The exocyclic functionalisation of bis(thiosemicarbazonate) complexes of zinc and copper: the synthesis of monomeric and dimeric species.

This paper reports the synthesis of bimetallic zinc thiosemicarbazone complexes with rigid aromatic linkers, using either 1,3- or 1,4- benzenediamines or 1,3- or 1,4- benzenedialdehydes as the basis of the linking groups. Non-rigid aliphatic diamines and dialdehydes were also used to link the zinc chelating units. Reaction of a bis(thiosemicarbazone) with a pendant NHNH(2) group with monoaldehydes or ketones gives a range of monomeric complexes with exocylic imine groups bearing a range of substituents. The zinc complexes can be quantitatively and rapidly transmetallated to the corresponding copper complexes and this route or direct reaction with the free ligand can be used to radiolabel the monomeric species with (64)Cu. In vivo and in vitro studies of one of the (64)Cu imine complexes shows substantial hypoxic selectivity and high tumour uptake in a murine model

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies

© 2017 American Chemical Society. Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co(III) to paramagnetic Co(II). Cobalt pro-drugs built upon the tris(2-pyridylmethyl)amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic 1H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co(II)TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies

In vitro and in vivo evaluations of a hydrophilic 64Cu-bis(thiosemicarbazonato)-glucose conjugate for hypoxia imaging.

UNLABELLED: A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. METHODS: The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. RESULTS: 64CuATSE/A-G showed oxygen concentration-dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. CONCLUSION: The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties

Functionalized bis(thiosemicarbazonato) complexes of zinc and copper: synthetic platforms toward site-specific radiopharmaceuticals.

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

Radiobiological effects of hypoxia-dependent uptake of (64)Cu-ATSM: enhanced DNA damage and cytotoxicity in hypoxic cells

PURPOSE: Hypoxia occurs frequently in cancers and can lead to therapeutic resistance due to poor perfusion and loss of the oxygen enhancement effect. (64)Cu-ATSM has shown promise as a hypoxia diagnostic agent due to its selective uptake and retention in hypoxic cells and its emission of positrons for PET imaging. (64)Cu also emits radiotoxic Auger electrons and beta(-) particles and may therefore exhibit therapeutic potential when concentrated in hypoxic tissue. METHODS: MCF-7 cells were treated with 0-10 MBq/ml (64)Cu-ATSM under differing oxygen conditions ranging from normoxia to severe hypoxia. Intracellular response to hypoxia was measured using Western blotting for expression of HIF-1alpha, while cellular accumulation of (64)Cu was measured by gamma counting. DNA damage and cytotoxicity were measured with, respectively, the Comet assay and clonogenic survival. RESULTS: (64)Cu-ATSM uptake in MCF-7 cells increased as atmospheric oxygen decreased (up to 5.6 Bq/cell at 20.9% oxygen, 10.4 Bq/cell at 0.1% oxygen and 26.0 Bq/cell at anoxia). Toxicity of (64)Cu-ATSM in MCF-7 cells also increased as atmospheric oxygen decreased, with survival of 9.8, 1.5 and 0% in cells exposed to 10 MBq/ml at 20.9, 0.1 and 0% oxygen. The Comet assay revealed a statistically significant increase in (64)Cu-ATSM-induced DNA damage under hypoxic conditions. CONCLUSION: The results support a model in which hypoxia-enhanced uptake of radiotoxic (64)Cu induces sufficient DNA damage and toxicity to overcome the documented radioresistance in hypoxic MCF-7 cells. This suggests that (64)Cu-ATSM and related complexes have potential for targeted radionuclide therapy of hypoxic tumours