DFT-guided discovery of bisureido ester of 2-(methylthio)ethanol: A novel lead against triple-negative breast cancer inducing apoptosis via pH-sensitive chloride transport

Dysregulation of transmembrane chloride flux has emerged as a promising strategy for inducing cancer-cell death through disruption of intracellular pH gradients and subsequent apoptosis. Herein, we report the design and synthesis of novel ortho-phenylenediamine (o-PDA) bisurea derivatives bearing carboxyl (5a–f) and 2-(methylthio)ethyl ester (6a,b) groups for pH-sensitive anion transport and resultant anticancer activity. Esterification of 5c–f proved synthetically challenging due to an unprecedented CDI-catalyzed intramolecular cyclization that yielded the 4-oxo-benzo[d][1,3,6]oxadiazepines 8a,b, rationalized via a DFT-supported four-step mechanism involving hydrogen-borrowing catalysis. All target compounds demonstrated 1:1 chloride ion-binding as shown by 1H NMR titration results and demonstrated a pH-gradient-induced transmembrane chloride transport (as shown by vesicular transport studies) which was enhanced by electron-withdrawing substituents on the outer phenyl rings. Ester derivatives 6a,b exhibited significantly higher chloride binding and up to ~67-fold increased transport efficiency over their carboxylic acid analogs. Compound 6b emerged as the lead, combining efficient chloride binding (Ka = 163 M⁻¹), potent transport (EC50 = 0.007 mol%), and superior antiproliferative activity (IC50 = 6.34 µM) against MDA-MB-231 cells via apoptosis induction. These findings establish 6b as a promising lead for chloride-transport-mediated cancer therapy, with further optimization recommended via EWG diversification and strategic consideration of the cyclization pathway in retrosynthetic design.