Novel Potent and Selective Dopamine D4 Receptor Piperidine Antagonists as Potential Alternatives for the Treatment of Glioblastoma

<b>Background/Objectives</b>: D4R antagonists have recently been suggested as potential therapeutic alternatives to the standard treatments of glioblastoma (GBM). In this study, new piperidine-based ligands, analogs of the potent and selective D4R compounds 77-LH-28-1 (<b>7</b>) and its 4-benzyl analog <b>8</b>, were synthesized and studied to investigate the effects produced by variations in the distances between the pharmacophoric features on the D4R affinity and selectivity. <b>Methods</b>: All the new compounds <b>9-20</b> were evaluated for their radioligand binding affinity at D2-like receptor subtypes and the results were rationalized by docking studies and molecular dynamics (MD) simulations. The functional profiles of the most interesting derivatives were assessed at D4R Go and Gi protein and β-arrestin by BRET assay and their potential anticancer activity was determined in GBM cell lines. <b>Results</b>: Radioligand binding results highlighted that the derivatives bearing a terminal butyl chain showed structure-activity relationships different from those with a benzyl terminal. From functional studies performed on the best derivatives <b>12</b> and <b>16</b>, the response profiles of both compounds were more robust in antagonist mode, with derivative <b>16</b> showing higher antagonist potency than <b>12</b> across all three transducers. Interestingly, <b>12</b> and <b>16</b> dose-dependently decreased the cell viability of GBM cells, inducing cell death and cell cycle arrest, promoting an increase in ROS production, causing mitochondrial dysfunction, and significantly inhibiting colony formation. <b>Conclusions</b>: The promising biological profiles of <b>12</b> and <b>16</b> make them new lead candidates that warrant further investigation to gain a better understanding of the mechanism behind their antitumor activity and better evaluate their potential for GBM treatment.