Synthesis and anti breast cancer activity of biphenyl based chalcones

Article abstract of DOI:10.1016/j.bmc.2010.05.015

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl) -bis(3-pheylprop-2-ene-1-ones (5-33) were prepared by the reaction of 1,3-diacetyl biphenyls (1-4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellen

Full text of DOI:10.1016/j.bmc.2010.05.015

Bioorganic & Medicinal Chemistry 18 (2010) 4711–4720
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti breast cancer activity of biphenyl based chalcones
Anindra Sharma a, Bandana Chakravarti b, , Munna Prasad Gupt a, Jawed A. Siddiqui b, Rituraj Konwar b,à
,
Rama P. Tripathi a,
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute (CSIR), Lucknow 226 001, India
b Endocrinology Division, Central Drug Research Institute (CSIR), Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
A series of (2E,20E)-1,10-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33)
were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of cat-
alytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer
activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and
MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer)
cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited
Article history:
Received 16 March 2010
Revised 4 May 2010
Accepted 5 May 2010
Available online 16 May 2010
Keywords:
the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 lM. The
Breast cancer
Biphenyl chalcones
KOH
two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against
the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell
cycle arrest and loss of mitochondrial membrane potential.
Piperidine
Ó 2010 Elsevier Ltd. All rights reserved.
Flavanoids
1. Introduction
dous amount of work is reported on the synthesis, bio-evaluations,
and mechanism of action of these compounds including their inter-
1,3-Diaryl-2-propen-1-ones, commonly known as chalcones are
prominent secondary metabolites and precursors of flavonoids and
isoflavonoids in plants. Structurally in such compounds two phenyl
rings are flanked by 2-propenone moiety and this arrangement
makes them ‘privileged structure’ as described by Evans et al.1 The
‘enone’ moiety is present in many biologically active molecules
and it is considered to be primarily responsible for eliciting the bio-
logical response in such molecule. Chalcones in general are reported
to exhibit various pharmacological activities such as anticancer,
antimalarial, antiinflammatory, immunomodulatory, antibacterial,
immunosuppressive, antiprotozan, trypanocidal, and leismanicidal
properties.2–8 Licochalcone-A, a natural product, isolated from the
licorice root, is known to have a wide variety of anticancer effects.9
They have recently been reported as antiproliferative and antitumor
agents and interest in this class of molecules in identifying potent
anticancer agents is renewed. These molecules with enone moiety
inhibit several enzymes which render them the therapeutic poten-
tial. The ease of preparation, the potential of oral administra-
tion,10–13 and safety13 also support the feasibility of chalcone-
based compounds to be used as chemotherapeutic agents. Tremen-
ference in microtubule formation14–24 and many cellular signaling
pathways,25–27 such as nuclear factor kappa B (NF-
and inhibition of IKB Kinaseb (IKKb). Any alteration in the 3-carbon
jB) inhibition
a
propenone moiety is known to lose the biological activities, the two
aromatic rings, therefore have extensively been modified to get a
variety of biologically active chalcones and few of them are shown
in Figure 1. A number of chalcones with hydrophobic moieties and
hydrophilic substituents attached to the aromatic rings such as
adamantanyl and steroidal substituents were prepared and showed
potent anticancer activity against different cancer cell lines.28 Re-
cently, we have reported
a,
a0-(EE)-bis(benzylidene)-cycloalka-
nones as potent antitubercular and based on reports that such
compounds may act as anticancer agents as well.29–31 We thought
to synthesize hitherto unreported chalcones and see their antican-
cer effects. Herein we have synthesized yet another type of novel
chalcones based on a 1,3-diacetyl biphenyl system with an extra
phenyl-2-propenone moiety (Fig. 2), which is a pharmacophore in
this class of compounds, and subsequently evaluated their antican-
cer activities against human breast cancer. The mode of action of the
two potent compounds was also established.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +91 0522 2612411; fax: +91 522 2623405/
2623938/2629504.
E-mail addresses: r_konwar@cdri.res.in (R. Konwar), rpt.cdri@gmail.com (R.P.
Tripathi).
Equal contribution first author.
Correspondence (Biological Activity) Tel.: +91 0522 2613894; fax: +91 522
à
The starting pentasubstituted 1,3-diacetyl biphenyls (1–4) were
prepared by reaction of different aromatic aldehydes and
2223405/2223938.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.015

4712
A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
O
O
O
OH
HO
OH
butein
OH
HO
H3CO
OH
HO
OH
OCH3
xanthohumol
licochalcone A
O
O
O
O
COOH
N
COOH
O
COOH
O
BMS 181156
AGN193198
MX781
O
O
R
O
COOH
R
n
HO
Where n= 1, 2, 3.
CD2325
Antitubercular and Anticancer activity
Figure 1. Chemical structure of some biologically active chalcone.
Pharmacophore
O
OH
Pharmacophore
R
Biphenyl system
O
A
CH3
B
R'
Earlier work
O
Pharmacophore
Our work
Figure 2. Earlier prepared chalcones and designed molecules with two propenone moieties as pharmacophore.
acetylacetone in presence of piperidine followed by iodine cata-
lyzed oxidative aromatization of the intermediate cyclohexanones
as recently reported by us.32 The reaction of the biphenyls (1–4)
separately with different aldehydes in the presence of catalytic
amount of solid KOH in ethanol resulted in the title compounds
5–33 in excellent yields (Scheme 1, Table 1). The structures of
these compounds were established on the basis of their spectro-
scopic data and microanalyses. The IR spectra of the compounds,
in general, exhibited the absorption band at around 3450 cmꢀ1
indicating the presence of phenolic hydroxyl group, alkene C–H
stretching vibration at around 3021 cmꢀ1, absorption band of con-
jugated carbonyl group at around 1638–1640 cmꢀ1. The ESMS
(mass spectra) of the compounds showed [M+H]+ peaks corre-
sponding to their molecular formulae. The NMR spectra (1H and
13C) are consistent with the proposed structures. The E geometry
of both the olefinic bonds was decided on the basis of the coupling
constant (J value) of 15.5 Hz proving the trans-relationship be-
tween the two olefinic protons. The 1H NMR spectrum of a proto-
type of these chalcones, compound 5, displayed exchangeable O–H
proton signal as a singlet at d 11.84, while the olefinic proton adja-
cent to phenyl group appeared as doublet at d 7.51 having a J value
of 15.54 Hz. Aromatic proton and another olefinic proton adjacent
to phenyl ring were observed as multiplet at d 7.33–6.92. The other
two olefinic protons adjacent to the two carbonyl groups appeared
O
OH
O
OH
Ar
H3C
+
KOH (5 mol%), Ethanol
rt, 7-8h
ArCHO
CH3
Ar
CH3
R
O
R
O
CH3
1- R = H
2- R = Br
5-33
3- R = OCH2Ph
4- R = Cl
Scheme 1. Synthesis of (2E,20E)-1,10-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones) (5–33).

A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
4713
M) in
Table 1
Table 2
Synthesis of biphenyl chalcones (5–33) with different biphenyls and aromatic
Cell growth inhibitory effect of compounds (5–33) and their IC50 values (in l
aldehydes
different cell lines
Entry
R
Ar
Product Time
(h)
Isolated yield
(%)
S.No. Compound MCF-7 SD
value
MDA-
MB231 SD value
value
Hela SD
HEK293 SD
value
1
2
3
4
5
6
7
H
H
H
H
H
H
H
Phenyl
5
6
7
8
9
7
7
7
7
8
8
7
93
92
93
94
92
93
94
4-Bromophenyl
4-Fluorophenyl
4-Chlorophenyl
3-Chlorophenyl
2-Chlorophenyl
4-Benzyloxy-
phenyl
4-Methoxyphenyl
3,4-Dimethoxy-
phenyl
1-Napthyl
2-Napthyl
Phenyl
4-Bromophenyl
4-Fluorophenyl
4-Chlorophenyl
4-Benzyloxy-
phenyl
1
2
3
4
5
5
6
7
8
9
10 0.02
10 0.01
20 0.006
10 0.01
20 0.02
Inactive
8.5 0.06
10 0.035
20 0.034
7.9 0.02
4.67 0.01
Inactive
10 0.023
Inactive
Inactive
Inactive
33.5 0.045 10 0.089
Inactive
30 0.03
30 0.07
22 0.021
Inactive
20 0.02
Inactive
Inactive
Inactive
Inactive
30 0.02
30 0.01
Inactive
10 0.13
15 0.02
Inactive
30 0.021
Inactive
Inactive
Inactive
Inactive
30 0.02
30 0.03
20 0.01
25 0.01
25 0.11
Inactive
Inactive
20(0.02)
30 0.01
Inactive
30 0.04
28 0.01
30 0.02
10
11
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
7
8
9
10 0.02
Inactive
Inactive
30 0.03
31.5 0.035 30 0.031
10 0.022
15 0.03
20 0.04
20 0.02
20 0.023
Inactive
8
9
H
H
12
13
7
8
94
92
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Inactive
10
11
12
13
14
15
16
H
H
14
15
16
17
18
19
20
8
8
7
7
8
7
8
92
94
92
94
92
94
92
8
0.032
Br
Br
Br
Br
Br
18 0.05
22.3 0.056 Inactive
15 0.04
20 0.01
Inactive
32 0.021
Inactive
Inactive
Inactive
Inactive
25 0.043
Inactive
Inactive
28 0.02
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
12.5 0.01
Inactive
Inactive
17
18
19
20
21
22
23
24
Br
4-Methoxyphenyl
21
22
23
24
25
26
27
28
7
8
8
7
7
7
8
8
91
90
93
95
93
92
91
93
30 0.08
28 0.02
Inactive
30 0.036
15 0.034
30(0.045)
20 0.01
12 0.03
22 0.02
4.4 0.02
30 0.03
29 0.06
30 0.01
Inactive
OCH2Ph Phenyl
OCH2Ph 4-Bromophenyl
OCH2Ph 4-Fluorophenyl
OCH2Ph 4-Chlorophenyl
OCH2Ph 4-Benzyloxyphenyl
OCH2Ph 4-Methoxy phenyl
OCH2Ph 3,4-Dimethoxy
phenyl
Cl
Cl
Cl
Cl
Cl
26 0.04
30 0.05
30 0.021
15 0.06
28 0.032
30 0.02
30 0.02
Inactive
5
0.04
Inactive
30 0.05
30 0.02
Inactive
10 0.06
25
26
27
28
29
Phenyl
29
30
31
32
33
7
7
8
7
7
92
95
91
92
93
4-Chlorophenyl
2-Chlorophenyl
1-Napthyl
Tamoxifen 8.9 0.02
10 0.02
2-Napthyl
A closure look into the structure activity relationship indicates
that out all the compounds (phenyl ring A common to all), com-
pounds having no substituent in the phenyl (naphthyl) ring B or
the phenyl rings (C) of the two cinnamoyl parts (compounds 5,
14, and 15) do not show any significant activity against the Hela
and HEK cell lines at the tested concentration. Although compound
5 displayed significant activity against MCF-7 and MDA-MB-231
cell lines, yet the other two compounds 14 and 15 with unsubsti-
tuted naphthyl moieties did not show any significant inhibition de-
spite the presence of the required phenyl propenone moieties,
indicating that substituents in the phenyl (naphthyl) rings are
important. The methoxy substituents in the phenyl rings (com-
pounds 12, 13, and 21) of the cinnamoyl phenyls offer no advan-
tage in improving the activity against different cell lines
screened. However, compounds with benzyloxy substituents
(compounds 11 and 20) showed activity against MCF7, MDA-
MB231, and HEK cell lines. Compound 20 was inactive against Hela
cells. The substitution of the phenyl ring A at the para-position
with Br (compound 16) as compared to unsubstituted compound
5 resulted in significant improvement in the inhibitory activities
against the above four cell lines and was better than tamoxifen
in inhibiting the MDB-MB231 cell line. Among the halogens as sub-
stituents in the phenyl ring of the cinnamoyl moiety both the nat-
ure and position of substituents affect the biological activities. The
trend in inhibitory activities was found to be Cl > Br > F and m-
> p > o. Two of the above compounds 9 and 29 with 4-chloro sub-
stituents in the phenyl rings C and B, respectively, were better than
the standard drug tamoxifen against the MDB-MB231 cell line. The
other halogens at different positions resulted in compounds which
did not display significant improvement in the activities as com-
pared to tamoxifen. Further, as compared to MCF-7 and HEK-293,
these two compounds inhibited growth of MDA-MB-231 cell lines
comparatively at lower concentrations. This may indicate that
as doublet at d 6.29 and 6.27, respectively, with J value of 16.12 and
15.52 Hz. The protons of the methyl group appeared as singlet at d
2.36.
In the 13C NMR spectrum, the two carbonyl carbons appeared at
d 198.2 and 195.9, while the quaternary aromatic carbon attached
to the hydroxyl group was observed at d 162.4. The olefinic carbons
were visible at d 145.2, 142.6, 128.3, and 126.7 ppm. The aromatic
quaternary carbons (ArC) were observed at their usual chemical
shifts of d 162.4, 143.0, 140.9, 139.6, 135.0, 134.7, 133.2, and
120.2 ppm whereas the tertiary aromatic carbons (ArCH) appeared
at d 131.2, 130.8, 130.6, 129.7, 129.1, 128.9, 128.7, 128.5, and
119.3 ppm. The methyl carbon was visible at d 20.8. Almost similar
patterns were observed in 1H NMR and 13C NMR spectra of other
compounds 6–33 of the series.
2.2. Biology
2.2.1. Cell growth inhibitory activity in breast cancer cells
The above compounds 5–33 were evaluated against breast can-
cer cell lines MCF-7 (estrogen responsive proliferative breast can-
cer model) and MDA-MB-231 (estrogen independent aggressive
breast cancer model), HeLa (cervical cancer) cell line, and human
embryonic kidney (HEK-293) cells using MTT assay to assess cell
proliferation. The results are shown in Table 2. As evident from
the results almost all of the compounds except compounds 10,
12, 13, 21, 22, and 33 were active at one or the other concentration
against one or the other cell lines. Among the active compounds,
the compounds 5, 8, 9, 16, and 29 were found to inhibit growth
of MDA-MB-231 cells at IC50 significantly better than the standard
anticancer agent, tamoxifen. Out of these five potent anticancer
compounds, two compounds 9 and 29 exhibited relatively lesser
toxicity in non-malignant cell line, HEK-293.

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A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
these compounds have more specific activity for inhibiting growth
of MDA-MB-231 cells.
Compounds 9 and 29 induced cell death significantly in a dose
dependent manner in MDA-MB231 cells as shown in Figure 3.
200
180
160
140
120
100
80
**
*
2.2.2. Compounds 9 and 29 induced apoptosis analyzed by flow
cytometry
60
As the two compounds 9 and 29 were the most potent com-
40
20
0
pounds of the series with IC50 values of 4.67 and 4.44 lM, respec-
tively, against MDA-MB-231 cells, we investigated whether the
inhibition of cell growth is associated with physiological apoptosis
or non-specific necrosis. The ability of compounds 9 and 29 to in-
duce apoptosis was measured by Annexin V-FITC (fluorescein iso-
thiocyanate) and propidium iodide (PI) labeling of MDA-MB231
Control
9
29
Figure 4. Apoptosis analysis in MDA-MB231 cells: MDA-MB-231 cells were treated
with 9 and 29 compounds at 5 M and their effects on Annexin-V positive cells
were measured with flow cytometry.
l
after treatment with 5
lM for 24 h. Compounds 9 and 29 showed
significant (p <0.05) increase in early apoptotic cells at 5
l
M con-
centration indicating significant induction of apoptosis as com-
pared to untreated control in MDA-MB-231 cells (Fig. 4).
Therefore, we determined
DWm of compounds 9 and 29 treated
MDA-MB-231 cells at 24 h. We used the mitochondrial membrane
potential-sensitive probe JC-1, which forms monomers (green fluo-
rescence) at low membrane potential and J-aggregates (red fluo-
rescence) at higher membrane potential. The ratio between the
2.2.3. Compounds 9 and 29 altered cell cycle
The tumor suppressor gene p53 is a multifunctional protein
responsible for maintaining genomic integrity and its mutation is
known to cause tumors in human.33 In response to DNA damage,
aberrant growth signals, or chemotherapeutic drugs, p53 activates
signaling for DNA repair initially, and in failure of DNA repair, in-
duces apoptosis and/or cell cycle arrest.34–36 The p53 exerts its
control on apoptosis by interacting with other important apoptotic
molecules, including members of the bcl-2 family.37 Data from
MTT assay showed that compounds 9 and 29 induced significant
inhibition of breast cancer cells. We tried to investigate whether
this inhibition of cell growth was due to cell cycle arrest. For this,
we studied cell cycle distribution of compound treated cells with
propidium iodide-labeling and then fluorescence-activated cell
sorting analysis. Results showed that compounds 9 and 29 at
red and the green signals is indicative of the
DWm. Compounds 9
and 29 caused decrease in 590/530 nm ratio indicating loss of
mitochondrial membrane potential in MDA-MB231 cells (Fig. 7).
Caspases, the serine proteases are involved in downstream of
apoptotic pathway which upon activation leads to DNA fragmenta-
tion. Therefore, we were interested to measure the caspase in the
cells treated with the compounds 9 and 29 using homogenous cas-
pase assay. A 5 lM concentration of the compounds 9 and 29 sig-
nificantly induced basal caspase level in MDA-MB-231 cells (Fig. 8).
3. Conclusion
In our study, we have prepared and characterized a series of
chalcones based on biphenyl system with two cinnamoyl moieties
as pharmacophores. Few of these compounds inhibited the cell
growth of human breast cancer cell lines, MCF-7, and MDA-MB-
231. Two of the compounds 9 and 29 appeared to be promising
lead against aggressive breast cancer. This inhibition of cell growth
is due to significant reduction in cells at S phase of cell cycle and
increase in sub-diploid apoptotic population of MDA-MB-231 cells.
Changes in mRNA expression level measured by real time PCR
showed that compounds 9 and 29 increased level of p53, p21,
and bax. The compounds led to caspase activation by and cause
DNA fragmentation in MDA-MB231 cells. In short, the active bis-
5 lM concentration resulted in an increase of sub-G1 phase and
decrease of cells in S phase of cell cycle as compared with un-
treated control (Fig. 5). Further, we analyzed the relative mRNA
expression level of cell cycle regulatory gene Cyclin A1 and impor-
tant apoptotic pathway gene such as bax, bcl-2, p21, and p53. Both
the compounds 9 and 29 reduced expression level of cyclinA1, bcl-
2 and increased bax, p53, and p21 (Fig. 6).
2.2.4. Compounds 9 and 29 induced loss of mitochondrial
membrane potential and caspase activation
A decline of the mitochondrial membrane potential
DWm is an
early event in the process of mitochondrial-mediated cell death.
120
100
80
60
40
20
0
*
Compound 9
Compound29
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Control
1
5
7.5
10
15
20
30
50
M
µ
Figure 3. Effect of compounds 9 and 29 on MDA-MB 231 cell proliferation. MDA-MB 231 cells were cultured in 10% FBS medium and treated with 0, 1, 5, 7.5, 10, 15, 20, 30,
and 50 M of compounds 9 and 29 for 24 h and cell viability was monitored by MTT assay. The percent viable cells were calculated in comparison to untreated cells taken as
100%. Values were expressed as mean SD of three independent experiments, each performed in triplicate (p <0.05).
l

A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
4715
80
70
60
50
40
30
20
10
0
G1
S
G2
Apoptosis
*
*
*
*
Control
29
9
Figure 5. Analysis of cell cycle in MDA-MB231 cells: MDA-MB-231 cells were treated with 5
lM of compounds 9 and 29 and their effect on phases of cell cycle was analyzed
by flow cytometry using PI stain.
Contol
9
3
***
2.5
29
**
2
1.5
1
*
*
*
*
*
*
*
0.5
0
CyclinA1
Bax
Bcl-2
p21
p53
Figure 6. Compounds 9 and 29 alters relative mRNA expression level of various genes such as cyclin A1, Bax, Bcl-2, p21, and p53, analyzed by real-time PCR.
chalcones cause mitochondrial-mediated apoptosis in MDA-MB-
231 cells. These studies led to identification of new prototypes
for further optimization and development to get novel compounds
for the treatment of aggressive breast cancer.
(100–200 mesh E. Merck). IR spectra were recorded as thin films
or in KBr solution with a Perkin–Elmer Spectrum RX-1 (4000–
450 cmꢀ1) spectrophotometer. 1H and 13C NMR spectra were re-
corded on a Brucker DRX-200 in CDCl3 and CDCl3 + CCl4. Chemical
shift values are reported in ppm relative to TMS (tetramethylsil-
ane) as internal reference, unless otherwise stated; s (singlet), d
(doublet), m (multiplet); J in hertz. ESI mass spectra were per-
formed using Quattro II (Micromass). Elemental analyses were per-
formed on a Perkin–Elmer 2400 II elemental analyzer.
Compound 9 and 29
p21
p53
4.1.1. General procedure for the synthesis of biphenyl based
chalcones (5–33)
To a stirring solution of the biphenyl (1 mmol) and aromatic
aldehyde (2.1 mmol) in minimum amount of ethanol, solid KOH
(5 mol %) was added. The reaction mixture was stirred at ambient
temperature till the disappearance of the starting material. After
Bcl-2
Loss of MMP
Caspase
Bax
Cell cycle arrest/Apoptosis
Apoptosis
14
12
10
Degradation of cellular DNA
8
6
4
2
0
*
Summary of anticancer effects of compounds 9 and 29
4. Experimental
**
4.1. Chemistry
Control
29
9
Commercially available reagent grade chemicals were used as
received. All reactions were followed by TLC on E. Merck Kieselgel
60 F254, with detection by UV light, spraying a 20% KMnO4 aq
solution. Column chromatography was performed on silica gel
Figure 7. Loss of mitochondrial membrane potential in MDA-MB-231 cells:
Compounds 9 and 29 at 5 lM for 24 h in MDA-MB-231 cells decreased ratio at
590/530 nm.

4716
A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
143.1, 141.2, 140.8, 139.7, 133.3, 133.2, 131.2, 130.9, 130.8,
160
140
120
100
80
*
130.6, 130.4, 130.3, 129.2, 129.1, 128.6, 128.0, 126.5, 120.1,
119.3, 116.6, 116.4, 116.2, 116.0, 20.8; MS (ESI+): m/z:
481[M+H]+. Elemental Anal. Calcd for C31H22O3F2: C, 77.49; H,
4.61. Found: C, 77.38; H, 4.57.
*
*
60
4.1.5. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(4-chlorophenyl)prop-2-en-1-one) (8)
40
20
0
It was obtained as yellow solid, mp 190–192 °C, in 94% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3857,
3020, 2358, 1640, 1518, 1216, 764, 671; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.87 (s, 1H, OH), 7.45 (d, J = 15.5 Hz, 1H, @CH),
7.31–7.13 (m, 11H, 10 ꢁ Ar-H and @CH), 6.97–6.87 (m, 4H, ArH),
6.33 (d, J = 16.1 Hz, 1H, @CH), 6.23 (d, J = 15.5 Hz, 1H, @CH), 2.31
(s, 3H, CH3). 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.2, 195.9
(2 ꢁ CO), 162.5, 143.8, 143.4, 141.1, 139.5, 136.9, 136.7, 133.4,
133.2, 133.1, 131.2, 129.8, 129.6, 129.5, 129.4, 129.3, 129.2,
128.5, 127.1, 120.1, 119.3, 20.9; MS (ESI+): m/z: 513[M+H]+. Ele-
mental Anal. Calcd for C31H22O3Cl2: C, 72.52; H, 4.32. Found: C,
72.50; H, 4.28.
Control
9
29
Figure 8. Induction of caspase in MDA-MB-231. Cells were treated with com-
pounds 9 and 29 for 24 h at 5 M 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 three independent experiments.
l
completion of reaction (TLC), the reaction mixture was neutralized
with dil. HCl (30%). The ethanol from the reaction mixture was
evaporated under reduced pressure and the reaction mixture was
partitioned between chloroform and water. The chloroform layer
was separated and washed with water, dried over anhydrous so-
dium sulfate (Na2SO4) and concentrated under reduced pressure.
The crude product was purified by column chromatography over
SiO2 (60–120 mesh) using appropriate hexane/ethylacetate as elu-
ent to give the respective chalcones.
4.1.6. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(3-chlorophenyl)prop-2-en-1-one) (9)
It was obtained as yellow solid, mp 190–192 °C, in 92% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3780,
3435, 3021, 2926, 2357, 1596, 1217, 1020, 770, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.92 (s, 1H, OH), 7.42–7.12 (m,
12H, 10 ꢁ ArH and @CH), 6.96–6.85 (m, 4H, ArH), 6.34 (d, 1H, J
= 16.5 Hz, @CH), 6.24 (d, 1H, J = 15.7 Hz, @CH), 2.32 (s, 3H, CH3);
13C NMR (50 MHz, CDCl3 + CCl4): d = 197.5, 195.4 (2 ꢁ CO), 162.8,
143.4, 143.1, 141.0, 140.7, 139.5, 136.9, 136.5, 135.3, 135.1,
133.1, 131.2, 130.6, 130.4, 130.3, 130.1, 129.6, 129.4, 129.3,
128.3, 128.0, 127.9, 127.0, 126.4, 119.9, 119.5, 20.9; MS (ESI+): m/
z: 513[M+H]+. Elemental Anal. Calcd for C31H22O3Cl2: C, 72.52; H,
4.32. Found: C, 72.50; H, 4.29.
4.1.2. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
phenylprop-2-en-1-one) (5)
It was obtained as yellow solid, mp 148–150 °C, in 93% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3619,
3450, 3021, 2360, 1635, 1563, 1216, 1039, 762, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.84 (s, 1H, OH), 7.51 (d,
J = 15.5 Hz, 1H, @CH), 7.33–7.14 (m, 13H, 12 ꢁ ArH and @CH),
7.00–6.92 (m, 4H, ArH), 6.37, (d, J = 16.1 Hz, 1H, @CH), 6.27 (d,
J = 15.5 Hz, 1H, @CH), 2.32 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.2, 195.9 (2 ꢁ CO), 162.4, 145.2, 143.0,
142.6, 140.9, 139.6, 135.0, 134.7, 133.2, 131.2, 130.8, 130.6,
129.2, 129.1, 128.9, 128.7, 128.5, 128.3, 126.7, 120.2, 119.3, 20.8).
MS (ESI+): m/z: 445[M+H]+. Elemental Anal. Calcd for C31H24O3:
C, 83.76; H, 5.44. Found: C, 83.69; H, 5.40.
4.1.7. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(2-chlorophenyl)prop-2-en-1-one (10)
It was obtained as yellow solid, mp 172–174 °C, in 93% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3880,
3381, 2926, 2364, 1851, 1634, 1572, 1198, 977, 768, 579; 1H
NMR (200 MHz, CDCl3 + CCl4): d = 11.82 (s, 1H, OH), 7.93 (d,
J = 15.5 Hz, 1H, @CH), 7.40–6.97 (m, 14H, 13 ꢁ Ar-H and @CH),
6.62–6.58 (m, 1H, ArH), 6.31 (d, J = 12.2 Hz, 1H, @CH), 6.23 (d,
J = 11.6 Hz, 1H, @CH), 2.33 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.0, 195.4 (2 ꢁ CO), 162.6, 143.3, 140.9,
140.8, 139.6, 137.9, 135.8, 135.4, 133.2, 133.0, 131.6, 131.4,
131.2, 130.4, 129.2, 129.1, 128.8, 127.9, 127.2, 126.9, 120.0,
119.4, 20.9; MS (ESI+): m/z: 513[M+H]+. Elemental Anal. Calcd for
C31H22O3Cl2: C, 72.52; H, 4.32. Found: C, 72.46; H, 4.23.
4.1.3. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(4-bromophenyl)prop-2-en-1-one) (6)
It was obtained as yellow solid, mp 188–190 °C, in 93% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3842,
3445, 3021, 2361, 1638, 1216, 765, 670; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.80 (s, 1H, OH), 7.42–7.17 (m, 10H, 8 ꢁ ArH
and @CH), 7.09–7.05 (m, 2H, ArH), 6.95 (m, 1H, ArH), 6.92–6.80
(m, 3H, ArH), 6.30–6.15 (m, 2H, @CH), 2.31 (s, 3H, CH3); 13C NMR
(50 MHz, CDCl3 + CCl4): d = 197.6, 195.5 (2 ꢁ CO), 162.6, 143.4,
141.0, 140.9, 139.6, 133.9, 133.5, 133.1, 132.4, 132.3, 131.2,
129.9, 129.7, 129.3, 129.1, 128.6, 127.2, 125.2, 125.1, 120.0,
119.4, 20.9; MS (ESI+): m/z: 601[M+H]+. Elemental Anal. Calcd for
C31H22O3Br2: C, 61.82; H, 3.68. Found: C, 61.81; H, 3.65.
4.1.8. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(4-benzyloxy)phenyl)prop-2-en-1-one) (11)
It was obtained as yellow solid, mp 163–165 °C, in 94% yield;
Rf = 0.4 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3856,
3449, 3021, 2362, 1635, 1524, 1216, 1039, 764, 670; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.92 (s, 1H, OH), 7.48–7.17 (m,
18H, 16 ꢁ ArH and @CH), 6.97–6.76 (m, 8H, ArH), 6.27 (d, 1H,
J = 16.0 Hz, @CH), 6.14 (d, 1H, J = 15.4 Hz, @CH), 5.04 (s, 2H,
OCH2), 5.03 (s, 2H, OCH2), 2.31 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.3, 195.8 (2 ꢁ CO), 162.3, 161.2, 161.1,
145.2, 142.7, 142.4, 140.7, 139.8, 136.7, 133.4, 131.1, 130.5,
130.3, 129.0, 128.9, 128.5, 128.0, 127.7, 127.6, 126.4, 124.8,
120.3, 119.2, 115.5, 115.3, 70.3, 20.8; MS (ESI+): m/z: 657[M+H]+.
Elemental Anal. Calcd for C45H36O5: C, 82.29; H, 5.52. Found: C,
82.10; H, 5.50.
4.1.4. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(4-fluorophenyl)prop-2-en-1-one (7)
It was obtained as yellow solid, mp 152–154 °C, in 93% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3862,
3429, 3021, 2359, 1639, 1515, 1216, 1043, 766, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.82 (s, 1H, OH), 7.46 (d,
J = 15.5 Hz, 1H, @CH), 7.32–7.21 (m, 7H, 6 ꢁ Ar-H and @CH),
7.00–6.84 (m, 8H, ArH), 6.27 (d, J = 16.1 Hz, 1H, @CH), 6.17 (d,
J = 15.5 Hz, 1H, @CH), 2.32 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 197.8, 195.6 (2 ꢁ CO), 162.5, 161.9, 143.7,

A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
4717
4.1.9. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis(3-
(4-methoxyphenyl)prop-2-en-1-one) (12)
3021, 2364, 1592, 1432, 1216, 1096, 764, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 12.00 (s, 1H, OH), 7.54–7.43 (m, 3H,
2 ꢁ ArH and @CH), 7.30–7.17 (m, 10H, 9 ꢁ ArH and @CH), 7.01–
6.91 (m, 4H, ArH), 6.46 (d, J = 16.2 Hz, 1H,@CH), 6.17 (d,
J = 15.4 Hz, 1H, @CH), 2.31 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.1, 195.5 (2 ꢁ CO), 162.8, 146.1, 143.1,
143.0, 139.4, 138.5, 134.8, 134.5, 133.0, 132.7, 132.2, 131.1,
130.9, 129.3, 129.2, 128.7, 128.6, 128.5, 126.5, 123.9, 119.8,
119.7, 20.7). MS (ESI+): m/z: 523[M+H]+. Elemental Anal. Calcd
for C31H23BrO3: C, 71.13; H, 4.43. Found: C, 71.06; H, 4.40.
It was obtained as yellow solid, mp 172–174 °C in 94% yield;
Rf = 0.4 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3756,
3020, 2360, 1598, 1513, 1216, 1170, 1034, 761, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.91 (s, 1H, OH), 7.48 (d, 1H,
J = 15.4 Hz, @CH), 7.31–7.17 (m, 7H, 6 ꢁ ArH and @CH), 6.96–
6.68 (m, 8H, ArH), 6.26 (d, 1H, J = 16.1 Hz, @CH), 6.13 (d, 1H,
J = 15.4 Hz, @CH), 3.79 (s, 6H, 2 ꢁ OCH3), 2.31 (s, 3H, CH3); 13C
NMR (50 MHz, CDCl3 + CCl4): d = 198.3, 195.8 (2 ꢁ CO), 162.3,
162.0, 161.9, 145.3, 142.7, 142.4, 140.7, 139.8, 133.3, 131.1,
130.5, 130.2, 128.9, 127.8, 127.3, 126.2, 124.7, 120.3, 119.1,
114.6, 114.4, 55.5, 20.8; MS (ESI+): m/z: 505[M+H]+. Elemental
Anal. Calcd for C33H28O5: C, 78.55; H, 5.59. Found: C, 78.51; H, 5.49.
4.1.14. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(4-bromophenyl)prop-2-en-1-one) (17)
It was obtained as yellow solid, mp 221–223 °C in 94% yield;
Rf = 0.5 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3426,
3021, 2363, 1633, 1584, 1216, 761, 671; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.94 (s, 1H, OH), 7.46–7.38 (m, 7H, 5 ꢁ ArH
and @CH), 7.26–7.12 (m, 4H, ArH), 6.97–6.82 (m, 4H, ArH), 6.40
(d, J = 16.1 Hz, 1H, @CH), 6.11 (d, J = 15.4 Hz, 1H, @CH), 2.30 (s,
3H, CH3). 13C NMR (50 MHz, CDCl3 + CCl4): d = 197.6, 195.2
(2 ꢁ CO), 162.9, 144.3, 143.3, 141.6, 139.4, 138.4, 133.7, 133.3,
132.9, 132.7, 132.6, 132.5, 132.2, 129.9, 128.8, 127.0, 125.6,
125.4, 124.0, 119.8, 119.7, 20.8; MS (ESI+): m/z: 679[M+H]+. Ele-
mental Anal. Calcd for C31H21Br3O3: C, 54.66; H, 3.11. Found: C,
54.50; H, 3.09.
4.1.10. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis
(3-(4 methoxyphenyl)prop-2-en-1-one) (13)
It was obtained as yellow solid, mp 90–92 °C, in 92% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3779,
3019, 2363, 1630, 1593, 1512, 1262, 1140, 1023, 759, 667; 1H
NMR (200 MHz, CDCl3 + CCl4): d = 11.93 (s, 1H, OH), 7.49 (d, 1H,
J = 15.4 Hz, @CH), 7.31–7.24 (m, 5H, 4 ꢁ ArH and @CH), 6.94–6.70
(m, 7H, ArH), 6.36 (s, 1H, ArH), 6.26 (d, 1H, J = 16.0 Hz, @CH), 6.10
(d, 1H, J = 15.3 Hz, @CH), 3.85 (s, 6H, 2 ꢁ OCH3), 3.81 (s, 3H,
OCH3), 3.76 (s, 3H, OCH3), 2.31 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.3, 195.6 (2 ꢁ CO), 162.3, 151.8, 149.5, 149.4,
145.7, 142.8, 140.6, 140.0, 133.2, 131.2, 128.8, 128.0, 127.5, 126.4,
124.6, 124.2, 123.3, 120.2, 119.2, 111.2, 111.1, 109.9, 109.4, 56.2,
56.1, 20.8; MS (ESI+): m/z: 565[M+H]+. Elemental Anal. Calcd for
C35H32O7: C, 74.45; H, 5.71. Found: C, 74.39; H, 5.67.
4.1.15. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(4-fluorophenyl)prop-2-en-1-one) (18)
It was obtained as yellow solid, mp 168–170 °C in 92% yield;
Rf = 0.5 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3449,
2929, 2367, 1634, 1506, 1359, 1223, 1160, 829, 773; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.94 (s, 1H, OH), 7.47–7.43 (m, 3H,
2 ꢁ ArH and @CH), 7.31–7.15 (m, 4H, 3 ꢁ ArH and @CH), 7.04–
6.87 (m, 8H, ArH), 6.36 (d, J = 16.1 Hz, 1H, @CH), 6.06 (d,
J = 15.4 Hz, 1H, @CH), 2.30 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 197.8, 195.3 (2 ꢁ CO), 162.8, 144.6, 143.2,
141.7, 139.3, 138.5, 132.7, 132.2, 130.6, 130.4, 128.1, 126.3,
123.9, 119.8, 116.8, 116.3, 20.8); MS (ESI+): m/z: 559[M+H]+. Ele-
mental Anal. Calcd for C31H21BrF2O3: C, 66.56; H, 3.78. Found: C,
66.50; H, 3.71.
4.1.11. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis
(3-(naphthalen-1-yl)prop-2-en-1-one) (14)
It was obtained as yellow solid, mp 180–181 °C, in 92% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3776,
3052, 2927, 2365, 1630, 1560, 1344, 1127, 974, 773; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.83 (s, 1H, OH), 8.40 (d, J = 15.2 Hz,
1H, @CH), 8.04 (d, J = 7.3 Hz, 1H, ArH), 7.86–7.77 (m, 6H, 5 ꢁ ArH
and @CH), 7.45–7.17 (m, 12H, Ar-H), 7.00 (s, 1H, ArH), 6.76–6.72
(d, J = 7.2 Hz, 1H, ArH), 6.43–6.30 (m, 2H, @CH), 2.39 (s, 3H, CH3);
13C NMR (50 MHz, CDCl3 + CCl4): d = 197.9, 195.7, 162.4, 143.1,
141.7, 140.9, 139.8, 134.0, 133.6, 132.1, 132.0, 131.7, 131.6,
131.0, 130.9, 129.2, 129.1, 129.0, 127.2, 126.6, 126.5, 125.6,
125.4, 123.7, 123.6, 120.4, 119.4, 21.0; MS (ESI+): m/z:
545[M+H]+. Elemental Anal. Calcd for C39H28O3: C, 86.01; H, 5.18.
Found: C, 85.91; H, 5.15.
4.1.16. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(4-chlorophenyl)prop-2-en-1-one) (19)
It was obtained as yellow solid, mp 174–176 °C in 94% yield;
Rf = 0.5 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3395,
3021, 2363, 1591, 1216, 1096, 766, 671; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.94 (s, 1H, OH), 7.47–7.39 (m, 3H, 2 ꢁ ArH
and @CH), 7.30–7.15 (m, 8H, 7 ꢁ ArH and @CH), 6.97–6.85 (m,
4H, ArH), 6.39 (d, J = 16.1 Hz, 1H, @CH), 6.10 (d, J = 15.4 Hz, 1H,
@CH), 2.30 (s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4);
d = 197.7, 195.2 (2 ꢁ CO), 162.9, 144.3, 143.3, 141.6, 138.4, 137.3,
137.0, 133.3, 132.9, 132.7, 132.2, 129.7, 129.6, 129.0, 128.7,
128.5, 126.9, 124.0, 119.8, 20.8; MS (ESI+): m/z: 591[M+H]+. Ele-
mental Anal. Calcd for C31H21BrCl2O3: C, 62.86; H, 3.57. Found: C,
62.80; H, 3.43.
4.1.12. (2E,20E)-1,10-(3-Hydroxy-5-methylbiphenyl-2,6-diyl)bis
(3-(naphthalen-2-yl)prop-2-en-1-one) (15)
It was obtained as yellow solid, mp 184–185 °C, in 94% yield;
Rf = 0.4 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3409,
3052, 2378, 1721, 1632, 1565, 1358, 1259, 1168, 977, 817, 747;
1H NMR (200 MHz, CDCl3 + CCl4): d = 11.99 (s, 1H, OH), 7.77–6.91
(m, 22H, 20 ꢁ Ar-H and @CH), 6.50, (d, J = 16.1 Hz, 1H, @CH),
6.38 (d, J = 15.4 Hz, 1H, @CH), 2.37 (s, 3H, CH3); 13C NMR
(50 MHz, CDCl3 + CCl4): d = 198.2, 195.8 (2 ꢁ CO), 162.6, 145.4,
143.1, 142.6, 141.0, 139.8, 134.7, 134.6, 133.6, 133.3, 132.6,
132.2, 131.3, 130.7, 130.6, 129.2, 129.1, 129.0, 128.9, 128.7,
128.4, 128.1, 127.7, 127.6, 127.1, 127.0, 126.9, 124.1, 123.7,
120.2, 119.3, 20.9.); MS (ESI+): m/z: 545[M+H]+. Elemental Anal.
Calcd for C39H28O3: C, 86.01; H, 5.18. Found: C, 85.89; H, 5.15.
4.1.17. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(4-(benzyloxy)phenyl)prop-2-en-1-one) (20)
It was obtained as yellow solid, mp 192–195 °C in 92% yield;
Rf = 0.5 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3752,
3454, 3021, 2364, 1563, 1216, 1017,761; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 12.07 (s, 1H, OH), 7.51–7.16 (m, 17H, 15 ꢁ ArH
and @CH), 6.98–6.87 (m, 8H, ArH), 6.35 (d, J = 16.1 Hz, 1H, @CH),
6.03 (d, J = 15.3 Hz, 1H, @CH), 5.06 (s, 4H, 2 ꢁ OCH2), 2.30 (s, 3H,
CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.3, 195.4 (2 ꢁ CO),
162.6, 161.4, 161.2, 146.1, 143.0, 142.7, 139.3, 138.7, 136.7,
4.1.13. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-phenylprop-2-en-1-one) (16)
It was obtained as yellow solid, mp 168–170 °C in 92% yield;
Rf = 0.6 (8:2 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3417,

4718
A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
136.6, 133.1, 132.7, 132.1, 130.6, 130.5, 129.0, 128.5, 127.8, 127.7,
127.3, 126.5, 124.5, 123.7, 119.9, 119.6, 115.6, 70.4, 20.7; MS
(ESI+): m/z: 735[M+H]+. Elemental Anal. Calcd for C45H35O5Br: C,
73.47; H, 4.80. Found: C, 73.41; H, 4.75.
4.1.22. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbi-
phenyl-2,6-diyl)bis(3-(4-chlorophenyl)prop-2-en-1-one) (25)
It was obtained as yellow solid, mp 164–165 °C in 93% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 13779,
3409, 3021, 2923, 1595, 1432, 1216, 1018, 929, 761, 671; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.79 (s, 1H, OH), 7.43 (d, J = 15.9 Hz,
1H, @CH), 7.24–7.18 (m, 13H, 12 ꢁ ArH and @CH), 6.99–6.85 (m,
6H, ArH), 6.34 (d, J = 16.3 Hz, 1H, @CH), 6.23 (d, J = 15.7 Hz, 1H,
@CH), 4.85 (s, 2H, OCH2), 2.31 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 197.8, 195.7 (2 ꢁ CO), 162.6, 159.8, 143.3, 143.2,
140.7, 140.5, 136.8, 136.5, 133.6, 133.2, 132.6, 131.8, 129.7 129.6,
129.5, 129.3, 129.0, 128.6, 128.5, 127.6, 127.3, 119.1, 115.4, 70.4,
20.9; MS (ESI+): m/z: 619[M+H]+. Elemental Anal. Calcd for
C38H28O4Cl2: C, 73.67; H, 4.56. Found: C, 73.60; H, 4.52.
4.1.18. (2E,20E)-1,10-(40-Bromo-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(4-methoxyphenyl)prop-2-en-1-one) (21)
It was obtained as yellow solid, mp 178–180 °C in 91% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3756,
3019, 2364, 1598, 1216, 1168, 1030, 763; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 12.04 (s, 1H, OH), 7.51–7.42 (m, 3H, 2 ꢁ ArH
and @CH), 7.26–7.16 (m, 4H, 3 ꢁ ArH and @CH), 6.99–6.95 (m,
4H, ArH), 6.83–6.76 (m, 4H, ArH), 6.35 (d, J = 16.0 Hz, 1H, @CH),
6.03 (d, J = 15.2 Hz, 1H, @CH), 3.80 (s, 6H, 2 ꢁ OCH3), 2.30 (s, 3H,
CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.3, 195.4 (2 ꢁ CO),
162.6, 162.2, 162.1, 146.2, 143.1, 142.7, 139.2, 138.7, 133.1, 132.7,
132.0, 130.5, 130.4, 127.6, 127.1, 126.4, 123.6, 119.9, 119.6, 114.7,
55.6, 20.7; MS (ESI+): m/z: 583[M+H]+. Elemental Anal. Calcd for
C33H27O5Br: C, 67.93; H, 4.66. Found: C, 67.80; H, 4.55.
4.1.23. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbi-
phenyl-2,6-diyl)bis(3-(4-(benzyloxy)phenyl)prop-2-en-1-one)
(26)
It was obtained as yellow solid, mp 64–65 °C in 92% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3781,
3324, 3036, 2362, 1602, 1505, 1247, 1016, 829, 731, 607; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.92 (s, 1H, OH), 7.46–7.22 (m, 20H,
18 ꢁ ArH and @CH), 7.03–6.77 (m, 10H, ArH), 6.30 (d, J = 16.0 Hz,
1H, @CH), 6.12 (d, J = 15.3 Hz, 1H, @CH), 5.04 (s, 2H, OCH2), 4.97
(s, 2H, OCH2), 4.84 (s, 2H, OCH2), 2.31 (s, 3H, CH3); 13C NMR
(50 MHz, CDCl3 + CCl4): d = 198.0, 195.7 (2 ꢁ CO), 162.4, 161.1,
161.0, 159.6, 144.7, 142.6, 141.8, 140.4, 136.9, 136.7, 133.3, 132.5,
132.3, 130.4, 130.3, 128.9, 128.8, 128.4, 128.2, 127.7, 127.6, 127.4,
126.5, 125.1, 120.3, 118.9, 115.5, 115.3, 70.3, 20.8; MS (ESI+): m/z:
763[M+H]+. Elemental Anal. Calcd for C52H42O6: C, 81.87; H, 5.55.
Found: C, 81.80; H, 5.53.
4.1.19. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-
methylbiphenyl-2,6-diyl)bis(3-phenylprop-2-en-1-one) (22)
It was obtained as yellow solid, mp 168–169 °C in 90% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3779,
3380, 3021, 2359, 1596, 1216, 762, 671; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.86 (s, 1H, OH), 7.51 (d, J = 15.5 Hz, 1H, @CH),
7.28–7.22 (m, 15H, 14 ꢁ ArH and @CH), 7.07–7.03 (m, 2H, ArH),
6.94–6.86 (m, 4H, ArH), 6.42 (d, J = 16.0 Hz, 1H, @CH), 6.28 (d,
J = 15.5 Hz, 1H, @CH), 4.84 (s, 2H, OCH2), 2.32 (s, 3H, CH3); 13C
NMR (50 MHz, CDCl3 + CCl4): d = 198.3, 196.0 (2 ꢁ CO), 162.5,
159.8, 145.0, 143.0, 142.1, 140.7, 136.7, 135.2, 134.8, 133.3,
132.5, 132.0, 130.8, 130.6, 129.2, 129.0, 128.8, 128.7, 128.6,
128.4, 128.3, 127.5, 126.9, 120.3, 118.9, 115.5, 70.4, 20.8); MS
(ESI+): m/z: 551[M+H]+. Elemental Anal. Calcd for C38H30O4: C,
82.89; H, 5.49. Found: C, 82.70; H, 5.45.
4.1.24. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbi-
phenyl-2,6-diyl)bis(3-(4-methoxyphenyl)prop-2-en-1-one) (27)
It was obtained as yellow solid, mp 69–70 °C in 91% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3783,
2838, 2362, 1628, 1604, 1509, 1248, 1169, 1027, 830, 728, 561;
1H NMR (200 MHz, CDCl3 + CCl4): d = 11.95 (s, 1H, OH), 7.49 (d,
J = 15.4 Hz, 1H, @CH), 7.25 (s, 9H, 8 ꢁ ArH and CH), 7.05–6.72 (m,
10H, ArH), 6.34 (d, J = 16.1 Hz, 1H, @CH), 6.15 (d, J = 15.5 Hz, 1H,
@CH), 4.84 (s, 2H, OCH2), 3.79 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),
2.31 (s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.8,
196.1 (2 ꢁ CO), 162.2, 162.1, 161.9, 159.6, 145.4, 142.8, 142.2,
140.6, 136.9, 133.4, 132.4, 132.2, 130.5, 130.4, 128.9, 128.3,
127.9, 127.6, 127.4, 126.4, 124.9, 120.5, 118.8, 115.4, 114.7,
114.5, 70.4, 55.7, 55.6, 20.8; MS (ESI+): m/z: 611[M+H]+. Elemental
Anal. Calcd for C40H34O6: C, 78.67; H, 5.61. Found: C, 78.65; H, 5.58.
4.1.20. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbiphe-
nyl-2,6-diyl)bis(3-(4-bromophenyl)prop-2-en-1-one) (23)
It was obtained as yellow solid, mp 170–171 °C in 93% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3778,
3376, 2927, 2365, 1632, 1236, 1009, 821, 771, 487; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.79 (s, 1H, OH), 7.43–7.09 (m, 14H,
12 ꢁ ArH and @CH), 6.92–6.88 (m, 6H, ArH), 6.35 (d, J = 16.5 Hz,
1H, @CH), 6.24 (d, J = 15.6 Hz, 1H, @CH), 4.85 (s, 2H, OCH2), 2.31
(s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 197.8, 195.7
(2 ꢁ CO), 162.6, 159.8, 143.4, 143.2, 140.5, 136.5, 134.1, 133.6,
132.6, 132.4, 132.3, 131.8, 129.9, 129.8, 129.0, 128.7, 128.5,
127.6, 127.4, 125.2, 125.0, 120.2, 119.1, 115.4, 109.9, 70.5, 20.9;
MS (ESI+): m/z: 707[M+H]+. Elemental Anal. Calcd for C38H28O4Br2:
C, 64.42; H, 3.98. Found: C, 64.39; H, 3.88.
4.1.25. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbi-
phenyl-2,6-diyl)bis(3-(3,4-dimethoxyphenyl)prop-2-en-1-one)
(28)
4.1.21. (2E,20E)-1,10-(40-(Benzyloxy)-3-hydroxy-5-methylbi-
phenyl-2,6-diyl)bis(3-(4-fluorophenyl)prop-2-en-1-one) (24)
It was obtained as yellow solid, mp 64–65 °C in 93% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3783,
3364, 3167, 2364, 1637, 1507, 1236, 1170, 827, 741, 504; 1H NMR
(200 MHz, CDCl3 + CCl4): d = 11.78 (s, 1H, OH), 7.44 (d, J = 15.4 Hz,
1H, @CH), 7.22 (s, 9H, 8 ꢁ ArH and @CH), 7.00–6.85 (m, 10H,
ArH), 6.29 (d, J = 16.1 Hz, 1H, @CH), 6.17 (d, J = 15.4 Hz, 1H, @CH),
4.86 (s, 2H, OCH2), 2.31 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 197.5, 195.5 (2 ꢁ CO), 166.8, 166.7, 162.6, 161.8,
159.8, 143.2, 143.1, 140.6, 140.5, 136.6, 133.2, 132.5, 132.0, 131.4,
131.1, 131.0, 130.5, 130.4, 130.3, 130.2, 128.9, 128.4, 128.1, 127.4,
126.7, 120.2, 119.0, 116.6, 116.4, 116.1, 116.0, 115.4, 70.4, 20.8;
MS (ESI+): m/z: 587[M+H]+. Elemental Anal. Calcd for C38H28O4F2:
C, 77.80; H, 4.81. Found: C, 77.62; H, 4.78.
It was obtained as yellow solid, mp 138–140 °C in 93% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3783,
2928, 2368, 1629, 1509, 1260, 1136, 1018, 843, 735, 569; 1H NMR
(300 MHz, CDCl3 + CCl4): d = 11.93 (s, 1H, OH), 7.45 (d, J = 11.6 Hz,
@CH), 7.25–7.17 (m, 7H, 6 ꢁ ArH and @CH), 6.90–6.84 (m, 5H,
ArH), 6.75–6.67 (m, 4H, ArH), 6.46 (d, J = 1.17 Hz, 1H, ArH), 6.28
(d, J = 12.0 Hz, 1H, @CH), 6.10 (d, J = 11.5 Hz, 1H, @CH), 4.81 (s,
2H, OCH2), 3.85 (s, 3H, OCH3), 3.81 (s, 6H, 2 ꢁ OCH3), 3.76 (s, 3H,
OCH3), 2.29 (s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4):
d = 198.3, 195.7 (2 ꢁ CO), 162.4, 159.5, 151.9, 151.8, 149.6, 149.5,
145.4, 142.8, 142.4, 140.4, 136.6, 133.3, 132.5, 132.3, 128.8, 128.3,
128.2, 127.7, 127.5, 126.6, 124.8, 124.0, 123.3, 120.4, 118.9, 115.2,
111.3, 111.1, 110.1, 109.5, 70.4, 56.2, 56.1, 56.0, 20.8). MS (ESI+):
m/z: 671[M+H]+. Elemental Anal. Calcd for C42H38O8: C, 75.21; H,
5.71. Found: C, 75.12; H, 5.69.

A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
4719
4.1.26. (2E,20E)-1,10-(40-Chloro-3-hydroxy-5-methylbiphenyl-2,-
6-diyl)bis(3-phenylprop-2-en-1-one) (29)
ArH), 7.15–6.93 (m, 3H, ArH), 6.58 (d, J = 16.4 Hz, 1H, @CH), 6.31
(d, J = 15.4 Hz, 1H, @CH), 2.35 (s, 3H, CH3); 13C NMR (50 MHz,
CDCl3 + CCl4): d = 198.3, 195.5 (2 ꢁ CO), 162.8, 146.3, 143.2, 143.1,
139.5, 138.1, 135.7, 134.8, 134.7, 133.6, 133.2, 132.5, 132.4, 132.0,
130.9, 130.7, 129.3, 129.1, 129.0, 128.9, 128.6, 128.2, 128.1, 127.9,
127.7, 127.2, 127.0, 126.8, 123.9, 123.6, 120.0, 119.7, 20.8; MS
(ESI+): m/z: 579[M+H]+. Elemental Anal. Calcd for C39H27O3Cl: C,
80.89; H, 4.70. Found: C, 80.82; H, 4.66.
It was obtained as yellow solid, mp 160–161 °C in 92% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3780,
3409, 3020, 2359, 1631, 1216, 726, 627; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.92 (s, 1H, OH), 7.53 (d, J = 15.4 Hz, 1H, @CH),
7.39–7.26 (m, 12H, 11 ꢁ Ar-H and @CH), 7.02–6.91 (m, 4H, ArH),
6.44, (d, J = 16.1 Hz, 1H, @CH), 6.18 (d, J = 15.5 Hz, 1H, @CH), 2.31
(s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.1, 195.5
(2 ꢁ CO), 162.7, 146.0, 143.1, 139.4, 138.0, 135.7, 134.8, 134.5,
133.1, 132.4, 131.1, 130.9, 129.3, 129.2, 128.6, 128.4, 126.5,
119.9, 119.6, 20.8; MS (ESI+): m/z: 479[M+H]+. Elemental Anal.
Calcd for C31H23O3Cl: C, 77.74; H, 4.84. Found: C, 77.69; H, 4.81.
4.2. Biology
4.2.1. MTT assay in breast cancer cell line
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.38 1 ꢁ 104 cells/well were
4.1.27. (2E,20E)-1,10-(40-Chloro-3-hydroxy-5-methylbiphenyl-2,-
6-diyl)bis(3-(4-chlorophenyl)prop-2-en-1-one) (30)
seeded in 100 ll DMEM (Dulbecco’s Modified Eagle’s Medium)
It was obtained as yellow solid, mp 162–163 °C in 95% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3776,
3021, 2359, 1592, 1215, 764, 621; 1H NMR (200 MHz,
CDCl3 + CCl4): d = 11.89 (s, 1H, OH), 7.50 (d, J = 15.4 Hz, 1H, @CH),
7.29–7.22 (m, 10H, 9 ꢁ Ar-H and @CH), 6.99–6.88 (m, 4H, ArH),
6.40 (d, J = 16.2 Hz, 1H, @CH), 6.15 (d, 15.4 Hz, 1H, @CH), 2.34 (s,
3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 197.7, 195.2
(2 ꢁ CO), 162.7, 144.3, 143.3, 141.5, 139.3, 137.9, 137.3, 137.0,
135.8, 133.3, 132.9, 132.5, 129.7, 129.5, 129.3, 128.6, 126.9,
119.8, 20.8; MS (ESI+): m/z: 547[M+H]+. Elemental Anal. Calcd for
C31H21O3Cl3: C, 67.94; H, 3.86. Found: C, 67.90; H, 3.81.
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 24 h of incubation, media were removed and to each well
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
4.2.2. Flow cytometric evaluation of apoptosis
MDA-MB231 cells (1 ꢁ 106) were seeded in six-well plates and
allowed to grow overnight. The medium was then replaced with
4.1.28. (2E,20E)-1,10-(40-Chloro-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(2-chlorophenyl)prop-2-en-1-one) (31)
complete medium containing 5 lM final concentrations of com-
pounds 9 and 29 or with vehicle alone (0.001% DMSO) as control.
After 24 h treatment, cells from the supernatant and adherent
monolayer cells were harvested by trypsinization, washed with
PBS at 300ꢁg. Then the cells (1 ꢁ 105) were stained with Annexin
V-FITC and propidium iodide using the Annexin-V-PI apoptosis
detection kit (Sigma). Flow cytometry was performed using a FAC-
Scan (Becton Dickinson) equipped with a single 488-nm argon la-
ser as described earlier.39 Annexin V-FITC were analyzed using
excitation and emission settings of 488 nm and 535 nm (FL-1
channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and
clumps were gated out using forward and orthogonal light scatter.
The experiment was repeated three times.
It was obtained as yellow solid, mp 190–192 °C in 91% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3779,
3404, 3061, 2923, 2367, 1595, 1564, 1437, 1351, 1312, 1162,
1026, 970, 858, 747, 580, 517; 1H NMR (200 MHz, CDCl3 + CCl4):
d = 11.91 (s, 1H, OH), 7.94 (d, J = 15.4 Hz, 1H, @CH), 7.39–6.99 (m,
13H, 12 ꢁ Ar-H and @CH), 6.64–6.60 (d, J = 7.4 Hz, 1H, ArH), 6.39
(d, J = 16.1 Hz, 1H, @CH), 6.17 (d, J = 15.4 Hz, 1H, @CH), 2.32 (s,
3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 198.0, 195.2
(2 ꢁ CO), 162.8, 143.3, 141.7, 139.4, 138.5, 137.9, 135.7, 135.4,
133.2, 132.8, 131.7, 131.4, 130.9, 130.6, 130.5, 130.1, 129.2,
128.7, 128.1, 128.0, 127.8, 127.5, 127.4, 127.2, 119.8, 20.8; MS
(ESI+): m/z: 547[M+H]+. Elemental Anal. Calcd for C31H21O3Cl3: C,
67.94; H, 3.86. Found: C, 67.88; H, 3.85.
4.2.3. Cell cycle analysis
4.1.29. (2E,20E)-1,10-(40-Chloro-3-hydroxy-5-methylbiphenyl-2,-
6-diyl)bis(3-(naphthalen-1-yl)prop-2-en-1-one) (32)
To determine the effect of compound on the stages of cell cycle,
MDA-MB231 cells (1 ꢁ 106) were seeded in six-well plates and
treated with compounds 29 and 9 at a final concentration of
It was obtained as yellow solid, mp 184–185 °C in 923% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3784,
3431, 2923, 1666, 1635, 1593, 1563, 1347, 1166, 973, 773; 1H
NMR (200 MHz, CDCl3 + CCl4): d = 11.95 (s, 1H, OH), 8.44 (d,
J = 15.3 Hz), 8.07–7.81 (m, 7H, 6 ꢁ ArH and @CH) 7.53–7.04 (m,
10H, Ar-H), 6.76–6.73 (m, 1H, ArH), 6.57 (d, J = 9.9 Hz, 1H, ArH),
6.52 (d, J = 15.8 Hz, 1H, @CH), 6.31 (d, J = 15.1 Hz, 1H, @CH), 2.40
(s, 3H, CH3); 13C NMR (50 MHz, CDCl3 + CCl4): d = 197.8, 195.4,
162.7, 143.2, 142.5, 139.9, 139.4, 138.2, 135.8, 134.0, 133.4, 132.8,
132.1, 131.9, 131.7, 131.3, 131.0, 129.4, 129.2, 129.1, 128.8, 127.4,
127.3, 126.7, 126.6, 125.7, 123.7, 123.4, 120.1, 119.8, 20.9). MS
(ESI+): m/z: 579[M+H]+. Elemental Anal. Calcd for C39H27O3Cl: C,
80.89; H, 4.70. Found: C, 80.81; H, 4.67.
5
l
M for 24 h. After 24 h treatments, both floating and trypsinized
adherent cells were collected and fixed with 70% ethanol. After fix-
ation cells were washed with PBS and stained with 50 g/ml pro-
l
pidium iodide in hypotonic lysis buffer (0.1% sodium citrate, 0.1%
Triton X-100) containing DNase-free RNase A for 20 min. Stained
cells were analyzed using fluorescence-activated cell sorter caliber
(Becton Dickinson).40
4.2.3.1. Quantitative Real Time PCR
Trizol reagent (Invitrogen) following the manufacturer’s instruc-
tions. Total RNA (1 g) was digested using DNase I (Invitrogen).
.Total RNA was isolated using
l
The DNase-treated RNA was then reverse transcribed using cDNA
synthesis kit (Fermentas). Triplicate samples of cDNA were ampli-
fied by PCR using the ABI Prism 7300 Real-Time PCR System (Ap-
plied Biosystems, Foster City, CA, USA). The amplification reaction
4.1.30. (2E,20E)-1,10-(40-Chloro-3-hydroxy-5-methylbiphenyl-
2,6-diyl)bis(3-(naphthalen-2-yl)prop-2-en-1-one) (33)
It was obtained as yellow solid, mp 179–180 °C in 93% yield;
Rf = 0.4 (7:3 hexane/ethylacetate); IR (KBr): mmax in cmꢀ1 3779,
3630, 2923, 2363, 1625, 1550, 1357, 1163, 979, 815, 745, 474; 1H
NMR (200 MHz, CDCl3 + CCl4): d = 12.06 (s, 1H, OH), 7.77–7.66 (m,
8H, 6 ꢁ ArH and @CH) 7.58–7.40 (m, 6H, Ar-H), 7.32–7.26 (m, 4H,
mixture (25 ll total volume) contained 1 ll cDNA, 20 pmol forward
primer (p21 F primer gaccgattttaccgatccatt, Bax F primer atgttttct-
gacggcaacttc, Bcl-2 F primer agtacctgaaccggcacct, cyclinA F primer
aatgggcagtacaggaggac, p53 F primer ctttccacgacggtgaca and GAPDH
F primer gctctctgctcctcctgttc, 20 pmol reverse primer (p21 R primer

4720
A. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 4711–4720
Cerino, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J.
aatctctggccgctctttct, Bax
R
primer atcagttccggcaccttg, bcl-2
primer ccacagtcagg-
gagtgcttt, p53 R primer tcctccatggcagtgacc and GAPDH R primer
acgaccaaatccgttgactc, 12.5 l iTaq SYBR Green Supermix (Bio-
Rad), and 9.5 l DEPC-H2O. RRNA (18S) was used as an endogenous
R
Med. Chem. 1988, 31, 2235.
2. Nowakowska, Z. Eur. J. Med. Chem. 2007, 42, 125.
primer ggccgtacagttccacaaa, cyclin
A R
3. Boumendjel, A.; Boccard, J.; Carrupt, P.-A.; Nicolle, E.; Blanc, M.; Geze, A.;
Choisnard, L.; Wouessidjewe, D.; Matera, E.-L.; Dumontet, C. J. Med. Chem. 2008,
51, 2307.
4. Cabrera, M.; Simoens, M.; Falchi, G.; Lavaggi, M. L.; Piro, O. E.; Castellano, E. E.;
Vidal, A.; Azqueta, A.; Monge, A.; Lopez de Cerain, A.; Sagrera, G.; Seoane, G.;
Cerecetto, H.; Gonzalez, M. Bioorg. Med. Chem. 2007, 15, 3356.
5. Sabzevari, O.; Galati, G.; Moridani, M. Y.; Siraki, A.; O’Brien, P. J. Chem. Biol.
Interact. 2004, 148, 57.
6. Rao, Y. K.; Fang, S.-H.; Tzeng, Y. M. Bioorg. Med. Chem. 2004, 12, 2679.
7. Liu, M.; Wilairat, P.; Croft, S. L.; Tan, A. L.-C.; Go, M. L. Bioorg. Med. Chem. 2003,
11, 2729.
8. Lunardi, F.; Guzela, M.; Rodriguez, A. T.; Correa, R.; Eger-Mangrich, I.; Steindel,
M.; Grisard, E. C.; Assreuy, J.; Calixto, J. B.; Santos, A. R. S. Antimicrob. Agents
Chemother. 2003, 47, 1449.
l
l
control to normalize the quantification of the target transcripts in
each sample.
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Acknowledgments
Authors thank, CSIR and DRDO New Delhi for financial assis-
tance. Anindra and Munna are thankful to CSIR New Delhi for
SRF and JRF. It is a CDRI Communication No. 7908.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.015.
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References and notes
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