Bioorganic Chemistry 118 (2022) 105464

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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Design, synthesis and anticancer activity of 2-arylimidazo[1,2-a]

pyridinyl-3-amines
Umesh Prasad Yadav a, e, Arshad J. Ansari b, Sahil Arora c, Gaurav Joshi c, Tashvinder Singh a,
Harsimrat Kaur a, Nilambra Dogra d, Raj Kumar c, *, Santosh Kumar e, *, Devesh M. Sawant b, *,
Sandeep Singh a, *
a
Department of Human Genetics and Molecular Medicine, School of Health Sciences Central University of Punjab, Bathinda 151401, India
b
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer 305817, India
c
Department of Pharmaceutical Sciences and Natural Products, School of Pharmaceutical Sciences, Central University of Punjab, Bathinda 151401, India
d
Centre for Systems Biology & Bioinformatics, Panjab University, Chandigarh 160014, India
e
Department of Biochemistry, All India Institute of Medical Sciences, Patna, India

A R T I C L E I N F O A B S T R A C T

Keywords: A series of imido-heterocycle compounds were designed, synthesized, characterized, and evaluated for the
2-Arylimidazo[1,2-a]pyridinyl-3-amines anticancer potential using breast (MCF-7 and MDA-MB-231), pancreatic (PANC-1), and colon (HCT-116 and HT-
Topoisomerase inhibitors 29) cancer cell lines and normal cells, while normal cells showed no toxicity. Among the screened compounds,
Anticancer
4h exhibited the best anticancer potential with IC50 values ranging from 1 to 5.5 μM. Compound 4h caused G2/M
Western blotting
p53
phase arrest and apoptosis in all the cell lines except MDA-MB-231 mammosphere formation was inhibited. In-
vitro enzyme assay showed selective topoisomerase IIα inhibition by compound 4h, leading to DNA damage as
observed by fluorescent staining. Cell signalling studies showed decreased expression of cell cycle promoting
related proteins while apoptotic proteins were upregulated. Interestingly MDA-MB-231 cells showed only
cytostatic effects upon treatment with compound 4h due to defective p53 status. Toxicity study using over­
expression of dominant-negative mutant p53 in MCF-7 cells (which have wild type functional p53) showed that
anticancer potential of compound 4h is positively correlated with p53 expression.

1. Introduction elevated in different cancers, including breast, gastric, colon, ovarian,

and prostate [10–13].
Cancer, an uncontrolled cellular proliferative disease, is one of the The high expression of Topo IIα in breast cancer cells is positively
major causes of mortality worldwide [1,2]. Multiple chemotherapeutic correlated to tumor size, tumor grade, and metastatic status [14]. Topo
agents targeting different cancer cell pathways are in clinical use, and IIα expression in tumor cells is considered an excellent target for cancer
efforts are ongoing to improve upon almost all of these [3–5]. Among treatment as knocking down Topo IIα in cancer cells prevents cancer
several druggable targets, topoisomerases are the most promising ones, cells proliferation and invasion [11]. Targeted therapies such as
subdivided into two types, topoisomerase 1 (Topo I) and topoisomerase monoclonal antibodies and small-molecule inhibitors are being used to
II (Topo II), both being targets of several current chemotherapeutics. treat different forms of cancer [15–18]. The currently FDA approved
Topo II is further subdivided into two isoforms, Topo IIα and Topo IIβ. Topo IIα inhibitors have several side effects, and thus efforts are ongoing
Topoisomerase is a protein that preserves the topology of DNA mole­ to develop better small molecule inhibitors with minimal side effects
cules by adjusting supercoiling in double strands. It is involved in and optimal inhibitory potential.
transcription, replication, recombination, and DNA repair [3,6–9]. Recently imidazo-heterocycles, especially imidazopyridines, have
Human Topo IIα cleaves the double strand of DNA to assist with DNA gained interest due to their diverse pharmacological activities, including
physiology and overcome DNA tension. The enzyme Topo IIα is highly cancer therapeutics (Fig. 1) [19,20]. Moreover, Nitrogen heterocycles
expressed in proliferating normal cells, and its expression is often contribute with their major share in US FDA-approved drugs [21]. To

* Corresponding authors.
E-mail addresses: raj.khunger@cup.edu.in (R. Kumar), santoshnccs@gmail.com (S. Kumar), dms@curaj.ac.in (D.M. Sawant), sandeepsingh82@cup.edu.in
(S. Singh).

https://doi.org/10.1016/j.bioorg.2021.105464
Received 9 August 2021; Received in revised form 26 October 2021; Accepted 28 October 2021
Available online 1 November 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.
U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

develop better anticancer agents derived from imidazopyridines, we aminoazines, aldehydes, and isocyanides were examined under the
explored imidazo[1,2-a]pyridine scaffold (Compound 5) as Topo IIα optimized conditions 2.5 mol% calcium triflate 160 ◦ C MW (scheme1).
inhibitors reported by Baviskar et al., [20] and designed and synthesized It is noteworthy that the current methodology was not influenced by the
new analogues (Fig. 2; 4a-4n) by varying substitutions at 2, 3, and 7-po­ stereo-electronic factors of the substituents on benzaldehydes with
sitions of compound 5. The preliminary docking of a representative electron-withdrawing substituents bromo, nitro, and electron-donating
compound (4h) into the active site of Topo II (PDB ID- 4R1F) revealed substituent methoxy furnished imidazopyridines in good to excellent
that it occupied the ADP binding site perfectly and groups at 2, 3, and 7- yield (4a-4d). Interestingly, the enolizable aldehyde was well tolerated
positions can be probed to tune the inhibition. to deliver the desired products 4e with remarkable yield. Hetero­
Traditionally imidazopyridines have been synthesized by the aromatic aldehydes produced the desired product 4f with excellent
condensation of 2-aminoazines with α-halo ketones and surrogates isolated yield. Various aliphatic isocyanides also successfully delivered
[22,23]. Recently, isocyanide-based Multi-component reaction with 2- the desired products 4g-4h and 4j (80–90% yield range), expecting to
aminopyridines and aldehydes, known as Groeb­ generate a small library of fused-imidazobi tricyclic heterocycles by
ke–Bienaymé–Blackburn reaction (GBB reaction), provides a viable using hetero-aminoazines diversity. To further investigate the generality
alternative to produce diversified fused imidazole heterocycles [24]. of the reaction, we tried to synthesize pyrazine and thiazole substrate to
Several synthetic strategies for GBB reaction based on Brønsted acids provide the fused-bicyclic heterocycles 4i-4m in good to excellent yield.
[24–31], Lewis acids [24,32–41], ionic liquids [42], solid-supported The optimized reaction condition also furnished the desired fused-tri­
reagents [43–46], organic bases [46], inorganic salts, etc. [47–49] cyclic heterocycle 4n with benzothiazole derivative with exceptional
have been reported recently. Nevertheless, some of these strategies are conversion. It was observed that the methodology was found to be easy,
associated with many limitations in terms of longer reaction time, high solvent-free, general, and convenient for the synthesis of medicinally
catalyst loading, poor yield, expensive solvents, harsher reaction con­ relevant scaffolds.
ditions, etc. Over the past few years, the recent upsurge in using calcium
complexes as Lewis acid catalysts for organic transformation has seen 3. Biological section
upward growth [50]. Low toxicity and cost-effectiveness make Ca-cat­
alysts an effective alternative over heavy metal catalysts [51,52]. 3.1. Antiproliferative effects of compounds (4a-4n)
Herein, we report the solvent-free Ca [53] catalyzed microwave-
assisted synthesis of imidazo[1,2-a]pyridines by GBB reaction and After synthesis and detailed chemical evaluation, all compounds (4a-
their biological evaluation as anticancer agents via multiple mecha­ 4n) were screened against a panel of cancer cell lines, such as different
nisms, including Topo IIα inhibition. phenotypic/genotypic breast carcinoma (non-metastatic cells MCF-7
and metastatic triple-negative cells MBA-MB-231), colorectal carci­
2. Results noma cells (HCT-116 and HT-29), lung carcinoma cells (A549), and
pancreatic carcinoma cells (PANC-1) by performing MTT assay, using
2.1. Chemistry etoposide and camptothecin as positive controls. The result suggested
that compounds (4a-4n) had varying antiproliferative efficacy in
2.1.1. Optimization of reactions conditions different cancer cell lines (Table 2). Among these compounds, 4h has the
We embarked our studies by reacting 2-amino 4-methyl pyridine most potent antiproliferative activity against both MCF-7 and MDA-MB-
(1a), benzaldehyde (2a), and t-butyl isocyanide (3a) in the presence of 231 breast cancer cell lines, with the lowest IC50 values of 1.04 ± 0.06
Ca(OTf)2 (10 mol%) and MeOH at room temperature (Table 1) to furnish μM and 1.08 ± 0.04 μM, respectively, as well as against cell lines A549,
the title compound 4a in 35% isolated yield. The yield was improved to HCT-116, HT-29, and (PANC-1) (Table 2). Interestingly, 4h was found to
80% by heating the reaction mixture at 160 ◦ C (entry 3). Further, mi­ be approximately 15 times more cytotoxic to MCF-7 cells as compared to
crowave irradiation improved the overall yield of 4a to 95% and the reported compound 5 (IC50 = 15 µM)[13]. MTT assay result of
fastened the progress of the reaction, thus being time-saving with compound 4h against normal cell line HEK-293 and Rat cardiac cell line
excellent yield output. Additionally, microwave irradiation allowed the H9C2 exhibited no antiproliferative nor cytotoxicity effects (Fig. 3B).
decrease in catalyst loading to 2.5 mol% without significant yield loss The ability of cancer cells to form the mammosphere is a possible
(entry 7). property of stemness and self-renewal [54]. From day 5 to day 15 of
To investigate the scope of this three-component reaction, various 2- culture, mammosphere culture of MCF-7 and MDA-MB-231 cells with or

N N
N
Cl N
N N
Cl HN
O
O O
N CN
N
Zolpidem Olprinone
Alpidem

N
N
N
N N
O
NH O
N
O S O
O Ph
Necopidem HS-173

Fig. 1. Clinically used imidazopyridine derivatives.

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

N G N
COOH R1
N N
NH NH
5 R2
Cl N
(J Med. Chem, 54(14), 5013-5030) Target compounds 4a-4n
N
N
NH
4h

Overlay of 4h and ADP

Fig. 2. Design of target compounds (4a-4n) as Topo II inhibitors.

DNA, inhibition of its activity leads to greater DNA damage in the cell.
Table1
To assess the functional implication of Topo IIα inhibition by compound
Optimization table for the synthesis of
4h, we sought to measure DNA damage in the treated cells [55]. Double
strand breaks (DSBs) in eukaryotes have been shown to cause serine 139
4a. to be phosphorylated at the carboxy terminus of histone H2A.X,
resulting in the formation of γH2A.X. Immunofluorescence may expose
the frequency of DSBs by detecting γH2A.X phosphorylation. Anti-
Entry Ca(OTf)2 (mol%) Condition Yielda 4a (%) histone γH2A.X antibody was used in an immunocytochemistry assay
1 10 rt, 24 h, MeOH 35
to determine the extent of formation γH2A.X in 4h treated MDA-MB-231
2 10 Neat, 24 h, rt Trace and MCF-7 cells at 0.5 M and 1 M concentrations. The fluorescence
3 10 160 ◦ C, 3 h 80 microscopic result after a 24 h treatment indicates that γH2A.X is
4 10 MW, 160 ◦ C, 5 min 95 detected in both the cell types indicating elevated DNA damage
5 MW, 160 ◦ C, 50 min 77
–
compared to the untreated cells (Fig. 3F).
6 5 MW, 160 ◦ C, 5 min 95
7 2.5 MW, 160 ◦ C, 5 min 93 Furthermore, the double strand-specific DNA breaks were ascer­
a
tained using comet assay, and the result indicated no single-strand DNA
Isolated yield after purification.
damage while double-strand damage was observed in 4h treated cells
(Figure S2; Supplementary material). In order to investigate the binding
without treatment with 1 μM 4h showed that control cells produced a mode of 4h, the molecular docking of 4h was carried out into Topo II
more prominent and greater number of mammospheres in comparison (PDB ID- 4R1F) (Fig. 4A) and compared with standard etoposide
to treated cells (Fig. 3C). Moreover, there was no formation of mam­ (Fig. 4B). The docking of 4h into Topo IIα disclosed that N–N dimethyl
mosphere by both the MCF-7 and MDA-MB-231 cells in a culture treated substituted phenyl ring displayed π-cation interactions with ARG98 and
with 3 μM of 4h (data not shown). Similar to stemness, cell migration is hydrogen bonding with SER149. The cyclohexyl ring of compound 4h
crucial in natural physiological processes and pathological conditions showed hydrogen bonding with ASN91, further linked with magnesium
such as cancer. Cancer cell movement is essential for cancer invasion (MG501) nearby. Also, the chlorine group of 4h molecule showed
and metastatic cascade. Wound healing scratch assay was used to halogen bonding with ALA92.
evaluate the anti-metastatic activity of synthetic 4h. The scratch assay Interestingly, the imidazopyridine nucleus showed conserved inter­
result showed that cells treated with 1 μM of compound 4h showed little action with Walker A motif followed by ARG162, ASN163, GLY164,
or no cell migration at 24 h treatment compared to the untreated cells TYR165, GLY166, and ALA167, account for Topo II inhibition. While
(Fig. 3D). few similar interactions observed with etoposide were seen (Fig. 4B),
like with SER149 and ARG98, the magnesium and Walker A were not in
3.2. Topoisomerase II inhibition and DNA damage induced by compound close proximity compared to 4h. Moreover, the docking score of the
4h compound 4h was found much better (-4.635) than standard etoposide
(-2.848). Furthermore, 4h did not exhibit much convincing binding af­
We used the topoisomerase II assay kit to analyze the impact of finity towards Too I (Figure S3; Supplementary material).
compound 4h on topoisomerase activity. Topo IIα enzymatic reaction
products run in agarose gel-based electrophoresis (non-EtBr gel) along 3.3. Cytostatic and cytotoxic activity of compound 4h
with marker molecule catenated DNA (kDNA) and relax (kDNA). The
band intensity of kDNA varies, as decatenated kDNA has a higher band One of the characteristics of cancer cells is their resistance to
intensity in negative control and a lower band intensity in positive apoptosis signals by inhibiting the host’s anti-tumor immune response.
control of Topo IIα inhibitor, i.e., etoposide. In the 4h treated reaction at Apoptosis assay was used to show the apoptotic activity of 4h against
100 μM, the relaxed de-catenated product has a much lower band in­ tumour cells. The PI/Annexin V apoptotic assay using a flow cytometer
tensity than the positive control. There were multiple bands of kDNA showed that 4h causes apoptosis in MCF-7 cell lines in a concentration-
topology present in the negative control, but such bands are not present dependent manner. In contrast to untreated cells, the late apoptotic cell
in both positive control and 4h (Fig. 3E). Since Topo IIα helps in DNA population is 49.2%, and the early apoptotic cell population is around
relaxation during various events involving uncoiling of double-stranded 21% at a concentration of 3 μM after 24 h (Fig. 5A & C). The viability/

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

N
NH2 2.5 mol% Ca(OTf)2 R1 Ar R1
R1 Ar R2 CHO R3 NC o N
N 160 C, MW,
neat, 5 min HN R3
1 2 3 4

Imidazopyridine
N N R2
N
N N
N
NH NH 4b, R2 = 4-OMe, 91%
4c, R2 = 2-Br, 90% NH
4a, 93% 4e, 91%
4d, R2 = 2-NO2, 90%

N Cl N
N
NO2 N
N N N
N
NH NH NH

4f, 84% 4g, 89% 4h, 90%

Fused-imidazo bi- and tricyclic heterocycles

N N N N
N N
F N
N N
NH HN HN

4i, 87% 4j, 91% 4k, 88%

S
N S N N
N S
N N
N N
NH NH NH

4l, 89% 4m, 92% 4n, 83%

Scheme 1. Scope of three-component reaction to yield.

death assay results on MDA-MB-231 and MCF-7 cells indicate that the in MCF-7 cells.
compound did not trigger apoptosis in MDA-MB-231 cells but did cause We further analyzed the status of various apoptotic markers in MCF-
apoptosis in MCF-7 cells (Fig. 5B). Further, cell cycle analysis of com­ 7 cells upon treatment with compound 4h. Antiapoptotic molecules such
pound 4h on MCF-7 cell line showed that compound induces cell cycle as Bcl-2 and Bcl-xl were found to be decreased, while apoptotic markers
arrest at G2/M phase that may later lead to cell death (Fig. 5D). such as cleaved Poly (ADP-ribose) Polymerase (PARP) were found to be
increased at higher concentrations of 4h at 3 μM and 5 μM. In both 3 μM
3.4. Cell signalling studies reveal p53 dependent function of compound 4h and 5 μM 4h treated MCF-7cells, pro-caspase levels of caspase-9, cas­
pase-7, and caspase-8 decreased. While the extent of cleaved caspase-8
To further understand the impact of the compound on breast cancer and cleaved caspase-9 conversion was increased, the extent of cleaved
cell lines, we performed a western blot analysis of various proteins caspase-8 and cleaved caspase-9 were negligible or undetectable in 4h
involved in signaling pathways regulating the cell cycle and apoptosis. treated MDA-MB-231 cells (Fig. 6B).
After 24 h of treatment with varying concentrations of 1 μM, 3 μM, and The kinetic studies of ERK1 signalling molecules in MDA-MB-231
5 μM, cell cycle regulatory molecules in both MDA-MB-231 and MCF-7 cells treated with 3 μM of 4h at 1 h, 6 h, 9 h, 12 h, and 24 h showed
cells were changed. Both the cell lines showed a decrease in various cell that after treatment, expression of both Phospho-ERK1 and ERK1 sig­
cycle markers (cyclin D3 & D1, CDK 2, 4 & 6, and p21 at all concen­ nalling molecules increased and continued to increase until 24 hr.
trations (Fig. 6A), but the change in protein expression was much more Increased ERK1 signalling activity inhibits the cell cycle in the G2/M
profound in MCF-7 compare to the MDA-MB-231 cells. Moreover, we phase in response to the DNA damage and is independent of p53. Other
analysed the potential of compound 4h to inhibit CDK4 or CDK6, the researchers discovered a similar result in Topo IIα inhibitors [56,57]. In
data indicated that there was no inhibition of kinase activity by the treated cells, ERK1 signalling inhibits cell cycle arrest and induces
compound (data not shown). Interestingly, in MDA-MB-231 cells, the apoptosis in response to latent DNA damage. 4h inhibits cell cycle arrest
compound did not have an impact on the p53 expression, while in MCF- while also suppressing oncogenic signalling molecules such as STAT-3,
7 cells, p53 expression was getting upregulated upto 3 μM concentration pSTAT3, AKT, and P-AKT. Thus, 4h acts as a potent anticancer agent

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

Table 2 by inhibiting cell proliferation and regulating oncogenic signalling, both

IC50 values of synthetics (4a-4n) against a panel of cancer cell lines. of which are required for cancer cell survival and growth (Fig. 6C).
Compounds IC50 value (μM) As shown earlier, the compound was able to efficiently inhibit Topo
IIα activity in both MCF-7 as well as MDA-MB-231 cell lines but was able
MDA-MB- MCF-7 PANC-1 HCT-116 HT-29
231 to induce apoptosis only in MCF-7 cells. Since both the cell lines
represent distinctive stages of breast cancer, MCF-7 as non-metastatic
4a 4.20 ± 5.40 ± 6.55 ± 5.20 ± 6.20 ±
0.06 0.04 0.06 0.08 0.06
while MDA-MB-231 as metastatic triple-negative breast cancer, we
4b 7.39 ± 4.98 ± 6.35 ± 0. 5.89 ± 4.75 ± 0. initially hypothesized that the difference in their behaviour was attrib­
0.07 0.10 03 0.05 09 uted to the different phenotypes. But since results also indicated upre­
4c 8.0 ± 0.12 8.33 ± 0. 6.65 ± 6.35 ± 5.96 ± gulation of p53 in MCF-7 cells, we sought to investigate the possible role
08 0.15 0.16 0.20
of p53 in the differential response of both the cell lines. The hypothesis
4d 7.98 ± 0. 5.93 ± 5.86 ± 6.29 ± 7.37 ±
21 0.12 0.21 0.09 0.07 stems from earlier reports that p53 is mutant in the MDA-MB-231 cell
4e 5.49 ± 6.69 ± 8.04 ± 4.22 ± 7.45 ± line. We transfected MCF-7 cells with mutant p53 vector with or without
0.11 0.13 0.03 0.11 0.06 treatment with compound 4h followed by western blot analysis of key
4f 8.83 ± 4.89 ± 7.0 ± 0.14 5.75 ± 4.5 ± molecules. The results reveal that MCF7 cells treated with 4h compound
0.19 0.15 0.23 0.13
4g 6.0 ± 0.19 6.45 ± 7.84 ± 5.78 ± 4.94 ±
showed higher PARP cleavage (Fig. 6D; lane 3) compared to mutant p53
0.12 0.16 0.19 0.12 overexpressing MCF-7 cells (Fig. 6D; lane 4), indicating p53 has a central
4h 1.08 ± 1.04 ± 5.54 ± 1.20 ± 2.43 ± role in the induction of apoptosis in MCF-7 cells upon treatment with
0.04 0.06 0.10 0.03 0.09 compound 4h. We further transfected MCF-7 cells with mutant p53 or
4i 5.28 ± 7.84 ± 7.22 ± 9.86 ± 8.88 ±
empty vector followed by MTT analysis with or without treatment with
0.06 0.14 0.09 0.17 0.18
4j 8.54 ± 10.44 ± 9.34 ± 7.98 ± 5.33 ± the compound. Our results indicate that MCF-7 cells containing mutant
0.12 0.09 0.11 0.17 0.11 p53 showed lower cell death than empty vector cells (wild type p53)
4k 3.32 ± 3.21 ± 4.85 ± 2.12 ± 3.45 ± upon treatment with compound 4h indicating p53 plays a central role in
0.02 0.04 0.08 0.07 0.06 inducing apoptosis (Fig. 6E). Overall, compound 4h is a very potent
4l 4.45 ± 3.23 ± 5.65 ± 3.60 ± 4.15 ±
0.08 0.10 0.06 0.05 0.03
anticancer compound against various cell lines, inhibits Topo -IIα ac­
4m 7.85 ± 5.65 ± 6.02 ± 5.8 ± 6.4 ± tivity, and can induce p53 mediated cancer cell-specific apoptosis.
0.18 0.12 0.20 0.24 0.17
4n 7.82 ± 9.26 ± 5.78 ± 5.6 ± 5.8 ±
4. Discussion
0.06 0.16 0.12 0.09 0.07
Etoposide 9.1 ± 0.07 3.1 ± 0.07 11.32 ± 2.8 ± 5.75 ±
0.15 0.03 0.11 The initial biological evaluation showed that all the synthetics
Camptothecin 3.95 ± 3.7 ± 0.15 12.15 ± 4.6 ± 3.43 ± showed good cytotoxic potential against various cancer cell lines, but
0.11 0.09 0.07 0.14 compound 4h showed the best activity, especially against cancer cell
lines. Different biological assays showed cell cycle arrest at the G2/M
phase, greater DNA damage and apoptosis upon treatment of MCF-7

Fig. 3. Cytotoxicity assay of compound 4h against (A) cancer cells, as well as (B) primary cell lines (C) Mammosphere assay in MCF-7 and MDA-MB-231 cell lines
(D), Wound healing scratch assay (E) Topoisomerase IIα based in-vitro DNA relaxation assay, (F) DNA damage assay using yH2AX in MCF-7 and MDA-MB-231
cell lines.

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

Fig. 4. Three-dimensional (3D) diagram of the potent molecule (4 h) (A) and etoposide (B) within the binding pocket of topoisomerase IIα (PDB ID: 4R1F).

Fig. 5. PI-Annexin V-based apoptosis assay using (A) flow cytometer as well as (B & C) using confocal imaging, (D) cell cycle analysis using compound 4h.

cells with compound 4h. All these parameters are hallmarks of Topo-II growth and self-renewal of cancer stem cells and can inhibit the for­
inhibition mediated cytotoxic response [58,59]. Further, docking mation of new tumour mass at distant places by cancer stem cells after
studies revealed Topo-II as a potential target for binding the compound, metastasis.
later validated using enzyme assays. Prior studies have shown that Topo IIα inhibitors arrest cells in the
The anticancer potential of 4h further tested against mammosphere G2/M phase of the cell cycle [60–65]. Similarly to this conclusion, the
culture showed that it effectively inhibits mammosphere formation in 4h prolongs the G2/M step of the cell cycle, according to western blot
both MDA-MB-231 and MCF-7 cell lines. The inhibition of mammo­ analysis of cell cycle regulatory molecules CDKs and cyclins. In MDA-
sphere formation is concentration-dependent, with smaller mammo­ MB-231 cells, cyclin D1 and CDK2 levels are higher in treated cells
spheres forming at 1 μM but no mammospheres forming at 3 μM in both than in control cells. In comparison to control, the expression of cell
cell lines. The mammosphere formation ability of cancer stem cells de­ cycle regulatory proteins p21 and p27 was not altered at 1 μM. CDK2
pends on their self-renewal and stemness properties [54]. So, inhibition and cyclin D1 aid the cell cycle transition from G1 to S phase, but not in
of mammosphere formation by 4h shows its potential to resist the p53 mutant MDA-MB-231 cells. CDK2 and cyclin D1 are responsible for

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

Fig. 6. (A) Western blot panel of various cell cycle-related proteins upon treatment with compound 4 h in MCF-7 as well as MDA-MB-231 cell lines. Western blot
analysis of (B) apoptosis-related proteins and (C) important cancer related signaling proteins in MDA-MB-231 cells after treatment with compound 4 h at 3 μM
concentration, (D) western blot analysis of various proteins upon expression of mutant p53 in MCF-7 cells with or without 4h treatment, (E) cytotoxicity assay at
different concentrations of compound 4h after expression of mutant p53 in MCF-7 cells.

the G2/M DNA damage checkpoint response and cell cycle exit in the pathways are impacted by compound 4h treatment to cancer cell lines
G2/M process. CDK2 and cyclin D1 levels in MCF-7 cells are lower than irrespective of p53 status.
control, whereas CDK4 and cyclin D3 levels are comparable to control at Overall, the present study described the synthesis and anticancer
1 μM concentration. In relation to control, the p53 and p21 molecules biological properties of compound 4h.
increase at 1 μM and 3 μM. In MCF-7 cells, the DNA damage response
mediated by the p53 and p21 axis inhibits CDK2 and causes cell cycle 5. Conclusion
arrest and apoptosis [60].
Interestingly biological studies in MCF-7 and MDA-MB-231 breast Thus, we have rationally designed and synthesized a series of 2-
cancer cell lines showed that the compound 4h acts in p53 dependent arylimidazo[1,2-a]pyridinyl-3-amines (4a-4i) through a new method­
manner. Our results indicate that mutant p53 (MDA-MB-231 cell line) ology. Among the series, 4h exhibited the strongest anticancer activity
leads to only cytostatic impact of the compound, while in wild type p53 against different cancer cell lines. Because 4h prevented mammosphere
cell line MCF-7, it is cytotoxic. Overexpression of (dominant-negative) formation, it had an excellent antiproliferative effect against cancer
mutant p53 in MCF-7 cells leads to a 4h induced cytotoxicity decrease. stem cells. 4h impaired Topo IIα functioning activity, as evidenced by in-
Protein p53 is probably the most studied human protein, having been vitro enzyme assay, computation findings (molecular docking) and
implicated in various cellular and physiological processes, including induced DNA damage in treated cells. In cancer cells, 4h prolonged the
development, growth and apoptosis [66–68]. Due to its involvement in G2/M phase of the cell cycle and reduced the cellular proliferation and
DNA damage detection and repair and regulation of apoptosis and survival signalling cascades while activating apoptotic signaling. 4h
cellular differentiation, it is often implicated as the tumor suppressor induced p53-mediated cellular cytotoxicity, as it promoted cytotoxicity
protein [69,70]. Researchers have reported direct interaction and in MCF-7 (p53 wild) cells and cytostatic action in MDA-MB-231 (p53
elevated catalytic activity of Topo IIα upon interaction with wild type mutant) and mutant p53 overexpressing MCF-7 cells. The overall data
p53, though mutant p53 may or may not exert a similar impact on its indicated that functional p53 is required for anticancer effect of 4h,
activity. p53 status also determines the effectiveness of anticancer drugs though it impacts several other signalling pathways independent of p53
in-vitro where treatments are given for shorter durations, while this as well. Drugs are dependent on cellular machinery for exerting their
impact is usually not observed in-vivo where anticancer chemotherapies impact on individual cell, thus understanding of exact cellular pathways
are usually administered for longer durations [71] where researchers by a compound is of utmost important. In this context, the present study
have shown that cancer cells die of mitotic catastrophe rather than highlights p53 dependent anticancer response of 4h, highlighting
apoptosis [72]. The signal transduction data show that expression of importance of multi-directional investigations during drug character­
Cyclins and CDKs is downregulated in both the cell lines, albeit more in ization. Understanding the involvement of various major proteins in
MCF-7 cells. The plausible reason of the profound effect on MCF-7 cells eliciting anticancer response of a drug adds to the existing pharmaco-
is due to the presence of wild type p53, which is known to regulate the genetics as well.
expression of cyclin and CDKs, still, it can be deduced that multiple

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U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

6. Experimental section 7.2. Measurement of cell growth by MTT assay

6.1. Chemistry Cells were trypsinized and plated in 96-well plates with 5000 cells
per well. After 24 h, serum-free media was added to synchronize the
All the reactions were carried out in a Microwave vial using standard cells in a single phase. After 6–8 h, cells were treated with 4h (working
techniques. All solvents used in synthesis were purchased from Spec­ concentration dissolved in DMEM complete media) at varying concen­
trochem. Aliphatic isocyanates were purchased from sigma; other re­ trations for different periods. 100 µl MTT (0.5 mg/ml) was added to each
agents were purchased from Aldrich or Spectrochem used without well after the stipulated time intervals were met, and the wells were
purification. Analytical TLC was performed using 2 × 4 cm plated coated incubated for 3 h in a CO2 incubator to enable formazan formation. The
with 0.25 mm thickness of silica gel (60F-254 Merck), and visualization formazan was dissolved in DMSO, and the colour intensity was
was accomplished with UV light or I2/ KMnO4 staining. 1H and 13C NMR measured at 570 nm after 30 min of incubation on the rocker. The IC50
spectra were obtained from Bruker’s Ascend 500 MHz spectrophotom­ value of the compound was estimated and analyzed using statistical and
eter operating at 500.3 MHz for 1H and 125.8 MHz for 13C experiments. graphical methods [73].
The chemical Shifts are reported in ppm scale with respect to CDCl3
(7.269 ppm) for 1H and (77.00 ppm) for 13C NMR as internal standard. 7.3. Migration assay

Cells were grown in six-well plates and treated with various con­
6.2. General procedure for the synthesis of compounds 4a-4n centrations for 24 h after cells reaching 70% confluence. Treatment was
given at the previous concentration, and the cells were incubated for a
To a standard microwave vial (10 mL), the specified amount of fixed period of time. After incubation, a sterile 200 µl pipette tip was
substituted amine (1), substituted aldehyde (2), substituted isonitrile used to finish the scratch and gently wash the cell to remove debris. At
(3), and Ca(OTf)2 was taken and heated under microwave irradiation (at 0 and 24 h, images of the wound area were taken using a (Nikon Eclipse
160 ◦ C, 40 W) for specified time duration mentioned at Table 1 and Ti) microscope.
Schemes 1. The reaction mixture was then cooled to room temperature
and triturated with n-pentane (5 mL) to obtain solid. The solid was then 7.4. Mamo-Sphere growth assay
washed with deionized water, dried, and used as such (without any type
of purification) for analytical characterization. The physical data of all Polyhydroxyethylmethacrylate (Poly-HEMA/pHEMA) was coated to
compounds 4a-g, 4i-k, 4m, and 4n were in accordance with previously multiwell plates and dried in a CO2 incubator at 37 0C. The cells were
reported data [24] disclosed by our group. The physical data of un­ trypsinized and suspended in stem cell media containing cortisone,
known compounds are reported below: heparin, and a proliferation supplement. In each of the Multiwell plates,
7-chloro-N-cyclohexyl-2-(4-(dimethylamino)phenyl)imidazo 15000–20000 cells/ml were plated, and cells were allowed to form a
[1,2-a]pyridin-3-amine (4h) Light green solid, yield: (90%); 1H NMR mammosphere. After 15 days, treatment with 4h was subjected to 3D
(500 MHz, CDCl3) δ: 8.03 (d, J = 7.0 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), cell mesh at various concentrations, and later the development of
7.55 (s, 1H), 6.80 (d, J = 8.3 Hz, 2H), 6.76 – 6.70 (m, 1H), 2.98 (d, J = mammosphere was observed under the microscope (Nikon Eclipse Ti).
31.3 Hz, 7H), 2.11 (s, 1H), 1.79 (d, J = 10.1 Hz, 2H), 1.68 (s, 2H), 1.58
(s, 1H), 1.25 – 1.10 (m, 4H). 13C NMR (126 MHz, CDCl3) δ: 149.8, 7.5. Viability/Cytotoxic assay
140.6, 130.1, 127.9, 123.7, 122.9, 121.7, 115.4, 112.9, 112.3, 56.9,
40.4, 34.1, 25.7, 24.8. HRMS (EI) calcd for C21H26ClN4 (M + H+): Cells were seeded in 30 mm dishes and given different doses of 4h for
369.1846. 24 h. Cells were washed in PBS after a specific period of drug incubation
time. Again, the cells were suspended in PBS/media, and 500 µl of
Calcein AM and 500 µl of EthD-1 were added from the supplied kit
6.3. N-cyclohexyl-2-(4-(dimethylamino)phenyl)imidazo[1,2-a]pyridin- (Invitrogen Viability/Cytotoxic kit). Cells were incubated in the dark for
3-amine (4l) 30 min before being examined under a fluorescence microscope for
viability and cytotoxicity (Nikon Eclipse Ti). Apoptotic cells retain EtBr
Green solid, yield: (94%); 1H NMR (500 MHz, CDCl3) δ: 8.96 (d, J = and emit red fluorescent light, while live cells containing Calcein AM
1.1 Hz, 1H), 8.03 – 7.98 (m, 1H), 7.93 – 7.88 (m, 2H), 7.84 (d, J = 4.6 emit green fluorescent light [73].
Hz, 1H), 6.81 (d, J = 8.9 Hz, 2H), 3.03 (s, 7H), 2.11 (s, 2H), 1.82 (d, J =
12.1 Hz, 2H), 1.74 – 1.66 (m, 2H), 1.61 – 1.55 (m, 1H), 1.24 (dd, J = 7.6. Cell cycle assay
22.0, 11.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ: 175.3, 150.6, 142.1,
140.9, 139.9, 136.5, 133.6, 133.2, 128.5, 128.2, 125.3, 121.0, 115.5, After reaching 70% confluence, cells were grown in 35 mm dishes for
112.2, 56.8, 40.4, 34.2, 25.6, 24.8, 21.1. HRMS (EI) calcd for C20H26N5 22–24 h and synchronized the cells after adding serum-free media for
(M + H+): 366.2188. 10–12 h. 4h was added to cells at various concentrations and incubated
for a stipulated period. The cells were then trypsinized, washed in cold
7. Biology PBS, and fixed in 70% ethanol for 3–4 h at 4 ◦ C. The fixed cells were
centrifuged and washed in cold PBS before being resuspended in (1X)
7.1. Cell culture PBS (106 cells per ml) and treated with RNase for 20–30 min before
being stained with Propidium Iodide (Sigma) and incubated for another
All the cell lines were obtained from NCCS Pune National Cell Re­ 30 min in the dark. BD Accuri C6 flow cytometer was used for aspiration,
pository and cultured in DMEM media supplemented with 10% Fetal and BD Accuri software was used for cell cycle analysis [73].
Bovine Serum and 1X penicillin–streptomycin antibiotic solution. Inside
the CO2 incubator, cell growth was maintained at 37 ◦ C under normal 7.7. Apoptosis assay
conditions of 5% CO2 and 95% humidity. After the cells had reached
70%-75% confluency, they were trypsinized. Cells were stored at − 80 ◦ C After reaching 70% confluence, cells were grown in 35 mm dishes for
for long term cryopreservation, and they were thawed before setting up 22–24 h and synchronized the cells after adding serum-free media for
the experiment. After thawing, the cells were seeded after 3–4 passages, 10–12 h. 4h was added to cells at various concentrations and incubated
and trypsinization was done normally at 70 percent confluency [73]. for a stipulated period. The cells were then trypsinized, washed in cold

8
U.P. Yadav et al. Bioorganic Chemistry 118 (2022) 105464

PBS, and fixed in 70% ethanol for 3–4 h at 4 ◦ C. The cells were sus­ drawn using ChemDraw Professional. The geometries of all the ligands
pended in 1X Annexin binding buffer and processed according to the were subsequently optimized and prepared by OPLS3 force field into the
catalogue instructions (Invitrogen). The cells were incubated in the dark 3D form using the LIGPREP module. The ligands were docked at the
for 30 min with Propidium Iodide and Annexin solutions. The cells were active site of topoisomerase II using the GLIDE module of Schrodinger
kept on ice while the samples were processed using a BD Accuri C6 flow software. GLIDE generates the results in the form of glide-dock using the
cytometer, and live, apoptotic, and dead cells were analyzed [73]. Lamarckian genetic algorithm, resulting in different conformations of a
ligand and an extra precision method. On the basis of dock score, the
7.8. Immunocytochemistry assay inhibitors were ranked accordingly with respect to their binding affinity
[74].
Untreated control cells and cells treated with different concentra­
tions of 4h for a set period of time were washed in cold PBS, then fixed in Declaration of Competing Interest
2% formaldehyde, and further membranes were permeabilized in 1%
Triton X-100. The primary antibody (CST) was incubated for 2 h after The authors declare that they have no known competing financial
blocking in 5% FBS. After washing the cells with PBS, they were incu­ interests or personal relationships that could have appeared to influence
bated with a secondary antibody that was fluorochrome FITC conju­ the work reported in this paper.
gated. Washed the sample again and mounted it in DAPI-containing
mounting media, with the coverslips sealed with nail polish. Fluores­
Acknowledgements
cence was observed under fluorescence microscopy.
UPY and TS are thankful to CSIR for SRF and JRF fellowships,
7.9. Topoisomerase II assay
respectively. UPY, TS, GJ, HK, RK and SS are thankful to the Professor R.
P. Tiwari, Vice Chancellor, Central University of Punjab. AJA and DMS
The activity was carried out in a 20 µl volume with kDNA as the
are thankful to the Central University of Rajasthan. The authors
substrate. As kDNA was incubated with human Topoisomerase II, it
acknowledge DST-SERB (EMR/2016/008016) to DMS, CSIR for Senior
forms decatenated kDNA. Etoposide (a Topo II inhibitor) was used as a
Research Associate grant (SB/S2/RJN-103/2015) to ND, DST-SERB
control, and 4h was used at a concentration of 100 µM. The kDNA does
Ramanujan fellowship (SB/S2/RJN-103/2015) to SK and DST-SERB
not migrate in non-EtBr agarose gel electrophoresis, but concatenated
(SR/SO/AS-31/2014) and DST-FIST [SR/FST/LSI-I/2017/49(C)] to SS,
kDNA does [8].
CSIR-Senior Research Associate- 13 (9027-A) to ND.

7.10. Transfection of MCF-7 with mutant P53

Appendix A. Supplementary data
After reaching 70% confluency, cells were transfected with mutant
Supplementary data to this article can be found online at https://doi.
p53, and empty vector using lipofectamine (Invitrogen) according to the
org/10.1016/j.bioorg.2021.105464.
instructions in the catalogue. 2 μg of the plasmid is placed in a micro­
centrifuge tube with 150 μl of media, while 12 μl of lipofectamine is
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