Original articleSynthesis, evaluation and metabolic studies of radiotracers containing a 4-(4-[18F]-fluorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B NMDA receptors
Graphical abstract
Highlights
► Fluorinated antagonists for the NR2B subunit of the NMDA receptors were developed. ► Two PET radiotracers containing the 4-(4-[18F]fluorobenzyl)piperidine moiety were synthesized. ► The in vivo evaluation by PET imaging demonstrated radiodefluorination. ► Metabolism of the 4-(4-[18F]fluorobenzyl)piperidine was studied by LC-MS.
Introduction
The N-methyl-d-aspartate (NMDA) receptors are glutamate-gated ion channels that regulate excitatory synaptic transmission in mammals [1], [2]. The channel activation of the NMDA receptors requires the binding of two different agonists, glutamate and glycine [3]. The important permeability to calcium ions confers on NMDA receptors a central role in a number of physiological processes involved in long-term potentiation, memory and cognition [4]. Moreover, over-activation of NMDA receptors has been implicated in several neuropathological and psychiatric disorders including neuropathic pain, epilepsy, neurodegenerative diseases (Alzheimer’s, Parkinson’s and Huntington’s) as well as stroke and brain trauma [5], [6], [7].
The functional NMDA receptor is a complex consisting of two obligatory NR1 subunits and two NR2 subunits. The NR1 subunit is widely expressed within the central nervous system and exists in eight isoforms (NR1a–h) encoded by alternative splicing of a unique gene [1]. NR2 subunits exist in four forms produced from distinct genes (2A, 2B, 2C and 2D) each of which shows a characteristic distribution and confers different pharmacological properties to the receptor [8], [9]. The NR2A subunit is expressed in all brain regions but to a greater degree in the cerebral cortex, hippocampus and cerebellum. NR2B expression is largely limited to forebrain regions, including the hippocampus, cortex and striatum [5]. NR2C subunit is expressed exclusively in the cerebellum whereas the midbrain contains high levels of NR2D subunits. The NR2B subunit containing NMDA receptors are the most studied and therefore the best known subtype. The NR2B receptors have become a therapeutic target for stroke, neurodegenerative diseases, schizophrenia and neuropathic pain [5], [10]. Ifenprodil (Fig. 1) was the first antagonist that showed selectivity for the NR2B NMDA receptor [11]. This property improved the neuroprotective potential, and reduced side-effects, compared to non-selective, first generation NMDA receptor inhibitors [3].
The in vivo quantification of the NR2B subunit containing NMDA receptors by positron emission tomography (PET) could be of great interest to understand the contribution of this complex to neurological disorders or brain pathologies [12], [13], [14]. Several NR2B subtype selective antagonists, such as [11C]EMD-95885, N-[11C]-(2-methoxy)benzyl-3-phenyl-amidines or a [11C]-methylated analog of CP101,606, have been labeled with the short-lived positron emitter, carbon-11 (T1/2: 20.4 min) or fluorine-18 (T1/2: 109.7 min) and their potential to image NR2B containing NMDA receptors in the living brain has been investigated [15], [16], [17]. In spite of promising in vitro binding data for some compounds, an inadequate brain penetration and/or a uniform distribution of tracer throughout brain structures have been reported for all the radiotracers so far developed [18]. Therefore, an effective PET radiotracer to image NR2B containing NMDA receptors is still needed.
Since the discovery of the NMDA related actions of ifenprodil, considerable efforts have been pursued to develop structurally-related compounds with higher affinity and selectivity for NR2B subtype [10], [19], [20], [21], [22]. As the 4-benzylpiperidine moiety was considered as an essential pharmacophore for the NR2B subunit receptor site, novel antagonists containing the 4-benzylpiperidine pattern were investigated including different substitutions on the aromatic ring [21], [22]. Among the substituted benzylpiperidine derivatives described, the 4-(4-fluorobenzyl)piperidine moiety is present in a number of NR2B antagonists, the most known ligands being eliprodil, besonprodil and EMD-95885 (Fig. 1). The fluorine atom on the aromatic ring led us to envisage the radiolabeling of such structures with fluorine-18 to obtain a radiotracer for PET imaging.
From a recent series of N-oxamide ligands (1a–f) (Fig. 2), two compounds (1a, 1c) bearing the 4-(4-fluorobenzyl)piperidine moiety, have been described with high affinity and selectivity for the NR2B subunit (IC50 = 6 ± 1 and 5 ± 1 nM, respectively for 1a and 1c) [23]. The addition of a fluorine atom in the para-position on the benzyl ring did not induce any loss of affinity compared to the non-fluorinated analogs (IC50 = 4 ± 1 and 8 ± 1 nM for 1b and 1d) in binding assay with [3H]Ro-25,6981. The compounds 1a–f (excepted for 1d) presented in vivo analgesic activities in the mouse formalin test with an efficacy dose (ED50) varying from 0.5 to 14 mg/kg (p.o.). The benzoxazolone derivative of this series (1a), namely RGH-896 or radiprodil, is currently in clinical trials [24]. From a second series of antagonists, based on the 4-(substitutedbenzyl)piperidinyl-1-methylbenzimidazol structure [25], the 4-(4-fluorobenzyl)piperidinyl-1-methyl-2-benzimidazol-5-ol (2a) (Fig. 2) showed an excellent affinity (Ki = 1.1 ± 0.1 nM) and selectivity for NR2B subunit containing NMDA receptors vs. human ether-a-go-go (hERG) potassium channel (inhibitory potency, IP = 500 nM) and α-adrenergic receptors (IC50 = 620 nM). In this series, the non-fluorinated analog of 2a presented interesting pharmacokinetic properties as an activity in carrageenan-induced mechanical hyperalgesia in rats with an ED50 of 3.3 mg/kg i.v. and a plasma half-life superior to 8 h. The 4-benzylpiperidine-1-methyl-2-benzimidazol-5-methylsulfamide (3a) demonstrated a high affinity (Ki = 0.99 ± 0.4 nM) and selectivity for NR2B [26].
From these structure–activity relationship (SAR) studies and pharmacological data, we have investigated the preparation of several ligands for the NR2B receptor that belong to these structural classes of compounds and bear the 4-(4-fluorobenzyl)piperidine moiety. The in vitro inhibitory activities were determined and two ligands were selected to be labeled with fluorine-18. The radiosynthesis of [18F]1a and [18F]2a was developed using a two-step labeling method. Their in vivo behavior, including tissue uptake, kinetics and radiometabolism were investigated in rats. A complementary mass spectroscopy study was undertaken to corroborate the in vivo metabolic pathway of the 4-(4-[18F]fluorobenzyl)piperidine moiety ([18F]6) observed with [18F]1a and [18F]2a.
Section snippets
Chemistry
The syntheses of fluorinated ligands 1a, 1g, 1h, 2a and 3b and non-fluorinated analogs 2b and 3a are showed in Scheme 1, Scheme 2. They were prepared by synthetic pathway differing from those described previously for the corresponding series within the objective to get a converging approach with the benzylpiperidines [23], [25]. The oxalamic acids 4a–c were obtained in high yield by addition of ethyl chlorooxoacetate to the corresponding amine [27] followed by basic hydrolysis of the ethyl
Conclusion
Several fluorinated ligands were synthesized and their antagonist effects for the NR2B NMDA receptor were measured by an in vitro functional assay. Two NR2B NMDA antagonists from two different series of compounds and presenting high affinity and selectivity vs. NR2A, [18F]1a and [18F]2a, were labeled by a multistep radiosynthesis. The syntheses of the reference compounds and of the different labeling precursors were optimized with the converging synthesis used by coupling the different
General methods
All reagents were purchased from Fluka or Sigma–Aldrich (Saint-Quentin Fallavier, France) and were used without further purification. Anhydrous THF, DMF and acetonitrile were obtained from a Mbraun SPS-800 solvent delivery system. HPLC quality solvents were purchased from Merck (France) or SDS (Peypin, France). [18F]-fluoride was produced using a Cyclone 18/9 (IBA) cyclotron at the Cyceron PET Center, via the 18O(p,n)18F nuclear reaction. Melting points were determined in open capillary tubes
Acknowledgments
Romain Labas received a bursary from the CEA and the Conseil Régional de Basse-Normandie. Gwénaëlle Gilbert was financed by the Ministère de l’Enseignement Supérieur et de la Recherche. The authors thank Eric T. MacKenzie for the proof-reading of the manuscript.
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These authors have contributed equally to this work.