Synthesis and anticancer evaluation of novel triazole linked N-(pyrimidin-2-yl)benzo[d]thiazol-2-amine derivatives as inhibitors of cell survival proteins and inducers of apoptosis in MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.11.083

A series of novel triazole linked N-(pyrimidin-2yl)benzo[d]thiazol-2-amine 5a-k were synthesized and evaluated for anticancer activity against breast (MCF-7), lung (A549) and skin (A375) cancer cell lines and their cytotoxic effects were compared against

Full text of DOI:10.1016/j.bmcl.2014.11.083

Accepted Manuscript
Synthesis and anticancer evaluation of novel triazole linked N-(pyrimidin-2-
yl)benzo[d]thiazol-2-amine derivatives as inhibitors of cell survival proteins and
inducers of apoptosis in MCF-7 breast cancer cells
Ravindra M. Kumbhare, Tulshiram L. Dadmal, M. Janaki Ramaiah, K.S.V.
Kishore, S.N.C.V.L. Pushpa Valli, Sudheer Kumar Tiwari, K. Appalanaidu, Y.
Khageswar Rao, Manika pal Bhadra
PII:
S0960-894X(14)01290-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.11.083
BMCL 22238
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 September 2014
27 November 2014
29 November 2014
Please cite this article as: Kumbhare, R.M., Dadmal, T.L., Janaki Ramaiah, M., Kishore, K.S.V., Pushpa Valli,
S.N.C.V.L., Tiwari, S.K., Appalanaidu, K., Khageswar Rao, Y., Bhadra, M.p., Synthesis and anticancer evaluation
of novel triazole linked N-(pyrimidin-2-yl)benzo[d]thiazol-2-amine derivatives as inhibitors of cell survival proteins
and inducers of apoptosis in MCF-7 breast cancer cells, Bioorganic & Medicinal Chemistry Letters (2014), doi:
http://dx.doi.org/10.1016/j.bmcl.2014.11.083
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Synthesis and anticancer evaluation of novel triazole linked N-(pyrimidin-2-yl)benzo[d]thiazol-2-
amine derivatives as inhibitor of cell survival proteins and inducers of apoptosis in MCF-7 breast
cancer cells
Ravindra M. Kumbharea,∗, Tulshiram L. Dadmala, M. Janaki Ramaiahb, K.S.V. Kishorea, S.N.C.V.L. Pushpa
Vallic, Sudheer Kumar Tiwaric, K. Appalanaidua, Y. Khageswar Raoa and Manika pal Bhadrac,∗
1

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and anticancer evaluation of novel triazole linked N-(pyrimidin-2-
yl)benzo[d]thiazol-2-amine derivatives as inhibitors of cell survival proteins and
inducers of apoptosis in MCF-7 breast cancer cells
Ravindra M. Kumbharea,∗, Tulshiram L. Dadmala, M. Janaki Ramaiahb, K.S.V. Kishorea, S.N.C.V.L.
Pushpa Vallic, Sudheer Kumar Tiwaric, K. Appalanaidua, Y. Khageswar Raoa and Manika pal Bhadrac,∗
aFluoroorganics Division, Indian Institute of Chemical Technology, Tarnaka Hyderabad 500607, India
bSchool of Chemical and Biotechnology, SASTRA University, Tirumalaisamudram, Thanjavur 613401, Tamilnadu, India
cChemical Biology Laboratory, Indian Institute of Chemical Technology, Tarnaka Hyderabad 500607, India
ARTICLE INFO
ABSTRACT
A
series of novel triazole linked N-(pyrimidin-2yl)benzo[d]thiazol-2-amine 5a-k were
Article history:
Received
synthesized and evaluated for anticancer activity against breast (MCF-7), lung (A549) and skin
(A375) cancer cell lines and their cytotoxic effects were compared against normal breast
epithelial cells. The effect of compounds on cell cycle of MCF-7 breast cancer cell line was
investigated by FACS. Result indicated G2/M cell cycle arrest of MCF-7 cells. Further
promising compounds 5b, 5g, 5h and 5i were tested for their apoptosis inducing ability as well
as inhibitory activity against key proteins NF-kB, Survivin, CYP1A1, and ERK1/2 which help
in cancer cell survival and proliferation. The apoptotic aspect of these compounds is further
evidenced by increase in the activity 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 highly
malignant breast cancers and can be selected for further biological studies.
Revised
Accepted
Available online
Keywords:
Triazole
N-(pyrimidin-2yl)benzo[d]thiazol-2-amine
Sharpless conditions
Cytotoxicity
Breast cancer cells
Cancer as a second cause of death after heart disease in the
world has posed a great challenge to the field of medicine and
immunology.1,2 Chemotherapy has been widely employed for
various cancer treatments. The toxicity and resistance of
traditional chemotherapeutic drugs makes it critical to develop
new targets and novel drugs for cancer therapy. Despite extensive
research and rapid progress in cancer treatment, there is a
growing demand for new therapies. It is important to identify
new agents and targets for the treatment of cancer. Benzothiazole
nucleus attracted considerable attention to anticancer research.3-8
Recent studies have reported that pyridinyl-2-amine linked
benzothiazole-2-thiol compounds exhibit potent and broad
spectrum inhibitory activities against human tumor cell lines.9
Various benzothiazoles.10,11 possess potent anticancer properties.
Currently, benzothiazole derivative, 2-(4-amino-3-
methylphenyl)-5-fluorobenzthiazole acting through novel
a
a
mechanism, has entered the clinical trial phase I in UK. It shows
high sensitivity towards the tumor cells by acting through aryl
hydrocarbon receptor (AhR) signaling pathway. The activation of
AhR pathway in sensitive tumor cells leads to induction of the
microsomal enzyme CYP1A1. CYP1A1 belongs to cytochrome
p450 family and has been implicated in cancer cell response to
the therapeutic agents. The selective over expression of
cytochrome p4501A1 (CYP1A1) in tumor cell induces
metabolism of benzothiazole and aminoflavone compounds to
this reactive species which are responsible for DNA adduct
formation and cell death.12
Among the antitumor drugs discovered in the recent years,
———
∗ Corresponding author. Tel.: +91-402-719-1776; fax: +91-402-719-3185; e-mail: kumbhare@iict.res.in; rakumbhare@yahoo.com
∗ Corresponding author. Tel.: +91-402-719-3236; e-mail: manika@iict.res.in.
1

Benzothiazole based hybrids were found to cause apoptosis in
cancer cells. Apoptosis is a controlled process by which cells
signal their own termination and is crucial for normal tissue
development and homeostasis.13,14 In contrast to normal cells,
cancer cells ‘switch off’ apoptosis, develop and divide
indefinitely.15,16 Impaired apoptosis is therefore critical in cancer
development and is a major barrier to effective treatment.
Apoptosis is executed by intracellular cysteine proteases
called caspases. Apoptosis occurs through two broad pathways
known as an intrinsic and an extrinsic pathways. Intrinsic
pathway involves mitochondria, while extrinsic pathway is
mediated by cell death receptors such as Fas, TNF α after
receiving the death signal.17,18 Molecules and signaling events
that regulates apoptosis affect disease progression and efficacy of
chemotherapy because most chemotherapy kills cancer cells by
inducing apoptosis.19 Thus, understanding the actual mechanism
of apoptosis plays a vital role in anticancer therapy.
Figure 1. Chemical Structures of biologically important compounds.
In the present study, a random screen of these analogues on a
panel of four selected cancer cell lines displayed higher antitumor
activity on MCF-7 breast cancer cells line, than others as
observed by studies such as cytotoxicity assay, cell cycle arrest
and inhibitory activity on levels of expression of key proteins
such ERK1/2, NF-kB, surviving and CYP1A1 which play a vital
role in estrogen positive breast cancer cell proliferation.
It is well known phenomenona that apoptosis occurs via death
receptors causing activation of caspases, the cysteine proteases.
But in some cases, depending upon cell and tissue, stimulation of
death receptors can also activate the non-apoptotic signaling
pathways mediated by transcription factors such as extracellular
signal regulated kinase (ERK1/2) and nuclear factor kappa B
(NF-kB).20-22
All the present triazole derivatives were synthesized from
appropriate azides 11 and alkynes 10 as shown in scheme 2 using
methods describes previously in detail by us.33 First we designed
the compound 9 via chemoselective oxidative cyclization of
thiourea 8 as per our previously reported method32 using the
readily available starting material 6. The compound 10 were
synthesized by alkylation of compound 9 using two equivalents
of inorganic base in DMF solvent at ambient temperature
(Scheme 1). Followed by the reaction with various azides in
THF, using Cu (I) catalysed [3+2] azide-alkyne cycloaddition
reaction through click chemistry under Sharpless conditions.35,36
as a result an exclusive formation of 1,4-disubstituted-1,2,3-
During the last two decades, several pyrimidine derivatives
have been developed as chemotherapeutic agents and have found
wide clinical applications. Pyrimidines have
a long and
distinguished history extending from the days of their discovery
as important constituents of nucleic acids to their current use in
the chemotherapy of AIDS.23-25 Cycloaddition reactions
involving heteroatoms, such as 1,3-dipolar cycloaddition,
provides rapid access to a wide variety of interesting five and six-
membered heterocycles, which were found to possess wide
applications in medicinal chemistry and material sciences26.
Among them, five-membered heterocycles namely, 1,4-
disubstituted 1,2,3-triazoles exhibit a remarkably broad scope of
selectivity, with a wide range of applications across a broad range
of therapeutic areas.27-29
triazole tagged N-(pyrimidin-2-yl)benzo[d] thiazol-2-amine
derivatives 5, are formed (Scheme 2).
In recent years, several attempts have been to modify the
benzothiazole and triazole pharmacophores to improve their
biological activities. Modifications on the benzothiazole nucleus
have resulted in a large number of compounds having diverse
pharmacological activities. Among them structurally related
benzothiazole
such
as
2-(3,4-dimethoxyphenyl)-5-
fluorobenzothiazole (PMX 610) 1 (Fig. 1) has been shown to
exhibit remarkable in vitro antitumor activity against malignant
cell lines. Also, 2-(4-amino-3-methylphenyl) benzothiazole (DF
203) 2a and the 5-fluoro analogue (5F203) 2b (Fig. 1) are
effective ligands for the aryl hydrocarbon receptor (AhR).
Among the fluorinated triazole, difluoride 3 is an anticonvulsive
agent.30 Voriconazole 4 (Fig.1) is a triazole antifungal
medication that is generally used to treat serious,
invasive fungal infections. Voriconazole has become the new
standard of care in the treatment of invasive aspergillosis, which
may occur in immuno compromised patients, including
Scheme 1. Synthesis of Propargylated N-(Pyrimidin-2-yl)benzo[d]thiazol-
2-amine 10.
allogeneic BMT,
transplants.31
other
hematologic,
and
solid organ
In continuation of our ongoing efforts on the design of novel
antitumor agents and realizing the importance of benzothiazoles,
pyrimidine, triazole and their derivatives in chemotherapy,32-34 it
was planned to synthesize and perform biological evaluation of
certain novel triazole linked N-(pyrimidin-2yl)benzo[d]thiazol-2-
amine derivatives.
Scheme 2. Synthesis of triazole derivatives of N-(Pyrimidin-2-yl)benzo[d]
thiazol-2-amine 5.
2

The new series of 1,2,3-triazole linked N-(pyrimidin-2-
yl)benzo[d]thiazol-2-amine Derivatives derivatives (5a–k) were
evaluated for their cytotoxic activity and observed the effect on
cell proliferation against four cancer cell lines such as human
breast cancer (MCF-7) cell line, Human lung cancer (A549) cell
line, Human melanoma (A375) cell line and normal breast
epithelial (MCF-10A) cell line by MTT assay. MTT assay is a
mitochondrial function assay that is based on the ability of viable
cells to reduce the MTT to insoluble formazan crystals by
Previous studies have revealed that derivatives of
benzothiazole control cell growth by affecting the cell cycle.39-41
Therefore in order to study the effect of these N-(pyrimidin-2-
yl)benzo[d]thiazol-2-amine derivatives on human breast cancer
(MCF-7) cells, FACS analysis was conducted. Here MCF-7 cells
were treated with compounds 5b, 5g, 5h and 5i at 2 and 4 µM
concentrations for 48h (Fig. 2, supplementary table 1). It is
observed that at less concentration (2µM) compounds caused
G2/M cell cycle arrest but on increasing the concentration (4µM)
lead to increase the percentage of apoptotic cells as compared to
untreated control cells. In FACS study, we have observed that
compounds induce apoptotic cell death of 8%, 33% (5b), 3%,
19% (5g), 17%, 29% (5h), and 5%, 22% (5i) at 2 and 4 µM
concentrations respectively. These results clearly demonstrate
that the identified compounds (5b, 5g, 5h and 5i) are more
effective in inducing apoptosis than Doxorubin (Doxo) and
Paclitaxel (Pacli) in MCF-7 cells (supplementary Table-2).
mitochondrial
dehydrogenase.37
2-(4-amino
phenyl)
benzothiazole (CJM-126) elicits potent growth inhibition in
human derived breast carcinoma cell lines including estrogen
receptor positive wild-type MCF-7.38 Here cell lines were treated
with compounds (5a-k) along with standard positive control such
as Doxorubin (Doxo) and Paclitaxel (Pacli). All these compounds
showed significant cytotoxicity with 5b, 5g, 5h and 5i being the
most effective molecules of all the molecules (Table 1;
supplementary Fig. 1, supplementary Table-1). Based on the IC50
value, it is observed that among all these cancer cell lines, human
breast cancer (MCF-7) cells are most sensitive to these
compounds. Also, these compounds did not cause significant
cytotoxicity in normal breast cells, indicating their specificity
towards cancerous cells.
Recent studies have revealed that molecules containing the
benzothiazole moiety exhibit anticancer activities through
inhibition of Raf/MEK/ERK signal cascade39-42. In addition,
studies have clearly demonstated that activation of key survival
proteins such as ERK1/2, NF-kB and surviving (IAP) cause rapid
cancer cell proliferation, survival, migration and tumor
progression.43-47 Among these, NF-kB protein acts as
transcription factor and regulates genes that control cell
proliferation and confer resistance to radiation and
chemotherapeutic agents while surviving inhibits apoptotic
process by inhibiting caspase 9, required for initiation of
mitochondrial mediated intrinsic pathway. Interestingly,
benzothiazole based small molecules were reported to influence
the CYP1A1, the microsomal enzyme that belongs to cytochrome
p450 family has been implicated in cancer cell response to the
therapeutic agents.48 Therefore herein we have made an attempt
to study the effect of these compounds on expression of ERK1/2,
NF-kB, surviving and CYP1A1. MCF-7 cells were treated with
compounds (5b, 5g, 5h and 5i) at 2 µM for 24 h. and we
observed a decrease in expression of these proteins especially in
cells treated with 5h and 5i. Thus, these molecules were found to
be effective targeting the survival proteins expression in MCF-7
cells (Fig. 3).
Table 1. In vitro cytotoxicity of triazole derivatives of N-
against
(pyrimidin-2-yl)benzo[d]thiazol-2-amine
human
breast cancer (MCF-7), Human lung cancer (A549), Human
melanoma (A375) and normal breast epithelial (MCF-10A)
cell line.
IC50 (µM)
Compound
MCF10A
24 ± 2.25
MCF7
A549
A375
5.2 ± 0.023
6.17 ± 0.5
5a
5b
5c
5d
5e
8.38 ± 1.13
4.45 ± 0.24
5.42 ± 0.13
6.4 ± 0.09
7.14 ± 0.17
6.02 ± 0.56
9.54 ± 0.87
8.59 ± 0.43
22 ± 0.172
21.33 ± 0.18
24 ± 2.45
3.0 ± 0.041
3.52 ± 0.11
4.64 ± 0.12
5.90 ± 0.051
4.46 ± 0.17
3.2 ± 0.123
2.52 ± 0.121
2.12 ± 0.18
4.68 ± 0.03
5.4 ± 0.023
3.4 ± 0.21
35 ± 1.23
9.16 ± 1.21
8.4 ± 1.09
Apoptosis is executed by a subfamily of cysteine proteases
known as caspases. Out of all caspases, caspase-8 was found to
be the initiator caspase and caspase-3 and caspase-9 were found
to be effector caspases. In contrast, inhibitor of apoptosis (IAP)
functions in an antagonistic manner to caspases.
5f
5g
5h
5i
31 ± 1.16
5.14 ± 0.22
4.62 ± 0.15
4.97 ± 0.22
5.48 ± 0.27
6.85 ± 0.118
4.97 ± 0.31
26 ± 1.23
6.028 ± 0.81
4.67 ± 0.45
4.7 ± 0.34
25 ± 2.62
It is well established fact that survivin, the inhibitor of
apoptosis, function by inhibiting caspase-9 that is required for
apoptosis. In general MCF-7 breast cancer cells lack endogenous
caspase-3 protein49,50 and thus caspase-9 is the effector caspase
that plays a role in apoptosis due to chemotherapeutic agents.
Thus in this study MCF-7 cells were treated with conjugates 5b,
5g, 5h and 5i at 2 µM concentration for 24 h and the lysates were
subjected to caspase-9 assay. As expected, we observed an
increase in caspase-9 activity in compound treated cells than in
untreated cells (Fig. 4), revealing the apoptosis-inducing ability
of these compounds.
29.33 ± 1.32
22.66 ± 3.42
21 ± 1.91
5j
9.38 ± 0.576
7.72 ± 0.028
6.83 ± 0.015
5k
10
4.62 ± 0.87
21 ± 1.28
Doxo
Pacli
0.12 ± 0.06
2.58 ± 0.03
24.0 ± 0.54
38.0 ± 1.15
3.13 ± 0.05
4.9 ± 0.25
7.2 ± 1.2
8.0 ± 1.1
In the present study, a series of novel triazole linked N-
(pyrimidin-2yl)benzo[d]thiazol-2-amine 5a-k were synthesized.
All these synthesized compound show significant anticancer
activity. Among the compounds tested, compounds 5b, 5g, 5h
and 5i have been identified to be the most effective molecules. In
all these selected cancer cell lines, mainly MCF-7 breast cancer
cells show high sensitivity with these compounds. Interestingly
these compounds did not cause significant cytotoxicity in normal
a IC50 is defined as the concentration, which results in a 50% decrease in cell
number as compared with that of the control cultures in the absence of an
inhibitor. The values represent the mean SE of four individual observations.
Doxo: Doxorubicin, Pacli: Paclitaxel.
3

breast cells. Further compound 5b, 5g, 5h and 5i affect the
expression of key proteins such as ERK1/2, NF-kB and Survivin
that cause abnormal cell proliferation. Hence compounds 5b, 5g,
5h and 5i could be a promising compound can be taken up for
further in vivo cancer studies that may be of interest in cancer
chemoprevention.
Figure 2. Effect of benzothiazole conjugates on cell cycle: MCF-7 cells were treated with compounds 5a-5k at 2 and 4 µM concentrations for 48h by flow
cytometry. Cells exhibited G2/M cell cycle arrest upon treatment with these compounds (5a-5k). Here Doxorubicin, paclitaxel and 10 were used as positive
controls.
4

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Authors are thankful to the CSIR New Delhi, India for the
financial support through XII five year plan project DITSF code:
CSC-0204, T.D. thanks to UGC, UK, K.N and K.R. thanks to
CSIR, New Delhi, for award of fellowships and R.M.K. thanks
S.E.R.C., Department of Science & Technology, Government of
India for financial assistance under the Fast Track Scheme for
young scientists (SR/FTP/CS-03/2010). MPB thanks CSIR 12th
FYP SMILE CSC0111 for providing financial support for
performing the biological evaluation of the work.
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6

Table 1. In vitro cytotoxicity of triazole derivatives of N-(pyrimidin-2-yl)benzo[d]thiazol-2-amine against
human breast cancer (MCF-7), Human lung cancer (A549), Human melanoma (A375) and normal breast
epithelial (MCF-10A) cell line.
IC 50 ( Um)
Compound
MCF 7
A549
A375
MCF 10 A
24 2.25
22 0.17
21.33 0.18
24 2.45
35 1.23
31 1.16
26 1.23
25 2.62
29.33 1.32
22.66 3.42
21 1.91
21 1.28
24.0 0.54
38.0 1.15
5a
5.2 0.023
3.0 0.041
3.52 0.11
4.64 0.12
5.90 0.051
4.46 0.17
3.2 0.123
2.52 0.121
2.12 0.18
4.68 0.03
5.4 0.023
3.4 0.21
0.12 0.06
2.58 0.03
6.17 0.5
4.45 0.24
5.42 0.13
6.4 0.09
8.38 1.13
6.02 0.56
9.54 0.87
8.59 0.43
9.16 1.21
8.4 1.09
6.02 0.81
4.67 0.45
4.7 0.34
9.38 0.57
7.72 0.02
6.83 0.01
7.2 1.2
5b
5c
5d
5e
7.14 0.17
5.14 0.22
4.62 0.16
4.97 0.22
5.48 0.27
6.85 0.11
4.97 0.31
4.62 0.87
3.13 0.05
4.9 0.25
5f
5g
5h
5i
5j
5k
10
Doxo
Pacli
8.0 1.1
a IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control cultures in the absence of an
inhibitor. The values represent the mean SE of four individual observations. Doxo: Doxorubicin, Pacli: Paclitaxel.