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S.E. As´ıs et al. / Il Farmaco 54 (1999) 517–523
able solvent (Table 1). In order to obtain compounds 4
and 5 a catalytic amount of acetic acid was necessary.
1H NMR and IR spectral data of representative
compounds are as follows:
hydrate was prepared (40% yield, m.p. 155–160°C) and
0.60 g (2.4 mmol) of this compound was dissolved in 20
ml water. To this solution 0.39 g (4.2 mmol) of sodium
acetate was added. Then a solution of 0.67 g (4.8
mmol) of 4-chlorobenzaldehyde in 5 ml EtOH was
added dropwise at r.t. Immediately a white solid precip-
itated. The mixture reaction was heated under reflux for
1 h to complete the reaction. The product was filtered
off and washed with EtOH, the crude was pure enough
1
Compound 6. H NMR l (ppm): 10.84 (s, 1H, CH),
9.19 (s, 1H, NHCO), 8.04–7.42 (m, 13H, arom. and
CONH). IR w (cm−1): 1700, 1580, 1400, 1300, 820.
1
Compound 11. H NMR l (ppm): 10.85 (s, 1H, CH),
9.15 (s, 1H, NHCO), 8.05–7.45 (m, 12H, arom. and
CONH). IR w (cm−1): 1720, 1700, 1470, 1300, 820, 710.
(90% yield, m.p. 225–228°C). H NMR l (ppm): 10.31
1
(s, 1H, CH), 8.72 (s, 1H, NHCO), 7.93–7.41 (m, 12H,
arom. and CONH), 6.53 (s, 2H, CH2). IR w (cm−1):
1680, 1520, 1460, 820.
4.5.2. Compounds 13–19: general procedure —
Method B
To a solution of the desired monoarylhydrazone (2.5
mmol) in benzene with stirring at r.t., was added drop-
wise a solution of arylisocyanate (3.0 mmol) in benzene.
The product that precipitated was filtered off and re-
crystallized from a suitable solvent (Table 1).
4.8. Binding to DNA
DNA solution: Calf thymus DNA (12.5 mg) was
slowly magnetically stirred in 5 ml Tris–HCl buffer (10
mM, pH 7.4) for 24 h at 4°C. From this solution, 0.6
ml were taken off and diluted with the same buffer to
25 ml.
1
The H NMR and IR spectral data of representative
compounds are as follows:
1
Compound 12. H NMR l (ppm): 10.70 (s, 1H, CH),
9.13 (s, 1H, NHCO), 7.98–6.99 (m, 12H, arom. and
CONH), 3.81 (s, 3H, OCH3). IR w (cm−1): 1690, 1530,
1240, 820.
Test compound solution: it was prepared at 10−4
M
concentration using a minimal ethanol volume and
water, then it was diluted to a concentration of 2×
10−5 M. A 3 ml sample of this resulting solution was
mixed with 3 ml of DNA solution described above and,
after slowly rotating the mixture for 24 h, its UV
spectra were recorded using a 1 cm cell at 20°C.
1
Compound 19. H NMR l (ppm): 11.04 (s, 1H, CH),
9.54 (s, 1H, NHCO), 8.25–7.24 (m, 11H, arom. and
CONH), 3.91 (s, 3H, OCH3). IR w (cm−1): 1700, 1600,
1510, 1330, 1220, 850.
4.6. 2-(4-Chlorophenylmethyl)-N-(1-naphthyl)hydra-
zinecarboxamide (20)
4.9. Cyclic 6oltammetry
4-Chlorobenzaldehyde monohydrazone (1.5 g) was
dissolved in 30 ml EtOH and 5 mg of 10% Pd/C were
added as catalyst. Then the mixture was hydrogenated
for 3 h at 30 psi. The catalyst was filtered off and the
solvent was vacuum evaporated; 1.0 g of 4-chloroben-
zylhydrazine was obtained (66% yield, m.p. B50°C),
which was dissolved in 10 ml benzene and then a
solution of 0.9 ml of commercial 1-naphthylisocyanate
in 3 ml benzene was added dropwise at r.t. The white
product that immediately precipitated was collected and
washed with the same solvent. Then it was recrystal-
lized from EtOH (39% yield, m.p. 220–222°C). 1H
NMR l (ppm): 9.20–9.08 (m, 2H, NHNH), 8.31–7.30
(m, 12H, arom. and CONH), 4.07–3.99 (m, 2H, CH2).
IR w (cm−1): 1680, 1560, 1460, 760.
Potentiometer: EQMAT-S1; glassy carbon working
electrode, calomel reference electrode, gold wire
antielectrode.
Supporting electrolyte: tetrabutylammonium perchlo-
rate (TBAP) 50 mM; sample concentration: 0.5 mM.
Sensibility: 25 mA/div. Solvent: acetonitrile of electro-
chemical purity. Inert bubbling gas: nitrogen. Sweep
rate: 50 mV/s. Potential scanning: between +2000 and
−2000 mV.
4.10. Topoisomerase I inhibition assay
Inhibition of topoisomerase I activity was determined
using a relaxation assay described [16] with modifica-
tions. Plasmid pHOT1 (Topogen, USA) was used as a
substrate in a final reaction volume of 20 ml containing
10 mM Tris–HCl, 1 mM EDTA, 0.15 M NaCl, 0.1%
BSA, 0.1 mM spermidine, 5% glycerol and 2 units of
purified human topoisomerase I (Topogen, USA). The
drug was pre-dissolved in DMSO and tested at doses of
100, 10 and 1 mg/ml. The reaction was carried out at
37°C for 30 min and terminated by addition of 10%
SDS and proteinase K, 50 mg/ml (Sigma, USA). After
4.7. 2-(4-Chlorophenylmethylene)-N-(1-naphthyl-
methyl)hydrazinecarboxamide (21)
N-(1-Naphthylmethyl)hydrazinecarboxamide
was
synthesized according to the method described above
for preparing N-phenylhydrazinecarboxamide, from N-
(1-naphthylmethyl)urea and hydrazine hydrate, then N-
(1-naphthylmethyl)hydrazinecarboxamide
chloro-