Computer-aided design, molecular dynamics simulations and in-vitro inhibitory potential of piperazine bridged bis-thiadiazole benzothioate derivatives against platelet-derived endothelial cell growth factor.

Dysregulated thymidine phosphorylase (TP) activity has been closely associated with pathological angiogenesis, tumor progression and chronic inflammation. This enzyme underscores as attractive therapeutic target for anticancer intervention. In the present study, we describe the rational design and multi-step synthesis of a focused library of piperazine-linked bis-thiadiazole dibenzothioate (1-6) scaffolds. The chemical structures of all synthesized analogues were spectroscopically confirmed by 1H-NMR, 13C-NMR, and high-resolution electrospray ionization mass spectroscopy (HREIMS). All synthesized compounds were evaluated for their in-vitro inhibitory activities against thymidine phosphorylase. Most analogues exhibited moderate to promising enzyme inhibition relative to the reference inhibitor 7-deazaxanthine (7-DX) with IC50 18.32 ± 1.04 µM. However, compound-5 emerged as the most active inhibitor showed an IC50 value of 7.67 ± 1.00 µM and compound-1 demonstrated with an IC50 of 8.13 ± 1.18 µM for thymidine phosphorylase. Moreover, the structure activity relationship (SAR) analysis indicates that enzymatic inhibition is strongly influenced by the nature, size, and positional orientation of aryl substituents to highlight the roles of electronic and steric effects in modulating ligand-enzyme complementarity. Similarly, molecular dynamics simulations also supported the stability of the highest-ranked complexes and reinforced confidence in the predicted binding modes. The most promising analogues also exhibit low binding free energies calculations supported the observed inhibitory trends that further reinforce their thermodynamic favorability of the ligand association. The integrated computational and biological evidence highlights a prioritized lead series with compound-1 at the forefront. These results identify piperazine-linked bis-thiadiazole dibenzothioate derivatives (1-6) as a promising chemical class for thymidine phosphorylase inhibition to offer a viable molecular framework for the development of next generation therapeutics in drug discovery.