Stereochemical optimization of N,2-substituted cycloalkylamines as norepinephrine reuptake inhibitors†

The norepinephrine transporter (NET), encoded by the SLC6A2 gene, is one of three key monoamine neurotransmitter transporters. Inhibition of NET-mediated reuptake of norepinephrine by monoamine reuptake inhibitors has been the main therapeutic strategy to treat disorders such as depression, ADHD and Parkinson's disease. Nevertheless, lack of efficacy as well as risk of adverse effects are still common for these treatments underscoring the necessity to improve drug discovery efforts for this target. In this study, we developed new inhibitors based on 4-((2-(3,4-dichlorophenyl)cyclopentyl)amino)butan-1-ol (8), a potent NET inhibitor, which emerged from earlier virtual screening efforts using a predictive proteochemometric model. Hence, we optimized the N,2-substituted cycloalkylamine scaffold in three regions to design twenty new derivatives. To establish structure–activity relationships for these NET inhibitors, all novel compounds were tested utilizing an impedance-based ‘transporter activity through receptor activation’ assay. Moreover, all stereoisomers of the most potent compound (27) were synthesized and evaluated for their inhibitory potencies. Initial screening indicated that modifications in the cyclopentylamine moiety and phenyl substitutions decreased NET inhibition compared to 8, emphasizing the importance of the five-membered ring, secondary amine and dichloro-substitution pattern in NET binding. Substituting the original butylalcohol at the R² position with a rigid cyclohexanol yielded lead compound 27, with potency similar to reference inhibitor nisoxetine. Pharmacological characterization of all eight stereoisomers of 27 revealed varying inhibitory potencies, favoring a trans-orientation of the N,2-substituted cyclopentyl moiety. Molecular docking highlighted key interactions and the impact of a hydrophilic region in the binding pocket. This study presents a novel set of moderate to highly potent NET inhibitors, elucidating the influence of molecular orientation in the NET binding pocket and offering valuable insights into drug discovery efforts for monoamine transport-related treatments.