Substituent effect of a fluorine atom on the mutagenicity of nitroquinolines

Article abstract of DOI:10.1016/S1383-5718(99)00049-2

Some 16 nitroquinolines (NQs) and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity. These NQs consist of 5-NQs, 5-nitroquinoline N-oxides (5-NQOs), N-methyl-5-nitroquinolinium methanesulfonates (N-Me-5-NQs) and 8-NQs, including three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs. For this purpose, eight F-NQs were newly synthesized. The data indicated that the ratio of the mutagenic activities (revertants/plate/nmol) of fluorinated NQs to those of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold. The fluorine atom located para to the nitro group markedly enhanced the mutagenicity (24-fold and more), while three ortho-fluorinated derivatives showed no significant increase in mutagenicity (enhancement ratio were 0.6, 0.8 and 1.7). With respect to 8-NQs, its meta-fluorinated derivative also had an enhanced mutagenicity over the parent compound (53-fold). In addition, although N-Me-5-NQ was less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-substitution. These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation. Copyright (C) 1999 Elsevier Science B.V.

Full text of DOI:10.1016/S1383-5718(99)00049-2

Ž
.
Mutation Research 441 1999 205–213
www.elsevier.comrlocatergentox
Community address: www.elsevier.comrlocatermutres
Substituent effect of a fluorine atom on the mutagenicity of
nitroquinolines
a
a
a
Ken-ichi Saeki a,), Ryuichi Murakami , Arihiro Kohara , Naoaki Shimizu ,
a
a
b,1
Hiroshi Kawai , Yutaka Kawazoe , Atsushi Hakura
Faculty of Pharmaceutical Sciences, Nagoya City UniÕersity, 3-1 Tanabedori, Mizuho-ku, Nagoya 467-8603, Japan
a
b
Drug Safety Research Laboratories, Eisai, Takehaya-machi, Kawashima-cho, Hashima-gun, Gifu 501-6195, Japan
Received 23 October 1998; received in revised form 16 March 1999; accepted 18 March 1999
Abstract
Ž
.
Some 16 nitroquinolines NQs and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium
TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity. These NQs consist of 5-NQs,
Ž
.
Ž
.
5-nitroquinoline N-oxides 5-NQOs , N-methyl-5-nitroquinolinium methanesulfonates N-Me-5-NQs and 8-NQs, including
three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs. For this purpose, eight F-NQs were newly
Ž
.
synthesized. The data indicated that the ratio of the mutagenic activities revertantsrplaternmol of fluorinated NQs to those
of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold. The fluorine atom located para to the
Ž
.
nitro group markedly enhanced the mutagenicity 24-fold and more , while three ortho-fluorinated derivatives showed no
Ž
.
significant increase in mutagenicity enhancement ratio were 0.6, 0.8 and 1.7 . With respect to 8-NQs, its meta-fluorinated
Ž
.
derivative also had an enhanced mutagenicity over the parent compound 53-fold . In addition, although N-Me-5-NQ was
less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-sub-
stitution. These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to
modify their mutagenic potency and to better understand the mechanism of mutation. q 1999 Elsevier Science B.V. All
rights reserved.
Keywords: Nitroquinoline; Fluoroquinoline; Mutagenicity; Ames test
1. Introduction
biological effects. Replacement of a hydrogen atom
with a fluorine results not only in changes in elec-
tron-density distribution within the molecule but also
in electric repulsiverattractive interactions with in-
trarintermolecular environments. These changes may
significantly affect such interactions as those be-
tween an enzyme and its substrate or between a
receptor and its ligand. In addition, the metabolism
of xenobiotics, including drugs, food-additives and
We have investigated fluorine-substituted het-
eroaromatic compounds with special attention to their
)
Corresponding author. Fax: q81-52-834-9309; E-mail:
saeki@phar.nagoya-cu.ac.jp
1 Second corresponding author. Fax: q81-586-89-5292; E-mail:
a1-hakura@eisai.co.jp.
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
Ž
.
PII: S1383-5718 99 00049-2

(
)
206
K. Saeki et al.rMutation Research 441 1999 205–213
other environmental chemicals, is sometimes cru-
cially altered by fluorine-substitution, rendering these
substances generally resistant to enzymic oxidation
genicity, and at better understanding the mechanism
of induction of the mutation. In this study, we used
12 fluorinated derivatives of 5- and 8-NQs for the
above purpose, and the Ames test was conducted for
measurement of the mutagenicity.
w
x
at the site of substitution 1–5 . This may result in
either a desirable or undesirable modification of the
w
x
biological activity. As we previously reported 6–11 ,
one example of this is the abolishment of genotoxic-
w
x
w x
ity in carcinogenic 12–14 and mutagenic 15
quinoline by fluorine-substitution at position-3, prob-
ably because of an inhibition of the metabolic trans-
formation of quinoline to its active metabolite re-
sponsible for the production of mutagenic DNA le-
2. Materials and methods
2.1. Materials
Ž .
sions. Introduction of fluorine atom s to a molecule
in question may also allow us to better understand
the mechanism of induction of the mutation by these
mutagens. In fact, we have proposed the enamine
epoxide hypothesis of quinoline mutagenicity, i.e.,
the ultimate mutagenic structure of the 2,3-epoxide
of 1,4-hydrated quinoline, through our studies with
5-NQ, 8-NQ, m-fluoroaniline and 2,4-difluoro-
aniline were purchased from Tokyo Kasei Kogyo
Ž
.
Tokyo ; 4-fluoro-2-nitroaniline from Aldrich Chem-
Ž
.
ical Milwaukee, USA . 3-Fluoroquinoline, 6-fluoro-
quinoline, 7-fluoroquinoline, 8-fluoroquinoline, 5-
w
Ž
.
various fluorine-substituted quinoline derivatives 6–
nitroquinoline N-oxide 5-NQO , 6-fluoroquinoline
N-oxide, 8-fluoroquinoline N-oxide and 6-fluoro-8-
x
9 .
Ž
.
Ž
Nitroquinolines NQs , nitro derivatives of quino-
nitroquinoline Registry Nos. 396-31-6, 396-30-5,
line, form a group of the most simple structural
nitroazaarenes. Nitroazaarenes are poorly understood
except a limited number of compounds such as
396-32-7, 394-68-3, 7613-19-6, 2338-74-1, 2795-43-
.
9 and 343-26-0, respectively were synthesized ac-
w
x
cording to the reported methods 20–23 . 3-Fluoro-
5-nitroquinoline, 5-fluoro-8-nitroquinoline and 8-flu-
w x
4-nitroquinoline N-oxide and azabenzo a pyrenes,
Ž
oro-5-nitroquinoline Registry Nos. 191861-20-8,
although they are distributed ubiquitously as potent
w
x
.
environmental mutagensrcarcinogens 16–19 . The
present study was undertaken to investigate struc-
ture–mutagenicity relationships by fluorine-substitu-
tion on the molecules of NQs, aiming at investigat-
ing the fluorine-substitution effects on the muta-
152167-85-6 and 94832-39-0, respectively were
w x
synthesized in our laboratory 4 . Melting points
were determined with a YANACO MP-500D micro
melting point apparatus without correction. Mass
spectra were measured by a JEOL AX 505HA spec-
Scheme 1. List of 5-nitroquinolines and 8-nitroquinolines examined.

(
)
K. Saeki et al.rMutation Research 441 1999 205–213
207
Ž
.
trometer. UV spectra were recorded on a SHI-
MADZU UV-2100 spectrophotometer. 1H NMR
spectra were measured by a JEOL JMN-EX 270 or
JMN-GSX 400 spectrometer in CDCl3 or Me2 SO-d6
using tetramethylsilane as an internal standard.
The following compounds were newly synthe-
poured into ice water 300 ml , neutralized with
Ž
.
Na2CO3, and extracted with CHCl3 200 ml=2 .
Recrystallization from benzene yielded 7 as brown
needles in 30% yield: mp 170–1738C; MS mrz: 208
1
q
Ž
.
Ž
.
Ž
M
; H NMR Me2 SO-d6 d 8.89 dd, 1H, J6F – 8
.
Ž
s5.1 Hz, J7 – 8 s9.5 Hz, H-8 , 8.73 d, 1H, J2 – 3
s
Ž
.
.
Ž
.
sized in this study Scheme 1 .
5.9 Hz, H-2 , 8.04 t, 1H, J6F – 7 s9.9 Hz, H-7 , 7.89
Ž
.
Ž
.
d, 1H, J3 – 4 s9.2 Hz, H-4 , 7.71 dd, 1H, H-3 ;
Anal. Calcd. for C9 H5FN2O3: C, 51.93; H, 2.42; N,
(
) ( )
2.1.1. 6-Fluoro-5-nitroquinoline 6-F-5-NQ
3
13.46. Found: C, 52.13; H, 2.47; N, 13.45.
Ž
.
6-Fluoroquinoline 309 mg was nitrated with 61%
nitric acid and conc. sulfuric acid at room tempera-
ture for 12 h. Purification of the reaction mixture by
2.1.4. 8-Fluoro-5-nitroquinoline N-oxide
Ž
.
column chromatography aluminium oxide, benzene
(
) ( )
8-F-5-NQO
8
yielded 3 in 38% yield: mp 93–968C; 1H NMR
Ž
.
8-Fluoroquinoline N-oxide 200 mg was nitrated
Ž
.
.
Ž
Me2 SO-d6 d 9.02 dd, 1H, J2 – 3 s4.3 Hz, J2 – 4
s
with 61% nitric acid and conc. sulfuric acid at room
temperature for 3 days. The reaction mixture was
Ž
.
.
1.2 Hz, H-2 , 8.37 d, 1H, J3 – 4 s8.5 Hz, H-4 , 8.35
Ž
Ž
dd, 1H, J7 – 8 s10.4 Hz, J6F – 8 s4.9 Hz, H-8 , 7.64
Ž
.
poured into ice water 400 ml , neutralized with
.
Ž
.
dd, 1H, H-3 , 7.63 t, 1H, J6F – 7 s9.8 Hz, H-7 ;
Ž
.
K2CO3, and extracted with CHCl3 200 ml=2 .
HR-MS mrz: 192.033, Calcd. for C9 H5FN2O2:
Purification of the extract by column chromatogra-
192.032.
Ž
.
phy silica gel, benzene:MeOHs2:1 and recrystal-
lization from benzene–hexane yielded 8 as yellow
powder in 53% yield: mp 178–1808C; MS mrz: 208
2.1.2. 3-Fluoro-5-nitroquinoline N-oxide
1
q
Ž
M
.
Ž
.
Ž
; H NMR Me2 SO-d6 d 8.63 d, 1H, J2 – 3
s
s
s
(
) ( )
3-F-5-NQO
6
.
Ž
6.4 Hz, J2 – 4 s1.5 Hz, H-2 , 8.49 dd, 1H, J3 – 4
Ž . Ž
.
3-Fluoro-5-nitroquinoline 4 300 mg was al-
lowed to react with m-chloroperbenzoic acid 70%,
576 mg, 1.5 eq in 10 ml of CHCl3 at room tempera-
ture for 45 h. The reaction mixture was washed with
.
Ž
9.3 Hz, H-4 , 8.24 dd, 1H, J6 – 7 s8.8 Hz, J6 – 8F
Ž
.
Ž
.
Ž
3.9 Hz, H-6 , 7.69 dd, 1H, H-3 , 7.70 dd, 1H,
.
.
J7 – 8F s12.2 Hz, H-7 ; Anal. Calcd. for C9 H5FN2O3:
C, 51.93; H, 2.42; N, 13.46. Found: C, 51.82; H,
Ž
.
5% Na2CO3 20 ml=2 , dried with MgSO4 anhy-
2.64; N, 13.54.
drous, filtered and evaporated in vacuo. The residue
Ž
was purified by column chromatography silica gel,
.
CHCl3 . Recrystallization from hexane yielded 6 as
pale yellow needles in 80% yield: mp 182–1838C;
2.1.5. N-methyl-5-nitroquinolinium methanesulfonate
1
q
Ž
.
Ž
.
(
) ( )
MS mrz: 208 M ; H NMR Me2 SO-d6 d 9.12
N-Me-5-NQ
9
Ž
Ž
.
Ž .
dd, 1H, J2 – 3F s4.9 Hz, J2 – 4 s2.4 Hz, H-2 , 8.90
5-NQ 352 mg was allowed to react with methyl-
.
Ž
Ž
.
dd, 1H, J6 – 7 s8.6 Hz, H-6 , 8.61 d, 1H, J7 – 8 s7.9
methanesulfonic acid 1.71 ml, 10 eq at 808C for 1
day. After addition of a small amount of hexane, the
reaction mixture was left standing in a freezer. Re-
crystallization of the crude 9, thus obtained, from
.
Ž
.
Hz, H-8 , 8.18 dd, 1H, J3F – 4 s9.8 Hz, H-4 , 7.95
Ž
.
t, 1H, H-7 ; Anal. Calcd. for C9 H5FN2O3: C, 51.93;
H, 2.42; N, 13.46. Found: C, 51.65; H, 2.51; N,
13.22.
ethanol yielded 9 as pale yellow solid in 64% yield:
1
Ž
.
Ž
mp 127–1318C; H NMR Me2 SO-d6 d 9.68 d,
.
Ž
1H, J2 – 3 s5.6 Hz, H-2 , 9.53 d, 1H, J3 – 4 s8.9
.
Ž
.
Ž
2.1.3. 6-Fluoro-5-nitroquinoline N-oxide
Hz, H-4 , 8.92 d, 1H, J6 – 7 s9.2 Hz, H-6 , 8.78 d,
(
) ( )
.
Ž
.
Ž
6-F-5-NQO
7
1H, J7 – 8 s7.6 Hz, H-8 , 8.43 dd, 1H, H-7 , 8.37
Ž
.
Ž
.
Ž
.
6-Fluoroquinoline N-oxide 264 mg was nitrated
with 61% nitric acid and conc. sulfuric acid at room
temperature for 4 days. The reaction mixture was
dd, 1H, H-3 , 4.72 s, 3H, N-CH3 , 2.30 s, 3H,
CH3SOy3 ; Anal. Calcd. for C11H12 N2O5S: C, 46.47;
H, 4.25; N, 9.85. Found: C, 46.22; H, 4.22; N, 9.96.
.

(
)
208
K. Saeki et al.rMutation Research 441 1999 205–213
Fig. 1. Mutagenicity of 5-NQs in S. typhimurium TA100 without
S9 mix. The symbols shown indicate the means of at least three
independent experiments.
Fig. 3. Mutagenicity of N-Me-5-NQs in S. typhimurium TA100
without S9 mix. The symbols shown indicate the means of at least
three independent experiments.
2.1.7. 8-Fluoro-N-methyl-5-nitroquinolinium
2.1.6. 3-Fluoro-N-methyl-5-nitroquinolinium
(
) ( )
methanesulfonate 8-F-N-Me-5-NQ 11
(
) ( )
methanesulfonate 3-F-N-Me-5-NQ 10
Ž . Ž
.
8-Fluoro-5-nitroquinoline 9 100 mg was al-
Ž . Ž
.
3-Fluoro-5-nitroquinoline 2 300 mg was al-
Ž
lowed to react with methylmethanesulfonic acid 0.44
ml, 10 eq in 1 ml of dimethylformamide at 858C for
Ž
lowed to react with methylmethanesulfonic acid 1.32
ml, 10 eq in 1 ml dimethylformamide at 808C for 1
.
.
18 h. The reaction mixture was left standing in a
freezer. The precipitate was washed with ether. 11
was obtained as brown solid in 74% yield: mp
day. The reaction mixture was left standing in a
freezer. The precipitate was washed with hexane and
ether. 10 was obtained as brown solid in 90% yield:
1
Ž
.
Ž
122–1238C; H NMR Me2 SO-d6 d 9.67 d, 1H,
1
Ž
.
Ž
mp 162–1638C; H NMR Me2 SO-d6 d 10.12 t,
.
Ž
J2 – 3 s5.5 Hz, H-2 , 9.54 d, 1H, J3 – 4 s9.2 Hz,
.
Ž
1H, J2 – 3F s3.1 Hz, H-2 , 9.45 dd, 1H, J3F – 4 s9.2
.
.
Ž
Ž
H-4 , 8.80 d, 1H, J6 – 7 s8.8 Hz, J6 – 8 Fs4.0 Hz,
.
Ž
.
.
Ž
Ž
Hz, H-4 , 8.95 d, 1H, J6 – 7 s8.8 Hz, H-6 , 8.83 d,
.
Ž
H-6 , 8.41 dd, 1H, H-3 , 8.31 dd, 1H, J7 – 8F s13.6
.
Ž
1H, J7 – 8 s7.9 Hz, H-8 , 8.40 t, 1H, H-7 , 4.76 s,
.
Ž
.
Ž
Hz H-7 , 4.81 d, 3H, N-CH3 , 2.30 s, 3H, CH3SO3
y
.
Ž
.
3H, N-CH3 , 2.30 s, 3H, CH3SO3 ; Anal. Calcd.
for C11H11FN2O5SPH2O: C, 41.25; H, 4.09; N,
8.75. Found: C, 41.12; H, 4.24; N, 8.99.
.
y ; Anal. Calcd. for C11H11FN2O5SP2H2O: C,
39.05; H, 4.47; N, 8.28. Found: C, 38.74; H, 4.74;
N, 8.71.
Fig. 2. Mutagenicity of 5-NQOs in S. typhimurium TA100 with-
out S9 mix. The symbols shown indicate the means of at least
three independent experiments.
Fig. 4. Mutagenicity of 8-NQs in S. typhimurium TA100 without
S9 mix. The symbols shown indicate the means of at least three
independent experiments.

(
)
K. Saeki et al.rMutation Research 441 1999 205–213
209
(
) ( )
2.1.8. 3-Fluoro-8-nitroquinoline 3-F-8-NQ 13
ture for 6 days. Purification of the reaction mixture
Ž
.
Ž
.
3-Fluoroquinoline 10.3 g was nitrated with 61%
nitric acid and conc. sulfuric acid at room tempera-
ture for 4.5 h. Purification of the reaction mixture by
by column chromatography silica gel, CHCl3 and
recrystallization from benzene–hexane yielded 16 as
white needles in 81% yield: mp 149–1508C; 1H
Ž
Ž . Ž
column chromatography aluminium oxide,
NMR CDCl3 d 9.05 dd, 1H, J2 – 3 s4.0 Hz,
.
Ž
. Ž
J2 – 4 s1.3 Hz, H-2 , 8.26 dd, 1H, J3 – 4 s8.4 Hz,
benzene:hexanes1:1 yielded 13 in 24% yield 3-F-
5-NQ was also obtained in 38% yield : mp 131–
.
. Ž
H-4 , 8.00 dd, 1H, J5– 6 s9.2 Hz, J5– 7F s5.5 Hz,
1
Ž
.
Ž
.
Ž
.
Ž
1338C; H NMR CDCl3 d 8.97 d, 1H, J2 – 3F s;0
H-5 , 7.56 dd, 1H, H-3 , 7.50 t, 1H, J6 – 7F s8.8
.
Ž
.
Hz, J2 – 4 s3.0 Hz, H-2 , 8.01–8.05 m, 2H, H-5 and
Hz, H-6 ; Anal. Calcd. for C9 H5FN2O2: C, 56.26;
.
Ž
.
Ž
H-7 , 7.90 dd, 1H, J3F – 4 s8.3 Hz, H-4 , 7.68 t,
H, 2.62; N, 14.58. Found: C, 56.25; H, 2.87; N,
14.65.
.
1H, J5– 6 sJ6 – 7 s7.9 Hz, H-6 ; HR-MS mrz:
192.033, Calcd. for C9 H5FN2O2: 192.030.
2.2. Mutation assay
(
) ( )
2.1.9. 7-Fluoro-8-nitroquinoline 7-F-8-NQ 16
Chemicals were tested for mutagenicity using
Salmonella typhimurium TA100 without S9 mix ac-
cording to the procedure of the Ames test with a
Ž
.
7-Fluoroquinoline 251 mg was nitrated with 61%
nitric acid and conc. sulfuric acid at room tempera-
Table 1
Mutagenicity of NQs in S. typhimurium TA100 without S9 mix
Substituent
Revertants per
plate per nmola
Maximum
F-substitution effect
revertants per plateb
on mutagenicityc
5-NQs
Ž .
Ž .
3-F 2
Ž
.
none 1
0.71
1.43
0.45
17.0
1321
3011
268
1.0
2.0
0.6
Ž . Ž
.
.
6-F 3 o-
Ž . Ž
8-F 4 p-
2528
24
5-NQOs
Ž .
Ž .
3-F 6
Ž
.
none 5
0.83
0.70
1.41
22.6
1295
833
539
1.0
0.8
1.7
Ž . Ž
.
.
6-F 7 o-
Ž . Ž
8-F 8 p-
2533
27
N-Me-5-NQs
Ž .
Ž
.
.
none 9
0.32
2.31
38.1
270
578
2551
1.0
7.2
Ž
.
. Ž
3-F 10
Ž
.
8-F 11 p-
119
8-NQs
Ž
.
Ž
1.0
2.2
none 12
0.10
0.22
2.61
5.29
0.08
281
364
3221
747
Ž
.
3-F 13
. Ž
Ž
.
5-F 14 p-
26
53
0.8
Ž
. Ž
.
6-F 15 m-
. Ž
Ž
.
7-F 16 o-
436
aCalculated from the slope of the linear portion of each curve near the origin. The numbers indicate the means of at least three independent
experiments.
bObtained from the assay up to 5 mmolrplate. The numbers indicate the means of at least three independent experiments.
c
Ž
.
The ratio of the mutagenic activity revertantsrplaternmol of the fluorinated quinoline to that of the corresponding parent non-fluorinated
compound.

(
)
210
K. Saeki et al.rMutation Research 441 1999 205–213
modification of pre-incubation at 378C for 20 min as
5-NQOs
w
x
previously reported 24–26 . Briefly, The chemical
to be tested was dissolved in 0.1 ml of dimethyl
sulfoxide and added to a mixture of 0.5 ml of 100
8-F-5-NQO para-position
Ž
.
Ž
.
mM sodium phosphate buffer pH 7.4 and 0.1 ml of
the overnight cell culture. After being incubated at
378C for 20 min under gentle shaking, 2.0 ml of 0.05
mM L-histidine–0.05 mM biotin molten top agar was
added. The mixture was layered on minimal glu-
cose–agar plates. The number of histidine prototroph
revertants was counted after incubation at 378C for 2
days. In this study, each compound was assayed in
triplicate or more replicate at each dose level. The
mean number of revertants obtained at each dose is
illustrated in Figs. 1–4.
46-F-5-NQO ortho-position
Ž
.
)5-NQOf3-F-5-NQf 5-NQ
Ž
.
N-Me-5-NQs
8-F-N-Me-5-NQ para-position
Ž
.
43-F-N-Me-5-NQ
) 5-NQ )N-Me-5-NQ.
Ž
.
In all the three 5-nitro groups, the fluorine atom
located para to the nitro group greatly enhanced the
mutagenicity. 3-Fluorinated derivatives, in which the
fluorine atom was located on the pyridine ring,
showed mutagenicities slightly more potent than, or
almost the same as, the corresponding non-fluorin-
ated derivatives. Ortho-fluorine-substitution on the
5-NQ molecule decreased the mutagenicity, while
the same substitution on the 5-NQO molecule slightly
enhanced the mutagenicity, compared to their respec-
tive parent molecules. Although N-Me-5-NQ was
less mutagenic than 5-NQ and 5-NQO, its muta-
genicity was most markedly enhanced by fluorine-
substitution among the above three parent com-
pounds.
3. Results
The NQs listed in Chart 1 were tested for muta-
genicity in S. typhimurium TA100 in the absence of
S9 mix according to the procedure of the Ames test
w
x
24–26 . The results are summarized in Table 1,
where the number of revertants per plate per nmol
was calculated from the slope of the initial linear
portion of each dose–response curve shown in Figs.
1–4, and the ratio of the number of revertants per
plate per nmol for fluorinated NQs to that for non-
fluorinated parent NQs is termed as the F-substitu-
tion effect on mutagenicity. In addition, the maximal
number of revertants per plate obtained from the
assay up to 5 mmolrplate were included in Table 1.
3.2. Mutagenicity of 8-NQs
3.1. Mutagenicity of 5-NQs
As shown in Fig. 4 and Table 1, the mutagenic
As shown in Figs. 1–3 and Table 1, mutagenic
Ž
.
potencies revertantsrplaternmol of the 8-NQs in
the Ames assay system decrease in the following
order:
Ž
.
potencies revertantsrplaternmol of the isomers ex-
amined in the Ames assay system decrease in the
following order:
5-NQs
6-F-8-NQ meta-position
Ž
.
8-F-5-NQ para-position
Ž
.
)5-F-8-NQ para-position
Ž
.
43-F-5-NQ
)5-NQ
43-F-8-NQ
)6-F-5-NQ ortho-position
Ž
.
)8-NQf7-F-8-NQ ortho-position .
Ž .

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)
K. Saeki et al.rMutation Research 441 1999 205–213
211
The fluorine atom located at the meta- or para-
position of the nitro group markedly enhanced the
mutagenicity. Fluorine-substitution in the pyridine
atom at position-3 of NQs probably inhibits the
formation of the enamine epoxides responsible for
production of mutagenic DNA lesions. The second
mechanism described above is a hypothesis based on
DNA-adduct formation analogous to dinitrophenyla-
tion of amino acids with dinitrofluorobenzene, which
is often used in identification of the N-terminal of
proteins. In fact, we observed that similar rapid
substitution reactions proceeded between the ortho-
and para-fluorine-substituted NQs and N-acetyl-L-
cysteine at 378C in phosphate buffer solution, and
meta- and 3-fluorinated NQs did not cause this sub-
Ž
.
moiety i.e., 3-F-8-NQ slightly potentiated the mu-
tagenicity of 8-NQ. Ortho-fluorine-substitution
showed no remarkable effect on the mutagenicity of
8-NQ.
4. Discussion
Ž
stitution under the same conditions unpublished re-
.
We measured the mutagenicity of 16 5- and 8-NQs
and their fluorinated derivatives in the absence of S9
sult . As shown in Table 1, the ortho-fluoro-NQs are
about one order less mutagenic than the correspond-
ing para-fluoro-NQs. In addition, the meta- and
3-fluorinated NQs are almost as mutagenic as, or
more mutagenic than, the corresponding ortho-flu-
oro-NQs. These findings may suggest that the muta-
genicity of F-NQs is not mainly due to the mecha-
nism based on the nucleophilic substitution reaction
of the F-NQs with the target DNA molecule. Fur-
thermore, these aromatic nucleophilic substitutions
will selectively occur with highly nucleophilic sub-
stances such as proteins or amino acids containing
sulfur atom. Such compounds will be so efficiently
quenched by cellular proteins or amino acids that
they will not reach the target DNA. Although the
possibility of mutation by this mechanism cannot
completely be excluded, we believe that the muta-
genicity of NQs was induced by the mechanism
widely accepted with nitro aromatic compounds.
The enhancing effect of para-fluorine substitu-
tion on the mutagenicity of 5- and 8-NQs might be
explained by their potent electron-donating R-effect,
which leads to the formation of a more stable nitre-
nium cation, an ultimate active form of NQs gener-
ated by ionic cleavage of the N–O bond of O-
acetylhydroxylaminoquinolines. Although the
ortho-fluorines of 6-F-5- and 7-F-8-NQs may exert
the enhancing R-effects similar to those of the
para-fluorines, the mutagenicities of ortho-fluorin-
ated 5- and 8-NQs were ca. one order lower than
those of the corresponding para-fluorinated com-
pounds, as described before. One possible reason for
this is that the ortho-fluorines to the nitro group
hinder access of the enzymes involved in mutagenic
activation, which include bacterial nitroreductase and
Ž
.
mix without metabolic activation systems using the
Ames test with a modification of pre-incubation at
378C for 20 min to investigate the structure-mutagen-
icity relationship after fluorine-substitution.
Many nitroarenes are transformed to their proxi-
mate active forms, O-acetylarylhydroxylamines, by
bacterial nitroreductase and O-acetyltransferase
without help of S9 mix to exert their mutagenicity
27–30 . 5- and 8-NQs and their derivatives tested in
this study were all mutagenic in S. typhimurium
TA100 without S9 mix. The ratio of the mutagenic
activities revertantsrplaternmol of the fluorinated
NQs to those of the corresponding parent non-fluo-
rinated NQs ranged from 0.6- to 119-fold Table 1 .
Greatest enhancing effect 24- to 119-fold on the
mutagenicity was obtained by para-fluorine-substitu-
tion to the nitro group compounds 4 , 8 , 11 and
w
x
Ž
.
Ž
.
Ž
.
Ž
Ž . Ž . Ž .
Ž
..
14 . With regard to 8-NQs, the mutagenicity was
Ž
.
also enhanced by meta-substitution 15 .
The structure of NQs may remind us of three
possible hypotheses of the mechanism of mutagenic-
Ž .
ity; 1 a mechanism with nitro aromatic compounds,
Ž .
2 a mechanism based on an aromatic nucleophilic
substitution, and
metabolically activated 1,2-epoxide-1,4-hydrated
Ž .
the mechanism involving
3
Ž
.
quinoline enamine epoxide in the case of quinoline
mutagenicity. Firstly, the involvement of enamine
epoxides can be denied in NQs mutagenicity by the
fact that the 3-fluorinated derivatives of NQs are
mutagenic in the absence of S9 mix as well as in the
presence of S9 mix data not shown . Because the
formation of arene oxides requires a metabolic acti-
vation by S9 mix and the introduction of a fluorine
Ž
.

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)
212
K. Saeki et al.rMutation Research 441 1999 205–213
assessed by an in vivo mutagenesis assay using lacZ trans-
O-acetyltransferase. In any way, further studies are
needed for better understanding the mechanism of
induction of the NQ mutagenicity.
In conclusion, the present study with a series of
fluorine-substituted NQs provides further basic data
for possible mutagenic modification by fluorine-sub-
stitution and may offer a tool for mechanistic studies.
Ž
.
genic mice, Mutat. Res. 414 1998 165–169.
w
w
x
12 K. Hirao, Y. Shinohara, H. Tsuda, S. Fukushima, M. Taka-
hashi, N. Ito, Carcinogenic activity of quinoline on rat liver,
Ž
.
Cancer Res. 36 1976 329–335.
x
13 Y. Shinohara, T. Ogiso, M. Hananouchi, K. Nakanishi, T.
Yoshimura, N. Ito, Effect of various factors on the induction
of liver tumors in animals by quinoline, Jpn. J. Cancer Res.
Ž
.
68 1977 785–796.
w
x
14 E.H. Wayand, J. Defauw, C.A. McQueen, C.L. Meschter,
S.K. Meegalla, E.J. LaVoie, Bioassay of quinoline, 5-fluoro-
quinoline, carbazole, 9-methylcarbazole and 9-ethylcarbazole
Ž
.
in newborn mice, Food Chem. Toxicol. 31 1993 707–715.
References
w
w
w
x
15 M. Nagao, T. Yahagi, Y. Seino, T. Sugimura, N. Ito, Muta-
genicities of quinoline and its derivatives, Mutat. Res. 42
w x
1
Ž
.
E.J. LaVoie, L. Tulley-Freiler, V. Bedenko, D. Hoffmann,
Mutagenicity of substituted phenanthrenes in Salmonella ty-
1977 335–342.
x
16 M. Nagao, T. Sugimura, Molecular biology of the carcino-
Ž
.
phinurium, Mutat. Res. 116 1983 91–102.
Ž
.
gen, 4-nitroquinoline 1-oxide, Adv. Cancer Res. 23 1976
w x
2
S.S. Hecht, E.J. LaVoie, V. Bedenko, L. Pingaro, S.
Katayama, D. Hoffmann, D.J. Sardella, E. Boger, R.E. Lehr,
Reduction of tumorigenicity and of dihydrodiol formation by
131–169.
x
17 M. Tada, K.H. Kohda, Y. Kawazoe, Biomimetic preparation
and structure determination of QG, one of the quinoline-DNA
base adducts formed in cells treated with 4-nitroquinoline
1-oxide, Jpn. J. Cancer Res. Gann 75 1984 976–985.
18 K. Fukuhara, A. Hakura, N. Sera, H. Tokiwa, N. Miyata, 1-
w
x
fluorine substitution in the angular rings of dibenzo a,i -
pyrene, Cancer Res. 41 1981 4341–4345.
Ž
.
Ž
.
Ž
.
w x
3
w
x
E. Hubenman, T.J. Slag, Mutagenicity and tumor-initiating
activity of fluorinated derivatives of 7,12-dimethyl-
w x
and 3-nitro-6-azabenzo a pyrene and their N-oxide; highly
mutagenic nitrated azaarenes, Chem. Res. Toxicol. 5 1992
w x
Ž
.
benz a anthracene, Cancer Res. 39 1979 411–414.
Ž
.
w x
4
L. Diamond, K. Chain, R.G. Harvey, J. DiGiovanni, Muta-
genic activity of methyl- and fluoro-substituted derivatives of
polycyclic aromatic hydrocarbons in a human hepatoma
149–153.
w
w
x
19 N. Sera, K. Fukuhara, N. Miyata, K. Horikawa, H. Tokiwa,
w x
Mutagenicity of nitro-azabenzo a pyrene and its related com-
Ž
.
cell-mediated assay, Mutat. Res. 136 1984 65–72.
Ž
.
pounds, Mutat. Res. 280 1992 81–85.
w x
5
E.C. Miller, J.A. Miller, The carcinogenicity of fluoro deriva-
tives of 10-methyl-1,2-benzanthracene: 3- and 4-monofluoro
x
20 A. Roe, G.F. Hawkins, The preparation of heterocyclic fluo-
rine compounds by the Schiemann reaction: II. The monoflu-
Ž
.
derivatives, Cancer Res. 20 1960 133–137.
Ž
.
oroquinolines, J. Am. Chem. Soc. 71 1949 1785–1786.
w x
6
K. Takahashi, M. Kamiya, Y. Sengoku, K. Kohda, Y. Kawa-
zoe, Deprivation of the mutagenic property of quinoline:
inhibition of mutagenic metabolism by fluorine substitution,
w
w
x
21 M.H. Palmer, The Skraup reaction: formation of 5- and
7-substituted quinolines, J. Chem. Soc. 1962 3645–3652.
22 M. Bellas, H. Suschitzky, Heterocyclic fluorine compounds:
Ž
.
x
Ž
.
Chem. Pharm. Bull. 36 1988 4630–4633.
V. Fluororyridine oxides and fluoroquinoline N-oxides, J.
w x
7
M. Kamiya, Y. Sengoku, K. Takahashi, K. Kohda, Y. Kawa-
zoe, Antimutagenic structure modification of quinoline: Fluo-
rine-substitution at position-3, in: Y. Kuroda, D.M. Shankel,
Ž
.
Chem. Soc. 1963 4007–4009.
w
w
x
23 J.H. Wilkinson, I.L. Finar, Properties of fluorine-substituted
5-aminoacridines and related compounds: III. Some 5-amino-
1,2,2 ,3 -pyridoacridines, J. Chem. Soc. 1948 288–291.
24 B.N. Ames, J. McCann, E. Yamazaki, Methods for detecting
X
X
Ž
.
M.D. Waters Eds. , Antimutagenesis and Anticarcinogene-
sis, Vol. II, Plenum, New York, 1990, pp. 441–446.
Ž
.
x
w x
8
K. Saeki, K. Takahashi, Y. Kawazoe, Metabolism of muta-
genicity-deprived 3-fluoroquinoline: comparison with muta-
carcinogens and mutagens with the Salmonellarmamma-
lian-microsome mutagenicity test, Mutat. Res. 31 1975
Ž
.
Ž
.
genic quinoline, Biol. Pharm. Bull. 16 1993 232–234.
K. Saeki, H. Kawai, Y. Kawazoe, A. Hakura, Dual stimula-
tory and Inhibitory effects of fluorine-substitution on muta-
genicity: an extension of the enamine epoxide theory for
activation of the quinoline nucleus, Biol. Pharm. Bull. 20
347–363.
w x
9
w
x
25 K. Takahashi, T. Kaiya, Y. Kawazoe, Structure–mutagenic-
ity relationship among aminoquinolines, aza-analogues of
naphthylamine, and their N-acetyl derivatives, Mutat. Res.
Ž
.
187 1987 191–197.
Ž
.
1997 646–650.
w
x
26 A. Hakura, Y. Tsutsui, H. Mochida, Y. Sugihara, T. Mikami,
F. Sagami, Mutagenicity of dihydroxybenzenes and dihy-
droxynaphthalenes for Ames Salmonella tester strains, Mu-
tat. Res. 371 1996 293–299.
27 H.S. Rosenkranz, R. Mermelstein, Mutagenicity and geno-
w
w
x
10 K. Saeki, M. Kadoi, Y. Kawazoe, M. Futakuchi, D. Ti-
wawech, T. Shirai, Modification of the carcinogenic property
of quinoline, a hepatocarcinogen, by fluorine atom substitu-
tion: evaluation of carcinogenicity by a medium-term assay,
Ž
.
w
x
Ž
.
Biol. Pharm. Bull. 20 1997 40–43.
toxicity of nitroarenes: all nitro-containing chemicals were
x
11 Y. Miyata, K. Saeki, Y. Kawazoe, M. Hayashi, T. Sofuni, T.
Suzuki, Antimutagenic structural modification of quinoline
Ž
.
not created equal, Mutat. Res. 114 1983 217–267.
w
x
28 E.C. McCoy, H.S. Rosenkranz, R. Mermelstein, Evidence for

(
)
K. Saeki et al.rMutation Research 441 1999 205–213
213
the existence of a family of bacterial nitroreductases capable
of activating nitrated polycyclics to mutagens, Environ. Mu-
DNP6 to the mutagenic action of nitroarenes, Mutat. Res.
Ž
.
121 1983 17–23.
Ž
.
w x
tagen. 3 1981 421–427.
30 D.W. Bryant, D. McCalla, P. Lultschik, M. Quilliam, B.
w
x
29 E.C. McCoy, M. Anders, H.S. Rosenkranz, The basis of the
McCarry, Metabolism of 1,8-dinitropyrene by Salmonella
Ž .
insensitivity of Salmonella typhimurium strain TA98r1,8-
typhimurium, Chem.-Biol. Interact. 49 1984 351–368.