In vitro antiproliferative effect of β-phenylethylamine derivatives and doxorubicin combinations on MCF/ADR cell lines

Article abstract of DOI:10.1007/s00044-012-0050-x

A series of 3,4-dimethoxyphenylethylamine derivatives was prepared from Baylis-Hillman (BH) adducts. The effect of these compounds on the proliferation of breast cancer cell lines (MCF-7) and the corresponding Doxorubicin (Dox) resistant cell lines (MCF-7

Full text of DOI:10.1007/s00044-012-0050-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:548–557
DOI 10.1007/s00044-012-0050-x
ORIGINAL RESEARCH
In vitro antiproliferative effect of b-phenylethylamine derivatives
and doxorubicin combinations on MCF/ADR cell lines
•
Wanda P. Almeida Paula C. Huber
•
•
Luciana K. Kohn Joa˜o E. de Carvalho
Received: 9 January 2012 / Accepted: 27 March 2012 / Published online: 11 April 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of 3,4-dimethoxyphenylethylamine
derivativeswaspreparedfrom Baylis–Hillman(BH)adducts.
The effect of these compounds on the proliferation of breast
cancer cell lines (MCF-7) and the corresponding Doxorubi-
cin (Dox) resistant cell lines (MCF-7/ADR) was studied.
Aminoalcohols 1a–d were obtained from the Michael addi-
tion reaction of 3,4-dimethoxyphenylethylamine and BH
adducts. For the preparation of unsaturated amines 2a–d, BH
adducts were converted into allylic bromides by treatment
with HBrin acidicsolution. N-nucleophileintroductioncould
be controlled by the choice of solvent furnishing the corre-
sponding amines 2a–d. A third class of amines (3a–c) was
prepared by hydrogenation reaction of amines 2a–d. In
respect of the biological evaluation, some of the synthesized
compounds exhibited moderate inhibitory effect on the cell
growth. In addition, in MCF-7/ADR cell lines, the antipro-
liferative effect of Dox shifted from 5 to 88 % when
co-administrated with compound 2b.
Keywords Phenylethylamines ꢀ Multidrug resistance ꢀ
Chemosensitizers
Introduction
Resistance to chemotherapy represents a major obstacle in
the treatment of cancer. Many tumors are intrinsically
resistant to chemotherapy, whereas others initially respond
to treatment, but acquire resistance to selected cytotoxic
drugs during the therapy. Multidrug resistance (MDR) can
be broadly defined as a phenomenon by which the tumor
cells show resistance simultaneously to a variety of struc-
turally and functionally dissimilar cytotoxic and xenobi-
otics compounds. Although MDR can have several causes,
one major form of resistance to chemotherapy has been
correlated with the presence of at least three molecular
‘‘pumps’’ that actively transport drugs out of the cell
(Szakacs et al., 2006). The most prevalent of these trans-
porters is P-glycoprotein (P-gp), a 170 kDa protein that
belongs to the ATP-binding cassette (ABC) superfamily of
transporters (Sharom, 2008). P-gp both modulates the
efflux of many structurally different drugs and controls
their intestinal absorption. Traditional P-gp substrates
include, e.g., cytotoxic agent Doxorubicin (Dox), Vincris-
tine and Vinblastine, Paclitaxel, Docetaxel, Colchicine and
Epipodophyllotoxins (Seelig, 1998). P-gp activity inhibi-
tion is a way of reversing MDR, and its mechanism of
extrusion has been extensively investigated for more than
two decades. (Higgins, 1994; Stenham et al., 2003).
W. P. Almeida (&) ꢀ P. C. Huber
Institute of Chemistry, University of Campinas, Cidade
´
Universitaria Zeferino Vaz, P.O. Box 6154, Campinas,
SP 13083-970, Brazil
e-mail: wanda@iqm.unicamp.br
L. K. Kohn ꢀ J. E. de Carvalho
´
Centro Pluridisciplinar de Pesquisas Quımicas, Biologicas e
Agrıcolas, University of Campinas, P.O. Box 6171, Paulınia,
´
´
´
As soon as P-gp was recognized as the main responsible
of MDR, blocking the efflux of drugs by inhibition of the
functions of this transporter has become a way to circum-
vent MDR. Several chemicals, already known or used as
drugs for others purposes, have been tested in vitro and in
SP 13081-970, Brazil
Present Address:
L. K. Kohn
Institute of Biology, University of Campinas, Rua Monteiro
Lobato 255, Campinas, SP 13083 862, Brazil
123

Med Chem Res (2013) 22:548–557
549
vivo on resistant tumor cells. Verapamil, a calcium channel
antagonist, was the first compound found to be active in
reversing MDR and has been used as a prototype to
develop new generations of MDR modulators (Toffoli
et.al., 1995; Colabufo et al., 2008; Bisi et al., 2006). In
order to establish a molecular approach for the design of
new substrates for P-gp, different attempts have been made
to find a common set of structural and functional features
required for a compound to interact with P-glycoprotein
(Pajeva and Wiese, 2001; Huber et al., 2010). The recog-
nition elements are formed by two (type I unit) or three
(type II unit) electron donors groups with a fixed spatial
4a–d (Basavaiah et al., 2010; Almeida and Coelho, 2000) as
depicted in Scheme 1.
BH adducts 4a–d were treated with 3,4-dimethox-
yphenylethylamine to afford the corresponding amines 1 in
good to moderate yields. For adducts 4b and 4c, the 1,4-
addition led to a mixture of diastereoisomers in a 3:1 ratio in
both cases. In the case of adducts 4a and 4d, a complete lack
of selectivity was observed. Unlike diastereoisomers 1a, 1c,
and 1d, isomers 1b were separated by column chromatog-
raphy and each of them was investigated in this study.
Unfortunately, the configuration of the major isomer could
not be determined. For the preparation of the unsaturated
amines 2a–d, compounds 1a–d were treated with phosphoric
acid, but this procedure was not effective. Other conditions,
involving the preparation of more reactive derivatives, were
attempted, but they all failed. In view of this, we decided to
prepare amines 2a–d through a nucleophilic substitution
reaction, using allylic bromides as electrophiles. Thus,
adducts 4a–d were treated with a solution of HBr (Hoffmann
and Buchholz, 1991; Ameer et al., 1983) leading to the
corresponding bromides in moderate to good yields. This
reaction has aconsiderable thermodynamicdriving forceand
proceeded with allylic rearrangement giving the trisubsti-
tuted double bond. The (Z) selective formation of the olefin
was confirmed by 1HNMR spectroscopy using NOE exper-
iment. Irradiation of the signal of vinyl proton shows no
increments of the signals corresponding to the methylene
protons. This observation is in accordance with previous
˚
separation of 2.5 0.3 and 4.6 0.6 A, respectively. The
binding to P-gp increases with the strength and the number
of electron donor or hydrogen bonding acceptor groups
with a fixed spatial separation. Furthermore, lipophilicity
appears as a general requirement for the MDR-related
drugs. Lipophilic molecules that minimally contain these
groups are predicted to be substrates for P-glycoprotein.
In this article, we report the synthesis and in vitro data of
compounds 1a–d, 2a–c, and 3a–c, chosen as P-gp inhibi-
tors candidates (Fig. 1). All compounds have the 3,4-
dimethoxyphenylethylamine moiety, the corresponding
side chain of verapamil. Moreover, these structures were
selected considering the presence of two aromatic residues,
carbonyl and ether groups, providing good p orbital over-
lapping and H-bond acceptor properties, respectively
(Ha et al., 2007).
´
results published in the literature (Sa et al., 2004). Bromides
5 were then converted into the corresponding allylic amines
2a–d, by reacting with 3,4-dimethoxyphenylethylamine.
The formation of SN2 products (2a–d) showed a remarkable
solvent dependence and could be achieved when THF,
CH3CN, or DMF was used (Scheme 2). In contrast, SN20
displacement was observed when no polar solvents were
employed leading to the corresponding amines 6a–d
Results and discussion
Chemistry
The compounds were synthesized by a short-step sequence
from the corresponding Baylis–Hillman (BH) adducts
Fig. 1 Verapamil and target
compounds
OH
O
CN
O
O
O
O
O
R
N
O
O
N
H
Verapamil
1a-d
O
O
O
O
O
O
O
O
R
R
N
H
N
H
2a-d
3a-d
a: R = H; b: R = 3,4-dimethoxy; c: R = 3,4-methylenedioxy; d: R = NO2
123

550
Med Chem Res (2013) 22:548–557
Scheme 1 Reagents and
conditions. i 3,4-
OH
O
OH
O
O
O
i
dimethoxyphenylethylamine,
THF (58–72 % yields); ii HBr,
H2SO4, CH2Cl2, 0 °C ? rt
(40–82 % yields); iii 3,4-
dimethoxyphenylethylamine,
THF (70–85 % yields); iv: H2,
Pd/C 10 %, MeOH (64–90 %
yields)
O
O
R
R
N
H
4a-d
1a-d
ii
O
O
iii
O
O
O
O
R
R
Br
N
H
2a-d
5a-d
iv
O
O
O
O
R
N
H
3a-d
a: R = H; b: R = 3,4-dimethoxy; c: R = 3,4-methylenedioxy; d: R = NO2
(Table 1). Amines 6a–d have been studied in our laboratory
for other purposes, which are not presented here.
have not been reported in the literature. All products were
characterized by 1H NMR, 13C NMR, and IR spectroscopy,
and Elemental Analyses was also included for the new
compounds.
Amines 2a–c were then hydrogenated to furnish the
saturated derivatives 3a–c in moderate to good yields.
Otherwise, amine 2d led to an untreatable mixture of
compounds. Compounds of the series 1 and 3 possess a
chiral center and are therefore racemic mixtures of two
enantiomers. Although it is important to test each enan-
tiomer, at this first stage the stereoselective synthesis of
these compounds was neglected and the results of their
biological evaluation refer to the racemic mixture. At the
best of our knowledge, compounds 1a–d, 2a–d, and 3a–c
Antiproliferative assays
The in vitro antiproliferative effect of amines 1a–d, 2a–d,
and 3a–c on the MCF-7/ADR cell line at 0.1, 1.0, 2.5, 5.0,
10.0, 25.0, 50.0, and 100 lmol/L concentrations was
examined and the results are given in Table 2. The IC10
and IC50 for all the compounds lie, respectively, in the
Scheme 2 Conversion of
Bromides 5a–d into the allylic
amines 2a–d and 6a–
d. Reagents and conditions:
(i) 3,4-
O
O
R
Br
dimethoxyphenylethylamine,
K2CO3, THF or DMF or
CH3CN, 0 °C ? rt, 24–48 h;
(ii) 3,4-
5a-d
ii
i
dimethoxyphenylethylamine,
K2CO3, cyclohexane, or
toluene, 0 °C ? rt, 24–48 h
O
O
O
O
O
O
NH
O
R
N
H
O
R
2a-d
6a-d
a: R = H; b: R = 3,4-dimethoxy; c: R = 3,4-methylenedioxy; d, R = NO2.
123

Med Chem Res (2013) 22:548–557
551
Verapamil was utilized as reference compound. The results
are summarized in Table 3, and the antiproliferative effect
is expressed as the percentage of GI. Among the drug
combinations, 2b ? Dox (entry 8) showed a fairly signif-
icant sensitizing effect on the MCF-7/ADR cell lines. In
the presence of verapamil, the antiproliferative effect of
Dox was moderately increased. In turn, the presence of
compound 2c has promoted a slight increase in the anti-
proliferative activity of Dox. When cells were pre-treated
with the studied amines, no significant variation in these
values were observed.
Table 1 Regioselectivity of the reactions of bromides 5 with (3,4-
dimethoxyphenyl)ethylamine
Bromides
Solvent
Yield (%)
2a–d
6a–d
5a
5b
5c
5d
a
Cyclohexanea
THFb
0
88
0
52
0
Cyclohexane
THF
37
0
65
25
72
28
45
Cyclohexane
THF
48
0
All the tested amines (1a–d, 2a–c, and 3a–c) have two
hydrophobic groups represented by the aromatic rings,
linked by a chain with six atoms, including one basic
nitrogen atom. Since only 10 compounds were employed in
the sensitization experiments, a concluding structure–
activity relationship could not be established, but structural
features of the most active amine are consistent with those
reported in the literature (Pajeva and Wiese, 2001) for
compounds which have demonstrated effect on the P-gp
activity, including verapamil. Compounds 2 present a
double bond in the carbon chain that could contribute to
locate ligand groups in a suitable distance to interact with a
biological target. The presence of dimethoxyl groups in
both aromatic rings of 2b seems to be essential for the
sensitization.
Cyclohexane
THF
23
0
Cyclohexane or toluene
b
THF, CH3CN or DMF
range 0.05–38.2 and 0.89–371 lmol/L. The analysis of cell
growth inhibition (GI) was performed using the sulforho-
damine B (SRB) assay. Compounds 1a–c, 2a–c, and 3a–
c showed no significant antiproliferative activity against
both drug sensitive cell line (MCF-7, data not showed) and
its drug resistant cell sublines MCF-7/ADR. In contrast, the
nitro derivative 2d exhibited good inhibitory activity on
the growth of cell lines and because of that was not used in
the MDR experiments. The importance of the nitro group
to the antiproliferative activity of acrylates was previously
demonstrated by our research group (Kohn et al., 2006).
The sensitization of Dox-resistant cell lines is generally
associated to the inhibition of drug efflux mediated by the
membrane proteins P-gp and MRP-1, which belong to the
ABC family of proteins (Boumendjel et al., 2005). P-gp is
well studied, but the exact mechanism of MRP-1 in MDR
is not clear. It is known that MRP-1 differs from P-gp in
Sensitization of MDR cells
Cells were treated with a combination of compounds 1a–d,
2a–c, and 3a–c an Dox in a sub-inhibitory concentration.
Table 3 Antiproliferative effect of drug combinations on MCF-7/
ADR cell line
Table 2 Antiproliferative effect of the synthesized compounds on
the human MDR cells
Entry
Compound
% GIb
Compound
IC10 (lmol/L)
IC50 (lmol/L)
1
Doxa
5.1 1.2
0
1aa
1b
1ca
1da
2a
14.0
238
371(298)
238
2
Verapamil
38.2 (30.1)
20.7
3
1a Dox
11.8 1.8
6.8 2.3
10.4 1.7
12.9 1.8
16.1 1.1
87.7 1.4
43.3 1.9
3.2 1.2
6.9 2.3
5.8 2.5
58.1 1.4
4
1b Dox
1c Dox
15.7
85.1
5
12.9
75.8
6
1d Dox
2b
2c
7.47
112
7
2a Dox
13.8
248
8
2b Dox
2d
3a
0.05
0.89
9
2c Dox
15.2
193
10
11
12
13
a
3a Dox
3b
3c
2.39
28.9
3b Dox
10.5
188
3c Dox
Verapamil Dox
IC10 and IC50 of the minor diastereoisomer are in parentheses. IC10
and IC50 are the concentrations required to inhibit 10 and 50 % of cell
growth
b
Dox was employed at 5 lmol/L concentration; Growth inhibition
(%); Data ? SEM are the mean of three independent determinations
(samples in triplicate)
a
Refers to the diastereomeric mixture
123

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Med Chem Res (2013) 22:548–557
several aspects, including structure, localization, and sub-
strates affinity. In this context, P-gp targets hydrophobic
substrates, while MRP-1 binds to hydrophilic molecules,
organic anions, glutathione (GSH), glucorunide, or sulfate
conjugates. Furthermore, a remarkable reduction in GSH
levels and activity of GSTs (glutathione transferases) have
been reported in Dox-resistant cancer cell lines (Meijer
et al., 1991; Peters and Roelofs, 1992).Thus, GSH-deplet-
ing cells are more susceptible to the lipid peroxidation
(Townsend et.al., 2011). Since the most active compounds
are Michael acceptor and could interact with thiol group of
GSH, the participation of GSH in the mechanism of sen-
sitization of cell lines could not be rejected.
points were measured in open capillary tubes using an
Electrothermal model 9100, and are uncorrected. Yields
were determined from GC analyses on a HP 6890 equip-
ment with a flame ionization detector, using a HP-5 cap-
illary (crosslinked 5 % Phenyl methyl siloxanes, 28 m)
column. Elemental analyses were described for unknown
compounds and are performed at CHNS 2400 Perkin Elmer
Analyser equipment. Analyses indicated by the symbols of
the elements were within 0.4 % of the theoretical values.
Manipulations and reactions were not performed under dry
atmospheres or employing dry solvents, unless otherwise
specified. Purification and separations by column chroma-
tography were performed on silica gel, using flash chro-
matography. TLC visualization was achieved by spraying
with 5 % ethanolic phosphomolybdic acid and heating.
Reactions were monitored by TLC/GC analyses. All
reagents were purchased from Aldrich and were used
without previous purification.
Conclusions
In this study, 11 new dimethoxyphenylethylamine-based
structure compounds were synthesized from BH adducts in
moderate to good yields and their antiproliferative effect on
the MCF-7/ADR cell line was evaluated. Unlike nitroaro-
matic derivative 2d that showed significant antiprolifera-
tive effect, amines 1a–d, 2a–c, and 3a–c were evaluated
for their ability to sensitize the MDR cells to Dox. Among
these compounds, the unsaturated amine 2b was more
effective. Once the studied compounds showed no intrinsic
antiproliferative activity on MDR cells, the pronounced
effect of Dox in the presence of 2b may be due to inhibi-
tion of P-gp.
General procedures for the preparation of amines 1a–d
To a stirred solution of 1.0 mmol of the BH adduct (5) in
THF (1 mL) was added 1.5 mmol of 3,4-dimethox-
yphenylethylamine and 2.0 mmol of powdered K2CO3.
The reaction was monitored by TLC (5 % MeOH–
CH2Cl2), and after completion the solvent was removed
under reduced pressure. The residue was partitioned
between ethyl acetate and brine. The organic layer was
washed twice with brine and then dried over Na2SO4.
Filtration and solvent removal furnished a residue that was
purified by flash chromatography (1 % MeOH–CH2Cl2).
Reaction time ranged from 30 to 72 h.
Aiming to clarify the mechanism by which these com-
pounds enhanced the effectiveness of Dox, flow cytometry
experiment and measurement of ATPase activity have been
carried out in our laboratory and preliminary results sug-
gest that the mechanism of cells sensitizing is correlated
with the Dox accumulation. The promising results reported
here prompt us to further investigate this new class of
compounds by the introduction of different residues from
other molecules showing important reverting activities.
Further pharmacological studies are in progress to better
evaluate the mechanism by which these classes of com-
pounds sensitize MCF-7/ADR cells to Dox, and exclude
adverse effects.
4-[2-(3,4-Dimethoxy-phenyl)-ethylamino]-2-(hydroxy-phe-
nyl-methyl)-butyric acid methyl ester (1a) Colorless oil;
68 % yield; obtained as a diastereomeric mixture in a 1:1
ratio. Reaction time: 48 h. IR (film) mmax/cm-1: 3467,
3043, 3001, 2998, 2837, 2093, 1733, 1517, 1262; 1H NMR
(CDCl3, 300 MHz, d, ppm): 7.37–7.27 (m, 5H, aromatic),
6.79–6.72 (m, 3H, aromatic), 5.12 (d, J = 6.3 Hz, 1H,
CHOH), 3.85 (s, 6H, OCH3 92), 3.62 (s, 3H, OCH3),
3.12–2.97 (m, 1H, CHC=O), 2.92–2.67 (m, 6H, CH2NH-
CH2CH2Ar), 2.31 (br s, 1H, exchangeable with D2O); 13C
NMR (75.4 MHz, CDCl3) d 173.1, 149.5, 147.7, 147.6,
137.6, 134.2, 128.1 (92), 127.1 (92), 126.8, 120.1; 111.9,
71.1, 55.9, 55.8, 51.4, 51.2, 48.3, 35.8, 25.8; Anal. Calcd
for C21H27NO5: C, 67.54; H, 7.29; N, 3.75; Found: C,
67.52; H, 7.27; N, 3.71.
Experimental
Chemistry
General
[2-(3,4-Dimethoxy-phenyl)-ethylamino]-2-[(3,4-dimethoxy-
phenyl)-hydroxy-methyl]-butyric acid methyl ester (1b)
Colorless oil, yield 58 % yield; obtained as a diastereo-
meric mixture in a 3:1 ratio. Reaction time: 72 h. IR (film)
1H and 13C spectra were recorded on a Varian GEMINI
BB-300 at 300 and 75.4 MHz, respectively. IR spectra
were obtained with a Nicolet model Impact 410. Melting
123

Med Chem Res (2013) 22:548–557
553
1
max/cm-1: 3056, 3003, 2950, 2837, 1733, 1517, 1262; H
General procedures for the synthesis of amines 2a–d
m
NMR (CDCl3, 300 MHz, d, ppm), major diastereoisomer
6.81–6.69 (m, 6H, aromatic), 5.07 (d, J = 6.1 Hz, 1H,
CHOH), 3.81 (s, 6H, OCH3 92), 3.73 (s, 6H, OCH3 92),
3.63 (s, 3H, CO2CH3), 3.20–3.16 (m, 3H, CHC=O,
CH2NH), 3.14 (br s, 1 H, exchangeable with D2O),
2.91–2.86 (m, 2H, NHCH2CH2Ar), 2.76–2.68 (m, 2H,
CH2Ar); 13C NMR (75.4 MHz, CDCl3) d 173.5, 149.4,
148.8, 148.5, 147.6, 147.5, 137.4, 134.1, 120.7, 120.5,
120.2, 111.9, 111.4, 67.9, 55.9, 55.8, 51.9, 51.8, 51.4, 51.2,
48.3, 35.8, 25.6; minor diastereoisomer: 6.67–6.63 (m,
6H), 5.20 (d, J = 5.1 Hz, 1H), 3.81 (s, 6H), 3.72 (s, 3H),
3.62 (s, 3H), 3.21 (br s, 1H), 3.08–3.01 (m, 2H), 2.74 (m,
1H), 2.66 (m, 2H); 13C NMR (75.4 MHz, CDCl3) d 172.9,
148.9, 148.5, 148.2, 147.7, 134.2, 131.8, 130.7, 120.7,
111.9, 111.4, 110.7, 108.9, 67.9, 55.9, 55.8, 55.7, 51.4,
48.4, 35.6, 25.6; Anal. Calcd for C23H31NO7: C, 63.73; H,
7.21; N, 3.23; Found: C, 63.74; H, 7.27; N, 3.25.
A mixture of 3,4-dimethoxyphenylethylamine (3 mmol)
and powdered K2CO3 (3 mmol) in 3 mL of THF was
cooled to 0 °C. Then, a solution of bromides 5a–d
(1 mmol) in THF, DMF, or acetonitrile (2 mL) was drop-
ped into the mixture and stirring was continued at room
temperature. The reaction was monitored by TLC and
when the reaction is complete, the inorganic salts were
filtered off. The filter cake was washed with small portions
of THF, and the filtrate was evaporated. The residual oil
was purified by flash chromatography.
2-Benzylidene-4-[2-(3,4-dimethoxy-phenyl)-ethylamino]-
butyric acid methyl ester (2a) Eluent: hexane/acetate
70:30, viscous oil, 88 % yield. IR (film): mmax/cm-1: 3082,
1
2967, 2835, 1732, 1593, 1518, 1260; H NMR (CDCl3,
300 MHz, d, ppm): 7.80 (s, 1H, CH=C), 7.39–7.21 (m, 5H,
aromatic), 6.79–6.72 (m, 3H, aromatic), 3.87 (s, 6H, OCH3
9 2), 3.85 (s, 3H, CO2CH3), 2.92–2.73 (m, 6H,
CH2NHCH2CH2Ar); 13C NMR (CDCl3, 75 MHz): 166.7,
149.5, 147.6, 146.4, 134.4, 128.1, 127.4, 127.1, 124.2,
120.1, 111.9, 55.9, 55.7, 51.2, 48.9, 35.8, 26.3; Anal. Calcd
for C22H27NO4: C, 71.52; H, 7.37; N, 3.79; Found: C,
71.50; H, 7.39; N, 3.74.
2-(Benzo[1,3]dioxol-5-yl-hydroxy-methyl)-4-[2-(3,4-dime-
thoxy-phenyl)-ethylamino]-butyric acid methyl ester
(1c) Colorless oil, 61 % yield; obtained as a diastereo-
meric mixture in a 3:1 ratio. Reaction time: 60 h. IR (film)
m
max/cm-1: 3051, 3002, 2949, 2837, 1730, 1516, 1263;
1H NMR (CDCl3, 300 MHz, d, ppm): 6.84–6.71 (m,
6H, aromatic), 5.98 (s, 2H, OCH2O), 4.87 (d, J =
6.2 Hz, 1H, CHOH), 3.72 (s, 6H, OCH3 9 2), 3.55 (s,
3H, CO2CH3), 3.21–3.17 (m, 3H, CHC=O, CH2NH), 3.1
(br s, 1 H, exchangeable with D2O), 2.90–2.82 (m, 2H,
NHCH2CH2Ar), 2.74–2.66 (m, 2H, CH2Ar); 13C NMR
(75.4 MHz, CDCl3) d 175.5, 149.3, 147.8, 147.6, 147.3,
146.3, 135.9, 134.2, 120.2, 111.8, 110.3, 108.1, 100.8,
73.8, 55.9, 55.8, 51.2, 51.1, 48.1, 35.6, 25.7; Anal. Calcd
for C22H27NO7: C, 63.30; H, 6.52; N, 3.38; Found: C,
63.33; H, 6.56; N, 3.35.
2-(3,4-Dimethoxy-benzylidene)-4-[2-(3,4-dimethoxy-phenyl)-
ethylamino]-butyric acid methyl ester (2b) Eluent: hex-
ane/acetate 70:30, viscous oil, 65 % yield. IR (film): mmax
/
cm-1: 3055, 2960, 2837, 1734, 1593, 1517, 1262; 1H NMR
(CDCl3, 300 MHz, d, ppm): 7.78 (s, 1H, CH=C), 7.34–7.24
(m, 6H, aromatic), 3.88 (s, 6H, OCH3 92), 3.86 (s, 3H,
CO2CH3), 2.89–2.67 (m, 6H, CH2NHCH2CH2Ar); 13C
NMR (CDCl3, 75 MHz): 167.1, 150.6, 149.5, 149.3, 147.6,
146.4, 143.6, 134.4, 127.2, 126.5, 124.4, 120.1, 112.7,
111.9, 111.4, 55.9, 55.7, 51.9, 51.8, 51.2, 48.9, 35.8, 26.3;
Anal. Calcd for C24H31NO6: C, 67.11; H, 7.27; N, 3.26;
Found: C, 67.15; H, 7.31, N, 3.29.
[2-(3,4-Dimethoxy-phenyl)-ethylamino]-2-[hydroxy-(4-nitro-
phenyl)-methyl]-butyric acid methyl ester (1d) Pale yel-
low oil, 72 % yield; obtained as a diastereomeric mixture
2-Benzo[1,3]dioxol-5-ylmethylene-4-[2-(3,4-dimethoxy-
phenyl)-ethylamino]-butyric acid methyl ester (2c) Elu-
ent: hexane/acetate 60:40, colorless oil, 72 % yield. IR
(film) mmax/cm-1: 3043, 2960, 2941, 2831, 1730, 1519,
in a 1:1 ratio. Reaction time: 30 h. IR (film) mmax/cm-1
:
3420, 3036, 3002, 2935, 2830, 1732, 1515, 1260; 1H NMR
(CDCl3, 300 MHz, d, ppm): 7.52 (d, J = 8.3 Hz, 2H,
aromatic), 7.14 (d, J = 8.4 Hz, 2H, aromatic), 6.81–6.62
(m, 3H, aromatic), 4.58 (d, J = 6.9 Hz, 1H, CHOH), 3.83
(s, 6H, OCH3 92), 3.58 (s, 3H, CO2CH3), 3.4 (broad s, 1H,
exchangeable with D2O), 3.05–2.86 (m, 4H, CH2NHCH2),
2.74–2.69 (m, 2H, CH2Ar); 13C NMR (75.4 MHz, CDCl3)
d 176.1, 149.8, 149.5, 147.6, 146.4, 138.9, 134.4, 127.1,
120.1, 111.9, 73.4, 55.9, 55.7, 51.2, 48.9, 44.4, 26.3. Anal.
Calcd for C21H26N2O7: C, 60.23; H, 6.26; N, 6.69; Found:
C, 60.24; H, 6.27; N, 6.64.
1
1260; H NMR (CDCl3, 300 MHz, d, ppm) 7.76 (s, 1H
CH=C), 6.74–6.65 (m, 6H aromatic), 5.95 (s, 2H, OCH2O),
3.81 (s, 6H, OCH3 92), 3.79 (s, 3H, CO2CH3), 2.84–2.51
(m, 6H, CH2NHCH2CH2Ar), 2.9 (broad s, 1H, exchange-
able with D2O); 13C NMR (CDCl3, 75 MHz): 166.9, 149.5,
147.6, 146.4, 141.8, 135.1, 134.4, 120.2, 120.1, 111.9,
111.6, 108.2, 107.9, 107.4, 107.1, 100.8, 55.9, 55.7, 51.2,
48.8, 35.7, 26.3; Anal. Calcd for C23H27NO6: C, 66.81; H,
6.58; N, 3.39; Found: C, 66.78; H, 6.55, N, 3.36.
123

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Med Chem Res (2013) 22:548–557
4-[2-(3,4-Dimethoxy-phenyl)-ethylamino]-2-(4-nitro-ben-
zylidene)-butyric acid methyl ester (2d) Eluent: hexane/
46.8, 35.8, 25.8; Anal. Calcd for C24H33NO6: C, 66.80; H,
7.71; N, 3.25; Found: C, 66.82; H, 7.78; N, 3.28.
acetate 50:50, pale yellow oil, 35 % yield. IR (film) mmax
/
cm-1: 3190, 2958, 2941, 2837, 1733, 1590, 1517, 1498,
1260; 1H NMR (CDCl3, 300 MHz, d, ppm): 8.14 (d,
J = 8.7 Hz, 2H, aromatic); 7.78 (s, 1H, CH=C); 7.31 (d,
J = 8.6 Hz, 2H, aromatic), 6.84–6.64 (m, 3H, aromatic),
3.85 (s, 6H, OCH3 92), 3.59 (s, 3H, CO2CH3), 2.81–2.46
(m, 6H, CH2NHCH2CH2Ar), 2.1 (broad s, 1H, exchange-
able with D2O). 13C NMR (CDCl3, 75 MHz): 166.8, 149.5,
148.6, 147.6, 147.5, 146.4, 141.9, 134.4, 127.7, 127.6,
123.9, 120.1, 112.8, 111.9, 55.9, 55.8, 52.3, 48.9, 35.8,
26.3. Anal. Calcd for C22H26N2O6: C, 63.76; H, 6.32; N,
6.76; Found: C, 63.79; H, 6.39; N, 6.36.
2-Benzo[1,3]dioxol-5-yl-methyl-4-[2-(3,4-dimethoxy-phenyl)-
ethylamino]-butyric acid methyl ester (3c) IR (film):
m
max/cm-1: 3423, 2949, 2835, 1732, 1590, 1516, 1463, 1262,
1238, 1141; 1H NMR (CDCl3, 300 MHz, d, ppm): 6.74–6.65
(m, 6H aromatic), 6.04 (s, 2H, OCH2O), 3.81 (s, 6H, OCH3
92), 3.62 (s, 3H, OCH3), 2.99–2.86 (m, 3H, CHC=O and
CH2Ar), 2.82–2.62 (m, 6H, CH2NHCH2CH2Ar); 13C NMR
(CDCl3, 75 MHz): 171.9, 149.1, 148.5, 148.2, 147.6, 147.3,
136.1, 134.4, 120.2 (92), 119.7, 119.6, 111.9, 100.4, 55.9,
55.8, 51.3, 48.9, 48.2, 46.2, 35.9, 25.7; Anal. Calcd for
C23H27NO6: C, 66.49; H, 7.04; N, 3.37; Found: C, 66.45; H,
7.03; N, 3.31.
General procedure for the preparation of amines
3a–c (Hydrogenation reaction)
General procedure for the preparation of the BH adducts
(Compounds 4a–d)
To a suspension of 5 % Pd–C (10 mol%) in ethyl acetate
(5 mL) was added, under nitrogen, a solution of amines
2a–d (1 mmol) in ethyl acetate (5 mL). Then, the reaction
atmosphere was changed by hydrogen, and the reaction
mixture stirred at room temperature. After a period for
reaction completion (20 min to 2 h), the suspension was
filtered over a pad of Celite and the solvent removed under
reduced pressure. Analysis by TLC did not indicate the
presence of the starting material.
A mixture of the aromatic aldehyde (1 equiv.), an excess of
methyl acrylate (20 equiv., used as solvent and reagent),
and 1,4-diazabicyclo[2.2.2.]octane, DABCO (0.65 equiv.)
was sonicated (1000 W, 25 kHz) for a certain period of
time. The ultrasound bath temperature was constantly
monitored and kept at 30–40 °C during the reaction,
through ice addition or by using a refrigerated circulator.
After a period for reaction completion, the mixture was
evaporated under reduced pressure to remove the excess of
acrylate. The residue was diluted with ethyl acetate
(30 mL). The organic solution was washed with 10 %
aqueous HCl (29 10 mL), saturated NaHCO3 (20 mL),
brine (20 mL), and dried over Na2SO4. After filtration and
solvent removal, the residue was filtered through a pad of
gel of silica. Spectral data are in according to preceding
literature (Coelho et al., 2002).
2-Benzyl-4-[2-(3,4-dimethoxy-phenyl)-ethylamino]-butyric
acid methyl ester (3a) IR (film): mmax/cm-1: 3323, 2949,
2835, 1732, 1590, 1516, 1463, 1262, 1238, 1141; 1H NMR
(CDCl3, 300 MHz, d, ppm), 7.18–7.21 (m, 5H, aromatic),
6.77–6.71 (m, 3H aromatic), 3.81 (s, 6H), 3.85 (s, 6H,
OCH3 92), 3.62 (s, 3H, OCH3), 3.09–2.92 (m, 1H,
CHC=O), 2.87–2.83 (d, J = 6.5Hz, 2H, CH2Ar), 2.81–2.67
(m, 6H, CH2NHCH2CH2Ar); 13C NMR (CDCl3, 75 MHz):
172.8, 149.4, 147.6, 137.2, 134.2, 128.2, 127.1 (92), 126.8,
120.2, 120.1 (92), 55.9, 55.8, 51.2, 49.8, 49.6, 48.3, 35.8,
26.1; Anal. Calcd for C22H29NO4: C, 71.13; H, 7.87; N,
3.77; Found: C, 71.17; H, 7.88; N, 3.75.
Methyl 2-[hydroxy(phenyl)methyl]acrylate (4a) Eluent:
hexane/acetate 70:30, viscous oil, 74 % yield. IR (film)
1
m
max/cm-1: 3482, 3062, 1714, 1336; H NMR (300 MHz,
CDCl3, d, ppm): 7.4–7.2 (m, 5H, aromatic), 6.33 (s, 1H,
CHOH), 5.83 (s, 1H, CHa=C), 5.55 (s, 1H, CHb=C), 3.71
(s, 3H, OCH3), 3.14 (br s, 1H, exchangeable with D2O);
13C NMR (75.4 MHz, CDCl3) d 166.9, 142.1, 141.4, 128.5,
127.9, 126.7 (92), 126.1, 73.2, 51.9.
2-(3,4-Dimethoxy-benzyl)-4-[2-(3,4-dimethoxy-phenyl)-eth-
ylamino]-butyric acid methyl ester (3b) IR (film): mmax
/
cm-1: 3413, 2945, 2834, 1732, 1591, 1515, 1463, 1261,
1236, 1140; 1H NMR (CDCl3, 300 MHz, d, ppm):
6.72–6.63 (m, 6H aromatic), 3.81 (s, 6H, OCH3 9 2), 3.73
(s, 6H, OCH3 92), 3.61 (s, 3H, CO2CH3), 2.99–2.94 (m,
1H, CHC=O), 2.91–2.86 (m, 4H, CH2NHCH2), 2.74–2.66
(m, 2H, CH2Ar), 2.4 (broad s, 1H, exchangeable with
D2O); 13C NMR (CDCl3, 75 MHz): 171.9, 148.7; 148.9,
148.5, 148.2, 147.6 (92), 147.3, 136.9, 134.2, 120.4, 120.3,
119.8, 119.7, 111.9, 55.8, 55.7, 51.9, 51.8, 51.3, 49.1, 48.3,
Methyl 2-[3,4-dimethoxy-5-yl(hydroxy)-methyl]acrylate (4b)
Eluent: hexane/acetate 80:20, white solid, mp: 55–54 °C,
Lit. (Coelho et al., 2002): 56–54 °C, 63 % yield. IR (KBr)
m
max/cm-1: 3490, 3100, 1708, 1622; 1H NMR (CDCl3,
300 MHz, d, ppm): 6.87–6.84 (m, 2H, aromatic), 6.77 (d,
J = 8Hz, 1H aromatic), 5.9 (s, 2H, OCH2O), 5.85 (broad s,
2H), 3.45 (d, J = 5Hz, 1H, CHOH), 3.7 (s, 3H, OCH3), 3.1
(d, J = 4.8 Hz, 1H, exchangeable with D2O); 13C NMR
123

Med Chem Res (2013) 22:548–557
555
(75.4 MHz, CDCl3) d 166.7, 147.7, 147.2, 141.9, 135.2,
125.9, 120.1, 108.1, 107.1, 101.1, 51.9.
aromatic), 6.88 (dd, J = 8,4 and 4.1 Hz, 1H aromatic),
6.02 (s, 2H, OCH2O), 4.25 (s, 2H, CH2Br), 3.87 (s, 3H,
OCH3); 13C NMR (CDCl3, 75 MHz): d 166.9, 141.8,
135.1, 120.2, 111.6, 108.2, 107.9, 107.4, 107.1, 101.2,
52.4, 28.2.
Methyl 2-[1,3-benzodioxol-5-yl(hydroxy)-methyl]acrylate
(4c) Eluent: hexane/acetate 50:50, viscous oil, 61 %
yield. IR (film) mmax/cm-1: 3442, 3119, 1706, 1620, 1480;
1H NMR (CDCl3, 300 MHz, d, ppm): 6.91 (dd, J = 8.0
and 2.1 Hz, 1H aromatic), 6.88 (d, J = 2.2 Hz, 1H aro-
matic), 6.83 (d, J = 7.9 Hz, 1H aromatic), 6.33 (d,
J = 1.1 Hz, 1H, CHa=C), 5.84 (dd, J = 1.5 and 1.1 Hz,
1H, CHb=C) 5.53 (br s, 1H, CHOH), 3.88 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.74 (9s, 3H, CO2CH3); 13C NMR
(75.4 MHz, CDCl3) d 166.4, 148.5, 148.2, 141.6, 125.4,
118.5, 110.5, 109.3, 72.6, 55.5, 55.4, 51.5.
Methyl (Z)-2-(bromomethyl)-3-(3,4-dimethoxyphenyl)-2-
propenoate (5c) Eluent: hexane/acetate 50:50, white
´
solid, 72 % yield, mp: 69–71 °C, Lit. (Sa et al., 2004):
70–71 °C. IR (KBr): mmax/cm-1: 2954, 2835, 1710, 1614,
1597, 1513, 1454, 1241, 1187, 1083; H NMR (CDCl3,
1
300 MHz, d, ppm): 7.80 (s, 2H, CH=C), 7.27 (d,
J = 1.9Hz, 1H, aromatic), 7.19 (dd, J = 8.4 and 2.1 Hz,
1H aromatic), 6.96 (d, J = 8.3 Hz, 1H aromatic), 4.48 (s,
2H, OCH2O), 3.94 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.88
(s, 3H, OCH3); 13C NMR (CDCl3, 75 MHz): 167.1, 150.6,
149.3, 143.6, 127.2, 126.5, 112.7, 111.4, 56.3, 56.2, 52.7,
28.1.
Methyl 2-[hydroxy(4-nitrophenyl)methyl]-acrylate (4d) Elu-
ent: hexane/acetate 70:30, yellow tinged solid, 88 % yield,
mp: 73–74 °C, Lit. (Coelho et al., 2002): 74 °C. IR (KBr)
m
max/cm-1: 3510, 2991, 1722, 1632; 1H NMR (CDCl3,
300 MHz, d, ppm): 8.71 (d, J = 8.8 Hz, 2H aromatic), 7.56
(d, J = 8.8 Hz, 2H aromatic), 6.40 (s, 1H, CHa=C), 5.89 (s,
1H, CHb=C), 5.64 (d, J = 5.9 Hz, 1H, CHOH), 3.75 (s, 3H,
OCH3), 3.40 (d, J = 6.1 Hz, 1H, exchangeable with D2O);
13C NMR (75.4 MHz, CDCl3) d 166.6, 148.7, 147.6, 141.1,
127.5, 127.4, 126.8, 123.7 (92), 72.8, 52.2.
Methyl (Z)-2-(bromomethyl)-3-(4-nitrophenyl)-2-propeno-
ate (5d) Yellow solid, 52 %, mp: 123–124 °C, Lit.
(Hoffmann and Buchholz, 1991): 123 °C. IR (KBr): mmax
/
cm-1: 3005, 2950, 1719, 1626, 1518, 1349; 1H NMR
(CDCl3, 300 MHz, d, ppm): 8.33 (m, 2H, aromatic), 7.83
(s, 1H, CH=C), 7.78 (m, 2H, aromatic), 4.32 (s, 2H,
CH2Br), 3.91 (s, 3H, OCH3); 13C NMR (CDCl3, 75 MHz):
166.8, 148.6, 147.5, 141.9, 127.7, 127.6, 123.9 (92), 112.8,
52.3, 28.6.
Synthesis of Bromide 5a–d
To a stirred solution of the BH adduct (3.0 mmol) in
1.5 mL of CH2Cl2 at 0 °C was carefully added 0.3 mL
(19.95 mmol) of 48 % HBr followed by concentrated
H2SO4 (0.15 mL, 9.75 mmol). The reaction was allowed to
warm to 25 °C and stirring was continued overnight. After
this period, the mixture was carefully diluted with CH2Cl2
and water. The aqueous phase was extracted twice with
CH2Cl2 and the combined organic layers washed twice
with water and then dried over Na2SO4 and evaporated.
The residue was purified by flash chromatography.
Biological assays
Cytotoxic assay
The anticancer activity was performed according to the in
vitro cancer screen methodology of the National Cancer
Institute, USA (Alley et al., 1988). The cells were main-
tained in RPMI-1640 medium, supplemented with 10 %
fetal calf serum (FCS) at 37 °C with 5 % CO2. The med-
ium was changed every 2 days until the cells reached
confluence, at which point they were subcultured. The
adherent cell lines were detached from the culture flasks by
adding 0.5 mL trypsin solution 0.025 %. The trypsin was
inactivated by adding 5 mL of 5 % FCS in RPMI-1640
medium. After counting, the cells were diluted to appro-
priate seeding densities and transferred to 96-well micro-
titer plates in a fixed volume of 100 lL per well. The
seeding densities were 6.5 9 104 and 5 9 104 cells/mL,
for MCF-7 and MCF/ADR, respectively. This initial cell
concentration was determined from individual growth
curves and ensured that the cells would be in the loga-
rithmic growth throughout the experiment. The microtiter
plates containing cells were pre-incubated for 24 h at
37 °C to allow stabilizations before addition (100 lL) of
Methyl (Z)-2-(bromomethyl)-3-phenyl-2-propenoate (5a)
Eluent: hexane/acetate (90:10), 65 % yield, colorless oil.
1
IR (film): mmax/cm-1: 2952, 1715, 1625; H NMR (CDCl3,
300 MHz, d, ppm): 7.80 (s, 1H, CH=C), 7.39–7.21 (m, 5H
aromatic), 4.38 (s, 2H, CH2Br), 3.87 (s, 3H, OCH3); 13C
NMR (CDCl3, 75 MHz): d 166.7, 128.5, 128.4, 128.3,
128.2, 127.4, 126.5, 124.2, 52.8, 28.3; Anal. Calcd for
C11H11BrO2: C, 51.79; H, 4.35; Found: C, 51.81; H, 4.37.
Methyl (Z)-2-(bromomethyl)-3(-3,4-methylenedioxyphenyl)-
2-propenoate (5b) Pale yellow solid, mp: 70–71 °C, Lit.
´
(Sa et al., 2004): 70–71 °C, 78 % yield. IR (KBr): mmax
/
cm-1: 2957, 1711, 1625; H NMR (CDCl3, 300 MHz, d,
ppm): 7.72 (s, 1H, CH=C), 7.15 (d, J = 8.1 Hz, 2H
1
123

556
Med Chem Res (2013) 22:548–557
samples as well as Dox. The plates were incubated with the
samples for 48 h at 37 °C and 5 % CO2. Compounds were
tested at eight concentrations (0.1, 1.0, 2.5, 5.0, 10.0, 25.0,
50.0, and 100 lmol/L), each in triplicate wells. The stock
solution was diluted with complete medium containing
fold potentiation was calculated by dividing the IC50 value
for Dox in the absence of modulator by the IC50 in the
presence of modulating agent (Colabufo et al., 2008).
Statistical analysis
50 lg of gentamicin mL-1
.
The IC50 values of the compounds reported in Tables and
were determined by nonlinear curve fitting utilizing the
Graph Pad Prism program.
SRB assay
This non-clonogenic methodology permits a highly sensi-
tive protein with a straight relationship to cell culture. The
SRB assay was done as described by Skehan et al. (1990).
Briefly, the cells were fixed with 50 % TCA (sigma) at
4 °C (50 lL per well, final concentration 10 %) for 1 h.
The supernatant was then discarded, and the plates were
washed five times with tap water. The cells were stained
for 30 min with 0.4 % SRB in 1 % acetic acid (50 lL per
well) and subsequently washed 49 with 1 % acetic acid to
remove unbound dye. The plates were air dried, and pro-
tein-bound dye was solubilized with 150 lL (100 mM) of
Trizma buffer. The resulting optical density was read in a
multiwell plate reader at 540 nm.
Acknowledgments W. P. Almeida is grateful to Brazilian National
Council of Research (CNPq). We also thank Prof. Jonathan Bradnick
for helpful suggestions about English grammar and style.
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