Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development SlusarczykMagdalena LopezMonica Huerta BalzariniJan MasonMalcolm JiangWen G. BlagdenSarah ThompsonEmely GhazalyEssam McGuiganChristopher 2014 Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, <b>6f</b>) was shown to be potent <i>in vitro</i>. Importantly, compared with gemcitabine, <b>6f</b> activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, <b>6f</b> showed a significant reduction in tumor volumes <i>in vivo</i> in pancreatic cancer xenografts. The ProTide <b>6f</b> is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents.