Synthesis and biological evaluation of 3,4,6-triaryl-2-pyranones as a potential new class of anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2009.04.032

A series of 3,4,6-triaryl-2-pyranones, new class of anti-breast cancer agents, have been synthesized as a structural variants of cyclic triphenylethylenes by replacing the fused benzene ring with pendant phenyl ring to mimic the phenolic A ring of estradi

Full text of DOI:10.1016/j.bmc.2009.04.032

Bioorganic & Medicinal Chemistry 17 (2009) 3847–3856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 3,4,6-triaryl-2-pyranones as a
potential new class of anti-breast cancer agents
Ravi Shankar a, Bandana Chakravarti b, Uma Sharan Singh a, Mohd. Imran Ansari a, Shreekant Deshpande a,
Shailendra Kumar Dhar Dwivedi b, Hemant Kumar Bid b, Rituraj Konwar b, Geetika Kharkwal b,
Vishal Chandra b, Anila Dwivedi b, K. Hajela a,
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute , Lucknow 226 001, UP, India
b Endocrinology Division, Central Drug Research Institute, Lucknow 226 001, UP, 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 3,4,6-triaryl-2-pyranones, new class of anti-breast cancer agents, have been synthesized as a
structural variants of cyclic triphenylethylenes by replacing the fused benzene ring with pendant phenyl
ring to mimic the phenolic A ring of estradiol. Nine of these newly synthesized pyranones exhibited sig-
nificant anti-proliferative activity in both ER+ve and ERÀve breast cancer cell lines. Four active non-cyto-
toxic compounds 5c, 5d, 5g and 5h showed specific and selective cytotoxicity and two compounds 5d
and 5h induced significant DNA fragmentation in both MCF-7 and MDA-MB-231 cell lines. Based on
RBA studies, the molecules probably act in an ER-independent mechanism. The involved pathway was
observed as caspase-dependant apoptosis in MCF-7 cells. However, the particular caspases involved
and the possible cellular target through which this series of compounds mediate cell death are not
known.
Received 25 February 2009
Revised 15 April 2009
Accepted 16 April 2009
Available online 22 April 2009
Keywords:
Triaryl-2-pyranones
Anti-proliferative
Apotosis
DNA fragmentation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
as nafoxidine and lasofoxifene, diarylbenzopyrans,7 for example,
ormeloxifene, benzopyranones8 like SP500263, benzothiophenes9
Breast cancer is one of the most commonly diagnosed cancer,
comprising 23% of all the female cancers and the second leading
cause of cancer deaths in women worldwide today.1,2 Several stud-
ies have established that estrogens are predominantly involved in
the initiation and proliferation of breast cancer and much efforts
are now being devoted to block estrogen formation and action.3
The most widely used strategy to treat breast cancer is to disrupt
estrogen mediated cancer proliferation through targeted antago-
nism of estrogen receptor by antiestrogens. Tamoxifen is the best
estrogen receptor modulator capable of exhibiting breast cancer
specific activity,4 but still its therapeutic potential is limited in
the sense that it is not effective against ER negative tumors as it
being an ER agonist and mostly acts via ER receptor. Therefore, cur-
rent need is to develop novel agents active against both estrogen
dependent and independent breast cancers.
such as raloxifene have formed the backbone of a multitude of
non-steroidal estrogen agonists as well as antagonists (Fig. 1).
As part of our drug development program, we aimed to explore
and develop new ER recognition motifs. Functionalized 2-pyranon-
es are key structural motifs found in a large number of biologically
important natural products10 that form a family of active com-
pounds with a wide range of biological activities like, anti-cancer,
antimicrobial, phytotoxic and HIV-1 protease inhibitors.11–14
Recently, a series of arylated 2-pyranones have shown cyclooxy-
genase-2(COX-2) inhibitory activity. One of the compounds,
3,4,6-triphenyl-2-pyranone (Fig. 1), was found to be a potent and
selective COX-2 inhibitor with an IC50 = 0.02 l
M.15 Structural
resemblance of 3,4,6-triaryl-2-pyranones to triarylethylene based
tamoxifen and ormeloxifene, a potent SERM developed by this
institute and exhibiting antifertility and antibreast cancer activi-
ties,16 prompted us to explore this pharmacophore as potential
lead for developing new anti-breast cancer agents.
Since the development of triaryl template-based tamoxifen
estrogen receptor modulator,5 substituted diarylnapthalenes6 such
The rationale to design the 2-pyranones was to build a scaffold
with specific alignment of three aryl substituents based on prece-
dented ligands or leads17 that are suitable to estrogen receptor
binding and activity. The 2-pyranone ring served as the central
core and the three aryl rings were suitably aligned in the desired
molecular geometry as to mimic the triaryl template of TAPs, the
only difference is the presence of pendent, that is, 6-aryl ring in
Abbreviations: ER, estrogen receptor; MCF-7, Michigan cancer foundation-7;
MDA-MB-231, MD Anderson metastatic breast-231; RBA, receptor binding affinity.
* Corresponding author. Tel.: +91 522 2612411 18x4463; fax: +91 522 2623405/
3938.
E-mail address: hajelak@yahoo.com (K. Hajela).
CDRI comm. No. 7469.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.032

3848
R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
N
N
N
N
O
O
O
O
O
HO
MeO
MeO
Tamoxifene
N
Ormeloxifene
Lasofoxifene
Nafoxidine
N
O
O
SO2Me
O
O
O
OH
S
O
O
HO
MeO
HO
Raloxifene
Ring 2
3,4,6-Triphenyl-2-pyranone
SP500263
Figure 1. Structures of non-steroidal estrogen antagonists.
OR
Ring 3
D
C
4
B
3
A
O
2
Ring 1
Ring 2
1
OR
Ring 3
Ormeloxifene
OH
C
D
D
C
4
3
5
B
A
B
2 O
6
HO
O
Ring 1
Ring 3
A
1
Ring 2
OR
Estradiol
3,4,6-Triaryl-2-pyranone
C
D
A
B
Ring 1
Tamoxifen
Figure 2. Structural resemblance of 3,4,6, triaryl-2-pyranones to tamoxifen, estradiol and ormeloxifene.
the place of fused benzopyran (Fig. 2). The essential requirement of
basic amino alkyl chain for inducing the favorable conformational
change to elicit the desired antagonistic effect18,19 to facilitate
interaction of ER with co-activator or co-repressor proteins was
introduced at the para position of 2-ring to give the target
molecules.
achieved by condensation of phenylacetylene or 4-methoxy phen-
ylacetylene with benzaldehyde or anisaldehyde in presence of an-
hyd zinc chloride) with activated manganese dioxide in 50%
yield.20,21 Since only moderate yields of 1,3-diarylpropynones
could be obtained by this method, Sonogashira coupling22 between
a substituted alkyne and substituted benzoyl chloride in presence
of cuprous iodide formed propynones in good yields of 70–75%
(Scheme 1).
2. Results and discussion
2.1. Chemistry
The methodology21,23 used to prepare the regioisomeric 3,4,6-
triphenyl-2-pyranones (5a–h) is outlined as shown in Scheme 2.
Simple and 4-methoxyphenyl ethyl phenylacetate (3a, b) when
condensed with 1,3-diarylpropynones (2a–c) in presence of so-
dium hydride at ambient temperature formed the desired methoxy
At the outset, 1,3-diarylpropynones (2a–c) were synthesised by
oxidation of 1,3-diarylprop-2-yn-1-ols (1a–c, which in turn were

R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
R2 R2
3849
O
H
O
Cl
c
a
b
+
+
OH
O
R1
R2
R2
R1
R1
R1
2a, R1=OCH3, R2=H
2b, R1=H, R2=OCH3
2c, R1 & R2=OCH3
1a, R1=OCH3, R2=H
1b, R1=H, R2=OCH3
1c, R1 & R2=OCH3
Scheme 1. Synthesis of 1,3-diarylpropynones. Reagents and conditions: (a) ZnCl2, Et3N, dry toluene, rt, 1 h (b) MnO2, dry acetone, rt, 5 h (c) CuI, Et3N, rt, 6 h.
R1
R1
R3
R3
a
+
O
O
O
EtO
O
R2
R2
3a, R3=H
3b, R3=OCH3
4a, R1=OCH3, R2 & R3=H
4b, R1 & R3= H, R2 = OCH3,
4c, R1 & R2 =H, R3 = OCH3
4d, R1 & R3 =OCH3, R2 =H
4e, R1=H, R2 & R3=OCH3
4f, R1 & R2 = OCH3, R3 =H
2a, R1= OCH3, R2=H
2b, R1 =H, R2 =OCH3
2c, R1& R2=OCH3,
b
R1
R1
R3
R3
c
O
O
O
O
R2
R2
5a, R1=OH, R2 & R3=H
5b, R1 & R3=H, R2=OH,
5c, R1 & R2=H, R3=OH
5d, R1 & R3=OH, R2=H
5e, R1=OCH3, R3=OH, R2=H
5f, R1=H, R2 & R3=OH
5g, R1=H, R2=OCH3, R3=OH
5h, R1 & R2=OH, R3=H
6a, R2 & R3=H,R1= ethoxypyrrolidine
6b, R2 & R3 =H, R1 = ethoxypiperidine
6c, R1& R3 =H, R2 =ethoxypyrrolidine
6d, R1& R3=H, R2 = ethoxypiperidine
6e, R1& R2 =H, R3 = ethoxypyrrolidine
6f, R1& R2 =H, R3 = ethoxypiperidine
6g, R1=OCH3, R 2=H, R3 = ethoxypiperidine
Scheme 2. Synthesis of 3,4,6-triphenyl-2-pyranones and its derivatives. Reagents and conditions: (a) NaH/DMSO, 25 °C, 1 h; (b) pyridine hydrochloride, 220 °C, 30 min or
HBr/gl.CH3COOH, 120 °C; (c) 1-(2-chloroethyl)pyrrolidine/piperidine hydrochloride, K2CO3, dry DMF, 70 °C.
substituted 3,4,6-triphenyl-2-pyranones(4a–f). Demethylation of
monomethoxy pyranones (4a–c) was carried out either with pyr-
idine hydrochloride or HBr in glacial acetic acid. The reaction
was neat and monophenolic derivatives (5a–c) were obtained
in good yields. Dimethoxypyranone 4f on demethylation under
similar conditions formed the single dihydroxy compound 5h
in 60% yield. However, demethylation of dimethoxy pyranones
4d and 4e under similar conditions formed two products each:
with 4d, 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-6-phenyl-
2H-pyran-2-one (5e) was formed as the major product (75%)
along with 3,4-bis(4-hydroxyphenyl)-6-phenyl-2H-pyran-2-one
(5d) as the minor product (25%), obtained after purification by
column chromatography, similarly 4e on demethylation again
formed two products 5g (major) and 5f (minor).
7.9 for the phenolic OH. On comparison with the proton spectra
of dimethoxy derivative 4d with 5e, the absence of peak at d
3.78 in 5e which corresponds to the methoxy group at the para po-
sition of 3-aryl ring confirmed the demethylation of this methoxy
group. This selectivity in demethylation is due to lactone ring in
conjugation with aryl rings at 4 and 6 positions, as a result, the
electron density is decreased at the oxygen of these methoxy
groups as compared to the para methoxy of 3-aryl ring. The attack
of Lewis acid is therefore preferential at this methoxy and leads to
its demethylation. The same was observed in case of 4e, where the
para methoxy of 3-aryl ring is again preferentially demethylated in
comparison to para methoxy of 6-aryl ring, forming compound 5g
showing the same phenolic absorption at d 7.9 and the dihydroxy
compound 5f. In case of demethylation of dimethoxy pyranone 4f,
only the dihydroxy compound 5h was obtained showing phenolic
absorptions a little downfield at d 8.8 and 9.2 ppm because here
both para methoxy groups on 4 and 6 aryl rings are almost equally
The position of hydroxyl groups in compounds 5e and 5d was
confirmed by NMR spectra. The proton spectrum of 5e showed a
single resonance peak at d 3.81 for OCH3 and a sharp singlet at d

3850
R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
conjugated with the lactone ring. Finally, alkylation of phenolic
derivatives (5a–c, e) with 1-(2-chloro ethyl) pyrrolidine/piperdine
hydrochlorides formed the target molecules (6a–g). All the synthe-
sized compounds were well characterized by spectroscopic
analyses.
ing affinity of each test compound was calculated from the graph
plotted between percent bound radioactivity verses molar concen-
tration of the test substance. At 50% inhibition, log of the compet-
itor concentration relative to that of estradiol expressed the affinity
of the test compound to ER relative to estradiol. This when multi-
plied with 100 gave the% value designated as RBA.
2.2. Biological activity
Based on RBA studies, all the compounds appeared to exhibit
weak ER ligand binding affinity. The highest RBA was observed in
compound 5h (RBA, 0.272% of 17-b estradiol) which is very low
in comparison to anticancer drug, tamoxifen (RBA, 2.2% of 17-b
estradiol) as shown in Table 1. From these data, it can be concluded
that the series is safe in terms of activating ER with some com-
pounds showing better activity in ERÀve cell line.
2.2.1. Anti-proliferative activity against breast cancer
The target compounds (5a–h) and (6a–g) were evaluated for
anti-proliferative activity in both MCF-7 (ER+ve) and MDA-MB-
231 (ERÀve) cells using MTT assay. Nine of these, 5b–d, g, h, 6a–
c and 6g, were found to inhibit growth of MCF-7 and MDA-MB-
231 cells at IC50 of less than 50.0
pounds, 5g, 5h and 6a showed maximum inhibition of cell growth
with an IC50 of 14 M (Table 1). In addition to MCF-7 cells, 6a also
exhibited potent cell growth inhibition in MDA-MD-231 cells with
an IC50 of 9.0 M, which is better than that observed with MCF-7
l
M. In MCF-7 cells, three com-
We next assessed whether the molecules 5c, 5d, 5g and 5h, that
specifically exert anti-proliferative activity on breast cancer cells
l
exhibit ER
7 cells transiently transfected with ER
a
or ERb activation. Consistent with our RBA data, Cos-
or ERb did not exhibit
a
l
any transcriptional activation by any of the compounds when com-
pared with E2 treatment (Fig. 4). This further confirms that the ac-
tive molecules act in an ER-independent mechanism or if at all act
through ER in MCF-7 then activity is also being mediated through
some non-ER target in MDA-MB-231.
cells (Table 1). From these data, we may conclude that 6a is the
most effective and potent molecule for anti-proliferative activity
in breast cancer cells.
2.2.2. Cytotoxicity towards normal cells
Specific killing of cancer cells without affecting normal cell
growth is a key safety feature of cancer chemotherapy. The nine ac-
tive compounds were therefore evaluated for possible cytotoxicity
in normal cells, for example, Vero and osteoblasts and five of these
compounds, 5b, 6a–c and 6g induced significant cell death in both
Vero and osteoblast cells (Fig. 3a and b). Compounds 6a, 6b and 6g
which exhibited greater anti-proliferative activity in cancer cells
are thus unsuitable as potential drug candidate as they are cyto-
toxic in normal cells. On the other hand, compounds, 5c, 5d, 5g
and 5h did not significantly affect growth of Vero and osteoblasts,
suggesting that these molecules have selectivity for inducing
growth inhibition of cancer cells.
2.2.4. Induction of apoptosis
We next investigated the mechanism of inhibition of cell
growth of the breast cancer cells by 5c, 5d, 5g and 5h and studied
whether or not apoptosis was involved in such process. This was
studied by DNA fragmentation and caspase activation assays.
2.2.4.1. DNA fragmentation. DNA fragmentation was measured
with BrdU-labeling (5-bromo-2-deoxyuridine) of fragmented
DNA (Roche, USA) with the four active compounds, 5c, 5d, 5g
and 5h. The results showed that compound 5d at 10
lM
(p = 0.034) and 20 M (p = 0.035), 5h at 20 M (p = 0.042) induced
l
l
significant DNA fragmentation in MCF-7 cell line (Fig. 5a). With the
other two compounds 5c and 5g, the DNA fragmentation was not
statistically significant. Similarly, in MDA-MB-231 cell line com-
2.2.3. Relative binding affinity to ER
In order to confirm if the action of active compounds is through
ER, their ER binding potential was checked using competitive bind-
ing assay, employing radio labeled estradiol (3H–E2) as the refer-
ence compound. Briefly, the uterine cytosol (obtained from
immature 20–21 days old E2 primed rats) was incubated with
3H-estradiol in the absence or presence of various concentrations
of test compounds for 22 h at 4 °C. 3H–E2 bound and free fractions
were separated by charcoal adsorption method. The relative bind-
pounds 5d at 20
lM (p = 0.025) and 5h at 10 lM (p = 0.044) and
at 20 M (p = 0.028) induced significant DNA fragmentation (Fig.
l
5b). Further, the four active and non-cytotoxic compounds (5d,
5h, 5c and 5g) also induced significant qualitative changes in per-
centage of apoptotic cells in treated MDA-MB-231 cells through
morphological analysis for apoptosis with Hoechst staining.
2.2.4.2. Induction of caspase. Caspases are a family of cysteine
proteases which sequentially get activated and are therefore used
as a measure of induction of caspase-mediated apoptosis. To assess
whether the apoptosis induced by these four compounds was cas-
pase mediated, evaluation of the four active compounds showed
Table 1
Antiproliferative activity of compounds against MCF-7 and MDA-MB-231 cell lines
that 5d at 20
lM (p = 0.033), 5h at 20 lM (p = 0.018) and 5g at
Compound no.
IC50
(lM)
RBA (% of estradiol-17b)
20 M (p = 0.039) significantly induced basal caspase level in
l
MCF-7
MDA-MB-231
MCF-7 signaling for caspase dependent apoptosis. However, 5c
caused induction of caspase only at a higher concentration of
40 lM (p = 0.021) (Fig. 6). Further, compounds did not induce cas-
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
6g
38
21
25
24
>50
26
14
14
14
26
25
24
23
>50
16
10
>100
13
38
0.015
0.143
0.107
0.150
0.093
0.125
0.03
0.272
0.02
0.091
<0.001
<0.001
0.12
pase activation in MDA-MB-231 cells because DNA fragmentation
and apoptosis do not always require caspase activation and induc-
tion of apoptosis may be due to oxidative stress or through apop-
tosis inducing factors (AIF) where caspase activation is not
essential.
From the results, it is clearly observed that 3,4,6-triarylpyra-
nones with phenolic substituent groups showed significant anti-
proliferative activity, diphenolic compounds showing better
activity than monophenolic molecules. Incorporation of tertiary
alkoxy amino groups, like ethoxypyrrolidine or piperidine an
essential requisite of SERM molecules at any of the phenolic group
rendered the compound toxic or diminished the antiproliferative
27
>100
>100
17
26
9
11
34
>100
>100
>100
12
0.08
0.02
2.2
Tamoxifen
20

R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
3851
Figure 3. Cytotoxicity of pyranone compounds towards normal cells. (a) Vero cell lines and (b) primary cultured osteoblasts were treated with active pyranone compounds at
varying concentrations for 12 h and induced cell death was measured with MTT assay. The data presented are average of three independent experiments.
activity, thereby giving credence to the low ER binding and an ER
independent pathway.
Furthermore, to rationalize the activity profile, we performed
comparative docking study of computationally energy minimized
conformation of active molecule 5h with structurally similar drug,
4-hydroxy tamoxifen (OHT) and 17b-estradiol in estrogen receptor
(ER a). The modeling study was carried out in molecular operating
environment (MOE) software using a systematic conformational
search with energy minimization. The crystal structure of estrogen
receptor ligand binding domain (LBD) in complex with the endog-
enous estrogen, 17b-estradiol, 4-hydroxytamoxifen and reference
protein coordinates used for structural alignment were obtained
from the Protein Data Bank (http://www.rcsb.org/pdb/home, entry
codes 1ERE and 3ERT, respectively).
Figure 7a and b, shows the orientation and interaction of the ac-
tive molecule 5h within the ER cavity in comparison to 4-hydroxy
tamoxifen and 17b-estradiol. As is evident from Figure 7a, of the
three aryl rings in 5h, 3 and 4-aryl rings are more favorably aligned
with the aryls of 4-hydroxy tamoxifen as rationalized. Deviation of
the pendant aryl ring from the planarity of the stilbene core of
tamoxifen and also with that of 17b-estradiol (Fig. 7b) may be
responsible for weak binding interaction of the ligand with the
Figure 4. Trans-activation of ER
a and ERb in Cos-7. COS-7 cells were transfected
with plasmids and treated with compounds as described in experimental section.
Results represent mean SD of relative luciferase activities obtained from three
independent experiments.

3852
R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
Figure 5. DNA Fragmentation induced in breast cancer cells. (a) MCF-7 and (b) MDA-MB-231. Cells were treated with 10, 20 and 30 lM concentrations of pyranone
compounds for 12 h and labeled fragmented DNA was measured at OD 370 nm. Positive and negative reference controls were used as per manufacturer’s instruction. Data
presented are mean of duplicates from two independent experiments.
Figure 6. Induction of caspase in MCF-7. Cells were treated with compounds for 12 h at various concentrations, and then incubated with DEVD-rhodamine-110. Fluorescence
from the activated caspases-mediated release of rhodamine-110 was measured at 560 nm. Data presented are mean of duplicates from two independent experiments.
Compounds did not induce caspase activation in MDA-MB-231cells (data not shown).
salient amino acids essential for receptor binding affinity. How-
ever, the similarity in orientation of aryl bearing the antagonistic
side chain of 4-hydroxy tamoxifen over the 4-aryl ring of 5h may
conform for the antagonistic profile shown in MCF-7 cells.

R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
4. Experimental
3853
4.1. General
All the glass apparatus were oven dried prior to use. Melting
points were determined on COMPLAB melting point apparatus
and are uncorrected. IR spectra were recorded on a Perkin–Elmer
FT-IR RXI spectrophotometer. 1H NMR and 13C NMR spectra were
recorded on Bruker DPX-200 (200 MHz for 1H and at 50 MHz for
13C) or DRX-300 (300 MHz for 1H and at 75 MHz for 13C) spec-
trometers using CDCl3 as solvent. Tetramethylsilane served as
an internal standard in 1H NMR and CDCl3 in 13C NMR spectra.
(FAB-MS) spectra were obtained on a JEOL SX 102 spectrometer
using argon/xenon (6 kV, 10 mA) as the FAB gas. Glycerol or m-
nitro benzyl alcohol was used as matrix. High-resolution electron
impact mass spectra (HR-EIMS) were obtained on a JEOL MS
route 600H instrument. Reactions were monitored on precoated
silica gel TLC plates (Merck). Detecting agents used (for TLC)
were iodine vapors and or spraying with an aqueous solution
of ceric sulfate followed by heating at 150 °C. Column chroma-
tography was performed over silica gel (60–120 Mesh) procured
from Qualigens (India) using freshly distilled solvents. All chem-
icals and reagents were obtained from Aldrich (Milwaukee, WI),
Lancaster (England), or Spectrochem (India) and were used with-
out further purification.
The synthesis of 3-(4-methoxyphenyl)-1-phenyl-prop-2-yn-1-
one(2a),
1-(4-methoxyphenyl)-3-phenyl-prop-2-yn-1-one(2b),
and 1,3-bis(4-methoxyphenyl)-prop-2-yn-1-one(2c) were pre-
pared following the reported procedure.21
4.1.1. 4-(4-Methoxyphenyl)-3,6-diphenyl-pyran-2-one (4a)
To a suspension of NaH (60% oil dispersion, 32.8 mg, 1.2 mmol)
in dry DMSO (5 mL) was added a solution of the ethyl phenyl ace-
tate (3a, 164 mg, 1 mmol) and the reaction mixture was stirred for
15 min. at rt. A solution of 2a (236 mg, 1 mmol) in DMSO (1 mL)
was added dropwise to the above solution and the mixture was
stirred at rt for 1 h. On completion, the reaction mixture was
quenched with 1 N HCl, the solid precipitated was filtered, washed
with water and crystallized from ethanol to afford yellow colored
crystals of 4a, yield (80%), mp 192–195 °C, IR (KBr)
mmax: 1705,
1450 cmÀ1 1H NMR (CDCl3):
;
d
3.8 (s, 3H, OCH3), 6.77 (d,
J = 8.4 Hz, 2H), 6.80 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.22–7.28 (m,
5H, ArH), 7.49–7.51 (m, 3H, ArH), 7.87–7.92 (m, 2H, ArH); 13C
NMR (CDCl3): d 55.66, 105.41, 123.56, 126.02, 128.13, 128.40,
128.81, 129.12, 129.39, 131.19, 131.30, 131.79, 134.23, 138.22,
153.18, 158.69, 163.15; MS(ESI) m /z 355 (M++1).
Figure 7. Comparative docking orientation and predicted receptor interactions. (a)
Compound 5h (green), 4-hydroxytamoxifen (OHT, red) and ER ; (b) compound 5h
(green), 17b-estradiol (red) and ERa (PDB code 3ERE).
a
4.1.2. 6-(4-Methoxyphenyl)-3,4-diphenyl-pyran-2-one (4b)
3. Conclusion
Yellow solid; yield (78%); mp 180–182 °C; IR (KBr)
1442 cmÀ1 1H NMR (CDCl3): d 3.87 (s, 3H, OCH3), 6.73 (s, 1H),
6.94 (d, J = 8.7 Hz, 2H), 7.20–7.26 (m, 10H, ArH), 7.90 (d,
J = 8.7 Hz, 2H); 13C NMR (CDCl3):
55.88, 103.99, 114.30,
mmax: 1716,
;
This is the first report highlighting the significance of 3,4,6-tri-
aryl pyranones as potential antiproliferative agents in breast can-
cer cells. This is confirmed by pharmacological results which
show that compounds 5c, 5d, 5g and 5h exhibit specific and selec-
tive cytotoxicity against both ER positive and ER negative breast
d
122.79, 124.34, 127.72, 127.96, 128.35, 128.75, 129.10, 131.35,
134.42, 138.42, 153.55, 158.88, 162.15, 163.29; MS(ESI) m /z
354(M+).
cancer cell lines. Their low relative binding affinity for ER
a and fail-
ure to cause trans-activation of both ER and ERb clearly demon-
a
strate their ER-independent nature of action in inducing cell
death. Two compounds 5d and 5h induced significant DNA frag-
mentation in both MCF-7 and MDA-MB-231 cell lines and the
induction of cell death by these compounds in MCF-7 cells is prob-
ably through caspase-dependant apoptosis. The active compounds
could be speculated to employ some non-ER dependant pathway,
the identification of the exact mechanism of action or cellular tar-
get utilized by these compounds are under investigation.
4.1.3. 3-(4-Methoxyphenyl)-4,6-diphenyl-pyran-2-one (4c)
Yellow solid; yield (77%); mp 203–205 °C; IR (KBr)
mmax: 1708,
1454 cmÀ1 1H NMR (CDCl3): d 3.77 (s, 3H, OCH3), 6.77 (d,
;
J = 8.6 Hz, 2H), 6.83 (s, 1H), 7.13 (d, J = 8.7 Hz, 2H), 7.22–7.26 (m,
5H, ArH), 7.42–7.47 (m, 3H, ArH), 7.90 (d, J = 8.8 Hz, 2H);.13C
NMR (CDCl3): d 55.58, 105.49, 109.30, 123.21, 125.94, 128.86,
129.03, 129.11, 129.35, 131.05, 131.68, 132.42, 132.56, 152.25,
158.53, 159.43, 163.15; MS (ESI) m/z 355 (M++H).

3854
R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
4.1.4. 3,4-Bis-(4-methoxyphenyl)-6-phenyl-pyran-2-one (4d)
Yellow solid; yield (75%), mp 210–212 °C; IR (KBr) max: 1685,
1415, 1440 cmÀ1 1H NMR (CDCl3): d 3.78 (s, 3H, OCH3), 3.79 (s,
6.76 (m, 4H, ArH), 7.42–7.45 (m, 3H, ArH), 7.46–7.48 (m, 2H, ArH),
m
7.79 (s, 1H, OH); MS (ESI) m/z 371 (M++1).
;
3H, OCH3), 6.70–6.80 (m, 4H+1H), 6.81–6.83 (m, 2H, ArH), 7.42–
7.46 (m, 3H, ArH), 7.87 (d, J = 8.8 Hz, 2H); 13C NMR (CDCl3): d
55.60, 55.69, 105.5, 114.0, 114.2, 122.46, 125.9, 126.73, 129.32,
130.7, 130.89, 131.94, 132.53, 138.68, 152.07, 158.08, 159.32,
160.25, 163.58; MS (ESI) m/z: 385 (M++H).
4.1.12. 3,6-Bis-(4-hydroxyphenyl)-4-phenyl-pyran-2-one (5f)
Yellow solid; yield (71%); mp >250 °C; IR (KBr)
mmax: 3407,
1684 cmÀ1 1H NMR (CD3COCD3): d 6.72–6.78 (m, 4H, ArH),
;
7.04–7.08 (m, 3H, ArH), 7.14–7.17 (m, 2H, ArH), 7.50–7.56 (m,
3H, ArH), 7.98–8.01 (m, 2H, ArH), 8.48 (s, 1H), 8.77 (s, 1H); MS
(ESI) m/z 357 (M++1).
4.1.5. 3,6-Bis-(4-methoxyphenyl)-4-phenyl-pyran-2-one (4e)
Yellow solid; yield (80%); mp 180–182 °C; IR (KBr)
mmax: 1709,
4.1.13. 3-(4-Hydroxyphenyl)-6-(4-methoxyphenyl)-4-phenyl-
pyran-2-one (5g)
1465, 1441 cmÀ1 1H NMR (CDCl3): d 3.79 (s, 3H, OCH3), 3.89 (s,
;
3H, OCH3), 6.71 (s, 1H), 6.73 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz,
2H), 7.22–7.26 (m, 7H, ArH), 7.84 (d, J = 8.8 Hz, 2H); 13C NMR
(CDCl3): d 55.57, 55.86, 104.06, 113.87, 114.00, 122.00, 124.42,
126.53, 127.63, 128.80, 128.92, 129.08, 132.57, 138.68, 152.90,
158.46, 159.30, 162.04, 163.56; MS (ESI) m/z 385 (M++H).
Yellow solid; yield (70%); mp >250 °C; IR (KBr)
mmax: 3405,
1683, 1432 cmÀ1 1H NMR (CD3COCD3): d 3.89 (s, 3H), 6.73 (s,
;
1H), 6.71 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 7.12–7.17 (m,
2H, ArH), 7.20–7.38 (m, 3H, ArH), 7.70 (d, J = 8.0 Hz, 2H), 7.89 (d,
J = 8.0 Hz, 2H); MS(ESI) m/z 371 (M++1), HRMS (ESI): calculated
370.1205 and measured mass 370.1205.
4.1.6. 4,6-Bis-(4-methoxyphenyl)-3-phenyl-pyran-2-one (4f)
Yellow solid; yield (85%); mp 195–197 °C; IR (KBr)
m
max: 1699,
4.1.14. 4,6-Bis-(4-hydroxyphenyl)-3-phenyl-pyran-2-one (5h)
1466, 1417 cmÀ1 1H NMR (CDCl3): d 3.79 (s, 3H, OCH3), 3.86 (s,
;
Yellow solid; yield (63%), mp >250 °C; IR (KBr)
mmax: 3426,
3H, OCH3), 6.82 (s, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.4 Hz,
2H), 7.39–7.44 (m, 7H, ArH), 7.89 (d, J = 8.8 Hz, 2H); 13C NMR
(CDCl3): d 55.69, 55.85, 105.63, 114.27, 114.94, 125.94, 127.97,
128.41, 128.99, 129.34, 130.75, 131.00, 132.59, 122.41, 127.04,
131.93, 137.31, 152.15, 158.11, 158.61, 160.29, 163.55; MS (ESI)
m/z 385 (M++H).
1670 cmÀ1 1H NMR (CD3COCD3): d 6.72 (d, J = 8.7 Hz, 2H), 6.91
;
(s, 1H), 6.99 (d, J = 8.7 Hz, 2H), 7.11 (d, J = 8.7 Hz, 2H), 7.20–7.23
(m, 5H, ArH), 7.88 (d, J = 8.7 Hz, 2H), 8.8 (s, 1H, OH), 9.2 (s, 1H,
OH); MS(ESI) m/z 357 (M++1), HRMS (ESI): calculated 356.1049
and measured mass 356.1075.
4.1.15. 3,6-Diphenyl-4[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]pyran-
2-one (6a)
4.1.7. 4-(4-Hydroxyphenyl)-3,6-diphenyl-pyran-2-one (5a)
A mixture of 4a and pyridine hydrochloride (1:3 w/w) was
heated at 200 °C for 2 h, cooled, diluted with water, and extracted
with EtOAc. The organic layer was washed once with an equal vol-
ume of 1 N HCl followed by water, dried (Na2SO4), and concen-
trated to obtain the crude product. It was purified over a short
column of silica gel to give pure 5a as yellow solid, yield (70%);
A mixture of 5a (1 mmol), 2-pyrrolidinoethyl chloride hydro-
chloride (1.5 mmol), anhydrous K2CO3 (2.5 mmol) in DMF
(10 mL) was heated at 70 °C for overnight. On completion the reac-
tion was cooled and filtered. The filtrate was concentrated in vacuo
and subjected to chromatography over a column of basic alumina
to furnish 6a, yellow solid; yield (63%); mp 117–119 °C; 1H NMR
(CDCl3): d 1.75–1.83 (m, 4H), 2.59–2.63 (m, 4H), 2.89 (t, 2H),
4.08 (t, 2H), 6.78 (d, J = 8.2 Hz, 2H), 6.85 (s, 1H), 7.12 (d,
J = 8.2 Hz, 2H,), 7.24–7.30 (m, 5H, ArH), 7.47–7.49 (m, 3H, ArH),
7.19 (d, J = 8.2 Hz, 2H); 13C NMR: d 21.31, 54.72, 54.97, 64.22,
105.26, 112.07, 119.48, 125.77, 126.67, 129.01, 129.72, 151.20,
156.92, 159.23; MS (ESI) m /z 438(M++H). Anal. Calcd for
C29H27NO3: C, 79.61; H, 6.22; N, 3.20. Found: C, 79.49; H, 6.32;
N, 3.18.
mp >250 °C; IR (KBr) mmax
:
3405, 1680 cmÀ1
;
1H NMR
(CDCl3 + DMSO-d6): d 6.63 (d, J = 8.4 Hz, 2H), 6.9 (s, 1H), 7.0 (d,
J = 8.4 Hz, 2H), 7.14–7.19 (m, 5H, ArH), 7.44–7.49 (m, 3H, ArH),
7.87–7.91 (m, 2H, ArH), 8.56 (s, OH); MS (ESI) m/z 341 (M++1).
4.1.8. 6-(4-Hydroxyphenyl)-3,4-diphenyl-pyran-2-one (5b)
Yellow solid; yield (67%); mp >250 °C; IR (KBr)
mmax: 3438,
1670 cmÀ1 1H NMR (CD3COCD3): d 6.52 (d, J = 8.7 Hz, 2H), 6.7 (s,
;
1H), 7.26 (d, J = 8.7 Hz, 2H), 7.35–7.39(m, 5H, ArH), 7.40–7.43 (m,
3H, ArH), 7.92–8.01 (m, 2H, ArH), 8.62 (s, OH); MS (ESI) m/z 341
(M++1).
4.1.16. 3,6-Diphenyl-4[4-(2-piperidin-1-yl-ethoxy)-phenyl]-pyran-2-
one (6b)
Yellow solid; yield (70%); mp 147–149 °C; 1H NMR (CDCl3): d
1.43–1.46 (m, 6H), 2.45–2.49 (m, 4H), 2.77(t, 2H), 4.09 (t, 2H),
6.78 (d, J = 8.8 Hz, 2H), 6.83 (s, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.23–
7.26 (m, 5H, ArH), 7.46–7.48 (m, 3H, ArH), 7.91 (d, J = 8.8 Hz,
2H); 13C NMR: d 21.92, 53.11, 61.98, 98.73, 103.86, 112.80,
113.27, 114.05, 122.43, 124.43, 125.99, 126.53, 127.05, 127.33,
127.45, 127.68, 127.95, 129.95, 130.03, 130.87, 131.14, 136.46,
151.38, 155.33; MS (ESI) m /z 452 (M++H). Anal. Calcd for
C30H29NO3: C, 79.80; H, 6.47; N, 3.10. Found: C, 79.69; H, 6.38; N,
3.16.
4.1.9. 3-(4-Hydroxyphenyl)-4,6-diphenyl-pyran-2-one (5c)
Yellow solid; yield (72%); mp >250 °C; IR (KBr)
mmax: 3430,
1684 cmÀ1 1H NMR (CD3COCD3): d 6.70 (d, J = 8.7 Hz, 2H), 7.0 (s,
;
1H), 7.08 (d, J = 8.7 Hz, 2H), 7.29–7.31 (m, 5H, ArH), 7.54–7.56
(m, 3H, ArH), 8.01–8.02 (m, 2H, ArH), 8.42 (s, OH); MS (ESI) m/z
341(M++1).
4.1.10. 3,4-Bis-(4-hydroxyphenyl)-6-phenyl-pyran-2-one (5d)
Yellow solid; yield (70%); mp >250 °C; IR (KBr)
mmax: 3332,
1680 cmÀ1 1H NMR (CD3COCD3): d 6.77–6.83 (m, 4H), 7.10–7.13
;
(m, 3H), 7.20–7.23 (m, 2H, ArH), 7.55–7.58 (m, 3H, ArH), 8.00–
8.04 (m, 2H, ArH), 8.58 (s, OH), 8.87 (s, OH); MS (ESI) m/z
357(M++1), HRMS (ESI): calculated 356.1049 and measured mass
356.1025.
4.1.17. 3,4-Diphenyl-6-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-
pyran-2-one (6c)
Yellow solid; yield (65%); mp 121–123 °C; 1H NMR (CDCl3): d
1.79–1.84 (m, 4H), 2.61–2.67 (m, 4H), 2.97 (t, 2H), 4.21 (t, 2H),
6.73 (s, 1H), 6.99 (d, J = 8.8 Hz, 2H), 7.16–7.25 (m, 10H, ArH),
7.28 (d, J = 8.8 Hz, 2H); 13C NMR: d 22.24, 53.45, 65.98, 103.55,
113.70, 120.67, 122.76, 125.99, 126.61, 127.02, 127.37, 129.64,
132.74, 136.78, 151.80, 157.21, 159.73, 161.52; MS (ESI) m/z 438
4.1.11. 3-(4-Hydroxyphenyl)-4-(4-methoxyphenyl)-6-phenyl-
pyran-2-one (5e)
Yellow solid; yield (68%); mp >250 °C; IR (KBr)
mmax: 3426, 1688,
1455 cmÀ1; 1H NMR (CD3COCD3): d 3.79 (s, 3H), 6.69 (s, 1H), 6.74–

R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
3855
(M++H). Anal. Calcd for C29H27NO3: C, 79.61; H, 6.22; N, 3.20.
Found: C, 79.72; H, 6.14; N, 3.32.
4.2.2. Cytotoxicity assay in Vero cell line and osteoblast
Cytotoxic effects of the compounds were determined using MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
4.1.18. 3,4-Diphenyl-6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-
pyran-2-one (6d)
reduction assay.25 1 Â 104 cells/well were seeded in 100
ll DMEM
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 °C in a CO2 incubator. Com-
pounds, diluted to the desired concentrations in culture medium.
After 48 h of incubation, media were removed and to each well
Yellow solid; yield (69%); mp 180–182 °C; 1H NMR (CDCl3): d
1.46–1.50 (m, 2H), 1.52–1.68 (m, 4H), 2.56–2.59 (m, 4H), 2.82(t,
2H), 4.12 (t, 2H), 6.77 (d, J = 8.6 Hz, 2H), 6.82 (s, 1H), 7.23–7.33
(m, 3H, ArH), 7.47–7.49 (m, 7H, ArH), 7.90 (d, J = 8.6 Hz, 2H). 13C
10
bated for 4 h. Supernatant from each well was carefully removed,
formazon crystals were dissolved in 100 l of DMSO and absor-
ll MTT (5 mg/mL) was added and the plates were further incu-
NMR:
d 23.49, 54.75, 54.97, 67.12, 105.02, 114.39, 125.59,
127.58, 128.08, 128.98, 129.86, 130.31, 130.66, 130.88, 134.21,
152.26, 158.08, 159.27, 162.65; MS (ESI) m /z 452(M++H). Anal.
Calcd for C30H29NO3: C, 79.80; H, 6.47; N, 3.10. Found: C, 79.73;
H, 6.34; N, 3.21.
l
bance at 540 nm wavelength were recorded.
4.2.3. Dual-luciferase reporter assay
40,000 cells Cos-7cells were seeded in 24-well plates 24 h be-
fore transfection. Cells were co-transfected with 200 ng of reporter
plasmid (pGL3-Gal4Tk-Luc), 200 ng of expression plasmid (pVP-
4.1.19. 4,6-Diphenyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-
pyran-2-one (6e)
Yellow solid; yield (60%); mp 110–112 °C; 1H NMR (CDCl3): d
1.80–1.83 (m, 4H), 2.60–2.66 (m, 4H), 2.75 (t, 2H), 4.06 (t, 2H),
6.74 (d, J = 8.8 Hz, 2H), 6.82 (s, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.25–
7.28 (m, 5H, ArH), 7.39–7.43 (m, 3H, ArH), 7.90 (d, J = 8.8 Hz,
2H); 13C NMR: d 22.36, 23.22, 27.7, 28.26, 53.57, 65.88, 103.68,
113.82, 120.86, 122.92, 126.02, 126.73, 127.13, 127.56, 129.86,
132.85, 136.96, 152.52, 157.36, 159.84, 161.65; MS(ESI) m /z
438(M++H); HRMS (ESI): calculated 437.2481 and measured mass
437.2443.
16/VP-16-ERa/VP-16-ERb), 200 ng of co-activator (pM-Tif-2),
100 ng of pRL-CMV, 100 ng of pEGFPC-1, using transfection reagent
(Lipofectamine-2000). After 24-h posttransfection, cells were har-
vested for luciferase reporter assay using the dual-luciferase repor-
ter assay system.26 The activated luciferase activity was
normalized to that of the internal control Renilla luciferase activity
by pRL-CMV as relative luciferase unit as well as by GFP and fold of
relative luciferase activation unit were compared to VP-16 control
transfection. All observations were performed as duplicate in three
independent experiments.
4.1.20. 4,6-Diphenyl-3-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-
pyran-2-one (6f)
4.2.4. DNA fragmentations assay
Yellow solid; yield (71%); mp 150–152 °C; 1H NMR (CDCl3): d
1.44–1.49 (m, 2H), 1.59–1.64 (m, 4H), 2.57 (t, 2H), 2.81 (t, 2H),
4.12 (t, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.78 (m, 2H, ArH), 6.84 (s,
1H), 7.28–7.30 (m, 5H, ArH), 7.44–7.48 (m, 3H, ArH), 7.92 (d,
J = 8.8 Hz, 2H); 13C NMR: d 22.02, 51.07, 60.86, 101.62, 105.52,
113.06, 114.72, 115.02, 120.23, 125.54, 126.07, 127.00, 128.22,
129.43, 129.47, 129.75, 129.89, 129.97, 133.25, 133.78, 134.14,
138.52, 153.24, 156.89; MS (ESI) m/z 452(M++H); HRMS (ESI): cal-
culated 451.2147 and measured mass 451.2148.
MCF cells were labeled with BrdU by incubating overnight. Then
the labeled cells were incubated for 24 h in the presence of com-
pound for test or without for untreated control. At the end of the
incubation, cells were treated with lysis buffer for 30 min at rt,
centrifuged at 250g for 10 min and supernatant was prepared.
The anti-DNA antibody was adsorbed onto the wells of a micro-
plate for 1h at rt. Supernatant of the cell lysate was added to the
pre-adsorbed microplate. BrdU-labeled DNA fragments in the sam-
ple bind to the immobilized anti-DNA antibody. The immunocom-
plex of BrdU-labeled DNA-fragments were fixed by microwave
irradiation for 5 min for the accessibility of the BrdU antigen. Then
peroxidase conjugated anti-BrdU antibodies were added for react-
ing with the BrdU-labeled DNA to form an immuno-complex by
incubating 90 min. The bound anti-BrdU-conjugates in the immu-
no-complex were then measured using a peroxidase substrate in
ELISA reader.
4.1.21. 4-(4-Methoxy-phenyl)-6-phenyl-3[4-(2-piperidin-1-yl-
ethoxy)-phenyl]-pyran-2-one (6g)
Oil, yield (70%); 1H NMR (CDCl3): d 1.55–1.59 (m, 2H), 1.61–1.64
(m, 4H), 2.47–2.52 (m, 4H), 2.79 (t, 2H), 3.81 (s, 3H), 4.12 (t, 2H),
6.68–6.72 (m, 5H, ArH), 6.80 (s, 1H), 7.08–7.15 (m, 4H, ArH),
7.42–7.48 (m, 3H, ArH), 7.79–7.84 (m, 2H, ArH); 13C NMR: d
23.94, 25.53, 30.88, 55.25, 57.68, 65.49, 105.10, 113.86, 114.25,
121.98, 125.49, 126.50, 128.90, 130.03, 130.29, 130.54, 131.52,
132.12, 151.73, 157.65, 158.03, 159.88, 163.10; MS(ESI) m/z 482
(M++H) Anal. Calcd for C31H31NO4: C, 77.31; H, 6.49; N, 2.91.
Found: C, 77.22; H, 6.59; N, 2.78.
4.2.5. Morphological analysis for apoptosis with Hoechst
staining
Cells were seeded at a density of 10,000 cells over 18-mm cov-
erslips and incubated for 24 h. Then, the medium was replaced,
and cells were treated with 10
lM of compounds for 18 h. Cells
4.2. Experimental protocols
treated with vehicle (0.001% DMSO) were included as controls
for all experiments. After overnight treatment, Hoechst 33342 (Sig-
4.2.1. MTT assay in Breast cancer cell line
ma–Aldrich) were added to medium at a concentration of 0.5 lg/
The antiproliferative activities of the compounds were deter-
mined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
razolium bromide) reduction assay.24 1 Â 104 cells/well were
ml containing 4% paraformaldehyde. After incubation for 30 min
at 37 °C, cells from each dish were captured from randomly se-
lected fields under fluorescent microscope (Leica, Germany) to
qualitatively determine the proportion of viable and apoptotic cells
based on their relative fluorescence and nuclear fragmentation.
seeded in 100 ll DMEM (Dulbecco’s Modified Eagle’s Medium)
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 °C in a CO2 incubator. Com-
pounds, diluted to the desired concentrations in culture medium.
After 48 h of incubation, media were removed and to each well
4.2.6. Caspase assay
The homogeneous caspase assay kit (Roche, Germany) that in-
cludes caspases 2, 3, 6, 7, 8, 9 and 10 was used to measure caspase
activities after apoptosis induction according to manufacturer’s
instructions. Camptothecin treated U937 cells were used as posi-
tive control. MCF-7 cells were cultured under serum-deprived con-
10
bated for 4 h. Supernatant from each well was carefully removed,
formazon crystals were dissolved in 100 l of DMSO and absor-
bance at 540 nm wavelength was recorded.
ll MTT (5 mg/mL) was added and the plates were further incu-
l

3856
R. Shankar et al. / Bioorg. Med. Chem. 17 (2009) 3847–3856
6. Rosati, R. L.; Da Silva Jardine, P.; Cameron, K. O.; Thompson, D. D.; Ke, H. Z.;
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C.; Newhouse, B. N.; Tjoa, C. M.; Sweetnam, P. M.; Cole, M. J.; Arriola, M. W.;
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concentrations. Cells were then incubated with DEVD-rhodamine-
110((tetra-peptide sequence ‘aspartic acid–glutamic acid–valine–
aspartic acid’ recognized by caspases).Upon cleavage of the rhoda-
mine substrate by activated caspases, fluorescence from the re-
leased rhodamine-110 was measured.
4.2.7. Data analysis
All data were expressed as mean SD with at least three sepa-
rate experiments. The data were compared using Microsoft Excel
or Prism statistical program, with Student’s t-test for unpaired
variants. Values of P < 0.05 were regarded as statistically
significant.
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Acknowledgments
The authors, Ravi Shankar is thankful to CSIR for SRF fellowship
and Uma Sharan Singh is grateful to Ministry of Health and Family
Welfare for financial assistance. All are thankful to SAIF for spectro-
scopic analyses of the compounds. The authors are also thankful to
Dr. S. Sanyal. DTDD, CDRI, for providing constructs lab facility and
guidance for conducting Dual luciferace reporter assay for ER
transactivation, Dr. N. Chattopadhyay, Head, Endocrinology for
his critical review of manuscript and suggestions, Dr. Y.S. Prabha-
kar, MPC, for comparative computational studies and Dr. Brijesh
Kumar, SAIF, for HRMS data.
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