Design and synthesis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.04.013

1,2,3-Triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a–v) were designed, synthesized and evaluated for their cytotoxic potency against some human cancer cell lines like DU-145 (prostate), HeLa (cervical), MCF-7 (breast) HepG2 (liver) and A549 (lung). Preliminary results revealed that some of these conjugates like 5f and 5k exhibited significant antiproliferative effect against human breast cancer cells (MCF-7) with IC₅₀ values of 0.60 and 0.78?μM respectively. Flow cytometric analysis of the cell cycle demonstrated an increase in the percentage of cells in the G₂/M phase which was further authenticated by elevation of cyclin B1 protein levels. Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 5f and 5k, and western blot analysis revealed that these conjugates accumulated more tubulin in the soluble fraction. Moreover, the conjugates caused apoptosis of the cells that was confirmed by mitochondrial membrane potential and Annexin V-FITC assay. Molecular docking studies indicated that these conjugates occupy the colchicine binding site of the tubulin protein.

Full text of DOI:10.1016/j.bmc.2017.04.013

Accepted Manuscript
Design and Synthesis of 1,2,3-Triazolo Linked Benzo[d]imidazo[2,1-b]thiazole
Conjugates as Tubulin Polymerization Inhibitors
Siddiq Pasha Shaik, M.V.P.S. Vishnuvardhan, Faria Sultana, A.V. Subba Rao,
Chandrakant Bagul, Debanjan Bhattacharjee, Jeevak Sopanrao Kapure, Nishant
Jain, Ahmed Kamal
PII:
S0968-0896(16)31384-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.013
BMC 13683
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
6 December 2016
11 April 2017
Please cite this article as: Shaik, S.P., Vishnuvardhan, M.V.P.S., Sultana, F., Subba Rao, A.V., Bagul, C.,
Bhattacharjee, D., Kapure, J.S., Jain, N., Kamal, A., Design and Synthesis of 1,2,3-Triazolo Linked
Benzo[d]imidazo[2,1-b]thiazole Conjugates as Tubulin Polymerization Inhibitors, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.013
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Design and Synthesis of 1,2,3-Triazolo Linked Benzo[d]imidazo[2,1-b]thiazole
Conjugates as Tubulin Polymerization Inhibitors
Siddiq Pasha Shaik,a,b M.V.P.S. Vishnuvardhan,a Faria Sultana,a A.V. Subba Rao,a,b Chandrakant
Bagul,d Debanjan Bhattacharjee,c Jeevak Sopanrao Kapure,d Nishant Jainc and Ahmed
Kamal*a,b,d,e
aMedicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical
Technology (IICT), Hyderabad-500007, India
b Academy of Scientific and Innovative Research, New Delhi, 110 025, India
cCentre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad
500007, India
dDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education &
Research (NIPER), Hyderabad 500 037, India
eCatalytic Chemistry Research Chair, Chemistry Department, College of Science, King Saud
University, Riyadh 11451, Saudi Arabia
Abstract:
1,2,3-Triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v) were designed,
synthesized and evaluated for their cytotoxic potency against some human cancer cell lines like
DU-145 (prostate), HeLa (cervical), MCF-7 (breast) HepG2 (liver) and A549 (lung). Preliminary
results revealed that some of these conjugates like 5f and 5k exhibited significant
antiproliferative effect against human breast cancer cells (MCF-7) with IC50 values of 0.60 and
0.78 µM respectively. Flow cytometric analysis of the cell cycle demonstrated an increase in the
percentage of cells in the G2/M phase which was further authenticated by elevation of cyclin B1
protein levels. Immunocytochemistry revealed loss of intact microtubule structure in cells treated
with 5f and 5k, and western blot analysis revealed that these conjugates accumulated more
tubulin in the soluble fraction. Moreover, the conjugates caused apoptosis of the cells that was
confirmed by mitochondrial membrane potential and Annexin V-FITC assay. Molecular docking
studies indicated that these conjugates occupy the colchicine binding site of the tubulin protein.
Keywords: benzo[d]imidazo[2,1-b]thiazole; 1,2,3-triazole; cytotoxicity; tubulin
depolymerization; apotosis
Corrosponding author: Dr. Ahmed Kamal
Email: ahmedkamal@iict.res.in
Tel: 040-27191633

1. Introduction
Antimitotic agents are a major class of cytotoxic drugs for the treatment of cancer and
tubulin is a target for numerous small natural and synthetic molecules that inhibit the formation
of the mitotic spindle.1,2 Tubulin-containing structures, such as microtubules are responsible for
the formation of the mitotic spindle during the process of mitosis. The microtubule system of
eukaryotic cells is a critical element in a variety of essential cellular processes in addition to
mitotic spindle assembly, including determination and maintenance of cell shape, regulation of
motility, cell signaling, secretion and intracellular transport.3 The drugs that target microtubules
and bind to the tubulin binding sites either stabilize or destabilize the microtubule assembly.
There are four main binding sites of tubulin4 which includes taxane and laulimalide/pelouside A
sites for the microtubule stabilizing agents,5-7 vinca8 and colchicine domains9,10 for the
destabilizing agents. They interfere with the dynamic stability of microtubules and act as spindle
poisons arresting the dividing cells in G2/M phase of the cell cycle, causing mitotic catastrophy
and finally leading to apoptotic cell death.11 Therefore, there is a growing interest in identifying
and developing newer molecules that could inhibit tubulin polymerization.
Imidazole ring is an important five-membered aromatic heterocycle which is widely
present in natural products and synthetic molecules. The unique structural feature of the
imidazole ring is that it allows to readily bind with a variety of enzymes and receptors in
biological systems through diverse weak interactions, thereby exhibiting broad bioactivities like
antimicrobial,12 anticonvulsant,13 sodium channel blocking,14 anti-inflammatory,15 CB1 receptor
inverse agonist16 and anticancer.17-19 In contrast, among the benzoheterocycles, benzothiazole has
a considerable place in the synthetic as well as in pharmaceutical chemistry because of its potent
and significant pharmacological activities including antimicrobial,20 anti-inflammatory,21
immunosuppressive,22 antiallergenic23 and anticancer.24,25 Nitrogen-bridge head fused
heterocycles containing an imidazole and benzothiazole ring are a common structural moiety in
many pharmacologically important molecules that display a wide range of activities for diverse
targets. Among them, benzo[d]imidazo[2,1-b]thiazole class of compounds, 2-aryl
benzo[d]imidazo[2,1-b]thiazole derivative (1, YM-201627, Figure 1) was found to be potent
orally active antitumor agent useful for the treatment of solid tumors.26 Another derivative (2,
AC220, Figure 1) was found to be highly active against FMS-like tyrosine kinase-3 (FLT3) and
has entered phase III clinical trials.27 Recently, a series of Mannich bases of 2-
arylbenzo[d]imidazo[2,1-b]thiazoles28 and earlier studies in this laboratory on 3-substituted 2-
phenylbenzo[d]imidazo[2,1-b]thiazole 29 (3, Figure 1) showed significant cytotoxic activity with

the ability to induce apoptosis through inhibition of tubulin polymerization. Whereas, 1,2,3-
triazoles have attracted the medicinal chemists,30-32 due to their wide range of biological
activities and high metabolic stability as well as hydrogen-bonding capability with bimolecular
targets.33-37 Recently, we reported 1,2,3-triazolo linked imidazo[2,1-b]thiazole conjugates with
potential cytotoxic and inhibition of tubulin polymerization activity (4, Fig. 1).38
In continuation of our ongoing efforts on the benzo[d]imidazo[2,1-b]thiazole scaffold as
tubulin inhibitors and apart from the encouraging results obtained by us for the 1,2,3-triazolo
linked imidazo[2,1-b]thiazole compounds,38 herein we report the synthesis of 1,2,3-triazolo
linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v) and the strategy of design is described
in Figure 2. These conjugates were evaluated for their cytotoxy potential and effect on tubulin
polymerization, apart from the cell cycle analysis to understand their possible mechanism of
action.
Figure 1: Chemical structures of some anticancer molecules: YM-201627 (1), AC220 (2), 2-
phenylbenzo[d]imidazo[2,1-b]thiazole derivative (3), 1,2,3-triazolo linked imidazo[2,1-
b]thiazole conjugates (4) and 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates
(5a-v).
Figure 2: Design strategy of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates.

2. Results and Discussion
2.1. Chemistry
These 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v) were
synthesized as depicted in Scheme 1. First we have initiated our synthesis with the preparation of
benzo[d]imidazo[2,1-b]thiazole derived alkyne motif’s. The condensation of substituted 2-
aminobenzothiazoles (6a-d) with the ethylbromopyruvate (7) in 1,2-dimethoxyethane (DME)
under reflux conditions afforded substituted benzo[d]imidazo[2,1-b]thiazole ester (8a-d), which
upon sodium hydroxide (NaOH) mediated ester hydrolysis gave the corresponding
benzo[d]imidazo[2,1-b]thiazole acids (9a-d).39 These were coupled with propargylamine
hydrochloride in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDCI), triethyl amine (TEA) and 1-hydroxybenzotriazole (HOBT) in dry
dimethylformamide (DMF) under nitrogen atmosphere to provide the corresponding
benzo[d]imidazo[2,1-b]thiazole terminal alkynes (10a-d). The other subunit, benzyl azides (13a-
f) were prepared from benzyl bromides (12a-f) with sodium azide (NaN3) in DMSO.40 Finally,
we obtained the desired 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v)
in high regioselectively by exposing benzo[d]imidazo[2,1-b]thiazole terminal alkynes (10a-d) to
benzyl azides (13a-f) in the presence of catalytic copper(׀׀) sulfate pentahytrate (CuSO4·5H2O)
and sodium ascorbate in a H2O⁄t-butyl alcohol (t-BuOH) mixture at room temperature in good to
excellent yields (65-90%). All the structures of these newly synthesized conjugates (5a-v) were
confirmed by spectral data (1H NMR, 13C NMR, MS and HRMS).
Scheme 1:

Scheme 1: Synthesis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates:
Reagents and conditions: (a) DME, reflux, 8 h, 55-67%; (b) ∆, EtOH (hydrolysis: (i) NaOH,
H2O, EtOH, (ii) HCl), 70-75%; (c) EDCI, HOBt, propargylamine hydrochloride, Et3N, DMF, rt,
8 h, 75-79%; (d) PBr3, CH2Cl2, 0 °C, 30 min; (e) NaN3, DMSO, rt, 12 h; (f) sodium ascorbate
(10 mol%), CuSO4.5H2O (5 mol %), H2O/t-BuOH, (2 : 1), rt, 5 h, 65-90%.
2.2. Biological activity
2.2.1. Antiproliferative activity
MTT assay41 was performed to evaluate the cytotoxic effects of these conjugates against
selected human cancer cell lines, namely, prostate (DU-145), cervical (HeLa), breast (MCF-7),
liver (HepG2) and lung adenocarcinoma (A549) using doxorubicin as a reference compound and
the IC50 values are presented in Table 1. All the synthesized conjugates (5a-v) displayed
significant antiproliferative activity against MCF-7 cell line when compared to other tested cell
lines in micro to submicromolar range except 5b, 5c and 5r. Amongst them 5d, 5f, 5i, 5k and 5q
showed impressive cytotoxicity with IC50 values of 1.412, 0.607, 1.061, 0.781 and 0.977 µM
respectively against MCF-7 cell line.
In order to investigate the structure activity relationship (SAR), we have varied the
substitution pattern of the ring A of benzo[d]imidazo[2,1-b]thiazole and benzyl ring (ring D)
linked to the 1,2,3-triazole ring of these conjugates. The cytotoxicity data of compounds 10a-d

deciphered that, by increasing the electron-donating nature on ring A of the
benzo[d]imidazo[2,1-b]thiazole scaffold decreases the activity particularly againt MCF-7 cell
line when compared to other tested cell lines. In contrast, the benzyl ring (ring D) of these
conjugates that has an electron withdrawing group like p-F (5f, 5k, 5q and 5v) as well as
electron donating groups like 3,4,5-trimethoxy (5d, 5i and 5o) showed significant
antiproliferative activity when compared to m-mono and disubstituted (3,4 and 3,5) electron
donating group on these conjugates. It was observed that electron withdrawing p-F group showed
slightly improved activity in comparison to electron donating group like 3,4,5-trimethoxy
substituted conjugates. Most of the corresponding conjugates showed enhanced cytotoxicities
compared to benzo[d]imidazo[2,1-b]thiazole alkynes (10a-d) particularly againt MCF-7 cell line
except 5a, 5b, 5c and 5r. The activity of these conjugates like 5f, 5k and 5q improved almost by
10 folds than their alkyne intermediates (10a-d). Some of the 1,2,3-triazolo linked
benzo[d]imidazo[2,1-b]thiazole conjugates like 5d, 5f, 5i, 5k, 5o, 5q and 5v showed comparable
or improved activity than YM-201627.28 And 5f and 5k conjugates displayed promising
cytotoxicity (IC50 values of 0.667 and 0.781 µM), these are superior or comparable to compound
3 (IC50 values of 0.778 µM). These results suggest that electron-donating substituents like H and
Me on ring A and electron withdrawing group like p-F and electron donating group like 3,4,5-
trimethoxy on ring D are essential for the cytotoxic activity as shown in Figure 3. Moreover
from the MTT assay, the most cytotoxic conjugates that is 5f and 5k were taken up for further
mechanistic studies, such as tubulin polymerization inhibition, immunohistochemistry and
apoptosis induction. In addition, these conjugates were also investigated for molecular docking
studies to understand their mode of binding.
Table 1: IC50 valuesa (in µM) for compounds 10a-d and 5a-v in human cancer cell lines.
Compound
10a
10b
10c
10d
5a
DU-145b
19.95
25.11
23.89
22.19
14.77
29.83
30.90
3.715
21.69
HeLac
16.25
14.79
20.05
20.74
12.41
15.31
27.58
3.311
14.79
MCF-7d
8.749
10.03
15.51
17.48
9.246
10.28
12.13
1.412
6.180
HepG2e
20.48
A549f
12.88
11.74
17.58
16.31
9.549
14.120
19.05
2.041
10.96
27.58
28.78
24.23
16.54
31.620
34.667
7.943
21.88
5b
5c
5d
5e

2.570
8.709
31.18
4.466
27.54
4.265
18.75
14.27
13.59
9.772
13.08
3.38
1.629
7.244
21.38
3.630
17.15
2.691
16.98
11.48
11.74
8.542
12.58
3.235
31.62
7.762
9.54
0.607
6.251
6.501
1.061
7.362
0.781
6.606
6.165
8.120
2.818
3.630
0.977
24.50
7.244
5.370
6.683
3.311
1.659
3.59
7.413
16.980
8.10
1.570
8.128
11.22
2.824
10.71
1.288
9.332
10.47
14.79
4.897
6.367
2.511
22.85
14.93
8.912
8.053
8.317
1.445
-
5f
5g
5h
4.897
35.77
6.606
26.895
22.450
15.031
17.370
30.19
6.688
34.60
15.963
18.70
18.14
17.310
2.511
2.02
5i
5j
5k
5l
5m
5n
5o
5p
5q
32.27
9.935
15.40
12.02
14.41
0.895
-
5r
5s
5t
15.10
12.58
2.089
4.30
5u
5v
Doxorubicin
YM-20162728
Note: a50% Inhibitory concentration and the values are average of three individual experiments
after 48 h of drug treatment. bprostate cancer, ccervical cancer, dbreast cancer, eliver cancer,
flung cancer.
Figure 3: SAR analysis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates.
2.2.2. Cell cycle analysis
To investigate the mechanism underlying the cytotoxic activity of 5f and 5k, the cell
cycle distribution in MCF-7 cancer cell line was analyzed by flow cytometry.42 In this study
MCF-7 cells were treated with 0.5 µM concentration for 48 h. The data obtained clearly
indicated that they show G2/M cell cycle arrest. Both 5f and 5k showed 33.9% and 32.6% of cell

accumulation in G2/M phase respectively, whereas in control (untreated cells) 2.6% of G2/M
phase was observed (Figure 4 and Table 2).
Figure 4: Flow cytometric analysis in MCF-7 cancer cell lines after treatment with compounds
at 0.5 µM concentrations for 48 h.
Table 2: Distribution of cells at G0/G1, S phase and G2/M phase of cell cycle.
Compound
% of cells in % of cells % of cells % of cells
Sub-
G1phase
3.2
in
in S phase in G2/M
phase
G1phase
83.2
54.4
54.1
Control
5f
11.2
8.1
2.6
3.6
33.9
32.6
2.9
9.4
5k
2.2.3. Effect on cellular cyclin-B1, CDK1 and Aurora B levels by immunoblot analysis
Cyclin-B1 is induced at the G2/M boundary to promote cell division. This protein is one
of the important regulatory proteins of mitosis and accumulation of cyclin-B1 is an indication for
G2/M arrest.43 Since these compounds arrest the cells at G2/M phase, we investigated their effect
on cyclin B1 protein levels. Thus, we treated the MCF-7 cells with the lead compounds 5f and
5k at 1 µM concentrations for 24 h and performed the immunoblot analysis for cyclin-B1 and

tubulin as loading control. Therefore, the accumulation of cyclin-B1 levels suggests that these
conjugates demonstrate cytotoxic effect through cell cycle arrest at mitosis. This data suggests
that treatment by 5f and 5k induces a mitotic arrest, with the activation of spindle checkpoint in
24 h as shown in Figure 5. We have also analyzed Cdk1 and Aurora B kinases levels in MCF-7
cells which are the key kinases involved in G2/M cell cycle transition in mammalian cells. Cdk1
is activated by the cyclin-B1 and involved in the metaphase by activating motor proteins required
for spindle separation. Similarly Aurora B kinase is also involved in phosphorylation of histone
H3 protein and localization of some proteins at kinetochore which recognizes the spindle
attachment at chromosome. It also binds to EB1 protein which regulates the microtubule
dynamics at spindle 44,45. Accumulation of Cdk1 and aurora B in MCF-7 cells treated with 5f and
5k is comparable with the positive control nocodazole. Therefore, the increased levels of these
two kinases in comparision to the untreated control cells (Figure 5) demonstrate that MCF-7
cells treated with 5f and 5k arrest the cells at G2/M phase due to the inhibition of metaphase to
anaphase transition.
Figure 5: Western blot analysis of Cyclin-B1, CDK1 and Aurora B: Treatment of MCF-7 cells
with 1 µM concentrations of 5k and 5k for 24 h resulted an increase in cyclin B1, CDK1 and
Aurora B levels significantly. Tubulin was employed as loading control and DMSO as negative
control.
2.2.4. Effect on tubulin polymerization
To investigate whether the anti-proliferative activities of these conjugates were related to
the interaction with tubulin, we studied their effect on the tubulin polymerization in a cell-free

system. Moreover these conjugates showed significant effect on the G2/M cell cycle arrest, hence
it was considered of interest to investigate the tubulin polymerization aspect.46,47 As tubulin
subunits heterodimerize and self-assemble to form microtubules in a time dependent manner, we
have investigated the progression of tubulin polymerization by monitoring the increase in
fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate for 30 min
at 37 °C with and without the compounds at 3 µM concentration. Conjugates 5f and 5k inhibited
tubulin polymerization by 49.9%, 52.3 %, compared to the control (Figure 6). Tubulin
polymerization inhibition was also observed in case of the standards like nocodazole (51.7%).
Furthermore, these two potential conjugates (5f and 5k) were evaluated for their in vitro tubulin
polymerization assay at different concentrations. They showed potent inhibition of tubulin
polymerization with IC50 values 1.23 and 1.65 µM respectively compared to the control, where
as nocodazole was used as the positive control that showed an IC50 value of 1.32 µM (Table 3).

Figure 6: Effect of compounds on the tubulin polymerization: tubulin polymerization was
monitored by the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 30
min at 37 °C. All the compounds were included at a final concentration of 3 µM.
Table 3: Inhibition of tubulin polymerization (IC50) for conjugates 5f and 5k.
a
IC50 +S.D(µM)
S.No
Compound
1.23+0.29
1
2
3
5f
1.65+0.99
5k
Nocodazole
1.32+0.85
a Concentration of drug to inhibit 50% of tubulin assembly.
2.2.5. Immunohistochemistry studies on tubulin
To validate the effect of these compounds on the disruption of microtubule dynamics in
the living cells, studies were carried out to examine the in situ effects of 5f and 5k on cellular
microtubules. MCF-7 cells seeded on sterile cover slips were treated with these compounds and
nocodazole was employed as a standard at a concentration of 0.5 µM for 48 h. The confocal
images depicted in Figure 7 showed that the untreated lung cancer cell line displayed the normal
distribution of microtubules. However, cells treated with compounds 5f and 5k showed disrupted
microtubule organization as seen in Figure 7, thus demonstrating the inhibition of tubulin
polymerization. The density of microtubules was pronounced at the cell periphery with
disorganized central networks and the standard nocodazole also showed disrupted microtubule
organization. These data suggested that both 5f and 5k acts as a mitotic inhibitors by blocking
cell cycle progression and metaphase by its ability to disrupt spindle assembly.

Figure 7: Effect on microtubules and nuclear condensation: MCF-7 cells were independently
treated with 5f, 5k and nocodazole at 0.5 µM concentration for 24 h. Following the termination
of the experiment, cells were fixed and stained for tubulin. DAPI was used as the counter stain
and the merged images of cells stained for tubulin and DAPI are seen.
2.2.6. Distribution of soluble versus polymerized tubulin in cells
Since inhibition of tubulin polymerization disturbs the microtubule dynamics, we
evaluated the levels of soluble versus polymerized forms of tubulin in MCF-7 cells following
treatment with 1 µM of 5k and 5f for 24 h. In addition, cells were treated with nocodazole, as
positive and taxol as negative controls in parallel experiments. Western blot analysis revealed
that the amount of tubulin protein in both soluble and polymerized fractions was approximately
the same in DMSO treated cells. Nocodazole treated cells exhibited a shift of tubulin from the
polymerized fraction into the soluble fraction. In comparison, paclitaxel a microtubule
polymerization agent showed more amount of tubulin in the polymerized fraction. As expected
the cells treated with 5k and 5f significantly increased the tubulin content in the soluble fraction,
with almost all the tubulin present in the soluble fraction similar to that of the positive control.
Therefore, increased tubulin in soluble fraction of cells treated by these conjugates corroborated
with the inhibition of tubulin assembly and arrest of cells in G2/M phase as shown in Figure 8.

Figure 8: MCF-7 cells were treated with 1 µM of 5k and 5f for 48 h. Nocodazole and taxol were
used as reference standards. Levels of tubulin were detected by Western blot analysis.
2.2.7. Measurement of mitochondrial membrane potential (∆Ψm)
Mitochondria plays an essential role in the propagation of apoptosis and its dysfunction
within the apoptotic process, which is often associated with loss or depolarization of
mitochondrial membrane potential (DΨm) leading to collapse of mitochondrial functions
ensuing cell death.48 Therefore, in this study we examined the effect of conjugates 5f and 5k on
DΨm. In control cells the dye concentrates in the mitochondrial matrix where it forms red
fluorescent aggregates (J-aggregates) because of the electrochemical potential gradient. In cells
treated with compounds which induce apoptosis, the mitochondrial membrane is depolarized
thus preventing the accumulation of the JC-1 dye in the mitochondria. Therefore, the dye in the
monomeric form is dispersed throughout the entire cell leading to a shift from red (J-aggregates)
to green fluorescence (JC-1 monomers). Whereas JC-1 displays a red fluorescence (590 nm) with
normal cells (high ∆ψmt), which is caused by the impulsive and local formation of aggregates
that are related with a large shift in the emission. In contrast, when the mitochondrial membrane
is depolarized (low ∆ψmt), JC-1 forms monomers that emit at 530 nm. In this study MCF-7 cells
were treated with 5f and 5k at 0.5 µM concentration for 48 h and stained with JC-1 dye. Further
the loss of mitochondrial membrane potential was quantified by flow cytometry. From the data
the results displayed that there is a remarkable shift of JC-1 aggregates to JC-1 monomers of the
treated conjugates 5f and 5k that has increased to 24.6% and 23.0% respectively. Whereas the
control cells showed 4.7% as seen in Figure 9, thus indicating that both the conjugates induce
apoptosis through depolarization of the mitochondrial membrane potential in MCF-7 cells.

Figure 9: Effect on disruption of mitochondrial function and induction of apoptosis in cancer
cells. Breast cancer cells were exposed to 5f and 5k for 48 h at 0.5 µM concentration and stained
with JC-1 dye for 20 min at room temperature.
2.2.8. Annexin-V FITC assay
The apoptotic inducing ability is also investigated by Annexin V FITC/PI (AV/PI) dual
staining assay to examine the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis.49 In this study
MCF-7 cells were treated with compounds 5f and 5k for 48 h at 0.5µM concentration to examine
the apoptotic effect. The results suggested that 94.0% cells were live, 3.7% and 2.1% cells were
in early and late apoptosis stage respectively and 0.1% cells were dead in the untreated MCF-7
cells (Figure 10). However, in the presence of 0.5 µM 5f and 5k, 13.7,8.2% of cells were found
to be live while 32.5,36.6 and 53.6,54.9% of cells were in the early and late apoptosis stages and
0.2,0.3% cells were necrotic as shown in Figure 10. Similarly treatment with etoposide (0.5
µM), 8.5% of cells were live where as 36.1% and 55.0% of cells were in the early and late
stages of apoptosis respectively and 0.4% of cells were necrotic. This experiment suggests that
they significantly induce apoptosis in MCF-7 cells.

Figure 10: Flow cytometric analysis of cells stained with annexin V and PI showing the
distribution of Breast cancer cells in different phases of apoptosis. Cells were incubated with
with 0.5µM of 5f and 5k for 48 h.
2.3. Molecular docking studies
To rationalize the experimental data obtained, molecular docking studies were performed
on this series of conjugates (5f and 5k) and to elucidate the possible binding mode on tubulin
protein. Coordinates of protein structure of tubulin-colchicine were obtained from the Protein
Data Bank (PDB ID 3E22).50 Docking was carried out into the colchicine binding site of the
tubulin using AutoDock 4.2 software51 and analyzed by PYMOL software.52 As seen from
Figure 11A amide group of conjugate 5f established the hydrogen binding interactions (red
dotted lines) in which carbonyl oxygen interacts with βAla354 and –NH interacts with βLeu248.
The benzo[d]imidazo[2,1-b]thiazole ring of the conjugate 5f buried in the hydrophobic pocket
and surrounded by βLeu255, βLys241, βLeu252, βLeu242, βVal348 and βAla316 amino acid
residues. The conjugate 5f interacts with the α and β tubulin interface wherein the 1,2,3-triazole
ring is surrounded by αSer148, αAla180, αSer178, βAsn258, βLeu255 and 4-fluorophenyl ring
by αAsn101, αTyr224 and βLeu248 amino acid residues. Similarly from Figure 11B, the amide
group of conjugate 5k established the hydrogen binding interactions like in case of conjugate 5f,
in which carbonyl oxygen interacts with βAla354 and -NH interacts with βLeu248. Conjugate 5k
establishes similar binding position like conjugate 5f and is surrounded by similar amino acid

residues. Figure 11C shows overlapping of molecular docking pose of conjugate 5f (yellow) and
5k (red) whereas Figure 11D shows that the proposed binding pose of conjugate 5f (yellow) and
5k (red). It is observed that they share similar binding site as colchicine (brown) with different
orientation at interface of α-tubulin (blue) and β-tubulin (pink). Overall, docking investigation
showed that conjugate 5f and 5k could interact with the colchicine binding site between the α
and β subunits of tubulin.
(A)
(B)
(C)
(D)
Figure 11: (A) and (B) Interaction of the conjugate 5f and 5k with colchicines binding site of
tubulin. This figure has been generated using the software PYMOL from the tubulin-colchicine
crystal structure. (C) Superimposition of conjugate 5f (yellow) and 5k (red) (D) Overlapping of
conjugate 5f (yellow), 5k (red) and colchicine (brown) which shows 5f and 5k share the similar
binding site of colchicine on tubulin. β-tubulin shows in pink and α-tubulin shows in blue color.

3. Conclusion
In the present investigation, a new class of 1,2,3-triazolo linked benzo[d]imidazo[2,1-
b]thiazole conjugates were synthesized and evaluated for their cytotoxic activity. Among them,
conjugates 5f and 5k showed significant cytotoxic activity against the human breast cancer cell
line (MCF-7). The SAR provided an insight that could be utilized in developing improved leads
based on these scaffolds. The flow cytometric analysis revealed that these conjugates cause cell
cycle arrest at G2/M phase. The presence of increased levels of the cyclin B1 protein and tubulin
in the soluble fraction of cells corroborate well with the tubulin polymerization inhibition.
Furthermore, they effectively inhibit microtubule assembly and disrupt the microtubule
organization in the breast cancer cells. The molecular docking studies revealed that these
conjugates bind at the colchicine site of the tubulin. The induction of apoptosis is associated with
mitochondrial membrane potential and Annexin V FITC assay. Thus these conjugates could be
considered as potential scaffolds that are useful in the development of new leads as
chemotherapeutics for breast cancer.
4. Experimental Section
4.1. Chemistry
All the chemicals and reagents, which were commercially received from Aldrich (Sigma-
Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill,
MA, USA), or Spectrochem Pvt. Ltd (Mumbai, India) used directly without further purification
unless otherwise noted. The reaction courses and product mixtures were routinely checked by
TLC performed on silica gel glass plates containing 60 GF-254, and visualized under UV
irradiation and iodine. Column chromatography was performed using Merck 60-120 mesh silica
gel. Proton(1H) NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or
Inova Varian-VXRunity (400, 500 MHz) instruments. Carbon (13C) NMR spectra were recorded
on a Bruker UXNMR/XWIN-NMR (75 MHz) instrument. Chemical shifts (δ) are reported in δ
units, parts per million (ppm) downfield from an internal standard TMS and coupling constants
(J) were expressed in hertz (Hz). Signal multiplicity patterns are designed as follows: s (singlet),
d (doublet), t (triplet), quartet (q) ds (double singlet), dd (double doublet), m (multiplet) and br s
(broad singlet). ESI spectra were recorded on a Micro mass Quattro LC using ESI+ software
with a capillary voltage of 3.98 kV and an ESI mode positive ion trap detector. High-resolution
mass spectra (HRMS) were recorded on a QSTAR XL hybrid MS-MS mass spectrometer.

Melting points were determined in open glass capillary with an electrothermal melting point
apparatus and were uncorrected.
4.1.1. General method for the synthesis of 7-Substituted-N-(prop-2-yn-1-
yl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamides (10a-d): The corresponding carboxylic acid
(9a-d; 1.0 equiv) was dissolved in dry DMF, cooled to 0 °C (ice bath). EDCI (1.5 equiv), HOBt
(1.2 equiv), propargylamine hydrochloride (1.2 equiv) and triethylamine (1.2 equiv) were added
slowly. After 10 min removed the ice bath and the mixture was stirred at room temperature for 8
h until the starting materials were consumed. Then ice cold water was added to the reaction
mixture and extracted with dichloromethane (DCM). The combined organic extracts were
washed with aq NaHCO3 and dried with Na2SO4 and concentrated in vacuo. The residue was
purified by column chromatography by using an ethyl acetate and hexane solvent system to
afford the title compounds (10a-d).
4.1.1.1. N-(prop-2-yn-1-yl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (10a). Pale yellow
solid; 79% yield; mp: 217-219 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6) δ 8.58 (s, 1H), 8.03
(s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.42 (t, J =
7.5 Hz, 1H), 4.19 (dd, J = 5.6, 2.2 Hz, 2H), 2.45 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ
161.20, 146.37, 131.48, 129.46, 126.77, 125.86, 125.02, 115.75, 114.18, 81.48, 72.44, 27.89; MS
(ESI): m/z 256 [M + H]+; HRMS calcd for C13H10N3OS+ [M + H]+ 256.0539, found 256.0554.
4.1.1.2. 7-methyl-N-(prop-2-yn-1-yl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (10b).
White solid; 75% yield; mp: 224-226 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (s, 1H), 7.56 - 7.48
(m, 2H), 7.33 (s, 1H), 7.28 (s, 1H), 4.26 (dd, J = 5.5, 2.5 Hz, 2H), 2.48 (s, 3H), 2.26 (t, J = 2.5
Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 161.74, 147.31, 141.27, 136.20, 130.51, 129.61, 127.59,
124.55, 113.90, 112.95, 79.49, 71.61, 28.86, 21.44; MS (ESI): m/z 270 [M + H]+; HRMS calcd
for C14H12N3OS+ [M + H]+ 270.0696, found 270.0700.
4.1.1.3. 7-methoxy-N-(prop-2-yn-1-yl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (10c).
White solid; 78% yield; mp: 215-217 °C; 1H NMR (500 MHz, CDCl3) δ 8.25 (s, 1H), 7.55 (d, J
= 8.8 Hz, 1H), 7.31 (t, J = 5.4 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.03 (dd, J = 8.9, 2.5 Hz, 1H),
4.26 (dd, J = 5.5, 2.6 Hz, 2H), 3.88 (s, 3H), 2.26 (t, J = 2.5 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 161.88, 158.05, 146.96, 141.20, 131.88, 125.88, 114.00, 113.90, 108.96, 79.62, 71.72,
56.12, 28.96; MS (ESI): m/z 286 [M + H]+; HRMS calcd for C14H12N3O2S+ [M + H]+ 286.0645,
found 286.0649.
4.1.1.4. 7-ethoxy-N-(prop-2-yn-1-yl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (10d).
White solid; 76% yield; mp: 159-161 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6) δ 8.30 (s, 1H),
7.60 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 8.9, 2.4 Hz, 1H), 4.24 (dd, J =

5.6, 2.5 Hz, 2H), 4.10 (q, J = 7.0 Hz, 2H), 2.29 (t, J = 2.5 Hz, 1H), 1.46 (t, J = 7.0 Hz, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 161.27, 156.63, 153.47, 140.85, 130.78, 125.40, 118.04, 113.38,
109.77, 106.20, 81.26, 72.38, 63.52, 14.75; MS (ESI): m/z 300 [M + H]+; HRMS calcd for
C15H14N3O2S [M + H]+ 300.0801, found 300.0813.
4.1.2. General method for the synthesis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole
conjugates (5a-v).
To a solution of 7-Substituted-N-(prop-2-yn-1-yl)benzo[d]imidazo[2,1-b]thiazole-2-
carboxamides (10a-d, 1.0 equiv ) and benzyl azides (13a-f, 1.10 equiv) in 2 : 1 mixture of water
and tert-butyl alcohol, sodium ascorbate (0.1 equiv) and copper(II) sulfate (0.05 equiv) were
added sequentially. The reaction was stirred at room temperature for 5 h and TLC analysis
indicated completion of reaction. The solvent was concentrated under vacuum and the resulting
mixture was extracted with dichloromethane (CH2Cl2), dried with Na2SO4 and concentrated in
vacuo. The residue was purified by column chromatography by using an ethyl acetate and
hexane solvent system to afford the pure title compounds (5a-v).
4.1.2.1. N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5a). White solid; 67% yield; mp: 179-181 °C; 1H NMR (300 MHz,
CDCl3+DMSO-d6) δ 9.10 (s, 1H), 8.86 (s, 1H), 8.69 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H),
8.04 (d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 6.87 (d, J
= 8.6 Hz, 1H), 6.74 (s, 2H), 5.39 (s, 2H), 4.50 (d, J = 5.8 Hz, 2H), 3.72 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 161.34, 147.62, 146.60, 146.32, 145.41, 141.51, 131.52, 129.46, 128.48,
126.77, 125.85, 125.04, 122.56, 119.08, 115.59, 115.38, 114.15, 112.22, 55.61, 52.48, 34.24; MS
(ESI): m/z 435 [M + H]+; HRMS calcd for C21H19N6O3S [M + H]+ 435.1234, found 435.1235.
4.1.2.2. N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5b). White solid; 79% yield; mp: 219-221 °C; 1H NMR (400 MHz,
CDCl3) δ 8.29 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.9 Hz, 2H), 7.53 - 7.45 (m, 2H),
7.40 (t, J = 7.7 Hz, 1H), 6.90 - 6.80 (m, 2H), 6.79 (s, 1H), 5.42 (s, 2H), 4.72 (d, J = 6.0 Hz, 2H),
3.87 (s, 3H), 3.83 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.33, 148.70, 146.29, 145.43,
141.52, 131.50, 129.44, 128.25, 126.76, 125.83, 125.02, 122.58, 120.67, 120.25, 115.56, 114.14,
112.11, 111.85, 55.48, 52.62, 34.24; MS (ESI): m/z 449 [M + H]+; HRMS calcd for
C22H21N6O3S [M + H]+ 449.1390, found 449.1390.
4.12.3. N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5c). White solid; 72% yield; mp: 199-201 °C; 1H NMR (300 MHz,
DMSO-d6) δ 8.87 (s, 1H), 8.72 (t, J = 5.9 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.05 (d, J = 7.9 Hz,
1H), 7.99 (s, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 6.51-6.41 (m, 3H), 5.48 (s,

2H), 4.52 (d, J = 5.9 Hz, 2H), 3.71 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ 161.33, 160.62,
145.48, 141.52, 138.19, 131.51, 129.45, 126.77, 125.84, 125.04, 122.93, 115.57, 114.15, 106.10,
99.49, 55.20, 52.67, 34.23; MS (ESI): m/z 449 [M + H]+; HRMS calcd for C22H21N6O3S [M +
H]+ 449.1390, found 449.1394.
4.1.2.4. N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5d). White solid; 89% yield; mp: 210-212 °C; 1H NMR (300 MHz,
CDCl3+DMSO-d6) δ 8.79 (s, 1H), 8.57 (t, J = 5.7 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.98 - 7.91
(m, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 6.66 (s, 2H), 5.46 (s, 2H), 4.57 (d, J =
5.7 Hz, 2H), 3.76 (s, 6H), 3.67 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.34, 152.96, 146.29,
145.47, 141.53, 137.29, 131.45, 129.45, 126.77, 125.85, 125.04, 122.80, 115.54, 114.16, 105.74,
59.95, 55.88, 52.96, 34.25; MS (ESI): m/z 479 [M + H]+; HRMS calcd for C23H23N6O4S [M +
H]+ 479.1496, found 479.1505.
4.1.2.5. N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-b]thiazole-
2-carboxamide (5e). White solid; 85% yield; mp: 199-201 °C; 1H NMR (500 MHz, CDCl3) δ
8.30 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.9 Hz, 2H), 7.54 (s, 1H), 7.48 (dt, J = 8.24,
1.06 Hz, 1H), 7.39 (dt, J = 8.08, 1.06 Hz, 1H), 7.35 - 7.31 (m, 2H), 7.30 - 7.26 (m, 1H), 7.11 (dt,
J = 7.47, 1.06 Hz, 1H), 6.99 (dd, J = 8.6, 1.0 Hz, 2H), 6.94 (dt, J = 8.2, 2.2 Hz, 2H), 6.91 (d, J =
1.7 Hz, 1H), 5.45 (s, 2H), 4.73 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 162.21,
158.17, 156.56, 147.49, 145.57, 141.83, 136.54, 131.76, 130.58, 129.99, 126.68, 125.95, 124.62,
123.91, 122.62, 122.33, 119.40, 118.68, 118.21, 113.88, 113.39, 53.96, 34.88; MS (ESI): m/z
481 [M + H]+; HRMS calcd for C26H21N6O2S [M + H]+ 481.1441, found 481.1444.
4.1.2.6. N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-b]thiazole-2-
carboxamide (5f). White solid; 78% yield; mp: 237-239 °C; 1H NMR (300 MHz, DMSO-d6) δ
8.87 (s, 1H), 8.71 (t, J = 6.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.99 (s,
1H), 7.57 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.39 (dd, J = 8.4, 5.6 Hz, 2H), 7.19 (t, J =
8.8 Hz, 2H), 5.55 (s, 2H), 4.51 (d, J = 6.0 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.83 (JC-
= 244 Hz), 161.35, 146.29, 145.54, 141.51, 132.41, 131.51, 130.28 (JC-F = 8 Hz), 129.44,
F
126.77, 125.84, 125.04, 122.83, 115.63 (JC-F = 6 Hz),115.39, 114.15, 51.87, 34.23; MS (ESI):
m/z 407 [M + H]+; HRMS calcd for C20H16FN6OS [M + H]+ 407.1085, found 407.1090.
4.1.2.7. N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
methylbenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5g). White solid; 78% yield; mp: 179-
181 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.67 (t, J = 5.9 Hz, 1H), 8.00 (d, J = 8.2
Hz, 1H), 7.93 (s, 1H), 7.82 (s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.00 (s, 1H), 6.95 - 6.82 (m, 2H),
5.45 (s, 2H), 4.49 (d, J = 5.9 Hz, 2H), 3.71 (s, 6H), 2.41 (s, 3H); 13C NMR (75 MHz, DMSO-d6)

δ 161.34, 148.70, 146.06, 145.44, 141.33, 135.61, 129.43, 128.25, 127.59, 124.80, 122.55,
120.66, 115.43, 113.77, 112.11, 111.86, 55.48, 52.60, 34.22, 20.88; MS (ESI): m/z 463 [M +
H]+; HRMS calcd for C23H23N6O3S [M + H]+ 463.1547, found 463.1548.
4.1.2.8. N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
methylbenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5h). Light brown solid; 72% yield; mp:
229-231 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.69 (t, J = 5.9 Hz, 1H), 8.04 - 7.95
(m, 2H), 7.81 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 6.50 - 6.39 (m, 3H), 5.47 (s, 2H), 4.51 (d, J = 5.9
Hz, 2H), 3.70 (s, 6H), 2.41 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.38, 160.62, 146.07,
145.52, 141.33, 138.19, 135.60, 129.43, 127.58, 124.78, 122.93, 115.44, 113.77, 106.10, 99.49,
55.19, 52.68, 34.23, 20.88; MS (ESI): m/z 463 [M + H]+; HRMS calcd for C23H23N6O3S [M +
H]+ 463.1547, found 463.1531.
4.1.2.9. 7-methyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5i). White solid; 85% yield; mp: 175-
177 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.68 (t, J = 5.9 Hz, 1H), 8.02-7.99 (m,
2H), 7.82 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 6.69 (s, 2H), 5.46 (s, 2H), 4.50 (d, J = 5.9 Hz, 2H),
3.73 (s, 6H), 3.61 (s, 3H), 2.41 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.36, 152.96, 146.07,
145.49, 141.34, 137.31, 135.61, 131.44, 129.43, 127.58, 124.78, 122.79, 115.41, 113.77, 105.73,
59.94, 55.88, 52.96, 34.23, 20.88; MS (ESI): m/z 493 [M + H]+; HRMS calcd for C24H25N6O4S
[M + H]+ 493.1653, found 493.1651.
4.1.2.10. 7-methyl-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5j). Brown solid; 81% yield; mp: 132-134 °C; 1H NMR (300 MHz,
DMSO-d6) δ 8.81 (s, 1H), 8.70 (t, J = 5.8 Hz, 1H), 8.02-7.99 (m, 2H), 7.82 (s, 1H), 7.41 - 7.31
(m, 4H), 7.11 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 3H), 6.92 (d, J =
8.0 Hz, 1H), 5.55 (s, 2H), 4.51 (d, J = 5.8 Hz, 2H), 2.41 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
δ 161.38, 156.89, 156.16, 145.57, 141.33, 138.28, 135.61, 130.38, 130.06, 129.43, 127.60,
124.80, 123.65, 122.97, 122.83, 118.77, 117.98, 117.88, 115.46, 113.77, 52.26, 34.23, 20.89; MS
(ESI): m/z 495 [M + H]+; HRMS calcd for C27H23N6O2S [M + H]+ 495.1598, found 495.1592.
4.1.2.11. N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methylbenzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5k). White solid; 73% yield; mp: 243-245 °C; 1H NMR (400 MHz,
CDCl3) δ 8.25 (s, 1H), 7.65 (s, 1H), 7.53 (s, 1H), 7.51 (s, 2H), 7.30 – 7.23 (m, 3H), 7.05 (t, J =
8.6 Hz, 2H), 5.46 (s, 2H), 4.72 (d, J = 6.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
163.0 (JC-F = 247 Hz), 162.34, 147.44, 145.74, 141.56, 136.31, 130.64, 130.14 (JC-F = 8 Hz),
129.71, 127.68, 124.69, 122.23, 116.26 (JC-F = 22 Hz), 113.84, 113.01, 53.58, 34.87, 21.55; MS
(ESI): m/z 421 [M + H]+; HRMS calcd for C21H18FN6OS [M + H]+ 421.1241, found 421.1244.

4.1.2.12. N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
methoxybenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5l). White solid; 65% yield; mp: 208-
210 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.77 (s, 1H), 8.64 (t, J = 5.9 Hz, 1H),
8.04 (d, J = 8.9 Hz, 1H), 7.88 (s, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.15 (dd, J = 8.8, 2.1 Hz, 1H),
6.88 (d, J = 8.7 Hz, 1H), 6.75 (s, 2H), 5.39 (s, 2H), 4.49 (d, J = 5.7 Hz, 2H), 3.83 (s, 3H), 3.73 (s,
3H); 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 161.41, 160.56, 158.39, 157.39, 147.62, 146.58,
130.82, 128.48, 125.55, 122.53, 119.08, 115.37, 114.77, 113.97, 112.23, 109.35, 55.87, 55.62,
52.47, 34.20; MS (ESI): m/z 465 [M + H]+; HRMS calcd for C22H21N6O4S [M + H]+ 465.1340,
found 465.1343.
4.1.2.13. N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
methoxybenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5m). White solid; 74% yield; mp: 199-
201 °C; 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.62 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.50
(s, 1H), 7.21 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.89-6.80 (m, 2H), 6.78 (s, 1H), 5.42 (s, 2H), 4.72
(d, J = 5.8 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
162.29, 158.03, 149.58, 146.93, 145.54, 141.45, 131.90, 127.03, 125.88, 122.10, 121.04, 113.92,
113.76, 111.44, 111.35, 108.94, 56.09, 54.26, 34.89; MS (ESI): m/z 479 [M + H]+; HRMS calcd
for C23H23N6O4S [M + H]+ 479.1496, found 479.1498.
4.1.2.14. N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
methoxybenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5n). Brown solid; 79% yield; mp: 208-
210 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.66 (t, J = 5.8 Hz, 1H), 8.04 (d, J = 8.9
Hz, 1H), 7.97 (s, 1H), 7.67 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.9, 2.3 Hz, 1H), 6.46 (s, 2H), 6.44
(d, J = 1.8 Hz, 1H), 5.47 (s, 2H), 4.50 (d, J = 5.8 Hz, 2H), 3.82 (s, 3H), 3.70 (s, 6H); 13C NMR
(75 MHz, DMSO-d6) δ 163.40, 161.44, 160.63, 157.38, 141.12, 138.19, 130.81, 125.56, 122.93,
115.38, 114.78, 113.97, 109.33, 106.10, 99.50, 55.87, 55.20, 52.69, 34.22; MS (ESI): m/z 501
[M + H]+; HRMS calcd for C23H22N6NaO4S [M + Na]+ 501.1315, found 501.1317.
4.1.2.15. 7-methoxy-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5o). Light brown solid; 90% yield;
mp: 201-203 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.64 (t, J = 5.9 Hz, 1H), 8.04
(d, J = 8.9 Hz, 1H), 7.99 (s, 1H), 7.67 (d, J = 2.3 Hz, 1H), 7.13 (dd, J = 8.9, 2.4 Hz, 1H), 6.69 (s,
2H), 5.46 (s, 2H), 4.50 (d, J = 5.9 Hz, 2H), 3.82 (s, 3H), 3.72 (s, 6H), 3.61 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 161.41, 157.38, 152.95, 145.76, 145.49, 141.11, 137.31, 131.43, 130.79,
125.52, 122.78, 115.33, 114.76, 113.95, 109.31, 105.74, 59.95, 55.88, 52.96, 34.22; MS (ESI):
m/z 509 [M + H]+; HRMS calcd for C24H25N6O5S [M + H]+ 509.1602, found 509.1600.

4.1.2.16. 7-methoxy-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5p). White solid; 81% yield; mp: 211-213 °C; 1H NMR (300 MHz,
CDCl3+DMSO-d6) δ 8.75 (s, 1H), 8.58 (t, J = 5.9 Hz, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.97 (s, 1H),
7.62 (d, J = 2.1 Hz, 1H), 7.40 - 7.30 (m, 3H), 7.12 (d, J = 7.6 Hz, 2H), 7.05 (d, J = 7.8 Hz, 1H),
6.98 (d, J = 7.6 Hz, 3H), 6.91 (d, J = 8.4 Hz, 1H), 5.55 (s, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.84 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 161.43, 157.39, 156.89, 156.17, 145.77, 145.60, 141.11,
138.29, 130.81, 130.40, 130.08, 125.55, 123.67, 122.97, 122.84, 118.78, 117.98, 117.89, 115.41,
114.78, 113.99, 109.35, 55.88, 52.26, 34.22; MS (ESI): m/z 511 [M + H]+; HRMS calcd for
C27H23N6O3S [M + H]+ 511.1547, found 511.1548.
4.1.2.17. N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methoxybenzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5q). White solid; 75% yield; mp: 253-255 °C; 1H NMR (300 MHz,
CDCl3+DMSO-d6) δ 8.74 (s, 1H), 8.57 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.96 (s, 1H),
7.60 (s, 1H), 7.42 – 7.33 (m, 2H), 7.13 (dd, J = 15.0, 7.4 Hz, 3H), 5.55 (s, 2H), 4.52 (d, J = 5.7
Hz, 2H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 161.43, 157.39, 145.78, 145.59,
141.10, 132.41, 130.81, 130.29 (JC-F = 8 Hz), 125.54, 122.82, 115.69, 115.40, 114.78, 113.98,
109.34, 55.88, 51.89, 34.21; MS (ESI): m/z 437 [M + H]+; HRMS calcd for C21H18FN6O2S [M +
H]+ 437.1190, found 437.1194.
4.1.2.18. N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
ethoxybenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5r). White solid; 80% yield; mp: 214-
216 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.64 (t, J = 5.8 Hz, 1H), 8.03 (d, J = 8.9
Hz, 1H), 7.93 (s, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.9, 2.1 Hz, 1H), 7.00 (s, 1H), 6.93-
6.85 (m, 2H), 5.46 (s, 2H), 4.49 (d, J = 5.8 Hz, 2H), 4.09 (q, J = 6.8 Hz, 2H), 3.72 (s, 6H), 1.36
(t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.41, 156.60, 148.69, 145.80, 141.07,
130.75, 128.24, 125.42, 122.55, 120.66, 115.31, 114.74, 114.34, 112.11, 111.85, 109.81, 63.89,
55.48, 52.61, 34.21, 14.54; MS (ESI): m/z 493 [M + H]+; HRMS calcd for C24H25N6O4S [M +
H]+ 493.1653, found 493.1651.
4.1.2.19. N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
ethoxybenzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5s). Light brown solid; 85% yield; mp:
223-225 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.63 (t, J = 6.0 Hz, 1H), 8.03 (d, J =
8.9 Hz, 1H), 7.97 (s, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.13 (dd, J = 8.9, 2.4 Hz, 1H), 6.46 (d, J = 1.9
Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 5.47 (s, 2H), 4.50 (d, J = 6.0 Hz, 2H), 4.10 (q, J = 6.9 Hz, 2H),
3.70 (s, 6H), 1.36 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 161.41, 160.62, 156.61,
145.52, 141.09, 138.19, 130.76, 125.44, 122.92, 115.35, 114.75, 114.35, 109.84, 106.09, 99.49,

63.90, 55.19, 52.67, 34.20, 14.55; MS (ESI): m/z 493 [M + H]+; HRMS calcd for C24H25N6O4S
[M + H]+ 493.1653, found 493.1653.
4.1.2.20. 7-ethoxy-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5t). White solid; 88% yield; mp: 208-
209 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.65 (t, J = 5.9 Hz, 1H), 8.08 - 7.96 (m,
2H), 7.67 (d, J = 2.3 Hz, 1H), 7.14 (dd, J = 8.9, 2.4 Hz, 1H), 6.70 (s, 2H), 5.46 (s, 2H), 4.50 (d, J
= 5.9 Hz, 2H), 4.09 (q, J = 6.9 Hz, 2H), 3.73 (s, 6H), 3.62 (s, 3H), 1.36 (t, J = 6.9 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 162.32, 157.38, 153.78, 146.94, 145.66, 141.36, 138.38, 131.78,
130.13, 125.71, 122.29, 114.40, 113.90, 113.73, 109.49, 105.41, 64.51, 60.96, 56.31, 54.58,
34.88, 14.85; MS (ESI): m/z 523 [M + H]+; HRMS calcd for C25H27N6O5S [M + H]+ 523.1758,
found 523.1764.
4.1.2.21. 7-ethoxy-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5u). White solid; 83% yield; mp: 191-193 °C; 1H NMR (400 MHz,
CDCl3) 13C NMR (75 MHz, DMSO-d6) δ 161.45, 156.90, 156.63, 156.18, 145.77, 145.59,
141.09, 138.29, 130.78, 130.40, 130.07, 125.45, 123.67, 122.97, 122.84, 118.78, 117.99, 117.90,
115.37, 114.77, 114.38, 109.85, 63.93, 52.27, 34.22, 14.57; MS (ESI): m/z 525 [M + H]+; HRMS
calcd for C28H25N6O3S [M + H]+ 525.1703, found 525.1706.
4.1.2.22. 7-ethoxy-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzo[d]imidazo[2,1-
b]thiazole-2-carboxamide (5v). White solid; 76% yield; mp: 255-257 °C;1H NMR (300 MHz,
DMSO-d6) δ 8.78 (s, 1H), 8.65 (t, J = 6.0 Hz, 1H), 8.12 – 7.95 (m, 2H), 7.67 (s, 1H), 7.38 (t, J =
6.8 Hz, 2H), 7.88 – 7.09 (m, 3H), 5.55 (s, 2H), 4.50 (d, J = 4.6 Hz, 2H), 4.10 (d, J = 6.8 Hz, 2H),
1.36 (t, J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 163.46, 161.44, 156.63,
145.77, 145.59, 141.08, 132.38, 130.77, 130.29 (JC-F = 8 Hz), 125.44, 122.83, 115.69, 115.40,
114.77, 114.37, 109.85, 63.92, 51.89, 34.21, 14.56; MS (ESI): m/z 461 [M + H]+; HRMS calcd
for C22H20FN6O2S [M + H]+ 451.1347, found 451.1350.
4.2. Biology
4.2.1. Anticancer activity MTT Assay
The cytotoxic activity of the compounds was determined using MTT assay. 1×104
cells/well were seeded in 200 µl DMEM, supplemented with 10% FBS in each well 96 well
microculture plates and incubated for 24 h at 37 °C 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, 10 µL of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium 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 µL of DMSO and absorbance at 540 nm wavelengths was recorded.
4.2.2. Cell cycle analysis
Flow cytometric analysis (FACS) anlaysis was performed to evaluate the distribution of
the cells through the cell cycle phases. Human breast cancer cells (MCF-7) were incubated with
compounds 5f and 5k at 0.5 µM concentration for 48 h. Control and treated cells were harvested,
washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide (Sigma
Aldrich). Cell cycle anlaysis was performed by flow cytometryR (Becton Dickinson FACS
Caliber) as described earlier.53
4.2.3. Dot-blot assay
Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 6-
well plates. These conjugates (5f and 5k) were evaluated for their activity against Cyclin B1.
MCF-7 cells were treated with 1 µM concentrations of 5f and 5k for 24 h. Subsequently, cells
were harvested and proteins were quantified using Amido Black followed by densitomerty
analysis. Equal amount of protein were blotted on nitrocellulose membrane using Bio-Dot SF
microfiltration apparatus (Bio-Rad). Briefly, nitrocellulose membrane and 3 filters papers
(Whatmann 3) were soaked in IX TBS solution for 10 min. Later, the filter papers, membrane
were arranged in the apparatus and connected to vacuum pump (Millipore). The membranes
were rehydrated using 100 µl of 1X TBS by vacuum filtration. Subsequently, 50 µl volumes of
equal protein samples were blotted on the membrane and washed with 200 µl of 1X TBS through
application of vaccum. The blot was blocked with 5% blotto for 1 h at room temperature.
Immunoblot analysis was performed using UVP, biospectrum 810 imaging system.
4.2.4. In vitro tubulin polymerization
A fluorescence based tubulin polymerization assay was performed according to the
manufacturers protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture was taken
within a total volume of 10 µL contained PEM buffer, GTP (1µM) in the presence or absence of
test compounds 5f and 5k at a final concentration of 3 µM. In vitro 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 420 nm
(excitation wavelength is 360 nm) was measured by using a Biotek multimode plate reader.
Polymerization was finally monitored by increase in the fluorescence mentioned above at 37 0C.
The IC50 value was defined as the drug concentration required for inhibiting 50% of tubulin
assembly compared to the control.
4.2.5. Immunohistochemistry of tubulin and Analysis of nuclear morphology

MCF-7 cells were seeded on glass cover slip, incubated for 24 h in the presence or
absence of test conjugates 5k and 5f (0.5 µM). Cells grown on cover slips were fixed in 3.5%
formaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 minutes at room temperature.
Cells were permeabilized for 6 minutes in PBS containing 0.5%Triton X-100 (Sigma) and 0.05%
Tween-20 (Sigma). The permeabilized cells were blocked with 2% BSA (Sigma) in PBS for 1h.
Later, the cells were incubated with primary antibody for tubulin from (sigma) at (1:200) diluted
in blocking solution for 4 h at room temperature. Subsequently the antibodies were removed and
the cells were washed thrice with PBS. Cells were then incubated with FITC labeled anti-mouse
secondary antibody (1:500) for 1h at room temperature. Cells were washed thrice with PBS and
mounted in medium containing DAPI. Images were captured using the Olympus confocal
microscope and analyzed with Provision software.
4.2.6. Western blot Analysis of Soluble versus Polymerized Tubulin
Cells were seeded in 12-well plates at 1×105 cells per well in complete growth medium.
Following treatment of cells with respective compounds (5f, 5k and nocodazole and paclitaxel)
for a duration of 24 h, cells were washed with PBS and subsequently soluble and insoluble
tubulin fractions were collected. To collect the soluble tubulin fractions, cells were
permeabilized with 200 µL of pre-warmed lysis buffer [80 mM Pipes-KOH (pH 6.8), 1 mM
MgCl2, 1 mM EGTA, 0.2% Triton X-100, 10% glycerol, 0.1% protease inhibitor cocktail
(Sigma-Aldrich)] and incubated for 3 min at 30 oC. Lysis buffer was gently removed, and mixed
with 100 µL of 3×Laemmli’s sample buffer (180 mM Tris-Cl pH 6.8, 6% SDS, 15% glycerol,
7.5% β-mercaptoethanol and 0.01% bromophenol blue). Samples were immediately heated to 95
oC for 3 min. To collect the insoluble tubulin fraction, 300 µL of 1×Laemmli’s sample buffer
was added to the remaining cells in each well, and the samples were heated to 95 oC for 3 min.
Equal volumes of samples were run on an SDS-10 % polyacrylamide gel and were transferred to
a nitrocellulose membrane employing semidry transfer at 50 mA for 1h. Blots were probed with
mouse anti-human α-tubulin diluted 1:2,000 ml (Sigma) and stained with rabbit anti-mouse
secondary antibody coupled with horseradish peroxidase, diluted 1:5000 ml (Sigma).Bands were
visualized using an enhanced Chemiluminescence protocol (Pierce) and radiographic film
(Kodak).
4.2.7. Measurement of the mitochondrial membrane potentiaL
The mitochondrial membrane potential was measured with lipophilic cationic dye JC-1
(Molecular Probes), as described.54 Cultures were treated with the test drugs 5f and 5k at 0.5 µM
for 48 h. After drug treatment the MCF-7 cells were incubated with JC-1 dye for 20 min at 37
°C. After incubation cultures were used for measuring the mitochondrial membrane potiential

(∆Ψm),according to the manufacturer’s instructions. The shift of the membrane potential was
measured by flow cytometry using JC-1 (Molecular Probes), as previously described.
4.2.8. Flow cytometric evaluation of apoptosis
MCF-7 were seeded in six-well plates and allowed to grow overnight. The medium was
then replaced with complete medium containing 0.5 µM concentration of compounds, 5f and 5k
for 48 h along with vehicle alone (0.001% DMSO) as the control. After 48 h of drug treatment,
the cells from the supernatant and adherent monolayer cells were harvested by trypsinization,
and washed with PBS at 3000 rpm. Then the cells were stained with annexin V-FITC and
propidium iodide using the annexin-V-PI apoptosis detection kit (Sigma Aldrich). Flow
cytometry was performed using a FACScan (Becton Dickinson)equipped with a single 488 nm
argon laser as described earlier.55 Annexin V-FITC was analyzed using excitation and emission
settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel).
Acknowledgments
S.P.S. and F.S., thank CSIR and the UGC, New Delhi for the award of fellowships. We
acknowledge funding received from the project entitled ‘Affordable Cancer Therapeutics (ACT)’
under XIIth five year plan and extend our appreciation to the International Scientific Partnership
Program ISPP at King Saud University for funding this research work through ISPP#0054.
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