Synthesis of [¹¹C]NR2B-SMe for evaluation as a PET radioligand for NR2B subunits in NMDA receptors

Objectives: NR2B is the most studied NMDA receptor subunit within the NMDA complex, and its expression is largely limited to forebrain regions and dorsal horn of the spinal cord [1]. NR2B is considered to be a therapeutic target for schizophrenia, stroke, and neurodegenerative diseases, especially neuro-pain. Therapeutics targeting NR2B rather than the NMDA channel have fewer side-effects [1]. The quantification of NR2B subunits within NMDA receptors could help to elucidate the contribution of this receptor to neuropsychiatric disorders and also assist in drug develoment [2]. Currently, no PET radioligand is available for such quantification [3]. Here, within a broader medicinal chemistry campaign, we discovered 7-methoxy-3-(4-(4-(methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (NR2B-SMe), which shows affinity for NR2B be in the nM range, moderate computed lipophilicity (clogD = 3.7), and amenability to labelling with carbon-11. We therefore prepared [¹¹C]NR2B-SMe from an S-methyl propionate precursor for evaluation as an NR2B PETradioligand in rat. Methods: The precursor for labelling, namely methyl 3-((4-(4-(1-hydroxy-7-methoxy-1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)butyl)phenyl)thio)propanoate, was synthesized in 4 steps. Treatment of this precursor (0.5 mg, 1.1 μmol) with tetrabutylammonium hydroxide (1 M, 5 μL, 5 μmol) and [¹¹C]MeI in DMF (400 μL) at RT for 5 min gave [¹¹C]NR2B-SMe, which was purified with HPLC on a Waters X-Bridge C18 column (250 × 10 mm) eluted with 0.1% TFA in H₂O/MeCN (65:35 v/v) and then formulated for intravenous injection. PET imaging of brain was performed after intravenous administration of [¹¹C]NR2B-SMe to rats at baseline and preblocked with NR2B-SMe, the NR2B-selective ligands eliprodil or ifenprodil, or the sigma-1 receptor-selective compound, SA4503 (each at 0.01−3 mg/kg, i.v. at 15 min before radioligand injection). Displacement studies where NR2B-SMe or eliprodil was given at 15 min after radioligand were also performed.

Results: The precursor was obtained in 38% overall yield. [¹¹C]NR2B-SMe was obtained in 20% yield from cyclotron-produced [¹¹C]CO₂ and with a radiochemical purity of >98% and a mean molar activity of 58 GBq/μmol. PET imaging of [¹¹C]NR2B-SMe in rats at baseline revealed very high brain radioactivity uptake, reaching 3.5 SUV at 5 min followed by very low washout over 90 min. NR2B-SMe, and SA 4503 pretreatment blocked brain radioactivity uptake in a dose-dependent manner. At high dose of pretreatment agent, a high proportion of brain radioctivity uptake (~80%) was blocked. Eliprodil and ifendopril (3 mg/kg i.v) also blocked brain radioactivity uptake to the similar high extent. Eliprodil failed to displace radioligand, wheras NR2B-SMe gave only slow and low displacecement over 90 min.

Conclusions: [¹¹C]NR2B-SMe was readily synthesized, and showed high brain uptake in rat, which could be pre-blocked by NR2B-SMe itself, elipodil, ifenprodil, and by a selective sigma-1 receptor ligand. Because sigma-1 receptors serve as multitasking ion channel protein chaperones [4] with involvement in modulation of NMDA activity [5], and are now known to interact directly with the N-terminal domains of NR1 and NR2B subunits [6], further study is required to characterize the nature of the specific binding of [¹¹C]NR2B-SMe in rat brain and its utility in brain research. These studies are ongoing. Research Support: Intramural Research Program of the National Insitutes of Health (NIMH). References: [1] Zhuo M, Neuropharmacology, 2017, 112, 228. [2] Kassenbrock A, Vasdev N, Liang SH. Curr. Top. Med. Chem. 2016; 16, 1830. [3] Krämer SD et al.,J. Nucl. Med. doi:10.2967/jnumed.117.200451. [4] Chu UB, Ruoho AE, Mol Phamrmacol. 2016, 89, 143. [5] Zhang XJ et al., Neurosignals 2011, 19, 110. [6] Balasuriya D et al., J. Neurosci 2013, 33, 18219.

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