Synthesis and evaluation of a 18F-labeled 4-phenylpiperidine-4-carbonitrile radioligand for σ1 receptor imaging

Article abstract of DOI:10.1002/jlcr.3408

We report the design and synthesis of several 4-phenylpiperidine-4-carbonitrile derivatives as σ₁ receptor ligands. In vitro radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ₁ r

Full text of DOI:10.1002/jlcr.3408

Research article
Received 16 January 2016,
Revised 10 March 2016,
Accepted 03 May 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3408
1
8
Synthesis and evaluation of a F-labeled 4-
phenylpiperidine-4-carbonitrile radioligand
for σ receptor imaging
1
a
a
b
c
Jiajun Ye, Xia Wang, Winnie Deuther-Conrad, Jinming Zhang,
a
c
a
b
Jianzhou Li, Xiaojun Zhang, Liang Wang, Jörg Steinbach,
b
a
Peter Brust and Hongmei Jia *
We report the design and synthesis of several 4-phenylpiperidine-4-carbonitrile derivatives as σ
1
receptor ligands. In vitro
receptors
radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ
1
1
8
(
K (σ ) = 1.22–2.14 nM) and extremely high subtype selectivity (K (σ ) = 830–1710 nM; K (σ )/K (σ ) = 680–887). [ F]9 was
i 1 i 2 i 2 i 1
prepared in 42–46% isolated radiochemical yield, with a radiochemical purity of >99% by HPLC analysis after purification,
via nucleophilic F substitution of the corresponding tosylate precursor. Biodistribution studies in mice demonstrated high
1
8 -
1
8
initial brain uptakes and high brain-to-blood ratios. Administration of SA4503 or haloperidol 5 min prior to injection of [ F]9
significantly reduced the accumulation of radiotracers in organs known to contain σ receptors. Two radioactive metabolites
were observed in the brain at 30 min after radiotracer injection. [ F]9 may serve as a lead compound to develop suitable
radiotracers for σ receptor imaging with positron emission tomography.
1
1
8
1
Keywords: fluorine-18; σ receptor; positron emission tomography; 4-phenylpiperidine-4-carbonitrile derivatives; molecular probe
1
Introduction
and 1600-fold subtype selectivity as well as no affinity for opioid
receptors. Thus, compound 1 appears to be an ideal lead
compound to design radionuclide-labeled probes to investigate
24
Sigma-1 (σ
operated molecular chaperone recently. This protein consists of
23 amino acids with a 25 kDa molecular mass and possesses
1
) receptor has been identified as a unique ligand-
1
σ
1
receptor function. In this paper, we replaced the hydrogen atom
at the para-position of the N-benzyl moiety of compound 1 with F, I,
or OCH CH F group for assessment of maintained affinity and
2
2
,3
two trans-membrane domains. It serves as a modulator of
inter-organelle communications and regulates a variety of cellular
2
2
18
selectivity (Figure 2). Moreover, we synthesized a F-labeled
radiotracer and evaluated its potential as a PET probe for imaging
4–6
function. Increasing evidence has shown that σ
1
receptors play
an important role in neuronal plasticity and are associated with
various neuropsychiatric diseases. Moreover, they are involved
σ
1
receptor through biodistribution and blocking studies in mice
4
–8
and radiometabolite studies.
9
10–12
in cardiovascular disease and human tumors.
of ligands and molecular imaging agents with high affinity and
selectivity for σ
Development
receptors will help improve our ability to track, Results and discussion
1
monitor, and understand σ receptor-related diseases.
1
Chemistry
In the past decades, many radionuclide-labeled ligands such as
C, F, or Br-labeled σ receptor positron emission tomography
11
18
76
1
The synthetic routes of 4-phenylpiperidine-4-carbonitrile derivatives
are illustrated in Scheme 1. 4-Hydroxybenzaldehyde reacted with
9
9m
123
(PET) imaging agents and
Tc,
I-labeled single-photon
emission computed1 tomography (SPECT) imaging agents
4
1-bromo-2-fluoroethane, followed by reduction with NaBH and
3
have been reported. Among them, only a few ligands such as
11
14–16 18
17
123
18
[
C]SA4503,
been evaluated in humans. Moreover, [ F]FPS and [ I]TPCNE
have shown irreversible kinetics in human studies. For the first
[ F]FPS, and [ I]TPCNE (Figure 1) have
1
8
19
123
18
a
Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of
Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
1
1
useful radiotracer [ C]SA4503, its use in clinic needs an on-site
18
20,21
18
22,23
b
cyclotron. More recently, [ F]fluspidine
Figure 1) have proved to be promising PET radiotracers for
visualizing σ1 receptors. However, further investigation is still
and [ F]FTC-146
Helmholtz-Zentrum Dresden-Rossendorf, Department of Neuroradiopharmaceuticals,
Institute of Radiopharmaceutical Cancer Research, 04318 Leipzig, Germany
(
c
Nuclear Medicine Department, Chinese PLA General Hospital, Beijing 100853,
required for their clinical translation. Therefore, there is a need China
18
for the development of F-labeled radiotracers with appropriate
affinity and high specificity for σ receptors.
It was reported that 1-benzyl-4-phenylpiperidine-4-carbonitrile
compound 1) showed subnanomolar affinity for σ
*
(
Correspondence to: Hongmei Jia, Key Laboratory of Radiopharmaceuticals
Beijing Normal University), Ministry of Education, College of Chemistry, Beijing
Normal University, Beijing 100875, China.
1
receptors E-mail: hmjia@bnu.edu.cn
1
(
J. Label Compd. Radiopharm 2016
Copyright © 2016 John Wiley & Sons, Ltd.

J. Ye et al.
1
Figure 1. Potent σ receptor radiotracers developed recently.
In vitro radioligand competition studies
The competition binding assays for σ
1
and σ
2
receptors of
4
-phenylpiperidine-4-carbonitrile derivatives were performed as
27
previously reported. The binding assays used rat brain with
3
(
+)-[ H]pentazocine as radioligand for the σ
1
receptors and rat
liver membranes with [ H]DTG (in the presence of 10 μM
dextrallorphan) as radioligand for the σ receptors. The results
are shown in Table 1. Replacement of hydrogen atom in the
phenyl group with I, F, or OCH CH F at the para-position of
compound 1 slightly decreased the affinity for σ receptors
compounds 7, 8, and 9 vs. compound 1). However, they still
3
Figure 2. Design concept of 4-phenylpiperidine-4-carbonitrile derivatives.
2
2
2
1
(
1
possessed low nanomolar affinity for σ receptors (1.22–2.14 nM)
and high subtype selectivity (680–887 fold).
For compounds 7–9, calculated log P (clog P) values obtained
by the CHEMDRAW software are 4.52, 3.54, and 3.57, respectively. It
is well-known that lipophilicity is a critical physicochemical
parameter for a radiotracer, as it impacts upon its ability to cross
the blood–brain barrier (BBB), the free fraction in the plasma and
28,29
in the brain, as well as non-specific binding.
Considering the
relatively lower lipophilicity of compound 9 with fluoroethoxy
moiety compared with compound 7 with I atom, and the simpler
radiolabeling method compared with that required for
Scheme 1. Synthetic routes of 4-phenylpiperidine-4-carbonitrile derivatives.
Regents and conditions: (a) 1-bromo-2-fluoroethane, K CO , dimethylformamide,
00 °C, 99%; (b) ethanol, NaBH , 85%; (c) CH Cl , PBr , 0 °C, 93%; (d) for 7, 4-
iodobenzyl bromide, acetonitrile, K CO , KI, 90 °C, 95%; for 8, 4-fluorobenzyl
bromide, acetonitrile, K CO , KI, 90 °C, 75%; for 9, 5, CH Cl , NaH, r.t., 62%.
18
2
3
compound 8, the corresponding radioligand [ F]9 was prepared
1
4
2
2
3
2
3
2
3
2
2
Table 1. Binding affinities of the 4-phenylpiperidine-4-
a
carbonitrile derivatives for σ
1
2
receptors and σ receptors
Compound
K (σ ) (nM)
K (σ ) (nM)
K (σ )/K (σ )
i 2 i 1
bromination to obtain intermediate 5. Compared with the reported
i
1
i
2
25,26
method in the literature,
the synthetic method of compound
b
1
0.41 ± 0.08
2.14 ± 0.38
1.22 ± 0.44
1.47 ± 0.89
4.95 ± 1.74
657 ± 19
1710 ± 650
830 ± 370
1304 ± 475
20.7 ± 0.07
1602
799
680
887
4
5
in this paper can avoid use of toxic carbon tetrachloride.
7
8
9
Moreover, this method has the advantages of high yields and easy
separation between the reactants and the products. N-Alkylation
of compound 6 with 4-iodobenzyl bromide, 4-fluorobenzyl
bromide, or intermediate 5 under basic conditions provided
target compounds 7, 8, and 9, respectively.
c
Haloperidol
a
Values are means ± standard deviation (SD) of at least two
experiments performed in triplicate.
The target compounds (7, 8, and 9) were analyzed by high-
performance liquid chromatography (HPLC) with purity of more
b
c
24
From reference.
From reference.
1
13
than 95%. They were characterized by H NMR, C NMR, and
high-resolution mass spectrometry.
26
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J. Label Compd. Radiopharm 2016

J. Ye et al.
1
and evaluated for its potential as a σ receptor radiotracer for
PET imaging.
Radiolabeling
18
The synthesis of the precursor and [ F]9 is depicted in Scheme 2.
Reductive amination of compound 6 with 4-hydroxybenzaldehyde
3
in the presence of NaBH(OAc) led to intermediate 10, which
reacted with ethylene glycol bistosylate to give the tosylate
18
precursor 11 in 71% yield. The radioligand [ F]9 is obtained
through a direct SN2 displacement of the tosylate group in
1
8
precursor 11 with [ F]fluoride (in the formation of kryptofix
+
18
À
2
.2.2/K /[ F]F complex). After removing inorganic salts via a
Waters C18 plus Sep-Pak cartridge, the crude product was purified
by an isocratic semi-preparative radio-HPLC using a reverse-phase
column and a mobile phase consisting of acetonitrile and water
Figure 3. High-performance liquid chromatography (HPLC) co-elution profile of 9
(
containing 10 mM NH
4
OAc) (65:35, v/v) at a flow rate of
18
18
and [ F]9, 9 t
R
R
= 12.77 min, [ F]9 t = 12.83 min (65% acetonitrile and 35% water
18
4
mL/min. The radioactive peak corresponding to [ F]9 was
containing 10 mM NH
4
OAc, 1 mL/min).
collected, diluted with water, and passed across a C18 Sep-Pak
cartridge. The product was eluted off with ethanol and diluted with
18
sterile saline to provide a solution with approximately 300 kBq of Table 2. [ F]9 exhibited high initial brain uptake with 11.10
radioactivity per 0.1 mL.
± 1.28% ID/g at 2 min after radiotracer injection. The
18
In order to identify the radiotracer, [ F]9 and the corresponding accumulation in the brain was highest at 2 min after radiotracer
unlabeled compound 9 were co-injected and co-eluted. Their HPLC injection and slowly decreased thereafter with 9.32 ± 0.92%
profiles using acetonitrile and water (containing 10 mM NH OAc) ID/g at 15 min, 7.49 ± 1.01% ID/g at 30 min, 4.84 ± 1.17% ID/g at
4
(
65/35, v/v) as mobile phase at a flow rate of 1 mL/min are presented 60 min, and 3.30 ± 0.22% ID/g at 120 min. The radiotracer levels
18
in Figure 3. The retention times of unlabeled compound 9 and [ F]9 in the blood were relatively low with 1.03 ± 0.08% ID/g at
were 12.77 and 12.83 min, respectively. The difference in retention 2 min, 1.01 ± 0.15% ID/g at 15 min, and 1.47 ± 0.23% ID/g at
time was corresponding to the time lag as a result of the volume 30 min, resulting in high brain-to-blood ratios of 10.8, 9.2, and
and flow rate within the distance between the UV and radioactivity 5.1 at 2, 15, and 30 min, respectively. The accumulation in the
detectors of our HPLC system. After purification by semi-preparative blood was increased with time, especially at 30 min after
18
18
radio-HPLC, the radiochemical purity (RCP) of [ F]9 was >99%. The radiotracer injection. [ F]9 also showed relatively fast washout
overall isolated radiochemical yield was 42–46% (n= 3, corrected for in the organs known to contain σ receptors, including the lungs,
1
decay). The total synthesis time was approximately 1 h. The specific kidneys, heart, spleen, and liver. Finally, the accumulation of
18
activity of [ F]9 was 23.56 GBq/μmol.
radiotracer in the bone is almost the same within 30 min and
The in vitro stability of [ F]9 in saline was evaluated by increased slightly thereafter with 6.37 ± 0.90% ID/g at 120 min,
18
18
measuring the RCP at different time points. After 1, 2, and 3 h, indicating that [ F]9 may undergo defluorination in vivo.
1
8
18
the RCPs of [ F]9 were still >99%, indicating high stability of
[
In order to verify the specific binding of [ F]9 to σ receptors
1
18
F]9 in vitro (Figure 4).
in vivo, the effects of preadministration of haloperidol (0.1 mL,
A shake-flask method was employed for the determination of 1 mg/kg) and SA4503 (3 μmol/kg) on the biodistribution of
lipophilicity of the radiotracer in terms of the apparent radiotracer in various organs of male ICR mice were investigated.
distribution coefficient in a 1-octanol and 0.05 mol/L potassium The blocking agent was injected 5 min prior to the radiotracer
2
6,30
phosphate buffer system at pH 7.4 as previously reported.
injection (0.1 mL, about 300 kBq). Blocking results of organ
1
8
18
The log D value of [ F]9 was 3.29 ± 0.15 (n = 3), which is within distribution of [ F]9 at 15 and 30 min after radiotracer injection
the range expected to have good BBB permeability.
in ICR mice are summarized in Figure 5. Pretreatment of animals
with haloperidol and SA4503 resulted in significant reduction of
radiotracer uptake in organs known to contain σ receptors at
1
Biodistribution and blocking studies in male ICR mice
15 min, including the brain (65–66%, p < 0.001), heart (35–37%,
18
In order to evaluate the kinetics of [ F]9, biodistribution studies p < 0.001), liver (36–51%, p < 0.001), spleen (56–58%, p < 0.001),
were performed in male ICR mice. The results are summarized in kidney (45–49%, p < 0.001), and lungs (44–49%, p < 0.01). The
1
8
Scheme 2. Synthesis of the precursor and [ F]9. Reagents and conditions: (a) 4-hydroxybenzaldehyde, 1,2-dichloroethane, NaBH(OAc)
3
, 72%; (b) ethylene glycol
1
8
À
2 3 2 3
bistosylate, acetonitrile, K CO , 90 °C, 71% ; (c) [ F]F , acetonitrile, Kryptofix 2.2.2, K CO , 90 °C, 7 min.
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J. Ye et al.
1
8
Figure 4. Analytical radio-HPLC chromatograms of the in vitro stability of [ F]9 in saline at 1, 2, and 3 h (65% acetonitrile and 35% water containing 10 mM NH
mL/min).
4
OAc,
1
18
a
Table 2. Biodistribution of [ F]9 in male ICR mice
Organ
2 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Small intestine
Stomach
Muscle
1.03 ± 0.08
11.10 ± 1.28
19.11 ± 2.52
5.17 ± 1.46
3.46 ± 1.66
41.14 ± 12.32
18.15 ± 3.25
5.62 ± 1.37
1.54 ± 0.14
4.36 ± 0.81
3.04 ± 0.54
10.8
1.01 ± 0.15
9.32 ± 0.92
5.76 ± 0.31
8.14 ± 0.76
7.47 ± 1.14
8.56 ± 1.09
9.57 ± 0.93
7.17 ± 1.48
1.86 ± 0.53
2.98 ± 0.15
2.89 ± 0.52
9.2
1.47 ± 0.23
7.49 ± 1.01
3.90 ± 0.41
7.74 ± 1.02
7.29 ± 0.95
6.18 ± 1.24
6.84 ± 0.36
5.11 ± 0.86
1.55 ± 0.21
2.54 ± 0.14
2.79 ± 0.74
5.1
2.35 ± 0.20
4.84 ± 1.17
3.28 ± 0.45
6.17 ± 1.02
4.88 ± 0.69
4.42 ± 0.70
4.59 ± 0.62
7.08 ± 1.31
1.96 ± 0.69
2.53 ± 0.30
3.95 ± 0.46
2.1
2.73 ± 0.27
3.30 ± 0.22
3.11 ± 0.34
4.00 ± 0.33
3.74 ± 0.70
3.46 ± 0.27
3.23 ± 0.41
8.56 ± 1.62
1.26 ± 0.13
2.64 ± 0.32
6.37 ± 0.90
1.2
b
b
Bone (femur)
Brain/Blood
Brain/Bone
3.7
3.2
2.7
1.2
0.5
a
Data are expressed as percentage of injected dose per gram, means ± SD, n = 5.
Percentage of injected dose per organ.
b
1
8
Figure 5. Effects of pretreatment with haloperidol (0.1 mL, 1 mg/kg, 2.7 μmol/kg) or SA4503 (0.1 mL, 3 μmol/kg) 5 min prior to the injection of radiotracer [ F]9 (0.1 mL,
about 300 kBq) at 15 min (A) and 30 min (B) after intravenous administration. Multiple tests including Student’s t-test (independent, two-tailed) and one-way ANOVA with a
post-hoc test were performed, and p < 0.05 (except for SA4503 in the heart at 30 min). Values are means ± SD, n = 5.
1
8
blocking rate at 30 min was slightly less than that at 15 min, In vivo metabolic stability of [ F]9
including the brain (59–61%, p < 0.001), heart (14–16%, p < 0.05
It is well known that the metabolism profile of the radiotracer
except for SA4503), liver (40–52%, p < 0.001), spleen (60–61%,
in vivo is a very important issue for brain imaging. The metabolic
p < 0.001), kidney (35%, p < 0.001), and1 lungs (37–41%,
8
18
p < 0.01). These data demonstrated that [ F]9 binds to σ
profile of [ F]9 was investigated in plasma and brain samples
1
receptors specifically in vivo.
obtained from mice at 30 min after radiotracer injection as
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J. Ye et al.
1
8
Figure 6. Analytical radio-HPLC chromatograms of the plasma and brain samples at 30 min after administration of [ F]9 in mice.
30
previously reported. Protein precipitation was performed by (t
= 3.0 min) and another compound (t =3.6min), were observed
R
R
in the brain and blood, indicating that radioactive metabolites may
cross the BBB. To investigate the effects of radioactive metabolites
on the accuracy of the brain PET signal, identification of the
using ice-cold acetonitrile. Acetonitrile extracts were analyzed and
quantified by radio-HPLC. Representative HPLC chromatograms
are given in Figure 6.
radioactive metabolites and measurement of their affinities for σ
receptors need to be performed. We will use such information to
1
At 30 min after radiotracer injection, 47% of the total
18
radioactivity was represented by the parent radiotracer [ F]9
with the retention time of 12.8 min in plasma samples. Two
hydrophilic radioactive metabolites M1 (9%) and M2 (44%) were
observed with retention times of 3.0 and 3.6 min, respectively. In
the brain samples, 74% of the total radioactivity was represented
18
refine the structure of [ F]9 for improved metabolic stability in the
future.
1
8
Conclusion
by [ F]9. Similar to what was found in plasma, two hydrophilic
radioactive metabolites M1 (5%) and M2 (21%) were observed
with retention times of 3.0 and 3.6 min, respectively.
We have synthesized three 4-phenylpiperidine-4-carbonitrile
1
derivatives with high affinity for σ receptors and very high
18
Currently, more and more studies have proved that the σ1
subtype selectivity. [ F]9 has been obtained in good
radiochemical yield and high radiochemical purity. The log D
receptors are linked to various human brain diseases. Neuroimaging
18
of σ receptors with optimal radiotracers provides an important tool
1
value of [ F]9 was within the range expected to show excellent
18
to investigate the σ receptors in the pathophysiology of neuro-
1
brain uptake. In biodistribution studies in mice, [ F]9 displayed
psychiatric diseases. The decreased density of σ receptors was
1
high initial brain uptake and high brain-to-blood ratios. Blocking
1
1
18
observed using [ C]SA4503 imaging studies in patients with
Parkinson’s disease and Alzheimer’s disease. However, [ C]
studies confirmed high specific binding of [ F]9 to σ
1
receptors
1
5
14
11
in vivo. Identification of two radioactive metabolites in the brain
18
SA4503 needs an on-site cyclotron which limited its applications in
is useful to refine the structure of [ F]9 for improved metabolic
18
clinic. Among the potential σ
1
receptor radiotracers up to date,
stability. [ F]9 may serve as a lead compound to develop
18
123
[
F]FPS and [ I]TPCNE were not optimal as a result of their
suitable PET radiotracers for σ receptor imaging. Radiolabeled
1
1
8
18
irreversible kinetics in humans. [ F]fluspidine and [ F]FTC-146 are
still waiting for human studies. In this study, compounds 7, 8, and
7
and 8 would be of future interests.
9
1
exhibited low nanomolar affinity for σ receptors and extremely
Experimental section
high subtype selectivity. Considering higher detection sensitivity
and resolution of imaging with PET than that with SPECT and simple
synthesis of
substitution of the corresponding tosylate precursor, [ F]9 was All reagents and chemicals were obtained from commercial suppliers
1
synthesized and evaluated for its potential as σ receptor radiotracer.
High in vitro stability of [ F]9 in saline was observed. The lipophilicity
of [ F]9 (log DpH 7.4 = 3.29) is appropriate for BBB penetration.
Compared with [ C]SA4503 and [ F]fluspidine, [ F]9 showed
higher initial brain uptake, higher brain-to-blood ratios within
General method
18
18 À
F-labeled radiotracers via nucleophilic
F
18
and used without further purification unless otherwise stated. Thin-layer
18
chromatography (silica gel 60 F254 plates Merck) was used to monitor the
reactions. Flash column chromatography was carried out on silica gel
(200–400 mesh) using the mobile phase indicated in the experimental
procedure. Melting points (Mps) were measured using a WRX-4 micro
melting point apparatus (Shanghai Yice Apparatus & Equipment Co.,
18
11
31
18
20 18
15 min, and lower brain-to-blood ratios thereafter. Consistent with
1
13
LTD, China) and were uncorrected. H and C NMR spectra were
18
1
defluorination in vivo, [ F]9 exhibited higher brain-to-bone ratios
recorded on a Bruker Avance III NMR spectrometer at 400 MHz ( H) and
1
3
within 30 min and lower brain-to-bone ratios thereafter. 100 MHz ( C). Chemical shifts (δ) are reported in ppm downfield from
Pretreatment of animals with haloperidol and SA4503 resulted in tetramethylsilane and coupling constants (J) in Hertz (Hz). Mass spectra
significant reduction of radiotracer uptake in organs known to were acquired by Quattro micro API ESI/MS (Waters, USA). High-
contain σ receptors at 15 and 30 min, indicating specific binding
of [ F]9 in vivo. Moreover, this radiotracer exhibited comparable
resolution mass spectrometry was performed on a LCT Premier XE
ESI-TOF mass spectrometry instrument (Waters, USA).
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 × 10 mm, 5 μm) using 65% acetonitrile
1
18
1
1
31
specific binding to σ receptors in the brain to [ C]SA4503 and
1
1
8
20
18
20
[
F]fluspidine.
Compared with 1[ F]fluspidine (98%),
the
8
percentage of parent radiotracer [ F]9 in the brain (74%) at
and 35% water (containing 10 mM NH
rate of 4 mL/min. HPLC analyses were performed on a Shimadzu SCL-
0 AVP HPLC system (Shimadzu Corporation, Japan) equipped with a
two radioactive SPD-M20A UV–VIS detector operating at a wavelength of 254 nm and a
metabolites M1 and M2, which could be attributed to radiofluoride Bioscan Flow Count 3200 NaI/PMTc-radiation scintillation detector. The
4
OAc) as mobile phase at a flow
30 min after radiotracer injection was lower, suggesting that the
18
18
stability of [ F]9 in the brain is apparently poorer than [ F]
2
18
20
fluspidine. Different with [ F]fluspidine,
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J. Ye et al.
samples were analyzed on an Agela Venusil MP C18 column
The mixture was stirred at 90 °C for 60 min. The mixture was
(
1
250 × 4.6 mm, 5 μm) using 65% acetonitrile and 35% water (containing concentrated under vacuum, and the residue was dissolved in CH
2
Cl
, and concentrated under
vacuum. The residue was purified by column chromatography (silica gel,
river experimental animal technical Co., LTD. All procedures of the animal petroleum ether/ethyl acetate = 4: 1, v/v) to provide 8 as a white solid
2
,
0 mM NH
4
OAc) as mobile phase at a flow rate of 1 mL/min.
washed with saturated NaCl, dried with MgSO
4
Male ICR mice (22–24 g, 4–5 weeks) were purchased from Beijing Vital
1
experiments were performed in compliance with relevant laws and (0.353 g, 75%). Mp: 82.0–82.5 °C. H NMR (400 MHz, CDCl
3
) δ (ppm):
institutional guidelines. All of the animal experiments were approved 7.54–7.49 (m, 2H), 7.45–7.38 (m, 2H), 7.38–7.30 (m, 3H), 7.08–6.98 (m,
by the Institutional Animal Care and Use Committee of Beijing Normal
University.
2H), 3.58 (s, 2H), 2.99 (d, J = 12.2 Hz, 2H), 2.60–2.48 (m, 2H), 2.15–2.06
13
(m, 4H). C NMR (101 MHz, CDCl
3
) δ (ppm): 162.24 (d, JC-F = 243.5 Hz),
40.44, 134.10 (d, JC-F = 3.1 Hz), 130.71 (d, JC-F = 7.9 Hz), 129.20, 128.28,
25.81, 122.25, 115.32 (d, JC-F = 21.1 Hz), 62.22, 50.81, 42.92, 36.75. HRMS
1
1
4-(2-Fluoroethoxy)benzaldehyde (3)
+
(
2
ESI-Tof), [M + H] : m/z calcd for C19H20FN 295.1605, found 295.1615.
A mixture of 4-hydroxybenzaldehyde (2, 1.01 g, 8.28 mmol), 1-bromo-2-
fluoroethane (2.05 g, 16.1 mmol), and K CO (2.49 g, 18.0 mmol) in
2
3
dimethylformamide (20 mL) was stirred at 100 °C for 5 h. The mixture
was concentrated under vacuum, and the residue was dissolved in
1
-(4-(2-Fluoroethoxy)benzyl)-4-phenylpiperidine-4-carbonitrile (9)
CH
2
Cl
2
, washed with water, dried with MgSO
4
, and concentrated under
Compounds 5 (0.103 g, 0.442 mmol) and 6 (0.0815 g, 0.438 mmol) were
vacuum to provide 3 as a yellow solid (1.37 g, 99%). Mp: 49.5–51.1 °C. dissolved in anhydrous CH
H NMR (400 MHz, CDCl ) δ (ppm): 9.90 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H),
3
2
Cl
(0.0180 g, 0.750 mmol). The mixture was stirred at room temperature for
.04 (d, J = 8.6 Hz, 2H), 4.80 (dt, J = 47.3, 4.0 Hz, 2H), 4.31 (dt, J = 27.5, 24 h. Then the reaction was quenched with water, extracted with ethyl
2
(10 mL), followed by addition of NaH
1
7
4
+
.1 Hz, 2H). ESI-MS, [M + H] : m/z = 169.1.
4
acetate. The combined organic layer was dried with MgSO , concentrated
under vacuum, and purified by column chromatography (silica gel,
petroleum ether/ethyl acetate/triethylamine = 20: 1 : 1, v/v/v) to provide
(
4-(2-Fluoroethoxy)phenyl)methanol (4)
1
9
as a colorless solid (0.0929 g, 62%). Mp: 58.6–60.0 °C. H NMR
(
(
(
(
1
6
400 MHz, CDCl
3
) δ (ppm): 7.53–7.25 (m, 7H), 6.90 (d, J = 8.6 Hz, 2H), 4.76
To a solution of compound 3 (1.45 g, 8.63 mmol) in ethanol (20 mL),
NaBH (0.813 g, 21.4 mmol) was added. The mixture was stirred at room
temperature for 15 min. Then the reaction was quenched with water,
neutralized with dilute hydrochloric acid, and extracted with CH Cl
The combined organic layer was dried with MgSO and concentrated
under vacuum. The residue was purified by column chromatography
silica gel, petroleum ether/ethyl acetate = 10: 1, v/v) to provide 4 as a
ddd, J = 47.4, 5.4, 4.2 Hz, 2H) , 4.22 (ddd, J = 27.8, 4.2, 3.0 Hz, 2H), 3.55
4
13
s, 2H), 2.99 (d, J = 12.2 Hz, 2H), 2.55–2.43 (m, 2H), 2.10 (s, 4H). C NMR
100 MHz, CDCl ) δ (ppm): 157.74, 140.30, 130.76, 130.33, 128.98, 128.04,
25.63, 122.11, 114.52, 81.92 (d, JC-F = 169.7 Hz), 67.20 (d, JC-F = 10.3 Hz),
2
2
.
3
4
+
2.24, 50.61, 42.82, 36.58. ESI-MS, [M + H] : m/z = 339.3. HRMS (ESI-Tof),
+
[
2
M + H] : m/z calcd for C21H24FN O 339.1873, found 339.1874.
(
1
white solid (1.24 g, 85%). Mp: 45.3–48.8 °C. H NMR (400 MHz, CDCl
ppm): 7.31 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 4.3 Hz, 2H), 4.76 (dt, J = 47.3,
.1 Hz, 2H), 4.63 (s, 2H), 4.22 (dt, J = 27.7, 4.1 Hz, 2H).
3
) δ
(
4
1-(4-Hydroxybenzyl)-4-phenylpiperidine-4-carbonitrile (10)
To a solution of compound 6 (0.192 g, 1.03 mmol) in 1,2-dichloroethane
1
-(2-Fluoroethoxy)-4-(bromomethyl)benzene (5)
(
(
5 mL), 4-hydroxybenzaldehyde (0.257 g, 2.10 mmol), and NaBH(OAc)
0.609 g, 2.87 mmol) were added. The mixture was stirred at room
3
To a solution of compound 4 (0.206 g, 1.21 mmol) in anhydrous CH
10 mL) at 0 °C, PBr (0.475 g, 1.75 mmol) was added. The mixture was
stirred at 0 °C for 3 h and then quenched with saturated NaHCO and
extracted with CH Cl . The combined organic layer was dried with
MgSO and concentrated under vacuum to provide 5 as a colorless oil
2 2
Cl
temperature for 24 h. Then the reaction was quenched with saturated
NaHCO , extracted with CH Cl . The combined organic layer was dried
with MgSO and concentrated under vacuum. The residue was purified
(
3
3
2
2
3
4
2
2
by column chromatography (silica gel, petroleum ether/ethyl
4
1
acetate/triethylamine = 4: 1, v/v) to provide 10 as a white solid (0.218 g,
3
(0.262 g, 93%). H NMR (400 MHz, CDCl ) δ (ppm): 7.33 (d, J = 8.6 Hz,
1
7
2%). Mp: 104.7–107.2 °C. H NMR (400 MHz, CDCl
3
) δ (ppm): 7.49 (d,
2
4
H), 6.89 (d, J = 8.6 Hz, 2H), 4.76 (dt, J = 47.4, 4.1 Hz, 2H), 4.50 (s, 2H),
.21 (dt, J = 27.8, 4.1 Hz, 2H).
J = 7.7 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.19 (d,
J = 8.2 Hz, 2H), 6.73 (d, J = 8.3 Hz, 2H), 3.57 (s, 2H), 3.06 (d, J = 11.7 Hz,
+
2
H), 2.54 (t, J = 11.7 Hz, 2H), 2.25–2.00 (m, 4H). ESI-MS, [M + H] :
1-(4-Iodobenzyl)-4-phenylpiperidine-4-carbonitrile (7)
m/z = 293.3.
A mixture of 4-phenylpiperidine-4-carbonitrile (6, 0.391 g, 2.10 mmol), 4-
iodobenzyl bromide (0.624 g, 2.10 mmol), K CO (2.89 g, 20.9 mmol), and
KI (0.265 g, 1.60 mmol) in acetonitrile (100 mL) was stirred at 90 °C for
0 min. The mixture was concentrated under vacuum. The residue was ethyl-4-methylbenzenesulfonate (11)
dissolved in CH Cl , washed with saturated NaCl, dried with MgSO
concentrated under vacuum and purified by column chromatography
silica gel, petroleum ether/ethyl acetate = 4: 1, v/v) to provide 7 as a
2
3
2-(4-((4-Cyano-4-phenylpiperidin-1-yl)methyl)phenoxy)
6
2
2
4
,
A mixture of 10 (0.0651 g, 0.223 mmol), ethylene glycol bistosylate
0.187 g, 0.505 mmol), and K CO (0.0721 g, 0.522 mmol) in anhydrous
acetonitrile (8 mL) was stirred at 90 °C for 24 h. The mixture was
(
2
3
(
1
white solid (0.804 g, 95%). Mp: 105.3–105.5 °C. H NMR (400 MHz, CDCl
δ (ppm): 7.67 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.5 Hz,
H), 7.34 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.9 Hz, 2H), 3.55 (s, 2H), 2.99
3
)
concentrated under vacuum, dissolved in CH Cl , washed with saturated
2
2
2
NaCl, dried with MgSO , and concentrated under vacuum. The residue
4
13
(d, J = 11.9 Hz, 2H), 2.58–2.46 (m, 2H), 2.17–2.08 (m, 4H). C NMR (101
was purified by column chromatography (silica gel, petroleum
MHz, CDCl ) δ (ppm): 140.24, 137.67, 131.28, 129.26, 128.37, 125.82,
1
for C19H20IN 403.0666, found 403.0672.
2
3
ether/ethyl acetate/triamine = 4: 1, v/v) to provide 11 as a white solid
+
22.16, 93.04, 62.31, 50.86, 42.80, 36.62. HRMS (ESI-Tof), [M + H] : m/z calcd
1
(
0.0775 g, 71%). Mp: 91.0–92.4 °C. H NMR (400 MHz, CDCl
3
) δ (ppm):
7
.83 (d, J = 8.3 Hz, 2H), 7.44–7.30 (m, 7H), 7.23 (d, J = 7.9 Hz, 2H), 6.76 (d,
J = 8.5 Hz, 2H), 4.36 (dd, J = 4.9, 3.4 Hz, 2H), 4.15 (dd, J = 4.9, 3.4 Hz, 2H),
1
-(4-Fluorobenzyl)-4-phenylpiperidine-4-carbonitrile (8)
13
3
.53 (s, 2H), 2.98 (d, J = 11.3 Hz, 2H), 2.54–2.38 (m, 5H), 2.09 (s, 4H).
NMR (100 MHz, CDCl ) δ (ppm): 157.31, 144.92, 140.23, 132.95, 130.91,
.10 mmol) were dissolved in anhydrous acetonitrile (100 mL), followed 130.31, 129.85, 128.99, 128.03, 125.63, 122.12, 114.42, 68.12, 65.51,
C
Compound 6 (0.298 g, 1.60 mmol) and 4-fluorobenzyl bromide (0.397 g,
2
by addition of K
3
+
2 3
CO (2.17 g, 15.7 mmol) and KI (0.199 g, 1.20 mmol). 62.22, 50.60, 42.81, 36.56, 21.66. ESI-MS, [M + H] : m/z = 491.9.
www.jlcr.org
Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016

J. Ye et al.
Radiochemistry
Biodistribution and blocking studies in male ICR mice
1
8
18
18
18
A saline solution of [ F]9 (0.1 mL, about 300 kBq) was intravenously
[
F]fluoride was produced through the nuclear reaction of O(p, n)
F
using proton beam bombardment of the target (20 MeV, 65 μA) for
injected into mice (five groups, n = 5 in each group) via the tail vein.
18
15 min in a Sumitomo HM-20S cyclotron. Then [ F]fluoride was The mice were sacrificed by decapitation at 2, 15, 30, 60, and 120 min
transported to the QMA column via nitrogen carrier gas and eluted into after radiotracer injection. Samples of blood, whole brain, heart, liver,
a reaction vessel using a solution of Kryptofix 2.2.2 (13 mg) and spleen, lungs, kidneys, muscle, and bone (femur) were removed,
potassium carbonate (1.1 mg) in CH
was removed at 120 °C under a stream of nitrogen. The residue was dried
three times with 1 mL of anhydrous acetonitrile each at 120 °C, followed was calculated by a comparison of the tissue count to suitably diluted
3
CN/H
2
O (0.8 mL/0.2 mL). The solvent weighed, and counted in an automatic counter (Wallac 1470 Wizard,
USA). The percentage of injected dose per gram of wet tissue (% ID/g)
by addition of a solution of the tosylate precursor 11 (6 mg/mL) in
aliquots of the injected radiotracer as counting standards. All
radioactivity measurements were corrected for decay. The results are
given as means ± SD.
anhydrous acetonitrile (0.6 mL). The mixture was stirred at 90 °C for
1
8
7
min to provide [ F]9. After being diluted with water and trapped on
a Waters C18 plus Sep-Pak cartridge, the product was eluted off the
cartridge with anhydrous acetonitrile and loaded onto a sample loop in
For the blocking studies, the mice were intravenously injected via the
tail vein with haloperidol (0.1 mL, 1 mg/kg, 2.7 μmol/kg) or SA4503
1
8
the isocratic semi-preparative radio-HPLC for purification (Agela Venusil (0.1 mL, 3 μmol/kg) 5 min prior to the injection of [ F]9 (0.1 mL, about
MP C18 column, 250 mm × 10 mm, 5 μm, eluent 65% CH CN and 35% 300 kBq). The mice were decapitated at 15 or 30 min after radiotracer
O containing 10 mM NH OAc, flow rate 4 mL/min). The product peak
was collected, diluted with water, and trapped on a Waters C18 plus
3
H
2
4
injection. The blood and organs of interest were isolated and analyzed
as described in the preceding texts. Significant differences between
Sep-Pak cartridge. The cartridge was washed with water. The product control and test groups were determined by multiple tests including
was eluted off the cartridge with ethanol. Radiochemical purity was Student’s t-test (independent, two-tailed) and one-way ANOVA with a
analyzed by analytical radio-HPLC (Agela Venusil MP C18 column, post-hoc test. The criterion for significance was p ≤ 0.05. Data given in
2
1
50 × 4.6 mm, 5 μm, eluent 65% CH
3
CN, and 35% H O containing the figures are means ± SD.
2
18
0 mM NH OAc, flow rate 1 mL/min). For animal experiments, [ F]9
4
was formulated as a saline solution containing no more than 7% ethanol.
18
In vivo metabolic stability of [ F]9
1
8
The in vivo metabolic fate of [ F]9 was performed in male ICR mice. The
Determination of log D value
1
8
mice were intravenously injected with a saline solution of [ F]9 (0.1 mL,
The distribution coefficient of [ F]9 was determined by measuring the 11.1 MBq) via the tail vein and sacrificed by decapitation at 30 min after
18
distribution of the radiotracer between 1-octanol and potassium injection. The plasma and brain were collected. The brain was washed
18
phosphate buffer (PBS, 0.05 mol/L, pH 7.4). The radiotracer [ F]9 (10 μL,
100 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
min, followed by centrifugation 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
with saline. The samples were placed separately in 2 mL of ice-cold
1
CH CN and homogenized with a homogenizer (LabGEN 7) for 2 min.
The mixture was centrifuged at 14000 rpm for 5 min (Eppendorf
3
3
Centrifuge 5418). The combined supernatants were collected and passed
through a 0.22 μm organic 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 (Shimadzu system, Agela Venusil MP C18 column,
250 × 4.6 mm, 5 μm, eluent 65% CH CN, and 35% H O containing
3
2
an automatic gamma-counter (Wallac 1470 Wizard, USA). The log D value 10 mM NH OAc, flow rate 1 mL/min).
4
was calculated as the ratio of the cpm/mL of 1-octanol to that of PBS and
expressed as log D = log [cpm/mL(1-octanol)/cpm/mL(PBS)]. The samples
from the remaining organic layer were repartitioned until consistent
Acknowledgement
distribution coefficient values were obtained. The measurement was
carried out in triplicate and repeated three times.
This work was supported by the National Natural Science
Foundation of China (no. 21471019).
In vitro stability studies
18
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Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016