DEVELOPMENT AND BIOLOGICAL INVESTIGATION OF NOVEL [DIARYLSALENE]- AND [SALOPHENE]PLATINUM(II) COMPLEXES WITH TUMOUR INHIBITING PROPERTIES Dissertation zur Erlangung des akademisches Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie, der Freien Universität Berlin verlegt von MARIA TERESITA PROETTO aus San Carlos de Bariloche 2011 Die vorliegende Arbeit wurde von Juni 2008 bis September 2011 unter der Leitung von Prof. Dr. Ronald Gust (Institut für Pharmazie, Freie Universität Berlin) angefertigt. 1. Gutachter: Prof. Dr. Ronald Gust 2. Gutachter: Prof. Dr. Gerhard Wolber Disputation am 27.01.2012 Dedicated to my parents, Teresita Neumann and César Proetto, and to the memory of Guillermo Neumann Acknowledgement First of all, I would like to thank my supervisor Prof. Dr. Ronald Gust for giving me the invaluable opportunity to start a Ph.D., for his guidance and for trusting in my potential during all these years. I would like to show my gratitude to Frau Veronique vom Bauer, for helping me with many different issues always in a very kind way and wearing a smile. I would also like to thank Dr. Adelheid Hagenbach for providing the crystal structures published in this thesis, Dr. Ruth Bieda for the DNA interaction studies and Prof. Dr. Christoph Schalley group for performing the mass spectrometry analyses. I would especially like to thank Dr. Anja Schäfer, Prof. Dr. César Proetto and Dr. Jean-Philippe Monserrat for their help and support during the writing of this dissertation. I am truly indebt and thankful to all my colleagues, for providing me a nice work environment during the past years. In particular I would like to thank Andrey Molchanov, Elena Mazzanti, Ewelina Fogelström, Magnus Krüger, Murat Üstünel, Sandra Alscher, Sandra Meieranz, Silke Bergemann and Thomas Rudolf. Wukun Liu deserves special thanks for helping me with my research and for being such a good friend. This thesis would not have been possible without the support and encouragement of all my people in Argentina. Although they were physically more than 11.000 km far from Berlin, they were always at my side, through the good, not so good and the winter times. Finally, I want to thank my mother, father and brother for their patience and unconditional presence. Table of Contents Abbreviations ................................................................................ 1 Introduction and aims of the project ............................................. 3 1 Introduction ........................................................................................... 4 1.1 Cancer ................................................................................................... 4 1.2 Metal complexes in medicine ..................................................................... 7 1.2.1 Platinum complexes as anticancer agents ...........................................10 1.2.2 Non-platinum metal complexes as antitumour drugs ...........................16 1.2.2.1 Ruthenium-based compounds ...........................................................16 1.2.2.2 Titanium and gallium compounds ......................................................18 2 Background and aims of the research project ...................................... 20 Results and discussion ................................................................. 23 3 Synthesis.............................................................................................. 24 3.1 Overview of the synthesized compounds ....................................................24 3.2 Substituted [diarylsalene]platinum(II) complexes .......................................25 3.3 Substituted [salophene]platinum(II) complexes ..........................................26 3.3.1 Complex with a different main structure .............................................26 3.4 Overview on the synthetic route ...............................................................27 3.5 Synthesis of the [diarylsalene]- and [salophene]platinum(II) complexes .............................................................................................28 3.5.1 Synthesis of substituted 1,2-diamino-1,2-diarylethane.........................28 3.5.1.1 Synthesis of meso-1,2-diamino-1,2-bis(2-hydroxyphenyl)ethane ..........28 3.5.1.2 Synthesis of meso-1,2-diamino-1,2-diarylethane ................................29 3.5.1.3 Synthesis of d,l-1,2-diamino-1,2-diarylethane ....................................32 3.5.2 Synthesis of substituted phenylenediamine ........................................33 3.5.2.1 Synthesis of 1,1,1-trimethylhydrazinium iodide ...................................34 3.5.2.2 Synthesis of 2-fluoro-6-nitroaniline ...................................................34 3.5.2.3 Synthesis of 3-fluoro-1,2-phenylenediamine .......................................36 3.5.3 Synthesis of the diarylsalene and the salophene ligands .......................36 3.5.4 Synthesis of the [diarylsalene]- and [salophene]platinum(II) complexes 37 4 Spectrometric Analysis ......................................................................... 39 4.1 1H-NMR Spectroscopy ..............................................................................39 4.1.1 Diarylsalene and salophene ligands ...................................................39 4.1.2 [Diarylsalene]- and [salophene]platinum(II) complexes .......................45 4.2 Mass spectrometry ..................................................................................47 4.3 Atomic absorption spectroscopy ................................................................48 4.4 X-ray crystallographic studies ...................................................................51 5 In-vitro chemosensitivity assay ........................................................... 57 5.1 Breast cancer cell lines ............................................................................57 5.2 Crystal violet chemosensitivity assay .........................................................57 5.3 IC50 determination ..................................................................................59 5.3.1 Compared effect on MCF-7 and MDA-MB-231 breast cancer cells lines ...60 5.3.2 [Diarylsalene]platinum(II) complexes ................................................62 5.3.3 [Salophene]platinum(II) complexes ...................................................63 5.3.4 Conclusion on the results of the determination of IC50 values of [diarylsalene]- and [salophene]platinum(II) complexes .......................68 5.4 Time- and concentration-dependent cytotoxicity .........................................69 6 Cellular accumulation ........................................................................... 72 6.1 Cell entry mechanisms of platinum drugs ...................................................72 6.2 Cell uptake determination: method and conditions ......................................74 6.3 Cellular accumulation of [salophene]- and [saldach]platinum(II) complexes after 6 and 24 h of drug exposure .............................................................75 6.4 Time-dependent cellular accumulation of [salophene]platinum(II) complexes 77 6.5 Efflux studies .........................................................................................80 6.6 Relationship between cellular accumulation and cytotoxicity .........................81 6.7 Conclusion on the accumulation studies .....................................................83 7 Analysis of physicochemical properties of the [salophene]platinum(II) complexes in relation to their biologic activities .................................. 84 7.1 Lipophilicity ............................................................................................84 7.1.1 Lipophilicity determination: procedure and results ...............................85 7.1.2 Relationship between lipophilicity and biological properties ...................89 7.2 Aqueous solubility ...................................................................................90 7.2.1 Solubility determination: procedure and results ..................................91 7.2.2 Relationship between solubility and biological properties ......................92 7.3 Conclusion on the solubility and lipophilicity results .....................................93 8 DNA interaction studies of the [salophene]platinum(II) complexes .... 96 8.1 DNA binding modes for metal complexes ...................................................96 8.2 Interaction of the platinum(II) complexes with isolated DNA ........................98 8.2.1 Circular dichroism studies ................................................................98 8.2.2 Thermal denaturation studies ...........................................................99 8.2.3 Viscosity measurements ................................................................. 101 8.2.4 Atomic absorption studies .............................................................. 102 8.3 Conclusion on the results of the interaction of the analyzed [salophene]platinum(II) complexes with isolated DNA ............................... 104 Conclusions ................................................................................ 107 9 Conclusions and outlook ..................................................................... 108 Experimental part ...................................................................... 113 10 Materials ............................................................................................ 114 10.1 Instrumentation