Teresa Esteves

99mTc-Tricarbonyl Complexes Functionalized with Anthracenyl Fragments: Synthesis, Characterization, and Evaluation of Their Radiotoxic Effects in Murine Melanoma Cells

Different pyrazolyl-diamine ligands bearing anthracenyl or anthrapyrazole functionalities as DNA-binding groups, at different positions of the chelator framework, were labeled with the fac-[(99m)Tc(CO)(3)](+) core. The resulting complexes, 1-4, are highly stable in vitro under physiologic conditions; all of them have been identified by high-performance liquid chromatography comparison with the Re congeners, with the exception of 3, that is anchored by an anthrapyrazole diamine ligand. Aiming to assess the ability of these complexes to target the cell nucleus and to induce enhanced cell death by effect of the Auger electrons emitted by (99m)Tc, the intracellular distribution and radiotoxicity of 1-4 were evaluated by using B16F1 murine melanoma cells. The radiotoxic effects depend very much on the position used to introduce the DNA-binding group and are well correlated with the nuclear uptake of the compounds. Complex 2, having the anthracenyl substituent at the 4-position of the pyrazolyl ring, rapidly entered the cells and accumulated inside the nucleus, exhibiting the highest radiotoxic effects. This compound induced an apoptotic cellular outcome, and its enhanced radiotoxic effects were certainly due to the Auger electrons emitted by the radiometal in close proximity to DNA.

Synthesis, characterization and biological evaluation of tricarbonyl M(I) (M = Re, 99mTc) complexes functionalized with melanin-binding pharmacophores

Aiming to evaluate their potential as radioactive probes for in vivo targeting of melanotic melanoma and its metastases, we have synthesized 99mTc(I) tricarbonyl complexes (Tc1–Tc8) anchored by pyrazolyl-containing chelators with (N3) or (N2O) donor atom sets and functionalized with 2-aminoethyldiethylamine and 4-amino-N-(2-diethylaminoethyl)benzamide groups as melanin-binding pharmacophores. The chemical identification of the several 99mTc complexes has been accomplished by HPLC comparison with the Re congeners (Re1–Re8), which were synthesized at the macroscopic level and fully characterized by common analytical techniques. The biological evaluation of the 99mTc(I) complexes comprised the determination of their in vitro binding to synthetic melanin, measurement of cellular uptake in B16F1 murine melanoma cells, as well as biodistribution studies in B16F1 melanoma-bearing mice. All the tested complexes have shown a moderate to high in vitro affinity to melanin, with percentages of binding spanning between 60 and 94%. In agreement with the poor cellular uptake measured in vitro, the in vivo tumor uptake of the complexes was in general relatively low, ranging between 0.12 and 1.69% ID g−1 at 4 h p.i. However, some complexes have shown favorable tumor-to-organ ratios with values as high as 28 and 5.3 for tumor–muscle and tumor–blood ratios, respectively. This seems to indicate that some selectivity towards melanoma tissue was conserved, and encourages further optimization of the in vitro/in vivo biological properties of this type of complexes aimed at finding novel radioactive probes for non-invasive imaging of melanoma.

Synthesis and Biological Studies of Pyrazolyl-Diamine PtII Complexes Containing Polyaromatic DNA-Binding Groups

New [PtCl(pz*NN)]n+ complexes anchored by pyrazolyl‐diamine (pz*NN) ligands incorporating anthracenyl or acridine orange DNA‐binding groups have been synthesized so as to obtain compounds that would display synergistic effects between platination and intercalation of DNA. Study of their interaction with supercoiled DNA indicated that the anthracenyl‐containing complex L2Pt displays a covalent type of binding, whereas the acridine orange counterpart L3Pt shows a combination of intercalative and covalent binding modes with a strong contribution from the former. L2Pt showed a very strong cytotoxic effect on ovarian carcinoma cell lines A2780 and A2780cisR, which are, respectively, sensitive to and resistant to cisplatin. In these cell lines, L2Pt is nine to 27 times more cytotoxic than cisplatin. In the sensitive cell line, L3Pt showed a cytotoxic activity similar to that of cisplatin, but like L2Pt was able significantly to overcome cisplatin cross‐resistance. Cell‐uptake studies showed that L2Pt accumulates preferentially in the cytoplasm, whereas L3Pt reaches the cell nucleus more easily, as clearly visualized by time‐lapse confocal imaging of live A2870 cells. Altogether, these findings seem to indicate that interaction with biological targets other than DNA might be involved in the mechanism of action of L2Pt because this compound, despite having a weaker ability to target the cell nucleus than L3Pt, as well as an inferior DNA affinity, is nevertheless more cytotoxic. Furthermore, ultrastructural studies of A2870 cells exposed to L2Pt and L3Pt revealed that these complexes induce different alterations in cell morphology, thus indicating the involvement of different modes of action in cell death.