Synthesis and Anticancer Activity of New Quinazolin-4(3H)-one Derivatives: Identification of a Tumor-Selective Anticancer Agent with Potential Inhibition of TGF-βRI (ALK5)

Background/Objectives: Cancer is a multifactorial disease in which drug resistance and limited selectivity remain major therapeutic challenges, highlighting the need for novel anticancer agents. As a privileged scaffold for multitarget anticancer drug discovery, quinazolin-4(3H)-one was selected for the design, synthesis, and evaluation of new derivatives as potential anticancer agents, together with investigation of their mechanisms of action and molecular targets. Methods: Fifteen new quinazolin-4(3H)-one derivatives were synthesized and screened using the NCI-60 human cancer cell line panel. The mechanism of action of the most active compound was investigated through cell cycle, apoptosis, and RT-qPCR analyses. A potential molecular target was identified from transcriptomic data in the Human Protein Atlas, focusing on highly expressed cancer-implicated genes in the most responsive cell lines, followed by molecular docking, molecular dynamics simulations, and in vitro kinase studies. Safety and pharmacokinetic properties were evaluated using an MTT cytotoxicity assay in normal WI-38 fibroblasts and in silico ADME analyses. Results: Compound 3e emerged as the most active and tumor-selective derivative, exhibiting GI50 values ranging from 2.63 to 17.12 µM across 31 cancer cell lines. In A549 cells, selected as a representative responsive model, 3e (GI50 = 10.8 µM, 72 h) induced G2/M cell-cycle arrest (59.58% vs. 26.96% in control), increased early apoptosis (43.94% vs. 0.11% in control), reduced viable cells (49.71% vs. 98.66%), elevated the Bax/Bcl-2 ratio (7.91), and upregulated the expression of caspase-9 and caspase-3 by 2.5- and 4.6-fold, respectively. Integrated target identification studies and an in vitro kinase assay (IC50 = 21.34 nM) suggested TGF-βRI (ALK5) as a plausible molecular target. Compound 3e also showed low cytotoxicity toward WI-38 fibroblasts (IC50 = 88.3 µM) and favorable predicted pharmacokinetic properties; nevertheless, high plasma protein binding and potential CYP2C9 inhibition are anticipated. Conclusions: Compound 3e is a promising tumor-selective anticancer lead with potential TGF-βRI inhibitory activity. Its antiproliferative effects in A549 cells appear to be mediated through G2/M cell-cycle arrest and activation of the intrinsic apoptotic pathway, supporting further development and pharmacokinetic optimization of this scaffold for anticancer therapy.