386
precipitate was collected via filtration and air dried to
give 3.61 g (95%) of the title compound 5a as an
off-white solid. The product was used to prepare 3,5,7-
trimethoxy-2-(3,4-dimethoxyphenyl)-4-oxoquinoline
(6a) without further purification.
posed of 1.4 mM ATP, 1.8 mM spermidine, 5 mM DTT,
0.14 mM Na2EDTA, 6.5% glycerol, 24 mM KCl, 4 mM
MgCl2, 0.36 µg/ml bovine serum albumin (mol. biol.
grade), and 35 mM Tris-HCl, pH 7.5. To this reaction,
drug was added from DMSO-solubilized stocks (such
that the final concentration of DMSO is < 3.5%), fol-
lowed by 1 unit of gyrase holoenzyme (reconstituted Gyr
A and Gyr B subunits). Each reaction was incubated for
30 min at 37 °C, and reactions were stopped by the
addition of SDS (to 0.5%), Na2EDTA (to 6 mM), and
5.35% glycerol containing 0.013% bromophenol blue (as
tracking dye). The total reaction mixture was loaded onto
either a 1% TAE or TBE agarose gel and was electro-
phoresed in a horizontal submarine apparatus to separate
different DNA topoisomers. Gels were stained with EtBr,
and visualized by Polaroid film 667 photography of
fluoresced gels. The percent supercoiling was determined
by the densitometric tracing (Collagey, Image Dynamics
Corporation) of supercoiling versus relaxed DNA, nor-
malized against no-drug control lanes.
5.3.
3,5,7-trimethoxy-2-(3,4-dimethoxyphenyl)-4-oxo-
quinoline (6a), a general procedure for the preparation
of the aza-flavone precursors 6a–6z
Sodium ethoxide solution, prepared from sodium
(0.30 g, 13.0 mmol) and ethanol (100 mL), was trans-
fered into a Parr bomb containing a solution of N-(3,5-
dimethoxy-2-methoxyacetylphenyl)-3,4-dimethoxy-ben
zamide (3.60 g, 9.24 mmol) in ethanol (absolute, 50 mL).
The bomb was sealed and heated at 160–165 °C, 180 psi
for 3 h. The reaction vessel was allowed to cool to room
temperature and the solvent was removed under vacuum.
Column chromatography (silica gel, 10% MeOH in
EtOAc) gave 1.23 g (36%) of 6a as a brown solid, m.p.
134–135 °C, MS (CI, CH4): MH+ at 372. 1H-NMR
(DMSO-d6): δ 10.93 (br, s, 1H, NH), 7.22 (s, 1H, ArH),
7.20 (d, J = 8 Hz, 1H, ArH), 7.11 (d, J = 8 Hz, 1H, ArH),
6.67 (s, 1H, ArH), 6.26 (s, 1H, ArH), 3.83 (s, 6H,
ArOCH3), 3.80 (s, 3H, ArOCH3), 3.78 (s, 3H, ArOCH3),
3.60 (s, 3H, ArOCH3). Anal. (C20H21NO6•0.50 H2O) C,
H, N.
-
6.2. Topoisomerase II assay [27, 28]
Each reaction contained an equal volume of Kineto-
plast DNA (kDNA, TopoGen, Inc., Columbus, OH,
1.1–1.8 µg/reaction), 67 mM Tris-HCl, pH 8, 160 mM
KCl, 13 mM MgCl2, 0.7 mM ATP, 0.7 mM dithiothreitol,
0.06 µg BSA, with an equal volume of test compound,
and 10 units of p170 human topoisomerase II enzyme
(TopoGen, Inc.). Each reaction mixture was incubated for
5 min at 37 °C, and the reaction was stopped with 0.5%
Sarkosyl, 0.0025% bromophenol blue, and 25% glycerol.
Reaction samples were electrophoresed in 0.6% TBE-
agarose gels at 30 mA. The gel was stained with EtBr,
and visualized by Polaroid film 667 or 665 (Polaroid
Corporation, Cambridge, MA) photography of fluoresced
gels at 300 nm. The decatenated kDNA band was quan-
tified by densitometric analysis (Collegey, Image Dy-
namics Corporation).
5.4.
3,5,7-Trihydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-
quinoline (2a), a general procedure for the preparation
of the aza-flavones 2a–2z
A
mixture of 3,5,7-trimethoxy-2-(3,4-dimethoxy-
phenyl)-4-oxoquinoline (0.70 g, 1.88 mmol) in aqueous
48% HBr (10 mL) was heated at reflux for 3.5 h. The
reaction mixture was then cooled and the solvent was
removed under vacuum. The residue was recrystallized
from H2O and EtOH and gave 0.35 g (49%) of the title
compound 2a as a dark yellow solid, m.p. > 300 °C,
MS(CI, CH4): MH+ at 302. 1H-NMR (DMSO-d6): δ
11.32 (s, 1H, NH), 10.5–8.50 (br, s, 5H, ArOH), 7.20 (d,
J = 2Hz, 1H, ArH), 7.04 (dd, J = 8, 2Hz, 1H, ArH), 6.87
(d, J = 8Hz, 1H, ArH), 6.44 (d, J = 2Hz, 1H, ArH), 5.97
(d, J = 2Hz, 1H, ArH). Anal. (C15H11NO6•0.50 H2O) C,
H, N.
References
[1] Wang J.C., Annu. Rev. Biochem. 54 (1985) 665–697.
[2] Allan Y.C., Leroy F.L., Annu. Rev. Pharmacol. Toxicol. 34 (1994)
191–218.
6. Biology
[3] Mitschner L.A., Zavod R.M., Shen L.L., Alfred Benzon Symp. 33
(1992) 60–77.
6.1. DNA gyrase supercoiling inhibition assay [26]
[4] Tewey K.M., Chen G.L., Nelson E.M., Liu L.F., J. Biol. Chem. 259
(1984) 9182–9187.
0.25–0.40 µg pBR322 DNA (previously relaxed with
topoisomerase I) was added to a reaction mixture com-
[5] Elsea S.H., McGuirk P.R., Gootz T.D., Moynihan M.S., Osheroff N.,
Antimicrob. Agents Chemother. 37 (1993) 2179–2186.