Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

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 Jain c, Ahmed Kamal a,b,d,e,⇑
a
Medicinal 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
c
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India
d
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education & Research (NIPER), Hyderabad 500 037, India
e
Catalytic Chemistry Research Chair, Chemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

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

Article history: 1,2,3-Triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a–v) were designed, synthesized and
Received 6 December 2016 evaluated for their cytotoxic potency against some human cancer cell lines like DU-145 (prostate),
Accepted 11 April 2017 HeLa (cervical), MCF-7 (breast) HepG2 (liver) and A549 (lung). Preliminary results revealed that some
Available online 12 April 2017
of these conjugates like 5f and 5k exhibited significant antiproliferative effect against human breast can-
cer cells (MCF-7) with IC50 values of 0.60 and 0.78 mM respectively. Flow cytometric analysis of the cell
Keywords: cycle demonstrated an increase in the percentage of cells in the G2/M phase which was further authen-
Benzo[d]imidazo[2,1-b]thiazole
ticated by elevation of cyclin B1 protein levels. Immunocytochemistry revealed loss of intact microtubule
1,2,3-Triazole
Cytotoxicity
structure in cells treated with 5f and 5k, and western blot analysis revealed that these conjugates accu-
Tubulin depolymerization mulated more tubulin in the soluble fraction. Moreover, the conjugates caused apoptosis of the cells that
Apoptosis was confirmed by mitochondrial membrane potential and Annexin V-FITC assay. Molecular docking stud-
ies indicated that these conjugates occupy the colchicine binding site of the tubulin protein.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Antimitotic agents are a major class of cytotoxic drugs for the
treatment of cancer and tubulin is a target for numerous small nat-
ural and synthetic molecules that inhibit the formation of the
mitotic spindle.1,2 Tubulin-containing structures, such as micro-
tubules are responsible for the formation of the mitotic spindle
during the process of mitosis. The microtubule system of eukary-
otic cells is a critical element in a variety of essential cellular pro-
cesses in addition to mitotic spindle assembly, including
determination and maintenance of cell shape, regulation of motil-
ity, 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
⇑ Corresponding author at: Medicinal Chemistry and Pharmacology Division,
CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
and act as spindle poisons arresting the dividing cells in G2/M
phase of the cell cycle, causing mitotic catastrophe and finally lead-
ing 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 hetero-
cycle 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 recep-
tors in biological systems through diverse weak interactions,
thereby exhibiting broad bioactivities like antimicrobial,12 anticon-
vulsant,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,

http://dx.doi.org/10.1016/j.bmc.2017.04.013
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
3286 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297

S O S
NEt 2.HCl N
N.HCl O N
N N
O
O N O

AC220, 2 N N
YM-201627, 1 (in phase III clinical trials) H H

O S R1
S
N N N H S
N N A B N
N N N N N
N C N
O NH
D
OMe O R2
3 OMe 4 5a-v
MeO

Fig. 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).

S R1
N N S
H N N
N N N N N
N N
O NH

OMe O R2
OMe
MeO

Fig. 2. Design strategy of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates.

2-aryl benzo[d]imidazo[2,1-b]thiazole derivative (1, YM-201627,
Fig. 1) was found to be potent orally active antitumor agent useful
for the treatment of solid tumors.26 Another derivative (2, AC220,
Fig. 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]thiazole29 (3, Fig. 1) showed
significant cytotoxic activity with the ability to induce apoptosis
through inhibition of tubulin polymerization. Whereas, 1,2,3-tria-
zoles 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 poly-
merization 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 synthe-
sis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conju-
gates (5a–v) and the strategy of design is described in Fig. 2.
These conjugates were evaluated for their cytotoxic potential and
effect on tubulin polymerization, apart from the cell cycle analysis
to understand their possible mechanism of action.
2. Results and discussion
2.1. Chemistry
These 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole con-
jugates (5a–v) were synthesized as depicted in Scheme 1. First we
have initiated our synthesis with the preparation of benzo[d]imi-
dazo[2,1-b]thiazole derived alkyne motif’s. The condensation of
substituted 2-aminobenzothiazoles (6a–d) with the ethylbro-
mopyruvate (7) in 1,2-dimethoxyethane (DME) under reflux condi-
tions 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)-N0 -
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 sub-
unit, 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]im-
idazo[2,1-b]thiazole terminal alkynes (10a–d) to benzyl azides
(13a–f) in the presence of catalytic copper(II) sulfate pentahydrate
(CuSO45H2O) 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).
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 syn-
thesized 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 cytotoxic-
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297 3287

R1 S
R1 O
S (a)
Br OEt N (b)
NH2 N
N OEt
O
6a-d 7 8a-d O

R1 S
R1 S
(c) N
N H
N N
N
OH 10a-d
O
9a-d R1 S
O 10a ; R 1=H
N
10b ; R1 =Me (f) N N N
N
10c; R 1=OMe NH
1 0d; R1 =OEt
O R2
5a-v
OH Br
N3 5a; R 1 =H,R2 =3-OH;4-OMe 5l; R1 =OMe,R 2 =3-OH;4-OMe
(d) (e) 5b ; R 1=H,R2 =3,4-diOMe 5m ; R 1=OMe,R 2 =3,4-diOMe
5c; R 1 =H,R2 =3,5-diOMe 5n; R 1=OMe,R 2 =3,5-diOMe
R2 R2 5d ; R 1=H,R2 =3,4,5-triOMe 5o ; R 1=OMe,R 2 =3,4,5-triOMe
R2 5e; R 1 =H,R2 =3-OPh 5p; R 1=OMe,R 2 =3-OPh
11a-f 12a-f
13a-f 5f ; R 1 =H,R2 =4-F 5q ; R 1=OMe,R 2 =4-F
5g; R 1=Me,R 2 =3,4-diOMe 5r; R 1=OEt,R 2=3,4-diOMe
13a ; R2 =3-OH;4-OMe
5h ; R 1=Me,R 2 =3,5-diOMe 5s ; R 1 =OEt,R2=3,5-diOMe
13b; R 2 =3,4-diOMe
5i; R1 =Me,R 2=3,4,5-triOMe 5t; R 1 =OEt,R2=3,4,5-triOMe
13c; R2 =3,5-diOMe
5j; R1 =Me,R 2=3-OPh 5u; R 1=OEt,R 2=3-OPh
13d ; R 2 =3,4,5-triOMe
5k ; R 1 =Me,R2 =4-F 5v ; R 1 =OEt,R2=4-F
13e; R2 =3-OPh
13f ; R2 =4-F

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) D, 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%), CuSO45H2O (5 mol%), H2O/t-BuOH, (2: 1), rt, 5 h, 65–90%.

Table 1
IC50 valuesa (in mM) for compounds 10a–d and 5a–v in human cancer cell lines. ity with IC50 values of 1.412, 0.607, 1.061, 0.781 and 0.977 mM
respectively against MCF-7 cell line.
Compound DU-145b HeLac MCF-7d HepG2e A549f
In order to investigate the structure activity relationship (SAR),
10a 19.95 16.25 8.749 20.48 12.88 we have varied the substitution pattern of the ring A of benzo[d]
10b 25.11 14.79 10.03 27.58 11.74
imidazo[2,1-b]thiazole and benzyl ring (ring D) linked to the
10c 23.89 20.05 15.51 28.78 17.58
10d 22.19 20.74 17.48 24.23 16.31 1,2,3-triazole ring of these conjugates. The cytotoxicity data of
5a 14.77 12.41 9.246 16.54 9.549 compounds 10a–d deciphered that, by increasing the electron-
5b 29.83 15.31 10.28 31.620 14.120 donating nature on ring A of the benzo[d]imidazo[2,1-b]thiazole
5c 30.90 27.58 12.13 34.667 19.05
scaffold decreases the activity particularly againt MCF-7 cell line
5d 3.715 3.311 1.412 7.943 2.041
5e 21.69 14.79 6.180 21.88 10.96
when compared to other tested cell lines. In contrast, the benzyl
5f 2.570 1.629 0.607 7.413 1.570 ring (ring D) of these conjugates that has an electron withdrawing
5g 8.709 7.244 6.251 16.980 8.128 group like p-F (5f, 5k, 5q and 5v) as well as electron donating
5h 31.18 21.38 6.501 8.10 11.22 groups like 3,4,5-trimethoxy (5d, 5i and 5o) showed significant
5i 4.466 3.630 1.061 4.897 2.824
antiproliferative activity when compared to m-mono and disubsti-
5j 27.54 17.15 7.362 35.77 10.71
5k 4.265 2.691 0.781 6.606 1.288 tuted (3,4 and 3,5) electron donating group on these conjugates. It
5l 18.75 16.98 6.606 26.895 9.332 was observed that electron withdrawing p-F group showed slightly
5m 14.27 11.48 6.165 22.450 10.47 improved activity in comparison to electron donating group like
5n 13.59 11.74 8.120 15.031 14.79
3,4,5-trimethoxy substituted conjugates. Most of the correspond-
5o 9.772 8.542 2.818 17.370 4.897
5p 13.08 12.58 3.630 30.19 6.367
ing conjugates showed enhanced cytotoxicities compared to
5q 3.38 3.235 0.977 6.688 2.511 benzo[d]imidazo[2,1-b]thiazole alkynes (10a–d) particularly
5r 32.27 31.62 24.50 34.60 22.85 againt MCF-7 cell line except 5a, 5b, 5c and 5r. The activity of these
5s 9.935 7.762 7.244 15.963 14.93 conjugates like 5f, 5k and 5q improved almost by 10 folds than
5t 15.40 9.54 5.370 18.70 8.912
their alkyne intermediates (10a–d). Some of the 1,2,3-triazolo
5u 12.02 15.10 6.683 18.14 8.053
5v 14.41 12.58 3.311 17.310 8.317 linked benzo[d]imidazo[2,1-b]thiazole conjugates like 5d, 5f, 5i,
Doxorubicin 0.895 2.089 1.659 2.511 1.445 5k, 5o, 5q and 5v showed comparable or improved activity than
YM-20162728 – 4.30 3.59 2.02 – YM-201627.28 And 5f and 5k conjugates displayed promising cyto-
Note: toxicity (IC50 values of 0.667 and 0.781 mM), these are superior or
a
50% Inhibitory concentration and the values are average of three individual comparable to compound 3 (IC50 values of 0.778 mM). These results
experiments after 48 h of drug treatment. suggest that electron-donating substituents like H and Me on ring
b
Prostate cancer. A and electron withdrawing group like p-F and electron donating
c
Cervical cancer.
d
Breast cancer.
group like 3,4,5-trimethoxy on ring D are essential for the cytotoxic
e
Liver cancer. activity as shown in Fig. 3. Moreover from the MTT assay, the most
f
Lung cancer. cytotoxic conjugates that is 5f and 5k were taken up for further
3288 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297

Hydrophobic region 2.2.3. Effect on cellular cyclin-B1, CDK1 and Aurora B levels by
R1 Tubulin binding region immunoblot analysis
S
N Cyclin-B1 is induced at the G2/M boundary to promote cell divi-
N N N sion. This protein is one of the important regulatory proteins of
N
Cytotoxicity order NH mitosis and accumulation of cyclin-B1 is an indication for G2/M
H>Me>OMe>OEt arrest.43 Since these compounds arrest the cells at G2/M phase,
O
Hydrogen R2 we investigated their effect on cyclin B1 protein levels. Thus, we
bonding region "F and tri OMe" gps treated the MCF-7 cells with the lead compounds 5f and 5k at
crucial for cytotoxicity 1 lM concentrations for 24 h and performed the immunoblot anal-
ysis for cyclin-B1 and tubulin as loading control. Therefore, the
Fig. 3. SAR analysis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole
conjugates.
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
mechanistic studies, such as tubulin polymerization inhibition, arrest, with the activation of spindle checkpoint in 24 h as shown
immunohistochemistry and apoptosis induction. In addition, these in Fig. 5. We have also analyzed Cdk1 and Aurora B kinases levels
conjugates were also investigated for molecular docking studies to in MCF-7 cells which are the key kinases involved in G2/M cell
understand their mode of binding. cycle transition in mammalian cells. Cdk1 is activated by the
cyclin-B1 and involved in the metaphase by activating motor pro-
2.2.2. Cell cycle analysis teins required for spindle separation. Similarly Aurora B kinase is
To investigate the mechanism underlying the cytotoxic activity also involved in phosphorylation of histone H3 protein and local-
of 5f and 5k, the cell cycle distribution in MCF-7 cancer cell line ization of some proteins at kinetochore which recognizes the spin-
was analyzed by flow cytometry.42 In this study MCF-7 cells were dle attachment at chromosome. It also binds to EB1 protein which
treated with 0.5 mM concentration for 48 h. The data obtained regulates the microtubule dynamics at spindle.44,45 Accumulation
clearly indicated that they show G2/M cell cycle arrest. Both 5f of Cdk1 and aurora B in MCF-7 cells treated with 5f and 5k is
and 5k showed 33.9% and 32.6% of cell accumulation in G2/M comparable with the positive control nocodazole. Therefore, the
phase respectively, whereas in control (untreated cells) 2.6% of increased levels of these two kinases in comparison to the
G2/M phase was observed (Fig. 4 and Table 2). untreated control cells (Fig. 5) demonstrate that MCF-7 cells

Fig. 4. Flow cytometric analysis in MCF-7 cancer cell lines after treatment with compounds at 0.5 mM 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 Sub-G1phase % of cells in G1phase % of cells in S phase % of cells in G2/M phase
Control 3.2 83.2 11.2 2.6
5f 3.6 54.4 8.1 33.9
5k 2.9 54.1 9.4 32.6
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297 3289

treated with 5f and 5k arrest the cells at G2/M phase due to the
inhibition of metaphase to anaphase transition.

2.2.4. Effect on tubulin polymerization

Fig. 5. Western blot analysis of Cyclin-B1, CDK1 and Aurora B: Treatment of MCF-7
cells with 1 mM 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.
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 tubu-
lin 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 mM
concentration. Conjugates 5f and 5k inhibited tubulin polymeriza-
tion by 49.9%, 52.3%, compared to the control (Fig. 6). Tubulin poly-
merization 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 mM respectively compared to the control, where as

Fig. 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 mM.
3290 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
Table 3
Inhibition of tubulin polymerization (IC50) for conjugates 5f and 5k.
S. no. Compound IC50a + S.D(mM)
1 5f 1.23 + 0.29
2 5k 1.65 + 0.99
3 Nocodazole 1.32 + 0.85
a
Concentration of drug to inhibit 50% of tubulin assembly.
nocodazole was used as the positive control that showed an IC50
value of 1.32 mM (Table 3).
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 con-
centration of 0.5 mM for 48 h. The confocal images depicted in Fig. 7
showed that the untreated lung cancer cell line displayed the nor-
mal distribution of microtubules. However, cells treated with com-
pounds 5f and 5k showed disrupted microtubule organization as
seen in Fig. 7, thus demonstrating the inhibition of tubulin poly-
merization. The density of microtubules was pronounced at the
cell periphery with disorganized central networks and the stan-
dard nocodazole also showed disrupted microtubule organization.
These data suggested that both 5f and 5k acts as a mitotic inhibi-
tors by blocking cell cycle progression and metaphase by its ability
to disrupt spindle assembly.
2.2.6. Distribution of soluble versus polymerized tubulin in cells
Since inhibition of tubulin polymerization disturbs the micro-
tubule dynamics, we evaluated the levels of soluble versus poly-
merized forms of tubulin in MCF-7 cells following treatment
Fig. 7. Effect on microtubules and nuclear condensation: MCF-7 cells were independently treated with 5f, 5k and nocodazole at 0.5 mM 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.
Fig. 8. MCF-7 cells were treated with 1 mM of 5k and 5f for 48 h. Nocodazole and
taxol were used as reference standards. Levels of tubulin were detected by Western
blot analysis.
with 1 mM 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 poly-
merization agent showed more amount of tubulin in the polymer-
ized 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 sol-
uble 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 Fig. 8.
2.2.7. Measurement of mitochondrial membrane potential (DWm)
Mitochondria plays an essential role in the propagation of apop-
tosis and its dysfunction within the apoptotic process, which is
often associated with loss or depolarization of mitochondrial mem-
brane potential (DWm) leading to collapse of mitochondrial func-
tions ensuing cell death.48 Therefore, in this study we examined
the effect of conjugates 5f and 5k on DWm. In control cells the
dye concentrates in the mitochondrial matrix where it forms red
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
fluorescent aggregates (J-aggregates) because of the electrochemi-
cal 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 fluo-
rescence (590 nm) with normal cells (high Dwmt), 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 mitochon-
drial membrane is depolarized (low Dwmt), JC-1 forms monomers
that emit at 530 nm. In this study MCF-7 cells were treated with
5f and 5k at 0.5 mM 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 mono-
mers 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 Fig. 9, thus indicating that both the conjugates
induce apoptosis through depolarization of the mitochondrial
membrane potential in MCF-7 cells.
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 necro-
sis.49 In this study MCF-7 cells were treated with compounds 5f
Fig. 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 mM concentration
and stained with JC-1 dye for 20 min at room temperature.
3291
and 5k for 48 h at 0.5 mM 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 (Fig. 10). How-
ever, in the presence of 0.5 mM 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 Fig. 10. Similarly treatment with etoposide (0.5 mM),
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.
2.3. Molecular docking studies
To rationalize the experimental data obtained, molecular dock-
ing 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 Fig. 11A amide group of conjugate 5f established the
hydrogen binding interactions (red dotted lines) in which carbonyl
oxygen interacts with bAla354 and –NH interacts with bLeu248.
The benzo[d]imidazo[2,1-b]thiazole ring of the conjugate 5f buried
in the hydrophobic pocket and surrounded by bLeu255, bLys241,
bLeu252, bLeu242, bVal348 and bAla316 amino acid residues.
3292 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
Fig. 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 0.5 mM of 5f and 5k for 48 h.
The conjugate 5f interacts with the a and b tubulin interface
wherein the 1,2,3-triazole ring is surrounded by aSer148, aAla180,
aSer178, bAsn258, bLeu255 and 4-fluorophenyl ring by aAsn101,
aTyr224 and bLeu248 amino acid residues. Similarly from
Fig. 11B, the amide group of conjugate 5k established the hydrogen
binding interactions like in case of conjugate 5f, in which carbonyl
oxygen interacts with bAla354 and -NH interacts with bLeu248.
Conjugate 5k establishes similar binding position like conjugate
5f and is surrounded by similar amino acid residues. Fig. 11C
shows overlapping of molecular docking pose of conjugate 5f (yel-
low) and 5k (red) whereas Fig. 11D shows that the proposed bind-
ing 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 a-tubulin (blue) and b-tubulin (pink).
Overall, docking investigation showed that conjugate 5f and 5k
could interact with the colchicine binding site between the a and
b subunits of tubulin.
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 scaf-
folds. 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 inhibi-
tion. 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 poten-
tial 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), Lan-
caster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA,
USA), or Spectrochem Pvt. Ltd (Mumbai, India) used directly with-
out further purification unless otherwise noted. The reaction
courses and product mixtures were routinely checked by TLC per-
formed on silica gel glass plates containing 60 GF-254, and visual-
ized 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 (d) are reported in d
units, parts per million (ppm) downfield from an internal standard
TMS and coupling constants (J) were expressed in Hertz (Hz). Sig-
nal 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
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297 3293

(A) (B)

(C) (D)

Fig. 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. b-tubulin shows in pink and a-tubulin shows in blue color.

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 dis-
solved in dry DMF, cooled to 0 °C (ice bath). EDCI (1.5 equiv), HOBt
(1.2 equiv), propargylamine hydrochloride (1.2 equiv) and triethy-
lamine (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 dichloro-
methane (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-carbox-
amide (10a). Pale yellow solid; 79% yield; mp: 217–219 °C; 1H
NMR (300 MHz, CDCl3 + DMSO-d6) d 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) d 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) d 8.28 (s, 1H), 7.56–7.48 (m, 2H), 7.33 (s,
3294 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
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) d 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]thia-
zole-2-carboxamide (10c). White solid; 78% yield; mp: 215–
217 °C; 1H NMR (500 MHz, CDCl3) d 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) d 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) d 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) d 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]imi-
dazo[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) d 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) d 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) d 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) d 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.
4.1.2.3. 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) d 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) d 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) d 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) d 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) d 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) d 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 C26H21N6-
O2S [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) d 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) d 161.83 (JC-
F = 244 Hz), 161.35, 146.29, 145.54, 141.51, 132.41, 131.51,
130.28 (JC-F = 8 Hz), 129.44, 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)
d 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) d 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 C23H23N6-
O3S [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,
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
DMSO-d6) d 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) d 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)
d 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) d 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)
d 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)
d 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-methyl-
benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5k). White solid;
73% yield; mp: 243–245 °C; 1H NMR (400 MHz, CDCl3) d 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) d 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) d 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) d 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)
d 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) d 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
3295
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) d 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) d 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) d 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) d 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) d 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) d 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-methoxy-
benzo[d]imidazo[2,1-b]thiazole-2-carboxamide (5q). White solid;
75% yield; mp: 253–255 °C; 1H NMR (300 MHz, CDCl3 +
DMSO-d6) d 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) d 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)
d 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) d 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.
3296 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297
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) d 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) d 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) d 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) d 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) d 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)
d 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);
13
C NMR (75 MHz, CDCl3 + DMSO-d6) d 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 ml DMEM, supple-
mented 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 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium 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; formazan crystals were dissolved in
100 mL of DMSO and absorbance at 540 nm wavelengths was
recorded.
4.2.2. Cell cycle analysis
Flow cytometric analysis (FACS) analysis was performed to
evaluate the distribution of the cells through the cell cycle phases.
Human breast cancer cells (MCF-7) were incubated with com-
pounds 5f and 5k at 0.5 lM 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 analysis was performed by flow cytometryR (Becton
Dickinson FACS Caliber) as described earlier.42
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 lM concentrations of 5f and 5k
for 24 h. Subsequently, cells were harvested and proteins were
quantified using Amido Black followed by densitometry analysis.
Equal amount of protein were blotted on nitrocellulose membrane
using Bio-Dot SF microfiltration apparatus (Bio-Rad). Briefly, nitro-
cellulose membrane and 3 filters papers (Whatmann 3) were
soaked in 1 TBS solution for 10 min. Later, the filter papers, mem-
brane were arranged in the apparatus and connected to vacuum
pump (Millipore). The membranes were rehydrated using 100 ll
of 1 TBS by vacuum filtration. Subsequently, 50 ll volumes of
equal protein samples were blotted on the membrane and washed
with 200 ll of 1 TBS through application of vacuum. 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 per-
formed according to the manufacturers protocol (BK011,
Cytoskeleton, Inc.). Briefly, the reaction mixture was taken within
a total volume of 10 mL contained PEM buffer, GTP (1 mM) in the
presence or absence of test compounds 5f and 5k at a final concen-
tration of 3 mM. 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 pro-
ceeds. 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 fluores-
cence mentioned above at 37 °C. 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 lM).
Cells grown on cover slips were fixed in 3.5% formaldehyde in
phosphate-buffered saline (PBS) pH 7.4 for 10 min at room temper-
ature. Cells were permeabilized for 6 min in PBS containing 0.5%
Triton X-100 (Sigma) and 0.05% Tween-20 (Sigma). The permeabi-
lized cells were blocked with 2% BSA (Sigma) in PBS for 1 h. Later,
the cells were incubated with primary antibody for tubulin from
(sigma) at (1:200) diluted in blocking solution for 4 h at room tem-
perature. 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 1 h 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.
 S.P. Shaik et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3285–3297 3297

4.2.6. Western blot analysis of soluble versus polymerized tubulin Supplementary data
Cells were seeded in 12-well plates at 1  105 cells per well in
complete growth medium. Following treatment of cells with Supplementary data associated with this article can be found, in
respective compounds (5f, 5k and nocodazole and paclitaxel) for the online version, at http://dx.doi.org/10.1016/j.bmc.2017.04.013.
a duration of 24 h, cells were washed with PBS and subsequently
soluble and insoluble tubulin fractions were collected. To collect References
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