Novel Thiazolyl‐2‐( N ‐Sulfonyl)Guanidines as Potential Anticancer Leads Targeting Caspase‐9: Synthesis, Apoptosis Induction, and In Silico Profiling

ABSTRACT A novel series of thiazolyl‐2‐( N ‐sulfonyl)guanidines was synthesized and biologically evaluated as potential anticancer agents. The target guanidine derivatives were synthesized via coupling of 4‐arylamino thiazoles with N ‐sulfonyl carbodiimides in toluene at 80°C using KOH as a base. The cytotoxic potential of the synthesized compounds was assessed against the MDA‐MB‐231 triple‐negative breast cancer (TNBC) cell line. Initial SRB assay screening revealed dose‐dependent growth inhibition, with compounds N ‐(( tert ‐butylamino)((4‐(4‐fluorophenyl)thiazol‐2‐yl)amino)methylene)‐4‐methylbenzenesulfonamide (3g) and N ‐(( tert ‐butylamino)((4‐(2‐chlorophenyl)thiazol‐2‐yl)amino)methylene)‐4‐chlorobenzenesulfonamide (3i) exhibiting the most significant cytotoxic activity. Compound 3i showed strong antiproliferative activity, with a GI 50 value of < 10 µg/mL. Selectivity studies using normal CC1 hepatocytes confirmed that both compounds were non‐toxic up to 100 µM. Flow cytometric analysis demonstrated dose‐dependent apoptosis induction, with compounds 3g and 3i inducing 40% and 27.55% apoptotic cell death, respectively, at 180 µM. Mechanistic investigations revealed upregulation of caspase‐3, caspase‐9, and Bax, indicating activation of the intrinsic apoptotic pathway. Enhanced intracellular reactive oxygen species (ROS) generation further suggested oxidative stress‐mediated apoptosis. In silico ADME, bioactivity, and toxicity analyses supported favorable drug‐like properties, with compound 3i emerging as the most promising lead. Molecular docking and dynamics simulations against caspase‐9 (PDB ID: 1NW9) revealed stronger, more stable binding for 3i than for 3g, corroborating its superior biological performance.