Synthesis of imidazothiazole-chalcone derivatives as anticancer and apoptosis inducing agents

Article abstract of DOI:10.1002/cmdc.201000346

A new class of imidazo[2,1-b]thiazole chalcone derivatives were synthesized and evaluated for their anticancer activity. These chalcone derivatives show promising activity, with logGI₅₀ values ranging from -7.51 to -4.00. The detailed biological aspects of these derivatives toward the MCF-7 cell line were studied. Interestingly, these chalcone derivatives induced G ₀/G₁-phase cell-cycle arrest, down-regulation of G ₁-phase cell-cycle regulatory proteins such as cyclin D1 and cyclin E1, and up-regulation of CDK4. Moreover, these compounds elicit the characteristic features of apoptosis such as enhancement in the levels of p53, p21, and p27, suppression of NF-κB, and up-regulation of caspase-9. One of these chalcone derivatives, 3d, is potentially well suited for detailed biological studies, either alone or in combination with existing therapies. Breaking the cycle: We undertook an extensive examination of the ability of a series of new chalcone derivatives to regulate the cell cycle and to induce apoptosis in various cancer cell lines. Compound 3d is a particularly suitable candidate for further detailed biological investigations, especially in the treatment of breast cancer.

Full text of DOI:10.1002/cmdc.201000346

DOI: 10.1002/cmdc.201000346
Synthesis of Imidazothiazole–Chalcone Derivatives as
Anticancer and Apoptosis Inducing Agents
Ahmed Kamal,* D. Dastagiri, M. Janaki Ramaiah, J. Surendranadha Reddy, E. Vijaya Bharathi,
Chatla Srinivas, S. N. C. V. L. Pushpavalli, Dhananjaya Pal, and Manika Pal-Bhadra*[a]
A new class of imidazo[2,1-b]thiazole chalcone derivatives were
synthesized and evaluated for their anticancer activity. These
chalcone derivatives show promising activity, with logGI50
values ranging from ꢀ7.51 to ꢀ4.00. The detailed biological as-
pects of these derivatives toward the MCF-7 cell line were
studied. Interestingly, these chalcone derivatives induced G0/
G1-phase cell-cycle arrest, down-regulation of G1-phase cell-
cycle regulatory proteins such as cyclin D1 and cyclin E1, and
up-regulation of CDK4. Moreover, these compounds elicit the
characteristic features of apoptosis such as enhancement in
the levels of p53, p21, and p27, suppression of NF-kB, and up-
regulation of caspase-9. One of these chalcone derivatives, 3d,
is potentially well suited for detailed biological studies, either
alone or in combination with existing therapies.
Introduction
Chalcones are naturally occurring compounds that belong to
the flavonoid family and have been associated with a wide va-
riety of anticancer effects.[1] These compounds have also been
reported to possess various other biological activities, such as
antimalarial,[2,3] anti-HIV,[4] and tyrosinase inhibition.[5] The re-
markable biological potential of these chalcones is due to their
possible interactions with various proteins related to apoptosis
and cell proliferation. Recent studies have shown that these
chalcones induce apoptosis in a variety of cell types, including
breast cancers.[6–9] Over the last few years, research into the an-
titumor properties of chalcones has received significant atten-
tion, and the majority of the naturally occurring chalcones con-
tain either hydroxy or methoxy groups in both the A and B
rings. The 3,4,5-trimethoxyphenyl ring is thought to be essen-
tial for the anticancer activity of chalcones. Some of the recent
advances in the development of anticancer agents involve
structural modification of chalcones to improve their bioavaila-
bility and to study the role of various substituents on aryl or
heteroaryl rings.[10] Moreover, heterocyclic derivatives of chal-
cones in which the B ring is replaced by a heterocyclic ring
have been systematically investigated. Imidazothiazoles are
also well-known compounds, and many derivatives of this
fused ring system have been evaluated for potential biological
activity, particularly antitumor activity.[11,12] Some representative
chalcones and imidazothiazole derivatives are illustrated in
Figure 1.
Figure 1. Structures of chalcone 1, imidazothiazole guanylhydrazones 2, and
imidazothiazole–chalcone derivatives 3a–p.
Results and Discussion
Chemistry
The imidazothiazole–chalcone derivatives 3a–p were prepared
by Claisen–Schmidt condensation[13] of the appropriately sub-
stituted acetophenones upon treatment with imidazo[2,1-
b]thiazole aldehydes 8a–i in the presence of 10% sodium hy-
droxide, as shown in Scheme 1. The imidazo[2,1-b]thiazole al-
dehydes 8[14] were obtained by Vilsmeier reaction with the cor-
responding imidazo[2,1-b]thiazoles 7, which were prepared
from the appropriate 2-aminothiazoles 5 and bromoketones 4.
The intermediate compounds 6 were isolated and used in the
subsequent step without further purification.
Considering the potent bioactivities of compounds that pos-
sess an imidazothiazole core, we synthesized new chalcone de-
rivatives that incorporate an imidazothiazole skeleton and eval-
uated their anticancer activity. The promising activity observed
prompted us to investigate the role of these new compounds
in the proliferation and apoptosis of a human breast cancer
cell line (MCF-7). We also investigated the effect of these com-
pounds on proteins that regulate cell-cycle progression.
[a] Dr. A. Kamal, D. Dastagiri, Dr. M. J. Ramaiah, J. S. Reddy, E. V. Bharathi,
C. Srinivas, S. N. C. V. L. Pushpavalli, D. Pal, Dr. M. Pal-Bhadra
Chemical Biology Laboratory
Indian Institute of Chemical Technology, Hyderabad 500607 (India)
Fax:(+91)40-2719-3189
E-mail: ahmedkamal@iict.res.in
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1937

A. Kamal, M. Pal-Bhadra, et al.
MED
cancer), IGROV1, NCI/ADR-RES, OVCAR-8 (ovarian cancer), UO-
31 (renal cancer), and MDA-MB-435, SK-MEL-2, SK-MEL-5 (mela-
noma). Compound 3d also showed promising activity against
the SR (leukemia) and HCT-116 (colon cancer) cell lines. More-
over, the other compounds also showed significant activity in
the sub-micromolar range against all the cell lines tested. Com-
pounds with trifluoromethyl and 2-thienyl substituents at the
6-position are the most active derivatives. However, a methyl
group at the 2-position of the imidazothiazole system (com-
pounds 3g–i) leads to a decrease in activity. Moreover, removal
of one methoxy group (compounds 3d and 3 f) in the A ring
also decreases activity.
Cell viability assays were carried out to identify the cytotoxic
effects of some of these chalcone derivatives (compounds 3b–
e and 3h) toward MCF-7 cells at a concentration of 4 mm. The
activities of parent chalcone 1 and doxorubicin as a positive
control were also examined to substantiate the activities the
chalcone derivatives tested (3b–e and 3h). Interestingly, the
test compounds show higher cytotoxicity than chalcone 1
toward MCF-7 cells, as shown in Figure 2, and the data for
these chalcone derivatives are statistically significant relative to
those of chalcone 1 (Table 2).
Scheme 1. Reagents and conditions: a) acetone, reflux, 6–8 h; b) 2n HCl,
reflux, 1 h, 85–95%; c) POCl3, DMF, reflux, 1 h, 70–80%; d) 10% NaOH(aq)
12 h, room temperature, 75–85%.
,
Biological results
Anticancer activity
Figure 2. Effect of chalcone derivatives 3b–e and 3h on cell viability. MCF-7
cells were treated with chalcone compounds at a concentration of 4 mm as
indicated for 24 h in 96-well plates seeded with ~104 cells per well. Optical
density (OD) readings were taken at l 420 nm. Doxorubicin was used as pos-
itive control; negative control: cells treated with DMSO vehicle alone.
The synthesized compounds[15] 3a–p were evaluated for their
anticancer activity by the National Cancer Institute (NCI) in a
60-cell-line screen. In a preliminary test at a single concentra-
tion (100 mm), a given compound was considered active if it
decreased the growth of any of the cell lines down to 32% or
less; from there it was included for evaluation against the full
panel of 60 cell lines. This panel is organized into sub-panels
representing leukemia, melanoma, and cancers of the lung,
colon, kidney, ovary, breast, prostate, and central nervous
system (CNS). Among the 19 derivatives tested by the NCI, 16
were active in the primary test, and nine were evaluated in a
panel of 60 cell lines; the results are listed in Table 1. Specifical-
ly, compound 3c exhibited excellent cytotoxicity, with GI50
values ranging from 0.26 to 4.74 mm in tests against leukemia
(CCRF-CEM, K-562, RPMI-8226, SR), colon (HCC-2998, HCT-15,
KM12), prostate (PC-3, DU-145), and breast (MCF-7, T-47D,
MDA-MB-468) cancer cell lines, as well as NCI-H23, NCI-H460,
NCI-H522 (non-small-cell lung cancer), SF-295, U251 (CNS
Chalcone-based compounds have been shown to exhibit
promising anticancer activity through various mechanisms.
These mechanisms include interference with microtubule for-
mation,[16–18] promotion of Bax protein expression and activa-
tion of caspases,[18,19] and inhibition of cell signaling path-
ways[9,20] such as blockage of nuclear factor kB (NF-kB). There
are also reports on mitochondria-mediated apoptosis, and
apoptosis involving the down-regulation of anti-apoptotic pro-
teins such as Bcl-2 and Bcl-XL.[21] In the present investigation,
as some of the chalcones exhibited promising anticancer activ-
ity, we were interested in getting a better understanding of
their mode of action, particularly for the most potent com-
pounds (Table 3).
1938
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Imidazothiazole–Chalcone Derivatives
Table 1. Anticancer activity of imidazothiazole–chalcone derivatives 3a–i in human cancer cell lines.
Cancer panel
Growth inhibition: logGI50 (m)
Cell line
3a
3b
3c
3d
3e
3 f
3g
3h
3i
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
ꢀ5.51
ꢀ5.78
ꢀ5.76
ꢀ5.60
–
ꢀ5.49
ꢀ5.84
ꢀ5.66
ꢀ5.65
ꢀ5.59
ꢀ5.95
ꢀ6.25
ꢀ5.93
ꢀ6.35
ꢀ5.99
ꢀ6.35
ꢀ6.58
ꢀ5.91
ꢀ5.74
ꢀ5.76
ꢀ5.72
ꢀ5.97
ꢀ6.27
ꢀ5.58
ꢀ5.74
ꢀ5.62
ꢀ5.68
ꢀ5.93
ꢀ5.89
ꢀ5.63
ꢀ6.00
ꢀ5.65
ꢀ5.65
ꢀ5.79
ꢀ6.31
ꢀ5.51
ꢀ5.60
ꢀ5.50
ꢀ5.63
ꢀ5.65
ꢀ5.68
ꢀ5.14
ꢀ5.54
ꢀ5.26
ꢀ5.79
ꢀ5.64
ꢀ6.10
ꢀ5.71
ꢀ5.57
ꢀ5.68
ꢀ5.89
ꢀ5.78
ꢀ5.95
ꢀ5.63
Non-small-cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
ꢀ5.65
ꢀ5.51
ꢀ5.66
ꢀ6.61
ꢀ5.59
ꢀ5.66
ꢀ5.43
ꢀ5.57
ꢀ5.71
ꢀ5.51
ꢀ5.48
ꢀ5.77
ꢀ5.56
ꢀ5.63
ꢀ5.62
ꢀ5.81
ꢀ5.78
ꢀ5.12
ꢀ5.80
ꢀ5.62
ꢀ5.62
–
ꢀ5.61
ꢀ6.09
ꢀ5.48
ꢀ6.22
ꢀ6.59
ꢀ5.46
ꢀ5.57
ꢀ5.71
ꢀ5.15
ꢀ5.66
ꢀ5.76
ꢀ5.71
ꢀ5.78
ꢀ5.69
ꢀ5.58
ꢀ5.41
ꢀ5.59
–
ꢀ5.66
ꢀ5.67
ꢀ5.37
ꢀ5.70
ꢀ5.90
ꢀ5.33
ꢀ5.35
ꢀ5.22
ꢀ5.23
ꢀ5.16
ꢀ5.56
ꢀ5.50
ꢀ5.71
ꢀ5.54
ꢀ5.47
ꢀ5.39
ꢀ5.37
–
ꢀ5.38
ꢀ5.56
ꢀ5.25
ꢀ5.50
ꢀ6.06
ꢀ5.03
ꢀ5.64
ꢀ4.07
ꢀ6.81
ꢀ5.02
ꢀ5.65
ꢀ5.06
ꢀ5.41
ꢀ5.53
ꢀ5.57
ꢀ5.70
ꢀ5.24
ꢀ6.33
ꢀ5.34
ꢀ5.30
ꢀ5.17
ꢀ5.73
ꢀ5.87
Colon
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ5.60
ꢀ5.75
ꢀ5.72
ꢀ5.46
ꢀ5.51
ꢀ5.58
ꢀ5.50
ꢀ5.73
ꢀ5.71
ꢀ5.64
ꢀ5.55
ꢀ5.65
ꢀ5.66
ꢀ5.55
ꢀ5.78
ꢀ6.09
ꢀ5.96
ꢀ6.08
ꢀ5.95
ꢀ6.47
–
ꢀ5.71
ꢀ5.77
ꢀ6.00
ꢀ5.74
ꢀ5.74
ꢀ5.83
ꢀ5.78
ꢀ5.62
ꢀ5.57
ꢀ5.64
ꢀ5.49
ꢀ5.46
ꢀ5.69
–
ꢀ5.34
ꢀ5.29
ꢀ5.85
ꢀ5.59
ꢀ5.70
ꢀ5.70
ꢀ5.83
ꢀ5.33
ꢀ5.43
ꢀ5.42
ꢀ5.47
ꢀ5.40
ꢀ5.57
–
ꢀ5.31
ꢀ5.00
ꢀ5.53
ꢀ5.43
ꢀ5.16
ꢀ5.44
ꢀ5.27
ꢀ5.40
ꢀ5.41
ꢀ5.42
ꢀ5.47
ꢀ5.41
ꢀ5.55
–
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ5.54
ꢀ5.56
ꢀ5.78
ꢀ5.45
ꢀ5.64
ꢀ5.52
ꢀ5.65
ꢀ5.51
ꢀ5.84
ꢀ5.52
ꢀ5.87
ꢀ5.58
ꢀ5.88
ꢀ6.02
ꢀ5.76
ꢀ5.67
ꢀ5.89
ꢀ6.04
ꢀ5.76
ꢀ5.59
ꢀ5.81
ꢀ5.57
ꢀ5.73
ꢀ5.86
ꢀ5.63
ꢀ5.51
ꢀ5.75
ꢀ5.61
ꢀ5.70
ꢀ5.59
ꢀ5.46
ꢀ5.50
ꢀ5.63
ꢀ5.37
ꢀ5.16
ꢀ5.73
ꢀ5.45
ꢀ5.35
ꢀ5.37
ꢀ5.34
ꢀ5.39
ꢀ5.39
ꢀ4.82
ꢀ5.63
ꢀ5.21
ꢀ4.06
ꢀ4.30
ꢀ5.39
ꢀ5.42
ꢀ5.43
ꢀ5.67
ꢀ5.54
ꢀ5.60
ꢀ5.43
Ovarian
IGROV1
ꢀ6.66
ꢀ5.65
ꢀ5.39
ꢀ5.70
ꢀ5.70
ꢀ5.59
ꢀ5.48
ꢀ4.93
ꢀ5.62
ꢀ5.55
ꢀ5.75
ꢀ5.54
ꢀ5.70
ꢀ5.62
ꢀ6.34
ꢀ5.99
ꢀ5.71
ꢀ5.32
ꢀ6.19
ꢀ6.33
ꢀ5.67
ꢀ5.57
ꢀ5.73
ꢀ5.52
–
ꢀ5.51
–
5.92
ꢀ5.50
ꢀ5.67
ꢀ5.39
ꢀ5.38
ꢀ5.62
ꢀ5.73
ꢀ5.47
ꢀ5.78
ꢀ5.63
ꢀ5.50
ꢀ4.00
ꢀ5.49
ꢀ5.55
ꢀ5.38
ꢀ5.06
ꢀ5.44
ꢀ5.64
ꢀ4.00
ꢀ5.31
ꢀ5.58
ꢀ5.21
ꢀ5.60
ꢀ5.55
ꢀ5.54
ꢀ4.09
ꢀ5.92
ꢀ5.68
ꢀ5.47
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
ꢀ5.66
ꢀ5.54
ꢀ5.61
ꢀ5.61
ꢀ5.64
ꢀ5.54
ꢀ5.54
Renal
786-0
A498
ꢀ5.59
ꢀ5.52
ꢀ5.39
ꢀ5.92
–
ꢀ5.54
ꢀ5.30
ꢀ5.95
ꢀ5.59
ꢀ5.69
ꢀ5.52
ꢀ5.46
ꢀ6.02
ꢀ5.67
ꢀ5.65
ꢀ5.39
5.82
–
ꢀ5.67
ꢀ5.75
–
ꢀ5.72
ꢀ5.68
ꢀ6.11
5.75
ꢀ5.47
ꢀ5.59
ꢀ5.49
ꢀ5.52
–
ꢀ5.51
ꢀ5.32
ꢀ6.19
ꢀ5.25
ꢀ5.12
ꢀ5.29
ꢀ5.61
ꢀ5.60
ꢀ5.53
ꢀ5.06
ꢀ5.07
ꢀ5.28
–
ꢀ5.38
ꢀ5.29
–
ꢀ5.39
ꢀ5.26
ꢀ5.64
ꢀ5.05
–
ꢀ5.43
ꢀ5.82
ꢀ5.29
ꢀ5.53
–
ꢀ5.28
ꢀ5.21
ꢀ5.74
ꢀ5.72
ꢀ5.74
ꢀ5.75
5.69
ꢀ5.81
ꢀ5.64
ꢀ5.73
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
ꢀ5.23
ꢀ5.75
ꢀ5.24
ꢀ5.05
ꢀ4.55
ꢀ5.53
Prostate
PC-3
DU-145
ꢀ5.51
ꢀ5.58
ꢀ5.43
ꢀ5.59
ꢀ6.14
ꢀ6.29
ꢀ5.58
ꢀ5.85
ꢀ5.66
ꢀ5.63
ꢀ5.27
ꢀ5.79
ꢀ5.64
ꢀ5.43
ꢀ5.62
ꢀ5.12
ꢀ5.55
ꢀ5.52
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1939

A. Kamal, M. Pal-Bhadra, et al.
MED
Table 1. (Continued)
Cancer panel
Growth inhibition: logGI50 (m)
Cell line
3a
3b
3c
3d
3e
3 f
3g
3h
3i
Breast
MCF7
MDA-MB-231/ATCC
HS-578T
BT-549
T-47D
ꢀ5.70
ꢀ5.59
ꢀ5.72
ꢀ5.46
ꢀ5.65
ꢀ5.53
ꢀ5.79
ꢀ5.66
ꢀ5.75
ꢀ7.51
ꢀ5.79
ꢀ5.60
ꢀ6.14
ꢀ5.81
–
ꢀ5.75
ꢀ6.10
ꢀ6.03
ꢀ5.90
ꢀ5.53
–
ꢀ5.54
ꢀ5.67
–
ꢀ5.65
ꢀ5.54
–
ꢀ5.61
ꢀ5.76
ꢀ5.67
ꢀ6.12
ꢀ5.23
ꢀ5.60
ꢀ5.49
ꢀ5.54
ꢀ5.21
ꢀ5.50
ꢀ5.42
–
ꢀ5.38
ꢀ5.62
ꢀ5.56
ꢀ5.74
ꢀ5.27
ꢀ5.08
ꢀ5.43
ꢀ5.53
ꢀ5.62
ꢀ5.63
ꢀ5.53
–
ꢀ5.42
ꢀ5.75
ꢀ5.65
MDA-MB-468
Melanoma
LOX-IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ5.62
ꢀ5.27
ꢀ5.63
ꢀ5.55
ꢀ5.62
ꢀ5.39
ꢀ5.89
–
ꢀ5.76
ꢀ5.72
ꢀ5.70
ꢀ5.60
ꢀ4.91
ꢀ5.25
ꢀ5.95
ꢀ4.85
ꢀ5.55
ꢀ5.97
ꢀ5.48
ꢀ5.60
ꢀ6.23
ꢀ6.23
–
ꢀ6.50
ꢀ5.94
ꢀ5.86
ꢀ5.87
ꢀ5.80
–
ꢀ5.79
ꢀ5.65
ꢀ5.77
ꢀ5.80
ꢀ5.72
ꢀ5.82
ꢀ5.59
ꢀ5.48
ꢀ5.51
ꢀ5.55
ꢀ5.73
–
ꢀ5.81
ꢀ5.58
ꢀ5.67
ꢀ6.22
ꢀ5.23
ꢀ5.57
ꢀ5.73
ꢀ5.39
ꢀ5.07
ꢀ5.38
ꢀ5.11
ꢀ5.31
ꢀ5.45
ꢀ5.59
ꢀ5.40
ꢀ5.58
–
ꢀ5.60
ꢀ5.57
5.29
ꢀ5.76
ꢀ5.24
ꢀ5.15
ꢀ6.29
ꢀ5.32
ꢀ5.54
ꢀ5.42
ꢀ5.50
ꢀ5.25
ꢀ5.57
ꢀ5.56
–
ꢀ6.02
ꢀ5.41
ꢀ5.60
–
ꢀ5.82
ꢀ5.60
ꢀ5.55
ꢀ5.68
Table 2. Inhibition of MCF-7 cell viability by chalcone derivatives 3b–e
and 3h at 4 mm.
[a]
Compd
Viability [OD420]
control
1
3b
3c
3d
1.843ꢁ0.037
1.013ꢁ0.021
0.873ꢁ0.005
0.808ꢁ0.016
0.794ꢁ0.038
0.871ꢁ0.007
0.862ꢁ0.006
0.623ꢁ0.025
3e
3h
doxorubicin
[a] Values represent the mean ꢁSD of three independent experiments
and are statistically significant compared with 1 by Student’s t-test (p<
0.001).
Figure 3. Histogram depicting the percentage of cells in G0 phase, an indica-
tor of percent apoptosis for the chalcone derivatives 3b–e and 3h.
Effect of imidazothiazole–chalcone derivatives on cell cycle
and 3d at 4 mm resulted in a decreased incorporation of BrdU
into cellular DNA. This level of inhibition of BrdU incorporation
is indicative of a G1-phase cell-cycle arrest by these chalcone
derivatives, as shown in Figure 4.
To investigate the effect of these chalcones on the cell cycle
phase distribution of MCF-7 cells, flow cytometry analysis was
performed at a test compound concentration of 4 mm for 24 h
exposure. Results indicate that the chalcone derivatives induce
G0/G1 cell-cycle arrest (Table 4), as the majority of cells in each
case were found to be in the G0/G1 phase: 67% (3b), 73%
(3c), 91% (3d), 58% (3e), 58% (3h), and 93% (doxorubicin)
relative to control (DMSO vehicle-treated) cells (55%). Thus,
modulation of G1 phase cell-cycle regulators may be a key
mechanism of action of these chalcones in MCF-7 cells. A de-
crease in the S-phase population was observed, indicating a
block in the G1!S transition. The percentage of apoptosis
mediated by compound 3d is greater than that induced by
doxorubicin (Figure 3).
Effect of chalcone derivatives on cell-cycle regulatory proteins
On the basis of the results of cell-cycle distribution, we next in-
vestigated the cell-cycle regulatory proteins related to G1
phase,[22] such as cyclins D1, E1, and A, and their associated
CDK2 and CDK4/CDK6, which are essential for cell-cycle pro-
gression from G1 to S phase. MCF-7 cells were treated with
compounds 3c and 3d at 4 mm for 24 h, and western blot
analysis was then carried out. As shown in Figure 5, the protein
levels of cyclin D1, cyclin A, and cyclin E1 were decreased sig-
nificantly, and their associated CDK2 levels remain unchanged,
whereas the CDK4 levels increased significantly relative to con-
trol. These results indicate that both chalcones 3c and 3d in-
duced cell-cycle arrest at G0/G1 phase.
In subsequent studies, compounds 3c and 3d were evaluat-
ed for G1 arrest in a 5-bromo-2’-deoxyuridine (BrdU) incorpora-
tion assay. This assay determines a compound’s ability to block
the G1!S transition. Treatment of cells with compounds 3c
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Imidazothiazole–Chalcone Derivatives
Table 3. Compounds selected by the National Cancer Institute (NCI) for
the 60-cell-line panel.
Table 4. Cell-cycle distribution of MCF-7 cells in the presence of com-
pounds 3b–e, 3h, and doxorubicin at 4 mm.[a]
Compd
NSC number[a]
Structure
Compd
G0/G1 [%]
S [%]
G2/M [%]
control
3b
3c
3d
3e
55ꢁ1.2
67ꢁ0.8
73ꢁ1.0
91ꢁ0.9
58ꢁ0.5
58ꢁ0.2
93ꢁ0.4
16ꢁ0.7
21ꢁ0.5
20ꢁ0.5
6ꢁ0.7
29ꢁ0.5
12ꢁ0.2
7.0ꢁ0.9
3ꢁ0.3
3a
748733
28ꢁ0.8
20ꢁ0.5
4.0ꢁ0.2
14ꢁ0.5
22ꢁ0.2
3ꢁ0.2
3h
doxorubicin
[a] Values represent the mean ꢁSEM of three determinations.
3b
748734
3c
750743
749801
3d
3e
750741
Figure 4. The BrdU cell proliferation assay. MCF-7 cells were seeded in 96-
well plates at a density of ~104 cells per well and were grown for 24 h. After
24 h the compounds (doxorubicin, 3c, and 3d) were added to the respec-
tive wells at a final concentration of 4 mm. The colorimetric intensity at
l 450 nm was measured and is proportional to the amount of BrdU incorpo-
rated in proliferating cells. Lower OD values denote lower BrdU concentra-
tions in the sample, thus an indirect reflection of a lower cell proliferation
rate.
3 f
749802
750742
3g
3h
749804
750744
3i
[a] NSC=National Service Center.
Phosphorylation of the retinoblastoma (Rb) protein, mediat-
ed by both cyclin D/CDK4 and cyclin E/CDK2, is required for
cells to progress from G1 to S phase in those cells possessing
functional Rb; phospho-Rb releases (thereby activating) the
previously Rb-bound transcription factor E2F-1, thus permitting
cell-cycle progression.[23] To elucidate the arrest point of these
chalcones (3c and 3d), western blot analysis was carried out
Figure 5. Effect of chalcone derivatives on the expression of cyclin and asso-
ciated proteins. MCF-7 cells were treated with doxorubicin, 3c, and 3d at
4 mm. Western blot analysis was carried out with antibodies against cyclins
(cyclin E1, cyclin A, cyclin D1), CDKs (CDK2 and CDK4), Rb, phospho-Rb
(Ser780), and E2F-1; b-actin was used as a loading control.
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1941

A. Kamal, M. Pal-Bhadra, et al.
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at 4 mm for 24 h. From Figure 5, it is apparent that the expres-
sion level of Rb protein increased, and the amount of phos-
pho-Rb (Ser780) decreased significantly, and thus the release
of E2F-1 also decreased significantly relative to control. These
results indicate that compound 3d is more effective and there-
by blocks the G1!S-phase transition.
suppress the activation of NF-kB and its inhibitory protein
Ikka, western blot analysis was carried out with test com-
pounds at 4 mm for 24 h as shown in Figure 6. Ikka protein
levels were significantly increased, and NF-kB levels were sig-
nificantly decreased after treatment with compounds 3c and
3d. Our data suggest that these chalcone derivatives affect cell
growth by inhibiting the NF-kB signaling pathway and that
compound 3d has potential for further development.
Effect on p53, p21, p27, and chk2 protein expression
Furthermore, induction of apoptotic cell death is mediated
by caspases; hence the involvement of various caspases and
their role in the process of apoptosis was also investigated.
The aspartic acid specific protease caspase-9 has been linked
to the mitochondrial death pathway, which is activated during
programmed cell death (apoptosis). Caspase-9 is also the effec-
tor caspase in the case of the intrinsic pathway. MCF-7 cells
were treated with compounds 3c and 3d at 4 mm for 24 h,
and western blot analysis was carried out. Caspase-9 levels
were up-regulated with the treatment, particularly with com-
pound 3d. However, the TNFR1 levels remained unchanged
(data not shown). These data indicate the presence of an in-
trinsic pathway for these new chalcone derivatives (Figure 6).
The activity of tumor suppressors p53, p21, and p27 blocks the
formation of tumors.[24] To understand the mechanism underly-
ing the G1 cell-cycle arrest in our chalcone derivatives, we ex-
amined the G1/S checkpoint-associated tumor suppressor pro-
teins p53, p21, p27, as well as chk2.
The activation of tumor suppressor genes such as p53, p21,
p27 and chk2 was found to be important for the regulation of
apoptotic pathways induced by various stimuli.[25–27] In order to
understand the effect of chalcone derivatives on p53-, p21-,
p27-, and chk2-dependent apoptotic pathways, western blot
analysis was carried out with test compounds at a concentra-
tion of 4 mm for 24 h. As shown in Figure 6, the expression
Conclusions
In summary, we synthesized a new class of chalcones, wherein
the B ring is replaced by an imidazo[2,1-b]thiazole scaffold.
Compounds 3c and 3d showed significant anticancer activity
in the NCI 60-cell-line panel and cell viability assays; these two
compounds also showed cytotoxicity toward MCF-7 cells at a
concentration of 4 mm. The flow cytometry analysis also
showed a greater population of cells in the sub-G1 phase, indi-
cating that these chalcone derivatives have the ability to
induce apoptosis. These effects were accompanied by changes
in expression of key proteins in the G1 phase of the cell cycle.
Further modulation of the expression and function of the cell-
cycle regulatory proteins provide the mechanism for the inhibi-
tion of growth. From the results, we also observed down-regu-
lation of cyclins and up-regulation of CDK4, thereby resulting
in down-regulation of phospho-Rb (Ser780), suggesting cell-
cycle block in the G1 phase. Furthermore, we observed that
the levels of G1/S checkpoint-associated tumor suppressor pro-
teins such as p53, p21, p27, and chk2 were up-regulated, thus
inducing cell-cycle arrest in the G1 phase. Moreover, the bal-
ance between p53 and NF-kB were found to control the cell
cycle and thus cell proliferation, as reported in earlier stud-
ies.[31] Our results also support that the up-regulation of p53
and concomitant down-regulation of NF-kB by these chalcones
ultimately leads to apoptosis. This study involved an extensive
examination into the cell-cycle regulatory role as well as apop-
tosis-inducing ability of these new chalcones. Compound 3d is
a particularly suitable candidate for detailed biological investi-
gations, especially in the treatment of breast cancer.
Figure 6. Effect of chalcone derivatives 3c and 3d on the expression of
tumor suppressor proteins (p53, p21, p27, and chk2), apoptotic proteins,
and their interactive partners (NF-kB, Ikka). MCF-7 cells were treated with
compounds 3c and 3d at 4 mm, and doxorubicin was used as a positive con-
trol. Cell lysates were collected, and western blot analysis was carried out
with the aforementioned antibodies; b-actin was used as a loading control.
levels of p53, p21, p27, and chk2 were up-regulated in the
case of compounds 3c and 3d relative to the untreated con-
trol; indeed, the effects of compound 3d were even more
prominent than those observed with the doxorubicin positive
control. Therefore, up-regulation of these tumor suppressor
genes is involved in G1 cell-cycle arrest.
Activation of p53 induces apoptosis in many tumor cells,
and this provides an effective anticancer therapy. One of the
key proteins that modulate the apoptotic response is NF-kB, a
transcription factor that can protect or contribute to apoptosis.
Suppression of NF-kB activity is required for the induction of
an apoptotic response, which is a prerequisite in the selection
of drugs for the treatment of cancer.[28,29] NF-kB is bound to an
inhibitory protein (Ikka)[30] in the cytoplasm when it is in an in-
active form. To investigate whether chalcone derivatives can
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Imidazothiazole–Chalcone Derivatives
1
108–1108C; H NMR (300 MHz, CDCl3): d=2.40 (s, 3H), 3.82 (s, 3H),
6.90 (d, J=9.0 Hz, 2H), 7.07 (s, 1H), 7.47 (s, 1H), 7.69 ppm (d, J=
9.0 Hz, 2H); ESI-MS: m/z 245 [M+1]+.
Experimental Section
Chemistry
All chemicals and reagents were obtained from Sigma–Aldrich
(St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Com-
pany, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd. (Mumbai,
India), and were used without further purification. Reactions were
monitored by TLC performed on glass plates coated with silica gel
containing 60 GF254, and visualization was carried out by UV light
or iodine indicator. Column chromatography was performed with
Merck 60–120 mesh silica gel. 1H NMR spectra were recorded on
Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR-Unity
(400 MHz) instruments. Chemical shifts (d) are reported in ppm
downfield from internal (CH3)4Si standard. ESI-MS data were record-
ed on a Micromass Quattro LC instrument using ESI+ software
with a capillary voltage of 3.98 kV and an ESI mode positive ion
trap detector. HRMS data were recorded on a QSTAR XL Hybrid
MS–MS mass spectrometer. Melting points were determined with
an Electrothermal melting point apparatus and are uncorrected.
6-(4-fluorophenyl)-2-methylimidazo[2,1-b][1,3]thiazole
(7g):
White solid (209 mg, 90% yield): Rf =0.34 (EtOAc/Hex 2:3); mp:
1
177–1798C; H NMR (300 MHz, CDCl3): d=2.41 (s, 3H), 7.05 (s, 1H),
7.08–7.12 (m, 2H), 7.53 (s, 1H), 7.74 ppm (dd, J=13.5, J=3.0 Hz,
2H); ESI-MS: m/z 233 [M+1]+.
6-(thien-2-yl)-2-methyl-imidazo[2,1-b]thiazole(7h): White solid
(187 mg, 85% yield): Rf =0.37 (EtOAc/Hex 3:7); mp: 177–1798C;
1H NMR (400 MHz, CDCl3): d=2.40 (s, 3H), 7.01 (t, J=3.9 Hz, 1H),
7.06 (s, 1H), 7.17 (d, J=4.8 Hz, 1H), 7.26 (d, J=3.9 Hz, 1H),
7.46 ppm (s, 1H); ESI-MS: m/z 221 [M+1]+.
General procedure for the synthesis of aldehydes 8a–h
The Vilsmeier reagent was prepared at 0–58C by dropping POCl3
(20 mmol) into a stirred solution of DMF (25 mmol) in CHCl3 (5 mL).
Compound 9 (10 mmol) in CHCl3 (60 mL) was added dropwise to
the Vilsmeier reagent while maintaining stirring and cooling. The
reaction mixture was kept at room temperature for 3 h and under
reflux for 1–24 h (according to a TLC test). CHCl3 was removed
under reduced pressure, and the resulting oil was poured onto ice.
The crude aldehyde 8 thus obtained was collected by filtration and
crystallized from ethanol with a yield of 70–80%.
General procedure for the synthesis of imidazo[2,1-b]thiazoles
7a–h
The appropriate 2-aminothiazole 4 (20 mmol) was dissolved in ace-
tone (100 mL) and treated with the appropriate 2-bromoacetophe-
none 5 (20 mmol, 1 equiv). The mixture was held at reflux for 6–
8 h, and the resulting salt was collected by filtration, washed with
acetone, and held at reflux for 1 h with 200 mL 2n HCl. Before
complete cooling, the resulting solution was cautiously basified
with 15% NH4OH, in order to precipitate the crude compound 9,
which was crystallized from ethanol at yields of 85–95%.
6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-carbaldehyde (8a):
Brown solid (206 mg, 80% yield): Rf =0.28 (EtOAc/Hex 3:7); mp:
147–1498C; 1H NMR (300 MHz, CDCl3): d=3.87 (s, 3H), 6.96–7.01
(m, 3H), 7.72 (d, J=8.3 Hz, 2H), 8.38 (d, J=4.5 Hz, 1H), 9.86 ppm
(s, 1H, CHO); ESI-MS: m/z 259 [M+1]+.
6-(4-methoxyphenyl)imidazo[2,1-b][1,3]thiazole (7a): White solid
(218 mg, 95% yield): Rf =0.4 (EtOAc/Hex 2:3); mp: 176–1788C;
1H NMR (300 MHz, CDCl3): d=3.83 (s, 3H), 6.78 (d, J=3.9 Hz, 1H),
6.93 (d, J=8.5 Hz, 2H), 7.40 (d, J=4.6 Hz, 2H), 7.63 (s, 1H),
7.74 ppm (d, J=8.5 Hz, 1H); ESI-MS: m/z 231 [M+1]+.
6-(4-fluorophenyl)imidazo[2,1-b]thiazol-5-carbaldeyde
(8b):
Yellow solid (192 mg, 78% yield): Rf =0.32 (EtOAc/Hex 2:3); mp:
169–1718C; 1H NMR (300 MHz, CDCl3): d=7.05 (d, J=4.5 Hz, 1H),
7.20 (t, J=8.3 Hz, 2H), 7.78 (dd, J=13.5, J=3.0 Hz, 2H), 8.39 (d, J=
4.5 Hz, 1H), 9.87 ppm (s, 1H, CHO); ESI-MS: m/z 247 [M+1]+.
6-(4-fluorophenyl)imidazo[2,1-b][1,3]thiazole (7b): White solid
(200 mg, 92% yield): Rf =0.35 (EtOAc/Hex 1:3); mp: 114–1168C;
1H NMR (CDCl3, 300 MHz): d=6.77 (s, 1H), 7.04 (t, J=7.6 Hz, 2H),
7.37 (s, 1H), 7.60 (s, 1H), 7.73 ppm (d, J=6.7 Hz, 2H); ESI-MS: m/z
218 [M+1]+.
6-(thien-2-yl)imidazo[2,1-b]thiazol-5-carbaldehyde (8c): Brown
solid (171 mg, 73% yield): Rf =0.24 (EtOAc/Hex 1:3); mp: 129–
1
1318C; H NMR (300 MHz, CDCl3): d=7.02 (d, J=4.3 Hz, 1H), 7.15
(dd, J=8.7, J=1.4 Hz, 1H), 7.48 (d, J=5.1 Hz, 1H), 7.56 (d, J=
3.6 Hz, 1H), 8.37 (d, J=4.3 Hz, 1H), 10.1 ppm (s, 1H, CHO); ESI-MS:
m/z 235 [M+1]+.
6-(thien-2-yl)imidazo[2,1-b]thiazole (7c): White solid (185 mg,
90% yield): Rf =0.38 (EtOAc/Hex 2:3); mp: 134–1368C; 1H NMR
(400 MHz, CDCl3): d=6.80 (d, J=4.5 Hz, 1H), 7.02 (dd, J=8.4 Hz,
J=1.3 Hz, 1H), 7.20 (dd, J=6.0 Hz, J=3.9 Hz, 1H), 7.30 (dd, J=
4.7 Hz, J=2.4 Hz, 1H), 7.38 (d, J=4.5 Hz, 1H), 7.61 ppm (s, 1H);
ESI-MS: m/z 207 [M+1]+.
6-(trifluoromethyl)imidazo[2,1-b]thiazol-5-carbaldehyde
(8d):
White solid (154 mg, 70% yield): Rf =0.22 (EtOAc/Hex 3:7); mp:
110–1128C; 1H NMR (300 MHz, CDCl3): d=7.24 (d, J=4.5 Hz, 1H),
8.42 (d, J=4.5 Hz, 1H), 10.01 ppm (s, 1H, CHO); ESI-MS: m/z 221
[M+1]+.
6-(trifluoromethyl)imidazo[2,1-b][1,3]thiazole (7d): White solid
(173 mg, 90% yield): Rf =0.4 (EtOAc/Hex 1:2); mp: 133–1358C;
1H NMR (300 MHz, CDCl3): d=6.97 (d, J=4.5 Hz, 1H), 7.47 (d, J=
4.5 Hz, 1H), 7.75 ppm (s, 1H); ESI-MS: m/z 193 [M+1]+.
6-(3,4-dimethoxyphenyl)imidazo[2,1-b]thiazol-5-carbaldehyde
(8e): White solid (216 mg, 75% yield): Rf =0.28 (EtOAc/Hex 2:3);
1
mp: 183–1858C; H NMR (300 MHz, CDCl3): d=3.94 (s, 3H), 3.96 (s,
6-(3,4-dimethoxyphenyl)imidazo[2,1-b]thiazole (7e): White solid
(226 mg, 87% yield): Rf =0.35 (EtOAc/Hex 2:3); mp: 101–1038C;
1H NMR (300 MHz, CDCl3): d=3.88 (s, 3H), 3.95 (s, 3H), 6.76 (d, J=
4.5 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H), 7.22 (dd, J=9.8 Hz, J=6.7 Hz,
1H), 7.37 (d, J=4.5 Hz, 1H), 7.40 (d, J=1.5 Hz, 1H), 7.59 ppm (s,
1H); ESI-MS: m/z 261 [M+1]+.
3H), 6.94 (d, J=8.7 Hz, 1H), 7.01 (d, J=4.3 Hz, 1H), 7.28 (dd, J=
9.5, J=6.5 Hz,1H), 7.36 (d, J=2.1 Hz, 1H), 8.39 (d, J=4.3 Hz, 1H),
9.89 ppm (s, 1H, CHO); ESI-MS: m/z 289 [M+1]+.
6-(4-methoxyphenyl)-2-methylimidazo[2,1-b]thiazol-5-carbalde-
hyde (8 f): White solid (190 mg, 70% yield): Rf =0.3 (EtOAc/Hex
1:4); mp: 160–1628C; 1H NMR (300 MHz, CDCl3): d=2.51 (s, 3H),
3.86 (s, 3H), 6.98 (d, J=8.9 Hz, 2H), 7.70 (d, J=8.9 Hz, 2H), 8.09 (s,
1H), 9.82 ppm (s, 1H, CHO); m/z ESI-MS: 273 [M+1]+.
6-(4-methoxyphenyl)-2-methylimidazo[2,1-b][1,3]thiazole
(7 f):
White solid (207 mg, 85% yield): Rf =0.32 (EtOAc/Hex 1:4); mp:
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A. Kamal, M. Pal-Bhadra, et al.
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6-(4-fluorophenyl)-2-methylimidazo[2,1-b]thiazol-5-carbaldehyde
(8g): White solid (190 mg, 73% yield): Rf =0.32 (EtOAc/Hex 1:4);
mp: 157–1598C; H NMR (300 MHz, CDCl3): d=2.52 (s, 3H), 7.17 (t,
J=8.7 Hz, 2H), 7.75 (dd, J=13.6, J=3.9 Hz, 2H), 8.09 (s, 1H),
9.82 ppm (s, 1H, CHO); ESI-MS: m/z 261 [M+1]+.
188.6, 163.3, 153.0 (2C), 147.0, 142.3, 135.9, 133.5, 129.9 (2C), 128.3,
127.9, 127.2, 126.8, 119.8, 119.2, 116.5 (2C), 114.1, 105.8 (2C), 60.8,
56.3 ppm (2C); IR (KBr): n˜ =3528, 3113, 2969, 2934, 1650, 1568,
1502, 1456, 1417, 1376, 1331, 1255, 1189, 1161, 1123, 1069, 997,
967, 937, 831, 773, 712 cmꢀ1; ESI-MS: m/z 427 [M+1]+; HRMS (ESI
m/z) for C21H18N2O4NaS2 calcd: 449.0605, found: 449.0604 [M+
Na]+.
1
6-(thien-2-yl)-2-methyl-imidazo[2,1-b]thiazol-5-carbaldehyde
(8h): Yellow solid (186 mg, 75% yield): Rf =0.26 (EtOAc/Hex 2:3);
mp: 111–1138C; 1H NMR (300 MHz, CDCl3): d=2.53 (s, 3H), 7.13
(dd, J=8.8, J=1.3 Hz, 1H), 7.45 (dd, J=6.0, J=4.1 Hz, 1H), 7.52
(dd, J=4.7, J=2.6 Hz, 1H), 8.08 (d, J=1.3 Hz, 1H), 10.04 ppm (s,
1H, CHO); ESI-MS: m/z 249 [M+1]+.
(E)-3-(6-(trifluoromethyl)imidazo[2,1-b]thiazol-5-yl)-1-(3,4,5-trime-
thoxyphenyl)prop-2-en-1-one (3d): This compound was prepared
according to the method described for compound 3a, employing
compound 9a (210 mg, 1 mmol) and 6-(trifluoromethyl)imidazo-
[2,1-b]thiazol-5-carbaldehyde (8d) (220 mg, 1 mmol) to obtain the
pure product 3d as a yellow solid (350 mg, 85% yield): Rf =4.00
(EtOAc/Hex 7:3); mp: 177–1798C; 1H NMR (300 MHz, CDCl3): d=
3.89 (s, 3H), 3.94 (s, 6H), 7.21 (d, J=4.5 Hz, 1H), 7.22 (s, 2H), 7.34
(d, J=15.8 Hz, 1H), 7.82 (d, J=4.5 Hz, 1H), 7.90 ppm (d, J=
16.6 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=186.7, 151.8 (2C), 151.4,
141.3, 130.2, 125.2, 121.3, 119.9, 119.4, 118.8, 115.9, 105.1 (2C),
104.3, 59.4, 55.1 ppm; IR (KBr): n˜ =3448, 3105, 2939, 1667, 1583,
1503, 1463, 1413, 1333, 1283, 1234, 1161, 1123, 1072, 1006, 965,
941, 833, 733 cmꢀ1; ESI-MS: m/z 413 [M+1]+; HRMS (ESI m/z) for
C18H15N2O4F3NaS calcd: 435.0602, found: 435.0605 [M+Na]+.
Synthesis of (E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-
yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3a): A 10% aque-
ous solution of NaOH (5 mL) was added to a stirred solution of
3,4,5-trimethoxyphenyl acetophenone (9a) (210 mg, 1 mmol) and
6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-carbaldehyde
(8a)
(258 mg, 1 mmol) in ethanol (20 mL). The reaction mixture was
stirred at room temperature 278C for 4 h and the reaction was
monitored by TLC using EtOAc/Hex (5:5) as a solvent system. The
solvent was evaporated under vacuum, then the residue was dis-
solved in EtOAc/H2O. The organic layer was washed with brine and
evaporated. This was further purified by column chromatography
using EtOAc/Hex (5:5) as a solvent system to obtain the pure prod-
uct as yellow solid (361 mg, 80% yield): Rf =0.36 (EtOAc/Hex 1:2);
(E)-3-(6-(3,4-dimethoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3e): This compound
was prepared according to the method described for compound
3a, employing compound 9a (210 mg, 1 mmol) and 6-(3,4-
1
mp: 177–1798C; H NMR (300 MHz, CDCl3): d=3.86 (s, 3H), 3.92 (s,
9H), 6.96–7.06 (m, 4H), 7.18 (s, 2H), 7.19 (d, J=15.5 Hz, 1H), 7.66
(d, J=8.5 Hz, 1H), 7.83 (d, J=3.8 Hz, 1H), 8.04 ppm (d, J=15.5 Hz,
1H); 13C NMR (75 MHz, CDCl3): d=188.5, 160.1, 153.4, 153.3 (2C),
142.2, 140.1, 133.6, 130.7, 130.2 (2C), 126.1, 119.1, 115.7 (2C), 114.1,
113.7, 105.8 (2C), 60.8, 56.3 (2C), 55.2 ppm; IR (KBr): n˜ =3420, 3120,
2924, 2834, 1647, 1557, 1501, 1455, 1382, 1330, 1298, 1243, 1162,
1121, 1005, 933, 841, 809, 767, 711 cmꢀ1; ESI-MS: m/z 451 [M+1]+;
HRMS (ESI m/z) for C24H23N2O5S calcd: 451.1327, found: 451.1338
[M+1]+.
dimethoxyphenyl)imidazo[2,1-b]thiazol-5-carbaldehyde
(8e)
(288 mg, 1 mmol) to obtain the pure product 3e as a yellow solid
(360 mg, 75% yield): Rf =0.38 (EtOAc/Hex 1:2); mp: 179–1818C;
1H NMR (300 MHz, CDCl3): d=3.91 (s, 9H), 3.93 (s, 3H), 3.94 (s, 3H),
6.95 (d, J=8.2 Hz, 1H), 7.04 (d, J=4.1 Hz, 1H), 7.17 (s, 2H), 7.22 (d,
J=8.2 Hz, 2H), 7.29 (d, J=17.1 Hz, 1H), 7.83 (d, J=4.8 Hz, 1H),
8.07 ppm (d, J=15.8 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=188.5,
153.0 (2C), 149.6, 133.6, 130.8, 126.2, 122.0, 121.8, 120.1, 119.2,
118.4, 115.8, 113.9, 111.5, 111.0, 110.7, 105.8 (2C), 60.9, 56.3 ppm
(3C); IR (KBr): n˜ =3437, 3107, 2924, 2843, 1649, 1584, 1557, 1500,
1455, 1409, 1378, 1327, 1230, 1160, 1125, 1024, 995, 904, 862, 806,
766, 707 cmꢀ1; ESI-MS: m/z 481 [M+1]+; HRMS (ESI m/z) for
C25H24N2O6NaS calcd: 503.1252, found: 503.1262 [M+Na]+.
(E)-3-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)-1-(3,4,5-trime-
thoxyphenyl)pro-2-en-1-one (3b): This compound was prepared
according to the method described for compound 3a, employing
compound 9a (210 mg, 1 mmol) and 6-(4-fluorophenyl)imidazo-
[2,1-b]thiazol-5-carbaldehyde (8b) (246 mg, 1 mmol) to obtain the
pure product 3b as a yellow solid (359 mg, 78% yield): Rf =0.38
(EtOAc/Hex 1:2); mp: 222–2258C; 1H NMR (400 MHz, CDCl3): d=
3.89 (s, 3H), 3.91 (s, 6H), 7.04 (d, J=4.3 Hz, 1H), 7.12 (s, 2H), 7.15
(d, J=15.8 Hz, 1H), 7.17 (d, J=8.0 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H),
7.71 (dd, J=13.9, J=3.6 Hz, 2H), 7.83 (d, J=4.3 Hz, 1H), 7.96 ppm
(d, J=15.8 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=188.2, 164.6,
161.3, 153.0 (2C), 152.0, 142.4, 133.4, 130.7, 129.7 (2C), 120.3, 119.1,
116.3 (2C), 115.8, 115.5, 114.1, 105.8 (2C), 60.8, 56.2 ppm (2C); IR
(KBr): n˜ =3434, 3099, 2927, 1649, 1569, 1531, 1456, 1381, 1332,
(E)-3-(6-(trifluoromethyl)imidazo[2,1-b]thiazol-5-yl)-1-(3,4-dime-
thoxyphenyl)prop-2-en-1-one (3 f): This compound was prepared
according to the method described for compound 3a, employing
compound 9b (180 mg, 1 mmol) and 6-(trifluoromethyl) imidazo-
[2,1-b]thiazol-5-carbaldehyde (8d) (220 mg, 1 mmol) to obtain the
pure product 3 f as a yellow solid. (325 mg, 85% yield): Rf =0.42
(EtOAc/Hex 2:3); mp: 166–1698C; 1H NMR (300 MHz, CDCl3): d=
3.97 (s, 6H), 6.93 (d, J=9.0 Hz, 1H), 7.19 (d, J=4.5 Hz, 1H), 7.41 (d,
J=15.8 Hz, 1H), 7.59 (dd, J=6.7, J=2.2 Hz, 2H), 7.83 (d, J=4.5 Hz,
1H), 7.91 ppm (d, J=15.8 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=
187.3, 153.5, 151.8, 149.3, 138.0, 130.6, 126.3, 122.9, 122.5, 121.5,
119.9, 118.6, 116.6, 110.6, 109.9, 56.0, 55.9 ppm; IR (KBr): n˜ =3448,
3124, 3006, 2954, 1658, 1600, 1575, 1511, 1467, 1419, 1379, 1328,
1250, 1226, 1191, 1157, 1118, 1068, 990, 938, 846, 773, 709 cmꢀ1
;
ESI-MS: m/z 439 [M+1]+; HRMS (ESI m/z) for C23H19N2O4FNaS
calcd: 461.0947, found: 461.0930 [M+Na]+.
1298, 1257, 1167, 1116, 1019, 971, 922, 875, 804, 757, 705 cmꢀ1
;
(E)-3-[6-(2-thienyl)imidazo[2,1-b][1,3]thiazol-5-yl]-1-(3,4,5-trime-
thoxyphenyl)-2-propen-1-one (3c): This compound was prepared
according to the method described for compound 3a, employing
compound 9a (210 mg, 1 mmol) and 6-(thien-2-yl)imidazo[2,1-
b]thiazol-5-carbaldehyde (8c) (234 mg, 1 mmol) to obtain the pure
product 3c as a yellow solid (349 mg, 82% yield): Rf =0.32 (EtOAc/
ESI-MS: m/z 383 [M+1]+; HRMS (ESI m/z) for C17H13N2O3F3NaS
calcd: 405.0496, found: 405.0488 [M+Na]+.
(E)-3-(6-(4-methoxyphenyl)-2-methylimidazo[2,1-b]thiazol-5-yl)-1-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3g): This compound
was prepared according to the method described for compound
3a, employing compound 9a (210 mg, 1 mmol) and 6-(4-methoxy-
1
Hex 3:2); mp: 178–1808C; H NMR (300 MHz, CDCl3): d=3.92–3.95
(br, 9H), 7.05 (d, J=4.3 Hz, 1H), 7.14 (d, J=4.3 Hz, 1H), 7.16 (d, J=
16.8 Hz, 1H), 7.20 (s, 2H), 7.44 (t, J=3.6 Hz, 2H), 7.82 (d, J=4.3 Hz,
1H), 8.19 ppm (d, J=16.1 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=
phenyl)-2-methylimidazo[2,1-b]thiazol-5-carbaldehyde
(8 f)
(272 mg, 1 mmol) to obtain the pure product 3g as a yellow solid
1944
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1937 – 1947

Imidazothiazole–Chalcone Derivatives
(362 mg, 78% yield): Rf =0.42 (EtOAc/Hex 2:3); mp: 193–1958C;
1H NMR (400 MHz, CDCl3): d=2.53 (s, 3H), 3.85 (s, 6H), 3.91 (s, 6H),
6.80 (d, J=8.0 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.11 (d, J=14.6 Hz,
1H), 7.15 (s, 2H), 7.51 (s, 1H), 7.63 (d, J=7.3 Hz, 2H), 7.96 ppm (d,
J=16.8 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=196.7, 188.7, 152.9
(2C), 133.7, 131.4, 130.9, 129.9 (2C), 127.8, 126.2, 122.7, 119.8, 115.7
(2C), 114.7, 114.0, 113.3, 105.8 (2C), 60.8, 56.2 (2C), 55.2 ppm; IR
(KBr): n˜ =3420, 3101, 2937, 2834, 1671, 1650, 1567, 1533, 1502,
1486, 1462, 1411, 1381, 1327, 1300, 1269, 1241, 1192, 1159, 1127,
1070, 1027, 996, 977, 932, 864, 842, 793, 737, 710 cmꢀ1; ESI-MS: m/
z 465 [M+1]+; HRMS (ESI m/z) for C25H24N2O5NaS calcd: 487.1303,
found: 487.1317 [M+Na]+.
(E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1-(thien-2-
yl)prop-2-en-1-one (3k): This compound was prepared according
to the method described for compound 3a, employing compound
9d (126 mg, 1 mmol) and 6-(4-methoxyphenyl)imidazo[2,1-
b]thiazol-5-carbalhedyde (8a) (258 mg, 1 mmol) to obtain the pure
product 3k as a yellow solid (275 mg, 75% yield): Rf =0.38 (EtOAc/
Hex 1:2); mp: 169–1728C; 1H NMR (300 MHz, CDCl3): d=3.86 (s,
3H), 7.00 (d, J=8.3 Hz, 2H), 7.03 (s, 1H), 7.11 (d, J=15.8 Hz, 1H),
7.15 (d, J=4.5 Hz, 1H), 7.62–7.68 (m, 3H), 7.76 (d, J=3.0 Hz, 1H),
7.84 (d, J=4.5 Hz, 1H), 8.06 ppm (d, J=15.8 Hz, 1H); 13CNMR
(75 MHz, CDCl3): d=181.2, 160.0, 153.7, 145.5, 133.2, 131.0, 130.2,
128.1, 125.9, 119.8, 119.4, 115.2, 114.1, 113.7, 55.2 ppm; IR (KBr): n˜ =
3424, 3136, 3111, 2932, 1633, 1555, 1517, 1452, 1376, 1331, 1286,
1196, 1180, 1026, 977, 891, 852, 771, 726 cmꢀ1; ESI-MS: m/z 367
[M+1]+; HRMS (ESI m/z) for C19H15N2O2S2 calcd: 367.0574, found:
367.0568 [M+1]+.
(E)-3-(6-(4-fluorophenyl)-2-methylimidazo[2,1-b]thiazol-5-yl)-1-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3h): This compound
was prepared according to the method described for compound
3a, employing compound 9a (210 mg, 1 mmol) and 6-(4-fluoro-
phenyl)-2-methylimidazo[2,1-b]thiazol-5-carbaldehyde
(8g)
(E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazo-5-yl)-1-(3,5-di-
fluorophenyl)prop-2-en-1-one (3l): This compound was prepared
according to the method described for compound 3a, employing
compound 9e (156 mg, 1 mmol) and 6-(4-methoxyphenyl)
imidazo[2,1-b]thiazol-5-carbalhedyde (8a) (258 mg, 1 mmol) to
obtain the pure product 3 L as a yellow solid (325 mg, 82% yield):
Rf =0.32 (EtOAc/Hex 3:7); mp: 234–2368C; 1H NMR (300 MHz,
CDCl3): d=3.87 (s, 3H), 7.02 (d, J=9.0 Hz, 2H), 7.06 (d, J=15.8 Hz,
1H), 7.46 (dd, J=10.5, J=6.0 Hz, 3H) 7.65 (d, J=8.3 Hz, 2H), 7.86
(d, J=4.5 Hz, 2H), 8.08 ppm (d, J=15.1 Hz, 1H); 13CNMR (75 MHz,
CDCl3): d=188.7, 165.7 (2c), 160.6, 141.1, 136.5, 135.9, 128.5, 127.3,
125.3, 122.5, 117.2, 114.8 (2c), 112.1, 110.5, 55.8 ppm; IR (KBr): n˜ =
3421, 3105, 2924, 2852, 1652, 1566, 1453, 1389, 1310, 1254, 1194,
1096, 1032, 974, 858, 805 cmꢀ1; ESI-MS: m/z 397 [M+1]+; HRMS
(ESI m/z) for C21H15N2O2F2S calcd: 397.0822, found: 397.0827 [M+
1]+.
(260 mg, 1 mmol) to obtain the pure product 3h as a yellow solid
(362 mg, 80% yield): Rf =0.4 (EtOAc/Hex 2:3); mp: 159–1628C;
1H NMR (400 MHz, CDCl3): d=2.54 (s, 3H), 3.79 (s, 3H), 3.90 (s, 6H),
7.31 (t, J=8.6 Hz, 3H), 7.33 (s, 2H), 7.50 (d, J=15.5 Hz, 1H), 7.68
(dd, J=13.8, J=3.4 Hz, 2H), 7.82 (d, J=15.5 Hz, 1H), 8.22 ppm (s,
1H); 13C NMR (75 MHz, CDCl3): d=186.8, 151.5 (2C), 149.4, 140.7,
132.1, 129.3 (2C), 128.7, 128.5, 126.6, 118.8, 115.8 (2C), 114.8, 114.3,
114.0, 104.6 (2C), 59.3, 54.9 ppm; IR (KBr): n˜ =3432, 2937, 1644,
1591, 1569, 1531, 1506, 1485, 1452, 1412, 1382, 1353, 1325, 1275,
1230, 1170, 1136, 1038, 994, 972, 939, 864, 836, 810, 721, 709 cmꢀ1
ESI-MS: m/z 453 [M+1]+; HRMS (ESI m/z) for C24H21N2O4FNaS
calcd: 475.1103, found: 475.1120 [M+Na]+.
;
(E)-3-(6-(thien-2-yl)-(2-methylimidazo[2,1-b]thiazol-5-yl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (3i): This compound was pre-
pared according to the method described for compound 3a, em-
ploying compound 9a (210 mg, 1 mmol) and 6-(thien-2-yl)-2-
(E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1-(Benzo
[d]1,3]dioxol-5-yl)prop-2-en-1-one (3m): This compound was pre-
pared according to the method described for compound 3a, em-
methyl-imidazo[2,1-b]thiazol-5-carbaldehyde
(8h)
(248 mg,
1 mmol) to obtain the pure product 3i as a yellow solid (361 mg,
82% yield): Rf =0.44 (EtOAc/Hex 2:3); mp: 169–1718C; 1H NMR
(400 MHz, CDCl3): d=2.51 (s, 3H), 3.91 (s, 3H), 3.93 (s, 6H), 7.07 (s,
1H), 7.09 (d, J=15.6 Hz, 1H), 7.11 (dd, J=8.8, J=1.3 Hz, 1H), 7.17
(s, 2H), 7.39 (dd, J=7.3, J=0.9 Hz, 1H), 7.50 (d, J=1.3 Hz, 1H),
8.10 ppm (d, J=15.6 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=188.6,
176.8, 153.0 (2C), 147.0, 135.9, 133.5, 129.9, 127.9, 126.8, 119.8,
116.5, 114.1, 105.8 (2C), 60.8, 56.3 ppm (2C); IR (KBr): n˜ =3426,
3100, 2937, 2833, 1660, 1570, 1504, 1461, 1411, 1331, 1259, 1222,
1190, 1159, 1124, 1000, 930, 851, 810, 707 cmꢀ1; ESI-MS: m/z 441
[M+1]+; HRMS (ESI m/z) for C22H20N2O4NaS2 calcd: 463.0762,
found: 463.0755 [M+Na]+.
ploying
compound
9 f
(164 mg,
1 mmol)
and
6-(4-
methoxyphenyl)imidazo[2,1-b]thiazol-5-carbalhedyde (8a) (258 mg,
1 mmol) to obtain the pure product 3m as a yellow solid (323 mg,
80% yield): Rf =0.30 (EtOAc/Hex 3:2); mp: 189–1918C; 1H NMR
(300 MHz, CDCl3): d=3.86 (s, 3H), 6.05 (s, 2H), 6.86 (d, J=8.0 Hz,
1H), 7.01 (dd, J=8.8, J=4.4 Hz, 3H), 7.17 (d, J=16.1 Hz, 1H), 7.46
(d, J=2.2 Hz, 1H) 7.55 (d, J=8.0 Hz, 2H), 7.65 (d, J=8.8 Hz, 1H),
7.84 (d, J=4.4 Hz, 1H), 8.03 ppm (d, J=15.4 Hz, 1H); 13CNMR
(75 MHz, CDCl3): d=189.7, 160.6, 154.6, 149.3, 145.7, 135.7, 131.5,
128.3, 125.3, 123.2, 121.6, 116.5, 113.9 (2c), 112.6, 101.5, 54.8 ppm;
IR (KBr): n˜ =3419, 3122, 2912, 1654, 1563, 1533, 1500, 1440, 1388,
1352, 1290, 1257, 1172, 1143, 1092, 1034, 956, 923, 832, 798,
756 cmꢀ1
;
ESI-MS: m/z 405 [M+1]+; HRMS (ESI m/z) for
(E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1-(1H-
pyrrol-2-yl)prop-2-en-1-one (3j): This compound was prepared ac-
cording to the method described for compound 3a, employing
compound 9c (109 mg, 1 mmol) and 6-(4-methoxyphenyl)imidazo
[2,1-b]thiazol-5-carbalhedyde (8a) (258 mg, 1 mmol) to obtain the
pure product 3p as a yellow solid (286 mg, 82% yield): Rf =0.34
(EtOAc/Hex 3:2); mp: 177–1798C; 1H NMR (300 MHz, CDCl3): d=
3.86 (s, 3H), 6.25 (dd, J=5.8, J=1.4 Hz, 2H), 7.04 (d, J=8.7 Hz, 3H),
7.29 (d, J=16.0 Hz, 1H), 7.36 (d, J=5.1 Hz, 1H), 7.63 (d, J=8.7 Hz,
2H), 7.89 (d, J=16.0 Hz, 1H), 8.45 ppm (d, J=4.3 Hz, 1H); 13CNMR
(75 MHz, CDCl3): d=177.0, 160.1, 157.0, 154.9, 148.7, 136.3, 129.7,
126.9, 124.4, 122.9, 120.7, 119.5, 116.3, 114.6, 113.7, 109.6,
54.7 ppm; IR (KBr): n˜ =3237, 3113, 2957, 1643, 1609, 1565, 1456,
1407, 1381, 1327, 1296, 1240, 1177, 1135, 1110, 1050, 1032, 943,
866, 835, 771, 742 cmꢀ1; ESI-MS: m/z 350 [M+1]+; HRMS (ESI m/z)
for C19H15N3O2NaS calcd: 372.0782, found: 372.0792 [M+Na]+.
C22H17N2O4S calcd: 405.0909, found: 405.0915 [M+1]+.
(E)-3-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)-1-(Benzo[d]-
[1,3]dioxol-5-yl)prop-2-en-1-one (3n): This compound was pre-
pared according to the method described for compound 3a, em-
ploying
compound
9 f
(164 mg,
1 mmol)
and
6-(4-
fluorophenyl)imidazo[2,1-b]thiazol-5-carbalhedyde (8b) (246 mg,
1 mmol) to obtain the pure product 3n as a yellow solid (323 mg,
80% yield): Rf =0.38 (EtOAc/Hex 3:2); mp: 205–2078C; 1H NMR
(300 MHz, CDCl3): d=6.07 (s, 2H), 6.88 (d, J=8.1 Hz, 1H), 7.07 (d,
J=4.5 Hz, 1H), 7.18 (d, J=15.6 Hz, 1H), 7.20 (d, J=8.6 Hz, 1H),
7.47 (d, J=2.2 Hz, 1H), 7.55 (dd, J=8.1 Hz, 2H), 7.70 (d, J=8.8 Hz,
2H), 7.86 (d, J=4.5 Hz, 1H), 8.01 ppm (d, J=15.6 Hz, 1H); 13CNMR
(75 MHz, CDCl3): d=185.6, 158.5, 152.3, 151.7, 150.0, 146.7, 131.4,
128.8 (2c), 128.4, 124.7, 123.3, 119.6, 118.7, 118.3, 113.8 (2c), 112.7,
ChemMedChem 2010, 5, 1937 – 1947
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1945

A. Kamal, M. Pal-Bhadra, et al.
MED
106.8, 106.5, 100.5, 53.9 ppm; IR (KBr): n˜ =3368, 3136, 1653, 1574,
1528, 1496, 1440, 1392, 1354, 1284, 1258, 1159, 1092, 1038, 923,
837, 807, 782, 757 cmꢀ1; ESI-MS: m/z 393 [M+1]+; HRMS (ESI m/z)
for C21H14N2O3FS calcd: 393.0709, found: 393.0713 [M+1]+.
doxorubicin at a concentration of 4 mm were added to the culture
media, and the cells were incubated for an additional 24 h. Cell
harvest was carried out with trypsin–EDTA, fixed with ice-cold 70%
ethanol at 48C for 30 min, washed with PBS, and incubated with a
1 mgmLꢀ1 solution of RNase A (Sigma) at 378C for 30 min. Cells
were collected by centrifugation at 2000 rpm (fixed angle rotor,
24ꢁ4g) for 5 min and further stained with 250 mL DNA staining so-
lution [10 mg propidium iodide (PI), 0.1 mg trisodium citrate, and
0.03 mL Triton X-100 dissolved in 100 mL sterile Milli-Q water at
room temperature for 30 min in the dark]. The DNA content of
20000 events were measured by flow cytometer (Dako Cytoma-
tion, Beckman Coulter, Brea, CA, USA). Histograms were analyzed
using Summit Software.
(E)-3-(6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-1-(3,4-di-
methoxyphenyl)prop-2-en-1-one (3o): This compound was pre-
pared according to the method described for compound 3a, em-
ploying compound 9b (180 mg, 1 mmol) and 6-(4-
methoxyphenyl)imidazo[2,1-b]thiazol-5-carbalhedyde (8a) (258 mg,
1 mmol) to obtain the pure product 3p as a yellow solid (328 mg,
78% yield): Rf =0.30 (EtOAc/Hex 3:7); mp: 195–1978C; 1H NMR
(300 MHz, CDCl3): d=3.86 (s, 3H), 3.95 (s, 6H), 6.87 (d, J=8.3 Hz,
1H), 6.97 (s, 1H), 7.01 (d, J=4.5 Hz, 2H), 7.23 (d, J=15.1 Hz, 1H),
7.51 (d, J=1.5 Hz, 1H), 7.54 (s, 1H), 7.65 (d, J=9.0 Hz, 2H), 7.85 (d,
J=4.5 Hz, 1H), 8.02 ppm (d, J=15.8 Hz, 1H); 13CNMR (75 MHz,
CDCl3): d=187.6, 160.0, 153.0, 152.4, 149.1, 131.2, 130.0, 125.5,
122.4, 120.0, 119.4, 115.8, 114.1, 113.9, 110.6, 109.8, 55.9, 55.2 ppm;
IR (KBr): n˜ =3422, 3035, 2923, 1656, 1575, 1518, 1451, 1381, 1335,
1274, 1248, 1160, 1028, 966, 822, 792, 767 cmꢀ1; ESI-MS: m/z 421
[M+1]+; HRMS (ESI m/z) for C23H20N2O4NaS calcd: 443.1041, found:
443.1046 [M+Na]+.
Cell proliferation assays: These assays were carried out by using
the 5-bromo-2’-deoxyuridine (BrdU) cell proliferation assay kit (Mil-
lipore) to asses the effect of doxorubicin and compounds 3c and
3d on the proliferation of MCF-7 cells. Cells (~104) were seeded
and allowed to grow for 24 h. BrdU was added and allowed to in-
corporate for 5 h followed by the addition of test compound at a
concentration of 4 mm for 24 h. Fixation was done for 30 min at
room temperature. The cells were then washed, anti-BrdU antibody
was added which binds to BrdU that was incorporated in the cells.
After 1 h incubation, 100 mL anti-BrdU goat anti-mouse horseradish
peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution)
was added and incubated for 30 min. Washing procedures were
followed according to the manufacturer’s instructions. TMB sub-
strate (100 mL) was added, incubated for another 30 min at room
temperature, and OD values were read at l 450 nm. Lower OD
values reflect lower BrdU concentrations in the sample, and thus
an indirect depiction of a low cell proliferation rate.
(E)-3-(6-(4-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)-1-(3,4-dime-
thoxyphenyl)prop-2-en-1-one (3p): This compound was prepared
according to the method described for compound 3a, employing
compound 9b (180 mg, 1 mmol) and 6-(4-fluorophenyl)-imidazo-
[2,1-b]thiazol-5-carbalhedyde (8b) (246 mg, 1 mmol) to obtain the
pure product 3o as a yellow solid (306 mg, 75% yield): Rf =0.38
(EtOAc/Hex 2:3); mp: 221–2238C; 1H NMR (300 MHz, CDCl3): d=
3.96 (s, 6H), 6.91 (d, J=8.3 Hz, 1H), 7.07 (d, J=4.5 Hz, 1H), 7.17 (d,
J=8.3 Hz, 2H), 7.29 (d, J=15.8 Hz, 1H), 7.54 (d, J=2.2 Hz, 1H),
7.57 (s, 1H), 7.72 (dd, J=9.0, J=8.3 Hz, 2H), 7.86 (d, J=4.5 Hz, 1H),
8.02 ppm (d, J=15.8 Hz, 1H); 13CNMR (75 MHz, CDCl3): d=187.0,
163.8, 152.7, 148.5, 130.6, 129.8, 128.7, 127.9, 122.7, 120.3, 115.8,
115.3, 114.2, 111.5, 110.3, 109.9, 55.5 ppm; IR (KBr): n˜ =3368, 3124,
2994, 2920, 1639, 1595, 1555, 1464, 1420, 1373, 1329, 1299, 1244,
1166, 1148, 1013, 981, 870, 837, 802, 768, 742 cmꢀ1; ESI-MS: m/z
409 [M+1]+; HRMS (ESI m/z) for C22H17N2O3FNaS calcd: 431.0841,
found: 431.0857 [M+Na]+.
Protein extraction and western blot analysis: Total cell lysates
were isolated from cultured MCF-7 cells after treatments with test
compounds as mentioned above by lysing the cells in ice-cold
RIPA buffer (1ꢁPBS, 1% NP-40, 0.5% sodium deoxycholate, and
0.1% SDS containing protease inhibitors). After centrifugation at
12000 rpm (fixed angle rotor, 24ꢁ4g) for 10 min, the protein in
the supernatant was quantified by the Bradford method (Bio-Rad)
using a Multimode Varioskan instrument (Thermo-Fischer Scientif-
ic). Protein (30 mg per lane) was subjected to 12% SDS-PAGE. After
electrophoresis, the protein was transferred to a polyvinylidine di-
fluoride (PVDF) membrane (Amersham Biosciences). The mem-
brane was blocked at room temperature for 2 h in TBS + 0.1%
Tween-20 (TBST) containing 5% blocking powder (Santa Cruz Bio-
technology). The membrane was washed with TBST for 5 min, and
primary antibody was added and incubated at 48C overnight.
Rabbit polyclonal antibodies p53 (53 kDa), CDK2 (32 kDa), E2F-1
(50 kDa), and cyclin D1 (35 kDa) were purchased from Santa Cruz
Biotechnology. Rabbit polyclonal antibodies cyclin E1, (40 kDa), b-
actin (38–53 kDa), Rb (110 kDa); mouse monoclonal antibodies
chk2 (60 kDa), cyclin A (50 kDa), NF-kB (65 kDa); and goat polyclo-
nal antibodies p27 (27 kDa) were purchased from Imgenex Pvt.
Ltd. (Orissa, India). Mouse monoclonal Ikka antibody was pur-
chased from Abcam Co. Mouse monoclonal p21 antibody (21 kDa)
was purchased from Ana Spec Co. Phospho-Rb (Ser780; 110 kDa)
rabbit polyclonal antibody was purchased from Cell Signalling
Technology. Rabbit polyclonal antibody CDK4 was purchased from
Pro Scientifics. After three TBST washes, the membrane was incu-
bated with corresponding HRP-labeled secondary antibody
(1:2000; Santa Cruz Biotechnology) at room temperature for 1 h.
Membranes were washed with TBST three times for 15 min, and
the protein blots were visualized with chemiluminescence reagent
Biology
Cell lines: The MCF-7 (human breast cancer) cell line was obtained
from the American Type Culture Collection (ATCC, USA). Cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitro-
gen), supplemented with 10% fetal calf serum and 100 UmLꢀ1
penicillin and 100 mgmLꢀ1 streptomycin sulfate (Sigma). The cells
were passaged and maintained at 378C in a humidified atmos-
phere containing 5% CO2.
Cell viability (MTT-based assays): Cell viability was assessed by
the MTT-based assay using WST-1 (premixed WST-1 cell prolifera-
tion assay system, Takara), which is more sensitive than MTT. Briefly,
MCF-7 cells were seeded in a 96-well plate (TPP) at a cell density of
~104 cells per well. After incubation overnight, the cells were treat-
ed with compounds 3b–e, 3h, or doxorubicin and incubated for a
further 24 h. The medium was then discarded and replaced with
fresh medium (100 mL) followed by the addition of WST-1 dye
(10 mL). Plates were incubated at 378C for 30 min. Optical density
(OD) was read at l 420 nm using a Multimode Varioskan FLASH in-
strument (Thermo-Fischer Scientific).
Cell-cycle analysis: MCF-7 cells (~5ꢁ105) were seeded in a 60 mm
dish and were allowed to grow for 24 h. Compounds 3b–e, 3h, or
1946
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ChemMedChem 2010, 5, 1937 – 1947

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We thank the National Cancer Institute (NCI), USA, for providing
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www.chemmedchem.org
1947