Synthesis and biological evaluation of combretastatin-amidobenzothiazole conjugates as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.08.021

A series of combretastatin-amidobenzothiazole conjugates have been synthesized and evaluated for their anticancer activity. All these compounds exhibited significant anticancer activity and the most potent compound (11a) showed GI₅₀ values ranging 0.019-11 μM. Biological studies such as cell cycle distribution, effect on tubulin polymerization and effect on ERK signalling pathway have been examined in MCF-7 cell line. FACS analysis revealed that these compounds induced cell cycle arrest at G2/M phase. Compound 11a showed significant effect on tubulin polymerization and affected the ERK signalling pathway that result in the decreased levels of ERK1/2, p-ERK and c-Jun proteins. Docking experiments have shown that the active molecules interact and bind well in the ATP binding pocket of ERK protein.

Full text of DOI:10.1016/j.ejmech.2012.08.021

European Journal of Medicinal Chemistry 56 (2012) 166e178
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of combretastatin-amidobenzothiazole
conjugates as potential anticancer agents
,
a
b
b
a
Ahmed Kamal a , Adla Mallareddy , M. Janaki Ramaiah , S.N.C.V.L. Pushpavalli , Paidakula Suresh ,
*
Chandan Kishor b, J.N.S.R.C. Murty a, N. Sankara Rao a, Sowjanya Ghosh a, Anthony Addlagatta b
,
,
*
Manika Pal-Bhadra b
,
*
a Division of Organic Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of combretastatin-amidobenzothiazole conjugates have been synthesized and evaluated for their
Received 29 March 2012
Received in revised form
6 July 2012
Accepted 15 August 2012
Available online 23 August 2012
anticancer activity. All these compounds exhibited significant anticancer activity and the most potent
compound (11a) showed GI50 values ranging 0.019e11
mM. Biological studies such as cell cycle
distribution, effect on tubulin polymerization and effect on ERK signalling pathway have been
examined in MCF-7 cell line. FACS analysis revealed that these compounds induced cell cycle arrest at
G2/M phase. Compound 11a showed significant effect on tubulin polymerization and affected the ERK
signalling pathway that result in the decreased levels of ERK1/2, p-ERK and c-Jun proteins. Docking
experiments have shown that the active molecules interact and bind well in the ATP binding pocket of
ERK protein.
Keywords:
CA-4
Cell cycle
Anticancer activity
ERK
Ó 2012 Elsevier Masson SAS. All rights reserved.
Tubulin
Molecular modeling
1. Introduction
proliferative disorders cause abnormal phosphorylation of ERK and
MAPK [5,6]. The ERK signalling as well as c-Jun are known to be the
Mitogen-activated protein kinases (MAPKs) transduce extra-
cellular stimuli from the cell surface to the nucleus, leading to
alterations in gene expression and cell functions. To date, three
major subfamilies of MAPKs have been identified: c-Jun N-terminal
kinases/stress-activated protein kinases (JNK/SAPKs), extracellular
signal-regulated kinases (ERK1/2 or p42/44 MAPK) and p38 MAPK
[1]. The extracellular signal-regulated kinase (ERK) signalling
pathway controls proliferation, differentiation and cell survival
processes [2e4]. ERK1/2 is primarily activated (phosphorylated) by
mitogens (MEK1/2) and growth factors and plays a key role in
transmission of proliferative signals in mammalian cells. Activated
ERK1/2 then phosphorylate serine/threonine residues of more than
50 downstream cytosolic and nuclear substrates, leading to alter-
ations in gene expression profiles that are implicated in prolifera-
tion, differentiation and cell survival [2e4]. Tyrosine kinase
mediated signalling, which exists in cancers as well as other
key molecules that regulate breast cancer cell migration [7]. This
prompted us to design and synthesize molecules based on com-
bretastatin scaffold that are likely to mediate the above pathways
and could generate agents useful towards the treatment of partic-
ularly breast cancers.
Combretastatin A-4 (2) which is isolated from the bark of the
South African Cape Bush willow (Combretum caffrum), exhibits
potent anticancer activity against a number of human cancer cell
lines [8] (Fig. 1). It acts as a microtubule destabilizing agent by
binding to the tubulin at the site similar to that of colchicine (1) [9].
A water-soluble prodrug, combretastatin A-4-phosphate (3) is now
in clinical trials for thyroid cancer [10e12] and in patients with
advanced cancer [13]. Interestingly, the cis form of 2 alone is bio-
logically active, with the trans form showing little or no activity
[14]. Various structural modifications to CA-4 have been reported
including variation of the A- and B-ring substituents [15e17]. It has
been observed that most of the modifications of the B-ring result
in decreased bioactivity; however, substitution of the 30-OH with
an amino group results in the enhancement of bioactivity and
better water solubility [18]. This amino derivative of CA-4 (4) is
* Corresponding authors. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in
(M. Pal-Bhadra).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.021

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
167
MeO
MeO
the presence of K2CO3 gives ester 9 followed by its hydrolysis with
LiOH.H2O to afford the acid 10. The synthesis of amidobenzothia-
zole analogues of combretastatin A-4 (11aek) was achieved by
the formation of amide bond between this acid (10) and a variety
of substituted 2-aminobenzothiazoles by using EDCI/HOBt.
Similarly, other analogues (12a,b) were synthesized using 6-
aminobenzothiazoles as shown in Scheme 1.
MeO
MeO
NHCOCH3
O
OMe
OMe
R
OMe
OMe
1
2 : R = OH, combretastatin A-4
3 : R = OPO3Na2, combretastatin A-4P
4 : R = NH2
5 : R = NH-CO-CH-(NH2)(CH2OH), AVE8062
2.2. Biological evaluation
MeO
MeO
MeO
2.2.1. Anticancer activity
MeO
The synthesized compounds were first evaluated for their
anticancer activity against selected human cancer cell lines of lung,
colon, leukaemia, CNS, breast, liver and prostate by using sulfo-
rhodamine B (SRB) method [36]. The results are expressed in % of
H
N
H
OMe
OMe
N
S
S
N
O
O
R
N
O
O
OMe
11a-k
OMe
R
12a-b
growth inhibition values at concentration of 10 mM as shown in
Fig. 1. Chemical structures of colchicine (1), combretastatins (2e5) and
combretastatin-benzothiazole conjugates (11aek and 12aeb).
Table 1. The compounds exhibiting % of growth inhibition values
>50 are considered to be active on the respective cell lines. It is
observed that all the compounds exhibit significant activity with
>50% of growth inhibition values. Amongst the nine compounds
synthesized, six compounds were evaluated under the 60 cell line
screening program by the National Cancer Institute (NCI), Bethesda,
USA. After preliminary screening on the tumour cell lines, these
compounds were tested for five dose concentration on a panel of 59
human tumour cell lines derived from nine different cancer types:
leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and
breast. The results expressed as GI50 values [37] for the test
compounds are illustrated in Table 2. All the compounds exhibited
potential anticancer activity with GI50 values ranging from 0.019 to
also in clinical trials as a water-soluble amino acid prodrug (5) [19].
The 3,4,5-trimethoxy substituted pattern in ring A, resembling the
trimethoxyaryl ring of colchicine, is considered optimal for the
bioactivity of CA-4 [20]. Many conformationally restricted
analogues of CA-4 have been reported, wherein the cis-double
bond has been replaced by a heterocyclic rigid ring scaffold
structure. Being a potent inhibitor of colchicine binding, CA-4 has
also shown to inhibit the growth and development of blood vessels
[21e24]. It is also demonstrated that mitogen-activated protein
kinases are involved in the cytotoxicity of CA-4 [25]. Benzothia-
zoles constitute an important scaffold of drugs, possessing several
pharmacological functions [26,27], and exhibit potent and
intriguing antitumor properties [28e30]. It is reported that some
of the 6-substituted 2-phenylbenzothiazole derivatives have
shown good anticancer activity [31,32]. Apart from this, some
benzothiazole derivatives as sub structure have shown to inhibit
ERK phosphorylation [33].
It was reported [25] that the cytotoxicity of CA-4 also caused by
affecting MAPK family of proteins and benzothiazole derivatives
exerts inhibitory activity on ERK phosphorylation [33]. The
encouraging anticancer profile of combretastatin and benzothia-
zole scaffolds and their effect on MAP kinases led us to synthesize
new conjugates of combretastatin. Herein we report the synthesis
of combretastatin-benzothiazole conjugates fused with amide
bond and evaluation of their anticancer potential. The synthesized
compounds were also evaluated for their effect on tubulin poly-
merization and ERK signalling. Moreover, the ERK inhibition
exhibited by selective compounds is rationalized by molecular
docking investigations.
18.6
mM. It is observed that the anticancer activity for these
combretastatin congeners is dependent on the nature of
substituent present on the benzothiazole ring system. Amongst
all the compounds tested for five dose studies, compound 11a
which does not have a substituent on the benzene ring of
benzothiazole showed potent anticancer activity as compared to
the compounds that had different substituent on 6-position of
benzothiazole ring. The anticancer activity (GI50) of this potential
compound 11a was in the range of 0.019e11 mM. This compound
exhibited remarkable growth inhibition (19 and 51 nM) towards
MDA-MB-435 and MCF-7 cell lines, respectively. This most active
compound 11a showed nano molar level activity against twenty
three cancer cell lines and sub-micro molar level activity in twenty
two cell lines. The other compounds showed micro molar level
activity except one cell line in case of 11b and 12b which showed
sub-micro molar activity against MDA-MB-435 and MCF-7 cell
lines, respectively. It is observed that compound 11b with 6-
fluorobenzothiazole moiety showed better anticancer activity
(0.33 mM) against MDA-MB-435 melanoma human cancer cell line
in comparison to the other compounds (11eeg) that have methoxy,
trifluoromethoxy and trifluoromethyl substitutions on the benzo-
thiazole moiety. The analogue 12b that has 6-amidobenzothiazole
group also exhibited promising anticancer activity (GI50, 0.74e
2. Results and discussion
2.1. Chemistry
9.41 mM) and showed activity of 0.74 mM against MCF-7 cell line.
The synthesis of new combretastatin-amidobenzothiazole
conjugates is shown in Scheme 1 from CA-4. The synthesis of
natural product CA-4 is shown in Scheme 1 and compound 7 was
prepared according to the reported procedure [34,35]. Compound 7
on Wittig reaction conditions at 0 ꢀC with trimethoxybenzaldehyde
gives geometrical isomers (8a and 8b) in the ratio of 3:2. The cis and
trans isomers were separated by flash column chromatography by
using 5% ethyl acetate in hexane as eluent. The conversion of trans
isomer to cis isomer was achieved in good yield under photo-
chemical conditions [34]. The cis isomer 8a upon treatment with
TBAF undergoes deprotection to afford the compound 2, as shown
Significant anticancer activity showed by these compounds
prompted us to evaluate the cell viability on MCF-7 cells. The MCF-7
cells were treated with compounds 11a, 11b, 11e, 11f, 11g, 11h, 11i,
11j,11k,12b and starting materials CA-4 and 2-aminobenzothiazole
(BT) at 4 mM concentration for 24 h. The viable cells after compound
treatment were assayed by MTT based cell viability studies. It was
observed that the compounds 11a and 11b showed slightly better
activity as compared to other compounds (Fig. 2).
2.2.2. Effect of compounds on cell cycle
It is well established that CA-4 shows G2/M arrest whereas
benzothiazoles cause G1 cell cycle arrest [38]. In order to
in Scheme 1. CA-4 upon etherification with a-bromoethyl acetate in

168
A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
OTBDMS
OMe
CHO
CH2PPh3Br
MeO
MeO
MeO
MeO
MeO
i
+
OMe
+
MeO
OTBDMS
OMe
OMe
OMe
OTBDMS
6
OMe
7
8a
8b : trans
8a : cis
ii
MeO
MeO
MeO
MeO
iii
iv
8a
OMe
OMe
OEt
OH
O
9
OMe
O
O
O
OMe
2
v
MeO
MeO
MeO
MeO
vi
H
N
OMe
S
O
OMe
OH
O
OMe
O
N
10
OMe
vii
R
11a-g
11a: R = H
11b: R = 6-fluoro
11c: R = 6-chloro
11d: R = 6-nitro
MeO
MeO
11e: R = 6-methoxy
H
N
11f: R = 6-trifluoromethoxy
11g: R = 6-trofluoromethyl
11h: R = 6-methyl
OMe
S
R
N
O
OMe
11i: R = 6-ethoxy
12a-b
11j: R = 4-methyl
11k: R =5,6-di methyl
12a: R = H
12b: R = Me
Scheme 1. Reagents and conditions: (i) NaH, CH2Cl2, 0 ꢀC, 14 h, 40%; (ii) EtOH, sunlight, 6e7 h, 72%; (iii) TBAF, THF, 0 ꢀC, 20 min, 79%; (iv) 2-bromoethyl acetate, K2CO3, DMF, 24 h,
94%; (v) LiOH, THF, H2O, 12 h, 80%; (vi) 2-aminobenzothiazoles, EDCI, HOBt, CH2Cl2, 24 h, 83e93%; (vi) 6-aminobenzothiazoles, EDCI, HOBt, CH2Cl2 24 h, 88e89%.
Table 2
Anticancer activity (GI50
the NCI human cancer cell lines.
a
of compounds 11a, 11b, 11e, 11f, 11g, 12b and 2b against
Table 1
)
Percentage of growth inhibitiona for selected cancer cell lines by combretastatine
benzothiazole conjugates at 10 mM concentration.
Panel/cell line
11ac
11bd
11ee
11ff
11gg
12bh
2
Comp
A549b
Colo205c
THP-1d
IMR32e
MCF-7f
Hep-2g
PC-3h
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
11a
11b
11c
11d
11e
11f
11g
11h
11i
44
44
naj
50
45
40
30
37
27
na
41
na
na
52
55
60
50
17
46
56
41
25
33
45
24
51
11
17
26
63
na
54
70
80
72
66
29
27
37
19
76
41
59
83
87
91
77
51
88
73
44
23
na
54
34
55
46
62
73
89
72
56
21
69
53
59
62
57
35
29
68
19
52
45
71
na
64
47
na
71
78
76
44
49
na
23
20
40
56
64
44
32
41
47
33
34
42
22
37
24
36
58
61
75
76
0.078
0.042
0.34
0.15
0.037
3.70
3.62
3.07
6.36
2.19
3.87
3.6
3.87
5.43
3.69
4.12
3.43
4.21
5.83
3.55
4.27
3.3
3.75
5.48
3.35
3.41
2.19
2.11
1.98
2.93
0.002
0.002
0.003
0.003
0.003
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0.34
0.038
nti
0.039
0.27
0.11
nt
5.99
5.72
5.70
5.73
5.60
4.26
naj
5.19
5.11
23
6.28
4.51
3.91
na
6
4.46
6.25
5.22
2.88
3.17
3.9
5.38
3.55
2.89
2.1
0.015
0.079
0.002
0.002
0.003
0.003
0.003
0.007
0.001
7.89
7.09
2.74
4.3
4.39
na
4.05
3.09
2.38
2.75
2.53
6.74
3.36
2.28
11j
11k
12a
12b
ADRi
CA-4
0.052
0.079
4.36
2.92
3.73
3.09
4.11
3.16
a
Inhibition of cell proliferation after an incubation time of 48 h.
Lung cancer cell line.
Colon cancer cell line.
Leukemia cancer cell line.
Neuro cancer cell line.
Breast cancer cell line.
Liver cancer cell line.
Prostate cancer cell line.
Adriamycin.
b
c
d
e
f
Colon
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
2.83
0.37
0.046
0.048
3.16
7.78
na
4.48
8.19
9.63
4.78
3.89
7.34
3.7
2.89
3.34
3.12
43.3
17.1
3.92
3.23
na
17.1
4.23
3.29
3.24
14.9
3.14
4.4
6.9
3.57
2.8
3.31
3.79
0.1
0.063
0.003
0.003
0.1
g
h
i
KM12
0.058
4.48
0.005
SW-620
nt
na
5.1
5.65
4.65
4.7
0.003
j
Not active.

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
169
Table 2 (continued )
Panel/cell line
11ac
11bd
11ee
11ff
11gg
12bh
2
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.18
0.22
0.045
nt
0.046
0.048
7.23
4.29
4.51
na
4.39
4.33
4.31
1.84
2.84
6.3
1.91
4
6.01
3.7
2.28
7.08
1.56
3.37
3.85
3.36
2.66
5.03
1.58
3.67
3.61
2.45
2.56
3.89
1.55
3.59
0.006
0.003
0.002
0.003
0.007
0.007
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.067
1.99
0.072
0.019
0.18
nt
0.036
11
0.063
3.23
5.22
6.82
0.33
8.46
na
1.76
8.69
4.05
4.17
na
4.77
nt
4.01
na
3.58
1.84
3.98
3
2.84
40
3.9
2.64
2.84
3.38
2.13
3.15
3.22
1.97
1.73
2.19
0.003
0.019
0.002
0.001
0.003
0.007
0.003
0.003
0.005
4.33
1.16
4.07
4.41
2.68
63
4.36
1.93
3.29
4.57
3.26
na
5.93
5.11
Fig. 2. Effect of combretastatin-benzothiazole conjugates on cell viability. MCF-7 cells
were treated with compounds (11a, 11b, 11e, 11f, 11g, 11h, 11i, 11j, 11k and 12b) at 4 mM
Ovarian
IGROV1
concentration for 24 h in 96 well plates at 10,000 cells per well followed by MTT assay.
Here the O.D directly indicates the cell viability after compound treatment. CA-4 was
used as standard control. Control indicates the untreated cells. BT indicates 2-
aminobenzothiazole.
0.21
0.043
0.37
nt
0.25
0.033
0.34
4.17
9.99
7.30
na
7.15
2.55
8
7
5.64
3.37
5.34
3.12
5
4.48
2.11
4.66
5.98
3.86
2.12
3.29
3.72
6.32
2.64
6.69
2.12
2.36
5.27
0.015
0.001
0.015
0.1
0.003
0.001
0.063
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
2.17
5.31
5.89
5.28
2.3
2.7
8.42
4.69
11k. It is obvious that the variation in cell cycle arrest depends on
the nature of compound as well as response of the cell type towards
the specific compound of interest.
Renal
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
7.3
7.12
6.25
3.33
2.78
6.29
4.61
9.89
2.56
7.52
3.5
7.77
3.2
na
6.96
2.81
4.82
3.1
2.27
4.43
7.03
3.59
7.55
4.18
3.12
2.19
5.06
3.53
4.21
2.41
0.1
0.36
0.093
0.41
0.05
0.37
nt
3.61
8.31
4.6
2.65
6.42
18.6
4.37
0.006
0.006
0.025
0.002
0.006
0.1
2.2.3. Effect of compound 11a on tubulin polymerization
In General G2/M cell cycle arrest is strongly associated with
inhibition of tubulin polymerization [39] and compound 11a is
most effective in causing G2/M cell cycle arrest, it was considered of
interest to understand the microtubule inhibitory function of 11a.
To evaluate inhibitory activity of compound 11a on tubulin poly-
merization, we have employed the HTS tubulin polymerization
assay, where inhibition was shown as decrease in optical density
(O.D). Here tubulin is dissolved in G-PEM buffer and incubated with
compounds (11a, CA-4, Noc, and Pac), and readings were taken by
photometry based method at a wavelength of 340 nm. It was
2.5
5.75
7.54
4.31
0.58
0.019
Prostate
PC-3
DU-145
0.18
0.1
6.74
8.97
4.45
2.04
5.12
4.11
4.32
2.51
2.8
3.81
0.001
0.001
Breast
MCF7
MDA-MB-231
HS 578T
BT-549
0.051
0.19
nt
0.41
1.0
2.3
9.34
na
16.7
3.39
3.68
2.88
5.4
4.15
21.2
4.03
2.29
2.89
5.16
4.33
10.5
3.95
2.3
3.16
3.38
4.18
5.23
4.79
2.16
0.74
5.45
4.32
3.05
9.41
2.39
0.005
0.001
0.001
10
10
na
observed that compound 11a showed good tubulin inhibition (IC50
,
5
mM) that is comparable to CA-4. Here paclitaxel (Pac) was used as
T-47D
MDA-MB-468
0.1
tubulin polymerization stabilizer (Fig. 4). Additionally, immuno-
fluoresence analysis against tubulin was also examined for
compound 11a in MCF-7 cells. Therefore, the cells were treated
a
Values are reported as GI50, the micro molar concentration of the compound
required to cause 50% inhibition of cell growth after an incubation time of 48 h.
b
NSC 613729 (http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp).
NSC 753574.
NSC 753576.
NSC 753578.
NSC 753581.
NSC 753584.
NSC 753587.
with
CA-4,
nocodozole
(Noc),
paclitaxel
(Pac),
2-
c
aminobenzothiazole (BT) and 11a at 4
mM concentration for 24 h.
d
e
Compound 11a has shown dispersed microtubule organization
when compared to control (untreated cells). Nocodazole, a tubulin
polymerization inhibitor and paclitaxel, a tubulin stabilizer were
employed as negative and positive controls respectively (Fig. 5).
f
g
h
i
nt ¼ not tested.
j
na ¼ not active.
2.2.4. Effect of conjugates on c-Jun and ERK
ERK protein was found to be associate with kinetochore of
mitotic spindles [40e44] and is needed for Mitotic Progression
[45,46]. Previous studies [47,48] have shown that microtubule
inhibitors cause specific as well as distinct effects on ERK pathway
and combined effect leads to cause effective cell death [49]. c-Jun is
a component of the activator protein (AP-1) and is associated with
angiogenesis that is dispensable for the invasiveness in breast
cancer [50]. Whereas ERK1/2 are the members of MAPK family
proteins that control the cell division and are considered to provide
survival signals which ultimately end in increased proliferation rate
of the tumour [51]. Thus these proteins (ERK and c-Jun) contribute
to metastasis in breast cancer [7]. Thus it was considered of interest
to understand the molecular mechanism behind the cytotoxic
nature of these conjugates. In this pursuit, MCF-7 cells were treated
understand the effect of these conjugates on cell cycle, we have
performed FACS analysis. In this study, MCF-7 cells were treated
with compounds 11a, 11b, 11e, 11f, 11g, 11h, 11i, 11j, 11k, 12b, CA-4
and BT for 24 h at 4 mM concentration. CA-4 and BT compounds
serves as starting materials. Compounds 11a, 11b, 11g, 11h, 11i and
12b have shown 46%, 42%, 37%, 43%, 38% and 37% of cell accumu-
lation in G2/M phase (G2/M arrest). Whereas 11e, 11f, 11j and 11k
exhibit 63%, 59%, 68% and 80% of cell accumulations in G1 phase
respectively (G1 arrest). In control (untreated cells) 58% and 35% of
accumulation in G1 and G2/M phases were observed, respectively
(Fig. 3). This data clearly indicated that majority of the compounds
showed G2/M cell cycle arrest with the exception of 11e,11f,11j and

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A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
Fig. 3. FACS analysis of cell cycle distribution of MCF-7 cells after treatment with combretastatin-benzothiazole conjugates at 4
mM concentration for 24 h. CA-4 and BT
(2-aminobenzothiazole) are the precursor molecules employed. Compounds 11a, 11b, 11g, 11h, 11i and 12b show G2/M arrest whereas compounds 11e, 11f, 11j and 11k show G1
arrest.

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
171
The trimethoxy phenyl ring of these compounds is surrounded
by Cys241, Lys254, Leu255, Asn258, Leu248, Val318, Ala354 and
Ile378. The Cys241 forms a hydrogen bond (SeHeO) with one of
the methoxy oxygen atoms of trimethoxyphenyl ring of 11a or
11b. The benzothiazole amide moiety of both compounds move
towards interface of
amide forms a series of hydrogen bonds with Ser178 and Tyr224
of -chain of tubulin while nitrogen atom of amide also partici-
pates in hydrogen bond with main chain of Gln247 of
-chain. Based on the modeling, it is clear that these molecules fit
aeb chain of tubulin. The keto group of
a
b
well in the tubulin
interactions.
aeb interface thereby allowing favorable
3. Conclusion
Fig. 4. Inhibitory activity of 11a on tubulin polymerization. In vitro HTS tubulin
polymerization assay was carried out using 4 M concentration of compounds CA-4,
nocadazole, paclitaxel and 11a. Compounds were incubated with 100 l of tubulin
(dissolved in GPEM buffer) provided in the kit. The O.D was taken at 340 nm and is
measure for the tubulin inhibitory activity.
m
In the present study,
amidobenzothiazole conjugates
compounds (11a, 11b, 11e, 11f, 11g and 12b) showed potent anti-
cancer activity against different human cancer cell lines. The cell
viability assay of the two promising compounds 11a and 11b have
a
new series of combretastatin-
were synthesized. The
m
with compounds CA-4, BT, 11a and 11b at 4 mM for 24 h followed by
shown potent cytotoxicity in MCF-7 cells at 4
mM. The FACS
Western blot analysis. It was observed that there was a decrease in
the level of ERK and c-Jun proteins in compound treated cells
(Fig. 6).
analysis of these compounds caused accumulation of cells in G2/M
phase. It was observed from the results of biological assays that
the active compounds have showed significant effect on tubulin
polymerization as well as ERK signaling pathway. The c-Jun and
ERK1/2 protein levels decreased upon treatment with compounds
11a and 11b in MCF-7 cells and more pronounced in case of 11a.
Interestingly, these compounds caused the decrease in phos-
phorylated forms of ERK1/2 protein and also prevented phospho-
ERK to enter the nucleus which resulted in retention of this
phospho-ERK in cytoplasm, thereby controlling the abnormal cell
proliferation [52]. Moreover these conjugates also exhibited
inhibitory activity on c-Jun protein. From the studies of biological
assays, it may conclude that the significant anticancer activity
exhibited by this compound is combined effect of both tubulin
polymerization inhibition and ERK inhibition. From molecular
modeling studies it was demonstrated that these molecules bound
well in the ATP binding pocket and were involved in a series of
interactions with the protein providing the molecular basis for
inhibition of ERK. The modeling study on tubulin demonstrated
that these molecules bind well with the tubulin and are involved
in a series of interactions with the protein. Thus these conjugates
could be considered as potential leads in the development of
a new class of anticancer therapeutics [53].
Further, the effect of these compounds on phosphorylated
(activated) form of ERK1/2 was examined, which up on entry in to
the nucleus phosphorylate many proteins that cause rapid prolif-
eration [2e4]. In this study as well, we found a decrease in
phosphorylated form of ERK1/2 (Fig. 6). Additionally, immunoflu-
orescence analysis against phosphorylated ERK1/2 in 11a treated
MCF-7 cells was also examined. It was surprising to observe that
the retention of phosphorylated ERK1/2 form in the cytoplasm as
shown in Fig. 7 revealed the inhibitory role of 11a on ERK pathway.
2.2.5. Molecular modeling
To determine the possible binding modes of this series of
compounds with ERK kinase, docking studies were carried out
with the most potent compounds. These conjugates were docked
in ATP binding pocket of ERK2. Biological activity studies sug-
gested that compounds 11a and 11b were potent, hence we
examined their interaction in ATP binding pocket of ERK2 (Fig. 8).
Docking followed by molecular dynamics of the ATP binding
pocket of ERK2 suggested that benzothiazole moiety was buried in
the gorge of the ATP binding pocket forming several hydrophobic
interactions with surrounding residues such as Val39, Ala52,
Leu107, Met108, Leu156 and a polar interaction with Gln105. The
combretastatin moiety also gets well tugged in the ATP binding
pocket and provided a series of polar interactions with several
residues that included Arg67, Lys54 and Cys166, while residues
Tyr113 and Lys114 were involved in hydrophobic interactions.
Interestingly, the interactions with gate keeper residues Val39,
Lys54 and Gln105 were considered to be critical for the inhibitor
4. Experimental section
All chemicals and reagents were obtained from Aldrich (Sigmae
Aldrich, 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
60GF-254, and visualization on TLC was achieved by UV light or
iodine indicator. Column chromatography was performed with
Merck 60e120 mesh silica gel. 1H NMR and 13C NMR spectra were
recorded on Gemini Varian-VXR-unity (200 MHz) or Bruker
ꢀ
selectivity. During molecular dynamics, 1.5e2.0 A movement was
observed in the loop near Cys166 towards the inhibitor, while
ꢀ
region around Tyr36 moved away by 2.5e3.0 A. Tight binding of
benzothiazole moiety deep inside the ATP binding pocket sug-
gested that any small change in the substituent could prove
sensitive towards the inhibition of the enzyme. This modeling data
provides a basis for possible interaction between the ERK and
inhibitors.
The modeling studies on tubulin protein were carried out to
evaluate binding modes in order to support the inhibition of
tubulin polymerization. Compounds (11a and 11b) were directly
docked in the colchicine binding site of tubulin and the proposed
UXNMR/XWIN-NMR (300 MHz) instruments. 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 positive
ion trap detector. High-resolution mass spectra (HRMS) were
recorded on QSTAR XL Hybrid MS/MS mass spectrometer. Melting
points were determined with an Electro thermal melting point
apparatus, and are uncorrected.
binding sites located at the interface with the
a-b subunit (Fig. 9).

172
A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
Fig. 5. Effect of compound on the microtubule network: MCF-7 cells were treated with compound 11a, combretastatin (CA-4), 2-aminobenzothiazole (BT), nocodazole (Noc), and
paclitaxel (Pac) at 4 M concentration for 24 h. This is followed by staining with -tubulin antibody. Microtubule organization was clearly observed by green colored tubulin
m
a
network like structures in control cells and was found to be disrupted in nocodazole and 11a treated cells. Paclitaxel works as microtubule stabilization (i.e. bundling of tubulin). BT
did not show any effect on tubulin polymerization and acted like control untreated cells. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
173
Fig. 8. Molecular modeling of 11a (yellow) and 11b (green) in complex with ERK2. A).
Overall structure is shown in grey cartoon diagram while, compounds and the
surrounding residues are displayed in sticks. B). Represented MD trajectory snapshot of
ERK2-11a complex. C) Represented MD trajectory snapshot of ERK2-11b complex. For
the sake of clarity all amino acids.
4.1. (Z)-tert-butyl(2-methoxy-5-(3,4,5-rimethoxystyryl)phenoxy)
dimethylsilane (8a)
Fig. 6. Compounds 11a and 11b inhibit c-Jun, ERK and ERK-p (phosphorylated ERK)
protein expression. Here MCF-7 cells were treated with compounds CA-4, BT
(2-aminobenzothiazole), 11a and 11b at 4 mM concentration for 24 h before subjected
to Western blot analysis.
To a solution of compound 7 (593 mg, 1.0 mmol) and trime-
thoxybenzaldehyde (196 mg, 1.0 mmol) in dry dichloromethane
(15 mL) was cooled to 0 ꢀC and added NaH (29 mg, 1.2 mmol). The
reaction mixture was stirred for 14 h at room temperature. The
reaction was monitored by TLC using ethyl acetate and hexane.
After completion of the reaction, the reaction mixture was
Fig. 7. Compound 11a inhibits the nuclear translocation of phosphorylated form of ERK. MCF-7 cells were treated with 4
to immunofluorescence procedure using phospho-ERK antibody.
mM concentration of compound 11a for 24 h and subjected

174
A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
Fig. 9. (A) Docking position of 11a (yellow) in the colchicine-binding site of tubulin; (B) Docking position of 11b (green) in the colchicine-binding site of tubulin. Blue cartoon
represent -chain of tubulin while orange represent -chain of tubulin. Red dotted lines represent possible hydrogen bonds between protein and ligands. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
a
b
quenched with ice water and extracted with ethyl acetate
(2 ꢁ 20 ml). The combined organic phases were washed with
water followed by brine solution, dried over Na2SO4 and evapo-
rated under vacuum to afford crude product. The crude product
was a mixture of Z- and E- isomers. The Z- isomer was separated
by flash column chromatography using hexane:ethyl acetate
(19:1) as a solvent system to obtain the pure product 8a (120 mg,
40% yield) as viscous liquid followed by compound 8b (241 mg,
60% yield) as white solid. Compound 8a: 1H NMR (200 MHz,
(200 MHz, CDCl3):
d
¼ 6.92 (d, 1H, J ¼ 2.2 Hz), 6.77e6.83 (dd, 1H,
J ¼ 8.3, 1.5 Hz), 6.73 (d, 1H, J ¼ 8.3 Hz), 6.53 (s, 2H), 6.47 (d, 1H,
J ¼ 12.0 Hz), 6.41 (d, 1H, J ¼ 12.0 Hz), 5.57 (br s, 1H), 3.86 (s, 3H),
3.84 (s, 3H), 3.7 (s, 6H); MS (ESI): m/z 317 [Mþ1]þ.
4.4. (Z)-ethyl-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)
acetate (9)
To a solution of compound 2 (316 mg, 1.0 mmol) in dry DMF
(10 mL) was added anhydrous K2CO3 (276 mg, 2.0 mmol) and
CDCl3):
d
¼ 6.76e6.82 (dd, 1H, J ¼ 8.3, 2.2 Hz), 6.73 (d, 1H,
J ¼ 2.2 Hz), 6.69 (d, 1H, J ¼ 8.3 Hz), 6.39e6.46 (m, 3H), 6.36 (d, 1H,
J ¼ 12.0 Hz), 3.79 (s, 3H), 3.77 (s, 3H), 3.69 (s, 6H), 0.96 (s, 9H), 0.14
(s, 6H); MS (ESI): m/z 431 [Mþ1]þ; Compound 8b: 1H NMR
a-bromoethyl acetate (250 mg, 1.5 mmol) and the mixture was
stirred at room temperature for 24 h. The reaction was monitored
by TLC using ethyl acetate:hexane (6:4). After completion of the
reaction as indicated by the TLC, K2CO3 was removed by filtration,
diluted with water and extracted with dichloromethane
(2 ꢁ 20 ml). The combined organic phases were washed with water
followed by brine solution, dried over Na2SO4 and evaporated
under vacuum. The residue, thus obtained was purified by column
chromatography using ethyl acetate and hexane (7:3) to afford pure
compound 9 (380 mg, 94% yield); 1H NMR (300 MHz, CDCl3):
(200 MHz, CDCl3):
d
¼ 6.71 (s, 2H), 6.91 (d, 1H, J ¼ 15.8 Hz), 6.85 (d,
1H, J ¼ 15.8 Hz), 6.83 (d, 1H, J ¼ 8.3 Hz), 7.02e7.09 (m, 2H), 3.91 (s,
6H), 3.87 (s, 3H), 3.82 (s, 3H), 1.03 (s, 9H), 0.19 (s, 6H); MS (ESI): m/
z 431 [Mþ1]þ.
4.2. Trans to cis isomerization of compound (8b) [34]
The trans compound 8b (2 g) was dissolved in ethanol (1 L). The
solution was kept under sunlight for 6e7 h. After completion of the
reaction as indicated by TLC, ethanol was distilled under reduced
pressure and purified by flash column chromatography using
hexane:ethyl acetate (19:1) as a solvent system to obtain the pure
cis isomer 8a as viscous liquid (1.44 g, 72% yield).
d
¼ 6.83e6.89 (dd, 1H, J ¼ 8.3, 1.5 Hz), 6.75 (d, 1H, J ¼ 8.3 Hz), 6.7 (d,
1H, J ¼ 1.5 Hz), 6.4e6.46 (m, 3H), 6.47 (d, 1H, J ¼ 12.0 Hz), 4.44 (s,
2H), 4.11e4.20 (q, 2H), 3.85 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H), 1.24 (t,
3H, J ¼ 7.5, 6.7 Hz); MS (ESI): m/z 403 [Mþ1]þ.
4.5. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)acetic
acid (10)
4.3. (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol (2)
To a solution of compound 9 (402 mg, 1.0 mmol) in THF (10 ml)
and water (2 ml), LiOH.H2O (84 mg, 2.0 mmol) was added, and the
mixture was stirred at room temperature for 12 h. The reaction was
monitored by TLC using ethyl acetate. After completion of the
reaction as indicated by the TLC, the solvent was removed under
vacuum and neutralized with dilute HCl up to pH 7. After neutral-
ization the reaction mixture was extracted with dichloromethane
(2 ꢁ 20 ml). The combined organic phases were washed with water
followed by brine solution, dried over Na2SO4 and evaporated
under vacuum to obtain crude compound 10. This compound was
purified by recrystallization by using ethyl acetate as solvent to
obtain the pure product 10 (302 mg, 80% yield); 1H NMR (200 MHz,
To a solution of compound 8a (430 mg, 1.0 mmol) in dry THF
(15 mL) was added TBAF solution (1.5 ml, 1 M solution in THF)
under cooling conditions and stirred for 20 min at 5e10 ꢀC. The
reaction was monitored by TLC using ethyl acetate:hexane. After
completion of the reaction as indicated by the TLC, the reaction
mixture was quenched with sodium bicarbonate solution and
extracted with ethyl acetate (2 ꢁ 20 ml). The combined organic
phases were washed with water followed by brine solution,
dried over Na2SO4 and evaporated under vacuum to afford
crude product of 2. This was further purified by column
chromatography using hexane:ethyl acetate (2:8) as a solvent
system to obtain the pure product 2 (251 mg, 79% yield); 1H NMR
CDCl3):
d
¼ 7.96 (br s, 1H), 6.84e6.9 (dd, 1H, J ¼ 8.3, 1.5 Hz), 6.77 (d,

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
175
1H, J ¼ 8.3 Hz), 6.72 (d, 1H, J ¼ 1.5 Hz), 6.31e6.49 (m, 4H), 4.47 (s,
2H), 3.86 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H); MS (ESI): m/z 375 [Mþ1]þ.
129.94, 129.72, 128.65, 127.22, 125.03, 121.72, 121.08, 118.10, 111.76,
105.96, 70.21, 60.91, 56.0; IR (KBr) (nmax/cmꢂ1): 3360, 2931, 1714,
1597, 1543, 1511, 1442, 1426, 1325, 1262, 1235, 1124, 1023, 996, 879,
818, 765, 668; MS (ESI): m/z 541 [M]þ; HRMS (ESI m/z) [Mþ1]þ
calcd for C27H26N2O6SCl: 541.1200, found: 541.1224.
4.6. (Z)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3,4,5-
trimethoxy styryl)phenoxy)acetamide (11a)
To a solution of compound 2-aminobenzothiazole (150 mg,
1.0 mmol) in dichloromethane (20 ml) was added 1-(3-Dimethyl
aminopropyl)-3-ethylcarbodiimide (EDCI) (210 mg, 1.1 mmol)
and 1-hydroxy-1,2,3-benzotriazole (HOBt) (13.5 mg, 0.1 mmol).
Then added (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl) phe-
noxy)acetic acid 10 (374 mg, 1.0 mmol) and the reaction mixture
was stirred at room temperature for 24 h and the reaction was
monitored by TLC. After completion of reaction, water was added
to reaction mixture and extracted with dichloromethane
(2 ꢁ 30 ml). The solvent was evaporated under vacuum to afford
the crude product. This was further purified by column chroma-
tography using ethyl acetate and hexane (1:1) as solvent system to
obtain the pure product 11a (449 mg, 88% yield) as white solid;
4.9. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
nitrobenzo[d]thiazol-2-yl) acetamide (11d)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-nitro benzothiazole (195 mg, 1.0 mmol)
to obtain the pure product 11d (500 mg, 90% yield) as yellow solid;
mp: 117e119 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 11.03 (s, 1H), 8.77
(d, 1H, J ¼ 2.1 Hz), 8.32e8.36 (dd, 1H, J ¼ 8.7, 2.1 Hz), 7.88 (d, 1H,
J ¼ 8.7 Hz), 7.03e7.07 (dd,1H, J ¼ 8.0, 2.1 Hz), 6.97 (d, 1H, J ¼ 2.1 Hz),
6.87 (d, 1H, J ¼ 8.0 Hz), 6.52 (d, 1H, J ¼ 12.4 Hz), 6.49 (s, 2H), 6.46 (d,
1H, J ¼ 12.4 Hz), 4.68 (s, 2H), 4.02 (s, 3H), 3.86 (s, 3H), 3.71 (s, 6H); IR
(KBr) (nmax/cmꢂ1): 3353, 2927, 1708, 1575, 1533, 1510, 1446, 1427,
1337, 1265, 1124, 1043, 878, 751, 659; MS (ESI): m/z 552 [Mþ1]þ;
HRMS (ESI m/z) [M þ Na]þ calcd for C27H25N3O8SNa: 574.1260,
found: 574.1279.
mp: 113e115 ꢀC; 1H NMR (200 MHz, CDCl3):
d
¼ 7.74e7.82 (m, 2H),
7.37e7.44 (m, 1H), 7.29 (d, 1H, J ¼ 8.3 Hz), 6.96e7.01 (d, 1H, J ¼ 8.3,
2.2 Hz), 6.93 (d, 1H, J ¼ 2.2 Hz), 6.82 (d, 1H, J ¼ 8.3 Hz), 6.46 (d, 1H,
J ¼ 12.0 Hz), 6.38e4.3 (m, 3H), 4.60 (s, 2H), 4.02 (s, 3H), 3.83 (s,
3H), 3.69 (s, 6H); 13C NMR (75 MHz, CDCl3):
d
¼ 167.54, 156.73,
152.98, 149.06, 148.47, 146.60, 137.29, 132.45, 132.20, 130.43,
129.70, 128.61, 126.19, 124.99, 124.05,121.36, 121.18, 118.14, 111.62,
105.81, 70.40, 60.92, 55.90; IR (KBr) (nmax/cmꢂ1): 3390, 2924, 1696,
1582, 1548, 1508, 1422, 1325, 1261, 1130, 1017, 882, 801, 755; MS
(ESI): m/z 507 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for
C27H27N2O6S: 507.1589, found: 507.1585.
4.10. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
methoxybenzo[d]thiazol-2-yl)acetamide (11e)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-methoxy benzothiazole (180 mg,
1.0 mmol) to obtain the pure product 11e (480 mg, 89% yield) as
4.7. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
fluorobenzo[d]thiazol-2-yl) acetamide (11b)
white solid; mp: 114e117 ꢀC; 1H NMR (300 MHz, CDCl3):
1H, J ¼ 9.0 Hz), 7.30 (d, 1H, J ¼ 2.2 Hz), 6.99e7.1 (m, 2H), 6.94 (d, 1H,
J ¼ 2.2 Hz), 6.84 (d, 1H, J ¼ 8.3 Hz), 6.43e6.53 (m, 4H), 4.63 (s, 2H),
3.98 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H); 13C NMR (75 MHz,
d
¼ 7.71 (d,
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-fluoro benzothiazole (168 mg, 1.0 mmol)
to obtain the pure product 11b (428 mg, 93% yield) as white solid;
CDCl3):
d
¼ 186.55, 176.15, 174.09, 172.20, 168.29, 165.82, 161.62,
156.45, 152.54, 151.72, 149.63, 148.92, 147.88, 144.14, 140.90, 137.24,
134.54, 130.86, 125.04, 123.38, 89.50, 80.16, 75.15, 75.02, 48.87; IR
(KBr) (nmax/cmꢂ1): 3381, 2927, 1688, 1606, 1579, 1550, 1508, 1429,
1325, 1262, 1125, 1059, 879, 820, 784; MS (ESI): m/z 537 [M þ 1]þ;
HRMS (ESI m/z) [Mþ1]þ calcd for C28H29N2O7S: 537.1695, found:
537.1677.
mp: 115e117 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 7.71e7.76 (m, 1H),
7.47e7.53 (dd, 1H, J ¼ 8.1, 2.4 Hz), 7.13e7.2 (m, 1H), 7e7.04 (dd, 1H,
J ¼ 8.1, 1.6 Hz), 6.93 (d, 1H, J ¼ 1.6 Hz), 6.83 (d, 1H, J ¼ 8.1 Hz), 6.46e
6.51 (m, 3H), 6.44 (d, 1H, J ¼ 12.2 Hz), 4.63 (s, 2H), 3.98 (s, 3H), 3.85
(s, 3H), 3.70 (s, 6H); 13C NMR (75 MHz, CDCl3):
d
¼ 167.63, 161.26,
158.04, 156.41, 152.99, 149.04, 146.60, 144.91, 137.24, 132.47, 130.49,
129.75, 128.58, 125.07, 121.99, 118.25, 114.77, 111.66, 107.47, 105.81,
70.46, 60.9, 55.89; IR (KBr) (nmax/cmꢂ1): 3360, 2929, 1712, 1608,
1578, 1553, 1513, 1458, 1261, 1126, 1024, 996, 883, 812, 674; MS
(ESI): m/z 525 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for
C27H26N2O6FS: 525.1495, found: 525.1483.
4.11. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
trifluoromethoxybenzo[d] thiazol-2-yl)acetamide (11f)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl)phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-trifluoromethoxy benzothiazole
(234 mg, 1.0 mmol) to obtain the pure product 11f (550 mg, 93%
yield) as white solid; mp: 122e124 ꢀC; 1H NMR (300 MHz, CDCl3):
4.8. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
chlorobenzo[d]thiazol-2-yl) acetamide (11c)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-chloro benzothiazole (184 mg,
1.0 mmol) to obtain the pure product 11c (502 mg, 92% yield) as
d
¼ 7.8 (d, 1H, J ¼ 8.8 Hz), 7.69 (s, 1H), 7.29e7.36 (dd, 1H, J ¼ 8.4,
2.2 Hz), 7.01e7.06 (dd, 1H, J ¼ 8.4, 1.7 Hz), 6.95 (d, 1H, J ¼ 1.7 Hz),
6.85 (d, 1H, J ¼ 8.3 Hz), 6.42e6.53 (m, 4H), 4.65 (s, 2H), 3.99 (s, 3H),
3.85 (s, 3H), 3.7 (s, 6H); 13C NMR (75 MHz, CDCl3):
d
¼ 167.82,
white solid; mp: 114e117 ꢀC; 1H NMR (200 MHz, CDCl3):
d
¼ 7.8 (d,
157.64, 153.03, 149.08, 147.14, 146.66, 145.5, 137.29, 133.09, 132.5,
130.57, 129.82, 128.59, 125.2, 121.96, 120.16, 118.42, 114.21, 111.70,
105.8, 103.38, 70.59, 60.96, 55.95; IR (KBr) (nmax/cmꢂ1): 3389, 2924,
1697, 1613, 1580, 1551, 1508, 1457, 1261, 1130, 10174, 868, 811, 783,
669, 638; MS (ESI): m/z 591 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd
for C28H26N2O7F3S: 591.1412, found: 591.1384.
1H, J ¼ 2.2 Hz), 7.73 (d, 1H, J ¼ 9 Hz), 7.38e7.44 (dd, 1H, J ¼ 8.3,
2.2 Hz), 7.0e7.06 (dd, 1H, J ¼ 8.3, 2.2 Hz), 6.94 (d, 1H, J ¼ 2.2 Hz),
6.84 (d, 1H, J ¼ 9.0 Hz), 6.42e6.54 (m, 4H), 4.64 (s, 2H), 3.98 (s, 3H),
3.85 (s, 3H), 3.7 (s, 6H); 13C NMR (75 MHz, CDCl3):
d
¼ 167.94,
160.37, 153.0, 149.02, 146.53, 145.93, 137.31, 132.85, 132.43, 130.53,

176
A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
4.12. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
trifluoromethylbenzo[d] thiazol-2-yl)acetamide (11g)
white solid; mp: 113e114 ꢀC; 1H NMR (300 MHz, CDCl3):
7.69 (m, 1H), 7.31e7.44 (m, 2H), 7.08 (d, 1H, J ¼ 8.3 Hz), 6.91 (d, 1H,
J ¼ 2.3 Hz), 6.82 (d, 1H, J ¼ 8.3 Hz), 6.41e6.52 (m, 4H), 4.61 (s, 2H),
3.88 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H), 2.62 (s, 3H); 13C NMR
d
¼ 7.62e
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl)phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-trifluoromethyl benzothiazole
(218 mg, 1.0 mmol) to obtain the pure product 11g (480 mg, 83%
yield) as white solid; mp: 119e122 ꢀC; 1H NMR (200 MHz,
(75 MHz, CDCl3):
d
¼ 186.1, 175.33, 174.23, 171.54, 167.65, 165.44,
161.12, 156.52, 152.97, 151.05, 149.27, 148.45, 147.64, 144.12, 140.87,
137.23, 134.57, 131.05, 125.43, 123.87, 80.97, 75.78, 74.25, 48.56,
27.38; MS (ESI): m/z 521 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for
C28H28N2O6S: 521.1473, found: 521.1454.
CDCl3):
d
¼ 8.12 (s, 1H), 7.93 (d, 1H, J ¼ 8.4 Hz), 7.69e7.75 (m, 1H),
7.01e7.06 (dd, 1H, J ¼ 8.3, 1.7 Hz), 6.95 (d, 1H, J ¼ 1.7 Hz), 6.85 (d,
1H, J ¼ 8.3 Hz), 6.43e6.53 (m, 1H), 4.68 (s, 2H), 3.99 (s, 3H), 3.83
4.16. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-
(5,6-dimethylbenzo[d] thiazol-2-yl)acetamide (11k)
(s, 3H), 3.7 (s, 6H); 13C NMR (75 MHz, CDCl3):
d
¼ 168.02, 1549.30,
153.02, 150.77, 149.07, 146.66, 137.32, 132.48, 132.31, 130.59,
129.80, 128.56, 126.01, 125.19, 123.29, 121.35, 119.10, 118.46,
111.72, 105.89, 103.43, 70.60, 60.91, 55.90; IR (KBr) (nmax/cmꢂ1):
3377, 2925, 1706, 1580, 1535, 1508, 1465, 1320, 1267, 1164, 1126,
1084, 863, 824, 782, 668; MS (ESI): m/z 597 [M þ Na]þ; HRMS
(ESI m/z) [Mþ1]þ calcd for C28H26N2O6F3: 575.1463, found:
575.1446.
This compound was prepared according to the method described
for compound 11a by employing (Z)-2-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)acetic acid (10) (374 mg, 1.0 mmol), and
2-amino-5,6-dimethyl benzothiazole (178 mg, 1.0 mmol) to
obtain the pure product 11k (472 mg, 83% yield) as white solid; mp:
119e120 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 8.14 (s, 1H), 8.02 (s, 1H),
6.75(d,1H, J ¼ 2.2 Hz), 6.87 (d,1H, J ¼ 8.3 Hz), 6.46e6.57 (m, 4H), 4.63
(s, 2H), 3.88 (s, 3H), 3.84 (s, 3H), 3.72 (s, 6H), 2.35 (s, 3H), 2.36 (s, 3H);
MS (ESI): m/z 535 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for
C29H30N2O6S: 535.1569, found: 535.1578.
4.13. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
methylbenzo[d] thiazol-2-yl)acetamide (11h)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl)phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-6-methyl benzothiazole (164 mg,
1.0 mmol) to obtain the pure product 11h (451 mg, 81% yield) as
4.17. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-
(benzo[d]thiazol-6-yl) acetamide (12a)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 6-aminobenzothiazole (150 mg, 1.0 mmol) to obtain
the pure product 12a (451 mg, 89% yield) as white solid; mp: 111e
white solid; mp: 112e114 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 7.64
(d, 1H, J ¼ 9.1 Hz), 7.28 (d, 1H, J ¼ 2.1 Hz), 6.91e7.02 (m, 2H), 6.89 (d,
1H, J ¼ 2.2 Hz), 6.84 (d, 1H, J ¼ 8.3 Hz), 6.42e6.55 (m, 4H), 4.63 (s,
2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.71 (s, 6H), 2.49 (s, 3H); 13C NMR
114 ꢀC; 1H NMR (200 MHz, CDCl3):
d
¼ 9.19 (br s, 1H), 9.15 (s, 1H),
(75 MHz, CDCl3):
d
¼ 185.9, 175.42, 174.01, 171.22, 168.21, 165.78,
161.44, 156.34, 152.54, 151.62, 149.54, 148.85, 147.75, 144.13, 140.92,
137.34, 134.65, 130.86, 125.12, 123.38, 80.16, 75.25, 75.02, 48.78,
28.3; MS (ESI): m/z 521 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for
C28H28N2O6S: 521.1362, found: 521.1344.
8.69 (d, 1H, J ¼ 2.4 Hz), 8.24 (d, 1H, J ¼ 8.1 Hz), 7.34e7.37 (dd, 1H,
J ¼ 8.9, 2.4 Hz), 6.97e7.02 (dd, 1H, J ¼ 8.1, 1.6 Hz), 6.92 (d, 1H,
J ¼ 2.4 Hz), 6.84 (d, 1H, J ¼ 8.1 Hz), 6.5 (d,1H, J ¼ 12.2 Hz), 6.44e6.48
(m, 3H), 4.53 (s, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.68 (s, 6H); 13C NMR
(75 MHz, CDCl3):
d
¼ 166.99, 156.49, 153.01, 148.84, 148.04, 146.71,
4.14. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-
ethoxybenzo[d] thiazol-2-yl)acetamide (11i)
135.58, 134.46, 132.51, 130.70, 129.81, 128.67, 124.65, 123.17, 119.51,
117.54, 112.39, 111.83, 105.86, 70.41, 60.95, 56.13, 55.93; MS (ESI): m/
z 507 [Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for C27H27N2O6S:
507.2369, found: 507.2345.
This compound was prepared according to the method described
for compound 11a by employing (Z)-2-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)acetic acid (10) (374 mg, 1.0 mmol), and
2-amino-6-ethoxy benzothiazole (194 mg, 1.0 mmol) to obtain the
pure product 11i (475 mg, 85% yield) as white solid; mp: 116e117 ꢀC;
4.18. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(2-
methylbenzo[d]thiazol-6-yl) acetamide (12b)
1H NMR (300 MHz, CDCl3):
J ¼ 2.4 Hz), 6.97e7.05 (m, 2H), 6.93 (d, 1H, J ¼ 2.2 Hz), 6.83 (d, 1H,
J ¼ 8.3 Hz), 6.42e6.51 (m, 4H), 4.62 (s, 2H), 4.02e3.98 (q, 2H), 3.88 (s,
3H), 3.86 (s, 3H), 3.72 (s, 6H), 1.47 (t, 3H); 13C NMR (75 MHz, CDCl3):
d
¼ 7.69 (d, 1H, J ¼ 9 Hz), 7.29 (d, 1H,
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl) phenoxy)acetic acid (10) (374 mg,
1.0 mmol) and 6-amino-2-methylbenzothiazole (164 mg,
1.0 mmol) to obtain the pure product 12b (460 mg, 88% yield); mp:
d
¼ 186.42,176.34,174.11,172.22,168.3,165.82,161.63,156.51,152.53,
151.72, 149.62, 148.9, 147.89, 144.23, 140.91, 137.21, 134.54, 130.81,
125.1,123.37, 89.51, 80.19, 75.45, 75.09, 48.53, 31.2; MS (ESI): m/z 551
[Mþ1]þ; HRMS (ESI m/z) [Mþ1]þ calcd for C29H30N2O7S: 551.1473,
found: 551.1455.
112e114 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 9.24 (s, 1H), 8.2 (d, 1H,
J ¼ 1.7 Hz), 7.79 (d, 1H, J ¼ 8.9 Hz), 7.62e7.67 (dd, 1H, J ¼ 8.9,
1.7 Hz), 6.98e7.03 (dd, 1H, J ¼ 8.9, 1.7 Hz), 6.95 (d, 1H, J ¼ 1.7 Hz),
6.87 (d, 1H, J ¼ 8.9 Hz), 6.44e6.54 (m, 4H), 4.54 (s, 2H), 3.95 (s, 3H),
3.82 (s, 3H), 3.69 (s, 6H), 2.85 (s, 3H); 13C NMR (300 MHz, CDCl3):
4.15. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(4-
methylbenzo[d] thiazol-2-yl)acetamide (11j)
d
¼ 167.48, 166.10, 153.13, 152.23, 148.33, 146.41, 136.45, 135.61,
131.95, 130.34, 129.70, 128.85, 128.43, 123.36, 120.89, 117.13, 116.34,
112.64, 111.37, 105.40, 69.39, 60.02, 55.46, 55.24, 19.55; IR (KBr)
This compound was prepared according to the method
described for compound 11a by employing (Z)-2-(2-methoxy-5-
(3,4,5-trimethoxystyryl)phenoxy)acetic acid (10) (374 mg,
1.0 mmol), and 2-amino-4-methyl benzothiazole (164 mg,
1.0 mmol) to obtain the pure product 11j (450 mg, 80% yield) as
(
nmax/cmꢂ1): 3388, 2936, 1690, 1576, 1518, 1464, 1431, 1416, 1325,
1251, 1124, 1057, 1003, 882, 803, 777; MS (ESI): m/z 521 [Mþ1]þ;
HRMS (ESI m/z) [Mþ1]þ calcd for C28H29N2O6S: 521.1746, found:
521.1733.

A. Kamal et al. / European Journal of Medicinal Chemistry 56 (2012) 166e178
177
5. Materials and methods
sodium deoxycholate and 0.1% SDS) and containing 100
mg/mL
PMSF, 5
and 100
m
g/mL Aprotinin, 5
mg/mL leupeptin, 5 mg/mL pepstatin
5.1. Cell culture
mg/mL NaF. After centrifugation at 12,000 rpm for 10 min,
the 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 (Amersham Biosciences). The membrane was blocked
at room temperature for 2 h in TBS þ 0.1% Tween20 (TBST) con-
taining 5% blocking powder (Santacruz). The membrane was
washed with TBST for 5 min, and primary antibody was added and
incubated at 4 ꢀC overnight (O/N). Antibodies like c-Jun, ERK and
phosphorylated form of ERK were purchased from Cell Signalling
technology, USA. The membrane was incubated with correspond-
ing 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.
MCF-7 cells (human breast cancer) were incubated by using
Dulbecco’s modified Eagle’s medium (DMEM), supplemented with
10% fetal calf serum, 100 mg/mL pencillin-G and 100 mg/mL strep-
tomycin sulfate (Sigma). The cell lines were maintained at 37 ꢀC in
a humidified atmosphere containing 5% CO2 in the incubator.
5.2. In vitro anticancer evaluation
All the synthesized compounds were evaluated for their in vitro
anticancer activity in different cancer cell lines by using sulfo-
rhodamine B (SRB) protein assay in 96 well plate under 10% fetal
bovine serum at 37 ꢀC, 5% CO2, and 100% relative humidity in
DMEM medium. Cancer cells were seeded in 96 well plate and
drugs of 10 mM concentration were added after 24 h of incubation.
The treated cells were incubated for 48 h and then cells were
processed according to the standard protocol. The absorbance was
read on an ELISA plate reader at a wavelength of 560 nm. Growth
percentage was calculated for test wells relative to control wells.
5.7. Immunofluorescence
5.3. Cell viability
Human breast cancer (MCF-7) cells were seeded on cover slips
Cell viability was assessed by the MTT based assay. MCF-7 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 CA-4, BT,11a,11b, 11e, 11f,11g, 11h, 11i,11j,11k and 12b
and were incubated for 24 h. The medium was then discarded and
and treated with compounds at concentration of 4 mM for 24 h.
After treatment, cover slips were fixed with a paraformaldehyde
solution (4% in 1ꢁ PBS) for 20 min at room temperature. Cell per-
meabilization was achieved by administration of a Triton X-100
solution (0.2% in 1ꢁ PBS) for 5min. Then cover slips were kept in
100% methanol at 4 ꢀC overnight. Subsequently, cover slips were
blocked with a 1% BSA solution for 60 min and then incubated with
anti-ERK phosphorylated antibody (or) a-tubulin antibody (1:100)
at room temperature for 2 h. The slides were washed three times
each of 5 min with PBST. Then cover slips were incubated with
replaced with fresh 100 mL media followed by addition of 10 mL of
MTT dye. Plates were incubated at 37 ꢀC for 2 h. The absorbance
(O.D) was measured at 570 nm using Multimode Varioskan Flash
(Thermo Fisher Scientifics).
5.4. Cell cycle analysis
a
FITC-conjugated anti-rabbit secondary antibody (Jackson
Immuno Research Laboratories Inc., Pennsylvania, USA) for 1 h and
cover slips were washed three times with PBST solution and
mounted with DAPI/PI solution. Finally, cells were observed under
confocal microscope (Olympus FV1000). Images taken were pro-
cessed with the support of the flow view version 1.7c software
program.
5 ꢁ 105cells each of MCF-7 seeded in 60 mm dish and were
allowed to grow for 24 h. A concentration of 4 mM of CA-4, BT, 11a,
11b, 11e, 11f, 11g, 11h, 11i, 11j, 11k and 12b was added to the culture
media, and the cells were incubated for an additional 24 h. Cells
were harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol
at 4 ꢀC for 30 min, washed with PBS and incubated with 1 mg/ml
RNaseA solution (Sigma) at 37 ꢀC for 30 min. Cells were collected by
centrifugation at 2000 rpm for 5 min and further stained with
5.8. Docking
250
m
L of DNA staining solution [10 mg of propidium iodide (PI),
ERK2 with small molecule inhibitor (PDB code: 1TVO) and
Tubilin bound with colchicine (PDB code: 3E22) was selected as the
receptor for docking simulation. Ligand bound in the ATP binding
site and colchicine pocket nearby solvent molecules were removed
followed by addition of hydrogen atoms to the entire protein.
Coordinates for each compound in the current study were obtained
using Chemdraw11 followed by MM2 energy minimization. Dock-
ing was carried out by AutoDock4 [54] in the ATP-binding pocket of
ERK2. Grid map in Autodock that defines the interaction of protein
and ligands in binding pocket was defined. The grid map was used
0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dis-
solved in 100 mL of sterile MilliQ water at room temperature for
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.
5.5. Tubulin polymerization assay
ꢀ
Tubulin polymerization assay was conducted with cytoskeleton
with 60 points in each x, y and z direction, equally spaced at 0.375 A.
kit (BK-004). The compounds 11a, CA-4, nocadazole, paclitaxel was
incubated with tubulin (100 ml) that was provided in the kit and the
readings were taken for 30 min to 1 h. The O.D at 340 nm was taken
and inhibition graph was plotted.
Docking was performed using the Lamarckian genetic algorithm in
AutoDock4 [55]. Each docking experiment was performed 10 times,
yielding 10 docked conformations. Parameters used for the docking
were as follows: population size of 150; random starting position
ꢀ
ꢀ
and conformation; maximal mutation of 2 A in translation and 50
5.6. Protein extraction and Western blot analysis
in rotations; elitism of 1; mutation rate of 0.02 and crossover rate of
0.8; and local search rate of 0.06. Simulations were performed with
a maximum of 1.5 million energy evaluations and a maximum of
27,000 generations. Final docked conformations were clustered
Total cell lysates from cultured MCF-7cells were obtained by
lysing the cells in ice-cold RIPA buffer (1xPBS, 1% NP-40, 0.5%

178
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ꢀ
distance of any non-water atom to the edge of the box was 4.0 A;
typical calculations included w10,000 water molecules. After 1000
cycle of energy minimization to remove bad Van der Waals
contacts, we equilibrated the system beginning with 100 ps
equilibration dynamics of solvent molecules at 300 K. The next step
involved equilibration of solute at 100-ps pressure. Then, the
equilibration dynamics of the entire system was performed at 1 ns
using periodic boundary condition.
Acknowledgments
The author A.M.R is thankful to CSIR, India and, P.S. & C.K. are
thankful to UGC, India for the award of research fellowships.
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