Synthesis and preclinical evaluation of ¹¹C-labeled (6-aryl-1H-pyrrolo[3,2-b]pyridin-1-yl)acetamide for imaging GluN2B subunit of NMDA receptors

Objectives: GluN2B is one of the most studied subunits of N‑methyl‑_D‑aspartate receptors (NMDARs), which are related to various central nervous system disorders and neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Schizophrenia and neuropathic pain [1]. Herein we developed two novel ¹¹C-labeled GluN2B-selected probes and performed their preliminary evaluations in rodents, including PET imaging and in vitro autoradiography.

Methods: The non-radioactive compounds, N2B-1810 (24) and N2B-1903 (32), were synthesized following the modified methods in the literature [2]. By replacing methylamine with H₂NMe·HCl,two ¹¹C-labeling precursors, 24 and 31 were obtained over 3 steps. Radiosynthesis of [¹¹C]N2B-1810 and [¹¹C]N2B-1903 were achieved using [¹¹C]CH₃I with NaOH in DMSO at 80 ^oC for 5 min. Dynamic PET imaging (60 min) were carried out in rats and in vitro autoradiography on the slides of rat brain were also performed to validate the binding specificity.

Results: The non-radioactive compounds, N2B-1810 and N2B-1903, were obtained in ~20% yields over 3 steps. Meanwhile the corresponding precursors 24 and 31 were achieved in total yield of 18% and 16%, respectively. N2B-1810 and N2B-1903 show strong in vitro GluN2B binding potency with K_i = 11 nM and 4.3 nM, respectively [3].¹¹C-methylation provided desired [¹¹C]N2B-1810 and [¹¹C]N2B-1903 in an average of 28% and 32% decay-corrected radiochemical yield (RCY) respectively, relative to starting [¹¹C]CO₂ at the end of synthesis, with high radiochemical purity (>99%) and high molar activity (>74 GBq/μmol). [¹¹C]N2B-1810 exhibited heterogeneous distribution of radioactivity in rat brain sections, in which the uptake was in decreasing order in the hippocampus, striatum, thalamus, cortex and cerebellum, in accordance with the expression pattern of GluN2B subunits in rodents. Unfortunately, neither of ligands displayed heterogeneous distribution in the PET images in the whole brain, and time-activity curves indicated that the uptake of [¹¹C]N2B-1810 and [¹¹C]N2B-1903 in the rat brains were limited to peak SUV of 0.5-0.7 at ~2 min, respectively.

Conclusions: We have successfully synthesized and radiolabeled two potent GluN2B-selective NMDA receptor antagonists, namely [¹¹C]N2B-1810 and [¹¹C]N2B-1903, with [¹¹C]CH₃I. In particular, [¹¹C]N2B-1810 demonstrated moderate-to-high specific binding to GluN2B subtype by in vitro autoradiography studies. However, further medicinal chemistry and ADME optimization are necessary for this chemotype attributed to low binding specificity in vivo. References: [1] Cur Top Med. Chem. 2016, 16, 1830-1842; [2] US2018208595, 2018; [3] ACS Med. Chem. Lett. 2019, 10, 261−266.