Anti-Cancer N-Heterocyclic Carbene Complexes of Gold(III), Gold(I) and Platinum(II) [recurso electrónico] Thiol "Switch-on" Fluorescent Probes, Thioredoxin Reductase Inhibitors and Endoplasmic Reticulum Targeting Agents

This thesis focuses on the development of gold- and non-classical platinum-based anti-cancer agents that display distinctively different anti-cancer mechanisms compared to the commonly used cisplatin. These metal complexes contain N-heterocyclic carbene (NHC) ligands which are able to form strong M-C(NHC) bonds, conferring high stability and favorable lipophilicity, reactivity and binding specificity of metal complexes on biomolecules. The author demonstrates significant advances made in anti-cancer gold(III), gold(I) and platinum(II) complexes. Detailed chemical synthesis, in vitro and/or in vivo anti-cancer activities are clearly presented including: (i) a class of Au(III) complexes containing a highly fluorescent N^N^N ligand and NHC ligand that simultaneously act as fluorescent thiol “switch-on” probes and anti-cancer agents; (ii) a dinuclear gold(I) complex with a mixed diphosphine and bis(NHC) ligand displaying favorable stability and showing significant inhibition of tumor growth in two independent mice models with no observable side effects; and (iii) a panel of stable luminescent cyclometalated platinum(II) complexes exhibiting high specificity to localize to the endoplasmic reticulum (ER) domain, inducing ER stress and cell apoptosis. These works highlight the clinical potential that gold and platinum complexes offer for cancer treatment. Parts of this thesis have been published in the following journal articles: 6 Supervisor's Foreword 7 Acknowledgments 9 Contents 11 1 Introduction 14 1.1 Inorganic Medicines 14 1.2 Lessons from Cisplatin and Its Derivatives 16 1.2.1 Overview of Platinum Drugs 16 1.2.2 Anticancer Mechanism of Cisplatin 17 1.2.3 Transplatin and Its Analogues 22 1.2.4 Cisplatin Resistance and Side Effect Problems 23 1.3 The Chemistry and Anticancer Properties of Gold 24 1.3.1 The Chemical Properties of Gold Complexes [32] 24 1.3.1.1 Gold(I) Complexes 24 1.3.1.2 Gold(III) Complexes 25 1.3.2 Gold Complexes Inhibit Thiol-Containing Enzyme Activities 26 1.3.2.1 Inhibition of Disulfide Reductase 27 1.3.2.2 Inhibition of Cysteine Protease 29 1.3.2.3 Inhibition of Other Thiol-Containing Enzymes 29 1.3.2.4 Inhibition via Au--His Binding 33 1.3.3 Current Status of Gold(I) Anticancer Agents 34 1.3.3.1 Gold(I)--Phosphine Complexes 34 1.3.3.2 Clinically Used Antiarthritis Drugs with Anticancer Properties 36 1.3.3.3 Gold(I)--NHC Complexes as Anticancer Agents 36 1.3.3.4 Gold(I)--Alkyne Complexes as Anticancer Agents 37 1.3.3.5 Gold(I)--Thiourea Complexes as Anticancer Agents 38 1.3.4 Current Status of Anticancer Gold(III) Complexes 39 1.3.4.1 Gold(III)--Porphyrin Complexes as Anticancer Agents 39 1.3.4.2 Cyclometalated Gold(III) Complexes Bearing Tridentate C-Deprotonated C^N^C Ligands as Anticancer Agents 40 1.3.4.3 Cyclometalated Gold(III) Complexes Bearing C-Deprotonated C^N and C^N^N Ligands as Anticancer Agents 41 1.3.4.4 Gold(III) Complexes Containing Bidentate N^N and Tridentate N^N^N Ligands as Anticancer Agents 43 1.3.4.5 Gold(III) Complexes Containing Dithiocarbamate Ligand as Anticancer Agents 43 1.4 The Chemistry and Anticancer Properties of Platinum 44 1.4.1 The Chemical Properties of Platinum Complexes 44 1.4.1.1 Pt(II) Complexes 44 1.4.1.2 Pt(IV) Complexes 45 1.4.2 Physiologically Stable Platinum(II) Complexes as Anticancer Agents 47 1.4.2.1 Platinum(II) Complexes Target Nucleic Acids 47 1.4.2.2 Platinum Complexes with Non-DNA Targets 52 1.5 The Chemistry of N-Heterocyclic Carbene Ligands 53 1.5.1 Electronic Properties of NHC Ligands 54 1.5.2 Steric Effects of NHC Ligands 54 1.5.3 Synthesis of NHC Ligands [125] 55 1.5.4 Synthesis of Metal--NHC Complexes 56 1.6 Luminescent Properties of Platinum(II) and Gold(I)/(III) Complexes 58 1.7 Fluorescent Thiol Probes 60 References 60 2 Experimental Section 68 2.1 Materials and Instrumentation 68 2.2 X-Ray Crystallography 68 2.2.1 Crystal Growth 68 2.2.2 Single Crystal Analysis 69 2.3 Stability Testing 70 2.3.1 UV--Vis Absorption Measurements 70 2.3.1.1 Beer-Lambert Law 70 2.3.1.2 Instrumentation 70 2.3.2 ESI-MS Measurements 70 2.3.3 1H NMR Measurements 71 2.4 Emission Measurements 71 2.4.1 Emission Spectra Measurements 71 2.4.2 Emission Lifetime Measurements 72 2.5 Fluorescence Microscopy 72 2.6 Cell Culture and Cytotoxicity Studies 73 2.6.1 Cell Subculture 73 2.6.2 MTT Assay 73 2.7 Gel Mobility Shift Assay 74 2.8 Spectroscopic Binding Studies 75 2.8.1 Determination of DNA-Binding Constants 75 2.8.2 Binding with Proteins 75 2.9 Transfection 75 2.10 Western Blot 76 2.11 Inductively Coupled Plasma Mass Spectrometry 78 2.12 Tube Formation Assay 78 2.13 In Vivo Antitumor Study 79 References 80 3 Gold(III) Complexes Containing N-Heterocyclic Carbene Ligand Serve as Dual Fluorescent Thiol ``Switch-On'' Probe and Anticancer Agent 81 3.1 Introduction 81 3.2 Experimental Section 82 3.2.1 Materials and Instrumentation 82 3.2.2 Synthesis and Characterization of Gold Complexes 83 3.2.2.1 Synthesis of Gold Complexes 83 3.2.2.2 X-Ray Crystallographic Analysis 89 3.2.3 Reactions with GSH 89 3.2.3.1 UV--Visible Spectroscopic Experiments 89 3.2.3.2 ESI-MS Experiments 89 3.2.3.3 1H NMR Experiments 90 3.2.4 Luminescent Properties of 3.9 90 3.2.4.1 In the Presence of Different Analysts 90 3.2.4.2 Fluorescence Microscopic Examination 90 3.2.5 Anticancer Properties 91 3.2.5.1 Cell Lines and Growth Inhibitory Assay 91 3.2.5.2 Cellular Activities of Thioredoxin Reductase (TrxR) 91 3.2.5.3 Lipophilicity Measurement 91 3.2.5.4 Cellular Uptake Experiments 92 3.2.5.5 Western Blot Analysis 92 3.2.5.6 In Vivo Tumor Growth Inhibition Experiments 92 3.3 Results and Discussion 93 3.3.1 Synthesis and Characterization 93 3.3.2 Reactions with GSH 95 3.3.3 Emission Properties of 3.9 Toward Thiols 99 3.3.4 Anticancer Properties 101 3.4 Conclusion 108 References 108 4 A Binuclear Gold(I) Complex with Mixed Bridging Diphosphine and Bis(N-Heterocyclic Carbene) Ligands Shows Favorable Thiol Reactivity and Effectively Inhibits Tumor Growth and Angiogenesis In Vivo 112 4.1 Introduction 112 4.2 Experimental Section 113 4.2.1 Materials and Instrumentation 113 4.2.2 Synthesis and Characterization of Complexes 114 4.2.2.1 Synthesis of Complexes 114 4.2.2.2 X-ray Crystallographic Analysis 116 4.2.3 Biological Application 117 4.2.3.1 Characterization of Reaction with Thiols 117 ESI-MS (Q-TOF) Experiment of Reaction of 4.1-PF6 with GSH 117 Interaction with Bovine Serum Albumin 117 ESI-MS (LTQ Orbitrap) Experiment of Reaction of 4.1-PF6 with Tetrapeptide GCUG 118 1H NMR Experiment of Reaction of 4.1-PF6 with GCUG 118 4.2.3.2 Anticancer Properties 118 Cell Lines and Growth Inhibitory Assay 118 Inhibition of Purified Thioredoxin Reductase (TrxR) 119 Inhibition of Purified Glutathione Reductase (GR) 120 TrxR Inhibition on Trx System 120 Sphere Formation Assay [53] 120 In Vivo Tumor Growth Inhibition Experiments 120 In Vivo Toxicology Studies 121 Determination of Blood Vessel Density in the Tumor Tissues 122 Tube Formation Assay 122 4.3 Results and Discussion 123 4.3.1 Synthesis and Characterization 123 4.3.2 Stability Toward Blood Thiols and In Vitro Cytotoxicity 123 4.3.3 Inhibition of Thioredoxin Reductase 127 4.3.4 Inhibition of Cancer Stem Cell Activity 130 4.3.5 In Vivo Antitumor Activities 132 4.3.6 Safety Pharmacology Study 138 4.4 Conclusion 142 References 142 5 Luminescent Organoplatinum(II) Complexes Containing Bis(N-Heterocyclic Carbene) Ligands Selectively Target Endoplasmic Reticulum and Induce Potent Phototoxicity 146 5.1 Introduction 146 5.2 Experimental Section 147 5.2.1 Materials and Methods 147 5.2.2 Experimental Procedure and Compound Characterization 147 5.2.3 Stability Toward Physiological Thiols 150 5.2.3.1 UV--Vis Absorption Experiment 150 5.2.3.2 1H NMR Experiment 151 5.2.4 Photo-physical Properties and Application in Protein Binding and Cell Imaging 151 5.2.4.1 Absorption and Emission 151 5.2.4.2 Protein Binding 151 5.2.4.3 Emission Responses Toward Different Analytes 151 5.2.4.4 Absorption-Titration Experiment 152 5.2.4.5 Fluorescence Quenching Experiment 152 5.2.4.6 Fluorescence Microscopic Examination 152 5.2.5 Anticancer Properties 153 5.2.5.1 Cell Lines and Growth Inhibitory Assay 153 5.2.5.2 Lipophilicity Determination 153 5.2.5.3 Cell Transfection and Fluorescence Microscopic Analysis 153 5.2.5.4 Western Blot Analysis 154 5.2.5.5 Mitochondrial Membrane Depolarization Assay 154 5.2.5.6 Determination of Phototoxicity 154 5.3 Results and Discussion 155 5.3.1 Synthesis of the Complexes 155 5.3.2 Stability Test 158 5.3.2.1 Photo-Physical Properties and Cell Imaging Studies 158 5.3.3 Anticancer Properties 164 5.3.4 Phototoxicities 169 5.4 Conclusion 170 References 172 6 Summary and Evaluation 174 Front Matter....Pages i-xiii Introduction....Pages 1-54 Experimental Section....Pages 55-67 Gold(III) Complexes Containing N-Heterocyclic Carbene Ligand Serve as Dual Fluorescent Thiol “Switch-On” Probe and Anticancer Agent....Pages 69-99 A Binuclear Gold(I) Complex with Mixed Bridging Diphosphine and Bis(N-Heterocyclic Carbene) Ligands Shows Favorable Thiol Reactivity and Effectively Inhibits Tumor Growth and Angiogenesis In Vivo....Pages 101-134 Luminescent Organoplatinum(II) Complexes Containing Bis(N-Heterocyclic Carbene) Ligands Selectively Target Endoplasmic Reticulum and Induce Potent Phototoxicity....Pages 135-162 Summary and Evaluation....Pages 163-164