Synthesis and biological evaluation of cytotoxic properties of stilbene-based resveratrol analogs

Article abstract of DOI:10.1016/j.ejmech.2008.05.003

This work deals with the preparation of stilbene-based resveratrol analogs by employing the Perkin reaction, aiming at synthesizing potential antitumor lead compounds and evaluating their pharmacological activities. The proliferation inhibitor test against tumor cell lines identified analogs 9 and 11 as the most active among all synthesized derivatives, presenting IC₅₀ in micromolar range for certain cell lines. For study on the embryonic development, compounds 8 and 9 at the lowest tested concentration (41.7 μM) that inhibited sea urchin egg development, but only after third cleavage were used. Both the compounds inhibited 100% of normal development since first cleavage. These data partially corroborated the results obtained with MTT assay using tumor cell lines. None of the tested compounds revealed hemolytic action in assay with mouse erythrocytes.

Full text of DOI:10.1016/j.ejmech.2008.05.003

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 701e707
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of cytotoxic properties
of stilbene-based resveratrol analogs
a,
Denis Pires de Lima , Rodrigo Rotta , Adilson Beatriz , Maria Rita Marques ,
a
a
b
*
Raquel C. Montenegro , Marne C. Vasconcellos , Claudia Pessoa , Manoel O. de Moraes ,
ˆ
c
c
c
c
´
c
d
´
Letıcia V. Costa-Lotufo , Alexandra Christine Helena Frankland Sawaya ,
Marcos Nogueira Eberlin d
a Departamento de Qu´ımica, Universidade Federal de Mato Grosso do Sul, CP 549, 79070-900 Campo Grande, MS, Brazil
b Departamento de Morfofisiologia, Universidade Federal de Mato Grosso do Sul, CP 549, 79070-900 Campo Grande, MS, Brazil
c Laborato´rio de Oncologia Experimental, Universidade Federal do Ceara´, Fortaleza, CE, Brazil
d Laborato´rio Thomson de Espectrometria de Massas, Instituto de Qu´ımica, Universidade Estadual de Campinas,
UNICAMP, Campinas, SP 13083-970, Brazil
Received 29 October 2007; received in revised form 2 May 2008; accepted 7 May 2008
Available online 15 May 2008
Abstract
This work deals with the preparation of stilbene-based resveratrol analogs by employing the Perkin reaction, aiming at synthesizing potential
antitumor lead compounds and evaluating their pharmacological activities. The proliferation inhibitor test against tumor cell lines identified analogs
9 and 11 as the most active among all synthesized derivatives, presenting IC50 in micromolar range for certain cell lines. For study on the embryonic
development, compounds 8 and 9 at the lowest tested concentration (41.7 mM) that inhibited sea urchin egg development, but only after third
cleavage were used. Both the compounds inhibited 100% of normal development since first cleavage. These data partially corroborated the results
obtained with MTT assay using tumor cell lines. None of the tested compounds revealed hemolytic action in assay with mouse erythrocytes.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Stilbenes; Resveratrol analogs; Cytotoxic properties; Embryonic development
1. Introduction
Resveratrol is a polyphenolic compound produced by
several species and found especially in Polygonum roots,
peanuts, seeds, berries and also grapes, and consequently pres-
ent in human diet or beverages (red wine, for example) [6].
It provides cancer chemopreventive effects for different
systems based on its striking inhibition of diverse cellular
events associated with tumor initiation, promotion, and
progression. It has also been shown that the physiological
quantities of resveratrol can modulate multiple cellular path-
ways relevant for tumorigenesis, among them include phase
I and phase II drug-metabolizing enzymes, DNA-synthesis
and inflammatory response, as well as cell survival, cell death
and cell cycle [7]. A majority of in vitro studies and some in
vivo studies show that resveratrol can affect proliferation
and survival through the inhibition of cell cycle, stimulation
Nature has been a source of medicinal agents for thousands of
years and an impressive number of modern drugs have been
isolated from natural sources or derived from natural product
molecules, especially in cancer therapy. Stilbene derivatives,
such as the plant microcomponents resveratrol (3,5,40-trihy-
droxy-trans-stilbene) and combretastatin A-4 (Fig. 1), exhibit
a variety of biological activities including antineoplastic, chemo-
preventive, antioxidant and antiestrogenic [1e4]. Combretastatin
A-4 is the most potent antimitotic agent isolated from Combre-
tum caffrum and it interacts with the colchicine site on tubulin [5].
* Corresponding author. Tel.: þ55 67 3345 3578; fax: þ55 67 3345 3552.
E-mail address: dlima@nin.ufms.br (D.P. de Lima).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.003

702
D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
OH
The procedures were performed in conformity with a syn-
4'
MeO
MeO
thetic study regarding the re-investigation of the Perkin
reaction that was recently re-investigated [10]. Structures of
these compounds were characterized using IR, HRMS (see
5
HO
OMe
OH
3
OH
1
Section 4), H NMR (Table 1), and 13C NMR (Table 2) anal-
OMe
Combretastatin A-4
Fig. 1. Structure of bioactive stilbenes.
yses alongside comparison with data from literature for similar
compounds [10e20].
Resveratrol
2.2. Cancer cell assays
of differentiation and induction of apoptosis in cancer cell
lines [7,8].
The cytotoxicity on tumor cell lines was evaluated by the
MTT assay and the results are summarized in Table 3. Resver-
atrol inhibited the proliferation of all tested cell lines with IC50
ranging from 10.74 mM in HL-60 to 62.91 mM in B-16 cells.
As previously mentioned, many studies had emphasized the
great potential of resveratrol as a potent chemotherapeutic
agent leading cancer cells to apoptosis [9,21e23]. Previous
data from studies using HL-60 leukemia cell line as a model
demonstrated IC50 values for resveratrol of the same order
of magnitude as the present study (12.1 mM) [23]. Addition-
ally, it was demonstrated that the antileukemic activity is
related to the depolarization of mitochondrial membranes
and activation of caspase 9, triggering apoptosis [21]. The
cytotoxic effects of resveratrol on MDA-MB-435 and MCF-
7 included a dramatic G0/G1 arrest, alterations in the
expression of Cyclin D1 and Stat3 phosphorylation [22].
To further enhance the cytotoxicity of resveratrol, a few
analogs have been synthesized and investigated in the search
for an anticancer agent with higher efficacy than resveratrol.
Among the analogs obtained in the present study, only
compounds 9 and 11 presented a weak to moderate cytotoxic-
ity. Compound 11 inhibited only MDA-MB-435 proliferation
with an IC50 of 58.8 mM, while compound 9 was active against
HL-60, K-562 and MDA-MB-435 cells, with IC50 values of
26.71, 26.55 and 25.30 mM, respectively.
In fact, the anticancer potential of resveratrol has been
widely studied, but it is still important to contribute for an
ample knowledge on the biological effects of related com-
pounds [9]. With this purpose, this work has been aimed at
synthesizing selected stilbene-based resveratrol analogs and
evaluating the cytotoxic potential of these compounds, thus
giving a platform for the correlation between structure and
activity using three different cell models: tumor cell lines,
sea urchin eggs and mouse erythrocytes.
2. Results and discussion
2.1. Chemistry and spectroscopic means
Our research group has decided to employ simple method-
ology to rapidly obtain molecules for screening of cytotoxic
properties. Perkin reaction seemed to be very efficient and
useful to assemble the correct units for building these types
of carbogenic frameworks given that it has been applied to
synthesize resveratrol itself [10].
As starting materials we have chosen compounds 1e4
(Scheme 1). The Perkin type condensation was employed as
the first step for the preparation of compounds 5e8. After
decarboxylation of compound 5, catalyzed by copper chromite
in quinoline, compound 9 was formed and treated with
methanol and concentrated hydrochloric acid to afford its
isomer 10. Compound 6 was submitted to hydrolysis under
basic conditions giving compound 11.
Kang et al. [9] have described that low concentrations of
resveratrol dimers and trimers, unlike resveratrol, strongly
decreased the MTT-reducing activity by more than 3-fold in
HL-60 leukemia cell line. Also, Cai et al. [24] have found
that 3,4-dihydroxyl groups are important to enhance
O
R2
CO2H
OH
R1
R
NEt3/ Ac2O
H
O
+
7: R= OMe; R1= R2= H
8: R= R1= H; R2= OH
reflux
R2
R
phenylacetic
acid
R1
5: R= R1= OMe; R2= H 6: R= OAc; R1= OMe; R2= H
1: R= OH; R1= OMe; R2= H
2: R=R1= OMe; R2= H
3: R= OMe; R1 = R2= H
4: R= R1= H; R2= OH
Quinoline
NaOH
CuCr2O
r. t.
260ºC
CO2H
MeO
HO
MeO
MeO
HCl / MeOH
r. t.
MeO
11
9
MeO
10
Scheme 1. Synthesis of resveratrol analogs.

D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
703
Table 1
1H NMR spectral data of synthesized compounds
Compound
1H NMR (CDCl3) d
5
3.35 (s, 3H), 3.83 (s, 3H), 6.41 (d, 1H, J ¼ 1.6 Hz), 6.72 (d, 1H, J ¼ 8.4 Hz), 6.86 (dd, 1H, J ¼ 8.4, 1.6 Hz),
7.24e7.43 (m, 5H), 7.87 (s, 1H)
6
2.27 (s, 3H), 3.35 (s, 3H), 6.53 (d, 1H, J ¼ 1.7 Hz), 6.84 (dd, 1H, J ¼ 8.2, 1.7 Hz), 6.91 (d, 1H, J ¼ 8.2 Hz),
7.26e7.45 (m, 5H), 7.89 (s, 1H)
7
8
9
3.75 (s, 3H), 6.68 (d, 2H, J ¼ 8.7 Hz), 7.01 (d, 2H, J ¼ 8.7 Hz), 7.23e7.39 (m, 5H), 7.89 (s, 1H)
7.13e7.54 (m, 7H), 7.70 (d, J ¼ 6.5 Hz, 2H), 7.80 (s, 1H)
3.57 (s, 3H), 3.81 (s, 3H), 6.53 (s, 2H), 6.71 (d, 1H, J ¼ 8.2 Hz), 6.78 (d, 1H, J ¼ 1.6 Hz), 6.82 (dd, 1H, J ¼ 8.2, 1.6 Hz),
7.16e7.31 (m, 5H)
10
11
3.89 (s, 3H), 3.93 (s, 3H), 6.85 (d, 1H, J ¼ 8.0 Hz), 6.95 (d, 1H, J ¼ 16.1 Hz), 7.05 (d, 1H, J ¼ 16.1 Hz), 7.06 (s, 1H),
7.22 (t, 1H, J ¼ 7.4 Hz), 7.33 (t, 2H, J ¼ 7.4 Hz), 7.48 (d, 2H, J ¼ 7.4 Hz)
3.28 (s, 3H), 6.26 (d, 1H, J ¼ 1.5 Hz), 6.65 (d, 1H, J ¼ 8.3 Hz), 6.70 (dd, 1H, J ¼ 1.5, 8.3 Hz), 7.13e7.33 (m, 5H), 7.74 (s, 1H)
anticancer activity of trans-resveratrol analogs, what corrobo-
rates with Murias et al. [23] who demonstrated that hydroxyl-
ation of resveratrol at 3, 40 and 5 positions led to a higher
cytotoxicity in HL-60 cells than resveratrol. Moreover, several
studies demonstrated that the introduction of methoxy groups
into resveratrol, as observed in trans-3,4,5,40-tetramethoxystil-
bene, enhances its antiproliferative effects against several
cancer cell lines and may have different cellular targets than
resveratrol, inhibiting microtubule assembly [22].
It is difficult to accurately elucidate a structureeactivity
relationship that satisfies the disclosed results with respect to
reduced activity of the obtained analogs when compared to
resveratrol. Gaukroger et al. [25] have pointed out that anti-
ring configuration of stilbenes shows a remarkable decrease
in their inhibitory effects on cancer cell growth when
compared to their corresponding syn isomers. Initially, this
information suggested that the prepared compounds with the
exception of compound 10 would present a better activity
than resveratrol. Presumably, the absence of a C-40 hydroxyl
group in all compounds might have caused diminution in the
cytotoxicity. Nevertheless, compounds 9 and 11 have no great
variation in molecular framework from compounds 5e8 what
places an intriguing question with respect to distinct biological
behavior. The activity is not entirely predictable. The addi-
tional hydroxyl group in the B ring seemed to disproportion-
ately enhance activity in some cases as in resveratrol or
combretastatin A-4. This could also be derived from confor-
mational issues [5] leading to a poor interaction with key
amino acid residues in the receptor site, and this in turn results
in poor cytotoxicity.
2.3. Antimitotic activity
The antimitotic activity was determined as the ability to
inhibit sea urchin egg development. The sea urchin egg
development has some peculiarities, making it possible to
suggest how the test substances acted. The sea urchin cell
cycle is highly abbreviated, essentially cycling from S
(synthesis) to M (mitosis) and the S with no G1 phase and
a relatively short G2 phase [26]. The inhibition of the first
cleavage in these cells is related to DNA and/or protein syn-
thesis or microtubule assembling, once RNA synthesis is very
slow or absent after fertilization [27]. At this time, the rapid
increase in the rate of protein synthesis is largely due to the
recruitment of material mRNA into polysomes [28]. How-
ever, when the substance interacts with microtubule assembly,
clear spots indicating nucleus duplication can be observed in
the cytoplasm. In the present work (Table 4), resveratrol at
43.8 mM inhibited the sea urchin egg development since the
first cleavage showing that this compound may be interacting
with DNA and/or protein synthesis. In addition, compounds 8
and 9 at the lowest tested concentration (41.7 mM) also
Table 2
13C NMR spectral data of synthesized compounds
Compound
13C NMR (CDCl3) d
5
55.1 (CH3), 55.8 (CH3), 110.4 (CH), 112.3 (CH), 126.1 (CH), 127.1 (C), 127.9 (CH), 128.9 (2CH), 129.0 (C), 130.0 (2CH), 136.0 (C),
142.4 (CH), 148.2 (C), 150.4 (C), 172.5 (C)
6
20.6 (CH3), 55.2 (CH3), 113.7 (CH), 122.6 (CH), 124.9 (CH), 125.9 (C), 128.1 (CH), 128.9 (2CH), 129.8 (2CH), 132.9 (C), 135.4 (C),
140.7 (CH), 141.6 (C), 150.5 (C), 168.7 (C), 172.1 (C)
7
55.2 (CH3), 113.8 (2CH), 126.9 (C), 127.9 (CH), 128.8 (2CH), 129.1 (C), 129.8 (2CH), 132.7 (2CH), 135.8 (C),
142.1 (CH), 160.6 (C), 173.0 (C)
8
116.0 (CH), 119.6 (C), 124.1 (CH), 127.5 (CH), 128.1 (2CH), 128.1 (2CH), 128.3 (C), 128.4 (CH), 131.4 (CH), 134.7 (C),
139.8 (CH), 153.4 (C), 160.6 (C)
9
54.7 (CH3), 55.1 (CH3), 110.4 (CH), 111.3 (CH), 121.4 (CH), 126.4 (CH), 127.7 (2CH), 128.3 (2CH), 128.4 (CH), 129.3 (C), 129.5 (C),
137.2 (C), 147.8 (C), 147.8 (C)
10
11
55.9 (CH3), 56.0 (CH3), 108.8 (CH), 111.2 (CH), 119.9 (CH), 126.3 (2CH), 126.8 (CH), 127.3 (CH), 128.5 (CH), 128.7 (2CH),
130.5 (C), 137.5 (C), 148.9 (C), 149.1 (C)
55.2 (CH3), 111.9 (CH), 112.9 (CH), 126.6 (C), 126.8 (CH), 127.9 (CH), 128.6 (C), 128.9 (2CH), 130 (2CH), 136.1 (C), 142.5 (CH),
145.9 (C), 147.3 (C), 172.0 (C)

704
D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
Table 3
Inhibitory effect on cultured cell growth of resveratrol and its analogs on tumor cell lines
Compound
B-16
HCT-8
HL-60
CEM
K-562
MBA-MD-435
PC3
Doxorubicin
0.03 (0.05)
0.02e0.04
14.35 (62.91)
11.97e17.21
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
>25 (104)
0.04 (0.07)
0.03e0.05
5.82 (25.51)
5.14e6.59
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
>25 (104)
0.02 (0.03)
0.01e0.02
2.45 (10.74)
1.95e3.09
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
6.42(26.71)
0.02 (0.03)
0.01e0.02
5.91 (25.91)
4.39e7.97
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
>25 (104)
0.14 (0.24)
0.14e0.21
9.62 (42.18)
3.48e2.65
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
6.38 (26.55)
3.13e13.01
>25 (104)
>25 (82.8)
0.47 (0.81)
0.34e0.65
0.45 (0.77)
0.38e0.68
3.51 (15.39)
2.99e2.45
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
>25 (104)
Resveratrol
13.81 (60.55)
10.44e18.25
>25 (88.0)
>25 (80.1)
>25 (98.4)
>25 (104)
5
6
7
8
9
6.08 (25.30)
2.22e16.70
>25 (104)
10
11
>25 (104)
>25 (82.8)
>25 (104)
>25 (82.8)
>25 (104)
>25 (82.8)
>25 (104)
>25 (82.8)
>25 (104)
>25 (82.8)
17.76 (58.8)
16.36e19.30
Data are presented as IC50 mg/mL (mM) values and their 95% confidence interval (CI, 95%) obtained by nonlinear regression. Doxorubicin was used as a positive
control.
inhibited sea urchin egg development, but only after third
cleavage. At 41.7 mM, both the compounds inhibited 100%
of normal development since first cleavage. These data
partially corroborated the results obtained with MTT assay
using tumor cell lines, where compound 9 was the most
active among all synthesized derivatives. On the other hand,
compound 11 was poorly active in this model, indicating
that the structural requirements for cytotoxic action will
depend upon the model used.
This is the first report on resveratrol’s effects on sea urchin
eggs. This model has been used for decades to detect cyto-
toxic, teratogenic and antineoplastic activities of new
compounds [29e31]. It is worthwhile mentioning that stilbene
effects on sea urchin eggs are linked to antioxidant properties.
Tamoxifen e the selective estrogen receptor modulator used in
the treatment of breast cancer e effects on sea urchin eggs
included early embryonic mortality to exposed embryos and
to offspring exposed eggs, developmental defects to the
offspring of exposed sperm, decrease in sperm fertilization
success and cytogenetic effects in the offspring of exposed
sperm or eggs [32].
2.4. Hemolytic activity
In order to verify whether the observed cytotoxicity is
related to membrane disruption, resveratrol analogs were
tested for their ability to induce lysis of mouse erythrocytes.
The erythrocyte membrane is a dynamic structure that can
show significant changes on interaction with drugs [33]. How-
ever, these substances show no lytic activity, suggesting that
the mechanism of cytotoxicity is not a result of membrane
damage. Getting all the results together, the cytotoxicity of
resveratrol and its analogs may be caused by more specific
pathway probably involving DNA and protein synthesis
instead of membrane damage.
Table 4
Percentage of the inhibition of the cell proliferation by the resveratrol and its
analogs on the embryos of the sea urchin Lytechinus variegatus on the 1st and
3rd cleavage and blastulae stages
3. Conclusion
Compound Concentration 1st Cleavage
3rd Cleavage Blastulae
mg/mL (mM) (mean ꢀ SEM) (mean ꢀ SEM) (mean ꢀ SEM)
The Perkin reaction showed to be a feasible method to
prepare stilbene analogs. By using the procedure along
with other simple methods it was possible to access the
syntheses of seven resveratrol analogs. The compounds
revealed fairly in vitro activity against cancer cell lines,
particularly compounds 9 and 11. In addition, compounds
8 and 9 strongly inhibited sea urchin egg development.
The cytotoxic effect of the stilbene-based resveratrol ana-
logs may be connected with different cellular pathways
relating to proliferation reduction such as cell cycle inhibi-
tion, differentiation or apoptosis induction, as shown for
resveratrol, however, generally, with a reduced antiprolifer-
ative action. Resveratrol showed to be largely more active
to the performed biological tests than all the prepared
compounds. However, the presented results may be used
to draw relevant information, such as SARs that might drive
future research.
Doxorubicin 10 (18.4)
100 (184)
100 ꢀ (0)
100 ꢀ (0)
97.9 ꢀ 1.1
4.3 ꢀ (1.5)
3 ꢀ (2.1)
0 ꢀ (0)
100 ꢀ (0)
100 ꢀ (0)
100 ꢀ 0
100 ꢀ (0)
100 ꢀ (0)
100 ꢀ 0
Resveratrol
5
10 (43.8)
10 (35.2)
100 (352)
10 (32.0)
100 (320)
10 (39.4)
100 (394)
10 (41.7)
100 (417)
10 (41.7)
100 (417)
10 (41.7)
100 (417)
10 (37.0)
100 (370)
0 ꢀ (0)
0.7 ꢀ (0.7)
0.7 ꢀ (0.7)
5 ꢀ (2.5)
0 ꢀ (0)
6
2.3 ꢀ (1.2)
100 ꢀ (0)
3.6 ꢀ (3.2)
9.3 ꢀ (5.5)
96.3 ꢀ 1.6
100 ꢀ 0
100 ꢀ (0)
0 ꢀ (0)
100 ꢀ (0)
0.3 ꢀ (0.3)
68.7 ꢀ (3)
97.3 ꢀ 2.7
95.4 ꢀ 2
7
0 ꢀ (0)
8
12.3 ꢀ 3.6
44.5 ꢀ 1.6
0 ꢀ (0)
9
100 ꢀ (0)
100 ꢀ (0)
6 ꢀ (2.3)
100 ꢀ (0)
7.6 ꢀ (3.8)
7.3 ꢀ (1.6)
100 ꢀ (0)
100 ꢀ (0)
83 ꢀ (5.5)
100 ꢀ (0)
5.3 ꢀ (2.7)
99.7 ꢀ (0.3)
100 ꢀ (0)
0 ꢀ (0)
10
11
0 ꢀ (0)
0 ꢀ (0)
0 ꢀ (0)
The inhibition was obtained considering negative control as 100% of cell
proliferation. Doxorubicin was used as a positive control.

D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
705
4. Materials and methods
4.2.4. (E )-3-(2-Hydroxyphenyl)-2-phenylacrylic acid (8)
Gray solid (0.84 g, 48% yield). M.p. ¼ 137e139 ꢁC. HRMS
[ESI(ꢃ)-MS]: C15H11O3 [M ꢃ H]ꢃ m/z, calcd 239.0708, found
239.0721.
4.1. Chemistry
All melting points were determined using Uniscience of
Brazil Mod. 498 equipment. Absorption FT-IR spectra were
obtained using the KBr pellet method or in chloroform
solution performed with a Perkin Elmer Mod. 783-FT spec-
trometer. NMR spectra were recorded on Bruker DPX-300
spectrometer, the chemical shifts were presented in ppm (d)
relative to TMS (d ¼ 0.0) and CDCl3 was employed as
a solvent. High resolution electrospray ionization-mass spec-
trometry (ESI-MS) analyses were performed using a Q-TOF
Micromass spectrometer in both positive and negative ion
modes with capillary set at ꢀ3000 V and cone voltage set at
ꢀ40 V, and de-solvation temperature at 100 ꢁC.
4.2.5. (Z )-1-(3,4-Dimethoxyphenyl)-2-phenyl-ethene (9)
Compound 5 (0.5 g, 1.7 mmol) and copper chromite (30 mg,
0.2 mmol) were added to quinoline (2.45 g, 19 mmol). The
reaction mixture was stirred and heated to reflux for 2 h. The
mixture was filtered over Celite and washed with ethyl acetate
(10 mL). The organic layer was washed with 2 M hydrochloric
acid (3 ꢂ 10 mL), dried over anhydrous magnesium sulfate and
concentrated to dryness in a rotaevaporator under vacuum at
45 ꢁC. A brown solid was obtained, which was purified by
chromatography on silica gel employing hexane/ethyl acetate
(3/1) as the eluent. Compound 9 was obtained as yellow oil
(0.32 g, 78% yield). FT-IR (CHCl3, cmꢃ1) nmax: 1600, 1514,
1270. HRMS [ESI(þ)-MS]: C16H17O2 [M þ H]þ m/z, calcd
241.1229, found 241.2111.
The solvents employed in the reactions and silica gel column
chromatography were previously purified and dried according
to the procedures found in literature [34]. Thin-layer chroma-
tography (TLC) was carried out on silica gel plates with a fluo-
rescence indicator F254 (0.2 mm, E. Merck); the spots were
visualized in UV light, and by spraying with 1% ethanol
solution of vanillin or by charring reagent. Purification of com-
pounds was performed using column chromatography; the
stationary phase was silica gel 60 (80e230 mesh) from ACROS
(Brazil), silica gel 60 (230e400 mesh) from Merck and Celite.
All reagents used in the present study were of analytical grade.
4.2.6. (E )-1-(3,4-Dimethoxyphenyl)-2-phenyl-ethene (10)
Compound 9 (0.020 g, 0.083 mmol) was dissolved in methyl
alcohol (5 mL) and concentrated hydrochloric acid (1 mL) was
added to the solution. The reaction mixture was allowed to stir
at room temperature for 48 h. Water was added (10 mL) and the
mixture was extracted with ethyl acetate (3 ꢂ 5 mL). The
organic layer was dried over anhydrous magnesium sulfate
and concentrated to dryness in a rotaevaporator under vacuum
at 45 ꢁC, resulting in a white solid (0.019 g, 98% yield). M.p. ¼
94e97 ꢁC. FT-IR (KBr, cmꢃ1) nmax: 1591, 1514, 1267. HRMS
[ESI(þ)-MS]: C16H17O2 [M þ H]þ m/z, calcd 241.1229, found
241.2111.
4.2. General procedure for Perkin’s reaction
Under argon, the corresponding aldehyde (1 equiv.), phenyl-
acetic acid (1 equiv.), acetic anhydride (2 equiv.) and triethyl-
amine (0.7 equiv.) were heated under reflux for 12 h, cooled at
room temperature, diluted with 50 mL of water and 50 mL of
ethyl acetate. The aqueous layer was extracted with ethyl acetate
(3 ꢂ 30 mL). The organic layer was washed with brine (50 mL),
dried over MgSO4 and concentrated under vacuum. A yellowish
solid was obtained most of the times and was purified by recrys-
tallization or chromatography on silica gel (hexane/ethyl acetate).
4.2.7. (E )-3-(4-Hydroxy-3-methoxyphenyl)-2-
phenylacrylic acid (11)
Compound 6 (0.010 g, 0.035 mmol) was added to a 10%
solution of sodium hydroxide (15 mL). The mixture was stirred
for 24 h and then diluted with ethyl acetate (3 ꢂ 10 mL). The
water layer was acidified in an ice bath. Vacuum filtration
followed by washing with cooled water (3 ꢂ 15 mL) resulted
in a white solid (0.0095 g, 95% yield). M.p. ¼ 182e185 ꢁC.
HRMS [ESI(ꢃ)-MS]: C16H13O4 [M ꢃ H]ꢃ m/z, calcd
269.0814, found 269.1530.
4.2.1. (E )-3-(3,4-Dimethoxyphenyl)-2-phenylacrylic
acid (5)
White solid (0.69 g, 49% yield). M.p. ¼ 223e225 ꢁC. FT-IR
(KBr, cmꢃ1) nmax: 3444, 1666, 1595, 1424, 1266, 1147. HRMS
[ESI(ꢃ)-MS]: C17H15O4 [M ꢃ H]ꢃ m/z, calcd 283.0970, found
283.0624.
4.3. MTT assay
The cytotoxicity of the compounds was tested against
CEM, HL-60 and K-562 (human leukemias), B-16 (murine
melanoma), HCT-8 (human colon), MBA-MD-435 (human
melanoma) and PC3 (human prostate) cell lines obtained
from the National Cancer Institute, Bethesda, MD, USA.
Cells were grown in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 2 mM glutamine, 100
mg/mL streptomycin and 100 U/mL penicillin and incubated
at 37 ꢁC with a 5% CO2 atmosphere. Cells were plated in
96-well plates (105 cells/well for adherent cells or 0.5 ꢂ
105 cells/well for suspended cells in 100 mL of medium).
4.2.2. (E )-3-(4-Acetoxy-3-methoxyphenyl)-2-
phenylacrylic acid (6)
White solid (0.75 g, 48% yield). M.p. ¼ 191e194 ꢁC. FT-IR
(KBr, cmꢃ1) nmax: 3444, 1773, 1673. HRMS [ESI(ꢃ)-MS]:
C18H15O5 [M ꢃ H]ꢃ m/z, calcd 311.0919, found 311.0972.
4.2.3. (E )-3-(4-Methoxyphenyl)-2-phenylacrylic acid (7)
White solid (0.62 g, 49% yield). M.p. ¼ 183e185 ꢁC. FT-IR
(KBr, cmꢃ1) nmax: 3000, 1671, 1603. HRMS [ESI(ꢃ)-MS]:
C16H13O3 [M ꢃ H]ꢃ m/z, calcd 253.0865, found 253.0693.

706
D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
After 24 h, the compounds (0.39e25.0 mg/mL) dissolved in
1% DMSO were added to each well and incubated for
Acknowledgements
´
72 h. Control group received the same amount of DMSO.
Ò
We wish to thank CNPq, Kardol Ind. Quımica Ltda., Insti-
tuto Claude Bernard, FUNCAP, Banco do Nordeste, FINEP
and PROPP-UFMS for the financial support in the form of
grants and fellowship awards. The authors thank Silvana
ˆ
Doxorubicin (Doxolem , Zodiac Produtos Farmaceuticos S/
A, Brazil) was used as a positive control. Tumor cell growth
was quantified by the ability of the living cells to reduce the
yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) to a purple formazan product
[35]. At the end of the incubation, the plates were centrifuged
and the medium was replaced by fresh medium (200 mL)
containing 0.5 mg/mL MTT. Three hours later, the MTT
formazan product was dissolved in 150 mL DMSO, and the
absorbance was measured using a multiplate reader (Spectra
Count, Packard, Ontario, Canada). The drug effect was
quantified as the percentage of the absorbance of reduced
dye at 550 nm in relation to control wells.
´
Franc¸a dos Santos, Luciana Franc¸a, and Maria de Fatima
Teixeira for technical assistance. We are also indebted to
Nanocore Biotecnologia Ltda. for logistic support and for
making proposals for future projects in this field.
References
[1] D. Simoni, M. Roberti, F.P. Invidiata, E. Aiello, S. Aiello, P. Marchetti,
R. Baruchello, M. Eleopra, A. Di Cristina, S. Grimaudo, N. Gebbia,
L. Crosta, F. Dieli, M. Tolomeo, Bioorg. Med. Chem. Lett. 16 (2006)
3245e3248.
[2] J. Griggs, J.C. Metcalfe, R. Hesketh, Lancet Oncol. 2 (2001) 82e87.
[3] M. Mokni, S. Elkahoui, F. Limam, M. Amri, E. Aouani, Neurochem.
Res. 32 (2007) 981e987.
4.4. Assay on sea urchins
[4] A. Cirla, J. Mann, Nat. Prod. Rep. 20 (2003) 558e564.
[5] G.R. Pettit, M.R. Rhodes, D.L. Herald, E. Hamel, J.M. Schmidt,
R.K. Pettit, J. Med. Chem. 48 (2005) 4087e4099.
[6] N. Latruffe, D. Delmas, B. Jannin, M. Cherkaoui Malki, P. Passilly-
Degrace, J.P. Berlot, Int. J. Mol. Med. 10 (2002) 755e760.
[7] K.P. Bhat, J.M. Pezzuto, Cancer Res. 61 (2001) 6137e6144.
[8] H.P. Ciolino, G.C. Yeh, Adv. Exp. Med. Biol. 492 (2001) 183e193.
[9] J.H. Kang, Y.H. Park, S.W. Choi, E.K. Yang, W.J. Lee, Exp. Mol. Med.
35 (2003) 467e474.
The assay was performed following the method described
by Jimenez et al. [36]. Adult sea urchins (Lytechinus variega-
tus) were collected at Lagoinha beach, on the northeastern
coast of Brazil. Gamete elimination was induced by injecting
3 mL of 0.5 M KCl into the urchin’s coelomic cavity. For
fertilization, 1 mL of a sperm suspension (0.05 mL of concen-
trated sperm in 2.45 mL of filtered sea water) was added to
every 50 mL of egg solution. The assay was carried out in
24-multiwell plates. The compounds were added immediately
after fecundation (within 2 min) to get concentrations of 10
´
´
[10] G. Solladie, Y. Pasturel-Jacope, J. Maignan, Tetrahedron 59 (2003)
3315e3321.
[11] C.J. Kelley, J.R. Mahajan, L.C. Brooks, L.A. Neubert, W.R. Breneman,
M. Carmack, J. Org. Chem. 40 (1975) 1804e1815.
[12] C.J. Kelley, R.C. Harruff, M. Carmack, J. Org. Chem. 41 (1976)
449e455.
and 100 mg/mL in a final volume of 2 mL. Doxorubicin
Ò
ˆ
(Doxolem , Zodiac Produtos Farmaceuticos S/A, Brazil)
[13] M. Bartok, G. Wittmann, G. Gondos, G.V. Smith, J. Org. Chem. 52
(1987) 1139e1141.
was used as a positive control. At appropriate intervals,
aliquots of 200 mL were fixed in the same volume of 10%
formaldehyde to obtain first and third cleavages and blastulae.
One hundred eggs were counted for each concentration to
obtain the percentage of normal cells.
[14] T. Sugimura, J. Watanabe, T. Okuyama, Y. Nitta, Tetrahedron: Asymme-
try 16 (2005) 1573e1575.
[15] T. Majima, S. Tojo, A. Ishida, S. Takamuku, J. Org. Chem. 61 (1996)
7793e7800.
[16] J.C. Roberts, J.A. Pincock, J. Org. Chem. 69 (2004) 4279e4282.
[17] J.C. Roberts, J.A. Pincock, J. Org. Chem. 71 (2006) 1480e1492.
[18] J.J. Heynekamp, W.M. Weber, L.A. Hunsaker, A.M. Gonzales,
R.A. Orlando, L.M. Deck, D.L.V. Jagt, J. Med. Chem. 49 (2006)
7182e7189.
4.5. Hemolytic assay
The test was performed in 96-well plates using a 2% mouse
erythrocyte suspension in 0.85% NaCl containing 10 mM
CaCl2, following the method described by Jimenez et al.
[36]. The compounds were tested at concentrations ranging
from 1.5 to 200 mg/mL. After incubation at room temperature
for 30 min and centrifugation, the supernatant was removed
and the liberated hemoglobin was measured spectrophotomet-
rically as the absorbance at 540 nm.
[19] J. Liu, Y. Zhao, Y. Zhou, L. Li, T.Y. Zhang, H. Zhang, Org. Biomol.
Chem. 1 (2003) 3227e3231.
[20] V. Kumar, A. Sharma, A. Sharma, A.K. Sinha, Tetrahedron 63 (2007)
7640e7646.
[21] J. Dorrie, H. Gerauer, Y. Wachter, S.J. Zunino, Cancer Res. 61 (2001)
4731e4739.
[22] Z. Ma, O. Molavi, A. Haddadi, R. Lai, R.A. Gossage, A. Lavasanifar,
Cancer. Chemother. Pharmacol., in press. doi:10.1007/s00280-008-
0704-z, <http://www.springerlink.com/content/8w31220m82583497/>
[23] M. Murias, W. Jager, N. Handler, T. Erker, Z. Horvath, T. Szekeres,
H. Nohl, L. Gille, Biochem. Pharmacol. 69 (2005) 903e912.
[24] Y.J. Cai, J.G. Fang, L.P. Ma, L. Yang, Z.L. Liu, Biochim. Biophys. Acta
1637 (2003) 31e38.
4.6. Statistical analysis
[25] K. Gaukroger, J.A. Hadfield, L.A. Hepworth, N.J. Lawrence,
A.T. McGown, J. Org. Chem. 66 (2001) 8135e8138.
[26] R.S. Jacobs, L. Wilson, in: A. Aszalor (Ed.), Modern Analysis of
Antibiotics, Marcel Dekker Inc., New York, 1986, pp. 481e493.
[27] P.R. Gross, L.I. Malkin, W.A. Moyer, Proc. Natl. Acad. Sci. U S A 51
(1964) 407e414.
Data are presented as mean ꢀ SEM. The IC50 or EC50 and
their confidence intervals were obtained by nonlinear regres-
sion using the GRAPHPAD program (Intuitive Software for
Science, San Diego, CA).

D.P. de Lima et al. / European Journal of Medicinal Chemistry 44 (2009) 701e707
707
[28] B.P. Brandhorst, Development Biology: A Comprehensive Synthesis, in:
L.W. Browder (Ed.), Oogenesis, Plenum, New York, 1985, pp. 525e576.
[29] L.V. Costa-Lotufo, P.C. Jimenez, D.V. Wilke, L.K. Leal, G.M. Cunha,
E.R. Silveira, K.M. Canuto, G.S. Viana, M.E. Moraes, M.O. de Moraes,
C. Pessoa, Z. Naturforsch. C 58 (2003) 675e680.
[30] L.V. Costa-Lotufo, M.T. Khan, A. Ather, D.V. Wilke, P.C. Jimenez,
C. Pessoa, M.E. de Moraes, M.O. de Moraes, J. Ethnopharmacol. 99
(2005) 21e30.
[32] G. Pagano, A. de Biase, I.B. Deeva, P. Degan, Y.K. Doronin,
M. Iaccarino, R. Oral, N.M. Trieff, M. Warnau, L.G. Korkina, Life
Sci. 68 (2001) 1735e1749.
[33] P. Sharma, J.D. Sharma, J. Ethnopharmacol. 74 (2001) 239e243.
[34] D.D. Perrin, W.L.F. Armarego (Eds.), Purification of Laboratory
Chemicals, Pergamon Press, Oxford, 1988.
[35] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
[36] P.C. Jimenez, S.C. Fortier, T.M.C. Lotufo, C. Pessoa, M.E.A. Moraes,
M.O. de Moraes, L.V. Costa-Lotufo, J. Exp. Mar. Biol. Ecol. 287
(2003) 93e101.
[31] N. Fusetani, in: P. Scheuer (Ed.), Bioorganic Marine Chemistry, Springer
Verlag, Berlin, 1987, pp. 61e91.