Design, synthesis, thymidylate synthase docking, and biological evaluation of Pyrazolone- and Benzothiazole-functionalized Bis(acetamide) derivatives in Caco-2 and HDF cells.

Abstract

Three phenylene-linked bis(acetamide) compounds were synthesized and evaluated to determine how pyrazolone or benzothiazole terminal groups alter chemical radical-scavenging, cellular metabolic viability, and predicted accommodation within human thymidylate synthase (hTS). Spectroscopic characterization supported the proposed structures, while RP-HPLC indicated purities of 94.80% for R1, 96.72% for R2, and 90.16% for R3. Redocking of co-crystallized dUMP reproduced its experimental pose with an RMSD of 1.3 Å, supporting pose recovery by the docking setup. R2 and R3 produced AutoDock Vina scores of -9.2 and - 9.4 kcal/Mol, respectively, compared with -7.45 kcal/Mol for FdUMP; these values were interpreted as exploratory scoring outputs rather than evidence of hTS inhibition. In the cell-free DPPH assay, R3 showed the strongest response among the synthesized compounds, with an IC₅₀ of 28.95 μg/mL, whereas R1 and R2 gave 46.29 and 47.64 μg/mL, respectively, and ascorbic acid gave 19.79 μg/mL. In Caco-2 cells, R2 and R3 yielded comparable MTT-derived IC₅₀ values of 59.94 and 60.43 μg/mL, both lower than R1 at 148.7 μg/mL. Their corresponding HDF IC₅₀ values were 141.2, 109.5, and 161.7 μg/mL, giving HDF/Caco-2 indices of 2.36, 1.81, and 1.09. 5-fluorouracil showed an index of 3.16. Comparisons between Caco-2 and HDF fitted logIC₅₀ values were significant for R2 and R3 but not R1, and no compound reached the stringent SI threshold of 10. Thus, DPPH reactivity and cellular response followed different trends, and the modest differential sensitivity, limited compound series, and suboptimal purity of R3 support cautious interpretation and further mechanistic validation using adequately purified compound batches.