3,4-Dihydropyrimidin-2(1H)-one Hybrids as Phenotypic Anti-Breast Cancer Agents: Synthesis, Colony-Formation Profiling and Computational Target Deconvolution

3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) are privileged heterocyclic scaffolds with broad biological activities. Sixteen DHPM hybrids (4a–p) carrying para- or ortho-aryloxy linkers and diverse terminal amines were prepared through a modular propargylation, Mannich-type, and Biginelli sequence. The compounds were ranked by NIH/3T3 cytotoxicity, MCF-7 antiproliferative activity (SRB assay), and colony formation under continuous exposure at the MCF-7 IC25. Compounds 4a and 4m were the most potent in MCF-7 cells (IC50 = 10.7 and 11.6 µM; preliminary selectivity indices of 9.6 and 9.1), but 4m inhibited colony formation by 68.3% compared with 25.3% for 4a, supporting its prioritization as the most balanced lead. Ligand-based target prediction followed by GNINA screening of seven candidate targets identified TOP2A as the best match to the experimental pIC50 ranking (Pearson r = 0.883, Spearman ρ = 0.893, n = 7), with Eg5/KIF11 as a secondary comparator. Docking, 200 ns molecular dynamics, and MM/PBSA estimates were compatible with this hypothesis but did not constitute experimental target validation. These data identify 4m as a starting point for DHPM-based antiproliferative agents and TOP2A as a hypothesis to be tested experimentally.