NUC1031 is a gemcitabine ProTide prodrug which is currently undergoing phase III. CD13 inhibitor bestatin is utilized as an adjunct therapy in conjunction with chemotherapy for cancer treatment, which has limitations in cytotoxic efficacy. In this study, we designed and synthesized a novel series of bestatin-gemcitabine's ProTide prodrug conjugates aimed at enhancing the antitumor efficacy of NUC1031. The representative compound 5f demonstrates a 10-fold increase in anti-proliferative activity compared to NUC-1031, with an IC