Synthesis and evaluation of a 18F-labeled spirocyclic piperidine derivative as promising σ1receptor imaging agent

Article abstract of DOI:10.1016/j.bmc.2014.08.003

Several spirocyclic piperidine derivatives were designed and synthesized as σ₁receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ₁rece

Full text of DOI:10.1016/j.bmc.2014.08.003

Bioorganic & Medicinal Chemistry 22 (2014) 5270–5278
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of a 18F-labeled spirocyclic piperidine
derivative as promising r1 receptor imaging agent
Yuan-Yuan Chen a, Xia Wang a, Jin-Ming Zhang b, Winnie Deuther-Conrad c, Xiao-Jun Zhang b,
Yiyun Huang d, Yan Li a, Jia-Jun Ye a, Meng-Chao Cui a, Jörg Steinbach c, Peter Brust c, Bo-Li Liu a,
Hong-Mei Jia a,
⇑
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
b Nuclear Medicine Department, Chinese PLA General Hospital, Beijing 100853, China
c Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, 04318 Leipzig, Germany
d PET Center, Department of Diagnostic Radiology, Yale University School of Medicine, New Haven, CT, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several spirocyclic piperidine derivatives were designed and synthesized as
r1 receptor ligands. In vitro
Received 16 April 2014
Revised 18 July 2014
Accepted 5 August 2014
Available online 12 August 2014
competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed
high affinity towards r1 receptors and subtype selectivity. Particularly for ligand 10-((6-(2-fluoro-
ethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofuran-1,40-piperidine] (2), high r1 receptor affinity
(Ki = 2.30 nM) and high r1/r2 subtype selectivity (142-fold) as well as high r1/VAChT selectivity
(234-fold) were observed. [18F]2 was synthesized using an efficient one-pot, two-step reaction method
Keywords:
in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8–10%, a
PET
radiochemical purity of higher than 99%, and specific activity of 56–78 GBq/
lmol. Biodistribution studies
18F
of [18F]2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of
Sigma-1 receptors
Imaging agent
Spirocyclic piperidine derivatives
haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 lmol/kg) 5 min prior to
injection of [18F]2 significantly decreased the accumulation of radiotracer in organs known to contain
r1 receptors. Ex vivo autoradiography in Sprague–Dawley rats demonstrated high accumulation of
radiotracer in brain areas with high expression of r1 receptors. These encouraging results prove that
[
18F]2 is a suitable candidate for r1 receptor imaging with PET in humans.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
been shown to be involved in cardiovascular disease9,10 and in the
development of human tumors,11,12 especially those with positive
progesterone receptor status.13 Therefore, the r1 receptor repre-
sents a potential target for both diagnosis and therapeutic develop-
ment of a wide range of diseases.
Sigma-1
(r1) receptors are intracellular transmembrane
proteins, consisting of 223 amino acids and assumed to possess
two transmembrane domains.1,2 They function as ‘ligand-operated
receptor chaperones’ and serve as an inter-organelle signaling
modulator locally at the mitochondrion-associated endoplasmic
reticulum (ER) membrane (MAM) and remotely at the plasma
The noninvasive imaging of r1 receptors by positron emission
tomography (PET) has potential not only to provide an innovative
pharmacological approach for the diagnosis of diseases but also to
contribute to a better understanding of the physiological and
pathophysiological role of r1 receptors.14 However, only a few
membrane.3,4 Increasing evidence suggests that
r1 receptors inter-
act with pharmacologically different drugs and are involved in var-
ious human diseases including, in particular, protein conformation
diseases.4 In the central nervous system (CNS), r1 receptors regu-
late a variety of signal transduction processes, ER stress, cellular
redox, cellular survival, and synaptogenesis,5–7 and are thus impli-
cated in anxiety, depression, schizophrenia, drug addiction, Alzhei-
mer’s disease, and neuroinflammation.5–8 Moreover, they have also
radiotracers such as [11C]SA4503,15–17 18F]FPS18 and [123I]TPCNE19
[
(Fig. 1) have been evaluated in humans until now. Among these,
[
18F]FPS20 and [123I]TPCNE19 were found to have irreversible kinet-
ics in humans. SA4503 showed affinity for the vesicular acetylcho-
line transporter (VAChT) (Ki = 50.2 nM),21 although [11C]SA4503
did not seem to bind to VAChT in the rat brain in vivo.22 In
addition, clinical use of [11C]SA4503 needs an on-site cyclotron.
More recently, [18F]fluspidine23 and [18F]FTC-14624,25(Fig. 1) have
been shown to be promising PET radiotracers for visualizing r1
⇑
Corresponding author. Tel./fax: +86 10 58808891.
E-mail address: hmjia@bnu.edu.cn (H.-M. Jia).
http://dx.doi.org/10.1016/j.bmc.2014.08.003
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
5271
Figure 1. The structures of potential r1 receptor radiotracers.
receptors, but their prospects for eventual clinical translation
require further investigation. Therefore, there is still need for the
development of radiotracers with appropriate affinity and high
specificity for r1 receptors to accurately quantify r1 receptors
and provide useful diagnostic tools for investigation of diseases
related to r1 receptors.
10-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofu-
ran-1,40-piperidine] (2) with 18F and evaluated the potential of
[
18F]2 as a putative
r1 receptor imaging agent in vivo.
2. Results and discussion
2.1. Organic chemistry
In the past decade, a novel class of spirocyclic piperidine com-
pounds were reported as potent
compounds to develop radiotracers for
r
1 receptor ligands and used as lead
1 receptor imaging.23,26–28
The synthetic routes of spirocyclic piperidine derivatives are
showed in Scheme 1. The intermediates 6, 7, 8, 9 and 10 were
synthesized based on the methods reported in the literature.29–31
Compound 2 was synthesized according to the method reported
previously.29 Reductive amination of compound 8 in the presence
of NaBH(OAc)3 with 4-(hydroxymethyl)benzaldehyde led to com-
pound 11. Reaction of intermediate 9 or 10 with 3-fluoropropyl
4-methylbenzenesulfonate (7) under basic condition (K2CO3) in
acetonitrile provided compound 3 or 4 in 61% or 33% yield, respec-
tively. Intermediate 11 reacted with 1-bromo-2-fluoroethane in
acetone at 60 °C to give compound 5 in 32% yield. The target com-
pounds (3, 4 and 5) were characterized and shown to have >95%
purity.
r
In our previous work,29 we have found that [18F]10-(4-(2-fluoroeth-
oxy)benzyl)-3H-spiro[2-benzofuran-1,40-piperidine] ([18F]1) is a
promising r1 receptor imaging agent with subnanomolar affinity
for r1 receptors (Ki = 0.79 nM) and excellent r1
tivity (350-fold), as well as high 1/VAChT selectivity (799-fold).
This radiotracer displayed high initial brain uptake and high accu-
mulation in brain areas known to express high levels of 1 receptors.
/r2 subtype selec-
r
r
However, its clearance rate was very slow in the brain. In order to
avoid irreversible kinetics and find radiotracers with relatively fast
washout rate in the brain, we modified the N-benzyl group and
designed analogs of spirocyclic piperidine as shown in Figure 2. It
was previously reported that replacement of the aromatic ring with
a pyridine at the N-benzyl moiety only slightly decreased the affinity
for r1 receptors.29 Moreover, such modification could lower the
lipophilicity and accordingly decrease the non-specific binding
in vivo. In an attempt to modify the affinity and pharmacokinetic
properties, we added a carbon atom at the para-substituent of the
N-benzyl group to investigate the effects of an elongation of the flu-
oroalkoxy group as well as the effects of the position of the oxygen
atoms. Herein we report on the synthesis and evaluation of the
above spirocyclic piperidine derivatives as r1 receptor ligands
in vitro. In addition, we labeled the previously reported ligand
2.2. Radiochemistry
We employed an efficient one-pot, two-step reaction method to
synthesize [18F]2 with a home-made automated synthesis module.
The synthetic routes towards [18F]2 are outlined in Scheme 2. In
the first step, ethane-1,2-diyl bis(4-methylbenzenesulfonate) 12
reacted with dried Kryptofix 2.2.2/K+/[18F]Fꢀ complex in acetoni-
trile at 90 °C to provide 2-[18F]fluoroethyl-1-tosylate 13. The
desired radiotracer [18F]2 was obtained via alkylation reaction
between the precursor 10 and 13 in the presence of NaOH for
20 min at 110 °C. After cooling to room temperature, the mixture
was passed through a C18 Sep-Pak cartridge to remove inorganic
salts including [18F]KF. The crude product was eluted and purified
by an isocratic semi-preparative radio-HPLC using a reverse-phase
column and a mobile phase consisting of acetonitrile and water
(containing 10 mM NH4OAc) (40:60, v/v) at a flow rate of 4 mL/
min. Retention time of [18F]2 was 33.4 min (Fig. S1, Supplementary
data). The radioactive peak corresponding to [18F]2 was collected,
diluted with water and passed across a C18 Sep-Pak cartridge.
The cartridge was washed with water. The product was eluted
off with ethanol and diluted with sterile saline to provide a solu-
tion with approximately 370 kBq of radioactivity per 0.1 mL.
In order to identify the radiotracer, [18F]2 and reference com-
pound 2 were co-injected and their HPLC profiles assessed using
Figure 2. Design concept of spirocyclic piperidine derivatives.

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Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
Scheme 1. Synthetic routes of spirocyclic piperidine derivatives. Regents and conditions: (a) TsCl, KOH, CH2Cl2, 0 °C, 83%; (b) TBAF (1 mol/L in THF), CH3CN, reflux, 41%; (c) for
9, (i) 4-hydroxybenzaldehyde, 1,2-dichloroethane, 2 h; (ii) NaBH(OAc)3, 6 h, 69%; for 10, (i) 6-hydroxynicotinaldehyde, 1,2-dichloroethane, 2 h; (ii) NaBH(OAc)3, 24 h, 24%; (d)
(i) 4-(hydroxymethyl)benzaldehyde, 1,2-dichloroethane, 2 h, (ii) NaBH(OAc)3, 48 h, 33%; (e) 7, CH3CN, K2CO3, reflux, 61% for 3, 33% for 4; (f) 1-bromo-2-fluoroethane, K2CO3,
acetone, 60 °C, 10 h, 32%.
of our HPLC system. After purification by semi-preparative radio-
HPLC, the radiochemical purity (RCP) of [18F]2 was higher than
99%. The overall isolated radiochemical yield was 8–10% (n = 3,
corrected for decay). The total synthesis time was about 1 h. The
specific activity was 56–78 GBq/lmol.
The in vitro stability of [18F]2 in mouse plasma was evaluated
by measuring the RCP at different time points. After 2 h and 4 h
of incubation with mouse plasma at 37 °C, the RCP of [18F]2 was
96% and 95%, respectively (Fig. S2, Supplementary data), suggest-
ing high stability of [18F]2 in vitro.
Lipophilicity in terms of the apparent distribution coefficient at
pH 7.4 was determined by shake-flask method in a 1-octanol and
0.05 mol/L potassium phosphate buffer system. The logD value of
Scheme 2. Synthesis of the radiotracer
Kryptofix 2.2.2, K2CO3, CH3CN, 90 °C, 15 min; (b) 13, 2 mol/L NaOH, DMSO,
110 °C, 20 min.
[
18F]2. Reagents and conditions: (a)
[
18F]2 was determined to be 2.58 0.01 (n = 3), which is within
the range expected to have good blood–brain barrier permeability.
In addition, the retention times of unlabeled 1 and 2 using acetoni-
trile and water (containing 10 mM NH4OAc) (60:40, v/v) as mobile
phase at a flow rate of 1 mL/min were 10.32 and 10.18 min, respec-
tively (Fig. S3, Supplementary data). These data indicate that the
lipophilicity of [18F]2 is slightly lower than that of [18F]1, which
is in good agreement with our expectation that replacement of a
benzene ring with pyridine decreases the lipophilicity of the
compound.
acetonitrile and water (containing 10 mM NH4OAc) (60:40, v/v) as
mobile phase at a flow rate of 1 mL/min are presented in Figure 3.
The retention times of 2 and [18F]2 were observed to be 10.88 and
10.97 min, respectively. The difference in retention times was in
accordance with the time lag due to the volume and flow rate
within the distance between the UV and radioactivity detectors
2.3. Biological studies
2.3.1. In vitro radioligand competition studies
The competition binding assays for r1 and r2 receptors were
carried out as previously reported.32 The r1 and r2 receptor
affinities of the new compounds were determined with the radio-
ligands (+)-[3H]pentazocine and [3H]DTG (in the presence of 10
lM
dextrallorphan) using rat brain and rat liver membranes, respec-
tively. The Ki values of the compounds are listed in Table 1.
Replacement of the benzene ring with pyridine decreased the affin-
ity for r1 receptors slightly (1 vs 2, 3 vs 4). Substitution at para-
position of the benzyl moiety with increased chain length also
led to decreased r1 receptor affinity (1 vs 3). Moreover, the posi-
tion of the oxygen atom in the substituent at the para-position of
the benzyl moiety has much influence on the r1 receptor affinity.
Replacement of the OCH2CH2CH2F with a CH2OCH2CH2F group dra-
matically decreased the r1 receptor affinity (3 vs 5).
Figure 3. HPLC co-elution profiles of 2 and [18F]2, with retention time of 10.88 and
10.97 min, respectively. Conditions: CH3CN/H2O (containing 10 mM NH4OAc) = 60/
40, v/v, flow rate = 1 mL/min.
In addition, the affinities of compound 2 for VAChT were also
determined in radioligand competition experiments as previously

Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
5273
30 min, respectively. However, the brain-to-blood ratios of [18F]2
were lower than those of [18F]1. Similar to the kinetic profile in
the brain, [18F]2 also showed relatively fast washout in the organs
Table 1
Binding affinities of the spirocyclic piperidine derivatives towards r1 and r2
receptorsa
Compound
Ki (
r
1) (nM)
Ki (
r
2) (nM)
Ki (r2)/Ki (r1)
known to contain
r1 receptors, including the lungs, kidneys, heart,
1b
0.79 0.11
2.30 0.69
3.32 0.71
4.00 1.14
57.5 3.6
277 71
327 47
143 11
131
2803 202
20.7 0.07
350
142
43
33
49
4
spleen, and liver. Finally, the accumulation of radiotracer in the
bone increased slightly with time with 7.45% ID/g at 120 min,
which was a little higher than that of [18F]1 with 6.13% ID/g, indicat-
ing that [18F]2 may undergo minimal defluorination in vivo.
In order to verify the specific binding of [18F]2 to r1 receptors
in vivo, the effects of preadministration of haloperidol (0.1 mL,
1 mg/kg), compound 1 (0.1 mL, 1 mg/kg) and different concentra-
2b
3
4
5
8
Haloperidol
4.95 1.74
a
Values are means SD of three experiments performed in triplicate.
From Ref. 29.
b
tions of SA4503 (3, 5, or 10 lmol/kg) on the biodistribution of
radiotracer in various organs of male ICR mice were investigated.
Either saline or the blocking agents was injected 5 min prior to
the radiotracer injection (0.1 mL, about 370 kBq). Blocking results
of organ distribution of [18F]2 at 30 and 60 min postinjection in
ICR mice are summarized in Figure 4. Pretreatment of animals with
haloperidol, compound 1 and different concentrations of SA4503
resulted in significant reduction of radiotracer uptake in organs
known to contain r1 receptors at 30 min, including the brain
(72–74%, p < 0.001), heart (22–32%, p < 0.05), liver (32–50%,
p < 0.05), spleen (47–70%, p < 0.05) and lungs (48–59%, p < 0.05
except for compound 1). The corresponding blocking rate at
60 min was less than that at 30 min, including the brain (64–67%,
p < 0.001), spleen (49–58%, p < 0.05) and lungs (34–43%, p < 0.05).
reported.33 Compound
(Ki = 538 14 nM) and was thus characterized by an excellent
selectivity for r1 receptors (Ki(VAChT)/Ki( 1) = 234).
Considering compound 2 possesses nanomolar affinity for r1
receptors and high selectivity for r2 receptors and VAChT as well
as the lowest lipophilicity within this series, it was selected for
18F-labeling and [18F]2 evaluated for its potential as r1 receptor
radiotracer.
2 displayed low affinity for VAChT
r
2.3.2. Biodistribution and blocking studies in male ICR mice
In order to evaluate the pharmacokinetics of [18F]2, we per-
formed in vivo biodistribution studies in male ICR mice. Experimen-
tal data are summarized in Table 2. [18F]2 showed a high initial
brain uptake with radioactivity concentration of 8.39 1.54% ID/g
at 2 min post-injection (p.i.). The accumulation in the brain was
highest at 15 min p.i. (8.56 0.34% ID/g), and slowly decreased
thereafter with 6.61 1.22% ID/g at 30 min, 4.62 0.17% ID/g at
60 min, and 3.31 0.33% ID/g at 120 min. Since [123I]TPCNE suffers
from irreversible binding in the human brain,19 a radiotracer with
These data demonstrated that [18F]2 binds to
r1 receptors specifi-
cally in vivo.
2.3.3. Ex vivo autoradiography in Sprague–Dawley rats
To evaluate the potential use of [18F]2 for r1 receptor imaging
in the brain, an ex vivo autoradiography at 1 h after injection of
[
18F]2 (0.3 mL, 74.0 MBq) was performed to investigate the distri-
bution of [18F]2 in different brain regions in Sprague–Dawley rats.
less avid binding to
for clinical applications. In our previous work, the accumulation of
18F]1 in the brain was found to be highest at 30 min p.i. with
r1 receptors was suggested to be better suited
The results are presented in Figure 5. Similar to what was found for
[
18F]1,29 high accumulation of radiotracer was observed in the
[
cortex, particularly in the temporal cortex auditory area. In the
cerebellum, comparably high uptake was found in the vermian lob-
ule and low uptake in the paramedian lobule. In the brain areas
known to possess high expression of r1 receptors, such as thala-
mus, hypothalamus, midbrain, red nucleus, and facial nucleus, high
accumulation of the radiotracer was also observed, which is consis-
tent with the brain distribution of [11C]SA4503.35 In addition, high
accumulation was observed in the medulla oblongata. Moderate
accumulation of radiotracer was observed in the hippocampus
and striatum. Comparably low accumulation was observed in the
nucleus accumbens and olfactory bulb. Taken together, the
accumulation of the radiotracer in the rat brain is in good
agreement with the density profile of r1 receptors.
9.32 0.50% ID/g, and decreased very slowly to 8.99 0.37% ID/g
at 120 min p.i.29 Replacement of the benzene ring in [18F]1 with a
pyridine resulted in [18F]2 with about 3 times lower affinity (0.79
vs 2.30 nM for 1 and 2, respectively) and lower lipophilicity (The
retention times of unlabeled 1 and 2 were 10.32 and 10.18 min,
respectively). Consistent with our expectations, [18F]2 exhibited a
faster clearance from the brain than [18F]1. In addition, the initial
brain uptake of [18F]2 was higher than that of [11C]SA4503
(3.61 0.39% ID/g at 5 min),34 18F]fluspidine (3.88 0.92% ID/g at
[
5 min),23 and [18F]FP-fluspidine (3.66 0.43% ID/g at 5 min).27 Sec-
ond, the radiotracer levels in the blood were relatively low with
1.62 0.18% ID/g at 15 min and 1.38 0.18% ID/g at 30 min p.i.,
resulting in high brain-to-blood ratios of 5.28 and 4.79 at 15 and
Table 2
Biodistribution of [18F]2 in male ICR micea
Organ
2 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Small intestine
Stomach
Muscle
2.59 0.28
8.39 1.54
13.29 2.49
6.97 1.15
4.10 0.56
38.45 8.14
23.60 1.23
5.07 1.33
1.41 0.27
2.78 0.14
2.64 0.72
3.24
1.62 0.18
8.56 0.34
6.66 1.06
6.72 1.29
8.67 1.51
10.26 1.41
13.00 1.94
6.43 0.60
1.84 0.44
2.62 0.48
3.70 0.66
5.28
1.38 0.18
6.61 1.22
3.49 0.52
6.95 1.35
6.76 1.11
6.23 1.89
7.83 1.14
4.73 1.86
1.23 0.49
1.98 0.24
5.61 0.89
4.79
1.43 0.19
4.62 0.17
3.08 0.86
5.42 0.89
5.95 0.81
4.34 0.30
6.97 2.36
5.74 1.67
1.08 0.23
2.10 0.30
6.28 1.47
3.23
1.90 0.08
3.31 0.33
2.83 0.78
3.74 0.37
4.13 0.37
2.65 0.30
3.79 0.28
6.34 1.61
0.89 0.14
1.61 0.29
7.45 0.75
1.74
b
b
Bone
Brain/blood
a
Data are means of % ID/g of tissue SD, n = 5.
% ID/organ.
b

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Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
Figure 4. (A) Effects of preadministration of haloperidol (0.1 mL, 1 mg/kg), compound 1 (0.1 mL, 1 mg/kg) and different concentrations of SA4503 (3, 5, or 10
lmol/kg) on
organ distribution of [18F]2 (0.1 mL, about 370 kBq) 30 min after intravenous injection. Student’s t test (independent, two-tailed) was performed, and p < 0.05 (except for
compound 1 in the lungs and in the kidney, SA4503 (3 lmol/kg) in the kidney). (B) Effects of preadministration of haloperidol (0.1 mL, 1 mg/kg) and SA4503 (0.1 mL, 3 lmol/
kg) 5 min prior to the injection of radiotracer [18F]2 (0.1 mL, about 370 kBq) 60 min after intravenous administration. Student’s t test (independent, two-tailed) was
performed, and p < 0.05 (except for haloperidol and SA4503 in the heart, haloperidol in the liver). Values are means SD, n = 5.
Figure 5. Ex vivo autoradiograms of the rat coronal brain slices at 60 min after intravenous injection of [18F]2 (0.3 mL, 74.0 MBq). (A) Olfactory bulb; (B) Frontal cortex; (C)
Cerebral cortex; (D) Nucleus accumbens; (E) Striatum; (F) Cingulate cortex (G) Temporal cortex auditory; (H) Hippocampus; (I) Thalamus; (J) Hypothalamus; (K) Midbrain;
(L) Red nucleus; (M) Vermian lobule; (N) Paramedian lobule (O) Paraflocculus; (P) Facial nucleus; (Q) Medulla Oblongata.
2.3.4. In vivo metabolism of [18F]2
2.93, 4.86 and 7.81 min, respectively. Similar to what were found
for [18F]fluspidine,23 there were two radiometabolites (M3 and
M4) not detected in plasma samples. In the brain samples, about
93% of the total radioactivity was represented by the parent radio-
tracer [18F]2.
At 60 min p.i., the percentage of parent tracer was decreased
rapidly to 3% in plasma samples. The percentage of the major
metabolite M1 increased to 87% (Fig. S4, Supplementary data).
Only 38% of the parent radiotracer remained in the brain. Identities
of the radioactive metabolites of [18F]2 formed in the periphery
needs to be investigated further.
For development of a radiotracer for brain imaging, it is neces-
sary to investigate the metabolism profile of the radiotracer
in vivo. Therefore, the metabolic profile of [18F]2 was investigated
in plasma, liver and brain samples obtained from mice at 30 and
60 min p.i., respectively. Protein precipitation was performed by
using ice-cold acetonitrile. Acetonitrile extracts were analyzed
and quantified by radio-HPLC. Representative HPLC chromato-
grams are given in Figure 6. Additional HPLC chromatograms are
presented in Table S1 (Supplementary data).
At 30 min p.i., about 77% of the total radioactivity was repre-
sented by [18F]2 (retention time of 10.76 min) in plasma samples.
Two hydrophilic radioactive metabolites M1 (19%) and M2 (4%)
were observed with retention times of 2.18 and 2.92 min, respec-
tively. In liver homogenates, 49% of total radioactivity accounted
for [18F]2 at 30 min p.i., while 8%, 2%, 13% and 29% accounted for
metabolites M1, M2, M3 and M4 with retention times of 2.18,
Recently, more and more evidence suggests that r1 receptors
are linked to many human brain diseases.5–8 Neuroimaging of r1
receptors with PET radiotracers provides an important tool to
investigate the involvement of
of neuropsychiatric diseases.14 However, among the potential PET
radiotracers, use of
11C]SA4503 needs an on-site cyclotron.
r1 receptors in the pathophysiology
[

Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
5275
Figure 6. Analytical radio-HPLC chromatograms of the plasma and brain extracts at 30 and 60 min after administration of [18F]2 in mice.
[
18F]FPS20 was found to have irreversible kinetics in humans.
and SA4503 as blocking agents for assessing the binding specificity
of [18F]2 to
1 receptors in vivo. It is encouraging that the percent-
age of specific uptake of [18F]2 in the brain was higher than that of
[
18F]1 (67% vs 58% for [18F]2 and [18F]1 at 60 min p.i.), which is
consistent with our expectations that lower lipophilicity could
decrease the non-specific binding in vivo. To evaluate the potential
use of [18F]2 for imaging r1 receptor density in the brain, we
performed ex vivo autoradiography study to investigate the
accumulation of [18F]2 in different brain regions in Sprague–Dawley
rats. High accumulation of radiotracer was observed in brain areas
known to have high expression of r1 receptors.
Prospects of [18F]fluspidine23 and [18F]FTC-14624,25 for eventual
clinical translation require further investigation. In our previous
r
work,29 we have found that [18F]1 is a promising
r
1 receptor imag-
ing agent with subnanomolar affinity for r1 receptors, excellent
r2 subtype selectivity, and high 1/VAChT selectivity. More-
over, no binding to EBP, dopamine D2/D3 receptors and 5-HTT of
r1
/
r
[
18F]1 was observed. However, [18F]1 was found to clear rather
slowly from the brain. In order to find suitable 18F-labeled radio-
tracers, especially to avoid irreversible binding kinetics, we designed
and synthesized some fluoroalkoxy analogues. Among them, com-
pound 2 possesses nanomolar affinity for r1 receptors and high
It is well known that investigation of a radioligand’s metabo-
lism profile in vivo is also very important. Therefore, in vivo meta-
bolic fate of [18F]2 in the plasma, brain and liver of male ICR mice at
30 and 60 min was investigated. Results showed that the parent
compound [18F]2 and two likely polar radioactive metabolites were
found in the mouse plasma. As indicated in the literature for the
plasma metabolic profile of [18F]AV-45,38 in addition to the parent
radiotracer, three metabolite peaks were observed in plasma. How-
ever, it seemed unlikely that plasma levels of radioactive metabo-
lites could be high enough to account for the observed tracer
activity in the brain. In this study, only two likely polar metabolites
were observed both at 30 and 60 min after injection of [18F]2. Thus,
if radioactive metabolites of [18F]2 may make some contribution,
particularly to the nontarget activity, they have minimal influence
selectivity over
philicity. So [18F]2 was synthesized and evaluated for its potential
as
1 receptor radiotracer. High in vitro stability of [18F]2 in mouse
r2 receptors and VAChT as well as the lowest lipo-
r
plasma was observed. In addition, replacement of the benzene ring
with pyridine slightly decreases the lipophilicity of the compound.
In the biodistribution studies, the initial brain uptake of [18F]2 was
observed to be a little lower than that of [18F]1. But it was still
higher than that of [11C]SA4503,34 18F]fluspidine,23 and [18F]FP-
[
fluspidine.27 More importantly, [18F]2 exhibited a faster clearance
from the brain and other organs known to contain r1 receptors,
which was in good agreement with the lower affinity of [18F]2.
Currently, haloperidol is recognized as the most widely used r1
antagonist in research on r1 receptors. Based on the results
reported in the literature, SA4503 possesses high affinity for r1
receptors and low to negligible affinity for 36 other receptors, ion
channels and second messenger systems.36 So SA4503 has also
been used as blocking agent to determine target specificity.37 In
addition, compound 1 proved to possess excellent affinity and
selectivity for r1 receptors. So we used haloperidol, compound 1
on the specific binding signal of [18F]2 to
r1 receptors in the brain.
In the brain, the percentage of [18F]2 at 30 min p.i. was higher than
that at 60 min (93% vs 38%), which was in accord with higher per-
centage of specific binding of [18F]2 at 30 min.
Currently, the identities of the radioactive metabolites have not
been determined. Moreover, selectivity over other neuroreceptors

5276
Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
of [18F]2 should be taken into consideration. These further investi-
gations and small animal PET imaging studies are in progress to
evaluate [18F]2 as a candidate for r1 receptor imaging with PET
in humans.
and K2CO3 (41.0 mg, 0.30 mmol). The mixture was heated to reflux
for 18 h. The mixture was concentrated under vacuum and the res-
idue was dissolved in CH2Cl2, washed with water, dried with
MgSO4 and concentrated under vacuum. The crude product was
purified by silica gel chromatography (ethyl acetate/petroleum
ether = 1/1, v/v) to provide 3 as colorless oil (33.0 mg, 61%). 1H
NMR (400 MHz, CDCl3) d (ppm): 7.28–7.26 (m, 4H), 7.21–7.16
(m, 2H), 6.89 (s, 1H), 6.87 (s, 1H), 5.07 (s, 2H), 4.71 (t, J = 5.8 Hz,
1H), 4.60 (t, J = 5.8 Hz, 1H), 4.10 (t, J = 6.1 Hz, 2H), 3.57 (s, 2H),
2.86 (s, 2H), 2.42 (s, 2H), 2.24–2.11 (m, 2H), 2.02 (s, 2H), 1.79 (s,
1H), 1.75 (s, 1H). 13C NMR (100 MHz, CDCl3) d 157.98, 145.77,
138.98, 130.65, 130.44, 127.59, 127.38, 121.08, 120.89, 114.24,
84.80, 81.64, 80.00, 77.30, 70.77, 63.53, 63.48, 62.83, 50.05,
36.61, 30.60, 30.40. ESI-TOF MS calcd for C22H27FNO2 [M+H]+:
356.2068; found: 356.2087.
3. Conclusion
We have successfully synthesized [18F]2 as a novel radiotracer
for r1 receptor imaging using an efficient one-pot, two-step reac-
tion sequence in a home-made automated synthesis module. In
biodistribution studies in mice [18F]2 displayed high initial brain
uptake and relatively fast clearance from the brain. Blocking stud-
ies confirmed high specific binding of [18F]2 to
r1 receptors in vivo.
Ex vivo autoradiography indicated that accumulation of the radio-
tracer in the rat brain was in good agreement with the density
profile of r1 receptors. In vivo metabolic studies suggested that
radioactive metabolites of [18F]2 have minimal influence on the
specific binding signal to r1 receptors in the brain. These findings
warrant further evaluation of the potential of [18F]2 as a radio-
tracer for investigation of r1 receptors with PET in humans.
4.3. 10-((6-(3-Fluoropropoxy)pyridin-3-yl)methyl)-3H-spiro[2-
benzofuran-1,40-piperidine] (4)
The procedure described for the synthesis of 3 was applied to 10
to provide 4 as colorless oil (12.0 mg, 33%). 1H NMR (400 MHz,
CDCl3) d (ppm): 8.08 (s, 1H), 7.74 (s, 1H), 7.29–7.21 (m, 4H),
6.76–6.74 (d, J = 8.1 Hz, 1H), 5.07 (s, 2H), 4.70 (t, J = 5.8 Hz, 1H),
4.58 (t, J = 5.8 Hz, 1H), 4.43 (t, J = 6.2 Hz, 2H), 3.62 (s, 2H), 2.92 (s,
2H), 2.54 (s, 2H), 2.22–2.05 (m, 4H), 1.80 (s, 1H), 1.77 (s, 1H).
ESI-TOF MS calcd for C21H26FN2O2 [M+H]+: 357.1973; found:
357.1996.
4. Experimental section
4.1. General method
All reagents and solvents were obtained in high purity from
commercial sources and used without further purification unless
otherwise stated. Reactions were monitored by thin-layer chroma-
tography (TLC) (TLC silica gel 60 F254 plates Merck). Flash column
chromatography was carried out on silica gel (200–400 mesh)
using the solvent system indicated in the experimental procedure.
HPLC methods were used to determine the purity of the test com-
pounds used in the binding assays. 1H NMR spectra were recorded
on a Bruker Avance III (400 MHz) NMR spectrometer. Chemical
shift (d) are reported in ppm downfield from tetramethylsilane
and coupling constants (J) in Hertz (Hz). 13C NMR spectra were
recorded on a Bruker Avance III (100 MHz) NMR spectrometer.
MS spectra were obtained by microTOF-QII ESI/MS (Bruker, USA).
HPLC separations were performed in the PET-MF-2V-IT-1 multi-
functional synthesis module. The semi-preparative radio-HPLC was
equipped with an Alltech 626 pump and UVSI 200 detector. Samples
were separated on an Agela Venusil MP C18 column (250 mm ꢁ
4.4. (4-((3H-Spiro[2-benzofuran-1,40-piperidin]-10-yl)methyl)
phenyl)methanol (11)
Compound 8 (50.0 mg, 0.26 mmol) was dissolved in 1,2-dichlo-
roethane (10 mL), followed by addition of 4-(hydroxymethyl)benz-
aldehyde (40.0 mg, 0.29 mmol). The mixture was stirred at room
temperature for 2 h, followed by addition of NaBH(OAc)3
(63.0 mg, 0.30 mmol). The mixture was stirred for an additional
48 h. Then the reaction was quenched with saturated NaHCO3,
evaporated to remove the organic solvent, and extracted with ethyl
acetate. The combined organic layers were dried with MgSO4, and
concentrated under vacuum. The residue was purified by silica gel
chromatography (ethyl acetate/petroleum ether = 2/1, v/v) to pro-
vide 11 as colorless oil (27.0 mg, 33%). 1H NMR (400 MHz, CDCl3) d
(ppm): 7.26 (s, 3H), 7.20–7.19 (m, 3H), 7.18–7.16 (m, 2H), 6.79 (br
s, 1H), 5.04 (s, 2H), 3.79 (s, 2H), 3.08 (s, 1H), 3.05 (s, 1H), 2.65 (td,
J = 4.32, 13.68 Hz, 2H), 2.06 (s, 2H), 1.76 (s, 1H), 1.73 (s, 1H). ESI-
MS, [M+H]+: m/z = 311.0.
10 mm, 5 lm) using acetonitrile and water (containing 10 mM
NH4OAc) (40:60, v/v) as mobile phase at a flow rate of 4 mL/min.
HPLC analyses for the identification of the radiotracer and for the
investigation of in vitro stability and in vivo metabolism of [18F]2
were performed on a Waters 600 system (Waters, corporation,
USA) equipped with a Waters 2489 UV–VIS detector and a Raytest
Gabi NaI (Tl) scintillation detector (Raytest, Germany). Samples
were analyzed on an Agela Venusil MP C18 column (250 mm ꢁ
4.5. 10-(4-((2-Fluoroethoxy)methyl)benzyl)-3H-spiro[2-
benzofuran-1,40-piperidine] (5)
A mixture of 11 (27.0 mg, 0.09 mmol), 1-bromo-2-fluoroethane
(218.0 mg, 1.72 mmol) and K2CO3 (194.0 mg, 1.40 mmol) in ace-
tone (30 mL) was heated at 60 °C for 10 h, followed by addition
of saturated NH4Cl to neutralize the mixture. The aqueous phase
was extracted with ethyl acetate. The combined organic phases
were dried over MgSO4 and concentrated under vacuum. The crude
product was purified by silica gel column chromatography (ethyl
acetate/petroleum ether = 1/4, v/v) to provide 5 as a white solid
(10.0 mg, 32%). 1H NMR (400 MHz, CDCl3) d (ppm): 7.37–7.31 (m,
4H), 7.27–7.26 (m, 2H), 7.21–7.19 (m, 1H), 7.16–7.14 (m, 1H),
5.07 (s, 2H), 4.65 (t, J = 4.1 Hz, 1H), 4.59 (s, 2H), 4.54 (t, J = 4.1 Hz,
1H), 3.77 (t, J = 4.1 Hz, 1H), 3.69 (t, J = 4.1 Hz, 1H), 3.60 (s, 2H),
2.87 (s, 1H), 2.84 (s, 1H), 2.44 (t, J = 11.6 Hz, 2H), 2.21 (t,
J = 12.0 Hz, 2H), 1.77 (s, 1H), 1.74 (s, 1H). 13C NMR (100 MHz,
CDCl3) d 145.71, 138.96, 136.66, 129.52, 127.81, 127.59, 127.37,
121.07, 120.87, 84.72, 84.02, 82.34, 77.27, 73.26, 70.26, 69.33,
4.6 mm, 5 lm) using acetonitrile and water (containing 10 mM
NH4OAc) (60:40, v/v) as mobile phase at a flow rate of 1 mL/min.
Male ICR mice (4–5 weeks, 22–25 g) and Sprague–Dawley rats
(male, 9 weeks old, 220–250 g) were purchased from Peking Uni-
versity Health Science Center. All procedures of the animal exper-
iments were performed in compliance with relevant laws and
institutional guidelines. All of the animal experiments were
approved by the Institutional Animal Care and Use Committee of
Beijing Normal University.
4.2. 10-(4-(3-Fluoropropoxy)benzyl)-3H-spiro[2-benzofuran-
1,40-piperidine] (3)
Compound 9 (45.0 mg, 0.15 mmol) was dissolved in anhydrous
CH3CN (10 mL), followed by addition of 7 (35.0 mg, 0.15 mmol)

Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
5277
69.13, 63.12, 50.14, 36.58, 30.96. ESI-TOF MS calcd for C22H27FNO2
[M+H]+: 356.2020; found: 356.2074.
were passed through an organic Millipore filter (0.22
l
m) and ana-
lyzed by radio-HPLC (Water 600 system, Agela Venusil MP C18 col-
m, eluent 60% CH3CN and 40% H2O
umn, 250 mm ꢁ 4.6 mm, 5
l
4.6. Radiochemistry
containing 10 mM NH4OAc, flow rate 1 mL/min).
The synthesis of radiotracer [18F]2 was performed in a home-
made synthesis module. [18F]fluoride was produced through the
nuclear reaction of 18O(p, n)18F using proton beam bombardment
4.9. In vitro binding assays
All the procedures for the
r
receptor competition studies were
1 receptor assay was carried out
of the target (20 MeV, 65
lA) for 15 min in a Sumitomo HM-20S
previously described. Briefly, the r
cyclotron. Then [18F]fluoride was transported to the QMA column
of the multifunctional synthesis module (PET-MF-2V-IT-I) via
nitrogen carrier gas, and eluted into a reaction vessel using a solu-
tion of Kryptofix 2.2.2 (13 mg) and potassium carbonate (1.1 mg)
in CH3CN/H2O (0.8 mL/0.2 mL). The solvent was removed at
115 °C under a stream of nitrogen. The residue was azeotropically
dried three times with 1 mL of anhydrous acetonitrile each at
115 °C, followed by adding a solution of the tosylate precursor 12
(8 mg/mL) in anhydrous acetonitrile (1 mL). The mixture was stir-
red at 90 °C for 15 min to provide compound 13. After the solvent
was removed under a stream of nitrogen, a solution of compound
with (+)-[3H]-pentazocine as the radioligand using rat brain mem-
brane homogenates. The r2 receptor affinity was performed using
rat liver membrane homogenates with the radioligand [3H]DTG in
the presence of 10
receptor binding sites. Nonspecific binding was determined with
addition of 10 M haloperidol. Ki values were calculated according
lM dextrallorphan for selective masking of r1
l
to the Cheng-Prusoff equation and represent data from at least two
independent experiments, each performed in triplicate. The results
are given as mean standard deviation (SD).
4.10. Biodistribution and blocking studies in male ICR mice
10 (7 mg) and NaOH (20 lL, 2 mol/L) in DMSO (0.5 mL) was added
to the reaction vessel. The mixture was heated at 110 °C for 20 min,
followed by addition of the HPLC eluent (2 mL) to quench the reac-
tion, and loaded onto a sample loop in the isocratic semi-prepara-
tive radio-HPLC for purification (Agela Venusil MP C18 column,
A saline solution of [18F]2 (0.1 mL, about 370 kBq) was intrave-
nously injected into mice (five groups, n = 5 in each group) via the
tail vein. The mice were sacrificed by decapitation at 2, 15, 30, 60
and 120 min p.i. Samples of blood, whole brain, heart, liver, spleen,
lungs, kidneys, muscle and bone were removed, weighed and
250 mm ꢁ 10 mm, 5
lm, eluent 40% CH3CN and 60% H2O contain-
ing 10 mM NH4OAc, flow rate 4 mL/min). The product peak was
collected, diluted with water and trapped on a Waters C18 plus
Sep-Pak cartridge. The product was eluted off the cartridge with
ethanol. Radiochemical purity was analyzed by analytical radio-
counted in an automatic c-counter (Wallac 1470 Wizard, USA).
The percentage of injected dose per gram of wet tissue (% ID/g)
was calculated by a comparison of the tissue count to suitably
diluted aliquots of the injected radiotracer as counting standards.
All radioactivity measurements were corrected for decay. The
results are given as mean standard deviation (SD).
For the blocking studies, the mice were injected via the tail
vein with either saline (0.1 mL) or a blocking agent, including
haloperidol (0.1 mL, 1 mg/kg), compound 1 (0.1 mL, 1 mg/kg),
HPLC (Agela Venusil MP C18 column, 250 mm ꢁ 4.6 mm, 5
lm,
eluent 60% CH3CN and 40% H2O containing 10 mM NH4OAc, flow
rate 1 mL/min). Specific activity was determined with a HPLC-
based method. For animal experiments, [18F]2 was formulated as
a saline solution containing no more than 8% ethanol.
SA4503 (0.1 mL,
SA4503 (0.1 mL, 10
18F]2 (0.1 mL, about 370 kBq). The mice were sacrificed by decap-
itation at 30 or 60 min after radiotracer injection. The blood and
organs of interest were isolated and analyzed as described above.
Significant differences between control and test groups were
determined by Student’s t test (independent, two-tailed). The
criterion for significance was p 6 0.05. Data given in the figures
are mean values standard deviation (SD).
3
l
mol/kg), SA4503 (0.1 mL,
5 lmol/kg) or
4.7. Determination of logD value
lmol/kg) 5 min prior to the injection of
[
The apparent distribution coefficient of [18F]2 was determined
by measuring the distribution of the radiotracer between 1-octanol
and potassium phosphate buffer (PBS, 0.05 mol/L, pH 7.4). The
radiotracer [18F]2 (10
lL, 540 kBq) was mixed with 1-octanol
(3 mL) and potassium phosphate buffer (3 mL) in a centrifuge tube
(15 mL). The tube was vortexed for 3 min, followed by centrifuga-
tion at 3500 rpm for 5 min (AnkeTDL80-2B, China). About 0.05 mL
of 1-octanol layer was weighed in a tared tube. About 0.5 mL of the
PBS layer was weighed in a second tared tube. After addition of
0.5 mL of buffer to the 1-octanol fraction and 0.05 mL of 1-octanol
to the aqueous fraction, activity in both tubes was measured in an
4.11. Ex vivo autoradiography of [18F]2 in rat brain
Ex vivo autoradiography was carried out in male Sprague–
Dawley rats (9 weeks, 220–250 g). A solution of [18F]2 (0.3 mL,
74.0 MBq) was intravenously injected into conscious rats via the
tail vein. The rats were killed by cervical dislocation at 60 min after
administration of the radioligand. The brain was rapidly removed,
frozen at ꢀ80 °C in a liquid nitrogen bath. The coronal brain sec-
automatic
c-counter (Wallac 1470 Wizard, United States). The
logD value was calculated as the ratio of the cpm/mL of 1-octanol
to that of PBS and expressed as logD = log[cpm/mL(1 ꢀ octanol)/
cpm/mL(PBS)]. Samples from the remaining organic layer were
repartitioned until consistent distribution coefficient values were
obtained. The measurement was carried out in triplicate and
repeated three times.
tions (20 lm) were consecutively cut on a cryotome (CM1900,
Leica, Germany). The brain slices were dried at room temperature
and exposed to a phosphor plate film (PerkinElmer, USA) for 2 h.
Autoradiographic images were obtained using a phosphor imaging
system (Cyclone Plus, PerkinElmer, USA) and analyzed with the
AIDA 2.31 software (Raytest, Straubenhardt, Germany).
4.8. In vitro stability studies
The in vitro stability of [18F]2 was evaluated by monitoring the
radiochemical purity at different time points. To 0.3 mL of freshly
prepared mouse plasma, 0.2 mL of [18F]2 solution was added and
incubated at 37 °C in an Electric Thermostatic Incubator (DH-
250). At 2 and 4 h, the mixture was treated with 0.4 mL of ice-cold
CH3CN, and vortexed for 1 min. The plasma samples were centri-
fuged at 14000 rpm for 5 min. The supernatants of each sample
4.12. In vivo metabolism of [18F]2
The in vivo metabolism of [18F]2 was performed in male ICR
mice. The mice were intravenously injected with a saline solution
of [18F]2 (0.1 mL, 18.5 MBq) via the tail vein and sacrificed by
decapitation at 30 or 60 min postinjection, respectively. The

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Y.-Y. Chen et al. / Bioorg. Med. Chem. 22 (2014) 5270–5278
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Millipore filter. The filtrates were concentrated to 0.1 mL under a
stream of nitrogen gas flow and injected into the radio-HPLC for
analysis (Water 600 system, Agela Venusil MP C18 column,
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Acknowledgment
24. James, M. L.; Shen, B.; Zavaleta, C. L.; Nielsen, C. H.; Mesangeau, C.; Vuppala, P.
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This work was supported by the national Natural Science
Foundation of China (No. 21071023).
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C.; Zavaleta, C.; Vuppala, P. K.; Jamalapuram, S.; Avery, B. A.; Lyons, D. M.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.08.003.
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