Stereoisomers of Chiral Methyl-Substituted Symmetric and Asymmetric Aryl Piperazinium Compounds Exhibit Distinct Selectivity for α9 and α7 Nicotinic Acetylcholine Receptors

We have characterized families of phenylpiperazine (PP) compounds, studying their relative activity with α7 and α9* nicotinic acetylcholine receptors (nAChRs) and focusing on the effects of side groups on the phenyl ring (R₁) and the effects of different alkyl groups on the base nitrogen. In this study, we evaluated the impact of methyl substitution on the piperazine ring, which introduced a chiral center, enabling the generation and separation of stereoisomers. Methyl groups were added to either the C2 or C3 positions on the piperazine of the α9α10 agonist/α7 partial agonist PA-EMPP. Additions at the C3 position greatly reduced activity, while additions at the C2 position had selective effects on either α7 or α9/α10 activity. The 2-methyl <i>S</i> and <i>R</i> isomers of PA-EMPP contain a second chiral center at the nitrogen. Notably, replacing the terminal substitution with <i>N</i>,<i>N</i>-dimethyl abolished α9/α910 agonist activity, rendering the compound selective for α7. We also tested 2M isomers of the α9α10 agonist <i>p</i>CN-EMPP and obtained similar enantioselective activity as observed with the PA-EMPP isomers. Compounds were studied for their ability to reduce the ATP-dependent release of IL-1β from monocytes, one aspect of the cholinergic anti-inflammatory activity. Results were consistent with their apparent activation or antagonism of α9* receptors. These findings underscore the critical role of chirality and structural modifications in fine-tuning receptor selectivity, offering valuable insights for the rational design of selective nicotinic therapeutics.