Synthesis and biological evaluation of novel Mannich bases of 2-arylimidazo[2,1-b]benzothiazoles as potential anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.06.031

A new series of Mannich bases of 2-arylimidazo[2,1-b]benzothiazoles were synthesized and evaluated for their anti-cancer activity. These compounds showed better cytotoxicity activity with IC₅₀ values ranging from 2.8 to 8.0 μM in HepG2, MCF-7 and HeLa cell lines. Further mechanism aspects responsible for the anti-cancer activity of two promising compounds 3c and 3f in HepG2 cell line were studied. Interestingly, 3c, 3f induced G2/M cell cycle arrest with down regulation of cyclin B and up regulation of Chk2 protein. Moreover, compounds 3c, 3f also showed the characteristic features of apoptosis such as enhancement in the levels of caspase-3. Treatments with compounds led to a decrease in levels of vital cell proliferation proteins such as Jun (C-Jun, JunB), p38 MAPK, p-JNK and PKCα. The compound 3f of the series could be considered as the potential lead for its development as a novel anti-cancer agent.

Full text of DOI:10.1016/j.ejmech.2011.06.031

European Journal of Medicinal Chemistry 46 (2011) 4258e4266
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel Mannich bases
of 2-arylimidazo[2,1-b]benzothiazoles as potential anti-cancer agents
,
a
b
a
Ravindra M. Kumbhare a , K. Vijay Kumar , M. Janaki Ramaiah , Tulshiram Dadmal ,
*
S.N.C.V.L. Pushpavalli b, Debasmita Mukhopadhyay b, B. Divya b, T. Anjana Devi b, Umesh Kosurkar a,
**
Manika Pal-Bhadra b
,
a Fluoroorganic Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
b Chemical biology Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of Mannich bases of 2-arylimidazo[2,1-b]benzothiazoles were synthesized and evaluated for
Received 11 March 2011
Received in revised form
16 June 2011
Accepted 23 June 2011
Available online 1 July 2011
their anti-cancer activity. These compounds showed better cytotoxicity activity with IC50 values ranging
from 2.8 to 8.0 mM in HepG2, MCF-7 and HeLa cell lines. Further mechanism aspects responsible for the
anti-cancer activity of two promising compounds 3c and 3f in HepG2 cell line were studied. Interestingly,
3c, 3f induced G2/M cell cycle arrest with down regulation of cyclin B and up regulation of Chk2 protein.
Moreover, compounds 3c, 3f also showed the characteristic features of apoptosis such as enhancement in
the levels of caspase-3. Treatments with compounds led to a decrease in levels of vital cell proliferation
Keywords:
proteins such as Jun (C-Jun, JunB), p38 MAPK, p-JNK and PKC
considered as the potential lead for its development as a novel anti-cancer agent.
Ó 2011 Elsevier Masson SAS. All rights reserved.
a. The compound 3f of the series could be
2-arylimidazo[2,1-b]benzothiazoles
2-Aminobenzothiazole
Cytotoxicity
Cell cycle effects
Tubulin depolymerization
1. Introduction
powerful antitumour agents particularly for the treatment of hep-
atocarcinoma as observed in our study. Also, in this context the study
of signalling pathways and key molecules that regulate cell prolifer-
ation, oncogenesis, invasion and metastasis of liver cancer such as AP-
1 (C-Jun, JunB), NF-kB, p38 MAPK would drive the way to the devel-
opment of selective targeted therapies for hepatocarcinoma [4].
Earlier studies on benzothiazoles demonstrated interesting
pharmacological activities including antitubercular [5], antima-
larial [6], anticonvulsant [7,8], antihelmintic [9,10], analgesic [11],
anti-inflammtory [12], antidiabetic [13] and antitumor activities
[14e17]. In recent years, several attempts were made for modifying
the benzothiazole nucleus to improve their antitumor activities.
Modifications on the benzothiazole nucleus have resulted in a large
number of compounds having diverse pharmacological activities.
Among them, imidazo benzothiazoles, as well as, polymerized
benzothiazoles and other substituted benzothiazoles such as
2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) (Fig. 1)
has been shown to exhibit exquisitely potent (GI50 < 0.1 nM) and
selective in vitro antitumor properties in human cancer cell lines
(e.g., colon, nonsmall- cell lung and breast subpanels) of the
National Cancer Institute (NCI) 60 human cancer cell line screen
[18] and also exhibited remarkable antitumor activity against
malignant cell lines [19]. Among the imidazo benzothiazole class
Cancer chemotherapy represents one of the most relevant
challenges of the chemists and oncologist. In order to gain new
insights into the complexity of the disease, robust screening
methods for evaluating different natural or synthetic drugs have
been carried out from the scientific community. Numerous studies
have been carried out to screen compounds with antitumour
activity against different cancer cell lines including hepatocellular
carcinoma, breast cancer, acute myelogenous leukaemia, non-small
cell lung carcinoma etc.
World statistics on cancers have revealed that hepatocarcinoma is
the sixth most common malignancy which is highly resistant to
chemotherapeutic treatment resulting in increased mortality rates
[1e3]. Therefore, in this respect, the benzothiazoles constitutes
an important scaffold of drugs, possessing several pharmacological
functions, rendering this molecule and its derivatives as
* Corresponding author. Tel.: þ91 40 27191776; fax: þ91 40 27193185.
** Corresponding author. Tel.: þ91 40 27193236.
E-mail addresses: kumbhare@iict.res.in (R.M. Kumbhare), manika@iict.res.in
(M. Pal-Bhadra).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.031

R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
4259
.
NEt2 HCl
R1
O
R2N
OCH3
OCH3
N
F
N
S
N
N
S
N
.HCl
S
R
3a−h
610 (1)
PMX
−201627(2)
YM
Fig. 1. Structure of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (1), N,N-Diethyl-2-(3-imidazo [2,1-b] [1,3]benzothiazole-2-ylphenoxy)ethnamine dihydrochloride (2), Mannich
bases of 2-arylimidazo[2,1-b]benzothiazoles derivatives (3).
R1
O
Br
N
S
N
S
a
N
NH2
R1
R
R
4
5
6 a e
−
R1
R2N
b
3a:
3b:
3c:
3d:
3e:
3f:
R = CH3; R1 = H; R2 = Morpholino
R = OC2H5; R1 = H; R2 = Morpholino
R = OC2H5; R1 = H; R2 = 4-(2-pyridinyl)piperazino
R = H; R1 = H; R2 = Pyrrolidino
N
S
N
R
3 a h
−
R = CH3; R1 = H; R2 = Pyrrolidino
R = F; R1 = H; R2 = Pyrrolidino
3g:
3h:
R = OC2H5; R1 = H; R2 = Pyrrolidino
R = H; R1 = Cl; R2 = Pyrrolidino
Scheme 1. Reagents and conditions: (a) Ethanol, MW, 180 W, 4e5 Min, 77ꢀ82%; (b) ZnCl2, secondary amine, HCHO, Ethanol, 4e5 h, rt, 84ꢀ91%.
of compounds, 2-arylimidazo [2,1-b] [1,3]benzothiazole derivative
(YM-201627) (Fig. 1) was found to be a potent and orally active
antitumor agent and useful for treatment of solid tumors [20].
Aminoalkylation of aromatic substrates by the Mannich reaction is
of considerable importance for the synthesis and modification of
biologically active compounds [21,22]. Further, the presence of
nitrogen atom may impart interesting biological activities to the
parent compound [23e25]. Hence in continuation of our efforts on
the design of novel anti-cancer agents [26e28] and keeping in
mind the medicinal importance of imidazo benzothiazole moiety, it
was thought worthwhile to synthesize certain novel derivatives of
benzothiazoles like Mannich bases of 2-arylimidazo[2,1-b]benzo-
thiazoles and screen them for their biological activities particularly
for their anti-cancer activity. Representatives of some biologically
important benzothiazole compounds have been illustrated in Fig. 1.
2. Chemistry
The 2-aryl-imidazo(2,1-b) benzothiazole derivatives (6aee)
were prepared by the condensation of substituted 2-aminobenzo-
thiazoles 4 with the appropriate 2-bromoacetophenone 5 under
Table 1
IC50 values for compounds 3aeh in selected cancer cell lines as well as normal cells
(HEK).
IC50
(m
M)
IC50
(
m
M)
IC50
(m
M)
IC50
(
m
M)
HEKa
HepG2b
MCF-7c
HeLad
YM
3a
3b
3c
3d
3e
3f
17.5 ꢁ 0.2
19 ꢁ 0.5
2.02 ꢁ 0.136
3.288 ꢁ 0.112
3.52 ꢁ 0.232
2.84 ꢁ 0.039
3.01 ꢁ 0.12
3.6 ꢁ 0.072
2.8 ꢁ 0.031
3.76 ꢁ 0.16
3.464 ꢁ 0.224
3.59 ꢁ 0.13
3.8 ꢁ 0.064
4.2 ꢁ 0.052
4.45 ꢁ 0.139
3.869 ꢁ 0.004
3.8 ꢁ 0.13
4.3 ꢁ 0.08
7.59 ꢁ 0.11
5.89 ꢁ 0.11
4.36 ꢁ 0.13
4.65 ꢁ 0.196
8.09 ꢁ 0.138
3.82 ꢁ 0.1
21 ꢁ 0.25
16 ꢁ 0.5
18.5 ꢁ 0.75
17.5 ꢁ 0.53
15 ꢁ 0.22
18.80 ꢁ 0.42
21.2 ꢁ 0.75
3.60 ꢁ 0.043
3.76 ꢁ 0.13
4.07 ꢁ 0.121
3g
3h
4.3 ꢁ 0.08
Fig. 2. Cell viability observed after the treatment with compounds at 4
tration by MTT assay. Here HepG2, MCF-7 and HeLa cells were treated with various
compounds (3aeh) at 4 M concentration as indicated for 24 h in 96 well plates at
10,000 cells per well. Compound YM-201627 (YM) was used as standard control.
Control indicates the untreated cells.
mM concen-
4.33 ꢁ 0.15
m
HEKa ¼ Human embryonic kidney cells; HepG2b¼ Hepatoma; MCF-7c ¼ breast
cancer cells; HeLad [ Human cervical cancer cells. The data obtained here is from
three independent experiments and Standard deviation (S.D) values were derived.

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R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
Fig. 3. FACS analysis of cell cycle distribution of HepG2 cells after treatment with compounds 3c, 3d and 3f at 4
standard control. Control ¼ untreated cells.
mM concentration for 24 h. Compound YM-201627 (YM) was used as

R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
4261
microwave irradiation conditions at 180 W for 4 min. Alkylation of
compounds (6aee) by reacting with cyclic secondary amines and
formic aldehyde in the presence of ZnCl2 as catalyst provided the final
compounds 3aeh as shown in Scheme 1.
series of compounds are in the range of 4.86e6.14, 3.59e5.2 and
2.163e4.89 in HepG2, MCF-7 and HeLa cell lines respectively,
Where as standard positive control YM-201627 has shown SI values
of 8.663, 4.87 and 4.069 in HepG2, MCF-7 and HeLa cell lines
respectively. Among all the compounds synthesized, compounds
3c, 3d and 3f were found to be more toxic than others of this series.
The structure - activity relationship studies revealed that the
introduction of a fluorine atom in the 7th position of the target
compounds (3f) enhanced the cytotoxic activity. Similarly, the
Mannich base of imidazobenzothiazole at the 3rd position derived
from various secondary cyclic amines also lead to an increase in
cytotoxic activity. Further, Trypan blue exclusion assay was con-
ducted and is based on the principle that live cells posseses intact
cell membranes that excludes the trypan blue dye where as dead
cells do not. Our results revealed the superior cytotoxic activity of
3c, 3d and 3f compared to other compounds of the same series
(data not shown).
3. Result and discussion
3.1. Biological evaluation
3.1.1. Anti-proliferative activity
The representative compounds 3aeh were evaluated for their
in vitro antiproliferative activity against three cancer cell lines such
as Hepato carcinoma cell line (HepG2), Breast carcinoma cell line
(MCF-7) and Human cervical carcinoma cell line (HeLa) by MTT
assay. MTT assay, is a mitochondrial function assay that is based on
the ability of viable cells to reduce the MTT to insoluble formazan
crystals by mitochondrial dehydrogenase [29]. Treatment of these
three cancer cell lines with compounds (3aeh) at a concentration
of 4
mM for 24 h inhibited cell proliferation and particularly the
3.1.2. Cell cycle effects
effect is more pronounced in HepG2 cells. (Fig. 2). IC50 values were
shown in Table 1. From these IC50 values, the selectivity index (SI)
was calculated for each of the cell line tested. Selectivity Index (SI)
for a compound is calculated as the ratio of IC50 for the normal cell
line to the IC50 for the cancer cell line. The selectivity index of this
To further investigate the differential growth inhibition mech-
anism mediated by benzothiazole compounds on cell cycle
dynamics. HepG2 cells were synchronized with Nocodazole treat-
ment and compounds 3c, 3d and 3f were tested. The cell cycle
distribution was analyzed by treating HepG2 cells at 4
mM
Fig. 4. Effect of compounds on the microtubule network in HepG2 cells. Cells were treated with compounds Nocodazole, 3f at 4 mM for 24 h. The cells were stained with a-tubulin
antibody. The tubulin pattern was observed as red coloured because secondary antibody used here was Cy3 labelled.

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R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
concentration for 24 h by flow cytometry. Results indicated 32%
(3c), 35% (3f), 28% (3d) of cells in G2/M phase respectively, where
as, the positive control YM-201627(YM) and control i.e untreated
cells showed 25% and 15% of G2/M phase cells respectively as
shown in Fig. 3a and b. Among all the compounds tested
compounds 3c and 3f could be considered as the most effective
derivatives to produce cell cycle arrest Fig. 3.
3.1.3. Effect on tubulin polymerization
Previous studies [30] showed that the inhibition of tubulin
polymerization is associated with G2-M phase cell cycle arrest. As
we have observed the G2/M phase arrest, it was considered of
interest to understand the underlying mechanism of these
compounds. Since 3f, is the most effective compound in causing
G2/M arrest among all the compounds tested the effect of this
compound on tubulin polymerization was studied by immuno-
Fig. 6. Effect of compounds on cell cycle and cell proliferation related proteins. HepG2
Cells were treated with 4 M concentration of compounds (3c, 3f) for 24 h. YM was
used as standards control. The cell lysates have been collected and observed the levels
of NF-kB (p65), PKC , p38 MAPK, C-Jun, JunB, p-JNK protiens using specific antibody.
-actin was used as loading control. C: control (untreated). Compound YM were used
as standard control.
m
a
b
fluoroscence microscopy studies using a-tubulin antibody. These
3.1.6. Effect on proteins that regulate cell proliferation
in HepG2 cells
results showed that 3f functions similar to Nocodazole (the stan-
dard tubulin polymerization inhibitor), and effected tubulin cyto-
skeleton as shown in Fig. 4.
Further interested to study the effect of these compounds on
other protein factors (c-Jun, JunB and NF-kB) that play vital role in
active cell proliferation and drug resistance [35,36]. Moreover, NF-
kB (p65) is a key molecule that is constitutively activated and
caused the hepatocarcinoma the suppression of which leads to
abrogation of cell proliferation [37,38]. c-Jun/JunB inactivation
induces apoptosis in cancer cells. In the absence of c-Jun, JunB can
act as a tumor promoter and inactivation of both, c-Jun and JunB,
could provide a valuable strategy for antitumor intervention [39].
Hence, the HepG2 cells were treated with these compounds (3c and
3.1.4. Effect on CyclinB1and check point 2 (Chk2) proteins
It is known from the previous studies that the cell cycle
progression is regulated by the expression of some cell cycle
specific cyclins. Hence, to understand the mechanism underlying
the G2/M cell cycle arrest in these compounds, we examined the
effect of compounds (3c, 3f) on the expression of Cyclin B1, which
controls cell cycle progression from G2 to M phases [31]. It is
observed from the results that there is down regulation of Cyclin B1,
particularly in case of compound 3f treated HepG2 cells as shown in
Fig. 5. G2/M phase arrest provides sufficient time for cells to repair
its damaged DNA before proceeding to the next phase of cell cycle.
Thus it was considered of interest to study the expression levels of
important check point protein such as Check point 2 (Chk2) that
play a vital role in the G2/M phase. HepG2 cells were treated with
3f) at 4 mM concentration for 24 h and western analysis was carried
out. Surprisingly from the results it is observed that there is down
regulation of c-Jun and its other isoform B-Jun and NF-kB proteins,
particularly in compound 3f treated cells as shown in Fig. 6. Thus,
this reveals the antiproliferative action of these compounds on
HepG2 cells. Further it is considered of interest to evaluate the
levels of up-stream activators of JNK pathway such as PKCa control
the processes such as cell proliferation and apoptosis [40]. Here, the
compounds (3c and 3f) at 4
mM concentration for 24 h. It was
observed that there is an up-regulation of Chk2 protein in case of
compounds 3c and 3f, especially in 3f compound treated cells as
shown in Fig. 5.
3.1.5. Effect on proteins that regulate cell cycle in HepG2 cells
Previous studies [32] revealed that MAPK family of proteins
such as JNK, ERK and p38 are involved in processes such as DNA
damage. Moreover, MAPK family proteins such as MAPK and
Phosphorylated forms of c-Jun terminal Kinase (p-JNK) regulate
the proteins that are tightly associated with G2/M arrest [33,34].
Hence to understand the role and possible involvement of MAPK
and JNK in this event, HepG2 cells were treated with compounds
3c and 3f at 4 mM concentration for 24 h. The cell lysates were
subjected to Western analysis and a down regulation of these two
proteins (MAPK and p-JNK) suggested a G2/M arrest as shown in
Fig. 6.
Fig. 7. Effect of compound on caspase-3 activation in HepG2 cells. The increased
enzymatic activity of caspase-3, in apoptosis after the treatment of compounds (3c,
and 3f) was determined by flourimetry. The cleavage of peptide by caspase-3 releases
the fluorophore AFC that was quantified at their excitation wavelength of 400 nm and
emission wave length of 505 nm. Y-axis represents relative caspase-3 activity and
X-axis represents compound treatments (YM, 3c and 3f). Compound YM-201627 (YM)
was used as standard. Control indicates theuntreated cells.
Fig. 5. Effect of compounds on Cyclin B1 and Chk2. HepG2 Cells were treated with
4
mM concentration of compounds (3c, 3f) for 24 h. The cell lysates have been collected
and observed the levels of Cyclin B and Chk2 proteins using specific antibody. -actin
b
was used as loading control. C: Control (untreated). Compound YM was used as
standard control.

R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
4263
HepG2 cells were treated with compounds (3c and 3f) at 4
concentration for 24 h and observed for the level of PKC protein.
The down regulation of PKC protein was observed in compound
treated cells as shown in Fig. 6. This revealed that these compounds
affect the PKC dependent pathway in HepG2 cells.
m
M
positive ion trap detector. Melting points were determined with an
Electro thermal melting point apparatus, and are uncorrected.
a
a
5.1. Synthesis of 2-phenyl imidazo [2,1-b]benzothiazole under
microwave heating
a
3.1.7. Effect on caspases
5.1.1. 2-Phenyl imidazo [2,1-b]benzothiazole (6a)
Previous studies [41] reported that caspase-3 is required for
DNA fragmentation and morphological changes associated with
apoptosis. Programmed cell death or apoptosis was mediated by
death proteases known as caspases. Out of all caspases the Caspase-
3 is a frequently activated death protease and is indispensible for
DNA fragmentation, chromatin condensation and the formation of
apoptotic bodies in all the cell types tested [42]. To understand the
apoptosis inducing nature of these compounds the caspase-3
activity was carried out by Fluorescence based method. In
compound treated cells particularly 3c and 3f the caspase-3 acti-
vation was up-regulated more than two-fold in comparison to
control. The order of caspase-3 activation was 3f > 3c (Fig. 7). Here
YM (YM-201627) was used as standard.
A mixture of 2-aminobenzothiazole 4a (1.00 g, 6.67 mmol), 2-
bromo acetophenone 5a (1.32 g, 6.67 mmol), and ethanol (8 mL)
were taken in a 20 mL pressure tube and were subjected to
microwave irradiation (CEM Discover, 180 W, 250 psi, 100 ꢂC) for
4 min the reaction mixture was cooled to room temperature and
the resulting white precipitate was filtered, and air-dried to give
3.29 g of 2-phenylimidazo [2,1-b]benzothiazolehydrobromide. The
hydrobromide was suspended in ethanol (20 mL) in a 100 ml of two
necked flask equipped with a reflux condenser and dropping fun-
nel. To this, triethylamine (5 mL) was added drop wise and refluxed
for 5 min. The solution was cooled to room temperature and poured
into ice water (50 mL). The resulting white precipitate was filtered
and air-dried to obtained 6a. Yield 2.05 g, 82%; mp 98e100 ꢂC. IR
(KBr, nmax cmꢀ1): 3041 (AreH), 1667 (C]N), 678 (CeSeC); 1H NMR
(CDCl3, 300 MHz):
d 8.26 (s, 1H, imidazole-H), 7.67e7.88 (m, 3H,
4. Conclusion
ArH), 7.47 (dt, 1H, J ¼ 8.30 Hz and J ¼ 0.75 Hz, ArH), 7.32e7.42 (m,
4H, ArH), 7.24 (t, 1H, J ¼ 7.36 Hz, ArH); MS (ESI): m/z 251 (M þ 1)þ.
A new class of Mannich bases of 2-arylimidazo[2,1-b]benzo-
thiazoles compounds were synthesised and evaluated for their
antiproliferative activities in three cancer cell lines. From the MTT
proliferation assay, it was observed that compound 3f isthe most
effective antiproliferative agent than the other compounds of the
series against all three cell lines (HepG2, MCF-7 and HeLa) when
5.1.2. 7-Methyl-2-phenyl imidazo [2,1-b] benzothiazole (6b)
This compound was prepared according to the method
described for compound 6a, employing Compound 5a (1.32 g,
6.67 mmol) and compound 4b (1.09 g, 6.67 mmol) to obtain the
pure product 6b as a white solid, yield 2.11 g, 80%; mp 160ꢀ162 ꢂC.
IR (KBr, nmax cmꢀ1):3072 (AreH), 1678 (C]N), 697 (CeSeC); 1H
tested at 4 mM concentration. The FACS analysis also showed more
population in G2/M phase in compounds 3c, 3d and 3f indicating
that these compounds caused G2/M cell cycle arrest with 3f being
the most effective. From the results of the detailed biological assays,
drastic down regulation of cell cycle regulatory protein such as
Cyclin B as well up-regulation DNA damage specific check point
protein (Chk2) protein was observed, suggesting that these proteins
are responsible for cell cycle blockade at G2/M phase in compound
3f. Moreover, caspase-3 protein level was increased that indicate
apoptotic inducing nature of the compounds 3c and 3f. Further
downstream proteins which have a potential role in cell cycle as
well as proliferation such as MAPK family (p38 and p-JNK) and Jun
family proteins were down regulated. These findings concretely
support the important cytotoxic as well as cell cycle regulatory role
of these Mannich bases of 2-arylimidazo[2,1-b]benzothiazoles
compounds. In this study an insight into the cell cycle regulatory
role as well as apoptotic inducing ability of the compound 3f was
elucidated. These studies suggest that compound 3f has the
potential to be taken up for further, particularly against
hepatocaricinoma.
NMR (CDCl3, 300 MHz):
d 8.28 (s, 1H, imidazole-H), 7.94 (d, 1H,
J ¼ 7.95 Hz, ArH), 7.64 (d, 2H, J ¼ 7.16 Hz, ArH), 7.49 (s, 1H, ArH), 7.41
(t, 2H, J ¼ 7.95 Hz, ArH), 7.32 (t, 1H, J ¼ 7.16 Hz, ArH), 7.19 (dd, 1H,
J ¼ 7.95 Hz and J ¼ 0.79 Hz, ArH), 2.48 (s, 3H, -CH3); MS (ESI): m/z
269 (M þ 1)þ.
5.1.3. 7-Fluoro-2-phenyl imidazo [2,1-b] benzothiazole (6c)
This compound was prepared according to the method
described for compound 6a, employing Compound 5a (1.32 g,
6.67 mmol) and compound 4c (1.12 g, 6.67 mmol) to obtain the
pure product 6b as a white solid, yield 2.06 g, 77%; mp 156ꢀ159 ꢂC.
IR (KBr, nmax cmꢀ1): 3088 (AreH), 1643 (C]N), 1340 (CeF), 654
(CeSeC); 1H NMR (CDCl3, 300 MHz):
d 8.27 (s, 1H, imidazole-H),
8.08 (q, 1H, J ¼ 4.53 Hz, ArH), 7.63 (d, 2H, J ¼ 7.55 Hz, ArH),
7.36e7.45 (m, 3H, ArH), 7.27e7.34 (m, 1H, ArH), 7.11 (dt, 1H,
J ¼ 8.30 Hz and J ¼ 2.26 Hz, ArH); MS (ESI): m/z 269 (M þ 1)þ.
5.1.4. 7-Ethoxy-2-phenyl imidazo [2,1-b] benzothiazole (6d)
This compound was prepared according to the method
described for compound 6a, employing Compound 5a (1.32 g,
6.67 mmol) and compound 4d (1.29 g, 6.67 mmol) to obtain the
pure product 6b as a white solid, yield 2.38 g, 81%; mp 179e181 ꢂC.
IR (KBr, nmax cmꢀ1): 3097 (AreH), 1658 (C]N), 1080 (CeOeC), 647
5. Experimental Protocols
All chemicals and reagents were purchased from Aldrich (Sig-
maeAldrich, 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 on TLC was achieved
by UV light or iodine indicator. Column chromatography was per-
formed with Merck 60e120 mesh silica gel. 1H and 13C spectra were
recorded Bruker UXNMR/XWIN-NMR (300 MHz) instruments.
(CeSeC); 1H NMR (CDCl3, 300 MHz):
d 8.24 (s, 1H, imidazole-H),
7.96 (d, 1H, J ¼ 8.87 Hz, ArH), 7.64 (d, 2H, J ¼ 7.36 Hz, ArH), 7.40 (t,
2H, J ¼ 7.17 Hz, ArH), 7.30 (t, 1H, J ¼ 7.17 Hz, ArH), 7.16 (d, 1H,
J ¼ 2.455 Hz, ArH), 6.91 (dd, 1H, J ¼ 9.06 Hz and J ¼ 2.45 Hz, ArH),
4.08 (q, 2H, J ¼ 6.98, eOCH2), 1.48 (t, 3H, J ¼ 6.98 Hz, -CH3); MS
(ESI); m/z 295 (M þ 1)þ.
5.1.5. 2-(4-Chlorophenyl) imidazo [2,1-b]benzothiazole (6e)
This compound was prepared according to the method
described for compound 6a, employing Compound 5b (1.89 g,
6.67 mmol) and compound 4a (1.0 g, 6.67 mmol) to obtain the pure
Chemical shifts (d) are reported in ppm downfield from internal
TMS standard. ESI spectra were recorded on Micro mass, Quattro LC
using ESI þ software with capillary voltage 3.98 kV and ESI mode

4264
R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
product 6b as a white solid, yield 2.21 g, 78%; mp 165ꢀ168 ꢂC. IR
(KBr, nmax cmꢀ1): 3057 (AreH), 1677 (C]N), 688 (CeSeC), 647
(q, 2H, J ¼ 6.79 Hz, eOCH2), 4.02 (s, 2H, methelene-H), 3.56 (t, 4H,
J ¼ 4.53 Hz, pyridinylpiperzine-H), 2.70 (t, 4H, J ¼ 4.53 Hz, pyr-
idinylpiperzine-H), 1.46 (t, 3H, J ¼ 6.79 Hz, eCH3); 13C NMR
(CeCl); 1H NMR (CDCl3, 300 MHz):
d 8.26 (s, 1H, imidazole-H), 8.07
(d, 1H, J ¼ 9.06 Hz, ArH), 7.69 (d, 1H, J ¼ 8.34 Hz, ArH), 7.63 (d, 2H,
J ¼ 8.78 Hz, ArH), 7.39 (t, 3H, J ¼ 8.34 Hz, ArH), 7.31 (t, 1H,
J ¼ 8.34 Hz, ArH); (ESI): m/z 285.
(75 MHz, CDCl3): d 147.9, 138.4, 133.9, 133.1, 130.1, 129.7, 125.8,
124.9, 123.8, 115.9, 113.8, 107.6, 77.9, 77.4, 76.9, 52.4, 51.2, 45.3, 33.7,
20.3; ESI-MS: 470 (M þ 1)þ; HRMS (ESI m/z) for C27H27N5OS, calcd
470.1911, found 470.1897.
5.1.6. Synthesis of 3-(morpholinomethyl)-2-phenyl-7-methyl-
imidazo [2,1-b]benzothiazole (3a)
5.1.9. 3-(pyrrolidinomethyl)-2-phenyl-imidazo [2,1-b]
A mixture of 7-methyl-2-phenyl imidazo [2,1-b] benzothiazole
6b (264 mg, 1 mmol), morpholine (241 mg, 3 mmol), formalin
(1 mL) and catalytic amount of anhydrous ZnCl2 (0.135 mg,
0.1 mmol) in ethanol was stirred at room temperature for 4 h. The
progress of the reaction was monitored with TLC. After completion
of the reaction, ethanol was removed under reduced pressure and
the reaction mixture was extracted with ethyl acetate (3 ꢃ 10 mL).
The combined extract was washed with water (1 ꢃ 5 mL) and brine
(1 ꢃ 5 mL) and was dried over anhyd. MgSO4. The organic layer was
concentrated under reduced pressure and the crude product was
purified by column chromatography silica gel (60e120 mesh) using
ethyl acetate-hexane(1:9) as eluent to afford compound 3a as
a white solid, yield 330 mg, 91%; mp 132ꢀ134 ꢂC; IR (KBr,
nmax cmꢀ1): 3064 (AreH), 2885 & 2811 (CeH), 1651 (C]N), 1067
benzothiazole (3d)
This compound was prepared according to the method
described for compound 3a, employing compound 2-phenyl imi-
dazo [2,1-b]benzothiazole 6a (250 mg, 1 mmol) and compound
pyrolidine (213 mg, 3 mmol) to obtain the pure product 3d as
a white solid, yield 286 mg, 86%; mp 127 ꢂC. IR (KBr, nmax cmꢀ1):
3067 (AreH), 2887 & 2763 (CeH), 1678 (C]N), 684 (CeSeC); 1H
NMR (CDCl3, 300 MHz):
d 7.71e7.89 (m, 3H, ArH), 7.48 (dt, 1H,
J ¼ 8.43 Hz and J ¼ 1.12 Hz, ArH), 7.32e7.42 (m, 4H, ArH), 7.24 (t, 1H,
J ¼ 7.43 Hz, ArH), 4.07 (s, 2H, methelene-H), 2.62 (t, 4H, J ¼ 8.43 Hz,
pyrolidine-H), 1.81 (t, 4H, J ¼ 8.43 Hz, pyrolidine-H); 13C NMR
(75 MHz, CDCl3):
d 149.1, 145.9, 137.8, 135.1, 133.8, 130.5, 129.7,
128.4, 123.1, 121.4, 115.8, 60.9, 51.7, 29.7; ESI-MS: 334 (M þ 1)þ;
HRMS (ESI m/z) for C20H19N3S, calcd 334.1353, found 334.1364.
(CeOeC), 729 (CeSeC); 1H NMR (CDCl3, 300 MHz):
d 7.94 (d, 1H,
J ¼ 7.87 Hz, ArH), 7.64 (d, 2H, J ¼ 7.11 Hz, ArH), 7.49 (s, 1H, ArH), 7.41
(t, 2H, J ¼ 7.87 Hz, ArH), 7.32 (t, 1H, J ¼ 7.11 Hz, ArH), 7.19 (dd, 1H,
J ¼ 7.87 Hz and J ¼ 0.78 Hz, ArH), 4.0 (s, 2H, methelene-H), 3.64 (t,
4H, J ¼ 3.96 Hz, morpholine-H), 2.56 (t, 4H, J ¼ 3.96 Hz, morpho-
5.1.10. 3-(pyrrolidinomethyl)-2-phenyl-7-methyl-imidazo [2,1-b]
benzothiazole (3e)
This compound was prepared according to the method
described for compound 3a, employing compound 7-methyl-2-
phenyl imidazo [2,1-b]benzothiazole 6b (264 mg, 1 mmol) and
compound pyrolidine (213 mg, 3 mmol) to obtain the pure product
3e as a white solid, yield 305 mg, 88%; mp 129 ꢂC. IR (KBr, nmax
cmꢀ1): 3067 (AreH), 2958 & 2867 (CeH), 1674 (C]N), 701
line-H), 2.48 (s, 3H, eCH3); 13C NMR (CDCl3, 75 MHz):
d 148.5,145.7,
137.9, 134.7, 133.5, 130.5, 129.7, 128.5, 124.8, 122.5, 115.6, 60.4, 51.8,
45.3, 28.7; ESI-MS: 364 (M þ 1)þ; HRMS (ESI m/z) for C21H21N3OS,
calcd 364.1545, found 364.1561.
(CeSeC); 1H NMR (300 MHz, CDCl3):
d
8.01 (d,1H, J ¼ 7.86 Hz, ArH),
5.1.7. 3-(morpholinomethyl)-2-phenyl-7-ethoxy-imidazo [2,1-b]
benzothiazole (3b)
7.68 (d, 2H, J ¼ 7.14 Hz, ArH), 7.52 (s, 1H, ArH), 7.44 (t, 2H,
J ¼ 7.86 Hz, ArH), 7.34 (t, 1H, J ¼ 7.14 Hz, ArH), 7.19 (dd, 1H,
J ¼ 7.86 Hz and J ¼ 1.24 Hz, ArH), 4.06 (s, 2H, methelene-H), 2.56 (t,
4H, J ¼ 4.04 Hz, pyrolidine-H), 2.42 (s, 3H, -CH3), 1.80 (t, 4H,
This compound was prepared according to the method
described for compound 3a, employing compound 7-Ethoxy -2-
phenyl imidazo [2,1-b]benzothiazole 6d (294 mg, 1 mmol) and
compound morpholine (241 mg, 3 mmol) to obtain the pure
product 3b as a white solid, yield 334 mg, 85%; mp 169 ꢂC. IR (KBr,
nmax cmꢀ1): 3062 (AreH), 2892 & 2813 (CeH), 1677 (C]N), 1087
J ¼ 4.04 Hz, pyrolidine-H); 13C NMR (75 MHz, CDCl3):
d 148.9, 145.7,
136.9, 134.7, 134.1, 131.7, 129.7, 128.5, 124.9, 122.8, 121.5, 115.6, 60.6,
51.9, 45.2, 28.7; ESI-MS: 348 (M þ 1)þ; HRMS (ESI m/z) for
C21H21N3S, calcd 348.1502, found 348.1514.
&1073 (CeOeC), 678 (CeSeC); 1H NMR (CDCl3, 300 MHz):
d 7.94 (d,
1H, J ¼ 8.79 Hz, ArH), 7.62 (d, 2H, J ¼ 7.24 Hz, ArH), 7.41 (t, 2H,
J ¼ 7.08 Hz, ArH), 7.32 (t, 1H, J ¼ 7.08 Hz, ArH), 7.15 (d, 1H,
J ¼ 2.39 Hz, ArH), 6.89 (dd, 1H, J ¼ 8.99 Hz and J ¼ 2.39 Hz, ArH),
4.06 (q, 2H, J ¼ 6.88 Hz, ArH), 3.98 (s, 2H, methelene-H), 3.62 (t, 4H,
J ¼ 3.94 Hz, morpholine-H), 2.61 (t, 4H, J ¼ 3.94 Hz, morpholine-H),
5.1.11. 3-(pyrrolidinomethyl)-2-phenyl-7-fluoro-imidazo [2,1-b]
benzothiazole (3f)
This compound was prepared according to the method
described for compound 3a, employing compound 7-Fluoro-2-
phenyl imidazo [2,1-b]benzothiazole 6c (268 mg, 1 mmol) and
compound pyrolidine (213 mg, 3 mmol) to obtain the pure product
3f as a white solid, yield 298.35 mg, 85%; mp 136 ꢂC. IR (KBr, nmax
cmꢀ1): 3054 (AreH), 2876 & 2799 (CeH), 1632 (C]N), 1151 (CeF),
1.48 (t, J ¼ 6.88 Hz, 3H, eCH3); 13C NMR (75 MHz, CDCl3):
d 148.9,
145.9, 138.9, 134.7, 133.8, 130.7, 129.9, 128.7, 125.1, 122.9, 115.9, 62.4,
51.8, 42.1, 34.7, 21.5; ESI-MS: 394 (M þ 1)þ; HRMS (ESI m/z) for
C22H23N3O2S, calcd 394.1527, found 394.1518.
678 (CeSeC); 1H NMR (300 MHz, CDCl3):
d
8.08 (q, 1H, J ¼ 5.48 Hz,
ArH), 7.64 (d, 2H, J ¼ 8.34 Hz, ArH), 7.35e7.47 (m, 3H, ArH),
7.27e7.32 (m, 1H, ArH), 7.11 (dt, 1H, J ¼ 7.96 Hz and J ¼ 2.15 Hz,
ArH), 4.05 (s, 2H, methelene-H), 2.52 (t, 4H, J ¼ 4.53 Hz, pyrolidine-
H), 1.75 (t, 4H, J ¼ 4.53 Hz, pyrolidine-H); 13C NMR (75 MHz, CDCl3):
5.1.8. 3-(4-(2-pyridinyl)piperazinomethyl)-2-phenyl-7-ethoxy-
imidazo [2,1-b]benzothiazole (3c)
This compound was prepared according to the method
described for compound 3a, employing compound 7-Ethoxy-2-
phenyl imidazo [2,1-b] benzothiazole 6d (294 mg, 1 mmol) and
compound pyridinyl piperzine (326 mg, 2 mmol) to obtain the pure
product 3c as a white solid, yield 408 mg, 87%; mp 160 ꢂC. IR (KBr,
nmax cmꢀ1): 3057 (AreH), 2857 & 2799 (CeH), 1681 (C]N), 1063
d
149.7, 145.7, 137.6, 134.8, 133.6, 130.4, 129.7, 128.4, 121.9, 115.8,
60.9, 45.4; ESI-MS: 352 (M þ 1)þ; HRMS (ESI m/z) for C20H18FN3S,
calcd 352.1263, found 352.1247.
5.1.12. 3-(pyrrolidinomethyl)-2-phenyl-7-ethoxy-imidazo [2,1-b]
benzothiazole (3g)
(CeOeC), 737 (CeSeC); 1H NMR (CDCl3, 300 MHz):
d 8.11 (dd, 1H,
J ¼ 5.28 Hz, and J ¼ 1.51 Hz, pyridinyl-H), 8.04 (d, 1H, J ¼ 9.06 Hz,
pyridinyl-H), 7.59e7.69 (m, 2H, pyridinyl-H), 7.39 (t, 3H, J ¼ 7.55 Hz,
ArH), 7.30 (d, 1H, J ¼ 6.79 Hz, ArH), 7.14e7.19 (m, 1H, ArH), 6.90 (dd,
1H, J ¼ 9.06 Hz and J ¼ 3.022 Hz, ArH), 6.51e6.59 (m, 2H, ArH), 4.08
This compound was prepared according to the method
described for compound 3a, employing compound 7-ethoxy-2-
phenyl imidazo [2,1-b]benzothiazole 6d (294 mg, 1 mmol) and
compound pyrolidine (213 mg, 3 mmol) to obtain the pure product

R.M. Kumbhare et al. / European Journal of Medicinal Chemistry 46 (2011) 4258e4266
4265
3g as a white solid, yield 327 mg, 87%; m.p 147 ꢂC. IR (KBr, nmax
cmꢀ1): 3065 (AreH), 2877 & 2763 (CeH), 1669 (C]N), 1079
30 min in the dark]. The DNA contents of 20,000 events were
measured by flow cytometer (DAKO CYTOMATION, Beckman
Coulter, Brea, CA). Histograms were analyzed using Summit
Software.
(CeOeC), 678 (CeSeC); 1H NMR (300 MHz, CDCl3):
d 7.97 (d, 1H,
J ¼ 9.06 Hz, ArH), 7.67 (d, 2H, J ¼ 8.75 Hz, ArH), 7.44 (t, 2H,
J ¼ 7.84 Hz, ArH), 7.31 (t, 1H, J ¼ 7.84 Hz, ArH), 7.16 (d, 1H,
J ¼ 3.45 Hz, ArH), 6.91 (dd, 1H, J ¼ 9.54 Hz, and J ¼ 3.45 Hz, ArH),
4.08 (q, 2H, J ¼ 7.65 Hz, -OCH2), 4.02 (s, 2H, methelene-H), 2.62 (t,
4H, J ¼ 4.47 Hz, pyrolidine-H), 1.80 (t, 4H, J ¼ 4.47 Hz, pyrolidine-H),
5.5. Protein extraction and Western blot analysis
Total cell lysates from cultured HepG2cells were obtained by
lysing the cells in ice-cold RIPA buffer (1XPBS, 1% NP-40, 0.5%
1.49 (t, 3H, J ¼ 7.65 Hz, eCH3), 13C NMR (75 MHz, CDCl3):
d 149.3,
146.1, 135.7, 133.8, 130.5, 128.7, 124.7, 122.9, 121.9, 116.5, 60.9, 51.6,
45.2, 29.7, 19.6; ESI-MS: 378 (M þ 1)þ; HRMS (ESI m/z) for
C22H23N3OS, calcd 378.1614, found 378.1628.
sodium deoxycholate and 0.1% SDS) and containing 100
PMSF, 5 g/mL Aprotinin, 5 g/mL leupeptin, 5 g/mL pepstatin and
100 g/mL NaF. After centrifugation at 12,000 rpm for 10 min, the
mg/mL
m
m
m
m
protein in supernatant was quantified by Bradford method (BIO-
RAD) using Multimode varioskan instrument (Thermo-Fischer
Scientifics). Fifty micrograms of protein per lane was applied in 12%
SDS-polyacrylamide gel. After electrophoresis, the protein was
transferred to polyvinylidine difluoride (PVDF) membrane (Amer-
sham Biosciences). The membrane was blocked at room tempera-
ture for 2 h in TBS þ 0.1% Tween20 (TBST) containing 5% blocking
powder (Santacruz). The membrane was washed with TBST for
5 min, and primary antibody was added and incubated at 4 ꢂC
overnight (O/N). Mouse monoclonal antibodies Chk2, CyclinB1, NF-
5.1.13. 3-(pyrrolidinomethyl)-2-(4-chlorophenyl) imidazo- [2,1-b]
benzothiazole (3h)
This compound was prepared according to the method
described for compound 3a, employing compound 2-(4-
chlorophenyl) imidazo [2,1-b]benzothiazole 6e (284 mg, 1 mmol)
and compound pyrolidine (213 mg, 3 mmol) to obtain the pure
product 3h as a white solid, yield 308 mg, 84%; mp 139 ꢂC. IR (KBr,
nmax cmꢀ1): 3057 (AreH), 2887 & 2789 (CeH), 1681 (C]N), 741
(CeSeC), 674 (CeCl); 1H NMR (300 MHz, CDCl3):
d 8.11 (d, 1H,
J ¼ 8.30 Hz, ArH), 7.70 (d, 1H, J ¼ 7.55 Hz, ArH), 7.63 (d, 2H,
J ¼ 8.30 Hz, ArH), 7.39 (t, 3H, J ¼ 7.55 Hz, ArH), 7.31 (t, 1H,
J ¼ 7.55 Hz, ArH), 4.07 (s, 2H, -CH2), 2.61 (t, 4H, J ¼ 4.53 Hz, pyro-
lidine-H), 1.80 (t, 4H, J ¼ 4.53 Hz, pyrolidine-H); 13C NMR (75 MHz,
kB (p65) and rabbit polyclonal antibodies MAPK, PKCa, JunB and b-
actin antibodies were purchased from Imgenex, USA. Rabbit poly-
clonal antibodies C-Jun and p-JNK was purchased from Santacruz
Company. The membrane was incubated with corresponding
horseradish peroxidase-labeled secondary antibody (1:2000)
(Santa Cruz) at room temperature for 1 h. Membranes were washed
with TBST three times for 15 min and the blots were visualized with
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The
X-ray films were developed with developer and fixed with fixer
solution.
CDCl3):
d 148.4, 145.7, 137.8, 134.9, 133.6, 130.5, 129.6, 128.1, 124.5,
122.3, 115.6, 60.5, 51.8, 29.7; ESI-MS: 368 (M þ 1)þ; HRMS (ESI m/z)
for C20H18ClN3S, calcd 368.1108, found 368.1121.
5.2. Cell culture
The HepG2 (human hepato-carcinoma), MCF-7 cells (human
breast cancer) and HeLa (human cervical cancer) were incubated by
using Dulbecco’s modified Eagle’s medium (DMEM), supplemented
5.6. Caspase-3 assay
with 10% fetal calf serum, 100 mg/mL pencillin-G and 100 mg/mL
The caspase-3 fluorescent assay kit (Clonetech,CA) was applied
to evaluate the caspase-3 activity, using the procedures provided by
the manufacturer HepG2 cells were treated with compounds YM-
streptomycin sulfate (Sigma). The cell lines were maintained at
37 ꢂC in a humidified atmosphere containing 5% CO2 in the
incubator.
201627(YM), 3c and 3f at 4 mM concentration as obtained from
FACS analysis. Cell lysates were added to the 2ꢃ reaction buffer
containing containing DTT and caspase substrate was added and
incubation was carried out at 37 ꢂC for 1 h. Readings were taken at
excitation wavelength 400 nm and emission wavelength 505 nm.
5.3. Cell viability
Cell viability was assessed by the MTT based assay HepG2, MCF-
7 and HeLa cells were seeded in a 96-well plate (TPP) at a cell
density of 10,000 cells/well. After overnight incubation, the cells
were treated with compounds 3aꢀh and YM-201627 (YM) was
incubated for 24 h. The medium was then discarded and replaced
Acknowledgment
We are thankful to Dr. J. S. Yadav, Director, IICT for providing
facilities, T.D. thank for UGC, and U.K. thank for CSIR, New Delhi, for
the award of a fellowship, and R.M.K thanks S.E.R.C., Department of
Science & Technology, Government of India for financial assistance
under the Fast Track Scheme for young scientists (SR/FTP/CS-93/
2006).
with fresh 100 mL media followed by addition of 10 mL of MTT dye.
Plates were incubated at 37 ꢂC for 2 h. The absorbance (O.D) was
measured at 570 nm using Multimode Varioskan Flash (Thermo
Fisher Scientifics).
5.4. Cell cycle analysis
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