Abstract
A new series of acridin-4-yl-based thiazolidinone derivatives was synthesized and structurally characterized using NMR, IR, HRMS, and single-crystal X-ray diffraction. NMR analysis in solution revealed signal multiplicity suggestive of isomeric or conformational heterogeneity. To investigate this behavior, we employed GFN2-xTB, DFT (PBE0-D4 and revDSD-PBEP86-D4), and ab initio molecular dynamics simulations. Theoretical results indicated a preference for nonplanar conformers due to steric hindrance and internal rotations, in agreement with experimental NMR and crystallographic data. Conformational searches and NMR prediction further supported the predominance of E (C1N2) Z (N3C4) Z (C7C8) and Z (C1N2) E (N3C4) Z (C7C8) isomers in solutions. Biological evaluation revealed selective cytotoxicity of compound 4e against HeLa and A549 cell lines (IC(50) = 14.75 and 17.75 μM, respectively). Mechanistic studies in HeLa cells demonstrated dose-dependent apoptosis induction, mitochondrial membrane hyperpolarization, cytochrome c release, S-phase cell cycle arrest, and elevated intracellular ROS. Co-treatment with the antioxidant N-acetylcysteine (NAC) significantly mitigated these effects, suggesting a ROS-mediated mitochondrial apoptotic pathway. This integrated experimental-theoretical study highlights the importance of conformational dynamics in modulating biological activity and provides valuable insights into the structure-activity relationship of acridine-thiazolidinone hybrids. The results support their potential as modular scaffolds for further development of anticancer agents.