DOI: 10.1002/cbic.201200472 Synthesis and Biological Studies of Pyrazolyl-Diamine Pt II Complexes Containing Polyaromatic DNA-Binding Groups Sofia Gama, [a] Filipa Mendes, [a] Teresa Esteves, [a] Fernanda Marques, [a] António Matos, [b] JosØ Rino, [c] Joana Coimbra, [d] Mauro Ravera, [e] Elisabetta Gabano, [e] Isabel Santos, [a] and António Paulo* [a] Introduction Cisplatin [cis-DDP, cis-diamminedichloroplatinum(II)] has a cen- tral role in cancer chemotherapy, being the most widely used platinum-based anticancer drug. It is currently prescribed in the treatment of bladder, ovarian, cervical, head and neck, non-small-cell lung, and other cancers, being curative in nearly all cases of testicular cancer. [1–2] Although cisplatin can induce apoptosis in cancer cells through binding to DNA, the drug also undergoes many non-selective reactions with a variety of biomolecules, such as proteins and phospholipids. Further- more, the drug is rapidly distributed throughout the whole body upon administration, interacting both with healthy and with cancerous tissues. [3] Treatment is thus limited by the side effects of nephrotoxicity, emetogenesis, and neurotoxicity. [1] To overcome these drawbacks it is fundamental to under- stand the mechanism of action of cisplatin and, probably even more importantly, the mechanisms by which cells process cis- platin. The major cellular processes by which cisplatin enters and damages cancer cells include uptake and transport, formation of DNA adducts and their recognition by damage-response proteins, and signal transduction leading to cell-cycle arrest, repair, and/or death. Any factor that interferes with these path- ways can lead to drug resistance. [1] With the aim of circumventing cisplatin ’s toxicity and drug resistance, new and selective anti-tumoral platinum com- pounds have been pursued. One of the strategies envisaged for the design of more selective platinum anticancer agents has been the improvement of the formation of DNA adducts. This can be achieved by different approaches, such as regulat- ing tumor-selective uptake and/or increasing the affinity of the drug to nuclear DNA through a DNA targeting approach. [3–4] The rationale behind this approach is that the incorporation into the platinum complex of a functional group that will inter- act or intercalate with DNA might lead to an increase in the 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-contain- ing complex L 2 Pt displays a covalent type of binding, whereas the acridine orange counterpart L 3 Pt shows a combination of intercalative and covalent binding modes with a strong contri- bution from the former. L 2 Pt 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, L 2 Pt is nine to 27 times more cytotoxic than cisplatin. In the sensitive cell line, L 3 Pt showed a cytotoxic ac- tivity similar to that of cisplatin, but like L 2 Pt was able signifi- cantly to overcome cisplatin cross-resistance. Cell-uptake stud- ies showed that L 2 Pt accumulates preferentially in the cyto- plasm, whereas L 3 Pt 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 interac- tion with biological targets other than DNA might be involved in the mechanism of action of L 2 Pt because this compound, despite having a weaker ability to target the cell nucleus than L 3 Pt, as well as an inferior DNA affinity, is nevertheless more cytotoxic. Furthermore, ultrastructural studies of A2870 cells exposed to L 2 Pt and L 3 Pt revealed that these complexes induce different alterations in cell morphology, thus indicating the involvement of different modes of action in cell death. [a] Dr. S.Gama, Dr. F. Mendes, Dr. T. Esteves, Dr. F. Marques, Dr. I.Santos, Dr. A. Paulo Instituto Superior TØcnico, Instituto Tecnológico e Nuclear Unidade de CiÞncias Químicas e RadiofarmacÞuticas Estrada Nacional 10, 2686-953 SacavØm (Portugal) E-mail : apaulo@itn.pt [b] Dr. A. Matos Centro Hospitalar de Lisboa Central Hospital Curry Cabral ServiÅo de Anatomia Patológica R. da BeneficÞncia 8 1069-166 Lisboa (Portugal) [c] Dr. J. Rino Faculdade de Medicina da Universidade de Lisboa– Instituto Medicina Molecular Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal) [d] Dr. J. Coimbra Universidade de Aveiro, Laboratório Central de Anµlises Campo de Santiago 3810–193 Aveiro Campus de Santiago Aveiro (Portugal) [e] Dr. M. Ravera, Dr. E. Gabano Dipartimento di Scienze e Innovazione Tecnologica Università del Piemonte Orientale “Amedeo Avogadro” Viale Michel 11, 15100, Alessandria (Italy) 2352  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2012, 13, 2352 – 2362