Amino acid tethered benzoxazolone as highly potent inhibitors of O-glycosylation

Novel inhibitors of GlcNAc-Ts have been reported based on benzoxazolone appended amino acids/esters which showed a competitive inhibition of the enzyme with IC50 values in lower micromolar range. The compounds 1b–5b showed a better inhibition profile against GlcNAc-Ts as compared to 1a–5a as evidenced by the in vitro 96 well plate ELISA experiment and the in vitro cytotoxicity studies on the cancer cell lines PDAC, Caco-2, MDA-MB-231, HT-29, PC-3, and A549. The execution of ELISA and cytotoxicity experiments on the compounds 1a–5a in the presence of esterase enzyme along with the appraisal of the lipophilicity index, and calculation of Ki and Ka values for the enzyme-inhibitor complex further indicated that the compounds 1a–5a serve as prodrugs of the compounds 1b–5b owing to a better lipophilicity index of the former. Double reciprocal plots were constructed for 2 different concentrations of the test compounds 1b–5b and that of the natural substrate of OGT. A competitive mode of inhibition was observed for the test molecules which was further confirmed by molecular docking analysis of these molecules in the active site of OGT in the presence of its natural inhibitor. Mechanistic evaluation of the inhibition of OGT by test molecules was performed by NMR and HPLC experiments where hydrolysis of exocyclic urea linkage in the test molecules was observed on their incubation with the enzyme over a period.