Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.02.037

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human canc

Full text of DOI:10.1016/j.ejmech.2013.02.037

European Journal of Medicinal Chemistry 63 (2013) 501e510
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell
migration inhibitors
Lívia B. Salum a, Wanessa F. Altei a, Louise D. Chiaradia b, Marlon N.S. Cordeiro b,
Rafael R. Canevarolo d, Carolina P.S. Melo d, Evelyn Winter c, Bruno Mattei c,
Hikmat N. Daghestani e, Maria Cláudia Santos-Silva f, Tânia B. Creczynski-Pasa c,
Rosendo A. Yunes b, José A. Yunes d, Adriano D. Andricopulo a, Billy W. Day g,
,
h,i,
*
Ricardo J. Nunes b , Andreas Vogt
**
a Laboratório de Química Medicinal e Computacional, IFSC e USP, São Carlos, SP, Brazil
b Laboratório Estrutura e Atividade, Departamento de Química, UFSC, Florianópolis, SC, Brazil
c Departamento de Ciências Farmacêuticas, UFSC, Florianópolis, SC, Brazil
d Centro Infantil Boldrini, Campinas, SP, Brazil
e Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
f Departamento de Análises Clínicas, UFSC, Florianópolis, SC, Brazil
g Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
h Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
i University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on classical colchicine site ligands and a computational model of the colchicine binding site on
beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition
of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone
scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In
particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand
etubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent
with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine
acute lymphoblastic leukemia cells compared with a previously described parent compound, and
inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the
growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization
and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in
scratch wound and Boyden chamber assays.
Received 22 November 2012
Received in revised form
22 February 2013
Accepted 25 February 2013
Available online 6 March 2013
Keywords:
Chalcones
Microtubule perturbing agents
High-content screening
Mitotic index
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Microtubules (MTs) are cytoskeletal polymers of
a and b tubulin
Abbreviations: Cy3, indocarbocyanine dye with three-methine linker; DMEM,
Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; FITC, fluorescein
isothiocyanate; HBSS, Hank’s balanced salt solution; HCS, high-content screening;
HUVEC, human umbilical vein endothelial cells; MDEC, minimal detectable effec-
tive concentration; MT, microtubules; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PBMC, peripheral blood mononuclear cells; RPMI,
Roswell Park Memorial Institute medium.
heterodimers involved in several critical cellular functions,
including motility, division and intracellular transport. Interference
with either assembly or disassembly of MTs in dividing cells pro-
vokes cell cycle arrest, triggering signals that induce apoptosis.
Therefore, the design of MT perturbing agents is one of the current
front-line approaches to anticancer therapy [1].
MT-targeting agents usually bind to one of three major binding
sites on tubulin, named taxane, vinca and colchicine sites. While
taxanes and vinca alkaloids have tremendously impacted the
treatment of human cancers, the high toxicity of colchicine (1a) and
podophyllotoxin (1b) (Fig. 1) has limited their therapeutic
* Corresponding author. Department of Computational and Systems Biology,
10047 Biomedical Science Tower 3, 3501 Fifth Avenue, University of Pittsburgh,
Pittsburgh, PA 15260, USA. Tel.: þ1 412 383 5856; fax: þ1 412 648 9009.
** Corresponding author. Laboratório Estrutura e Atividade, Departamento de
Química, Universidade Federal de Santa Catarina, Campus Trindade, CEP 88040-900
Florianópolis, SC, Brazil. Tel.: þ55 48 3721 6844x236.
E-mail addresses: ricardo.nunes@ufsc.br (R.J. Nunes), avogt@pitt.edu (A. Vogt).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.037

502
L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
and apoptosis [12]. As part of our ongoing efforts to design
improved inhibitors of tubulin polymerization, we synthesized a
series of type 1 chalcones (2ae2d) (Fig. 2) with aromatic substi-
tution patterns mimicking those in classical colchicine site ligands
(Fig. 1). Consistent with molecular modeling predictions, the tri-
methoxy derivative 2c was more potent than the parent compound
(2b) in L-1210 proliferation and tubulin assembly inhibition assays,
motivating the synthesis and assessment of a novel series of 3,4,5-
trimethoxychalcones (type 2 chalcones) 3 (Fig. 2). In the new series,
the majority of analogs retained growth inhibitory activity against
L-1210 cells (Supplemental Table S1). The most potent type 1 and
type 2 chalcones were further evaluated for cytotoxicity in REH and
Jurkat human leukemia cells, for mitotic arrest and alterations in
cellular MT morphology using high-content analysis in human
cervical cancer cells, and for antimigratory activity in human breast
cancer cells using scratch wounding and Boyden chamber cell
migration assays. The most active analogs, 2c and 3a, inhibited
tubulin assembly in vitro as potently as colchicine and showed
submicromolar activity in the human cancer cell assays.
Fig. 1. Representative microtubule-interacting agents that bind to the colchicine site
on tubulin.
application for the therapy of cancer. Because the utility of clinically
used MT perturbing agents is compromised by drug resistance,
adverse side effects, complex formulations, synthetic complexity,
scarceness and limited bioavailability, the search for novel, struc-
turally simpler small molecule modulators of the tubulin/MT sys-
tem has been a priority for many years [1e3]. Lately, the biological
properties of the colchicine site ligand combretastatin A-4 (1c)
(Fig. 1), a potent inhibitor of tubulin assembly and cell growth that
also has antiangiogenic activity [4,5] have renewed interest in the
development of colchicine analogs as potential anticancer agents.
Moreover, the fact that combretastatin A-4 is not recognized by P-
glycoprotein, which is related to multidrug resistance, has rein-
forced the significance of colchicine site ligands as antitubulin
agents [4,5]. In this context, the chemically versatile and easily
synthesized chemical class of chalcones 2 (Fig. 2) represents an
attractive scaffold for the design of novel colchicine site ligands that
inhibit tubulin assembly. In fact, several studies have demonstrated
that chalcone derivatives are cytotoxic against a variety of cell lines
and may have potential applications in cancer treatment (reviewed
in Ref. [6]).
Although antimitotic chalcones have been identified in the
1980’s (reviewed in Ref. [7]), considerable efforts are being dedi-
cated to identify new chalcone-based leads within the oncology
field, due to their anticancer properties and simple synthesis. The
most recent advances include photodynamic agents [8], dihalo-
genated chalcones [9], and peptidyl [10] and boronic acid de-
rivatives [11]. Work within our own group has led to the
development of the chalcone 2b (Fig. 2), derived from 2-
naphtaldehyde, that was selectively cytotoxic to L-1210 murine
acute lymphoblastic leukemia cells and promoted cell cycle arrest
2. Material and methods
2.1. Synthesis
2.1.1. Preparation of compounds
Reagents used were obtained commercially from Sigmae
Aldrich. Type 1 and 2 chalcones were prepared by aldol conden-
sation of acetophenones (1 mmol) with aldehydes (1 mmol), in
methanol (15 mL), KOH (50% v/v), at room temperature with
magnetic agitation for 24 h. The volume of KOH varied according to
the different inductive and mesomeric effects for the various sub-
stituents of the aromatic rings: 2 mL for compounds 2a, 2b and 2c;
40 drops for compounds 2d and 3p; 15 drops for compounds 3d
and 3q; 13 drops for compounds 3a, 3h, 3j and 3o; 10 drops for
compounds 3b, 3c, 3e, 3f, 3i and 3k; 8 drops for compounds 3n, 3r
and 3s; and 5 drops for compounds 3g, 3l and 3m. KOH addition
was stopped at the first sign of precipitation. Distilled water and
10% hydrochloric acid were added to the reaction for total precip-
itation of the compounds, which were then obtained by vacuum
filtration and later recrystallized in dichloromethane, with forced
precipitation by hexane. The purity of the synthesized compounds
was analyzed by thin-layer chromatography (TLC) using Merck
silica pre-coated aluminum plates of 200
several solvent systems of different polarities. Compounds were
visualized with ultraviolet light (
¼ 254 and 360 nm) and using
mm thickness, with
l
sulfuric anisaldehyde solution followed by heat application as the
developing agent. The chalcones were soluble in dimethyl sulf-
oxide, acetone, acetyl acetate, chloroform and dichloromethane.
Compounds 3ae3o and 3r were previously cited in the literature
[6,7,16e21]. Chalcone derivatives 2a, 2b and 2c were previously
synthesized by our group [13e15] and 2d, 3p, 3q and 3s are novel
compounds.
2.1.2. Physicoechemical data of the compounds
The structures were confirmed by melting points (m.p.), infrared
spectroscopy (IR) and 1H and 13C nuclear magnetic resonance
spectroscopy (NMR), as well as elementary analysis for previously
undescribed structures. Melting points were determined with a
Microquímica MGAPF-301 apparatus and are uncorrected. IR
spectra were recorded with an Abb Bomen FTLA 2000 spectrometer
on KBr disks. Elementary analysis was carried out using a CHNS EA
1110; percentages of C and H were in agreement with the product
formula (within ꢀ0.4% of theoretical values for C). NMR (1H and
13C) spectra were recorded on a Varian Oxford AS-400 (400 MHz)
instrument, using tetramethylsilane as an internal standard. 1H
Fig. 2. Type 1 and type 2 chalcones.

L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
503
NMR spectra revealed that structures were geometrically pure and
configured E (JH
¼ w16 Hz), except 3a, which is a mixture of E
2.1.2.5. 3q e (2E)-1-(30,40,50-trimethoxyphenyl)-3-(3-trifluoromethyl-
4-chlorophenyl)-2-propen-1-one. Light yellow solid, m.p. 146e
a
ꢁH
b
(JH
¼ 16 Hz) 2:1 Z (JH
¼ 12 Hz) isomers.
147 ꢂC; 1H NMR (CDCl3)
d 3.95 (s, 3H, p-OCH3), 3.95 (s, 6H, m-
a
ꢁHb
a
ꢁHb
OCH3), 7.26 (2, 2H, H20, H60), 7.32 (dd, 1H, J ¼ 8.0/1.0 Hz, H5), 7.38 (d,
2.1.2.1. 2c e (2E)-1-(30,40,50-trimethoxyphenyl)-3-(2-naphthyl)-2-
propen-1-one. Light yellow solid, m.p. 131e132 ꢂC; 1H NMR
1H, J ¼ 16.0 Hz, H ), 7.48 (s, 1H, H2), 7.68 (d, 1H, J ¼ 8.0 Hz, H6), 8.08
a
(d, 1H, J ¼ 16.0 Hz, H
OCH3), 106.49 (C20, C60), 125.32 (C
(C3), 130.38 (C6), 132.13 (C4), 133.17 (C10), 136.19 (CF3), 136.70 (C1),
b
). 13C NMR (CDCl3)
), 127.80 (C2), 128.78 (C5), 130.34
d 56.61 (m-OCH3), 61.16 (p-
(CDCl3)
7.54 (m, 2H, H3, H6), 7.60 (d, 1H, J ¼ 15.6 Hz, H
J ¼ 8.0 Hz, H7), 7.87 (m, 2H, H5, H8), 7.89 (m, 1H, H4), 7.99 (d, 1H,
J ¼ 15.6 Hz, H 56.66 (m-OCH3),
), 8.05 (s, 1H, H1). 13C NMR (CDCl3)
61.28 (p-OCH3), 106.29 (C20, C60), 122.04 (C3), 123.86 (C
), 127.07
(C1), 127.68 (C6), 128.07 (C7), 128.90 (C5), 128.98 (C8), 130.99 (C4),
d
3.96 (s, 6H, OCH3), 3.98 (s, 3H, OCH3), 7.32 (s, 2H, H20, H60),
a
a
), 7.83 (d, 1H,
139.53 (Cb /
), 141.59 (C40), 153.41 (C30, C50), 189.40 (C]O). IR nmax
b
d
cmꢁ1 1658 (C]O), 1574 (C]C), 1230, 1001 (CeO), 1161 (CeF), 1125
(CeCl), 3080, 2938, 2838, 1598, 1505, 1451, 1413, 1343, 1314, 1104,
816, 648 (Ar) (KBr). Anal. Calcd for C19H16ClF3O4: C 56.94, H 4.02.
Found: C 57.20, H 4.16. Yield: 90%.
a
132.59 (C10), 133.59 (C2),133.84 (C4a), 134.61 (C8a), 145.14 (C40, C
b),
153.40 (C30, C50),189.47 (C]O). IR nmax/cmꢁ11656 (C]O),1582 (C]
C), 1230 (CeO), 2834, 1502, 1458, 1411, 996, 813 (Ar) (KBr). Anal.
Calcd for C22H20O4: C 75.84, H 5.79. Found: C 75.53, H 5.81.
Yield ¼ 97%.
2.1.2.6. 3s e (2E)-1-(30,40,50-trimethoxyphenyl)-3-(3,5-
dichlorophenyl)-2-propen-1-one. Cream solid, m.p. 136e138 ꢂC; 1H
NMR (CDCl3)
d 3.95 (s, 3H, OCH3), 3.96 (s, 6H, OCH3), 7.27 (2, 2H,
H20, H60), 7.40 (m, 1H, H4), 7.46 (d, 1H, J ¼ 16.0 Hz, H
a
), 7.50e7.51 (s,
56.74
), 127.29
(C4), 132.38 (C10, C2, C6), 133.16 (C3, C5), 134.10 (C1), 141.79 (C
b),
2.1.2.2. 2d e (2E)-1-(10,30-benzodioxol-5-yl)-3-(2,3,4-
trimethoxyphenyl)-2-propen-1-one. Light yellow solid, m.p. 100e
2H, H2, H6), 7.66 (d, 1H, J ¼ 16.0 Hz, H
b d
). 13C NMR (CDCl3)
(m-OCH3), 61.74 (p-OCH3), 106.44 (C20, C60), 123.85 (C
a
101 ꢂC; 1H NMR (CDCl3)
d
3.89 (s, 3H, OCH3), 3.90 (s, 3H, OCH3),
3.94 (s, 3H, OCH3), 6.05 (s, 2H, eOCH2Oe), 6.71 (d, 1H, J ¼ 8.0 Hz,
143.16 (C40), 153.47 (C30, C50), 188.66 (C]O). IR nmax/cmꢁ1 1664 (C]
O), 1584 (C]C), 1230, 1003 (CeO), 1132 (CeCl), 3069, 2945, 2831,
1610, 1564, 1508, 1467, 1418, 1340, 1164, 973, 838, 803, 660 (Ar)
(KBr). Anal. Calcd for C18H16Cl2O4: C 58.87, H 4.39. Found: C 58.61, H
5.00. Yield: 92%.
H5), 6.88 (d, 1H, J ¼ 8.0 Hz, H50), 7.36 (d, 1H, J ¼ 8.0 Hz, H6), 7.51 (d,
1H, J ¼ 16.0 Hz, H
a
), 7.53 (s, 1H, H20), 7.64 (dd, 1H, J ¼ 8.0/1.0 Hz,
H60), 7.97 (d, 1H, J ¼ 16.0 Hz, H
b d 56.07 (OCH3),
). 13C NMR (CDCl3)
60.92 (OCH3), 61.39 (OCH3), 101.78 (eOCH2Oe), 107.56 (C5), 107.84
(C20),108.45 (C50),121.00 (C6),122.09 (C1),123.88 (C60),124.49 (C
a
),
133.32 (C10), 139.58 (C
b
), 142.48 (C3), 148.19 (C30), 151.45 (C2),
2.2. Antiproliferative assays
153.75 (C40), 156.67 (C4), 188.65 (C]O). IR nmax/cmꢁ1 1652 (C]O),
1583 (C]C), 1249, 1041 (CeO), 3079, 2976, 2942, 2901, 2837, 1492,
1484, 1464, 1446, 1413, 1329, 1301, 1282, 1115, 1095, 988, 942, 916,
815, 697, 506 (Ar) (KBr). Anal. Calcd for C19H18O6: C 66.66, H 5.30.
Found: C 66.91, H 5.27. Yield: 94%.
2.2.1. L-1210 mouse leukemia cell assays
2.2.1.1. Cell culture. Murine lymphoblastic leukemia (L1210) cells
were obtained from American Type Culture Cell (ATCC). The cells
were cultured in RPMI (Roswell Park Memorial Institute medium)
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
2.1.2.3. 3a e (2E,Z)-1-(30,40,50-trimethoxyphenyl)-3-(4-
dimethylaminophenyl)-2-propen-1-one. Golden yellow solid, m.p.
100 U/mL penicillin, 100 mg/mL streptomycin and 10 mM HEPES.
The cell culture was maintained at 37 ꢂC in a 5% CO2 humidified
atmosphere and pH 7.4. Every 2e3 days, cells were passaged by
removing 90% of the supernatant and replacing it with fresh me-
dium. In all experiments, viable cells were checked in the beginning
of the experiment by the Trypan Blue exclusion test.
148e149 ꢂC; 1H NMR (CDCl3)
d
2.98 (s, 3H, Z-CH3), 3.06 (s, 3H, E-
CH3), 3.88 (s, 6H, Z-m-OCH3), 33.90 (s, 3H, Z-p-OCH3), 3.93 (s, 3H, E-
p-OCH3), 3.95 (s, 6H, E-m-OCH3), 6.44 (d,1H, J ¼ 12.0 Hz, Z-H ), 6.58
(d, 1H, J ¼ 8.0 Hz, Z-H3, Z-H5), 6.71 (d, 1H, J ¼ 8.0 Hz, E-H3, E-H5),
a
6.90 (d, 1H, J ¼ 12.0 Hz, Z-H
b
), 7.19 (2, 2H, Z-H20, Z-H60), 7.27 (2, 2H,
E-H20, E-H60), 7.32 (d, 1H, J ¼ 16.0 Hz, E-H
a
), 7.56 (m, 2H, E-H2, E-
2.2.1.2. Cytotoxicity. Cytotoxicity was evaluated with the MTT ((3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
[37]. 1 ꢃ 105 cells/well were incubated for 24 h in triplicate with
compounds (solubilized in a maximum of 1% DMSO), at different
H6), 7.5e (m, 2H, Z-H2, Z-H6), 7.80 (d, 1H, J ¼ 16.0 Hz, E-H
b
). 13C
NMR (CDCl3)
(C20, C60), 112.04 (C3, C5), 116.77 (C1), 122.84 (C
134.71 (C10), 146.04 (C
d
40.36 (CH3), 56.57 (m-OCH3), 60.80 (p-OCH3), 106.06
), 130.66 (C2, C6),
), 141.20 (C40), 152.29 (C4), 153.27 (C30, C50),
a
b
concentrations (1e100
at 37 ꢂC, cells were washed with fresh culture medium, and 10
MTT (5 mg/mL) were added followed by incubation at 37 ꢂC for 2 h.
The precipitated formazan was dissolved in 100 L of DMSO, and the
mM) in 96-well microplates. After incubation
189.70 (C]O); in the RMN 13C was possible observe only the E-
isomer. IR nmax/cmꢁ1 1650 (C]O), 1563 (C]C), 1228, 1006 (CeO),
1153 (CeN), 2942, 2845, 1613, 1549, 1524, 1506, 1432, 1413, 1377,
1347, 1302, 1191, 1126, 1073, 984, 817, 669 (Ar) (KBr). Anal. Calcd for
mL of
m
absorbance was measured at 540 nm using a micro-well system
reader. The IC50 values were calculated from a four parameter logistic
equation. The chalcones were dissolved in DMSO, and in order to
verify if the solvent itself could affect the cells, in all experiments,
control curves without chalcones and in the presence of the cells and
thesolventwerecarriedoutinparallel.Thecontrolswithsolventwere
not statistically different from control cells alone (data not shown).
C
20H23NO4: C 70.36, H 6.79, N 4.10. Found: C 70.05, H 6.83, N 4.08.
Yield: 45%.
2.1.2.4. 3p e (2E)-1-(30,40,50-trimethoxyphenyl)-3-(5-methylfuran-2-
yl)-2-propen-1-one. Dark yellow solid, m.p. 89e90 ꢂC; 1H NMR
(CDCl3)
d 2.41 (s, 3H, CH3), 3.94 (s, 3H, p-OCH3), 3.95 (s, 6H, m-
OCH3), 6.14 (d, 1H, J ¼ 4.0 Hz, H4), 6.44 (d, 1H, J ¼ 4.0 Hz, H5), 7.29
(2, 2H, H20, H60), 7.32 (d, 1H, J ¼ 16.0 Hz, H
a
), 7.55 (d, 1H, J ¼ 16.0 Hz,
2.2.2. Cytotoxicity in tumorigenic and non-tumorigenic cell lines
2.2.2.1. Cell culture. REH and JURKAT human acute lymphoblastic
leukemia cell lines of precursor B-cell and T-cell immunopheno-
types, respectively, were cultured in RPMI-1640 medium supple-
mented with 10% FBS (RPMI-10), 100 U/mL penicillin and 100 pg/
mL streptomycin. Cultures were maintained at 37 ꢂC in a 5% CO2
humidified atmosphere. NIH3T3 and HUVEC were cultured under
the same conditions in RPMI-1640 and DMEM, respectively. Normal
Hb d 14.30 (CH3), 56.66 (m-OCH3), 61.17 (p-
). 13C NMR (CDCl3)
OCH3), 106.17 (C20, C60), 109.64 (C4), 117.23 (C5), 118.67 (C
a), 131.07
(Cb
), 133.96 (C10), 142.48 (C40), 150.65 (C1), 153.32 (C30, C50), 156.20
(C3), 188.85 (C]O). IR nmax/cmꢁ1 1653 (C]O), 1565 (C]C), 1227,
1012 (CeO), 2941, 2839, 1606, 1582, 1503, 1414, 1335, 1158, 1126,
796, 698, 625 (Ar) (KBr). Anal. Calcd for C17H18O5: C 67.54, H 6.00.
Found: C 67.76, H 6.42. Yield: 78%.

504
L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
human mononuclear cells were isolated from blood of a healthy
individual using FicollePaque (GE Healthcare) gradient, washed
twice with PBS buffer and cultured in 96 well plates at 200,000 cell/
treated with vehicle (DMSO 0.1% final concentration) or 10 two-fold
concentration gradients of test agents within 4e6 h of seeding.
Cells were incubated for 18 h at 37 ꢂC and 5% CO2, fixed with
well in RPMI-10 supplemented with 20
mL/mL of phytohemagglu-
formaldehyde, and labeled with 10 mg/mL Hoechst 33342 in Hank’s
tinin (Cultilab, Campinas, SP, Brazil) and 50 IU/mL of interleukin-2
(Proleukin, ZODIAC) to induce proliferation of T lymphocytes.
Cytotoxicity assays were initiated after 24 h of PHA/interleukin-2
balanced salt solution (HBSS). Cells were permeabilized with 0.5%
(w/w) Triton-X-100 for 5 min at room temperature and incubated
with a primary antibody cocktail consisting of an HBSS solution
containing rabbit polyclonal antiphosphohistone H3 (Ser10, 1:500,
stimulation by adding 20
mL of serial chalcone dilutions (see
below). For comparison, JURKAT cells (30,000/well) were seeded in
the same 96 well plates and cultured for 24 h in RPMI-10 (without
PHA or interleukin-2) before treatment.
Upstate, Charlottesville, VA, USA), and mouse monoclonal anti-a-
tubulin (1:3000, Sigma, St. Louis, MO, USA), followed by a mixture
of FITC-labeled donkey anti-mouse IgG (1:500) and Cy3-labeled
donkey anti-rabbit IgG (1:500) as secondary cocktail. Cells were
rinsed once with HBSS and analyzed with an ArrayScanII high-
content reader (Thermo Fisher Cellomics, Pittsburgh, PA, USA) us-
ing the Target Activation Bioapplication. Within the application,
1000 individual cells in each well were imaged at three different
wavelengths using an Omega XF93 filter set (Omega Optical, Inc.,
Brattleboro, VT, USA) at excitation/emission wavelengths of 350/
461 nm (Hoechst), 494/519 nm (FITC), and 556/573 nm (Cy3). The
following parameters were used for data analysis: average nuclear
intensity, nuclei per field, average nuclear FITC intensity, and
average nuclear Cy3 intensity. A nuclear mask was generated from
Hoechst 33342-stained nuclei. MT density and histone H3 phos-
phorylation were measured in the FITC and Cy3 channel, respec-
tively. MT density was defined as the average green (FITC) pixel
intensity in an area defined by the nuclear mask. For determination
of mitotic index and nuclear condensation, thresholds for Hoechst
33342 and phosphohistone-H3 intensities were defined as the
average Hoechst 33342 or Cy3 intensity plus 1 SD from 28 vehicle-
treated wells located in the center of the microplate. Cells were
classified as positive if their average Hoechst 33342 or Cy3 intensity
exceeded this threshold. EC50 values from cell density curves were
obtained by a four-parameter logistic equation (GraphPad Prism).
The shape of the chromatin condensation, MT density and mitotic
index graphs precluded EC50 determinations. Accordingly, we
determined the minimum detectable effective concentration
(MDEC) as an alternative measurement of chalcones and colchicine
activities as described previously [38]. To visually illustrate the ef-
fects of test agents on cellular MT morphology and mitotic arrest,
the identical 384-well plates were then used to acquire higher
resolution images of selected wells.
2.2.2.2. Cytotoxicity assay. For the cytotoxic assay, chalcones were
dissolved in DMSO to obtain a stock solution of 20 mM. Further
dilutions were made in culture medium (RPMI-10) immediately
before use. REH and JURKAT cells were plated at 3 ꢃ 104 cells/well
in 80 mL RPMI-10. Immediately, 20 mL of 10-fold serial chalcone
dilutions were added to each well, in triplicate, at final concen-
trations of 0.01e100 M. DMSO used as a vehicle was included as
control at a final concentration of 0.5%. After 24e48 h of culture,
cell number and viability were measured by adding 20 L of MTT
reagent (5 mg/mL). After 4.5 h of incubation at 37 ꢂC and 5% CO2,
the precipitated formazan crystals were dissolved by adding 100
m
m
mL
of an acidic sodium dodecyl sulfate solution (10% SDS, 0.01 mol/L
HCl) or DMSO. Absorbance was measured at 570 nm. IC50 values
were calculated with GraphPad Prism software.
2.3. Molecular modeling
The structures of the type 1 chalcones were constructed using
the standard tools available in SYBYL 8.0 and the structures were
energy-minimized employing the Tripos force field and Gasteigere
Hückel charges. Docking protocols were used as implemented in
GOLD 3.1. The X-ray crystal structures of the tubulin-colchicine and
tubulin-ABT751 complexes (PDB IDs 1SA0 and 3HKC, respectively)
were retrieved from the Protein Data Bank. For the calculations, the
ligands were removed and hydrogen atoms were added using the
Biopolymer module (SYBYL 8.0). Residues in the binding site were
manually checked for possible flipped orientation, protonation and
tautomeric states. The binding cavity was centered on the C22 atom
ꢀ
of DAMA-colchicine and a radius sphere of 8 A was considered for
the docking procedures, which were repeated 10 times for the s-cis
and s-trans conformations of the type 1 chalcones.
2.6. Cell migration assays
2.4. Inhibition of tubulin assembly
2.6.1. Cell culture
The human breast cancer cell line MDA-MB-231 was kindly
provided by Prof. Heloisa Sobreiro Selistre de Araujo (Departa-
mento de Ciências Fisiológicas, Universidade Federal de São Carlos).
Electrophoretically homogenous bovine brain tubulin (final con-
centration 10 mM; 1 mg/mL) was pre-incubated with test agents
dissolved in DMSO (1% v/v final concentration) and monosodium
glutamate (0.8 M final concentration) at 30 ꢂC in 96-well plates. The
reactionmixtureswere cooled on icefor10 minand GTP(0.4mMfinal
concentration) was added. The absorbance at 350 nm was immedi-
ately followed in a spectrophotometer. Baselines were established
and temperature was quickly raised to 37 ꢂC. The turbidity value after
20 min at 30 ꢂC for 1% DMSO was assigned as 100% assembly, and for
colchicine (10
mM) as 0% assembly. The IC50 was calculated by
nonlinear regression of the percent assembly values at the 20 min
time point obtained with 0.625, 1.25, 2.5, 5 and 10 mM test agents.
2.5. Automated high-content cellular analysis
The effects of chalcones 2ae2d and 3a on mitotic arrest, nuclear
morphology and cellular microtubules were studied as previously
described [28,29]. HeLa human cervical carcinoma cells (8000/
well) were plated in collagen-1-coated 384-well microplates and
Fig. 3. Preparation of type 2 chalcones. a) KOH 50% w/v, methanol, r.t., 24 h. *Novel
compounds, Ph ¼ phenyl.

L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
505
Table 1
Cancer cell selective cytotoxicity and tubulin polymerization inhibition.
Compound
IC50 (mM)
L-1210a
REHb
JURKATb
NIH3T3a
HUVECa
PBMCb
Tubulin assemblyc
1a (Colchicine)
NDd
>100
54.0 ꢀ 0.5
26.0 ꢀ 1.0
47.5 ꢀ 1.5
30.0 ꢀ 0.8
0.0095 ꢀ 0.005
ND
2.58 ꢀ 0.58
0.73 ꢀ 0.15
23.6 ꢀ 1.1
1.05 ꢀ 0.11
0.0077 ꢀ 0.003
17.1 ꢀ 1.1
0.0026 ꢀ 0.0012
0.023 ꢀ 0.0011
>100
2.3
>10
3.6
2.2
>10
2.8
2a
2b
2c
2d
3a
ND
ND
27.0 ꢀ 1.3
35.6 ꢀ 1.1
18.0 ꢀ 1.2
81.2 ꢀ 1.1
92.5 ꢀ 1.3
5.90 ꢀ 2.04
0.50 ꢀ 0.15
30.0 ꢀ 0.8
ND
ND
34 ꢀ 1.5
37 ꢀ 1.8
ND
ND
1.20 ꢀ 0.15
66 ꢀ 1.4
>100
a
24 h MTT assay.
b
48 h MTT assay. Data are the averages ꢀ SD of triplicate determinations.
c
Average of duplicate determinations from a single experiment that was repeated once.
ND, not determined.
d
The cells were maintained in DMEM (Dulbecco’s Modified Eagle’s
Medium) supplemented with 10% FBS at 37 ꢂC in 5% CO2 humidified
atmosphere. Cell viability was tested by Trypan blue exclusion
before experiments.
with a cotton swab and cells on the underside of the insert were
fixed with methanol and stained with toluidine blue stain (Sigma).
Transmigrated cells were counted with a light microscope, using six
random regions of the membrane. The NIS Elements software
(NIKON) was used to count the cells. Two independent experiments
were carried out in duplicate for each compound.
2.6.2. Wound healing assay
For cell motility determination, cells were seeded in 24-well
tissue culture plates (Becton Dickinson) and grown to 80e90%
confluence. After aspirating the medium, the centers of the cell
monolayers were scraped with a sterile micropipette tip to create a
denuded zone. Subsequently, cellular debris was removed by
washing with DMEM (without FBS supplementation) and cells
were incubated at 37 ꢂC with growth media containing different
concentrations of compounds (1a, 2c and 3a) for 22 h. Pictures of
the initial wounded monolayers were taken at 0 and 22 h, and the
percentage of wound closure was quantified with ImageJ software.
3. Results and discussion
3.1. Chemistry
The type 1 chalcones 2a, 2b and 2c (Fig. 2) were prepared as
previously described [13e15] with yields between 39 and 97%.
Chalcone 2d is a novel compound, obtained by the same method-
ology as for the other type 1 chalcones, with yield of 94%. The type 2
chalcones (3ae3s) were prepared by condensation between 3,4,5-
trimethoxyacetophenone and corresponding aldehydes (Fig. 3),
with yields ranging from 20 to 92%.
2.6.3. Boyden chamber migration assay
Cell migration was assayed with Boyden chambers [8.0 mm pore
All reagents used were available commercially. Synthesized
compounds that had been previously reported [7,13e21] were
characterized by 1H NMR, 13C NMR and IR (see Supplementary
data). Detailed characterization (1H NMR, 13C NMR, IR and
elemental analysis) for novel compounds (2d, 3p, 3q and 3s) is
presented in the Material and Methods section. Previous studies
size, polyethylene terephthalate membrane, FALCON cell culture
insert (BectoneDickinson)]. MDA-MB-231 cells were cultured in
DMEM until 80e90% confluency. For migration assay, cells were
detached with trypsin and resuspended in serum free medium.
After cell counting, 4 ꢃ 104 cells in serum free media (350
m
L) were
added to the upper chamber. In the lower chamber, 700
mL of cul-
have demonstrated that
a-substituted E-chalcones may be more
ture medium containing 10% FBS (chemoattractant) were added.
The transwells were incubated for 6 h at 37 ꢂC with different
concentrations (usually 5e6) of the compounds in both the upper
and lower chambers. The control was the vehicle given in the same
method. After incubation, cells inside the inserts were removed
potent inhibitors of tubulin polymerization than the corresponding
Z geometric isomers [6,22]. 1H NMR spectra revealed that, as ex-
pected, structures were configured E (JH
for 3a, which was a 2:1 mixture of E (JH
¼ w16 Hz) [6] except
a
ꢁH
b
¼ 16 Hz) and Z
a
ꢁHb
(JH
¼ 12 Hz) isomers (see Materials and methods Section).
aꢁHb
Fig. 4. Molecular modeling studies comparing chalcone 2c with DAMA colchicine. (A) s-cis and s-trans conformations of 2c. (B) Crystallographic positioning of DAMA-colchicine
(gray) and (C) docking pose of chalcone 2c (green) in the colchicine binding site. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

506
L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
Table 2
3.2.2. Molecular modeling studies
High-content analysis of mitotic arrest in HeLa cells.a
Classical ligands of the colchicine site on tubulin (Fig. 1)
frequently contain the 3,4,5-trimethoxyphenyl group [6,7,16,23,24],
which has been linked to increased antiproliferative potency in
type 1 chalcones [6]. This is thought to occur through specific in-
teractions between the trimethoxy groups and colchicine site res-
idues in tubulin, which force the ligand into a curved, s-trans
conformation. While crystallographic studies suggest that the most
probable configuration of chalcones in solution is s-cis [25], sub-
stitutions at the double bond that force the molecule into an s-trans
conformation enhance biological activity [6,16]. The crystallo-
graphic structure of tubulin complexed with N-deacetyl-N-(2-
mercaptoacetyl)colchicine (DAMA-colchicine, PDB ID 1SA0) was
the starting point for docking studies, which were performed to
explore possible binding modes for the series of type 1 chalcones at
Compound
EC50 (nM)b
cell density
MDEC (nM)b,c
Nuclear
condensation
Tubulin
intensity
Mitotic
indexd
1a
2a
2b
2c
2d
3a
32.5 ꢀ 4.2
>50,000
3300 ꢀ 600
684 ꢀ 139
>50,000
12.0 ꢀ 4.8
>50,000
2200 ꢀ 700
317 ꢀ 23
>50,000
10.2 ꢀ 3.8
>50,000
1100 ꢀ 200
283 ꢀ 52
>50,000
15.9 ꢀ 2.9
>50,000
1400 ꢀ 100
397 ꢀ 24
>50,000
357 ꢀ 35
217 ꢀ 67
139 ꢀ 13
200 ꢀ 15
a
24 h HCS assay.
b
c
Average of three independent experiments (ꢀSD).
MDEC, Minimum detectable effective concentration.
Percentage of phospho-histone H3-positive cells.
d
the tubulin a/b intradimer interface. The results suggested that the
3.2. Biological evaluation of type 1 chalcones
curved s-trans conformer of 2c overlapped well with DAMA-
colchicine within the colchicine binding domain, with their 3,4,5-
trimethoxyphenyl groups buried in the same hydrophobic sub-
cavity. The C4-methoxy substituent of 2c superposed with the C2-
methoxy group of colchicine and similarly established a polar
3.2.1. Mouse leukemia cell growth inhibitory activity
The type 1 chalcones (2ae2d) were evaluated for their anti-
proliferative activities against L-1210 murine acute lymphoblastic
leukemia cells (Table 1). While the general non-substituted 1,3-
diphenylprop-2-en-1-one backbone 2a was inactive in our tests
(2a, IC50 > 100 mM), the addition of a 3,4,5-trimethoxybenzoyl group
to the A-ring of the previously characterized 2-naphtaldehyde de-
interaction with Cys b241 (Fig. 4) [6]. In support of this model,
docking of the non-substituted inactive chalcone 2a did not
generate a clearly favored orientation (data not shown). The
chalconeetubulin complex was further stabilized by two hydrogen
bonds, involving the carbonyl oxygen of 2c and the backbone NH of
rivative 2b resulted in the most potent compound among the type 1
Asp b251 and Leu b255 (Fig. 4). Together, the described intermo-
chalcones (2c, IC50 ¼ 26
m
M). 2c showed two-fold improved activity
compared with 2b (IC50 ¼ 54
mM) [12]. Consistent with findings by
lecular interactions are consistent with the higher potency
observed in compounds having the carbonyl group in a adjacent
position to the trimethoxybenzene A-ring, instead of the B-ring as
in 2d.
Kong et al. [11], the presence of the 2,3,4-trimethoxyphenyl group in
the B-ring did not significantly augment cytotoxic potency (2d,
IC50 ¼ 47.5
mM).
Fig. 5. High-content analysis of mitotic arrest. Cells were treated in 384-well plates with 10 twofold dilutions of colchicine (triangles) and chalcones 2c (squares) and 3a (circles),
and analyzed by high-content analysis for (A) cell density, (B) Mitotic index, (C) chromatin condensation, and (D) microtubule density. All agents caused cell loss, enhanced mitotic
index and nuclear condensation, and provoked an initial increase in microtubule density that plateaued and reversed at higher concentrations. Data are the average ꢀ SEM of
quadruplicate wells from a single experiment repeated three times with similar results.

L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
507
The s-cis conformations of the synthesized E-chalcones might be
more stable than the (more bioactive) s-trans conformations [25].
Consistent with this, we observed internal ligand torsion strain
penalties for the s-trans binding conformations. These penalties
were, however, offset by additional van der Waal’s interactions
contributed by the 2-naphtaldehyde moiety in 2c, which was
positioned at the opposite side of the binding cavity, in the same
region as the tropone ring of colchicine [26] (Fig. 4C). Taken
together, the data indicate that besides the relative position of the
aromatic rings and the carbonyl group, specific substituents on the
chalcone moiety are important to correctly anchor the bioactive
s-trans conformation of chalcone derivatives into the colchicine
binding site.
Consistent with the tubulin assembly assays, the 3,4,5-
trimethoxyphenyl derivative 2c was more toxic to L-1210 cells
than either 2a, 2b or 2d (Table 1).
3.3. Biological evaluation of type 2 chalcones
The importance of the 3,4,5-trimethoxy substitution for both
inhibition of tubulin assembly and cytotoxic activity encouraged
the synthesis of a novel series of type 2 chalcones having the
general backbone 3 (Fig. 2).
3.3.1. In vitro cell growth inhibitory activity and inhibition of
tubulin polymerization
The type 2 chalcones were first screened for inhibition of cell
growth against murine L-1210 cells (see Supplementary data
Table S1). Of the nineteen analogs, fourteen (74%) retained anti-
proliferative activity; the derivative 3a was the most active (IC50
3.2.3. Inhibition of tubulin polymerization
The molecular modeling results suggested that, once bound to
the colchicine pocket, cytotoxic type 1 chalcones, like colchicine,
could prevent curved tubulin from assuming a straight conforma-
tion, thus resulting in cellular MT disassembly and cytotoxic ac-
tivity [27]. We therefore examined the ability of type 1 chalcones to
inhibit tubulin polymerization in vitro (Table 1). The 3,4,5-
trimethoxyphenyl derivative 2c had slightly improved ability to
inhibit tubulin polymerization compared with the parent 2d,
approaching the potency of colchicine (1a). In contrast, the
unsubstituted general scaffold 2a and the 1,3-dioxole derivative 2d
¼ 30
m
M). Further evaluation in REH and Jurkat human leu-
(L1210)
kemia cells (Table 1) demonstrated that the chalcones 2c (IC50
¼ 0.73
m
M and IC50
¼ 0.50
m
M) and 3a (IC50
(REH)
(REH)
(JURKAT)
¼ 1.05
m
M and IC50 (JURKAT) ¼ 1.20
mM) were at least an order of
magnitude more potent against human leukemia cells compared
with the murine L-1210 line. 2c and 3a also represented a three to
twenty five-fold improvement over the type 1 chalcones 2b (IC50
¼ 2.58
m
m
M and IC50
M and IC50
¼ 5.90
m
M) and 2d (IC50
(REH)
(REH)
(JURKAT)
(JURKAT)
did not inhibit tubulin assembly at concentrations up to 10 mM.
¼ 23.55
¼ 30.04
mM). Cytotoxicity
Fig. 6. Mitotic arrest phenotype. HeLa cells were treated with (A) vehicle (dimethyl sulfoxide, DMSO 0.1%), (B) colchicine 1a (62 nM), (C) 2c, the most potent type 1 chalcone
(940 nM), or (D) 3a, the most potent type 2 chalcone (470 nM), followed by simultaneous, immunostaining of -tubulin (green), phosphohistone H3 (red), and Hoechst 33342
a
(blue). Vehicle-treated cells have highly organized microtubules and a low percentage of mitotic cells. Colchicine 1a and the chalcones 2c and 3a caused a heterogenous response of
tubulin disorganization, increased number of phosphohistone H3-positive cells, chromatin condensation, and nuclear fragmentation. Images shown are representative fields from a
single experiment repeated three times with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

508
L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
Table 3
mitotic arrest, we conducted our previously described multi-
parameter high-content analysis [28,29]. We investigated and
compared the effects of colchicine 1a, the type 1 chalcones 2ae2d,
and the most potent type 2 chalcone 3a on MT perturbation,
apoptotic morphology, cell cycle arrest and histone H3 phosphor-
ylation. Asynchronously growing HeLa cells were treated for 18 h
with each compound or dimethyl sulfoxide (DMSO, 0.1%) as vehicle
in collagen-coated 384-well microplates, fixed, and incubated with
primary antibodies against tubulin and the mitotic marker protein
phosphohistone H3, followed by fluorescein isothiocyanate (FITC)
and Cy3-conjugated secondary antibodies, respectively. Cells were
detected by nuclear counterstaining with Hoechst 33342, which
also provided information about chromatin condensation and cell
density as markers of cell death.
Multi-parametric analysis of mitotic arrest showed that like
colchicine (1a), chalcones 2b, 2c and 3a provoked cell loss, chro-
matin condensation, and an increase in the percentage cells with
elevated phosphohistone H3 levels (Table 2). These same com-
pounds also showed a biphasic response in the measurements of
cellular MTs, previously observed for MT-destabilizing agents
(Fig. 5) [28,29]. At lower concentrations, tubulin staining intensity
increased due to cell rounding and concentration of MTs in a
smaller area. At higher concentrations, MT density measurements
plateaued and subsequently decreased because of the drug-
induced shift of the dynamics of MTs toward tubulin monomers
and extraction of monomeric tubulin during the permeabilization
process [28]. 2c and 3a inhibited attachment and survival of HeLa
Effect of chalcones 2c and 3a on MDA-MB-231 human breast cancer cell migration.
Compound
Scratch assay
Boyden chamber
assay IC50 (nM)b
(% inhibition at 1 m
M)a
1a
2c
3a
74 ꢀ 2
46 ꢀ 2
54 ꢀ 3
475 ꢀ 106
1095 ꢀ 29
656 ꢀ 119
a
Average ꢀ SD of three independent experiments.
Average ꢀ range of two independent experiments.
b
evaluations in NIH3T3 immortalized mouse fibroblasts, human
umbilical vein endothelial cells (HUVEC), and peripheral blood
mononuclear cells (PBMC) revealed that 2c and 3a were consider-
ably less toxic to nontransformed cells compared with the two
human tumor cell lines (45e100-fold selectivity, Table 1). In
contrast, colchicine showed only a 2e3 fold preference for the
transformed phenotype, although it was very well tolerated by
PBMC (Table 1). Moreover, 3a (IC50 ¼ 2.8
M) and compound 2c, the most potent
M), in its ability to inhibit tubulin
mM) was similar to
colchicine 1a (IC50 ¼ 2.3
m
type 1 chalcone (IC50 ¼ 2.2
assembly in vitro (Table 1).
m
3.3.2. High-content analysis of mitotic arrest
MT-interacting agents typically cause cell cycle arrest and
cellular MT perturbations. To investigate the effects of the most
potent type 1 and 2 chalcones on cellular MT morphology and
Fig. 7. Inhibition of MDA-MB-231 human breast tumor cell migration in a scratch wound assay. (Upper panel) Pictures of wound healing assay at 0 h and 22 h, comparing the wound
closure by colchicine 1a and the chalcones 2c and 3a. (Lower panel) Quantitative measurements of gap closure inhibition by 2c and 3a at three different concentrations. Data are the
average ꢀ SEM of triplicate wells from a single experiment repeated twice with similar results.

L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
509
cells at submicromolar concentrations, representing a five to ten-
fold improvement over the parent chalcone 2d, and a mere ten-
fold loss of potency compared with the natural product, colchi-
cine (1a) (Fig. 5). Moreover, 2c and 3a induced mitotic arrest as
measured by chromatin condensation and increasing percentages
of cells with elevated phosphohistone H3 as well as perturbed
cellular MTs. In contrast, agents that did not inhibit tubulin as-
sembly (2a and 2d) did not cause mitotic arrest in HeLa cells at
and 2 chalcones, 2c and 3a, on cell migration (Table 3 and Fig. 7).
For cellular assays, we selected the MDA-MB-231 human breast
cancer cell line, an estrogen independent breast cancer model that
we have used previously in cell migration assays because of its high
metastatic potential [34]. Initially, chalcones 2c and 3a were eval-
uated at three different concentrations in a scratch wounding assay,
which involves the response of confluent monolayer cells to me-
chanical injury [35]. All three compounds significantly inhibited
cell migration in the scratch wound assay (Fig. 7). Based on the
results from the wound healing experiments, we performed
concentration-dependence experiments in transwell cell culture
chambers, which confirmed the antimigratory activities of the two
chalcones (Fig. 8). 2c and 3a inhibited MDA-MB-231 cell migration
in both wound healing and transwell assays with IC50 values
approaching that of colchicine (Table 3). These results suggest that
chalcones 2c and 3a should be explored as anti-metastatic agents.
Our findings are consistent with studies that demonstrated the
ability of combretastatin A-4 to inhibit the migration of bladder
cancer cells [36]. In this study, the reduction of cell migration by
combretastatin A-4 was attributed in part to decreased AKT protein
expression, which is involved with tumor cell survival, prolifera-
tion, migration and invasiveness [36]. In this context, our results
generate enthusiasm for further investigations of the exact mo-
lecular mechanisms of the antimigratory activity of the developed
chalcones.
concentrations up to 50 mM (Table 2). Fluorescence micrographs of
representative images of nuclei (blue), tubulin (green), and phos-
phohistone H3 (red) staining (Fig. 6) confirmed that, while vehicle-
treated cells had organized MTs and a low percentage of mitotic
cells, cells treated with colchicine showed disorganized MTs and a
high percentage of cells with elevated histone H3 phosphorylation
levels. High-nanomolar concentrations of 2c and 3a caused similar
MT disorganization and an increase in the percentage of mitotic
cells. Hoechst 33342 counterstaining revealed the presence of
condensed and fragmented nuclei characteristic of apoptosis. These
results validated the chalcone core structure as a bona fide inhibitor
of MT assembly in mammalian cells.
3.3.3. Effects on cell migration
Besides their effects on cell division, MT-interacting agents can
also interfere with cell migration [30e33]. Directional migration of
cancer cells requires changes in cell polarity and remodeling of the
cytoskeleton, including the regulation of MTs, actin, and interme-
diate filaments at the leading edge of the cell. We therefore
examined the effects of colchicine 1a, and the most potent type 1
4. Conclusions
We designed, synthesized, and evaluated the biological activ-
ities of type 1 and 2 chalcones as potential anticancer agents. The
most active agents inhibited tubulin assembly in vitro with po-
tencies comparable to colchicine and were only an order of
magnitude less potent than colchicine in human cancer cell toxicity
assays. The presence of a 3,4,5-trimethoxyphenyl group in ring A
appeared beneficial for tubulin interaction, antiproliferative, and
antimitotic effects. Our data suggest that the most cytotoxic type 1
and 2 chalcones kill cancer cells by inducing apoptosis and/or
mitotic catastrophe through a mechanism that involves MT desta-
bilization. Furthermore, our chalcones showed selective cytotox-
icity to cancer cells compared with non-tumorigenic cells and
inhibited the motility of a highly metastatic breast cancer cell line
in two different types of migration assays. These properties make
the 3,4,5-trimethoxyphenyl chalcone pharmacophore an attractive
scaffold for further development. We are currently pursuing new
analogs of compounds 2c and 3a with structural modifications
predicted to increase potency, such as restricting the conformation
of the double bond to the s-trans configuration. Future studies will
focus on additional cancer-related activities such as anchorage in-
dependent cell growth and invasion, on the elucidation of intra-
cellular signaling pathways that mediate compound toxicity, and
on preclinical testing in human tumor xenograft models.
Acknowledgments
We would like to thank CNPq, CAPES and FAPESP for financial
support and fellowships. We also thank the Departamento de
Química da Universidade Federal de Santa Catarina for equipment
used in chemical analysis and the Departamento de Ciências
Fisiológicas, Universidade Federal de São Carlos for the cell mate-
rial. For technical support, we thank Fernanda Spezia Pedrini
(Universidade Federal de Santa Catarina) and Laura Lee Vollmer
(The University of Pittsburgh Drug Discovery Institute). This project
was supported in part by National Institutes of Health grant
CA78039, and used the UPCI Chemical Biology Facility that is sup-
ported in part by award P30CA047904.
Fig. 8. Effect of chalcones on MDA-MB-231 cell migration using Boyden chamber as-
says. (A) Representative micrographs of cells that migrated through the membrane
insert after staining with toluidine. (B) Quantitation of antimigratory response. Data
are from a single experiment that has been repeated once with identical results.

510
L.B. Salum et al. / European Journal of Medicinal Chemistry 63 (2013) 501e510
T. Sarkar, E. Hamel, Design, synthesis, and biological evaluation of thiophene
Appendix A. Supplementary data
analogues of chalcones, Bioorg. Med. Chem. 16 (2008) 5367e5376.
[20] D. Batovska, S. Parushev, B. Stambohyska, I. Tsvetkova, M. Ninova,
H. Najdenski, Examination of growth inhibitory properties of synthetic chal-
cones for which antibacterial activity was predicted, Eur. J. Med. Chem. 44
(2009) 2211e2218.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.02.037.
[21] Y.K. Rao, S.H. Fang, Y.M. Tzeng, Synthesis and biological evaluation of 3 ’,4 ’,5
’-trimethoxychalcone analogues as inhibitors of nitric oxide production and
tumor cell proliferation, Bioorg. Med. Chem. 17 (2009) 7909e7914.
[22] D.J. Kerr, E. Hamel, M.K. Jung, B.L. Flynn, The concise synthesis of chalcone,
indanone and indenone analogues of combretastatin a4, Bioorg. Med. Chem.
15 (2007) 3290e3298.
[23] N.J. Lawrence, R.P. Patterson, L.L. Ooi, D. Cook, S. Ducki, Effects of alpha-
substitutions on structure and biological activity of anticancer chalcones,
Bioorg. Med. Chem. Lett. 16 (2006) 5844e5848.
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