Complexes of Cu(II), Ni(II) and Zn(II) have been prepared with bis(thiosemicarbazonate) ligands bearing a pendant Bodipy fluorophore both with (ATSMBodipy) and without (GTSBodipy) backbone methyl groups. Biophysical analysis using steady state confocal laser scanning fluorescence images and excited state fluorescence lifetime imaging microscopy (FLIM) data obtained from live cells under aerobic uptake conditions has uniquely allowed both the spatial distribution and degree of dissociation of the complexes to be assessed. The CuATSMBodipy complex is virtually intact after 20 min and 1 h whereas the corresponding CuGTSBodipy complex is totally dissociated. The complexes ZnATSMBodipy and NiATSMBodipy show little evidence for dissociation. The relevance of this data to the biomedical applications of bis(thiosemicarbazonate) complexes for imaging applications and implications for the treatment of disease such as Alzheimer's and the PET imaging of hypoxia are discussed.

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. 64Cu-ATSM) and nitroimidazoles (e.g. 18F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H2ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) – H2ATSM/en. Oxygen-dependent uptake studies were performed using the 64Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted 64Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of 64Cu-ATSM/en demonstrated superior hypoxia selectivity to 64Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.

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