Synthesis and biological evaluation of radio-iodinated benzimidazoles as SPECT imaging agents for NR2B subtype of NMDA receptor

Article abstract of DOI:10.1016/j.bmc.2010.08.053

In this study, the benzimidazole derivatives were synthesized and evaluated as imaging agents for the NR2B subtype of NMDA receptor. Among these ligands, 2-{[4-(4-iodobenzyl)piperidin-1-yl]methyl}benzimidazol-5-ol (8) and N-{2-[4-(4-iodobenzyl)-piperidin-

Full text of DOI:10.1016/j.bmc.2010.08.053

Bioorganic & Medicinal Chemistry 18 (2010) 7497–7506
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of radio-iodinated benzimidazoles
as SPECT imaging agents for NR2B subtype of NMDA receptor
Takeshi Fuchigami a,c, Hiroshi Yamaguchi b, Mikako Ogawa a, Le Biao a, Morio Nakayama c,
Mamoru Haratake c, Yasuhiro Magata a,b,
⇑
a Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
b Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
c Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Revised 27 August 2010
Accepted 28 August 2010
Available online 25 September 2010
In this study, the benzimidazole derivatives were synthesized and evaluated as imaging agents for the
NR2B subtype of NMDA receptor. Among these ligands, 2-{[4-(4-iodobenzyl)piperidin-1-yl]methyl}ben-
zimidazol-5-ol (8) and N-{2-[4-(4-iodobenzyl)-piperidin-1-ylmethyl]benzoimidazol-5-yl}-methanesul-
fonamide (9) exhibited high affinity for the NR2B subunit (Ki values; 7.28 nM for 8 and 5.75 nM for 9).
In vitro autoradiography experiments demonstrated high accumulation in the forebrain regions but
low in the cerebellum for both [125I]8 and [125I]9. These regional distributions of the radioligands corre-
lated with the expression of the NR2B subunit. The in vitro binding of these ligands was inhibited by
NR2B antagonist but not by other site ligands, which suggested the high selectivity of [125I]8 and
Keywords:
NMDA receptor
NR2B subtype
Benzimidazole
SPECT
[
125I]9 for the NR2B subunit. In mice, the regional brain uptakes of [125I]8 and [125I]9 at 5–180 min after
administration were 0.42–0.56% and 0.44–0.67% dose/g, respectively. The brain-to-blood ratio of [125I]8
at 180 min was reduced by 34% in the presence of non-radioactive ligands and by 59% in the presence
of the NR2B ligand Ro-25,6981. These results indicated that [125I]8 could be partially bound to the
NR2B subunit in vivo. Although the brain uptake of these benzimidazole derivatives was too low to allow
for in vivo SPECT imaging, these compounds might be useful scaffolds for the development of imaging
probes specific for the NMDA receptors.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
subunits in the adult forebrain.8 It is suggested that NR2A-contain-
ing NMDA receptors are involved in prosurvival signaling; thereby,
The N-methyl-
D
-aspartate (NMDA) receptor, a member of the
they exert a neuroprotective action against glutamate receptor-
dependent apoptotic neuronal injuries.9–11 On the other hand, an
increasing number of reports have demonstrated the importance
of the NR2B subunit in determining the pharmacological and func-
tional properties of the NMDA receptor. The NR2B has been impli-
cated in modulating functions, such as learning, memory
processing, and feeding behaviors, as well as being involved in a
number of human disorders.12 It is suggested that activation of
the NR2B-containing NMDA receptor initiates apoptotic signaling
cascades and promotes neuronal death.9–11 Recent progress in
molecular biology revealed the in vitro function of the NR2B-con-
taining NMDA receptor considerably. However, its function in the
living brain is not well understood due to the lack of visualizing
method in vivo. Positron emission tomography (PET) and single
photon emission computed tomography (SPECT) are the most effi-
cient imaging methods for in vivo measurement of neurotransmit-
ter receptors and enzymes in the brain. Our study, as well as other
studies, has developed several radioligands for the NR2B subunit of
the NMDA receptor (Fig. 1). These ligands showed similar accumu-
lation pattern with the localization of expression of the NR2B
glutamate receptors, plays an important role in the neurotransmis-
sion function of the central nervous system (CNS).1 Overactivation
of the NMDA receptor, however, is thought to cause various disor-
ders, such as ischemia, stroke, Parkinson’s Disease, Alzheimer’s
Disease, Huntington’s Disease, and schizophrenia.2–6 NMDA recep-
tors are heteromers, which contain an NR1 subunit and one or
more types of the different NR2 subunits (NR2A-D).7 NR1 subunit
has only one gene (eight splice variants) with ubiquitous distribu-
tion in the brain. NR2 subunits, on the other hand, exhibit distinct
distribution in the brain, which determine the synaptic localization
and function of the NMDA receptor. The NR2A subunit is widely
distributed throughout the brain; but, the NR2B subunit is con-
fined in the forebrain region, including the cerebral cortex, hippo-
campus, and olfactory bulb. The NR2C and NR2D are localized in
the cerebellum and diencephalus/lower brain stem, respectively.
The NR2A and NR2B are known to be the predominant NR2
⇑
Corresponding author. Tel./fax: +81 53 435 2398.
E-mail address: magata@hama-med.ac.jp (Y. Magata).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.053

7498
T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
O
OH
OH
NH
O
11CH3
O11CH3
O
*
N
N
O
N
N
H
H
HO
*= 11
C
F
11C]CP-101,606 derivative
[
11C]Benzylamidine derivative
[
11C]EMD-95885
[
Figure 1. Structure of radioligands for imaging of the NR2B subtype of the NMDA receptor.
subunit only under in vitro condition but not in vivo.13–19 Among
these ligands, only [11C]EMD-95885 showed significant reduction
of the in vivo brain uptake by treatment with the NR2B ligand.16
Although the inhibition pattern was inconsistent with NR2B
expression, this chemical structure might be a useful lead for the
development of potential imaging probes. Recently, a new series
of benzimidazole derivatives 1 and 2 are developed as novel
NR2B antagonists (Fig. 2),20 which have similar chemical structures
with EMD-95885. For example, compound 1a showed excellent
affinity for the NR2B subunit (Ki = 1.5 nM). Introduction of a hydro-
phobic substituent into the R2-position of benzimidazoles, such as
compound 1, maintained receptor affinity.20–22 On the bases, we
designed new SPECT imaging agents, which introduced 125I atom
into the R2-position of benzimidazoles. In this study, [125I] labeled
benzimidazole derivatives were synthesized and the in vitro bind-
ing properties and in vivo brain uptake characteristics in rodents
were examined. It is reported that 11C labeled 4-acetoxy derivative
of L-703,717, which is a PET ligand for glycine site of NMDA recep-
tor, can be a substrate of P-gp.23 However it is not clear if the NR2B
ligands are substrates of P-gp. Some of NR2B ligands such as ifen-
prodil and benzidazoles in this series have acidic phenol group. It
has been shown that organic anion transporter is responsible for
the elimination of several acidic drugs from the brain across the
BBB.24 Therefore the effect of P-gp and OAT on the in vivo uptake
of [125I]benzimidazoles were also investigated.
synthesized as shown in Scheme 1. Alkylation of compound 325 with
ethyl bromoacetate gave 4 (71% yield), followed by a bromo-to-tri-
butyltin exchange reaction gave tributyltin derivative 5 for yields
of 53%. Compound 5 was allowed to react with iodine in CCl4 to give
the iodo derivative 6 (68% yield). Compound 6 was hydrolyzed, fol-
lowed by general amide coupling reaction with the corresponding
phenylenediamines. Dehydration of the amidated products in acetic
acid at reflux gave the benzimidazoles 7 and 9 for yields of 55% and
53%, respectively. Compound 7 was converted to 8 by desmethyla-
tion with HBr (63% yield). The target iodo phenoxypiperidine deriv-
ative (13) was synthesized as shown in Scheme 2. Phenoxypi
peridine 11 was synthesized according to the method of Scheme 1
starting from compound 1026 for yield of 45%. Benzimidazole 12
was obtained by the same procedure for the synthesis of compound
7 for yield of 73%. Finally, compound 12 was desmethylated by BBr3
to give 13 (77% yield).
2.2. In vitro binding assays
The binding affinities (Ki values) of benzimidazole derivatives
for the NR2B subunit of NMDA receptor were evaluated with dis-
placement studies of the typical NR2B antagonist, [3H]ifenprodil,
to rat cortical synaptic membranes according to the literature.27
Trifluoroperazine was used to block the binding of [3H]ifenprodil
in rat brain membranes to sites other than the NMDA receptors.
As shown in Table 1, lead compound 1a showed excellent affinity
(Ki = 3.09 nM) for the NR2B subunit, which was consistent with
the results by using hNR1a/NR2B receptors expressed in Ltk-cells
(Ki = 1.50 nM) reported in the literature.20 Introduction of an iodine
atom in R2 resulted in a slight reduction of binding affinity; how-
ever, high affinity for the NR2B subunit (Ki values; 7.28 and
5.75 nM for 8 and 9, respectively) was maintained. On the other
hand, phenoxypiperidine benzimidazole 13 exhibited a 4.5-fold
lower affinity (Ki = 32.5 nM) than compound 8. These results sug-
gested that the presence of phenoxypiperidine frame, instead of
the benzylpiperidine, negatively influenced the in vitro binding
affinity for this class of compound. In the recent report, the in-
dole-carboxamide derivatives, with an oxygen atom substituted
in the X-position, have several-fold lower affinity than the com-
pounds with methylene group. On the contrary, comparable affin-
ities were shown between benzimidazole-carboxamide derivatives
substituted with methylene group and oxygen atom in X-posi-
tion.22 Therefore, it might be difficult to fit the binding space for
phenoxypiperidine derivatives in the NR2B antagonist binding site.
Since 8 and 9 had high affinity for the NR2B subunit of the NMDA
receptor, these ligands were selected to carry out further biological
evaluations on the corresponding I-125-labeled probes.
2. Results and discussion
2.1. Chemistry
Based on the previous structure–activity relationship studies,20–
22 the SPECT imaging agent candidates 8, 9, and 13 were designed, in
which an iodine atom was introduced into the 40-position (R2-posi-
tion) of benzyl- or phenoxy-piperidine group of benzimidazoles.
Lead compound 1a was synthesized according to the literature.20
The 40-iodo substituted benzylpiperidine derivatives (8 and 9) were
H
N
H
N
R1
R1
N
N
N
O
R2
1
2
1a; R1= OH, R2= H
2.3. Radiochemistry
R1= OH, NHSO2Me
R2= Me, F, Cl
Trimethyltin precursors 14 and 15 were synthesized from 8 and
9, respectively, by an iodo-to-trimethyltin exchange reaction (53%
yield for 14 and 97% yield for 15). To obtain [125I]8 and [125I]9, the
Figure 2. Structure of benzimidazoles as NR2B antagonists.

T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
7499
a
b
c
HN
EtO C
2
N
EtO C
N
2
Br
Br
SnBu
3
5
3
4
H
N
R
1
N
N
d
EtO C
N
2
I
I
6
R = OMe
7
8
9
1
e
R = OH
1
R = NHSO Me
1
2
Scheme 1. Preparation of benzylpiperidine derivatives (8 and 9). Reagent and conditions: (a) ethyl bromoacetate, diisopropylethylamine, DMF, rt, 71%; (b) (Bu3Sn)2,
(Ph3P)4Pd, toluene, 110 °C, 53%; (c) I2, CCl4, rt, 68%; (d) (1) HCl, 100 °C, (2) EDC, HOAt, triethylamine, 4-substituted phenylenediamine, DMF, rt, (3) acetic acid, 140 °C, 55% for
7, 53% for 9; (e) HBr/H2O, 100 °C, 63%.
H
MeO
N
O
O
c
b
a
N
N
EtO C
N
N
2
I
Boc
HO
I
O
I
10
11
12
H
N
N
N
O
I
13
Scheme 2. Preparation of phenoxypiperidine derivative (13). Reagent and conditions: (a) (1) TFA, CH2Cl2, rt, (2) ethyl bromoacetate, diisopropylethylamine, DMF, rt, 45%; (b)
(1) HCl, 100 °C, (2) EDC, HOAt, triethylamine, 4-substituted phenylenediamine, DMF, rt, (3) acetic acid, 140 °C, 73%; (c) BBr3, CH2Cl2, rt, 77%.
Table 1
corresponding trimethyltin precursors were allowed to react with
In vitro binding affinity (Ki) of new benzimidazole derivatives for the NR2B subtype of
the NMDA receptor and octanol/phosphate buffer distribution co-efficient (log D)
[
125I]NaI (carrier-free) in the presence of H2O2 and HCl at room
temperature for 40 min by a similar method in the literature28
(Scheme 3). The resulting I-125 labeled crude products were puri-
fied by HPLC. The radiochemical yields based on [125I] NaI were
85–90% and 78–81% for [125I]8 and [125I]9, respectively.
H
R1
N
N
N
2.4. In vitro autoradiography
X
R2
In vitro regional distributions of [125I]benzimidazoles on the rat
brain sections were evaluated by a method mentioned in the liter-
ature.13 As shown in the autoradiogram in Figure 3, both [125I]8
and [125I]9 showed high accumulation in the forebrain region
(cerebral cortex and hippocampus), which is an NR2B-rich region,
and lower uptake in the cerebellum, which is an NR2B-poor region
(Fig. 3A and B). These distribution patterns were similar to [3H]CP-
101,606,29 as well as to the mRNA distribution of the NR2B sub-
unit.30 Non-specific binding was determined in the presence of
c
Compounds
R1
R2
X
Kia (nM)
Kib (nM)
log D7.4
Ifenprodil
1a
8
9
13
20.0 4.89
3.09 0.22
7.28 2.93
5.75 1.19
32.5 16.3
OH
OH
NHSO2Me
OH
H
I
I
1.50
CH2
CH2
O
3.95
3.54
3.74
I
Data from Ref. 20. The assay was inhibition study of 3H-[(E)-N1-(2-methoxy-
benzyl)cinnamamidine] binding to hNR1a/NR2B receptors expressed in Ltk-cells.
The Ki values were obtained by the method in Ref. 25. Each value (mean SD)
was determined triplicate using rat cortical membrane homogenates in HEPES-KOH
buffer.
a
b
corresponding non-radioactive benzimidazoles (10 lM), which
resulted in a significant decrease in [125I]8 and [125I]9 accumula-
tions in the forebrain region compared with total binding as visu-
alized in Figure 3C and D and quantified in Figure 3E and F,
c
The partition co-efficient between n-octanol and sodium phosphate buffer at
pH 7.4 (log D7.4) was determined by conventional shake-flask method (n = 3).

7500
T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
H
N
H
H
N
R1
R1
N
R1
a
b
N
N
N
N
N
N
125I
I
SnMe3
125
R = OH
1
8
R = OH
1
[
I]8
I]9
R = OH
1
14
125
R = NHSO Me
9
R = NHSO Me
[
R = NHSO Me 15
1
2
1
2
1
2
Scheme 3. Preparation of [125I]8 and [125I]9. Reagent and conditions: (a) (Me3Sn)2, (Ph3P)4Pd, toluene, 110 °C, 53% for 14 and 97% for 15; (b) [125I]NaI, H2O2, HCl, EtOH, rt,
85–90% for [125I]8 and 78–81% for [125I]9.
respectively. Almost no specific binding of both ligands exists in
tor antagonist) in blocking these receptors in the inhibition studies
were examined. In result, the in vitro bindings of [125I]8 and [125I]9
in the cerebral cortex and hippocampus were inhibited only by the
NR2B selective antagonist, Ro-25,6981, as shown in Table 2. On the
contrary, no ligands inhibited the bindings in the cerebellum.
These results indicated that [125I]8 and [125I]9 selectively bind to
the NR2B subunit. Therefore, this study attempted to carry out
in vivo studies to validate the effectiveness of these [125I] benzim-
idazoles as SPECT imaging agents for the NR2B-subunit-containing
NMDA receptor.
the cerebellum. The specific binding of [125I]8 was higher than
[
125I]9 in the forebrain regions (Fig. 3E and F). These [125I]benzim-
idazoles showed similar binding property with C-11-labeled ana-
log of CP-101,606, which is a high-affinity radioligand for the
NR2B subunit.13 However, their specific bindings were lower than
the [11C]CP-101,606 analog. The high lipophilic property of [125I]8
and [125I]9 was attributed to. The log D7.4 values of 8 and 9 were
3.95 and 3.54, respectively, as shown in Table 1, which are higher
than the [11C] CP-101,606 analog (log P = 0.75).
In order to investigate the specific binding of [125I]8 and [125I]9
to the NR2B subunit of the NMDA ion channel, in vitro inhibition
studies on rat brain region using Ro-25,6981 (NR2B antagonist)
were carried out. Since it is reported that several ifenprodil-like
2.5. In vivo pharmacology
The biodistribution studies of [125I]8 and [125I]9 were performed
by using normal mice and the results were shown in Table 3. Initial
brain uptake of [125I]8 was 0.21% dose/g at 0.5 min after intravenous
antagonists bind to
a
1-adrenergic and
r
receptor,31,32 the effects
recep-
of prazosin ( 1-adrenergic receptor antagonist) and DTG (
a
r
Figure 3. Autoradiogram of in vitro total binding (TB) of [125I]8 (A), non-specific binding (NBS) of [125I]8 (B), total binding of [125I]9 (C), and non-specific binding of [125I]9 (D)
to rat brain sagittal sections. The quantified values of [125I]8 (E) and [125I]9 (F) in the cerebral cortex, hippocampus, and cerebellum were expressed as (PSL-BG)/mm2
*
(mean SD, n = 4–6). Non-specific binding was determined in the presence of corresponding non-radioactive benzimidazoles (10
group (Mann–Whitney U-test).
l
M). P <0.01 in comparison to the control

T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
7501
Table 2
Effects of drugs on in vitro binding of [125I]benzimidazoles to rat brain slices
Ligands
% control bindinga
[
125I]8
[
125I]9
Cerebral cortex
Hippocampus
Cerebellum
Cerebral cortex
Hippocampus
Cerebellum
Ro-25,6981
Prazosin
DTG
58.3
95.6
93.5
50.9
92.6
104.6
91.8
102.9
101.2
58.1
92.8
93.0
59.0
95.5
107.6
89.3
97.0
103.6
a
Average values obtained by at least triplicate slices from three rats.
injection; whereas, the uptake of [125I]9 was 0.32% dose/g at the cor-
responding time. Both ligands showed very low brain-to-blood ratio
(0.07) at 0.5 min. Radioactivity of [125I]8 and [125I]9 in the brain in-
creased with time and peaked at 180 min after administration
(0.56% and 0.67% dose/g, respectively). Accordingly, brain-to-blood
ratio of these ligands rose to 1.99 and 1.23 at 180 min, respectively.
High hepatic uptake of [125I]8 and [125I]9 was observed, which
peaked to 32.22% and 17.81% dose/g at 180 min, respectively. The
amount of radioactivity of these tracers in the kidney was also high
(5.35–12.20% and 3.26–15.23% dose/g, respectively). The distribu-
tion patterns in peripheral tissues could be attributed to the high
lipophilic property of these [125I]benzimidazoles. These ligands
showed almost homogenous brain distribution at 5–360 min after
intravenous administration (Fig. 4), which was inconsistent with
the localization of expression of the NR2B subunit. The results for
and Ro-25,6981 (3 mg/kg, intravenously), which were adminis-
tered 30 min prior to injection of the radioligand. Since brain up-
take of [125I]8 peaked at 180 min as shown in Table 3, regional
brain distribution at 180 min after the radiotracer injection was
determined. As shown in Figure 5, pre-injection of non-radioactive
8 resulted in a significantly increased blood radioactivity of [125I]8
(P <0.01) compared with the control group. Altogether, the liver
uptake was significantly decreased (P <0.01). On the other hand,
the brain uptake was increased slightly. Pre-administration of the
NR2B selective antagonist, Ro-25,6981, had no effect on blood
radioactivity of [125I]8. The hepatic uptake of [125I]8 was slightly
increased but the regional brain uptake was slightly decreased. It
should be noted that both groups treated with cold 8 and Ro-
25,6981 showed a markedly lower brain-to-blood ratio of [125I]8
than the control group (Fig. 5D). Considering these results, [125I]8
could have specific binding component in the NR2B subunit under
in vivo condition, which was similar to [11C]EMD-95885.16 How-
ever, both [125I]8 and [11C]EMD-95885 demonstrated reduced
radioactivity in the NR2B poor region, cerebellum, even in the pres-
ence of NR2B ligands, which was unexplainable.
[
125I]benzimidazoles were similar to other radioligands for the
NR2B subunit.13–16 Metabolisms of [125I]8 and [125I]9 in the mouse
brain were analyzed by radio-TLC of brain homogenates obtained
at 180 min post-injection; and 77% and 85%, respectively, of the par-
ent compounds remained unchanged (data not shown). Thus,
metabolism could have minimal effect the distribution pattern of
On the other hand, the brain uptake of commonly used PET
radioligands, such as [11C]raclopride and [11C]DASB, was higher
than [125I]8.33,34 Considering these facts, the brain permeability
of [125I]8 could be inadequate for the visualization of NR2B subtype
of the NMDA receptor by SPECT. All of the radioligands for NR2B
subunit including benzidazoles in this report were inadequate
BBB permeability. Log P (or log D7.4) values of [125I]8, [125I]9, and
[
125I]8 and [125I]9. The results of this study showed that in vitro spe-
cific binding and in vivo brain uptake to blood radioactivity ratio of
[
[
125I]8 were higher than [125I]9. Therefore, in vivo experiments on
125I]8 were further performed.
The specificity of regional brain binding of [125I]8 for the NR2B
subunit was studied by blocking studies using non-radioactive 8
Table 3
Biodistribution data for 125I labeled benzimidazoles in normal ddY micea
Organ
125I]8
0.5 min
5 min
30 min
60 min
180 min
360 min
[
Blood
Liver
Kidney
Intestine
Stomach
Spleen
Pancreas
Heart
3.22 0.58
14.68 3.50
5.35 0.91
4.02 0.64
0.72 0.16
0.50 0.32
0.49 0.13
1.47 0.29
6.60 0.95
0.21 0.03
0.07 0.02
1.15 0.06
25.03 4.20
12.20 3.18
1.83 0.33
1.93 0.40
5.66 1.25
5.74 2.45
5.39 0.33
14.48 2.52
0.42 0.06
0.36 0.03
0.67 0.11
28.99 6.31
11.20 2.65
2.39 0.79
2.54 0.81
5.91 1.25
6.10 1.42
2.31 0.48
8.16 2.37
0.44 0.07
0.67 0.12
0.75 0.13
30.50 6.60
10.34 1.60
2.35 0.47
2.16 0.24
4.65 0.51
6.35 1.25
1.87 0.41
7.54 2.19
0.48 0.09
0.64 0.06
0.30 0.11
32.22 6.56
7.74 1.32
4.55 1.03
2.51 0.55
4.25 0.69
6.53 1.95
1.58 0.16
4.75 0.33
0.56 0.06
1.99 0.57
0.28 0.11
30.16 1.83
5.46 0.67
5.89 1.05
2.17 0.75
3.79 0.43
8.76 0.37
1.58 0.11
3.44 0.80
0.55 0.14
2.23 0.99
Lung
Brain
Brain-to-blood ratio
[
125I]9
Blood
Liver
Kidney
Intestine
Stomach
Spleen
Pancreas
Lung
4.74 1.59
4.60 2.69
3.26 1.64
0.55 0.31
0.42 0.22
0.90 0.61
0.92 0.46
9.07 5.18
3.82 1.65
0.32 0.12
0.07 0.01
0.95 0.40
19.35 9.22
10.99 4.46
1.58 0.75
1.28 0.47
3.19 1.43
3.09 1.59
5.83 1.29
3.52 2.19
0.44 0.19
0.46 0.09
0.53 0.08
16.67 2.84
8.79 0.91
3.07 0.50
2.24 0.46
2.91 0.42
3.71 0.69
4.29 0.32
1.86 0.94
0.50 0.07
0.94 0.13
0.54 0.08
13.74 1.72
8.52 1.98
3.79 0.69
2.40 1.05
2.78 0.57
4.55 1.14
4.35 0.38
1.70 0.73
0.52 0.07
1.00 0.26
0.55 0.12
17.81 4.07
15.23 2.31
7.82 1.22
4.04 0.83
2.85 0.74
5.39 1.18
6.93 0.36
1.61 2.29
0.67 0.08
1.23 0.16
0.36 0.10
10.93 2.51
10.51 1.97
6.49 1.77
3.56 1.28
1.60 0.26
3.72 0.59
3.62 0.21
0.93 0.55
0.47 0.09
1.34 0.28
Heart
Brain
Brain-to-blood ratio
a
[
125I]Benzimidazoles (14.8 kBq) were 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).

7502
T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
30min
5min
60min 180min
360min
A
B
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Cerebral
cortex
Cerebral
cortex
Cerebellum
Cerebellum
Hippocampus
Hippocampus
Figure 4. Regional brain uptake of [125I]8 (A) and [125I]9 (B) in normal ddY mice. [125I]Benzimidazoles (148 kBq) were injected intravenously via mice tail vein.
11C labeled CP-101,606 analog were 3.95, 3.54, and 0.75.13 Log P
the brain uptake of [125I]8 is effluxed by OAT. Therefore, the effect
of P-gp and OAT to the in vivo brain uptake of [125I]8 was analyzed.
Pretreatment with CsA (Cyclosporine A) (substrate of P-gp,
50 mg/kg) or probenecid (substrate of OAT, 200 mg/kg) by intrave-
nous injection on mice tail vein 30 min before [125I]8 administra-
tion was followed by examination of biodistribution 180 min
later. As shown in Table 4, pretreatment with CsA resulted in the
reduction of blood radioactivity level of [125I]8 but increase in
the brain uptake in the CsA group compared with the control
group. Therefore, the brain-to-blood ratio was 1.6-fold higher than
the control group. Although P-gp is known to be present in the li-
ver and kidney,39 the effect of CsA to the uptake of [125I]8 in the li-
ver and kidney is nil. In contrast, pretreatment with probenecid
increased the brain uptake but decreased the blood radioactivity
level of [125I]8. The brain-to-blood ratio in the probenecid group
was 1.3-fold higher than the control group. The uptake of [125I]8
in the kidney was significantly reduced relative to the control
group. The hepatic uptake of [125I]8, on the other hand, demon-
values of 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydro-
benzoimidazol-2-[11C]one14 and
[
11C]EMD-9588516 were not
reported, however, calculated by ChemDraw Ultra to be 3.36 and
3.40, respectively. These ligands are not within the optimal range
(1.0–3.0) for compounds expected to penetrate the BBB.35 There-
fore log P value might be one of the reasons that the ligands
showed low brain uptake. Another reason was thought to be high
uptake of NR2B ligands in lung. The basic piperidine group in the
ligands might cause these accumulation proclivities. Alternatively,
lower molecular weight derivatives might be necessary to increase
BBB permeability.
Another likely reason for the low uptake could be the excretion
from brain tissue by the efflux systems of the BBB. It has been
shown that many radioligands for mapping neuroreceptors are
substrates for P-gp.36–38 It is reported that several acidic drugs
are eliminated by the OAT from the brain across the BBB.24 Since
the phenolic group in 8 has acidic moiety, there is a possibility that
Control
8
Ro,25,6981
2.5
2.0
1.5
1.0
0.5
0.0
40
30
20
10
0
*
*
Blood
Liver
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
*
*
*
Cerebral
cortex
Cerebral
cortex
Cerebellum
Hippocampus
Hippocampus
Cerebellum
Figure 5. Effects of non-radioactive 8 and Ro-25,6981 (NR2B antagonist) on the blood concentration (A), liver uptake (B), regional brain uptake (C), and brain tissue to blood
ratio (D) of [125I]8 in mice at 180 min after injection. Drugs (3 mg/kg) were given as pre-treatment by intravenous injection 30 min before [125I]8 (148 kBq) administration.
*
Values were presented as mean SD (% dose/g, n = 5). P <0.01 in comparison to the control group (Kruskal–Wallis test, post-test was Dunn’s test).

T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
7503
than normal region.51 In lieu with these reports, it is conjectured
that the different biodistribution patterns of radioligands for the
NR2B subtype of the NMDA receptor could be observed between
normal condition and overactivated state, such as pathological
stage.
Table 4
The modulation of BBB permeability of [125I]8 by P-glycoproteina
Control
Cyclosporin A (50 mg/kg)
Blood
Liver
Kidney
Brain
Brain/blood
0.78 0.27
16.20 1.34
4.49 1.34
0.50 0.04
0.74 0.41
0.52 0.26
15.20 5.71
4.80 0.69
0.66 0.22
1.16 0.36
Since [125I]8 and [125I]9 showed low brain uptake and homoge-
neous brain distribution, these ligands were unsuitable for SPECT
imaging of cerebral NMDA receptors. However, it should be noted
that [125I]8 may have specific binding component in the NR2B sub-
unit in vivo. The present study indicated that benzimidazole deriv-
atives may be potential lead compounds for developing novel
imaging agents for the NR2B subtype of NMDA receptors. Further
structure–activity relationship studies are required.
a
Regional organ uptake at 180 min after [125I]8 injection. Cyclosporin A (50 mg/kg)
was given as pre-treatment by intravenous injection 30 min before [125I]8 (148 kBq)
administration. Values were presented as mean SD (% dose/g, n = 5).
Table 5
The modulation of BBB permeability of [125I]8 by organic anion transportera
3. Conclusion
Control
Probenecid (200 mg/kg)
Blood
Brain
Liver
Kidney
Brain/blood
0.46 0.07
0.44 0.06
19.13 2.09
6.29 1.47
0.98 0.13
0.41 0.11
0.57 0.19
20.03 5.81
4.20 0.97
1.43 0.37
Our study has developed high-affinity benzimidazole deriva-
tives 8 and 9 as new SPECT ligand candidates for NR2B subunit
of the NMDA receptor. In vitro autoradiography experiments dem-
onstrated that [125I]8 and [125I]9 showed NR2B selective binding in
the rat brain slices. Although [125I] benzimidazoles showed poor
BBB permeability and inconsistent distribution with NR2B subunit,
*
Values were presented as mean SD (% dose/g, n = 5). P <0.05 in comparison to the
control group (Mann–Whitney U-test).
Regional organ uptake at 180 min after [125I]8 injection. Probenecid (200 mg/kg)
[
125I]8 may be partially bound to the NR2B subunit under in vivo
a
was given as pre-treatment by intravenous injection 30 min before [125I]8 (148 kBq)
condition. Compound 8 may be a potential scaffold for further
structural modification to develop novel imaging probes of cere-
bral NMDA receptors.
administration.
strated no reduction by treatment with probenecid (Table 5). It is
reported that probenecid is the substrate of OAT1 and 3. OAT1 is
mainly expressed in the kidney and OAT3 in the kidney and brain.
OATs play a role in the renal influx of probenecid.40,41 Therefore,
this study indicated that [125I]8 may be a substrate of OATs in
the brain and kidney. Although the extent of transport via P-gp
and OAT is thought to be minimal, a certain amount of [125I]8 that
enters the brain might serve as substrate of P-gp and OAT. Since it
is reported that numerous structurally and pharmacologically
unrelated lipophilic and amphipathic organic compounds are sub-
strate of P-gp,42 the structural relationship between benzimidaz-
oles and P-gp substrate is not elucidated. The introduction of a
non-acidic group instead of a hydroxyl group in the benzimidazole
skeleton of [125I]8 is expected to reduce the efflux by OAT. Further-
more, reduced lipophilic property of [125I]8 should be necessary to
increase BBB permeability.
4. Experimental section
4.1. General information
1H NMR spectra were recorded on a JNM-GSX-270WB spec-
trometer (270 MHz; JEOL, Tokyo, Japan), using tetramethylsilane
as an internal standard. Fast atom bombardment mass spectra
(FAB-MS) were obtained on a JMS-AX505H spectrometer (JEOL).
Electrospray ionization mass spectra (ESI-MS) were obtained on a
TSQ 7000 (Thermo Fisher Scientific, California, USA). High-resolu-
tion mass spectra (HRMS) were obtained on a QSTARÒXL MS/MS
System with the use of ESI. [125I]NaI was purchased from MP Bio-
medicals (California, USA). All other chemicals used were reagent
grade. All animals were supplied by Japan SLC (Hamamatsu, Japan).
An automated gamma counter with a NaI(Tl) detector (ARC-2000;
Aloka, Tokyo, Japan) was used to measure radioactivity. The pres-
ent animal study was approved by the Animal Care and Use Com-
mittee of the Hamamatsu University School of Medicine.
Another problem is the homogeneous distribution of [125I]8 and
[
125I]9 observed in the brain tissues under in vivo condition, which
was quite different from in vitro results. Various factors might be
present under in vivo condition such as non-specific binding to
capillary endothelial cells or other region. It is unclear whether
the radioactivity of regional brain is present in the target site or
peripheral site. However the benzimidazoles with optimized log P
values might decrease an accumulation in peripheral region.
Similar to this study, previously reported imaging agents for
NR2B subunit of NMDA receptor showed consistent accumulation
pattern with the expression of NR2B subunit and high specificity
to NR2B rich region only under in vitro condition but not
in vivo.13–19 Moreover, similar binding characters were observed
in the radioligands for other binding sites of NMDA receptor, such
as ion-channel site43,44 and glycine site.45–47 Kew et al. suggested
that ifenprodil might exhibit higher affinity for the agonist-bound
activated and desensitized states of the NMDA receptor, respec-
tively, relative to the resting, agonist-unbound state.48 Further-
more, Mott et al. demonstrated that NR2B antagonists were
much more potent as a neuroprotectant at pH 6.5 than at pH
7.5.49 The pH of ischemic tissue is as low as 6.5.50 In like manner,
it is reported that [125I]CNS-1261, a radioligand for ion-channel site
of NMDA receptor, showed higher uptake in the ischemic rat brain
4.1.1. Ethyl 2-{4-(4-bromobenzyl)piperidin-1-yl} acetate (4)
To a solution of 323 (13.0 g, 51.15 mmol) in DMF (8.0 ml), diiso-
propylethylamine (10.65 ml, 61.26 mmol), and ethyl bromoacetate
(6.84 ml, 61.26 mmol) were added, and the reaction mixture was
stirred at room temperature for 3 h. EtOAc (50 ml) was added to
the mixture, and the organic layers were washed with aqueous
NaHCO3 and brine, dried with Na2SO4, and evaporated to dryness.
The crude product was chromatographed on silica gel with hexane/
EtOAc = 2:1 to provide 4 (12.4 g, 36.44 mmol, 71%) as a colorless
oil, 1H NMR (CDCl3) d: 7.38 (2H, d, J = 8.43 Hz), 7.01 (2H, d,
J = 8.42 Hz), 4.17 (2H, q, J = 7.33 Hz), 3.17 (2H, s), 2.90 (2H, d,
J = 11.92 Hz), 2.48 (2H, d, J = 6.59 Hz), 2.10 (2H, t, J = 11.54 Hz),
1.58–1.33 (5H, m), 1.26 (3H, t, J = 7.15 Hz), ESI-MS m/z; 340
(M+H)+.
4.1.2. Ethyl 2-{4-(4-tributylstannylbenzyl)piperidin-1-yl}
acetate (5)
To a solution of 4 (5.0 g, 14.69 mmol) in toluene (50 ml), bistrib-
utyltin (14.7 ml, 29.38 mmol), and tetrakis (triphenylphosphine)
palladium (509 mg, 0.44 mmol) were added, and the reaction mix-

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T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
ture was refluxed for 36 h. After cooling, the reaction mixture was
filtered through Celite. The filtrate was evaporated to dryness. The
crude product was chromatographed on silica gel with hexane/
EtOAc = 3:1 to provide 5 (4.30 g, 7.75 mmol, 53%) as a colorless
oil, 1H NMR (CDCl3) d: 7.35 (2H, d, J = 7.35 Hz), 7.09 (2H, d,
J = 7.90 Hz), 4.17 (2H, q, J = 7.16 Hz), 3.17 (2H, s), 2.91 (2H, d,
J = 11.54 Hz), 2.50 (2H, d, J = 6.61 Hz), 2.10 (2H, t, J = 11.43 Hz),
1.66–1.23 (20H, m), 1.06–0.85 (15H, m), FAB-MS m/z; 552 (M+H)+.
s), 2.50 (2H, d, J = 6.62 Hz), 2.11 (2H, t, J = 10.69 Hz), 1.64–1.20 (5H,
m), ESI-HRMS (m/z) calcd for C21H26IN4O2S, 525.0821 (M+H)+, obsd
525.0769.
4.1.7. Ethyl 2-{4-(4-Iodophenoxy)piperidin-1-yl} acetate (11)
Compound 10 (686 mg, 1.70 mmol)25 was dissolved in 30% TFA/
CH2Cl2 (5 ml), and the mixture was stirred at room temperature for
5 h followed by evaporated to dryness. The crude product was con-
verted to the title compound 11 using the above procedure for
compound 4. In result, compound 11 (300 mg, 45%) was obtained
as a colorless oil, 1H NMR (CDCl3) d: 7.53 (2H, d, J = 8.98 Hz), 6.68
(2H, d, J = 8.98 Hz), 4.30 (1H, quin, J = 3.85 Hz), 4.19 (2H, q,
J = 7.09 Hz), 3.24 (2H, s), 2.83–2.75 (2H, m), 2.58–2.47 (2H, m),
2.05–1.97 (2H, m), 1.91–1.84 (2H, m), ESI-MS m/z; 390 (M+H)+.
4.1.3. Ethyl 2-{4-(4-iodobenzyl)piperidin-1-yl} acetate (6)
To a stirred solution of 5 (2.85 g, 5.17 mmol) in carbon tetra-
chloride (135 ml), 50 mM solution of iodine in dry carbon tetra-
chloride (135 ml, 6.72 mmol) was added dropwise, and the
reaction mixture was allowed to stand at room temperature for
1 h. The mixture was evaporated to dryness. The crude product
was chromatographed on silica gel with hexane/EtOAc = 1:1 to
provide 6 (1.36 g, 3.51 mmol, 68%) as a colorless oil, 1H NMR
(CDCl3) d: 7.58 (2H, d, J = 8.33 Hz), 6.88 (2H, d, J = 8.33 Hz), 4.17
(2H, q, J = 7.08 Hz), 3.17 (2H, s), 2.90 (2H, d, J = 11.54 Hz), 2.47
(2H, d, J = 6.41 Hz), 2.10 (2H, t, J = 11.54 Hz), 1.62–1.34 (5H, m),
1.26 (3H, t, J = 7.16 Hz), FAB-MS m/z; 388 (M+H)+.
4.1.8. 2-{[4-(4-Iodophenoxy)piperidin-1-yl]methyl}-5-meth
oxybenzimidazole (12)
Using the above procedure for 7 starting from compound 11,
the title compound 12 (96 mg, 0.207 mmol, 73%) was obtained as
a yellow solid, mp = 50–52 °C, 1H NMR (CD3OD) d: 7.53 (2H, d,
J = 8.97 Hz), 7.41 (1H, d, J = 8.76 Hz), 7.04 (1H, d, J = 2.13 Hz), 6.86
(1H, dd, J = 8.76, 2.57 Hz), 6.73 (2H, d, J = 8.98 Hz), 4.38 (1H, quin,
J = 3.74 Hz), 3.83 (3H, s), 3.79 (2H, s), 2.85–2.77 (2H, m), 2.51–
2.41 (2H, m), 2.07–1.98 (2H, m), 1.87–1.77 (2H, m), ESI-MS m/z;
164 (M+H)+.
4.1.4. 2-{[4-(4-Iodobenzyl)piperidin-1-yl]methyl}-5-
methoxybenzimidazole (7)
Compound 6 (804 mg, 2.08 mmol) was dissolved in 6 M HCl
(5 ml), and the mixture was heated to 100 °C for 1 h. The reaction
mixture was cooled and evaporated to dryness. The residue was
dissolved in DMF (6 ml), followed by EDC (518 mg, 2.70 mmol),
1-hydroxy-7-azabenzotriazole (367 mg, 2.70 mmol), triethylamine
4.1.9. 2-{[4-(4-Iodophenoxy)piperidin-1-yl]methyl}benzimid
azol-5-ol (13)
Using the above procedure for 8 starting from compound 12,
the title compound 13 (108 mg, 0.241 mmol, 77%) was obtained
as a white solid, mp = 63–65 °C, 1H NMR (CD3OD) d: 7.53 (2H, d,
J = 8.98 Hz), 7.34 (1H, d, J = 8.55 Hz), 6.90 (1H, d, J = 2.25 Hz), 6.74
(1H, dd, J = 8.45, 2.68 Hz), 6.73 (2H, d, J = 8.97 Hz), 4.38 (1H, quin,
J = 3.63 Hz), 3.76 (2H, s), 2.86–2.76 (2H, m), 2.49–2.41 (2H, m),
2.07–1.99 (2H, m), 1.86–1.77 (2H, m), ESI-HRMS (m/z) calcd for
(570 ll, 4.16 mmol), and 4-methoxy-1,2-phenylenediamine
(287 mg, 2.08 mmol). The reaction mixture was stirred at room
temperature for 3 h followed by quenching with aqueous NaHCO3
and ethyl acetate. Organic layer was separated and washed by
brine, dried with Na2SO4, and evaporated to dryness. The crude
oil was dissolved in acetic acid (6 ml) and heated to 140 °C for
4 h. The reaction mixture was cooled and evaporated to dryness.
The crude product was chromatographed on silica gel with
CHCl3/MeOH = 20:1 to provide 7 (530 mg, 1.15 mmol, 55%) as a
colorless oil, 1H NMR (CDCl3) d: 7.59 (2H, d, J = 8.12 Hz), 7.46
(1H, d, J = 7.91 Hz), 7.04 (1H, s), 6.88 (1H, d, J = 8.12 Hz), 6.87
(1H, d, J = 7.98 Hz), 3.85 (5H, s), 2.93 (2H, d, J = 12.18 Hz), 2.51
(2H, d, J = 6.41 Hz), 2.21 (2H, t, J = 11.33 Hz), 1.68–1.25 (5H, m),
ESI-MS m/z; 462 (M+H)+.
C
19H21IN3O2, 450.679 (M+H)+, obsd 450.0621.
4.1.10. 2-{[4-(4-Trimethylstannylbenzyl)piperidin-1-yl]methyl}
benzimidazol-5-ol (14)
To a solution of 8 (50 mg, 0.125 mmol) in toluene (3 ml), bis-
trimethyltin (104 ll, 0.502 mmol), and tetrakis (triphenylphos-
phine) palladium (15 mg, 0.013 mmol) were added, and the
reaction mixture was refluxed for 6 h. After cooling, the reaction
mixture was filtered through Celite. The filtrate was evaporated
to dryness. The crude product was chromatographed on silica gel
with CHCl3/MeOH = 15:1 to provide 14 (32 mg, 0.066 mmol, 53%)
as a colorless oil, 1H NMR (CDCl3) d: 7.41 (1H, d, J = 8.80 Hz), 7.39
(2H, d, J = 8.06 Hz), 7.08 (2H, d, J = 7.69 Hz), 7.04 (1H, d,
J = 2.20 Hz), 6.83 (1H, dd, J = 8.80, 2.20 Hz), 4.09 (2H, s), 3.11 (2H,
d, J = 12.09 Hz), 2.53 (2H, d, J = 5.87 Hz), 2.47 (2H, t, J = 11.73 Hz),
1.77–1.51 (5H, m), 0.27 (9H, s), FAB-MS m/z; 486 (M+H)+.
4.1.5. 2-{[4-(4-Iodobenzyl)piperidin-1-yl]methyl}benzimidazol-
5-ol (8)
Compound 7 (530 mg, 1.15 mmol) was dissolved in HBr/H2O
(48%, 10 ml), and the mixture was heated to 100 °C for 6 h. The
reaction mixture was cooled and evaporated to dryness. The crude
product was chromatographed on silica gel with CHCl3/MeOH/
NH4OH = 200:10:1 to provide 8 (324 mg, 0.724 mmol, 63%) as a
white solid, mp = 117–118 °C, 1H NMR (CDCl3) d: 7.67 (1H, dd,
J = 8.66, 1.01 Hz), 7.63 (2H, d, J = 8.55 Hz), 7.16–7.12 (2H, m), 6.99
(2H, d, J = 8.34 Hz), 4.77 (2H, s), 3.63 (2H, d, J = 11.97), 3.26–3.17
(1H, m), 2.58 (d, J = 6.84 Hz), 1.95–1.90 (4H, m), 1.66–1.60 (2H,
m), ESI-HRMS (m/z) calcd for C20H23IN3O, 448.0880 (M+H)+, obsd
448.0829.
4.1.11. N-{2-[4-(4-Trimethylstannylbenzyl)-piperidin-1-ylmeth
yl]benzoimidazol-5-yl}-methanesulfonamide (15)
Using the above procedure for 14 starting from compound 9,
the title compound 15 (5.2 mg, 9.26 lmol, 97%) was obtained as
a colorless oil, 1H NMR (CDCl3) d: 7.57–7.53 (2H, m), 7.40 (2H, d,
7.91 Hz), 7.18 (1H, d, 8.33 Hz), 7.10 (2H, d, 7.70 Hz), 3.78 (2H, s),
2.97 (3H, s), 2.86 (2H, d, J = 11.12 Hz), 2.54 (2H, d, J = 6.62 Hz),
2.14 (2H, t, J = 10.15 Hz), 1.41–1.22 (5H, m), 0.27 (9H, s), FAB-MS
m/z; 563 (M+H)+.
4.1.6. N-{2-[4-(4-Iodobenzyl)-piperidin-1-ylmethyl]benoimida
zol-5-yl}-methanesulfonamide (9)
Using the above procedure for 7 with N-(3,4-diaminophe-
nyl)methanesulfonamide,20 the title compound
9
(27 mg,
0.052 mmol, 53%) was obtained as a white solid, mp = 92–94 °C, 1H
NMR (CD3OD) d: 7.58 (2H, d, J = 8.34 Hz), 7.50 (1H, d, J = 8.12 Hz),
7.49 (1H, dd, J = 2.08, 0.48 Hz), 7.13 (1H, dd, J = 8.66, 2.04 Hz), 6.93
(2H, d, J = 7.91 Hz), 3.75 (2H, s), 3.32 (2H, d, J = 10.47 Hz), 2.90 (3H,
4.2. Radiosynthesis
[
125I]8 and [125I]9 were prepared by similar method described in
the literature.26 In brief, 30% aqueous hydrogen peroxide (100
l)
l

T. Fuchigami et al. / Bioorg. Med. Chem. 18 (2010) 7497–7506
7505
was added to a mixture of [125I] NaI (60
free), 1% HCl (40 l), and trimethylstannyl precursor 14, 15 (0.1 mg
in 100 l of ethanol) in a sealed vial. The mixture was allowed to
react for 40 min at room temperature and then quenched with
10% aqueous sodium bisulfite (100 l). EtOAc (5 ml) was added
to the mixture, and the organic layers were washed with aqueous
brine, and evaporated to dryness. The crude products were purified
by HPLC (column; Nacalai Cosmosil 5C18-AR II, 10 ꢀ 250 mm, mo-
bile phase; MeOH: 0.1% TFA in H2O = 60:40, flow rate: 3.0 ml/min).
Each fraction was collected and the solvent was removed in vacuo.
Radiochemical purity was assayed to be >98% by analytical HPLC
(column; Nacalai Cosmosil 5C18-ARII, 4.6 ꢀ 250 mm, mobile
phase; MeOH: 0.1% TFA in H2O = 55:45, flow rate: 1.0 ml/min).
The radiochemical yields based on [125I] NaI were 85–90% and
78-81% for [125I]8 and [125I]9, respectively.
ll, 19.4–41.4 MBq, carrier-
divided into the cerebral cortex, hippocampus, and cerebellum.
The tissues were then weighed and the radioactivity was measured
by automated gamma counting. Data were calculated as the per-
centage of the injected dose per gram of tissue (% dose/g).
l
l
l
To assess the in vivo specific binding of [125I]8, the non-radioac-
tive 8 or NR2B antagonist Ro-25,6981 (3 mg/kg in 10% DMSO/sal-
ine) was given as pre-treatment by intravenous injection 30 min
before [125I]8 administration. Mice were killed 180 min after injec-
tion. The modulation of [125I]8 by P-gp and OAT in vivo was eval-
uated according to the literature.36,52,53 In brief, CsA {50 mg/kg in
a mixture of cremophor EL (140 mg/ml) and ethanol (5%)/saline}
or probenecid (200 mg/kg in 5% sodium bicarbonate/saline) was gi-
ven as pre-treatment by intravenous injection 30 min before
[
[
125I]8 administration. Mice were killed 180 min after injection of
125I]8. In each of the control mouse, the same amount of drug-free
corresponding solvent was injected.
4.3. In vitro binding assays
To assess the metabolism of [125I] benzimidazoles in the mouse
brain, the mice were killed at 180 min after [125I] benzimidazoles
(185 kBq) injection. Whole brains were quickly removed, homoge-
nized with ice-cooled MeOH (1.0 ml), and centrifuged at 1750g for
10 min at 4 °C. The metabolites in MeOH extract were analyzed by
radio-TLC using CHCl3/MeOH = 9:1 and EtOAc/EtOH = 1:1 for
The Ki values of benzimidazoles for binding of [3H]ifenprodil to
rat cortical membrane homogenates were determined by the
method mentioned in the literature.25 In brief, well washed mem-
branes (200–300
7.4), were incubated with [3H]ifenprodil (4.0 nM) with either buf-
fer or displacing drug added to make a final volume of 500 l for
lg of protein) in 20 mM HEPES-KOH buffer (pH
[
125I]8, CHCl3/EtOH = 4:1 and EtOAc/EtOH = 4:1 for [125I]9, as mo-
l
bile phases.
120 min at 25 °C. In each experiment the assay buffer contained
100 mM trifluoroperazine. Non-specific binding was defined by
the addition of unlabeled ifenprodil at 1 mM. 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. 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.
4.6. Partition co-efficient determination
Partition co-efficient between n-octanol and buffer was mea-
sured by a conventional shake flask method in triplicate. The benz-
imidazoles (1–2 mg) were well shaken in a mixture of n-octanol
(1 ml) and 10 mM phosphate buffer (pH 7.4, 1 ml) for 15 min at
ambient temperature. After centrifugation (2500 rpm for 5 min)
of the mixture, the two layers were separated and analyzed by
HPLC (column; Nacalai Cosmosil 5C18-AR 300, 4.6 ꢀ 250 mm, mo-
bile phase; MeOH: 0.1% TFA in H2O = 50:50, flow rate: 1.0 ml/min)
to obtain log D (pH 7.4) values (Table 1).
4.4. In vitro receptor autoradiography
The brain sagittal sections, obtained by the method described in
the literature,13 were pre-incubated for 30 min at 25 °C in 50 mM
Tris–HCl buffer (pH 7.4 at 25 °C) containing 5% bovine serum albu-
min (BSA). These were subsequently incubated in the same buffer
containing [125I]8 or [125I]9 (300 kBq, 0.09–0.14 nM) at 25 °C for
30 min. The slices were rinsed triplicate for 3 min each with cold
(5 °C) incubation buffer containing 1% BSA, and subsequently
dipped into cold water. The sections were dried under a steam of
warm air and placed in contact with 125I-sensitive imaging plates
(BAS-SR 2040; Fuji Photo. Film, Tokyo, Japan) for 3 days. Distribu-
tions of radioactivity on the plates were analyzed by Bio-Image
Analyzer (FLA-3000; Fuji Photo. Film, Tokyo, Japan), which were
photographically visualized as shown in Figure 3A–D. Regions of
interest (ROIs) on the slices were placed on the cerebral cortex,
hippocampus, and cerebellum; and the radioactivities in these re-
gions were expressed as photostimulated luminescence (PSL) val-
ues on ROI-background PSL value per square millimeter. The
specific binding was determined as the difference between total
binding and binding in the presence of the corresponding non-
4.7. Data analysis
Data were expressed as the mean standard deviation. In vitro
experiments of [125I]8 and [125I]9 were statistically analyzed by
Mann–Whitney U-test as shown in Figure 2. In vivo experiments
of [125I] 8 were analyzed by the Kruskal–Wallis test with Dunn’s
multiple comparison post-test as shown in Figure 4 or Mann–
Whitney U-test as shown in Table 4. A value of P <0.05 was consid-
ered statistically significant.
Acknowledgments
The authors gratefully acknowledge our colleague, Mutsumi
Kosugi, for her technical help in the animal experiments. This work
was supported by the Grant-in-Aid for Young Scientists (Start-up)
(18890080) and Hamamatsu University School of Medicine Re-
search Grant for Young Researchers.
radioactive benzimidazoles (10
were performed as similar procedure in the presence of several
drugs (10 M).
lM). Receptor selectivity studies
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