Design, synthesis and characterization of versatile copper complexes with anticancer and catalytic activity.

Although copper has a long history of medical application, copper coordination compounds have been investigated as potential anticancer agents only in the last few decades, particularly after the discovery of cisplatin, the most widely used antitumor metallodrug. Copper, as an essential cofactor in a number of enzymes and physiological processes, may be less toxic than non-essential metals, such as platinum. Up to now, a great variety of copper complexes have been tested as cytotoxic agents and found to be endowed with an antitumor activity in several in vitro tests and few in vivo experiments. Based on these assumptions, in my PhD research work copper was selected for the synthesis of potential metal-based anticancer drugs, that could be suitable alternative to platinum-based drugs that are hampered by marked side effects and chemoresistance. An important aim of this work, in the inorganic chemistry research field, was to synthesize new species able to coordinate metals useful to obtain Cu(I) and Cu(II) complexes with potential anticancer activity. Copper complexes were synthesized employing bis(pyrazolyl)acetic acids (belonging to the family of the heteroscorpionate) as ligands. This class of ligands was selected due to their stability, flexibility and ease to be functionalized and derivatized. In fact, they were used as they are (with the carboxylic acid group) or esterified with aliphatic alcohols (branched and not) or bioconjugated with several biologically active compounds. For this purpose, the ligands were conjugated with metronidazole (an antibiotic agent investigated for hypoxia-selective cytotoxicity), NMDA-ANT (an antagonist for the N-methyl-D-aspartate receptor endowed with micromolar cytotoxic activity on a panel of solid tumor cell lines) and lonidamine (an antineoplastic drug) in order to obtain Cu(I) and Cu(II) complexes potentially able to exert an anticancer activity through synergistic mechanisms of action. Additionally, to stabilize copper in the +1 oxidation state (avoiding its oxidation) and to modulate the solubility profile of the related copper(I) complexes, hydrophilic or lipophilic phosphanes, such as 1,3,5-triaza-7- phosphaadamantane (PTA) and triphenylphosphine (PPh3) respectively, were used as coligands. My main work was the design, synthesis and characterization of the ligands and related copper complexes, both in solid state (FT-IR, elemental analysis and melting point) and in solution (1H-, 11B-, 13C-, 31P-NMR and ESI-MS) to confirm their structure, stoichiometry and purity. In parallel, X-ray diffraction studies were conducted on single crystals of ligands and complexes,[b,e,g,s] showing in some cases new and unexpected dimeric structures.[g] In addition, the structural characterization and the study of the local coordination environment of several complexes were exploited by X-ray photoelectron spectroscopy (XPS) and X-ray absorption fine structure (XAFS) spectroscopy (in the near edge and in the extended regions).[d,f,h,l,m,p,t] In all the cases, the structural investigations confirmed the hypothesized geometry of the metal center and the coordinative fashion of the ligands. Several new complexes and the corresponding uncoordinated ligands were evaluated for their ability to promote cell death against a panel of human cancer cell lines, cisplatin resistant tumor cell lines and spheroids, evaluating also cellular uptake, mechanism of action and morphological modifications induced by the complexes once inside the tumor cells. [a,c,h,i,j,k,n,o,q,r,t,u] In detail, for a series of selected precursors, ligands and Cu(I) and Cu(II) complexes the biological activity was evaluated by means of MTT test, cellular uptake, reactive oxygen species (ROS) production, comet assay and/or transmission electron microscope (TEM) analyses. Summarizing, even if with slight differences, it was possible to state that the complexes were, in general, more active that cisplatin (the drug used as reference compound), both in 2D and 3D cell cultures, showing their effect in the low micromolar concentration, or even lower. On the contrary, the related free ligands and precursors did not show relevant cytotoxic activity. Interestingly, the fact that the complexes proved to be significantly more active than cisplatin, even against three-dimensional spheroids of selected cancer cells, increased the relevance of the in vitro results. In fact, 3D spheroids of cancer cells more closely mimic the heterogeneity and complexity of in vivo tumors, being consequently more predictive for in vivo results than conventional 2D cell cultures. The most frequent mechanism of action for the tested complexes was the paraptotic one, a type of programmed cell death different from the classical apoptosis induced by drugs such as cisplatin. This alternative programmed cell death leads to the overcoming of the inherited or acquired cisplatin or multi-drug resistance. Regarding the ligands esterified with aliphatic alcohols and the related copper complexes the biological studies are still in progress but, according to the preliminary data, these complexes seem to be very promising. Another field of interest of my research work was the investigation of the catalytic activity of new copper(II) compounds in the Kharasch-Sosnovsky reaction,[d,g,n] that is a useful reaction for the synthesis of protected allylic alcohols, via radical oxidation of olefins leaving the double bond in its original position. In particular, the catalytic activity of the copper(II) complexes, containing the isopropyl or hexyl ester chain, was evaluated in the Kharasch-Sosnovsky reaction. The original reaction conditions were optimized changing several parameters and very high yields were obtained employing the compounds containing bromide as counterion. The major limitations of this reaction, such as long reaction times, employ of benzene as solvent and high waste of olefins, were overcome replacing the original cheap but no-so-effective CuBr with the new synthesized Cu(II) complexes. In order to validate the generality of the method, different olefin substrates were tested (obtaining excellent yields) and other promising Cu(II) complexes are under evaluation as catalysts.