Two new types of unsymmetrical bis(thiosemicarbazone) proligands and their neutral zinc(II) and copper(II) complexes have been synthesized. These bifunctional ligands both chelate the metal ions and provide pendent amino groups that can be readily functionalized with biologically active molecules. Functionalization has been demonstrated by the synthesis of three water-soluble glucose conjugates of the new zinc(II) bis(thiosemicarbazonato) complexes, and their copper(II) analogues have been prepared in aqueous solution via transmetalation. A range of techniques including NMR, electron paramagnetic resonance, cyclic voltammetry, high-performance liquid chromatography (HPLC), UV/vis, and fluorescence emission spectroscopy have been used to characterize the complexes. Four compounds, including two zinc(II) complexes, have been characterized by X-ray crystallography. The connectivity and conformation of the glucose conjugates have been assigned by NMR spectroscopy. Time-dependent density functional theory calculations have been used to assign the electronic transitions of the copper(II) bis(thiosemicarbazonato) chromophore. Two copper-64-radiolabeled complexes, including one glucose conjugate, have been prepared and characterized using radio-HPLC, and transmetalation is shown to be a viable method for radiolabeling compounds with copper radionuclides. Preliminary cell washout studies have been performed under normoxic conditions, and the uptake and intracellular distribution have been studied using confocal fluorescence microscopy.

64 Cu-diacetyl-bis(N 4 -methylthiosemicarbazonate), 64 Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. Methods: The retention of radioactivity in tumors after administration of 64 Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared 64 Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of 64 Cu-ATSM or 64 Cu-acetate. Results: In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for 64 Cu-ATSM and 64 Cu-acetate. Copper retention in tumors at 15 min is higher after injection of 64 Cu-acetate than 64 Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for 64 Cuacetate and 64 Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O 2 . In vitro, substantially less uptake is observed for 64 Cu-acetate, although this uptake had some hypoxia selectivity. Although 64 Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both 64 Cu-ATSM and 64 Cu-acetate. Conclusion: That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from 64 Cu-ATSM in tumors essentially mirrors that of 64 Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.

New M(II) bis(thiosemicarbazonato) complexes (M = Ni(II), Cu(II) and Zn(II)) featuring allyl groups at the exocyclic nitrogens have been synthesised. The complexes were characterised in solution by spectroscopic methods and their solid state structures determined by single crystal X-ray diffraction using synchrotron radiation. The Zn(II) complex was found to be intrinsically fluorescent and soluble in biocompatible media. The uptake of this Zn(II) complex in HeLa, MCF-7 and IGROV cancer cells was monitored by fluorescence microscopies (epi- and confocal fluorescence imaging). The radiolabelling to (64)Cu(II) bis(thiosemicarbazonato) complex was performed cleanly by transmetallation from the corresponding Zn(II) species using (64)Cu(OAc)(2).