Article pubs.acs.org/jmc
1‑(4‑[18F]Fluorobenzyl)-4-[(tetrahydrofuran-2-yl)methyl]piperazine: A Novel Suitable Radioligand with Low Lipophilicity for Imaging σ1 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*,† †
Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China ‡ Helmholtz-Zentrum Dresden-Rossendorf, 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, Connecticut 06520-8048, United States S Supporting Information *
ABSTRACT: We have designed and synthesized novel piperazine compounds with low lipophilicity as σ1 receptor ligands. 1-(4-Fluorobenzyl)-4-[(tetrahydrofuran-2yl)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 A2A, adrenergic α2, cannabinoid CB1, dopamine D1, D2L, γaminobutyric acid A (GABAA), 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.
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INTRODUCTION
such as positron emission tomography (PET) and single photon emission computed tomography (SPECT). In the past several 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,16−18 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 on-site cyclotron because of the short half-life of 11C (t1/2 = 20 min). More recently, [18F]FTC-146 ([18F]4)20−22 has been reported to be a promising PET radiotracer for visualizing σ1 receptors. However, further investigation is still required for its clinical
The σ1 receptor consists of 223 amino acids with a molecular weight of 25 kDa.1 Contrary to the prevailing model 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 “ligandoperated receptor chaperone”4 and interacts with other functional proteins in the plasma membrane, endoplasmic reticulum, mitochondria, and even cytosol.5 A growing body of evidence has 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.5−12 Molecular probes with appropriate affinity, high selectivity, and specificity for σ1 receptors will be useful in the understanding and monitoring of σ1 receptor-related diseases using noninvasive imaging techniques © 2017 American Chemical Society
Received: November 24, 2016 Published: April 14, 2017 4161
DOI: 10.1021/acs.jmedchem.6b01723 J. Med. Chem. 2017, 60, 4161−4172
Journal of Medicinal Chemistry
Article
Figure 1. Structures of σ1 receptor radiotracers investigated in human or non-human primates.
Figure 2. Design concept for target compounds.
Scheme 1. Synthetic Route of Target Compoundsa
a Reagents and reaction conditions: (a) K2CO3, KI, CH3CN, 80 °C, overnight; for 9, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene, 83%; for 10, 4fluorobenzyl bromide, 66%; for 11, 4-iodobenzyl bromide, 63%; for 12, 4-fluorobenzyl bromide, 63%.
The objective of this study was 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 radiotracer with low lipophilicity described above, we eliminated the aromatic ring in the 2,3-dihydrobenzofuran moiety of compound 8 and introduced a 2-fluoroethoxy group or halogen atoms at the para position 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 18 F-labeled 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 studies in rats.
translation. It is interesting that several potential radiotracers are spirocyclic piperidine compounds.23−28 The lead compound, 1′-benzyl-3-methoxy-3H-spiro[2-benzofuran-1,4′-piperidine], possessed nanomolar affinity for σ1 receptors and excellent selectivity over the σ2 receptor and more than 60 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 non-human primates.28 The radiation risk of (S)-[18F]5 imaging proved to be within acceptable limits in the first-in-human study,30 but its performance in humans has not yet been demonstrated. In our previous work, elimination of the aromatic ring in [18F]7 led to a selective σ1 ligand as a tumor imaging agent, 8[4-(2-[ 18 F]fluoroethoxy)benzyl]-1,4-dioxa-8-azaspiro[4.5]decane, with a 2-fold 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 of haloperidol/kg of body weight at 60 min) and thus failed to meet the requirements of a radiotracer for neuroimaging. 4162
DOI: 10.1021/acs.jmedchem.6b01723 J. Med. Chem. 2017, 60, 4161−4172
Journal of Medicinal Chemistry
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Article
RESULTS Chemistry. The synthetic route for compounds 9−12 is shown in Scheme 1. Compound 15 was obtained according to the literature method.33 N-Alkylation of compound 13 or 14 with the corresponding bromide 15−17 provided compounds 9−12 in 63−83% yields. The target compounds (9−12) were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) as having chemical purities of ≥95% as shown in Table S1. 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 and 10 μM dextrallorphan] as reported previously.34 The Ki values for σ1 and σ2 receptors are listed in Table 1. In general, elimination of
Table 2. Binding Affinities of Compound 10 for Other Receptorsa
b
8 9 10 11 12 1c 1d
Ki(σ1) (nM)
Ki(σ2) (nM)
7 (IC50) 15.8 ± 2.20 3.22 ± 0.87 1.21 ± 0.05 198 ± 90.0 4.40 ± 1.00 3.33 ± 0.12
− 652 168 23.4 6856 242 50.7
± ± ± ± ± ±
129 56.0 4.90 276 17.0 1.30
percent inhibition 7 6 1 −5 −1 −4 0 2 3 1
a The percent inhibition was determined at a concentration of 0.1 μM; ≥50% inhibition or stimulation for binding assays is regarded as a significant response.
Scheme 2. Synthesis of Radiotracer [18F]10a
Table 1. Binding Affinities of 1-[(Tetrahydrofuran-2yl)methyl]piperazine Derivatives for σ1 and σ2 Receptorsa compd
receptor adenosine A2A adrenergic α2, nonselective cannabinoid CB1 dopamine D1 dopamine D2L γ-aminobutyric acid A (GABAA) NMDA melatonin MT1 melatonin MT2 serotonin 5-HT1, nonselective
Ki(σ2)/Ki(σ1) − 41 52 19 35 55 15.2
a
a Reagents and reaction conditions: (a) [18F]F−, K2.2.2, K2CO3, DMSO, 140 °C, 2.5 min; (b) 1-[(tetrahydrofuran-2-yl)methyl]piperazine (13), NaBH3CN, DMSO, CH3COOH, 40 °C, 5 min.
the aromatic ring in the 1-[(2,3-dihydrobenzofuran-2-yl)methyl]piperazine moiety preserved preferential binding to σ1 receptors. Introduction of a small group at the para position 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, the affinity of compound 10 for the vesicular acetylcholine transporter (VAChT) was also determined using radioligand competition assays [(−)-[3H]vesamicol].35 Compound 10 demonstrated an 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, γ-aminobutyric acid A (GABAA), NMDA, melatonin MT1 and MT2, and serotonin 5-HT1 receptors as shown in Table 2. Hence, compound 10 was labeled with 18F for further evaluation. Radiochemistry. The synthetic route for [18F]10 is presented in Scheme 2. A two-step procedure was used to prepare the radiotracer, starting from 4-(N,N,Ntrimethylamino)benzaldehyde iodide. In the first step, 4[18F]fluorobenzaldehyde was prepared from the precursor through displacement 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 4-[ 18 F]fluorobenzaldehyde using NaBH3CN as a reducing agent and CH3COOH as a catalyst provided the desired radiotracer. After purification via semi-
preparative high-performance liquid chromatography (HPLC), [18F]10 was obtained in 20−30% radiochemical yield (n = 5; decay-corrected) with a radiochemical purity (RCP) of >99% and a specific activity (SA) of 54−86 GBq/μmol (n = 3). The total synthesis time was ∼80 min. Evaluation of the Radiolabeled Compound. Lipophilicity. The apparent distribution coefficient of the 18Flabeled radiotracer was determined using the shake-flask method as previously reported.26 The log D7.4 value of [18F] 10 was 0.76 ± 0.01. In Vitro Stability. The in vitro stability of [18F]10 in saline and mouse serum was evaluated by measuring the RCP at different time points using HPLC. After incubation with saline or mouse serum for 2 h, the RCP of [18F]10 was still >99% as shown in 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 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 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 the liver with retention times of 70%) in the level of accumulation of the radiotracer in the brain was observed after 15 min, indicating high specific binding of [18F]10 to σ1 receptors in rat brain in vivo. Ex Vivo Autoradiography in Senescence-Accelerated Prone Mice. The senescence-accelerated prone mouse (SAMP8) has been proposed as a naturally derived animal model for AD.41 It was reported that an increase in the level of hyperphosphorylated tau in SAMP8 occurred at 5 months of age, and an abnormal β-amyloid (Aβ) was deposited in the hippocampus at 6 months of age.42,43 To investigate the potential use of the σ1 receptor radiotracer for early diagnosis of Alzheimer’s disease, an ex vivo autoradiography study in SAMP8 and its corresponding senescence-accelerated resistant mouse (SAMR1) was performed. Similar to the results of ex vivo autoradiography in rats, a high level of accumulation of [18F]10 was found in areas of the brain known to have a high level of expression of σ1 receptors, such as cortex, hippocampus, thalamus, and cerebellum of the SAMR1 mice (3 months of age). However, a reduced level of accumulation of the radiotracer was observed in these regions of the SAMP8 mice at the same age (Figure 8A). 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 near-infrared fluorescent probe, (E)-2-{3-[6-(dimethylamino)naphthalen-2-yl]allylidene}malononitrile, reported previously for the detection of Aβ plaques.44 As shown in Figure 8B, no deposit of Aβ plaques was detected in the SAMP8 or SAMR1 mice.
Table 4. Effects of SA4503 on the Biodistribution of [18F]10 in Male ICR Micea organ 15 min
30 min
60 min
blood brain heart liver spleen lung kidney blood brain heart liver spleen lung kidney blood brain heart liver spleen lung kidney
control 0.50 11.2 10.6 6.16 8.61 20.5 13.3 0.50 10.6 6.62 9.96 10.8 15.2 11.9 0.42 8.29 4.43 10.0 8.51 8.61 8.45
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.86 0.72 0.57 1.24 5.35 1.29 0.11 1.31 1.00 1.21 3.90 2.55 2.22 0.09 0.66 0.55 0.84 0.74 2.38 0.94
blocking
blocking (%)
pb
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
269 −79 −78 283 −58 −82 −26 192 −88 −72 146 −76 −84 −25 129 −86 −69 96 −77 −77 −39
