Design, synthesis and anticancer properties of novel oxa/azaspiro[4,5]trienones as potent apoptosis inducers through mitochondrial disruption

Article abstract of DOI:10.1016/j.ejmech.2015.06.050

Abstract A series of twenty seven oxa/azaspiro[4,5]trienone derivatives were synthesized and their anticancer properties have been explored. GI₅₀ values of all these compounds were evaluated against four types of human cancer cell lines, i.e. MCF-7 (breast), DU-145 (prostate), A549 (lung) and HepG2 (liver). Five compounds of the series exhibited good anticancer potential against MCF-7 with GI₅₀ values less than 2 μM. Detailed biological studies of the two representative compounds 9b and 9e revealed that they arrest cell cycle in G0/G1 phase and induce mitochondria mediated apoptosis, that was further confirmed by measurement of mitochondrial membrane potential (ΔΨm), intracellular ROS generation, caspase 9 activity and Annexin V-FITC assay. Furthermore, western blot analysis suggested that these compounds up-regulate the levels of p53, p21, p27 and Bax, and down-regulate the level of Bcl-2 confirming the apoptosis inducing properties.

Full text of DOI:10.1016/j.ejmech.2015.06.050

European Journal of Medicinal Chemistry 101 (2015) 348e357
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and anticancer properties of novel oxa/azaspiro[4,5]
trienones as potent apoptosis inducers through mitochondrial
disruption
D. Yugandhar a, b, V. Lakshma Nayak , Sivakumar Archana , Kunta Chandra Shekar ,
Ajay Kumar Srivastava
a
a
a
a
a, b, *
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India
Academy of Scientific and Innovative Research (AcSIR), NewDelhi, 110 025, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 March 2015
Received in revised form
A series of twenty seven oxa/azaspiro[4,5]trienone derivatives were synthesized and their anticancer
properties have been explored. GI50 values of all these compounds were evaluated against four types of
human cancer cell lines, i.e. MCF-7 (breast), DU-145 (prostate), A549 (lung) and HepG2 (liver). Five
compounds of the series exhibited good anticancer potential against MCF-7 with GI50 values less than
23 June 2015
Accepted 27 June 2015
Available online 2 July 2015
2 mM. Detailed biological studies of the two representative compounds 9b and 9e revealed that they
arrest cell cycle in G0/G1 phase and induce mitochondria mediated apoptosis, that was further confirmed
by measurement of mitochondrial membrane potential (DJm), intracellular ROS generation, caspase 9
activity and Annexin V-FITC assay. Furthermore, western blot analysis suggested that these compounds
up-regulate the levels of p53, p21, p27 and Bax, and down-regulate the level of Bcl-2 confirming the
apoptosis inducing properties.
Keywords:
Ipsocyclization
Oxaspiro[4,5]trienone
Azaspiro[4,5]trienone
Anticancer activity
Cell cycle analysis
© 2015 Elsevier Masson SAS. All rights reserved.
Mitochondrial membrane potential (DJm)
Western blot analysis
Apoptosis
1. Introduction
[3]. In our efforts to study the pharmaceutical potential of new
scaffolds, we have identified novel oxaspiro[4,5]trienones; II pos-
Identification of novel scaffolds with improved pharmaceutical
properties is always exciting to medicinal chemists towards the
development of new chemical entities (NCEs). There are several
scaffolds of significant synthetic interests whose pharmaceutical
properties need to be explored [1]. The newly designed molecules,
mimicking drug skeletons with additional functionalities, can
display improved or reduced pharmaceutical potential depending
on their interactions with biological system. Hence, a detailed
mechanistic investigation and structure activity relationship (SAR)
studies of the new skeletons are required for developing pharma-
cophores with improved efficacy [2]. With the exceeding cases of
cancer patients every year and lesser success in anticancer drug
development, new scaffolds need to be identified as potential leads
sessing anti-proliferative properties [4]. These spiro[4,5]trienones
were designed as constrained tamoxifen; I mimic to develop new
selective estrogen receptor modulators (SERMs), however in vitro
screening of these compounds revealed that they are not selective
towards ER positive breast cancer cell line (MCF-7) and were found
to be equally potent against ER negative breast cancer cell line
(MDA-MB-231) [Fig. 1] [4]. In continuation to our previous work,
herein we report the detailed biological profiling of oxaspiro[4,5]
trienones; II and azaspiro[4,5]trienones; III along with their
structure activity relationship (SAR) studies. The literature survey
shows that structurally related antitumor compounds bearing
enone moiety may facilitate ROS production which in turn can
induce apoptosis [Fig. 1] [5e8]. Anthracenyl amino acid IV [5],
Dehydroaltenusin V [6]; a natural product isolated from a fungus
Alternaria tennuis, and its tautomer VI, inhibit DNA polymerase
a
and induce apoptosis. Increase in ROS production by small mole-
cules triggers the cancer cell death [7], for example, a dienone
containing natural product derivative Deoxynyboquinone VII
*
Corresponding author. Medicinal Chemistry and Pharmacology Division, CSIR-
Indian Institute of Chemical Technology, Hyderabad, 500 007, India.
E-mail address: aksrivastava@iict.res.in (A.K. Srivastava).
http://dx.doi.org/10.1016/j.ejmech.2015.06.050
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
349
boronic acids; 8 produced 10a-o in good yields [Scheme 1]. All the
1
13
compounds were characterized by H, C NMR and HRMS analysis.
Purity of all the newly synthesized compounds was checked by
HPLC.
2
2
.2. Biology
.2.1. Anticancer activity
In vitro cytotoxicity of compounds 9a-h and 10a-o were evalu-
ated against four types of human cancer cell lines; MCF-7 (Breast-
ER positive), DU-145 (Prostate), A549 (Lung) and HepG2 (Liver) by
using sulforhodamine B (SRB) method [11]. There are several rapid
colorimetric assays described for in vitro chemo sensitivity testing
of tumor cell lines. While tetrazolium [MTT; 3-(4,5-
dimethylthiazolyl-2)-2, 5 diphenyltetrazoliumbromide] assay be-
ing the most widely used, recently the US National Cancer Institute
(
NCI) recommended use of the sulforhodamine B (SRB) protein
Fig. 1. Recently reported apoptosis inducing agents and our designed prototypes.
stain for in vitro chemo sensitivity testing. The SRB assay appeared
to be more sensitive than MTT assay, with better linearity with cell
number and higher reproducibility [12]. The compounds exhibiting
exhibits excellent tumoristatic potential in animal models through
ROS generation [8].
GI50 ꢀ 10
mM were considered to be active against the respective
cancer cell lines. The growth inhibition data (expressed as GI50) of
compounds 9a-h and 10a-o are shown in Table 1. The results of the
cytotoxicity assay showed that compounds 9b and 9e possess sig-
nificant anti-proliferative properties against the human breast
cancer cell line MCF-7. The promising activity of 9b and 9e
prompted us to examine their pharmacological properties in detail.
ROS production in cancer cells regulates cellular growth, cell
signaling and synthesis of important substances. Furthermore,
mitochondria plays an important role for the survival of cancer
cells, hence disrupting mitochondrial function by small molecules
induces apoptosis [9,10]. Considering these findings from literature,
we became interested to examine the apoptosis inducing proper-
ties of oxa/azaspiro[4,5]trienones.
2.2.2. Cell cycle analysis
2
. Results and discussion
In general, anticancer agents prevent cell division at various
checkpoints of cell cycle, thereby decreasing the growth and pro-
liferation of cancerous cells [13]. Cell cycle analysis after treatment
with potent anticancer agents shows the distinguish cells in
different phases of the cell cycle. In this study, MCF-7 cells were
2
.1. Chemistry
As reported in our previous communication, oxaspiro[4,5]tri-
enones 9a-g were obtained by coupling of compound 6 with
various boronic acids; 8 under Suzuki conditions [4]. Compound 9h
was prepared by Heck reaction of 6 with methylacrylate. Azaspiro
treated with compound, 9b and 9e at 0.5 and 1 mM concentrations
for 48 h. The data obtained clearly indicated that these compounds
(9b and 9e) showed G0/G1 cell cycle arrest when compared to
untreated control (Fig. 2, Table 2).
[4,5]trienones 10a-o were synthesized from N-methyl-p-anisidine;
2
by following similar synthetic strategy. N-methyl-p-anisidine; 2
was coupled with 3-phenylpropiolic acid; 3 to yield amide; 5 which
was subjected to iodine mediated ipso-iodocyclization for the
construction of compound 7. Suzuki coupling of 7 with various
2.2.3. Measurement of mitochondrial membrane potential (DJm)
The maintenance of mitochondrial membrane potential (DJm)
is important for mitochondrial integrity and bioenergetic function
Scheme 1. Synthesis of spirotrienediones.

350
D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
Table 1
Anticancer activity data of compounds 9a-h and 10a-o.
Entry
Compound
R
GI50
(
m
M)
MCF-7
DU-145
A549
6.2 ± 0.03
HepG2
1
2
3
4
5
7
8
9
9a
9b
9c
9d
9e
9f
9g
9h
4-OH-Ph
3.4 ± 0.17
1.0 ± 0.04
1.8 ± 0.02
1.7 ± 0.15
1.0 ± 0.02
3.5 ± 0.13
3.4 ± 0.01
1.2 ± 0.25
7.6 ± 0.09
4.2 ± 0.16
8.1 ± 0.07
2.4 ± 0.08
8.2 ± 0.04
3.6 ± 0.31
6.7 ± 0.16
9.9 ± 0.12
52.7 ± 1.93
71.7 ± 7.27
11.1 ± 0.15
13.61 ± 0.61
45.4 ± 5.14
7.4 ± 0.14
15.8 ± 0.37
1.6 ± 0.08
8.0 ± 0.20
2.7 ± 0.05
1.9 ± 0.32
4.1 ± 0.10
2.4 ± 0.14
0.9 ± 0.15
4.8 ± 0.04
4.0 ± 0.05
4.3 ± 0.37
11.5 ± 0.56
9.0 ± 0.31
17.6 ± 0.36
6.3 ± 0.20
15.5 ± 0.66
8.2 ± 0.11
16.3 ± 1.25
14.0 ± 0.35
91.2 ± 7.33
62.7 ± 5.83
18.2 ± 1.82
11.6 ± 0.40
55.7 ± 0.47
12.1 ± 1.19
26.4 ± 4.27
0.95 ± 0.20
19.3 ± 0.79
3.5 ± 0.15
1.5 ± 0.04
3.4 ± 0.23
3.3 ± 0.15
1.4 ± 0.12
6.3 ± 0.21
2.6 ± 0.02
6.3 ± 0.18
7.7 ± 0.54
7.2 ± 0.10
13.7 ± 0.16
6.12 ± 0.23
9.2 ± 0.11
13.9 ± 0.22
19.6 ± 0.20
10.5 ± 0.63
62.8 ± 1.76
69.1 ± 6.64
12.7 ± 0.19
16.6 ± 0.64
61.7 ± 8.30
10.4 ± 0.04
18.7 ± 0.20
1.4 ± 0.27
21.7 ± 0.97
4-NO
4-OMe-Ph
4-CH OH-Ph
2-OMe,5-F-Ph
4-CO H-Ph
5-CHO-Thiophenyl
eCH]CHeCO Me
4-CH OH-Ph
2-benzothiophenyl
4-CF Ph
2
-Ph
4.0 ± 0.01
7.3 ± 0.16
8.1 ± 0.04
4.6 ± 0.14
6.3 ± 0.02
2
2
4.6 ± 0.38
7.8 ± 0.04
2
10
13
14
15
16
17
18
19
20
21
22
23
24
25
27
28
29
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
Doxorubicin
Tamoxifen
2
13.6 ± 0.47
18.8 ± 1.87
30.3 ± 2.83
17.3 ± 0.90
22.5 ± 1.91
16.6 ± 0.21
30.2 ± 1.34
27.6 ± 1.06
126.0 ± 3.18
100.6 ± 7.61
36.2 ± 1.36
25.4 ± 2.13
98.0 ± 0.70
21.1 ± 0.91
50.4 ± 0.27
1.9 ± 0.12
3
1-Naphthyl
Ph
5-CHO-2-thiophenyl
3,4-Methylenedioxy-Ph
2,3-Cl-Ph
3-Furyl
2,4-OMePh
4-CHO-Ph
4-CN-Ph
4-OMe-Ph
3-Cl,4-Me-Ph
4-SMe-Ph
e
e
24.4 ± 0.26
GI50 values represent as mean ± SD of three determinations.
[
14]. Mitochondrial changes, including loss of mitochondrial
significant apoptosis against MCF-7 cells as shown in Fig. 5 and
Table 3. Results indicated that compounds 9b and 9e showed 12.63
and 11.47% and 39.0 and 30.85% apoptosis respectively at 0.5 and
membrane potential (DJm), are key events that take place during
drug-induced apoptosis. Mitochondrial injury by 9b and 9e was
evaluated by detecting drops in mitochondrial membrane potential
1 mM concentrations for 48 h.
(DJm). In this study, we have investigated the involvement of
mitochondria in the induction of apoptosis by these compounds.
After 48 h of treatment with compounds 9b and 9e, reduction in
mitochondrial membrane potential (DJm) of MCF-7 cells was
observed, that was assessed by JC-1 staining [Fig. 3].
2.2.6. Activation of caspase-9
The activation of caspases plays an important role in the process
of programmed cell death or apoptosis. Caspases or cys-
teineaspartic proteases are crucial mediators of apoptosis. The
MCF-7 cells lack endogenous caspase-3, whereas caspase-9 plays
an important role in mediating drug-induced apoptosis [17]. MCF-7
2.2.4. Effect on intracellular ROS generation
Many anticancer agents have demonstrated to exert their
cells were treated with compounds 9b and 9e at 1 mM concentra-
cytotoxic effects by the generation of reactive oxygen species (ROS)
15], which is considered as one of the key mediators of apoptotic
tion for 48 h. The results demonstrate that there was 2e3 fold in-
duction in caspase-9 activity when compared to untreated control
[Fig. 6] which suggested that they have the ability to induce cell
death by apoptosis in MCF-7 cells.
[
signaling. Therefore, we decided to investigate the role of com-
pounds 9b and 9e in inducing the production of ROS that could
potentially lead to the cytotoxic effect in the MCF-7 cells. In order to
demonstrate the role of compounds on ROS generation during
apoptosis process, production of ROS was examined by using an
2.2.7. Western blot analysis of Bcl-2, Bax, p53, p21 and p27
The members of the Bcl-2 family play a pivotal role in the
regulation of the mitochondrial apoptotic pathway. Among these,
Bcl-2 inhibits apoptosis, whereas Bax counterbalances the Bcl 2
effect and stimulates apoptosis [18]. Activation of tumor sup-
pressor genes such as p53 and p27 is found to be important in the
regulation of apoptotic pathway induced by various stimuli [19].
The p53 gene, is a tumor suppressor gene and its activity stops
the formation of tumors. p27 is considered the main regulator of
G1 phase activity, and p21 function as a sensor of cytostatic sig-
nals and is mainly regulated by the p53 tumor suppressor gene
oxidant-sensitive
(
fluorescent
2 ,7 dichlorofluorescindiacetate). After treatment with com-
pounds 9b and 9e at 0.5 and 1 M concentrations for 48 h, the level
probe,
DCFDA
0
0
m
of ROS was significantly increased [Fig. 4]. The ratio of DCF-positive
cells for compounds 9b and 9e was 30.4 and 16.2% and 78.5 and
57.4% respectively at 0.5 and 1 mM concentrations for 48 h. Un-
treated control cells showed only 1.2% DCF positive cells. The results
confirmed that these compounds had enhanced the generation of
ROS in MCF-7 cells.
[
20]. In the cell cycle study, we observed that these compounds
2
.2.5. Annexin V-FITC for apoptosis
The apoptotic effects of 9b and 9e were further evaluated by
caused cell cycle arrest at G1 phase. To further evidence the
expression of possible G1 cell cycle-associated regulators (p53,
p21 and p27) along with proapoptotic protein bax and anti-
apoptotic protein bcl-2 were detected by western blot analysis. As
shown in Fig. 7, we observed that compounds 9b and 9e induced
G1 cell cycle arrest in MCF-7 cells might be through induction of
p53, p21, p27 and bax followed by a decrease in the expression of
Bcl-2.
Annexin V FITC/PI (AV/PI) dual staining assay [16] to examine the
occurrence of phosphatidylserine externalization and also to un-
derstand whether it is due to physiological apoptosis or nonspecific
necrosis. In this study MCF-7 cells were treated with compounds 9b
and 9e for 48 h at 0.5 and 1
apoptotic effect. It was observed that these compounds showed
mM concentrations to examine the

D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
351
Fig. 2. Flow cytometric analysis in MCF-7 breast cancer cell lines after treatment with compounds. A: Control cells (MCF-7), B: 9b (0.5
m
M), C: 9b (1
mM), D: 9e (0.5
mM) and E: 9e
(1 mM).
Table 2
Considering the better growth inhibition shown by oxaspiro[4,5]
trienones over azaspiro[4,5]trienones, it was inferred that a lipo-
philic replacement of oxygen might improve the anticancer po-
tential of these scaffolds. Hit optimization with various possible
derivatives of spiro[4,5]trienones is currently underway and will be
reported in due course.
Effect of compounds 9b and 9e on cell-cycle phase distribution in MCF-7 cells.
Sample
Sub G1%
G0/G1%
S %
G2/M %
A: Control (MCF-7)
2.75
4.38
3.69
2.89
3.25
77.79
84.21
89.15
81.44
88.42
0.99
0.34
1.75
1.12
1.30
11.98
3.35
0.76
9.63
2.20
B: 9b (0.5
C: 9b (1
D: 9e (0.5
E: 9e (1 M)
m
M)
M)
M)
m
m
m
3.1. Experimental
3
. Conclusions
In summary, we have discovered oxa/azaspiro[4,5]trienones as
3.1.1. General
Compound 6 and 7 were synthesized from commercially avail-
able chemicals using previously reported procedures. Reagents and
solvents were purchased from locally available commercial sup-
pliers and were used without further purification. H and C NMR
spectra were obtained on Bruker DRX-300 and Avance-500.
Chemical shifts were reported in ppm on the basis of a compari-
son with tetramethylsilane (TMS) as an internal standard or re-
sidual solvent peak (for H NMR spectra, CDCl
NMR spectra, CDCl : 77.16). Multiplicity was indicated as follows: s
(singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd
novel scaffolds for anticancer drug development. The synthesis is
straightforward and high yielding. The biological studies revealed
that these compounds cause cell cycle arrest at G0/G1 phase and
induced mitochondria mediated apoptosis. Studies like, measure-
ment of mitochondrial membrane potential, ROS generation, Cas-
pase 9 activity, Annexin V-FITC assay and western blot analysis
demonstrated that these compounds disrupt mitochondrial po-
tential by increasing ROS production in order to induce apoptosis.
1
13
1
13
3
: 7.26 and for
C
3

352
D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
Fig. 3. Compounds 9b and 9e triggers mitochondrial injury. Drops in membrane potential (DJm) was assessed by JC-1 staining of MCF-7 cells treated with test compound and
samples were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1, FL2 channel to detect mitochondrial potential. A: Control cells (MCF-7), B: 9b
(
0.5 mM), C: 9b (1 mM), D: 9e (0.5 mM) and E: 9e (1 mM).
(
doublet of doublet), bs (broad singlet), etc., and coupling constants
temperature and reaction mixture dissolve in Ethylacetate (5 mL),
then crushed ice was added to the mixture, compound was
extracted with ethylacetate (3 ꢂ 30 mL). The organic layers were
collected, combined, washed with saturated aq NaCl (2 ꢂ 25 mL),
are given in hertz (Hz). The low-resolution mass spectrometric
analysis was performed at National Center for Mass Spectrometry,
CSIReIICT. The conversion of starting materials was monitored by
thin layer chromatography (TLC) using silica gel plates (silica gel 60
2 4
dried over anhydrous Na SO , filtered and concentrated under
F254 0.25 mm), and the reaction components were visualized by
reduced pressure. The crude compound was purified by column
observation under UV light (254 and 365 nm) or by treatment of
TLC plates with visualizing agents such as KMnO4, anisaldehyde
and ceric sulfate which was followed by heating. All reactions were
carried out in oven-dried glassware or 20 mL vials with silicon caps.
Products were purified by column chromatography on silica gel
chromatography on silica gel to afford 9h (132 mg). Yield ¼ 74%;
1
R
f
¼ 0.43 (30% EtOAc in hexane); H NMR (500 MHz, CDCl
3
)
d
7.51e7.47 (m, 1H), 7.43 (td, J ¼ 1.37 Hz, 2H), 7.27 (d, J ¼ 1.52 Hz,
1H), 7.27 (s, 1H), 7.24 (d, J ¼ 8.08 Hz, 2H), 6.65 (dt, J ¼ 1.83, 2.89 Hz,
13
2H), 6.40 (dd, J ¼ 1.67, 10.07 Hz, 2H), 3.77 (s, 3H), C NMR
(125 MHz, CDCl 183.5, 168.9, 166.8, 163.7, 142.0, 132.1, 131.2,
(
100e200 mesh) using a mixture of EtOAc/hexane or MeOH/DCM
3
) d
as eluents.
130.9, 129.2, 128.8, 128.0, 125.9, 124.1, 81.2, 52.0; ESIMS (m/z): calcd
for C19H
14
O
5
¼ 322, found [MþH]þ ¼ 323.
3.1.2. Methyl (E)-3-(2,8-dioxo-4-phenyl-1-oxaspiro[4.5]deca-3,6,9-
trien-3-yl)acrylate 9h
3.1.3. General procedure for the Suzuki coupling
To a solution of Compound 6 (200 mg, 0.549 mmol) in DMF
To a solution of compound 6 or 7 (0.274 mmol) in DMF (4 mL)
(
1
1
8
4 mL), Pd(OAc)
.09 mmol) was added, followed by methyl acrylate (110
.09 mmol) was added and the mixture was stirred under reflux at
2
(0.0005 mmol) was added, and K
2
CO
3
(152 mg,
and water (1 mL) was added Pd(OAc)
boronic acid 8 (0.548 mmol) and K CO
the mixture was stirred under reflux at 85 C. The reaction progress
was monitored by TLC. After completion of the reaction, the
mixture was allowed to cool at room temperature and the mixture
2
(6 mg, 0.027 mmol), then
mL,
2
3
(75.6 mg, 0.548 mmol) and
ꢁ
ꢁ
0 C. The reaction progress was monitored by TLC. After comple-
tion of the reaction, the mixture was allowed to cool at room

D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
353
Fig. 4. The effect of 9b and 9e on the ROS production in human breast cancer cells (MCF-7). A: Control cells (MCF-7), B: 9b (0.5 mM), C: 9b (1 mM), D: 9e (0.5 mM) and E: 9e (1 mM).
was extracted with ethylacetate (3 ꢂ 30 mL). The organic layers
(m, 2H), 7.17 (dd, J ¼ 1.06, 8.08 Hz, 2H), 6.59 (d, J ¼ 10.22 Hz, 2H),
13
were collected, combined, washed with saturated aq NaCl
6.44 (d, J ¼ 10.22 Hz, 2H), 3.02 (s, 3H); C NMR (125 MHz, CDCl
3
)
(
2 ꢂ 25 mL), dried over anhydrous Na
2
SO
4
, filtered and concen-
d 183.8, 168.5, 148.0, 144.9, 140.2, 138.7, 133.3, 132.3, 130.8, 129.8,
trated under reduced pressure. The crude compound was purified
by column chromatography on silica gel to afford the coupled
product.
128.9, 128.8, 126.3, 125.3, 124.3, 124.3, 121.8, 67.5, 29.6; HRMS (ESI)
calcd for C24
purity ¼ 94.09%.
H
18NO
2
S [MþH]þ ¼ 384.1058, found ¼ 384.1060; HPLC
3
.1.4. 3-(4-(Hydroxymethyl)phenyl)-1-methyl-4-phenyl-1-azaspiro
3.1.6. 1-Methyl-4-phenyl-3-(4-(trifluoromethyl)phenyl)-1-azaspiro
[4.5]deca-3,6,9-triene-2,8-dione 10c
[4.5]deca-3,6,9-triene-2,8-dione 10a
Yield ¼ 71.3%; R
f
¼ 0.29 (30% EtOAc in hexane); 1H NMR
Yield ¼ 78%; R
f
¼ 0.37 (30% EtOAc in hexane); 1H NMR
7.70 (s, 1H), 7.60 (d, J ¼ 7.78 Hz, 1H), 7.52 (d,
(
300 MHz, DMSO- d
6
)
d
7.39 (d, J ¼ 8.30 Hz, 2H), 7.31e7.27 (m, 2H),
(500 MHz, CDCl
3
) d
7
.23e7.20 (m, 3H), 7.10 (dd, J ¼ 1.51, 8.30 Hz, 2H), 6.59 (d,
J ¼ 7.78 Hz, 1H), 7.38 (t, J ¼ 7.78 Hz, 1H), 7.33 (tt, J ¼ 1.22, 2.89 Hz,
J ¼ 10.19 Hz, 2H), 6.47 (d, J ¼ 10.19 Hz, 2H), 4.62 (s, 2H), 2.95 (s, 3H);
1H), 7.28 (d, J ¼ 7.62 Hz, 2H), 7.10 (d, J ¼ 7.17 Hz, 2H), 6.60 (dt,
13
13
C NMR (75 MHz, CDCl
3
& DMSO- d
6
)
d
183.3, 168.8, 148.8, 145.1,
J ¼ 1.98, 2.74 Hz, 2H), 6.51 (dt, J ¼ 1.98, 2.74 Hz, 2H), 2.98 (s, 3H);
C
141.9, 134.5, 132.3, 131.1, 128.6, 128.6, 128.4, 127.9, 127.7, 125.7, 66.4,
3
NMR (75 MHz, CDCl ) d 184.0, 169.1, 151.5, 145.0, 138.9, 133.4, 132.6,
2
9.3; HRMS (ESI) calcd for C23
H
20NO
3
[MþH]þ ¼ 358.1443,
131.2, 131.0, 129.7, 128.8, 128.7, 128.2, 126.4, 126.3, 125.3, 125.3, 67.2,
found ¼ 358.1445.
29.6; HRMS (ESI) calcd for C23
found ¼ 396.1213; HPLC purity ¼ 97.33%.
H
17
F
3
NO
2
[MþH]þ ¼ 396.1211,
3.1.5. 3-(Benzo[b]thiophen-2-yl)-1-methyl-4-phenyl-1-azaspiro
[
4.5]deca-3,6,9-triene-2,8-dione 10b
3.1.7. 1-Methyl-3-(naphthalen-1-yl)-4-phenyl-1-azaspiro[4.5]
Yield ¼ 79.2%; R
f
¼ 0.31 (30% EtOAc in hexane); 1H NMR
deca-3,6,9-triene-2,8-dione 10d
(
7
500 MHz, CDCl
3
)
d
8.14 (s, 1H), 7.73 (dd, J ¼ 1.83, 5.95 Hz, 1H),
Yield ¼ 63.7%; R
f
¼ 0.31 (30% EtOAc in hexane); 1H NMR
.66e7.64 (m, 1H), 7.48e7.44 (m, 1H), 7.41e7.38 (m, 2H), 7.31e7.27
(500 MHz, CDCl
3
)
d
7.84 (d, J ¼ 8.24 Hz, 1H), 7.67 (d, J ¼ 8.39, 1H),

354
D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
Fig. 5. Annexin V-FITC staining. A: Control cells (MCF-7), B: 9b (0.5 mM), C: 9b (1 mM), D: 9e (0.5 mM) and E: 9e (1 mM).
7
7
.46e7.42 (m, 2H), 7.41e7.37 (m, 1H), 7.35 (dd, J ¼ 1.22, 7.01 Hz, 1H),
.13 (tt, J ¼ 1.37, 1.98 Hz, 1H), 7.09 (d, J ¼ 6.86 Hz, 2H), 7.03 (t,
J ¼ 7.93 Hz, 2H), 6.78 (dd, J ¼ 3.20, 10.22 Hz, 2H), 6.74 (dd, J ¼ 3.05,
13
10.22 Hz, 2H), 6.61e6.58 (m, 2H), 3.00 (s, 3H); C NMR (75 MHz,
CDCl 184.2, 169.9, 151.8, 146.3, 145.9, 133.7, 133.1, 131.3, 131.0,
3
) d
1
6
29.4, 129.3, 128.6, 128.4, 128.2, 127.8, 126.3, 126.0, 125.4, 124.9,
7.0, 29.7; HRMS (ESI) calcd for C26
H20NO
2
[MþH]þ ¼ 378.1494,
found ¼ 378.1496.
Table 3
Distribution of apoptotic cells in Annexin-V FITC experiment.
Sample
UL %
UR %
LL%
LR%
3.57
5.87
10.13
4.22
A: Control
B: 9b (0.5
C: 9b (1
D: 9e (0.5
E: 9e (1
0.86
1.96
3.77
1.69
4.77
3.08
6.76
28.87
7.25
92.50
85.41
57.24
86.85
64.39
m
M)
M)
M)
M)
m
Fig. 6. Effect of compounds 9b and 9e on caspase-9 activity; MCF-7 breast cancer cells
were treated with these compounds at 1 M concentration for 48 h. Values indicated
are the mean ± SD of two different experiments performed in triplicates.
m
m
m
24.21
6.64

D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
355
13C NMR (100 MHz, CDCl
)
d
184.0, 169.5, 148.4, 147.8, 147.3, 145.6,
3
1
6
34.6,133.0, 131.8, 129.1, 128.6,128.2, 124.1, 123.8,109.6, 108.1,101.0,
þ
6.8, 26.1; HRMS (ESI) calcd for C23
H
18NO
4
[MþH] ¼ 372.1236,
found ¼ 372.1240; HPLC purity ¼ 93.13%.
3.1.11. 3-(2,3-Dichlorophenyl)-1-methyl-4-phenyl-1-azaspiro[4.5]
deca-3,6,9-triene-2,8-dione 10h
Yield ¼ 68.6%; R
f
¼ 0.28 (30% EtOAc in hexane); 1H NMR
(
1
500 MHz, CDCl
3
)
d
7.45 (dd, J ¼ 1.52, 7.93 Hz, 1H), 7.28 (dt, J ¼ 1.22,
.98 Hz, 1H), 7.21e7.17 (m, 3H), 7.11 (dq, J ¼ 1.52 Hz 3H), 6.65 (dd,
13
J ¼ 1.67, 10.22 Hz, 2H), 6.58e6.53 (m, 2H), 2.95 (s, 3H); C NMR
100 MHz, CDCl 183.9, 168.4, 152.4, 145.5, 144.9, 135.1, 133.5,
33.3, 133.0, 132.7, 131.9, 130.8, 130.7, 129.7, 129.4, 128.5, 127.5,
27.4, 67.0, 26.0; HRMS (ESI) calcd for
(
1
1
[
3
) d
22 16 2
C H Cl2NO
þ
MþH] ¼ 396.0558, found ¼ 396.0563; HPLC purity ¼ 98.50%.
3
3
.1.12. 3-(Furan-3-yl)-1-methyl-4-phenyl-1-azaspiro[4.5]deca-
,6,9-triene-2,8-dione 10i
Yield ¼ 83.3%; R
f
¼ 0.40 (30% EtOAc in hexane); 1H NMR
(
(
500 MHz, CDCl
3
)
d
8.28 (q, J ¼ 0.76 Hz, 1H), 7.41e7.34 (m, 3H), 7.22
t, J ¼ 1.67 Hz, 1H), 7.14 (dt, J ¼ 1.37 Hz, 2H), 6.55 (dt, J ¼ 1.98,
.74 Hz, 2H), 6.42 (d, J ¼ 1.98, 2.74 Hz, 2H), 5.85 (dd, J ¼ 0.76,
2
13
3
1.98 Hz, 1H), 2.96 (s, 3H); C NMR (125 MHz, CDCl ) d 169.4, 145.9,
1
45.3, 143.7, 142.4, 133.1, 131.7, 129.3, 128.7, 128.4, 108.5, 67.7, 29.6;
HRMS (ESI) calcd for
found ¼ 318.1131; HPLC purity ¼ 97.35%.
C
20
H16NO
3
[MþH]þ
¼
318.1130,
3.1.13. 3-(2,4-Dimethoxyphenyl)-1-methyl-4-phenyl-1-azaspiro
[
4.5]deca-3,6,9-triene-2,8-dione 10j
Yield ¼ 69.3%; R
f
¼ 0.18 (30% EtOAc in hexane); 1H NMR
(
(
6
2
500 MHz, CDCl
3
)
d
7.29 (d, J ¼ 8.39 Hz, 1H), 7.23e7.20 (m, 1H), 7.16
Fig. 7. The effect of compounds on p53, p21, p27, Bax and Bcl-2 levels: MCF-7 cells
were treated with compounds 9b and 9e at 1 M concentration for 48 h. Cell lysates
were collected, and the expression levels of p53, p21, p27, Bax and Bcl-2 were deter-
mined by western blot analysis. -Actin was used as a loading control.
t, J ¼ 7.78, 2H), 7.13 (d, J ¼ 7.17 Hz, 2H), 6.63 (d, J ¼ 9.91 Hz, 2H),
m
.52e6.49 (m, 3H), 6.34 (d, J ¼ 2.28 Hz,1H), 3.80 (s, 3H), 3.37 (s, 3H),
13
3
.93 (s, 3H); C NMR (125 MHz, CDCl ) d 173.9, 161.4, 157.9, 133.2,
b
1
32.7, 131.7, 131.5, 128.9, 128.2, 127.2, 112.9, 111.7, 104.5, 98.9, 55.3,
54.8, 29.7; HRMS (ESI) calcd for C24
H22NO
4
[MþH]þ ¼ 388.1549,
3
.1.8. 1-Methyl-3,4-diphenyl-1-azaspiro[4.5]deca-3,6,9-triene-2,8-
dione 10e
Yield ¼ 64.9%; R
500 MHz, CDCl 7.42e7.40 (m, 2H), 7.31e7.27 (m, 3H), 7.23 (t,
found ¼ 388.1548; HPLC purity ¼ 97.79%.
1
¼ 0.31 (30% EtOAc in hexane); H NMR
3.1.14. 4-(1-Methyl-2,8-dioxo-4-phenyl-1-azaspiro[4.5]deca-3,6,9-
trien-3-yl)benzaldehyde 10k
f
(
3
)
d
Yield ¼ 70.2%; R
f
¼ 0.18 (30% EtOAc in hexane); 1H NMR
J ¼ 7.62 Hz, 2H), 7.11 (d, J ¼ 7.17 Hz, 2H), 6.60 (d, J ¼ 10.07 Hz, 2H),
1
3
6
d
1
C
.48 (d, J ¼ 10.22 Hz, 2H), 2.96 (s, 3H); C NMR (75 MHz, CDCl
184.2, 169.6, 149.7, 145.7, 135.6, 133.2, 132.7, 130.5, 129.4, 129.3,
28.6, 128.4, 128.2, 128.0, 67.0, 29.7; HRMS (ESI) calcd for
3
)
(500 MHz, CDCl
3
)
d
9.97 (d, J ¼ 3.66 Hz,1H) 7.77 (dt, J ¼ 1.67 Hz, 2H),
7.59 (dd, J ¼ 2.13, 8.39 Hz, 2H), 7.35e7.31 (m, 1H), 7.26 (t, J ¼ 7.62 Hz,
2H), 7.10 (d, J ¼ 8.54 Hz, 2H), 6.60 (dd, J ¼ 2.59, 12.66 Hz, 2H), 6.50
þ
13
22
H
18NO
2
[MþH]
¼
328.1338, found
¼
328.1335; HPLC
(dt, J ¼ 2.74 Hz, 2H), 2.98 (s, 3H); C NMR (75 MHz, CDCl
3
) d 191.8,
purity ¼ 98.60%.
183.9, 168.9, 152.1, 145.0, 136.6, 135.8, 134.4, 133.4, 131.2, 130.1,
29.8,129.4,128.9,128.2, 67.1, 29.6; HRMS (ESI) calcd for C23
1
H18NO
3
þ
[MþH] ¼ 356.1287, found ¼ 356.1288; HPLC purity ¼ 96.35%.
3.1.9. 5-(1-Methyl-2,8-dioxo-4-phenyl-1-azaspiro[4.5]deca-3,6,9-
trien-3-yl)thiophene-2-carbaldehyde 10f
3
.1.15. 4-(1-Methyl-2,8-dioxo-4-phenyl-1-azaspiro[4.5]deca-3,6,9-
Yield ¼ 73.8%; R
f
¼ 0.21 (30% EtOAc in hexane); 1H NMR
trien-3-yl)benzonitrile 10l
(
500 MHz, CDCl
3
)
d
9.84 (s, 1H), 7.63 (d, J ¼ 4.12 Hz, 1H), 7.56 (d,
Yield ¼ 72.1%; R
f
¼ 0.33 (30% EtOAc in hexane); 1H NMR
J ¼ 4.12 Hz, 1H), 7.46 (tt, J ¼ 1.37, 2.74 Hz, 1H), 7.41 (tt, J ¼ 1.37,
(300 MHz, CDCl
3
)
d
7.54 (s, 4H), 7.35 (t, J ¼ 7.62 Hz, 1H), 7.29 (d,
1
6
.98 Hz, 2H), 7.12 (dt, J ¼ 1.98 Hz, 2H), 6.56 (dt, J ¼ 1.98, 2.74 Hz, 2H),
J ¼ 7.62 Hz, 2H), 7.08 (d, J ¼ 7.32 Hz, 2H), 6.58 (d, J ¼ 10.22 Hz, 2H),
13
.45 (dt, J ¼ 1.98, 2.74 Hz, 2H), 3.00 (s, 3H); C NMR (125 MHz,
187.3, 183.2, 168.8, 144.1, 135.3, 133.6, 130.2, 129.4129.2,
29.1, 128.1, 67.7, 26.5; HRMS (ESI) calcd for
13
6
d
.51 (d, J ¼ 10.22 Hz, 2H), 2.97 (s, 3H); C NMR (125 MHz, CDCl
3
)
3
CDCl ) d
183.9, 168.8, 152.6, 144.8, 135.3, 133.8, 133.7, 132.0, 131.1, 130.2,
1
[
21 3
C H16NO S
130.0, 129.1, 128.3, 118.6, 112.3, 67.3, 29.8; HRMS (ESI) calcd for
þ
MþH] ¼ 362.0851, found ¼ 362.0850; HPLC purity ¼ 79.78%.
C
23
17
H N
2
O
2
[MþH]þ
¼
353.1290, found
¼
353.1290; HPLC
purity ¼ 94.26%.
3.1.10. 3-(Benzo[d][1,3]dioxol-5-yl)-1-methyl-4-phenyl-1-azaspiro
[
4.5]deca-3,6,9-triene-2,8-dione 10g
3.1.16. 3-(4-Methoxyphenyl)-1-methyl-4-phenyl-1-azaspiro[4.5]
Yield ¼ 68%; R
f
¼ 0.28 (30% EtOAc in hexane); 1H NMR
deca-3,6,9-triene-2,8-dione 10m
(
(
500 MHz, CDCl
3
)
d
7.30e7.24 (m, 3H), 7.12 (d, J ¼ 7.62 Hz, 2H), 6.97
Yield ¼ 78.5%; R
f
¼ 0.25 (30% EtOAc in hexane); 1H NMR
d, J ¼ 8.24 Hz, 1H), 6.90 (s, 1H), 6.70 (d, J ¼ 8.24 Hz, 1H), 6.57 (d,
(500 MHz, CDCl
3
)
d
7.39 (dd, J ¼ 2.74, 11.59 Hz, 2H), 7.31e7.23 (m,
J ¼ 8.54 Hz, 2H), 6.46 (d, J ¼ 9.00 Hz, 2H), 5.92 (s, 2H), 2.95 (s, 3H);
3H), 7.12 (d, J ¼ 7.17 Hz, 2H), 6.78 (dd, J ¼ 2.74, 11.59 Hz, 2H), 6.59

356
D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
ꢁ
JC-1 (5 mg/mL) and incubated at 37 C for 15 min. Cells were rinsed
(
3
1
1
dd, J ¼ 2.59, 12.66 Hz, 2H), 6.47 (dd, J ¼ 2.59, 12.66 Hz, 2H), 3.77 (s,
H), 2.96 (s, 3H); 13C NMR (125 MHz, CDCl
184.2, 169.8, 159.7,
47.7, 145.9, 134.6, 133.0, 132.1, 130.8, 129.0, 128.6, 128.3, 122.9,
13.6, 66.9, 55.1, 29.6; HRMS (ESI) calcd for
3
)
d
three times with medium and suspended in pre warmed medium.
The cells were then subjected to flow cytometric analysis on a flow
cytometer (Becton Dickinson) in the FL1, FL2 channel to detect
mitochondrial potential [17].
23 3
C H20NO
þ
[MþH] ¼ 358.1443, found ¼ 358.1443; HPLC purity ¼ 99.53%.
3
.1.17. 3-(3-Chloro-4-methylphenyl)-1-methyl-4-phenyl-1-
3
.1.22. ROS generation
The production of ROS (reactive oxygen species) was measured
azaspiro[4.5]deca-3,6,9-triene-2,8-dione 10n
Yield ¼ 71.1%; R ¼ 0.37 (30% EtOAc in hexane); H NMR
500 MHz, CDCl
7.48 (d, J ¼ 1.67 Hz, 1H), 7.32 (tt, J ¼ 1.37,
.28 Hz, 1H), 7.28e7.24 (m, 2H), 7.15 (dd, J ¼ 1.67, 7.93 Hz, 1H),
1
f
0
0
by flow cytometry using DCFDA (2 ,7 dichlorofluorescindiacetate)
as previously described [18]. In this study MCF-7 cells were treated
with compound 9b and 9e at 0.5 and 1
After treatment, cells were incubated with DCFDA (2
for 30 min and then measured with the flow cytometer (FACS).
(
2
3
) d
mM concentrations for 48 h.
7.12e7.08 (m, 3H), 6.58 (dt, J ¼ 1.98, 2.74 Hz, 2H), 6.48 (dt, J ¼ 1.98,
2
d
ꢁ
mM) at 37 C
13
.74 Hz, 2H), 2.96 (s, 3H), 2.32 (s, 3H); C NMR (125 MHz, CDCl
3
)
184.0, 169.2, 150.0, 145.4, 136.9, 134.2, 134.0, 133.2, 131.4, 130.6,
1
29.7, 129.5, 129.4, 128.7, 128.2, 127.5, 67.0, 29.6, 19.9; HRMS (ESI)
calcd for C23
purity ¼ 98.89%.
H
19ClNO
2
[MþH]þ ¼ 376.1104, found ¼ 376.1105; HPLC
3
.1.23. Annexin staining assay for apoptosis
MCF-7 (1 ꢂ 10 ) were seeded in six-well plates and allowed to
6
grow overnight. The medium was then replaced with complete
3
.1.18. 1-Methyl-3-(4-(methylthio)phenyl)-4-phenyl-1-azaspiro
medium containing compounds 9b and 9e at 0.5 and 1 mM con-
[4.5]deca-3,6,9-triene-2,8-dione 10o
centrations. After 48 h of drug treatment, cells from the superna-
tant and adherent monolayer cells were harvested by
trypsinization, washed with PBS at 3000 rpm. Then the cells were
stained with Annexin VFITC and propidium iodide using the
Annexin-V-FITC apoptosis detection kit (Sigma Aldrich). Flow
cytometry was performed for this study as described earlier [19].
Yield ¼ 70.7%; R
f
¼ 0.25 (30% EtOAc in hexane); 1H NMR
(
500 MHz, CDCl
3
)
d
7.36 (d, J ¼ 8.54 Hz, 2H), 7.30 (t, J ¼ 7.62 Hz, 1H),
7
6
.25 (d, J ¼ 7.93 Hz, 2H), 7.12e7.10 (m, 4H), 6.58 (d, J ¼ 9.91 Hz, 2H),
13
.47 (d, J ¼ 10.07 Hz, 2H), 2.96 (s, 3H), 2.44 (s, 3H); C NMR
(
125 MHz, CDCl
3
)
d
184.0, 169.5, 148.9, 145.6, 139.6, 134.6, 133.1,
131.8, 129.6, 129.2, 128.6, 128.3, 126.9, 125.5, 66.9, 29.6, 15.1; HPLC
purity ¼ 99.17%.
3
.1.24. Activation of caspase 9
Caspase-9 assay was conducted for detection of apoptosis in
3.1.19. In vitro anti-proliferative assay
The anti-proliferative activities of the compounds were calcu-
breast cancer cell line (MCF-7). The commercially available
apoptosis detection kit (Sigma Aldrich) was used. MCF-7 cells were
treated with compounds 9b and 9e at 1 mM concentration for 48 h.
After 48 h of treatment, cells were collected by centrifugation,
washed once with PBS, and cell pellets were collected. Suspended
the cell pellet in lysis buffer and incubated for 15 min. After incu-
bation, cells were centrifuge at 20,000 rpm for 15 min and collected
the supernatant. Supernatants were used for measuring caspase 9
activity using an ELISA-based assay, according to the manufac-
turer's instructions.
lated from the growth inhibition data at three different concen-
trations (1, 5 and 10 M) by using Sulphorhodamine B (SRB) assay
15]. Cells grown in DMEM, supplemented with 10% FBS were
m
[
seeded in each well of 96 well microculture plates and incubated
ꢁ
for 24 h at 37 C in a CO
2
incubator. Compounds, diluted to the
M) in DMSO, were added to the
desired concentrations (1, 5 and 10
m
wells. All the working solutions were prepared from stock solution
of DMSO (10 mM). The maximum concentration of DMSO used for
the assay was 0.1%. After 48 h, cells were fixed with 10% trichloro
acetic acid (TCA) solution and were further incubated for 60 min at
ꢁ
4
C. The plates were washed with tap water and air dried. Later
l) at 0.4% (w/v) in 1% acetic
acid was added to each well, and plates were incubated for
0 min at room temperature. The residual dye was removed by
Sulforhodamine B (SRB) solution (50
m
3.1.25. Protein extraction and western blot analysis
MCF-7 cells were treated with compounds 9b and 9e at 0.5 and
2
1 mM concentrations for 48 h. The cell lysates were obtained by
washing with 1% acetic acid and the plates were air dried. Bound
stain was subsequently eluted with 10 mM trizma base, and the
absorbance was recorded on multimode reader (TECAN) at a
wavelength of 540 nm. All experiments were performed in
triplicate.
lysing the cells in ice-cold radioimmunoprecipitation assay (RIPA)
buffer (1 ꢂ PBS,1% NP-40 detergent, 0.5% sodium deoxycholate, and
0.1% SDS) containing 100 mg/mL phenylmethanesulfonyl fluoride
(PMSF), 5 mg/mL aprotinin, 5 mg/mL leupeptin, 5 mg/mL pepstatin,
and 100 mg/mL NaF. After centrifugation at 12000 rpm for 10 min,
the protein in the supernatant was quantified by the Bradford
method (BIO-RAD) by using a Multimode Varioskan instrument
3.1.20. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the
(Thermo Fischer Scientifics Ltd.). Protein (50 mg per lane) was
distribution of the cells through the cell-cycle phases. MCF-7 cells,
breast cancer cells were incubated for 48 h with compounds 9b and
applied in 12% SDS polyacrylamide gel. After electrophoresis, the
protein was transferred to a polyvinylidine difluoride (PVDF)
membrane (Thermo Scientific Inc.). The membrane was blocked at
room temperature for 2 h in Tris-buffered saline (TBS) with 0.1%
Tween 20 (TBST) containing 5% blocking powder (Santa Cruz). The
membrane was washed with TBST for 5 min, then primary antibody
was added and the membrane was incubated for overnight. After
incubation the membrane was incubated with the corresponding
horseradish peroxidase labeled secondary antibody at room tem-
perature for 1 h. Membranes were washed with TBST three times
for 15 min, and the blots were visualized with chemiluminescence
reagent. Images were captured by using the chemiluminescence
(vilber lourmat) [20].
9
e at concentrations of 0.5 and 1 mM. Untreated and treated cells
were harvested, washed with phosphate-buffered saline (PBS),
fixed in ice-cold 70% ethanol, and stained with propidium iodide
(
SigmaeAldrich). Cell-cycle analysis was performed by flow
cytometry (Becton Dickinson FACS Caliber instrument) [16].
3
.1.21. Mitochondrial membrane potential
MCF-7 (1 ꢂ 10 cells/well) cells were cultured in six-well plates
6
after treatment with compounds 9b and 9e at 0.5 and 1 mM con-
centrations for 48 h. After 48 h of treatment, cells were collected by
trypsinization and washed with PBS followed by resuspending in

D. Yugandhar et al. / European Journal of Medicinal Chemistry 101 (2015) 348e357
357
Acknowledgment
deoxynyboquinone, a potent inducer of cancer cell death, J. Am. Chem. Soc.
32 (2010) 5469e5478.
9] D.C. Wallace, Mitochondria and cancer, Nat. Rev. Cancer 12 (2012) 685e698.
10] S. Fulda, L. Galluzzi, G. Kroemer, Targeting mitochondria for cancer therapy,
Nat. Rev. Drug Dis. 9 (2010) 447e464.
1
[
This work was funded by CSIR, New Delhi under the XIIth Five
year plan project ‘Affordable Cancer Therapeutics-ACT’ (CSC-0301).
SA thanks CSIR-New Delhi and DY thanks UGC-New Delhi for
providing fellowships.
[
[
11] (a) B. Antonsson, Mitochondria and the Bcl-2 family proteins in apoptosis
signaling pathways, Mol. Cell Biochem. 256e257 (2004) 141e155;
(b) T. Taguchi, Y. Kato, Y. Baba, G. Nishimura, Y. Tanigaki, C. Horiuchi,
I. Mochmatsu, M. Tsukuda, Protein levels of p21, p27, cyclin E and Bax predict
sensitivity to cisplatin and paclitaxel in head and neck squamous cell carci-
nomas, Oncol. Rep. 11 (2004) 421.
Appendix A. Supplementary data
[
12] (a) P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.050.
1107e1112;
(
b) L.V. Rubinstein, R.H. Shoemaker, K.D. Paull, R.M. Simon, S. Tosini,
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