Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies,and thus has been validated as a promising therapeutic target for cancer therapy.To date,only few USP28 inhibitors with moderate inhibitory activity have been reported,highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase.In this current study,we reported the synthesis and biological evaluation of new[l,2,3]triazolo[4,5-d]pyrimidine derivatives as potent USP28 inhibitors.Especially,compound 19 potently inhibited USP28 (IC50 =1.10 ± 0.02 μmol/L,Kd =40 nmol/L),showing selectivity over USP7 and LSD1 (IC50 ＞ 100 μmol/L).Compound 19 was cellularly engaged to USP28 in gastric cancer cells.Compound 19 reversibly bound to USP28 and directly affected its protein levels,thus inhibiting the proliferation,cell cycle at S phase,and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines.Docking studies were performed to rationalize the potency of compound 19.Collectively,compound 19 could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers.