Synthesis and study of benzothiazole conjugates in the control of cell proliferation by modulating Ras/MEK/ERK-dependent pathway in MCF-7 cells

Article abstract of DOI:10.1016/j.bmcl.2013.07.068

By applying a methodology, a series of benzothiazole-pyrrole based conjugates (4a-r) were synthesized and evaluated for their antiproliferative activity. Compounds such as 4a, 4c, 4e, 4g-j, 4m, 4n, 4o and 4r exhibited significant cytotoxic effect in the MCF-7 cell line. Cell cycle effects were examined for these conjugates at 2 μM as well as 4 μM concentrations and FACS analysis show an increase of G2/M phase cells with concomitant decrease of G1 phase cells thereby indicating G2/M cell cycle arrest by them. Interestingly 4o and 4r are effective in causing apoptosis in MCF-7 cells. Moreover, 4o showed down regulation of oncogenic expression of Ras and its downstream effector molecules such as MEK1, ERK1/2, p38 MAPK and VEGF. The apoptotic aspect of this conjugate is further evidenced by increased expression of caspase-9 in MCF-7 cells. Hence these small molecules have the potential to control both the cell proliferation as well as the invasion process in the highly malignant breast cancers.

Full text of DOI:10.1016/j.bmcl.2013.07.068

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5733–5739
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and study of benzothiazole conjugates in the control of cell
proliferation by modulating Ras/MEK/ERK-dependent pathway
in MCF-7 cells
a
b
a
a
Ahmed Kamal a, , Shaikh Faazil , M. Janaki Ramaiah , Md. Ashraf , M. Balakrishna ,
⇑
S.N.C.V.L. Pushpavalli b, Nibedita Patel b, Manika Pal-Bhadra b,
⇑
a Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
By applying a methodology, a series of benzothiazole–pyrrole based conjugates (4a–r) were synthesized
and evaluated for their antiproliferative activity. Compounds such as 4a, 4c, 4e, 4g–j, 4m, 4n, 4o and 4r
exhibited significant cytotoxic effect in the MCF-7 cell line. Cell cycle effects were examined for these
Received 29 April 2013
Revised 12 July 2013
Accepted 30 July 2013
Available online 8 August 2013
conjugates at 2 lM as well as 4 lM concentrations and FACS analysis show an increase of G2/M phase
cells with concomitant decrease of G1 phase cells thereby indicating G2/M cell cycle arrest by them.
Interestingly 4o and 4r are effective in causing apoptosis in MCF-7 cells. Moreover, 4o showed down reg-
ulation of oncogenic expression of Ras and its downstream effector molecules such as MEK1, ERK1/2, p38
MAPK and VEGF. The apoptotic aspect of this conjugate is further evidenced by increased expression of
caspase-9 in MCF-7 cells. Hence these small molecules have the potential to control both the cell prolif-
eration as well as the invasion process in the highly malignant breast cancers.
Keywords:
Ras/MEK/ERK
Cell cycle
Caspase-9
Benzothiazole–pyrrole hybrid
Ó 2013 Elsevier Ltd. All rights reserved.
Amplification of Ras proto oncogene and activating mutations
that lead to the expression of constitutively active Ras proteins
are observed in approximately 30% of human cancers.1 ERK/MAPK
signaling promotes cell proliferation, cell survival and metastasis
along with the overwhelming frequency in which this pathway is
aberrantly activated in cancer, in particular by upstream activation
by the epidermal growth factor receptor (EGFR) and Ras small gua-
nosine triphosphatases (GTPases).2 Extra cellular-signal-regulated
kinases (ERK1/2) are Serine/Threonine kinases and their activities
are positively regulated by phosphorylation mediated by MEK1
and MEK2.3 It is well known that phosphorylated ERK (pERK) is a
key downstream component of the RAF/MEK/ERK signaling path-
way. It can be translocated to the nucleus after phosphorylation,
where it leads to changes in gene expression by phosphorylating
and regulating various transcription factors such as Ets family tran-
scription factors (Elk-1).2 The Ras/Raf/MEK/ERK signaling cascades
play a critical role in the transmission of signals from growth factor
receptors to regulate gene expression and prevent apoptosis.4 Be-
cause the expression is often aberrant in tumours it is a popular
target for small molecule inhibition.5 Over the years two main
strategies have been vigorously pursued to identify anti-Ras inhib-
itors. First approach has been the development of inhibitors of Ras
downstream effector signaling, with efforts focused on the ERK
MAPK pathway. The second approach focused in blocking the
post-translational modifications that promote Ras membrane asso-
ciation.2 There has been considerable impetus for developing
inhibitors of PI3K/AKT/mTOR and Ras/MEK/ERK pathways as these
O
N
F
N
S
NH2
O
S
(2)
(1)
O
F
R1
R2
I
N
S
N
NH
O
N
H
OH
R3
N
R6
R4
O
R5
(4a-r)
(3)
Figure 1. Chemical structure of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole
(1), 2-(4-aminophenyl)benzothiazoles (2), 5-acetyl-2-(2-fluoro-4-iodophenylami-
no)-N-(2-hydroxyethoxy)-1-methyl-1H-pyrrole-3-carboxamide (3), benzothiazole–
pyrrole hybrid derivatives (4a–r).
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.068

5734
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
Cl
O
NO2
ii
NO2
R1
R2
NH2
H
N
H
N
R1
R2
R1
R2
i
+
O
S
R3
R3
R3
8a-d
NO2
6
7a-d
5a-d
iii
5a: R1 = R2 = R3 = H
R1
R2
R1
R2
5b: R1 = R3 = H; R2 = F
N
S
N
iv
5c: R1 = R3 = H; R2 = OMe
NH2
NO2
S
1
: R = R2 = R3 = OMe
5d
R3
R3
9a-d
10a-d
10a: R1 = R2 = R3 = H
10b: R1 = R3 = H; R2 = F
10c: R1 = R3 = H; R2 = OMe
: R1 = R2 = R3 = OMe
10d
Scheme 1. Reagents and conditions: (i) pyridine, reflux, 2 h; (ii) Lawesson’s reagent, toluene, reflux, 8 h; (iii) K3Fe(CN)6, aq NaOH, 90 °C, 3 h; (iv) SnCl2Á2H2O, EtOH, reflux, 2 h.
MEK/ERK signal cascade.7,8 Literature survey revealed that these
O
CHO
R5
O
R6
R5
simple structures possess remarkable and intriguing anti-leukemic
properties,9,10 and their biological profile is unlike that of any
known new anticancer agent. A variety of benzothiazole deriva-
tives were synthesized with structural modifications and were
studied for their diverse biological activities, like antimicrobial,11
antiallergenic,12 anti-inflammatory,13 immunosuppressive,14 anti-
convulsant,15 and antitumor activities through inhibiting different
types of enzymes that play important roles in cell division.16
It is reported that some of the substituted 2-phenylbenzothiaz-
ole derivatives have revealed good anticancer activity.17,18 More-
over, modifications on the benzothiazole nucleus have resulted in
a large number of compounds having diverse pharmacological
activities. Earlier studies on modified benzothiazoles demonstrated
interesting pharmacological activities and thus represent an
important scaffold of drugs.19,20 Among them, imidazobenzothiaz-
oles, as well as polymerized benzothiazoles and other substituted
benzothiazoles such as 2-(3,4-dimethoxyphenyl)-5-fluorobenzo-
thiazole (1) exhibited potent cytotoxicity (GI50 <0.1 nM). 2-(4-Ami-
nophenyl)benzothiazoles (2) the original lead compound from the
benzothiazole series exhibited nanomolar activity against certain
breast cancer cell lines.21 Similarly, pyrrole based series (3) are
reported22 as selective MEK kinase inhibitors show promising
i
+
O
R6
R4
R4
13a-e
12
11a-e
: R4 = R5 = R6 = Me
: R4 = R6 = H ; R5 = Me
11a
13a
13b: R4 = R6 = H, ,R5 = F
13c: R4 = R6 = H, ,R5 = OMe
13d: R4 = R6 = H, ,R5 = NO2
11b: R4 = R6 = H, ,R5 = F
11c: R4 = R6 = H, ,R5 = OMe
11d: R4 = R6 = H, ,R5 = NO2
: R4 = R6 = R5 = OMe
: R4 = R6 = R5 = OMe
13e
11e
Scheme 2. Reagents and conditions: (i) 3-ethyl-5(2-hydroxyethyl)-4-meth-
ylthiazolium bromide, triethyl amine, EtOH, reflux, 6–8 h.
are considered to be the central transducers of oncogenic signals in
solid tumors.6
Benzothiazoles is a privileged heterocyclic system with exten-
sive pharmaceutical importance due to its diverse chemotherapeu-
tic potential including versatile antineoplastic properties.
Molecules containing the benzothiazole moiety have been recently
reported to have anticancer activities through inhibition of the Raf/
R1
R2
O
N
R1
R2
N
S
R6
R5
N
NH2
i
S
+
O
R3
R3
R4
13a-e
R6
10a-d
R4
R5
4a-r
4a: R1 = R2 = R3 = R4 = R6 = H; R5 = Me
4b: R1 = R2 = R3 = R4 = R6 = H; R5 =F
4j: R1 = R3 = H; R2 = F; R4 = R5 = R6 = 3,4,5-trimethoxy
4k: R2 = OMe; R1 = R3 = R4 = R6 = H; R5 = Me
4l: R2 = OMe; R1 = R3 = R4 = R6 = H; R5 = F
4c: R1 = R2 = R3 = R4 = R6 = H; R5 =OMe
1
2
3
4
6
5
4d
2
5
1
3
: R = R = OMe; R = R = R4 = R6 = H
: R = R = R = R = R = H; R = NO2
4m
1
2
3
R = R = R = H; R4 = R5 = R6 = 3,4,5- trimethoxy
4e:
: R2 = OMe; R1 = R3 = R4 = R6 = H; R5 = NO2
4n
4f: R2 = F; R1 = R3 = R4 = R6 = H; R5 = Me
4g: R2 = R5 = F; R1 = R3 = R4 = R6 = H
4h: R1 = R3 = R4 = R6 = H; R2 = F; R5 =OMe
4o: R1 = R3 = H; R2 = OMe; R4 = R5 = R6 = 3,4,5-trimethoxy
4p: R1 = R2 = R3 = 3,4,5-trimethoxy; R4 = R6 = H; R5 = Me
4q: R1 = R2 = R3 = 3,4,5-trimethoxy; R4 = R6 = H; R5 = F
1
3
4
6
2
5
4i
1
2
R = R = R3 = 3,4,5-trimethoxy; R4 = R6 = H; R5 = OMe
: R = R = R = R = H; R = F; R = NO2
4r:
Scheme 3. Reagents and conditions: (i) KHSO4, EtOH, reflux, 1 h.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
5735
Table 1
activity against colon cancer (Fig. 1). Pyrrole nucleus is well known
to exhibit anticancer activity23–25 and their structurally modified
derivatives have encouraged current attention as effective antican-
cer agents.26,27 A number of mechanisms are involved in their cyto-
toxic action, like dihydrofolate reductase inhibitors,27 cyclin
dependant kinase inhibitors,28 tyrosine kinase inhibitors.29
Moreover, the ERK inhibition exhibited by selective compounds
is rationalized by molecular docking investigations. Modified ben-
zothiazole derivatives constitute a class of powerful antitumour
agents particularly for the treatment of hepatocarcinoma by mod-
ulating signaling pathways and key molecules that regulate cell
proliferation, oncogenesis, invasion and metastasis of liver cancer
such as AP1(C-Jun, JunB), NF-kB and p38MAPK.8,30 It is not only ac-
tive against malignant cell but as well as useful for the treatment of
solid tumors.31
Anti-proliferative (GI50 values) activity of compounds (4a–r) against breast cancer cell
line (MCF-7) by SRB assay
Compound
GI50 for MCF-7 (
l
M)
GI50 for MDA-MB-231 (lM)
Etoposide
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
0.87 0.07
2.5 0.12
1.67 0.08
4.4 0.24
2.62 0.32
2.78 0.23
2.54 0.11
2.34 0.26
2.15 0.08
1.79 0.17
2.12 0.05
0.918 0.06
1.41 0.16
2.57 0.25
2.48 0.05
3.09 0.24
3.17 0.244
0.92 0.04
2.91 0.138
2.67 0.062
1.23 0.16
4.34 0.37
3.99 0.40
3.90 0.27
3.45 0.35
3.14 0.06
3.03 0.28
3.07 0.25
1.67 0.21
3.0 0.453
4.01 0.38
4.06 0.19
4.12 0.15
4.56 0.26
1.76 0.352
4.14 0.65
3.94 0.17
2.24 0.18
In continuation of our efforts on the design of new anti-cancer
agents32–34 and keeping in mind the medicinal importance of ben-
zothiazole moiety for its impressive anticancer profile and their ef-
fect on MAP kinases led us to synthesize new conjugates of
benzothiazole. Herein we report the synthesis of benzothiazole–
pyrrole conjugates and their evaluation as potential anticancer
Figure 2. MCF-7 cells were treated with conjugates (4a–r) and etoposide (Eto) at 0.25–8 lM concentrations for 72 h. The MTT cell viability assay was conducted and optical
density (O.D) was observed at 570 nm. Control indicates untreated cells.
Table 2
IC50 values for conjugates (4a–r) were derived by conducting MTT assay against tumor and nontumor cells
Compound
IC50 for MCF-7a
(l
M)
IC50 for MDA-MB-231b
(lM)
IC50 for MCF-10Ac
(l
M)
SI for MCF-7
SI for MDA-MB-231
Etoposide
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
1.2
3.1
1.5
36.26
30.56
40
36.26
38.80
38.80
33.58
37.77
37.80
39
40
34
38.3
30.56
34.87
37.77
42.5
43.17
43.87
30.21
9.1
12.57
11.13
12.47
8.87
11.30
13.48
14.85
16.4
16.93
11.92
13.67
6.36
24.17
1.71
3.4
2.44
3.2
17.77
11.76
14.81
12.12
16
3.181
3.255
3.11
4.37
2.97
2.8
2.545
2.372
2.362
2.85
2.80
4.827
4.516
1.52
3.11
2.91
2.2
2.42
2.01
2.68
1.98
2.29
2.60
1.65
2.77
2.23
2.74
2.92
3.45
3.29
3.5
16.66
14.06
19.04
17.02
15.38
20.51
13.79
12.69
12.70
12.90
12.30
13.11
12.50
25.3
24.84
13.66
14.83
19.945
SI = selective index and is obtained by dividing IC50 of the normal cells to IC50 of cancer cells; IC50 is a measure of the effectiveness of a compound in inhibiting biological or
biochemical function. IC50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the
agonist.
a
Estrogen positive breast cancer cells.
Estrogen negative breast cancer cells.
Normal mammary epithelial cells.
b
c

5736
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
Figure 3. MCF-7 were treated with conjugates 4a, 4c, 4e, 4m, 4n, 4o, 4r and etoposide at 2 and 4 lM concentrations for 24 h. The cells were further processed for FACS
analysis and control indicates untreated cells.
agents. The synthesized compounds were evaluated for their effect
on MEK1, ERK1/2, p38 MAPK and VEGF signaling. Few representa-
tives of some biologically important benzothiazole and pyrrole re-
lated compounds have been illustrated in Figure 1.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
5737
Figure 4. Treatment of MCF-7 cells with etoposide (E) and 4o at 2 and 4 lM
concentration for 24 h and the cell lysates were subjected to Western blot analysis
Figure 6. Model proposing the mode of action of compound 4o.
using antibodies against VEGF-A, H-Ras, MEK1, phospho p38 MAPK, phospho ERK1/
2 and integrin
concentration for cells is 2
Compound 4o (2): compound (4o) concentration for cells is
compound (4o) concentration for cells is 4 M.
a
Vb3 proteins. Beta-actin is used as loading control. E (2): Etoposide
M, E (4): Etoposide concentration for cells is 4 M.
M, 4o (4):
l
l
methyl vinyl ketone (12) and 3-ethyl-5(2-hydroxyethyl)-4-meth-
ylthiazolium bromide was refluxed at 80 °C for 6 h, quenched with
2
l
l
aqueous acetic acid to yield
c-diketones (13a–e) as shown in
Scheme 2. These -diketones (13a–e) on treatment with different
c
substituted 2-(4-aminophenyl)benzothiazoles (10a–d) by using
catalytic potassium bisulphate (KHSO4) in ethanol upon heating
afforded the desired conjugates (4a–r) in good yields, as shown
in Scheme 3.
All the synthesized compounds have displayed significant anti-
proliferative activity against breast cancer cell line (MCF-7) with
GI50 values in the range of 1–4 lM. The results are summarized
in Table 1. With a view to understand the cytotoxic nature of these
compounds, MTT assay was performed by treating MCF-7 cells
with compounds 4a–r at different concentrations ranging from 1
to 8
lM for 72 h. It is observed that compounds 4o and 4r cause al-
most 50% of cell death at 4
lM. The cytotoxic effect of these conju-
gates was comparable to etoposide, the standard drug used in the
study. It is found that most of the compounds induce cytotoxicity
without effecting cell cycle. Although 4g–j have shown the signif-
icant cytotoxicity, however they did not exhibit effect on cell cycle
and thus these conjugates have not been evaluated for further
studies. The IC50 values were deduced for MCF-7, MDA-MB-231
and MCF-10A (normal cells) to show the specificity of conjugates
towards the breast cancer cells Figure 2, Table 2.
Figure 5. The effect of compounds etoposide (Eto) and 4o on caspase-9 activity.
MCF-7 cells were treated with etoposide (Eto) and 4o at 2 M concentrations for
24 h. The cell lysates were subjected to fluorescence based caspase-9 assay. The
compound treatment resulted in increase in caspase-9 activity. Con: indicates
untreated cells.
l
In order to understand the effect of these conjugates in cell cy-
cle, FACS analysis was conducted for some representative com-
pounds in MCF-7 breast cancer cells. The cells were treated for
A green chemistry protocol was employed for the synthesis of
the target compounds, benzothiazole–pyrrole hybrid (4a–r) via
Paal knorr pyrrole methodology. The synthesis of the final com-
pounds was achieved by condensation reaction between amin-
24 h with compounds at 2 and 4 lM concentrations. It was ob-
served that cells showed G2/M cell cycle arrest (i.e., cells accumu-
lated in G2/M phase). One of the compounds 4o has shown highest
G2/M cell cycle arrest (i.e., 48%) whereas etoposide, a standard
drug has shown 67% of G2/M cell cycle arrest. There was not much
change in cell cycle arrest upon increasing the concentration of the
compounds; however the percentage of apoptotic cells enhanced
Figure 3.
Combretastatin-amidobenzothiazole affected the ERK activity
as observed by protein activity studies in MCF-7 cells and docking
studies.7 Furthermore Mannich bases of 2-arylimidazo[2,1-b]ben-
zothiazoles caused G2/M cell cycle arrest and affected the MAPK
proteins in HepG2 hepatocarcinoma.8 It is evident from studies
on EphB4 protein the involvement of Ras/MEK/ERK dependent
pathway in breast cancer and as well as endothelial cell
obenzothiazoles and
c-diketones. Nitrobenzoyl chloride was
added slowly to a solution of the appropriately substituted aniline
(5a–d) in pyridine and the solution was stirred under reflux for 2 h
to afford the benzanilides (7a–d) as solids. Treatment of benzani-
lides with Lawesson’s reagent in refluxing toluene yielded substi-
tuted thiobenzanilide (8a–d), which on treatment with aqueous
sodium hydroxide containing ethanol (3 mL) was added dropwise
to a pre-heating solution of potassium ferricyanide in water at
90 °C over a period of 3 h, to afford substituted 2-(4-nitro-
phenyl)-benzothiazoles (9a–d). Further, substituted 2-(4-nitro-
phenyl)-benzothiazoles with tin(II) chloride dihydrate in ethanol
under reflux reduced to amines (10a–d) as shown in Scheme 1.
The
mixture of the appropriate benzaldehyde (11a–e), triethylamine,
c a
-diketones (13a–e) were prepared by Stetter reaction,35

5738
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
proliferation and invasion.36 MCF-7 cells treated with compounds
4o and etoposide caused decrease in the expression of oncogenic
proteins such as Ras, MEK1 and phosphorylated forms of ERK1/2
7. Kamal, A.; Mallareddy, A.; Ramaiah, M. J.; Pushpavalli, S. N. C. V. L.; Paidakula,
S.; Chandan, K.; Murty, J. N. S. R. C.; Rao, N. S.; Sowjanya, G.; Anthony, A.;
Manika, P. E. J. Med. Chem. 2012, 56, 166.
8. Ravindra, M. K.; Kumar, K. V.; Ramaiah, M. J.; Tulshiram, D.; Pushpavalli, S. N. C.
V. L.; Debasmita, M.; Divya, B.; Devi, T. A.; Umesh, K.; Manika, P. E. J. Med. Chem.
2011, 46, 4258.
9. Odlo, K.; Hentzen, J.; Fournier dit Chabert, J.; Ducki, S.; Gani, O. A. B. S. M.; Sylte,
I.; Skrede, M.; Florenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829.
10. Zhang, Q.; Peng, Y.; Wang, X. I.; Keeman, S. M.; Aurora, S.; Welsh, W. J. J. Med.
Chem. 2007, 50, 749.
11. Bradshaw, T. D.; Wrigley, S.; Shi, D.-F.; Schultz, R. J.; Paull, K. D.; Stevens, M. F.
G. Br. J. Cancer 1998, 77, 745.
and proteins that help in angiogenesis such as integrin
aVb3 and
VEGF proteins. Similar to our results sorafenib (Nexavar) blocks
the tumor cell proliferation and tumor angiogenesis by targeting
Raf/MEK/ERK and VEGF signaling.37 Thus the data obtained sug-
gests the inhibitory role of these compounds on tumor prolifera-
tion and invasion Figure 4.
Raf/MEK/ERK and PI3K/Akt affect the phosphorylation of apop-
totic regulatory molecules that exert their effects on mitochondrial
membrane potential and thereby preventing apoptosis. Moreover
the Ras/Raf/MEK/ERK pathway aids in the assembly of the protein
translation complex responsible for the translation of ‘weak’
mRNAs (MCl-1) important in the prevention of apoptosis.38 Thus
we were interested to observe caspase-9 expression as a measure
of cell death. Treatment of cells with Eto and 4o for 24 h. The
lysates were then subjected to caspase-9 assay. As expected we ob-
served increased caspase-9 activity Figure 5.
Conjugate decreased the growth factor dependent expression
of Ras and its downstream effectors of Ras such as MEK1 and
ERK1/2 was decreased. The decreased level of MEK1 inhibited
the phosphorylation of ERK1/2 which suppressed the rapid cell
proliferation and induce apoptosis affecting both nuclear and
mitochondrial genes (caspase-9). The schematic diagram pro-
posing the mode of action of compound 4o has shown in
Figure 6.
12. Bradshaw, T. D.; Shi, D.-F.; Schultz, R. J.; Paull, K. D.; Kelland, L.; Wilson, A.;
Fiebig, H. H.; Wrigley, S.; Stevens, M. F. G. Br. J. Cancer 1998, 78, 421.
13. Bryson, M.; Fulton, B.; Benfield, P. Drugs 1996, 52, 549.
14. (a) Al-Tel, T. H.; Al-Qawasmeh, R. A.; Zaarour, R. Eur. J. Med. Chem. 1874, 2011,
46; (b) Palkar, M.; Noolvi, M.; Sankangoud, R.; Maddi, V.; Gadad, A.; Nargund, L.
V. Arch. Pharm. 2010, 343, 353.
15. Ager, I. R.; Barnes, A. C.; Danswan, G. W.; Hairsine, P. W.; Kay, D. P.; Kennewell,
P. D.; Matharu, S. S.; Miller, P.; Robson, P.; Rowlands, D. A. J. Med. Chem. 1988,
31, 1098.
16. el-Shorbagi, A. N.; Sakai, S.; el-Gendy, M. A.; Omar, N.; Farag, H. H. Chem.
Pharm. Bull. 1989, 37, 2971; (b) Papadopoulou, C.; Geronikaki, A.; Hadjipavlou-
Litina, D. Il Farmaco 2005, 60, 969.
17. Kisak, E. T.; Coldren, B.; Evans, C. A.; Boyer, C.; Zasadzinski, J. A. Curr. Med.
Chem. 2004, 11, 1241.
18. Kok, S. H. L.; Gambari, R.; Chui, C. H.; Yuen, M. C. W.; Lin, E.; Wong, R. S. M.; Lau,
F. Y.; Cheng, G. Y. M.; Lam, W. S.; Chan, S. H.; Lam, K. H.; Cheng, C. H.; Lai, P. B.
S.; Yu, M. W. Y.; Cheung, F.; Tanga, J. C. O.; Chana, A. S. C. Bioorg. Med. Chem.
2008, 16, 3626.
19. Mizoule, J.; Meldrum, B.; Mazadier, M.; Croucher, M.; Ollat, C.; Uzan, A.;
Legrand, J. J.; Gueremy, C.; Le Fur, C. G. Neuropharmacology 1985, 24, 767.
20. Tanaka, T.; Umekawa, H.; Saitoh, M.; Ishikawa, T.; Shin, T.; Ito, M.; Kawamatsu,
Y.; Sugihara, H.; Hidaka, H. Mol. Pharmacol. 1986, 29, 264.
21. Shi, D. F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.; Lelieveld, P.; Fichtner, I.;
Stevens, M. F. J. Med. Chem. 1996, 39, 3375.
22. Mark, E. A.; Michael, B. W.; Toufike, K.; Nicholas, S.; O’Connell, S. M.; Hiroshi,
M.; Lihong, S.; Petro, H.; Lilly, Z.; Qing, D. Bioorg. Med. Chem. Lett. 2012, 22, 241.
23. Hanauske, A. R.; Chen, V.; Paoletti, P.; Niyikiza, C. Oncologist 2001, 6, 363.
24. Lee, M.; Rhodes, A. L.; Wyatt, M. D.; DIncalci, M. D.; Forrow, S.; Hartley, J. A. J.
Med. Chem. 1993, 36, 863.
25. Wyatt, M. D.; Garbiras, B. J.; Haskell, M. K.; Lee, M.; Souhami, R. L.; Hartley, J. A.
Anti-Cancer Drug Des. 1994, 9, 511.
26. Bennasroune, A.; Gardin, A.; Aunis, D.; Cremel, G.; Hubert, P. Crit. Rev. Oncol.
Hematol. 2004, 50, 23.
27. Moriarty, K. J.; Koblish, H. K.; Garrabrant, T.; Maisuria, J.; Khalil, E.; Ali, F.;
Petrounia, I. P.; Crysler, C. S.; Maroney, A. C.; Johnson, D. L.; Galemmo, R. A.
Bioorg. Med. Chem. Lett. 2006, 16, 5778.
28. Choi, H. S.; Wang, Z.; Richmond, W.; He, X.; Yang, K.; Jiang, T.; Karanewsky, D.;
Gu, X. J.; Zhaou, V.; Liu, Y.; Che, J.; Lee, C. C.; Caldwell, J.; Kanazawa, T.;
Umemura, I.; Matsuura, N.; Ohmori, O.; Fonda, T.; Gray, N.; He, H. Bioorg. Med.
Chem. Lett. 2006, 16, 2689.
29. Rosowsky, A. A.; Chen, H.; Fu, H.; Queener, S. F. Bioorg. Med. Chem. 2003, 11, 59.
30. Weng, C. J.; Chau, C. F.; Hsieh, Y. S.; Yang, S. F.; Yen, G. C. Carcinogenesis 2008,
29, 147.
In summary, the synthesis and evaluation of antiproliferative
activity of a new series of benzothiazole–pyrrole conjugates has
been investigated.39 Most of these conjugates have exhibited po-
tent antiproliferative activity against selected human cancer cell
lines MCF-7 and MDA-MB-231. Cell cycle analysis indicated G2/
M cell cycle arrest with increased cytoxicity at 2 as well as 4 lM
concentrations. Thus, we provided the evidence that these newly
synthesized hybrid molecules inhibited growth of human breast
cancer MCF-7 cells by inducing apoptosis.40 The apoptotic death
was associated with down regulation of gene expression of onco-
gene such as Ras as well as activation of MEK1, ERK1/2, p38 MAPK
and VEGF and integrin. In addition, we have observed increased
expression of active caspase-9 protein in MCF-7 cells as observed
by ELISA.
31. Nobuaki, A.; Yukitaka, I.; Mayumi, Y.; Sadao, K.; Katsunori, T.; Kiyohiro, S.;
Akira, M.; Masafumi, K.; Masayuki, S. Cancer Lett. 2006, 238, 119.
32. Kamal, A.; Srikanth, Y. V. V.; Khan, M. N. A.; Ashraf, Md.; Reddy, M. K.; Sultana,
F.; Kaur, T.; Chashoo, G.; Suri, N.; Sehar, I.; Wani, Z. A.; Saxena, A.; Sharma, P. R.;
Bhushan, S.; Mondhe, D. M.; Saxena, A. K. Bioorg. Med. Chem. 2011, 19, 7136.
33. (a) Kamal, A.; Sultana, F.; Ramaiah, M. J.; Srikanth, Y. V. V.; Viswanath, A.;
Kishore, C.; Sharma, P.; Pushpavalli, S. N. C. V. L.; Anthony, A.; Bhadra, M. P.
ChemMedChem. 2012, 7, 292; (b) Kamal, A.; Khan, M. N. A.; Reddy, K. S.;
Srikanth, Y. V. V.; Sridhar, B. Chem. Biol. Drug Des. 2008, 71, 78.
34. (a) Kamal, A.; Reddy, K. S.; Khan, M. N. A.; Shetti, R. V. C. R. N. C.; Ramaiah, M. J.;
Bhadra, M. P. Bioorg. Med. Chem. 2010, 18, 4747; (b) Kamal, A.; Khan, M. N. A.;
Srikanth, Y. V. V.; Shetti, R. V. C. R. N. C. Chem. Biol. Drug Des. 2009, 73, 687.
35. Stetter, H. Angew. Chem., Int. Ed. Engl. 1976, 15, 639.
Acknowledgments
The authors (S.F., M.A. and M.B.) are thankful to Council of Sci-
entific and Industrial Research (CSIR) for financial support under
the XIIth Five year plan Project ‘Affordable Cancer Therapeutics’
and University Grant Commission (UGC) for the award of
Fellowship.
Supplementary data
36. Xiao, Z.; Carrasco, R.; Kinneer, K.; Sabol, D.; Jallal, B.; Coats, S.; Tice, D. A. Cancer
Biol. Ther. 2012, 13, 630.
37. Liu, L.; Cao, Y.; Chen, C.; Zhang, X.; Mc Nabola, A.; Wilkie, D.; Wilhelm, S.;
Lynch, M.; Carter, C. Cancer Res. 2006, 24, 11851.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
07.068.
38. Steelma, L. S.; Chappell, W. H.; Abrams, S. L.; Kempf, R.; Long, J.; Laidler, P.;
Mijatovic, S.; Maksimovic-Ivanic, D.; Stivala, F.; Mazzarino, M. C.; Donia, M.;
Fagone, P.; Malaponte, G.; Nicoletti, F.; Libra, M.; Milella, M.; Tafuri, A.; Bonati,
A.; Basecke, J.; Cocco, L.; Evangelisti, C.; Martelli, A. M.; Montalto, G.; Cervello,
M.; Mc Cubrey, J. A. AGING 2011, 3, 192.
39. General method for the synthesis of compounds 4a–r: A mixture of substituted 2-
(4-aminophenyl)benzothiazole (10a–d) (0.5 mmol), diketone (13a–e) (1 equiv)
in 50 mL of ethanol with a catalytic amount (25 mol %) of potassium bisulfate
was heated under reflux for 2 h. The reaction was monitored by TLC. After the
completion of the reaction, the solvent was removed under vaccum, water was
added to reaction mixture and extracted with ethyl acetate (2Â 30 mL). The
solvent was evaporated under the reduced pressure to afford the crude product
which was purified by flash chromatography on silica gel eluting with EtOAc
and hexane to give the pure compounds. 2-(4-(2-Methyl-5-p-tolyl-1H-pyrrol-1-
References and notes
1. Stirewalt, D. L.; Kopecky, K. J.; Meshinchi, S.; Appelbaum, F. R.; Slovak, M. L.;
William, C. L. Blood 2001, 97, 3589.
2. Roberts, P. J.; Der, C. J. Oncogene 2007, 26, 3291.
3. Chang, F.; Lee, J. T.; Novalanic, P. M. Leukemia 2003, 17, 590.
4. Mc Cubrey, J. A.; Steelman, L. S.; Chappel, W. L.; Abrams, S. L.; Wong, E. W. T.;
Chang, F.; Lehmann, B.; Terrian, D. M.; Milella, M.; Tafuri, A.; Stivala, F.; Libra,
M.; Basecke, J.; Camilla, E.; Alberto, M. M.; Franklin, R. A. Biochim. Biophys. Acta
2007, 1773, 1263.
5. Ripple, M. O.; Kim, N.; Springett, R. J. Biol. Chem. 2013, 288, 5374.
6. Elina, J.; Niina, L.; Jussi, P. K. BMC Cancer 2012, 12, 612.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5733–5739
5739
yl)phenyl)benzo[d]thiazole (4a) Yellow solid; Yield 86%, 162 mg; 1H NMR
(CDCl3, 300 MHz) d 8.03 (d, 2H, J = 8.30 Hz), 7.89 (d, 1H, J = 8.67 Hz), 7.82 (d,
1H, J = 8.30 Hz), 7.43 (t, 1H, J = 8.3, 6.73 Hz), 7.36 (t, 1H, J = 8.3, 6.73 Hz), 7.25
(d, 2H, J = 8.30 Hz), 7.07 (d, 2H, J = 8.67 Hz), 6.96 (d, 2H, J = 8.60 Hz), 6.26 (d,
1H, J = 2.83 Hz), 5.94 (d, 1H, J = 2.89 Hz), 2.13 (s, 3H), 1.97 (s, 3H); 13C NMR
(CDCl3, 300 MHz) d 13.55, 17.10, 96.65, 96.98, 99.28, 105.84, 115.79, 125.26,
127.67, 128.03, 128.60, 130.18, 132.27, 132.92, 137.68, 138.01, 153.72, 155.25,
159.95, 160.30, 166.73; ESI–MS: m/z 381 [M+H]+. 2-(4-(2-(4-Fluorophenyl)-5-
methyl-1H-pyrrol-1-yl)phenyl)benzo[d]thiazole (4b) Yellow solid; Yield 90%,
173 mg; 1H NMR (CDCl3, 300 MHz) d 8.19 (d, 2H, J = 8.30 Hz), 7.88 (d, 1H,
J = 8.67 Hz), 7.85 (d, 1H, J = 8.30 Hz), 7.47 (t, 1H, J = 8.3, 6.73 Hz), 7.38 (t, 1H,
J = 8.3, 6.73 Hz), 7.27 (d, 2H, J = 8.30 Hz), 7.07–6.94 (4H,m), 6.29 (d, 1H,
J = 2.83 Hz), 5.98 (d, 1H, J = 2.89 Hz), 2.11 (s, 3H); 13C NMR (CDCl3, 300 MHz) d
14.13, 97.25, 97.48, 105.49, 116.35, 124.41, 125.99, 128.17, 128.78, 132.87,
133.43, 137.66, 138.38, 153.12, 153.18, 154.28, 154.24, 155.68, 160.38, 167.17;
ESI–MS: m/z 385 [M+H]+. 2-(4-(2-(4-Methoxyphenyl)-5-methyl-1H-pyrrol-1-
yl)phenyl)benzo[d]thiazole (4c) Yellow solid; Yield 182 mg, 91%, mp 140 °C;
1H NMR (CDCl3, 300 MHz) d 8.06 (d, 2H, J = 8.3 Hz), 7.9 (d, 1H, J = 8.6 Hz), 7.8 (d,
1H, J = 8.3 Hz), 7.41 (t, 1H, J = 8.3, 6.7 Hz), 7.3 (t, 1H, J = 8.3, 6.7 Hz), 7.2 (d, 2H,
J = 8.3 Hz), 7.02 (d, 2H, J = 8.6 Hz), 6.9 (d, 2H, J = 8.6 Hz), 6.2 (d, 1H, J = 2.8 Hz),
5.9 (d, 1H, J = 2.8 Hz), 3.8 (s, 3H), 2.1 (s, 3H); 13C NMR (CDCl3, 300 MHz) d 12.11,
55.65, 97.05,97.43, 114.82, 116.22, 118.78, 125.67, 127.64, 128.12, 128.72,
129.56, 133.00, 133.21, 137.60, 1378.47, 146.12, 154.18, 155.58, 160.38; ESI-
MS: m/z 397 [M+H]+. The detail spectral data for other compounds are
available in Supplementary data.
FACS analysis: 5 Â 105 MCF-7 were seeded in 60 mm dish and were allowed to
grow for 24 h. Further compounds (4a,4c,4e,4m,4n,4o,4r) were added at a final
concentration of 2 and 4 lM 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 RNase A solution (Sigma) at 37 °C for 30 min. Cells were collected by
centrifugation at 2000 rpm for 5 min and further stained with 250 mL of DNA
staining solution [10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate,
and 0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ water at
room temperature for 30 min in the dark]. These cells were analysed to observe
the changes in the cellcycle pattern and apoptotic death. The DNA contents of
20,000 events were measured by flow cytometer (DAKO CYTOMATION,
Beckman Coulter, Brea, CA). Histograms were analyzed using Summit Software.
Western blot analysis: Total cell lysates from cultured MCF-7 cells were
obtained by lysing the cells in ice-cold RIPA buffer (1Â PBS, 1% NP-40, 0.5%
sodium deoxycholate and 0.1% SDS) and containing 100 mg/mL PMSF, 5 mg/
mL, Aprotinin, 5 mg/mL leupeptin, 5 mg/mL pepstatin and 100 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 (GE
Biosciences). The membrane was blocked at room temperature for 2 h in TBS
+0.1%, Tween20 (TBST) containing 5% blocking powder (Santacruz). The
membrane was washed with TBST for 5 min, primary antibody was added
and incubated at 4 °C overnight (O/N). VEGF-A, H-Ras, MEK-1, ERK1/2 phospho,
40. Evaluation of in vitro anti-cancer activity
Cell culture: Human breast carcinoma cells (MCF-7), MDA-MB-231 and MCF-10
were purchased from American Type culture collection was maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen), supplemented with
2 mM glutamax (Invitrogen), 10% fetal calf serum and 100 U/ml Pencillin and
100 mg/ml streptomycin sulfate (Sigma). The cell line was maintained at 37 °C
in a humidified atmosphere containing 5% CO2 in the incubator.
MTT assay: Cell viability was assessed by the MTT assay, a mitochondrial
function assay. It is based on the ability of viable cells to reduce the MTT to
insoluble formazan crystals by mitochondrial dehydrogenase. MCF-7 cells
were seeded in a 96-well plate at a density of 10,000 cells/well. After overnight
p38 MAPK phospho, aVb3 integrin were purchased from Cell Signalling,
Abbiotec and Millipore companies. 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.
Caspase-9 assay: The Apoalert caspase-9/6 fluorescent assay kit (Clonetech, CA,
USA) was used according to the manufacturer’s recommendations. MCF-7 cells
were treated with compounds Etoposide (Eto), 4o at 2 lM. Here the substrate
incubation MCF-7 (breast cancer cells) were treated with 4a–r at 1–8
concentration and incubated for 72 h. Medium was then discarded and
replaced with 10 L MTT dye. Plates were incubated at 37 °C for 2 h. The
resulting formazan crystals were solubilized in 100 L extraction buffer. The
l
M
used is LEHD–AMC, which was added to the cell lysates, and incubation was
carried out at 378 °C for 1 h. Readings were taken at k excitation 400 nm and k
emmission 505 nm.
l
l
optical density (OD) was read at 570 nm with micro plate reader (Multi-mode
Varioskan instrument-Themo Scientific).