Carboxamides and Naphthyridin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2491
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PBS and stained with 50 µg/mL propidium iodide (PI) with the
addition of 0.2 µg/µL RNase A. The stained cells were then
analyzed with Becton Dickinson FACScalibur (Becton Dicken-
son) flow cytometer (20 000 counts were measured). The
percentage of the cells in each cell cycle phase was determined
using the ModFit LT software (Verity Software House) based
on the DNA histograms. The tests were performed in duplicate
and repeated at least twice.
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Topoisomerase I DNA Unwinding/Cleavage Assay. In vitro
assay using purified calf thymus topoisomerase I (Invitrogen) was
used in order to check selected compounds for their inhibitory effect
on topoisomerase I according to Bailly,34 whereby two possible
modes of inhibition can be detected: nonspecific effect, due to DNA
intercalation, and specific effects resulting from the poisoning of
topoisomerase I. Briefly, the reaction mixture contained 0.8 µg of
negatively supercoiled plasmid pCI (Promega, kindly provided by
Dr. Andreja Ambriovic´ Ristov, Ru{er Bosˇkovic´ Institute, Zagreb,
Croatia) and 6 U of topoisomerase I, with or without tested
inhibitors in 40 µL of relaxation buffer (10 mM Tris-HCl, pH 7.5,
175 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 2.5% glycerol). The
mixtures were incubated at 37 °C for 30 min, and the reaction was
terminated by the addition of 2.5 µL of 10% SDS, followed by
proteinase K (50 µg/ml) digestion at 37 °C for 15 min. After
extraction with a mixture of phenol, chloroform, and isoamyl
alcohol (25:24:1), aqueous samples were removed and an amount
of 2 µL of gel-loading buffer (0.25% bromphenol blue, 0.25%
xylene cyanol, 60% glycerol, 150 mM Tris, pH 7.6) was added.
The samples were divided in two and loaded onto either 1% agarose
gel containing 0.5 µg/mL ethidium bromide or 1% agarose gel
without ethidium bromide. Gels were run at 80 V constant voltage
in a horizontal electrophoresis system (BIO-RAD). Resulting
products were visualized and documented with UV light at 254
nm (Uvitec, Cambridge, U.K.).
Cellular Tubulin Staining. MiaPaCa-2 cells (2 × 105) were
grown overnight on 18 mm × 18 mm coverslips and placed in
each well of the six-well plate. Our tested compounds, paclitaxel
(Sigma) and colchicine (Sigma), were then added at various
concentrations as described in the results section and Supporting
Information. After 24 h, coverslips were washed with PBS and cells
were fixed in 4% formaldehyde in PBS for 10 min, washed twice
with PBS, and permeabilized with 0.1% Triton-X 100 in PBS for
10 min. Coverslips were than washed with PBS and blocked with
4% FCS in PBS for 30 min. After that, coverslips were incubated
with a mouse monoclonal anti-R-tubulin antibody (100 µg/mL,
Calbiochem) diluted 1/200 in blocking solution for 2 h, washed five
times in PBS, and incubated with FITC-goat antimouse IgG/IgM
1
secondary antibody (0.5 mg/mL, BD Pharmingen) diluted /500 in
blocking solution for 1 h. After being washed with PBS, coverslips
were washed with water and placed on microscope slide in 1 drop
of fluorescence mounting medium (DAKO). DAPI was added to
the mounting medium in a final concentration of 100 ng/mL.
Immunofluorescence was analyzed on an Olympus BX51 micro-
scope with an Olympus DP51 camera.
Acknowledgment. Support for this study by the Ministry
of Science, Education and Sport of Croatia is gratefully
acknowledged (Projects 125-0982464-1356, 098-0982464-2514,
98-0982914-2918, 098-0982464-2393). The authors are grateful
to Dr. Mirko Hadzˇija for his assistance at the fluorescence
microscope.
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W. A. Synthesis and cytotoxic activity of carboxamide derivatives of
benzo[b][1,6]naphthyridines. J. Med. Chem. 2003, 46, 1049–1054.
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Chovan, L. E.; Kalvin, D.; Merta, P. J.; Nilius, A. M.; Pratt, S. D.;
Soni, N. B.; Wagenaar, F. L.; Weitzberg, M.; Wagner, R.; Beutel,
B. A. Novel antibacterial class: a series of tetracyclic derivatives.
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Different modes of action of naphthyridones in Gram-positive and
Gram-negative bacteria. Antimicrob. Agents Chemother. 2006, 50, 385–
387.
(18) Tabart, M.; Picaut, G.; Desconclois, J. F.; Dutka-Malen, S.; Huet, Y.;
Berthaud, N. Synthesis and biological evaluation of benzo[b]naph-
thyridones, a series of new topical antibacterial agents. Bioorg. Med.
Chem. Lett. 2001, 11, 919–921.
Supporting Information Available: Experimental details, spec-
troscopic data, and elemental analysis results for compounds 4-7,
10-13, and 14-17; spectroscopy details; results of DNA binding
studies; additional photos of tubulin immunofluorescence staining
of MiaPaCa-2 cells. This material is available free of charge via
(19) Dogan Koruznjak, J.; Slade, N.; Zamola, B.; Pavelic´, K.; Karminski-
Zamola, G. Synthesis, photochemical synthesis and antitumor evalu-
ation of novel derivatives of thieno[3′,2′: 4,5]thieno[2,3-c]quinolones.
Chem. Pharm. Bull. 2002, 50, 656–660.
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