Anti-mutagenic structural modification by fluorine-substitution in highly mutagenic 4-methylquinoline derivatives

Article abstract of DOI:10.1016/S1383-5718(99)00226-0

We have previously shown that fluorine-substitution at position 3 of quinoline deprived this molecule of mutagenicity, possibly due to interference with the yield of its metabolically activated form, the 1,4-hydrated 2,3-epoxide (enamine epoxide), which is directly responsible for the mutagenic modification of DNA. To further explore the possibility of a method for anti-mutagenic modification of mutagens by fluorine-substitution, 4-methylquinoline (4-MeQ), the most mutagenic form of all the quinoline derivatives examined so far, was used as a target in the present study. Five mono- and di-fluorinated derivatives of 4-MeQ, 2-fluoro-4-methylquinoline (2-F-4-MeQ), 6-F-4-MeQ, 7-F-4-MeQ, 2,6-difluoro-4-methylquinoline (2,6-diF-4-MeQ), and 2,7-diF-4-MeQ, were subjected to analysis of their structure-mutagenicity relationships. The 2-fluorinated derivatives (2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ) were all non-mutagenic in the Ames test. 7-F-4-MeQ was as highly mutagenic as, and 6-F-4-MeQ was less mutagenic than non-fluorinated 4-MeQ. Metabolic studies were also conducted with 4-MeQ, 2-F-4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ, using a liver microsomal enzyme fraction prepared from the 3-methylcholanthrene-treated rat. The HPLC analytical data showed that, although the metabolic patterns (hydroxylation at 4-methyl group as a main metabolic pathway and 3-hydroxylation as a minor pathway) of these four F-MeQs were similar to one another, only the 3-hydroxy metabolite of 2-F-4-MeQ was not produced under the present experimental conditions employed. These results suggest that fluorine-substitution at position 2 of 4-MeQ inhibited the formation of the enamine epoxide in the pyridine moiety and deprived this molecule of mutagenicity as in the case of quinoline. Copyright (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S1383-5718(99)00226-0

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Mutation Research 465 2000 173–182
www.elsevier.comrlocatergentox
Community address: www.elsevier.comrlocatermutres
Anti-mutagenic structural modification by fluorine-substitution in
highly mutagenic 4-methylquinoline derivatives
a
b
a
a,)
Taka-aki Kato , Atsushi Hakura , Takaharu Mizutani , Ken-ichi Saeki
a
Faculty of Pharmaceutical Sciences, Nagoya City UniÕersity, 3-1 Tanabedori, Mizuho-ku, Nagoya 467-8603, Japan
Drug Safety Research Laboratories, Eisai 1, Takehaya-machi, Kawashima-cho, Hashima-gun, Gifu 501-6195, Japan
b
Received 7 October 1999; received in revised form 15 November 1999; accepted 15 November 1999
Abstract
We have previously shown that fluorine-substitution at position 3 of quinoline deprived this molecule of mutagenicity,
Ž
possibly due to interference with the yield of its metabolically activated form, the 1,4-hydrated 2,3-epoxide enamine
.
epoxide , which is directly responsible for the mutagenic modification of DNA. To further explore the possibility of a
Ž
.
method for anti-mutagenic modification of mutagens by fluorine-substitution, 4-methylquinoline 4-MeQ , the most
mutagenic form of all the quinoline derivatives examined so far, was used as a target in the present study. Five mono- and
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.
di-fluorinated derivatives of 4-MeQ, 2-fluoro-4-methylquinoline 2-F-4-MeQ , 6-F-4-MeQ, 7-F-4-MeQ, 2,6-difluoro-4-
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.
methylquinoline 2,6-diF-4-MeQ , and 2,7-diF-4-MeQ, were subjected to analysis of their structure-mutagenicity relation-
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.
ships. The 2-fluorinated derivatives 2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ were all non-mutagenic in the Ames
test. 7-F-4-MeQ was as highly mutagenic as, and 6-F-4-MeQ was less mutagenic than non-fluorinated 4-MeQ. Metabolic
studies were also conducted with 4-MeQ, 2-F-4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ, using a liver microsomal enzyme
fraction prepared from the 3-methylcholanthrene-treated rat. The HPLC analytical data showed that, although the metabolic
Ž
.
patterns hydroxylation at 4-methyl group as a main metabolic pathway and 3-hydroxylation as a minor pathway of these
four F-MeQs were similar to one another, only the 3-hydroxy metabolite of 2-F-4-MeQ was not produced under the present
experimental conditions employed. These results suggest that fluorine-substitution at position 2 of 4-MeQ inhibited the
formation of the enamine epoxide in the pyridine moiety and deprived this molecule of mutagenicity as in the case of
quinoline. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Anti-mutagenic structural modification; Fluorine-substitution; 4-methylquinoline; Mutagenicity; Metabolism
1. Introduction
genicity and metabolism of aza-aromatic compounds.
It is well known that, when an aromatic nucleus is
fluorinated, enzymatic oxidation is generally inhib-
ited at the site of F-substitution due to its electron-
Our continued interest in biological effects of a
Ž .
Ž .
fluorine F atom s has prompted us to investigate
Ž .
the substituent effect of an F atom s on the muta-
w
x
withdrawing nature 1–4 . Therefore, F-substitution
at the activation site may decrease the mutagenicity
of aromatic compounds. For example, we previously
)
Corresponding author. Tel.: q81-52-836-3485; fax: q81-52-
w x x
w
reported that mutagenic 5 and carcinogenic 6,7
834-9309; e-mail: saeki@phar.nagoya-cu.ac.jp
1383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
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PII: S1383-5718 99 00226-0

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T. Kato et al.rMutation Research 465 2000 173–182
w
x
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)
quinoline was deprived of both in vitro 8,9 and in
2.1.1. 2-Fluoro4-methylquinoline 2-F-4-MeQ
w
x
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.
vivo 10,11 genotoxicity by F-substitution at posi-
tion 3, whereas 5-fluoroquinoline was as genotoxic
as quinoline in the same assay systems. These previ-
ous findings have revealed that the genotoxicity of
quinoline may be attributable to the formation of an
enamine epoxide, 2,3-epoxide of 1,4-hydrated quino-
2-OH-4-MeQ 499 mg was dissolved in POCl3
5 ml , and PCl5 804 mg was added to this solu-
tion. After refluxing for 24 h, the reaction mixture
was poured into water, neutralized with sodium car-
bonate, extracted with CHCl3, and evaporated. 2-
Ž
.
Ž
.
Ž
Chloro-4-methylquinoline 2-Cl-4-MeQ, CAS Reg-
w
x
.
line 8–15 .
istry No. 634-47-9 was obtained as a white solid in
q
q
Ž
.
Ž
. Ž .
4-Methylquinoline 4-MeQ was found to be much
more mutagenic in Salmonella typhimurium TA100
when compared to quinoline, in the presence of rat
liver S9 mix 9,16 . Our previous studies showed that
4-MeQ was metabolized by the liver microsomal
96% yield. MS mrz: 177
M
, 179 M q2 .
1
Ž
.
Ž
.
Ž
H-NMR CDCl3 d: 2.69 s, 3H, CH3 , 7.25 s,
H-3 , 7.57 ddd, H-6 , 7.73 ddd, H-7 , 7.96 dd,
H-8 , 8.02 dd, H-5 ; J5– 6 s7.9, J6 – 7 s6.9, J7 – 8
.
Ž
.
Ž
.
Ž
w
x
.
Ž
.
s
8.3 Hz.
Ž
.
Ž
.
preparation from the 3-methylcholanthrene 3-MC -
treated rat mainly to non-mutagenic 4-hydroxymeth-
2-Cl-4-MeQ 296 mg was dissolved in 4 ml of
tetraethylammonium fluoride-5.5 mol eq. of hydro-
gen fluoride. Then the solution was kept stirring for
20 min at 1608C. The reaction mixture was poured
into 100 ml of water, neutralized with sodium car-
bonate, and extracted with CHCl3. Purification of the
Ž
.
ylquinoline 4-HMeQ and a small amount of 3-hy-
Ž
.
droxy-4-methylquinoline 3-OH-4-MeQ , which is
possibly derived from the corresponding 2,3-oxide
derivative, and that this compound was deprived of
its potent mutagenicity by introduction of a chlorine
atom to position 3 16 . From these findings, we
proposed that the mutagenic activation of 4-MeQ is
achieved through a mechanism common to that of
quinoline itself. To confirm this hypothesis, the pre-
sent study was undertaken to investigate the effect of
fluorine-substitution on the mutagenicity and
metabolism of 4-MeQ, using three mono- and two
di-fluorinated derivatives.
Ž
extract by column chromatography silica gel,
CHCl3 yielded 2-F-4-MeQ as colorless oil in 36%
yield. MS mrz: 161 M
2.69 s, 3H, CH3 , 6.89 s, H-3 , 7.52 ddd, H-6 ,
w
x
.
1
q
Ž
.
Ž
.
.
H-NMR CDCl3 d:
Ž
Ž
.
Ž
.
Ž
.
s
.
Ž
.
7.70 ddd, H-7 , 7.93 m, 2H, H-5 and H-8 ; J5– 6
8.3, J6 – 7 s6.8, J7 – 8 s8.3 Hz. Anal. Calcd. for
C10 H8 FN: C, 74.52; H, 5.00; N, 8.69. Found: C,
74.15; H, 4.96; N, 8.64.
(
2.1.2. 6-Fluoro-4-methylqunoline 6-F-4-MeQ, CAS
)
Registry No. 31598-65-9
2. Materials and methods
Ž
Ž
.
p-Fluoroaniline 2.35 g was allowed to react with
methylvinylketone 1.8 ml, 1.0 eq in 50 ml of EtOH
at 08C for 1 h. Then the solution was kept stirring at
.
2.1. Materials
Ž
.
room temperature for 5 h. ZnCl2 2.98 g , FeCl3 P
Ž
.
Ž
Ž
.
Ž
.
4-MeQ CAS Registry No. 491-35-0 , 3-MC CAS
6H2O 4.47 g and c-HCl 2.0 ml were mixed in the
reaction mixture. After stirring at 708C for 13 h, the
reaction mixture was poured into water, neutralized
with sodium carbonate, and extracted with CHCl3.
Purification of the extract by column chromatogra-
.
Registry No. 56-49-5 , fluorinated anilines, and 2-
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.
hydroxy-4-methylquinoline 2-OH-4-MeQ were
purchased from Aldrich. Tetraethylammonium fluo-
ride-5.5 mol eq. of hydrogen fluoride was from
Morita Chemical, Osaka, Japan. Melting points were
determined with a Yamato MP-500D micro melting
point apparatus without correction. Mass spectra were
measured with a JEOL JMS-SX102A spectrometer.
1H-NMR spectra were recorded with a JEOL JNM-
EX270 or JNM-GSX400 spectrometer in CDCl3 us-
ing tetramethylsilane as an internal standard. The
following compounds were synthesized in this study.
Ž
phy aluminium oxide, hexane:CHCl3 s 1:1
CHCl3, and silica gel, hexane:CHCl3 s 1:1
.
CHCl3 yielded 6-F-4-MeQ as a white solid in 22%
q
Ž
.
Ž
Ž
.
yield. mp 1808C sublimated . MS mrz: 161 M
H-NMR CDCl3 d: 2.62 s, 3H, CH3 , 7.21 d,
H-3 , 7.45 ddd, H-7 , 7.54 dd, H-5 , 8.08 dd,
H-8 , 8.72 d, H-2 ; J2 – 3 s4.4, J5– 6F s9.9, J5– 7
2.8, J6F – 7 s11.2, J6F – 8 s5.6, J7 – 8 s8.9 Hz. Anal.
.
1
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.
.
Ž
.
.
Ž
.
Ž
.
Ž
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.
s

(
)
T. Kato et al.rMutation Research 465 2000 173–182
175
Ž .
6-F-2-OH-4-MeQ 500 mg was dissolved in
Calcd. for C10 H8 FNPHClP3r2 H2O: C, 53.46; H,
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.
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.
5.38; N, 6.23. Found: C, 53.42; H, 5.40; N, 6.29.
POCl3 5 ml , and PCl5 804 mg was added to this
solution. After refluxing for 12 h, the reaction mix-
ture was poured into water, neutralized with sodium
carbonate, extracted with CHCl3, and evaporated.
(
)
2.1.3. 7-Fluoro-4-methylquinoline 7-F-4-MeQ
Ž
.
Ž
m-Fluoroaniline 3.74 g was allowed to react
2-Chloro-6-fluoro-4-methylquinoline 2-Cl-6-F-4-
Ž
.
.
MeQ was obtained as a white solid in 99% yield.
with methylvinylketone 3.2 ml, 1.0 eq in 50 ml of
EtOH at 08C for 1 h. Then the solution was kept
stirring at room temperature for 18 h. ZnCl2 4.1 g
and FeCl3 P6H2O 14.2 g were added to the reac-
tion mixture. After stirring at 708C for 13 h, the
reaction mixture was poured into water, neutralized
with sodium carbonate, and extracted with CHCl3.
The extract was crudely purified by column
q
q
Ž . Ž .
mp 112–1188C. MS mrz: 195 M , 197 M q2 .
1
Ž
.
Ž . Ž . Ž
H-NMR CDCl3 d: 2.65 s, 3H, CH3 , 7.26 s,
Ž
.
. Ž . Ž . Ž
H-3 , 7.49 ddd, H-7 , 7.55 dd, H-5 , 8.01 dd,
.
H-8 ; J5– 6F s9.5, J5– 7 s2.7, J6F – 7 s11.0, J6F – 8
5.4, J7 – 8 s9.0 Hz. Anal. Calcd. for C10 H7ClFN: C,
61.40; H, 3.61; N, 7.16. Found: C, 61.41; H, 3.66;
N, 7.14.
s
Ž
Ž
.
chromatography alumina, hexane:CHCl3 s1:1
2-Cl-6-F-4-MeQ 279 mg was dissolved in 4 ml
of tetraethylammonium fluoride-5.5 mol eq. of hy-
drogen fluoride. Then the solution was kept stirring
for 20 min at 1608C. The reaction mixture was
poured into 100 ml of water, neutralized with sodium
carbonate, and extracted with CHCl3. Purification of
.
CHCl3 . The crude mixture of 7-F-4-MeQ and 5-F-
4-MeQ was purified by recrystallization as a dichro-
mate salt from EtOH. Further purification of the
7-F-4-MeQ dichromate salt by recrystallization as a
sulfate salt from EtOH yielded 7F-4-MeFQPH2 SO4
as white needles in 21% yield. mp 142–1448C. MS
Ž
the extract by column chromatography silica gel,
CHCl3 and recrystallization from hexane yielded
2,6-diF-4-MeQ as white needles in 29% yield. mp
1
q
Ž
.
Ž
.
.
mrz: 161 free form, M
.
H-NMR CDCl3 d:
Ž
Ž
.
Ž
.
Ž
.
s
s
2.83 s, 3H, CH3 , 7.65 d, H-3 , 7.73 ddd, H-6 ,
1
q
.
Ž
.
Ž
.
Ž
Ž
.
Ž
.
.
7.84 dd, H-8 , 8.37 dd, H-5 , 8.98 d, H-2 ; J2 – 3
97–1008C. MS mrz: 179 M . H-NMR CDCl3
Ž
.
.
Ž
4.9, J5– 6 s9.0, J5– 7F s6.1, J6 – 7F s11.5, J6 – 8
2.4, J7 – 8 s9.8 Hz. Anal. Calcd for C10 H8 FNP
H2 SO4: C, 46.33; H, 3.89; N, 5.40. Found: C, 46.14;
H, 3.91; N, 5.62.
d:2.69 s, 3H, CH3 , 6.97 s, H-3 , 7.45 ddd, H-7 ,
Ž . Ž .
7.58 dd, H-5 , 7.94 dd, H-8 ; J5– 6F s9.5, J5– 7 s
2.8, J6F – 7 s11.0, J6F – 8 s5.4, J7 – 8 s9.1 Hz. Anal.
Calcd for C10 H7 F2 N: C, 67.04; H, 3.94; N, 7.82.
Found: C, 66.91; H, 3.93; N, 7.60.
(
2.1.4. 2,6-Difluoro-4-methylquinoline 2,6-diF-4-
)
(
2.1.5. 2,7-Difluoro-4-methylquinoline 2,7-dif-4-
MeQ
p-Fluoroaniline 5.0 g was refluxed with diketene
Ž
.
)
MeQ
m-Fluoroaniline 5.0 g was refluxed with diketene
Ž
.
Ž
.
Ž
.
4.2 ml in THF 100 ml . After 24 h, this solution
Ž
. Ž .
was evaporated. The resulting residue was kept at
808C for 30 min in c-H2 SO4. The reaction mixture
was poured into 100 ml of water, neutralized with
sodium carbonate and extracted with CHCl3. The
organic layer was dried over anhydrous MgSO4 and
evaporated. 6-Fluoro-2-hydroxy-4-methylquinoline
4.2 ml in THF 100 ml . After 12 h, this solution
was evaporated. The resulting residue was kept at
808C for 30 min in c-H2 SO4. The reaction mixture
was poured into 100 ml of water, and the precipitates
were collected by suction. 7-Fluoro-2-hydroxy-4-
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.
methylquinoline 7-F-2-OH-4-MeQ was obtained as
Ž
.
6-F-2-OH-4-MeQ was obtained in 91% yield. mp
a white solid in 66% yield. mp 251–2538C. MS
1
1
q
q
Ž
.
.
.
Ž
.
Ž
.
Ž
.
Ž
1808C sublimated . MS mrz: 177 M . H-NMR
mrz: 177 M . H-NMR CDCl3 d: 2.41 s, 3H,
Ž
Ž
Ž
.
Ž
.
. Ž . Ž .
CH3 , 6.35 s, H-3 , 7.05 m, 2H, H-6 and H-8 ,
CDCl3 d: 2.40 s, 3H, CH3 , 6.45 s, H-3 , 7.32
dd, H-8 , 7.40 ddd, H-7 , 7.51 dd, H-5 , 11.65 br,
Ž
.
Ž
.
Ž
Ž
.
Ž
.
7.75 dd, H-5 , 11.66 br, –OH ; J5– 6 s9.5, J5– 7F
s6.1 Hz. Anal. Calcd. for C10 H8 FNO: C, 67.79;
H, 4.55; N, 7.91. Found: C, 67.72; H, 4.48; N, 7.67.
.
–OH ; J5– 6F s10.0, J5– 7 s2.8, J6F – 7 s11.7, J6F – 8
s4.9, J7 – 8 s8.9 Hz. Anal. Calcd for C10 H8 FNO:
C, 67.79; H, 4.55; N, 7.91. Found: C, 67.67; H, 4.76;
N, 7.61.
Ž
.
7-F-2-OH-4-MeQ 805 mg was dissolved in
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.
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.
POCl3 10 ml , and PCl5 1.27 g was added to this

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T. Kato et al.rMutation Research 465 2000 173–182
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.
solution. After refluxing for 20 h, the reaction mix-
ture was poured into water, neutralized with sodium
carbonate, extracted with CHCl3, and evaporated.
was hydrolyzed in c-NH3 aq. 10 ml at 808C for 2
h. The resulting mixture was extracted with CHCl3.
Purification of the extract by column chromatogra-
Ž
Ž
.
2-Chloro-7-fluoro-4-methylquinolin 2-Cl-7-F-4-
phy silica gel, CHCl3: MeOHs19:1 yielded 6-F-
4-HMeQ and 6-F-3-OH-4-MeQ in 49% and 36%
yield, respectively.
.
MeQ was obtained as a white solid quantitatively.
mp 100–1068C. MS mrz: 195 M , 197 M q2 .
H-NMR CDCl3 d: 2.69 s, 3H, CH3 , 7.22 s,
q
q
Ž
.
Ž
.
.
1
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
H-3 , 7.36 ddd, H-6 , 7.65 dd, H-8 , 7.97 dd,
2.1.6.1. 6-F-4-HMeQ. mp 146–1508C. MS mrz:
1
q
.
Ž
.
Ž
.
Ž
.
H-5 ; J5– 6 s9.1, J5– 7F s5.9, J6 – 7F s11.0, J6 – 8
2.7, J7F – 8 s10.0 Hz. Anal. Calcd for C10 H7ClFN:
C, 61.40; H, 3.61; N, 7.16. Found: C, 61.24; H, 3.60;
N, 6.88.
2-Cl-7-F-4-MeQ 105 mg was dissolved in 2 ml
of tetraethylammonium fluoride-5.5 mol eq. of hy-
drogen fluoride. Then the solution was kept stirring
for 20 min at 1608C. The reaction mixture was
poured into 100 ml of water, neutralized with sodium
carbonate, and extracted with CHCl3. Purification of
the extract by column chromatography silica gel,
CHCl3 and recrystallization from hexane yielded
2,7-diF-4-MeQ as white needles in 14% yield. mp
H-NMR CDCl3
d: 2.73 s, 3H, CH3 , 6.90 s, H-3 , 7.33 ddd, H-6 ,
7.58 dd, H-8 , 7.97 dd, H-5 ; J5– 6 s9.0, J5– 7F
5.9, J6 – 7F s11.0, J6 – 8 s2.7, J7F – 8 s10.0 Hz.
Anal. Calcd for C10 H7 F2 N: C, 67.04; H, 3.94; N,
7.82. Found: C, 66.82; H, 4.00; N, 7.81.
s
177 M . H-NMR CDCl3 d: 2.41 s, CH2OH ,
Ž . Ž . Ž
5.16 s, 2H, C H2OH , 7.49 ddd, H-7 , 7.57 m, 2H,
.
Ž
.
Ž
.
H-3 and H-5 , 8.14 dd, H-8 , 8.86 d, H-2 ; J2 – 3
4.4, J5– 7 s2.7, J6F – 7 s12.9, J6F – 8 s5.6, J7 – 8
9.5 Hz. Anal. Calcd. for C10 H8 FNO: C, 67.79; H,
s
s
Ž
.
4.55; N, 7.91. Found: C, 67.81; H, 4.76; N, 8.01.
2.1.6.2. 6-F-3-OH-4-MeQ. mp 2108C. MS mrz: 177
1
q
Ž
M
Ž
.
Ž
.
Ž
Ž
.
. H-NMR CDCl3 d: 2.41 s, 3H, CH3 , 7.29
.
Ž
.
.
Ž
ddd, H-7 , 7.64 dd, H-5 , 7.93 dd, H-8 , 8.55 s,
Ž
.
H-2 ; J5– 6F s11.0, J5– 7 s2.8, J6F – 7 s11.5, J6F – 8
s5.9, J7 – 8 s9.3 Hz. Anal. Calcd for C10 H8 FNO:
C, 67.79; H, 4.55; N, 7.91. Found: C, 67.77; H, 4.79;
N, 7.76.
.
1
q
Ž
.
Ž
.
75–768C. MS mrz: 179 M
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
(
s
2.1.7. 7-Fluoro-4-hydroxymethylquinoline 7-F-4-
HMeQ and 7-fluoro-3-hydroxy-4-methylquinoline
7-F-3-OH-4-MeQ
7-F-4-MeQ 396 mg was allowed to react with
m-chloroperoxybenzoic acid 462 mg in 30 ml of
CHCl3 at room temperature for 24 h. The reaction
mixture was washed with 5% sodium carbonate aq.
The organic layer was dried over anhydrous MgSO4
and evaporated. 7-F-4-MeQ N-oxide was obtained in
)
(
)
Ž
.
Ž
.
(
2.1.6. 6-Fluoro-4-hydroxymethylquinoline 6-F-4-
HMeQ and 6-fluoro-3-hydroxy-4-methylquinoline
)
(
)
6-F-3-OH-4-MeQ
Ž
.
6-F-4-MeQ 182 mg was allowed to react with
1
q
Ž
.
Ž
.
Ž
.
m-chloroperoxybenzoic acid 323 mg in 30 ml of
CHCl3 at room temperature for 40 h. The reaction
mixture was washed with 5% sodium carbonate aq.
The organic layer was dried over anhydrous MgSO4
and evaporated. 6-F-4-MeQ N-oxide was obtained in
79% yield. MS mrz: 177 M . H-NMR CDCl3
d: 2.60 s, 3H, CH3 , 7.15 d, H-3 , 7.52 ddd, H-7 ,
7.57 dd, H-5 , 8.39 d, H-8 , 8.84 dd, H-2 ; J2 – 3
6.0, J5– 6F s9.3, J5– 7 s2.7, J6F – 8 s5.6, 76 – 8 s9.5
73% yield. MS mrz: 177 M . H-NMR CDCl3
d: 2.75 s, 3H, CH3 , 7.11 d, H-3 , 7.46 m, H-6 ,
Ž
.
Ž
Ž
.
Ž
.
.
Ž
.
8.00 dd, H-5 , 8.47 m, 2H, H-2 and H-8 ; J2 – 3
s
6.1, J5– 6 s9.0, J5– 7F s5.4, J6 – 8 s2.7 Hz.
Ž
.
7-F-4-MeQ N-oxide 318 mg was refluxed in
1
q
Ž
.
Ž
.
Ž
.
acetic anhydride 20 ml for 10 h. After the solvent
was evaporated, the residue was dissolved in water,
neutralized with sodium carbonate, and extracted
with CHCl3. The CHCl3 layer was dried over anhy-
drous MgSO4 and evaporated. The resulting residue
Ž
.
Ž
Ž
.
Ž
Ž
.
Ž
.
.
.
s
Hz.
Ž
.
Ž
.
6-F-4-MeQ N-oxide 150 mg was refluxed in
was hydrolyzed in c-NH3 aq. 10 ml at 608C for 2
h. The resulting mixture was extracted with CHCl3.
Purification of the extract by column chromatogra-
Ž
.
acetic anhydride 20 ml for 4 h. After the solvent
was evaporated, the residue was dissolved in water,
neutralized with sodium carbonate, and extracted
with CHCl3. The CHCl3 layer was dried over anhy-
drous MgSO4 and evaporated. The resulting residue
Ž
.
phy silica gel, CHCl3: MeOHs19:1 yielded 7-F-
4-HMeQ and 7-F-3-OH-4-MeQ in 38% and 55%
yield, respectively.

(
)
T. Kato et al.rMutation Research 465 2000 173–182
177
Ž
.
2.1.7.1. 7-F-4-HMeQ. mp 142–1448C. MS mrz:
ture was combined with a half volume 50 ml of
EtOH and centrifuged at 10,000=g for 15 min. The
supernatant was analyzed by HPLC.
q
Ž
M
177
1H-NMR CDCl3 d: 5.22 s, 2H,
. Ž . Ž
.
.
Ž
.
Ž
.
Ž
C H2OH , 7.51 d, H-3 , 7.37 m, H-6 , 7.76 dd,
.
Ž
.
Ž
.
H-8 , 8.00 dd, H-5 , 8.89 d, H-2 ; J2 – 3 s4.4,
J5– 6 s8.9, J5– 7F s5.9, J6 – 8 s2.7, J7 – 8 s10.0 Hz.
Anal. Calcd. for C10 H8 FNO: C, 67.79; H, 4.55; N,
7.91. Found: C, 67.79; H, 4.69; N, 8.01.
2.3.2. Isolation and characterization of 2-fluoro-4-
(
)
hydroxymethylquinoline 2-F-4-HMeQ
For structural characterization of the metabolite of
2-F-4-MeQ, the above incubation mixture was scaled
up 40-fold and incubated at 378C for 120 min. The
resulting reaction mixture was extracted with 2 vol-
umes of ethyl acetate, and the organic solvent layer
was dried over MgSO4 and evaporated. The major
metabolite of 2-F-4-MeQ, 2-F-4-HMeQ, was isolated
from the ethyl acetate extract by preparative thin-
2.1.7.2. 7-F-3-OH-4-MeQ. mp 210–2158C. MS mrz:
1
q
Ž
.
Ž
.
Ž
.
177 M . H-NMR CDCl3 d: 2.66 s, 3H, CH3 ,
Ž
.
Ž
.
Ž
.
7.37 m, H-6 , 7.76 dd, H-8 , 7.96 dd, H-5 , 8.80
Ž
.
s, H-2 ; J5– 6 s9.3, J5– 7F s5.9, J5– 8 s2.7, J7F – 8
s9.8 Hz. Anal. Calcd for C10 H8 FNO: C, 67.79; H,
4.55; N, 7.91. Found: C, 68.01; H, 4.75; N, 7.78.
Ž
layer chromatography silica gel, CHCl3:MeOHs
1
.
2.2. Isolation of the microsomal fraction
19:1 . Its structure was identified by H-NMR and
high resolution mass spectroscopy: 1 H-NMR
Ž
.
Ž
.
Ž
.
Preparation of the microsomal fraction was per-
CDCl3 d: 5.07 s, 2H, C H2OH , 7.32 s, H-3 ,
w
x
Ž
.
Ž
.
Ž
.
formed according to the previous report 14,16 after
minor modification with respect to the dose of the
7.64 dd, H-6 , 7.82 dd, H-7 , 7.90 d, H-8 , 8.04
Ž
.
d, H-5 ; J5– 6 s8.3, J6 – 7 s7.2, J7 – 8 s8.3, J6 – 8
s
Ž
inducer. Briefly, male Sprague–Dawley rats 6 weeks
2.7, J7 – 8 s10.0 Hz; HR-MS mrz: 177.059, Calcd.
for C10 H8 FNO: 177.059.
.
old were intraperitoneally injected with 3-MC dis-
solved in corn oil at a daily dose of 50 mgrkg body
weight for three consecutive days. The rats were
fasted for 18 h and sacrificed by decapitation 48 h
after the last 3-MC injection. The liver was rapidly
2.4. HPLC analysis
HPLC analysis was performed using a Shimadzu
liquid chromatograph equipped with a Model LC-10A
solvent delivery system, a Model SPD-M6A UV-VIS
photodiode array detector and a Merck LiChrospher
Ž
excised and homogenized in 0.15 M KCl 3 mlrg
.
liver . The homogenate was centrifuged at 9000=g
for 20 min and the supernatant was used as the S9
fraction. The microsomal fraction was prepared by
centrifugation of the S9 fraction at 105,000=g for
90 min. The pellet containing the microsomal frac-
tion obtained from 3 ml of the S9 was suspended in
1 ml of 0.02 M potassium phosphate buffer solution
Ž
.
Ž
.
100-18e ODS column 4 mm=250 mm . The
solvent system consisted of 30% MeOH in 1r15 M
Ž
.
phosphate buffer pH 6.8 at zero time, followed by
a linear gradient of MeOH increasing up to 80% in a
40 min period. The flow rate was 0.8 mlrmin.
Ž
.
Ž
0.15 M KCl, 20% glycerol, pH 7.5 . The microso-
mal protein content was determined by Lowry’s
Decreases of the starting materials 4-MeQ, 2-F-4-
.
MeQ, 6-F-4-MeQ, and 7-F-4-MeQ and increases of
hydroxymethyl metabolites 4-HMeQ, 2-F-4-HMeQ,
6-F-4-HMeQ, and 7-F-4-HMeQ were quantified
w
x
Ž
method 17 . The cytochrome P-450 content was
w
x
.
measured as described by Omura and Sato 18 .
2.3. Metabolism with microsomal P-450
2.3.1. HPLC analysis of 4-MeQ metabolites
from the peak areas measured by UV absorption at
290 nm with reference to their respective authentic
samples. 3-Hydroxy metabolites of 4-MeQ, 6-F-4-
MeQ, and 7-F-4-MeQ were quantified from the peak
areas measured by UV absorption at 330 nm with
reference to their respective authentic samples, be-
cause the lmax values of 3-OH-4-MeQ, 6-F-3-OH-4-
MeQ, and 7-F-3-OH-4-MeQ are 331, 326, and 332
nm, respectively. Retention times of the 4-MeQs and
their metabolites are given in Table 1, and examples
Ž
.
The incubation mixture 1.0 ml contained 1 nmol
Ž
.
of cytochrome P-450 ca. 1 mg of protein , 0.25 mM
4-methylquinolines, 4 mM NADPH, 4 mM NADH,
5 mM G-6-P, 32.8 mM KCl, 8 mM MgCl2 , and 100
Ž
.
mM phosphate buffer pH 7.4 . After incubation at
378C for an appropriate period, 100 ml of this mix-

(
)
178
T. Kato et al.rMutation Research 465 2000 173–182
Table 1
dose–response curves for the test compounds are
shown in Fig. 2. 7-F-4-MeQ as well as 4-MeQ was
highly mutagenic, while 6-F-4-MeQ was less muta-
genic than 4-MeQ. None of the 2-F-derivatives, i.e.,
2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ,
showed mutagenicity in the whole dose range exam-
ined.
Retention times of 4-MeQs and their metabolites in HPLC
4-hydroxymethyl 3-OH Starting material
Ž
.
Ž
.
Ž
.
min
min
min
4-MeQ
15.4
18.7
16.9
17.0
29.0
31.8
32.2
32.8
31.6
2-F-4-MeQ
6-F-4-MeQ
7-F-4-MeQ
n.d.
30.2
29.5
HPLC analysis was performed as described in Section 2.
n.d.; not detected.
3.2. Metabolism of 4-MeQ and its monofluorinated
deriÕatiÕes
of the HPLC profiles of 2-F-4-MeQ and 7-F-4-MeQ
metabolites are shown in Fig. 1.
Ž
4-MeQ, and its monofluorinated derivatives 2-F-
4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ were exam-
.
2.5. Mutation assay
ined for their metabolism profiles using liver micro-
somes from the 3-MC-treated rat. Liver microsome
prepared from rats pretreated with 3-MC was used in
the present study for metabolism studies to compare
Chemicals were tested for mutagenicity as previ-
w
x
ously reported 19–21 , using S. typhimurium TA100
Ž
.
in the presence of the S9 fraction 50 ml and
w
x
with our previous data 16 . On the other hand, liver
S9 fraction pretreated with phenobarbital and 5,6-
Ž
.
cofactors Cofactor Ie . The S9 fraction and Cofac-
tor Ie were purchased from Oriental Yeast, Tokyo.
The S9 fraction was prepared from the male
Sprague–Dawley rat liver pretreated with pheno-
Ž
barbital i.p. injected at a daily dose of 30, 60, 60,
.
and 60 mgrkg for four consecutive days and 5,6-
benzoflavone i.p. injected at a single dose of 80
Ž
mgrkg 2 days after the first injection of pheno-
.
barbital . Cofactor Ie consisted of 4 mM NADPH, 4
mM NADH, 5 mM G-6-P, 32.8 mM KCl, 8 mM
Ž
.
MgCl2 , and 100 mM phosphate buffer pH 7.4 . The
TA100 strain was the gift of Dr. Matsushima of the
University of Tokyo, and were originally provided
by Dr. B.N. Ames of the University of California,
Berkeley. Assays were carried out after preincuba-
tion of the test chemical with S9 mix at 378C for 20
min.
3. Results
3.1. Mutagenicity of 4-MeQ and its fluorinated
deriÕatiÕes
The 4-MeQs listed in Scheme 1 were tested for
mutagenicity in S. typhimurium TA100 in the pres-
ence of S9 mix according to the procedure of the
Fig. 1. HPLC profiles of 2-F- and 7-F-4-MeQ metabolites after 30
min incubation in the presence of the microsomal fraction. 2-F-
and 7-F-4-MeQ were metabolized for 30 min. The detection
wavelengths were 290 and 330 nm. Details are described in
w
x
Ames test 19–21 . In the present paper, derivatives
inducing less than twice the number of revertants in
the background were considered non-mutagenic. The
Ž .
a
Ž .
Section 2.
metabolites of 2-F-4-MeQ.
b metabolites of
7-F-4-MeQ.

(
)
T. Kato et al.rMutation Research 465 2000 173–182
179
Scheme 1. List of 4-methylquinolines examined.
Ž
derivatives were almost same as for parent com-
pounds , or slightly longer than for the two metabo-
benzoflavone was used for mutagenicity assay since
there is no significant difference in the mutagenicity
of the quinoline derivatives between the two exoge-
.
Ž
.
lites of each parent compound , those of their respec-
Ž
.
Ž
.
nous metabolic systems data not shown . The
metabolites were identified and quantified by reverse
phase HPLC analysis with reference to their respec-
tive authentic samples other than 2-F-4-HMeQ and
tive non-fluorinated analogs Table 1 .
Fig. 3 shows the metabolism of 4-MeQ and its
monofluorinated derivatives in the presence of the
microsomal fraction. 6-F-4-MeQ was metabolized by
the rat liver microsomal fraction to two metabolites:
6-F-4-HMeQ as a major metabolite and 6-F-3-OH-4-
MeQ as a minor one. The amount of 6-F-4-HMeQ
increased with time up to 120 min after incubation,
while that of 6-F-3-OH-4-MeQ reached a maximum
in 30 min, followed by a gradual decrease. The
metabolism was almost completed in 120 min under
the reaction conditions employed. 4-MeQ and 7-F-4-
MeQ showed metabolic patterns similar to that ob-
served with 6-F-4-MeQ except for the lesser amounts
of the corresponding 4-hydroxymethyl derivatives
Ž
2-F-3-hydroxy-4-methylquinoline 2-F-3-OH-4-
.
MeQ , which were difficult to synthesize chemically.
2-F-4-HMeQ was isolated as a metabolite from the
Ž
.
large-scale 40-fold reaction mixture. The retention
Ž
times of the metabolites 4-hydroxymethyl and 3-hy-
droxy derivatives and the starting materials of these
.
4-MeQs are given in Table 1, and representative
charts of HPLC analysis for 2-F-4-MeQ and 7-F-4-
MeQ metabolism are also shown in Fig. 1a and b,
respectively. The retention times of the fluorinated
Ž
produced 6-F-4-MeQ: 93%, 4-MeQ: 85%, and 7-F-
.
4-MeQ: 60% .
On the other hand, 2-F-4-MeQ produced only one
peak corresponding to 2-F-4-HMeQ other than the
starting material on the HPLC chart. Since the reten-
tion time of 2-F-3-OH-4-MeQ was predicted to be
slightly shorter than that of the starting material, we
concluded that 2-F-3-OH-4-MeQ was not produced
under the present experimental conditions employed.
4. Discussion
Fig. 2. Mutagenicity of 4-methylquinolines in S. typhimurium
TA100 in the presence of S9 mix. The symbols shown indicate the
means of at least three independent experiments.
We previously proposed that the genotoxicity of
quinoline may be attributable to formation of an

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)
180
T. Kato et al.rMutation Research 465 2000 173–182
Fig. 3. Metabolism of 4-MeQ and its monofluorinated derivatives in the presence of the microsomal fraction.
enamine epoxide, 2,3-epoxide of 1,4-hydrated quino-
Actually, it is the most mutagenic form of all the
quinoline derivatives examined so far. As we re-
w
x
line 8,9 . This hypothesis is supported by the fact
that its mutagenicity was eliminated by fluorine-sub-
stitution at position 3 but not by substitution at
w
x
ported 16 , the major metabolite of 4-MeQ in the
3-MC-induced rat liver microsomal enzyme system
was identified as non-mutagenic 4-HMeQ and other
w
x
position 5, 6, 7, or 8 8,9,22 . 4-MeQ, a 4-methyl
derivative of quinoline, is found to be highly muta-
genic in S. typhimurium TA100 when compared to
Ž
metabolites identified were 4-MeQ N-oxide 4-
.
MeQO , 3-OH-4-MeQ, and 3-hydroxy-4-hydroxy-
methylquinoline 3-OH-4-HMeQ , while 5,6-epoxi-
w
x
Ž .
quinoline in the presence of rat liver S9 mix 9,16 .
Fig. 4. Proposed mutagenic mechanism of 4-MeQ.

(
)
T. Kato et al.rMutation Research 465 2000 173–182
181
dation, a major detoxification metabolic pathway of
quinoline, barely proceeded with 4-MeQ. In addi-
tion, our previous studies showed that 4-MeQ was
deprived of its potent mutagenicity by introduction
the 3-hydroxy metabolites may be further metabo-
lized to 3-hydroxy-4-hydroxymethyl derivatives etc.
In fact, we detected the 3-OH-4-HMeQ metabolite in
the previous study, where the liver microsomal en-
zyme fraction was prepared from rats treated with
3-MC for 1 day instead of 3 days as in the present
w
x
of a chlorine atom to position 3 16 . These findings
suggest that the mechanism of mutation induction by
4-MeQ is the same as that applied to mutagenicity of
quinoline, which appears to be mediated through the
w
x
study 16 . The longer treatment of rats with 3-MC
may have affected the expression of cytochrome
P-450 proteins.
Ž
.
formation of enamine epoxide of 4-MeQ Fig. 4 .
The enhancement of mutagenicity of 4-MeQ may be
due to its 4-methyl group that interfered with the
detoxification process of 5,6-epoxidation at the peri-
position andror due to retarded enzymatic epoxide-
ring opening of 1,4-hydrated 2,3-epoxide by epoxide
hydrolase or glutathion-S-transferase. As a result, the
1,4-hydrated 2,3-epoxide might be stable enough to
In conclusion, the present study affords further
evidence to support the enamine epoxide theory as a
common activation mechanism of the genotoxicity of
the quinoline nucleus and, at the same time, suggests
that 2-r3-F-substitution in the pyridine moiety may
be responsible for anti-mutagenic structural modifi-
cation in the quinoline nucleus.
w
x
have greater chance to react with DNA 16 .
In the present study, 2-F-4-MeQ, in which an F
atom was substituted for H at the position of
metabolism in the pyridine moiety, was compared
with 6-F- and 7-F-4-MeQ which were fluorinated at
the non-metabolic positions, in order to analyze F-
substitution effects on the mutagenicity and
metabolism of 4-MeQ. The mutagenic activity of
4-MeQ was totally lost by introduction of an F atom
to position 2, while 6-F-4-MeQ and 7-F-4-MeQ
showed mutagenic potency. In the latter two
monofluorinated 4-MeQs, a second F-substitution at
position 2 also deprived both compounds of their
Acknowledgements
The authors are greatly indebted to Dr. Yutaka
Kawazoe, emeritus professor, at Nagoya City Uni-
versity, for helpful advice and encouragement. We
also wish to thank Dr. Kunitaka Momota of Morita
Chemical, Osaka, for kindly supplying us with the
fluorination reagent.
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