Towards new photodynamic therapy agents: synthesis, biological evaluation and targeting to tumor cells

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.