3388
H. J. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3385–3388
10. Beall, H. D.; Siegel, D.; Liu, Y.; Bolton, E. M.; Gibson, N.
W.; Ross, D. Biochem. Pharmacol. 1996, 51, 645.
their cytotoxic effects, and also other mechanisms of
action could not be excluded.
11. Kim, Y.; Park, S.; Lee, H.; Suh, M.; Schollmeyer, D.; Lee,
C. Bioorg. Med. Chem. 2003, 11, 1709.
12. Liu, L. F. Annu. Rev. Biochem. 1989, 58, 351.
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In conclusion, a series of 6-arylamino-7-chloro-quinazo-
line-5,8-dione derivatives has been prepared and evalu-
ated for their cytotoxic activities with the goal of
developing potent antitumor agents. We found that
most of compounds synthesized in this study generally
showed potent cytotoxic effects and also more suscep-
tible to human stomach cancer cells compared to lung or
colon cancer cells tested. Among them 7c could serve as
a good lead in these series of compounds. Further, based
on the information of DNA intercalating potential of
these series of compounds, the mechanism of action
study was employed by topoisomerase activity. Several
compounds exhibited the potential inhibitory activity
against either topo I or topo II, suggesting that the
inhibitory activity of topo I or II might be one possible
mechanism for their cytotoxic activity against cancer
cells. Further studies for more potent cytotoxic com-
pounds, based on the above findings, are in progress in
our laboratory.
15. Lee, S. K.; Cui, B.; Mehta, R. R.; Kinghorn, A. D.;
Pezzuto, J. M. Chem. Biol. Interact. 1998, 115, 215.
16. Park, H. J.; Lee, H.; Lee, E.; Hwang, H. J.; Shin, S.; Suh,
M.; Kim, C.; Kim, H. J.; Seo, E.; Lee, S. K. Biosci.
Biotechnol. Biochem. 2003, 67, 1944. (a) Measurement of
topoisomerase I catalytic activity: An assay was done using
supercoiled pHOT1 DNA as a substrate according to the
protocol provided by TopoGEN, Inc. (Columbus, USA).
Supercoiled (form I) plasmid substrate DNA was used in a
reaction volume of 20 lL containing the following: 10 mM
Tris–HCl, pH 7.9, 1 mM ethylene diamine tetraacetic acid
(EDTA), 150 mM NaCl, 0.1% bovine serum albumin
(BSA), 0.1 mM spermidine, 5% glycerol, and 1 unit of
purified human topo I (TopoGEN, Inc.). The appropriate
inhibitor was added as indicated, and the reaction was
started by the addition of the enzyme. After 10 min of
incubation at 37 °C, the reaction was stopped by addition
of 5 lL of stop buffer containing the loading dye (1%
sarkosyl, 0.025% bromophenol blue, 5% glycerol), and
then the reaction mixture was analyzed on a 1% agarose
gel by running at 40 V for 3.5 h in TBE buffer (89 mM Tris,
89 mM borate, 2 mM Na-EDTA, pH 8.3). Gels were
stained with SYBR Green I (Molecular Probes, Eugene,
OR), and observed under UV illumination. For the
quantitative determination of topo I activity, photo-
graphic negatives were densitometrically scanned using
AlphaImager 2200 (AlphaEase version 5.5). The inhibition
of topo I was calculated from the equation: %Inhibi-
tion ¼ [Intensity of sample-treated DNA/Intensity of vehi-
cle-treated control DNA]ꢀ100; (b) Decatenation assay for
topo II activity: The total reaction volume of the topo II-
mediated cleavage reaction was fixed at 20 lL. Briefly,
assay buffer [50 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM
MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, 30 lg/mL
bovine serum albumin (BSA)] containing 200 ng of
KDNA (TOPOgen), and a solution of the test drugs were
added to 1 unit of the human recombinant topo II (the
amount of enzyme, which resulted in the complete
decatenation of 200 ng of KDNA). After 10 min of
incubation at 37 °C, the reaction was stopped by addition
of 5 lL of stop buffer containing the loading dye (1%
sarkosyl, 0.025% bromophenol blue, 5% glycerol), and
then the reaction mixture was analyzed on a 1% agarose
gel by running at 40 V for 3.5 h in TBE buffer (89 mM Tris,
89 mM borate, 2 mM Na-EDTA, pH 8.3). Gels were made
visible as described above for the relaxation assay. Gels
were photographed, and remaining KDNA from photo-
graphic negatives was scanned and calculated the inhibi-
tion activity as mentioned above.
Acknowledgements
This study was supported in part by grant KRF-2003-
E00003 funded by Korea Research Foundation.
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