Peter J. Barnard

Spectroelectrochemical and computational studies on the mechanism of hypoxia selectivity of copper radiopharmaceuticals

Detailed chemical, spectroelectrochemical and computational studies have been used to investigate the mechanism of hypoxia selectivity of a range of copper radiopharmaceuticals. A revised mechanism involving a delicate balance between cellular uptake, intracellular reduction, reoxidation, protonation and ligand dissociation is proposed. This mechanism accounts for observed differences in the reported cellular uptake and washout of related copper bis(thiosemicarbazonato) complexes. Three copper and zinc complexes have been characterised by X-ray crystallography and the redox chemistry of a series of copper complexes has been investigated by using electronic absorption and EPR spectroelectrochemistry. Time-dependent density functional theory (TD-DFT) calculations have also been used to probe the electronic structures of intermediate species and assign the electronic absorption spectra. DFT calculations also show that one-electron oxidation is ligand-based, leading to the formation of cationic triplet species. In the absence of protons, metal-centred one-electron reduction gives the reduced anionic copper(I) species, [CuIATSM](-), and for the first time it is shown that molecular oxygen can reoxidise this anion to give the neutral, lipophilic parent complexes, which can wash out of cells. The electrochemistry is pH dependent and in the presence of stronger acids both chemical and electrochemical reduction leads to quantitative and rapid dissociation of copper(I) ions from the mono- or diprotonated complexes, [CuIATSMH] and [Cu(I)ATSMH2]+. In addition, a range of protonated intermediate species have been identified at lower acid concentrations. The one-electron reduction potential, rate of reoxidation of the copper(I) anionic species and ease of protonation are dependent on the structure of the ligand, which also governs their observed behaviour in vivo.

In Vitro and In Vivo Evaluations of a Hydrophilic 64Cu-Bis(Thiosemicarbazonato)–Glucose Conjugate for Hypoxia Imaging

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.

Controlled axial coordination: solid-phase synthesis and purification of metallo-radiopharmaceuticals.

Solid-supported reagents show great potential for improving the synthesis of radiodiagnostic agents, in terms of radiochemical yield and purity, as well as convenience and safety. The preparation of the positron emission tomography (PET) imaging agent [F]-2-fluoro-2-deoxy-d-glucose has recently been demonstrated using a solid-bound substrate which is selectively cleaved from the solid support upon reaction with [F]-fluoride ions. Whilst this covalent approach is appropriate for the nucleophilic substitution chemistry of fluoride ions, its utility with metallonuclides is limited and the only examples to date have used Tc in conjunction with proligands attached to resins and gold surfaces. Conventional solid-phase synthesis is therefore limited by the requirement for proligands possessing a donor group which can both be covalently attached to the solid support and cleaved from it upon coordination to the desired metal ion. Herein, we report a novel strategy for solid-phase synthesis based on selective axial coordination of Zn substrates to 4-(dimethylamino)pyridine(DMAP)-functionalized polystyrene resin and exemplify its use in the preparation and purification of known and potential Cu and Tc radiopharmaceuticals. This strategy is applicable to a wide range of metallic radionuclides and is suitable for the macrocyclic ligand systems that are favored in nuclear medicine because of their high in vivo stability. Zn complexes of tetradentate ligands that are constrained in pseudo-square-planar conformations can potentially bind a fifth donor atom in an axial coordination site. Jahn–Teller distortion disfavors the coordination of a fifth donor atom in the axial site of analogous Cu complexes. A Zn precursor can be bound to polymer-supported DMAP by axial coordination. Upon transmetalation of the Zn complex with Cu, the coordinate bond to the solid support is broken and only the transmetalated complex is released from the resin (Scheme 1 a). Similarly, polystyrene-supported DMAP can be used to selectively bind the Zn complex from a solution-phase reaction mixture leaving only the radiolabeled complex in solution (Scheme 1b).

(2-Hydroxyphenylimido-κN)(methanolato-κO)[2-(2-oxidobenzylideneamino)phenolato-κ2O,N,O′](triphenylphosphine-κP)rhenium(V)

In the neutral title compound, [Re(C6H5NO)(C13H9NO2)(CH3O)(C18H15P)], an 18-valence-electron complex, the ReV ion lies in an octahedral coordination geometry with the tridentate dianionic Schiff base 2-(2-oxidobenzylideneamino)phenolate ligand occupying three equatorial coordination sites, and with the triphenylphosphine ligand situated trans to the imine N atom. The ReV coordination is completed with a methanolate ligand and a 2-hydroxyphenylimido(2-) ligand. There are two molecules in the asymmetric unit. The crystal structure involves O—H⋯O and C—H⋯O hydrogen bonds. One N and one C atom are disordered over two positions; the site occupancy factors are ca 0.7 and 0.3.

(2-Hydroxy­phenyl­imido-κN)(methano­lato-κO)[2-(2-oxidobenzyl­ideneamino)phenolato-κ2O,N,O′](triphenyl­phosphine-κP)rhenium(V)

In the neutral title compound, [Re(C6H5NO)(C13H9NO2)(CH3O)(C18H15P)], an 18-valence-electron complex, the ReV ion lies in an octahedral coordination geometry with the tridentate dianionic Schiff base 2-(2-oxidobenzylideneamino)phenolate ligand occupying three equatorial coordination sites, and with the triphenylphosphine ligand situated trans to the imine N atom. The ReV coordination is completed with a methanolate ligand and a 2-hydroxyphenylimido(2-) ligand. There are two molecules in the asymmetric unit. The crystal structure involves O—H⋯O and C—H⋯O hydrogen bonds. One N and one C atom are disordered over two positions; the site occupancy factors are ca 0.7 and 0.3.