Synthesis and characterization of [125I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl} (phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

Article abstract of DOI:10.1016/j.bmc.2011.09.010

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methyl

Full text of DOI:10.1016/j.bmc.2011.09.010

Bioorganic & Medicinal Chemistry 19 (2011) 6245–6253
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of [125I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}
(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine
transporter 1
Takeshi Fuchigami a,b, , Mamoru Haratake a, Yasuhiro Magata b, Terushi Haradahira c, Morio Nakayama a,
⇑
⇑
a Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
c Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, 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, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and
evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which pre-
dominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoro-
methyl-benzamide (5) showed excellent inhibitory activity of [3H]glycine uptake (IC50 = 2.4 nM).
Saturation assay in rat cortical membranes revealed that [125I]5 had a single high affinity binding site
with a Kd of 1.54 nM and a Bmax of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that
Received 16 August 2011
Revised 6 September 2011
Accepted 7 September 2011
Available online 10 September 2011
Keywords:
[
125I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited
Glycine transporter 1 (GlyT1)
by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [125I]5 with
GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [125I]5 showed high blood–
brain barrier permeability (1.68–2.17% dose/g at 15–60 min) and similar regional brain distribution pat-
tern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of
125I
Single photon emission computed
tomography (SPECT)
Schizophrenia
[
125I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5
is a promising SPECT imaging probe for GlyT1.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
disease, and Alzheimer’s disease.4 Alterations in the ambient gly-
cine concentration are known to have modulatory effects on the
NMDAR.3 Specifically, pharmacological modulation of glycine-
mediated neurotransmission by recently developed various
GlyT-1 inhibitors (Fig. 1) revealed beneficial effect to the negative
and cognitive symptoms of schizophrenia.5–7 First generation
GlyT1 inhibitors are glycine derivatives, such as ALX5407 and
Org24461 with high inhibitory potency.8 Unfortunately, none of
these compounds are expected to be promising drug candidates
because of their safety profiles and pharmacokinetics.9 More
recently, second generation nonamino acid type series of com-
pounds have been developed as potent GlyT1 inhibitors. Some of
them showed positive results for clinical use in the treatment of
schizophrenia.6,9 In addition, recent reports suggested that GlyT1
inhibitors could be also applied for various disorders such as
depression, anxiety, seizures, and neuropathic pain by modulating
NMDA receptor or inhibitory GlyRs.10,11 However, the existence of
a correlation between the progress of such diseases and the change
of GlyT1 expression and activity remains to be solved.
Glycine has dual physiological functions as an inhibitory neuro-
transmitter for strychnine-sensitive glycine receptors (GlyRs) and
as a co-agonist for NMDA receptors which play key roles in excit-
atory glutamatergic transmission.1 Glycine transporters (GlyTs)
modulate these neurotransmissions by controlling glycine concen-
tration in the synaptic cleft of glial and neuronal cells. GlyTs are
classified into two sodium/chloride dependent transporters, GlyT1,
and GlyT2.2 It is considered that GlyT2 participate in inhibitory gly-
cinergic neurotransmission, because they are mainly expressed by
glycinergic neurons in the caudal regions, such as the spinal cord
and brain stem. In contrast, GlyT1 are widely expressed in many
brain regions, such as the forebrain and caudal regions. Therefore
GlyT1 are thought to regulate the glycine concentration in both
excitatory NMDA receptors and inhibitory GlyRs.3
Dysfunction of the NMDA receptor is thought to be involved in
various disorders, such as schizophrenia, stroke, Parkinson’s
Positron emission tomography (PET) and single photon emission
computed tomography (SPECT) are the efficient imaging methods
for noninvasive visualization of target proteins. Development of no-
vel imaging probes for GlyT1 is, thus, considered useful for obtaining
⇑
Corresponding authors. Tel./fax: +81 95 819 2443 (T.F.); tel./fax: +81 95 819
2441 (M.N.).
E-mail addresses: t-fuchi@nagasaki-u.ac.jp (T. Fuchigami), morio@nagasaki-u.ac.
jp (M. Nakayama).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.010

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T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
of SSR504734 derivatives. Hence, we present the synthesis of
Amino acid type
new GlyT1 ligands, 125I labeling, and preliminary in vitro and
in vivo binding characterization in rodents.
CF3
2. Results and discussion
2.1. Chemistry
O
O
N
CO2H
N
CO2H
F
F
The synthetic scheme of N-Me-SSR504734 (4) and 2-iodo-
substituted SSR504734 derivatives (3 and 5) were presented in
Scheme 1. (S)-{(S)-1-allylpiperidin-2-yl}(phenyl)methanamine (1)
was prepared according to the literature.18 General coupling reac-
tion of 1 with 3-trifluoromethyl-2-iodobenzoic acid resulted in 71%
yield of amide 2. Subsequent Pd(0) catalyzed deprotection of allyl
group provided compound 3 (88% yield). Finally, reductive amina-
tion of SSR50473418 and 3 yielded 94% of N-Me-SSR504734 (4) and
95% of compound 5.
Org24461
ALX5407
Nonamino acid type
N
H
O
N
O
CF3
HN
O
2.2. [3H]glycine uptake assay
Cl
N
N
F
SO2Me
Assays for the inhibitory activities of GlyT1 were performed
based on the reported method utilizing [3H]glycine uptake into
JAR cells.19 The inhibitory activity of each compound was ex-
pressed as IC50 values as shown in Table 1. Whereas SSR504734
showed modest inhibitory activity (IC50 = 80.1 nM), its N-methyl
derivative 4 showed 11-fold higher activity (IC50 = 7.51 nM). These
results are consistent with the literature; wherein, GlyT1 ex-
pressed in CHO cells resulted in the respective IC50 of 314 and
2.5 nM for SSR504734 and 4.16 Introduction of an iodine atom into
the X-position of SSR504734 resulted in the improvement of inhib-
itory activity (3, 43.0 nM). Notably, compound 5 exhibited excel-
lent inhibitory activity (IC50 = 2.4 nM), which was 18-fold and
triplicate higher than 3 and 4, respectively. These results indicate
the importance of the methyl group in R-position for GlyT1 inhibi-
tion and the iodine atom is preferred over the chloride atom in the
X-position to manifest strong inhibitory activity. On the other
hand, a first generation GlyT1 inhibitor, ALX5407, showed sixfold
higher inhibitory activity than 5 (IC50 = 0.39 nM). This extraordi-
nary potent inhibitory activity of ALX5407 correlates with the
result of a previous report (IC50 = 0.3 nM).20
CF3
F3C
SSR504734
RG1678
Figure 1. Chemical structure of GlyT1 selective inhibitors.
information about such diseases and occupancy of GlyT1 inhibitors
in vivo. Most recently, several nonamino acid type 11C or 18F radio-
tracers were developed as PET potential ligands for GlyT1 as shown
in Figure 2. The [18F]MK-6577 and [11C]GSK931145 had been dem-
onstrated to be promising GlyT1 imaging agents because of good
blood–brain barrier permeability, consistent accumulation with
GlyT1 expression, and displacement to homogenous level by GlyT1
ligands in the pig or rhesus monkey brain in vivo.12,13 Furthermore
[
11C]GSK931145 was applied for human PET studies which resulted
in similar bindingproperty with nonhuman brain. However, this tra-
cer showed unsatisfactory signal-to-noise ratio and reproducibility
because of significantly lower plasma free fraction and K1 value in
humans.14 On the other hand, SSR504734 and its derivatives have
been reported that selective and competitive inhibitors of GlyT-1 re-
cently.15,16 Since these compounds have molecular of approxi-
mately 400 and possess moderate lipophilicity, they may be useful
lead compounds for the development of imaging agents. In a most
recent report, SSR504734-related radioligands such as [11C]SA1
showed high brain uptake and consistent accumulation with GlyT1
distribution in the monkey brain; however, the total volume of dis-
tribution (Vt) was not significantly decreased by GlyT1 ligands.17
The clinical usefulness of recently developed PET ligands re-
mains to be investigated. The need to develop new imaging agents
with optimal in vivo binding property still arises. In addition, there
is still no SPECT imaging agent for GlyT1 reported. Accordingly, the
aim of this study was to develop new SPECT imaging agents;
wherein, a radioiodine atom was introduced into the 2-position
2.3. Radiochemistry
According to the [3H]glycine uptake assay, this study investi-
gated the potential of radio-iodinated 5 as a candidate SPECT imag-
ing agent for GlyT1. Preparation of [125I]5 is shown in Scheme 2.
The synthesis of [125I]5 from tributyltin precursor was initially
carried out; however, radioiodination of the tributyltin precursor
did not proceed. The reason was probably due to steric hindrance
of the precursor (data not shown). Therefore, trimethyltin precur-
sor 6 was prepared from compound 5 by an iodo-to-trimethyltin
exchange reaction (67% yield). Then, [125I]5 was successfully
synthesized by the reaction of 6 and [125I]NaI (carrier-free) in the
O
Cl
S
H
H3C
N
18F
N
N
H
N
H311C
N
HN
O
H311C
O
Cl
N
Cl
O2S
CF3
11C]SA1
[
11C]GSK931145
[
18F]MK-6577
Figure 2. Chemical structure of PET probes for GlyT1.
[

T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
6247
N
H
N
HN
O
HN
O
a
b
N
I
I
NH2
1
CF3
CF3
2
3
N
N
H
HN
O
HN
O
c
X
X
CF3
CF3
4: X= Cl
5: X= I
SSR504734: X= Cl
3: X= I
Scheme 1. Preparation of N-{phenyl (piperidin-2yl)methyl}benzamides (4 and 5). Reagents and conditions: (a) 3-trifluoromethyl-2-iodobenzoic acid, HOAt, EDC,
triethylamine, DMF, rt, 3 h, 71%; (b) 1,3-dimethylbarbituric acid Pd(PPh3)4, CH2Cl2, 35 °C, 2 h, 88%; (c) HCHO, NaBH(OAc)3, EtOH, rt, 6–8 h, 94% for compound 4, 95% for
compound 5.
presence of chloramine-T and HCl at 60 °C for 40 min. The radio-
chemical yield based on [125I]NaI was 13–19% and the radiochem-
ical purity was >98%.
Table 1
IC50 value of compounds calculated from inhibition assay of [3H]glycine uptake in
GlyT1 expressing cells (JAR cells)
2.4. In vitro binding characterization
N
R
In order to investigate the binding property of [125I]5, saturation
assays in the rat cortical brain homogenates were carried out. The
saturation curve and scatchard plot are shown in Figure 3. The
binding of [125I]5 was calculated to be a well-fitted one binding site
model (R2 = 0.985) with high affinity (Kd = 1.58 0.34 nM). The
Bmax value was estimated to be 3.40 0.18 pmol/mg protein which
is consistent with the results of other radioligands for GlyT1.21,22
To characterize in vitro regional brain distributions of [125I]5,
autoradiography experiments using the rat brain sections were
performed. As shown in the autoradiogram in Figure 4, [125I]5
showed high accumulation in the corpus callosum, thalamus, mid-
brain, medullary, and cerebellar white matter. On the other hand,
low uptake of [125I]5 was observed in the cerebral cortex, hippo-
campus, striatum, and cerebellar gray matter (Fig. 4A and C). It
should be noted that the accumulation pattern of [125I]5 was quite
similar to the distribution of GlyT1 immunoreactivity in the rat
brain.23 Nonspecific binding was determined in the presence of
HN
O
X
CF3
Compounds
R
X
Inhibition of [3H]glycine uptake (IC50, nM)
hGlyT1c/CHO cellsa
JAR cellsb
SSR504734
3
4
5
H
H
CH3
CH3
Cl
l
Cl
l
314
80.1 7.55
43.0 4.12
7.51 1.91
2.35 0.35
0.39 0.05
2.5
ALX5047
Data from Ref. 16. The assay was inhibition study of [3H]glycine uptake to
a
recombinant mammalian cells with stable expression of human GlyT1c (hGlyT1c/
CHO cells).
Inhibition assay of [3H]glycine uptake was carried out using JAR cells. IC50
b
values are averages of at least three measurements. Values were presented as
mean SEM.
corresponding nonradioactive 5 (10 lM), which resulted in a
N
N
N
a
b
HN
O
HN
O
HN
O
125I
I
SnMe3
CF3
CF3
CF3
[
125I]5
5
6
Scheme 2. Preparation of [125I]5. Reagents and conditions: (a) (Me3Sn)2, (Ph3P)4Pd, triethylamine, dioxane, 110 °C, 67%, (b) [125I]NaI, chloramine-T, HCl, EtOH, 80 °C, 40 min,
13–19%.

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T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
cerebellum (21ꢀ27% and 20ꢀ29% compared with control, respec-
tively). In contrast, a relatively lower reduction of radioactivity
was observed from the GlyT1-poor regions, such as the cerebral
cortex, hippocampus, and striatum (32ꢀ45% and 32ꢀ46%, respec-
tively). HPCP, which has a different structure from [125I]5, also
showed strong inhibition of [125I]5 binding to GlyT-rich regions
(11ꢀ23% compared with control) and moderate reduction of radio-
activity accumulation in GlyT1-poor regions (31ꢀ56%). Treatment
with ALX5407 decreased [125I]5 binding in all brain regions; how-
ever, the extent was lower than other GlyT1 inhibitors. The reason
might be caused by allosteric inhibition of ALX5047 to the [125I]5
binding for GlyT1. Indeed, it is reported that the SSR504734 deriv-
atives have been identified as competitive inhibitors of GlyT1;
whereas, ALX5407 exhibits a noncompetitive GlyT1 inhibitory
activity.16 While 1 mM of glycine had almost no effect on the
[
125I]5 binding, the increase to 10 mM caused reduction of accu-
Figure 3. Saturation curve and scatchard plot of [125I]5 binding to rat forebrain
membranes. Kd and Bmax values were determined by saturation analysis using
increasing concentrations of [125I]5 (0.1–20 nM). Nonspecific binding was deter-
mulated radioactivity in all brain regions. These results suggest
that compound 5 is a competitive inhibitor to the glycine binding
site of GlyT1 and the binding affinity of glycine to the [125I]5 bind-
ing site is much lower than the GlyT1 inhibitors. This binding prop-
erty is similar to 4 (N-Me-SSR504734) as reported previously.16
ORG25543 and L-701,324 did not affect the [125I]5 binding in all
brain regions.
mined with 10 lM of unlabeled 5.
significant reduction of radioactivity accumulation in the whole
brain compared with total binding. Additionally, regional differ-
ences in the [125I]5 binding disappeared in the brain regions
(Fig. 4B and C). These results indicate that the specific binding of
The results of in vitro experiments indicate that [125I]5 selec-
tively binds to GlyT1. Therefore, further in vivo studies were per-
formed in order to validate the effectiveness of radio-iodinated 5
as a SPECT imaging agent for GlyT1.
[
125I]5 in the rat brain slices correlates with GlyT1 expression level.
In order to investigate the specificity of [125I]5 binding to GlyT1,
in vitro inhibition studies were performed using GlyT1 inhibitors
[SSR504734, 4 (N-Me-SSR504734), ALX5047, and HPCP {(+)-N-
[cis-1-(2-Hydroxy-2-phenyl-cyclohexyl)-piperidin-4-yl]-4-meth-
oxy-N-phenyl-benzenesulfonamide: potent GlyT1 inhibitor24}] and
2.5. In vivo studies
The biodistribution studies of [125I]5 were carried out using nor-
mal mice and the results are shown in Table 3. High pulmonary up-
take of [125I]5 was observed at all points in time (9.34ꢀ17.19%
dose/g), which could be attributed to the piperidine moiety of this
tracer. Initial brain uptake of [125I]5 was 0.51% dose/g at 0.5 min
after intravenous injection. Radioactivity of [125I]5 in the brain
tissue increased with time and peaked to 2.42% dose/g at 30 min,
a
GlyT1 substrate (glycine), as well as a GlyT2 inhibitor
(ORG25543) and an NMDA receptor glycine site antagonist
(L-701,324) as shown in Table 2. In the presence of SSR504734
and its N-methyl derivative 4 (10 lM), the regional accumulation
of [125I]5 was dramatically decreased in the GlyT1-rich regions,
such as the corpus callosum, thalamus, midbrain, medullary, and
Figure 4. Autoradiograms of in vitro total binding of [125I]5 (A), nonspecific binding of [125I]5 (B), were visualized. The quantified values of [125I]5 in the cerebral cortex (CTX),
corpus callosum (CC), hippocampus (HIP), thalamus (THA), midbrain (MID), medullary (MED), and cerebellum (CB) were expressed as (PSL-BG)/mm2 (mean SD, n = 4–5
from at least three rats). (C), Nonspecific binding was determined in the presence of corresponding nonradioactive 5 (10
(Mann–Whitney U-test).
l
M). ⁄P <0.05 in comparison to the control group

T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
6249
Table 2
Effects of drugs on in vitro binding of [125I]5 to rat brain slices
Drugs
Concentration (mM)
% Control binding of [125I]5b
STR THA
CTX
CC
HIP
MID
MED
CB
GlyT1
Inhibitor
SSR504734
4 (N-Me-SSR504734)
HPCPa
0.01
0.01
0.01
0.01
29.3
37.5
31.0
57.6
21.8
20.2
19.7
27.8
46.3
50.2
29.6
28.9
31.7
20.6
20.5
25.3
27.7
45.8
55.9
63.2
32.2
31.8
46.7
25.1
22.5
33.2
25.3
19.7
36.2
28.9
23.0
43.1
ALX5407
Substrate
Glycine
1
10
89.6
43.7
94.1
39.6
107.6
71.5
121.5
47.6
107.6
52.7
105.8
52.1
104.1
61.2
97.4
44.9
GlyT2
ORG25543
0.01
0.01
95.1
92.5
98.2
92.5
104.9
90.9
103.6
96.8
99.7
87.1
115.0
81.3
91.4
123.8
87.1
NMDA receptor glycine site
L-701324
104.1
a
HPCP; (+)-N-[cis-1-(2-Hydroxy-2-phenyl-cyclohexyl)-piperidin-4-yl]-4-methoxy-N-phenyl-benzenesulfonamide.24
% Contro binding was calculated from average values obtained by at least triplicate slices from three rats.
b
Table 3
Biodistribution data for [125I]5 in normal ddY micea
Organ
0.5 min
5 min
15 min
30 min
60 min
180 min
Blood
Liver
Kidney
Intestine
Spleen
1.35 0.37
1.82 0.62
4.05 1.14
0.81 0.24
0.97 0.25
17.19 3.91
0.83 0.33
1.18 0.43
8.19 1.58
0.51 0.16
0.38 0.05
0.87 0.22
9.25 1.98
10.71 2.20
2.47 0.60
5.11 1.27
19.30 4.14
3.30 0.73
6.40 1.60
5.17 0.59
1.33 0.15
1.57 0.25
0.69 0.09
11.51 1.85
8.86 1.96
3.25 0.64
5.50 0.68
15.24 2.03
5.22 1.81
7.93 1.42
3.38 0.59
1.68 0.30
2.42 0.18
0.90 0.17
14.74 1.93
10.22 1.00
5.76 1.21
7.68 1.59
17.07 3.03
8.66 1.75
12.02 1.09
3.95 0.45
2.17 0.40
2.46 0.41
0.80 0.09
16.04 1.53
8.27 0.50
9.59 1.06
5.86 1.17
12.09 2.47
8.26 2.97
9.51 1.13
2.79 0.30
1.85 0.21
2.32 0.12
0.85 0.15
15.05 3.49
6.97 1.73
17.13 1.82
3.41 1.15
9.34 3.82
12.73 4.64
4.84 1.21
1.79 0.38
0.73 0.23
0.85 0.15
Lung
Stomach
Pancreas
Heart
Brain
Brain to blood
a
[
125I]5 (11.1 kBq) was injected intravenously via tail vein into ddY mice (Male, 6 W, 30–35 g). Values were presented as mean SD (% dose/g, n = 5–6).
suggesting the good blood–brain barrier permeability of [125I]5.
Although brain uptake of
125I]5 gradually decreased after
relatively lower than the GlyT1-rich regions (Fig. 5A and C). Pre-
administration of 4 also caused a significant decrease of regional
radioactivity uptake in the thalamus (56% and 56% compared with
control at 30 and 60 min, respectively), midbrain (55% and 48%),
and medullary (59% and 47%); whereas, little displacement of
radioactivity from the hippocampus and striatum was observed
as shown in Figure 5A and C. The estimated brain regions to blood
ratios were also significantly decreased by nonradioactive 5 and
compound 4 in the brain regions with high GlyT1 density at 30
and 60 min (Fig. 5B and D). The radioactivity level of [125I]5 in all
brain regions became homogenous by these blockers both at 30
and 60 min in agreement with the in vitro autoradiography exper-
iments. These results indicate that in vivo binding of [125I]5 is spe-
cifically mediated by GlyT1. When SSR504734 was administered as
pretreatment, the uptake of [125I]5 reduced slightly but the change
was not significant in all brain regions both at 30 and 60 min
(Fig. 5A and C), in which the reason could be due to the much lower
inhibitory activity (Table 1) and binding affinity16 for GlyT1 of
SSR504734 than its N-methyl analogs, compounds 4 and 5. Pre-
treatment with ALX5407 showed almost no effect to the regional
brain uptake of [125I]5 at 30 min. On the other hand, the brain up-
take of [125I]5 was decreased at 60 min; however, the reduction
was not significant in most regions. The slow and weak inhibitory
effects of ALX5407 for [125I]5 binding agree with in vitro results
(Table 2).
[
30 min, brain to blood ratios remained at the same level at
60 min (2.46% and 2.32% dose/g at 30 and 60 min, respectively).
It is suggested that radiotracers with moderate lipophilicity
(logD7.4 values; 2.0–3.5) show optimal passive brain entry under
in vivo condition.25,26 Indeed, the experimental log D7.4 value of
[
[
[
125I]5 was 2.86 (data not shown); therefore, high brain uptake of
125I]5 can be partially explained in terms of ideal lipophilicity of
125I]5. Metabolisms of [125I]5 in the mice brain were analyzed by
radio-TLC of brain homogenates obtained at 5, 30, and 60 min
post-injection; and 92%, 90%, and 89%, respectively, of the parent
compounds remained unchanged (data not shown). Thus, metabo-
lism should have little or no influence on the in vivo brain distribu-
tion pattern of [125I]5.
Next, the specificity of regional brain binding of [125I]5 toward
GlyT1 was studied by blocking studies using nonradioactive 5,
compound 4, SSR504734, and ALX5407 (2 mg/kg, intravenously),
which were administered 15 min prior to injection of the radioli-
gand. Because brain/blood ratio of [125I]5 peaked at 30 and
60 min as shown in Table 3, regional brain distribution studies
were conducted at 30 and 60 min after the radiotracer injection.
The results of in vivo blocking studies are shown in Figure 5. Pre-
injection of nonradioactive 5 resulted in significant reduction of
[
125I]5 accumulation in the GlyT1-rich regions, such as the thala-
mus (45% and 58% compared with control at 30 and 60 min,
respectively), midbrain (48% and 51%), and medullary (53% and
52%). In contrast, the extent of reduction of [125I]5 uptake in the
GlyT1-poor regions, such as the hippocampus and striatum was
To investigate further in vivo binding property of [125I]5 to
GlyT1, we performed ex vivo autoradiography studies. Mice were
injected intravenously with [125I]5 and sacrificed 60 min after
administration. As shown in Figure 6, a high uptake of [125I]5

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T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
Figure 5. Effects of nonradioactive 5, 4 (N-Me-SSR504734), SSR504734, and ALX504734 of [125I]5 in mice at 30 or 60 min after injection. Drugs (2 mg/kg) were given as pre-
treatment by intravenous injection 15 min before [125I]5 (33.3 kBq) administration. Figure 4A and B represent the percentage of the injected dose per gram of tissue (% dose/g)
and organ to blood ratio of % dose/g at 30 min after injection, respectively. Figure 4C and D represent % dose/g and organ to blood ratio at 60 min after injection, respectively.
Values were presented as mean SD (% dose/g, n = 5–6). WB represents whole brain. ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001 in comparison to the control group (Kruskal–Wallis test,
post-test was Dunn’s test).
was clearly visualized in the GlyT1-rich regions (thalamus, mid-
brain, cerebellum, and medullary). On the other hand, the accumu-
lation was low in the GlyT1-poor regions (cerebral cortex,
hippocampus, and striatum). The low radioactivity of [125I]5 in
the corpus callosum was inconsistent with that shown in the
in vitro autoradiography studies. The in vivo results might be
due to the influence of regional blood flow in the brain. It is well
known that blood flow in the white matter, such as the corpus cal-
losum was lower than that of other brain regions.27,28 To further
confirm the in vivo specificity of [125I]5 for GlyT1, blocking studies
were performed in which nonradioactive 5 or compound 4 (2 mg/
kg body weight) was given as pretreatment before [125I]5 injection.
As shown in the autoradiogram images in Figure 6B and C, both 5
and 4 abolished the heterogenous distribution of [125I]5 in the
brain regions. Quantified accumulation of [125I]5 was significantly
reduced by these compounds only in the GlyT1-rich regions
(Fig. 6D). These results strongly suggest that [125I]5 could recognize
GlyT1 protein under in vivo conditions.
Since the accumulation of [125I]5 was dependent on GlyT1
expression and specific binding to GlyT1, this tracer is expected
to be a promising SPECT probe for targeting GlyT1 in the living
brain. Additionally, the N-methyl group of 5 could be labeled with
carbon-11; therefore, compound 5 could be employed as a PET li-
gand. Further in vivo imaging studies of radio-labeled 5 are neces-
sary to clarify the potential of this compound as a clinical useful
imaging probe for GlyT1.

T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
6251
Figure 6. Ex vivo autoradiograms of [125I]5 in the sagittal sections of mouse brains at 60 min after injection (200 kBq). In the blocking studies, drugs (2 mg/kg) were given as
pre-treatment by intravenous injection 15 min before the tracer administration. (A) control [125I]5 binding; (B) [125I]5+ nonradioactive 5; (C) [125I]5 + 4; (D) the quantified
radioactivity of [125I]5 in the brain regions. The radioactivity level in each region was determined from the brain images (n = 5) and was expressed by as photostimulated-
luminescence (PSL)/mm2 region. ⁄P <0.05, ⁄⁄P <0.01, in comparison to the control group (Kruskal–Wallis test, post-test was Dunn’s test).
room temperature for 30 min. To this mixture, a solution of 118
3. Conclusion
(127 mg, 0.55 mmol) and triethylamine 430 (199 ll, 1.43 mmol)
In this study, [125I]5 was developed as a candidate SPECT ligand
for GlyT1. Compound 5 was discovered to possess potent GlyT1
inhibitory activity. In addition, [125I]5 showed consistent binding
with GlyT1 expression in the rodent brain and the accumulated
radioactivity was significantly decreased by GlyT1 inhibitors in
the GlyT1-rich regions both in vitro and in vivo. Altogether, the
radio-iodinated 5 could be a promising SPECT probe for neuroim-
aging of central GlyT1.
in DMF (2.5 mL) was added and stirred further for 3 h. The reaction
mixture was added aqueous NaHCO3 and ethyl acetate. The organic
layer was separated and washed by brine, dried with Na2SO4, and
evaporated to dryness. The crude product was chromatographed
on silica gel with hexane/EtOAc = 1:2 to provide 2 (205 mg,
0.388 mmol, 71%) as a white solid. 1H NMR (300 MHz CDCl3) d
7.67 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.42–7.28 (m, 6H),
5.78–5.65 (m, 1H), 5.12 (d, J = Hz, 16.8 Hz), 5.09 (d, J = Hz,
9.3 Hz), 4.96 (d, J = 6.6 Hz, 9.3 Hz), 3.33–3.18 (m, 2H), 3.05–2.98
(m, 1H), 2.87–2.79 (m, 1H), 2.62–2.53 (m, 1H), 1.85–1.73 (m,
4. Experimental
1H), 1.56–1.28 (m, 5H), ½a D20
ꢁ
= +16.2 (c = 1.0; CHCl3)}, MS (FAB)
m/z: 529 (M+).
4.1. General information
4.1.2. 2-Iodo-N-[(S)-{(S)-1-piperidin-2-yl}(phenyl)methyl]3-
trifluoromethyl-benzamide (3)
All reagents were commercially available products and were used
without further purification unless otherwise indicated. 1H NMR
spectra were obtained on a Varian Gemini 300 spectrometer with
TMS as internal standard. Mass spectra were obtained on JEOL IMS-
DX or JMS-T100TD instruments. HPLC analysis was performed on a
Shimadzu HPLC system (LC-20AT pump with a SPD-20A UV detector,
k = 254 nm). An automated gamma counter with a NaI(Tl) detector
(PerkinElmer, 2470 WIZARD2) was used to measure radioactivity.
(+)-N-[cis-1-(2-Hydroxy-2-phenyl-cyclohexyl)-piperidin-4-yl]-
4-methoxy-N-phenyl-benzenesulfonamide (HPCP) was prepared
according to the literature.24 ORG 25543 {4-Benzyloxy-N-[1-
(dimethylamino)cyclopentylmethyl]-3,5-dimethoxybenzamide}
was synthesized based on a reported procedure.29 [3H]glycine was
purchased from Morarek Biochemicals (Brea, CA, USA). [125I]NaI
was obtained by MP biomedicals (Costa Mesa, CA, USA).
To the stirred solution of compound 2 (172 mg, 0.33 mmol) in
CH2Cl2 (5 mL), 1,3-dimethylbarbituric acid (153 mg, 0.98 mmol)
and Pd(PPh3)4 (19.2 mg, 0.017 mmol) were added at room temper-
ature. The reaction mixture was stirred for 2 h followed by quench-
ing with saturated NaHCO3 solution, extracted with CH2Cl2,
washed by brine, dried with Na2SO4, and evaporated to dryness.
The crude product was chromatographed on silica gel with
CHCl3/MeOH = 10:1 to provide 3 (142 mg, 0.29 mmol, 88%) as a
white solid. 1H NMR (300 MHz CDCl3) d 7.68 (dd, J = 7.0, 2.3 Hz,
1H), 7.53–7.47 (m, 2H), 7.43–7.35 (m, 4H), 7.34–7.30 (m, 1H),
5.16 (dd, J = 7.5, 3.4 Hz, 1H), 3.00 (d, J = 11.0 Hz, 1H), 2.58–2.45
(m, 1H), 2.11–1.85 (m, 6H), 1.84–1.52 (m, 2H), 1.48–1.25 (m,
2H), ½a 2D3
ꢁ
= +61.9 (c = 0.75; CH3OH), HRMS (FAB) m/z: calcd for
C
20H21F3IN2O (M+) 489.0651, found 489.0660.
The experiments with animals were conducted in accordance
with our institutional guidelines and were approved by Nagasaki
University Animal Care Committee. All animals were supplied by
Tagawa Experimental Animal Laboratory (Nagasaki, Japan).
4.1.3. 2-Chloro-N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)-
methyl]3-trifluoromethyl-benzamide (4)
To the stirred solution of SSR50473418 (26 mg, 0.070 mmol) in
CH3CN (1 mL), 37% formaldehyde solution (19 ll, 0.246 mmol)
4.1.1. N-[(S)-{(S)-1-allylpiperidin-2-yl}(phenyl)methyl]-2-iodo
3-trifluoromethyl-benzamide (2)
and NaBH(OAc)3 (60 mg, 0.281 mmol) were added and the reaction
mixture was stirred for 3 h at room temperature. The mixture was
added with water and extracted with CHCl3. The organic layer was
washed by brine, dried with Na2SO4, and evaporated to dryness.
The crude product was chromatographed on silica gel with
2-Iodo-3-trifluoromethylacetic acid30 (228 mg, 0.72 mmol) in
DMF (2.5 mL) was mixed with EDC (138 mg, 0.72 mmol) and 1-hy-
droxy-7-azabenzotriazole (97 mg, 0.72 mmol), then stirred at

6252
T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
CHCl3/MeOH = 10:1 to provide 4 (29 mg, 0.065 mmol, 94%) as a
white solid. 1H NMR (300 MHz CDCl3) d 9.29 (s, 1H), 7.73 (d,
J = 7.6 Hz, 1H), 7.65 (d, J = 6.6 Hz, 1H), 7.48–7.27 (m, 6H), 5.12
(dd, J = 9.4, 8.7 Hz, 1H), 3.72–3.58 (m, 3H), 3.20–3.15 (m, 2H),
2.75 (s, 3H), 1.87–1.67 (m, 2H), 1.66–1.52 (m, 1H), 1.44–1.30 (m,
ice-cooled TB1A buffer (2.0 mL ꢂ 2). The cells were then solubi-
lized with 1.0 mL of 0.5 M NaOH solution and placed at room tem-
perature overnight. Radioactivity in the cells was measured by
liquid scintillation counting.
1H), ½a 2D3
ꢁ
= +46.8 (c = 1.0; CH3OH). HRMS (FAB) m/z calcd for
4.4. In vitro saturation assays
C
21H23ClF3N2O (M+) 411.1451, found 411.1482.
The in vitro saturation assays of [125I]5 to rat cortical membrane
4.1.4. 2-Iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)-
methyl]3-trifluoromethyl-benzamide (5)
homogenates were performed according to the method in the liter-
ature.21 In brief, well washed membranes (25
l
g protein) were
Using the above procedure for 4 starting from compound 3, the
title compound 5 (75 mg, 95%) was obtained as a white solid. 1H
NMR (300 MHz CDCl3) d 9.14 (s, 1H), 7.65 (t, J = 5.0 Hz, 1H),
7.51–7.47 (m, 2H), 7.43–7.30 (m, 5H), 5.10 (dd, J = 9.1, 8.0 Hz),
3.71–3.49 (m, 1H), 3.28–3.06 (m, 2H), 2.78 (s, 3H), 1.87–1.74 (m,
incubated with [ ll of TB1 buffer
125I]5 (0.1–20 nM) in 250
(120 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES,
pH 7.5) for 60 min at room temperature. Nonspecific binding was
defined by the addition of unlabeled 5 at 10 lM. Bound ligands
were collected by rapid filtration using a Brandel cell harvester
onto glass fiber filter (GF/B). The filters were washed rapidly three
times with 2.5 mL of ice-cold assay buffer. The radioactivity of fil-
ters was measured by automated gamma counter.
2H), 1.64–1.27 (m, 4H), ½a D23
ꢁ
= + 50.4 (c = 1.0; CH3OH). HRMS
(FAB) m/z calcd for C21H23F3IN2O (M+) 503.0807, found 503.0819.
4.1.5. N-[(S)-{(S)-1-Methylpiperidin-2-yl}(phenyl)methyl]-3-
trifluoromethyl-2-trimethylstannyl-benzamide (6)
4.5. In vitro autoradiography
A mixture of 5 (6.0 mg, 0.012 mmol), bis(trimethyltin) (13
ll,
0.060 mmol) and Pd(PPh3)4 (0.7 mg, 0.595 mol) in a mixed sol-
l
In vitro autoradiography studies were carried out according to the
vent (1.5 mL, 2:1 dioxane/triethylamine mixture) was stirred for
4 h under reflux. The solvent was removed, and the residue was
purified by silica gel chromatography CHCl3/MeOH = 10:1, which
gave 6 (4.4 mg, 0.008 mmol, 67%) as a colorless oil.
previous reports.31 The brain sagittal sections (20
lm)werepre-incu-
bated for 20 min at 25 °C in binding buffer (120 mM NaCl, 2 mM KCl,
1 mM MgCl2, 1 mM CaCl2, and 50 mM Tris–HCl, pH 7.5). These were
subsequently incubated in the same buffer containing
[
125I]5
1H NMR (300 MHz CDCl3) d 8.97 (s, 1H), 8.28 (d, J = 7.4 Hz, 1H),
7.52–7.46 (m, 3H), 7.39–7.29 (m, 3H), 5.12 (d, J = 7.4 Hz, 1H), 3.81–
3.70 (m, 1H), 3.67–3.63 (m, 1H), 3.48–3.30 (m, 3H), 2.78 (s, 3H),
(100 kBq, 0.03–0.04 nM) at 25 °C for 30 min. The slices were rinsed
triplicate for 3 min each with cold (5 °C) in washing buffer (120 mM
NaCl, 50 mM Tris–HCl, pH 7.5), and subsequently dipped into cold
water. The sections were dried under cold air and placed in contact
with 125I-sensitive imaging plates (BAS-SR 2040; Fuji Photo, Film, To-
kyo, Japan) for 3 days. Distributions of radioactivity on the plates
were analyzed byBio-ImageAnalyzer(FLA-5100;FujiPhoto, Film, To-
kyo, Japan), which were photographically visualized as shown in Fig-
ure 4. Regions of interest (ROIs) on the slices were placed on the
cerebral cortex, hippocampus, striatum, thalamus, midbrain, medul-
lary, and cerebellum. The radioactivities in these regions were ex-
pressed as photostimulated luminescence (PSL) values on ROI-
background PSL value per square millimeter. The specific binding
was determined as the difference between total binding and binding
1.97–1.51 (m, 2H), 1.53–1.26 (m, 2H), ½a D23
ꢁ
= +10.5 (c = 0.4; CHCl3),
MS (DART) m/z 541.09 (M+).
4.2. Radiosynthesis of [125I]5
An aqueous solution of chloramine-T (20
added to a mixture of [125I] NaI (5–10
l, 18.5–37.0 MBq, carrier-
free), 1 M HCl (50 l), and trimethylstannyl precursor 6 (0.2 mg
in 100 l of ethanol) in a sealed vial. The mixture was heated to
60 °C for 40 min and then quenched with 10% aqueous sodium
bisulfite (100 l) and saturated NaHCO3 (500 l). The mixture
was extracted with EtOAc (500
l ꢂ3) was added to the mixture
ll, 15 mg/mL) was
l
l
l
l
l
l
in the presence of the correspondingnonradioactive5 (10
tivity studies were performed as similar procedure in the presence of
several drugs (10 M) except for glycine (1 or 10 mM).
lM). Selec-
and dried under N2 gas. The crude products were purified by HPLC
(column; Nacalai Cosmosil 5C18-AR II, 4.6 ꢂ 250 mm, mobile
phase; CH3CN/0.1% TFA in H2O = 35:65, flow rate: 1.0 mL/min).
Radiochemical purity was assayed to be >98% by HPLC. The radio-
chemical yields based on [125I] NaI were 13–19%.
l
4.6. In vivo experiments
The [125I]5 (0.1 mL, ca. 11.1 kBq for whole body distribution
studies, ca. 33.3 kBq for regional brain distribution studies) was in-
jected intravenously via the tail vein into ddY mice (male, 6 W, 30–
35 g). At the designated time intervals, the mice were killed and
the organs were dissected. The brain was further divided into the
cerebral cortex, corpus callosum, hippocampus, thalamus, mid-
brain, medullary, and cerebellum.
The tissues were then weighed and the radioactivity was mea-
sured by automated gamma counting. Data were calculated as the
percentage of the injected dose per gram of tissue (% dose/g). To as-
sess the in vivo specific binding of [125I]5, the nonradioactive 5 or
GlyT1 antagonists SSR504734, 4(N-Me-SSR504734), and ALX5407
(2 mg/kg in 30% DMSO/saline) were given as pre-treatment by
intravenous injection 15 min before [125I]5 administration. Mice
were killed 30 or 60 min after injection.
4.3. [3H]glycine uptake assay
[3H]Glycine uptake assay was carried out according to the
method in the literature19 with minor modification.
Human placental choriocarcinoma cells (ATCC No. HTB-144)
were obtained from the American Type Culture Collection. JAR cells
were cultured in six-well plates in RPMI 1640 medium containing
10% fetal bovine serum. Cells were plated at a density of 4.0 ꢂ 105
cells/well and grown at 37 °C in a humidified atmosphere of 5% CO2
for 48–60 h.
The culture medium was removed from the six-well plates and
the JAR cells were incubated with 1.0 mL of TB1A buffer (120 mM
NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 5 mM L-
alanine, pH 7.5 adjusted with 1 M Tris base) with or without drugs
for 1 min. Then, 1.0 mL of [3H]glycine (96.2 kBq, 4.8 GBq/mmol) in
TB1A buffer was added to each well to give a final concentration of
10 lM. Nonspecific uptake was defined by the addition of unla-
beled glycine at 10 mM. After incubation at room temperature
For ex vivo autoradiography studies, mice were injected intra-
venously with 200 kBq of [125I]5 and were sacrificed 60 min post-
injection. For the inhibition experiment, nonradioactive 5 or 4
(2 mg/kg) was injected by intravenous injection 15 min before
for 2 h, the incubation medium was removed and rinsed with
[
125I]5 administration. The whole brain was quickly removed and

T. Fuchigami et al. / Bioorg. Med. Chem. 19 (2011) 6245–6253
6253
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Acknowledgments
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