Synthesis and evaluation of 1-[2-(4-[11C]methoxyphenyl)phenyl] piperazine for imaging of the serotonin 5-HT7 receptor in the rat brain

Article abstract of DOI:10.1016/j.bmc.2013.06.020

1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT ₇ receptor antagonist (K_i = 2.6 nM) with a low binding affinity for the 5-HT_1A receptor (K_i = 476 nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT ₇ receptor, [¹¹C]4 was synthesized at high radiochemical yield and specific activity, by O-[¹¹C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [ ¹¹C]methyl iodide. Autoradiography revealed that [¹¹C]4 showed in vitro specific binding with 5-HT₇ in the rat brain regions, such as the thalamus which is a region with high 5-HT₇ expression. Metabolite analysis indicated that intact [¹¹C]4 in the brain exceeded 90% of the radioactive components at 15 min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [¹¹C]4 in the brain (1.2 SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT₇-selective antagonist SB269970 (3) did not decrease the uptake of [¹¹C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT₇ and higher in vivo stability in brain than 4.

Full text of DOI:10.1016/j.bmc.2013.06.020

Bioorganic & Medicinal Chemistry 21 (2013) 5316–5322
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 1-[2-(4-[11C]
methoxyphenyl)phenyl]piperazine for imaging
of the serotonin 5-HT7 receptor in the rat brain
Yoko Shimoda a, Joji Yui a, Lin Xie a, Masayuki Fujinaga a, Tomoteru Yamasaki a, Masanao Ogawa a,b
,
Nobuki Nengaki a,b, Katsushi Kumata a, Akiko Hatori a, Kazunori Kawamura a, Ming-Rong Zhang a,
⇑
a Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT7 receptor antagonist (Ki = 2.6 nM)
with a low binding affinity for the 5-HT1A receptor (Ki = 476 nM). As a potential positron emission
tomography (PET) radiotracer for the 5-HT7 receptor, [11C]4 was synthesized at high radiochemical yield
and specific activity, by O-[11C]methylation of 20-(piperazin-1-yl)-[1,10-biphenyl]-4-ol (6) with
Received 24 April 2013
Revised 6 June 2013
Accepted 7 June 2013
Available online 17 June 2013
[
11C]methyl iodide. Autoradiography revealed that [11C]4 showed in vitro specific binding with 5-HT7
in the rat brain regions, such as the thalamus which is a region with high 5-HT7 expression. Metabolite
analysis indicated that intact [11C]4 in the brain exceeded 90% of the radioactive components at 15 min
after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The
PET study of rats showed moderated uptake of [11C]4 in the brain (1.2 SUV), but no significant regional
difference in radioactivity in the brain. Pretreatment with 5-HT7-selective antagonist SB269970 (3) did
not decrease the uptake of [11C]4 in the rat brain. Further studies are warranted that focus on the devel-
opment of PET ligand candidates with higher binding affinity for 5-HT7 and higher in vivo stability in
brain than 4.
Keywords:
5-HT7 receptor
Carbon-11
PET
Autoradiography
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
in the rat and guinea pig brains is similar to the distribution pat-
tern of mRNA in the corresponding brain regions.15–17
Serotonin 5-hydroxytryptamine (5-HT) receptors are classified
into seven structurally and pharmacologically distinct suptypes.1
Of these subtypes, 5-HT7 receptor is the newest addition to the
family of 5-HT receptors.2,3 The 5-HT7 receptor has been cloned
from the cDNA of the mouse, rat, guinea pig, and human.3–5 In
the peripheral system of animals, stimulation of this receptor
caused relaxation of the blood vessels in animals.6 In the central
nervous system, the 5-HT7 receptor may play roles in mediating
5-HT-induced phase shifts of neuronal activity in the suprachias-
matic nucleus of the hypothalamus.7 This receptor is also involved
in the pathophysiology of sleep disorders, depression and schizo-
phrenia.8–11 In particular, some atypical antipsychotics, such as
clozapine and risperidone, showed high affinity for 5-HT7, suggest-
ing that the therapeutic effects of these drugs may be exerted par-
tially via this receptor.12,13 The expression of 5-HT7 mRNA is the
highest in the brain, where it is discretely located within the thal-
amus, hypothalamus, and various limbic and cortical regions.3–5,14
Autoradiography has shown that the distribution pattern of 5-HT7
Positron emission tomography (PET) is a useful modality for
in vivo imaging and evaluation study of receptors, enzymes and
plaques. In vivo visualization of the 5-HT7 receptor would be useful
to investigate its function and to elucidate its role in the patho-
physiology of brain disorders. Although many potent and selective
ligands for 5-HT7 exist,18–21 few have been labeled for use in PET
imaging studies. Ten years ago, we developed the first PET radio-
tracer 1-[11C]methyl-2a-[4-(4,5,6,7-tetrahydrothieno[3,2-c]pyri-
dine-5-yl)butyl]-2a,3,4,5-tetrahydro-1H-benz[cd]indole-2-one
([11C]DR4446, [11C]1; Scheme 1),22 which showed relatively high
affinity for 5-HT7 (Ki = 9.7 nM) and high selectivity over other 5-
HT receptors.23 A PET study using [11C]1 showed a high uptake of
radioactivity (3.8 SUV) in the monkey brain and stable metabolism
in the monkey plasma. However, only a low level of in vivo specific
binding of [11C]1 was observed in the brain.22 Recently, 1-(2-[(2R)-
1-[([18F]fluorophenyl)sulfonyl]pyrrodin-2-yl]ethyl)-4-methylpi-
peridine ([18F]2FP3, [18F]2; KB = 1.43 nM for 5-HT7), an analog of the
5-HT7-selective ligand SB269970 (3), has been developed.24–26
[
18F]2 is the first promising PET radiotracer to enable in vivo imag-
⇑
ing of 5-HT7 in the cat brain, and is likely to be a promising radio-
Corresponding author. Tel.: +81 43 382 3709; fax: +81 43 206 3261.
E-mail address: zhang@nirs.go.jp (M.-R. Zhang).
tracer for neuroimaging studies in the human brain.26 However, to
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.020

Y. Shimoda et al. / Bioorg. Med. Chem. 21 (2013) 5316–5322
5317
Scheme 1. Chemical structures of ligands for the 5-HT7 receptor.
our knowledge, thus far, no PET radiotracer has been used for
imaging and measurement studies of the 5-HT7 receptor in the hu-
man brain.
prepared according to the reaction sequences reported previously
with some modification.27 2-Bromo-1-iodobenzene (7) was treated
with tert-butyl piperazine-1-carboxylate in the presence of Pd2-
dba3 and Xantphos to obtain tert-butyl 4-(2-bromophenyl)pipera-
zine-1-carboxylate (8) in 46% yield. Coupling of 8 with (4-
methoxyphenyl)boronic acid, followed by treatment of 9 with
trifluoroaceic acid, produced 4 in 36% yield (two steps). A similar
coupling reaction of 8 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl acetate produced 10 in 59% yield, which was
converted to precursor 5 by hydrolyzing 10 with NaOH. Another
precursor 6 was synthesized by treating 5 with trifluoroacetic acid
in 83% yield.
Therefore, in this study, we developed 1-[2-(4-methoxy-
phenyl)phenyl]piperazine (4, Scheme 1) as a candidate of PET radi-
otracer for 5-HT7. This compound showed high bind affinity to
human recombinant 5-HT7 receptors expressing in mammalian
cells (Ki = 2.6 nM), whereas its binding affinity to 5-HT1A receptor
was 476 nM.19,27 It also displayed a weak affinity for adrenergic 1
receptor (Ki = 156 nM). Furthermore, compound 4 has a methoxyl
group in its molecule and can be easily labeled by 11C without
changing its chemical structure, pharmacokinetics and pharmaco-
logical functions.
Here, we describe (1) synthesis of the unlabeled compound 4
and two novel phenol precursors 5 and 6 for radiolabeling, (2)
radiosynthesis of [11C]4, and (3) in vitro autoradiography, metabo-
lite analysis and PET study of the rat brain for [11C]4.
2.2. Radiosynthesis
[
11C]4 was synthesized using a homemade automated synthesis
system.28 Considering the piperazine moiety in compound 4, we
first labeled 4 by reacting [11C]methyl iodide ([11C]CH3I) with 5,
in which the piperazine was protected by a Boc group, followed
by the deletion of the protective group in [11C]9 with trifluoroace-
tic acid (route 1 of Scheme 2).
2. Results and discussion
2.1. Chemical synthesis
The labeling agent [11C]CH3I was produced by reduction of
cyclotron-produced [11C]CO2 with LiAlH4, followed by iodination
with 57% HI (Scheme 2).29,30 Reaction of 5 with [11C]CH3I in the
For radiolabeling, the targeted compound 4 and two precursors
5 and 6 were synthesized as shown in Scheme 2. Compound 4 was
Route 1
Route 2
Scheme 2. Chemical synthesis and radiosynthesis: (a) tert-butyl piperazine-1-carboxylate, Pd2dba3, Xantphos, NaO(t-Bu), toluene, 80 °C, 16 h; (b) 4-methoxyphenylboronic
acid, Pd(PPh3)2Cl2, Na2CO3, DME–H2O, 80 °C, 10 h; (c) trifluoroacetic acid, CH2Cl2, room temperature, 2 h; (d) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl acetate,
Pd(PPh3)2Cl2, Na2CO3, DME–H2O, 80 °C, 2.5 h; (e) NaOH, H2O–MeOH, 80 °C, 5 h; (f) trifluoroacetic acid, CH2Cl2, room temperature, overnight; (g) LiAlH4, THF, ꢀ15 °C, 2 min;
(h) hydroiodic acid, 180 °C, 2 min; (i) NaOH, DMF, 80 °C, 5 min; (j) trifluoroacetic acid, 100 °C, 5 min; (k) NaOH, DMF, 80 °C, 3 min.

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Y. Shimoda et al. / Bioorg. Med. Chem. 21 (2013) 5316–5322
A: Route 1
B: Route 2
UV
RI
UV
RI
[
11C]4
[
11C]4
by-product
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
min
min
Figure 1. Chromatograms obtained on HPLC of the reaction mixtures by routes 1 (A) and 2 (B) used in the radiosynthesis of [11C]4. See Section 3 for the chromatographic
conditions.
presence of NaOH yielded [11C]9. Without separating [11C]9, the
reaction mixture was treated with trifluoroacetic acid. Reverse-
phase HPLC was performed to purify the reaction mixture and
yield [11C]4 (Fig. 1A). From 13.0 to 19.6 GBq of [11C]CO2, 1.4–
2.7 GBq of [11C]4 was produced in 34 2 min (n = 5) of synthesis
times from the end of bombardment (EOB), in 40 6% radiochem-
ical yield (decay-corrected, based on [11C]CO2).
ing problematic lipophilic brain-penetrating metabolites. The com-
puted values of lipophilicity at pH 7.4 (cLogD) for 4 were 3.34.
Following the labeling of 4 with 11C, its lipophilicity (LogD) was
measured using the shake flask method.31 The experimentally
determined LogD value of [11C]4 was 1.67 0.02 (n = 3). The pre-
cise reason for the difference between the cLogD and LogD values
of [11C]4 remains unclear.
Despite the high radiolabeling efficiency, it took 5 min to re-
move the Boc group in [11C]9. More importantly, utilization of tri-
fluoroacetic acid damaged the synthetic module. Therefore, we
attempted to directly react another precursor 6 with [11C]CH3I
(route 2 of Scheme 2). Without the protection of piperazine in 6,
O-[11C]methylation mainly occurred on the hydroxy group, and
the putative N-[11C]methylated product (tR = 4.5 min) was found
on the HPLC separation chart as a by-product (Fig. 1B). From 7.4
to 17.0 GBq of [11C]CO2, 0.8–2.8 GBq of [11C]4 was produced in
2.4. In vitro autoradiography
Figure 2 shows representative in vitro autoradiography for
[
11C]4 in the brain sections of rats (n = 4). In the control image
(Fig. 2A), the distribution pattern of [11C]4 was heterogeneous with
high and moderate radioactivity in the thalamus, cerebellum,
pons/medulla and striatum. Low radioactivity was observed in
the hippocampus and cerebral cortex. The distribution pattern of
36 9% radiochemical yield (decay-corrected), 54 14 GBq/lmol
specific activity, >98% radiochemical purity within a synthetic time
of 27 2 min (n = 3) from EOB.
[
11C]4 in the autoradiogram of brain regions was similar to the pre-
viously reported distribution pattern of 5-HT7 receptor.15–17 In par-
ticular, the thalamus, which was indicated to have high 5-HT7
density in the brain, displayed the highest radioactivity level in
the brain sections. On the other hand, the hippocampus and cere-
bral cortex, two regions with high expression of 5-HT1A receptor,32
only showed a low radioactivity, suggesting that [11C]4 did not
have significant specific binding for the 5-HT1A receptor. This result
suggested the specificity of [11C]4 for 5-HT7, which was supported
After comparing the radiolabeling efficiency, synthesis times
and convenience of operating the synthetic module for the two
synthetic routes, we chose to synthesize [11C]4 by using route 2
for the animal experiment. At EOS, the radioactivity amount, radio-
chemical purity, and specific activity of [11C]4 as an intravenously
injectable solution were sufficient for the evaluation. The analyti-
cal results of [11C]4 were in compliance with our in-house quality
control/assurance specifications for radiopharmaceuticals. This
product did not show radiolysis at room temperature until
100 min after formulation, indicating that [11C]4 is radiochemically
stable within the duration of at least one PET scan.
by the weak binding affinity (Ki = 476 nM) of 4 for 5-HT1A
.
Incubation with unlabeled 4 at 1 M decreased the radioactive
l
signals in the brain sections to 60–80% of the total radioactivity in
the corresponding regions of the brain (Fig. 2B). By co-incubation
with the 5-HT7-selective ligand 3, the radioactive signals in the
brain section were also reduced (Fig. 2C). The decrease in the per-
centage of the radioactive signals caused by 3 was similar to that
caused by 4. These results suggested that [11C]4 had in vitro spe-
cific binding to 5-HT7 in the rat brain section, although the
level was not as high as the specific binding of [18F]2 in the cat
brain.
2.3. Computation and measurement of lipophilicity
Moderate lipophilicity is an important physiochemical property
required for the development of a promising radiotracer, which
facilitates high brain uptake via passage through the blood-brain
barrier (BBB), without incurring nonspecific binding and for avoid-
A: [11C]4
Hippocampus
B: [11C]4 + 4 (1 µM)
C: [11C]4 + 3 (1 µM )
Cerebral
Cortex
Cerebellum
Thalamus
Striatum
Pons /
Medulla
Figure 2. Representative in vitro autoradiograms of [11C]4 in the brain sections. (A) [11C]4 alone; (B) [11C]4 with 4 (1 M); (C) [11C]4 with 3 (1
l lM).

Y. Shimoda et al. / Bioorg. Med. Chem. 21 (2013) 5316–5322
5319
2.5. Determination of the intact form of [11C]4 in rat plasma and
brain
A: [11C]4
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Frontal cortex
Hippocampus
Thalamus
Because metabolites of a radiotracer may be present in the plas-
SUV
1.5
ma, which may eventually enter the brain and confound the results
of the PET imaging study of neuroreceptors, irrespective of
whether the radiolabeled metabolites bind to the targeted neuro-
receptor, we performed metabolite analysis of [11C]4 in the plasma
and brain of rats prior to the PET study.
Cerebellum
0
The plasma and extract of homogenized brain tissues were ana-
lyzed by HPLC coupled to a highly sensitive radioactivity detector.
Figure 3 shows the percentages of the intact form of [11C]4 in the
plasma and brain of rat at 15 and 60 min after injection. The fraction
corresponding to the intact form (tR: 7.7 min) of [11C]4 in the plasma
decreased to 51% at 15 min and 30% at 60 min. Two major radiola-
beled metabolites (tR: 3.3 and 5.8 min) with higher polarity than
0
10 20 30 40 50 60 70 80 90
min
B: [11C]4 + 3 (3 mg/kg)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Frontal cortex
Hippocampus
Thalamus
[
11C]4 were observed on the HPLC charts. On the other hand, un-
changed [11C]4 in the brain homogenate remained at 91% and 69%
of the total radioactivity at 15 and 60 min, respectively. The radiola-
beled metabolites in the brain showed the same retention times with
the metabolites in the plasma. This result indicates that metabolism
of [11C]4 was relatively slow in the brain, or that only a limited level
of radiolabeled metabolites originating from the plasma enter the
brain. Moreover, this result suggests that the brain uptake was
mainly attributed to [11C]4 itself, but the radiolabeled metabolites
in the brain would affect the specific binding of [11C]4 to a low extent.
SUV
1.5
Cerebellum
0
0
10 20 30 40 50 60 70 80 90
min
2.6. Small-animal PET study
Figure 4. PET study using [11C]4 in rats. Representative PET images of rat brains
were showed in transverse (upper) and coronal (bottom) views. PET images were
generated by summing the whole scan (0–90 min). Time-activity curves in the rat
brain regions after injection of [11C]4. (A) [11C]4 alone; (B) [11C]4 after treatment
with 3 (3 mg/kg). The uptakes of radioactivity were decay-corrected to the injection
time and are expressed as the standardized uptake value (SUV), normalized for the
injected radioactivity and body weight. Data are the means SD (n = 3 in each
group).
Figure 4 shows representative PET images and time-activity
curves in the rat brains using PET with [11C]4. In the baseline mea-
surement (Fig. 4A), [11C]4 entered the rat brain rapidly after injec-
tion, and uptake of radioactivity (approximately 1.2 SUV) peaked at
3–5 min in all the regions examined, indicating that [11C]4 could
pass across the BBB. The uptake of radioactivity in the brain was
probably relative to the lipophilicity of LogD 1.67. This suggests
that transporters at the BBB might not significantly limit the en-
trance of [11C]4 into the brain. After the initial uptake, the radioac-
tivity began to decrease in all the brain regions and was at 20–30%
of the peak uptake by 90 min after the injection of [11C]4. The base-
line PET images showed that the radioactive signals spread
throughout the brain without any significant regional differences.
This distribution pattern of radioactivity was different from that
observed on the in vitro autoradiogram. On the other hand, in
the blocking experiment (Fig. 4B), pretreatment with 5-HT7-selec-
tive 3 increased the uptake of [11C]4. The radioactivity level in the
examined regions was increased to approximately two-fold of that
of the corresponding controls.
The present PET study showed a relatively homogenous distri-
bution of radioactivity in the whole brain, which was not consis-
tent with the distribution pattern of 5-HT7 in the brain.15–17 In
particular, the uptake in the 5-HT7-rich thalamus was not higher
than that in the other brain regions. Moreover, the blocking exper-
iment using 5-HT7-selective 3 did not reduce but increased the
brain uptake. Thus, it was difficult to determine whether the brain
uptake was due to blood flow or due to specific binding of [11C]4 in
the brain based on the visual difference in PET images and kinetics
between the baseline and blocking experiment. A quantification
approach using brain kinetics is warranted to validate the presence
of in vivo specific binding of [11C]4 to 5-HT7 in the brain. To this
end, arterial input measures in rat using quantitative analysis
method, such as Lassen graphic analysis, should be performed.
Taken together, although [11C]4 exhibited in vitro binding with
5-HT7, moderate brain uptake and relatively stable metabolism in
the brain, in vivo evaluation showed limited specificity of [11C]4 for
5-HT7. The level of specific binding in the brain appeared to be low-
er than that of [18F]2, the most promising radiotracer for 5-HT7 to
date. Therefore, further studies should be conducted that focus on
the development of ligand candidates with more potent binding
affinity for 5-HT7 than that of 4.
100
15 min
60 min
80
60
40
20
0
In general, the ratio of density (Bmax) of a targeted receptor in
the brain tissues to the equilibrium dissociation constant (KD) of
a radiotracer is used to predict the magnitude of specific binding
in a PET imaging study. The ratio of Bmax/KD, also indicated as bind-
ing potential, should preferably be >2 to elucidate specific binding
on the basis of individual experiences.33 Although no data regard-
ing the Bmax of 5-HT7 have been reported, the density of the 5-HT7
receptor was determined to be less than one-tenth of that of the
Blood
Brain
Figure 3. Metabolite analysis of [11C]4 in the brain and plasma of rat (n = 3 in each
group).

5320
Y. Shimoda et al. / Bioorg. Med. Chem. 21 (2013) 5316–5322
other 5-HT7 subtypes.34 The 5-HT1A Bmax, for example, was mea-
sured to be 5–10 nM in the hippocampus and frontal cortex of
the rat brain,35 therefore, the Bmax of 5-HT7 in the brain could be
assumed to be 0.5–1 nM. Considering the ratio of Bmax/KD, the
binding affinity of 4 (Ki = 2.6 nM) may be not sufficient to elucidate
in vivo specific binding to the 5-HT7 receptor in the brain. There-
fore, to visualize this receptor in the brain, novel candidates for
PET tracers with higher binding affinity for 5-HT7 and high
in vivo stability in brain than 4 are required. Consequently, new
compounds are being designed, with modified chemical structures
for the optimization of a PET radiotracer.
s), 2.98 (4H, t, J = 4.8 Hz), 3.61 (4H, t, J = 4.8 Hz), 6.90–6.96 (1H,
m), 7.02 (1H, dd, J = 1.6, 8.1 Hz), 7.25–7.30 (1H, m), 7.57 (1H, d,
J = 7.9 Hz). 13C NMR (75 MHz, CDCl3): d 28.4, 43.9 (br), 51.6, 79.8,
120.0, 121.0, 124.7, 128.3, 133.9, 150.4, 154.9. FAB-MS: m/z 341
(M+H).
3.1.2. tert-Butyl 2-[(4-methoxyphenyl)phenyl]piperazine-1-
carboxylate (9)
A mixture of 8 (252 mg, 0.74 mmol), 4-methoxyphenylboronic
acid (136 mg, 0.89 mmol), Na2CO3 (239 mg, 2.25 mmol) and
Pd(PPh3)2Cl2 (17 mg, 3 mol % Pd) in dimethyl ether (DME)/water
(8 mL, 3/1, v/v) was heated at 80 °C for 10 h under N2 gas. The reac-
tion mixture was poured into water and extracted with ether. The
organic layer was washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. Purification of the resi-
due by silica gel column chromatography (EtOAc/n-hexane, 1/10,
v/v) yielded 9 (191 mg, 70%) as a colorless crystal; mp, 153–
155 °C. 1H NMR (300 MHz, CDCl3): d 1.44 (9H, s), 2.78 (4H, br),
3.32 (4H, br), 3.85 (3H, s), 6.93 (2H, d, J = 8.4 Hz), 6.99 (1H, d,
J = 7.5 Hz), 7.08–7.10 (1H, m), 7.22–7.26 (2H, m), 7.57 (2H, d,
J = 7.5 Hz). 13C NMR (75 MHz, CDCl3): d 28.4, 43.1 (br), 50.9, 55.2,
79.6, 113.6, 118.4, 123.1, 127.9, 129.9, 131.4, 133.2, 134.9, 150.0,
154.9, 158.5. FAB-MS: m/z 369 (M+H).
In conclusion, this study describes the synthesis and evaluation
of [11C]4 as a potential PET radiotracer for the imaging of the 5-HT7
receptor in the rat brain. [11C]4 was synthesized by O-[11C]methyl-
ation of the desmethyl precursor 6 with [11C]CH3I, producing a
high radiochemical yield and specific activity. Although [11C]4
showed in vitro specific binding in the brain, the present evalua-
tion could not show the presence of in vivo specific binding of
[
11C]4 to the 5-HT7 receptor. Further optimization of the structure
of [11C]4 will thus be required to achieve in vivo imaging of the 5-
HT7 receptor in the brain.
3. Experimental section
3.1.3. 1-[2-(4-Methoxyphenyl)phenyl]piperazine (4)
A mixture of 9 (104 mg, 0.30 mmol) and trifluoroacetic acid
(460 lL, 5.97 mmol) in CH2Cl2 (3 mL) was stirred at room temper-
Melting points (mp) were uncorrected in this study. 1H NMR
spectra were recorded on a JNM-AL-300 spectrometer (JEOL; To-
kyo, Japan) using tetramethylsilane as an internal standard. All
chemical shifts (d) were reported in parts per million (ppm) down-
field from the standard. High resolution (HR) fast atom bombard-
ment-mass spectrometry (FAB-MS) was performed using JEOL
NMS-SX102 spectrometer (JEOL). Column chromatography was
performed on Wakogel C-200 (Wako Pure Chemical Industries,
Ltd.; Osaka, Japan). HPLC was performed using a JASCO HPLC sys-
tem (JASCO; Tokyo, Japan): effluent radioactivity was monitored
using a NaI (Tl) scintillation detector system. SB269970 (3) was
purchased from ALEXIS BIOCHEMICALS (San Diego, CA). All chem-
ical reagents of the highest grade commercially available were pur-
chased from Sigma–Aldrich Corporation (Milwaukee, WI) or Wako
Pure Chemical Industries, Ltd. or Tokyo Chemical Industries, Ltd.
(Tokyo, Japan). 11C was produced using a CYPRIS HM-18 cyclotron
(Sumitomo Heavy Industry; Tokyo, Japan). Unless otherwise sta-
ted, radioactivity was measured using an IGC-3R Curiemeter (Hit-
achi Aloka Medical, Ltd., Tokyo, Japan). All animal experiments
were performed according to the recommendations of the Com-
mittee for the Care and Use of Laboratory Animals, National Insti-
tute of Radiological Sciences (Chiba, Japan). Animals were
maintained and handled in accordance with the recommendations
of the National Institute of Health and the institutional guidelines
of the National Institute of Radiological Sciences. Sprague–Dawley
male rats (220–240 g, 7 weeks) were provided by Japan SLC (Shi-
zuoka, Japan).
ature for 2 h. The reaction mixture was poured into saturated
aqueous NaHCO3 and extracted with CH2Cl2. The organic layer
was washed with brine, dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to obtain 4 (43 mg, 52%) as a pale
yellow oil. 1H NMR (300 MHz, CDCl3): d 1.68 (1H, br, D2O ex-
changed), 2.78 (8H, s), 3.85 (3H, s), 6.94 (2H, d, J = 8.4 Hz), 7.03–
7.08 (2H, m), 7.21–7.29 (2H, m), 7.59 (2H, d, J = 8.4 Hz). 13C NMR
(75 MHz, CDCl3): d 46.1, 52.4, 55.2, 113.5, 118.3, 122.6, 127.9,
129.9, 131.3, 133.5, 134.7, 150.7, 158.4. HRMS (FAB) calcd for
C17H21ON2, 269.1654; found, 269.1640.
3.1.4. tert-Butyl 2-[(4-acetoxyphenyl)phenyl]piperazine-1-
carboxylate (10)
A mixture of 8 (282 mg, 0.83 mmol), 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl acetate (260 mg, 0.99 mmol), Na2-
CO3 (261 mg, 2.46 mmol) and Pd(PPh3)2Cl2 (18 mg, 3 mol % Pd) in
DME/water (8 mL, 3/1, v/v) was heated at 80 °C for 2.5 h under
N2 gas. The reaction mixture was poured into saturated aqueous
NaHCO3 and extracted with ether. The organic layer was washed
with brine, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Purification of the residue by silica gel column
chromatography (EtOAc/n-hexane, 1/10, v/v) yielded 10 (193 mg,
59%) as a colorless crystal; mp, 123–124 °C. 1H NMR (300 MHz,
CDCl3): d 1.43 (9H, s), 2.31 (3H, s), 2.77 (4H, br), 3.31 (4H, br),
7.01 (1H, d, J = 7.7 Hz), 7.06–7.16 (3H, m), 7.23–7.31 (2H, m),
7.63 (2H, d, J = 8.4 Hz). 13C NMR (75 MHz, CDCl3): d 21.2, 28.4,
44.1 (br), 51.2, 79.7, 118.7, 121.3, 123.4, 128.6, 129.9, 131.5,
134.4, 138.3, 149.6, 149.7, 154.8, 169.5. FAB-MS: m/z 397 (M+H).
3.1. Chemical synthesis
3.1.1. tert-Butyl 4-(2-bromophenyl)piperazine-1-carboxylate (8)
A mixture of 1-bromo-2-iodobenzene (7; 626 lL, 5.08 mmol),
3.1.5. tert-Butyl 2-[(4-hydroxyphenyl)phenyl]piperazine-1-
carboxylate (5)
tert-butyl piperazine-1-carboxylate (942 mg, 5.06 mmol), NaO
(t-Bu) (739 mg, 7.69 mmol), Xantphos (292 mg, 10 mol %) and Pd2-
dba3 (115 mg, 5 mol % Pd) in toluene (14 mL) was heated at 80 °C
for 16 h under N2 gas. The reaction mixture was poured into water
and extracted with CH2Cl2. The organic layer was washed with
brine, dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. Purification of the residue by silica gel column
chromatography (EtOAc/n-hexane, 1/10, v/v) yielded 8 (786 mg,
46%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3): d 1.49 (9H,
A mixture of 8 (190 mg, 0.49 mmol) and aqueous 1 M NaOH
(1 mL) in MeOH/water (7 mL, 6/1, v/v) was heated at 80 °C for
5 h. After cooling to room temperature, MeOH was removed under
reduced pressure. The reaction mixture was adjusted to pH 7 with
aqueous 0.1 M HCl, and extracted with CH2Cl2. The organic layer
was washed with saturated aqueous NaHCO3, dried over anhy-
drous Na2SO4 and concentrated under reduced pressure to obtain
5 (137 mg, 80%) as a colorless crystal; mp, 208–209 °C. 1H NMR

Y. Shimoda et al. / Bioorg. Med. Chem. 21 (2013) 5316–5322
5321
(300 MHz, CDCl3): d 1.38 (9H, s), 2.69 (4H, br), 3.23 (4H, br), 6.80
(2H, d, J = 8.4 Hz), 6.99–7.07 (2H, m), 7.14–7.22 (2H, m), 7.45
(2H, d, J = 8.4 Hz), 9.43 (1H, s). 13C NMR (75 MHz, CDCl3): d 28.4,
44.0 (br), 50.8 (br), 80.0, 115.1, 118.3, 123.0, 127.9, 130.1, 131.3,
132.9, 134.9, 150.0, 155.0, 155.1. HRMS (FAB) calcd for
radiochemical purity: >99%; and specific activity at EOS: 55 GBq/
mol.
l
3.3. Measurement of partition co-efficients (Log D)
C21H27O3N2, 355.2022; found, 355.2041.
Partition co-efficient values were measured by mixing [11C]4
(radiochemical purity: 100%; approximately 150,000 cpm) with
n-octanol (3.0 g) and sodium phosphate-buffered saline (PBS,
3.0 g; 0.1 M, pH 7.40) in a test tube. The tube was vortexed for
3 min at room temperature, followed by centrifugation at
3500 rpm for 5 min. An aliquot of 1 mL PBS and 1 mL n-octanol
was removed, weighted, and counted. Samples from the remaining
organic layer were removed and re-partitioned until consistent
LogD values were obtained. The LogD value was calculated by
comparing the ratio of cpm/g of n-octanol to that of PBS and ex-
pressed as LogD = Log[cpm/g (n-octanol)/cpm/g(PBS)]. All assays
were performed in triplicate. Meanwhile, the value of cLogD of
3.1.6. 1-[2-(4-Hydroxyphenyl)phenyl]piperazine (6)
A mixture of compound 5 (99 mg, 0.28 mmol) and trifluoroace-
tic acid (862 lL, 11.2 mmol) was stirred overnight at room temper-
ature. After removal of trifluoroacetic acid under reduced pressure,
purification of the residue by silica gel column chromatography
(EtOAc/MeOH/Et3N, 9/1/0.001, v/v/v) to give trifluoroacetate of 6
(85 mg, 83%) as a pale pink crystal; mp, 177–179 °C. 1H NMR
(300 MHz, DMSO-d6): d 2.93 (4H, s), 3.01 (4H, s), 6.82 (2H, d,
J = 8.4 Hz), 7.02–7.10 (2H, m), 7.16–7.27 (2H, m), 7.44 (2H, d,
J = 8.4 Hz), 8.88 (2H, br), 9.58 (1H, br). 13C NMR (75 MHz, DMSO-
d6): d 43.0, 47.4, 115.3, 118.4, 123.4, 127.8, 129.4, 130.5, 130.9,
134.2, 148.6, 156.5. HRMS (FAB) calcd for C16H19ON2, 255.1497;
found, 255.1485.
[
11C]4 was computed using Pallas 3.4 software (CompuDrug; Sedo-
na, AZ).
3.4. In vitro autoradiography
3.2. Radiosynthesis of 1-[2-(4-[11C]
methoxyphenyl)phenyl]piperazine ([11C]4)
Four rat brains were quickly removed and frozen on powdered
dry ice. Sagittal sections (20 lm) of the brain were cut using a
3.2.1. Route 1
cryostat microtome (HM560; Carl Zeiss, Germany) and thaw-
mounted on glass slides, which were then dried and stored at
ꢀ80 °C until use. Brain sections were preincubated (3 ꢁ 5 min) in
Tris–HCl (50 mM, pH 7.4) at room temperature. After preincuba-
tion, these sections were incubated for 30 min at room tempera-
After irradiation, the cyclotron-produced [11C]CO2 was bubbled
into 0.4 M LiAlH4 in anhydrous THF (300
THF, the remaining complex was treated with 57% HI (300
produce [11C]CH3I, which was distilled and transferred under N2
gas flow into a solution of 5 (1.0 mg) and NaOH (6 L, 0.5 M) in
anhydrous DMF (100
l
L). After evaporation of
lL) to
l
ture in fresh buffer with
Unlabeled 4 or 3 (1 M) was used to elucidate the specific binding
[
11C]4 (1 nM, 37 MBq/200 mL).
l
L) at ꢀ15 to ꢀ20 °C. When the radioactivity
l
reached a plateau, the reaction mixture was subsequently heated
of [11C]4 for 5-HT7. After incubation, the sections were washed
(3 ꢁ 5 min) with cold buffer, dipped in cold distilled water, and
dried with cold air. These sections were placed in contact with
imaging plates (BAS-MS2025; FUJIFILM, Tokyo, Japan). Autoradio-
grams were obtained and photo-stimulated luminescence values
(PSL) in the regions of interest (ROIs) were measured using a Bio-
Imaging Analyzer System (BAS5000, FUJIFILM).
at 80 °C for 5 min. After [11C]methylation, trifluoroacetic acid
(200 lL) was added and this resulting solution was heated at
100 °C for another 5 min. After adding NaOAc (1.5 mL, 2.5 M), the
final mixture was subjected to HPLC. HPLC separation was com-
pleted on a Capcell Pak C18 column (10 mm id ꢁ 250 mm; Shiseido,
Tokyo, Japan) using MeCN/H2O/trifluoroacetic acid (4/6/0.05, v/v/
v) at 5.0 mL/min. The radioactive fraction corresponding to [11C]4
(tR: 8.8 min) was collected in a sterile flask, evaporated to dryness
under reduced pressure, redissolved in 3 mL of sterile normal sal-
3.5. Metabolite assay for rat plasma and brain homogenate
ine, and passed through a 0.22
l
m Millipore filter to obtain
The rat brain was dissected and the brain sections were homog-
enized in an ice-cooled MeCN/H2O (1:1, 1.0 mL) solution, respec-
tively. The homogenate was centrifuged at 15,000 rpm for 2 min
at 4 °C and the supernatant was collected. The recovery of radioac-
tivity into the supernatant was 68–87% based on the total radioac-
tivity in the brain homogenate.
2.7 GBq of [11C]4.
The radiochemical purity of [11C]4 (tR: 7.8 min) was analyzed
using HPLC with a detector for monitoring radioactivity under
the following conditions: Capcell Pak C18 column, 4.6 mm
id ꢁ 250 mm; MeCN/H2O/trifluoroacetic acid (4/6/0.05, v/v/v),
1.0 mL/min. The identity of [11C]4 was confirmed by HPLC analysis
with unlabeled 4. The specific activity was calculated by comparing
the assayed radioactivity to the mass measured at UV 254 nm. The
synthesis time was 35 min from EOB; radiochemical yield (decay-
corrected): 46% based on [11C]CO2; radiochemical purity: >99%;
An aliquot of the supernatant (100–500 lL) obtained from the
plasma or brain homogenate was injected into the radio-HPLC sys-
tem, and analyzed under the same analytical conditions described
above. The percentage of [11C]4 to total radioactivity (corrected for
decay) on the HPLC charts was calculated as (peak area for [11C]4/
total peak area) ꢁ 100.
and specific activity at EOS: 100 GBq/lmol.
3.2.2. Route 2
3.6. PET imaging
[
11C]CH3I was produced and transferred into anhydrous DMF
(300
l
L) containing 6 (0.8 mg) and NaOH (10
l
L, 0.5 M) at ꢀ15
PET scans were performed using a small-animal Inveon PET
scanner (Siemens Medical Solutions USA, Knoxville, TN), which
provides 0.796 mm (center-to-center)-apart 159 transaxial slices,
10 cm transaxial FOV, and 12.7 cm axial FOV. Before the scans,
the rats were anesthetized with 5% (v/v) isoflurane, and main-
tained thereafter with 1–2% (v/v) isoflurane. Emission scans were
acquired for 90 min in a 3-dimensional list mode with an energy
window of 350–750 keV, immediately after an intravenous injec-
to ꢀ20 °C. This reaction mixture was heated to 80 °C and main-
tained for 3 min. After MeCN/H2O/trifluoroacetic acid (1 mL, 4/6/
0.05, v/v/v) was added to the reaction vessel, the radioactive mix-
ture was transferred into a reservoir for HPLC purification (Capcell
Pak C18 column, 10 mm id ꢁ 250 mm; MeCN/H2O/trifluoroacetic
acid = 4/6/0.05, 5 mL/min). The desired radioactive fraction was
treated as described above to obtain [11C]4 (1.84 GBq at EOS) as
an injectable solution. The synthesis time was 29 min from EOB;
radiochemical yield (decay-corrected): 45% based on [11C]CO2;
tion of [11C]4 (16–18 MBq/200
in vivo specific binding, unlabeled 3 (3 mg/kg dissolved in 300
lL, 0.07–0.11 nmol). To evaluate
lL

5322
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saline containing 10% ethanol and 5% polysorbate 80) was injected
1 min before injection of [11C]4. Three rats were used for each
experiment. All list-mode acquisition data were sorted into 3-
dimensional sinograms, which were then Fourier rebinned into
2-dimensional sinograms (frames ꢁ min: 4 ꢁ 1, 8 ꢁ 2, 14 ꢁ 5). Dy-
namic images were reconstructed with filtered back-projection
using a Hanning’s filter, with a Nyquist cutoff of 0.5 cycle/pixel.
ROIs were placed on the striatum, hippocampus, cerebral cortex,
thalamus, medulla, and cerebellum using ASIPro VM™ (Analysis
Tools and System Setup/Diagnostics Tool, Siemens Medical Solu-
tions, USA) with reference to the MRI template. The uptake of
radioactivity in the brain was decay-corrected to the injection time
and was expressed as the standardized uptake value (SUV), which
was normalized to the injected radioactivity and body weight.
SUV = (radioactivity per milliliter tissue/injected radioactiv-
ity) ꢁ gram body weight.
Acknowledgments
We thank the crew of the Cyclotron Operation Section and
Molecular Imaging Center of National Institute of Radiological Sci-
ences for their help with the operation of the cyclotron, production
of the radioisotope and animal experiments.
26. Lemonine, L.; Andriès, J.; Le Bars, D.; Billard, T.; Zimmer, L. J. Nucl. Med. 2011,
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