The metabotropic glutamate receptor 2 (mGluR) has emerged as a potential therapeutic target for the treatment of various neurological diseases, prompting substantial interest in the development of mGluR-targeted drug candidates. As part of our medicinal chemistry program, we synthesized a series of isoindolone derivatives and assessed their potential as mGluR positive allosteric modulators (PAMs). Notably, AZ12559322 exhibited high affinity ( mGluR = 1.31 nM) and an excellent in vitro binding specificity of 89% while demonstrating selectivity over other mGluR subtypes (>4000-fold). Autoradiography with the radiolabeled counterpart, [H]AZ12559322, revealed a heterogeneous accumulation with the highest binding in mGluR-rich brain regions. Radioligand binding was significantly reduced by pretreatment with nonradioactive mGluR PAMs in brains of rats and nonhuman primates. Although positron emission tomography imaging of [C]AZ12559322 () revealed low brain uptake in a nonhuman primate, this study provides valuable guidance to further design novel isoindolone-based mGluR PAMs with improved brain exposure.