Benzodiazepine-based N,S-heterocyclic hybrids as multi-target therapeutic candidates for cancer and inflammation: enzymatic evaluation, cellular mechanistic studies, in vivo anti-inflammatory assessment, and computational investigations.

A series of eleven novel benzodiazepine-based N, S-heterocyclic hybrids incorporating thienothiazine and thienopyrimidine scaffolds was designed, synthesized, and characterized by spectroscopic and analytical techniques. Compounds 4 and 6 showed favorable selectivity against HCT-116 and MDA-MB-231 cells relative to normal WI-38 fibroblasts. Compound 6 exhibited potent EGFR and VEGFR-2 inhibition (IC50 = 0.071 and 0.181 μM, respectively) and COX-2 inhibition (IC50 = 4.74 μM), while compound 4 showed the highest COX-2 selectivity index (SI = 4.56). Compound 6 induced G2/M arrest and increased total apoptosis from 2.69% to 34.76%, accompanied by 6.251-fold upregulation of Bax and reduction of Bcl-2 expression to 0.327-fold. In vivo, compound 6 attenuated carrageenan-induced paw edema by 39% at 2 mg/kg and reduced serum TNF-α, MAPK, COX-2, and PGE₂ levels by 86%, 34%, 52%, and 69%, respectively. Molecular docking showed binding scores of -9.91 and - 10.15 kcal/mol for compounds 4 and 6, respectively, against EGFR, -9.36 and - 9.52 kcal/mol against VEGFR-2, and - 10.62 and - 10.18 kcal/mol against COX-2. In 100 ns molecular dynamics simulations, compound 6 maintained 2-4 hydrogen bonds in the EGFR complex and 2-4 in the COX-2 complex, while showing an EGFR RMSD of 0.40-0.55 nm and VEGFR-2 RMSD of 0.16-0.23 nm. DFT calculations gave ΔE values of 1.065 and 2.216 eV for compounds 4 and 6, respectively. ADMET predictions indicated low intestinal absorption and no BBB permeation, with compound 6 predicted AMES-positive and hERG II-positive. Computational findings were complementary and exploratory and did not establish a causal relationship between kinase inhibition and antiproliferative activity. Histopathological examination showed dose-dependent improvement in inflammation-associated tissue alterations, with high-dose compound 6 approaching the control architecture. Overall, compound 6 warrants further investigation as a potential anticancer lead with anti-inflammatory activity.