Background/Objectives: 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 (7) and its 4-benzyl analog 8, were synthesized and studied to investigate the effects produced by variations in the distances between the pharmacophoric features on the D4R affinity and selectivity. Methods: All the new compounds 9–20 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. Results: 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 12 and 16, the response profiles of both compounds were more robust in antagonist mode, with derivative 16 showing higher antagonist potency than 12 across all three transducers. Interestingly, 12 and 16 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. Conclusions: The promising biological profiles of 12 and 16 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.

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (K_i = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.