Development of a New Generation of Metal-Based Anticancer Drugs Dissertation In partial fulfilment for the degree of Doctor of natural sciences (Dr. rer. nat.) Presented to the Department of Chemistry of the Philipps-Universität Marburg by Elisabeth Katharina Martin, M.Sc. Gießen, Germany Marburg/Lahn 2017 The experimental work presented in this thesis was prepared from April 2012 to December 2015 under the supervision of Prof. Dr. Eric Meggers at the Department of Chemistry of the Philipps- Universität Marburg. Submission date: 29.05.2017 Accepted by the Department of Chemistry of the Philipps-Universität Marburg (University ID: 1180) as dissertation: 23.06.2017 Date of oral examination: 29.06.2017 Supervisor: Prof. Dr. Eric Meggers Second reviser: Prof. Dr. Armin Geyer Acknowledgement I would like to thank first and foremost Prof. Dr. Eric Meggers for giving me the opportunity to work on this great topic for the last couple of years, and for giving me the support and freedom, whatever needed at the moment, to fully drive my project and as well as my personal development forward. I also need to thank Prof. Dr. Armin Geyer for kindly taking over the role as second referee as well as teaching me many basics in organic chemistry during my studies. Thank you Prof. Dr. Ulrich Tallarek for taking part in the examination board and teaching me that mass spectrometry is the universal weapon in analytical chemistry. Thank you to Angela for giving me the great opportunity to spend some months in your former lab in Groningen and for giving me the chance to gain a deeper understanding of all that in vitro/ex vivo stuff. Thank you as well to everybody welcoming me in the lab and helping me out with everything Natalia, Sophie, Sarah, Andreia, Viktoriia, Suresh, Mirja, Gerian, Margot, Bernard, Ming, Vivianna and Sylvia. Thanks for the great time! A big thank you goes to Dr. Stefan Peukert for not only being a great collaborator, but also for giving me the chance to come to Cambridge (MA, USA) and get to know all the basic assays for ADME prediction at NIBR and to learn soooo many things during my stay there. A big thank you also goes to everyone involved in the project who helped me so much in learning the basics for interpretation and understanding of the data (Doug, Bing, Sandrine, Suzanne, Dallas, Carrie, Gina, Phong and Brad and everybody else). I also need to thank all the department staff, be it from the MS, NMR or X-ray department, for all the help and support with the measurements. A special thanks goes to Dr. Uwe Linne and Jan Bamberger from the MS department for the ICP-MS measurements and all the work I was loading on them besides all the trouble the MS department was running through anyways. Thank you to Dr. Klaus Harms for X-ray structure determination and to Michael Hellwig for SEM measurements. A huge thank you to each and every member and former member of our group for all the help, support and fun times! Katja, Ina and Andrea I am forever grateful for all the support with everything organisational! A special thanks goes to my lab husbands and wife Manu, Flo and Conni for keeping motivation up and for day to day support! Cheers to everybody else from “der harte Kern” Katja, Sandra, Kathrin, Anja, Janne, Melanie, Jens, Netti, Sabine and Henne. I will never forget the great time we had together! Thank you guys from the big lab, especially Raji, Markus and die Thomase, and of course to Sabrina and Olalla for all the support you gave me. Also a big thanks to all my Schäfchen and Bääätchis (Johanna, Henne times two, Matthias, Bene, Felix, Sebweb and Maddie) for their support in my various projects. Thanks to everybody from my semester: Even “Versuchskaninchen” can survive, and: We did it! I can’t forget my non-chemist friends at that point (Mella, der Alex, Anika, Martin and everybody else)! Thank you for keeping me in “the real life” and sorry for always being the smart-arse. But I am sure you can cope with that, you had enough practice over the last couple of years. Thanks to all my besties who supported me with proofreading of the thesis, you will of course not stay unmentioned! Conni, Manu, Martin, Raji, Nina, Jens, Netti, Sabine, Sabrina, Thomas C., Thomas M., Sabrina, Olalla. Thank you very much indeed. Last but not least: A huge thanks goes to each and every member my family! I would never ever have been able to do all this without you guys!!! Thanks for the support, however little time there was left for you! The same holds true for Sam. Thank you for being there and accepting me the way I am. I know it hasn’t been always easy, so thank you so much for your support and love! Publication list Abstracts of this work have previously been published as peer-review articles or in conferences. Peer-Review Articles 1. R. Rajaratnam, E. K. Martin, M. Dörr, K. Harms, A. Casini, E. Meggers, Nonracemic Octahedral Rhodium-Prolinato Complexes as Protein Kinase Inhibitors, Inorg. Chem., 2015, 54(16), 8111-8120. 2. E. K. Martin, N. Pagano, M. E. Sherlock, K. Harms, E. Meggers, Synthesis and anticancer activity of ruthenium half-sandwich complexes comprising combined metal centrochirality and planar chirality, Inorg. Chim. Acta, 2014, 423, 530-539. Conference Activities 1. E. K. Martin, E. Meggers Drug-like properties of inert metal-based kinase inhibitors. Poster Presentation. Frontiers in Medicinal Chemistry. (Marburg, Germany, 15.03.2015 - 18.03.2015). 2. E. K. Martin, E. Meggers Development of inert rhodium complexes as potential drug candidates. Poster Presentation. MCB Symposium: Joining forces in pharmaceutical analysis and medicinal chemistry. Awarded the RSC books poster price (Groningen, The Netherlands, 25.08.2014 - 26.08.2014). 3. S. Peukert*, E. Martin, D. Chin, F. Lombardo, E. Meggers. Expanding the druggable chemical space with inert metal-organic complexes. Poster. Gordon Research Conference: Metals in Medicine: Defining the Future of Medicinal Inorganic Chemistry. (Andover, MA, USA, 22.06.2014 - 27.06.2014) * presenting author 4. E. K. Martin, E. Meggers Development of inert rhodium complexes as potential drug candidates. Poster Presentation. Protein Kinases in Drug Discovery - Europe. (Berlin, Germany, 08.05.2014 - 09.05.2014). 5. E. K. Martin Development of a new generation of metal-based anticancer drugs. Oral presentation during the 2nd Whole Action Meeting of the COST Action CM1105 at the 1st International Symposium on Functional Metal Complexes that Bind to Biomolecules. (Barcelona, Spain, 09.09.2013 - 10.09.2013). 6. E. K. Martin, K. Harms, E. Meggers Development of a new generation of metal-based anticancer drugs. Poster presentation. Selected for oral poster presentation. 20th EuCheMS Conference on Organometallic Chemistry. (St. Andrews, Scotland, 30.06.2014 - 04.07.2014). Zusammenfassung Seit der Entdeckung der zentralen Rolle von Kinasen in der intrazellulären Signaltransduktion und der Entdeckung von Naturstoffen wie Staurosporin, die als unselektive Inhibitoren für Kinasen fungieren können, hat sich die Entwicklung von potenten Kinaseinhibitoren zu einem elementaren Teil der biologischen und medizinischen Forschung entwickelt. Während der letzten zwei Jahrzehnte hat die MEGGERS-Gruppe eine große Auswahl an selektiven und hoch-spezifischen ATP-kompetitiven Kinaseinhibitoren entwickelt, die auf inerten Koordinationskomplexen mit Metallzentren basieren, welche als strukturelles Templat zur Nachahmung des zuvor erwähnten Staurosporins dienen. Die generelle Struktur dieser Metallo-Pyridocarbazole ist in Abbildung 1 dargestellt. Es konnte gezeigt werden, dass diese Verbindungen sehr potent sind und oft selektiv Kinasen in Enzymassays, in Krebszelllinien in vitro und einfachen eukaryotischen Systemen in vivo inhibieren können. Dabei hat die Gruppe neue und innovative Chemotypen entwickelt, welche einen weitgehend unerforschten Bereich des chemischen Raums einnehmen, und kann damit die stark besiedelten urheberrechtlich geschützten Gebiete umgehen. Basierenden auf dieser Grundlage werden diese Verbindungen als potentielle Krebstherapeutika angesehen. Abbildung 1: Strukturelles Templat der ATP-kompetitiven Kinaseinhibitoren, welche in der Meggers-Gruppe entwickelt wurden. Zusätzlich sind die zwei entwickelten Beispielkomplexe np829 und RR97a dargestellt. Weiterführende Untersuchungen ihrer Eignung als potentielle Therapeutika, basierenden auf u.a. ihren pharmakokinetischen und pharmakodynamischen Eigenschaften, wurden dabei in der Vergangenheit nicht verwirklicht. Daher beschäftigt sich diese Arbeit mit der Eignung dieser Verbindungsklasse als potentielle Krebstherapeutika. Dabei sollte die beste Verbindung identifiziert und als Leitstruktur zur Optimierung der benötigten Eigenschaften verwendet werden. Im ersten Ansatz wurden dabei, basierend auf der bereits bekannten Funktion als Kinase- bzw. ATPase-Inhibitoren und der damit inhärenten Eigenschaft als zielgerichtete molekulare Agenzien, die Verbindungen in einer Substanzbibliothek von 441 Verbindungen zusammengefasst und in einem klassischen empirischen Screening bezüglich ihrer Wirkung gegen Krebszellen in einem MTT-basierten Zytotoxizitätsassay in HT-29 Zellen getestet. Die dabei erfolgversprechendste Verbindung np829 (Abbildung 1), sowie attraktive synthetisierte Derivate, zeigten aber Toxizität, die vermutlich nicht alleine durch die Inhibition von Kinasen zu begründen ist. Daher wurden in einem zweiten Ansatz selektierte Verbindungen auf ihre Wirkstoffartigkeit in Bezug auf Löslichkeit, Permeabilität und metabolische Stabilität in Kollaboration mit den