A multi-computational and crystallographic investigation for the antibiotic mechanism of two 2,4-bis(4-methoxyphenamino)pyrimidines

•The synthesized 2,4-bis(4-methoxyphenamino)pyrimidines exist in two distinct conformers as determined by single-crystal X-ray crystallography.•Hirshfeld surface analysis, docking, MM-GB/SA, MD, and DFT are used to investigate the mechanism of compounds against Salmonella Typhimurium.•They bind at the pore of the Salmonella SseF virulent protein.•The presence of electron-rich phenyl rings and H-bond donor and acceptor atoms enhances the binding of the molecules to the protein through various interactions.•A methyl group in 1b results in a higher molecular volume than 1a for the same conformer, thereby impacting its fit within the SseF pore. 2,4-bis(4-methoxyphenamino)pyrimidines are effective anti-bacterial against various bacteria. The compounds 1a and 1b displayed IC50 values of 3 µM and 6.1 µM, respectively, against Salmonella Typhimurium. The research aims to comprehensively understand the mechanisms of action of these compounds against bacteria using a diverse array of experimental and computational techniques. The findings from the single-crystal X-ray crystallographic investigation reveal several key insights. First, compounds 1a and 1b adopt distinct conformations of the same structure. Second, the amine-pyrimidine resonance is favored over the amine‑methoxy aryl resonance. Third, H···H, O···H, and N···H interactions are the primary driver of molecular assembly. Lastly, Van der Waals and hydrophobic interactions significantly influence the crystal architecture and are conformation-dependent. Computational results suggest that, depending upon the molecular volume, both compounds can inhibit SseF with a marginal difference by stable binding with the same conformer observed in the solid state of 1a through hydrophobic interactions due to the presence of electron-rich phenyl rings. The compound 1b exhibited greater structural variability in the binding site compared to 1a, leading to lower stability. Moreover, the higher molecular volume of 1b, attributed to the methyl group, results in its comparatively lesser fitting in the SseF pore compared to 1a. [Display omitted]