Identification and preclinical evaluation of a radiofluorinated benzazepine derivative for imaging the GluN2B subunit of the Ionotropic NMDA receptor

The previously reported carbon-11 labeled GluN2B PET radioligand C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analogue designated (R)-F-OF-Me-NB1. Given the short physical half-life of 20.3 min for carbon-11, (R)-F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. Upon chiral resolution, the respective enantiomers were radiolabeled with carbon-11 and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. Based on the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution and metabolite studies in Wistar rats. To rule out off-target binding to the σ1 receptor, the brain uptake of (R)-F-OF-Me-NB1 in wild-type mice was compared with σ1 receptor knock-out mice. Autoradiographic assessment revealed that both enantiomers of C-PF-Me-NB1 distributed homogenously across all brain regions on rodent brain sections. In contrast, the two enantiomers of C-OF-Me-NB1 exhibited an entirely different behaviour. While (S)-C-OF-Me-NB1 bound virtually to all brain regions with considerable σ1 receptor binding, (R)-C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analogue (R)-C-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13-25% and molar activities ranging from 61-168 GBq/µmol. PET imaging and biodistribution studies in Wistar rats indicated appropriate pharmacokinetic profile and high in vivo specific binding of (R)-F-OF-Me-NB1 as revealed by blocking studies with GluN2B-antagonist CP101,606. Off-target binding to the σ1 receptor was excluded by PET imaging with σ1 receptor knock-out mice. Receptor occupancy experiments with CP101,606 revealed a D50-value of 8.3 µmol/kg (intravenous). (R)-F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-D-aspartate (NMDA) complex and enables in vivo target occupancy studies in rodents