Synthesis and evaluation of novel 18F-labeled spirocyclic piperidine derivatives as σ1 receptor ligands for positron emission tomography imaging

Article abstract of DOI:10.1021/jm301734g

A series of spirocyclic piperidine derivatives were designed and synthesized as σ₁ receptor ligands. In vitro competition binding assays showed that 1′-(4-(2-fluoroethoxy)benzyl)-3H-spiro[2- benzofuran-1,4′-piperidine] (19) possessed high σ₁ receptor affinity (K_i = 0.79 nM) and excellent σ₁/ σ₂ subtype selectivity (350-fold) as well as high σ₁/VAChT selectivity (799-fold). The radiolabeled compound [¹⁸F]19 was synthesized by substitution of the tosylate precursor 24 with [¹⁸F]fluoride, with an isolated radiochemical yield of 35-60%, a radiochemical purity of >99%, and a specific activity of 30-55 GBq/μmol. Biodistribution studies in imprinting control region mice indicated that [ ¹⁸F]19 displayed excellent initial brain uptake and slow washout. Ex vivo autoradiography in Sprague-Dawley rats demonstrated high accumulation of the radiotracer in brain areas known to express high levels of σ₁ receptors. Micro positron emission tomography imaging and blocking studies confirmed the specific binding of [¹⁸F]19 to σ₁ receptors in vivo.

Full text of DOI:10.1021/jm301734g

Article
pubs.acs.org/jmc
18
Synthesis and Evaluation of Novel F‑Labeled Spirocyclic Piperidine
Derivatives as σ1 Receptor Ligands for Positron Emission
Tomography Imaging
Yan Li,† Xia Wang,† Jinming Zhang,‡ Winnie Deuther-Conrad,§ Fang Xie,† Xiaojun Zhang,‡ Jian Liu,‡
Jinping Qiao,† Mengchao Cui,† Jorg Steinbach,§ Peter Brust,§ Boli Liu,† and Hongmei Jia*,†
̈
†Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing 100875, People’s Republic of China
‡Nuclear Medicine Department, Chinese PLA General Hospital, Beijing 100853, People’s Republic of China
§Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Department of
Neuroradiopharmaceuticals, 04318 Leipzig, Germany
S
* Supporting Information
ABSTRACT: A series of spirocyclic piperidine derivatives were designed and
synthesized as σ1 receptor ligands. In vitro competition binding assays showed
that 1′-(4-(2-fluoroethoxy)benzyl)-3H-spiro[2-benzofuran-1,4′-piperidine] (19)
possessed high σ1 receptor affinity (Ki = 0.79 nM) and excellent σ1/σ2 subtype
selectivity (350-fold) as well as high σ1/VAChT selectivity (799-fold). The
radiolabeled compound [18F]19 was synthesized by substitution of the tosylate
precursor 24 with [18F]fluoride, with an isolated radiochemical yield of 35−60%,
a radiochemical purity of >99%, and a specific activity of 30−55 GBq/μmol.
Biodistribution studies in imprinting control region mice indicated that [18F]19
displayed excellent initial brain uptake and slow washout. Ex vivo autoradiography in Sprague−Dawley rats demonstrated high
accumulation of the radiotracer in brain areas known to express high levels of σ1 receptors. Micro positron emission tomography
imaging and blocking studies confirmed the specific binding of [18F]19 to σ1 receptors in vivo.
and SPECT imaging agents for σ1 receptors have been
developed. Among these, [11C]SA4503 ([11C]1),15−19
[18F]FPS ([18F]2),20,21 and [123I]TPCNE ([123I]3)22 (Figure
1) have been evaluated in human studies. However, [18F]2 and
INTRODUCTION
■
σ1 receptors, containing 223 amino acids with two trans-
membrane domains, are a distinct class of intracellular
membrane proteins.1−3 They have a characteristic distribution
in the central nervous system (CNS) and in peripheral organs
such as the liver, spleen, lungs, heart, and kidneys.4,5 The σ1
receptors function as “receptor chaperones” and are believed to
be involved in the regulation of various ion channels, G-
protein-coupled receptors, lipids, and other signaling proteins
through a direct protein−protein interaction with another
endoplasmic reticulum (ER) chaperone binding immunoglo-
bulin protein/78 kDa glucose-regulated protein (BiP/GRP-
78).2,3,6 Recently, more and more evidence suggests that the σ1
receptors are implicated in a myriad of CNS diseases, such as
anxiety, depression, schizophrenia, drug addiction, and
Alzheimer’s disease (AD).7−11 In addition, the σ1 receptors
are believed to be linked to a number of tumors as well as heart
failure.12−14
Positron emission tomography (PET) and single-photon
emission computed tomography (SPECT) are noninvasive
imaging modalities useful in the investigation of receptor status
in vivo. PET or SPECT imaging with σ1 receptor selective
radiotracers could quantitatively visualize σ1 receptors in the
living human brain, thus providing a valuable tool to investigate
the function of this receptor and its dysregulation under
diseased conditions. In the past few years, a number of PET
[
123I]3 have been found to display irreversible kinetics and thus
are not suitable for the quantification of σ1 receptors in
vivo.20,22 [11C]1, as the first useful PET radiotracer, showed
high affinity for σ1 receptors and selectivity toward 36 other
receptors, ion channels, and second messenger systems.23 The
in vivo [11C]1 imaging studies showed that the density of σ1
receptors was decreased in the brain of Parkinson’s disease
(PD) and AD patients.15,16 Due to the short half-life of 11C
(t1/2 = 20 min), use of [11C]1 needs an on-site cyclotron. The
longer half-life 18F-labeled compound seems more promising
for σ1 receptor imaging.
In the past decade, a novel class of spirocyclic piperidine
compounds were reported as potent σ1 receptor ligands. From
this class compound 4 (1′-benzyl-3-methoxy-3H-spiro[2-
benzofuran-1,4′-piperidine]; Figure 2) was shown to possess
nanomolar affinity for σ1 receptors and excellent selectivity over
the σ2 and more than 60 other receptors, transporters, and ion
channels.24−26 As a result, spirocyclic piperidines have been
used as lead compounds to develop radiotracers for σ1 receptor
Received: November 25, 2012
Published: April 8, 2013
© 2013 American Chemical Society
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Figure 1. Structures of σ1 receptor radiotracers investigated in human studies.
Figure 2. Spirocyclic piperidine σ1 receptor ligands and radiotracers.
imaging. The first approach in these development efforts was
the direct incorporation of a fluorine atom into the benzene
ring of the N-benzyl moiety (e.g., ligands [18F]5a and [18F]5b,
Figure 2).27 Unfortunately, these tracers were metabolically
unstable in vivo and hence not suitable for imaging applications.
The second approach was to introduce the fluorine atom into
the C-3 side chain of the spirocyclic benzofuran system with a
1−4 carbon length (e.g., [18F]6, Figure 2).28−32 From this
approach, [18F]fluspidine ([18F]6b) emerged as a potential
radiotracer with favorable target affinity and specificity as well
as metabolic stability in vivo.
Figure 3. Design concept of 18F-labeled spirocyclic piperidine
compounds.
We took a third approach to the development of imaging
agents from the spirocyclic piperidine series of compounds, i.e.,
structural modifications on the N-benzyl moiety while
eliminating the substituent on the spirocyclic benzofuran
section. We have previously reported the discovery of Spiro-I
(7), with an iodine at the para-position of the benzyl moiety, as
a ligand with high affinity and subtype selectivity for the σ1
receptors (σ1, Ki = 2.75 nM; σ2, Ki = 340 nM).33 The
radioligand [125I]7 (Figure 2) exhibited high initial brain uptake
and specific binding to σ1 receptors in vivo. To develop a PET
radiotracer as a σ1 receptor imaging agent with suitable
properties, we replaced the iodine atom at the para-position of
the N-benzyl moiety with either fluorine or a methoxy group
for initial assessment of preserved affinity and selectivity. We
also elongated the para-substituent with a short fluorooligoe-
thoxylated chain (mono-, di-, and triethoxy) in an attempt to
modify the affinity, lipophilicity, and pharmacokinetic proper-
ties of the resulting compounds. Moreover, we replaced the
aromatic ring in the N-benzyl moiety with a pyridine group to
further reduce the lipophilicity of this series of derivatives. The
design concept is shown in Figure 3. Herein we report the
synthesis and evaluation of spirocyclic piperidine derivatives as
σ1 receptor ligands for imaging of σ1 receptor density in the
brain.
RESULTS
■
Chemistry. The synthetic routes for the novel spirocyclic
piperidine ligands are depicted in Scheme 1. All compounds
were prepared from the key intermediate 8 (3H-spiro[2-
benzofuran-1,4′-piperidine]), which was synthesized according
to a published method.34 Preparation of intermediates 9 and 10
followed the method in the literature; i.e., p-cresol was reacted
with 1-bromo-2-fluoroethane to obtain compound 9, followed
by radical bromination.35 Another intermediate, 6-(2-
fluoroethoxy)nicotinaldehyde (11), was obtained by reaction
of 6-chloronicotinaldehyde with 2-fluoroethanol in the
presence of t-BuOK in DMF. The intermediates 15 and 16
were obtained by fluorination of the oligoethylene glycol
ditosylate esters 13 and 14 using anhydrous tetra-n-
butylammonium fluoride (TBAF) in THF.36 N-Alkylation of
the intermediate 8 with 1-(bromomethyl)-4-fluorobenzene, 1-
(bromomethyl)-4-methoxybenzene, or compound 10 provided
compounds 17, 18, and 19, respectively, with yields of 30%,
40%, and 57%. Reductive amination of compound 8 in the
presence of NaBH(OAc)3 with 11 or 4-hydroxybenzaldehyde
led to the N-methylpyridinyl derivative 20 or compound 21,
respectively, with yields of 58% and 69%. The fluoroalkoxy
analogues 22 and 23 were synthesized by heating the
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a
Scheme 1. Synthetic Routes of Compounds 17−23
a
Reagents and conditions: (a) 1-bromo-2-fluoroethane, K2CO3, acetone, 60 °C, 10 h, 60%; (b) NBS, AIBN, CCl4, 80 °C, 4 h, 36%; (c) 2-
fluoroethanol, t-BuOK, DMF, rt, 23%; (d) TsCl, KOH, CH2Cl2, 0 °C to rt, 75−80%; (e) TBAF (1 M in THF), THF, reflux, 42−58%; (f) K2CO3,
KI, CH3CN; for 17, 1-(bromomethyl)-4-fluorobenzene, reflux, 30%; for 18, 1-(bromomethyl)-4-methoxybenzene, rt, 40%; (g) 10, NaH, THF, rt,
57%; (h) (i) 11, 1,2-dichloroethane, 2 h; (ii) NaBH(OAc)3, 6 h, 58%; (i) (i) 4-hydroxybenzaldehyde, 1,2-dichloroethane, 2 h; (ii) NaBH(OAc)3, 6
h, 69%; (j) CH3CN, K2CO3, reflux, 67% for 22, 74% for 23.
Figure 4. Crystal structure of compound 19.
intermediate 21 with tosylate derivatives 15 and 16 under basic
condition (K2CO3) in acetonitrile with yields of 67% and 74%,
respectively.
In Vitro Radioligand Competition Studies. Affinities of
the new compounds for the σ1 and σ2 receptors were
determined in radioligand competition binding assays.37 The
results are listed in Table 1. In vitro competition binding assays
indicated that, similarly to the previously reported spirocyclic
piperidine derivatives,24,25 small substituents at the para-
position of the N-benzyl moiety, such as F, OCH3, or
OCH2CH2F, preserved high affinity and selectivity for σ1
receptors (17−19). In particular, compound 19 exhibited
subnanomolar affinity for σ1 receptors (Ki = 0.79 0.11 nM)
and excellent subtype selectivity (σ2 receptor, Ki = 277 71
nM; Ki(σ2)/Ki(σ1) = 350). Moreover, introduction of a
fluoroethoxy group appeared to lead to an increase in subtype
selectivity. On the other hand, substitution at the same position
To examine the binding model of spirocyclic piperidine
compounds, the single X-ray crystal structure analysis of
compound 19 was carried out. Crystal data together with
details of the determinations are summarized in the Supporting
Information. The crystal structure with the atomic numbering
scheme of compound 19 is shown in Figure 4. The oxygen
atom of the benzofuran ring adopts the axial position at the
piperidine chair. The distance between the N-atom and the
center of the aromatic ring of the N-benzyl residue is 3.8 Å. The
distance between the N-atom and the center of the aromatic
ring of the benzofuran system is 5.7 Å.
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D7.4 values for [18F]19 and [18F]23 were 2.41 0.09 and 1.90
0.06, respectively (Table 2), indicating that the lipophilicity
of the radiotracers decreases with the introduction of extra
ethoxy units at the para-position of the N-benzyl moiety.
The in vitro stability of [18F]19 was evaluated by measuring
the RCP under different conditions. [18F]19 proved to be
chemically stable in 0.9% NaCl solution and in 80% ethanol at
room temperature for up to 4 h. Furthermore, after incubation
with mouse plasma at 37 °C for 4 h, [18F]19 retained a RCP of
>99%, indicating an excellent stability in vitro.
Biodistribution Studies in Mice and Blocking Studies. To
evaluate the pharmacokinetics of the two radiotracers [18F]19
and [18F]23, biodistribution studies were performed in male
imprinting control region (ICR) mice at 2, 30, 60, and 120 min
postinjection. The results are presented in Table 2. [18F]19
displayed high initial brain uptake and a slow clearance rate
with 8.07% injected dose (ID)/g at 2 min, 9.32% ID/g at 30
min, and 8.99% ID/g at 120 min. Among peripheral organs, the
liver displayed a kinetic profile similar to that in the brain, with
peak uptake of 15.30% ID/g at 30 min. The lungs and heart
exhibited a significantly high uptake at 2 min (47.91% and
21.71% ID/g, respectively), which decreased gradually (16.20%
and 8.54% ID/g at 120 min, respectively). The high uptake in
these organs is consistent with the expression of σ1 receptors
and similar to the tissue distribution pattern of [18F]6b.31
Compared with [18F]19, [18F]23 displayed lower brain
uptake and relatively fast washout with 5.79% ID/g at 2 min
and 3.91% ID/g at 120 min following tracer injection. [18F]23
also showed similarly fast washout in the peripheral organs
known to contain σ1 receptors, such as the lungs and kidneys,
which may reflect the lower affinity of this radiotracer for σ1
receptors. In addition, accumulation of [18F]19 and [18F]23 in
the bone increased only slightly over time, suggesting that
defluorination was minimal in mice and that both radiotracers
were fairly stable in vivo.
Table 1. Binding Affinities of the Spirocyclic Piperidine
Derivatives toward σ1 and σ2 Receptors
a
compd
Ki(σ1) (nM)
Ki(σ2) (nM)
Ki(σ2)/Ki(σ1)
17 (Spiro-F)
0.58 0.02
0.18 0.09
1.70 1.74
0.79 0.11
2.30 0.69
52.9 2.33
37.5 2.54
2.75 0.12
4.95 1.74
106 21
131
182
728
162
350
142
16
b
Spiro-F
18
19
20
22
23
276 115
277 71
327 47
860 4.99
592 123
340
16
c
7
123
4
haloperidol
20.7 0.07
a
Values are means SD of three experiments performed in triplicate.
From ref 27. From ref 33.
b
c
with an increased length of the fluorooligoethoxylated chain (n
= 2, 3) led to decreased affinities for σ1 receptors (compounds
22 and 23). Finally, replacement of the benzene ring with
pyridine slightly decreased the affinity for σ1 receptors and
subtype selectivity (20 vs 19).
Affinities of compound 19 for the vesicular acetylcholine
transporter (VAChT) were also determined in radioligand
competition binding assays.38 Compound 19 exhibited low
affinity for VAChT (Ki = 631 50.2 nM) and thus resulted in
excellent selectivity for σ1 receptors (Ki(VAChT)/Ki(σ1) =
799).
Radiochemistry. The synthesis of the precursors and
radioligands is outlined in Scheme 2. The tosylate precursors
Scheme 2. Synthetic Route of the Precursors 24 and 25 and
a
Radioligands [18F]19 and [18F]23
To detect the specific binding of [18F]19 to σ1 receptors in
vivo, the effects of preinjection of haloperidol (0.1 mL, 1.0 mg/
kg) on the biodistribution of radioactivity in various organs of
male ICR mice were examined. Either saline or haloperidol was
injected 5 min prior to the radiotracer injection. The results of
blocking studies at 60 min postinjection are listed in Table 3. A
significant reduction of 58% (p < 0.001) in the accumulation of
radiotracer was observed in the brain. Moreover, accumulation
of radiotracer in other organs known to contain σ1 receptors
was also significantly reduced by haloperidol pretreatment
(reduction of 61%, 76%, 47%, and 64% in the heart, lungs,
kidneys, and liver, respectively). The same blocking study was
carried out with [18F]23. Little reduction in radiotracer
accumulation was seen in the brain (16%). These results
demonstrated that specific binding of this radiotracer is small,
reflecting its lower affinity for σ1 receptors.
On the basis of the results reported in the literature,
compound 1 had weak binding affinities for α1 adrenergic,
dopamine D2, serotonin (5-HT)1A, 5-HT2, histamine H1,
muscarinic M1, and muscarinic M2 receptors at a concentration
of 10 μM. However, these binding affinities were about 100
times lower than that for the σ1 receptor subtype. 1 had no
affinity for 29 other receptors, ion channels, and second
messenger systems.23 These data indicated that 1 is a better σ1
receptor ligand for demonstrating the specificity of the novel
ligands compared to haloperidol. To further investigate the
specific binding of [18F]19 to σ1 receptors in vivo, blocking
studies were carried out by preinjection of different
a
Reagents and conditions: (a) K2CO3, CH3CN, reflux, 67% for 24,
61% for 25; (b) Kryptofix 2.2.2, K2CO3, 18F−, CH3CN, 95 °C, for
[18F]19 35−60%, for [18F]23 20−40%.
24 and 25 were prepared from the coupling of 21 with ditosyl
ethers 12 and 14 in yields of 67% and 61%, respectively. The
formation of the desired compounds [18F]19 and [18F]23 was
achieved through a direct SN2 displacement of the tosylate
group in 24 and 25 with [18F]fluoride. After purification by
semipreparative HPLC, [18F]19 and [18F]23 were obtained in
35−60% (n = 5) and 20−40% (n = 3) decay-corrected
radiochemical yields, respectively, with a radiochemical purity
(RCP) of >99%. The specific activity was 30−55 GBq/μmol at
the end of the synthesis. The total synthesis time was about 1 h.
Evaluation of Radiolabeled Compounds. Lipophilicity
and in Vitro Stability. For compounds 19 and 23, calculated
log P (clogP) values obtained by the Chemdraw software are
3.14 and 2.83, respectively, lower than those of compounds
from the [18F]6 series (clogP of 3.25−4.23 for 6a−6d, Figure
2).32 The log D7.4 values of the two radiotracers [18F]19 and
[18F]23 were determined using a shake flask method.39 The log
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a
Table 2. Biodistribution of Radiotracers [18F]19 and [18F]23 in Male ICR Mice
organ
2 min
30 min
60 min
120 min
[18F]19 (log D = 2.41 0.09)
blood
1.71 0.26
8.07 1.00
21.71 2.57
8.46 0.55
4.84 0.52
47.91 8.41
18.76 2.04
1.45 0.22
6.62 0.57
5.45 0.44
2.74 0.63
4.7
0.72 0.12
0.80 0.08
9.10 0.60
10.74 1.31
13.87 1.80
8.52 0.89
25.21 6.99
10.87 1.29
0.90 0.12
7.81 0.67
4.41 0.88
4.27 0.49
11.4
1.20 0.15
8.99 0.37
8.54 0.36
brain
9.32 0.50
heart
14.39 1.51
15.30 2.14
8.20 1.82
liver
13.60 1.09
9.09 0.93
16.20 1.59
9.55 0.80
0.89 0.10
7.17 0.94
4.01 0.54
6.13 0.86
7.5
spleen
lung
25.89 3.30
13.16 1.80
1.09 0.21
kidney
stomach
b
b
intestine
muscle
8.02 0.38
4.59 0.44
bone
3.30 0.15
brain/blood
12.9
[18F]23 (log D = 1.90 0.06)
blood
brain
heart
liver
1.97 0.23
5.79 0.76
7.69 0.69
5.83 0.75
5.35 1.25
20.68 1.55
16.45 1.56
2.19 0.26
6.21 0.97
3.22 0.57
3.38 0.79
2.9
2.96 0.15
3.35 0.36
4.63 0.65
3.43 0.38
6.32 0.76
5.43 0.91
3.73 0.40
4.37 0.70
1.45 0.52
4.68 0.48
2.64 0.26
4.51 0.51
1.4
3.52 1.40
3.91 0.74
3.25 0.37
4.14 0.68
3.91 0.93
3.16 0.35
3.96 0.91
2.03 0.35
4.04 0.96
2.62 0.34
5.28 1.49
1.1
5.20 0.32
2.95 0.37
8.67 0.82
spleen
lung
7.64 0.79
4.46 0.36
kidney
5.09 0.45
b
stomach
2.00 0.64
b
intestine
6.72 1.13
muscle
2.42 0.36
bone
4.10 0.52
brain/blood
1.8
a
b
Data are expressed as percentage of injected dose per gram, mean SD, n = 5. Percentage of injected dose per organ.
concentrations of 1 (2, 5, or 10 μmol/kg) 5 min prior to the
radiotracer injection. Blocking results of organ distribution of
[18F]19 at 60 min postinjection in ICR mice are summarized in
Figure 5A. Pretreatment of animals with different concen-
trations of 1 resulted in a significant reduction in radiotracer
uptake in organs known to contain σ1 receptors, including the
brain (46−56%), heart (60−63%), lungs (71−76%), kidneys
(57−64%), and spleen (29−55%). Moreover, with pretreat-
ment of animals with a higher concentration of 1, higher
reduction of radiotracer accumulation was observed in organs
known to contain σ1 receptors. At the same time, higher
radiotracer accumulation was observed in blood. Since our aim
is to develop σ1 receptor ligands for imaging σ1 receptor density
in the brain, we also use other receptor ligands, including
tamoxifen (a ligand with high affinity for the emopamil binding
protein (EBP) receptor), (S)-(−)-raclopride (dopamine D2/D3
receptor ligand), or fluvoxamine (SSRI) as well as haloperidol
and 1, as blocking agents. The blocking results in the brain are
presented in Figure 5B. In contrast with haloperidol and 1, no
significant reduction was observed by preinjection of tamoxifen,
(S)-(−)-raclopride, and fluvoxamine, indicating that [18F]19
does not bind to EBP, dopamine D2/D3 receptors, and
serotonin transporter (5-HTT) in the brain at the low doses
used for imaging.
observed in the hypothalamus and thalamus. As presented in
Figure 6, there was no remarkable higher accumulation in the
hippocampus. There was negligible uptake of [18F]19 in the
lateral ventricle and white matter (internal capsule). In the
cerebellum, regions with comparably high uptake (vermian
lobule) and comparably low uptake (paramedian lobule) were
observed. Taken together, the distribution of [18F]19 in the rat
brain is consistent with the density profile of σ1 receptors. To
compare our results with those of [11C]1,40 we selected the
cerebellum as a reference region and calculated the region to
cerebellum activity ratios for both tracers. The results are
shown in Table 4. The rank order of [18F]19 uptake in various
brain regions is in good agreement with that of [11C]1. When
the results of [18F]19 were compared with those of [18F]6b,31
where ex vivo autoradiography was performed in the brain of
the CD-1 mouse, both radiotracers displayed high accumulation
in the cerebellum, cortex, and superficial gray layer of the
superior colliculus and moderate accumulation in the hippo-
campus. However, [18F]6b showed an extremely high
accumulation in the facial nucleus, while moderate accumu-
lation in this region was found for [18F]19. The accumulation
of [18F]19 in the cortex and thalamus is slightly higher than that
of [18F]6b.
MicroPET Studies. To further evaluate the in vivo
distribution of [18F]19 and confirm its specific binding to σ1
receptors, microPET/computed tomography (CT) studies
were carried out in anaesthetized Sprague−Dawley rats. In
vivo binding specificity was evaluated in a blocking study with
preadministration of haloperidol (1 mg/kg) 5 min prior to
radiotracer injection. Sagittal and coronal brain images at 60
min postinjection of the tracer are shown in Figure 7. [18F]19
crossed the blood−brain barrier (BBB) rapidly and accumu-
Ex Vivo Autoradiography in Sprague−Dawley Rats. To
investigate the accumulation of [18F]19 in different brain
regions, an ex vivo autoradiography study was carried out in
Sprague−Dawley rats. The results are presented in Figure 6. It
is notable that a distinctly high accumulation was observed in
the cortex, especially in the temporal cortex auditory area
(TeAud). The accumulation was a little lower in the primary
visual cortex. Moderate accumulation of radiotracer was
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[18F]19 decreased in all brain regions, consistent with the
results from the biodistribution study.
Table 3. Effects of Preinjection of Haloperidol (0.1 mL, 1.0
mg/kg) 5 min Prior to the Injection of Radiotracer [18F]19
or [18F]23 on the Biodistribution of Radioactivity in Male
Additional Biological Studies. In Vitro Metabolism of
Compound 19 by Rat Liver Microsomes. Metabolites of
compound 19 in the rat liver microsomes were determined by
ultraperformance liquid chromatography (UPLC)−MS. They
were identified by comparing the incubation samples with the
blank samples (incubation in the absence of 19). Figure 8
presents the total ion chromatography (TIC) of metabolites
from compound 19 incubated with rat liver microsomes at 37
°C for 30 min. Three metabolites were observed. The retention
time of compound 19 was 1.82 min. Metabolites M1 and M2
eluted at 1.74 and 1.28 min, respectively. Metabolite M1
showed a protonated molecular ion at m/z 296, a loss of 46 Da
from the parent molecule, suggesting an O-dealkylated product.
Metabolite M2 displayed a protonated molecular ion of m/z
358, an increase of 16 Da from the parent molecule. Since M2
eluted off earlier than both M1 and the parent, it was postulated
as an oxidized species of the parent. The third metabolite M3
presented a protonated molecular ion of m/z 360, an increase
of 18 Da from 19, with a retention time of 1.42 min, so
metabolite M3 was considered as the furan ring-opening
product. The proposed metabolic pathways for ligand 19 are
given in Figure 8.
a
ICR Mice
60 min
blocking
(%)
b
organ
blood
60 min (control)
(blocking)
p
[18F]19
0.94 0.37
9.65 0.70
10.15 1.29
15.86 2.21
8.36 0.17
23.92 1.73
10.79 0.87
1.09 0.20
7.38 1.20
4.27 0.17
4.69 0.01
4.25 0.70
4.06 0.65
3.99 0.53
5.72 0.80
8.75 1.21
5.71 0.96
5.69 1.02
3.36 0.67
6.85 2.02
3.41 0.51
5.54 0.88
[18F]23
352
−58
−61
−64
5
<0.001
<0.001
<0.001
<0.001
0.611
brain
heart
liver
spleen
lung
−76
−47
208
−7
−20
18
<0.001
<0.001
0.001
kidney
stomach
c
c
intestine
muscle
bone
0.699
0.032
0.254
blood
brain
heart
liver
3.84 0.22
4.51 0.89
3.39 0.42
5.61 1.27
6.50 1.28
3.72 0.43
4.07 0.64
2.16 0.19
4.83 0.21
2.99 0.23
6.47 1.04
4.51 0.36
3.80 0.51
3.97 0.70
3.61 0.29
4.40 0.32
3.93 0.34
4.53 0.36
2.77 0.92
6.30 0.56
3.51 0.63
4.79 0.73
17
−16
17
0.013
0.171
0.245
0.010
0.009
0.432
0.216
0.314
0.006
0.159
0.024
−36
−32
6
spleen
lung
In Vivo Metabolic Stability of [18F]19. The metabolic fate of
[18F]19 was investigated in the brain and liver of male ICR
mice 30 min after administration of the radiotracer. Figure 9
shows that the parent compound [18F]19 was the only
radioactive species present in the mouse brain, indicating no
entry of radioactive metabolites into the brain. HPLC analyses
of mouse liver extract revealed two less lipophilic radio-
metabolites, which correspond to the two F-containing
metabolites found from the metabolic studies with microsomes
in vitro. The retention times of the parent tracer and these two
radiometabolites are 7.72 min (40.3%), 5.70 min (32.8%), and
4.68 min (26.9%), respectively.
kidney
11
c
stomach
intestine
muscle
bone
28
c
30
17
−26
a
Data are expressed as percentage of injected dose per gram, mean
b
SD, n = 5. Values for the control versus blocking group at 60 min
postinjection by Student’s t test (independent, two-tailed). Percent-
c
age of injected dose per organ.
lated in the midbrain, thalamus, and hypothalamus. High
uptakes were also seen in the cortex, pontine reticular nucleus,
and cerebellum. Lower uptakes were observed in the internal
capsule and olfactory bulb, areas with negligible levels of σ1
receptors and thus a representation of nonspecific binding.
When rats were pretreated with haloperidol, uptake levels of
DISCUSSION AND CONCLUSION
■
Neuroimaging with σ1 receptor PET radiotracers provides an
important tool to investigate the involvement of this receptor
subtype in the pathophysiology of neuropsychiatric diseases
such as schizophrenia, depression, anxiety, and dementia.9,11,41
Figure 5. (A) Effects of preinjection of different concentrations of 1 (2, 5, or 10 μmol/kg) on organ distribution of [18F]19 (370 kBq, 0.1 mL) 60
min after intravenous injection. Student’s t test was performed, and p < 0.05. (B) Effects of preinjection of haloperidol, 1, (S)-(−)-raclopride,
fluvoxamine, or tamoxifen (2.2 μmol/kg) on brain accumulation of [18F]19. Student’s t test was performed, and p < 0.001 for haloperidol and 1.
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Figure 6. Ex vivo autoradiography of coronal brain slices of the rat brain obtained 60 min after intravenous injection of [18F]19 (12.2 MBq, 0.2 mL).
(A), (B), (C), and (D) are the slices from the anterior to the posterior.
Table 4. Comparison of the ex Vivo Autoradiography Results
The distance between the N-atom and the center of the
aromatic ring of the benzofuran system is a little shorter than
the postulated distance based on Glennon’s pharmacophore
model (6−10 Å).
a
of [18F]19 with Those of [11C]1
region
TeAud
[11C]1 [18F]19
region
[11C]1 [18F]19
2.06
1.59
1.78
1.90
1.50
1.30
thalamus
1.70
0.96
1.38
1.22
In vitro binding assay showed that all new compounds
displayed selective binding to the σ1 receptors. Among them,
compound 19 possessed the highest affinity and subtype
selectivity for σ1 receptors. Moreover, compound 19 displayed
799-fold less affinity for VAChT. Thus, compound 19 is a more
promising σ1 receptor ligand compared to 17 and 7. To define
the lower affinity limit of a ligand for imaging of σ1 receptors
and to detect the pharmacokinetics profile of the influence of
the oligoethoxylated chain, we decided to carry out the
radiolabeling of 19 and 23 with 18F and evaluation of the
corresponding 18F-labeled radiotracers [18F]19 and [18F]23 for
in vitro and in vivo studies.
For in vitro properties, the lipophilicity of the radioligand is
important in the development of brain PET tracers. Moderate
lipophilicity (log D = 1−3) is considered desirable for adequate
blood−brain barrier penetration, increased free (protein-
unbound) fraction in the plasma, and decreased nonspecific
binding in the brain, which all lead to increased brain uptake
and enhanced specific binding signals.44,45 For compounds 19
and 23, clogP values obtained by the Chemdraw software are
lower than those of compounds from the [18F]6b series.32 In
addition, the lipophilicity of [18F]19 and [18F]23 is lower than
that of [125I]7 (log D7.4 = 3.06 0.11).33 The lipophilicity of
[18F]19 and [18F]23 is within the optimal range for adequate
brain entry and reduced nonspecific binding to brain tissue.
Furthermore, [18F]19 displayed remarkable stability in vitro at
room temperature for up to 4 h.
frontal cortex
hippocampus
interior colliculus
a
Uptakes of [11C]1 and [18F]19 in each region are expressed as ratios
(with the cerebellum as the reference region).
Since spirocyclic piperidine compound 4 possessed nanomolar
affinity for σ1 receptors and excellent selectivity over the σ2 and
more than 60 other receptors, transporters, and ion
channels,24−26 we use this compound as the lead compound
to develop radiotracers for σ1 receptor imaging. In our previous
work, we introduced iodine at the para-position of the benzyl
moiety and found compound 7 possessed high affinity and
subtype selectivity for the σ1 receptors.33 In addition, the
radioligand [125I]7 exhibited high initial brain uptake and
specific binding to σ1 receptors in vivo. Therefore, to develop a
suitable PET radiotracer as a σ1 receptor imaging agent, we
replaced the iodine atom at the para-position of the N-benzyl
moiety with fluorine, a methoxy group, and a short
fluorooligoethoxylated chain (mono-, di-, and triethoxy). The
single X-ray crystal structure analysis of compound 19 showed
that the oxygen atom of the benzofuran ring adopted the axial
position at the piperidine chair, which confirmed the calculation
results by Rack et al. that the conformation with the oxygen
atom in the axial orientation was more stable than that with the
oxygen atom in the equatorial orientation.42 The distance
between the N-atom and the center of the aromatic ring of the
N-benzyl residue is exactly within the range of the σ1 receptor
pharmacophore model postulated by Glennon (2.5−3.9 Å).43
Figure 7. Sagittal and axial CT and microPET images in rats. PET images are summed from 60 to 90 min after intravenous injection with [18F]19
(23 MBq, 0.2 mL) under control and blocking conditions.
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Figure 8. Proposed metabolic pathways of compound 19 in rat liver microsomes (left) and total ion chromatography recorded after incubation of
compound 19 with rat liver microsomes at 37 °C for 30 min (right).
Figure 9. Analytical HPLC chromatograms of the mouse brain (left) and liver (right) extracts, taken 30 min after administration of [18F]19 (18.5
MBq, 0.2 mL).
Following these encouraging in vitro properties, biological
experiments were carried out to evaluate the labeled
compounds for their potential as PET imaging tracers. In
biodistribution studies in mice, the brain uptake of [18F]19
(9.32% ID/g at 30 min) is higher than that of [11C]1 (3.61%
ID/g at 5 min),46 [18F]6b (3.88% ID/g at 5 min),31 or [125I]7
(5.28% ID/g at 15 min).33 [18F]19 also exhibited a high brain-
to-blood ratio of 12.9 at 30 min postinjection, which is
comparable to that of [11C]1 (11.6 at 30 min)46 but somewhat
lower than that of [18F]6b (18.8 at 120 min).31 It needs to be
pointed out that [18F]19 displayed little defluorination in vivo,
which is also important in 18F-labeled tracer development.
Currently, haloperidol is the most widely used σ1 antagonist
in research on σ1 receptors, and its affinity is high enough to
bind σ1 receptors in humans and small animals. Therefore, in
the first step, we use haloperidol as the blocking agent to
investigate the binding specificity of the radiotracers to the σ1
receptors in vivo. With administration of haloperidol (0.1 mL, 1
mg/kg) 5 min prior to [18F]19 injection, significant reduction
of the accumulation of radiotracer in organs known to contain
σ1 receptors was observed. A marked reduction of 58% in the
brain was comparable to that obtained with [18F]6b (62%
reduction at 60 min)31 and [11C]1 (60−70% reduction at 30
min).46 These data suggest the specific binding of [18F]19 to σ1
receptors in vivo. On the other hand, [18F]23 displayed lower
brain uptake and relatively fast washout compared with [18F]19,
which may result from the lower lipophilicity and lower affinity
of [18F]23 for σ1 receptors. Little reduction of radiotracer
accumulation in organs known to contain σ1 receptors was
observed, reflecting that [18F]23 is not suitable for further
investigation owing to its lower affinity (Ki = 37.5 nM) for σ1
receptors.
It is well-known that haloperidol binds with similarly high
affinity to dopamine D2/D3 receptors and σ1 receptors.47,48
Haloperidol also binds adrenergic and serotonergic receptors
with moderate affinity.49 Furthermore, the reduced metabolite
of haloperidol has high affinity for σ1 and σ2 receptors while
showing much lower affinity for dopamine receptors than
haloperidol itself.50 As indicated in the literature, 1 had low
affinity or no affinity for 36 other receptors, ion channels, and
second messenger systems.23 Therefore, 1 was synthesized in
our laboratory and used as a better σ1 receptor ligand for
demonstrating the specificity of [18F]19 to σ1 receptors in vivo.
Significant reduction in radiotracer uptake in organs known to
contain σ1 receptors, including the brain, heart, lungs, kidneys,
and spleen, was observed by pretreatment of animals with
different concentrations of 1 (2, 5, or 10 μmol/kg). Because
our aim is to develop a σ1 receptor brain imaging agent,
interaction of [18F]19 with other receptors in the brain needs to
be considered. It was reported that 1 has high affinity for EBP
(Ki = 1.72 nM)51 and for VAChT (Ki = 50.2 nM).52 Our ligand
19 displayed 799 times lower affinity for VAChT with a Ki
value of 631
50.2 nM. To further investigate the specific
binding of [18F]19 to σ1 receptors in the brain, tamoxifen, (S)-
(−)-raclopride, and fluvoxamine were used as blocking agents
to investigate the possible interaction of [18F]19 with EBP,
dopamine D2/D3 receptors, and 5-HTT. It was encouraging
that pretreatment of tamoxifen, (S)-(−)-raclopride, and
fluvoxamine resulted in no significant reduction in radiotracer
accumulation in the brain. Thus, [18F]19 does not bind to EBP,
dopamine D2/D3 receptors, and 5-HTT in the brain at the low
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doses used for imaging. These data indicated that [18F]19
accumulation in the mouse brain could represent specific
binding to σ1 receptors.
monitored by thin-layer chromatography (TLC) (TLC silica gel 60
F254, E. Merck). Flash column chromatography was performed on
silica gel (200−400 mesh) using the solvent system indicated.
Elemental analysis and HPLC methods were used to determine the
purity of the test compounds that were used for the binding assay. All
the final compounds were tested with a purity of more than 95%
(Supporting Information).
HPLC analyses were performed on a Waters 600 system (Waters
Corp., United States) equipped with a Waters 2489 UV−vis detector
and a Raytest Gabi NaI(Tl) scintillation detector (Raytest, Germany).
Analyses were carried out on an Agela Venusil MP C18 column (250
× 4.6 mm, 5 μm) using acetonitrile (0.1% TFA)/water (0.1% TFA)
(50:50) as the mobile phase at a flow rate of 1 mL/min.
Semipreparative HPLC separations were performed on a Shimadzu
SCL-20AVP system consisting of a binary pump with an online
degasser, a model SPD-20AVP UV−vis detector operating at a
wavelength of 254 nm, and a Bioscan Flow Count 3200 NaI/PMT γ-
radiation scintillation detector. Samples were separated on an Alltech
Alltima RPC-18 column (250 mm × 10 mm, 5 μm) using acetonitrile
(0.1% TFA)/water (0.1% TFA) (60:40) as the mobile phase at a flow
rate of 4 mL/min.
The UPLC−MS/MS system consisted of a Waters Acquity UPLC
system equipped with an Acquity ethylene-bridged (BEH) C18
column (2.1 × 55 mm, 1.7 μm) and a mass spectrometer. The mass
spectrometric detection was performed using a Quattro micro triple-
quadrupole instrument and an electrospray ionization (ESI) interface
in positive ionization mode. The capillary voltage was set at 3.0 kV.
The source temperature was 110 °C, and the desolvation temperature
was maintained at 500 °C. Nitrogen was used both as a cone gas and
as a desolvation gas, with flow rates of 30 and 600 L/h, respectively.
Argon was used as a collision gas. Data processing was performed with
the Mass Lynx 4.1 software.
ICR mice (male, 4 weeks old, 18−22 g) and Sprague−Dawley rats
(male, 9 weeks old, 220−250 g) were purchased from the Laboratory
Animal Centre of the Peking University Health Science Centre
(Beijing, China). All animal experiments were approved by the
Institutional Animal Care and Use Committee of Beijing Normal
University and performed in compliance with relevant laws and
institutional guidelines.
To evaluate the potential use of [18F]19 for imaging σ1
receptor density in the brain, we performed an ex vivo
autoradiography study to investigate the accumulation of [18F]
19 in different brain regions in Sprague−Dawley rats. [18F]19
accumulated in brain areas known to have high expression of σ1
receptors, such as the cortex, cerebellum, and thalamus. The
rank order of [18F]19 uptake in various brain regions is in good
agreement with that of [11C]1.40 To further evaluate [18F]19 as
a promising radiotracer for investigating σ1 receptors in living
subjects, microPET/CT studies were carried out in anaes-
thetized Sprague−Dawley rats. [18F]19 crossed the BBB rapidly
and accumulated in the midbrain, thalamus, hypothalamus,
cortex, pontine reticular nucleus, and cerebellum, which are
known to contain high expression of σ1 receptors, while lower
uptakes were observed in the areas with negligible levels of σ1
receptors. It should be pointed out that the brain regional
distribution of [18F]19 evaluated by microPET was slightly
different from that of ex vivo autoradiography studies, which
may result from the different conditions of the animals (awake
vs anesthetized).53,54 Pretreatment with haloperidol resulted in
the reduction of radiotracer accumulation in all brain regions,
consistent with the results from the biodistribution study.
Taken together, these results suggest that the novel radiotracer
[18F]19 may be useful for quantitative imaging of σ1 receptors
in vivo.
Another important point for development of a brain
radiotracer is to investigate the radioligand’s metabolism profile
in vivo. If radiolabeled metabolites could enter the brain, they
would confound the binding signals in the brain. Therefore,
studies of the in vitro metabolism of compound 19 by rat liver
microsomes and in vivo metabolic fate of [18F]19 in the brain
and liver of male ICR mice at 30 min were carried out. The
results showed that the parent compound [18F]19 was the only
radioactive species present in the mouse brain, indicating no
entry of radioactive metabolites into the brain.
Chemistry. 6-(2-Fluoroethoxy)nicotinaldehyde (11). To a sol-
ution of 6-chloronicotinaldehyde (200.0 mg, 1.4 mmol) in DMF (10
mL) were added 2-fluoroethanol (360.0 mg, 5.6 mmol) and t-BuOK
(320.0 mg, 2.8 mmol). The mixture was stirred at rt for 6 h, poured
into water, and extracted with CHCl3. The combined organic layers
were dried (Na2SO4), filtered, and evaporated. Flash chromatography
on silica gel (hexane:ethyl acetate = 5:1) gave 11 as a white solid (55.0
In conclusion, the novel radiotracer [18F]19 warrants further
evaluation as a promising imaging agent for investigation of the
σ1 receptors in humans.
1
mg, 23%). Mp: 48−49 °C. H NMR (400 MHz, CDCl3): δ (ppm)
EXPERIMENTAL SECTION
9.97 (s, 1H), 8.62 (d, J = 2.1 Hz, 1H), 8.10 (dd, J1 = 8.6 Hz, J2 = 2.3
Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 4.86−4.83 (m, 1H), 4.74−4.71 (m,
2H), 4.67−4.65 (m, 1 H). ESI-MS: [M + H]+ (m/z = 170.1).
1′-(4-Fluorobenzyl)-3H-spiro[2-benzofuran-1,4′-piperidine] (17).
To a solution of 8 (160.0 mg, 0.85 mmol) in MeCN (10 mL) were
added K2CO3 (1.17 g, 8.5 mmol), KI (133.0 mg, 0.85 mmol), and 1-
(bromomethyl)-4-fluorobenzene (160.0 mg, 0.85 mmol). The mixture
was refluxed for 8 h. After filtration, the solvent was removed in vacuo
and the residue purified by silica gel chromatography (hexane:ethyl
acetate = 4:1) to provide 17 as a white solid (75.0 mg, 30%). Mp: 61−
■
General Procedures. All reagents and solvents were obtained
from commercial sources and used without further purification unless
otherwise stated. Tamoxifen (Toronto Research Chemicals Inc.,
Canada) was purchased from J & K Scientific Ltd. Haloperidol, (S)-
(−)-raclopride (+)-tartrate salt and fluvoxamine maleate were
obtained from Sigma-Aldrich Co. Ltd. (Beijing, China). Compound
1 was synthesized according to the method in the literature with some
modifications.55 The synthetic route and characterization are shown in
1
the Supporting Information. H NMR spectra were recorded on a
1
62 °C. H NMR (400 MHz, CDCl3): δ (ppm) 7.35−7.32 (m, 2H),
Bruker Avance III (400 MHz) instrument. Chemical shifts are
reported as δ (ppm) relative to tetramethylsilane (s = singlet, br s =
broad singlet, d = doublet, dd = double doublet, t = triplet, m =
multiplet). 13C NMR spectra were recorded on a Bruker Avance III
(100 MHz) spectrometer. MS spectra were obtained with a Quattro
micro API ESI/MS instrument (Waters, United States). High-
resolution mass spectrometry (HRMS) was performed on a JEOL-
NMS-SX102 spectrometer (JEOL). Melting points were measured on
a SGW X-4 micro melting point apparatus (Shanghai Precision
Scientific Instrument Co., Ltd., China). Elemental analyses (C, H, N)
were determined on an Elementar 240C device (PerkinElmer, United
States). X-ray crystallography data were collected on a Bruker Smart
Apex II crystal diffractometer (Bruker Co., Germany). Reactions were
7.28−7.26 (m, 2H), 7.21−7.19 (m, 1H), 7.16−7.14 (m, 1H), 7.04−
6.99 (m, 2H), 5.07 (s, 2H), 3.56 (s, 2H), 2.84 (s, 1H), 2.81 (s, 1H),
2.42 (t, J = 11.6 Hz, 2H), 1.99 (br s, 2H), 1.78 (s, 1H), 1.75 (s, 1H).
13C NMR (100 MHz, CDCl3): δ 163.23, 160.79, 145.71, 138.95,
130.77, 130.69, 127.55, 127.33, 121.05, 120.80, 115.10, 114.89, 84.70,
70.72, 62.61, 50.06, 36.62. ESI-MS: [M + H]+ (m/z = 298.0).
1′-(4-Methoxybenzyl)-3H-spiro[2-benzofuran-1,4′-piperidine]
(18). To a solution of 8 (500.0 mg, 2.64 mmol) in CH3CN (5 mL)
were added 1-(bromomethyl)-4-methoxybenzene (580.0 mg, 2.90
mmol), K2CO3 (2.0 g, 14.5 mmol), and KI (200.0 mg, 1.20 mmol).
The mixture was stirred at rt for 1 h. After filtration, the solvent was
removed in vacuo and the residue purified by silica gel chromatog-
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raphy (hexane:ethyl acetate = 1:2) to provide 18 as a white solid
(300.0 mg, 40%). Mp: 76−77 °C. H NMR (400 MHz, CDCl3): δ
residue was purified by silica gel chromatography (hexane:ethyl acetate
= 1:1) to provide 22 as a yellow oil (120.0 mg, 67%). H NMR (400
1
1
(ppm) 7.30−7.24 (m, 4H), 7.22−7.19 (m, 1H), 7.16−7.14 (m, 1H),
6.88 (d, J = 9.2 Hz, 2H), 5.07 (s, 2H), 3.81(s, 3H), 3.55 (s, 2H), 2.86
(s, 1H), 2.84 (s, 1H), 2.41 (t, J = 11.2 Hz, 2H), 2.03 (br s, 2H), 1.78−
1.74 (m, 2H). ESI-MS: [M + H]+ (m/z = 309.7). Anal. Calcd for
C20H23NO2: C, 77.64; H, 7.49; N, 4.53. Found: C, 77.66; H, 7.51; N,
4.50.
MHz, CDCl3): δ (ppm) 7.28−7.23 (m, 4H), 7.21−7.18 (m, 1H),
7.15−7.13 (m, 1H), 6.89 (d, J = 8.5 Hz, 2H), 5.06 (s, 2H), 4.65 (t, J =
4.1 Hz, 1H), 4.53 (t, J = 4.1 Hz, 1H), 4.16−4.13 (m, 2H), 3.90−3.88
(m, 2H), 3.87−3.85 (m, 1H), 3.80−3.78 (m, 1H), 3.54 (s, 2H), 2.85
(s, 1H), 2.82 (s, 1H), 2.42 (t, J = 11.6 Hz, 2H), 2.02−1.97 (m, 2H),
1.77 (s, 1H), 1.74 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 157.91,
145.78, 138.97, 130.59, 127.54, 127.34, 121.04, 120.86, 114.41, 84.78,
84.02, 82.34, 77.19, 70.73, 70.51, 70.00, 67.52, 62.79, 50.03, 36.59. ESI-
MS: [M + H]+ (m/z = 386.1).
1′-(4-(2-Fluoroethoxy)benzyl)-3H-spiro[2-benzofuran-1,4′-piperi-
dine] (19). Compound 8 (267.8 mg, 1.42 mmol) was dissolved in
anhydrous THF (18.8 mL) under a N2 atmosphere, followed by
addition of NaH (120.3 mg, 5.00 mmol). The mixture was stirred at rt
for 15 min, followed by addition of 10 (330.1 mg, 1.42 mmol). The
mixture was then stirred at rt for an additional 15 h, poured into water,
and extracted with CH2Cl2. The combined organic layers were dried
(MgSO4), filtered, and evaporated in vacuo. Purification by silica gel
chromatography (CH2Cl2:CH3OH = 20:1) gave 19 (276.0 mg, 57%)
as a white solid. The solid was crystallized from a mixture of diethyl
ether and petroleum ether as white crystals. Mp: 52−53 °C. 1H NMR
(400 MHz, CDCl3): δ (ppm) 7.37 (d, J = 8.5 Hz, 2H), 7.29−7.25 (m,
2H), 7.21−7.18 (m, 2H), 6.92 (d, J = 8.6 Hz, 2H), 5.06 (s, 2H), 4.82
(t, J = 4.1 Hz, 1H), 4.70 (t, J = 4.1 Hz, 1H), 4.25 (t, J = 4.1 Hz, 1H),
4.18 (t, J = 4.1 Hz, 1H), 3.71 (s, 2H), 3.01 (s, 1H), 3.00 (s, 1H), 2.61
(t, J = 10.9 Hz, 2H), 2.21 (br s, 2H), 1.80 (s, 1H), 1.77 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 157.58, 145.84, 138.98, 130.53, 127.51,
127.30, 121.02, 120.81, 114.41, 84.80, 82.78, 81.09, 70.68, 67.28,
67.08, 62.77, 50.04, 36.66. ESI-MS: [M + H]+ (m/z = 341.7). Anal.
Calcd for C21H24FNO2·1/2H2O: C, 71.98; N, 4.00; H, 7.19. Found: C,
71.49; N, 3.50; H, 7.15.
1′-(4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)benzyl)spiro[2-benzo-
furan-1(3H),4′-piperidine] (23). The procedure described for the
synthesis of 22 was applied to 16 to give 23 as a yellow oil (74%). 1H
NMR (400 MHz, CDCl3): δ (ppm) 7.27−7.23 (m, 4H), 7.20−7.18
(m, 1H), 7.15−7.13 (m, 1H), 6.88 (d, J = 8.5 Hz, 2H), 5.06 (s, 2H),
4.62 (t, J = 4.1 Hz, 1H), 4.50 (t, J = 4.1 Hz, 1H), 4.13 (t, J = 4.9 Hz,
2H), 3.86 (t, J = 4.9 Hz, 2H), 3.79 (t, J = 4.1 Hz, 1H), 3.75−3.71 (m,
5H), 3.53 (s, 2H), 2.85 (s, 1H), 2.82 (s, 1H), 2.41 (t, J = 11.6 Hz, 2H),
2.06−1.93 (m, 2H), 1.77 (s, 1H), 1.74 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 157.95, 145.79, 138.97, 130.52, 127.53, 127.32, 121.03,
120.84, 114.36, 84.79, 84.00, 82.32, 70.89, 70.72, 70.57, 70.37, 69.86,
67.45, 62.81, 50.03, 36.60. ESI-MS: [M + H]+ (m/z = 430.2).
2-(4-((3H-Spiro[2-benzofuran-1,4′-piperidin]-1′-yl)methyl)-
phenoxy)ethyl 4-Methylbenzenesulfonate (24). Compound 21
(150.0 mg, 0.51 mmol) was added to a solution of 1,2-bis(4-
methylbenzenesulfonate) (12) (563.9 mg, 1.524 mmol) in acetonitrile
(10 mL), followed by K2CO3 (70.0 mg, 0.51 mmol). The mixture was
refluxed for 12 h, then poured into water, and extracted with CH2Cl2.
The combined organic layers were dried (Na2SO4) and filtered, and
the solvent was removed in vacuo. The residue was purified by silica
gel chromatography (CH2Cl2:CH3OH = 10:1) to provide 24 as a
1′-((6-(2-Fluoroethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofur-
an-1,4′-piperidine] (20). To a solution of 11 (50.0 mg, 0.29 mmol) in
1,2-dichloroethane (10 mL) was added compound 8 (50.0 mg, 0.26
mmol). The mixture was reacted for 2 h, followed by addition of
NaBH(OAc)3 (65.0 mg, 0.42 mmol). The mixture was reacted for
another 6 h. Then the reaction was quenched with saturated NaHCO3,
and the solvent was removed in vacuo. Following extraction with ethyl
acetate, the combined organic layers were dried (Na2SO4) and filtered,
and the solvent was removed in vacuo. The residue was purified by
silica gel chromatography (hexane:ethyl acetate = 1:1) to give 20 as a
1
white solid (169 mg, 67%). Mp: 90−92 °C. H NMR (400 MHz,
CDCl3): δ (ppm) 7.82 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H),
7.28−7.24 (m, 4H), 7.22−7.15 (m, 2H), 6.75 (d, J = 8.4 Hz, 2H), 5.06
(s, 2H), 4.38−4.35 (m, 2H), 4.15−4.13 (m, 2H), 3.53 (s, 2H), 2.80
(br s, 2H), 2.45−2.35 (m, 5H), 2.04−1.98 (m, 2H), 1.77 (s, 1H), 1.74
(s, 1H). ESI-MS: [M + H]+ (m/z = 493.6).
2-(2-(2-(4-((3H-Spiro[2-benzofuran-1,4′-piperidin]-1′-yl)methyl)-
phenoxy)ethoxy)ethoxy)ethyl 4-Methylbenzenesulfonate (25). The
procedure described for the synthesis of 24 was applied to 14 to give
25 as a yellow oil (61%). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.80
(d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.27−7.24 (m, 4H),
7.20−7.18 (m, 1H), 7.16−7.12 (m, 1H), 6.88 (d, J = 8.3 Hz, 2H), 5.06
(s, 2H), 4.17−4.15 (m, 2H), 4.11−4.09 (m, 2H), 3.83−3.80 (m, 2H),
3.71−3.80 (m, 2H), 3.67−3.65 (m, 2H), 3.62−3.60 (m, 2H), 3.53 (s,
2H), 2.85 (s, 1H), 2.82 (s, 1H), 2.43−2.38 (m, 5H), 2.01−1.96 (m,
2H), 1.77 (s, 1H), 1.74 (s, 1H). ESI-MS: [M + H]+ (m/z = 582.1).
X-ray Crystallography. Single-crystal X-ray diffraction measure-
ments were carried out on a Bruker Smart Apex II crystal
diffractometer at 150(2) K using graphite-monochromated Mo Kα
radiation (λ = 0.71073 Å). An empirical absorption correction was
applied using the SADABS program.56 All structures were solved by
direct methods and refined by full-matrix least-squares on F2 using the
SHELXL-97 program package.57 All of the hydrogen atoms (except
solvent H2O) were geometrically fixed using the riding model.
In Vitro Binding Assays. σ Receptor Binding Assays. Radioligand
competition binding assays were performed with rat brain homoge-
nates for σ1 receptors and rat liver homogenates for σ2 receptors.37 For
σ1 receptors, (+)-[3H]pentazocine was employed as the competing
radioligand. For σ2 receptors, the nonselective radioligand [3H]-1,3-di-
o-tolylguanidine (DTG) was used in the presence of 10 μM
dextrallorphan for selective masking of σ1 receptor binding. Non-
specific binding was determined with 10 μM haloperidol. Ki values
were calculated according to the Cheng−Prusoff equation and
1
white solid (52.0 mg, 58%). Mp: 73−74 °C. H NMR (400 MHz,
CDCl3): δ (ppm) 8.05 (s, 1H), 7.66−7.64 (m, 1H), 7.28−7.26 (m,
2H), 7.21−7.19 (m, 1H), 7.15−7.14 (m, 1H), 6.80 (d, J = 8.5 Hz,
1H), 5.07 (s, 2H), 4.82 (t, J = 4.0 Hz, 1H), 4.70 (t, J = 4.0 Hz, 1H),
4.60 (t, J = 4.4 Hz, 1H), 4.53 (t, J = 4.3 Hz, 1H), 3.52 (s, 2H), 2.83(s,
1H), 2.80 (s, 1H), 2.42 (t, J = 10.6 Hz, 2H), 1.97 (br s, 2H), 1.78 (s,
1H), 1.75 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 162.63, 147.00,
145.63, 140.22, 138.95, 127.59, 127.36, 121.07, 120.80, 110.96, 84.61,
82.11, 81.27, 70.75, 64.86, 64.66, 59.90, 49.96, 36.59. HRMS (EI): m/z
calcd for C20H24FN2O2 [M + H]+ 343.1822, found 343.1832.
4-((3H-Spiro[2-benzofuran-1,4′-piperidine]-1′-yl)methyl)phenol
(21). Compound 8 (500.0 mg, 2.64 mmol) was dissolved in 1,2-
dichloroethane (15 mL) under a N2 atmosphere, followed by addition
of 4-hydroxybenzaldehyde (355.0 mg, 2.89 mmol), and the mixture
was stirred at rt for 2 h. Then NaBH(OAc)3 (650.0 mg, 3.07 mmol)
was added, and the mixture was stirred for an additional 6 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 (MgSO4) and filtered, and the solvent was
removed in vacuo. Then the residue was purified by silica gel
chromatography (CH2Cl2:CH3OH = 5:1) to provide 21 as a white
solid (539.6 mg, 69%). Mp: 192−194 °C. 1H NMR (400 MHz,
CDCl3): δ (ppm) 9.34 (s, 1H), 7.26 (s, 4H), 7.13 (d, J = 7.5 Hz, 2H),
6.73 (d, J = 8.0 Hz, 2H), 4.96 (s, 2H), 3.37 (s, 2H), 2.75 (s, 2H), 2.33
(s, 2H), 1.89 (br s, 2H), 1.62 (s, 1H), 1.59 (s, 1H). ESI-MS: [M + H]+
(m/z = 295.7).
represent the mean
standard deviation (SD) from at least three
1′-(4-(2-(2-Fluoroethoxy)ethoxy)benzyl)spiro[2-benzofuran-1-
(3H),4′-piperidine] (22). A mixture of 21 (88.0 mg, 0.30 mmol), 2-(2-
fluoroethoxy)ethyl 4-methylbenzenesulfonate (15) (130 mg, 0.47
mmol), and potassium carbonate (138.0 mg, 1 mmol) in CH3CN (5
mL) was refluxed for 6 h. After the solvent was removed in vacuo, the
independent experiments, each performed in triplicate.
VAChT Binding Assays. Radioligand competition binding assays
were performed using membrane homogenates obtained from PC12
cells stably transfected with rat VAChT (obtained from Ali Roghani,
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Department of Pharmacology and Neuroscience, Texas Tech
University Health Sciences Center, Lubbock, TX) and (−)-[3H]-
vesamicol (1 nM working concentration). Assays were performed with
compound 19 (at concentrations of 10−11 to 10−5 mol/L) in 50 mM
Tris−HCl, pH 7.4, by incubation at room temperature for 120 min.
Nonspecific binding was determined in the presence of 10 μM
(−)-vesamicol. Ki values were calculated according to the Cheng−
Prusoff equation and represent the mean from two single experiments,
each performed in triplicate.
In Vitro Stability of [18F]19. [18F]19 (3.7 MBq, in 100 μL of water)
was added to 80% EtOH (1 mL) or 0.9% sodium chloride (1 mL).
The solutions were vortexed and then allowed to stand at room
temperature for 4 h. To test the plasma stability, [18F]19 was added to
the freshly prepared murine plasma (1 mL), and the solution was
incubated at 37 °C for 4 h. The radiochemical purity of [18F]19 in the
solutions was assayed by HPLC at 1, 2, and 4 h.
Biological Experiments. Biodistribution Studies in Male ICR
Mice and Blocking Studies. Four groups of ICR mice (n = 5 in each
group) were injected intravenously with a saline solution of [18F]19 or
[18F]23 (370 kBq, 0.1 mL). The mice were sacrificed by decapitation
at 2, 30, 60, and 120 min postinjection. Whole brain, heart, lungs, liver,
spleen, and other organs of interest were removed, weighed, and
counted in an automatic γ-counter. The percentage of injected dose
per gram of wet tissue (% ID/g) was calculated by a comparison of the
tissue counts to suitably diluted aliquots of the injected material. All
radioactivity measurements were corrected for decay. Averages and SD
were calculated.
For the initial blocking studies, the mice were injected via tail vein
with either saline (0.1 mL) or haloperidol (0.1 mL, 1.0 mg/kg) 5 min
prior to [18F]19 or [18F]23 injection. Sixty minutes after radiotracer
administration, the animals were sacrificed by decapitation. Tissues of
interest were excised and analyzed as described. Significant differences
between control and test groups were determined by Student’s t test
(independent, two-tailed). The criterion for significance was p ≤ 0.05.
For the further blocking studies, ICR male mice were injected via
tail vein with either saline (0.1 mL) or a blocking agent, including 1
(0.1 mL, 2 μmol/kg), 1 (0.1 mL, 5 μmol/kg), 1 (0.1 mL, 10 μmol/
kg), haloperidol (0.1 mL, 2.2 μmol/kg), 1 (0.1 mL, 2.2 μmol/kg), (S)-
(−)-raclopride (0.1 mL, 2.2 μmol/kg), fluvoxamine (0.1 mL, 2.2
μmol/kg), or tamoxifen (0.1 mL, 2.2 μmol/kg), 5 min prior to the
injection of [18F]19 (0.1 mL, 370 kBq). Sixty minutes after radiotracer
administration, the animals were sacrificed by decapitation. Tissues of
interest were excised and analyzed as described. Significant differences
between control and test groups were determined by Student’s t test
(independent, two-tailed). The criterion for significance was p ≤ 0.05.
Ex Vivo Autoradiography. A solution of [18F]19 (0.2 mL, 12.2
MBq) was intravenously injected into conscious Spraque−Dawley rats
(n = 3). The rat was killed by cervical dislocation 60 min after injection
of the radioligand. The brain was removed and frozen in a liquid
nitrogen bath, and coronal brain sections (20 μm) were cut on a
cryostat microtome (CM1900, Leica, Germany). The brain slices were
dried at room temperature and were exposed to an imaging plate
(PerkinElmer, United States) for 2 h. Autoradiographic images were
obtained using a phosphor imaging system (CyclonePlus, PerkinElm-
er, United States) and analyzed with the AIDA 2.31 software (Raytest,
Straubenhardt, Germany).
MicroPET Imaging Experiment. The Sprague−Dawley rats were
anesthetized with isoflurane (1.5−2.5%) in oxygen at a flow rate of 1−
2 L/min and injected with 23 MBq of [18F]19 (0.2 mL in saline) via
tail vein. Blocking studies involved preinjection with haloperidol (0.2
mL, 1 mg/kg) 5 min prior to tracer administration. MicroPET imaging
was performed on an eXplore VISTA-CT scanner (GE Healthcare,
Spain) equipped with a computer-controlled bed. The field of view
(FOV) was 6.8 cm transaxial and 4.8 cm axial with an image resolution
of <1 mm. Imaging data were acquired for 30 min, starting 60 min
after tracer injection, followed by a 5 min CT scan for anatomical
information. For image reconstruction, list-mode data were sorted into
3-D sinograms, transformed by Fourier rebinning, and reconstructed
by 2-D ordered-subset expectation maximization reconstruction with 2
iterations and 50 subsets. Corrections for dead time, random
scattering, and attenuation were made for each scan.
Radiochemistry. 1′-(4-(2-[18F]Fluoroethoxy)benzyl)-3H-spiro[2-
benzofuran-1,4′-piperidine] ([18F]19). [18F]fluoride was produced
via the 18O (p,n) 18F nuclear reaction with 20 MeV protons in a
Sumitomo HM-20PS cyclotron and transferred into a reaction vessel
containing a solution of Kryptofix 2.2.2 (13 mg) and K2CO3 (1.1 mg)
in MeCN/water (0.8 mL/0.2 mL). The solvent was removed at 115
°C under a stream of nitrogen gas, and the residue was azeotropically
dried with 1.0 mL of anhydrous acetonitrile, repeated three times. A
solution of the corresponding tosylate precursor 24 (3.0 mg) in
MeCN (1.0 mL) was added to the reaction vessel. The mixture was
heated at 95 °C for 5 min, diluted with water (20 mL), and passed
through a C-18 Sep-Pak cartridge. The crude product was eluted off
the cartridge with MeCN (1 mL) and the solution loaded onto a
semipreparative HPLC column for purification (Alltech Alltima RPC-
18, 250 × 10 mm, 5 μm, eluent 60% CH3CN and 40% water with 0.1%
TFA, flow rate 4 mL/min). The total synthesis time was about 1 h.
The radiochemical yield was 35−60% (n = 5, decay corrected), and the
specific activity at the end of synthesis was 30−55 GBq/μmol,
determined with an HPLC-based method. For animal experiments, the
radiotracer was formulated as a saline solution containing no more
than 8% ethanol.
1′-(4-(2-(2-(2-[18F]Fluoroethoxy)ethoxy)ethoxy)benzyl)spiro[2-
benzofuran-1(3H),4′-piperidine] ([18F]23). The procedure described
for the synthesis of [18F]19 was applied to 25 to give [18F]23. The
total synthesis time was about 1 h. The radiochemical yield was 20−
40% (n = 3, decay corrected). The radiochemical purity was >99%.
The specific activity at the end of the synthesis was 30−55 GBq/μmol,
determined with an HPLC-based method. For animal experiments, the
radiotracer was formulated as a saline solution containing no more
than 8% ethanol.
To identify the radiotracer, [18F]19 and [18F]23 were coinjected
and coeluted with the corresponding unlabeled compounds (Shimadzu
SCL-20AVP, Agela Venusil MP C18, 250 mm × 4.6 mm, 5 μm). Their
HPLC profiles using acetonitrile (0.1% TFA)/water (0.1% TFA)
(50:50, v/v) at a flow rate of 1 mL/min are presented in the
Supporting Information (Figure S2). The retention times of unlabeled
19 and [18F]19 were observed to be 5.31 and 5.83 min, respectively.
The retention times of unlabeled 23 and [18F]23 were observed to be
4.81 and 5.30 min, respectively. The difference in retention time was in
good agreement with the time lag, which corresponds to the volume
and flow rate within the distance between the UV and radioactivity
detector of our HPLC system.
Determination of the Partition Coefficient. Partition coefficients
for [18F]19 and [18F]23 were determined by measuring the
distribution of the radiotracers between 1-octanol and sodium
phosphate buffer (PBS, 0.05 M, pH 7.4). The radiolabeled compound
(370 kBq, 10 μL) was mixed with 1-octanol (3.0 mL) and sodium
phosphate buffer (3.0 mL) in a centrifuge tube. The tube was vortexed
for 3 min at room temperature, followed by centrifugation at 3500 rpm
for 5 min. About 50 μL of the 1-octanol layer was weighed in a tared
tube. The 1-octanol layer was removed, and about 0.5 mL of the buffer
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 automatic
γ-counter (Wallac 1470 Wizard, United States). The log D value was
calculated by comparing 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)]. 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.
In Vitro Metabolism Studies of 19 in Liver Microsomes. Unlabeled
compound 19 was incubated with rat liver microsomes. The
incubation mixture consisted of 2 mg of protein/mL of liver
microsomes, 100 μM 19, and an NADPH-generating system with 2
mM β-NADPH, 20 mM glucose 6-phosphate, 2 U of glucose 6-
phosphate dehydrogenase, and 12 mM MgCl2·12H2O, all in 0.05 M
Tris−HCl buffer (pH 7.4). The total volume was 500 μL. The reaction
was started by the addition of 19 after preincubation of the reaction
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mixture for 3 min at 37 °C. Blank incubations were performed in the
absence of 19. The reaction was stopped after 30 min by the addition
of 1.5 mL of ice-cold methanol. The reaction mixture was vortexed and
centrifuged at 15000 g for 15 min. The supernatant was collected and
filtered through a 0.22 μm microbore cellulose membrane, and
aliquots were analyzed by UPLC−MS.
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collected and washed with saline. The samples were placed separately
in 2 mL of ice-cooled phosphate-buffered saline (0.01 M, pH 7.4) and
homogenized for 2 min with a LabGEN 7 homogenizer. Ice-cold
MeCN (4 mL) was added to the homogenates, and the mixtures were
vortexed first and then centrifuged at 13 200 rpm for 5 min. The
supernatants were collected and passed through a 0.22 mm Millipore
filter. The filtrates were concentrated to 0.2 mL under a stream of
nitrogen gas flow and then injected onto the radio-HPLC system
(Waters 600 system, Agela Venusil MP C18, 250 mm × 4.6 mm, 5
μm) for analysis using a flow rate of 1 mL/min, and the eluent was
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ASSOCIATED CONTENT
* Supporting Information
■
S
Purity of key target compounds, HPLC chromatograms of 19,
[18F]19, 23, and [18F]23, HPLC analysis of the stability of [18F]
19 in vitro, X-ray crystallographic data for compound 19
(including a CIF file), and synthetic route and characterization
of compound 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-10-58808891. Fax: +86-10-58808891. E-mail:
hmjia@bnu.edu.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We give special thanks to Dr. Xuebing Deng (College of
Chemistry, Beijing Normal University) for assistance with the
X-ray diffraction. We are grateful to Prof. Henry Huang for his
helpful suggestions and valuable comments on this manuscript.
This work was supported by the National Natural Science
Foundation of China (Grant 21071023).
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; DMF, dimethylformamide; EBP,
emopamil binding protein; NADPH, nicotinamide adenine
dinucleotide phosphate; PBS, phosphate-buffered saline; PET,
positron emission tomography; PD, Parkinson’s disease; RCP,
radiochemical purity; SSRI, selective serotonin reuptake
inhibitor; 5-HTT, serotonin transporter; SPECT, single-photon
emission computed tomography; TBAF, tetra-n-butylammo-
nium fluoride; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TLC, thin-layer chromatography; VAChT, vesicular acetylcho-
line transporter
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