Synthesis and anticancer potential of benzothiazole linked phenylpyridopyrimidinones and their diones as mitochondrial apoptotic inducers

Article abstract of DOI:10.1016/j.bmcl.2013.11.057

A series of benzothiazole linked phenylpyridopyrimidinones (8a-g) and their diones (9a-g) have been designed, synthesized and evaluated for their anticancer activity. Among the series one of the conjugate 8b showed significant cytotoxicity against human cervical cancer cell line ME-180 with IC ₅₀ value of 4.01 μM. This compound was tested on the cell cycle perturbations and DNA damage. Flow cytometry analysis revealed that the compound 8b showed drastic cell cycle perturbations due to concentration dependent increase in the sub-G0 phase in ME-180 cell line. DNA fragmentation and Hoechst staining reveals that this compound induced cell death by apoptosis. Further caspase-3 and loss of mitochondrial membrane potential suggested that the compound induces cell death by apoptosis.

Full text of DOI:10.1016/j.bmcl.2013.11.057

Bioorganic & Medicinal Chemistry Letters 24 (2014) 147–151
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer potential of benzothiazole linked
phenylpyridopyrimidinones and their diones as mitochondrial
apoptotic inducers
⇑
Ähmed Kamal , Md. Ashraf, M. V. P. S. Vishnu Vardhan, Shaikh Faazil, V. Lakshma Nayak
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Tarnaka, 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:
A series of benzothiazole linked phenylpyridopyrimidinones (8a–g) and their diones (9a–g) have been
designed, synthesized and evaluated for their anticancer activity. Among the series one of the conjugate
8b showed significant cytotoxicity against human cervical cancer cell line ME-180 with IC50 value of
Received 28 August 2013
Revised 6 November 2013
Accepted 22 November 2013
Available online 1 December 2013
4.01 lM. This compound was tested on the cell cycle perturbations and DNA damage. Flow cytometry
analysis revealed that the compound 8b showed drastic cell cycle perturbations due to concentration
dependent increase in the sub-G0 phase in ME-180 cell line. DNA fragmentation and Hoechst staining
reveals that this compound induced cell death by apoptosis. Further caspase-3 and loss of mitochondrial
membrane potential suggested that the compound induces cell death by apoptosis.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Benzothiazole linked-
phenylpyridopyrimidinone
Cytotoxicity
Cell cycle arrest
Cancer, the uncontrolled growth of cells, is a major cause of
death throughout the world. Currently chemotherapy is one of
the treatments for cancer and its ultimate goal is to produce a drug
which can specifically destroy cancer cells without having any sig-
nificant effect on the normal cells. Cancer chemotherapy has
achieved significant success through the discovery of various
new drugs. E7010 (1), a sulphonamide class of molecule, inhibits
tubulin polymerization1 and causes cell cycle arrest in M phase.2,3
This compound exhibits good in vivo antitumor activity against
several rodent as well as human tumours and is presently in phase
II clinical trials.4 Based on E7010 (1) we have previously designed
and synthesized a series of 2-anilinonicotinyl linked aminobenzo-
thiazoles (2) which showed potent cytotoxicity, cell cycle arrest
and DNA fragmentation.5 Similarly, 2-anilinonicotinyl sulfonylhyd-
razide conjugates (3) also showed promising anticancer activity,6
however 2-anilinonicotinyl linked oxadiazoles (4) derivatives
proved to be good inhibitors of tubulin polymerization.7 Benzo-
thiazoles are known to exhibit various biological properties includ-
ing antimicrobial, anticancer, anti-amyloid, anti-rheumatic and
anti-glutamate activities.8–11 Various modified aryl benzothiazoles
and substituted 2-aminobenzothiazoles are also known to possess
significant anticancer activity both in vitro and in vivo models
(Fig. 1).12–16
agents.17–21 Based on these successes an attempt has been made
to further focus on 2-anilino-pyridyl structural component of
E7010. These newly designed benzothiazole linked phenylpyrido-
pyrimidinones (8a–g) and their diones (9a–g) were synthesized
by cyclization of 2-anilinonicotinyl linked aminobenzothiazoles
by inserting of CH2 group and carbonyl group, respectively. These
newly designed compounds were evaluated for their cytotoxic po-
tential on four different cancer cell lines with IC50 values lying in
the range of 4.01–44.7
8b has shown promising cytotoxicity specifically against human
cervical cancer cell line ME-180 with IC50 value of 4.01 M. Flow
lM. Among the series one of the compound
l
cytometric analysis of this compound shows arrest of cell cycle
in the G0/G1 phase. Hoechst 33258 staining and DNA fragmenta-
tion assay reveals that this compound induces cell death by apop-
tosis. Further, activation of caspase-3 and loss of mitochondrial
membrane potential also suggested that this compound produces
apoptotic cell death (Scheme 1).
2-Chloronicotinic acid was converted into ethyl 2-chloronicoti-
nate (2) by refluxing with ethanol and H2SO4 at 80 °C for 2 h. The
substituted ethyl 2-anilinonicotinate is prepared by using standard
method5 from ethyl 2-chloronicotinate by reaction with various
substituted anilines in ethylene glycol at 160 °C for 8 h. Further,
hydrolysis of ester (4a–d) by using solution of 2N NaOH in ethanol
for 2 h under reflux afforded corresponding substituted 2-anilinon-
icotinic acid (5a–d). Synthesis of 7a–g was carried out by the
reaction of respective 2-anilinonicotinic acids with 6-substituted
2-amino benzothiazoles (6a–d) by using EDCI/HOBt in dry
DMF as solvent. The target substituted benzothiazole linked
Our earlier efforts towards the synthesis of
a variety of
hybrid molecules led to the development of promising anticancer
⇑
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (Ä. Kamal).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.057

148
Ä. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 147–151
X
O
N
O
X
O
N
S
N
S
N
H
N
N
N
NH
R1
R2
R1
R3
R2
R4
R3
2
9a-g
O
X
O
N
N
H
N
X
S
S
N
O
O2
H
N
N
NH
N
N
H
S
E7010
O
O
Y
N
NH
R2
R1
R3
OH
1
R2
8a-g
R1
3
O
S
O
N
X
Y
N
N
Z
N
N
R
N
NH
N
NH
O
R1
R2
N
NH
R4
R3
R3
R1
5
R2
4
Figure 1. Flow chart of various series derived from E7010 in our laboratory.
phenylpyridopyrimidinones (8a–g) and diones (9a–g) are prepared
by the using standard methodology from 7a–g in good yields. Com-
pounds 8a–g obtained by the reaction of 7a–g with formaldehyde
in water and acetic acid (9:1) for 1 h and 9a–g were prepared by
the reaction of 7a–g in THF with triphosgene and triethylamine
was added as a base and the reaction mixture was refluxed at
60 °C for 2 h. The chemical structures of target substituted conju-
gates 8a–g and 9a–g were confirmed by IR, NMR and MS spectral
analysis and the molecular formula of all the compounds were
determined by elemental analysis.
The synthesized compounds have been tested for their cytotox-
icity against a different set of human cancer cell lines MCF-7
(breast), B-16 (melanoma), ME-180 (cervical), DU-145 (prostate
cancer) using MTT assay22 and the values obtained were compared
to the standard drug like 5-flurouracil as shown in Table 1. The
screening results suggest that 8b exhibits significant activity in
induces apoptosis and/or cell cycle arrest.23 Data from MTT assay
showed that conjugate 8b induced significant inhibition of cervical
cancer cells. It was of interest to understand whether this inhibi-
tion of cell growth was on account of cell cycle arrest. Hence, we
studied the cell cycle distribution of this conjugate by treating
the cells with propidium iodide-labelling and followed by fluores-
cence-activated cell sorting analysis. Results (Fig. 2 and Table 2)
indicated that 8b showed cell cycle arrest at G0/G1 phase in 48 h
as compared to the untreated control that activates signalling for
DNA repair initially and in failure of DNA as shown in Figure 3.
Apoptosis was also assessed by electrophoresis of extracted
genomic DNA from cells. Endonuclease mediated cleavage of
nuclear DNA results in the formation of oligonucleosomal DNA
fragment (180–200 base pairs long) a biochemical hallmark of
apoptosis in a number of cell types.24,25 DNA laddering assay was
performed with ME-180 cells by treatment of 8b at a concentration
ME-180 cell line with IC50 value 4.01
lM that is comparable to
of 4 lM for 24 h, then the genomic DNA was isolated and fallowed
the standard 5-flurouracil as shown in Table 1. To study the cyto-
toxic effect on normal cell lines, compound 8b was screened on
HEK-293 and the result suggested that this conjugate (8b) was
10 fold less toxic to normal cell exhibiting an IC50 value of
by electrophoresis in 1.8% agarose gel. The conjugate 8b induces
DNA fragmentation which is a characteristic ladder pattern in
ME-180 cells at 4 lM concentration whereas such laddering was
observed in the control as shown in Figure 4.
42.1
l
M. Therefore, it was considered of interest to examine the
Further confirmation of the ability of the most potent
compound 8b to induce apoptosis was obtained by analysis of
effect of 8b on cell cycle progression by FACS analysis.
The tumor suppressor gene p53 is a multifunctional protein
responsible for maintaining genomic integrity and its mutation is
known to cause tumors in humans. In response to DNA damage,
aberrant growth signals, or chemotherapeutic drugs, p53 repair,
drug-treated ME-180 cell populations (4 lM, 24 h) by fluorescence
microscopy.26 Following drug treatment morphological changes of
ME-180 cells was observed. The untreated cells possess large sized
nuclei, whereas the incubation for 24 h at 4 lM concentration of

Ä. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 147–151
149
COOEt
NH
NH2
COOH
Cl
COOEt
N
(i)
(ii)
+
N
N
Cl
R1
R3
R1
R3
R2
1
R2
2
3a-d
3a: R1 = R2 = R3 = H
3b: R1 = R2 = R3 = OMe
3c: R1 = R3 = H; R2 = F
4a-d
: R1 = R3 = H; R2 = OMe
(iii)
3d
X
O
N
COOH
NH
X
O
N
N
N
S
S
N
H
NH2
S
N
N
N
NH
X
(v)
(iv)
+
N
6a-d
X = NO2., F, OMe, OEt.
R1
R3
R1
R3
R2
R1
R3
R2
7a-g
5a-d
R2
8a-g
8a: R1 = R2 = R3 = H; X = F
(vi)
8b: R1 = R2 = R3 = H; X = OEt.
: R1 = R2 = R3 = OMe; X = NO2.
8c
8d
8e
X
O
N
N
N
O
1
: R = R3 = H, R2 = F, X = OMe
: R1 = R3 = H; R2 = F; X = OEt.
S
8f: R1 = R3 = H; R2 = F; X = NO2
8g: R1 = R3 = H; R2 = OMe; X=F
N
R1
R3
R2
9a-g
9a: R1 = R2 = R3 = H; X = F
9b: R1 = R2 = R3 = H; X = OEt.
: R1 = R2 = R3 = OMe; X = NO2.
9c
9d
9e
1
: R = R3 = H, R2 = F, X = OMe
: R1 = R3 = H; R2 = F; X = OEt.
9f: R1 = R3 = H; R2 = F; X = NO2
9g: R1 = R3 = H; R2 = OMe; X=F
Scheme 1. Reagents and conditions: (i) ethanol, H2SO4, 80 °C, 2 h; (ii) ethylene glycol, 160 °C, 8 h; (iii) 2N NaOH, ethanol, 80 °C 2 h; (iv) EDCI, HOBT, DMF, 8 h; (v)
formaldehyde, acetic acid, H2O (1:9), 1 h; (vi) triphosgene, THF, 60 °C, 2 h.
Table 1
8b revealed fragmentation of nuclei due to apoptosis in ME-180
cells (Fig. 5).
Cytotoxic effects of compounds 8a–g and 9a–g on various cancer cell lines. The bold
values signifies the Potent cytotoxic effect against the corresponding cell lines.
Most of the proteolytic cleavages during apoptosis result from
the activation of caspases, a family of cysteine-dependent prote-
ases.27 Caspases can initiate apoptotic signals and execute the
apoptotic programs. Hence, the involvement of various caspases
and their role in the process of apoptosis has been investigated.
Compounds that showed apoptosis induction in flow cytometric
assay was also evaluated for their ability to induce caspase activity
Compound
IC50
MCF-7b
(lM)
ME-180a
B-16c
DU-145d
8a
8.30
4.01
>20
9.9
>20
7.8
6.1
8.1
15.2
6.4
8be
12.1
>20
>20
5.1
12.1
13.8
15.2
12.5
19.9
17.5
>20
7.4
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
>20
>20
>20
14.2
>20
>20
>20
>20
12.3
11.6
13.7
19.9
3.5
5.1
6.10
13.23
16.2
>20
15.8
>20
>20
13.9
19.0
18.9
4.9
12.1
>20
5.2
12.5
19.9
>20
9.3
17.5
8.3
>20
—
in ME-180 cells. Conjugate 8b upon treatment at 2 and 4 lM con-
centrations for 48 h in ME-180 cells activated caspase-3 by 4–6
folds when compared to control as shown in the Figure 6. The acti-
vation of caspase-3 by 8b at both concentrations indicates that it
induces apoptosis through both the intrinsic as well as extrinsic
apoptotic pathway in ME-180 cells.
The loss of mitochondrial membrane potential is a major char-
acteristic of apoptosis.28 Our results have shown that 8b disrupts
mitochondrial membrane potential. The result of JC-1 staining
14.6
4.2
5-Fluorouracil
a
b
c
Cervical cancer.
Breast cancer.
Mouse macrophages cell line.
Prostrate cancer.
indicates loss of DWm which displays strong evidence of apoptosis
in this conjugate 8b. Thus, the data generated has shown high per-
centage of chromatin condensation, nucleus fragmentation, and
d
e
loss of
DWm in this conjugate.
Normal cell line HEK-293 (IC50 of 8b is 42.1 lM).

150
Ä. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 147–151
Figure 2. Flow-activating cell sorting (FACS) analysis of compounds 8b at 2 and 4 lM. Effect of compound 8b on cell cycle distribution of ME-180 cell line was examined by
staining cells with propidium iodide.
Table 2
Compound
Control
G0
2.97
2.38
13.18
G1
S
G2/M
69.80
79.78
79.54
3.40
1.57
1.49
23.82
16.41
5.79
8b (2
8b (4
l
l
M)
M)
% of DNA content in G0, G1, S and G2/M phase in ME-180 cells.
Figure 5. Effect of compound 8b on caspase-3, activities in ME-180 cells
determined by fluorimetry at and after 48 h incubation. Error bars
2
4 lM
represents data from two independent experiments with each performed in
duplicate. Data are mean SD from three independent experiments.
In the present study, two new series of benzothiazole linked
phenylpyridopyrimidinones 8a–g and their dione conjugates
9a–g have been designed, synthesized and evaluated for their cyto-
toxic activity against four human cancer cell lines (ME-180, DU-
145, MCF-7, B-16). Some of these compounds exhibit promising
cytotoxicity at micromolar (
one compound 8b exhibited significant anticancer activity (IC50
4.1 M) against ME-180 cancer cell line and flow cytometric anal-
lM) concentration. Among the series
,
l
ysis of this compound shows arrest of cell cycle in the G0/G1 phase.
Hoechst 33258 staining and DNA fragmentation assay reveals that
this conjugate induces cell death by apoptosis. Further, activation
of caspase-3 loss of mitochondrial membrane potential also sug-
gested that this compound produces apoptotic cell death. There-
fore this is an interesting lead to take up future structural
modifications on this scaffold.
Figure 3. Effect of compound 8b on DNA fragmentation on ME-180 cells. Cells were
incubated for 24 h with 4 M concentration.
l
Figure 4. Fluorescent microscopy pictures showing morphological changes in ME-180 cells treated with 4 lM, compound 8b for 24 h.

Ä. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 147–151
151
Figure 6. The loss of mitochondrial membrane potential was monitored by JC-1 stain. High
D
W
m in control mitochondria causes association with the dye in an aggregated
state leading to red-orange fluorescence emission. Compound 8b lead to dissipation of
DWm, and the dye is now partitioned into cytosol, resulting in green fluorescence.
9. Hutchinson, A.; Chua, M.; Browne, H. L.; Trapani, V.; Bradshaw, T. D.; Westwell,
Acknowledgments
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M.A. and S.F. acknowledge CSIR-UGC, New Delhi and for the
award of senior research fellowship. We also acknowledge CSIR
for financial support under the 12th Five Year plan project ‘Afford-
able Cancer Therapeutics (ACT)’ (CSC0301).
Supplementary data
Supplementary data (experimental procedures and spectro-
scopic characterization of the compounds) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2013.11.057.
17. Kamal, A.; Khan, M. N. A.; Srikanth, Y. V. V.; Reddy, K. S.; Juvekar, A.; Sen, S.;
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18. Kamal, A.; Tekumalla, V.; Krishnan, A.; Bhadra, M. P.; Bhadra, U. Chem. Med.
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