Synthesis and biological evaluation of imidazopyridinyl-1,3,4-oxadiazole conjugates as apoptosis inducers and topoisomerase IIα inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.09.002

A series of imidazopyridinyl-1,3,4-oxadiazole conjugates were synthesized and investigated for their cytotoxic activity and some compounds showed promising cytotoxic activity. Compound 8q (NSC: 763639) exhibited notable growth inhibition that satisfies th

Full text of DOI:10.1016/j.bioorg.2016.09.002

Accepted Manuscript
Synthesis and Biological Evaluation of Imidazopyridinyl-1,3,4-oxadiazole con-
jugates as Apoptosis Inducers and Topoisomerase IIα Inhibitors
A.V. Subba Rao, M.V.P.S. Vishnu Vardhan, N.V. Subba Reddy, T. Srinivasa
Reddy, Siddiq Pasha Shaik, Chandrakant Bagul, Ahmed Kamal
PII:
S0045-2068(16)30068-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.09.002
YBIOO 1948
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
10 June 2016
1 September 2016
2 September 2016
Please cite this article as: A.V. Subba Rao, M.V.P.S. Vishnu Vardhan, N.V. Subba Reddy, T. Srinivasa Reddy, S.P.
Shaik, C. Bagul, A. Kamal, Synthesis and Biological Evaluation of Imidazopyridinyl-1,3,4-oxadiazole conjugates
as Apoptosis Inducers and Topoisomerase IIα Inhibitors, Bioorganic Chemistry (2016), doi: http://dx.doi.org/
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0.1016/j.bioorg.2016.09.002
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Synthesis and Biological Evaluation of Imidazopyridinyl-1,3,4-oxadiazole
conjugates as Apoptosis Inducers and Topoisomerase IIα Inhibitors
a,b
a
a
c
A.V. Subba Rao, M.V.P.S. Vishnu Vardhan, N. V. Subba Reddy, T. Srinivasa Reddy,
a
d
*a,b,c,d
Siddiq Pasha Shaik, Chandrakant Bagul and Ahmed Kamal
a
b
Medicinal Chemistry and Pharmacology, Academy of Scientific and Innovative Research,
c
IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad
5
00007, India
d
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education &
Research (NIPER), Hyderabad 500 037, India
Abstract
A series of imidazopyridinyl-1,3,4-oxadiazole conjugates were synthesized and investigated for
their cytotoxic activity and some compounds showed promising cytotoxic activity. Compound
8
q (NSC: 763639) exhibited notable growth inhibition that satisfies threshold criteria at single
dose (10 µM) on all human cancer cell lines. This compound was further evaluated at five dose
levels (0.01, 0.1, 1, 10 and 100 µM) to obtain GI50 values ranging from 1.30-5.64 µM. Flow
cytometric analysis revealed that compound 8q arrests the A549 cells in sub G1 phase followed
by induction of apoptosis which was further confirmed by Annexin-V-FITC, Hoechst nuclear
staining, caspase 3 activation, measurement of mitochondrial membrane potential and ROS
generation. Topo II mediated DNA relaxation assay results showed that conjugate 8q could
significantly inhibit the activity of Topo II. Moreover, molecular docking studies also indicated
binding to the topoisomerase enzyme (PDBID 1ZXN).
Keywords: Imidazopyridine, oxadiazole, cytotoxicity, topoisomerase, molecular docking.
a,b,c,d
*
Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.);
E-mail: ahmedkamal@iict.res.in (A. Kamal).

1
. Introduction
Cancer is a group of heterogeneous diseases with dysregulation of cell functioning, which
ultimately alter the gene transcription programs in the cells which causing death. It is remained
as an unsolvable challenge to both physicians and scientists second leading cause of death in
developing as well as developed countries.[1,2] Although chemotherapy is the mainstay of
cancer therapy, the use of available chemotherapeutics is often limited mainly due to undesirable
side effects and it clearly underscores the need of developing novel chemotherapeutic agents for
more effective cancer treatments.[3] The search for such potential anticancer agents has been
designed to the discovery of small synthetic molecules with promising anticarcinogenic activity.
Apoptosis or programmed cell death is a critical cellular process for the maintenance of tissue
homeostasis and normal development of multicellular organisms. Inappropriate regulation of
apoptosis signaling pathways may lead to several diseases including cancer.[4] In tumor cells,
the pathways that lead to apoptosis are disrupted or impaired due to which cells undergo
malignant transformation. Therefore, targeting critical apoptosis regulators with a goal to induce
apoptosis in cancer cells has emerged as an attractive strategy in cancer chemotherapy.[5] DNA
topoisomerase (topo) is recognized as an important target in anticancer drug discovery.[6-10]
Topoisomerases I and II are nuclear enzymes that modulate in maintaining the topological
changes of DNA and play a crucial role in the cellular processes such as transcription, replication
and chromosome segregation to resolve the topological problems of DNA double helix during
the repeated cycles of DNA cleavage and ligation.[11] Human topo II exists in two isoforms
namely topo-IIα and topo-II and the level of topo-II is constant throughout the cell cycle,
whereas the level of topo-IIα is higher in rapidly proliferating cells.[12] From the recent
advances it has been evident that drug molecules that act on topo II enzyme can be divided into
topo-II poisons that form a stabilized cleavable covalent ternary complex and cause apoptosis by
blocking the relegation and enzyme release step. Whereas, topo-II catalytic inhibitors act on the
catalytic steps.[13] ATP sensitive topo-II poisons may specifically target on ATP-bound topo-II
and it works as a sliding protein clamp.[14] It has long been accepted that topoisomerases are
valuable therapeutic targets with the development of a number of topo II inhibitors, such as
etoposide (A, Figure 1).[15]

Nitrogen heterocycles are the most significant structural components of pharmaceuticals.
Analysis of database of U.S. FDA approved drugs reveal that 59% of unique small-molecule
drugs contain a nitrogen-bridgehead fused heterocycles.[16] Among synthesized small
molecules, imidazopyridine[17] is one of the most important heterocyclic systems from the
bicyclic N-fused imidazoles which are known to display a wide range of activities for diverse
number of targets. Imidazopyridines are known to display broad spectrum of biological activities
such as inhibitors of aromatase estrogen production suppressors,[18] platelet aggregation
inhibitors, positive inotropic agents,[19] thromboxane synthetase inhibitors,[20]
antibacterial,[21] antiviral,[22] hypnoselective and anxioselective activities.[23] Imidazole ring
containing N-fused bicyclic system has possessed marketed drugs, such as zolpidem (hypnotic,
B) and zolimidine (antiulcer, C), possess structural resemblance to these antitopoisomerase II
biaryls (Figure 1).[24,25] Moreover, 1,3,4-oxadiazoles are pharmalogically important molecules
that are known to possess significant biological activities such as antibacterial,[26] anti-
inflammatory,[27] anticonvulsant,[28] anticancer,[29] analgesic,[30] antiviral[31] and antifungal
properties.[32] Further, 1,3,4-oxadiazole heterocycles are very good bioisosteres of amides and
esters, which can contribute substantially in increasing pharmacological activity by participating
in hydrogen bonding interactions with the receptors.[33]
As part of our ongoing efforts to discover newer anticancer agents, we previously reported the
synthesis of a number of heterocyclics like imidazo[1,5-a]pyridine-benzimidazole hybrids,[34]
oxindole derived imidazopyridines, (D, Figure 1)[35] imidazo[1,5-a]pyridine–PBD
conjugates[36] and 2-aryl-1,2,4-oxadiazolo-benzimidazoles conjugates.[37] The promising
biological activity exhibited by such conjugates prompted us to explore some newer molecules
by linking the two pharmacophores such as imidazopyridine and oxadiazole scaffolds with a
view to enhance their anticancer activity. In this context, we have designed and synthesized
imidazopyridine-oxadiazole conjugates (E, 8a–s, Figure 1) that were evaluated for their
anticancer potential at the Development Therapeutic Program (DTP) of the National Cancer
Institute (NCI), USA, against a panel of sixty cancer cell lines and their detailed biological
implication relating to apoptotic cell death apart from their effect on DNA topoisomerase-IIa
inhibition.

Figure 1: Topoisomerase II inhibitor etoposide (A), imidazopyridine derivatives (B, C, D and
target molecules E, 8a-s).
2
2
. Results and discussions
.1 Chemistry
The synthesis of imidazopyridine oxadiazole conjugates (8a-s) was carried out by using
commercially available ethyl 2-(2-pyridyl)acetate (1) as the starting material and is illustrated in
Scheme 1. The reaction of 1 with sodium nitrite in acetic acid yielded 2 with 83% yield which
was reduced with Pd/C in methanol to give the amino intermediate (3) in 76% yield. Treatment
of this amine (3) with trimethoxybenzoylchloride and triethylamine in tetrahydrofuran at 0 °C
afforded the amide (4) with 74% yield and the cylization of this amide was carried out by using
phosphorous oxychloride at 120 °C to provide imidazo[1,5-a]pyridine intermediate (5) with 70%
yield.[38] The reaction of 5 with hydrazine hydrate in isopropanol afforded the corresponding
hydrazide in 82% yield. The desired oxadiazole conjugates (8a-s) were obtained by the reaction
of Schiff bases, which were obtained from the hydrazide and aldehydes (7a-s), in the presence of
iodine and potassium carbonate in dimethyl sulphoxide at 80 °C [39] with range of 63-74%
yields.

Scheme : Reagents and Conditions: a) aq.NaNO
2
, AcOH, 3 h, 83%; b) Pd/C, MeOH, 8 h, 76%;
c) 3,4,5-trimethoxy benzoyl chloride, TEA, Dry THF, 0 °C to rt, 3 h, 74%; d) POCl
3
, 120 °C, 3
.
h, 70%; e) NH
2
NH
2
H
2
O, isopropanol, 80 °C, 6 h, 82%; f) I
2
, K
2
CO
3
, DMSO, 80 °C, 4 h, 63-
7
4%.
3
.0 Biology
3
.1 Cytotoxic activity
All the synthesized oxadiazole linked imidazopyridines (8a-s) were submitted to the National
Cancer Institute (NCI), Bethesda for evaluation of cytotoxic activity. Initially these were
evaluated at a single concentration of 10 µM towards a panel of approximately sixty cancer cell
lines derived from nine different cancer types (leukemia, lung, colon, CNS, melanoma, ovarian,
renal, prostate and breast). Primary cytotoxic assays were performed according to the NCI
protocols.[40-42] These compounds were added at a single concentration and the cell culture was
incubated for 48 h and end point determinations were made by using SRB method. The results of

each compound are reported as the percentage of growth (GP) of the treated cells in comparison
to the untreated control cells as shown in Table 1. Some structure activity relationship could be
observed based on the number of cell lines that displayed sensitivity towards each of the newly
synthesized compounds. Among all compounds, the compounds 8c, 8k, and 8l having electron
releasing groups (Methoxy and N-Methyl) at para- position of aromatic ring have shown better
anti-proliferative activity with 63.05, 69.69 and 69.61 % mean growth. The activity was also
influenced by the presence of halogen substituents like fluorine, chlorine and bromine at para
position. For instance, compounds 8d (fluoro), 8e (chloro) and 8f (bromo) showed moderate
growth inhibition. In contrast, compounds having di substitution with fluoro group at para and
chloro at meta position resulted in most active compound 8q with 33.54 mean growth percent.
Based on the results of primary screening, 8q was selected for the five dose screening to
determine its GI50 (the molar concentration required to cause 50% growth inhibition) and the
data is shown in Table 2.

Table 1: Sensitivity, NSC: code, growth percent, mean growth percent of NCI cancer cell lines
treated with synthesized compounds (10 µM)
Growth % of
most sensitive
cell line
Mean
growth
Range of
growth
Compound
NSC code
Most sensitive cell line
8
8
a
NSC: 761871 / 1
NSC: 761874 / 1
NSC: 761861 / 1
NSC: 761872/ 1
NSC: 761860 / 1
NSC: 763709 / 1
81.62
78.69
63.05
78.96
77.54
86.46
14.74 to 116.16
37.16 to 115.02
18.57 to 105.78
45.80 to 113.02
34.82 to 126.65
38.71 to 121.17
OVCAR (Ovarian)
RPMI-8226 (Leukemia)
UO-31 (Renal)
14.74
37.16
18.57
45.80
34.82
b
8
c
8
d
RPMI-8226 (Leukemia)
RPMI-8226 (Leukemia)
8
e
8
f
HOP-62(Non-Small Cell
Lung Cancer)
38.71
8
h
NSC: 761124 / 1
NSC: 761877 / 1
NSC: 761875 / 1
NSC: 763622 / 1
72.12
100.09
69.69
69.61
35.10 to 115.79
60.85 to 129.20
22.10 to 106.60
8.84 to 105.54
UO-31 (Renal)
35.10
60.85
22.10
8
j
RPMI-8226 (Leukemia)
RPMI-8226(Leukemia)
8
k
8
l
EKVX(Non-Small Cell Lung
Cancer)
8.84
54.08
74.67
33.54
8
m
NSC: 763623 / 1
NSC: 761878 / 1
NSC: 763639 / 1
91.47
97.76
33.54
54.08 to 115.18
74.67 to 126.97
-65.54 to 72.94
CCRF-CEM(Leukemia)
8
o
SR(Leukemia)
8
q
HOP-62 (Non-Small Cell
Lung Cancer)
8
r
NSC: 761829 / 1
84.86
46.01 to 105.50
UO-31 (Renal)
68.52
This compound was dissolved in DMSO and evaluated for its cytotoxic activity at five
-4
-5
-6
-7
concentrations at 10 fold dilutions (the highest being 10 M and others 10 M, 10 M, 10 M
-8
and 10 M). The results showed that the 8q exhibited remarkable cytotoxic activity against most
of the tested tumor cell lines with GI50 values in the range of 1.30-5.46 µM. This compound
exhibited a distinctive pattern of sensitivity against some individual cell lines (Table 2), as well
as a broad spectrum of cytotoxic activity. With regard to the sensitivity against some individual
cell lines, it exhibited remarkable growth inhibition against SNB-75 (CNS cancer), HOP-92
(
non-small cell lung cancer), A498 (renal), HCT-116 (colon) BT-549 (breast) cancer cell lines
with GI50 values 1.30, 1.55, 1.55, 1.60 and 1.67 µM and less sensitive against HCC-2998

(
colon) , CAKI-1 (renal) and NCI-H322M (non-small cell lung) cancer cells with GI50 5.64,
.46, 5.05 µM. This data revealed an obvious sensitivity profile towards CNS cancer subpanel
GI50 value ranging from 1.30 to 3.09 µM), least for SNB-75 and maximum for SF-295 cell line.
5
(
This compound also proved to be sensitive towards all the tested breast cancer cell lines with
GI50 less than 3.39 µM.
Table 2: Cytotoxicity of conjugate 8q against a panel of 60 human cancer cell lines.
a
a
a
Panel/Cell Line
GI50 (µM)
Panel/Cell Line
GI50( µM)
Panel/Cell Line
GI50 (µM)
Leukemia
K-562
RPMI-8226
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
DU-145
Breast Cancer
MCF7
MDA-MB-
231/ATCC
HS 578T
BT-549
1.69
1.88
2.10
4.34
1.79
2.03
3.09
2.59
1.93
1.30
1.86
Non-Small Cell
Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
2.93
1.55
2.29
5.46
2.62
2.42
2.40
2.20
1.95
4.19
2.19
1.55
2.38
4.00
5.05
1.99
1.92
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
3.83
3.83
3.27
3.09
2.27
2.11
3.03
2.23
2.03
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
2.11
2.56
4.68
5.64
1.60
4.55
2.20
2.48
3.05
2.06
2.15
3.61
2.76
1.83
4.57
2.51
1.67
2.10
3.39
HT29
KM12
SW-620
T-47D
MDA-MB-468
[
a]
b
Compound concentration required to decrease cell growth to half that of untreated cells. (8q,
NSC 763639)
3
.2 Cell cycle analysis
Cell cycle analysis plays an important role in studies for anticancer screening, mechanism and
cytokinetic research.[43] To gain further insight into the mode of action of 8q as the most potent
compound, we examined its effect on the cell cycle by flow cytometry in A549 cells.
Interestingly, a concentration dependent change was observed in the cell cycle pattern (Figure 2

and Table 3). These results clearly demonstrate that the treatment of cells for 48 h with 2 and 4
µM of compound 8q causes cell cycle arrest at sub G1 phase from 29.52% to 41.70% as well as
subsequent reduction of cell population in G1 phase from 60.72% to 49.30% as seen in Figure 2
and Table 3.
Figure 2: Flow cytometric analysis in A549 lung cancer cell lines after treatment with compound
8
q at 2 and 4 µM concentrations for 48 h.
Table 3: Effect of compound 8q on cell cycle phase distribution in A549 cells.
Compound Conc. Cell cycle phase [%]
[
µM] Sub G1
G1
S
G2/M
7.39
7.56
5.53
Control
7.05
29.52
41.70
78.03
60.72
49.30
7.26
2.20
3.47
8
8
q
q
2
4

3
.3 Caspase-3 Activation
Activation of caspases plays a vital role for the initiation and execution of the apoptotic process.
Among the caspases, caspase-3 is one of the key effector caspases which cleave multiple
proteins in cells, leading to apoptotic cell death.[44] It is well known that the cell cycle arrest at
Sub G1 phase is shown to induce cellular apoptosis,[45] hence it was considered of interest to
examine whether the cytotoxicity of 8q is by virtue of apoptotic cell death. We examined that
activation of caspase-3 in A549 cells treated with compound 8q at 2 and 4 µM concentration.
The results indicate that there is nearly 3 to 4-fold induction in caspase-3 activity relative to the
control as shown in Figure 3. This indicates that 8q activates caspase activity and has the ability
to induce apoptosis in A549 cancer cell lines.
Figure 3: Effect of the compound 8q on caspase-3 activity. A549 cells were treated with the
conjugate 8q for 48 h with 2 and 4 µM concentrations. Values indicated are the mean ± SD of
two different experiments performed in triplicate.
3
.4 Effect on DNA (Tunel assay and Hoechst staining)
A terminal transferase dUTP nick end labelling (TUNEL) assay[46] was carried out with a view
to understand the effect of 8q on DNA by treating with A549 cells at 2 µM for 24 h. The Figure
4
a illustrates that DNA fragmentation (green FITC staining) was observed in the treated cells
whereas there is no fragmentation in the untreated cells, thereby suggesting apoptotic cell death.
Moreover, Hoechst DNA staining also indicated the formation of apoptotic bodies (blebbing) in

cells treated with conjugate 8q, which is more prominent than untreated cells, as apparent in
Figure 4b and the results demonstrated that 8q have apoptotic-inducing ability.
4
a)
Figure 4a: Effect of compound on DNA fragmentation. A549 cells were treated with conjugate
q at 2 µM for 24 h. DNA fragmentation was monitored by primary antibody binding to
8
fragmented DNA, followed by detection with FITC-labeled secondary antibody. Apoptotic cells
are thus stained green. Control cells yield only back ground color. Apoptotic nature of the
conjugate 8q was detected by fragmented nucleus in the compound treated cells.
4
b)
Figure 4b: Effect of conjugate 8q on apoptosis. A549 cells were treated with conjugate 8q at 2
µM for 24 h. Hoechst 33258 was used to stain the nuclei of both control and conjugate 8q-

treated cells. As above, the apoptotic nature of the conjugate 8q is apparent by fragmented nuclei
in compound-treated cells.
3
.5 Measurement of mitochondrial membrane potential ((ΔΨm)
Mitochondrial membrane potential regulates the mitochondrial permeability and plays an
important role in triggering apoptosis. Loss of ∆Ψm leads to depolarization of mitochondrial
membrane leading to collapse of mitochondrial functions ensuing cell death.[47] In order to
further investigate the apoptosis-inducing effect of 8q, changes in mitochondrial membrane
potential was measured by using JC-1 staining. Thus A549 cells were treated with 8q at 2 µM
concentration for 24 h and were analyzed for aggregates to monomers by using JC-1 dye. The
percentages of JC-1 aggeregates in control cells was 6.8 % whereas in the treated compound 8q
the percentage increases to 21.5 % thus indicating loss of ∆Ψm and provides evidence of
apoptosis for 8q treated cells with low mitochondrial potential as shown in Figure 5. These
results demonstrate that mitochondrial pathway is probably playing a key role in modulating cell
apoptosis caused by 8q.

Figure 5: The loss of mitochondrial membrane potential was monitored by JC-1 staining of
A549 cells treated with compound 8q and samples were then subjected to flow cytometry
analysis on a FACScan (Becton Dickinson).
3
.6 Effect on intracellular ROS generation
Mitochondrial membrane depolarization is associated with mitochondrial production of
ROS.[45] Therefore, we investigated whether ROS production has increased after treatment with
the test compounds. Apoptosis can be initiated by oxidative stress which may be mediated by the
generation of reactive oxygen species (ROS). [48,49] As 8q is cytotoxic and induces apoptosis in
A549 cells, it was considered of interest to understand its effect of 8q on the generation of ROS.
Therefore, cells were incubated with 8q for 24 h at 2 µM concentration and the intracellular
generation of ROS was examined using DCFH-DA dye by fluorescent microscopy. As shown in
Figure 6, there was a significant increase in DCF positive cell population by 8q as compared to
the control which reveals that it induces apoptosis through intracellular ROS in A549 cells.
Altogether, these results indicate that this compound induced apoptosis through the
mitochondrial pathway.
Figure 6: The effect of conjugate 8q on the ROS production in A549 cells.
3
.7 Annexin V-FITC staining
The apoptotic effect of 8q was further evaluated by Annexin V FITC/PI (AV/PI) dual staining
assay[50] to examine the occurrence of phosphatidylserine externalization and also to understand

whether it is due to physiological apoptosis or nonspecific necrosis. Thus A549 cells were
treated with 8q for 48 h at 2 µM concentrations to examine the apoptotic inducing ability. From
the results it was observed that 8q showed 22.9 % of apoptosis, whereas 14.9 % of apoptosis was
observed in case of control (untreated cells) as shown in Figure 7. Thus this experiment
suggested that 8q induces apoptotic cell death in A549 cells.
Figure 7: Annexin V-FITC staining. A549 cells were treated with compounds 8q at 2 µM
concentration for 48 h.
3
.8 Effect on topoisomerase - IIα protein on 8q
DNA Topoisomerase exists in two isoforms and it is well established that throughout the cell
cycle of rapidly proliferating cells topo IIα is relatively expressed higher than topo I.[51] DNA-
topoisomerase II is a nuclear enzyme that controls the DNA structure by catalyzing the DNA
cleavage and relegating the phosphodiester bonds. In the recent years, DNA topoisomerase II
inhibitors have gained importance as clinically useful chemotherapeutical agents. The protocols
described are of assays used to assess new chemical entities for their ability to inhibit both forms
of DNA topoisomerase [52]. The main factors, which must be considered for assaying enzymatic
activity, are temperature, pH, ionic strength and the proper concentrations of the essential

components like substrates and enzymes. Nevertheless, many enzymes, especially those from
mammalian sources, possess a pH optimum near the physiological pH of 7.5, and the body
temperature of about 37 °C can serve as assay temperature, although because of experimental
reasons frequently 25 °C is preferred. But in many cases the particular features of the individual
enzyme dictate special assay conditions, which can deviate considerably from recommended
conditions. In addition, exact values for the concentrations of assay components such as
substrates and enzymes cannot be given, unless general rules depending on the relative degree of
saturation can be stated. Enzymes display their highest activity at their respective optimum
conditions, deviations from the optimum conditions cause a reduction of the activity, depending
on the degree of the deviation. Based on the above information the concentration of the sample is
taken more for enzymatic assays to get the efficient results.[53] Topoisomerase II inhibition
studies were performed by using topoisomerase II Drug Screening Kit (TG 1009, Topogen,
USA),[54] which is suitable for screening the enzyme inhibition by both catalytic inhibitory
compounds (CICs) and interfacial poisons (IFPs). CICs inhibit the activity of the enzyme,
whereas IFP compounds stimulate the formation of the cleavage complexes and block the
relegation which leads to form linear DNA. Catenated DNA in the presence of topo II enzyme
incubated with etoposide and conjugate 8q at concentration of 100 µM (lane E, F in control,
Figure 8) did not show the formation of linear DNA, but most of the catenated DNA was highly
super coiled and remained in the wells documenting the formation of only nicked circular and
relaxed DNA, indicates that the conjugate 8q interference in the catalytic activity of the topo II
enzyme and suggesting that the role of 8q as catalytic inhibitory compound. This assay suggests
that conjugate 8q affect the topoisomerase II enzyme activity.

A
B
C
D
E
F
Figure 8: A: Linear DNA; B: Decatenated DNA; C: Catenated DNA; D: Catenated DNA+topo
IIα; E: Catenated DNA+topo IIα+etoposide (100 µM); F: Catenated DNA+topo IIα+8q (100
µM).
3
.9 Molecular Docking studies
The cell cycle analysis and topo-II assay prompted us to do molecular docking studies on
topo-II to understand the precise nature of binding and to develop an insight into the atomic level
interactions by such molecules. Docking studies were performed on 8q and topo-II structure was
obtained from Protein Data Bank (PDBID 1ZXN).[55] Necessary corrections to the protein were
carried out using Protein Preparation Wizard from Schrodinger package. Geometry of the
molecule was optimized in Gaussian 09 using PM3 semi-empirical method.[56] Additional
docking studies were performed using AutoDock 4.2 docking software.[57] Grids for the
docking studies were prepared around ATP binding site using ATP as a center for the grid. For
the validation of docking protocol, docking studies on reference ligand (ADP) were performed.
Binding pose of reference ligand shows RMSD 0.91 with docking score -8.84. The binding pose
(
as in Figure 9) shows that this molecule binds effectively in the ATP binding pocket with
docking score -8.29. The trimethoxyphenyl group is deeply buried in the ATP binding pocket
whereas 5-phenyloxadiazole ring is situated towards the surface. Superimposed pose of 8q with
cocrystal ligand shows that trimethoxyphenyl ring was near to the purine ring where nitrogen of
purine ring and methoxy group of trimethoxyphenyl ring forming hydrogen bonding interactions

with same amino acid Asn120. Sugar moiety of ADP is superimposed with imidazopyridine ring
of 8q. The trimethoxyphenyl ring shows the hydrophobic interactions with the Glu87, Ile88,
Asn91, Ala92, Asn95, Ile118, Asn120, Gly124, Ala167, Thr215, Cys216, Ile217 amino acids,
oxygen of the 3-methoxy substituent shows hydrogen binding interactions with the side chain of
Asn120 and phenyl ring shows π-π interactions with Phe142. The imidazopyridine ring exhibits
hydrophobic interactions with the Ile141, Thr147, Ser148, Ser149, Lys168 amino acids. The 5-
phenyloxadiazole ring was involved in the hydrophobic interactions with Asp94, Gln97, Arg98,
Asn150, Thr159, Gly161 amino acids. Further Lys157 involves in the cation-π interactions with
the phenyl ring and nitrogen of oxadiazole ring forms hydrogen bonding side chain of Asn150.
Thus, these molecular docking studies along with the biological assay data suggest that conjugate
8
q is a potential topo-II inhibitor.
Figure 9: (A) Binding pose of 8q with surface Reference Ligand: (B) Docking pose for 8q in
ATP binding site of Topo-II. Molecule shown in peru color, protein in green color, amino acid
forming hydrogen bond in magenta color and amino acid having π interaction are in violate
color. (C) Superimposed pose of 8q with cocrystal ligand ADP.

4
.0 Conclusion
In conclusion, a series of imidazopyridinyl-1,3,4-oxadiazole conjugates were synthesized
and evaluated for their cytotoxic activity against a panel of sixty human tumor cell lines. Among
them conjugate 8q exhibited significant cytotoxic activity with GI50 values ranging from 1.30-
5
.64 µM and arrests the cells at subG1 phase of the cell cycle. The Topo II inhibitory activity of
these conjugate was tested by relaxation and cleavage complex in vitro assay. Molecular docking
of 8q in the Topo II binding site reveals the possible binding mode with the target. Interestingly,
8
q induces apoptosis through caspase-3 activation, nuclear fragmentation condensation and
disruption of mitochondrial membrane potential as well as increase in the intracellular ROS
levels and Annexin-V. Overall, the present study demonstrates that the synthesis of
imidazopyridinyl-1,3,4-oxadiazole conjugates as promising anticancer agents with apoptotic-
inducing activity and inhibition of the topoisomerase IIα and it could be concluded that the 8q
may be used as a new template for development of chemotherapeutics.
5
5
.0 Experimental Section
.1. Chemistry
All chemicals and reagents are commercially available and were used directly without
further purification. Reactions were monitored by TLC performed on silicagel glass plate
containing 60 GF-254, and visualization was achieved by UV light or iodine indicator. Column
1
13
chromatography was performed with Merck 60–120 mesh silica gel. H and C NMR spectra
were recorded by using Varian and Avance instruments. Chemical shifts are expressed in parts
per million ( in ppm) downfield from internal TMS and coupling constants are expressed in Hz.
1
H NMR spectroscopic data are reported in the following order: multiplicity (s, singlet; brs,
broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet), coupling constants in
+
Hz, number of protons. ESI mass spectra were recorded on a Micro mass Quattro LC using ESI
software with capillary voltage 3.98 kV and an ESI mode positive ion trap detector. Melting
points were determined with an Electro thermal melting point apparatus, and are uncorrected.
5
.1.1. Ethyl 2-hydroxyimino-2-(2-pyridyl)acetate (2) [38]:
A solution of 5g (0.03 mol) of ethyl (2- pyridyl) acetate (1) in 10 mL of glacial acetic acid was
cooled in an ice bath and 2.20 g (0.032 mol) of sodium nitrite in 5 ml of water was added over a
period of 20 minutes, keeping the temperature between 15 to 25 °C. The mixture was stirred for

an additional 30 minutes, 50 mL of water was added, and the stirring continued for two hours.
The crystals were washed with water, washed three times with 5% sodium bicarbonate solution
and again with water. Recrystallization from methanol yielded 4.9 g (83%) of white needles. mp:
1
1
47−152 °C; H NMR (300 MHz, CDCl
3
): 7.65 (s, 1H), 6.95-6.89 (m, 2H), 6.75 (s, 1H), 3.98 (q,
+
J = 6.7 Hz, 2H), 1.54 (t, J = 6.7 Hz, 3H); MS (ESI, m/z): 195 (M+H) .
5
.1.2. Ethyl 2-amino-2-(2-pyridyl)acetate (3) [38]:
To a solution of ethyl 2-hydroxyimino-2- (2-pyridyl)acetate (2) (4 g, 0.02 mol) in 50 mL of
methanol was added catalytic amount of 10% palladium on activated carbon. The mixture was
stirred overnight under hydrogen, the reaction mixture was filtered, and the filtrate was
evaporated. The crude product 3 was used directly in next step without further purification. MS
+
(
ESI, m/z): 181 (M+H) .
5
.1.3. Ethyl 2-(pyridin-2-yl)-2-(3,4,5-trimethoxybenzamido)acetate (4) [38]:
To a stirred solution of ethyl 2-amino-2-(pyridin-2-yl)acetate 3 (3.8 g, 0.035 mol) in dry THF,
triethylamine (1.1 mmol) and trimethoxybenzoylchloride (4.9 g, 0.035 mol) were added at 0 °C.
The reaction mixture was stirred for 3 h and the reaction was monitored by TLC. After
completion of reaction, the solution was concentrated in vacuo. The residue was partitioned
between water and ethyl acetate, and the aqueous layer was extracted with ethyl acetate. The
combined extracts were dried over Na
2
SO and concentrated. The crude was further purified by
4
column chromatography using ethyl acetate/hexane (5:5) to obtain the pure product in 74% yield.
1
H NMR (300 MHz, CDCl ):  8.36 (m, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 6.7 Hz, 1H,)
3
7
6
5
.58 (m, 1H), 7.16 (dd, J = 9.8, 7.5 Hz, 1H), 6.98 (s, 2H), 5.47 (d, J = 6.5 Hz, 1H,), 4.32 (q, J =
+
.9 Hz, 2H), 3.92 (s, 3H), 1.52 (t, J = 6.9 Hz, 3H); MS (ESI, m/z): 375 [M+H] ;
.1.4. Ethyl 3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridine-1-carboxylate (5):
The reaction mixture of 4 (3g, 8 mmol) and POCl
3
was refluxed for 3h. After completion
reaction, the reaction mixture was poured in ice-water and quenched with saturated NaHCO
3
.
This water layer was extracted with ethyl acetate and the combined extracts were dried and
concentrated under vaccum. The crude obtained was further furified by column chromatography
1
to obtain pure product as yellow solid in 70% yield. H NMR (300 MHz, CDCl
3
): δ 8.29 (dd, J =
1
6.9, 8.2 Hz, 2H), 7.17 – 7.09 (m, 1H), 6.98 (s, 2H), 6.83 – 6.76 (m, 1H), 4.55 (q, J = 13.5, 6.7
+
Hz, 2H), 3.92 (s, 6H), 3.91 (s, 3H), 1.47 (t, J = 6.7 Hz, 3H); MS (ESI, m/z): 357 [M+H] . HRMS
+
(
ESI, m/z) calcd for C19
H
21
O
5
N
2
: 357.14463 [M+H] ; found: 357.14459.

5
.1.5. 3-(3,4,5-Trimethoxyphenyl)imidazo[1,5-a]pyridine-1-carbohydrazide (6):
To a solution of compound 5 (2 g, 5.6 mmol) in isopropanol, hydrazine hydrate (0.84 mL, 16.8
mmol) was added and the reaction mixture was heated at 80 °C. Then the reaction mixture was
cooled to room temperature and the solvent was removed. The residue was diluted with water
and extracted with ethyl acetate. The combined organic extracts were dried over Na
2
SO and
4
concentrated under vaccum. The crude obtained was further furified by column chromatography
1
to yield pure product in 82% yield. H NMR (300 MHz, CDCl
3
): δ 9.86 (s, 1H), 8.44 (d, J = 9.1
Hz, 1H), 8.26 (s, 1H), 7.11- 7.06 (m, 1H), 6.95 (s, 2H), 6.80-6.76 (m, 1H), 3.94 (s, 6H), 3.93 (s,
+
+
3
H); MS (ESI, m/z): 343 [M+H] . HRMS (ESI, m/z) calcd for C17
found: 343.13971.
.1.6. General procedure for the synthesis of imidazopyridine-oxadiazoles (8a-s)
H
19
O
4
N : 343.13976 [M+H] ;
4
5
A solution of acyl hydrazine 6 (1mmol) and aromatic aldehydes 7a-s (1 mmol) in i-PrOH (10
mL) was refluxed until the reaction was completed (monitored by TLC, 3−5 h), and then the
solvent was evaporated under reduced pressure. To the resulting residue dissolved in DMSO (5
mL), potassium carbonate (3 mmol) and iodine (1.2 mmol) were added in sequence. Then the
reaction mixture was heated to 80 °C and stirred until the completion of reaction. After being
cooled to room temperature, it was treated with 5% Na
2
S
2
O
3
(20 mL), extracted with ethyl
SO and concentrated under
acetate the combined organic extracts were dried over anhydrous Na
2
4
vaccum to obtain crude products. The crude obtained was purified by column chromatography
using ethyl acetate and hexane as eluent to afford pure products (8a-s).
5
.1.6.1. 2-Phenyl-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-oxadiazole
(
8a):
The title compound was obtained from compound (6), and benzaldehyde (7a) following the
1
general procedure described above. Yield 72%; yellow solid; mp: 230-233 °C; H NMR (300
MHz, CDCl
dd, J = 9.0, 6.0 Hz, 1H), 7.04 (s, 2H), 6.77 (t, J = 7.5 Hz, 1H), 3.96 (s, 6H), 3.91 (s, 3H); C
NMR (75 MHz, CDCl ): δ 163.31, 161.27, 153.79, 139.72, 132.41, 131.35, 128.85, 126.94,
24.21, 124.07, 123.49, 122.34, 119.81, 114.73, 105.98, 60.99, 56.41; MS (ESI, m/z): 429
3
): δ 8.36 (dd, J = 15.4, 8.2 Hz, 2H), 8.28 – 8.23 (m, 2H), 7.56 – 7.49 (m, 3H), 7.13
1
3
(
3
1
+
+
[
M+H] ; HRMS (ESI, m/z) calcd for C24
H
21
O
4
N
4
: 429.17753 [M+H] ; found: 429.17752.
5
.1.6.2.
2-p-Tolyl-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazole (8b):

The title compound was obtained from compound (6) and 4- methybenzaldehyde (7b) following
1
the general procedure described above. Yield: 69%; yellow solid; mp: 210-213 °C; H NMR
(
300 MHz, CDCl
3
): δ 8.39 (d, J = 9.2 Hz, 1H), 8.35 (d, J = 7.3, Hz, 1H), 8.12 (d, J = 8.2 Hz,
2
H), 7.34 (d, J = 8.0 Hz, 2H), 7.17-7.13 ( dd, J = 6.4, 2.2 Hz, 1H), 7.04 (s, 2H), 6.83 (t, J = 6.4
1
3
Hz, 1H), 3.96 (s, 6H), 3.94 (s, 3H), 2.44 (s, 3H); C NMR (75 MHz, CDCl
3
): δ 163.46, 160.98,
1
1
53.77, 141.80, 139.64, 139.28, 132.28, 129.59, 126.89, 124.21, 123.41, 122.31, 121.17, 119.80,
16.35, 114.70, 105.96, 60.93, 56.41, 21.59; MS (ESI, m/z): 443 [M+H]+; HRMS (ESI, m/z)
+
calcd for C25
H
23
N
4
O
4
: 443.17107 [M+H] ; found: 443.17102.
5
1
.1.6.3.
2-(4-Methoxyphenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-
,3,4-oxadiazole (8c):
The title compound was obtained from compound (6) and 4- methoxybenzaldehyde (7c)
1
following the general procedure described above. Yield: 71%; yellow solid; mp: 240-244 °C; H
NMR (300 MHz, CDCl
3
): δ 8.37 (dd, J = 16.8, 8.2 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.09 (dd, J
=
8.9, 6.4 Hz, 1H), 6.99 (d, J = 10.1 Hz, 4H), 6.77 (t, J = 6.7 Hz, 1H), 3.97 (s, 6H), 3.91 (s, 3H),
1
3
3
1
5
.89 (s, 3H); C NMR (75 MHz, CDCl ): δ 163.27, 162.01, 160.80, 153.77, 139.58, 139.27,
3
32.19, 128.71, 124.26, 123.31, 122.28, 119.85, 116.55, 114.69, 114.30, 105.96, 60.98, 56.41,
+
5.33; MS (ESI, m/z): 459 [M+H] ; HRMS (ESI, m/z) calcd for C25
H
23
N
4
5
O : 459.16607
+
[
M+H] ; found: 459.16595.
5
.1.6.4. 2-(4-Fluorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4
oxadiazole (8d):
The title compound was obtained from compound (6) and 4- fluorobenzaldehyde (7d) following
1
the general procedure described above. Yield: 67%; yellow solid; mp: 220-224 °C; H NMR
(
300 MHz, CDCl
3
): δ 8.40 (dd, J = 13.4, 8.2 Hz, 2H), 8.27 (dd, J = 8.8, 5.3 Hz, 2H), 7.23 (t, J =
8
3
1
5
.6 Hz, 2H), 7.14 (dd, J = 9.0, 6.5 Hz, 1H), 7.03 (s, 2H), 6.81 (t, J = 6.8 Hz, 1H), 3.99 (s, 6H),
1
3
.94 (s, 3H); C NMR (75 MHz, CDCl ): δ 166.28, 162.93, 162.59, 153.88, 139.82, 132.52,
3
29.32, 129.21, 124.24, 123.64, 122.43, 120.39, 119.88, 116.42, 116.13, 114.84, 106.05, 61.06,
+
6.49; MS (ESI, m/z): 447 [M+H] ; HRMS (ESI, m/z) calcd for C24
H
20
O
4
4
N F: 447.14624
+
[
M+H] ; found: 447.14616.
5
.1.6.5. 2-(2-Chlorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazole (8e):

The title compound was obtained from compound (6) and 2- chlorobenzaldehyde (7e) following
1
the general procedure described above. Yield: 66%; yellow solid; mp: 196-200 °C; H NMR
(
300 MHz, CDCl
3
): δ 8.40 (dd, J = 11.3, 8.2 Hz, 2H), 8.17 (dd, J = 7.4, 2.0 Hz, 1H), 7.60 (d, J =
7
.5 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.22 – 7.13 (m, 1H), 7.05 (s, 2H), 6.86 (t, J = 6.3 Hz, 1H), 3.97
1
3
(
s, 6H), 3.95 (s, 3H); C NMR (75 MHz, CDCl
3
): δ 164.48, 162.59, 153.58, 139.71, 132.37,
1
5
29.68, 129.27, 124.68, 123.11, 122.78, 120.29, 119.77, 116.82, 116.03, 114.94, 106.05, 60.56,
+
6.67; MS (ESI, m/z): 463 [M+H] ; HRMS (ESI, m/z) calcd for C24
H
20
O
4
4
N Cl: 463.11667
+
[
M+H] ; found: 463.11660.
5
.1.6.6. 2-(4-Bromophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazole (8f):
The title compound was obtained from compound (6) and 4- bromobenzaldehyde (7f) following
1
the general procedure described above. Yield: 74%; yellow solid; mp: 225-229 °C; H NMR
(
300 MHz, CDCl
3
): δ 8.41 (dd, J = 15.2, 8.2 Hz, 2H), 8.14 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.6
Hz, 2H), 7.24 – 7.15 (m, 1H), 7.04 (s, 2H), 6.86 (t, J = 6.7 Hz, 1H), 3.99 (s, 6H), 3.96 (s, 3H);
1
3
C NMR (75 MHz, CDCl
3
): δ 164.61, 162.43, 153.78, 139.61, 132.45, 129.86, 129.32, 124.32,
1
5
5
1
23.65, 122.26, 120.77, 119.78, 116.81, 116.00, 114.33, 105.89, 60.79, 55.89; MS (ESI, m/z):
+
+
07 [M+H] ; HRMS (ESI, m/z) calcd for C24
H
20
O
4
N Br: 507.06673 [M+H] ; found: 507.06669.
4
.1.6.7. 2-(4-(Trifluoromethyl)phenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-
-yl)-1,3,4-oxadiazole (8g):
The title compound was obtained from compound (6) and 4-(trifluoromethyl)benzaldehyde (7g)
1
following the general procedure described above. Yield: 69%; yellow solid; mp: 243-247 °C; H
NMR (300 MHz, CDCl
3
): δ 8.44 (d, J = 9.2 Hz, 1H), 8.39 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.3
Hz, 2H), 7.71 (dd, J = 5.7, 3.3 Hz, 1H), 7.53 (dd, J = 5.7, 3.3 Hz, 1H), 7.04 (s, 2H), 6.86 (t, J =
1
3
6
1
1
4
5
.8 Hz, 1H), 3.98 (s, 2H), 3.95 (s, 1H); C NMR (75 MHz, DMSO): δ 165.39, 162.59, 153.45,
39.87, 132.38, 129.72, 129.16, 124.81, 123.85, 122.71, 120.47, 119.58, 116.69, 116.19, 114.72,
+
06.09, 61.06, 56.49; MS (ESI, m/z): 497 [M+H] ; HRMS (ESI, m/z) calcd for C25
H
20
O
4
N
4
3
F :
+
97.14256 [M+H] ; found: 497.14259.
.1.6.8.
2-(4-(Trifluoromethoxy)phenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-
a]pyridin-1-yl)-1,3,4-oxadiazole (8h):
The title compound was obtained from compound (6) and 4-(trifluoromethoxy)benzaldehyde
7h) following the general procedure described above. Yield: 70%; yellow solid; mp: 220-225
(

1
°
C; H NMR (300 MHz, CDCl
3
): δ 8.40 (dd, J = 9.7, 8.6 Hz, 2H), 8.33 – 8.26 (m, 2H), 7.39 (d, J
=
8.1 Hz, 2H), 7.23 – 7.16 (m, 1H),7.05 (s, 2H), 6.90 – 6.84 (m, 1H), 3.98 (s, 6H), 3.96 (s, 6H);
1
3
C NMR (75 MHz, CDCl
3
): δ 162.21, 161.68, 153.93, 140.03, 139.49, 132.85, 127.25, 126.01,
+
1
24.15, 123.94, 122.51, 119.83, 114.93, 106.06, 61.08, 56.48; MS (ESI, m/z): 513 [M+H] ;
+
HRMS (ESI, m/z) calcd for C25
H
20
F
3
O
5
N
4
: 513.13862 [M+H] ; found: 513.13856.
5
.1.6.9. 2-(4-Nitrophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazole (8i):
The title compound was obtained from compound (6) and 4- nitrobenzaldehyde (7i) following
1
the general procedure described above. Yield: 71%; yellow solid; mp: 245-249 °C; H NMR
(
300 MHz, CDCl
3
): δ 8.48 – 8.37 (m, 6H), 7.23 (dd, J = 9.0, 6.5 Hz, 1H), 7.04 (s, 2H), 6.90 (t, J
13
=
6.8 Hz, 1H), 3.99 (s, 6H), 3.96 (s, 3H); C NMR (75 MHz, DMSO): δ 165.81, 162.57,
1
1
4
5
53.86, 144. 91, 139.86, 132.52, 129.21, 126.63, 124.24, 123.64, 120.39, 119.32, 116.13, 114.84,
+
06.11, 65.92, 56.89; MS (ESI, m/z): 474 [M+H] ; HRMS (ESI, m/z) calcd for C24
H
20
O
6
5
N :
+
74.14132 [M+H] ; found: 474.14119.
.1.6.10.
2-(Pyridin-3-yl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazole (8j):
The title compound was obtained from compound (6) and 3- pyridinecarboxaldehyde (7j)
following the general procedure described above. Yield: 64%; yellow solid; mp: 251-255 °C;
1
H NMR (300 MHz, DMSO): δ 9.45 (s, 1H), 8.75 (d, J = 4.3 Hz, 1H), 8.51 (d, J = 8.1 Hz, 1H),
8
.41 (dd, J = 17.9, 8.8 Hz, 2H), 7.46 (dd, J = 8.1,5.6 Hz, 1H), 7.15 (dd, J = 9.6, 6.7 Hz, 1H), 7.00
1
3
(
s, 2H), 6.87 (t, J = 6.98 Hz, 1H), 3.96 (s, 6H), 3.91 (s, 3H); C NMR (75 MHz, DMSO): δ
1
1
66.81, 163.89, 153.62, 146. 82, 144. 68, 139.46, 136.82, 127. 62, 124.24, 123.64, 120.39,
+
19.52, 116.37, 114.78, 106.09, 65.84, 56.75; MS (ESI, m/z): 430 [M+H] ; HRMS (ESI, m/z)
+
calcd for C23
H
20
N
5
O
4
: 430.15110 [M+H] ; found: 430.15103.
5
.1.6.11. N,N-Dimethyl-4-(5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazol-2-yl)aniline (8k):
The title compound was obtained from compound (6) and 4-(dimethylamino)benzaldehyde (7k)
1
following the general procedure described above. Yield: 69%; yellow solid; mp: 135-140 °C; H
NMR (300 MHz, CDCl ): δ 8.38 (dd, J = 15.9, 8.1 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 7.12 – 7.05
3
1
3
(
(
m, 1H), 7.04 (s, 2H), 6.76 (d, J = 8.9 Hz, 3H), 3.99 (s, 6H), 3.93 (s, 3H), 3.10 (s, 6H); C NMR
75 MHz, CDCl ): δ 164.04, 160.18, 153.74, 152.09, 139.39, 139.19, 131.92, 128.34, 124.38,
3

1
23.00, 122.18, 119.92, 114.60, 111.47, 111.09, 105.94, 60.97, 56.41, 40.06; MS (ESI, m/z): 472
+
+
[
M+H] ; HRMS (ESI, m/z) calcd for C26
H
26
N
5
O : 472.19730 [M+H] ; found: 472.19728.
4
5
1
.1.6.12. 2-(4-Methoxy-3-nitrophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-
-yl)-1,3,4-oxadiazole (8l):
The title compound was obtained from compound (6) and 4-methoxy-3-nitrobenzaldehyde (7l)
1
following the general procedure described above. Yield: 66%; yellow solid; mp: 248-251 °C; H
NMR (300 MHz, CDCl
Hz, 1H), 7.04 (s, 2H), 6.89 (t, J = 6.9 Hz, 1H), 4.10 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H); C NMR
75 MHz, DMSO): δ 164.31, 161.27, 153.79, 153.33, 143.93, 139.72, 132.41, 131.35, 128.85,
26.94, 124.21, 123.49, 122.34, 119.81, 114.73, 105.94, 60.99, 56.41, 54.98; MS (ESI, m/z): 504
3
): δ 8.40 (t, J = 8.4 Hz, 2H), 7.99-7.87 (m, 3H), 7.22 (dd, J = 8.7, 6.9
1
3
(
1
+
+
[
M+H] . HRMS (ESI, m/z) calcd for C25
H
21
N
5
O
7
Na: 526.13472 [M+Na] ; found:526.13470.
5
.1.6.13.
2-Methoxy-4-(5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-1,3,4-
oxadiazol-2-yl)phenol (8m):
The title compound was obtained from compound (6) and 4-hydoxy-3-methoxybenzaldehyde
1
(
(
7m) following the general procedure described above. Yield: 63%; yellow solid; mp: H NMR
300 MHz, CDCl
3
): δ 8.38 (dd, J = 12.5, 8.2 Hz, 2H), 7.78 (dd, J = 8.3, 1.7 Hz, 1H), 7.73 (d, J =
1
4
1
1
.6 Hz, 1H), 7.19 – 7.12 (m, 1H), 7.05 (d, J = 3.5 Hz, 1H), 7.04 (s, 2H), 6.84 (t, J = 6.4 Hz, 1H),
1
3
.02 (s, 3H), 3.96 (s, 6H), 3.94 (s, 3H); C NMR (75 MHz, CDCl ): δ 164.54, 162.45, 159.72,
3
58.64, 156.79, 156.12, 153.53, 150.39, 149.75, 139.64, 132.54, 125.73, 125.62, 120.62, 117.34,
+
13.91, 109.73, 106.47, 60.98, 56.41, 55.39; MS (ESI, m/z): 475 [M+H] ; HRMS (ESI, m/z)
+
calcd for C25
H
23
N
4
O
6
: 475.16118 [M+H] ; found: 475.16115.
5
.1.6.14.
2-(3,4-dimethoxyphenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-
yl)-1,3,4-oxadiazole (8n):
The title compound was obtained from compound (6) and 3,4-dimethoxybenzaldehyde (7n)
1
following the general procedure described above. Yield: 67%; yellow solid; mp: 248-252 °C; H
NMR (300 MHz, CDCl3): δ 8.43 (d, J = 9.1 Hz, 1H), 8.36 (d, J = 7.1 Hz, 1H), 7.81 (dd, J = 8.4,
1
.6 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.12 (dd, J = 9.0, 6.5 Hz, 1H), 7.03 (s, 2H), 6.96 (d, J = 8.4
1
3
Hz, 1H), 6.80 (t, J = 6.7 Hz, 1H), 4.02 (s, 3H), 3.98 (d, J = 3.7 Hz, 9H), 3.93 (s, 3H); C NMR
75 MHz, CDCl ): δ 163.32, 160.89, 153.80, 152.13, 151.65, 149.15, 139.59, 139.31, 132.27,
24.20, 123.38, 122.27, 120.52, 119.87, 116.62, 114.73, 111.96, 109.43, 105.96, 61.97, 56.40,
(
3
1

+
5
6.20, 55.96; MS (ESI, m/z): 489 [M+H] . HRMS (ESI, m/z) calcd for C26
H
25
N
4
6
O : 489.17615
+
[
M+H] ; found: 489.17607.
5
1
.1.6.15. 2-(2,4-difluorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-
,3,4-oxadiazole (8o)
The title compound was obtained from compound (6) and 2,4-difluorobenzaldehyde (7o)
following the general procedure described above. Yield: 67%; yellow solid; mp: 218-220 °C;
1
H NMR (300 MHz, CDCl
3
): δ 8.39 (d, J = 9.1 Hz, 2H), 8.24 (dd, J = 14.9, 8.6 Hz, 1H), 7.28 (s,
1
H), 7.19 (dd, J = 9.5, 6.6 Hz, 1H), 7.07 (dd, J = 13.1, 4.8 Hz, 3H), 6.86 (t, J = 6.8 Hz, 1H), 3.96
1
3
(
d, J = 4.9 Hz, 9H); C NMR (75 MHz, CDCl
3
): δ 163.77, 161.47, 153.84, 150.13, 149.75,
1
1
39.97, 139.48, 137.34, 137.22, 132.64, 131.21, 131.13, 124.16, 123.72, 122.45, 119.75, 116.05,
+
14.77, 112.42, 112.22, 106.10, 61.01, 56.47; MS (ESI, m/z): 465 [M+H] . HRMS (ESI, m/z)
+
calcd for C24
H
19
N
4
O
4
F
2
: 465.13658 [M+H] ; found: 465.13636.
5
1
.1.6.16. 2-(2-Chloro-4-fluorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-
-yl)-1,3,4-oxadiazole (8p)
The title compound was obtained from compound (6) and 2-chloro-4-fluorobenzaldehyde (7p)
following the general procedure described above. Yield: 69%; yellow solid; mp: 225-230 °C;
1
H NMR (500 MHz, CDCl
3
): δ 8.36 (dd, J = 15.4, 8.1 Hz, 2H), 8.14 (dd, J = 8.7, 5.9 Hz, 1H),
7
1
1
1
.31 (dd, J = 8.4, 2.3 Hz, 1H), 7.14 (dd, J = 14.1, 5.4 Hz, 2H), 7.01 (s, 2H), 6.82 (t, J = 6.5 Hz,
1
3
H), 3.93 (s, 6H), 3.92 (s, 3H); C NMR (75 MHz, CDCl ): δ 162.34, 160.82, 158.84, 157.25,
3
56.73, 156.12, 153.20, 150.42, 149.82, 139.82, 132.62, 125.88, 125.31, 120.74, 117.18, 113.82,
+
09.75, 106.17, 60.52, 56.37; MS (ESI, m/z): 481 [M+H] ; HRMS (ESI, m/z) calcd for
+
C
24
H
19
N
4
O
4
ClF: 481.10736 [M+H] ; found: 481.10739.
5
1
.1.6.17. 2-(3-Chloro-4-fluorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-
-yl)-1,3,4-oxadiazole (8q)
The title compound was obtained from compound (6) and 3-chloro-4-fluorobenzaldehyde (7q)
1
following the general procedure described above. Yield: 73%; yellow solid; mp: 220-224 °C; H
NMR (300 MHz, CDCl
3
): δ 8.42 (dd, J = 15.8, 8.1 Hz, Ar-H, 2H), 8.34 (dd, J = 6.9, 2.1 Hz, Ar –
H, 1H), 8.17 (ddd, J = 8.5, 4.5, 2.1 Hz, Ar-H, 1H), 7.33 (dd, J = 10.9, 6.2 Hz, Ar-H, 1H), 7.17
(
dd, J = 9.0, 6.6 Hz, Ar-H, 1H), 7.02 (s, Ar-H, 2H), 6.84 (t, J = 6.5 Hz, Ar-H, 1H), 3.99 (s,
1
3
2
×OMe, 6H), 3.94 (s, 1×OMe, 3H); C NMR (75 MHz, CDCl ): δ 161.51 (Ar-C), 160.13 (Ar-
3
C), 159.46 (Ar-C), 158.95 (Ar-C, d, J = 234.2 Hz, C-F), 158.43 (Ar-C), 157.92 (Ar-C), 153.57

(
(
(
Ar-C), 150.13 (Ar-C), 149.75 (Ar-C), 139.78 (Ar-C), 132.50 (Ar-C), 125.26 (Ar-C), 124.14
Ar-C), 120.96 (2×Ar-C), 117.15 (Ar-C), 113.33 (Ar-C), 109.52 (Ar-C), 106.17 (Ar-C), 60.28
+
2×OMe-C), 56.24 (1×OMe-C); MS (ESI, m/z): 481 [M+H] ; HRMS (ESI, m/z) calcd for
+
C
24
H
19
N
4
O
4
ClF: 481.10737 [M+H] ; found: 481.10720.
5
1
.1.6.18. 2-(2,4-dichlorophenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)-
,3,4-oxadiazole (8r)
The title compound was obtained from compound (6) and 2,4-dichlorobenzaldehyde (7r)
following the general procedure described above. Yield: 66%; yellow solid; mp: 220-224 °C;
1
H NMR (500 MHz, CDCl ): δ 8.39 (ddd, J = 14.8, 7.9, 4.1 Hz, 2H), 8.11 (d, J = 8.5 Hz, 1H),
3
7
.61 (d, J = 2.0 Hz, 1H), 7.42 (dd, J = 8.5, 2.1 Hz, 1H), 7.18 (ddd, J = 9.2, 6.5, 0.9 Hz, 1H), 7.03
+
(
(
s, 2H), 6.87 – 6.83 (m, 1H), 3.95 (s, 6H), 3.94 (s, 3H). MS (ESI, m/z): 498 [M+H] . HRMS
+
ESI, m/z) calcd for C24
H
19
N
4
O
4
Cl
2
: 498.07822 [M+H] ; found: 498.07794.
5
.1.6.19. 2-(3,4,5-Trimethoxyphenyl)-5-(3-(3,4,5-trimethoxyphenyl)imidazo[1,5-a]pyridin-1-
yl)-1,3,4-oxadiazole (8s):
The title compound was obtained from compound (6) and 3,4,5-trimethoxybenzaldehyde (7s)
following the general procedure described above. Yield: 72%; yellow solid; mp: 173-176 °C;
1
H NMR (300 MHz, CDCl
3
): δ 8.45 (d, J = 9.2 Hz, 1H), 8.38 (d, J = 7.2 Hz, 1H), 7.48 (s, 2H),
7
.19 (dd, J = 8.8, 6.1 Hz, 1H), 7.05 (s, 2H), 6.90 – 6.84 (m, 1H), 4.00 (s, 6H), 3.97 (s, 6H), 3.95
1
3
(
d, J = 1.6 Hz, 6H); C NMR (75 MHz,CDCl
3
): δ 163.31, 161.21, 153.88, 153.55, 139.67,
1
6
39.43, 132.49, 130.84, 128.74, 125.09, 123.57, 122.30, 119.97, 119.21, 114.85, 105.99, 104.18,
+
0.98, 56.44; MS (ESI, m/z): 519 [M+H] ; HRMS (ESI, m/z) calcd for C27
H
27
N
4
7
O : 518.18734
+
[
M+H] ; found: 519.18715.
6
6
.0 Biology
.1. In vitro growth inhibition
The screening of anticancer activity was evaluated by the NCI, USA, according to
standard procedures (http://dtp.nci.nih.gov/ branches/btb/ivclsp.html).[58]
.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
6
through the cell cycle phases. A549, lung cancer cells were incubated with compounds 8q at
concentrations of 2 and 4 μM for a period of 48 h. Untreated and treated cells were harvested,

washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide (Sigma
Aldrich). Cell cycle was performed by flow cytometry (Becton Dickinson FACS Caliber).[59]
6
.3. Caspase 3 activity
Caspase-3 assay was conducted for detection of apoptosis in lung cancer cell line (A549).
The commercially available apoptosis detection kit (Sigma-Caspase 3 Assay kit, Colorimetric)
was used. A549 cells were treated with the most active compound 8q at 2 and 4 μM for 48 h.
After 48 h of treatment, cells were collected by centrifugation, washed once with PBS, and cell
pellets were collected. Collected cell pellets were suspended in lysis buffer and incubated for 15
min. After incubation, cells were centrifuged at 20,000 rpm for 15 min and collected the
supernatant. Supernatants were used for measuring caspase 3 activity using an ELISA-based
assay, according to the manufacturer’s instructions.
6
.4. Tunel assay
After incubation with conjugate 8q at 2µM for 24 h, cells were rinsed three times with
phosphate-buffered saline (PBS) and fixed with methanol. Based on the TUNEL protocol
DeadEnd™ Fluorometric TUNEL System) cells were permeabilized with 0.1% Triton X-100 in
.1% sodium citrate and then rinsed twice with PBS. The DNA nick-labeling reaction was
(
0
performed using 50 μL TUNEL reaction mixture, including 45 μL enzyme solution and 5 μL
nucleotide mix at 37 ºC for 60 min. Then the samples were rinsed three times with PBS and
analyzed under a fluorescence microscope.
6
.5. Mesurement of Mitochondrial Membrane Potential
Mitochondrial membrane potential (ΔΨm) was determined by using JC-1 dye. Cell-
permeable cationic carbocyanine dye JC-1 (Sigma aldrich India, Catlog- CS0390), also known as
,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodine, emits green
5
fluorescence (525 nm) in its monomeric form. However, upon transfer to the membrane
environment of a functionally active mitochondrion, it exhibits an aggregation dependent orange-
red fluorescence (emission at 590 nm). Briefly, cultures were treated with the test drugs for 24 h.
After drug treatment the cells were incubated with JC-1 dye for 20 min at 37 °C. After
incubation cultures were used for the measuring mitochondrial membrane potiential (ΔΨm),
according to the manufacturer’s instructions
6
.6. ROS Generation

To evaluate intracellular reactive oxygen species (ROS) levels, 2’,7’-dichlorofluorescein
diacetate (DCFH-DA, Molecular Probes) fluorescent dye was used to clarify this issue. The
nonpolar DCFH-DA is converted to the polar derivative DCFH by esterases when it is taken up
by the cell. DCFH is nonfluorescent but is rapidly oxidized to the highly fluorescent DCF by
intracellular H
2
2
O or nitric oxide. A549 cells were treated with compound 8q at 2 µM
concentration for 24 h. To determine the production of ROS, cells were treated with 2 µM of
DCFH2-DA at 37°C for 30 min, and the fluorescence of DCF was measured at 530 nm after
excitation at 485 nm (DCFH2-DA, after deacetylation to DCFH2, is oxidized intracellularly to
its fluorescent derivative, DCF).[60]
6
.7. Flow cytometric evaluation of apoptosis
6
A549 (1×10 ) were seeded in six-well plates and allowed to grow overnight. The medium
was then replaced with complete medium containing 2 µM concentration of compound 8q for 48
h along with vehicle alone as control. After 48 h of drug treatment, cells from the supernatant
and adherent monolayer cells were harvested by trypsinization, washed with PBS at 3000 rpm.
6
Then the cells (1×10 ) were stained with Annexin V-FITC and propidium iodide using the
Annexin-V-PI apoptosis detection kit (Invitrogen). Flow cytometry was performed using a
FACScan (Becton Dickinson) equipped with a single 488-nm argon laser as described
earlier.[61] Annexin V-FITC was analyzed using excitation and emission settings of 488 nm and
5
35 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and clumps were gated
out using forward and orthogonal light scatter.
6
.8. Topoisomerase II inhibition assay
Topoisomerase II inhibition assay was determined by the ATP dependent decatenation of kDNA.
Topoisomerase-II activity was determined using kit (TG 1009, Topogen, USA)[62]. Reactions
were carried out in 20 µL and contained 120 mM KCl, 50 mM Tris–HCl, pH 8, 10 mM MgCl
2
,
0
.5 mM dithiothreitol, 0.5 mM ATP, 30 mg/ml bovine serum albumin, 200–300 ng of kDNA,
and topoisomerase II. The amount of topoisomerase II (5 units) was adjusted in preliminary
experiments to decatenate approximately 100% of the kDNA under our assay conditions. The
0
reactions were incubated at 37 C for 30 min and terminated by the addition of 2 µL of a stop
buffer containing 10% (w/v) SDS and 2 µL 0.5 mg/ml proteinaseK and then incubated for 10
0
min at 37 C. After completion of the reaction catenated and decatenated reaction products were
separated by 1% agarose gel electrophoresis stained with 0.5 µg/ml ethidium bromide (EtBr) in

TAE buffer (40 mM tris-acetate, pH 8.0, and 1 mMEDTA). Authentic decatenated kDNA and
linear kDNA (Topogen Inc.) were run as controls to identify decatenated kDNA. The gels were
run at 100 V for about 1hr and visualized under UV transillumination (BIO RAD gel doc XR+,
USA).
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