Tumor hypoxia has been widely explored over the years as a diagnostic and therapeutic marker. Herein, we synthesized an alkyne functionalized version of evofosfamide, a hypoxia-selective prodrug. The purpose of this effort was to investigate if this novel 2-nitroimidazole phosphoramide nitrogen mustard (2-NIPAM) retained hypoxia selectivity and could be utilized in radiopharmaceutical development to significantly increase retention of conjugated agents in hypoxic cells. 2-NIPAM demonstrated good hypoxia selectivity with a 62- and 225-fold increase in cytotoxicity toward PC-3 and DU145 human prostate cancer cell lines, respectively, under hypoxic conditions. Radiolabeling of 2-NIPAM with 125I was accomplished through a Cu(I)-mediated azide-alkyne cycloaddition reaction. The 125I-conjugate demonstrated 13.6 and 17.8% lower efflux rates for DU145 and PC-3 cells, correspondingly, under hypoxic conditions, suggesting that the increased retention is likely due to the known intracellular trapping mechanism. In conclusion, these studies demonstrate the potential of 2-NIPAM in serving as a trapping agent for radiopharmaceutical development.