Development of a New Radioligand, N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[ 18F]fluoroethyl-5-methoxybenzyl)acetamide, for PET Imaging of Peripheral Benzodiazepine Receptor in Primate Brain

Article abstract of DOI:10.1021/jm0304919

To develop a positron emission tomography (PET) ligand for imaging the 'peripheral benzodiazepine receptor' (PBR) in brain and elucidating the relationship between PBR and brain diseases, four analogues (4-7) of N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (2) were synthesized and evaluated as ligands for PBR. Of these compounds, fluoromethyl (4) and fluoroethyl (5) analogues had similar or higher affinities for PBR than the parent compound 2 (K_i = 0.16 nM for PBR in rat brain sections). Iodomethyl analogue 6 displayed a moderate affinity, whereas tosyloxyethyl analogue 7 had weak affinity. Radiolabeling was performed for the fluoroalkyl analogues 4 and 5 using fluorine-18 (¹⁸F, β⁺; 96.7%, T_1/2 = 109.8 min). Ligands [¹⁸F] 4 and [¹⁸F] 5 were respectively synthesized by the alkylation of desmethyl precursor 3 with [¹⁸F]fluoromethyl iodide ([¹⁸F]8) and 2-[¹⁸F] fluoroethyl bromide ([¹⁸F]9). The distribution patterns of [ ¹⁸F]4 and [¹⁸F]5 in mice were consistent with the known distribution of PBR. However, compared with [¹⁸F]5, [¹⁸F] 4 displayed a high uptake in the bone of mice. The PET image of [ ¹⁸F]4 for monkey brain also showed significant radioactivity in the bone, suggesting that this ligand was unstable for in vivo defluorination and was not a useful PET ligand. Ligand [¹⁸F]5 displayed a high uptake in monkey brain especially in the occipital cortex, a region with richer PBR than the other regions in the brain. The radioactivity level of [ ¹⁸F]5 in monkey brain was 1.5 times higher than that of [ ¹¹C]2, and 6 times higher than that of (R)-(1-(2-chlorophenyl)-N-[¹¹C]methyl,N-(1-methylpropyl)isoquinoline ([¹¹C]1). Moreover, the in vivo binding of [¹⁸F]5 was significantly inhibited by PBR-selective 2 or 1, indicating that the binding of [¹⁸F]5 in the monkey brain was mainly due to PBR. Metabolite analysis revealed that [¹⁸F]4 was rapidly metabolized by defluorination to [¹⁸F] F- in the plasma and brain of mice, whereas [¹⁸F]5 was metabolized by debenzylation to a polar product [ ¹⁸F]13 only in the plasma. No radioactive metabolite of [ ¹⁸F]5 was detected in the mouse brain. The biological data indicate that [¹⁸F]5 is a useful PET ligand for PBR and is currently used for imaging PBR in human brain.

Full text of DOI:10.1021/jm0304919

2228
J . Med. Chem. 2004, 47, 2228-2235
Develop m en t of a New Ra d ioliga n d ,
N-(5-F lu or o-2-p h en oxyp h en yl)-N-(2-[18F ]flu or oeth yl-5-m eth oxyben zyl)a ceta m id e,
for P ET Im a gin g of P er ip h er a l Ben zod ia zep in e Recep tor in P r im a te Br a in
Ming-Rong Zhang,*,†,§ J un Maeda,‡,§ Masanao Ogawa,†,§ J unko Noguchi,†,§ Takehito Ito,†,§ Yuichiro Yoshida,†,§
Takashi Okauchi,‡,§ Shigeru Obayashi,‡ Tetsuya Suhara,‡ and Kazutoshi Suzuki†
Department of Medical Imaging, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,
Chiba 263-8555, J apan; Brain Imaging Project, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,
Chiba 263-8555, J apan; and SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, J apan
Received October 1, 2003
To develop a positron emission tomography (PET) ligand for imaging the ‘peripheral benzo-
diazepine receptor’ (PBR) in brain and elucidating the relationship between PBR and brain
diseases, four analogues (4-7) of N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)-
acetamide (2) were synthesized and evaluated as ligands for PBR. Of these compounds,
fluoromethyl (4) and fluoroethyl (5) analogues had similar or higher affinities for PBR than
the parent compound 2 (Ki ) 0.16 nM for PBR in rat brain sections). Iodomethyl analogue 6
displayed a moderate affinity, whereas tosyloxyethyl analogue 7 had weak affinity. Radiola-
beling was performed for the fluoroalkyl analogues 4 and 5 using fluorine-18 (18F, â+; 96.7%,
T1/2 ) 109.8 min). Ligands [18F]4 and [18F]5 were respectively synthesized by the alkylation of
desmethyl precursor 3 with [18F]fluoromethyl iodide ([18F]8) and 2-[18F]fluoroethyl bromide
([18F]9). The distribution patterns of [18F]4 and [18F]5 in mice were consistent with the known
distribution of PBR. However, compared with [18F]5, [18F]4 displayed a high uptake in the
bone of mice. The PET image of [18F]4 for monkey brain also showed significant radioactivity
in the bone, suggesting that this ligand was unstable for in vivo defluorination and was not a
useful PET ligand. Ligand [18F]5 displayed a high uptake in monkey brain especially in the
occipital cortex, a region with richer PBR than the other regions in the brain. The radioactivity
level of [18F]5 in monkey brain was 1.5 times higher than that of [11C]2, and 6 times higher
than that of (R)-(1-(2-chlorophenyl)-N-[11C]methyl,N-(1-methylpropyl)isoquinoline ([11C]1).
Moreover, the in vivo binding of [18F]5 was significantly inhibited by PBR-selective 2 or 1,
indicating that the binding of [18F]5 in the monkey brain was mainly due to PBR. Metabolite
analysis revealed that [18F]4 was rapidly metabolized by defluorination to [18F]F- in the plasma
and brain of mice, whereas [18F]5 was metabolized by debenzylation to a polar product [18F]13
only in the plasma. No radioactive metabolite of [18F]5 was detected in the mouse brain. The
biological data indicate that [18F]5 is a useful PET ligand for PBR and is currently used for
imaging PBR in human brain.
Sch em e 1. Chemical Structures of [11C]1 and [11C]2
Analogues
In tr od u ction
‘Peripheral benzodiazepine receptor’ (PBR), which
was initially identified in the peripheral tissues, is
located on the mitochondrial outer membrane in several
organs including the kidney, nasal epithelium, lung,
heart, and endocrine organs such as the adrenal, testis,
and pituitary gland.1-3 Later studies further demon-
strated the presence of PBR in the central nervous
system.4-6 The PBR density in the brain was increased
in injured brain, and this increase has been used as an
indicator of neuronal damage7 and several neuroden-
erative disorders, such as Alzheimer’s disease,8 Hun-
tington’s disease,9 Wernicke’s encephalopathy,10 mul-
tiple sclerosis,11 and stroke.12 In addition, a relationship
between alterations of PBR and epileptic foci have been
proposed.13,14 These findings have resulted in the de-
velopment of a radioligand labeled by a positron-emitter,
which has made possible to visualize the distribution
of PBR in the brain.15-19
* Corresponding author: Tel: 81-43-206-4041; Fax: 81-43-206-3261;
E-mail: zhang@nirs.go.jp.
(R)-(1-(2-Chlorophenyl)-N-[11C]methyl,N-(1-methyl-
propyl)isoquinoline ([11C](R)-PK 11195, [11C]1, Scheme
1) is a clinically useful positron emission tomography
(PET) ligand for imaging PBR in cases of several brain
† Department of Medical Imaging, National Institute of Radiological
Sciences.
‡ Brain Imaging Project, National Institute of Radiological Sciences.
§ SHI Accelerator Service Co. Ltd.
10.1021/jm0304919 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/26/2004

Radioligand for PET Imaging of Benzodiazepine Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2229
Sch em e 2. Chemical Synthesis of Compounds 4-7a
diseases.20-23 However, the signal of [11C]1 in the
primate brain was not high enough for quantitative
analysis using PET because of its low uptake into the
brain.23 Therefore, the precise characterization of PBR
in primate brain awaits development of new PET
ligands with improvements over [11C]1. In 1998, N-(2,5-
dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)aceta-
mide (DAA1106, 2, Scheme 1) was reported as a potent
and selective ligand for PBR by the Taisho Pharmaceu-
tical’s group.24-27 Compound 2 had a high affinity for
PBR in mitochondrial fractions of rat (Ki ) 0.043 nM)
and monkey (Ki ) 0.188 nM) brains.25 Moreover, 2 had
weak or negligible affinities (IC50 > 10 µM) for other
receptors, ion channels, uptake/transporters, and second
messengers.26 Thus, we previously synthesized [11C]228
as a PET ligand for PBR by reacting the desmethyl
precursor N-(5-fluoro-2-phenoxyphenyl)-N-(2-hydroxy-
5-methoxybenzyl)acetamide24,27 (DAA1123, 3) with [11C]-
CH3I. The in vitro and in vivo evaluation using mouse,
rat and monkey demonstrated that [11C]2 had high
specific binding with PBR in the brains and about a
4-fold higher uptake of radioactivity in the monkey
brain than [11C]1.28,29 Now, [11C]2 is being used to
investigate PBR in the human brain in our facility.
a
Reagents and conditions: (a) NaH, DMF, 0 °C, 5 h, 79%; (b)
NaH, DMF, 80 °C, 8 h, 65%; (c) NaH, DMF, 25 °C , 3 h, 34%; (d)
NaH, DMF, 0 °C , 5 h, 46%.
Sch em e 3. Radiosynthesis of [18F]4 and [18F]5 by Two
Methodsa
In general, the short half-life of a 11C labeled ligand
(â+; 99.8%, T1/2 ) 20.4 min) often limits its usefulness
if a dynamic PET experiment has a turnover time longer
than 100 min. Since 18F has advantages over 11C, with
a longer half-life (110 min vs. 20 min) and a lower
positron energy (650 keV vs 960 keV), an 18F-labeled
ligand gives higher quality images with a higher spatial
resolution in PET measurements. Moreover, 18F is
convenient for long-time storage and long-distance
transportation to other facilities. To develop 18F-labeled
PET ligand with improved properties compared to
[11C]2, we previously prepared two [18F]fluoroalkyl
analogues of 2: N-(5-fluoro-2-phenoxyphenyl)-N-(2-
[18F]fluoromethyl-5-methoxybenzyl)acetamide ([18F]FM-
DAA1106, [18F]4) and N-(5-fluoro-2-phenoxyphenyl)-N-
(2-[18F]fluoroethyl-5-methoxybenzyl)acetamide ([18F]-
FEDAA1106, [18F]5) as putative radioligands for PBR
(Scheme 1).30 Preliminary studies revealed that ana-
logues 4 and 5 have similar or higher inhibitory activity
(IC50) for PBR in the rat brain than 2.30 The ex vivo
autoradiograms of [18F]4 and [18F]5 binding sites in rat
brain revealed significantly high radioactivity in the
olfactory bulb and cerebellum, the richer regions of PBR
compared with other brain regions.30
a
Reagents and conditions: (a) 110 °C, 10 min, 8%; (b) NaH,
DMF, -15 °C, 3 min, 90%; (c) o-dichlorobenzene, 130 °C, 5 min,
55%; (d) NaH, DMF, -15 °C, 10 min and then 120 °C, 10 min,
82%; (e) DMF, 30-120 °C, 1-15 min, 2-37%; (f) DMF, 30-120
°C, 1-15 min, 2-60%.
Resu lts a n d Discu ssion
Ch em istr y. The four analogues 4-7 were synthe-
sized according to the reaction sequences lineated in
Scheme 2. Reaction of the desmethyl precursor 324,27
with excess fluoromethyl iodide31 (FCH2I, 8) in the
presence of NaH at 0 °C for 5 h gave the fluoromethyl
analogue 4. Treatment of 3 with 2-fluoroethyl bromide
(FCH2CH2Br, 9) and NaH at 80 °C gave the fluoroethyl
analogue 5. The iodomethyl analogue 6 was prepared
by the reaction of 3 with diiodomethane (CH2I2, 10) and
NaH at 25 °C for 3 h. The tosyloxyethyl analogue 7 was
obtained by treating 3 with excess ethylene ditosylate
(11) and NaH at 0 °C.
In this study, we synthesized four analogues (4-7,
Scheme 2) including two novel compounds: (N-(5-fluoro-
2-phenoxyphenyl)-N-(2-iodomethyl-5-methoxybenzyl)ac-
etamide (6) and N-(5-fluoro-2-phenoxyphenyl)-N-(5-
methoxy-2-tosyloxyethylbenzyl)acetamide (7). Using
autoradiography technique, we measured the binding
affinities (Ki) of 4-7 to PBR and the central benzodi-
azepine receptor (CBR). We labeled the fluoroalkyl
analogues 4 and 5 using 18F and prepared the radioac-
tive products in reproducible radiochemical yields. We
examined the regional distribution of [18F]4 and [18F]5
in mice and measured the percentages of unmetabolized
[18F]4 and [18F]5 in the plasma and brain. Using PET,
we determined the uptake of [18F]4 and [18F]5 and
elucidated the specific binding of [18F]5 to PBR in the
monkey brain.
Ra d iosyn th esis. Ligands [18F]4 and [18F]5 were
prepared by two methods according to Scheme 3. The
first method was a two-step reaction sequence which
involved the preparation of the radioactive interme-
diate [18F]fluoromethyl iodide ([18F]FCH2I, [18F]8) or
2-[18F]fluoroethyl bromide ([18F]FCH2CH2Br, [18F]9),
followed by the alkylation of 3 with [18F]8 or [18F]9. The
second method was direct nucleophilic replacement of
the iodomethyl analogue 6 or tosyloxyethyl analogue 7

2230 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Zhang et al.
with [18F]F-. As for the two-step method, the alkylating
intermediate [18F]8 or [18F]9 was prepared by the
reaction of [18F]F- with 10 or 2-trifluoromethanesulfo-
nyloxyethyl bromide (BrCH2CH2OTf, 12) by using a
newly developed automated system.32,33 After the fluo-
rine reaction finished, [18F]8 or [18F]9 was purified by
distillation and directly trapped in a solution of DMF
containing 3 and NaH at -15 °C. The purification of
[18F]8 (bp: 53-54 °C) or [18F]9 (bp: 71.5 °C) by distil-
lation was effective, because this procedure can leave
behind all nonvolatile impurities such as metal ions
from the cyclotron target, the unreacted [18F]F-, and
the phase-transfer reagent Kryptofix 222/K2CO3. After
the radioactive [18F]8 or [18F]9 trapping ended, the
[18F]fluoromethylation finished perfectly, while the
[18F]fluoroethylation required a further 10 min at 120
°C. As for the direct method, heating 6 and 7 with
[18F]F- at 30-120 °C gave [18F]4 and [18F]5. However,
the radiochemical yields were not reproducible (2%-
60%), and purifying [18F]4 and [18F]5 from the reaction
mixtures was often difficult since the many impurities
resulting from the target and reaction greatly reduced
the efficiency. After comparing the reaction conditions
and radiochemical yields, we synthesized [18F]4 and
[18F]5 by the two-step reaction method (Method 1) via
the intermediates [18F]8 and [18F]9, respectively.
Ta ble 1. In Vitro Binding Affinity (Ki) for PBR and CBR, and
Octanol/Phosphate Buffer Distribution Coefficient (log D)
Ki(nM)a
ligand
R
PBRb
CBRc
log Dd
4
5
6
7
2
1
FCH2
FCH2CH2
ICH2
TsOCH2CH2
CH3
0.17 ( 0.02
0.078 ( 0.01
4.8 ( 0.68
18.1 ( 1.30
0.16 ( 0.02
0.83 ( 0.24
>1000
>1000
>1000
>1000
>1000
>1000
3.70
3.81
4.02
4.65
3.65
2.78
a
Values represent the mean obtained from 9 concentrations of
b
compound using at least 8 slices of rat brain (n ) 3).
[
11C]2
was incubated in the presence of the compounds examined.
c
[
11C]Flumazenil was incubated in the presence of the compounds
d
examined. 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%.
6 may become a suitable candidate of a SPECT ligand
for imaging PBR. Similarly, the binding affinities of
4-7 for CBR were measured using CBR-selective
[11C]flumazenil. As shown in Table 1, 4-7 did not
display significant inhibitory effects (Ki > 1 µM) on
[11C]flumazenil binding in the rat brain. The negligible
affinities of these analogues for CBR may be due to a
difference from the typical benzodiazepine structure,
which resulted in high selectivity for PBR.
Reversed phase semipreparative HPLC purification
of the reaction mixtures gave [18F]4 and [18F]5 in 45 (
10% (n ) 4) and 12 ( 4% (n ) 4) radiochemical yields
based on [18F]F-, corrected for the physical decay in a
synthesis time of 63 ( 4 min and 50 ( 3 min. The
identity of the desired product was confirmed by coin-
jection with the corresponding nonradioactive 4 or 5 on
reverse phase analytic HPLC. In the final product
solutions, the radiochemical purity of [18F]4 or [18F]5
was higher than 98% and the specific activity was 40-
65 GBq/µmol for [18F]4 and 110-145 GBq/µmol for
[18F]5 as determined from the mass measured by HPLC/
UV analysis. No significant peak corresponding to 3 was
observed in the HPLC for the final products. Moreover,
the radiochemical purities of [18F]4 and [18F]5 remained
>95% after 180 min at 25 °C, and they were stable as
PET ligands while performing the evaluation.
Biod istr ibu tion on Mice. The radioactivity distri-
bution of [18F]4 and [18F]5 was measured in mice over
120 min (the data are shown in Supporting Informa-
tion). These results revealed that [18F]4 and [18F]5
showed a similar distribution pattern of radioactivity
in the regions examined except bone. As can be seen,
high radioactivity levels (>5% ID/g) were found in
tissues such as the lung, heart, kidney, and adrenal
gland, organs known to be rich in PBR. The distribution
patterns of uptake were in agreement with that of PBR
in the peripheral system, which was consistent with the
distribution of [11C]2 or [11C]1 in the mouse. The highest
accumulations of [18F]4 and [18F]5 were found in the
lung and these high levels were probably due to the high
mitochondrial contents containing PBR in the organ. On
the other hand, high radioactivities (2.6-4.4% ID/g for
[18F]4 and 2.2-4.9% ID/g for [18F]5) were also found in
the brain, the target tissue in this experiment. The
values were about 1.3-1.6-fold higher than that of
[11C]2, and 2-3-fold higher than that of [11C]1, which
was consistent with their higher partition coefficients
as shown in Table 1. These findings suggested that
[18F]4 and [18F]5 do pass across the brain-blood barrier
(BBB), which is a prerequisite for a promising PET
ligand.
In Vitr o Bin d in g Assa ys. The in vitro binding
affinities (Ki) of the four analogues 4-7 for PBR were
determined from competition for the [11C]2 binding to
PBR using quantitative autoradiography of rat brain
sections34,35 (Table 1). Among these compounds, the
fluoroethyl analogue 5 was the most active toward PBR.
The Ki value (0.078 nM) of 5 was 2-fold higher than that
of 2, and 10-fold higher than that of 1. This result
suggested that substituting the OCH3 group with OCH2-
CH2F group was favorable for augmenting its binding
affinity for PBR. The fluoromethyl analogue 4 displayed
a similar affinity to 2, suggesting that substitution with
a OCH2F group did not obviously affect the affinity. This
may be due to the molecular similarity and bioisoteric
property of the OCH2F and OCH3 groups. The iodom-
ethyl analogue 6 had 30 times lower affinity than 2,
while the tosyloxyethyl analogue 7 was 110 times
weaker than 2. These results suggested that the relative
bulk groups in this series were not favorable for the
binding of PBR. However, although it was moderately
potent (Ki ) 4.8 nM) for PBR, the iodomethyl analogue
In the bone of mouse, a significant difference in
radioactivity was observed between [18F]4 and [18F]5.
The accumulation of [18F]5 in the bone was initially low
and showed no increase from 30 min (0.31% dose/g) to
120 min (0.09% dose/g), which demonstrated the ex-

Radioligand for PET Imaging of Benzodiazepine Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2231
F igu r e 1. PET summation images of the monkey brain acquired between 30 and 180 min after [18F]ligand injection. The images
were obtained from the same subject. (a) [18F]4; (b) [18F]5; (c) [18F]5 after treatment with 2 (1 mg/kg); (d) [18F]5 after treatment
with 1 (5 mg/kg). Significant radioactivity was observed in the bone after injection of [18F]4 (a). High radioactivity was observed
in the occipital cortex after injection of [18F]5 (b), which was significantly inhibited by 2 (c) and 1 (d).
pected stability of the fluoroethyl group against in vivo
defluorination. In contrast, after injection of [18F]4, the
radioactivity began to accumulate in the bone and
reached 5.6% dose/g at 120 min, which was 50 times
higher than that of [18F]5 in the bone. On consideration
of the characteristic of [18F]F- to readily accumulate in
bone, [18F]4 may be decomposed to [18F]F- by in vivo
defluorination. Our previous findings also demonstrated
that [18F]fluoromethyl compounds are relatively un-
stable in comparison with the corresponding [18F]fluo-
roethyl and [11C]methyl compounds.33 Therefore, [18F]4
may not be a useful PET ligand, even if it entered the
brain and had some binding sites with PBR.
Mon k ey P ET. The uptake of [18F]4 and [18F]5 in
monkey brain was examined using PET. In the previous
in vitro study for postmortem human brain, the highest
density of PBR was observed in the occipital cortex,36
so the region of interest (ROI) in this PET experiment
was placed on the occipital cortex. Figure 1 shows
typical PET summation images of the monkey brain
acquired from 30 to 180 min after [18F]4 (a) and [18F]5
(b) injection (80-85 MBq/1.5 mL). The two images
displayed a high accumulation of radioactivity in the
brain especially in the occipital cortex. However, in
comparison with [18F]5, a marked accumulation of [18F]4
was observed in the bone. This image is visual evidence
that [18F]4 was decomposed to [18F]F- in vivo as
reflected in the high accumulation of [18F]4 in the mouse
bone. Therefore, no further evaluation of [18F]4 was
carried out using PET in monkey brain.
F igu r e 2. Time-activity curves (TACs) of [18F]5 in the
occipital cortex of monkey brain. The radioactivity of the
control (circles) was strongly inhibited by pretreatment with
2 (triangles) and 1 (squares).
Figure 2 (circles) shows the time activity curve (TAC)
of [18F]5 in the occipital cortex of monkey brain after
intravenous (iv) injection. At 2 min after injection, a
high level of radioactivity was observed in the occipital
cortex, which then remained almost same level during
PET measurement (180 min). The radioactivity of [18F]5
was 1.5 times higher than that of [11C]2,29 and 6 times
higher than that of [11C]129 at 30 min after injection.
Pretreatment with nonradioactive 2 (1.0 mg/kg) gave a
marked reduction of uptake for the image (Figure 1c)
and TAC (Figure 2: triangles) of [18F]5 as compared to
the control experiment which was obtained under the
same conditions. As shown in Figure 2, pretreatment
with 1 enhanced the initial maximal uptake of [18F]5.
The increase may be derived from [18F]5 dispossessed
by mass nonradioactive 2 from lung abundant in PBR.
Similar cases have been reported in the PET studies of
F igu r e 3. Percent conversion of [18F]4 or [18F]5 to metabolite
in the mouse plasma and brain at several time points after iv
injection of ligand (5-10 MBq) into the mice (n ) 3). The
unchanged tracers and metabolites were analyzed by HPLC
for CH3CN extracts from the plasma and brain homogenate
prepared as described in the Experimental Section.
[11C]136 and [11C]2.29 From 30 min after injection to the
end (180 min) of the PET scan, pretreatment with 2
reduced the level of radioactivity to about 20% of the
control. This result suggested high specific binding of
[18F]5 present in the occipital cortex. Pretreatment with
the PBR-selective 1 (5 mg/kg) also produced a significant
reduction of radioactivity in the occipital cortex, as
shown in the image (Figure 1d) and TAC (Figure 2:
squares). The radioactivity was reduced to about 30%

2232 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Zhang et al.
Sch em e 4. Metabolism of [18F]4 and [18F]5 in the Plasma and Brain
of the control from 30 min after injection to the end of
the PET scan. The reduction percentage on the uptake
of [18F]5 by 1 was slightly lower that by 2, which was
probably due to the weaker affinity of 1 for PBR and
its lower penetration27 into the brain than that of 2.
These findings confirmed that [18F]5 may have high
specific binding for PBR in the monkey brain.
Meta bolite An a lysis. In the imaging brain study,
the presence of metabolites in the plasma of the subject
may preclude the evaluation of a PET ligand if the
metabolites enter the brain and are retained/bound at
the target sites. Therefore, metabolite analyses were
performed for [18F]5 in the plasma and brain of mouse,
and the plasma of monkey. For comparison, similar
analyses were also performed for [18F]4.
Figure 3 shows the percentages of unchanged [18F]4
and [18F]5 in the plasma and brain homogenate of mice
measured by HPLC. After injection into the mouse, the
fraction corresponding to the unmetabolized [18F]4 in
the plasma or brain rapidly decreased to 32% or 35%
at 5 min, and to 15% or 22% at 30 min. A major
radioactive metabolite with high polarity was observed
in the HPLC. Using ion exchange chromatography, the
metabolite was assigned to [18F]F-. Since [18F]F- is
probably not brain permeable, [18F]4 may be metabo-
lized in the plasma and brain, respectively (Scheme 4).
After its injection into the mouse, the amount of [18F]5
in the plasma continued to decrease during the entire
experiment. The fraction corresponding to the un-
changed [18F]5 in the plasma was 64% at 5 min, 29% at
30 min, and 25% of the total radioactivity at 60 min
after injection. No [18F]F- but another radioactive
metabolite was observed in the HPLC. As estimated by
its retention time (tR ) 1.9 min), the metabolite was
much more polar than [18F]5 (tR ) 11.8 min). On the
other hand, only [18F]5 was detected in the brain
homogenate with no evidence (<5%) of any radioactive
metabolites even at 60 min after injection.
with a similar profile to [11C]2 (Scheme 1). A radioactive
metabolite ([18F]13) with a benzyl moiety and nonra-
dioactive metabolite (N-(5-fluoro-2-phenoxyphenyl)ac-
etamide (14) may be the putative metabolite of [18F]5.
The debenzylated compound 14 had no affinity for PBR
and CBR (IC50 > 10 µM).25 Therefore, the presence of
the nonradioactive 14 could not interfere with the
specific binding of [18F]5 to PBR in the brain, even if it
passed the BBB and entered the brain. Since no
radioactive metabolites including [18F]13 were detected
in the mouse brain homogenates, the chemical structure
of [18F]13 was not further identified. These findings
could reveal that although [18F]5 was extensively me-
tabolized in the plasma, all of the specific binding
determined in the monkey brain may be due to this
ligand itself and not influenced by its radioactive or
nonradioactive metabolite.
Con clu sion
To develop PET ligands for imaging PBR in the pri-
mate brain, four analogues 4-7 for PBR were synthe-
sized and evaluated in this time. The two fluoroalkyl
analogues 4 and 5 had higher or same affinities for PBR
than the parent compound 2 and had no potency for
CBR. The iodomethyl analogue 6 had a moderate
affinity for PBR and may become a candidate for a
SPECT ligand. The PET ligands [18F]4 and [18F]5 were
respectively synthesized by the alkylation of the desm-
ethyl precursor 3 with [18F]8 and [18F]9 in reproducible
radiochemical yields. The distribution patterns of [18F]-
4 and [18F]5 in mice were consistent with the distribu-
tion of PBR. However, compared with [18F]5, [18F]4
exhibited a high accumulation of radioactivity in the
bone. The PET image of [18F]4 in the monkey brain also
showed significant radioactivity in the bone, indicating
that it was not a useful PET ligand because of its
remarkable in vivo defluorination. The uptake of [18F]5
in the occipital cortex of the monkey brain was 1.5 times
higher than that of [11C]2, and 6 times (maximum value)
higher than that of [11C]2. Pretreatment with 1 or 2
significantly reduced the radioactivity levels of [18F]5
to 20-30% of the control in the brain, suggesting a high
specific binding of [18F]5 to PBR in the monkey brain.
No radioactive metabolite of [18F]5 was detected in the
brain although it was metabolized in the plasma by
debenzylation. This study has succeeded in developing
a potential PET ligand [18F]5 which has high specific
binding for PBR in the monkey brain.
The metabolite analysis was also performed using
monkey plasma. After its injection into the monkey,
[18F]5 in plasma decreased to 77% at 5 min, 56% at 30
min, and 18% at 90 min. From then, the amount of
unchanged [18F]5 continued to decrease, while that of
a radioactive metabolite increased to 90% by the end
(180 min) of the PET experiment. The radioactive
metabolite in the monkey plasma was the same me-
tabolite determined in the mouse plasma.
As reported previously, debenzylation of 2 and [11C]2
was a main route of metabolism.25,28 Since the fluoro-
ethyl analogue [18F]5 had molecular similarity to and
the bioisoteric property of [11C]2, it may be metabolized
Exp er im en ta l Section
1H NMR spectra were recorded on a J NM-GX-270 spec-
trometer (J EOL, Tokyo) with tetramethylsilane as an internal

Radioligand for PET Imaging of Benzodiazepine Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2233
standard. All chemical shifts (δ) were reported in parts per
million (ppm) downfield from the standard. FAB-MS was
obtained on a J EOL NMS-SX102 spectrometer (J EOL, To-
kyo). Column chromatography was performed using Merck
Kieselgel gel 60 F254 (70-230 mesh). 18-Fluorine (18F) was
produced by the 18O (p, n) 18F nuclear reaction using a CYPRIS
HM-18 cyclotron (Sumitomo Heavy Industry, Tokyo). If not
otherwise stated, radioactivity was determined with an IGC-
3R Curiemeter (Aloka, Tokyo). HPLC was performed using a
J ASCO HPLC system (J ASCO, Tokyo): effluent radioactivity
was monitored using a NaI (Tl) scintillation detector system.
Samples 2 and 3 were kindly provided by Dr A. Nakazoto
(Taisho Pharmaceutical). All chemical reagents with the
highest grade commercially available were purchased from
Aldrich Chem. (Milwaukee, WI) and Wako Pure Chem. Ind.
(Osaka). The animal experiments were carried out according
to the recommendations of the committee for the care and use
of laboratory animals, National Institute of Radiological Sci-
ences (NIRS).
using 18 MeV protons (14.2 MeV on target) from the cyclotron
and separated from [18O]H2O using Dowex 1-X8 anion-
exchange resin in an irradiating room. The [18F]F- was eluted
from the resin with aqueous K2CO3 (3.3 mg/0.3 mL) into a vial
containing CH3CN (1.5 mL)/4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8,8,8]hexacosane (Kryptofix 222, 25 mg) and
transferred into a reaction vessel in a hot cell.
N-(5-F lu or o-2-p h en oxyp h en yl)-N-(2-[18F ]flu or om eth yl-
5-m eth oxyben zyl)a ceta m id e ([18F ]4). [18F]F- from the ir-
radiating room was transported to a Pyrex glass vessel (5 mL)
containing 100 µL of o-dichlorobenzene (o-DCB), and the
[18F]F- was dried to remove H2O and CH3CN at 120 °C for 15
min. After CH2I2 (10, 100 µL) was added to the radioactive
mixture by a helium flow (50 mL/min) at 120 °C, [18F]FCH2I
([18F]8) resulted in this vessel was distilled at once under
helium for 3 min and bubbled into another Pyrex glass vessel
containing the demethyl precursor (3, 1.5 mg) and NaH (10
µL, 1.5 g/20 mL DMF) in anhydrous DMF (300 µL) at -15 °C.
After maximum radioactivity was bubbled into the solution,
the reaction was terminated by adding CH3CN/H2O (6/4, 500
µL). The radioactive mixture was applied to a semipreparative
HPLC column. HPLC semipreparative purification was com-
pleted on YMC J ’sphere ODS-H80 column (10 mm ID × 250
mm) using a mobile phase of CH3CN/H2O (60/40) at a flow
rate of 6.0 mL/min. The retention time (tR) for [18F]4 was 11.2
min, whereas that for 3 was 6.7 min. The radioactive fraction
corresponding to [18F]4 was collected in a sterile flask contain-
ing polysorbate (80) (75 µL) and ethanol (150 µL), evaporated
to dryness under vacuum, redissolved in 7 mL of sterile normal
saline, and passed through a 0.22 µm Millipore filter to obtain
the final product. At the end of synthesis (EOS), 180-300 MBq
of [18F]4 was obtained as an iv injectable solution at a beam
current of 10-15 µA and 20-25 min proton bombardment.
Ch em istr y. N-(5-F lu or o-2-p h en oxyp h en yl)-N-(2-flu o-
r om eth yl-5-m eth oxyben zyl)a ceta m id e (4). A mixture of 3
(19 mg, 0.05 mmol), FCH2I (4, about 20 mg in 1 mL of CH3CN),
and NaH (4 mg, 0.1 mmol) in DMF (2 mL) was stirred at 0 °C
for 5 h. The reaction mixture 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. Column chromatography of the residue on
silica gel with CHCl3/hexane (1/9) gave 4 (16 mg, 79%) as a
1
white powder; mp: 71-72 °C; H NMR (300 MHz, CDCl3) δ:
7.18-7.40 (2H, m), 6.71-7.13 (8H, m), 6.28-6.44 (1H, m), 5.30
(2H, d, J ) 49 Hz), 4.80 (2H, dd, J ) 2, 12 Hz), 3.71 (3H, s),
2.12 (3H, s); FABMS (m/z): 414.2 (M+ + 1); Anal. (C23H21F2NO4)
C, H, N.
N-(5-F lu or o-2-p h en oxyp h en yl)-N-(2-[18F ]flu or oeth yl-5-
m eth oxyben zyl)a ceta m id e ([18F ]5). After the [18F]F- was
dried, BrCH2CH2OTf (12, 8 µL) in o-dichlorobenzene (400 µL)
was added to the radioactive mixture. The [18F]FCH2CH2Br
([18F]9) in this vessel was distilled under a helium flow (90-
100 mL/min) at 130 °C for 5 min and bubbled into another
vessel containing 3 (1.5 mg) and NaH (10 µL, 1.5 g/20 mL
DMF) in anhydrous DMF (300 µL) at -15 °C, and the reaction
mixture was heated and kept at 120 °C for 10 min. HPLC
semipreparative purification was performed on a YMC J’sphere
ODS-H80 column (10 mm ID × 250 mm) using a mobile phase
of CH3CN/H2O (55/45) at a flow rate of 6.0 mL/min. The tR for
[18F]5 was 14.2 min. At EOS, 570-780 MBq of [18F]5 was
obtained as an iv injectable solution at a beam current of 10-
15 µA and 20-25 min proton bombardment.
Ra d ioch em ica l P u r ity a n d Sp ecific Activity Deter m i-
n a tion s. Radiochemical purity was assayed by analytical
HPLC (column: CAPCELL PAK C18, 4.6 mm ID × 250 mm,
UV at 254 nm; mobile phase: CH3CN/H2O ) 6/4). The tR for
[18F]4 and [18F]5 was 6.1 and 6.3 min at a flow rate of 2.0 mL/
min. The specific activity of [18F]4 and [18F]5 was determined
by comparison of the assayed radioactivity to the mass
associated with the carrier UV peak at 254 nm.
In Vitr o Bin d in g Assa ys. 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 Co.,
Germany) and thaw-mounted on glass slides (Matsunami
Glass Ind., Tokyo), which were then dried at room temperature
and stored at -18 °C until use for experiments. The brain
sections were preincubated at 25 °C for 20 min in 50 mM Tris-
HCl (pH 7.4) buffer. After the preincubation, these sections
were incubated at 37 °C for 30 min in the assay buffer
containing [11C]2 (1 nM, specific activity: 110 GBq/µmol) or
[11C]flumazenil (1 nM, specific activity: 220 GBq/µmol). To
determine the IC50 values of 1, 2, 4-7 for the [11C]2 (for PBR)
or [11C]flumazenil (for CBR) binding, the brain sections were
incubated with [11C]2 or [11C]flumazenil in the presence of
increasing concentrations of the corresponding 1, 2, 4-7 (0.1-
1000 nM). Nonradioactive 2 or flumazenil (10 µM) of was used
N-(5-Flu or o-2-ph en oxyph en yl)-N-(2-flu or oeth yl-5-m eth -
oxyben zyl)a ceta m id e (5). A mixture of 3 (38 mg, 0.1 mmol),
FCH2CH2Br (9, 25 µL, 0.2 mmol) and NaH (8 mg, 0.2 mmol)
in DMF (5 mL) was heated at 80 °C for 8 h. The reaction
mixture 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.
Column chromatography of the residue on silica gel with
CHCl3/hexane (1/13) gave 5 (27 mg, 65%) as a white powder;
mp: 54-56 °C; 1H NMR (300 MHz, CDCl3) δ: 7.26-7.59 (2H,
m), 6.22-7.19 (9H, m), 4.88 (2H, dt, J ) 4, 41 Hz), 4.65 (2H,
dd, J ) 2, 12 Hz), 4.12 (2H, dt, J ) 4, 27 Hz), 3.80 (3H, s),
2.15 (3H, s); FABMS (m/z): 428.2 (M+ + 1); Anal. (C24H23F2NO4)
C, H, N.
N-(5-Flu or o-2-ph en oxyph en yl)-N-(2-iodom eth yl-5-m eth -
oxyben zyl)a ceta m id e (6). A mixture of 3 (19 mg, 0.05 mmol)
and CH2I2 (10, 11 µL, 0.15 mmol) in DMF (5 mL) was stirred
at 25 °C for 3 h. The reaction mixture 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. Column chromatography of the
residue on silica gel with CHCl3/hexane (1/15) gave 6 (8.9 mg,
1
34%) as a colorless oil; H NMR (300 MHz, CDCl3) δ: 7.18-
7.40 (2H, m), 6.15-7.33 (9H, m), 4.81 (dd, J ) 2, 12 Hz), 4.70
(2H, s), 3.78 (3H, s), 2.20 (3H, s); FABMS (m/z): 524.1 (M+
1); Anal. (C23H21FINO4) C, H, N.
+
N-(5-F lu or o-2-p h en oxyp h en yl)-N-(5-m et h oxy-2-t osy-
loxyeth ylben zyl)a ceta m id e (7). A mixture of 3 (19 mg, 0.05
mmol), TsOCH2CH2OTs (11, 37 mg, 0.1 mmol) and NaH (8
mg, 0.2 mmol) in DMF (5 mL) was stirred at 0 °C for 5 h. The
reaction mixture 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. Column chromatography of the residue on silica gel
with CHCl3/hexane (1/20) gave 7 (14 mg, 46%) as a colorless
1
oil; H NMR (300 MHz, CDCl3) δ: 6.52-7.48 (15H, m), 4.62
(2H, dd, J ) 2, 12 Hz), 4.43-4.28 (2H, m), 4.12-3.92 (2H, m),
3.80 (3H, s), 3.36 (3H, s), 2.32 (3H, s); FABMS (m/z): 580.3
(M+ + 1); Anal. (C31H30FNSO7) C, H, N.
Ra d iosyn th esis. [18F ]F lu or id e ([18F ]F -). [18F]F- was
produced by the 18O (p, n) 18F reaction on 10-20 at. % H218O

2234 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Zhang et al.
to determine the nonspecific binding for PBR or CBR. After
the incubation, the sections were washed three times for 2 min
each time with the cold assay buffer, dipped into cold distilled
water, and dried with a stream of warm air (about 50 °C).
These sections were then placed in contact with imaging plates
(BAS-SR 127, Fuji Photo Film Co. Ltd., Tokyo, J apan) for 60
min to analyze the distribution of their radioactivity with a
FUJ IX BAS 3000 bioimaging analyzer (Fuji). The region of
interest (ROI) on the sections was placed on the cerebellum.
PSL data corresponding to the radioactivity on the cerebellum
in the presence and absence of the displacement 1, 2, 4-7 were
determined. Specific binding for PBR or CBR was defined as
total binding minus binding in the presence of 2 or flumazenil
(10 µM). The PSL data corresponding to the specific binding
at each compound concentration was calculated as a percent-
age in relation to the control specific binding, and which were
converted to probit values to determine the IC50 of each
compound. The IC50 value was further converted to Ki accord-
ing to the Cheng-Prusoff equation.36
system for radioactivity and analyzed under the same condi-
tions described above except that the mobile phase was CH3-
CN/H2O with a ratio of 1/1. The percent ratio of [18F]ligand
(tR ) 10.6 min for [18F]4 and 11.8 min for [18F]5) to total
radioactivity (corrected for decay) on the HPLC chromatogram
was calculated as % ) (peak area for [18F]ligand/total radio-
active peak area) × 100.
Meta bolite An a lysis for Mon k ey P la sm a . After iv injec-
tion of [18F]5 (80 MBq) into the monkey, arterial blood samples
(1 mL) were collected at 2, 10, 30, 60, 90, 120, and 180 min.
All samples were centrifuged at 15 000 rpm for 1 min at 4 °C
to separate plasma (250 µL), which was collected in a test tube
containing CH3CN (0.5 mL). The tube was vortexed for 15 s
and centrifuged at 15 000 rpm for 1 min for deproteinization.
The extraction efficiency of radioactivity into the CH3CN
ranged from 73% to 94% of the total radioactivity in the
plasma. The radioactive fractions in these samples were
determined using HPLC.
Biod istr ibu tion on Mice. A saline solution of 18F-ligand
(average of 8 MBq/200 µL) was injected into ddy mice (30-40
g, 9 weeks, male) through the tail vein. Four mice for each
time point were sacrificed by cervical dislocation at 5, 15, 30,
60 and 120 min after injection. Whole brain, liver, lung, heart,
kidney, adrenal, bone, and blood samples were quickly re-
moved and weighed. The radioactivity present in the various
tissues was measured in a Packard autogamma scintillation
counter and expressed as a percentage of the injected dose per
gram of wet tissue (% ID/g). All radioactivity measurements
were corrected for decay.
P ET on Mon k eys. The PET scan was performed using a
high-resolution SHR-7700 PET camera (Hamamatsu Photo-
nics, Hamamatsu, J apan) designed for laboratory animals,
which provides 31 transaxial slices 3.6 mm (center-to-center)
apart and a 33.1 cm field of view. A male rhesus monkey
(macaca mulatta) weighing about 5 kg was repeatedly anes-
thetized with ketamine (Ketalar, 10 mg/kg/h, im) every hour
through the session. After transmission scans for attenuation
correction were performed for 1 h using a 74 MBq 68Ge-68Ga
source. A dynamic emission scan in the 3D acquisition mode
was performed for 90 min (2 min × 5 scans, 4 min × 10 scans,
10 min × 4 scans). All emission scan images were recon-
structed with a Colsher filter of 4 mm, and circular regions of
interest (ROIs) with a 5-mm diameter were placed over the
occipital cortex using an image analysis software.35,36 A solu-
tion of [18F]4 and [18F]5 (80-85 MBq) was injected iv into the
monkey, and time-sequential tomographic scanning was per-
formed on a transverse section of the brain for 180 min. In
pretreatment experiments, nonradioactive 2 (1 mg/kg) or 1 (5
mg/kg) was injected at 2 min before the [18F]5 injection. The
time activity curves (TACs) in the occipital cortex were
obtained for each scan of the brain.
Ack n ow led gm en t. The authors are grateful to Dr
A. Nakazoto (Taisho Pharmaceutical Co., Ltd.) for
providing the samples (2 and 3) and helpful suggestions.
We also thank the crew of the Cyclotron Operation
Section and Radiopharmaceutical Chemistry Section of
National Institute of Radiological Sciences (NIRS) for
support in operation of the cyclotron and production of
radioisotopes.
Su p p or tin g In for m a tion Ava ila ble: Radioactivity dis-
tribution of [18F]4 and [18F]5. This material is available free
of charge via the Internet at http://pubs.acs.org.
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