Coordination Chemistry of Antimicrobial and Anticancer Agents by Katja Dralle Mjos M.Sc. (Dipl.-Chem.), Carl von Ossietzky University, Oldenburg, Germany, 2007 a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the faculty of graduate and postdoctoral studies (Chemistry) The University of British Columbia (Vancouver) August 2015 c Katja Dralle Mjos, 2015 Abstract The World Health Organization has named the resistance of microbes to known antimi- crobial drugs as an increasingly serious threat to global public health. Isolates of the ESKAPE pathogens (E. faecium, S. aureus, K. pneumonia, A. baumanii, P. aeruginosa, and Enterobacter species) are responsible for many nosocomial infections each year that require complicated, and therefore expensive, medical treatment, often leading to death in immune-compromised patients. Over the past 50 years, (fluoro-)quinolone antimicrobial agents have been widely used in the clinic as broad-spectrum antibiotics, but lately growing resistance against this drug class has been reported. Combining metal ions with known organic small-molecule drugs is one strategy to over- come such developed resistances. Previously, the antimicrobial properties of copper(II) and gallium(III) had been investigated, leading to Greek mythology comparisons for their mechanism of action: Cu2+ as the "Achilles Heel", Ga3+ as the "Trojan Horse" sub- terfuge for Fe3+. In this thesis, gallium(III) and copper(II) coordination complexes of (fluoro-)quinolone antimicrobial agents, and derivatives thereof, were synthesized in an at- tempt to combine the antimicrobial potency of Cu2+ and Ga3+ with that of the quinolone antimicrobial agents in one molecule. The antimicrobial susceptibility of these coordination complexes was evaluated against five isolates of the ESKAPE pathogens; combinational effects between the metals and the quinolone ligands were not observed. ii While the combination of metal ions with small, organic drug molecules may lead to novel potent metallodrugs, the interaction of metal ions with drugs in vivo is often associated with toxic side-effects of medical treatment, for which the iron(III)-mediated cumulative-dose cardiotoxicity of doxorubicin is one example. Vosaroxin is a first-in-class anticancer quinolone derivative in clinical trials. Unlike the anthracycline anticancer drug doxorubicin, vosaroxin is minimally metabolized in vivo. Spectrophotometric titrations and further studies of the isolated tris(vosaroxino)iron(III) and -gallium(III) complexes supported a strong coordination of the metal ion suggesting that vosaroxin treatment may not result in cardiotoxicity. iii Preface The research work for this thesis has been conducted by Katja D. Mjos (KDM) under the immediate supervision of Dr. Chris Orvig and co-supervision of Dr. Michael J. Abrams from September 2009 until August 2014 at The University of British Columbia (UBC). KDM designed the projects and performed all syntheses, analytical and biological experi- ments, if not stated otherwise below. Characterization data of all compounds were obtained by KDM, besides high-resolution mass spectrometry and elemental analyses, which were conducted at UBC's Mass Spectrometry Centre, as well as 600 MHz nuclear magnetic resonance (NMR) spectra, which were recorded by Dr. Maria Ezhova at UBC's NMR Facilities. Chapters 1 and 6 are an adaptation of published work, reproduced in part, with per- mission from Mjos, K.D. and Orvig, C.: Metallodrugs in Medicinal Inorganic Chemistry; Chem. Rev. 2014, 114, 4540-4563, Copyright 2014 The American Chemical Society. The manuscript benefited from discussions between KDM and Dr. Michael J. Abrams; it was written by KDM with editing from Dr. Chris Orvig. Chapters 2 and 3 are an adaptation of the manuscript in preparation for publication, Mjos, K.D.; Cawthray, J.F.; Polishchuk, E.; Abrams, M.J.; Orvig, C.: Testing the "Trojan Horse" Theory: Gallium(III) and Iron(III) Complexes of Quinolone Antimicrobials. The first idea for this project, to synthesize a tris(ciprofloxacino)gallium(III) complex and test iv its antimicrobial properties, came from Dr. Michael J. Abrams. KDM expanded the project from there, planned and performed all experiments. Dr. Jacqueline F. Cawthray modelled one possible stereoisomer of the tris(ciprofloxacino)gallium(III) complex using density functional theory (DFT) calculations. The manuscript was written by KDM with editing from Dr. Chris Orvig. Chapter 4 is an adaptation of the manuscript in preparation for publication, Mjos, K.D.; Polishchuk, E.; Abrams, M.J.; Orvig, C.: Syntheses, Characterization, and Evalua- tion of the Antimicrobial Potential of Copper(II) Coordination Complexes with Quinolone and Xylenyl-Linked Quinolone Ligands. The manuscript was written by KDM with editing from Dr. Chris Orvig. Chapter 5 is an adaptation of published work, and is reproduced in part, with permis- sion from Mjos, K.D.; Cawthray, J.F.; Jamieson, G.; Fox, J.A. and Orvig, C. Iron(III)- Binding of the Anticancer Agents Doxorubicin and Vosaroxin; Dalton Trans., 2015, 44, 2348-2358, Copyright 2015 The Royal Society of Chemistry. KDM and Dr. Jacqueline F. Cawthray designed the spectrophotometric titration experiment and titrated the iron(III)- vosaroxin system, KDM recorded all data for the iron(III)-doxorubicin system, accordingly. Data fitting and DFT calculations were performed by Dr. Jacqueline F. Cawthray, who prepared the respective figures. Dr. Maria Ezhova carried out the temperature dependent NMR-study at 400 MHz. Together with Dr. Zhicheng (Paul) Xia, Dr. Maria Ezhova sup- ported KDM with the design and execution of the NMR titration experiment. Caterina Ramogida and Jeff Therien assisted KDM with the cyclic voltammetry (CV) measure- ments. The results of this project were disseminated to Sunesis Pharmaceuticals, Inc. in form of a technical report in March 2014, which was written by KDM with input from Dr. Jacqueline F. Cawthray. This technical report laid the foundation for the manuscript which was written by KDM with input from Dr. Gene Jamieson and Dr. Judith A. Fox, v Sunesis Pharmaceuticals, Inc.. Dr. Jacqueline F. Cawthray edited earlier versions of the manuscript; Dr. Chris Orvig edited the final version. vi Table of Contents Abstract . ii Preface . iv Table of Contents . vii List of Tables . xv List of Figures . .xvii List of Abbreviations . xxi Acknowledgments . .xxix Dedication . .xxxii 1 Introduction . 1 1.1 Medicinal Inorganic Chemistry . 2 1.2 Diagnostic Metallodrugs . 4 1.3 Therapeutic Metallodrugs . 9 1.3.1 Anticancer Metallodrugs . 9 1.3.1.1 Anticancer Therapeutics . 9 1.3.1.2 Therapeutic Radiopharmaceuticals . 14 vii 1.3.1.3 Photochemotherapeutic Metallodrugs . 15 1.3.2 Antimicrobial and Antiparasitic Metallodrugs . 16 1.3.3 Antiarthritic Metallodrugs . 20 1.3.4 Antidiabetes Metallodrugs . 22 1.3.5 Antiviral Metallodrugs . 24 1.3.6 Metallodrugs Addressing Deficiencies . 26 1.3.7 Metallodrugs for the Treatment of Cardiovascular Disorders . 29 1.3.8 Metallodrugs for the Treatment of Gastrointestinal Disorders . 30 1.3.9 Metallodrugs as Psychotropics . 31 1.3.10 Chelating Proligand Drugs . 33 1.3.10.1 In the Treatment of Overload Disorders . 33 1.3.10.2 In the Treatment of Cancer, Microbial and Parasitic Infections 38 1.4 Strategies for the Design of Metallodrugs . 40 1.4.1 Finding a Druggable Target . 41 1.4.2 The Advantage of Variety: Designing Metal Complexes for the Per- fect Fit . 44 1.4.3 Exploring the Druggability of the Target . 48 1.4.4 Pharmacokinetics: Thermodynamic Stability and Kinetic Lability . 49 1.4.5 Pre-Clinical Studies . 51 1.4.6 Clinical Studies . 52 1.5 Conclusion & Thesis Outlook . 53 2 Introduction to Quinolone Antimicrobial Agents . 55 2.1 Quinolone Antimicrobial Agents . 55 2.2 Materials & Methods . 60 2.2.1 Chemicals . 60 viii 2.2.2 Instrumentation . 60 2.2.3 Biological Studies . 61 2.2.4 Chemical Characterization . 62 2.2.4.1 Ciprofloxacin, Hcipro . 62 2.2.4.2 Enoxacin, Henox . 63 2.2.4.3 Fleroxacin, Hflex . 63 2.2.4.4 Levofloxacin, Hlevox . 64 2.2.4.5 Lomefloxacin, Hlomx . 65 2.2.4.6 Nalidixic acid, Hnxa . 66 2.2.4.7 Norfloxacin, Hnofx . 67 2.2.4.8 Oxolinic acid, Hoxa . 67 2.2.4.9 Pipemidic acid, Hpia . 68 2.2.5 Stability in Iso-Sensitest Broth . 69 2.2.6 Antimicrobial Susceptibility Single-Disk Test in Iso-Sensitest Medium 70 2.3 Results & Discussion . 71 2.3.1 Chemical Characterization . 71 2.3.2 Stability in Iso-Sensitest Broth . 83 2.3.3 Antimicrobial Susceptibility Disk Test . 86 2.4 Conclusion . 88 3 Testing the "Trojan Horse Theory": Gallium(III) and Iron(III) Com- plexes of Quinolone Antimicrobials . 90 3.1 A Bioinorganic Approach: Fighting the Growing Antimicrobial Resistance With Metallodrugs . 90 3.2 Materials & Methods . 97 3.2.1 Chemicals . 97 ix 3.2.2 Instrumentation . 97 3.2.3 Thermogravimetric Analysis . 99 3.2.4 Potentiometry . 99 3.2.5 Computational Details . 100 3.2.6 Antimicrobial Susceptibility Studies . 100 3.2.7 Synthesis & Characterization of Tris(quinolono)metal(III) Complexes 100 3.2.7.1 Tris(ciprofloxacino)gallium(III), [Ga(cipro)3] . 102 3.2.7.2 Tris(enoxacino)gallium(III), [Ga(enox)3] . 103 3.2.7.3 Tris(fleroxacino)gallium(III), [Ga(flex)3] . 104 3.2.7.4 Tris(levofloxacino)gallium(III), [Ga(levox)3] . 105 3.2.7.5 Tris(lomefloxacino)gallium(III), [Ga(lomx)3] . 106 3.2.7.6 Tris(nalidixo)gallium(III), [Ga(nxa)3] . 107 3.2.7.7 Tris(norfloxacino)gallium(III), [Ga(nofx)3] . 107 3.2.7.8 Tris(oxalino)gallium(III), [Ga(oxa)3] . 108 3.2.7.9 Tris(pipemido)gallium(III), [Ga(pia)3] . 109 3.2.7.10 Tris(ciprofloxacino)iron(III), [Fe(cipro)3] . 110 3.2.7.11 Tris(enoxacino)iron(III), [Fe(enox)3] . 110 3.2.7.12 Tris(fleroxacino)iron(III), [Fe(flex)3] . 110 3.2.7.13 Tris(levofloxacino)iron(III), [Fe(levox)3] . 111 3.2.7.14 Tris(lomefloxacino)iron(III), [Fe(lomx)3] .