince Sattam bin Abdulaziz University, Suadia Arabia; Integrative
b
c
d
Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam,
e
Selangor Darul Ehsan, Malaysia; Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
f
Puncak Alam, Selangor Darul Ehsan, Malaysia; Institute of Advance Research Studies in Chemical Sciences, University of Sindh
g
h
Jamshoro, Hyderabad 76080, Pakistan; Depatment of Chemistry, Hazara University, Mansehra, Pakistan; H. E. J. Research Institute
of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
Compound 10 showed 0.95 ± 0.01 µM as compare to standard drug pentamidine (IC = 7.02 ± 0.09 µM)
50

ACCEPTED MANUSCRIPT
Synthesis and Molecular Modelling Studies of Phenyl Linked Oxadiazole-
Phenylhydrazone Hybrids as Potent Antileishmanial Agents
∗
a,b
a,b
a,b
c
Muhammad Taha , Nor Hadiani Ismail , Syahrul Imran , El Hassane Anouar , Manikandan
d,e
f
g
f
h
Selvaraj , Waqas Jamil , Muhammad Ali Syed Muhammad Kashif , Fazal Rahim , Khalid
i
a,b
Mohammed Khan , Mohd Ilham Adenan
a
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam
Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia; Faculty of Applied Science UiTM, 40450 Shah Alam,
Selangor, Malaysia; Chemistry Department, College of Sciences and Humanities,Prince Sattam bin Abdulaziz
University, Suadia Arabia; Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA
b
c
d
e
(
UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; Faculty of
Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor
f
Darul Ehsan, Malaysia; Institute of Advance Research Studies in Chemical Sciences, University of Sindh Jamshoro,
g
Hyderabad 76080, Pakistan; Department of Chemistry, COMSATS Institute of Information Technology, University
h
i
Road, Abbottbad-22060, KPK, Pakistan, Depatment of Chemistry, Hazara University, Mansehra, Pakistan; H. E. J.
Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-
7
5270, Pakistan
Abstract:
Molecular hybridization yielded phenyl linked oxadiazole-benzohydrazones hybrids 6-35 and
were evaluated for their antileishmanial potentials. Compound 10, a 3,4-dihydroxy analog with
IC value of 0.95 ± 0.01
µ
M, was found to be the most potent antileishmanial agent (7 times
more active) than the standard drug pentamidine (IC = 7.02 ± 0.09 M). The current series 6-
5 conceded in the identification of thirteen (13) potent antileishmanial compounds with the IC50
values ranging between 0.95 ± 0.01 - 78.6 ± 1.78 M. Molecular docking analysis against
5
0
µ
5
0
3
µ
pteridine reductase (PTR1) were also performed to probe the mode of action. Selectivity index
showed that compounds with higher number of hydroxyl groups have low selectivity index.
Theoretical stereochemical assignment was also done for certain derivatives by using density
functional calculations.
Keywords: Oxadiazole-benzohydrazone hybrids, leishmaniasis, SAR study, Molecular Docking
Studies, DFT, Selectivity index
∗
Correspondence and reprints
E-mail: taha_hej@yahoo.com and muhamm9000@puncakalam.uitm.edu.my, Tel: 0060182901765
1

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1
.0 Introduction
About 12 million people in developing world are affected by leishmaniasis while more than 350
million are at danger. Two million new cases are registered each year while estimated 51,000
annual deaths had been recorded and reported [1-3].
Leishmaniasis is a parasitic infestation manifested by four central clinical syndromes, i.e.
cutaneous leishmaniasis (CL), visceral leishmaniasis/kala azar (VL), mucocutaneous
leishmaniasis (MCL) and post ‘kala azar’ dermal leishmaniasis (PKDL). Nearly 90% of all VL
cases occur in 3 endemic foci: 1. India, Bangladesh, and Nepal; 2. East Africa; and 3. Brazil.
Albeit of high pervasiveness, existing squads to battle against leishmaniasis are inadequate and
limited. The use of pentavalent antimonials as standard treatment for leishmaniasis for many
decades is not influential in nearly 60% of VL cases worldwide due to widespread drug
resistance. In last decade, several new VL treatments have emerged but each of that appeared
with some serious limitations including the patients with HIV/VL coinfections, which is more
difficult to treat [4]. Pentamidine and amphotericin B, the second line of drugs, due to their
severe toxicities and economic inviability, had to suffer from a limited utility [5, 6]. Further to
this, studies revealed the evaluation of some vaccine candidates against leishmanial infection.
These vaccines could be considered promising and low-priced with reasonable immunogenicity,
however, a successful prophylactic vaccine is yet to be recognized [7, 8].
Survival and growth of leishmanial parasite is dependent upon a polyamine bases particularly
spermidine. In turn, the polyamine bases are synthesized in a metabolic process that involves the
enzyme arginase (ARG) and polyamine precursors present in microenvironment [9]. Interfering
with either polyamine bases directly or with the biosynthetic pathways of these bases chemically
could result in a potential solution to halt the leishmanial infection.
At the periphery of bio-inspired and logical drug design, it has become crucial to synthesize
hybrid molecules with two or more structural domains having different biological roles. These
hybrid motifs may act as two distinct pharmacophoric units [10, 11]. Keeping in view of these
fundamentals, the synthesis of new antileishmanial agents is centered on the concept of
molecular hybridization. The notion has also successfully been employed and earned much
attention in our research group to mature the hybrid motifs for a particular biological target [12].
2

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Our search for the development of new antileishmanial agents [13] lead us to design new and
unique structural motifs consisting of phenyl linked oxadiazole-benzohydrazone hybrids (Fig. 1).
Figure1: Design of new hybrid structures as potent antileishmanial scaffolds
Most of the hydrazone derivatives are known to be associated with a broad spectrum of
biological potentials [14-20] while 1,3,4-oxadiazole derivatives, an important class of
heterocyclic compounds has attracted tremendous attention in drug development [21-32]. With
this background, we hereby present the synthesis of new phenyl linked oxadiazole-
phenylhydrazone hybrids 6-35 as new antileishmanicidal agents (Scheme 2).
2
.1.0 Chemistry
The synthetic route towards 6-35 is shown in Scheme-1 and 2. Hydrazide 2 was synthesized
from its corresponding methyl 2-methoxybenzoate ester upon reflux with hydrazine hydrate. The
resulting hydrazide 2 was then treated with different aromatic aldehydes to yield hydrazone 3,
which upon oxidative cyclization with phenyliododiacetate [PhI(OAc) ] in dry chloroform
2
afforded oxadiazole 4. Compound 4 was further converted into its corresponding hydrazide by
treating it with hydrazine hydrate to afford phenyl linked oxadiazole-hydrazide hybrid 5 (Scheme
1
).
3

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Scheme-1. Synthesis of oxadiazole based hydrazide analog 5
In order to achieve the target molecules 6-35, phenyl linked oxadiazole-hydrazide hybrid 5 was
then reacted with various aromatic aldehydes in the presence of catalytic amount of acetic acid
(Table-1). Structures of the all newly synthesized hybrid analogs 6-35 were confirmed through
spectroscopic techniques such as NMR, MS and were further confirmed using CHN analysis.
Scheme-2. Synthesis of phenyl linked oxadiazole-benzohydrazone hybrids 6-35
4

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Table-1. Oxadiazole-benzohydrazones hybrids 6-35, their antileishmanial potential and
Selectivity Index (SI)
IC50
IC50
IC50
Mean ± SEM )
(
Mean ±
(
Mean ±
a
R
SI
No.
a
SEM ) [
cell line U-
37
µM]
a
(
SEM ) [
µ
M]
[µM]
THP-1 cells
9
b
b
6
7
4
.14 ± 0.06
50
22
207.2 ± 2.5
NA
NA
1
8.12 ± 0.100
398.1 ± 3.1
260.7 ± 2.6
b
b
8
9
7
.45 ± 0.11
35
NA
NA
OH
HO
1
.40 ± 0.02
72.6
101.6 ± 1.4
b
b
1
1
0
1
0
.95 ± 0.01
105.3
22.44
100.0 ± 1.2
100.1 ± 1.1
NA
NA
4.46 ± 0.04
b
1
1
1
2
3
4
3
.78 ± 0.03
27.5
5.5
103.9 ± 1.2
5.40 ± 0.1
12.40 ± 0.2
NA
0
.98 ± 0.01
7.46 ± 0.1
4
.78 ± 0.06
2.6
15.20 ± 0.1
5

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b
b
1
1
5
6
6
.6 ± 0.08
46.6
40.6
307.5 ± 3.2
260.1 ± 2.6
NA
NA
6
.40 ± 0.12
b
b
1
1
7
8
1
6.3 ± 0.68
18.6
60.4
303.2 ± 2.2
313.0 ± 2.3
NA
NA
5.18 ± 0.08
b
b
1
2
2
9
0
1
6
.35 ± 0.02
55.6
30.1
353.0 ± 2.7
427.4 ± 4.0
-
NA
NA
NA
1
2
3
4.20 ± 0.18
9.10 ± 1.12
4.10 ± 1.02
b
b
NA
b
b
b
2
2
2
2
2
3
4
5
NA
-
NA
b
2
0.7 ± 0.61
NA
-
NA
5.12 ± 0.06
15.4
7.4
78.8 ± 0.8
67.0 ± 0.7
75.60 ± 0.9
68.60 ± 0.8
9
.06 ± 0.1
6

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b
b
2
2
6
7
4
6.48 ± 1.15
8.48 ± 1.02
3.6
4.5
167.3 ± 1.2
173.1 ± 1.4
NA
NA
3
b
b
2
8
7
8.6 ± 1.78
NA
NA
NA
-
-
-
NA
NA
NA
b
b
b
b
2
3
9
0
7
4.20 ± 1.46
3
4
4.12 ± 1.05
0.20 ± 1.20
b
b
3
3
1
2
NA
NA
-
-
NA
NA
b
b
29.6 ± 0.85
b
3
3
64.5 ± 1.61
2.5
161.2 ± 1.3
NA
b
b
3
3
4
5
7
4.2 ± 1.8
1.6
3.6
118.7 ± 1.0
203.2 ± 2.1
7.7 ± 0.1
NA
NA
5
6.45 ± 0.16
Standard:
7
.02 ± 0.09
1.10
6.02 ± 0.1
Pentamidine
a
b
SEM standard error of the mean. NA Not Active; Selectivity index (SI) is the ratio of IC50 of THP-1 cells
and to that of antileishmanial IC50 values. Cytotoxcity is also measured against cell line U-937 of all
compounds upto 50 µM.
7

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2
.2. In Vitro Antileishmanial Activity
At present, chemotherapy is the leading armory to confront the issue of Leishmaniasis.
Many commercially available regiments that comprise of pentavalent antimonials, amphotericin
B, pentamidine, aminosidine, miltefosine, allopurinol, some azole derivatives, sitamaquine and
immunomodulators are the standard choice of drugs to counter the infection [33]. However, the
increasing occurences of drug resistance in leishmanial infections, made it imperative to develop
new remedies that are economical, possibly orally available, and safer. Pentamidine that belongs
to diamidine class of drugs is one of the available regiment against leishmaniasis in present day
(
Figure 2). The compound is known to exert antiparasitic activity by binding to DNA. Presence
of highly electronegative atoms such as nitrogen and oxygen on this molecules are proposed to
deter with polyamine metabolism and disruption of mitochondrial membrane potential [34].
Figure 2. (a) Pentamidine, first-line antileishmanial drug belonging to diamidine class of
compounds and (b) Spermidine, an agent that assists growth and survival of leishmanial parasites
The newly synthesized hybrid analogs 6-35 exhibited varying degree of antileishmanial potential
with IC50 values ranging between 0.95 ± 0.01 – 78.6 ± 1.78
pentamidine (IC = 7.02 ± 0.09 M) (Table-1). Compound 10, a 3,4-dihydroxy analogue, with
M, was found to be the most potent antileishmanial agent that is seven
7) times more active than the standard drug pentamidine (IC = 7.02 ± 0.09 M). Other
compounds that showed antileishmanial potential are 13, 9, 12, 6, 11, 14, 24, 18, 16, 19, 15 and 8
with IC50 values ranging between 0.98 ± 0.01 - 7.45 ± 0.11 M. Thus the current series 6-35
conceded in the identification of thirteen (13) new but exceptionally potent antileishmanial
compounds while compound 8 (IC = 7.45 ± 0.11 M) is equivalent in potential with that of
pentamidine (IC = 7.02 ± 0. 09µM). These compounds may serve as viable leads and could be
µM, as compared to standard
µ
5
0
IC value of 0.95 ± 0.01
µ
5
0
(
µ
5
0
µ
µ
5
0
5
0
convincing starting points to convert them into new antileishmanial era of therapies.
8

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Among halogen substituted analogs, compound 18 with ortho fluoro substitution (IC50
=
5
.18 ± 0.08 µM) and 19 with meta fluoro substitution (IC = 6.35 ± 0.02 µM) were found better
50
active than the standard while compound 20, a para fluoro analog (IC50 = 14.20 ± 0.18 µM)
displayed weaker inhibitory activity. However, mono chloro substituted analogues 21 (IC50
=
2
9.10 ± 1.12 µM), 22 (IC = 34.10 ± 1.02 µM), and 23 (IC = 20.7 ± 0.61 µM) were found to
50
50
be weaker than the flouro substituted compounds. Compound 24, a 2,4-di-Cl derivative (IC50
.12 ± 0.06 µM), was found to be equivalent in leishmanicidal potential with that of analog 18.
When mono hydroxyl substitution was replaced with methoxy group, as in compound 28
=
5
and 29, the activity potential decrease five to ten times, respectively. Nitro substituted
compounds exhibited even more weaker potential in the order para- nitro (35) > ortho- nitro (33)
and then meta- nitro (34).
Concisely, it is unlikely to corroborate these results on the basis of structure-activity relationship
because of some unique features in all compounds, i.e. a biologically active oxadiazole moiety
attached with equally important benzohydrazone moiety with a broad range of substitutions and
then a phenyl linker. These groups/moieties/substitutions are capable of forming strong
organization of hydrogen bonds with metals and polyamines present in the microenvironments of
the leishmanial parasite. The machinery of leishmanial parasite operates through its enzyme’s
metabolic process that involves a binuclear manganese enzyme arginase (ARG) for its survival
and propagation. In turn, the metabolic activity produces polyamine bases that empower cell
growth and endurance [35]. Interfering either with the catalytic role of arginase or with
polyamine products, or binding with DNA terminates the leishmanial life cycle. Compounds 6-
3
5 bear such substitutions/groups that facilitate the rest of molecular structure to form strong
hydrogen bonds with polyamines to halt the scavenging ability of leishmanial parasite.
Nevertheless, further studies are vital to explore the mechanism of action of the newly
synthesized compounds on the machinery of parasite to convert these leads into new
antileishmanial targets.
2
.3. Selectivity Index (SI)
Selectivity index (SI) is the ratio of IC50 values of THP-1 cells to that of antileishmanial IC50
values. Based on IC values, the selectivity index for each compound is determined while this
5
0
parameter reflects the quantity of compound that is active against the target but is not toxic
9

ACCEPTED MANUSCRIPT
towards the host cell. In our investigations, we found that compounds with higher number of
hydroxyl groups are more active but having low selectivity index which can be seen in
compounds 13 and 14, both trihydroxyl derivatives, showed SI of 5.5 and 2.6, respectively. In
dihydroxy analogues 9, 10, 11 and 12 showed better selectivity index 72.6, 105.3, 22.6 and 27.5
respectively as compared to trihydroxy analogues with good activity as well. Furthermore, the
compounds having adjacent dihydroxy groups such as in 9 and 10 showed far better activity and
selectivity as compare to isolated dihydroxy compounds 11 and 12. When we compare
monohydroxy analogues 6, 7 and 8, we found that compound 6 is better in terms of selectivity
index as well as activity. In case of 3-substituted hydroxyl analogue 7, and 4-substituted
hydroxyl analogue 8, the activity and selectivity are both dropped. Therefore, the selectivity
index in monosubstituted hydroxyl analogues is in the order of 2-OH> 4-OH > 3-OH. Among
halogenated compounds fluorinated compounds 18, 19 and 20 showed very good activity with
very high selectivity index ranging between 60-30 with order of 2-F >3-F >4-F. The chlorinated
compounds 21, 22 and 23 showed no cytotoxicity. The nitro compounds 33, 34 and 35 showed
very weak activity with low selectivity index. The methyl substituted compounds 30, 31 and 32
showed no cytotoxicity and similar results obtained for methoxy substituted compounds 28 and
2
9. Different pyridine substituted compounds 25, 26 and 27 showed moderate selectivity index
with good to moderate activity Table-1.
2
.4. Molecular docking studies of oxadiazole-benzohydrazone hybrids on Pteridine
reductase
Leishmania species requires reduced pteridines (pterins and folates) for biosynthesis of nucleic
acid and protein and therefore they uptake biopterin or folate from their host body. Futher these
two compounds undergo two successive reductions to generate the active form of tetrahydro-
species. Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine
reductase (PTR1) are the two enzymes carry out these reactions. PTR1 is the major enzyme for
the reduction of folate and 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) [36]. PTR1
is the only enzyme that has been reported to reduce biopterin in Leishmania species and have
been shown to be vital for growth in vivo [37]. Fortunately, PTR1 is less susceptible to inhibition
by clinical DHFR inhibitors like methotrexate and it is therefore likely to be accountable for the
failure of antifolate therapeutic agents targeting DHFR by acting as a metabolic bypass [38, 36].
10

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In this perspective, PTR1 represents as an attractive new antileishmanial drug target for
leishmaniasis. The present study endeavor the probable binding modes of benzohydrazone
derivatives.
From molecular docking of the hybrid derivatives, only four potent compounds (10, 13, 9
and 12) were selected for binding mode analysis and are discussed in detail in the following
section. Figure-3 shows that the potent compounds of the oxadiazole-benzohydrazone hybrids
adopt binding mode orientation in such a way that the Ar substituted group projects into the
narrow tunnel zone of the Pteridine reductase active site (black dotted circled area in Figure-3)
establishing stable interaction, while the standard drug (Pentamidine) binding mode was unable
to reach this zone, making Pentamidine 7 times less active than the four potent compounds.
Figure-3: Shows the binding mode of the active benzohydrazone derivatives (blue color line) in
comparison with the binding mode of standard drug (Pentamidine) in magenta color stick.
Figure-4a shows the binding mode of the most potent compound 10 (IC = 0.95 ± 0.01µM)
5
0
among the benzohydrazone derivatives. The binding orientation shows that the 3,4-dihydroxy
group over the aryl group forms stable hydrogen bond with the backbone oxygen of Gly13. In
addition the aryl ring projected to the deep narrow into the pocket forms well established π- π
stacking and cation-π interaction with the guanidinium group of Arg17 and hydrophobic
interaction with Ala15 and Leu18, respectively. Subsequently, rest of the moieties of the
compound10 establishes hydrophobic interaction, where the benzene ring interacts with Phe113,
11

ACCEPTED MANUSCRIPT
Tyr194, Pro224, Val228 and Leu229. Additionally, the methoxyphenyl oxadiazole ring forms
hydrophobic interactions with Met183, Pro187, Leu226, Met233, Leu188 and Tyr283.
Likewise in the case of compound 13 (IC50: 0.98 ± 0.01µM) the aryl substituted 2,4,5-
trihydroxy groups play the central role in the activity profile, by establishing hydrogen bonding,
2
5
-hydroxy with NH2 of Leu18 main chain, 4-hydroxy with the main chain oxygen of Gly13 and
-hydroxy with the main chain oxygen of Ser111, respectively (Fig.4.b). Meanwhile a cation- π
interaction is observed between the aryl ring and the NH3 side chain group of Lys16.
Consequently, the benzene ring of the compound13 establishes hydrophobic interaction with
Ala110 and Val228. While the methoxyphenyl oxadiazole ring forms hydrophobic interactions
with Leu18, Phe113, Tyr194, Leu229, Met183, Pro187, Leu226, Met233, Leu188, Tyr283 and
Pro224.
Fig.4: Illustrates the predicted binding modes of active benzohydrazone derivatives. Binding
modes of (a) compound 10, (b) compound 13, (c) compound 9 and (d) compound 12. Key
residues are represented in spear and labeled. The hydrogen bond interactions are represented by
12

ACCEPTED MANUSCRIPT
magenta color arrow (dotted with side chain and continues with main chain residues
respectively), π-π interactions are shown in green color lines and cation–p interactions are shown
in red color lines. Compounds are shown in line form.
Subsequently in the case of compound 9 (IC = 1.40 ± 0.02 µM), both hydroxyl group
5
0
2
on the aryl ring form hydrogen bond with the backbone NH of Gly19 and oxygen of Gly13,
respectively. While the amino group of Lys16 forms cation-π interaction with the aryl group.
There is also a hydrogen bond interaction between the carbohydrazide oxygen and the side chain
NH2 of Arg17. Additionally, there is a π-π stacking between the benzene ring and Phe113. The
complex is stabilized by hydrophobic interaction with methoxyphenyl oxadiazole ring and
Leu229, Met233, Leu226, Tyr194, Pro187, Met183, Leu188 and Tyr283, respectively (Figure-
4
c).
Figure-4d show the binding mode of compound 12 with fairly good activity (IC = 3.78 ± 0.03
5
0
µM). The aryl ring substituted with 2,4-dihydroxy forms a hydrogen bond with main chain
oxygen of Gly19 and with main chain oxygen of Gly13, respectively. In addition, there is also a
hydrogen bond between the side chain NH of Arg17 and carbohydrazide oxygen of compound
1
2. Similar to that of compound 9 there is π-π stacking between benzene ring and the Phe113.
The complex is also stabilized by hydrophobic interaction between the methoxyphenyl
oxadiazole ring moiety and hydrophobic restudies such as Met233, Leu188, Tyr283, Leu226,
Met183 and Pro224, respectively.
Among the oxadiazole-benzohydrazone hybrid analogs, the presence of hydroxyl group
substituted on the aryl ring is the key determinant of the potency of the compound and has been
observed among compound 6-16. Substitution of the aryl ring with methoxy (compound 15, 16,
1
7, 28, 29) or a methyl group (compound 30, 31, 32), nitro (compound 33-35) or halogen
(compound 18-24) moieties eventually decrease the activity profile. This could be due to the
presence of hydrophilic residues at the narrow core pocket of the Pteridine reductase active site
that interact with the aryl substituted moieties.
4
.0 Conclusion
In the current study, we have identified new hybrid structures as potent antileishmanial
scaffolds. It was found that the antileishmanial potential of hybrid analogs was more promising
then the antileishmnial potentials of individual components such as oxadiazoles and
13

ACCEPTED MANUSCRIPT
benzohydrazones. Selectivity index showed that compounds with higher number of hydroxyl
groups have low selectivity index. The dihydroxy compounds showed good activity and
selectivity index. Among halogenated compounds, compounds with fluorine substitution
displayed good activity and selectivity index with the order of 2-F >3-F >4-F. DFT calculations
confirmed the presence of E-configurations while theoretical IR and NMR analysis corroborated
with experimental data.
Acknowledgments
Muhammad Taha would like to acknowledge the Ministry of Higher Education (MOHE) for
financial support under the Fundamental Research Grant Scheme (FRGS) with sponsorship
reference numbers FRGS/1/2016/STG01/UiTM/02/2 and Atta-ur-Rahman Institute for Natural
Product Discovery (RiND) for providing excellent laboratory and facility for the research and all
technical and non-technical staff for a lot of support for this work.
5
.0 General Experimental
NMR experiments were performed on Ultra Sheild Bruker FT NMR 500 MHz. IR experiments
were performed on Perkin Elmer FT-IR and UV, Perkin Elmer Lambda 35 UV-Vis
Spectrometer. CHN analysis was performed on a Carlo Erba Strumentazion-Mod-1106, Italy.
Ultraviolet Electron impact mass spectra (EI MS) were recorded on a Finnigan MAT-311A,
Germany. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum
plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms were visualized by using UV at
2
54 and 365 nm.
5
.1. In vitro Leishmaniasis Assay:
The assay was carried out according to Seifert & Croft (2006). Briefly, THP-1 cells (ATCC)
were cultured in RPMI-1640 (R5886 Sigma) supplemented with 1% L-glutamine and 10%
6
HIFBS (complete medium) before harvested at 1.0 x 10 cells/mL. Cells were diluted to 2.0 x
5
1
0 cells/mL with the complete medium, seeded in 16-well Lab Tek tissue culture chamber slide
4
(
Fisher Scientific) at a seeding density of 5.0 x 10 macrophage/well (100 µL) and allowed to
adhere by the addition of PMA (Phorbol -12 myristate Acetate P8139 Sigma) for 3 days at 37 °C
in a 5% CO – 95% air mixture. Macrophages were then infected with long-slender (stationary
2
stage) of Leishmania major promastigote (JISH118) obtained from The London School of
14

ACCEPTED MANUSCRIPT
Hygiene and Tropical Medicine (LSHTM) United Kingdom, which were cultured at 26°C in
Schneiders Drosophila Medium (S0146 Sigma), at a macrophage-promastigote ratio of 1:5.
Infected macrophages were maintained at 34 °C in a 5% CO – 95% air mixture. After 48 hours,
2
extracellular parasites were removed by substituting the overlay with new fresh RPMI-1640
medium supplemented with 1% L-glutamine. Fresh Pentamidine and test compounds with
various concentrations were added and drug or compound activity was determined from the
percentage of infected cells in drug-treated cultures in relation to non-treated cultures using
GraphPad Prism after methanol fixation and Giemsa staining. All testing were triplicated.
5
.1.1 Detection of Cytotoxicity on Vero Cells.
The cytotoxicity assay was performed as described by [12c]. Briefly, Vero (African green
monkey kidney) cells were cultivated in tissue culture flasks with Eagle’s modified essential
medium (MEM; Gibco), supplemented with 5% heat-inactivated fetal bovine serum in 37ºC, 5%
CO
2
/95% air in carbon dioxide incubator. Confluent monolayers were removed with trypsin
5
EDTA, resuspended to approximately 1 x 10 cells/ml in MEM and 0.1mL of cells were pipetted
into each well of a 96-well microtiter plate. After incubation at 37ºC in 5% CO /95% air for
2
overnight, the cells were treated with compounds. Pentamidine was used as positive control and
MEM with 1% DMSO was used as negative control. Each sample was tested in duplicate.
Cytotoxic activity of Vero cells was measured via Alamar Blue Assay and IC50 value was
determined using Excel and GraphPad Prism.
5
.2. Synthesis of 2-methoxybenzohydrazide (2)
Methyl 2-methoxybenzoate (1) (50 mmol, 8.31g) and 50 ml of hydrazine hydrate were mixed in
methanol (100 mL). The mixture was refluxed for 6 hours. Methanol was then evaporated and
the product formed was being rinsed with plenty of water to remove excess hydrazine hydrate.
The product formed was left to dry at room temperature and yielded 94% (7.82g)
5
.3. Synthesis of (E)-methyl 4-((2-(2-methoxybenzoyl)hydrazono)methyl)benzoate (3)
15

ACCEPTED MANUSCRIPT
A mixture of compound 2 (7.00 g, 46 mmol), methyl 4-formylbenzoate (7.56 g, 46 mmol) and
catalytic amount of acetic acid in methanol (50 mL) was refluxed for 3 hours. The solvent was
evaporated and the residue (3) was washed with diethyl ether, filtered, dried, and then
crystallized from ethanol and gives white solid, (12.8 g, 93%).
5
.4.
Synthesis of methyl 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzoate (4)
A mixture of compound 3 (11.0 g, 37 mmol) and equivalent amount of PhI(OAc) was stirred in
2
dichloromethane (100 ml) at room temperature overnight. The solvent was evaporated and the
residue (4) was washed with diethyl ether, filtered, dried, and then crystallized from ethanol to
gives white solid, (9.8 g, 89 %)..
5
.5. Synthesis of 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzohydrazide (5)
Compound 4 (9.00 g, 30 mmol) and 15 ml of hydrazine hydrate were mixed in methanol (50
mL). The mixture was refluxed for 6 hours. Methanol was then evaporated and the product
formed was being rinsed with plenty of water to remove excess hydrazine hydrate. The product
formed (5) was left to dry at room temperature and yielded 8.50 g (94 %).
5
.6. General procedure for synthesis of oxadiazole benzohydrazones (6-35)
Equimolar quantities (1 mmol) of compound 5 and substituted benzaldehydes (1 mmol) in
methanol (25 mL) were refluxed for 3 h, in the presence of catalytic amount of glacial acetic
acid. The resulting solid was filtered and recrystallized from methanol in good yields.
6
.0. N'-(2-hydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (6)
1
Yellowish white. Yield: 72.9 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.31 (s,
6
1
H), 11.20 (s, 1H), 8.68 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.01 (dd, J =
.0, 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.34-7.30 (m, 2H), 7.20 (t, J
2
1
3
=
8.0 Hz, 1H), 6.96-6.93 (m, 2H), 3.90 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.23,
1
64.45, 158.31, 158.17, 156.24, 151.67, 133.86, 132.19, 131.91, 131.29, 129.82, 128.74, 128.21,
16

ACCEPTED MANUSCRIPT
1
28.21, 126.72, 126.72, 121.03, 120.25, 120.19, 118.41, 117.16, 115.35, 56.72; HR-ESI-MS:
m/z calcd for C H N O , [M]+ 414.1328; Found 414.1335;
2
3
18
4
4
6
.1. N'-(3-hydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzohy-
drazide (7)
1
White. Yield: 82.6%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 11.99 (s, 1H), 9.65
(s, 1H), 8.41 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.15 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 7.5 Hz, 1.5
Hz, 1H), 7.66 (t, J = 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.24 (s, 1H),
1
3
7
.19 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 3.97 (s, 3H); C
NMR (150 MHz, DMSO-d6): δ 165.37, 164.52, 158.41, 156.72, 156.63, 148.58, 137.18, 133.12,
1
1
4
6
32.49, 131.92, 131.39, 130.29, 128.31, 128.31, 126.72, 126.72, 120.45, 120.13, 119.26, 118.31,
15.39, 114.74, 56.71; HR-ESI-MS: m/z calcd for C H N O , [M]+ 414.1328; Found
2
3
18
4
4
14.1324;
.2. N'-(4-hydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (8)
1
Yellowish white. Yield: 76.2 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 11.84 (s,
1
H), 9.96 (s, 1H), 8.39 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 7.5
Hz, 1H), 7.66 (t, J = 7.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J =
13
7
1
1
.5 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.42,
64.57, 158.38, 158.21, 156.64, 149.34, 133.41, 132.09, 131.98, 131.69, 129.62, 129.62, 128.47,
28.47, 126.84, 126.84, 126.68, 120.35, 118.13, 115.83, 115.83, 115.35, 56.68; HR-ESI-MS:
m/z calcd for C H N O , [M]+ 414.1328; Found 414.1321;
2
3
18
4
4
6
.3. N'-(2,3-dihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (9)
Yellowish white. Yield: 94.3 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
2
1
6
): δ 12.35 (s,
H), 9.24 (s, 1H), 8.65 (s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 8.5 Hz, 2H), 8.01 (dd, J =
.5 Hz, 1.5 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H),
1
7
7
1
3
.01 (d, J = 7.5 Hz, 1H), 6.88 (d, J = 7.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 3.97 (s, 3H); C
NMR (150 MHz, DMSO-d6): δ 165.47, 164.32, 158.21, 156.64, 149.93, 146.94, 144.69, 133.41,
17

ACCEPTED MANUSCRIPT
1
5
32.09, 131.98, 131.59, 128.38, 126.89, 122.00, 121.33, 120.55, 119.93, 119.85, 118.38, 115.30,
6.78; HR-ESI-MS: m/z calcd for C H N O , [M]+ 430.1277; Found 430.1272.
2
3
18
4
5
6
.4. N'-(3,4-dihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (10)
Light yellow. Yield: 74.7 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
.40 (s, 1H), 9.27 (s, 1H), 8.31 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.13 (d, J = 8.0 Hz, 2H), 8.01
d, J = 7.5 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.19 (d, J =
2
1
6
): δ 11.79 (s, 1H),
9
(
1
3
7
.0 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 3.97 (s, 3H); C NMR (150
MHz, DMSO-d6): δ 165.67, 164.31, 158.26, 156.67, 148.28, 148.17, 145.32, 133.61, 132.01,
1
1
31.68, 131.39, 128.32, 128.32, 127.72, 126.89, 126.89, 121.14, 120.75, 118.28, 116.43, 115.98,
15.36, 56.38; HR-ESI-MS: m/z calcd for C H N O , [M]+ 430.1277; Found 430.1283.
2
3
18
4
5
6
.5. N'-(2,5-dihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (11)
White. Yield: 78.6 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
2
1
6
): δ 12.17 (s, 1H), 10.31
(s, 1H), 8.98 (s, 1H), 8.63 (s, 1H), 8.24 (d, J = 7.0 Hz, 2H), 8.15 (d, J = 7.0 Hz, 2H), 8.01 (dd, J
=
8.0 Hz, 1.5 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H),
1
3
7
1
1
.03 (d, J = 2.5 Hz, 1H), 6.74-6.79 (m, 2H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ
65.53, 164.52, 158.11, 156.84, 152.73, 151.29, 149.51, 133.42, 132.03, 131.74, 131.29, 128.34,
28.34, 126.79, 126.79, 122.17, 121.32, 120.69, 118.27, 118.12, 116.38, 115.32, 56.72; HR-ESI-
MS: m/z calcd for C H N O , [M]+ 430.1277; Found 430.1273.
2
3
18
4
5
6
.6. N'-(2,4-dihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (12)
Light yellow. Yield: 89.2 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
2
1
6
): δ 12.18 (s, 1H),
1.56 (s, 1H), 8.60 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 7.5 Hz,
H), 7.66 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.5
1
1
1
3
Hz, 1H), 6.55(dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.52 (d, J = 2.0 Hz, 1H), 3.97 (s, 3H); C NMR (150
MHz, DMSO-d6): δ 165.47, 164.32, 162.58, 161.62, 158.24, 156.37, 151.21, 133.63, 132.29,
18

ACCEPTED MANUSCRIPT
1
1
31.74, 131.15, 130.86, 128.48, 128.48, 126.79, 126.79, 120.51, 118.36, 115.20, 113.16, 109.25,
03.14, 56.58; HR-ESI-MS: m/z calcd for C H N O , [M]+ 430.1277; Found 430.1282.
2
3
18
4
5
6
.7. N'-(2,4,5-trihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (13)
White. Yield: 67.4 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
2
1
6
): δ 11.99 (s, 1H), 10.54
(s, 1H), 9.57 (s, 1H), 8.57 (s, 1H), 8.51 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H),
8
1
.01 (d, J = 7.0 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz,
13
H), 7.19 (t, J = 8.5 Hz, 1H), 6.93(s, 1H), 6.36 (s, 1H), 3.97 (s, 3H); C NMR (150 MHz,
DMSO-d6): δ 165.41, 164.36, 158.71, 156.43, 153.74, 150.81, 149.41, 140.51, 133.48, 132.13,
1
5
31.84, 131.51, 128.47, 128.47, 126.75, 126.75, 120.58, 118.25, 117.21, 115.19, 112.86, 102.84,
6.36; HR-ESI-MS: m/z calcd for C H N O , [M]+ 446.1226; Found 446.1235.
2
3
18
4
6
6
.8. N'-(2,4,6-trihydroxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-
yl)benzohydrazide (14)
Yellow. Yield: 94.1%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
2
1
6
): δ 12.08 (s, 1H), 11.10
s, 1H), 9.84 (s, 1H), 8.85 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 7.5 Hz, 2H), 8.01 (d, J =
.5 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 5.87 (s,
(
7
1
1
1
4
1
3
H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.45, 164.38, 163.12, 161.46, 161.46,
58.31, 156.69, 144.76, 133.44, 132.16, 131.85, 131.52, 128.46, 128.46, 126.84, 126.84, 120.52,
18.34, 115.31, 106.49, 95.87, 95.87, 56.75; HR-ESI-MS: m/z calcd for C23
46.1226; Found 446.1234.
18 4 6
H N O , [M]+
6
.9. N'-(2-hydroxy-4-methoxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (15)
1
Brown. Yield: 74.5 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.32 (s, 1H), 8.62
(s, 1H), 8.59 (s, 1H), 8.17-8.29 (m, 7H), 8.01 (d, J = 7.5 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.66
1
3
(
t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150
MHz, DMSO-d6): δ 165.43, 164.36, 162.02, 161.13, 158.31, 156.68, 151.19, 133.51, 132.01,
1
1
31.78, 131.53, 130.62, 128.45, 128.45, 126.72, 126.72, 120.35, 118.32, 115.37, 113.74, 107.36,
02.15, 56.74, 56.23; HR-ESI-MS: m/z calcd for C24 , [M]+ 444.1434; Found 444.1429.
20 4 5
H N O
19

ACCEPTED MANUSCRIPT
6
.10. N'-(3-hydroxy-4-methoxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2
yl)benzohydrazide (16)
1
Dark brown. Yield: 74.4%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.32 (s, 1H),
8
7
.62 (s, 1H), 8.59 (s, 1H), 8.17-8.29 (m, 7H), 8.01 (d, J = 7.5 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H),
1
3
.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR
(150 MHz, DMSO-d6): δ 165.42, 164.62, 158.31, 156.68, 149.60, 148.63, 146.85, 133.43,
1
1
4
32.49, 131.72, 131.51, 129.36, 128.24, 128.24, 126.73, 126.73, 120.53, 120.45, 118.42, 115.57,
15.24, 115.09, 56.72; HR-ESI-MS: m/z calcd for C24
44.1432.
20 4 5
H N O , [M]+ 444.1434; Found
6
.11. N'-(2-hydroxy-5-methoxybenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (17)
Yellowish white. Yield: 86.2 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
1
6
): δ 11.89 (s,
H), 9.39 (s, 1H), 8.32 (s, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.13 (d, J = 8.0 Hz, 2H), 8.01 (dd, J =
.0, 8.0 Hz, 1H), 7.67 (dt, J = 2.0, 8.5 Hz, 1H), 7.32 (s, 1H), 7.30 (dd, J = 2.0, 2.5 Hz, 1H), 7.20
1
2
(t, J = 8.0 Hz, 1H), 6.94 (dd, J = 2.0, 9.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 3.95 (s, 3H), 3.82 (s,
1
3
3
1
1
4
H); C NMR (150 MHz, DMSO-d6): δ 165.38, 164.36, 158.25, 156.63, 154.48, 154.34,
49.65, 133.46, 132.19, 131.46, 131.63, 128.63, 128.63, 126.82, 126.82, 121.75, 120.58, 118.33,
16.51, 116.34, 115.32, 112.87, 56.74, 56.13; HR-ESI-MS: m/z calcd for C H N O , [M]+
2
4
20
4
5
44.1434; Found 444.1439.
6
.12. N'-(2-fluorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (18)
White. Yield: 68.9 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.18 (s, 1H), 8.75
1
6
(s, 1H), 8.24 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 8.7 Hz, 2H), 8.01 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 7.98
(d, J = 6.0 Hz, 2H), 7.66 (t, J = 8.5 Hz, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H),
1
3
7
.32 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-
d6): δ 165.41, 164.37, 161.41, 159.33, 158.26, 156.62, 149.38, 149.18, 133.51, 132.29, 131.72,
20

ACCEPTED MANUSCRIPT
1
1
31.49, 130.51, 130.73, 129.63, 129.17, 128.24, 128.24, 126.82, 126.82, 125.45, 125.21, 124.76,
24.24, 120.75, 118.32, 117.43, 117.23, 115.28, 56.73; HR-ESI-MS: m/z calcd for C H FN O ,
2
3
17
4
3
[M]+ 416.1285; Found 416.1281.
6
.13. N'-(3-fluorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (19)
1
Light yellow. Yield: 74.1 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.17 (s, 1H),
6
8
7
1
.51 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 7.5 Hz, 1.5 Hz, 1H),
.66 (t, J = 9.0 Hz, 2H), 7.61 (d, J = 9.5 Hz, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 6.0 Hz,
1
3
H), 7.32 (d, J = 8.5 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz,
DMSO-d6): δ 165.43, 164.36, 163.61, 161.75, 158.26, 156.68, 148.62, 148.61, 138.67, 138.53,
1
1
33.46, 132.02, 131.68, 131.39, 130.12, 129.92, 128.38, 128.38, 126.81, 126.81, 121.79, 121.35,
20.58, 118.18, 117.08, 116.72, 115.26, 114.36, 114.12, 56.72; HR-ESI-MS: m/z calcd for
23 4 3
C H17FN O , [M]+ 416.1285; Found 416.1279.
6
.14. N'-(4-fluorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (20)
1
Yellow. Yield: 78.6 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.07 (s, 1H), 8.50
6
(s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 7.83
(t, J = 6.0 Hz, 2H), 7.66 (t, J = 9.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H),
1
3
7
1
1
.19 (t, J = 8.0 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.45, 164.36,
62.31, 160.27, 158.23, 156.64, 149.34, 133.41, 132.09, 131.98, 131.59, 130.73, 130.70, 130.40,
30.14, 128.32, 128.32, 126.69, 126.69, 120.51, 118.48, 115.67, 115.67, 115.28, 115.06, 56.68;
4 3
HR-ESI-MS: m/z calcd for C23H17FN O , [M]+ 416.1285; Found 416.1289.
6
.15. N'-(2-chlorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzohyd-
razide (21)
White. Yield: 93.8 %. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.27 (s, 1H), 8.92
1
6
(s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 6.0 Hz, 1H), 8.01 (dd, J =
8
7
.0 Hz, 1.5 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 6.5 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H),
1
3
.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6):
21

ACCEPTED MANUSCRIPT
δ 165.49, 164.37, 158.26, 156.34, 149.92, 133.45, 132.82, 132.18, 131.82, 131.49, 131.16,
1
30.91, 128.62, 128.31, 128.31, 127.53, 127.36, 126.81, 126.81, 120.35, 118.32, 115.28, 56.71;
HR-ESI-MS: m/z calcd for C H ClN O , [M]+ 432.0989; Found 432.0993.
2
3
17
4
3
6
.16. N'-(3-chlorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (22)
1
White. Yield: 77.3%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 11.87 (s, 1H), 9.32
6
(s, 1H), 8.35 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 7.0 Hz, 1H),
7
.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 9.0 Hz, 1H), 7.30 (s, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.09 (d, J
13
=
8.0 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 3.97 (s, 3H), 3.82 (s, 3H); C NMR (150 MHz, DMSO-
d6): δ 165.42, 164.22, 158.18, 156.67, 148.63, 135.38, 134.19, 133.31, 132.79, 131.81, 131.49,
30.19, 128.28, 128.28, 128.05, 127.63, 126.84, 126.84, 125.15, 120.51, 118.32, 115.26, 56.58;
HR-ESI-MS: m/z calcd for C H ClN O , [M]+ 432.0989; Found 432.0984.
1
2
3
17
4
3
6
.17. N'-(4-chlorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (23)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.12 (s,
6
1
1
H), 8.49 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 1.5, 7.5 Hz,
H), 7.79 (d, J = 8.0 Hz, 2H), 7.66 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.5
1
3
Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.41,
1
1
64.38, 158.11, 156.61, 149.32, 135.19 134.13, 133.45, 132.29, 131.89, 131.52, 129.43, 129.43,
29.03, 129.03, 128.41, 128.41, 126.72, 126.72, 120.51, 118.31, 115.27, 56.71; HR-ESI-MS:
m/z calcd for C H ClN O , [M]+ 432.0989; Found 432.0982.
2
3
17
4
3
6
.18. N'-(2,4-dichlorobenzylidene)-4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (24)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.34 (s,
6
1
7
H), 8.83 (s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.16 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.5 Hz, 1H),
.730 (s, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 7.20 (t,
1
3
J = 7.5 Hz, 1H), 3.95 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.42, 164.35, 158.19,
56.68, 149.69, 134.68, 134.32, 133.40, 132.19, 131.91, 131.52, 131.09, 130.28, 129.07, 128.58,
1
22

ACCEPTED MANUSCRIPT
1
28.45, 128.45, 126.86, 126.86, 120.52, 118.36, 115.34, 56.71; HR-ESI-MS: m/z calcd for
C H Cl N O , [M]+ 466.0599; Found 466.0608.
23
16
2
4
3
6
.19. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-2-ylmethylene)benzohyd-
razide (25)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.26 (s,
1
7
7
H), 8.65 (d, J = 4.5 Hz, 1H), 8.53 (s, 1H), 8.26 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H),
.90-8.06 (m, 2H), 7.92 (t, J = 8.0 Hz, 1H), 7.66 (t, J = 8.5 Hz, 1H), 7.45 (t, J = 5.0 Hz, 1H),
1
3
.32 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6):
δ 165.46, 164.31, 158.26, 156.67, 154.74, 148.17, 146.15, 137.67, 133.45, 132.01, 131.28,
31.55, 128.34, 128.34, 126.75, 126.75, 123.06, 120.51, 119.03 118.33, 115.28, 56.72; HR-ESI-
MS: m/z calcd for C H N O , [M]+ 399.1331; Found 399.1327.
1
2
2
17
5
3
6
.20. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-3-ylmethylene)benzohyd-
razide (26)
1
Light yellow. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.22 (s, 1H),
8
2
.90 (s, 1H), 8.65 (d, J = 4.0 Hz, 1H), 8.56 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.20 (d, J = 10.0 Hz,
H), 8.17 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 7.66 (t, J = 8.5 Hz, 1H), 7.51 (d,
13
J = 8.0 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 3.97 (s, 3H); C NMR (150
MHz, DMSO-d6): δ 165.48, 164.34, 158.25, 156.69, 149.81, 146.34, 145.57, 135.56, 133.86,
1
5
33.47, 132.19, 131.68, 131.68, 128.35, 128.35, 126.75, 126.75, 124.38, 120.59, 118.28, 115.35,
6.72; HR-ESI-MS: m/z calcd for C H N O , [M]+ 399.1331; Found 399.1325.
2
2
17
5
3
6
.21. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(pyridin-4-
ylmethylene)benzohydrazide (27)
1
White. Yield: 87.3%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.32 (s, 1H), 8.69
(d, J = 3.5 Hz, 2H), 8.50 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.01 (d, J =
7
.5 Hz, 1H), 7.71 (d, J = 3.5 Hz, 2H), 7.66 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J
1
3
=
8.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.41, 164.36, 158.24, 156.67,
50.19, 150.19, 149.38, 140.76, 133.43, 132.09, 131.78, 131.69, 128.48, 128.48, 126.79, 126.79,
1
23

ACCEPTED MANUSCRIPT
1
3
22.37, 122.37, 120.53, 118.35, 115.27, 56.72; HR-ESI-MS: m/z calcd for C22
99.1331; Found 399.1337.
17 5 3
H N O , [M]+
6
.22. N'-(2-methoxybenzylidene)-4-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (28)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.07 (s,
1
1
H), 8.48 (s, 1H), 8.24 (d, J = 8.5 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H), 8.01 (dd, J = 7.5 Hz, 1.5 Hz,
H), 7.66 (t, J = 8.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.5
1
3
Hz, 1H), 7.05 (d, J = 6.5 Hz, 1H), 3.97 (s, 3H), 3.83 (s, 3H); C NMR (150 MHz, DMSO-d6): δ
1
1
65.81, 165.42, 164.37, 161.34, 159.78, 150.86, 133.46, 132.19, 128.91, 128.58, 128.31, 128.31,
27.91, 126.83, 126.83, 125.48, 125.22, 121.51, 120.57, 117.31, 114.21, 113.62, 56.98, 56.15;
HR-ESI-MS: m/z calcd for C H N O , [M]+ 428.1485; Found 428.1479.
2
4
20
4
4
6
.23. N'-(2-methoxybenzylidene)-4-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-
yl)benzohydrazide (29)
1
Light yellow. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 11.94 (s, 1H),
8
7
.42 (s, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.5 Hz, 2H), 8.01 (dd, J = 1.5, 7.5 Hz, 1H),
.72 (d, J = 8.5 Hz, 2H), 7.67 (dt, J = 2.0, 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.20 (t, J = 7.5
1
3
Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 3.96 (s, 3H), 3.82 (s, 3H); C NMR (150 MHz, DMSO-d6): δ
1
1
65.57, 164.28, 162.19, 159.54, 150.61, 133.42, 132.17, 128.28, 128.32, 128.32, 127.74, 127.17,
26.82, 126.82, 125.41, 121.64, 113.48, 113.63, 56.38, 56.23; HR-ESI-MS: m/z calcd for
C H N O , [M]+ 428.1485; Found 428.1488.
24
20
4
4
6
.24. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-
methylbenzylidene)benzohydrazide (30)
1
Brown. Yield: 84.7%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.02 (s, 1H), 8.80
(s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 8.5 Hz, 2H), 8.01 (dd, J = 7.5 Hz, 2.0 Hz, 1H), 7.66
(t, J = 8.5 Hz, 1H), 7.64 (s, 1H), 7.30-7.37 (m, 3H), 7.19 (t, J = 8.0 Hz, 1H), 3.97 (s, 3H), 2.48 (s,
1
3
3
1
H); C NMR (150 MHz, DMSO-d6): δ 165.48, 164.34, 158.26, 156.67, 145.54, 136.35,
33.61, 133.21, 132.19, 131.93, 131.45, 130.24, 128.36, 128.36, 128.16, 126.81, 126.63, 126.63,
24

ACCEPTED MANUSCRIPT
1
4
26.12, 120.45, 118.35, 115.26, 56.73, 20.43; HR-ESI-MS: m/z calcd for C24
12.1535; Found 412.1541.
20 4 3
H N O , [M]+
6
.25. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-
methylbenzylidene)benzohydrazide (31)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.03 (s,
1
7
8
H), 8.47 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 8.0 Hz, 1H),
.66 (t, J = 7.5 Hz, 1H), 7.64 (s, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.38 (t, J = 7.5, 1H), 7.30 (d, J =
1
3
.5 Hz, 1H), 7.28 (d, J = 7.5, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H), 2.38 (s, 3H); C NMR
(150 MHz, DMSO-d6): δ 165.43, 164.36, 158.24, 156.54, 148.68, 137.40, 136.63, 133.42,
1
1
4
32.13, 131.92, 131.48, 129.75, 128.51, 128.48, 128.48, 128.29, 126.72, 126.72, 123.52, 120.57,
18.48, 115.37, 56.68, 21.24; HR-ESI-MS: m/z calcd for C H N O , [M]+ 412.1535; Found
2
4
20
4
3
12.1527.
6
.26. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-
methylbenzylidene)benzohydrazide (32)
Light yellow. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 11.98 (s, 1H),
1
6
8
7
7
.47 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H), 8.01 (dd, J = 8.0 Hz, 1.5 Hz, 1H),
.64-7.68 (m, 3H), 7.32 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 10.0 Hz, 1H),
13
.19 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 3.97 (s, 3H), 2.48 (s, 3H); C NMR (150 MHz,
DMSO-d6): δ 165.43, 164.35, 158.27, 156.68, 149.37, 138.53, 133.51, 132.29, 131.91, 131.80,
1
5
31.52, 129.13, 129.13, 128.48, 128.48, 127.25, 127.25, 126.84, 126.84, 120.45, 118.34, 115.27,
6.71, 21.10; HR-ESI-MS: m/z calcd for C H N O , [M]+ 412.1535; Found 412.1542.
2
4
20
4
3
6
.27. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(2-nitrobenzylidene)benzohyd-
razide (33)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d ): δ 12.40 (s,
6
1
8
1
H), 8.93 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 7.5 Hz, 2H), 8.16 (d, J = 7.5 Hz, 1H),
.11 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 7.5 Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 7.66 (t, J = 7.5 Hz,
1
3
H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz,
DMSO-d6): δ 165.43, 164.31, 158.31, 156.61, 147.57, 142.92, 133.71, 133.46, 132.19, 131.94,
25

ACCEPTED MANUSCRIPT
1
5
31.67, 130.77, 129.30, 128.35, 128.35, 128.15, 126.78, 126.78, 125.22, 120.75, 118.34, 115.36,
6.71; HR-ESI-MS: m/z calcd for C H N O , [M]+ 443.1230; Found 443.1234.
2
3
17
5
5
6
.28. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(3-
nitrobenzylidene)benzohydrazide (34)
1
Yellowish white. Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
6
): δ 12.35 (s,
1
8
8
H), 8.59 (s, 1H), 8.30 (d, J = 7.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 2H), 8.21 (d, J = 7.0 Hz, 1H),
.16 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 2.0, 7.5 Hz, 2H), 7.79 (t, J = 7.5 Hz, 1H), 7.32 (dt, J = 2.0,
1
3
.5 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 3.96 (s, 3H); C NMR (150 MHz,
DMSO-d6): δ 165.42, 164.34, 158.18, 156.63, 148.65, 147.41, 137.78, 133.42, 132.69, 132.29,
1
5
31.68, 131.39, 129.64, 128.28, 128.28, 126.73, 126.73, 124.56, 122.37, 120.52, 118.32, 115.31,
6.73; HR-ESI-MS: m/z calcd for C H N O , [M]+ 443.1230; Found 443.1226.
2
3
17
5
5
6
.29. 4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-N'-(4-
nitrobenzylidene)benzohydrazide (35)
Light yellow, Yield: 72.9%. m.p.: 254-255 °C; H NMR (500 MHz, DMSO-d
.60 (s, 1H), 8.33 (d, J = 8.0 Hz, 2H), 8.23 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 7.5 Hz, 2H), 8.04 (d,
J = 8.5 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19
1
6
): δ 12.35 (s, 1H),
8
13
(
t, J = 7.5 Hz, 1H), 3.97 (s, 3H); C NMR (150 MHz, DMSO-d6): δ 165.43, 164.36, 158.28,
1
1
56.44, 149.37, 148.07, 139.83, 133.46, 132.19, 131.48, 131.29, 128.32, 128.32, 127.74, 127.74,
26.69, 126.69, 124.58, 124.58, 120.65, 118.37, 115.31, 56.79; HR-ESI-MS: m/z calcd for
C H N O , [M]+ 443.1230; Found 443.1238.
23
17
5
5
6
.3 Molecular Docking studies
Through the present molecular docking studies Pteridine reductase (PTR) an essential enzyme
responsible for Pteridine salvage in Leishmania and other trypanosomatid protozoans has been
used as an anti-leishmaniasis drug target. Further, we have explored the binding orientation of
the synthesized benzohydrazone derivatives 6-35 as potential anti-leishmanial agents using PTR.
The Crystal structure of Pteridine reductase of Leishmania major with PDB id: 1E7W have be
used for the docking study [39].
26

ACCEPTED MANUSCRIPT
Pteridine reductase structure preparation for docking
The Leishmania major Pteridine reductase (PTR) structure (PDB code: 1E7W) was retrieved
from the Protein database and structural optimization was done using Protein Preparation Wizard
in Maestro [40]. The initial crystal structure was preprocessed for assigning bond order, the
addition of hydrogen, adding missing disulfide bonds and deleting B-chain, water molecules and
co-factors. Further, the structure was refined by minimizing hydrogens of altered species, pH 7
was set and a restrained energy minimization was done with converge heavy atoms to RMSD
0
.30Å.
Ligand preparation for synthesized benzohydrazone derivatives
All the thirty five benzohydrazone derivatives 3D structures were drawn using the Schrodinger
Maestro [40] using build option. Further the benzohydrazone derivatives were optimized using
Ligprep application with OPLS_2005 forcefeld, generation of possible states of pH 7.0 _ 2.0.
Generating possible tautomers with all combinations of stereoisomer and with lowest energy
conformation.
Docking studies of benzohydrazone derivatives with Pteridine reductase
Molecular docking studies were carried out using Glide: a complete solution for ligand-receptor
docking in small molecule drug discovery suite [40]. Initially, receptor grid generation was done
by generating grid on the Pteridine reductase structure were the grid box was centered on
methotrexate (MTX) complexed ligand with 12 °A radius respectively. Both standard precision
(SP) mode and Extra precision (XP) mode was chosen during the Glide docking process and
Glide gscore was considered for analysis. Further top rank scored binding mode analyzed in
Pymol [41].
27

ACCEPTED MANUSCRIPT
References
1
2
3
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4
5
. A. S. Nagle, S. Khare, A. B. Kumar, F. Supek, A. Buchynskyy, C.J. Mathison, N.K.
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4
9 (2002) 81-86.
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7
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Oswaldo Cruz, 105 (2010) 818-822.
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