Nitroimidazole and glucosamine conjugated heteroscorpionate ligands and related copper(II) complexes. Syntheses, biological activity and XAS studies.

New nitroimidazole and glucosamine conjugated heteroscorpionate ligands, namely 2,2-bis(3,5-dimethyl-1H-pyrazol-1-yl)-N-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)acetamide (L(MN)) and 1,3,4,6-tetra-O-acetyl-2-{[bis(3,5-dimethyl-1H-pyrazol-1-yl)acetyl]amino}-2-deoxy-ß-D-glucopyranose (L(DAC)), respectively, were synthesized by direct coupling of preformed side chain acid and amine components. The related copper(II) complexes {[(L(MN))(2)Cu]Cl(2)}, and {[(L(DAC))(2)Cu]Cl(2)} have been prepared from the reaction of CuCl(2)*2H(2)O with L(MN) or L(DAC) ligand in methanol solution. Single crystal structural characterization was undertaken for the L(MN) ligand. In the absence of a coordinated metal core, the overall arrangement of the ligand is determined by some loose intra- and inter-molecular nonbonding contacts. X-Ray Absorption Spectroscopy (XAS) has been used to probe the local structure of the two copper(II) complexes, {[(L(MN))(2)Cu]Cl(2)} and {[(L(DAC))(2)Cu]Cl(2)}. The EXAFS analysis has permitted the identification of the local environment of the copper site. Copper interacts with 2 units of ligand in both complexes, and it is found to be 6-fold coordinated. Its local structure is described by four Cu-N and two Cu-O interactions to form a pseudo-octahedron core, with a 0.14 Å lengthening of the Cu-O bond length in the case of L(DAC) complex with respect to the L(MN) one, likely due to the higher steric hindrance of the glucosamine moiety. The XANES analysis agrees with these results, also confirming the Cu(II) formal copper oxidation state for both complexes. The new copper(II) complexes {[(L(MN))(2)Cu]Cl(2)} and {[(L(DAC))(2)Cu]Cl(2)} as well as the corresponding uncoordinated ligands were evaluated for their cytotoxic activity towards a panel of several human tumour cell lines. The results reported here indicate that both copper(II) complexes show similar spectra of cytotoxicity and very low resistance factors (RF < 2) against C13* ovarian cancer cells which have acquired resistance to cisplatin.

In vitro antitumour activity of water soluble Cu(I), Ag(I) and Au(I) complexes supported by hydrophilic alkyl phosphine ligands.

Hydrophilic, monocationic [M(L)(4)]PF(6) complexes (M = Cu or Ag; L: thp = tris(hydroxymethyl)phosphine, L: PTA = 1,3,5-triaza-7-phosphaadamantane, L: thpp = tris(hydroxypropyl)phosphine) were synthesized by ligand exchange reaction starting from [Cu(CH(3)CN)(4)]PF(6) or AgPF(6) precursors at room temperature in the presence of an excess of the relevant phosphine. The related [Au(L)(4)]PF(6) complexes (L = thp, PTA or thpp) were synthesized by metathesis reactions starting from [Au(L)(4)]Cl at room temperature in the presence of equimolar quantity of TlPF(6). The three series of complexes [M(L)(4)]PF(6) were tested as cytotoxic agents against a panel of several human tumour cell lines also including a defined cisplatin resistant cell line. These investigations have been carried out in comparison with the clinically used metallodrug cisplatin and preliminary structure-activity relationships are presented. The best results in terms of in vitro antitumour activity were achieved with metal-thp species and, among the coinage metal complexes, copper derivatives were found to be the most efficient drugs. Preliminary studies concerning the mechanism of action of these [M(L)(4)]PF(6) species pointed to thioredoxin reductase as one of the putative cellular targets of gold and silver complexes and provided evidence that copper derivatives mediated their cytotoxic effect through proteasome inhibition.

The relationship between the electrospray ionization behaviour and biological activity of some phosphino Cu(I) complexes.

Electrospray ionization mass spectrometry was usefully employed for the characterization of three phosphino copper(I) complexes of medicinal interest. This technique revealed that the original [CuL(4)](+) pro-drugs (L = hydrophilic tertiary phosphine) underwent dissociation with production of coordinative unsaturated [CuL(3)](+) and [CuL(2)](+) species, which represented key intermediates for the activation of potential biological properties. The more favoured was the displacement of the ligands from the [CuL(4)](+) parent complex, the more favoured was in turn the possibility for the metal ion to directly interact with biological substrates, including pharmacological targets related to cancer proliferation. An inverse correlation between the stability and the cytotoxic activity of the three copper(I) complexes investigated has been clearly established.

In vitro antitumor activity of the water soluble copper(I) complexes bearing the tris(hydroxymethyl)phosphine ligand.

Monocationic hydrophilic complexes [Cu(thp)4](+) 3 and [Cu(bhpe)2](+) 4 were synthesized by ligand exchange reactions starting from the labile [Cu(CH3CN)4][PF6] precursor in the presence of an excess of the relevant hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines. Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

Synthesis, in vitro and in vivo characterization of (64)Cu(I) complexes derived from hydrophilic tris(hydroxymethyl)phosphane and 1,3,5-triaza-7-phosphaadamantane ligands.

Four novel (64)Cu complexes ([(64)Cu(thp)(4)](+) (1), [(64)Cu(TPA)(4)](+) (2), [HC(CO(2))(pz(Me2))(2) (64)Cu(thp)(2)] (3) and [HC(CO(2))(tz)(2) (64)Cu(thp)(2)] (4), [where thp is tris(hydroxymethyl)phosphine, TPA is 1,3,5-triaza-7-phosphaadamantane, pz(Me2) is 3,5-dimethylpyrazole and tz is 1,2,4-triazole] were successfully synthesized and characterized. The complexes were produced in high radiochemical purity and yield (more than 98%) without the need for further purification. Their logP values and serum stabilities were measured and in vitro behavior was observed in cultured EMT-6 cells. The logP values (+/- standard deviation) obtained were -2.26 +/- 0.04 (1), 0.01 +/- 0.01 (2), -1.24 +/- 0.03 (3) and -2.06 +/- 0.03 (4). Complex 3 demonstrated the highest serum stability, with approximately 33% of the complex still intact after 1-h incubation. Complex 2 showed a rapid cell-association with EMT-6 cells, with more than 8.5% association at 2 h. This association was significantly higher (P < 0.001) than for the other three compounds after a 2-h incubation (1, 1.21%; 3, 0.63%; 4, 2.75%). Biodistribution and small-animal positron emission tomography/computed tomography was undertaken with 1 in mice bearing EMT-6 tumors. EMT-6 tumor uptake was high at 1 h (7.71 +/- 2.17 %ID/g) and decreased slowly over 24 h (4 h, 4.90 +/- 0.78 %ID/g; 24 h, 3.74 +/- 0.73 %ID/g). The PET/CT images show that the EMT-6 tumors can be visualized at all time points. In this proof-of-concept study, we have successfully synthesized and characterized a novel series of versatile water-soluble Cu(I) complexes containing monophosphine ligands. We also report the use of 1 as a building block for new radiopharmaceuticals, perhaps the first time such a method has been used in the production of copper radiopharmaceuticals.