Design, synthesis and biological evaluation of substituted naphthalene diimides as anticancer agents

It has been proved that naphthalene diimide (NDI) derivatives display anticancer properties as intercalators and G-quadruplex-binding ligands, leading to DNA damage, senescence and down-regulation of oncogene expression. This thesis deals with the design and synthesis of disubstituted and tetrasubstituted NDI derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development. Disubstituted NDI compounds have been designed with the aim to provide potential multitarget directed ligands (MTDLs), in order to create molecules able to simultaneously interact with some of the different targets involved in this pathology. The most active compound, displayed antiproliferative activity in submicromolar range, especially against colon and prostate cancer cell lines, the ability to bind duplex and quadruplex DNA, to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Tetrasubstituted NDI compounds have been designed as G-quadruplex-binding ligands endowed with anticancer activity. In order to improve the cellular uptake of the lead compound, the N-methylpiperazine moiety have been replaced with different aromatic systems and methoxypropyl groups. The most interesting compound was 1d, which was able to interact with the G-quadruplexes both telomeric and in HSP90 promoter region, and it has been co-crystallized with the human telomeric G-quadruplex, to directly verify its ability to bind this kind of structure, and also to investigate its binding mode. All the morpholino substituted compounds show antiproliferative activity in submicromolar values mainly in pancreatic and lung cancer cell lines, and they show an improved biological profile in comparison with that of the lead compound. In conclusion, both these studies, may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Development of novel multi-substituted apatite nanophases with advanced functionalities for bone regeneration

In the field of bone substitutes is highly researched an innovative material able to fill gaps with high mechanical performances and able to stimulate cell response, permitting the complete restoration of the bone portion. In this respect, the synthesis of new bioactive materials able to mimic the compositional, morphological and mechanical features of bone is considered as the elective approach for effective tissue regeneration. Hydroxyapatite (HA) is the main component of the inorganic part of bone. Additionally ionic substitution can be performed in the apatite lattice producing different effects, depending from the selected ions. Magnesium, in substitution of calcium, and carbonate, in substitution of phosphate, extensively present in the biological bones, are able to improve properties naturally present in the apatitic phase, (i.e. biomimicry, solubility e osteoinductive properties). Other ions can be used to give new useful properties, like antiresorptive or antimicrobial properties, to the apatitic phase. This thesis focused on the development of hydroxyapatite nanophases with multiple ionic substitutions including gallium, or zinc ions, in association with magnesium and carbonate, with the purpose to provide double synergistic functionality as osteogenic and antibacterial biomaterial. Were developed bioactive materials based on Sr-substituted hydroxyapatite in the form of sintered targets. The obtained targets were treated with Pulsed Plasma Deposition (PED) resulting in the deposition of thin film coatings able to improve the roughness and wettability of PEEK, enhancing its osteointegrability. Were investigated heterogeneous gas-solid reactions, addressed to the biomorphic transformations of natural 3D porous structures into bone scaffolds with biomimetic composition and hierarchical organization, for application in load-bearing sites. The kinetics of the different reactions of the process were optimized to achieve complete and controlled phase transformation, maintaining the original 3-D morphology. Massive porous scaffolds made of ion-substituted hydroxyapatite and bone-mimicking structure were developed and tested in 3-D cell culture models.

Development of thermochemiluminescence-based sensitive probes: synthesis, optimization, and characterization of c2- and c7-substituted acridine-containing 1,2-dioxetanes

After initial efforts in the late 1980s, the interest in thermochemiluminescence (TCL) as an effective detection technique has gradually faded due to some drawbacks, such as the high temperatures required to trigger the light emission and the relatively low intensities, which determined a poor sensitivity. Recent advances made with the adoption of variably functionalized 1,2-dioxetanes as innovative luminophores, have proved to be a promising approach for the development of reagentless and ultrasensitive detection methods exploitable in biosensors by using TCL compounds as labels, as either single molecules or included in modified nanoparticles. In this PhD Thesis, a novel class of N-substituted acridine-containing 1,2-dioxetanes was designed, synthesized, and characterized as universal TCL probes endowed with optimal emission-triggering temperatures and higher detectability particularly useful in bioanalytical assays. The different decorations introduced by the insertion of both electron donating (EDGs) and electron withdrawing groups (EWGs) at the 2- and 7-positions of acridine fluorophore was found to profoundly affect the photophysical properties and the activation parameters of the final 1,2-dioxetane products. Challenges in the synthesis of 1,2-dioxetanes were tackled with the recourse to continuous flow photochemistry to achieve the target parent compound in high yields, short reaction time, and easy scalability. Computational studies were also carried out to predict the olefins reactivity in the crucial photooxygenation reaction as well as the final products stability. The preliminary application of TCL prototype molecule has been performed in HaCaT cell lines showing the ability of these molecules to be detected in real biological samples and cell-based assays. Finally, attempts on the characterization of 1,2-dioxetanes in different environments (solid state, optical glue and nanosystems) and the development of bioconjugated TCL probes will be also presented and discussed.