Development of N-[11C]methylamino 4-hydroxy-2(1H)-quinolone derivatives as PET radioligands for the glycine-binding site of NMDA receptors

Article abstract of DOI:10.1016/j.bmc.2009.06.014

In this study, we synthesized and evaluated several amino 4-hydroxy-2(1H)-quinolone (4HQ) derivatives as new PET radioligand candidates for the glycine site of the NMDA receptors. Among these ligands, we discovered that 7-chloro-4-hydroxy-3-{3-(4-methylam

Full text of DOI:10.1016/j.bmc.2009.06.014

Bioorganic & Medicinal Chemistry 17 (2009) 5665–5675
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Bioorganic & Medicinal Chemistry
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Development of N-[11C]methylamino 4-hydroxy-2(1H)-quinolone derivatives
as PET radioligands for the glycine-binding site of NMDA receptors
Takeshi Fuchigami a,c, Terushi Haradahira b, Noriko Fujimoto a, Yumiko Nojiri a, Takahiro Mukai a,
Fumihiko Yamamoto a, Takashi Okauchi b, Jun Maeda b, Kazutoshi Suzuki b, Tetsuya Suhara b,
a
Hiroshi Yamaguchi d, Mikako Ogawa c, Yasuhiro Magata c,d, , Minoru Maeda
*
a Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
b Molecular Imaging Center, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan
c Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
d Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized and evaluated several amino 4-hydroxy-2(1H)-quinolone (4HQ) derivatives
as new PET radioligand candidates for the glycine site of the NMDA receptors. Among these ligands, we
discovered that 7-chloro-4-hydroxy-3-{3-(4-methylaminobenzyl) phenyl}-2-(1H)-quinolone (12) and 5-
ethyl-7-chloro-4-hydroxy-3-(3-methylaminophenyl)-2(1H)-quinolone (32) have high affinity for the gly-
cine site (Ki values; 11.7 nM for 12 and 11.8 nM for 32). In vitro autoradiography experiments indicated
that [11C]12 and [11C]32 showed high specific binding in the brain slices, which were strongly inhibited
by both glycine agonists and antagonists. In vivo brain uptake of these 11C-labeled 4HQs were examined
in normal mice. Cerebellum to blood ratio of accumulation, of both [11C]12 and [11C]32 at 30 min were
0.058, which were slightly higher than those of cerebrum to blood ratio (0.043 and 0.042, respectively).
These results indicated that [11C]12 and [11C]32 have poor blood brain barrier permeability. Although the
plasma protein-binding ratio of [11C]32 was much lower than methoxy analogs (71% vs 94–98%, respec-
tively), [11C]32 still binds with plasma protein strongly. It is conjectured that still acidic moiety and high
affinity with plasma protein of [11C]32 may prevent in vivo brain uptake. In conclusion, [11C]12 and
Received 5 May 2009
Revised 4 June 2009
Accepted 6 June 2009
Available online 13 June 2009
Keywords:
Glycine-binding site
NMDA receptor
4-Hydroxy-2(1H)-quinolone
PET
[
11C]32 are unsuitable for imaging cerebral NMDA receptors.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
treatment of schizophrenia.4 Imaging of the glycine site coupled
to the NMDA ion channel by positron emission tomography (PET)
is, thus, considered useful for obtaining information about the
functional mechanism of the NMDA ion channel in the living brain
and in the diagnosis of various neurological disorders.
The N-methyl-D-aspartate (NMDA) receptor is known to play a
central role in excitatory neurotransmission, such as learning,
memory, and synaptic plasticity. Dysfunction of this receptor is
thought to be involved in various disorders, including epilepsy,
ischemic brain damage, and neurodegenerative disorders, such as
Parkinson’s and Alzheimer’s diseases. The NMDA ion channel has,
therefore, been considered to be a prime therapeutic target in
the development of useful neuroprotective strategies.1–3 Since gly-
cine is an essential co-agonist for NMDA ion channel activation,
various classes of glycine-binding site antagonists with anticonvul-
sant and neuroprotective properties have been developed.1,2 On
the other hand, it is suggested that impairment in the NMDA
receptor function is associated with the pathophysiology of schizo-
phrenia. Consistent with this theory, clinical trials have demon-
strated that the enhancement of NMDA receptor function by
potentiating the glycine site of the receptor is efficacious in the
We recently developed the 11C-labeled 4-hydroxy-2(1H)-quino-
lone (4HQ) derivatives, acetyl-[11C]L-703,717 ([11C]2)5,6 (a
prodrug of [11C]L-703,717 ([11C]1)7–9 and 3-[11C]methoxy-MDL-
104,653 (or 4-hydroxy-3-(3-[11C]methoxyphenyl)-2(1H)-quino-
lone) ([11C]3),10–12 as PET radioligands for the glycine-binding site
(Fig. 1). [11C]2 is a potential radioligand for use in PET investiga-
tions of the cerebellar NMDA ion channel and has recently been ap-
plied in the first human trial. In human brain, although, [11C]2
showed the highest accumulation in the cerebellum, the radioac-
tivity concentration was too low to visualize the cerebellar NMDA
receptors by PET, which is indicative of poor brain penetration of
[
11C]2.6 More recently we developed high affinity ethyl- and io-
dine-C-5 substituted analogs of [11C]3 {Ki values were 7.2 and
10.3 nM for 4 and 5, respectively}. Although [11C]4 and [11C]5 have
moderate lipophilicity, these ligands did not result in any signifi-
cant increase in brain uptake.13 The 4HQs are all acidic with pKa
* Corresponding author. Tel./fax: +81 53 435 2398.
E-mail address: magata@hama-med.ac.jp (Y. Magata).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.014

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T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
11
11
O
CH
O CH
3
3
OH
OAc
R
OH
2
11
O
CH
3
Cl
N
H
O
Cl
N
H
O
Cl
N
H
O
[11C]Ac-L-703,717 (2)
[11C]L-703,717 (1)
R2= H
R2= Et
R2= I
[11C] 3
[11C] 4
[11C] 5
Figure 1. Structure of 11C labeled 4-hydroxy-2-quinolone derivatives.
values of around 5 or below,12 and more amenable to binding with
plasma proteins than non-acidic compounds, resulting in poor
blood brain barrier (BBB) penetration. Introduction of basic amino
group into 4HQ may increase the pKa value and reduce unsuitable
protein bindings, which could increase the brain uptake. Therefore,
in order to develop the glycine/NMDA receptor radioligands with
reduced plasma protein binding affinity, we synthesized several
amino analogs of 4HQs and examined the in vitro binding proper-
ties and in vivo brain uptake characteristics in rodents.
substituted at X-position with amino group, was prepared as
shown in Scheme 2. Coupling of methyl anthranilate derivative
14 with acid chloride 15 provided amide derivative 16, then the
reduction of nitro group formed 17. Copper-catalyzed coupling15
of 17 with 4-methoxyphenylboronic acid gave compound 18. Sub-
sequent cyclization of 18 using potassium hexamethyldisilazide
afforded the desired compound 19. The 5-substituted (R2-substi-
tuted) 3 derivatives were prepared as shown in Scheme 3. Starting
with compound 20,16 5-amino derivative 24 was obtained by sim-
ilar procedure as described above. The 5-methylamino derivative
25 and 5-dimethylamino derivative 27 were synthesized by using
the general reductive amination. The compound 4 derivatives with
methoxy group at R3-position replaced with amino group were
prepared as shown in Scheme 4. Starting with compound 28,17
the desired 4HQs, 32 and 33, were obtained by the similar reaction
steps as described above.
2. Results and discussion
2.1. Chemistry
The L-703,717 (1) derivatives substituted at R1 position with
amino groups were prepared as shown in Scheme 1. Palladium-
catalyzed Suzuki coupling of 614 with 4-nitrophenylboronic acid
formed nitro benzyl derivative 7, followed by the reduction of
the nitro group using TiCl3 formed amino derivative 8. Methylation
of 8 using methyl iodide gave methylamino derivative 10 and
dimethylamino derivative 11. Cyclization of 8, 10, and 11 using
potassium carbonate afforded the desired 4HQs 9, 12, and 13,
respectively. Compound 19, which is the L-703,717 (1) derivative
2.2. In vitro binding assays
The binding affinities (Ki values) of 4HQs for the glycine-binding
sites of the NMDA ion channel were measured by displacement of a
typical glycine-site antagonist, [3H]MDL-105,519, to rat cortical
synaptic membranes.18 The pKa values of 4-hydroxy group in
O
O
O
O
a
NO2
Cl
NH
Cl
NH
Br
O
O
O
7
6
NH2
OH
O
NH
b
c
NH2
Cl
Cl
N
H
O
O
9
8
d
O
R1
OH
O
NH
c
R1
Cl
Cl
N
H
O
O
R = NHMe 12
R = NHMe 10
1
1
R = NMe
13
R = NMe
11
1
2
1
2
Scheme 1. Preparation of 400-methyl amino derivatives of 1. Reagents and conditions: (a) Pd(Ph3)4, p-nitrophenylboronic acid, Na2CO3, DME, 90 °C, 95%; (b) TiCl3, THF, rt, 85%;
(c) K2CO3, DMSO, 90 °C, then MeOH, HCl, 77% for 9, 97% for 12, 95% for 13; (d) CH3I, EtOH, DMF, rt, 23% for 10, 32% for 11.

T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
5667
O
O
O
O
O
a
b
O
Cl
NH
Cl
NH
Cl
NH
2
O
d
NO
2
O
O
NH
2
ClOC
O
NO
2
14
17
16
15
OH
O
c
O
N
Cl
NH
H
Cl
N
H
O
O
N
H
18
19
Scheme 2. Preparation of diphenylamino derivative of 1. Reagent and conditions: (a) ClCH2CH2Cl, 80 °C, 86%; (b) Fe, AcOH, EtOH, 80 °C, 97%, (c) Cu(OAc)2, myristic acid, 2,6-
lutidine, 4-methoxyphenylboronic acid, rt, 74%; (d) KHMDS, THF, rt, then TFA, 98%.
NO O
NH O
2
2
NO O
2
O
O
a
b
O
Cl
NH
Cl
NH
Cl
NH
2
O
O
O
O
ClOC
O
20
23
22
21
NH OH
NH OH
2
c
d
O
O
Cl
N
H
O
Cl
N
H
O
25
24
N
O
NH O
2
N
OH
O
O
e
c
O
Cl
NH
Cl
NH
Cl
N
H
O
O
O
O
O
23
27
26
Scheme 3. Preparation of 5-amino derivatives of 3. Reagent and conditions : (a) ClCH2CH2Cl, 80 °C, 95%; (b) Fe, AcOH, EtOH, 80 °C, 95%; (c) K2CO3, DMSO, 90 °C, 98% for 24,
98% for 27; (d) HCHO, NaBH3CN, MeOH, rt, 71%; (e) HCHO, NaBH4, H2SO4, THF, rt, 86%.
4HQs and log D7.4 values of 4HQs were calculated on SPARK on line
calculator.19 As shown in Table 1, replacement of the methoxy
group at R1-position of 1 with amino groups maintained high
receptor affinity. The calculated pKa value of 12 was comparable
with 1 (5.20). Methyl amino derivative 12 had approximately two-
fold higher affinity than dimethyl amino derivative 13 (Ki values;
11.7 nM for 12, 22.2 nM for 13). Introduction of amino group into
X-position of 1 also maintained high binding affinity (Ki value;
23.6 nM for 19), which was comparable with that of 13. The calcu-
lated pKa values of 13 and 19 were 5.25 which were slightly higher
than that of 12. These results indicate that the pKa values of 4-hy-
droxy group and the binding affinity for the glycine site of 4HQs
may not change significantly, when the amino group is introduced
at R1-position of L-703,717 (1). Introduction of NH2 group into the
5-position (R2-position) of 3 (Ki = 167 nM) resulted in slightly
enhancement of the affinity (Ki = 119 nM for 24). However, 5-
methylamino derivative 25 and 5-dimethylamino derivative 27
exhibited lower affinity than 24 (Ki values; 715 nM for 25,
566 nM for 27). Next, we attempted to introduce the amino groups
into R3-position of high affinity 5-ethyl derivative
(Ki = 7.20 nM).13 Therefore, methylamino derivative 32 had a high
affinity (Ki = 11.8 nM), which is comparable with that of
4
4
(Ki = 7.20 nM). On the other hand, dimethylamino derivative 33
had approximately threefold lower affinity (Ki = 34.3 nM) relative
to 32. We previously reported that introduction of ethyl group
and iodine atom into the 5-position of 3 resulted in a significant
enhancement of the affinity (Ki values; 7.2 nM for 4 and 10.3 nM
for 5, respectively) as compared with the parent 3 (Ki = 167 nM).13
Although binding affinity for glycine site of 4 and 5 were compara-
ble, the pKa values of 4 and 5 differ greatly. The pKa value of 5-ethyl
derivative 4 is much higher than that of 3, on the other hand, the
pKa value of 5-iodo derivative 5 is lower than that of 3 (pKa values;
5.35 for 3, 5.51 for 4, and 4.97 for 5). Introduction of NH2 or NHMe
group into the 5-position of 3 greatly increase pKa values (pKa val-

5668
T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
O
O
OH
O
a
b
NO
O
2
Cl
NH
Cl
N
H
O
Cl
NH
2
O
NO
2
ClOC
OH
NO
2
28
30
29
15
OH
OH
d
N
H
N
c
NH
2
+
Cl
N
H
O
Cl
N
H
O
Cl
N
H
O
32
31
33
Scheme 4. Preparation of 30-methyl amino derivatives of 4. Reagent and conditions: (a) ClCH2CH2Cl, 80 °C, 65%; (b) KHMDS, THF, rt, 87%; (c) Fe, AcOH, EtOH, 80 °C, 91%; (d)
CH3I, EtOH, DMF, 80 °C, 17% for 32, 8% for 33.
Table 1
affinity for the glycine site of the NMDA receptor among the amino
derivatives newly synthesized in this study, we chose these ligands
to carry out further biological evaluations on the corresponding C-
11 labeled probes.
In vitro binding affinity (Ki) of new 4-hydroxy-2-quinolone derivatives for glycine site
of the NMDA receptor, experimental octanol/phosphate buffer distribution coefficient
(log D7.4), calculated log D7.4, and calculated pKa of 4-hydroxy group
R
1
OH
X
2.3. Radiochemistry
Cl
N
H
O
To obtain [11C]12 and [11C]32, the corresponding amino precur-
sors, 9 and 31, were reacted with [11C]CH3OTf in acetone at 60 °C
for 1.5 min (Scheme 5). The resulting C-11 labeled crude products
were purified by HPLC. The desired products were obtained in
30 min; and the specific activities at the end of synthesis were
c
d
e
Compounds
X
R1
Kia (nM)
log D7.4
log D7.4
pKa
1
CH2
CH2
CH2
NH
OMe
5.12 0.99b
11.7 1.41
22.2 3.62
23.6 9.89
3.47
3.00
3.84
3.08
3.53
2.63
5.20
5.20
5.25
5.25
12
13
19
NHMe
NMe2
OMe
47 GBq/l l
mol for [11C]12 and 63 GBq/ mol for [11C]32, respec-
tively. The radiochemical purities were >98% for both ligands,
and the radiochemical yields were 8% for [11C]12 and 27% for
[
R
OH
2
11C]32 (decay-corrected).
R
3
Cl
N
H
O
2.4. In vitro autoradiography
In vitro regional distributions of [11C]12 and [11C]32 on the rat
brain sections were evaluated by a method similar to that in the
literature.20 The results are summarized in Figures 2 and 3. As
shown, the autoradiogram in Figure 2, both [11C]12 and [11C]32
showed the highest accumulation in the hippocampus, followed
by the cerebral cortex, thalamus, striatum, and lowest uptake in
the cerebellum (Fig. 2A and C). Nonspecific binding was deter-
mined in the presence of corresponding nonradioactive 4HQ
R2
R3
3
4
5
24
25
27
32
33
H
Et
I
NH2
NHMe
NMe2
Et
OMe
OMe
OMe
OMe
OMe
OMe
NHMe
NMe2
167 23.0b
7.20 1.56b
10.3 1.30b
119 456
715 206
566 197
11.8 3.32
34.3 12.9
1.75
2.84
2.46
2.09
2.35
2.84
2.16
2.60
5.35
5.51
4.97
5.67
5.67
5.48
5.59
5.59
2.78
1.92
Et
(10
lM), which resulted in a significantly lower radioactivity of
11C]12 and [11C]32 in the whole brain compared with total bind-
a
[
The Ki values were obtained by the method in Ref. 18. Each value (mean SD)
was determined three times with triplicate using rat cortical membrane homoge-
nates in Tris–HCl buffer.
ing (Fig. 2B and D). These results are consistent with the brain dis-
tributions of other high-affinity glycine-site antagonists.21,22 The
specific bindings of [11C]12 and [11C]32 on the slices were 30% (cer-
ebellum) to 70% (hippocampus) and 75% (cerebellum) to 89% (hip-
pocampus), respectively. Although, both ligands showed high
specific bindings in forebrain regions (cerebral cortex, hippocam-
pus), the specific binding ratio of [11C]12 was higher than that of
b
The Ki values have been reported in our previous studies from Ref. 13.
The partition coefficient between n-octanol and sodium phosphate buffer at pH
c
7.4 (log D7.4) was determined by conventional shake-flask method (n = 3).
d
The log D7.4 values of 4HQs were calculated on SPARK on line calculator.19
The pKa values of 4-hydroxy group in 4HQs were calculated on SPARK.19
e
[
11C]32 in all brain regions (Fig. 2E and F).
In order to investigate the specificity of [11C]12 and [11C]32
ues; 5.67 for 24 and 25, 5.35 for 3). However binding affinity for
the glycine site of these ligands was quite different from each other
(Ki values; 119 nM for 24, 715 nM for 25). Introduction of the ami-
no groups into R3-position of 4 slightly increase pKa values (pKa
values; 5.59 for 32 and 33). However binding affinity for the gly-
cine site of 33 was threefold lower than that of 32. The experimen-
tal results indicated that the binding affinity of 3 derivatives for the
glycine site may have no correlation with calculated pKa values of
4-hydroxy group in 4HQs. Since 12 and 32 had the highest binding
binding to the glycine site of the NMDA ion channel, we carried
out in vitro inhibition studies on rat hippocampal region using gly-
cine-site agonists (glycine and
D-serine) and antagonists
{L
-703,717 (1); methoxy-MDL-104,653 (3), MDL-105,519}, with
the results being summarized in Figure 3A (agonists) and B (antag-
onists). The corresponding methoxy derivatives [11C]1 and [11C]4
were also investigated for comparison of binding characters with
amino derivatives. The in vitro total bindings of [11C]12 were

T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
5669
H
N
NH2
OH
OH
11CH3
a
Cl
N
H
Cl
N
H
O
O
11
[
C]12
9
OH
OH
11CH3
a
N
NH2
H
Cl
N
H
O
Cl
N
H
O
11
31
[
C]32
Scheme 5. Radiosynthesis of [11C]12 and [11C]32. Reagent and conditions: (a) [11C]CH3OTf, acetone, 60 °C, 1.5 min, 8% for [11C]12, 27% for [11C]32 (decay-corrected).
HIP
HIP
A
C
THA
CTX
THA
CB
CTX
STR
CB
STR
TB of [11C]12
TB of [11C]32
B
E
D
F
NSB of [11C]32
NSB of [11C]12
TB of [11C]32
NSB of [11C]32
TB of [11C]12
800
600
400
200
0
1000
NSB of [11C]12
800
600
400
200
0
CTX
HIP
STR
THA
CB
CTX
HIP
STR
THA
CB
Figure 2. Autoradiogram of in vitro total binding of [11C]12 (A), nonspecific binding of [11C]12 (B), total binding of [11C]32 (C), and nonspecific binding of [11C]32 (D) to rat
brain sagittal sections. The quantified values of [11C]12 (E) and [11C]32 (F) in frontal cortex (CTX), hippocampus (HIP), striatum (STR), thalamus (THA), and cerebellum (CB) are
expressed as (PSL-BG)/mm2 (mean SD, n = 4–6). TB, total binding; NSB, nonspecific binding determined in the presence of corresponding nonradioactive 4HQ (10
lM).
strongly inhibited by both glycine-site agonists (46–53%) and
antagonists (58–93%), respectively. The bindings of [11C]32 were
also inhibited by both agonists (59–66%) and antagonists (64–
70%). The inhibition characteristics of 11C labeled amino deriva-
tives 12 and 32 were similar to corresponding methoxy derivatives
1 and 4 (Fig. 3A and B).
binding throughout the brain which was inhibited by antagonists
alone.10,13 Previous experimental data, including mutational analy-
sis, support the evidence that glycine-site agonists and antagonists
bind to overlapping but different sites on the NMDA ion chan-
nel.23,24 The existence of multiple glycine agonist domains has also
been reported on the NMDA ion channel.25 Thus, it is considered
that two binding sites for [11C]4HQs on the NMDA ion channel
may be existed, the agonist-sensitive and agonist-insensitive (or
antagonist-preferring) sites as described in our previous study.13
It is suggested that the agonist-sensitive sites located on the func-
tional NMDA ion channel are formed by the combination of heter-
omeric NR1 and NR2 subunits. In contrast, the agonist-insensitive
Recently, we reported that the binding characters of 4HQs var-
ied according to the substituent groups. The [11C]1 and 5-ethyl
analog [11C]4 showed higher in vitro binding in the forebrain re-
gions than in the cerebellum, in which the binding was strongly
inhibited by both glycine-site agonists and antagonists. In contrast,
[
11C]3 and 5-iodo analog [11C]5 showed a homogeneous in vitro

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T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
A 100
B 100
80
80
60
40
20
0
60
40
20
0
Glycine
3
D-Serine
11C]1
1
MDL-105,519
[
[
11C]12
[
11C]4
[
11C]32
Figure 3. Effects of NMDA agonists (A) and antagonists (B) on in vitro total [11C]4HQ bindings to hippocampus. The PSL data in the presence of drugs were converted to the
percentage of control PSL data in the absence of drugs (mean SD, n = 4–6).
sites may exit on homomeric NR1 receptors or desensitized or
inactivated NMDA ion channel.26 In fact, the accumulation of 11C
4HQs in the brain tissue tended to be inhibited by antagonists
stronger than agonists (Fig. 3). These experiments indicate that
most binding site of [11C]12 and [11C] 32 may overlap with the gly-
cine antagonist binding site. In these studies, [11C]12 and [11C]32
showed high specific binding in the forebrain regions than cerebel-
lum, in which the binding on the latter area was strongly inhibited
by both glycine-agonists and antagonists. These results strongly
suggest that 4HQs, which have bulky 3-benzyl phenyl group in
3-position, such as 1 and 12, or ethyl group in 5-position, such as
4 and 32, demonstrate high specific binding for the functional het-
eromeric NR1 and NR2 subunits.
2.5. In vivo pharmacology
The biodistribution studies of [11C]1, [11C]4, [11C]12, and [11C]32
were performed by using normal mice and the results were shown
in Table 2. All of these ligands exhibited very high blood radioactiv-
ity level. The radioactivity of 5-ethyl substituted analogs ([11C]4
and [11C]32) at 1 min were 20.91–29.16% of injected dose per gram
of tissue (% dose/g), which were much higher than those of L-
703,717 analogs ([11C]1 and [11C]12; 8.37–13.08% dose/g). The
radioactivity concentration of [11C] 4HQs in blood were still high
level (9.08–10.66% dose/g for 5-ethyl analog and 3.58–3.93%
dose/g for L-703,717 analogs) at 30 min. The brain uptake of
[
11C]12 at 1 min after intravenous injection was 0.53% dose/g in
The in vitro binding experiments demonstrate that [11C]12 and
the cerebellum, which was higher than cerebral uptake (0.40%
dose/g). After 30 min, the uptake of [11C]12 in the cerebrum and
cerebellum were 0.17% and 0.23% dose/g, respectively. The brain
to blood ratios of [11C]12 at 1 min and 30 min were 0.031–0.037
and 0.043–0.058, respectively. These results indicate that [11C]12
has poor BBB permeability. The brain uptake of [11C]32 at 1 min
after intravenous injection was 0.87% dose/g in the cerebellum,
which was much higher than the cerebral uptake (0.47% dose/g).
At 30 min, the uptake of [11C]32 in the cerebrum and cerebellum
were 0.36% and 0.53% dose/g, respectively. The brain to blood ra-
[
11C]32 are useful as PET imaging agents for the functional NMDA
receptor. As shown in Table 1, the experimental log D7.4 value of 12
and 32 were well correlated with calculated one (3.00 vs 3.08 for
compound 12 and 1.92 vs 2.16 for compound 32, respectively).
The lipophilicity of these ligands are in the optimal range (1.0–
3.0) for compounds expected to penetrate the BBB.27 Thus, we at-
tempted further in vivo experiments on these radioligands. We
previously demonstrated that 4HQs are selective ligands for the
glycine site of the NMDA receptor.13
Table 2
Brain and blood uptake of [11C]4HQs in mice at 1 min and 30 min after injection
Region
1 min
Tissue/bloodb
30 min
Tissue/bloodb
30 min Blockinga
Tissue/bloodb
% Dose/gb
% Dose/gb
% Dose/gb
[
11C]1
Blood
Cerebrum
Cerebellum
8.37 0.60d
0.32 0.03d
0.36 0.05d
3.46 0.53
0.20 0.01
0.24 0.05
0.038 0.004
0.043 0.005
0.051 0.004
0.068 0.007
[
11C]12
Blood
Cerebrum
Cerebellum
13.08 0.76
0.40 0.07
0.48 0.10
3.93 0.25
0.17 0.01
0.23 0.02
0.031 0.007
0.037 0.006
0.043 0.003
0.058 0.004
[
11C]4
11C]32
a
Blood
Cerebrum
Cerebellum
29.16 6.03
0.50 0.11
0.72 0.09
10.66 1.36
0.32 0.03
0.44 0.05
0.018 0.006
0.026 0.007
0.030 0.003
0.042 0.008
[
Blood
Cerebrum
Cerebellum
20.91 2.34
0.47 0.10
0.87 0.03
9.08 0.70
0.38 0.06
0.53 0.03c,e
7.77 0.72
0.34 0.05
0.34 0.04f
0.022 0.003
0.042 0.011
0.042 0.008
0.058 0.006
0.044 0.004
0.044 0.002f
Non-radioactive 32 was co-administrated with [11C]32.
Mean SD (n = 3).
b
c
d
e
f
Mean SD (n = 5).
Data from Ref. 13.
P < 0.01 in comparison to the cerebrum (Mann–Whitney U-test).
P < 0.05 in comparison to the control group (Mann–Whitney U-test).

T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
5671
tios of [11C]32 at 1 min and 30 min were 0.022–0.042 and 0.038–
*
0.053, respectively. Considering these results, [11C]32 may also
have low BBB permeability. The uptake of [11C] 4HQs in the cere-
bellum tend to be slightly higher than in the cerebrum. These
in vivo results are inconsistent with in vitro brain distribution of
radioligands. Previously, we demonstrated that the agonist-sensi-
tive [11C]1 and its pro-drug [11C]2 show high in vivo specific bind-
ing in the cerebellum.5,9 The different brain uptake of [11C]1
between in vitro and in vivo condition may be due to the regional
brain differences in endogenous agonist levels. It is known that
100
80
60
40
20
0
endogenous glycine agonists, such as glycine and
in
M quantities in the brain.1 Glycine is high in the brainstem/
spinal cord but low in the cerebellum/forebrains. On the contrary,
D-serine, exist
l
D
-serine is high in the forebrains but is undetectable in the cerebel-
lum.28 Thus, it is considered that the existence of endogeneous gly-
cine agonists in cerebellum is lower than in the cerebrum under
in vivo condition. In fact, we demonstrated a greatly diminished
1
3
4
32
Figure 4. The plasma protein-binding ratio of 4HQs was obtained by ultrafiltration
method after incubation of radioligand in rat plasma/buffer at 37 °C. Data are
presented as the percentage (mean SD, n = 3). * P < 0.01 (Kruskal–Wallis test, post
test was Dunn’s test).
cerebellar localization of [11C]1 in mutant mice lacking the
D-ami-
no acid oxidase (DAO); wherein, the
D-serine content in the cere-
bellum is drastically increased from a nondetectable level in
normal mice.29 Therefore, it is suggested that agonist-sensitive li-
gands, [11C]12 and [11C]32, showed high cerebellar accumulation
for a similar reason.
[
11C]32 still binds with plasma protein strongly and free fraction
may be very low level.
Although the uptake of 11C amino derivatives of 4HQs were low
level, the radioactivity of [11C]32 in brain and blood are much high-
er than that of [11C]12 as shown in Table 2. In addition, the calcu-
lated pKa value of [11C]32 was much higher than that of [11C]12
(5.59 and 5.20, respectively). Therefore we performed further
experiments of [11C]32. Metabolisms of [11C]32 in mouse brain
and blood were analyzed by radio-TLC of brain homogenates ob-
tained at 30 min post-injection; and 70% and 93%, respectively, of
the parent compounds remained unchanged (data not shown).
The in vivo stability of [11C]32 is comparable with methoxy deriv-
atives.13 Next, we performed blocking studies of [11C]32. Co-injec-
tion of non-radioactive 32 (2 mg/kg) with [11C]32 resulted in
significant inhibition of radioactivity in cerebellum (P < 0.05) as
shown in Table 2. On the other hand, the uptake of radioactivity
in cerebrum was not inhibited significantly. Furthermore cerebel-
lum to blood ratio of [11C]32 was also significantly different be-
tween control and blocking studies. On the contrary, no
significant difference was observed in cerebrum to blood ratio. It
cannot be regarded that [11C]32 shows in vivo specific accumula-
tion in cerebellum because of poor BBB permeability of [11C]32.
However certain specific binding site of [11C]32 may exist in the
cerebellar glycine-binding site of the NMDA receptor. Finally, we
examined the plasma protein-binding ratio of 4HQs, which was ob-
tained by ultrafiltration method as shown in Figure 4. The plasma
protein-binding ratio of [11C]32 was much lower than the methoxy
analogs (71% vs 94–98%, respectively). Although these results indi-
cate that the free fraction of [11C]32 is several fold higher than
those of methoxy analogs, the brain uptake of [11C]32 was as low
as those of methoxy analogs. It would appear that the reasons
are in part because [11C]32 might be excreted from brain tissue
by the efflux systems of BBB. The BBB permeability of various
endogenous and exogenous ligands is regulated by efflux trans-
porters such as P-glycoprotein (P-gp), organic anion transporter
(OAT), and multidrug resistance protein.30 It is reported that some
radioligands for mapping neuroreceptors are substrates for P-gp.31
It has been shown that OAT3 is responsible for the elimination of
several acidic drugs from the brain across the BBB.32 Although
the pKa value of 32 is calculated to be 5.59 which is much lower
than those of methoxy analogs, 32 still has acidic moiety. It is un-
clear that which systems affect the permeability of [11C]32 across
BBB. However it is possible that [11C]32 may be preferentially ex-
creted from brain tissue by OAT. Another reason may be because
Introduction of multiple basic groups and other electron-donat-
ing groups near the 4-position or non-acidic substituent in 4-posi-
tion of 4HQs may increase significant higher pKa values of 4-
hydroxy group of 4HQs. As the results, plasma protein binding
and efflux from organic anion transporter of 4HQs will be reduced,
which is expected to improve BBB penetration. Further investiga-
tion is required for developing suitable in vivo imaging agents for
the glycine site of the NMDA receptor.
3. Conclusion
We have developed high-affinity N-[11C]Methylamino 4HQ
derivatives 12 and 32 as new PET radioligand candidates for the
glycine site of the NMDA receptor. In vitro autoradiography exper-
iments imply that [11C]12 and [11C]32 showed high specific bind-
ing to the glycine site in the rat brain slices. In vivo studies using
mice showed that both of these ligands have poor BBB permeabil-
ity. Although the plasma protein-binding ratio of [11C]32 was much
lower than the methoxy analogs, [11C]32 still binds with plasma
protein strongly. Still acidic moiety and high affinity for plasma
protein of [11C]32 may prevent the BBB penetration of [11C]32. Fur-
ther SAR studies are required for developing favorable PET ligands
for the glycine site of the NMDA receptor with improved BBB
permeability.
4. Experimental
4.1. General information
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded on a JEOL GX-270 spectrometer and a Varian UNITY INO-
VA 400. Mass spectra (MS) were obtained on a JOEL TMS-D300
spectrometer or an Applied Biosystem Mariner System 5299
spectrometer by use of electrospray ionization (ESI) or fast atom
bombardment (FAB). High-resolution mass spectra (HRMS) were
obtained on a QSTARÒXL MS/MS System by use of ESI. Infrared
(IR) spectra were recorded with a JASCO IR Report-100 spectrom-
eter. Column chromatography was done on Merck Kieselgel 60
(70–230 mesh), and analytical thin layer chromatography (TLC)
was carried out on Silica Gel 60 F254 plates (Merck). In the syn-
thetic procedures, organic extracts were routinely dried over
anhydrous Na2SO4 and evaporated with a rotatory evaporator

5672
T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
under reduced pressure. High-performance liquid chromatogra-
phy (HPLC) was done using a Waters HPLC system for non-radio-
active runs or a JASCO HPLC system for radioactive runs. Effluent
radioactivity from the HPLC was determined using a NaI (Tl)
scintillation detector system. Carbon-11 was generated by the
J = 8.6 Hz), 7.30 (1H, d, J = 1.5 Hz), 7.25 (1H, t, J = 7.4 Hz), 7.19
(1H, dd, J = 8.6, 1.9 Hz), 7.16 (1H, s), 7.13 (1H, d, J = 7.6 Hz), 7.10
(1H, d, J = 7.5 Hz), 6.90 (2H, d, J = 8.5 Hz), 6.48 (2H, d, J = 8.2 Hz),
3.77 (3H, s), IR (KBr) cmꢀ1: 3400–3000, 2922, 1650, 1582, FAB-
MS (m/z); 377 (M+H)+.
14N(p,
a)
11C nuclear reaction using a CYPRIS HM-18 cyclotron
11C]CH3I,
(Sumitomo Heavy Industries, Ltd). Preparation of
[
4.1.4. Methyl 4-chloro-2-(3-(4-methylaminobenzyl)phenylacet-
amide) benzoate (10) and methyl 4-chloro-2-(3-(4-dimethyl-
aminobenzyl)phenylacetamide) benzoate (11)
[
11C]CH3OTf and subsequent 11C-methylation were carried out
automatically by using a synthetic apparatus for 11C-labeled
compounds in National Institute of Radiological Sciences (NIRS).
Radioactivity was quantified with an IGC-3R Curiemeter (Aloka).
To a solution of 8 (947 mg, 2.32 mmol) in DMF/EtOH (1:1,
10 ml), CH3I (0.14 ml, 2.32 mmol) was added and stirred at room
temperature. After 5 h, EtOAc (50 ml) was added to the mixture,
washed with brine, dried with Na2SO4, and evaporated to dryness.
The crude product was chromatographed on silica gel with hexane/
EtOAc = 3:1 to provide 10 (176 mg, 23%) as a colorless oil and 11
(220 mg, 32%) as a colorless oil.
[
11C]1, [11C]3, and [11C]4 were prepared as described in the liter-
ature.7,13 All reagents were used as received unless otherwise
stated. The animal experiments were carried out according to
the recommendations of the committee for the care and use of
laboratory animals, NIRS.
Compound 10: 1H NMR (CDCl3) d: 11.03 (1H, br s), 8.82 (1H, d,
J = 2.1 Hz), 7.90 (1H, d, J = 8.7 Hz), 7.27 (1H, t, J = 7.5 Hz), 7.19
(1H, s), 7.17 (1H, d, J = 7.6 Hz), 7.10 (1H, d, J = 7.5 Hz), 7.02 (1H,
dd, J = 8.6, 2.1 Hz), 7.01 (2H, d, J = 8.5 Hz), 6.53 (2H, d, J = 8.5 Hz),
4.1.1. Methyl 4-chloro-2-(3-(4-nitrobenzyl)phenylacetamide)
benzoate (7)
Methyl 4-chloro-2-(3-(bromomethyl) phenylacetamido) benzo-
ate (6)14 (2.4 g, 6.03 mmol), 4-nitrophenylboronic acid (1.51 g,
9.05 mmol), and tetrakistriphenylphosphine palladium(0) (70 mg,
0.06 mmol) were stirred in dimethoxyethane (10 ml) for 10 min,
then 2 M aqueous sodium carbonate (9.7 ml, 18.09 mmol) was
added, and stirred at 90 °C. After 4 h, EtOAc (30 ml) was added to
the mixture, the organic layer was separated, and the aqueous
layer extracted with EtOAc (20 ml). The combined organic layers
were washed with water and brine, dried with Na2SO4, and evap-
orated to dryness. The crude product was chromatographed on sil-
ica gel with hexane/EtOAc = 4:1 to provide 7 (2.5 g, 95%) as a white
solid. mp = 106–107 °C, 1H NMR (CDCl3) d: 11.10 (1H, s), 8.81 (1H,
d, J = 2.0 Hz), 8.36 (2H, d, J = 8.7 Hz), 8.12 (1H, d, J = 8.6 Hz), 7.92
(1H, d, J = 8.6 Hz), 7.78 (2H, d, J = 8.8 Hz), 7.35 (1H, d, J = 8.6 Hz),
7.32 (1H, t, J = 7.5 Hz), 7.20 (1H, s), 7.04 (1H, dd, J = 8.6, 2.1 Hz),
4.09 (2H, s), 3.86 (3H, s), 3.73 (2H, s), IR (CHCl3) cmꢀ1: 2910,
1687, 1590, ESI-MS m/z; 349 (M+H)+.
3.89 (2H, s), 3.84 (3H, s), 3.71 (2H, s), 2.81 (3H, s), IR (KBr) cmꢀ1
:
3200, 2916, 1689, 1578, FAB-MS (m/z); 423.2 (M+H)+.
Compound 11: 1H NMR (CDCl3) d: 11.04 (1H, br s), 8.82 (1H, d,
J = 2.0 Hz), 7.91 (1H, d, J = 8.6 Hz), 7.27 (1H, d, J = 7.5 Hz), 7.21
(1H, s), 7.19 (1H, d, J = 7.4 Hz), 7.11 (1H, d, J = 7.3 Hz), 7.06 (2H,
d, J = 8.7 Hz), 7.03 (1H, dd, J = 8.6, 2.1 Hz), 6.66 (2H, d, J = 8.6 Hz),
3.90 (2H, s), 3.83 (3H, s), 3.71 (2H, s), 2.90 (6H, s), IR (KBr) cmꢀ1
:
3300, 2900, 1690, 1578, FAB-MS (m/z); 437 (M+H)+.
4.1.5. 7-Chloro-4-hydroxy-3-{3-(4-
methylaminobenzyl)phenyl}-2-(1H)-quinolone (12)
Following the above procedure for 9 starting from compound
10, the title compound 12 (96 mg, 97%) was obtained as a white so-
lid, mp = 261–263 °C, 1H NMR (DMSO) d: 11.44 (1H, s), 10.19 (1H,
br s), 7.90 (1H, d, 8.6 Hz), 7.29 (1H, d, J = 1.9 Hz), 7.26 (1H, t,
J = 7.7 Hz), 7.20–7.17 (3H, m), 7.14 (1H, d, J = 7.3 Hz), 7.09 (1H, d,
J = 7.6 Hz), 6.97 (2H, d, J = 8.4 Hz), 6.45 (2H, d, J = 8.4 Hz), 5.38
(1H, br s), 3.79 (2H, s), 2.61 (3H, s), IR (KBr) cmꢀ1: 3421, 3200–
2800, 1647, 1581, ESI-HRMS (m/z) calcd for C23H20ClN2O2,
391.121 (M+H)+, obsd 391.1221.
4.1.2. Methyl 4-chloro-2-(3-(4-aminobenzyl)phenylacetamide)
benzoate (8)
To a solution of 7 (2.4 g, 5.47 mmol) in THF (100 ml) was added
dropwise a solution of 20% TiCl3 in hydrochloric acid (40 ml,
62.17 mmol) and stirred at room temperature. After 24 h, the mix-
ture was cooled in an ice bath and neutralized with aqueous 10%
NaOH. The resulting dark slurry was diluted with EtOAc (500 ml)
and the mixture was stirred vigorously until the precipitation be-
came yellow. The suspension was filtered, and the residue was
washed with EtOAc. The filtrate was washed with water and brine,
dried with Na2SO4, and evaporated to dryness. The crude product
was chromatographed on silica gel with hexane/EtOAc = 2:1 to
provide 8 (1.97 g, 85%) as a colorless oil, 1H NMR (CDCl3) d: 11.04
(1H, br s), 8.82 (1H, d, J = 2.0 Hz), 7.91 (1H, d, J = 8.6 Hz), 7.26
(1H, t, J = 7.3 Hz), 7.19 (1H, d, J = 7.1 Hz), 7.18 (2H, d, J = 7.3 Hz),
7.10 (1H, d, J = 7.3 Hz), 7.03 (1H, dd, J = 8.6, 2.1 Hz), 6.98 (2H, d,
J = 8.2 Hz), 6.60 (2H, d, J = 8.2 Hz), 3.89 (2H, s), 3.84 (3H, s), 3.71
(2H, s), 3.56 (2H, br s), IR (CHCl3) cmꢀ1: 3246, 3001, 1687, 1578,
FAB-MS m/z; 409 (M+H)+.
4.1.6. 7-Chloro-4-hydroxy-3-{3-(4-dimethylaminoben-
zyl)phenyl}-2-(1H)-quinolone (13)
Following the above procedure for 9 starting from compound
11, the title compound 13 (143 mg, 95%) was obtained as a white
solid, mp = 253–255 °C, 1H NMR (DMSO) d: 11.48 (1H, s), 10.21
(1H, br s), 7.91 (1H, d, 8.6 Hz), 7.30 (1H, d, J = 1.9 Hz), 7.28 (1H, t,
J = 7.5 Hz), 7.19 (1H, dd, J = 8.8, 2.0 Hz), 7.17 (1H, s), 7.14 (1H, d,
J = 8.1 Hz), 7.11 (1H, d, J = 7.9 Hz), 7.06 (2H, d, J = 8.4 Hz), 6.64
(2H, d, J = 8.6 Hz), 3.83 (2H, s), 2.82 (6H, s), IR (KBr) cmꢀ1: 3410,
3300–2800, 1647, 1581, ESI-HRMS (m/z) calcd for C24H22ClN2O2,
405.1370 (M+H)+, obsd 405.1369.
4.1.7. Methyl 2-(3-nitrophenyl) acetamide-4-chlorobenzoate
(16)
To a solution of 3-nitrophenyl acetic acid (1.63 g, 9.81 mmol) in
CH2Cl2 (10 ml), 2 M dichloromethane solution of oxalyl chloride
(6.5 ml, 13.0 mmol) was added and stirred at room temperature.
After 6 h, the mixture was evaporated and the residue was dis-
solved in dichloroethane (10 ml); and methyl-2-amino-4-chloro-
benzoate (14) (800 mg, 4.31 mmol) was added. The mixture was
refluxed for 5 h, cooled to room temperature, quenched by the
addition of saturated aqueous sodium hydrogen carbonate, and ex-
tracted with CHCl3. The organic layer was washed with brine, dried
with Na2SO4, and evaporated to dryness. The crude product was
chromatographed on silica gel with hexane/CHCl3 = 1:3 to provide
4.1.3. 7-Chloro-4-hydroxy-3-{3-(4-aminobenzyl)phenyl}-2-
(1H)-quinolone (9)
To a solution of 8 (135 mg, 0.33 mmol) in DMSO (3 ml) was
added anhydrous potassium carbonate (227 mg, 1.64 mmol) and
stirred at 80 °C. After 6 h, MeOH (2 ml) was added to the mixture,
followed by 1 M HCl to maintain at pH 7–8 in order to give a white
precipitate. The solid was collected, washed with ethanol and
ether, and dried to give
9 (96 mg, 77%) as a white solid,
mp = 228–230 °C, 1H NMR (DMSO) d: 11.49 (1H, s), 7.91 (1H, d,

T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
5673
16 as (1.29 g, 86%) a white solid, mp = 125–126 °C, 1H NMR (CDCl3)
d: 11.30 (1H, s), 8.78 (1H, d, 2.1 Hz), 8.27 (1H, t, J = 1.8 Hz), 8.19
(1H, d, J = 7.9 Hz), 7.94 (1H, d, J = 8.6 Hz), 7.72 (1H, d, J = 7.5 Hz),
7.56 (1H, t, 8.0 Hz), 7.07 (1H, dd, 8.6, 2.6 Hz), 3.89 (3H, s), 3.87
(2H, s). IR (KBr) cmꢀ1: 3249, 1701, 1689, 1521, FAB-MS (m/z);
349 (M+H)+.
cmꢀ1: 3250, 1736, 1670, 1652, ESI-MS (m/z); 379 {(M+H)+ for
35Cl}, 381 {(M+H)+ for 37Cl}.
4.1.12. Methyl 2-(3-methoxyphenyl)acetamide-6-amino-4-
chlorobezoate (23)
Following the above procedure for 17 starting from compound
22, the title compound 23 (1.76 g, 95%) was obtained as a white so-
lid, mp = 90–93 °C, 1H NMR (CDCl3) d: 10.52 (1H, br s), 8.01 (1H, d,
J = 1.9 Hz), 7.29 (1H, t, 7.8 Hz), 6.93 (1H, d, J = 7.5 Hz), 6.89 (1H, s),
6.86 (1H, d, J = 8.1 Hz), 6.37 (1H, d, J = 2.0 Hz), 5.44 (2H, s), 3.82
(3H, s), 3.73 (3H, s), 3.71 (2H, s), IR (KBr) cmꢀ1: 3500, 3363,
1734, 1670, 1609, ESI-MS (m/z); 349 {(M+H)+ for 35Cl}, 351
{(M+H)+ for 37Cl}.
4.1.8. Methyl 2-(3-aminophenyl) acetamide-4-chlorobenzoate
(17)
To a solution of 16 (3.5 g, 10.04 mmol) in AcOH/EtOH (2:7,
45 ml), iron powder (3.36 g, 60.16 mmol) was added and stirred
at 80 °C. After 6 h, the mixture was cooled to room temperature,
added to water (200 ml), and filtered. The filtrate was extracted
with EtOAc, washed with brine dried with Na2SO4, and evaporated
to dryness. The crude product was chromatographed on silica gel
with CHCl3/EtOAc = 10:1 to provide 17 (3.1 g, 97%) as a yellow
oil, 1H NMR (DMSO) d: 10.69 (1H, s), 8.46 (1H, d, 2.0 Hz), 7.91
(1H, d, J = 8.7 Hz), 7.24 (1H, dd, J = 8.6, 2.0 Hz), 6.98 (1H, d,
J = 8.6 Hz), 6.53–6.46 (3H, m), 5.14 (2H, br s), 3.79 (3H, s), 3.56
(2H, s). IR (KBr) cmꢀ1: 3362, 3263, 1684, 1652, ESI-MS (m/z); 319
(M+H)+.
4.1.13. 5-Amino-7-chloro-4-hydroxy-3-(3-methoxyphenyl)-
2(1H)-quinolone (24)
Following the above procedure for 9 starting from compound
23, the title compound 24 (89 mg, 98%) was obtained as a white so-
lid, mp = 263–265 °C, 1H NMR (DMSO) d: 11.15 (1H, br s), 7.79 (2H,
br s), 7.29 (1H, d, J = 7.7 Hz), 6.87–6.84 (3H, m), 6.40 (1H, d,
J = 1.8 Hz), 6.32 (1H, d, J = 1.7 Hz), 3.32 (3H, s), IR (KBr) cmꢀ1
:
3503, 3396, 3200–2700, 1647, 1605, ESI-HRMS (m/z) calcd for
C16H14ClN2O3; 317.0693 (M+H)+, obsd 317.0700 (M+H)+.
4.1.9. Methyl 4-chloro-2–3-{4-methoxyphenylamino}-
phenylacetamido}-benzoate (18)
4.1.14. 5-Methylamino-7-chloro-4-hydroxy-3-(3-methoxy-
phenyl)-2(1H)-quinolone (25)
To a solution of 24 (52 mg, 0.16 mmol) in MeOH (1 ml), 37%
formaldehyde/H2O (9.8 ll, 0.131 mmol) was added, followed by
sodium cyanoborohydride (8.5 mg, 0.131 mmol) and stirred at
room temperature. After 10 h, the reaction mixture was quenched
with water (1 ml), extracted with EtOAc, washed with brine, dried
with Na2SO4, and evaporated to dryness. The crude product was
chromatographed on silica gel with CHCl3/MeOH = 15:1 to provide
25 (38 mg, 71%) as a yellow solid, mp = 261–263 °C, , 1H NMR
(DMSO) d: 11.00 (1H, s), 7.24 (1H, t, 7.7 Hz), 6.93–6.79 (3H, m),
6.44 (1H, d, J = 1.9 Hz), 6.08 (1H, d, J = 1.8 Hz), 3.73 (3H, s), 2.77
(3H, s), ESI-HRMS (m/z) calcd for C17H16ClN2O3, 331.0849 (M+H)+,
obsd 331.0869 (M+H)+.
To
a
solution of 4-methoxyphenylboronic acid (2.14 g,
solution of 17 (3.0 g,
14.08 mmol) in toluene (10 ml),
a
9.41 mmol) in toluene (5 ml), copper(II) acetate (171 mg,
0.94 mmol), myristic acid (430 mg, 1.88 mmol), and 2,6-lutidine
(1.1 ml, 9.42 mmol) was mixed. The mixture was stirred for 24 h
at room temperature, then EtOAc (50 ml) was added, washed with
brine dried with Na2SO4, and evaporated to dryness. The crude
product was chromatographed on silica gel with hexane/
EtOAc = 3:1 to provide 18 (2.96 g, 74%) as
a brown solid,
mp = 89–90 °C, 1H NMR (CDCl3) d: 11.05 (1H, br s), 8.82 (1H, d,
J = 2.1 Hz), 7.91 (1H, d, 8.6 Hz), 7.24–7.06 (3H, m), 7.03 (1H, dd,
J = 8.6, 2.1 Hz), 7.00–6.84 (5H, m), 3.86 (3H, s), 3.76 (3H, s), 3.67
(2H, s), IR (KBr) cmꢀ1: 3412, 2916, 1705, 1598, ESI-MS (m/z);
425 (M+H)+.
4.1.15. Methyl 2-(3-methoxyphenyl)acetamide-4-chloro-6-
dimethylamino benzoate (26)
4.1.10. 7-Chloro-4-hydroxy-3-{3-(4-methoxyphenylamino)-
phenyl}-2(1H)-quinolone (19)
To an ice cooled solution of 37% formaldehyde/H2O (0.145 ml,
1.9 mmol) 3 M of aqueous H2SO4 (0.26 ml) was added a slurry of
25 (110 mg, 0.32 mmol) and sodium borohydride (84 mg,
2.22 mmol) in THF (1 ml). The mixture was stirred at room temper-
ature for 2 h, then quenched with 10% aqueous NaOH (5 ml), ex-
tracted with EtOAc, washed with brine, dried with Na2SO4, and
evaporated to dryness. The crude product was chromatographed
on silica gel with CHCl3/MeOH = 40:1 to provide 26 (102 mg,
86%) as a colorless oil, 1H NMR (CDCl3) d: 9.16 (1H, br s), 7.95
(1H, d, 1.9 Hz), 7.30 (1H, t, J = 7.5 Hz), 6.91 (1H, d, J = 7.1 Hz),
6.87 (1H, d, J = 6.7 Hz), 6.60 (1H, d, J = 1.9 Hz), 3.83 (3H, s), 3.74
(3H, s), 3.68 (2H, s), 2.78 (6H, s), IR (KBr) cmꢀ1: 3363, 2939,
1684, 1595, ESI-MS (m/z); 377 {(M+H)+ for 35Cl}, 379 {(M+H)+ for
37Cl}.
To a solution of 18 (890 mg, 2.09 mmol) in THF (15 ml), potas-
sium hexamethyldisilazide (9.1 ml of 15% solution in toluene,
6.27 mmol) was added and stirred at room temperature. After
5 h, the reaction mixture was quenched by the addition of MeOH
(5 ml) followed by TFA to maintain at pH 7–8, then EtOAc
(20 ml) was added, washed with brine, dried with Na2SO4, and
evaporated to dryness. The crude product was chromatographed
on silica gel with CHCl3/MeOH = 15:1 to provide 19 (780 mg,
98%) as a white solid, mp = 269–271 °C, 1H NMR (DMSO) d: 11.48
(1H, s), 10.13 (1H, br s), 7.89 (1H, d, J = 8.6 Hz), 7.83 (1H, d,
J = 2.1 Hz), 7.29 (1H, d, J = 2.0 Hz), 7.19 (1H, d, J = 8.6 Hz), 7.18
(1H, t, J = 7.0 Hz), 7.08–7.01 (2H, m, 6.92–6.83 (3H, m, 6.70 (1H,
d, J = 7.5 Hz), 3.69 (3H, s), IR (KBr) cmꢀ1: 3383, 2918, 1598, ESI-
HRMS (m/z) calcd for C22H18ClN2O3; 393.1006 (M+H)+, obsd
393.1012.
4.1.16. 5-Dimethylamino-7-chloro-4-hydroxy-3-(3-methoxy-
phenyl)-2(1H)- quinolone (27)
Following the above procedure for 9 starting from compound
26, the title compound 27 (89 mg, 98%) was obtained as a white so-
lid, mp = 208–210 °C, 1H NMR (DMSO) d: 11.42 (1H, br s), 7.49 (1H,
t, 1.9 Hz), 7.24 (1H, t, J = 8.0 Hz), 7.21 (1H, d, J = 1.8 Hz), 7.02 (1H, d,
J = 7.9 Hz), 7.00 (1H, d, J = 2.4 Hz), 6.81 (1H, dd, J = 8.2, 2.1 Hz), 3.74
(3H, s), 2.76 (6H, s), IR (KBr) cmꢀ1: 3380, 2927, 1635, ESI-HRMS (m/
z) calcd for C18H18ClN2O3, 345.1006 (M+H)+, obsd 345.1003
(M+H)+.
4.1.11. Methyl 2-(3-methoxyphenyl)acetamide-4-chloro-6-
nitrobezoate (22)
Following the above procedure for 16 starting from compound
2016 and 21, the title compound 22 (1.76 g, 95%) was obtained as
a white solid, mp = 96–98 °C, 1H NMR (CDCl3) d: 9.19 (1H, br s),
8.78 (1H, d, J = 2.1 Hz), 7.51 (1H, d, 2.0 Hz), 7.34 (1H, t,
J = 1.9 Hz), 6.93–6.85 (3H, m), 3.84 (3H, s), 3.74 (5H, s), IR (KBr)

5674
T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
4.1.17. Methyl 2-(3-nitrophenyl)acetamide-4-chloro-6-
ethylbenzoate (29)
lytical HPLC (column; Nacalai Cosmosil 5C18-ARII, 4.6 ꢁ 250 mm,
mobile phase; CH3CN/H2O = 50:50, flow rate; 1.0 ml/min). The spe-
cific activity of purified [11C]2 was calculated by UV spectroscopy
(254 nm) to be 47 GBq/lmol. The total synthesis and purification
time was 27 min with an overall decay corrected yield of 8%.
Following the above procedure for 16 starting from compound
2817 and 15, the title compound 29 (334 mg, 65%) was obtained
as a white solid, mp = 85–86 °C, 1H NMR (DMSO) d: 9.72 (1H, s),
8.34 (1H, d, J = 2.2 Hz), 8.22–8.19 (2H, m), 7.71 (1H, d, J = 8.0 Hz),
7.57 (1H, d, J = 7.9 Hz), 6.99 (1H, d, J = 2.1 Hz), 3.85 (3H, s), 3.82
(2H, s), 2.74 (2H, q, J = 7.5 Hz), 1.17 (3H, t, J = 7.5 Hz), IR (KBr)
cmꢀ1: 3244, 1722, 1670, 1529, ESI-MS (m/z); 377 (M+H)+.
4.2.2. 5-Ethyl-7-chloro-4-hydroxy-3-(3-[11C]methylaminophe-
nyl)-2(1H)-quinolone ([11C]32)
[
11C]Methylation of 31 (0.7 mg, 2.2
lmol) was carried out by
the same manner as described above. The radioactive fraction hav-
4.1.18. 5-Ethyl-7-chloro-4-hydroxy-3-(3-nitrophenyl)-2(1H)-
quinolone (30)
ing a retention time of 13 min from HPLC (column; Nacalai COS-
MOSIL 5C18
-
ARII, 10 ꢁ 250 mm, mobile phase; CH3CN/
Following the above procedure for 19 starting from compound
29, the title compound 30 (193 mg, 87%) was obtained as a white
solid, mp = 232–233 °C, 1H NMR (DMSO) d: 11.62 (1H, s), 10.42
(1H, br s), 8.20–8.17 (2H, m), 7.80 (1H, d, J = 7.6 Hz), 7.69 (1H, t,
J = 7.9 Hz), 7.23 (1H, d, J = 2.2 Hz), 7.03 (1H, d, J = 2.2 Hz), 3.14
(2H, q, J = 7.4 Hz), 1.20 (3H, t, J = 7.4 Hz), IR (KBr) cmꢀ1: 3377,
1681, 1635, FAB-MS (m/z); 345 (M+H)+.
H2O = 50:50, flow rate; 4.0 ml/min) was collected, evaporated,
and dissolved in sterile distilled water to give radiochemically pure
(>98%) [11C]32. The specific activity of purified was 63 GBq/
lmol.
The total synthesis and purification time was 29 min with an over-
all decay corrected yield of 27%.
4.3. In vitro binding assays
4.1.19. 3-(3-Aminophenyl)-7-chloro-4-hydroxy-2(1H)-
quinolone (31)
The purity of test compounds were assayed to be >97% by ana-
lytical HPLC (column; Nacalai Cosmosil 5C18-AR 300,
4.6 ꢁ 250 mm, mobile phase; MeOH/H2O = 80:20–45:55, flow rate;
1.0 ml/min). The Ki values of 4HQs for binding of [3H]MDL-105,519
to rat cortical membrane homogenates in 50 mM Tris–HCl buffer
were determined by the method in the literature.18 In brief, well-
Following the above procedure for 17 starting from compound
30, the title compound 31 (489 mg, 91%) was obtained as a white
solid, mp = 178–179 °C, 1H NMR (DMSO) d: 11.44 (1H, br s), 9.92
(1H, s), 7.62 (1H, d, J = 8.1 Hz), 7.30 (1H, t, J = 7.8 Hz), 7.19 (1H, t,
J = 2.2 Hz), 7.05–6.96 (2H, m), 3.12 (2H, q, J = 7.3 Hz), 1.18 (3H, t,
J = 7.2 Hz), IR (KBr) cmꢀ1: 3480, 3300, 1683, 1635, FAB-MS (m/z);
315 (M+H)+.
washed membranes (125
pH 8.0, were incubated with [3H]MDL-105,519 (2 nM) with either
buffer or displacing drug to a final volume of 500 l for 45 min at
4 °C. Non-specific binding was defined by the addition of unlabeled
glycine at 100 M. Bound ligand was collected by rapid filtration
lg protein) in 50 mM Tris–HCl buffer,
l
4.1.20. 5-Ethyl-7-chloro-4-hydroxy-3-(3-methylaminophenyl)-
2(1H)-quinolone (32) and 5-ethyl-7-chloro-4-hydroxy-3-(3-
dimethylaminophenyl)-2(1H)-quinolone (33)
Following the above procedure for 10 and 11 starting from com-
pound 31, the title compounds 32 (3.5 mg, 17%) and 33 (1.8 mg,
8%) were obtained as white solid, respectively.
l
using a Brandel cell harvester onto glass fiber filter (GF/B). The fil-
ters were washed rapidly three times with 2.5 ml of ice-cold assay
buffer. Following separation and rinsed, the filters were placed into
scintillation liquid (4 ml; ASCII, GE Healthcare Bio-Sciences) and
radioactivity was determined with a liquid scintillation counter.
Compound 32: mp = 196–198 °C, 1H NMR (DMSO) d: 11.31 (1H,
s), 9.47 (1H, s), 7.17 (1H, s), 7.11 (1H, t, J = 2.5 Hz), 6.95 (1H, s),
6.49–6.47 (3H, m), 5.55 (1H, s), 3.11 (2H, q, J = 7.0 Hz), 2.67 (3H,
s), 1.17 (3H, t, J = 7.4 Hz), IR (KBr) cmꢀ1: 3280, 3000, 1670, 1590,
ESI-HRMS (m/z) calcd for C18H18ClN2O2, 329.1057 (M+H)+, obsd
329.1069.
4.4. In vitro receptor autoradiography
The brain sagittal sections obtained by the method in the liter-
ature21 were pre-incubated for 30 min at 25 °C in 50 mM Tris–HCl
buffer (pH 7.4 at 25 °C), and subsequently incubated in the same
buffer containing [11C]12 or [11C]32 (1.59–2.13 nM) at 25 °C for
30 min. The slices were rinsed twice for 3 min each with cold
(5 °C) incubation buffer, and subsequently dipped into cold water.
The sections were dried under a steam of warm air and placed in
contact with 11C-sensitive imaging plates (BAS-SR 127, Fuji Photo
Film) for 60 min. Distributions of radioactivity on the plates were
analyzed by FUJIX BAS 5000 bioimaging analyzer (Fuji Photo Film
Co, Ltd), which was photographically visualized as shown in Figure
2A–D. Regions of interest (ROIs) on the slices were placed on the
cerebral cortex, hippocampus, striatum, thalamus, and cerebellum;
and the radioactivities in these regions were expressed as photo-
stimulated luminescence (PSL) values on ROI-background PSL va-
lue per square millimeter. The specific binding was determined
as the difference between total binding and binding in the pres-
ence of the corresponding non-radioactive 4-hydroxyquinolones
Compound 33: mp = 203–205 °C, 1H NMR (DMSO) d: 11.40 (1H,
s), 9.48 (1H, s), 8.36 (1H, s), 7.19 (1H, s), 6.98 (1H, d, J = 2.3 Hz), 6.71
(1H, d, J = 7.6 Hz), 6.64 (1H, s), 6.59 (1H, d, J = 7.1 Hz), 3.11 (2H, q,
J = 7.2 Hz), 2.89 (6H, s), 1.18 (3H, t, J = 7.2 Hz), IR (KBr) cmꢀ1: 3350,
3010, 1692, 1610, ESI-HRMS (m/z) calcd for C19H20ClN2O2,
343.1213 (M+H)+, obsd 343.1219.
4.2. Radiosynthesis
4.2.1. 7-Chloro-4-hydroxy-3-{3-(4-
[
11C]methylaminobenzyl)phenyl}-2-(1H)-quinolone ([11C]12)
[
11C]CH3OTf was prepared using general method as described in
the literature.33 11C]CH3OTf was trapped in an anhydrous acetone
(250 l) containing 9 (0.5 mg, 1.34
mol) at ꢀ15 to ꢀ20 °C. The
reaction vessel was heated at 60 °C and kept there for 1.5 min.
After addition of an HPLC mobile phase (600 l) and DMF
(400 l), the radioactive mixture was transferred onto an HPLC
[
l
l
l
(10 lM).
l
(Cosmosil 5C18-AR II, 10 ꢁ 250 mm, Nacalai) and eluted with
CH3CN/H2O = 50:50 at a flow rate of 4.0 ml/min. A radioactive frac-
tion having a retention time of 11 min was collected in a flask con-
4.5. Drug inhibition for in vitro binding
The brain slices were incubated at 25 °C for 30 min in 50 mM
Tris–HCl buffer (pH 7.4 at 25 °C) containing [11C]4HQ (1.59–
taining Tween 80 (75 ll) and ethanol (150 ll). After evaporation of
the solvents under reduced pressure, the radioactive residue was
dissolved in sterile distilled water (5 ml) to give [11C]12 as an aque-
ous solution. Radiochemical purity was assayed to be >98% by ana-
2.13 nM) with or without drugs (10 lM for antagonist and
1 mM for agonist). Following the incubation, the sections were
rinsed, dried, and apposed to the imaging plates for 60 min by

T. Fuchigami et al. / Bioorg. Med. Chem. 17 (2009) 5665–5675
5675
the same manner as described above. The PSL data corresponding
Acknowledgments
to the hippocampus in the presence of drugs were divided by the
respective control PSL data in the absence of drugs. The obtained %
total binding values were averaged in at least triplicate sections
from three animals and shown in Figure 3A (agonist) and B
(antagonist).
The authors gratefully acknowledge the assistance of Mr. Nen-
gaki and Mr. Ogawa, Sumitomo Accelerator Services, and Mr. Takei,
Tokyo Nuclear Service, in conducting the radiosyntheses. We are
also grateful to the cyclotron crew of the NIRS for their technical
support. The studies were supported by Grant-in-Aid for Young
Scientists (Start-up) (18890080).
4.6. In vivo brain distribution and metabolism
The [11C]12 or [11C]32 (0.2 ml, ca. 7.4 MBq) was injected intra-
venously via tail vein into ddY mice (35–45 g). These mice were
killed at 1 min or 30 min by decapitation. The whole brains were
rapidly removed; and the cerebrum and cerebellum were dis-
sected. The obtained tissue and blood samples were weighed and
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4.7. Partition coefficient determination
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ambient temperature. After centrifugation (2500 rpm for 5 min)
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4.9. Data analysis
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shown in Figure 4. A value of P < 0.05 was considered statistically
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