Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter: Part 3. A potential 5-HT transporter imaging agent, 3-(3-[18F]Fluoropropyl)-6-nitroquipazine

Article abstract of DOI:10.1016/j.bmc.2003.09.009

3-(3-[¹⁸F]Fluoropropyl)-6-nitroquipazine ([¹⁸F]FPNQ) as a 5-HT transporter imaging agents was designed, synthesized, and evaluated. FPNQ was selected due to its potent in vitro biological activity (K _i=0.32 nM) in rat brain cortical membranes. The ¹⁸F- labeled FPNQ was prepared by reaction of the propyl mesylate as a precursor with tetra-n-butylammonium [¹⁸F]fluoride generated under NCA conditions. The precursor mesylate was synthesized from commercially available hydrocarbostyril in nine steps in 21% overall yield. The specific activity of the [¹⁸F]FPNQ determined by radioreceptor assay was 27.0 GBq/μmol. Tissue distribution studies in mice showed the highest uptake in the frontal cortex (5.79%ID/g) at 60 min post-injection.

Full text of DOI:10.1016/j.bmc.2003.09.009

Bioorganic & Medicinal Chemistry 11 (2003) 4949–4958
Syntheses and Binding Affinities of 6-Nitroquipazine Analogues
for Serotonin Transporter: Part 3.y A Potential 5-HT Transporter
Imaging Agent, 3-(3-[18F]Fluoropropyl)-6-nitroquipazine
Byoung Se Lee,a Soyoung Chu,a Kyo Chul Lee,a Bon-Su Lee,a Dae Yoon Chi,a,*
Yearn Seong Choe,b,* Sang Eun Kim,b Yun Seon Songc and Changbae Jinc
aDepartment of Chemistry, Inha University, 253 Yonghyundong Namgu, Inchon 402-751, South Korea
bDepartment of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine,
50 Ilwon-dong Kangnam-ku, Seoul 135-710, South Korea
cBioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 130-650, South Korea
Received 23 July 2003; revised 27 August 2003; accepted 4 September 2003
Abstract—3-(3-[18F]Fluoropropyl)-6-nitroquipazine ([18F]FPNQ) as a 5-HT transporter imaging agents was designed, synthesized,
and evaluated. FPNQ was selected due to its potent in vitro biological activity (Ki=0.32 nM) in rat brain cortical membranes. The
18F-labeled FPNQ was prepared by reaction of the propyl mesylate as a precursor with tetra-n-butylammonium [18F]fluoride gen-
erated under NCA conditions. The precursor mesylate was synthesized from commercially available hydrocarbostyril in nine steps
in 21% overall yield. The specific activity of the [18F]FPNQ determined by radioreceptor assay was 27.0GBq/ mmol. Tissue dis-
tribution studies in mice showed the highest uptake in the frontal cortex (5.79 %ID/g) at 60min post-injection.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
On the other hand, [11C]McN-5652,16 5-[123I]iodo-6-
nitroquipazine (1a),17À20 and 5-[76Br]bromo-6-nitroqui-
pazine (1b),21 showed somewhat encouraging results.
The recently developed ADAM, ODAM and IDAM
series22À24 are also attractive lead compounds in the
development of new PET and SPECT radioligands for
the understanding of dysfunction in psychiatric dis-
eases as well as pathological studies of 5-HT neuro-
transmission.
Development of radiopharmaceuticals for positron
emission tomography (PET) and single photon emis-
sion computed tomography (SPECT) as diagnostic
imaging tools, especially for the central nervous system,
is an active and emerging field in nuclear medicine.2
Many dopamine transporter (reuptake site) imaging
agents, such as cocaine derivatives b-CFT,3À5 b-CIT,6À10
IPT,11 and TRODAT,12À15 enabled one to gain a better
understanding of Parkinson’s disease and to help in
staging patients with Parkinson’s disease. Serotonin
(5-hydroxyltryptamine or 5-HT) transporter which is
related to depression and other psychiatric diseases in
the mammalian brain has also been studied extensively
using radiotracers such as [3H]imipramine, [3H]parox-
etine, and [3H]citalopram. Most of these agents, how-
ever, did not reach the suitable target-to-non-target
uptake ratios in vivo for serotonin transporter imaging.
Although 6-nitroquipazine (1, 6-NQ) has been known
as one of the most potent and selective inhibitors for the
5-HT transporter (SERT) in vitro25,26 and in vivo,27,28
the structure–activity relationship of its derivatives has
not been extensively studied. Recently, we reported the
first structure–activity relationship29 of 6-NQ deriva-
tives and the first regioselective synthetic route30 to
6-NQ.
*Corresponding author. Tel.: +82-32-860-7686; fax: +82-32-867-
5604; e-mail: dychi@inha.ac.kr
yPart 2. See ref 1.
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.009

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B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
In this paper, we synthesized 3-alkyl-6-NQ derivatives
and measured their biological activities for the 5-HT
transporter. Among these derivatives, 3-(3-fluoro-
propyl)-6-NQ (6e) was selected for 18F-labeling and tis-
sue distribution in mice.
of hydrocarbostyril and alkyl halides using lithiumdii-
sopropylamide (LDA) as a base at À78 ꢀC. When
n-butyllithium was used instead of LDA, many side
products that lowered the reaction yield were formed.
The nitration of 3a–e gave two isomers, 6-nitro as a
major and 8-nitro as a minor compound. Compounds
5a–e were obtained by the reaction of 4a–e with one
equivalent of DDQ and three equivalents of phosporous
oxychloride in DMF under very mild conditions (rt, 3
h). Nucleophilic aromatic substitution of the chloride
on C2 position with 1-piperazinecarboxaldehyde pro-
vided piperazin-1-yl quinoline analogues. Without fur-
ther purification, direct deformylation under acidic
conditions provided yellow solids 6a–e which were fur-
ther treated with 4 N NaOH until basic.
Results and Discussion
Synthesis of 3-alkylated 6-nitroquipazines 6a–e
We reported the regioselective synthetic route of
6-nitroquipazine from commercially available hydro-
carbostyril (2).30 Five, 3-alkylated 6-NQ derivatives 6a–
e were prepared by applying this route as shown in
Scheme 1. Several alkyl groups were introduced to C3
position of 6-nitroquipazine by the reaction of dianion
Synthesis of the mesylate precursor 14
The mesylate 14 was used as the precursor for the
synthesis of 18F-labeled quipazine derivative 6e. The
synthetic route for 14 is shown in Scheme 2. Although
14 was synthesized via the same synthetic route shown
in Scheme 1, several steps were modified for the protec-
tion of the alcohol group due to the introduction of
30-hydroxyl group. As the nitration reaction of 7 failed
to give the desired product, an additional removal step
of the t-butyldimethylsilyl group was necessary. To
introduce a chlorine atom selectively on C2 position, the
propyl alcohol had to be protected. The nitration of 8
followed by acetylation gave the compound 10. In early
attempts, the nitrated compound 9 was obtained in 40%
yield, because TLC analysis of the nitration reaction
showed the multiple spots at the beginning of the reac-
tion. The number of spots were, however, reduced as the
reaction mixture was allowed to stand overnight, which
also improved the yield up to 55%. Treatment of 12
with methanolic potassium carbonate furnished com-
pound 13 that was further reacted with methanesulfonyl
chloride to give the corresponding mesylate 14. FPNQ
was prepared by fluorination of 14 with tetra-n-butyl-
ammonium fluoride hydrate followed by direct defor-
mylation under acidic conditions.
Scheme 1. Reagents and conditions: (a) LDA, R-X, THF, À78 ꢀC;
(b) c. H2SO4, c. HNO3, 0 ꢀC; (c) POCl3, DDQ, DMF, rt; (d) 1-piper-
azinecarboxaldehyde, DMF, 80 ꢀC; (e) 4 M H2SO4, 80 ꢀC.
Scheme 2. Reagents and conditions: (a) LDA, Br(CH2)3OTBDMS, THF, À78 ꢀC; (b) n-Bu4NF, THF, rt, 12 h; (c) c. H2SO4, c. HNO3, 0 ꢀC, 10min;
(d) Ac2O, pyridine, rt, 12 h; (e) POCl3, DDQ, DMF, rt, 3 h; (f) 1-piperazinecarboxaldehyde, DMF, 100 ꢀC, 3 h; (g) K2CO3, MeOH/CH2Cl2, rt, 15 h;
ꢀ
(h) MsCl, Et3N, CH2Cl2, 0 ꢀC, 1 h; (I) nBu4NF, CH3CN, reflux, 1 h; (j) 4 M H2SO4, 10 0 C, 2 h.

B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
4951
Radiochemical synthesis of [18F]FPNQ
3-ethyl- (6b) and 3-propyl-6-nitroquipazine (6c), the
values of the inhibition binding constant dramatically
increased to those similar to 6-nitroquipazine. This sig-
nificant improvement in the binding affinities indicates
that alkyl chains longer than a methyl group have a
favorable lipophilic interaction and thus compensates
for electron-donating effects by the alkyl group on C3
position of 6-nitroquipazine derivatives. It is likely that
there are London forces between 3-alkyl chains of
6-nitroquipazine derivatives and the corresponding
residue in the binding pocket of the 5-HT transporter.
The target compound, 3-(3-fluoropropyl)-6-nitroquipa-
zine, has the binding affinity similar to 6c. These results
suggest that [18F]6e can be a good imaging agent for the
5-HT transporter.
The [18F]FPNQ was prepared by fluoride ion displace-
ment reaction of the mesylate 14 (2.0mg, 5.0 mmol) at
130 ꢀC for 30min with tetra- n-butylammonium
[18F]fluoride generated under no-carrier-added (NCA)
conditions, followed by direct deformylation of [18F]15
under acidic conditions at 130 ꢀC for 15 min as shown in
Scheme 3. Resolubilization of tetra-n-butylammonium
[18F]fluoride prepared from the [18F]fluoride ion and
tetra-n-butylammonium hydroxide (40% aq, 2.6 mL, 4.0
mmol) was obtained in 70–90% yield and took 10–12 min.
The isolated radiochemical yield of [18F]15 and [18F]6e
were 16–28 and 85–89% (decay-corrected), respectively.
After workup and removal of the solvent, [18F]FPNQ
was purified by a semipreparative HPLC column before
testing its specific activity, binding affinity, and biodis-
tribution in mice.
Specific activity and in vivo tissue distribution studies of
[18F]6e
The [18F]6e collected from HPLC was dissolved in 10%
ethanol-saline and the specific activity determined by
radioreceptor assay was 27.0ꢁ4.4 GBq/mmol (Fig. 1).
The [18F]6e solution was injected into mice (25–30g,
n=4). Tissue distribution data were obtained at 5, 15, 30,
60, 90 and 120 min post-injection and are shown in Fig-
ure 2 and Table 2. The brain uptake of this radioligand
reached 1.8 %ID/g at 60min similar to [ 125I]5-iodo-6-
nitroquipazine (1.9 %ID/g at 5 min). The [18F]6e showed
the highest uptake (5.79 %ID/g) in the frontal cortex and
low uptake (2.73 %ID/g) in the cerebellum at 60min
In vitro serotonin transporter bindings studies
Table 1 shows the in vitro binding affinities, expressed
as the inhibition binding constant, of 6a–e for the 5-HT
transporter including 6-NQ, fluoxetine, and paroxetine.
In comparison with 6-nitroquipazine, the binding affi-
nity of 3-methyl-6-nitroquipazine (6a) decreased by
approximately 50-fold, but was slightly higher than that
of 3-bromo-6-nitroquipazine. The methyl group itself
donates electrons to the quinoline ring, consequently
decreasing the binding affinity. However, in cases of
Figure 1. HPLC profiles of the reaction mixture containing
18F]FPNQ.
Scheme 3. Reagents and conditions: (a) n-Bu4N[18F]F, CH3CN,
130 ꢀC, 20min; (b) 4 M H 2SO4, 130 ꢀC, 10min.
[
Table 1. Binding affinities (Ki) for 5-HT transportera
Compd
Ki (nM)
6a
6b
6c
6d
8.45ꢁ0.62
0.36ꢁ0.02
0.26ꢁ0.01
1.08ꢁ0.17
0.31ꢁ0.01
12.62ꢁ1.44
22.13ꢁ1.77
0.53ꢁ0.08
0.17ꢁ0.03
6e
3-Bromo-6-nitroquipazine
Fluoxetine
Paroxetine
6-Nitroquipazine
aThe inhibition of [3H]citalopram binding was determined at 1 nM
[3H]citalopram for the various compounds listed. Binding data are the
means of three independent experiments. Eleven concentrations of
displacer were used for each determination. The Kd value of [3H]cita-
lopram measured by Scatchard analysis of the equilibrium-saturation
experiment was 1.12 nM.
Figure 2. Tissue distribution of [18F]FPNQ in mouse brain (n=4). CB,
cerebellum; F. Ctx, frontal cortex; Olf, Olfactory tubercle; Hypo,
hypothalamus; Hippo, hippocampus.

4952
B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
Table 2. Tissue distribution (% ID/g) of [18F]FPNQ in mouse brain and bone (n=4)a
Min
CB
F. Cox
Olf
Hypo
Hippo
Str
Ctx
S. Colli
I. Colli
Thal
Bone
5
15
302.68
602.73
902.14
2.21ꢁ0.33 2.25ꢁ0.52 1.90ꢁ0.87 1.89ꢁ0.61 1.40ꢁ0.45 2.05ꢁ0.30 2.29ꢁ0.38 2.26ꢁ0.63 1.97ꢁ0.46 2.02ꢁ0.48 2.49ꢁ0.20
2.95ꢁ0.36 3.82ꢁ0.54 2.99ꢁ0.60 3.06ꢁ0.68 2.63ꢁ0.37 3.00ꢁ0.43 3.31ꢁ0.41 3.79ꢁ0.46 3.27ꢁ0.32 3.57ꢁ0.67 2.66ꢁ0.21
ꢁ0.69 4.04ꢁ1.22 3.95ꢁ1.21 3.37ꢁ0.94 2.93ꢁ0.88 3.21ꢁ0.82 3.54ꢁ0.80 3.64ꢁ0.97 3.44ꢁ0.94 3.75ꢁ0.94 2.63ꢁ0.42
ꢁ0.21 5.79ꢁ0.41 5.00ꢁ0.64 4.66ꢁ0.48 4.45ꢁ0.50 4.24ꢁ0.42 4.30ꢁ0.58 4.36ꢁ0.62 4.65ꢁ0.31 4.17ꢁ2.16 3.51ꢁ0.44
ꢁ0.16 5.47ꢁ0.73 4.99ꢁ0.99 4.81ꢁ1.04 4.33ꢁ0.72 4.12ꢁ0.76 4.24ꢁ0.84 4.03ꢁ1.08 4.70ꢁ1.605.12 ꢁ1.17 4.58ꢁ0.76
1201.76 ꢁ0.17 4.76ꢁ0.35 4.72ꢁ0.33 4.23ꢁ0.38 3.76ꢁ0.30 3.34ꢁ0.27 3.08ꢁ0.15 3.32ꢁ0.30 3.80ꢁ0.33 4.17ꢁ0.45 5.41ꢁ0.64
aCB, cerebellum; F. Cox, frontal cortex; Olf, Olfactory tubercle; Hypo, hypothalamus; Hippo, hippocampus; Str, striatum; Ctx, cortex; S. Colli:, I.
Colli:, Thal: thalamus.
Conclusions
Table 3. In vivo blocking studies at 60min postinjection. Ratio of
%ID/g of organ/cerebellum [18F]FPNQ in mouse brain (n=4)
We synthesized five, high affinity ligands for the 5-HT
trnasporter, 3-alkyl-6-nitroquipazine (6a–e) and one
18F-labeled analogue [18F]FPNQ ([18F]6e) for tissue
distribution studies in vivo. The [18F]FPNQ was pre-
pared by 18F-labeling of the propyl mesylate 14 fol-
lowed by deformylation in high radiochemical yield.
Tissue distribution studies of this radioligand in mice
showed the highest uptake (5.79 %ID/g) in the frontal
cortex at 60min post-injection, although bone uptake
increased slowly over time. This result suggested that the
investigation of this radioligand at 60min post-injection
may be suitable for 5-HT imaging in mice. Further stud-
ies on the syntheses and biological studies of fluor-
oalkylated 6-nitroquipazines at different positions such
as C4, C5 and C7 are in progress in our laboratories to
develop radioligands with lower bone uptake.
Saline Paroxetine Fluoxetine GBR12909 Cold FPNQ
F. Ctx
Olf
Hypo
Hippo
Str
2.201.57
1.92
1.75
2.01
1.63
1.72
1.95
1.55
1.24
1.17
1.41
1.27
1.47
1.42
1.23
1.43
1.72
1.66
1.42
1.06
1.00
1.19
1.301.53
1.101.58
1.17
1.39
Ctx
1.63
postinjection, although the region-to-cerebellum ratios
increased as a function of time. The temporal distribu-
tion pattern of this radioligand in brain regions was
quite different from that of [125I]5-iodo-6-nitroquipazine
which was taken up quickly to the brain (ꢂ1.5 %ID/g
in the prefrontal cortex at 5 min post-injection) and
washed out over time.18 Bone uptake slowly increased
over time, ranging from 2.49 %ID/g at 5 min, 3.51
%ID/g at 60min to 5.41 %ID/g at 120min, suggesting
slow defluorination in vivo.
Experimental
General procedure for the synthesis of compounds 3a–3e
In vivo blocking studies
The [18F]6e was injected alone or with various drugs to
mice to investigate the effects of these drugs on the in
vivo binding of this radioligand. Displacement of the
binding of this radioligand was measured at 60min
postinjection, the time point when a maximal uptake to
the cortex and relatively low level of bone uptake were
observed (Table 3). Both fluoxetine and paroxetine,
serotonin reuptake blockers, inhibited the in vivo
binding of the [18F]6e in the frontal cortex by 11–31%,
although higher levels of inhibition of [125I]5-iodo-6-
nitroquipazine by these two blockers has been repor-
ted.18 The difference in the level of inhibition may be
explained by the different uptake patterns between
these two radioligands.18 In contrast, no inhibition was
detected in the cerebellum where low level of serotonin
reuptake sites is located.
Under N2 atmosphere, to a solution of hydrocarbostyril
(2, 1.0g, 6.79 mmol) in 15 mL of sodium-dried THF
was dropwise added 2.0M LDA solution (7.47 mL,
14.95 mmol) at À78 ꢀC. The mixture was stirred for 30
min at À78 ꢀC and the cooling bath was removed. The
mixture was stirred for additional 1 h at rt, and then
cooled to À78ꢀC. Alkyl halide (7.13 mmol) was added
dropwise to the mixture at À78ꢀC, which was stirred for
90min from À78ꢀC to rt. The reaction was quenched by
adding of 1 mL of methanol and 200 mL of water in suc-
cession. The organic compounds were extracted with 70
mL of EtOAc three times. The combined extract was dried
over Na2SO4 and concentrated with 1.0g of silica gel. The
product was isolated by flash column chromatography
with 20% EtOAc/hexane and recrystallized from
EtOAc/hexane as a white solid.
Coadministration of the unlabeled 6e showed higher
level of inhibition than paroxetine in all regions studied.
Meanwhile, a dopamine reuptake blocker, GBR-12909
did not inhibit the in vivo binding of [18F]6e. This study
demonstrates specific binding of [18F]6e to the 5-HT
transporter in vivo. At this point, it is not clear whether
[18F]6e has the selective binding toward SERT or not,
because SERT presents high similarities to dopamine
transporter as well as norepinephrine transporter.31
3 - Methyl - 3,4 - dihydro - 2(1H) - quinolinone (3a). 60%,
white solid, mp 85–87 ꢀC; H NMR (CDCl3) d 8.38 (br
1
s, 1H), 7.13–7.20(m, 2H), 6.93–7.01 (m, 1H), 6.78 (d,
J=7.6 Hz, 1H), 2.62–3.02 (m, 3H), 1.29 (d, J=6.6 Hz,
3H); 13C NMR (CDCl3) d 173.0, 135.6, 126.3, 125.8,
121.9, 121.2, 113.5, 33.3, 31.8, 13.7; MS (EI) m/z 161
(M+, 100), 132, 118, 92. Anal. calcd for C10H11NO: C,
74.50; H, 6.88; N, 9.93. Found: C, 74.23; H, 6.99; N,
10.21.

B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
4953
3-Ethyl-3,4-dihydro-2(1H)-quinolinone (3b). 61%, white
solid, mp 79–81 ꢀC; 1H NMR (CDCl3) d 9.09 (br s, 1H),
7.12–7.19 (m, 2H), 6.92–7.00 (m, 1H), 6.82 (d, J=7.8
Hz, 1H), 3.04 (dd, J=15.8, 6.0Hz, 1H), 2.75 (dd,
J=15.6, 8.6 Hz, 1H), 2.40–2.55 (m, 1H), 1.79–1.99 (m,
1H), 1.40–1.62 (m, 1H), 1.02 (t, J=7.3 Hz, 3H); 13C
NMR (CDCl3) d 172.2, 135.4, 126.5, 125.7, 121.6, 121.2,
113.4, 39.9, 28.5, 21.0, 9.8; MS (EI) m/z 175 (M+), 146
(100), 132, 118, 106. Anal. calcd for C11H13NO: C,
75.39; H, 7.48; N, 8.00. Found: C, 75.15; H, 7.80; N,
8.31.
3-Methyl-6-nitro-3,4-dihydro-2(1H)-quinolinone (4a).
Mp 213–214 ꢀC; H NMR (CDCl3) d 8.92 (br s, 1H),
1
8.09–8.12 (m, 2H), 6.90 (d, J=9.0Hz, 1H), 3.12 (dd,
J=15.0, 5.2 Hz, 1H), 2.66–2.90 (m, 2H), 1.32 (d, J=6.6
Hz, 3H); 13C NMR (DMSO-d6) d 170.1, 122.0, 121.0,
120.9, 112.3, 112.3, 95.5, 31.6, 30.2 12.0; MS (EI) m/z
206 (M+, 100), 178, 131, 117. Anal. calcd for
C10H10N2O3: C, 58.24; H, 4.89; N, 13.59. Found: C,
58.46; H, 4.70; N, 13.91.
3 - Ethyl - 6 - nitro - 3,4 - dihydro - 2(1H) - quinolinone (4b).
50%, white solid, mp 191–192 ꢀC; H NMR (CDCl3) d
1
3-Propyl-3,4-dihydro-2(1H)-quinolinone (3c). 44%, white
9.35 (br s, 1H), 8.10(br s, 2H), 6.91 (d, J=9.2 Hz, 1H),
3.14 (dd, J=16.2, 6.2 Hz, 1H0, 2.86 (dd, J=16.2, 9.0Hz,
1H), 2.47–2.61 (m, 1H), 1.83–1.96 (m, 1H), 1.43–1.65 (m,
1H), 1.04 (t, J=7.5 Hz, 3H); 13C NMR (DMSO-d6) d
171.4, 143.3, 140.6, 123.2, 122.6, 113.8, 39.4, 28.0, 21.2,
10.1; MS (EI) m/z 220( M +) 191 (100), 177, 145, 130,
117. Anal. calcd for C11H12N2O3: C, 59.99; H, 5.49; N,
12.72. Found: C, 60.03; H, 5.58; N, 12.49.
solid, mp 105–107 ꢀC; H NMR (CDCl3) d 8.74 (br s,
1
1H), 7.12–7.20(m, 2H), 6.96 (td, J=7.2, 1.3 Hz, 1H),
6.80(d, J=7.6 Hz, 1H), 3.11 (dd, J=15.6, 3.0Hz, 1H),
2.73 (dd, J=15.8, 8.5 Hz, 1H), 2.52–2.59 (m, 1H),
1.74–1.88 (m, 1H), 1.37–1.58 (m, 3H), 0.93 (t, J=7.0
Hz, 3H); 13C NMR (CDCl3) d 172.6, 135.4, 126.5,
125.7, 121.6, 121.2, 113.3, 38.2, 30.0, 29.0, 18.5, 12.3;
MS (EI) m/z 191 (M+, 100), 161, 145, 130, 117. Anal.
calcd for C12H15NO: C, 76.15; H, 7.99; N, 7.40. Found:
C, 76.05; H, 8.12; N, 7.41.
3-Propyl-6-nitro-3,4-dihydroꢀ-2(1H)-quinolinone (4c).
1
84%, white solid, mp 207–208 C; H NMR (CDCl3) d
9.07 (br s, 1H), 8.08–8.11 (m, 2H), 6.89 (d, J=0.2 Hz,
1H), 3.15 (dd, J=16.2, 5.8 Hz, 1H), 2.84 (dd, J=16.1,
8.5 Hz, 1H), 2.59–2.66 (m, 1H), 1.79–1.85 (m, 1H),
1.39–1.55 (m, 3H), 0.95 (t, J=6.8 Hz, 3H); 13C NMR
(DMSO-d6) d 172.3, 141.5, 141.0, 122.5, 122.2, 122.2,
113.3, 37.6, 30.0, 28.7, 18.4, 12.2; MS (EI) m/z 234
(M+), 191 (100), 161, 145, 130, 117. Anal. calcd for
C12H14N2O3: C, 61.53; H, 6.02; N, 11.96. Found: C,
61.05; H, 6.17; N, 11.92.
3-(3-Chloropropyl)-3,4-dihydro-2(1H)-quinolinone (3d).
ꢀ
1
75%, white solid, mp 97–100 C; H NMR (CDCl3) d
9.13 (br s, 1H), 7.13–7.21 (m, 2H), 6.97 (td, J=7.3,
1.2 Hz, 1H), 6.83 (d, J=8.0Hz, 1H), 3.54–3.60(m,
2H), 3.05 (dd, J=15.4, 6.0Hz, 1H), 2.76 (dd,
J=15.3, 9.1 Hz, 1H), 2.53–2.61 (m, 1H), 1.86–2.04
(m, 3H), 1.62–1.71 (m, 1H); 13C NMR (CDCl3) d
172.1, 140.7, 135.2, 126.5, 125.9, 121.4, 113.5, 43.1,
37.8, 29.4, 28.5, 25.5; MS (EI) m/z 225 (M++2), 223
(M+), 188, 160, 146 (100), 132. Anal. calcd for
C12H14ClNO: C, 64.43; H, 6.31; N, 6.26. Found: C,
64.35; H, 6.52; N, 6.17.
3-(3-Chloropropyl)-6-nitro-3,4-dihydro-2(1H)-quinolinone
(4d). 89%, white solid, mp 206–208 ꢀC; 1H NMR
(DMSO-d6) d 10.69 (br s, 1H), 8.11 (s, 1H), 8.05 (d,
J=8.8 Hz, 1H), 6.98 (d, J=8.8 Hz, 1H), 3.64 (t, J=6.1
Hz, 2H), 3.13 (dd, J=15.9, 6.1 Hz, 1H), 2.79 (dd,
J=16.2, 10.2 Hz, 1H), 2.48–2.60 (m, 1H), 1.77–1.91 (m,
3H), 1.37-1.52 (m, 1H); 13C NMR (DMSO-d6) d 176.3,
148.2, 145.6, 128.1, 127.6, 127.6, 118.8, 49.2, 41.9, 33.5,
33.4, 30.6; MS (EI) m/z 270(M ++2), 268 (M+), 233,
205, 191 (100), 177, 145, 130, 117. Anal. calcd for
C12H13ClN2O3: C, 53.64; H, 4.88; N, 10.43. Found: C,
53.28; H, 4.95; N, 10.34.
3-(3-Fluoropropyl)-3,4-dihydro-2(1H)-quinolinone (3e).
ꢀ
1
61%, white solid, mp 92–93 C; H NMR (CDCl3) d
8.44 (br s, 1H), 7.14–7.21 (m, 2H), 6.98 (t, J=7.5 Hz,
1H), 6.78 (d, J=7.6 Hz, 1H), 4.47 (dt, J=47.1, 5.7 Hz,
2H), 3.05 (dd, J=15.6, 5.6 Hz, 1H), 2.77 (dd, J=15.4,
9.4 Hz, 1H), 2.55–2.63 (m, 1H), 1.73–2.00 (m, 3H),
1.58–1.66 (m, 1H); 13C NMR (CDCl3) d 172.1, 135.2,
126.5, 125.9, 121.4, 121.4, 113.5, 82.2 (d, J=164 Hz),
38.0, 29.3, 26.3 (d, J=19 Hz), 24.0(d, J=5 Hz); MS
(EI) m/z 207 (M+), 146 (100), 132, 117, 106. Anal. calcd
for C12H14FNO: C, 69.55; H, 6.81; N, 6.76. Found: C,
69.61; H, 6.97; N, 6.75.
3-(3-Fluoropropyl)-6-nitro-3,4-dihydro-2(1H)-quinolinone
(4e). 77%, white solid, mp 208–209 ꢀC; 1H NMR
(CDCl3) d 8.65 (br s, 1H), 8.10–8.14 (m, 2H), 6.88 (d,
J=9.2 Hz, 1H), 4.40(dt, J=47.1, 5.5 Hz, 2H), 3.71 (dd,
J=15.8, 5.8 Hz, 1H), 2.88 (dd, J=18.5, 9.8 Hz, 1H),
2.05–2.73 (m, 1H), 2.05–1.57 (m, 4H); 13C NMR
(CDCl3) d 171.3, 143.2, 140.6, 123.1, 122.6, 122.5, 113.9,
82.6 (d, J=161 Hz), 37.1, 28.5, 26.2 (d, J=19 Hz),
24.0(d, J=6 Hz); MS (EI) m/z 252 (M+), 232, 205,
191 (100), 177, 145, 130, 117. HRMS (FAB) m/
General procedure for the synthesis of compounds 4a–4e
To a solution of 3a (586 mg, 3.64 mmol) in 10mL of
concd H2SO4 and 2 mL of water at 0 ꢀC was dropwise
added 6l% HNO3 (0.30 mL, 4.00 mmol). After stirred
for 5 min at 0 ꢀC, the reaction mixture was carefully
poured into 400 mL of ice-water. The resulting pre-
cipitate was filtered and washed with 300 mL of water
and dried under suction for 3 h. The solid was dissolved
in 250mL of EtOAc, which was concentrated with 1.0g
of silica in vacuo. The product 4a (582 mg, 78%) was
obtained by flash column chromatography with 20%
EtOAc/hexane as a white solid.
z
253.0988.
C12H14FN2O3 (MH+) calcd: 253.0988; found:
General procedure for the synthesis of compounds 5a–5e
To a solution of 4a (586 mg, 2.84 mmol) and DDQ (724
mg, 3.13 mmol) in 15 mL of DMF was dropwise added

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B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
phosphorus oxychloride (0.41 mL, 4.26 mmol) at rt. The
mixture was stirred for 2 h at rt and then poured into
ice-water. The resulting precipitate was filtered and
washed with 50mL of water and dried under suction
for 3 h. The solid was dissolved in EtOAc and the
concentrated with 1.0g of silica gel in vacuo. The pro-
duct 5a (609 mg, 96%) was obtained by flash column
chromatography with 10% EtOAc/hexane as a white
solid.
General procedure for the synthesis of compounds 6a–6e
The mixture of 5a (609 mg, 2.74 mmol) and 1-piper-
azinecarboxaldehyde (1.00 mL, 8.21 mmol) in 30 mL of
DMF was stirred for 3 h at 100 ꢀC and then cooled to rt.
The reaction was quenched by adding 100 mL of water.
The resulting precipitate was filtered, washed with 50
mL of water and dried under suction for 1 h. The solid
was dissolved in 4 M H2SO4, which was stirred for 3 h at
80 ꢀC and then cooled to rt. After adding of 50mL of
water into mixture, aqueous 4 N NaOH was slowly
added to the mixture until basic to litmus. The resulting
precipitate was filtered, washed with 50mL of water
and dried under suction for 1 h to give 6a (402 mg,
56%) as a yellow solid.
2-Chloro-3-methyl-6-nitroquinoline (5a). Mp 145–147 ꢀC;
1H NMR (CDCl3) d 8.72 (d, J=2.2 Hz, 1H), 8.44 (dd,
J=9.3, 2.3 Hz, 1H), 8.15 (s, 1H), 8.11 (d, J=10.0 Hz,
1H), 2.60(s, 3H); 13C NMR (CDCl3) d 156.9, 146.7,
144.1, 137.2, 131.2, 128.3, 124.7, 121.8, 121.3, 18.5; MS
(EI) m/z 224 (M++2), 222 (M+, 100), 192, 176, 164,
155, 140, 113. Anal. calcd for C10H7ClN2O2: C, 53.95;
H, 3.17; N, 12.59. Found: C, 54.01; H, 3.50; N, 12.52.
3-Methyl-6-nitro-2-(piperazin-1-yl)quinoline (6a). Mp
164–166 ꢀC; H NMR (CDCl3) d 8.55 (d, J=1.8 Hz,
1
1H), 8.29 (dd, J=9.2, 1.8 Hz, 1H), 7.87 (s, 1H), 7.83 (d,
J=10.2 Hz, 1H), 3.40–3.44 (m, 4H), 3.04–3.09 (m, 4H),
2.46 (s, 3H); 13C NMR (CDCl3) d 161.5, 147.3, 141.6,
137.6, 126.5, 125.0, 122.0, 121.5, 120.5, 48.8, 44.4, 18.0;
MS (CI) m/z 273 (M++1), 257, 243 (100), 173. Anal.
calcd for C14H16N4O2: C, 61.78; H, 5.93; N, 20.59.
Found: C, 61.91; H, 6.26; N, 20.52.
2-Chloro-3-ethyl-6-nitroquinoline (5b). 75%, white solid,
mp 141–142 ꢀC; 1H NMR (CDCl3) d 8.75 (d, J=2.6 Hz,
1H), 8.44 (dd, J=9.1, 2.5 Hz, 1H), 8.14 (s, 1H), 8.11 (d,
J=9.0Hz, 1H), 2.96 (q, J=7.5 Hz, 2H), 1.40(t, J=7.3
Hz, 3H); 13C NMR (CDCl3) d 153.9, 146.5, 144.1,
136.5, 135.7, 128.2, 124.8, 122.0, 121.3, 24.8, 11.4; MS
(EI) m/z 238 (M++2), 236 (M+, 100), 221, 206, 191,
175, 155, 140, 127. Anal. calcd for C11H9ClN2O2: C,
55.82; H, 3.83; N, 11.84. Found: C, 55.80; H, 4.07; N,
11.63.
3-Ethyl-6-nitro-2-(piperazin-1-yl)quinoline (6b). 66%,
yellow solid, mp 130–131 ꢀC; H NMR (CDCl3) d 8.60
1
(d, J=2.6 Hz, 1H), 8.30(dd, J=9.2, 2.2 Hz, 1H), 7.94 (s,
1H), 7.85 (d, J=9.2 Hz, 1H), 3.35–3.40(m, 4H),
3.04–3.09 (m, 4H), 2.80 (q, J=7.2 Hz, 2H), 1.61 (br s,
1H), 1.37 (t, J=7.4 Hz, 3H); 13C NMR (CDCl3) d 161.6,
147.0, 141.6, 135.4, 131.3, 126.6, 122.4, 121.8, 120.5,
49.5, 44.4, 22.8, 12.3; MS (CI) m/z 287 (M++1), 271,
257 (100), 187. Anal. calcd for C15H18N4O2: C, 62.92; H,
6.34; N, 19.57. Found: C, 63.25; H, 6.08; N, 19.63.
2 - Chloro - 3 - propyl - 6 - nitroquinoline (5c). 95%, white
solid, mp 133–134 ꢀC; 1H NMR (CDCl3) d 8.74 (d,
J=2.6 Hz, 1H), 8.44 (dd, J=9.5, 2.5 Hz, 1H), 8.12 (s,
1H), 8.13 (d, J=9.5 Hz, 1H), 2.90(t, J=7.7 Hz, 2H),
1.71–1.89 (m, 2H), 1.06 (t, J=7.3 Hz, 3H); 13C NMR
(CDCl3) d 154.0, 146.6, 144.1, 136.7, 135.1, 128.3, 124.7,
122.0, 121.4, 33.6, 20.5, 12.0; MS (EI) m/z 252 (M++2),
250(M +, 100), 236, 220, 192, 176, 155. Anal. calcd for
C12H11ClN2O2: C, 57.50; H, 4.42; N, 11.17. Found: C,
57.17; H, 4.57; N, 11.22.
3-Propyl-6-nitro-2-(piperazin-1-yl)quinoline (6c). 54%,
ꢀ
1
yellow solid, mp 140–141 C; H NMR (CDCl3) d 8.58
(d, J=2.4 Hz, 1H), 8.30(dd, J=9.1, 2.5 Hz, 1H), 7.92
(s, 1H), 7.84 (d, J=9.2 Hz, 1H), 3.35–3.39 (m, 4H),
3.04–3.09 (m, 4H), 2.72 (t, J=7.7 Hz, 2H), 1.72–1.83
(m, 2H), 1.67 (br s, 1H), 1.01 (t, J=7.3 Hz, 3H); 13C
NMR (CDCl3) d 161.8, 147.1, 141.8, 136.0, 129.9, 126.7,
122.3, 121.8, 120.5, 49.6, 44.4, 32.1, 21.4, 12.4; MS (CI)
m/z 301 (M++1), 285, 271 (100), 201. Anal. calcd for
C16H20N4O2: C, 62.18; H, 6.14; N, 17.07. Found: C,
62.08; H, 6.41; N, 17.26.
2-Chloro-3-(3-chloropropyl)-6-nitroquinoline (5d). 99%,
white solid, mp 137–138 ꢀC; H NMR (CDCl3) d 8.76
1
(d, J=2.6 Hz, 1H), 8.46 (dd, J=9.2, 2.2 Hz, 1H), 8.20
(s, 1H), 8.13 (d, J=10.0 Hz, 1H), 3.62 (t, J=6.3 Hz,
2H), 3.13 (t, J=7.5 Hz, 2H), 2.17–2.31 (m, 2H); 13C
NMR (CDCl3) d 153.6, 146.8, 144.2, 137.3, 133.4, 128.3,
124.6, 122.0, 121.7, 42.1, 29.7, 28.9; MS (EI) m/z 288
(M++4), 286 (M++2), 284 (M+), 256, 221 (100), 175,
140. Anal. calcd for C12H10Cl2N2O2: C, 50.55; H, 3.54;
N, 9.82. Found: C, 49.98; H, 3.58; N, 9.67.
3-(3-Chloropropyl)-6-nitro-2-(piperazin-1-yl)quinoline
(6d). 42%, yellow solid, 132–133 ꢀC; H NMR (CDCl3)
1
d 8.58 (d, J=2.6 Hz, 1H), 8.30(dd, J=9.2, 2.6 Hz, 1H),
7.93 (s, 1H), 7.85 (d, J=9.2 Hz, 1H), 3.59 (t, J=6.3 Hz,
2H), 3.34–3.41 (m, 4H), 3.05–3.10 (m, 4H), 2.91–2.99
(m, 2H), 2.17–2.24 (m, 2H), 1.98 (br s, 1H); 13C NMR
(CDCl3) d 161.6, 147.2, 142.0, 136.6, 128.5, 126.9, 122.3,
121.8, 120.8, 49.6, 44.3, 42.7, 30.7, 27.6; MS (CI) m/z
337 (M++3), 335 (M++1), 285, 271, 257 (100). Anal.
calcd for C16H19ClN4O2: C, 57.40; H, 5.72; N, 16.74.
Found: C, 57.46; H, 6.08; N, 16.72.
2-Chloro-3-(3-fluoropropyl)-6-nitroquinoline (5e). 80%,
white solid, mp 117–119 ꢀC; H NMR (CDCl3) d 8.75
1
(d, J=2.6 Hz, 1H), 8.46 (dd, J=9.2, 2.6 Hz, 1H), 8.18
(s, 1H), 8.12 (d, J=9.6 Hz, 1H), 4.55 (dt, J=47.1, 5.7
Hz, 2H), 3.09 (t, J=7.7 Hz, 2H), 2.03–2.29 (m, 2H); 13C
NMR (CDCl3) d 153.6, 146.8, 144.2, 137.2, 133.8, 128.3,
124.7, 122,0, 121.7, 80.9 (d, J=166 Hz), 27.9 (d, J=24
Hz), 27.7; MS (EI) m/z 270(M ++2), 268 (M+), 238,
221 (100), 191, 175, 155, 140. Anal. calcd for
C12H10ClFN2O2: C, 53.65; H, 3.75; N, 10.43. Found: C,
53.29; H, 3.66; N, 10.43.
3-(3-Fluropropyl)-6-nitro-2-(piperazin-1-yl)quinoline (6e).
53%, yellow solid, mp 136–138 ꢀC; 1H NMR (CDCl3) d

B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
4955
8.58 (d, J=2.4 Hz, 1H), 8.31 (dd, J=9.2, 2.6 Hz, 1H),
7.93 (s, 1H), 7.85 (d, J=9.2 Hz, 1H), 4.52 (dt, J=47.4,
5.7 Hz, 2H), 3.35–3.40(m, 4H), 3.05–3.10(m, 4H), 2.92
(t, J=7.9 Hz, 2H), 2.32 (br s, 1H), 2.13 (dm, J=26.0
Hz, 2H); 13C NMR (CDCl3) d 161.6, 147.2, 141.9,
136.4, 128.8, 126.8, 122.3, 121.8, 120.8, 81.5 (d, J=165
Hz), 49.5, 44.2, 28.8 (d, J=20Hz), 26.1. HRMS (FAB)
m/z C16H20FN4O2 (MH+) calcd: 319.1578; found:
319.1574.
and concentrated with 2.0g of silica gel in vacuo. Flash
column chromatography with 70% EtOAc/hexane gave
ꢀ
1
9 (1.032 g, 55%) as a white solid: mp 157–158 C; H
NMR (CDCl3) d 8.36 (br s, 1H), 8.10(br s, 2H), 6.86 (d,
J=9.0Hz, 1H), 3.65–3.74 (m, 2H), 3.16 (dd, J=16.0,
6.0Hz, 1H), 2.89 (dd, J=15.9, 9.7 Hz, 1H), 2.71–2.63
(m, 1H), 2.04–1.61 (m, 4H); 13C NMR (DMSO-d6) d
171.6, 143.3, 140.5, 123.1, 122.7, 122.6, 113.8, 59.6, 37.5,
28.7, 28.5, 24.9. Anal. calcd for C12H14N2O4: C, 57.59;
H, 5.64; N, 11.19. Found: C, 57.47; H, 5.77; N, 11.23.
3 - (3 - tert - Butyldimethylsilyloxypropyl) - 3,4 - dihydro -
2(1H)-quinolinone (7). Under N2 atmosphere, to a solu-
tion of hydrocarbostyril (3.0g, 20.38 mmol) in 60mL of
sodium-dried THF was dropwise added 2.0M LDA
solution (22.42 mL, 44.85 mmol) at À78 ꢀC. The mix-
ture was stirred for 30min at À78 ꢀC and cooling bath
was removed. The mixture was stirred for additional 1 h
at rt, and then cooled to À78 ꢀC. (3-Bromopropoxy)-1-
tert-butyldimethylsilane (5.42 g, 21.40mmol) was drop-
wise added to the mixture at À78 ꢀC, which was stirred
for 90min at À78 ꢀC to rt. The reaction was quenched
by adding of 3 mL of MeOH and 500 mL of water in
succession. The organic compounds were extracted
three times with 100 mL of EtOAc. The combined
extract was dried over Na2SO4 and concentrated with
5.0g of silica gel in vacuo. The product was isolated by
flash column chromatography with 20% EtOAc/hexane
to give 7 (5.86 g, 90%) of as a white amorphous solid:
1H NMR (CDCl3) d 9.00 (s, 1H), 7.19–7.12 (m, 2H),
6.95 (t, J=7.3 Hz, 1H), 6.81 (d, J=7.2 Hz, 1H), 3.63 (t,
J=6.2 Hz, 2H), 3.05 (dd, J=15.4, 6.0Hz, 1H), 2.75 (dd,
J=15.4, 8.8 Hz, 1H), 2.63–2.52 (m, 1H), 1.94–1.81 (m,
1H), 1.74–1.46 (m, 3H), 0.86 (s, 9H), 0.01 (s, 6H); 13C
NMR (CDCl3) d 172.6, 135.4, 126.5, 125.7, 121.6, 121.2,
113.4, 61.3, 38.2, 29.1, 28.6, 24.4, 24.3, 16.6, À7.0.
HRMS (FAB) m/z C18H30NO2Si (MH+) calcd:
320.2030; found: 320.2038.
3-(3-Acetoxypropyl)-6-nitro-3,4-dihydro-2(1H)-quinoli-
none (10). To a solution of 9 (362 mg, 1.47 mmol) in 7
mL of pyridine was dropwise added acetic anhydride
(0.42 mL, 4.41 mmol) at rt. The mixture was stirred for
12 h at rt and poured into 50mL of ice-water. 4 M
H2SO4 was added to the mixture until acidic to litmus.
The resulting precipitate was filtered, washed with water
and dried under suction for 1 h to give 10 (362 mg,
84%) as a white solid: mp 143 ꢀC; H NMR (CDCl3) d
1
8.14–8.10(m, 3H), 6.85 (d, J=9.2 Hz, 1H), 4.10(t,
J=6.2 Hz, 2H), 3.16 (dd, J=15.8, 5.8 Hz, 1H), 2.87 (dd,
J=15.9, 9.7 Hz, 1H), 2.59–2.67 (m, 1H), 2.04 (s, 3H),
2.00–1.54 (m, 4H); 13C NMR (CDCl3) d 171.9, 169.4,
141.5, 140.8, 122.4, 122.4, 122.0, 113.5, 62.3, 37.4, 28.9,
24.5, 24.4, 19.3. HRMS (FAB) m/z C14H17N2O5 (MH+)
calcd: 293.1129; found: 293.1133.
2-Chloro-3-(3-acetoxypropyl)-6-nitroquinoline (11). The
mixture of 10 (547 mg, 1.87 mmol), DDQ (433 mg, 1.87
mmol) and phosphorus oxychloride (0.27 mL, 2.81
mmol) in 10mL of DMF was stirred for 3 h at rt and
then poured into 100 mL of ice-water. The resulting
precipitate was filtered, washed with water and dried
under suction for 1 h. The solid was dissolved in 100 mL
of EtOAc, concentrated with 1.0g of silica gel in vacuo.
The product 11 (525 mg, 91%) was obtained by flash
column chromatograꢀphy with 10% EtOAc/hexane as a
1
3-(3-Hydroxypropyl)-3,4-dihydro-2(1H)-quinolinone (8).
The mixture of 7 (3.31 g, 10.37 mmol) and tetra-
butylammonium fluoride hydrate (4.07 g, 15.55 mmol)
in 65 mL of THF was stirred for 12 h at rt and then
washed with 100 mL of water twice, dried over Na2SO4,
concentrated in vacuo. The product 8 (2.11 g, 99%) was
obtained by flash column chromatography with EtOAc
white solid: mp 114 C; H NMR (CDCl3) d 8.75 (d,
J=2.6 Hz, 1H), 8.46 (dd, J=9.2, 2.6 Hz, 1H), 8.17 (s,
1H), 8.12 (d, J=9.0Hz, 1H), 4.19 (t, J=6.4 Hz, 2H),
3.01 (t, J=7.9 Hz, 2H), 2.04–2.15 (m, 2H), 2.08 (s, 3H);
13C NMR (CDCl3) d 169.3, 153.7, 146.7, 144.1, 137.0,
133.9, 128.3, 124.6, 122.0, 121.4, 61.6, 28.3, 26.2, 19.3.
Anal. calcd for C14H13ClN2O3: C, 54.47; H, 4.24; N,
9.07. Found: C, 54.11; H, 4.09; N, 8.89.
1
only as a white amorphous solid; H NMR (CDCl3) d
8.49 (br s, 1H), 7.13–7.20(m, 2H), 6.97 (t, J=7.5 Hz, 1H),
6.78 (d, J=7.4 Hz, 1H), 3.66 (t, J=5.6 Hz, 2H), 3.03 (dd,
J=15.4, 5.8 Hz, 1H), 2.77 (dd, J=15.4, 9.6 Hz, 1H),
2.67–2.57 (m, 1H), 2.24 (br s, 1H), 1.95–1.58 (m, 4H); 13C
NMR (CDCl3) d 172.8, 135.2, 126.5, 125.8, 121.6, 121.4,
113.5, 60.6, 38.0, 29.3, 28.3, 24.2. HRMS (FAB) m/z
C12H16NO2 (MH+) calcd: 206.1181; found: 206.1182.
3-(3-Acetoxypropyl)-6-nitro-2-(4-formylpiperazin-1-yl)-
quinoline (12). The mixture of 11 (436 mg, 1.41 mmol)
and 1-piperazinecarboxaldehyde (0.51 mL, 2.12 mmol)
in 9 mL of DMF was stirred for 3 h at 100 ꢀC and then
cooled to rt, poured into 100 mL of ice-water. The
resulting precipitate was filtered, washed with 50mL of
water and dried under suction for 1 h. The solid was
dissolved in 100 mL of CH2Cl2, concentrated with 1.0g
of silica gel in vacuo. The product 12 (452 mg, 83%)
was obtained by flash column chromatography with 3%
3-(3-Hydroxypropyl)-6-nitro-3,4-dihydro-2(1H)-quinoli-
none (9). To a solution of 8 (1.56 g, 7.59 mmol) in 30
mL of concd H2SO4 and 6 mL of water was dropwise
added 61% HNO3 (0.68 mL, 9.10 mmol) at 0 ꢀC. After
stirring for 12 h at rt, the reaction mixture was carefully
poured into 500 mL of ice-water and neutralized with
potassium carbonate to pH 3. Organic compounds were
extracted from aqueous phase two times with 70mL of
CH2Cl2. The combined extract was dried over Na2SO4
MeOH/CH2Cl2 as a yellow solid: mp 142 ꢀC; H NMR
1
(CDCl3) d 8.58 (d, J=2.6 Hz, 1H), 8.29 (dd, J=9.2, 2.6
Hz, 1H), 8.13 (s, 1H), 7.99 (s, 1H), 7.85 (d, J=9.2 Hz,
1H), 4.13 (t, J=6.4 Hz, 2H), 3.72–3.74 (m, 2H),
3.57–3.62 (m, 2H), 3.40–3.47 (m, 2H), 3.30–3.35 (m,
2H), 2.85 (t, J=7.9 Hz, 2H), 1.99–2.16 (m, 2H), 2.03 (s,

4956
B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
3H); 13C NMR (CDCl3) d 169.2, 161.0, 159.3, 146.8,
142.3, 136.7, 128.7, 127.1, 122.7, 121.8, 120.9, 62.0, 48.7,
48.1, 43.8, 38.3, 27.0, 26.5, 19.2. HRMS (FAB) m/z
C15H13N2O3 (MH+) calcd: 387.1656; found: 387.1662.
(40% aq., 2.6 mL, 4.0 mmol). Three azeotropic distilla-
tions were carried out each time with 100–200 mL of
CH3CN at 90 ꢀC (oil bath) under a gentle stream of N2.
The radioactivity was resolublized and transferred to a
reaction vial containing a magnetic stirrer and 14 (2.0
mg, 5.0 mmol) using 2ꢃ100 mL of CH3CN. The mixture
was stirred at 130 ꢀC for 20min and then cooled. The
solvent was removed under a gentle stream of N2 at
90 ꢀC (water bath). To the residue was added H2SO4
(4 M, 0.3 mL), and the reaction mixture was stirred at
130 ꢀC for 10min. The reaction was quenched by add-
ing 0.1 mL of water to the mixture, prior to the addition
of NaOH (4 N) until basic to litmus. The organic com-
pounds were extracted from aqueous phase with
CH2Cl2 (1.0mL ꢃ3). The combined extracts (396 MBq;
not decay-corrected) was dried over sodium sulfate and
concentrated (N2 flow). The residue was redissolved in 1
mL of CH2Cl2 and injected onto a semipreparative
HPLC column (Alltech Econosil silica gel, 10 m, 10ꢃ250
mm), which was eluted with a 95:5 mixture of CH2Cl2
and MeOH (1% NH4OH) at a flow 4 mL/min. HPLC
purification of [18F]6e (tR=17–19 min) and removal of
the solvents gave the desired compound (241 MBq) in
17.3% radiochemical yield (decay-corrected). Authenti-
city of [18F]FPNQ was confirmed by coinjection with
unlabeled standard FPNQ on HPLC. The overall yield
was 14–25%, based on the initial radioactivity, and
total time including HPLC purification was 120min.
3-(3-Hydroxypropyl)-6-nitro-2-(4-formylpiperazin-1-yl)-
quinoline (13). The mixture of 12 (380mg, 0.98 mmol)
and K2CO3 (408 mg, 2.95 mmol) in 8 mL of MeOH and
8 mL of CH2Cl2 was stirred for 15 h at rt. The reaction
was quenched by adding 100 mL of water. Organic
compounds were extracted from aqueous phase three
times with 30mL of CH 2Cl2. The combined extract was
dried over Na2SO4 and concentrated with 0.5 g of silica
gel in vacuo. The product 13 (242 mg, 72%) was obtained
by flash column chromatography with 3% MeOH/
CH2Cl2 as yellow solid: mp 131–132 ꢀC; 1H NMR
(CDCl3) d 8.59 (d, J=2.4 Hz, 1H), 8.30(dd, J=9.2, 2.6
Hz, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.87 (d, J=9.2 Hz,
1H), 3.78–3.56 (m, 6H), 3.41–3.48 (m, 2H), 3.32–3.37 (m,
2H), 2.91 (t, J=7.7 Hz, 2H), 2.25 (br s, 1H), 1.92–2.06 (m,
2H); 13C NMR (CDCl3) d 161.1, 159.4, 146.7, 142.2,
136.8, 129.6, 127.1, 122.8, 121.8, 120.7, 59.8, 48.8, 48.1,
43.8, 38.3, 31.0, 26.0. HRMS (FAB) m/z C17H21N4O4
(MH+) calcd: 345.1562; found: 345.1555.
3-(3-Methanesulfonyloxypropyl)-6-nitro-2-(4-formylpi-
perazin-1-yl)quinoline (14). To a solution of 13 (206 mg,
0.60 mmol) and triethylamine (0.13 mL, 0.90 mmol) in 5
mL of CH2Cl2 was added dropwise methane sulfonyl
chloride (0.05 mL, 0.66 mmol) at 0 ꢀC. After stirring for
1 h at 0 ꢀC, the mixture was washed with 10mL of water
twice and dried over Na2SO4. The product 14 (238 mg,
94%) was obtained by short column chromatography
In vitro serotonin transporter bindings studies
Binding of [3H]citalopram to the 5-HT transporter was
measured according to the method of Pahkla et al.32 with
slight modifications using crude synaptic membranes
prepared from the rat cerebral cortex.33 Saturation
binding assays were performed to measure the equili-
brium dissociation constant (Kd) and the density of
binding sites (Bmax) using 15 concentrations of [3H]cita-
lopram between 0.01 and 30 nM. Nonspecific binding
was defined as that determined in the presence of 10 mM
fluoxetine. Scatchard analysis of the saturation binding
data indicated a single population of binding sites with
a Kd of 1.12ꢁ0.01 nM and a Bmax of 704ꢁ47 fmol/mg
protein (n=3). Competition binding assays were per-
formed to measure the concentrations of test com-
pounds which inhibited the specific binding by 50%
(IC50 values) using 1 nM [3H]citalopram and 11 con-
centrations of the unlabeled compounds between 10À11
and 10À5 M. IC50 values were determined from the
competition binding data using computer-assisted curve
fitting with the GraphPad Prism 3.0program. Inhibi-
tion binding constant (Ki) values were subsequently
calculated from IC50 values using the Cheng–Prusoff
equation.34
with 3% MeOH/CH2Cl2 as
a yellow solid: mp
145–151 ꢀC; H NMR (CDCl3) d 8.63 (d, J=2.6 Hz,
1H), 8.36 (dd, J=9.6, 2.6 Hz, 1H), 8.14 (s, 1H), 8.01 (2,
1H), 7.90(d, J=9.0Hz, 1H), 4.28 (t, J=6.0Hz, 2H),
3.75–3.80(m, 2H), 3.59–3.64 (m, 2H), 3.45–3.32 (m,
4H), 3.02 (s, 3H), 2.97 (t, J=8.0Hz, 2H), 2.18–2.26 (m,
2H); 13C NMR (CDCl3) d 161.0, 159.3, 146.9, 142.4,
136.8, 128.1, 127.2, 122.6, 121.9, 121.1, 67.3, 48.8, 48.1,
43.7, 38.2, 35.9, 27.6, 26.0. HRMS (FAB) m/z
C18H23N4O6S (MH+) calcd: 423.1338; found: 423.1340.
1
3-(3-Fluoropropyl)-6-nitro-2-(piperazin-1-yl)quinoline
(6e). The mixture of 14 (80mg, 0.19 mmol) and tetra-
butylammonium fluoride hydrate (59 mg, 0.23 mmol) in
2 mL of acetonitrile was heated under reflux for 1 h and
then cooled to rt. The reaction was quenched by adding
100 mL of water. The resulting precipitate was filtered,
washed with 50mL of water and dried under suction for
1 h. The solid was dissolved in 4 M H2SO4, which was
stirred for 3 h at 80 ꢀC and then cooled to rt. After
adding of 50mL of water to the mixture, aqueous 4 N
NaOH was slowly added to the mixture until basic to
litmus. The resulting precipitate was filtered, washed
with 50mL of water and dried under suction for 1 h to
give product 6e (402 mg, 42%) as a yellow solid.
Specific activity and in vivo tissue distribution studies of
[18F]6e
The radioligand collected from HPLC was concentrated,
redissolved in ethanol, and diluted with saline to give a
final solution of 10% ethanol-saline. The specific activity
of [18F]6e was determined by radioreceptor assay as
described by Fowler et al.35 In brief, aliquots of 200 mL
3-(3-[18F]Fluoropropyl)-6-nitro-2-(piperazin-1-yl)quino-
line ([18F]6e). [18F]Fluoride ion (1.91 GBq) produced
on a medical cyclotron was transferred to a Vacutai-
ner1 containing tetra-n-butylammonium hydroxide

B. S. Lee et al. / Bioorg. Med. Chem. 11 (2003) 4949–4958
4957
solution of [18F]6e were assayed for radioactivity with a
NaI(TI) well counter. After allowing the F-18 to decay,
successive dilutions of this solution were made in incu-
bation buffer just before aliquoting for determination of
the concentration of 6e in each dilution. The competi-
tive binding of each dilution of the [18F]6e solution with
1 nM [3H]citalopram was determined in duplicate and
compared with that of a standard competitive binding
curve made up of a series of concentrations of non-
radioactive 6e. Only those dilutions producing 20–80%
inhibition of the specific binding were used for calcula-
tion, since they represent the most accurate values. The
concentration of 6e in each of those dilutions was
determined from the standard competitive binding
curve using computer-assisted curve fitting with the
GraphPad Prism 3.0program. Together with the origi-
nal radioactivity determinations of the batch prepara-
tion, the average of three separate determinations was
used to determine the specific activity of the batch. Male
ICR mice (25–30g, four mice per time points) were
injected with the radioligand (1.5–1.9 MBq) in 0.2 mL of
10% ethanol–saline via tail vein. At the indicated time
points (5, 15, 30, 60, 90 and 120 min), the mice were
sacrificed by cervical dislocation, and samples of bone
and brain tissue (striatum, frontal cortex, hypothala-
mus, hippocampus, olfactory tubercle, cerebellum, and
the rest of the brain) were removed, weighed, and
counted. The data were expressed as percent injected
dose per gram of tissue (%ID/g).
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In vivo blocking studies
The mice (n=4) were injected with the radioligand
[18F]6e alone or with the following compounds: parox-
etine (2 mg/kg), fluoxetine (5 mg/kg), GBR-12909 (RBI,
2 mg/kg) or unlabeled 6e (2 mg/kg). These compounds
were dissolved in saline prior to the injection, except the
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ine due to the insolubility in saline alone. The mouse
brain tissues were dissected at 60min postinjection as
previously described. The data were expressed as per-
cent injected dose per gram of tissue (%ID/g).
Acknowledgements
We are grateful for the support of this work through a
grant from National Research Laboratory Program in
Korea and the Korea Science and Engineering Foun-
dation (No 97-03-01-01-5-L). We also thank Prof.
Cheon-Gyu Cho for the optimization of nitration step
for the synthesis of compound 9.
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