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T. Higashi et al. / Bioorg. Med. Chem. 17 (2009) 3568–3571
bromosuccimide3 or 1,3-dichloro-5,5-dimethylhydantoin.11 The
quinoline skeletons in 14 was prepared under improved conditions
for Skraup synthesis.12 The cell line used in antiproliferative activ-
ity study, CCRF-HSB-2 was provided by RIKEN Cell Bank (Tsukuba,
Japan).
4.2. Analytical methods
Gel electrophoreses were performed on ADVANCE Mupid-2plus.
HPLC data were recorded with HITACHI L-4000 UV detector.
4.3. Crosslinking assay
DNA interstrand crosslinking activity was assayed as follows.
Linearized pBluescriptÒ DNA (460 ng) was treated with the sam-
ples dissolved in DMSO at 37 °C for 6 h. Then, DNA was precipi-
tated with EtOH (95%) and cooled for 24 h before being
centrifuged for 20 min. The supernatant was removed, and the pel-
let was washed with EtOH (75%), and further spun for 20 min. The
pellet was lyophilized to dryness, and the resulted DNA was then
dissolved in separation buffer [EDTA (1 mM) in aqueous DMSO
(30% v/v) solution]. The mixture was heated at 95 °C for 5 min
and immediately placed in an ice bath. The DNA sample was sep-
arated by 1% agarose gel electrophoresis, and the DNA bands were
visualized by staining with ethidium bromide. The results were
shown in Figure 3. In the case of 3a–12, plasmid was treated at
Figure 6. Substitution of naphthalene in 3a and 6a to quinoline to 138 and 14.9
tuted form 14 were quite stable (half-life: 13: 770 min; 14:
630 min), they no longer exhibited significant activity ( Fig. 7).
Although hydrogen bond formation between the lone pair electron
on the nitrogen and certain protic hydrogens in DNA had been sug-
gested, it did not work advantageously in the present quinoline
derivatives 13 and 14.
3. Conclusion
We found highly active bis-bromomethylated aromatic mole-
cule 3a with crosslinking activity on DNA. The unique behavior,
judged from the contrasting results between the expression of
microbial mutagenicity and double-strand formation on linearized
plasmid DNA, suggests intrastrand crosslinking bond formation,
which is well known as the major crosslinking mechanism of cis-
platin. The two remotely located bromomethyl groups, which eas-
ily suffer from nucleophilic attack, are the origin of the high
activity. The antiproliferative activity of a wide range of substi-
tuted naphthalenes and quinolines towards leukemia cells as men-
tioned above is currently being investigated, and the results will be
reported in due course.
10
der 1
l
M of each sample. As the reference, cisplatin was applied un-
M.
l
4.4. Ames test
S. typhimurium TA92 was grown in the medium [5 mL, nutrient
broth (6 g) and of NaCl (5 g) in H2O (1000 mL)] for 15.5 h at 37 °C.
The samples for assay were diluted in DMSO. In a tube containing
sodium phosphate buffer (0.1 M, pH 7.4, 0.5 mL), the bacterial cul-
ture as above (0.1 mL), and the sample solution (50 lL) were mixed,
then the mixture was incubated at 37 °C for 20 min. This was mixed
with separately prepared soft agar solution (0.6% agar in 0.6% NaCl,
2 mL) at 45 °C, and the total mixture was overlaid on a minimal agar
plate. Each agar plate (90 mm diameter) contained the minimal
medium (30 mL) with Bacto agar (1.5%). After incubation at 37 °C
for 2 days, the number of colonies on the plate, which indicates
the number of revertants caused by crosslinking was counted man-
ually. The mean of the numbers from six plates for each experiment
was calculated, and presented in Figures 4 and 7.
4. Experimental
4.1. Materials and methods
Linearized pBluescriptÒ DNA was the product from STRATA-
GENE. Nutrient broth and Bacto agar were the products from DIF-
CO Laboratories. DMSO was freshly distilled from CaH2 before use
(bp14 76 °C). The minimal media for mutation assays were pre-
pared according to the reported procedure.10 The samples 3a–13
were provide by a halogenations of the benzylic position with N-
4.5. Cell proliferation assay
CCRF-HSB-2 cells were cultured in RPMI1640 supplemented
with 10% fetal calf serum at 37 °C in an atmosphere containing
5% CO2. Cells were collected when they were nearly confluent
and diluted to a concentration of 3 Â 105 cell/mL with culture med-
ium. Aliquots (3 mL) of cell suspension were dispensed into each
well of a 6-well plate. After incubation for 24 h at 37 °C in an atmo-
sphere containing 5% CO2, the cells were treated with the test sam-
ples as solution in DMSO. Each well received 3 lL of the sample
solutions. After incubation for 24 h at 37 °C in an atmosphere con-
taining 5% CO2, Trypan blue-excluding cells were counted.
4.6. Stability in aqueous solution
The stability of the samples was examined by measuring the
decomposition rate in aqueous solution. Each sample was dis-
solved in a mixture of sodium phosphate buffer (0.01 M, pH 7.4)
and MeCN (4:1, final concentration: 10
(2 lL) was analyzed on a LiChrosorb RP-18 column (5
l
M). The resulting mixture
Figure 7. Mutagenicity of substituted naphthalenes and quinolines, solid circle: 3a;
open circle: 13; solid triangle: 6a; open triangle: 14.
lm particle
Higashi, Toshinori
