R. Martí-Centelles et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Table 2
ized by comparison of their physical and spectral data with those
in the literature.22,23
Primers used and sizes of the PCR products
Conditions B.24 The synthesis of 2-hydroxystilbene 3a from 2-
iodophenol 2f is described as a representative example: 2-iodo-
VEGF
Sense: 50-CCTGATGAGATCGAGTACATCTT-30
379
287
Antisense: 50-ACCGCCTCGGCTTGTCAC-30
b-Actin
Sense: 50-TCATGAAGTGTGACGTTGACATC CGT-30
Antisense: 50-CGTAGAAGCATTTGCGGTGCAC GATG-30
phenol (115
lL, ca. 1 mmol), styrene (172
lL, 1.5 mmol), tri-n-
butylamine (480
l
L, 2 mmol), tetra-n-butylammonium bromide
(322 mg, 1 mmol) were suspended in water (4 mL). The mixture
was the stirred to homogeneity and then treated with Pd(NH3)2Cl2
(3 mg, 0.015 mmol). The mixture was heated at reflux (bath tem-
perature, 140 °C) for 24 h, then cooled and extracted three times
with EtOAc. The organic layers were dried on anhydrous MgSO4
and filtered. Removal of volatiles under reduced pressure gave a
solid (119 mg, 86%), which was shown to be 3a by comparison of
their physical and spectral data with those in the literature.
The same conditions were used for the synthesis of stilbenes 3b
and 3c from the corresponding iodoarenes 2g and 2h. Yields were:
3b (65%) and 3c (97%). For stilbenes 3d and 3e, bromoarenes 2d
and 2e were used instead of the corresponding iodoarenes. Yields
were: 3d (65%) and 3e (78%).
Some 3 ꢂ 103 BAE cells or 5 ꢂ 103 in case of HT-29 cells in a total
volume of 100 lL of their respective growth media were incubated
with serial dilutions of the tested compounds. After 3 days of incu-
bation (37 °C, 5% CO2 in a humid atmosphere) 10 l of MTT (5 mg/
ml in PBS) were added to each well and the plate was incubated for
further 4 h (37 °C). The resulting formazan was dissolved in 150
l
lL
of 0.04 N HCl/2-propanol and read at 550 nm. All determinations
were carried out in triplicate. IC50 values mean the concentration
of compound yielding a 50% of cell survival.
6.3.3. Endothelial cell differentiation assay: tube formation on
Matrigel
6.2.2. O-Allylation of phenols
A solution of 3a (196 mg, 1 mmol) in acetone (15 mL) was trea-
Matrigel (50
lL of about 10.5 mg/mL) at 4 °C was used to coat
ted with allyl bromide (260 lL, 3 mmol) and K2CO3 (415 mg,
each well of a 96-well plate and allowed to polymerize at 37 °C
for
a
minimum of 30 min as previously described.30 Some
3 mmol). The mixture was stirred at reflux under N2 for 24 h. Sub-
sequently, the mixture was filtered through silica gel, with addi-
tional washing of the silica gel pad with EtOAc. Removal of all
volatiles under reduced pressure gave 4a (187 mg, 79%). The prod-
uct was characterized by means of comparison of its physical and
spectral data with those in the literature.27
Under the same conditions, stilbene 3c was converted into its
O-allyl derivative 4b in 90% yield: white solid, mp 120–121 °C;
1H NMR (500 MHz) d 7.48 (2H, br d, J ꢁ 7.4 Hz), 7.43 (2H, br d,
J ꢁ 8.8 Hz), 7.33 (2H, t, J ꢁ 7.6 Hz), 7.22 (1H, tt, J ꢁ 7.6, 1.5 Hz),
7.05 (1H, d, J = 16.5 Hz), 6.96 (1H, d, J = 16.5 Hz), 6.90 (2H, br d,
J ꢁ 8.8 Hz), 6.05 (1H, ddt. J = 17.5, 10.4, 5.5 Hz), 5.42 (1H, dq,
J = 17.5, 1.5 Hz), 5.29 (1H, dd, J = 10.4, 1.5 Hz), 4.54 (2H, dt,
J = 5.5, 1.5 Hz); 13C NMR (125 MHz) d 158.3, 137.6, 130.3 (C),
133.2, 128.6 (2ꢂ), 128.2, 127.7 (2ꢂ), 127.2, 126.7, 126.3 (2ꢂ),
115.0 (2ꢂ) (CH), 117.7, 68.9 (CH2); HR EIMS m/z 236.1206 (M+).
Calcd for C17H16O, 236.1201.
5 ꢂ 104 BAE cells were added with 200
lL of DMEM. Finally, differ-
ent amounts of the tested compounds were added and incubated
at 37 °C in a humidified chamber with 5% CO2. After incubation
for 7 h, cultures were observed (200ꢂ magnifications) and photo-
graphed with a NIKON inverted microscope DIAPHOT-TMD (NI-
KON Corp., Tokyo, Japan). Two different observers evaluated the
results of tube formation inhibition.
6.3.4. RT-PCR analysis
HT-29 cells at 70–80% confluence were collected after serum
starvation for 24 h. Cells were incubated with 20
lg/mL resveratrol
in DMSO and with 10 g/mL stilbene 3b in DMSO for 48 h. Cells
l
were collected and the total cellular RNA from HT-29 cells was iso-
lated using Ambion RNA extraction Kit according to the manufac-
turer’s instructions. The cDNA was synthesized by MMLV-RT
with 1–21 lg of extracted RNA and oligo(dT)15 according to the
manufacturer’s instructions. Gene-specific PCR primers (see Ta-
ble 2) were then added for amplification. PCR products were ana-
lyzed by electrophoresis on 1.5% agarose gels and visualized by
ethidium bromide staining under UV transillumination. The se-
quences of primers used in the RT-PCR are listed in Table 2. The
PCR conditions were as follows: VEGF at 94 °C for 30 s, at 58 °C
for 1 min, and at 72 °C for 1 min 50 s; and b-actin at 94 °C for
30 s, at 58 °C for 50 s, and at 72 °C for 50 s. Analysis of b-actin
was used to monitor RNA integrity and accuracy of loading.31
6.3. Biological procedures
6.3.1. Reagents and cell culture
Cell culture media were purchased from Gibco (Grand Island,
NY, USA) and Biowhittaker (Walkersville, MD, USA). Fetal bovine
serum (FBS) was a product of Harlan-Seralab (Belton, U.K.). Matri-
gel was purchased from Becton Dickinson (Bedford, MA, USA). Sup-
plements and other chemicals not listed in this section were
obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Plastics
for cell culture were supplied by NUNC (Roskilde, Denmark). Stilb-
enes 3a–e and 4a–b (samples purified by crystallization) as well as
resveratrol were dissolved in DMSO at a concentration of 10 mg/
mL and stored at ꢀ20 °C until use.
6.3.5. ELISA analysis
HT-29 cells at 70–80% confluence were collected after serum
starvation for 24 h. Cells were incubated with 20
lg/mL resveratrol
in DMSO and with 10 g/mL of the corresponding stilbene in
l
BAE cells were obtained as reported28 by collagenase digestion.
Human colon adenocarcinoma (HT-29) cells were obtained from
American Type Culture Collection. Both cell lines were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) containing glu-
cose (1 g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin
DMSO for 72 h. Culture supernatants were collected and VEGF se-
creted by HT-29 cells was determined using Invitrogen Human
Vascular Endothelial Growth Factor ELISA Kit according to the
manufacturer’s instructions.
(50 lg/mL) and amphoterycin (1.25 lg/mL), supplemented with
10% FBS.
Acknowledgments
Financial support has been granted to M.C. by the Spanish Min-
istry of Education and Science (Projects CTQ2008-02800 and
CTQ2011-27560), by the Consellería dEmpresa, Universitat i Cien-
6.3.2. Cell proliferation assay
The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
´
bromide (MTT; Sigma Chemical Co., St. Louis, MO) dye reduction
assay in 96-well microplates was used, as previously described.29
cia de la Generalitat Valenciana (ACOMP09/113) and by the BAN-