Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin

Article abstract of DOI:10.1016/j.ejmech.2013.04.064

A series of chalcone and flavonol derivatives were synthesized in good yield by an eco-friendly approach. A pharmacological evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed that the anticancer activity of flavonols was higher when compared with that of the respective chalcone precursors. The antiproliferative activity of halogenated derivatives increases as the substituent in the 3- or 4-positon of the B-ring goes from F to Cl and to Br. In addition, halogens in position 3 enhance anticancer activity in chalcones whereas for flavonol derivatives the best performance was registered for the 4-substituted derivatives. Flow cytometry analysis showed that compounds 3p and 4o induced cell cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were corroborated by western blot and fluorescence microscopy analysis. In summary, halogenated chalcones and flavonols were successfully prepared and presented high anticancer activity as shown by their cell growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a positive control.

Full text of DOI:10.1016/j.ejmech.2013.04.064

European Journal of Medicinal Chemistry 65 (2013) 500e510
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Superior anticancer activity of halogenated chalcones and flavonols
over the natural flavonol quercetin
,
Tatiana A. Dias a, Cecília L. Duarte b, Cristovao F. Lima b, M. Fernanda Proença a
**
,
*
,
Cristina Pereira-Wilson c
a Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
b CITAB e Centre for the Research and Technology of Agro-Environmental and Biological Sciences, Department of Biology, University of Minho, Campus of
Gualtar, 4710-057 Braga, Portugal
c CBMA e Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of chalcone and flavonol derivatives were synthesized in good yield by an eco-friendly approach.
A pharmacological evaluation was performed with the human colorectal carcinoma cell line HCT116 and
revealed that the anticancer activity of flavonols was higher when compared with that of the respective
chalcone precursors. The antiproliferative activity of halogenated derivatives increases as the substituent
in the 3- or 4-positon of the B-ring goes from F to Cl and to Br. In addition, halogens in position 3 enhance
anticancer activity in chalcones whereas for flavonol derivatives the best performance was registered for
the 4-substituted derivatives. Flow cytometry analysis showed that compounds 3p and 4o induced cell
cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were
corroborated by western blot and fluorescence microscopy analysis. In summary, halogenated chalcones
and flavonols were successfully prepared and presented high anticancer activity as shown by their cell
growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a
positive control.
Received 16 January 2013
Received in revised form
29 April 2013
Accepted 30 April 2013
Available online 14 May 2013
Keywords:
Chalcone
Flavonol
Colorectal carcinoma
HCT116 cells
Anticancer activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Extensive research on the biological activity of chalcones
included anti-inflammatory, antibacterial [8], antileishmanial,
Cancer is one of the leading causes of death in the contemporary
western society [1]. Colorectal cancer is the most frequently diag-
nosed type of cancer in Europe and the second most common cause
of cancer death [2]. The search for new molecules with the capacity
to inhibit tumour initiation as well as tumour progression is an
important contribution to overcome this problem [3,4]. Natural
products have been an effective source of bioactive molecules,
where the flavonoid scaffold is a central group. This moiety is
widely present in fruits, vegetables, tea and certain spices, and has
been associated with a range of health benefits due, in part, to its
radical quenching properties [5].
antimalarial, antifungal, antituberculosis, antioxidant, antiviral and
anticancer activity, a subject that has been reviewed over the years
[9]. The chemopreventive potential of some chalcone derivatives is
often emphasized [10,11] and includes a number of natural prod-
ucts incorporating this core structure (Fig. 1) [12e14].
Among the bioactive natural products of the flavonol family
(Fig. 1), quercetin, a major dietary flavonoid [15e17], has been
widely studied, considering the potent antioxidant activity of this
compound [18]. A recent report demonstrated that flavonols, in
particular those which are less methoxylated, present greater
antiproliferative activity than chalcones [19]. At least part of this
activity was attributed to the induction of an apoptotic response
through caspase activation, an effect which appears to be restricted
to cancer cells [20]. The anticancer potential of natural flavonols
[21e23] including colon cancer [24,25], is well known, mainly due
to the ability of these compounds to interact with several molecular
targets important for cancer progression and response to chemo-
therapy. This comprises the ability to decrease the levels of onco-
genes (like K-ras, H-ras and c-myc), to increase the levels of the p53
Chalcones can be envisaged as open-chain flavonoids, where the
two aromatic rings are linked by a conjugated three-carbon system,
with a carbonyl and an alkene group. They are often used as pre-
cursors in the synthesis of flavonoid derivatives [6,7].
* Corresponding author.
** Corresponding author.
E-mail address: fproenca@quimica.uminho.pt (M.F. Proença).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.064

T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
501
Fig. 1. Naturally occurring chalcone and flavonol derivatives recognized as bioactive molecules.
tumour suppressor and cell cycle control proteins such as p21 and
p27, as well as to inhibit tyrosine and serineethreonine kinases
(like MAPKs, PI3K and CDKs), important mediators in pathways of
cell survival and growth [26e31].
Inspired by the important biological activity of flavonoids, a
series of chalcones and flavonols were synthesized using an eco-
friendly approach and a panel of 33 compounds was tested in an
anticancer screening with HCT116 colon cancer cells.
towards CNE2 cell growth, unlike the halogenated analogues, but
the substituents in the bi-aryl system also played an important role
for anticancer activity.
In the present work and contrarily to these previous reports,
molecules with multiple methoxyl substituents in the B-ring (3iej
and 4gei) did not show an improved activity in relation to the
analogues with one methoxyl substituent (3eef and 4def) and
therefore they were not used in the following experiments. When
the potential anticancer activity of chalcones and flavonols with
the same substituents were compared (Table 1 vs Table 2, respec-
tively) the cyclic structure showed lower IC50 values, with the
exception of compounds 3f and 4f. In general, molecules with
halogen substituents in the B-ring showed improved activity
relative to the hydroxylated, methylated or methoxylated ana-
logues. Consequently, only halogenated chalcones 3 and flavonols 4
proceeded to further studies where the IC50 concentration was
calculated from the doseeresponse curves. The results from the
doseeresponse experiment of the different halogenated com-
2. Results and discussion
2.1. Chemistry
The 2-hydroxychalcones 3aer were synthesized from the re-
action of 2-hydroxyacetophenone 2 with the appropriate aromatic
aldehyde 1, using typical procedures for the aldol condensation.
The reaction was carried out in aqueous base (3 M NaOH), at room
temperature or at 40 ꢀC for chalcone 3i. The solid product usually
precipitated from the reaction media and was used without further
purification (Scheme 1). Chalcones 3aeh, 3j and 3ler were con-
verted to the corresponding flavonols 4aeo, using the Flynne
AlgareOyamada method for epoxidation [32,33] and subsequent
intramolecular cyclization of the open-chain structure. The reaction
was performed with hydrogen peroxide in aqueous base (4 M
NaOH) [34], an eco-friendly approach previously used for the
synthesis of analogous structures.
pounds with the IC50 values and the anticancer activity at 10 mM
were summarized in Table 3. These data confirm that flavonols
displayed a higher activity as compared with the corresponding
chalcones. Chalcones and flavonols presented different kinetics in
the doseeresponse curves, as can be seen in Fig. 2 for compounds
3l and 4j shown as an example. Contrarily to flavonols, chalcones
are non-active until higher concentrations are reached then, the
number of viable cells decreases abruptly with increasing con-
centration of the compound. Chalcones have higher slope values in
the doseeresponse curves when compared with the corresponding
cyclic product (Fig. 2).
2.2. Biological assays
The chalcones 3 and flavonols 4 were tested at 15
m
M in a first
This structureeactivity analysis also revealed an enhancement
in the inhibition of cell growth as the B-ring substituent goes from F
to Cl and to Br, either in the 3- or in the 4-position (Table 3). The size
of the halogen atom increases in that order, but also the polariz-
ability, a measure of the dynamic response of the electron cloud of
the atom to the effect of a nearby ion or dipole. Chalcones with a 40-
halogen substituent (3m, 3o and 3q) were the only exception as the
round of anticancer screening in order to select the most active
compounds. HCT116 cells were incubated for 48 h with each of the
test compounds and the final number of viable cells was estimated
by the MTT assay. The results for the chalcone (Table 1) and flavonol
derivatives (Table 2) indicated that most of the compounds
decreased cell viability below that of quercetin. This natural
flavonol was tested at 30
mM, twice the concentration of the syn-
cell viability remained basically unchanged at 10 mM.
thetic compounds and the number of viable cells decreased to 60%
as compared to control cells.
Likewise, the results evidenced that chalcones with the halogen
substituent at the 3-position of the aromatic B-ring are more active
than those substituted at the 4-position. The opposite happens for
brominated flavonols, where the substituent at the 4-position en-
hances the anticancer activity. This difference in activity related
with the halogen substitution pattern of the B-ring may be asso-
ciated with a more flexible structure of the chalcone, where a
distinct spatial arrangement of the molecule could lead to an
improved interaction with the active site of the receptor.
In a recent work by Wiese et al. [35] a selection of methoxylated
or chlorinated chalcones, in the B-ring, were tested as inhibitors of
breast cancer resistance protein (BCRP/ABCG2), using two different
cell lines e MCF-7 MX and MDCK BCRP. The authors clearly observed
that compounds with 3,4-dimethoxy substituents were the most
active. A different work by Bu et al. [36] reported the synthesis
of biaryl-based chalcones with hydroxyl/methoxyl groups or
halogen atoms in the B-ring. Compounds with the 4-methoxy and
3,4-dimethoxy substituents exhibited potent inhibitory activities
A comparison between the anticancer activity of flavonol 4o at
10 mM (cell viability of 33%) and quercetin at 30 mM (cell viability of

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T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
Scheme 1. Syntheses of chalcones 3aer and flavonols 4aeo.
60%), showed that this newly synthesized molecule is around 6
times more potent than the natural flavonol. The polyhydroxylated
structure of quercetin results in a higher hydrophilic character and
lower cell bioavailability and this may contribute to the lower
biological effect of quercetin compared with the halogenated
analogues.
Considering the superior anticancer potential of these haloge-
nated compounds and their ability to inhibit cell growth, the most
potent chalcone 3p and flavonol 4o were selected to evaluate their
capacity to induce cell cycle arrest and apoptosis. The halogenated
chalcone 3r, with low anticancer activity, was used for comparison.
The morphological changes in cells after treatment with these
compounds evidenced a different outcome between chalcones and
flavonols. Flavonols, whose cell viability stabilizes at 30e40% for
higher concentrations, seem to be more potent at inhibiting cell
growth (even at lower concentrations). In contrast, at higher con-
centrations, chalcones are better at inducing cell death (as shown
by the appearance of floating cells e see Fig. 3), resulting in lower
values of cell viability as compared with that induced by flavonols
(see Fig. 2).
Table 1
Potential anticancer activity of chalcones 3aec, 3eef, 3ier
against HCT116 cells by the MTT assay.
Table 2
Potential anticancer activity of flavonols 4aeo against HCT116
cells by the MTT assay.
Comp
Cell viability (%) at 15 m
Ma
Comp
Cell viability (%) at 15 m
Ma
3a
3b
3e
3i
3k
3l
3n
3p
3c
3f
91.7 ꢁ 4.4NS
85.9 ꢁ 3.5NS
89.6 ꢁ 2.8NS
91.1 ꢁ 3.4NS
84.7 ꢁ 3.5*
45.3 ꢁ 4.0***
37.5 ꢁ 2.9***
29.6 ꢁ 3.8***
89.1 ꢁ 3.9NS
89.1 ꢁ 1.1NS
83.7 ꢁ 3.0*
85.1 ꢁ 2.1*
74.9 ꢁ 4.2**
69.1 ꢁ 7.3*
68.4 ꢁ 6.8**
4a
4b
4e
4d
4h
4j
64.6 ꢁ 1.5***
38.7 ꢁ 0.7***
39.5 ꢁ 7.3***
89.1 ꢁ 4.7NS
78.1 ꢁ 8.8NS
45.9 ꢁ 4.1***
32.9 ꢁ 1.9***
29.2 ꢁ 3***
4l
4n
4c
4f
4g
4i
4k
4m
4o
59.3 ꢁ 6.3**
96.0 ꢁ 1.8NS
76.6 ꢁ 4.4*
3j
3r
3m
3o
3q
70.0 ꢁ 5.1**
49.2 ꢁ 1.6***
38.0 ꢁ 1.5***
29.2 ꢁ 2.3***
NS
not significant (P > 0.05), *P ꢂ 0.05, **P ꢂ 0.01, ***P ꢂ 0.001,
when compared with negative control (100 ꢁ 4.7; cells incubated
without compound, 0.5% DMSO only). Quercetin at 30 M was
NS
not significant (P > 0.05), *P ꢂ 0.05, **P ꢂ 0.001, when
m
compared with negative control (100 ꢁ 4.9; cells incubated
used as positive control; number of viable cells was 59.7 ꢁ 4.5***.
without compound, 0.5% DMSO only).
a
a
Values are mean ꢁ SEM of 4 independent experiments.
Values are mean ꢁ SEM of 4 independent experiments.

T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
503
Table 3 (continued )
Comp
Cell viability (%) at 10
m
Ma
IC50
4.6
(mM)
Table 3
Evaluation of the anticancer activity of halogenated chalcones 3 and flavonols 4
against the colorectal carcinoma cell line HCT116 by the MTT assay.
4o
32.8 ꢁ 1.7**
Comp
Cell viability (%) at 10
m
Ma
IC50 (mM)
Chalcones
NS not significant (P > 0.05), *P ꢂ 0.05, **P ꢂ 0.01, ***P ꢂ 0.001, when compared with
negative control (100 ꢁ 4.7; cells incubated without compound, 0.5% DMSO only).
3l
90.2 ꢁ 6.2NS
14.5
Quercetin at 30
mM was used as positive control; number of viable cells was
59.7 ꢁ 4.5***.
a
Values are mean ꢁ SEM of 4 independent experiments.
3m
85.2 ꢁ 4.5NS
20.5
Cell cycle analysis by flow cytometry for both compounds 3p
and 4o (Fig. 4) showed that they induced cell cycle arrest at S and
G2/M phases. As expected, compound 3r displayed a much lower
activity. Contrarily to chalcone 3p, flavonol 4o already attained
maximal inhibition of cell cycle at its IC50 concentration, which may
indicate a higher specificity of the cyclic derivative to induce cell
cycle arrest. Analysis by western blot confirmed the higher ability
of flavonol 4o to inhibit cell cycle, in view of a higher induction of
the levels of the tumour suppressor p53 and the cell cycle inhibitor
p27 (Fig. 5). Cell cycle analysis also revealed that both 3p and 4o
induced an increase in cells at sub-G1 phase (Fig. 4), typical of
apoptosis. This increase was higher for 3p which agrees with the
morphological observations and emphasizes that chalcones 3 have
a higher potential to induce cell death than flavonols 4 at concen-
trations higher than their IC50. To further demonstrate the ability of
these compounds to induce cell death by apoptosis, the presence of
nuclear condensation was assessed by fluorescence microscopy.
This assay showed an increase, in a concentration-dependent
manner, for both 3p and 4o (Fig. 6). Western blot evidenced that
the chalcone and the flavonol also induced the cleavage of
poly(ADP-ribose) polymerase 1 (PARP-1) as well as a decrease of
total PARP-1 levels (Fig. 5), a marker of induction of apoptosis.
Considering the higher levels of these parameters in the tested
chalcone, these results corroborate the higher ability of compound
3p to induce apoptosis as compared with 4o.
3n
3o
75.8 ꢁ 7.1*
13.2
20.9
84.8 ꢁ 4.8NS
3p
48.4 ꢁ 6.7**
10.9
3q
85.5 ꢁ 6.4NS
60.9 ꢁ 3.8**
19.1
13.0
Flavonols
4j
3. Conclusions
A selection of chalcone derivatives was prepared, oxidized by
H2O2 in aqueous base to generate the corresponding flavonols
which were isolated in good yield. Both chalcones and their cyclic
4k
61.1 ꢁ 3.8**
14.4
4l
43.8 ꢁ 4.4**
40.8 ꢁ 0.6**
8.1
6.8
4m
4n
37.6 ꢁ 2.7**
7.5
Fig. 2. Doseeresponse effects of compounds 3l and 4j in the number of viable cells
after 48
h of incubation. The IC50 concentration of tested compounds is also
represented.

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T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
Fig. 3. Effect of compounds 3r, 3p and 4o on cell morphology after 48 h of incubation with HCT116 cells. Bar indicates 100 mm.
derivatives were evaluated for their anticancer activity in human
colorectal carcinoma HCT116 cells.
400 MHz and 13C: 100 MHz) and deuterated DMSO was used as
solvent. The chemical shifts were expressed in parts per million
(ppm scale) and the coupling constants, J, were reported in Hertz
(Hz). The peak patterns were indicated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet and br, broad. The reactions were
monitored by Thin Layer Chromatography (TLC) using silica gel 60
F254 plates (Merck). The melting points were determined on a
Stuart SMP3 melting point apparatus. Elemental analyses were
performed on a LECO CHNS-932 instrument.
A first roundofanticancerscreeningshowed enhancedactivityfor
chalcones 3 and flavonols 4 with halogen substituents in the B-ring
when compared with methoxyl/methyl group analogues. In general,
the effects observed are related to those identified for the natural
flavonoid quercetin, with the advantage of enhanced activity at a
much lower concentration range, for the halogenated compounds.
The halogenated compounds were selected for further studies
and displayed an enhanced antiproliferative activity as the sub-
stituent of the B-ring changed from F to Cl and to Br. The 3-
substituted derivatives in chalcones and the 4-substituted prod-
ucts in flavonols exhibited higher anticancer activity. Flow cytom-
etry analysis of the most active chalcone 3p and flavonol 4o
revealed an induction of cell cycle arrest at S and G2/M phases as
well as an increase of sub-G1 cells, typical of apoptosis. These facts
were confirmed by western blot and nuclear condensation and
indicated that although flavonol 4o demonstrated a higher cyto-
static (growth inhibitory) activity, chalcone 3p revealed a superior
cytocidal (death inducer) capacity by apoptosis.
4.1.1. General procedure for the synthesis of (2E)-1-(2-hydroxy-
phenyl)-3-phenylprop-2-en-1-ones 3
An aqueous solution of NaOH (3 M, 2e10 mL) was added to a
solution of 2-hydroxyacetophenone 2 (1.22e4.88 mmol) and aro-
matic aldehyde 1 (1.2 equiv.), in EtOH (1e12 mL). The reaction was
stirred at room temperature or at 40 ꢀC, for compound 3i, until the
starting material was totally consumed, as evidenced by TLC. The
reaction mixture was cooled in an ice-water bath and acidified to
pH 2 with concentrated HCl (37%). The solid formed was filtered
and washed with distilled water.
Compound 4o proved to be around six times more potent when
compared with quercetin. Therefore, this compound family de-
serves to be explored in further studies to investigate its biological
potential and to understand in more detail their anticancer mo-
lecular mechanisms of action.
4.1.1.1. (2E)-1-(2-Hydroxyphenyl)-3-phenylprop-2-en-1-one
Isolated as a yellow solid in 77.4% yield. Mp 87e88 ꢀC. 1H NMR
(300 MHz, DMSO-d6):
(3a).
d
7.00 (dd, J ¼ 8.4, 1.5 Hz,1H), 7.02 (td, J ¼ 8.4,
1.2 Hz, 1H), 7.46e7.48 (m, 3H), 7.56 (td, J ¼ 8.4, 1.5 Hz, 1H), 7.83 (d,
J ¼ 15.3 Hz, 1H), 7.89e7.92 (m, 2H), 8.03 (d, J ¼ 15.3 Hz, 1H), 8.24
(dd, J ¼ 8.4, 1.5 Hz, 1H), 12.49 (br s, 1H) ppm; 13C NMR (75 MHz,
4. Experimental section
DMSO-d6):
d 117.7, 119.2, 120.8, 121.8, 129.0 (2C), 129.2 (2C), 130.9,
4.1. Chemistry
131.0, 134.4, 136.3, 144.7, 161.8, 193.6 ppm. Anal. Calcd. for C15H12O2
(224.08): C, 80.34; H, 5.39, found: C, 80.48; H, 5.52.
Solvents and chemicals were of analytical grade and were pur-
chased from commercial sources and used without further
purification.
4.1.1.2. (2E)-1-(2-Hydroxyphenyl)-3-(3-methylphenyl)prop-2-en-1-
one (3b). Isolated as a yellow solid in 55.2% yield. Mp 62e64 ꢀC. 1H
All compounds were fully characterized by spectroscopic data
and compared with the data available in the literature. The NMR
spectra were recorded at room temperature, on a Varian Unity Plus
(1H: 300 MHz and 13C: 75 MHz) or on a Bruker Avance 3400 (1H:
NMR (300 MHz, DMSO-d6):
d
6.99 (t, J ¼ 8.4 Hz, 1H), 7.01 (d,
J ¼ 8.4 Hz, 1H), 7.28 (d, J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 7.8 Hz, 1H), 7.56 (t,
J ¼ 8.1 Hz, 1H), 7.67 (d, J ¼ 7.5 Hz, 1H), 7.75 (s, 1H), 7.79 (d,
J ¼ 15.9 Hz, 1H), 8.10 (d, J ¼ 15.9 Hz, 1H), 8.23 (d, J ¼ 8.1 Hz, 1H),

T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
505
Fig. 4. Effect of compounds 3p, 3r and 4o for 48 h in cell cycle progression of HCT116 cells, assessed by flow cytometry. (A) Distribution of single cells through the phases of cell
cycle of a representative experiment. (B) Percentage of cells in the different phases of cell cycle of 3 independent experiments. Values are mean ꢁ S.E.M. *P ꢂ 0.05, **P ꢂ 0.01,
***P ꢂ 0.001, when compared with control (cells incubated without compound, 0.5% DMSO only).
12.50 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
119.0, 120.9, 121.8, 123.7, 126.6, 128.8, 129.4, 130.9, 131.7, 136.2,
138.2, 144.8, 161.9, 193.5 ppm. Anal. Calcd. for C16H14O2.0.1H2O
(239.90): C, 80.04; H, 5.96, found: C, 79.95; H, 5.99.
d
20.8, 117.8,
(td, J ¼ 8.4, 1.5 Hz, 1H), 7.80 (d, J ¼ 7.8 Hz, 2H), 7.81 (d, J ¼ 15.6 Hz,
1H), 7.99 (d, J ¼ 15.6 Hz, 1H), 8.24 (dd, J ¼ 8.4,1.5 Hz, 1H), 12.57 (br s,
1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 21.1, 117.7, 118.0, 119.1,
120.6, 120.7, 129.2, 129.6, 130.8, 136.3, 141.2, 144.9, 161.9, 193.6 ppm.
4.1.1.3. (2E)-1-(2-Hydroxyphenyl)-3-(4-methylphenyl)prop-2-en-1-
4.1.1.4. (2E)-1-(2-Hydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-
one (3c). Isolated as a yellow solid in 86.1% (lit. [37] 74%) yield. Mp
1-one (3d). Isolated as a yellow solid in 79.5% (lit. [38] 46%) yield.
95e97 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
2.35 (s, 3H), 6.98 (t,
Mp 110e112 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
3.90 (s, 3H), 6.99
J ¼ 8.4 Hz, 1H), 6.99 (d, J ¼ 8.4 Hz, 1H), 7.28 (d, J ¼ 7.8 Hz, 2H), 7.54
(dd, J ¼ 8.4, 1.2 Hz, 1H), 7.04 (t, J ¼ 8.4 Hz, 1H), 7.05 (t, J ¼ 8.1 Hz, 1H),

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T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
1H NMR (300 MHz, DMSO-d6):
d
7.00 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.01 (t,
J ¼ 8.7 Hz,1H), 7.06 (d, J ¼ 0.9 Hz,1H), 7.38 (t, J ¼ 8.1 Hz,1H), 7.48 (d,
J ¼ 8.1 Hz, 1H), 7.50 (d, J ¼ 8.1 Hz, 1H), 7.57 (td, J ¼ 8.7, 1.8 Hz, 1H),
7.81 (d, J ¼ 15.6 Hz, 1H), 8.04 (d, J ¼ 15.6 Hz, 1H), 8.27 (dd, J ¼ 8.7,
1.8 Hz,1H),12.50 (s,1H); 13C NMR (75 MHz, DMSO-d6):
d 55.3,113.6,
117.7, 119.2, 120.7, 122.0 (2C), 130.0, 131.0, 135.8, 136.4, 144.8, 159.7,
161.9, 193.7 ppm. Anal. Calcd. for C16H14O3$0.1H2O (255.90): C,
75.04; H, 5.59, found: C, 75.20; H, 5.67.
4.1.1.6. (2E)-1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-
1-one (3f). Isolated as a yellow solid in 57.6% (lit. [37] 65%) yield.
Mp 94e96 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.83 (s, 3H), 6.99 (t,
Fig. 5. Effect of compounds 3p, 3r and 4o for 48 h on the expression of p53, p27 and
PARP in HCT116 cells, as assessed by western blot. Shown are representative images of
two independent experiments. b-actin was used as loading control.
J ¼ 8.7 Hz, 1H), 7.00 (d, J ¼ 8.7 Hz, 1H), 7.03 (d, J ¼ 9.0 Hz, 2H), 7.55
(td, J ¼ 8.7, 1.8 Hz, 1H), 7.89 (dd, J ¼ 9.0, 2.4 Hz, 2H), 7.92 (d,
J ¼ 15.6 Hz,1H), 7.82 (d, J ¼ 15.6 Hz,1H), 8.26 (dd, J ¼ 8.7, 1.8 Hz,1H),
12.71 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 55.4, 114.5
7.12 (d, J ¼ 8.1 Hz, 1H), 7.47 (dt, J ¼ 8.1, 1.5 Hz, 1H), 7.55 (td, J ¼ 8.4,
1.2 Hz, 1H), 7.98 (d, J ¼ 15.6 Hz, 1H), 7.99 (dd, J ¼ 8.1, 1.5 Hz, 1H), 8.15
(d, J ¼ 15.6 Hz,1H), 8.19 (dd, J ¼ 8.4,1.2 Hz,1H),12.50 (br s,1H) ppm;
(2C), 117.7, 118.9 (2C), 119.1, 120.6, 127.1, 130.8, 131.3, 136.2, 145.1,
161.7, 162.0, 193.6 ppm.
13C NMR (75 MHz, DMSO-d6):
d 55.8, 111.9, 117.8, 119.2, 120.8, 120.8,
4.1.1.7. (2E)-3-(2,5-Dimethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-
121.4, 122.7, 128.8, 130.7, 132.8, 136.2, 139.2, 158.4, 161.8, 193.7 ppm.
en-1-one (3g). Isolated as a yellow solid 74.7% (lit. [38] 38%) yield.
Mp 101e103 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.80 (s, 3H), 3.84
4.1.1.5. (2E)-1-(2-Hydroxyphenyl)-3-(3-methoxyphenyl)prop-2-en-
1-one (3e). Isolated as a yellow solid in 91.7% yield. Mp 93e96 ꢀC.
(s, 3H), 6.98 (d, J ¼ 7.8 Hz, 1H), 7.00 (t, J ¼ 7.8 Hz, 1H), 7.04 (d,
J ¼ 7.5 Hz, 1H), 7.06 (d, J ¼ 1.5 Hz, 1H), 7.57 (dt, J ¼ 7.8, 1.5 Hz, 1H),
7.58 (dd, J ¼ 7.5, 1.5 Hz, 1H), 8.00 (d, J ¼ 15.6 Hz, 1H), 8.14 (d,
J ¼ 15.6 Hz, 1H), 8.25 (dd, J ¼ 7.8, 1.5 Hz, 1H), 12.58 (s, 1H) ppm; 13
C
NMR (75 MHz, DMSO-d6): d 55.6, 55.8, 117.7, 112.7, 113.1, 118.8, 119.1,
120.7, 121.5, 123.2, 130.8, 136.3, 139.1, 152.9, 153.3, 163.0, 194.7 ppm.
4.1.1.8. (2E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-
en-1-one (3h). Isolated as a yellow solid 46.7% (lit. [38] 50%) yield.
Mp 112e114 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.82 (s, 3H), 3.86
(s, 3H), 6.99 (dd, J ¼ 8.1, 1.5 Hz, 1H), 7.02 (t, J ¼ 8.1 Hz, 1H), 7.03 (d,
J ¼ 8.7 Hz, 1H), 7.42 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.55 (dd, J ¼ 8.1, 1.5 Hz,
1H), 7.58 (d, J ¼ 1.8 Hz, 1H), 7.81 (d, J ¼ 15.6 Hz, 1H), 7.93 (d,
J ¼ 15.6 Hz, 1H), 8.29 (dd, J ¼ 8.1, 1.5 Hz, 1H), 12.79 (s, 1H) ppm; 13
C
NMR (75 MHz, DMSO-d6):
d 55.6, 55.8, 110.9, 111.5, 117.8, 118.8,
119.0, 120.5, 124.7, 127.2, 130.8, 136.2, 145.7, 149.1, 151.7, 162.2,
193.7 ppm.
4.1.1.9. (2E)-1-(2-Hydroxyphenyl)-3-(2,4,6-trimethoxyphenyl)prop-
2-en-1-one (3i). Isolated as a yellow solid in 58.0% yield. Mp 132e
134 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.86 (s, 3H), 3.93 (s, 6H),
6.32 (s, 2H), 6.96 (d, J ¼ 8.7 Hz, 1H), 6.99 (t, J ¼ 8.7 Hz, 1H), 7.52 (td,
J ¼ 8.7, 0.9 Hz, 1H), 7.94 (d, J ¼ 8.7 Hz, 1H), 7.97 (d, J ¼ 15.3 Hz, 1H),
8.24 (d, J ¼ 15.3 Hz, 1H), 12.89 (s, 1H) ppm; 13C NMR (75 MHz,
DMSO-d6): d 55.6, 56.2 (2C), 91.1 (2C), 105.0, 117.8, 118.8, 119.1, 120.7,
129.8, 135.8, 136.4, 161.8 (2C), 162.1, 164.0, 194.3 ppm. Anal. Calcd.
for C18H18O5.0.1H2O (315.92): C, 69.39; H, 5.80, found: C, 69.17; H,
5.94.
4.1.1.10. (2E)-1-(2-Hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (3j). Isolated as a yellow solid in 67.9% (lit. [37] 55%)
yield. Mp 156e158 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.71 (s, 3H),
3.86 (s, 6H), 6.98e7.03 (m, 2H), 7.26 (s, 2H), 7.56 (td, J ¼ 8.4, 1.5 Hz,
1H), 7.78 (d, J ¼ 15.6 Hz, 1H), 7.98 (d, J ¼ 15.6 Hz, 1H), 8.29 (dd,
J ¼ 8.4, 1.5 Hz, 1H), 12.63 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-
d6):
d 56.2 (2C), 60.2, 106.9 (2C), 117.7, 119.1, 120.7, 120.9, 130.0,
130.9, 136.3, 140.1, 145.4, 153.1, 161.9, 193.7 ppm.
4.1.1.11. (2E)-3-(2-Hydroxy-3-methoxyphenyl)-1-(2-hydroxyphenyl)
Fig. 6. Effect of compounds 3p, 3r and 4o for 48 h on apoptosis in HCT116 cells, as
assessed by nuclear condensation assay. (A) Results given as mean ꢁ SEM of at least 3
independent experiments. *P ꢂ 0.05, **P ꢂ 0.01, ***P ꢂ 0.001, when compared to
control. (B) Representative images where nuclear condensation is shown with an ar-
prop-2-en-1-one (3k). Isolated as a yellow solid in 3.5% yield. Mp
200 ꢀC (dec.). 1H NMR (300 MHz, DMSO-d6):
d 3.82 (s, 3H), 6.98 (t,
J ¼ 8.1 Hz, 1H), 7.05 (dd, J ¼ 8.1, 1.5 Hz, 1H), 7.49 (d, J ¼ 8.1 Hz, 1H),
row. Bar indicates 50 mm.
7.53 (td, J ¼ 8.1, 1.5 Hz, 1H), 7.91 (d, J ¼ 15.6 Hz, 1H), 8.14 (dd, J ¼ 8.1,

T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
507
1.5 Hz, 1H), 8.16 (d, J ¼ 15.6 Hz, 1H), 9.54 (br s, 1H), 12.52 (br s, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6):
56.00, 113.99, 117.71, 119.08,
2H), 8.06 (d, J ¼ 15.6 Hz, 1H), 8.24 (dd, J ¼ 8.4, 1.5 Hz, 1H), 12.44 (s,
1H) ppm; 13C NMR (75 MHz, DMSO-d6):
117.7, 119.2, 120.7, 122.6,
d
d
119.15, 119.93, 120.83, 121.09, 121.45, 130.57, 135.96, 139.90, 146.89,
148.06, 161.22, 193.68 ppm. Anal. Calcd. for C16H14O4.0.1H2O
(271.89): C, 70.63; H, 5.26, found: C, 70.44; H, 5.20.
124.4, 130.9, 131.0 (2C), 131.9 (2C), 133.7, 136.4, 143.4, 161.8,
193.5 ppm.
4.1.1.18. (2E)-1-(2-Hydroxyphenyl)-3-[4-(trifluoromethyl)phenyl]
prop-2-en-1-one (3r). Isolated as a yellow solid in 80.5% (lit. [39]
4.1.1.12. (2E)-3-(3-Fluorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (3l). Isolated as a yellow solid in 99.2% yield. Mp 109e110 ꢀC. 1H
46%) yield. Mp 100e102 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 7.00
NMR (300 MHz, DMSO-d6):
d
6.98 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.01 (t,
(d, J ¼ 8.4 Hz,1H), 7.02 (t, J ¼ 8.4 Hz, 1H), 7.57 (td, J ¼ 8.4, 1.5 Hz, 1H),
7.82 (d, J ¼ 8.7 Hz, 2H), 7.86 (d, J ¼ 16.0 Hz, 1H), 8.11 (d, J ¼ 8.7 Hz,
2H), 8.15 (d, J ¼ 16.0 Hz, 1H), 8.23 (dd, J ¼ 8.4, 1.5 Hz, 1H), 12.29 (s,
J ¼ 8.1 Hz, 1H), 7.29 (td, J ¼ 6.0, 1.8 Hz, 1H), 7.49 (dt, J ¼ 8.4, 1.8 Hz,
1H), 7.57 (dt, J ¼ 8.1, 1.8 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 1H), 7.81 (d,
J ¼ 15.6 Hz,1H), 7.86 (dd, J ¼ 8.4,1.8 Hz,1H), 8.03 (d, J ¼ 15.6 Hz,1H),
8.26 (dd, J ¼ 8.1, 1.8 Hz, 1H), 12.44 (br s, 1H) ppm; 13C NMR (75 MHz,
1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 117.7, 119.3, 120.9, 124.8,
125.7 (q, J ¼ 3.8 Hz, 2C), 129.6 (2C), 130.2 (q, J ¼ 31.7 Hz), 131.0,
DMSO-d6):
d
114.9 (d, J ¼ 21.8 Hz), 117.6 (d, J ¼ 21.2 Hz), 117.7, 119.2,
136.5, 138.5 (q, J ¼ 1.1 Hz), 142.4, 161.7, 193.4 ppm.1
120.7, 123.3, 125.8 (d, J ¼ 2.2 Hz), 130.9 (d, J ¼ 8.2 Hz), 131.0, 136.5,
137.0 (d, J ¼ 8.0 Hz), 143.2 (d, J ¼ 3.8 Hz), 161.9, 162.5 (d,
J ¼ 242.0 Hz),193.6 ppm. Anal. Calcd. for C15H11FO2.H2O (260.08): C,
69.22; H, 5.03, found: C, 69.06; H, 5.15.
4.1.2. General procedure for the synthesis of 3-hydroxy-2-phenyl-
4H-chromen-4-ones 4
An aqueous solution of H2O2 (30%) (151e430
mL) and 4 M NaOH
(120e300 L) was added to a suspension of chalcone 3 (0.44e
m
4.1.1.13. (2E)-3-(4-Fluorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
1.10 mmol) in MeOH (0.2e2 mL). The resulting mixture was stirred
at room temperature (compounds 4aed, 4fei) or at 55 ꢀC (com-
pounds 4e, 4jek), until the starting material was totally consumed
as evidenced by TLC. The reaction mixture was cooled in an ice bath
and distilled water (2e4 mL) was added. Concentrated HCl (37%)
was added until pH 2 and the precipitate was filtered and washed
with distilled water.
one (3m). Isolated as a yellow solid in 91.0% (lit. [37] 44%) yield. Mp
120e122 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d
6.99 (dd, J ¼ 8.4,
1.6 Hz, 1H), 7.00 (t, J ¼ 8.4 Hz, 1H), 7.30 (dt, J ¼ 8.8, 2.0 Hz, 2H), 7.57
(td, J ¼ 8.4, 1.6 Hz, 1H), 7.83 (d, J ¼ 15.6 Hz, 1H), 7.99 (dt, J ¼ 8.8,
2.0 Hz, 2H), 8.00 (d, J ¼ 15.6 Hz, 1H), 8.24 (dd, J ¼ 8.4, 1.6 Hz, 1H),
12.50 (br s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 116.0 (d,
J ¼ 22.0 Hz, 2C), 117.7, 118.5, 120.7, 121.6 (d, J ¼ 2.0 Hz), 130.9, 131.1
(d, J ¼ 3.0 Hz), 131.6 (d, J ¼ 9.0 Hz, 2C), 136.3, 143.5, 161.8, 163.6 (d,
J ¼ 248.0 Hz), 193.5 ppm.
4.1.2.1. 3-Hydroxy-2-phenyl-4H-chromen-4-one (4a). Isolated as a
white solid in 53.2% yield. Mp 169e170 ꢀC. 1H NMR (300 MHz,
DMSO-d6):
d
7.46 (t, J ¼ 8.7 Hz, 1H), 7.51 (t, J ¼ 7.5 Hz, 1H), 7.56 (t,
4.1.1.14. (2E)-3-(3-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (3n). Isolated as a yellow solid in 84.6% yield. Mp 105e107 ꢀC.
J ¼ 7.5 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 1H), 7.79 (td, J ¼ 8.7, 1.2 Hz, 1H),
8.11 (d, J ¼ 8.1 Hz, 1H), 9.61 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-
1H NMR (300 MHz, DMSO-d6):
d
7.00 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.01 (t,
d6): d 118.4, 121.3, 124.6, 124.8, 127.7 (2C), 128.5 (2C), 129.9, 131.3,
J ¼ 8.4 Hz, 1H), 7.58 (td, J ¼ 8.4, 1.8 Hz, 1H), 7.49 (t, J ¼ 7.2 Hz, 1H),
7.50 (dd, J ¼ 7.2, 2.1 Hz,1H), 7.79 (d, J ¼ 15.6 Hz,1H), 7.84 (dd, J ¼ 7.2,
2.1 Hz, 1H), 8.09 (d, J ¼ 2.1 Hz, 1H), 8.11 (d, J ¼ 15.6 Hz, 1H), 8.24 (dd,
J ¼ 8.4, 1.8 Hz, 1H), 12.44 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
133.7, 139.1, 145.2, 154.6, 173.0 ppm. Anal. Calcd. for C15H10O3
(238.06): C, 75.62; H, 4.23, found: C, 75.54; H, 4.41.
4.1.2.2. 3-Hydroxy-2-(3-methylphenyl)-4H-chromen-4-one
Isolated as a beige solid in 37.6% yield. Mp 143e145 ꢀC. 1H NMR
(300 MHz, DMSO-d6):
(4b).
d
111.8, 119.2, 120.7, 123.4, 128.2 (2C), 130.5, 130.8, 131.1, 133.9, 136.6,
136.7, 143.0, 161.9, 193.6 ppm. Anal. Calcd. for C15H11ClO2.0.1H2O
(259.85): C, 69.16; H, 4.33, found: C, 69.24; H, 4.52.
d
2.40 (s, 3H), 7.31 (d, J ¼ 7.5 Hz, 1H), 7.43 (t,
J ¼ 7.5 Hz, 1H), 7.47 (td, J ¼ 8.7, 1.5 Hz, 1H), 7.75 (t, J ¼ 8.7 Hz, 1H),
7.81 (dd, J ¼ 8.7, 1.5 Hz, 1H), 8.01 (d, J ¼ 7.5 Hz, 1H), 8.02 (s, 1H), 8.11
(dd, J ¼ 8.7, 1.5 Hz, 1H), 9.56 (s); 13C NMR (75 MHz, DMSO-d6):
4.1.1.15. (2E)-3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (3o). Isolated as a yellow solid in 75.3% (lit. [37] 85%) yield. Mp
d 21.2, 118.4, 121.3, 124.6, 124.8, 125.0, 128.0, 128.4, 130.6, 131.3,
143e145 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
6.99 (dd, J ¼ 8.1,1.8 Hz,
133.7, 137.7, 139.0, 145.3, 154.6, 173.0 ppm. Anal. Calcd. for C16H12O3
(252.08): C, 76.18; H, 4.79, found: C, 76.34; H, 4.56.
1H), 7.01 (t, J ¼ 8.1 Hz, 1H), 7.53 (d, J ¼ 6.6 Hz, 2H), 7.57 (dt, J ¼ 8.1,
1.8 Hz,1H), 7.81 (d, J ¼ 15.6 Hz,1H), 7.94 (dd, J ¼ 6.6,1.8 Hz, 2H), 8.05
(d, J ¼ 15.6 Hz, 1H), 8.24 (dd, J ¼ 8.1, 1.8 Hz, 1H), 12.44 (s, 1H) ppm;
4.1.2.3. 3-Hydroxy-2-(4-methylphenyl)-4H-chromen-4-one
Isolated as a white solid in 60.9% (lit. [34] 65%) yield. Mp 192e
194 ꢀC. 1H NMR (300 MHz, DMSO-d6):
2.37 (s, 3H), 7.36 (d,
(4c).
13C NMR (75 MHz, DMSO-d6):
d 117.7, 119.2, 120.8, 122.6, 129.0 (2C),
130.8 (2C), 130.9, 133.3, 135.5, 136.4, 143.3, 161.8, 193.5 ppm.
d
J ¼ 8.1 Hz, 2H), 7.47 (td, J ¼ 8.1, 1.2 Hz, 1H), 7.77 (dd, J ¼ 8.1, 1.2 Hz,
1H), 7.80 (dd, J ¼ 8.1, 1.2 Hz, 1H), 8.09 (dd, J ¼ 8.1, 1.2 Hz, 1H), 8.12 (d,
J ¼ 8.1 Hz, 2H) 9.53 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
4.1.1.16. (2E)-3-(3-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (3p). Isolated as a yellow solid in 60.8% (lit. [37] 66%) yield. Mp
108e110 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
6.99 (dd, J ¼ 6.9,
d 21.0, 118.4, 121.3, 124.5, 124.8, 127.6 (2C), 128.5, 129.1 (2C), 133.6,
0.9 Hz, 1H), 7.01 (t, J ¼ 6.9 Hz, 1H), 7.42 (t, J ¼ 7.8 Hz, 1H), 7.57 (dt,
J ¼ 6.9, 0.9 Hz, 1H), 7.64 (dd, J ¼ 7.8, 0.9 Hz, 1H), 7.85 (d, J ¼ 15.6 Hz,
1H), 7.87 (d, J ¼ 7.8 Hz,1H), 8.10 (d, J ¼ 15.6 Hz,1H), 8.23 (s, 1H), 8.29
(d, J ¼ 6.9 Hz, 1H), 12.45 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6)
138.8, 139.8, 145.4, 154.5, 172.8 ppm.
4.1.2.4. 3-Hydroxy-2-(2-methoxyphenyl)-4H-chromen-4-one (4d).
Isolated as a pale yellow solid in 74.8% (lit. [38] 74%) yield. Mp 211e
d
111.7, 119.2, 120.7, 122.4, 123.3, 128.6, 131.0 (2C), 131.1, 133.4, 136.6,
213 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 3.79 (s, 3H), 7.08 (t,
136.9, 143.0, 161.9, 193.5 ppm.
J ¼ 8.1 Hz, 1H), 7.18 (d, J ¼ 8.1 Hz, 1H), 7.46 (dd, J ¼ 8.4, 1.5 Hz, 1H),
7.48 (t, J ¼ 8.1 Hz,1H), 7.52 (dd, J ¼ 8.4,1.5 Hz,1H), 7.60 (d, J ¼ 8.4 Hz,
1H), 7.75 (dt, J ¼ 8.4, 1.5 Hz, 1H), 8.13 (dd, J ¼ 8.4, 1.5 Hz, 1H), 8.93 (s,
4.1.1.17. (2E)-3-(4-Bromophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (3q). Isolated as a yellow solid in 80.5% (lit. [37] 58%) yield. Mp
150e152 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
6.98 (d, J ¼ 8.4 Hz,1H),
1
7.00 (td, J ¼ 8.4, 1.5 Hz, 1H), 7.56 (td, J ¼ 8.4, 1.5 Hz, 1H), 7.67 (dd,
The chemical shift of the carbon atom attached to the fluorine atoms was not
J ¼ 6.9, 1.8 Hz, 2H), 7.79 (d, J ¼ 15.6 Hz, 1H), 7.87 (dd, J ¼ 6.9, 1.8 Hz,
visible in the spectrum.

508
T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
1H) ppm; 13C NMR (75 MHz, DMSO-d6):
120.2, 122.00, 124.5, 124.9, 131.1, 131.8, 133.9, 139.1, 147.1, 155.0,
157.1, 172.7 ppm.
d
55.8, 112.0, 118.4, 119.9,
1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.78 (dt, J ¼ 8.4, 1.2 Hz, 1H), 8.10 (dd,
J ¼ 8.4, 1.2 Hz, 1H), 8.27 (dt, J ¼ 6.6, 2.1 Hz, 2H), 9.69 (br s, 1H) ppm;
13C NMR (75 MHz, DMSO-d6):
d
118.4,115.6 (d, J ¼ 21.8 Hz, 2C),121.3,
124.6,124.8,127.8 (d, J ¼ 3.2 Hz),130.2 (d, J ¼ 8.6 Hz, 2C),133.8,138.9,
4.1.2.5. 3-Hydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one (4e).
Isolated as a beige solid in 39.2% yield. Mp 133e135 ꢀC. 1H NMR
144.4, 154.5, 162.6 (d, J ¼ 247.4 Hz), 173.0 ppm. Anal. Calcd. for
C15H9FO3.H2O (274.06): C, 65.89; H, 4.04, found: C, 66.03; H, 4.12.
(300 MHz, DMSO-d6):
d
7.09 (dd, J ¼ 8.4, 2.7 Hz,1H), 7.46 (td, J ¼ 8.4,
1.2 Hz, 1H), 7.49 (t, J ¼ 8.1 Hz, 1H), 7.75e7.82 (m, 4H), 8.12 (dd,
4.1.2.12. 2-(3-Chlorophenyl)-3-hydroxy-4H-chromen-4-one
Isolated as a white solid in 42.1% (lit. [34] 29%) yield. Mp 151e
153 ꢀC. 1H NMR (300 MHz, DMSO-d6):
(4l).
J ¼ 8.4, 1.2 Hz, 1H), 9.65 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-
d6):
d
113.4,115.2, 118.5, 120.0,121.2, 124.6,124.8, 129.6,132.5,133.8,
d
7.46 (td, J ¼ 7.5, 2.1 Hz, 1H),
139.2, 144.8, 154.5, 159.2, 173.0 ppm. Anal. Calcd. for C16H12O4
(284.10): C, 71.82; H, 5.67, found: C, 71.94; H, 5.49.
7.55 (d, J ¼ 6.9 Hz, 1H), 7.58 (t, J ¼ 6.9 Hz, 1H), 7.93 (s, 2H), 8.10 (d,
J ¼ 7.5 Hz, 1H), 8.18 (d, J ¼ 6.9 Hz, 1H), 8.26 (s, 1H), 9.92 (s, 1H) ppm;
13C NMR (100 MHz, DMSO-d6):
d 118.5, 121.2, 124.7, 124.8, 126.1,
4.1.2.6. 3-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one
Isolated as an off-white solid in 74.6% yield. Mp 234e236 ꢀC. 1H
NMR (300 MHz, DMSO-d6):
(4f).
127.1, 129.5, 130.5, 133.3 (2C), 133.9, 139.7, 143.4, 154.6, 173.1 ppm.
d
7.12 (dd, J ¼ 7.0, 2.0 Hz, 2H), 7.45 (td,
4.1.2.13. 2-(4-Chlorophenyl)-3-hydroxy-4H-chromen-4-one
Isolated as a white solid in 73.5% (lit. [34] 65%) yield. Mp 200e
202 ꢀC. 1H NMR (300 MHz, DMSO-d6):
(4m).
J ¼ 8.0, 1.6 Hz, 1H), 7.75 (dd, J ¼ 6.4, 1.6 Hz, 1H), 7.77 (dd, J ¼ 6.4,
1.6 Hz, 1H), 8.09 (dd, J ¼ 8.0, 1.6 Hz, 1H), 8.20 (d, J ¼ 7.0 Hz, 2H), 9.45
d
7.46 (td, J ¼ 8.1, 1.2 Hz, 1H),
(br s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
55.4, 114.0, 118.3,
7.63 (d, J ¼ 9.0 Hz, 2H), 7.75 (d, J ¼ 8.1 Hz,1H), 7.82 (dt, J ¼ 8.1,1.2 Hz,
1H), 8.10 (d, J ¼ 8.1, 1.2 Hz, 1H), 8.27 (d, J ¼ 9.0 Hz, 2H), 9.85 (s, 1H)
121.3, 123.6, 124.5, 124.7, 129.4, 133.8, 138.2, 145.6, 154.4, 160.4,
172.6 ppm. Anal. Calcd. for C16H12O4.0.1H2O (269.87): C, 71.16; H,
4.55, found: C, 71.31; H, 4.32.
ppm; 13C NMR (75 MHz, DMSO-d6):
d 118.5,121.3,124.7,124.8,128.7
(2C), 129.4 (2C), 130.2, 133.9, 134.5, 139.4, 144.0, 154.5, 173.0 ppm.
4.1.2.7. 2-(2,5-Dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one
4.1.2.14. 2-(3-Bromophenyl)-3-hydroxy-4H-chromen-4-one
Isolated as a white solid in 53.5% yield. Mp 162e164 ꢀC. 1H NMR
(100 MHz, DMSO-d6):
(4n).
(4g). Isolated as a pale yellow solid in 67.9% (lit. [38] 45%) yield. Mp
183e185 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
3.73 (s, 3H), 3.74 (s,
d
7.47 (m, 1H), 7.52 (d, J ¼ 7.8 Hz, 1H), 7.70 (d,
3H), 7.08 (d, J ¼ 7.8 Hz, 1H), 7.09 (d, J ¼ 2.7 Hz, 1H), 7.10 (dd, J ¼ 7.8,
2.7 Hz, 1H), 7.46 (dt, J ¼ 7.8, 1.2 Hz, 1H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.76
(dt, J ¼ 7.8, 1.2 Hz, 1H), 8.13 (dd, J ¼ 7.8, 1.2 Hz, 1H), 8.94 (s, 1H) ppm;
J ¼ 7.8 Hz,1H), 7.80 (d, J ¼ 3.0 Hz, 2H), 8.11 (d, J ¼ 7.5 Hz,1H), 8.22 (d,
J ¼ 7.8 Hz, 1H), 9.92 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
118.6, 121.3, 121.8, 124.7, 124.8, 126.4, 129.9, 130.7, 132.5, 133.6,
13C NMR (75 MHz, DMSO-d6):
d
55.6, 56.3, 113.3, 116.4, 116.7, 118.4,
134.0, 139.6, 143.3, 154.6, 173.1 ppm. Anal. Calcd. for C15H9BrO3
(315.97): C, 56.81; H, 2.86, found: C, 56.59; H, 2.92.
120.5, 122.0, 124.5, 124.9, 133.5, 139.1, 146.8, 155.0, 172.8 ppm.
4.1.2.8. 2-(3,4-Dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one
4.1.2.15. 2-(4-Bromophenyl)-3-hydroxy-4H-chromen-4-one
Isolated as an off-white solid in 68.2% (lit. [40] 76%) yield. Mp 197e
199 ꢀC. 1H NMR (300 MHz, DMSO-d6):
7.46 (t, J ¼ 8.1 Hz, 1H), 7.76
(d, J ¼ 8.1 Hz, 4H), 8.10 (d, J ¼ 8.1 Hz,1H), 8.17 (d, J ¼ 8.1 Hz, 2H), 9.84
(s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
118.4, 121.3, 123.3,
(4o).
(4h). Isolated as a pale yellow solid in 68.8% (lit. [38] 70%) yield. Mp
200e202 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
3.84 (s, 6H), 7.13 (d,
d
J ¼ 8.7 Hz, 1H), 7.45 (m, 1H), 7.78 (d, J ¼ 8.1 Hz, 1H), 7.79 (dd, J ¼ 8.1,
2.1 Hz, 1H), 7.81 (d, J ¼ 1.8 Hz, 1H), 7.86 (dd, J ¼ 8.7, 1.8 Hz, 1H), 8.09
(d, J ¼ 8.1 Hz, 1H), 9.46 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
124.6, 124.8, 129.5, 130.5 (2C), 131.6 (2C), 133.9, 139.4, 144.0, 154.5,
173.0 ppm.
d
55.6 (2C), 111.0, 111.5, 118.4, 121.3, 121.5, 123.6, 124.5, 124.7, 133.6,
138.3, 145.5, 148.4, 150.3, 154.4, 172.6 ppm.
4.2. Biological assays
4.1.2.9. 3-Hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-One
(4i). Isolated as a pale yellow solid in 39.0% (lit. [37] 38%) yield. Mp
4.2.1. Chemicals and cell culture
179e181 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
3.74 (s, 6H), 3.75 (s,
The 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bro-
mide (MTT), RPMI-1640, antibiotic/antimycotic solution, sodium
pyruvate, sodium bicarbonate, N-(2-hydroxyethyl)piperazine-N0-
(2-ethanesulfonic acid) (HEPES), propidium iodide (PI) and Ribo-
nuclease A (RNase A) were purchased from SigmaeAldrich (St.
Louis, MO, USA). Fetal bovine serum (FBS) was bought from Bio-
chrom AG (Berlin, Germany). All other reagents were of analytical
grade. HCT116 cells (human colorectal carcinoma cell line) were
kindly provided by Prof. Raquel Seruca from IPATIMUP, Porto. The
cell line was maintained in culture with RPMI 1640 medium con-
taining 6% FBS, 1% antibioticeantimycotic solution, 0.1 mM sodium
pyruvate and 10 mM HEPES under an atmosphere of 5% CO2 and
95% air at 37 ꢀC.
3H), 7.46 (m, 1H), 7.55 (s, 2H), 7.81 (d, J ¼ 3.3 Hz, 2H), 8.10 (d,
J ¼ 8.1 Hz, 1H), 9.59 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
50.1 (2C), 60.2, 105.6 (2C), 118.5, 121.2, 124.6, 124.7, 126.6, 133.6,
138.8, 139.1, 145.0, 152.7, 154.4, 172.8 ppm.
4.1.2.10. 2-(3-Fluorophenyl)-3-hydroxy-4H-chromen-4-one
(4j).
Isolated as a pale yellow solid in 48.9% yield. Mp 170e173 ꢀC. 1H
NMR (400 MHz, DMSO-d6):
d
7.34 (dt, J ¼ 6.8, 2.1 Hz, 1H), 7.46 (td,
J ¼ 8.4, 1.6 Hz, 1H), 7.60 (dt, J ¼ 6.8, 2.1 Hz, 1H), 7.77 (dd, J ¼ 8.4,
1.6 Hz, 1H), 7.81 (dd, J ¼ 8.4, 1.6 Hz, 1H), 8.02 (dt, J ¼ 6.8, 2.1 Hz, 1H),
8.07 (dt, J ¼ 6.8, 2.1 Hz, 1H), 8.10 (dd, J ¼ 8.4, 1.6 Hz, 1H), 9.89 (br s,
1H) ppm; 13C NMR (100 MHz, DMSO-d6):
d
114.1 (d, J ¼ 24.0 Hz),
116.6 (d, J ¼ 20.0 Hz), 118.5, 121.2, 123.6 (d, J ¼ 3.0 Hz), 124.6, 124.8,
130.6 (d, J ¼ 9.0 Hz), 133.5 (d, J ¼ 9.0 Hz), 133.9, 139.6, 143.8 (d,
J ¼ 3.0 Hz), 154.5, 162.0 (d, J ¼ 242.0 Hz), 173.1 ppm. Anal. Calcd. for
Stock solutions of quercetin and synthesized chalcones and
flavonols were made in dimethyl sulfoxide (DMSO) and kept in
aliquots at ꢃ20 ꢀC. Compounds were then added to the culture
medium just before use in order that the concentration of DMSO
was not higher than 0.5% (v/v); controls received DMSO only.
C
15H9FO3.0.8H2O (270.46): C, 66.57; H, 3.95, found: C, 66.71; H,
3.87.
4.1.2.11. 2-(4-Fluorophenyl)-3-hydroxy-4H-chromen-4-one
(4k).
4.2.2. MTT reduction assay
The MTT reduction assay was performed to study the potential
of test compounds to decrease the number viable cells, as
Isolated as a white solid in 72.8% yield. Mp 148e150 ꢀC. 1H NMR
(300 MHz, DMSO-d6):
d
7.40 (t, J ¼ 6.6 Hz, 2H), 7.46 (td, J ¼ 8.4,1.2 Hz,

T.A. Dias et al. / European Journal of Medicinal Chemistry 65 (2013) 500e510
509
previously described [41]. Briefly, HCT116 cells were plated in 24-
multiwell culture plates at 70 000 cells per mL (500 L/well) two
antibody conjugated with horseradish peroxidase and immunore-
active bands were detected using the Immobilon solutions (Milli-
pore) under a chemiluminescence detection system, the ChemiDoc
XRS (Bio-Rad Laboratories). Band area intensity was quantified
using the Quantity One software from Bio-Rad. The antibodies
against p53 (DO-1), p27 (C-19) and PARP-1 (F-2) were purchased to
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody
m
days before incubation with test compounds. Cells were then
treated with different concentrations of test compounds in fresh
complete medium for 48 h. One hour before the end of the incu-
bation period MTT (final concentration 0.5 mg/ml) was added to
each well. Then, the medium was removed, and the formazan
crystals formed by the cell’s capacity to reduce MTT were dissolved
against b-actin (SigmaeAldrich) was used as loading control.
with
a 50:50 (v/v) DMSO:ethanol solution, and absorbance
measured at 570 nm (with background subtraction at 690 nm). The
results were expressed as percentage relative to the control (cells
without any test compound). The concentration of test compound
that decreased the number of viable cells to 50% (IC50) relative to
control was calculated using the range of concentrations used (2.5e
4.2.6. Statistical analysis
Values are presented as mean ꢁ S.E.M. of at least 3 independent
experiments. Statistical analyses were performed by the Student’s
t-test using the GraphPad Prism 4.0 software (San Diego, CA, USA),
and P-values ꢂ 0.05 were considered statistically significant.
20 mM) for each compound.
Acknowledgements
4.2.3. Cell cycle analysis
HCT116 cells were plated in 6-multiwell culture plates at
70 000 cells per mL (2 mL/well) two days before incubation with
test compounds. After an incubation period with test compounds of
48 h, both floating and attached cells were harvested by tripsini-
zation, washed with PBS, and fixed with ethanol 70% at 4 ꢀC for
15 min. After rinsing fixed cells with PBS, they were incubated with
We gratefully acknowledge to the University of Minho and the
Foundation for Science and Technology (FCT, Portugal) for financial
support to the NMR Portuguese network (Bruker Avance III 400-
Univ Minho) and also the research grants PEst-C/QUI/UI0686/2011
and PEst-C/BIA/UI4050/2011, that are co-funded by the program
COMPETE from QREN with co-participation from the European
Community fund FEDER.
staining solution (50 mg/ml PI and 20 m
g/ml RNase A in PBS) at 37 ꢀC
for 15 min. Cell cycle progression was analysed by flow cytometry
using a Coulter Epics XL Flow Cytometer (Beckman Coulter Inc.,
Miami, FL, USA) counting at least 40 000 single cells per sample.
Phases of cell cycle were fitted using the mathematical Watson
Pragmatic model with the FlowJo Analysis Software (Tree Star, Inc.,
Ashland, OR, USA).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.04.
064. These data include MOL files and InChiKeys of the most
important compounds described in this article.
4.2.4. Nuclear condensation
HCT116 cells were plated in 12-multiwell culture plates at
70 000 cells per mL (1 mL/well) two days before incubation with
test compounds. After an incubation period with test compounds of
48 h, both floating and attached cells were harvested by tripsini-
zation, washed with PBS, and fixed with 4% (w/v) para-
formaldehyde in PBS for 15 min at room temperature. After rinsing
with PBS, cells were attached into a polylysine-treated slide using a
Shandon Cytospin 4 (Thermo Scientific, Waltham, MA, USA) and
nuclei stained with Hoechst. The percentage of apoptotic cells was
calculated from the ratio between cells presenting nuclear
condensation and the total number of cells (nuclei stained with
Hoechst), from a count higher than 500 cells per slide under a
fluorescence microscope. Results are presented as mean ꢁ SEM of
at least three independent experiments.
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