Synthesis, Characterization and Glyoxalase Inhibitory Activity of 4,6-Diheteroarylpyrimidine-2-amine Derivatives: In Vitro and In Silico Studies

New series of 4,6-diheteroarylpyrimidine-2ـamines (4a-e) and (5a-c) were prepared by cyclization of particular chalcones (2) or (3) with guanidine nitrate in the presence of potassium hydroxide. Chalcones (2) and (3) were prepared by base-catalyzed Claisen-Schmidt condensation of heteroaryl aldehydes (1) with 2-acetyl-5-chlorothiophene and 3-acetyl-2,5-dichlorothiophene, respectively. All the newly synthesized compounds were characterized by spectroscopic and spectrometric techniques such as IR, 1H NMR, 13C NMR and mass spectrometry. This study evaluated in vitro glyoxalase I (GLO-I) inhibitory activity, coupled with molecular docking analysis. Spectroscopic methods confirmed the structures, and structure-activity relationship was established, revealing the importance of chloro substitutions and a furan ring for enhanced inhibition. In vitro glyoxalase inhibitory activity showed that 5b has excellent inhibitory activity against the glyoxalase I enzyme, with an IC50 of 15 µM. Molecular docking using AutoDock 4.2 highlighted key interactions within the active site of the human glyoxalase I enzyme, underscoring the absence of direct Zn interactions in the synthesized compounds compared to the cocrystallized ligand and revealing the need for structural optimization to introduce metal-binding functionalities. This research paves the way for the rational design of more potent glyoxalase I inhibitors, contributing significantly to the field of enzyme inhibition and therapeutic agent development.