Solid-phase synthesis of 2′-hydroxychalcones. Effects on cell growth inhibition, cell cycle and apoptosis of human tumor cell lines

Article abstract of DOI:10.1016/j.bmc.2011.11.042

Thirty-one 2′-hydroxychalcones were prepared via solid-phase synthesis by base-catalyzed aldol condensation of substituted 2′-hydroxyacetophenones and benzaldehydes. Chalcones were tested for their growth inhibitory activity in three human tumor cell line

Full text of DOI:10.1016/j.bmc.2011.11.042

Bioorganic & Medicinal Chemistry 20 (2012) 25–33
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Solid-phase synthesis of 20-hydroxychalcones. Effects on cell growth
inhibition, cell cycle and apoptosis of human tumor cell lines
Marta Perro Neves a,b, Sara Cravo a,b, Raquel T. Lima a,c, M. Helena Vasconcelos c,d
,
f,
M. São José Nascimento a,d, Artur M.S. Silva e, Madalena Pinto a,b, Honorina Cidade a,b, , Arlene G. Corrêa
⇑
⇑
a Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
b Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164,
4050-047 Porto, Portugal
c Cancer Drug Resistance Group, IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal
d Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
e Departamento de Química & QOPNA, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
f Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235 – SP-310, CEP 13565-905, São Carlos, São Paulo, Brazil
a r t i c l e i n f o
a b s t r a c t
Thirty-one 20-hydroxychalcones were prepared via solid-phase synthesis by base-catalyzed aldol conden-
sation of substituted 20-hydroxyacetophenones and benzaldehydes. Chalcones were tested for their
growth inhibitory activity in three human tumor cell lines (MCF-7, NCI-H460 and A375-C5) using the
SRB assay. Results revealed that several of the tested compounds caused a pronounced dose-dependent
growth inhibitory effect on the tumor cell lines studied in the low micromolar range. To gain further
insight on the cellular mechanism of action of this class of compounds, studies of their effect on cell cycle
profile as well as on induction of cellular apoptosis were also carried out. Generally, the tested chalcones
interfered with the cell cycle profile and increased the percentage of apoptotic MCF-7 cells. The results
here presented may help to identify new chalcone-like structures with optimized cell growth inhibitory
activity which may be further tested as potential antitumor agents.
Article history:
Received 21 September 2011
Accepted 20 November 2011
Available online 30 November 2011
Keywords:
20-Hydroxychalcones
Solid-phase
Antitumor
Cell cycle
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Dihydroxychalcone is a naturally occurring compound which is
well known for its in vitro antitumor activities, namely by inducing
Flavonoids represent an outstanding class of naturally occurring
compounds, which reveal to be an important source of interesting
biological activities.1–3 Among this family of compounds, chalcones
represent one of the major classes of natural products and have
long been recognised for their myriad of biological activities,1–6
namely antitumor. Interestingly, the antitumor properties of chal-
cones were found to be related to the presence, the number and
the position of hydroxyl, methoxyl and halogen substituents in
both A and B rings.7 The occurrence of at least three adjacent meth-
oxyl groups appeared to be favourable for their growth inhibitory
effect in tumor cell lines.8 Moreover, several studies have demon-
strated an interesting association between interference in cell cycle
and the methoxylation pattern.5 Actually, a polymethoxyphenyl
moiety is commonly found in a number of naturally occurring anti-
cancer agents, such as colchicine and combretastatin A.5 20,40-
cytotoxicity in gastric cancer cells (MGC-803) in a dose- and time-
dependent manner, causing MGC-803 cell shrinkage and apoptotic
body formation, typical characteristics of apoptosis and cell cycle
arrest in the G2/M phase.9,10 Furthermore, 20,40-dihydroxychalcone
significantly increased caspase-3 activity and decreased survivin
mRNA expression suggesting that it induces apoptosis in gastric
cancer cells via down-regulation of survivin mRNA expression.9,10
According to these results the substitution profile 20,40-dihydroxy
in ring A of the chalcone scaffold seems to be interesting to obtain
chalcones with growth inhibitory activity in tumor cell lines.
Considering this, we planned to synthesize a series of struc-
tural related chalcones possessing both halogen and methoxyl
groups on B ring and hydroxyl and alkyl groups on A ring to fur-
ther investigate their ability to inhibit the in vitro growth of
three human tumor cell lines, MCF-7 (breast adenocarcinoma),
NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma).
To get some insights into the cellular mechanism of action of
these chalcones, the most active derivatives were also studied
regarding their capacity to cause cell cycle arrest and to induce
apoptosis.
⇑
Corresponding authors. Tel.: +351 222 078 916; fax: +351 222 003 977 (H.C.);
tel.: +55 16 3351 8215; fax: +55 16 3351 8350 (A.G.C.).
E-mail addresses: hcidade@ff.up.pt (H. Cidade), agcorrea@power.ufscar.br (A.G.
Corrêa).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.042

26
M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
1.0 mmol Cl/g. The flavanones 32 and 33 were obtained by cycliza-
2. Results and discussion
2.1. Chemistry
tion of the correspondent 20-hydroxychalcones, induced by the
acid conditions used during the cleavage. This kind of events has
already been described for similar conditions.13 In addition,
20,4,40-trihydroxy-2-methoxymethoxychalcone (1) was obtained
instead of 20,40-dihydroxy-2,4-bis(methoxymethoxy)chalcone
revealing that deprotection of position 4 occurred during the acidic
cleavage.
This article focuses on the synthesis of specific methoxylated
and halogenated chalcones. A series of thirty-one chalcones and
two flavanones was prepared in solid-phase via base-catalyzed al-
dol reaction using three 20-hydroxyacetophenones and twelve
benzaldehydes as building blocks. The synthesis was performed
according to the general reaction pathway outlined in Scheme 1.
The diversity of the present small library was planned based on
the scaffold of some bioactive chalcones,5,7,9,10 and on the commer-
cial availability of the building blocks. To our best knowledge
twenty-three (1, 12–33) of the thirty-three synthesized com-
pounds are being described for the first time.
The structure elucidation of 20-hydroxychalcones and flavanon-
es (purity P 95%) was established by IR, HRMS and NMR tech-
niques. On the other hand, compounds with lower purity were
only characterized by HRMS and 1H NMR. The spectroscopic data
of compounds 3 and 8–10 were in agreement with those found
in literature.14,15 The coupling constants for the protons of the
vinylic system (JH –Hb = 15.3–16.1 Hz) confirmed the (E)-configura-
a
Before carrying out the synthesis of the 20-hydroxychalcones
some tests were done in order to optimize the reaction conditions.
Once the best conditions had been set up, we went on performing
the solid-phase synthesis using a Büchi Syncore synthesizer. Thus,
the immobilization of 20-hydroxyacetophenones on the solid sup-
port was carried out using diisopropylethylamine (DIEA) in CH2Cl2,
and the condensation of the solid-supported acetophenones with
benzaldehydes was achieved using NaOH in THF/MeOH. The use
of 2-chlorotrityl chloride resin for the immobilization of 20-
hydroxyacetophenones was related to the high loading and mild
acidic cleavage conditions presented for this type of resins over
other polymeric supports.11
tion. In fact, this synthetic route of chalcones is quite attractive
since it generates the (E)-isomer in a diastereoselective way, usu-
ally in fairly good yields, from appropriate substituted 20-hydroxy-
acetophenones and benzaldehydes.
2.2. Biological activity
The effect of chalcones on the in vitro growth of three human
tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-
small cell lung cancer) and A375-C5 (melanoma) was evaluated
according to the procedure adopted by the National Cancer Insti-
tute (NCI, USA) which uses the protein-binding dye sulforhoda-
mine B (SRB) to assess cell growth.16,17 Based on this procedure,
a dose–response curve was obtained for each cell line with each
test compound and the concentration that caused cell growth inhi-
bition of 50% (GI50, corresponding to the concentration of com-
pound that inhibited 50% of net cell growth), was determined as
described elsewhere.17
The solid-phase synthesis of 20-hydroxychalcones began with
the treatment of the 2-chlorotrityl chloride resin with 20-hydroxy-
acetophenones under basic conditions, in order to provide the so-
lid-supported acetophenones. The resin-loading was 70–90%, as
determined by mass difference of vacuum dried resins. To confirm
the immobilization of acetophenones on the solid support, KBr pel-
lets of the solid-supported acetophenones were analysed by IR. The
appearance of a characteristic band of carbonyl stretch for ace-
tophenones at 1631–1625 cmꢀ1 revealed their immobilization on
the resin. The treatment of this solid-supported acetophenones
with substituted benzaldehydes under basic conditions afforded
the solid-supported chalcones. The exposure of these immobilized
chalcones to 2% TFA in CH2Cl2 promoted the cleavage of 2-chloro-
trityl resin to afford a series of thirty-one chalcones and two flav-
anones in moderate yields (Scheme 1).12 The cleavage product was
purified and the yield after two steps (loading and cleavage) was
determined based on an estimated resin functionalization of
To clarify the mechanism of action of the most active com-
pounds (GI50 <10.5 lM), the MCF-7 cell line was used for treatment
with the compounds, and cells were further processed in order to
analyze alterations in the cell cycle profile and in the levels of
apoptosis.
The synthesized chalcones and flavanones with purity values
greater than 95% were evaluated for their capacity to inhibit the
in vitro growth of three human tumor cell lines, MCF-7, NCI-
H460 and A375-C5. Results showed in Table 1 indicate that from
the studied chalcones the most potent were the 20,40-dihydroxy-
3,4,5-trimethoxychalcone (2), 4-benzyloxy-20,40-dihydrochalcone
Scheme 1. Synthetic strategy for solid-phase synthesis of 20-hydroxychalcones. Reagents and conditions: (a) DIEA, CH2Cl2/DMF, rt, 48 h; (b) NaOH, THF/MeOH, rt, 48 h; (c) 2%
TFA/CH2Cl2, rt, 30 min (the numbering used concerns the NMR assignments).

M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
27
Table 1
Effect of 20-hydroxychalcones (C1–C31) and flavanones (F1–F2) on the growth of three human tumor cell lines
Compound
R1
R2
R3
R4
R5
GI50 (lM)
MCF-7
NCI-H460
A375-C5
1
H
OCH2OCH3
H
H
H
ND
ND
ND
2
3
4
5
H
H
H
H
H
OCH3
OCH3
H
OCH3
H
H
OCH3
OCH3
OCH3
Br
OCH3
H
OCH3
H
7.8 0.5
19.5 1.0
31.8 2.4
ND
8.2 0.3
37.0 4.0
60.0 2.5
ND
8.1 1.6
30.3 3.9
34.8 7.1
ND
H
6
H
H
H
CF3
H
ND
ND
ND
7
8
H
H
H
H
H
H
OBn
Cl
H
H
8.0 0.5
ND
10.5 1.3
ND
9.0 1.1
ND
9
H
Cl
H
Cl
H
ND
ND
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
OCH3
F
H
H
H
OCH3
H
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
ND
ND
ND
7.3 0.6
18.2 2.1
4.1 0.5
ND
9.1 0.6
ND
15.3 2.2
24.3 2.2
8.3 0.9
ND
24.5 3.7
ND
ND
ND
24.7 1.9
ND
12.5 1.2
>150
ND
ND
ND
20.3 4.6
ND
43.0 4.1
22.5 1.3
ND
ND
47.0 10.1
>150
14.1 2.1
35.0 6.4
8.2 1.6
ND
23.0 1.9
ND
ND
ND
46.3 9.4
ND
14.2 1.7
>150
ND
ND
ND
24.7 4.7
ND
41.6 1.4
21.4 1.8
ND
ND
32.0 3.0
>150
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH2OCH3
H
OCH3
OCH3
H
H
H
H
OCH2OCH3
OCH3
OCH3
OCH3
Br
CF3
Cl
OCH3
F
H
OCH2OCH3
OCH3
OCH3
Br
CF3
OBn
Cl
Cl
OCH3
F
OCH3
OCH3
ND
ND
13.3 1.9
ND
7.1 0.6
32.7 5.4
ND
H
Cl
OCH2OCH3
H
OCH3
H
H
H
H
Cl
H
ND
ND
19.3 1.7
ND
34.5 2.7
10.5 2.7
ND
ND
18.8 1.1
12.5 1.2
H
H
H
H
H
H
H
H
H
H
OCH3
H
CH3
CH2CH2CH3
CH3
H
OCH3
OCH3
Results are given in concentrations that were able to cause 50% of cell growth inhibition (GI50) after continuous treatment for 48 h and represent mean + SEM of at least three
independent experiments performed in duplicated. Doxorubicin was used as positive control: GI50 (MCF-7) = 68.8 15.2 nM; GI50 (NCI-H460) = 86.0 9.6 nM; GI50 (A375-
C5) = 91.5 8.0 nM. ND = not determined.
(7), and 20,40-dihydroxy-3,4,5-trimethoxy-30-propylchalcone (13)
at C-2 (29) was associated with an increase of the growth inhibi-
tory effect in the three human tumor cell lines.
which presented GI50 values ranging from 4.1 to 10.5
three human tumor cell lines studied.
lM in the
Considering the substitution patterns of ring A, the comparison
of GI50 values of chalcones 4 and 15 revealed that the presence of a
30-propyl side chain was responsible for a substantial improvement
in the growth inhibitory activity in the three human tumor cell
lines studied. Additionally, the presence of a propyl side chain
(chalcone 12) instead of a 30-methyl group (chalcone 22) was also
associated with an increase in the growth inhibitory effect of MCF-
7 cells and an appearance of activity in NCI-H460 and A375-C5 cell
lines. These results suggest that the strategy of alkylation may play
an interesting role in the cell growth inhibitory activity caused by
this class of compounds in human tumor cell lines.
Considering the substitution patterns of ring B, some interesting
results were observed. Regarding the methoxylated chalcones,
when comparing GI50 values of 3,4,5-trimethoxychalcones 2 and
13 with the corresponding 2,4,5-trimethoxychalcones 4 and 15,
the results revealed that the presence of three methoxyl groups
on positions 3,4,5 is more favourable for their growth inhibitory
activity than the presence on positions 2,4,5. These results suggest
the importance of a trimethoxyphenyl moiety in positions 3,4,5 of
the chalcone scaffold for this activity. Moreover, the comparison of
the GI50 values of chalcone 15 (GI50 = 9.1–24.5
lM) with the corre-
sponding flavanone 32 (GI50 = 18.8–47.0 M) suggest the impor-
l
By comparing the GI50 values obtained for each compound in
the three human tumor cell lines, we observed that the MCF-7 cell
line was the most sensitive to the compounds studied, in particular
to compounds 22 and 33.
The most active compounds (GI50 <10.5 lM) were selected to
study their effect on cell cycle profile and apoptosis. Therefore,
tance of the chalcone scaffold for the cell growth inhibitory
effect. Regarding the growth inhibitory activity for the halogenated
chalcones, 26, 28 and 29, the results showed that the presence of 4-
trifluoromethyl substituent (26) was more favourable than a 4-
chloride substituent (28) for the studied effect. Furthermore, com-
paring compounds 28 and 29, the presence of additional chlorine
cells were treated for 48 h with compounds 2, 7, 11, 13, 15, 21

28
M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
Table 2
and 29 at their respective GI50 concentrations and then harvested
and further processed for cell cycle analysis and apoptosis detec-
tion by flow cytometry. Results showed that the majority of the
tested chalcones interfered with the cell cycle distribution of
MCF-7 cells. In addition, compounds 2, 7, 11 increased the sub-
G1 peak, which is indicative of DNA degradation, characteristic of
apoptosis (Fig. 1). Among the studied compounds, the 4-fluoro-
20,40-dihydroxychalcone (11) was the most potent, increasing the
sub-G1 peak more than 11% in relation to DMSO control. In order
to confirm this observation, the most active compounds (2, 7, 11,
13, 15, 21 and 29) were further evaluated concerning their capabil-
ity to induce apoptosis in the MCF-7 cell line. Results from the An-
nexin-V-FITC/PI assay showed that compounds 2 and 11 increased
the levels of apoptosis. Once more, the 4-fluoro-20,40-dihydroxy-
chalcone (11) was the most active compound, causing a significant
increase in the levels of apoptosis rising from 11.1% (DMSO con-
trol) to 23.9% (Table 2).
Apoptosis levels of MCF-7 cells treated with chalcones with GI50 6 10.5 lM
Compounds
Apoptosis (% of total cells)
Blank
DMSO
12.9 1.29
11.1
2 (7.8
7 (8.0
11 (7.3
13 (4.1
15 (9.1
21 (7.1
l
l
l
l
l
l
M)
M)
M)
M)
M)
M)
20.0 2.63
15.4 1.32
23.9 2.73*
14.0 1. 97
13.3 2.01
14.8 1.65
16.6 2.54
29 (10.5 lM)
Appropriate controls were included: untreated cells (blank) and cells treated with
the highest concentration of DMSO used to dissolve the compounds (DMSO).
Results represent the mean SEM of three independent experiments, except for
DMSO control which results from only two independent experiments.
*
Represents p 6 0.05 when comparing the effect of each compound with the blank
(control).
3. Conclusions
ride), the 20-hydroxyacetophenones and benzaldehydes were dried
over P2O5 in a desiccator under vacuum before use. IR was used to
monitor and optimize the reactions performed in solid-phase (Bo-
mem M102 spectrometer, software: ACD/Labs 6.00). Purifications
of 20-hydroxychalcones were carried out by flash chromatography
using GraceResolvÒ silica gel cartridges (5 g/25 mL). Melting points
were obtained in a Köfler microscope and are uncorrected. IR spec-
tra were measured on an ATI Mattson Genesis series FTIR (soft-
ware: WinFirst, v. 2.10) spectrophotometer in KBr microplates
(cmꢀ1). 1H and 13C NMR spectra were taken in CDCl3 at room tem-
perature, on Bruker Avance 300 and 500 instruments (300.13 or
500.13 MHz for 1H and 75.47 or 125.77 MHz for 13C). Chemical
shifts are expressed in d (ppm) values relative to tetramethylsilane
(TMS) used as an internal reference; 13C NMR assignments were
made by 2D (HSQC and HMBC) NMR experiments (long-range
13C–1H coupling constants were optimized to 7 Hz). ESI-HRMS
experiments were performed at C.A.C.T.I.—University of Vigo
(Spain) on an APEXQe FT-ICR MS (Bruker Daltonics, USA), equipped
with a 7T actively shielded magnet. Ions were generated using a
Combi MALDI-electrospray ionization source. Data acquisition
was performed using the ApexControl software version 3.0.0, and
data processing was performed using the Data Analysis software,
A series of thirty-one substituted 20-hydroxychalcones was syn-
thesized through a solid-phase approach. Most of the studied com-
pounds inhibited the growth of the three human tumor cell lines,
being 2, 7, 11, 13 and 21 the most active chalcones. In addition,
the growth inhibitory effect of chalcone 11 could be associated,
at least in part, to an induction of cellular apoptosis and/or to alter-
ations in the cell cycle profile of MCF-7 cells. In conclusion, the
present study allows the identification of compounds with antitu-
mor activity and brought some structure–activity data that will aid
in the design of more active chalcones.
4. Experimental
4.1. Chemistry
The phenol groups of 2,4-dihydroxybenzaldeyde were pro-
tected with chloromethylmethyl ether (MOMCl) according to the
procedure described elsewhere,18 to afford 2,4-bis(methoxyme-
thoxy)benzaldehyde in 59% yield. Solid-phase synthesis of chal-
cones was performed using a Büchi Syncore synthesizer. The
reaction vessels were oven-dried and the resin (2-chlorotritylchlo-
45
40
35
30
*
*
**
**
25
**
20
15
10
5
0
Blank
DMSO
2
7
11
(7.3 µM)
13
15
21
29
(7.8 µM)
(8.0 µM)
(4.1 µM)
(9.1 µM)
(7.1 µM)
(10.5 µM)
Sub-G1
G1
S
G2/M
Figure 1. Cell cycle analysis of MCF-7 cells treated with the most active chalcones (GI50 <10.5
lM). Cells were cultured with the GI50 concentration of the tested compounds
for 48 h. Appropriate controls were included: untreated cells (blank) and cells treated with the highest concentration of DMSO used to dissolve the compounds (DMSO).
Results represent the mean SEM of t⁄h⁄ree independent experiments, except for DMSO control and compound 7 in which case only two independent experiments were
⁄
performed. Represents p 6 0.05. and represents p 6 0.01 when comparing the effect of each compound with the blank (control).

M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
29
version 4.0 both from Bruker Daltonics. Analytical HPLC analyses
4.1.4. 20,40-Dihydroxy-2,4-dimethoxychalcone (3)
were performed on a Finnigan Surveyor (Thermo Electron Corpora-
tion, USA) equipped with a diode array detector TSP UV6000LP. The
separation was carried out on a 250 ꢁ 4.6 mm i.d. GraceSmart
Yield: 51% as yellow solid; mp:81–84 °C; IR (KBr)
m
max: 3461,
2930, 2817, 1622, 1601, 1548, 1499, 1450, 1351, 1302, 1255,
1196, 1138, 1106, 1016; 1H NMR (CDCl3, 500 MHz): d 13.69 (1H,
s, 20-OH), 8.11 (1H, d, J = 15.6 Hz, H-b), 7.81 (1H, d, J = 9.6 Hz, H-
RP18 (5 lm) (Grace Davison Discovery Sciences, Belgium). LC
analysis was performed by isocratic elution using a mixture of
MeOH:H2O as mobile phase and the flow rate was set at 1 mL/
min. The injected volume was 20 lL and the eluent was monitored
60), 7.60 (1H, d, J = 15.6 Hz, H-
a), 7.56 (1H, d, J = 8.8 Hz, H-6),
6.58–6.51 (2 ꢁ 1H, m, H-3,5), 6.49–6.44 (2 ꢁ 1H, m, H-30,50), 3.90
(3H, s, 4-OCH3), 3.86 (3H, s, 2-OCH3); 13C NMR (CDCl3, 125 MHz):
d 192.6 (C@O), 166.3 (C-20), 163.2 (C-4), 162.8 (C-40), 160.5 (C-2),
at 190–800 nm. Xcalibur 2.0 SUR 1 software (Thermo Electron Cor-
poration 1998–2005) managed chromatographic data. Methanol
(HPLC grade) was obtained from Carlo Erba Reagents, Val de Reuil,
Italy and HPLC grade water (SimplicityÒ UVUltrapure Water
System, Millipore Corporation, USA). Prior to use, mobile phase sol-
vents were degassed in an ultrasonic bath for 15 min. All commer-
cially available reagents were purchased from Sigma Aldrich Co.
Reagents and solvents were purified and dried according to the
usual procedures described elsewhere.19 The following materials
were synthesized and purified by the described procedures.
140.3 (C-b), 131.9 (C-60), 131.3 (C-6), 118.2 (C-
a), 117.0 (C-
1),114.5 (C-10), 110.5 (C-5), 107.6 (C-50), 105.5 (C-3), 103.6(C-30),
55.5 (2,4-OCH3); ESI-HRMS (+) m/z: Anal. Calcd for C17H17O5
(M+H)+: 301.1071; found: 301.1070.
4.1.5. 20,40-Dihydroxy-2,4,5-trimethoxychalcone (4)
Yield: 53% as orange solid; mp: 186–188 °C; IR (KBr) mmax: 3395,
2936, 2912, 2823, 2715, 1641, 1602, 1571, 1509, 1457, 1398, 1364,
1332, 1290, 1237, 1199, 1118, 1021; 1H NMR (CDCl3, 500 MHz): d
8.18 (1H, d, J = 15.4 Hz, H-b), 7.84 (1H, d, J = 8.3 Hz, H-60), 7.53 (1H,
4.1.1. General procedure for the solid-phase synthesis of 20-
hydroxychalcones and flavanones (1–33)
d, J = 15.4 Hz, H-a), 7.12 (1H, s, H-6), 6.53 (1H, s, H-3), 6.43 (1H, dd,
J = 8.3, 2.5 Hz, H-50), 6.42 (1H, s, H-30), 3.96 (3H, s, 2-OCH3), 3.93
(3H, s, 5-OCH3), 3.92 (3H, s, 4-OCH3); 13C NMR (CDCl3, 125 MHz):
d 192.4 (C@O), 166.4 (C-20), 162.3 (C-40), 154.9 (C-2), 152.7 (C-5),
In a 24 reaction vessels of Büchi Syncore synthesizer a mixture
of 20-hydroxyacetophenones (10 mmol), DIEA (2 mmol) and 2-
chlorotritylchloride resin (1.0–1.5 mmol Cl/g) in anhydrous
CH2Cl2/DMF (3:0.5 mL) was stirred at room temperature under
nitrogen atmosphere for 48 h to provide the solid-supported ace-
tophenones. These immobilized acetophenones were washed with
anhydrous THF (2 ꢁ 3 mL), anhydrous MeOH (3 ꢁ 3 mL) and anhy-
drous CH2Cl2 (4 ꢁ 3 mL) and dried under vacuum for 24 h. To the
solid-supported acetophenones (1 mmol) were added the appro-
priate benzaldehydes (10 mmol), a solution of 4% NaOH in anhy-
drous MeOH (2 mmol) and anhydrous THF (3 mL). The reaction
mixture was gentle stirred at room temperature under nitrogen
atmosphere for 48 h to give the solid-supported chalcones. These
immobilized chalcones were washed and dried in the same se-
quence as the first step described above. The solid-supported chal-
cones were exposed to 2% TFA in CH2Cl2 at room temperature
under nitrogen atmosphere for 30 min. The cleaved 2-chlorotrityl
resin was filtered and washed with 2% TFA:CH2Cl2 (2 ꢁ 1 mL) and
CH2Cl2 (5 ꢁ 3 mL) and the solvent was evaporated under reduced
pressure. The resulting residue was then purified by flash chroma-
tography cartridges (SiO2, n-hexane:EtOAc) to afford the 20-
hydroxychalcones 1–31 and flavanones 32 and 33.
143.4 (C-4), 140.0 (C-b), 131.8 (C-60), 118.0 (C-
a), 115.3 (C-1),
114.5 (C-10), 111,7 (C-6), 107.4 (C-50), 103.7 (C-30), 104.7 (C-3),
56.6 (2-OCH3), 56.3 (4-OCH3), 56.1 (5-OCH3); ESI-HRMS (+) m/z:
Anal. Calcd for C18H19O6 (M+H)+: 331.1176; found: 331.1175.
4.1.6. 4-Bromo-20,40-dihydroxychalcone (5)
1
Yield: 20%; H NMR (CDCl3, 300 MHz): d 13.28 (1H, s, 20-OH),
7.85 (1H, d, J = 8.3 Hz, H-60), 7.82 (1H, d, J = 15.5 Hz, H-b), 7.61–
7.46 (2 ꢁ 1H, m, H-2,6), 7.54 (1H, d, J = 15.5 Hz, H-
a), 7.34–7.26
(2 ꢁ 1H, m, H-3,5), 6.45 (1H, dd, J = 8.3, 2.0 Hz, H-50), 6.43 (1H, s,
H-30). ESI-HRMS (+) m/z: Anal. Calcd for C15H12BrO3 (M+H)+:
318.9964; found: 318.9964.
4.1.7. 20,40-Dihydroxy-4-trifluoromethylchalcone (6)
1
Yield: 9%; H NMR (CDCl3, 300 MHz): d 13.21 (1H, s, 20-OH),
7.88 (1H, d, J = 15.6 Hz, H-b), 7.84 (1H, d, J = 8.9 Hz, H-60), 7.77–
7.58 (2 ꢁ 1H, m, H-2,6), 7.63 (1H, d, J = 15.6 Hz, H-
a), 7.35
(2 ꢁ 1H, d, J = 8.5 Hz, H-3,5), 6.49 (1H, dd, J = 8.9, 2.4 Hz, H-50),
6.45 (1H, s, H-30); ESI-HRMS (+) m/z: Anal. Calcd for C16H12F3O3
(M+H)+: 309.0733; found: 309.0732.
4.1.2. 20,4,40-Trihydroxy-2-methoxymethoxychalcone (1)
4.1.8. 4-Benzyloxy-20,40-dihydroxychalcone (7)
1
Yield: 14%; H NMR (CDCl3, 300 MHz): d 13.55 (1H, s, 20-OH),
Yield: 58% as yellow solid; mp: 162–165 °C; IR (KBr) mmax:
8.20 (1H, d, J = 15.6 Hz, H-b), 7.82 (1H, d, J = 9.4 Hz, H-60), 7.58
3438–3383, 2930, 2847, 1625, 1594, 1556, 1503, 1355, 1216,
1
(1H, d, J = 8.5 Hz, H-6), 7.54 (1H, d, J = 15.6 Hz, H-
a), 6.71 (1H, d,
1202, 1124; H NMR (CDCl3, 300 MHz): d 13.47 (1H, s, 20-OH),
J = 2.4 Hz, H-3), 6.54 (1H, dd, J = 8.5, 2.4 Hz, H-5), 6.44–6.41 (1H,
m, H-50), 6.42 (1H, d, J = 2.3 Hz, H-30) 5.28 (2H, s, –OCH2), 3.52
(3H, s, –OCH3); ESI-HRMS (+) m/z: Anal. Calcd for C17H17O6
(M+H)+: 317.1020; found: 317.1020.
7.86 (1H, d, J = 15.3 Hz, H-b), 7.83 (1H, d, J = 9.4 Hz, H-60), 7.62
(2 ꢁ 1H, d, J = 8.7 Hz, H-2,6), 7.46 (1H, d, J = 15.3 Hz, H-
a), 7.46–
7.33 (5 ꢁ 1H, m, 4-OBn), 7.02 (2 ꢁ 1H, d, J = 8.7 Hz, H-3,5), 6.44
(1H, dd, J = 9.4, 2.4 Hz, H-50), 6.42 (1H, s, H-30), 5.88 (1H, br s, 40-
OH), 5.13 (2H, s, 4-OCH2); 13C NMR (CDCl3, 125 MHz): d 191.9
(C@O), 166.4 (C-20), 162.5 (C-40), 161.0 (C-4), 146.4 (C-b), 136.3
(C1-Bn), 131.8 (C-60), 130.4 (C-2,6),128.7 (C3,5-Bn), 128.2 (C4-
4.1.3. 20,40-Dihydroxy-3,4,5-trimethoxychalcone (2)
Yield: 35% as yellow solid; mp: 199–200 °C; IR (KBr) mmax: 3316,
2913, 2885, 2819, 1615, 1571, 1491, 1450, 1364, 1318, 1266, 1194,
1171, 1117; 1H NMR (CDCl3, 500 MHz): d 13.38 (1H, s, 20-OH), 7.85
(1H, d, J = 8.6 Hz, H-60), 7.81 (1H, d, J = 15.3 Hz, H-b), 7.45 (1H, d,
Bn), 127.7 (C-1), 127.5 (C2,6-Bn),117.9 (C-a), 115.3 (C-3,5), 114.6
(C-10), 107.6 (C-50), 103.7 (C-30), 70.1 (4-OCH2); ESI-HRMS (+) m/
z: Anal. Calcd for C22H19O4 (M+H)+: 347.1278; found: 347.1276.
J = 15.3 Hz, H-
a
), 6.87 (2 ꢁ 1H, s, H-2,6), 6.45 (1H, dd, J = 8.6,
2.5 Hz, H-50), 6.43 (1H, d, J = 2.5 Hz, H-30), 3.94 (2 ꢁ 3H, s, 3,5-
OCH3), 3.91 (3H, s, 4-OCH3); 13C NMR (CDCl3, 125 MHz): d 191.7
(C@O), 166.5 (C-20), 162.6 (C-40), 153.5 (C-3,5), 144.7 (C-b), 140.6
4.1.9. 4-Chloro-20,40-dihydroxychalcone (8)
1
Yield: 24%; H NMR (CDCl3, 300 MHz): d 13.29 (1H, s, 20-OH),
7.84 (1H, d, J = 15.9 Hz, H-b), 7.83 (1H, d, J = 7.5 Hz, H-60), 7.62–
(C-4), 131.9 (C-60), 130.2 (C-1), 119.4 (C-
a
), 114.5 (C-10), 107.7
7.57 (2 ꢁ 1H, m, H-2,6), 7.54 (1H, d, J = 15.9 Hz, H-
a), 7.42–7.30
(C-50), 105.8 (C-2,6), 103.8 (C-30), 61.0 (4-OCH3), 56.3 (3,5-OCH3);
ESI-HRMS (+) m/z: Anal. Calcd for C18H19O6 (M+H)+: 331.1176;
found: 331.1177.
(2 ꢁ 1H, m, H-3,5), 6.49–6.43 (1H, m, H-50), 6.43 (1H, s, H-30),
5.74 (1H, br s, 40-OH); ESI-HRMS (+) m/z: Anal. Calcd for C15H12ClO3
(M+H)+: 275.0470; found: 275.0469.

30
M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
4.1.10. 2,4-Dichloro-20,40-dihydroxychalcone (9)
4.1.15. 20,40-Dihydroxy-2,4-dimethoxy-30-propylchalcone (14)
1
Yield: 3%; 1H NMR (CDCl3, 500 MHz): d 13.20 (1H, s, 20-OH),
8.19 (1H, d, J = 15.5 Hz, H-b), 7.80 (1H, d, J = 9.5 Hz, H-60), 7.69
Yield: 51%; H NMR (CDCl3, 300 MHz): d 13.89 (1H, s, 20-OH),
8.12 (1H, d, J = 15.5 Hz, H-b), 7.70 (1H, d, J = 8.9 Hz, H-60), 7.62
(1H, d, J = 8.5 Hz, H-6), 7.54 (1H, d, J = 15.5 Hz, H-
a), 7.44 (1H, d,
(1H, d, J = 15.5 Hz, H-a), 7.57 (1H, d, J = 8.5 Hz, H-6), 6.58 (1H, dd,
J = 2.1 Hz, H-3), 7.32 (1H, dd, J = 8.5, 2.1 Hz, H-5), 6.44 (1H, dd,
J = 9.5, 2.5 Hz, H-50), 6.43 (1H, s,H-30); ESI-HRMS (+) m/z: Anal.
Calcd for C15H11Cl2O3 (M+H)+: 309.0080; found: 309.0079.
J = 8.5, 2.4 Hz, H-5), 6.48 (1H, d, J = 2.4 Hz, H-3), 6.40 (1H, d,
J = 8.9 Hz, H-50), 3.92 (3H, s, 2-OCH3), 3.87 (3H, s, 4-OCH3), 2.68–
2.61 (2H, m, 30-CH2CH2CH3), 1.63–1.58 (2H, m, 30-CH2CH2CH3);
1.02–0.92 (3H, m, 30-CH2CH2CH3); ESI-HRMS (+) m/z: Anal. Calcd
for C20H23O5 (M+H)+: 343.1540; found: 343.1538.
4.1.11. 20,40-Dihydroxy-4-methoxychalcone (10)
1
Yield: 54%; H NMR (CDCl3, 300 MHz): d 13.47 (1H, s, 20-OH),
7.87 (1H, d, J = 15.1 Hz, H-b), 7.84 (1H, d, J = 9.6 Hz, H-60), 7.62
4.1.16. 20,40-Dihydroxy-2,4,5-trimethoxy-30-propylchalcone (15)
(2 ꢁ 1H, d, J = 8.8 Hz, H-2,6), 7.46 (1H, d, J = 15.1 Hz, H-
a
), 6.95
Yield: 28% as orange solid; mp: 180–183 °C; IR (KBr) mmax: 3397,
(2 ꢁ 1H, d, J = 8.8 Hz, H-3,5), 6.44 (1H, dd, J = 9.6, 2.2 Hz, H-50),
6.42 (1H, s, H-30), 5.71 (1H, br s, 40-OH), 3.87 (3H, s, 4-OCH3);
ESI-HRMS (+) m/z: Anal. Calcd for C16H15O4 (M+H)+: 271.0965;
found: 271.0964.
2940, 2902, 2844, 1602, 1540, 1498, 1448, 1422, 1364, 1273, 1222,
1195, 1093; 1H NMR (CDCl3, 300 MHz): d 13.89 (1H, s, 20-OH), 8.17
(1H, d, J = 15.5 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60),7.55 (1H, d,
J = 15.5 Hz, H-a), 7.12 (1H, s, H-6), 6.53 (1H, s, H-3), 6.41 (1H, d,
J = 8.9 Hz, H-50), 5.44 (1H, br s, 40-OH), 3.96 (3H, s, 2-OCH3), 3.93
(3H, s, 5-OCH3), 3.92 (3H, s, 4-OCH3), 2.66 (2H, t, J = 7.7 Hz, 30-
CH2CH2CH3), 1.66–1.56 (2H, m, 30-CH2CH2CH3); 1.00 (3H, t,
J = 7.4 Hz, 30-CH2CH2CH3); 13C NMR (CDCl3, 125 MHz): d 192.6
(C@O), 164.4 (C-20), 159.8 (C-40), 154.8 (C-2), 154.6 (C-5), 143.2
4.1.12. 4-Fluoro-20,40-dihydroxychalcone (11)
Yield: 10% as yellow solid; mp: 178–180 °C; IR (KBr) mmax: 3438,
2912, 2846, 2730, 1630, 1592, 1550, 1500, 1353, 1311, 1279, 1221,
1
1140; H NMR (CDCl3, 300 MHz): d 13.38 (1H, s, 20-OH), 7.86 (1H,
d, J = 15.5 Hz, H-b), 7.83 (1H, d, J = 9.7 Hz, H-60), 7.65 (2 ꢁ 1H, d,
(C-4), 139.6 (C-b), 128.8 (C-60), 118.3 (C- ), 116.0 (C-30), 115.4
a
J = 8.6 Hz, H-2,6), 7.50 (1H, d, J = 15.5 Hz, H-
a
), 7.13 (2 ꢁ 1H, d,
(C-1), 114.3 (C-10), 111.7 (C-6), 106.8 (C-50), 96.7 (C-3), 56.6 (4-
OCH3), 56.3 (5-OCH3), 56.1 (2-OCH3), 24.5 (30-CH2CH2CH3), 21.9
(30-CH2CH2CH3), 14.2 (30-CH2CH2CH3); ESI-HRMS (+) m/z: Anal.
Calcd for C21H25O6 (M+H)+: 373.1646; found: 373.1643.
J = 8.6 Hz, H-3,5), 6.45 (1H, dd, J = 9.7, 2.5 Hz, H-50), 6.43 (1H, s,
H-30), 5.75 (1H, br s, 40-OH); 13C NMR (CDCl3, 125 MHz): d 191.7
(C@O), 166.5 (C-20), 162.7 (C-40), 143.2 (C-b), 131.9 (C-60), 132.6
(C-1), 131.0 (C-4), 130.5 (C-2,6), 120.0 (C-a), 116.2 (C-3,5), 114.4
(C-10), 107.7 (C-50), 103.8 (C-30); ESI-HRMS (+) m/z: Anal. Calcd
4.1.17. 4-Bromo-20,40-dihydroxy-30-propylchalcone (16)
for C15H12FO3 (M+H)+: 259.0765; found: 259.0764.
Yield: 11%; H NMR (CDCl3, 300 MHz): d 13.58 (1H, s, 20-OH),
1
7.80 (1H, d, J = 15.6 Hz, H-b), 7.73 (1H, d, J = 8.7 Hz, H-60), 7.54–
7.44 (2 ꢁ 1H, m, H-2,6), 7.34–7.26 (2 ꢁ 1H, m, H-3,5), 7.57 (1H, d,
4.1.13. 20,40-Dihydroxy-2,4-bis(methoxymethoxy)-30-propyl-
chalcone (12)
J = 15.6 Hz, H-a
), 6.45 (1H, d, J = 8.7 Hz, H-50), 5.77 (1H, br s, 40-
Yield: 12% as yellow solid; mp: 158–160 °C; IR (KBr)
mmax: 3439,
OH), 2.59–2.55 (2H, m, 30-CH2CH2CH3), 1.65–1.57 (2H, m, 30-
CH2CH2CH3), 1.02–0.93 (3H, m, 30-CH2CH2CH3); ESI-HRMS (+) m/
z: Anal. Calcd for C18H18BrO3 (M+H)+: 361.0434; found: 361.0432.
2952, 2926, 2866, 1614, 1542, 1474, 1437, 1370, 1311, 1293, 1236,
1154, 1107; 1H NMR (CDCl3, 500 MHz): d 13.75 (1H, s, 20-OH), 8.12
(1H, d, J = 15.6 Hz, H-b), 7.63 (1H, d, J = 8.8 Hz, H-60), 7.54 (1H, d,
J = 8.7 Hz, H-6), 7.52 (1H, d, J = 15.6 Hz, H-
a
), 6.79 (1H, d,
4.1.18. 20,40-Dihydroxy-30-propyl-4-trifluoromethylchalcone
(17)
J = 2.3 Hz, H-3), 6.68 (1H, dd, J = 8.7, 2.3 Hz, H-5), 6.33 (1H, d,
J = 8.8 Hz, H-50), 5.21 (2H, s, 2-OCH2), 5.14 (2H, s, 4-OCH2), 3.45
(3H, s, 2-OCH3), 3.43 (3H, s, 4-OCH3), 2.59 (1H, t, J = 7.7 Hz, 30-
CH2CH2CH3), 1.57–1.51 (1H, m, 30-CH2CH2CH3), 0.93 (1H, t,
J = 7.4 Hz, 30-CH2CH2CH3); 13C NMR (CDCl3, 125 MHz): d 192.6
(C@O), 164.4 (C-20), 160.5 (C-40), 159.9 (C-4), 157.8 (C-2), 139.3
1
Yield: 4%; H NMR (CDCl3, 300 MHz): d 13.53 (1H, s, 20-OH),
7.88 (1H, d, J = 15.6 Hz, H-b), 7.75 (2 ꢁ 1H, d, J = 8.4 Hz, H-2,6),
7.71 (1H, d, J = 9.0 Hz, H-60), 7.68 (2 ꢁ 1H, d, J = 8.4 Hz, H-3,5),
7.65 (1H, d, J = 15.6 Hz, H-a
), 6.43 (1H, d, J = 9.0 Hz, H-50), 2.69–
2.64 (2H, m, 30-CH2CH2CH3), 1.68–1.58 (2H, m, 30-CH2CH2CH3),
1.00 (3H, t, J = 7.4 Hz, 30-CH2CH2CH3); ESI-HRMS (+) m/z: Anal.
Calcd for C19H18F3O3 (M+H)+: 351.1203; found: 351.1201.
(C-b), 129.8 (C-6), 128.8 (C-60), 118.9 (C-
a), 118.5 (C-1), 116.0
(C-30), 114.3 (C-10), 109.4 (C-5), 106.9 (C-50), 103.3 (C-3), 94.7
(2-OCH2), 94.3 (4-OCH2), 56.5 (2-OCH3), 56.3 (4-OCH3), 24.5
(30-CH2CH2CH3), 21.8 (30-CH2CH2CH3), 14.2 (30-CH2CH2CH3); ESI-
HRMS (+) m/z: Anal. Calcd for C22H27O7 (M+H)+: 403.1751; found:
403.1749.
4.1.19. 4-Chloro-20,40-dihydroxy-30-propylchalcone (18)
Yield: 12%; 1H NMR (CDCl3, 300 MHz): d 7.84 (1H, d, J = 15.5 Hz,
H-b), 7.70 (1H, d, J = 8.8 Hz, H-60), 7.58 (2 ꢁ 1H, d, J = 8.5 Hz, H-
2,6), 7.56 (1H, d, J = 15.5 Hz, H-
a
), 7.41–7.26 (2 ꢁ 1H, m, H-3,5),
4.1.14. 20,40-Dihydroxy-3,4,5-trimethoxy-30-propylchalcone (13)
6.42 (1H, d, J = 8.8 Hz, H-50), 2.62–2.55 (2H, m, 30-CH2CH2CH3),
1.65–1.57 (2H, m, 30-CH2CH2CH3), 1.02–0.93 (3H, m, 30-CH2CH2-
CH3); ESI-HRMS (+) m/z: Anal. Calcd for C18H18ClO3 (M+H)+:
317.0939; found: 317.0938.
Yield: 34% as yellow solid; mp: 165–167 °C; IR (KBr) mmax
:
3215–3170, 2944, 2917, 2859, 2826, 1702, 1621, 1569, 1542,
1494, 1440, 1411, 1362, 1318, 1274, 1212, 1148, 1120, 1098; 1H
NMR (CDCl3, 300 MHz): d 13.68 (1H, s, 20-OH), 7.80 (1H, d,
J = 15.4 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60), 7.46 (1H, d,
4.1.20. 20,40-Dihydroxy-4-methoxy-30-propylchalcone (19)
J = 15.4 Hz, H-
a
), 6.86 (2 ꢁ 1H, s, H-2,6), 6.43 (1H, d, J = 8.9 Hz, H-
Yield: 26% as yellow solid; mp: 177–180 °C; IR (KBr) mmax: 3355,
50), 5.78 (1H, br s, 40-OH), 3.93 (2 ꢁ 3H, s, 3,5-OCH3), 3.91 (3H, s,
4-OCH3), 2.67 (2H, t, J = 7.7 Hz, 30-CH2CH2CH3), 1.68–1.56 (2H, m,
30-CH2CH2CH3), 1.00 (3H, t, J = 7.4 Hz, 30-CH2CH2CH3); 13C NMR
(CDCl3, 125 MHz): d 192.0 (C@O), 164.5 (C-20), 160.5 (C-40), 153.4
(C-3,5), 144.3 (C-b), 140.4 (C-4), 130.4 (C-1), 128.9 (C-60), 119.7
2945, 2924, 2851, 1618, 1594, 1542, 1501, 1476, 1413, 1359, 1279,
1226, 1196, 1161, 1096; 1H NMR (CDCl3, 300 MHz): d 13.78 (1H, s,
20-OH), 7.86 (1H, d, J = 15.4 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60),
7.61 (2 ꢁ 1H, d, J = 8.8 Hz, H-2,6), 7.24 (1H, d, J = 15.4 Hz, H-
a),
6.95 (2 ꢁ 1H, d, J = 8.8 Hz, H-3,5), 6.41 (1H, d, J = 8.9 Hz, H-50),
5.39 (1H, br s, 40-OH), 3.87 (3H, s, 4-OCH3), 2.66 (2H, t, J = 7.7 Hz,
30-CH2CH2CH3), 1.65–1.58 (2H, m, 30-CH2CH2CH3), 1.00 (3H, t,
J = 7.4 Hz, 30-CH2CH2CH3); 13C NMR (CDCl3, 125 MHz): d 192.2
(C@O), 164.4 (C-20), 161.7 (C-4), 160.0 (C-40), 144.1 (C-b), 130.3
(C-a
), 116.3 (C-30), 114.1 (C-10), 107.2 (C-50), 105.7 (C-2,6), 61.0
(4-OCH3), 56.2 (3,5-OCH3), 24.5 (30-CH2CH2CH3), 21.8 (30-
CH2CH2CH3), 14.2 (30-CH2CH2CH3); ESI-HRMS (+) m/z: Anal. Calcd
for C21H25O6 (M+H)+: 313.1646; found: 313.1643.

M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
31
(C-2–6), 128.8 (C-60), 127.6 (C-1), 118.0 (C-
a
), 116.1 (C-30), 114.4
(1H, d, J = 8.7 Hz, H-50), 5.55 (1H, br s, 40-OH), 3.96 (3H, s, 2-
OCH3), 3.93 (3H, s, 5-OCH3), 3.92 (3H, s, 4-OCH3), 2.17 (3H, s, 30-
CH3); ESI-HRMS (+) m/z: Anal. Calcd for C19H21O6(M+H)+:
345.1333; found: 345.1331.
(C-3,5), 114.2 (C-10), 107.0 (C-50), 55.4 (4-OCH3), 24.4 (30-
CH2CH2CH3), 21.8 (30-CH2CH2CH3), 14.2 (30-CH2CH2CH3); ESI-
HRMS (+) m/z: Anal. Calcd for C19H21O4 (M+H)+: 313.1434; found:
313.1435.
4.1.26. 4-Bromo-20,40-dihydroxy-30-methylchalcone (25)
4.1.21. 4-Fluoro-20,40-dihydroxy-30-propylchalcone (20)
1
Yield: 26%; H NMR (CDCl3, 300 MHz): d 13.64 (1H, s, 20-OH),
1
Yield: 4%; H NMR (CDCl3, 300 MHz): d 13.64 (1H, s, 20-OH),
7.82 (1H, d, J = 15.6 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60), 7.59
7.85 (1H, d, J = 15.5 Hz, H-b), 7.71 (1H, d, J = 8.9 Hz, H-60), 7.64
(1H, d, J = 15.6 Hz, H-
a
), 7.55–7.46 (2 ꢁ 1H, m, H-2,6), 7.32
(2 ꢁ 1H, d, J = 8.7 Hz, H-2,6), 7.52 (1H, d, J = 15.5 Hz, H-
a), 7.14
(2 ꢁ 1H, d, J = 8.6 Hz, H-3,5), 6.44 (1H, d, J = 8.9 Hz, H-50), 5.49
(1H, br s, 40-OH), 2.18 (3H, s, 30-CH3); ESI-HRMS (+) m/z: Anal. Calcd
for C16H14BrO3(M+H)+: 333.0121; found: 333.0119.
(2 ꢁ 1H, d, J = 8.7 Hz, H-3,5), 6.42 (1H, d, J = 8.9 Hz, H-50), 5.48
(1H, br s, 40-OH), 2.66 (2H, t, J = 7.7 Hz, 30-CH2CH2CH3), 1.65–1.59
(2H, m, 30-CH2CH2CH3), 1.03–0.95 (3H, m, 30-CH2CH2CH3); ESI-
HRMS (+) m/z: Anal. Calcd for C18H18FO3 (M+H)+: 301.1235; found:
301.1233.
4.1.27. 20,40-Dihydroxy-30-methyl-4-trifluoromethylchalcone
(26)
Yield: 6% as yellow solid; mp: 164–168 °C; IR (KBr) mmax: 3422–
4.1.22. 2-Chloro-20,40-dihydroxy-30-propylchalcone (21)
3393, 2909, 2843, 1691, 1621, 1551, 1473, 1426, 1354, 1309, 1279,
1224, 1162, 1095, 1052; 1H NMR (CDCl3, 300 MHz): d 13.55 (1H, s,
20-OH), 7.88 (1H, d, J = 15.5 Hz, H-b), 7.75 (2 ꢁ 1H, d, J = 8.6 Hz, H-
2,6), 7.71 (1H, d, J = 9.3 Hz, H-60), 7.68 (2 ꢁ 1H, d, J = 8.6 Hz, H-3,5),
Yield: 11% as yellow solid; mp: 184–188 °C; IR (KBr) mmax
:
3461–3389, 2940, 2911, 2842, 1611, 1544, 1474, 1449, 1424,
1351, 1295, 1258, 1221, 1100; 1H NMR (CDCl3, 300 MHz): d
13.56 (1H, s, 20-OH), 8.26 (1H, d, J = 15.5 Hz, H-b), 7.70 (1H, d,
J = 8.9 Hz, H-60), 7.77–7.73 (1H, m, H-3), 7.58 (1H, d, J = 15.5 Hz,
7.65 (1H, d, J = 15.5 Hz, H-a
), 6.45 (1H, d, J = 9.3 Hz, H-50), 5.50 (1H,
br s, 40-OH), 2.18 (3H, s, 30-CH3); 13C NMR (CDCl3, 75 MHz): d 191.5
(C@O), 164.5 (C-20), 160.0 (C-40), 142.2 (C-b), 138.9 (C-4), 132.4 (C-
1), 128.8 (C-60), 128.5 (C-2,6), 125.9 (C-3,5), 125.9 (4-CF3), 122.8
H-
a
), 7.47–7.44 (1H, m, H-6), 7.35–7.32 (2 ꢁ 1H, m, H-4,5), 6.42
(1H, d, J = 8.9 Hz, H-50), 5.51 (1H, br s, 40-OH), 2.66 (2H, t,
J = 7.7 Hz, 30-CH2CH2CH3), 1.65–1.58 (2H, m, 30-CH2CH2CH3), 1.00
(3H, t, J = 7.4 Hz, 30-CH2CH2CH3); 13C NMR (CDCl3, 125 MHz): d
191.9 (C@O), 164.2 (C-20), 160.4 (C-40), 140.0 (C-b), 135.5 (C-1),
133.2 (C-2), 131.2 (C-4), 130.4 (C-6), 129.0 (C-60), 127.9 (C-3),
(C-a
), 113.9 (C-10), 111.5 (C-30), 107.2 (C-50), 7.4 (30-CH3) ESI-HRMS
(+) m/z: Anal. Calcd for C17H14F3O3 (M+H)+: 323.0890; found:
323.0888.
127.0 (C-5), 123.3 (C-a
), 116.2 (C-30), 114.1 (C-10), 107.3 (C-50),
4.1.28. 4-Benzyloxy-20,40-dihydroxy-30-methylchalcone (27)
24.4 (30-CH2CH2CH3), 21.8 (30-CH2CH2CH3), 14.2 (30-CH2CH2CH3);
ESI-HRMS (+) m/z: Anal. Calcd for C18H18ClO3 (M+H)+: 317.0939;
found: 317.0938.
1
Yield: 26%; H NMR (CDCl3, 300 MHz): d 13.81 (1H, s, 20-OH),
7.87 (1H, d, J = 15.4 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60), 7.62
(2 ꢁ 1H, d, J = 8.8 Hz, H-2,6), 7.48 (1H, d, J = 15.4 Hz, H-
a), 7.43–
4.1.23. 20,40-Dihydroxy-30-methyl-2,4-bis(methoxymethoxy)-
chalcone (22)
7.33 (5H, m, 4-OBn), 7.02 (2 ꢁ 1H, d, J = 8.8 Hz, H-3,5), 6.42 (1H,
d, J = 8.9 Hz, H-50), 5.36 (1H, br s, 40-OH), 5.13 (2H, s, 4-OCH2),
2.18 (3H, s, 30-CH3); ESI-HRMS (+) m/z: Anal. Calcd for
Yield: 15% as yellow solid; mp: 150–153 °C; IR (KBr) mmax
:
C
23H21O4(M+H)+: 361.1434; found: 361.1432.
3460–3403, 2907, 2839, 1711, 1602, 1530, 1484, 1439, 1362,
1285, 1218, 1138, 1098, 1057, 972; 1H NMR (CDCl3, 300 MHz): d
13.89 (1H, s, 20-OH), 8.20 (1H, d, J = 15.6 Hz, H-b), 7.71 (1H, d,
J = 8.9 Hz, H-60), 7.62 (1H, d, J = 8.7 Hz, H-6), 7.59 (1H, d,
4.1.29. 4-Chloro-20,40-dihydroxy-30-methylchalcone (28)
Yield: 9% as yellowsolid; mp: 204–207 °C; IR (KBr) max: 3442,
m
J = 15.6 Hz, H-a), 6.87 (1H, d, J = 2.3 Hz, H-3), 6.75 (1H, dd, J = 8.7,
2923, 2862, 1709, 1633, 1553, 1487, 1441, 1367, 1317, 1239,
2.3 Hz, H-5), 6.42 (1H, d, J = 8.9 Hz, H-50), 5.29 (2H, s, 2-OCH2),
5.21 (2H, s, 4-OCH2), 3.52 (3H, s, 2-OCH3), 3.50 (3H, s, 4-OCH3),
2.17 (3H, s, 30-CH3); 13C NMR (CDCl3, 125 MHz): d 192.6 (C@O),
164.3 (C-20), 160.5 (C-40), 159.9 (C-4), 157.9 (C-2), 139.4 (C-b),
1
1107; H NMR (CDCl3, 300 MHz): d 13.63 (1H, s, 20-OH), 7.83
(1H, d, J = 15.5 Hz, H-b), 7.71 (1H, d, J = 8.8 Hz, H-60), 7.59
(2 ꢁ 1H, d, J = 8.5 Hz, H-2,6), 7.56 (1H, d, J = 15.5 Hz, H-
a), 7.40
(2 ꢁ 1H, d, J = 8.5 Hz, H-3,5), 6.43 (1H, d, J = 8.8 Hz, H-50), 5.41
(1H, br s, 40-OH), 2.17 (3H, s, 30-CH3); 13C NMR (CDCl3, 125 MHz):
d 191.5 (C@O), 163.9 (C-20), 162.4 (C-40), 142.1 (C-b), 136.1 (C-1),
129.8 (C-6), 128.6 (C-60), 118.9 (C- ), 118.5 (C-1), 114.1 (C-10),
a
110.0 (C-30), 109.4 (C-5), 106.7 (C-50), 103.3 (C-3), 94.7 (2-OCH2),
94.3 (4-OCH2), 56.5 (2-OCH3), 56.3 (4-OCH3), 7.4 (30-CH3); ESI-
HRMS m/z: Anal. Calcd for C20H23O7 (M+H)+: 375.1438; found:
375.1436.
133.3 (C-4), 129.4 (C-2,6), 129.1 (C-3,5), 128.5 (C-60), 121.2 (C-
a),
113.0 (C-10), 111.8 (C-30), 107.1 (C-50), 7.2 (30-CH3); ESI-HRMS (+)
m/z: Anal. Calcd for
C
16H14ClO3(M+H)+: 289.0626; found:
289.0624.
4.1.24. 20,40-Dihydroxy-30-methyl-3,4,5-trimethoxychalcone
(23)
4.1.30. 2,4-Dichloro-20,40-dihydroxy-30-methylchalcone (29)
Yield: 6% as yellowsolid; mp: 194–197 °C; IR (KBr) max: 3462–
1
Yield: 3%; H NMR (CDCl3, 300 MHz): d 13.73 (1H, s, 20-OH),
m
7.82 (1H, d, J = 15.4 Hz, H-b), 7.73 (1H, d, J = 8.9 Hz, H-60), 7.47
3399, 2923, 2845, 1623, 1480, 1456, 1432, 1356, 1303, 1252, 1225,
1204, 1099; 1H NMR (CDCl3, 300 MHz): d 13.54 (1H, s, 20-OH), 8.18
(1H, d, J = 15.5 Hz, H-b), 7.69 (1H, d, J = 8.4 Hz, H-6), 7.68 (1H, d,
(1H, d, J = 15.4 Hz, H-
a
), 6.87 (2 ꢁ 1H, s, H-2,6), 6.44 (1H, d,
J = 8.9 Hz, H-50), 3.94 (2 ꢁ 3H, s, 3,5-OCH3), 3.91 (3H, s, 4-OCH3),
2.18 (3H, s, 30-CH3); ESI-HRMS (+) m/z: Anal. Calcd for
J = 8.9 Hz, H-60), 7.56 (1H, d, J = 15.5 Hz, H-
a), 7.48 (1H, d,
C
19H21O6(M+H)+: 345.1333; found: 345.1332.
J = 2.1 Hz, H-3), 7.31 (1H, dd, J = 8.4, 2.1 Hz, H-5), 6.43 (1H, d,
J = 8.9 Hz, H-50), 5.46 (1H, br s, 40-OH), 2.17 (3H, s, 30-CH3); 13C
NMR (CDCl3, 125 MHz): d 191.5 (C@O), 164.5 (C-20), 160.5 (C-40),
138.8 (C-b), 136.5 (C-4), 136.1 (C-2), 131.8 (C-1),130.2 (C-3),
4.1.25. 20,40-Dihydroxy-30-methyl-2,4,5-trimethoxychalcone
(24)
1
128.8 (C-60), 128.6 (C-6),127.5 (C-5), 123.5 (C- ), 114.0 (C-10),
a
Yield: 36%; H NMR (CDCl3, 300 MHz): d 13.95 (1H, s, 20-OH),
111.5 (C-30), 107.1 (C-50), 7.3 (30-CH3); ESI-HRMS (+) m/z: Anal.
8.18 (1H, d, J = 15.5 Hz, H-b), 7.75 (1H, d, J = 8.7 Hz, H-60), 7.55
Calcd for C16H13Cl2O3 (M+H)+: 323.0236; found: 323.0235.
(1H, d, J = 15.5 Hz, H-a), 7.12 (1H, s, H-6), 6.54 (1H, s, H-3), 6.42

32
M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
4.1.31. 20,40-Dihydroxy-30-methyl-4-methoxychalcone (30)
Final concentrations of DMSO did not interfere with the biological
activity tested.
1
Yield: 69%; H NMR (CDCl3, 300 MHz): d 13.81 (1H, s, 20-OH),
7.87 (1H, d, J = 15.4 Hz, H-b), 7.72 (1H, d, J = 8.9 Hz, H-60), 7.62
(2 ꢁ 1H, d, J = 8.7 Hz, H-2,6), 7.48 (1H, d, J = 15.4 Hz, H-
a
), 6.95
4.2.2. Cell culture
(2 ꢁ 1H, d, J = 8.7 Hz, H-3,5), 6.42 (1H, d, J = 8.9 Hz, H-50), 5.41
(1H, br s, 40-OH), 3.87 (3H, s, 4-OCH3), 2.18 (3H, s, 30-CH3); ESI-
HRMS (+) m/z: Anal. Calcd for C17H17O4 (M+H)+: 285.1121; found:
285.1120.
The following three human tumor cell lines were used: MCF-7
(breast adenocarcinoma, ECACC, UK), NCI-H460 (non-small cell
lung cancer, a kind gift from NCI, Bethesda, USA) and A375-C5
(melanoma, ECACC, UK). All cell lines were grown as monolayer
and routinely maintained in RPMI-1640 medium with ultragluta-
mine supplemented with 5% heat-inactivated fetal bovine serum
(FBS) at 37 °C in a humidified atmosphere containing 5% CO2.
4.1.32. 4-Fluoro-20,40-dihydroxy-30-methylchalcone (31)
1
Yield: 34%; H NMR (CDCl3, 300 MHz): d 13.67 (1H, s, 20-OH),
7.85 (1H, d, J = 15.5 Hz, H-b), 7.71 (1H, d, J = 8.9 Hz, H-60), 7.64
4.2.3. Growth inhibition assay
(2 ꢁ 1H, d, J = 8.7 Hz, H-2,6), 7.52 (1H, d, J = 15.5 Hz, H-
a), 7.14
(2 ꢁ 1H, d, J = 8.7 Hz, H-3,5), 6.34 (1H, d, J = 8.9 Hz, H-50), 5.47
(1H, br s, 40-OH), 2.17 (3H, s, 30-CH3); ESI-HRMS (+) m/z: Anal. Calcd
for C16H14FO3 (M+H)+: 273.0922; found: 273.0920.
Cells were plated in 96-well plates at appropriate densities in
order to ensure exponential growth throughout the experimental
period (5.0 ꢁ 103 cells/well for MCF-7 and NCI-H460 and
7.5 ꢁ 103 cells/well for A375-C5) and then allowed to adhere over-
night. Cells were then treated for 48 h with five serial dilutions of
each compound (1:2 or 1:3). Following this incubation period, cells
were fixed in situ with trichloroacetic acid, washed and stained
with SRB.17,20 The bound stain was then solubilised with Tris and
the absorbance was measured at 492 nm in a plate reader (Biotek
Instruments Inc. PowerWave XS, Winooski, USA). The effect of the
vehicle solvent (DMSO) on the growth of these cell lines was eval-
uated by exposing untreated control cells to the maximum concen-
tration of DMSO used in each assay (0.25%).
4.1.33. 7-Hydroxy-20,40,50-trimethoxy-8-propylflavanone (32)
Yield: 5% as yellow solid; mp: 186–189 °C; IR (KBr) mmax: 3422,
2930, 2902, 2833, 1640, 1563, 1496, 1417, 1266, 1187, 1081, 1011;
1H NMR (CDCl3, 300 MHz): d 7.75 (1H, d, J = 8.6 Hz, H-5), 7.20 (1H,
s, H-60), 6.56 (1H, s, H-30), 6.53 (1H, d, J = 8.6 Hz, H-6), 5.74 (1H, dd,
J = 12.9, 3.4 Hz, H-2), 3.94 (3H, s, 20-OCH3), 3.90 (3H, s, 40-OCH3),
3.83 (3H, s, 50-OCH3), 2.92 (1H, dd, J = 16.9, 3.4 Hz, H-3eq), 2.81
(1H, dd, J = 16.9, 12.9 Hz, H-3ax), 2.69–2.63 (2H, m, 8-CH2CH2CH3),
1.67–1.58 (2H, m, 8-CH2CH2CH3), 0.97 (3H, t, J = 7.4 Hz, 8-
CH2CH2CH3); 13C NMR (CDCl3, 125 MHz): d 192.1 (C-4), 161.7 (C-
7), 159.9 (C-8a), 150.1 (C-50), 149.4 (C-20), 143.1 (C-40), 126.1 (C-
5), 119.3 (C-10), 116.3 (C-8), 115.2 (C-4a), 110.0 (C-60), 109.8 (C-
6), 97.0 (C-30), 74.6 (C-2), 56.5 (40-OCH3), 56.2 (20-OCH3), 56.1 (50-
OCH3), 43.5 (C-3), 24.9 (8-CH2CH2CH3), 22.2 (8-CH2CH2CH3), 14.1
(8-CH2CH2CH3); ESI-HRMS (+) m/z: Anal. Calcd for C21H25O6
(M+H)+: 373.1646; found: 373.1645.
4.2.4. Cell cycle analysis
MCF-7 cells were plated in 6-well plates (5.0 ꢁ 103 cells/well)
and incubated at 37 °C for 24 h. Exponentially growing cells were
then incubated with the compounds (2, 7, 11, 13, 15, 21 and 29)
at their respective GI50 concentrations (previously determined
with the SRB assay, Table 1). Untreated cells (control) or cells
treated with the compound’s solvent (DMSO) were included.
DMSO was used at the highest concentration used in the experi-
ments. Following 48 h treatment, cells were centrifuged and fixed
in 70% ethanol at 4 °C for at least 12 h and subsequently resus-
4.1.34. 7-Hydroxy-8-methyl-20,40-dimethoxyflavanone (33)
Yield: 56% as yellow solid; mp: 206–209 °C; IR (KBr) mmax
:
3422–3147, 2907, 2815, 1695, 1595, 1528, 1489, 1445, 1414,
1351, 1305, 1281, 1256, 1199, 1143, 1088, 1058, 1010; 1H NMR
(CDCl3, 300 MHz): d 7.74 (1H, d, J = 8.7 Hz, H-5), 7.53 (1H, d,
J = 8.5 Hz, H-60), 6.58 (1H, dd, J = 8.5, 2.3 Hz, H-50), 6.52 (1H, d,
J = 8.7 Hz, H-6), 6.50 (1H, d, J = 2.3 Hz, H-30), 5.73 (1H, dd,
J = 11.3, 4.7 Hz, H-2), 5.48 (1H, br s, 7-OH), 3.84 (3H, s, 20-OCH3),
3.82 (3H, s, 40-OCH3), 2.92–2.78 (2H, m, H-3), 2.15 (3H, s, 8-CH3);
13C NMR (CDCl3, 125 MHz): d 192.4 (C-4), 161.8 (C-7), 160.8 (C-
20), 160.1 (C-8a), 157.1 (C-40), 127.0 (C-60), 125.9 (C-5), 120.4 (C-
10), 115.0 (C-4a), 111.6 (C-8), 109.4 (C-6), 104.3 (C-50), 98.4 (C-30),
74.6 (C-2), 55.4 (20-OCH3), 55.3 (40-OCH3), 43.3 (C-3), 7.9 (8-CH3);
ESI-HRMS (+) m/z: Anal. Calcd for C18H19O5 (M+H)+: 315.1227;
found: 315.1225.
pended in PBS containing 0.1 mg/mL RNase A and 5 lg/mL propi-
dium iodide (PI). Cellular DNA content, for cell cycle distribution
analysis, was measured by flow cytometry using an Epics XL-MCL
Coulter flow cytometer (Brea, CA, USA) plotting at least 20,000
events per sample, as previously described.21,22 The percentage
of cells in the G1, S and G2/M phases of the cell cycle and the per-
centage of cells in the sub-G1 peak were determined using the
FlowJo 7.2 software (Tree Star, Inc., Ashland, OR, USA) after cell
debris exclusion.
4.2.5. Analysis of cellular apoptosis
MCF-7 cells were incubated with the compounds as described
above. Cells were then harvested and the Human Annexin-V-
FITC/PI apoptosis kit (Bender MedSystems, Vienna, Austria) was
used according to the manufacturer’s instructions in order to de-
tect apoptotic cells. Flow cytometry was carried out using an Epics
XL-MCL Coulter flow cytometer (Brea, CA, USA) plotting at least 20
000 events per sample. Data obtained from the flow cytometer was
analyzed using the FlowJo 7.2 software (Tree Star, Inc., Ashland,
OR, USA), as previously described.23,24
4.2. Biological activity
4.2.1. Reagents and stock solutions of compounds for biological
assays
Fetal bovine serum (FBS), phosphate buffer saline (PBS), and
trypsin were purchased from Gibco Invitrogen Co. (Scotland, UK).
RPMI-1640 medium with ultraglutamine was purchased from Lon-
za (Basel, Switzerland). Dimethylsulfoxide (DMSO), doxorubicin,
ethylenediaminetetracetic acid (EDTA), propidium iodide (PI),
RNase, sulforhodamine B (SRB) and trypan blue were purchased
from SigmaChemical Co. (Saint Louis, MO, USA). Trichloroacetic
acid (TCA) and Tris were purchased from Merck (Darmstadt, Ger-
many). Stock solutions of tested compounds were prepared in
DMSO and stored at ꢀ20 °C. Appropriate dilutions of the com-
pounds were freshly prepared in medium just prior to the assays.
4.2.6. Statistical analysis
All experimental data are presented as means SEM from at
least three independent experiments (most of them performed in
duplicate). Statistical analysis was carried out using an unpaired
student’s t-test. All analysis were performed comparing cells trea-
ted with compounds with blank cells (cells incubated with med-
⁄
ium only). Indicates p 6 0.05 and ⁄⁄p 6 0.01.

M. P. Neves et al. / Bioorg. Med. Chem. 20 (2012) 25–33
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This work is funded through national funds from FCT—Fundação
para a Ciência e a Tecnologia under the projectCEQUIMED—PEst-OE/
SAU/UI4040/2011, the Portuguese National NMR Network (RNRMN)
and 4.1.3/CAPES/CPLP-GRICES 00770 29/05/08. This work is also
funded by FEDER funds through the COMPETE program under the
project FCOMP-01-0124-FEDER-011057. M.P.N. has a PhD grant
(SFRH/BD/21770/2005) and R.T.L. a Post-doc grant (SFRH/BPD/
28787/2010) from FCT. The authors are also indebted to the National
Cancer Institute, Bethesda, MD, USA, for the generous gift of the
NCI-H460 cell line. IPATIMUP is an Associate Laboratory of the
Portuguese Ministry of Science, Technology and Higher Education
and is partially supported by FCT, the Portuguese Foundation for
Science and Technology.
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