1-(4-[18F]Fluorobenzyl)-4-[(tetrahydrofuran-2-yl)methyl]piperazine: A Novel Suitable Radioligand with Low Lipophilicity for Imaging σ1 Receptors in the Brain

Article abstract of DOI:10.1021/acs.jmedchem.6b01723

We have designed and synthesized novel piperazine compounds with low lipophilicity as σ₁ receptor ligands. 1-(4-Fluorobenzyl)-4-[(tetrahydrofuran-2-yl)methyl]piperazine (10) possessed a low nanomolar σ₁ receptor affinity and a high selectivity toward the vesicular acetylcholine transporter (>2000-fold), σ₂ receptors (52-fold), and adenosine A_2A, adrenergic α₂, cannabinoid CB₁, dopamine D₁, D_2L, γ-aminobutyric acid A (GABA_A), NMDA, melatonin MT₁, MT₂, and serotonin 5-HT₁ receptors. The corresponding radiotracer [¹⁸F]10 demonstrated high brain uptake and extremely high brain-to-blood ratios in biodistribution studies in mice. Pretreatment with the selective σ₁ receptor agonist SA4503 significantly reduced the level of accumulation of the radiotracer in the brain. No radiometabolite of [¹⁸F]10 was observed to enter the brain. Positron emission tomography and magnetic resonance imaging confirmed suitable kinetics and a high specific binding of [¹⁸F]10 to σ₁ receptors in rat brain. Ex vivo autoradiography showed a reduced level of binding of [¹⁸F]10 in the cortex and hippocampus of the senescence-accelerated prone (SAMP8) compared to that of the senescence-accelerated resistant (SAMR1) mice, indicating the potential dysfunction of σ₁ receptors in Alzheimer’s disease.

Full text of DOI:10.1021/acs.jmedchem.6b01723

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Article
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1-(4-[F]Fluorobenzyl)-4-((tetrahydrofuran-2-
yl)methyl)piperazine: A novel suitable radioligand with
low lipophilicity for imaging sigma-1 receptors in the brain
Yingfang He, Fang Xie, Jiajun Ye, Winnie Deuther-Conrad, Bixiao Cui, Liang
Wang, Jie Lu, Jörg Steinbach, Peter Brust, Yiyun Huang, Jie Lu, and Hongmei Jia
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01723 • Publication Date (Web): 14 Apr 2017
Downloaded from http://pubs.acs.org on April 16, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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1ꢀ(4ꢀ[18F]Fluorobenzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine: A novel
suitable radioligand with low lipophilicity for imaging sigmaꢀ1 receptors in the
brain
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⊥
⊥
Yingfang He† , Fang Xie† , Jiajun Ye†, Winnie DeutherꢀConrad‡, Bixiao Cui§, Liang Wang†, Jie
Lu†, Jörg Steinbach‡, Peter Brust‡, Yiyun Huangꢁ, Jie Lu§*, Hongmei Jia†*
† Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education,
College of Chemistry, Beijing Normal University, Beijing, China;
‡ HelmholtzꢀZentrum DresdenꢀRossendorf, Institute of Radiopharmaceutical Cancer Research,
Department of Neuroradiopharmaceuticals, 04318 Leipzig, Germany;
§ Department of Nuclear Medicine, Xuanwu Hospital Capital Medical University, Beijing, China;
ꢁ Yale PET Center, Department of Radiology and Biomedical Imaging, Yale University School of
Medicine, New Haven, CT, USA.
Corresponding author:
*To whom correspondence should be addressed: Phone: +86ꢀ10ꢀ58808891; Fax: +86ꢀ10ꢀ58808891.
Email: hmjia@bnu.edu.cn (H Jia), imaginglu@hotmail.com (J Lu)
Author Contributions:
⊥Both authors contributed equally.
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ABSTRACT
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We have designed and synthesized novel piperazine compounds with low lipophilicity as σ1
receptor ligands. 1ꢀ(4ꢀFluorobenzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine (10) possessed low
nanomolar σ1 receptor affinity and high selectivity toward the vesicular acetylcholine transporter
(>2000ꢀfold), σ2 receptors (52ꢀfold), adenosine A2A, adrenergic α2, cannabinoid CB1, dopamine D1,
D2L, γꢀaminobutyric acid A (GABAA), NMDA, melatonin MT1, MT2 and serotonin 5ꢀHT1
receptors. The corresponding radiotracer [18F]10 demonstrated high brain uptake and extremely
high brainꢀtoꢀblood ratios in biodistribution studies in mice. Pretreatment with the selective σ1
receptor agonist SA4503 significantly reduced radiotracer accumulation in the brain. No
radiometabolite of [18F]10 was observed to enter the brain. PET/MRI imaging confirmed suitable
kinetics and high specific binding of [18F]10 to σ1 receptors in rat brain. Ex vivo autoradiography
showed reduced binding of [18F]10 in the cortex and hippocampus of the senescenceꢀaccelerated
prone (SAMP8) compared to the senescenceꢀaccelerated resistant (SAMR1) mice, indicating
potential dysfunction of σ1 receptors in Alzheimer’s disease.
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Keywords: Brain, sigmaꢀ1 receptors, piperazine compounds, Alzheimer’s disease, PET/MRI
imaging.
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INTRODUCTION
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The sigmaꢀ1 (σ1) receptor consists of 223 amino acids with molecular weight of 25 kDa.1
Contrary to the prevailing models of two transmembrane segments,2 the recently reported crystal
structures of the human σ1 receptor revealed a trimeric architecture with a single transmembrane
domain in each protomer.3 Most importantly, this receptor is a unique “ligandꢀoperated receptor
chaperone”4 and interacts with other functional proteins in the plasma membrane, endoplasmic
reticulum, mitochondria, and even the cytosol.5 Increasing evidences have indicated the
involvement of the σ1 receptors in the pathophysiology of a number of human diseases, including
Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS),
Huntington's disease, stroke/ischemia, pain/neuropathic pain, cocaine addiction, myocardial
hypertension, and cancers.5ꢀ12 Molecular probes with appropriate affinity, high selectivity and
specificity for σ1 receptors will be useful in the understanding and monitoring of σ1 receptorꢀrelated
diseases using noninvasive imaging techniques such as positron emission tomography (PET) and
single photon emission computed tomography (SPECT).
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In the last decades, many PET and SPECT radiotracers for σ1 receptors have been reported.13
However, to date there have been no suitable σ1 receptor radiotracers for clinical use. Among the
existing radiotracers investigated thus far in humans, [18F]FPS ([18F]2) 14 and [123I]TPCNE ([123I]3)
15 (Figure 1) displayed irreversible kinetics in the brain. [11C]SA4503 ([11C]1, Figure 1), as the first
useful PET radiotracer for imaging σ1 receptors in humans,16ꢀ18 possessed nanomolar affinity for σ1
receptors and high selectivity toward 36 other receptors, ion channels, and second messenger
systems.19 However, the use of [11C]1 needs an onꢀsite cyclotron due to the short halfꢀlife of 11C
(t1/2 = 20 min). More recently, [18F]FTCꢀ146 ([18F]4)20ꢀ22 has been reported to be a promising PET
radiotracer for visualizing σ1 receptors. However, further investigation is still required for its
clinical translation. It is interesting that several potential radiotracers are spirocyclic piperidine
compounds.23ꢀ28 The lead compound, 1’ꢀbenzylꢀ3ꢀmethoxyꢀ3Hꢀspiro[2ꢀbenzofuranꢀ1,4’ꢀpiperidine],
possessed nanomolar affinity for σ1 receptors and excellent selectivity over the σ2 and more than 60
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other receptors, transporters, and ion channels.29 (S)ꢀ[18F]Fluspidine ((S)ꢀ[18F]5) and [18F]6 are
viable σ1 receptor tracers with favorable kinetics in nonhuman primates.28 The radiation risk of
(S)ꢀ[18F]5 imaging proved to be within acceptable limits in the firstꢀinꢀhuman study.30 But its
performance in humans is yet to be further demonstrated.
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In our previous work, elimination of the aromatic ring in [18F]7 led to a selective σ1 ligand as a
tumor imaging agent, 8ꢀ(4ꢀ(2ꢀ[18F]fluoroethoxy)benzyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane, with
2ꢀfold lower lipophilicity.31 Nonetheless, the latter displayed relatively low specific binding to σ
receptors in the brain (only 25% reduction with simultaneous injection of 1 mg/kg body weight of
haloperidol at 60 min) and thus failed to meet the requirements of a radiotracer for neuroimaging.
The objective of the current study has been to develop a potential σ1 receptor radioligand with low
lipophilicity, high in vivo stability and high specific binding.
Compound 8 was reported to have nanomolar affinity for σ1 receptors.32 Similar to the design
concept of the above radiotracer with low lipophilicity, we eliminated the aromatic ring in the
2,3ꢀdihydrobenzofuran moiety of compound 8 and introduced a 2ꢀfluoroethoxy group or halogen
atoms at the paraꢀposition of the benzyl group for radionuclide labeling. We also incorporated a
carbonyl group between the piperazine and tetrahydrofuran moieties to form an amide group, as
shown in Figure 2, to further decrease the lipophilicity. Herein we report on the synthesis of a novel
18Fꢀlabeled radiotracer with low lipophilicity and its evaluation as a suitable candidate PET
radioligand for imaging σ1 receptors in the brain by radiometabolite analysis, biodistribution studies
in mice, and ex vivo autoradiography and PET/MRI imaging studies in rats.
RESULTS
Chemistry. The synthetic route for compounds 9–12 are shown in Scheme 1. Compound 15 was
obtained according to the literature method.33 NꢀAlkylation of compound 13 or 14 with the
corresponding bromide 15–17 provided compounds 9–12 in yields of 63–83%.
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The target compounds (9–12) were characterized by H NMR, 13C NMR, and highꢀresolution
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mass spectrometry (HRMS) as to have chemical purities of ≥ 95% as shown in the Supporting
Information (Table S1).
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In Vitro Radioligand Competition Studies. The affinities of the novel ligands 9–12 for the σ1 and
σ2 receptors were determined with radioligand competition assays (σ1 receptors:
(+)ꢀ[3H]pentazocine; σ2 receptors: [3H]DTG + 10 ꢂM dextrallorphan) as reported previously.34 The
Ki values for σ1 and σ2 receptors are provided in Table 1. In general, elimination of the aromatic ring
in the 1ꢀ((2,3ꢀdihydrobenzofuranꢀ2ꢀyl)methyl)piperazine moiety preserved preferential binding to
σ1 receptors. Introduction of a small group at the paraꢀposition of the benzyl group also maintained
the affinity for σ1 receptors (Ki values of 1.21–15.8 nM for compounds 9–11) and subtype
selectivity (Ki(σ2)/Ki(σ1) = 19–52). Introduction of a carbonyl group between the piperazine and the
tetrahydrofuran moieties led to decreased affinity and subtype selectivity (compare ligands 10 and
12).
Considering its higher affinity and subtype selectivity for σ1 receptors, affinity of compound 10
for the vesicular acetylcholine transporter (VAChT) was also determined using radioligand
competition assays ((ꢀ)ꢀ[3H]vesamicol).35 Compound 10 demonstrated extremely low affinity for
VAChT (Ki = 10.4 ± 0.2 µM) and thus resulted in an excellent selectivity (Ki(VAChT)/Ki(σ1) =
2819). Moreover, this ligand also displayed negligible affinity for adenosine A2A, adrenergic α2,
cannabinoid CB1, dopamine D1 and D2L, γꢀaminobutyric acid A (GABAA), NMDA, melatonin MT1
and MT2, and serotonin 5ꢀHT1 receptors as shown in Table 2. Hence, compound 10 was labeled
with Fꢀ18 for further evaluation.
Radiochemistry. The synthetic route for [18F]10 is presented in Scheme 2. A twoꢀstep procedure
was used to prepare the radiotracer, starting from 4ꢀ(N, N, Nꢀtrimethylamino)benzaldehyde iodide.
In the first step, [18F]4ꢀfluorobenzaldehyde was prepared from the precursor through displacement
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of the trimethylammonium iodide group. Similar to the method developed for the radiosynthesis of
dopamine D4 receptor ligands,38,39 reductive amination of compound 13 with [18F]4ꢀ
fluorobenzaldehyde using NaBH3CN as a reducing agent and CH3COOH as catalyst provided the
desired radiotracer. After purification via semipreparative high performance liquid chromatography
(HPLC), [18F]10 was obtained in 20–30% radiochemical yield (n = 5, decayꢀcorrected) with
radiochemical purity (RCP) of > 99% and specific activity (SA) of 54−86 GBq/ꢂmol (n = 3). The
total synthesis time was about 80 min.
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Evaluation of the Radiolabeled Compound.
Lipophilicity. The apparent distribution coefficient of the 18Fꢀlabeled radiotracer was determined
using the shakeꢀflask method as previously reported.26 The log D7.4 value of [18F]10 was 0.76 ±
0.01.
In Vitro Stability. In vitro stability of [18F]10 in saline and mouse serum was evaluated by
measuring the RCP at different time points using HPLC. After 2 h of incubation with saline or
mouse serum, the RCP of [18F]10 was still higher than 99% as shown in the Supporting Information
(Figure S2).
In Vivo Metabolic Stability. The metabolic stability of [18F]10 was investigated in brain, plasma,
and liver samples of male ICR mice at 15, 30 and 60 min after radiotracer injection (18.5–37.7
MBq, 0.15 mL). Acetonitrile extracts were analyzed by HPLC. Analytical HPLC chromatograms of
radioactive compounds in mouse brain and liver samples at 15 and 30 min are shown in the
Supporting Information (Figure S3). Representative HPLC chromatograms of samples obtained at
60 min are presented in Figure 3. In the brain samples, ≥ 95% of the radioactivity signal represented
the intact tracer [18F]10. In plasma samples, only ~10% of the radioactivity was from [18F]10, along
with one major radiometabolite (tR = 4.6 min). Several more hydrophilic metabolites were found in
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the liver with retention times of less than 6.0 min. The intact tracer and the major radiometabolite
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(tR = 3.4 min) accounted for about 49% and 44%, respectively, of the radioactivity at 60 min.
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Biodistribution Studies in ICR Mice. The kinetics of [18F]10 were investigated in vivo by
biodistribution studies in male ICR mice. Shown in Tables 3 and 4 are the organ distribution data
obtained at 2, 15, 30, 60 and 120 min after radiotracer injection. [18F]10 showed a high initial brain
uptake with radioactivity levels of 12.8 ± 0.79% ID/g at 2 min, which decreased to 11.2 ± 0.86%
ID/g at 15 min, 10.6 ± 1.31% ID/g at 30 min, 8.29 ± 0.66% ID/g at 60 min and 5.40 ± 0.62% ID/g
at 120 min, respectively. On the other hand, radiotracer levels in the blood were very low, at 0.50%
ID/g or lower after 15 min. Therefore, high brainꢀtoꢀblood ratios were obtained (ratios of 22, 21 and
20, respectively, at 15, 30 and 60 min after injection). Moreover, radiotracer accumulation in the
bone was low and remained constant with time, indicating no defluorination of the radiotracer.
To determine the binding specificity of [18F]10 in vivo, blocking studies with the σ1ꢀselective
agonist SA4503 were conducted. SA4503 (5 µmol/kg body weight) was injected at 5 min prior to
radiotracer injection. As shown in Table 4, significant reduction in radiotracer accumulation was
observed in the brain, with 79%, 88% and 86% blockade, respectively, at 15, 30 and 60 min after
radiotracer injection. Radiotracer accumulation in the heart, lungs, and spleen, which are known to
have high expression of σ1 receptors, were also significantly reduced. These results indicated high
specific binding of [18F]10 to σ1 receptors in vivo.
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Effect of Pꢀgp on Brain Uptake of [18F]10. Pꢀglycoprotein (Pꢀgp) is highly expressed on the
bloodꢀbrain barrier and can block entry of compounds into the brain. To test whether [18F]10 is a
substrate for Pꢀgp in vivo, biodistribution of [18F]10 was investigated in ICR mice pretreated with
50 mg/kg of the Pꢀgp inhibitor cyclosporine A. Slight increases in radiotracer uptake were observed
in both the brain and blood (Figure 4). However, there were no significant differences in
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brainꢀtoꢀblood ratios between the control and treated groups (p > 0.05), suggesting that [18F]10 is
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unlikely a substrate for Pꢀgp.
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Ex Vivo Autoradiography in Rats. To investigate the distribution of [18F]10 in different brain
regions, an ex vivo autoradiography study was performed in SpragueꢀDawley rats (n = 2). Animals
were injected with the radiotracer (0.4 mL, 24.9 MBq) and sacrificed at 30 min after injection.
Brains were excised, frozen, and sliced with a cryostat, and slices exposed to phosphor films.
Results are presented in Figure 5. Similar to what we found before, high accumulation of the
radiotracer was observed in the cortex, particularly in the temporal cortex auditory area. Distinctly
high accumulation of [18F]10 was also observed in other brain areas known to possess high levels of
σ1 receptors, including the midbrain, red nucleus, vermian lobe, paramedian lobe and hippocampus.
Moderate accumulation was found in the striatum, and low accumulation of the radiotracer was
observed in the nucleus accumbens and olfactory bulb. It is notable that high accumulation was
concentrated in the pineal gland, which is a small endocrine gland in the vertebrate brain, indicating
that σ1 receptors may play a role in the endocrine system as previously reported.40
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PET/MRI Imaging in Rats. To further evaluate the kinetics and confirm the specific binding of
[18F]10 in the brain, PET/MRI studies were carried out in anaesthetized Sprague−Dawley rats. In
vivo binding specificity was evaluated with administration of SA4503 (5 µmol/kg) at 5 min prior to
radiotracer injection. Dynamic brain PET scanning was started immediately after injection of
[18F]10 and lasted for 90 min. The timeꢀactivity curves (TACs) in Figure 6 represented the
radioactivity in the whole rat brain as a function of time for both the baseline and blocking studies.
Depicted in Figure 7 are coronal PET/MRI images summed from 15−30 min, 30−45 min, 45−60
min and 60−75 min after intravenous injection of [18F]10. The baseline TAC demonstrated that
[18F]10 entered the brain rapidly, reached peak concentration within 2 min, and then washed out
steadily over time, consistent with the results from biodistribution studies. Pretreatment with
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SA4503 led to a decreased brain uptake and much faster washout within 10 min. Significant
reduction (> 70%) of radiotracer accumulation in the brain was observed after 15 min, indicating
high specific binding of [18F]10 to σ1 receptors in rat brain in vivo.
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Ex Vivo Autoradiography in Senescenceꢀaccelerated Prone Mice. The senescenceꢀaccelerated
prone mouse (SAMP8) has been proposed as a naturally derived animal model for AD.41 It was
reported that an increase in hyperphosphorylated tau in SAMP8 occurred at 5 months of age and an
abnormal βꢀamyloid (Aβ) was deposited in the hippocampus at 6 months of age.42, 43 To investigate
the potential use of σ1 receptor radiotracer for early diagnosis of Alzheimer’s disease, an ex vivo
autoradiography in SAMP8 and its corresponding senescenceꢀacceleratedꢀresistant mouse (SAMR1)
was performed. Similar to the results of ex vivo autoradiography in rats, high accumulation of
[18F]10 was found in brain areas known to have high expression of σ1 receptors, such as cortex,
hippocampus, thalamus and cerebellum of the SAMR1 mice (3 months of age). However, reduced
accumulation of the radiotracer was observed in the above regions of the SAMP8 mice at the same
age (Figure 8(A)). Moreover, there was a significant difference (p < 0.001) between the digital light
unit density (DLUD) in the cortex, striatum, thalamus, hippocampus and cerebellum of SAMP8
mice and SAMR1 mice, but no significant difference in DLUD (p > 0.05) was found in the
olfactory bulb (Figure 9). Finally, we used a highly sensitive nearꢀinfrared fluorescent probe,
(E)ꢀ2ꢀ(3ꢀ(6ꢀ(dimethylamino)naphthalenꢀ2ꢀyl)allylidene)ꢀmalononitrile, reported previously for the
detection of Aβ plaques.44 As shown in Figure 8(B), no deposit of Aβ plaques was detected in either
the SAMP8 or SAMR1 mice.
DISCUSSION
The σ1 receptor has been proposed as a pluripotent modulator implicated in a number of CNS
diseases.5 Molecular imaging with positron emission tomography (PET) is a powerful tool to
investigate the pathophysiology of CNS diseases. An ideal radiotracer for imaging σ1 receptors in
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the brain is required to possess characteristics such as nanomolar affinity for σ1 receptors and high
selectivity over other receptors, transporters and ion channels, high brain uptake, high
brainꢀtoꢀblood ratios, high stability in the brain, high specific binding and suitable tissue kinetic
profiles. Moderate lipophilicity (log D = 1–3) is desirable for adequate BBB penetration of the
imaging agents, as high lipophilicity is believed to increase nonꢀspecific binding in vivo.45 However,
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a
recent
σ1
receptor
radioligand
(8ꢀ(4ꢀ(2ꢀ[18F]fluoroethoxy)benzyl)ꢀ1,4ꢀdioxaꢀ8ꢀ
azaspiro[4.5]decane) with low lipophilicity (log D7.4 = 0.81) was found to cross the BBB rapidly
with high initial accumulation in the brain (SUV 2.89 in rats).31 To further explore the appropriate
lipophilicity required for brain radiotracer, we aim to develop a novel radiotracer with low
lipophilicity combined with high in vivo stability and high specific binding signal for imaging σ1
receptors in the brain. We chose compound 8 as a lead compound and designed a new series of σ1
receptor ligands by elimination of the benzene ring in the 2,3ꢀdihydrobenzofuran moiety.
In vitro binding assays showed that compounds 9–11 with the 1ꢀ((tetrahydrofuranꢀ2ꢀ
yl)methyl)piperazine moiety still maintained high affinity and subtype selectivity for σ1 receptors.
Moreover, compound 10 displayed >2000ꢀfold lower affinity for VAChT and negligible affinity for
adenosine A2A, adrenergic α2, cannabinoid CB1, dopamine D1 and D2L, γꢀaminobutyric acid A
(GABAA), NMDA, melatonin MT1 and MT2, and serotonin 5ꢀHT1 receptors. Therefore, it appears
that the smaller tetrahydrofuran moiety with less lipophilicity can also serve as a “primary
hydrophobic region” on the substituted piperazines, confirming again that the “primary
hydrophobic region” in Glennon’s pharmacophore model is more flexible than reported
previously.31
Consistent with our expectation, the corresponding radiotracer [18F]10 (log D7.4 = 0.76) exhibited
lower lipophilicity than [18F]2 (log P7.5 = 2.93),46,47 123I]3 (log P7.5 = 3.36),47 [18F]5 (log D7.2
[ =
1.90)23 and [11C]1 (log P7.4 = 2.52).48 Moreover, [18F]10 displayed high in vitro stability in saline
and mouse serum at room temperature.
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For a brain radiotracer, the appearance of radiometabolites in the brain, i.e. the generation of
brainꢀpenetrating radiometabolites by peripheral metabolism will confound the binding signals in
the brain. To test the suitability of [18F]10 as a radiotracer for imaging σ1 receptors in the brain, the
in vivo metabolic profile of [18F]10 was investigated. At 60 min after injection, the parent tracer
[18F]10 accounted for ~95% of the total radioactivity (n = 2) in the mouse brain, indicating that
[18F]10 was the predominant radioactive species and no entry of radioactive metabolites into the
brain.
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In biodistribution studies in mice, [18F]10 exhibited higher brain uptake (12.8, 11.2 and 10.6 %
ID/g at 2, 15 and 30 min, respectively) than [11C]1 (3.61% ID/g at 5 min),49 [18F]4 (4.85% ID/g at
25 min),22 [18F]5 (4.71% ID/g at 30 min),23 and [18F]6 (8.39% ID/g at 2 min).27 It then gradually
washed out and decreased to 42% of its maximum activity levels at 120 min after injection. At the
same time, [18F]10 exhibited very low blood accumulation after 15 min, resulting in higher
brainꢀtoꢀblood ratios (22, 21, 20 and 14, respectively, at 15, 30, 60 and 120 min after tracer
injection) than [11C]1 (11 at 30 min),49 [18F]6 (5 at 15 min) 27 and [18F]7 (13 at 30 min).26
To confirm the binding specificity of [18F]10 to σ1 receptors in vivo, pretreatment study was
performed using the selective σ1 receptor agonist SA4503 as a blocking agent. Pretreatment with
SA4503 led to significant reduction of radioactivity in the brain (79%, 88% and 86% at 15, 30 and
60 min), respectively, indicating higher specific binding of [18F]10 than that of [11C]1 (30‒40%
reduction at 30 min with 2 µmol/kg of SA4503 or haloperidol),49 [18F]5 (62% reduction at 60 min
with 1 mg/kg of haloperidol),23 [18F]6 (74% reduction at 30 min with 5 ꢂmol/kg of SA4503),27
[18F]7 (58% reduction at 60 min with 1 mg/kg of haloperidol).26 Significant reductions of
radioactivity uptake were also observed in other organs known to contain σ1 receptors, including the
heart, lungs and spleen.
Besides lipophilicity, Pꢀgp expression at the BBB also influences the brain uptake of radiotracers.
The lipophilicity of [18F]10 (log D7.4 = 0.76) is not within the proposed optimal range for adequate
bloodꢀbrain penetration. However, the brain uptake in mice is extremely high. To investigate the
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relative importance of lipophilicity and interaction with Pꢀgp of the radiotracer on its brain uptake,
effect of the Pꢀgp inhibitor cyclosporine A on the biodistribution of [18F]10 was investigated in ICR
mice. For [18F]10, pretreatment of the animals with cyclosporine A (50 mg/kg body weight, given at
60 min before tracer injection) slightly increased the accumulation of [18F]10 in both the blood and
brain at 2 min after tracer injection. However, the difference in brainꢀtoꢀblood ratio between the
control and treated groups is not significant (p > 0.05), suggesting that [18F]10 is not a substrate for
Pꢀgp. These results are in good agreement with the higher brain uptake of [18F]10 even though its
log D value is less than 1.
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Distribution of [18F]10 in regions of brain was further investigated in ex vivo autoradiography
study in Sprague−Dawley rats. High accumulation was found in the cortex, cerebellum, and
thalamus, all of which are known to have high levels of σ1 receptors, a result in good agreement
with those of other σ1 receptor radiotracers reported in the literature.26,27
To further confirm the time course and specific binding of [18F]10 in vivo and test its potential as
σ1 receptor radiotracer in the living system, PET/MRI studies were carried out in anaesthetized
SpragueꢀDawley rats. [18F]10 crossed the BBB rapidly, reached peak concentration in the brain
within 2 min, then washed out steadily with time. Pretreatment with SA4503 resulted in a slight
decrease in initial brain uptake, and much faster washout rate with 77% reduction of radioactivity at
30 min, indicating high specific binding of [18F]10 to σ1 receptors in the brain.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of
senile dementia. In postmortem study reduction of σ1 receptors has been observed in the
hippocampus of patients with AD.50 Similarly, a recent PET study with [11C]1 found lower levels of
σ1 receptors in early AD patients compared to ageꢀmatched controls.51 Moreover, increasing
evidence demonstrated that σ1 receptor agonists exhibited neuroprotective effect.52 Donepezil, an
acetylcholinesterase (AChE) inhibitor approved for AD treatment,53 was found to bind to σ1
receptors in the living human brain in a doseꢀdependent manner.18 To investigate the involvement
of σ1 receptor in AD, we used SAMP8 mice, which displayed ageꢀrelated cognitive decline closely
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matched to that in humans,41 as a model of AD and investigated the changes in σ1 receptors. As an
increase in hyperphosphorylated tau was reported to occur at 5 months and abnormal Aβ deposition
in the hippocampus at 6 months of age in SAMP8 animals,42, 43 we used SAMP8 mice at 3 months
of age and the control SAMR1 animals in ex vivo brain autoradiography study with [18F]10 to
investigate whether σ1 receptors are changed in the preꢀAD stage and thus might be a useful
biomarker for the early diagnosis of AD. Significant reduction in [18F]10 uptake was found in the
cortex, striatum, hippocampus and cerebellum of SAMP8 compared to SAMR1 mice. Moreover,
absence of Aβ plaques in both SAMP8 and SAMR1 mice was confirmed using a highly sensitive
nearꢀinfrared fluorescent probe for the detection of Aβ plaques.44 This finding suggests a possible
molecular link between σ1 receptors and preclinical AD before Aβ deposition and thus imaging of
σ1 receptors might be an alternative biomarker for early AD diagnosis. The role of σ1 receptor in
AD progression warrants further investigation.
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CONCLUSION
The novel ligand 1ꢀ(4ꢀfluorobenzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine (10) with a small
tetrahydrofuran moiety as “primary hydrophobic region” possesses nanomolar affinity for σ1
receptors, good subtype selectivity, and high selectivity over VAChT and other receptors, including
adenosine A2A, adrenergic α2, cannabinoid CB1, dopamine D1 and D2L, GABAA, NMDA, melatonin
MT1 and MT2, and serotonin 5ꢀHT1 receptors. The corresponding radiotracer [18F]10 exhibits
favorable characteristics in vivo, including high stability in the brain, high brain uptake, high
brainꢀtoꢀblood ratios and specific binding to σ1 receptors. Thus the new radioligand [18F]10 is a
suitable probe for imaging σ1 receptors in the brain. In addition, when compared to normal mice,
uptake of [18F]10 was found to be significantly reduced in the cortex, striatum, hippocampus and
cerebellum of an AD mouse model before emergence of Aβ deposition, indicating that imaging of
σ1 receptors might be a potential method for early diagnosis of AD in its preclinical/prodromal
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stage. Further investigation is warranted to understand the role of σ1 receptor in AD progression and
its use as a biomarker for AD neurodegeneration.
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EXPERIMENTAL SECTION
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General Information. All reagents and solvents were purchased from commercial resources and
used without further purification. [18F]Fluoride was produced using 18O(p,n)18F reaction with a
onꢀsite cyclotron at the Department of Nuclear Medicine, Xuanwu Hospital of Capital Medical
University. SA4503 was synthesized according to the method in the literature.26 Reactions were
monitored by thin layer chromatography (TLC) (TLC silica gel 60 F254 plates, E. Merck). Flash
column chromatography was conducted on silica gel (45–75 µm, Qingdao Haiyang Chemical Co.,
Ltd). 1H NMR spectra and 13C NMR spectra were recorded on a Bruker Avance Ⅲ spectrometer
1
(400 MHz for H, 100 MHz for 13C). Chemical shift (δ) are reported in ppm downfield from
tetramethylsilane and coupling constants (J) are reported in Hertz (Hz). Mass spectrometry (MS)
spectra were obtained with the Quattro micro API ESI/MS instrument (Waters, USA).
Highꢀresolution mass spectrometry (HRMS) was performed on a LCT Premier XE ESIꢀTOF mass
spectrometer (Waters, USA). The purity of compounds 9–12 was analyzed by high performance
liquid chromatography (HPLC). All final compounds were tested with a purity of > 95%.
HPLC purification of the radiotracer was performed on a Shimadzu SCLꢀ20 AVP system
(SHIMADZU Corporation, Japan) equipped with a SPDꢀM20A UVꢀVIS detector operating at a
wavelength of 254 nm, a Bioscan Flow Count 3200 NaI/PMT γꢀradiation scintillation detector, and
an Agela Venusil MP C18 column (250 × 10 mm, 5 ꢂm) eluting with 15% CH3CN and 85% water
containing 50 mM NaH2PO4 at a flow rate of 4 mL/min. HPLC analyses were performed on a
Waters 600 system (Waters, corporation, USA) equipped with Waters 2489 UVꢀVIS detector, and a
Raytest Gabi NaI(Tl) scintillation detector (Raytest, Germany). Purity of the target compounds and
radiotracer was determined by HPLC analysis on an Agela Venusil MP C18 column (250 × 4.6 mm,
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5 ꢂm) using 25% CH3CN and 75% water containing 50 mM NaH2PO4 as mobile phase at a flow
rate of 1 mL/min.
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9
Male ICR mice (4–5 weeks, 23 ± 2 g), Spraque−Dawley rats (6−7 weeks, 220 ± 10 g), SAMR1
mice (3 months, 30 ± 2 g) and SAMP8 mice (3 months, 30 ± 2 g) were purchased from Vital
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River Laboratory Animal Technology Co. Ltd. Procedures related to animal experiments were
performed in compliance with relevant laws and institutional guidelines. All of the animal
experiments were approved by the Institutional Animal Care and Use Committee of Beijing Normal
University.
Chemistry.
1ꢀ(4ꢀ(2ꢀFluoroethoxy)benzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine (9). To a solution of
1ꢀ(bromomethyl)ꢀ4ꢀ(2ꢀfluoroethoxy)benzene (260 mg, 1.12 mmol) in CH3CN (10 mL) were added
compound 13 (330 mg, 1.34 mmol), K2CO3 (231 mg, 1.67 mmol), and NaI in catalytic amount. The
mixture was stirred at 80 °C overnight. After filtration, the solvent was removed under reduced
pressure and the residue purified by column chromatography (silica gel, methanol/dichloromethane
= 1:20, v/v) to afford 9 as yellow oil (300 mg, 83%). 1H NMR (400 MHz, CDCl3): δ 7.17 (d, J = 8.5
Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 4.67 (dt, J = 47.5, 4.1 Hz, 2H), 4.11 (dt, J = 28.0, 4.1 Hz, 2H),
4.04–3.95 (m, 1H), 3.83–3.74 (m, 1H), 3.70–3.61 (m, 1H), 3.43 (s, 2H), 2.73–2.27 (m, 10H),
1.98–1.85 (m, 1H), 1.83–1.72 (m, 2H), 1.49–1.33 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 157.66,
130.60, 129.73, 114.33, 81.88 (d, JCꢀF = 169.59 Hz), 76.19, 68.11, 67.09 (d, JCꢀF = 20.35 Hz), 62.91,
61.96, 53.25, 52.20, 30.29, 25.26. MS (ESI+): m/z 322.90 [M+H]+. HRMS (EI)m/z: calcd for
C18H28FN2O2 [M+H]+ 323.2129, found 323.2127.
1ꢀ(4ꢀFluorobenzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine (10). The procedure described
for the synthesis of 9 was applied to 4ꢀfluorobenzyl bromide (208 mg, 1.10 mmol), compound 13
(330 mg, 1.34 mmol), K2CO3 (190 mg, 1.37 mmol), and NaI in catalytic amount to afford 10 as
lightꢀyellow oil (203 mg, 66%). 1H NMR (400 MHz, CDCl3): δ 7.26 (dd, J = 8.5, 5.6 Hz, 2H), 6.97
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(dd, J = 8.7, 8.7 Hz, 2H), 4.07–3.97 (m, 1H), 3.90–3.81 (m, 1H), 3.79–3.68 (m, 1H), 3.45 (s, 2H),
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2.67–2.37 (m, 10H), 2.03–1.91 (m, 1H), 1.89–1.77 (m, 2H), 1.52–1.41 (m, 1H). 13C NMR (100
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MHz, CDCl3) δ 161.94 (d, JCꢀF = 243.18 Hz), 133.79, 130.60 (d, JCꢀF = 7.82 Hz), 114.90 (d, JCꢀF
=
8
9
21.07 Hz), 77.17, 68.08, 63.31, 62.16, 53.70, 52.79, 30.38, 25.36. 19F NMR (376 MHz, CDCl3): δ
–116.50. MS (ESI+): m/z 278.81 [M+H]+. HRMS (EI)m/z: calcd for C16H24FN2O [M+H]+ 279.1873,
found 279.1864.
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1ꢀ(4ꢀIodobenzyl)ꢀ4ꢀ((tetrahydrofuranꢀ2ꢀyl)methyl)piperazine (11). The procedure described for
the synthesis of 9 was applied to 4ꢀiodobenzyl bromide (234 mg, 0.78 mmol), compound 13 (111
mg, 0.65 mmol), K2CO3 (107 mg, 0.65 mmol), and NaI in catalytic amount to afford 11 as a white
solid (159 mg, 63%). 1H NMR (400 MHz, CDCl3): δ 7.55 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.2 Hz,
2H), 4.07–3.97 (m, 1H), 3.84–3.77 (m, 1H), 3.73–3.63 (m, 1H), 3.39 (s, 2H), 2.70–2.38 (m, 10H),
2.00–1.89 (m, 1H), 1.87–1.73 (m, 2H), 1.48–1.34 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 137.55,
137.23, 131.08, 92.45, 76.11, 68.15, 62.91, 62.09, 53.45, 52.32, 30.23, 25.27. MS (ESI+): m/z
386.88 [M+H]+. HRMS (EI)m/z: calcd for C16H24IN2O [M+H]+ 387.0927, found 387.0929.
(4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl)(tetrahydrofuranꢀ2ꢀyl)methanone (12). The procedure
described for the synthesis of 9 was applied to 4ꢀfluorobenzyl bromide (208 mg, 1.10 mmol),
compound 14 (227 mg, 1.23 mmol), K2CO3 (190 mg, 1.38 mmol) and NaI in catalytic amount to
1
afford 12 as yellow oil (203 mg, 63%). H NMR (400 MHz, CDCl3): δ 7.26–7.18 (m, 2H),
7.01–6.91 (m, 2H), 4.57–4.49 (m, 1H), 3.96–3.86 (m, 1H), 3.82–3.76 (m, 1H), 3.68–3.57 (m, 2H),
3.55–3.45 (m, 2H), 3.43 (s, 2H), 2.43–2.32 (m, 4H), 2.27–2.18 (m, 1H), 2.06–1.79 (m, 3H). 13C
NMR (100 MHz, CDCl3): δ 169.75, 162.05 (d, JCꢀF = 243.92 Hz), 133.19, 130.54 (d, JCꢀF = 7.97
19
Hz), 115.10 (d, JCꢀF = 21.11 Hz), 75.77, 69.02, 61.99, 53.06, 52.63, 45.27, 41.90, 28.42, 25.66. F
NMR (376 MHz, CDCl3) δ –116.10. MS (ESI+): m/z 292.93 [M+H]+. HRMS (EI)m/z: calcd for
C16H22N2O2F2 [M+H]+ 293.1665, found 293.1664.
In Vitro Radioligand Competition Studies
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σ Receptor Binding Assays. All the procedures for the radioligand competition studies were
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previously described.34 Detailed procedures are shown in the Supporting Information.
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VAChT Binding Assays. The determination of the affinity for VAChT was conducted by the
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method in the literatures.35 Detailed procedures are shown in the Supporting Information.
Adenosine A2A, Adrenergic α2, Cannabinoid CB1, Dopamine D1 and D2L, γꢀAminobutyric
acid A (GABAA), NMDA, Melatonin MT1 and MT2, and Serotonin 5ꢀHT1 Receptors Binding
Assays. The affinities of compound 10 for the above receptors are presented in a percentage of
inhibition form under certain concentration. Detailed procedures are shown in the Supporting
Information.
Radiochemistry. [18F]Fluoride was produced via the 18O(p,n)18F reaction with 11 MeV protons in
RDS111 cyclotron (CTI, Germany). [18F]Fluoride collected on a QMA cartridge (Waters) was
eluted off with 1 mL of Kryptofix 222/K2CO3 solution (13 mg of Kryptofix 222 and 1.1 mg of
K2CO3 in CH3CN/H2O, 4: 1 (v/v)) into a clean glass vial. The solvent was removed at 110 oC under
a stream of nitrogen gas. The residue was azeotropically dried with anhydrous acetonitrile added in
three portions (~0.7 mL each). A solution of 4ꢀ(N, N, Nꢀtrimethylamino)benzaldehyde iodide (5.0
o
mg) in 0.5 mL of dry DMSO was added to the reaction vessel. The mixture was heated at 140 C
for 2.5 min, diluted with 10.0 mL of water and the solution passed through a SepꢀPak C18 cartridge
(Waters). The intermediate [18F]ꢀ4ꢀfluorobenzaldehye was eluted off the cartridge with 1 mL of
DMSO. To the solution were then added compound 13 (5.0 mg), CH3COOH (40 ꢂL) and
NaBH3CN (3.0 mg). The mixture was heated at 40 oC for 5 min with gentle shaking. After cooled to
room temperature, the crude product was loaded onto a semipreparative HPLC column for
purification (Agela Venusil MP C18 column, 250 × 10 mm, 5 ꢂm; mobile phase: 15% CH3CN and
85% water containing 50 mM NaH2PO4; flow rate: 4 mL/min). The product peak was collected,
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diluted with water (10 mL) and passed through a second SepꢀPak C18 cartridge. After washing with
water, the product was eluted off with 1 mL of ethanol and dried under a nitrogen stream. For the in
vivo experiments, the product was formulated in sterile saline containing no more than 7% ethanol.
To identify the radioactive product, [18F]10 and the corresponding unlabeled compound 10 were
coꢀinjected and coꢀeluted for analysis (Waters 600 system, Agela Venusil MP C18 column, 250 ×
4.6 mm, 5 ꢂm; mobile phase: 25% CH3CN and 75% water containing 50 mM NaH2PO4, flow rate:
1 mL/min). HPLC chromatograms are presented in the Supporting Information (Figure S1). The
retention times of [18F]10 and 10 were 8.71 and 8.61 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 detectors of our HPLC system.
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Evaluation of the Radiolabeled Compound.
Lipophilicity. The log D value of [18F]10 was determined by measuring the distribution of the
radiotracer between 1ꢀoctanol and 0.05 mol/L potassium phosphate buffer at pH 7.4 according to
literature procedures.26 Detailed procedures are provided in the Supporting Information.
In Vitro Stability. The in vitro stability of radiotracer was evaluated in saline or freshly prepared
mouse serum at 37 °C for 2 h according to literature procedures.26 Detailed procedures are provided
in the Supporting Information.
In Vivo Metabolic Stability. The in vivo metabolism of [18F]10 (18.5–37.7 MBq, 0.15 mL) was
studied in male ICR mice according to the method previously reported.26, 37 Detailed procedures are
shown in the Supporting Information.
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Biodistribution Studies in ICR mice. Biodistribution and blocking studies of HPLCꢀpurified
[18F]10 (185 kBq, 0.1 mL) were carried out based on the method reported previously.26 Detailed
procedures are shown in the Supporting Information.
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Effect of Pꢀgp on the Brain Uptake of [18F]10 in Mice. To investigation of the effect of Pꢀgp on
the brain uptake of [18F]10, mice were injected via the tail vein with either saline (0.1 mL) or
cyclosporine A (0.1 mL, 50.0 mg/kg) 60 min prior to [18F]10 injection (370 kBq, 0.1 mL saline)
according to the method in the literature.37 Detailed information is provided in the Supporting
Information.
Ex Vivo Autoradiography in Rats. A solution of [18F]10 (0.4 mL, 24.9 MBq) was intravenously
injected into conscious Spraque−Dawley rats (6−7 weeks, 220 ± 10 g, n = 2). Ex vivo
autoradiography was then performed following literature methods.26 Detailed information is
provided in the Supporting Information.
PET/MRI Imaging in Rats. Rats were anesthetized by intraperitoneal administration of chloral
hydrate (10% aqueous solution, 1 mL) and injected with [18F]10 (22.1‒23.7 MBq, 0.6‒0.7 mL) via
tail vein. Blocking studies involved preinjection with SA4503 (0.1 mL, 5 µmol/ kg) 5 min prior to
tracer administration. PET/MRI imaging was performed on a hybrid TOFꢀPET/MR (Signa, GE
Healthcare, USA) equipped with a computerꢀcontrolled bed and acquisition of dynamic PET data
(10×6, 3×60, 3×120, and 16×300 s) was started immediately after radiotracer injection.
T2ꢀweighted coronal MR images were acquired using a fast spinꢀecho sequence (TR/TE = 5150/72
ms). The field of view (FOV) was 6 × 4.8 mm2 and the matrix size was 192 × 192 with slice
thickness = 1 mm and slice gap = 2.2 mm. Region of interest (ROI) analysis was performed by
spatial transformation of a predetermined ROI template to the imaging data. The ROI template was
generated by drawing the whole brain on a single representative brain template based on
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T2ꢀweighted MR images. Registration parameters were obtained to apply ROIs to individual PET
scans, and time–activity curves were generated.
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Ex Vivo Autoradiography in Senescenceꢀaccelerated Prone Mice. Similar to the ex vivo
autoradiography experiments in the rats, a solution of [18F]10 (0.1 mL, 25.0 MBq) was
intravenously injected into conscious SAMR1 (3 months, 30 ± 2 g, n = 2) and SAMP8 mice (3
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months, 30 ± 2 g, n = 3) to investigate the relationship between σ1 receptor and early stage of AD.
The experiments were performed following the literature proedures.26 Detailed information is
provided in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
Some parts of evaluation of the radiotracer in experimental section, purity of key target
compounds, HPLC coꢀelution profiles of 10 and [18F]10, radiochemical purity of [18F]10 in saline
and mouse serum at 2 h, analytical HPLC chromatograms of radioactive compounds in mouse brain
and liver samples at 15 and 30 min after intravenous injection of [18F]3. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION`
Corresponding Author
*To whom correspondence should be addressed: Phone: +86ꢀ10ꢀ58808891, Fax:
+86ꢀ10ꢀ58808891. Eꢀmail: hmjia@bnu.edu.cn (H Jia), imaginglu@hotmail.com (J Lu)
Author Contributions
#H.Y. and X.F. contributed equally to this work.
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We are grateful to Professor Zhang Xianzhong (Center for Molecular Imaging and Translational
Medicine, School of Public Health, Xiamen University) for his valuable suggestions of in vivo
studies. This work was supported by the National Natural Science Foundation of China (Grant No.
21471019).
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ABBREVIATIONS USED
ID, injected dose; BBB, bloodꢀbrain barrier; CNS, central nervous system; [11C]SA4503,
1ꢀ(3ꢀmethoxyꢀ4ꢀ[11C]methoxyphenethyl)ꢀ4ꢀ(3ꢀphenylpropyl)piperazine;
SAMP8,
senescenceꢀaccelerated prone mouse; SAMR1, senescenceꢀaccelerated mouse resistant; PET,
positron emission tomography; MRI, magnetic resonance imaging; SPECT, single photon emission
computed tomography; PD, Parkinson disease; AD, Alzheimer’s disease; [18F]Fluspidine,
[18F]1’ꢀbenzylꢀ3ꢀ(2ꢀfluoroethyl)ꢀ3Hꢀspiro[[2]benzofuranꢀ1,4’ꢀpiperidine];
6ꢀ(3ꢀ[18F]fluoropropyl)ꢀ3ꢀ(2ꢀ(azepanꢀ1ꢀyl)ethyl)benzo[d]thiazolꢀ2(3H)ꢀone;
[18F]FTCꢀ146,
[18F]FPS,
1ꢀ3ꢀ[18F]fluoropropylꢀ4ꢀ((4ꢀcyanophenoxy)ꢀmethyl)
piperidine;
[
123I]TPCNE,
1ꢀ(transꢀ[123I]iodopropenꢀ2ꢀyl)ꢀ4ꢀ[(4ꢀcyanophenoxy)methyl] piperidine; HRMS, highꢀresolution
VAChT,
mass
spectrometry;
vesicular
acetylcholine
transporter;
[3H]DTG,
[3H]ꢀ1,3ꢀdi(2ꢀtolyl)guanidine; DMSO, dimethylsulfoxide; SA, specific activity; HPLC, high
performance liquid chromatography; RCP, radiochemical purity; Pꢀgp, Pꢀglycoprotein; TAC,
timeꢀactivity curve; DLUD, digital light unit density; TLC, thin layer chromatography; MS, mass
spectrometry; δ, chemical shift; Hz, Hertz; PBS, phosphateꢀbuffered saline.
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