Guanidine-Based DNA Binders and Strategies Toward their Platinum Complexes as Anticancer Dual Agents A thesis presented to the University of Dublin for the degree of Doctor of Philosophy by Patrick O’Sullivan, B. Sc. Under the supervision of Prof. Isabel Rozas School of Chemistry Trinity College Dublin March 2016 Declaration This work comprises a doctoral thesis submitted for the consideration of Trinity College Dublin. I declare that this thesis has not been submitted as an exercise for a degree at this or any other university and it is entirely my own work, with due acknowledgement and reference given to the work of others, where appropriate. I agree to deposit this thesis in the University’s open access institutional repository or allow the library to do so on my behalf, subject to Irish Copyright Legislation and Trinity College Library conditions of use and acknowledgement. ………………………………………………………………………………………. Patrick O’Sullivan March 2016 Trinity College Dublin Acknowledgements First and foremost, I would like to thank Prof. Isabel Rozas for your help over the last four and a half years. You have guided me on the journey from student to scientist and for that I will forever be grateful. You have been a boss, a teacher and a friend and I thank you from the bottom of my heart. To all the members of the Rozas group- past and present- you have been an inspiration throughout. I would like to specifically thank Caitríona for being my biophys guru and great friend, Brendan for his expertise in docking, especially for the detailed study in Section 3.1.2, and his valuable insights into chemistry and life in general, Elena Diez for her inspirational work hard-play hard attitude, and Julian for rekindling my love of chemistry and being the resident Pd expert. I also thank the current Rozas group members, Dr Cristina Trujillo for the nicest and most patient computational chemistry expert in the world, Viola for being my biochemistry saviour, my councillor and a true friend, and Aaron for his work on Pt complexation as my summer student and his inquisitiveness and boundless enthusiasm as a fellow PhD student. I would also like to thank my 4th year project students Maeve and Ayat for their work on the aminoguanidine section and my summer student Rory for helping out with some Pt complexes. Thanks also to Ale and Pilar for their work in the very beginning! A special mention goes to the most efficient, dedicated, and craziest chemist I know. Michela, you have been with me pretty much from the beginning to the end of my PhD and I have become a better chemist by following your example. More than that, you have been a cherished friend and companion throughout. I could not in a million years have picked a better person to share the last four years with. To the many people in the School of Chemistry who helped me, from the staff in the office to the technicians to fellow postgrads, postdocs and lecturers, I thank you all. To Dr John O’Brien for your encyclopaedic NMR knowledge, friendship and willingness to always run “just one more sample” for me, Dr Manuel Ruether for help on NMR, UV/vis, CD and HPLC, Dr Brendan Twamley the king of crystallography and Dr Martin Feeney and Dr Gary Hessman for running the mass spectrometry facility. There are too many fellow students to name individually but Helen, Andy and Elena de Calatrava deserve a special mention. Thanks guys for your humour, companionship and chemistry tips. We have lifelong friendships to look forward to! I would like to thank Prof. Aidan McDonald and Prof. Bob Baker for their advice on Pt chemistry and inorganic chemistry in general. Through your insightful questioning and suggestion I learned a great deal. Thanks to Fergus in the Gunnlaugsson group for help with the biophysical measurements and Aoife in the Senge group for her expertise in measuring 1 O2 generation. To Prof. Celine Marmion in the RCSI and your students James, Ziga and Reece, I would like to thank you for your help with Pt complexation and ideas for malonate- based ligand synthesis. To Celine I also thank you for initially conceiving these parts of the project with Isabel. Thanks are also due to Maite Garcia and Prof. Daniela Zisterer in the School of Biochemistry for help with the cytotoxicity section of the project. To each and every member of the Trinity Trampoline Club, especially fellow postgrads Danny, Aideen and Naoise, I cannot explain in words how much you all mean to me. Without you, I would not have lasted more than three months in Dublin. I wish everybody every possible success in everything you do. A special mention is due to my secondary school chemistry teacher, Liz O’Riordan for igniting the first flame in my love of chemistry in Coláiste an Chraoibhín. I cannot thank you enough for all you have done. To all my friends and family, thank you for being so supportive in the last few years. Your encouragement has meant everything to me. I would like to especially thank all my previous housemates for putting up with me, and in particular my Corkonian companions Anne-Marie and Mary. Both of you were constant sources of assistance, reassurance and necessary distraction and it has been my pleasure to live with you guys. Finally, to my parents, Michael and Patsy, and my brother James, your incessant love and support have made me everything I am. Thank you for believing in me, for lifting me up when I was down, and for bringing me down to earth when I needed it! No matter how far from home I am, you are always in my heart. I dedicate this work to you. Ní neart go cur le chéile Abstract One in three people will be diagnosed with cancer in their lifetime. One of the main targets of chemotherapy is DNA. Platinum-based anti-cancer agents form covalent bonds with bases in the major groove of DNA in cancer cells, causing death of these cells. The platinum-based coordination compound cisplatin (Platinol®) and its derivatives carboplatin (Paraplatin®) and oxaliplatin (Eloxatin®) are used in 50% of chemotherapies and are especially efficacious against testicular cancer, where platinum-based drugs have increased the five-year survival rate from 10% in the 1960s to 98% today. One of the most recent chemotherapies to gain approval for cancer treatment is trabectedin (Yondelis®), a natural product that works as a DNA minor groove binder (MGB) and specifically treats liposarcomas by binding covalently to the minor groove of DNA. Over the past 10 years, Rozas’ group has developed dicationic guanidinium-like DNA MGBs that strongly bind to AT-rich DNA sequences and have demonstrated varying cytotoxic activity in cancer cells. In this work, we sought to improve the cytotoxicity of these MGBs by enhancing their lipophilicity, constraining their structures in a planar conformation, or by coordinating them to Pt to create covalent DNA binders. Additionally, aryl guanidine- and aminoguanidine-based platinum complexes were synthesised to explore their cytotoxic effect. Firstly, a family of diaryl bis-(2-amino-1,4,5,6-tetrahydropyridinium) MGBs was prepared. Secondly, a method to synthesise N-amino-N’-arylguanidinium salts was developed. Thirdly, we created new bis-(2-aminoimidazolinium)-containing malonic ester derived MGBs using either a flexible diaryl scaffold or a conformationally restricted and planar 3,6-substituted fluorene motif. Furthermore, using a variety of Pt precursor complexes and neutral N’- arylguanidine ligands, we discovered a wide range of cyclometalated and non-cyclometalated structural motifs containing Pt-arylguanidine and N-amino-N’-arylguanidine complexes. We then applied this knowledge to the synthesis of Pt-MGB complexes bound via a guanidine. The above compounds were tested using biophysical measurements to evaluate DNA binding and using biochemical measurements to assess cytotoxicity on the HL-60 leukemia cell line. Abbreviations ALL Acute Lymphocytic Leukemia AML Acute Myeloid Leukaemia ATP Adenosine Triphosphate BCL-2 B-cell Lymphoma gene 2 Boc tert-Butoxycarbonyl br s broad singlet CD Circular Dichroism CML Chronic Myleoid Leukaemia DACH 1,2-diaminocylohexane DACP 1,2-diaminocyclopentane DFT Density Functional Theory DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic Acid ER Endoplasmic Reticulum FDA Food and Drug Administration HAPT1 High-Affinity Pentamidine Transporter HB Hydrogen bond IC50 Half Maximal Inhibitory Concentration IR Infrared kDNA kinetoplast DNA LUMO Lowest Unoccupied Molecular Orbital NER Nucleotide Excision Repair PCD Programmed Cell Death PDT Photodynamic Therapy PSA Polar Surface Area PTA 1,3,5-triaza-7-phophoadamantane RNS Reactive Nitrogen Species ROS Reactive Oxygen Species rt Room Temperature SOMO Singly Occupied Molecular Orbital TM Melting Temperature TFAA Trifluoroacetic Anhydride TPA Trans Planar Amine UV Ultraviolet Table of Contents 1.0 Introduction ............................................................................................................................ 1.1 Cancer Overview ............................................................................................................. 1 1.2 Overview of cancer treatments ........................................................................................ 4 1.3 Chemotherapy: Drugs in the clinic that kill cancer cells ................................................. 5 1.3.1 Chemotherapy invokes Cell death ............................................................................ 7 1.4 DNA targeting .................................................................................................................