N-(5-fluoro-2-phenoxyphenyl)-N-(2-[131I]iodo-5-methoxybenzyl) acetamide: A potent iodinated radioligand for the peripheral-type benzodiazepine receptor in brain

Article abstract of DOI:10.1021/jm061127n

To image the peripheral-type benzodiazepine receptor (PBR) in vivo, we previously developed two positron emission tomography (PET) ligands, N-(2-[ ¹¹C],5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([ ¹¹C]1a) and its [

Full text of DOI:10.1021/jm061127n

848
J. Med. Chem. 2007, 50, 848-855
N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[131I]iodo-5-methoxybenzyl)acetamide: A Potent Iodinated
Radioligand for the Peripheral-type Benzodiazepine Receptor in Brain
Ming-Rong Zhang,*,† Katsushi Kumata,† Jun Maeda,‡ Terushi Haradahira,† Junko Noguchi,† Tetsuya Suhara,‡
Christer Halldin,§ and Kazutoshi Suzuki†
Radiochemistry Section, Department of Molecular Probe, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1
Anagawa, Inage-ku, Chiba 263-8555, Japan, Molecular Neurobiology Section, Department of Molecular Neuroimaging, Molecular Imaging
Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, and Psychiatry Section, Department of
Clinical Neuroscience, Karolinska Institute, S17176 Stockholm, Sweden
ReceiVed September 28, 2006
To image the peripheral-type benzodiazepine receptor (PBR) in vivo, we previously developed two positron
emission tomography (PET) ligands, N-(2-[11C],5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide
([11C]1a) and its [18F]fluoroethyl analogue ([18F]1b), for the investigation of PBR in the living human brain.
This time, using 1a as a leading compound, we designed two novel iodinated analogues, N-(5-fluoro-2-
phenoxyphenyl)-N-(2-iodo-5-methoxybenzyl)acetamide (3a) and N-(2,5-dimethoxybenzyl)-N-(5-iodo-2-
phenoxyphenyl)acetamide (3b) for the PBR imaging. Ligands 3 were synthesized by the iodination of
tributystannyl precursors 10. Radiolabeling for 3 with 131I was carried out by the reaction of 10 with [131I]-
NaI using H2O2 as an oxidizing agent. In vitro competition experiments determined that 3a exhibited both
high affinity and selectivity for PBR (IC50: 7.8 nM) vs CBR (>1 µM). Biodistribution study in mice
determined that [131I]3a had a high radioactivity level (1.69% dose/g) in the brain, and its distribution pattern
in the brain was consistent with the known distribution of PBR in rodents. Ex vivo autoradiography of the
rat brain gave visual evidence that [131I]3a was a potent and specific radioligand for PBR.
Table 1. In Vitro Binding Affinity (IC50) for Peripheral Benzodiazepine
Receptor (PBR) and Central Benzodiazepine Receptor (CBR), and
Octanol/Phosphate Buffer Distribution Coefficient (logD)
Introduction
The peripheral-type benzodiazepine receptor (PBR),1 which
is an 18-kDa protein as a critical functional unit of a multimeric
140-200-kDa complex,2 is mainly distributed in the outer
mitochondrial membrane at the cellular level.3,4 PBR, which
was initially identified in the peripheral tissues, was also found
in the central nervous system (CNS).5 In the CNS, PBR is
mainly located in glial cells, which are distributed in the
olfactory bulb and cerebellum of rodents, and in the occipital
cortex, etc., of primates.6-10 It has been demonstrated that glial
cells play a crucial role in neurodegenerative process in the
CNS.11 When a brain was injured, glial cells in the brain were
activated, which led to increased PBR receptor density;12
therefore, PBR has been determined as a sensitive marker protein
for activated glial cells in various experimental models of brain
injury.12-14 So far, clinical investigations for PBR have revealed
that this receptor correlates with various brain injuries12-14 and
neurodegenerative disorders such as Alzheimer’s disease15-17
and multiple sclerosis.18,19 These findings have prompted the
development of radioligands for PBR, which can be used to
visualize this receptor and to measure its level in vivo in both
humans and animals.20-24
IC50 (nM)a
PBR
for [11C]1ab for [11C]2ab CBRc logDd
7.8 ( 0.4 4.4 ( 0.1 >10000 4.42
ligand
R1
R2
3a
3b
F
I
I
OCH3 69.2 ( 5.9 60.1 ( 6.3 >10000 4.03
1a (DAA1106)
2a (PK11195)
1.6 ( 0.1
8.3 ( 1.2
1.3 ( 0.7 >10000 3.65
9.2 ( 0.4 >10000 2.78
a Values (mean ( SD) represent the mean obtained from nine concentra-
tions of compound using at least eight slices of rat brain (n ) 3). b [11C]1a
and [11C]2a were incubated in the presence of the compounds examined,
respectively. c [11C]Flumazenil was incubated in the presence of the
compounds examined. d The logD values were determined in the phosphate
buffer (pH ) 7.4)/octanol system using the shaking flask method. All results
were presented as mean values (n ) 3) with a maximum range of (5%.
PBR than 1-(2-chlorophenyl)-N-methyl,N-(1-methylpropyl)-
isoquinoline (PK11195, 2a), a standard ligand for PBR, and
had remarkable selectivity against other receptors including the
classical central benzodiazepine receptor (CBR).27-29 In vivo
study determined that [11C]1a and [18F]1b had higher uptake
and better specific binding in rodent and primate brains than
[11C]PK11195 ([11C]2a).25-27 Now, [11C]1a and [18F]1b are
being used to investigate PBR in human brains in our laboratory
to elucidate the relationship between PBR and brain diseases.31,32
In addition to PET, single photoemission computed tomog-
raphy (SPECT) is another radioimaging modality for in vivo
investigation and diagnosis. Although PET provides functional
information with higher resolution and better selectivity than
SPECT, SPECT imaging is a more practical and routine method
for clinical usefulness due to the convenient production of
radiopharmaceuticals. So far, several radioiodinated ligands for
PBR imaging have been developed and evaluated in rodents
and primates.33-35 Of these ligands, [123I]2b, an iodinated
Recently, we have developed two novel PET ligands:
N-(2-[11C], 5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)-
acetamide ([11C]DAA1106, [11C]1a, Scheme 1) and N-(5-fluoro-
2-phenoxyphenyl)-N-(2-[18F]fluoroethoxy-5-methoxybenzyl)-
acetamide ([18F]FEDAA1106, [18F]1b) for PBR imaging.25-27
The two ligands had more potent in vitro binding affinities for
* Corresponding author. Tel: 81-43-206-4041; Fax: 81-43-206-3261;
E-mail: zhang@nirs.go.jp.
† Department of Molecular Probes, National Institute of Radiological
Sciences.
‡ Department of Molecular Neuroimaging, National Institute of Radio-
logical Sciences
§ Karolinska Institute.
10.1021/jm061127n CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/11/2007

Iodinated Radioligand for PBR in Brain
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 849
Scheme 1. Chemical Structures of DAA1106 (1) and PK11195 (2) Analogues
Scheme 2. Chemical Structures of Iodinated Analogues 3a
and 3b
proceeded rapidly even at room temperature to afford 3 at high
chemical yields of >80%, respectively.
The labeling of 3a and 3b using 131I was carried out as shown
in Scheme 3. The [131I]iodination of the tributylstannyl precursor
10a or 10b with [131I]NaI was performed by regioselective
destannylation under mild reaction conditions using H2O2 as
an oxidizing agent in acetic acid. After the reaction was
completed, the radioactive mixture was applied onto a semi-
preparative HPLC system with a radioactive detector. Within
8-11 min of starting HPLC purification, the radioactive fraction
corresponding to [131I]3a or [131I]3b was collected and the
solvents were removed by nitrogen gas flow at room temper-
ature. The obtained residue was redissolved in saline containing
10% ethanol to give the desired radioactive product as an
intravenously injectable solution. Using HPLC and radio-TLC,
the identity of [131I]3 was confirmed by comparison with the
non-radioactive 3, and the radiochemical purity in the products
was determined to be >98%, respectively. The specific activity
of [131I]3 was assumed to >30 GBq/µmol based on the specific
activity of the commercial [131I]NaI. In the final products, no
significant chemical impurities were determined. Moreover, the
radiochemical purity of [131I]3 remained >95% after mainte-
nance of the products at room temperature for 24 h, and they
were stable for performing the evaluation on animals.
analogue of 2a (Scheme 1), has been used for the assessment
of neuroinflammation and microglial activation in Alzheimer’s
disease.17
Here, using 1a as a lead compound, we designed two novel
iodinated ligands: N-(5-fluoro-2-phenoxyphenyl)-N-(2-iodo-5-
methoxybenzyl)acetamide (3a) and N-(2,5-dimethoxybenzyl)-
N-(5-iodo-2-phenoxyphenyl)acetamide (3b) (Scheme 2). These
compounds could be labeled not only by the gamma emitter
123I for SPECT, but also by the positron emitter 124I for PET
and other radioiodines without changing their chemical struc-
tures and pharmacological profiles. In this paper, we report the
(1) chemical synthesis of 3a and 3b starting from commercial
materials, (2) radiolabeling of 3a and 3b using easily handled
131I, and (3) in vitro binding affinity to PBR and biodistribution
of these ligands in rodent brains.
In Vitro Binding Assays. The in vitro binding affinities
(IC50) of ligands 3a and 3b for PBR were determined from
competition against [11C]1a and [11C]2a binding to PBR by
using quantitative autoradiography27 on rat brain sections. As
shown in Table 1, replacement of the methoxyl group by an
iodine atom onto the benzyl moiety maintained the potency of
3a for PBR. Although the binding affinity of 3a (IC50: 7.8 nM
for [11C]1a or 4.4 nM for [11C]2a) was slightly lower than that
of 1a (1.6 nM or 1.3 nM), 3a showed a similar potency with
2a (8.3 nM or 9.2 nM), the mostly used standard ligand for
PBR. This result suggests that the electron density and size of
the substitution group in the benzene ring of the benzyl could
not affect interaction between PBR and 3a significantly. This
finding was in agreement with our previous results that
replacement of the methoxyl group (1a) by several alkoxyl
groups such as OCH2F, OCD2F, OCH2CH2F (1b), OCH2CH3,
and OCH(CH3)2 groups could maintain their binding affinities
for PBR.24,27 Contrary to 3a, substitution of the fluorine with
an iodine atom onto the acetanilide weakened the binding
affinity of 3b for PBR. The affinity of 3b was 40-, 8-, and 10-
fold lower than those of 1a, 2a, and 3a, respectively. The iodine
bulky in the benzene ring of acetanilide appeared unfavorable
for expressing the affinity for PBR, despite the some similarity
of iodine and fluorine atoms. This finding suggests that the
conformation of 3b while interacting with PBR was affected
negatively due to iodine hindrance. Considering that these
Chemistry. The synthetic route chosen for the preparation
of ligands 3a and 3b involved introduction of the iodine onto
the benzene ring by the iodination of tributylstannyl precursors.
They were prepared in a six-step sequences of reactions
delineated in Scheme 3, respectively. In this synthetic approach,
the reactions of 2-bromonitrobenzenes 4 with phenol under basic
conditions afforded 2-phenoxynitrobenzenes 5, which were
reduced with powdered iron and then treated with acetyl chloride
to give acetanilides 7. Alkylations of 7 with benzyl halides 8
using sodium hydride as a base afforded bromoacetanilides 9,
which were treated with bistributyltin in the presence of tetrakis-
(triphenyphosphine)palladium (0) to give tributylstannyl precur-
sors 10. The stannylation of 9a to form 10a required much
longer reaction time (90 h) than that of 9b to form 10b (18 h).
This difference relates steric hindrance in 9a to restricted rotation
of the benzyl group as inferred form the two signals for its
1
methylene protons in H NMR spectrum. Finally, the desired
products 3a and 3b were prepared by iodination of the
tributylstannyl 10 with iodine powder. The two reactions

850 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Scheme 3. Chemical Synthesis and Radiosynthesis
Zhang et al.
(a)K2CO3, DMF, 80 °C, 3 h; b) NH4Cl, EtOH, H2O, 80 °C, 5 h; (c) Et3N, CH2Cl2, room temperature, 3 h; (d) NaH, DMF, room temperature, 6 h; (e) NaH,
DMF, room temperature, 3 h; (f) Pd(PPh3)4, toluene, reflux, 90 h; (g) Pd(PPh3)4, toluene, reflux, 18 h; (h) CH2Cl2, 25 °C, 3 h; (i) H2O2, AcOH, EtOH,
25 °C, 30 min.
derivatives evolved from the classical benzodiazepine structure,
the in vitro binding affinities of 3a and 3b for the central
benzodiazepine receptor (CBR) were measured by competition
against the CBR-selective [11C]flumazenil binding. As can be
seen in Table 1, the two ligands did not display remarkable
inhibitory effects (IC50>1 µM) on the [11C]flumazenil binding
in the rat brain. Their high selectivity for PBR over CBR may
be due to their drastically structural difference from the classical
benzodiazepine ring. The in vitro binding experiment demon-
strated that 3a had both high affinity and selectivity for PBR
vs CBR.
dose/g) had decreased 44% when compared to the uptake at 15
min. The result suggests that this ligand does pass across the
brain-blood barrier (BBB) into the brain, which is a prerequisite
for a promising radioligand for brain imaging. This finding was
also compatible with its high lipophilicity expressed as a
distribution coefficient (logD: 4.42), as shown in Table 1.
The regional distribution of [131I]3a in the mouse brains was
determined over 60 min (Table 2). The radioactivity of [131I]3a
accumulated with time in all regions examined, and the uptake
in these regions peaked during 15-30 min and then declined
until 60 min. Among the brain regions examined, the highest
uptake (3.06% dose/g at 15 min) was observed in the olfactory
bulb, the highest PBR density area in the mouse brain. Following
the olfactory bulb, a high radioactivity level (2.32% dose/g
at 15 min) was also detected in the cerebellum, whereas a
moderate or low uptake was observed in the cerebral cortex,
hypothalamus, thalamus, striatum, and hippocampus. The uptake
pattern of radioactivity was consistent not only with [3H]1a28
Brain Uptake in Mice. The uptake of [131I]3a and [131I]3b
was examined in mouse brains and the radioactivity levels are
expressed as percent dose per gram of tissue weight (% dose/
g) as shown in Table 2. [131I]3a showed a rapid penetration
into the brain and exhibited high initial uptake (1.06% dose/g)
in the entire brain at 1 min after intravenous injection. At 15
min after injection, the uptake of [131I]3a reached the maximum
value (1.69% dose/ g) and then displayed gradual washout from
the brain over 60 min. After 60 min, the brain uptake (0.75%

Iodinated Radioligand for PBR in Brain
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 851
Table 2. Brain Regional Distribution (% Injected Dose/g Tissue: Mean ( SD, n ) 3) of [131I]3a and [131I]3b in Mice at 1, 5, 15, 30, 60 min after
Injection
ligand
tissue
1 min
5 min
15 min
30 min
60 min
3a
whole brain
cerebellum
olfactory bulb
hippocampus
striatum
cerebral cortex
hypothalamus
thalamus
1.06 ( 0.25
1.21 ( 0.49
1.49 ( 0.51
0.53 ( 0.21
0.71 ( 0.15
0.82 ( 0.26
0.78 ( 0.18
0.67 ( 0.20
2.34 ( 0.89
0.87 ( 0.39
0.92 ( 0.45
1.15 ( 0.34
0.25 ( 0.15
0.47 ( 0.17
0.60 ( 0.12
0.22 ( 0.06
0.53 ( 0.04
2.67 ( 1.01
1.38 ( 0.32
1.48 ( 0.38
2.24 ( 0.34
0.62 ( 0.49
0.71 ( 0.23
0.91 ( 0.35
0.99 ( 0.34
0.49 ( 0.10
1.85 ( 0.23
1.15 ( 0.36
1.08 ( 0.24
1.35 ( 0.29
0.67 ( 0.41
0.46 ( 0.05
0.48 ( 0.32
0.39 ( 0.03
0.61 ( 0.08
1.99 ( 0.58
1.69 ( 0.21
2.32 ( 0.28
3.06 ( 0.39
0.75 ( 0.32
0.69 ( 0.24
0.84 ( 0.26
0.97 ( 0.18
0.48 ( 0.25
1.11 ( 0.46
0.71 ( 0.30
0.79 ( 0.09
1.02 ( 0.51
0.37 ( 0.25
0.55 ( 0.22
0.36 ( 0.23
0.45 ( 0.10
0.35 ( 0.19
1.38 ( 0.51
1.28 ( 0.44
1.63 ( 0.24
2.42 ( 0.43
0.59 ( 0.26
0.60 ( 0.31
0.55 ( 0.12
0.73 ( 0.29
0.53 ( 0.16
0.76 ( 0.35
0.38 ( 0.12
0.44 ( 0.06
0.58 ( 0.13
0.23 ( 0.17
0.26 ( 0.19
0.13 ( 0.30
0.21 ( 0.22
0.20 ( 0.01
0.41 ( 0.38
0.75 ( 0.17
1.00 ( 0.12
1.17 ( 0.56
0.55 ( 0.21
0.23 ( 0.16
0.24 ( 0.09
0.41 ( 0.13
0.32 ( 0.07
0.25 ( 0.24
0.18 ( 0.17
0.16 ( 0.08
0.24 ( 0.12
0.08 ( 0.13
0.12 ( 0.14
0.16 ( 0.09
0.21 ( 0.19
0.14 ( 0.02
0.29 ( 0.20
blood
3b
whole brain
cerebellum
olfactory bulb
hippocampus
striatum
cerebral cortex
hypothalamus
thalamus
blood
brain regions compared with the control group. The most
significant decrease was found in the olfactory bulb (33% of
control) and cerebellum (40% of control). Other brain regions
(striatum, hippocampus, hypothalamus, and cerebral cortex)
showed slow decreases (60-90%) in the percent uptake of [131I]-
3a. The inhibition experiment determined that some specific
bindings of [131I]3a occurred in the all brain regions, and high
levels (>60% of total binding) were present in the olfactory
bulb and cerebellum. PBR-selective 2a (1 mg/kg) also signifi-
cantly reduced radioactivity in these regions of the mouse brain,
to an extent similar to that obtained using the same amount of
1a. The largest decrease in the radioactivity occurred in the
olfactory bulb (40%) and cerebellum (48%) compared with the
control group. These results indicate that the uptake of
radioactivity in PBR-rich regions after the injection of [131I]3a
was due to PBR sites in the mouse brain. Moreover, [131I]3a
exhibited potentially favorable kinetic properties for the in vivo
imaging of PBR in the brain.
Figure 1. Effects of the PBR-selective 1a (1 mg/kg) and 2a (1 mg/
kg) on [131I]3a concentrations (mean ( SD, n ) 3) in selected regions
of mouse brains at 30 min after intravenous injection (0.4 MBq).
Ex Vivo Autoradiography. The ex vivo autoradiography
technique cannot only provide visual evidence of radioligand
binding with a receptor, but also afford statistical information
about ligand distribution in the regions of interest (ROI). Figure
2 (a) shows an ex vivo autoradiogram of [131I]3a for the rat
brain at 30 min after intravenous injection. This image revealed
the relatively high radioactivity level of [131I]3a in the rat brain,
as detected in the mouse brain. Among the regions examined,
the highest radioactivity was observed in the choroids plexus,26,28
which is a structure related to the secretion of cerebrospinal
fluid containing a large concentration of PBR. In addition, a
high level of radioactivity was also seen in the olfactory bulb.
Following the olfactory bulb, moderate radioactivity was
detected in the cerebellum, whereas low uptake was detected
in other regions such as the cerebral cortex and striatum. The
distribution order reflected the known distribution of PBR in
the rodent brain. In the previous studies, autoradiograms of [3H]-
1a26 and [11C]1a28 also demonstrated that a high density of PBR
binding is localized in the olfactory bulb and choroids plexus.6,7
Quantitative analysis was performed for all autoradiograms (n
) 15) obtained from the rat brain. The radioactivity in each
brain region was determined and expressed as a photostimulated-
luminescence (PSL)/mm2 region. As shown in Figure 2 (c),
using the striatum as a reference region with low PBR density,28
the ratios of choroids plexus and olfactory bulb to the striatum
were 3.2 and 1.5, respectively, for [131C]3a, whereas the ratios
of other brain regions to the striatum were about 1.0-1.5. These
and [11C]1a25,26 binding sites in the rat or mouse brain, but also
with the known regional distribution of PBR in the rodent
brain.6,7
On the other hand, although [131I]3b had rapid and high initial
uptake into the brain, its radioactivity peaked (1.15% dose/g)
at 5 min after injection, which was 67% of [131I]3b. From 5
min, [131I]3b displayed a gradual washout of radioactivity from
various regions. At 15 min after injection, the radioactivity of
[
131I]3b in the mouse brain had decreased by 0.91% dose/g (54%
of [131I]3a at the same time point). Moreover, the highest levels
of radioactivity for [131I]3b in the olfactory bulb and cerebellum
were 33% and 30% of those for [131I]3a, respectively. The
relatively lower uptake of [131I]3b in the brain was due to lower
in vitro binding affinity with PBR. Although 3b was also highly
lipophilic (logD: 4.03), its binding affinity was >10 fold lower
than that of 3a. These results demonstrated that [131I]3b did
not display a more favorable property as a ligand for PBR
imaging in the brain than [131I]3a, therefore, no further evalu-
ation of [131I]3b was carried out.
The in vivo specificity of [131I]3a for PBR was tested by co-
injecting PBR-selective 1a and 2a at a dose of 1 mg/kg body
weight, respectively. The result of the blocking study at 30 min
after injection is presented in Figure 1. Co-injection with
nonradioactive 1a reduced the radioactivity of [131I]3a in the

852 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Zhang et al.
Figure 2. Ex vivo autoradiograms of [131I]3a in the sagittal sections of rat brains at 30 min after injection (2 MBq): (a) [131I]3a; (b) [131I]3a + 1a
(1 mg/kg); (c) the radioactivity distribution of [131I]3a in the brain regions. The radioactivity level in each region was determined from the brain
images (n ) 10-15) and was expressed by as photostimulated-luminescence (PSL)/mm2 region.
visual results indicate that the uptake of radioactivity in PBR-
rich regions after the injection of [131I]3a was due to PBR sites
in the rat brain.
brains. This novel ligand can be labeled not only by 123I for
SPECT but also by 124I or 11C for PET and then can be evaluated
in rodents and primates. These studies are in progress in our
laboratory.
To further determine the in vivo specificity of [131I]3a in the
rat brain, a blocking study was performed in which non-
radioactive 1a (1 mg/kg body weight) was injected simulta-
neously with [131I]3a. As shown in Figure 2 (b), PBR-selective
1a significantly reduced radioactivity in the brain regions. This
high dose of 1a abolished regional differences in the uptake of
radioactivity in the olfactory bulb, choroids plexus, and other
brain regions. Quantitative analysis of these images (n ) 10)
revealed the most evident decreases in the choroids plexus (43%
of the control) and olfactory bulb (43%) (Figure 2 (c)). Other
brain regions (cerebellum, striatum, and frontal cortex, etc.)
showed moderate or low decreases in the percent uptake (63-
85%) of [131I]3a. These results further support that high specific
binding of [131I]3a to PBR occurred in the olfactory bulb and
choroids plexus of the rat brain.
Experimental Section
All chemicals and solvents of the highest grade commercially
available were purchased from Aldrich Chem. (Milwaukee, WI)
and Wako Pure Chem. Ind. (Osaka) without further purification in
all cases. The melting points (mp) were determined using a Yanaco
1
MP-500D melting point apparatus and are uncorrected. H NMR
spectra were recorded on a JNM-GX-300 spectrometer (JEOL,
Tokyo) with tetramethylsilane as an internal standard. All chemical
shifts (δ) were reported in parts per million (ppm) downfield from
the standard. FAB-MS was obtained on a JEOL NMS-SX102
spectrometer (JEOL, Tokyo). Column chromatography was carried
out using silica gel (WAKO Wakogel C-200). Thin-layer chroma-
tography analyses (TLC) were performed using silica gel F254 PLC
plates (20 × 20, 0.5 µm; Merck). Radioactive mixtures were
purified and analyzed by high-performance liquid chromatography
(HPLC) with an in-line UV (254 nm) detector in series with a NaI
crystal radioactivity detector. Isolated radioactivity was measured
with an IGC-3 Curiemeter (Aloka, Tokyo). All animal experiments
were carried out according to the recommendations of the committee
for the care and use of laboratory animals, National Institute of
Radiological Sciences (NIRS).
5-Bromo-2-phenoxynitrobenzene (5b). A mixture of 5-bromo-
2-fluoronitrobenzene (4b; 1.10 g, 5.0 mmol), phenol (0.52 g, 5.5
mmol), and K2CO3 powder (0.83 g, 5.0 mmol) in DMF (10 mL)
was stirred at 80 °C for 3 h. The mixture was concentrated under
reduced pressure, and the residue was partitioned between AcOEt
and water. The separated organic layer was washed with 1 M
aqueous HCl and saturated NaCl solution. After the organic layer
was dried over Na2SO4, the solvent was removed to give 5b (1.44
g, 98%) as a yellowish oil. 1H NMR (CDCl3) δ: 8.08 (1H, d, J )
2.4 Hz), 7.58 (1H, dd, J ) 2.4, 8.8 Hz), 7.40 (2H, t, J ) 7.7 Hz),
7.21 (1H, t, J ) 7.7 Hz), 7.05 (2H, d, J ) 7.7 Hz), 6.89 (1H, d, J
) 8.8 Hz). HRMS (FAB) Calcd for C12H8BrNO3: 293.9766.
Found: 293.9754.
The in vivo stability of [131I]3a in the mouse brains (n ) 3)
was determined. At 30 min after injection, 90-94% of total
radioactivity in the brain homogenate represented unchanged
[
131I]3a, and no significant radioactive metabolites were detected
in the brain homogenate. Thus, all specific [131I]3a binding
determined in the brain might be due to this ligand itself and
not influenced by its radioactive metabolites. This result suggests
that [131I]3a could serve as an in vivo imaging agent for PBR
in the brain.
Conclusion
In this study, two novel iodinated compound, 3a and 3b, were
designed, synthesized, and evaluated for PBR imaging in rodent
brains. [131I]3a and [131I]3b were prepared by no carrier-added
[
131I]NaI treatment of their corresponding tributylstannyl precur-
sors (10a and 10b) with I+ generated in situ by H2O2 oxidation
of 131I-. In vitro binding assays for the rat sections determined
that 3a had potent affinity and exhibited high selectivity for
PBR over CBR, whereas 3b was weaker than 3a. A biodistri-
bution study determined that [131I]3a had high uptake in the
mouse brain and good specific binding in regions rich in PBR.
An ex vivo autoradiography study provided visual evidence that
specific binding of [131I]3a to PBR occurred in the rat brain.
This study has succeeded in developing a potential iodinated
ligand [131I]3a which has high specific binding for PBR in rodent
5-Bromo-2-phenoxyaniline (6b). A mixture of 5b (1.0 g, 3.4
mmol), Fe powder (0.63 g, 11.0 mmol), and NH4Cl (91 mg, 1.7
mmol) in a mixture of EtOH (10 mL) and water (3 mL) was stirred
at 80 °C for 5 h. The reaction mixture was quenched with AcOEt,
and the separated organic layer was washed with water. After the
organic layer was dried over Na2SO4, the solvent was removed to
1
give 6b (0.84 g, 94%) as a pale brown solid. Mp: 47-48 °C. H

Iodinated Radioligand for PBR in Brain
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 853
NMR (CDCl3) δ: 7.30 (2H, t, J ) 7.9 Hz), 7.07 (1H, t, J ) 7.9
Hz), 6.94-6.97 (3H, m), 6.80 (1H, dd, J ) 2.2, 8.4 Hz), 6.70 (1H,
d, J ) 8.4 Hz), 3.85 (2H, br). FABMS (m/z): 264 (M+ + 1), 266
(M+ + 3). HRMS (FAB) Calcd for C12H10BrNO: 263.9958.
Found: 263.9991.
1.43 (12H, m), 0.81-0.95 (15H, m). HRMS (FAB) Calcd for
C34H46FNO3Sn: 654.2489. Found: 654.2451.
N-(2,5-Dimethoxybenzyl)-N-(2-phenoxy-5-tri-n-butylstannylphe-
nyl)acetamide (10b). The solid 9b (584 mg, 1.5 mmol) was
dissolved in dry toluene (10 mL) which had been degassed by
bubbling nitrogen gas through it for 5 min. Tetrakis(triphenyphos-
phine)palladium (0) (173 mg, 150 µmol) and bis(tri-n-butyl)tin (0.76
mL, 1.51 mmol) were successively added. The reaction mixture
was refluxed for 18 h under an argon atmosphere. After cooling,
the volatiles were evaporated under reduced pressure. The residue
was chromatographed on silica gel with AcOEt/hexane (1/5) to
afford 10b (450 mg, 45%) as a yellowish oil. 1H NMR (300 MHz,
CDCl3) δ: 7.32 (1H, t, J ) 7.8 Hz), 7.22 (1H, d, J ) 7.9 Hz), 7.14
(1H, t, J ) 7.3 Hz), 6.86-6.99 (5H, m), 6.81 (1H, d, J ) 7.9 Hz),
6.62-6.71 (2H, m), 5.20 (1H, d, J ) 14.5 Hz), 4.65 (1H, d, J )
14.5 Hz), 3.66 (3H, s), 3.50 (3H, s), 1.94 (3H, s), 1.25-1.51 (12H,
m), 0.84-0.98 (15H, m). HRMS (FAB) Calcd for C35H49NO4Sn:
666.2693. Found: 666.2726.
N-Acetyl-5-bromo-2-phenoxyaniline (7b). To a solution of 6b
(0.70 g, 2.7 mmol) and Et3N (0.46 mL, 3.3 mmol) in CH2Cl2 (6
mL) was added acetyl chloride (0.45 mL, 3.0 mmol) in a dropwise
manner under cooling in an ice-bath, and the mixture was stirred
at room temperature for 3 h. After the reaction mixture was
concentrated under reduced pressure, the residue was quenched with
AcOEt and washed with water and a saturated NaCl solution. After
the organic layer was dried over Na2SO4, the solvent was removed
to give a residue. The residue was chromatographed on silica gel
with AcOEt/hexane (1/5) to obtain 7b (0.69 g, 85%) as a pale
1
yellow oil. H NMR (CDCl3) δ: 8.66 (1H, d, J ) 2.0 Hz), 7.72
(1H, br), 7.38 (2H, t, J ) 8.0 Hz), 7.18 (1H, t, J ) 7.4 Hz), 7.10
(1H, dd, J ) 2.4, 11.0 Hz), 7.01 (2H, d, J ) 7.9 Hz), 6.68 (1H, d,
J ) 8.6 Hz), 2.18 (3H, s). FABMS (m/z): 306 (M+ + 1); 308 (M+
+ 3). HRMS (FAB) Calcd for C14H12BrNO2: 306.0201. Found:
306.0165.
N-(5-Fluoro-2-phenoxyphenyl)-N-(2-iodo-5-methoxybenzyl)-
acetamide (3a). To a solution of 10a (230 mg, 0.35 mmol) in CH2-
Cl2 (5 mL) was added iodine solid (260 mg, 1.0 mmol) in a
dropwise manner at room temperature. The reaction mixture was
stirred at room temperature for 3 h. After the reaction was
completed, the saturated sodium metabisulfite solution was added
until the color of the reaction mixture disappeared entirely. The
organic layer was collected, and the reaction mixture was extracted
with CH2Cl2 for a further two times. After the combined organic
layer was washed with water and saturated NaCl solution and dried
over Na2SO4, the solvent was removed to give a residue. The residue
was chromatographed on silica gel with AcOEt/hexane (1/5) to give
3a (140 mg, 81%) as a yellowish oil. HPLC determination: (1)
11.2 min; 3.9 mm ID × 150 mm, µ-Porasin (Waters); 1 mL/min,
hexane/AcOEt, 9/1, UV at 254 nm. (2) 8.6 min; 4.6 mm ID × 250
mm, Capcell Pak C18 (SHISEIDO); 1.5 mL/min, CH3CN/H2O, 8/2,
N-(2-Bromo-5-methoxybenzyl)-N-(5-fluoro-2-phenoxypheny-
l)acetamide (9a). To a solution of 7a36 (4.52 g, 18.4 mmol) in
DMF (20 mL) was added NaH (60% dispersion in mineral oil, 0.80
g, 20 mmol), and the mixture was stirred for 1 h at room
temperature. To the mixture was added 2-bromo-5-methoxybenzyl
bromide (8a; 5.04 g, 18 mmol), which was prepared by reacting
2-bromo-5-methoxytoluene with N-bromosuccinimide. The reaction
mixture was stirred for 5 h at room temperature. The mixture was
poured into ice water and extracted with AcOEt three times. The
combined organic layer was washed with 0.5 M aqueous HCl and
saturated NaCl solution. After the organic layer was dried over Na2-
SO4, the solvent was removed to give a residue. This residue was
chromatographed on silica gel with AcOEt/hexane (1/5) to afford
1
UV at 254 nm. H NMR (CDCl3) δ: 7.57 (1H, d, J ) 8.6 Hz),
1
9a (5.33 g, 65%) as a yellowish oil. H NMR (CDCl3) δ: 7.29-
7.32 (2H, t, J ) 7.9 Hz), 7.12 (1H, t, J ) 7.3 Hz), 7.04 (1H, d, J
) 2.9 Hz), 6.93-6.99 (1H, m), 6.80-6.89 (4H, m), 6.49 (1H, dd,
J ) 2.9, 8.6 Hz), 5.15 (1H, d, J ) 15.1 Hz), 4.69 (1H, d, J ) 15.1
Hz), 3.66 (3H, s), 2.00 (3H, s). Anal. (C22H19FINO3) C, H, N.
HRMS (FAB) Calcd for C22H19FINO3: 492.0574. Found: 492.0523.
N-(2,5-Dimethoxybenzyl)-N-(5-iodo-2-phenoxyphenyl)aceta-
mide (3b). To a solution of 10b (300 mg, 0.45 mmol) in CH2Cl2
(5 mL) was added iodine solid (345 mg, 1.38 mmol) in a dropwise
manner at room temperature. The reaction mixture was stirred at
room temperature for 3 h. After the reaction was completed, the
saturated sodium metabisulfite solution was added until the color
of the reaction mixture disappeared. The organic layer was collected,
and the reaction mixture was extracted with CH2Cl2 for further two
times. After the combined organic layer was washed with water
and saturated NaCl solution and dried over Na2SO4, the solvent
was removed to give a residue. The residue was chromatographed
on silica gel with AcOEt/hexane (1/5) to give 3b (180 mg, 80%)
as a white solid. HPLC determination: (1) 10.0 min; 3.9 mm ID
× 150 mm, µ-Porasin (Waters); 1 mL/min, hexane/AcOEt, 9/1,
UV at 254 nm. (2) 9.5 min; 4.6 mm ID × 250 mm, Capcell Pak
C18 (SHISEIDO); 1.5 mL/min, CH3CN/H2O, 8/2, UV at 254 nm.
7.31 (3H, m), 7.12 (1H, t, J ) 7.3 Hz), 6.92-7.02 (2H, m), 6.81-
6.89 (4H, m), 6.62 (1H, d, J ) 8.8 Hz), 5.18 (1H, d, J ) 15.1 Hz),
4.74 (1H, d, J ) 15.1 Hz), 3.66 (3H, s), 2.00 (3H, s). HRMS (FAB)
Calcd for C22H19BrFNO3: 444.0520. Found: 444.0565.
N-(5-Bromo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)ace-
tamide (9b). To a solution of 7b (3.58 g, 11.7 mmol) in DMF (20
mL) was added NaH (60% dispersion in mineral oil, 0.80 g, 20
mmol), and the mixture was stirred for 1 h at room temperature.
To this mixture was added 2,5-dimethoxybenzyl chloride (8b; 3.02
g, 16.5 mmol), which was prepared by reacting 2,5-dimethoxy-
benzyl alcohol with concentrated HCl. The reaction mixture was
stirred for 2 h at room temperature. The mixture was poured into
ice-water and extracted with AcOEt three times. The combined
organic layer was washed with 0.5 M aqueous HCl and saturated
NaCl solution. After the organic layer was dried over Na2SO4, the
solvent was removed to give a residue. This residue was chro-
matographed on silica gel with AcOEt/hexane (1:5) to obtain 9b
1
(5.06 g, 94.8%) as a white solid. Mp: 118.5-119.5 °C. H NMR
(CDCl3) δ: 7.29-7.35 (3H, m), 7.12-7.21 (2H, m), 6.95 (1H, d,
J ) 2.8 Hz), 6.84 (2H, d, J ) 7.7 Hz), 6.66-6.75 (3H, m), 5.05
(1H, d, J ) 14.3 Hz), 4.73 (1H, d, J ) 14.3 Hz), 3.67 (3H, s), 3.55
(3H, s), 1.97 (3H, s). HRMS (FAB) Calcd for C23H22BrNO4:
456.0851. Found: 456.0831.
1
Mp: 114.5-115.0 °C. H NMR (CDCl3) δ: 7.45 (1H, d, J ) 8.6
Hz), 7.30-7.38 (3H, m), 7.14 (1H, t, J ) 7.3 Hz), 6.95 (1H, d, J
) 2.8 Hz), 6.84 (2H, d, J ) 7.9 Hz), 6.66-6.75 (2H, m), 6.55
(1H, d, J ) 8.6 Hz), 5.03 (1H, d, J ) 14.3 Hz), 4.73 (1H, d, J )
14.3 Hz), 3.66 (3H, s), 3.56 (3H, s), 1.97 (3H, s). Anal. (C23H22-
INO4) C, H, N. HRMS (FAB) Calcd for C23H22INO4: 504.0672.
Found: 504.0673.
N-(5-Fluoro-2-phenoxyphenyl)-N-(5-methoxy-2-tri-n-butyl-
stannylbenzyl)acetamide (10a). The oil 9a (1.02 g, 2.30 mmol)
was dissolved in dry toluene (20 mL) which had been degassed by
bubbling nitrogen gas through it for 5 min. Tetrakis(triphenylphos-
phine)palladium (0) (266 mg, 230 µmol) and bis(tri-n-butyl)tin (1.16
mL, 2.31 mmol) were successively added. The reaction mixture
was refluxed for 90 h under a nitrogen atmosphere. After cooling,
the volatiles were evaporated under reduced pressure. The residue
was chromatographed on silica gel with AcOEt/hexane (1/5) to
afford 10a (320 mg, 21%) as a yellowish oil. 1H NMR (CDCl3) δ:
7.10-7.34 (4H, m), 6.72-7.00 (7H, m), 5.25 (1H, d, J ) 15.2
Hz), 4.37 (1H, d, J ) 15.2 Hz), 3.70 (3H, s), 2.03 (3H, s), 1.18-
N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[131I]iodo-5-methoxyben-
zyl)acetamide ([131I]3a). Aqueous hydrogen peroxide (10 µL, 30%
w/v) was added to a mixture of 10a (100 µL, 1 mg/mL in ethanol),
acetic acid (100 µL), and [131I]NaI (18 MBq, 10 µL in 0.1M NaOH,
specific activity: >30 GBq/µmol; Perkin-Elmer, Yokohama) in a
sealed vial. The reaction was allowed to proceed for 30 min at
room temperature, after which it was terminated by the addition of
sodium metabisulfite (100 µL, 100 mg/mL in water). The reaction

854 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Zhang et al.
mixture was injected onto a reverse-phase HPLC column (10 mm
ID × 250 mm, HDS80 C18 (YMC); 4 mL/min, CH3CN/H2O, 9/1,
UV at 254 nm). The fraction eluted at 11.4 min containing [131I]3a
was collected and evaporated with a nitrogen gas flow. The final
product [131I]3a was taken up in an isotonic saline solution (1 mL)
and passed through a 0.22 µm filter into a sterile, pyrogen-free
bottle with a radiochemical purity of >98% (10 MBq).
Biodistribution Study. A saline solution of [131I]3a or [131I]3b
(average of 0.5 MBq/100 µL), was injected into ddy mice (n )
3/[131I]ligand; 30-40 g, 9 weeks, male, SLC, Shizuoka, Japan)
through the tail vein. The animals were sacrificed by cervical
dislocation at 1, 5, 15, 30, or 60 min after injection. The whole
brain was quickly removed and bisected, and one-half was weighed.
The radioactivity present in the brains was determined with a
Packard autogamma scintillation counter. The percent dose per gram
of brain (% dose/g) was calculated by comparison of the decay-
corrected brain counts with the decay-corrected counts of suitably
diluted aliquots of the injected material.
From another half brain, the cerebellum, olfactory bulb, striatum,
hippocampus, thalamus, hypothalamus, and cerebral cortex were
further dissected and weighed. The radioactivity present in these
tissues was measured as described above.
Blocking Study. To determine in vivo specificity and selectivity
of [131I]3a binding to PBR, 1a or 2a at a dose of 1 mg/kg each was
mixed with [131I]3a (0.5 MBq/100 µL) and injected into ddy mice
(n ) 3; 30-40 g, 9 weeks, male, SLC, Shizuoka, Japan),
respectively. At 30 min after injection, these mice were sacrificed
by cervical dislocation and the whole brains were removed quickly.
The brain tissue samples (cerebellum, olfactory bulb, striatum,
hippocampus, thalamus, hypothalamus, and cerebral cortex) were
dissected and treated as described above.
N-(2,5-Dimethoxybenzyl)-N-(5-[131I]iodo-2-phenoxyphenyl)-
acetamide ([131I]3b). Aqueous hydrogen peroxide (10 µL, 30% w/v)
was added to a mixture of 10b (100 µL, 1 mg/mL in ethanol), acetic
acid (100 µL), and [131I]NaI (18 MBq, 10 µL in 0.1M NaOH,
specific activity: >30 GBq/µmol; Perkin-Elmer, Yokohama) in a
sealed vial. The reaction was allowed to proceed for 30 min at
room temperature, after which it was terminated by the addition of
sodium metabisulfite (100 µL, 100 mg/mL in water). The reaction
mixture was injected onto a reverse-phase HPLC column (10 mm
ID × 250 mm, HDS80 C18 (YMC); 4 mL/min, CH3CN/H2O, 9/1,
UV at 254 nm). The fraction eluted at 9.5 min containing [131I]3b
was collected and evaporated with a nitrogen gas flow. The final
product [131I]3b was taken up in an isotonic saline solution (1.0
mL) and passed through a 0.22 µm filter into a sterile, pyrogen-
free bottle with a radiochemical purity of > 98% (8 MBq).
Identity Confirmation and Radiochemical Purity Determi-
nation. A solution of [131I]3a or [131I]3b (1 mL) containing
nonradioactive 3a or 3b (1 mg/5 mL CH3CN) was spotted on a
silica gel TLC plate, which was then developed with AcOEt/hexane
(1/1) as a mobile phase. The TLC plate was air-dried and placed
in contact with a phosphor imaging plate (BAS-SR 127, Fuji Photo
Film, Tokyo) for 10 min, and the radioactivity distribution on the
plate was analyzed using a FUJI BAS 1800II bioimaging analyzer
(Fuji Photo Film, Tokyo). [131I]3a or [131I]3b was identified by
comparing the Rf with the nonradioactive 3a (0.57) or 3b (0.53),
visualized by UV lamp at 254 nm.
Aliquots of the formulated solutions were used to establish
chemical and radiochemical purity employing analytical HPLC (4.6
mm ID × 250 mm, LiChrosphor RP-18e (Merck); CH3CN/H2O,
7/3, UV at 254 nm). The retention times for [131I]3a and [131I]3b
were 10.3 and 9.9 min at a flow rate of 1.0 mL/min, respectively.
In Vitro Binding Assays. Male Sprague-Dawley rats (n ) 4)
weighing 220-250 g were killed by decapitation under ether
anesthesia, and their brains were quickly removed and frozen on
powdered dry ice. Brain sagittal sections (20 µm) were cut on a
cryostat microtome (HM560, Carl Zeiss, Germany) and thaw-
mounted on glass slides (Matsunami Glass Ind., Tokyo), which were
then dried at room temperature and stored at -18 °C until used for
experiments. The brain sections were preincubated at 25 °C for 20
min in 50 mM Tris-HCl (pH 7.4) buffer. After preincubation, these
sections were incubated at 37 °C for 30 min in the assay buffer
containing [11C]1a (1 nM, specific activity: 110 GBq/µmol), [11C]-
2a (1 nM, 50 GBq/µmol), or [11C]flumazenil (1 nM, 210 GBq/
µmol). To determine the IC50 values of 1a, 2a, 3a, and 3b for PBR)
or CBR binding, brain sections were incubated with [11C]ligands
in the presence of increasing concentrations of the corresponding
ligands (0.1 nM-1 µM). Nonradioactive 1a and flumazenil (1 µM)
were used to determine nonspecific bindings for PBR and CBR,
respectively. After incubation, the sections were washed three times
for 2 min each time with cold assay buffer, dipped in cold distilled
water, and dried with warm air. These sections were then placed
in contact with imaging plates (BAS-SR 127, Fuji Photo Film,
Tokyo) for 60 min to analyze the distribution of their radioactivity
with a FUJIX BAS 1800II bioimaging analyzer (Fuji). The region
of interest (ROI) on the sections was placed on the cerebellum and
the radioactivity in ROI was expressed as photostimulated-
luminescence (PSL) values. PSL data corresponding to the radio-
activity on the cerebellum in the presence and absence of the
displaced ligand were determined. Specific binding for PBR or CBR
was defined as total binding minus binding in the presence of 1a
or flumazenil (1 µM). The PSL data corresponding to specific
binding at each compound concentration were calculated as
percentages in relation to control specific bindings, which were
converted to probit values to determine the IC50 of each compound.
Ex Vivo Autoradiography. A saline solution of [131I]3a (2 MBq/
100 µL) was injected into a male Sprague-Dawley rat (220 g, 8
weeks, SLC, Shizuoka, Japan) through the tail vein. At 30 min
after injection, the rat was sacrificed by decapitation under ether
anesthesia. The brain was quickly removed and frozen on powdered
dry ice. Brain sagittal sections (20 µm) were cut on a cryostat
microtome (HM560), thaw-mounted on glass slides, and dried with
warm air. These sections were then placed in contact with imaging
plates (BAS-SR 127) for one week to analyze radioactivity
distribution with a bioimaging analyzer. The radioactivity in the
region of interest was expressed as photostimulated-luminescence
(PSL) value per mm2 region.
For the inhibition experiment, a mixture of [131I]3a (2 MBq/100
µL) and 1a (1 mg/kg, 10% EtOH in 200 µL saline) was used as
described above.
In Vivo Stability of [131I]3a in the Mouse Brains. After iv
injection of [131I]3a (0.2 MBq/100 µL) into ddy mice (n ) 3), these
mice were sacrificed by cervical dislocation at 30 min. The whole
brain samples were removed quickly. The cerebellum and forebrain
including the olfactory bulb were dissected from the mouse brain
and homogenized together in an ice-cooled CH3CN/H2O (1/1, 1.0
mL) solution. The homogenate was centrifuged at 15 000 rpm for
10 min at 4 °C, and the supernatant was collected.
An aliquot of the supernatant (10 µL) containing trace of
nonradioactive 3a was spotted on a silica gel TLC plate, which
was then developed with AcOEt/hexane (1/ 1) as a mobile phase.
The TLC plate was air-dried and placed in contact with a phosphor
imaging plate (BAS-SR 127) for 10 days, and the radioactivity
distribution on the plate was analyzed using a FUJI BAS 1800II
bioimaging analyzer. [131I]3a was identified by comparing the Rf
with the nonradioactive 3a (0.57) visualized by UV lamp at 254
nm. The percent ratio of the unchanged [131I]3a to the total
radioactivity (corrected for decay) on the TLC chromatogram was
calculated as % ) (peak area for [131I]3a/total radioactive peak
area) × 100.
Acknowledgment. We thank Taisho Pharmaceutical Co.,
Ltd. (Tokyo, Japan) for providing DAA1106 (1).
Supporting Information Available: Elemental analyses, HPLC
purities, and analytical HPLC charts, using reversed-phase and
normal-phase solvents, of the target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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