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.

Background: Glioblastoma (GBM) is the most aggressive subtype of malignant glioma. Current therapeutic options remain limited, highlighting the need for the development of novel compounds capable of improving the efficacy of standard treatments. This study aimed to evaluate the biological activity of a series of pyrazolone-based hydrazone compounds (TPPs) in vitro GBM cell lines. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylethan-1-one and tested on two human GBM cell lines, T98 and U87. The cytotoxic effects were evaluated via MTT assay. The most active compound was further investigated at IC₅₀ and IC₂₅ concentrations to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨ_m) changes. Additional assays included colony formation, cell cycle analysis, and evaluation of DNA damage and apoptosis markers. Results: TPP25 exhibited the highest activity with IC₅₀ values of 11.01 μM (95% CI: 10.42 to 11.64) and 13.12 μM (95% CI: 10.23 to 16.87) on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization, along with a significant reduction in colony formation. Cell cycle analysis revealed accumulation in the sub-G0 phase, consistent with increased cell death, supported by propidium iodide uptake. Furthermore, the results suggest the involvement of an apoptotic-like mechanism as supported by Annexin V positivity, γ-H2AX upregulation and transient caspase-3 activation. Conclusions: TPP25 demonstrates significant in vitro cytotoxicity, likely driven by a pro-apoptotic mechanism. This profile positions it as a potential lead compound for further preclinical evaluation, supporting its future transition into in vivo GBM models.

Background: Glioblastoma (GBM) is the most aggressive subtype of malignant glioma. Current therapeutic options remain limited, highlighting the need for the development of novel compounds capable of improving the efficacy of standard treatments. This study aimed to evaluate the biological activity of a series of pyrazolone-based hydra-zone compounds (TPP) against GBM. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with the 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylethan-1-one, and tested on two human GBM cell lines, T98 and U87. The cytotoxic effects were evaluated via MTT assay. The most active compound was further investigated at IC₅₀ and IC₇₅ concentra-tions, to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔѰm) changes. Additional assays included colony formation, cell cycle analysis, and evaluation of DNA damage and apoptosis markers. Results: TPP25 exhibited the highest activity with IC₅₀ values of 11 and 13 µM on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization within 24 h, along with a significant reduction in colony formation. Cell cycle analysis revealed sub-G0 accumulation, consistent with increased cell death, confirmed by propidium iodide uptake. TPP25 also increased phosphorylated ɣ-H2A.X and cleaved Caspase-3 expression. Conclusions: TPP25 emerges as a promising candidate for GBM treatment, demonstrating significant cytotoxic activity.