Copper complexes coupled to nucleosides, bile acids and histone deacetylase inhibitors.

Abstract

The aim of this project is to synthesize and characterise novel inorganic complexes which consist of metal-based DNA cleavage entities coupled to biomolecules. It is hoped that the coupling of known DNA cleavage agents to molecules which can form specific binding interactions with biological targets will lead to improved anti-cancer therapeutics. The selection of copper as the metal investigated was deliberate since copper has been shown to be less toxic and better tolerated than heavier metals, such as platinum. Furthermore, the biologically accessible redox potential of copper could be exploited to cause oxidative damage DNA and induce strand breakage in cancer cells. The metal binding entities that were selected were the 1,10-phenanthroline derivative 5-amino-1,10-phenanthroline, with 1,10-phenanthroline as a co-ligand and 1,4,7-triazacyclononane. Precious work carried out in the group showed that copper bound to each of these entities, and that the copper complexes bearing these ligands showed anti-cancer activity in vitro. Furthermore, Cu complexes in which a histone deacetylase inhibitor was linked to the metal binding entity were examined as potential mediators to be used in ‘full-copper’ dye sensitized solar cells. The biomolecules selected belong to three distinct classes; nucleosides, bile acids and histone deacetylase inhibitors. Each class of biomolecule was chosen to allow for the targeting of distinct cellular components; nucleoside-derivatives were designed to interact with both nuclear DNA as well as extra-nuclear nucleic acid species such as those found in the mitochondria or ribozymes. The bile acids were chosen to stimulate bile acid-receptors, which have been shown to be overexpressed on the surface of a number of types of cancer cells, including breast and colon cancer cells. It was intended that through bile acid receptor activation, assimilation of the DNA cleavage agent into cancer cells would be more efficient. Finally, the histone deacetylase inhibitor was selected in an effort to selectively inhibit histone deacetylase, an important target in the treatment of cancer. Modelling studies were carried out on the complexes [Cu(HL1)(1,10-phen)]2+ and [Cu(H2L2)(H2O)n]2+ (where HL1 = N1-(2-(2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)-N5-(1,10-phenanthrolin-5-yl)glutaramide and H2L2 = N-(2-(2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)-5-oxo-5-(1,4,7-triazonan-1-yl)pentanamide) using the Spartan14 software. These studies were intended to investigate the binding of the Cu2+ to the metal-binding entity of the ligands and identify any H-bonding interactions within the molecule. The [Cu(H2L2)(H2O)n]2+ complex revealed the potential binding of more than one H2O ligands to the Cu2+-centre. We found that Cu2+ did bind to the metal-binding entities of the ligands and intermolecular H-bonding interactions were likely to occur. The synthesis of ligands HL1 and H2L2 involved a redesign of the strategy to synthesize 2’-aminodeoxyuridine. We successfully synthesized the nucleoside without the need for the chlorinated intermediate reported in the literature. We attempted to show metal binding to ligands via UV-Vis spectroscopy, but were unable due to the absorption maximum of Cu(NO3)2 overlapping with the absorption maximum of the 2’-aminodeoxyuridine. Cyclic voltammetry was then employed in an attempt to show metal binding to the complexes. We successfully showed binding of [Cu(1,10-phen)]2+ to the ligand HL1 to form [Cu(HL1)(1,10-phen)]2+. The ΔEp of the complex was found to be 213 mV and the E1/2 was found to be 106.5 mV. We were unbale to show Cu2+ binding to the ligand H2L2, since above pH 6, Cu(NO3)2 exchanges the nitrate ligands and forms the insoluble precipitate Cu(OH)2. The complexes [Cu(HL3)(1,10-phen)]2+, [Cu(HL4)(1,10-phen)]2+, [Cu(H2L5)(H2O)]2+ and [Cu(H2L6)(H2O)]2+ (where HL3 = N-(1,10-phenanthrolin-5-yl)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide, HL4 = 4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-(1,10-phenanthrolin-5-yl)pentanamide, H2L5 = 1-(1,4,7-triazonan-1-yl)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one and H2L6 = 4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(1,4,7-triazonan-1-yl)pentan-1-one) were modelled using the Spartan14 program. The purpose of the modelling studies was to investigate whether the addition of the metal binding entity to the bile acid would restrict the H-bonding interactions between the bile acid-hydroxyl groups to the amino acid residues of the receptors which are necessary for receptor activation. We found that the Cu2+- bile acid derivatives did not interfere with the OH groups on the steroid cores of the ligands and so would not prevent the OH group from interacting with their complimentary amino acid residue on the receptor surface. Furthermore, the free rotation around the bile acid alkyl chain linking the steroid core to the metal binding entity would prevent the metal-binding moiety of the complex from inhibiting docking to the receptor. We were unable to isolate pure samples of any of the cholic acid-derivatives [Cu(HL3)(1,10-phen)]2+ and [Cu(H2L5)(H2O)]2+. We believe that the decreased Cu2+ affinity of the phenanthroline core is due to the 5’-amide on the phenanthroline moiety in complex [Cu(HL3)(1,10-phen)]2+ as well as formation of supramolecular assemblies of the ligands which made Cu2+-binding difficult in these cases. The deoxycholic acid derivatives [Cu(HL4)(1,10-phen)]2+ and [Cu(H2L6)(NO3)](NO3) were isolated in sufficient purity for cellular testing. The histone deacetylase inhibitor 4-phenylbutyric acid (PBA) was selected and coupled to metal-binding entities 5’-amino-1,10-phenanthroline and 1,4,7-triazacyclononane. The complexes [Cu(PBA-)(1,10-phen)2](NO3), [Cu(HL7)(1,10-phen)](NO3)2 and [Cu(H2L8)(NO3)](NO3) (where HL7 = N-(1,10-phenanthrolin-5-yl)-4-phenylbutanamide and H2L8 = 4-phenyl-1-(1,4,7-triazonan-1-yl)butan-1-one) were synthesized and isolated in sufficient purity to be sent for evaluation as anti-cancer therapeutics. The redox activity of the complexes [Cu(PBA-)(1,10-phen)2](NO3), [Cu(HL7)(1,10-phen)](NO3)2 and [Cu(H2L8)(NO3)](NO3) was investigated by cyclic voltammetry to investigate whether the complexes could be used as dye mediators in ‘full-copper’ dye-sensitized solar cells. These types of solar cells are a ‘green chemistry’ alternative which would lower the cost of production of the cells as well as eliminate the need for extraction of heavy metals such as ruthenium. Our complexes were found to have quais-reversible redox behaviour, with E1/2OX values that were equal to or lower than those of the dye and electrolytes reported in the first ‘full-copper’ dye sensitized solar cell. Experiments where electron-withdrawing groups were added to the phenanthroline rings were carried out and the dinuclear complex [Cu2(PBA-)(NO3)(H2O)(μ-OH)]NO¬3 was synthesized and characterised by X-ray diffraction. The E1/2OX value of the complex was found to be the same as one of the electrolyte mixtures in the first ‘full-copper’ cell (110 mV). Based on the distorted geometry of the Cu-centres of these ligands, which would make cycling between Cu(I) and Cu(II) easier, and the redox activity of the complexes investigated by cyclic voltammetry, we believe these complexes merit further investigation as mediators in dye sensitized solar cells.