R.B. de Oliveira et al. / European Journal of Medicinal Chemistry 41 (2006) 756–760
759
J = 9.0 Hz, 2 × H-arom.); 7.88 (2H, d, J = 9.0 Hz, 2 × H-
arom.); high-resolution ms calcd for C18H16N2O3: 308.11609.
Found: 308.11507.
Unbound SRB was removed by washing five times with 1%
acetic acid and the plates dried at room temperature overnight.
The plates were read at 515 nm after dissolution of the dye
with Tris buffer. Etoposide was used as positive controls and
DMSO 0.2% as negative control. Each experiment was per-
formed in triplicate. Representative data of mean and S.D. of
at two independent experiments.
6.1.1.3. 2-(4-Carboxyphenyl)furan (11). The arylfuran 11 was
prepared from 4-aminobenzoic acid using the method de-
scribed above. Yield: 19%; white solid; m.p. 228.1–229.0 °C
(hexane–AcOEt) (Ref. [19]: 226 °C); 1H-NMR (200 MHz,
DMSO-d6) δ 6.63 (1H, dd, J = 3.4 and 1.8 Hz, H-furan); 7.12
(1H, d, J = 3.4 Hz, H-furan); 7.78–7.83 (3H, m, H-furan and
2 × H-arom.); 7.98 (2H, d, J = 8.5 Hz, 2 × H-arom.).
6.3. Lymphocyte assays
Assay with PBMC was run as previously, with modifica-
tions [13]. Shortly, PBMC of health adults were separated by
Ficoll gradient. PBMC (1.5 × 105 cells per well) were cultured
in RPMI-1640 medium, supplemented with 5% (v/v) heat-in-
activated, pooled AB serum and 2 mM L-glutamine and anti-
biotic/antimicotic solution containing 1000 U/ml penicillin,
1000 μg/ml streptomycin and 25 μg/ml fungisone in flat bot-
tomed microtiter plates. The cultures were stimulated with
2.5 μg/ml of phytohemaglutinin (PHA) in presence of different
compounds at a final concentration of 20 μg/ml and incubated
for 72 h at 37 °C in a humidified atmosphere containing 5%
CO2. Proliferation was quantified using MTT [14]. Dexa-
methasone was used as positive controls in this assay. PBMC
assay were performed in quadruplicate.
6.1.1.4. 3,4-Dicarboxyethyl-2-(4-sulfonamidephenyl)furan
(14). The arylfuran 14 was prepared from sulfanilamide and
diethyl 3,4-furandicarboxylate using the method described
above. Yield: 39%; white solid; m.p. 137.4–137.8 °C
(hexane–CH2Cl2); 1H-NMR (200 MHz, DMSO-d6) δ 1.27
(6H, t, J = 6.9 Hz, 2 × CH3CH2O); 4.4–4.21 (4H, m, 2 ×
CH3CH2O); 7.49 (2H, sl, NH2); 7.82 (2H, d, J = 8.3 Hz,
2 × H-arom.); 7.94 (2H, d, J = 8.3 Hz, 2 × H-arom.); 8.59
(1H, s, H-furan); EM: m/z 367, 322, 294, 213, 184, 60, 44, 28.
6.1.1.5. 4-(2-Furyl)-N-(6-methoxy-3-pyridazinyl)-benzenosul-
fonamide (16). The arylfuran 16 was prepared from sulfa-
methoxypiridazine using the method described above. Yield:
36%; white solid; m.p. 164–165 °C (hexane–AcOEt); 1H-
NMR (200 MHz, DMSO-d6) δ 3.82 (3H, s, OCH3); 6.63
(1H, dd, J = 3.3 and 1.7 Hz, H-furan); 7.10 (1H, d,
J = 3.3 Hz, H-furan); 7.37 (1H, d, J = 9,8 Hz, H-pyridazinyl);
7.7–7.9 (7H, m, H-furan, NH, 4 × H-arom., H-pyridazinyl);
high-resolution ms calcd for C15H13N3O4S: 331.06268. Found:
331.06207.
6.4. Statistical analysis
Data were analyzed with the Student’s t-test when appropri-
ate or by one-way analysis of variance and Bonferroni’s multi-
ple comparison test. P values < 0.05 were taken to be signifi-
cance.
Acknowledgements
6.2. Assay with tumor cells
The authors are grateful to Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG) and Conselho Nacio-
nal de Desenvolvimento Científico e Tecnológico (CNPq), for
financial support.
The effect of arylfurans on the growth of tumor cell lines
was evaluated according to the procedure adopted by the Na-
tional Cancer Institute for the in vitro anticancer drug screening
that uses the protein-binding dye SRB to estimate cell growth.
The methodology used was the same as originally published by
the NCI team [12]. Three human tumor cell lines were used,
MCF-7 (breast cancer), TK-10 (renal cancer), and UACC-62
(melanoma). All cells were cultured in RPMI medium supple-
mented with 5% FCS and gentamicin. Shortly before reaching
confluence the cells were detached with trypsin-EDTA and
seeded into 96-well plates so that 100 μl per well contained
10,000 UACC-62 and MCF-7 cells, and 15,000 TK-10 cells.
After 24 hours of incubation, 80 μl of medium and 20 μl of the
compound solutions (200 μg/ml in 2% aqueous DMSO) were
added. After 48 hours in the presence of the compounds, the
cells were fixed by adding 50 μl of cold 50% (w/v) trichlora-
cetic acid (TCA) to each well and incubating the plate at 4 °C
for 1 h. The supernatant was then discarded and the cells
washed five times with water. After drying at room tempera-
ture, 50 μl of SRB solution (0.4% w/v in 1% acetic acid) was
added to each well, and the plate incubated for 30 min at 4 °C.
References
[1] H. Zhai, T. Inoue, M. Moriyama, T. Esumi, Y. Mitsumoto, Y. Fukuya-
ma, Biol. Pharm. Bull. 28 (2005) 289–293.
[2] T. Konishi, T. Konoshima, A. Daikonya, S. Kitanaka, Chem. Pharm.
Bull. (Tokyo) 53 (2005) 121–124.
[3] C. Kraft, K. Jenett-Siems, I. Köhler, B. Tofern-Reblin, K. Siems, U. Bi-
enzle, E. Eich, Phytochemistry 60 (2002) 167–173.
[4] N.P. Lopes, M.J. Kato, M. Yoshida, Phytochemistry 51 (1999) 29–33.
[5] N.P. Lopes, R. Chicaro, M.J. Kato, S. Albuquerque, M. Yoshida, Planta
Med. 64 (1998) 667–669.
[6] N.N. Girotra, T. Biftu, M.M. Ponpipom, J.J. Acton, A.W. Alberts, T.N.
Bach, R.G. Ball, R.L. Bugianesi, W.H. Parsons, J.C. Chabala, P. Davies,
T.W. Doebber, J. Doherty, D.W. Graham, S.-B. Hwang, C.H. Kuo,
M.-H. Lam, S. Luell, D.E. MacIntyre, R. Meurer, C.D. Roberts, S.P. Sa-
hoo, M.S. Wu, J. Med. Chem. 35 (1992) 3474–3482.
[7] T. Biftu, N.F. Gamble, T. Doebber, S.-B. Hwang, T.-Y. Shen, J. Snyder,
J.P. Springer, R. Stevenson, J. Med. Chem. 29 (1986) 1917–1921.