Synthesis and biological evaluation of curcumin inspired imidazo[1,2-a]pyridine analogues as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.11.010

With an aim to develop new curcumin inspired analogues as potent anticancer agents, we synthesized a series of (1E,4E)-1-phenyl-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-3-ones (12a–t) as tubulin polymerization inhibitors. An initial screening was carried out to evaluate their cytotoxic potential on a panel of six cancer cell lines namely, cervical (HeLa), gastric (HGC-27), lung (NCI-H460), prostate (DU-145 and PC-3) and breast (4T1), using MTT assay. Among the compounds tested, compounds 12e, 12r and 12t showed potent growth inhibition and 12t {(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-one} being the most active member of the series inhibited the growth of all the tested cell lines with IC₅₀ values varying from 1.7 – 2.97 μM. Moreover, 12t showed promising cytotoxicity on PC-3, HGC-27 and HeLa cell lines with IC₅₀ values of 2.11 ± 0.27 μM, 2.21 ± 0.25 μM and 2.53 ± 0.01 μM respectively. The results from aqueous solubility test showed that compounds 12e and 12t have 1.7 and 2.8 times more aqueous solubility than curcumin. Interestingly, the most active compound 12t was found to be nearly 2 times more selective on PC-3 cells as well as safe on normal human prostate (RWPE-1) cells. In addition, compound 12t efficiently inhibited tubulin polymerization with IC₅₀ value of 8.44 ± 0.13 μM and molecular modelling studies disclosed that 12t binds at the colchicine binding site of the tubulin. Cell cycle analysis revealed that 12t arrests PC-3 cells in G2/M phase in a dose dependant manner. Further, treatment of PC-3 cells with 12t showed typical apoptotic morphology, also led to the impairment of mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS). Altogether, the results from acridine orange/ethidium bromide (AO-EB) and DAPI staining studies, annexin V-FITC/propidium iodide staining assay, analysis of mitochondrial membrane potential (DΨm) and reactive oxygen species (ROS) levels undoubtedly demonstrated the induction of apoptosis in PC-3 cells by compound 12t.

Full text of DOI:10.1016/j.ejmech.2017.11.010

Accepted Manuscript
Synthesis and biological evaluation of curcumin inspired imidazo[1,2-a]pyridine
analogues as tubulin polymerization inhibitors
P.V.Sri Ramya, Lalita Guntuku, Srinivas Angapelly, Chander Singh Digwal, Uppu
Jaya Lakshmi, Dilep Kumar Sigalapalli, Bathini Nagendra Babu, V.G.M. Naidu,
Ahmed Kamal
PII:
S0223-5234(17)30895-4
10.1016/j.ejmech.2017.11.010
EJMECH 9885
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 October 2017
Revised Date: 3 November 2017
Accepted Date: 3 November 2017
Please cite this article as: P.V.S. Ramya, L. Guntuku, S. Angapelly, C.S. Digwal, U.J. Lakshmi, D.K.
Sigalapalli, B.N. Babu, V.G.M. Naidu, A. Kamal, Synthesis and biological evaluation of curcumin inspired
imidazo[1,2-a]pyridine analogues as tubulin polymerization inhibitors, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.010.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and biological evaluation of curcumin inspired imidazo[1,2-
a]pyridine analogues as tubulin polymerization inhibitors
P. V. Sri Ramya,a Lalita Guntuku,b Srinivas Angapelly,a Chander Singh Digwal,a Uppu Jaya
Lakshmi,b Dilep Kumar Sigalapalli,a Bathini Nagendra Babu,a V.G.M. Naidu,b Ahmed
Kamala*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500037, India.
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500037, India.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of curcumin inspired imidazo[1,2-
a]pyridine analogues as tubulin polymerization inhibitors
P. V. Sri Ramya,a Lalita Guntuku,b Srinivas Angapelly,a Chander Singh Digwal,a Uppu Jaya
Lakshmi,b Dilep Kumar Sigalapalli,a Bathini Nagendra Babu,a V.G.M. Naidu,b Ahmed Kamala*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500037, India.
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad 500037, India.
Abstract
With an aim to develop new curcumin inspired analogues as potent anticancer agents, we
synthesized a series of (1E,4E)-1-phenyl-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-
3-ones (12a‒t) as tubulin polymerization inhibitors. An initial screening was carried out to
evaluate their cytotoxic potential on a panel of six cancer cell lines namely, cervical (HeLa),
gastric (HGC-27), lung (NCI-H460), prostate (DU-145 and PC-3) and breast (4T1), using MTT
assay. Among the compounds tested, compounds 12e, 12r and 12t showed potent growth
inhibition and 12t {(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4,6-
trimethoxyphenyl)penta-1,4-dien-3-one} being the most active member of the series inhibited the
growth of all the tested cell lines with IC50 values varying from 1.7 – 2.97 µM. Moreover, 12t
showed promising cytotoxicity on PC-3, HGC-27 and HeLa cell lines with IC50 values of 2.11 ±
0.27 µM, 2.21 ± 0.25 µM and 2.53 ± 0.01 µM respectively. The results from aqueous solubility
test showed that compounds 12e and 12t have 1.7 and 2.8 times more aqueous solubility than
curcumin. Interestingly, the most active compound 12t was found to be nearly 2 times more
selective on PC-3 cells as well as safe on normal human prostate (RWPE-1) cells. In addition,
compound 12t efficiently inhibited tubulin polymerization with IC50 value of 8.44 ± 0.13 µM
and molecular modelling studies disclosed that 12t binds at the colchicine binding site of the
tubulin. Cell cycle analysis revealed that 12t arrests PC-3 cells in G2/M phase in a dose
dependant manner. Further, treatment of PC-3 cells with 12t showed typical apoptotic
morphology, also led to the impairment of mitochondrial membrane potential (DΨm) and
increased levels of reactive oxygen species (ROS). Altogether, the results from acridine

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orange/ethidium bromide (AO-EB) and DAPI staining studies, annexin V-FITC/propidium
iodide staining assay, analysis of mitochondrial membrane potential (DΨm) and reactive oxygen
species (ROS) levels undoubtedly demonstrated the induction of apoptosis in PC-3 cells by
compound 12t.
Keywords: Curcumin inspired analogues, imidazo[1,2-a]pyridine, cytotoxic, apoptosis, Claisen-
Schmidt condensation.
*Corresponding author: Tel.: +91 40 27193157; fax: +91 40 27193189. E-mail:
ahmedkamal@iict.res.in
1. Introduction
Natural products have traditionally been a magnificent source of novel medicinal leads, and their
contribution in drug discovery is uniquely pronounced in the domain of cancer pharmacology,
where the fraction of the drugs derived from natural products amounts to 60% [1]. Curcumin
(1,7-bis(4-hydroxy 3-methoxy phenyl)-1,6-heptadione-3,5-dione or diferuloylmethane) (Figure
1, 1) is one of such natural products, obtained from the rhizomes of the plant Turmeric (Curcuma
longa) [2]. Curcumin was found to be a highly pleiotropic molecule that interacts with a diverse
variety of molecular targets and impede with multiple cell signaling pathways, including
proliferation (HER-2, AP-1 and EGFR), angiogenesis (VEGF), apoptosis (activation of caspases
and down regulation of antiapoptotic gene products), and inflammation (NF-kB, TNF, IL-1, IL-
6, COX-2, and 5-LOX) [3-12]. Therefore it possess a diverse range of biological activities such
as anti-carcinogenic [13], antioxidant [14], antitumor [15], anti-angiogenesis [16], anti-
inflammatory [17], antithrombotic [18], antihepatotoxic [19], hypoglycemic [20] and
antihyperlipidaemic [21]. Additionally, numerous studies have shown that curcumin has
beneficial effects in the treatment of myocardial infarction, Alzheimer's disease, rheumatoid
arthritis, human immunodeficiency virus (HIV) syndrome and psoriasis [22]. Further, curcumin
is non-toxic even at high dosages and has been categorized as ‘generally recognized as safe’
(GRAS) by the National Cancer Institute [23,24]. These multiple biological properties attracted
clinicians to investigate the therapeutic potential of curcumin and nearly 65 clinical trials are
being carried out worldwide in order to accelerate this lead molecule from kitchen to clinic
[21,25,26]. Despite of the broad spectrum activities and safety profile, it is not yet approved as a

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therapeutic agent because of its poor oral bioavailability, poor water and plasma solubility as
noticeable from the data of pharmacokinetic and pharmacodynamics studies [23,24]. Hence,
there have been significant efforts to synthesize analogues of curcumin with a similar safety
profile, besides increased activity and improved oral bioavailability and the process is quite on
the way of progress, with a huge number of successes.
Curcumin has substantially been used as a lead compound to design and synthesize analogs with
improved activity. The reported studies focused mainly on modifications in the β-diketone
structure and aryl substitution pattern of curcumin. As evident from the reported studies, the β-
diketone moiety of curcumin seems to be a specific substrate of a series of aldo-keto reductases
and can be metabolised rapidly in vivo [27,28] whereas monoketone analogs generally have
improved pharmacokinetic profiles over curcumin. Moreover, monoketone analogues (Figure 1,
curcumin analogues 2-7) containing 5/6 membered heterocycles such as quinazoline [29],
thiazole [30], imidazole [31], chromone [32] and indole [33] moieties have been well studied in
the literature for their improved cytotoxic potency than curcumin. Thus, the 1,5-
diaryl/diheteroaryl/arylheteroaryl penta-1,4-dien-3-one moiety portrayed by these compounds,
can be considered as an optimal scaffold for developing novel curcumin mimics/analogues as
potent anticancer agents. It is hence worthwhile to instigate extensive investigation of this
category of curcumin analogues as potential cytotoxic agents.
<Insert Figure 1 here>
On the contrary, imidazopyridine is one of the important fused bicyclic 5-6 heterocycles, with a
wide range of biological activities such as antitumor, anti-apoptotic, hypnoselective,
antibacterial, antifungal, antiviral, antiprotozoal and anti-inflammatory. Furthermore,
imidazo[1,2-a]pyridine is regarded as a “drug prejudice” moiety because of its unavoidable
occurrence in numerous clinical drugs that includes zolpidem, saripidem, olprinone, zolimidine,
rifaximin and drug candidates such as ND-09759 and Q203, which are at present in pre-clinical
and phase I clinical development respectively for tuberculosis (Figure 2) [34,35]. In recent
years, imidazopyridine/pyrimidine chalcones [36], 2-phenyl-imidazo[1,2-a]pyridine analogues
(tubulin polymerization inhibitors) [37], sulfonylhydrazone-substituted imidazo[1,2-a]pyridines
(PI3 kinase p110α inhibitors) [38] and the other imidazo-[1,2-a]pyridine derivatives acting on

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Nek2 [39] and c-Met kinases [40] (Figure 2) have been extensively studied in the literature for
their excellent antitumor activity.
<Insert Figure 2 here>
Inspired by the synthetic feasibility and biological activities of imidazo[1,2-a]pyridine
derivatives, it would thus be considering beneficial to synthesize curcumin inspired imidazo[1,2-
a]pyridine analogues, possessing a five-carbon dienone linear linker attached to substituted
imidazo[1,2-a]pyridine and phenyl motifs on either sides of it and evaluate their anticancer
activity (Figure 3). In line with our interest in the development of novel curcumin inspired
analogues [33,41], herein we wish to report curcumin inspired imidazo[1,2-a]pyridine
derivatives as tubulin polymerization inhibitors.
<Insert Figure 3 here>
2. Results and discussion
2.1. Chemistry
The target compounds (1E,4E)-1-phenyl-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-
3-ones (12a‒t) were synthesized by employing Claisen-Schmidt condensation between (i) 3-
substituted imidazo[1,2-a]pyridine-2-carbaldehyde (11), and (ii) a substituted chalcone (3) as
depicted in Scheme 1. Initially, chalcones (3) were prepared by NaOH-catalysed Claisen-
Schmidt condensation of appropriately substituted benzaldehydes (1) with acetone [42] (a,
Scheme 1). The next core structural element, aldehyde (11) (b, Scheme 1) was synthesized by
following a 5-step scheme, starting from 2-amino pyridine and ethylbromopyruvate to afford
ester intermediate (6) [43]. Later, it was iodinated with N-iodosuccinimide [44] to form
intermediate 7 and in the next step, it underwent Suzuki coupling with different phenyl boronic
acids in the presence of Pd(PPh3)4/Na2CO3 [45] to give intermediate 9. Further, it was reduced to
the corresponding alcohol (10) by using LiAlH4 [43] and then oxidized to the respective
aldehyde (11) in the presence of Dess-Martin periodinane [46]. Finally, CH3ONa-catalysed
Claisen-Schmidt condensation of chalcone (3) and aldehyde (11) furnished the target compounds
12a‒t in moderate to very good yields (c, Scheme 1) [47].
<Insert Scheme 1 here>

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All the synthesized compounds (12a‒t) were unambiguously characterized by using FT-IR,
HRMS, 1H and 13C NMR spectroscopy. The 1H NMR spectrum of 12a showed a sharp singlet of
methoxy protons at δ 3.85 and rest all protons appeared in the range of δ 8.10 ‒ 6.78. In the 13C
NMR spectrum of 12a, the carbonyl and methoxy carbons appeared at δ 189.0 and 55.4
respectively and the remaining carbons appeared in the range of δ 161.5 ‒ 112.8. Nearly similar
1
patterns were noticed in H and 13C NMR spectra of all the compounds (12a-t). The FT-IR
spectrum of 12a showed a sharp band at 1651.8 cm-1 which confirmed the presence of ketone
functionality. The HRMS (ESI) of 12a‒t showed characteristic [M+H]+ corresponding peaks
equivalent to their molecular formulae.
2.2. Biological evaluation
2.2.1. In vitro anticancer activity
The newly synthesized curcumin inspired imidazo[1,2-a]pyridine analogues (12a‒t) were
screened for their in vitro cytotoxicity against selected cancer cell lines viz. cervical (HeLa),
gastric (HGC-27), lung (NCI-H460), prostate (DU-145 and PC-3), breast (4T1) and one normal
human prostate epithelial cell (RWPE-1) using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay [48]. The IC50 (µM) values (concentration required to
cause 50% inhibition of the viability of cancer cells) of test compounds 12a‒t and the standard,
curcumin and vincristine sulphate are shown in Table 1.
In order to understand the structure-activity relationship (SAR), we used diversified substitutions
on the phenyl ring of the chalcone as well as the phenyl ring attached to the imidazo[1,2-
a]pyridine moiety of these curcumin inspired analogues. From the preliminary results, it is
evident that some of the synthesized compounds exhibited moderate to potent cytotoxicity
against the tested cancer cell lines with IC50 values in the range of 1.67 ± 0.34 to 10.27 ± 0.25
µM. From the close analysis of Table 1, it can be observed that compounds 12c, 12e, 12n, 12o,
12q, 12r, 12s and 12t were the most active members of this series and the remaining compounds
were almost inactive against the tested cell lines. Compounds 12a, 12b, 12d and 12f carrying 3-
phenylimidazo[1,2-a]pyridine moiety and 4-methoxy/2,4-dimethoxy/3,4-dimethoxy/2,4,6-
trimethoxy substitutions respectively on the phenyl ring of the chalcone were found to be
inactive on the cell lines screened (IC50 values of > 10 µM), whereas 12c and 12e possessing 2,5-

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dimethoxy and 2,4,5-trimethoxy substitutions respectively exhibited potent cytotoxicity on
nearly all the cell lines examined with IC50 values ranging from 1.67 – 7.08 µM. Surprisingly,
compounds 12g, 12h, 12i and 12j which contain 3-(3-methoxy)phenylimidazo[1,2-a]pyridine
moiety and 4-methoxy/2,4-dimethoxy/2,5-dimethoxy/3,4-dimethoxy substitutions respectively,
did not show significant cytotoxicity at 10 µM. Similarly, compounds 12k, 12l and 12m having
3-(4-methoxy)phenylimidazo[1,2-a]pyridine
moiety
and
4-methoxy/2,4-dimethoxy/2,5-
dimethoxy substitutions respectively on the phenyl ring of the chalcone were found to be
inactive. Interestingly, compounds 12n and 12o containing 3-(4-methoxy)phenylimidazo[1,2-
a]pyridine moiety and 3,4-dimethoxy or 2,4,5-trimethoxy substitutions respectively on the
phenyl ring of the chalcone displayed considerable cytotoxicity with IC50 values ranging from
3.64 – 9.41 µM. Compound 12p with 3-(3,4-difluoro)phenylimidazo[1,2-a]pyridine moiety and
4-methoxy substitution on the chalcone showed IC50 of > 10 µM, while 12q with 2,4-dimethoxy
group on the chalcone showed cytotoxicity in the range of 8.41 – 10.27 µM. Importantly, 12r,
12s and 12t holding 3-(3,4-difluoro)phenylimidazo[1,2-a]pyridine moiety and 2,5-
dimethoxy/3,4-dimethoxy/2,4,6-trimethoxy substitutions respectively on the phenyl ring of the
chalcone displayed potent cytotoxicity on all the cell lines screened with IC50 varying from 1.71
– 6.35 µM. Compound 12r was active on all cell lines tested, especially PC-3 with IC50 of 2.84 ±
0.58 µM. Further, compound 12t, the most active member of the series exhibited remarkable
cytotoxicity on HeLa (2.53 ± 0.01 µM), HGC-27 (2.21 ± 0.25 µM), NCI-H460 (2.63 ± 0.05 µM),
DU145 (2.97 ± 0.08 µM), PC-3 (2.11 ± 0.27 µM) and 4T1 (1.71 ± 0.11 µM) cell lines.
<Insert Table 1 here>
The impact of substitution on chalcones and imidazo[1,2-a]pyridine is interesting and the
following conclusions have been drawn from the structure-activity relationship (SAR) analysis
(Figure 4) of these compounds:
a.
From the IC50 values, in general, it can be noted that the presence of electron donating (3-
methoxy or 4-methoxy) groups on the phenyl ring attached to imidazo[1,2-a]pyridine did not
produce the bioactive compounds (12g‒m) with the exception for compounds 12n and 12o.
b.
Compounds without any substitution on the phenyl ring linked to imidazo[1,2-a]pyridine
and with mono or di or trimethoxy substitutions on the chalcone (12a, 12b, 12d and 12f) were

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nearly inactive at 10 µM, while compounds with 2,5-dimethoxy (12c) or 2,4,5-trimethoxy (12e)
substitutions showed significant cytotoxicity.
c.
In general, compounds with electron withdrawing (fluoro) groups on the phenyl ring
attached to imidazo[1,2-a]pyridine (12q‒t) were more potent, compound 12p being the
exceptional case, than compounds having unsubstituted phenyl or phenyl ring with electron
donating groups (methoxy): the order may be electron withdrawing > neutral > electron donating.
d.
Additionally, most of the compounds containing dimethoxy or trimethoxy group on the
chalcone showed enhanced cytotoxicity than mono methoxy compounds.
<Insert Figure 4 here>
By comparing the IC50 values of compounds with that of curcumin, 12c, 12e, 12n‒o, 12q‒t were
considered as more potent than curcumin (Table 2). They are 1.8 to 6.5 times, 3.9 to 11.1 times,
and 2.1 to 8.9 times, respectively, more potent than curcumin on human lung cancer cell line
(NCI-H460) and two human prostate cancer cell lines (DU145 and PC-3). Further, the most
active compound 12t is 6.5 times, 11.1 times and 8.9 times more cytotoxic on NCI-H460, DU-
145 and PC-3 cell lines, respectively, than curcumin.
<Insert Table 2 here>
The aqueous solubility of the most active curcumin inspired analogues 12e and 12t was
measured in comparison to curcumin by making their saturated aqueous solutions in milli-Q
water [49]. The amount of compound solubilized was determined spectrophotometrically by
scanning at 200 ‒ 600 nm. Figure 5 shows the UV-visible spectra recorded for compounds 12e
and 12t. The results demonstrate that 12e and 12t have 1.7 and 2.8 times more aqueous solubility
than curcumin.
<Insert Figure 5 here>
To find out the selectivity towards cancer cells, the most active compound 12t was tested on
normal human prostate epithelial cell line (RWPE-1). Interestingly, compound 12t was found to
be almost 2 times more selective for PC-3 cells compared to normal RWPE-1 cells. The
promising cytotoxicity of compound 12t on PC-3 cells motivated us to investigate its effects at
cellular level.

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2.2.2. Effect of compound 12t on tubulin polymerization
Tubulin is the principal component of the eukaryotic cytoskeleton and function in several
crucial cellular processes including cell replication. As evident from the literature, chalcones
shows cytotoxic activity through the inhibition of tubulin polymerization, hence impede with
microtubule formation [50]. Moreover, chalcones and curcumin analogues have a great structural
resemblance, thus, to investigate the correlation between cytotoxicity of these compounds and
tubulin, compound 12t was examined for its effects on tubulin polymerization in a cell-free in
vitro assay and nocodazole was chosen as the positive control. Initially, 12t was screened at 10
µM and % inhibition was found to be 51.8%. As depicted in Figure 6, 12t efficiently inhibited
tubulin polymerization in a dose-dependent manner with IC50 value of 8.44 ± 0.13 µM. These
results stipulate that the indicative molecular target of these curcumin analogues might be
tubulin.
<Insert Figure 6 here>
2.2.3. Cell cycle analysis
Most of the cytotoxic compounds elicit their growth inhibitory effect either by arresting the cell
cycle at a specific checkpoint or by inducing apoptosis [51]. In vitro screening results disclosed
that compound 12t showed significant cytotoxicity against PC-3 cells. Therefore, we examined
the effect of compound 12t on cell cycle progression of PC-3 cells by using flow cytometry
analysis [52]. The PC-3 cells were treated with 1 µM and 2 µM of compound 12t for 24 h and
stained with propidium iodide. The results from Figure 7 clearly shows that the ratio of PC-3
cells in G2/M phase was increased from 33.1% in control (DMSO) to 44.6% at 1 µM and 55.8%
at 2 µM of 12t. Concomitantly, there was reduction in the ratio of cells in G0/G1 phase in a dose
dependent manner. Henceforth, these results clearly indicate that the treatment of PC-3 cells with
12t led to G2/M cell cycle arrest.
<Insert Figure 7 here>
2.2.4. Acridine orange-ethidium bromide (AO-EB) staining
The induction of apoptosis by chemotherapeutic agents has always been a preferred choice in the
development of new anticancer agents [53]. The morphological changes induced by the most

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active compound 12t in PC-3 cells were further examined by using acridine orange-ethidium
bromide (AO-EB) staining to differentiate the live, apoptotic and necrotic cells [54]. AO-EB
staining method discerns the live cells from dead cells, because AO permeates the intact cell
membrane and stains the cells green, while ethidium bromide can stain only the cells with lost
membrane integrity in orange. From Figure 8, it can be inferred that the control cells exhibited
normal morphology whereas compound 12t treated PC-3 cells were characterized by the early
signs of apoptosis such as condensed chromatin and blebbing of cell membrane at 0.5 µM
concentration of compound 12t. Apoptotic body formation, irregular distribution of chromatin
that marginated into horse-shoe shaped nuclei and destructive fragmentation of nuclei were
observed at 1 µM and 2 µM respectively.
<Insert Figure 8 here>
2.2.5. Annexin V-FITC/Propidium iodide staining assay
In order to quantify the percentage of cells undergoing apoptosis by the treatment with
compound 12t, Annexin V-FITC/propidium iodide dual staining assay was carried out according
to the known procedure. This assay facilitates the detection of live cells (Q1-LL; AV‒/PI‒), early
apoptotic cells (Q2-LR; AV+/PI‒), late apoptotic cells (Q3-UR; AV+/PI+) and dead cells (Q4-
UL; AV‒/PI+). PC-3 cells were treated with 2 µM and 4 µM of compound 12t for 24 h and
stained with Annexin V-FITC/propidium iodide. As shown in Figure 9, the compound 12t
increased the percentage of late apoptotic cells [from 5.7% (control) to 27.9% (at 2 µM) and
48.3% (at 4 µM)] which suggests that the compound 12t induced apoptosis in PC-3 cells in a
dose dependent manner.
<Insert Figure 9 here>
2.2.6. DAPI staining
DAPI (4',6-Diamidino-2-phenylindole) is a nuclear stain that strongly binds to adenine-thymine
clusters of the minor groove of double-stranded DNA and thus it can visualize nuclear
morphological changes. DAPI stains the apoptotic cells bright coloured because of the
condensed nucleus which is a characteristic feature of apoptosis. Therefore, DAPI staining was
performed as per the procedure available in the literature to detect nuclear damage and chromatin
condensation induced by compound 12t in PC-3 cells. As shown in the Figure 10, compound

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12t treated PC-3 cells displayed condensed, fragmented and horse-shoe shaped nuclei while the
untreated cells exhibited intact nucleus.
<Insert Figure 10 here>
2.2.7. Effect on mitochondrial membrane potential (DΨm)
Mitochondria are cellular power house of energy and also plays crucial role in apoptotic
processes. As evident from the literature, the damage of mitochondrial membrane integrity, loss
or collapse of mitochondrial membrane potential and increase of intracellular ROS levels are
closely connected processes that happen during apoptosis [55]. Hence, we tested this possibility
by investigating the effect of compound 12t on DΨm of PC-3 cells using lipophilic cationic JC-1
dye [56]. Healthy polarised mitochondria exhibits red to orange colour because of potential
dependent formation of J-aggregates, whereas depolarised mitochondria emits green due to the
presence of J-monomers. PC-3 cells were treated with 2 µM and 4µM of 12t for 24 h and stained
with JC-1 dye. As evident from Figure 11, there was increment in depolarised cell population
(P2) from 11.9% (control) to 23.67% (at 2 µM) and 52.30% (at 4 µM) in concentration
dependent manner. Hence, these results clearly indicate that compound 12t induced cytotoxicity
through mitochondria dependent apoptosis.
<Insert Figure 11 here>
2.2.8. Measurement of reactive oxygen species (ROS) levels
High levels of reactive oxygen species (ROS) causes oxidative damage to mitochondrial
permeability transition pore leading to dissipation of mitochondrial membrane potential which
further leads to the initiation of intrinsic apoptotic cascade [57]. Hence, we evaluated the
elevation of intracellular reactive oxygen species by compound 12t in PC-3 cells using DCFDA
staining method. Treatment with compound 12t for 6 h resulted in significant increase in
DCFDA fluorescence compared to control cells, representing ROS accumulating property of
compounds (Figure 12), which indicates that compounds induced apoptosis through ROS
generation.
<Insert Figure 12 here>

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2.3. Molecular docking simulation study
To elucidate the binding mode and type of interactions with tubulin (PDB ID: 1SA0) [58], we
have performed molecular docking simulation studies with the most active compound 12t using
the GLIDE docking module of Schrödinger suite 2014-3 [59]. From the docking study, it was
observed that the top ranked conformation of compound 12t was well accommodated inside the
colchicine binding site of the tubulin. As shown in Figure 13a, docking studies suggest that the
compound 12t binds well in the colchicine-binding domain at the α/β-tubulin interface. More
detailed analysis of the inhibitor–tubulin complex (Figure 13b) revealed various hydrogen
bonding interactions that appear to play a key role in the binding mode. Compound 12t showed
four hydrogen bond interactions with the catalytically active residues Asn101, Gly144, Val181
and Lys254. The nitrogen atom at N-1 position of imidazo[1,2-a]pyridine ring acts as hydrogen
bond acceptor and involved in the hydrogen bond interaction with back bone NH2 of Val181 (d =
3.7 Å). The oxygen atom of methoxy group at the second position of chalcone phenyl moiety
established two hydrogen bond interactions with side chain NH2 of Asn101 (d = 1.9 Å) and main
chain NH2 of Gly144 (d = 3.5 Å). Similarly, the oxygen atom of dienone linker showed a
hydrogen bonding interaction with the side chain NH2 of Lys254 (d = 3.6 Å). Additionally,
several hydrophobic interactions were observed between the compound 12t and the active site
residues, e.g., Val74, Ala180, Val181, Cys241, Leu242, Leu248, Ala250, Leu252, Leu255,
Met259, Val315, Ala316 and Val351 which stabilizes the binding of the compound 12t in the
colchicine-binding domain of α/β-tubulin interface.
<Insert Figure 13 here>
To acquire further intuition, we demonstrated the superimposition of co-crystallized ligand and
the best docked pose of compound 12t in the colchicine binding site of α/β-tubulin interface. The
superimposition poses recommended that 12t occupies the binding pocket in a similar fashion as
that of co-crystallized ligand and certainly the 2,4,6-trimethoxy group in 12t superimposes with
the trimethoxy phenyl moiety of colchicine as seen in Figure 14. Overall, these molecular
docking simulation results provided us a rational justification for why compound 12t had the
good tubulin polymerization inhibitory activity and some important directions for future
structural modifications.

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<Insert Figure 14 here>
3. Conclusion
In the course of the development of potent cytotoxic agents, a series of some new curcumin
inspired imidazo[1,2-a]pyridine analogues (12a‒t) were synthesized and characterized by IR,
1
13
HRMS, H and C NMR spectral data. Initially, these compounds were evaluated for their in
vitro cytotoxic potential on cervical (HeLa), gastric (HGC-27), lung (NCI-H460), prostate (DU-
145 and PC-3) and breast (4T1) cancer cell lines and one normal human prostate (RWPE-1) cell
line using MTT assay. Among the compounds tested, compounds 12e, 12r and 12t exhibited
significant cytotoxicity and 12t [(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-
5-(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-one] being the most active member of the series
showed excellent antiproliferative activity on all the tested cell lines, especially PC-3, HGC-27
and HeLa (IC50 of 2.11 ± 0.27 µM, 2.21 ± 0.25 µM, 2.53 ± 0.01 µM respectively). Interestingly,
the most active compound 12t was found to be less cytotoxic on normal human prostate (RWPE-
1) cells and almost 2 times more selective on PC-3 cells. The results from aqueous solubility test
showed that compounds 12e and 12t have 1.7 and 2.8 times more aqueous solubility than
curcumin. Moreover, compound 12t efficiently inhibited tubulin polymerization with IC50 value
of 8.44 ± 0.13 µM and molecular modelling studies disclosed that 12t binds at the colchicine
binding site of the tubulin. Also, 12t arrests PC-3 cells in G2/M phase as evident from the cell
cycle analysis. Further, treatment of PC-3 cells with 12t displayed typical apoptotic morphology,
also led to the impairment of mitochondrial membrane potential (DΨm) and increased levels of
reactive oxygen species (ROS). Overall, the results from acridine orange/ethidium bromide (AO-
EB) and DAPI staining studies, annexin V-FITC/propidium iodide staining assay, analysis of
mitochondrial membrane potential (DΨm) and reactive oxygen species (ROS) levels certainly
demonstrated the induction of apoptosis in PC-3 cells by compound 12t. In conclusion, curcumin
inspired imidazo[1,2-a]pyridine analogues described in the present work indicate exciting
possibilities of developing new cancer therapeutics via their structural modifications.
4. Experimental section
4.0. General

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All the chemicals, reagents, starting materials and solvents were procured from commercial
suppliers and were 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) or neutral alumina. Ethyl acetate
and hexane were used as eluents. Melting points were checked using Stuart digital SMP 30
melting point apparatus and were uncorrected. FT-IR spectra for all the compounds were
1
13
recorded on a Perkin Elmer instrument by using ATR method. H and C NMR spectra were
recorded on an Avance 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.
4.1. Synthetic procedures and spectral data
4.1.1. General synthetic procedure for the synthesis of chalcones 3
To a magnetically stirred solution of substituted benzaldehyde (1, 1 mmol) in ethanol (3 mL)
were added 0.5 mL of acetone and 1 mL of 15% aqueous NaOH (1 mL) solution at 0 ºC. The
reaction was allowed to stir at room temperature till it was completed. The reaction mixture was
evaporated to dryness, extracted twice with ethyl acetate, the combined organic layers were dried
over anhydrous Na2SO4 and concentrated under vacuum. The crude residue was purified by
column chromatography (Silica gel, 60-120 mesh, 9:1 hexane/ethyl acetate) to afford the desired
chalcone (3) in good to very good yields.
4.1.2. General synthetic procedure for the synthesis of 6
A solution of 2-amino pyridine (4, 1 mmol) and ethyl bromopyruvate (5, 1.2 mmol) in ethanol
(15 mL) was stirred under reflux for 1 h, and concentrated in vacuo to remove the solvent. The
residue was partitioned between dichloromethane (15 mL) and saturated aqueous solution of
sodium carbonate (10 mL). The organic layer was separated, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography on

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neutral alumina to afford imidazo[1,2-a]pyridine-2-carboxylic acid ethyl ester as a light brown
solid (6) in good yield.
4.1.3. General procedure for the synthesis of 7
N-Iodosuccinimide (1.5 mmol) was added to a magnetically stirred solution of imidazo[1,2-
a]pyridine-2-carboxylic acid ethyl ester (6, 1 mmol) in dry CH3CN (5 mL). The reaction mixture
was stirred at rt for 1 h. The filtrate was concentrated to remove the solvent, and the residue was
purified by column chromatography on neutral alumina to afford ethyl 3-iodoimidazo[1,2-
a]pyridine-2-carboxylate (7) in moderate yield.
4.1.4. General procedure for the synthesis of 9
To a mixture of ethyl 3-iodoimidazo[1,2-a]pyridine-2-carboxylate (7, 1 mmol) and Pd(PPh3)4
(0.05 mmol) in dioxane (8 mL) was added the corresponding phenyl boronic acid (1.1 mmol)
followed by the addition of sodium carbonate (2 mmol) in water (2 mL). The reaction mixture
was heated at 75 °C with vigorous stirring under nitrogen atmosphere, and the reaction was
monitored by TLC. After the aryl halide (7) was consumed completely, the reaction mixture was
concentrated to remove the solvent and then extracted with dichloromethane (2 x 20 mL). The
combined organic extracts were washed with water (20 mL), dried over anhydrous Na2SO4, and
concentrated to dryness under vacuo. The crude product (9) was purified by column
chromatography on neutral alumina.
4.1.5. General procedure for the synthesis of 10
To a slurry of lithium aluminium hydride (2 mmol) in anhydrous tetrahydrofuran (3 mL), which
was cooled to 0 °C, was added a solution of ethyl 3-arylimidazo[1,2-a]pyridine-2-carboxylate (9,
1 mmol) in anhydrous tetrahydrofuran (2 mL) dropwise. After being stirred at 0 °C for 1 h, the
reaction was quenched by adding 1N aqueous solution of hydrochloride (0.8 mL) slowly. After
being stirred for 10 min, the mixture was basified by the addition of saturated aqueous solution
of sodium bicarbonate (1 mL), and filtered through celite. The filter cake was washed with
dichloromethane (10 mL). The organic layer was separated and the aqueous layer was extracted
with dichloromethane (2 x 10 mL). The organic layers were combined, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was purified by column

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chromatography on neutral alumina to afford (3-arylimidazo[1,2-a]pyridin-2-yl)methanol (10) in
moderate to good yields.
4.1.6. General procedure for the synthesis of 11
o
To a magnetically stirred solution of 10 (1 mmol) in DCM (10 mL) at 0 C were added water
(100 µL) and Dess-Martin periodinane (2.5 mmol) sequentially. The reaction was monitored by
TLC until the alcohol was consumed. The reaction mixture was filtered through a cartridge of
SiO2 (eluting with Et2O) and solvent was removed in vacuo. The residue was purified by column
chromatography on neutral alumina to afford 3-arylimidazo[1,2-a]pyridin-2-carbaldehyde (11) in
moderate to good yields.
4.1.7. General procedure for the synthesis of 12a‒t
To a stirred solution of chalcone 3 (0.5 mmol) in ethanol (5 mL) was added 15% aqueous
CH3ONa (1-2 mL) solution and aldehyde 11 (0.5 mmol) at 0 ºC. The resulting solution was
stirred at room temperature till the reaction was completed. The reaction mixture was evaporated
to dryness, partitioned between ethyl acetate and water, the combined organic layers were dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The crude mass was purified
either by recrystallization in ethanol or by column chromatography on neutral alumina to furnish
the final compounds 12a‒t in moderate to very good yields.
4.1.8. Spectral data
4.1.8.1. (1E,4E)-1-(4-methoxyphenyl)-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-3-
one (12a)
Yellow solid, yield 73.1%; mp: 152‒153 ºC; FT-IR (cm-1): 3047.4, 2932.3, 1651.8, 1620.5,
1
1510.8, 1450.3, 1352.2, 1249.9, 1096.5, 763.0; H NMR (500 MHz, CDCl3): δ 8.10 (d, J = 6.9
Hz, 1H), 7.77 (s, 1H), 7.73 (d, J = 7.0 Hz, 2H), 7.66 (d, J = 9.1 Hz, 1H), 7.63 ‒ 7.43 (m, 8H),
13
6.93 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 16.1 Hz, 1H), 6.78 (t, J = 6.7 Hz, 1H), 3.85 (s, 3H); C
NMR (125 MHz, CDCl3): δ 189.0, 161.5, 145.5, 143.0, 139.1, 133.2, 130.2, 130.1, 129.5, 129.3,
127.7, 127.6, 126.3, 125.9, 125.1, 123.6, 117.8, 114.4, 112.8, 55.4; HRMS (ESI): m/z calcd for
C25H21N2O2 381.1598, found 381.1606 [M+H]+.

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4.1.8.2. (1E,4E)-1-(2,4-dimethoxyphenyl)-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-
3-one (12b)
Yellow solid, yield 68.7%; mp: 141‒142 ºC; FT-IR (cm-1): 2935.0, 1644.6, 1600.7, 1503.2,
1454.9, 1348.8, 1290.9, 1094.7, 751.4; 1H NMR (500 MHz, CDCl3): δ 8.10 (d, J = 6.9 Hz, 1H),
8.02 (d, J = 16.2 Hz, 1H), 7.74 (d, J = 1.1 Hz, 2H), 7.66 (d, J = 9.2 Hz, 1H), 7.58 (d, J = 7.0 Hz,
2H), 7.54 ‒ 7.43 (m, 5H), 7.03 (d, J = 16.2 Hz, 1H), 6.77 (t, J = 6.5 Hz, 1H), 6.53 (d, J = 8.6, 2.2
13
Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H); C NMR (125 MHz, CDCl3): δ
189.6, 162.9, 160.2, 145.5, 139.2, 138.8, 132.8, 130.8, 130.2, 129.5, 129.2, 127.6, 127.5, 126.4,
126.2, 125.7, 123.6, 117.7, 117.1, 112.7, 105.4, 98.4, 55.5, 55.4; HRMS (ESI): m/z calcd for
C26H23N2O3 411.1703, found 411.1712 [M+H]+.
4.1.8.3. (1E,4E)-1-(2,5-dimethoxyphenyl)-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-
3-one (12c)
Yellow solid, yield 63.3%; mp: 129‒130 ºC; FT-IR (cm-1): 2930.1, 1663.4, 1618.9, 1535.6,
1461.8, 1350.2, 1291.9, 1091.1, 751.3; 1H NMR (500 MHz, CDCl3): δ 8.11 ‒ 8.07 (m, 1H), 8.04
(s, 1H), 7.75 (s, 2H), 7.67 (d, J = 9.2 Hz, 1H), 7.63 ‒ 7.42 (m, 6H), 7.12 (d, J = 3.0 Hz, 1H), 7.07
(d, J = 16.3 Hz, 1H), 6.93 (dd, J = 9.0, 3.0 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 6.78 (t, J = 6.8 Hz,
1H), 3.87 (s, 3H), 3.80 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 189.5, 153.5, 153.2, 145.5,
139.1, 138.4, 133.4, 130.2, 129.5, 129.3, 128.2, 127.6, 126.3, 125.9, 124.4, 123.7, 117.8, 117.6,
113.1, 112.8, 112.4, 56.0, 55.8; HRMS (ESI): m/z calcd for C26H23N2O3 411.1703, found
411.1709 [M+H]+.
4.1.8.4. (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-
3-one (12d)
Yellow solid, yield 79.2%; mp: 102‒104 ºC; FT-IR (cm-1): 2936.6, 1668.0, 1650.2, 1621.4,
1
1507.3, 1466.0, 1348.9, 1265.0, 1101.4, 751.1; H NMR (500 MHz, CDCl3): δ 8.10 (d, J = 7.0
Hz, 1H), 7.79 ‒ 7.71 (m, 3H), 7.66 (d, J = 9.1 Hz, 1H), 7.59 (t, J = 7.3 Hz, 2H), 7.52 (dd, J = 9.1,
7.9 Hz, 3H), 7.31 ‒ 7.27 (m, 1H), 7.19 (dd, J = 8.3, 1.6 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H), 6.89
13
(dd, J = 16.2, 8.2 Hz, 2H), 6.78 (t, J = 6.8 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H); C NMR (125
MHz, CDCl3): δ 188.9, 151.3, 149.2, 145.5, 143.2, 139.0, 133.3, 130.2, 129.5, 129.3, 127.9,
127.7, 127.5, 126.3, 125.7, 125.5, 123.7, 123.1, 117.8, 112.8, 111.1, 109.7, 56.0, 55.9; HRMS
(ESI): m/z calcd for C26H23N2O3 411.1703, found 411.1712 [M+H]+.

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4.1.8.5. (1E,4E)-1-(3-phenylimidazo[1,2-a]pyridin-2-yl)-5-(2,4,5-trimethoxyphenyl)penta-1,4-
dien-3-one (12e)
Yellow solid, yield 73.5%; mp: 244‒246 ºC; FT-IR (cm-1): 3046.2, 1657.3, 1601.0, 1505.1,
1447.6, 1349.3, 1284.1, 1094.7, 754.1; 1H NMR (500 MHz, CDCl3): δ 8.13 ‒ 8.02 (m, 2H), 7.75
(s, 2H), 7.66 (d, J = 9.2 Hz, 1H), 7.64 ‒ 7.55 (m, 3H), 7.54 ‒ 7.47 (m, 3H), 7.09 (s, 1H), 6.96 (d,
J = 16.2 Hz, 1H), 6.78 (t, J = 6.8 Hz, 1H), 6.51 (s, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H);
13C NMR (125 MHz, CDCl3): δ 189.4, 154.3, 152.3, 145.5, 143.2, 139.2, 138.3, 132.9, 130.2,
129.5, 129.2, 127.7, 127.6, 126.2, 126.0, 125.7, 123.6, 117.7, 115.4, 112.7, 110.8, 96.7, 56.3,
56.2, 56.0; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1813 [M+H]+.
4.1.8.6. (1E,4E)-1-(3-phenylimidazo[1,2-a]pyridin-2-yl)-5-(2,4,6-trimethoxyphenyl)penta-1,4-
dien-3-one (12f)
Yellow solid, yield 76.7%; mp: 202‒205 ºC; FT-IR (cm-1): 2972.0, 1660.1, 1598.7, 1554.4,
1469.1, 1350.9, 1285.2, 1090.9, 752.1; 1H NMR (500 MHz, CDCl3): δ 8.20 (d, J = 16.2 Hz, 1H),
8.10 (d, J = 6.9 Hz, 1H), 7.76 ‒ 7.62 (m, 3H), 7.60 ‒ 7.55 (m, 2H), 7.51 (d, J = 7.3 Hz, 3H), 7.42
(d, J = 16.2 Hz, 1H), 7.27 ‒ 7.20 (m, 1H), 6.76 (dd, J = 9.8, 3.8 Hz, 1H), 6.12 (s, 2H), 3.88 (s,
6H), 3.85 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 190.5, 163.0, 161.6, 145.4, 139.4, 134.5,
132.1, 130.2, 129.4, 129.1, 127.7, 127.6, 127.3, 127.0, 126.1, 123.6, 117.7, 112.6, 106.4, 90.4,
55.7, 55.4; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1813 [M+H]+.
4.1.8.7. (1E,4E)-1-(4-methoxyphenyl)-5-(3-(3-methoxyphenyl)imidazo[1,2-a]pyridin-2-yl)penta-
1,4-dien-3-one (12g)
Yellow solid, yield 64.8%; mp: 113‒114 ºC; FT-IR (cm-1): 2969.6, 1647.1, 1599.9, 1509.7,
1422.1, 1346.0, 1288.4, 1091.7, 754.0; 1H NMR (500 MHz, CDCl3): δ 8.12 (d, J = 6.9 Hz, 1H),
7.81 ‒ 7.67 (m, 3H), 7.66 (d, J = 9.1 Hz, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.50 (t, J = 7.9 Hz, 1H),
7.29 ‒ 7.24 (m, 1H), 7.11 ‒ 7.04 (m, 2H), 7.02 (s, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 16.1
13
Hz, 1H), 6.78 (t, J = 6.7 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H); C NMR (125 MHz, CDCl3): δ
189.0, 161.5, 160.3, 145.5, 143.0, 139.1, 133.2, 130.6, 130.1, 128.8, 127.6, 127.5, 126.3, 125.9,
125.1, 123.8, 122.5, 117.8, 115.7, 114.8, 114.4, 112.7, 55.5, 55.4; HRMS (ESI): m/z calcd for
C26H23N2O3 411.1703, found 411.1709 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.8.8.
(1E,4E)-1-(2,4-dimethoxyphenyl)-5-(3-(3-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12h)
Yellow solid, yield 76.4%; mp: 138‒140 ºC; FT-IR (cm-1): 2972.2, 1662.4, 1597.5, 1505.4,
1466.1, 1335.1, 1275.2, 1099.4, 751.9; 1H NMR (500 MHz, CDCl3): δ 8.12 (d, J = 6.9 Hz, 1H),
8.02 (d, J = 16.2 Hz, 1H), 7.81 ‒ 7.69 (m, 2H), 7.66 (d, J = 9.1 Hz, 1H), 7.55 ‒ 7.45 (m, 2H),
7.27 ‒ 7.23 (m, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.07 ‒ 7.00 (m, 3H), 6.77 (d, J = 6.8 Hz, 1H), 6.53
13
(dd, J = 8.6, 2.2 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H); C
NMR (125 MHz, CDCl3): δ 189.5, 162.9, 160.3, 145.5, 139.2, 138.8, 132.8, 130.7, 130.5, 128.8,
127.3, 126.4, 126.2, 125.7, 123.8, 122.5, 117.7, 117.1, 115.7, 114.8, 112.7, 105.3, 98.4, 55.5,
55.4; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1813 [M+H]+.
4.1.8.9.
(1E,4E)-1-(2,5-dimethoxyphenyl)-5-(3-(3-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12i)
Yellow solid, yield 67.3%; mp: 170‒171 ºC; FT-IR (cm-1): 2971.8, 1648.7, 1587.2, 1493.8,
1348.9, 1286.3, 1092.3, 750.2; 1H NMR (500 MHz, CDCl3): δ 8.12 (d, J = 6.9 Hz, 1H), 8.06 (d,
J = 16.3 Hz, 1H), 7.76 (q, J = 15.3 Hz, 2H), 7.66 (d, J = 9.1 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H),
7.28 ‒ 7.24 (m, 1H), 7.14 ‒ 7.04 (m, 4H), 7.02 (s, 1H), 6.93 (dd, J = 9.0, 2.9 Hz, 1H), 6.86 (d, J
13
= 9.0 Hz, 1H), 6.78 (t, J = 6.8 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.80 (s, 3H); C NMR (125
MHz, CDCl3): δ 189.5, 160.3, 153.5, 153.2, 145.5, 139.1, 138.4, 133.4, 130.6, 128.8, 128.1,
127.5, 126.3, 125.9, 124.4, 123.8, 122.5, 117.8, 117.5, 115.7, 114.9, 113.1, 112.7, 112.4, 56.0,
55.8, 55.4; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1817 [M+H]+.
4.1.8.10.
(1E,4E)-1-(3,4-dimethoxyphenyl)-5-(3-(3-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12j)
Yellow solid, yield 77.2%; mp: 170‒172 ºC; FT-IR (cm-1): 2967.8, 1642.2, 1621.6, 1600.3,
1
1510.1, 1465.5, 1358.0, 1295.9, 1088.1, 750.3; H NMR (500 MHz, CDCl3): δ 8.11 (d, J = 7.0
Hz, 1H), 7.80 ‒ 7.70 (m, 3H), 7.65 (d, J = 9.2 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.28 ‒ 7.24 (m,
1H), 7.19 (dd, J = 8.3, 1.4 Hz, 1H), 7.14 (s, 1H), 7.11 ‒ 7.04 (m, 2H), 7.02 (s, 1H), 6.92 ‒ 6.85
13
(m, 2H), 6.78 (t, J = 6.8 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.88 (s, 3H); C NMR (125 MHz,
CDCl3): δ 188.9, 160.3, 151.3, 149.2, 145.4, 143.2, 139.0, 133.3, 130.5, 128.7, 127.9, 127.5,

ACCEPTED MANUSCRIPT
126.3, 125.7, 125.5, 123.8, 123.0, 122.5, 117.7, 115.7, 114.8, 112.8, 111.1, 109.7, 56.0, 55.8,
55.4; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1818 [M+H]+.
4.1.8.11.
(1E,4E)-1-(4-methoxyphenyl)-5-(3-(4-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12k)
Yellow solid, yield 63.7%; mp: 126‒128 ºC; FT-IR (cm-1): 2988.5, 1654.7, 1590.0, 1509.5,
1347.2, 1300.1, 1249.9, 1081.9, 750.3; 1H NMR (500 MHz, CDCl3): δ 8.06 (d, J = 6.9 Hz, 1H),
7.80 ‒ 7.75 (m, 1H), 7.72 (s, 2H), 7.67 (d, J = 8.9 Hz, 1H), 7.56 (d, J = 8.7 Hz, 2H), 7.43 (d, J =
8.4 Hz, 2H), 7.30 ‒ 7.27 (m, 1H), 7.13 ‒ 7.08 (m, 2H), 6.93 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 16.1
13
Hz, 1H), 6.79 (t, J = 6.8 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H); C NMR (125 MHz, CDCl3): δ
189.0, 161.5, 160.4, 145.4, 142.9, 138.9, 133.4, 131.6, 130.0, 128.5, 127.7, 126.1, 125.6, 125.2,
123.7, 119.6, 117.7, 115.0, 114.4, 112.6, 55.5, 55.4; HRMS (ESI): m/z calcd for C26H23N2O3
411.1703, found 411.1706 [M+H]+.
4.1.8.12.
(1E,4E)-1-(2,4-dimethoxyphenyl)-5-(3-(4-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12l)
Yellow solid, yield 69.5%; mp: 165‒166 ºC; FT-IR (cm-1): 2988.2, 1644.4, 1590.0, 1503.2,
1455.6, 1345.6, 1289.5, 1091.5, 762.5; 1H NMR (500 MHz, CDCl3): δ 8.02 (d, J = 17.5 Hz, 2H),
7.73 ‒ 7.40 (m, 6H), 7.25 ‒ 7.18 (m, 1H), 7.10 (d, J = 7.3 Hz, 2H), 7.02 (d, J = 16.0 Hz, 1H),
6.81 ‒ 6.72 (m, 1H), 6.53 (d, J = 6.7 Hz, 1H), 6.47 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.85 (s,
13
3H); C NMR (125 MHz, CDCl3): δ 189.6, 162.9, 160.3, 160.2, 145.3, 139.0, 138.7, 133.0,
132.1, 131.5, 130.7, 128.5, 128.4, 127.5, 126.0, 125.8, 123.6, 119.6, 117.7, 117.1, 115.0, 112.6,
105.3, 98.4, 55.5, 55.4; HRMS (ESI): m/z calcd for C27H25N2O4 441.1809, found 441.1815
[M+H]+.
4.1.8.13.
(1E,4E)-1-(2,5-dimethoxyphenyl)-5-(3-(4-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12m)
Yellow solid, yield 63.0%; mp: 96‒98 ºC; FT-IR (cm-1): 2971.3, 1645.5, 1610.4, 1492.8, 1347.3,
1286.6, 1089.8, 753.5; 1H NMR (500 MHz, CDCl3): δ 8.08 (d, J = 15.9 Hz, 2H), 7.81 ‒ 7.51 (m,
4H), 7.50 ‒ 7.30 (m, 3H), 7.15 ‒ 7.03 (m, 3H), 6.93 (d, J = 6.4 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H),
13
6.82 ‒ 6.76 (m, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.81 (s, 3H); C NMR (125 MHz, CDCl3): δ

ACCEPTED MANUSCRIPT
189.5, 160.4, 153.5, 153.2, 145.4, 138.9, 138.3, 133.6, 131.6, 128.2, 127.8, 126.1, 125.5, 124.4,
123.7, 119.5, 117.7, 117.5, 115.0, 113.1, 112.6, 112.4, 56.0, 55.8, 55.4; HRMS (ESI): m/z calcd
for C27H25N2O4 441.1809, found 441.1813 [M+H]+.
4.1.8.14.
(1E,4E)-1-(3,4-dimethoxyphenyl)-5-(3-(4-methoxyphenyl)imidazo[1,2-a]pyridin-2-
yl)penta-1,4-dien-3-one (12n)
Yellow solid, yield 65.4%; mp: 116‒118 ºC; FT-IR (cm-1): 2988.6, 1656.2, 1620.1, 1508.3,
1439.4, 1248.6, 1101.9, 752.0; 1H NMR (500 MHz, CDCl3): δ 8.05 (d, J = 6.9 Hz, 1H), 7.76 (s,
1H), 7.73 (d, J = 2.9 Hz, 2H), 7.65 (d, J = 9.1 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.25 ‒ 7.22 (m,
1H), 7.19 (d, J = 8.2 Hz, 1H), 7.16 ‒ 7.07 (m, 3H), 6.92 ‒ 6.85 (m, 2H), 6.77 (t, J = 6.7 Hz, 1H),
13
3.94 (s, 3H), 3.93 (s, 3H), 3.91 (s, 3H); C NMR (125 MHz, CDCl3): δ 188.9, 160.4, 151.3,
149.2, 145.4, 143.1, 138.9, 133.5, 131.6, 127.9, 127.8, 126.1, 125.6, 125.3, 123.7, 123.0, 119.5,
117.7, 115.0, 112.6, 111.1, 109.7, 56.0, 55.8, 55.5; HRMS (ESI): m/z calcd for C27H25N2O4
441.1809, found 441.1814 [M+H]+.
4.1.8.15.
(1E,4E)-1-(3-(4-methoxyphenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4,5-
trimethoxyphenyl)penta-1,4-dien-3-one (12o)
Yellow solid, yield 63.8%; mp: 194‒195 ºC; FT-IR (cm-1): 2971.0, 1658.7, 1606.0, 1507.5,
1463.7, 1349.2, 1282.8, 1090.3, 751.9; 1H NMR (500 MHz, CDCl3): δ 8.13 ‒ 8.05 (m, 2H), 7.83
‒ 7.66 (m, 4H), 7.43 (d, J = 8.5 Hz, 2H), 7.15 ‒ 7.07 (m, 3H), 6.96 (d, J = 16.2 Hz, 1H), 6.80 (t,
13
J = 6.8 Hz, 1H), 6.51 (s, 1H), 3.94 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H); C NMR (125 MHz,
CDCl3): δ 189.4, 160.4, 154.3, 152.3, 145.3, 143.3, 139.0, 138.2, 133.0, 131.6, 127.6, 126.0,
125.8, 125.7, 123.7, 119.6, 117.7, 115.4, 115.0, 112.6, 110.9, 96.7, 56.3, 56.2, 56.0, 55.4; HRMS
(ESI): m/z calcd for C28H27N2O5 471.1914, found 471.1919 [M+H]+.
4.1.8.16.
(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(4-
methoxyphenyl)penta-1,4-dien-3-one (12p)
Yellow solid, yield 71.9%; mp: 136‒138 ºC; FT-IR (cm-1): 2988.5, 1651.7, 1600.8, 1511.1,
1495.6, 1346.0, 1306.5, 1115.5, 752.4; 1H NMR (500 MHz, CDCl3): δ 8.02 (d, J = 6.9 Hz, 1H),
7.79 ‒ 7.64 (m, 4H), 7.56 (d, J = 8.6 Hz, 2H), 7.44 ‒ 7.28 (m, 3H), 6.94 (d, J = 8.7 Hz, 2H), 6.89
13
(s, 1H), 6.84 (dd, J = 12.7, 6.0 Hz, 2H), 3.85 (s, 3H); C NMR (125 MHz, CDCl3): δ 188.8,

ACCEPTED MANUSCRIPT
161.6, 145.6, 143.3, 139.5, 132.3, 130.1, 127.5, 126.9, 126.6, 126.5, 125.1, 125.0, 124.5, 123.3,
119.2, 119.1, 118.8, 118.6, 117.9, 114.4, 113.2, 55.4; HRMS (ESI): m/z calcd for C25H19F2N2O2
417.1409, found 417.1406 [M+H]+.
4.1.8.17.
(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4-
dimethoxyphenyl)penta-1,4-dien-3-one (12q)
Yellow solid, yield 73.0%; mp: 195‒196 ºC; FT-IR (cm-1): 2972.3, 1639.7, 1602.6, 1503.6,
1454.2, 1346.7, 1305.0, 1140.4, 756.3; 1H NMR (500 MHz, CDCl3): δ 8.06 ‒ 7.99 (m, 2H), 7.74
(d, J = 15.2 Hz, 1H), 7.70 ‒ 7.63 (m, 2H), 7.53 (d, J = 8.6 Hz, 1H), 7.44 ‒ 7.27 (m, 4H), 7.02 (d,
J = 16.2 Hz, 1H), 6.82 (t, J = 6.8 Hz, 1H), 6.53 (dd, J = 8.6, 2.2 Hz, 1H), 6.47 (d, J = 2.2 Hz,
1H), 3.90 (s, 3H), 3.85 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 189.4, 163.0, 160.3, 145.6,
139.6, 139.1, 131.8, 130.8, 127.0, 126.9, 126.5, 125.6, 124.9, 124.5, 123.2, 119.2, 119.1, 118.8,
118.6, 117.9, 117.0, 113.1, 105.4, 98.4, 55.5, 55.4; HRMS (ESI): m/z calcd for C26H21F2N2O3
447.1515, found 447.1519 [M+H]+.
4.1.8.18.
(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,5-
dimethoxyphenyl)penta-1,4-dien-3-one (12r)
Yellow solid, yield 79.7%; mp: 177‒179 ºC; FT-IR (cm-1): 2988.3, 1667.5, 1617.8, 1493.4,
1349.1, 1289.6, 1092.9, 1025.9, 748.2; 1H NMR (500 MHz, CDCl3): δ 8.07 (d, J = 16.3 Hz, 1H),
8.03 (d, J = 6.9 Hz, 1H), 7.76 (d, J = 15.2 Hz, 1H), 7.70 ‒ 7.65 (m, 2H), 7.44 ‒ 7.27 (m, 4H),
7.12 (d, J = 3.0 Hz, 1H), 7.06 (d, J = 16.3 Hz, 1H), 6.94 (dd, J = 9.0, 3.0 Hz, 1H), 6.87 (d, J =
9.0 Hz, 1H), 6.83 (t, J = 6.8 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H); 13C NMR (125 MHz, CDCl3): δ
189.3, 153.5, 153.2, 145.6, 139.5, 138.7, 132.5, 128.1, 126.9, 126.6, 126.5, 125.2, 124.5, 124.3,
123.3, 119.2, 119.1, 118.8, 118.6, 117.9, 117.7, 113.2, 113.1, 112.4, 56.0, 55.8; HRMS (ESI):
m/z calcd for C26H21F2N2O3 447.1515, found 447.1522 [M+H]+.
4.1.8.19.
(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(3,4-
dimethoxyphenyl)penta-1,4-dien-3-one (12s)
Yellow solid, yield 82.0%; mp: 180‒182 ºC; FT-IR (cm-1): 2959.0, 1658.2, 1617.3, 1508.1,
1469.7, 1347.6, 1285.2, 1107.3; 1H NMR (500 MHz, CDCl3): δ 8.03 (d, J = 6.9 Hz, 1H), 7.75 (d,
J = 15.5 Hz, 2H), 7.71 ‒ 7.63 (m, 2H), 7.44 ‒ 7.28 (m, 3H), 7.20 (dd, J = 8.3, 1.5 Hz, 1H), 7.14

ACCEPTED MANUSCRIPT
(s, 1H), 6.97 ‒ 6.87 (m, 2H), 6.84 (dd, J = 12.7, 5.8 Hz, 2H), 3.94 (s, 3H), 3.93 (s, 3H); 13C NMR
(125 MHz, CDCl3): δ 188.7, 151.4, 149.2, 145.6, 143.5, 139.5, 132.3, 127.7, 126.9, 126.6, 126.3,
125.4, 125.2, 124.4, 123.3, 123.1, 119.2, 119.1, 118.8, 118.7, 117.9, 113.2, 111.1, 109.7, 56.0,
55.8; HRMS (ESI): m/z calcd for C26H21F2N2O3 447.1515, found 447.1519 [M+H]+.
4.1.8.20.
(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4,6-
trimethoxyphenyl)penta-1,4-dien-3-one (12t)
Yellow solid, yield 67.5%; mp: 240‒241 ºC; FT-IR (cm-1): 2972.0, 1658.6, 1639.2, 1621.3,
1601.2, 1511.2, 1465.4, 1351.1, 1294.5, 1099.7, 752.5; 1H NMR (500 MHz, CDCl3): δ 8.22 (d, J
= 16.2 Hz, 1H), 8.03 (d, J = 6.8 Hz, 1H), 7.73 (d, J = 15.3 Hz, 2H), 7.63 (d, J = 15.3 Hz, 1H),
7.49 ‒ 7.27 (m, 5H), 6.84 (t, J = 6.6 Hz, 1H), 6.12 (s, 2H), 3.90 (s, 6H), 3.86 (s, 3H); 13C NMR
(125 MHz, CDCl3): δ 190.3, 163.1, 161.7, 145.6, 139.8, 134.9, 131.1, 128.3, 127.0, 126.9, 126.8,
126.4, 124.7, 123.2, 119.2, 119.1, 118.7, 118.6, 117.9, 113.1, 106.4, 90.4, 55.7, 55.4; HRMS
(ESI): m/z calcd for C27H23F2N2O4 477.1620, found 477.1625 [M+H]+.
4.2. Biological evaluation
4.2.0. Cell cultures
The cervical (HeLa), gastric (HGC-27), lung (NCI-H460), prostate (DU-145, PC-3) and breast
(4T1) tumor cell lines were procured from National center for Cell science (NCCS), Pune, India.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], trypsin-EDTA were
bought from Sigma Chemicals Co (St. Louis, MO). DMEM, RPMI 1640 medium, fetal bovine
serum were purchased from GIBCO-Invitrogen. The 12, 24 and 96 well flat bottom tissue culture
plates were procured from Corning.
4.2.1. MTT assay
The anticancer activity of the compounds (12a‒t) was determined by using MTT assay. 2.5‒5 x
103 cells per well were seeded in 100 µL DMEM or RPMI , supplemented with 10% FBS in each
well of 96-well microculture plates and incubated for 24 h at 37 ºC in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture medium, were added to the wells
with respective vehicle control. After 48 h of incubation period, medium was discarded, 100 µL
of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (0.5 mg/mL)

ACCEPTED MANUSCRIPT
containing medium was added to each well and the plates were incubated for 4 h. The
supernatant from each well was removed carefully, formazan crystals were dissolved in 200 µL
of DMSO and the absorbance was recorded at 570 nm wavelength with multimode plate reader
(Spectramax M4, Molecular devices, USA).
4.2.2. Evaluation of tubulin polymerization inhibitory activity
Tubulin polymerization kit was procured from Cytoskeleton, Inc. (BK011). To assess the effect
of compound 12t on tubulin polymerization, a fluorescence based in vitro tubulin polymerization
assay was carried out according to the manufacturer’s protocol and Nocodazole was used as
positive control. The reaction mixture having porcine brain tissue (2 mg/mL) in 80 mM PIPES at
pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, 1.0 mM GTP and glycerol in the presence and absence
of test compound (12t, at final concentration of 10 µM) was prepared and added to each well of
96-well plate. Tubulin polymerization was followed by a time dependent increase in
fluorescence due to the incorporation of a fluorescence reporter into microtubules as
polymerization proceeds. Fluorescence emission at 440 nm (excitation wavelength is 360 nm)
was measured using a Spectramax M4 Multi mode Micro plate Detection System. The IC50 value
was calculated from the compound concentration required to inhibit 50% of tubulin assembly in
comparison to control.
4.2.3. Cell cycle analysis
To examine the effect of compound 12t on cell cycle, cells were seeded in 12-well plates at a
density of 1 x 105 cells/mL and allowed to attach for 24 h. Cells were treated with required
concentrations (1 µM and 2 µM) of compound 12t and incubated further for 24 h. The cells were
collected, washed and fixed in 70% ethanol in PBS at -20 ºC. After leaving overnight, the fixed
cells were pelleted and stained with Propidium Iodide (25 µg/mL) in the presence of RNase A
(40 µg/mL) containing 0.1% Triton X-100 for 0.5 h, at 37 °C in dark, and about 10000 events
were analyzed using flow-cytometer (FACS verse, Becton Dickinson, US).
4.2.4. Acridine orange/ethidium bromide (AO/EB) staining
The morphological changes induced in PC-3 cells by compound 12t were examined by Acridine
Orange/Ethidium Bromide (AO/EB) staining. PC-3 cells were grown in 24 well plates (25000

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cells/well) for 24 h and were treated with 0.5 µM, 1 µM and 2 µM concentration of compound
12t for 48 h. After the incubation period, medium was discarded and cells were washed with
PBS and treated with 10 µg/mL of acridine orange and 10 µg/mL of ethidium bromide.
Morphological features were observed and photographs were taken immediately under
fluorescence inverted microscope (Model: Nikon, Japan) using 488 nm excitation and 530 nm
emission at 200x maginification.
4.2.5. Annexin V binding assay
PC-3 cells (1 x 105) were seeded in 12 well plates and allowed to grow overnight. Then, the
medium was replaced with medium containing compound 12t at 2 µM and 4 µM concentrations.
After 24 h of treatment, cells from the supernatant and adherent monolayer cells were harvested
by trypsinization, washed with PBS at 3000 rpm. Then the cells were processed using Annexin
V-assay kit (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen™) according to the
manufacturer's protocol. Further, flow cytometric analysis was performed using a flow-
cytometer (BD FACSVerse™, USA).
4.2.6. DAPI staining
PC-3 cells were seeded on 24 well plates at the density of 25000 cells/well and allowed to adhere
overnight. Then, the cells were treated with 1 µM and 2 µM concentration of compound 12t for
48 h, washed with PBS and fixed with 4% neutral buffered formalin solution for 20 min. Again,
the cells were washed twice with PBS and permeabilized with 0.2% triton-x for 5 min and
stained with DAPI (1 µg/mL) for 10 min at room temperature. Cells were examined for
morphological changes under fluorescence microscope (Model: Nikon, Japan) using 350 nm
excitation and 460 nm emission at 200x magnification.
4.2.7. Measurement of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential (MMP) was determined using JC-1 dye as per method given
by Atulya et al., with minor modifications [49]. PC-3 cells were cultured in 12 well plates at a
density of 1 x 105 cells/mL and allowed to grow overnight. Then, the cells were treated with 2
µM and 4 µM of compound 12t for 24 h. After treatment, cells were treated with JC-1 (2 µM)

ACCEPTED MANUSCRIPT
dye and incubated for 45 min in dark at 37 °C in an incubator. Subsequently, cells were washed
twice with PBS, trypsinized and analyzed by flow-cytometer (BD FACSVerse™, USA).
4.2.8. Measurement of reactive oxygen species (ROS) levels
PC-3 cells were seeded in 12 well plates at a density of 1 x 105 cells/mL and allowed to adhere
for overnight. The cells were treated with 1 µM and 2 µM concentrations of compound 12t for
24 h. The medium was replaced with culture medium containing DCFDA dye (10 µM) and
incubated for 0.5 h at room temperature in dark. The fluorescence intensity from each sample
was analyzed at excitation wavelength of 488 nm and emission wavelength of 530 nm by flow-
cytometer.
4.2.9. Computational methods
The 3D structure of compound 12t was built on Maestro Molecule Builder of Schrödinger. The
built molecule was optimized using OPLS_2005 force field in LigPrep module of Schrödinger
software. Docking procedure was followed using the standard protocol implemented in Maestro,
version 9.9 and the compound 12t was docked against the colchicine binding site of α/β-tubulin
interphase. The ligand –protein complex was analyzed for interactions and the 3D pose of most
active compound 12t was taken using Schrödinger and PyMOL v0.99.
4.2.10. Water solubility test
2 mg of curcumin/12e/12t was mixed with 2.0 mL of milli-Q water and the mixture was
vortexed for 5 min, sonicated for 1 min, and incubated at 37 °C for 24 h. Then, the mixture was
centrifuged at 14,000 rpm for 15 min and the water layer was separated and scanned in a
spectrophotometer at a wavelength range of 200 – 600 nm.
Acknowledgements
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi, for
the award of NIPER fellowship.
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