Design, Synthesis, and Characterization of Novel, Subtype-Selective Fluorescent Antagonists Targeting the Nociceptin/Orphanin FQ Opioid Peptide Receptor

The nociceptin/orphanin FQ opioid peptide receptor (NOPr) is a member of the opioid receptor family under investigation for the treatment of depression, Parkinson's disease, addiction, and pain. Opioid analgesics such as morphine act through μ-opioid receptor (MOPr) activation but cause MOPr-driven side effects that include respiratory depression, tolerance, addiction, and constipation. Bivalent NOPr/MOPr agonists have been shown to confer effective analgesia with an improved side effect profile. However, the development of new NOPr-targeting drugs is challenged by a paucity of pharmacological tools to characterize NOPr-ligands and visualize receptor expression. We report the design, synthesis, and pharmacological evaluation of the first high affinity small molecule NOPr-targeting fluorescent ligands, based on the antagonist: (2<i>R</i>)-1-(phenylmethyl)-<i>N</i>-(3-spiro[1<i>H</i>-2-benzofuran-3,4'-piperidine]-1'-ylpropyl)pyrrolidine-2-carboxamide (<b>C24</b>). These ligands display excellent selectivity for the NOPr against MOPr, δ (DOPr), and κ (KOPr) opioid receptors and are effective tracers for competition binding assays to evaluate NOPr-ligand affinity and in live cell imaging to visualize NOPr expression.