3-Substituted 2-Phenylimidazo[2,1-b]benzothiazoles: Synthesis, Anticancer Activity, and Inhibition of Tubulin Polymerization

Article abstract of DOI:10.1002/cmdc.201100511

A new series of 3-substituted 2-phenylimidazo[2,1-b]benzothiazoles (3a-h) were synthesized by C-arylation of 2-arylimidazo[2,1-b]benzothiazoles using palladium acetate as catalyst, and the resulting compounds were evaluated for their anticancer activity.

Full text of DOI:10.1002/cmdc.201100511

MED
DOI: 10.1002/cmdc.201100511
3-Substituted 2-Phenylimidazo[2,1-b]benzothiazoles:
Synthesis, Anticancer Activity, and Inhibition of Tubulin
Polymerization
Ahmed Kamal,*[a, c] Farheen Sultana,[a] M. Janaki Ramaiah,[b] Y. V. V. Srikanth,[a] A. Viswanath,[a]
Chandan Kishor,[b] Pranjal Sharma,[b] S. N. C. V. L. Pushpavalli,[b] Anthony Addlagatta,*[b] and
Manika Pal-Bhadra*[b]
A new series of 3-substituted 2-phenylimidazo[2,1-b]benzothia-
zoles (3a–h) were synthesized by C-arylation of 2-arylimidazo-
[2,1-b]benzothiazoles using palladium acetate as catalyst, and
the resulting compounds were evaluated for their anticancer
activity. Compounds 3a, 3e, and 3h exhibited good antiproli-
ferative activity, with GI50 values in the range of 0.19–83.1 mm.
Compound 3h showed potent anticancer efficacy against 60
human cancer cell lines, with a mean GI50 value of 0.88 mm.
This compound also induced cell-cycle arrest in the G2/M
phase and inhibited tubulin polymerization followed by activa-
tion of caspase-3 and apoptosis. A high-throughput tubulin
polymerization assay showed that the level of inhibition for
compound 3h is similar to that of combretastatin A-4. Molecu-
lar modeling studies provided a molecular basis for the favora-
ble binding of compounds 3a, 3e, and 3h to the colchicine
binding pocket of tubulin.
Introduction
Microtubules are composed of dynamic polymers of tubulin
that are involved in cellular processes and play an essential
role in mitosis, especially in induction of apoptosis,[1] thus
making them an important target in the development of com-
pounds for the treatment of cancer.[2] Cells that divide rapidly
have impaired microtubule dynamics, and are thus more sus-
ceptible to tubulin polymerization inhibitors and subsequent
arrest during mitosis than non-dividing cells.[1] The hallmark of
microtubule-interfering drugs is mitotic arrest, which ultimately
leads to apoptosis.[3] Tubulin polymerization inhibitors specifi-
cally bind to any one of the three reported binding sites,[4]
thereby stabilizing or destabilizing microtubule assembly.[5] Dis-
ruption of microtubule formation leads to cell-cycle arrest in
the G2/M phase, followed by apoptotic cell death.[4] Combre-
tastatin A-4 (CA-4), a naturally occurring compound isolated
from Combretum caffrum, binds to the colchicine binding site
and exhibits potent anticancer activity against a wide variety
of human cancer cell lines, including MDR cancer cell lines
which possess tubulin polymerization-inhibiting properties.[6,7]
Combretastatin A-4 phosphate (CA-4P), a prodrug of CA-4 is
presently undergoing clinical trials as a tumor vasculature-tar-
geting agent.[7–9] A number of CA-4 derivatives, containing
five-membered heterocycles that are considered good surro-
gates for the CA-4 double bond as they retain the cis-alkene
configuration, have been reported as potent tubulin inhibitors
that exhibit enhanced cytotoxicity.[10]
nist,[15] and anticancer activities.[16–18] In various imidazole-based
compounds, the position of a substituent has been observed
to have a significant effect on the mode of action. Various 1,5-
and 1,2-diarylimidazoles exhibit anticancer activity through in-
hibition of COX-2.[19–21] Similarly, CA-4-based compounds such
as 4,5-diaryl-1H-imidazole (1) (Figure 1) and 2-aryl-4-benzoyl-
imidazoles exhibit anticancer activity through inhibition of tu-
bulin polymerization.[22–24]
Benzothiazole, with its simple heterocyclic scaffold, has re-
cently emerged as an important pharmacophore with potential
anticancer activity.[25–29] Moreover, structural modification of the
benzothiazole scaffold results in a variety of biological proper-
ties including antimicrobial,[30] anti-inflammatory,[31] immuno-
suppressive,[32] antiallergenic,[33] and anticancer activities.[34,35] 2-
Arylimidazo[2,1-b][1,3]benzothiazole derivative YM-201627 (2),
for example, is a fused imidazobenzothiazole compound that
has shown potent anticancer activity against a number of solid
[a] Dr. A. Kamal, F. Sultana, Dr. Y. V. V. Srikanth, A. Viswanath
Division of Organic Chemistry
Indian Institute of Chemical Technology, Hyderabad 500607 (India)
E-mail: ahmedkamal@iict.res.in
[b] Dr. M. J. Ramaiah, C. Kishor, P. Sharma, Dr. S. N. C. V. L. Pushpavalli,
Dr. A. Addlagatta, Dr. M. Pal-Bhadra
Chemical Biology Laboratory
Indian Institute of Chemical Technology, Hyderabad 500607 (India)
E-mail: manika@iict.res.in
anthony@iict.res.in
Imidazoles are common scaffolds in highly significant bio-
molecules such as biotin, histamine, and in a number of alka-
loids that exhibit a broad spectrum of biological properties in-
cluding antimicrobial,[11] anticonvulsant,[12] sodium channel
blocking,[13] anti-inflammatory,[14] CB1 receptor inverse ago-
[c] Dr. A. Kamal
Catalytic Chemistry Chair, Chemistry Department
College of Science, King Saud University, Riyadh (Saudi Arabia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100511.
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Benzothiazoles Targeting Cancer and Tubulin Polymerization
2-aminobenzothiazole (4) with 2-bromoacetophenone (5) in
ethanol under reflux conditions for 1 h provided 7-methoxy-2-
phenyl imidazo[2,1-b]benzothiazole (6). Upon C-arylation[45]
with aryl and heteroaryliodo compounds 7a–h in the presence
of palladium acetate, copper iodide, and triphenyl phosphine,
using cesium carbonate as a base and 1,4-dioxane as a solvent,
afforded the desired benzothiazoles 3a–h after 16 h at reflux.
Biological activity
Anticancer activity
Fused benzothiazoles 3a–h were evaluated with regard to an-
ticancer activity against 60 cancer cell lines derived from nine
types of human cancer (lung, leukemia, colon, melanoma,
ovarian, renal, prostate, and breast cancer) at the National
Cancer Institute (NCI, Bethesda, MD, USA). Results are ex-
pressed as a percentage of growth inhibition (GI50) determined
relative to that of untreated control cells (Table 1). Among the
Figure 1. 4,5-diaryl-1H-imidazole (1), YM-201627 (2), and 3-substituted 2-
phenylimidazo[2,1-b]benzothiazoles (3).
tumors and is considered useful in the treatment of cancer,[36]
as well as being reported as an amyloid binding agent.[37]
Other imidazo[2,1-b]benzothiazole derivatives have been iden-
tified as p53 inhibitors.[38] Recently, a new series of Mannich
bases of 2-arylimidazo[2,1-b]benzothiazoles have been shown
to possess anticancer activity, with the ability to induce apop-
tosis through inhibition of tubulin polymerization.[39]
Table 1. Growth inhibition for selected cancer cell lines by 3-substituted
2-phenylimidazo[2,1-b]benzothiazoles.
Our earlier efforts toward the synthesis of a variety of novel
molecules led to the development of efficient anticancer
agents.[40–44] In the present investigation, an attempt has been
made, in view of the biological importance of both diarylimida-
zoles and imidazobenzothiazole, to synthesize 3-substituted 2-
phenylimidazo[2,1-b]benzothiazoles (3) using palladium-cata-
lyzed arylation and heteroarylation of 2-arylimidazo[2,1-b]ben-
zothiazoles. These compounds were evaluated for in vitro anti-
cancer activity, cell-cycle effects, and potential to inhibit tubu-
lin polymerization.
Compound
Cell line
Inhibition [%][a]
3b (NSC755298)
K-562
MDA-MB-435
HCT-116
MDA-MB-435
NCI-H522
53.21
84.92
51.81
86.37
À22.74[b]
3d (NSC755297)
3 f (NSC755303)
[a] Determined at 10 mm; data were obtained from the NCI in vitro dis-
ease-oriented human tumor cell screen. [b] Negative value indicates cell
death.
eight benzothiazoles synthesized, three were active in the pri-
mary screen and were further evaluated against a panel of 60
cell lines at five concentrations, with results as shown in
Table 2. These three compounds, 3a, 3e, and 3h, exhibited a
wide spectrum of activity against various cancer cell lines with
mean GI50 values of 21.8, 5.12, and 0.88 mm, respectively. Spe-
cifically, compound 3h exhibited excellent anticancer activity
against sixty cancer cell lines, with GI50 values ranging from
0.19–3.30 mm. Compound 3e also showed promising anticanc-
er activity, particularly against MDA-MB-435 cell line (GI50 value
of 0.32 mm). Compound 3a exhibited anticancer activity in the
micromolar range against certain cell lines tested. In this inves-
tigation, the phenyl ring remained unchanged while modifying
the aryl/heteroaryl substitution of the imidazobenzothiazole
ring system in an attempt to understand the SAR. It was ob-
served that compounds having 2-naphthyl and 3-thienyl
groups were the most active among the series. In contrast, aryl
rings with electron-donating substituents and heteroaromatic
groups such as 3-pyridyl and 2-pyrazinyl in the imidazobenzo-
thiazole system (3b–d and 3 f–g) displayed lower activity in a
majority of the cell lines. However, compound 3a, with a 4-hy-
droxy-3-methoxyphenyl group, exhibited moderate anticancer
activity with GI50 values ranging from 1.4 to 83.1 mm.
Results and Discussion
Chemistry
3-Substituted 2-phenylimidazo[2,1-b]benzothiazoles 3a–h were
prepared as shown in Scheme 1. Condensation of 6-methoxy-
Scheme 1. Reagents and conditions: a) EtOH, reflux, 6–8 h, 65–80%;
b) Pd(OAc)2, CuI, PPh3, aryl/heteroaryliodo compounds 7a–h, Cs2CO3, 1,4-di-
oxane, reflux, 16 h, 74–88%.
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Table 2. Anticancer activity of 3-substituted 2-phenylimidazo[2,1-b]benzothiazole derivatives in human cancer cell lines.
Cancer
Panel/cell line
GI50 [mm][a]
3e[c]
Cancer
Panel/cell line
GI50 [mm][a]
3a[b]
3h[d]
3a[b]
3e[c]
3h[d]
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Ovarian
IGROV1
NT[e]
NT[e]
3.90
0.87
4.20
NT[e]
2.02
0.45
>100
6.53
2.04
0.37
3.10
2.10
0.92
0.51
0.28
5.33
4.53
12.8
NT[e]
0.26
0.38
0.93
0.99
0.35
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
10.1
2.15
3.71
4.49
5.95
2.53
3.48
39.1
33.7
41.2
4.5
3.34
19.6
Non-small-cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Renal
786-0
A498
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
9.91
74.7
37.8
28.0
22.05
95.5
5.02
7.76
7.86
9.89
2.17
0.81
3.30
0.29
30.9
28.3
38.9
8.54
4.9
7.58
6.26
6.76
5.04
1.66
12.1
37.7
6.03
0.89
0.46
0.84
0.79
0.18
0.68
3.00
1.80
NT[e]
0.50
0.95
5.17
0.35
0.43
4.0
80.5
>100
>100
6.50
15.0
>100
4.40
81.0
2.90
Colon
Breast
COLO205
HCC-2998
HCT-116
HCT-15
HT29
5.30
>100
2.20
4.80
4.18
2.91
2.95
3.70
3.84
0.25
1.50
0.62
0.46
0.33
0.39
0.31
MCF7
28.6
9.8
>100
3.78
5.46
>100
0.35
1.10
0.48
1.80
1.40
1.10
MDA-MB-231
HS578T
BT-549
T-47D
MDA-MB-468
4.90
4.03
3.56
4.81
5.14
17.5
83.1
24.3
6.55
6.27
1.98
KM12
SW-620
CNS
Prostate
PC-3
DU-145
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
28.8
8.00
6.60
32.5
11.1
5.28
8.54
3.06
2.62
6.90
2.2
0.82
0.50
0.36
0.64
0.20
0.40
26.3
35.4
4.02
3.45
0.50
0.80
3.72
Melanoma
LOXIMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
Melanoma
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
7.6
>100
5.25
>100
2.64
0.58
>100
8.9
3.6
>100
8.2
4.90
3.34
>100
3.52
0.41
0.20
0.49
0.39
4.3
1.4
21.3
0.55
0.19
0.35
0.32
9.72
[a] Compound concentration required to decrease cell growth to half that of untreated cells. [b] 3a (NSC755302). [c] 3e (NSC755308). [d] 3h (NSC755307).
[e] Not tested.
In vitro cytotoxicity
3a–g caused G1 phase cell-cycle arrest, while compound 3h
To understand the cytotoxic nature of these compounds, an
MTT cytotoxicity assay was performed by treating A549 human
lung cancer cell line with compounds 3a–h at 4 mm for 24 h. It
was observed that compounds 3e and 3h were found to
cause significant cytotoxicity in cancer cells. Interestingly, com-
pound 3h showed cytotoxicity similar to that of CA-4, which
was employed as a positive control in this assay (Figure 2).
surprisingly caused G2/M cell-cycle arrest with a large accumu-
lation of cells (69%) in the G2/M phase. Similar effects were ob-
served for CA-4-treated cells (65%), as shown in Figure 3A.
Furthermore, apoptosis-inducing ability was also observed for
these compounds (3e, 3h, and CA-4) with an increase in the
percentage of cells in the G0 phase (Figure 3B).
Effect on inhibition of tubulin polymerization
Effect on cell cycle
Compounds that alter cell-cycle parameters with preferential
G2/M blockade are known to exhibit effects on tubulin assem-
bly. Moreover, inhibition of tubulin polymerization is strongly
associated with G2/M cell-cycle arrest.[46] As compound 3h ex-
Flow cytometry analysis was performed to investigate the
mechanism of cell death. A549 cells were treated with 4 mm of
3a–h, using CA-4 as the positive control, for 24 h. Compounds
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Benzothiazoles Targeting Cancer and Tubulin Polymerization
Moreover, these results confirmed the transient transfection
of tubulin–GFP-expressing plasmid in A549 cells following
treatment with compounds 3h, CA-4, and noc, whereas com-
pound 3h exhibited an inhibitory effect on tubulin polymeri-
zation (Figure 4B). We were therefore interested in investigat-
ing the level of tubulin inhibition for this compound (3h), for
which we employed a high-throughput tubulin polymerization
assay. Interestingly, 3h demonstrated tubulin inhibition similar
to that of CA-4. Pac was used as a tubulin polymerization sta-
bilizer control (Figure 4C).
Figure 2. A549 cell viability observed by MTT assay following treatment with
compounds 3a–h for 24 h at 4 mm; 10000 cells per well were seeded in 96-
well plates. CA-4 was used as a positive control; negative control (ctrl):
DMSO alone without test compound. Error bars represent SD from the
mean of triplicate measurements.
Activation of caspases
From previous reports, it is well established that molecules af-
fecting microtubule polymerization cause mitotic arrest and ul-
timately lead to apoptosis.[47] It is also known that caspases
play a vital and active role for the initiation and execution of
the apoptotic process.[48] Among the caspases, caspase-3 is
one of the key effector caspases which cleave multiple pro-
teins in cells, leading to apoptotic cell death.[49] In this context,
the effect of these conjugates in causing effective apoptosis in
A549 cells was examined. Cells were treated with 3h, CA-4,
noc, and pac at 4 mm, and cell lysates were subjected to West-
ern blot analysis. Results indicated that increased expression of
active caspase-3 protein corresponds to the cell death-induc-
ing nature of fused benzothiazole 3h, as shown in Figure 5.
hibited G2/M cell-cycle arrest, it was considered of interest to
understand the microtubule inhibitory role of this compound.
Therefore, A549 cells were treated with 3h, CA-4, nocodozole
(noc, a known tubulin inhibitor), and paclitaxel (pac, a known
tubulin stabilizer) at 4 mm for 24 h. Compound 3h resulted in
dispersed microtubule organization relative to control cells, as
observed by immunofluorescence and microscopy studies
using anti a-tubulin antibody (Figure 4A).
Figure 3. A) Effect of 3-substituted 2-phenylimidazo[2,1-b]benzothiazoles on cell cycle. A549 cells were treated with compounds 3a–h at 4 mm for 24 h. CA-4
was used as positive control. B) Percent apoptosis following treatment with compounds 3a–h at 4 mm for 24 h.
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Figure 4. A) Effect of compounds on the microtubule network. A549 cells were treated with CA-4, nocodazole (noc), paclitaxel (pac), and 3h at 4 mm for 24 h.
Microtubule organization can be observed in green in control cells; this was found to be disrupted in cells treated with CA-4, noc, and 3h. B) Effect of com-
pound 3h on ectopically expressed tubulin. A549 cells were transfected with tubulin–GFP-expressing plasmid and treated with 3h, CA-4, and noc at 4 mm for
24 h. Treatment with these compounds resulted in disrupted microtubule patterns relative to untreated control cells. C) Inhibitory activity of compound 3h
on tubulin polymerization. An in vitro high-throughput tubulin polymerization assay was carried out using compounds CA-4, noc, pac, and 3h at 4 mm. OD
readings were taken at l 340 nm and were used to measure tubulin inhibitory activity.
drophobic contacts with the binding pocket of the b-chain
(Figure 6). The benzothiazole ring is shown to bind to the
same position as ring A of colchicine. Amino acids in proximity
to the benzothiazole moiety include Cys241, Leu242, Ala250,
Leu255, Val318, and Ile378. In addition, hydrophobic contacts
were also observed for the phenyl group buried within the
Figure 5. Effect of conjugates on the expression of caspase-3 protein. A549
cells were treated with compounds CA-4, noc, pac, or 3h for 24 h, and cell
lysates were subjected to Western blot for active caspase-3. b-Actin was
region including Ala80 (from the a-chain), Val181 (a-chain),
Met259, Ala316, and the hydrophobic region of Lys352. The
variable moiety of compounds 3a–h extends into the interface
of the a- and b-chains. The naphthyl group of the most active
compound (3h) is surrounded by Leu248, the hydrophobic
portion of Lys254, the side chain peptide p-region of Asn258,
Ser178 and Ala180 from the a-chain. Compound 3a, which
contains a 4-hydroxy substituent on the phenyl ring, likely
forms a hydrogen bond with Lys254 while the 3-methoxy sub-
stituent binds within the hydrophobic region similar to the
naphthyl ring of compound 3h. The thienyl group in com-
pound 3e is smaller in size than the naphthyl group of com-
used as a loading control.
Docking studies
To elucidate the mode of binding to tubulin and consequent
microtubule formation, it was considered of interest to exam-
ine these fused benzothiazoles (3a–h) by directly docking to
the colchicine binding site of b-tubulin. AutoDock results sug-
gest that the docking position of the benzothiazole moiety is
in the colchicine binding pocket and results in extensive hy-
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Benzothiazoles Targeting Cancer and Tubulin Polymerization
ed for their anticancer and tubulin polymerization inhibitory
potential. These compounds exhibited significant anticancer
activity, with GI50 values ranging from 0.19 to 83.1 mm. Com-
pound 3h, containing a naphthyl ring, was the most active
amongst this series and showed potent anticancer efficacy
against sixty human cancer cell lines with a mean GI50 value of
0.88 mm. Moreover, studies showed that compound 3h arrest-
ed cells in the G2/M phase of the cell cycle, followed by the ac-
tivation of caspase, as the mechanism of cytotoxicity. A high-
throughput tubulin polymerization assay showed that the level
of tubulin inhibition for compound 3h was similar to that of
CA-4. The biochemical data presented suggests that com-
pounds 3a and 3e arrest the cell cycle in the G1 phase to a
greater extent than the G2/M phase, while the opposite is true
of 3h. The modeling data further confirms that all of these
compounds are able to bind to tubulin; however, there may
be other mechanistic aspects by which they arrest the cell
cycle. Biochemical data of tubulin polymerization inhibition by
these fused benzothiazoles corroborates well with the molecu-
lar docking studies. The results suggest that these new 3-sub-
stituted 2-phenylimidazo[2,1-b]benzothiazoles, particularly 3h,
have the potential to be developed as a new class of tubulin
polymerization inhibitors for the treatment of cancer.
Experimental Section
Chemistry
Figure 6. Molecular modeling. A) Binding of compound 3h to the b-chain
(shown as a grey surface) where colchicine is known to bind. The structure
in light blue represents the a-chain at Ala180. Note that common regions of
the inhibitors are buried inside the hydrophobic pocket. B) Compound 3h is
shown in magenta, while the b-chain is shown in green and the a-chain in
blue. Amino acids surrounding the inhibitor are shown labeled in single-
letter code for clarity.
All chemicals and reagents were obtained from Aldrich (Sigma–Al-
drich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai,
India) and were used without further purification. Reactions were
monitored by TLC performed on silica gel glass plates containing
60 GF-254, and visualization was achieved by UV light or iodine in-
dicator. Column chromatography was performed with Merck 60–
1
120 mesh silica gel. H spectra were recorded on Bruker UXNMR/
pound 3h and is therefore buried favorably within the a-/b-
chain interface. Compounds 3b, 3c, and 3d are variations of
methoxy substitution on the phenyl ring. Dimethoxy-contain-
ing compounds 3b and 3c have unfavorable interactions with
the hydrophobic pocket and do not fit well. Similar interac-
tions were observed with compound 3d. Although the 4-hy-
droxy group on phenyl ring is quite favorable, the methoxy
group leads to unfavorable interaction with Lys254, Leu255,
and Asn258 residues of the b-chain due to the increased steric
bulk on the phenyl ring. Both the pyridyl and pyrazinyl rings of
compound 3 f and 3g pack perpendicularly to the naphthyl
group, and it is possible that the ring nitrogen groups do not
find favorable hydrogen bonding partners, resulting in their in-
significance as inhibitors in this series of fused benzothiazoles.
Overall, modeling studies carried out not only provide a molec-
ular basis for the mode of binding but also show the specific
mode of inhibition of tubulin polymerization.
XWIN-NMR (300 MHz) or Inova Varian-VXR-unity (400, 500 MHz) in-
struments. Chemical shifts (d) are reported in ppm downfield from
an internal TMS standard. ESI spectra were recorded on a Micro-
mass Quattro LC using ESI+ software with capillary voltage 3.98 kV
and 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 with an Electro
thermal melting point apparatus, and are uncorrected.
7-Methoxy-2-phenylimidazo[2,1-b]benzothiazole (6): A mixture
of 6-methoxy-2-aminobenzothiazole (4, 1.00 g, 5.55 mmol), 2-bro-
moacetophenone (5, 1.20 g, 6.02 mmol), were dissolved in 10 mL
EtOH and stirred for 1 h at reflux. The solution was cooled to room
temperature and concentrated under vacuum to remove the EtOH.
The resulting crude mass was diluted with H2O, neutralized with
saturated NaHCO3 solution, and extracted with EtOAc to obtain 6
as a white solid. Yield: 1.32 g (84%); mp: 110–1118C; 1H NMR
(300 MHz, CDCl3): d=8.26 (s, 1H), 7.88–7.67 (m, 3H), 7.47 (dt, J=
8.3, 0.7 Hz, 1H), 7.42–7.32 (m, 4H), 7.24 (t, J=7.4 Hz, 1H), 3.81 ppm
(s, 3H); MS (ESI) m/z: 281 [M+1]+.
2-Methoxy-4-(7-methoxy-2-phenylbenzo[d]imidazo[2,1-b]-
Conclusions
[1,3]thiazol-3-yl)phenol (3a):
A
mixture of 7-methoxy-2-
A series of 3-substituted 2-phenylimidazo[2,1-b]benzothiazoles
were synthesized using palladium-catalyzed arylation/heteroar-
ylation of 2-arylimidazo[2,1-b]benzothiazoles and were evaluat-
phenylimidazo[2,1-b]benzothiazole (6, 280 mg, 1 mmol), 4-hydroxy-
3-methoxyiodobenzene (7a, 250 mg, 1 mmol), Pd(OAc)2 (10 mol%),
PPh3 (290 mg, 0.5 mmol), and Cs2CO3 (390 mg, 1.2 mmol) in 1,4-di-
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A. Kamal, A. Addlagatta, M. Pal-Bhadra, et al.
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oxane was stirred at reflux for 16 h. Progress of the reaction was
monitored by TLC. Following completion of the reaction, the mix-
ture was cooled to room temperature, dioxane was removed
under reduced pressure, and the reaction mixture was extracted
with EtOAc (3ꢁ10 mL). The combined extract was washed with
H2O (1ꢁ5 mL) and brine (1ꢁ5 mL) and was dried over anhydrous
Na2SO4. The organic layer was concentrated under reduced pres-
sure, and the crude product was purified by column chromatogra-
phy using EtOAc/hexane (2:8) as the eluent to afford compound
(300 MHz, CDCl3): d=7.51–7.48 (m, 4H), 7.27–7.13 (m, 5H), 6.80–
6.77 (d, J=9.0 Hz, 1H), 6.72–6.70 (d, J=9.0 Hz, 1H), 3.81 ppm (s,
3H); MS (ESI) m/z: 363 [M+1]+; HRMS (ESI m/z) calcd for
C20H15N2OS2: 363.06258, found: 363.05778 [M+1]+; anal. calcd for
C20H14N2OS2: C 66.27, H 3.89, N 7.73, found: C 66.29, H 3.93, N 7.72.
7-Methoxy-2-phenyl-3-(3-pyridyl)benzo[d]imidazo[2,1-b]-
[1,3]thiazole (3 f): This compound was prepared according to the
method described for compound 3a, employing compound 7-me-
thoxy-2-phenylimidazo[2,1-b]benzothiazole (6, 280 mg, 1 mmol)
and 3-iodopyridine (7 f, 205 mg,1 mmol) to obtain pure product 3 f
as a white solid. Yield: 270 mg (76%); mp: 166–1678C; 1H NMR
(300 MHz, CDCl3 +[D6]DMSO): d=8.78–8.76 (d, J=5.0 Hz, 1H), 8.74
(s, 1H), 7.92–7.91 (d, J=8.0 Hz, 1H), 7.56–7.54 (m, 1H), 7.43–7.41
(m, 2H), 7.33 (s, 1H), 7.23–7.15 (m. 3H), 6.76–6.71 (m, 2H),
3.81 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=151.7, 150.3, 138.7,
128.9, 127.2, 127.1, 123.9, 113.5, 112.9, 108.8, 55.8 ppm; MS (ESI) m/
z: 358 [M+1]+; HRMS (ESI m/z) calcd for C21H16ON3S: 358.10086,
found: 358.09944 [M+1]+; anal. calcd for C21H15N3OS: C 70.57, H
4.23, N 11.76, found: C 70.60, H 4.19, N 11.73.
1
3a as a white solid. Yield: 300 mg (74%); mp: 170–1718C; H NMR
(500 MHz, CDCl3 +[D6]DMSO): d=9.23 (s, 1H), 8.01 (s, 1H), 7.48–
7.40 (m, 2H), 7.32–7.27 (m, 2H), 7.19–7.13 (m, 4H), 6.68–6.62 (m,
2H), 3.78 (s, 3H), 3.45 ppm (s, 3H); MS (ESI) m/z: 403 [M+1]+;
HRMS (ESI m/z) calcd for C24H21O3N2S: 403.11164, found: 403.11089
[M+1]+; anal. calcd for C23H18F3N2O3S: C 68.64, H 4.51, N 6.96,
found: C 68.62, H 4.54, N 6.97.
3-(2,4-Dimethoxyphenyl)-7-methoxy-2-phenylbenzo[d]imidazo-
[2,1-b][1,3]thiazole (3b): This compound was prepared according
to the method described for compound 3a, employing compound
7-methoxy-2-phenylimidazo[2,1-b]benzothiazole
(6,
280 mg,
7-Methoxy-2-phenyl-3-(2-pyrazinyl)benzo[d]imidazo[2,1-b]-
[1,3]thiazole (3g): This compound was prepared according to the
method described for compound 3a, employing compound 7-me-
thoxy-2-phenylimidazo[2,1-b]benzothiazole (6, 280 mg, 1 mmol)
and 2-iodopyrazine (7g, 206 mg,1 mmol) to obtain pure product
1 mmol) and 2,4-dimethoxyiodobenzene (7b, 264 mg, 1 mmol) to
obtain pure product 3b as a white solid. Yield: 330 mg (80%); mp:
1
165–1668C; H NMR (300 MHz, CDCl3): d=7.60–7.56 (m, 2H), 7.27–
7.15 (m, 5H), 6.76–6.55 (m, 4H), 3.93 (s, 3H), 3.68 ppm (s, 6H); MS
(ESI) m/z: 417 [M+1]+; HRMS (ESI m/z) calcd for C24H21O3N2S:
417.12674, found: 417.12556 [M+1]+; anal. calcd for C24H20N2O3S:
C 69.21, H 4.84, N 6.73, found: C 69.24, H 4.82, N 6.74.
1
3g as a white solid. Yield: 300 mg (83%); mp: 169–1708C; H NMR
(500 MHz, CDCl3 +[D6]DMSO): d=8.63 (d, J=7.5 Hz, 1H), 8.43 (s,
1H), 7.45 (d, J=7.5 Hz, 1H), 7.38–7.33 (m, 2H), 7.30–7.20 (m, 3H),
7.18 (s, 1H), 6.89–6.78 (m, 2H), 3.79 ppm (s, 3H) MS (ESI) m/z: 359
[M+1]+; HRMS (ESI m/z) calcd for C20H15N4OS: 359.09666, found:
359.09586 [M+1]+; anal. calcd for C20H14N4OS: C 67.02, H 3.94, N
15.63, found: C 67.00, H 3.96, N 15.65.
3-(2,6-Dimethoxyphenyl)-7-methoxy-2-phenylbenzo[d]imidazo-
[2,1-b][1,3]thiazole (3c): This compound was prepared according
to the method described for compound 3a, employing compound
7-methoxy-2-phenylimidazo[2,1-b]benzothiazole
(6,
280 mg,
1 mmol) and 2,6-dimethoxyiodobenzene (7c, 264 mg,1 mmol) to
obtain pure product 3c as a white solid. Yield: 340 mg (81%); mp:
175–1768C; H NMR (500 MHz, CDCl3): d=7.54–7.52 (m, 2H), 7.46–
7.42 (m, 1H), 7.19–7.16 (m, 2H), 7.12–7.08 (m, 2H), 6.66–6.61 (m,
4H), 3.79 (s, 3H), 3.60 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=
156.5, 134.6, 133.6, 131.9, 130.3, 128.1, 126.4, 113.6, 112.8, 108.2,
105.1, 99.1, 55.7, 55.5, 55.4 ppm; MS (ESI) m/z: 417 [M+1]+; HRMS
(ESI m/z) calcd for C24H21O3N2S: 417.12674, found: 417.12638 [M+
1]+; anal. calcd for C24H20N2O3S: C 69.21, H 4.84, N 6.73, found: C
69.19, H 4.85, N 6.76.
7-Methoxy-3-(2-naphthyl)-2-phenylbenzo[d]imidazo[2,1-b]-
[1,3]thiazole (3h): This compound was prepared according to the
method described for compound 3a, employing compound 7-me-
thoxy 2-phenylimidazo[2,1-b]benzothiazole (6, 280 mg, 1 mmol)
and 1-iodonaphthalene (7h, 254 mg,1 mmol) to obtain pure prod-
uct 3h as a white solid. Yield: 360 mg (88%); mp: 170–1718C;
1H NMR (500 MHz, CDCl3): d=7.85 (s, 1H), 7.82–7.80 (m, 2H), 7.66–
7.59 (m, 1H), 7.55–7.51 (m, 1H), 7.48–7.45 (m, 2H), 7.39–7.36 (m,
3H), 7.26–7.23 (m, 1H), 7.19–7.18 (m. 1H), 7.14–7.07 (m, 2H), 6.97–
6.95 (m, 1H), 3.31 ppm (s, 3H)); 13C NMR (75 MHz, [D6]DMSO): d=
154.1, 148.8, 143.9, 143.4, 133.7, 132.8, 132.1, 132.0, 129.8, 129.1,
128.2, 128.1, 128.0, 127.2, 126.8, 126.2, 123.2, 121.6, 120.4, 101.2,
56.1, 55.6 ppm; MS (ESI) m/z: 407 [M+1]+; HRMS (ESI m/z) calcd
for C26H19ON2S: 407.12069, found: 407.12126 [M+1]+; anal. calcd
for C26H18N2OS: C 76.82, H 4.46, N 6.89, found: C 76.80, H 4.49, N
6.86.
1
7-Methoxy-2-phenyl-3-(3,4,5-trimethoxyphenyl)benzo[d]imidazo-
[2,1-b][1,3]thiazole (3d): This compound was prepared according
to the method described for compound 3a, employing compound
7-methoxy-2-phenylimidazo[2,1-b]benzothiazole
(6,
280 mg,
1 mmol) and 3,4,5-trimethoxyiodobenzene (7d, 294 mg,1 mmol) to
obtain pure product 3d as a white solid. Yield: 360 mg (80%); mp:
1
170–1718C; H NMR (300 MHz, CDCl3): d=7.61–7.59 (m, 2H), 7.29–
7.24 (m, 2H), 7.21–7.17 (m, 2H), 6.80 (s, 1H), 6.77–6.74 (m, 3H),
4.00 (s, 3H), 3.83 (s, 3H), 3.81 ppm (s, 6H); 13C NMR (75 MHz,
CDCl3): d=156.7, 153.8, 131.7, 128.2, 126.8, 126.5, 125.4, 123.9,
113.9, 112.9, 108.6, 107.9, 104.5, 61.1, 60.9, 56.2, 55.8 ppm; MS (ESI)
m/z: 447 [M+1]+; HRMS (ESI m/z) calcd for C25H23O4N2S:
447.13730, found: 447.13729 [M+1]+; anal. calcd for C25H22N2O4S:
C 67.25, H 4.97, N 6.27, found: C 67.27, H 4.93, N 6.24.
Biology
Cell culture: The A549 human lung cancer cell line was purchased
from American Type Culture Collection and maintained in Dulbec-
co’s modified Eagle’s medium (DMEM) (Invitrogen), supplemented
with 2 mm glutamax (Invitrogen), 10% fetal calf serum, 100 UmLÀ1
penicillin and 100 mgmLÀ1 streptomycin sulfate (Sigma). The cell
line was maintained at 378C in a humidified atmosphere contain-
ing 5% CO2.
7-Methoxy-2-phenyl-3-(3-thienyl)benzo[d]imidazo[2,1-b]-
[1,3]thiazole (3e): This compound was prepared according to the
method described for compound 3a, employing compound 7-me-
thoxy-2-phenylimidazo[2,1-b]benzothiazole (6, 280 mg, 1 mmol)
and 3-iodothiophene (7e, 210 mg,1 mmol) to obtain pure product
MTT assay: Cell viability was assessed using an MTT mitochondrial
function assay, based on the ability of viable cells to reduce MTT
to insoluble formazan crystals by mitochondrial dehydrogenase.
A549 cells were seeded in a 96-well plate at a density of 10000
1
3e as a white solid. Yield: 300 mg (83%); mp 168–1698C; H NMR
298
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 292 – 300

Benzothiazoles Targeting Cancer and Tubulin Polymerization
cells per well. Following overnight incubation, cells were treated
with compounds 3a–h and CA-4 at 4 mm and were incubated for
24 h. The medium was then discarded and replaced with 10 mL
MTT dye. Plates were incubated at 378C for 2 h. The resulting for-
mazan crystals were solubilized in 100 mL extraction buffer. The op-
tical density (OD) was read at l 570 nm with a microplate reader
(Multi-mode Varioskan Instrument, Thermo Scientific).
12000 rpm (8064 g) for 10 min, protein in the supernatant was
quantified by the Bradford method (BioRad) using a Multimode
VarioSkan instrument (Thermo Scientific); 50 mg protein per lane
was loaded on a 12% SDS-polyacrylamide gel. After electrophore-
sis, the protein was transferred to a polyvinylidine difluoride
(PVDF) membrane (Amersham Biosciences). The membrane was
blocked at room temperature for 2 h in TBS containing 0.1%
Tween 20 (TBST) with 5% blocking powder (Santa Cruz Biotech-
nology). The membrane was washed with TBST for 5 min, then pri-
mary antibody was added and incubated at 48C overnight. Active
caspase-3 and b-actin were purchased from Imgenex Corporation
(San Diego, CA, USA). Membranes were washed three times with
TBST for 15 min, and the blots were visualized with chemilumines-
cence reagent (Thermo Scientific). X-ray films were developed and
fixed using solutions from Kodak.
Immunofluorescence assay: A549 cells were seeded on cover slips
and treated with 4 mm 3h, noc, CA-4, or pac for 24 h. After treat-
ment, cover slips were fixed with a paraformaldehyde solution (4%
in 1ꢁ PBS) for 20 min at room temperature. Cell permeabilization
was achieved by administration of a Triton X-100 solution (0.2% in
1ꢁ PBS) for 5 min. The cover slips were left in 100% MeOH over-
night at 48C. Subsequently, cover slips were blocked with a 1%
BSA solution for 60 min, then incubated with anti a-tubulin anti-
body (1:100) at room temperature for 2 h. The slides were washed
three times for 5 min each with PBST. Next, cover slips were incu-
bated with a FITC-conjugated anti-rabbit secondary antibody (Jack-
son Immuno Research Laboratories Inc., Pennsylvania, USA) for 1 h
then were washed three times with PBST solution and mounted
with DAPI/PI solution. Finally, cells were observed under a confocal
microscope (Olympus FV1000). Collected images were processed
with the support of Flow View version 1.7c software [DakoCytoma-
tion (Beckman Coulter, Brea, CA, USA)].
Molecular modeling
Tubulin bound to colchicine (PDB code: 3E22) was selected as the
receptor for docking simulations. After removing the ligand and
solvent molecules, hydrogen atoms and Kollman charges were
added to each protein atom. Coordinates for each compound
were obtained from Chemdraw11 followed by MM2 energy mini-
mization. Docking was carried out by AutoDock4 in the colchicine
binding pocket.[50–52] Grid map in AutoDock was used to define the
interaction of protein and ligand in the binding pocket. The grid
map was used with 60 points in each x, y, and z direction, equally
spaced at 0.375 ꢂ. Docking was performed using the Lamarckian
genetic algorithm.[53] Each docking experiment was performed 100
times, yielding 100 docked conformations. Parameters used for the
docking were as follows: population size of 150; random starting
position and conformation; maximal mutation of 2 ꢂ translation
and 50degrees rotation; elitism of 1; mutation rate of 0.02 and
crossover rate of 0.8; and local search rate of 0.06. Simulations
were performed with a maximum of 1.5 million energy evaluations
and a maximum of 50000 generations. Final docked conformations
were clustered using a tolerance of 1 ꢂ root mean square deviation
(RMSD). The best model was chosen based on the most favorable
stabilization energy.
Tubulin transfection studies: Human A549 cells were seeded on
cover slips. The tubulin-expressing plasmid construct was transient-
ly transfected with lipofectamine 2000. After 24 h, the A549 cells
were treated with compound 3h, CA-4, noc, or pac at 4 mm for
24 h. Cells were then examined using confocal microscopy and
images were collected.
Tubulin polymerization assay: A tubulin polymerization assay was
conducted using cytoskeleton kit BK-004 (Lab-pro Company,
Denver, CO, USA). Compounds 3h, CA-4, noc, and pac were incu-
bated with tubulin (100 mL) as provided in the kit, and readings
were taken for 30 min to 1 h. The OD values at l 340 nm were
measured, and inhibition graphs were plotted (MS Office Excel
2007).
Cell-cycle analysis: 5ꢁ105 A549 cells were seeded in 60 mm dishes
and were allowed to grow for 24 h. Compounds 3a–h or CA-4
(positive control) at were added to the culture media to a final
concentration of 4 mm, and the cells were incubated for an addi-
tional 24 h. Cells were harvested with Trypsin-EDTA, fixed with ice-
cold 70% EtOH at 48C for 30 min, washed with PBS, and incubated
with 1 mgmLÀ1 RNAse solution (Sigma) at 378C for 30 min. Cells
were collected by centrifugation at 2000 rpm for 5 min and were
further stained with 250 mL DNA staining solution [10 mg propidi-
um iodide (PI), 0.1 mg trisodium citrate, and 0.03 mL Triton X-100,
dissolved in 100 mL sterile water at room temperature for 30 min
in the dark]. The DNA contents of 20000 events were measured
using a DakoCytomation flow cytometer (Beckman Coulter, Brea,
CA, USA). Histograms were analyzed using Summit Software.
Acknowledgements
The authors are thankful to the Council of Scientific and Industri-
al Research (CSIR), India (F.S. and A.V.) and the University Grants
Commission (UCG), New Delhi, India (Y.V.V.S. and C.K.) for the
award of research fellowships.
Keywords: antitumor agents · benzothiazoles · caspases ·
molecular modeling · tubulin
Protein extraction and Western blot analysis: A549 human lung
cancer cells were seeded in 60 mm dishes and were allowed to
grow to attain 80% confluency for 24 h. Compounds (3h, CA-4,
noc, or pac) were added to the culture media for a final concentra-
tion of 4 mm, and the cells were incubated with compounds for
24 h. After 24 h, total cell lysates from cultured A549 cells were ob-
tained by lysing the cells in ice-cold RIPA buffer (1ꢁ PBS, 1% NP-
40, 0.5% sodium deoxycholate, and 0.1% SDS) containing
100 mgmLÀ1 PMSF, 5 mgmLÀ1 aprotinin, 5 mgmLÀ1 leupeptin,
5 mgmLÀ1 pepstatin, and 100 mgmLÀ1 NaF. After centrifugation at
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Received: November 3, 2011
Revised: December 7, 2011
Published online on January 12, 2012
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