Ga-68 is an attractive radiometal for use in positron emission tomography (PET) imaging. The success of Ga-68-based agents is dependent on a chelator that exhibits rapid radiometal incorporation, and strong kinetic inertness to prevent transchelation of Ga-68 in vivo. The linear chelating agents H(2)dedpa (1,2-[[6-carboxypyridin-2-yl]methylamino]ethane) and H2CHXdedpa (CHX = cyclohexyl/cyclohexane) (N4O2) have recently been developed that bind Ga3+ quickly and under mild conditions, ideal properties to be incorporated into a Ga-68 PET imaging agent. Herein, nitroimidazole (NI) derivatives of H(2)dedpa and H(2)CHXdedpa to investigate specific targeting of hypoxic tumor cells are investigated, given that NI can be reduced and retained exclusively in hypoxic cells. Nine N,N'-bis-alkylated derivatives of H(2)dedpa and H2CHXdedpa have been synthesized; they have been screened for their ability to bind gallium, and cyclic voltammetry of nonradioactive complexes was performed to probe the redox cycling mechanism of NI. The compounds were radiolabeled with Ga-67 and Ga-68 and show promising radiolabeling efficiencies (>99%) when labeled at 10(-5) M for 10 min at room temperature. Moreover, stability studies (via apo-transferrin challenge, 37 degrees C) show that the Ga-67 complexes exhibit exceptional stability (86-99% intact) after 2 h. In vitro uptake studies under hypoxic (0.5% O-2) and normoxic (21% O-2) conditions in three cancerous cell lines [HT-29 (colon), LCC6HER-2 (breast), and CHO (Chinese hamster ovarian)] were performed. Of the four H(2)dedpa or H(2)CHXdedpa NI derivatives tested, all showed preferential uptake in hypoxic cells compared to normoxic cells with hypoxic/normoxic ratios as high as 7.9 +/- 2.7 after 120 min. The results suggest that these novel bis-alkylated NI-containing H(2)dedpa and H(2)CHXdedpa ligands would be ideal candidates for further testing in vivo for PET imaging of hypoxia with Ga-68.