Synthesis and biological evaluation of novel imidazo[1,2-a]pyridine-oxadiazole hybrids as anti-proliferative agents: Study of microtubule polymerization inhibition and DNA binding

Article abstract of DOI:10.1016/j.bmc.2021.116277

Efforts towards the development of potential anticancer agents, a new series of imidazo[1,2-a]pyridine-oxadiazole hybrids were synthesized and evaluated for their in vitro anticancer activity against lung cancer (A549) and prostate cancer (PC-3, DU-145) cell lines. Amongst the compounds tested, 6d showed the highest potency on A549 cells with an IC₅₀ value of 2.8 ± 0.02 μM. Flow cytometric analysis of compound 6d treated A549 cells showed apoptosis induction by annexin-v/PI dual staining assay and the effect of 6d on different phases of cell cycle was also analyzed. Target based studies demonstrated the inhibition of tubulin polymerization by 6d at an IC₅₀ value of 3.45 ± 0.51 μM and its effective binding with CT-DNA. Further, the molecular modelling studies revealed that 6d has a prominent binding affinity towards α/β-tubulin receptor with admirable physico-chemical properties.

Full text of DOI:10.1016/j.bmc.2021.116277

Bioorg. Med. Chem. 43 (2021) 116277
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel imidazo[1,2-a]
pyridine-oxadiazole hybrids as anti-proliferative agents: Study of
microtubule polymerization inhibition and DNA binding
a
b
c
a
Dilep Kumar Sigalapalli , Gaddam Kiranmai , G. Parimala Devi , Ramya Tokala ,
a
b
a
a
Sravani Sana , Chaturvedula Tripura , Govinda Shivaji Jadhav , Manasa Kadagathur ,
Nagula Shankaraiah , Narayana Nagesh , Bathini Nagendra Babu
Neelima D. Tangellamudi
a,
*
b,
*
c,*
,
a,*
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, India
CSIR-Centre for Cellular and Molecular Biology, Medical Biotechnology Complex, ANNEXE II, Uppal Road, Hyderabad 500007, India
Department of Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Efforts towards the development of potential anticancer agents, a new series of imidazo[1,2-a]pyridine-oxa-
diazole hybrids were synthesized and evaluated for their in vitro anticancer activity against lung cancer (A549)
and prostate cancer (PC-3, DU-145) cell lines. Amongst the compounds tested, 6d showed the highest potency on
Imidazo[1,2-a]pyridine
1
,3,4-oxadiazole
Cytotoxicity
A549 cells with an IC50 value of 2.8 ± 0.02
μ
M. Flow cytometric analysis of compound 6d treated A549 cells
Apoptosis
showed apoptosis induction by annexin-v/PI dual staining assay and the effect of 6d on different phases of cell
cycle was also analyzed. Target based studies demonstrated the inhibition of tubulin polymerization by 6d at an
Tubulin polymerization
Molecular docking
ADME/T
IC50 value of 3.45 ± 0.51
μ
M and its effective binding with CT-DNA. Further, the molecular modelling studies
revealed that 6d has a prominent binding affinity towards
α/β-tubulin receptor with admirable physico-chemical
properties.
1
. Introduction
antifungal, antibacterial, hypnoselective, antiprotozoal, anti-
9
inflammatory and anticancer, etc. Imidazo[1,2-a]pyridines are also
Cancer is the second most deadly disease after cardiovascular dis-
good bioisosteres for indoles and azaindoles. Some of the marketed
drugs such as zolpidem, necopidem, minodronic acid and zolimidine
consist of imidazo[1,2-a]pyridine as core moiety.10 In the recent years, a
variety of imidazo[1,2-a]pyridine containing molecules were reported
as promising anticancer agents, in particular, imidazo[1,2-a]pyridines
eases. Cancer is known as a worldwide killer for the last few decades. It is
a life-threatening disease, which is the result of abnormal cell cycle and
apoptosis regulation, characterized by uncontrolled cell growth and cell
division.1 Effective cancer treatment with fewer side effects is the
major topic of research in the pharmaceutical industry and academia for
the last few decades.4 In this vision, microtubule modulators have
played an essential role in cancer chemotherapy over the past de-
–3
substituted at C2 and C3 position showed remarkable anticancer activ-
11,12
ity.
Imidazo[1,2-a]pyridines show anticancer activity via acting on
CDK, VEGFR, PI3K, EGFR, topoisomerase II , micro-tubulin protein,
α
5
,6
13–15
cades.
In this connection, the development of chemotherapeutic
etc.
Weiteng et al. reported novel 2-phenyl-imidazo[1,2-a]pyridine
agents that inhibit the function of tubulin leads to metaphase arrest, and
DNA binding ability will ultimately help in the activation process of
apoptosis.7
analogs that act as anti-proliferative agents by inhibiting tubulin poly-
merization.16 Recently, Kamal et al. synthesized new curcumin inspired
,8
17
imidazo[1,2-a]pyridine analogs as tubulin polymerization inhibitors.
Imidazo[1,2-a]pyridines are fused bicyclic 5–6 membered hetero-
cycles with a wide range of pharmacological activities such as antiviral,
On the other side, oxadiazole is one of the important structural
components in several pharmaceutical products. Among all the
*
Corresponding authors.
E-mail addresses: shankar@niperhyd.ac.in (N. Shankaraiah), nagesh@ccmb.res.in (N. Nagesh), bathini@iict.res.in (B.N. Babu), tdneelima@gmail.com
N.D. Tangellamudi).
(
https://doi.org/10.1016/j.bmc.2021.116277
Received 10 April 2021; Received in revised form 7 June 2021; Accepted 9 June 2021
Available online 16 June 2021
0
968-0896/© 2021 Elsevier Ltd. All rights reserved.

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
oxadiazole isoforms, 1,3,4-oxadiazole displays good stability, remark-
able hydrogen-bonding capability with receptor/drug target and con-
tributes to the lipophilicity of the drug, thus providing an easy access to
the drug target.18 The azole functionality in the 1,3,4-oxadiazole is
responsible for diversified biological activities such as hypoglycemic,
antidepressant, antitubercular, antianxiety, anticancer, antifungal,
1
9
antibacterial and anti-inflammatory, etc. Zibotentan, raltegravir and
furamizole are some established drugs that contain 1,3,4-oxadiazole
scaffold.20 1,3,4-Oxadiazoles act as a potential anticancer agents by
inhibiting the different enzymes which are essential for cell growth, such
as telomerase, histone deacetylase (HDAC), topoisomerase, epidermal
growth factor (EGF), vascular endothelial growth factor (VGFR), etc.21
In recent years, Vaidya et al. published a review on recent progress in
anticancer activities of 1,3,4-oxadiazole and its derivatives.22 Kamal et
al. reported combretastatin-linked 1,3,4-oxadiazole conjugates and 2-
anilinonicotinyl-linked 1,3,4-oxadiazole derivatives as potent tubulin
polymerization inhibitors.2
3,24
In the past few decades, molecular hybridization has been an
important technique used in medicinal chemistry to develop molecular
hybrids with enhanced biological activities by combining two or more
pharmacophores/scaffolds. The challenges associated with the present
drugs such as selectivity, specificity and multidrug resistance may be
circumvented by molecular hybridization technique for the develop-
ment of molecular hybrids as anticancer agents.25 Earlier, we reported
various biologically active molecular hybrids as tubulin inhibitors.2
In continuation of our efforts in the direction of the development of
bioactive tubulin inhibitors, we designed and synthesized certain imi-
dazo[1,2-a]pyridine-1,3,4-oxadiazole hybrids by linking imidazo[1,2-a]
pyridine with 1,3,4-oxadiazole by using the hybridization technique
Scheme 1. Synthesis of imidazo[1,2-a]pyridine-1,3,4-oxadiazole hybrids 6a–p.
corresponding to the methoxy groups appeared at δ 4.12 and 4.09 and
the rest of the aromatic protons appeared in the range of δ 8.87–7.69. In
6–28
1
3
C NMR of 6d, the carbons of 1,3,4-oxadiazole fragment gave two peaks
at δ 167.12 and 155.16. While the two aromatic carbons (C3 and C5) of
the trimethoxy benzene motif attached the methoxy groups gave a peak
at δ 153.7, the carbon – C4 gave a peak at 137.7. The C2 and C6 carbons
the benzene ring gave a peak at 105.5. The bridged carbon and the C3
carbon of the imidazopyridine gave peaks at δ 141.4 and 112.2,
respectively. The ether carbons of the trimethoxy group gave peaks at
(
Fig. 1), and further evaluated them for in vitro anticancer activity.
2
. Chemistry
6
0.89 and 55.7.
To achieve the designed molecules, we began with the condensation
of 2-aminopyridines (1) with ethyl bromo pyruvate (2) in ethanol,
which resulted in ethyl imidazo[1,2-a]pyridine-2-carboxylate (3) fol-
lowed by reaction with hydrazine hydrate to get imidazo[1,2-a]pyri-
dine-2-carbohydrazide intermediate 4 with good yields.17,29 Finally, the
oxidative cyclization between imidazo[1,2-a]pyridine-2-carbohy-
3. Pharmacology
3.1. In vitro antiproliferative activity
The synthesized imidazo[1,2-a]pyridine-1,3,4-oxadiazole hybrids
6a-p were screened for their in vitro cytotoxicity against a panel of
human cell lines such as lung cancer (A549), prostate cancer (PC-3, DU-
145) and normal embryonic kidney (HEK-293) using 3-(4,5-dime-
drazide (4) and aromatic acids 5 in the presence of POCl
3
yielded imi-
dazo[1,2-a]pyridine-1,3,4-oxadiazole hybrids 6a-p in good to excellent
2
9
yields (Scheme 1). The synthesized analogs were characterized by
1
13
1
30
utilizing HRMS, H and C NMR spectroscopic techniques. The H NMR
spectrum of the representative compound 6d showed a singlet of imi-
dazopyridine at δ 8.98 and a sharp singlet of the two aromatic protons
belonging to the trimethoxy benzene ring at δ 7.57. The protons
thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The
IC50(µM) values of compounds 6a-p and reference standard are dis-
played in Table 1.The initial cytotoxic results indicated that some of the
imidazo[1,2-a]pyridine-1,3,4-oxadiazole hybrids exhibit moderate to
Fig. 1. Examples of bioactive imidazo[1,2-a]pyridine and 1,3,4-oxadiazole-containing tubulin polymerization inhibitors.
2

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Table 1
In vitro anticancer activity of compounds 6a-p.
a
Compound
IC50 (µM)
b
c
d
e
A549
PC-3
DU-145
HEK-293
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
23.4 ± 0.12
32.4 ± 1.2
11.9 ± 0.01
2.8 ± 0.02
>50
22.2 ± 0.14
33.7 ± 0.34
13.4 ± 0.13
3.5 ± 0.12
>50
23.4 ± 0.23
32.8 ± 0.89
12.9 ± 0.13
4.5 ± 0.2
–
–
–
89 ± 1.23
>50
–
–
–
–
–
–
–
–
–
–
–
–
–
23.4 ± 0.56
34.7 ± 0.56
12.8 ± 0.43
12.3 ± 0.12
10.9 ± 0.02
33.4 ± 0.24
9.9 ± 0.23
14.5 ± 0.25
43.5 ± 1.2
23.4 ± 1.5
10.8 ± 0.12
0.09 ± 0.01
22.8 ± 0.99
37.8 ± 0.67
11.8 ± 0.12
11.6 ± 0.24
11.5 ± 0.22
33.5 ± 0.21
10.9 ± 0.13
17.8 ± 1.4
42 ± 0.23
21.8 ± 0.89
38.9 ± 0.78
10.8 ± 0.11
10.8 ± 0.23
11.8 ± 0.45
31.5 ± 0.12
11.2 ± 0.13
18.9 ± 1.54
46 ± 1.1
g
h
i
Fig. 3. Selectivity Index (SI) of lead molecule 6d.
j
k
l
found to reduce the activity of the compounds (6b, 6e, 6g, 6k, 6n
and 6o).
m
n
o
p
c. Unsubstituted imidazo[1,2-a]pyridines (6d) were relatively more
active in comparison to the methyl substituted imidazo[1,2-a]pyri-
dine containing compound (6l).
24.5 ± 0.75
11.2 ± 0.56
0.06 ± 0.89
22.3 ± 0.67
12.8 ± 0.13
0.06 ± 0.08
Podophyllotoxin
a
5
0% Inhibitory concentration after 48 h of drug treatment.
b
Human lung cancer.
4. Flow cytometric analysis
c, d
Human prostate cancer.
e
Humannormal embryonic kidney.
4.1. Apoptosis detection study by annexin V/PI dual staining assay
good cytotoxic activity against the tested cancer cells with IC50 values in
M.
The results from the MTT assay demonstrated that 6d is cytotoxic in
A549 cells. Annexin V-FITC/Propidium iodide dual staining assay expe-
dites the quantification of apoptotic cell death using flow cytometry. This
assay identifies the percentage of necrotic cells, late apoptotic cells, early
apoptotic cells and live cells after 48 h treatment of HEK and A549 cells
with 6d compound. The apoptotic assay was performed to verify the
percentage of cancer cells undergoing apoptosis upon treatment of about
2.8 µM concentration of 6d. When A549 cells were harvested in the
absence of 6d, viable cells are ~ 83.1% and only 0.1% apoptotic (both pre
and post apoptotic cells) and 0.9% necrotic cells are observed. Similarly, in
HEK cell line in the absence of 6d, viable cells are ~80.2% and only 0.2%
apoptotic (both pre and post apoptotic cells) and 0.5% necrotic cells are
the range of 2.8 ± 0.02 to 18.9 ± 1.54
μ
The preliminary screening results indicated that compounds 6c, 6d,
h, 6i, 6j, 6l and 6p were the most active, the remaining are moderately
6
active to less active against the tested cancer cells. Captivatingly, the
compound 6d was found to be most active in the lung (A549) and
prostate (PC-3, DU-145) cell lines and showed distinct cytotoxic activity
with IC50 of 2.8 ± 0.02
fold less active towards normal human embryonic kidney cells (HEK-
M in contrast to A549 cells (Fig. 2), which
μM towards A549 cells and also found to be 29-
2
93) with IC50 of 89 ± 1.23 μ
indicates the selectivity of newly synthesized compounds towards
cancerous cells and the SI was presented in Fig. 3.
observed. But when HEK and A549 cells were treated with 2.8
compound, the percentage of viable and apoptotic cells are 18.5% and
4.5% (both pre and post apoptotic cells) respectively among HEK cells
μ
M of 6d
3
.2. Structure-Activity relationship (SAR)
7
and the viable and apoptotic cells are 4.3% and 86.5% (both pre and post
apoptotic cells) respectively among A549 cells. From the assay, it is
evident that the 6d compound will induce apoptosis among A549 cells
much effectively compared to control HEK cells. The details are shown in
Fig. 4a and quantitatively expressed as a bar graph in Fig. 4b.
The following conclusions have been drawn from the structur-
e–activity relationship (SAR) analysis of the synthesized compounds
based on the substitution on phenyl and imidazo[1,2-a]pyridine
moieties:
a. From the preliminary cytotoxicity screening results (Table 1), it can
be noted that the presence of electron-donating (4-methoxy, 3,4-
dimethoxy, 3,4,5-trimethoxy and 4-N,N-dimethyl) groups on the
phenyl ring attached to 1,3,4-oxadiazole enhanced the bioactivity of
the compounds 6c, 6d, 6h-j, 6l and 6p as compared to the unsub-
stituted counterparts (6a, 6f and 6m).
4
.2. Cell cycle analysis
Cells undergo spontaneous cell divisions in a systematic manner that
is called the cell cycle. Most of the chemotherapeutic agents exhibit their
growth inhibitory potential by the obstruction of the cell cycle at a
specific checkpoint. In vitro cytotoxicity suggests that the compound 6d
displayed significant cytotoxicity against the A549 cell line. A cell cycle
analysis study was performed to understand the role of 6d in different
phases of cell cycle, wherein untreated HEK cells were considered as a
control. The distribution percentages of untreated A549 cells in sub-G1,
G0/G1, S, and G2/M phases were 4.2%, 73.0%, 12.7 %and 10.1%,
respectively. Upon treatment of A549 cells, with 2.8 µM of 6d, there is a
slight change in the distribution of cells in G0/G1 and a significant
change in the G2/M phase compared to treated HEK cells. After treat-
ment, the percentages of cells in sub-G1, G1, S, and G2/M phases were
b. The presence of electron-withdrawing (4-chloro, 4-bromo and 2-
iodo) groups on the phenyl ring linked to 1,3,4-oxadiazole were
1
.3%, 74.5%, 6.9% and 17.1%, respectively. After the treatment of A549
cells with 6d, the percentage of cells in G0/G1 and G2/M phase has
increased from 73 to 74.5% and 10.1% to 17.1% respectively. Whereas
when HEK cells were treated with 2.8 µM of 6d, the percentages of cells
in sub-G1, G1, S, and G2/M phases are 2.1%, 63.2%, 18.8% and 15.7%,
respectively, compared to untreated HEK cells (sub-G1, G1, S, and G2/M
Fig. 2. Cytospecificity exhibited by compound 6d towards human cancer cell
lines tested in comparison to normal human embryonic kidney cells.
3

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Fig. 4. (a) Annexin-V/PI dual staining assay of compound 6d in human cancer and normal human cell lines. Flow cytometric analysis and the percentage of Annexin
V-FITC/PI positive cells were represented in four quadrants. Images A and C depict the control HEK-293 and A549 cells respectively. Images B and D represent the 6d
treated HEK-293 and A549 cells respectively. (b) Bar graph quantifying the viable and apoptotic (early and late) cells.
phases were 1%, 64.1%, 18.8% and 16.1%, respectively). In HEK cells,
the cell cycle did not show accumulation of cells in the G0/G1 and G2/M
phase. The cell cycle and apoptosis assay indicate that compound 6d has
moderate effect on apoptosis induction and cell cycle inhibition. The
details of the distribution of cells in different phases of the cell cycle are
shown in Fig. 5a and quantified as bar graphs in Fig. 5b.
are considered positive controls. Confocal microscopic results suggested
that untreated cancer cells displayed the normal distribution of micro-
tubules, whereas cells treated with 6d compound as well as podo-
phyllotoxin showed the disrupted microtubule organization as shown in
Fig. 7, demonstrating the inhibition of tubulin polymerization.
5
5
.2. DNA binding studies
5
. Target assays
.2.1. UV–Visible spectral studies
The interaction of compound 6d with CT-DNA was studied to un-
Results from the previous experiments indicate that the compound
6
d is efficient in inducing apoptosis among cancer cells. Cell cycle re-
derstand the mode of DNA interaction with the selected ligand. The
compound 6d–DNA complex UV–visible spectra displayed a prominent
absorption peak at around 301 nm and 312 nm. In the present study,
when equal proportions of different DNA were added to a fixed con-
centration of the compound, the complex absorption band displayed a
continuous increase in its intensity (hyperchromicity). The 6d–DNA
complex absorption band has not shown any shift. Continuous hyper-
chromicity of the complex absorption band with the compound 6d with
the addition of DNA solution is an indication of very good interaction of
the compound with CT-DNA. After each successive addition of CT-DNA
to 6d solution, samples were equilibrated for 5 min before measure-
ment. The data obtained were fitted to equation (1), to obtain the
veals the ability of compound 6d to arrest cell cycle at both G0/G1and
G2/M phase in A549 cells. Therefore to evaluate the efficiency of
compound 6d on different targets in cancer, further target based anal-
ysis (tubulin & DNA based studies) was performed.
5
.1. Tubulin polymerization inhibition study
Further, to check the probable mechanism of action of these mole-
cules in inhibition of tubulin polymerization, an enzyme based inhibi-
tory assay along with immunohistochemistry assay was performed to
examine the in situ effects on cellular microtubules in A549 cells.
dissociation constant (K
d
) taking into account the total amount of 1:1
5
.1.1. Enzyme based assay
complex formed, independently of the derivative binding site(s).
To identify the tubulin inhibitory potential of novel imidazo[1,2-a]
K = [CT - DNA] [LC/LMcom pound] / [CT - DNA.LC/LMcom pound]
d
pyridine-oxadiazole hybrids, the different concentrations (0.5, 1, 2, 4
and 8 M) of compound 6d along with 3 M of podophyllotoxin were
(
1)
Based on the absorbance values obtained, the 6d CT-DNA dissocia-
tion constant(K ) was calculated using equation (1). The K values ob-
tained on the interaction of 6d with CT- DNA is 42 nM. Lower values K
μ
μ
used for the cell-free in vitro tubulin polymerization assay by examining
the change in fluorescence emission at 440 nm (excitation wavelength is
d
d
◦
3
60 nm) for 1 h at 37 C (Fig. 6a). The assay was performed in duplicates
d
and it was observed that at concentrations of 0.5, 1, 2, 4 and 8
μ
M, there
values indicate that 6d has a better binding affinity with CT-DNA. The
UV–Visible spectra profile obtained when 6d interacts with that of CT-
DNA is shown in Fig. 8.
is 4.6%, 11.5%, 21.7%, 60.1% and 81.2% of inhibition of tubulin
polymerization respectively, in comparison to the vehicle group (DMSO)
(
Fig. 6b). Remarkably, the compound 6d exhibited noteworthy inhibi-
tion of tubulin polymerization in dose dependent mode with an IC50
value of 3.45 ± 0.51 M.
5.2.2. Fluorescence studies
μ
Fluorescence study is an additional important method to scrutinize
the mode of binding of small molecules with the DNA and to study the
electronic environment around the DNA-complex at comparatively
lower concentrations. Since the complexes are fluorescent, their inter-
action with DNA can be monitored at low concentration with this study.
6d compound is excited at 250 nm and the emission spectra obtained
after the interaction with CT-DNA show a prominent peak at around
360 nm. When an equal quantity of DNA is added to the 6d, the emission
5
.1.2. Immunohistochemical assay
The effect of 6d was considered in A549 cells only because in the
previous experiments, the effect of 6d was maximum on A549 cells only
compared to other cells. Since the effect was high in A549 cells, they
were treated with 6d compound at half maximal inhibitory concentra-
tion for 48 h. The cells that are not treated with 6d and podophyllotoxin
4

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Fig. 5. (a) Flow cytometric analysis of cell cycle distribution. Dot plots were obtained on the treatment of HEK and A549 cells with 2.8 µM of compound 6d. Image A.
HEK cells before the treatment of 6d. Image B. HEK cells after the treatment of 6d. Image C. A549 cells before the treatment of 6d. Image D. A549 cells after the
treatment of 6d. (b) The bar graph represents the quantification of cells at different phases of the cell cycle. The untreated controls were compared with compound
6
d treated respective cells and cell cycle arrest in human normal kidney cells (HEK-293) was compared with human lung cancer cells (A549).
peak intensity reduced gradually, indicating that the 6d compound may
be binding to the surface of DNA or binding to the groove of double
helical DNA. With the increase in DNA concentration, the emission in-
tensity gradually decreased may be due to self-stacking of the compound
and binding to the DNA surface.31 The fluorescence spectral profile
obtained on the addition of DNA to 6d is shown in Fig. 9.
conformation of DNA can be evaluated with circular dichroism spectral
studies only. To understand it better, CD spectroscopy experiments are
performed. These studies will further indicate the conformational
changes of double stranded CT-DNA during interaction with ligands like
6d. CT-DNA exhibited a characteristic DNA conformation with a posi-
tive peak around 270 nm and a negative peak around 240 nm. But the
CT-DNA showed positive peaks at 279 and 260 nm and negative peak at
245 nm, whereas dsDNA has shown a positive peak at 290 nm and a
negative peak at around 238 nm. On addition of 6d compound, the
positive peak decreased its intensity and the negative peak intensity has
reduced (Fig. 10). This indicates that on interaction with 6d, CT- DNA
5
.2.3. Circular dichroism (CD) spectroscopic studies
In spectroscopic studies like UV–visible and fluorescence studies has
indicated that the compound 6d is interacting with DNA, which may be
through minor groove binding. But the effect of 6d interaction on the
5

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Fig. 6. (a) Effect of compound 6d on the tubulin polymerization: tubulin polymerization was monitored by the increase in fluorescence at 360 nm (excitation) and
4
40 nm (emission) for 1 h at 37 ◦C. (b) % Dose-response inhibition of tubulin polymerization by compound 6d at different concentrations 0.5, 1, 2, 4 and 8 µM.
Podophyllotoxin was used as standard. Data expressed as mean ± SEM (n = 3).
Fig. 7. A. Status of tubulin polymerization in untreated A549 cells. B. Status of tubulin polymerization in the presence of podophyllotoxin in A549 cells. C. Status of
tubulin polymerization in the presence of 6d molecule in A549 cells.
Fig. 9. Fluorescence spectra of 6d on interaction with CT-DNA. The fluores-
Fig. 8. UV–visible spectra of 6d on interaction with CT-DNA. The absorption
cence spectra exhibited slow hypochromicity on interaction with CT-DNA.
spectra exhibited slow hyperchromicity on interaction with CT-DNA. Concen-
Concentration of 6d is maintained at 10 µM throughout the fluorescence
tration of 6d is 10 µM and each time about 0.5 µM of CT-DNA was added.
titration. Each time about 0.5 µM of CT-DNA was added.
started melting and the change in the negative peak intensity indicates
As shown in Fig. 11, the interaction of 6d with DNA resulted in a slight
that there is a change in the helix of CT-DNA.
enhancement in viscosity. The increase in viscosity of DNA and ligand
complex is moderate. The result shows that variation in DNA viscosity is
5
.2.4. Relative viscosity experiment
between ethidium bromide (EtBr) and Hoechst 33342, but closer to
Hoechst 33342 indicating that the molecules 6d may be binding to the
minor groove or to the surface of DNA. All the spectroscopic and vis-
cosity studies results indicate that the molecule 6d exhibited moderate
interaction with DNA.
From the spectroscopic studies, it is seen that the potent 6d deriva-
tive interacted well with DNA. Further, to understand the nature and
mode of interaction of 6d with double helical DNA, viscosity studies are
carried out. When a molecule intercalates between the DNA, the relative
viscosity increases due to the increase in the axial length, whereas, the
reduction is observed with covalent DNA binding.32,33 Furthermore,
DNA surface binding results in a slight change in the relative viscosity.34
6

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
3
5
Schr o¨ dinger Suite release 2019–1. The protein crystal structure of
/β-tubulin receptor was retrieved from the RCSB Protein Data Bank
PDB ID: 3E22).36 The docking procedure was followed using the stan-
dard protocol implemented in Maestro and the ligand was docked
against the active site of
/β-tubulin. Compound 6d was well accom-
modated at the active pocket of
α
(
α
α
/β-tubulin and has shown a good
number of interactions with the active site residues. Fig. 12 illustrates
the predicted binding mode and the detailed protein-ligand interactions
of 6d with the active site residues of
α/β-tubulin receptor. The nitrogen
atom of the oxadiazole moiety of compound 6d acts as hydrogen bond
acceptor and formed two H-bond interactions with Asn249, Ala250 with
a distance of 3.54 Å and 3.17 Å, respectively. The 3,4,5-trimethoxy-
Fig. 10. CD spectra of 6d upon interaction with CT-DNA. The spectra shown
are recorded when 1:1 and 1:2 ratios CT-DNA: compound 6d is added.
phenyl ring of the compound 6d established a
π – cation (arene-
cation) interaction with the active site residue Lys254 (d = 3.95 Å).
Additionally, several hydrophobic interactions were observed between
the compound 6d and the active site residues, i.e. Ala250, Ala180,
Val181, Tyr224, Leu248, Leu255, Met259, Val315 and Ala316. Based on
the molecular docking studies, it is clear that compound 6d well lodged
in the tubulin
α and β interface thereby allowing favorable interactions
followed by the inhibition of tubulin polymerization.
6
.2. Prime MM/GBSA binding energy calculations
MM-GBSA (Molecular mechanics generalized born surface area)
approach available in the Prime module was employed to calculate the
binding free energy of 6d with protein using the OPLS3e force field.
Compound 6d energy was minimized and simulation was performed
using the VSGB energy model. VSGB energy model incorporates an
implicit solvation system, which is priorly optimized and performs
physics-based corrections for various interactions.
From the MM-GBSA binding free energy calculations, compound 6d
has shown comparable binding energy with that of co-crystalized ligand
Fig. 11. Viscosity variation in CT-DNA occurred when it is allowed to interact
with 6d, EtBr and Hoechst 33342.
(
Table 2). The study shows that the compound 6d has capable of binding
6
. Molecular modelling studies
and inhibiting tubulin enzyme.
6
.1. Molecular docking
6
.3. In silico ADME/T studies
The molecular docking study was carried out to explore the binding
QikProp program of Schr o¨ dinger software was utilized to assess the
mode of 6d within the active pocket of the
α/β-tubulin receptor using
drug-like
physicochemically
important
descriptors
and
Fig. 12. Predicted docking pose of compound 6d (purple color stick) and its ligand-protein interactions in the binding site of the
α/β-tubulin receptor (PDB ID:
3
E22). The red dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Asn249 (3.54),
Ala250 (3.17). Green color wire indicates residue (Lys254) involved in arene-cation interaction with 6d.
7

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Table 2
The average binding (ΔGBind) free energy (Kcal/mol) results from MM-GBSA calculations.
a
b
c
d
e
f
g
h
S. No
ΔGBind
ΔGBind
ΔGBind
ΔGBind
ΔGBind
ΔGBind
ΔGBind
ΔGBind
Coulomb
Covalent
H-Bond
Lipo
Solv GB
packing
vdW
6
d
ꢀ 73.462
ꢀ 84.640
ꢀ 19.563
ꢀ 16.229
6.970
6.757
ꢀ 0.400
ꢀ 0.127
ꢀ 31.959
ꢀ 52.160
24.370
28.196
ꢀ 1.257
ꢀ 51.622
ꢀ 51.078
Co-crystal
0.000
a
b
c
d
e
f
Free energy of binding; Coulomb energy; Covalent energy (internal energy); Hydrogen-bonding correction; Lipophilic energy; electrostatic solvation energy;
g
h
Pi-pi packing correction; Van der Waals energy.
pharmacokinetically key properties of 6d. Some of the computed
8. Experimental section
ADME/T parameters and their recommended ranges are mentioned in
Table 3. ADME/T prediction studies reveal that 6d conforms to Lip-
inski’s rule of five and has an appropriate logP value. Additionally, the
recommended ranges of physicochemical properties were also in the
acceptable range.
8.1. Chemistry
Materials and methods: All the starting materials, chemicals and
solvents were procured from commercial suppliers and used without
further purification. Analytical thin layer chromatography (TLC) was
performed using MERCK® pre-coated silica gel 60-F-254 (0.5 mm)
aluminum plates. Visualization of spots on TLC plates was achieved by
UV light. Wherever required, column chromatography was performed
with silica gel (60–120 mesh). Ethyl acetate and hexane were used as
eluents. Melting points were checked using Stuart digital SMP 30
7
. Conclusion
In conclusion, we have successfully synthesized, characterized and
evaluated a series of novel imidazo[1,2-a]pyridine-oxadiazole hybrids
and studied their anti-cancer potential. Anti-proliferative activities of
the synthesized compounds were evaluated in lung cancer (A549) and
prostate cancer (PC-3, DU-145) cell lines using MTT assay. Amongst the
synthesized compounds, 6d showed excellent anti-proliferative activity
on all the tested cell lines, particularly in lung cancer cell lines A549
1
13
melting point apparatus and were uncorrected. H and C NMR spectra
were recorded on Bruker NMR instrument operated at 500 and 125
MHz, respectively using tetra methyl silane (TMS) as the internal
reference. Chemical shift values were given in ppm and J values were
documented in Hertz. Spin multiplicities were explained as s (singlet),
d (doublet), t (triplet), dd (doublet of doublet), q (quartet) and m
(multiplet). HRMS were obtained with Agilent QTOF mass spectrometer
6540 series instrument and were performed in the ESI technique at 70
eV.
(
IC502.8 ± 0.02). The cell cycle analysis disclosed that 6d showed sig-
nificant G2/M phase arrest in A549 cells. Additionally, an immunohis-
tochemistry assay on A549 cell lines showed, compound 6d significantly
disrupted the microtubule organization. Further, target based studies
demonstrated the tubulin polymerization inhibition of compound 6d at
an IC50 value of 3.45 ± 0.51
with CT-DNA. The molecular modelling studies inferred that 6d binds at
the active site of
/β-tubulin with prominent binding affinity. Further-
more, we have examined physico-chemical and ADME/T properties of
d, which are in the acceptable range.
μ
M and also exhibited effective binding
8.1.1. General procedure for the synthesis of substituted ethyl imidazo[1,2-
a]pyridine-2-carboxylate (3)
α
A solution of 2-amino pyridine (1) (1 mmol) in ethanol (10 mL) was
◦
6
stirred at 0–5 C. To this solution, ethylbromopyruvate/3-bromo-2-oxo-
propionicacidethylester (2, 1.5 mmol) was added drop-wise within 15
min. During the addition, reaction mixture turns white due to the for-
mation of pyridinium salt. On completion of addition, the reaction
mixture was stirred under reflux for 12 h. After the completion of the
reaction, as monitored by TLC, the reaction mixture was cooled to room
temperature. The reaction mixture was partitioned between ethyl ace-
tate (3 × 30 mL) and water (150 mL). The organic layer was separated,
Table 3
ADME/T profile of 6d.
S.
ADME/T Parameters
Recommended
6d
No.
Values or Range
dried over anhydrous Na
to obtain the desired imidazo[1,2-a]pyridine-carboxylic acid ethyl ester
3) as a light brown solid.
2 4
SO and concentrated under reduced pressure
1
2
Rule of five
Maximum is 4
No
violation
75.245
(
PSA (van der Waals surface area of
polar nitrogen and oxygen atoms and
carbonyl carbon atoms)
SASA (Total solvent accessible surface
area)
7.0–200.0
8.1.2. General procedure for the synthesis of substituted imidazo[1,2-a]
pyridine-2-carbohydrazide (4)
3
300.0–1000.0
612.224
The crude imidazo[1,2-a]pyridine-carboxylic acid ethyl ester (3, 1
mmol) was dissolved in ethanol and hydrazine hydrate (2 mmol) was
added. Then, the mixture was refluxed for about 12 h. After completion
of the reaction as monitored by TLC, the reaction mixture was extracted
with ethyl acetate (3 × 30 mL). The combined organic layers were
4
5
6
7
8
Dipole Moment
1.0–12.5
9.361
Molecular Volume
500.0–2000.0
0.0–6.0
1077.231
0.000
Donor HB
Acceptor HB
2.0–20.0
ꢀ 1.5 to 1.5
6.250
QPlogKhsa (Prediction of binding to
human serum albumin)
QPlogPo/w (Predicted octanol/water
partition coefficient)
ꢀ 0.054
2 4
washed with brine solution, dried over anhydrous Na SO and concen-
9
ꢀ 2.0 to 6.5
3.122
trated under reduced pressure to yield imidazo[1,2-a]pyridine-2-car-
bohydrazide (4) in good yields.
10
11
12
13
14
15
QPpolrz (Predicted polarizability in
cubic angstroms)
13.0–70.0
37.953
ꢀ 0.337
ꢀ 1.525
ꢀ 5.741
1950.450
100.000
QPlogBB (Predicted brain/blood
partition coefficient)
ꢀ 3.0 to 1.2
8
.1.3. General procedure for the synthesis of 2-(imidazo[1,2-a]pyridin-2-
yl)-5-phenyl-1,3,4-oxadiazole (6a-p)
QPlogKp (Predicted skin
permeability)
ꢀ 8.0 to ꢀ 1.0
concern below ꢀ 5
A solution of imidazo[1,2-a]pyridine-2-carbohydrazide (4, 1 mmol),
QPlogHERG (Predicted IC50 value for
3
substituted benzoic acid (5) (1 mmol) and POCl (8 mL) was refluxed
+
blockage of HERG K channels.)
with stirring for 10–12 h. The reaction mixture was cooled and poured
over crushed ice and quenched by saturated ice-cold solution of NaOH.
The precipitate thus obtained was filtered washed with sodium bicar-
bonate. When the precipitate was not formed, the mixture was extracted
QPPCaco (Predicted apparent Caco-2
cell permeability in nm/sec.)
POA (Predicted human oral
<25 is poor, >500
great
>80% is high <25%
is poor
absorption on 0–100% scale)
8

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
with ethyl acetate (3 × 30 mL). The combined organic layers were
[M+H]+.
2 4
washed with brine solution, dried over anhydrous Na SO and concen-
trated under reduced pressure to give desired product in good yields.
Further compounds were purified by using column chromatography.
8.1.3.4. 2-(Imidazo[1,2-a]pyridin-2-yl)-5-(3,4,5-trimethoxyphenyl)-
1,3,4-oxadiazole (6d)
8
.1.3.1. 2-(Imidazo[1,2-a]pyridin-2-yl)-5-phenyl-1,3,4-oxadiazole (6a)
Dark grey solid, yield 93%; mp: 245–247 ◦C; H NMR (500 MHz,
1
White solid, yield 96%; mp: 218–220 ◦C; H NMR (500 MHz,
1
TriFluro Acetic Acid (TFA)): δ 8.98 (s, 1H), 8.87 (d, J = 6.7 Hz, 1H),
DMSO‑d
6
): δ 8.81 (s, 1H), 8.67 (d, J = 6.8 Hz, 1H), 8.12 (dd, J = 7.5, 1.8
8
2
.28–8.20 (m, 1H), 8.15–8.08 (m, 1H), 7.70 (t, J = 6.9 Hz, 1H), 7.57 (s,
Hz, 2H), 7.73 (d, J = 9.1 Hz, 1H), 7.66 (dd, J = 10.4, 5.1 Hz, 3H),
13
H), 4.12 (s, 3H), 4.09 (s, 6H); C NMR (125 MHz, TriFluro Acetic Acid
1
3
7
.45–7.40 (m, 1H), 7.07 (t, J = 6.6 Hz, 1H); C NMR (125 MHz,
DMSO‑d ): δ 164.2 (C5), 161.4 (C2), 145.8 (C12), 132.5 (C9), 130.0 (C8,
C10), 129.9 (C19), 128.0 (C6), 127.3 (C16), 127.1 (C7, C11), 123.9
(
(
TFA)): δ 167.1 (C5), 155.2 (C2), 153.7 (C8, C10), 141.8 (C12), 141.4
C9), 137.7 (C19), 129.5 (C6), 119.4 (C16), 119.5 (C14), 117.0 (C13),
6
1
16.5 (C20), 112.2 (C15), 105.5 (C7, C11), 60.9 (C22), 55.7 (C25, C26);
(
C14), 117.9 (C13), 115.3 (C20), 114.2 (C15); HRMS (ESI): m/z calcd
HRMS (ESI): m/z calcd for
M+H]+.
18 17 4 4
C H N O 353.1250, found 353.1290
for C15
H
11
N
4
O263.0933, found 263.0930 [M+H]+.
[
8
.1.3.2. 2-(4-Chlorophenyl)-5-(imidazo[1,2-a]pyridin-2-yl)-1,3,4-oxadia-
8
.1.3.5. 2-(4-Bromophenyl)-5-(imidazo[1,2-a]pyridin-2-yl)-1,3,4-oxadia-
zole (6b)
zole (6e)
White solid, yield 88%; mp: 250–253 ◦C; H NMR (500 MHz,
1
Light grey solid, yield 82%; mp: 260–262 ◦C; H NMR (500 MHz,
TriFluro Acetic Acid (TFA)): δ 9.42 (s, 1H), 9.31 (d, J = 6.7 Hz, 1H),
8.74–8.66 (m, 1H), 8.58 (d, J = 9.1 Hz, 1H), 8.52 (d, J = 8.4 Hz, 2H),
1
DMSO‑d
H), 7.76–7.70 (m, 3H), 7.45–7.39 (m, 1H), 7.07 (t, J = 6.7 Hz, 1H);
NMR (125 MHz, DMSO‑d ): δ 163.4 (C5), 161.5 (C2), 145.8 (C12), 137.2
C9), 130.16 (C8, C11), 129.8 (C19), 128.9 (C7, C11), 128.1 (C6), 127.3
6
): δ 8.81 (s, 1H), 8.66 (d, J = 6.8 Hz, 1H), 8.12 (d, J = 7.3 Hz,
13
2
C
6
1
3
(
(
(
8.31 (d, J = 8.4 Hz, 2H), 8.16 (t, J = 6.9 Hz, 1H); C NMR (125 MHz,
TriFluro Acetic Acid (TFA)): δ167.7 (C2), 155.7 (C5), 142.0 (C6), 138.3
(C13), 133.8 (C19, C17), 130.8 (C18), 130.0 (C15), 129.2 (C20, C16),
C16), 122.7 (C14), 117.9 (C13), 115.4 (C20), 114.2 (C15); HRMS
ESI): m/z calcd for C15
H10ClN
4
O297.0543, found 297.0593 [M+H]+.
1
20.2 (C10), 120.1 (C8), 119.4 (C7), 117.6 (C14), 112.8 (C9); HRMS
(
ESI): m/z calcd for C15
H10BrN
4
O 341.0038, found 341.0124 [M+H]+.
8
.1.3.3. 2-(Imidazo[1,2-a]pyridin-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxa-
diazole (6c)
8
.1.3.6. 2-(8-Methylimidazo[1,2-a]pyridin-2-yl)-5-phenyl-1,3,4-oxadia-
zole (6f)
Dark grey solid, yield 91%; mp: 233–235 ◦C; H NMR (500 MHz,
1
TriFluro Acetic Acid (TFA)): δ 9.46 (s, 1H), 9.35 (d, J = 6.2 Hz, 1H), 8.71
(
dd, J = 18.5, 8.1 Hz, 3H), 8.62 (d, J = 9.0 Hz, 1H), 8.20 (t, J = 6.5 Hz,
1
1
3
White solid, yield 89%; mp: 226–232 ◦C; H NMR (500 MHz,
DMSO‑d
): δ 8.80 (s, 1H), 8.51 (d, J = 6.6 Hz, 1H), 8.12 (d, J = 6.1 Hz,
H), 7.66 (d, J = 6.3 Hz, 3H), 7.22 (d, J = 6.6 Hz, 1H), 6.97 (t, J = 6.7
1
H), 7.76 (d, J = 8.4 Hz, 2H), 4.54 (s, 3H); C NMR (125 MHz, TriFluro
6
Acetic Acid (TFA)): δ 168.1 (C5), 165.7 (C9), 155.2 (C2), 142.1 (C12),
1
2
38.4 (C19), 130.9 (C7, C11), 130.1 (C16), 120.1 (C14), 117.6 (C6),
1
3
Hz, 1H), 2.58 (s, 3H); C NMR (125 MHz, DMSO‑d
6
): δ 164.1 (C5),
61.5 (C2), 146.4 (C12), 132.5 (C9), 130.0 (C8, C10), 129.5 (C19),
27.5 (C14), 127.1 (C7, C11), 125.7 (C6), 125.5 (C13), 123.8 (C16),
1
16.1 (C13), 112.9 (C8, C10), 112.4 (C20), 111.6 (C15), 55.6 (C22);
1
1
HRMS (ESI): m/z calcd for
16 13 4 2
C H N O 293.1039, found 293.1058
9

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
1
15.8 (C20), 114.2 (C15), 17.0 (C21); HRMS (ESI): m/z calcd for
(C6, C4), 127.1 (C15), 124.4 (C14), 124.1 (C17), 121.8 (C3, C7), 119.7
(C5), 119.3 (C21), 118.1 (C16), 47.2 (C23, C24), 14.5 (C22); HRMS
C
16
H
13
N
4
O 277.1108, found 277.1108 [M+H]+.
(
ESI): m/z calcd for C18
H
18
N
5
O 320.1511, found 320.1530 [M+H]+.
8
1
.1.3.7. 2-(4-Bromophenyl)-5-(8-methylimidazo[1,2-a]pyridin-2-yl)-
,3,4-oxadiazole (6g)
8.1.3.10. 2-(3,4-Dimethoxyphenyl)-5-(8-methylimidazo[1,2-a]pyridin-2-
yl)-1,3,4-oxadiazole (6j)
◦
1
Light grey solid, yield 76%; mp: 242–243.5 C; H NMR (500 MHz,
DMSO‑d
): δ 8.80 (s, 1H), 8.50 (t, J = 6.6 Hz, 1H), 8.05 (d, J = 8.5 Hz,
H), 7.87 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 6.8 Hz, 1H), 6.97 (t, J = 6.8
White solid, yield 90%; mp: 257–258 ◦C; H NMR (500 MHz, Tri-
1
6
2
Fluro Acetic Acid (TFA)): δ 8.90 (s, 1H), 8.64 (d, J = 6.3 Hz, 1H), 7.94 (t,
J = 13.9 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.72 (s, 1H), 7.54 (t, J = 6.8
Hz, 1H), 7.18 (t, J = 9.9 Hz, 1H), 4.03 (d, J = 8.9 Hz, 6H), 2.82 (s, 3H);
1
3
Hz, 1H), 2.57 (s, 3H); C NMR (125 MHz, DMSO‑d
61.6 (C5), 146.4 (C6), 133.1 (C18, C20), 129.3 (C13), 129.0 (C17,
C21), 127.5 (C8), 126.1 (C19), 125.7 (C7), 125.6 (C16), 123.1 (C10),
6
): δ 163.5 (C2),
1
13
C NMR (125 MHz, TriFluro Acetic Acid (TFA)): δ 167.3 (C2), 154.9
1
16.0 (C14), 114.2 (C9), 17.1 (C15); HRMS (ESI): m/z calcd for
(
C5), 154.2 (C19), 149.1 (C18), 141.8 (C6), 137.0 (C13), 127.0 (C8),
C
16
H
14BrN
4
O 357.0335, found 357.0177 [M+3H]+.
1
24.3 (C7), 123.4 (C10), 119.6 (C21), 119.3 (C16), 112. 5 (C14), 111.8
(
(
C9, C20), 110.1 (C17), 55.5 (C25), 55.2 (C23), 14.4 (C15); HRMS
8
1
.1.3.8. 2-(4-Methoxyphenyl)-5-(8-methylimidazo[1,2-a]pyridin-2-yl)-
337.1301, found 337.1314 [M+H]+.
17 4 3
ESI): m/z calcd for C18H N O
,3,4-oxadiazole (6h)
8
1
.1.3.11. 2-(2-Iodophenyl)-5-(8-methylimidazo[1,2-a]pyridin-2-yl)-
,3,4-oxadiazole (6k)
Dark grey solid, yield 84%; mp: 175–178 ◦C; H NMR (500 MHz,
1
DMSO‑d
6
): δ 8.76 (s, 1H), 8.50 (d, J = 5.7 Hz, 1H), 8.05 (d, J = 7.8 Hz,
13
1
White solid, yield 85%; mp: 214–216 ◦C; H NMR (500 MHz,
2
H), 7.20 (d, J = 7.8 Hz, 3H), 6.96 (s, 1H), 3.89 (s, 3H), 2.57 (s, 3H);
NMR (125 MHz, DMSO‑d ): δ 164.1 (C2), 162.6 (C10), 161.0 (C5), 146.3
C6), 129.6 (C13), 129.0 (C17, C21), 127.5 (C8), 125.7 (C7), 125.5
C10), 116.1 (C16), 115.6 (C14), 115.4 (C18, C20), 114.1 (C9), 56.0
C
6
DMSO‑d ): δ 8.80 (s, 1H), 8.51 (d, J = 6.8 Hz, 1H), 8.17 (dd, J = 8.0, 0.8
6
(
(
(
Hz, 1H), 7.91 (dd, J = 7.7, 1.5 Hz, 1H), 7.66 (td, J = 7.6, 1.1 Hz, 1H),
7.41 (td, J = 7.7, 1.6 Hz, 1H), 7.24–7.21 (m, 1H), 6.98 (t, J = 6.8 Hz,
1H), 2.56 (s, 3H);13C NMR (125 MHz, DMSO‑d ): δ 164.0 (C2), 161.7
C23), 17.0 (C15); HRMS (ESI): m/z calcd for C17
H
15
N
4
O
2
307.1195,
6
+
found 307.1211 [M+H] .
(C5), 146.4 (C6), 141.1 (C18), 133.6 (C13), 132.0 (C19), 129.4 (C21),
1
29.3 (C20), 129.1 (C8), 127.5 (C7), 125.7 (C10), 125.6 (C16), 116.0
8
.1.3.9. N,N-Dimethyl-4-(5-(8-methylimidazo[1,2-a]pyridin-2-yl)-1,3,4-
(C9), 114.2 (C14), 96.6 (C17), 17.0 (C15); HRMS (ESI): m/z calcd for
oxadiazol-2-yl)aniline (6i)
C
16
H12IN
4
O 403.0053, found 403.0081 [M+H]+.
8
.1.3.12. 2-(8-Methylimidazo[1,2-a]pyridin-2-yl)-5-(3,4,5-trimethox-
yphenyl)-1,3,4-oxadiazole (6l)
Dark grey solid, yield 78%; mp: 247–250 ◦C; H NMR (500 MHz,
1
TriFluro Acetic Acid (TFA)): δ 8.94 (s, 1H), 8.63 (d, J = 6.6 Hz, 1H), 8.47
(
(
d, J = 8.5 Hz, 2H), 7.95 (t, J = 8.6 Hz, 3H), 7.55 (t, J = 7.0 Hz, 1H), 3.51
s, 6H), 2.83 (s, 3H); 13C NMR (125 MHz, TriFluro Acetic Acid (TFA)): δ
White solid, yield 79%; mp: 216–218 ◦C; H NMR (500 MHz,
1
1
65.2 (C9), 156.1 (C12), 145.7 (C2), 141.9 (C13), 137.1 (C20), 130.5
1
0

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
1
TriFluro Acetic Acid (TFA)): δ 8.95 (s, 1H), 8.69 (d, J = 5.9 Hz, 1H), 7.99
Light grey solid, yield 80%; mp: 258–260 ◦C; H NMR (500 MHz,
(
d, J = 7.0 Hz, 1H), 7.62–7.51 (m, 3H), 4.10 (d, J = 2.2 Hz, 3H), 4.07 (s,
TriFluro Acetic Acid (TFA)): δ 9.83 (s, 1H), 9.51 (s, 1H), 9.19 (s, 1H),
6
1
1
H), 2.86 (s, 3H); 13C NMR (125 MHz, CDCl
3
): δ 165.2 (C5), 159.5 (C2),
8.95 (d, J = 8.3 Hz, 2H), 8.75 (d, J = 8.3 Hz, 2H), 3.52 (s, 3H); C NMR
13
53.7 (C8, C10), 145.5 (C18), 145.4 (C9), 141.3 (C25), 128.1 (C20),
(125 MHz, TriFluro Acetic Acid (TFA)): δ 168.2 (C5), 156.0 (C2), 143.5
(C13), 140.5 (C15), 134.2 (C20), 133.4 (C8, C10), 131.2 (C9), 129.6
(C7, C11), 127.0 (C6), 121.2 (C16, C17), 119.8 (C21), 105.8 (C14), 17.2
27.2 (C19), 124.4 (C22), 118.6 (C6), 115.2 (C26), 114.4 (C21), 104.9
(
C7, C11), 61.0 (C15), 56.7 (C13, C17), 17.5 (C27); HRMS (ESI): m/
z calcd for C19
H
19
N
4
O
4
367.1406, found 367.1412 [M+H]+.
2 4
(C22); HRMS (ESI): m/z calcd for C16H13Br N O 434.9456, found
4
34.9298 [M+3H]+.
8
1
.1.3.13. 2-(8-Bromo-6-methylimidazo[1,2-a]pyridin-2-yl)-5-phenyl-
,3,4-oxadiazole (6m)
8.1.3.16. 4-(5-(8-Bromo-6-methylimidazo[1,2-a]pyridin-2-yl)-1,3,4-oxa-
diazol-2-yl)-N,N-dimethylaniline (6p)
Light grey solid, yield 87%; mp: 231–232 ◦C; H NMR (500 MHz,
1
DMSO‑d
.75–7.57 (m, 4H), 2.32 (s, 3H); C NMR (125 MHz, DMSO‑d
C5), 161.0 (C2), 142.8 (C12), 132.6 (C14), 132.5 (C9), 130.0 (C19),
6
): δ 8.83 (s, 1H), 8.48 (s, 1H), 8.12 (d, J = 6.3 Hz, 2H),
White solid, yield 76%; mp: 255–259 ◦C; H NMR (500 MHz, Tri-
1
13
7
6
): δ 164.2
Fluro Acetic Acid (TFA)): δ 9.01 (s, 1H), 8.61 (s, 1H), 8.48 (s, 2H), 8.24
(
1
3
(
s, 1H), 7.95 (t, J = 13.3 Hz, 2H), 3.52 (s, 6H), 2.58 (s, 3H); C NMR
1
1
30.0 (C8, C10), 127.2 (C7, C11), 125.0 (C6), 124.1 (C15), 123.7 (C16),
(
125 MHz, TriFluro Acetic Acid (TFA)): δ 165.2 (C11), 155.7 (C14),
16.9 (C20), 110.3 (C13), 17.7 (C21); HRMS (ESI): m/z calcd for
O 355.0194, found 355.0196 [M+H]+.
145.7 (C2), 142.6 (C15), 139.6 (C17), 132.3 (C22), 130.6 (C4, C6),
16 4
C H12BrN
1
1
26.2 (C18), 124.1 (C19), 121.9 (C3, C7), 120.0 (C5), 118.7 (C23),
04.8 (C16), 47.2 (C8, C9), 16.2 (C24); HRMS (ESI): m/z calcd for
8
.1.3.14. 2-(8-Bromo-6-methylimidazo[1,2-a]pyridin-2-yl)-5-(4-chlor-
C
18
H19BrN
5
O400.0773, found 400.0689 [M+3H]+.
ophenyl)-1,3,4-oxadiazole (6n)
8
8
.2. Biological evaluation
.2.1. Cell culture
Human cancer cell lines such as lung cancer (A549), prostate cancer
(
PC-3, DU-145) and normal human embryonic kidney (HEK-293) were
obtained from National Centre for Cell Science, Pune, India. Cells were
maintained in appropriate media supplemented with 10% fetal bovine
serum (FBS) stabilized with 1% antibiotic-antimycotic solution (Sigma
◦
Aldrich) in the incubator at 37 C. When the cells reached up to 80–90%
of confluency, sub-culturing was performed using a 0.25% trypsin/1
mM EDTA solution for further passage.
Light grey solid, yield 83%; mp: 253–255 ◦C; H NMR (500 MHz,
1
TriFluro Acetic Acid (TFA)): δ 9.70 (s, 1H), 9.37 (s, 1H), 9.04 (s, 1H),
1
3
8.2.2. MTT assay
8
.89 (d, J = 8.5 Hz, 2H), 8.43 (d, J = 8.4 Hz, 2H), 3.37 (s, 3H); C NMR
The cytotoxicity of all newly synthesized compounds was deter-
mined by using MTT assay, where, MTT (3-(4,5-dimethylthiazol- 2-yl)-
(
(
(
(
125 MHz, TriFluro Acetic Acid (TFA)): δ 167.9 (C5), 155.8 (C2), 143.5
C13), 143.0 (C9), 140.4 (C15), 133.3 (C20), 131.1 (C8, C10), 129.7
C7, C11), 127.0 (C6), 121.1 (C16), 119.2 (C17), 118.9 (C21), 105.7
2
,5-diphenyl tetrazolium bromide) is a dye that converts into insol-
uble formazan by mitochondrial succinate dehydrogenase enzyme. In
brief, cells were seeded in 96-well plates at a density of 1000–4000 cells
per well in 100 µL of complete medium and allowed to grow overnight.
Further, the media was replaced with fresh media and cells were treated
with various concentrations of the compounds for a period of 48 h. After
incubation, the media was aspirated and 100 µL of MTT (0.5 mg/mL)
C14), 17.2 (C22); HRMS (ESI): m/z calcd for C16
H
13BrClN
4
O 390.9961,
+
found 390.9832 [M+3H] .
8
.1.3.15. 2-(8-Bromo-6-methylimidazo[1,2-a]pyridin-2-yl)-5-(4-bromo-
phenyl)-1,3,4-oxadiazole (6o)
◦
was added and incubated at 37 C for 4 h. After that, MTT reagent was
aspirated and the formazan crystals formed were dissolved in DMSO
◦
(
200 µL) for 20 min at 37 C. Finally, the formed formazan was measured
by using a spectrophotometric microtiter plate reader at 570 nm
wavelength.
8
.2.3. Flow cytometric analysis
8
.2.3.1. Apoptosis detection study byAnnexin V/PI dual staining assay.
This assay was performed by the method given by Rieger et al. with
slight modifications.37 Briefly, 1 × 10 A549 and HEK cells were seeded
5
in a 12-well plate and treated with 2.8 M 6d compound for 48 h. The
μ
1
1

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
collected cells were washed twice with ice-cold PBS, then incubated
100 mM Tris-HCl (pH 7.0) each time to the quartz cuvette containing
about 10 µM of 6d compound solution. Each time about 0.5 µM of CT-
DNA was added to cuvette containing fixed concentration of com-
pound 6d. Titrations were carried out until the complex absorption band
remained at a fixed wavelength upon five successive additions of CT-
DNA. UV–vis spectra were recorded from 200 to 400 nm range.
8.2.4.2.2. Fluorescence titration studies. Fluorescence emission
with 200
μL of 1 × binding buffer containing 1
μ
L propidium iodide (PI)
for 15 min at room temperature in the dark. After incubation, cells were
analyzed for apoptosis using a flow cytometer (BD FACSVerseTM, USA).
Apoptosis and necrosis were analyzed with quadrant statistics on pro-
pidium iodide-negative cells, fluorescein positive cells, and propidium
iodide (PI)-positive cells, respectively.
◦
spectra were measured at 25 C using a Hitachi F7000 spectrofluorim-
8
.2.3.2. Cell cycle assay. To validate the results obtained from the MTT
eter (Maryland, USA) using a 1 cm path length quartz cuvette.
Throughout the fluorescence experiment, the concentration of the 6d
compound was kept constant (10 µM) and titrated with the addition of
equal quantities of CT-DNA. Fluorescence spectra were recorded after
each addition of CT-DNA to the compound containing fluorescent
cuvette. Each time about 0.5 µM of CT-DNA was added to cuvette con-
taining fixed concentration of compound 6d. After each experiment, the
quartz cuvettes were thoroughly washed with distilled water and dilute
nitric acid (approximately 0.1 N, nitric acid) to remove traces of com-
pound binding to the walls of the quartz cuvette. 6d compound was
excited at 250 nm, and emission spectra for each titration were collected
in the range from 300 to 500 nm and the emission peak was monitored at
assay as well as to understand the effect of 6d compound on cell cycle
progression, cell cycle analysis was performed. Flow cytometry experi-
ments were performed according to the reported protocol.38 A549 and
HEK cells were incubated with 2.8 M of 6d compound for 48 h. Un-
μ
treated A549 and HEK cells were used as a control. Untreated and
treated cells were harvested, washed with phosphate-buffered saline
(
(
PBS), fixed in ice-cold 70% alcohol, and stained with propidium iodide
PI) (Sigma-Aldrich). The cell cycle assay was performed using a Becton
Dickinson FACSCalibur flow cytometer.
8
8
.2.4. Target based assays
3
60 nm. Each spectrum was recorded three times and an average of
three scans was taken.
.2.4.2.3. Circular dichroism studies. Circular dichroism experi-
.2.4.1. Tubulin polymerization assay
8
8
.2.4.1.1. Enzyme based assay. Tubulin polymerization kit was
ments were carried out using JASCO 815CD spectropolarimeter (Jasco,
Tokyo, Japan). CD spectrum was recorded from 220 to 320 nm to find
the conformational changes in CT-DNA after DNA-compound interac-
procured from Cytoskeleton, Inc. (BK011). To study the effect of com-
pound 6d, fluorescence based in vitro tubulin polymerization assay was
performed following the manufacturer’s protocol. The reaction mixture
having porcine brain tissue (2 mg/mL) in 80 mM PIPES at pH 6.9, 2.0
ꢀ 6
tion. For CD experiments, 10 × 10
M of CT-DNA was used. For
characterizing compound-CT-DNA interaction, CD spectra were recor-
2
mM MgCl , 0.5 mM EGTA,1.0 mM GTP and glycerol in the presence and
ded in 1:0, 1:1 and 1:2 M ratio of CT-DNA: 6d compound. CD titrations
absence of test compound 6d (final concentration of 10 mM) was pre-
pared and added to each well of 96-well plate. Tubulin polymerization
was followed by a time dependent increase in fluorescence due to the
insertion of a fluorescence reporter into microtubules as polymerization
takes place. Spectramax M4 Multi mode Micro plate Detection System
was used to measure Fluorescence emission at 440 nm (excitation
wavelength is 360 nm). Podophyllotoxin was used as positive control in
◦
were performed in 100 mM Tris-HCl (pH 7.0) at 25 C. Each CD spectrum
was recorded thrice and the average of three scans was considered.
8
.2.4.2.4. Relative viscosity studies. Viscosity experiments were
conducted using an Ostwald viscometer immersed in a water bath and
◦
maintained at 25 C. Viscosity experiments were performed for 6d
compound (15 µM), after mixing them with CT-DNA solution (150 µM).
Before mixing CT-DNA and 6d compound, viscosity measurements were
performed with CT-DNA alone. EtBr and Hoechst 33342 complex with
CT-DNA were considered as control. The DNA solution was prepared in
the assay at 3 M final concentration. The IC50 value was calculated
μ
from the drug concentration required for inhibiting 50% of tubulin as-
sembly compared to control.
1
/3
1
00 mM Tris-HCl (pH 7.0). The graph was drawn by plotting (ŋ/ŋ
versus compound/CT-DNA, where ŋ is the viscosity of CT-DNA in the
is the viscosity of CT-DNA alone.
o
)
8
.2.4.1.2. Immunohistochemistry assay. A549 cells were seeded on
glass cover slips and incubated for 48 h in the presence or absence of 6d
compound and podophyllotoxin at 2 µM concentration. Podophyllotoxin
was reported to show tubulin polymerization inhibition.39 After treat-
ment, the cover slips were fixed with a paraformaldehyde solution (4%
in 1 × PBS) for 20 min at room temperature. Cell permeabilization was
achieved by administration of a Triton X-100 solution (0.2% in 1 × PBS)
presence of 6d compound and ŋ
o
Viscosity values were calculated according to the protocol mentioned by
Tan et al.40
8
.3. Molecular modelling
◦
for 5 min. The cover slips were left in 100% MeOH overnight at 4 C.
The tubulin crystal structure has been taken from the Protein Data
Subsequently, the cover slips were blocked with a 1% bovine serum
Bank (PDB ID: 3E22). The protein preparation tool was used for the
preparation of the tubulin protein. The grid is generated by picking the
active site where the co-crystal is located and grid box of 10 × 10 × 10 Å
albumin (BSA) solution for 60 min and then incubated with -tubulin
α
antibody (1:1000) at room temperature for 2 h. The slides were washed
three times for 5 min each with PBST. Next, the cover slips were incu-
bated with FITC conjugated anti-mouse secondary antibody (Cell
signaling technology) for 1 h and then washed three times with PBST
solution. Finally, the cells were observed under a confocal microscope,
and the pictures were analyzed for the integrity of the microtubule
network.
(
Schr o¨ dinger 2019–1). By using 2D sketcher the potent hybrid 6d was
sketched and energy minimized, ligand preparation was performed for
the generation of different conformers (Schr o¨ dinger 2019–1). The
various conformers thus obtained were subjected to molecular docking
with SP Glide (Schr o¨ dinger 2019–1). The poses generated were assessed
and the best one was reported.
8
.2.4.2. DNA binding studies
Declaration of Competing Interest
8
.2.4.2.1. UV–Vis spectroscopic studies. UV–visible spectroscopy ti-
trations were performed using ABI Lambda 40 UV–Vis spectrophotom-
eter (Foster City, USA) at 25 C using a 1 cm path length quartz cuvette.
◦
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Stock solutions of 1 mM of CT-DNA (calf thymus DNA, which can form
perfect double stranded DNA structure) were prepared in 100 mM Tris-
HCl (pH 7.0). 1 mM stock solution of the synthesized 6d compound was
prepared by dissolving them in 1:1 DMSO: Milli Q water. UV–visible
absorption titrations are performed by adding 10 µM CT-DNA solution in
Acknowledgments
The authors are thankful to Department of Pharmaceuticals (DoP),
1
2

D.K. Sigalapalli et al.
Bioorganic & Medicinal Chemistry 43 (2021) 116277
Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi, for the
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