Bioorganic Chemistry 77 (2018) 515–526

Contents lists available at ScienceDirect

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

Synthesis and biological evaluation of imidazo[2,1-b]thiazole-

benzimidazole conjugates as microtubule-targeting agents
Mirza Feroz Baig a,b, V. Lakshma Nayak a, Prasad Budaganaboyina a, Kishore Mullagiri a, Satish Sunkari a,b,
Jitendra Gour c, Ahmed Kamal a,b,d,⇑
a
Medicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India
b
Academy of Scientific and Innovative Research, New Delhi 110 025, India
c
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, India
d
School of Pharmaceutical Education and Research(SPER), Jamia Hamdard, New Delhi 110062, India

a r t i c l e i n f o a b s t r a c t

Article history: A series of imidazo[2,1-b]thiazole-benzimidazole conjugates were synthesized and evaluated for their
Received 27 November 2017 antiproliferative activity against four human cancer cell lines i.e.; HeLa (cervical), A549 (lung), MCF-7
Revised 6 February 2018 (breast) and DU-145 (prostate) along with normal HEK-293 cell line. Amongst them, conjugate 6d dis-
Accepted 9 February 2018
played significant cytotoxicity against human lung cancer cell line, A549 with IC50 value 1.08 mM.
Available online 12 February 2018
Further, cell cycle analysis revealed that this compound arrested the cell cycle at G2/M phase in A549
cells. Furthermore, the tubulin polymerization assay results suggest that this conjugate (6d) exhibits sig-
Keywords:
nificant inhibitory effect on the tubulin assembly with an IC50 value of 1.68 mM. Moreover, the apoptotic
Imidazo[2,1-b]thiazoles
Benzimidazole
inducing properties of compound 6d was confirmed by Hoechst staining, measurement of mitochondrial
Cytotoxicity membrane potential (DWm) and annexin V-FITC assay. Further, molecular docking studies revealed that
Cell cycle compound 6d occupied the colchicine binding site.
Apoptosis Ó 2018 Elsevier Inc. All rights reserved.

1. Introduction
Cancer is a major health problem across the world and the sec-
ond leading cause of death in developed countries and most upset-
ting disease in humans [1] Improvement in the treatment and
awareness regarding the disease has led to reduce the cancer
deaths, but the number of new diagnoses has been persistent. Pre-
sently, chemotherapy is one of the regimens for the cancer which
includes the use of one or more anti-cancer drugs which can
specifically destroy cancer cells without having any significant
effect on the normal cells. The microtubule is the mitotic spindle,
used by eukaryotic cells to keep apart their chromosomes during
cell division [2]. Microtubules that consist of a- and b-tubulin het-
erodimers play a crucial role in various cellular processes, such as
guideline of motility, cell signaling, formation and maintenance of
cell shape, secretion and intracellular transport [3].
Generally, drugs target microtubules through their binding
sites, thereby stabilizing (Taxol 1) or destabilizing like (nocodazole
2) microtubule assembly. There are four main binding sites of
tubulin [4] which includes the taxane and laulimalide/peloruside
⇑ Corresponding author at: Medicinal Chemistry and Biotechnology Division,
CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
sites for the microtubule stabilizing agents [5,6] whereas the vinca
[7] and colchicine domain[8,9] sites for the destabilizing agents.
Interruption to the microtubule network by these agents act as
spindle poisons arresting the dividing cell in G2/M phase of the cell
cycle which is the hallmark of microtubule-interfering drugs that
finally lead to apoptotic cell death [10]. Naturally occurring tubulin
binding ligand that targets the microtubule dynamics by binding to
distinct colchicine domain of tubulin is colchicines (3) [11] Noco-
dazole is well known inhibitor of tubulin polymerization and
mostly used as pharmaceutical tool and positive control [12]. Many
existing anticancer drugs act through different mechanisms, How-
ever the action of some of them is not very clear. In this regard,
development of target based libraries of different chemical entities
is under investigation across the globe.
In recent years, there is considerable interest in the
development and pharmacology of different benzimidazole linked
heterocyclic compounds [13,14] due to their wide range of
biological activities such as antitumor [15,16], antibacterial
[17,18], anti-angiogenesis [19], antivascular [20] and antiviral
[21,22]. Moreover, benzimidazole scaffold is a component of vita-
min B12, that supports its potential use as therapeutics and the
structural similarity of benzimidazole moiety with some naturally
occurring compounds such as purine, encourages that it can easily

https://doi.org/10.1016/j.bioorg.2018.02.005
0045-2068/Ó 2018 Elsevier Inc. All rights reserved.
516 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526

interact with biomolecules of the living system. Additionally, ben- from appropriate 2-bromo-1-arylethanone (2a–c) and 2-
zimidazole based pharmacophores such as Hoechst 33258 (4), dis- aminothiazole (1).
played a wide spectrum of antiproliferative activity with DNA
minor groove binding and inhibition of topoisomerase ability
2.2. Biological activity
(Fig. 1) [23]. As reported in the literature, imidazo-thiazole exhib-
ited tubulin polymerization inhibitory activity [24–26]. Moreover,
2.2.1. Cytotoxic activity
benzimidazole based other heterocyclic moieties including fused
These compounds (6a–t) were evaluated for their cytotoxic
ring resulted in improved pharmacological profiles [27]. Thus,
activity in tested human cancer cell lines, namely, cervical (HeLa),
excellent biological activity profile of these hybrids has prompted
A549 (lung), breast (MCF-7) and prostate (DU-145), by employing
us to explore a new benzimidazole linked imidazothiazole which
MTT assay [31], wherein nocodazole was used as the reference
are prepared through chemical hybridization strategy. In this con-
compound. The results are summarized in Table 1 and expressed
text, we have designed and synthesized imidazothiazole-
as IC50 values. The results revealed that these compounds exhibit
benzimidazole derivatives (6a–t) and their biological evaluation
promising anticancer activity against the tested cell lines. Among
as tubulin targeting agent have been demonstrated (Fig. 1).
them compound, 6d showed significant cytotoxic activity against
In continuation to our ongoing research efforts for the develop-
A549 cell line, (IC50 values of 1.08 and 1.13 mM). To understand
ment of cytotoxic agents, we synthesized a number of pharmaco-
the structure activity relationship (SAR) we synthesized twenty
logically important heterocyclic compounds [28–30]. Our
imidazothiazole linked benzimidazole conjugates (6a–t) consisting
approach, as outlined in this paper is to discover new microtubule
of A-, B-, C-, and D-rings (Fig. 3) derived from Scheme 1. These
inhibitors that contain the benzimidazole core structure. We
compounds prepared with respect to different modification that
herein designed and synthesized a library of compounds based
was carried out on the C- and D-rings. The C-ring constituted the
on imidazothiadiazole-oxindole and imidazol-2-yl)-2H-chromen-
electron-neutral/donating group like H, CH3, OCH3 and 3, 4-
2-one moieties. They possess potent cytotoxicity profile wherein
(OCH2O) and electron withdrawing substituents like 2-bromo
the benzimidazole structural motif of 3-(1H-benzo[d]imidazol-2-
and whereas the D-ring possessed both the electron donating as
yl)-7-(bis(2-hydroxyethyl)amino)-2H-chromen-2-one has been
well as withdrawing groups like OCH3, H, F, 3,4-diCl, COPh and CF3.
directly attached to an imidazothiazole moiety resulting in 5-
The in vitro screening results revealed that these conjugates
(1H-benzo[d]imidazol-2-yl)-6-phenylimidazo[2,1-b]thiazole (6a–
acquire considerable cytotoxicity with IC50 values ranging from
t) and strategy of design is described in Fig. 2. Moreover, in order
1.08 lM to 48.70 lM. Similarly, conjugates 6c–6e, 6g–6h, and
to establish the SAR of this series and to investigate the possibility
6m–6n with electron-neutral/donating substituents on d-ring
of rising new potent antitumor agents several substituents were
showed significant anticancer activity with IC50 values in micro
introduced. Conjugates 6a–t were subjected to biological evalua-
to submicromolar range, in contrast electronegative substituent
tion in order to understand their possible mechanism of action
like chloro and bromo (6a, 6s and 6t) decreases the cytotoxicity.
which is discussed herein.
In particular, the conjugates 6d having electron-withdrawing sub-
stituents such as para-trifluoromethyl group on C-rings and para-
methoxy group on D-rings acquire significant cytotoxic activity
2. Results and discussion
against lung cancer cells with IC50 values of 1.08 lM. These are
superior to compound 5 (IC50 values of 1.48 mM) [32]. However,
2.1. Chemistry
imidazothiazole conjugates (6c, 6d, 6e, 6g, 6h, 6m, and 6n) exhib-
ited better cytotoxicity compared with imidazothiazole conjugates
The imidazothiazole-benzimidazole conjugates (6a–u)
(6a and 6o–t) against tested cell lines.
were prepared by the oxidative cyclization of substituted
o-phenylenediamine (5a–i) and imidazo[2,1-b]thiazole-5-
carbaldehyde (4a–d) with sodium metabisulphite in ethanol, as 2.2.2. Cell cycle analysis
shown in scheme 1. The imidazo[2,1-b]thiazole-5-carbaldehyde Many cytotoxic compounds exert their growth inhibitory effect
(4a–d) were obtained by the Vilsmeier-Haack reaction with the either by arresting the cell cycle at a particular checkpoint of cell
corresponding imidazo[2,1-b]thiazole (3a–d), that were obtained cycle or by induction of apoptosis or a combined effect of both

Fig. 1. Structures of some anticancer compounds.

 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526 517

Fig. 2. Design of imidazothiazole-benzimidazole conjugates (6a–t).

Scheme 1. Reagents and Conditions: a) EtOH, 60 °C, 8 h 82–90%; b) POCl3, DMF, 0 °C-rt, 8 h 80–88%; c) Na2S2O5, EtOH, 60 °C, 65–86%.

cycle block and apoptosis [33,34]. The in vitro screening results was on account of cell cycle arrest. In this study, A549 cells were
revealed that compound 6d showed significant activity against treated with this compound at 1 and 2 mM concentrations for 48
human lung cancer cell line (A549). Therefore, it was considered h. Results revealed that this compound arrested the cell cycle at
of interest to understand whether this inhibition of cell growth G2/M phase (Fig. 4 and Table 2). The conjugate 6d showed 15.1%
518 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526

Table 1
IC50a values (in mM) for compounds 6a–t in selected cancer cell lines.

Compound Helab A549c MCF-7d DU-145e HEK-293f

6a 48.70 15.07 19.09 17.57 94.64
6b 24.59 3.214 4.074 10.51 101.46
6c 14.47 1.849 2.344 8.714 143.71
6d 7.552 1.086 1.652 2.004 124.34
6e 10.32 1.197 1.803 8.111 112.33
6f 13.47 3.882 4.285 3.715 77.68
6g 11.16 1.130 3.236 3.162 120.55
6h 13.90 1.291 2.964 10.25 121.47
6i 28.76 8.781 14.25 15.28 53.30
6j 39.67 10.24 16.19 16.00 133.97
6k 34.90 9.345 11.94 16.65 103.18
6l 18.95 7.367 12.57 18.04 79.20
6m 12.97 1.153 1.419 14.32 112.27
6n 11.82 1.413 1.556 9.636 85.07
6o 31.67 15.81 13.41 20.17 91.71
6p 44.90 12.03 14.33 22.47 75.55
6q 33.13 13.28 16.16 22.16 126.67
6r 12.82 9.781 11.59 15.43 107.30
6s 46.30 16.18 20.39 31.93 93.24
6t 16.35 15.21 16.03 23.10 136.24
Nocodazole 1.778 1.807 1.585 1.811 –
a
50% Inhibitory concentration after 48 h of drug treatment and the values are average of three individual experiments.
b
Cervical cancer.
c
Lung cancer
d
Breast cancer.
e
Prostate cancer.
f
Normal cells

2.2.4. Measurement of mitochondrial membrane potential (DWm)

The maintenance of mitochondrial membrane potential (DWm)
is significant for mitochondrial integrity and bioenergetic function
[38]. Mitochondrial changes, including loss of mitochondrial mem-
brane potential (DWm), are key events that take place during drug-
induced apoptosis. Mitochondrial injury by compound 6d was
evaluated by detecting drops in mitochondrial membrane poten-
tial (DWm). In this study, we have investigated the involvement
of mitochondria in the induction of apoptosis by this compound.
After 48 h of drug treatment with this compound, it was observed
that reduced mitochondrial membrane potential (DWm) of A549
Fig. 3. Structural activity relationship of imidazothiazole-benzimidazole cells, assessed by JC-1 staining (Fig. 6).
conjugates.

2.2.5. Annexin V-FITC for apoptosis

of cell accumulation in G2/M phase at 1 mM concentration, whereas
they exhibited 56.3% of cell accumulation at 2 lM concentration
(Fig. 4, Table 2).
2.2.3. Effect of compounds on tubulin polymerization
In general G2/M cell cycle arrest is strongly associated with
inhibition of tubulin polymerization [35] and since compound 6d
cause cell cycle arrest at G2/M phase, it was considered of interest
to investigate their microtubule inhibitory function. Tubulin sub-
units are known to heterodimerize and self-assemble to form
microtubules in a time dependent manner. The progression of
tubulin polymerization [36,37] was thus examined by monitoring
the increase in fluorescence emission at 420 nm (excitation wave-
length is 360 nm) in 384 well plate for 1 h at 37 °C with and with-
out the conjugate at 3 mM concentration in comparison with
reference compound nocodazole. The test compound 6d signifi-
cantly inhibited tubulin polymerization by 68.08%, whereas the
reference compound (nocodazole) exhibited 64.65% inhibition
(Fig. 5).
This was followed by the evaluation of IC50 values for this com-
pound and results indicate that 6d showed better tubulin assembly
inhibition with an IC50 value of 1.68 ± 0.03 mM and was comparable
to nocodazole (IC50 = 1.99 ± 0.06 mM).
The apoptotic effect of 6d was further evaluated by Annexin V
FITC/PI (AV/PI) dual staining assay [39] to examine the occurrence
of phosphatidylserine externalization and also to understand
whether it is due to physiological apoptosis or nonspecific necrosis.
In this study, A549 cells were treated with this compound for 48 h
at 1 and 2 mM concentrations to examine the apoptotic effect. It
was observed that this compound showed significant apoptosis
against A549 cells as shown in Fig. 7. Results indicated that com-
pound 6d showed 14.94 and 43.62% at 1 and 2 mM concentrations
respectively for 48 h (Fig. 7 and Table 3).
2.2.6. Hoechst staining
Apoptosis is one of the major pathways that lead to the process
of cell death. Chromatin condensation and fragmented nuclei are
known as the classic characteristics of apoptosis. It was considered
of interest to investigate the apoptotic inducing effect of the
derivatives by Hoechst staining (H33258) method in A549 cell line.
Therefore, A549 cells were treated with 6d at 1 and 2 mM concen-
trations for 48 h. Manual field quantification of apoptotic cells
based on cytoplasmic condensation, presence of apoptotic bodies,
nuclear fragmentation and relative fluorescence of these deriva-
tives (6d) revealed that there was significant increase in the per-
centage of apoptotic cells (Fig. 8).
 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526 519

Fig. 4. Flow cytometric analysis in A549 lung cancer cell lines after treatment with conjugates, 6d at 1 and 2 mM concentrations for 48 h; A: Control cells (A549), B:
Nocodazole (2 mM), C: 6d (1 mM), and D: 6d (2 mM).

with tubulin, we performed molecular docking studies of the most

Table 2 active compound 6d at colchicine binding site in tubulin dimmer,
Cell cycle distribution of A549 cell line treated with 6d. the colchicines binds at the interface of a b-tubulin subunits, both
Sample Sub G1% G0/G1% S% G2/M% the chains were considered for molecular docking studies The
tubulin crystal structures were obtained from Protein Data Bank
A: Control (A549) 1.80 81.24 5.42 11.96
B: Nocodazole (2 mM) 2.06 38.93 3.34 54.72 (PDB code: 3E22) [40] and necessary corrections to the protein
C: 6d (1 mM) 1.98 76.63 6.79 15.18 were carried out using Protein Preparation Wizard from the Schro-
D: 6d (2 mM) 1.68 36.39 2.55 56.36 dinger Suite 2014-3. Compound 6d was sketched by using 2D
sketcher and Schrodinger’s LigPrep program was used to generate
different conformations of the ligand. Molecular docking studies
were performed by using a GLIDE docking module of Schrodinger
suite and the results were analyzed on the basis of the GLIDE dock-
ing score and molecular recognition interactions. The 3D figures
were obtained using Schrodinger Suite 2014-3 [41].
The close view of the potential binding pose of compound 6d is
illustrated Fig. 9 and it binds at the interface of a- and b-tubulin.
The docking results into colchicine binding site were consistent
with previous results found for colchicine and other colchicine
binding site inhibitors [42,43]. The interaction was strongly stabi-
lized by two hydrogen bonds, the first one by imidazo[2,1-b]thia-
zole with ASNa101 at distance of 2.31 Å. The second formed
between benzimidazole NH and Tyra224 at distance of 2.01 Å.
Fig. 5. Effect of compounds on tubulin polymerization: tubulin polymerization was
Most interactions of compound 6d was driven mainly by
monitored by the increase in fluorescence at 360 nm (excitation) and 420 nM
(emission) for 1 h at 37 °C. Nocodazole was used as the reference compound in this hydrophobic interactions with Ala12, Ser178, Thr179, Ala180,
study. Values indicated are the mean ± SD of two different experiments. and Gln183 amino acids of the a-tubulin and Leu248, Lys254,
Leu255, Asn258, Met325 and Lys 352 amino acids of the b-tubulin.

2.2.7. Molecular modeling 3. Conclusion

Tubulin polymerization assay suggests that imidazothiazole-
benzimidazole series exert their effects by inhibiting tubulin poly- In conclusion, we have designed, synthesized a series of
merization. To better understand how these analogues interact imidazothiazole-benzimidazole conjugates (6a–t) and evaluated
520 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526
Fig. 6. Compound 6d triggers mitochondrial injury. Drops in membrane potential (DWm) was assessed by JC-1 staining of A549 cells treated with test compound and
samples were then subjected to flow cytometry analysis on a FAC Scan (Becton Dickinson) in the FL1, FL2 channel to detect mitochondrial potential. A: Control cells (A549), B:
Nocodazole (1 mM) and C: 6d (1 mM) and D: 6d (2 mM).
then for their cytotoxic activity against four human cancer cell
lines, namely Hela (cervical), A549 (lung), MCF-7 (breast) and
DU-145 (prostate). Most of the compounds showed promising
cytotoxicity, whereas compound 6d showed significant cytotoxic-
ity against human lung cancer cell line (A549) with IC50 value
1.08 mM. The tubulin polymerization assay showed that compound
6d disrupted microtubule dynamics and induced abnormal spindle
structure and centrosome which causes cell cycle arrest at G2/M
phase. Moreover, biological studies that is mitochondrial mem-
brane potential and annexin V-FITC assay suggested that this com-
pound induced cell death by apoptosis in human lung cancer cells.
In addition, molecular docking studies rationalized the binding
modes of conjugate at the colchicine site of the tubulin. These
results suggest that imidazothiazole-benzimidazole conjugates
are potential cytotoxic agents that could be useful in the develop-
ment of newer leads for the treatment of lung cancer.
4. Experimental section
4.1. Chemistry
All reagent, starting materials, and solvents were purchased
from commercial sources and used without further purification.
1
H and 13C NMR spectra were recorded with 300, 400, and 500
MHz spectrometer in CDCl3 solutions with tetramethylsilane
(TMS) as an internal standard. High-resolution mass spectra (ESI-
HRMS) were obtained by using ESI-QTOF mass spectrometer. All
reactions were monitored by thin-layer chromatography (TLC)
using precoated silica gel 60 F254 Merck and components were
visualized by exposure to UV light. Column chromatography was
performed using silica gel (60–120 mesh). Melting points were
determined on an electrothermal melting point apparatus and
are uncorrected.
4.1.1. 6-Phenylimidazo[2,1-b]thiazole-5-carbaldehyde (4a)
The Vilsmeier reagent was prepared at 0–5 °C by dropping
POCl3 (2.8 mL, 18 mmol) into a stirred solution of DMF (2.2 mL)
in CHCl3 (8 mL). Compound 3 (1.5 g, 7.5 mmol) in CHCl3 (40 mL)
was added drop wise to the Vilsmeier reagent while maintaining
stirring and cooling. The reaction mixture was quenched with iced
water and neutralized with aq. 2 N NaOH solution at 0 °C. The solid
was filtered and dried to furnish 4a as a pale brown solid, 1.5 g,
88%. (20% ethyl acetate in petroleum ether); Melting point: 139–
141 °C. 1H NMR (400 MHz, CDCl3) d 9.91 (s, 1H), 8.40 (d, J = 4.4 H
z, 1H), 7.82–7.79 (m, 2H), 7.57–7.46 (m, 3H), 7.06 (d, J = 4.4 Hz,
1H); 13C NMR (125 MHz, CDCl3) d 178.1, 156.5, 155.1, 133.5,
133.3, 132.6, 130.9, 127.5, 124.3, 123.3, 121.2, 115.2; HRMS (ESI)
calcd for C12H9ON2S [M+H]+ 229.04249, found 229.04301.
4.1.2. 6-(4-Methoxyphenyl)imidazo[2,1-b]thiazole-5-carbaldehyde
(4b)
Yellow solid, 1.3 g, 84%; (25% ethyl acetate in Petroleum ether);
Melting point: 138–140 °C; 1H NMR (500 MHz, CDCl3) d 9.88 (s,
1H), 8.38–8.37 (m, 1H), 7.77–7.73 (m, 2H), 7.10–6.89 (m, 3H),
3.88 (s, 3H); 13C NMR (125 MHz, CDCl3) d 177.9, 160.9, 158.2,
155.7, 130.4, 125, 123.7, 121.6, 114.5, 114.3, 55.4; MS (ESI): m/z
259 [M+H]+.
 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526 521

Fig. 7. Annexin V-FITC staining. A: Control cells (A549), B: Nocodazole (1 mM) and C: 6d (1 mM) and D: 6d (2 mM).

Table 3
Annexin V-FITC assay. benzene-1,2-diamine (5a, 37.8 mg, 0. 350 mmol) and sodium
Sample UL% UR% LL% LR% metabisulfite (66.6 mg, 350 mmol) were added. The reaction mix-
A: Control (A549) 0.35 9.45 84.74 5.46 ture was allowed to stir at 80 °C for 8 h, and was monitored by TLC
B: Nocodazole (2 mM) 0.06 31.77 58.51 9.66 using 50% ethyl acetate in hexanes as a solvent system. After com-
C: 6d (1 mM) 0.32 10.71 84.74 4.23 pletion of the reaction the solution was cooled to room tempera-
D: 6d (2 mM) 0.01 31.70 56.39 11.92 ture. The obtained precipitate was filtered and washed with
ethanol. The crude product was purified by using column chro-
matography using (40% ethyl acetate in Petroleum ether) as the
4.1.3. 6-(2-Bromophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde (4c) eluent to afford compound 6a as a Yellow solid, 83 mg, 77%; (40%
Pale yellow solid, 1.6 g, 81%; (20% ethyl acetate in Petroleum ethyl acetate in Petroleum ether); Melting point: 189–191 °C; 1H
ether); Melting point: 153–155 °C; 1H NMR (400 MHz, CDCl3) d NMR (500 MHz, CDCl3) d 9.19 (s, 1H), 8.72 (d, J = 4.4 Hz, 1H), 7.75
9.62 (s, 1H), 8.37 (d, J = 4.4 Hz, 1H), 7.74 (dd, J = 8.0, 1.1 Hz, 1H), (dd, J = 21.5, 8.1 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.34–7.27 (m,
7.55 (dd, J = 7.6, 1.7 Hz, 1H), 7.44 (td, J = 7.5, 1.2 Hz, 1H), 7.35 (td, 1H), 7.05 (t, J = 11.7 Hz, 2H), 6.94 (dd, J = 14.5, 6.7 Hz, 1H), 6.74–
J = 7.8, 1.8 Hz, 1H), 7.11 (d, J = 4.4 Hz, 1H); 13C NMR (125 MHz, 6.68 (m, 2H), 3.89 (s, 3H); 13C NMR (125 MHz, CDCl3) d 160.2,
CDCl3) d 178, 156.4, 155.1, 133.5, 133.3, 132.6, 130.9, 127.5, 151.6, 147.2, 143.7, 134.7, 130.1, 126.5, 121.7, 120.3, 116.8,
124.3, 123.3, 121.2, 115.2. MS (ESI): m/z 308 [M+H]+. 114.7, 112.4, 55.4; HRMS (ESI) calcd for C19H15ON4S [M+H]+
347.09657, found 347.09611.
4.1.4. 6-(Benzo[d][1,3]dioxol-5-yl)imidazo[2,1-b]thiazole-5-
carbaldehyde (4d)
Yellow solid, 1.5 g, 83%; (30% ethyl acetate in Petroleum ether); 4.1.6. (2-(6-(4-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1H-benzo
Melting point: 145–147 °C; 1H NMR (400 MHz) d 9.87 (s, 1H), [d]imidazol-6-yl)(phenyl)methanone (6b)
8.38–8.37 (m, 1H), 7.30–7.26 (m, 2H), 7.05–7.03 (m, 1H), 6.95– Yellow solid, 103 mg, 74%; (40% ethyl acetate in Petroleum
6.92 (m, 1H), 6.05 (s, 2H); 13C NMR (100 MHz, CDCl3) d 177.9, ether); Melting point: 196–198 °C; 1H NMR (400 MHz) d 9.39 (s,
158, 155.5, 149.2, 148.3, 126.5, 123.8, 123.5, 121.6, 114.4, 109.2, 1H), 8.74 (dd, J = 24.3, 4.2 Hz, 1H), 8.20 (s, 1H), 7.81 (dd, J = 15.3,
108.7, 101.6; MS (ESI): m/z 273 [M+H]+. 7.9 Hz, 3H), 7.66 (d, J = 8.1 Hz, 2H), 7.54–7.43 (m, 3H), 7.08–6.96
(m, 3H), 6.69 (d, J = 8.1 Hz, 1H), 3.89 (s, 3H); 13C NMR (100 MHz,
4.1.5. 5-(1H-Benzo[d]imidazol-2-yl)-6-(4-methoxyphenyl)imidazo CDCl3) d 196.5, 160.5, 138.8, 138.4, 132.1, 131.4, 130.1, 129.9,
[2,1-b]thiazole (6a) 129.6, 128.2, 128.1, 126.5, 126.2, 125.4, 121.8, 119, 114.8, 114.4,
To the stirred solution of 6-phenylimidazo[2,1-b]thiazole-5- 113.6, 112.8, 55.4; HRMS (ESI) calcd for C26H19O2N4S [M+H]+
carbaldehyde (3a, 80 mg, 0.350 mmol) in ethanol (10 mL), 451.12229, found 451.12232.
522 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526

Fig. 8. Hoechst staining in A549 lung cancer cell line. A: Control cells, B: Nocodazole (2 mM), C: 6d (1 mM) and D: 6d (2 mM).

Lys 352
Asn 258
Leu 255

Thr 179
Ala 180

Ser 178

Glh 183

Met 325
Asn 101

Leu 248

Lys 254

Tyr 224

Ala 12

Fig. 9. Binding modes of 6d in the colchicine binding site of tubulin (PDB ID code: 3E22) shown as a ball and stick. Interacting residues of a-and b-tubulin are shown as sticks
and pink dotted lines: hydrogen bond interaction.

4.1.7. 6-(4-Methoxyphenyl)-5-(6-methyl-1H-benzo[d]imidazol-2-yl)
imidazo[2,1-b]thiazole (6c)
Pale Yellow solid, 96 mg, 86%; (40% ethyl acetate in Petro-
leum ether); Melting point: 151–153 °C; 1H NMR 1H(500 MHz,
CDCl3) d 8.98 (s, 1H), 8.71 (d, J = 4.5 Hz, 1H), 7.66 (d, J = 8.7 H
z, 3H), 7.05 (dd, J = 15.8, 8.3 Hz, 4H), 6.95 (d, J = 4.5 Hz, 1H),
3.89 (s, 3H), 2.46 (s, 3H); 13C NMR (125 MHz, CDCl3) d 160.3,
151.5, 146.9, 130.1, 126.6, 121.7, 114.7, 114.2, 112.4, 55.4,
21.7; HRMS (ESI) calcd for C20H17ON4S [M+H]+ 361.11227, found
361.11176.
 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526
4.1.8. 6-(4-Methoxyphenyl)-5-(6-(trifluoromethyl)-1H-benzo[d]
imidazol-2-yl)imidazo[2,1-b]thiazole (6d)
Brown solid, 91 mg, 71%; (40% ethyl acetate in Petroleum
ether); Melting point: 136–138 °C; 1H NMR (400 MHz) d 9.86 (s,
1H), 8.72 (d, J = 4.3 Hz, 1H), 8.37 (d, J = 4.4 Hz, 1H), 7.73 (d, J = 8.
7 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.49 (s, 1H), 7.09–6.96 (m, 3H),
3.88 (s, 3H); 13C NMR (100 MHz) d 177.9, 160.4, 148.1, 147.7,
144.2, 137.7, 130.5, 130.1, 125.7, 121.7, 117.6, 114.4, 113.1, 55.4;
HRMS (ESI) calcd for C20H16ON4F3S [M + 2]+ 417.09885, found
417.09914.
4.1.9. 5-(6-Bromo-1H-benzo[d]imidazol-2-yl)-6-(4-methoxyphenyl)
imidazo[2,1-b]thiazole (6e)
Brown solid, 91 mg, 69%; (40% ethyl acetate in Petroleum
ether); Melting point: 141–143 °C; 1H NMR (400 MHz) d 9.87 (s,
1H), 8.74–8.30 (m, 2H), 7.77–7.59 (m, 3H), 7.33 (d, J = 8.1 Hz,
1H), 7.02 (d, J = 5.8 Hz, 3H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 178, 160.4, 151.9, 147.6, 147.2, 146.7, 130.5, 130.2, 130.1, 126.5,
126.3, 125.9, 121.7, 118.5, 114.4, 112.6, 55.4; HRMS (ESI) calcd for
C19H14ON4BrS [M]+ 425.00713, found 425.00662.
4.1.10. 5-(6-Chloro-1H-benzo[d]imidazol-2-yl)-6-(4-methoxyphenyl)
imidazo[2,1-b]thiazole (6f)
Yellow solid, 78 mg, 67%; (40% ethyl acetate in Petroleum
ether); Melting point: 148–150 °C; 1H NMR (400 MHz) d 8.64–
8.52 (m, 1H), 7.66 (t, J = 8.8 Hz, 2H), 7.26 (s, 1H), 6.98 (dt, J =
18.0, 10.4 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.80–6.64 (m, 1H), 3.87
(s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO) d 164.9, 150.7, 148.3,
141.1, 136.6, 134.8, 131.1, 130.7, 127.5, 126.7, 125.7, 122.1,
121.7, 120.8, 119.1, 118.2, 117.6, 60.1; HRMS (ESI) calcd for C19H14-
ON4BrS [M]+ 425.00713, found 425.00662.
4.1.11. 5-(1H-Benzo[d]imidazol-2-yl)-6-(p-tolyl)imidazo[2,1-b]
thiazole (6g)
Yellow solid, 90 mg, 82%; (40% ethyl acetate in Petroleum
ether); Melting point: 175–177 °C; 1H NMR (400 MHz) d 9.24 (s,
1H), 8.74 (d, J = 4.5 Hz, 1H), 8.66 (s, 1H), 8.62 (d, J = 4.4 Hz, 1H),
7.64 (d, J = 8.1 Hz, 2H), 7.31 (dd, J = 16.3, 7.9 Hz, 2H), 7.06 (td, J =
7.8, 1.3 Hz, 1H), 6.98–6.93 (m, 1H), 6.82–6.70 (m, 1H), 2.42 (s,
3H); 13C NMR (100 MHz, CDCl3) d 153.5, 146.6, 141.4, 138.7,
138.3, 130.6, 129.9, 129.5, 128.7, 128.6, 127.2, 121.8, 118.8,
117.4, 115.4, 112.9, 21.2; HRMS (ESI) calcd for C19H15N4S [M+H]+
331.10031, found 331.10119.
4.1.12. 5-(5-Methyl-1H-benzo[d]imidazol-2-yl)-6-(p-tolyl)imidazo
[2,1-b]thiazole(6h)
Brown solid, 87 mg, 77%; (40% ethyl acetate in Petroleum
ether); Melting point: 172–174 °C; 1H NMR (500 MHz, CDCl3) d
9.90 (s, 1H), 8.73–8.61 (m, 2H), 7.64 (d, J = 6.5 Hz, 2H), 7.40–7.18
(m, 2H), 7.00–6.80 (m, 2H), 6.67–6.51 (m, 1H), 2.44 (d, J = 19.2 H
z, 3H), 2.27 (d, J = 16.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
153.1, 145.6, 141.3, 138.6, 137.3, 135.8, 130.7, 130, 129.5, 128.7,
122.1, 121.8, 119.6, 117.1, 116.1, 112.8, 112.4, 21; HRMS (ESI)
calcd for C20H17N4S [M+H]+ 345.11603, found 345.11684.
4.1.13. 5-(5,6-Dichloro-1H-benzo[d]imidazol-2-yl)-6-(p-tolyl)imidazo
[2,1-b]thiazole (6i)
Brown solid, 90 mg, 68%; (30% ethyl acetate in Petroleum
ether); Melting point: 171–172 °C; 1H NMR (400 MHz) d 9.89 (s,
1H), 8.57–8.52 (m, 2H), 7.61 (t, J = 6.0 Hz, 1H), 7.31 (d, J = 7.8 Hz,
1H), 6.98 (d, J = 5.3 Hz, 2H), 6.85 (s, 2H), 2.43 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 154.7, 154.1, 147.3, 140.9, 139.2, 137.6,
129.7, 128.8, 121.7, 121.1, 118.6, 115.9, 113.3, 21.4; HRMS (ESI)
calcd for C19H15Cl2N4S [M + 2]+ 401.03751, found 401.03890.
523
4.1.14. 6-(3,4-Dichlorophenyl)-5-(6-methyl-1H-benzo[d]imidazol-2-
yl)imidazo[2,1-b]thiazole (6j)
Green solid, 79 mg, 73%; (30% ethyl acetate in Petroleum ether);
Melting point: 181–183 °C; 1H NMR (400 MHz) d 8.61 (d, J = 4.4 Hz,
1H), 7.85 (t, J = 9.4 Hz, 1H), 7.57–7.50 (m, 2H), 6.98 (dd, J = 8.3, 4.5
Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.66–6.54 (m, 2H), 2.29 (s, 3H); 13C
NMR (100 MHz) d 153.38, 149.85, 144.34, 141.45, 137.91, 135.34,
133.55, 132.8, 130.7, 130.4, 127.7, 121.8, 119.2, 116.9, 113.5,
113.2, 21.3; HRMS (ESI) calcd for C19H13N4Cl2S [M+H]+
399.02257, found 399.02325
4.1.15. 6-(3,4-Dichlorophenyl)-5-(5-methoxy-1H-benzo[d]imidazol-
2-yl)imidazo[2,1-b]thiazole (6k)
Brown solid, 73 mg, 65%; (30% ethyl acetate in Petroleum
ether); Melting point: 147–149 °C; 1H NMR (500 MHz, CDCl3) d
9.90 (s, 1H), 8.61–8.50 (m, 2H), 7.85 (dd, J = 7.7, 1.6 Hz, 1H),
7.54–7.47 (m, 2H), 6.99–6.92 (m, 2H), 6.41–6.29 (m, 1H), 3.79 (s,
3H); 13C NMR (101 MHz, CDCl3) d 159.8, 143.1, 142.8, 134.1,
132.7, 130.8, 130.7, 130.3, 127.7, 127.3, 121.7, 121.2, 117.7,
113.5, 113.3, 104.3, 100.9, 55.8; HRMS (ESI) calcd for C19H13ON4OS
[M+H]+ 415.01709, found 415.01816.
4.1.16. 5-(6-Fluoro-1H-benzo[d]imidazol-2-yl)-6-phenylimidazo[2,1-
b]thiazole (6l)
Yellow solid, 90 mg, 77%; (40% ethyl acetate in Petroleum
ether); Melting point: 137–139 °C; 1H NMR (400 MHz, CDCl3) d
9.13 (s, 1H), 8.70 (dd, J = 15.4, 5.3 Hz, 1H), 8.64–8.57 (m, 1H),
7.75–7.72 (m, 1H), 7.57–7.42 (m,5H), 6.99 (dd, J = 5.8, 4.5 Hz,
1H), 6.52–6.39 (m, 1H); 13C NMR (126 MHz, CDCl3) d 145.98,
134.2, 129.3, 129.2, 128.9, 128.8, 128.7, 121.7, 117.9, 114.2,
113.3, 112.8, 104.8, 102; HRMS (ESI) calcd for C18H12N4FS [M+H]+
335.07526, found 335.07612.
4.1.17. Phenyl(2-(6-phenylimidazo[2,1-b]thiazol-5-yl)-1H-benzo[d]
imidazol-6-yl)methanone (6m)
Brown solid, 115 mg, 78%; (40% ethyl acetate in Petroleum
ether); Melting point: 143–145 °C; 1H NMR (400 MHz) d 9.90 (s,
1H), 9.44 (d, J = 15.1 Hz, 1H), 8.73 (dt, J = 9.6, 3.7 Hz, 1H), 8.61
(dd, J = 12.2, 4.5 Hz, 1H), 7.85–7.70 (m, 5H), 7.60–7.41 (m, 5H),
7.00 (d, J = 4.4 Hz, 1H), 6.72 (dd, J = 30.7, 8.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 195.4, 178.2, 154.2, 153.8, 147.7, 146.1,
140.7, 138.7, 138.4, 137.4, 134.1, 132.1, 131.5, 131, 129.9, 128.8,
127.6, 125.6, 122.5, 121.7, 119.6, 118.9, 114.4, 113.39, 110.5;
HRMS (ESI) calcd for C25H17ON4S [M+H]+ 421.11180, found
421.11176.
4.1.18. 6-(Benzo[d][1,3]dioxol-5-yl)-5-(1H-benzo[d]imidazol-2-yl)
imidazo[2,1-b]thiazole (6n)
Yellow solid, 79 mg, 75%; (40% ethyl acetate in Petroleum
ether); Melting point: 177–179 °C; 1H NMR (400 MHz, CDCl3) d
9.39 (s, 1H), 8.71 (d, J = 4.4 Hz, 1H), 7.26–7.21 (m, 2H), 7.19 (d, J
= 6.6 Hz, 2H), 6.96 (d, J = 4.5 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.71
(s, 1H), 6.03 (s,2H); 13C NMR (100 MHz, CDCl3) d 151.5, 148.4,
148.3, 146.8, 143.5, 128, 122.4, 121.7, 120.3, 116.8, 114.1, 112.6,
109.2, 108.8, 101.5; HRMS (ESI) calcd for C19H13O2N4S [M+H]+
361.07473, found 361.07537
4.1.19. 6-(Benzo[d][1,3]dioxol-5-yl)-5-(5,6-dichloro-1H-benzo[d]
imidazol-2-yl)imidazo[2,1-b]thiazole (6o)
Brown solid, 89 mg, 70%; (40% ethyl acetate in Petroleum
ether); Melting point: 194–196 °C; 1H NMR (400 MHz, CDCl3) d
9.87 (s, 1H), 8.53 (t, J = 2.2 Hz, 1H), 7.27–7.25 (m, 1H), 7.25–7.17
(m, 1H), 6.99 (t, J = 2.2 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.85 (s,
1H), 6.75 (s, 1H), 6.05 (d, J = 2.2 Hz, 2H). 13C NMR (100 MHz, CDCl3)
d 177.93, 154.32, 148.29, 147.22, 140.96, 137.63, 134.32, 129.87,
127.25, 123.13, 121.56, 118.61, 117.49, 115.94, 113.31, 109.13,
524 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526
108.79, 101.50; HRMS (ESI) calcd for C19H13O2N4Cl2S [M + 2]+
431.01252, found 431.01308.
4.1.20. 6-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methyl-1H-benzo[d]
imidazol-2-yl)imidazo[2,1-b]thiazole (6p)
Brown solid, 85 mg, 77%; (40% ethyl acetate in Petroleum
ether); Melting point: 148–150 °C; 1H NMR (500 MHz, CDCl3) d
9.26 (s, 1H), 8.68 (s, 1H), 7.76–7.46 (m, 1H), 7.17 (s, 2H), 7.07 (d,
J = 7.8 Hz, 1H), 6.94 (s, 1H), 6.89 (s, 1H), 6.03 (s, 2H), 2.47 (s, 3H);
13
C NMR (100 MHz) d 151.3, 148.3, 148.2, 146.5, 143.2, 127.9,
124.3, 122.3, 121.6, 114.1 112.5, 109.2, 108.8, 101.5, 21.7. MS
(ESI): m/z 375 [M+H]+.
4.1.21. 5-(1H-Benzo[d]imidazol-2-yl)-6-(2-bromophenyl)imidazo
[2,1-b]thiazole (6q)
Yellow solid, 71 mg, 69%; (30% ethyl acetate in Petroleum
ether); Melting point: 148–150 °C; 1H NMR (500 MHz, CDCl3) d
8.82 (d, J = 4.4 Hz, 1H), 8.46 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.76
(s, 1H), 7.61 (dd, J = 7.5, 1.7 Hz, 1H), 7.51 (dd, J = 10.6, 4.3 Hz,
1H), 7.45–7.41 (m, 1H), 7.24 (s, 2H), 7.04 (d, J = 4.5 Hz, 1H), 6.71
(s, 1H); 13C NMR (100 MHz) d 151.4, 145.3, 144.8, 143.2, 135.1,
133.8, 132.5, 131.1, 128.2, 124.3, 121.8, 120.3, 116.8, 115.8,
113.1; HRMS (ESI) calcd for C18H12N4BrS [M]+ 394.99556, found
394.99606.
4.1.22. (4-(5-(1H-benzo[d]imidazol-2-yl)imidazo[2,1-b]thiazol-6-yl)
phenyl)(phenyl)methanone (6r)
Yellow solid, 81 mg, 78%; (30% ethyl acetate in Petroleum
ether); Melting point: 154–156 °C; 1H NMR (400 MHz, CDCl3) d
8.57 (d, J = 4.3 Hz, 1H), 8.40 (s, 1H), 7.85–7.75 (m, 2H), 7.74–7.68
(m, 2H), 7.54 (t, J = 7.5 Hz, 4H), 7.45 (d, J = 7.6 Hz, 2H), 7.05 (d, J
= 4.4 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H); 13C NMR (100 MHz, 3) d
195.4, 147.5, 146.1, 138.7, 137.3, 133.9, 133.5, 132.7, 132.4,
132.1, 131.5, 131.1, 130.5, 129.9, 129.5, 128.2, 128.1, 127.5,
123.4, 122.6, 121.4, 119.8, 113.8, 113.4; H RMS (ESI) calcd for C25-
H16ON4BrS [M]+ 499.02151, found 499.02227.
4.1.23. 6-(2-Bromophenyl)-5-(6-chloro-1H-benzo[d]imidazol-2-yl)
imidazo[2,1-b]thiazole (6s)
Yellow solid, 73 mg, 65%; (30% ethyl acetate in Petroleum
ether); Melting point: 159–161 °C; 1H NMR (500 MHz, CDCl3) d
8.77 (dd, J = 4.1, 2.2 Hz, 1H), 8.47 (d, J = 14.1 Hz, 1H), 7.82 (dd, J =
8.0, 0.8 Hz, 1H), 7.73 (s, 1H), 7.68–7.58 (m, 1H), 7.51 (td, J = 7.5,
1.1 Hz, 1H), 7.43 (td, J = 7.8, 1.7 Hz, 1H), 7.21 (dd, J = 8.5, 1.5 Hz,
1H), 7.16 (s, 1H), 7.05 (d, J = 3.9 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d 151.6, 145.6, 134.9, 133.9, 132.4, 131.2, 128.2, 124.2, 123.5,
121.7, 119.9, 119.1, 115.4, 113.3, 111.3, 110.7; HRMS (ESI) calcd
for C18H11N4BrClS [M]+ 428.95578, found 428.95708.
4.1.24. 6-(2-Bromophenyl)-5-(6-methyl-1H-benzo[d]imidazol-2-yl)
imidazo[2,1-b]thiazole (6t)
Yellow solid, 86 mg, 81%; (30% ethyl acetate in Petroleum
ether); Melting point: 153–155 °C; 1H NMR (400 MHz, CDCl3) d
8.79 (d, J = 4.5 Hz, 1H), 8.50–8.28 (m, 1H), 7.81 (dd, J = 8.0, 1.1
Hz, 1H), 7.59 (dt, J = 15.1, 7.5 Hz, 2H), 7.49 (td, J = 7.5, 1.2 Hz,
1H), 7.41 (td, J = 7.7, 1.8 Hz, 1H), 7.05 (s, 1H), 7.01 (d, J = 4.5 Hz,
1H), 2.43 (s, 3H); 13C NMR (100 MHz, CDCl3) d 151.3, 135.2,
133.8, 133.3, 132.5, 131.1, 128.1, 124.7 124.4, 124.1, 121.8, 119.1,
118.7, 115.9, 112.9, 110.54, 110.1, 21.7; HRMS (ESI) calcd for C19-
H14N4BrS [M+H]+ 409.01042, found 409.01171.
4.2. Biology
4.2.1. MTT assay
The cytotoxic activity of the compounds was determined using
MTT assay [32] 1  104 cells/well were seeded in 100 ml DMEM
(Dulbecco’s Modified Eagle’s medium)/MEM (Minimum Essential
Medium), supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 °C in a CO2 incu-
bator. After 24 h of incubation, all the synthesized compounds
were added to the cells and incubated for 48 h. After 48 h of drug
treatment, 10 ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) (5 mg/mL) was added to each well and the
plates were further incubated for 4 h. Then the supernatant from
each well was carefully removed, formazon crystals were dissolved
in 100 ml of DMSO and absorbance at 570 nm wavelength was
recorded.
4.2.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the
distribution of the cells through the cell cycle phases. A549 cells
were treated with compound 6d at 1 and 2 mM concentrations
for 48 h. Untreated and treated cells were harvested, washed with
phosphate buffered saline (PBS), fixed in ice-cold 70% ethanol, and
stained with propidium iodide (Sigma–Aldrich). Cell cycle analysis
was performed by flow cytometry (Becton Dickinson FACS Caliber
instrument) [34].
4.2.3. Tubulin polymerization assay
A fluorescence based in vitro tubulin polymerization assay was
performed according to the manufacturer’s protocol (BK011,
Cytoskeleton, Inc.). Briefly, the reaction mixture in a total volume
of 10 ml contained PEM buffer, GTP (1 mM) in the presence or
absence of test compounds (final concentration of 3 mM). Tubulin
polymerization was followed by a time dependent increase in flu-
orescence due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission
at 420 nm (excitation wavelength is 360 nm) was measured by
using a Varioscan multimode plate reader (Thermo scientific
Inc.). Nocodazole was used as reference compound. The IC50 value
was defined as the drug concentration required inhibiting 50% of
tubulin assembly compared to control. The reaction mixture for
these experiments include: tubulin (3 mg/ml) in PEM buffer, GTP
(1 mM), in the presence or absence of test compounds at varying
concentrations. Polymerization was monitored by increase in the
fluorescence as mentioned above at 37 °C [36,37].
4.2.4. Measurement of mitochondrial membrane potential (DWm)
A549 (1  106 cells/well) cells were cultured in six well plates.
After plating, cells were treated with compound 6d at 1 and
2 mM concentrations for 48 h. After 48 h of treatment, cells were
collected by trypsinization and washed with PBS followed
by resuspending in JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-
benzimidazolocarbocyanine iodide) and incubated at 37 °C for
15 min. The cells were then subjected to flow cytometric analysis
on a flow cytometer (Becton Dickinson) in the FL1, FL2 channel
to detect mitochondrial potential [38].
4.2.5. Annexin V-FITC assay for apoptosis
A549 (1  106) cells were seeded in six well plates and allowed
to grow overnight. The medium was then replaced with complete
medium containing compound 6d at 1 and 2 mM concentrations for
48 h. After 48 h of drug treatment, cells from the supernatant and
adherent monolayer cells were harvested by trypsinization,
washed with PBS at 5000 rpm. Then the cells were stained with
Annexin VFITC and propidium iodide using the Annexin-V-FITC
apoptosis detection kit (Sigma aldrich). Then the samples were
analyzed by flowcytometry as described earlier [44].
4.2.6. Hoechst staining
A549 cells were seeded at a density of 10,000 cells over 18 mm
cover slips and incubated for 24 h. After incubation, cells were
 M.F. Baig et al. / Bioorganic Chemistry 77 (2018) 515–526
treated with the compound 6d at 1 and 2 lM concentration for 48
h. Hoechst 33258 (Sigma Aldrich) was added to the cells at a con-
centration of 0.5 mg/mL and incubated for 30 min at 37 °C. Later,
the cells were washed with phosphate buffered saline (PBS). Cells
from each cover slip were captured from randomly selected fields
under fluorescent microscope (Olympus microscope) to qualita-
tively determine the proportion of viable and apoptotic cells based
on their relative fluorescence and nuclear fragmentation [45].
4.2.7. Molecular modeling
The 3D coordinates of the tubulin crystal structures was
obtained from Protein Data Bank (PDB ID code: 3E22) [46]. The
PDB protein-ligand structures were processed with the Protein
Preparation Wizard in the Schrödinger suite. The protein structure
integrity was checked and adjusted, and missing residues and loop
segments near the active site were added using Prime. A 3D box
was generated around each ligand to enclose the entire vicinity
of active site. The receptor grid for each target was prepared with
the help of OPLS-2005 force field. The grid center was set to be the
centroid of the co-crystallized ligand, and the cubic grid had a size
of 20 Å. 3D structures were generated by Schrödinger suite.
Schrödinger’s LigPrep program was used to generate different con-
formations of ligands. Molecular docking studies were performed
by using a GLIDE docking module of Schrodinger suite. For the val-
idation of docking protocol, the cocrystalized ligand (Colchicine in
3E22) was subjected to re-docking into the tubulin dimer (PDB
code: 3E22) using GLIDE. The bound and docked conformations
of Colchicine showed similar interactions and binding pose at their
respective binding sites. Finally, prepared ligands were docked into
the generated receptor grids using Glide SP docking precision. The
results were analyzed on the basis of the GLIDE docking score and
molecular recognition interactions.
Acknowledgements
The authors thanks CSIR – India and UGC, New Delhi, India for
the award of fellowships. We also thanks funding received from
the project, entitled ‘Affordable Cancer Therapeutics (ACT)’ under
XIIth five-year plan.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bioorg.2018.02.005.
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