Synthesis, in Vitro, and in Vivo Biological Evaluation and Molecular Docking Analysis of Novel 3-(3-oxo-substitutedphenyl-3-)4-(2-(piperidinyl)ethoxy)phenyl)propyl)-2H-chromen-2-one Derivatives as Anti-breast Cancer Agents

Article abstract of DOI:10.1111/cbdd.12696

The analogs of coumarin-chalcones have been reported to exhibit antineoplastic, anti-allergic, antihepatoprotective, and estrogenic activity. Herein, we have reported 3-(3-oxo-substitutedphenyl-3-)4-(2-(piperidinyl)ethoxy)phenyl)propyl)-2H-chromen-2-one derivatives as a new class of compounds that exhibit selectivity for ER-α binding along with antiproliferative and cytotoxic activity on human breast cancer cell line. The active compounds which show prominent activity against estrogen receptor-alpha-positive (ER+) human breast cancer cell lines MCF-7 and Zr-75-1 are subjected to in vivo screening. The Glide XP docking was performed for designed scaffold to optimize its structural requirement for ER-α inhibition.

Full text of DOI:10.1111/cbdd.12696

Chem Biol Drug Des 2016; 87: 608–617
Research Article
Synthesis, in Vitro, and in Vivo Biological Evaluation
and Molecular Docking Analysis of Novel 3-(3-oxo-
substitutedphenyl-3-)4-(2-(piperidinyl)ethoxy)phenyl)
propyl)-2H-chromen-2-one Derivatives as Anti-breast
Cancer Agents
human cancer, prompting the search for new antitumor
agents with improved tumor selectivity, efficiency, and
safetya.
Pritam N. Dube, Madhuri N. Waghmare and
Santosh N. Mokale*
Department of Pharmaceutical Chemistry, Y. B. Chavan
College of Pharmacy, Aurangabad, Maharashtra 431001,
India
*Corresponding author: Santosh N. Mokale,
santoshmokale@rediffmail.com
Among all cancers, breast cancer is the most common
type of cancer affecting more than one million women and
accounting for the highest mortality worldwideb. Even
though large number of breast cancers express estrogen
receptor (ER) and respond to therapy with hormones or
aromatase inhibitors, there is a group of patients (12–
17%) who do not respond to such treatment due to lack
of ER. These are known as triple-negative breast cancers
(TNBC) which represent a highly aggressive subtype of
breast cancer that is difficult to treat. ER ligands that
show differential responses within different tissues have
been more accurately described as selective estrogen
receptor modulators (SERMs). The tamoxifen (TAM) and
raloxifene act as an ER antagonist in breast tissue from
differential activation of multiple estrogen responsive path-
ways (1–3).
The analogs of coumarin–chalcones have been
reported to exhibit antineoplastic, anti-allergic, anti-
hepatoprotective, and estrogenic activity. Herein, we
have
reported
3-(3-oxo-substitutedphenyl-3-)4-(2-
(piperidinyl)ethoxy)phenyl)propyl)-2H-chromen-2-one
derivatives as a new class of compounds that exhibit
selectivity for ER-a binding along with antiproliferative
and cytotoxic activity on human breast cancer cell line.
The active compounds which show prominent activity
against estrogen receptor-alpha-positive (ER+) human
breast cancer cell lines MCF-7 and Zr-75-1 are sub-
jected to in vivo screening. The Glide XP docking was
performed for designed scaffold to optimize its struc-
tural requirement for ER-a inhibition.
Epidemiological and animal studies have demonstrated
that plant-derived dietary constituents of food play an
important role in the prevention of disease. A number of
food components that inhibit the initiation and progres-
sion of cancer or otherwise influence the potential for
disease outcome have been identified (4). The beneficial
effects of these dietary compounds have been attributed
partly to the presence of numerous chalcone analogs
with antioxidant and free radical scavenging properties.
The natural and synthetic analogs of chalcones and cou-
marins have been reported to exhibit antineoplastic,
antibacterial, antifungal, antimalarial, antiviral, antitubercu-
lar, anti-allergic, anti-inflammatory, antihepatoprotective,
and estrogenic activity. The natural flavonoid and isofla-
vonoid compounds also have potent antitumor activities
(4–6).
Key words: breast cancer, Coumarin–chalcone, docking,
estrogen receptor
Received 4 July 2015, revised 8 October 2015 and accepted
for publication 24 November 2015
Cancer is a notably complex, widespread, and lethal
disease that are projected to continue rising, with an
estimated 13.1 million deaths in 2030 (1). Cancer can
affect almost every tissue lineage in the human body
and poses great challenges to medical science. Most
cancers were characterized by uncontrolled cell prolifer-
ation, lack of cell differentiation, and loss of contact
inhibition, which confers upon the tumor cell, a capabil-
ity to invade local tissues, and metastasize. The non-
selectivity and acute toxicity of many anticancer agents
beside the development of cellular drug resistance have
been the major deterrent in their usage for treating
Herein, we have designed and synthesized coumarin–chal-
cone analogs (Figure 1, Scheme 1). The synthesized
derivatives ware screen for their anticancer activity on
human breast cancer cell line and animal model.
608
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12696

Synthesis of Anti-Breast Cancer Agents
R
HO
O
O
Estrogen receptor alpha
selectivity
O
S
N
N
O
O
O
OH
Raloxifene
Designed pharmacophore
Figure 1: Designed pharmacophore.
quenched by adding of ice-cold water (20 mL) and was
dried and recrystalized by methanol.
Experimental Section
General
Melting points were recorded in open capillaries with elec-
trical melting point apparatus and were uncorrected. IR
spectra of all synthesized compound in KBr were recorded
using a (JASCO FT-IR 4000) spectrophotometer. 1H and
13C NMR spectra were recorded on Bruker Avance
(300 MHz) Spectrometer in CDCl3 solutions, with TMS as
an internal reference. Mass spectra were recorded on a
Varian Inc., 410 Prostar Binary LC with 500 MS IT PDA
Detectors. All the reagents and solvents used were of ana-
lytical grade.
3-(3-oxo-1-phenyl-3-(4-(2-(piperidin-1-yl)ethoxy)
phenyl)propyl)-2H-chromen-2-one (BI-1)
% Yield: 54.86; MW: 481.23; MF: C31H31NO4; MP: 128–
130 °C; IR (KBr): 678 (Ar-H), 1183 (C-O), 1341 (C-N), 1533
(C=C), 1724 (C=O) cmꢀ1 1H NMR (DMSO, 400 MHz):
;
0
0
d = 7.7–7.4 (m, 14H, Ar-H), 4.3 (t, 2H, H11 ), 4.1 (t, 1H, H1 ),
0
0
3.3 (d, 2H, H2 ), 2.7 (t, 2H, H12 ), 2.3–1.6 (m, 10H, piperi-
13
dine); C NMR (DMSO, 100 MHz): d = 201.5 (C30, C=O),
165.2 (C70), 161.1 (C2, C=O), 154.6 (C10), 143.8 (C1″),
140.1 (C4), 129.8–129.4 (C50, C60, C80, C90), 128.2–126.9
(Ar-C), 120.3 (C5), 116.4 (C9), 69.2 (C110), 58.6 (C120),
57.2–57.0 (C140, C180), 42.3 (C20), 28.5 (C10), 25.9 (C150,
C170), 23.8 (C160); MS: m/z = 482.2 [M + 1].
General procedure for synthesis of substituted
p-hydroxy chalcone (3)
An equimolar mixture of 4-hydroxy acetophenone, substi-
tuted benzaldehydes, and KOH (2 mmol) was stirred in
PEG-400 (15 mL) at 40 °C for 2–3 h. After the completion
of the reaction (monitored by TLC), the crude mixture was
worked up in ice-cold water (100 mL). The resultant pro-
duct was separated out and recrystallized from absolute
ethanol (7).
3-(1-(4-chlorophenyl)-3-oxo-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-2)
% Yield: 60.34; MW: 515.19; MF: C31H30ClNO4; MP: 142–
144 °C; IR (KBr): 623 (Ar-H), 751 (C-Cl), 1174 (C-O), 1309
(C-N), 1541 (C=C), 1737 (C=O) cmꢀ1
;
1H NMR (DMSO,
0
400 MHz): d = 7.8–7.2 (m, 13H, Ar-H), 4.1 (t, 2H, H11 ), 3.9 (t,
0
0
0
1H, H1 ), 3.3 (d, 2H, H2 ), 2.6 (t, 2H, H12 ), 2.2–1.7 (m, 10H,
13
piperidine); C NMR (DMSO, 100 MHz): d = 199.3 (C30,
General procedure for synthesis of substituted [(4-
(2-piperidine-1-yl)ethoxy)] chalcone
C=O), 164.8 (C70), 160.2 (C2, C=O), 153.7 (C10), 144.2 (C1″),
139.9 (C4), 132.5 (C4″, C-Cl), 129.6–129.3 (C50, C60, C80,
C90), 128.1–126.5 (Ar-C), 121.4 (C5), 114.8 (C9), 67.6 (C110),
59.8 (C120), 57.3–57.1 (C140, C180), 44.3 (C20), 29.9 (C10),
24.2 (C150, C170), 21.3 (C160); MS: m/z = 517.2 [M + 2].
A mixture of substituted chalcone (0.625 mmol), anhy-
drous K2CO3 (3.12 mmol), 1-(2-chloroethyl) piperidine (0.9
3 mmol), and dry acetone (10 mL) was refluxed for 24 h.
K2CO3 was filtered off, and acetone was distilled out. The
residue was diluted with water and extracted with ethyl
acetate. The organic layer was washed with water, brine
and dried over anhydrous Na2SO4. The precipitate was
recrystallized from absolute ethanol (7).
3-(1-(4-fluorophenyl)-3-oxo-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-3)
% Yield: 58.76; MW: 499.22; MF: C31H30FNO 4; MP:
112–114 °C; IR (KBr): 682 (Ar-H), 1028 (C-F), 1216 (C-O),
1289 (C-N), 1520 (C=C), 1721 (C=O) cmꢀ1 1H NMR
;
General procedure for synthesis of substituted [3-
(3-oxo-1-phenyl-3-)4-(2-(piperidinyl)ethoxy)phenyl)
propyl)-2H-chromen-2-one
To the stirred solution of coumarin (0.46 mmol) in dry THF
(10 mL), NaH (60%) was added and washed with n-hex-
ane (0.6 mmol) at 0 °C. The mixture was stirred at 0 °C
for 30 min. A solution of substituted chalcone (1.0 mmol)
in dry THF (10 mL) was added drop wise at 0 °C, and the
mixture was warmed to room temperature for 20 min. The
resulting mixture was refluxed for 24 h. The mixture was
(DMSO, 400 MHz): d = 7.8–7.3 (m, 13H, Ar-H), 4.2 (t, 2H,
0
0
0
0
H11 ), 3.8 (t, 1H, H1 ), 3.1 (d, 2H, H2 ), 2.7 (t, 2H, H12 ),
2.3–1.8 (m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 189.8 (C30, C=O), 163.7 (C70), 161.5 (C4″, C-F), 157.8
(C2, C=O), 152.3 (C10), 142.4 (C1″), 138.6 (C4), 129.5–
129.3 (C50, C60, C80, C90), 127.8–126.4 (Ar-C), 120.9 (C5),
112.1 (C9), 66.7 (C110), 60.1 (C120), 57.2–57.0 (C140,
C180), 43.7 (C20), 30.1 (C10), 23.8 (C150, C170), 21.5
(C160); MS: m/z = 501.2 [M + 1].
Chem Biol Drug Des 2016; 87: 608–617
609

Dube et al.
O
O
O
O
H
+
+
CH3
R
H
R
HO
HO
p-hydroxy-Acetophenone
Substituted aldehyde
(2)
Substituted acetophenone
(2')
p-hydroxy benaldehyde
(1')
(1)
Stirred for 2-3 hr
at 40oC
Stirred for 2-3 hr
at 40oC
KOH,
PEG-400
KOH,
PEG-400
O
O
R
R
OH
OH
p-hydroxy Substituted Chalcone (3')
p-hydroxy Substituted Chalcone (3)
K2CO3,
Cl
K2CO3,
Cl
N
Acetone
reflux for 24 hrs
N
Acetone
reflux for 24 hrs
O
O
R
R
N
N
O
O
Substituted[(4-(2-(piperidine-1-yl)ethoxy)]chalcone (4')
Substituted[(4-(2-(piperidine-1-yl)ethoxy)]chalcone (4)
NaH
THF
NaH
THF
Stirr at 00C for 30 min
Stirr at 00C for 30 min
Reflux for 2-3 hr
O
O
O
O
Reflux for 2-3 hr
2H-chromen-2-one
2H-chromen-2-one
R
O
R
O
O
O
N
O
O
O
N
O
BI-1 to BI-12
BI-13 to BI-15
Scheme 1: Synthesis of designed compounds.
3-(1-(2,3-dichlorophenyl)-3-oxo-3-(4-(2-(piperidin-
1-yl)ethoxy)phenyl)propyl)-2H-chromen-2-one
(BI-4)
% Yield: 68.48; MW: 549.15; MF: C31H29Cl2NO4; MP:
127–129 °C; IR (KBr): 629 (Ar-H), 746 (C-Cl), 1169 (C-O),
1311 (C-N), 1474 (C=C), 1722 (C=O) cmꢀ1
(DMSO, 400 MHz): d = 7.7–7.1 (m, 12H, Ar-H), 4.4 (t, 2H,
;
1H NMR
0
0
0
0
H11 ), 3.9 (t, 1H, H1 ), 3.3 (d, 2H, H2 ), 2.8 (t, 2H, H12 ),
2.4–1.9 (m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 194.3 (C30, C=O), 165.8 (C70), 160.2 (C2, C=O), 153.7
610
Chem Biol Drug Des 2016; 87: 608–617

Synthesis of Anti-Breast Cancer Agents
(C10), 144.2 (C1″), 139.9 (C4), 133.6 (C3″, C-Cl), 131.4
(C2″, C-Cl), 129.2–128.7 (C50, C60, C80, C90), 128.0–126.4
(Ar-C), 120.9 (C5), 113.5 (C9), 66.7 (C110), 60.6 (C120),
57.5–57.3 (C140, C180), 43.8 (C20), 29.5 (C10), 23.9 (C150,
C170), 20.3 (C160); MS: m/z = 551.1 [M + 2].
3-(1-(3-methoxyphenyl)-3-oxo-3-(4-(2-(piperidin-1-
yl)ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-8)
% Yield: 72.38; MW: 511.24; MF: C32H33NO5; MP: 114–
116 °C; IR (KBr): 664 (Ar-H), 1166 (C-O), 1297 (C-N),
1534 (C=C), 1725 (C=O) cmꢀ1
;
1H NMR (DMSO,
0
400 MHz): d = 7.8–7.1 (m, 13H, Ar-H), 4.4 (t, 2H, H11 ),
0
0
4.1 (t, 1H, H1 ), 3.8 (s, 3H, OCH3), 3.5 (d, 2H, H2 ), 2.9 (t,
0
3-(1-(2,3-dimethoxyphenyl)-3-oxo-3-(4-(2-
(piperidin-1-yl)ethoxy)phenyl)propyl)-2H-chromen-
2-one (BI-5)
2H, H12 ), 2.3–1.8 (m, 10H, piperidine); 13C NMR (DMSO,
100 MHz): d = 199.7 (C30, C=O), 168.1 (C70), 161.6 (C2,
C=O), 157.2 (C10), 147.1 (C3″, C-O), 144.3 (C1″), 140.9
(C4), 129.8–129.3 (C50, C60, C80, C90), 128.3–127.4 (Ar-
C), 121.2 (C5), 111.7 (C9), 63.2 (C110), 60.4 (OCH3), 56.5
(C120), 57.1–56.9 (C140, C180), 42.9 (C20), 30.2 (C10), 24.5
(C150, C170), 21.6 (C160); MS: m/z = 512.3 [M + 1].
% Yield: 62.44; MW: 541.25; MF: C33H35NO6; MP: 152–
154 °C; IR (KBr): 662 (Ar-H), 1182 (C-O), 1296 (C-N),
1527 (C=C), 1719 (C=O) cmꢀ1
;
1H NMR (DMSO,
0
400 MHz): d = 7.9–7.5 (m, 12H, Ar-H), 4.3 (t, 2H, H11 ),
0
0
4.1 (t, 1H, H1 ), 3.9 (s, 6H, OCH3), 3.5 (d, 2H, H2 ), 2.8 (t,
2H, H12 ), 2.4–1.8 (m, 10H, piperidine); 13C NMR (DMSO,
0
100 MHz): d = 198.5 (C30, C=O), 167.3 (C70), 162.4 (C2,
C=O), 156.8 (C10), 151.2 (C2″, C-O), 146.4 (C3″, C-O),
143.7 (C1″), 141.2 (C4), 129.8–129.5 (C50, C60, C80, C90),
128.2–127.1 (Ar-C), 121.1 (C5), 114.8 (C9), 67.3 (C110),
61.2 (OCH3), 58.6 (C120), 57.2-57.0 (C140, C180), 53.4
(OCH3), 43.5 (C20), 29.6 (C10), 24.9 (C150, C170), 21.8
(C160); MS: m/z = 542.3 [M + 1].
3-(3-oxo-3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-1-
(2,3,4-trimethoxyphenyl)propyl)-2H-chromen-2-one
(BI-9)
% Yield: 75.88; MW: 571.26; MF: C34H37NO7; MP: 162–
164 °C; IR (KBr): 644 (Ar-H), 1167 (C-O), 1307 (C-N),
1498 (C=C), 1727 (C=O) cmꢀ1
;
1H NMR (DMSO,
0
400 MHz): d = 8.1–7.4 (m, 11H, Ar-H), 4.3 (t, 2H, H11 ),
0
0
4.0 (t, 1H, H1 ), 3.9 (s, 9H, OCH3), 3.4 (d, 2H, H2 ), 2.8 (t,
2H, H12 ), 2.4–1.9 (m, 10H, piperidine); 13C NMR (DMSO,
0
3-(1-(2,4-dichlorophenyl)-3-oxo-3-(4-(2-(piperidin-
1-yl)ethoxy)phenyl)propyl)-2H-chromen-2-one
(BI-6)
100 MHz): d = 192.5 (C30, C=O), 167.4 (C70), 162.4 (C2,
C=O), 156.9 (C10), 152.3 (C2″, C-O), 147.5 (C3″, C-O),
146.9 (C4″, C-O), 143.5 (C1″), 141.3 (C4), 129.9–129.6
(C50, C60, C80, C90), 128.2–127.3 (Ar-C), 122.4 (C5), 114.7
(C9), 67.8 (C110), 61.6 (OCH3), 58.5 (C120), 57.2–57.0
(C140, C180), 53.7 (OCH3), 43.5 (C20), 29.6 (C10), 24.9
(C150, C170), 21.8 (C160); MS: m/z = 572.3 [M + 1].
% Yield: 54.90; MW: 549.15; MF: C31H29Cl2NO4; MP:
139–141 °C; IR (KBr): 628 (Ar-H), 786 (C-Cl), 1163 (C-O),
1291 (C-N), 1524 (C=C), 1747 (C=O) cmꢀ1 1H NMR
;
(DMSO, 400 MHz): d = 7.8–7.2 (m, 12H, Ar-H), 4.3 (t, 2H,
0
0
0
0
H11 ), 3.6 (t, 1H, H1 ), 3.1 (d, 2H, H2 ), 2.9 (t, 2H, H12 ),
2.3–1.9 (m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 197.3 (C30, C=O), 167.3 (C70), 161.5 (C2, C=O), 155.2
(C10), 144.8 (C1″), 138.1 (C4), 132.8 (C4″, C-Cl), 132.0
(C2″, C-Cl), 129.2–128.5 (C50, C60, C80, C90), 127.6–126.2
(Ar-C), 121.2 (C5), 116.5 (C9), 68.9 (C110), 61.5 (C120),
57.6–57.4 (C140, C180), 44.2 (C20), 30.4 (C10), 23.8 (C150,
C170), 20.1 (C160); MS: m/z = 551.1 [M + 2].
3-(1-(furan-2-yl)-3-oxo-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-10)
% Yield: 62.24; MW: 471.20; MF: C29H29NO5; MP: 130–
132 °C; IR (KBr): 673 (Ar-H), 1172 (C-O), 1289 (C-N),
1519 (C=C), 1731 (C=O) cmꢀ1
;
1H NMR (DMSO,
400 MHz): d = 7.7–7.4 (m, 10H, Ar-H), 6.1–6.0 (m, 2H,
0
0
furan ring), 4.3 (t, 2H, H11 ), 4.0 (t, 1H, H1 ), 3.4 (d, 2H,
H2 ), 2.7 (t, 2H, H12 ), 2.2–1.6 (m, 10H, piperidine); 13C
NMR (DMSO, 100 MHz): d = 188.2 (C30, C=O), 165.2
(C70), 161.1 (C2, C=O), 154.6 (C10), 149.8 (C1″), 140.1
(C4), 129.8–129.5 (C50, C60, C80, C90), 128.2–126.9 (Ar-
C), 120.3 (C5), 116.4 (C9), 109.4, 105.6 (C-furan), 69.7
(C110), 58.4 (C120), 57.2-57.0 (C140, C180), 42.3 (C20),
28.5 (C10), 25.9 (C150, C170), 23.8 (C160); MS: m/
z = 472.2 [M + 1].
0
0
3-(1-(2,6-dichlorophenyl)-3-oxo-3-(4-(2-(piperidin-
1-yl)ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-
7)
% Yield: 65.96; MW: 549.15; MF: C31H29Cl2NO4; MP:
144–146 °C; IR (KBr): 651 (Ar-H), 773 (C-Cl), 1176 (C-O),
1288 (C-N), 1536 (C=C), 1739 (C=O) cmꢀ1 1H NMR
;
(DMSO, 400 MHz): d = 7.8–7.3 (m, 12H, Ar-H), 4.4 (t, 2H,
0
0
0
0
H11 ), 3.6 (t, 1H, H1 ), 3.0 (d, 2H, H2 ), 2.8 (t, 2H, H12 ),
2.3–1.7 (m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 196.8 (C30, C=O), 165.2 (C70), 161.4 (C2, C=O), 153.7
(C10), 142.6 (C1″), 138.1 (C4), 133.4 (C6″, C-Cl), 131.9
(C2″, C-Cl), 129.3–128.5 (C50, C60, C80, C90), 127.5–126.2
(Ar-C), 120.7 (C5), 112.1 (C9), 66.9 (C110), 61.2 (C120),
57.6–57.4 (C140, C180), 41.3 (C20), 32.5 (C10), 24.2 (C150,
C170), 20.3 (C160); MS: m/z = 551.1 [M + 2].
3-(3-oxo-3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-1-(p-
tolyl)propyl)-2H-chromen-2-one (BI-11)
% Yield: 76.74; MW: 495.24; MF: C32H33NO4; MP: 164–
166 °C; IR (KBr): 668 (Ar-H), 1182 (C-O), 1311 (C-N),
1517 (C=C), 1724 (C=O), 2912 (C-H) cmꢀ1 1H NMR
;
(DMSO, 400 MHz): d = 7.8–7.1 (m, 13H, Ar-H), 4.4 (t, 2H,
Chem Biol Drug Des 2016; 87: 608–617
611

Dube et al.
0
0
0
0
H11 ), 4.1 (t, 1H, H1 ), 3.5 (d, 2H, H2 ), 2.9 (t, 2H, H12 ), 2.4
(s, 3H, CH3), 2.3–1.8 (m, 10H, piperidine); 13C NMR
(DMSO, 100 MHz): d = 198.6 (C30, C=O), 167.2 (C70),
162.2 (C2, C=O), 155.4 (C10), 148.2 (C3″, C-O), 145.1
(C1″), 141.7 (C4), 136.3 (C4″), 129.5–129.2 (C50, C60, C80,
C90), 128.2–127.1 (Ar-C), 121.4 (C5), 113.8 (C9), 65.9
(C110), 57.3–57.0 (C140, C180), 56.5 (C120), 43.2 (C20),
31.4 (C10), 25.3 (C150, C170), 21.1 (C160), 20.3 (CH3); MS:
m/z = 496.2 [M + 1].
3-(3-(4-methoxyphenyl)-3-oxo-1-(4-(2-(piperidin-1-
yl)ethoxy)phenyl)propyl)-2H-chromen-2-one (BII-3)
% Yield: 77.34; MW: 511.24; MF: C32H33NO5; MP: 114–
116 °C; IR (KBr): 673 (Ar-H), 1188 (C-O), 1319 (C-N),
1535 (C=C), 1742 (C=O) cmꢀ1
;
1H NMR (DMSO,
0
400 MHz): d = 7.8–7.0 (m, 13H, Ar-H), 4.3 (t, 2H, H11 ),
0
0
4.0 (t, 1H, H1 ), 3.7 (s, 3H, OCH3), 3.4 (d, 2H, H2 ), 2.9 (t,
2H, H12 ), 2.3–1.8 (m, 10H, piperidine); 13C NMR (DMSO,
0
100 MHz): d = 200.3 (C30, C=O), 169.0 (C70), 162.8 (C2,
C=O), 157.3 (C10), 147.5 (C4″, C-O), 144.4 (C1″), 140.8
(C4), 129.8–129.3 (C50, C60, C80, C90), 128.3–127.4 (Ar-
C), 121.9 (C5), 113.7 (C9), 64.6 (C110), 60.4 (OCH3), 56.5
(C120), 57.1–56.9 (C140, C180), 44.8 (C20), 31.3 (C10), 25.1
(C150, C170), 22.7 (C160); MS: m/z = 512.3 [M + 1].
3-(1-(2-hydroxyphenyl)-3-oxo-3-(4-(2-(piperidin-1-
yl)ethoxy)phenyl)propyl)-2H-chromen-2-one (BI-12)
% Yield: 70.68; MW: 497.22; MF: C31H31NO5; MP: 158–
160 °C; IR (KBr): 653 (Ar-H), 1171 (C-O), 1296 (C-N),
1526 (C=C), 1737 (C=O), 3245 (O-H) cmꢀ1 1H NMR
;
Anticancer screening
(DMSO, 400 MHz): d = 7.6–7.0 (m, 13H, Ar-H), 5.1 (s,
In vitro screening
0
0
0
1H, OH), 4.3 (t, 2H, H11 ), 4.1 (t, 1H, H1 ), 3.5 (d, 2H, H2 ),
2.8 (t, 2H, H12 ), 2.4–2.0 (m, 10H, piperidine); 13C NMR
In vitro testing is performed using SRB assay protocols (8),
each drug is tested at four dose levels (1 9 10ꢀ7 M,
1 9 10ꢀ6 M, 1 9 10ꢀ5 M, and 1 9 10ꢀ4 M, or 10, 20, 40,
and 80 lg/mL). Appropriate positive controls are run in each
experiment, and each experiment is repeated thrice. Results
are given in terms of GI50, TGI, and LC50 values. The com-
pounds were tested for their cytotoxic assay using MCF-7
and Zr-75-1 estrogen receptor-positive cancer cell lines.
0
(DMSO, 100 MHz): d = 190.4 (C30, C=O), 163.7 (C70),
161.6 (C2, C=O), 153.1 (C10), 147.0 (C3″, C-O), 144.9
(C1″), 140.5 (C4), 132.8 (C2″, C-O), 129.7–129.3 (C50,
C60, C80, C90), 128.4–127.2 (Ar-C), 121.6 (C5), 116.2
(C9), 66.4 (C110), 58.8 (C120), 57.4–57.2 (C140, C180),
45.6 (C20), 30.9 (C10), 24.8 (C150, C170), 22.7 (C160); MS:
m/z = 498.2 [M + 1].
In vivo screening
3-(3-oxo-3-phenyl-1-(4-(2-(piperidin-1-yl)ethoxy)
phenyl)propyl)-2H-chromen-2-one (BII-1)
The active compounds which show prominent activity
against MCF-7 and Zr-75-1 cancer cell line are subjected
for in vivo screening.
% Yield: 61.74; MW: 481.23; MF: C31H31NO4; MP: 128–
130 °C; IR (KBr): 681 (Ar-H), 1189 (C-O), 1339 (C-N),
1541 (C=C), 1730 (C=O) cmꢀ1
;
1H NMR (DMSO,
Female virgin Sprague Dawley rats were obtained from
Wockhardt Pvt Ltd (Aurangabad) at 35 days of age. Rats
were housed at 4 per cage and maintained at (25 ꢁ 2) °C
under 12-h dark/light cycle with access to standard diet
and water ab libitum. Animals were experimented with
prior approval from the institutional ethics committee.
0
400 MHz): d = 7.8–7.4 (m, 14H, Ar-H), 4.4 (t, 2H, H11 ),
0
0
0
4.2 (t, 1H, H1 ), 3.2 (d, 2H, H2 ), 2.7 (t, 2H, H12 ), 2.4–1.6
(m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 202.8 (C30, C=O), 166.1 (C70), 163.4 (C2, C=O), 154.7
(C10), 143.2 (C1″), 141.0 (C4), 129.9–129.4 (C50, C60,
C80, C90), 128.2–126.9 (Ar-C), 120.3 (C5), 116.4 (C9),
69.2 (C110), 58.6 (C120), 57.2–57.0 (C140, C180), 42.3
(C20), 27.9 (C10), 25.9 (C150, C170), 23.8 (C160); MS: m/
z = 482.2 [M + 1].
The MNU (methyl nitrosourea) was purchased from
Sigma-Aldrich (USA). An aqueous solution at a concentra-
tion of 10 mg/mL was made by wetting the MNU powder
with 3% acetic acid and then dissolving it in 0.9% NaCl
solution; a fresh solution was prepared for each injection.
Rats were given intraperitoneally (i.p.) 50 mg/kg of MNU
on the 50th and 57th day. Animals were divided into differ-
ent groups with six animals in each group. Group I (intact
control) received 0.9% NaCl with 3% acetic acid i.p.
Groups II–VI were introduced with MNU. After 60 days,
animals were treated with synthesized compounds and
tamoxifen (TAM) (5 mg/kg and 10 mg/kg in 1% Tween-80
by gavage once per day) for four weeks. Animals in intact
control group and untreated MNU group were given vehi-
cle (Tween-80) according to experimental protocol.
3-(3-(4-chlorophenyl)-3-oxo-1-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)propyl)-2H-chromen-2-one (BII-2)
% Yield: 74.96; MW: 515.19; MF: C31H30ClNO4; MP:
142–144 °C; IR (KBr): 625 (Ar-H), 756 (C-Cl), 1174 (C-O),
1311 (C-N), 1545 (C=C), 1734 (C=O) cmꢀ1 1H NMR
;
(DMSO, 400 MHz): d = 7.8–7.2 (m, 13H, Ar-H), 4.2 (t, 2H,
0
0
0
0
H11 ), 3.9 (t, 1H, H1 ), 3.4 (d, 2H, H2 ), 2.6 (t, 2H, H12 ),
2.2–1.7 (m, 10H, piperidine); 13C NMR (DMSO, 100 MHz):
d = 199.9 (C30, C=O), 165.1 (C70), 160.6 (C2, C=O), 154.7
(C10), 143.7 (C1″), 139.8 (C4), 132.5 (C4″, C-Cl), 129.6–
129.3 (C50, C60, C80, C90), 128.1–126.5 (Ar-C), 121.4 (C5),
114.8 (C9), 67.6 (C110), 59.8 (C120), 57.3–57.1 (C140,
C180), 44.3 (C20), 30.2 (C10), 24.5 (C150, C170), 22.4
(C160); MS: m/z = 517.2 [M + 2].
After completion of treatment, blood was collected from
retro orbital puncture and analyzed for estrogen-level mea-
612
Chem Biol Drug Des 2016; 87: 608–617

Synthesis of Anti-Breast Cancer Agents
Table 1: Anticancer activity of designed compounds
Cell line MCF7
Cell line Zr-75-1
Comp code
BI-1
R
LC50
TGI
45.6
GI50
<10
LC50
TGI
70.3
GI50
21.1
>80
>80
BI-2
BI-3
67.9
33.9
61.7
<10
49.3
23.6
<0.1
36.8
Cl
>80
28.2
>80
>80
F
Cl
BI-4
BI-5
>80
>80
47.2
<10
>80
>80
65.4
13.4
Cl
OCH3
>80
51.9
>80
>80
H3CO
Cl
BI-6
BI-7
64.8
60.2
35.1
18.1
<10
<10
51.1
21.9
56.1
<0.1
18.6
Cl
Cl
>80
Cl
H3CO
BI-8
BI-9
>80
78.5
45.3
37.2
13.7
>80
>80
>80
>80
OCH3
77.0
60.1
18.8
H3CO
H3CO
BI-10
BI-11
>80
>80
40.6
36.4
<10
<10
>80
54.8
26.2
8.6
O
57.0
<0.1
H3C
Chem Biol Drug Des 2016; 87: 608–617
613

Dube et al.
Table 1: continued
Cell line MCF7
LC50
Cell line Zr-75-1
LC50
Comp code
BI-12
R
TGI
29.2
GI50
<10
TGI
34.6
GI50
0.9
OH
67.4
68.3
BII-1
BII-2
BII-3
68.5
>80
31.8
67.9
42.1
<10
26.1
<10
>80
>80
65.2
62.4
22.07
43.6
16.9
>80
Cl
77.2
41.1
H
3CO
TAM
ADR
–
–
29.4
68.5
11.2
31.5
<10
<10
61.8
79.9
36.6
35.8
<0.1
<0.1
surement. Serum hormone value for estrogen was aver-
aged from blood samples collected on 30th and 45th the
day of treatment.
NMR, and LC-MS spectroscopy. The first step in the syn-
thetic route consisted of the Claisen–Schmidt condensa-
tion of 40-hydroxyl acetophenone with substituted
benzaldehydes and 40-hydroxyl benzaldehydes with substi-
tuted acetophenone in basic conditions (10% aqueous
KOH) to give a, b-unsaturated ketones (chalcones,
Scheme 1) (7). The second step consisted of the conden-
sation of hydroxyl group by amino side chain. The target
compounds (BI–1 to BI–15) were synthesized by Michael
addition of coumarin to chalcone derivatives. The substitu-
tions of target compounds are shown in Table 1.
Docking study
Docking procedure aims to identify the correct binding poses
within the binding site of the protein. To locate the appropri-
ate binding orientations and conformations of chalcone com-
pounds interacting with human estrogen receptor-a (hER-a),
molecular docking was performed with the GLIDE-5.8 pro-
€
gram interfaced with MAESTRO-9.3 of Schrodinger 2012. The
flexible docking method has been shown to be very efficient
on numerous protein receptors. To better describe the pos-
sible binding modes of chalcone compounds with hERa,
RAL was used. We examined the ability of our procedure in
reproducing the binding position for RAL in PDB entry 1ERR,
when docked in another X-ray structure (PDB: 1ERR) with a
cocrystalized antagonist. The procedure was successful in
reproducing the binding position for RAL. The crystal struc-
tures of hER-a (PDB entry code: 1ERR) was extracted from
Brookhaven Protein Database (PDB http://www.rcsb.org/
pdb). Prior to docking calculations, ligand preparation was
performed with the LigPrep program using OPLS_2005
forcefield. Default parameters were used for docking. The
docking poses are ranked according to Glide score.
Anticancer screening
In vitro screening
The target compounds were evaluated for anticancer
activity against estrogen receptor-alpha-positive (ER+)
human breast cancer cell lines MCF-7 and Zr-75-1 (8).
The in vitro activity profile is shown in Table 1. The GI50
concentration for each compound was calculated with ref-
erence to a control sample, which represents the concen-
tration that results in a 50% decrease in cell growth/
proliferation after 48-h incubation in the presence of drug.
The total growth inhibition (TGI) is the concentration of test
drug which signifies a cytostatic effect. The LC50 is con-
centration of compound that produces 50% cytocidal
effect. Tamoxifen and adriamycin were used as reference.
Results and Discussion
Chemistry
In vivo screening
The proposed derivatives were synthesized in three steps
and their structures were verified by IR, 1H-NMR, 13C-
The active compounds BI–2, BI–6, and BI–11 which show
prominent activity against MCF-7 and Zr-75-1 cancer cell
614
Chem Biol Drug Des 2016; 87: 608–617

Synthesis of Anti-Breast Cancer Agents
lines are subjected to in vivo screening. In vivo study was
carried out using M-methyl-nitrosourea (MNU)-induced
mammary carcinoma in female Sprague Dawley rats. MNU
is most often used to model mammary tumor initiation and
progression. The compounds which were shown promi-
nent activity against MCF-7 and Zr-75-1 cancerous cell
lines are also shows marked modulator activity on estro-
gen receptor (Table 2). All the treated groups show
marked antagonistic activity on estrogen receptor which
results in decrease level of estrogen to normal range as
compared to group II.
tion has been focused on protein–ligand interactions of the
dichloro-substituted compounds BI–4 and BI–6, and the
furan-substituted compound BI–10. The chemical struc-
Table 3: Binding interaction of compounds with estrogen receptor-a
Docking No. of
Compound ID
score
H-bonds Hydrogen bond details
+
ꢀ
BI-1
BI-2
BI-3
BI-4
BI-5
BI-6
BI-7
BI-8
ꢀ7.768
ꢀ8.058
ꢀ8.399
ꢀ9.401
ꢀ8.493
ꢀ9.031
ꢀ7.972
ꢀ4.622
ꢀ8.133
ꢀ8.850
ꢀ8.199
ꢀ6.271
ꢀ9.028
1
0
1
1
2
1
0
1
0
1
0
1
1
2
NH -ASP 351 (2.108 A)
–
+
ꢀ
NH -ASP 351 (1.729 A)
+
ꢀ
NH ASP 351 (1.646 A)
+
ꢀ
ꢀ
NH -ASP 351 (1.867 A)
+
NH -ASP 351 (2.168 A)
SAR analysis
–
NH+ GLU380 (1.674 A)
ꢀ
The SAR analysis suggested that the all compounds
showed moderate to good anticancer activity. Among the
synthesized derivatives, BI–2, BI–6, BI–7, BI–10, and BI–
11 show good antiproliferative activity as compared to
standard tamoxifen. The compounds BI–2, BI–6, and BI–
11 showed potent cytotoxic activity on both cell lines.
This indicates the substitution of chloro or methyl group
on para position of substituted phenyl ring increases
activity. Whereas substitution of methoxy group on sub-
stituted phenyl ring decreases activity. The structural dif-
ference between raloxifene (RAL) and synthesized
compounds showed that the presence of hydrophobic
thiophene ring in RAL is responsible for strong ER-a
binding. The modification at propenone linkage of chal-
cone by any hydrophobic ring and hydrogen bond donor
or acceptor group may cause increase in antiproliferative
activity in comparison with RAL.
BI-9
–
+
ꢀ
BI-10
BI-11
B1-12
Tamoxifene
NH -ASP 351 (1.664 A)
–
ꢀ
ꢀ
OH-TRH 347 (2.005 A)
+
NH -ASP 351 (1.936 A)
+
ꢀ
4-OH Tamoxifen ꢀ9.321
NH -ASP 351 (1.645 A)
ꢀ
OH-HID 524 (2.437 A)
ꢀ
Raloxifene
ꢀ11.99
4
OH-GLU 353 (1.596 A)
ꢀ
OH-ARG394 (1.887 A)
ꢀ
OH-HID 524 (2.411 A)
+
ꢀ
NH -ASP 351 (1.646 A)
Molecular docking analysis
To explore the main interactions with the target hER-a
receptor of the chalcone derivatives, we performed molec-
ular docking studies. These results indicated an accept-
able reliability of the parameters specified in Maestro-Glide
in reproducing the binding mode for this compounds (9).
After successful reproduction of the binding mode of RAL,
the docking method was used to search for the binding
modes of the whole data set. All the compounds were
successfully docked into the binding pocket of ER-a, and
their binding interactions are tabulated in Table 3. In this
research, to illustrate the interaction mechanism, the atten-
Table 2: Estrogen levels (pg/mL) of animals on day 30 and 45
Estrogen level (mean ꢁ SE)
Group
Compound no.
On 30th day
On 45th day
I
II
III
IV
V
VI
Control
MNU
BI-2
BI-6
BI-11
TAM
9.9 ꢁ 1.22
18.96 ꢁ 2.09
11.16 ꢁ 1.42
11.18 ꢁ 1.09
12.91 ꢁ 1.62
11.06 ꢁ 1.18
9.7 ꢁ 1.36
20.77 ꢁ 1.98
11.08 ꢁ 1.22
10.74 ꢁ 1.76
13.12 ꢁ 1.37
11.28 ꢁ 1.69
Figure 2: Docking pose of compounds (A) BI–4 (purple) with
raloxifene (green) and (B) BI–7 (yellow) with tamoxifen (maroon).
Chem Biol Drug Des 2016; 87: 608–617
615

Dube et al.
tures and binding modes of BI–4 with standard RAL and
BI–7 with standard TAM are displayed in Figure 2. It can
be seen that BI–4 and BI–7 are located at the active site
of the receptor. Specifically, the tertiary nitrogen group
(piperidine ring) acted as a hydrogen bond donor and
formed a hydrogen bond with the oxygen atom of Asp351
same as that of RAL as show in Figure 3. Compounds
BI–5 and BI–7 form p–p stacking interaction with Phe404,
whereas compound BI–5 forms p–p stacking interaction
with Hid525.
His524. The results of this study may conclude that further
structural modification is required for the development of
new therapeutic agent.
Conclusion
It can be concluded that the 3-(1-(4-substituted phenyl)-3-(3-
oxo-1,3-diphenylpropyl)-2H-chromen-2-one
derivatives
designed are a new class of estrogen receptor modulators
endowed with significant antitumor activity on MCF-7 and Zr-
75-1 cell line culture. Several compounds are interesting as
they display favorable inhibitory potency on estrogen recep-
tor. Molecular docking suggested that multiple hydrophobic
and hydrogen bond interactions are two predominant factors
that affect the binding process. The decomposition of binding
free energy to each residue revealed that the most favorable
contributions came from Asp351, Phe404, Phe425, Leu354,
Met388, Met421, and His524. The results of this study may
conclude that further structural modification is required for
the development of new therapeutic agent.
In summary, we have prepared substituted chalcones and
coumarins (BI–1 to BI–15), and evaluated for their anti-
cancer activity using MCF-7 and Zr-75-1 cell line culture.
In vivo study shows the prominent estrogen receptor mod-
ulator activity. Molecular docking suggested that multiple
hydrophobic and hydrogen bond interactions are two pre-
dominant factors that affect the binding process. The
decomposition of binding free energy to each residue
revealed that the most favorable contributions came from
Asp351, Phe404, Phe425, Leu354, Met388, Met421, and
A
B
Figure 3: Ligand interaction diagram of (A)
compound BI–5 and (B) compound BI–6.
616
Chem Biol Drug Des 2016; 87: 608–617

Synthesis of Anti-Breast Cancer Agents
€
€
6. Bjornstrom L., Sjoberg M. (2005) Mechanisms of estro-
Acknowledgments
gen receptor signaling: convergence of genomic and
nongenomic actions on target genes.
Endocrinol;19:833–842.
M
Mol
This work was supported by the Department of Science
and Technology (DST), New Delhi, India [Project File No.
SR/FT/LS-132/2012 & Sanction Diary No. SERB/F/4302/
2012-13]. The authors are thankful to Tata Memorial Cen-
tre, Advanced Centre for Treatment, Research and Educa-
tion in Cancer (ACTREC) Kharghar, Navi Mumbai for
in vitro anti-breast cancer activity.
7. Mokale S.N., Dube P.N., Bhavale S.A., Sayed I., Begum
A. (2015) Synthesis, in-vitro screening, and docking
analysis of novel pyrrolidine and piperidine-substituted
ethoxy chalcone as anticancer agents. Med Chem
Res;24:1842–1856.
8. Skehan P., Storeng R., Scudiero D., Monks A., McMo-
han J., Vistica D., Warren J.T., Bokesch H., Kenney S.,
Boyd M.R. (1990) New colorimetric cytotoxicity assay
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