Anticancer activities of novel chalcone and bis-chalcone derivatives

Article abstract of DOI:10.1016/j.bmc.2006.01.003

A series of novel chalcones and bis-chalcones containing boronic acid moieties has been synthesized and evaluated for antitumor activity against the human breast cancer MDA-MB-231 (estrogen receptor-negative) and MCF7 (estrogen receptor-positive) cell lin

Full text of DOI:10.1016/j.bmc.2006.01.003

Bioorganic & Medicinal Chemistry 14 (2006) 3491–3495
Anticancer activities of novel chalcone and bis-chalcone derivatives
Aneta Modzelewska,a Catherine Pettit,a Geetha Achanta,b Nancy E. Davidson,a
Peng Huangb and Saeed R. Khana,*
aDivision of Chemical Therapeutics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
bDepartment of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
Received 10 November 2005; revised 3 January 2006; accepted 4 January 2006
Available online 24 January 2006
Abstract—A series of novel chalcones and bis-chalcones containing boronic acid moieties has been synthesized and evaluated for
antitumor activity against the human breast cancer MDA-MB-231 (estrogen receptor-negative) and MCF7 (estrogen receptor-posi-
tive) cell lines and against two normal breast epithelial cell lines, MCF-10A and MCF-12A. These molecules inhibited the growth of
the human breast cancer cell lines at low micromolar to nanomolar concentrations, with five of them (1–4, 9) showing preferential
inhibition of the human breast cancer cell lines. Furthermore, bis-chalcone 8 exhibited a more potent inhibition of colon cancer cells
expressing wild-type p53 than of an isogenic cell line that was p53-null.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
pharmaceutical agents such as enzyme inhibitors,11 sac-
charide sensors,12 and boron neutron capture therapy
(BNCT) agents.13
Despite recent advances in our understanding of the bio-
logical processes leading to the development of cancer,
there is still a need for new and effective agents to help
bring this disease under control. Among the currently
identified antitumor agents, chalcones represent an
important class of molecules that are abundant in edible
plants. Chemically, they are open-chained molecules
bearing two aromatic rings linked by a three-carbon en-
one fragment. Many of these molecules exhibit beneficial
biological activities, including anti-inflammatory,1–3 anti-
pyretic,4 and anti-invasive5,6 properties.
In the present study, we have further demonstrated the
ability of these novel chalcones, appended to a boronic
acid group, to effectively inhibit breast cancer cell lines.
Biological evaluation of these molecules led us to exam-
ine an unusual class of synthetic molecules containing
a pair of a,b-unsaturated groups; in these studies, we
found that two boronic acid moieties exhibited a
higher in vitro efficacy than did a single boronic acid
group. Although similar compounds featuring diene-
one groups have been described in the literature, infor-
mation about their biological activity is relatively
scarce.14,15
Recent reports have focused on the antiproliferative,7,8
and tumor-reducing9 activities of some chalcones, caus-
ing renewed interest in this class of molecules. These
broad antitumor properties have prompted us to synthe-
size a series of novel boronic chalcones and to evaluate
these compounds at the molecular level. We have previ-
ously shown10 that a set of chalcones containing the
boronic acid moiety possesses selective antiproliferative
activity against breast cancer cell lines when compared
to normal epithelial cell lines. In addition, the boronic
acid moiety has already shown promise in potential
A series of novel chalcone derivatives were synthesized for
use in the present study. Compounds 1–11 (Scheme 1)
O
O
O
H
Ar
Ar
R'
A, B or C
R'
R
R
Keywords: Anticancer; Chalcone; Bis-chalcone; Claisen–Schmidt
reaction.
Scheme 1. Procedure A = KOH(aq), EtOH, reflux. Procedure
B = KOH, EtOH, RT. Procedure C = (1) KOH, EtOH, reflux (2)
KH, Et2O, BrCH2B(OR)2.
*
Corresponding author. Tel.: +1 410 614 0200; fax: +1 410 614
8397; e-mail: khansa@jhmi.edu
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.003

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A. Modzelewska et al. / Bioorg. Med. Chem. 14 (2006) 3491–3495
were synthesized according to the Claisen–Schmidt con-
densation protocol.16
Compound 1 was treated with pinacol (bromometh-
yl)boronate in the presence of KOH and a catalytic
amount of 18-crown-6. After a standard workup, the
product obtained was purified by crystallization
(Scheme 2).
2. Results and discussion
2.1. Chemistry
Synthesis of compounds 6, 7, and 8 (Scheme 3, Table 2)
required the use of 3 equiv of the appropriate aldehydes
and 4-t-Bu-cyclohexanone or N-methylpiperidine-4-one
in the presence of an ethanolic solution of KOH. The
residual solid was purified by crystallization. Treatment
of two of the bis-chalcones, one bearing an N-Me unit
(7) and the other containing a 4-t-Bu group (6), with
NaBH4 generated bis-a,b-unsaturated alcohols 9 and
10. Compound 10 was further esterified using benzoyl
chloride to afford compound 11. The whole series was
synthesized and evaluated to address the question of
whether higher potency and specificity could be achieved
by introducing multiple boronic acid fragments into a
Compounds 1, 3, 4, and 5 (Table 1) were prepared from
substituted acetophenones and appropriate aldehydes
in the presence of ethanolic KOH. After a standard
work-up (dilution with water, acidification with 1 M
HCl to pH 3, and extraction with ethyl acetate), the
resulting solid was purified by column chromatography
to yield the desired product.
Compound 2 was prepared in a two-step process
that involved formation of chalcone 1 and then incorpo-
ration of the boronic acid moiety into the molecule.
Table 1. Novel chalcone derivatives (1–5)
Compound
R
R0
Ar
Procedure
Yield (%)
1
2
3
4
5
OH
H
PhCl
PhCl
A
C
B
B
A
92
81
98
97
85
OCH2B(OH)2
B(OH)2
B(OH)2
Cl
H
H
Thiophene
Benzothiophene
PhOH
H
COOH
Scheme 2. Reagents and condition: (a) KOH, MeOH, reflux; (b) KH, pinacol (bromomethyl)boronate, Et2O, 18-crown-6.
O
O
OHC
R'
KOHaq, MeOH
R'
X
R
6-8
R'
R'
X
R
O
OH
NaBH4
R'
X
R
R'
R'
X
R
6, 7
9, 10
OCOPh
OH
PhCOCl
Py
Cl
N
Cl
Cl
N
Cl
Me
Me
10
11
Scheme 3.

A. Modzelewska et al. / Bioorg. Med. Chem. 14 (2006) 3491–3495
Table 4. Inhibition of colon cancer cell lines by bis-chalcone 8a
3493
Table 2. Novel chalcone derivatives (6–11)
Compound
X
R
R0
Yield (%)
Cell line
IC50
SD
Replicates (n)
6
7
C
N
N
C
N
N
t-Bu
Me
B(OH)2
Cl
87
96
93
85
78
90
HCT116 p53+/+
HCT116 p53ꢀ/ꢀ
2.8
5.5
0.32
0.23
3
3
8
Me
B(OH)2
B(OH)2
Cl
a IC50 values expressed in lM; see biology section for details of the
MTT assay.
9
t-Bu
Me
10
11
Me
Cl
lar concentrations. Reduction of the carbonyl group (9,
10) led to a higher selectivity for the malignant over the
normal cell lines. Compound 9 was completely selective
for the cancer cell lines, inhibiting only the MCF7 and
MDA-MB-231 cell lines, with IC50 values of 16.5 and
15, respectively. However, conversion of the free alcohol
to an ester caused a complete lack of sensitivity at
25 lM, with none of the treated cell lines reaching IC50s.
molecule. A study regarding the importance of the en-
one unit was performed by converting the dienone to a
dienol and subsequently masking the free hydroxyl
group as an ester.
2.2. Biology
Chalcones 1–11 were then evaluated against the breast
cancer cell lines MCF-7 and MDA-MB-231. The normal
breast cell lines MCF-10A and MCF-12A were included
in the panel as controls. The cell lines were incubated
with each of the chalcones 1–11 for 96 h, and an MTT
assay was performed to determine the IC50 for each
chalcone against the panel of cell lines (Table 3).
To explore the potential growth inhibitory effect of these
chalcones on colon cancer cell lines and to determine
whether the growth inhibition was p53-dependent, we
evaluated the effect of bis-chalcone 8 on a pair of isogen-
ic HCT116 colon cancer cell lines that differed only in
p53 expression. An initial screening suggested that cells
with wild-type p53 (HCT116 p53+/+) were more sensi-
tive to the cytotoxic effects of bis-chalcone 8 than
were cells that lacked p53 (HCT116 p53ꢀ/ꢀ) (Table
4). Further studies addressing these considerations are
underway.
2.2.1. Growth inhibition studies. IC50 values were used to
determine growth inhibition in the presence of the chal-
cone derivatives. Of particular interest were the com-
pounds that were able to preferentially inhibit the
growth of human breast cancer cell lines over the normal
breast epithelial cell lines. Compounds 1 and 2 inhibited
the cancer cell lines at low micromolar concentrations,
whereas the normal breast cell lines were 4- to 6-fold less
sensitive to these chalcones. In the presence of compounds
3 and 4, growth of the human breast cancer cell lines
MDA-MB-231 and MCF7 was inhibited, with low IC50
values ranging from 3.3 to 15.1 lM. Cell growth in the
normal breast epithelial cell lines MCF-10A and MCF-
12A appeared unaffected at the maximum concentration
used in this study (25 lM). Chalcone 5 was toxic only to
MCF7; it had no effect on eitherMBA-MD-231or normal
breast cells (MCI-10A and -12A).
3. Conclusions
In summary, we have prepared and characterized a
series of chalcones and bis-chalcones that inhibit the
growth of human breast and colon cancer cells in culture
at low micromolar to nanomolar concentrations. Some
of them are particularly exciting, as they preferentially
inhibited the growth of human breast cancer cell lines,
as compared to normal epithelial cells.
The long-term objective of these studies is to develop
highly potent and selective inhibitors of the MDM2-
p53 interaction for use as anticancer drugs. Our goal
in this specific proposal is to develop and characterize
In a related series, bis-chalcones 6, 7, and 8 were found
to inhibit all the cell lines at low micromolar to nanomo-
Table 3. Inhibition of human breast cell lines by chalcones (1–11)a
Compound
Wt-MCF7
SD
MDA-MB-231
SD
MCF-10A
SD
MCF-12A
SD
1
2
2.3
3.1
0.76
1.65
3.18
1.61
2.12
0.94
2.12
3.33
1.06
3.89
0
3.8
2.0
1.06
1.15
2.18
1.0
25.0
13.0
1.41
8.0
0
21.2
22.5
2.54
2.08
0
b
b
3
7.0
3.3
15.1
5.5
—
—
b
b
4
—
b
3.17
0
—
b
0
b
—
5
22.0
1.3
0
—
—
0
6
0.7
0.4
0.25
0.14
1.05
1.06
2.12
0
7.0
2.5
1.5
2.75
1.87
0.58
0.35
0.35
0
8.0
4.8
4.0
1.77
2.40
2.42
0.35
0
7
2.0
8
0.35
16.5
20
0.35
15
20.3
b
b
9
—
22.3
—
b
10
11
—
—
b
—
b
—
b
—
b
0
a IC50 values expressed in lM; see Section 2.2 for details of the MTT assay.
b IC50 > 25 lM.

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A. Modzelewska et al. / Bioorg. Med. Chem. 14 (2006) 3491–3495
novel boronic chalcone compounds that can prevent the
interaction between the tumor-suppressor p53 and the
oncogene MDM2, as a means of activating p53 tumor-
suppressor activity in tumors. Therefore, we have used
computer-aided drug design techniques followed by vir-
tual screening to design and evaluate a preliminary series
of novel boronic chalcones. Molecular modeling studies
have suggested that boronic acid efficiently binds to ly-
sine residues K51 and/or K94 of MDM2 oncoprotein.
These boronic-chalcone derivatives can therefore serve
as potential platforms for the design of clinically useful
MDM2 antagonists. Structure–activity relationship
studies of this class of compounds are continuing and
will be reported in due course.
in cells treated with bis-chalcone 8 was plotted as a per-
centage of the value obtained for untreated control cells,
and the IC50 values (or the concentration of the drug
that caused a 50% inhibition in cell growth) were calcu-
lated for the two cell lines.
4.2. Chemistry
4.2.1. General method for syntheses of chalcones
4.2.1.1. Procedure A. To a solution of substituted
acetophenone in ethanol (10 ml/mmol) was added the
appropriate aldehyde (1.5 equiv). The mixture was
stirred for 15 min, followed by the addition of an aque-
ous solution of KOH (3 equiv) at 0 °C. The mixture was
refluxed overnight. After the reaction reached
completion, it was quenched with water (20 ml). The
resulting mixture was concentrated in vacuo, diluted
with water (50 ml), acidified with 1 M HCl to pH 3,
and extracted with ethyl acetate. The organic layer was
dried over MgSO4 and concentrated in vacuo. The resid-
ual solid was purified by column chromatography to
yield the desired product.
4. Experimental
4.1. Biology
The human breast cancer cell lines MDA-MB-231
(estrogen receptor-negative) and MCF7 (estrogen recep-
tor-positive) were maintained in DMEM supplemented
with 5% fetal bovine serum, 2 mM glutamine, and 100
units/mL penicillin/streptomycin. The normal human
mammary epithelial cell lines MCF-10A and MCF-
12A were maintained in 5% and 10% horse serum,
respectively, supplemented with 2 mM glutamine, 100
units/mL penicillin/streptomycin, 0.02 lg/mL EGF,
0.01 mg/mL insulin, and 0.1 lg/mL cholera toxin. Cells
were incubated at 37 °C in a 5% CO2 atmosphere. The
MTT colorimetric assay was used to determine growth
inhibition.17 Cells were plated in 96-well plates and al-
lowed to attach for 24 h. Chalcone derivatives were dis-
solved in DMSO at 10 mM. Cells in quadruplicate wells
were exposed to the individual chalcone derivatives at
0.5–25 lM for 96 h. After 96 h, the medium was aspirat-
ed, and 100 lL of 1 mg/mL MTT solution (Sigma
Chemical Co.) diluted in serum-free medium was added
to each well. After 4 h of incubation, the MTT solution
was removed, and 200 lL of a 1:1 (v/v) solution of
DMSO/ethanol was added to each well to dissolve the
formazan crystals. The absorbance at A540nm was deter-
mined on a plate reader. IC50 values were determined
from log plots of percent of control vs concentration.
Each compound was assayed twice in quadruplicate.
4.2.1.2. Procedure B. To a solution of substituted ace-
tophenone in ethanol (10 ml/mmol) was added the
appropriate aldehyde (3 equiv). The mixture was stirred
for 15 min, followed by the addition of an ethanolic
solution of KOH (6 equiv) at 0 °C. The mixture was stir-
red at room temperature overnight. After the reaction
reached completion, it was quenched with water
(20 ml). The resulting mixture was concentrated in vac-
uo, diluted with water (50 ml), acidified with 1 M HCl to
pH 3, and extracted with ethyl acetate. The organic layer
was dried over MgSO4 and concentrated in vacuo. The
residual solid was purified by crystallization to yield
the desired product.
4.2.1.3. Procedure C. An ethereal solution of com-
pound 1 was added dropwise to a cooled solution of
KH (1.8 equiv) in diethyl ether, and the reaction mixture
was stirred at room temperature for 40 min. The mixture
was cooled to 0 °C, and a solution of pinacol (bromo-
methyl)boronate in diethyl ether was added dropwise,
followed by a catalytic amount of 18-crown-6. The mix-
ture was stirred at room temperature overnight. After a
standard workup, the product obtained was purified by
crystallization.
HCT116 p53+/+ and HCT116 p53ꢀ/ꢀ colon cancer
cells (kindly provided by Dr. Bert Vogelstein, Johns
Hopkins University, Baltimore) were maintained in
McCoy’s 5A medium containing 10% FBS. Cell growth
inhibition was evaluated using the MTT assay. The
p53+/+ and p53ꢀ/ꢀ cells were plated in 96-well plates
at the density of 1500 cells/well. The cells were allowed
to attach overnight and then treated with various con-
centrations of bis-chalcone 8 (0–10 lM) in triplicate.
At the end of 72 h, 50 ll of 3 mg/ml of MTT was added
to each well and mixed. The plates were incubated for a
further 4 h. The supernatant from each well was then
carefully removed, and 200 ll of DMSO was added to
each well and mixed thoroughly. The plate was read
with a 540-nm filter using a Multiskan Ascent plate
reader (Thermo Electron, WI). The growth inhibition
4.2.1.4.
3-(4-Chloro-phenyl)-1-(4-hydroxy-phenyl)-
propenone (1). Procedure A: yield 92%; 1H NMR
(CDCl3) d 8.00 (d, 2H, J = 8 Hz), 7.76 (d, 1H,
J = 16 Hz), 7.57 (d, 2H, J = 8 Hz), 7.50 (d, 1H,
J = 16 Hz), 7.39 (d, 2H, J = 8 Hz), 6.93 (d, 2H,
J = 8 Hz); 13C NMR (MeOD) r 191.2, 164.9, 144.4,
138.0, 136.1, 133.3, 133.2, 131.4, 130.6, 124.5, 117.9;
HRMS (FAB-MS) m/z 258.04476 (M+).
4.2.1.5.
3-(4-Chloro-phenyl)-1-(phenyl-4-hydroxy-
methylboronic acid)-propenone (2). Procedure C: yield
81%; 1H NMR (CDCl3) d 8.04 (d, 2H, J = 8.8 Hz),
7.76 (d, 1H, J = 16 Hz), 7.57 (m, 3H), 7.40 (d, 1H,
J = 8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 3.89 (s, 2H); 13C
NMR (MeOD) r 190.6, 166.0, 144.0, 137.2, 135.2,

A. Modzelewska et al. / Bioorg. Med. Chem. 14 (2006) 3491–3495
3495
132.4, 132.2, 131.0, 130.0, 124.0, 75.1; MS (ESI) m/z
J = 16 Hz), 2.50 (s, 3H); 13C NMR (MeOD) d 136.1,
135.6, 134.7, 133.1, 131.2, 130.3, 75.5, 53.4, 42.6; HRMS
(FAB-MS) m/z 359.08437 (M+).
316.9 (M+).
4.2.1.6. 1-(Phenyl-4-boronic acid)-3-thiophen-2-yl-
propenone (3). Procedure B: yield 98%; 1H NMR
(MeOD) d 8.0 (br d, 2H), 7.95 (d, 1H, J = 15.2 Hz),
7.77 (br d, 2H), 7.62 (d, 1H, J = 5.2 Hz), 7.52 (d, 1H,
J = 3.2 Hz), 7.48 (d, 1H, J = 15.2 Hz), 7.16 (dd, 1H,
J = 4.2, 3.6 Hz); 13C NMR (MeOD) d 192.0, 141.6,
138.9, 135.2, 134.1, 133.4, 131.0, 130.6, 130.3, 128.5,
121.3; MS (ESI) m/z 258.9 (MH+).
4.2.1.14. Benzoic acid 3,5-bis-(4-chloro-benzylidene)-1-
methyl-piperidin-4-yl ester (11). Yield 90%; 1H NMR
(CDCl3) d 7.30–7.40 (m, 13H), 6.38 (br s, 2H), 5.22 (s,
1H), 3.32–3.50 (m, 2H), 2.70–3.10 (m, 2H), 2.34 (s,
3H); 13C NMR (CDCl3) d 167.0, 137.5, 133.0, 132.8,
130.5, 129.7, 128.8, 128.4, 127.6, 119.2, 84.8, 54.0,
39.8; HRMS (FAB-MS) m/z 463.11058 (M+).
4.2.1.7.
3-Benzo[b]thiophen-2-yl-1-(phenyl-4-boronic
1
acid)-propenone (4). Procedure B: yield 97%; H NMR
(MeOD) d 8.06 (m, 3H), 7.87 (m, 2H), 7.81 (br d, 2H),
7.76 (s, 1H), 7.52 (d, 1H, J = 15.2 Hz), 7.42 (m, 2H);
13C NMR (CDCl3) d 191.5, 141.8, 141.4, 141.2, 139.1,
134.6, 131.7, 131.4, 128.5, 128.2, 127.3, 126.5, 125.7,
124.4, 123.5; MS (ESI) m/z 308.9 (MH+).
Acknowledgments
We gratefully acknowledge the financial support of
FAMRI, as well as grants CA100428 and CA 88843
from the National Cancer Institute, National Institutes
of Health.
4.2.1.8.
2-(4-Chloro-benzoyl)-3-(4-hydroxy-phenyl)-
acrylic acid (5). Procedure A: yield 85%; 1H NMR
(MeOD) d 7.94 (d, 2H, J = 8.8 Hz), 7.88 (s, 1H), 7.50
(d, 2H, J = 8.8 Hz), 7.22 (d, 2H, J = 8.8 Hz), 6.68 (d,
2H, J = 8.8 Hz); 13C NMR (MeOD) d 197.5, 168.1,
141.5, 136.2, 134.1, 133.0, 132.0, 130.1, 128.9, 125.4,
117.3, 116.2; HRMS (FAB-MS) m/z 302.03459 (M+).
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1
4.2.1.12. 4-tert-Butyl-2,6-bis-(4-boronic acid-benzyli-
1
dene)-cyclohexanol (9). Yield 85%; H NMR (MeOD) d
7.60 (m, 4H), 7.25–7.40 (m, 4H), 6.38 (br s, 2H), 4.78 (s,
1H), 3.62 (m, 2H), 3.33 (m, 2H), 0.85 (s, 9H); 13C NMR
(MeOD) d 144.6, 140.9, 135.8, 135.0, 130.5, 129.2, 80.8,
40.7, 33.8, 20.8, 20.7; MS (ESI) m/z 419.1 (MH+).
4.2.1.13. 3,5-Bis-(4-chloro-benzylidene)-1-methyl-pip-
1
eridin-4-ol (10). Yield 78%; H NMR (MeOD) d 7.40
(d, 4H, J = 9 Hz), 7.25 (d, 4H, J = 9 Hz), 6.97 (s, 2H),
4.89 (s, 1H), 4.01 (d, 2H, J = 16 Hz), 3.86 (d, 2H,