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Porphyrins and metalloporphyrins have been extensively investigated as potential anticancer drugs working as DNA binding agents as well as photosensitizer in the photodynamic therapy (PDT)​. Conjugation of the macrocycle with organo-​metallic complexes have been considered a powerful tool to implement the soly. of the porphyrin and to provide addnl. cytotoxic properties. Here, we prepd. six four-​fold sym. cationic porphyrin-​ruthenium(II) conjugates which differ one from each other's for the nature of the linker, the substituents on the Ru(II) fragments and the total pos. charge and we investigated their interaction with DNA templates different from the B form. Our data suggested that the tested conjugated are not selective toward the G-​quadruplex form of the human telomeric sequence. Indeed, unfolded DNA efficiently works as matrix for the cationic conjugates. Nevertheless, they are extremely efficient in cleaving the macromol. upon irradn., irresp. of its structural arrangement.

The synthesis of three complexes, which bear tumor-targeting peptide and which are suitable for imaging applications might act at the same time, is reported. Their structure contain a PS (a porphyrin or a fulleropyrrolidine derivative), a [1,4,7]-triazacyclononane (TACN) chelator suitable for coordination, and a bombesin BN[7-14] as a targeting peptide. Overall, this work paves the way for the preparation of theranostic complexes although the yields will need to be seriously improved and the synthetic process optimized. Targeting peptides are very promising molecules to increase tumor selectivity of anticancer drugs. Herein, we report the synthesis of three complexes, which might act at the same time as photosensitizers (PSs) for cancer photodynamic therapy (PDT), as delivery systems of Re(I)/99mTc(I) fragments into tumor cells as well as targeting agents for prostate cancer cells. To this aim, we designed a structure containing a PS such as a water soluble +3-charged tris-methylpyridinium porphyrin or a fulleropyrrolidine derivative, a [1,4,7]-triazacyclononane (TACN) chelator suitable for coordination, and a bombesin BN[7-14] as a targeting peptide. Their syntheses were performed using two different approaches: the first one was based on a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC, “click reaction”) in solution, that led to compound 1, and the second one based on coupling reactions on the solid phase, that led to compounds 2 and 3. We could successfully prepare molecules bearing tumor-targeting PSs and which are suitable for imaging applications. Overall, this work paves the way for the synthesis of theranostic complexes although the yields will need to be seriously improved and the synthetic process optimized.

This PhD thesis reports the synthesis, the phototoxic activity, the DNA interactions of new porphyrins and their Re(I) conjugates and a first study to the targeting of porphyrins to tumor cells. The thesis is divided in to two sections. In the first one the synthesis of two new water soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivatives, in which either one or four [1,4,7]-triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker, is described. The correspondent Re(I) conjugates are also reported. The Re(I) metal was inserted at the periphery of the macrocycle both to provide a possible additive therapeutic effect and to obtain a molecule with diagnostic feature. In fact, since the properties of the “cold” Re and of the “hot” 99mTc complexes are assumed to be very similar, the hot 99mTc(I) conjugate can be synthesized from the correspondent cold Re(I) conjugate. These compounds were evaluated as photosensitizers for photodynamic therapy (PDT). The in vitro (photo)toxic effects were evaluated towards the human cell lines HeLa (cervical cancer), H460M2 (non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic epithelial cells). Three of the compounds were not cytotoxic in the dark up to 100 µM and upon Re(I) coordination an enhancement in the phototoxicity activity was observed only for the fourfold-symmetric compound. The intracellular localization of these compounds was studied on HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA and a significant selectivity in the tris-methylpyridinium derivates for G-quadruplex was observed. In the second section a preliminary study on the targeting of photosensitizers towards cancer cells is described. Indeed, it is well known that the accumulation of porphyrins in the tumor tissues is only preferential and not selective. For this reason the use of targeting peptides is very promising in order to increase the tumor selectivity of the photosensitizer. The fundamental concept of the project was to synthesize a molecule that could be a good photosensitizer in PDT treatment, delivery into the tumor a Re(I) fragment and finally accumulate in a specific tumor. A molecule containing a water soluble +3-charged tris-methylpyridinium porphyrin, a [1,4,7]-triazacyclononane (TACN) unit and the targeting peptide bombesin BN[7-14] was synthesized in order to increase the selectivity on prostate cancer cells. The synthesis was performed both in solution and in solid phase using different approaches. Unfortunately, several synthetic problems were found concerning the reaction with the porphyrin unit. For this reason a really small amount of the final compound was obtained and the correspondent Re(I) conjugate was not synthesized. Since fullerene derivatives are good photosensitizers the correspondent molecule containing a fullerene unit, the BN[7-14] and the chelator TACN was synthesized, but also in this case the yield was very low and the product was difficult to purify. The results of this PhD thesis show that a neutral fourfold-symmetric and a +3-charged tris-methylpyridiniumporphyrins can bind the fac-{Re(CO)3}+ fragment in good yields. Considering the biological evaluations, the possibility of obtaining the correspondent 99mTc conjugates, these results are interesting in order to develop new therapeutic agents. Furthermore, a new targeted porphyrin and fullerene containing a TACN unit, as a chelator for Re(I) metal fragments, were synthesized. A new synthetic strategy to obtain a single molecule able to target the charged tris-methylpyridinium porphyrin and a Re(I) fragment to prostate cancer cell is still to be discovered in order to overcome low yield and hard purification procedures.

In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water-soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivative, in which either four or one [1,4,7]-triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker; we also report their corresponding tetracationic Re(I) conjugates. The in vitro (photo)toxic effects of the compounds toward the human cell lines HeLa (cervical cancer), H460M2 (non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic epithelial cells) are reported. Three of the compounds are not cytotoxic in the dark up to 100 μm, and the fourfold-symmetric couple revealed very good phototoxic indexes (PIs). The intracellular localization of all derivatives was studied in HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA; we observed significant selectivity in the tris-methylpyridinium derivatives for G-quadruplex interactions.

We recently prepared two novel water soluble porphyrins bearing a single peripheral chelator, either diethylenetriamine (1) or bipyridyl (2), tethered to one meso position. The preparation of their conjugates with a fac-{(99m) Tc(CO)3 }(+) fragment and the potential of these resulting conjugates as fluorescence and radio imaging tools were also described. In this work, we focused on the corresponding non-radioactive analogues that bear the fac-{Re(CO)3 }(+) fragment (diethylenetriamine 3 and bipyridyl 4). We report on the uptake, in vitro PDT activity, and cellular localization of Re(I) conjugates 3 and 4 in comparison to the parent porphyrins 1 and 2. Compounds 1-4 have modest or negligible cytotoxicity in the dark against HeLa human cervical cancer cells but become remarkably cytotoxic after exposure to moderate doses of red visible light (590-700 nm). This phototoxicity was found to be directly proportional to the total light dose. Although the four compounds show distinct uptake patterns, they have comparable PDT activity. Confocal fluorescence measurements showed that porphyrin 1 and its Re(I) conjugate 3 have different cellular localization patterns in HeLa cells.