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Coordination chemistry of antimicrobial and anticancer agents Mjos, Katja Dralle 2015

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Coordination Chemistry ofAntimicrobial and Anticancer AgentsbyKatja Dralle MjosM.Sc. (Dipl.-Chem.), Carl von Ossietzky University, Oldenburg, Germany, 2007a thesis submitted in partial fulfillmentof the requirements for the degree ofDoctor of Philosophyinthe faculty of graduate and postdoctoral studies(Chemistry)The University of British Columbia(Vancouver)August 2015c© Katja Dralle Mjos, 2015AbstractThe 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 theESKAPE pathogens (E. faecium, S. aureus, K. pneumonia, A. baumanii, P. aeruginosa,and Enterobacter species) are responsible for many nosocomial infections each year thatrequire complicated, and therefore expensive, medical treatment, often leading to death inimmune-compromised patients. Over the past 50 years, (fluoro-)quinolone antimicrobialagents have been widely used in the clinic as broad-spectrum antibiotics, but lately growingresistance 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 theirmechanism 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 quinoloneantimicrobial agents in one molecule. The antimicrobial susceptibility of these coordinationcomplexes was evaluated against five isolates of the ESKAPE pathogens; combinationaleffects between the metals and the quinolone ligands were not observed.iiWhile the combination of metal ions with small, organic drug molecules may leadto novel potent metallodrugs, the interaction of metal ions with drugs in vivo is oftenassociated with toxic side-effects of medical treatment, for which the iron(III)-mediatedcumulative-dose cardiotoxicity of doxorubicin is one example. Vosaroxin is a first-in-classanticancer quinolone derivative in clinical trials. Unlike the anthracycline anticancer drugdoxorubicin, vosaroxin is minimally metabolized in vivo. Spectrophotometric titrationsand further studies of the isolated tris(vosaroxino)iron(III) and -gallium(III) complexessupported a strong coordination of the metal ion suggesting that vosaroxin treatment maynot result in cardiotoxicity.iiiPrefaceThe research work for this thesis has been conducted by Katja D. Mjos (KDM) under theimmediate supervision of Dr. Chris Orvig and co-supervision of Dr. Michael J. Abramsfrom 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 obtainedby KDM, besides high-resolution mass spectrometry and elemental analyses, which wereconducted at UBC’s Mass Spectrometry Centre, as well as 600 MHz nuclear magneticresonance (NMR) spectra, which were recorded by Dr. Maria Ezhova at UBC’s NMRFacilities.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. Themanuscript benefited from discussions between KDM and Dr. Michael J. Abrams; it waswritten 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 ”TrojanHorse” Theory: Gallium(III) and Iron(III) Complexes of Quinolone Antimicrobials. Thefirst idea for this project, to synthesize a tris(ciprofloxacino)gallium(III) complex and testivits antimicrobial properties, came from Dr. Michael J. Abrams. KDM expanded theproject from there, planned and performed all experiments. Dr. Jacqueline F. Cawthraymodelled one possible stereoisomer of the tris(ciprofloxacino)gallium(III) complex usingdensity functional theory (DFT) calculations. The manuscript was written by KDM withediting 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 Quinoloneand Xylenyl-Linked Quinolone Ligands. The manuscript was written by KDM with editingfrom 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, whoprepared the respective figures. Dr. Maria Ezhova carried out the temperature dependentNMR-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. CaterinaRamogida and Jeff Therien assisted KDM with the cyclic voltammetry (CV) measure-ments. The results of this project were disseminated to Sunesis Pharmaceuticals, Inc. inform of a technical report in March 2014, which was written by KDM with input fromDr. Jacqueline F. Cawthray. This technical report laid the foundation for the manuscriptwhich was written by KDM with input from Dr. Gene Jamieson and Dr. Judith A. Fox,vSunesis Pharmaceuticals, Inc.. Dr. Jacqueline F. Cawthray edited earlier versions of themanuscript; Dr. Chris Orvig edited the final version.viTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xviiList of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiAcknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxixDedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxxii1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Medicinal Inorganic Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Diagnostic Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.3 Therapeutic Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.1 Anticancer Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . . Anticancer Therapeutics . . . . . . . . . . . . . . . . . . . Therapeutic Radiopharmaceuticals . . . . . . . . . . . . . . 14vii1.3.1.3 Photochemotherapeutic Metallodrugs . . . . . . . . . . . . 151.3.2 Antimicrobial and Antiparasitic Metallodrugs . . . . . . . . . . . . . 161.3.3 Antiarthritic Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . 201.3.4 Antidiabetes Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . 221.3.5 Antiviral Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . 241.3.6 Metallodrugs Addressing Deficiencies . . . . . . . . . . . . . . . . . . 261.3.7 Metallodrugs for the Treatment of Cardiovascular Disorders . . . . . 291.3.8 Metallodrugs for the Treatment of Gastrointestinal Disorders . . . . 301.3.9 Metallodrugs as Psychotropics . . . . . . . . . . . . . . . . . . . . . 311.3.10 Chelating Proligand Drugs . . . . . . . . . . . . . . . . . . . . . . . 331.3.10.1 In the Treatment of Overload Disorders . . . . . . . . . . . 331.3.10.2 In the Treatment of Cancer, Microbial and Parasitic Infections 381.4 Strategies for the Design of Metallodrugs . . . . . . . . . . . . . . . . . . . 401.4.1 Finding a Druggable Target . . . . . . . . . . . . . . . . . . . . . . . 411.4.2 The Advantage of Variety: Designing Metal Complexes for the Per-fect Fit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441.4.3 Exploring the Druggability of the Target . . . . . . . . . . . . . . . . 481.4.4 Pharmacokinetics: Thermodynamic Stability and Kinetic Lability . 491.4.5 Pre-Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 511.4.6 Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521.5 Conclusion & Thesis Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Introduction to Quinolone Antimicrobial Agents . . . . . . . . . . . . . . 552.1 Quinolone Antimicrobial Agents . . . . . . . . . . . . . . . . . . . . . . . . 552.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60viii2.2.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602.2.3 Biological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612.2.4 Chemical Characterization . . . . . . . . . . . . . . . . . . . . . . . 622.2.4.1 Ciprofloxacin, Hcipro . . . . . . . . . . . . . . . . . . . . . 622.2.4.2 Enoxacin, Henox . . . . . . . . . . . . . . . . . . . . . . . . 632.2.4.3 Fleroxacin, Hflex . . . . . . . . . . . . . . . . . . . . . . . . 632.2.4.4 Levofloxacin, Hlevox . . . . . . . . . . . . . . . . . . . . . . 642.2.4.5 Lomefloxacin, Hlomx . . . . . . . . . . . . . . . . . . . . . 652.2.4.6 Nalidixic acid, Hnxa . . . . . . . . . . . . . . . . . . . . . . 662.2.4.7 Norfloxacin, Hnofx . . . . . . . . . . . . . . . . . . . . . . . 672.2.4.8 Oxolinic acid, Hoxa . . . . . . . . . . . . . . . . . . . . . . 672.2.4.9 Pipemidic acid, Hpia . . . . . . . . . . . . . . . . . . . . . 682.2.5 Stability in Iso-Sensitest Broth . . . . . . . . . . . . . . . . . . . . . 692.2.6 Antimicrobial Susceptibility Single-Disk Test in Iso-Sensitest Medium 702.3 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712.3.1 Chemical Characterization . . . . . . . . . . . . . . . . . . . . . . . 712.3.2 Stability in Iso-Sensitest Broth . . . . . . . . . . . . . . . . . . . . . 832.3.3 Antimicrobial Susceptibility Disk Test . . . . . . . . . . . . . . . . . 862.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883 Testing the ”Trojan Horse Theory”: Gallium(III) and Iron(III) Com-plexes of Quinolone Antimicrobials . . . . . . . . . . . . . . . . . . . . . . 903.1 A Bioinorganic Approach: Fighting the Growing Antimicrobial ResistanceWith Metallodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 903.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97ix3.2.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973.2.3 Thermogravimetric Analysis . . . . . . . . . . . . . . . . . . . . . . . 993.2.4 Potentiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993.2.5 Computational Details . . . . . . . . . . . . . . . . . . . . . . . . . . 1003.2.6 Antimicrobial Susceptibility Studies . . . . . . . . . . . . . . . . . . 1003.2.7 Synthesis & Characterization of Tris(quinolono)metal(III) Complexes 1003.2.7.1 Tris(ciprofloxacino)gallium(III), [Ga(cipro)3] . . . . . . . . 1023.2.7.2 Tris(enoxacino)gallium(III), [Ga(enox)3] . . . . . . . . . . . 1033.2.7.3 Tris(fleroxacino)gallium(III), [Ga(flex)3] . . . . . . . . . . . 1043.2.7.4 Tris(levofloxacino)gallium(III), [Ga(levox)3] . . . . . . . . . 1053.2.7.5 Tris(lomefloxacino)gallium(III), [Ga(lomx)3] . . . . . . . . 1063.2.7.6 Tris(nalidixo)gallium(III), [Ga(nxa)3] . . . . . . . . . . . . 1073.2.7.7 Tris(norfloxacino)gallium(III), [Ga(nofx)3] . . . . . . . . . . 1073.2.7.8 Tris(oxalino)gallium(III), [Ga(oxa)3] . . . . . . . . . . . . . 1083.2.7.9 Tris(pipemido)gallium(III), [Ga(pia)3] . . . . . . . . . . . . 1093.2.7.10 Tris(ciprofloxacino)iron(III), [Fe(cipro)3] . . . . . . . . . . 1103.2.7.11 Tris(enoxacino)iron(III), [Fe(enox)3] . . . . . . . . . . . . . 1103.2.7.12 Tris(fleroxacino)iron(III), [Fe(flex)3] . . . . . . . . . . . . . 1103.2.7.13 Tris(levofloxacino)iron(III), [Fe(levox)3] . . . . . . . . . . . 1113.2.7.14 Tris(lomefloxacino)iron(III), [Fe(lomx)3] . . . . . . . . . . . 1113.2.7.15 Tris(nalidixo)iron(III), [Fe(nxa)3] . . . . . . . . . . . . . . 1123.2.7.16 Tris(norfloxacino)iron(III), [Fe(nofx)3] . . . . . . . . . . . . 1123.2.7.17 Tris(oxalino)iron(III), [Fe(oxa)3] . . . . . . . . . . . . . . . 1123.2.7.18 Tris(pipemido)iron(III), [Fe(pia)3] . . . . . . . . . . . . . . 1133.2.8 Synthesis & Characterization of Tris(maltolato)metal(III) Complexes 113x3.2.8.1 Tris(maltolato)gallium(III), [Ga(ma)3] . . . . . . . . . . . . 1133.2.8.2 Tris(maltolato)iron(III), [Fe(ma)3] . . . . . . . . . . . . . . 1143.3 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1143.3.1 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1143.3.2 Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163.3.3 Solid State Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.3.4 Thermal Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263.3.5 Stability in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283.3.6 Antimicrobial Susceptibility Testing . . . . . . . . . . . . . . . . . . 1303.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1394 Syntheses, Characterization, and Evaluation of the Antimicrobial Po-tential of Copper(II) Coordination Complexes with Quinolone andXylenyl-Linked Quinolone Ligands . . . . . . . . . . . . . . . . . . . . . .1404.1 Mixing Things Up: Another Metal, a Modified Ligand . . . . . . . . . . . . 1414.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1434.2.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1444.2.3 Antimicrobial Susceptibility Studies . . . . . . . . . . . . . . . . . . 1444.2.4 Synthesis & Characterization . . . . . . . . . . . . . . . . . . . . . . 1454.2.4.1 α,α’-Xylenyl-Linked Ciprofloxacin Dimer, H2ciproXcipro . 1454.2.4.2 α,α’-Xylenyl-Linked Pipemidic Acid Dimer, H2piaXpia . . 1454.2.4.3 [Cu2(ciproXcipro)2] . . . . . . . . . . . . . . . . . . . . . . 1464.2.4.4 [Cu2(piaXpia)2] . . . . . . . . . . . . . . . . . . . . . . . . 1474.2.4.5 Bis(ciprofloxacino)copper(II), [Cu(cipro)2] . . . . . . . . . . 1474.2.4.6 Bis(pipemido)copper(II), [Cu(pia)2] . . . . . . . . . . . . . 148xi4.2.4.7 Bis(maltolato)copper(II), [Cu(ma)2] . . . . . . . . . . . . . 1494.3 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1494.3.1 Synthesis & Characterization . . . . . . . . . . . . . . . . . . . . . . 1494.3.2 Antimicrobial Susceptibility Testing . . . . . . . . . . . . . . . . . . 1534.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575 Iron(III)-Binding of the Anticancer Agents Doxorubicin and Vosaroxin 1595.1 The Two Anticancer Agents: Doxorubicin & Vosaroxin . . . . . . . . . . . . 1595.2 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645.2.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645.2.3 Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655.2.4 Computational Details . . . . . . . . . . . . . . . . . . . . . . . . . . 1665.2.5 Synthesis & Characterization . . . . . . . . . . . . . . . . . . . . . . 1675.2.5.1 Vosaroxin, Hvox . . . . . . . . . . . . . . . . . . . . . . . . 1675.2.5.2 Tris(vosaroxino)iron(III), [Fe(vox)3] . . . . . . . . . . . . . 1675.2.5.3 Tris(vosaroxino)gallium(III), [Ga(vox)3] . . . . . . . . . . . 1685.2.6 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1695.3 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705.3.1 Stability Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705.3.2 Synthesis & Characterization of Tris(vosaroxino)iron(III) and-gallium(III) Complexes . . . . . . . . . . . . . . . . . . . . . . . . . 1755.3.3 Cyclic Voltammetry Studies . . . . . . . . . . . . . . . . . . . . . . . 1815.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1826 Conclusion & Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185xiiBibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192A Antimicrobial Susceptibility Testing By Single-Disk Method . . . . . . .224A.1 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225A.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225A.3 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228A.3.1 Purchased Items . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228A.3.2 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230A.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231A.4.1 Personal Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231A.4.2 Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231A.4.3 Important Pathogen Safety Information by the Public Health Agencyof Canada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231A.4.3.1 Bacterium Enterococcus faecalis . . . . . . . . . . . . . . . 231A.4.3.2 Bacterium Escherichia coli . . . . . . . . . . . . . . . . . . 232A.4.3.3 Bacterium Klebsiella pneumonia . . . . . . . . . . . . . . . 232A.4.3.4 Bacterium Pseudomona aeruginosa . . . . . . . . . . . . . 232A.4.3.5 Bacterium Staphylococcus aureus . . . . . . . . . . . . . . . 233A.5 Test Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233A.5.1 Marking of Petri Dishes . . . . . . . . . . . . . . . . . . . . . . . . . 233A.5.2 Preparation of Agar Plates . . . . . . . . . . . . . . . . . . . . . . . 233A.5.3 Preparation of Broth Storage . . . . . . . . . . . . . . . . . . . . . . 235A.5.4 Transferring of Bacteria Culture . . . . . . . . . . . . . . . . . . . . 235A.5.5 Preparations for the Actual Test Day (Day 1) . . . . . . . . . . . . . 236A.5.6 Growing Bacteria in Broth (Day 1) . . . . . . . . . . . . . . . . . . . 236A.5.7 Setting up the Biosafety Cabinet (Day 2) . . . . . . . . . . . . . . . 237xiiiA.5.8 Preparation and Standardization of Inoculum Suspension (Day 2) . 237A.5.9 Preparation of Test Solutions (Day 2) . . . . . . . . . . . . . . . . . 237A.5.10 Inspection of Agar Plates (Day 2) . . . . . . . . . . . . . . . . . . . 238A.5.11 Loading of Filter Disks with Test Compound (Day 2) . . . . . . . . 239A.5.12 Inoculation of Plates (Day 2) . . . . . . . . . . . . . . . . . . . . . . 239A.5.13 Placement of Loaded Disks (Day 2) . . . . . . . . . . . . . . . . . . 239A.5.14 Incubation of Test Plates (Day 2) . . . . . . . . . . . . . . . . . . . . 240A.5.15 Interpretation and Measurement of Zone Sizes (Day 3) . . . . . . . . 240A.5.16 Reporting of Measured Zone Sizes . . . . . . . . . . . . . . . . . . . 241A.5.17 Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . 243B Supplementary Information to Chapter 5 . . . . . . . . . . . . . . . . . .244B.1 UV-Vis Titration of the Vosaroxin-Iron(III) System . . . . . . . . . . . . . . 245B.2 UV-Vis Titration of the Doxorubicin-Iron(III) System . . . . . . . . . . . . 249B.3 Cyclic Voltammograms of Hvox and [Ga(vox)3] . . . . . . . . . . . . . . . . 253B.4 NMR Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255xivList of TablesTable 1.1 FDA approved diagnostic metalloradiopharmaceuticals . . . . . . . . . . 8Table 1.2 Approved therapeutic metalloradiopharmaceuticals . . . . . . . . . . . . 14Table 2.1 UV-Vis absorbance maxima (Amax [nm]) . . . . . . . . . . . . . . . . . . 72Table 2.2 1H NMR data in δH [ppm] . . . . . . . . . . . . . . . . . . . . . . . . . . 80Table 2.3 13C NMR data in δC [ppm] (d, JC,F [Hz]) . . . . . . . . . . . . . . . . . 81Table 2.4 19F NMR data in δF [ppm] . . . . . . . . . . . . . . . . . . . . . . . . . . 82Table 2.5 Results of antimicrobial susceptibility study of nine quinolones . . . . . . 88Table 3.1 Carboxylate stretching frequencies [cm−1]. . . . . . . . . . . . . . . . . . 120Table 3.2 Comparison of the determined pKa values of ciprofloxacin. . . . . . . . . 128Table 3.3 Results of antimicrobial susceptibility study. . . . . . . . . . . . . . . . . 136Table 4.1 Selected IR stretching frequencies [cm−1] and their assignments. . . . . . 151Table 4.2 Inhibition zone sizes [mm] of copper(II) complexes. . . . . . . . . . . . . 154Table 5.1 Protonation and Fe3+ formation constants for doxorubicin and vosaroxin. 171Table A.1 Synthetic formula of Iso-Sensitest agar. . . . . . . . . . . . . . . . . . . . 227Table A.2 Approximate formula of Mueller-Hinton II agar. . . . . . . . . . . . . . . 228Table A.3 Selected pathogenic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 228xvTable A.4 List of Gram-positive and Gram-negative bacteria tested . . . . . . . . . 231Table A.5 List of items needed for the transfer of bacterial cultures . . . . . . . . . 235Table A.6 Items to incubate on day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 237Table A.7 Items needed in biosafety cabinet on day 2 . . . . . . . . . . . . . . . . . 238xviList of FiguresFigure 1.1 Selected examples of successful therapeutic and diagnostic metallodrugs. 4Figure 1.2 Anticancer platinum metallodrugs approved and in clinical trials in theUSA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Figure 1.3 Anticancer metallodrugs in clinical trials. . . . . . . . . . . . . . . . . . 13Figure 1.4 Metallodrugs for photodynamic therapy. . . . . . . . . . . . . . . . . . . 16Figure 1.5 Antimicrobial and antiparasitic drugs approved and in clinical trials. . . 19Figure 1.6 Approved gold(I) antiarthritis metallodrugs. . . . . . . . . . . . . . . . . 22Figure 1.7 Promising metallodrug candidates for the treatment of diabetes. . . . . 24Figure 1.8 CTC-96 and sodium thiomersal. . . . . . . . . . . . . . . . . . . . . . . 26Figure 1.9 M40403 and BSS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Figure 1.10 Approved metal chelating prodrugs. . . . . . . . . . . . . . . . . . . . . 35Figure 1.11 Two metal chelators in clinical trials for the treatment of neurodegener-ative diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Figure 1.12 Metal-chelators for cancer therapy. . . . . . . . . . . . . . . . . . . . . . 40Figure 1.13 Overview of various design possibilities for metallodrugs. . . . . . . . . 45Figure 1.14 Organometallic ruthenium(II) complexes with promising anticancer-activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Figure 2.1 Molecular structures of nine selected quinolones. . . . . . . . . . . . . . 56xviiFigure 2.2 pH dependency of ciprofloxacin . . . . . . . . . . . . . . . . . . . . . . . 73Figure 2.3 Protonation equilibria of ciprofloxacin. . . . . . . . . . . . . . . . . . . . 74Figure 2.4 IR spectra of the nine quinolones. . . . . . . . . . . . . . . . . . . . . . 75Figure 2.5 UV-Vis study to monitor the stability of nine selected quinolones inIso-Sensitest broth and in water. . . . . . . . . . . . . . . . . . . . . . . 85Figure 3.1 Overview of the synthesized and characterized tris(quinolono)metal(III)complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Figure 3.2 Synthetic pathway to tris(quinolono)metal(III) complexes. . . . . . . . . 115Figure 3.3 Low-resolution MS spectra (ES+) of [Ga(nxa)3] and [Fe(nxa)3]. . . . . . 118Figure 3.4 IR spectra of norfloxacin, [Ga(nofx)3], and [Fe(nofx)3]. . . . . . . . . . . 121Figure 3.5 19F NMR spectrum of [Ga(enox)3]. . . . . . . . . . . . . . . . . . . . . . 123Figure 3.6 Solid state structure of [La(cipro)4]−. . . . . . . . . . . . . . . . . . . . 125Figure 3.7 Result of the DFT calculation of (fac, ∆)-[Ga(cipro)3]. . . . . . . . . . . 126Figure 3.8 TGA/DTA results for [Ga(cipro)3], [Fe(cipro)3], and Hcipro. . . . . . . 127Figure 3.9 Initial spectrophotometric study of the Ga3+:Hcipro system. . . . . . . 130Figure 3.10 Comparison of inhibition zone sizes in Iso-Sensitest and Mueller-Hintonmedia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133Figure 4.1 Pipemidic acid, ciprofloxacin, and their respective (α,α’-)xylenyl-linkeddimers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142Figure 4.2 IR spectra of ciprofloxacin, [Cu(cipro)2], H2ciproXcipro, and[Cu2(ciproXcipro)2]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Figure 4.3 Photo of a growth plate of P. aeruginosa. . . . . . . . . . . . . . . . . . 156Figure 5.1 Chemical structures of doxorubicin, vosaroxin, and dexrazoxane. . . . . 160Figure 5.2 Doxorubicin affects Fe3+ homeostasis in vivo. . . . . . . . . . . . . . . . 161xviiiFigure 5.3 Comparison of the changes in absorbance at 400 nm. . . . . . . . . . . . 173Figure 5.4 Representative fit at 400 nm for Fe3+:Hvox = 1:3. . . . . . . . . . . . . 174Figure 5.5 Interaction of the Fe3+ ion with vosaroxin. . . . . . . . . . . . . . . . . 175Figure 5.6 Species distribution curves for the iron(III)-doxorubicin system. . . . . . 176Figure 5.7 Species distribution curves for the iron(III)-vosaroxin system. . . . . . . 176Figure 5.8 Synthetic route to tris(vosaroxino)metal(III) complexes. . . . . . . . . . 178Figure 5.9 IR spectra of Hvox, [Fe(vox)3], and [Ga(vox)3]. . . . . . . . . . . . . . . 180Figure 5.10 Cyclic voltammogram of [Fe(vox)3]. . . . . . . . . . . . . . . . . . . . . 182Figure 6.1 Effect of metal ion intake on overall health. . . . . . . . . . . . . . . . . 190Figure A.1 Template for plate marking (numerical values in [cm]). . . . . . . . . . . 234Figure A.2 Bacteria culture plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 236Figure A.3 Example photo for test plate documentation. . . . . . . . . . . . . . . . 241Figure A.4 Example of inhibition zone size recording sheet. . . . . . . . . . . . . . . 242Figure B.1 UV-Vis spectra of one titration run of Hvox. . . . . . . . . . . . . . . . 245Figure B.2 UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 1:1. . 246Figure B.3 UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 2:1. . . 247Figure B.4 UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 3:1. . . 248Figure B.5 UV-Vis spectra of one titration run of Hdox. . . . . . . . . . . . . . . . 249Figure B.6 UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 1:1. . 250Figure B.7 UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 2:1. . . 251Figure B.8 UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 3:1. . . 252Figure B.9 Cyclic voltammogram of Hvox. . . . . . . . . . . . . . . . . . . . . . . . 253Figure B.10 Cyclic voltammogram of [Ga(vox)3]. . . . . . . . . . . . . . . . . . . . . 254Figure B.11 NMR titration of Hvox with Fe3+. . . . . . . . . . . . . . . . . . . . . . 255xixFigure B.12 Temperature dependent NMR study of [Ga(vox)3]. . . . . . . . . . . . . 256xxList of AbbreviationsA adenine.AAS atomic absorption standard.AD Alzheimer disease.ADME concept concept of absorption, distribution, metabolism and excretion.ADP adenosine triphosphate.AIDS acquired immunodeficiency syndrome acquired through infection with human im-munodeficiency virus (HIV).APL acute promyelocytic leukaemia.AQD anticancer quinolone aromatic ring system.ATO arsenic trioxide.ATP adenosine triphosphate.ATRA all-trans retinoid acid.BAL 2,3-dimerceptopropanol, H2DMPA.xxiBBB blood brain barrier.BEOV bis(ethylmaltolato)oxovanadium(IV).BMOV bis(maltolato)oxovanadium(IV).BP bipolar broad.BSAC British Society for Antimicrobial Chemotherapy.BSS bismuth subsalicylate.Caco-2 cells human colon colorectal adenocarcinoma cells.CBS colloidal bismuth sub-citrate.CDC U.S. Centers for Disease Control and Prevention.CKD-MBD chronic kidney disease-mineral and bone disorder.CLSI U.S. Clinical and Laboratory Standards Institute.CNS central nervous system.COST European Cooperation in Science and Technology.COSY correlated spectroscopy.CTCL cutaneous T-cell lymphoma.CV cyclic voltammetry.d doublet.xxiidd doublet of doublets.DFO desferrioxamine B.DFP deferiprone, 1,2-dimethyl-3-hydroxypyridin-4-one.DFT density functional theory.DM diabetes mellitus.DMARD disease-modifying anti-rheumatic drugs.DMSO dimethyl sulfoxide.DNA deoxyribonucleic acid.DSB double/strand breaks.DTA differential thermal analysis.E. coli Escherichia coli .E. faecalis Enterococcus faecalis.EA elemental analysis.ECDC European Centre for Disease Prevention and Control.EMA European Medicines Agency.ESCI electrospray and chemical ionization.EU European Union.EUCAST European Committee for Antimicrobial Susceptibility Testing.xxiiiFDA U.S. Food and Drug Administration.FT Fourier transformation.G guanine.GI gastrointestinal.H. pylori Helicobacter pylori .H3DMPS D,L-2,3,-dimercaptopropane-1-sulfonic acid.H4DMSA meso-2,3,-dimercaptosuccinic acid.H4DOTA 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetracetic acid.H4EDTA ethylenediaminetetraacetic acid.H5DTPA diethylenetriaminepentaacetic acid.Hcipro ciprofloxacin.HD Huntington disease.Hdox doxorubicin.Henox enoxacin.Hflex fleroxacin.HFR High Flux Reactor.HIV human immunodeficiency virus.Hlevox levofloxacin.xxivHlomx lomefloxacin.Hma maltol.HMBC heteronuclear multiple bond correlation.Hnofx norfloxacin.Hnxa nalidixic acid.Hoxa oxolinic acid.Hpia pipemidic acid.HPLC high performance liquid chromatography.HR-ESI high-resolution electrospray ionization.HSA human serum albumin.HSQC heteronuclear single quantum coherence spectroscopy.HSV-1 herpes simplex virus type 1.Hvox vosaroxin.ICL670 deferasirox.IDSA Infectious Disease Society of America.IL-2 interleukin-2.IR infrared.IRTK insulin receptor tyrosine kinase.xxvK. pneumonia Klebsiella pneumonia.KDM Katja D. Mjos.m multiplet.m/z mass-to-charge ratio.MD Menkes moderate.MDR multi-drug resistant.MIC minimum inhibitory melting point.MPAC metal-protein attenuating compound.MRI magnetic resonance imaging.MS mass spectrometry.MTD maximum tolerated dose.NCCLS U.S. National Committee for Clinical Laboratory Standards.NCI U.S. National Cancer Institute.NIH U.S. National Institutes of Health.NMR nuclear magnetic resonance.NRU National Research Universal.xxviP. aeruginosa Pseudomona aeruginosa.PD Parkinson disease.PDT photodynamic therapy.PET positron emission tomography.pip 1,4-piperazinyl ring in C7 position on aromatic ring system.POM polyoxometalates.prop propyl ring in N1 position on aromatic ring system.PTH parathyroid hormone.q quartet.RA rheumatoid arthritis.RBC ranitidine bismuth citrate.RNA ribonucleic acid.ROS reactive oxygen species.s singulet.S. aureus Staphylococcus aureus.SALEN 2,2’-ethylenebis(nitrilomethylidene) shoulder.siRNA small interfering ribonucleic acid.xxviiSNP sodium nitroprusside, Na2[Fe(CN)5NO] · 2 H2O.SOD superoxide dismutase.SPECT single photon emission computed strong.TETA 2,2,2-trientine.Tf transferrin.TGA thermogravimetric analysis.TSS toxic shock triplet of triplets.UBC The University of British Columbia.USA United States of America.UV-Vis ultraviolet-visible.w weak.WD Wilson disease.WHO World Health Organization.xxviiiAcknowledgmentsIn dependence on an African proverb which says that it takes a village to raise a child, Iwant to thank my personal village of people who helped me become a PhD chemist.First and foremost, the late Professor Dr. John R. Moss, who, in 2004, when I was afourth-year student, invited me to join his research group at The University of Cape Town.It was there, that I worked independently on a research project for the first time, and thatI was left in the lab with a package from Canada one afternoon. The package containedsamples for a biological study. Because I was basically too slow to hide from John, I wasleft with the tedious task to weigh in test amounts of these compounds (to the amusementof my fellow lab mates). So, I spent the rest of the day in front of the analytical balancewith lots of time to think about the place, where this package came from − on the senderlabel it read: Medicinal Inorganic Chemistry Group, The University of British Columbia,Vancouver, Canada.Motivated through my great experience in South Africa, I started my personal endeav-our of travelling and working as a chemist with stops along the way at EMD Merck (Ger-many), in the research group of Professor Dr. Ru¨diger Beckhaus at the Carl von OssietzkyUniversity in Oldenburg (Germany), and at DNV Energy (Norway) gaining experience ina variety of areas, thanks to many skilled colleagues who shared their knowledge with me.The idea of doing my PhD in Canada, however, had manifested itself in my mind. Thanksxxixto Professor Dr. Chris Orvig, my dream came true in 2009, when he accepted me intohis group. Boss, thank you very much for letting me run freely in all directions. Thanksto your support of my career aspirations, I was able to not only deepen my knowledge ofchemistry but to learn more about intellectual property law and technology transfer in myPhD years, too. I am as well grateful that Dr. Michael J. Abrams came on board officiallyas my co-supervisor in 2012. Mike, whenever we met, I left our discussion motivated, in-spired and ready to return to the academic lab with a clearly defined goal in mind. Bothof you, thank you very much for being my Doktorva¨ter.I as well would like to thank the members of my supervisory committee, ProfessorsDrs. Raymond J. Andersen and Laurel L. Schafer, for their support throughout my PhDtime, with an extra big Thank You for Professor Dr. Kristin Orians, who watchfully readmy thesis from cover to cover.I feel honoured that I was able to spend five years in a team of talented young re-searchers. Working together with people from six continents on a daily basis furtheredmy cultural understanding and my intercultural communication skills. In the beginning,Yasmin, Paloma, and Eszter, helped me with setting up my hood, getting started in thelab and maneuvering UBC’s Chemistry Department. Later, Caterina, Gwen, and Karenjoined the early bird brigade, and it is simply nice to meet smiling faces at work. Alongthe way, I as well had the pleasure of working together with several postdocs, especiallydiscussions with Adriana, Cristina, Lisa, and Jacquie furthered my science.UBC Chemistry Research Facilities are the backbone of the department, and all unitshave been genuinely supporting me, especially Dr. Maria Ezhova (NMR), Dr. Yun Lingand Mr. Marshall Lapawa (MS), and Dr. Elena Polishchuk and Jessie Chen (BiologicalServices). The optimism of Dr. Brian O. Patrick who, even after running more thantwenty crystals on the X-ray diffractometer and always having to evaluate the obtainedxxxdata unsuitable afterwards, still got excited, when I came into his office with new crystalattempts of my (quinolono)metal complexes, is unsurpassed.I say Thank You, Vielen Dank & tusen takk to...The many fabulous professional women who I met through the Society for Canadian Womenin Science and Technology and the Women in Leadership Mentoring Program. Yoga teach-ers, Gloria and Max, who showed me that I am much stronger than I would have everthought I was. My UBC friends, Merill, Kerry and Jennifer, for many interesting luncheons.My fellow expats, Eva Kathrin and Sarah, for trusting friendship and solid support throughall ups and downs. My big brothers and sisters in chemistry, Drs. Thomas, Corinna, Nils,Do¨rte and Frank, for good times, three fine weddings, and hour-long Skype talks about lifeand science. The honourable maids of the cyber knitting club, Alice, Imke and Kathrin, formany cultural field trips, always open ears, and enriching my life monthly. My family, pastand present, in Germany and Norway, who love(d) their scientist. My soulmate Anders,for looking at my data with the mind of a physicist, for being pragmatic as an engineer,for patiently teaching me LATEX, for being on my team at all times, and for never doubtingthat this all was worth five years of our lives.xxxiDedicationFu¨r vier starke Frauen— Four Women —Edith, Elfriede, Else, GerdaxxxiiChapter 1IntroductionThis thesis describes studies of the coordination chemistry of antimicrobial and anticanceragents. The aim of this research is to find novel metallodrugs to fight the growing resistanceof bacteria to common antibiotics, as well as to understand the interaction of small-organicmolecule drugs with metals present in the human body, which can reduce the potencyof an administered drug or even lead to toxic side-effects. In this chapter, an impressionof the current field of metallodrugs in the discipline of medicinal inorganic chemistry isgiven. The focus lies on therapeutic metallodrugs which are currently approved in theUnited States of America (USA) and/or countries of the European Union (EU), but someof the most widely used diagnostic metallodrugs are briefly introduced as well. In addition,promising novel metallodrugs which are currently in clinical trials are discussed, next togeneral strategies and challenges of metallodrug research and development. Numerousreview articles and books have been published on medicinal inorganic chemistry,1 2 thefield of metallodrugs,3 4 5 6 7 8 9 and especially on anti-cancer treatments;10 11 12 13, therefore,the objective of this chapter is to be neither repetitive nor comprehensive, it is merelysetting the stage for the following chapters of research on this topic.11.1 Medicinal Inorganic ChemistryMetal ions play important roles in biological processes,14 and the field of knowledge con-cerned with the application of inorganic chemistry to therapy or diagnosis of disease ismedicinal inorganic chemistry.15 Among the natural sciences, medicinal inorganic chem-istry is still considered a rather young discipline by many, but this is contrary to the his-torically proven use of metals in pharmaceutical potions, which traces back to the ancientcivilizations of Mesopotamia, Egypt, India and China.16 17 18The introduction of metal ions or metal ion binding components into a biological sys-tem for the treatment of diseases is one of the main sub-divisions in the field of bioinorganicchemistry.19 Such an intentional introduction of metal ions into the human biological sys-tem has proven to be useful for both diagnostic and therapeutic purposes. Figure 1.1presents selected examples of some successful therapeutic and diagnostic metallodrugs.The latter have led to an increased understanding and early detection of diseases throughthe imaging of the living body. Nowadays, contrast agents containing radioactive metalisotopes are produced and administered daily in many medium sized hospitals around theworld in single photon emission computed tomography (SPECT) scans of the human body.Magnetic resonance imaging (MRI) contrast also uses metal ions (Gd3+). In Canada 1.7million MRI scans, 63,000 positron emission tomography (PET) scans and over a millionSPECT scans were performed in 2011−2012, and the numbers are growing internation-ally.20 Thanks to these diagnostic methods malignant growth, cardiologic diseases andatherosclerosis in patients can be detected early; furthermore, such imaging agents en-hance research as they, for example, enable researchers to visualize the activity of thebrain in vivo.One of the first therapeutic metallodrugs was salvarsan, an arsenic-based antimicrobialagent developed by Paul Ehrlich under the working name 606, a mixture of 3-amino-24-hydroxyphenyl-arsenic(III) compounds. In 1912, Paul Ehrlich published his results ofsalvarsan as an effective treatment against syphillis.21 Salvarsan provided an effectivedemonstration for Ehrlich’s belief that it is possible to fight infectious diseases througha systematic search for drugs that kill invading microorganisms without damaging thehost, his idea of ”Magic Bullets”. Although model structures for salvarsan have been elu-cidated recently,22 the exact composition of salvarsan is still unknown; despite that fact,it has been used widely in humans. With the addition of mercury and bismuth, salvarsanremained the standard remedy for syphilis until it was replaced by penicillin after WorldWar II.23Although Ehrlich’s Salvarsan is widely regarded as the birth of modern chemother-apy and often cited as the beginning of modern research and development of metallo-drugs, the star drug of the field until today is the anticancer agent cisplatin (Platinol),which was discovered serendipitously in 1965 while Barnett Rosenberg and Loretta VanCamp at Michigan State University were studying the effect of an electric current onEscherichia coli.24 It was found that cell division was inhibited by the production of cis–diamminedichloroplatinum(II) from the platinum electrodes.24 25 Further studies on thisplatinum-agent indicated that it possessed antitumor activity, and this finding led to on-going research and development of anticancer metallodrugs.26Despite the immense success of cisplatin and the fact that some inorganic formulateddrugs such as dietary supplements and antacids have been readily available over the counterfor centuries, the majority of all drugs on the market today is of organic or biological origin.It seems that from historical experience the know-how and expertise of the pharmaceuticalindustry rest almost entirely in these areas. Even today metal containing medicinal agentsare often discovered in an academic research setting, before risk friendly start-up companiesdevelop the actual metallodrug candidate further, moving it into first clinical trials.273PtClClH3NH3NcisplatinAsAs AsAsAsOHOHOHHOHONH2NH2H2NH2NH2NAsAs AsOHNH2OHNH2HOH2Ncyclic species as models for salvarsanTherapeuticDiagnostic[99mTc(sestamibi)]+TcCC CCCCNNNNNN OOOOOO[Gd(DOTA)]-N NNNOOOOOOO OGd3+Gd3+[Gd(DTPA)]2-NOON OOOOOOOO NFigure 1.1: Selected examples of successful therapeutic and diagnostic metallodrugs.1.2 Diagnostic MetallodrugsRadiopharmaceuticals play an important role in medical diagnostics and therapy (see aswell Section Diagnostic radiopharmaceuticals are a powerful tool in the diagnosisof cancer, cardiological disorders, infections, kidney or liver abnormalities, and neurologi-cal disorders.29 30 For imaging specific biological targets at low concentrations, they haveunprecedented advantages over other less sensitive diagnostic methods. Over the past fiftyyears the imaging quality of medical scans has improved tremendously through novel di-4agnostic metallodrugs entering the market as well as through the development of imagingdevices with higher sensitivity and enhanced resolution. Abnormal growth can now beeasily detected, and in the diagnosis of cancer, for example, it is often possible to differen-tiate between carcinogenic tissue and healthy tissue based on the visual imaging impressionbefore an actual tissue sample is taken.To image a variety of medical conditions, a diversity of different imaging agents canbe employed that specifically target a certain organ or body fluid. Table 1.1 presents anoverview of the diagnostic radiopharmaceuticals currently approved by the U.S. Food andDrug Administration (FDA). The dominant isotope in diagnostic imaging is technetium-99m, which has been called the ”Workhorse of Diagnostic Nuclear Medicine”.31 A total ofsixty-seven 99mTc imaging agents have been approved over the years by the FDA alone;currently, a total of twenty-eight 99mTc imaging agents are FDA-approved.32 Its dominanceof the imaging market is reflected in the annual sales of the two leading 99mTc diagnos-tic imaging agents Cardiolite, and Myoview, both heart imaging agents, which amountedto 675 million USD in 2007.33 In general, worldwide sales for a diagnostic drug vary be-tween 100−400 million USD per year,34 which makes this area one of the financially mostrewarding in the field of metallodrugs.Since the first technetium-99m radiotracers were developed at the University of Chicagoin 1964, 99mTc has revealed itself to be the optimal metal isotope for imaging with com-mercial γ-cameras, because it conveniently emits a 140 keV γ-ray with 89% abundanceand activities of > 1.11 GBq, and it can be injected with a low radiation exposure to thepatient.35 The nine different oxidation states of technetium, from −I (d8) to +V II (d0),together with its diversified stereochemistry spanning from coordination number 4 to 9,open up a variety of target-specific tuneable platforms for the development of radiophar-maceuticals.36 375A key challenge of the technetium-99m isotope, however, is the ongoing shortage in itsproduction. Long blackout periods in the two obsolete nuclear reactors which have beengenerating more than 70% of the global market of molybdenum-99, the parent nuclideof technetium-99m, have contributed to a medical isotope crisis. The National ResearchUniversal (NRU) reactor, built in 1957 in Chalk River, Canada, has been providing 45%of the world’s supply of 99Mo; the High Flux Reactor (HFR), built in 1961 in Petten, TheNetherlands, has been supplying 30%. The reduced availability of 99mTc has sparked thesearch for possible future alternatives in radiochemistry.One alternative to technetium-99m are isotopes gallium-67 for SPECT and gallium-68 for PET imaging. The many advantages of 68Ga radiopharmaceuticals35 and espe-cially the easy generation of 68Ga through mobile 68Ge/68Ga-generator systems have beendiscussed38 and questioned for many years.39 Although no such generator is currentlyapproved by the FDA or the European Medicines Agency (EMA), the pre´paration magis-trale of imaging agents is possible in many European countries. German authorities havegranted manufacturing authorization for pharmacological 68Ge/68Ga generators for use inclinical studies in 2012.40 Already in 2011 the EMA had given orphan drug designation togallium-68 pasireotide tetraxetan (SOMscan), which is developed as a PET imaging agentfor gastro-entero-pancreatic neuroendrocrine tumors.41Metal chelating agents such as diethylenetriaminepentaacetic acid (H5DTPA) and1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetracetic acid (H4DOTA) complexed to thehighly paramagnetic 4f7 Gd3+ ion are used as injectable macrocyclic contrast agents forMRI scans (Figure 1.1);42 the imaging agent [Gd(DTPA)]2− (Magnevist, Magnegita) ob-tained FDA approval in 1988, while [Gd(DOTA)]− (Dotarem, Gadovist) was approvedin March 2013. The use of these macrocyclic chelators in SPECT or PET radiopharma-ceuticals opens the gateway to theranostic agents. Theranostics implies the quantitative6molecular imaging diagnosis of a disease with a diagnostic pharmaceutical followed by apersonalized treatment with a therapeutic radiopharmaceutical analog.43 Preliminary clin-ical results for an example of such a theranostic approach based on the 68Ga radionuclidefor diagnostic imaging followed by therapeutic treatment with 90Y have been successful,and European wide trials will start soon.417Table 1.1: FDA approved diagnostic metalloradiopharmaceuticals32radioisotope radiation active ingredient trade name diagnostic imaging67Ga γ Ga-67 citrate Hodgkin’s disease, lymphoma, bronchogenic car-cinoma82Rb β+ Rb-82 chloride Cardiogen-82 myocardium99mTc γ Tc-99m bicisate Neurolite stroke99mTc γ Tc-99m disofenin Hepatolite cholecystitis99mTc γ Tc-99m exametazime Ceretec stroke, abdominal infection99mTc γ Tc-99m macroaggregated albumin pulmonary perfusion, shunt patency99mTc γ Tc-99m mebrofenin Choletec hepatobiliary system99mTc γ Tc-99m medronate MDP-Bracco bone99mTc γ Tc-99m mertiatide Technescan MAG3 kidney99mTc γ Tc-99m oxidronate Technescan HDP bone99mTc γ Tc-99m pentetate brain, kidney99mTc γ Tc-99m pyrophosphate Technescan PYP bone, myocardium, blood pool99mTc γ Tc-99m red blood cells UltraTag blood pool99mTc γ Tc-99m sestamibi Cardiolite myocardium, breast99mTc γ Tc-99m sodium pertechnetate Technelite brain, thyroid, blood pool, urinary tract, naso-lacrimal drainage system99mTc γ Tc-99m succimer kidney99mTc γ Tc-99m sulfur colloid lymphatic system, liver99mTc γ Tc-99m tetrofosmin Myoview myocardium99mTc γ Tc-99m tilmanocept Lymphoseek lymphatic system111In γ In-111 capromab pendetide ProstaScint prostate cancer111In γ In-111 chloride Indiclor radiolabeling of ProstaScint111In γ In-111 oxyquinoline leukocytes, inflammation111In γ In-111 pentetate brain, spinal canal111In γ In-111 pentetreotide Octreoscan neuroendocrine tumors201Tl γ Tl-201 chloride myocardium, thyroid81.3 Therapeutic MetallodrugsThis section provides an overview of metallodrugs which have been approved for the medicaltreatment of human diseases or are currently in clinical trials.1.3.1 Anticancer Metallodrugs1.3.1.1 Anticancer TherapeuticsThe World Health Organization (WHO) names cancer as a leading cause of death world-wide, accounting for 7.6 million deaths (around 13 % all deaths) in 2008 and projected torise above 13.1 million deaths in 2030.44One of the oldest and best-known metallodrugs is the anticancer drug cisplatin, cis-diammine-dichloroplatinum(II) (Platinol), a square planar Pt2+ complex (Figures 1.1 and1.2).45 First synthesized by Peyrone in 1844,46 its anticancer properties were discoveredby Rosenberg and co-workers in the 1960s,24 47 further explored,25 48 49 patented,50 andapproved by the FDA in December 1978; cisplatin was the first metal-based medicinalagent to enter into worldwide clinical use for the treatment of cancer. Used alone or incombination against different types of cancers, cisplatin is a blockbuster drug and oneof the most successful therapeutic metallodrugs even today;26 it was amongst the toprevenue-generating licensed products,51 which provided Michigan State University with alarge gross revenue from licensing royalties52 until its second patent53 was invalidated inlitigation on the ground of obviousness-type double patenting.54These days, cisplatin therapy can be considered part of a standard treatment againstmany forms of cancer. After the initial surgical removal of malignant tissue, the patientundergoes cycles of intravenous injections of cisplatin. During treatment the patient ex-periences major unpleasant side effects of the drug because cisplatin is highly cytotoxic.The Pt2+ of the {Pt(NH3)2}2+ unit binds covalently to deoxyribonucleic acid (DNA),9more specifically, to the N-7 of either guanine (G) or adenine (A) in the dinucleotide se-quences GG and AG to form interstrand crosslinks and 1,2- or 1,3-intrastrand crosslinks.55Such cisplatin-DNA adducts, together with cellular pathways activated in response to cis-platin, lead to replication arrest, transcription inhibition, cell-cycle arrests, DNA repairand apoptosis.56 For many years, cisplatin’s mechanism of action has been described toinvolve activation by aquation inside cells due to varying Cl− concentration.57 58 Researchon platinum drugs for anticancer therapy embraced this concept and tried to overcomeits drawbacks, mainly lowering the level of cytoxicity, with second generation platinumdrugs as oxaliplatin and carboplatin. All anticancer platinum-metallodrugs that have beenapproved by the FDA or are currently in clinical trials in the USA are presented in Fig-ure 1.2. Carboplatin, cis-diammine-dicyclobutane-1,1-dicarboxylato-platinum(II) (Para-platin), was reported by Cleare and Hoeschele in 1973,59 60 patented in 197961 and ap-proved by the FDA in 1989. The chelate effect of the six-membered ring reduces itschemical reactivity and possible side effects as well as damage to the ear (otoxicity) andthe kidneys (nephrotoxicity). Oxaliplatin, (1R, 2R)-(N, N’ -1,2-diamminocyclohexane)-(O-O’ )-ethanedioato)platinum(II) (Eloxatin),62 63 received European approval in 1999 andapproval by the FDA in 2002. Drugs of a similar design are nedaplatin, lobaplatin andheptaplatin, which are currently in clinical trials in the USA but are already in clinical usein Japan, China and South Korea, respectively. In addition, novel liposome nanoparticleformulations of cisplatin (Lipoplatin) and oxaliplatin (Lipoxal), which appear to reduceserious adverse reactions allowing a better exploitation of the anticancer activity of theplatinum agent,64 65 are currently undergoing clinical trials.66 67The attractive advantage of satraplatin, bis(acetato)amminedichloro(cyclohexylamine)-platinum(IV) (JM216, Orplatna), is its oral availability; it can be administered in pill formwhich is convenient for the patient and reduces health care costs. JM216 contains the10mononuclear platinum(IV) core, which in the blood stream is reduced by metal-containingredox proteins68 to the active Pt(II) complex (JM118).69 Presently, satraplatin is still inclinical trials against various common cancers.BBR3464, triplatin tetra nitrate, is an unusual trinuclear platinum complex with anoverall charge of +4.70 In phase II clinical trials, lung cancer patients did not show asignificant response to BBR3464 while experiencing toxicity associated side-effects such asneutropenia and diarrhea, therefore, further clinical development was stopped.71Nevertheless, satraplatin as well as BBR3464 have proven that breaking with the lim-iting conditions initially set for platinum drugs for cancer therapy (platinum(II) and cis-conformation) can open up ways to novel lead compounds. Developing novel nonclassicalstructures among current platinum complexes72 and fully understanding their mechanismof action might be the solution to the problem of acquired or intrinsic resistance facingall platinum formulations currently on the market.73 Recent advances in cancer researchhave shown that even the most successful targeted therapies lose potency with time. Evenif an initial response occurs, acquired resistance due to mutations and epigenetic eventslimits efficacy.74 Combination therapy or ”Cocktail Therapy”, the co-administration of twoor more drugs simultaneously, is another approach to promising results; in the majorityof cases an additive therapeutic effect is achieved, because each agent acts via a differentmechanism of action or targets different pathways.75In addition to the vast amount of research that is still undertaken on platinum-basedanticancer drugs, coordination complexes of gallium and organometallic complexes of ruthe-nium have moved into the focus for anticancer therapy since the 1990s. KP46, tris(8-hydroxyquinolinato)gallium(III),76 contains the metal chelating agent 8-hydroxyquinoline,which itself has anticancer properties.77 An oral formulation of KP46 (NKP2235) is sched-uled to start phase I clinical trials in the USA soon.78 An advantage of ruthenium-based11anticancer agents is their effectiveness against metastasis and their potency against a widerange of tumors, which might be due to their two core properties: ruthenium agents are ac-tivated by reduction of the ruthenium(III) core and selectively transported via the transfer-rin pathway,79 their exact mechanism of action, however, remains elusive despite numerousmechanistic hypotheses.80 81Already in the 1950s Dwyer started working on bacteriostatic and anticancer rutheniumcoordination complexes.82 The anticancer agent NAMI-A, imidazolium trans-tetrachloro-(dimethylsulfoxide)imidazole-ruthenate(III), developed by Alessio, Mestroni, Sava andco-workers was the first ruthenium compound to enter into clinical trials followed bythe coordination compound of the Keppler group, KP1019, trans-tetrachlorobis(1H-indazole)ruthenate(III) or its 35-fold better soluble sodium salt (N)KP1339 which is usedin clinical trials for the preparation of KP1019.83 84 Figure 1.3 shows the anticancer met-allodrugs which are currently in clinical trials.With ruthenium and gallium compounds still in clinical trials, arsenic is the only othernon-radioactive metal ion approved for the treatment of cancer. In traditional Chinesemedicine, solutions containing crude arsenic oxide have been administered for thousands ofyears to treat different illnesses. Since the 20th century, injectable solutions of arsenic triox-ide (ATO), commercially sold as Trisenox, are used in the treatment of acute promyelocyticleukaemia (APL).85 Until now ATO is the treatment of choice for APL patients who relapseafter the first line treatment of all-trans retinoid acid (ATRA) combined with chemother-apy, but recent clinical studies have shown that the novel, chemotherapy-free combinationtherapy of ATRA and ATO is not inferior to the standard ATRA-chemotherapy treat-ment in non-high-risk APL patients.86 Darinaparsin, S-dimethylarsino-glutathione (DAR,ZIO101), is a novel arsenic-based anticancer agent currently in clinical trials.8712PtOOH3NH3NPtOOH2NNH2OOOOcarboplatin oxaliplatinApprovedClinical trials (U.S.)PtOOH3NH3NOnedaplatinPtOOH2NNlobaplatinOPtOONNheptaplatinOOsatraplatinPtClCl NH3NH2OOOOH2H2 OOH2PtClClH3NH3NcisplatinFigure 1.2: Anticancer platinum metallodrugs approved and in clinical trials in theUSA.HOHNNHHOOH2NOOOS AsDarinaparsinKP1019NAMI-ARuClCl ClClSNNHORuClCl ClClNNNHHNHNHNNHHN(N)KP1339RuClCl ClClNNNHHNNaFigure 1.3: Anticancer metallodrugs in clinical trials.13Table 1.2: Approved therapeutic metalloradiopharmaceuticalsradioisotope radiation active ingredient trade name indications89Sr β Sr-89 chloride Metastron skeletal metastases90Y β Y-90 ibritumomab tiux-etanZevalin non-Hodgkin’s lymphoma153Sm β Sm-153 lexidronam pen-tasodiumQuadramet osteoblastic skeletal metastases223Ra α Ra-223 dichloride Xofigo castration-resistant prostate can-cer, symptomatic bone metas-tases1.3.1.2 Therapeutic RadiopharmaceuticalsApproved metal-based therapeutic radiopharmaceuticals are often employed as a measureof last resort in advanced stages of prostate cancer, breast cancer, lung cancer, bladdercancer and thyroid cancer where the cancer has spread to the bone tissue, as they areable to deliver cytotoxic doses of ionizing radiation directly to the local targeted tissue.88Metastatic bone cancer is extremely painful and restricts the mobility of patients. Table1.2 provides an overview of injectable salt solutions of radium-223 dichloride, pentasodiumsamarium-153 N,N,N’,N’-tetrakis(phosphonato-methyl)ethane-1,2-diamine and strontium-89 chloride approved for the palliative pain treatment of metastatic bone cancer; yttrium-90is a conjugated antibody used in the treatment of non-Hodgkin’s lymphoma. Furthermore,several formulations of holmium-166, rhenium-186, rhenium-188, bismuth-213, actinium-225 and lutetium-288 are currently in clinical trials against a variety of cancers. β-emittingradionuclides 153Sm, 89Sr, 90Y, 186/188Re and 213Bi have traditionally been used in clinicalradionuclear therapy, because β-particles range far in biological tissue (50−1000 cell diam-eters) which makes them suitable for treating larger or poorly vascularized tumors.89 Incontrast, 223Ra and 225Ac are α-emitting radionuclides with a much shorter effective range(<10 cell diameters).90141.3.1.3 Photochemotherapeutic MetallodrugsPhotochemotherapy is as well referred to as photoradiation therapy, phototherapy, or pho-todynamic therapy (PDT). Currently, PDT is used clinically for the treatment of obstruct-ing esophageal cancer, and obstructing or microinvasive endobronchial non-small-cell lungcancer; additionally, several indications such as prostate cancer and non-resectable or in-operable cholangiocarcinoma are under investigation. As compared to traditional invasivecancer treatments, such as surgery and radiotherapy, PDT is not associated with radicalside-effects, such as surgical removal of parts of the lung or complete excision of the bladder,and can be seen as a quite effective treatment option for localized cancers.91 92PDT is a two-step treatment. First, a photosenitizer agent is administered eithertopically or intravenously. Secondly, after a couple of hours or days, depending on thedrug-to-light interval of the drug, light of a specific wavelength is shone on the area tobe treated. Because the light has to reach the deeper tissue layers, red light is usuallychosen over short wavelength light in PDT.91 The light photoactivates the photosensitizingagent, and via its excited triplet state the agent generates highly reactive singlet oxygen(1O2) from ground state oxygen (3O2) within the tumor blood vessels.1O2 reacts furtherand a variety of reactive oxygen species (ROS) are produced which subsequently reactwith cellular components, leading to vasoconstriction, platelet aggregation, clotting and,ultimately, tumor vascular occlusion.93Porfimer sodium (Photofrin) has been approved as a photosensitizing agent by theFDA in 1995, and the palladium-based padeliporfin (WST11, Tookad Soluble) is currentlyin phase III clinical trials (Figure 1.4).9415padeliporfinporfimer sodiumHNNNHNHOHOOOOONaNaNNNNOPdOOKOO NHSO3OKApproved Clinical trialsFigure 1.4: Metallodrugs for photodynamic therapy (PDT).1.3.2 Antimicrobial and Antiparasitic MetallodrugsSome of the first metallodrugs used in therapy were antimicrobial and antiparasitic agentsbased on arsenic.95 96 In 1907 Breinl and Thomas studied the use of atoxyl, arsanilicacid, for the treatment of trypanosomiasis (sleeping sickness).97 Inspired by their find-ings, Ehrlich and co-workers began their work on arsenic antimicrobials,98 which led tothe discovery of salvarsan and marks the beginning of chemotherapy as outlined in 1.1.Although arsenicals, arsenic-based pharmaceuticals, were widely used in medicine in thebeginning of the 20th century, most of them have been superseded by less toxic drugs. Onearsenic drug that is still used against trypanosomiasis today, despite its severe side effect ofencephalopathy, is melarsoprol, 2-(4-amino)-(4,6-diamino-1,3,5-triazin-2-yl)-phenyl-1,2,3-dithiarsolan-4-methanol (Mel B, Arsobal), discovered in 1949. The WHO lists melarsoprolas a second stage treatment for both forms of human African sleeping sickness.99The other two heavier pnictogens, antimony and bismuth, have been in medical useagainst microbes and parasites as well. Antimony-based drugs have been prescribed againstcutaneous and mucocutaneous leishmaniasis since the parasitic transmission of the tropical16disease was understood in the beginning of the 20th century. The Brazilian physician Gas-par Vianna was the first to treat mucocutaneous leishmaniasis with antimony(III) tartarmetic, potassium antimony tartrate.100 Shortly afterwards, the activity of arsenic againstvisceral leishmanisis was confirmed in Italy and India, which led to the synthesis of anarray of arsenic containing parasitic agents, among them the less toxic pentavalent antimo-nials: Stibosan, Neostibosan and Ureastibamine.100 Other antimony(IV) drugs followed:sodium stibogluconate (Pentostam) and melglumine antimoniate (Glucantim, or Glucan-time); both continue to be in use today despite their toxic side effects and increasing lossin potency due to the growing resistance of the parasite against antimony.101 102While one has to weigh the toxicity against the therapeutic benefit for arsenic andantimony, bismuth is nontoxic and well tolerated at high doses.103 Since the 18th centurybismuth has been used internally as its subnitrate or subcitrate. The history of bismuthdrugs is closely connected to gastrointestinal disorders (Section 1.3.8), but bismuth is alsoco-administered in the fight against the bacterium Helicobacter pylori (H. pylori). A H.pylori infection can lead to gastritis (type-B, bacterial), ulcers in the gastrointestinal tract,and gastric cancer. Bismuth preparations as colloidal bismuth sub-citrate (CBS), sold asDe-Nol, or ranitidine bismuth citrate (RBC), sold as Pylorid or Tritec, are used to treatpeptic ulcers that are often associated with H. pylori. Clarithromycin has been the an-tibiotic of choice to kill the bacterium, but its strength is diminishing with an increasingresistance of the bacterium. This acquired resistance can be partly overcome throughthe co-administration of clarithromycin together with CBS or RBC alone, or in combina-tion with a second antibiotic (amoxicillin) and a proton pump inhibitor (omeprazole). Inthe so-called bismuth-based triple therapy bismuth subcitrate potassium (Pylera) or bis-muth subsalicylate (Helidac) are included in the cocktail together with metronidazole andtetracycline hydrochloride.104 In cases where the two described first-line treatments have17failed, the quadruple therapy can be highly effective against H. pylori : bismuth subcitratepotassium is administered together with metronidazole, tetracycline and omeprazole in onesingle capsule.105 106The external use of tribromophenatebismuth(III), xeroform, because of its antimicro-bial properties was first described at the end of the 19th century. In the past, xeroformwas often used as a substitute for iodoform in the treatment of wounds. Nowadays, oc-clusive petrolatum gauze readily impregnated with 3% bismuth tribromophenate is soldunder the name Xeroform. Bismuth-thiol compounds have been widely studied for theirantimicrobial properties and are currently marketed as a treatment of chronic wounds, suchas diabetic foot ulcers; Microbion, is supposed to prevent the formation of biofilm growthin wounds.107Another metal that has been widely used in the treatment of wounds and their infec-tion managements is silver.108 Topical sulphonamide ointments such as silver sulphadiazine(Silvadene, Silverex, Silvazine, SSD, Thermazene) are applied as a cream formulation oraqueous solution (1% silver salt) to prevent and treat infections of second or third de-gree burns, although it appears that the use of silver preparations in burn treatment istraditionally rooted, and its effectiveness has been questioned109 and criticized110 lately.Since 1976, cerium nitrate-silver sulphadiazine (Flammacerium) has been employed as atopical treatment for most cutaneous burns not undergoing immediate excision;111 it is be-lieved to reduce the inflammatory response to burn injury, decrease bacterial colonizationand provide a firm eschar for easier wound management.112 Figure 1.5 presents approvedantimicrobial and antiparasitic drugs as well as drug candidates currently in clinical trials.Many different applications of silver drugs are currently in clinical trials. Tested treat-ments range from the use of silver fluoride to treat hypersensitivity in teeth to the use ofsilver nitrate in the healing of cysts and abcesses. Silver ions are incorporated into surgical18NNNH2NNH2HN AsSS OHmelarsoprol meglumine antimoniate sodium stibugluconateApprovedClinical trialsxeroformsilver sulphadiazineOBrBrBrOBrBr BrOBr BrBr BiNClHNFeNferroquineCBSBiO OO OOO OO O BiOOOOOnSbOOHONH OHOHOHOHOH OSbOOONaHOOHHOOSb OOHOHOHOOO OONa NaNNH2N SOO NAgAgAgN OONN SOONH2NNAgnFigure 1.5: Antimicrobial and antiparasitic drugs approved and in clinical trials.wound dressing cloths (e.g., Acticoat) and catheters (e.g., SilverSoaker) for infection pre-vention or into textiles for the treatment of acute neurodermitis. Silver alginate (Algidex)is even studied for the prevention of central line infections in very low birth weight infants,while at the same time the toxicity of such silver biomaterials for clinical applications isstill under evaluation.113Two other promising metallodrug candidates that are currently undergoing phase II19clinical trials are the antimalaria agent ferrochloroquine (ferroquine, SSR97193, Figure1.5) and the antifungal agent VT1161. Through the combination of ferrocene with theknown antimalaria drug chloroquine, the resistance against chloroquine, which the malariapathogen Plasmodium falciparum has developed, can be overcome.114 115 VT1161 is cur-rently in phase II clinical trials for the oral treatment of onychomycosis and candidiasis, thischelating agent of unspecified structure selectively inhibits the microbial metalloenzymelanosterol demethylase (CYP51) involved in the synthesis of fungal cell wall sterols.1161.3.3 Antiarthritic MetallodrugsUp to 2% of the global population is affected by the chronic, systemic, inflammatoryautoimmune disorder rheumatoid arthritis (RA). Although the aetiology of arthritis is notcompletely elucidated, it is a complex interplay of environmental and genetic factors thateventually leads to the inflammation of joints, which marks the beginning of the disease.Over time, the inflammatory condition leads to the progressive destruction of the joints,which restricts the movement of patients and leaves them in pain.117In the 1930s, Forestier realized the potential of gold compounds in the treatment ofRA.118 This is another example of a lucky drug discovery: during the years of 1925−1935,which have been described as the ”Gold Decade”,119 gold compounds, mainly gold(I)cyanide and thiosulfates, were used for the treatment of pulmonary tuberculosis, a medi-cal approach that was more based on hope than on evidence.120 Back then, arthritis wasalso believed to be a bacterial infection. Many of the gold thiosulfates still in clinical usetoday were introduced into therapy during the early 20th century: sodium aurothiomalate(Myochrysine, Myocrisin, Tauredon), aurothioglucose (Aureotan, Solganal, Solganol, Au-romyose), sodium aurothiopropanol sulfonate (Allochrysine) and sodium aurothiosulfate(Sanochrysin). All of the named gold(I) compounds are charged, polymeric, and adminis-tered as water-soluble injectables directly into muscle tissue, while auranofin, tetraacetyl-20beta-D-thioglucose-gold(I)-thioethylphosphine (Crisinor, Crisofin, Ridaura), a much newergold(I) compound that received FDA approval in 1985, is a monomeric, neutral coordi-nation compound that is lipophilic and administered orally in capsule form. These golddrugs, classified as disease-modifying anti-rheumatic drugs (DMARD), slow the progres-sion of RA and act by inhibiting several cathepsins implicated in RA, depending on theligand system.121 Approved gold(I) antiarthritis metallodrugs are shown in Figure 1.6.Despite the good therapeutic response gold drugs have shown in the clinic, chrysother-apy, the use of gold compounds in medical therapy, has been controversial over the manyyears gold drugs have been in use. In 1960, the Empire Rheumatism Council conducteda study on the efficacy of chrysotherapy and came to the conclusion that gold drugs dohave a medicative effect,122 but the toxicity of gold(I), its slow clearance from the body,the not clearly defined structure of the intramuscular gold solutions, and the still not fullyelucidated mode of action of gold(I) compounds against arthritis are often cited as coun-terarguments.123 The market of DMARD has seen many new additions during the past tenyears, these are mainly drugs based on active small organic molecules, or biologicals such asmonoclonal antibodies or proteins. This development has seen traditional gold drugs beingpushed down in priority and being prescribed for patients when other drugs have failedto provide sufficient relief. In these cases, practising physicians prefer intramuscular goldpreparations over the orally available auranofin for the treatment of RA,124 solely or incombination with other DMARD,125 because gold is readily absorbed intramuscularly.126It remains to be seen if nanotechnology can revive the area of gold-pharmacology,127and if gold beads in knee osteoarthritis128 or chemo-photothermal treatments129 will reju-venate gold treatment of arthritis.During the 1950s, the intra-articular injection of aqueous solutions of osmium tetroxide,osmic acid, as a chemical synovectomy procedure for the treatment of RA in the knees,21NaOONaOSOAunOHOOHSOHHO Aunsodium aurothiomalate aurothioglucose sodium aurothiopropanolsulfonateSAuSO3NaOHnAu SSO3SSO33 Na+sodium aurothiosulfate auranofinOAcOOAcSOAcAcO Au PFigure 1.6: Approved gold(I) antiarthritis metallodrugs.moved into focus in Scandinavian countries.130 131 This beneficial procedure has been inclinical practice ever since,132 133 and lately it was shown that osmium tetroxide, as a fastmimic of superoxide dismutase, very efficiently catalyzes the dismutation of superoxideanion radical, one of the primary inflammatory species.1341.3.4 Antidiabetes MetallodrugsAn estimated 347 million people worldwide have diabetes mellitus (DM), and the num-bers are increasing globally with more than 80% of diabetes deaths occurring in low- andmiddle-income countries.135 Vanadium salts and coordination compounds have demon-strated various insulin-enhancing and antidiabetic effects; although they are not able tofundamentally substitute for the lack of insulin necessary in type I diabetes, they haveshown to manage blood sugar levels in type II diabetes patients in a convenient oral for-mulation.136In 1899, Lyonnet recorded that the administration of sodium vanadate to his22diabetes patients had a positive effect on their health.137 In 1977, Josephsonet al. realized that vanadate has an inhibitory effect towards phosphatases.138In 1985, McNeill and co-workers reported that adding sodium orthovanadate todrinking water of experimentally diabetic rats could reverse most of the diabeticsymptoms.139 These findings triggered extensive research on the biological func-tions of vanadium itself,140 as well as on vanadium(IV, V) coordination complexeswith a variety of organic ligands such as naglivan,141 maltol, kojic acid, picolinicacid, acetylacetonate, dicarboxylate esters or 2,2’-ethylenebis(nitrilomethylidene)diphenol(SALEN).142 Bis(maltolato)oxovanadium(IV) (BMOV)143 and its ethylmaltol analogbis(ethylmaltolato)oxovanadium(IV) (BEOV)144, depicted in Figure 1.7, arose as leadcompounds, showing an increased bioavailability over vanadyl sulfate in vivo. Both werecarefully studied in animals and BEOV (AKP020) completed clinical trials phases I andII.145 146 Their insulin-enhancing effect is thought to originate from the activation of the in-sulin receptor through the inhibition of insulin receptor tyrosine kinase (IRTK) associatedphosphatases. Unfortunately, although nowadays many vanadium preparations are avail-able over the counter, for example, vanadyl sulfate is advertised as sports supplement (VanaTrace) and even available on, the story of an BMOV antidiabetes vanadiumdrug ends here due to patent expiry and side effects affecting the kidney of the patients;147however, the story of BMOV continues. Under the management of CFM Pharma, BMOV(compound CFM10, Vanadis) is currently being developed into a therapeutic for the pre-vention, stoppage and reparation of secondary tissue injury caused by fire, accidents (roadtraffic, brain trauma) or a heart attack.148A challenge with the first generation vanadium complexes BMOV and BEOV has beenthe high dose necessary to achieve a therapeutic effect. Further generation ligand systemssuch as that in bis((5-hydroxy-4-oxo-4H-pyran-2-yl)-methyl benzoatato) oxovanadium(IV)23BMOV BEOVOOOO OVOOOOO OVOFigure 1.7: Promising metallodrug candidates for the treatment of diabetes.(BBOV) show half the acute oral toxicity compared to BMOV,149 and through novel for-mulations the dose can be apparently lowered by a factor of 1000.150 Vanadium formulatedwith Aonys, for the treatment of metabolic discorders has successfully completed phase Iclinical trials in the European Union. The reverse-micelle emulsion containing vanadiumis applied to the mucous membranes lining the inside of the mouth (buccal mucosa) witha spray pump, which reduces active doses from the mg/kg to the µg/kg level and avoidsside effects associated with high doses of vanadium in the earlier oral formulations.150 Inanimal models, sodium tungstate (Na2WO4) reduced glycemia151 and adiposity152 with-out any significant side effects associated with long-term applications;153 however, sodiumtungstate did not show any efficacy as a pharmacological agent in the treatment of humanobesity.1541.3.5 Antiviral MetallodrugsThere are currently no metallodrugs approved for the treatment of virus diseases, al-though two compounds have successfully proven to be effective against viri in theclinic. Bis(2-methylimidazole)-[(bis(acetylacetone)(ethylenediimine)]cobalt(III), CTC-96(Doxovir) shown in Figure 1.8, has successfully completed phase II clinical trials for thetreatment of Herpes simplex labialis and phase I clinical trials for the treatment of twoviral eye infections (ophthalmic herpetic keratitis, adenoviral conjunctivitis).155 In in vitrostudies CTC-96 has shown to be active against herpes simplex virus type 1 (HSV-1) by24preventing the entry of virus into cells through inhibition of membrane fusion events;156this resonates with findings that (acacen)cobalt(II) complexes bind covalently to histidineresidues of zinc finger domains, and therewith prevent binding of the protein to its recog-nition oligonucleotide.157In 1985, Rozenbaum et al. were the first to administer polynuclear, transition-metaloxyanions, so-called polyoxometalates (POM), to patients with an acquired immunode-ficiency syndrome acquired through infection with HIV (AIDS), and their study showedthat the therapy with compound HPA-23 (ammonium-21-tungsto-9-antimonate) decreasedlevels of HIV in the patients.158 POM are globular or spherical polyanionic structures con-taining bridging oxygen atoms, where the individual anionic charge is carried by the oxygenatoms on the periphery. A variety of POM structures exists (Lindquist, Keggin, Dawson,Anderson, Waugh and Silverton) incorporating a variety of transition-metals (vanadium,tungsten, molybdenum, niobium), all of which inhibit different families of enzymes.159 Thishas shown to decrease activities of HIV, severe acute respiratory syndrome coronavirus,influenza virus, herpes simplex virus and hepatitis B virus in vivo.160 161 Despite their ac-tivity against ribonucleic acid viri and their favourable selectivity profile in vitro, so far noPOM have been advantageous enough to surpass small organic molecule drugs currently inclinical use (such as aztreonam or ribavirin)162, and toxicity, especially deposition in theliver during long-term treatments, has been a concern.163It should also be noted that aluminium and mercury have been used as adjuvants invaccines since the beginning of the 20th century. Aluminium hydroxide, aluminium phos-phate and potassium alum (KAl(SO4)2 · 12 H2O) help to stimulate the immune responsevia poorly understood mechanisms while displaying an excellent safety profile.164 Sodium-2-ethylmercurithio-benzoate (Thiomersal, Thimerosal) is mainly added as a preservative(see Figure 1.8). The ethylmercurithio cation of thiomersal, binds readily to thiol-groups25CoON NONNNHHNCTC-96 sodium thiomersalSOOHgNaFigure 1.8: A promising metallodrug candidate (CTC-96) and a long-time vaccineadjuvant (sodium thiomersal) for antiviral protein structures blocking their enzymatic activity. The many applications of mercuryand its high neurotoxicity have been controversial for years, culminating in January 2013,when governments participating in the WHO Intergovernmental Negotiating Committeeagreed to the text of the ”Minamata Convention on Mercury”, a global legally bindinginstrument on mercury use, opened for signature October 2013.165 Yet, like ”large measur-ing devices where currently there are no mercury-free alternatives,”165 vaccines and dentalfillings will be excluded from the treaty, and the debate on the use of mercury in medicalapplications continues.1.3.6 Metallodrugs Addressing DeficienciesInsufficient concentrations of essential metals lead to deficiency syndromes. Mild forms ofnutrient deficiency caused mostly by micronutrient malnutrition can be treated temporarilyor over longer periods of time with dietary supplements comprising one single metal ion ora mix of several essential metal ions, until levels considered as normal by the medical com-munity are reached. Worldwide, iron deficiency is the most prevalent nutritional deficiencyaffecting more than 2 billion people and is a priority area within the global micronutri-tient initiative program.166 It should also be noted that in industrialized countries, many26dietary supplements are taken as self-medication and not under medical surveillance. Alarge collection of dietary supplements is available in a variety of convenient oral prepara-tions (capsule, drink powder, chewy tablet) with some appearing almost too convenient, asespecially children can be in danger of acute metal intoxication from such preparations.167The demand for dietary supplements for medical and increasingly personal reasons is highand the market lucrative: the vitamin and supplement manufacturing industry is expectedto grow its revenue with a rate of 2.4% annually to a total of 15.8 billion U.S. dollars in2018.168Certain metal deficiencies result from genetic metabolic disorders (acrodermatitis en-teropathica, Menkes disease (MD)) or arise as complications in cases of gastric atrophy orchronic kidney disease. Acrodermatitis enteropathica is an autosomal-recessive metabolicdisorder affecting the uptake of zinc; there is no cure, and patients depend lifelong on zincsupplements to survive.MD is caused by a mutation on the gene encoding Cu2+-transporting ATPase thatleads to a dysfunction of several copper-dependent enzymes and overall copper deficiency.Treatment must start in the first 2−3 months of life to avoid brain damage. Copperhistidine is currently in phase II clinical trials for therapy in Menkes Disease; the copperreplacement is injected directly into the body to bypass the normal route of absorptionthrough the gastrointestinal tract, though severe cases of MD do not gain a therapeuticeffect from copper-replacement therapy.169Severe iron-deficiency (anemia) or vitamin B12-deficiency (Biermer-Addison’s anemia,older name: pernicious anemia) can arise from chronic kidney disease or gastric bypasssurgery, respectively. Treatment options for both diseases are based on replacing themissing metal ion (Fe2+) and coordination complex (vitamin B12) through intravenousinjections. Iron dextran (Proferdex, Dexferrum, InFed) or iron sucrose (Venofer) are ad-27ministered intravenously to treat severe iron-deficiency, while the cobalt(III)-containingcyanocobalamin (CN-Cbl) and hydroxycobalamin (OH-Cbl) are available in form of anasal spray (Nascobal) or parenteral injection (Vibisone) for the therapy of vitamin B12-deficiency.A common problem in hospitalized cancer patients is hypercalcemia, the imbalancebetween the net resorption of bone and urinary excretion of calcium. Through infusions ofgallium(III) nitrate (Ganite) the calcium resorption from bone is reduced, as gallium(III)exerts a hypocalcemic effect.170Osteoporosis is a disease characterized by low bone mass and microarchitectural de-terioration of bone tissue leading to enhanced bone fragility and consequently a higherrisk in bone fractures.171 In the majority of cases, such osteoporotic fractures affect thehips and knees of postmenopausal women, but men and children can as well be struckby osteoporosis. In the treatment of this chronic disease, a variety of nutrients are moni-tored and adjusted as necessary: calcium, magnesium, phosphorous, fluorine, vitamin D,and proteins. Besides the common hormone therapy, calcium supplements (e.g., Calci-trate) and strontium ranelate (Osseor, Protelos) are metal-based drugs employed in themanagement of osteoporosis. Strontium ranelate is approved in some European countriesand Australia for the treatment and prevention of osteoporosis in postmenopausal women,but its use is becoming increasingly restricted after complications in patients with acutevenous thromboemboli,172 hypertonus or other cardiovascular diseases.173 174 Because cal-cium preparations can cause hypercalcemia in patients (possible complications resultingfrom hypercalcemia are discussed in the context of hyperphosphatemia in kidney diseasein Section, new treatment options are needed.281.3.7 Metallodrugs for the Treatment of Cardiovascular DisordersMetallodrugs for the treatment of cardiovascular diseases focus on the regulation of nitricoxide (NO) and dioxygen (O2) in the blood vessels. Vasolidation, the widening of bloodvessels, increases the blood flow in the body. Nitric oxide can be used therapeutically toadjust vasolidation. Sodium nitroprusside, Na2[Fe(CN)5NO] · 2 H2O (SNP), sold as Nitro-press, rapidly decreases arterial pressure and total peripheral resistance.175 One downsideof SNP is the fact that, in parallel with NO, toxic cyanide (CN−) is released into theblood system as well. New NO coordination complexes of ruthenium176 and photoactiveiron complexes177 might eventually overcome this unwanted side effect, and ruthenium NOdonor complexes have also been explored for the treatment of parasitic diseases.178 In somemedical conditions, such as toxic shock syndrome (TSS), the blood pressure is extremelylow and needs to be raised quickly to stabilize the patient. Here, metal complexes thatabsorb excess NO in a swift manner might be useful.179Dioxygen is essential for our survival, but failures in processing of O2 can lead to theformation of superoxide anion (O−·2 ) or hyperoxyl (HO·2) in acidic regions. Both O−·2 andHO·2, are highly damaging ROS that not only protect the cells from invading organismsbut also initiate auto-oxidation reactions in vivo that damage membrane lipids, tissue andDNA. To avoid any of the detrimental chain reactions, the superoxide dismutase (SOD)carefully control and limit O−·2 levels in the cells by catalytically disproportionating it intomolecular oxygen and hydrogen peroxide, the latter being further disproportionated towater and molecular oxygen by glutathione peroxidase or catalase.180 Three types of thesefirst-line-of-defense-metalloenzymes have been characterized: two isoforms of CuSOD/Zn-SOD are located either intracellularly in cytoplasm and nucleus (SOD1) or extracellularly(ECSOD, SOD3), while MnSOD (SOD2) acts in the mitochondria and appears to be theSOD most critical for mammals.181 However, in cases of disease or trauma, the production29of harmful superoxide species might increase above the capacity of allocatable SODs toenforce dismutation.In such cases of extreme oxidative stress, SOD-mimicking metallodrugs may assistthe autoimmune defense of the body in disarming superoxide species. Macrocycles ofporphyrins, phthalocyanines, porphyryzines as well as cyclic polyamines and SALEN co-ordinated to iron(II), copper(II), and manganese(II) have been widely studied as SOD-mimics.182 Compared to copper(II) and iron(II), manganese(II) macrocycles seem less frag-ile because toxic side effects, such as radical formation or Fenton chemistry starting from”free” iron or copper ions, have not been observed for manganese, and the overall toxicityof manganese(II) macrocycles is lower as compared to free aquatic forms of manganese.182Compound M40403, a SOD mimicking manganese(II) (pentaaza)macrocycle shown in Fig-ure 1.9, possesses advanced selectivity, as it can quench superoxide anions while not im-pacting NO, H2O2 or hypochlorite;183 furthermore, it displayed the highest SOD-activityin a comparison study with other manganese(II) macrocycles.184 Phase I/II clinical trialsfor the prevention or reduction of hypotension in patients receiving interleukin-2 (IL-2)therapy with M40403 have been suspended for now, but the possible application of man-ganese(II) macrocycles in pain management in vivo has gained some attention already.1851.3.8 Metallodrugs for the Treatment of Gastrointestinal DisordersMinor stomach pain and digestion problems have been treated with metallodrugs forcenturies. Oral antacid preparations of sodium(I), magnesium(II), calcium(II), and alu-minum(III) as their basic carbonate, hydrogen carbonate or hydroxide salts increase thepH in the stomach and reduce the secretion of acid by gastric cells leading to a neutral-ization of excessive acidity in the stomach and a relief from heartburn symptoms. Brandproducts, for example, Alka-Seltzer (chew tablet containing NaHCO3 and KHCO3), Maalox(solid or liquid formulation of Al(OH)3 and Mg(OH)2), or Rennie (chew tablet containing30M40403MnHN NHHN NHClClNBSSOBiOOOHFigure 1.9: SOD-mimicking macrocycle (M40403), a promising metallodrug candi-date for the treatment of cardiovascular disorders (left); bismuth sub-salicylate(BSS), a widely used metallodrug for the treatment of gastrointestinal disor-ders (right).CaCO3 and MgCO3), as well as a variety of generic antacid products are available over thecounter worldwide and are safe to use even for pregnant women. Magnesium hydroxide, invernacular language known as ”Milk of Magnesia”, is both an antacid as well as a laxative;epsom salt (Mg2SO4) helps in cases of constipation, too. Known in many countries aroundthe world as the ”Pink Stuff”, bismuth subsalicylate (BSS) or Pepto-Bismol, shown inFigure 1.9, was developed in 1901 and is still used to self-medicate an upset stomach andsymptoms of diarrhea, heartburn indigestion and nausea. Despite the fact that BSS is soldacross the globe and has been used safely by many people for over 100 years, its chemicalstructure and mechanism of action are still not fully understood.1.3.9 Metallodrugs as PsychotropicsBipolar disorder (BP) is a psychiatric disorder that demonstrates itself as times of maniaalternating with episodes of depression. Patients showing such strong mood disorders havebeen treated and maintained with lithium carbonate since lithium became recognized as amodern psychopharmacological agent in the 1950s.186 While Eskalith, Lithane are listed bythe FDA as discontinued, Lithobid, an oral lithium carbonate formulation first approved in311979 is still on the market in the USA. Lithium cations have proven to reduce suicide riskand mood swings in bipolar disorder patients; however, the high dose causes a variety of un-pleasant side effects which leave lithium drugs with a narrow therapeutic window betweenbeneficial therapeutic and detrimental toxic effects.187 Moreover, treatment responses tolithium drugs vary, and a genetic component to this has been discussed.188 Offspring ofbipolar parents often inherit their manifested classical mood disorders,189 and response tolithium appears as well to be a family trait.190 Although the mechanism of action of lithiumions has not been completely illuminated, it is understood that they act on multiple levelsregulating neurotransmission and actively modulating cellular and intracellular changes inthe second messenger systems.191 Furthermore, lithium ions exert a neuroprotective effecton amygdala, hippocampus and prefrontal cortical regions.192 This neuroprotective role oflithium bears a tremendous benefit for the treatment of neurodegenerative diseases; such atreatment however would require the life-long intake of sufficient amounts of lithium, andthis could not be reached with the current lithium carbonate preparations without serioustoxic interferences. A novel Aonys, formulation of lithium citrate tetrahydrate claims toachieve similar therapeutic effects as traditional oral lithium carbonate preparations con-taining a 150−400 times lower dose of lithium cation and is currently undergoing clinicaltrials for the treatment of Huntington disease (HD).150 HD is an inherited neurodegenera-tive disorder affecting muscle coordination and cognitive abilities which leads to long-timephysical deterioration accompanied by emotional turmoil and eventually death. Today’sapproved treatment options for HD can only relieve the symptoms of the disease such asinvoluntary movements, anxiety or depression, but NP03, an Aonys, water-in-oil microemulsion drug delivery vector in which a low-dose of lithium has been incorporated, provedto be successful in a HD mouse model,193 and phase I clinical trials have been completed.150321.3.10 Chelating Proligand Drugs1.3.10.1 In the Treatment of Overload DisordersIn all living organisms, metal ion homeostasis consists of a variety of highly complex trans-actions, and some metals are essential for surviving.194 In cases of acute intoxication orchronic disease, the concentration of foreign or essential metal ions increases above nor-mal values recommended by the medical community. Such unwanted metal ions can beredistributed or removed through chelation therapy, which refers to the administrationof chelating agents as drugs. Different from all other metallodrug examples presented inthis chapter, these chelating agents are in principle proligand drugs. To effectively treata metal sequestering disorder, the chelating ligand prodrug finds a free metal ion in thebody, complexes it strongly (sequestering it), and promotes its excretion from the body.Because chelating agents do not selectively complex unwanted metal ions, problems duringthe treatment may arise, because biologically essential metal ions are excreted from thebody as well. In metal intoxication therapy, one differentiates between two medical condi-tions: while the proligand drug should not compete with any natural metal binding sitesin case of chronic intoxication diseases, the drug must excel in its metal-binding propertiesabove any of the natural metal-binding sites in cases of acute metal intoxications to avoidany further toxic uptake of unwanted metal.Acute intoxication is often caused through the adventitious exposure to metals andmetalloids such as aluminum, antimony, arsenic, bismuth, cadmium, cobalt, copper, gold,iron, lead, mercury, nickel, organic tin compounds, thallium or zinc; the acute over-load usually occurs by overnutrition, exposure to pesticides, or environmental or occu-pational exposure.195 167 Research on possible treatments for metal intoxication was inthe beginning fuelled by the need to mitigate the toxicity of lead and of arsenic com-pounds, which were the standard prescription against syphillis in the first half of the3320th century. First, intravenous infusions of calcium or zinc polyamine carboxylic acidssuch as ethylenediaminetetraacetic acid (H4EDTA), and Ca2(EDTA) respectively, shownin Figure 1.10, or H5DTPA, and ZnNa3(DTPA) respectively, were developed, but theirmetal-complexes were poorly absorbed in the gastrointestinal tract. During World WarII, British Anti-Lewisite, 2,3-dimerceptopropanol, H2DMPA (BAL), was used as an anti-dote to the chemical weapon Lewisite, dichlorovinylarsine, the so-called ”Dew of Death”.Meso-2,3,-dimercaptosuccinic acid (H4DMSA) and D,L-2,3,-dimercaptopropane-1-sulfonicacid (H3DMPS) from the 1950s are known for their high biological stability. With thegrowing understanding of chronic intoxication diseases, sequestering agents for iron andcopper ions moved into focus. Desferrioxamine B (DFO), sold as Desferal, a siderophoreisolated from Streptomycin pilosus by the Ciba-Geigy AG in 1960,196 and the orally activedeferiprone, 1,2-dimethyl-3-hydroxypyridin-4-one (DFP), sold as Ferriprox, penicillamine(H2DPA, Cuprimine, Depen) and 2,2,2-trientine (TETA), sold as Syprine, chelate excesscopper and iron well; moreover, they have been used successfully to treat aluminium197and arsenic intoxications.198 Other chelating agents are more specific; in severe cases ofcyanide poisoning the patient is given hydroxycobalamin, a precursor to cyanocobalaminwhich binds cyanide ion and forms cyanocobalamin which is then excreted by the kidneys(Cyanokit, FDA-approved since 2006). The newest iron-chelator on the market approvedby the FDA (2005) and by the EU (2006) for use in children is deferasirox (ICL670), soldas Exjade.199Chronic metal intoxications are genetically conditioned (Thalassemia, Wilson Disease),have been connected to neurodegenerative diseases (Alzheimer Disease, Parkinson Disease),and often eventuate as a side effect of organ failure (e.g., chronic kidney disease-mineralbone disorder).Thalassemia is known as an autosomal-recessive bequeathed disorder manifesting it-34H4(EDTA)H5(DTPA)H3(DMPA), BAL H4(DMSA)H3(DMPS)DFOTETAD-penicillamineICL670OHNHOONHOO OHOOH2NHN NHNH2OHONH2HSDFPNHOOHN N OHOONOOHNHO N NH3+OHONNNOH HOHOOOOOHHSHSOHSO3HHSHSOHHSHSN N NHO OHOOO OOHO HO HOFigure 1.10: Approved metal chelating prodrugs.self in the insufficient production of hemoglobin. Depending on the levels of hemoglobinone distinguishes between a mild disorder or a major defect causing severe anemia; bothlead to iron overload either from iron-rich foods or from complications of frequent bloodtransfusions during treatment. Iron-chelating therapies with multidentate ligands are thetreatment of choice for β-thalassemia, which is also known as transfusion-dependent tha-lassemia.200 Parenteral administered desferrioxamine and oral doses of deferiprone, aloneor combined, are the first line treatment.201Wilson disease (WD) is caused by homozygous or compound heterozygous mutationsin the ATP7B gene (OMIM-606882) on chromosome 13q14 (OMIM-277900). It is an au-tosomal recessive disorder characterized by a disfunction of several copper-dependent en-zymes that leads to a toxic accumulation of copper primarily in the liver and the brain,35resulting in growth defects, neurological defects and psychiatric symptoms.202 Currently,three treatment options for WD are available: two are chelating agents (D-penicillamineor TETA) assisting with the excretion of copper, while oral preparations of zinc acetate(Galzin, Wilzin) work as transmetallation agents and successfully block the absorption ofcopper ions in the intestinal tract.203 Promising metallodrug agents in development, whichinhibit copper trafficking proteins through metal cluster formation, are based on the activecopper-depleting agent tetrathiomolybdate (TM, MoS42−). Ammonium tetrathiomolyb-date [(NH4)2(MoS4)]204 and bis(choline)-tetrathiomolybdate (ATN-224, Decuprate) havebeen tested in clinical trials against WD and cancer, and the latter has received orphandrug designation from the EU in 2013.Many questions and uncertainties are still surrounding neurodegenerative diseases suchas Alzheimer disease (AD) and Parkinson disease (PD). Worldwide nearly 36 million peoplelive with dementia, and this number is expected to grow rapidly over the next forty years;2051% of the world population suffer from motor impairment and dementia caused by PD.Although AD and PD are connected to the longevity of the population and aging processesin the brain and have been assumed to occur sporadically, a monogenic form of PD existswhich occurs in about 5−10% of PD patients and their families as a genetic disorder.206Nowadays, it is widely accepted that dyshomeostasis and overall miscompartmentalizationof metals such as copper, zinc, and iron lead to disfunctions in the AD and the PD brain,with accumulation of copper and zinc in amyloid-β deposits and accumulation of iron inplaque-associated neurons, while the influence of aluminum in AD is still a controversialsubject of ongoing debate.207 There is no cure for these neurodegenerative diseases; currenttherapies merely aim at symptomatic relief (e.g., reduction of tremor), and in good casesthe cognitive decline is decelerated. Potential medicinal inorganic treatment options focuson the chelation and removal of copper, zinc and iron from the brain.208 The fact that the36clioquinolNClOHIPBT2NClCl NOHFigure 1.11: Two metal chelators in clinical trials for the treatment of neurodegen-erative diseases.proligand drugs have to pass the blood brain barrier (BBB) to be able to reach the brain isa major challenge. Otherwise successful classic iron-chelators based on the desferrioxaminemoiety fail to stand up to this challenge, and novel ideas209 such as Feralex, DP-109, JKL-169, or ligands designed the basis of natural products210 have been investigated. Clioquinol,5-chloro-7-iodo-quinolin-8-ol, a known oral antifungal and antiprotozoal drug, crosses theBBB and inhibits zinc and copper ions from binding to amyloid-β (Figure 1.11).211 It hascompleted a pilot phase II clinic trial for the treatment of AD through chelation therapy,in which patients reported improved cognition and showed lower plasma levels of amyloid-β42.212 In addition, its metal-sequestering action is useful in the managing of PD, becausechelating free metals in the brain prevents metal-mediated production of hydrogen peroxideand other radical oxygen species. A second generation of such a metal-protein attenuatingcompound (MPAC) with improved metal-peptide attenuating effects is the orally available8-hydroxyquinoline (PBT2, Figure 1.11) which has completed phase II clinical trials213without showing a significant reduction in the levels of β-amyloid plaques in the brains ofAD patients, but will proceed into phase IIb trials for the treatment of HD in the USA,while clinical trails against PD are in preparation.214 PBT2 however is not acting as ametal chelator but rather as an ionophore; it increases the permeability of membranesleading to a more normal neuronal function.21537Patients with a chronic kidney disease-mineral and bone disorder (CKD-MBD) showabnormalities in their calcium and phosphorus metabolism as well as in their parathy-roid hormone (PTH) and vitamin D levels; in addition, they show abnormalities in boneturnover, mineralization, strength and growth (e.g., calcifications of adjacent tissue).216The progressive loss of kidney function leads to increased serum phosphate levels, andhyperphosphatemia is one of the clinical consequences that accompany end stage renaldisease. A variety of treatment options for CKD-MBD are available targeting the downregulation of phosphate levels without disturbing levels of calcium ion; many of these phar-macological treatments are metal-based.217 Aluminium hydroxide (Alu-Cap) is a potentand cheap phosphate binder but highly insoluble, often leading to constipation and an over-all increased risk of aluminium toxicity; therefore, calcium salts such as calcium carbonate(Calcichew, Titralac) and calcium acetate alone (Phosex, PhosLo) or in combination withmagnesium carbonate (Renepho, OsvaRen) have almost superseded aluminium hydrox-ide in the treatment of hyperphosphatemia, but such associated risks as hypercalcemiaand calcification are observed in the clinic.218 Lanthanum carbonate (Fosrenol) avoids anyproblems of calcium overloading or aluminium toxicity and does not cause digestive issues;in addition, it conveniently requires the intake of fewer pills per day than the leading smallorganic molecule drug sevelamer (Renagel, Renvela).219 It should be noted that all thesemetallodrug therapies focus on binding any excess phosphate, while several biological ther-apeutics are available on the market that selectively target the vitamin D receptor and theparathyroid gland (e.g., Zemplar). In the Treatment of Cancer, Microbial and Parasitic InfectionsAccording to the nutritional immunity theory, parasites or bacteria in a host can be killedby reducing nutrients and therewith depriving the invading organism, which under suchlimiting conditions cannot proliferate, and eventually dies. The use of iron-deficiency,38especially in the context of malaria prevention, has been the controversial for more thanforty years.220 221 Although such iron chelating prodrugs as desferrioxamine and deferipronehave been used against malaria in clinical studies, the data has been evaluated as insufficientfor supporting the use of iron-chelating agents as adjuncts in the treatment of malaria.222In addition, macrocyclic chelating agents such as nonactin and valinomycin take thisline of defence by complexing potassium ion, while crown ethers such as 15-crown-5,dibenzo-18-crown-6, and 24-crown-8 provide a good fit for the smaller sodium ion. Af-ter the macrocycle has wrapped up the metal ion, the coordination complex is transportedthrough the cell membrane. In vitro studies have shown that in this way these agentschange the permeability of the membrane to potassium ions, disrupting oxidative phospho-rylation and inhibiting the processing of some proteins resulting in an overall antimicrobialeffect.223A spin-off from the POM research presented in Section 1.3.5 led via the de-tected anti-HIV activity of bicyclams to another serendipitous drug discovery; themetal-chelating agent AMD3100, [1,1’-[1,2-phenylene-bis(methylene)]-bis(1,4,8,11-tetra-azacyclotetradecane)octahydrochloride dihydrate] (JM3100, Plerixafor, Mozobil) depictedin Figure 1.12, is an EMA and FDA approved selective CXCR4 chemokine receptorantagonist used to mobilize hematopoietic stem cells to the peripheral blood for collectionand autologous transplantation in patients with non-Hodgekin’s lymphoma or multi-ple myeloma.224 Vorinostat, N-hydroxy-N’-phenyloctanediamide (Zolinza), is a histonedeacetylase inhibitor approved for the treatment of cutaneous T-cell lymphoma (CTCL).225Other Zn-chelating agents currently in clinical trials against cancer are PXD101,(2E)-3-[3-(anilinosulfonyl)phenyl]-N-hydroxyacrylamide (Belinostat), and givinostat,6-[(diethylamino)methyl]-naphthalen-2-yl-[methyl(4-hydroxycarbamoyl)phenyl]-carbamate.39NH HNNNHNH HNHNNAMD3100NHOHOSNHO OPXD101O NHNO NHOHOgivinostatHN NHOHOOvorinostatApprovedClinical trialsFigure 1.12: Metal-chelators for cancer therapy.1.4 Strategies for the Design of MetallodrugsMany of the metallodrugs currently on the market have been discovered by chance, asthe discovery stories for some of the prominent metallodrugs in previous sections reflect.On the basis of such first generation serendipious hits (e.g., cisplatin), further generationsof drugs have been developed by carefully studying, analyzing, and partly guessing themechanism of action as well as reasons for unwanted side effects of the first generationdrug to be able to iron out these flaws in the second and often third generation of drugmolecules. Staying with the example of platinum drugs for cancer therapy, these would becarboplatin (second generation), satraplatin (third generation) and subsequent agents suchas nedaplatin, lobaplatin and heptaplatin. During the past years, medicinal bioinorganicchemists have focused strongly on moving the drug development process from the initialserendipity discoveries, which undoubtedly laid the foundation for this field, to a morerational drug design process. This section describes the drug design and development40process in general including advantages and challenges that arise from bringing a metal ioninto the game.1.4.1 Finding a Druggable TargetA rational approach of designing a metallodrug is in its principles not very different fromdesigning a drug based on a small organic molecule or a biological molecule. The first stepis the overall identification of a disease target and the specific elucidation of a moleculartarget associated with this disease’s etiology and pathology.To define putative targets, traditional medicinal chemists employ the disciplines of ge-nomics and proteomics. Complementary to genomics and proteomics, bioinorganic medic-inal chemists call on the growing field of metallomics to support their target validation.Metallomics refers to the characterization of the entirety of metal and metalloid speciespresent in a cell or tissue type, as well as their interactions with the genome, transcrip-tome, proteome, and metabolome.226 The ultimate goal of this novel field is to understandcomprehensively metal uptake, trafficking, function, and excretion in biological systems.227Species of interest for metallomics are complexes of trace elements and their compoundswith endogenous or bioinduced biomolecules such as organic acids, proteins, sugars, orDNA fragments.228Seven of the twenty-one amino acids, the building blocks of peptides and proteins,possess appropriate donor atoms such as nitrogen, oxygen, or sulfur in their side-chains,giving them the opportunity to interact with a metallodrug. These seven amino acids areaspartic acid, cysteine, glutamic acid, histidine, lysine, methionine, and tyrosine. Moreover,specific metal-binding sites are located in the N-terminus of many naturally occurringproteins; one of these is the amino terminal copper(II)- and nickel(II)-binding ATCUN-motif, which is formed in proteins from a histidine in the third position, its proceedingresidue, and the free N-terminus, providing a total of three N-atoms for interaction with a41metal.229 230 If a metal ion is purposefully administered to the human body in form of ametallodrug, the metal ion can bind to a protein, possibly resulting in an altered proteinstructure and therewith loss or alteration of its function. On the other hand, the metal ionat the core of metalloenzymes is essential for their catalytic activity, for example, Zn2+ inzinc-enzymes or Cu2+;231 if a proligand drug is administered, the chelating agent can bindstrongly to the metal ion at the center of the metalloenzyme and remove it, which rendersthe metalloenzyme inactive. Emerging protein targets for metallodrugs have been recentlyreviewed.232Another target for metallodrugs is DNA itself. All four bases contain nitrogen andoxygen as donor atoms to which a metal ion can bind, the N-7 of adenine and guanine inthe major groove of double-helical DNA being among the most important binding sites. In acoordinative, covalent binding interaction with DNA, a metal ion can connect both strandsto form an intrastrand crosslink, bind solely to bases on the same strand in an interstrandcrosslink fashion or build a link with amino acid side-chains of a neighbouring protein, aso-called protein-DNA crosslink. Moreover, small, planar and mostly hydrophobic drugmolecules can slide into the inside of the helix where they can intercalate between the basepairs in a non-covalent fashion. So-called dual mode DNA binding metallodrugs not onlybind covalently to the DNA but additionally intercalate as well, while other metallodrugsselectively target a specific sequence.Biological targets of metallodrugs have been comprehensively reviewed and criticallyevaluated quite recently.233 234 Nucleic acids, proteins, and DNA are commonly expressedby all kind of cells and are, therefore, rather unselective targets. In the current post-genomic era, in which the life sciences are being transformed by gene sequencing knowledgeand advanced techniques, metallodrug research is as well progressing towards selective tar-geting. A specific tumor type and its unique chemical pathways235 or one molecular target42in parasite biology236 can be clearly identified and selectively attacked with a specificallydesigned metal-based molecule.Besides macromolecular structures such as proteins and DNA, metal ions can reactwith various other small molecules contained in the body’s fluids.237 In human blood theconcentration of chloride amounts to 104 mM. In addition to chloride, human body fluidscontain phosphate and carbonate in high concentrations, two other anions which couldpotentially bind the metal ion delivered with the metallodrug molecule. Because manymetallodrugs are administered intravenously, it is important to understand what happensto the metallodrug molecule once it is surrounded by a variety of small anions such aschlorides, phosphates or carbonates.Research in the area of biophysical chemistry increasingly focuses on the improvedunderstanding of metal ion metabolism in the human body, often coupled with a varietyof pharmacological methods. This has led to diverse novel bioanalytical methodologiesfor studying the mode of action of metallodrugs and therewith identifying their specifictargets.238 For example, such approaches that comprise a variety of biophysical and phar-macological techniques span probing the interaction of metal ions with proteins239 to theapplication of different analytical techniques such as mass spectrometry,240 other hyphen-ated techniques,241 and capillary electrophoresis.242In classic drug development, it is critical to gain as much detailed information aboutone specific target as possible, because all this information can flow into the design ofa drug molecule, which has a perfect fit and therefore preference for binding to a singletarget instead of interacting with various other molecules and their competing bindingsites in the body, resulting in less unwanted side effects. On the other hand, such a onemolecule-one target approach not only limits the number of possible side effects, but aswell limits the ability to combat complex neurodegenerative diseases such as Alzheimer or43Parkinson disease, for which more radical approaches of multifunctional metal chelatorsaiming at multiple neurological targets are needed.243 Another recent example from ADresearch has shown the danger that lies in developing a drug candidate on a diffuselydefined target: the drug candidate tramiprosate (Alzhemed), which had been designed toselectively block the aggregation of amyloid-β plagues, was stopped in phase III clinicaltrial stage, when the statistical model for evaluating the drug based on cognitive efficiencydata and brain volume data showed large variations and was therefore unable to supportclinical efficacy.2441.4.2 The Advantage of Variety: Designing Metal Complexes for thePerfect FitCompared to the structural features that can be built around a metal ion, the possibil-ities of small organic molecules and biological molecules seem almost drab. While suchdrug molecules rely purely on carbon, their binding geometry in space is dictated by theprinciples of hybridization − sp (linear), sp2 (trigonal-planar) and sp3 (tetrahedral) −compared to the diverse geometry in 3D space open to metal ion-containing drugs. Besideslinear, square-planar, and tetrahedral geometries, pyramidal, trigonal bipyramidal, andoctahedral shapes can be created, the latter being of tremendous importance for biologicalprocesses. With the growing number of substituents around the metal center, the varietyin stereoisomers and stereochemical flexibility in general increases to open up a diversityin 3D structures.245 Modification of these substituents or ligands tailors them to manifoldfunctions and specific targets.246 Although ligand exchange reactions are often calculatedmechanisms of action in medicinal inorganic chemistry, the metal ion itself is at the heartof action. The metal ion orchestrates the ligand coordination according to precise 3D con-figurations. With its fine-tuned redox-chemistry, the metal ion can participate in biologicalredox actions, and transition metals such as ruthenium or iron, which have multiple stable44Figure 1.13: Overview of various design possibilities for metallodrugs.oxidation states, offer catalytic potential. Moreover, the metal ion introduces a distinctspectroscopic handle that can be exploited in a variety of techniques, some of which arenot accessible for purely organic molecules, for example, Mo¨ssbauer spectroscopy. In addi-tion, a metal ion can add magnetic properties to the metal-ligand complex and, if needed,radioactivity for utilizing elements with appropriate isotopes. Despite their described struc-tural complexity, metal-ligand complexes are still quite small and lightweight as comparedto some macrocyclic biological organic molecules. All of these tuneable design components(see Figure 1.13) create indefinite possibilities for metal-ligand complexes with novel andunprecedented properties.247An extensive study by the U.S. National Cancer Institute (NCI) from 2005 mirrors the45design diversity for metallodrugs for the treatment of cancer. About one-thousand metal ormetalloid containing compounds with potential anticancer activity were included. The aimof this study was to establish correlations between specific cytotoxic responses and differ-ential gene expression profiles to expand the knowledge base for evaluating, designing, anddeveloping new target-specific metallo-anticancer drugs. Although the study confirmed alarge variety of possible mechanisms of action for metal-based compounds, four fundamen-tal response classes were identified on the basis of preference of (1) binding to biologicalsulfhydryl groups, (2) chelation, (3) generation of reactive oxygen species and (4) produc-tion of lipophilic ions.248 These four categories are extremely broad, but only demonstrateonce more the variety of targets affected by metallodrugs. Similarly, one metallodrugmight be active against a variety of diseases. Gold(I) compounds (sodium aurothiomalate,auranofin) that have been traditionally employed in the therapy of rheumatoid arthritis(Section 1.3.3) are becoming more and more known for their anticancer properties, whichare currently being tested in clinical trials.Instead of such drug repositioning, one can take inspiration from naturally occurringmolecules and carefully study the binding pocket of proteins to which they bind, perhapswith the assistance of computational methods,249 to discover a specific synthetic structurethat nicely docks onto the protein. For example, PIM kinases are enzymes located on theproviral insertion site of the moloney murine leukaemia virus that can be selectively inhib-ited by inert half-sandwich ruthenium-indolocarbazole complexes.250 These organoruthe-nium complexes have demonstrated an extremely good fit in the adenosine triphosphate(ATP) binding pockets of PIM1 and PIM2 inactivating these PIM kinases, which in returnleads to restored apoptosis in drug-resistant cancer cells.251 The high potential of neutralor cationic arena ruthenium complexes for the development of anticancer metallodrugs hasbeen widely discussed;252 organometallic arene-ruthenium(II) complexes such as RM175,46RM175 RAPTA-CClRuNH2H2N PRuClClN NNNP309RuCON NFHOHNO OPF6Figure 1.14: Organometallic ruthenium(II) complexes with promising anticancer-activity.[(η6-biphenyl)-(ethylene-diamine)ruthenium(II)chloride],253 254 RAPTA-C, [(η6-para-cymene-(1,3,5-triaza-7-phosphaada-mantane)-ruthenium(II)dichloride],255 256 and NP309,[(η6-cyclopentadiene)-[N,N-(9-hydroxy-pyridol)-(2,3-a-pyrrolo)-(3,4-c-carbazole)-(5,7-dione)]-ruthenium(II)carbonyl],250, depicted in Figure 1.14, have shown promising resultsin various in vitro and in vivo studies and continue to fuel research into organometallicOs(II) and Ir(III) complexes.257Certainly, the perfect fit for a defined target in the body is important, but first thedrug molecule has to reach its target. Many metallodrugs are injected into the bloodstreamor the muscle tissue, and during their passage through the blood and eventually intothe cells, the drug molecule comes in contact with biological substances that can modifyits composition through ligand exchange reactions; often serum proteins are their firstbinding partners.242 For many metallodrugs, human serum albumin (HSA), 0.65 mM, actsas a reservoir, which can be exploited for delivery purposes. HSA-conjugates have beenshown to accumulate in tumor tissue due to their enhanced permeability and clearanceretention effect.258 Another protein with a strong affinity for metal ions is apo-transferrin(Tf), 0.037 mM, which can not only bind two equivalents of iron(III) but interacts witha variety of main group,259 transition group260 and lanthanide261 metal ions. When the47concentration of a drug in the plasma does not correlate with its expected therapeuticeffect, it must be assumed that the supposed drug molecule was only a prodrug and theactive drug metabolite was only generated in a biological interaction in vivo.262 This is notnecessarily negative, as prodrugs can be an efficient way to deliver an active compoundacross barriers,263 for example, the successful cis-platinum drugs used in cancer therapyare prodrugs.Because of their diverse structures, metallodrugs can act through different mechanismsof action compared to small organic molecule or biological drugs, such as targeted ligand-exchange with biological molecules in vivo, giving a variety of novel drug targets andtransport pathways. This notion gives hope that for therapeutic areas in which drug resis-tance is growing, metallodrugs can overcome developed resistance;264 examples of overcomedrug resistance from malaria research265 and cancer research266 suggest this hope may notbe in vain. In Chapters 3 and 4 of this thesis, the possibility of overcoming the growingresistance of microbes to known antibiotic drugs with coordination complexes of Ga3+ andCu2+ will be explored.1.4.3 Exploring the Druggability of the TargetOnce a target is found, its druggability needs to be explored. Therefore, screening sys-tems are established that test the novel potential therapeutic agent against the desiredtarget. In drug research, such testing of larger compound libraries is often performed inthe form of high-throughput screening or high content cell-based assays. There are twocomplementary common approaches to validate a target. In the chemical approach, smallmolecule inhibitors can be used to modulate the functional activity of a target, provid-ing insights into chemical evidence for druggability of the target or favourable selectivetoxicity against the pathogen versus the host (cell, tissue or whole animal). The geneticapproach can be classified into target gene knockout (often mouse models) and target ri-48bonucleic acid (RNA) knockdown methodologies, often using small interfering ribonucleicacid (siRNA).267Because drug discovery is in general an expensive process, it is important to recog-nize early problematic drug candidates and undruggable molecular targets that will mostprobably fail (many genes are for example not druggable) to save costs and in return allowthese resources to be used for the drug candidates that will most probably succeed. Themost critical point in this regard is improving the suitability and robustness of the agentsthat enter the clinic.268 This relates directly to the thermodynamic and kinetic stability invivo. It is essential to understand how the drug molecule affects the body, as well as how inreturn the body effects the drug molecule. Developing such an understanding is even morechallenging in metallodrug-research, as metallodrugs can interact with a variety of biolog-ical molecules inside the body as was illustrated in Section 1.4.2. As an example from theclinic, the in vivo interaction of Fe3+ with the FDA approved anticancer drug doxorubicinand the newly developed anticancer agent vosaroxin, which is currently in phase III clinicaltrials, will be discussed in detail in chapter Pharmacokinetics: Thermodynamic Stability and Kinetic LabilityA drug that is unable to reach its molecular target in the body possesses poor pharma-cokinetics. The pharmacokinetic characteristics are defined by the concept of absorption,distribution, metabolism and excretion (ADME concept) properties of the potential drugmolecule. Knowledge of ADME concept properties of the drug and its metabolites in hu-mans (as well as in animals used for the toxicology assessments) is crucial to understanddifferences in effect among species and to optimize drug dosing in general.It appears that the pharmacokinetic characteristics of a drug are strongly related toits physicochemical properties such as solubility, lipophilicity and stability, which can bedetermined by measuring the octanol-water partition coefficient (log P ) and pKas. These49measurements are useful in predicting protein binding, tissue distribution, and absorptionin the gastrointestinal tract.269Lipinski defined270 five rules for the lipophilicity, and therewith a measurable value forhow easily a molecule can pass through the blood brain barrier, from empirical experience.According to ”Lipinski’s Rule of 5”,270 poor absorption and permeation are more likelywhen the molecule has (I) more than 5 hydrogen bond donors, expressed as the sum of OHsand NHs, (II) more than 10 hydrogen bond acceptors, expressed as the sum of nitrogen andoxygen atoms in the molecule, (III) a molecular weight of over 500, and (IV) a partitioncoefficient of log P > 5.270 Although Lipinski’s rules are helpful to evaluate and identifyorally bioavailable drugs, bioinorganic medicinal chemists should bear in mind that theserules have been empirically found in approved organic small-molecule drugs and may notnecessarily apply to metallodrugs in the same way.271The possibility of interactions of a metallodrug molecule with other biomolecules whichare available at high concentrations in the human body has been discussed in Section1.4.2. How likely a metallodrug is to undergo a structure-altering process such as ligandexchanges or transmetallations is determined by the strength of the metal-ligand bond(s)under physiological conditions.272 Stability constants (log βn), as defined in equation 1.1,are a measure of metal chelation, in which M represents the metal ion and L symbolizesthe free ligand.log βn = log ([MLn][M ][L]n) (1.1)This principle relationship can as well be expressed as protonation constants (Ka, pKa),dissociation constants (Kd, pKd), effective binding constants (Keff ) or free metal ionconcentration pM .273During the drug development process the stability of the metallodrug candidate againstthe two dominant proteins, apo-Tf and HSA, under biological conditions in 0.15−0.16 M50aqueous sodium chloride solution at 37 oC can be measured and compared with evaluationsfrom potentiometry or spectrophotometric studies.For orally administered drugs, adequate absorption and bioavailability must beachieved,274 which seems to be a challenge for metallodrugs. Many metallodrugs are givenintravenously due to their limited solubility in oral formulation, the need to administeronly small amounts of metal ion to avoid toxic side effects, and the lack of stability ofmetal-ligand complexes on their way through the various pH levels in the stomach and in-testines. Novel approaches for the delivery of metallodrugs are required and have recentlybeen reviewed;275 among them nanoparticles open up new vistas of improved delivery, celluptake, and targeting.234 276 Micelle emulsions150 and liposomal formulations also appearpromising.277 2781.4.5 Pre-Clinical StudiesBesides target validation and pharmacological assessment, a first set of studies on the invitro metabolism of the drug candidate and some initial toxicity studies are often includedin the preclinical assessment of whether a drug candidate is suitable for the clinic or not.Drug metabolism can be studied on liver cells (heptatocytes) and cytochrome P450 en-zymes, while cell permeability is often tested on MDCK and/or human colon colorectaladenocarcinoma cells (Caco-2 cells). The Caco-2 cells permeability assay has been widelyadopted for understanding the gastrointestinal drug absorption process. At this stage, tox-icity is evaluated in the in vitro cytotoxicity studies and eventually single acute dose studiesin animals (mouse, rat, dog) to establish the maximum tolerated dose (MTD).274 Drug de-velopment candidates that satisfy these initial tests and any further extensive toxicologicalstudies are deemed safe enough to proceed into clinical trials.511.4.6 Clinical StudiesTesting of the drug candidate in the clinic starts with phase 0. This exploratory inves-tigational new drug study of a few healthy individuals, in which these volunteers receiveless than 1% of the therapeutic dose of the investigational drug over the course of max-imum seven days, is followed by phase I during which the preliminary pharmacokineticsand toxicology are evaluated in healthy individuals in a primarily safety screening.The drug candidate is tested for the first time in patients suffering from the targeteddisease in phase II clinical trials. At this stage, the efficacy of the investigational drug isestablished against a placebo. The decisive challenge in phase II clinical trials lies in thedesign of the study itself. How can the desired outcome of the study be clearly described?What is the definite endpoint of success? Which patients can be recruited for the study?Often these questions are heatedly discussed until the respective proof-of-concept criteriafor a clinical study finally can be clearly defined. Especially in oncology and in diseases ofthe central nervous system (CNS), it has proven to be difficult to establish clear efficacysignals. For example, in the early times of anticancer drug research, the efficacy goal wasto shrink the tumor tissue, and for metallodrugs such as cisplatin this was an acceptable(and facile) way to evaluate the drug’s performance. In contrast, new drug developmentssuch as the ruthenium-based compounds, NAMI-A and KP1019, do not aim exclusively atreducing the malignant tissue but, moreover, are targeting angiogenesis to avoid metastasis.In addition, financial factors must be considered, because investors may fear that narrowlydefined indications translate into a narrow market for the drug, which, coupled with safetyconcerns, was the reason the clinical development of MRI contrast agent ferumoxide, basedon iron oxide nanoparticles (Combidex, Sinerem), was halted.34These practical examples illustrate that the design of a clinical trial to prove the princi-ple action of the new drug is of vital importance and must be addressed already in the early52stages of the drug development process. Should it prove to be impossible to demonstratethe desired action of the drug in the clinic through a carefully defined screening procedure,the best idea for a drug is worthless, because government agencies such as the FDA or EMAexpect clear and complete data to grant approval. Attrition rates in 2011−2012 show thatefficacy was stated as the cause of failure in 59% of all drug development projects killedin phase II clinical trials and 52% in phase III clinical trials, while the overall failure rateswere highest in the therapeutic areas of oncology (29.5%) and CNS (14%),279 which oncemore illustrates the difficulty to establish clear efficacy signals in these therapeutic areas.The major costs of clinical trials occur in phase III studies that are performed to confirmthe safety, established in phase I, and the efficacy, established in phase II. This is usuallythe final step before the application for approval of the drug candidate can be filed withthe respective government agencies.1.5 Conclusion & Thesis OutlookWith a good overview of the diagnostic and therapeutic metallodrugs currently approved bythe FDA and EMA, an impression of the biological challenges of metallodrug research anddevelopment as well as potential strategies to overcome these, we now set out to study thecoordination chemistry of antimicrobial and anticancer agents. In Chapter 2, the drug classof the (fluoro-)quinolones is introduced, and the properties of nine members of this classare discussed in detail. In addition, the question of their stability in Iso-Sensitest broth, agrowth medium for bacteria, is addressed, and their antimicrobial susceptibility against thefive most commonly reported causative pathogens in nosocomial diseases is tested in vitro.Results once more show the growing resistance of microbes against commonly prescribedantibiotic drugs, and we, therefore, take these alarming results as motivation to developnovel antimicrobial agents based on a coordination chemistry approach in the following53two chapters.In an attempt to combine the anticipated antimicrobial properties of gallium(III) ionswith the antimicrobial potential of the nine quinolones presented in Chapter 2, noveltris(quinolono)gallium(III) complexes and their respective iron(III) analogs are synthe-sized, chemically characterized, and their antimicrobial properties tested against the fiveselected bacteria, in Chapter 3. The following chapter, Chapter 4, expands on this strategyby acting on novel designs in quinolone antimicrobial research and complexing these ligandsto copper(II), another metal ion that has been explored for its antimicrobial potential.The agent vosaroxin is currently under development by Sunesis Pharmaceuticals, Inc.for the treatment of cancer; it is as well a quinolone. The leading anticancer drug onthe market nowadays is doxorubicin, which is not only known for its anticancer potencybut as well for its cardiotoxicity, a common side-effect of treatment with this drug that isbelieved to be partially caused through the interaction with the essential metal iron in vivo.Chapter 5 discusses the behaviour of vosaroxin and doxorubicin towards iron(III) basedon results of a comparative spectrophotometric stability study under in vivo conditions.Moreover, the coordination chemistry of vosaroxin is explored further through the noveltris(vosaroxacino)iron(III) and its respective gallium(III) complex.Chapter 6, summarizes the research results of this thesis and modestly addresses thefuture of the field of metallodrugs in medicinal inorganic chemistry.54Chapter 2Introduction to Quinolone AntimicrobialAgentsIn this chapter, the drug class of quinoline antimicrobial agents will be introduced. Nineselected quinolones will be characterized, their stability in Iso-Sensitest medium will bediscussed, and their antimicrobial susceptibility will be evaluated.2.1 Quinolone Antimicrobial AgentsWith his discovery of the bactericidal properties of the naphthyridine agent nalidixic acidin 1962, George Y. Lesher laid the foundation for a highly successful class of antimicrobialagents.280 Quinolones have become a major group of popular synthetic antibacterial agentswith activity against a diversity of Gram-positive and Gram-negative bacteria.281 Due totheir excellent penetration of most bodily tissue fluids, their clinical use dominates inbacterial infections of the genitourinary, respiratory, and gastrointestinal tracts,282 whileciprofloxacin hydrochloride is also approved by the FDA for treatment of the inhaled formof anthrax.283Quinolones have either a quinoline or a 1,8-naphthyridine aromatic ring at their core55Core structuresN NHOOOHNHOOOH1st generation 2nd generation 3rd generation1,8-naphthyridine quinolineN NOOOHnalidixic acid (Hnxa)NOOOHoxolinic acid (Hoxa)OONN NOOOHpipemidic acid (Hpia)NHNNOOOHnorfloxacin (Hnofx)NN NOOOHenoxacin (Henox)NOOOHciprofloxacin (Hcipro)NHNHNFFNHNFNOOOHlomefloxacin (Hlomx)NHNFFNOOOHfleroxacin (Hflex)NNOOOHlevofloxacin (Hlevox)NFFNN OFF2344'56788'567 8' 2344'Figure 2.1: Aromatic core structures of quinolone antimicrobial drugs and molecularstructures of the nine quinolone agents selected for this study.56(Figure 2.1). The carboxylic acid group on Car3, together with the carbonyl group onCar4 are key in their antimicrobial mode of action.284 Although their exact mechanisms ofaction still remain elusive, the quinolones most probably interact through hydrogen bind-ing via the 3-carboxyl-4-oxo modality with their microbial enzymatic targets, DNA gyrase(topoisomerase II) or/and topoisomerase IV, depending on the bacterium.285 iThese enzymes are essential for orchestrating the supercoiling of cellular DNA.286 287 288Quinolones bind to the formed enzyme-DNA-complex rendering the respective enzyme in-active,289 and this disruption of the supercoiling process by the quinolone is bactericidal.290The influence of the bivalent metal cations Mg2+ and Ca2+ in the process of establishingcontact between the quinolone molecule and the enzyme-DNA-complex has been discussedfor many years;291 292 293 recent studies state a water-metal bridge between the quinolonemolecule and the topoisomerase IV to be crucial for this interaction.294Over the past fifty years, a large array of activity related quinolone-core based struc-tures has been developed with fine-tuned differences in their spectrum of activity and po-tency as well as adverse side-effects.295 296 297 298 These are commonly sorted into differentdrug generations. In the late 1970s, quinolones of the second generation, such as norfloxacinand enoxacin, bearing a fluorine in the 6-position, showed improved enzyme inhibition anda broadened spectrum of activity, leading to the dogma that the 6-fluorine was an essentialfeature and stamping another name for this drug class, fluoroquinolones.ii Even nowadays,quinolone antibacterial agents still gain attention,299 and medicinal chemists continue totweak their chemical structure, and therewith pharmacological properties, to develop noveliFor quinolone targets, bacteria can be sorted into three categories (I) Gyrase only: human pathogensthat lack a close homologue to topoisomerase IV, e.g., Mycobacterium tuberculosis, Helicobacter pylori ; (II)Gyrase more sensitive a target than topoisomerase IV: Gram-negative bacteria, e.g., Escherichia coli ; (III)Equal sensitivity for Gyrase and toposimerase IV: Gram-positive bacteria, e.g., Staphylococcus aureus.285iiBecause this study comprises (fluoro-)quinolones from different generations, including partly fluorinatedand non-fluorinated derivatives, compounds based on the core structures shown will be simply referred toas quinolones, including mono-, bi- and tri-fluorinated species (Figure 2.1).57antibiotic drugs against the looming threat of growing antimicrobial resistance.300Although common clinical quinolone drugs have been widely reviewed, and their synthe-ses, determination, and pharmacological data, as well as clinical data have been discussedfor the past fifty years, we were surprised to find no complete set of chemical characteriza-tion data in the literature. Searching the published data, it proved difficult to find reliablechemical characterizations, as reported data came from many different sources and had beenobtained in different laboratories, on a variety of instruments, under various conditions,and in different decades according to the best analytical standards of that time. A combi-nation of all these factors stands presumably behind the fact that conflicting informationon quinolone compounds has been published, particularly for ultraviolet-visible (UV-Vis)and NMR measurements. For these reasons, we saw the need for a single source collectionof chemical characterization. In this chapter, the obtained spectroscopic and spectromet-ric data, including results of elemental analysis for sake of completion, of nine selectedquinolones from three drug generations is reported (Figure 2.1): ciprofloxacin (Hcipro),enoxacin (Henox), fleroxacin (Hflex), levofloxacin (Hlevox), lomefloxacin (Hlomx), nalidixicacid (Hnxa), norfloxacin (Hnofx), oxolinic acid (Hoxa), and pipemidic acid (Hpia).Another issue that has been raised in regard to quinolone antibiotic agents is theirstability in standardized susceptibility testing settings, especially their behaviour towardsmetal ions in the test medium, and how such metal ions might affect their bactericidal per-formance. In their comparison of cation-adjusted Mueller-Hinton broth with Iso-Sensitestbroth following the broth micro dilution method of the U.S. Clinical and Laboratory Stan-dards Institute (CLSI), formerly known as U.S. National Committee for Clinical Labora-tory Standards (NCCLS), Koeth et al. found that the four quinolone antimicrobial agentsthat they tested, amongst others ciprofloxacin and levofloxacin, showed slightly higherminimum inhibitory concentration (MIC) values in Iso-Sensitest than in Mueller-Hinton58medium.301 It has been known for over forty years that the Ca2+ and Mg2+ content of bi-ological media can have a major effect on the bactericidal activity of antimicrobial agents,e.g., against Pseudomonas aeruginosa,302 and can therefore significantly influence the re-sults of susceptibility tests.303 When Turel et al. directly compared the antimicrobialactivity (MIC) of their synthesized bis(ciprofloxacino)magnesium(II) complex with that ofciprofloxacin, they had to conclude that the magnesium(II)-quinolone complex possesseda two fold lower activity than the reference compounds ciprofloxacin and ciprofloxacinhydrochloride.304 Before the CLSI defined a specific concentration range for cations andother possible inhibitors in Mueller-Hinton media, batch variance, and therewith varianceof metal ion concentrations, had been one of the core issues with Mueller-Hinton media.Although batch variance and too high metal ion concentrations are an issue in in vitrolaboratory practice, current research results report as well that Mg2+ and Ca2+ play animportant role in the mechanism of action of quinolone antimicrobial agents,291 292 294 aswas mentioned earlier.Compared to Mueller-Hinton, Iso-Sensitest is a synthetic and therewith chemically well-defined medium containing only minimal amounts of variable nutrients (for media formulaesee Table A.1 and A.2). Traditionally, Iso-Sensitest has been used in Europe, where it hasproven itself as a reliable medium with lesser reported problems than Mueller-Hinton.305Several European National Committees have been advising the use of Iso-Sensitest, suchas the British Society for Antimicrobial Chemotherapy (BSAC), but lately the EuropeanCommittee for Antimicrobial Susceptibility Testing (EUCAST) has been recommendingMueller-Hinton in an attempt to unify susceptibility testing procedures to reach comparabletest results across Europe and the world.306 Most probably, this decision will only feed thefire of the ongoing controversial discussion of Iso-Sensitest vs. Mueller-Hinton media.307 308In our lab, we prefer the use of Iso-Sensitest over Mueller-Hinton, because of its better59defined chemical content. To ensure the stability of the nine selected quinolones (Figure 2.1)throughout the antibacterial susceptibility single-disk test procedure, we have studied theirbehaviour in Iso-Sensitest broth via UV-Vis spectroscopy over time. Moreover, the resultsof antimicrobial susceptibility single-disk tests of these nine quinolones in Iso-Sensitestmedium against five pathogens are reported, including Gram-positive and Gram-negativemicroorganisms, which are a common cause of nosocomial infections.2.2 Materials & Methods2.2.1 ChemicalsAll chemicals were purchased from commercial sources: ciprofloxacin, enoxacin, lev-ofloxacin, norfloxacin, oxolinic acid, and pipemidic acid were from Sigma-Aldrich, whilefleroxacin, nalidixic acid, and lomefloxacin hydrochloride were from TCI America. Aque-ous solutions were prepared from deionized water, purified through a ELGA PURELABultrapure water system with a resistivity of 18 MΩ·cm (25◦C).2.2.2 InstrumentationAll melting point (mp) measurements were conducted in triplicate on a DigiMelt SRS melt-ing point apparatus by Stanford Research Systems and are uncorrected. Ultaviolet-Visiblespectra were obtained on a Hewlett Packard 8453 instrument run by UV-Vis ChemStationSoftware (version B.04.01[61], Agilent Technologies, 2001−2010) in methanol with up to2% dimethyl sulfoxide (DMSO), in water, in Iso-Sensitest broth, and in aqueous sodiumchloride solution (0.16 M). All maximum absorption bands and extinction coefficients ()are listed. Infrared (IR) spectra were recorded neat in the solid state on a Thermo ScientificNicolet 6700 Fourier transformation (FT) IR spectrometer in the range of 4000−450 cm−1and analyzed with OMNIC software (version 7.4.127, Thermo Scientific). Bands were in-60terpreted using the following abbreviations: strong (st), moderate (md), weak (w), broad(br), and shoulder (sh). Nuclear magnetic resonance spectroscopy was conducted on BrukerAvance 300 and 600 spectrometers running Topspin 2.1 software on Redhat Linux. TheBruker Avance 600 spectrometer contained a Bruker TCI-Z-5mm cryoprobe for detectionof the 13C and the 1H nuclei with high sensitivity at signal-to-noise ratios of 6000/1 (1H)and 600/1 (13C). 1H NMR, 13C NMR spectra as well as correlated spectroscopy (COSY),heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiplebond correlation (HMBC) spectra, and 19F spectra where applicable, were recorded at roomtemperature with the residual solvent signal of the deuterated solvent (d6-DMSO or D2O)as internal standard,309 referencing all chemical shifts (δ) in ppm against tetramethylsilane(δ= 0) and against trichlorofluoromethane (δ= 0 ppm) as applicable. The software iNMR(version 5.1.2, Mestrelab Research) was employed for spectral analysis, and the followingabbreviations were used for description: aromatic ring system (ar), doublet (d), doublet ofdoublets (dd), multiplet (m), 1,4-piperazinyl ring in C7 position on aromatic ring system(pip), propyl ring in N1 position on aromatic ring system (prop), quartet (q), singulet (s),triplet of triplets (tt). Low-resolution mass spectrometry (MS) was performed on a WatersZQ spectrometer equipped with an electrospray and chemical ionization (ESCI) source andMassLynx Mass Spectrometry software (version 4.00.00, Waters). Characteristic signalsare listed as dimensionless numbers of their mass-to-charge ratio (m/z ), with their intensityrelated to the base signal. Microanalyses for C, H, and N (elemental analysis (EA)) wererecorded at the UBC Mass Spectrometry Centre on a Carlo Erba Elemental Analyzer EA1108.2.2.3 Biological StudiesAll biological experiments were performed in UBC’s Biological Services Laboratory.Iso-Sensitest agar and broth were manufactured by Oxoid. All one-time-use articles61were bought from Fisher-Scientific, only the filter disks (1/4 inch diameter, approx.0.6 mm) were obtained from Schleicher & Schu¨ll, Germany. Antimicrobial susceptibilitysingle-disk tests were performed against Enterococcus faecalis (ATCC-51575), methicillin-susceptible Staphylococcus aureus (MSSA-476, ATTC-BAA-1721), both Gram-positive; Es-cherichia coli (ATCC-25922), Klebsiella pneumonia (ATCC-13883), Pseudomona aerugi-nosa (ATCC-27853), all Gram-negative. Methanol and DMSO were purchased from com-mercial sources, the DMSO was only used after filtration through Millex-FG (0.20 µm).Please see Appendix A for a detailed test procedure.2.2.4 Chemical Characterization2.2.4.1 Ciprofloxacin, HciproAppearance: off-white solid. Mp: 253−255◦C (brown). UV-Vis (CH3OH with 1.5%DMSO): λ [nm] () [M−1cm−1] = 289 (19900), 317 (12500), 331 (11800). IR (neat):ν˜ [cm−1] = 3044 (m, br), 2844 (w, br) 1614 (st), 1587 (st), 1540 (md), 1498 (st), 1472(md), 1448 (sh), 1372 (st, br), 1329 (md), 1310 (w), 1284 (st), 1260 (sh), 1172 (md), 1146(st), 1130 (sh), 1102 (w), 1076 (w), 1035 (st), 1022 (st), 978 (w), 934 (st), 891 (md), 868(st), 833 (st), 822 (md), 784 (st), 721 (st), 707 (md), 652 (md), 622 (st), 565 (st), 553 (sh),543 (st), 494 (st), 479 (sh), 443 (md). NMR: δH (600 MHz, 298 K, d6-DMSO) [ppm]= 14.68 (br s, 1H, COOH); 8.67 (s, 1 H, Car2H); 7.93 (d, J3H,F= 13.0 Hz, 1 H, Car5H);7.60 (d, J4H,F= 7.4 Hz, 1 H, Car8H); 3.86 (tt, J3H,H= 7.2, 3.7 Hz, 1 H, CpropH); 3.54 (t,J3H,H= 5.0 Hz, 4 H, Cpip2,6H2); 3.27 (t, J3H,H= 5.0 Hz, 4 H, Cpip3,5H2); 1.34−1.31 (m, 2 H,CpropHb,b′);310 1.20−1.17 (m, 2 H, CpropHa,a′). δC (125 MHz, 298 K, d6-DMSO) [ppm] =176.4 (s, Car4); 165.9 (s, COOH); 152.9 (d, J1C,F= 207.9 Hz, Car6); 148.2 (s, Car2); 144.3(d, J2C,F= 7.9 Hz, Car7); 139.1 (s, Car8′); 119.2 (d, J3C,F= 5.9 Hz, Car4′); 111.2 (d, J2C,F=19.1 Hz, Car5); 108 (from HMBC, Car3); 106.8 (s, Car8); 46.8 (s, Cpip2,6); 42.8 (s, Cpip3,5);6236.0 (s, CpropH); 7.6 (s, CpropH2). δF (282 MHz, 298 K, d6-DMSO) [ppm] = -121.8 (s, 1F, Car6F ). MS (ES+, CH3OH): m/z (%) = 332 (100) [HL + H+]. m/z (%)= 685 (100)[(HL)2 + Na+]. EA: Anal. Calcd. (found) [%] for C17H18FN3O3: C, 61.62 (61.76); H,5.48 (5.52); N, 12.68 (12.46). Enoxacin, HenoxAppearance: off-white, fine crystalline solid. Mp: 226−228◦C (yellow). UV-Vis(CH3OH with 1.1% DMSO): λ [nm] () [M−1cm−1] = 287 (14900), 345 (17800). IR (neat):ν˜ [cm−1] = 3390 (md, br), 2835 (st, br), 2773 (md, br), 2556 (md, br), 1625 (st), 1577(st), 1468 (sh), 1440 (st), triple crown motiv [1403 (md), 1365 (st), 1340 (st)], 1271 (st,br), 1172 (md), 1144 (md), 1107 (md), 1037 (md, br), 942 (st, br), 918 (sh), 826 (st), 790(md), 729 (md, br), 681 (md), 639 (w), 622 (st), 546 (w), 561 (w), 524 (w), 474 (md, br).NMR: δH (300 MHz, 298 K, d6-DMSO) [ppm] = 8.93 (s, 1 H, Car2H); 7.98 (d, J3H,F=13.8 Hz, 1 H, Car5H); 4.45 (q, J3H,H= 7.1 Hz, 2 H, CH2CH3); 3.73 (dd, J3H,H= 5.9, 4.1Hz, 4 H, Cpip2,6H2); 2.84 (dd, J3H,H= 5.9, 4.1 Hz, 4 H, Cpip3,5H2); 1.37 (t, J3H,H= 7.1 Hz,3 H, CH2CH3). δC (75 MHz, 298 K, d6-DMSO) [ppm] = 176.2 (d, J4C,F= 2.3 Hz, Car4);168.0 (s, Car8′); 165.9 (s, COOH); 149.9 (d, J2C,F= 9.0 Hz, Car7); 147.5 (s, Car2); 146.2(d, J1C,F= 204.2 Hz, Car6); 119.2 (d, J2C,F= 22.0 Hz, Car5); 112.2 (d, J3C,F= 3.6 Hz, Car4′);108.0 (s, Car3); 48.2 (d, J4C,F= 7.9 Hz, Cpip2,6); 47.1 (s, CH2CH3); 45.6 (s, Cpip3,5); 14.6 (s,CH2CH3). δF (282 MHz, 298 K, d6-DMSO) [ppm] = -127.3 (s, 1 F, Car6F ). MS (ES+):m/z (%) = 321 (100) [HL + H+]. m/z (%)= 664 (100) [(HL)2 + Na+]. EA: Anal. Calcd.(found) [%] for C15H17FN4O3·1.5 H2O: C, 51.87 (51.60); H, 5.80 (5.67); N, 16.13 (15.86). Fleroxacin, HflexAppearance: white solid. Mp: 260◦C (decomposed, pale yellow). UV-Vis (CH3OHwith 1.5% DMSO): λ [nm] () [M−1cm−1] = 294 (27500), 320 (12400), 330 (11500). IR63(neat): ν˜ [cm−1] = 3054 (md), 2941 (md), 2796 (md), 1716 (st), 1622 (st), 1556 (md), 1542(sh), 1513 (md), 1474 (st, br), 1447 (st), 1408 (md), 1390 (w), 1375 (md), 1360 (md), 1327(md), 1279 (st, br), 1244 (md), 1228 (md), 1214 (md), 1205 (md), 1142 (st), 1122 (sh),1098 (md), 1061 (st), 1036 (st), 1019 (sh), 1010 (st), 970 (st), 941 (st), 925 (st), 869 (md),852 (md), 816 (sh), 806 (st), 783 (md), 754 (sh), 741 (st), 672 (w), 656 (md, br), 573 (md),550 (md), 532 (w), 504 (md), 450 (md, br). NMR: δH (600 MHz, 298 K, d6-DMSO)[ppm] = 14.77 (br s, 1 H, COOH); 8.84 (s, 1 H, Car2H); 7.86 (d, J3H,F= 11.9 Hz, 1 H,Car5H); 4.97−4.84 (m, 4 H, (CH2)2); 3.34 (br s, 4 H, Cpip2,6H2, overlaid with water); 2.45(br s, 4 H, Cpip3,5H2); 2.23 (s, 3 H, CH3). δC (125 MHz, 298 K, d6-DMSO) [ppm] = 175.6(s, Car4); 165.5 (s, COOH); 154.5 (d, J1C,F= 208.2 Hz, Car6); 152.1 (s, Car2); 146.0 (d,J1C,F= 212.2 Hz, Car8); 133.7 (two overlapping d, J2C,F= 13.9, 11.7 Hz, Car7); 127.3 (d,J2C,F= 9.1 Hz, Car8′); 120.1 (d, J3C,F= 35.1 Hz, Car4′); 107.0 (d, J2C,F= 19.2 Hz, Car5); 106(from HMBC, Car3); 82.1 (d, J1C,F= 138.2 Hz, CH2CH2F); 57.8 (two overlapping d, JC,F=15.8, 12.2 Hz, CH2CH2F); 55.1 (s, Cpip2,6); 50.3 (s, Cpip3,5); 46.0 (s, CH3). δF (282 MHz,298 K, d6-DMSO) [ppm] = -119.2 (d, J4F,F= 11.9 Hz, 1 F, Car6F ); -127.6 (q, JF,F= 5.9 Hz,1 F, Car8F ); -224.1 (d, J6F,F= 5.9 Hz, 1 F, (CH2)2F ). MS (ES+): m/z (%) = 370 (100)[HL + H+], 762 (80) [(HL)2 + Na+]. EA: Anal. Calcd. (found) [%] for C17H18F3N3O3:C, 55.28 (55.14); H, 4.91 (4.90); N, 11.38 (10.98). Levofloxacin, HlevoxAppearance: pale yellow solid. Mp: 224−226◦C (dark brown). UV-Vis (CH3OH with1.5% DMSO): λ [nm] () [M−1cm−1] = 299 (25100), 318 (10200). IR (neat): ν˜ [cm−1] =3247 (md, br), 2935 (md), 2884 (md), 2848 (md), 2802 (md), 1720 (st), 1619 (st), 1538(md), 1518 (md), 1492 (md), 1468 (sh), 1439 (st, br), 1414 (sh), 1394 (st), 1359 (md), 1340(st), 1315 (sh), 1289 (st), 1240 (st), 1207 (sh), 1195 (md), 1163 (md), 1136 (st), 1116 (md),1086 (st, br), 1066 (sh), 1048 (md), 1004 (st), 963 (sh), 951 (md, br), 903 (sh), 873 (st),64839 (md), 800 (st), 778 (md), 755 (md), 741 (st), 727 (md), 695 (w), 666 (w), 650 (st), 578(w), 559 (st), 490 (md), 459 (md). NMR: δH (600 MHz, 298 K, d6-DMSO) [ppm] = 15.20(br s, 1 H, COOH); 8.96 (s, 1 H, Car2H); 7.56 (d, J3H,F= 12.4 Hz, 1 H, Car5H); 4.91 (d,JH,H= 6.8 Hz, 1 H, CH); 4.58 (dd, JH,H= 11.5, 1.7 Hz, 1 H) and 4.36 (dd, JH,H= 11.5,2.3 Hz, 1 H) (OCH2CH); 3.33−3.25 (m, 4 H, Cpip2,6H2); 2.43 (br s, 4 H, Cpip3,5H2); 2.23(s, 3 H, NCH3); 1.44 (d, J3H,H= 3.3 Hz, 3 H, CHCH3). δC (125 MHz, 298 K, d6-DMSO)[ppm] = 176.4 (s, Car4); 166.1 (s, COOH); 155.5 (d, J1C,F= 206.3 Hz, Car6); 146.2 (s, Car2);140.1 (d, J3C,F= 5.7 Hz, Car8); 132.1 (d, J2C,F= 11.9 Hz, Car7); 124.8 (s, Car8′); 119.6 (d,J3C,F= 7.7 Hz, Car4′); 106.6 (s, Car3); 103.3 (d, J2C,F= 20.4 Hz, Car5); 68.0 (s, OCH2CH);55.3 (s, Cpip2,6); 54.8 (s, CH); 50.1 (s, Cpip3,5); 46.1 (s, NCH3); 17.9 (s, CH(CH3)). δF(282 MHz, 298 K, d6-DMSO) [ppm] = -120.2 (s, 1 F, Car6F ). MS (ES+): m/z (%) = 362(100) [HL + H+]. m/z (%)= 541 (100), 745 (60) [(HL)2 + Na+], 1173 (30). EA: Anal.Calcd. (found) [%] for C18H20FN3O4: C, 59.83 (59.44); H, 5.58 (5.66); N, 11.63 (11.43). Lomefloxacin, HlomxAppearance: white solid. Mp: >260◦C. UV-Vis (CH3OH with 1.1% DMSO): λ [nm]() [M−1cm−1] = 291 (34500), 320 (15800), 332 (13100). IR (neat): ν˜ [cm−1] = 3055 (w),2936 (md), 2842 (w), 2756 (sh), 2698 (st, br), 2456 (md), 1721 (st), 1611 (st), 1543 (sh),1524 (md), 1491 (st), 1471 (sh), 1448 (st, br), 1411 (w), 1392 (st), 1328 (st), 1299 (md),1281 (md), 1253 (st), 1205 (st), 1182 (w), 1166 (w), 1141 (md), 1114 (md), 1093 (st), 1065(w), 1041 (st), 1021 (md), 1006 (st), 979 (md), 928 (st), 892 (st), 844 (md), 821 (md),807 (st), 791 (sh), 756 (sh), 738 (st), 653 (md), 578 (w), 555 (md), 545 (md), 534 (md),513 (st), 488 (md), 476 (md), 452 (md). NMR: δH (600 MHz, 298 K, D2O) [ppm] =8.55 (s, 1 H, Car2H); 7.46 (d, J3H,F= 11.4 Hz, 1 H, Car5H); 4.48 (d, J3H,H= 6.0 Hz, 2H,CH2CH3); 3.70−3.53 (m, 5 H, Cpip2,6H2 and Cpip3H); 3.42−3.38 (m, 2 H, Cpip4H2); 1.49(t, J3H,H= 7.1 Hz, 3 H, CH2CH3); 1.40 (d, J3H,H= 6.6 Hz, 3 H, CH3). δC (125 MHz, 29865K, d6-DMSO) [ppm] = 175.3 (s, Car4); 168.3 (COOH); 154.9 (d, J1C,F= 208.6 Hz, Car6);150.6 (s, Car2); 146.1 (d, J1C,F= 210.3 Hz, Car8); 133.0 (two overlapping d, J2C,F= 11.6,11.6 Hz, Car7); 127.0 (d, J2C,F= 5.6 Hz, Car8′); 120.8 (d, J3C,F= 7.3 Hz, Car4′); 106.8 (d,J2C,F= 19.1 Hz, Car5); 106.1 (s, Car3); 55.0 (d, J4C,F= 13.5 Hz, Cpip2); 53.4 (s, CH2CH3);51.7 (s, Cpip6); 46.7 (s, Cpip3); 43.5 (s, Cpip5); 15.3 (s, CH2CH3); 14.9 (s, CH3). δF (282MHz, 298 K, d6-DMSO) [ppm] = -118.6 (d, J4F,F= 10.7 Hz, 1 F, Car6F ); -128.6 (d, J4F,F=11.3 Hz, 1 F, Car8F ). MS (ES+): m/z (%) = 352 (100) [HL + H+]. m/z (%)= 769 (100),725 (60) [(HL)2 + Na+], 1143 (30). EA: Anal. Calcd. (found) [%] for C17H20F2N3O3·1HCl: C, 52.65 (52.79); H, 5.20 (5.17); N, 10.84 (10.56). Nalidixic acid, HnxaAppearance: white solid. Mp: 228−230◦C (soft pink). UV-Vis (CH3OH with 1.1%DMSO): λ [nm] () [M−1cm−1] = 320 (13500), 328 (13700). IR (neat): ν˜ [cm−1] = 3044(md, br), 2987 (w, br), 2948 (w), 1707 (st, br), 1614 (st, br), 1562 (w), 1538 (w), 1518(md), 1465 (sh), 1440 (st, br), 1384 (w), 1370 (md), 1353 (md), 1327 (w), 1294 (md), 1270(sh), 1252 (st), 1227 (st), 1129 (st), 1102 (w), 1051 (w), 1034 (w), 971 (st, br), 875 (md),803 (st), 777 (sh), 706 (md), 656 (md), 634 (md), 563 (w), 539 (md), 505 (w), 485 (st),454 (md). NMR: δH (300 MHz, 298 K, d6-DMSO) [ppm] = 9.18 (s, 1 H, Car2H); 8.60(d, J3H,H= 8.2 Hz, 1 H, Car5H); 7.59 (d, J3H,H= 8.2 Hz, 1 H, Car6H); 4.64 (q, J3H,H= 7.1Hz, 2 H, CH2CH3); 2.71 (s, 3 H, CH3); 1.42 (t, J3H,H= 7.1 Hz, 3 H, CH2CH3). δC (75MHz, 298 K, d6-DMSO) [ppm] = 178.2 (s, Car4); 165.6 (s, Car8′); 164.7 (s, COOH); 149.7(s, Car2); 148.3 (s, Car7); 135.6 (s, Car5); 122.6 (s, Car6); 118.3 (s, Car4′); 108.6 (s, Car3);46.8 (s, CH2CH3); 25.1 (s, CH3); 15.0 (s, CH2CH3). MS (ES+): m/z (%) = 255 (100)[HL + Na+], 233 (60) [HL + H+]. m/z (%)= 786 (100) [(HL)3 + Na+], 1040 (30) [(HL)4+ Na+], 1294 (10) [(HL)5 + Na+]. EA: Anal. Calcd. (found) [%] for C12H12N2O3: C,62.06 (62.32); H, 5.21 (5.18); N, 12.06 (11.94).662.2.4.7 Norfloxacin, HnofxAppearance: pale yellow solid. Mp: 221−223◦C (yellow). UV-Vis (CH3OH with 1.5%DMSO): λ [nm] () [M−1cm−1] = 290 (21900), 317 (9900), 330 (8700). IR (neat): ν˜ [cm−1]= 3046 (w, br), 2944 (md), 2827 (md, br), 1722 (st), 1614 (st), 1519 (md), 1471 (st), 1439(st), 1401 (w), 1373 (md), 1350 (md), 1323 (w), 1300 (md), 1272 (w), 1248 (st), 1210 (md),1198 (st), 1148 (md), 1127 (md), 1102 (st), 1090 (sh), 1050 (w), 1025 (md), 978 (w), 945(st, br), 885 (st), 838 (st), 826 (sh), 804 (st), 783 (md), 748 (st), 712 (w), 698 (st), 664(md), 638 (md), 620 (md), 557 (md), 514 (md), 498 (md), 485 (w), 450 (md). NMR: δH(300 MHz, 298 K, d6-DMSO) [ppm] = 8.92 (s, 1 H, Car2H); 7.85 (d, J3H,F= 13.5 Hz, 1H, Car5H); 7.12 (d, J4H,F= 7.3 Hz, 1 H, Car8H); 4.57 (q, J3H,H= 7.1 Hz, 2 H, CH2CH3);3.22 (dd, J3H,H= 5.9, 3.8 Hz, 4 H, Cpip2,6H2); 2.89 (dd, J3H,H= 5.9, 3.8 Hz, 4 H, Cpip3,5H2);1.41 (t, J3H,H= 7.1 Hz, 3 H, CH2CH3). δC (75 MHz, 298 K, d6-DMSO) [ppm] = 176.1 (d,J4C,F= 2.7 Hz, Car4); 166.1 (s, COOH); 152.5 (d, J1C,F= 207.5 Hz, Car6); 148.3 (s, Car2);145.9 (d, J2C,F= 9.7 Hz, Car7); 137.2 (s, Car8′); 118.9 (d, J3C,F= 7.7 Hz, Car4′); 111.0 (d,J2C,F= 23.1 Hz, Car5); 107.0 (s, Car3); 105.4 (d, J3C,F= 3.7 Hz, Car8); 50.8 (d, J4C,F= 4.8Hz, Cpip2,6); 49.0 (s, CH2CH3); 45.4 (s, Cpip3,5); 14.3 (s, CH2CH3). δF (282 MHz, 298 K,d6-DMSO) [ppm] = -121.3 (s, 1 F, Car6F ). MS (ES+): m/z (%) = 320 (100) [HL + H +].m/z (%)= 662 (70) [2 HL + Na+]. EA: Anal. Calcd. (found) [%] for C16H18FN3O3: C,60.18 (60.02); H, 5.68 (5.75); N, 13.16 (12.92). Oxolinic acid, HoxaAppearance: white solid. Mp: >260◦C. UV-Vis (CH3OH with 2.2% DMSO): λ [nm]() [M−1cm−1] = 322 (7600), 336 (7700). IR (neat): ν˜ [cm−1] = 3061 (md), 2984 (md),2930 (md, br), 1698 (st), 1632 (st), 1573 (st), 1504 (md), 1440 (st, br), 1384 (md), 1350(sh), 1301 (md) 1259 (st), 1222 (w), 1204 (w), 1186 (md), 1127 (md), 1094 (w), 1075 (md),671036 (st), 936 (st, br), 876 (st), 856 (st), 807 (st), 773 (md), 754 (md), 690 (md), 645(st), 605 (md), 556 (md), 498 (md), 447 (md). NMR: δH (300 MHz, 298 K, d6-DMSO)[ppm] = 8.89 (s, 1 H, Car2H); 7.63 (s, 1 H, Car8H); 7.61 (s, 1 H, Car5H); 6.29 (s, 2 H,OCH2O); 4.53 (q, J3H,H= 7.1 Hz, 2 H, CH2CH3); 1.38 (t, J3H,H= 7.1 Hz, 3 H, CH2CH3).δC (75 MHz, 298 K, d6-DMSO) [ppm] = 176.0 (s, Car4); 166.3 (s, COOH); 153.7 (s, Car7);147.10 (s, Car6); 147.0 (s, Car2); 136.9 (s, Car8′); 121.3 (s, Car4′); 107.3 (s, Car3); 103.3 (s,OCH2O); 101.8 (s, Car5); 97.2 (s, Car8); 49.6 (s, CH2CH3); 14.6 (s, CH2CH3). MS (ES+):m/z (%) = 284 (100) [HL + Na+], 262 (10) [HL + H+]. m/z (%)= 589 (90) [(HL)2 +Na+], 873 (100) [(HL)3 + Na+], 1156 (30) [(HL)4 + Na+]. EA: Anal. Calcd. (found) [%]for C13H11NO5: C, 59.77 (59.83); H, 4.24 (4.24); N, 5.36 (5.37). Pipemidic acid, HpiaAppearance: white, fine powdered solid. Mp: 258−260◦C (orange-brown). UV-Vis(CH3OH with 2.2% DMSO): λ [nm] () [M−1cm−1] = 288 (13500), 325 (7300), 342 (5400).IR (neat): ν˜ [cm−1] = 3365 (md, br), 3028 (w), 2979 (w), 1615 (st), 1577 (st), 1532 (md),1510 (md), 1471 (st), 1429 (st), 1378 (sh), 1357 (st, br), 1309 (md), 1279 (md), 1238 (st,br), 1259 (w), 1159 (w), 1147 (w), 1127 (st), 1092 (md), 1078 (md), 1044 (md) 1022 (st),975 (md), 940 (md), 914 (st), 903 (md), 867 (md), 832 (st), 802 (md), 783 (md), 743 (st),715 (md), 655 (w), 608 (w), 540 (st), 489 (md), 453 (md). NMR: δH (300 MHz, 298 K,d6-DMSO) [ppm] = 9.15 (s, 1 H, Car5H); 8.93 (s, 1 H, Car2H); 4.36 (q, J3H,H= 7.1 Hz, 2H, CH2CH3); 3.84 (d, J3H,H= 17.1 Hz, 4 H, Cpip2,6H2); 2.78 (br s, 4 H, Cpip3,5H2); 1.35 (t,J3H,H= 7.1 Hz, 3 H, CH2CH3). δC (75 MHz, 298 K, d6-DMSO) [ppm] = 177.1 (s, Car4);165.3 (s, COOH); 160.5 (Car7); 160.1 (s, Car5); 155.1 (s, Car8′); 150.6 (s, Car2); 109.5 (s,Car4′); 108.3 (s, Car3); 45.8 (s, CH2CH3); 45.3 (br s, Cpip2,3,5,6); 14.4 (s, CH2CH3). MS(ES+): m/z (%) = 304 (50) [HL + H+], 326 (70) [HL + Na+], 348 (100) [HL + CO2 +H+]. m/z (%)= 673 (40) [(NaL)2 + Na+], 999 (95) [(NaL)3 + Na+]. EA: Anal. Calcd.68(found) [%] for C14H17N5O3: C, 55.44 (55.10); H, 5.65 (5.63); N, 23.09 (22.96).2.2.5 Stability in Iso-Sensitest BrothOn the day of the experiment, stock solutions of the quinolones were prepared in methanolwith small amounts of DMSO (≤ 2%) to ensure full dissolution. From these stock solutions,test solutions were prepared in Iso-Sensitest broth (5.9 g in 500 mL deionized water, auto-claved) via dilution to a final concentration of 0.1 mM quinolone. The amount of DMSOand the final concentration of the test solutions matched the conditions of the single-diskdiffusion test (Section 2.2.6). UV-Vis spectra of each of the respective quinolone test solu-tions (in alphabetical order) were recorded at the following time points: 20 min, 1 h, 2 h,4 h, 8 h, 18 h, 20 h, and 24 h. The UV-Vis spectrum of the Iso-Sensitest broth servedas the blank. To avoid any contamination of the biological growth medium, which couldalter the UV-Vis test results, the Iso-Sensitest test solutions were prepared in a biologicalsafety cabinet in UBC’s Biological Services Laboratory. Each test solution was transferredinto a UV-Vis cuvette, and the cuvette opening was tightly covered with parafilm. Allsingle-steps (dilution, mixing, transferring, parafilm wrapping, labelling, transport to UV-Vis spectrophotometer) took some time so that the first time point could only be measuredapproximately 20 min after the initial dilution step. On the following day, respective testsolutions in water (0.1 mM) were made up from the same quinolone stock solutions, and theUV-Vis stability study was repeated with these quinolone-water solutions at time pointsof 0 min (immediately after dilution), 30 min, 1 h, 2 h, 4 h, 18 h, 20 h, and 24 h. Be-cause the quinolone-water test solutions were not as sensitive to biological contaminationas were the test solutions in Iso-Sensitest broth, these test solutions were diluted, mixed,and transferred into the cuvette directly next to the UV-Vis spectrometer, which allowedthe measurement of an approximate 0 min time point.692.2.6 Antimicrobial Susceptibility Single-Disk Test in Iso-SensitestMediumThe antimicrobial activities of the selected quinolones were evaluated according to theagar diffusion single-disk testing method. The work was performed in UBC’s BiologicalServices Laboratory, a biological level II facility, following respective operating and safetyprocedures. Iso-Sensitest media were prepared according to the manufacturer’s specifica-tions. Agar plates of 150 mm diameter were poured with an approximate height of 4 mm.Bacteria were grown in 5 mL broth (Falcon tube) at 37 ◦C on a shaker to an OD600 of≥ 1. On the day of the experiment, the quinolone test solutions were prepared in methanoland DMSO to ensure complete solubility (max. 2% DMSO) alongside one pure methanoland one 2%-DMSO-methanol solution as controls. Agar plates were inspected for signs ofdegradation, and placement positions of the disks were marked at the bottom of the petridish with a minimum distance between each disk (center to center) of at least 24 mm andwith not more than 14 disk positions total. The following steps were done in triplicate.Paper filter disks were loaded with 20 µL of each test and control solution and left todry for about 5 min. While these were drying, the previously prepared agar plates wereinoculated with 0.5 mL bacteria growth broth that was spread evenly across the plate. Theloaded filter disks, including the two control disks, were placed on the marks and carefullypressed onto the agar. The lid was put back onto the petri dish, the sides of the petri dishwere sealed with parafilm, and the petri dish was placed up-side-down in the incubatorat 37 ◦C for 20 h. After this time, the plates were taken from the incubator, placed on anonreflecting black surface, and the no-growth zone around each disk on each of the plateswas measured with a ruler with the naked eye. As it is convention, the inhibition zone sizeswere recorded as diameters rounded to the nearest millimeter with the diameter of eachdisk being included in the measurement. Growth up to the edge of a disk was evaluated70as 0 mm.2.3 Results & Discussion2.3.1 Chemical CharacterizationAll quinolones were dissolved in methanolic solutions with 20% DMSO content and dilutedin methanol to appropriate UV-Vis concentrations. Depending on the nature of the aro-matic core and the number of fluorine-substituents, a quinolone test concentration between3·10−5 M to 6·10−5 M resulted in absorbance maxima between 1.3−0.5 AU over the studiedrange from 190 to 1100 nm wave numbers. The absorbance maxima of the selected ninequinolones are summarized in Table 2.1. All quinolones gave a broad absorbance band be-tween 300−380 nm with a long tail. In addition, the quinolones with a 1,4-piperazinyl ringin Car7 position on the condensed aromatic ring system showed a second sharp absorbanceband of high intensity at lower wave numbers between 280−300 nm. These observationsare not surprising, as from the molecular structure of the quinolones (Figure 2.1), onechromophore is expected for the condensed aromatic ring system with Nar1 (chromophoreI), plus the 1,4-piperazinyl ring on Car7 represents a second chromophore in respectivelysubstituted quinolones (chromophore II).311The first absorbance maximum (280−300 nm) is related to the energy absorption of thearomatic core, while the second absorbance maximum (300−380 nm) is composed of twosub-peaks and has been assigned to the n→ pi∗ (HOMO-LUMO) electronic transition.312These two sub-peaks reflect two different types of hydrogen bonds forming, an intramolec-ular hydrogen bond between the carbonyl group in Car4 position and the carboxylic acidgroup in Car3 position as well as an intermolecular one between the quinolone molecule andresidual water molecules in the organic solvent.312 313 The 1,4-piperazinyl ring on Car7 hasa strong effect on the electronics of the condensed aromatic ring system, as in quinolones71Table 2.1: UV-Vis absorbance maxima (Amax [nm]) in methanol (≤2% DMSO) so-lutionquinolone Amax1 [nm] Amax2 [nm]Hcipro 289 317 331Henox 287 * 345Hflex 294 320 330Hlevox 299 318 *Hlomx 291 320 332Hnxa * 320 328Hnofx 290 317 330Hoxa * 322 336Hpia 288 325 342*not observed. [Hquino] = 3−6 10−5 M for intensity Amax2= 0.5−0.8 AU.without this substituent, such as nalidixic acid and oxolinic acid, the strong first absorbanceband was not observed. The second absorption maximum is highly affected by the chosenUV-Vis solvent,314 because acetonitrile, methanol, water, or any mixtures thereof alter therequisite for hydrogen bond formation, which is as well influenced by pH. In Figure 2.2,the recorded UV-Vis spectra of ciprofloxacin over the pH range from 2 to 11 in an aque-ous sodium chloride solution (0.16 M) are presented. Although hypso- and hyper-chromiceffects can be observed for the first absorbance maximum between 260 to 290 nm wave-length from acidic to basic pH, the second absorbance band is the most affected by thepH changes. Here, bathochromic and hyperchromic effects are dominant when comparingacidic to basic pH; in addition, the two subpeaks become more defined and of equal valueat a pH of 7 and higher. At 275 nm, 305 nm, and 346 nm, lie isobestic points. During thetitration with sodium hydroxide, the deprotonation equilibria between pH 4.5 to 8 wereslow. The acid-base equilibria of ciprofloxacin are drawn in Figure 2.3. Because the de-protonation of the N-atom in 4-position of the 1,4-piperazinyl ring in ciprofloxacin leads toa zwitterionic state in the neutral pH range (Figure 2.3),315 this observation correspondswell to the major changes of the molecular structure occurring in this pH region, which72260 280 300 320 340 360 380 400Wavelength [nm] [AU]pH 2pH 3pH 4pH 5pH 6pH 7pH 8pH 9pH 10pH 11Figure 2.2: pH dependency of ciprofloxacin ([Hcipro] = 2·10−5 M, pH 2−11,INaCl= 0.16 M).are as well reflected in the changes dominating the 300 to 340 nm region in the recordedUV-Vis spectra (Figure 2.2). Previously reported additional protonation of the Nar1-atomin the acidic pH range (pH ≤ 3) was not observed.316The molecular structure of the quinolone drugs with the carboxyl functional groupin Car3-position and the carbonyl functional group in Car4-position lends nice handles tospectroscopic analysis in the mid-infrared region (4000−400 cm−1, Figure 2.4). The COstretching vibration of the carboxyl group, νCOOH , was observed around 1715 cm−1 and theCO deformation vibration, δCOOH , around 1350 cm−1. As previously clarified,317 ionic car-boxylates, such as ciprofloxacin in its zwitterionic state (Figure 2.3), do not show a νCOOHstretching vibration;318 instead, two characteristic bands in the range of 1650−1510 cm−1and 1400−1280m−1 were observed that were assigned as the asymmetric and symmetricνOCO stretching vibrations in agreement with the literature: 1587 cm−1 and 1372 cm−173NFHNNOOONFHNNOOONFH2NNOOONFH2NNOOOHHLH2+LH±LHL-Figure 2.3: Protonation equilibria of ciprofloxacin.for ciprofloxacin,317 1577 cm−1 and 1365 cm−1 for enoxacin,319 as well as 1577 cm−1 and1357 cm−1 for pipemidic acid,320 respectively.Other highly characteristic IR features of these drug molecules are the C=C stretchingvibration of the conjugated aromatic ring system, νC=C , around 1620 cm−1 as well as thestretching of the aromatic quinolone core, νC=N , around 1400 cm−1 and the C-H bendingstretch, δC−H , in the range of 1440−1500 cm−1. Oxolinic acid possesses with the penta-cyclic ether (1,3-dioxolane) a unique structural feature, which gives a strong absorbancestretch of the C−O vibration at 1036 cm−1. For a detailed discussion of FT-Raman spectro-scopic characterizations of quinolones, the avid reader is referred to Neugebauer et al.312.The 1H, 13C, and 19F NMR spectral assignments of the selected nine quinolone antimi-crobial agents are presented in Tables 2.2, 2.3, and 2.4 respectively. The resonances wereallocated with confidence from recorded data at 300 or 600 MHz for the 1H nucleus, 75 or125 MHz for the 13C nucleus, and 128 MHz for the 19F nucleus as applicable, in additionto COSY, HSQC, and HMBC 2D-experiments. All samples were dissolved in deuterated74Figure 2.4: IR spectra of the nine quinolones.75dimethyl sulfoxide with the help of sonicating and heating, only for lomefloxacin hydrochlo-ride deuterated water seemed to be the more appropriate solvent, in which it dissolvedreadily.In 1H NMR measurements, carboxylic protons protons were not observed in the stan-dard range from 0 to 10 ppm, but could be detected in the spectra recorded in d6-DMSO at600 MHz frequency in the lower field range as extremely broad singlets around 14.68 ppm(Hcipro), 14.77 ppm (Hflex), and 15.20 ppm (Hlevox); in contrast to literature reports,321no tertiary nitrogen protons from the piperazinyl-substituent in Car7 could be noted up to23 ppm. In an earlier NMR study of selected gyrase inhibitors in acidic and basic solu-tions, Holzgrabe et al. showed that the deprotonation of the carboxyl group only affectsthe Car3 atom and the carboxyl-C itself, while the protonation of the nitrogen atom of thepiperazinyl-group only influences the C-atoms of the 1,4-piperazinyl-ring on Car7.322From the 1H NMR data summary in Table 2.2, three major observations can be made.Firstly, the introduction of a second nitrogen atom in Car8-position has only a small in-fluence on the aromatic proton on Car5, as the comparison of norfloxacin (quinoline core,Car5H at 7.85 ppm) vs. enoxacin (naphthyridine core, Car5H at 7.98 ppm) reveals. Sec-ondly, in the 6-fluoroquinolones, the fluorine couples not only with the vicinal aromatic pro-ton on Car5 (J3H,F= 11.9−13.8 Hz) but as well with long-range Car8H (J4H,F= 7.3−7.4 Hz).Thirdly, cyclic alkyl-substituents on Nar1 rotate fast in solutions at room temperature, re-flected in the more complex coupling patterns observed for ciprofloxacin and levofloxacin.The methylene groups in Hcipro are chemically not equivalent, as they give each one mul-tiplet in the 1H NMR measurements, resulting in the triplet of triplet pattern of the vicinalmethine proton (J3H,H= 7.2, 3.7 Hz). According to earlier NMR studies by Zieba et al., themethylene protons were assigned as Ha,a′= 1.20−1.17 ppm and Hb,b′= 1.34−1.31 ppm.310In Hlevox, the methylene protons on the hexane ring, which connects to the quinoline76aromatic core through Nar1 and Car8, gave each a doublet of doublets with 4.58 ppm (dd,J3H,H= 11.5, 1.7 Hz) and 4.36 (dd, J3H,H= 11.5, 2.3 Hz). This indicates a rapid foldingmovement of the hexane ring in solution at room temperature; an observation that can aswell be noted in the six-membered piperazinyl-ring on Car7, which gives doublet of doubletsin 1H NMR spectra of Henox and Hnofx (J3H,H= 5.9, 3.8−4.1 Hz) or more complex cou-pling patterns resulting in multiplets in 1H NMR spectra of Hlevox or Hlomx (Table 2.2).Opposite to the cyclic alkyl-substituents, the ethyl-chains on Nar1 move freely and weredetected in the 1H NMR measurements as characteristic quintet pattern of the methyleneprotons (q, J3H,H= 7.1 Hz) and triplet pattern of the methyl protons (t, J3H,H= 7.1 Hz)with matching coupling constants.323The summarized results of the conducted 13C NMR measurements (Table 2.3) showclearly the difference in electronegativity of the condensed aromatic ring system depend-ing on, if the aromatic core is a quinoline, a naphthyridine, or a [2,3-d]-pyrimidine, andthe introduction of possibly one or two fluorine substituents on Car6 and Car8. While thecarboxylic-C and Car2 to Car4 are barely affected, the largest changes in chemical shifts wererecorded for Car6 and Car8′ . The latter is shifted downfield in naphthyridines (>160 ppm)compared to quinolines (around 100 ppm). Although the effect of the 6-fluorine sub-stituent is most strongly felt on Car6 (J1C,F around 207 ppm), its influence spreads overthree bonds across the substituted ring and even into the piperazinyl-substituent on Car7in bi-fluorinated species (Car6, Car8), as the respective coupling constants (JC,F ) reveal.Overall, the determined JC,F coupling constants correspond well with reported litera-ture values.322 324 Comparing the coupling of the fluorine atoms with the carbon atoms,the fluorine-substituted Car6-atom possesses a high carbon-fluorine coupling constant ofJ1Car6,F= 206.3−207.9 Hz (lit. 245.3 Hz)324 in mono-fluorinated species. The vicinal Car5-atom couples to fluorine at J2Car5,F= 19.1−23.1 Hz (lit. 21.0 Hz)324, while the coupling with77the other vicinal Car7-atom is slightly reduced in frequency probably due to the piperizyl-subsituent in 7-position J2Car7,F= 9.7−11.9 Hz. Moreover, the Car4′-atom couples to fluorineat J3Car4′ ,F= 5.9−7.7 Hz (lit. 7.7 Hz)324, while the fluorine coupling constants of the Car8-atom with J3Car8,F=3.7−5.7 Hz are slightly lower, possibly an influence of the Nar1-atomclose by. In cases where a coupling to fluorine could be detected for the carbonyl-Car4, itscoupling constant was J3Car4,F=2.3−2.7 Hz (lit. 3.3 Hz).324Furthermore, it should be noted that the detection of the Car3 signal even at highfrequency (600 MHz) proved to be difficult for Hcipro and Hflex. Unfortunately, evenvarying the NMR parameters did not lead to improved signal strengths. The standardpulse program configuration for 13C measurements at 600 MHz frequency was at 1D-sequence with power gated decoupling with spin echo at a sleeve angle of 90◦. Anotherdata set recorded at 1D-sequence with a larger spin-lattice relaxation time (t1) of 30 sec-onds (normally in the range from 10−4 to 102 seconds)325 without spin echo and witha flip angle of 30◦ for 6.5 hours did not show any new signals either. The additionof chromium(III)acetylacetonate to the deuterated test solution, a standard relaxationagent in NMR spectroscopy, was redeemed unsuitable in this situation, because an ear-lier coordination chemistry experiment with Cr3+ had led to the formation of a greentris(quinolono)chromium(III) complex, which had been isolated in solid form; therefore,a reaction between Cr3+ and the quinolone seemed likely under the given conditions (d6-DMSO, ambient temperature). In addition to changing the 1D-sequence parameters, threedifferent sets of HMBC spectra at different coupling constants were recorded. The standardsetting with a long-range coupling constant of J= 8.0 Hz showed all correlations withinthe range of J= 8 ± 3 Hz after recording data for 1 hour. Much more correlations could bedetected in HMBC measurements with a coupling constant reduced by 50% (J= 4.0 Hz)for 2 hours. A third one-hour long run, with an even lower coupling constant of J= 2.0 Hz,78however, did not reveal any further correlations between the 1H and 13C nuclei. Finally,the reported signals for the Car3 atoms in Hcipro and Hflex are based on estimated valuesresting upon weak interactions in the HMBC spectra that were recorded with set couplingconstants at J= 8.0 Hz and J= 4.0 Hz. In case of Hcipro, the proton of the carboxylic acidsubstituent on Car3 is coupling to a C-signal at 108 ppm, which corresponds to J3H,C ; but,due to the extreme broadness of the hydroxyl proton signal, this gives only a faint signal inthe corresponding HMBC spectra. In case of Hflex, the Car3 signal could be estimated at106 through a faint coupling signal with Car2 (J2H,C) in the HMBC spectra. The signals forCar3 extracted from the respective HMBC spectra correspond well with those of quinolonesof similar chemical structure, such as Hlomx in case of Hflex as well as Hnofx in case ofHcipro (Table 2.3).79Table 2.2: 1H NMR data in δH [ppm]quinolone solvent Car2H Car5H Car6H Car8H N1 −R1 Car7 −R2Hcipro d6-DMSO 8.67 7.93 (d, 13.0)a n/a 7.60 (d, 7.4)a 3.86 (tt, 7.2, 3.7)b,1.34−1.31 (m)b, 1.20−1.17(m)b3.54 (t, 5.0)b, 3.27 (t, 5.0)bHenox d6-DMSO 8.93 7.98 (d, 13.8)a n/a n/a 4.45 (q, 7.1)b, 1.37 (t, 7.1)b 3.73 (dd, 5.9, 4.1)b, 2.84 (dd, 5.9, 4.1)bHflex d6-DMSO 8.84 7.86 (11.9)a n/a n/a 4.97−4.84 (m)a,b 3.34, 2.45, 2.23Hlevox d6-DMSO 8.96 7.56 (d, 12.4)a n/a n/a 4.91 (d, 6.8)b, 4.58 (dd, 11.5,1.7)b, 4.36 (dd, 11.5, 2.3)b,1.44 (d, 3.3)b3.33−3.25 (m)b, 2.43 (br s)a, 2.23Hlomx D2O 8.55 7.46 (d, 11.4)a n/a n/a 4.48 (6.0)b, 1.49 (t, 7.1)b 3.70−3.53 (m)b, 3.42−3.38 (m)b, 1.40(d, 6.6)bHnxa d6-DMSO 9.18 8.60 (d, 8.2)b 7.59 (d, 8.2)b n/a 4.64 (q, 7.1)b, 1.42 (t, 7.1)b 2.71Hnofx d6-DMSO 8.92 7.85 (d, 13.5)a n/a 7.12 (d, 7.3)a 4.57 (q, 7.1)b, 1.41 (t, 7.1)b 3.22 (dd, 5.9, 3.8)b, 2.89 (dd, 5.9, 3.8)bHoxa d6-DMSO 8.89 7.61 n/a 7.63 4.53 (q, 7.1)b, 1.38 (t, 7.1)b 6.29Hpia d6-DMSO 8.93 9.15 n/a n/a 4.36 (q, 7.1)b, 1.35 (t, 7.1)b 3.84 (d, 17.1)b, 2.78 (br s)baJH,F [Hz], bJ3H,H [Hz]80Table 2.3: 13C NMR data in δC [ppm] (d, JC,F [Hz])quinolone solvent COOH Car2 Car3 Car4 Car4′ Car5 Car6 Car7 Car8 Car8′ N1 −R1 Car7 −R2Hcipro d6-DMSO 165.9 148.2 108 176.4 119.2(5.9)111.2(19.1)152.9(207.9)144.3(7.9)106.8 139.1 36.0, 7.6 46.8, 42.8Henox d6-DMSO 165.9 147.5 108.0 176.2(2.3)112.2(3.6)119.2(22.0)146.2(204.2)149.9(9.0)n/a 168.0 47.1, 14.6 48.2 (7.9),45.6Hflex d6-DMSO 165.5 152.1 106 175.6 120.1(35.1)107.0(19.2)154.5(208.2)133.7(13.9, 11.7)146.0(212.2)127.3(9.1)82.1 (138.2),57.8 (15.8,12.2)55.1, 50.3,46.0Hlevox d6-DMSO 166.1 146.2 106.6 176.4 119.6(7.7)103.3(20.4)155.5(206.3)132.1(11.9)140.1(5.7)124.8 68.0, 54.8,17.955.3, 50.1,46.1Hlomx D2O 168.3 150.6 106.1 175.3 120.8(7.3)106.8(19.1)154.9(208.6)133.0(11.6, 11.6)146.1(210.3)127.0(5.6)53.4, 15.3 55.0 (13.5),51.7, 46.7,43.5, 14.9Hnxa d6-DMSO 164.7 149.7 108.6 178.2 118.3 135.6 123.6 148.3 n/a 165.6 46.8, 15.0 25.1Hnofx d6-DMSO 166.1 148.3 107.0 176.1(2.7)118.9(7.7)111.0(23.1)152.5(207.5)145.9(9.7)105.4(3.7)137.2 49.0, 14.3 50.8 (4.8)a,45.4Hoxa d6-DMSO 166.3 147.0 107.3 176.0 121.3 101.8 147.1 153.7 97.2 136.9 49.6, 14.6 103.3Hpia d6-DMSO 165.3 150.6 108.3 177.1 109.5 160.1 n/a 160.5 n/a 155.1 45.8, 14.4 45.381Table 2.4: 19F NMR data in δF [ppm] (referenced against δ(C6F6)= -164.9 ppm vs.δ(CFCl3)= 0 ppm)quinolone solvent Car6F Car8F N1 −R1Hcipro d6-DMSO -121.8 n/a n/aHenox d6-DMSO -127.4 n/a n/aHflex d6-DMSO -127.6 -119.2 -224.1Hlevox d6-DMSO -120.2 n/a n/aHlomx D2O -128.6 -118.6 n/aHnofx d6-DMSO -121.3 n/a n/aThe 19F measurements support this chemical similarity argument (Table 2.4). Forquinoline-based, mono-fluorinated (Car6) quinolones, such as Hcipro, Hlevox, Hnofx, theshift of the 19F appears at -121.8, -120.2, and -121.3 ppm, respectively. For enoxacin withits naphthyridine core, the chemical shift of 19F is shifted to high-field at -127.4 ppm dueto the increased electronegativity in the condensed aromatic ring system accompanyingthe introduction of the second N-atom in 8-position. On the other hand, for quinoline-based, bi-fluorinated (Car6, Car8) lomefloxacin, the electronegativity seems to be slightlydispersed, which manifests itself in an upshifted Car6F (-128.6 ppm) and a low-field shiftedCar8F (-118.6 ppm). The 19F NMR measurements of fleroxacin correspond to this with-127.6 ppm (Car6F ) and -119.2 ppm (Car8F ), in addition to -224.0 ppm for the fluorineatom at the end of the alkyl chain substituent on Nar1.Mass spectrometry revealed various re-combinations of single quinolone molecules(HL) with one sodium cation in the higher mass range, [(HL)2−4 + Na+], next to onesingle quinolone molecule plus either one proton, [(HL) + H+], or one sodium cation,[(HL) + Na+], as parent peak(s) in the lower mass range. No solvent influences were foundin MS-spectra recorded in methanol, acetonitrile or aqueous mixtures of these solvents.To complete the chemical characterization and to control the quality of the purchasedchemicals, elemental analyses for the elements C, H, and N were performed as well. All82analyzed quinolone drugs matched the calculated C, H, and N percentage values withinan average difference of ∆ =0.21. The largest differences between the analytically cal-culated and found values of C, H, and N were observed for levofloxacin (C, 0.39) andlomefloxacin hydrochloride (H, 0.28). These were the only two quinolone molecules out ofnine that contain a stereocenter. In the levofloxacin molecule, the chiral center sits at themethyl-subsituted C-atom of the condensed hexane ring connecting to the quinoline corethrough Nar1 and Car8. In the lomefloxacin hydrochloride molecule, it is located in the1,4-piperazinyl-ring at Cpip3 attached to the condensed aromatic core in 7-position.2.3.2 Stability in Iso-Sensitest BrothTest solutions of the nine selected quinolones in Iso-Sensitest broth were monitored employ-ing UV-Vis spectroscopy over 24 hours. In addition, test solutions in water were preparedfrom the same quinolone stock solutions and monitored with UV-Vis over 24 hours to al-low for a direct comparison. In both experimental set-ups, the quinolone concentrationin the final test solutions was 0.1 mM, the same concentration at which the quinolonesentered the antimicrobial susceptibility disk test (Section 2.3.3). Sample spectra of allnine selected quinolones in Iso-Sensitest broth as well as water for comparison, includingrespective spectra of the solvent media themselves, are presented in Figure 2.5.Before the UV-Vis spectra of the quinolones were recorded in alphabetical order ateach time point, spectra of the initial blank sample of water and Iso-Sensitest broth werecollected; these spectra have been drawn in subfigure (a) of Figure 2.5. Strikingly, theseUV-Vis spectra showed an increase in absorbance by 0.02 AU with time in Iso-Sensitestbroth as well as in water. The fact that such changes appeared not only in Iso-Sensitestbroth but as well in pure water, resolved any immediate assumptions that these changesmight reflect chemical alterations or biological degradations in the Iso-Sensitest broth.Because the trend in increasing absorbance from one UV-Vis measurement to the next83immediately stopped after a new blank of these two solutions was recorded for all UV-Vismeasurements to follow the 20 h time point, and the absorbance intensity fell back onto theinitial absorbance curves recorded at the first time point, the observed trend of increasingabsorbance can only be implicated in an effect of the UV-Vis machine related to the blankfunction of the instrument.326 Furthermore, the observed absorbance changes over time arenot due to temperature or pH changes, as the room temperature as well as the pH valuesof the test solutions were monitored and provided constant values (data not shown) overthe duration of both studies.Comparing the recorded UV-Vis spectra for the nine selected quinolones in water andIso-Sensitest medium over 24 hours (Figure 2.5 (b) to (d)), no changes between the freshlyprepared test solutions and the 24 hour old test solutions are visible, neither in waternor in the Iso-Sensitest medium. Any alteration of the quinolone molecule through achemical reaction with any of the ingredients of the iso-Sensitest broth would have resultedin detectable changes in the UV-Vis spectrum. As has been discussed in Section 2.3.1, thesecond absorbance maximum at higher wavelength (300−380 nm) is especially sensitiveto pH changes, as these affect the intramolecular hydrogen bond formation between thecarbonyl group in Car4 position and the carboxylic acid group in Car3 position. Thequinolones are known to interact with metal ions through the same binding modality;327 328therefore, if the quinolones were to react with metals from the Iso-Sensitest medium, therespective molecular changes would be reflected in major changes in the second absorbancemaximum. When Song et al. studied the effect of copper(II) and magnesium(II) ionson nalidixic acid in water (20 µM), they saw a distinct hyperchrome shift in the secondabsorption maximum of Hnxa at a metal ion concentration of 0.4 mM and higher, whichcorresponded to an excess factor of 20x.313It must be mentioned that it was not possible to record the UV-Vis spectra in Iso-84(a) (b)(c) (d)Figure 2.5: UV-Vis study to monitor the stability of nine selected quinolones inIso-Sensitest broth and in water. (A) Recorded blanks of water (top) and Iso-Sensitest broth (bottom) over time. (B) UV-Vis spectra of Hcipro, Henox, andHflex in water (top) and Iso-Sensitest broth (bottom). (C) UV-Vis spectra ofHlevox, Hlomx, and Hnxa in water (top) and Iso-Sensitest broth (bottom).(D) UV-Vis spectra of Hnofx, Hoxa, and Hpia in water (top) and Iso-Sensitestbroth (bottom). 85Sensitest broth prepared according to the manufacturer’s specification due to limitationsof the UV-Vis technique. Instead it was necessary to dilute its concentration by a fac-tor of two to limit the noise caused by the biological growth medium to an acceptablerange and to ensure sufficient UV-Vis sensitivity towards the studied quinolones, as themedium itself absorbed UV light up to 340 nm wavelength, but any absorption above320 nm was not larger than 0.02 AU (Figure 2.5a). According to the product data sheetof the Iso-Sensitest broth,329 the used product (OXOID CM0473) contains 0.2 g/L mag-nesium glycerophosphate and 0.1 g/L calcium gluconate, corresponding to a concentrationof c100%= 1.03·10−3 M (c50%= 0.515 mM) and c100%= 2.32·10−4 M (c50%= 0.116 mM), re-spectively. Even at 50%- concentration, the concentration of metal cations in Iso-Sensitestbroth exceeds the test concentration of the quinolones (0.1 mM), as the medium containsMg2+ in 5.5x higher and Ca2+ in 1.2x higher concentration. Therewith, the concentrationof Mg2+ in 50% Iso-Sensitest broth is with 0.515 mM as well already larger than in thestudy of Song et al. (0.4 mM);313 therefore, any extra amounts of nutrients included in100% Iso-Sensitest broth can be regarded as true excess, and it is highly unlikely thatthese should have any further effect on the quinolone molecules during the antimicrobialsusceptibility test. Because no changes indicating a chemical modification of the quinolonemolecule through interaction with metal ions present in Iso-Sensitest medium can be seenin the UV-Vis spectra, there is no evidence for chemical or biological degradation or de-composition of the selected nine quinolones in the tested solvent media over the monitoredtime frame of 24 hours.2.3.3 Antimicrobial Susceptibility Disk TestQuninolones demonstrate good in vitro activity against a range of Gram-positive andGram-negative bacteria.284 285 The in vitro activities of the nine selected quinolones, ata test concentration of 0.1 mM, against strains of some of the most reported causative86pathogens330 are listed in Table 2.5. Their antimicrobial susceptibility was tested ac-cording to the single-disk method331 332 on a selection of organisms comprising Gram-positive, Enterococcus faecalis (E. faecalis) and Staphylococcus aureus (S. aureus), andGram-negative bacteria, Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia),and Pseudomona aeruginosa (P. aeruginosa).The results of the antimicrobial susceptibility single-disk test show various resistancesagainst the quinolones (0.1 mM), which partly can be related to the length of time thatthe drug has been in use and therewith the drug generations (Figure 2.1). Nalidixic acid,the oldest quinolone and the foundation of this drug class, has no efficacy anymore againstany of the tested microbes. The same holds true for pipemidic acid, another member ofthe first generation of quinolone drugs. Oxolinic acid, on the other hand, is not potentagainst the tested Gram-positive bacteria strains, but it does inhibit the growth of E. coliand K. pneumonia, although it does show no efficacy against P. aeruginosa.Enoxacin, an early member of the second generation of quinolone drugs, is not effec-tive anymore against the Gram-positive bacteria included in the study. Norfloxacin andlomefloxacin hydrochloride do not inhibit the growth of E. faecalis, and Hlomx is as wellnot potent against P. aeruginosa at 0.1 mM concentration. The tested strain of E. fae-calis appears to be resistant against the majority of quinolones included in this study,even against the third-generation fleroxacin, only ciprofloxacin (second generation) andlevofloxacin (third generation) show efficacy against it. This comes as no surprise, as the(fluoro-)quinolones are known to exert a reduced rate of kill against enterococcal species.333Overall, the results indicate that the selected quinolones are not as potent against theGram-positive bacteria as they are against the Gram-negative organisms selected for thisstudy. Once more, ciprofloxacin and levofloxacin proved to be the best all-round quinoloneantimicrobial drugs available against a variety of pathogens with an activity relationship87Table 2.5: Results of antimicrobial susceptibility study of nine quinolonesBacteria Hcipro Henox Hflex Hlevox Hlomx Hnxa Hnofx Hoxa HpiaE. faecalis 9 (0) 0 0 8 (1) 0 0 0 0 0S. aureus 12 (1) 0 10 (1) 14 (1) 7 (1) 0 7 (1) 0 0E. coli 22 (1) 16 (1) 19 (1) 20 (0) 17 (2) 0 19 (1) 14 (1) 0K. pneumonia 18 (0) 14 (1) 19 (1) 18 (0) 16 (1) 0 15 (1) 11(1) 0P. aeruginosa 18 (1) 7 (1) 7 (0) 9 (0) 0 0 6 (0) 0 0Reported inhibition zones [mm] are averaged values from three plates (standard devi-ation). Disk diameter 0.6 mm. Loading volume 20 µL. Concentration quinolone testsolution 0.1 mM. Disks loaded with solutions of methanol and 2% DMSO in methanolserved as controls, all of these showed no inhibition (0 mm).that compares well to previous literature reports: Hlevox > Hcipro in S. aureus,334 andHcipro > Hlevox in P. aeruginosa;335 however, this should not mask the fact that theoverall bacterial susceptibility to quinolone antimicrobial agents continues to decrease,336and Table 2.5 does identify patterns of resistance.2.4 ConclusionThis chapter presented a comprehensive chemical characterization of nine selectedquinolone antimicrobial drug molecules from three generations of quinolone drugs.Nalidixic acid, oxolinic acid, and pipemidic acid from the first generation; ciprofloxacin,enoxacin, lomefloxacin hydrochloric, and norfloxacin from the second generation, as wellas fleroxacin and levofloxacin from the third generation of quinolone drugs. Melting pointmeasurements, UV-Vis and IR spectroscopy data, 1H NMR, 13C NMR, and 19F NMRmeasurements, have been reported and discussed, in addition to mass spectrometry andelemental analysis data. During a 24 hour-long UV-Vis study, the nine selected quinolonesshowed no signs of degradation or decomposition in Iso-Sensitest broth compared to testsolutions in water.Antimicrobial susceptibility single-disk test studies in this medium were performed88against the most commonly reported pathogens associated with nosocomical infections330.The results proved once more the growing resistance of bacteria against commonly usedantimicrobial drugs.337 Nalidixic acid and pipemidic acid, both from the first generation ofquinolones, were not effective against any of the tested Gram-positive and Gram-negativebacteria strains at the chosen test concentration of 0.1 mM. Only ciprofloxacin and lev-ofloxacin were successful in killing all of the five tested pathogens in vitro.89Chapter 3Testing the ”Trojan Horse Theory”:Gallium(III) and Iron(III) Complexes ofQuinolone AntimicrobialsIn this chapter, the ”Trojan Horse Theory” will be tested. Will the combination of gal-lium(III) ion with quinolone antimicrobial agents, which were introduced in the previouschapter, have a combinational, or maybe even a synergistic effect, leading to increasedantimicrobial efficacy of such novel complexes?3.1 A Bioinorganic Approach: Fighting the GrowingAntimicrobial Resistance With Metallodrugs”The world is on the brink of losing (...) miracle cures,” with these words Director-Generalof the WHO, Dr. M. Chang, summarized the growing resistance of microbes to known an-timicrobial drugs on World Health Day 2011.338 The WHO has rated these developmentsas one of the greatest threats to human health, because through antimicrobial resistancethe control of infectious diseases is impeded, the achievements of modern medicine (e.g.,90surgeries) are jeopardized, and the costs of health care are rising globally.339 337 All of thesedevelopment are only a weak foretaste of life in the post-antibiotic era that is near.337 Forthe trend of growing resistance of microbes, the annual antimicrobial resistance surveillancereports of the European Centre for Disease Prevention and Control (ECDC) provide evi-dence beyond doubt.340 According to the U.S. Centers for Disease Control and Prevention(CDC), each year at least 2 million patients in the US are infected with multi-drug resistant(MDR) bacteria, often in a hospital setting; the numbers of deaths vary between 23,000341to 99,000.342 This also costs the U.S. economy from 20 billion USD in excess direct healthcare costs to 35 billion USD including additional loss of productivity (2008).341 343 In thebeginning of the 21st century, the glory days of antibacterial drug research seem to be com-ing to an end, as the Infectious Disease Society of America (IDSA) reports that only twonew antibiotics have been approved, since their trans-Atlantic initiative with the EMA todevelop 10 new antibiotic drugs by 2020 (titled ”10x’20”) was rolled out six years ago,344and the number of new antimicrobial agents approved by the FDA continues to decline.345Scientists from academia and industry, health agencies, and policy makers need to takeaction to build a sustainable research and development structure for antimicrobial drugs,as well as global resistance surveillance systems to overcome the yawning innovation gap,manage the cost-benefit equation longterm, fight the imminent health crisis, and protectfuture generations against the ever-evolving resistance in microbes.343 346 347 348 349 350 351In the field of bioinorganic chemistry, the application of metal complexes to the ther-apy and diagnosis of developed drug resistance is an accepted concept,264 which holds hopefor novel parasitic236 and antibacterial352 drugs. In addition, a number of antimicrobialagents exact metal ions for their mechanism of action (see Chapter 2 for Mg2+ example),and an improved understanding of the structure, function, and actions of such ”metal-loantibiotics”353 is essential to design metal complexes with new mechanisms of action to91overcome growing antimicrobial resistance.The transition metal iron is critical for the metabolism and growth of most organisms,with the possible exemption of some lactobacilli.354 About 0.3 to 4.0 µM concentrations ofiron are required for the growth of cells of animals (mouse), plants (algae), and microor-ganisms (fungi, Gram-positive/-negative bacteria).355 Hypoferraemia, the limitation of ironavailability in vivo, is utilized by many species, including humans, as an autoimmune hostdefence.356 357 To be successful, such defence systems require extremely low levels of freeFe3+ ions of ≤10−18M in normal tissue fluids of the host;358 however, microbial pathogenshave learned to counteract this strategy and to scavenge iron from the host sources (ferritin,lactoferrin, transferrin, and heme compounds)359 by secreting siderophores,360 361 362 a crit-ical step in bacterial infections.363 364 The fact that bacterial virulence is highly enhanced,if free iron is widely available, was first recognized in the clinic365 and has been furtherexemplified in medical practice.366 On the other hand, iron metabolism has been exploredas a target for antimicrobial strategies367 and even for the treatment of cancer.368 Besidesreducing the availability of free iron with chelating agents or inhibiting iron metabolism inthe infected host, a third antimicrobial strategy utilizes the pathogen’s own iron transportsystem for the delivery of bactericidal agents, and has been named the ”Trojan Horse”strategy.367 The quintessence of this strategy lies in deceiving bacteria to take up antimi-crobial agents that then kill them. The idea of linking antimicrobial agents to siderophoreshas been around for forty years, and various structures linking sulfonamides or β-lactamantibiotics to siderophores have been successfully realized,369 e.g., a recent example ofenterobactin-ampicillin conjugates showed a 1000-fold decrease in MIC against E. coli.370In medicinal bioinorganic chemistry it is commonly accepted that biological systemscannot distinguish Ga3+ from the essential element iron in its tripositive ionic form due totheir similarities in charge (both 3+), ionic radii (Fe3+= 0.65 A˚, Ga3+= 0.62 A˚), preferred92coordination number (CN= 6) and chemical behaviour (both hard Lewis acids). Oneimportant difference of both tripositive metals lies in their redox chemistry. In aqueousmedia, iron commonly exists in two stable oxidation states Fe2+ (d6) and Fe3+ (d5); invivo the redox potential (E◦ = 0.771 V, 25◦C)371 between these two states enables a widerange of metabolic activities that are carefully regulated to prevent cytotoxic reactions.As a group 13 metal, gallium lacks such interesting redox chemistry, which allows itsuse as a redox-inactive Fe3+ substitute in vivo. In biology this relationship is useful forstudying metal complexation in proteins and bacterial populations;372 however, the bindingstrength between biological (macro-)molecules and these two metals might differ slightly,e.g., gallium(III) binds to transferrin with a 300-fold less affinity than iron(III).373 Thesubstitution of Ga3+ into metalloenzymes (at sufficient excess of Ga3+ over Fe3+) canresult in a loss of enzymatic function, because the proteins are rendered inactive due totheir inability to access the essential 3 + /2+ redox chemistry, with cellular toxicity as aresult, which has stamped Ga3+ the ”Trojan Horse” in biological systems.374As described in Chapter 1, gallium(III) has been in therapeutic use for the past thirtyyears and is, in general, considered safe.375 376 Its coordination chemistry377 is utilized inradioimaging, e.g., 68Ga pasireotide tetraxetan (SOMscan) is a PET imaging agent forgastroentero-pancreatic neuroendocrine tumors in clinical phase I/II trials,378 where 68Gaseems a convenient PET alternative to 99mTc.39 379 In therapy, intravenous gallium nitrateis already approved by the FDA for the treatment of cancer-associated hypercalcemia (Gan-ite),170 the oral tris-(8-quinolinolato)gallium(III) (KP46) and tris(maltolato)gallium(III)are in clinical trials against cancer,380 and recently, the antimicrobial effect of 69Ga3+ hasgained attention. Schlesinger and co-workers reported that gallium(III) nitrate and Ga-transferrin inhibit the growth of Mycobacterium tuberculosis and Mycobacterium aviumextracellularly and within human macrophages.381 382 A pharmacokinetic and safety study93of Ganite in cystic fibrosis patients is underway,383 and a phase II study (IGNITE) hasbeen scheduled.384 The use of gallium(III) salts at physiological pH, however, is consid-ered a problem, because Ga3+ ions, similar to Fe3+ ions, are prone to hydrolysis, formingmono- and polynuclear oxo/hydroxo species of low solubility.385 386 This might as well beone of the reasons why Beraldo and co-workers realized that gallium(III) nitrate was notpotent against the studied strain of Pseudomona aeruginosa but observed an increase inactivity upon coordination to thiosemicarbazone ligands.387 Chelated to ligands, the gal-lium(III) at the center of the complex is sheltered from the otherwise inevitable hydrolysisin vivo. Other examples of gallium(III) coordination complexes with antimicrobial proper-ties are: gallium-citrate,388 gallium-desferroxamine B,388 389 and gallium-maltol,390 whichall have been primarily tested against Pseudomona aeruginosa. Tris(maltolato)gallium(III)has gained special attention for the treatment of infections associated with bacterialbiofilms.391 392 393 394 Besides complexation, co-administration of gallium(III) salts withknown antimicrobial agents is another way to circumvent unwanted hydroxide formationin vivo, especially in smart formulations, such as gallium-gentamicin in liposomes.395 Thecombination of antimicrobial and non-antimicrobial agents has been described as a generalconcept to enhance antimicrobial potency.396The quinolone antimicrobial agents were introduced in Chapter 2 (Figure2.1), where we as well saw the developed bacterial resistance against some ofthe members of these drug class, although the resistance situation comparedto other antimicrobial drug classes is still fortunate.397 Numerous metal com-plexes of main group and transition metals with a diversity of quinolone ligandshave been reported,327 328 as have been the syntheses of the iron(III) complexestris(nalidixido)iron(III),398 tris(norfloxacino)iron(III),399 tris(enrofloxacino)iron(III),400tris(ciprofloxacino)iron(III),401 bis(sparfloxacino)iron(III),402 and tris(lomefloxacino)-94iron(III),403 as well as some mixed complexes of iron(III) with quinolones and a secondligand, such as bispyrazolones404 or the nitrilotriacetic anion.405 Research in this area hasbeen motivated by the clinical observation that bioavailability and bactericidal efficacy ofquinolone antimicrobial agents are reduced through the interaction with cations in the hu-man body, and has, therefore, focused on metals generally included in antacid preparationsor vitamin supplements.406 407 408 Bivalent or trivalent metal ions included in such prepa-rations bind to one or up to three quinolone molecules through the ionized carboxylate onCar3 and the adjacent keto group on Car4 in vivo,409 occupying the actual binding sideto DNA gyrase or/and topoisomerase IV, therewith perturbing the mechanism of action(Section 2.1). Several studies support this observation, in in vitro tests the activity ofciprofloxacin was reduced upon complexation to Mg2+,304 while in in vivo tests (dogs)only a small extend of complexation of norfloxacin with Ca2+, Mg2+, Zn2+, Fe2+, Al3+resulted in disproportionately large reductions (60−80%) in bioavailability.410In an attempt to develop novel antimicrobial agents, we have explored the ”Tro-jan Horse” bactericidal concept with a coordination chemistry approach and synthesizedtris(quinolono)gallium(III) complexes of nine selected quinolone antimicrobial drugs (Fig-ure 3.1). In addition, we have prepared the analogous tris(quinolono)iron(III) complexesto be able to directly compare the effect of the Ga3+ versus the Fe3+ ion in these otherwiseidentical compounds. In first in vitro studies against pathogens associated with hospital-acquired diseases, we tested all tris(quinolono)metal(III) complexes (0.1 mM) along sidethe free quinolone ligands (0.1 mM and 0.3 mM) to determine if the combination of aquinolone antimicrobial agent with Ga3+ in a 3:1 ratio exhibits a combinational or even ananticipated synergistic effect. To further study the antimicrobial potency of Ga3+ versusFe3+, complexes with maltol (Hma), a widely used food additive, have been synthesizedand tested as well.95Figure 3.1: Overview of the synthesized and characterized tris(quinolono)metal(III)complexes (M= Ga3+, Fe3+).963.2 Materials & Methods3.2.1 ChemicalsThe majority of chemicals used were purchased from Sigma-Aldrich: ciprofloxacin,enoxacin, levofloxacin, norfloxacin, oxolinic acid, pipemidic acid, and gallium(III)nitratenonahydrate as well as iron(III)chloride hexahydrate. TCI America supplied theciprofloxacin hydrochloride, nalidixic acid, and lomefloxacin hydrochloride. Organic sol-vents and sodium salts were obtained from Fisher Scientific. The 50 % sodium hydroxidesolution came from ACROS. Atomic absorption standard (AAS) solutions of iron(III) andgallium(III) (1000 mg/L ±4 mg/L) were obtained from Fluka. Potassium hydrogen ph-thalate from BDH Chemicals, Ltd. was recrystallized, and deionized water was purifiedusing a ELGA MAXIMA ultra pure water system (resistivity 18 MΩ·cm, 25◦C); all otherchemicals and solvents were used without further purification.3.2.2 InstrumentationDuring the synthetic preparation of the metal complexes, the pH of the reaction mixturewas monitored with a Metrohm 6.0234.110 electrode connected to a Metrohm 713 pH me-ter. The electrode was filled with 3.0 M potassium chloride solution as the electrolyteand calibrated against reference buffer solutions (4.00, 7.00, 10.00) from the FisherScien-tific Buffer-Pac on a regular basis. Mp determinations were performed in triplicate on aDigiMelt SRS Stanford Research Systems melting point apparatus and are uncorrected.UV-Vis spectra were recorded on a Hewlett Packard 8453 instrument running UV-VisChemStation software (version B.04.01[61], Agilent Technologies, 2001−2010). IR spectrawere obtained neat in the solid state on a Thermo Scientific Nicolet 6700 FT-IR spec-trometer using OMNIC (version 7.4.127, Thermo Fisher Scientific Inc.) in the range of4000−500 cm−1. For the interpretation of bands, the following abbreviations were used:97st, md, w, br, and sh. NMR spectroscopy was performed at the UBC NMR Facility. 1HNMR and 13C spectra, as well as 19F NMR spectra as applicable, were recorded in addi-tion to COSY, HSQC and HMBC experiments at ambient temperature on Bruker Avance300 and 600 spectrometers running Topspin software (version 3.2, Bruker). The BrukerAvance 600 spectrometer was equipped with a Bruker TCI-Z-5mm cryoprobe, which al-lowed for the detection of 13C and 1H nuclei at a low signal-to-noise ratio of 6000/1 (1H)and 600/1 (13C). For the NMR experiments, the tris(quinolono)gallium complexes weredissolved in deuterated DMSO with the help of sonication and heating at a final con-centration of approximately 6 mM. The residual solvent signal of d6-DMSO was used asthe internal standard.309 Chemical shifts are referenced in ppm against tetramethylsilane(δC,H= 0 ppm) and trichlorofluoromethane (δF= 0 ppm), respectively. Multiplicities aredescribed as: s, d, q, and m. Aromatic protons are abbreviated ar, while pip representsthe 1,4-piperazinyl ring in C7 position on the aromatic ring system and prop the propylring in N1 position on the aromatic ring system. All NMR spectral analysis were per-formed with the software iNMR (version 5.1.2, Mestrelab Research). For low-resolutionmass spectrometry, a Water ZQ spectrometer equipped with an electrospray and chemi-cal ionization source was used. All samples showed acceptable solubility in methanol atvarying pH values; MS experiments were conducted in methanol or nitromethane. High-resolution mass spectrometry was performed at the UBC Mass Spectrometry Centre ona Waters Micromass LCT employing electrospray-ionization. Characteristic signals havebeen listed as dimensionless mass-to-charge ratios with the intensity related to the basesignal. The UBC Mass Spectrometry Centre as well determined the elemental compositionof the synthesized compounds. Microanalyses for the elements C, H and N were preparedon a Carlo Erba Elemental Analyzer EA 1108.983.2.3 Thermogravimetric AnalysisCombined thermogravimetric analysis (TGA) and differential thermal analysis (DTA) mea-surements were performed in minimum duplicate on a simultaneous thermal analyser PerkinElmer STA 6000 running Pyris Manager (version, Perkin Elmer) over the tem-perature range from 25−900◦C under a stream of nitrogen gas (19.8 mL/min). The programstarted by stabilizing the sample at 25◦C for 3 min, before it was heated up to 900◦C inintervals of 5◦C/min, held at 900◦C for 3 min, and finally cooled down to 25◦C at a rateof 50◦C/min.3.2.4 PotentiometryPotentiometric titrations were carried out using a Metrohm 809 Titrando system with aMetrohm 800 Dosino unit and a Metrohm 801 Stirrer interfaced to a PC computer runningTitrando PC Control (Version 5.0, Metrohm). The system was equipped with a ThermoScientific ORION 8103BN combination electrode (precision: ± 0.1 mV). The referencecompartment of the electrode was filled with 0.16 M aqueous sodium hydroxide solutionas the electrolyte. All titrations were carried out under an inert atmosphere by bubblingnitrogen through the cell for at least five minutes prior to proceeding and also during thetitration. To exclude any carbon dioxide the nitrogen was washed with 2.0 M aqueoussodium hydroxide solution prior to entering the cell. The titrations were performed at25◦C (± 0.1◦C) in a 10 mL water-jacketed vessel. All solutions were prepared in ultra purewater having a constant ionic strength (I= 0.16 M) using sodium chloride.Carbonate-free solutions of the titrant, sodium hydroxide (NaOH), were prepared bydilution of 50% solution with freshly boiled ultra pure water under a stream of nitrogengas. The aqueous NaOH solution was standardized with potassium hydrogen phthalate.Prior to each potentiometric equilibrium study, electrode calibration was accomplished by99titrating the sodium hydroxide stock solution into the standardized hydrochloric stock so-lution. Calibration data were analyzed by standard computer treatment provided withinMacCalib411 to obtain the calibration parameter E◦ and pKw. For the autoprotolysis con-stant of water at 25◦C, the following diffusion correction terms were used: E◦= 2.463 V andpKw= 1.057. Protonation constants of ciprofloxacin and iron(III) as well as gallium(III)complexation constants were obtained from titrations performed in triplicate with allowedequilibration times varying to 60 minutes maximum; data fitting was performed with thesoftware Hyperquad2008 (Protonic Software).3.2.5 Computational DetailsWith DFT, one of four possible isomer structures of gallium(III) coordinatedto three ciprofloxacin anions was calculated at the B3LYP level utilizing the6-31+G(d,p); LANL2DZ mixed basis set as implemented in Gaussian412 The optimizedgeometry is characterized as an energetic minimum, indicated by the absence of imaginaryfrequencies.3.2.6 Antimicrobial Susceptibility StudiesThe biological study was performed in UBC’s Biological Service Laboratory, a biologicalsafety level II facility, according to respective operating and safety protocols. Please seeAppendix A for the detailed test procedure.3.2.7 Synthesis & Characterization of Tris(quinolono)metal(III)ComplexesThe tris(quinolino)metal(III) complexes were synthesized according to the following threegeneral synthetic methods:Method (a): To a solution of metal(III)nitrate nonahydrate (0.1 mmol) in water (2 mL),an acidified aqueous solution (8 mL) of the quinolone (0.3 mmol) was added dropwise. Dur-100ing the addition the pH was carefully monitored and kept below pH 5; finally, raising thepH to pH 7.5 with aqueous sodium hydroxide (1.0 M and 0.1 M) resulted in a character-istically colored solution (yellow for Ga3+, red-brown for Fe3+) that was stirred rigorouslyat room temperature for 20 min, before the vial was closed tightly and placed in the fridgeat 4◦C. After 3−5 days the desired product had precipitated. The solid was separated byfiltration (glass frit size F), thoroughly washed with water (2 mL) and methanol (2 mL),and dried in vacuo.Method (b) is a modification of the reported synthesis of tris(nalidixido)iron(III):398The quinolone (0.3 mmol) was heated with sodium bicarbonate or sodium hydroxide(0.3 mmol) in water (10 mL) until the initially white suspension had turned into a clearsolution, which was then added onto the solid metal(III) nitrate nonahydrate (0.1 mmol).Upon addition the pH was kept at pH≤5, the desired product started forming immediatelyand precipitated as solid (final pH∼7). The suspension was stirred rigorously until cooledto room temperature (for a minimum of 30 min, often overnight). The desired product,which precipitated often at room temperature or otherwise after placing the reaction vialin the fridge (4◦C) was separated by filtration (glass grit, size F) as a solid, washed withwater (2 mL) and methanol (2 mL), and dried in vacuo.Method (c) is a modification of the reported synthesis of tris(ciprofloxacino)iron(III):401A suspension of quinolone (0.31 mmol) with sodium hydroxide or potassium hydroxide(0.33 mmol) in methanol (15 mL) was refluxed until it turned into a clear, colorless solu-tion. The hot methanolic solution was added onto the solid metal(III)nitrate nonahydrate(0.1 mmol), and the resulting colored solution was refluxed further for thirty minutes. Thereaction solution was left to cool in air. Evaporation of the solvent in air, or a reduction ofthe solvent by at least 50% volume, led to precipitation of a colored solid, which was sepa-rated by filtration (glass frit, size F), thoroughly washed with water (2 mL) and methanol101(2 mL), and dried in vacuo.The method that gave the highest product purity, as determined by EA and high-resolution electrospray ionization (HR-ESI) mass spectrometry, at a 0.1 mmol scale isreported. Tris(ciprofloxacino)gallium(III), [Ga(cipro)3]Method (c) gave a pale yellow solid (99 mg, 0.094 mmol, 94%). Mp: ≥220◦C, decomposi-tion to brown solid. IR (neat): ν˜ [cm−1] = 3418 (md, br, water), 2846 (w, br) 1620 (st),1545 (w), 1516 (sh), 1472 (st), 1451 (sh), 1373 (st, br), 1287 (sh), 1252 (st), 1182 (md)1146 (md), 1106 (sh), 1025 (st), 949 (st), 893 (md), 810 (md), 787 (md), 765 (sh), 740(st, br), 704 (md), 627 (st), 540 (md), 505 (st). NMR: δH (600 MHz, 298 K, d6-DMSO)[ppm] = 8.93 (s), 8.84 (s), 8.80 (s), (3 H, Car2H); 7.65 (d, J3H,F= 13.8 Hz), 7.57−7.53 (m),7.41 (d, J4H,F= 7.2 Hz), (6 H, Car5H and Car8H); 3.95−3.81 (m, 3 H, CpropH); 3.29−3.20(m, 12 H overlaid with water, Cpip2,6H2); 2.96−2.91 (m, 12 H, Cpip3,5H2); 1.36−1.32 (m,6 H, CpropHb,b′); 1.23−0.82 (m, 6 H, CpropHa,a′). δC (125 MHz, 298 K, d6-DMSO) [ppm]= 173.7 (s), 173.6 (s), 173.2 (s), (Car4); 165.6 (s), 165.5 (s), 165.3 (s), (COOH); 153.1 (d,J1C,F= 206.1 Hz), 153.0 (d, J1C,F= 207.1 Hz), 152.9 (d, J1C,F= 207.3 Hz), (Car6); 149.8 (s),149.6 (s), 149.3 (s), 149.2 (s), (Car2); 145.4 (d, J2C,F= 21.0 Hz), 145.3 (d, J2C,F= 21.8 Hz),145.2 (d, J2C,F= 19.5 Hz), (Car7); 139.0 (s), 138.8 (s), 138.7 (s), (Car8′); 118.7 (d, J3C,F= 6.9Hz), 118.6 (d, J3C,F= 7.0 Hz), 118.4 (d, J3C,F= 7.8 Hz), (Car4′); 111.8 (s), 111.7 (s), 111.5(s), (Car5); 110.5 (s), 110.3 (s), 110.2 (s), (Car3); 106.3 (s), 105.7 (s), 105.3 (s), (Car8); 50.1(s), 50.0 (s), 49.9 (s), (Cpip2,6); 45.0 (s), 44.9 (s), 44.8 (s), (Cpip3,5); 36.2 (s), 36.0 (s), 35.9(s), 35.8 (s), (CpropH); 7.7 (s), 7.6 (s), 7.5 (s), (CpropH2). δF (282 MHz, 298 K, d6-DMSO)[ppm] = -121.1 (s), -121.15 (s), -121.21 (s), (3 F, Car6F ). MS (ES+, CH3OH): m/z (%) =1083 (40) [ML3 + Na+], 730 (100) [ML2]+. HR-ESI-MS: m/z for C51H51F3 69GaN9O9+ H+ calcd. (found): 1060.3096 (1060.3073); for C34H34F2 69GaN6O6+ calcd. (found):102729.1764 (729.1785). EA: Anal. Calcd. (found) [%] for C51H51F3GaN9O9·8 H2O: C, 50.84(50.61); H, 5.60 (4.80); N, 10.46 (10.79). Tris(enoxacino)gallium(III), [Ga(enox)3]Method (b) gave a pale yellow solid (85 mg, 0.082 mmol, 82%). Mp: ≥200◦C, decomposi-tion to light brown solid. IR (neat): ν˜ [cm−1] = 3404 (md, br, water), 3045 (w, br), 2977(w, br), 1625 (st), 1562 (md), 1519 (md), 1469 (sh), 1434 (st, br), 1369 (md), 1346 (st),1323 (md), 1276 (st), 1253 (st, br), 1185 (md), 1153 (w), 1119 (md), 1092 (md), 1039 (md,br), 972 (md), 941 (md, br), 908 (sh), 812 (st), 789 (md), 766 (md), 746 (md), 677 (w),651 (md), 626 (st), 563 (md), 516 (st), 453 (w). NMR: δH (300 MHz, 298 K, d6-DMSO)[ppm] = 9.18 (s), 9.13 (s), 9.08 (s), 9.04 (s), 8.99 (s) (3 H, Car2H); 8.12 (d, J3H,F= 13.2Hz), 7.75 (d, J3H,F= 13.2 Hz), 7.72 (d, J3H,F= 14.0 Hz), 7.69 (d, J3H,F= 13.8 Hz), (3 H,Car5H); 4.59−4.43 (m, 6 H, CH2CH3); 3.92−3.85 (m, 12 H, Cpip2,6H2); 3.12−3.02 (m, 12H, Cpip3,5H2); 1.43−1.23 (m, 9 H, CH2CH3). δC (75 MHz, 298 K, d6-DMSO) [ppm] =173.9 (s), 173.5 (s), 173.4 (s), (Car4); 168.9 (s), 168.8 (s), 168.7 (s), (Car8′); 165.8 (s), 165.6(s), 165.5 (s), 165.3 (s), (COOH); 149.6 (d, J2H,F= 90.3 Hz), 149.4 (d, J2H,F= 85.1 Hz),149.0 (d, J2H,F= 89.4 Hz), (Car7); 147.2 (d, J1H,F= 207.0 Hz), 147.1 (d, J1H,F= 202.8 Hz),147.0 (d, J2H,F= 214.6 Hz), 146.9 (d, J1H,F= 215.4 Hz), (Car6); 144.7 (s), 144.3 (s), 144.1(s), (Car2); 119.6 (d, J2C,F= 18.1 Hz), 119.0 (d, J2C,F= 17.5 Hz), 118.70 (d, J2C,F= 19.6Hz), (Car5); 113.2−112.6 (m, Car4′); 108.1 (s), 108.0 (s), 109.9 (s), (Car3); 47.5 (s), 47.3(s), 47.2 (s), (Cpip2,6); 46.2 (s), 45.9 (s), 45.6 (s), (CH2CH3); 44.2 (s), 44.0 (s), 43. 7 (s),(Cpip3,5); 14.9 (s), 14.8 (s), 14.7 (s), (CH2CH3). δF (282 MHz, 298 K, d6-DMSO) [ppm]= -126.4, -126.5, -126.6, -126.8 (3 F, Car6F ). MS (ES+, CH3OH): m/z (%) = 1050 (20)[ML3 + Na+], 708 (100) [ML2]+. HR-ESI-MS: m/z for C45H48F3 69GaN12O9 + Na+calcd. (found): 1049.2773 (1049.2797).1033.2.7.3 Tris(fleroxacino)gallium(III), [Ga(flex)3]Method (b) gave a pale yellow solid (85 mg, 0.082 mmol, 82%). Mp: ≥230◦C, decompo-sition to orange-brown solid. IR (neat): ν˜ [cm−1] = 3392 (w, br, water), 3054 (md), 2943(md), 2848 (w), 2796 (md), 1622 (st), 1556 (md), 1514 (md), 1475 (st, br), 1449 (sh), 1409(w), 1391 (w), 1376 (md), 1360 (md), 1328 (md), 1280 (st), 1245 (md), 1230 (w), 1214(w), 1206 (w), 1143 (st), 1123 (md), 1099 (w), 1077 (w), 1062 (md), 1037 (md), 1020 (st),1010 (st), 970 (md), 942 (md), 926 (md), 869 (md), 853 (md), 807 (st), 783 (md), 754 (sh),741 (st), 672 (w), 656 (st), 573 (st), 550 (md), 532 (w), 505 (w, br), 450 (md, br). NMR:δH (600 MHz, 298 K, d6-DMSO) [ppm] = 8.86 (s, 3 H, Car2H); 7.87 (d, J3H,F= 11.4 Hz,3 H, Car5H); 4.98−4.96 (m), 4.93−4.90 (m), 4.86−4.84 (m) (12 H, (CH2)2); 3.36 (br s,12 H overlaid with water, Cpip2,6H2); 2.44 (s, 12 H, Cpip3,5H2); 2.23 (s, 9 H, CH3). δC(125 MHz, 298 K, d6-DMSO) [ppm] = 175.8 (s, Car4); 165.5 (s, COOH); 154.6 (d, J1C,F=205.9 Hz), 154.5 (d, J1C,F= 206.5 Hz), (Car6); 152.1 (s, Car2); 146.1 (d, J1C,F= 205.9 Hz,Car8), 146.0 (d, J1C,F= 205.9 Hz, (Car8); 133.9 (two overlapping d, J2C,F= 11.3, 11.5 Hz,Car7); 127.4 (d, J2C,F= 9.1 Hz, Car8′); 120.1 (d, J3C,F= 6.8 Hz, Car4′); 107.1 (d, J2C,F= 18.9Hz, Car5); 106.6 (s, Car3); 82.0 (d, J1C,F= 137.0 Hz, CH2CH2F); 58.0 (d, JC,F= 11.9 Hz),57.8 (d, JC,F= 16.4 Hz), (CH2CH2F); 55.1 (s, Cpip2,6); 50.3 (s, Cpip3,5); 46.0 (s, CH3). δF(282 MHz, 298 K, d6-DMSO) [ppm] = -119.2 (d, J4F,F= 11.0 Hz, 3 F, Car6F ); -127.6 (q,JF,F= 5.6 Hz, 3 F, Car8F ); -224.2 (d, J6C,F= 6.0 Hz, 3 F, (CH2)2F ). MS (ES+, CH3OH):m/z (%) = 1197 (80) [ML3 + Na+], 806 (100) [ML2]+. HR-ESI-MS: m/z for C51H51F969GaN9O9 + Na+ calcd. (found): 1196.2820 (1196.2838); for C34H34F6 69GaN6O6+ calcd.(found): 805.1700 (805.1719).1043.2.7.4 Tris(levofloxacino)gallium(III), [Ga(levox)3]Method (a) gave a yellow solid (47 mg, 0.041 mmol, 41%). Mp: ≥190◦C, decompositionto orange-brown solid. IR (neat): ν˜ [cm−1] = 3426 (md, br, water), 3033 (w, br), 2931(w), 2848 (w), 2794 (w), 1616 (st), 1519 (st), 1447 (st), 1385 (md, sh), 1331 (st, br), 1261(st), 1150 (sh), 1129 (md), 1093 (md), 1047 (st), 1003 (md), 978 (st), 898 (md), 866 (md),844 (w), 810 (st), 763 (md), 744 (st), 696 (md), 638 (w), 554 (md), 510 (st), 463 (md,br). NMR: δH (600 MHz, 298 K, d6-DMSO) [ppm] = 9.25 (s), 9.19 (s), 9.13 (s), 9.01(s), 9.98 (s), 9.93 (s), 8.98 (s), (3 H, Car2H); 7.60 (d, J3H,F= 12.0 Hz), 7.48−7.45 (m),7.42 (d, J3H,F= 12.0 Hz), 7.29 (d, J3H,F= 12.0 Hz), 7.24−7.211, 7.14 (d, J3H,F= 12.6 Hz),(3 H, Car5H); 5.08−4.88 (m, 3 H, CH); 4.64−4.27 (m, 6 H, OCH2CH); 3.50−3.26 (m,12 H overlaid with water, Cpip2,6H2); 2.76−2.61 (m, 12 H, Cpip3,5H2); 2.46−2.38 (m, 9 H,NCH3); 1.52−1.42 (m), 1.23 (br s), 1.14−1.08 (m), (9 H, CHCH3). δC (125 MHz, 298 K,d6-DMSO) [ppm] = 176.4 (s), 173.6 (s), 173.4 (s), 173.2 (s), (Car4); 166.0 (s), 165.8 (s),165.6 (s), 165.4 (s), (COOH); 155.5 (d, J1C,F= 204.8 Hz), 155.4 (d, J1C,F= 203.3 Hz), 155.2(d, J1C,F= 200.0 Hz), (Car6); 148.2 (s), 147.9 (s), 147.7 (s), 147.5 (s), 146.3 (s), (Car2); 140.4(d, J3C,F= 5.8 Hz), 139.8 (d, J3C,F= 8.9 Hz), 139.2 (d, J3C,F= 10.4 Hz), (Car8); 131.1−130.8(m, Car7); 124.9 (s), 124.6 (s), 124.5 (s), 124.2 (s), (Car8′); 120.0 (s), 119.8 (s), 119.5 (s),119.4 (s) (Car4′); 112.6 (s), 112.5 (s), 111.8 (s), 111.6 (s), 106.7 (s), (Car3); 103.3 (d, J2C,F=20.4 Hz), 102.9 (d, J2C,F= 24.5 Hz), 102.3 (d, J2C,F= 22.8 Hz), (Car5); 68.2 (s), 68.0 (s),67.9 (s), (OCH2CH); 55.2 (s), 55.0 (s), 54.8 (s), (Cpip2,6); 54.7 (s), 54.6 (s), 54.5 (s), (CH);49.4−48.8 (m, Cpip3,5); 45.2−44.4 (m, NCH3); 18.2−17.7 (m, CH(CH3)). δF (282 MHz,298 K, d6-DMSO) [ppm] = -119.4 (s), -119.5 (s), -119.7 (s), -119.8 (s), -119.86 (s), -119.93(s), (3 F, Car6F ). MS (ES+, CH3OH): m/z (%) = 1173 (40) [ML3 + Na+], 790 (100)[ML2]+. HR-ESI-MS: m/z for C54H57F3 69GaN9O12 + Na+ calcd. (found): 1172.3232(1172.3248); for C36H38F2 69GaN6O+8 calcd. (found): 789.1975 (789.1989).1053.2.7.5 Tris(lomefloxacino)gallium(III), [Ga(lomx)3]Method (a) gave a pale yellow solid (40 mg, 0.035 mmol, 35%). Mp: ≥200◦C, decompo-sition to brown solid. IR (neat): ν˜ [cm−1] = 3419 (md, br, water), 2980 (w, br), 2848(w, br), 2478 (w, br), 1619 (st), 1556 (w), 1523 (st), 1452 (st), 1354 (md, sh), 1323 (st,br), 1277 (md), 1247 (st), 1123 (md), 1090 (md), 1050 (st), 1001 (st), 932 (st), 886 (md),811 (st), 776 (w), 742 (md), 651 (md), 542 (sh), 505 (md, br). NMR: δH (600 MHz,298 K, D2O) [ppm] = 9.18 (s), 9.15 (s), 9.01 (s), 9.05 (s), 8.94 (s), (3 H, Car2H); 7.87 (d,J3H,F= 11.4 Hz), 7.66 (d, J3H,F= 11.4 Hz), 7.61 (d, J3H,F= 10.8 Hz), 7.51 (d, J3H,F= 10.2Hz), (3 H, Car5H); 4.68−4.58 (m, 6 H, CH2CH3); 3.43−3.28 (m, 12 H, Cpip2,6H2, and 3H, Cpip3H); 3.10−2.91 (m, 12 H, Cpip4H2); 1.48−1.34 (m, 9 H, CH2CH3); 1.10−1.07 (m, 9H, CH3). δC (125 MHz, 298 K, d6-DMSO) [ppm] = 175.7 (s), 173.1 (s), 172.8 (s), (Car4);165.7 (s), 165.3 (s), 165.0 (s), 164.1 (s), (COOH); 154.8 (d, J1C,F= 207.1 Hz), 154.6 (d,J1C,F= 205.8 Hz), 154.6 (d, J1C,F= 207.8 Hz), (Car6); 153.4 (s), 153.1 (s), 152.8 (s), 151.4(s), (Car2); 146.2 (d, J1C,F= 207.0 Hz); 146.1 (d, J1C,F= 207.4 Hz); 145.5 (d, J1C,F= 212.2Hz), (Car8); 133.7 (s), 133.6 (s), 133.5 (s), 133.4 (s), (Car7); 127.4 (d, J2C,F= 5.5 Hz), 127.2(d, J2C,F= 5.0 Hz), (Car8′); 120.8 (d, J3C,F= 6.3 Hz), 120.4 (d, J3C,F= 6.0 Hz), (Car4′); 112.3(s), 112.2 (s), 112.1 (s), (Car5); 107.2 (s), 107.1 (s), 106.6 (s), (Car3); 56.5 (s), 56.4 (s),56.3 (s), (Cpip2); 54.2 (s), 54.1 (s), 53.9 (s), 53.8 (s), (CH2CH3); 51.0 (s), 50.9 (s), 49.5(s), (Cpip6); 49.5 (br s, Cpip3); 44.9 (br, s, Cpip5); 17.9 (s), 17.7 (br s) (CH2CH3); 16.3(s), 16.2 (s), 16.1 (s), (CH3). δF (282 MHz, 298 K, d6-DMSO) [ppm] = (-118.8)−(-118.9)(m), -119.4 (d, J4F,F= 10.7 Hz), (3 F, Car6F ); -129.4 (d, J4F,F= 10.4 Hz), (-129.9)−(-130.4)(m), (3 F, Car8F ). MS (ES+, CH3OH): m/z (%) = 1143 (30) [ML3 + Na+], 770 (100)[ML2]+. HR-ESI-MS: m/z for C51H54F6 69GaN9O9 + Na+ calcd. (found): 1142.3102(1142.3093).1063.2.7.6 Tris(nalidixo)gallium(III), [Ga(nxa)3]Method (b) gave an off-white solid (37 mg, 0.046 mmol, 46%). Mp: ≥190◦C, decompositionto beige-brown solid. IR (neat): ν˜ [cm−1] = 3443 (md, br, water), 3025 (md, br), 2985(w), 1676 (sh), 1608 (st, br), 1557 (st), 1518 (sh), 1490 (st), 1440 (st), 1380 (w), 1365(md), 1348 (md), 1320 (md), 1293 (st), 1256 (st), 1227 (sh), 1168 (w), 1130 (st), 1109(w), 1089 (md), 991 (w, br), 944 (w), 898 (md), 843 (w), 807 (st), 776 (st), 702 (w), 663(md), 639 (md), 561 (sh), 543 (md), 506 (st), 452 (st). NMR: δH (600 MHz, 298 K,d6-DMSO) [ppm] = 9.45 (s), 9.35 (s), 9.27 (s), 9.19 (s), (3 H, Car2H); 8.62 (d, J3H,H= 8.2Hz), 8.43−8.31 (m) (3 H, Car5H); 7.61 (d, J3H,H= 8.2 Hz), 7.56 (d, J3H,H= 8.4 Hz), 7.51(d, J3H,H= 7.8 Hz), (3 H, Car6H); 4.76−4.57 (m, 6 H, CH2CH3); 2.69 (d, J4H,H= 30.0 Hz,9 H, Car7CH3); 1.45 (t, J3H,H= 6.9 Hz), 1.42 (t, J3H,H= 7.1 Hz), 1.31 (t, J3H,H= 7.1 Hz),(9 H, CH2CH3). δC (125 MHz, 298 K, d6-DMSO) [ppm] = 178.1 (s), 175.6 (s), 175.4 (s),175.2 (s), (Car4); 165.6 (s), 165.3 (s), 165.1 (s), 165.0 (s), (Car8′) 164.9 (s), 164.8 (s), 164.7(s), (COOH) 151.7 (s), 151.4 (s), 151.3 (s), 151.1 (s), (Car2); 149.7 (s), 148.4 (s), 147.7 (s),147.4 (s), (Car7); 135.9 (s), 135.7 (s), 135.4 (s), 135.3 (s), (Car5); 123.0 (s), 122.9 (s), 122.6(s), (Car6); 118.4 (s), 118.2 (s), 118.1 (s), 118.0 (s), (Car4′); 113.7 (s), 113.6 (s), 113.3 (s),(Car3); 47.1 (s), 47.0 (s), 46.9 (s), 46.8 (s), (CH2CH3); 25.1 (br s) (CH3); 15.2 (s), 15.1(s), 15.0 (s), (CH2CH3). MS (ES+, CH3OH): m/z (%) = 1296 (10) [M2L5]+, 785 (100)[ML3 + Na+], 531 (20) [ML2]+. HR-ESI-MS: m/z for C36H33 69GaN6O9 + Na+ calcd.(found): 785.1463 (785.1479). EA: Anal. Calcd. (found) [%] for C36H33GaN6O9·1.5 H2O:C, 54.70 (54.59); H, 4.59 (4.42); N, 10.63 (10.24). Tris(norfloxacino)gallium(III), [Ga(nofx)3]Method (a) gave a pale yellow solid (38 mg, 0.036 mmol, 36%). Mp: ≥200◦C, decompo-sition to orange-brown solid. IR (neat): ν˜ [cm−1] = 3391 (md, br, water), 2839 (w, br),1071615 (st, br), 1551 (w), 1519 (md), 1471 (st, br), 1377 (md), 1320 (md), 1255 (st, br), 1188(md), 1129 (md), 1089 (w), 1037 (md), 971 (w), 930 (st), 889 (sh), 812 (st), 788 (md),769 (md), 747 (st), 697 (w), 628 (st), 561 (md), 513 (st). NMR: δH (600 MHz, 298 K,d6-DMSO) [ppm] = 9.19 (s), 9.09 (s), 9.05 (s), 8.97 (s), (3 H, Car2H); 7.94 (d, J3H,F= 13.2Hz), 7.75 (d, J3H,F= 13.2 Hz), 7.57 (d, J3H,F= 13.8 Hz), 7.53 (d, J3H,F= 13.2 Hz), (3 H,Car5H); 7.26 (d, J4H,F= 6.6 Hz), 7.21 (d, J4H,F= 7.8 Hz), 7.19 (d, J4H,F= 7.2 Hz), 7.13 (d,J4H,F= 7.2 Hz), (3 H, Car8H); 4.70−4.48 (m, 6 H, CH2CH3); 3.44−3.31 (m, 12 H overlaidwith water, Cpip2,6H2); 3.09−3.02 (m, 12 H, Cpip3,5H2); 1.46−1.40 (m), 1.30 (t, J3H,H=7.2 Hz), (9 H, CH2CH3). δC (125 MHz, 298 K, d6-DMSO) [ppm] = 173.5 (s), 172.9 (s),172.8 (s), (Car4); 165.7 (s), 165.6 (s), 165.4 (s), (COOH); 152.9 (d, J1C,F= 208.4 Hz), 152.7(d, J1C,F= 206.9 Hz), (Car6); 150.5 (s), 150.0 (s), 149.9 (s), (Car2); 145.5−145.0 (m, Car7);136.9, 136.8, 136.6 (Car8′); 119.3 (d, J3C,F= 7.8 Hz), 119.2 (d, J3C,F= 8.2 Hz), 119.0 (d,J3C,F= 7.4 Hz), (Car4′); 112.0−110.4 (m, Car5 and Car3); 105.5 (s), 105.3 (s), 105.0 (s),(Car8); 49.4−48.5 (m, Cpip2,6 and CH2CH3); 44.4 (s), 44.2 (s), 44.1 (s), 44.0 (s), (Cpip3,5);14.62 (s), 14.60 (s), 14.5 (s), 14.4 (s), (CH2CH3). δF (282 MHz, 298 K, d6-DMSO) [ppm]= -120.9 (s), -121.0 (s), -121.1 (s), -121.2 (s), (3 F, Car6F ). MS (ES+, CH3OH): m/z (%)= 1047 (10) [ML3 + Na+], 706 (100) [ML2]+. HR-ESI-MS: m/z for C48H51F3 69GaN9O9+ Na+ calcd. (found): 1046.2915 (1046.2925). Tris(oxalino)gallium(III), [Ga(oxa)3]Method (b) gave an off-white solid (69 mg, 0.082 mmol, 82%). Mp: ≥240◦C, decompositionto beige-brown solid. IR (neat): ν˜ [cm−1] = 3402 (md, br), 3060 (w, br), 2979 (w, br),2918 (w,br), 1637 (st), 1599 (st), 1567 (md), 1539 (st), 1463 (st, br), 1412 (sh), 1387 (md),1329 (md), 1258 (st, br), 1193 (md), 1158 (w), 1126 (w), 1087 (w), 1029 (st, br), 933 (md),904 (md), 846 (w), 812 (st), 777 (st, br), 656 (md), 618 (md), 563 (w). NMR: δH (600MHz, 298 K, d6-DMSO) [ppm] = 8.90 (s, 3 H, Car2H); 7.64 (s, 3 H, Car8H); 7.63 (s, 3 H,108Car5H); 6.30 (s, 6 H, OCH2O); 4.53 (q, J3H,H= 7.1 Hz, 6 H, CH2CH3); 1.37 (t, J3H,H= 7.1Hz, 9 H, CH2CH3). δC (125 MHz, 298 K, d6-DMSO) [ppm] = 176.0 (s, Car4); 166.3 (s,COOH); 153.7 (s, Car7); 147.1 (s, Car6); 147.0 (s, Car2); 136.9 (s, Car8′); 121.3 (s, Car4′);107.4 (s, Car3); 103.3 (s, OCH2O); 101.9 (s, Car5); 97.3 (s, Car8); 49.6 (s, CH2CH3); 14.6(s, CH2CH3). MS (ES+, CH3OH): m/z (%) = 873 (100) [ML3 + Na+], 589 (80) [ML2]+.HR-ESI-MS: m/z for C39H30 69GaN3O15 + Na+ calcd. (found): 872.0830 (872.0822).EA: Anal. Calcd. (found) [%] for C39H30GaN3O15·3.5 H2O: C, 51.28 (51.37); H, 4.08(3.97); N, 4.60 (4.70). Tris(pipemido)gallium(III), [Ga(pia)3]Method (b) gave an off-white solid (64 mg, 0.066 mmol, 66%). Mp: ≥190◦C, decompositionto beige-brown solid. IR (neat): ν˜ [cm−1] = 3373 (st, br, water), 3029 (w), 2980 (w), 1616(st), 1578 (st), 1536 (md), 1510 (md), 1471 (st), 1430 (st), 1378 (sh), 1358 (st, br), 1310(md), 1280 (md), 1249 (st, br), 1159 (w), 1148 (w), 1128 (st), 1092 (md), 1079 (md), 1045(md), 1024 (st), 976 (md), 940 (md), 915 (st), 903 (md) 868 (md), 832 (st), 802 (md),744 (st), 715 (md), 703 (md), 657 (w), 609 (w), 541 (md), 489 (md), 463 (st). NMR: δH(600 MHz, 298 K, d6-DMSO) [ppm] = 9.17 (s, 3 H, Car5H); 8.94 (s, 3 H, Car2H); 4.37 (q,J3H,H= 7.1 Hz, 6 H, CH2CH3); 3.85 (d, JH,H= 40.1 Hz, 12 H, Cpip2,6H2); 2.78 (d, JH,H=16.2 Hz, 12 H, Cpip3,5H2); 1.35 (t, J3H,H= 6.9 Hz, 9 H, CH2CH3). δC (125 MHz, 298 K,d6-DMSO) [ppm] = 177.1 (s, Car4); 165.4 (s, COOH); 160.6 (s, Car7); 160.1 (s, Car5);155.1 (s, Car8′); 150.6 (s, Car2); 109.7 (s, Car4′); 108.3 (s, Car3); 45.9 (s, CH2CH3); 45.6(s), 45.3 (s), (Cpip2,3,5,6); 14.4 (s, CH2CH3). MS (ES+, CH3OH): m/z (%) = 999 (100)[ML3 + Na+], 673 (80) [ML2]+. HR-ESI-MS: m/z for C42H48 69GaN15O9 + Na+ calcd.(found): 998.2913 (998.2939); for C28H32 69GaN10O+6 calcd. (found): 673.1762 (673.1776).EA: Anal. Calcd. (found) [%] for C42H48GaN15O9·12.5 H2O: C, 41.97 (41.70); H, 6.12(5.85); N, 17.48 (17.26).1093.2.7.10 Tris(ciprofloxacino)iron(III), [Fe(cipro)3]Method (c) gave a red-brown solid (51 mg, 0.049 mmol, 49%). Mp: ≥220◦C, decompositionto black-brown solid. IR (neat): ν˜ [cm−1] = 3411 (w, br, water), 2846 (w, br), 1610(st), 1543 (w), 1513 (sh), 1450 (st, br), 1371 (md, br), 1285 (sh), 1252 (st), 1182 (md),1129 (md), 1108 (sh), 1026 (st), 949 (st), 890 (md), 809 (md), 788 (md), 761 (sh), 738(st), 702 (md), 627 (st), 577 (md), 556 (w), 538 (w), 506 (st). MS (ES+, CH3OH):m/z (%) = 1763 (≤10) [M2L5]+, 716 (100) [ML2]+. HR-ESI-MS: m/z for C51H51F356FeN9O9 + Na+ calcd. (found): 1069.3009 (1069.3007). EA: Anal. Calcd. (found) [%]for C51H51F3FeN9O9·2.5 H2O: C, 56.10 (56.04); H, 5.17 (4.80); N, 11.55 (11.27). Tris(enoxacino)iron(III), [Fe(enox)3]Method (b) gave a red-orange solid (59 mg, 0.058 mmol, 58%) Mp: ≥200◦C, decompositionto black brown solid. IR (neat): ν˜ [cm−1] = 3403 (md, br, water), 3039 (w, br), 2976 (w,br), 1626 (st), 1565 (md), 1516 (md), 1441 (st, br), 1368 (md), 1347 (md), 1323 (md), 1275(st), 1251 (st, br), 1184 (md), 1153 (w), 1119 (md), 1093 (md), 1039 (md, br), 971 (md),943 (md, br), 907 (sh), 812 (st), 788 (md), 763 (md), 744 (md), 677 (w), 650 (md), 625 (st),562 (md), 518 (st). MS (ES+, CH3NO2): m/z (%) = 694 (100) [ML2]+ HR-ESI-MS:m/z for C45H48F3 56FeN12O9 + Na+ calcd. (found): 1036.2866 (1036.2866). EA: Anal.Calcd. (found) [%] for C45H48F3FeN12O9·3 H2O: C, 50.62 (50.48); H, 5.10 (4.82); N, 15.74(16.07). Tris(fleroxacino)iron(III), [Fe(flex)3]Method (b) gave a red-brown solid (91 mg, 0.078 mmol, 78%). Mp: ≥230◦C, decomposi-tion to brown solid. IR (neat): ν˜ [cm−1] = 3054 (w), 2930 (md), 2848 (md), 2792 (md),1625 (st), 1548 (w), 1526 (md), 1471 (sh), 1447 (st, br), 1413 (md), 1389 (w), 1377 (md),1358 (md), 1325 (md), 1295 (st), 1245 (md), 1236 (sh), 1203 (md), 1160 (md), 1144 (st),1101121 (md), 1097 (w), 1075 (w), 1045 (md), 1026 (st), 1002 (st), 963 (md), 949 (md, br),896 (md), 861 (md), 803 (st), 779 (md), 752 (md), 736 (md), 714 (md), 648 (st), 599 (md),568 (st), 536 (w, br), 517 (w), 491 (w), 474 (w). MS (ES+, CH3OH): m/z (%) = 1184(20) [ML3 + Na+], 793 (100) [ML2]+. HR-ESI-MS: m/z for C51H51F9 56FeN9O9 + Na+calcd. (found): 1183.2913 (1183.2938); for C34H34F6 56FeN6O+6 : 792.1793 (792.1785). EA:Anal. Calcd. (found) [%] for C51H51F9FeN9O9: C, 52.77 (52.78); H, 4.43 (4.53); N, 10.86(10.49). Tris(levofloxacino)iron(III), [Fe(levox)3]Method (a) gave a red-brown solid (102 mg, 0.090 mmol, 90%). Mp: ≥190◦C, decompo-sition to black-brown solid. IR (neat): ν˜ [cm−1] = 3427 (water), 3041 (w, br), 2935 (w),2846 (w), 2797 (w), 1615 (st), 1518 (st), 1443 (st), 1382 (md, br), 1330 (st, br), 1291 (w),1256 (st, br), 1150 (sh), 1129 (md), 1094 (md), 1048 (st), 1003 (md), 978 (st), 896 (md),864 (w), 843 (w), 826 (sh), 810 (st), 759 (md), 742 (md), 693 (md), 637 (w), 553 (md, br),508 (st), 443 (st, br). MS (ES+, CH3OH): m/z (%) = 1160 (40) [ML3 + Na+], 777 (100)[ML2]+. HR-ESI-MS: m/z for C54H57F3 56FeN9O12 + Na+ calcd. (found): 1159.3326(1159.3328); for C36H38F2 56FeN6O8 calcd. (found): 776.2069 (776.2065). Tris(lomefloxacino)iron(III), [Fe(lomx)3]Method (b) gave a red-brown solid (80 mg, 0.072 mmol, 72%). Mp: ≥210◦C, decompo-sition to black-brown solid. IR (neat): ν˜ [cm−1] = 3423 (w, br), 2979 (w, br), 2844 (w,br), 2725 (w, br), 2467 (w, br), 1616 (st), 1552 (w), 1520 (st), 1446 (st), 1358 (md, sh),1321 (st, br), 1275 (w), 1244 (st), 1122 (md), 1089 (md), 1049 (st), 1002 (st), 932 (st),880 (md), 810 (st), 760 (md, br), 738 (md), 654 (md), 541 (sh), 502 (md), 449 (md). MS(ES+, CH3OH): m/z (%) = 1130 (10) [ML3 + Na+], 756 (100) [ML2]+. HR-ESI-MS:m/z for C51H54F6 56FeN9O9 + Na+ calcd. (found): 1129.3196 (1129.3193).1113.2.7.15 Tris(nalidixo)iron(III), [Fe(nxa)3]Method (b) gave a red-brown solid (66 mg, 0.089 mmol, 89%). Mp: ≥180◦C, decomposi-tion to black-brown solid. IR (neat): ν˜ [cm−1] = 3436 (water), 3023 (md, br), 2983 (w),1668 (sh), 1606 (st, br), 1556 (st), 1515 (sh), 1486 (st), 1440 (st), 1366 (md), 1347 (md),1313 (md), 1289 (st), 1255 (st), 1226 (sh), 1168 (w), 1129 (st), 1089 (md), 990 (w, br),942 (w), 895 (md), 842 (w), 806 (st), 769 (st), 702 (w), 663 (md), 639 (md), 559 (w), 545(md), 503 (st), 442 (st). MS (ES+, CH3OH): m/z (%) = 1268 (≤10) [M2L5]+, 772 (100)[ML3 + Na+], 518 (40) [ML2]+. HR-ESI-MS: m/z for C36H33 56FeN6O9 + Na+ calcd.(found): 772.1556 (772.1562). EA: Anal. Calcd. (found) [%] for C36H33FeN6O9·1 H2O:C, 56.33 (56.62); 4.60 (4.80); N, 10.95 (11.05). Tris(norfloxacino)iron(III), [Fe(nofx)3]Method (b) gave a red-brown solid (68 mg, 0.067 mmol, 67%). Mp: ≥190◦C, decompo-sition to black-brown solid. IR (neat): ν˜ [cm−1] = 3371 (md, br, water), 2838 (w, br),1610 (st, br), 1547 (w), 1516 (md), 1453 (st, br), 1380 (st), 1324 (md), 1282 (sh), 1252(st, br), 1186 (st), 1124 (md, br), 1024 (md), 923 (st, br), 891 (sh), 875 (w), 810 (md),786 (md), 760 (md), 738 (st), 694 (w), 624 (md), 558 (md), 512 (md), 443 (md, br). MS(ES+, CH3OH): m/z (%) = 692 (100) [ML2]+, 1033 (20) [ML3 + Na+]. HR-ESI-MS:m/z for C48H51F3 56FeN9O9 + Na+ calcd. (found): 1033.3009 (1033.3002). EA: Anal.Calcd. (found) [%] for C48H51F3FeN9O9·6 H2O: C, 51.52 (51.42); H, 5.67 (5.72); N, 11.27(10.89). Tris(oxalino)iron(III), [Fe(oxa)3]Method (b) gave a golden-brown solid solid (70 mg, 0.084 mmol, 84%). Mp: ≥240◦C,decmposition to brown solid. IR (neat): ν˜ [cm−1] = 3030 (w, br), 2979 (w, br), 2913 (w,br), 1633 (st), 1604 (st), 1563 (md), 1536 (md), 1493 (sh), 1460 (st, br), 1410 (sh), 1385112(md), 1319 (md), 1288 (md), 1259 (st, br), 1198 (st), 1155 (w), 1130 (w), 1079 (w), 1041(st, br), 978 (w), 934 (md), 899 (w), 886 (w), 875 (w), 849 (w), 813 (st), 775 (st), 759 (sh),714 (w), 656 (md), 621 (md), 570 (md), 524 (st), 504 (sh), 455 (st). MS (ES+, CH3OH):m/z (%) = 860 (≤10) [ML3 + Na+], 576 (100) [ML2]+. HR-ESI-MS: m/z for C39H3056FeN3O15 + Na+ calcd. (found): 859.0924 (859.0914). EA: Anal. Calcd. (found) [%] forC39H30FeN3O15·1.5 H2O: C, 54.24 (54.24); H, 3.85 (3.72); N, 4.87 (4.74). Tris(pipemido)iron(III), [Fe(pia)3]Method (b) gave a brown solid (78 mg, 0.081 mmol, 81%). Mp: ≥200◦C, decomposition tobrown solid. IR (neat): ν˜ [cm−1] = 3406 (md, br), 3028 (w), 2979 (w), 1615 (st), 1578 (st)1534 (md), 1510 (md), 1471 (st), 1429 (st), 1377 (sh), 1357 (st, br), 1310 (md), 1280 (md),1248 (st, br), 1158 (w), 1147 (w), 1128 (st), 1092 (md), 1078 (md), 1045 (md), 1023 (st),975 (md), 940 (md), 914 (st), 903 (md), 867 (w), 832 (st), 802 (md), 784 (md), 744 (st), 715(md), 704 (sh), 656 (w), 609 (w), 540 (md), 489 (md), 454 (st). MS (ES+, CH3OH): m/z(%) = 986 (20) [ML3 + Na+], 715 (100) [ML2·3H2O]+, 661 [ML2]+ (10). HR-ESI-MS:m/z for C42H48 56FeN15O9 (M + Na+) calcd. (found): 985.3007 (985.3017). EA: Anal.Calcd. (found) [%] for C42H48FeN15O9·4 H2O: C, 48.75 (48.94); H, 5.45 (6.23); N, 20.30(20.33).3.2.8 Synthesis & Characterization of Tris(maltolato)metal(III)Complexes3.2.8.1 Tris(maltolato)gallium(III), [Ga(ma)3]The synthesis followed the literature procedure.413 Scale: 3-hydroxy-2-methyl-4H-pyran-4-one (2.60 g, 20.55 mmol), gallium(III) nitrate nonahydrate (2.86 g, 6.85 mmol), water(40 mL). Yield: off-white solid (1.957 g, 4.40 mmol, 64%). IR (neat): ν˜ [cm−1] = 3027(w), 1611 (sh), 1568 (st), 1514 (st), 1456 (st), 1295 (sh), 1277 (st), 1241 (md), 1192 (st),1131087 (w), 1043 (md), 921 (md), 850 (st), 830 (st), 745 (st), 720 (st), 664 (md), 617 (md),557 (sh), 528 (sh), 511 (w), 493 (md). MS (ES+, CH3OH): m/z (%) = 765 (100) [M2L5]+,445 (10) [ML3H+], 319 (40) [ML2]+. HR-ESI-MS: m/z for C18H15 69GaO9 (M + Na+)calcd. (found): 466.9870 (466.9866). EA: Anal. Calcd. (found) [%] for C18H15GaO9·1H2O: C, 46.69 (46.77); H, 3.70 (3.34). Tris(maltolato)iron(III), [Fe(ma)3]The synthesis followed the literature procedure.414 Scale: 3-hydroxy-2-methyl-4H-pyran-4-one (3.792 g, 3.0 mmol), iron(III) nonahydrate (4.037 g, 1.0 mmol), water and ethanol(100 mL each). Yield: ruby-red solid (0.354 g, 0.82 mmol, 82%). IR (neat): ν˜ [cm−1] =3024 (w), 1605 (sh), 1564 (st), 1505 (st), 1455 (st), 1293 (sh), 1272 (st), 1238 (md), 1190(st), 1084 (w), 1038 (md), 920 (md), 849 (st), 829 (st), 745 (st), 719 (st), 664 (md), 607(md), 536 (st), 470 (st). MS (ES+, CH3OH): m/z (%) = 737 (20) [M2L5]+, 454 (70)[ML3Na+], 306 (100) [ML2]+. HR-ESI-MS: m/z for C18H15 56FeO9 (M + Na+) calcd.(found): 453.9963 (453.9963). EA: Anal. Calcd. (found) [%] for C18H15FeO9·2 H2O: C,46.28 (46.19); H, 4.10 (3.86).3.3 Results & Discussion3.3.1 SynthesisThe tris(quinolono)metal(III) complexes were derived through the reaction of quinoloneand gallium(III) or iron(III) nitrate in a 3:1 ratio (Figure 3.2). Similar to the reactionpathways reported to give a diversity of metal-quinolone complexes,398 401 415 the synthesiscan be carried out in three principle steps. First, the respective quinolone was dissolvedin water or methanol. Most quinolones exist in a zwitterionic state of neutral charge inthe neutral pH-range (Chapter 2), which makes their dissolution difficult. Upon proto-114XZR6R7R8 R1OOOH3 +  M(NO3)3  9 H2OpH 5-7r.t. or ∆H2O/CH3OH[ML3]Figure 3.2: Synthetic pathway to tris(quinolono)metal(III) complexes, M= Ga3+,Fe3+.nation (HCl) or deprotonation (NaHCO3, NaOH, KOH) at room temperature or in heat(60−100◦C), depending on the chemical nature of the quinolone, it dissolves more easily;Hcipro·HCl and Hlomx·HCl were readily dissolved in the solvent of choice at room tem-perature. The dissolved quinolone was then added dropwise to the respective metal(III)nitrate, which was either solid or previously had been dissolved in water or methanol aswell. Throughout the addition, the pH was carefully monitored and kept below pH 3 toavoid the formation of insoluble metal(III) hydroxides (Ga(OH)3(s), Fe(OH)3(s)). Finally,the pH of the reaction solution was adjusted to pH 5−7 and stirred rigorously. The desiredcomplex of the general form [M(quino)3] with M= Ga3+, Fe3+ was obtained as a coloredsolid, which in most cases readily precipitated from the aqueous reaction mixture at roomtemperature and was isolated on a fine glass frit; in some reaction mixtures with a highmethanol content, the precipitate started to form after reduction of the initial reactionvolume by at least 50% (in vacuo or evaporation in air). The precipitate was washedthoroughly with water and methanol and dried in vacuo. In general, the yields were be-tween 40−90% but varied with the employed method and the respective quinolone ligand.Analyses of the obtained compounds via HR-ESI mass spectrometry were consistent withthe formation of tris(quinolono)metal(III) formulations. Elemental analyses for the threeelements C, H, and N supported these findings successfully for the majority of compounds.Besides the nine novel tris(quinolono)gallium(III) complexes, [Ga(quino)3], and their115nine iron(III) analogs, [Fe(quino)3], tris(maltolato)gallium(III) and tris(maltolato)iron(III)were synthesized following the published procedures.413 414 In addition, several syntheticattempts were made to prepare mixed ligand complexes of the general form [GaLaxLby](x+ y = 3 and x, y 6= 0) with La,b being potentially bidentate ligands such as anions of thequinolones, maltol, acetylacetonate,405 or other heterochelates.404 Today, a combination ofdifferent bactericidal drugs is often administered to the patient in severe cases of infections,and such treatment showed potential to overcome bacterial resistance;396 likewise, the com-plexation of different quinolones to Ga3+ might lead to a deadly combination cocktail forbacteria, which could be conveniently administered in a single dose. Moreover, introducingmaltol, a widely used food-additive, into such a mixed ligand complex, the pharmaco-logical properties could be altered further. Unfortunately, such mixed ligand complexeswere difficult to realize because the ligand exchange rates of Ga3+ as well as Fe3+ are fast(kH2O= 103−102 s−1),416 and only a cocktail of respective metal(III)-complexes with vari-ous ratios of 2:1 ligands coordinated to the central metal ion could be detected in the massspectra. Compared to Ga3+ and Fe3+, the ligand exchange rate of Cr3+ is slow (kH2O=10−6 s−1);416 however, not even in the attempted chromium(III) mixed-ligand complexesone single species formed dominantly (data not shown).3.3.2 CharacterizationTris(quinolono)gallium(III) complexes are off-white to yellow in color, whiletris(quinolono)-iron(III) complexes come in various shades of red-brown; the inten-sity in color is directly related to the electronegativity present at the core aromatic ringsystem, as highly fluorinated quinolone-ligands, such as lomefloxacin or fleroxacin, givegallium(III) and iron(III) complexes of more intense color. All synthesized complexes arenonvolatile and stable. When stored in a desiccator at ambient temperature in darkness(cupboard), the complexes did not degrade over the course of four years; however, melting116point measurements and TGA revealed that they start to decompose when heated to250◦C or higher. The solubility of these complexes is low and highly pH dependent. Uponheating and sonicating, they dissolve in DMSO, which was therefore the chosen solventfor structure analyses by NMR spectroscopy at 600 MHz, while MS spectra were recordedat much lower compound concentration in methanol or acetonitrile, if necessary underpH adjustments. Of course, the lack of solubility as well affected the growth of crystalssuitable for X-ray analysis (Section 3.3.3).The recorded mass spectra were diagnostic of the complex formulations being 3:1quino:metal. In all cases, loss of one ligand from a [ML3] unit was observed as the [ML2]+fragment with a mass-to-charge ratio of 100%. With the exception of [Fe(enox)3], alliron(III) and gallium(III) complexes gave [NaML3]+ or [KML3]+ as the parent peak. Inaddition, [M2L5]+ peaks of low intensity (≤10%) were observed, which occur throughthe cationization of the molecular unit by recombination of one [ML2]+ fragment withone [ML3] unit and have been previously reported as a characteristic MS feature oftris(maltolato)gallium(III).413 Due to limitations of the mass spectrometer, these recombi-nation peaks could be only observed for complexes of nalidixic acid, a low-mass quinoloneligand (232.24 g/mol). Figure 3.3 shows the ES+ spectra of the novel [Ga(nxa)3] complexand its iron(III) derivative.Elemental analyses have been performed on all eighteen tris(quinolono)metal(III) com-plexes. Similar to literature reports of other tris(quinolono)iron(III) complexes,398 417 theycontain several water molecules in their elemental formulae. This holds especially true forthe gallium(III) derivatives, of which only [Ga(nxa)3], [Ga(oxa)3] and [Ga(pia)3] match thecalculated results for C,H, and N with 1.5, 3.5, and 12.5 molecules of water included inthe elemental formula, respectively. Theses three quinolone ligands all belong to the firstgeneration of quinolone antimicrobial agents (Chapter 2) and their molecular structures do117Figure 3.3: Low-resolution MS spectra (ES+) of [GaL3] and [FeL3] (L = nxa).not contain any bulky substituents, fluorine atoms, or stereocenters. Numerous attemptsfrom many different compound batches have been made for the tris(quinolono)gallium(III)complexes to pass EA, but over and over only the same three representatives matchedtheir calculated result. For the tris(quinolono)iron(III) complexes, the situation is differ-ent. With the exception of [Fe(lomx)3] and [Fe(levox)3], the iron(III) analogs match thecalculated values for C, H, and N. Although various different batches of [Fe(lomx)3] and[Fe(levox)3] compounds were submitted for elemental analysis, the analytical result for theamount of carbon in in elemental formula did never match, which is probably related to thefact that the ligands, levofloxacin as well as lomefloxacin, contain a stereocenter. Alreadyin Chapter 2, these two quinolones showed the largest differences between the analyticaland found values of C, H, and N.Next to mass spectrometry and elemental analysis confirming the total mass of thecompound and therewith the metal-to-ligand ratio, infrared spectroscopy was employed tostudy the metal-ligand coordination. Although the IR spectra of the quinolones are quitecomplex, due to the varied C−H and C−N vibrations as well as various functional sub-118stituents on the condensed aromatic ring system, the stretching frequencies of the carboxy-late on Car3 and the carbonyl on Car4-position are strong and lend themselves as suitableIR-handles to characterize the chelation of the metal (Figure 3.4). The IR spectrum of thefree ligand (HL) shows the intense stretching vibration of the dimeric carboxylate group(νCOOH) between 1722−1707 cm−1 with the exception of quinolones in their ionic form, inwhich the carboxylate group is deprotonated, because ionic carboxylates show no carbonylstretching.317 318 When the quinolone ligand is fully coordinated to the metal, this strongband disappears completely, indicating that no free HL is present in the sample and thatthe quinolone anion binds to the metal through one carboxylato-O. Furthermore, the twodistinct bands in the range of 1637−1606 cm−1 and 1400−1300 cm−1 are assigned as νCO2asymmetric and symmetric stretching vibrations, being characteristic for the complexationof a quinolone ligand to a metal.328 In the vast amount of IR data on metal-quinolone com-plexes described in the literature, there is almost complete agreement about the assignmentof the asymmetric stretching frequency (νasym(CO2)), however, the frequency of the sym-metric stretch (νsym(CO2)) has been assigned quite ambiguously. From our own experience,we know that the difficulty in identifying νsym(CO2) with certainty lies with the numberof bands appearing in the IR spectrum between 1400 cm−1 and 1300 cm−1, in additionstretches in this region are often quite broad. In the case of the tris(levofloxacino)metal(III)and tris(lomefloxacino)metal(III) complexes, only a single broad band with several weakshoulder stretches is observed in this specific 100 cm−1 range. After carefully consideringthe IR stretches of the nine free ligands in this region (Chapter 2) as well as comparing theIR spectra of the eighteen synthesized [M(quino)3] complexes (M= Ga3+, Fe3+), we havedecided to include an overview of the single carboxylato stretching frequencies in our discus-sion despite the previously mentioned controversy and described challenges. The difference∆ = νasym(CO2)−νsym(CO2) varies within values from minimum 230 cm−1 for [Fe(nofx)3]119to maximum 265 cm−1 for [Ga(lomx)3] with an average value of ∆Ga,average= 248 cm−1for all nine tris(quinolono)gallium(III) complexes and ∆Fe,average= 246 cm−1 for all ninetris(quinolono)iron(III) complexes (Table 3.1). The similarities between the ∆ values forcomplexes with the same ligand, which is further reflected in the conformity of the ∆averagevalues, demonstrate the uniformity of the studied [Ga(quino)3] and [Fe(quino)3] originatingfrom the chemical similarity of the central metals. According to the ∆ values, the centralmetal is chelated in a monodentate mode,318 418 chemically bound through the deproto-nated carboxylate group on Car3 and coordinated through the carbonyl group on Car4,which is the most common coordination mode in quinolone chelates.327 The cooperationof the carbonyl group in the chelation of the metal is further supported by a shift to lowerwave numbers for the Car4=O band upon complexation, a critical observation that has beenpreviously made for tris(norfloxacino)aluminium(III).419 Furthermore, the coordination ofthe metal(III) ion via the carbonyl-O and the carboxylato-O is reflected in the ν(M−O)stretching vibrations dominant between 600−500 cm−1.420Figure 3.4 shows the recorded IR spectra of [Ga(nofx)3] and [Fe(nofx)3] together withTable 3.1: Carboxylate stretching frequencies [cm−1] of the synthesizedtris(quinolino) gallium(III) and iron(III) complexes. IR spectra were recordedneat in solid state.complex νasym(CO2) νsym(CO2) ∆(a) complex νasym(CO2) νsym(CO2) ∆(a)Ga(cipro)3 1620 1373 247 Fe(cipro)3 1610 1371 239Ga(enox)3 1625 1369 256 Fe(enox)3 1626 1368 258Ga(flex)3 1622 1376 246 Fe(flex)3 1625 1377 248Ga(levox)3 1616 1385 231 Fe(levox)3 1615 1382 233Ga(lomx)3 1619 1354 265 Fe(lomx)3 1616 1358 258Ga(nxa)3 1608 1365 243 Fe(nxa)3 1606 1366 240Ga(nofx)3 1615 1377 238 Fe(nofx)3 1610 1380 230Ga(oxa)3 1637 1387 250 Fe(oxa)3 1633 1385 248Ga(pia)3 1616 1358 258 Fe(pia)3 1615 1357 258(a)∆ = νasym(CO2) − νsym(CO2).120Figure 3.4: IR spectra of norfloxacin as free ligand (HL, black), [Ga(nofx)3] (green),and [Fe(nofx)3] (red).that of the free ligand norfloxacin. Characteristic is the intense band at 1722 cm−1 of thearomatic carbonyl functionality in the spectrum of the free ligand, which disappears uponcoordination to the metal. Other major differences in the spectrum of Hnofx compared toits metal(III) complexes are the single broad stretch in the range from 1400−1300 cm−1 forthe metal complexes, in addition to the shift of the Car4=O band from 1614 cm−1 (Hnofx)to 1615 cm−1 ([Ga(nofx)3]) and 1610 cm−1 ([Fe(nofx)3]), in addition to new bands occurring121in the low finger-print region (800−400 cm−1) that are related to the ν(M−O) stretchingvibrations. The broad absorption around 3400 cm−1 in both tris(norfloxacino)metal(III)complexes once more shows the presence of water in the compound, as discussed in con-nection with the EA and TGA results (Section 3.3.4).To record nuclear magnetic resonance spectra of the 1H and 13C nuclei, as well asof the 19F nucleus where applicable, the isolated complexes were dissolved in d6-DMSO,heated with a heat gun, and sonicated until the test solutions turned clear (∼4 mg/mL). Inthe tris(quinolono)iron(III) complexes, the central Fe3+ ion retains a paramagnetic high-spin state upon complexation, therefore, NMR signals for all three nuclei are substantiallybroadened and impossible to assign with certainty. While MS and EA data proved theformation of ML3 complexes, and IR data showed that the central metal(III) ion was coor-dinated from three quinolone bidentate anions, the NMR data offered a first impression intothe stereochemistry and solution chemistry of the diamagnetic tris(quinolono)gallium(III)complexes. Four stereoisomers are possible for the coordination of three bidentate ligandsin an octahedral fashion: ∆-fac, Λ-fac, ∆-mer, and Λ-mer. In the 1H and 13C spec-tra recorded at 298 K, the stereoisomers gave a multitude of signals.i Figure 3.5 showsthe recorded 19F spectra for tris(enoxacino)gallium(III). Enoxacin contains one fluorineatom on Car6 that gives one single peak at -127.3 ppm in the 19F spectrum (Section2.2.4.2), which upon complexation of the metal multiplies to four signals for the respective[Ga(enox3] complex with an integration of 1:1:0.8:0.2 adding up to a total of 3 19F. Inter-estingly, [Ga(oxa)3] as well as [Ga(pia)3] do not show multiple signals in the 1H and 13CNMR measurements; either these complexes possess a unique stereochemistry, because ofthe condensed cyclic ether or the pyrido[2,3]pyrimidine aromatic core, respectively, or theysimply cannot stand the rather rough conditions necessary to dissolve them in the NMRiPlease see Appendix B for a temperature dependent 1H NMR study of tris(vosaroxacino)gallium(III),where the interchange happened rapidly on the NMR time scale at 393 K.1220.2430.2430.7640.7641.011.0111ppm-127.5 -127.5-127.0-127.0-126.5-126.5-126.0-126.0-349.467-127.412-126.768-126.586-126.498-126.374Figure 3.5: 19F NMR spectrum of [Ga(enox)3].solvent, however, both complexes were successfully analyzed by HR-ESI mass spectrometryand elemental analysis.3.3.3 Solid State StructureTo further characterize the coordination complexes, attempts were made to grow a singlecrystal of a tris(quinolono)metal(III) complex suitable for X-ray diffraction. Numerouscrystallization experiments were performed with all nine quinolones and their respectivegallium(III) and iron(III) complexes trying various organic solvents (acetone, acetonitrile,chloroform, DMSO, dimethyl formamide, ethanol, ethyl acetate, methanol) and aqueoussolvents as well as mixtures thereof following many different crystallization procedures (con-centration gradients, layering of solvents, reactive crystallization, diffusion in solution andin air with different glass ware set-ups, open to air or tightly/partly capped) in a multitudeof environmental settings (window sill at all four seasons, fume hood, shelf, dark cupboard,123fridge, freezer); unfortunately, these only yielded crystals of tris(quinolono)metal(III) com-plexes that were not suitable for X-ray diffraction, such as very fragile needles, or crystalsof the free quinolone ligand that were suitable for X-ray diffraction, but had already beenreported. Although reactive crystallization attempts were not successful with Ga3+, Fe3+,or Al3+, crystals were grown by layering lanthanum(III) nitrate (44 mg, 0.1 mml) withwater (1 mL) and a solution of cipro− (deprotonated with NaOH) in methanol/DMSO/di-ethyl ether, giving the solid-state structure depicted in Figure 3.6. Unfortunately, thecounter cation Na+ could not be detected with certainty in this solid state structure, there-fore, further reactive crystallization attempts were made employing bulky cations, such astetrabutyl- or tetraethyl-ammonium; however, these attempts only resulted in structuresfurther complicated through the coordination of multiple molecules of methanol solventand nitrate (data not shown). In the [La(cipro)4]− complex (Figure 3.6), the lanthanumion at the center is coordinated by four ciprofloxacin ligands via the carboxylate-O on Car3and the carbonyl-O on Car4. Bond lengths between the carboxylate-O and La(1) vary from2.26(2) A˚ (O(8)) to 2.480(14) A˚ (O(2)) and are therewith shorter than the bond contactsbetween the carbonyl-O and La3+, which lie in the range from 2.538(12) A˚ (O(12)) to2.564 (12) A˚ (O(6)).As crystals of a tris(quinolono)gallium(III) could not be obtained, DFT calculationswere run on one of the four possible stereoisomers of [Ga(cipro)3] to propose a possible3D structure model of the complex. The result of the DFT calculation is graphically pre-sented in Figure 3.7. Earlier, Psomas had demonstrated that the average energies of allfour stereoisomers of tris(ciprofloxacino)iron(III) were almost equal (∼150 kcal mol−1) andthat the difference in minimum energy between the fac and mer isomers of [Fe(cipro)3]was 0.7 kcal mol−1, essentially negligible;401 therefore, it was deemed sufficient to calculateonly one of the possible four stereoisomers of the gallium(III) analog. The three carbonyl-O124Figure 3.6: Solid state structure of [La(cipro)4]−. Hydrogen atoms omit-ted for clarity. The La3+ ion at the center is coordinated by fourciprofloxacin ligands through one O-atom of the carboxylate group onCar3 and the carbonyl-O on Car4 with the following respective bondcontacts [A˚]: O(2)−La(1) 2.480(14), O(3)−La(1) 2.470(10), O(5)−La(1)2.449(14), O(6)−La(1) 2.564(12), O(8)−La(1) 2.26(2), O(9)−La(1) 2.540(13),O(11)−La(1) 2.471(11), O(12)−La(1) 2.538(12).atoms and the three carboxylate-O atoms are arranged around the central Ga3+ atom inan octahedral fashion. The calculated bond angles reveal a slight distortion that consti-tutes the difference in bond lengths between the Ga3+ atom the O-atoms of the carbonyland carboxylate groups. The calculated bond lengths of the Ga−O(carbonyls) and theGa−O(carboxylates) in the equatorial plane are 2.167 A˚ and 1.679 A˚, respectively. A sim-ilar trend can be observed along the vertical axis of the distorted octahedron, where the125Figure 3.7: Result of the DFT calculation of (fac, ∆)-[Ga(cipro)3]. Graphi-cal presentation with Avogadro as stick-model (hydrogen atoms omittedfor clarity). Calculated bond lengths [A˚] and bond angles [◦]: Ga−O(1)1.671, Ga−O(2) 2.167, Ga−O(3) 2.167, Ga−O(4) 1.679, Ga−O(5) 1.679,Ga−O(6) 2.165, O(1)−Ga−O(2) 87.4, O(1)−Ga−O(4) 107.9, O(2)−Ga−O(5)85.3, O(5)−Ga−O(4) 107.3, O(4)−Ga−O(3) 87.2, O(3)−Ga−O(2) 74.4,O(1)−Ga−O(6) 155.3.Ga−O(carbonyl) bond was 2.165 A˚ longer than the Ga−O(carboxylate) bond of 1.67 A˚,proving a very slight compression along the vertical axis. This difference in bond lengthsbetween the O-atoms of the carbonyl and the carboxylate groups corresponds well withthe experimental result of [La(cipro)4]− (Figure 3.6).3.3.4 Thermal StabilityTGA/DTA measurements were carried out for tris(ciprofloxacino)gallium(III), its iron(III)analog, and free ciprofloxacin ligand to investigate and compare the thermal stability ofthese three compounds (Figure 3.8). Around 250◦C, ciprofloxacin starts to show weightloss that finishes around 450◦C, resulting in a total weight reduction of about 76%. Two en-dothermic peaks appear during the single weight loss step, the first one at 255◦C matches126Figure 3.8: TGA/DTA results of [Ga(cipro)3] (green), [Fe(cipro)3] (red), andciprofloxacin (Hcipro) (gray). Measurements were taken in the tempera-ture range of 25−900◦C with a heating rate of 5◦C/min under N2-flow(19.8 mL/min).well with the previously determined melting point of ciprofloxacin (252−255◦C, Section2.2.4.1), while the second one at 415◦C can be interpreted as the boiling point, abovewhich only the bare aromatic core remains as a graphite fragment [C9H3N] correspond-ing to 37.8% (calc.) of the total molecular mass. The observed weight loss curves oftris(ciprofloxacino)gallium(III) and its iron(III) complex analog proceed similarly up toabout 600◦C, when the iron(III) complex begins to decline at a faster rate than the cor-responding gallium(III) complex. In the initial heating step from up to 100◦C, both com-plexes shed water molecules, [Ga(cipro)3] one and [Fe(cipro)3] four. This again furthersupports the observation that quinolone-metal complexes have numerous water moleculesin their lattice,421 422 423 as was previously discussed for FT-IR spectra and EA results.Corresponding well with the behaviour of ciprofloxacin, the respective metal complexesare thermally stable from 25◦C to approximately 260◦C. At 263◦C, [Ga(cipro)3] starts de-127composing in one single step resulting in a total weight loss of 62.2% (expected weight loss64.5%), corresponding to the remaining fragment [GaC9H3N]. At 258◦C, [Fe(cipro)3] startsdecomposing in one single step, however, here the total weight loss is almost complete to4.22%, which corresponds well with the formation of FeO at temperatures above 575◦C.Both tris(ciprofloxacino)metal(III) complexes possess similar thermal stability, which ul-timately arises from the thermal stability of the free ligand itself; possible degradationproducts (metal oxides) depend on the accessible chemistry of the metal ion.3.3.5 Stability in SolutionSolution thermodynamic investigations of ciprofloxacin in aqueous sodium chloride solution(0.16 M) have provided pKa values of 6.40 (1) and 8.65 (1), obtained by potentiometrictitration. These are in good accordance with values reported for similar systems (Table3.2); as discussed in Chapter 2 (see Figure 2.3), in the neutral pH-range the zwitterionicspecies is dominant.Although potentiometric titration curves, starting at pH 2 and ending at pH 12, wereobtained in triplicate for the Ga3+:Hcipro as well as the Fe3+:Hcipro system in ratiosof 1:1, 1:2, 1:3, and 1.4:1, the fitting of the obtained data with the Hyperquad softwareremains a challenge. All three potentiometric curves obtained under the same conditionsmatch beautifully. Throughout the titration process, the solution remains clear, but afterTable 3.2: Comparison of the determined pKa values of ciprofloxacin with literaturevalues.conditions (a) (b)424 (c)424 (d)315pKa1 6.40 (1) 6.18 (1) 6.17 (2) 6.17 (1)pKa2 8.65 (1) 8.5 (1) >8.2 8.54 (1)(a) [Hcipro]= 8·10−4M, 298 K, INaCl= 0.16 M. (b) [Hcipro]= 10−3M, 298 K, INaCl=0.15 M. (c) [Hcipro]=10−3M, 310 K, INaCl=0.15 M. (d) [Hcipro]=8·10−4M, 298 K,IKCl= 0.20 M.128reaching the end point (pH 12) and standing overnight, insoluble hydroxides precipitatedfrom the test solution of the Fe3+:Hcipro system (Fe(OH)3, red). Equilibria from pH 5 topH 8 were slow and the maximum wait time of 1 h was reached in some cases. In this pHrange, the obtained titration curve is not as smooth as in the lower and higher pH ranges,which could indicate some small amounts of insoluble hydroxides forming, however, notenough to be detected by human eye. Numerous attempts have been made to slow downthe titration. The concentration of ciprofloxacin ligand in the system was reduced from8·10−4 M to 7·10−4 M and 5·10−4 M. Furthermore, the concentration of sodium hydroxidebase was reduced from 0.1597 M to 0.1006 M, while the volume of the titration vessel wasincreased from 5 mL to 10 mL to ensure that with the smallest increment of base addition(2 µL) less hydroxide ions entered the test solution. The tardiness of the equilibria betweenHquino and metal ions is a known fact; a previous study of the Fe3+:Hcipro system reportedwaiting times of seven days until the pH of the test solution was stable (within ±0.01 pHunit).425 Apart from the slow equilibria, the representation of zwitterionic species in thefitting model is a complex issue, in addition to the myriad of possible hydroxide species,which need to be included in the solution as well. Previously reported calculated logβvalues for the Fe3+:Hcipro system did not lead to a mathematical fit or a chemically sensiblesolution of the potentiometric data either.426 My colleague Dr. Jacqueline F. Cawthray,who is highly experienced in the field of potentiometric titrations, who has trained me inthis technique, and who has attempted to fit the data herself, contacted Dr. Peter Gansabout this, a renowned analytical chemist and the software engineer of the Hyperquadprogram; however, even insights from these discussions did not guide us to a good fit. Dr.Cawthray repeated some of the titrations of the Ga3+:Hcipro system herself and got thesame results than I had previously obtained, therefore, potential operator errors can beexcluded with confidence as well.129Figure 3.9: Initial spectrophotometric study of the Ga3+:Hcipro system. [Hcipro]=8.0·10−4 M, [Ga3+]= 6.7·10−4 M, INaCl= 0.16 M, ambient temperature.Some of my preliminary spectrophotometric studies proved that UV-Vis titrationsmight be a suitable avenue to determine the stability constants (Figure 3.9), which has ledto success in case of the iron(III)-vosaroxin system discussed in Chapter 5. Spectropho-tometric measurements were made on solutions of ciprofloxacin (8·10−4 M) with varyingamounts of Ga3+ to give ratios of Ga3+:Hcipro= 1:1, 1:2, 1:3. For each ratio, differenttest solutions were prepared with a pH value from pH 2 to pH 12. Similar to the UV-Visspectra of the free ligand (Hcipro) discussed in Section 2.3.1, both absorbance maximashow a strong pH dependence; isobestic points exist at 268 nm, 317 nm, 345 nm.3.3.6 Antimicrobial Susceptibility TestingThe antimicrobial activities of the synthesized tris(quinolono)metal(III) complexes wereevaluated according to the single-disk method against pathogens associated with nosoco-130mial diseases. E. faecalis and methicillin-susceptible S. aureus (both Gram-positive), aswell as E. coli, K. pneumonia, and P. aeruginosa (all Gram-negative).330 The metal com-plexes were tested at a concentration of 0.1 mM against the respective free quinolone ligandat 0.1 mM as well as at 0.3 mM corresponding to the ratio of ligand to metal (3:1) in thecomplexes.The susceptibility of pathogens against various quinolone-metal and various gal-lium(III) complexes has been previously evaluated using the single-disk method in a vari-ous growth media, such as Bactec,381 Bacto,372 Iso-Sensitest,427 428 429 Lauria304, Mueller-Hinton,430 and modified personal recipes.431 In their antimicrobial susceptibility single-disktest procedure, the CLSI recommends Mueller-Hinton medium,432 but Iso-Sensitest is asynthetic and chemically reliable medium, which has been widely used in Europe. As eluci-dated further in Chapter 2, Iso-Sensitest is our medium of choice for these tests; however, tobe able to exclude potential effects of the test medium on our metal complexes, e.g., cross-metallation, we examined the antimicrobial potency of all metal complexes in Iso-Sensitestmedia (Table 3.3) and additionally chose to test ciprofloxacin, levofloxacin, nalidixic acid,and their respective metal compounds in Mueller-Hinton media as well (Figure 3.10). Thesethree quinolones were selected, as they represent three different generations of quinoloneantimicrobial agents (Chapter 2). Their respective gallium(III) and iron(III) complexesperformed comparably well in both media giving similar inhibition zone sizes against thepathogens, with K. pneumonia being an exception to the rule. This microorganism had al-ready developed a resistance against nalidixic acid and was only susceptible to ciprofloxacinand levoxfloxacin and their respective gallium(III) and iron(III) complexes. All four com-plexes seemed to perform slightly better against K. pneumonia in Mueller-Hinton thanin Iso-Sensitest medium with recorded inhibition zone sizes of >25 mm over 20 mm, re-spectively (Figure 3.10). Because the pathogen grew on both agar medium plates in an131off-white, pale yellow color, the evaluation was not influenced by any differences in bac-terium growth color but, measuring the inhibition zone sizes of K. pneumonia growth waschallenging due to the frayed edges of the inhibition zones and several different inhibitionrings of weakening intensity around each disk, and both effects have potentially led toa larger divergence of the measured inhibition zone size values in this organism. ATCCrecommends to grow the chosen strain of K. pneumonia (ATCC-13883) on a nutrient agarcomposition of 3.0 g beef extract, 5.0 g peptone, and 15.0 g agar, which is neither the exactrecipe of Mueller-Hinton nor Iso-Sensitest medium (Appendix A);433 therefore, any furtherdifferences in growth related to the chosen biological growth medium, which further couldhave affected the size of the inhibition zone, can be ruled out.To further study the stability of tris(ciprofloxacino)gallium(III) andtris(ciprofloxacino)-iron(III), solutions of both compounds (0.1 mM) in 50% Iso-Sensitestbroth were monitored via UV-Vis spectroscopy over the course of 24 hours (experimentalset-up as described in Chapter 2, data not shown). No changes in the UV-Vis spectrawere detected, which would have indicated potential chemical alterations or degradation,and the complexes are therewith considered stable in Iso-Sensitest medium. In summary,we are confident that the tris(quinolono)metal(III) complexes stay intact over the courseof the antimicrobial single-disk test and are not affected by ingredients of the Iso-Sensitestmedium.The results of the antimicrobial susceptibility study of the tris(quinolono)gallium(III)and -iron(III) complexes (0.1 mM) in direct comparison to the respective free quinoloneligand at single (0.1 mM) and triple concentration (0.3 mM) are summarized as averagevalues of recorded inhibition zone sizes from three independent plates in Table 3.3. Threeconclusions can be drawn from these results. First, bacteria that have developed a re-sistance against quinolone antimicrobial agents do not become susceptible to these again132(a)(b)Figure 3.10: Comparison of inhibition zone sizes measured for six differenttris(quinolono)metal(III) complexes in Iso-Sensitest medium (a) and Mueller-Hinton medium (b).133upon complexation of the quinolone to Ga3+ or Fe3+. Second, the recorded inhibition zonesizes of the tris(quinolono)metal complexes (0.1 mM) are in the same range than those ofthe respective free quinolone ligands at 0.3 mM. Third, there are no differences in recordedinhibition zone sizes between the gallium(III) and the iron(III) complexes of the samequinolone ligand. Hence, complexation of Ga3+ to a quinolone does not have a synergisticeffect, or even only a combinational effect, that would lead to increased antimicrobial po-tency compared to the quinolone on its own. Moreover, the observed increase in measuredinhibition zone sizes of the tris(quinolono)metal(III) complexes is solely related to the factthat there are three quinolone molecules coordinated as ligands to the metal, whether Ga3+or Fe3+ is at the center of the complex does not fortify or weaken the antimicrobial effect.According to the ”Trojan Horse Theory”, the tris(quinolono)gallium(III) complexes shouldhave had superior antimicrobial powers compared to the respective tris(quinolono)iron(III)complexes, however, such an effect was not observed (Table 3.3). Control disks loaded withgallium(III) nitrate (0.1 mM) were placed on each test plate. These Ga3+ controls nevershowed any inhibition, which might be due to the formation of gallium(III) hydroxides(Section 3.1); therefore, we included tris(maltolato)gallium(III) and its iron(III) analog inthis study. Because the maltol ligand does not possess any antimicrobial properties, anyantimicrobial effect of these complexes could be solely associated with the respective metalion; however, no growth inhibition was observed (Table 3.3).Although the single-disk test has been successfully used to evaluate the performanceof metal-quinolone complexes many times before,304 372 381 427 428 429 430 431 additional MICstudies were performed to rule out any doubts regarding the suitability of the evaluationmethod. In a growth assay, varying ratios of ciprofloxacin and gallium(III) (Ga3+:Hcipro =0:1, 1:1, 1:2, 1:3, 1:4) were tested against P. aeruginosa in cation-adjusted Mueller-Hintonbroth, however, these test results did not show a combinational effect for the interaction134of quinolone and Ga3+ cation (MIC) either.434Although no combinational effect for the coordination of one Ga3+ cation with threefree quinolone ligands was observed, neither did the complexation to Ga3+ or Fe3+ leadto a reduction in quinolone antimicrobial activity, as it had been previously reported forsome synthesized Mg2+-quinolone complexes.304135Table 3.3: Results of antimicrobial susceptibility study in Iso-Sensitest medium of tris(quinolono)- andtris(maltolato)gallium(III) and -iron(III) complexes in comparison to free ligands at two concentrations.bacteria Hcipro(0.3 mM)Hcipro(0.1 mM)[Ga(cipro)3](0.1 mM)[Fe(cipro)3](0.1 mM)Henox(0.3 mM)Henox(0.1 mM)[Ga(enox)3](0.1 mM)[Fe(enox)3](0.1 mM)E. faecalis 13 (0) 9 (0) 12 (1) 12 (1) 8 (1) 0 (0) 7 (0) 7 (0)S. aureus 16 (1) 12 (1) 16 (2) 16 (1) 12 (0) 0 (0) 11 (1) 10 (1)E. coli 24 (1) 22 (1) 24 (1) 24 (1) 20 (0) 16 (1) 19 (1) 19 (1)K. pneumonia 20 (0) 18 (0) 20 (0) 20 (0) 19 (1) 14 (1) 18 (1) 18 (0)P. aeruginosa 25 (1) 18 (1) 24 (1) 24 (1) 15 (3) 7 (1) 12 (2) 12 (2)bacteria Hflex(0.3 mM)Hflex(0.1 mM)[Ga(flex)3](0.1 mM)[Fe(flex)3](0.1 mM)Hlevox(0.3 mM)Hlevox(0.1 mM)[Ga(levox)3](0.1 mM)[Fe(levox)3](0.1 mM)E. faecalis 9 (1) 0 (0) 14 (1) 8 (1) 13 (1) 8 (1) 13 (1) 13 (1)S. aureus 18 (1) 10 (1) 19 (2) 16 (1) 19 (1) 14 (1) 18 (1) 18 (1)E. coli 23 (1) 19 (1) 24 (1) 23 (1) 22 (1) 20 (0) 22 (0) 23 (0)K. pneumonia 24 (1) 19 (1) 25 (1) 22 (1) 20 (1) 18 (0) 20 (1) 20 (0)P. aeruginosa 12 (1) 7 (0) 18 (2) 10 (1) 18 (1) 9 (0) 15 (2) 16 (2)136bacteria Hlomx(0.3 mM)Hlomx(0.1 mM)[Ga(lomx)3](0.1 mM)[Fe(lomx)3](0.1 mM)Hnxa(0.3 mM)Hnxa(0.1 mM)[Ga(nxa)3](0.1 mM)[Fe(nxa)3](0.1 mM)E. faecalis 8 (0) 0 (0) 7 (0) 7 (0) 0 (0) 0 (0) 0 (0) 0 (0)S. aureus 15 (1) 7 (1) 12 (1) 12 (1) 0 (0) 0 (0) 0 (0) 0 (0)E. coli 20 (1) 17 (2) 20 (1) 19 (1) 0 (0) 0 (0) 0 (0) 0 (0)K. pneumonia 20 (1) 16 (1) 18 (1) 19 (1) 0 (0) 0 (0) 0 (0) 0 (0)P. aeruginosa 14 (2) 0 10 (1) 10 (1) 0 (0) 0 (0) 0 (0) 0 (0)bacteria Hnofx(0.3 mM)Hnofx(0.1 mM)[Ga(nofx)3](0.1 mM)[Fe(nofx)3](0.1 mM)Hoxa(0.3 mM)Hoxa(0.1 mM)[Ga(oxa)3](0.1 mM)[Fe(oxa)3](0.1 mM)E. faecalis 9 (1) 0 (0) 9 (1) 9 (1) 0 (0) 0 (0) 0 (0) 0 (0)S. aureus 15 (1) 7 (1) 12 (1) 13 (1) 0 (0) 0 (0) 0 (0) 0 (0)E. coli 21 (1) 19 (1) 21 (1) 21 (0) 19 (1) 14 (1) 19 (1) 15 (1)K. pneumonia 16 (2) 15 (1) 15 (0) 15 (1) 15 (1) 11 (1) 15 (1) 10 (1)P. aeruginosa 16 (1) 6 (0) 12 (1) 14 (1) 0 (0) 0 (0) 0 (0) 0 (0)137bacteria Hpia(0.3 mM)Hpia(0.1 mM)[Ga(pia)3](0.1 mM)[Fe(pia)3](0.1 mM)Hma(0.3 mM)Hma(0.1 mM)[Ga(ma)3](0.1 mM)[Fe(ma)3](0.1 mM)E. faecalis 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)S. aureus 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)E. coli 11 (1) 0 (0) 11 (1) 11 (1) 0 (0) 0 (0) 0 (0) 0 (0)K. pneumonia 6 (0) 0 (0) 6 (0) 6 (0) 0 (0) 0 (0) 0 (0) 0 (0)P. aeruginosa 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)Reported inhibition zones [mm] are averaged values from three plates (standard deviation). Disk diameter0.6 mm. Loading volume 20 µL. Disks loaded with solutions of methanol and 2% DMSO in methanolserved as controls, all of these showed no inhibition (0 mm).1383.4 ConclusionIn this chapter, the potential of a combinational or even synergistic effect between Ga3+and quinolone antimicrobial agents was investigated. According to the ”Trojan HorseTheory”, the Ga3+ ion does kill bacteria by tricking them into uptake in that they getthe Fe3+ ion necessary for their own growth. Nine tris(quinolono)gallium(III) coordinationcomplexes and their respective iron(III) analogs were synthesized and characterized (Figure3.1). A comparison of their stability in solution and in heat did reveal slight differencesbetween the gallium(III) and the iron(III) complexes, however, their antimicrobial efficacywas similar against five of the most common nosocomial pathogens, following the single-disktest procedure. Further competing antimicrobial studies with tris(maltolato)gallium(III)and its iron(III) analog were not able to confirm the claimed antimicrobial efficacy of Ga3+either, and neither did MIC studies in solution; however, the quinolones did not lose theirantimicrobial activity upon complexation to Ga3+, as it had been reported earlier for Mg2+-quinolone complexes.304. The antimicrobial effect of the eighteen tris(quinolono)metal(III)[ML3, M= Ga3+, Fe3+] complexes correspond well with the effect of the free quinoloneligand (HL) at triple concentrations. In conclusion, the ”Trojan Horse Theory” and the”hype” around the antimicrobial properties of Ga3+ do not hold any truth according tothe results of this study.139Chapter 4Syntheses, Characterization, andEvaluation of the Antimicrobial Potentialof Copper(II) Coordination Complexeswith Quinolone and Xylenyl-LinkedQuinolone LigandsIn this chapter, the focus of the antimicrobial project will be broadened. On the metalside, the antimicrobial properties of copper(II) will be explored. In addition, modifiedquinolone ligands will be introduced, in which the secondary amine of the piperazinyl ringsof two quinolone drug molecules will be joined with a α,α’-xylenyl-linker. The antimicrobialsusceptibility of these α,α’-xylenyl-linked quinolone copper(II) sandwich complexes will becompared to the activity of the bis(quinolono)copper(II) complexes.1404.1 Mixing Things Up: Another Metal, a Modified LigandThe growing resistance of bacteria to commonly used antimicrobial drugs is a global healthconcern that threatens the effective prevention and therapy of bacterial and fungal infec-tions, possibly leading to a post-antibiotic era (see as well Section 3.1).337 339 For the past 50years, quinolone antimicrobial agents have been widely used in the clinic for the treatmentof bacterial infections, but bacterial resistance against this drug class is rising.435 436 437For example, pipemidic acid (Figure 4.1), one of the first generation quinolone agents, isnot effective against many bacterial strains anymore, as data presented in Table 2.5 and3.3 show. The development of novel quinolone agents is ongoing with the aim to modifythe aromatic core structure of the quinolones in a way that makes bacteria susceptibleagain to new members of this drug class and that increases the ability of the agents to killbacteria cells.299 438 439 440 441 442 Previous research in the groups of Drlica and Kerns has fo-cused on crosslinking two molecules of ciprofloxacin, norfloxacin, or pipemidic acid via the1,4-piperazinyl-group in Car7-position with crosslinking moieties, such as trans-butenyl,2,6-pyridinyl, meta-xylenyl, and para-xylenyl (Figure 4.1).443 444 445 446 447 Depending onthe linker, these crosslinked dimers displayed a higher antimicrobial activity than the sin-gle quinolones.446 447 In the case of virtually inactive pipemidic acid, the crosslinkage led toincreased antimicrobial susceptibility against the tested strains of Staphylococcus aureus,indicating a unique, non-equivalent interaction of this crosslinked dimer with the bacterialtarget site (topoisomerase/gyrase, DNA).447These findings suggest that novel mechanisms of action are needed to regain a lead overbacteria. Because many fundamental processes in the pathogen, as well as the host, are reg-ulated by metal-requiring cofactors,448 the targeting of metal transport pathways opens updiverse mechanisms of action. In the area of tropical parasitic diseases, metallodrugs haveshown potential to overcome drug resistance by novel modes of action.236 264 Moreover, as141NN NOOOHNHNHpiaNOOOHNHNHciproFNN NOOOHNNH2piaXpiaNNNOOOHNNNOOOHNNH2ciproXciproNOOOHNNF FFigure 4.1: Pipemidic acid (Hpia, 1st generation), ciprofloxacin (Hcipro, 2nd gen-eration), and their respective α,α’-xylenyl-linked dimers: H2piaXpia andH2ciproXcipro.discussed in Chapter 3, iron metabolism in vivo has been the target of antibacterial strate-gies for many years367 and can be seen as the starting point for the growing discussion ofnutrient metal homeostasis at the pathogen-host interface.449 The ancient Egyptians valuedthe antimicrobial properties of copper, using copper formulations in personal hygiene andfor the treatment of wounds.450 Over the past years, there has been a trend in chemistry touse copper for a variety of antimicrobial applications: antifouling coatings for marine envi-ronments,451 452 antimicrobial alloys for use in healthcare settings,453 454 surface treatmentsfor joint replacement implants,455 antimicrobial nanomaterials,456, and medical uses,457 458have been reported. Into the latter category fall a multitude of copper-quinolone com-plexes,459 327 such as [Cu(Hcipro)2]Cl2·6H2O460 and [Cu(pia)2(H2O)]·2H2O.461 Recently,142Wolschendorf et al. observed that guinea pigs increased their levels of copper(II) in thelung tissue upon infection with Mycobacterium tuberculosis in an autoimmune response,462similar to the lowering of iron(III) levels at the site of infection widely observed in mam-mals.358 Although the mechanism of copper-dependent bacterial killing remains unclear,the Cu(II)/Cu(I) redox couple, which could catalyze the formation of toxic radical oxygenspecies as well as the disruption of iron-sulfur cluster proteins through the formation ofCu(I)-thiolate bonds, appears to play a key role in the bactericidal action.463 In dependenceon Greek methology, copper has been named the ”Achilles Heel” of bacteria.462 464In an attempt to combine the antimicrobial properties of copper(II) with the in-creased antimicrobial potency of α,α’-xylenyl-linked quinolones, this chapter describesthe synthesis and characterization of copper(II)-sandwich complexes with α,α’-xylenyl-linked quinolone dimers of ciprofloxacin and pipemidic acid. The antimicrobial efficacy ofthese complexes was evaluated in direct comparison to bis(ciprofloxacino)copper(II) andbis(pipemido)copper(II), as well as copper(II) chloride and bis(maltolato)copper(II), fol-lowing the single-disk test procedure in Iso-Sensitest medium.4.2 Materials & Methods4.2.1 ChemicalsChemicals and materials were purchased from commercial suppliers (Alfa-Aesar, BD BBL-Difco, Cambridge Isotope Laboratories, Inc., Fischer-Scientific, Sigma-Aldrich) and usedwithout further purification. Water was purified through a Elga Purelab Pure WaterSystem to 18 MΩ·cm.1434.2.2 InstrumentationReversed-phase high performance liquid chromatography (HPLC) was conducted on Phe-nomenex Synergi Hydro-RP 80 A˚ columns (250 mm x 4.6 mm analytical or 250 mm x21.2 mm semipreparative) on a Waters WE 600 HPLC system consisting of a Waters600 controller running Empower Pro software (version, 2002), a Waters 2478dual wavelength absorbance detector, and a Waters delta 600 pump. Melting points weretaken on a Stanford Research Systems DigiMelt SRS melting point apparatus and areuncorrected. UV-Vis spectra were recorded on a Hewlett Packard 8453 instrument op-erated by ChemStation Software (version B.04.01[61], Agilent Technologies, 2001-2010).IR spectra were recorded in solid state on a PerkinElmer Frontier FT-IR spectrometer(4000−650 cm−1) running PerkinElmer Spectrum Software (version 10.03.02, 2011); char-acteristic bands were interpreted using the abbreviations: st, md, w, sh. At the UBC MassSpectrometry Centre, low-resolution mass spectra were recorded on a Water ZQ spectrom-eter equipped with an electrospray and chemical ionization source, while high-resolutionmass spectra were obtained on a Waters Micromass LCT (electrospray-ionization), andelemental analyses (C, H, N) were performed on a Carlo Erba Elemental Analyzer EA1108.4.2.3 Antimicrobial Susceptibility StudiesThe following bacteria strains were used in the antimicrobial susceptibility single-disk test:Gram-positive E. faecalis (ATCC-51575) and S. aureus (MSSA-476, ATTC-BAA-1721);Gram negative E. coli (ATCC-25922), K. pneumonia (ATCC-13883), and P. aeruginosa(ATCC-27853). Filter disks (1/4 inch, approx. 0.6 mm, diameter) were purchased fromSchleicher & Schu¨ll, Germany. The study was performed in UBC’s Biological ServicesLaboratory (biological safety level II), according to the procedure presented in Appendix A.1444.2.4 Synthesis & Characterization4.2.4.1 α,α’-Xylenyl-Linked Ciprofloxacin Dimer, H2ciproXciproThe synthesis was based on the reported synthetic scheme by Kerns and co-workers.447Ciprofloxacin (0.663 g, 2.0 mmol), α,α’-chloro-xylene (0.175 g, 1.0 mmol) and sodiumbicarbonate (0.401 g, 4.8 mmol) were transferred into a 100 mL round-bottom flask, sus-pended in dimethylformamide (60 mL) and heated to 80◦C for 24 hours. An off-whitesolid was isolated from the reaction mixture after cooling on a fine glass frit, and washedwith water and methanol (yield: 0.686 g, 89%). HPLC (linear gradient of acetonitrile in0.1% TFA/water) afforded separation of the desired product as a single peak at 220 nmand 254 nm. Removal of solvents and drying in vacuo yielded an off-white solid. Mp:≥230◦C, decomposition to pale brown solid. IR (neat): ν˜ [cm−1] = 3047 (w), 2937 (w),2810 (md), 2772 (w), 1721 (st), 1627 (st), 1607 (sh), 1545 (md), 1495 (st), 1450 (st), 1437(sh), 1384 (md), 1334 (md), 1293 (w), 1255 (st), 1203 (sh), 1110 (md), 1047 (w), 1030 (w),1005 (st), 945 (st), 892 (st), 831 (st), 807 (st), 781 (w), 747 (md), 709 (md), 667 (md).NMR: δH (300 MHz, 298 K, d6-DMSO) [ppm] = 8.64 (s, 2 H, Car2H); 7.88 (d, J3H,F=13.5 Hz, 2 H, Car5H); 7.55 (d, J4H,F= 7.5 Hz, 2 H, Car8H); 7.33 (s, 4 H, Car,linkerH);3.81−3.76 (m, 2 H, CpropH); 3.57 (s, 4 H, ClinkerH2); 3.33 (br s, 8 H overlaid with water,Cpip2,6H2); 2.89−2.60 (m, 8 H, Cpip3,5H2); 1.32−1.15 (m, 8 H, CpropH2). δF (282 MHz, 298K, d6-DMSO) [ppm] = -121.8 (s, Car6F ). MS (ES+, CH3OH): m/z (%) = 765 (100) [M+ H+], 787 (40). HR-ESI-MS: m/z for C42H42F2N6O6 + H+ calcd. (found): 765.3212(765.3210). α,α’-Xylenyl-Linked Pipemidic Acid Dimer, H2piaXpiaThe synthesis followed the procedure described in Section using pipemidic acid(0.608 g, 2.0 mmol), α,α’-chloro-xylene (0.177 g, 1.0 mmol), sodium bicarbonate (0.349 g,1454.0 mmol), and 60 mL dimethylformamide. Yield: off-white solid (0.570 g, 80%). The de-sired product eluted from HPLC column (linear gradient of acetonitrile in 0.1% TFA/wa-ter) as a single peak at 220 nm and 254 nm. Mp: ≥245◦C, decomposition to pale brownsolid. IR (neat): ν˜ [cm−1] = 3047 (w), 2982 (w), 2934 (w), 2826 (w), 1725 (st), 1637(st), 1569 (w), 1549 (md), 1518 (md), 1483 (st), 1452 (md), 1376 (md), 1359 (md), 1305(w), 1264 (md), 1226 (w), 1200 (w), 1127 (md), 1114 (md), 1092 (w), 1053 (w), 1007 (st),971 (md), 854 (w), 830 (w), 812 (st), 793 (w), 763 (w), 737 (w), 718 (md), 698 (w), 667(w). NMR: δH (300 MHz, 298 K, d6-DMSO) [ppm] = 9.02 (s, 2 H, Car5H); 8.48 (s, 2H, Car2H); 7.34 (s, 4 H, Car,linkerH); 4.23−4.20 (m, 4 H, CH2CH3); 3.90−3.86 (m, 4 H,ClinkerH2); 3.81−3.78 (m, 8 H, Cpip2,6H2); 2.77−2.71 (m, 8 H, Cpip3,5H2); 1.32−1.23 (m, 6H, CH2CH3). MS (ES+, CH3OH): m/z (%) = 710 (100) [M + H+], 731 (80) [M + Na+].HR-ESI-MS: m/z for C36H40N10O6 + H+ calcd. (found): 709.3211 (709.3214). [Cu2(ciproXcipro)2]In a warm solution (50◦C) of methanol (15 mL), H2ciproXcipro (0.077 g, 0.1 mmol) wasdissolved with two drops of triethylamine under sonication and rigorous stirring. Thewarm solution was filtered and added dropwise to the blue solution of copper(II) chloride(0.017 g, 0.1 mmol) in 2 mL methanol, which resulted in a color change via green toturquoise. The reaction mixture was warmed up to 50◦C once more and stirred over nightwith the flask open to air. After removal of half of the remaining solvent in vacuo, theflask was left standing for further evaporation, which resulted in a light turquoise solidthat was collected on a glass frit (size F), washed with cold water and diethyl ether, anddried in vacuo (0.042 g, 25%, first crop). Mp: ≥234◦C, black-brown decomposition melt.IR (neat): ν˜ [cm−1] = 3042 (w), 2944 (w), 2821 (md), 2791 (w), 1625 (st), 1592 (sh),1545 (md), 1520 (sh), 1471 (st), 1451 (md), 1397 (w), 1372 (md), 1352 (w), 1285 (st), 1256(st) 1224 (sh), 1180 (md), 1144 (w), 1089 (w), 1030 (w), 1001 (st), 948 (st), 890 (st), 836146(md), 811 (st), 788 (w), 765 (md), 743 (md), 708 (md). MS (ES+): m/z (%) = 1715[Cu3(LXL)2]++, 1654 [Cu2(LXL)2 + H+], 826 [Cu(LXL)]+. [Cu2(piaXpia)2]H2piaXpia (0.071 g, 0.1 mmol) was dissolved in a warm solution (50◦C) of methanol:water(70:30, 20 mL) with two drops of triethylamine and the help of sonication and rigorousstirring. The warm solution was filtered and added dropwise to a solution of copper(II)chloride (0.017 g, 0.1 mmol) in 2 mL methanol. After an immediate color change togreen, the solution turned blue-turquoise. Re-heating the solution again to 50◦C, stirringit overnight in open air, and leaving the flask standing for further evaporation, resulted ina turquoise solid, which was washed with cold water and diethyl ether, and dried in vacuo(0.055 g, 36%, first crop). Mp: ≥246◦C, black decomposition melt. IR (neat): ν˜ [cm−1]= 3414 (br, water), 3046 (w), 2987 (w), 2937 (w), 2811 (w), 1608 (st), 1540 (md), 1476(st), 1446 (st), 1386 (sh), 1355 (st), 1312 (md), 1254 (st), 1128 (st), 1093 (sh), 1054 (w),1002 (st), 926 (md), 854 (w), 817 (st), 785 (md), 769 (md), 742 (w), 718 (st), 699 (w). MS(ES+): m/z (%) = 1604 [Cu3(LXL)2]++, 1542 [Cu2(LXL)2 + H+], 771 [Cu(LXL)]+. Bis(ciprofloxacino)copper(II), [Cu(cipro)2]Ciprofloxacin (0.077 g, 0.2 mmol) was suspended in methanol (15 mL), and one drop oftriethylamine was added. The reaction mixture was refluxed at 80◦C for 30 min, before thestill warm, clear solution was added dropwise to a solution of copper(II) chloride (0.017 g,0.1 mmol) in methanol (2 mL). A color change to grass-green and then to blue was observed.Once the addition was completed, the reaction mixture was refluxed at 80◦C for 30 min.Upon heating it turned into a clear, turquoise solution, from which a light turquoise solidprecipitated. The solid was separated from the cold reaction mixture by filtration (glassfrit, size F), washed with water and diethyl ether, and dried in vacuo (0.0461 g, 64%).147Mp: ≥237◦C, black decomposition melt. IR (neat): ν˜ [cm−1] = 3396 (br, water), 3198(w), 2935 (w), 2844 (w), 1622 (st), 1571 (st), 1538 (w), 1491 (st), 1465 (sh), 1447 (sh),1433 (sh), 1372 (md), 1335 (md), 1297 (st), 1280 (w), 1262 (st), 1193 (w), 1172 (w), 1116(w), 1060 (w), 1019 (st), 946 (st), 889 (md), 830 (st), 812 (st), 788 (md), 749 (st), 702 (w),670 (w). MS (ES+, CH3OH): m/z (%) = 724 (100) [ML2 + H+], 747 (40) [ML2 + Na+];1119 (10) [M2L3]+. HR-ESI-MS m/z for C34H34 63CuF2N6O6 + Na+ calcd. (found):746.1702 (746.1702). EA: Anal. Calcd. (found) [%] for C34H34CuF2N6O6·2 H2O [%]: C,53.71 (53.62); H, 5.04 (5.14); N, 11.05 (11.14). Bis(pipemido)copper(II), [Cu(pia)2]A solution of pipemidic acid trihydrate (0.752 g, 2.1 mmol) and sodium hydroxide (0.096 g,2.4 mmol) in water (75 mL) was added dropwise to a solution of copper(II) chloride (0.171 g,1.0 mmol) dissolved in water (5 mL), while the pH was kept at pH∼4 (HCl(aq)). Once theaddition was completed, the pH was adjusted to 7.4 (NaOH(aq)). While stirring the reactionsolution rigorously overnight, a deep turquoise solid began to form, which was isolated byfiltration (glass frit, size F), washed with methanol, and dried in vacuo (0.229 g, 34%).Mp: ≥209◦C, black-brown decomposition melt. IR (neat): ν˜ [cm−1] = 3283 (w), 2812(w), 1609 (st), 1559 (w), 1537 (md), 1476 (st), 1444 (sh), 1380 (md), 1359 (st), 1308 (md),1286 (w), 1245 (st), 1147 (w), 1114 (md), 1060 (md), 987 (md), 921 (md), 814 (st), 787(st), 770 (st), 741 (w), 717 (md), 618 (md). MS (ES+, CH3OH): m/z (%) = 668 (100)[ML2 + H+]; 690 (40) [ML2 + Na+]. HR-ESI-MS m/z for C28H32 63CuN10O6 + H+calcd. (found): 668.1881 (668.1882). EA: Anal. Calcd. (found) [%] for C28H32CuN10O6·2H2O: C, 47.76 (48.17); H, 5.15 (5.15); N, 19.89 (19.49).1484.2.4.7 Bis(maltolato)copper(II), [Cu(ma)2]The reaction has been previously reported.465 Maltol (0.689 g, 5.5 mmol), copper(II) sul-phate (0.662 g, 2.65 mmol) in 40 mL water gave a green solid (0.569 g, 68%). Mp: ≥250◦C,decomposition to olive-brown solid. IR (neat): ν˜ [cm−1] = 3118 (w), 3087 (w), 2951 (w),2911 (w), 1906 (w), 1604 (md), 1563 (st), 1507 (st), 1470 (st), 1361 (w), 1275 (st), 1240(md), 1198 (st), 1085 (w), 1039 (md), 955 (w), 923 (md), 849 (st), 826 (st), 764 (w), 680(w), 719 (st), 626 (md). MS (ES+, CH3OH): m/z (%) = 651 (100) [M2L4 + Na+], 336(80) [ML2 + Na+]. HR-ESI-MS m/z for C12H10 63CuO6 + H+ calcd. (found): 313.9852(313.9863). EA: Anal. Calcd. (found) [%] for C12H10CuO6: C, 45.94 (45.92); H, 3.21(3.24).4.3 Results & Discussion4.3.1 Synthesis & CharacterizationThe copper(II) complexes were synthesized by combining the copper(II) salt (chloride,sulphate) with the respective ligand that was deprotonated with sodium hydroxide or tri-ethylamine. Water and methanol were the solvents; the higher the amount of water in thesolvent mixture, the easier the desired product formed as a solid precipitating from solu-tion. The resulting copper(II) complexes of the general formula [Cu(L)2] for ciprofloxacin,pipemidic acid, and maltol were neutral of charge and challenging to dissolve, prefer-ring polar-aprotic solvents such as DMSO; however, the copper(II) sandwich complexes,[Cu2(ciproXcipro)2] and [Cu2(piaXpia)2], were even harder to bring into and especially tokeep in solution. Because of the paramagnetic nature of the copper(II) center, respectiveNMR spectra of the complexes were extremely noisy. The isolated products were char-acterized through melting point determination, IR spectroscopy, mass spectrometry (low-and high-resolution), and elemental analysis.149Mass spectrometry confirmed the general composition of the complexes to be 2:1 and1:1 ligand:metal ratios for [Cu(L)2] and [Cu2(LXL)2], with [M + H+] or [M + Na+] asparent peaks of exact mass. In the low-resolution MS spectra, the complexes displayed thecharacteristic copper isotope distribution of 63Cu/65Cu with the peak of 100% intensitycorresponding to the 63Cu isotope of higher abundance, and the [M + 2] peak of approx-imately 50% intensity corresponding to the 65Cu isotope. For the [Cu(L)2] complexes,[Cu2L3]+ peaks of lower intensity were detected, formed via the recombination of a [CuL2]with a [CuL]+ fragment. The [Cu2(LXL)2] complexes, on the other hand, showed thedoubly-charged recombination peak of [Cu3(LXL)2]++.Spectroscopic analysis in the mid-infrared region (4000−600 cm−1) of the copper-quinolone complexes confirmed the complete coordination of the copper(II) ion throughthe carboxylate-O on Car3 and the carbonyl-O on Car4. Figure 4.2 shows the recordedIR spectra of ciprofloxacin, bis(ciprofloxacino)copper(II), the xylenyl-linked ciprofloxacindimer, and the respective copper(II) complex [Cu2(ciproXcipro)2]. In the spectrum ofH2ciproXcipro, the intense stretching vibration at 1721 cm−1 stems from the dimeric car-boxylate group (νCOOH); upon coordination to copper(II) this band disappears, and thespectrum of [Cu2(ciproXcipro)2] reveals full coordination of the ligand to the copper(II)ions. The spectrum of ciprofloxacin does not show the dominant stretch of the hydroxidegroup, because its proton from the carboxyl group on Car3 is free to move to the secondaryamine on the piperazinyl group on Car7 (Figure 4.1) resulting in the zwitterionic state(Figure 2.3) for which no OH-stretch can be expected.317 318 Following previous literaturereports,327 328 the two distinct bands in the range of 1650−1600 cm−1 and 1400−1300 cm−1were assigned as νCO2 asymmetric and symmetric stretching vibrations characteristic forthe metal complexation of deprotonated quinolone ligands. In addition, new peaks wereobserved in the low fingerprint region (800−700 cm−1) and assigned to metal-oxygen vibra-150Table 4.1: Selected IR stretching frequencies [cm−1] and their assignments.complex νasym(CO2) νsym(CO2) ∆(a) ν(CuO)[Cu(cipro)2] 1622 1372 250 749[Cu(pia)2] 1609 1359 250 770[Cu2(ciproXcipro)2] 1625 1372 253 765[Cu2(piaXpia)2] 1608 1355 253 769(a)∆ = νasym(CO2) − νsym(CO2).tions in the formed complexes. Selected IR bands and their assignments are summarizedin Table 4.1.To evaluate the behaviour of the complexes under bioreductive conditions, attemptswere made to record cyclic voltammograms of [Cu2(ciproXcipro)2], [Cu2(piaXpia)2], andthe respective free ligands (H2ciproXcipro, H2piaXpia). Unfortunately, due to lack ofsolubility of the linked-quinolone dimer ligands at suitable concentration in suitable solventsfor CV studies (acetonitrile, DMSO, methanol, tetrahydrofuran), these planned studiescould not be completed. Moreover, many attempts were made to grow single crystalsof the α, α’-xylenyl-linked quinolones and their copper(II) complexes, however, the lowsolubility of the free ligands and metal complexes hampered these attempts due to DMSObeing the only solvent in which a certain amount of solubility could be reached. Thecrystallization attempts ranged from slow diffusion in DMSO with acetone, acetonitrile,chloroform, diethyl ether, and methanol in various environmental settings (window sill,fume hood, dark cupboard, fridge, freezer) to reactive crystallization experiments, in whichthe xylenyl-linked quinolone dimer, dissolved in heat in DMSO and filtered, at the bottomof the vial was layered with 1 mL of copper(II) chloride in methanol. The latter resultedin crystals, but, unfortunately, these were unsuitable for X-ray diffraction.151Figure 4.2: IR spectra, from top to bottom, of ciprofloxacin (black),bis(ciprofloxacino)copper(II) (teal), H2ciproXcipro (navy), and[Cu2(ciproXcipro)2] (blue).1524.3.2 Antimicrobial Susceptibility TestingThe antimicrobial activity of the synthesized copper(II) complexes and their free ligandswas evaluated against the following pathogens associated with nosocomial diseases:330 E.faecalis and methicillin-susceptible S. aureus (both Gram-positive); E. coli, K. pneu-monia, and P. aeruginosa (all Gram-negative). Copper(II) complexes were tested at0.1 mM concentration, while the respective free ligands were investigated at 0.1 mM and0.2 mM concentrations to preclude any effects arising from the double concentration ofquinolones in the copper-complexes. Furthermore, solutions of copper(II) chloride as wellas bis(maltolato)copper(II) over the concentration range 10 mM−0.1 mM were included toallow the assessment of the antibacterial properties of copper(II) ions. Maltol is widely usedas a flavour enhancer in the food industry and considered non-toxic and safe,465 therefore,any antimicrobial effect of this complex would be directly arising from Cu2+.The results of the antimicrobial study, presented in Table 4.2, are averaged values of therecorded inhibition zone sizes from three independent test plates. Three conclusions can bedrawn from the data. Firstly, complexing pipemidic acid with copper(II) did not overcomethe developed resistance against this first generation quinolone. Secondly, the antimicro-bial potency of the bis(quinolono)copper(II) complexes is solely determined by the con-centration of quinolone, as the recorded inhibition zone sizes for 0.2 mM ciprofloxacin andpipemidic acid correspond well with those recorded for 0.1 mM bis(ciprofloxacin)copper(II)and bis(pipemido)copper(II), respectively.153Table 4.2: Inhibition zone sizes [mm] of copper(II) complexes.bacteria Hcipro(0.2 mM)Hcipro(0.1 mM)[Cu(cipro)2](0.1 mM)Hpia (0.2 mM) Hpia (0.1 mM) [Cu(pia)2](0.1 mM)E. faecalis 13 (0) 9 (0) 11 (1) 0 (0) 0 (0) 0 (0)S. aureus 17 (1) 12 (1) 15 (1) 0 (0) 0 (0) 0 (0)E. coli 25 (1) 22 (1) 24 (1) 10 (1) 0 (0) 7 (0)K. pneumonia 22 (1) 18 (0) 21 (1) 5 (4) 0 (0) 0 (0)P. aeruginosa 12 (2) 8 (1) 9 (1) 0 (0) 0 (0) 0 (0)154bacteria H2ciproXcipro(0.2 mM)H2ciproXcipro(0.1 mM)[Cu2(ciproXcipro)2](0.1 mM)H2piaXpia(0.2 mM)H2piaXpia(0.1 mM)[Cu2(piaXpia)2](0.1 mM)E. faecalis 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)S. aureus 5 (4) 0 (0) 8 (1) 3 (5) 0 (0) 8 (1)E. coli 8 (1) 7 (0) 0 (0) 0 (0) 0 (0) 0 (0)K. pneumonia 5 (4) 5 (4) 0 (0) 0 (0) 0 (0) 0 (0)P. aeruginosa 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)Reported inhibition zones [mm] are averaged values from three plates (standard deviation). Disk diameter0.6 mm. Loading volume 20 µL. Disks loaded with solutions of methanol and 2% DMSO in methanolserved as controls, all of these showed no inhibition (0 mm).1551234567891011121314Figure 4.3: Photo of a growth plate of P. aeruginosa after 20 h of incubation at37◦C during antimicrobial single-disk test. Disk 1, 2% DMSO in methanolcontrol; disk 2−7, copper(II) chloride (20 µL) in decreasing concentrations(10 mM−0.5 mM); disk 8−14, bis(maltolato)copper(II) (20 µL) in decreasingconcentrations (10 mM−0.1 mM). No test compound showed inhibition ofgrowth, recorded inhibition zone size: 0 mm.Thirdly, the xylenyl-linked quinolone dimers are not as potent as the quinolone monomers,which could suggest that due to the difference in size the quinolone-dimers are not diffusingas well into the agar medium as are the smaller free quinolone ligands. This observation issupported by the large differences between the recorded inhibition zone sizes from one plateto another, as reflected in the high values for the standard deviations for H2ciproXciproand H2piaXpia. The diffusion effect is even more prominent for the copper(II) sandwichcomplexes [Cu2(ciproXcipro)2] and [Cu2(piaXpia)2], which do not seem to move far on theplate. Both complexes appear to be only potent against the tested strain of methicillin-susceptible S. aureus, as depicted in the small inhibition zones of 8 (1) mm. This meansthat, although S. aureus showed resistance against pipemidic acid (0.1 and 0.2 mM) and156bis(pipemido)copper(II) (0.1 mM), linking two pipemidic acid molecules together via thexylene-linker makes the test strain susceptible again. This effect has been first reportedand studied by Kerns and co-workers.446 447 Further complexation of these xylenyl-linkedquinolones with copper(II) in a sandwich fashion seems to increase this effect (Table 4.2),however, we were unable to measure the antimicrobial effect of copper(II) alone. Anextensive single-disk study of solutions of copper(II) chloride and bis(maltolato)copper(II)ranging from 10 mM to 0.1 mM (loading volume 20 µL) was performed on all five bacteria intriplicate. Figure 4.3 shows a photo of one growth plate of P. aeruginosa as a representativeexample for the entire study. Neither the disks loaded with copper(II) chloride nor the onesloaded with bis(maltolato)copper(II) showed any growth inhibition against the five testedpathogens, which grew onto the edge of each disk seemingly unaffected by the presence ofcopper(II).4.4 ConclusionIn this chapter, the syntheses of bis(ciprofloxacino)copper(II) and [Cu2(ciproXcipro)2] aswell as bis(pipemido)copper(II) and [Cu2(piaXpia)2] were reported. The complexes werecharacterized by elemental analysis, IR spectroscopy, mass spectrometry, and melting pointdetermination; due to the paramagnetic nature of the copper(II) core, NMR spectroscopymeasurements were not possible. In a single-disk test procedure, the antimicrobial effi-cacy of these complexes was tested against five pathogens that are commonly associatedwith nosocomial diseases (E. faecalis, S aureus, E. coli, K. pneumonia, P. aeruginosa)and directly compared to the antimicrobial effect of the respective free ligands, as well ascopper(II) chloride, and bis(maltolato)copper(II). Maltol, a food additive does not possesany antimicrobial properties, but coordinates the copper(II) tightly,466 preventing the for-mation of insoluble metal hydroxides. The test results did not show a combinational an-157timicrobial effect between Cu2+ and the respective quinolone ligand, but the antimicrobialpotency of the synthesized copper(II) complexes rather depends solely on the concentra-tion of quinolone alone. Further studies of copper(II) chloride and bis(maltolato)copper(II)over the concentration range from 10 mM to 0.1 mM did not show any growth inhibitionof any of the five tested bacteria strains either.In conclusion, the ”Achilles Heel Theory” may be questioned, as the combination ofCu2+ with quinolone antimicrobial agents, and derivatives thereof, based on this theory,as well as high concentrations (10 mM) of copper(II) chloride alone or [Cu(ma)2] did notshow an improved antimicrobial effect, or any bacterial growth inhibitory effect at all.158Chapter 5Iron(III)-Binding of the AnticancerAgents Doxorubicin and VosaroxinThe importance of kinetic and thermodynamic stability in vivo has already been introducedin Section 1.4.3 and will be exemplified through the discussion of the interaction of iron(III)with two anticancer agents in vivo in this chapter.5.1 The Two Anticancer Agents: Doxorubicin & VosaroxinAnthracycline anticancer drugs, such as doxorubicin (Hdox) (Figure 5.1), are in broaduse clinically but are associated with cumulative-dose cardiomyopathy.467 468 469 470 471 472Molecular mechanisms for doxorubicin induced cardiomyopathy remain controversial,469 473despite decades of investigations that have been recently reviewed.471 474 These includeinteraction of doxorubicin with topoisomerase IIβ and induction of DNA damage,475 476accumulation in normal myocardium,477 metabolic conversion including the formation ofdamaging species,478 479 480 481 482 483 and the generation of oxidative stress resulting fromthe interaction of doxorubicin with oxygen catalyzed by iron.484 485 Through iron-mediatedinteractions, doxorubicin causes the formation of reactive oxygen species such as hydrogen159OOOOHOHOHOOOHO NH2doxorubicin (Hdox)N NOO OHN SNHNOvosaroxin (Hvox)HN NOON NHOOdexrazoxaneOHFigure 5.1: Chemical structures of doxorubicin, vosaroxin, and dexrazoxane.peroxide (H2O2) and superoxide radical anion (O·−2 ). As depicted in Figure 5.2, the uni-valent reduction (e−) of the aromatic core of the doxorubicin molecule gives the unstablesemiquinone free-radical, doxoquinone that in the presence of oxygen can auto-oxidize backto its parent quinone. The reduction of molecular oxygen leads to the formation of ROSwhich then can react further with free iron following the well-travelled pathways of Fentonchemistry, leading to an accumulation of iron in the mitochondria, increased levels of ROS,and overall impaired mitochondrial respiration.474 The bivalent reduction (2 e−) of theside-chain carbonyl group of the doxorubicin molecule converts Hdox irreversibly into itssecondary alcohol metabolite, doxol, which is slightly less redox active than the unstablesemiquinone free-radical, therefore able to accumulate; doxol’s disruptive effect on humanCa2+ and Fe3+ homeostasis appears stronger than the parent compound.473Reduction of Fe(III) to Fe(II) is an essential biological step that occurs widely in Na-ture.486 On the molecular level, in vivo iron homeostasis is heavily regulated because livingorganisms carefully sequester iron(II) in stable complexes with biomolecules such as trans-ferrin to prevent any toxicity arising from free iron overload. Reactive oxygen species, suchas O·−2 , H2O2 as well as the hydroxyl radical (OH·), dramatically affect iron homeostasis inNature. While ROS are involved in various essential biological functions, they can becomeharmful at higher concentrations, when their oxidation reaction of biomolecules increasesphysiological stress.487 ROS species directly interact with ferrous and ferric ions in vivo160Figure 5.2: Doxorubicin affects Fe3+ homeostasis in vivo: univalent reduction tosemiquinone (left), bivalent reduction to secondary alcohol (right).161according to Fenton oxidation chemistry.488 Superoxide radical anions reduce Fe(III) thatis coordinated to biological ligands to Fe(II) and dioxygen. In an inner-sphere electron-transfer mechanism, H2O2 oxidizes biologically-ligated Fe(II) to Fe(III) with concomitantformation of hydroxyl radical and hydroxide (HO−) as reductive side-products, which leadsto increased oxidative stress.Oxidative stress is directly involved in the pathogenesis of heart failure; it damagesthe mitochondria through excess formation of O·−2 , reduction of adenosine triphosphate(ADP), and transcriptional alteration of genes associated with heart failure.489 490 To cir-cumvent induction of oxidative stress and cardiomyopathy, doxorubicin is administeredwith radical scavenger drugs, such as dexrazoxane (5.1), which reduce mitochondrial iron-levels.491 492 493Vosaroxin (Hvox), presented in Figure 5.1, is a first-in-class anticancer quinolone deriva-tive (AQD) that induces DNA damage and inhibits topoisomerase II, inducing site-selectiveDNA damage, G2 arrest and apoptosis.295 297 494 495 Vosaroxin induces DNA double/strandbreaks (DSB) in cancer cells in guanine/cytosine rich sequences analogous to those causedby quinolone antimicrobials in bacteria.494 495 In contrast to doxorubicin, vosaroxin is nota substrate for the multidrug resistance protein P-glycoprotein496 and evades resistancemechanisms associated with p53 deficiencies.497 498 Vosaroxin has been studied in both solidtumor cancers,499 500 as well as hematologic malignancies,501 and it is currently completinga phase III clinical trial in patients with relapsed or refractory acute myeloid leukemia.502 Incontrast to doxorubicin, vosaroxin’s anticancer activity appears to result exclusively fromintercalation of DNA and inhibition of topoisomerase II. Unlike doxorubicin, vosaroxin isminimally metabolized,480 483 503 and thereby produces limited free radicals ROS via intrin-sic metabolic activation.494 As mentioned above, cardiomyopathy is a serious side-effectof treatment with doxorubicin, which has been associated with the formation of ROS and162other toxic metabolites partly catalyzed by iron.The fact that quinolones coordinate metals in various oxidation states in differentcoordination geometries327 is a known side-effect for antimicrobial therapy, as such co-ordination leads to a reduction in quinolone bioavailability.504 Iron(III) complexes withvarious commercially available quinolone antimicrobial drugs in which the iron coordi-nates the drug ligands in a stable octahedral 1:3 fashion have been reported and stud-ied.398 399 400 401 403 505 506 Iron(III) forms some of the most stable complexes with quinolonescompared to other bivalent and trivalent metals,426 and the determined stability constantsrange from logβFeL3 = 25.16 (1) for enoxacini to logβFeL3 = 46.94 for ciprofloxacin.iiAntimicrobial treatment with quinolone drugs is, in general, considered safe, as theyare known to be commonly well tolerated and have safety profiles that compare to those ofother antimicrobial drug classes.282 507 508 Frequently reported mild adverse reactions affectthe gastrointestinal (GI) tract (e.g., nausea, vomiting, diarrhea) and the central nervoussystem (e.g., dizziness, headache, drowsiness), while tendinitis and tendon rupture as wellas phototoxicity509 are more severe side-effects for which certain quinolone antimicrobialagents are widely known.282 295 297 507 508 510The molecular mechanisms of actions of vosaroxin, a quinolone derivative, and dox-orubicin, an anthracycline, are differentiable as a result of their distinct chemical scaffolds(Figure 5.1). In order to further understand the properties of these two compounds, theinteraction of vosaroxin with iron(III) has been characterized, and solution spectrophoto-metric studies of iron(III) coordination chemistry with vosaroxin and doxorubicin have beenconducted. In addition, the novel tris(vosaroxino)iron(III) complex has been synthesizedand characterized in order to examine the iron(III) coordination properties of vosaroxin indirect comparison to doxorubicin.iHL= enoxacin, determined potentiometrically: 22◦C, INaCl= 0.1 M, inert gas N2.426iiHL= ciprofloxacin, extrapolated from potentiometric data: 25◦C, IKCl= 0.2 M.315163Because the Ga3+ ion possesses many chemical similarities with the Fe3+ ion (seeChapter 3), their chemical binding properties are similar, especially in regard to ligandchelation or protein binding.376 Biological systems cannot distinguish between Fe3+ andGa3+, a fact exploited in Fe3+ transport studies in vivo 372 or imaging.377 Therefore, thediamagnetic tris(vosaroxino)gallium(III) analog has been synthesized as well, which allowedthorough NMR-studies of the isolated complex.5.2 Materials & Methods5.2.1 ChemicalsDoxorubicin and iron(III) nitrate nonahydrate were purchased from Sigma-Aldrich, gal-lium(III) nitrate nonahydrate was obtained from Alfa-Aesar, and dimethyl sulfoxide wasfrom Fisher Scientific. Sunesis Pharmaceuticals, Inc. provided the vosaroxin (referencestandard quality, lot# 12AK0025B). Deuterated dimethyl sulfoxide was purchased fromCambridge Isotope Laboratories, Inc., and Sigma-Aldrich delivered the deuterium oxide aswell as the phosphoric acid-d3 solution and the sodium deuteroxide. The atomic iron(III)standard solution for AAS (1000 mg/L ± 4 mg/L) was obtained from Fluka. In thepreparation of all aqueous solutions for spectrophotometric measurements and syntheses,only deionized water, purified through a ELGA PURELAB ultrapure water system with aresistivity of 18 MΩ·cm (25◦C), was used.5.2.2 InstrumentationMelting points were determined using a Stanford Research Systems DigiMelt SRS meltingpoint apparatus and are uncorrected. Ultraviolet-Visible spectrophotometry was performedon a Hewlett Packard 8453 instrument. Spectra were recorded using the UV-Vis Chem-Station Software (version B.04.01[61], Agilent Technologies, 2001−2010), and all maximum164absorption bands and extinction coefficients () are listed. Infrared spectra were recorded inthe solid state on a PerkinElmer Frontier FT-IR spectrometer in the range 4000−650 cm−1using the software PerkinElmer Spectrum (version 10.03.02, 2011). Only the most charac-teristic bands were interpreted using the following abbreviations: s, strong; m, moderate;w, weak; br, broad; sh, shoulder. Nuclear magnetic resonance data (1D, 2D) were col-lected on a BRUKER AV600 spectrometer (600 MHz). The residual solvent signal of thedeuterated solvent was used as the internal standard.309 Chemical shifts δ are referencedin ppm against tetramethylsilane (δ= 0). Multiplicities are abbreviated as: s, singlet; m,multiplet; br, broad. Ar represents aromatic protons. All spectra were analyzed with thesoftware inmr (version 5.3.4, Mestrelab Research). Low-resolution mass spectral analysiswas performed on a Water ZQ spectrometer equipped with ESCI sources. High-resolutionmass spectra were obtained at the UBC Mass Spectrometry Centre on a Waters MicromassLCT employing electrospray-ionization. Only characteristic signals have been listed as thedimensionless mass-to-charge ratio, with intensity related to the base signal.5.2.3 SpectrophotometryThe instrumental set-up comprised a Corning Hot Plate Stirrer PC-351, a Fisher ScientificAccumet Basic pH-meter and a ThermoFischer ORION 8103BN ROSS Semi-Micro Combi-national pH-electrode, which was calibrated before each titration using reference solutions(pH 4.00, 7.00, 10.00) from the FisherScientific Buffer-Pac. All solutions were prepared inaqueous sodium chloride (INaCl= 0.15 M). Stock solutions of vosaroxin and doxorubicinwere prepared as follows: about 1 mg of Hvox, or about 1.5 mg of Hdox, respectively, wasdissolved in 5.00 mL sodium chloride solution, and after 30 min of sonication the suspen-sion turned to a clear, colorless solution; these stock solutions were stable and used fortwo to three days. Solutions of ligand (Hvox, Hdox) only (∼2.0·10−5 M) or ligand (Hvox,Hdox) (∼2.0·10−5 M) mixed with iron(III) standard solutions in Fe3+:HL ratios of 1:1, 1:2,165or 1:3 in a total volume of 10.00 mL were freshly prepared on the day of the experiment.To ensure that the titration started with the fully protonated species of ligand, the lig-and solutions were prepared in aqueous hydrochloric acid (0.2 M). The freshly preparedsolution was sonicated for 15 min and transferred into a glass vial before the electrodewas submerged in the test solution. The solution was stirred and the electrode remainedsubmerged in the solution throughout the titration. The data were collected as pH vs.volume of titrant (2−20 µL). For each determination of ligand protonation constant orFe(III) complexation constant, titrations were conducted in triplicate. Equilibration timesbetween additions ranged from 3−10 min. All titration data were manipulated in MS Exceland plotted using Plot2 (version 2.0, Michael Wesemann). HypSpec (Protonic Software,Leeds) was used to fit the obtained UV-Vis curves to obtain the stability constants.5.2.4 Computational DetailsCalculations of vosaroxin with iron(III) were performed using DFT at the B3LYP level uti-lizing the 6-31+g(d,p) basis set as implemented in Gaussian.412 All optimised geometriesare characterised as minima as indicated by the absence of imaginary frequencies. Ge-ometry optimisations were performed on the systems and were evaluated to obtain stableFe3+:vosaroxin structures. Computational modelling was used to verify the binding site ofthe iron(III) ion to Hvox. The initial geometry of vosaroxin was optimised and was used ina series of subsequent calculations, whereby the Fe3+ was placed at various regions aroundthe vosaroxin ligand but at distances greater than ∼3.5 A˚ so as not to bias the interaction,if any, between the Fe3+ metal ion and the potential binding partner.1665.2.5 Synthesis & Characterization5.2.5.1 Vosaroxin, HvoxAppearance: Amorphous, off-white solid. Mp: ≥240◦C, decomposition to dark yellowsolid. UV-Vis (DMSO): λ [nm] () [M−1cm−1] = 275 (80000), 250 (50000). IR (neat):ν˜ [cm−1] = 3316 (w), 3088 (md), 3047 (sh), 2988 (w), 2940 (md), 2885 (md), 2819 (w),1728 (st), 1619 (st), 1548 (st), 1510 (sh), 1491 (st), 1439 (md), 1417 (md); 1386 (md),1327 (md), 1299 (md), 1261 (sh), 1251 (md), 1220 (w), 1185 (w), 1161 (w), 1113 (st), 1092(sh), 1016 (w), 958 (st), 871 (w), 854 (w), 830 (md), 799 (st), 764 (md), 736 (md), 698(w), 681 (md), 657 (md). NMR: δH (600 MHz, 298 K, d6-DMSO) [ppm] = 9.72 (s, 1 H,Car2H); 8.23 (d, J3HH= 9.1 Hz, 1 H, Car5H); 7.81−7.80 (m, 1 H, Ctaz4H); 7.78−7.76 (m,1 H, Car6H); 6.85−6.83 (m, 1 H, Ctaz5H); 3.96 (s, 1 H, Cazo3H); 3.88−3.80 (m, 1 H, NH);3.78−3.71 (m, 2 H, Cazo5H2); 3.66−3.63 (m, 1 H, Cazo4H); 3.52−3.43 (m, 2 H, Cazo2H2);3.31−3.28 (m, 3 H, OCH3); 2.37 (d, J3HH= 2.6 Hz, 3 H, NH(CH3)). δC (150 MHz, 298K, d6-DMSO) [ppm] = 176.8 (Car4); 165.3 (COOH); 157.3 (br, Car7); 155.2 (br, Ctaz2);147.7, 147.6 (Car8); 141.9 (br, Car2); 137.8, 137.7 (Ctaz4); 135.4, 135.3 (Car5); 121.6, 121.5(Car6); 110.0 (br, Car4); 109.3 (br, Ctaz5); 109.0, 108.9 (Car3); 82.0, 81.6 (Cazo3); 62.4, 62.1(OCH3); 56.3, 56.3 (Cazo2); 53.4, 53.1 (Cazo5); 51.2, 50.8 (Cazo4); 34.2, 34.1 (NCH3). MS(ES+, CH3OH): m/z (%) = 402 (100) [HL + H+]. HR-ESI-MS: m/z for C18H19N5O4S +H+ calcd. (found): 402.1236 (402.1242). EA: Anal. Calcd. (found) [%] for C18H19N5O4S:C, 53.86 (53.97); H, 4.77 (4.74); N, 17.45 (17.24); S, 7.99 (7.64). Tris(vosaroxino)iron(III), [Fe(vox)3]Dissolving vosaroxin (125 mg, 0.31 mmol) in deionized water (20 mL) and stirring for15 min at ambient temperature gave a clear, colorless solution of neutral pH. This solutionwas added dropwise into a previously prepared solution of iron(III) nitrate nonahydrate167(40 mg, 0.1 mmol) in deionized water (5 mL). During the addition, the pH was adjustedto <3, if necessary, with aqueous hydrochloride solution (0.1 M). Upon completion of theaddition, the pH was raised to pH 5 with aqueous sodium hydroxide (1.0 M), changing thecolor of the reaction mixture to red-brown. The reaction mixture was stirred at ambienttemperature overnight, before the solvent was removed in vacuo to result an amorphous,lustrous, dark red-brown solid, which was washed repeatedly with deionized water andmethanol, and then thoroughly dried in vacuo (108 mg, 0.086 mmol, 86%). Mp: ≥200◦C,decomposition to black-brown solid. UV-Vis (DMSO): λ [nm] () [M−1cm−1] = 275(80000), 250 (50000). IR (neat): ν˜ [cm−1] = 3433 (w, br), 3083 (w), 2997 (w), 2941 (w),2881 (w), 2832 (w), 2732 (w), 2462 (w, br), 1621 (st), 1562 (sh), 1494 (st), 1443 (md), 1419(md), 1317 (w), 1293 (w), 1276 (w), 1253 (st), 1178 (w), 1097 (st), 1038 (md), 968 (md),921 (md), 854 (w), 825 (w), 803 (st), 758 (st), 722 (w), 701 (w), 675 (md). MS (ES+):m/z (%) = 1280 (< 10) [ML3 + Na+], 857 (100) [ML2]+. HR-ESI-MS: m/z for C54H5456FeN15O12S3 39K+ calcd. (found): 1295.2225 (1295.2233). Tris(vosaroxino)gallium(III), [Ga(vox)3]Vosaroxin (125 mg, 0.31 mmol) was dissolved in deionized water (12 mL) and stirred atambient temperature for 10 min. The clear colorless vosaroxin solution was added drop-wiseinto a solution of gallium(III) nitrate nonahydrate in deionized water (5 mL). The reactionmixture turned pale yellow during the addition, and its pH increased to pH 5. Withoutfurther adjustments, the reaction solution was stirred at ambient temperature overnight.Removal of the solvent in vacuo gave an amorphous, lustrous, pale yellow solid that waswashed repeatedly with water and methanol, and then thoroughly dried in vacuo (69 mg,0.082 mmol, 82%). Mp: ≥190◦C, decomposition to brown solid. UV-Vis (DMSO): λ[nm] () [M−1cm−1] = 275 (80000), 250 (50000). IR (neat): ν˜ [cm−1] = 3421 (md, br),3086 (w), 3001 (w), 2937 (w), 2874 (w), 2832 (w), 2733 (w), 2473 (w, br), 1615 (st), 1564168(sh), 1494 (st), 1446 (md), 1423 (md), 1318 (w), 1277 (w), 1256 (st), 1179 (w), 1098 (st),1040 (md), 970 (md), 923 (st), 858 (w), 826 (w), 803 (st), 760 (st), 728 (w), 702 (w), 680(md). NMR: δH (400 MHz, 363 K, d6-DMSO) [ppm] = 10.05 (s), 9.96 (s), 9.79 (s), (3H, Car2H); 8.42 (d, J3HH= 8.9 Hz), 8.33 (d, J3HH= 9.1 Hz), 8.21 (d, J3HH= 7.8 Hz), (3 H,Car5H); 7.91-7.80 (m, 6 H, Ctaz4H and Car6H); 7.00 (d, J3HH= 9.1 Hz), 6.90 (d, J3HH= 7.2Hz), (3 H, Ctaz5H); 4.30-4.26 (m, 3 H, Cazo3H); 4.12-3.78 (m, 15 H, Cazo5H2, Cazo4H, 3.43Cazo2H2); 3.43 (s, 9 H, OCH3); 2.68 (d, J3HH= 17.4 Hz, 9 H, NH(CH3)). δC (150 MHz,298 K, d6-DMSO) [ppm] = 178.4, 176.1, 174.5 (Car4); 165.0, 164.9, 164.8 (COOH); 157.3,157.1, 157.0 (Car7); 155.6, 155.5, 155.4 (Ctaz2); 147.9, 147.7, 146.5 (Car8); 144.4, 144.1,143.4 (Car2); 138.4, 138.1, 137.9 (Ctaz4); 136.4, 136.2, 135.8 (Car5); 122.3, 122.1, 121.8(Car6); 110.7, 110.6, 110.4 (Car4); 110.0, 109.8, 109.6 (Ctaz5); 109.9, 109.0, 108.8 (Car3);79.5, 79.4, 79.3 (Cazo3); 60.7, 60.6, 60.5 (OCH3); 57.2, 57.1, 57.0 (Cazo2); 53.8, 53.7, 53.6(Cazo5); 50.7, 50.6, 50.5 (Cazo4); 31.9, 31.8, 31.7 (NCH3). MS (ES+): m/z (%) = 1272(50) [ML3 + H+], 869 (100) [ML2]+. HR-ESI-MS: m/z for C54H54 69GaN15O12S3 23Na+calcd. (found): 1292.2392 (1292.2397); m/z for C36H36 69GaN10O8S+2 calcd. (found):869.1415 (869.1414).5.2.6 ElectrochemistryCyclic voltammetry studies were performed with Hvox, [Fe(vox)3] and [Ga(vox)3] in adimethyl sulfoxide solution (V = 10.0 mL) containing 0.1 M of tetra(n-butyl)ammoniumperchlorate as the supporting electrolyte in a three electrode system composed of aplatinum-disk electrode as the working electrode, a platinum-mesh electrode as the counterelectrode and a silver electrode as the pseudo reference electrode. A potentiostat (PineAFCBP1, ID 23051890) was integrated into the electric circuit. The software AfterMath,Inc. (version 1.2.4532) was used for controlling the potentiostat and recording the data.CV measurements followed standard procedures. All glassware was dried in the oven at169100◦C for 24 hours before use. Tetra(n-butyl)ammonium perchlorate (0.342 g, 0.001 mol)was transferred into the electrochemical cell and dried in vacuo for 15 min. In parallel,dimethyl sulfoxide was degassed with N2 gas. Using Schlenk techniques, 10.0 mL of thedegassed dimethyl sulfoxide was transferred onto the tetra(n-butyl)ammonium perchlorateinside the electrochemical cell. The mixture was stirred vigorously until the electrolytesalt had completely dissolved. Throughout the experiment the electrochemical cell waskept under N2 gas at all times. Firstly, a blank voltammogram of the electrolyte solutionwas recorded. Secondly, the compound to be measured was added as a solid and dissolvedin the electrolyte solution (0.01 mmol, respectively: Hvox, 41 mg; [Fe(vox)3], 12.6 mg;[Ga(vox)3] 12.0 mg). Measurements on these compounds were performed in the generalvoltage range between +1.3 V and −2.3 V, starting from an initial voltage of 0 V andending at a final voltage of 0 V. The sweep rate was 100 mV·s−1, the electrode range wasvaried between 5−10 µA, and the number of segments was set to 5 as default. The exper-iment concluded with a reference measurement of ferrocene. All electrodes were cleanedaccordingly. The platinum-mesh electrode and the silver pseudo-reference electrode weresubmerged in methanol (separate vials) and sonicated for 30 min. The platinum-disk elec-trode was polished using Buehler MicroPolish II Alumina Powder (0.3 µm and 0.05 µm);polish residues were rinsed off with deionized water and the electrode was dried.5.3 Results & Discussion5.3.1 Stability ConstantsVosaroxin and doxorubicin each contain ionizable protons. The pKa values for each ligand,as determined by spectrophotometric titration, are given in Table 5.1. In acidic solutions,doxorubicin exists as the singly charged species [H2dox]+ with the positive charge at thesugar amino group. Initial dissociation (pKa 7.67) is assigned to the amino sugar group170Table 5.1: Protonation and Fe3+ formation constants for doxorubicin and vosaroxin.KFeLH doxorubicin vosaroxinlogK110 17.985a 16.31(3)logK120 11.049 8.70(2)logK130 4.379 7.80(3)logβ130 33.413 32.80(3)pMb 17.0 15.9pKa1 10.96(1) 9.97(2)pKa2 9.46(1) 7.091(4)pKa3 7.67(2) 5.125(4)pKa4 n/a 2.779(4)a37◦C, INaCl= 0.15 M.511 bpM= −log[Fe], pH 7.4, [L]T= 10 µM, [Fe]T= 1 µM.followed by the dissociation of the phenolic hydrogens (pKa 9.46 and 10.96). These as-signments are in agreement with those in the literature.511 It is interesting to note thatthere is some variation in literature data on the amino group pKa value 6.8−8.99,512 andpKa of 9.01−11.2 for the phenolic group. These differences can be attributed, in part, toself-association and decomposition of the drug at higher concentrations (>30 µM).513 Theconcentrations of doxorubicin used in the present studies were purposefully low enough toavoid self-association (∼20 µM). Vosaroxin has four ionizable protons, existing as the triplycharged species [H4vox]3+ under acidic conditions. The ionization process and pKa valuesdetermined in this study are in close agreement with those, which have been extrapolatedfrom a co-solvent system earlier.514 Small differences can be attributed to the use of sodiumchloride at biologically relevant concentrations (INaCl= 0.15 M) as background electrolyte.The formation of the Fe(III)-doxorubicin and Fe(III)-vosaroxin complexes as a functionof pH and [Fe3+]:[HL] molar ratios was investigated by spectrophotometric titration. Theformation of the Fe3+-doxorubicin complexes elicited broad absorption bands centered at600 nm (data presented in Appendix B), as previously reported.512 515 516 Experiments withFe(III)-doxorubicin proved problematic due to drifting electrode measurements, formation171and precipitation of Fe(OH)3 as well as low concentrations of complex formed under theexperimental conditions utilized. Despite the apparent distance between the amino groupand the iron binding site of doxorubicin, the charge on the amino group is known to in-fluence the strength of interaction with the metal ion.517 These problems interfered withconvergence within HypSpec software when fitting the data, and there were large uncer-tainties in the logK values that could be obtained. Consequently, the logK values fordoxorubicin presented in Table 5.1 have been taken from the literature.511The visible absorption spectra of solutions containing Fe(III) and vosaroxin as a func-tion of pH (Appendix B) are characterized by a new, broad absorption band centeredaround 400 nm as shown in Figures 5.3 and 5.4. In order to fit the iron(III)-vosaroxintitration data, it was necessary to select a model incorporating all possible species. Com-putational modelling was utilized in order to identify the preferred binding site of the Fe3+ion to vosaroxin.518 For this, geometry optimisation (energy minimization) calculationswere performed on different states, whereby the Fe3+ ion was placed at various locationsaround the vosaroxin ligand; however, not to bias the interaction, the Fe3+ ion was neverplaced in a distance closer than ∼3.5 A˚ to vosaroxin, the potential binding partner. Themost stable conformer involves the ferric ion being chelated by the ketone oxygen and thedeprotonated carboxylate moiety (Figure 5.5). It is interesting to note that energy min-imization of vosaroxin alone or in the presence of iron results in rotation of the thiazolering such that the orientation of this group differs between bound and unbound species.For unbound vosaroxin, the thiazole group is almost co-planar with the naphthyridine ringand the sulfur atom nearest the ring nitrogen atoms. Following complexation of Fe3+ atthe diketone, the thiazole group rotates so it is now almost at a right angle to the naph-thyridine ring. This was confirmed by scanning the potential energy surface for rotationaround the N−C bond of the thiazole, and is consistent with the 1H NMR data collected172Figure 5.3: Comparison of the changes in absorbance at 400 nm for a solution ofFe3+-vosaroxin ([Fe3+] = 5.8·10−6 M and [Hvox] = 2.24·10−5 M) () andvosaroxin only () with varying pH in aqueous electrolyte solution (INaCl=0.15 M).following titration of Fe3+ with vosaroxin. These data show that addition of Fe3+ influ-ences the chemical shift of neighboring hydrogens including those around the thiazole andnaphtyridine rings, not just those expected to be influenced by chelation (data shown inAppendix B).Speciation plots for solutions of doxorubicin and vosaroxin with Fe(III) as a functionof pH are shown in Figure 5.6 and Figure 5.7, respectively. At a ratio of 3:1 ligand tometal, the predominant species in the Fe(III)-doxorubicin system at pH 7.4 is the non-coordinated, singly charged ligand (H3dox+), in contrast to the Fe(III)-vosaroxin systemwhere [Fe(vox)3] is the predominant species. This reflects the differences in both stabilityof the metal-ligand and metal-protonated ligand complexes, and also the ionization stateof the ligands at physiological pH. For vosaroxin, the [Fe(vox)3] complex is the single,173Figure 5.4: Representative fit at 400 nm using obtained stability constants for asolution of Fe(III)-vosaroxin in 1:3 molar ratio: ◦ experimental data, fittedabsorbance.dominant species from pH 6.5 onwards into the basic pH range; however, the interactionbetween Fe3+ and doxorubicin appears to be more complex as various species of ironcoordinated and protonated doxorubicin are observed in the distribution. [Fe(vox)3] onlyslowly starts to form at pH 8 and higher, while at physiological pH free ligand in variousprotonation states (H2dox, H3dox+) exist next to the hydroxide adduct [Fe(dox)(OH)].The latter is a minor species (≤30%) between pH 6.5−9. In their study of the stability andiron coordination in DNA adducts of anthracycline based anticancer drugs, Eriksson andcoworkers519 found that the Fe3+ in the [dox-DNA]Fe3+ system was coordinated to fourO-atoms belonging to the [dox-DNA] adduct, and that it was in addition coordinating fivewater molecules as well. They suggested that the lower number of O-atoms and the highernumber of H2O molecules bound to the Fe3+ were related to a lower binding energy of the174Figure 5.5: Interaction of the Fe3+ ion (lavender) with vosaroxin, showing the moststable diketone-coordinated conformation obtained following energy minimiza-tion using B3LYP/6-31+g(d,p).metal ion possibly resulting in an increased production of hydroxyl radicals in vivo. Thissuggests that the [Fe(vox)3] species is potentially more thermodynamically stable, becausethe central Fe3+ is coordinated to a total of six O-atoms of the three vox-ligands, versus the[Fe(dox)(OH)] species, in which the metal is only coordinated to two ligand O atoms anda hydroxide. The coordination number of iron(III) is six. In the [Fe(vox)3] complex, all sixiron coordination positions are occupied leaving no access for further hydroxyl coordinationto the unoccupied iron orbitals.5.3.2 Synthesis & Characterization of Tris(vosaroxino)iron(III) and-gallium(III) ComplexesAqueous solutions of vosaroxin were mixed with aqueous solutions of iron(III) nitrate non-ahydrate, and gallium(III) nitrate nonahydrate respectively, in a 3:1 ratio (Figure 5.8).175Figure 5.6: Species distribution curves for the iron(III)-doxorubicin system.[Fe3+]T = 3.3·10−4 M, [L]T = 1·10−3 M.Figure 5.7: Species distribution curves for the iron(III)-vosaroxin system.[Fe3+]T = 3.3·10−4 M, [L]T = 1·10−3 M.176The coordination of vosaroxin was favoured over the formation of metal-hydroxide speciesaround pH 5, as indicated by the lack of precipitation due to hydroxide formation, andby MS-samples taken out of the reaction solution with a peak for [ML2]+ (100%). Al-lowing the reaction solution to stand at ambient temperature, or in the fridge at 4◦C,for several days did not promote precipitation, and the MS signal continued to show thatcharacteristic [ML2]+ peak (100%). Upon removal of the solvent in vacuo, and thoroughwashing of the obtained solid with small amounts of water and methanol, the respectivemetal-vosaroxin complexes were isolated and characterized by HR-ESI mass spectrometry.The data were consistent with the formation of tris(vosaroxino)metal(III). The compoundsare nonvolatile and stable, decomposing at approximately 200◦C. The solubility of theobtained [M(vox)3] (M= Fe3+, Ga3+) complexes is generally low and shows a high pHdependence; therefore, solution characterization of these complexes by MS was carried outwith solutions in methanol, acetonitrile and nitromethane. Structure analysis by 1H and13C NMR spectroscopy was only possible in d6-DMSO due to the extremely low solubil-ity of both complexes in D2O and other standard NMR-solvents. This low solubility, inaddition to the various stereoisomers, negatively impacted both elemental analyses andattempts to grow single crystals suitable for X-ray analysis of either complex. It has beenreported previously that it is challenging to grow single-crystals of quinolone-metal com-plexes.328 Over the duration of eighteen months, a multitude of attempts were undertakento grow single crystals according to various crystallization methods and personal tricks offellow researchers in the department. Diffusion methods in varying volumes, concentrationand glass ware set-ups were used, employing mainly acetonitrile, chloroform, diethyl ether,DMSO, methanol and water, according to solubility. Respective crystallization vials wereplaced in the freezer, the fridge, on the window sill and in a dark cupboard at room temper-ature, but crystals suitable for single x-ray diffraction did not grow. In addition, reactive177N NN SOOOHNH OHHN +     M(NO3)3  9 H2ONaOH(aq)H2O / CH3OH NNNSOOONHOHNHM33Figure 5.8: Synthetic route to tris(vosaroxino)metal(III) complexes [M(vox)3], M =Fe3+, Ga3+.crystallization experiments were conducted, in which the starting materials were dissolvedin different solvents over a range of pH conditions and layered on top of each other in onevial according to the density of the solvent. Unfortunately, the diffusion reaction did notlead to crystals but instead to an amorphous powder in some cases (systems of DMSO,methanol, water), or to no reaction at all.The mass spectra, however, were diagnostic of the complex formulations at a 3:1 ra-tio of vosaroxin:metal. With both metal ions (Fe3+, Ga3+), loss of one ligand from a[ML3] unit was observed giving the [ML2]+ fragment with 100% intensity. In addition, thetris(vosaroxino)iron(III) complex in methanol cationized in the high-resolution ES+ by at-tachment of one sodium or potassium cation to form [NaML3]+ or [KML3]+ as the parentpeak. [Ga(vox)3] was dissolved in low concentrations in various solvents (methanol, ace-tonitrile, nitromethane, DMSO) for further characterization with low- and high-resolutionES+ techniques. The spectra clearly reflected the effect of the different solvents on themass pattern. In addition to the [NaML3]+ parent peak, recombination signals correspond-ing to [M2L5]+ were observed for [Ga(vox)3] in nitromethane, which we have previouslyreported as characteristic for tris(ligand)metal(III) complexes.413Spectroscopic analysis in the mid-infrared region (4000−650 cm−1) supported the com-plete coordination of the respective metal through the carboxylate-O-atom. Although the178IR spectra of the quinolones are in general quite complex because of the numerous func-tional groups in the molecule, the stretching frequencies of the carbonyl and carboxylgroup are strong and can be identified as prominent absorption bands among the manyand varied Caryl−H and C−N vibrations in the same IR region.327 328 The IR spectrumof the free ligand showed a strong characteristic band at 1728 cm−1 attributed to thestretching frequency of the carboxyl-OH-group in Car3-position on the aromatic ring sys-tem; upon coordination of Fe3+ or Ga3+ it disappeared completely, as the IR-spectra ofthe respective metal-vosaroxin complexes show (Figure 5.9). In the IR spectra of bothvosaroxin-metal complexes, two distinct bands in the range 1620−1315 cm−1 could beassigned to the νCO2 asymmetric and symmetric stretching vibrations. The difference∆[cm−1] = νasym(CO2) − νsym(CO2) is quite large with ∆= 304 cm−1 for [Fe(vox)3], and∆= 297 cm−1 for [Ga(vox)3], likely characteristic for a monodentate coordination mode ofthe carboxyl group.318 4181H NMR spectra of vosaroxin were recorded in D2O as well as in d6-DMSO, showinga negligible solvent effect. Vosaroxin can form four different stereoisomers around themetal(III) center upon coordination of three bidentate anions in an octahedral fashion; thefour possible stereoisomers of [M3+(vox)3] are: ∆−fac; Λ−fac; ∆−mer; Λ−mer. In thecase of the diamagnetic [Ga(vox)3], the different stereoisomers gave a multitude of signals ina 1H NMR spectrum recorded at 298 K, but at 393 K the interchange happened so rapidlyon the NMR time scale that a separation of signals occurred and a clear assignment waspossible (T dependent NMR study presented in Appendix B). In the case of [Fe(vox)3],the Fe3+ ion retains a paramagnetic high-spin state upon complexation. As a result,the NMR signals are broadened considerably and impossible to assign with certainty. Inan attempt to support the DFT calculations, which favoured coordination through thecarboxyl-substituent and the carbonyl-group on the aromatic ring system, and to rule179Figure 5.9: IR spectra of vosaroxin (Hvox, black, top), [Fe(vox)3] (red, middle) and[Ga(vox)3] (green, bottom); the spectrum of Hvox shows the peak at 1728 cm−1disappearing upon coordination to Fe3+ or Ga3+.180out experimentally that the metal was not also coordinated to vosaroxin via the nitrogenatoms at the substituent on Nar1 and Car6, complexes were characterized by 1H NMR afterincremental addition of AAS-standard iron(III) solution. Vosaroxin (c= 5·10−4 M, V=5 mL) was dissolved in deuterated phosphate buffer pH 7.20 (pD 7.0), because the ligandwas insoluble in deuterated phosphate buffer at pH 2.15. Small increments (V= 2 µL) ofAAS-standard iron(III) solution were added to the titration solution, which was then stirredrigorously for three minutes, before a sample of the solution (V= 0.5 mL) was transferredinto a NMR tube. The titration was monitored via 1H NMR at 600 MHz (data presentedin Appendix B). Unfortunately, upon addition of iron(III), all NMR signals broadenedsignificantly; therefore, it was impossible to detect a measurable increased broadening inthe aromatic region, which would have supported coordination through the carboxyl-groupon Car3 and the carbonyl-group on Car4, over the aliphatic region. This would indicatecoordination via the substituent ring systems on Nar1 and Car6. The experiment wasfurther complicated by the precipitation of a dark red solid, (presumably [Fe(vox)3]) fromthe solution, although the chemical identity of the precipitate could not be determined toour full satisfaction.5.3.3 Cyclic Voltammetry StudiesTo evaluate redox/decomplexation of [Fe(vox)3] and [Ga(vox)3], the complexes were studiedvia cyclic voltammetry (CV curves of Hvox and [Ga(vox)3] are presented in Appendix B).For [Fe(vox)3] (Figure 5.10), the Fe(III)/Fe(II) couple at 0.771 V vs. NHE371 could not beclearly identified in the recorded cyclic voltammogram. The cyclic voltammogram, whileirreversible, was found to be reproducible over multiple cycles without a large decreaseof intensities of either peak. For the iron(III) as well as for the gallium(III) complex ofvosaroxin, a dissimilar peak shape was observed, which indicates a reorganization in the co-ordination sphere, and therewith the coordination symmetry, around the metal center upon181Figure 5.10: Cyclic voltammograms, 0.001 M [Fe(vox)3] in DMSO (red), 0.1 Mtetra(n-butyl)ammonium perchlorate, scan rate 100 mV·s−1 (background,grey).reduction of the metal, as had been previously observed for tris(ciprofloxacino)iron(III).4015.4 ConclusionThe Fe(III)-binding constant of vosaroxin, an anticancer quinolone derivative, has beendetermined spectrophotometrically and compared with the analogous iron(III) complexformed with doxorubicin, an anticancer agent widely used in the clinic. These spectropho-tometric titrations in 0.15 M NaCl, at ambient temperature, in the pH range from pH 2−12,showed that the two anticancer agents doxorubicin and vosaroxin bind Fe3+ with similarstrength: Hdox (logβFeL3= 33.41, pM= 17.0) and Hvox (logβFeL3= 33.80(3), pM= 15.9).At physiological pH, however, [Fe(vox)3] is the predominant species in contrast to the mix-ture of protonated ligand species observed for the Fe3+:doxorubicin system. Here, H2dox(∼30%) and H3dox+ (∼40%), in addition to the minor (∼30%) [Fe(dox)(OH)] species, areobserved, indicating a more labile interaction between Fe3+ and doxorubicin than between182Fe3+ and vosaroxin at physiological pH. Furthermore, two novel vosaroxin-metal(III) com-plexes were successfully synthesized from iron(III) nitrate and gallium(III) nitrate at a1:3 ratio. In tris(vosaroxino)iron(III) as well as in tris(vosaroxino)gallium(III), the metalion is coordinated through the deprotonated carboxylate oxygen on the Car3-atom of thenaphthyridine ring system in a monodentate coordination mode leading to the formationof four stereoisomers. Their redox behavior was studied by CV, and the stereochemistry ofthe gallium(III) analog was further explored in temperature dependent 1H NMR studies.For the [Fe(vox)3] complex, the iron redox couple was observed in the recorded CV spec-trum. Both complexes were fully characterized. The obtained results are consistent withthe well-studied clinical and chemical interaction between iron preparations (ferrous glu-conate/sulfate, various multivitamin preparations) and quinolone-based drug molecules.520When co-administered, the ferrous iron is oxidized to its ferric form, which rapidly formsquite stable tris(quinolono)iron(III) complexes. In vivo, the quinolones are very likely sta-ble in the presence of iron521 in contrast to the anthracyclines whose interaction with ironpresumably leads to the formation of free radicals and lipoperoxidation.484 485 The stable[Fe(vox)3], dominant at physiological pH, seems unlikely to produce toxic metabolites andROS associated with the more labile interaction from doxorubicin and Fe3+. The datapresented here suggest that the molecular pharmacology of their interaction with iron(III)may be one possible differentiation in the safety profile of quinolones compared to anthra-cyclines in relation to cardiotoxicity.Please see Appendix B for: UV-Vis spectra of one titration run of vosaroxin to deter-mine the pKas of the test solution, of Hvox:Fe3+ in the ratio of 1:1, of Hvox:Fe3+ in theratio of 2:1, and of Hvox:Fe3+ in the ratio of 3:1; as well as UV-Vis spectra of one titra-tion run of doxorubicin to determine the pKas of the test solution, of Hdox:Fe3+ in theratio of 1:1, of Hdox:Fe3+ in the ratio of 2:1, and of Hdox:Fe3+ in the ratio of 3:1; cyclic183voltammetric curves for 0.001 M of vosaroxin and [Ga(vox)3] in DMSO solution containingtetra(n-butyl)ammonium perchlorate 0.1 M at a scan rate of 100 mV·s−1; 1H NMR spectraof the titration of Hvox with Fe3+ at pD 7 (deuterated phosphate buffer) at 298 K; as wellas 1H NMR spectra of the temperature dependence study conducted on [Ga(vox)3] in therange from 298 K to 363 K.184Chapter 6Conclusion & OutlookThe field of metallodrugs in medicinal inorganic chemistry has grown constantly during thepast 50 years; however, despite the tremendous advancement of a few metallodrugs, thediscipline is still less fully developed compared to the traditional medicinal chemistry areasof small organic or biological drug molecules. Twelve metals522 are essential for the humanbody. For these metals, the human body has developed a sophisticated and sensitive systemof pathways for their transport as different and diverse as the essential metals themselves;consequently, this diversity amounts to a core challenge for the systematic development ofmetallodrugs. In addition, other nonessential metals can be used for therapy as well. Ofcourse, many great discoveries in science have been made by accident, and the serendipitousdiscovery of the anticancer activity of platinum or the antiarthritis activity of gold or theantidiabetic activity of vanadium are good examples. It might be surprising to somereaders that many metallodrugs on the market today are being used in patients withouta thorough understanding of the active structure, behaviour in the biological environmentor indeed the exact molecular mechanisms of action; the beneficial therapeutic effect ofthese metallodrugs is the sole sanction of their continuing use in the clinic. As debated inChapter 1, the majority of approved metallodrugs are either quite old (e.g., Pepto-Bismol,185aurothioglucose) or are, despite their toxic side effects, still in use for the treatment ofa neglected disease occurring in a developing country (e.g., melarsoprol against humanAfrican sleeping sickness) for which advanced treatment options with less side effects havenot yet been developed.To exploit fully the potential of metallodrugs, it is absolutely essential to understandwhat happens to the coordination complex and its components, the metal and the lig-and(s), once the metal-ligand-complex enters the body. To what extent can the activemetabolite be defined for drugs that are essentially delivery vehicles for metal ions to un-dergo dissociation and ligand exchange once administered? What role does the design ofthe ligand itself play in this? Are the pharmacological and toxicological properties of novelmetallodrugs predictable based on an improved understanding of metal ion speciation invivo? In what way does the oxidation state of the metal influence this? How importantare the thermodynamic versus kinetic considerations for metallodrugs in the body? Whatis there still to learn from the biochemistry of essential metals and metal ion distributionin the human body? These are questions that were raised years ago18 and the answers areslow in coming. Funding from research councils across the world, some of which seem torecognize the tremendous therapeutic potential of metallodrugs, brings the field of medici-nal inorganic chemistry closer to these answers. The European Cooperation in Science andTechnology (COST) has been funding research actions in the area of medicinal inorganicchemistry and metallodrugs for many years. Action ”CM1105 Functional Metal Complexesthat Bind to Biomolecules”, currently a four year long project from 2012−2016, aims ata structure-targeted approach to develop and evaluate new metal-based compounds thatexert their function as therapeutic metallodrugs, as research tools, or as diagnostic metal-lodrugs by binding to biomolecules, and to understand their modes of action.523 The U.S.National Institutes of Health (NIH) program ”Metals in Medicine” pursued a similar aim.186This thesis has presented FDA and EMA approved diagnostic and therapeutic met-allodrugs together with biological challenges of metallodrug research and development aswell as potential strategies to overcome these in the Introduction. In Chapter 2, nine com-mercially available quinolone antimicrobials of different drug generations − ciprofloxacin,enoxacin, fleroxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,oxolinic acid, and pipemidic acid − were introduced. For the first time, comprehensivechemical characterization data comprising infrared spectroscopy, mass spectrometry, melt-ing point determination, nuclear magnetic resonance spectroscopy (1D and 2D NMR spec-tra were recorded of 1H, 13C, 19F nuclei as applicable), and elemental analyses (C, H, N)was recorded and summarized. Furthermore, the long-time question of their stability inmetal ion containing biological medium (Iso-Sensitest) was addressed in a UV-Vis moni-toring study over 24 hours. No signs of decomposition or degradation of the nine quinoloneagents were observed. The antimicrobial susceptibility of these nine quinolones was testedagainst five of the most causative pathogens (E. faecalis, S. aureus, E. coli, K. pneumonia,P. aeruginosa), and revealed various patterns of resistance.Chapter 3 and 4 focused on the development of novel antimicrobial agents based on acoordination chemistry approach with gallium(III), iron(III), and copper(II) ions. Iron isan essential nutrient for many microbes. According to the ”Trojan Horse Theory,” biolog-ical systems cannot distinguish between Fe3+ and Ga3+, which constitutes the antimicro-bial efficacy of the gallium(III) ion. Based on the quinolone agents introduced in Chap-ter 2, nine novel tris(quinolono)gallium(III) complexes and their corresponding iron(III)analogs were synthesized and fully characterized, because a synergistic effect between theantimicrobial potency of Ga3+ and the antimicrobial effect of three quinolone ligands com-bined in one coordination complex was anticipated. The antimicrobial efficacy of thesetris(quinolono)gallium(III) complexes was studied against E. faecalis and S. aureus (both187Gram-positive), as well as E. coli, K. pneumonia, and P. aeruginosa (all Gram-negative)in direct comparison to the tris(quinolono)iron(III) complexes and the corresponding freequinolone ligands at various concentrations. For the tris(quinolono)gallium(III) complexes,no synergistic or even only combinational antimicrobial effects between Ga3+ and thequinolone antimicrobial agents were observed.The antimicrobial properties of copper have been known to mankind since the ancienttimes. In a coordination chemistry approach to develop novel antimicrobial agents, theantimicrobial properties of ciprofloxacin and pipemidic acid, as well as the xylenyl-linkeddimers thereof, were combined with copper(II) following the ”Achilles Heel Theory” inChapter 4. The preparation and antimicrobial evaluation of bis(ciprofloxacino)copper(II)[Cu(cipro)2], bis(pipemido)copper(II) [Cu(pia)2], and the corresponding dimer complexes[Cu2(ciproXcipro)2] and [Cu2(piaXpia)2] were reported. Again, no combinational antimi-crobial effect between Cu2+ and the respective quinolone ligands was observed.A pressing problem in the pharmacology of the anticancer agents doxorubicin andvosaroxin was tackled in Chapter 5 in a coordination chemistry approach. Although an-thracycline drugs, such as doxorubicin, are widely used in cancer therapy, the cumulative-dose cardiomyopathy associated with these anticancer agents remains a challenge duringoncological treatment. Through iron-mediated interactions, doxorubicin causes the forma-tion of ROS in vivo, which leads to oxidative stress affecting the heart. Vosaroxin is afirst-class anticancer quinolone derivative that is currently in clinical trials. The Fe(III)-binding constant of vosaroxin was determined spectrophotometrically and compared withthe analogous Fe(III) complex formed with doxorubicin. The in vivo metabolic stabilityand iron coordination properties of the quinolones compared to the anthracylines could pro-vide significant benefit to cardiovascular safety. Both doxorubicin (Hdox, logβFeL3= 33.41,pM= 17.0) and vosaroxin (Hvox, logβFeL3= 33.80(3), pM= 15.9) bind iron(III) with com-188parable strength; at physiological pH however, [Fe(vox)3] is the predominant species incontrast to a mixture of species observed for the Fe:dox system. Iron(III) nitrate and gal-lium(III) nitrate at a 1:3 ratio with vosaroxin formed stable tris(vosaroxino)iron(III) andtris(vosaroxino)gallium(III) complexes that were isolated and characterized. Their redoxbehavior was studied by CV, and their stereochemistry was further explored in tempera-ture dependent 1H NMR studies. The molecular pharmacology of their interaction withiron(III) could be one possible differentiation in the safety profile of quinolones comparedto anthracyclines in relation to cardiotoxicity.All of the discussed challenges so far have been scientific; however, another criticalaspect of metallodrugs is in perception. Although metallodrugs have been used for manyyears successfully in medical therapy, and self-medication with metal-containing dietarysupplements is widely accepted, it seems that there is still a lack of public acceptancefor the use of metal ions in the clinic. One of the greatest commonly espoused counter-arguments for metallodrugs is the ”toxicity associated with metals”. The general publichas only a basic understanding of chemistry and may know metals only from jewelry orhave read in the press about the harm of metals, such as aluminium(III) salts in antiper-spirants might be linked to Alzheimer Disease. The public often judges chemistry in anegative way. Chemists of all backgrounds must acknowledge and overcome this. Oneimportant aspect is to communicate to the public that, whatever we put into our bodies,the dosage determines if it harms or benefits us, or if it has any effect at all. This ideagoes back to the Middle Ages in Europe, when Paracelsus first described the concept ofdose-dependency of medical potions in his Defensiones.524 In the 20th century, Bertrandfollowed up on this concept with his work on the connection between the pharmaceuticaldose and the beneficial therapeutic effect or detrimental toxic effect.525 Figure 6.1 is a novelpresentation of the Bertrand diagram including the time component next to the dosage and189Figure 6.1: Effect of metal ion intake on overall health. Concentration of metalions in the body, represented on the y-axis, varies widely for different metalions. Following the traffic light principle, the optimal provision with metal ionsaccording to the guidelines of the medical community is shaded in green, whiledeficiencies or overload of metal ions can be harmful (yellow) to lethal (red).Another important factor in the dose-response scenario is the time duringwhich the body is exposed to conditions of metal ion deficiency or overload,shown on the x-axis.its effects. In dose-dependence, the beneficial versus the detrimental effect applies equallyto essential and non-essential metal ions. For the field of medicinal inorganic chemistryand metallodrugs to expand further, the medicinal inorganic chemistry community mustaddress public misapprehension.If the scientific community succeeds to communicate the benefit of metallodrugs tothe public, in addition to answering the questions raised above and gaining an increasedunderstanding of the metal homeostasis in the body, the chances that the public andthereby as well ”Big Pharma” will become more receptive to medicinal inorganic chemistryapproaches will improve. The revenue from such successful metallodrugs as imaging agents,190anticancer drugs, and metal supplements ought to be a persuasive argument to invest in thisinterdisciplinary area of medicinal chemistry. Particularly, metal coordination compoundsin therapy open an array of possibilities, which traditional organic or biological moleculescannot fulfill any longer due to growing drug resistance. 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Pathogen Safety Data Sheets: (accessed August06, 2014).[532] UBC Laboratory Biological Safety Course Manual; (accessedFebruary 01, 2015).223Appendix AProcedure: Antimicrobial SusceptibilityTesting By Single-Disk Method224A.1 ScopeTo test the antimicrobial activity of coordination complexes of gallium(III), iron(III) andcopper(II) with (fluoro-)quinolones, quinolone-dimers and maltol, and to compare theirbactericidal activity directly against commercially available (fluoro-)quinolone drugs.A.2 IntroductionSince the first antimicrobials have been developed, their potency to inhibit the growthof bacteria has been evaluated. The specific susceptibility of an antimicrobial agent to amicroorganism was originally tested using broth dilution methods. Because such brothdilution tests are time consuming to perform and pipetting errors occur easily in thistype of experiment, the disk diffusion procedure for the determination of susceptibility ofbacteria to antimicrobials was developed as a quick test alternative for the clinic.526 Bythe early 1950s, the majority of U.S. clinical microbiological laboratories had assumed theso-called disk diffusion method, and exactly therein lay the problem. As soon as differentlaboratories started using the disk diffusion method, they as well started adapting themethod to their own needs, using different test media, inoculum concentrations, incubationtimes, or incubation temperatures. In addition, many academic researchers publishedtheir own variations for the disk test procedure. It does not come as a surprise that thesedevelopments resulted in multiple protocols, most of them public, and therewith widespreadconfusion.The lack of standardization for the determination of bacterial susceptibility continuedto be a problem throughout the 1950s, until William M. M. Kirby and Alfred W. Bauerextensively reviewed the susceptibility testing literature, consolidated and updated all theprevious descriptions of the disk diffusion method.331 332 Around the same time, the WorldHealth Organization stressed the importance for the ”Standardization of Methods for Con-225ducting Microbic Sensitivity Tests” in regard to reliably measure and compare bacterialresistance to antimicrobials across the world.527 The report527 concluded that the methodin which single filter paper discs are placed on the surface of an inoculated culture mediumon a plate is suitable and recommended for general clinical use. Based on the WHO recom-mendation, standardized procedures for single antimicrobial disk testing, often referred toas ”Kirby-Bauer Single-Disk Diffusion Test”, were developed across the world on the na-tional level. In North America, the Clinical and Laboratory Standards Institute (CLSI) isresponsible for updating and modifying the original procedure of Kirby and Bauer througha global consensus process.432 528 In Europe, the European Committee on AntimicrobialSusceptibility Testing (EUCAST) has developed a common method calibrated to EuropeanMIC breakpoints over the past five years and with that harmonized an array of nationalprocedures across the European Union in 2014.306This procedure describes the antimicrobial susceptibility testing by single-disk methodfollowing a modified version of the original procedure by Kirby and Bauer331 332 takinginto account recommendations from the CLSI432 528 and EUCAST306 test procedures. Onemajor deviation from the CLSI and EUCAST test procedures is the use of Iso-Sensitestmedium instead of Mueller-Hinton medium. Please see Chapter 2 for an overview of thearguments used in the debate of Iso-Sensitest vs. Mueller-Hinton medium. Furthermore,several reports of potential metalloantimicrobials being tested in Iso-Sensitest mediumwithout any comments regarding cross-metalation can be found in the literature, includingcoordination complexes of gallium(III),388 529 iron(III),429 and copper(II).427 428It should be clarified that both biological growth media, Iso-Sensitest329 and Mueller-Hinton530, contain relatively large amounts of metal salts compared to the concentrations oftest compounds (0.1 mM), after all it is a nutrient rich medium to grow cultures. The fact,however, that Mueller-Hinton medium is less defined than the purely synthetic Iso-Sensitest226medium (media recipes are described in Tables A.1 and A.2) and that the compositionof Mueller-Hinton, especially in regard to cations, such as Ca2+ and Mg2+, has beenknown to vary widely across manufacturers and even across different batches from the samemanufacturer,303 made Iso-Sensitest medium occur as the better choice of the necessaryevil, as at least the ingredients of Iso-Sensitest medium are clearly defined.Table A.1: Synthetic formula of Iso-Sensitest agar (Oxoid, pH 7.4±0.2, 25◦C)329ingredients nutrient class amount [g/L]agar agar 8.0glucose sugar 2.0starch sugar 1.0casein (hydrolized) protein 11.0peptones peptides, amino acids 3.0cystein amino acid 0.02tryptophan amino acid 0.02adenine nucleotide 0.01guanine nucleotide 0.01uracil nucleotide 0.01xanthine nucleotide 0.01sodium chloride metal salt 3disodium hydrogen phosphate metal salt 2.0sodium acetate metal salt 1.0magnesium glycerophosphate metal salt 0.2calcium gluconate metal salt 0.1maganese(II) chloride metal salt 0.002cobalt(II) sulfate metal salt 0.001copper(II) sulfate metal salt 0.001ferrous sulfate metal salt 0.001zinc sulfate metal salt 0.001cyanocobalamine vitamin 0.001menadione vitamin 0.001nicotinamide vitamin 0.003panthothenate vitamin 0.003pyridoxin vitamin 0.003biotin vitamin 0.0003thiamine vitamin 0.00004227Table A.2: Approximate formula of Mueller-Hinton II (cation-adjusted) agar(BBL)530ingredients nutrient class amount [g/L]agar agar 17.0starch sugar 1.5casein (hydrolized) protein 17.5beef extract various 2.0The microorganisms selected for this study are summarized in Table A.3.531 Selectioncriteria were to include (a) a mix of Gram-positive and Gram-negative bacteria, (b) onlymicrobes that are pathogenic for humans, (c) only such pathogens that are commonlyassociated with hospital-acquired infections, so-called nosocomial diseases. Please refer tosection A.4 for the safe handling of the selected pathogens.Table A.3: Selected pathogenic bacteriabacterium strain nosocomialGram-positiveEnterococcus faecalis ATCC-51575 YesStaphylococcus aureus MSSA-476, ATTC-BAA-1721 YesGram-negativeEscherichia coli ATCC-25922 YesKlebsiella pneumonia ATCC-13883 YesPseudomonas aeruginosa ATCC-27853 YesA.3 EquipmentA.3.1 Purchased ItemsPurchased from manufacturer:• Mueller-Hinton II (Cation-Adjusted) Agar, BD BBL, product# 211441• Mueller-Hinton Broth, BD Difco, product# 272530228• Iso-Sensitest Agar, Fisher Scientific-Thermo Scientific Oxoid, product#OXCM0471B• Iso-Sensitest Broth, Fisher Scientific-Thermo Scientific Oxoid, product#OXCM0473B• Filter disks (1/4 inch diameter), Schleicher & Schu¨ll, product# 10328171Purchased from UBC-Chem Stores:• Disposable petri dishes (large 150 x 15 mm), UBC-Chem stores product# GL281005(25 dishes per sleeve)• Disposable petri dishes (small 60 x 15 mm), UBC-Chem stores product# GL28137F(25 dishes per sleeve)• Capped tubes (V = 1.5 mL), UBC stores product# EQ95515M• Falcon tubes (V = 15 mL), UBC stores product# GL95397R• Forceps, UBC stores product# EQ067761• Serological pipets (V = 10 mL), sterile, UBC-Chem stores product# GL68551J• Micro-Pipet filtertips, 20−200 and 100−1000 µL, UBC-Chem stores prod-uct#EQ67897Y and EQ67767U• Parafilm, UBC stores product# EQ072201Purchased from UBC-Biological Services (Jessie):• Inoculation loops (disposable, pack of 25), sterile packaged, UBC-Biological Servicesproduct229• Swabs (disposable), sterile packaged, UBC-Biological Services product• Syringe (V = 3 mL), sterile packaged, UBC-Biological Services product• Syringe filter (0.22 µm) -GP-, sterile packaged, UBC-Biological Services product• UV-cuvettes, UBC-Biological Services productObtained from various sources:• Black folder cover as photo background• Tooth picks (grocery store)• RulerA.3.2 Instruments• Autoclave• Balance• Biological safety cabinet (II)• Heatgun• Incubator (37◦C, non-CO2)• Shaker• UV-Vis spectrometer230A.4 SafetyA.4.1 Personal SafetyA laboratory coat with both hand openings taped tightly should be worn at all times.532All infectious material should be handled wearing nitrile gloves,532 wearing two pairs ofgloves is recommended when handling bacteria. The use of safety-googles is recommendedto avoid splashes into the eye even when working inside the biological safety cabinet.A.4.2 BiosafetyTable A.4: List of Gram-positive and Gram-negative bacteria testedorganism strain biosafety levelGram-positiveEnterococcus faecalis ATCC-51575 IIStaphylococcus aureus MSSA-476, ATTC-BAA-1721 IIGram-negativeEscherichia coli ATCC-25922 IKlebsiella pneumonia ATCC-13883 IIPseudomonas aeruginosa ATCC-27853 IIA.4.3 Important Pathogen Safety Information by the Public HealthAgency of CanadaFor detailed information, confer the Public Health Agency of Canada’s pathogen safetydata sheets.531A.4.3.1 Bacterium Enterococcus faecalisCharacteristics: streptococci, facultatively anaerobic, arranged in pairs and short chains.Host: normal human flora (intestinal tract, female genital tract, oral cavity), humans, pets,livestock. Pathogenicity: urinary tract, wound and soft tissue infection, bacteremia.231Susceptibility to disinfectants: susceptible to ethyl alcohol (70%). Physical inacti-vation: heat treatment >80◦C. Autoclave at standard solid program.A.4.3.2 Bacterium Escherichia coliCharacteristics: rod-shaped, strain ATCC 25922 is a recommended reference strain forantibiotic susceptibility testing. Host: humans, animals, lifestock. Pathogenicity: foodpoisoning, wound infection. Susceptibility to disinfectants: susceptible to ethyl alcohol(70%, 20◦C, 30 seconds contact time). Physical inactivation: Heat treatment >80◦C.Autoclave at standard solid program.A.4.3.3 Bacterium Klebsiella pneumoniaCharacteristics: rod-shaped Host: humans, animals, plants (flora). Pathogenicity:pneumonia, septicaemia, urinary tract infection, wound infection, intensive care unit in-fections, neonatal septicaemias. Susceptibility to disinfectants: susceptible to ethylalcohol (70%). Physical inactivation: heat treatment, autoclave at standard solid pro-gram.A.4.3.4 Bacterium Pseudomona aeruginosaCharacteristics: pseudomonadaceae, non-spore forming, pigmented. Host: humans,wild and domestic animals, plants (flora, fungi). Pathogenicity: infection of respiratoryand urinary tract, deep disseminated infections leading to pneumonia and bacteremia, eyeinfections; increasingly associated with bacterial meningitis, abscesses, endocarditis. Sus-ceptibility to disinfectants: susceptible to ethyl alcohol (70%); few reports of this bac-teria growing in disinfectant solutions, alcohol-containing disinfectants recommended forresistant strains. Physical inactivation: inactivated by moist heat (121◦C for >15 min),autoclave at standard solid program.232A.4.3.5 Bacterium Staphylococcus aureusCharacteristics: cocci, usually in clusters. Host: humans, wild and domestic animals.Pathogenicity: normal human flora (nose, skin), food intoxication, localized surface in-fections (from animal bites, impetigo, folliculitis, abscesses, boils, infected lacerations),deep infections include endocarditis, meningitis, septic arthritis, pneumonia, osteomyelitis.Susceptibility to disinfectants: susceptible to ethyl alcohol (70%). Physical inac-tivation: inactivated by moist heat (121◦C for >15 min), autoclave at standard solidprogram.A.5 Test ProtocolA.5.1 Marking of Petri DishesNo more than fourteen disks should be placed on a 150 mm diameter plate. The distancebetween each disk (center to center) should be at least 24 mm. All petri-dishes are to bemarked on the bottom according to the respective template shown in Figure A.1.A.5.2 Preparation of Agar PlatesThe dehydrated culture medium is prepared according to the manufacturer’s specifications.A volume of 1 L agar medium fills about twenty-two petri-dishes (100 x 15 mm) to a uniformdepth of 4 mm. After sterilization by autoclaving the medium is poured immediately intothe dishes to an approximate height of 4 mm on a level surface.i (Careful: The medium isvery hot, wear proper gloves!) The agar cools and solidifies in 30 minutes. A lid is placedon each dish and all dishes are stored in the original storage bag, which is closed tightlywith a rubber band or clip; single plates can be wrapped with parafilm for storage. Theplates are stored at ambient temperature and should be used within 14 days of preparation.iAgar deeper than 4 mm may cause false resistance results (excessively small zones), while agar lessthan 4 mm deep may be associated with excessively large zones and false susceptibility.233Figure A.1: Template for plate marking (numerical values in [cm]).234A.5.3 Preparation of Broth StorageThe dehydrated culture medium is prepared according to the manufacturer’s directions.After sterilization by autoclaving (liquid program), the medium is left to cool and trans-ferred into a storage bottle with a tightly closed cap that is wrapped in parafilm. Aftereach opening, the broth storage bottle must be autoclaved again.A.5.4 Transferring of Bacteria CultureThe bacteria cultures are kept growing on an agar plate for a maximum of seven daysbefore a few single colonies are transferred onto a fresh agar plate. To ensure a smoothwork flow, a list of items needed for this process has been compiled in Table A.5. Withan inoculation loop or a swab, bacteria samples from the old petri dish are collected andstreaked onto a fresh agar plate. The freshly inoculated plates are incubated at 37◦C for24 h, after which growth can be observed (Figure A.2). The growth culture plates are thenkept at room temperature. In addition to keeping the bacteria culture alive by growingthem on agar plates, it is as well advised to keep each culture growing in broth as a back-upas well: 5 mL fresh broth + 1 mL bacteria growth broth culture.Table A.5: List of items needed for the transfer of bacterial culturesitem checkmarkcultures on agar platesfresh agar platesinoculation loopsparafilm (stripes cut for wrapping)brothFalcon tubes (for grow up)serological pipets (to transfer broth into Falcon tubes)235Figure A.2: Bacteria culture plates.A.5.5 Preparations for the Actual Test Day (Day 1)On the day before the test, the items listed in Table A.6 need to be autoclaved. In addition,the agar plates should be counted and checked for first signs of contamination to ensure asufficient supply of intact plates for the following day.A.5.6 Growing Bacteria in Broth (Day 1)Four to five bacteria colonies are picked from the growth culture plates and inoculated into5−10 mL of broth in a Falcon tube. The inoculated broth tube is placed on a shaker at37◦C for about 16−24 hours prior to inoculation.236Table A.6: Items to incubate on day 1item checkmarkforceps (1)tin foil (several pieces about 15 x 10 cm in size)filter disks in glas bottle (unloaded)PCR tubes (1.5 mL)PCR tube rack (1)tips (1000 µL, 1 box)tips (200 µL, 2 boxes)tooth picks in glass bottleA.5.7 Setting up the Biosafety Cabinet (Day 2)To ensure a smooth work flow, a list of items needed on the test day has been compiled inTable A.7.A.5.8 Preparation and Standardization of Inoculum Suspension (Day2)The bacteria concentration in the growth broth is measured by UV (optical density, OD).(Reminder: The UV-lamp needs to warm up 15 minutes before use.) One single use cuvetteis filled with broth solution as the blank. Another single use cuvette is filled with thebacteria growth broth and its opening is tightly closed with parafilm. The UV reading istaken at 600 nm wave length. OD readings between 0.7−1.5 are acceptable. If the OD islower, the culture needs to grow longer; if the OD is higher, the culture needs to be dilutedwith fresh broth solution, and the UV-reading needs to be taken again until the OD is inthe acceptable range.A.5.9 Preparation of Test Solutions (Day 2)The test solutions are prepared in organic solvents that evaporate easily. One of thepreferred solvents is methanol, but often dimethyl sulfoxide has to be added to overcome237Table A.7: Items needed in biosafety cabinet on day 2item checkmarkpreviously incubated items:forcepstin foil piecesfilter disks in glas bottle (unloaded)PCR tubes and rackmicro-pipet tips (1000 µL)micro-pipet tips (200 µL)solvents:dimethyl sulfoxidemethanolsingle use items:agar platesswabs (sterile)Falcon tubes for preparation of test solutionsserological pipetssyringe and syringe filter for DMSOUV-cuvettesitems to wipe with 70% ethyl alcohol:micro-pipet (1000 µL volume)micro-pipet (200 µL volume)pipet helperlightersolubility issues. Falcon tubes are used for the stock solutions, PCR tubes are used for alldilutions and the final test solution (c= 0.1 mM). Due to the toxicity of DMSO on livingorganisms, the concentration of DMSO in all test solutions should be ≤2%. To ensurethat the DMSO is not toxic for the test organisms and therewith introducing an error intothe test results, a methanol solution of 2% DMSO as well as a pure methanol solution areincluded into the test as controls.A.5.10 Inspection of Agar Plates (Day 2)Before usage, all agar plates have to be inspected for cracks, biological contamination,and other irregularities. Only plates without detected abnormalities are used for the test,238others are discarded.A.5.11 Loading of Filter Disks with Test Compound (Day 2)The empty disks are carefully spread out on sterile tin foil. Each disk is loaded with 20 µLof test compound. About 5 minutes after loading, the methanol has evaporated; the driedand loaded disks are then ready for placement on the agar.A.5.12 Inoculation of Plates (Day 2)At the time the agar is inoculated, no droplets of moisture should be visible on its surfaceor on the petri dish cover.Method A: A sterile cotton swab is dipped into the growth broth solution, rotatedseveral times, and gently pressed onto the inside wall of the Falcon tube above the fluidlevel to remove excessive inoculum from the swab. The swab is then streaked over theentire surface of the agar plate three times, while the plate is being rotated 60◦ each timeto ensure even distribution of the inoculum. A final sweep of the swab is made aroundthe agar rim. If necessary, the lid may be left ajar for 3 minutes to allow excess surfacemoisture to be absorbed before the impregnated disks are applied.Method B: About 0.5 mL of growth broth solution are pipetted onto a fresh agarplate. The liquid is evenly distributed over the plate with a spatula or a cotton swab.The advantage of this dilution method is the even growth pattern compared to the visiblestreaks that remain from the swabbing method, however, this method bears the risk ofcontamination with accidental splashes of the broth solution.A.5.13 Placement of Loaded Disks (Day 2)Within 10 minutes after the plates have been inoculated, the impregnated disks are placedonto the surface in the previously marked positions (Section A.5.1). Each impregnated disk239is positioned with iso-prop flamed forceps and pressed down firmly on the agar to ensurecomplete, level contact. Once a disc has touched the agar surface, it is not to be relocated.When all disks have been placed, the petri dish is closed with the lid and wrapped inparafilm.A.5.14 Incubation of Test Plates (Day 2)Within 20 minutes of disk placement, all plates are inverted and placed in the air incubatorwith the agar side up. Incubation conditions: 37◦C for 20 hours.A.5.15 Interpretation and Measurement of Zone Sizes (Day 3)Immediately following the incubation, the zone sizes are measured using a ruler. Allmeasurements are made with the unaided eye on the backside of the petri dish on a black,nonreflecting surface illuminated with reflected light. The plate is viewed directly in avertical line of sight to avoid any parallax. The zone margin is considered to be the areashowing no obvious, visible growth that can be detected with the unaided eye. The diameterof the disk is included in the measurement, and the measurement is rounded to the nearestmillimeter. All zone sizes are recorded on the zone size recording sheet (Figure A.4), anda photo of each plate is taken for documentation (Figure A.3). Further clarification:• Growth up to the edge of a disk is reported as a zone of 0 mm.• If the placement of disk does not allow a direct reading of the zone diameter, thedistance from the centre of the disk to a point on the circumference with a distinctedge is measured; this radius measurement is then multiplied by factor x2 to indirectlydetermine the diameter.• Distinct, discrete colonies within an obvious zone of inhibition should not be con-sidered swarming. Should the repeated testing show the same growth pattern, the240Figure A.3: Example photo for test plate documentation.organism must be considered resistant to the antimicrobial agent loaded on the disk.A.5.16 Reporting of Measured Zone SizesAll tests are done in triplicate, and the inhibition zone sizes of one test compound againstthe same bacteria are measured on three different plates. From these three independentmeasurements, the average and the respective standard deviation are calculated and re-ported in tabular format.241Figure A.4: Example of inhibition zone size recording sheet.242A.5.17 Waste ManagementAll waste from the Biosafety Cabinet is handled in closed autoclavable plastic waste bags.These are autoclaved for 121◦C at 25 minutes, which decontaminates all organisms handledin this procedure. The autoclaved box content is then double-packaged: first in an orangeUBC Biohazard RG2 bag, then once more in a clear bag. Each waste bag is labelled witha red biological waste ”UBC Autoclaved Risk Group II” tag.532243Appendix BSupplementary Information to Chapter 5244B.1 UV-Vis Titration of the Vosaroxin-Iron(III) SystemFigure B.1: UV-Vis spectra of one titration run of Hvox to determine the pKas ofthe test solution.245Figure B.2: UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 1:1.246Figure B.3: UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 2:1.247Figure B.4: UV-Vis spectra of one titration run of Hvox:Fe3+ in the ratio of 3:1.248B.2 UV-Vis Titration of the Doxorubicin-Iron(III) SystemFigure B.5: UV-Vis spectra of one titration run of Hdox to determine the pKas ofthe test solution.249Figure B.6: UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 1:1.250Figure B.7: UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 2:1.251Figure B.8: UV-Vis spectra of one titration run of Hdox:Fe3+ in the ratio of 3:1.252B.3 Cyclic Voltammograms of Hvox and [Ga(vox)3]Figure B.9: Cyclic voltammogram of vosaroxin (0.001 M, solid line) in DMSOsolution; also shown is the blank voltammogram containing tetra(n-butyl)ammonium perchlorate 0.1 M (dotted line). Scan rate was 100 mV/s.Potential values are given with reference electrode Ag/AgCl(sat) and againstthe ferrocene couple Fc+/Fc = +0.64 V vs. SHE371.253Figure B.10: Cyclic voltammogram of [Ga(vox)3] (0.001 M, green) in DMSOsolution; also shown is the blank voltammogram containing tetra(n-butyl)ammonium perchlorate 0.1 M (dotted line). Scan rate was 100 mV/s.Potential values are given with reference electrode Ag/AgCl(sat) and againstthe ferrocene couple Fc+/Fc = +0.64 V vs. SHE371.254B.4 NMR StudiesFigure B.11: Titration of vosaroxin (5·10−4 M) in deuterated phosphate buffer(5·10−2 M) at pD 7.0 with increasing amounts of iron(III) nitrate in D2Omonitored via 1H NMR (600 MHz, D2O, 298 K) with a total increase in vol-ume throughout the titration of 0.6%. The 1H NMR spectra with differentratios of Fe3+:Hvox are shown with the same intensities for better compari-son. From bottom to top: Hvox (dark blue), Fe3+:Hvox = 1:50 (light blue),Fe3+:Hvox = 1:25 (intense blue), Fe3+:Hvox = 1:12.5 (teal), Fe3+:Hvox =1:6.25 (purple), Fe3+:Hvox = 1:6.25 after 60 min wait time (grey), Fe3+:Hvox= 1:3.33 (black). The NMR signals broaden with increasing amounts of Fe3+.At a ratio of Fe3+:Hvox = 1:6.25, one NMR spectrum was recorded after thestandard time of 3 min (purple), and a second spectrum was recorded afterthe sample had been stirred for 60 min at ambient temperature upon which aprecipitate had formed (grey). The second spectra showed a clear narrowingof signals again, which indicated that the amount of Fe3+ ions in solutionwas reduced and supported the assumption that the Fe[(vox)3] complex, acomplex not soluble in aqueous media, formed over the course of the titra-tion. This assumption is further supported by the fact that the precipitatedid not have the characteristic orange color of insoluble Fe(OH)3(s).255ppm1 122334455667788991010298 K308 K320 K331 K341 K353 K363 Kcool down, 298 KFigure B.12: Temperature dependent NMR study (400 MHz, d6-DMSO) of[Ga(vox)3]. The sample was heated inside the spectrometer from ambienttemperature (298 K, bottom, dark blue) to 363 K (red). To conclude theexperiment, the sample was cooled down again to ambient temperature (top,black). As it could be expected, the heat accelerated the interchanging of thevarious stereoisomers in solution, which was reflected in more defined signalsin the NMR spectra recorded at higher temperature. This phenomenon wassolely temperature dependent and fully reversible, as a comparison of thefirst spectrum (bottom, dark blue) and the final spectrum (top, black) show.256


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