Design, synthesis, docking and QSAR study of substituted benzimidazole linked oxadiazole as cytotoxic agents, EGFR and erbB2 receptor inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.12.014

The synthesis of benzimidazole linked oxadiazole derivatives designed as potential EGFR and erbB2 receptor inhibitors with anticancer and apoptotic activity were studied. Compounds 7a specifically inhibit EGFR and erbB2 receptor at 0.081 and 0.098 μM concentration. Some of the compounds showed strong, broad-spectrum antiproliferative activitiy when tested against five human cancer cell lines. Compounds 7a and 7n were more cytotoxic than 5-fluorouracil against MCF-7 cancer cell, with IC₅₀values of 5.0 and 2.55 μM whereas, only 7a led to cell cycle arrest at G₂/M phase accompanied by an increase in apoptosis. Compounds 7a and 7n showed normal architecture of myofibrils in cardiomyopathy study whereas only compound 7a showed nearly equal biochemical parameters (SGOT and SGPT) when compared to control. Molecular docking & 3D-QSAR studies were used to establish interactions of 7a and 7n within the active site of enzyme for ATP binding site of kinase domain.

Full text of DOI:10.1016/j.ejmech.2016.12.014

Accepted Manuscript
Design, synthesis, docking and QSAR study of substituted benzimidazole linked
oxadiazole as cytotoxic agents, EGFR and erbB2 receptor inhibitors
Md Jawaid Akhtar, Anees Ahmad Siddiqui, Ahsan Ahmed Khan, Zulphikar Ali,
Rikeshwer Prasad Dewangan, Santosh Pasha, M. Shahar Yar
PII:
S0223-5234(16)31020-0
10.1016/j.ejmech.2016.12.014
EJMECH 9105
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 October 2016
Revised Date: 22 November 2016
Accepted Date: 6 December 2016
Please cite this article as: M.J. Akhtar, A.A. Siddiqui, A.A. Khan, Z. Ali, R.P. Dewangan, S. Pasha, M.S.
Yar, Design, synthesis, docking and QSAR study of substituted benzimidazole linked oxadiazole as
cytotoxic agents, EGFR and erbB2 receptor inhibitors, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2016.12.014.
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ACCEPTED MANUSCRIPT

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Design, synthesis, docking and QSAR study of substituted benzimidazole linked
oxadiazole as cytotoxic agents, EGFR and erbB2 receptor inhibitors
1
1
1
1
Md Jawaid Akhtar , Anees Ahmad Siddiqui , Ahsan Ahmed Khan , Zulphikar Ali ,
2
2
1
Rikeshwer Prasad Dewangan , Santosh Pasha and M Shahar Yar*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi-110062, India
2
Lab no 405, Institute of Genomics and Integrative Biology, New Delhi, India
Corresponding Author; E-mail: yarmsy@rediffmail.com
*
ABSTRACT:
The synthesis of benzimidazole linked oxadiazole derivatives designed as potential EGFR and
erbB2 receptor inhibitors with anticancer and apoptotic activity were studied. Compounds 7a
specifically inhibit EGFR and erbB2 receptor at 0.081 and 0.098 µM concentration. Some of the
compounds showed strong, broad-spectrum antiproliferative activitiy when tested against five
human cancer cell lines. Compounds 7a and 7n were more cytotoxic than 5-fluorouracil against
MCF-7 cancer cell, with IC values of 5.0 and 2.55 µM whereas, only 7a led to cell cycle arrest at
5
0
G /M phase accompanied by an increase in apoptosis. Compounds 7a and 7n showed normal
2
architecture of myofibrils in cardiomyopathy study whereas only compound 7a showed nearly
equal biochemical parameters (SGOT and SGPT) when compared to control. Molecular docking &
3
D-QSAR studies were used to establish interactions of 7a and 7n within the active site of enzyme
for ATP binding site of kinase domain.
Key Words: Benzimidazole linked oxadiazole, Anticancer, Apoptosis, 3D-QSAR studies

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1
. Introduction
The EGFR (also known as erbB1 or HER1) and the related human epidermal growth factor receptor
HER2 (also known as erbB2) is a promising target for anticancer therapy because of its role in
tumor growth, metastasis and angiogenesis [1-4, 4a]. Targeted inhibition of this signaling pathway
has thereby become attractive therapeutics in cancer treatment and are distinct from the
conventional chemotherapy and radiotherapy [3,5]. The EGFR or related receptors are
overexpressed, dysregulated or mutated in many epithelial malignancies, and their activation
appears important in tumor growth and progression [6, 6a]. Two most clinically advanced
approaches for their inhibition includes tyrosine-kinase inhibitors and monoclonal antibodies
classes of anticancer therapy [6-8]. The difference exists in that the former functions at the
extracellular ligand-binding site, whereas the latter functions at the intracellular tyrosine-kinase
domain [6]. Tyrosine-kinase inhibitors compete with the ATP considering the fact that there are so
many kinases and all uses ATP as the phosphorylating agent. Thus in the past few years, there has
been major focus in the development of ATP-competitive inhibitors [9]. The recently launched
kinase inhibitor drugs dasatinib, sunitinib, sorafenib, pazopanib, lapatinib and afatinib inhibit a
broad array of cancer-related protein kinases [5, 5a]. These quinazoline-containing derivatives form
an important class of synthetic products and represent an attractive scaffold for EGFR and related
receptors. However, due to the recent findings of EGFR mutations and erbB2 toxicity-related dose
limitation which render the kinase ineffective to Gefitinib, Erlotinib and Afatinib there is an urgent
need for new scaffolds to solve this tough problem [9].
Benzimidazoles are structurally related to indole and are ligands for serotonin (5-HT) receptors,
histamine (H4) receptors, bradykinin (B2) receptors, and dopamine (D4) receptors [10].
Additionally, benzimidazole being an isostere of purine nucleosides and an important scaffold in
various biologically active molecules is widely explored for the development of anticancer agents
[11-12]. On the other hand, 1,3,4-oxadiazole heterocycles, recognized as very good bioisosteres of
amides and esters, which can contribute substantially in enhancing pharmacological activity by
participating in hydrogen bonding interactions with the receptors [13-25]. Some of the drugs under
clinical trials containing benzimidazole and oxadiazole moieties and rationally designed targeted
compounds are represented in Figure 1. In the year 2012 Rashid et al [26] synthesized
benzimidazole-2-substituted oxadiazole and compounds exhibited remarkable anticancer activity

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against most of the tested cell lines with GI50 values between 0.79 and 17.8 uM. Salahuddin et al in
2
014 [27] synthesized benzimidazole-1-oxadiazole derivatives and found compound active against
breast cancer cell lines (MDA-MB-468) and showed 72.85 growth percent (GP). Despite many 1-
substituted oxadiazole) benzimidazole derivatives (compound 1, Figure 2) have been synthesized
(
and evaluated for the anticancer and antimicrobial activities [27], 2-(substituted oxadiazole)
benzimidazole derivatives (compound 2, Figure 2) have been seldom reported in any literature. The
present work is directed towards the design and synthesis of benzimidazole linked oxadiazole
derivatives (Figure 3) utilizing easily accessible chemicals, facile synthetic pathways, and
evaluation of the EGFR and erbB-2 inhibition assay using gefitinib as a positive control. The
literature data concerning EGFR activation have been associated with the development and
progression of human tumors viz. breast, liver, lungs and immortalized human keratinocyte. Thus,
cytotoxicity studies were determined against five human tumor cell lines (MCF7 “breast”, HEPG2
“liver”, MDA-MB231 “breast”, HaCaT “skin” and A549 “human lung carcinoma” cancers) using
MTT assay. Apoptosis, necrosis, and cell cycle analysis were performed using FACS analysis
against MCF7 cell lines. Molecular docking studies and 3D-QSAR studies are also performed for
understanding the binding and confirming the pharmacological observations.
2
2
. Results and discussion
.1. Chemistry
As shown in scheme 1, 6-substituted/unsubstituted 2-Cyanomethyl-1H-benzimidazole (3a-b) was
prepared from refluxing from easily available o-phenylenediamine/4-chlorobenzene-1,2-diamine
(1a-b) and ethyl cyanoacetate (2) in anisole for 10h. Compounds (4a–b) were synthesized by acidic
hydrolysis of 6-substituted/unsubstituted 2-cyanomethyl-1H-benzimidazole (3a-b) using benzene
sulphonic acid as catalyst. Esterification of the intermediates (4a-b) was achieved with ethanol and
2 4
a catalytic amount of conc. H SO . The reaction of esters (5a-b) with 99% hydrazine monohydrate
in ethanol medium produced the corresponding hydrazides (6a-b). The (7a-x) and (8a-e)
derivatives were synthesized by reaction between different substituted aliphatic/aromatic acid with
the respective 2-(6-substituted-1H-benzo[d]imidazol-2-yl)acetohydrazide (6a-b) in phosphorous
o
°
oxychloride (POCl ) at 60 C [27]. Heating at temperature above 60 C resulted in breakdown of the
3

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compound 7a giving two spots in TLC, later characterized into benzimidazole 2-acetyl chloride and
benzimidazole 2-acetic acid.
In order to synthesize compounds (3a-b), the reaction of o-phenylene diamine with malonic
acid/ethyl cyanoacetate in different solvents (4N HCl, 6N HCl, phosphoric acid, PPA, phosphorous
oxychloride, potassium carbonate and PCl ) were carried out. It was found that end product was
3
disappointing mixture of its byproduct under the present experimental conditions. The synthesis of
the derivatives (3a-b) was optimized using anisole as solvent under refluxing through Dean-Stark
apparatus. However, the reaction was incomplete under normal refluxing conditions even at the
extended period of time.
Further, synthesis of substituted benzimidazole-2-acetic acid (4a-b) was carried out in different
media. At first instance, 3a was hydrolyzed in the presence of conc. sulphuric acid which yielded
desired pure product 4a with 50% yield. In the second stance compound 3a was hydrolyzed with
1
5% NaOH followed by neutralization yielded a pure product with 40% yield. Whereas the
compound 3a on hydrolyzing in the presence of benzene sulphonic acid and HCl-Water mixture
produced pure compound (92%) in the shortest period of time as compared to other two
conventional methods.
2
.2.
Pharmacology
2
.2.1 In vitro EGFR and erbB2 phosphorylation assay
Compound 7a (most active para chloro analog) and compound 7n (active para methoxy analog)
were tested for their binding affinity to EGFR; both compounds showed better binding to the
EGFR. Compounds 7a (0.081µM) and 7n (0.098µM) showed comparable EGFR binding affinity as
that of the standard drug gefitinib, whereas 2-Cl, 2,4-diCl substituted phenyl 7g and 7b and 1-
2
methylnapthyl 7u contributed moderate inhibition at 4.5, 3.7 & 5.7 µM. Comparing the five analog
of 6-chloro benzimidazole derivatives (8a-e), compound 8a bearing para chlorophenyl has the
highest binding affinity to the EGFR (0.98 µM). These findings demonstrate that the position and
numbers of chlorine affect the EGFR inhibitory activity. Many compounds such as 7k, 7l, 7o, and
7
p were found to be inactive which may be due to the replacement of the phenyl to heteroaromatic
rings/aliphatic group. This result demonstrated that phenyl ring was essential for the EGFR
bindings. However, substitution in phenyl ring other than chloro/p-methoxy groups in compounds
7
f, 7i, 7q, 7t, 8c and 8d did not exhibit any appreciable activity. Further, compounds having 2,3

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(OCH
3
)
2
(7j), and 5-NH
2
-2-OH (7s) substituted phenyl showed less activity. The inhibitory
activities (IC ) of the synthesized compounds are illustrated in Table 1.
5
0
Similarly for the selected compounds with significant EGFR activity, it was observed that IC50
value for the inhibition of erbB2 was higher than EGFR phosphorylation assay. Among them
compound 7a (0.61uM) displayed the most potent activity, comparable to the positive control
geftinib. Compound 7n (0.91) was also found to be good inhibitor erbB2 autophosphorylation in a
MCF7 breast carcinoma cell line engineered to overexpress erbB2 whereas 7b, 7g and 7u were
shown to be weak to moderate inhibitor. Alternative 6-chloro benzimidazole derivatives 8a (IC =
5
0
2
.5) showed favorable erbB2 selectivity.
2
.2.2 In Vitro Cytotoxicity
The high activity of EGFR signal pathway has been related with an increase in cell proliferation and
a poor prognosis in many human malignancies including breast cancer [35-37, 37a], hepatocellular
carcinomas [38-40] and 40% to 80% of NSCLC patients [41-45]. Additionally, epidermal growth
factor receptor (EGFR), located on the cellular membrane of the keratinocytes, is widely recognized
as a key regulator of numerous essential processes underlying skin development, homeostasis, and
repair [46]. Abnormalities in the expression and signaling pathways downstream of the epidermal
growth factor receptor (EGFR) contribute to malignant transformation in human cancers, including
those of the cutaneous epithelium [47]. All these findings indicate targeting EGFR as an effective
means to combat breast, liver, lungs and skin cancer.
To verify EGFR and erbB2 receptor tyrosine kinase induced tumorigenesis, in vitro MTT assay
against five cancer cell lines MCF7 (breast), HaCaT (human skin), MDA-MB231 (breast) HepG2
(liver) and A549 (lung) was performed. The IC50 values are reported in Table 1. The three
chlorosubstituted phenyl compounds 7a, 7b and 8a showed lower IC value (5.0µM to 7.0µM)
5
0
than the 5-FU (7.12µM), this indicates that on introduction of electron rich chloro group at the para
position of the phenyl ring have contributed better inhibitory activity against MCF7 cancer cell
lines. Comparing with the three analogs, compound 7a with the monosubstituted chloro derivatives
at the para position of the phenyl ring has the highest inhibition, whereas compound 8a with
th
additional chloro substitution at 6 position of benzimidazole ring and compound 7b with 2,4-diCl
2
substitution have lower activity as compared to compound 7a. Compound 7n (para methoxy)

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showed better inhibitory activity with an IC50 of 2.5µM as compared to the chloro derivatives
analogue against MCF7 cancer cell lines. Compounds 7a, 7b, 8a (chloro derivatives) and 7n
(methoxy derivatives) showed the similar pattern of inhibition against MDA-MB231 cancer cell
lines but at higher IC50 value (14.5µM to 25.9µM) except for compound 7n showed better activity
with much lower IC value (0.131µM), this fall in concentration is expected due to the presence of
5
0
electron donating methoxy group at p-position of phenyl ring.
Whereas, the activity against human liver cancer cell line HepG2, compound 8a was found most
active with IC50 = 11.2 µM followed by compounds 7a and 7n with IC50 values of 12.5 and 15.6
µM. The rest of the compounds showed activity at the higher IC50 values. Among the tested
compounds the order of antitumor activity against the Non-Small Cell Lung Cancer (A549) was 7u
>
7n > 7a > 8a since their IC values falls between 8.8 to 23.6 µM. Activity against HaCaT cell
50
line revealed that compound 7n showed higher activity (IC = 3.8 µM), followed by compound 7a,
5
0
7
u and 8e with IC50 values of 9.5, 12.7 and 19.5 µM respectively. Compound 7a was more
cytotoxic than 5-fluorouracil against MCF-7 cancer cell lines, while 7n was less active than 5-
fluorouracil only against the HepG2 and A549 cancer cell lines.
5
Structure-activity relationship revealed that the nature of substituent on the C -position of the
oxadiazole influences the activity. Phenyl ring at C -position showed better antitumor activity than
5
the ones with aliphatic chain (7p) or heteroaromatic ring (7k, 7l, 7o, 7v, and 7w). Secondly, an
introduction of methyl group between oxadiazole and aromatic ring 7c, 7m, 7u, 7x and 8c does not
show positive results except compound 7u which may be due to bulky lipophilic napthyl ring. From
the screening results, as shown in (Table 1), it was observed that different substituent on phenyl
ring has the varying degree of inhibition against the cancer cell lines. The most active compound of
the series 7a and 7n suggest that on introduction of electron withdrawing group -Cl and electron
donating group -OCH at the para position of phenyl ring contributed to anticancer activities,
3
whereas compounds with un-substituted (7d and 8d) causes the anticancer effects but at higher
IC50. Compound 7g and 8e with o-chloro-substituted retains some of the activity in MCF-7, HaCaT,
and MDA-MB231 cell lines. Furthermore, when the phenyl ring is disubstituted with -Cl (7b and
8
b) retains only its activity against MCF-7 and MDA-MB231 with less active on other cancer cell
lines. In the same way, disubstituted -OCH (7j) retains activity in all cancer cell lines but at higher
3
IC . Analogues having para substituted –OH (7f), -CH (7i), -F (7q) and ortho substituted -CH
3
5
0
3

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(
7h), -COCH
3
(7r), -COOH (7t) showed less cytotoxic activity against all tested cancer cell lines.
th
The introduction of the -Cl group at the 6 position of benzimidazole nucleus (8a-e), showed some
influence in the antitumor effect but found to be lower as compared with the compounds (7a-e).
In general, it was observed that the compounds substituted with chlorine atom/methoxy group at
para position of aryl ring showed better antitumor activity than the compounds substituted with an
aliphatic chain or heteroaromatic ring. Overall, compounds 7a and 7n were found active, whereas
compound 7n was most potent in the series which showed highest antitumor activity against MCF-7
and MDA-MB231 cancer cell lines.
2
.2.3 Cell Apoptosis
On the basis of EGFR and erbB2 inhibition activity of the compound 7a and 7n with broad
spectrum activity against the selected cancer cell lines (most potent against MCF7), it was
considered worthwhile to further investigate the mechanism of action. To evaluate the apoptosis
assay, flow cytometry using propidium iodide (PI) and annexin-V-FITC in MCF7 cells was
performed. After post-treatment with the compound 7a & 7n at a conc. of 5 µM for 24 h, cells were
labeled with the two dyes. The corresponding red (PI) and green (FITC) fluorescence were
observed with the flow cytometry. There was increased in the late/secondary cellular apoptosis
from 3.86 % (DMSO control) to 19.4 and 33.8 % for compound 7a and 7n respectively, whereas
there was only marginal increase in the early/primary apoptosis for compound 7a “i.e.” 1.8% (0.1%
DMSO control) to 1.9% and corresponding decrease for compound 7n. These results indicate that
compound 7a act through apoptosis mechanism (Figure 4).
2
.2.4 Cell Cycle Inhibition
To understand the effect of selected compound 7a and 7n in the cell cycle, FACS analysis was
performed. MCF7 cells were treated with the compound 7a and 7n at a concentration of 5 µM each
for 24 h. The results confirm 33.6% of cell cycle inhibition in the G /M phase (G /M arrest) with
2
2
compound 7a at a conc. of 5 µM as compared to 16.43% (0.1 % DMSO control), whereas for
compound 7n, the percentage of cells in G /M phase was 13.5 % (Figure 5).
2
2
.2.5 Molecular docking

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Docking studies were carried out by using ligand docking to understand the binding mode of
benzimidazole linked oxadiazole derivatives. To validate the ligand docking, the protein was
redocked with the co-crystallized ligand erlotinib. The designed molecules were docked into the
active site of EGFR receptor (PDB 1M17) [48-51]. The catalytic domain consists of an N-terminal
lobe, which consists mainly of β-strands but contains one α-helix and C helix. The C-terminal lobe
is mainly α-helical, and a short strand termed the hinge region connects the two lobes [9]. Figure 6
&
7 (left) give details of the docking analysis of the compound (7a). The results revealed that the
N1 of the benzimidazole showed interaction with Asp831. One hydrogen bond was observed
between nitrogen of the oxadiazole ring and Met769 and another water molecule mediated
hydrogen bond was observed between nitrogen of the oxadiazole ring and Thr830 side chain similar
to erlotinib. These interactions highlight the importance of nitrogen atoms for the inhibitory and
binding efficiency to EGFR enzymes. The oxadiazole moiety lies in deep hydrophobic pocket and
lay in the same site of quinazoline ring of erlotinib. Analysis of compound 7n binding mode in the
active binding site demonstrated that docking mode of compound 7n is similar to that of compound
7
a with same hydrogen bond between N1 of the benzimidazole and Asp831. The difference lies in
the area of oxadiazole ring which do not show hydrogen bond interaction as was seen in compound
a and erlotinib. Residues within 2.8 Å areas of erlotinib and benzimidazole linked oxadiazole (7a
7n) are shown in Figure 6 & 7. Figure 8 represent the compound (7a & 7n) binding along with
the ligand erlotinib inside the protein.
7
&
2
.2.6 3D QSAR analysis
The scatter plots of the predicted vs. actual inhibitory activity and the contour cubes of the models
describe the features essential for the interaction between the synthesized compounds and the
2
EGFR kinase protein. The predicted correlation coefficient (r ) for the selected model was 0.83 and
is shown in Figure 9. The image for the most active compounds 7a with the favorable (blue cubes)
and the unfavorable (red cubes) is presented in Figure 10 a-d. These figures provide the detailed
chemical features of the hydrogen bond donor, hydrophobic, electron withdrawing and the
combined effects of the substituents of the ligands which contribute to the inhibitory effects against
EGFR.
The presence of red cubes in Figure 10a at N3 of benzimidazole and aryl ring attached with the
oxadiazole ring showed the unfavorable sites for the hydrogen donor group. Figure 10b showed that

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the presence of hydrophobic groups at the benzimidazole ring responsible for the negative potential,
whereas addition of suitable hydrophobic groups in the phenyl ring attached to the oxadiazole ring
give rise the inhibitory effects against EGFR. In Figure 10c blue cubes to the phenyl ring explain
the favorable effects for the electron withdrawing group. The effect of the electron withdrawing
substituent at the para position of the phenyl group show pronounced inhibitory effects. Figure 10d
demonstrate that substitution on phenyl ring is favorable to enhance binding affinity for EGFR
whereas, benzimidazole part should be un-substituted. The training set composed 20 members and
9
members of test set with their actual and predicted EGFR kinase activity are presented in Table 2.
2
.2.7 Acute oral toxicity
The results showed no mortality in rats when the compound 7a and 7n were treated with the dose of
00 mg/kg body weight. Thus, the lethal dose (LD ) of the tested compounds are >500 mg/kg body
5
5
0
weight. Therefore, it is concluded that the compounds tested and are classified toxicity oral LD50
>500 <2000 mg/kg) which is recommended by Organization for Economic Co-operation and
Development [28].
(
2
.2.8 Cardiomyopathy studies and Hepatotoxicity studies
Cardiotoxicity is a serious side effect of drugs used to treat cancer patients. Cardiac toxicity can be
caused by the tyrosine kinase inhibitors imatinib mesylate, dasatinib, nilotinib, sunitinib, sorafenib
and lapatinib, while gefitinib and erlotinib have not been related to toxic effect on the heart [53].
Although these drugs are associated with adverse cardiac events (e.g., LV dysfunction [LVD], HF,
cardiac ischemia, and myocardial infarction), still granted approval because of the limited treatment
options and needs to achieve anticancer efficacy [54]. Therefore, the most potent compounds 7a
and 7n were further tested for its toxicity in rat (heart) and doxorubicin used as the standard drug.
The slide of the treated compounds 7a & 7n showed that normal architectures of myofibril in the
sub endothelial zone and well maintained architectures of myofibril. There is no significant
difference in architecture of cardiac fibre as compared to the normal control group. While group
treated with these synthesized compounds were significant altered in architecture of muscle fibre as
compared to the doxorubicin (DOX) treated group as shown in Figure 11. DOX control group
induced cardiomyopathy, shown by a loss cardiac fibre, vacoulation, inflammation in left ventricle
tissue.

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Tyrosine kinase inhibitors induce liver injury and liver toxicity which varies in incidence and
severity by drugs. Drug induced liver toxicity causes marked increase in SGOT and SGPT levels.
The potent compounds (7a ) when studied for their liver toxic effect showed nearly equal value (p <
0
.01) of biochemical parameters (SGPT and SGOT) when compared to control whereas compound
(7n) showed marked increase in the serum hepatic enzyme levels (SGOT and SGPT) (Table 3).
Doxorubicin treated animals showed significant increase (p < 0.01) in levels of SGOT and SGPT as
compared to control [57].
3
. Conclusion
A reagent based approach for benzimidazole synthesis coupled with POCl cyclization into
3
oxadiazole was used to construct benzimidazole linked oxadiazole. Among the newly synthesized,
compounds 7a inhibit EGFR and erbB2 receptor at 0.081 and 0.61 µM concentration. Moreover,
compounds 7a and 7n exhibited excellent broad spectrum in vitro anticancer activity. The results
showed that p-substituted chloro/methoxy phenyl at fifth position of oxadiazole (7a & 7n) was
found to be most active against MCF-7 (2.55-5.00 µM) and MDA-MB231 (0.131-14.5 µM) cancer
cell line. Apoptosis assay and cell cycle analysis result demonstrated that compound 7a inhibits the
MCF-7 cancer cells by inducing apoptosis and arresting the cell cycle at G /M phase. Furthermore,
2
molecular docking analysis of most active compound 7a and 7n in the active site of protein kinase
reveals that the binding pattern resembles as that of drug EGFR inhibitor (erlotinib). 3D QSAR
study showed the un-substituted benzimidazole ring and phenyl ring substituted with hydrogen-
bond donor, hydrophobic, electron withdrawing group is favorable for activity. Additionally, the
most active compounds are free from toxicity at a dose ranging from 50 to 500 mg/kg of body
weight of animals. Cardiomyopathy studies showed normal architecture of myofibrils for
compound 7a and 7n, whereas only compound 7a showed nearly equal biochemical parameters
(SGOT and SGPT) as compared to control in hepatoxicity studies. These compounds thus provide
the templates and encourage us to continue for further studies to obtain more potent anticancer
agents.
4
. Experimental section
.1. General
4

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All the chemicals used were of laboratory grade and procured from E. Merck (Darmstadt,
Germany) and S.D. Fine Chemicals (Mumbai, India). Melting points were determined by open
capillary tubes in a Hicon melting point apparatus (Hicon, New Delhi, India) and are uncorrected.
Purity of the compounds was checked by thin-layer chromatography (TLC) plates (silica gel G),
which were visualized by exposing to iodine vapours and UV (ultra violet) light. The FTIR spectra
were recorded on (IR affinity SHIMADZU) FTIR spectrophotometer using KBr pellets; υmax
-1
1
values are given in cm and The H NMR spectra were taken on a NMR spectra were recorded on
1
13
Bruker model DRX-400 MHz NMR spectrometer ( H at 400 MHz, C at 100 MHz) in DMSO-d .
6
Chemical shifts (δ) are expressed in ppm relative to tetramethylsilane (TMS) as an internal standard
and coupling constants (J values) are expressed in Hz. Mass spectra were recorded on LCMS/MS
(PerkineElmer and LABINDIA, Applied Biosystem) model no. API 3000, presented as m/z.
Elemental analysis (C, H and N) were undertaken with Perkin-Elmer model 240C analyzer.
Analyses for C, H, N were within ±0.4% of the theoretical values. The absorbance of the in vitro
assay was measured using an ELISA reader (BioTek Instrument) at a wavelength of 570 nm [28].
The DNA content of the stained cells was analyzed by flow cytometry (BD Biosciences) and the
cycle distribution was quantified [29]. Protocol of the apoptosis/necrosis by flow cytometer as per
the kit manufacturer’s instructions (SIGMA ALDRICH) and in vitro EGFR kinase assay by
reported procedures [29].
Thin-layer chromatography (TLC) was run throughout the reaction to optimize the reaction for
purity and completion. Analytical and spectral data of all the synthesized compounds were in good
agreement with the composition of synthesized compounds.
-1
The FT-IR bands at the 3312-3242, 2964-2855, 1689-1626 and 1292-1262 cm confirmed the
1
presence of -NH, -CH , -C=N and C-O-C functionalities respectively. The H NMR spectrum
2
showed singlet at δ3.65-3.99 and multiplet at δ7.06-8.11 confirming methylene proton and
benzimidazole aromatic protons, while broad singlet at δ12.08-12.95 confirms NH of
benzimidazole ring protons. The structures and purity of the final compounds were confirmed on
the basis of spectral and elemental analyses and the data were within ± 0.4% of the theoretical
values. See supplementary material for the general procedure and NMR results for 7a–x and 8a-e
derivatives.
4
.2. General procedure for the synthesis of compounds (3a-b)

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A mixture of 1.45 g (0.013mole) of 4-substitutedbenzene-1,2-diamine (1a-b) and 1.65g/1.55 ml
(0.014mole) of ethylcyanoacetate (2b) was heated under reflux for 10h in 6.7 ml (0.061mole). After
cooling to room temperature, the precipitate was filtered and washed with anisole. The product was
dried and recrystallized with ethanol/water mixture to get compound (3a) (88% yield). In the case
of chloro-substituted benzene-1,2-diamine (1b), a sticky product was obtained which was extracted
with ether to get the pure compound (3b).
0
4
.2.1
2-Cyanomethyl-1H-benzimidazole (3a, C H N ) brown; Yield 85%, mp 210-212 C; R =
9
7
3
f
-1
0
.60 [toluene: ethyl acetate: formic acid (5:4:1)]; IR (KBr) (cm ): 3383 (NH str), 2253 (C≡N str);
1
6 2
H NMR (300 MHz, DMSO-d ) δ(ppm): 4.37 (s, 2H, -CH CN), 7.18 (dd, J = 3.0, 3.3 Hz, 2H, H-5,6
benzimidazole), 7.55 (d, 2H, J = 8.6 Hz, H-4,7 benzimidazole), 12.63 (bs, 1H, -NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d
6
) δ(ppm): 153.88, 138.04, 132.80, 126.54, 122.15,
7.00. Anal. Calcd.: C, 68.78; H, 4.49; N, 26.74; Found: C, 68.75; H, 4.45; N, 26.76.
.2.2 6-Chloro-2-cyanomethyl-1H-benzimidazole (3b, C H ClN ) brown; Yield 70%, mp 105-
4
4
1
2
9
6
3
0
-1
06 C; R
f
= 0.55 [toluene: ethyl acetate: formic acid (5:4:1)]; IR (KBr) (cm ): 3366 (-NH str),
1
215 (-CN); H NMR (300 MHz, DMSO-d ) δ(ppm): 4.50 (s, 2H, -CH CN), 6.67 (d, 1H, J = 7.8
6
2
Hz, H-5 benzimidazole), 7.07 (d, 1H, J = 6.9, H-4 benzimidazole), 7.31 (s, 1H, H-7 benzimidazole),
1
3
1
1
3.32 (bs, 1H, -NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 145.32, 132.43,
2 6
31.32, 129.32, 127.16, 123.65, 122.67, 119.21, 41.29. Anal. Calcd.: C, 56.41; H, 3.16; N, 21.93;
Found: C, 56.40; H, 3.12; N, 21.96.
4
.3
General procedure for the synthesis of compounds (4a-b)
A mixture of 1g of (3a-b) and 2 ml of 37% hydrochloric acid in water using 1 ml of benzene
sulfonic acid was heated under reflux with stirring for 4-5 h. The reaction was monitored by TLC.
Upon completion, reaction mixture was cooled to room temperature, extracted with diethyl ether (3
x 30 ml) and the solvent was removed under reduced pressure to obtain the product (4a-b).
4
.3.1
1H-Benzimidazol-2-acetic acid (4a, C
9
H
8
N
2
O
2
) green solid; hygroscopic, Yield 81%,
0
-1
mp 240-242 C; R = 0.42 [toluene: ethyl acetate: formic acid (5:4:1)]; IR (KBr) (cm ): 3624 (-OH
f
1
str), 3230(-NH str), 1722 (-C=O str); H NMR (300 MHz, DMSO-d
6
) δ(ppm): 3.64 (s, 2H, -
CH COOH), 7.58 (dd, 2H, J = 7.5, 0.9 Hz, H-5,6 benzimidazole), 7.92 (d, 2H, J = 7.5 Hz, H-4,7
2
1
3
benzimidazole ), 10.59 (bs, 1H, -COOH), 12.03 (bs, 1H, -NH). C NMR (75 MHz, DMSO-d )
6

ACCEPTED MANUSCRIPT
δ(ppm): 164.20, 152.20, 131.95, 128.32, 122.50, 48.40. Anal. Calcd.: C, 61.36; H, 4.58; N, 15.90;
Found: C, 61.33; H, 4.56; N, 15.92.
4
1
3
7
1
.3.2 6-Chloro-1H-benzimidazol-2-acetic acid (4b, C
9
H
7
ClN
2
O
2
) green solid; Yield 71%, mp
0
-1
45-147 C; R = 0.49 [toluene: ethyl acetate: formic acid (5:4:1)]; IR (KBr) (cm ): 3620 (-OH str),
f
1
235(-NH str), 1720 (-C=O str); H NMR (300 MHz, DMSO-d
6
) δ(ppm): 3.84 (s, 2H, -CH
2
COOH),
.06 (d, 1H, J = 7.5 Hz, H-5 benzimidazole), 7.35 (d, 1H, J = 7.8 Hz, H-4 benzimidazole), 7.64 (s,
1
3
H, H-7 benzimidazole ), 11.20 (s, 1H, -COOH), 12.47 (bs, 1H, -NH).
) δ(ppm): 164.21, 152.63, 133.24, 132.32, 129.17, 127.13, 123.55, 122.76, 47.24. Anal.
Calcd.: C, 51.32; H, 3.35; N, 13.30; Found: C, 51.30; H, 3.32; N, 13.33.
C NMR (75 MHz,
DMSO-d
6
4
.4
General procedure for the synthesis of compounds (6a-b)
A solution of compound (4a-b) (4g, 0.022 mol) in absolute ethanol (15 mL) was refluxed for 7 h in
presence of conc. H SO (0.1 mL). After completion of the reaction, it was cooled to room
2
4
temperature, then 2.14 ml (0.044 mol) of hydrazine hydrate was added and the reaction was further
refluxed for another 8h. Detecting single-spot in TLC using the solvent system chloroform:
methanol (9:1), the reaction mixture was poured in ice. A precipitate formed (6a-b) which was
filtered, washed with water and crystallized from ethanol.
4
.4.1
2-(1H-benzo[d]imidazol-2-yl)acetohydrazide (6a, C
9
H
10
N
4
O) white flaky; Yield 86%,
0
-1
mp 120-122 C; R = 0.40 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3360 (NH str), 1733
f
1
(
C=O); H NMR (300 MHz, DMSO-d ) δ(ppm): 3.66 (s, 2H, NH ), 4.28 (s, 2H, CH C=O), 7.11-
6
2
2
7
.12 (d, 2H, J = 3.6, H-5,6 benzimidazole), 7.47 (s, 2H, H-4,7 benzimidazole), 9.36 (s, 1H, -
1
3
NHNH ), 12.22 (bs, 1H, NH benzimidazole, D O exchangeable). C NMR (75 MHz, DMSO-d )
2
2
6
δ(ppm): 164.38, 149.56, 137.68, 133.50, 125.50, 121.27, 45.06. Anal. Calcd.: C, 56.83; H, 5.30;
N, 29.46; Found: C, 56.85; H, 5.34; N, 29.43.
4
.4.2
2-(6-chloro-1H-benzo[d]imidazol-2-yl)acetohydrazide (6b, C H ClN O) white flaky;
9 9 4
0
-1
f
Yield 70%, mp 125-127 C; R = 0.45 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3343 (NH
1
str), 1738 (C=O); H NMR (300 MHz, DMSO-d ) δ(ppm): 3.65 (s, 2H, NH ), 4.37 (s, 2H,
6
2
2
CH C=O), 7.15 (s, 1H, H-5 benzimidazole), 7.38 (s, 1H, H-4 benzimidazole), 7.98 (s, 1H, H-7
1
3
benzimidazole), 9.28 (s, 1H, -NHNH ), 12.23 (bs, 1H, NH, D O exchangeable). C NMR (75
2
2

ACCEPTED MANUSCRIPT
MHz, DMSO-d
6
) δ(ppm): 165.30, 151.48, 134.30, 133.65, 129.29, 127.16, 123.50, 121.27, 46.60.
Anal. Calcd.: C, 48.12; H, 4.04; N, 24.94; Found: C, 48.10; H, 4.08; N, 24.96.
4
.5 General procedure for the synthesis of compounds (7a-x and 8a-e)
A mixture of (6a-b) (0.001 mol), an aliphatic/aromatic acid (equimolar, 0.001 mol) and POCl (5
3
0
mL) was heated under reflux along with stirring for 2 h at 60 C. After completion of the reaction,
checked by single-spot TLC using the solvent systems; chloroform: methanol (9:1), the reaction
mixture was cooled and poured slowly onto crushed ice, neutralized with sodium bicarbonate
solution. A solid mass precipitated out, which was filtered and washed with excess quantity of
water to remove the inorganic component. The compound was recrystallized with ethanol water
mixture.
4
.5.1
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole
(7a,
0
C H ClN O) brown; Yield 70%, mp 180-182 C; R = 0.62 [chloroform: methanol (9:1)]; IR
16
11
4
f
-1
1
(
KBr) (cm ): 3280 (NH str), 2860 (CH str), 1675, 1669 (C=N str), 1275 (C–O–C str); H NMR
2
(
300 MHz, DMSO-d ) δ(ppm): 3.82 (s, 2H, CH ), 7.13 (dd, 2H, J = 1.2, 2.1, H-5,6 benzimidazole),
6
2
7
.48-7.88 (m, 4H, phe), 7.91 (d, 2H, J = 8.7, H-4,7 benzimidazole), 12.36 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d
30.07, 129.85, 122.62, 121.93, 115.07, 35.02. ESI MS (m/z): 311 [M+H]; Anal. Calcd.: C, 61.84;
H, 3.57; N, 18.03; Found: C, 61.80; H, 3.54; N, 18.10.
6
) δ(ppm): 167.21, 164.96, 149.06, 137.20, 131.57,
1
4
.5.2
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(2,4-dichlorophenyl)-1,3,4-oxadiazole
(7b,
0
C H Cl N O) brown; Yield 68%, mp 140-142 C; R = 0.71 [chloroform: methanol (9:1)]; IR
16
10
2
4
f
-1
1
(
KBr) (cm ): 3267 (NH str), 2855 (CH
2
str), 1689, 1682 (C=N str), 1267 (C–O–C str); H NMR
(
300 MHz, DMSO-d ) δ(ppm): 3.65 (s, 2H, CH ), 7.19 (dd, 2H, J = 1.5, 2.5, H-5,6 benzimidazole),
6
2
7
.45-7.85 (m, 3H, phe), 7.90 (d, 2H, J = 7.5, H-4,7 benzimidazole), 12.82 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 168.21, 165.30, 142.62, 136.87, 135.08,
6
1
34.76, 133.56, 130.32, 130.00, 128.56, 122.45, 115.25, 34.62. ESI MS (m/z): 345 [M+H]; Anal.
Calcd.: C, 55.67; H, 2.92; N, 16.23; Found: C, 55.60; H, 2.96; N, 16.27.
.5.3 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-benzyl-1,3,4-oxadiazole (7c, C H N O) dark
brown; Yield 70%, mp 188-190 C; R = 0.82 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3242
4
17
14
4
0
-1
f

ACCEPTED MANUSCRIPT
1
(
NH str), 2849 (CH
2 6
str), 1688, 1681 (C=N str), 1284 (C–O–C str); H NMR (300 MHz, DMSO-d )
δ(ppm): 3.89 (s, 2H, CH ), 3.93 (s, 2H, CH -phe), 7.17 (dd, J = 1.2, 2.6, 2H, H-5,6 benzimidazole),
2
2
7
.24-7.59 (m, 5H, phe), 7.73 (d, J = 7.8, 2H, H-4,7 benzimidazole), 12.43 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d
6
) δ(ppm): 165.23, 164.60, 144.52, 139.13, 135.32,
29.64, 128.45, 125.52, 122.43, 117.26, 30.43, 35.48. ESI MS (m/z): 291 [M+H]; Anal. Calcd.: C,
0.33; H, 4.86; N, 19.30; Found: C, 70.36; H, 4.82; N, 19.36.
.5.4 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-phenyl-1,3,4-oxadiazole (7d, C H N O)
1
7
4
16
12
4
0
-1
white flaky; Yield 58%, mp 105-107 C; R = 0.65 [chloroform: methanol (9:1)]; IR (KBr) (cm ):
f
1
3
312 (NH str), 2964 (CH
2
str), 1685, 1674 (C=N str), 1275 (C–O–C str); H NMR (300 MHz,
DMSO-d ) δ(ppm): 3.79 (s, 2H, CH ), 7.21 (dd, 2H, J = 1.2, 2.3, H-5,6 benzimidazole), 7.41-7.51
6
2
2
(m, 5H, phe), 7.73 (d, 2H, J = 7.5, H-4,7 benzimidazole), 12.42 (bs, 1H, NH, D O exchangeable).
1
3
C NMR (75 MHz, DMSO-d ) δ(ppm): 167.26, 163.56, 143.22, 138.57, 129.53, 128.30, 127.05,
6
1
2
4
23.45, 123.63, 116.50, 32.99. ESI MS (m/z): 277 [M+H]; Anal. Calcd.: C, 69.55; H, 4.38; N,
0.28; Found: C, 69.59; H, 4.35; N, 20.23.
.5.5
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(phenoxymethyl)-1,3,4-oxadiazole
(7e,
0
C
17
H
14N4O
2
) flaky and brown; Yield 71%, mp 184-186 C; R
f
= 0.63 [chloroform: methanol (9:1)];
-1
1
IR (KBr) (cm ): 3310 (NH str), 2887 (CH str), 1668, 1659 (C=N str), 1292 (C–O–C str); H NMR
2
(
300 MHz, DMSO-d ) δ(ppm): 3.83 (s, 2H, CH ), 4.66 (s, 2H, CH -O-phe), 6.96-7.16 (m, 5H, phe),
6 2 2
7
.33 (dd, 2H, J = 4.8, 7.5, H-5,6 benzimidazole),7.50 (d, 2H, J = 3.3, H-4,7 benzimidazole), 12.35
1
3
(
bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 167.26, 167.12, 166.79,
2
6
1
58.17, 149.01, 129.92, 121.92, 121.25, 115.17, 114.82, 66.36, 34.87. ESI MS (m/z): 307 [M+H];
Anal. Calcd.: C, 66.66; H, 4.61; N, 18.29; Found: C, 66.62; H, 4.67; N, 18.26.
.5.6 4-(5-((1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazol-2-yl)phenol (7f, C H N O ) lt
brown; Yield 69%, mp 220-222 C; R
4
1
6
12
-1
4
2
0
f
= 0.82 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3286
1
(
NH str), 2874 (CH str), 1632, 1626 (C=N str), 1265 (C–O–C str); H NMR (300 MHz, DMSO-d )
2
6
δ(ppm): 3.97 (s, 2H, CH
2
), 6.51 (s, 1H, -OH), 7.14 (dd, 2H, J = 1.5, 2.7, H-5,6 benzimidazole),
6
.98-7.48 (m, 4H, phe), 7.86 (d, 2H, J = 7.5, H-4,7 benzimidazole),12.77 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 165.92, 164.58, 145.06, 143.37, 138.10,
6
1
22.30, 118.96, 116.95, 116.57, 115.30, 32.25. ESI MS (m/z): 293 [M+H]; Anal. Calcd.: C, 65.75;
H, 4.14; N, 19.17; Found: C, 65.79; H, 4.19; N, 19.15.

ACCEPTED MANUSCRIPT
4
.5.7
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(2-chlorophenyl)-1,3,4-oxadiazole
(7g,
0
C H ClN O) dark brown; Yield 67%, mp 130-132 C; R = 0.67 [chloroform: methanol (9:1)]; IR
16
11
4
f
-
1
1
(
(
KBr) (cm ): 3289 (NH str), 2868 (CH str), 1668, 1659 (C=N str), 1274 (C–O–C str); H NMR
2
300 MHz, DMSO-d
6 2
) δ(ppm): 3.77 (s, 2H, CH ), 7.06 (dd, 2H, J = 1.6, 2.9, H-5,6 benzimidazole),
7
.36-7.41 (m, 4H, phe), 7.52 (d, 2H, J = 7.6, H-4,7 benzimidazole), 12.22 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d
32.32, 130.20, 129.54, 128.80, 127.53, 123.66, 117.37, 34.36. ESI MS (m/z): 311 [M+H]; Anal.
Calcd.: C, 61.84; H, 3.57; N, 18.03; Found: C, 61.85; H, 3.60; N, 18.09
6
) δ(ppm): 165.59, 164.12, 142.40, 138.11, 135.69,
1
4
.5.8
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-o-tolyl-1,3,4-oxadiazole (7h, C17
H
14
N
4
O) white
0
-1
flaky; Yield 68%, mp 190-192 C; R = 0.82 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3267
f
1
(
NH str), 2889 (CH
2
str), 1672, 1661 (C=N str), 1270 (C–O–C str); H NMR (300 MHz, DMSO-d
6
)
δ(ppm): 2.35 (s, 3H, -CH ), 3.72 (s, 2H, CH ), 7.12 (dd, 2H, J = 7.5, 10.5, H-5,6 benzimidazole),
3
2
7
.33-7.38 (m, 4H, phe), 7.66 (d, 2H, J = 7.5, H-4,7 benzimidazole), 12.40 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d
36.15, 129.31, 128.52, 127.50, 126.31, 123.50, 116.91, 33.65. ESI MS (m/z): 291 [M+H]; Anal.
Calcd.: C, 70.33; H, 4.86; N, 19.30; Found: C, 70.38; H, 4.89; N, 19.35.
.5.9 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-p-tolyl-1,3,4-oxadiazole (7i, C H N O) brown;
6
) δ(ppm): 165.88, 164.50, 143.23, 138.88, 137.93,
1
4
17
14
-1
4
0
Yield 65%, mp 164-166 C; R = 0.85 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3292 (NH
f
1
str), 2895 (CH
2
str), 1677, 1669 (C=N str), 1263 (C–O–C str); H NMR (300 MHz, DMSO-d
6
)
δ(ppm): 2.35 (s, 3H, -CH ), 3.76 (s, 2H, CH ), 7.12 (dd, 2H, J = 1.5, 2.6, H-5,6 benzimidazole),
3
2
7
2
.29-7.90 (m, 4H, phe), 7.95 (d, 2H, J = 7.8, H-4,7 benzimidazole), 12.69 (bs, 1H, NH, D O
1
3
exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.56, 164.48, 143.65, 138.25, 130.63,
6
1
7
4
28.61, 127.54, 123.59, 121.58, 116.30, 32.13, 21.33. ESI MS (m/z): 291 [M+H]; Anal. Calcd.: C,
0.33; H, 4.86; N, 19.30; Found: C, 70.36; H, 4.82; N, 19.35.
.5.10 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(2,3-dimethoxyphenyl)-1,3,4-oxadiazole
(7j,
0
C
18
H
16
N
4
O
3
) brown; Yield 70%, mp 150-152 C; R
f
= 0.64 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3288 (NH str), 2890 (CH str), 1664, 1651 (C=N str), 1272 (C–O–C str); H NMR (300
2
MHz, DMSO-d ) δ(ppm): 3.36 (s, 6H, -OCH ), 3.84 (s, 2H, CH ), 7.20 (d, 2H, J = 6.6, H-4,7
6
3
2
benzimidazole), 7.30-7.37 (m, 3H, phe), 7.52 (dd, 2H, J = 2.1, 5.4, H-5,6 benzimidazole), 12.32
1
3
(
bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 165.72, 164.68, 148.35,
2
6

ACCEPTED MANUSCRIPT
1
45.52, 143.13, 138.52, 130.23, 123.57, 122.62, 120.53, 117.30, 116.29, 52.12, 32.78. ESI MS
(m/z): 337 [M+H]; Anal. Calcd.: C, 64.28; H, 4.79; N, 16.66; Found: C, 64.33; H, 4.74; N, 16.70.
4
.5.11 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(thiophen-2-yl)-1,3,4-oxadiazole
(7k,
0
C
14
H
10
N
4
OS) brown; Yield 55%, mp 110-112 C; R
f
= 0.71 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3273 (NH str), 2868 (CH str), 1678, 1664 (C=N str), 1269 (C–O–C str); H NMR (300
2
6 2
MHz, DMSO-d ) δ(ppm): 3.86 (s, 2H, CH ), 7.18 (dd, 2H, J = 1.8, 2.8, H-5,6 benzimidazole), 7.17-
7
.73 (m, 3H, thiophene), 7.92 (d, 2H, J = 7.6, H-4,7 benzimidazole), 12.76 (bs, 1H, NH, D O
2
1
3
exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.44, 164.49, 143.68, 138.60, 132.42,
6
1
31.21, 128.70, 127.70, 123.34, 116.52, 32.90. ESI MS (m/z): 283 [M+H]; Anal. Calcd.: C, 59.56;
H, 3.57; N, 19.85; Found: C, 59.51; H, 3.53; N, 19.88.
4
.5.12 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(furan-2-yl)-1,3,4-oxadiazole (7l, C14
H
10
N
4
O
2
)
0
-1
brown ; Yield 54%, mp 118-120 C; R = 0.68 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3293
f
1
(
NH str), 2858 (CH str), 1662, 1655 (C=N str), 1265 (C–O–C str); H NMR (300 MHz, DMSO-d )
2
6
δ(ppm): 3.93 (s, 2H, CH
2
), 7.13 (dd, 2H, J = 7.5, 7.5, H-5,6 benzimidazole), 7.37 (d, 2H, J = 7.5, H-
1
3
4
,7 benzimidazole), 7.50-7.68 (m, 3H, furan), 12.26 (bs, 1H, NH, D O exchangeable). C NMR
2
(
75 MHz, DMSO-d
6
) δ(ppm): 166.95, 164.62, 147.35, 143.58, 138.93, 138.33, 123.63, 116.69,
1
16.40, 112.32, 32.72. ESI MS (m/z): 267 [M+H]; Anal. Calcd.: C, 63.15; H, 3.79; N, 21.04;
Found: C, 63.19; H, 3.74; N, 21.06.
4
.5.13 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-benzhydryl-1,3,4-oxadiazole (7m, C23
H
18
N
4
O) lt
0
-1
brown; Yield 51%, mp 120-122 C; R = 0.68 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3274
f
1
(
NH str), 2860 (CH
2
str), 1673, 1662 (C=N str), 1262 (C–O–C str); H NMR (300 MHz, DMSO-d
6
)
δ(ppm): 3.65 (s, 2H, CH ), 4.50 (s, 1H, CH -diphenyl), 7.21 (dd, 2H, J = 1.8, 2.6, H-5,6
2
2
benzimidazole), 7.23-7.53 (m, 10H, diphenyl), 7.86 (d, 2H, J = 7.3, H-4,7 benzimidazole), 12.15
1
3
(
bs, 1H, NH, D
43.40, 138.37, 129.67, 128.36, 125.75, 123.44, 116.05, 41.60, 32.42. ESI MS (m/z): 367 [M+H];
Anal. Calcd.: C, 75.30; H, 4.95; N, 15.29 ; Found: C, 75.35; H, 4.93; N, 15.23.
2 6
O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.57, 164.52, 143.72,
1
4
.5.14 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole
(7n,
0
C H N O ) brown; Yield 72%, mp 160-168 C; R = 0.80 [chloroform: methanol (9:1)]; IR (KBr)
17
14
4
2
f
-1
1
(
cm ): 3263 (NH str), 2883 (CH
2
str), 1667, 1652 (C=N str), 1285 (C–O–C str); H NMR (300
MHz, DMSO-d ) δ(ppm): 3.80 (s, 3H, -OCH ), 5.02 (s, 2H, CH ), 7.12-7.15 (dd, 2H, J = 3.3, 3.6,
6
3
2
H-5,6 benzimidazole), 7.20-7.40 (m, 4H, phe), 7.51 (d, 2H, J = 3.3, H-4,7 benzimidazole), 12.35

ACCEPTED MANUSCRIPT
1
3
(
bs, 1H, NH, D
43.34, 138.70, 123.30, 122.40, 118.63, 116.17, 115.92, 52.08, 32.81. ESI MS (m/z): 307 [M+H];
Anal. Calcd.: C, 66.66; H, 4.61; N, 18.29; Found: C, 66.61; H, 4.65; N, 18.26.
2 6
O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.55, 163.94, 162.03,
1
4
.5.15 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(pyridin-3-yl)-1,3,4-oxadiazole (7o, C15
H
11
N
5
O)
0
-1
brown; Yield 67%, mp 160-162 C; R = 0.71 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3275
f
1
(
NH str), 2908 (CH
2
str), 1671, 1662 (C=N str), 1272 (C–O–C str); H NMR (300 MHz, DMSO-d
6
)
δ(ppm): 3.77 (s, 2H, CH ), 7.12 (dd, 2H, J = 7.2, 7.8, H-5,6 benzimidazole), 7.37 (d, 2H, J = 7.2,
2
1
3
H-4,7 benzimidazole), 7.50-7.68 (m, 4H, pyr), 12.25 (bs, 1H, NH, D O exchangeable). C NMR
2
(
75 MHz, DMSO-d
6
) δ(ppm): 166.81, 164.48, 150.09, 147.61, 142.89, 138.29, 134.56, 127.42,
1
2
4
25.42, 123.00, 116.64, 32.70. ESI MS (m/z): 278 [M+H]; Anal. Calcd.: C, 64.97; H, 4.00; N,
5.26; Found: C, 64.92; H, 4.04; N, 25.22.
.5.16 2-(5-((1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazol-2-yl)acetonitrile (7p, C H N O)
1
2
9
5
0
-1
brown; Yield 65%, mp 145-147 C; R = 0.68 [chloroform: methanol (9:1)]; IR (KBr) (cm ): 3273
f
1
(
NH str), 2866 (CH
2
str), 1679, 1665 (C=N str), 1282 (C–O–C str); H NMR (300 MHz, DMSO-d
6
)
δ(ppm): 3.94 (s, 2H, CH ), 4.21 (s, 2H, CH -CN), 7.05 (dd, 2H, J = 2.9, 3.5, H-5,6 benzimidazole),
2
2
1
3
7
2
.76 (d, 2H, J = 7.6, H-4,7 benzimidazole), 12.44 (bs, 1H, NH, D O exchangeable). C NMR (75
MHz, DMSO-d ) δ(ppm): 165.86, 164.70, 143.40, 138.72, 127.71, 123.32, 116.27, 32.83, 25.34.
6
ESI MS (m/z): 240 [M+H]; Anal. Calcd.: C, 60.25; H, 3.79; N, 29.27; Found: C, 60.20; H, 3.75; N,
2
9.31.
4
.5.17
2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole
(7q,
0
C
16
H
11FN
4
O) brown; Yield 62%, mp 165-167 C; R
f
= 0.66 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3269 (NH str), 2902 (CH str), 1688, 1674 (C=N str), 1289 (C–O–C str); H NMR (300
2
MHz, DMSO-d ) δ(ppm): 3.81 (s, 2H, CH ), 7.13 (dd, 2H, J = 2.7, 3.0, H-5,6 benzimidazole), 7.29-
6
2
7
.35 (d, 2H, phe), 7.44-7.54 (d, 2H, H-4,7 benzimidazole) 7.91-7.95 (d, 2H, phe), 12.31 (bs, 1H,
1
3
NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.53, 164.78, 160.90, 143.49,
2
6
1
6
4
38.70, 125.33, 123.36, 121.42, 117.61, 116.20, 33.04. ESI MS (m/z): 295 [M+H]; Anal. Calcd.: C,
5.30; H, 3.77; N, 19.04; Found: C, 65.35; H, 3.81; N, 19.09.
.5.18 1-(2-(5-((1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazol-2-yl)phenyl)ethanone
(7r,
0
C
18
H
14
N
4
O
2
) brown; Yield 75%, mp 215-217 C; R
f
= 0.64 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3289 (NH str), 2852 (CH str), 1662, 1654 (C=N str), 1268 (C–O–C str); H NMR (300
2
6 3 2
MHz, DMSO-d ) δ(ppm): 2.31 (s, 3H, -COCH ), 3.87 (s, 2H, CH ), 7.23 (dd, 2H, J = 1.5, 2.7, H-

ACCEPTED MANUSCRIPT
5
1
1
,6 benzimidazole), 7.41-7.66 (m, 4H, phe), 7.82 (d, 2H, J = 7.6, H-4,7 benzimidazole), 12.30 (bs,
1
3
H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 175.96, 165.96, 164.62,
2
6
43.85, 138.91, 137.92, 135.53, 134.21, 133.42, 128.72, 127.30, 122.99, 116.48, 32.70, 29.63. ESI
MS (m/z): 319 [M+H]; Anal. Calcd.: C, 67.91; H, 4.43; N, 17.60; Found: C, 67.95; H, 4.48; N,
1
7.64.
4
.5.19 2-(5-((1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazol-2-yl)-4-aminophenol
(7s,
0
C H N O ) lt brown; Yield 68%, mp 165-167 C; R = 0.61 [chloroform: methanol (9:1)]; IR
16
13
5
2
f
-1
1
(
KBr) (cm ): 3271 (NH str), 2901 (CH str), 1669, 1661 (C=N str), 1269 (C–O–C str); H NMR
2
(300 MHz, DMSO-d
6 2 2
) δ(ppm): 3.95 (s, 2H, CH ), 5.34 (s, 1H, -OH), 6.17 (s, 2H, -NH ), 6.51-6.69
(m, 3H, phe), 7.12 (dd, 2H, J = 1.5, 6.9, H-5,6 benzimidazole),7.34 (d, 2H, J = 7.8, H-4,7
1
3
2 6
benzimidazole), 12.95 (bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm):
1
3
4
4
66.33, 164.61, 143.75, 143.32, 141.32, 138.92, 123.49, 117.72, 116.76, 116.24, 115.76, 112.80,
2.68. ESI MS (m/z): 308 [M+H]; Anal. Calcd.: C, 62.53; H, 4.26; N, 22.79; Found: C, 62.59; H,
.29; N, 22.81.
.5.20 2-(5-((1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazol-2-yl)benzoic
acid
(7t,
0
C
17
H
12
N
4
O
3
) brown; Yield 75%, mp 176-178 C; R
f
= 0.58 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3262 (NH str), 2871 (CH str), 1672, 1661 (C=N str), 1285 (C–O–C str); H NMR (300
2
MHz, DMSO-d ) δ(ppm): 3.95 (s, 2H, CH ), 7.19 (dd, 2H, J = 3.5, 7.2, H-5,6 benzimidazole), 7.37
6
2
(d, 2H, J = 7.5, H-4,7 benzimidazole), 7.62-7.85 (m, 4H, phe), 11.42 (s, 1H, -COOH), 12.34 (bs,
1
3
1
1
H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.72, 165.73, 164.69,
2 6
43.70, 138.58, 138.34, 134.54, 130.21, 129.50, 128.15, 127.60, 123.35, 116.65, 32.53. ESI MS
(m/z): 321 [M+H]; Anal. Calcd.: C, 63.75; H, 3.78; N, 17.49; Found: C, 63.71; H, 3.72; N, 17.45.
4
.5.21 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(naphthalen-1-ylmethyl)-1,3,4-oxadiazole (7u,
0
C
21
H
16
N
4
O) lt brown; Yield 70%, mp 162-164 C; R
f
= 0.51 [chloroform: methanol (9:1)]; IR
-
1
1
(
(
KBr) (cm ): 3286 (NH str), 2881 (CH str), 1676, 1668 (C=N str), 1266 (C–O–C str); H NMR
2
300 MHz, DMSO-d
6
) δ(ppm): 3.94 (s, 2H, CH
2 2
), 4.21 (s, 2H, -CH ), 7.15 (dd, 2H, J = 1.9, 2.8, H-
5
1
1
1
,6 benzimidazole), 7.38 (d, 2H, J = 7.6, H-4,7 benzimidazole), 7.65-7.90 (m, 7H, biphenyl),
1
3
2.42 (bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.95, 164.50,
2
6
43.44, 138.51, 134.28, 133.05, 132.31, 131.40, 129.32, 128.53, 125.43, 125.16, 117.65, 126.75,
23.42, 116.67, 32.15. ESI MS (m/z): 341 [M+H]; Anal. Calcd.: C, 74.10; H, 4.74; N, 16.46;
Found: C, 74.17; H, 4.79; N, 16.51.

ACCEPTED MANUSCRIPT
4
.5.22 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-((2,7a-dihydro-1H-indol-3-yl)methyl)-1,3,4-
0
oxadiazole (7v, C H N O) yellow; Yield 68%, mp 180-182 C; R = 0.56 [chloroform: methanol
1
9
17
5
f
-1
1
(
9:1)]; IR (KBr) (cm ): 3267 (NH str), 2872 (CH str), 1672, 1664 (C=N str), 1281 (C–O–C str); H
2
6 2
NMR (300 MHz, DMSO-d ) δ(ppm): 3.23 (s, 2H, -CH -indole), 3.17-3.43 (m, 3H, pyrrole part of
indole), 3.99 (s, 2H, CH ), 7.01 (bs, 1H, -NH), 7.15 (dd, 2H, J = 3.7, 7.2, H-5,6 benzimidazole),
2
7
2
.38 (d, 2H, J = 7.6, H-4,7 benzimidazole), 7.87-7.84 (m, 4H, indole), 12.08 (bs, 1H, NH, D O
1
3
exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.47, 164.44, 143.36, 138.21, 138.76,
6
1
32.60, 131.32, 123.42, 128.92, 127.44, 116.12, 64.36, 52.13, 32.49, 29.23. ESI MS (m/z): 332
M+H]; Anal. Calcd.: C, 68.87; H, 5.17; N, 21.13; Found: C, 68.82; H, 5.13; N, 21.19.
.5.23 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(pyridin-2-yl)-1,3,4-oxadiazole (7w, C H N O)
[
4
15
11
5
0
-1
lt brown; Yield 58%, mp 172-174 C; R
f
= 0.65 [chloroform: methanol (9:1)]; IR (KBr) (cm ):
1
3
282 (NH str), 2891 (CH str), 1683, 1677 (C=N str), 1268 (C–O–C str); H NMR (300 MHz,
2
DMSO-d ) δ(ppm): 3.84 (s, 2H, CH ), 7.15 (dd, 2H, J = 3.0, 3.3, H-5,6 benzimidazole), 8.67 (d,
6
2
2
2
H, J = 7.7, H-4,7 benzimidazole), 7.51-8.04 (m, 4H, pyr), 12.23 (bs, 1H, NH, D O exchangeable).
1
3
C NMR (75 MHz, DMSO-d ) δ(ppm): 166.64, 164.32, 157.08, 149.76, 143.03, 138.97, 137.13,
6
1
25.30, 123.67, 123.32, 116.14, 32.80. ESI MS (m/z): 278 [M+H]; Anal. Calcd.: C, 64.97; H, 4.00;
N, 25.26; Found: C, 64.92; H, 4.04; N, 25.29.
4
.5.24 2-((1H-benzo[d]imidazol-2-yl)methyl)-5-(2-methylbenzyl)-1,3,4-oxadiazole
(7x,
0
C
18
H
16
N
4
O) grey; Yield 70%, mp 181-183 C; R
f
= 0.70 [chloroform: methanol (9:1)]; IR (KBr)
-1
1
(
cm ): 3272 (NH str), 2878 (CH str), 1665, 1656 (C=N str), 1273 (C–O–C str); H NMR (300
2
6 3 2 2
MHz, DMSO-d ) δ(ppm): 2.29 (s, 3H, CH ), 3.50 (s, 2H, CH ), 3.81 (s, 2H, CH phenyl), 6.91-7.13
(m, 4H, phe), 7.34 (dd, 2H, J = 7.5, 7.8, H-5,6 benzimidazole), 7.61 (d, 2H, J = 2.3, H-4,7
1
3
benzimidazole), 12.28 (bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm):
2
6
1
3
7
4
66.53, 164.34, 144.87, 138.40, 133.08, 130.93, 127.54, 126.73, 125.43, 123.01, 115.12, 32.43,
0.32, 21.10. ESI MS (m/z): 305 [M+H]; Anal. Calcd.: C, 71.04; H, 5.30; N, 18.41; Found: C,
1.05; H, 5.33; N, 18.45.
.5.25 2-((6-chloro-1H-benzo[d]imidazol-2-yl)methyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole (8a,
0
C H Cl N O) brown; Yield 55%, mp 185-187 C; R = 0.61 [chloroform: methanol (9:1)]; IR
16
10
2
4
f
-1
1
(
KBr) (cm ): 3260 (NH str), 2893 (CH
2
str), 1670, 1662 (C=N str), 1279 (C–O–C str); H NMR
(
300 MHz, DMSO-d ) δ(ppm): 3.97 (s, 2H, CH ), 6.95 (d, 1H, J = 7.5, H-5 benzimidazole), 7.12 (d,
6
2
1
H, J = 7.2, H-4 benzimidazole), 7.34 (s, 1H, H-7 benzimidazole), 7.50-7.67 (m, 4H, phe), 12.21

ACCEPTED MANUSCRIPT
1
3
(
bs, 1H, NH, D
40.43, 138.37, 132.70, 131.93, 131.31, 129.41, 128.27, 123.32, 116.20, 115.80, 30.70. ESI MS
m/z): 345 [M+H]; Anal. Calcd.: C, 56.84; H, 3.37; N, 15.60; Found: C, 56.79; H, 3.34; N, 15.64.
2 6
O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.56, 164.34, 143.42,
1
(
4
.5.26 2-((6-chloro-1H-benzo[d]imidazol-2-yl)methyl)-5-(2,4-dichlorophenyl)-1,3,4-oxadiazole
0
(
8b, C H Cl N O) brown; Yield 58%, mp 146-148 C; R = 0.70 [chloroform: methanol (9:1)]; IR
1
6
9
3
4
f
-1
1
(
KBr) (cm ): 3279 (NH str), 2883 (CH
2
str), 1671, 1664 (C=N str), 1276 (C–O–C str); H NMR
(
300 MHz, DMSO-d ) δ(ppm): 3.92 (s, 2H, CH ), 7.17 (d, 1H, J = 7.2, H-5 benzimidazole), 7.55 (d,
6
2
1
H, J = 7.4, H-4 benzimidazole), 7.11-7.70 (m, 3H, phe), 8.11 (s, 1H, H-7 benzimidazole), 12.77
1
3
(
2 6
bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.57, 164.40, 143.20,
1
2
2
4
40.86, 138.97, 136.40, 135.29, 132.52, 131.50, 130.53, 129.52, 126.16, 123.81, 116.28, 115.35,
7.70. ESI MS (m/z): 378 [M+H]; Anal. Calcd.: C, 51.87; H, 2.82; N, 14.23; Found: C, 51.82; H,
.85; N, 14.26.
.5.27 2-benzyl-5-((6-chloro-1H-benzo[d]imidazol-2-yl)methyl)-1,3,4-oxadiazole
(8c,
0
C
17
H
13ClN
4
O) brown; Yield 54%, mp 186-188 C; R
f
= 0.85 [chloroform: methanol (9:1)]; IR
-
1
1
(
(
KBr) (cm ): 3306 (NH str), 2872 (CH str), 1689, 1681 (C=N str), 1269 (C–O–C str); H NMR
2
6 2 2
300 MHz, DMSO-d ) δ(ppm): 3.80 (s, 2H, CH ), 3.93 (s, 2H, CH -phe), 7.10 (d, 1H, J = 7.4, H-5
benzimidazole), 7.26-7.33 (m, 5H, phe), 7.49 (d, 1H, J = 7.5, H-4 benzimidazole), 8.11 (s, 1H, H-7
1
3
benzimidazole), 12.31 (bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm):
2
6
1
3
4
4
66.32, 164.98, 143.31, 140.53, 138.29, 129.53, 129.20, 128.64, 125.57, 123.31, 116.17, 115.30,
0.08. ESI MS (m/z): 325 [M+H]; Anal. Calcd.: C, 62.87; H, 4.03; N, 17.25; Found: C, 62.82; H,
.07; N, 17.29.
.5.28 2-((6-chloro-1H-benzo[d]imidazol-2-yl)methyl)-5-phenyl-1,3,4-oxadiazole
(8d,
0
C H ClN O) brown; Yield 63%, mp 110-112 C; R = 0.64 [chloroform: methanol (9:1)]; IR
16
11
4
f
-1
1
(
KBr) (cm ): 3294 (NH str), 2883 (CH
2
str), 1671, 1662 (C=N str), 1282 (C–O–C str); H NMR
(
300 MHz, DMSO-d ) δ(ppm): 3.78 (s, 2H, CH ), 7.26 (d, 1H, J = 7.5, H-5 benzimidazole), 7.59 (d,
6
2
1
H, J = 7.5, H-4 benzimidazole), 8.12 (s, 1H, H-7 benzimidazole), 7.40-8.21 (m, 5H, phe), 12.82
1
3
(
bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 166.89, 164.83, 143.61,
2
6
1
40.63, 138.64, , 129.50, 129.31, 128.73, 127.56, 123.28, 122.38, 115.98, 115.31, 32.36. ESI
MS (m/z): 311 [M+H]; Anal. Calcd.: C, 61.84; H, 3.57; N, 18.03; Found: C, 61.80; H, 3.54; N,
8.07.
1

ACCEPTED MANUSCRIPT
4
.5.29 2-((6-chloro-1H-benzo[d]imidazol-2-yl)methyl)-5-(2-chlorophenyl)-1,3,4-oxadiazole (8e,
0
C H Cl N O) brown; Yield 65%, mp 135-137 C; R = 0.65 [chloroform: methanol (9:1)]; IR
16
10
2
4
f
-1
1
(
KBr) (cm ): 3298 (NH str), 2890 (CH str), 1668, 1661 (C=N str), 1290 (C–O–C str); H NMR
2
6 2
(300 MHz, DMSO-d ) δ(ppm): 3.67 (s, 2H, CH ), 7.15 (d, 1H, J = 7.6, H-5 benzimidazole), 7.58
(d, J = 7.9, 1H, H-4 benzimidazole), 7.18-7.60 (m, 4H, phe), 8.07 (s, 1H, H-7 benzimidazole),
1
3
1
1
1
5
2 6
2.42 (bs, 1H, NH, D O exchangeable). C NMR (75 MHz, DMSO-d ) δ(ppm): 165.99, 164.87,
43.32, 140.59, 138.48, 138.07, 134.59, 130.62, 129.66, 128.70, 127.15, 126.56, 123.35, 116.64,
15.58, 30.89. ESI MS (m/z): 345 [M+H]; Anal. Calcd.: C, 56.84; H, 3.37; N, 15.60; Found: C,
6.89; H, 3.33; N, 15.64.
4
.6
Anticancer activity assays
All of the target compounds were determined against MCF-7 (breast cancer cell line), HaCaT
(
human skin), MDA-MB231 (breast adenocarcinoma), A549 (human lung carcinoma) and HepG2
liver hepatocellular carcinoma). Exponentially growing cells were seeded into 96-well plates at a
(
3
0
concentration of 3 x 10 cells per well. After 24 h incubation at 37 C, the culture medium was
removed and replaced with fresh medium containing the candidate compounds in different
concentrations. The cells were incubated for another 72 h. After 72 h, media of each well was
aspirated and added 100 µL of MTT solution (1 mg/mL) in serum free media. The plate was further
0
incubated for 4 h at 37 C. Discarded the suspension and added 100 µL of dimethyl sulfoxide
(DMSO) to each well and swirled the plates to dissolve the dark blue crystals of formazan. The
absorbance was measured using an ELISA reader (BioTek Instrument) at a wavelength of 570 nm.
Each concentration was analyzed in triplicate and the experiment was repeated three times.
Percentage viability based was calculated based on following formula:
%
cell viability = 100 (A-A )/(A -A )
0
t
0
Where, A represents absorbance of compounds at 570nm and A
0
t
and A represent zero percent cell
viability and 100% cell viability determined in media only, respectively.
The average 50% inhibitory concentration (IC ) was calculated after plotting graph of dose-
5
0
response curves by using GraphPad Prism 5 [28].
4
.7 In vitro EGFR and erbB2 phosphorylation assay

ACCEPTED MANUSCRIPT
3
Exponentially growing KB cells [29] (seeded at 3x10 cells/well in 96-well plates in serum
containing medium for 72 h) and then were starved with serum free media for 24 h. Compound was
added and incubated for 90 min before the addition of EGF (100 ng/ml) for 5 min to increase EGFR
phosphorylation to 90% of max (ED 90) to allow interassay comparison. The cells were lysed in
RIRA buffer [50 mmol/L tris (pH 7.4), 1% NP40, 0.25% deoxycholate, 150 mmol/L NaCl, 1 mmol/
L sodium orthovanidate, 1 mmol/L EDTA, and Roche protease inhibitor cocktail] and a sandwich
ELISA was used to measure total EGFR phosphorylation.
In Vitro erbB2 phosphorylation studies:- MCF-7 cells were grown in RPMI 1640 containing 10%
FCS for 72 h, serum starved (2.5% stripped serum) for 24 h, and incubated with compound for 90
min before stimulating for 5 min with 35 ng/mL (ED90) HRG (Heregulin). Cells were lysed in a
radioimmunoprecipitation assay buffer (Sigma radioimmunoprecipitation assay buffer with protease
inhibitors) and analyzed using the human phospho-erbB2 ELISA kit (R&D systems). The MCF-7
cl24 line was created by transfecting parental MCF-7 cells with the full-length erbB2 receptor as
described previously [29].
4
.8
Cell Apoptosis Assay
6
Cells were seeded at 10 per well in 6-well plate and incubated 18 h. Cells were treated with
different concentrations of 7a and 7n for 24 h and harvested by using trypsin. Cell pellet were
washed twice with PBS and cellular apoptosis/necrosis were determined by flow cytometry using
annexin V-FITC Apoptosis kit (SIGMA ALDRICH). Protocol of this assay was adopted as per the
kit manufacturer’s instructions [30].
4
.9 Flow Cytometry Cell Analysis
6
MCF-7 Cells were seeded in density of 10 cells per well in 6-well plate and incubated for 18 h.
Cells were treated with various concentrations of 7a and 7n and incubated further for next 24 h.
Cells were washed with PBS and trypsinized to harvest from plates. After washing with PBS
fixation of cells was carried out in ice-cold 70% ethanol for 30 min. Cells were resuspended in PBS
containing RNase A for 10 min and then added 50 µL PI (500ug/ml) to stain the cellular DNA, the
staining process lasted 30 min at 4°C in darkness. The DNA content of the stained cells was

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analyzed by flow cytometry (BD Biosciences) and the cycle distribution was quantified. All the
experiments were performed in two independent times and representative data are presented here
(
Figure 5) [30].
4
.10
Molecular Docking
The ligand docking studies into tyrosine kinase receptor binding pocket were carried out using
Maestro 10.5 program (Schrodinger Inc. USA). The tyrosine kinase enzyme is a validated target for
anticancer drug and the crystal structure was downloaded from protein data bank (PDB 1M17) [31].
The protein preparation was done in three step “i.e.” preprocess, review & modify and refinement
using ‘protein preparation wizard’ in Maestro 10.5. In these steps, water molecules are deleted and
hydrogen atoms are added. The Energy of the structure was minimized using OPLS 2005 force
field. Similarly, ligands were prepared again using force field 2005. Receptor grid generation
program was run by clicking any atom of the ligand and the default box was prepared. The ligand
was docked into the grid generated from the protein using extra precision (XP). The results were
evaluated by glide score (docking score) [32].
4
.11 3D QSAR generation Model
The primary requirement for the 3D-QSAR generation is the alignment of the molecules.
Alignments of the molecules were obtained from running maximum common substructures (Canvas
alignment) [33]. Due to the common structural framework of the molecules, atom based 3D-QSAR
models were chosen to predict the structure-activity relationships (SAR). Atom based 3D-QSAR
were generated for the 20-members of the training set and 9-members of the test set molecules, grid
space of 1.0 Å and four PLS factor [34].
4
.12
Animal and treatment
The compounds which showed good anticancer activity against cancer cell lines 7a and 7n were
chosen for the acute toxicity (lethal dose) which is one of the basic requirements in fixing the
therapeutic dose in drug development [52]. The investigations were conducted on female albino rats
of (110-115 g). The female albino rats were kept under standard conditions at an ambient
o
temperature of 25 ± 2 C and allowed free access to food and water except at the time they were
brought out of the cage. Rats were allowed to acclimate to laboratory conditions for 7 days prior to

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dosing. All compounds were dissolved in DMSO and administered by gavages at a fixed volume of
0
.5 mL/ rats.
4
.12.1 Acute toxicity studies
Animals were randomly divided into three groups, five rats of each. Two groups were used for
single dose treatments of compounds 7a and 7n at 500 mg/kg body weight, and third group as
control (0.5 mL DMSO/rat). Then mortality of treated-rats was recorded after 24 h. All the
experimental protocols were carried out with the permission from Institutional Animal Ethics
committee (IAEC), form no. 1199. Animals were obtained from Central Animal House Facility,
Hamdard University, New Delhi-62. Registration no. and date of registration is 173/CPCSEA and
Jan 28, 2000.
4
.12.2 Cardiomyopathy and Hepatotoxicity studies
Animals were divided into the four groups; the group I and II is treated orally with the drug 7a and
n (500 mg/kg body weight each). Group III was treated orally with a dose (2 mg/kg daily for 5
7
days for a cumulative dose of 10 mg/kg) of standard drug doxorubicin [55] whereas, group IV
serves as control administered with a single dose of DMSO solution. The animals were observed for
a periods of 14 days. On the day 14 the animals were sacrificed and the heart were taken out and
preserved in 40% of formalin. Section were cut transversally and stained with hematoxylin and
eosin before being observed under an Olympus microscope at 100X and 400X magnifications [56].
For the liver enzyme, the active drugs were administered to each animal at a single dose treatment
of compounds 7a and 7n at 500 mg/kg body weight. After the stipulated period of 2 weeks, each
animal was anesthetized by anesthetic ether, and blood was collected from the liver to assess the
biochemical parameters such as SGOT, SGPT, alkaline phosphate, and total protein according to
the reported methods [58].
Acknowledgments
We are grateful to Jamia Hamdard (Hamdard University) for providing laboratory facilities. One of
the author (Md. Jawaid Akhtar, IF-130481) expresses thanks to Department of Science and
Technology (DST) New Delhi, India for INSPIRE fellowship. Authors also expresses their thanks
to All India Council for Technical Education (AICTE) New Delhi, India for RPS (8-

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9
9/RIFD/RPS/POLICY-3/2013-14) fellowship. Assistance provided by Vaibhav Jain and Adarsh
from CSIR- IGIB in FACS analysis thankfully acknowledged. Support rendered by Vinod Devaraji
and Pritesh Bhat of schrodinger is also acknowledged.
Abbreviation: QSAR, Quantitative structure-activity relationships; SGOT, Serum glutamic oxaloacetic transaminase;
SGPT, serum glutamic-pyruvic transaminase; ATP, Adenosine triphosphate; NSCLC, Non-small cell lung cancer;
MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; PPA, polyphosphoric acid; 5-FU, 5-Flurouracil;
FACS, Fluorescence-activated cell sorting; LVD, Left ventricular dysfunction; HF, Heart Failure; POCl , phosphorus
3
oxychloride; FTIR, Fourier transform infrared spectroscopy; KBr, Potassium Bromide; ELISA, enzyme-linked
1
immunosorbent assay; H NMR, Proton nuclear magnetic resonance; TLC, Thin layer chromatography; HCl,
Hydrochloric acid; NaOH, Sodium hydroxide; PBS, Phosphate-buffered saline.

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References
1
2
3
4
. J.J Liao, Molecular recognition of protein kinase binding pockets for design of potent and
selective kinase inhibitors, J. Med. Chem. 50 (2007) 409-424.
. J. Zhang, P.L Yang, Gray NS, Targeting cancer with small molecule kinase inhibitors,
Nature reviews/cancer, 9 (2009) 28-39.
. F. Zuccotto, E. Ardini, E. Casale, M. Angiolini, Through the “Gatekeeper Door”: Exploiting
the active kinase conformation, J. Med. Chem. 53 (2010) 2681-2694.
. R.B Bakr, E.K.A. Abdelall, M.K. Abdel-Hamid, M.M. Kandeel, Design and synthesis of
new EGFR-tyrosine kinase inhibitors containing pyrazol0[3-4-d] pyrimidine cores as
anticancer agents, Bull. Pharm. Sci. Assiut. Univ. 35 (2012) 27-42. [4a] J. Han, S.
Henriksen, K.G. Nørsett , E. Sundby , B.H. Hoff, Balancing potency, metabolic stability and
permeability in pyrrolopyrimidine-based EGFR inhibitors, Eur. J. Med. Chem. 124 (2016)
5
83-607.
5
. R. Determann, J. Dreher, K. Baumann, L. Preu, P.G. Jones, F. Totzke, C. Schächtele,
M.H.G. Kubbutat, C. Kunick, 2-Anilino-4-(benzimidazol-2-yl)pyrimidines - A multikinase
inhibitor scaffold with antiproliferative activity toward cancer cell lines, Eur. J. Med. Chem.
5
3 (2012) 254-263. [5a] J. Han, S.J. Kaspersen, S. Nervik, K.G. Nørsett, E. Sundby, B. H.
Hoff, Chiral 6-aryl-furo[2,3-d]pyrimidin-4-amines as EGFR inhibitors, Eur. J. Med. Chem.
19 (2016) 278-299.
1
6
. P.M. Harari, Epidermal growth factor receptor inhibition strategies in oncology, Endocrine-
Related Cancer, 11 (2004) 689–708. [6a] S. Yin, C. Tang, B. Wang, Y. Zhang, L. Zhou, L.
Xue, C. Zhang, Design, synthesis and biological evaluation of novel EGFR/HER2 dual
inhibitors bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold. Eur. J. Med. Chem. 120
(2016) 26-36.
7
8
. M. Zukin, Epidermal growth factor receptor inhibitors in non-small cell lung cancer: current
status and future perspectives, Rev. Assoc. Med. Bras. 58 (2012) 263-268.
. A. Díaz, A. Lage, Therapies based on inhibitors of the epidermal growth factor receptor:
reaching for the future, Biotecnología. Aplicada. 24 (2007)10-18.

ACCEPTED MANUSCRIPT
9
1
1
1
1
. J. Lin, W. Shen, J. Xue, J. Sun, X. Zhang, C. Zhang, Novel oxazolo[4,5-g]quinazolin-2(1H)-
ones: Dual inhibitors of EGFR and Src protein tyrosine kinases, Eur. J. Med. Chem. 55
(
2012) 39-48.
0. L. Chen, C. Chang, D.B. Salunke et al, Divergent Synthesis of Unsymmetrical Annulated
Biheterocyclic Compound Libraries: Benzimidazole Linked Indolo-
benzodiazepines/quinoxaline, ACS. Comb. Sci. 13 (2011) 391–398.
1. A. Kamal, P.P. Kumar, K. Sreekanth et al, Synthesis of new benzimidazole linked
pyrrolo[2,1-c][1,4]benzodiazepine conjugates with efficient DNA-binding affinity and
potent cytotoxicity, Bioorg. Med. Chem. Lett. 18 (2008) 2594–2598.
2. K. Shao, X. Zhang, P. Chen et al, Synthesis and biological evaluation of novel pyrimidine-
benzimidazol hybrids as potential anticancer agents, Bioorg. Med. Chem. Lett. 24 (2014)
3
877-3881.
3. N.C. Desai, N. Bhatt, H. Somani, A. Trivedi, Synthesis, antimicrobial and cytotoxic
activities of some novel thiazole clubbed 1,3,4-oxadiazoles, Eur. J. Med. Chem. 67 (2013)
5
4-59.
1
1
4. D. Kumar, G. Patel, A.K. Chavers, K. Chang, K. Shah, Synthesis of novel 1,2,4-oxadiazoles
and analogues as potential anticancer agents, Eur. J. Med. Chem. 46 (2011) 3085-3092.
5. N.B. Patel, A.C. Purohit, D.P. Rajani, R. Moo-Puc, G. Rivera, New 2-benzylsulfanyl-
nicotinic acid based 1,3,4-oxadiazoles: Their synthesis and biological evaluation, Eur. J.
Med. Chem. 62 (2013) 677-687.
1
6. N.I. Ziedan, F. Stefanelli, S. Fogli, A.D. Westwell, Design, synthesis and pro-apoptotic
antitumour properties of indole-based 3,5-disubstituted oxadiazoles, Eur. J. Med. Chem. 45
(2010) 4523-4530.
1
1
7. M.A. Ali, M. Shaharyar, Oxadiazole mannich bases: synthesis and antimycobacterial
activity, Bioorg. Med. Chem. Lett. 17 (2007) 3314-16.
8. R. Gitto, S. Ferro, S. Agnello et al, Synthesis and evaluation of pharmacological profile of
1
-aryl-6,7-dimethoxy-3,4-dihyroisoquinoline-2(1H)-sulfonamide, Bioorg. Med. Chem. 17
(2009) 3659-3664.
1
9. A. Zarghi, M. Faizi, B. Shafaghi et al. Design and synthesis of new 2-substituted-5-(2-
benzylthiophenyl)-1,3,4-oxa diazole as benzodiazepine receptor agonists, Bioorg. Med.
Chem. Letters. 15 (2009) 3126-3129.