Synthesis and biological evaluation of novel triazoles and isoxazoles linked 2-phenyl benzothiazole as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2012.07.041

A new series of isoxazoles and triazoles linked 2-phenyl benzothiazole were synthesized and evaluated for their anticancer activity. These compounds have been tested for their cytotoxicity three cancer cell lines. Among the compounds tested, compound 5d s

Full text of DOI:10.1016/j.bmcl.2012.07.041

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5424–5427
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel triazoles and isoxazoles linked
2-phenyl benzothiazole as potential anticancer agents
Ravindra M. Kumbhare a, , Umesh B. Kosurkar a, M. Janaki Ramaiah b, Tulshiram L. Dadmal a,
⇑
S. N. C. V. L. Pushpavalli b, 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 isoxazoles and triazoles linked 2-phenyl benzothiazole were synthesized and evaluated
for their anticancer activity. These compounds have been tested for their cytotoxicity against three cancer
cell lines. Among the compounds tested, compound 5d showed good cytotoxicity against Colo-205 and
A549 cells in comparison to standard control PMX 610(1). Further compound 5d has been tested for
its apoptotic activity and its inhibitory activity against caspase and PARP proteins. Hence this compound
has the potential that it can be selected for further biological studies.
Received 11 April 2012
Revised 22 June 2012
Accepted 10 July 2012
Available online 17 July 2012
Keywords:
Phenyl benzothiazole
Triazole
Ó 2012 Elsevier Ltd. All rights reserved.
Isoxazole
Cytotoxicity
Cell cycle effects
Over the last two decades, the synthesis and functionalization
of benzothiazole has become a major area of focus for synthetic or-
ganic chemists because of their several pharmacological functions
including antitumour activity,1–8 neurotransmission blockage,9–11
calmodulin antagonists12 and neuroprotective activity.13,14 In re-
cent years, extensive research has been carried out for modifying
benzothiazole nucleus to improve their antitumour activities.
Among the modified structures, especially interesting are 2-phenyl
CH3
OCH3
R
N
S
F
N
S
NH2
OCH3
R = H ( DF 203) 2a
R = F ( 5F 203) 2b
PMX610 (1)
N
S
F
O
HO
R1
O
N
N
N
3
O
N
O
N
N
S
R
S
4 a-g
5 a-f
Figure 1. Chemical structure of anticancer phenylbenzothiazole derivatives.
⇑
Corresponding authors. Tel.: +91 40 27191776; fax: +91 40 27193185 (R.M.K.); tel.: +91 40 27193236 (M.P.-B.).
E-mail addresses: rakumbhare@yahoo.com, kumbhare@iict.res.in (R.M. Kumbhare), manika@iict.res.in (M. Pal-Bhadra).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.041

R. M. Kumbhare et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5424–5427
5425
benzothiazoles that exhibit potent and selective antitumour activ-
ity. Some of the structurally related benzothiazoles such as 2-(3,4-
dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610)15 (1) (Fig. 1),
2-(4-amino-3-methylphenyl) benzothiazole (DF 203) and the 2-
(4-amino-3-methylphenyl)-5-fluoro benzothiazole (5F 203)16 (2a
and 2b of Fig. 1) have been reported to possess potent and selective
in vitro antitumour properties in human cancer cell lines particu-
larly against colon, non-small cell lung and breast cancer lines of
the National Cancer Institute (NCI) 60 human cancer cell line
screen. Other benzothiazole derivatives such as 4-(benzothiazol-
2-yl)-4-hydroxycyclohexa-2,5-dienones17 (PMX 464) (3) (Fig. 1)
exhibited potent and selective antitumour activity concentrated
in certain colon, renal and breast cancer cell lines, that acts via
inhibition of the cellular redox protein thioredoxin (Trx-1). On
the other hand, isoxazole and triazole derivatives have been re-
ported for their anticancer activity against human colon cancer cell
lines.18
Hence in continuation of our efforts for the structural modifica-
tions on benzothiazole moiety and thus to improve their anticancer
activity,19,20 new series of benzothiazoles have been synthesized by
HO
N
HO
NH2
H2O2 /CAN
OHC
50 0C
neat,
S
SH
6
O
N
Acetone,K2CO3
Reflux,2hrs
Br
6
S
7
7
i
ii
R1
N
O
N
N
N
O
O
N
S
N
S
R
5 a-f
4 a-g
1
4a
: R = 4-CH3
4e
5a
5b
5d:
: R = 4-Br
: R = C6H5
R
R
R
1 = 3CF3-C6H4
1
: R1 = 4-F-C6H4
4b
4c
4d
: R = 4-OCH3
: R = 2-F
5e:
5f:
4f
= C6F13-CH2-CH2-
: R = 4-OCF3
1
1 = C8F17-CH2-CH2-
4g
: R = 3,4,5-OCH3
5c
: R = 3-Cl,4-F-C6H3
: R = 4-Cl
Scheme 1. Reagents and conditions: (i) aq NaOCl (9–12%), DCM, Et3N, oxime, rt, 24 h, 65–75%; (ii) dry THF, CuI (5 mol %), R1-N3, rt, 24 h, 85–89%.
Table 1
IC50 values for compounds (4a–5f) in selected cancer cell lines (A549, colo-205, MCF-7) as well as normal cells MCF-10A
S. No.
Compound
IC in MCF-10A
50
IC
in A549
IC in Colo-205
50
IC
in MCF-7
50
50
1.
2.
3.
4.
5.
6.
7.
8.
Std
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
25.22 0.80
26.37 0.74
34.13 1.82
42.54 0.50
38.8 1.65
29.2 0.70
43.87 2.57
42.87 2.37
36.41 2.32
33.32 3.72
26.28 1.54
31.07 2.73
35.05 3.67
33.1 3.92
15.88 0.93
16.37 0.56
24.13 1.12
22.54 0.56
18.8 0.75
19.2 0.57
23.87 0.57
22.87 0.37
16.41 1.12
23.32 0.37
16.28 0.56
11.07 0.62
15.05 0.75
13.1 0.74
13.94 0.24
14.16 0.91
20.70 0.68
18.28 0.68
16.45 0.68
16.449 0.36
19.87 0.62
17.5 0.69
16.58 0.69
18.87 0.69
14.40 0.69
10.78 0.69
14.16 0.45
12.21 0.32
20.22 0.97
27.52 1.28
22.14 1.28
23.87 0.96
23.8 1.66
20.03 0.96
26.62 1.28
25.6 0.96
26.24 1.28
26.24 1.28
27.26 1.33
18.94 0.97
19.84 1.28
19.2 0.36
9.
10.
11.
12.
13.
14.
IC50 is concentration at which 50% of cells were undergo cytotoxic cell death due to compound treatment.

5426
R. M. Kumbhare et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5424–5427
6 coupled with propargyl bromide to provide compound 7 which
were reacted with oximes and aromatic/aliphatic azides produced
corresponding isoxazoles (4a–g) and triazoles (5a–f) respectively
as shown in Scheme 1.
The new series of isoxazoles and triazoles linked 2-phenyl ben-
zothiazole derivatives (4a–g and 5a–f) were evaluated for their
cytotoxic activity against three cancer cell lines such as human
lung adenocarcinoma (A549), human colon cancer cells (Colo-
205) and breast carcinoma cell line (MCF-7) by MTT assay. These
cancer cell lines were treated with compounds (4a–g and 5a–f)
along with PMX 610 (1) as a standard positive control at a concen-
tration ranging from 1 to 64 lM for 24 h .Most of these benzothiaz-
oles exhibited higher anticancer activity than 1, particular against
colon-205 and A549 cell lines. (Table 1 and Supplementary Figs. 1
and 3a–4c). The structure–activity relationship studies revealed
that the introduction of a fluorine atom particular the –CF3 group
in the 3rd position of the target compounds 5d enhanced the cyto-
toxic activity. Similarly, the triazole derivatives of phenyl benzothi-
azole series also lead to an increase in cytotoxic activity.
In order to study the effect of these benzothiazole derivatives
on cell cycle progression and apoptosis in human colon cancer cells
(Colo-205) were treated with compounds 1, 5d, 5e and 5f analyzed
using fluorescence activated cell sorter (FACS shown in Supple-
mentary Figs. 2, 5a and b). We have observed 3–4% of apoptotic
Figure 2. The graph representing the percentage of apoptotic cells accumulated
after the treatment of colo-205 cells with compounds 1, 5d, 5e and 5f at 8 and
16 lM concentrations for 24 h. control indicates the untreated cells.
Table 2
cells as indicated by G0 phase at
phenylbenzothiazoles.
Interestingly the percentage of apoptosis was observed to be 27,
40, 12 and 34 in case of compounds 1, 5d, 5e and 5f treated cells
8 lM concentration of
The cell cycle distribution of compounds
Compound
Control
1 (8 lM)
G0
G1
S
G2/M
2.28 0.25
4.15 0.30
27.17 2.45
4.02 0.46
40.70 1.12
2.72 0.25
11.91 0.37
3.30 0.26
34.45 0.50
71.02 1.00
70.04 0.96
52.82 2.45
70.91 1.01
45.77 0.69
73.66 0.57
61.36 1.18
72.0 0.56
3.56 0.40
3.21 0.25
4.31 0.59
3.37 0.33
2.68 0.27
3.09 0.15
4.42 0.51
4.43 0.51
3.79 0.35
23.09 1.01
22.5 0.92
15.6 0.54
21.68 0.59
10.67 0.58
20.53 0.84
22.3 1.08
20.22 0.69
15.18 1.40
respectively. Thus compounds at a concentration of 16 lM was
1 (16
lM)
considered as effective concentration and compounds have apop-
totic inducible nature (Fig. 2, Table 2 and Supplementary Figs. 2,
5a and b).
5d (8
lM)
5d (16
lM)
5e (8
lM)
5e (16
l
M)
One of the important characteristic features of apoptosis is the
degradation of procaspase-3 to active caspase-3 and cleavage of
DNA repair enzyme PARP [Poly (ADP-ribose) Polymerase] by cas-
pase-3.22 Early literature has shown that caspase-3 and caspase-9
plays a prominent role in mediating drug induced apoptosis.23 Cas-
pase-3 is considered to be the effector caspase and also considered
as the therapeutic target for the treatment of cancer.24 It was ob-
served in apoptosis pro-caspases are cleaved to active caspases.25
5f (8
lM)
5f (16
lM)
46.57 1.91
FACS analysis of compounds(1, 5d, 5e and 5f) at 8 and 16
205 cells for 24 h. Cell cycle distribution was conducted in three independent
experiments. Average and standard deviation was derived.
l
M concentration in Colo-
incorporating isoxazole and triazole moieties at the 2-phenyl group
of 2-phenyl benzothiazole. Further, these new series of compounds
have been tested for their in vitro cytotoxicity against three human
cancer cell lines. Representatives of some biologically important
phenylbenzothiazole compounds have been illustrated in Figure 1.
The synthesis of 2-(20-hydroxyphenyl) benzothiazole 6 was car-
ried out from the compounds thiophenol and 2-hydroxybenzalde-
hyde which were prepared by the literature method.21 Compound
Since compounds have shown apoptotic cell death at 16 lM con-
centration, the role of caspases and PARP was examined using
the effective compound of the series (5d). The positive control used
in this study is 1. Colo-205 cells were treated with compounds 1
and 5d at 8 and 16 lM for 24 h. The cell lysates was isolated and
Western blot analysis was carried out using antibodies against pro-
caspase-8, procaspase-9, active caspase-3 and cleaved PARP. It was
Figure 3. Effect of phenylbenzothiazole compounds on caspase and PARP proteins. The colo-205 cells were treated with 1, 5d compounds at 8 and 16
24 h. The cell lysates extracted were subjected to Western blot analysis using antibodies against procaspases-8, 9, active caspase-3 and active PARP (cleaved) proteins. b-Actin
was used as loading control. Compound 1 indicates the positive control. Here 16 M concentration was highly effective.
lM concentration for
l

R. M. Kumbhare et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5424–5427
5427
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Acknowledgements
We are thankful to Dr. J. S. Yadav, Director, IICT for providing
facilities, U.K. thank for CSIR, and T.D. thank for UGC, New Delhi,
for the award of a fellowship, and R.M.K thanks S.E.R.C., Depart-
ment of Science & Technology, Government of India for financial
assistance under the Fast Track Scheme for young scientists (SR/
FTP/CS-93/2006).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.041.
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