Neuropeptide FF is an endogenous RF-amide with two receptor subtypes originally described as having anti-opioid characteristics. While peptide work helped to elucidate key features for targeting the subtypes of the neuropeptide FF receptor, non-peptide small molecules offer a more refined tool to discover features that affect selectivity and affinity. Improvements in small molecule ligands for neuropeptide FF support lead development and offer a clearer understanding of the binding pocket of each receptor subtype. Previous work on the lead 4–anilindopiperidine structure clarified a key feature between agonist and antagonist behavior. Early modifications of substituents of the piperidine nitrogen were tolerated and created selective ligands. Therefore, syntheses of aliphatic and aromatic substitutions of the piperidine nitrogen of a 4–anilinopiperdine lead molecule were conducted. In vitro evaluation of the novel compounds was conducted through a collaborator. Binding affinities of the novel compounds were determined through displacement of a radioligand ([3H]-NPVF for NPFF1 receptors and [3H]-EYF for NPFF2 receptors) and were conducted for each subtype. Though some of the compounds, such as cycloheptylmethyl substituted analog, did not bind within the limits of the assay to either subtype, other compounds, such as the cyclopropylmethyl and methylindole derivatives, did bind. The cyclopentylmethyl and methylindole derivatives offered a weak preference for the second subtype. The formalin assay offers an opportunity to evaluate neuropeptide FF in a tonic pain model. In summation, selective non-peptide ligands for each neuropeptide FF receptor offer a key tool to elucidating the evolving role of neuropeptide FF and creating potentially useful therapeutics in the future
