New oxadiazoles with selective-COX-2 and EGFR dual inhibitory activity: Design, synthesis, cytotoxicity evaluation and in silico studies

Article abstract of DOI:10.1016/j.ejmech.2019.111693

Novel heterocyclic oxadiazoles viz. 2-subsituted-5-(4-pyridyl)-1,3,4-oxadiazoles, 2-subsituted-5-(3-pyridyl)-1,3,4-oxadiazoles and 2-subsituted-5-(phenyl or 4-chlorophenyl-1,3,4-oxadiazoles) were designed and synthesized as potential anticancer agents. In

Full text of DOI:10.1016/j.ejmech.2019.111693

Journal Pre-proof
New oxadiazoles with selective-COX-2 and EGFR dual inhibitory activity: Design,
synthesis, cytotoxicity evaluation and in silico studies
Nehad A. El-Sayed, Mai S. Nour, M. Alaraby Salem, Reem K. Arafa
PII:
S0223-5234(19)30843-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111693
EJMECH 111693
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2019
Revised Date: 14 August 2019
Accepted Date: 9 September 2019
Please cite this article as: N.A. El-Sayed, M.S. Nour, M.A. Salem, R.K. Arafa, New oxadiazoles
with selective-COX-2 and EGFR dual inhibitory activity: Design, synthesis, cytotoxicity evaluation
and in silico studies, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111693.
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New Oxadiazoles with Selective-COX-2 and EGFR Dual Inhibitory Activity:
Design, Synthesis, Cytotoxicity Evaluation and In Silico Studies
Nehad A. El-Sayeda, Mai S. Nourb, M. Alaraby Salemb, Reem K. Arafac,d,*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,
11562, Cairo-Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy , October University of Modern
Sciences and Arts (MSA), 6th of October City, Giza-Egypt.
c Biomedical Sciences Program, Zewail City of Science and Technology, University of Science and
Technology, October Gardens, 6th of October City, 12578, Giza-Egypt.
dDrug Design and Discovery Laboratory, Helmy Institute of Science and Technology, Zewail City of
Science and Technology, 12578, Cairo, Egypt
*Corresponding author:
Prof. Reem K. Arafa, Biomedical Sciences Program-University of Science and Technology; Director of the
Drug Design and Discovery Laboratory-Helmy Institute of Science and Technology; Zewail City of Science
and Technology, Ahmed Zewail Road, October Gardens, 6th of October City, Giza, Egypt, 12578, Mobile:
+2-01002074028, E-mail: rkhidr@zewailcity.edu.eg
Page 1 of 27

Abstract
Novel heterocyclic oxadiazoles viz. 2-subsituted-5-(4-pyridyl)-1,3,4-oxadiazoles, 2-subsituted-5-(3-
pyridyl)-1,3,4-oxadiazoles and 2-subsituted-5-(phenyl or 4-chlorophenyl-1,3,4-oxadiazoles) were
designed and synthesized as potential anticancer agents. In this investigation, all compounds were
evaluated for their COX-1 and COX-2 inhibitory activity in vitro as new therapeutic approaches assumed
cytotoxic effect associated with selective COX-2 inhibition. Results showed that most of the derivatives
demonstrated inhibition towards both isoforms of COX comparable to the standard reference drugs
indomethacin, diclofenac sodium and celecoxib. Then, nine selected compounds (IIId, VIb, VIIc, IX, XI,
XIIa, XIVa, XVIb and XVIIIb) were subjected to cytotoxic screening against UO-31 renal cancer cell line
using MTT assay. Compounds IIId, IX and XIIa displayed promising behavior by showing good anticancer
activity. Moreover, kinase inhibitory assay against the tyrosine kinase EGFR was performed for the three
compounds showing the highest cytotoxic activity. The tested compounds were potent against EGFR
with the highest activity being observed for compound IX showing nearly double the potency of the
reference drug Erlotinib. Moreover, molecular docking and molecular dynamics were performed for IIId,
IX and XIIa against EGFR, in an attempt to elucidate a model for their binding at the molecular level,
simulate and understand the possible binding interactions underlying the association between these
small molecules and the kinase enzyme ATP binding pocket essential amino acids. Finally, in silico
pharmacokinetic profile predication was investigated for IIId, IX and XIIIa using SWISS/ADME to identify
the most promising small-molecule cytotoxic agent on the basis of displaying the best drug-like
properties. Results indicated that compound IX has a potential to serve as a lead compound for
developing novel anticancer therapeutic agents.
Keywords: Oxadiazoles; Anticancer; Molecular docking; Molecular dynamics; EGFR; Cyclooxygenases; in
silico ADME.
Page 2 of 27

1. Introduction
Cancer is one of the global health issues raising various clinical challenges with increasing
frequency every year.1,2 Among the 10 most frequently occurring cancers in Western communities is
kidney cancer with approximately 90% of all kidney cancers being renal cell carcinomas (RCC). It was
estimated to effect 403,262 (2.2) new cases and 175,098 (1.8) deaths in 2018.3 On the molecular level,
chronic kidney diseases, amongst which is renal cancer, have been well linked to epidermal growth
factor receptor ligands’ induced renal inflammation.4 Despite that mass research was devoted and rapid
progress was achieved, there is yet a growing demand for finding new renal cancer therapies including
ones with multiple possible mechanisms of actions.5 Thus, in light of existing problems in cancer
therapy, discovery of novel, efficient and safe agents for renal cancer is still a thrust area for medicinal
chemists.
Much attention has been paid to the chemistry and biological activities of oxadiazole nucleus-
based molecules. Previous studies showed that compounds containing the 1,3,4-oxadiazole skeleton
exhibited a variety of biological activities such as insecticidal6, herbicidal7, antibacterial8, antifungal9,
analgesic10, anti-inflammatory11, anti-malarial12, anti-anxiety13, antituberculous14 and anticonvulsant
activities.15 Moreover, literature reports demonstrate the anticancer potential of 1,3,4-oxadiazoles and
high expectations for new therapies are attached to the extensive ongoing research based on the
diversity of action mechanisms associated with those molecules.16,17,18 Furthermore, ZD4054 (zibotentan,
1) (Figure 1), a drug in clinical trials containing 1,3,4-oxadiazole nucleus and showing high anticancer
activity, furnishes consideration for this moiety to be incorporated in the development of new
anticancer agents.19
Cyclooxygenase (COX), which is known for its rate-limiting role in the conversion of arachidonic
acid to prostaglandins (PG), is generally classified into three subtypes; COX-1, COX-2 and COX-3. The
significantly unregulated expression of the inducible isoform COX-2 in pathological processes revealed
its involvement in diseases such as inflammation and various types of cancer.20,21,22 It is well reported
that COX-2 overexpression in many different cancers is associated with cancer survival, evasion of
immunity, cancer stem cells repopulation during therapy and eventually resistance to chemo- and
radiotherapy.23,24,25 Besides, the National Cancer Institute has suggested, as a result of a conducted
review, that NSAIDs could have anticancer effects, especially those with more selective COX-2 inhibition
potentials. These compounds have shown capacity to restore the normal apoptosis in many cancer cell
lines, and inhibit the angiogenesis in cell culture and in rodent models.26 A variety of 5-membered
heterocycles can serve as a template for COX-2 inhibitors (Figure 1), i.e. pyrazole (celecoxib) (2),
isoxazole (valdecoxib) (3), furanone (rofecoxib) (4) and thiophene (DuP 697) (5) (Figure 1).27 Moreover
literature reports have advocated for 2,5-diaryl 1,3,4-oxadiazole derivatives as COX-2 inhibitors whereby
the 1,3,4-oxadiazole serves as a classical bioisostere for the aforementioned five membered
heterocycles.28
On the other hand, epidermal growth factor (EGF) plays a considerable role in tumor
development and progression, including cell proliferation, regulation of apoptotic cell death,
angiogenesis and metastatic spread by binding to its receptor, EGFR.29 Due to the overexpression of
EGFR in various types of epithelial cancers, like pancreatic, colorectal, breast, and lung cancer, it has
been thought that EGFR is an appropriate target for cancer therapy.30,31 Moreover, it is overexpressed in
both primary and metastatic RCC suggesting the potential of anti-EGFR agents as therapeutics for the
treatment of RCC.32 Literature survey confirmed the EGFR inhibitory potential of a variety of 1,3,4-
oxadiazole derivatives33,34 as exemplified by derivative (6) showing promising EGFR inhibitory activity
(Figure 1).35
Furthermore, it has been found that within the 1,3,4-oxadiazole derivatives, substitution at
position 5 of the oxadiazole moiety has great influence on the anticancer activity. Substitution with
various pharmacophores at this position may give rise to novel molecules with enhanced anticancer
Page 3 of 27

properties.36 Additionally, a conducted structure activity relationship (SAR) study revealed that the
activity of some 1,3,4-oxadiazole-2-thione derivatives increased due to the attachment of the phenyl
ring with substitution of an electronegative group at position 5 of the oxadiazole moiety.37 Another SAR
findings postulated the presence of the basic skeleton of 1,3,4-oxadiazole for the broad spectrum of
cytotoxic activity towards different cell lines.38
In light of the above findings and in continuation of our efforts to find potent EGFR
inhibitors,39,40 new hybrid molecules were designed based on reported EGFR and COX-2 inhibitors based
on the 1,3,4-oxadiazole moiety. Structure-activity relationship was probed through altering substituents
at C2 and C5 positions of the 1,3,4-oxadiazole skeleton (Figure 1). The COX activity for all compounds
was evaluated. Based on the COX results, some compounds were selected and evaluated for their RCC
cytotoxicity followed by EGFR inhibitory activity assessment for the top three compounds. Moreover, in
silico study was investigated for those compounds tested against EGFR tyrosine kinase using docking and
molecular dynamics (MD) to elucidate a postulated model for their binding at the molecular level.
Finally, in silico ADME profiles were predicted for those three compounds.
Figure 1. Structures of COX and EGFR inhibitors, and design strategy of the target 1,3,4-oxadiazoles.
2. Results and Discussion
2.1.Chemical synthesis
Synthesis pathways adopted for preparation of the novel 1,3,4-oxadiazoles are depicted in
Schemes 1-3. Scheme 1 illustrates the synthesis of 2-subsituted-5-(4-pyridyl)-1,3,4-oxadiazoles with
Page 4 of 27

flexible or rigid side chains at position 2 of the oxadiazole ring. First, the acid hydrazide (isoniazid) was
treated with carbon disulfide in alkaline medium affording, after acidic treatment, 5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-thiol I. This was followed by the reaction of I with hydrazine hydrate under different
conditions to furnish 2-hydrazinyl-5-(pyridin-4-yl)-1,3,4-oxadiazole II. Initially, the key intermediate II
was treated with different substituted aryl aldehydes to obtain oxadiazole derivatives IIIa-d. Moreover,
the reaction of phenylisocyanate with II resulted in the formation of the semicarbazide derivative IV
with a flexible short linker.
Furthermore, the thiol derivative I was treated with ethyl chloroacetate followed by reaction
with hydrazine hydrate to afford 2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide V. In an
attempt to increase the linker length, substituted aryl aldehydes were reacted with the hydrazide
derivative V to yield Schiff’s bases VIa,b. Moreover, in another attempt to introduce some rigidity to the
linker, the formohydrazino moiety of V was cyclized and various dioxadiazoles VIIa-c were obtained via
the reaction of compound V with different aromatic acids in the presence of phosphorous oxychloride.
Scheme 1. Synthesis of 2-subsituted-5-(4-pyridyl)-1,3,4-oxadiazoles
Additionally, intermediate VIII was employed for the synthesis of different 2-subsituted-5-(3-
pyridyl)oxadiazoles as demonstrated in Scheme 2. Initially, VIII was treated with phenylisocyanate and
Page 5 of 27

yielded the nucleophilic addition product IX. Moreover, the target compound X was obtained via
reacting VIII with ethyl cyanoacetate. Treatment of the key intermediates VIII with triethyl orthoformate
resulted in the formation of XI. Also, Schiff’s bases XIIa,b were obtained via condensation of the amino
group of VIII with the active carbonyl of p-chlorobenzaldehyde and p-nitrobenzaldehyde, respectively.
Scheme 2. Synthesis of 2-subsituted-5-(3-pyridyl)-1,3,4-oxadiazoles.
Finally, synthesis of the 5-(phenyl or 4-chlorophenyl)-1,3,4-oxadiazole derivatives is represented
in Scheme 3 employing the intermediates XIIIa,b. Treatment of either of XIIIa,b with phenylisocyanate
yielded the urea derivatives XIVa,b, respectively, while XV was obtained by reacting XIIIb with ethyl
cyanoacetate. Reaction of XIIIa,b with triethylorthoformate resulted in formation of the target
compounds XVIa,b respectively. Additionally, several Schiff’s bases XVIIa,b and XVIIIa-c were obtained
through the condensation reaction of intermediates XIIIa,b and different substituted aryl aldehydes.
Structures of the synthesized compounds were assigned on the basis of elemental analysis and spectral
data. The IR, 1H-NMR and 13C-NMR spectral data of compounds are given in the experimental protocols.
Page 6 of 27

Scheme 3. Synthesis of 2-subsituted-5-(phenyl or 4-chlorophenyl)-1,3,4-oxadiazoles.
2.2.Biological Activity
2.2.1 COX inhibitory activity
The synthesized compounds were aimed as COX-2 inhibitors. To gain an insight into the
plausible selective anti-COX-2 activity, the inhibitory concentration (IC50) values were determined
against both COX-1 and COX-2. While the IC50 values of the synthesized compounds against COX-1
ranged from 4.17 to 15.38 µM, they were more potent against COX-2 eliciting IC50 values between 0.038
and 0.56 µM. Interestingly, most of the new derivatives displayed prominent inhibition constants
towards both COX isoforms comparable to those of the standard drugs indomethacin, diclofenac sodium
and celecoxib (Table 1). Most notably, structural variations appeared to affect both the potency and
selectivity of these derivatives against the tested enzymes. This is a looked-for trait especially for the
development of selective anti-cancer agents to avoid undesirable side effects.
Table 1. IC50 values and Selectivity index (SI) of in vitro COX-1/COX-2 enzyme inhibition assay.
SI
SI
COX-1
(IC50 µM)*
COX-2
(IC50 µM)
COX-1
(IC50 µM)
COX-2
(IC50 µM)
Compd. No
(COX-1/
COX-2)
24.62
29.96
139.26
Compd. No
(COX-1/
COX-2)
IIIa
IIIb
IIIc
10.59±0.02
8.69±0.03
5.71±0.02
0.43±0.014
0.29±0.0003
0.11±0.007
XIVa
XIVb
XV
6.87±0.028 0.063±0.0014 109.04
10.87±0.035
13.74±0.1
0.29±0.009
0.25±0.005
37.48
53.88
Page 7 of 27

IIId
Via
VIb
VIIa
VIIb
VIIc
IX
X
XI
XIIa
XIIb
9.87±0.01
6.78±0.04
4.49±0.01
7.96±0.035
6.84±0.02
4.17±0.035 0.053±0.007
9.37±0.028
13.08±0.02
0.041±0.002
0.069±0.004
0.038±0.007 118.15
0.1±0.003
0.089±0.005
89.72
98.26
XVIa
XVIb
XVIIa
XVIIb
XVIIIa
14.21±0.035 0.11±0.0009
129.18
153.80
37.86
15.38±0.02
11.36±0.035
0.1±0.0005
0.3± 0.005
79.60
76.85
78.67
55.11
23.35
33.39
81.07
39.47
9.81±0.028 0.094±0.0006 104.36
7.54±0.07
0.081±0.0007
93.08
91.35
92.15
308.16
4.52
XVIIIb
XVIIIc
5.39±0.014 0.059±0.0006
11.98±0.042 0.13±0.0008
0.17±0.002
0.56±0.021
Celecoxib
Diclofenac Na
Indometh.
15.1±0.1
3.8±0.025
0.041±0.006
0.049±0.004
0.84±0.012
0.51±0.021
12.69±0.035 0.38±0.007
10.54±0.056 0.13±0.003
8.29±0.035
0.08
0.21±0.008
* Data are the average of 3 independent runs ± SD
With regards to the COX-2 enzymatic inhibitions assay results, among all studied derivatives,
compounds IIId and VIb were the most potent displaying lower IC50 values (IC50; 0.041 µM and SI; 89.7
and IC50; 0.038 µM and SI; 118.68, respectively) compared to the known COX-2 selective inhibitor
Celecoxib (IC50; 0.049 µM and SI; 308.16). Moreover, compounds VIIc, XIVa and XVIIIb displayed slightly
higher IC50 values and SI than celecoxib (IC50; 0.053 µM, SI; 79.62, IC50; 0.063 µM, SI; 109.6 and IC50;
0.059 µM, SI; 91.3, respectively). On the other hand, results showed that the cyanoacetamide derivative
X (IC50; 0.56 µM and SI; 23.41) has the least activity amongst all compounds yet still potent being active
against COX-2 at the sub-nanomolar level. Regarding the 5-(4-pyridyl)-1,3,4-oxadiazole series, it was
found that 4-nitro substitution in the phenyl ring with linkers of different length and rigidity; has
improved the inhibitory activity more than their corresponding 4-bromo, 4-chloro and un-substituted
analogs respectively. Moreover within the dioxadiazoles series, it has been found that the derivative
with 4-bromophenyl group VIIc has better inhibitory activity than chloro VIIb and hydroxy VIIa analogs.
Concerning the 5-(3-pyridyl)-1,3,4-oxadiazole series, results revealed that the presence of
cyanoacetamide or ethylformimidate showed comparatively lower inhibitory activities as demonstrated
in compounds X and XI (IC50; 0.56, µM, SI; 23.35 and IC50; 0.38 µM, SI; 33.39, respectively). Referring to
the 5-(phenyl or 4-chlorophenyl)-1,3,4-oxadiazole series, it was observed that substitution on position 2
of 1,3,4-oxadiazole ring with cyanoacetamide or ethylformimidate retained the same SAR findings of 5-
(4-pyridyl)-1,3,4-oxadiazole series, since compounds XVb, XVIa and XVIb showed relatively higher IC50
values (IC50; 0.25 µM, SI; 53.88; IC50; 0.11 µM, SI; 129.18 and IC50; 0.1 µM, SI; 153.80, respectively).
Moreover in the 5-phenyl-1,3,4-oxadiazole series, the nitro derivative XVIIb showed better
activity than the corresponding chloro analog XVIIa. On the other hand, for the 4-chlorophenyl
homologous series the 4-chloro derivative XVIIIb showed better activity than the un-substituted and the
nitro analogs XVIIIa and XVIIIc, respectively. Furthermore, the Schiff’s bases XVIIIc and XVIIIb in the 4-
chlorophenyl series showed enhanced activities when compared with their counterparts XIIa, XIIb
,XVIIa, and XVIIb in both the 3-pyridyl and phenyl series, respectively.
2.2.2. Cytotoxic activity
Based on the results of COX inhibitory activities, nine structurally diverse compounds (IIId, VIb,
VIIc, IX, XI, XIIa, XIVa, XVIb and XVIIIb) were selected and evaluated for their cytotoxic activity against
renal cancer cell line UO-31 using MTT cytotoxicity assay. Doxorubicin, one of the most effective broad-
spectrum anticancer drugs, was used as the reference drug.41 The concentrations required for 50%
inhibition of cell viability (IC50) were calculated and compared with that of doxorubicin (IC50; 7.4530 nM).
Page 8 of 27

The results are summarized in Table 2, most of the tested compounds exhibited significant cytotoxic
activity against the renal cancer cell line UO-31. Compound IX; with IC50 value of 5.7587 nM; showed
more potent cytotoxic activity than the reference drug doxorubicin. Alternatively compounds IIId and
XIIa showed slightly lower cytotoxic activity than doxorubicin with IC50 values of 8.6035 and 13.5670 nM
respectively.
SAR analysis revealed that the linker plays an important role in the observed cytotoxicity; where
derivatives with short linker as IIId, XIIa, IX, XVIIIb and XIVa displayed more potent activity than
derivatives with long chain whether the chain is flexible or rigid as VIb and VIIc. Moreover, presence of
pyridine ring at position 5-substitution of the 1,3,4-oxadiazole ring enhanced the anti-proliferative
activity, where compounds XIIa and IX showed better IC50 values than XVIIIb and XIVa. Besides, the
absence of phenyl ring at position 2 substitution of the 1,3,4-oxadiazole ring decreased the activity as
demonstrated by compound XI which displayed the lowest activity of all the tested compounds with IC50
value of 310.0395 nM. Nevertheless, the presence of 4-Cl benzene in such derivatives at position 2 of
the 1,3,4-oxadiazole ring improved the activity, where compound XVIb exhibited better activity than XI.
Table 2. IC50 values against renal cancer cell line UO-31.
Compd.
No.
IIId
VIb
VIIc
IX
IC50*
(nM)
Compd.
No.
IC50
(nM)
8.6035±0.05
36.7161±0.61
83.5060±0.94
5.7587±0.06
310.0395±1.8
XIIa
13.5670±0.09
33.1536±0.13
52.6648±0.24
29.6311±0.15
7.4530±0.03
XIVa
XVIb
XVIIIb
Dox
XI
* Data are the average of 3 independent runs ± SD
2.2.3. EGFR inhibitory activity
We had selected the top three compounds (IIId, IX and XIIa), demonstrating the best cytotoxic
activity against UO-31, and tested their EGFR inhibitory activity to confirm that these compounds act
through the initially postulated EGFR kinase activity inhibition pathway. As displayed in Table 3, the
EGFR inhibitory activities agreed with the SAR analysis of the anticancer activities. The results suggested
that the potent anticancer activities of the synthesized compounds were correlated with their EGFR
inhibitory activities according to the order IX>IId>XIIa. While, compound IX (IC50; 0.2757 µM) exhibited
nearly double the potency of the reference drug erlotinib (IC50; 0.4178 µM), compound IIId (IC50; 0.4123
µM) showed potency almost equal to erlotinib. On the other hand, compound XIIa (IC50; 1.1289 µM)
displayed lower EGFR inhibitory activity in consistency with the anti-proliferative profile.
Table 3. EGFR kinase inhibitory activity.
Compd. No.
IC50 (µM)
IIId
IX
XIIa
0.4123±0.022
0.2757±0.013
1.1289±0.045
0.4178±0.014
Erlotinib
* Data are the average of 3 independent runs ± SD
Page 9 of 27

Figure 2. EGFR inhibitory activity of IIId, IX and XIIa.
2.3.1. Molecular Docking with COX-2 and EGFR
Docking of the most potent COX-2 inhibitor in the enzymatic assay, derivative IIId, against COX-2
was performed to rationalize its relatively larger activity as compared to celecoxib. Docking scores for
celecoxib and IIId were -6.10 Kcal/mol and -8.83 Kcal/mol, respectively. As illustrated in Figure 3, IIId is
expected to have similar binding pose to celecoxib with some subtle differences. The nitro group of IIId
occupies the same region of the sulfonamide group of celecoxib, and forms similar interactions with the
Arg499 in the vicinity. In contrast to celecoxib, IIId displays additional hydrogen bonding interactions
with the active site amino acids of COX-2. First is the H-bond between the Val335 carbonyl oxygen and
the spacer NH group in IIId; in addition to another H-bond between the Ser516 hydroxyl group and one
of the oxadiazole ring nitrogens in IIId. These extra interactions can in part justify the enhanced activity
of IIId. In its current pose, however, IIId does not fully occupy the secondary pocket in COX-2, which
explains the comparatively poorer selectivity index.
Figure 3. Depiction of the best pose from the highest-populated cluster retrieved from the docking
experiment of compound IIId in the active site of COX-2 (PDB ID: 3LN1).The co-crystallized pose of
celecoxib is shown in yellow for reference. The predicted extra H-bonds of IIId are shown as dotted red
lines.
Moreover, the three compounds tested against EGFR tyrosine kinase were investigated using
docking and MD to elucidate a model for their binding at the molecular level. Docking was initially used
Page 10 of 27

to generate starting poses that were used to produce the initial coordinates for MD. Docking results are
summarized in Table 4 and Figure 4. In case of IIId, docking of all generated conformers yielded the
same pose. However, for IX and XIIa, docking yielded three and two poses of higher probability,
respectively. For IIId, it seems that the ligand is anchored in position via a H-bond to the neighboring
Gln767. A similar interaction exists for the first pose of XIIa, but with Met769. For IX, all poses seem to
rely on a H-bond between the oxadiazole ring and Met769 or Leu768. This is the same for the second
pose of XIIa. The scores for all poses of the three compounds were all comparable, ranging from -7.99
Kcal/mol for IIId to -7.09 Kcal/mol for the third pose of IX. Also, docking and MD showed that derivatives
IX (pose 3) and XIIa (pose 2), having a (5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl) moiety, displayed a more or
less similar binding pose in the EGFR binding site. On the other hand, derivative IIId with a 5-(pyridin-4-
yl)-1,3,4-oxadiazole had a flipped orientation relative to the other two explored derivatives (Figure 4).
Table 4. Docking scores in Kcal/mol of poses from the highest-populated clusters for each ligand.
Ligand
IIId
pose number
Single pose
pose 1
Score (Kcal/mol)
-7.99
-7.34
IX
pose 2
-7.69
pose 3
-7.09
pose 1
-7.10
XIIa
pose 2
-7.19
Page 11 of 27

Figure 4. Depiction of the poses from the highest-populated clusters retrieved from the docking
experiment of compounds IIId, IX and XIIa on the active site of 1m17. The co-crystallized pose of
Erlotinib is shown in yellow for reference. Hydrogen bonds to erlotinib are shown as dashed lines, while
those to the new compounds are shown as solid red lines. The active site is labeled for the second pose
of IX.
2.3.2. Molecular Dynamics (MD)
Seven trajectories in total were run: 6 for the previous poses obtained from the docking study and
one for the native Erlotinib structure as a reference. The aim for the MD simulation was to improve the
prediction of the binding poses made via docking, by exploring more conformational space and including
explicit waters.
Pose stability
The stability for each pose was compared to that of erlotinib as a reference over the trajectory of
100 ns. The RMSD for the ligand in each trajectory is plotted in Figure 5. With the exception of IIId, all
starting poses have fluctuations that are comparable to those of erlotinib. There is a larger average
deviation associated with IIId, together with large fluctuations around that average. Although the
several docking experiments led to the exact pose for IIId, it seems that the ligand tries to explore other
poses. The hydrogen bond analysis further strengthens this argument. For IX, poses 1 and 2 seem to be
more stable than pose 3. The fluctuations for the two poses of XIIa are comparable, although pose 2 is
associated with slightly larger average displacement from the initial pose over the dynamics trajectory.
Page 12 of 27

Figure 5. RMSD for the ligands from the starting poses given by the docking experiment over the course
of the MD simulation.
H-bond analysis
Hydrogen bonds that are formed in more than 10% snapshots of the 100-ns trajectory are given
along with their lifetimes in Table 4. For IIId, there is a very stable H-bond with Gln767 that is persistent
in 89% of the trajectory. Combining this with the RMSD behavior in Figure 5, one can deduce that the
ligand is trapped with this stable H-bond, in addition to being forced to expose the hydrophilic nitro
group to the solvent. Given this pose, the pyridine ring is directed towards the hydrophobic inside of the
protein. Anchored by the H-bond with Gln767, the IIId still significantly fluctuates away from its initial
position, which suggests ruling out the pose for IIId as a general pose for the ligands under study.
Nevertheless, other H-bonds of considerable strengths are also observed with Met769 and through a
water bridge to Thr766. These are the same amino acids which interact with erlotinib in the
experimentally determined crystal structure. For IX, pose 1 does not seem to form any stable H-bonds
and thus is ruled out as a potential pose. We are left with poses 2 and 3 that were predicted in the
docking experiment; see Figure 4. In case of pose 3, two H-bonds between the urea nitrogens and
Gln767 were observed in more than 60% of the snapshots each. Another weaker H-bond was observed
with Met769, but with the oxygen in the oxadiazole ring contributing as the acceptor and not the
nitrogen. This pose is considerably different from the starting docking pose 3; see Figure 4. The most
reliable pose in the case of IX is thus pose 2. In that pose, a stable H-bond is observed with Gln767 and
another two less stable H-bonds with Met769 from the hinge region and the gatekeeper Thr766. These
are the same residues with which erlotinib interacts, but with a major difference is that the interaction
of IX with Thr766 is direct and not through a water bridge. In the case of XIIa, no single pose can be
inferred from the H-bond analysis, as none of the predicted H-bonds through docking were persistent
during the MD trajectory. In pose 1, the halophenyl ring is exposed to the solution, while in pose 2, the
orientation is flipped exposing the pyridine while directing the halophenyl rings towards the inner
hydrophobic residues. Based on studying the pose of erlotinib in the PDB ID 1m17 crystal structure, it
Page 13 of 27

makes more sense to have the more hydrophobic parts of the molecule positioned towards the inside of
the protein. This suggests that pose 2 is more plausible. Nevertheless, the shorter linker between the
halophenyl ring and the oxadiazole does not allow for the positioning of the oxadiazole towards the
hinge region to make stable H-bonds while keeping the halophenyl ring close to the hydrophobic
residues.
Table 5. Hydrogen-bond analysis for the 100 ns of the respective MD trajectories, see Figure 5. Lifetime
refers to the percentage of snapshots in which the relevant hydrogen bond is formed. The dash
designates little incidence of hydrogen bonds; lifetime less than 10%.
Contributing amino acid
Life time of H-bond
IX
IIId
XIIa
-
pose1 pose2 pose3 pose1 pose2
Gln767
Met769
89%
-
56%
62%
13%
-
-
-
-
-
-
-
-
31%
-
30%
36%
-
-
-
-
-
-
-
Thr766
-
-
32%
-
-
-
-
-
-
Thr160
13%
-
-
-
-
-
-
-
CYS103
-
water bridge to THR766
water bridge to Asp161
Water bridge to Gln767
21%
-
-
-
31%
20%
Studying the superposition of pose 2 of IX and erlotinib adds more insight to the proposed
binding pose of this new series of oxadiazoles; see Figure 4. In that pose, the phenyl ring of IX occupies
the same region as that of erlotinib. The oxygen of the urea bridge overlapping with the oxygen of the
water bridges a hydrogen bond between erlotinib and the gatekeeper, Thr766. The oxadiazole nitrogen
overlaps with that in the quinazoline ring of erlotinib. In addition, the pyridinyl ring of IX is exposed to
the solution in a region similar to that occupied by the benzene of the quinazoline ring of erlotinib. In
conclusion, the combined docking and MD study serves to give a qualitative explanation of the observed
enzymatic activity. The deduced docking pose for the most active compound, IX, shows much
resemblance to erlotinib. This pose proves to be relatively stable as illustrated via RMSD and H-bond
analysis.
2.4. In silico pharmacokinetic predication
The pharmacokinetic profile i.e. absorption, distribution, metabolism and excretion (ADME) of
the three synthesized compounds were predicted with the help of SWISSADME
(http://www.swissadme.ch/index.php). The application was used for in silico prediction of drug-likeness
of the three compounds based on various molecular descriptors and the results are presented in Table
6.
The three compounds are in clear violation of Lipinski rule of five indicating good oral
bioavailability and permeability. Though values of TPSA; ranging from 64.17 to 122.02; and XLOGP3;
ranging from 1.24 to 2.60; makes the three compounds expected to have both drug and lead like
properties. Unfortunately the presence of alerts towards Brenk; two alerts and one alert in compounds
IIId and XIIa, respectively, results in making both compounds less favorable to drug-likeness. On the
other hand, the most potent compound IX in the biological experimental data has high GI absorption
Page 14 of 27

and does not pass BBB. Moreover it has zero alerts in PAINS and Brenk, identifying it as a promising drug
candidate among all the synthesized compounds for further research and development.
Table 6. Drug-likeness predication through SWISS/ADME.
Property
MW
IIId
IX
XIIa
284.70 g/mol
2.69
310.27 g/mol
281.27 g/mol
MLOGP
N or O
1.61
1.22
7
5
2
5
NH or OH
Lipinski rule
TPSA
1
0
Yes-0 violation
Yes-0 violation
92.94
Yes-0 violation
64.17
2.60
122.02
XLOGP3
GI Absorption
BBB
2.12
1.24
Yes
Yes (High)
No
Yes
No
Yes
PAINS
0 alert
2 alerts:
imine_1, nitro_group
0.55
0 alert
0 alert
1 alert:
imine_1
0.55
Brenk
0 alert
Bioavailability score
Lead likeness
0.55
Yes
Yes
Yes
3.
Conclusion
In conclusion, we have designed and synthesized a series of novel oxadiazole based molecules, all
the targeted molecules were tested in vitro for their COX-1 and COX-2 inhibitory activity. Most of the
compounds exhibited significant activities for both the isoforms of COX. Nine compounds were selected
and evaluated for their cytotoxic activity against UO-31 cell line. Compounds IIId, IX and XIIa displayed
remarkable cytotoxic activity and their EGFR kinase inhibitory efficiency was determined by comparing
them with a known kinase inhibitor erlotinib in vitro. The three compounds exhibited promising
activities and were in correlation with their cytotoxic activity; where compound IX (IC50; 0.2757357 µM)
showed nearly double the potency of erlotinib (IC50; 0.41785 µM). Furthermore in silico combined
docking and MD study was performed and the results were fundamentally in agreement with the
biological data. The study revealed the qualitative explanation of the observed enzymatic activity; where
the deduced docking pose for the most active compound, IX, shows much resemblance to Erlotinib. This
pose proves to be relatively stable via RMSD and H-bond analysis. Moreover, In silico ADME study was
conducted and predicted these molecules to exhibit good oral bioavailability. Finally and according to
the in vitro and in silico studies, compound IX stands out as a promising scaffold that can contribute to
the synthesis of compounds with potent anticancer activity and can be considered as promising lead for
further future optimization.
4.
Experimental section
4.1. Chemical synthesis
Melting points were determined on Electrothermal Stuart SMP3 digital melting point apparatus
and were uncorrected. Elemental microanalyses were performed at the regional center for Mycology
and Biotechnology, Al-Azhar University. Infrared spectra were determined (KBr) using Shimadzu Infrared
spectrometer (IR-435) and FT-IR 1650 (Perkin Elmer), Faculty of Pharmacy, Cairo University and MSA
1
University- Central Research laboratories. H NMR spectra were performed in DMSO-d6 using Joel, 400
MHz NMR spectrometer, Faculty of Pharmacy-Microanalytical Unit-Cairo University. 13C NMR spectra
were performed in DMSO-d6 Joel, 100 MHz NMR spectrometer-Faculty of Pharmacy-Microanalytical
Page 15 of 27

Unit-Cairo University. Thin-layer chromatography (TLC, Merck) was run throughout the reactions to
optimize the reactions for purity and completion. The spots were viewed under a UV lamp of
wavelength 254 nm. Compounds I42, II and V43 were prepared according to reported procedures.
4.1.1. General procedure for the preparation of (IIIa-d)
To equimolar solution of the hydrazide derivative (II) (10mmol) and the appropriate aldehyde in
absolute ethanol, drops of glacial acetic acid were added. The reaction mixture was refluxed and the
formed precipitate was filtered off while hot. The obtained solid was then re-crystallized from ethanol.
4.1.1.1. 2-(2-Benzylidenehydrazinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (IIIa) yield% 69; m.p. 123-1250C; IR
1
(KBr, cm-1): 3315 (NH), 3160 (CH arom.), 2971 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 5.86 (s,
1H, CH), 7.66–7.57 (m, 5H, Ar-H), 7.89 (d, 1H, J=6.9 Hz, pyridine CH), 7.95 (d, 1H, J=7.2 Hz, pyridine CH),
8.03 (d, 1H, J=6 Hz, pyridine CH), 8.77 (d, 1H, J=5.92 Hz, pyridine CH), 9.75 (s, 1H, NH exchanged with
D2O). 13C NMR (DMSO-d6, 100 MHz): δ 168.19, 167.52, 150.80, 150.65(2), 147.86, 133.52, 133.40,
132.24(2), 129.78(2), 122.29(2). Anal. Calcd. for C14H11N5O: C,63.93; H,4.18; N,26.40. Found: C,64.02;
H,4.25; N,26.73.
4.1.1.2. 2-(2-(4-Chlorobenzylidene)hydrazinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (IIIb) yield% 82; m.p. 155-
157 0C; IR (KBr, cm-1): 3271 (NH), 3061(CH arom.), 2881 (CH aliph.). 1H NMR (DMSO-d6, 400 MHz): δ ppm
5.86 (s, 1H, CH), 7.64-7.97 (m, 4H, Ar-H), 8.03 (d, 2H, J=5.24 Hz, pyridine 2 × CH), 8.77 (d, 2H, J=5.12 Hz,
pyridine 2 × CH), 9.81 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): δ 168.19, 165.93,
150.79(2), 147.85, 147.04, 138.19, 133.40, 133.10(2), 130.98(2), 122.33(2). Anal. Calcd. For C14H10ClN5O:
C,56.10; H,3.36; N,23.37. Found: C,56.38; H,3.61; N,23.40.
4.1.1.3. 2-(2-(4-Bromobenzylidene)hydrazinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (IIIc) yield% 67; m.p.140-
142 0C; IR (KBr, cm-1): 3392 (NH), 3061 (CH arom.), 2978 (CH aliph.). 1H NMR (DMSO-d6, 400 MHz): δ ppm
5.86 (s, 1H, CH), 7.74-7.89 (m, 4H, Ar-H), 8.03 (d, 2H, J=6.04 Hz, pyridine 2 × CH), 8.77 (d, 2H, J=6 Hz,
pyridine 2 × CH), 9.80 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): δ 169.39, 164.53,
149.85(2), 143.65, 144.76, 132.25, 131.47(2), 129.24(2), 125.78(C), 121.45(2). Anal. Calcd. for
C14H10BrN5O: C,48.86; H,2.93; N,20.35 Found: C,49.12; H,2.95; N,20.46.
4.1.1.4. 2-(2-(4-Nitrobenzylidene)hydrazinyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole (IIId) yield% 70 ; m.p. 138-
140 0C; IR (KBr, cm-1): 3267 (NH), 3059 (CH arom.), 2880 (CH aliph.). 1H NMR (DMSO-d6, 400 MHz): δ ppm
5.86 (s, 1H, CH), 7.87-8.04 (m, 4H, Ar-H), 8.21 (d, 2H, J=8.76 Hz, pyridine 2 × CH), 8.41 (d, 2H, J=8.72 Hz,
pyridine 2 × CH), 10.11 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): δ 168.19, 163.97,
150.80, 150.62(2), 147.85, 147.32, 138.14, 130.42(2), 124.83(2), 122.51(2). Anal. Calcd. for C14H10N6O3:
C,54.20; H,3.25; N,27.09 Found: C,54.38; H,3.61; N,27.11.
4.1.2. N-phenyl-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)hydrazinecarboxamide (IV)
To a solution of hydrazide derivative II (1.97 mmol) in absolute ethanol, few drops of triethylamine and
phenylisocyanate (2.37 mmol) were added. The reaction mixture was refluxed for 12h, then the solvent
was removed under reduced pressure and the formed residue was triturated with ether. The product
was re-crystallized from ethanol.
0
1
yield% 65; m.p. 130-132 C; IR (KBr, cm-1): 3332-3420 (NH), 3059 (CH arom.), 1716 (C=O). H NMR
(DMSO-d6, 400 MHz): δ ppm 5.31 (s, 1H, NH exchanged with D2O), 6.97 (s, 2H, NH exchanged with D2O),
7.50–7.23 (m, 5H, Ar-H), 8.82 (d, 2H, J=5.2 Hz, pyridine 2 × CH), 8.99 (d, 2H, J=5.6 Hz, pyridine 2 × CH). 13C
NMR (DMSO-d6, 100 MHz): δ 156.52, 153.11, 151.33, 150.59, 140.29(2), 140.21, 129.22, 129.07, 122.24,
Page 16 of 27

122.15(CH), 121.16, 118.85, 118.51. Anal. Calcd. for C14H12N6O2: C,56.75; H,4.08; N,28.36 Found:
C,56.92; H,4.11; N,28.40.
4.1.3. General procedure for the preparation of (VIa,b)
To a mixture of V (10mmol) and appropriate aromatic aldehyde (10mmol) in absolute ethanol,
drops of glacial acetic acid were added. Then the reaction mixture was refluxed for 10-12h. The formed
solid product was filtered off while hot, dried and then re-crystallized from ethanol.
4.1.3.1. N’-(4-chlorobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide(VIa) yield%
52; m.p. 222-2240C; IR (KBr, cm-1): 3332 (NH), 3098 (CH arom.), 2915 (CH aliph.), 1720 (C=O), 1540 (CS).
1H NMR (DMSO-d6,400 MHz): δ ppm 4.28 (s, 1H, CH), 4.70 (s, 2H, CH2), 7.49-7.60 (m, 4H, Ar-Cl ), 7.75 (d,
2H, J=8.44 Hz, pyridine 2 × CH), 8.82 (d, 2H, J=8.76 Hz, pyridine 2 × CH), 11.87 (s, 1H, NH exchanged with
D2O). 13C NMR (DMSO-d6, 100 MHz): δ 168.47, 165.48, 164.10, 151.41(2), 146.64, 143.44, 135.00,
133.39, 133.26(2), 129.57(2), 129.29(2), 52.49. Anal. Calcd. for C16H12ClN5O2S: C,51.41; H,3.24; N,18.73
Found: C,51.69; H,3.35; N,18.75.
4.1.3.2. N’-(4-nitrobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (VIb) yield%
55; m.p. 156-1580C; IR (KBr, cm-1): 3420 (NH), 3088 (CH arom.), 2939 (CH aliph.), 1690 (C=O), 1558 (CS).
1H NMR (DMSO-d6, 400 MHz): δ ppm 4.32 (s, 1H, CH), 4.74 (s,2H,CH2), 7.89 (d, 2H, J=5 Hz, pyridine 2 ×
CH), 7.97-8.34 (m, 4H, Ar-H), 8.81 (d, 2H, J=4.88 Hz, pyridine 2 × CH), 12.16 (s, 1H, NH exchanged with
D2O). 13C NMR (DMSO-d6, 100 MHz): δ 168.83, 165.39(2), 151.41(2), 148.31, 145.52, 142.39, 140.56,
128.61(2), 124.51(2), 120.43(2), 63.03. Anal. Calcd. for C16H12N6O4S: C,50.00; H,3.15; N,21.86 Found:
C,50.22; H,3.17; N,21.83.
4.1.4. General procedure for the preparation of (VIIa-c)
Compound V (10 mmol) was added to a solution of aromatic acid (10 mmol) in phosphorus
oxychloride (3 mL). The reaction mixture was heated for 12-18h in water bath and was cooled to R.T.
then poured onto crushed ice portion wise with continuous stirring. Then the mixture was treated with
ammonia solution, and the obtained solid was filtered off and re-crystallized from ethanol.
4.1.4.1. 4-(5-(((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1,3,4-oxadiazol-2-yl)phenol (VIIa) yield%
75; m.p. 151-1530C; IR (KBr, cm-1): ), 3404 (OH), 3075 (CH arom.), 2978 (CH aliph.), 1504 (CS). 1H NMR
(DMSO-d6, 400 MHz): δ ppm 4.06 (s, 2H, CH2), 7.41-7.57 (m, 4H, Ar-OH), 8.04 (d, 2H, J=9.28 Hz, pyridine
2 × CH), 8.19 (d, 2H, J=9.44 Hz, pyridine 2 × CH), 8.79 (1H, OH exchanged with D2O). 13C NMR (DMSO-d6,
100 MHz): δ 167.09(2), 164.03, 163.96, 150.22, 132.17, 131.42, 131.36, 129.97(2), 123.23(2), 122.68(2),
120.50, 35.22. Anal. Calcd. for C16H11N5O3S: C,54.38; H,3.14; N,19.82; Found: C,54.42; H,3.16; N,19.90.
4.1.4.2. 2-(4-Chlorophenyl)-5-(((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)1,3,4-oxadiazole (VIIb)
yield% 72 ; m.p. 145-147 0C; IR (KBr, cm-1): 3085 (CH arom.), 2935 (CH aliph.), 1540 (CS). 1H NMR (DMSO-
d6, 400 MHz): δ ppm 4.10 (s, 2H, CH2), 7.84-7.77 (m, 4H, Ar-Cl), 7.91 (d, 2H, J=8 Hz, pyridine 2 × CH), 8.12
(d, 2H, J=8 Hz, pyridine 2 × CH). 13C NMR (DMSO-d6, 100 MHz): δ 166.33(2), 164.03(2), 154.42, 136.09,
132.42, 130.36, 128.67(2), 122.23(2), 120.68(2), 118.67, 40.07.Anal. Calcd. for C16H10ClN5O2S: C,51.69;
H,2.71; N,18.84; Found: C,51.71; H,2.75; N,18.92.
Page 17 of 27

4.1.4.3. 2-(4-Bromophenyl)-5-(((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)1,3,4-oxadiazole (VIIc)
yield% 65; m.p. 153-1550C; IR (KBr, cm-1): 3090 (CH arom.), 2840 (CH aliph.), 1586 (CS). 1H NMR (DMSO-
d6, 400 MHz): δ ppm 4.34 (s, 2H, CH2), 7.70-7.91 (m, 4H, Ar-Br), 8.10 (d, 2H, J= 8.84, pyridine 2 × CH),
8.77 (d, 2H, J= 8.08, pyridine 2 × CH). 13C NMR (DMSO-d6, 100 MHz): δ 167.08(2), 163.38(2), 133.01(2),
132.43, 132.18(2), 131.76(2), 130.31, 130.02, 129.16(2), 30.31. Anal. Calcd. for C16H10BrN5O2S: C,46.17;
H,2.42; N,16.82; Found: C,46.23; H,2.65; N,17.12.
4.1.5. General procedure for preparation of (IX, XIVa,b)
A mixture of VIII or XIIIa,b (1.97 mmol) and phenylisocyanate (2.37 mmol) in absolute ethanol
with few drops of triethylamine was refluxed for 10h. The formed precipitate was filtered off and re-
crystallized from ethanol.
4.1.5.1. 1-Phenyl-3-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)urea (IX) yield% 82; m.p. 165-1670C; IR (KBr, cm-
1
1): 3392 (2NH), 3010 (CH arom.), 1694 (C=O). H NMR (DMSO-d6, 400 MHz): δ ppm 6.95 (t, 1H, J=7.36
Hz, Ar-H), 7.26 (t, 2H, J=8.2 Hz, Ar-H), 7.49 (dd, 2H, J=1.8, 6.88 Hz, Ar-H), 7.46-7.51 (m, 3H, pyridine 3×
CH), 7.53 (s, 1H, pyridine CH), 9.07(s, 2H, 2NH exchanged with D2O), 13C NMR (DMSO-d6, 100 MHz): δ
153.15, 149.43(2), 140.33, 135.28, 129.43, 129.35, 129.23(2), 122.15(2), 118.50(3). Anal. Calcd. for
C14H11N5O2: C,59.78; H,3.94; N,24.90; Found: C,59.79; H,3.99; N,25.12.
4.1.5.2. 1-Phenyl-3-(5-phenyl-1,3,4-oxadiazol-2yl)urea (XIVa) yield% 74; m.p. 173-1750C; IR (KBr, cm-1):
1
3303 (2NH), 3062 (CH arom.), 1700 (C=O). H NMR (DMSO-d6, 400 MHz): δ ppm 6.98 (t, 1H, J=6.88 Hz,
C4', Ar-H), 7.30 (t, 4H, J=7.52 Hz, C3,C5,C3',C5', Ar-H), 7.45-7.81 (m, 5H, C2',C6',C2, C4,C6, Ar-H), 8.65 (s,
2H, 2NH exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): δ 164.35, 157.80, 153.00, 140.17,
130.80(2), 129.68(2), 129.24(2), 129.16, 124.87, 122.27, 118.65(2). Anal. Calcd. for C15H12N4O2: C,64.28;
H,4.32; N,19.99; Found: C,64.35; H,4.48; N,20.14.
4.1.5.3. 1-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-3-phenylurea (XIVb) yield% 72; m.p. 210-2120C; IR
1
(KBr, cm-1): 3420 (2NH), 3059 (CH arom.), 1725 (C=O). H NMR (DMSO-d6, 400 MHz): δ ppm 7.31-7.24
(m, 3H, Ar-H), 7.45 (d, 2H, J=7.92 Hz, Ar-H), 7.60 (dd, 2H, J=2.2, 8.56 Hz, Ar-Cl), 7.78 (dd, 2H, J=1.8, 8.56
Hz, Ar-Cl), 8.65 (s, 1H, NH exchanged with D2O), 9.58 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-
d6, 100 MHz): δ 164.47, 157.03, 154.00, 139.69, 135.36, 129.83(2), 129.23, 129.15, 127.22(2), 123.70,
122.72, 118.64(2). Anal. Calcd. for C15H11ClN4O2: C,57.24; H,3.52;N,17,80; Found: C,57.41; H,3.63;
N,17,85.
4.1.6. General procedure for the preparation of (X, XV)
A mixture of compound VIII or XIIIb (10 mmol) and ethyl cyanoacetate (10 mmol) in glacial
acetic acid (10 mL) was heated under reflux for 8h. The reaction mixture was cooled to R.T. and poured
onto ice water (30 mL). The separated solid was filtered off, washed with water and re-crystallized from
ethanol.
4.1.6.1. 2-Cyano-N-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)acetamide (X) yield% 75; m.p. 235-2370C; IR
(KBr, cm-1): 3392 (NH), 3094 (CH arom.), 2837 (CH aliph.), 2225(CN), 1836 (C=O). 1H NMR (DMSO-d6, 400
MHz): δ ppm 4.35 (s, 2H, CH2), 7.39-8.97 (m, 4H, pyridine 4 x CH), 9.75 (s, 1H, NH exchanged with D2O).
Page 18 of 27

13C NMR (DMSO-d6, 100 MHz): δ 163.90, 162.55, 153.71, 149.30, 144.16, 130.84, 122.56, 119.16,
114.60, 23.50. Anal. Calcd. for C10H7N5O2: C,52.40; H,3.08; N,30.56; Found: C,52.65; H,3.22; N,30.58.
0
4.1.6.2. N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2-cyanoacetamide (XV) yield% 75; m.p. 225-227 C;
1
IR (KBr, cm-1): 3420 (NH), 3039 (CH arom.), 2919 (CH aliph.), 2225 (CN), 1782 (C=O). H NMR (DMSO-d6,
400 MHz): δ ppm 4.30 (s, 2H, CH2), 7.62 (dd , 2H, J=1.4, 8.48 Hz, Ar-Cl), 7.95 (dd, 2H, J=1.44, 8.44 Hz, Ar-
Cl), 8.41 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): δ 166.92, 154.81, 153.48,
138.26, 131.60(2), 129.20(2), 127.51(C), 123.35, 62.93. Anal. Calcd. for C11H7ClN4O2: C,50.30; H,2.69;
N,21.33; Found: C,50.67; H,2.72; N,21.38.
4.1.7. General procedure for the preparation of (XI, XVIa,b)
A mixture of compound VIII or XIIIa,b (2 mmol) and catalytic amount of acetic anhydride in
excess triethyl orthoformate was heated for 8h. The reaction mixture cooled and poured onto ice water.
The formed precipitate was re-crystallized from ethanol and dried.
0
4.1.7.1. Ethyl N-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)formimidate (XI) yield% 45; m.p. 210-212 C; IR
1
(KBr, cm-1): 3035(CH arom.), 2978 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 2.80 (t, 3H, J=8 Hz,
CH2-CH3), 3.43 (q, 2H, J=8 Hz, CH2-CH3), 4.11 (s, 1H, CH=N), 7.17-8.75 (m, 4H, pyridine 4 x CH). 13C NMR
(DMSO-d6, 100 MHz): δ 150.43, 149.19, 145.87, 143.47(2), 134.17, 124.40(2), 45.43, 28.12. Anal. Calcd.
for C10H10N4O2: C,55.04; H,4.62; N,25.68; Found: C,55.65; H,4.83; N,25.72.
0
4.1.7.2. Ethyl N-(5-phenyl-1,3,4-oxadiazol-2-yl)formimidate (XVIa) yield% 50; m.p. 270-271 C; IR (KBr,
1
cm-1): 3143 (CH arom.), 2935 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 2.66 (t, 3H, J=8 Hz, CH2-
CH3), 3.34 (q, 2H, J=8 Hz, CH2-CH3), 7.30 (s, 1H, CH=N), 7.59-7.93 (m, 5H, Ar-H). 13C NMR (DMSO-d6, 100
MHz): δ 160.13, 158.05, 145.07, 136.72(2), 130.23, 128.56(2), 122.78, 40.60, 23.15. Anal. Calcd. for
C11H11N3O2: C,60.82; H,5.10; N,19.34; Found: C,60.91; H,5.15; N,19.38.
4.1.7.3. Ethyl N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)formimidate (XVIb) yield% 52; m.p. Above 300
0C; IR (KBr, cm-1): 3133 (CH arom.), 2938 (CH aliph.) 1H NMR (DMSO-d6, 400 MHz): δ ppm 2.51 (t, 3H, J=8
Hz, CH2-CH3 ), 3.34 (q, 2H, J=8 Hz, CH2-CH3), 7.30 (s, 1H, CH=N), 7.66 (dd, 2H, J=1.28 , 8.6 Hz, Ar-Cl), 7.81
(dd,2H, J=1.12, 8.64 Hz, Ar-Cl). 13C NMR (DMSO-d6, 100 MHz): δ 160.13, 158.05, 138.26, 136.72,
131.61(2), 129.86(2), 123.71, 40.62, 23.89. Anal. Calcd. for C11H10ClN3O2: C,52.50; H,4.01; N,16.70;
Found: C,52.53; H,4.13; N,16.78.
4.1.8. General procedure for the preparation of (XIIa,b, XVIIa,b and XVIIIa-c)
To a solution of compound VIII or XIIIa,b (10 mmol) and the appropriate aldehyde (10 mmol) in
absolute ethanol, drops of glacial acetic acid were added. The reaction mixture was refluxed for 14 to
20h and the formed precipitate was filtered off while hot. The obtained solid was then re-crystallized
from ethanol.
4.1.8.1. N-(4-Chlorobenzylidene)-5-(pyridin-3-yl)-1,3,4-oxadiazol-2-amine (XIIa) yield% 64; m.p. 143-
1450C; IR (KBr, cm-1): 3097 (CH arom.), 2878 (CH aliph.). 1H NMR (DMSO-d6, 400 MHz): δ ppm 4.23 (s, 1H,
CH), 6.65 (t, 1H, J= 8 Hz, pyridine CH), 7.91 (dd, 2H, J=1.44, 8.36 Hz, Ar-Cl), 8.11 (dd, 2H, J=1.88, 8.32 Hz,
Ar-Cl), 8.18 (d, 1H, J= 8 Hz, pyridine CH), 8.33 (d, 1H, J=12 Hz,pyridine CH), 9.01 (s, 1H, C-2, pyridine CH).
13C NMR (DMSO-d6, 100 MHz): δ 156.09, 154.19, 140.86(2), 135.17(2), 130.04, 127.34, 124.74(2),
Page 19 of 27

124.59(2), 123.86, 120.19. Anal. Calcd. for C14H9ClN4O: C,59.06; H,3.19; N,19.68; Found: C,59.13; H,3.25;
N,19.72.
4.1.8.2. N-(4-Nitrbenzylidene)-5-(pyridin-3-yl)-1,3,4-oxadiazol-2-amine (XIIb) yield% 70; m.p. 165-167 0C;
1
IR (KBr, cm-1): 3057 (CH arom.), 2914 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 3.6 (s, 1H, CH),
6.95(t, 1H, J= 8.6 Hz, pyridine CH), 8.16 (d, 1H, J=10 Hz, pyridine CH), 8.41-8.18 (m, 4H, Ar-NO2), 8.43 (d,
1H, J=12 Hz, pyridine CH), 10.17 (s, 1H, pyridine CH). 13C NMR (DMSO-d6, 100 MHz): δ 151.09, 140.54,
139.86(2), 131.11(2), 130.04(2), 124.74(2), 124.59, 123.71, 121.15, 120.29. Anal. Calcd. for C14H9N5O3:
C,56.95; H,3.07; N,23.72; Found: C,56.97; H,3.12; N,23.76.
4.1.8.3. N-(4-Chlorobenzylidene)-5-phenyl-1,3,4-oxadiazol-2-amine (XVIIa) yield% 54; m.p. 175-1770C; IR
1
(KBr, cm-1): 3114(CH arom.), 2782 (CH aliph.) H NMR (DMSO-d6, 400 MHz): δ ppm 6.93 (s, 1H, CH=N),
7.25-7.60 (m, 5H, Ar-H), 7.89 (dd, 2H, J=2.24, 7.92, Ar-Cl), 8.80 (dd, 2H, J=1.6, 8 Hz, Ar-Cl). 13C NMR
(DMSO-d6, 100 MHz): δ 164.34, 161.06, 157.79, 136.50, 133.08, 130.81, 130.49(2), 129.69(2), 129.56(2),
125.49(2), 124.87. Anal. Calcd. for C15H10ClN3O: C,63.50; H,3.55; N,14.81; Found: C,63.55; H,3.59;
N,14.86.
4.1.8.4. N-(4-Nitrobenzylidene)-5-phenyl-1,3,4-oxadiazol-2-amine (XVIIb) yield% 58; m.p. 280-2820C; IR
1
(KBr, cm-1): 3107 (CH arom.), 2949 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 7.26 (s, 1H, CH=N),
7.45-7.53 (m, 5H, Ar-H), 7.80-8.42 (m, 4H, Ar-NO2). 13C NMR (DMSO-d6, 100 MHz): δ 164.47, 164.35,
157.78, 132.01, 131.11, 130.80(2), 129.84(2), 128.72(2), 128.00, 127.22, 125.47(2). Anal. Calcd. for
C15H10N4O3: C,61.22; H,3.43; N,19.04; Found: C,61.25; H,3.48; N,19.11.
4.1.8.5. N-Benzylidene-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (XVIIIa) yield% 60; m.p. above 3000C;
1
IR (KBr, cm-1): 3109 (CH arom.), 2890 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 6.04 (s, 1H,
CH=N), 7.55-7.63 (m, 5H, Ar-H), 7.81 (dd, 2H, J=1.8, 8.4 Hz, Ar-Cl), 7.95 (dd, 2H, J=2.2, 8.36 Hz, Ar-Cl). 13
C
NMR (DMSO-d6, 100 MHz): δ 164.76, 164.47, 159.50, 138.31, 136.88, 135.36, 132.04(2), 129.95(2),
129.85(2), 129.19(2), 128.66. Anal. Calcd. for C15H10ClN3O: C,63.50; H,3.55; N,14.81; Found: C,63.62;
H,3.58; N,14.90.
4.1.8.6. N-(4-Chlorobenzylidene)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (XVIIIb) yield% 65; m.p.
1
275-2770C; IR (KBr, cm-1): 3140 (CH arom.), 2930 (CH aliph.). H NMR (DMSO-d6, 400 MHz): δ ppm 5.61
(s, 1H, CH=N), 7.31-7.57 (m, 4H, Ar-Cl), 7.59-7.80 (m, 4H, Ar-Cl). 13C NMR (DMSO-d6, 100 MHz): δ
164.46(2), 157.03, 135.36(3), 129.82(2), 127.21(4), 123.69(2), 122.47. Anal. Calcd. for C15H9Cl2N3O:
C,56.63; H,2.85; N,13.21; Found: C,56.68; H,2.87; N,13.22.
4.1.8.7. 5-(4-Chlorophenyl)-N-(4-nitrobenzylidene)-1,3,4-oxadiazol-2-amine (XVIIIc) yield% 50 ; m.p.288-
2900C; IR (KBr, cm-1): 3107 (CH arom.), 2850 (CH aliph.). 1H NMR (DMSO-d6, 400 MHz): δ ppm 5.67 (s, 1H,
CH=N), 7.67 (dd, 2H, J=3.48 , 8.6 Hz, Ar-Cl), 7.84 (dd, 2H, J=4, 8.86 Hz, Ar-Cl), 8.14 -8.41 (m, 4H, Ar-NO2).
13C NMR (DMSO-d6, 100 MHz): δ 164.46(2), 157.02, 151.06, 147.84, 140.52, 135.35(2), 129.81(2),
127.19(2), 124.71(2), 123.81. Anal. Calcd. for C15H9ClN4O3: C,54.81; H,2.76; N,17.04; Found: C,54.85;
H,2.79; N,17.04.
4.2. Biological activity
4.2.1. COX inhibitory activity
Page 20 of 27

Synthesized compounds and the positive control drugs, Celecoxib, Diclofenac sodium and
Indomethacin, were tested for their ability to inhibit COX-1 and COX-2 using COX Inhibitor Screening
Assay kit (ACETM EIA kits, catalog no. 560131). The assay includes both ovine COX-1 and human
recombinant COX-2 enzymes. Various concentrations for each of the tested compounds’ stock solutions
(0.5, 1, 2.5, 5 and 10 μM) were used to obtain the concentration inhibition response curve and calculate
the concentration of the test compound causing 50% inhibition (IC50, μM).
4.2.2. Cytotoxic activity
Cell culture
Cell Line cells were obtained from American Type Culture Collection. Cells were cultured in
DMEM (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone,), 10 ug/mL of insulin
(Sigma), and 1% penicillin-streptomycin. All other chemicals and reagents were from Sigma, or
Invitrogen. Cells were plated (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of 100 µl complete
growth medium + 100 µL of the tested compound per well in a 96-well plate for 24 hours before the
MTT assay.
MTT-Cytotoxicity assay
Cell viability was assessed employing IN VITRO TOXICOLOGY ASSAY KIT-MTT BASED, Stock No.
TOX-1, Sigma. The key component is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
(MTT). Cells were treated with serial dilutions of the test compounds and the reference drug
doxorubicin. Mitochondrial dehydrogenases of viable cells cleaved the tetrazolium ring, yielding purple
formazan crystals which were insoluble in aqueous solutions. Crystals formed were dissolved in acidified
isopropanol. The resulting purple solution was spectrophotometrically measured at a wavelength of 570
nm. The intensity of the purple color is directly proportional to the number of viable cells. Dose-
response curves were plotted for each compound and IC50 values were determined and reported as the
average from 3 independent runs ± SD.
4.2.3. EGFR Inhibitory activity
To determine whether any of the selected compounds can affect EGFR, profiling of compounds
IIId, IX and XIIa against EGFR tyrosine kinase target was performed employing erlotinib as a positive
reference drug. PBS Bioscience EGFR Kinase Assay Kit, catalog # 40321 was utilized in the assay and the
standard protocol was followed.
All samples and controls were tested in triplicates and the data enlisted in Table 3 represent
IC50 values compared to that of the control. IC50s are the average of the three experiments ± SD.
4.3. In silico studies
4.3.1. Docking
For COX-2 docking study, protein data bank (PDB) co-crystal structure with the selective COX-2
inhibitor celecoxib was utilized (PDB ID 3LN1). Of the many EGFR crystal structures available on the PDB
for EGFR TK, the one with PDB ID 1M1744 was chosen for docking and dynamics, as it has erlotinib as the
co-crystallized ligand which is the reference molecule in the enzyme assay. In all dockings, a grid box of
dimensions 42x40x40 grid points and spacing 0.375 was centered on the given co-crystallized ligand.
Docking was performed via Autodock445 implementing 100 steps of genetic algorithm while keeping all
the default settings provided by Autodock Tools.45 Sixteen conformers were generated for each ligand
using OpenBabel.46 Visualization was done using Ligplot+ for 2D plots47 and VMD for 3D plots48.
4.3.2. Molecular Dynamics
Page 21 of 27

Topology and coordinate files were prepared via Amber tools 1449. During the preparation of all
starting files, the loop that connects the tyrosine kinase to the transmembrane domain of EGFR was
deleted. The 1M17 crystal structure was prepared using the pdb4 amber and reduce programs50. The
numbering of the protein is changed in the output of pdb4 amber such that Gly672 is number 2 after the
acetyl cap. The all-atom forcefield AMBER ff14SB51,52 was universally used. As the forcefield does not
contain parameters for the ligand, all ligands were parameterized using ANTECHAMBER53 to generate
parameters that are consistent with the General Amber Force Field (GAFF)54. The AM1-BCC method was
used to assign charges. Crystallographic waters were retained and each protein was solvated with
approximately 21144TIP3P water molecules in a 100x100x84 Å3 box. Three 3 Cl- ions were added to
neutralize the positively-charged protein and additional 57 NaCl ions were added to reach a salt
concentration of 0.15 mM.
MD simulations were carried out using the AMBER Molecular Dynamics package49 following a
protocol composed of minimization, heating, density equilibration and production. The AMBER input
files are the same as the ones provided in the supplementary information of the work of Salem and
Brown55 with minor changes regarding the number of residues. The trajectory lengths for heating,
density equilibration and production were 20 ps, 50 ps and 100 ns, respectively. As given in the input
files, Langevin dynamics were employed. Analysis was done using CPPTraj56, XMgrace57 and VMD48 .The
RMSD plot was calculated for residues ARG11 to VAL286 using backbone atoms, as the other residues
belong to highly flexible loops that are far from the binding site and add erroneous noise to the plots.
A reference MD run was done using the 1M17 initial coordinates and the co-crystallized
erlotonib that serves as a control reference. As all the dockings for IIId clustered around a single pose,
only this pose was used to generate the initial coordinates of the MD run for IIId. For IX and XIIa, MD
trajectories were run using three and two initial poses, respectively, as these represent the highest
populated clusters from docking. In total, 7 MD trajectories were run yielding a total of 0.7 μs.
Acknowledgement
This research was enabled in part by support provided by Westgrid (www.westgrid.ca) and
Compute/Calcul Canada (www.computecanada.ca). Access to Westgrid was provided through
collaboration with Professor Alex Brown, University of Alberta.
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Page 25 of 27

Figures and Tables Legends
Figure 1. Structures of COX and EGFR inhibitors and design strategy of the target 1,3,4-oxadiazoles.
Figure 2. EGFR inhibitory activity of IIId, IX and XIIa.
Figure 3. Depiction of the pose from the highest-populated cluster retrieved from the docking
experiment of compound IIId in the active site of COX-2 (PDB ID: 3LN1).The co-crystallized pose of
celecoxib is shown in yellow for reference. The predicted extra H-bonds of IIId are shown as dotted red
lines.
Figure 4. Depiction of the poses from the highest-populated clusters retrieved from the docking
experiment of compounds IIId, IX and XIIa on the active site of 1m17. The co-crystallized pose of
Erlotinib is shown in yellow for reference. Hydrogen bonds to erlotinib are shown as dashed lines, while
those to the new compounds are shown as solid red lines. The active site is labeled for the second pose
of IX.
Figure 5.RMSD for the ligands from the starting poses given by the docking experiment over the course
of the MD simulation.
Scheme 1. Synthesis of 2-subsituted-5-(4-pyridyl)-1,3,4-oxadiazoles
Scheme 2. Synthesis of 2-subsituted-5-(3-pyridyl)-1,3,4-oxadiazoles.
Scheme 3. Synthesis of 2-subsituted-5-(phenyl or 4-chlorophenyl)-1,3,4-oxadiazoles.
Table 1. IC50 values and Selectivity index (SI) of in vitro COX-1/COX-2 enzyme inhibition assay.
Table 2. IC50 values against renal cancer cell line UO-31.
Table 3. EGFR kinase inhibitory activity.
Table 4. Docking scores in Kcal/mol of poses from the highest-populated clusters for each ligand.
Table 5. Hydrogen-bond analysis for the 100 ns of the respective MD trajectories, see Figure 4. Lifetime
refers to the percentage of snapshots in which the relevant hydrogen bond is formed. The dash
designates little incidence of hydrogen bonds; lifetime less than 10%.
Table 6. Drug-likeness predication through SWISSADME.
Page 26 of 27

Graphical Abstract
Different chemical
scaffold
H
Cl
1,3,4-oxadiazole
Br
NO2
OH
N
N
CN
N
O
Linker
N
O
Hetero/Aryl
six membered
ring
H
Cl
Rigid cyclic
Rigid Acyclic
Flexible short
Flexible long
Compound IX
COX-1
COX-2
SI
9.37±0.028
0.17±0.007
55.11
UO-31 IC50 nM
EGFR IC50 µM
5.7587±0.05
0.2757
Page 27 of 27

Highlights
•
New cytotoxic molecules based on 1,3,4-oxadiazole scaffold with dual
activity.
•
•
•
•
•
Inhibitory COX-1 and COX-2 activity assessment with selectivity to COX-2.
Antiproliferative effect against UO-31 cell line.
Potent kinase inhibitory activity against EGFR.
Docking and molecular dymanics study against EGFR.
In silico ADME study displayed promising PK profiles for IIId, IX and XIIa.