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UBC Theses and Dissertations

Synthesis, characterization and anti-cancer activity of new ruthenium maltolato and imidazole complexes Kennedy, David Charles 2003

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SYNTHESIS, CHARACTERIZATION AND ANTI-CANCER ACTIVITY OF RUTHENIUM M A L T O L A T O AND IMIDAZOLE COMPLEXES by DAVID CHARLES KENNEDY B. Sc., The University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES (Department of Chemistry) e accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 2003 © 2003 David Kennedy Abstract Several new Ru(III) maltolato (ma) and ethylmaltolato (Ema) complexes have been synthesized, characterized, and tested for biological activity in vitro against M D A -MB-435S cells, a human breast cancer cell line. Ru(ma)3 (1) and Ru(Ema)3 (2) were studied in detail using a number of spectroscopic techniques. In particular, the solution ' H N M R spectra of these paramagnetic solids were investigated and, through the use of .o. NO, N \ N v O H r=\ O V 0H ,CFo NH C F , O Maltol (R = Me) (Hma) and Ethylmaltol (HEma) (R = Et) C H 3 Metronidazole N O ; EF5 2D N M R techniques, structural information about the complexes was determined. These two complexes were then used to synthesize bis-imidazole complexes via removal of one ma or Ema ligand with triflic acid. Trans-[Ru(ma)2(2MeIm)2]CF3S03 (10), -[Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8), and -[Ru(Ema)2(metro)2]CF3S03 (11), where 2MeIm = 2-methylimidazole, lMelm = 1-methylimidazole, and metro = metronidazole, were synthesized and characterized by X-ray crystallography, while trans-[Ru(ma)2(metro)2]CF3S03 (4) was also synthesized and subsequently characterized by X -ray crystallography by another member of our group. Comparison of the ' H N M R data of 8, 10, and 11 with those for the analogous complexes [Ru(ma) 2(4MeIm) 2]CF 3S03-CH 2Cl 2 (6), [Ru(Ema) 2(4MeIm) 2]CF 3S0 3 (12), and [Ru(Ema) 2(2MeIm) 2]CF 3S0 3 (13), where 4MeIm = 4-methylimidazole, implies that these last three complexes also have trans-configurations. By a similar comparison, [Ru(ma)2(Im)2]CF3S03 (7), where Im = imidazole, was found to be a mixture of cis- and trans-isomers that could not be separated. ii H H H 3 C' 1 0 (R = Me), 1 3 (R = Et) 6 (R = Me), 1 2 (R = Et) 8 The ma and Ema complexes were then tested in vitro using a so-called M T T assay against human breast cancer cells to evaluate their antiproliferatory activity. This assay determines cell viability as a measure of the cell's ability to reduce the yellow M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the purple formazan in the mitochondria. Complexes containing Ema exhibited lower IC50 values (complex concentration at which cell proliferation has been reduced by 50 % after 3 days), than the corresponding ma complexes, and 1 3 exhibited the lowest value of 500 n M , compared to that for cisplatin (IC50 = 40 pM) . Ru-uptake data showed that Ema complexes were taken into Chinese Hamster Ovarian (CHO) cells at levels 4-5 times greater than those for corresponding ma complexes. No significant difference in Ru-uptake was observed between ma complexes with different imidazole ligands when these lacked a nitro group. [Ru(ma)2(EF5) 2]CF 3S03-EtOH ( 1 4 ) , where EF5 = 2-(2-nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide, exhibited the highest Ru-uptake of the complexes tested, and was the only complex to exhibit DNA-binding in C H O cells over a 3h incubation period, although it exhibited very low activity in the M T T assay ( IC 5 0 value > 500 uM) . Day 1 - Plate cells Day 5 - Read plate Outline for the M T T assay iii Complexes containing the bidentate ligands 4,4'-biimidazole (biim) and 2,2'-dimethyl-4,4'-biimidazole (Me2biim) were also synthesized, characterized and tested by the M T T assay. The Ru(HT) complexes [Ru(ma)2(L)]CF3S03-2H20, where L = biim (24) or Me2biim (25), were synthesized from 1. The Ru(II) complexes [Ru(L)3][CF3S03]2 (L = biim (26) and L = Me2biim (27)) were synthesized from [Ru(DMF) 6][CF 3S0 3]3, while the complexes RuCl 2(DMSO) 2(L) (L = biim (20) and L = Me2biim (22)) and Ru 2Cl4(DMSO)4(L) (L = biim (21) and L = Me2biim (23)) were synthesized from cis-RuCl 2(DMSO) 4. 24 - 27 all exhibited significant activity in the M T T assay (IC 5 0 values 15-50 uM), but 20 and 22 were essentially inactive. The Ru-uptake values of 24 - 27 in CHO cells were comparable to those observed for the Ru-ma bis-imidazole complexes (4, 6, 7, 8 and 10). The new Ru complexes [Ru2(pic)4(EtOH)Cl]H20 (31) and Ru(Im-C02)(Im-C 0 2 H ) 2 C l 2 H 2 O E t O H (33), synthesized using the N-heterocyclic carboxylic acid ligands pyridine-2-carboxylic acid (Hpic) and imidazole-4-carboxylic acid (Im-C02H), were also tested using the M T T assay, but were considerably less active than either the biimidazole or bis-imidazole complexes (IC50 values = 100 and 400 uM for 31 and 33, respectively). iv Table of Contents Abstract i i Table of Contents v List of Figures x i i i List of Tables '. xix List of Symbols and Abbreviations xxi Key to Numbered Complexes ; xxiv Acknowledgements xxvi Chapter 1 Introduction 1 1.1 Introduction 1 1.2 Nitroimidazoles and Hypoxia 2 1.3 In Vitro Bioassays 4 1.4 Platinum Anti-Cancer Drugs 5 1.4.1 Cisplatin and Other Pt(II) Anti-Cancer Drugs 5 1.4.2 Platinum(IV) as a Pro-drug 7 1.5 Ruthenium Complexes and Their Potential as Anti-Cancer Drugs 8 1.5.1 Activation by Reduction 8 1.5.2 c w - R u C ] 2 ( D M S O ) 3 ( D M S O ) 9 1.5.3 Ru-imidazole Complexes 9 1.5.3.1 N A M I a n d N A M I - A ; 10 1.5.4 Ru-arene Complexes 11 1.5.5 Ru-azopyridine Complexes 13 1.5.6 Ru Intercalators 14 1.6 Maltolato (ma) Chemistry 15 1.6.1 Maltolato Complexes with Ru 15 1.6.2 Maltolato Complexes with other Transition Metals 16 1.7 Goals of this Thesis 17 1.8 References 18 v Chapter 2 General Experimental Procedures, and Syntheses of Ruthenium Precursor Complexes 23 2.1 Chemicals 23 2.1.1 Ligand Precursors 23 2.1.2 Solvents 23 2.1.3 Gases 24 2.1.4 Biological Reagents 24 2.1.5 Other reagents 24 2.2 Analytical Techniques and Instrumentation 24 2.2.1 Nuclear Magnetic Resonance Spectroscopy 24 2.2.2 Infrared Spectroscopy 26 2.2.3 Mass Spectrometry 27 2.2.4 UV-Visible Spectroscopy 27 2.2.5 Cyclic Voltammetry 27 2.2.6 Conductivity 28 2.2.7 X-Ray Analysis 28 2.2.8 Elemental Analysis 29 2.2.9 Biological Analysis 29 2.3 Chromatographic Techniques 29 2.4 Ruthenium Precursors 31 2.4.1 RuCl 3 -3H 2 0 31 2.4.2 [Ru(DMF)6][CF3S03]3 31 2.4.3 cw-RuCl2(DMSO)3(DMSO) 31 2.4.4 K 3 [RuCl 6 ] 32 2.4.5 Ru 2 (CH 3 COO) 4 Cl 32 2.5 References 33 Chapter 3 Synthesis and Characterization of Ruthenium(III) Maltolato and Imidazole Complexes 35 3.1 Introduction 35 3.2 Investigation of Ru(ma)3 and Ru(Ema)3 36 vi 3.2.1 Characterization and Solution NMR Data for Ru(ma)3 (1) 36 3.2.2 Characterization and Solution NMR Data for Ru(Ema)3 (2) 43 3.2.3 "Activation" of Ru(ma)3 with C F 3 S 0 3 H - Synthesis of [Ru(ma)2(EtOH)2]CF3S03 (3) 47 3.3 Reactions of Ru(ma)3 (1) and Ru(Ema)3 (2) with Imidazoles and Triazoles 49 3.3.1 Synthesis and Characterization of Imidazole and Triazole Complexes 49 3.3.2 Solution ! H NMR Spectroscopy of the Im, 2MeIm, 4MeIm and lMelm Complexes 55 3.3.3 Solution ] H NMR Spectroscopy of metro, 2N02Im, 4N02Im and 3N02tri Complexes 61 3.3.4 Compounds Synthesized Using 2,2,3,3,3-Pentafluoropropylamine (F5) 62 3.3.5 Synthesis, Characterization, and Solution 'H NMR Spectroscopy of [Ru(ma)2(EF5)2]CF3S03-EtOH (14) and [Ru(ma)2-(triF5) 2]CF 3S0 3 (18) 64 3.4 Synthesis of [Ru(HMepyr)3]Cl3 (19) 65 3.5 Cyclic Voltammetry of Complexes 66 3.6 Experimental Procedures for the Syntheses of Compounds Derived from F5 v , 68 3.6.1 N-(2,2,3,3,3-pentafluoropropyl) chloroacetamide (C1F5) 68 3.6.2 2-(2-Nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) 68 3.6.3 2-(3-Nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (triF5) 69 3.6.4 IMF10 69 3.7 Experimental Procedures for the Syntheses of Ru(III) Complexes 70 3.7.1 Ru(ma)3 (1) 70 3.7.2 Ru(Ema)3(2) 70 3.7.3 [Ru(ma)2(EtOH)2]CF3S03 (3) 71 vii 3.7.4 [Ru(ma)2(metro)2]CF3SC>3 (4) and [Ru(ma)2(metro)(EtOH)]CF3S03 (5) 71 3.7.5 [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) 72 3.7.6 [Ru(ma)2(Im)2]CF3S03 (7) 73 3.7.7 [Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 ( 8 ) 73 3.7.8 [Ru(ma)(lMeIm)4][CF3S03]2 (9) 74 3.7.9 [Ru(ma)2(2MeIm)2]CF3S03 (10) 75 3.7.10 [Ru(Ema)2(metro)2]CF3S03 (11) 75 3.7.11 [Ru(Ema)2(4MeIm)2]CF3S03 (12) 76 3.7.12 [Ru(Ema)2(2MeIm)2]CF3S03 (13) 76 3.7.13 [Ru(ma)2(EF5)2]CF3S03-EtOH (14) 77 3.7.14 [Ru(ma)2(2N02Im)2]CF3S03 (15) 77 3.7.15 [Ru(ma)2(4N02Im)2]CF3S03 (16) 78 3.7.16 [Ru(ma)2(3N02tri)2]CF3S03 (17) 78 3.7.17 [Ru(ma)2(triF5)2]CF3S03 ( 1 8 ) 78 3.7.18 [Ru(HMepyr)3]Cl3 (19) 78 3.8 References 79 Chapter 4 Synthesis and Characterization of Ru Complexes with 4,4'-Biimidazoles 81 4.1 Introduction 81 4.2 Synthesis and Reactions of 4,4'-Biimidazoles 82 4.2.1 Synthesis of 4,4'-Biimidazoles 82 4.2.2 Attempted Nitration of H 2biim 85 4.3 Attempted Synthesis of 5,5'-Dinitro-3,3'-bi(l,2,4-triazole) 86 4.4 Ru-biim Complexes Synthesized from cz's-RuCl2(DMSO)4 87 4.5 Complexes Synthesized from Ru(ma)3 95 4.6 Complexes Synthesized from [Ru(DMF) 6](CF 3S0 3)3 97 4.7 Complexes Synthesized from RuCl 3-3H 20 99 4.8 Experimental Preparation of Biimidazoles 100 4.8.1 Sources of Materials 100 viii 4.8.2 2,4,5-Triiodoimidazole 100 4.8.3 4(5)-Iodoimidazole 101 4.8.4 4-Iodo-l-(triphenylmethyl)imidazole 101 4.8.5 l,l'-Bis(triphenylmethyl)-4,4'-biimidazole 101 4.8.6 4,4'-Biimidazolium trifluoroacetate (H2biim) 102 4.8.7 4,5,-Diiodo-2-methylimidazole 102 4.8.8 4(5)-Iodo-2-methylimidazole 103 4.8.9 4-Iodo-2-methyl-l-(triphenylmethyl)imidazole 103 4.8.10 2,2'-Dimethyl -l,l'-bis(triphenylmethyl)-4,4'-biimidazole 103 4.8.11 2,2'-Dimethyl-4,4'-biimidazolium trifluoroacetate (H2Me2biim) 104 4.9 Experimental Syntheses of Complexes 104 4.9.1 m-RuCl 2(DMSO) 2(biim) (20) 104 4.9.2 Ru2Cl4(DMSO)4(biim) (21) 104 4.9.3 RuCl2(DMSO)2(Me2biim) (22) 105 4.9.4 Ru 2Cl 4(DMSO) 4(Me 2biim) (23) 105 4.9.5 [Ru(maltolato)2(biim)]CF3S03-2H20 (24) 105 4.9.6 [Ru(maltolato)2(Me2biim)]CF3S03-2H20 (25) 106 4.9.7 [Ru(biim)3][CF3S03]2 (26) 106 4.9.8 [Ru(Me2biim)3][CF3S03]2 (27) 107 4.9.9 [Ru(Hbiim)2Cl2]Cl2 (28) 107 4.9.10 [Ru((IiMe2biim)Me2biim)2(FIMe2biim)]Cl3 (29) 107 4.10 References 108 Chapter 5 Synthesis and Characterization of Ru Complexes with Chelating N-Heterocyclic Carboxylates 110 5.1 Introduction 110 5.2 Synthesis and Characterization of [Ru2(pic)4(EtOH)Cl]-H20 (31) 112 5.3 Complexes of Ru with Imidazole-4-carboxylic Acid 115 5.3.1 Synthesis and Characterization of Ru(Im-C0 2) 3-2H 20 (32) 115 ix 5.3.2 Synthesis and Characterization of Ru(Im-C02)(Im-C 0 2 H ) 2 C l 2 E t O H H 2 0 (33) 116 5.4 Synthesis of Nitroimidazole and Nitrotriazole Carboxylic Acids 119 5.4.1 Synthesis of 3-Nitro-l,2,4-triazole-5-carboxylic Acid (HCANT) 119 5.4.2 Attempted Synthesis of 2-Nitroimidazole-4-carboxylic Acid .... 119 5.4.3 Attempted Synthesis of 2-Nitroimidazole-4,5-dicarboxylic Acid 120 5.5 Attempted Reactions of HCANT with Ru Precursors 121 5.6 Experimental Procedures 122 5.6.1 3-Nitro-l,2,4-triazole-5-carboxylic Acid (HCANT) 122 5.6.2 Methyl imidazole-4-carboxylate 122 5.6.3 Methyl l-tritylimidazole-4-carboxylate 123 5.6.4 4-Hydroxymethyl-l-tritylimidazole 123 5.6.5 4-Hydroxymethyl-2-nitroimidazole 124 5.6.6 Dimethyl imidazole-4,5-dicarboxylate 125 5.6.7 Dimethyl l-tritylimidazole-4,5-dicarboxylate 125 5.6.8 4,5-Dihydroxymethyl-l-tritylimidazole 126 5.6.9 Ru(pic) 3H 20 (30) 126 5.6.10 [Ru2(pic)4(EtOH)Cl]-H20 (31) 127 5.6.11 Ru(Im-C0 2) 3-2H 20 (32) 127 5.6.12 Ru(Im-C0 2 )(Im-C0 2 H) 2 Cl 2 EtOHH 2 0 (33) 128 5.7 References 128 Chapter 6 Antiproliferatory Activity of Ruthenium Complexes Using the M T T Assay 130 6.1 Introduction 130 6.2 General Experimental Procedures 132 6.2.1 Experimental Media, Solutions, and Materials 132 6.2.2 Cell Preparation 133 6.2.3 Preparation of Ru Complexes 133 x 6.2.4 Preparation of Ligand Precursor Solutions 134 6.3 M T T Assay Procedure 134 6.3.1 Day 1 - Plating Cells 134 6.3.2 Day 2 - Addition of Ru Complexes and Ligand Precursors 135 6.3.3 Day 5 - Addition of M T T and Plate Reading 136 6.3.4 Data Analysis 137 6.4 Results and Discussion 138 6.4.1 Ligand Precursors 138 6.4.2 Ru Maltolato and Imidazole Complexes 139 6.4.3 Ru 4,4'-Biimidazole Complexes 145 6.4.4 Ru Carboxylate complexes 149 6.5 Summary 150 6.6 References 151 Chapter 7 In Vitro Toxicity, Ru-Uptake and Ru-DNA Binding Studies of Ru Complexes, and Antibody Recognition Studies of a New Nitrotriazole in Chinese Hamster Ovarian (CHO) Cells 153 7.1 Introduction ; : 153 7.2 General Experimental 154 7.2.1 Materials, Media, and Prepared Solutions 154 7.2.2 Cell Preparation 155 7.2.3 Stock Solutions of Ru Complexes 156 7.3 Hypoxia Selectivity Assays 156 7.3.1 Incubation of Complexes and Plating Efficiency 156 7.3.2 Ru-Uptake Determination 158 7.3.3 DNA Isolation 159 7.3.4 Monoclonal Antibody Binding Assay 160 7.4 Results and Discussion 162 7.4.1 Plating Efficiency of CHO Cells 162 7.4.2 Ru-Uptake 164 7.4.3 Ru-DNA Binding 167 xi 7.4.4 The Monoclonal Antibody Binding Assay 168 7.4.5 Reaction of [Ru(ma)2(2MeIm)2]CF3S03 (10) with L-cysteine ... 169 7.5 References 171 Chapter 8 Conclusions, and Direction of Future Work 173 8.1 Conclusions 173 8.2 New Complexes to Synthesize and Test 174 8.3 New Directions for Hypoxia Markers 175 8.4 Future Biotests 176 8.5 References 177 Structural Appendices Appendix A l Experimental Details for the X-ray Crystallographic Study of mer-Ru(ma)3 (1) 179 Appendix A2 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2 (8) 185 Appendix A3 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(ma)2(2MeIm)2]CF3S03 (10) 193 Appendix A4 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(Ema)2(metro)2]CF3S03 (11) 201 Appendix A5 Experimental Details for the X-ray Crystallographic Study of 1,1'-Bis(triphenylmethyl)-4,4'-biimidazole 209 xii List of Figures Figure 1.1 Gradient of oxygen concentration in cancerous tumours (adapted from ref. 6) 3 Figure 1.2 Molecular structures of metronidazole, EF5 and triF5 3 Figure 1.3 Proposed reduction pathway of nitroimidazoles (adapted from ref. 12) 4 Figure 1.4 Molecular structures of Pt(II) anti-cancer complexes 6 Figure 1.5 Pt-DNA adduct formed from a d(GpG) intrastrand cross-link 7 Figure 1.6 Molecular structures of Pt(IV) anti-cancer complexes 8 Figure 1.7 Molecular structures of Ru(III) anti-cancer complexes 10 Figure 1.8 Molecular structure of NAMI-A 11 Figure 1.9 Molecular structure of [RuCl(j9-cymene)(en)]PF6 12 Figure 1.10 Molecular structure of RuC10>cymene)(BESE)]PF6 12 Figure 1.11 Molecular structure of [Ru(p-cymene)Cl2(pta)] 13 Figure 1.12 Molecular structure of a-dichlorobis(2-phenylazopyridine)-ruthenium(II) 13 Figure 1.13 Molecular structure of [Ru(phen)2dppz](PF6)2 14 Figure 1.14 Molecular structures of maltol (R = Me) and ethylmaltol (R = Et) 15 Figure 1.15 Molecular structures of tropolone and catechol 15 Figure 1.16 Molecular structure of Ru(ma)2(DMSO)2 (R = Me) and Ru(Ema)2-(DMSO) 2 (R = Et) 16 Figure 2.1 NMR set-up for performing the Evans method determination of peff 25 Figure 2.2 Cyclic Voltammetry cell used with Pt wire counter and working electrodes, and an Ag wire reference electrode 28 Figure 2.3 Preparative T L C chamber with silica gel plate 30 Figure 3.1 Numbered molecular structures of imidazole and 3-nitro-l,2,4-triazole.. 36 Figure 3.2 Molecular structure of Ru(ma)3 (1) 36 Figure 3.3 ORTEP diagram of mer-Ru(ma)3 (1) with 33 % probability thermal ellipsoids (see Appendix A l for details) 37 Figure 3.4 'H NMR spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2 C1 2 38 xiii Figure 3.5 ' H - ' H COSY spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2 C1 2 39 Figure 3.6 ' H - ^ C H M Q C spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2 C1 2 with the 1 3 C spectrum on the left-hand side and the 'H spectrum at the top 40 Figure 3.7 Partial 1 3 C NMR spectrum of 1 (300 MHz, 298 K) in CD 2 C1 2 generated as a positive projection of the y-axis from the H - C H M Q C spectrum of 1 41 Figure 3.8 Plot of the chemical shift (ppm) vs. 1/T (K"1) for the H(5) and H(6) protons on each ma ring of Ru(ma)3 (1), here labelled H(a)-(f) 42 Figure 3.9 Plot of the chemical shift (ppm) vs. 1/T (K"1) for the Me groups of Ru(ma)3 (1) ....43 Figure 3.10 ! H NMR spectrum of Ru(Ema)3 (2) (300 MHz, 298 K) in CD 2 C1 2 44 Figure 3.11 ' H - ' H COSY spectrum of 2 (300 MHz, 298 K) in CD 2 C1 2 45 Figure 3.12 ! H - 1 3 C H M Q C spectrum (300 MHz, 298 K) of Ru(Ema)3 (2) in CD 2 C1 2 with the 1 3 C spectrum on the left-hand side and the : H spectrum at the top 46 Figure 3.13 In situ formation of [Ru(ma) 2(CD 3OD) 2]CF 3S0 3 as shown by 'H NMR spectroscopy, the broad downfield signal being characteristic of a trans-geometry for the maltolato ligands (see text) 48 Figure 3.14 Molecular structures for the zrans-isomers of 4, 6 - 8 and 10 49 Figure 3.15 Molecular structure of [Ru(ma)(lMeIm)4][CF3S03]2 (9) 50 Figure 3.16 ORTEP diagram of the cation of trans-[Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2 (8) with 50 % probability thermal ellipsoids (see Appendix A2 for details) 51 Figure 3.17 ORTEP diagram of the cation of frans-[Ru(ma)2(2MeIm)2]CF3S03 (10) with 50 % probability thermal ellipsoids (see Appendix A3 for details).. 52 Figure 3.18 Diagram depicting the potential steric interaction between an Im-Me group and the ma plane for 10 53 Figure 3.19 Template with numbering scheme for crystal structure information displayed in Table 3.2 53 xiv Figure 3.20 ORTEP diagram of the cation of rrans-[Ru(Ema)2(metro)2]CF3S03 (11) with 50 % probability thermal ellipsoids (see Appendix A4 for details).. 54 Figure 3.21 'H NMR spectrum of [Ru(ma)2(lMeIm)2]CF3S03 (8) in a) acetone-J6 (300 MHz, 298 K) and b) CDC1 3 (300 MHz, 298 K) 56 Figure 3.22 The effect of varying the temperature when reacting 1 with lMelm 57 Figure 3.23 'H NMR spectrum (300 MHz, 298 K) of [Ru(ma)(lMeIm)4][CF3S03]2 (9) in acetone-^ showing the ma-Me at 8 81.6 and lMelm H(2) protons at 5 -19.4 and -24.0 58 Figure 3.24 *H NMR spectrum (300 MHz, 298 K) of [Ru(ma)2(2MeIm)2]CF3S03 (10) in CDC1 3 58 Figure 3.25 'H NMR spectra (300 MHz, 298 K) of a) [Ru(Ema)2(2MeIm)2]CF3S03 (13) in CDCI3 and b) [Ru(Ema)2(4MeIm)2]CF3S03 (12) in C D c i 3 61 Figure 3.26 Molecular structures of three compounds synthesized from F5 62 Figure 3.27 Synthesis of 2,2,3,3,3-pentrafluoropropylamine (F5) from 2,2,3,3,3-pentafluoroproprionic acid 63 Figure 3.28 Molecular structure of IMF10 64 Figure 3.29 l 9 F NMR spectrum (300 MHz, 298 K) of [Ru(ma) 2(EF5) 2]CF 3S0 3EtOH (14) 65 Figure 3.30 Cyclic voltammogram of [Ru(metro)2(ma)2]CF3S03 • (4) in MeCN containing ferrocene 67 Figure 4.1 Molecular structure of 2,2'-biimidazole 81 Figure 4.2 Synthesis for H 2biim and H 2Me 2biim 82 Figure 4.3 ORTEP diagram of l,l'-bis(triphenylmethyl)-4,4'-biimidazole with 50 % probability thermal ellipsoids (see Appendix A5 for details) 84 Figure 4.4 Molecular structure of 5,5'-diamino-3,3'-bi(l,2,4-triazole) 86 Figure 4.5 ] H NMR spectra (300 MHz, 298 K) in C D 3 O D showing the imidazole protons of a) free H 2biim in CD 3 OD, b) 1:2 H 2biim:RuCl 2(DMSO) 4 after 4 h at 65 °C in CD 3 OD, and c) 1:1 H 2biim:RuCl 2(DMSO) 4 after 4 h at 65 °C in C D 3 O D 88 Figure 4.6 Proposed solid state structure for RuCl2(DMSO)2(biim) 88 Figure 4.7 ' H - ' H COSY NMR spectrum (300 MHz, 298 K) of 20 in CD 3 OD 89 xv Figure 4.8 Mass spectrum of RuCl2(DMSO)2(biim) (20): the most intense group of signals is for ( M + - CI - DMSO) 90 Figure 4.9 Structures (a) and (b) represent possible solution structures for 21, where all DMSO ligands are S-bound and S = C D 3 O D 92 Figure 4.10 *H NMR data (300 MHz, 298 K) for Me2biim complexes 93 Figure 4.11 Mass spectrum for Ru 2Cl 4(DMS0) 4(Me 2biim) (23); the most intense group of signals is for [M + - CI - 3 DMSO], centred at 551 94 Figure 4.12 1 H NMR spectra of 24 and 25 96 Figure 4.13 Proposed structure for the cation of 24 96 Figure 4.14 Three possible structures for the cation of 25 97 Figure 4.15 Possible structure for a bis-biimidazole species, from reduction of the Ru centre 98 Figure 4.16 Molecular structure for 28 99 Figure 5.1 Binding modes of carboxylate groups with transition metal complexes; a) Monodentate, b) Chelating, and c) Bridging 110 Figure 5.2 Molecular structure of pyridine-2-carboxylic acid, Hpic I l l Figure 5.3 ESI-MS spectrum (in MeOH) of [Ru2(pic)4(EtOH)Cl]-H20 (31) clearly showing the [M + - EtOH] fragment centered at 727 112 Figure 5.4 Theoretical isotopic distribution for the [M+] peak of Ru(Im-C02)(Im-C 0 2 H ) 2 C l 2 E t O H H 2 0 (33, left) (Section 5.3), and for the [ M + - EtOH] peak of [Ru2(pic)4(EtOH)Cl]-H20 (31, right) 113 Figure 5.5 'H NMR spectrum (300 MHz, 298 K) of [Ru2(pic)4(EtOH)Cl]-H20 (31) in D 2 0 114 Figure 5.6 Proposed solid state structures for 31 where N-0 = pic" 115 Figure 5.7 Molecular structure of Im-C0 2 H 116 Figure 5.8 One possible isomer for the proposed structure for Ru(Im-C02)(Im-C 0 2 H ) 2 C l 2 E t O H H 2 0 (33) (solvent molecules not shown) 117 Figure 5.9 Molecular structure of [Ru(PPh3)2(L-H)2] (L-H 2 = imidazole 4,5-dicarboxylic acid) 117 Figure 5.10 Mass spectrum of Ru(Im-C0 2)(Im-C0 2H) 2Cl 2-EtOH-H 20, showing M + at 508 118 xvi Figure 5.11 Molecular structure of HCANT 119 Figure 6.1 Mitochondrial reduction of M T T to Formazan 130 Figure 6.2 Pictures of cells on Day 5 of MTT assay before adding M T T in a control experiment with no complex (left), and in an experiment incubated for 69 h in 2 mM Ru(ma)3 (right) 131 Figure 6.3 Outline of M T T assay from Day 1 - Day 5; see Section 6.3 131 Figure 6.4 Experimental 96-well plate set-up showing the control column (dark grey), the blank (white), the wells with water (light grey) and those containing the complex solutions (speckled) 135 Figure 6.5 Image of plate after M T T treatment 137 Figure 6.6 Cell viability vs. concentration for [Ru(ma)2(2MeIm)2]CF3S03 (10), with error bars representing 95% confidence limit 140 Figure 6.7 Cell viability vs. concentration for Ru(ma)3 (1), with error bars representing 95% confidence limit 142 Figure 6.8 Cell viability vs. concentration for Ru(Ema)3 (2), with error bars representing 95% confidence limit 142 Figure 6.9 Cell viability vs. concentration for [Ru(Ema)2(2MeIm)2]CF3S03 (13), with error bars representing 95% confidence limit 144 Figure 6.10 Cell viability vs. concentration for cisplatin, with error bars representing 95% confidence limit 145 Figure 6.11 Cell viability vs. concentration for [Ru(ma)2(Me2biim)]CF3S03-2H20 (25), with error bars representing 95% confidence limit 147 Figure 6.12 Cell viability vs. concentration for [Ru(ma)2(biim)]CF3S03-2H20 (24), with error bars representing 95% confidence limit 147 Figure 6.13 Cell viability vs. concentration for [Ru(Me 2biim)3][CF 3S0 3] 2 (27), with error bars representing 95% confidence limit 148 Figure 6.14 Cell viability vs. concentration for [Ru(biim)3][CF3S03]2 (26), with error bars representing 95% confidence limit 149 Figure 6.15 Cell viability vs. concentration for [Ru2(pic)4(EtOH)Cl]-H20 (31), with error bars representing 95% confidence limit 150 Figure 7.1 CHO cells in a+/+ medium (~106 cells/mL) 156 xvii Figure 7.2 Conical tube set-up used for CHO experiments (gas = air or N 2) 157 Figure 7.3 Colony of CHO cells after 7 days, following staining with methylene blue 158 Figure 7.4 Structure of the indocarbocyanine fluorescent dye, Cy3, used to label the ELK3-51 MoAb 161 Figure 7.5 Large cell colony formations resulting from clumped cells (A and B) compared with a colony grown from a single cell over the same time period, C 163 Figure 7.6 Median fluorescence intensity of CHO cells incubated with EF5 or triF5 under N 2 or air and then treated with the ELK3-51/Cy3 antibody 169 Figure 7.7 Molecular structure of L-cysteine 170 xviii List of Tables Table 3.1 Variable temperature chemical shifts of Ru(ma)3 (300 MHz) in CD2CI2 including values at T = 00, as determined by linear regression 42 Table 3.2 Selected X-ray data from bis-imidazole derivative structures 53 Table 3.3 'H NMR data of Ru(III) maltolato-imidazole complexes in CDC1 3 at r.t 55 Table 3.4 *H NMR data for the Ema-Im complexes 12 and 13, compared with those for the ma-Im complexes 6 and 10 60 Table 3.5 Ru(III/II) reduction potentials of ma and Ema-containing complexes 66 Table 4.1 Ru (TH/II) reduction potentials for the biimidazole complexes 24 and 25, compared with those for 7 and 10 97 Table 5.1 Selected IR spectral data for Ru carboxylate complexes 113 Table 6.1 Dilution table for the addition of complexes using a 4 mM stock solution 136 Table 6.2 I C 5 0 values for the ligand precursors against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h 138 Table 6.3 IC50 values for Ru-maltolato complexes against MDA-MB-435S cells in L-15 medium and against MDA435/LCC6-WT cells in D M E M after incubation at 37 °C for 69 h 140 Table 6.4 IC50 values for cisplatin, and three Ru complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h 143 Table 6.5 IC50 values for ethylmaltolato complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h 143 Table 6.6 I C 5 0 values for biim and Me2biim complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h; data for new Ru complexes containing picolinic acid (Hpic) or imidazole-4-carboxylic acid (Im-CC^H) against the same cell line under the same conditions are also shown 146 xix Table 7.1 Plating efficiency of CHO cells after a 3 h incubation period under both aerobic and anaerobic conditions with several Ru(DI) complexes at 100 MM 164 Table 7.2 Uptake of Ru by CHO cells after a 3 h incubation period under both aerobic and anaerobic conditions with several Ru complexes at 100 uM 166 Table 7.3 Amount of Ru associated with DNA isolated from CHO cells after a 3 h incubation period under both aerobic and anaerobic conditions with several Ru(III) complexes at 100 uM 168 xx List of Symbols and Abbreviations acac acetylacetonate Anal. analysis Ar argon, or aryl atm atmosphere biim 4,4'-biimidazole br broad Calcd calculated cat. catalyst CHO Chinese Hamster Ovarian (cell line) COSY homonuclear correlation spectroscopy (NMR) cymene isopropyltoluene d doublet, deoxy dd doubly distilled D M E M Dubelco's modified essential medium DMSO S-bound DMSO ligand DMSO O-bound DMSO ligand e electron E1/2 electrochemical half-wave potential EDTA ethylenediaminetetraacetato EF5 2-(2-nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide Ema ethylmaltolato, 2-ethyl-3-hydroxy-4-pyronate equiv. equivalent(s) ESI electrospray ionization FBS fetal bovine serum FeCp 2 ferrocene FeCp*2 bis(pentamethylcyclopentadienyl)iron(II) G guanosine H 2biim 4,4'-biimidazolium trifluoroacetate H 2Me 2biim 2,2'-dimethyl-4,4'-biimidazolium trifluoroacetate xxi HCANT 3-nitro-1,2,4-triazole-5-carboxylic acid HMepyr 3-hydroxy-1,2-dimethyl-4-pyridone HMQC heteronuclear multiple quantum coherence Hpic pyridine-2-carboxylic acid, picolinic acid IC 5 0 concentration at which 50 % inhibition of cell proliferation is observed Im imidazole Im-C0 2H imidazole-4-carboxylic acid K Kelvin (T) LR low resolution LSIMS liquid secondary ion mass spectrometry m multiplet, or medium intensity or milli M molar (mol L" 1 ) ma maltolato, 3-hydroxy-2-methyl-4-pyronate Me2biim 2,2'-dimethyl-4,4'-biimidazole lMelm 1-methylimidazole 2MeIm 2-methylimidazole 4MeIm 4(5)-methylimidazole metro metronidazole MoAb monoclonal antibody mol mole MS mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium N M M N-methylmorpholine 2N02Im 2-nitroimidazole 4N02Im 4-nitroimidazole 3N02tri 3-nitro-1,2,4-triazole ORTEP Oakridge Thermal Ellipsoid Program P phosphodiester linkage in a DNA backbone PBS phosphate-buffered saline solution PE plating efficiency pF paraformaldehyde xxii q quartet r.t. room temperature s singlet solv solvent T Thymidine TBAP tetrabutylammonium hexafluorophosphate' TOF time of flight t triplet triF5 2-(3-nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide 8 chemical shift (ppm) X wavelength (nm) v wavenumber (cm"1) \i micro, or bridging coordination mode r| hapticity [ ] concentration (mol L"1) { } broadband decoupled (NMR) xxiii Key to Numbered Complexes 1 Ru(ma)3 2 Ru(Ema)3 3 [Ru(ma)2(EtOH)2]CF3S03 4 [Ru(ma)2(metro)2]CF3SC>3 5 [Ru(ma)2(metro)(EtOH)]CF3S03 6 [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 7 [Ru(ma)2(Im)2]CF3S03 8 [Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2 9 [Ru(ma)(lMeIm)4][CF3S03]2 10 [Ru(ma)2(2MeIm)2]CF3S03 11 [Ru(Ema)2(metro)2]CF3S03 12 [Ru(Ema)2(4MeIm)2]CF3S03 13 [Ru(Ema)2(2MeIm)2]CF3S03 14 [Ru(ma)2(EF5)2]CF3S03-EtOH 15 [Ru(ma)2(2N02Im)2]CF3S03 16 [Ru(ma)2(4N02Im)2]CF3S03 17 [Ru(ma)2(3N02tri)2]CF3S03 18 [Ru(ma)2(triF5)2]CF3S03 19 [Ru(HMepyr)3]Cl3 20 RuCl 2DMS0 2(biim) 21 Ru 2Cl 4DMS0 4(biim) 22 RuCl 2DMS0 2(Me 2biim) 23 Ru 2Cl 4DMS0 4(Me 2biim) 24 [Ru(ma)2(biim)]CF3S03-2H20 25 [Ru(ma)2(Me2biim)]CF3S03-2H20 26 [Ru(biim)3][CF3S03]2 27 [Ru(Me 2biim) 3][CF 3S0 3] 2 28 [Ru(Hbiim)2Cl2]Cl2 xxiv 29 [Ru(HMe2biim)(Me2biim)2]Cl3 30 Ru(pic) 3H 20 31 [Ru 2(pic) 4(EtOH)Cl]H 20 32 Ru(Im-C0 2) 3-2H 20 33 Ru(Im-C0 2)(Irn-C0 2H) 2Cl 2H 2OEtOH XXV Acknowledgements I would like to thank my supervisor Prof. Brian James for his help and guidance during the past five years, and for his encouragement in my development as a scientist. I would also like to thank Dr. Jorn Miiller for allowing me the opportunity of coming to working with his research group for a year at the Technisches Universitat (TU) in Berlin towards the completion of my degree here at UBC. I thank the remaining group members, Maria, Julio and Paolo, as well as the many past members I have had the honour of working with, particularly Jo Ling, Craig, Nathan, Paul, Cristina, Adam, Ian, Matt, Guibin, Hossein, Erin and Marcio. Without the assistance and helpful insight of my workmates in Berlin, the year I spent could not have been nearly as productive as it was, and so I would like to thank Mattel, Tews, Andy 'Mahlzeit' Hejl, Petra, Captain Schiller, and Speedy Kempf for all their help and the Polisches Frau for keeping me posted on travel deals and always reminding me that the end would eventually arrive. I would also like to acknowledge the work of Dr. Brian Patrick of this department in solving the X-ray crystal structures in this thesis work. Thank you as well to Marietta and Liane for all of their help with the NMR spectroscopy work I performed. I thank too all the other members of the UBC Chemistry Department with whom I have had the pleasure of working. Special thanks to the GSP: Gord, Pav, Joe and Pete for supporting me in all my endeavours and to Mark for nighttime blading and a friendship that has stood the test of time, some 22 odd years now. Finally I would like to thank my family who have supported me in so many ways and have never failed to be a guiding light in my life. My parents who have always encouraged me in everything I have done and my brothers, Chris and Jon, who through friendly competition and brotherly understanding have helped me strive to do my best. xxvi As a great French-Canadian once said... "when you have snow and ice for 6 months of the year, you might as well skate." xxvii Chapter 1 Introduction 1.1 Introduction Although often thought of as a single condition, cancer actually consists of more than 100 diseases, all of which result from the uncontrolled growth of abnormal cells. Such cell growth is the result of genetic mutations that disrupt the normal function of the cell, and disable the cell's own internal repair pathways. This, in turn, leads to uncontrolled growth and spread of these abnormal cells.1 Cancer can be divided into five major subclasses; carcinomas (cancer of the epithelial cells), sarcomas (cancer of the •muscle, bone, fat, or tendons), myelomas (cancer of the plasma cells of bone marrow), leukemias (cancer of the bone marrow), and lymphomas (cancer of the lymphatic system) with carcinomas accounting for 80-90 % of total cases. The Canadian Cancer Society estimates that in 2003 almost 140,000 new cases of cancer will be diagnosed in Canada and over 67,000 Canadians will die from the disease.2 Based on current trends it is estimated that approximately 38 % of men and 41 % of women will be diagnosed with some form of cancer in their lifetime.2 Although the death rate of cancer patients has been steadily decreasing over the last ten years, there is still a great demand for new drug treatments, particularly in light of the fact that some cancer tumours develop resistance to drug treatment over time, while other types of cancer have only a very limited response to chemotherapy and can only prolong the life of the patient at best.3 One major goal in cancer research is to develop new drugs that can selectively target cancer tumours and trigger apoptosis, cell death, while exhibiting only limited side-effects to normal healthy tissue. Research directed at understanding tumour biology now makes it possible to specifically target tumour cells.4 Some of the differences between normal tissue and cancerous tumours result from the rapid growth and division often characteristic of malignant tumour cells. This can lead to poor vasculature forming around the tissue, generating a local region of low extra-1 cellular pH and low oxygen concentration (hypoxia).5 Local regions of hypoxia have been exploited in the search of new cancer treatments (Section 1.6.1) and new cancer imaging agents (Section 1.2). The use of hypoxia selective imaging agents is important not only for the detection of tumours, but also to quantify the extent of hypoxia in a given tumour. The presence of hypoxic cells is a major limiting factor in the efficacy of radiotherapy and thus the diagnosis of such cells prior to prescribing a treatment is of the utmost importance.5 In this chapter the concept of hypoxia detection will be briefly introduced, followed by an introduction to the development of Pt anti-cancer compounds and a review of the rapidly evolving field of Ru anti-cancer compounds. 1.2 Nitroimidazoles and Hypoxia Hypoxia is defined as a condition in which a tissue receives a reduced supply of oxygen. As stated in Section 1.1, hypoxic regions often occur in tumours due to poor vasculature resulting from rapid growth of the tumour. Although cells deprived of an adequate oxygen supply will ultimately die, a tumour at any given time, may possess a region of viable, hypoxic cells that will be resistant to radiation treatment. O2 diffuses from the capillaries through tissues, and in tumours containing hypoxic cells, a gradient of [02] is established with healthy cells nearest the capillary, then a region of viable hypoxic cells as the [02] decreases, with necrotic cells being those farthest from the capillary (Figure l . l ) 6 . Regions of hypoxia in a tumour can cause problems in treatment, as hypoxic cells are considerably less responsive than normal tissue to radiation therapy. The effectiveness of radiation therapy is dependent on the partial pressure of oxygen inside the cell. This ratio of hypoxic to oxic doses of radiation required to produce the same biological effect is known as the oxygen enhancement ratio (OER). For sparsely ionizing radiation such as y-rays or X-rays, OER values are typically between 2.5 and 3.6 2 Figure 1.1 Gradient of oxygen concentration in cancerous tumours (adapted from ref. 6). Since the early 1970s, nitroimidazoles have been investigated as potential radiosensitizers. Radiosensitizers enhance the effectiveness of radiation therapy by binding to the damaged DNA caused by the free radicals that result from irradiation of the cell. In the case of nitroimidazoles, the nitro group is thought to react with damaged DNA under hypoxic conditions in a manner similar to that of oxygen under normal oxic conditions, making the DNA irreparable and thus leading to cell death. Metronidazole (Figure 1.2) was one of the first nitroimidazoles investigated as a potential radiosensitizer but was found to cause extreme gastrointestinal toxicity at biologically active doses.7 Since then, research has focused on the more electron affinic 2-nitroimdiazoles5 (E1/2 values typically between -360 and -400 mV for 2N02Im systems vs. -510 to -545 mV for 5NQ2Im and -540 to -685 mV for 4N0 2Im systems).8 C H 3 N 0 2 metronidazole EF5 triF5 Figure 1.2 Molecular structures of metronidazole, EF5 and triF5. Some nitroimidazoles are also -known to preferentially accumulate in hypoxic cells. 2-(2-Nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) (Figure 1.2) is currently undergoing clinical trials as a potential hypoxia imaging agent.9 Labelled with radioactive 1 9 F , this compound can be traced using positron emission topography (PET). 1 0 EF5 accumulation can also be monitored using invasive techniques by detection of adducts formed with a fluorescent antibody (ELK3-51) that is specific for EF5." Both the detection and treatment of hypoxic cells by nitroimidazoles result from the reducibility of the nitro group under physiological conditions. The higher activity of 2-nitroimidazoles over other nitroimidazoles is likely a result of their more positive reduction potentials. It has been proposed that nitroreductase enzymes inside the cell carry out the reduction, and that the selectivity for hypoxic cells is a result of a lack of oxygen to carry out the reverse oxidation (Figure 1.3).12'13 The radical anion formed as a result of the one-electron reduction can then undergo further reductions or react with other components inside the cell. In the presence of oxygen, the radical anion is reoxidized rapidly to the nitro group, and the compound can then diffuse back out of the cell. 1 3 The further reduction or other reactivity of the radical anion traps it inside the hypoxic cells and results in the accumulation of the reduced nitroimidazole species. r"\ e" r=\ e" i=\ f=\ NyNR Nitroreductase ' N y N R 2 H + " " " " N " N0 2 N<V NO N H 2 O2' O2 Radical Anion Nitroso Amine Figure 1.3 Proposed reduction pathway of nitroimidazoles (adapted from ref. 12). 1.3 In Vitro Bioassays A detailed introduction to the bioassays used in this thesis work will be presented at the beginning of both Chapters 6 and 7. The M T T assay, used here as a preliminary screening assay for antiproliferatory activity and cytotoxicity of some newly synthesized Ru complexes, has been used similarly by several groups with other Ru complexes before proceeding with in vivo experiments.14"20 Ru-uptake and Ru-DNA binding assays are 4 also performed to quantify the amount of metal accumulated in cells or in DNA, respectively.21'22 These assays can be modified to place the cells under anaerobic conditions, and thus can be used to investigate the uptake of Ru under both normal and hypoxic conditions. A monoclonal antibody specific for the side-chain of EF5 has been developed11 and is used in Chapter 7 to determine the hypoxic selectivity of 2-(3-nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (triF5, Figure 1.2) for comparison with that of EF5 under the same conditions (medium, cell line, concentration). 1.4 Platinum Anti-Cancer Drugs 1.4.1 Cisplatin and Other Pt(II) Anti-Cancer Drugs The first metal anti-cancer drug to be developed was cisplatin, discovered by Rosenberg et al. in 1965 after noting that the cell division of Escherichia coli bacteria in their reaction medium was being inhibited by an electrolysis product formed in situ from a Pt electrode. ' This product, identified as [NFL^tPtCle], was then converted to cis-PtCl4(NH3)2 via a photochemical process, while subsequent reduction gave the biologically active complex, cw-PtCl2(NH3)2 (cisplatin, Figure 1.4).25 Cisplatin, the most active of a series of cis-amine Pt species against sarcoma 180 and leukemia L1210 tumour models in mice, entered clinical trials in 1971 and was approved for the treatment of testicular and ovarian cancer in 1978.26 Unfortunately, there are drawbacks to cisplatin therapy: action against a limited range of tumours, poor solubility (thus the drug requires intravenous administration), and several toxic side-effects. The high incidence of nausea, vomiting, neuropathy, ototoxicity and nephrotoxicity has led to the search for new metal complexes having similar activity but with fewer side-effects. Carboplatin (Figure 1.4), a less toxic analogue of cisplatin, was introduced into clinical trials in 1981, and is the only other Pt complex that has received worldwide approval and regular clinical use.26 The major drawbacks of carboplatin therapy are that it is only applicable to the same narrow range of tumours as that of cisplatin and still must be administered intravenously. 5 Two other Pt(U)-amine complexes have received limited approval for use in some countries. Oxaliplatin ((rrans-L-diaminocyclohexane)oxalatoplatinum(II)) has been approved in France for the secondary treatment of metastatic colorectal cancer, while nedaplatin (c/s-diammine-glycolato-0,0'-platinum(II)) has received approval in Japan for the treatment of some cancers.26 cisplatin carboplatin AMD473 Figure 1.4 Molecular structures of Pt(II) anti-cancer complexes. More recently, the rational design of Pt(U) complexes with a greater steric bulk in the amine ligands in order to limit deactivation of the complex by glutathione has led to the development of AMD473, cw-(ammine)dichloro(2-methylpyridine)platinum(U) (Figure 1.4). AMD473 entered clinical trials after exhibiting improved activity over that of cisplatin against cisplatin-sensitive and -resistant cell lines; this complex is also sufficiently water-soluble that it can be administered orally,26 and is currently in Phase U clinical trials for treatment of hormone resistant prostate cancer (HRPC). 2 7 The mechanism by which cisplatin arrests cell division is well understood. In aqueous solution, cisplatin undergoes chloride dissociation to give both mono- and diaqua species. In the presence of DNA, these species react with the N(7) position on guanine bases, and give rise to either d(GpG) (Figure 1.5) or d(GpTpG) intrastrand cross-links (d = deoxy, G = guanosine, p = phosphodiester linkage and T = thymidine).28 The resulting intrastrand adduct can be removed and repaired by the cell; however, the intrastrand cross-link also results in a kink in the DNA that can then be recognized by damage-recognition proteins29 that may then block the repair enzymes from accessing the damaged D N A . 3 0 6 Figure 1.5 Pt-DNA adduct formed from a d(GpG) intrastrand cross-link. 1.4.2 Platinum(IV) as a Pro-Drug The anti-cancer activity of Pt(IV) complexes such as cw-PtCl 4(NH 3) 2 has been known since the discovery of cisplatin.23'25 Several Pt(IV) compounds have since been made in the hope that they might be activated by an in vivo reduction of the metal centre. At least three of these compounds (iproplatin,31 tetraplatin,32 and JM2 1 6,33 3 5 Figure 1.6) have entered clinical trials, but have ultimately failed to produce results better than those of cisplatin; trials with iproplatin and JM216 failed to produce significant in vivo effects, while treatment with tetraplatin gave acute toxic side-effects. Complexes that remain in the higher Pt(IV) oxidation state in the bloodstream have the potential advantage over Pt(II) complexes of undergoing fewer, unwanted side-reactions. This can result in fewer, toxic side-effects and thus a greater amount of the active drug arriving in the target cells.36 The higher lipophilicity of some Pt(IV) complexes can also lead to better cellular uptake.36 7 Iproplatin Tetraplatin JM216 Figure 1.6 Molecular structures of Pt(IV) anti-cancer complexes. The rate of in situ reduction of Pt(IV) to Pt(U) varies greatly depending on the ligands bound to the metal, and the ease at which the metal centre is reduced may greatly influence its biological activity.36 Direct correlations between the activity of a complex and its Pt(IV/U) reduction potential cannot always be made; the reduction potential of tetraplatin is about 300 mV more positive than that of JM216, yet both are highly active in vitro36 It may be possible, though, to tune the reduction potential of complexes already known to be active, to try and slow the in vivo reduction so that the more active complex reaches and is taken up into the cells.36 1.5 Ruthenium Complexes and Their Potential as Anti-Cancer Drugs Since the discovery of cisplatin, complexes with other transition metals have been investigated, and Ru complexes have emerged as strong candidates for potential therapeutic use. Some proposed mechanisms of action and potential anti-cancer drugs will be discussed in the following sections. 1.5.1 Activation by Reduction Ru(IU) complexes have been investigated as pro-drugs for potentially more active Ru(U) species, and a mechanism of activation by reduction may be the reason that several Ru(HI) complexes have shown high levels of in vivo activity against various types of cancerous tumours.37 The concept is the same as the use of Pt(IV) as a pro-drug, to 8 deliver a greater percentage of active complex to the cancerous cells by starting with a less reactive species that then becomes activated by reduction in the hypoxic regions of tumours. Ru(II) complexes are generally more substitutionally labile than those of 37 R u ( m r and are thus more likely to lose ligands in vitro, opening coordination sites for DNA or other cellular targets. 1.5.2 m-RuCl 2 (DMSO) 3 (DMSO) ds-RuCl 2(DMSO) 3(DMSO) (ds-DMSO) was first reported by our group in 1971.38 Studies suggesting that this complex could interact in vitro with DNA were reported in 1975,39 and this resulted in further studies comparing its antimutagenic properties with those of cisplatin.40 ds-DMSO, tested in vivo in mice implanted with Lewis lung carcinoma-, B16 melanoma-, and Mca mammary carcinoma tumour models, was as effective as cisplatin against primary tumour growth and lung metastases and significantly less toxic.40'41 Coluccia et al. have reported in vivo testing of ds-DMSO in mice bearing P388 and the cisplatin-resistant P388/DDP leukemia tumour models, and in the latter, ds-DMSO was more effective than cisplatin at reducing primary tumour growth.42 ds-DMSO likely acts via a DNA-binding mechanism, and several groups have reported its interactions with purines.43"45 Although the O-bound DMSO ligand of cis-DMSO rapidly dissociates in aqueous solution, followed by the slow loss of one CI" ligand,46 Farrell and De Oliveira have reported a dicationic Ru-bis(adenine) complex in which the O-bound DMSO is retained and the two CI" ligands are replaced by adenine.43 1.5.3 Ru-imidazole Complexes Keppler et al. first reported on the synthesis and biological activity of imidazolium [?ran5-tetrachlorobis(imidazole)ruthenate(IU)] (ICR) in 1987 (Figure 1.7).15 Since then, numerous studies on analogues of this complex using other N-heterocyclic ligands in place of imidazole have been reported.14'16"18 Preliminary studies of ICR against P388 leukemia-, Walker 256 carcinosarcoma-, and sarcoma 180 tumour-infected mice resulted in increased survival times (T/C values of up to 194 vs. 175 % for cisplatin,15 where T/C values are calculated as the survival time of mice treated with the 9 complex divided by the survival time of mice to which no complex was administered). The methylimidazole derivatives were less active than ICR against P388 leukemia (T/C values of 130 - 150 %), while (Htri)[rrans-Ru(tri)2Cl4] (tri = 1,2,4-triazole) also exhibited weaker activity (T/C = 138 %).14 (fflnd)[rrans-Ru(Ind)2Cl4] (Ind = indazole) (Figure 1.7) was as effective as ICR in reducing colorectal tumour growth (typically 50 - 80 % reduction in growth) and was devoid of any side-effects at active dosages.41'47 Imidazolium pentachloro(imidazole)ruthenate(IU) ((mm)2[Ru(Irn)Cl5])16 and the analogous triazole complex, (Htri)2[Ru(tri)Cl5],14 exhibit survival rates in P388 tumour-bearing mice that are lower than those for cisplatin and ICR (T/C = 150-162 % for (fflm)2[Ru(Im)Cl5]16 and 150 % for (Htri)2[Ru(tri)Cl5]).14 (fflm)[rrans-Ru(Im)2Cl4] (fflnd)[frans-Ru(Ind)2Cl4] Figure 1.7 Molecular structures of Ru(III) anti-cancer complexes. 1.5.3.1 NAMI and NAMI-A (fflm)[rratts-Ru(Im)(DMSO)Cl4] (NAMI-A) (Figure 1.8) exhibits an anti-metastatic effect on several tumour models and is the only Ru complex to date to enter into clinical trials.41 In vivo studies show that both NAMI-A and NAMI (Na[trans-Ru(Im)(DMSO)Cl4]), like cisplatin, are capable of reducing primary tumour growth in Lewis lung carcinoma-, B16 melanoma-, Mca mammary carcinoma tumour-models by up to 40 % 4 8 4 9 Unlike cisplatin, however, treatment with NAMI or NAMI-A also increased the life-span of the hosts, independent of the tumour model used, and reduced the 10 formation of lung metastases even when the inhibition of primary tumour growth was negligible.41 NAMI and NAMI-A are thought to arrest cells in the G2/M phase of the cell cycle, and do not appear to act via a DNA-binding mechanism, although they do bind DNA in vitro.50 The complexes may be activated by an in situ reduction to a Ru(U) species.51 The major advantage of NAMI-A over NAMI is that it is a more stable solid.51 Figure 1.8 Molecular structure of NAMI-A. 1.5.4 Ru-arene Complexes Morris et al. have reported the synthesis and in vitro testing of Ru(U)-arene complexes against A2780 human ovarian cancer cells.19 Complexes such as [RuX(p-cymene)(en)]PF6, where X = CI or I (Figure 1.9), exhibited I C 5 0 values (9 and 8 uM for CI and I, respectively) similar to that of carboplatin (6 uM) against the same cell line, but more than an order of magnitude higher than that of cisplatin (0.5 uM). 1 9 These complexes bind strongly to the guanine bases of D N A . 1 9 It is thought that the arene group not only stabilizes the Ru(U) metal centre but may also help in cellular uptake by increasing the hydrophobicity of the complex.19 The range of Ru-arene complexes studied was then expanded by the same group who then reported that [RuCl-(tetrahydroanthracene)(en)]PF6 exhibited the lowest I C 5 0 value, 0.5 uM, of a range of complexes tested, equal to that observed for cisplatin.52 11 Figure 1.9 Molecular structure of [RuCl(p-cymene)(en)]PF6. Ru-arene complexes have also been synthesized in our group and tested for anti-cancer activity against MDA-MB-435S cells, a human breast cancer cell line.53 The complex, [RuCl(p-cymene)(BESE)]PF6 (Figure 1.10), where BESE = 1,2-bis(ethylsulfinyl)ethane, and the dimeric complex, [RuCl2(/?-cymene)]2(u.-BESE), have been tested for activity using the M T T assay to determine their IC50 values. RuCl(p-cymene)(BESE)]PF6 exhibited significant activity (IC 5 0 = 55 uM) when compared with data for cisplatin (IC 5 0 = 10 uM). 5 3 C H 2 C H 3 Figure 1.10 Molecular structure of RuCl(p-cymene)(BESE)]PF6. 12 c A third group has reported on the pH dependent cytotoxicity of Ru(p-cymene)Cl2(pta) (Figure 1.11), where pta = l,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane, which may also prove useful as an anti-cancer agent.54 Figure 1.11 Molecular structure of Ru(p-cymene)Cl2(pta). 1.5.5 Ru-azopyridine Complexes Velders et al. have reported on the activity of the so-called a-isomer of dichlorobis(2-phenylazopyridine)ruthenium(II) (Figure 1.12) against 7 different tumour cell lines; the activity was invariably equal to or better than that of cisplatin, with IC50 Figure 1.12 Molecular structure of a-dichlorobis(2-phenylazopyridine)ruthenium(II). 13 values being an order of magnitude lower than that of cisplatin in both the EVSA-T (a human breast cancer cell line) and M19 (a Chinese hamster ovarian cell line) cell lines.20 Two geometric isomers, the so-called |3- (containing cis-pyridine and cis-Cl ligands) and y- (containing cis-pyridine and trans-Cl) isomers were considerably less active. Believing that the oc-isomer acts in a manner similar to that of cisplatin by binding to the guanine bases of DNA, the same group later showed that this complex reacts with 9-ethylguanine55 to form a-[Ru(2-phenylazopyridine)2(9-ethylguanine)(H20)](PF6)2, with the guanine bound through the N(7) position; 3-methyladenine, correspondingly formed a-[Ru(2-phenylazopyridine)2(3-methyl-adenine)](PF6)2, in which the adenine is chelated through the N(6) and N(7) positions.55 It was thought that such Ru-adducts might form in the presence of DNA and be responsible for the anti-tumour activity of oc-dichlorobis(2-phenylazopyridine)ruthenium(II). 1.5.6 Ru Intercalators Ru complexes with large conjugated ligands can also interact with the purine bases of DNA via 7X-stacking interactions. Such Ru intercalators are of particular interest in photodynamic therapy in which they can be activated to cause local damage to the surrounding D N A . 3 7 Both enantiomers of [Ru(phen)2dppz](PF6)2, where phen = 1,10-phenanthroline and dppz = 2,2'-dipyrido-3,3'-phenazine (Figure 1.13), exhibit a high affinity for DNA without loss of any chelating ligand.56 Figure 1.13 Molecular structure of [Ru(phen)2dppz](PF6)2. 14 1.6 Maltolato (ma) Chemistry Maltol (3-hydroxy-2-methyl-4-pyranone, Figure 1.14), is a water-soluble food additive with a pK a of 8.67 for the hydroxy group;57 the anionic form, maltolato (ma), can then chelate to transition metal centres. This is an attractive ligand to use in biological inorganic chemistry as complexes that contain one or more maltolato ligands are often water-soluble and the free ligand, if dissociated in vivo, is non-toxic.58 1 R = Me, Et Figure 1.14 Molecular structures of maltol (R = Me) and ethylmaltol (R = Et). 1.6.1 Maltolato Complexes with Ru Ru(ma)3, first reported by Griffith and Greaves in 198 8,59 was synthesized as part of a research project investigating the transition metal complexes of tropolone and catechol (Figure 1.15). Indeed, the assignment of the ! H NMR spectrum of tropolone catechol Figure 1.15 Molecular structures of tropolone and catechol. 15 Ru(tropolonato)3, first discussed by Eaton et al, shows similarities to that of Ru(ma)3 which will be discussed in Chapter 3. El-Hendawy and El-Shahawi reported later the synthesis of another Ru(ITI) maltolato species, RuCl2(ma)(PPh3)2,61 while other groups have reported Ru(U) complexes containing one or more maltolato ligands. Fryzuk et al. have reported on c«-Ru(ma) 2 (PPh 3 ) 2 , cw-Ru(ma)2(DMSO)2, and cz's-Ru(ma)2(COD)2,62 while Capper et al. have characterized Ru(mes)Cl(L) and [Ru(mes)(CO)(L)](BF4) where L = maltolato (ma) or ethylmaltolato (Ema) and mes = 1,3,5-trimethylbenzene.63 Our group has recently reported on the anti-cancer activity of Ru-maltolato-sulfoxide complexes of the type Ru(L) 2(DMSO) 2 where L = maltolato (ma) or ethylmaltolato (Ema) (Figure 1.16).64'65 These complexes were tested against the human breast cancer cell line MDA-MB-435S and exhibited I C 5 0 values of 190 (L = Ema) and 220 uM (L = ma), these data being taken from Wu's M.Sc. thesis.65 Replacement of the DMSO ligands by BESE (see Section 1.6.4) led to less active complexes.64'65 The higher stability of the chelated-BESE complexes in solution may limit the potential in vitro for the Ru to bind DNA or other cellular targets 6 4 , 6 5 D M S O Figure 1.16 Molecular structure of Ru(ma)2(DMSO)2 (R = Me) and Ru(Ema)2(DMSO)2 (R = Et). 1.6.2 Maltolato Complexes with Other Transition Metals Fe(ma)3 has been structurally characterized and proposed as a potential drug for iron-deficiency anaemia,66 while trivalent complexes of Cr, Mn, Rh, Ga, In, V and Al 16 have also been reported. ' VO(ma)2 and VO(Ema)2 are being studied as potential insulin mimetic drugs. 6 9 , 7 0 Greaves and Griffith have also reported the syntheses of trans-Os(0)2(ma)2, £rans-U(0) 2 (ma) 2 and cw-Mo(0)2(ma)2. Sn(ma)2Cl2 and Sn(ma)2 have been synthesized, and other divalent maltolato complexes of Co, Ni, Cu, and Zn have been reported.67 Numerous trivalent lanthanide complexes of the form M(ma)3-H20 have also been synthesized (M = La, Pr, Nd, Sm, Gd, Dy, and Yb). 5 7 1.7 Goals of this Thesis The goals at the onset of this thesis work were to synthesize and characterize new Ru-maltolato-imidazole and -nitroimidazoles complexes, and to investigate their toxicity, cellular uptake and interactions with DNA in cells using standard in vitro bioassays. Previous work in our group had shown that Ru-EF5 complexes could be used as potential radiosensitizers73 while other groups have shown that Ru(IJJ) imidazole complexes may be good anti-metastasis drugs50 or intercalators.52 A 3-nitro-1,2,4-triazole analogue of EF5 was proposed as a possible new hypoxia selective agent, and was thus synthesized, characterized and tested; this compound was examined in vitro for hypoxia selective activity but proved to be not as effective as EF5. Of the Ru(IU)-maltolato-imidazole complexes tested, several exhibited interesting biological activity, and as the biological testing proceeded, it became apparent that these complexes might also produce an antiproliferatory effect similar to that of NAMI-A, and thus the M T T assay was used as a general screening assay to probe complexes for anti-cancer activity. The range of Ru complexes synthesized and characterized was then expanded to investigate complexes of several, bidentate ligands, especially imidazole-4-carboxylic acid, 4,4'-biimidazole and 2,2'-dimethyl-4,4'-biimidazole. Complexes of these ligands with Ru(ma)3 and several other Ru starting materials afforded several new complexes that were characterized and tested in preliminary in vitro bio-assays. This thesis consists of 8 chapters, and appendices containing data for the 5 crystal structures presented. In Chapter 2, general experimental procedures and methods are discussed. The syntheses and characterization data of new Ru(ffl)-maltolato-imidazole 17 complexes are described in Chapter 3. The syntheses of 4,4'-biimidazole ligands, and the reactions of these ligands with both Ru(U) and Ru(III) precursors to afford new Ru-biimidazole complexes, are described in Chapter 4. These ligands were designed initially to synthesize a bidentate biimidazole analog of EF5. Ultimately, the complexes with the biimidazole ligands were themselves found to be worthy of study even without nitration to form the intended 2,2'-dintrobiimidazole species. The successful results from biotests with Ru-bis(2MeIm) complexes (Chapter 3), led to the development of the analgous 2,2'-dimethyl-4,4'-biimidazole complexes for study. In Chapter 5, the reactions of imidazole-4-carboxylic acid and other N-heterocyclic carboxylic acids with Ru(m) precursors are described, as are the attempted syntheses of new 2-nitroimidazole-4-carboxylic acid derivatives. In Chapter 6, the results from the M T T assay for many of the complexes described in Chapters 3-5 will be discussed, while in Chapter 7, the results of Ru-uptake and - D N A binding experiments for several Ru complexes are reported and discussed. A monoclonal antibody binding assay is also discussed and the results of this assay with both EF5 and triF5 are presented. 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I.; Burgess, J.; Fawcett, J.; Parsons, S. A.; Russell, D. R.; Laurie, S. H. Polyhedron 2000,19, 129. (73) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents , Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. 22 Chapter 2 General Experimental Procedures, and Syntheses of Ruthenium Precursor Complexes 2.1 Chemicals 2.1.1 Ligand Precursors Imidazole (Im), 1-methylimidazole (lMelm), 2-methylimidazole (2MeIm), 4-methylimidazole (4MeIm), metronidazole (metro), 2-nitroimidazole (2N02Im), 4-nitroimidazole (4N02Im), 3-nitro-1,2,4-triazole (3N02tri), 4-(hydroxymethyl)imidazole, imidazole-4-carboxylic acid (Im-C02H), pyridine-2-carboxylic acid (Hpic), 3-hydroxy-l,2-dimethyl-4-pyridone (HMepyr) and imidazole-4,5-dicarboxylic acid were purchased from Aldrich and used as received. 4,4'-Biimidazolium trifluoroacetate (H2biim) and 2,2'-dimethyl-4,4'-biimidazolium trifluoroacetate (H2Me2biim) were synthesized using modified literature procedures' (Sections 4.8.2 - 4.8.11). 2-(2-Nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) and 2-(3-nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (triF5) were synthesized using a modified literature procedure (Sections 3.6.2 and 3.6.3, respectively). 3-Nitro-l,2,4-triazole-5-carboxylic acid (HCANT) was also synthesized using a modified literature procedure3 (Section 5.6.1). 3-Hydroxy-2-methyl-4-pyrone (maltol) (Cultor Food Science) and 2-ethyl-3-hydroxy-4-pyrone (ethylmaltol) (Pfizer Food Science) were donated by Dr. C. Orvig. 2.1.2 Solvents Reagent grade DMF, DMSO, MeCN, CHC1 3, and EtOAc were purchased from Fisher Scientific and used as received. Other solvents (also purchased form Fisher) were dried and distilled prior to use: EtOH (distilled from Mg), MeOH (distilled from Mg), CH 2 C1 2 (distilled from CaH 2), Et 2 0 (distilled from Na), hexanes (distilled from Na), THF (distilled from Na), and acetone (distilled from K 2 C 0 3 ) . Deuterated solvents CDC1 3, 23 CD 2C1 2 , CD3OD, D 2 0 , acetone-ak, and dmso-Jg were purchased form Cambridge Isotope Laboratories and used without further drying or purification. 2.1.3 Gases N 2 (medical grade), Ar, compressed air, and H 2 (extra dry) were purchased from Praxair and used as received. 2.1.4 Biological Reagents Materials used in the biological experiments are listed in Sections 6.2.1 and 7.2.1. 2.1.5 Other reagents 2,2,3,3,3-Pentafluoropropylamine was purchased from Interchim and Lancaster Synthesis. It was also prepared in small amounts according to a literature procedure.4'5 Trifluoromethane sulphonic acid (triflic acid), ferrocene (FeCp2), bis(pentamethylcyclopentadienyl)iron(U) (FeCp*2), tetrabutylammonium hexafluorophosphate (TBAP), potassium re/t-butoxide, chlorotriphenylmethane (trityl chloride), N-methylmorpholine (NMM) and chloroacetyl chloride were purchased from Aldrich and used as received. RuCl 3-3H 20 was kindly donated by Colonial Metals Inc., and had a Ru composition of 39 %. All other chemicals were used as received unless otherwise stated. 2.2 Analytical Techniques and Instrumentation 2.2.1 Nuclear Magnetic Resonance Spectroscopy ID NMR spectra were recorded on Bruker AV300 (*H, 1 9 F , 1 3 C), Bruker AV400 ('H, 1 3 C), Bruker ARX200 ('H, 1 9F), Bruker ARX400 ('H, 1 9F) and Bruker AC200F ('H, 1 9 F , 1 3 C) spectrometers. The AC200F spectrometer was typically used for testing ligand samples as many of the complexes were paramagnetic and required a wider sweep width to record their NMR spectra, typically 150 ppm for spectra, than the AC200F could provide, and therefore their spectra were recorded on the AV300 and 400 spectrometers. All 'H NMR and 1 9 F NMR spectra performed during my stay at the T U Berlin were 24 recorded on the ARX200 or ARX400 spectrometers. 'H- 'H COSY and ' H - 1 3 C HMQC experiments were performed on the Bruker AV300 and AV400 spectrometers. Low temperature 'H NMR spectra were all recorded on the AV300 spectrometer. The peaks are described in this thesis as observed in the spectra: s = singlet, d = doublet, t = triplet, q = quartet, br = broad, and m = multiplet. 'H NMR spectra were referenced using the residual protonated solvent signals: 7.24 (CDC13), 5.32 (CD2C12), 4.65 (D 20), 3.30 (CD3OD), 2.49 (dmso-<4) and 2.04 (acetone-^). 1 3 C NMR spectra were referenced to the 1 3 C solvent signal: 77.0 (CDC13) and 53.8 (CD2C12). MestRe-C (Magnetic Resonance Companion, Version 1.5.0) was used to process the raw data for spectra obtained on the AC200F NMR spectrometer. For all other spectrometers, Win-NMR was used to analyze and process the spectral data. Determination of p,eff for Ru(III) paramagnetic complexes and the corresponding number of unpaired electrons for these complexes was performed using the Evans method at r.t.6 This technique measures the shift of the residual solvent signal due to the presence of the paramagnetic complex. The signal is typically shifted downfield. The complex solution was placed inside a melting point capillary that was subsequently flame-sealed and suspended inside a standard NMR tube containing only the deuterated solvent (Figure 2.1). Typically, the complex is in the NMR tube and the pure solvent capillary containing complex dissolved in deuterated solvent deuterated solvent Figure 2.1 NMR set-up for performing the Evans method6 determination of |!eff- The capillary is flame-sealed after being filled with a solution of known complex concentration, and lowered into an NMR tube using Teflon tape that allows for easy retrieval of the sample. 25 in the capillary; however, because of limited quantities of some samples and overlapping signals' in the solvent region from the complex, it was decided to place the complex solution in the capillary. Because of paramagnetic broadening of multiplet solvent signals, only solvents with sharp residual singlet signals were used for these measurements, i.e. CDC1 3 or CD 2 C1 2 . The values for Av were determined using the AV300 spectrometer and, from these values, peff was calculated using the following equations (Equations 2.1-2.4).6'7 The diamagnetic correction value, %L, was calculated from literature values for each ligand.8 m = concentration of sample (g/mL) Av = peak separation (Hz) r^q 2 1 ) v 0 = frequency of spectrometer (Hz) Xo = mass susceptibility of solvent X M = X G M M = compound molecular weight (Eq. 2.2) (g/mol) X M ' = X M _ ^JXL X M = mass susceptibility of metal ion %L = diamagnetic correction for ligands p e f f = 2.84^/XM'T = yjn(n + 2 ) T = absolute temperature (K) (Eq. 2.4) n = number of unpaired e" on metal ion X E 3Av 47tvom + X 0 2.2.2 Infrared Spectroscopy Infrared spectra were recorded at UBC on an ATI Matson Genesis or Bomem-Michelson MB-100 FT-IR spectrometer. At the T U Berlin, spectra were recorded by Nicolet on a Typ Magna-IR 750 spectrometer. Spectra were recorded as KBr pellets and are reported in wavenumbers (± 4 cm"1) with selected functional groups assigned based on data for similar, previously reported complexes (see, for example, Section 5.3.2),9 and on references from standard IR textbooks.10'11 26 2.2.3 Mass Spectrometry Mass spectral data were collected in the Mass Spectrometry laboratory at UBC under the supervision of Dr. G. Eigendorf and Dr. Y. Ling. The spectra were measured on a Kratos Concept IJHQ liquid secondary ion mass spectrometer, a Bruker Esquire electrospray (ESI ion trap) spectrometer and a Micromass L C T electrospray time of flight (ESI TOF) spectrometer. At the T U Berlin, mass spectra were collected on a Varian double focussed MS - Typ 311 A/AMD. M + is defined as the positive molecular ion of a given species and does not include the mass of solvated molecules or counter anions (e.g. for [Ru(ma)2(lMeIm)2]CF3SC>3-CH2Cl2 , M + = [Ru(ma)2(lMeIm)2]+). 2.2.4 UV-Visible Spectroscopy UV-Vis spectra were recorded on an HP 8452A diode array spectrophotometer and are reported as A m a x (± 3 nm) (em a x x 10"3, M"'cm''). 2.2.5 Cyclic Voltammetry Cyclic Voltammetry was performed on selected compounds in solutions of 0.1 M TBAP in degassed MeCN under a flow of N 2 . Voltammograms were recorded on a Pine Biopotentiostat, model AFCBP1, and processed using the accompanying software Pinechem v2.00. Compounds were scanned at 100 mV/s between -2 V and +2 V. The home-made cell used to perform these measurements (Figure 2.2) contained a Pt wire working electrode, a Pt wire counter electrode, and a silver wire reference electrode. FeCp 2 and FeCp*2 were used as internal standards ( E ] / 2 (Feni / I1) = 0.40 and -0.19 V vs. SCE in MeCN, respectively).12 The electrodes were cleaned with cone. HNO3 before use, then rinsed with water and acetone, and dried at 100 °C. A voltammogram was then recorded with just solvent and electrolyte to ensure a flat baseline before repeating the CV measurement with the sample. The sample solution was scanned once without FeCp 2, then again after FeCp2 had been added, this procedure being repeated to ensure values for the reduction potentials, E1/2, were reproducible. The E i / 2 values are calculated as the average of the oxidation and reduction peaks (Ei/ 2 = (E p c . + Eax.)/2), and are reported as the average of two or more values for each compound (± 4mV). The 27 reported values are calculated as follows: Ei/2(compound) in mV vs. SCE in MeCN = 400 - E 1 / 2(FeCp 2) + E1/2(observed). r \ Pt Counter Electrode Pt Working Electrode Ag Reference Electrode Figure 2.2 Cyclic Voltammetry cell used with Pt wire counter and working electrodes, and an Ag wire reference electrode. 2.2.6 Conductivity Conductivity measurements were performed using a Serfass conductance bridge model RCM151B (Arthur H. Thomas Co. Ltd.) connected to a 3403 cell (Yellow Springs Instrument Co.). The cell was calibrated using a 0.0100 M KC1 solution with a molar conductance (AM) of 141.3 Q'cn^mol"1 at 25 °C and a cell constant of 1.016 cm"1 (measurement of the cell constant was performed by another group member).13 ,14 Solutions to be tested were prepared at 10"3 M . Values in this thesis are reported as A M (SrWmor1).14 2.2.7 X-Ray Analysis Four X-ray crystal structures were determined by Dr. B. O. Patrick in the X-ray crystallographic laboratory at UBC on a Rigaku/ADSC CCD area detector with graphite 28 monochromated M o K a radiation (see Appendices 1-4). One X-ray crystal structure was determined by Dr. P. Escarpa at the TTJ Berlin on a Siemens Smart C C D area detector with graphite monochromated M o K a radiation (see Appendix 5). 2.2.8 Elemental Analysis Elemental analyses were performed at UBC by Dr. P. Borda and M . Lakha on a Carlo Erba Instruments E A 1106 CFIN-O elemental analyzer. Some samples were also analyzed by Mr. M . K. Yang of the SFU Chemistry Department and by Canadian Microanalytical Services Ltd. 2.2.9 Biological Analysis Biological assays were performed in the biological services laboratory of the chemistry department at UBC under the supervision of Dr. E . Polishchuk, with the exception of the antibody binding assay which was performed at the BC Cancer Research Centre in the laboratory of Dr. K Skov. A discussion of biological techniques will be reserved for Chapters 6 and 7, at which point the techniques used will be discussed in detail along with the general experimental procedures for the biological assays. 2.3 Chromatographic Techniques Reactions for the syntheses of both ligands and metal complexes were monitored by T L C . The reaction mixtures were typically viewed under a U V lamp (Mineralight® Lamp Model UVG-54 short-wave UV-254 nm). For imidazoles and other compounds that could not be seen under the U V lamp, the developed T L C strips were exposed to I2 to visualize the remaining bands. Solvent combinations, including volume ratios, are listed in brackets as follows: e.g. (CH 2 Cl 2 :MeOH, 10:1). Many samples were purified using column chromatography or, more commonly, preparative thin layer chromatography. For columns, silica gel (230-400 mesh, Silicycle) was added to the eluent, the mixture stirred for -30 seconds, and then slowly poured into the glass column (plugged with glass wool just above the stopcock to prevent silica from 29 pouring out the bottom) to avoid forming air bubbles. The column was then eluted under a positive pressure of N 2 until the solvent level reached the top of the silica gel. The compound to be purified was then dissolved in a minimum amount of eluent and the solution then added to the top of the column with care taken not to disturb the top of the column. The solvent level was again lowered to the top of the silica gel. A layer of approximately 5 mm of silica gel was then added to the top of the column to prevent the compound from migrating back up into the solvent reservoir before the eluent was added and the column eluted. For preparative T L C , a complex was dissolved in a minimum amount of eluent and the solution then loaded onto the T L C plate (20 cm x 20 cm x 1000 pm thick, Uniplate from Analtech). Loading consisted of slowly streaking the solution across the plate forming a line about 1 cm in height centred approximately 1 inch from the bottom of the plate, and leaving approximately 2-3 cm between the line edge and the plate edge on either side of plate. The compounds were loaded slowly to prevent broadening of the line of streaked solution, using up to 150 mg of sample on a single plate. The plates were dried in air for 30 min before being developed to prevent streaking and improve separation (Figure 2.3). 22 cm t 25 cm Figure 2.3 Preparative T L C chamber with silica gel plate. 30 2.4 Ruthenium Precursors 2.4.1 R u C V 3 H 2 0 This starting material, as stated in Section 2.1.5, was generously donated by Colonial Metals Inc. The actual oxidation state of the metal is likely a mixture of Ru1" and R u I v . 1 5 ' 1 6 The material comes with a certificate of authenticity stating a Ru content of 39 % which agrees with the formulation of RuCl 3-3H 20; however, the sample is highly hygroscopic and readily absorbs water if left exposed to air. 2.4.2 [Ru(DMD 6 ][CF 3 S0 3 ]3 This complex was synthesized using a modified version of the procedure published by Judd et al." A solution of RuCl 3-3H 20 (0.50 g, 1.9 mmol) in D M F (40 mL) was refluxed under a flow of H 2 for 3 h. The initially brown solution eventually became blue. To the blue solution was added AgCF 3 S0 3 (2.20 g, 8.6 mmol), and the resulting solution was then refluxed for 1 h under 1 atm N 2 . The mixture was then cooled in an ice-bath and then filtered to remove AgCl. The resulting filtrate was reduced in volume under vacuum at 80 °C to -10 mL, when 100 mL of EtOAc was added. The resulting yellow precipitate was then collected by filtration, washed with Et 2 0 ( 3 x 5 mL), and then dried under vacuum for 24 h at 78 °C . Yield: 1.60 g (85 %). ] H NMR (dmso-d6): 5 22.46, 19.73 (s, CH3). IR (KBr): 1650 (C=0). Anal. Calcd for C 2 1 H 4 2 F 9 N 6 O 1 5 S 3 R U : C, 25.56; H, 4.26; N, 8.52. Found: C, 25.22; H, 4.24; N, 8.46. The ' H NMR and IR data agree with those previously reported.17'18 2.4.3 m-RuCl 2 (DMSO) 3 (DMSO) This complex was synthesized according to the procedure of Evans et al}9 A -brown solution of R u C l 3 3 H 2 0 (0.80 g, 3.1 mmol) in DMSO (10 mL) was stirred and refluxed at 180 °C for 10 min, during which time the solution became red and then yellow. The yellow solution was then cooled to r.t., and acetone (50 mL) was added with continued stirring. Over 20 min a yellow precipitate formed. The mixture was filtered 31 and the yellow solid washed with acetone (3 x 10 mL), and then dried under vacuum at 78 °C for 48 h. Yield: 0.92 g (62 %). 'H NMR (CDC13): 8 2.70 (s, 6H, Cff3SO); 3.30, 3.41, 3.47, 3.50 (s, 18H, Cff 3SO). LR-MS (+LSIMS, thioglycerol): 485 (M+), 449 ( M + -CI), 371 (M + - CI - DMSO). IR (KBr): 916 (0=S); 1096, 1120 (S=0). Anal. Calcd for C8H2404S4Cl2Ru: C, 19.83; H, 4.99. Found: C, 20.04; H, 4.96. The ' H NMR and IR data are in agreement with those in the literature.19 2.4.4 K 3 [RuCl 6 ] This compound was synthesized from a literature procedure.20 A solution of RuCl 3-3H 20 (1.00 g, 3.82 mmol) in MeOH (50 mL) was refluxed under a stream of H 2 . After 3 h the brown solution became green. KC1 (0.90 g, 12 mmol) was then added and the mixture was refluxed in air for 3 h. As the KC1 dissolved, a brown precipitate formed, and this was collected by filtration and recrystalized from 12 M HCI. The product was then washed with MeOH (2 x 10 mL) and dried under vacuum for 24 h at 78 °C . Yield 1.22 g (74 %). UV-Vis (12 M HCI) 348 (3.41), 311 (3.31), 229 (4.38). The UV-Vis data agree with the literature values.20 2.4.5 R u 2 ( C H 3 C O O ) 4 C l The complex was synthesized via a modified version of the procedure by Stephenson and Wilkinson.21 A brown solution of RuCl 3-3H 20 (1.00 g, 3.82 mmol) in glacial acetic acid (30 mL) and acetic anhydride (5 mL) was refluxed in air for 2 h. The solution slowly developed a dark green colour and after 2 h was cooled to r.t. and filtered to remove a black precipitate. The solution was then refluxed for an additional 6 h at which time the green solution was cooled to r.t. and left standing in air for 16 h. Brown crystals were then removed from the solution by filtration, washed with cold acetic acid (2x5 mL, 0 °C) and Et 2 0 (2x5 mL) and then dried for 24 h at 78 °C . Yield: 0.65 g (72 %). LR-MS (+ESI): 440 ( M + - CI). IR (KBr): 1445, 1401 (n-MeC0 2). Anal. Calcd for C 8 H 1 2 0 8 C l R u 2 : C, 20.27; H, 2.53. Found: C, 20.43; H, 2.62. 32 2.5 References (1) Cliff, M. ; Pyne, S. Synthesis 1994, 681. (2) Baird, I. R.; Skov, K. A.; James, B. R.; Rettig, S. J.; Koch, C. J. Synth. Commun. 1998, 25, 3701. (3) Bagal, L. I.; Pevzner, M . S.; Frolov, A. N.; Sheludyakova, N. I. Khim. Geterotsikl. Soedin. [English Translation] 1970, 2, 240. (4) Haszeldine, R. N. J. Chem. Soc. 1953, 1548. (5) Husted, D. R.; Ahlbrecht, A. H. J. Am. Chem. Soc. 1953, 75, 1605. (6) Evans, D. F. J. Chem. Soc. 1959, 2003. (7) Crawford, T. H.; Swanson, J. J. Chem. Educ. 1971, 48, 382. (8) Figgis, B. N. Modern Coordination Chemistry; Lewis, J., Wilkins, R. G. ed.; Interscience Publishers, Inc.: London, 1960. (9) Sengupta, P.; Dinda, R.; Ghosh, S.; Sheldrick, W. Polyhedron 2001, 20, 3349. (10) Nakamoto, K.; McCarthy, P. J. Spectroscopy and Structure of Metal Chelate Compounds; John Wiley and Sons, Inc.: New York, 1968. (11) Pavia, D. L.; Lampman, G. M. ; Kriz, G. S. Introduction to Spectroscopy 2n^ ed.; Harcourt Brace & Company: Orlando, 1996. (12) Connelly, N. G.; Geiger, W. E. Chem. Rev. 1996, 96, 877. (13) Geary, W. J. Coord. Chem. Rev. 1971, 7, 81. (14) Huheey, J. E . Inorganic Chemistry: Principles of Structure and Reactivity, third ed.; Harper Collins Publishers: New York, 1983. (15) Hui, B. C ; James, B. R. Can. J. Chem. 1974, 52, 348. (16) Seddon, E. A.; Seddon, K. R. The Chemistry of Ruthenium; Elsevier: Amsterdam, 1984. (17) Judd, R. J.; Cao, R.; Biner, M. ; Armbruster, T.; Buergi, H ; Merbach, A. E.; Ludi, A. Inorg. Chem. 1995, 34, 5080. (18) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents , Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. 33 (19) Evans, I. P.; Spencer, A.; Wilkinson, G. J. J. Chem. Soc. Dalton Trans. 1973, 204. (20) James, B. R.; McMillan, R. S. Inorg. Nucl. Chem. Lett. 1975,11, 837. (21) Stephenson, T. A.; Wilkinson, G. / . Inorg. Nucl. Chem. 1966, 28, 2285. 34 Chapter 3 Synthesis and Characterization of Ruthenium(III) Maltolato and Imidazole Complexes 3.1 Introduction This chapter examines the synthesis and characterization of several new Ru(IU) maltolato and imidazole complexes. Ru(ma)3 was first synthesized by Greaves and Griffith in 1988,1 but the ' H NMR spectrum for this paramagnetic complex was not reported; such data are quite useful in determining structural information about this complex and its derivatives. The ! H NMR spectra of paramagnetic complexes can exhibit chemical shifts far removed from the "expected" diamagnetic ones.2 Paramagnetic shifts can range from a few Hz to > 100 ppm, and the signals are often broad and of relatively low intensity. Their appearance varies greatly depending on the geometry and nature of the ligands present.3'4 For these reasons, ! H NMR spectra of paramagnetic complexes can be difficult to interpret. In this Chapter, the ID *H NMR and 2D ' H - ' H COSY and ' H - I 3 C HMQC spectra of some Ru(JU) complexes are examined in order to elucidate structural information of the species in solution. Anderson and Beauchamp have studied the paramagnetic ] H NMR spectra of Ru(IU) complexes of imidazole and methyl-imidazoles,5'7 where 2-deuteroimidazole was used to make spectral assignments.8 A general trend was observed in the shift of the imidazole protons with H(5) < H(4) < H(2), the H(2) signal being shifted the farthest upfield. As will be discussed in Section 3.3, methyl signals on both maltolato and imidazole ligands shift downfield, while imidazole proton signals shift upfield. Eaton et al. have performed in depth studies of paramagnetic tropolonato species,9 and the 'H NMR data for Ru(tropolonato)3 have been used in assigning the maltolato and ethylmaltolato spectra presented in Section 3.2. In this thesis work, no distinct pattern has been observed for the chemical shifts of the maltolato ring protons; in fact, these proton signals are generally not observed in the spectra of the mixed maltolato imidazole complexes. 35 In addition to maltolato complexes with ancillary imidazole and nitroimidazole ligands, this work was expanded to investigate 3-nitro-l,2,4-triazoles (Figure 3.1) as well because nitrotriazoles, like nitroimidazoles, can be effective radiosensitizers.10 A triazole analogue of EF5 was synthesized and will be discussed in more detail in Section 3.3 and Chapter 7. 2 H 1 Imidazole 3-Nitro-l,2,4-triazole Figure 3.1 Numbered molecular structures of imidazole and 3-nitro-l,2,4-triazole. 3.2 Investigation of Ru(ma)3 and Ru(Ema)3 3.2.1 Characterization and Solution NMR Data for Ru(ma)3 (1) The synthesis of Ru(ma)3 (Figure 3.2) has been previously reported1 but, in this thesis work, its purification was modified to remove impurities (observed by TLC), thus improving the elemental analysis from that reported (Section 3.6.1). The IR data agreed with those reported1 and the mass spectrum showed the parent peak at 477. The Figure 3.2 Molecular structure of mer-Ru(ma)3 (1). 36 conductivity of an aqueous solution of 1 was 25 ^'cn^moi' 1, at the upper limit for a non-electrolyte,11 suggesting that a small amount of the maltolato ligand may be dissociating in solution. The structure of the red complex was determined in this department by X-ray crystallography (Figure 3.3), but disorder in the crystal (grown from slow evaporation of a CH2Cl2:acetone, 1:1 solution) prevented full refinement of the structure (see Appendix A l for structure details). Nevertheless, the complex crystallizes in the mer-configuration, and the solution 'H NMR spectrum at r.t. in CD 2 C1 2 also shows the mer-configuration as the primary isomer. C.12 Figure 3.3 ORTEP diagram of mer-Ru(ma)3 (1) with 33 % probability thermal ellipsoids (see Appendix A l for details). 37 The ! H NMR spectrum (Figure 3.4) clearly shows nine singlets assigned to mer-l and an additional 3 trace singlets that are assigned to the fac-isomer (8 33.8, 1.6, 1.0). 'H NMR was performed in CDC1 3, CD 2 C1 2 , CD3OD, D 2 0 and acetone-ek and in each case the mer-isomer was observed. The three resonances assigned to the fac-isomer are only observed in CDC1 3 and CD 2 C1 2 in which 1 is more soluble, and varied proportionally in intensity between different samples, giving further evidence that their assignment to a single species is correct. CDHC1 2 H(5<)/H(6) Figure 3.4 'H NMR spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2C1 2 . Resonances for the mer-isomer have been labelled and those for the fac-isomer have been identified with boxes. The signals shifted the farthest downfield, 8 43.2, 41.0 and 21.1, are for the Me groups on the maltolato ring, and this was confirmed by the 'H- 'H COSY spectrum (Figure 3.5); these 3 signals showed no crosspeaks as expected for the Me groups, while the remaining 6 resonances each showed a mutual correlation to one of the other resonances, giving 3 pairs of singlets (8 11.8 and 9.2, 3.1 and -4.6, 0.8 and -0.9), one pair for the H(5) and H(6) protons for each maltolato ring. The resonances assigned to the solvent, CDHC1 2, and to H 2 0 also gave no crosspeaks, suggesting that these assignments are correct. 38 Figure 3.5 ' H - ' H COSY spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2 C1 2 . The signals downfield of 8 20 are omitted as they showed no crosspeaks. The coupled pairs for H(5) and H(6) are highlighted with boxes. The ' H - 1 3 C H M Q C spectrum (Figure 3.6) provides further evidence for the ] H assignments, as the 6 maltolato ring proton signals each give a crosspeak between 8 75 and 175 in the 1 3 C NMR spectrum as expected for protons on an sp2-hybridized carbon.12 Two crosspeaks for the Me groups are also observed, that for the 'H signal at 8 21.1, giving a l 3 C crosspeak at 8 -7, the other for the trace fac-signal at 8 33.8 giving rise 39 Figure 3.6 ' H - 1 3 C H M Q C spectrum (300 MHz, 298 K) of Ru(ma)3 (1) in CD 2 C1 2 with the 1 3 C spectrum on the left-hand side and the ' H spectrum at the top. Some decomposition occurred in this sample leading to additional crosspeaks. The H(5) and H(6) protons, the solvent and two Me groups, which all show crosspeaks, are indicated with arrows. to a 1 3 C crosspeak at 5-52. Crosspeaks for the remaining two Me groups (8 41.0 and 43.2) may be shifted too far upfield in the 1 3 C NMR spectrum, beyond 8-75, to be detected. Additional signals in the ! H NMR spectrum between 8 0-5 result from slow decomposition of the sample in situ as the acquisition of the H M Q C spectrum takes several hours. Although the decomposition products may be present in very small amounts, they can still give rise to relatively intense signals compared to those observed 40 for the paramagnetic complex. 1 3 C signals for these decomposition products are also not observed, but crosspeaks are seen between 8 0-40 with the 1 3 C N M R spectrum. Only a signal (8 54) for C D 2 C 1 2 is observed in the 1 3 C N M R spectrum after 12 h; however, a partial spectrum can be generated from the crosspeaks of the ' H - 1 3 C H M Q C experiment, producing a spectrum that contains signals for those C-atoms that show correlation with signals in the ' H N M R spectrum (Figure 3.7). —| . | . | , | , | , | , , , 1 , , , , , ! , ! , f 180 160 140 120 100 80 60 40 20 0 -20 -40 Figure 3.7 Partial 1 3 C N M R spectrum of 1 (300 M H z , 298 K ) in C D 2 C 1 2 generated as a positive projection of the y-axis from the ' H - 1 3 C H M Q C spectrum of 1. Only carbons that gave rise to crosspeaks in the 2D spectrum are observed (o denotes carbons attached to H(6) protons and • denote those attached to H(5) protons). The ' H N M R spectrum of Ru(ma) 3 was also recorded at lower temperatures (Table 3.1). These shifts can be plotted vs 1/T (Figures 3.8 and 3.9) and the intercept at 1/T = 0 should give the value expected for a corresponding diamagnetic species. 1 3 From the intercepts of Figure 3.7, it can be determined which of the six signals correspond to H(5) and H(6), because for free maltol, H(5) is observed at 8 6.4 and H(6) at 8 7.7. For each pair of singlets observed in the ' H - ' H C O S Y spectrum of 1, one can be assigned as H(5) and the other as H(6). H(a) and H(b) show correlation in Figure 3.5, and in Figure 3.8 give X-intercepts at 8 5.2 and 8.2, respectively; from these values, H(a) can be 41 assigned as an H(5) proton, and H(b) as an H(6) proton. Similarly, H(c) and H(d) can be assigned as H(6) protons, and H(e) and H(f) can be assigned as H(5) protons. The X -intercepts for the Me groups in Figure 3.9 (8 14.3, 10.0 and 9.0) do not correlate well with the Me signal of free maltol (8 2.4). The ' H N M R shift data were also used to determine peff (1-52 B M at 22 °C) using Evan's method.14 Table 3.1 Variable temperature chemical shifts of Ru(ma)3 (300 MHz) in CD2CI2 including values at T = co, as determined by linear regression. Temp (K) 5 Mel 8Me2 5Me3 8H(a) 8H(b) 8H(c) 8H(d) 8H(e) 8H(f) 0 0 9.0 10.0 14.3 5.2 8.2 9.0 9.0 7.5 6.7 280 45.969 43.039 22.061 12.659 8.869 3.264 0.092 -1.368 -5.022 267 47.520 44.149 22.391 12.955 8.889 3.013 -0.327 -1.715 -5.488 256 49.163 45.936 22.807 13.269 8.932 2.722 -0.756 -2.080 -5.992 244 50.968 47.609 23.261 13.626 8.965 2.392 -1.223 -2.485 -6.546 233 52.986 49.384 23.719 14.023 9.002 2.044 -1.724 -2.930 -7.146 221 55.162 51.482 24.232 14.468 9.043 1.647 -2.312 -3.441 -7.837 212 57.6234 53.680 24.806 14.976 9.089 1.217 -2.953 -3.992 -8.574 200 60.351 56.057 25.379 15.521 9.134 0.702 -3.655 -4.632 -9.392 0.006 Figure 3.8 Plot of the chemical shift (ppm) vs. 1/T ( K 1 ) for the H(5) and H(6) protons on each ma ring of Ru(ma)3 (1), here labelled H(a)-(f). 42 0.006 3.2.2 Characterization and Solution N M R Data for Ru(Ema) 3 (2) The general synthesis for Ru(Ema)3 (2) is the same as for 1, and 2 has also been characterized by elemental analysis, MS and IR spectroscopy. The ID and 2D NMR spectra for 2 are more complicated than those for 1 because of the presence of an Et vs. Me group. Ru(Ema)3 also exists predominantly as the mer-isomer in solution based on comparison with the ' H NMR spectrum for 1. The r.t. 'H NMR spectrum for 2 (Figure 3.10) in CD2CI2 shows 6 resonances of roughly equal intensity shifted downfield past 5 15, as opposed to the 3 seen in the 'H NMR spectrum of 1 (Figure 3.4), and there are now two trace signals observed for the fac-isomer in the downfield region as opposed to the one seen for Ru(ma)3. The three resonances for the Me, H(5) and H(6) protons for the fac-isomer of 2 are not observed, and presumably overlap with signals for the mer-isomer. The 6 major downfield signals are assigned to the 6 inequivalent protons of the three C H 2 groups. 43 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 - 5 Figure 3.10 *H NMR spectrum of Ru(Ema)3 (2) (300 MHz, 298 K) in CD 2 C1 2 . The resonances for the C H 3 of the Et groups are also shifted downfield (8 4.9, 4.8, and 2.1 for 2 vs. 8 1.22 for free ethylmaltol in CD 2C1 2), but by a considerably less amount than are the C H 2 signals. The Me signals are distinguished from those of the maltolato ring protons and from the residual solvent signal using 2D NMR spectroscopy. The *H-'H COSY spectrum clearly shows that there are again 3 pairs of singlets for the maltolato ring protons (8 12.5 with 9.0, 4.9 with -4.9, and 1.2 with -0.8) (Figure 3.11). The spectrum also shows pairs of signals for the C H 2 groups (those signals shifted downfield of 8 15) correlating with signals for the corresponding Me groups for each Ema ligand (8 40.1 and 35.3 with 8 2.1, 8 38.8 and 33.4 with 8 4.9, and 8 21.7 and 18.9 with 8 4.8). The ' H - ' H COSY spectrum also shows correlation between the two C H 2 protons, but only for the signals at 8 21.7 and 18.9. Although the resonances at 8 40.1 and 35.3 do not give rise to mutual crosspeaks, they can be still be assigned to one C H 2 group as they correlate with the same Me group (8 2.1). The same holds true for the C H 2 resonances at 8 38.8 and 33.4 that give a crosspeak with the same Me resonance (8 4.9). As in the 1 3 C NMR spectrum of 1, that of 2 shows no signals for the complex, only a single resonance for CD 2 C1 2 (8 54). The ! H - 1 3 C H M Q C spectrum, however, 44 Figure 3.11 ' H - ' H COSY spectrum of 2 (300 MHz, 298 K) in CD 2 Cl 2 . .a) Complete spectrum, b) Expansion showing correlation between C H 2 protons shifted downfield for 2 of the ligands. c) Expansion showing correlation between C H 2 and Me protons for the same two C H 2 groups shown in (b). d) Expansion showing H(5) and H(6) correlations for all 3 ligands (blue boxes) as well as the CH 2 /Me and C H 2 / C H 2 correlations for the third ligand not observed in (b) or (c) (purple boxes). 45 shows crosspeaks for many of the 'H signals with the 1 3 C NMR spectrum of 2 (Figure 3.12). In particular, there are 6 signals observed between 8 75 and 175 for the H(5) and H(6) protons on the maltolato ring, as for Ru(ma)3. No crosspeaks are observed for either the C H 2 or C H 3 of the ethyl groups. The partial 1 3 C NMR spectrum, as for 1, can be generated for 2 from the H M Q C spectrum. H(5) 20 15 10 5 0 Figure 3.12 ' H - 1 3 C H M Q C spectrum (300 MHz, 298 K) of Ru(Ema)3 (2) in CD 2 C1 2 with the I 3 C spectrum on the left-hand side and the ' H spectrum at the top. Some decomposition occurred in this sample leading to additional crosspeaks. The H(5) and H(6) protons are indicated with arrows, as is the solvent. The 'H NMR resonances downfield of 8 20 have been omitted as no crosspeaks were observed in this region of the spectrum. 46 3.2.3 "Activation" of Ru(ma)3 with C F 3 S O 3 H - Synthesis of [Ru(ma)2(EtOH)2]CF3S03 (3) The reaction of Ru(ma)3 (1) with triflic acid under N 2 was monitored in CD 3 OD (Figure 3.13). The C D 3 O D was degassed for one minute with N 2 in an NMR tube, to which was added 10 mg of 1. The 'H NMR spectrum of the sample was recorded (Figure 3.12a), then the NMR tube was removed from the spectrometer and 1 equivalent of CF3SO3H was added to the solution of 1, still under N 2 . This mixture was shaken vigorously for 2 min, and the spectrum of the mixture was then recorded (Figure 3.13b). The total time lapse between adding the triflic acid and recording the spectrum was 10 min. The sample was then left sitting at r.t. for an additional 50 min before recording the 'H NMR spectrum of the solution mixture again (Figure 3.13c). In this reaction, loss of the signals for 1 was accompanied with generation of signals for a new Ru(UI) complex, as well as signals for free maltol [8 2.4 (s, Me), 6.4 (d, H(5)), and 7.7 (d, H(6))]. There is only one Ru(IJI) ma containing species present in solution after 1 h as evidenced by the single broad resonance at 8 45, likely for rrans-[Ru(ma)2(CD30D)2]CF3S03 (see below). When 1 was treated with triflic acid in EtOH in a synthetic reaction, the initially red solution darkened and, after 1 h, T L C analysis revealed that 1 had been quantitatively converted to a new product. The volume of this solution was then reduced and the complex precipitated from the solution on addition of Et 2 0. This complex has been characterized by elemental analysis, MS, IR and 'H NMR spectroscopies as [Ru(ma)2(EtOH)2]CF3S03 (3). The parent peak is found in the mass spectrum as are peaks for the subsequent fragmentation loss of one and two EtOH ligands. The IR spectrum shows strong bands for the C=0 of ma and for O-H of EtOH. The conductivity of an EtOH solution of 3 (36 Q^cn^mol"1) was in the range for a 1:1 electrolyte. When 3 is dissolved in CD3OD, the immediate ! H NMR spectrum shows a single species (as evidenced by a single resonance for the ma-Me group); this then decomposes over 15 min to yield a spectrum for a complex identical to that formed from the in situ reaction (Figure 3.13c), with the exception that the free maltol signals are now absent and free EtOH signals are now present. The presence of only one Me signal for the maltolato ligand is consistent with a trans-isomer as observed with all the structurally characterized, Im-ma complexes described in the next section. 47 a) b) c) 60 50 40 30 20 10 0 -10 -20 Figure 3.13 In situ formation of [Ru(ma)2(CD30D)2]CF3S03 as shown by *H NMR spectroscopy, the broad downfield signal being characteristic of a trans-geometry for the maltolato ligands (see text), a) Ru(ma)3 in CD 3 OD b) Ru(ma)3 in CD 3 OD.+ C F 3 C O O H after -10 min c) Ru(ma)3 in C D 3 O D + C F 3 C O O H after ~1 h. 48 3.3 Reactions of Ru(ma)3 (1) and Ru(Ema)3 (2) with Imidazoles and Triazoles 3.3.1 Synthesis and Characterization of Imidazole and Triazole Complexes Complexes of the form [Ru(ma)2(L)2]CF3S03 discussed in this section can be divided into two groups, those without nitro groups: L = 4MeIm (6), Im (7), lMelm (8), 2MeIm (10); and those with nitro groups: L = metro (4), EF5 (14), 2N02Im (15), 4N02Im (16), 3N02tri (17) and triF5 (18). Two additional complexes, [Ru(ma)(lMeIm)4][CF3S03]2 (9) and [Ru(ma)2(metro)(EtOH)]CF3S03 (5) were also isolated. These complexes have been characterized generally by elemental analysis, mass spectrometry, and NMR and IR spectroscopies. The complexes 15-18 were insoluble in all matrices for LSfMS, and lacked sufficient solubility in applicable solvents to examine them by ESI-MS. Solid state MS techniques were not available in the department MS facility. Complexes 6, 7, 8, and 10 (Figure 3.14) were synthesized by reacting 1 with one equivalent of triflic acid in EtOH at r.t., and then adding excess of the imidazole to the reaction mixture. All four complexes were purified using preparative T L C and isolated as red solids. MeOH solutions of each gave conductivity values in the range for 1:1 electrolytes.15 The elemental analyses for 6 and 8 indicate the presence of one CH 2 C1 2 solvate, and in the case of 8, the solvated CH 2 C1 2 was detected by X-ray crystallography (Figure 3.16, Appendix A2). L = metro (4), 4MeIm (6), Im (7), lMelm (8), and 2MeIm (10). Figure 3.14 Molecular structures for the trans-isomers of 4, 6 - 8 and 10. 49 The reaction between 1, CF3SO3H and 4 equivalents of l M e l m in refluxing E t O H was followed by T L C . A blue species that increased in intensity over time became the major product after 48 h, and was isolated by preparative T L C (18 % yield) and formulated as 9 (Figure 3.15). The reaction was repeated adding an 8-fold excess of l M e l m and two equivalents of CF3SO3H instead of one; the percent yield of 9 after 48 h increased to 51 %. The conductivity of a M e O H solution of 9 (273 Q^cn^mol" 1 ) is in the range for a 2:1 electrolyte. 1 5 Figure 3.15 Molecular structure of [Ru(ma)(lMeIm)4][CF 3 S0 3 ]2 (9). 4 (Figure 3.14) was synthesized by the same procedure as for 6, 7, 8, and 10, but with refluxing the reaction mixture for 6 h; the product then precipitated when the solution was cooled. Work-up of the filtrate led to the isolation of a second, green product in low yield, 5. Elemental analysis and M S confirmed the formulations of 4 and 5. 4 was then subsequently crystallized by another member of our group, and the X-ray structure determined that it was of a trans-configuration.1 6 2+ / (CF 3S0 3) 2 / 50 Complexes 8 and 10 have also been characterized by X-ray crystallography (Figures 3.16 and 3.17, respectively), and all three structures show trans-imidazole ligands and a centre of inversion at the Ru. 51 Figure 3.17 ORTEP diagram of the cation of rran5-[Ru(ma)2(2MeIm)2]CF3S03 (10) with 50 % probability thermal ellipsoids (see Appendix A3 for details). The Ru-N bond lengths for 10 might be expected to be longer than those for 8 as the 2-Me group could give rise to steric interactions with the ma ligands: in 8, a 1-Me derivative, the imidazole Me groups point away from the ma ligands and in 10 they point towards the ma ligands. However, there are no differences in the Ru-N bond lengths. Instead, the imidazole rings in 10 tilt to move the Me group away from the plane of the maltolato ligands (Figure 3.18). This can be seen by looking at the Ru-N(3)-C(2) and Ru-N(3)-C(4) angles (see Figure 3.19 for the atomic numbering scheme) in the structures of 8 and 10. For 8, these angles differ by less than one degree (Table 3.2), while for 10 these angles differ by 6.2 degrees. 52 H H Figure 3.18 Diagram depicting the potential steric interaction between an Im-Me group and the ma plane for 10. a) Steric interaction when coordinated symmetrically about N(3). b) Shows how tilting at the coordinated nitrogen removes this steric interaction by increasing the Ru-N(3)-C(2) bond angle and decreasing the Ru-N(3)-C(4) bond angle. Table 3.2 Selected X-ray data from bis-imidazole derivative structures. The numbering used is shown in Figure 3.16.a Complex Ru-N(3) bond length Ru-O(l) bond length Ru-0(2) bond length Ru-N(3)-C(2) bond angle Ru-N(3)-C(4) bond angle rran5-[Ru(ma)2( lMeIm)2]CF3S03 (8) 2.072(5) 2.012(4) 2.065(4) 126.7(4) 127.6(4) rranHRu(ma)2(2MeIm)2]CF3S03 (10) 2.078(2) 2.006(2) 2.061(2) 129.7(2) 123.5(2) ?rans-[Ru(Ema)2(metro)2]CF3S03 (11) 2.080(3) 1.998(3) 2.051(3) 130.6(3) 122.0(3) fra«HRu(ma) 2(metro) 2]CF 3S0 3 (4) 2.069(3) 2.010(3) 2.063(3) 130.1(3) 122.8(3) a - the data for 4 are taken from reference 15. Figure 3.19 Template with numbering scheme for crystal structure information displayed in Table 3.2. 53 Complexes of the form [Ru(Ema)2(L)2]CF3S03 were also synthesized from Ru(Ema)3 with L = metro (11), 4MeIm (12) and 2MeIm (13). Again, complexes were characterized by elemental analysis, MS, and NMR and IR spectroscopies, while 11 was also characterized by X-ray crystallography (Figure 3.20). 11 was synthesized in the same manner as was 4; however, the observed green T L C band, likely containing [Ru(Ema)2(metro)(EtOH)]CF3S03 (analogous to 5), did not generate an isolable solid. The structure of 11, like those for 4,1 6 8 and 10, shows trans-imidazole ligands with a centre of inversion at Ru, and the distortion of bond angles at N(3), to relieve the steric interaction between the 2-Me group and the ma-type ligands, is observed in the structures o f 4 1 6 a n d l l , as was observed for 10 (Table 3.2). Figure 3.20 ORTEP diagram of the cation of trans-[Ru(Ema)2(metro)2]CF3S03 (11) with 50 % probability thermal ellipsoids (see Appendix A4 for details). 54 [Ru(ma)2(L)2]CF3S03 (L = 2N02Im ( 1 5 ) , 4N0 2Im ( 1 6 ) , 3N02tri ( 1 7 ) ) were synthesized by adding an excess of the appropriate nitroimidazole or nitrotriazole to a solution of 1 and triflic acid in EtOH under N 2 and then refluxing the mixture for 24 h. Corresponding reactions at r.t. showed [Ru(ma)2(EtOH)2]CF3S03 as the major species present in solution after 24 h, as analyzed by TLC. The elemental analyses for 1 5 - 1 7 were consistently high in C, H and N and, as the ! H NMR spectra of suspensions of 1 5 and 1 6 in D 2 0 gave weak signals for free 2N02Im and 4N02lm, respectively, some free ligand might be present in these complexes. 3.3.2 Solution X H N M R Spectroscopy of the Im, 2MeIm, 4MeIm and lMelm Complexes The ' H NMR spectra of 6 - 1 0 were examined in CDCI3; the data for the structurally characterized complexes 8 and 1 0 are consistent with the primary isomer in solution being the trans-isomer (Table 3.3). Table 3.3 *H NMR data of Ru(IU) maltolato-imidazole complexes in CDC1 3 at r.t. Complex 8 ma-Me 8 Im-Me 8 Im H(5) 8 Im H(4) 8 Im H(2) rra«i-[Ru(ma)2(4MeIm)2]CF3S03 (6) 60.0 16.3 -4.5 - -20.0 rrans-[Ru(ma)2(Im)2]CF3S03 (7) 62.2 - -3.7 a -19.6 c/5-[Ru(ma)2(Im)2]CF3S03 (7) 60.0, 44.6 -1.5,-5.6 -15.5,-24.7 rrarc5-[Ru(ma)2(lMeIm)2]CF3S03 (8) 62.6 13.4 -3.8 a -19.0 [Ru(ma)(lMeIm)4][CF3S03]2 (9) 81.6 25.6,20.4 -0.5,5.7 a -18.4, 24.0 rra«5-[Ru(ma)2(2MeIm)2]CF3S03 (10) 67.0 42.0 -1.6 -4.7 -a -signals for these protons were not observed. For [Ru(ma)2(lMeIm)2]CF3S03 ( 8 ) , the ma-Me resonance is shifted downfield to 8 62.6 with respect to that of the Hma-Me (8 2.37), and the Im-Me resonance at 8 13.41 (Table 3.3) also shows a downfield shift with respect to that of free lMelm (8 3.64). The shift for the Im-Me resonance is consistent with data for other Ru(UI)-lMeIm complexes,5 and that for the ma-Me resonance is considered consistent with that observed for Ru(IU)-ma (Table 3.1). The ! H NMR spectrum of 8 was first studied in acetone-^ 55 (Figure 3.21a), following purification of the complex by preparative TLC: the data suggest the presence of a mixture of isomers. An in situ synthesis in C D 3 O D showed the formation of only the trans-isomer. When MeOH was used to extract the complex from the T L C plate, the ] H NMR spectrum of the isolated solid in CDCI3 showed numerous peaks between 8 1-5 that were not present in the *H NMR of the original crude mixture before purification; these can likely be assigned to the binder and other components of the T L C plate that may be soluble in MeOH. The workup procedure was then modified to use 1-2% MeOH in CH2CI2 to remove the complex from the silica scraped from the plate. The 'H NMR spectrum of 8 was then recorded in CDCI3 and showed the trans-isomer in almost 100% purity (Figure 3.21b). Extraction of the other TLC-isolated imidazole complexes (6, 7, 9, 10, 12 and 13) from the silica was subsequently performed using either neat CH 2 C1 2 or a mixture of MeOH (< 5%) and CH 2 C1 2 . ' ''0 ' 60 ' s o ' 10 30 20 10 0 -10 -20 Figure 3.21 *H NMR spectrum of [Ru(ma)2(lMeIm)2]CF3S03 (8) in a) acetone-d6 (300 MHz, 298 K) and b) CDC1 3 (300 MHz, 298 K). As discussed in Section 3.3.1, the complexes [Ru(ma)2(L)2]CF3S03, where L = Im, 2MeIm, 4MeIm and lMelm, were all synthesized via reactions at r.t. Heating the reaction mixtures led to the formation of numerous side-products. In the case of lMelm 56. (Figure 3.22), at least 11 signals for the ma-Me group were observed in the ! H NMR spectrum in CDC1 3 of a solid isolated after a reaction mixture of 1 in EtOH with four equivalents of lMelm and one equivalent of triflic acid had been refluxed for 6 h. Numerous additional signals were also observed in the regions where the imidazole methyl (5 21 to 12) and H(5) (8 2 to -8) proton resonances occur. After this reaction mixture was refluxed for 48 h, [Ru(ma)(lMeIm)4][CF3S03]2 (9) was isolated. The signal for the ma-Me of 9 (8 81.6, Figure 3.23) is shifted farther downfield than for the bis-imidazole complexes (Table 3.3). Two sharp resonances at 8 25.6 and 20.4, assigned as Im-Me groups, are shifted farther downfield than are those observed for the Im-Me groups of 8 (8 13.4). a) ill fl b) 70 60 50 40 30 20 i 10 1 1^  0 I -10 -20 p p n 70 60 50 40 30 20 10 0 -10 -20 Figure 3.22 The effect of varying the temperature when reacting 1 with lMelm. a) 'H NMR spectrum in CDC1 3 (300 MHz, 298 K) of the crude reaction mixture isolated after refluxing Ru(ma)3 with 4 equiv. of lMelm and 1 equiv. of triflic acid in EtOH under N 2 for 6 h. b) ] H NMR spectrum (300 MHz, 298 K) of [Ru(ma)2(lMeIm)2]CF3S03 (8) in CDC1 3. Attempts to form similar tetrakis(imidazole) complexes with Im, 2MeIm, and 4MeIm were unsuccessful. Attempts to form a mixed tetrakis(imidazole) complex by refluxing 6 equivalents of lMelm with one equivalent of [Ru(ma)2(2MeIm)2]CF3S03 (10) and one equivalent of C F 3 S 0 3 H in EtOH were also unsuccessful. 57 Im-H(5) ma-Me Im-H(2) -11) -20 Figure 3.23 'H NMR spectrum (300 MHz, 298 K) of [Ru(ma)(lMeIm)4][CF3S03]2 (9) in acetone-<2?6 showing the ma-Me at 8 81.6 and lMelm H(2) protons at 8 -19.4 and -24.0. Two sharp resonances at 8 25.6 and 20.4 are assigned as Im-Me groups. The H NMR spectrum of ?ran5-[Ru(ma)2(2MeIm)2]CF3S03 (10) shows a single resonance at 8 67.0 for the ma-Me groups (Figure 3.24); resonances for the H(4) (8 -4.7), H(5) (8 -1.6), and Im-Me (8 42.0) protons are also observed. 58 The *H NMR spectrum of [Ru(ma)2(4MeIm)2]CF3S03 (6) also shows only a single broad resonance for the ma-Me protons. Even though this complex has not been characterized crystallographically, it can be inferred from similarities between its *H NMR spectrum and those of trans-S and trans-10, that 6 is also in a trans-configuration. The resonance for the Im-Me group is shifted less downfield for 6 (5 16.3) than is observed for 10 (8 42.0). In some Ru(ITJ) complexes formed from 4MeIm, the 4MeIm isomerizes to 5MeIm upon coordination,5 to relieve steric interactions between the Me group and other coordinated ligands. Anderson and Beauchamp have reported the 'H NMR spectrum for the anion [RuCl4(5MeIm)2]" in D2O, attributing signals between 8 -0.2 and 2.3 to the Me group of coordinated 5MeIm.5 Previous work in our group has described the chemical shifts of the Im-Me groups of a mixed 4-Me/5-Me Ru(UI) cation, [Ru(acac)2(4MeIm)(5MeIm)]+, assigning a signal for the 4-Me group at 8 14.7 and one for the 5-Me group at 8 7.5.17 This suggests that [Ru(ma)2(4MeIm)2]CF3S03 (6) likely exists as the 4-Me species as its structure is analogous to that of the acac complex, and the chemical shift of the Im-Me falls close to that reported for the 4-Me group of the mixed-acac complex. It is difficult to determine if the chemical shift of the imidazole proton of 6 is definitive of either an H(4) or an H(5). Although the relative chemical shifts of the Im protons remain constant, H(5) < H(4) < H(2), H(2) being the farthest upfield, the absolute regions in which these resonances are found varies considerably and are dependent on the nature of the other coordinated ligands. Steric interactions between the Im-Me and the ma ligands of 10 are overcome as the 2MeIm tilts to move the Me away from the ma plane (Table 3.1, Section 3.3.1). The 4MeIm of 6 could tilt in a similar manner to remove steric interactions without isomerizing to 5MeIm. Without an X-fay structure, it is not possible to define this complex as the 4MeIm species, but this would agree with the literature observations and the data collected for this complex. The 'H NMR spectrum of [Ru(ma)2(Im)2]CF3S03 (7) showed both the cis- and trans-isomers in solution, despite using the modified isolation technique that succeeded in removing the cis-isomers for 6, 8, and 10. Attempts to separate the two isomers of 7 by chromatography were unsuccessful. The intensities of the 'H NMR signals for the cis-isomer varied between samples suggesting the absence of a solution equilibrium, and that two overlapping T L C bands may be the source of the two isomers. When silica taken 59 from the lower part of the red band was not included in the isolation, weaker signals for the cis-isomer were observed in the 'H NMR spectrum, but the 'H NMR spectrum taken from a sample isolated from a thin section (~1 mm in width) cut from the top of the red band still revealed the presence of two isomers in solution. Table 3.4 summarizes some 'H NMR signals for trans-[Ru(Ema)2(4MeIm)2]CF3S03 (12) and rran5-[Ru(Ema)2(2MeIm)2]CF3S03 (13), both synthesized from Ru(Ema)3, and the data for the corresponding ma complexes, 6 and 10; the chemical shifts for the Im-protons and -Me groups of 12 and 13 are almost identical to those of 6 and 10, respectively, while the Ema-CH 2 resonances are shifted upfield from those of the ma-Me resonances for 6 and 10, respectively. As only one resonance is observed for the Ema-CH 2 group, the Im-Me group, and each of the Im protons for both 12 and 13 (Figure 3.25), these complexes are likely trans-isomers. The chemical shift of the Im-Me group for 12 is almost identical to that of 6, and therefore, by the same reasoning discussed previously in this Section for 6, 12 also likely exists as a 4MeIm complex and not a 5MeIm one. The resonances for the Ema-CH 2-C7/ 3 is not observed and likely overlaps with the 8 0-10 region in the spectrum where the solvent signals likely overwhelm any paramagnetic resonances. Table 3.4 'H NMR data for the Ema-Im complexes 12 and 13, compared with those for the ma-Im complexes 6 and 10. Complex 8 Ema- 8 ma- 81m- 81m- 81m- 81m-C H 2 C H 3 C H 3 H(5) H(4) H(2) rra«HRu(Ema) 2(4MeIm) 2]CF 3S03 (12) 49.1 - 16.5 -4.6 - -19.0 franHRu(Ema)2(2MeIm)2]CF3S03(13) 51.7 - 43.7 -1.9 -5.2 -?ra«i-[Ru(ma) 2(4MeIm) 2]CF 3S0 3 (6) - 60.0 16.3 -4.5 - -20.0 rrart5-[Ru(ma)2(2MeIm)2]CF3SO3(10) _ 67.0 42.0 -1.6 -4.7 60 Figure 3.25 'H NMR spectra (300 MHz, 298 K) of a) [Ru(Ema)2(2MeIm)2]CF3S03 (13) in CDC1 3 and b) [Ru(Ema)2(4MeIm)2]CF3S03 (12) in CDC1 3. The H(2) signal in (b) is very broad and can only be observed when the y-axis is expanded. 3.3.3 Solution X H NMR Spectroscopy of the metro, 2N02Im, 4N02Im and 3N02tri Complexes Of the complexes with metro (4, 5, and 11), 2N0 2Im (15), 4N0 2Im (16) and 3N02tri (17), only those with metro were soluble in aqueous solution. Unlike the *H NMR spectra for 6-10, 12 and 13, which were measured in CDC1 3 (Section 3.3.2), the *H NMR spectrum of [Ru(ma)2(metro)2]CF3S03 (4) was measured in acetone-ak because of its insolubility in CDC1 3; observed were a broad resonance for the ma-Me group (5 70.0) and another for the Im-Me group (8 34.0), consistent with a trans-geometry (as discussed in Section 3.3.2 for 6, 8 and 10) and the solid state structure.16 Similarly, in the *H NMR spectrum of [Ru(Ema)2(metro)2]CF3S03 (11), broad signals for each of the ma-Me protons (8 65.3), the Im-Me protons (8 32.2), and the H(5) of metronidazole (8 -4.8) are consistent with the trans-geometry determined by X-ray crystallography (Section 3.3.1, Figure 3.20). 61 The *H NMR spectrum of [Ru(ma)2(metro)(EtOH)]CF3S03 (5) in acetone-^ shows 4 signals in the region where the ma-Me group is typically observed. Five singlets between 8 21 and 8, which are not present in the spectrum for 4, were also observed. These signals could be due to the H(5) or H(6) protons of ma, the metro-Me group, the metro 1-hydroxyethyl group, or bound EtOH; signals for free EtOH were not observed even after 1 h showing that the acetone-^ was not displacing the coordinated EtOH. The [Ru(ma)2(L)2]CF3S03 complexes, where L = 2N0 2Im (15), 4N0 2Im (16) and 3N02tri (17) were only readily soluble in dmso-6?6 for ] H NMR investigation. For each of these three complexes, no paramagnetic signals were detected; however, intense signals were observed for the free heterocyclic ligands, implying that the complexes dissociate the (N)-bound ligands in dmso-dg. Weak signals were also observed for free ma, suggesting further decomposition of the complexes. The 'H NMR spectrum of a suspension of 16 in acetone-^ was recorded (4096 scans over 2h) and a trace resonance was observed for the coordinated ma-Me group at 8 63.8. Attempts to study 15 and 17 in a similar manner were unsuccessful. Further study of 15 - 17 is needed to better understand their structures. 3.3.4 Compounds Synthesized Using 2,2,3,3,3-Pentafluoropropylamine (F5) Three heterocyclic compounds were prepared via reaction with 2,2,3,3,3-penta-fluoropropylamine (F5). An improved synthesis of EF5 (Figure 3.26) has been published o EF5 o triF5 N 0 2 Figure 3.26 Molecular structures of three compounds synthesized from F5. 62 by our group,18 but was further modified in this thesis work to limit the number of reagents required to convert F5 into EF5 and conserve the amount of F5 required because of the rapidly rising cost of this amine. In this work, F5 was purchased from Lancaster or Interchim or was prepared according to a published procedure (Figure 3.27).19'20 ' C F 2 ^ - O H C F 2 .OMe ^"2. CFo N H F 3 C ^ H 2 S Q 4 , M e Q H ^ N H 3 F 3 C ^ ^ L i A I H 4 j F 3 C ^ 2 P2O5 II E t 2 0 J O Figure 3.27 Synthesis of 2,2,3,3,3-pentrafluoropropylamine (F5) from 2,2,3,3,3-pentafluoroproprionic acid. The procedure for synthesizing EF5 used chloroacetyl chloride to form the precursor N-(2,2,3,3,3-pentafluoropropyl)chloroacetamide (C1F5, Figure 3.26) instead of the previously used iodoacetic acid to form N-(2,2,3,3,3-pentafluoropropyl)-iodoacetamide (IF5).18 The chloroacetyl chloride reaction proceeded more quickly and gave a higher yield (91 for C1F5 vs. 53 % for IF5) than the corresponding reaction in which iodoacetic acid is first activated with isobutyl chloroformate before being reacted with F5. 1 8 The reaction of C1F5 with 2N02lm to afford EF5 proceeded identically to 1 & that reported for the corresponding reaction with IF5. The procedure used to prepare EF5 was also used to synthesize the new ligand 2-(3-nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (triF5, Figure 3.26), but with use of 3N02tri instead of 2N02lm. Elemental analysis agreed with the formulation for triF5 that was further characterized by NMR and IR spectroscopies. A report investigating nitrotriazoles as potential anticancer agents10 led to the interest in developing this triazole analogue of EF5. Although complexes of this ligand were not tested for bioactivity in this work, preliminary biological results for triF5 itself will be discussed in Chapter 7. The compound lH-imidazole-4,5-N,N'-bis(2,2,3,3,3-pentafluoropropyl)-dicarboxamide, (IMF10, Figure 3.28), was synthesized by reacting imidazole-4,5-dicarboxylic acid with two equivalents of isobutyl chloroformate and then adding two 63 equivalents of F5; IMF10 was characterized by MS and NMR spectroscopy. Elemental analysis indicates the presence of a 0.5 H 2 0 solvate. The two IR bands between 3200-3400 cm"1 presumably arise from v(NH) and v(OH). Figure 3.28 Molecular structure of IMF10. 3.3.5 Synthesis, Characterization, and Solution X H NMR Spectroscopy of [Ru(ma)2(EF5)2]CF3S03EtOH (14) and [Ru(ma)2(triF5)2]CF3S03 (18) Complexes 14 and 18 were synthesized from [Ru(ma)2(EtOH)2]CF3S03 (3) instead of Ru(ma)3 (1), because (in the absence of excess ma) this allowed for the use of stoichiometric amounts of EF5. The reactions proceeded quantitatively, and purification of the complexes was simpler than when using 1 as a precursor. 18 was insoluble in all solvents tested and thus was only characterized by IR spectroscopy and elemental analysis that agrees well with the proposed formulation. 14, on the other hand, was soluble in both EtOH and H 2 0 and was therefore characterized by MS and NMR spectroscopy as well as IR spectroscopy and elemental analysis. The ' H NMR spectrum (r.t., CD 3 OD) of [Ru(ma)2(EF5)2]CF3S03-Et0H (14) showed a broad resonance for the ma-Me group (8 59.2) and two broad singlets at 8 -4.8 and -16.1 for the H(5) and H(4) protons, respectively. Resonances for the EF5 side-chain, and H(5) and H(6) of ma, are likely hidden in the 8 0-10 region where trace signals for diamagnetic impurities and residual solvents overwhelm any 'paramagnetic' signals. Although the solid was dried for 24 h under vacuum at 78 °C before the *H NMR spectrum was taken, signals for free EtOH are observed. The addition of one EtOH to the molecular formula fits well the elemental analysis. In the 1 9 F NMR spectrum, two broad signals for the bound EF5 (8 -8.9 and -46.1) (Figure 3.29) overlap with a triplet (8 -46.5) 64 and a multiplet (5 -9.4) that are for either free EF5 in solution or a diamagnetic impurity, although T L C analysis of the NMR sample showed no evidence of the free ligand. A singlet for the triflate counterion is also observed at 8 -3.52. The mass spectrum gives peaks for [M + -16] and [M + - 16 - EF5], consistent with the commonly observed loss of an O-atom from a nitroaromatic group. ' ~ 5 ~ 1 0 -15 -20 -25 -30 -35 -40 -45 p p m Figure 3.29 I 9 F NMR spectrum (300 MHz, 298 K) of [Ru(ma)2(EF5)2]CF3S03-EtOH (14). Both signals for bound EF5 are broadened, while the sharper, diamagnetic signals likely correspond to those of free EF5. 3.4 Synthesis of [Ru(HMepyr) 3]Cl 3 (19) Preliminary attempts to synthesize a complex analogous to 1 but with 3-hydroxy-l,2-dimethyl-4-pyridone led instead to the synthesis of 19 that was characterized by IR spectroscopy and the elemental analysis. The conductivity of an aqueous solution of 19 (451 i2"1cm2mol"1) is in the range for a 3:1 electrolyte.11 Attempts to deprotonate this complex and isolate a neutral species were unsuccessful. 65 3.5 Cyclic Voltammetry of Complexes The Ru(IJI/n) one-electron reduction potentials were determined for the complexes that were soluble in either MeCN or CH 2C1 2 . The potentials determined for Ru(ma)3 in CH 2 C1 2 agree with those reported in the literature,1 and those in MeCN are reported in Table 3.5. Of the mixed Im-ma complexes tested, 10 had the lowest reduction potential, and is also the most active ma complex from the M T T biological studies (Section 6.4.2). This observation counters the theory that to be biologically active, Ru(IH) complexes should have a more positive reduction potential to facilitate in situ reduction to potentially more labile Ru(U) species.23 There does not appear, however, to be any direct link between the reduction potential and the in vitro activity, as will be discussed in more detail in Section 6.4.2. Table 3.5 Ru(IJJ/]I) reduction potentials of ma and Ema-containing complexes. The Ru(IJJ/n) reduction potentials of the Ema complexes were some 14 - 90 mV more negative than those for the corresponding ma complexes. 13, the complex with the lowest IC 5 0 of all those tested for bioactivity (Section 6.4.2), had the lowest Ru(III/n) reduction potential of the all the bis-imidazole complexes. Complex Ru (m/II) Reduction Potential in MeCN vs SCE (mV) Ru(ma)3 (1) Ru(Ema)3 (2) [Ru(Ema)2(2MeIm)2]CF3S03 (13) [Ru(Ema)2(4MeIm)2]CF3S03 (12) [Ru(Ema)2(metro)2]CF3S03 (11) [Ru(ma)2(2MeIm)2]CF3S03 (10) [Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2(8) [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) [Ru(ma)2(Im)2]CF3S03 (7) [Ru(ma)2(metro)2]CF3S03 (4) [Ru(ma)2(metro)(EtOH)]CF3S03 (5) [Ru(ma)(lMeIm)4][CF3SQ3]2 (9) -1132 -1176 -889 -829 -568 -844 -765 -738 -705 -554 -496 -442 66 The voltammograms of the metro complexes were the only ones obtained for complexes with ligands containing a nitro group. With the exception of 14, the other nitro containing complexes were insoluble in 0.100 M TBAP solutions in either CH 2 C1 2 or MeCN. The C V of [Ru(ma)2(metro)2]CF3S03 (4) shows three reversible waves for Ru"™ (-554 mV), Ru I V / I " (1085 mV), and N0 2 /N0 2 _ (-1111 mV) (Figure 3.30). Free metronidazole was found to undergo a 1-electron reduction for the nitro group at -1154 mV, 43 mV more negative than for the coordinated ligand, and 17 mV more negative than for [Ru(Ema)2(metro)2]CF3S03 (11) (Ei / 2 (N02/N02") = -1137 mV). From these data it can be concluded (as expected) that coordination of metronidazole to Ru(IU) makes the reduction of the nitro group easier (i.e. gives a more positive potential). 14 was soluble in MeCN but no signals were observed in the voltammogram between -2 and 2 V. F eiii/n R u ,v/n. R u I M I ^ A \ NOVNCV f\ \ \ — / V _J , ^ -18 - i p s—•QQ/' -0.3 ^ ^ 0-2 0.7 1.2 1.7 / / V J I \ / v Figure 3.30 Cyclic voltammogram of [Ru(metro)2(ma)2]CF3S03 (4) in MeCN containing ferrocene. 67 3.6 Experimental Procedures for the Syntheses of Compounds Derived from F5 3.6.1 N-(2,2,3,3,3-pentafluoropropyl)chloroacetamide (C1F5) This compound was prepared via a modified reported procedure.17 Chloroacetyl chloride (0.375 mL, 4.71 mmol) was dissolved in THF at 0 °C under N 2 . To this solution was added 2,2,3,3,3-pentafluoropropylamine (F5) (0.500 mL, 4.70 mmol) and N-methylmorpholine (NMM) (0.410 mL, 4.70 mmol). The mixture was stirred at 0 °C for 30 min, at which time the ice-bath was removed and stirring was continued for an additional 30 min. The solution was filtered to remove a white precipitate and the filtrate was then evaporated under vacuum to leave a yellow, crystalline solid. This solid was dried under vacuum at 0 °C for 24 h. Yield: 0.96 g (91 %). *H NMR (acetone-rf6): 5 8.16 (s, 1H, NH); 4.26 (s, 2H, C\CH2); 4.15 (td, 2H CH2CF2). 1 9F{ ]H} NMR (acetone-d6): 5 -8.25 (t, CF 3 ) ; -45.27 (q, CF 2 ) . IR (KBr pellet): v 3320 (N-H, m), 1655 (C=0, s). ESI-MS (MeOH): 190 ( M + - CI). The spectroscopic data agree with those previously reported.17 3.6.2 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(2,2,3»3,3-pentafluoropropyI)acetamide (EF5) This ligand was synthesized using a modified procedure from that published by Baird et a/. 1 8 A solution of 2N0 2Im (0.10 g, 0.92 mmol) in D M F (5 mL) was degassed with N 2 for 10 min. To this solution was added C s 2 C 0 3 (0.30 g, 0.93 mmol). Stirring of this mixture at 50 °C for 2 h yielded a yellow solution and a white precipitate. To this mixture was added C1F5, and the reaction mixture was then stirred for 3 h at 50 °C. The solution was filtered and the precipitate washed with dry THF ( 3 x 5 mL). The filtrate was reduced in volume to 1 mL and then loaded onto a silica gel column. Elution of the column (CH2Cl2:acetone, 10:1) removed unreacted starting materials. The concentration of acetone was then increased to 40 % to elute EF5. The fractions containing the product were combined and evaporated under vacuum to yield EF5 as a white solid. Yield: 0.23 g (82 %). 'H NMR (CD 3OD): 5 7.48 (s, 1H, Im-r75); 7.21 (s, 1H, Im-r74); 5.29 (s, 2H C// 2 CO); 4.08 (t, 2H C/7 2CF 2). l9F{]H} NMR (CD 3OD): 5-10.58 (t, CF 3 ); -47.21 (q, CF 2 ) . IR (KBr pellet): v 3305 (N-H, m), 1686 (C=0, s), 1490 (N-O a s y m , m), 1363 (N-O s y m , s). ESI-MS (MeOH): 303 (M+). Anal. Calcd for C 8 H 7 N 4 0 3 F 5 : C, 31.80; H, 2.34; 68 N, 18.54. Found: C, 31.62; H, 2.44; N, 18.25. The data agree with those reported in the literature.18 3.6.3 2-(3-Nitro-l-H-triazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (triF5) This compound was prepared using the same procedure as that for EF5, but using 3-nitro-l,2,4-triazole (0.048 g, 0.42 mmol) in place of 2N02Im, and C s 2 C 0 3 (0.14 g, 0.43 mmol). The triF5 product was isolated as a pale yellow solid. Yield: 0.86 g (67 %). 'H NMR (aceton-^j): 5 8.61 (s, 1H, tri-//5); 8.17 (s, 1H N-H); 5.24 (s, 2H - C / / 2 - C O - ) ; 4.11 (td, 2H - C / / 2 - C F 2 - ) . 19F{'H} NMR (acetone-d6): 5-10.41 (t, -CF 3 ); -48.65 (q, -CF 2 -). IR (KBr pellet): v 3341 (N-H, m), 1701 (C=0, s), 1407 (N-O a s y m , m), 1318 (N-O s y m , s). ESI-MS (MeOH): 304 (M+). Anal. Calcd for C 7 H 6 N 5 0 3 F 5 : C, 27.72; H, 1.98; N, 23.10. Found: C, 27.62; H, 2.14; N, 22.87. 3.6.4 IMF10 To a suspension of imidazole-4,5,-dicarboxylic acid (0.10 g, 0.65 mmol) in THF (5 mL) under an atmosphere of N 2 was added N M M (0.115 mL, 1.32 mmol). The mixture was stirred for 10 min at which time isobutyl chloroformate was added (0.175 mL, 1.34 mmol). The mixture was stirred at r.t. for 1 h. Pentafluoropropyl amine (0.140 mL, 1.31 mmol) was then added and the mixture stirred under N 2 for 12 h at r.t. The mixture was then filtered to remove the white precipitate and the yellow filtrate was collected, and evaporated to leave a yellow oil. T L C analysis (CH 2 Cl 2 :MeOH, 10:1) showed a single major product, R f = 0.67. The oil was purified using column chromatography (CH 2 Cl 2 :MeOH, 10:1) and the combined fractions for this band were collected and evaporated under vacuum to yield a yellow solid. Yield 0.086 g (31 %). l H NMR (CD 3OD): 5 7.72 (s, 1H, Im-H2); 4.12 (td, 4H - C / / 2 - C F 2 - ) . 19F{'H} NMR (acetone-^): 5 -8.20 (t, -CF 3 ); -45.07 (q, -CF 2 -). IR (KBr pellet): v 3322 (N-H, m), 3226 (N-H, br s), 1648 (C=0, s). LR-MS (+LSIMS, thioglycerol): 418 (M+). Anal. Calcd for C, ^ 4 0 ^ , 0 - 0 . 5 ^ 0 : C, 30.91; H, 2.11; N, 13.11. Found: C, 30.86; H, 2.31; N, 12.47. 69 3.7 Experimental Procedures for the Syntheses of Ru(III) Complexes 3.7.1 Ru(ma)3 (1) This compound was synthesized by a literature procedure.1 To a brown solution of RuCl 3 3H 2 0 (1.01 g , 3.86 mmol) in water (80 mL) and sodium acetate (4.0 g, 30 mmol), maltol was added (2.50 g, 19.2 mmol) under N 2 , and the mixture refluxed for 6 h; a red precipitate was then collected by filtration. T L C analysis (CH 2 Cl 2 :MeOH, 20:1) revealed two spots; the product (Rf = 0.6) and an impurity (Rf = 0). The mixture was then dissolved in CH 2 C1 2 (40 mL) and filtered through Celite (2 g) to remove the black impurity. The filtrate was reduced in volume to ~5 mL at which time hexanes (30 mL) were added to yield a red precipitate. The product was isolated via filtration and dried at 78 °C under vacuum for 48 h. Yield: 0.95 g (52 %). ' H NMR (CD2C12): 8 43.2, 41.0, 21.1 (s, -C#3); 11.8 (s, Hs-ma), 9.2 (s, H6-ma); 3.1 (s, H6-ma), -4.6 (s, #5-ma); 0.8 (s, H6-ma), -0.9 (s, H5-ma). IR (KBr pellet): v 1600 (C=0, s), 1551 (s), 1466 (s), 1261 (s), 1199 (s). LR-MS (+LSIMS, thioglycerol): 477 (M+), 352 ( M + - ma). UV-vis (H20): 216 (45.4), 284 (14.1), 380 (10.2). C V (MeCN): E ! / 2 (RuIU/I1) = -1.132 V, E 1 / 2 (Ru l v / i n) = 0.524 V vs. SCE. C V (CH2C12): E 1 / 2 (Runi/") = -1.273 V, E 1 / 2 (RuIV/l") = 0.497 V vs. SCE. A M (H 20) = 25.Q"1cm2mol'1. Anal. Calcd for C i 8 H i 5 0 9 R u : C, 45.38; H, 3.17. Found: C, 45.32; H, 3.19. The C V (in CH 2C1 2) and IR data agree with those reported in the literature1 The X-ray structure shows a mer-configuration (Section 3.2.1). 3.7.2 Ru(Ema) 3(2) This complex was synthesized following the procedure outlined in Section 3.6.1, but using ethylmaltol in lieu of maltol (2.80 g, 20 mmol). T L C analysis (CH 2 Cl 2 :MeOH, 20:1) revealed two spots; the product (R f = 0.55) and an impurity (R f = 0). The mixture was then dissolved in CH 2 C1 2 (50 mL) and filtered through Celite (3 g) to remove the impurity. The filtered solution was reduced in volume to ~5 mL at which time hexanes (20 mL) were slowly added to yield a red precipitate. The precipitate was collected, and dried at 78 °C under vacuum for 48 h. Yield: 0.83 g (45 %). ' H NMR (CD2C12): 8 40.1, 38.8, 35.3, 33.4, 21.7, 18.9 (s, -C# 2 CH 3 ); 4.9, 4.8, 2.1 (s, -Cri2CH3); 12.5 (H5-Ema), 9.0 (#6-Ema); 4.9 (ff6-Ema), -4.9 (//5-Ema); 1.2 (#6-Ema), -0.8 (#5-Ema). IR (KBr pellet): v 70 1596 (C=0, s), 1550 (s), 1471 (s), 1258 (s), 1187 (s). LR-MS (+LSEVIS, thioglycerol): 519 (M+), 380 ( M + - Ema). UV-vis (H20): 216 (45.1), 284 (14.4), 382 (10.6). CV (MeCN): E 1 / 2 (Ru"17") = -1.176 V, Em ( R u ™ 1 ) = 0.540 V vs. SCE. C V (CH2C12): E 1 / 2 (Ru""") = -1.292 V, E1/2 (Ru l v / 1 1 1) = 0.493 V vs. SCE. A M (H 20) = 36 Q-Wmol" 1 . Anal. Calcd for C 2 ] H 2 1 0 9 R u : C, 48.65; H, 4.08. Found: C, 48.64; H, 4.09. 3.7.3 [Ru(ma)2(EtOH)2]CF3S03 (3) To a red solution of 1 (0.090 g, 0.19 mmol) in EtOH (5 mL), triflic acid was added (0.030 g, 0.20 mmol) under N 2 and the solution refluxed for 1 h. The solvent was reduced to ~1 mL under vacuum to which Et 2 0 (20 mL) was added. A red precipitate, which contained only one band by T L C analysis (CH 2 Cl 2 :MeOH, 20:1; Rf = 0.45), was collected and washed with Et 2 0 (2x5 mL) before being dried at r.t. under vacuum for 24 h. Yield: 0.048 g (43 %). ' H NMR (CD 3OD): 5 48.0 (br s, C//3-ma); 28.0, 26.0 (br s, C7/ 3C/7 2OH). IR (KBr pellet): v 3427 (O-H, m), 1608 (C=0, s), 1471 (s), 1261 (s). LR-MS (+LSIMS, thioglycerol): 444 (M+), 398 ( M + - EtOH), 352 ( M + - 2 EtOH). A M (EtOH) = 36 Q-Wmol" 1 . Anal. Calcd for C n H 2 2 F 3 O n R u : C, 34.46; H, 3.72. Found: C, 34.53; H, 3.75. 3.7.4 [Ru(ma) 2(metro) 2]CF 3S0 3 (4) and [Ru(ma)2(metro)(EtOH)]CF3S03 (5) To a red solution of 1 (0.090 g, 0.19 mmol) in EtOH (5 mL), triflic acid was added (0.030 g, 0.20 mmol) under N 2 and the solution was refluxed for 1 h. Metronidazole was then added (0.13 g, 0.76 mmol) and the solution was refluxed for an additional 6 h. After 2 h the solution was green and slowly turned blue over the next 4 h. The solvent was removed under vacuum and CH 2 C1 2 (lOmL) was added. The mixture was shaken vigorously to leave a blue solid suspended in a green solution. The solid (4) was removed by filtration and washed with CH 2 C1 2 (2x5 mL) before being dried at 78 °C under vacuum for 48 h. Yield: 0.080 g (50 %). 'H NMR (acetone-de): 5 70.0 (br s, CH3-ma); 34.0 (br s, C#3-metro); -6.3 (br s, #5-metro). IR (KBr pellet): v 3420 (O-H, m), 1604 (C=0, s), 1561 (N-O a s y m , m), 1367 (N-O s y m , m), 1446 (s), 1263 (s). LR-MS (+LSIMS, thioglycerol): 694 (M+), 523 (M + - metro), 352 ( M + - 2 metro). CV (MeCN): E 1 / 2 (N0 2/N0 2 _) = -1.111 V, Em (Ru"17") = -0.554 V, E 1 / 2 (Ru IV/111) = 1.085 V vs. SCE. 71 Anal. Calcd for C 2 5H 2 8F3N 6 0, 5 SRu (4): C, 35.63; H, 3.33; N, 9.98. Found: C, 35.68; H, 3.41; N, 9.76. After 4 was removed by filtration, the green solution was reduced in volume to ~2 mL and mounted on a 20 x 20 cm silica'preparative T L C plate. The plate was developed with CH 2 Cl 2 :MeOH, 25:1 (130 mL). A green band was cut from the silica plate, suspended in 20 mL of a solution of CH 2 Cl 2 :EtOH, 19:1, and the mixture was left stirring for 16 h. The green mixture was then filtered to remove the silica and the solvent was removed under vacuum. The green oil remaining was dissolved in ~3 mL CH 2 C1 2 and hexanes (10 mL) were then added. The resulting green product was collected, washed with hexanes (2x5 mL) and dried at 78 °C under vacuum for 24 h. Yield: 0.032 g (12 %). 'H NMR (acetone-d6): 5 70.4, 65.6, 61.7, 54.2 (br s, C7/3-ma); 41.3, 34.9 (br s, C/7 3-metro); 24.97, 20.99, 19.01 (br s, CH3CH2OH); -3.13, -6.29 (br s, metro-/Y5). IR (KBr pellet): v 3443 (N-H, m), 1701 (C=0, s), 1559 (N-O a s ym, m), 1363 (N-O s y m , s), 1446 (s), 1263 (s). LR-MS (+LSIMS, thioglycerol): 523 (M + - EtOH), 352 ( M + - EtOH - metro). CV (MeCN): E 1 / 2 (Ru"™) = -0.496 V. Anal. Calcd for C 2 , H 2 5 F 3 N 3 0 1 3 S R u (5): C, 35.15; H, 3.49; N, 5.86. Found: C, 34.87; H, 3.32; N, 6.09. 3.7.5 [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) To a red solution of 1 (0.050 g, 0.11 mmol) in EtOH (5 mL), triflic acid was added (0.020 g, 0.13 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, 4MeIm was added (0.041 g, 0.50 mmol) and the solution was stirred for an additional 12 h. After 4 h the solution was checked by T L C (CH 2 Cl 2 :MeOH, 10:1). The band for complex 5 had disappeared and been replaced by 3 major red. bands (Rf = 0.32, 0.44, 0.54). After 12 h, only one major red band was observed by T L C (Rf = 0.56). The solvent was removed under vacuum and the dark red residue was dissolved in CH 2 C1 2 (2 mL) and mounted on a 20 x 20 cm silica preparative T L C plate that was developed with CH 2 Cl 2 :MeOH, 20:1 (130 mL), when one major red band observed. This red band was cut out, added to 50 mL of CH 2 C1 2 , and the mixture was left stirring for 16 h. This was then filtered to remove the silica and the volume was reduced to ~5 mL. Hexanes (30 mL) were then added and the resulting red precipitate was collected, washed with 72 hexanes (2x5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.032 g (42 %). 'H NMR (CDC13): 5 60.0 (br s, C//3-ma); 16.3 (br s, C#3-Im); -4.5 (br s, #5-Im); -20.0 (br s, H2-lm). IR (KBr pellet): v 3224 (N-H, m), 1601 (C=0, s), 1446 (s), 1263 (s). LR-MS (+LSIMS, thioglycerol): 516 (M+), 434 (M + - 4MeIm), 352 ( M + - 2 (4MeIm)). CV (MeCN): E 1 / 2 (Ru I M I) = -0.738 V. A M (MeOH) = 107 Q-Wmol" 1 . Anal. Calcd for C 2 1 H 2 2 F 3 N 4 0 9 S R u C H 2 C l 2 : C, 35.25; H, 3.20; N, 7.48. Found: C, 35.63; H, 3.26; N, 7.55. 3.7.6 [Ru(ma)2(Im)2]CF3S03 (7) To a red solution of 1 (0.055 g, 0.12 mmol) in EtOH (5 mL), triflic acid was added (0.022 g, 0.14 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, Im was added (0.035 g, 0.51 mmol) and the solution was stirred for an additional 12 h. After 4 h the solution was checked by T L C (CH 2 Cl 2 :MeOH, 10:1). The band for complex 5 had disappeared and been replaced by 11 minor bands. After 12 h, a single major band was observed with 5 minor bands of lower R f. The solvent was removed under vacuum and the dark red residue was dissolved in CH 2 C1 2 (2 mL) and mounted on a 20 x 20 cm silica preparative T L C plate that plate was developed with CH 2 Cl 2 :MeOH, 15:1 (130 mL). When the major band had clearly separated, it was cut from the silica plate, and added to 50 mL of CH2CI2 with 1 mL MeOH; this mixture was left stirring for 16 h. The red mixture was then filtered to remove the silica and the filtrate was reduced in volume to ~5 mL. Hexanes (30 mL) were then added and the resulting red precipitate was collected, washed with hexanes ( 2 x 5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.038 g (51 %). 'H NMR (m-isomer, CDC13): 5 62.2, 44.6 (s, C//3-ma); -1.5. -5.6 (s, tf5-Im); -15.5, -24.7 (s, H2-Im). 'H NMR (frans-isomer, CDC13): 60.0 (s, C#3-ma); -3.7. (s, H5-Im); -19.6 (s, H2-lm). IR (KBr pellet): v 3145 (N-H, m), 1603 (C=0, s), 1467 (s), 1263 (s). LR-MS (+LSIMS, thioglycerol): 488 (M+), 420 ( M + - Im), 352 ( M + - 2 Im). CV (MeCN): E 1 / 2 (Ru1 I l / n) = -0.705 V. A M (MeOH) = 119 aWmol"1. Anal. Calcd for C 1 9 H 1 8 F 3 N40 9 SRu: C, 35.85; H, 2.83; N, 8.81. Found: C, 35.92; H, 2.92; N, 8.73. 3.7.7 [Ru(ma)2(lMeIm)2]CF3S03CH2Cl2 (8) To a red solution of 1 (0.080 g, 0.17 mmol) in EtOH (5 mL), triflic acid was added (0.028 g, 0.19 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, 73 lMelm was added (0.062 g, 0.76 mmol) and the solution was stirred for an additional 24 h. After 6 h the solution was checked by T L C (CH 2 Cl 2 :MeOH, 10:1): 6 bands were observed with the most intense being a red one at Rf = 0.6. After 18 h a similar analysis revealed two major bands, a red one at Rf = 0.6 and a less intense blue one at Rf = 0.35. After 24 h the solvent was removed under vacuum and the dark red residue was dissolved in CH 2 C1 2 (~2 mL) and mounted on a 20 x 20 cm silica preparative T L C plate that was developed with CH 2 Cl 2 :MeOH, 12:1 (130 mL). The red band was cut out, added to 50 mL of CH 2 C1 2 with 1 mL MeOH, and the mixture was left stirring for 16 h. Attempts to extract the blue band were unsuccessful. The red suspension was then filtered to remove the silica and the volume was reduced to ~3 mL. Hexanes (20 mL) were then added and the resulting red precipitate was collected, washed with hexanes (2x5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.060 g (48 %). 'H NMR (CDC13): 5 62.0 (br s, C7/3-ma); 13.4 (br s, C#3-lMeIm); -3.8 br (s, ff5-lMeIm); -19.0 (br s, /72-lMeIm). IR (KBr pellet): v 3458 (m), 1601 (C=0, s), 1467 (s), 1263 (s), 1031 (s). LR-MS (+LSMS, thioglycerol): 516 (M+), 434 (M + - lMelm), 352 ( M + - 2 (lMelm)). C V (MeCN): E , / 2 (Ru™ 1) = -0.765 V. A M (MeOH) = 109 S r W m o l " 1 . Anal. Calcd for C 2 1 H 2 2 F 3 N40 9 SRu-CH 2 Cl2: C, 35.25; H, 3.20; N, 7.48. Found: C, 35.71; H, 3.22; N, 7.86. 3.7.8 [Ru(ma)(lMeIm) 4][CF 3S03]2 (9) To a red solution of 1 (0.067 g, 0.14 mmol) in EtOH (5 mL), triflic acid was added (0.054 g, 0.36 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, lMelm was added (0.077 g, 1.1 mmol) and the solution was refluxed 48 h. After 24 h the solution was checked by T L C (CH 2 Cl 2 :MeOH, 10:1): 2 major bands were observed of approximately equal intensity; a red band at Rf = 0.6, and a blue band at Rf = 0.35. After 48 h, analysis revealed a major blue band (Rf = 0.35), several minor bands (Rf = 0.2 -0.7), and a black spot (Rf = 0). The solvent was removed under vacuum and the dark blue residue was dissolved in CH 2 C1 2 (-1.5 mL) and mounted on a 20 x 20 cm silica preparative T L C plate that was developed with CH 2 Cl 2 :MeOH, 10:1 (130 mL). The blue band was cut out, added to 50 mL of CH 2 C1 2 with 2 mL MeOH, and the mixture was left stirring for 16 h. The blue mixture was then filtered to remove the silica and the volume was reduced to ~1 mL. Hexanes (10 mL) were then added and the resulting blue 74 precipitate was collected, washed with hexanes ( 2 x 5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.055 g (46 %). 'H NMR (CDC13): 5 81.6 (br s, C#3-ma); 25.6, 20.4 (s, C// 3-lMeIm); -0.5, -5.7 (s, // 5-lMeIm); -18.4, -24.0 (s, // 2-lMeIm). IR (KBr pellet): v 3137 (m), 1601 (C=0, s), 1546 (m), 1470 (m), 1265 (s), 1031 (s). LR-MS (+LSIMS, thioglycerol): 704 ( M + + CF 3 S0 3 ) , 622 ( M f + C F 3 S 0 3 - lMelm), 540 ( M + + C F 3 S 0 3 - 2 lMelm), 458 ( M + + C F 3 S 0 3 - 3 lMelm), 391 ( M + - 2 lMelm), 309 (M + - 3 lMelm). CV (MeCN): Ei/2 (Ru1 I l / n) =-0.442 V. A M (MeOH) = 273 fl-Wmol'1. Anal. Calcd for C 2 4H29F 6 N 8 0 9 S 2 Ru: C, 33.80; H, 3.40; N, 13.14. Found: C, 33.23; H, 3.34; N, 12.73. 3 . 7 . 9 [Ru(ma) 2(2MeIm) 2]CF 3S0 3 (10) To a red solution of 1 (0.092 g, 0.193 mmol) in EtOH (5 mL), triflic acid was added (0.0320 g, 0.213 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, 2MeIm was added (0.066 g, 0.805 mmol) and the solution was stirred for an additional 12 h. The solvent was then removed under vacuum and the dark red residue was dissolved in CH 2 C1 2 (~2 mL) and mounted on a 20 x 20 cm silica preparative T L C plate that was developed with CH 2 Cl 2 :MeOH, 20:1 (130 mL). The resulting single, red band was cut out, added to CH 2 C1 2 (50 mL), and the mixture was left stirring for 16 h. The red mixture was then filtered to remove the silica and the volume was reduced to ~2 mL. Hexanes (10 mL) were then added and the resulting red product was collected, washed with hexanes (2x5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.063 g (56 %). 'H NMR (CDC13): 8 67.0 (br s, C//3-ma); 42.0 (br s C#3-Im); -1.6 (br s, Hs-lm); -4.7 (br s, H4-Im). IR (KBr pellet): v 3216 (N-H, m), 1602 (C=0, s), 1447 (s), 1265 (s). LR-MS (+LSEVIS, thioglycerol): 516 (M +), 434 (M + - 2MeIm), 352 (M +-2 (2MeIm)). C V (MeCN): E 1 / 2 (Ru i n / I 1) = -0.844 V. A M (MeOH) = 121 Q.Acm2moY\ Anal. Calcd for C 2 i H 2 2 F 3 N 4 0 9 S R u : C, 37.95; H, 3.31; N, 8.43. Found: C, 37.61; H, 3.38; N, 8.20. 3.7.10 [Ru(Ema)2(metro)2]CF3S03 (11) This complex was prepared using the same procedure as described for complex 3, but using 2 instead of 1 (0.065 g, 0.125 mmol), triflic acid ( 0.0213 g, 0.141 mmol) and metro (0.094 g, 549 mmol). The green species for this reaction could not be isolated as 75 was the case with 4. Yield: 0.0642 g (59 %).. *H NMR (acetone-^): 5 65.3 (br s, CH3Cr72-Ema); 32.2 (br s, C#3-metro); -4.8 (br s, /75-metro). IR (KBr pellet): v 3439 (O-H, m), 1600 (C=0, s), 1560 (N-O a s y m , m), 1367 (N-O s y m , m), 1265 (s). LR-MS (+LSFMS, thioglycerol): 722 (M +), 551 ( M + - metro), 380 ( M + - 2 metro). C V (MeCN): E 1 / 2 (N0 2/NOy) = -1.137 V, Ei/2 (Runi /11) = -0.568 V, E 1 / 2 (RuIV/I") = 0.967 V vs. SCE. Anal. Calcd for C 2 7 H 3 2 F 3 N 6 O i 5 S R u : C, 36.49; H, 3.86; N, 9.46. Found: C, 36.52; H, 3.72; N,9.55. 3.7.11 [Ru(Ema) 2(4MeIm) 2]CF 3S0 3 (12) To a red solution of 2 (0.060 g, 0.116 mmol) in EtOH (5 mL) was added triflic acid (0.0220 g, 0.146 mmol) under N 2 and the solution stirred at r.t. for 1 h. After 1 h, 4MeIm was added (0.038 g, 0.463 mmol) and the solution was stirred for an additional 16 h. After 3 h the solution was checked by T L C (CH 2 Cl 2 :MeOH, 10:1). A single major red band (Rf = 0.60) was present with a minor brown one (Rf = 0). After 16 h the solvent was removed under vacuum and the reddish-brown residue was dissolved in CH 2 C1 2 and mounted on a 20 x 20 cm silica preparative T L C plate that was developed with CH 2 Cl 2 :MeOH, 20:1 (130 mL). The major band was cut out, added to 50 mL of CH 2 Cl 2 :MeOH (49:1), and the mixture was stirred for 16 h. The red mixture was then filtered to remove the silica and the volume was reduced to ~1 mL. Hexanes (15 mL) were then added and the resulting dark red precipitate was collected, washed with hexanes (3x5 mL) and dried at 78 °C under vacuum for 48 h. Yield: 0.0449 g (56 %). 'H NMR (CD2C12): 5 49.1 (br s, CH 3C/7 2-Ema); 16.5 (br s, CH3-4MeIm); -4.6 (br s, H5-4MeIm); -19.0 (br s, 7/2-4MeIm). IR (KBr pellet): v 3439 (N-H, m), 1597 (C=0, s), 1547 (m), 1473 (m), 1259 (s), 1030 (s). LR-MS (+LSEVIS, thioglycerol): 544 (M+), 462 (M + -4MeIm), 380 ( M + - 2 (4MeIm)). C V (MeCN): E 1 / 2 (Ru""11) = -0.829 V. Anal. Calcd for C 2 3 H 2 6 F 3 N 4 0 9 S R u : C, 39.88; H, 3.76; N, 8.09. Found: C, 39.48; H, 3.88; N, 7.93. 3.7.12 [Ru(Ema) 2(2MeIm) 2]CF 3S0 3 (13) This complex was prepared using the same procedure as described for complex 12, but using 2 (0.082 g, 0.158 mmol), triflic acid (0.0270 g, 0.179 mmol), and 2MeIm (0.051 g, 0.622 mmol). R f = 0.55 for the major red band of 13. Yield: 0.0629 g (58 %). 76 'H NMR (CD 2CI 2): 5 51.7 (br s, Ctf 3C// 2-Ema); 43.7 (br s, C//3-2MeIm); -1.9 (br s, H5-2MeIm); -5.2 (br s, //4-2MeIm). IR (KBr pellet): v 3463 (N-H, m), 1597 (C=0, s), 1548 (m), 1472 (m), 1260 (s), 1031 (s). LR-MS (+LSEVIS, thioglycerol): 544 (M+), 462 ( M + -2MeIm), 380 ( M + - 2 (2MeIm)). CV (MeCN): Em (Ru i n / n) = -0.889 V. Anal. Calcd for R u C 2 3 H 2 6 F 3 N 4 0 9 S : C, 39.88; H, 3.76; N, 8.09. Found: C, 39.71; H, 3.87; N, 7.82. 3.7.13 [Ru(ma) 2 (EF5) 2 ]CF 3 S0 3 EtOH (14) To a solution of 3 (0.104g, 0.175 mmol) in 5 mL EtOH under N 2 was added EF5 (0.106g, 0.351 mmol). This solution, on refluxing for 48 h, slowly became brown and finally blue. T L C showed that almost all of the EF5 had been consumed in the reaction and that there was one major blue band, Rf = 0 (CH 2 Cl 2 :MeOH, 10:1). A weak intensity pink band Rf = 0.6 was also observed. The solvent was reduced in volume to ~1 mL and CH 2 C1 2 was added (10 mL) to yield a blue precipitate that was collected, and dried at 78 °C under vacuum for 24 h. Yield: 0.098 g (51 %). 'H NMR (CD 3OD): 5 59.2 (br s, C / / 3 -ma); -4.8 (br s, /75-EF5); -16.1 (br s, tf4-EF5). 1 9 F NMR (CD 3OD): 5 -3.52 (s, CF 3 S0 3 ) ; -8.9 (br s, C F 2 C F 3 ) ; -46.1 (br s, CF 2 CF 3 ) . IR (KBr pellet): v 3423 (N-H, s), 2924 (C-H, m), 1686 (C=0, s), 1602 (C=0), 1548 (m), 1473 (m), 1264 (s), 1202 (s). LR-MS (+LSIMS, thioglycerol): 940 ( M + - 16), 642 (M + - EF5 - 16), 352 ( M + - 2 EF5). Anal. Calcd for C 2 9 H 2 6 F 3 N 4 0 9 S R u C 2 H 6 0 : C, 32.34; H, 2.61; N, 9.74. Found: C, 32.17; H, 2.52; N, 9.97. 3.7.14 [Ru(ma) 2(2N0 2Im) 2]CF 3S0 3 (15) A solution of 1 (0.042 g, 0.088 mmol) in 5 mL EtOH and triflic acid (0.014 g, 0.093 mmol) was refluxed for 1 h under N 2 ; 2N02Im (0.040 g, 0.354 mmol) was then added and this solution was refluxed for 24 h. A blue precipitate that formed was then removed by filtration, washed with MeOH (2x5 mL) and dried under vacuum at 78 °C for 48 h. Yield: 0.017 g (27 %). IR (KBr pellet): v 3431 (N-H, s), 2967 (C-H, m), 1653 (C=0, m), 1542 (m), 1491 (N-O a s y m , s), 1367 (N-O s y m , s). Anal. Calcd for C i 9 H 1 6 F 3 N 6 0 , 3 S R u : C, 31.41; H, 2.20; N, 11.57. Found: C, 32.87; H, 2.32; N, 12.97. 77 3.7.15 [Ru(ma)2(4N02lm)2]CF3S03 (16) This complex was prepared using the same procedure as used for complex 15, except using 4N0 2Im (0.041 g, 0.363 mmol) in place of 2N0 2Im with complex 1 (0.043 g, 0.090 mmol). Yield: 0.016 g (24 %). 'H NMR (acetone-^): 5 63.8 (br s, C#3-ma). IR (KBr pellet): v 3471 (N-H, s), 2883, 2821 (C-H, m), 1556 (C=0, m), 1509 (m), 1496 (N-O a s y m , s), 1380 (N-O s y m , s). Anal. Calcd for C i 9 H i 6 F 3 N 6 0 1 3 S R u : C, 31.41; H, 2.20; N, 11.57. Found: C, 33.02; H, 2.37; N, 12.64. 3.7.16 [Ru(ma) 2(3N0 2tri) 2]CF 3S0 3 (17) This complex was prepared using the same procedure as used for complex 15, except using 3N02tri (0.044 g, 0.386 mmol) in place of 2N02Im with complex 1 (0.041 g, 0.086 mmol). Yield: 0.022 g (35 %). IR (KBr pellet): v 3471 (N-H, m), 2883, 2821 (C-H, m), 1556 (C=0, m), 1509 (m), 1496 (N-Oasym, s), 1380 (N-O s y m , s). Anal. Calcd for C 1 9 H 1 6 F 3 N 6 0 ] 3 S R u : C, 28.02; H, 1.92; N, 15.38. Found: C, 30.06; H, 2.40; N, 16.47. 3.7.17 [Ru(ma) 2(triF5) 2]CF 3S0 3 (18) This complex was prepared using the same procedure as used for complex 15, except using triF5 (0.068 g, 0.224 mmol) in place of 2N02Im with complex 1 (0.041 g, 0.086 mmol). Yield: 0.032 g (34 %). IR (KBr pellet): v 3435 (N-H, m), 2926 (C-H, m), 1601 (C=0, m), 1542 (m), 1466 (N-O a s y m , m), 1371 (N-O s y m , w), 1264 (m). Anal. Calcd for C 2 7 H 2 2 F, 3 Nio0 1 5 SRu: C, 29.29; H, 1.99; N, 12.66. Found: C, 29.43; H, 2.11; N, 12.24. 3.7.18 [Ru(HMepyr) 3]Cl 3 (19) To a solution of RuCl 3 3H 2 0 (0.103 g, 0.394 mmol) in 10 mL H 2 0 was added HMepyr (0.164 g, 1.18 mmol). This solution was stirred for 15 min until it had turned dark purple in colour. The volume was reduced to ~2 mL and acetone (100 mL) was added. The resulting purple precipitate was collected, and dried under vacuum at 78 °C for 24 h. Yield: 0.227 g (92 %). IR (KBr pellet): v 3419 (N-H, s), 1598 (C=0, m), 1497 (s), 1272 (s). A M (H 20) = 451 Q-Wmol" 1 . Anal. Calcd for C 2 1 H 2 7 N 3 0 6 C l 3 R u : C, 40.35; H, 4.32; N, 6.73. Found: C, 40.52; H, 4.47; N, 6.58. 78 3.8 References (1) Greaves, S. J.; Griffith, W. P. Polyhedron 1988, 7, 1973. (2) Nakamoto, K.; McCarthy, P. J. Spectroscopy and Structure of Metal Chelate Compounds; John Wiley & Sons, Inc.: New York, 1968. (3) Harris, R. K.; Mann, B. E. NMR and the Periodic Table; Academic Press Inc.: New York, 1978. (4) Lippard, S. J.; Berg, J. M . Principles of Bioinorganic Chemistry; University Science Books: Mill Valley, 1994. (5) Anderson, C ; Beauchamp, A. Can. J. Chem. 1995, 72, 471. (6) Anderson, C ; Beauchamp, A. Inorg. Chim. Acta 1995, 233, 33. (7) Anderson, C. Can. J. Chem. 2001, 79, 1477. (8) Anderson, C ; Beauchamp, A. Inorg. Chem. 1995, 34, 6065. (9) Eaton, S. S.; Eaton, G. R.; Holm, R. H.; Muetterties, E. L. J. Am. Chem. Soc. 1973,95, 1116. (10) Nagao, Y.; Sano, S.; Ochiai, M . Tetrahedron 1990, 46, 3211. (11) Huheey, J. E. Inorganic Chemistry: Principles of Structure and Reactivity, third ed.; HarperCollins Publishers: New York, 1983. (12) Pavia, D. L.; Lampman, G. M. ; Kriz, G. S. Introduction to Spectroscopy; Harcourt Brace College Publishers: Orlando, 1996. (13) Banci, L.; Bertini, I.; Luchinat, C ; Pierattelli, R.; Shokhirev, N. V.; Walker, F. A. J. Am. Chem. Soc. 1998,120, 8472. (14) Evans, D. F. J. Chem. Soc. 1959, 2003. (15) Geary, W. J. Coord. Chem. Rev. 1971, 7, 81. (16) Wu, A. Synthesis and Characterization of Ruthenium Maltolato, Sulfoxide, and Nitroimidazole Complexes as Potential Anticancer Agents , M . Sc. Dissertation, University of British Columbia: Vancouver, 2002. (17) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents , Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. 79 (18) Baird, I. R.; Skov, K. A.; James, B. R.; Rettig, S. J.; Koch, C. J. Synth. Commun. 1998,28,3701. (19) Haszeldine, R. N. / . Chem. Soc. 1953, 1548. (20) Husted, D. R.; Ahlbrecht, A. H. J. Am. Chem. Soc. 1953, 75, 1605. (21) McLafferty, F. W.; Turecek, F. Mass Spectrometry; University Science Books: Mill Valley, 1993. (22) Domingues, M . R. M. ; Marques, M . G. O. S.; Domingues, P.; Neves, M . G.; Cavaleiro, J. A. S.; Ferrer-Correia, A. J. J. Am. Soc. Mass Spectrom. 2001, 12, 381. (23) Clarke, M . J. Coord. Chem. Rev. 2002, 232, 69. 80 Chapter 4 Synthesis and Characterization of Ru Complexes with 4,4'-Biimidazoles 4.1 Introduction Although the coordination chemistry of 2,2'-biimidazole (Figure 4.1) with Ru and other transition metals has been extensively explored,1"4 no complexes have been reported in the literature containing the analogous 4,4'-biimidazole ligand (biim). Since biim was first synthesized in 1994,5 its only reported interaction with a transition metal ion was with Cu(II), to form in situ catalytic systems for making polymers from 2,6-dimethylphenol.6 No complexes, however, were isolated or characterized in situ. Figure 4.1 Molecular structure of 2,2'-biimidazole. It has been suggested that some Ru complexes that show anticancer activity may act through a mechanism similar to that of cisplatin, in which dissociation of N-donor ligands such as imidazoles (instead of CI" as is the case for cisplatin), inside the cell, renders coordination sites available for DNA bases to bind the metal.7 In such cases, however, any information contained in the imidazole ligand (e.g. radiolabels, fluorescent tags), is lost upon ligand dissociation. Bidentate imidazole ligands, with their ability to bind the metal centre more strongly through the chelate effect, could in principle remain bound in situ to a higher degree than their monodentate analogues and functional groups within the imidazole would then remain associated with the complex and could be targeted to the DNA. In this chapter, the synthesis and characterization of Ru complexes containing chelating 4,4'-biimidazole ligands will be discussed. 81 4.2 Synthesis and Reactions of 4,4'-Biimidazoles 4.2.1 Synthesis of 4,4'-Biimidazoles These compounds were synthesized using modified procedures of those published by Cliff and Pyne.5 The syntheses of both 4,4'-biimidazolium trifluoroacetate (H2biim) and 2,2'-dimethyl-4,4'-biimidazolium trifluoroacetate (H2Me2biim) require 5 steps each, when conducted from the starting materials Im and 2MeIm, respectively (Figure 4.2). While the synthesis of H 2biim is straightforward, that of H 2Me 2biim required several modifications to the published procedure. Some of the refinements made for the R R R R= H, M e R= I, M e R = H, M e R = H , M e Figure 4.2 Synthesis for H 2biim and H 2Me 2biim. 82 synthesis of H 2Me 2biim were then applied to give a higher yield synthesis of H2biim. In the first step of the H 2Me 2biim synthesis, formation of 4,5-diiodo-2-methylimidazole (Section 4.8.7), the reported rapid addition of I2 to the biphasic CHCl 3/aq. NaOH system led to the formation of a brown emulsion that did not separate into two phases again over 24 h. To overcome this problem, the total amount of I2 must be slowly added in 5 g increments over 20 min, allowing it to dissolve entirely after each addition. In the synthesis of H 2biim this modification was not required. A modified procedure was also adopted in the second step (Section 4.8.8), precipitation of the monoiodo-imidazole species, whereby addition of EtOH to the EtOH/water solution precipitated many of the Na salts which were then removed by filtration. The filtrate was then boiled to remove the EtOH and reduced in volume until the product, 4-iodo-2-methylimidazole, began to precipitate. This modification was also applied to give a higher yield synthesis of 4(5)-iodoimidazole (Section 4.8.3). As well, protection of the NH proton of 4-iodo-2-methylimidazole (third step), according to the literature that reported a 1:1, chlorotriphenylmethane:imidazole ratio, resulted in the isolation of a product containing a large excess of the trityl group (determined by 'H NMR spectroscopy), presumably present as triphenylmethanol, which can form in H 2 0 under basic conditions.8 Chromatographic separation of the product from this impurity could not be achieved because of their almost identical Rf values. By increasing the ratio of imidazole:trityl chloride to 1.5:1, it was found that 4-iodo-2-methyl-l-(triphenylmethyl)imidazole was isolable in pure form. The crude product containing the triphenylmethanol impurity does not undergo the next reaction step (Suzuki coupling); instead a black precipitate is formed which displays a single resonance for PPh3 in the 3 IP{ 1H} NMR spectrum in CDC1 3 and no evidence for either the methyl or H(5) imidazole signals in the *H NMR spectrum. It is important that the protected iodoimidazole species be pure before performing the coupling reaction. Purity was assessed by checking the relative integration values for the aromatic protons, Me protons, and H(5) proton of the imidazole species. As the residual CHC1 3 resonance in CDC1 3 overlaps with those of the aromatic signals, the 'H NMR spectra were recorded in CD 2 C1 2 in order to avoid inaccurate integrations. The 15:3 83 aromatic regiommethyl integration ratio for 4-iodo-2-methy]-l-(triphenylmethyl)-imidazole is consistent with the formulation.5 A singlet at 8 6.72 (6.75 in CDCI3, Section 4.8.9) integrating for one proton is observed for the imidazole H(5) proton. For 4-iodo-l-(triphenylmethyl)imidazole (Section 4.8.4), as the aromatic region overlaps the H(2) resonance of the imidazole, the aromatic region:H(5)-imidazole integration ratio is 16:1. The singlet for the H(5) proton at 5 6.87 (6.92 in CDC1 3, Section 4.8.4) is shifted out of the aromatic region. The Suzuki coupling for preparing both 4,4'-biimidazoles was performed according to the published procedure;5 however, considerably improved yield and purity in the desired product can be obtained (78 vs. 43% for l,r-bis(triphenylmethyl)-4,4'-biimidazole and 69 vs. 27% for 2,2'-dimethyl-l,r-bis(triphenylmethyl)-4,4'-biimidazole) when the catalyst, Pd(PPh3)4, is freshly prepared9 just prior to use. The protecting groups were removed in step 5 to afford H 2biim (Section 4.8.6) and H 2Me 2biim (Section 4.8.11) according to the reported procedures.5 X-ray quality crystals of l,r-bis(triphenylmethyl)-4,4'-biimidazole were grown from slow evaporation of a CH 2 C1 2 solution of the compound (Figure 4.3). The imidazole rings are essentially co-planar (the torsion angle between them is 0.8 degrees), and the molecule has a centre of inversion. The C4-C4' bond length is 1.448 A, intermediate between that observed for the C4=C5 double bond (1.353 A ) and the single bond between C-Ph (1.544 A) in the protecting group. Figure 4.3 ORTEP diagram of l,r-bis(triphenylmethyl)-4,4'-biimidazole with 50 % probability thermal ellipsoids (see Appendix A5 for details). 84 4.2.2 Attempted Nitration of H2biim 2-Nitroimidazoles are known to accumulate in hypoxic cells, and by nitrating H2biim, one could potentially synthesize new hypoxic markers.10 Such bidentate nitrobiimidazole ligands might also bind more strongly to metal centres than do nitroimidazoles, thus leading to increased stability of the nitroimidazole-type complexes in situ. Nitration at the 4,4' and 5,5' positions of 2,2'-biimidazole using cone. HNO3 has been reported, and 4,4',5,5'-tetranitro-2,2'-biimidazole has been characterized by X-ray diffraction.11'12 The di-substituted derivative, 4,4'-dinitro-2,2'-biimidazole, has also been synthesized, again using cone. HNO3 as the nitrating agent; however, its characterization solely by elemental analysis leaves some ambiguity as to the actual position of the nitro groups.13 In this thesis work, attempts were made to nitrate H 2biim selectively at the 2,2' positions, to obtain a bidentate analogue of 2N02Im. In the case of imidazole itself, nitration with cone. HNO3 takes place preferentially at the H(4) position, and in the case of 4MeIm, the H(5) position is nitrated preferentially over the H(2) position.1 4 , 1 5 In this thesis work, nitration of l,l'-bis(triphenylmethyl)-4,4'-biimidazole at the 2,2'-positions was attempted following the procedure described by Davis et al. for the nitration of 1-(triphenylmethyl)imidazole at the 2-position using rc-propyl nitrate.16 1,1'-Bis(triphenylmethyl)-4,4'-biimidazole, however, is insoluble in non-chlorinated solvents and no reaction was observed upon treatment of a suspension of this biimidazole in THF, dioxane or hexanes, with n-BuLi, even at refluxing temperatures. Attempts to synthesize the desired 2,2'-dinitro-4,4'-biimidazole via an alternate route, namely Suzuki coupling of two 2N0 2Im derivatives, were also unsuccessful. 4-Bromo-l-methyl-2-nitroimidazole has been reported17'18 but the described syntheses proceed in low yield (< 40 %). 4-Bromo-2-nitroimidazole has also been reported, but the pure compound could not be isolated from the reaction mixture.17 It was also determined (Section 4.3) that the Suzuki coupling step does not tolerate a nitro functional group on the reactant. 85 4.3 Attempted Synthesis of 5,5'-Dinitro-3,3'-bi(l,2,4-triazole) Work with 3-nitro-l,2,4-triazoles as potential anticancer agents suggests that nitrotriazoles can act in a manner similar to that of nitroimidazoles.19 The synthesis of 5,5'-diamino-3,3'-bi(l,2,4-triazole) (bisAT) (Figure 4.4) has been reported in a patent,20 and an improved procedure subsequently established by a member of our group led to bis AT being obtained in 76 % yield.21 The synthesis of the corresponding 5,5'-dinitro-3,3'-bi(l,2,4-triazole) from bisAT using a described literature procedure22 was unsuccessful. The procedure involved reacting the amino groups with cone. H 2 S 0 4 and NaNO"2 to form diazonium salts that can then undergo substitution reactions with NaN0 2 to form the dinitro species. Even after several extractions of the aqueous reaction mixture with ethyl acetate, no product was observed. Attempts to isolate the bitriazole from the aqueous mixture by neutralizing the solution, removing the solvent, and triturating the residue with ethyl acetate, yielded a solid which showed no evidence of any nitro groups by IR spectroscopy and no evidence for the bitriazole unit by ESI-MS. Conversely, the same procedure was successfully employed in the nitration of 3-amino-l,2,4-triazole-5-carboxylic acid (see Section 5.4) to yield the corresponding 3-nitro compound. Suzuki coupling of 3-iodo-5-nitrotriazole to afford the desired dinitro-bitriazole was also unsuccessful; the Pd(PPh3)4 catalyst may react with the nitro group itself, thus decomposing both reactant and catalyst. H H Figure 4.4 Molecular structure of 5,5'-diamino-3,3'-bi(l,2,4-triazole). 86 4.4 Ru-biim Complexes Synthesized from d,s-RuCl2(DMSO)4 Complexes of the type RuCl2(DMSO)2L2 (L = imidazole or nitroimidazole) have been synthesized from the precursor a'.y-RuCl2(DMSO)4 by our group,23 although no crystal structures were performed to determine unambiguously the geometries of the complexes. Similarly, the addition of one equiv. of a biimidazole to the same precursor was expected to lead to the displacement of 2 DMSO ligands with formation of the analogous complex containing one bidentate biimidazole in place of 2 monodentate imidazole ligands, and indeed a 1:1 reaction of H 2biim with RuCl 2 (DMSO) 4 gives the monomelic RuCl2(DMSO)2(biim) species (20). The 'H NMR spectrum of a solution species formed from 20 in C D 3 O D displays 4 resonances for the biimidazole protons indicating that each of the two rings inhabits a different chemical environment (Figure 4.5c, this region of the spectrum is identical for isolated and in situ formed 20); four different resonances are also detected for the DMSO methyl groups, indicating that each of them is inequivalent in solution. Only the all cis-isomer (Figure 4.6) in the solid state (as precursor of the solution species), perhaps accounts for these observations. Figure 4.5a shows the resonances for the free ligand, H2biim, in C D 3 O D to show how the spectrum of the ligand changes upon coordination to the metal centre (Figure 4.5c). The conductivity of 20 in MeOH (127 Q.'lcm2mo\']), is in the range for a 1:1 electrolyte24 thus showing that one CI" ligand is dissociated in solution. An all cis structure has been demonstrated crystallographically for the similar complex RuCl2(DMSO)2(l,2-dimethylimidazole)2,25 this finding contrasts, however, with those from our group for a related compound containing a chelating sulfoxide, trans-RuCl2[i?,^-l,2-bis(ethylsulfinyl)ethane](metro)2, in which the chloride ligands are mutually trans in the solid state structure as determined by X-ray diffraction, although the solution 'H NMR spectrum in D 2 0 shows a mixture of cis- and trans-isomers.26 The l H - ' H COSY NMR spectrum of 20 shows correlation between the two DMSO singlets at 8 2.29 and 3.01 and between those at 5 3.23 and 3.49 (Figure 4.7), each pair corresponding to the two Me groups within one DMSO ligand. Similarly, the resonances for the H(5) and H(2) imidazole protons on each ring also correlate with each 87 free H 2biim T • | • | • , . | . 1 , , , , 8 . 6 8 . 4 8 . 2 8 . 0 7 . 8 7 . 6 7 . 4 7 . 2 Figure 4.5 'H NMR spectra (300 MHz, 298 K) in C D 3 O D showing the imidazole protons of a) free H 2biim in CD 3 OD, b) 1:2 H 2biim:RuCl 2(DMSO) 4 after 4 h at 65 °C in CD 3 OD, and c) 1:1 H 2biim:RuCl 2(DMSO) 4 after 4 h at 65 °C in CD 3 OD. The spectra for isolated 21 and 20 in C D 3 O D are identical to those in (b) and (c), respectively, in this region. CI Figure 4.6 Proposed solid state structure for RuCl2(DMSO)2(biim). 88 a) _ J U L JLUL ppm 3 1 • / 8 7 6 5 4 3 2 1 0 ppm ppm Figure 4.7 'H- 'H COSY NMR spectrum (300 MHz, 298 K) of 20 in CD 3 OD. a) Complete spectrum, b) Expansion showing correlation of imidazole protons, c) Expansion showing correlation of DMSO protons. Small peaks at 8 3.38 and 2.65 are due to Ru2Cl4(DMSO)4(biim) (21) (see text). 89 other, showing that the signals at 8 8.39 and 7.67 belong to the H(2) and H(5) of one ring and those at 5 8.37 and 7.72 to the H(2) and H(5) on the other ring, respectively. From the ' H NMR spectrum , it appears as if there is one S-bound DMSO (8 3.23 and 3.49) and one O-bound DMSO (8 2.29 and 3.01) present; however, the IR shows two strong S=0 stretches at 1071 and 1067 cm"1, both in the region for S-bound DMSO. No IR bands are observed for O-bound DMSO (typically 900 - 1000 cm"1 for Ru(II) complexes).272 The possible upfield shift of the resonances at 8 3.01 and 2.29 may arise from an interaction between the Me protons on the S-bound DMSO (cis to biim) and the 7i-system of biim. The mass spectrum of this complex (Figure 4.8) is consistent with the proposed formulation, clearly showing the M + species, as well as fragments corresponding to the loss of one CI" and to the subsequent loss of one DMSO ligand. Elemental analysis was also consistent with the formulation. By increasing the H2biim:Ru ratio to 2:1, it was expected that two biim ligands might displace all 4 DMSO ligands of the starting material as is the case when RuCl2(DMSO)4 is reacted with pyridine to afford cis-Ru(pyridine)4Cl2;28 however, only 2 0 could be isolated from this reaction. Attempts to further substitute 2 0 with a 5-fold excess of FJ^biim were also unsuccessful. 3 4 8 . 9 3 5 0 9 4 2 6 . 9 '!l 4 2 5 . 9 1 ! :"' 4 2 4 . 9 1 3 5 2 . 9 , ' ' , • 3 8 3 - 9 | 3 5 0 ' 4OO* 4 CO ' SO Figure 4.8 Mass spectrum of RuCl2(DMSO)2(biim) (20): the most intense group of signals is for (M + - CI - DMSO). The parent peak is the small cluster centred at 463. I 463 4 3 0 . 9 ^ 90 Reaction of H 2biim with RuCl 2(DMSO) 4 (H2biim:Ru, 1:2) results in the formation of a bimetallic complex, Ru2Cl4(DMSO)4(biim) (21). Elemental analysis supports this formulation and an [ M + - CI] fragmentation splitting pattern characteristic for this formulation is observed in the mass spectrum (see Figure 5.4 for description of splitting pattern differences between Ru and Ru2). The splitting pattern differs from that of 20 as it clearly shows the isotopic distribution for a unit with two Ru atoms present. The ! H NMR spectrum of a solution form of 21, made in situ in C D 3 O D , displays two major resonances for the H(2) and H(5) protons of the bound biim (Figure 4.4b). This indicates that each set of protons within the two rings is in an equivalent chemical environment, and is consistent with a solid-state formulation in which the biim ligand is situated trans to two bridging chlorides (Figure 4.9). In the region of the 'H NMR spectrum corresponding to the DMSO protons, two major singlets are present, one for free DMSO at 8 2.65 and one at 8 3.38 for bound DMSO, integrating in a 1:1 ratio. A set of weaker intensity resonances is also detected in this region; however, they account for < 5 % of the total integration of the DMSO resonances. The presence of a resonance corresponding to free DMSO in a 1:1 ratio with that of bound DMSO indicates that two of the four DMSO ligands formulated for this species, are displaced in CD3OD. The conductivity of a MeOH solution of isolated 21 (112 Q^cr^rnol"1) is in the range for a 1:1 electrolyte, suggesting that one chloride is also dissociated in this solvent.24 The IR data show two strong stretches for S=0 at 1081 and 1086 cm"1, in the region for S-bound DMSO. From the 'H NMR data, two possible solution structures (Figures 9a and b) can account for the resonances observed in CD 3 OD; these two representations contain equivalent DMSO ligands and equivalent biim rings. Four possible solid state structures (Figure 9c-f) can give rise to the two solution structures (Figures 9a and b); 9e and 9f are preferred, as 9c and 9d are formally Ru(I/IU) spceies. Bimetallic species containing a Ru(I) metal centre are uncommon and almost exclusively involve a metal-metal bond. 2 7 b Precedence for the loss of DMSO and CI" ligands in solution has been shown with RuCl 2(DMSO) 4 which rapidly loses one O-bound DMSO ligand upon dissolution in D 2 0 , and then slowly loses a CI".29 91 D M S O C I C I D M S O e) C I D M S O Figure 4.9 Structures (a) and (b) represent possible solution structures for 21, where all DMSO ligands are S-bound and S = CD 3 OD. Structures (c), (d), (e) and (f) represent the four corresponding solid state structures which could give rise to (a) and (b) in CD3OD; again all DMSO ligands are S-bound. Similar to the biim system, carrying out a 1:1 reaction with H2Me2biim and RuCl 2(DMSO)4 gives formation of the monomeric RuCl 2(DMSO) 2(Me 2biim) species (22). ESI-MS and elemental analysis agree with the proposed formulation, and the ER. spectrum shows two distinct bands for S-bound DMSO (v = 1091 and 1099 cm"1). The "H NMR spectrum of 22 in CD 3 OD is more complicated than that for the corresponding biim complex, 20, in that 3 resonances are observed for the H(5) protons of the Me2biim ligand: 8 7.65, 7.46, and 7.25 (Figure 4.10b). The integrations at 8 7.65 and 7.25 are the same, while that at 8 7.47 is 1.4 times greater. Three resonances are also observed for the methyl protons of the Me2biim ligand: 8 1.66, 1.59, and 1.58 (Figure 4.10c). The integration of the resonances at 8 1.58 and 1.59 are the same, and each of 92 a) 7 . 8 Figure 4.10 7 . 6 7 . 4 7.2 ppm 1 . 7 1.6 ppm 'H NMR data (300 MHz, 298 K) for Me2biim complexes, a) H(5) protons of 23 (see text) in CD 3 OD. b) H(5) protons for 22 in CD 3 OD; weak resonances for 23 are observed as an impurity, c) Corresponding methyl signals for Me2biim of 22 in CD 3 OD. Resonances for single isomers are highlited in (b) and (c). these integrates in a 3:1 ratio with those at 8 7.65 and 7.25, suggesting that these four resonances correspond to a single isomer in which the two rings of the Me2biim ligand are inequivalent. The remaining two 1:3 resonances at 8 7.47 and 1.66 correspond to a second isomer in which the two Me2biim rings are equivalent. In the 'H NMR spectrum of 22 there are overlapping signals for bound DMSO between 8 3.51-2.23. The IR data suggest only S-bound DMSO is present; however, from the 'H NMR spectrum, no interpretation of the DMSO proton resonances can be made, other than that there is no evidence for free DMSO. The conductivity of 22 in MeOH solution (132 Q"'cm2 mol"1) is in the range for a 1:1 electrolyte,24 suggesting that one CI" is dissociated, probably from each isomer. The 93 isomer with the equivalent M e 2 b i i m rings must have trans-chloride and c i s -DMSO ligands in the solid state structure, and when one CI" dissociates and is replaced by M e O H in solution, the equivalence of the two Me 2 b i im rings is preserved. Two solid state structures can be proposed for the other isomer with inequivalent M e 2 b i i m rings. The first, a c i s -Cl , t rans-DMSO species, could produce the observed resonances in the ' H N M R spectrum upon dissociating on chloride in CD3OD; however, precedence in the literature for such a species with other N-donor ligands could not be found. It is more probable that this species is a c is -Cl , c i s -DMSO species as is the case for the analogous biim complex, 20. In the case of the reported RuCl 2 (DMSO) 2 (2MeIm) 2 , the monodentate analogue of 22, the ' H N M R spectrum (in CDCI3) is consistent with an all cis-30 structure. When H 2 M e 2 b i i m and R u C l 2 ( D M S O ) 4 are reacted in a 1:2 ratio, a bimetallic species analogous to that seen with bi im (21) is formed. Elemental analysis and mass spectral data of the isolated complex are consistent with the formulation R u 2 C l 4 ( D M S O ) 4 ( M e 2 b i i m ) (23). Although the parent peak is not seen, as for 21, intense signals for [ M + - CI] (785), and then fragments for the subsequent loss of 1, 2, and 3 551 811 82: 6 5 0 7 0 0 7 5 0 8 0 0 Figure 4.11 Mass spectrum for R u 2 C l 4 ( D M S O ) 4 ( M e 2 b i i m ) (23); the most intense group of signals is for [ M + - CI - 3 D M S O ] , centred at 551. 94 DMSO ligands are observed (Figure 4.11), all exhibiting an isotopic splitting pattern consistent with the presence of two Ru atoms in the complex. In the 'H NMR spectrum in CD3OD, two major resonances are observed for the H(5) proton at 8 7.38 and 7.33 (Figure 4.10a), that at 8 7.38 being roughly twice the size of that at 8 7.33; this suggests the presence of two isomers with equivalent Me2biim rings. There is also a pair of 1:1 singlets at 8 7.47 and 7.46, implying the presence of one isomer with inequivalent Me2biim rings. Between 8 3.55 and 2.54, numerous overlapping and unresolved resonances are observed for the Me resonances of the DMSO and Me2biim ligands, including an intense resonance for free DMSO. As for 21, the conductivity of 23 in MeOH solution (97 £2~'cm2 mol'1) is in the range for a 1:1 electrolyte,24 suggesting replacement of a terminal CI ligand by MeOH. 4.5 Complexes Synthesized from Ru(ma)3 In Chapter 3, the synthesis and characterization of Ru(III) complexes of the type [Ru(ma)2(L)2]CF3S03, where L is an N-bound imidazole or triazole derivative, were presented. With the same experimental procedure used for 14 (Section 3.3.5), analogous complexes of the type [Ru(ma)2(L)]CF3S03-2H20, where L = biim (24) or Me2biim (25), were synthesized. These were characterized by elemental analysis, mass spectrometry, and IR and NMR spectroscopies. The 'H NMR spectrum of 24 in CDCI3 (Figure 4.12a) shows a single resonance for the maltolato Me groups. As the biim rings are "locked" in a cis-configuration, the maltolato ligands must be coordinated so that the "same type" oxygen from each maltolato is trans to biim (Figure 4.13). In the corresponding *H NMR spectrum of 25, two resonances are detected for the maltolato Me groups as well as two resonances for the Me2biim Me groups. This is due either to inequivalent oxygen atoms bound trans to Me2biim (Figure 4.14b), or to the complex existing in solution as a combination of two different isomers with equivalent oxygen atoms bound trans to Me2biim (Figure 4.14b and c). Although the integrations of signals in paramagnetic 'H NMR spectra are generally poor, the near equal intensities and 95 peak widths of the two maltolato Me signals might indicate that they are for a single species, thus favouring the structure in Figure 4.14a. C//3-ma Figure 4.13 Proposed structure for the cation of 24. 96 (a) (b) (c) Figure 4.14 Three possible structures for the cation of 25. The elemental analyses for both complexes give formulations with two hydrated H 2 0 molecules. The IR spectra show strong, broad bands at 3514 and 3550 cm"1 for O-H for 24 and 25, respectively, consistent with the presence of H 2 0 in the solids. The cyclic voltammograms of the complexes in MeCN reveal markedly more positive reduction potentials than for the analogous bis(imidazole) complexes (7 and 10, Table 4.1). The difference in cis/trans geometry of the bis(imidazole) and biimidazole complexes can not account for such a large difference in potential; however, the more conjugated 7t-system of the biimidazole ligands may make these ligands better rc-acceptors and thus increase the reduction potentials for 24 and 25. Table 4.1 Ru (HUH.) reduction potentials for the biimidazole complexes 24 and 25, compared with those for 7 and 10. Complex Ru (mm) Reduction Potential in MeCN vs. SCE (mV) cw-[Ru(ma),(biim)][CF3S03] (24) -101 cis-[Ru(ma)2(Me2biim)] [CF 3S0 3] (25) -35 [Ru(ma)2(Im)2][CF3S03] (7) -705 trans-[Ru(ma)2(2MeIm)2] [CF 3S0 3] -844 4.6 Complexes Synthesized from [Ru(DMF) 6 ](CF 3 S0 3 ) 3 [Ru(DMF) 6](CF 3S0 3) 3 has been previously used in this group as a precursor for the synthesis of Ru(U) hexakis(imidazole) complexes.31 The same general synthetic 97 procedure with MeOH as solvent was employed in the synthesis of the analogous complexes [Ru(L)3](CF 3S0 3) 2, where L = biim (26) and Me2biim (27), which were isolated in 72 and 83 % yield, respectively. Four equivalents of ligand are required for the syntheses, whereby one equivalent of H2biim or H2Me2biim reduces the Ru centre (m^H). 3 2 Although MeOH can also act as a reducing agent,33 the same synthetic reaction with H 2biim performed in situ in CD 3 OD showed no trace of free H2biim after 12 h by *H NMR spectroscopy, but instead the spectrum of the product solution showed two singlets at 8 8.92 and 7.84, not present in the spectrum for the isolated complex, 26. These resonances may be due to a bis-biimidazole species (such as that shown in Figure 4.15), resulting from one equivalent of H2biim being oxidized by the Ru(DI). This type of Ru(UI)—+Ru(II) reduction in the presence of excess Im has been reported to occur in water at r.t. over several days.32 For both 26 and 27, the mass spectra clearly show both the Ru(L) 3 + peaks ( M + for 26 and 27 is defined as [Ru(L) 3][CF 3S0 3] 2, see Sections 4.9.7 and 4.9.8) as well as the [Ru(L) 3][CF 3S0 3] + peaks (L = biim or Me2biim, respectively). The *H NMR spectrum of the 26 displays 2 equal intensity singlets at 8 8.37 for H(2) and 7.62 for H(5). In the corresponding *H NMR spectrum of 27, two resonances at 8 7.43 for H(5) and 2.54 for the Me-group, integrating for 1 and 3 protons, respectively, are detected. Figure 4.15 Possible structure for a bis-biimidazole species, from reduction of the Ru centre. 98 4.7 Complexes Synthesized from R u C l 3 3H2O Ru0 3 -3H 2 0 was also used as a precursor for the synthesis of Ru-biim and Ru-Me2biim complexes; these were synthesized via the 'Ru blue' solution, formed upon reduction of RuCI 3-3H 20 by H 2 in M e O H . 3 4 Addition of 2 equivalents of H2biim to such a solution gives a complex formulated as [Ru(Hbiim)2Cl2]Cl2 (28), which was isolated as a brown precipitate from a MeOH/acetone solution and characterized by 'H NMR spectroscopy and ESI-MS. Only two broad singlets at 5 8.59 and 7.52 are observed in Figure 4.16 Molecular structure for 28. 2 cr the ' H NMR spectrum in D 2 0 , suggesting that the biim ligands are trans with 4 chemically equivalent heterocyclic rings. The two additional protons in the complex presumably exchange mutually in solution, thus broadening the proton resonances of H(2) and H(5). A potentiometric titration of a solution of 28 in H 2 0 at r.t. with 0.01 M AgN0 3 solution confirmed the presence of the two non-coordinated chlorides,. the solution conductivity increased sharply after 2 equivalents of AgN0 3 had been added, suggesting that two equivalents of chloride had been titrated. MS on the resulting solution, after the titration was completed, revealed that the complex was still intact (M + at 440) with two CI" bound. Titration of a solution of 28 in H 2 0 with 0.01 M NaOH gave two equivalence points for the two titratable protons with pK a values of 7.7 and 8.5. The conductivity of an aqueous solution of 28 was 265 Q"1 cm 2 mol"1, in the range for a 2:1 electrolyte.35 The corresponding "Ru blue" reaction with H 2Me 2biim in place of H 2biim gave a product consistent with the formulation for [Ru(HMe2biim)(Me2biim)2]Cl3 (29). Again, only two broad signals are observed in the [ H NMR spectrum in D 2 0 , one for the H(5) 99 protons, and one for the Me protons, consistent with three equivalent Me2biim ligands with the extra proton presumably exchanging between all 3 of them. Titration of a solution of 29 in H 2 0 with AgN0 3 confirmed the presence of 3 unbound CI" ions while titration with NaOH showed that there was one titratable proton with a pK a of 8.8. The conductivity of 29 in H 2 0 (385 Q.'1 cm 2 mol"1) is in the range for a 3:1 electrolyte.35 Unlike 28 where two biim ligands can adopt a trans-configuration, 2 Me2biim ligands are unlikely to be mutually trans due to the steric hindrance of the 2-methyl groups, and this results in the ready formation of the all-cis tris-substituted product. 4.8 Experimental Preparation of Biimidazoles 4.8.1 Sources of Materials The sources of solvents, some reagents and Ru precursors are reported in Chapter 2. Pd(PPh3)4 was prepared according to a literature procedure.9 PdCl 2 was obtained from Colonial Metals Inc. I2, NEt 3 , Na 2 S 2 0 3 and Na 2 S0 3 (not listed in Chapter 2) were all purchased from Fisher Scientific. 4.8.2 2,4,5-Triiodoimidazole This compound was synthesized according to literature procedures.36"39 To a solution of l2 (112.0 g, 441 mmol) in CHC1 3 (400 mL) was added Im (10.0 g, 147 mmol) and 2 M NaOH (400 mL). This mixture formed a two-phase system with the organic phase containing the I2 on the bottom, and the top aqueous phase being colourless. The mixture was stirred for 3 h at r.t. after which time the bottom phase had become colourless and the top phase had turned pink. The phases were separated and the organic phase was discarded. To the aqueous phase was added Na 2 S 2 0 3 (50.0 g) to prevent colouration of the product, before the aqueous phase was neutralized to pH 7.0 with AcOH. The resulting white precipitate was collected, washed with cold water (2 x 10 mL) and dried at 80 °C for 16 h. Yield: 46.42 g (70 %). FAB-MS: 446 (M+), 320 ( M + -I), 193 (M + -2I) . 100 4.8.3 4(5)-Iodoimidazole This compound was synthesized from a modified literature procedure.36 To a solution of Na 2 S0 3 (70.0 g, 555 mmol) in 30 % aq. EtOH (1.0 L), 2,4,5-triiodoimidazole (30.0 g, 67.3 mmol) was added. The mixture was heated at 50 °C for 1 h at which time the solid had completely dissolved affording a yellow solution that was refluxed for 24 h. The condenser was then removed and the solution concentrated until a precipitate started to form. The flask was then immediately removed from the heating mantel and allowed to cool slowly to r.t. The resulting pale yellow crystals were removed by filtration and dried under vacuum for 24 h. Yield: 8.42 g (64 %). 'H NMR (acetone-d6): 8 10.20 (br s, 1H, Im-//,), 7.80 (s, 1H, Im-H2), 7.28 (s, 1H, Im-//5). FAB-MS: 194 (M +) , 67 ( M + -1). Anal. Calcd for C 3 H 3 N 2 I : C, 18.56; H, 1.55; N, 14.43. Found: C, 18.52; H, 1.61; N, 14.21. 4.8.4 4-Iodo-l-(triphenylmethyl)imidazole This compound was synthesized from a literature procedure.36 To a solution of 4-iodoimidazole (4.00 g, 20.6 mmol) in D M F (35 mL) was added chlorotriphenylmethane (5.75 g, 20.6 mmol). The solution was degassed for 10 min and then stirred at r.t. for 1 h under N 2 . To this solution was then added NEt 3 (2.30 g, 22.7 mmol) and the resulting mixture was stirred for 24 h. The mixture was then poured into an ice slurry (150 mL) and the precipitate was collected via filtration. The solid was dried under vacuum at 78 °C for 24 h. Yield: 6.23 g (69 %). 'H NMR (CDC13): 5 7.37-7.29 (m, 10H, Im-// 2 and Ar//), 7.14-7.09 (m, 6H, Ar/7), 6.92 (d, 1H, Im-H5). FAB-MS: 436 (M +), 243 (CPh3), 194 (M + - CPh3). Anal. Calcd for C 2 2 Hi ? N 2 I : C, 60.55; H, 3.90; N, 6.42. Found: C, 60.69; H, 4.11; N, 6.18. The ! H NMR data for this compound agree with those reported.36 4.8.5 1,1 '-Bis(tripheny lmethyl)-4,41 -biimidazole This compound and the following six compounds were synthesized via modified literature procedures.5 A solution of 4-iodo-l-(triphenylmethyl)imidazole (4.00 g, 9.17 mmol) in D M F (15 mL) was degassed with Ar for 10 min. To this solution was added Pd(PPh3)4 (450 mg, 0.39 mmol) and NEt 3 (1.96 g, 18.4 mmol). The flask was then 101 wrapped in foil and heated to 120 °C for 48 h in the dark. The solution was cooled to r.t., and the resulting precipitate was collected by filtration, rinsed with acetone ( 3 x 5 mL), and dried under vacuum for 24 h at 78 °C. Yield: 2.21 g (78 %). 'H NMR (CDC13): 8 7.37 (d, 2H, // 2 i 2-biim), 7.32-7.29 (m, 18H, ArH), 7.27 (d, 2H, #5>5.-biim), 7.20-7.17 (m, 12H, ArH). FAB-MS: 618 (M +), 243 (CPh3). Anal. Calcd for C44H 3 4N 4: C, 85.43; H, 5.50; N, 9.06. Found: C, 85.61; H, 5.43; N, 8.75. The 'H NMR and MS characterization data for this compound and for the next six compounds agree with those reported.5 4.8.6 4,4'-Biimidazolium trifluoroacetate (H2biim) l,l'-Bis(triphenylmethyl)-4,4'-biimidazole (2.00 g, 3.23 mmol) was dissolved in 50% C F 3 C 0 2 H / H 2 0 (50 mL) and the solution was stirred at r.t. for 12 h. The resulting white precipitate (CPh3OH) was then removed by filtration and the yellow filtrate was evaporated under vacuum to yield a bright yellow oil. Addition of MeCN (2.0 mL) yielded a white solid that was collected, rinsed with acetone (3x5 mL) and dried under vacuum at 78 °C for 24 h. Yield: 0.96 g (82 %). *H NMR (CD 3OD): 8 8.74 (s, 2H, H2,2-biim), 7.79 (s, 2H, H 5 > 5-biim). ESI-MS: 135 (M+). Anal. Calcd for C 1 0 H 8 N 4 F 6 O 4 : C, 33.15; H, 2.21; N, 15.47. Found: C, 33.24; H, 2.12; N, 15.68. 4.8.7 4,5,-Diiodo-2-methylimidazole To a solution of 2MeIm (10.0 g, 122 mmol) in 2 M NaOH (300 mL) was added CHC1 3 (300 mL). I2 (62.0 g, 244 mmol) was then added to this mixture slowly over 20 min, adding 5 g at a time and waiting until it had completely dissolved before adding more. Adding the I2 too quickly led to the formation of a brown foam, from which the final product could not be separated. The two-phase system was then stirred for an additional 3 h at which time the lower phase had become colourless. The phases were separated and the aqueous phase neutralized according to the procedure described in Section 4.8.2. Yield: 28.10 g (69 %). *H NMR (CDC13): 8 10.72 (br s, Im-H,), 2.48 (s, Im-C#3). FAB: 335 (M+), 209 ( M + -1). 102 4.8.8 4(5)-Iodo-2-methyIimidazole 4,5,-Diiodo-2-methylimidazole (20.0 g, 59.9 mmol) was dissolved in 700 mL H 2 0 . To this solution was added Na 2 S0 3 (60.0 g, 476 mmol) and EtOH (300 mL). This mixture was heated for 1 h at 50 °C until the sulfite had completely dissolved. The yellow solution was then refluxed for 24 h, cooled to r.t., and 500 mL of EtOH were added. The resulting white precipitate (sodium salts) was removed by filtration and the filtrate was concentrated until a fine precipitate started to form. The filtrate was then cooled and the resulting yellow crystals were collected, washed with cold H 2 0 (2x5 mL) and dried at 78 °C for 24 h. Yield: 6.82 g (55 %). 'H NMR (CDC13): 5 11.08 (br s, 1H, Im-//,), 5 7.08 (s, 1H, Im-//5), 2.44 (s, 3H, Im-C/73). FAB: 209 (M+). 4.8.9 4-Iodo-2-methyl-l-(triphenylmethyl)imidazole 4(5)-Iodo-2-methylimidazole (4.69 g, 22.5 mmol) was added to a solution of chlorotriphenylmethane (4.18 g, 15.0 mmol) in D M F (40.0 mL). Under 1 atm N 2 , NEt 3 (1.95 g, 19.2 mmol) was added and the solution was stirred at r.t. for 24 h. The mixture was then poured onto an ice slurry (150 mL) and the resulting precipitate was collected and dried under vacuum for 24 h at 78 °C. Yield: 4.52 g (67 %). 'H NMR (CDC13): 5 7.36-7.33 (m, 9H, Ar//), 7.14-7.09 (m, 6H, Ar//), 6.75 (s, 1H, Im-/75), 1.58 (s, 3H, Im-C// 3 ) . FAB: 450 (M+), 243 (CPh3), 209 (M + - CPh3). Anal. Calcd for C 2 3 H 1 9 N 2 I : C, 61.33; H, 4.22; N, 6.22. Found: C, 61.25; H, 4.17; N, 6.02. 4.8.10 2,2'-Dimethyl -1,1'-bis(triphenylmethyl)-4,41 -biimidazole 4-Iodo-2-methyl-l-triphenylmethylimidazole(4.00 g, 8.89 mmol) was dissolved in DMF (15 mL), and the solution was degassed with Ar for 10 min. To this was added Pd(PPh3)4 (550 mg, 0.440 mmol) and NEt 3 (1.80 g, 17.8 mmol). The flask was then sealed under N 2 and wrapped in foil to exclude all light, and the reaction mixture then heated at 120 °C for 48 h. The flask was then cooled to r.t. and the resulting precipitate was collected, washed with acetone (3x5 mL), and dried under vacuum at 78 °C for 24 h. Yield: 1.97 g (69 %). 'H NMR (CDC13): 5 7.31-7.29 (m, 18H, Ar//), 7.22 (s, 2H, / / 5 , 5 -Me2biim), 7.17-7.15 (m, 12H, Ar//), 1.60 (s, 6H, C/73-Me2biim). LSfMS: 647 (M+), 243 103 (CPh3). Anal. Calcd for C 4 6 H 3 8 N 4 : C, 85.45; H , 5.88; N, 8.67. Found: C, 85.34; H , 5.75; N, 8.48. 4.8.11 2,2'-Dimethyl-4,4'-biimidazoIium trifluoroacetate (H2Me2biim) 2,2'-Dimethyl-l,r-bis(triphenylmethyl)-4,4'-biimidazole (1.90 g, 2.94 mmol) was suspended in 60 % CF3CO2H/H2O (50 mL), and the mixture was then refluxed for 6 h and cooled to r.t., when the resulting precipitate was removed by filtration. The filtrate was evaporated to give a yellow oil. MeCN (3 mL) was added affording a white precipitate that was collected, washed with acetone ( 3 x 5 mL), and dried under vacuum for 24 h at 78 °C. Yield: 0.89 g (78 %). 'H NMR (CD 3OD): 5 7.65 (s, 2H, tf5,5-Me2biim), 2.72 (s, 6H, C// 3-Me 2biim). ESI-MS: 163 (M+). Anal. Calcd for C 1 2 H i 2 N 4 F 6 0 4 : C, 36.92; H, 3.08; N, 14.36. Found: C, 37.19; H, 2.98; N, 14.38. 4.9 Experimental Syntheses of Complexes 4.9.1 m-RuCl2(DMSO)2(biim) (20) To a yellow solution of RuCl 2(DMSO) 4 (0.070 g, 0.15 mmol) in MeOH (10 mL), H2biim was added (0.054 g, 0.15 mmol) under N2 and the mixture refluxed for 4 h. The solvent was then reduced in volume to ~1 mL, followed by the addition of 10 mL of acetone that yielded a yellow precipitate that was collected, washed with acetone (2x5 mL), and dried in vacuo. Yield: 0.059 g (88 %). 'H NMR (CD 3OD): 8 8.39, 8.37 (s, 2H, //2,2-biim), 7.72, 7.67 (s, 2H, // 5, 5-biim), 3.49 (s, 3H, C//3SOC//3), 3.23 (s, 3H, C//3SOC//3), 3.01 (s, 3H, C//3SOC//3), 2.29 (s, 3H, C/ / 3 SOC// 3 ) . IR (KBr pellet): v 3451 (s, N-H), 1067 (s, S=0), 1020 (m). ESI-MS (MeOH): 463 (M+), 427 ( M + - CI), 349 (M + - CI - DMSO). A M (MeOH) = 127 frWmor 1. Anal. Calcd for C ,oH 1 8 Cl2N 4 02S2Ru: C, 25.87; H, 3.90; N, 12.12. Found: C, 25.67; H, 3.98; N, 11.99. 4.9.2 Ru2Cl4(DMSO)4(biim) (21) This complex was synthesized following the procedure outlined in Section 4.9.1, but using H 2biim (0.026 g, 0.074 mmol) and RuCl 2(DMSO) 4 (0.071 g, 0.15 mmol). Yield: 0.042 g (73 %). *H NMR (CD 3OD): 8 8.55 (s, 2H, //2,2-biim), 7.48 (s, 2H, H5tS>-104 biim), 3.46 (s, 6H, C / / 3 S O C H 3 ) , 2.65 (s, 6H, free DMSO). IR (KBr pellet): v 3449 (br s, N-H), 1086 (s, S=0), 1081 (s, S=0), 1013 (m). ESI-MS (MeOH): 757 (M +-C1), 679 ( M + - CI - DMSO). A M (MeOH) = 112 Q-Wmol" 1. Anal. Calcd for C14H30CI4N4O4S4R112: C, 21.27; H, 3.80; N, 7.09. Found: C, 21.36; H, 4.00; N, 7.51. 4.9.3 RuCl2(DMSO)2(Me2biim) (22) This complex was synthesized following the procedure outlined in Section 4.9.1, but using H 2Me 2biim (0.058 g, 0.15 mmol) and RuCl 2(DMSO) 4 (0.072 g, 0.15 mmol). Yield: 0.064 g (90 %). ] H NMR (CD 3OD): 8 7.65, 7.47, 7.25 (s, 2H, // 5 j 5.-Me 2biim), 3.54- 3.32, 3.25-2.73, 2.32 (s, 12H, C//3SOC//3), 1 66, 1.59, 1.58 (s, 6H, C// 3-Me 2biim). IR (KBr pellet): v 3455 (N-H), 1420 (s), 1091 (s, S=0), 1099 (s, S=0), 1014 (m). ESI-MS (MeOH): 491 (M+), 455 ( M + - CI), 377 ( M + - CI - DMSO). A M (MeOH) = 132 £2 W m o r 1 . Anal. Calcd for C 1 2 H 2 2 C l 2 N 4 0 2 S 2 R u : C, 29.39; H, 4.49; N, 11.43. Found: C, 29.58; H, 4.64; N, 11.82. 4.9.4 Ru 2Cl 4(DMSO) 4(Me 2biim) (23) This complex was synthesized following the procedure outlined in Section 4.9.1, but using H 2Me 2biim (0.029 g, 0.074 mmol) and RuCl 2(DMSO) 4 (0.070 g, 0.15 mmol). Yield: 0.049 g (81 %). *H NMR (CD 3OD): 8 7.47, 7.46, 7.38, 7.33 (s, H5^-Me2bnm), 3.55- 3.31, 3.05-2.75, 2.59, 2.54 (s, CH3SOCH3 and C// 3-Me 2biim), 2.65 (s, free DMSO). IR (KBr pellet): v 3457 (s, N-H), 1086(s, S=0), 1081 (s, S=0). ESI-MS (MeOH): 785 (M + - CI), 707 ( M + - CI - DMSO), 629 (M + - CI - 2 DMSO), 551 ( M + - CI - 3 DMSO). A M (MeOH) = 97 Q-Wmol" 1 . Anal. Calcd for Ci 6H34Cl 4N 404S 4Ru2: C, 23.47; H, 4.16; N, 6.85. Found: C, 23.58; H, 4.19; N, 6.65. 4.9.5 [Ru(maltolato)2(biim)]CF3S03-2H20 (24) To a solution of [Ru(maltolato)2(EtOH)2]CF3S03 (3) (0.088g, 0.15 mmol) in 5 mL EtOH was added H2biim (0.055g, 0.15 mmol), and the mixture was refluxed under N 2 for 24 h, when it slowly turned brown. After 24 h, the solvent was removed under vacuum and the brown residue was redissolved in acetone; this was cooled to 0 °C and then filtered through Celite (2g). The filtrate was warmed to r.t. and reduced in volume 105 to 2 mL. Hexanes (15 mL) were then added to yield a brown precipitate that was collected, washed with hexanes ( 2 x 5 mL) and dried at 78 °C under vacuum for 48 h. Yield 0.054 g (58 %). 'H NMR (CDC13): 5 52.7 (br s, C//3-ma), -7.0 (br s, //5,5-biim), -26.2 (br s, //2,2-biim). IR (KBr pellet): v 3514 (O-H, s), 2899 (C-H, m), 1602 (C=0, m), 1548 (m), 1467 (m), 1264 (m), 1031 (s). LR-MS (+LSIMS, thioglycerol): 486 (M +), 361 (M + - ma). A M (MeOH) = 106 QAcm2moY]. C V (MeCN): E 1 / 2 (Ru""") = -0.101 V. Anal. Calcd for C 1 9 Hi 6 F 3 N 4 09SRu-2H 2 0: C, 34.02; H, 2.99; N, 8.36. Found: C, 34.11; H, 2.89; N, 8.18. 4.9.6 [Ru(maltolato) 2(Me 2biim)]CF 3S0 3-2H 20 (25) This complex was prepared using the same procedure of section 4.9.5 but using 3 (0.090 g, 0.15 mmol) and H 2Me 2biim (0.060 g, 0.15 mmol). Yield 0.061 g (61 %). 'H NMR (CDC13): 5 64.8, 60.6 (br s, C//3-ma), 42.1, 37.5 (br s, C// 3-Me 2biim), -9.1 br (s, // 5, 5-Me 2biim). IR (KBr pellet): v 3550 (O-H, s), 2916 (C-H, m), 1602 (C=0, m), 1542 (m), 1468 (m), 1263 (m), 1030 (s). ESI-MS: 514 (M+), 389 ( M + - ma). A M (MeOH) = 127 a'cr^mor 1. C V (MeCN): E 1 / 2 (Ru I M I) = -0.035 V vs. SCE. Anal. Calcd for C 2 iH 2 oF 3 N 4 0 9 SRu- 2H 2 0: C, 36.00; H, 3.43; N, 8.00. Found: C, 35.92; H, 3.28; N, 8.17. 4.9.7 [Ru(biim) 3][CF 3S0 3] 2 (26) To a solution of [Ru(DMF) 6][CF 3S0 3] 3 (0.083 g, 0.085 mmol) in MeOH (5 mL) was added H 2biim (0.12 g, 0.34 mmol). This yellow solution was refluxed; after 10 h, the solution had turned green and after 24 h had become very dark green. The solvent was removed under vacuum and the green residue was redissolved in acetone (10 mL); the mixture was filtered through Celite, and the filtrate was then reduced in volume to ~1 mL. To this was added hexanes (8 mL) to yield a green precipitate that was collected, washed with hexanes (2x5 mL), and dried under vacuum at 78 °C for 24 h. Yield: 0.034 g (60 %). 'H NMR (CD 3OD): 5 8.37 (s, 6H, // 2, 2-biim), 7.62 (s, 6H, //5,5-biim). ESI-MS (MeOH): 652 ( M + - CF 3 S0 3 ) , 502 (M + - 2 CF 3 S0 3 ) , 369 ( M + - 2 C F 3 S 0 3 - biim). A M (MeOH) = 243 trWmor 1. Anal. Calcd for C 2 0 H i 8 F 6 N , 2 O 6 S 2 R u : C, 29.96; H, 2.25; N, 20.97. Found: C, 29.75; H, 2.31; N, 20.64. 106 4.9.8 [Ru(Me2biim)3][CF3S03]2 (27) To a solution of [Ru(DMF) 6][CF 3S0 3] (0.074 g, 0.075 mmol) in MeOH (5 mL) was added H 2Me 2biim (0.12 g, 0.30 mmol). This yellow solution was refluxed for 16 h; the solution turned brown after 2 h and slowly became dark blue in colour. After 16 h, the solvent was removed under vacuum and the black residue was redissolved in acetone (10 mL); this mixture was filtered through Celite, and the filtrate reduced in volume to ~1 mL. To this was added hexanes (8 mL) to yield a blue precipitate that was subsequently collected and dried under vacuum at 78 °C for 24 h. Yield: 0.042 g (63 %). *H NMR (CD 3OD): 8 7.43 (s, 6H, #5,5'-Me2biim), 2.54 (s, 18H, Cr73-Me2biim). ESI-MS (MeOH): 736 (M + - CF3SO3), 587 ( M + - 2 CF3SO3), 575 ( M + - C F 3 S O 3 - Me2biim), 425 (M + - 2 C F 3 S O 3 - Me2biim). Anal. Calcd for C 2 6 H3oF 6 N 1 2 0 6 S 2 Ru: C, 35.25; H, 3.39; N, 18.98. Found: C, 35.16; H, 3.32; N, 18.44. 4.9.9 [Ru(Hbiim)2Cl2]Cl2 (28) H 2 was bubbled through a solution of ,RuCl 3-3H 20 (0.054 g, 0.21 mmol) in refluxing MeOH for 3 h at which time the brown solution had become blue. To this solution was added H 2biim (0.15 g, 0.40 mmol). and the mixture was refluxed for 10 h under 1 atm H 2 when it slowly turned brown. The solvent was then reduced in volume under vacuum to ~1 mL and acetone (5 mL) was added to yield a brown precipitate that was collected, washed with acetone (2x5 mL), and dried under vacuum at 78 °C for 24 h. Yield: 0.042 g (42 %). J H NMR (D20): 8 8.59 (br s, H 2 ) 2-biim), 7.58 (br s, /7 5, 5-biim). ESI-MS (H 20): 440 ( M + - 2H), 404 (M + - H - CI). A M (H 20) = 265 Q-Wmoi' 1. Anal. Calcd for C 1 2 H , 4 C l 4 N 8 R u : C, 28.07; H, 2.73; N, 21.83. Found: C, 27.92; H, 2.79; N, 21.59. 4.9.10 [Ru(HMe2biim)(Me2biim)2]Cl3 (29) The procedure of Section 4.9.9 was followed but using RuCl 3 -3H 2 0 (0.042 g, 0.16 mmol) and H 2Me 2biim (0.13 g, 0.33 mmol); also Et 2 0 (5 mL) rather than acetone was added to give the brown product. Yield: 0.034 g (30 %). 'H NMR (D20): 8 7.71 (br s, 1H, /75,5.-Me2biim), 1.68 (br s, 3H, C// 3-Me 2biim). ESI-MS (H 20): 587 ( M + - H), 425 107 (M + - HMe2biim). A M (H 20) = 385 aWrnol"1. Anal. Calcd for C 2 4 H 3 i C l 3 N 1 2 R u : C, 41.47; H, 4.46; N, 24.19. Found: C, 41.25; H, 4.51; N, 23.93. 4.10 References (1) Goulle, V.; Thummel, R. Inorg. Chem. 1990, 29, 1767. (2) Rillema, D. P.; Sahai, R.; Matthews, P.; Edwards, A. K.; Shaver, R. J.; Morgan, L. Inorg. Chem. 1990, 29, 167. (3) Rau, S.; Buettner, T.; Temme, C ; Ruben, M. ; Goerls, H.; Walther, D.; Duati, M.; Fanni, S.; Vos, J. G. Inorg. Chem. 2000, 39, 1621. (4) Elgafi, S.; Field, L. D.; Messerle, B. A.; Hambley, T. W.; Turner, P. J. Chem. Soc., Dalton Trans. 1997, 2341. (5) Cliff, M. ; Pyne, S. Synthesis 1994, 681. (6) Gamez, P.; Simons, C ; Aromi, G.; Driessen, W.; Challa, G.; Reedijk, J. Appl. Catal. A 2001, 214, 187. (7) Clarke, M . J.; Zhu, F.; Frasca, D. R. Chem. Rev. 1999, 99, 2511. (8) McMurry, J. Organic Chemistry, third ed.; Brooks/Cole Publishing Co.: Pacific Grove, 1992. (9) Coulson, D. R. Inorg. Synth. 1972,13, 121. (10) Hodgkiss, R. J. Anti-Cancer Drug Des. 1998, 13, 687. (11) Cromer, D. T.; Storm, C. B. Acta Cryst. C 1990, 46, 1959. (12) Cromer, D. T.; Storm, C. B. Acta Cryst. C 1990, 46, 1957. (13) Melloni, P.; Dradi, E.; Logemann, W. J. Med. Chem. 1972,15, 926. (14) Novikov, S. S.; Khmel'nitskii, L. I.; Lebedev, O. V.; Sevast'yanova, V. V.; Epishina, L. V. Khim. Geterotsikl. Soedin. [English Translation] 1970, 6, 465. (15) Novikov, S. S.; Khmel'nitskii, L. I.; Lebedev, O. V.; Sevast'yanova, V. V.; Epishina, L. V. Khim. Geterotsikl. Soedin. [English Translation] 1970, 6, 614. (16) Davis, D. P.; Kirk, K. L ; Cohen, L. A. J. Heterocycl. Chem. 1982,19, 253. (17) Farah, S. F.; McClelland, R. A. Can. J. Chem. 1993, 71, 427. (18) Palmer, B. D.; Denny, W. A. J. Chem. Soc, Perkin Trans. 11989, 95. 108 (19) Nagao, Y.; Sano, S.; Ochiai, M . Tetrahedron 1990, 46, 3211. (20) Shreve, R. N.; Charlesworth, R. K. U.S. Patent 2,744,116 1956. (21) Tsang, J. Y. K. Synthesis of Ruthenium(II) Complexes Containing Biheterocycles, B.Sc. Dissertation, University of British Columbia: Vancouver, 2003. (22) Bagal, L. I.; Pevzner, M . S.; Frolov, A. N.; Sheludyakova, N. I. Khim. Geterotsikl. Soedin. [English Translation] 1970, 2, 240. (23) Chan, P. K. L.; Chan, P. K. H.; Frost, D. C ; James, B. R. Can. J. Chem. 1988, 66, 117. (24) Geary, W. J. Coord. Chem. Rev. 1971, 7, 81. (25) Iwamoto, M ; Alessio, E.; Marzilli, L. G. Inorg. Chem. 1996, 35, 2384. (26) Wu, A.; Kennedy, D. C ; Patrick, B. O.; James, B. R. Inorg. Chem. 2003, 42, 7579. (27) Seddon, E. A.; Seddon, K. R. The Chemistry of Ruthenium; Elsevier Sceince Publishers: New York, 1984. (a) p354, (b) p 891. (28) Evans, I. P.; Spencer, A.; Wilkinson, G. J. Chem. Soc., Dalton Trans. 1972, 204. (29) Alessio, E . ; Mestroni, G.; Nardin, G.; Attia, W. M . ; Calligaris, M . ; Sava, G.; Zorzet, S. Inorg. Chem. 1988, 27, 4099. (30) Chan, P. K. L. Ruthenium Nitroimidazole Complexes as Radiosensitizers, Ph. D. Dissertation, University of British Columbia: Vancouver, 1988. (31) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents, Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. (32) Anderson, C ; Beauchamp, A. Inorg. Chem. 1995, 34, 6065. (33) Hallman, P. S.; Stephenson, T. A.; Wilkinson, G. Inorg. Synth. 1970,12, 237. (34) Rose, D.; Wilkinson, G. J. Chem. Soc, Dalton Trans. 1970, 1791. (35) Huheey, J. E. Inorganic Chemistry: Principles of Structure and Reactivity, third ed.; Harper Collins Publishers: New York, 1983. (36) Kirk, K. L. J. Heterocycl. Chem. 1985, 22, 57. (37) Iddon, B.; Lim, B. L. J. Chem. Soc, Perkin Trans. 11983, 735. (38) Brunings, K. J. J. Am. Chem. Soc. 1947, 69, 205. (39) Pauly, H.; Arauner, E. J. Prakt. Chem. 1928,118, 33. 109 Chapter 5 Synthesis and Characterization of Ru Complexes with Chelating N-Heterocyclic Carboxylates 5.1 Introduction The coordination of carboxylate groups to transition metals is typically investigated using IR spectroscopy, the difference in the symmetric and asymmetric stretching frequencies being used to determine the coordination mode when X-ray structural data are not available (Figure 5.1).1'2 R R R a b c Figure 5.1 Binding modes of carboxylate groups with transition metal complexes; a) Monodentate, b) Chelating, and c) Bridging. The coordination chemistry of pyridine-2-carboxylic acid, more commonly referred to as picolinic acid (Hpic) (Figure 5.2), has been widely studied.3"9 Hpic is a tryptophan metabolite, and its transition metal complexes may have biological implications.10 For example, Cr(pic)3 is currently marketed as a nutritional supplement and weight-loss agent.8'11 Trace Cr is essential in the body, while Cr(pic)3 is readily absorbed by the body and shows a lack of toxicity at doses as high as 30 mg Cr per kg body weight in rats.12 The fate of the complex and nature of the absorbed active species, however, remain unknown.8 At a concentration of 120 uM, Cr(pic)3 on reduction can react with 0 2 to form hydroxyl radicals that possibly cleave D N A . 1 1 At pH < 1, aqueous 110 solutions of Cr(pic)3 form [Cr(pic)2(H20)]+, which may be the active species absorbed by the body.8 Figure 5.2 Molecular structure of pyridine-2-carboxylic acid, Hpic. Ru readily forms bridged-carboxylate bimetallic complexes when reacted with carboxylic acids,13 for example, the Ru(IJJ/n) bimetallic precursor used in the work discussed in this chapter, [Ru 2(|i-CH 3COO) 4Cl]. Related oxo-centered Ru trimetallic carboxylate complexes have also been reported.13'14 More recently there have been reports of N-heterocyclic carboxylates chelating via N- and O-atoms to Ru to form monomeric complexes. For example, mer-Ru(pic)3-H20 has been synthesized from K 2[RuCl 5(H 2Q)], 3 [Ru 2(u-CH 3COO) 4Cl], 4 and RuCl 3-3H 20; 5 however, this complex is only sparingly soluble in aqueous solution thus limiting its potential for biological applications. In Section 5.2, a new Ru(IJI/n) bimetallic picolinate complex is described; this complex is highly water-soluble, and may have useful biological applications. Reactions analogous to those reported in the literature with Ru and Hpic, but using instead imidazole-4-carboxylic acid (Im-C02H), will then be discussed in Section 5.3. Finally, some preliminary work with a third ligand, 3-nitro-l,2,4-triazole-5-carboxylic acid (HCANT), will be presented. In Section 3.3.3, it was noted that Ru-maltolato complexes containing 2N02Im, 4N02Im or 3N02tri ligands were not stable in DMSO solution and were insoluble in other solvents. It was thought, therefore, that as two of the carboxylate complexes discussed in this chapter ([Ru2(pic)4(Et0H)Cl]-H20 (31) and Ru(Im-C0 2 )(Im-C0 2 H) 2 Cl 2 EtOHH 2 0 (33)) exhibited high water-solubility, that Ru complexes with H C A N T might also be water-soluble and thus have potential for further biological testing. I l l 5.2 Synthesis and Characterization of [Ru 2(pic) 4(EtOH)CI]H 20 (31) mer-Ru(pic)3H20 (30) is a yellow solid, sparingly soluble in water and DMSO, and completely insoluble in other solvents tested.4 The synthesis of 30 was carried out using a modification of procedures reported by Ellis et al? and Barral et al.4 Both groups used base to deprotonate Hpic in solution; however, base was found to be unnecessary and yields obtained were similar to those reported. The reaction of Hpic with RuCl 3-3H 20, reported by Ghatak et al.,5 was also repeated. Again, the procedure was modified to omit base; however, this time 30 was not formed, and an orange solid (31) was isolated. The RuCl 3-3H 20 reaction was also repeated with base as reported,5 but neither 30 nor any other identifiable species could be isolated. A precipitated black solid had very low C (6.2 %) and N (1.4 %) content and thus contained minimal picolinate. Elemental analysis of 31 supports the formulation [Ru2(pic)4(EtOH)Cl]H20. The MS data (in MeOH), showing a Ru dinuclear splitting pattern with a parent peak for [M + - EtOH] at 727 (Figure 5.3), also support this formulation. This pattern is easily identified compared to that of a mononuclear species because of the distinct isotopic splitting for a Ru 2 species vs. that for one Ru atom (Figure 5.4). 7 2 7 7 0 0 Figure 5.3 ESI-MS spectrum (in MeOH) of [Ru2(pic)4(EtOH)Cl]-H20 (31) clearly showing the [M + - EtOH] fragment centered at 727. 112 100-] 9 0- : so: 7 0 : 60-: 50-; 40-Figure 5.4 Theoretical isotopic distribution for the [M+] peak of Ru(Im-C02)(Im-C 0 2 H ) 2 C l 2 E t 0 H H 2 0 (33, left) (Section 5.3), and for the [M + - EtOH] peak of [Ru2(pic)4(EtOH)Cl]-H20 (31, right), clearly showing the difference in isotopic splitting patterns between a Ru and a Ru 2 species. The IR data for 31 (Table 5.1) show that the carboxylate groups are not bridging, and that the bridging is likely via one O-atom and the pyridine-N. For bridging carboxylate groups, the difference between v s y m and v a s y m is typically < 150 cm"1 for Ru 2 complexes;1 for 31 Av is -300 cm"1, suggesting that the carboxylate group is binding in a monodentate fashion, similar to that observed for 30. Table 5.1 Selected ER spectral data for Ru carboxylate complexes. Complex Vasym ^ s y m (cm"1) (cm"1) mer-Ru(pic)3-H20 (30) 1661 1315 [Ru2(pic)4(EtOH)Cl]-H20 (31) 1672,1639 1396,1316 Ru(Im-C0 2) 3-2H 20 (32) 1642 1324 Ru(Im-C0 2)(Im-C0 2H) 2Cl 2-Et0HH 20 (33) 1718,1706, 1570 1384,1350, 1313 113 The strong ER band at 3468 cm - 1 is assigned to VOH and could be for either coordinated EtOH or the H 2 0 solvate. The bound EtOH is more easily identified by the *H NMR data (Figure 5.5): two broadened resonances (the Me-triplet at 8 1.45, and the C H 2 quartet at 8 4.53) are shifted downfield from those of free EtOH (with the Me-triplet at 8 1.12, and the C H 2 quartet at 8 3.55). The relative intensities of the free and bound EtOH resonances in D 2 0 imply that in this solvent, ~ 40 % of 31 is converted to a species with coordinated D 2 0 . a) pic 1 i 1 H O C H 2 C H 3 1 0 - 2 solvent I H O C H 2 C H 3 5 - 0 4 . 5 4 . 0 3 . 5 3 . 0 2 . 5 2 . 0 1 . 5 1 . O Figure 5.5 ' H NMR spectrum (300 MHz, 298 K) of [Ru2(pic)4(Et0H)Cl]-H20 (31) in D 2 0 . a) Complete spectrum, b) Expansion showing signals of the bound EtOH at 8 4.53 and 1.45 highlighted with boxes. Barral et al.4 have reported the synthesis of [Ru2(pic)4], with a proposed structure containing either four bridging N,0-pic ligands or two bridging N,0-pic ligands and two 114 non-bridging r^-pic ligands. Similar structures with four bridging pic ligands, each Ru having a terminal EtOH or CI (Figure 5.6a), or two p-pic ligands and two, non-bridging r| -pic ligands, one on each Ru (Figure 5.6b), are plausible for 31. The conductivity for 31 in MeOH is 112 fi'ciT^mor1, in the range for a 1:1 electrolyte,1516 suggesting CI" dissociation. If the CI" was bridging, its dissociation in solution would likely lead to decomposition of the bimetallic unit; however, ESI-MS data in MeOH show it to be still intact. a) b) Figure 5.6 Proposed solid state structures for 31 where N-0 = pic", a) unit with four bridging pic" ligands. b) unit with two bridging and two non-bridging pic" ligands. 5.3 Complexes of Ru with Imidazole-4-carboxylic Acid 5.3.1 Synthesis and Characterization of RufJm-COz^^E^O (32) In reactions similar to those reported for the synthesis of Ru(pic) 3H 20 (30) (Section 5.2), Ru(Im-C02)3 was synthesized from both K3RuCl6 and Ru2(acetate)4Cl, using Im-C0 2 H (Figure 5.7) in place of Hpic. 3' 4 The isolated, pale yellow product, was characterized by elemental analysis, MS, and IR and NMR spectroscopies. Elemental analysis was consistent with the formulation of Ru(Im-C02)3-2H20. The Av value of 318 cm"1 for Vasym - v s y m (Table 5.1) is consistent with a chelated structure with binding via the imidazole-N(3) and one carboxylate-0 atom as for Ru(pic) 3H 20; the presence of water is indicated by a VQH at 3423 cm"1, and by an increase in intensity of the residual 115 water signal in dmso-d6 in the spectrum of the complex compared with that of a blank of the solvent containing no complex. O 2 Figure 5.7 Molecular structure of Im-C0 2H. 32 is only sparingly soluble in DMSO, and is insoluble in other solvents. The 'H NMR spectrum in dmso-d6 (recorded over 6 h, -20,000 scans) shows 6 very weak resonances for the bound imidazoles at 8 0.31, 0.09 and -0.09 for H(5) and 8 -2.67, -13.55 and -17.87 for H(2), the assignments being based on those observed for monodentate imidazole ligands with Ru(UI) (Section 3.3.2). As for the synthesis of Ru(pic) 3H 20 (30), no base was used in the synthesis of 32. With the use of 3 equivalents of base to reproduce the reaction conditions reported for the synthesis of 30, 3 - 5 32 was not formed; the isolated products using Lm-C0 2 H under basic conditions were not identified but contained very low percentages of C and N, both below 10 %, as was found when RuCl 3 3H 2 0 was reacted under basic conditions with Hpic (Section 5.2). 5.3.2 Synthesis and Characterization of R u ( I m - C 0 2 ) ( I m - C 0 2 H ) 2 C l 2 E t O H H 2 0 (33) There are currently no literature examples of Ru complexes containing Im-C0 2H (Figure 5.7) or Im-C02~ as a ligand. The only such transition metal complexes reported are a pair of Mn clusters in which Im-C02" chelates through a carboxylate-0 and the imidazole-N(3).17 There is, however, one reported Ru complex with imidazole 4,5-dicarboxylic acid, where IR stretches for both bound - C O O - and free - C O O H are reported; these have proved useful in the formulation of Ru(Lm-C02)(Im-C 0 2 H ) 2 C l 2 E t O H H 2 0 (33). 116 Unlike the reaction of R u C l 3 - 3 H 2 0 with Hpic, the reaction with I m - C 0 2 H does not form a bimetallic complex. 33 is synthesized by refluxing I m - C 0 2 H and R u C l 3 - 3 H 2 0 in a 3:1 ratio in E t O H (Figure 5.8). The IR data show two distinct types of carboxylates with v a s y m = 1718, 1706, and 1570 (Table 5.1). For [Ru(PPh 3 ) 2 (L-H) 2 ] ( L - H 2 = imidazole 4,5-dicarboxylic acid) (Figure 5.9), v a s y m for the protonated carboxylic acid group was observed at 1720 cm"1 and for the bound carboxylate was observed at 1627 cm" 1 . 1 8 This suggests that for 33, the stretches at 1718 and 1706 cm"1 are for - C O O H groups, and the stretch at 1577 cm" 1 is for a bound - C O O " . O H Figure 5.8 One possible isomer for the proposed structure for Ru(Im-C0 2 )(Im-C 0 2 H ) 2 C l 2 E t 0 H H 2 0 (33) (solvent molecules not shown). Figure 5.9 Molecular structure of [Ru(PPh 3 ) 2 (L-H) 2 ] ( L - H 2 = imidazole 4,5-dicarboxylic acid). 1 8 117 ESI -MS data (in M e O H ) show a parent peak for 33 at 508 (Figure 5.10) with the same isotopic splitting pattern as that of a theoretically calculated spectrum (see Figure 5.4). In M e O H , the conductivity of 33 over 3h remained at 19-23 Q" 1 cm 2 mol ' 1 , consistent with a neutral species. 1 5 ' 1 6 3 0 8 3 4 2 | | 3 2 4 3 0 0 472 Figure 5.10 Mass spectrum of R u ( I m - C 0 2 ) ( I m - C 0 2 H ) 2 C l 2 E t O H H 2 0 , showing M + at 508. A more exact structural formulation for 33 cannot be given, but the elemental analysis requires E t O H and H 2 0 solvates. From the ' H N M R spectrum in C D 3 O D , both I m - C 0 2 H ligands are likely in different chemical environments as three signals are observed for both the H(2) and H(5) protons of the I m - C 0 2 H and I m - C 0 2 ligands. Resonances for free E t O H are also observed in the *H N M R spectrum, while a strong IR band at 3467 cm" 1 may show evidence for the water solvate. 118 5.4 Synthesis of Nitroimidazole and Nitrotriazole Carboxylic Acids 5.4.1 Synthesis of 3-Nitro-1,2,4-triazoIe-5-carboxylic Acid (HCANT) The synthetic procedure for HCANT was modified from a general procedure published by Pevzner and coworkers for nitrating numerous aminotriazole compounds.19,20 The precursor, 3-amino-l,2,4-triazole-5-carboxylic acid, was first synthesized using a reported procedure, but was later purchased from Aldrich when it became available commercially in 2002. The nitration procedure used is an example of a Sandmeyer reaction. The literature reports that the amino precursor is soluble in acetic acid; however this was found to be incorrect. In order to solubilize the amine-triazole, a higher H 2 S0 4 :CH3COOH ratio was used for formation of the diazonium salt. The amino group is first converted to a diazonium salt, followed by the facile replacement of the -N + =N group by N0 2 ' . Caution must be taken in adding the diazonium salt solution to the NaN0 2 solution as rapid addition can lead to a violent reaction, producing a foam that erupts from the flask; dropwise addition with rapid stirring and gentle heating prevents this. The isolated solid was characterized by elemental analysis, mass spectrometry and IR spectroscopy. 1 Figure 5.11 Molecular structure of HCANT. 5.4.2 Attempted Synthesis of 2-Nitroimidazole-4-carboxy!ic Acid 4-Hydroxymethyl-2-nitroimidazole was synthesized using a modified literature procedure to afford initially 4-hydroxymethyl-l-tritylimidazole, the imidazole precursor for the nitration step. Davis and coworkers report the synthesis of this compound in 4 steps from imidazole-4,5-dicarboxylic acid. The first step, the synthesis of Im-C0 2H, 119 was omitted as this material was purchased (Aldrich). Then the methyl ester, rather than the reported ethyl ester, was synthesized and characterized by 'H NMR and IR spectroscopies and mass spectrometry. As for the synthesis of H 2biim and H 2Me 2biim (Chapter 4), the trityl group was used to protect the imidazole-N(l) position, as this group is easily removed with dilute acid once the nitration at the C(2) position is complete. The reported procedure for reducing the ester group used LiAlFL;, 2 3 but attempts to repeat this were unsuccessful. The LiAlFL; decomposed rapidly during workup upon the addition of water, indicating that it was still active. Diisobutylaluminum hydride (Dibal-H), using two equivalents per ester, was subsequently used successfully for reduction of the ester group at r.t over 1 h, although the isolated yield of 4-hydroxymethyl-l-tritylimidazole was only 44 % compared to the reported yield, 86 %, using LiAlFL;. This compound was also synthesized directly by treatment of 4-(hydroxymethyl)imidazole with trityl chloride. The availability and cost of 4-(hydroxymethyl)imidazole vary, but the relatively low yield from the ester reduction makes this second route economically viable. The reported yield of 4-hydroxymethyl-2-nitroimidazole from 4-hydroxymethyl-1-tritylimidazole and n-propyl nitrate is 29 %; however, two attempts to reproduce this reaction yielded only ~3 % of the desired product. When the same nitration procedure was used to synthesize 2-nitroimidazole from 1-tritylimidazole, the yield of 2-nitroimidazole obtained was 39 %, while the reported yield was 35-50 %. 2 3 This implies that there is no problem with the n-BuLi or n-propyl nitrate being used, or with the chromatographic set-up. The low yield of 4-hydroxymethyl-2-nitroimidazole prevented any attempt to oxidize it to 2-nitroimidazole-4-carboxylic acid. Either more work on optimizing the synthesis of 4-hydroxymethyl-2-nitroimidazole or a new synthetic route all together is needed in order to synthesize 2-nitroimidazole-4-carboxylic acid. 5.4.3 Attempted Synthesis of 2-Nitroimidazole-4,5-dicarboxylic Acid As there is only one available position for substitution on the imidazole ring in imidazole-4,5-dicarboxylic acid, C(2), it was thought that introduction of a nitro group at this position might be facilitated by direct nitration using H N O 3 or a mixture of H N 0 3 and H 2 S0 4 . Such a nitration might hopefully proceed in much higher yield than that 120 discussed for 4-hydroxymethyl-2-nitroimidazole in Section 5.4.2. The nitration of 4-nitroimidazole to form 2,4-dintroimidazole has been reported,24 suggesting that nitration at this position is possible under certain conditions. Unfortunately, both imidazole-4,5-dicarboxylic acid and dimethyl imidazole-4,5-dicarboxylate did not react with HN0 3 . After imidazole-4,5-dicarboxylic acid or the dimethyl ester was mixed with cone. H N 0 3 for 12 h, and the mixtures neutralized with 5 M NaOH, only the unreacted imidazoles were collected by filtration. The reported procedure using n-propyl nitrate was then attempted with 4,5-bis(hydroxymethyl)-l-tritylimidazole; however, no nitrated species were observed and no products could be isolated from the reaction. 5.5 Attempted Reactions of H C A N T with Ru Precursors Reactions analogous to those discussed in Sections 5.2 and 5.3 with Hpic and Im-C 0 2 H were repeated using HCANT (Section 5.4.1) as a ligand. The reaction of three equivalents of H C A N T with K 3 [RuCl6] in H 2 0 gave a purple solution from which a purple solid was isolated. The MS showed a parent peak consistent with the formulation of Ru(CANT)(HCANT) 2Cl 2 , analogous to that of 33 synthesized from RuCly3H 20 (Section 5.3.2). In the ER spectrum, asymmetric C=0 stretches were observed at 1677, 1665, and 1652 cm"1, and there was also a nitro stretch at 1542 cm"1.25 An intense band at 1912 cm"1, however, suggests that either a coordinated C=0 or N=0 may be formed during the reaction. Elemental analysis (Anal. Calcd for C 9 H5N 1 2 0] 2 Cl 2 Ru: C, 16.74; H, 0.78; N, 26.05. Found: C, 11.49; H, 3.04; N, 21.62) of this purple solid was low in both C and N but high in H for the proposed formulation, possibly suggesting the presence of considerable water; consistent with the presence of a broad, intense band at ~ 3400 cm"1 in the ER spectrum. The reaction of H C A N T with Ru2(acetate)4Cl also gave a purple solution from which a purple solid was precipitated; its ER spectrum was the same as that mentioned above, except that the 1912 cm"1 band was now even more intense. ESI-MS (negative, MeOH) showed only one peak at 444 with a clear Ru splitting pattern, consistent with the formulation of Ru(CANT) 2(NO); the stretch at 1912 cm"1 is in the range for N=0 bound to Ru(UI).26"28 The elemental analysis (Anal. Calcd for C 6 H 2 N 9 0 9 R u : C, 16.18; H, 0.45; 121 N, 28.31. Found: C, 15.34; H, 2.56; N, 24.35) was also high in H and low in C and N for this proposed formulation. Both reactions with HCANT gave products with reproducible ER spectra that differ only in the intensity of the 1912 cm"1 band. The elemental analyses for the isolated purple solids, however, were not reproducible, but were always low in C and N and high in H. Further study on these complexes is needed to determine their nature. 5.6 Experimental Procedures 5.6.1 3-Nitro-1,2,4-triazole-5-carboxylic Acid (HCANT) This compound was synthesized by a modified literature procedure.19'20 Sodium nitrite (1.10 g, 15.9 mmol) was added to 7 mL of H 2 S 0 4 at -5 °C, and to this was added glacial acetic acid (15 mL) and finely ground 3-amino-l,2,4-triazole-5-carboxylic acid (2.00 g, 15.6 mmol). The mixture was stirred at -5 °C for 10 min until most of the triazole had dissolved; H 2 0 (25 mL) was then added with the temperature kept below 0 °C. The resulting yellow solution was then added dropwise to a sodium nitrite solution (200 g NaN0 2 in 200 mL H 2 0) at 50 °C. (If added too quickly, a foam of diazonium salts can form, causing the reaction to heat up out of control with contents erupting from the flask.) The green product solution was heated for 2 h at 50 °C, when the now colourless solution was extracted with EtOAc (4 x 50 mL), and the combined extracts were evaporated yielding the desired product. Yield: 1.41 g (57 %). IR (KBr pellet): v 3416 (N-H, s), 3257 (O-H, m), 1710 (C=0, s), 1574 (N0 2, m), 1383 (N0 2 , m), 1268 (m), 720 (m). ESI-MS: 159 (M+). Anal. Calcd for C 3 H 2 N 4 04: C, 22.78; H, 1.26; N, 35.43. Found: C, 22.52; H, 1.41; N, 35.40. The ER characterization data agree with those reported.20 5.6.2 Methyl imidazole-4-carboxylate This compound was prepared by modifying a reported procedure used to synthesize the analogous ethyl ester.23 To a suspension of imidazole-4-carboxylic acid (1.00 g, 8.92 mmol) in MeOH (20 mL) was added cone. H 2 S 0 4 (1.5 mL). The mixture was refluxed for 24 h, at which point the solid was completely dissolved and T L C 122 showed the absence of the carboxylic acid. The solution was then cooled to 0 °C and neutralized to pH 8 using 5 M NaOH. The solvent was removed under vacuum and the white residue redissolved in a minimal volume of boiling water. White crystals of the ester formed when the aqueous solution was cooled. After standing for 1 h, the mixture was filtered, and the crystals were washed with cold H 2 0 (1x5 mL). The solid was then dried under vacuum at r.t. for 24 h. Yield: 0.87 g (77 %). *H NMR (dmso-ck): 8 7.74 (s, 1H, H5-lm), 7.65 (s, 1H, H2-Im), 2.51 (s, 3H, C//3-Im). IR (KBr pellet): v 3105 (N-H, s), 2976, 2846 (C-H, m), 1619 (C=0, m), 1363 (s), 1156 (m), 864 (m). FAB (+): 127 (M+). Anal. Calcd for C 5 H 6 N 2 0 2 : C, 47.62; H, 4.76; N, 22.22. Found: C, 47.54; H, 4.85; N, 21.69. 5.6.3 Methyl l-tritylimidazole-4-carboxylate This compound was prepared by modifying a reported procedure used to synthesize the analogous ethyl ester.23 To a solution of methyl imidazole-4-carboxylate (0.80 g, 6.3 mmol) in D M F (20 mL), under N 2 , was added trityl chloride (1.77 g, 6.35 mmol), and the mixture was stirred at r.t. for 10 min until solution was complete. NEt 3 (0.98 mL, 7.0 mmol) was then added, and the mixture stirred for 16 h at r.t. The contents were then poured over ice, and the mixture left standing for 5 min. The cold mixture was then filtered and the precipitate was washed with H 2 0 ( 2 x 5 mL), and dried under vacuum at r.t. for 24 h. Yield: 2.01 g (86 %). ] H NMR (CDC13): 8 7.65 (s, 1H, H5-lm), 7.52 (s, 1H, H2-lm), 7.03-7.36 (m, 15H, Ph3C), 2.42 (s, 3H, C//3-Im). FAB (+): 369 (M+), 243 (CPh3). Anal. Calcd for C 2 4 H 2 0 N 2 O 2 : C, 78.26; H , 5.43; N, 7.61. Found: C, 78.35; H, 5.56; N, 7.37. 5.6.4 4-Hydroxymethyl-l-tritylimidazole Method A. This compound was prepared using a modified literature procedure.23 To a solution of methyl l-tritylimidazole-4-carboxylate (1.02 g, 2.72 mmol) in THF (15 mL) at r.t. under N 2 was added 5.50 mL of a 1.0 M diisobutylaluminum hydride (Dibal-H) solution in hexanes . The mixture was stirred for 1 h until the ester was no longer present by T L C . The mixture was then cooled to 0 °C and the following were slowly added in sequence: H 2 0 (0.8 mL) , 15 % aq. NaOH (1.0 mL), and H 2 0 (0.8 mL). The 123 mixture was then filtered, and the precipitate washed with THF (3 x 10 mL), and the filtrate and combined washings were then evaporated under vacuum. The residue was dissolved in CH2CI2; this solution was washed with H 2 0 to remove any remaining inorganic salts and then the CH2CI2 layer evaporated under vacuum. The white product was scraped from the flask and dried for an additional 24 h at r.t. under vacuum. Yield: 0.41 g (44 %). Method B. To a solution of 4-(hydroxymethyl)imidazole (0.52 g, 5.3 mmol) in DMF (10 mL), under N 2 , was added trityl chloride (1.48 g, 5.30 mmol). This mixture was stirred for 10 min at which time the solids had dissolved, and then NEt3 (0.90 mL, 6.4 mmol) was added. This mixture was stirred for 16 h at r.t., then poured onto ice and left standing for 5 min. The resulting precipitate was collected, washed with H 2 0 (2 x 10 mL), and dried under vacuum at r.t. for 24 h. Yield: 1.53 g (85 %). 'H NMR (CDCI3): 5 7.39 (s, 1H, H2-lm), 7.27-6.97 (m, 15H, CPh 3), 6.80 (s, 1H, H5-lm), 4.62 (s, 2H, -CH 2 OH). FAB (+): 341 (M+), 243 (CPh3). Anal. Calcd for C23H2oN20: C, 81.18; H, 5.88; N, 8.24. Found: C, 81.24; H, 5.91; N, 7.87. The *H NMR data agree with those reported.23 5.6.5 4-HydroxymethyI-2-nitroimidazole This compound was prepared according to a literature procedure.23 To a solution of 4-hydroxymethyl-l-tritylimidazole (0.99 g, 2.9 mmol) in THF (35 mL) under N 2 at 0 °C was added 4.05 mL (6.08 mmol) of a 1.5 M solution of n-BuLi in hexanes slowly over 1 min. The pale yellow solution, on being stirred for 2 h, became dark red and a white precipitate formed. n-Propyl nitrate (0.64 g, 6.1 mmol) was then added and gave immediately a dark brown mixture that was stirred for 1 h. The solution was then cooled to 0 °C, diluted with MeOH (40 mL), and then cone. HCI (5 mL) was added to remove the trityl group and to hydrolyze any remaining traces of nitrate esters. This mixture was stirred for 12 h, and then the solvent was removed under vacuum. The residue was triturated with 20 % aq. EtOH (10 mL) and then filtered. The filtrate was evaporated leaving a brown solid that was then chromatographed on silica gel (~ 80 g). The column was first eluted with EtOAc to remove impurities and then with 5 % MeOH in EtOAc to elute the desired product. The fractions were analyzed by T L C , and those containing the 124 product were combined and evaporated, and the resulting solid was dried at r.t. for 24 h. Yield: 12 mg (3 %). J H NMR (CD 3OD): 8 7.15 (s, 1H, H5-lm), 4.50 (s, 2H, -C// 2 OH). ESI-MS: 144 (M+). The *H NMR data agree with those reported.23 5.6.6 Dimethyl imidazole-4,5-dicarboxylate This compound was prepared according to a literature procedure. " To a suspension of imidazole-4,5-dicarboxylic acid (5.03 g, 32.2 mmol) in MeOH (80 mL) was added cone. H 2 S 0 4 (5 mL). The mixture was refluxed for 6 h when the solid had dissolved to give a colorless solution; T L C showed the absence of the dicarboxylic acid. The solution was then cooled at 0 °C and neutralized to pH 8 using 5 M NaOH. The solvent was removed under vacuum and the white residue redissolved in a minimal volume of boiling water. A white precipitate separated when the solution was cooled. The white precipitate was collected, washed with cold H 2 0 (1x5 mL), and dried under vacuum at r.t. for 24 h. Yield: 5.03 g (85 %). 'H NMR (dmso-rf6): 8 7.89 (s, 1H, H2-lm), 3.79 (s, 6H, -OCH3). FAB (+): 185 (M+). Anal. Calcd for C 7 H 8 N 2 0 4 : C, 45.65; H, 4.35; N, 15.22. Found: C, 45.42; H, 4.56; N, 15.01. 5.6.7 Dimethyl l-tritylimidazole-4,5-dicarboxylate This compound was prepared according to a literature procedure. To a solution of dimethyl imidazole-4,5-dicarboxylate (4.02 g, 21.8 mmol) in D M F (40 mL) under N 2 was added trityl chloride (6.09 g, 21.9 mmol), and NEt 3 (3.05 mL, 21.9 mmol); the mixture was stirred at r.t. for 6 h when T L C revealed that none of the precursor dicarboxylate remained. The mixture was then poured over crushed ice and, after 10 min, was filtered; the white precipitate was collected, washed with H 2 0 (2 x 10 mL), and dried under vacuum at r.t. for 24 h. Yield: 6.61 g (71 %). 'H NMR (CDC13): 8 7.55 (s, 1H, H2-hn), 7.37-7.17 (m, 15H, CPh3), 3.86 (s, 3H, -OCtf 3), 3.17 (s, 3H, -OC// 3 ) . FAB (+): 427 (M+), 243 (CPh3). Anal. Calcd for C 2 6 H 2 2 N 2 0 4 : C, 73.22; H, 5.19; N, 6.57. Found: C, 73.27; H, 5.23; N, 6.41. 125 5.6.8 4,5-Dihydroxymethyl-l-tritylimidazole This compound was prepared according to a modified literature procedure.29 To a solution of dimethyl l-tritylimidazole-4,5-dicarboxylate (1.03 g, 2.41 mmol) in THF (20 mL) at r.t. under N 2 was added 10.5 mL of a 1.0 M Dibal-H solution in hexanes. The mixture was stirred for 4 h, when the T L C band for the dicarboxylate was no longer present. The mixture was cooled at 0 °C and to it was added in sequence: H 2 0 (1.5 mL), 15 % aq. NaOH (2 mL), and H 2 0 (1.5 mL). The mixture was then filtered and the precipitate washed with warm THF (3 x 10 mL). The combined filtrate and washings were evaporated under vacuum, and the residue was dissolved in CH 2 C1 2 . This solution was filtered through Celite (~ 10 g) and the filtrate was evaporated. The resulting white solid was dried under vacuum at r.t. for 24 h. Yield: 0.42 g (44 %). *H NMR (CDC13): 5 7.67 (s, 1H, H2-Jm), 7.41-7.19 (m, 15H, CPh3), 4.83 (s, 2H, -CH2OH), 3.89 (s, 2H, -C// 2 OH). FAB (+): 371 (M+), 243 (CPh3). Anal. Calcd for C 2 6 H 2 2 N 2 0 4 : C, 77.81; H, 5.99; N, 7.56. Found: C, 77.74; H, 5.91; N, 7.39. 5.6.9 Ru(pic) 3 H 2 0 (30) Method A . This method was modified from a literature procedure.3 Picolinic acid (0.14 g, 1.2 mmol) was added to a red solution of K 3[RuCl 6] (Section 2.4.4) (0.17 g, 0.39 mmol) in 5 mL H 2 0 ; this was heated at 60 °C for 5 h when a yellow precipitate slowly formed. The mixture was then cooled to r.t. before isolation of the solid by filtration; the solid was washed with MeOH (2x5 mL) and H 2 0 ( 1 x 5 mL) and dried under vacuum at 78 °C for 48 h. Yield: 0.11 g (58 %). Method B. This method was performed as reported in the literature.4 Hpic (0.096 g, 0.78 mmol) was added to a brown solution of [Ru 2(CH 3COO) 4Cl] (0.062 g, 0.13 mmol) in water/MeOH, 1:1 (10 mL), and the mixture was refluxed for 8 h when a yellow precipitate formed. The reaction mixture was cooled to r.t. and the solid isolated by filtration, washed with H 2 0 ( 2 x 5 mL), and dried under vacuum at 78 °C for 24 h. Yield: 0.049 g (77 %). IR (KBr pellet): v 3443 (O-H, s), 1661 (C=O a s y m , s), 1565 (m), 1315 (C=O s y m, m), 1280 (s), 1057(m). ESI-MS: 468 (M+). Anal. Calcd for C 1 8 H 1 2 N 3 0 6 R u H 2 0 : C, 44.54; H, 2.89; N, 8.65. Found: C, 44.51; H, 2.74; N, 8.28. The ER data agree with those reported.3'4 126 5.6.10 [Ru 2 (pic) 4 (EtOH)Cl]H 2 0 (31) Hpic (0.072 g, 0.59 mmol) was added to a brown solution of RuCl 3 3H 2 0 (0.051 g, 0.19 mmol) in 10 mL EtOH. This solution was refluxed for 6 h, the colour becoming red and finally bright orange. The solvent was reduced in volume to ~ 1 mL, when Et 2 0 (10 mL) was added to give an orange precipitate that was collected, washed with Et 2 0 (2 x 5 mL), and then dried under vacuum at 78 °C for 24 h. Yield: 0.066 g (86 %). *H NMR (D20): 5 8.76-7.92 (m, pic-H), 4.53 (br q, coordinated HOC/7 2 CH 3 ) , 1.45 (br t, coordinated H O C H 2 C / / 3 ) , 0.2, -5.1 (br s, pic-H). IR (KBr pellet): v 3468 (O-H, s), 3124 (N-H, m), 1672 (C=O a s y m , s), 1639 (C=O a s y m , s), 1600 (m), 1456 (m), 1396 (C=O s y m, m), 1316 (C=O s y m, m), 1282 (m), 1147 (m), 856 (m), 759 (s), 691 (m). ESI-MS: 727 ( M + -EtOH), 382 (Ru(pic)2Cl+), 346 (Ru(pic)2+). A M (MeOH) = 112 aWmor1. Anal. Calcd for C 2 6 H 2 2 N 4 0 9 C l R u 2 H 2 0 : C, 39.52; H, 3.04; N, 7.09. Found: C, 39.38; H, 2.89; N, 6.94. 5.6.11 Ru(Im-C0 2 ) 3 -2H 2 0 (32) Method A. Imidazole-4-carboxylic acid (Im-C02H) (0.094 g, 0.84 mmol) was added to a red solution of K 3[RuCl 6] (0.12 g, 0.28 mmol) in H 2 0 (10 mL). The resulting red solution was refluxed for 6 h to afford a yellow precipitate that was collected at r.t., washed with MeOH (2x5 mL), and dried under vacuum at 78 °C for 48 h. Yield: 0.074 g (57 %). Method B. Im-C0 2 H (0.092 g, 0.82 mmol) was added to a brown solution of [Ru 2(CH 3COO) 4Cl] (0.065 g, 0.14 mmol) in water/MeOH, 1:1 (10 mL). The resulting brown solution was refluxed for 8 h to afford a yellow precipitate that was collected at r.t., washed with MeOH (2x5 mL), and dried under vacuum at 78 °C for 48 h. Yield: 0.043 g (67 %) 'H NMR (dmso-<4): 8 0.31, 0.09, -0.09 (s, H(5)-Im), -2.67, -13.55, -17.87 (s, H(2)-Im). IR (KBr pellet): v 3423 (O-H, s), 3109 (N-H, br s), 1642 (C=O a s y m , s), 1324 (C=O s y m, s), 1201 (m), 1090 (m), 1024 (m), 930 (m), 829 (m). ESI-MS: 435 (M+). Anal. Calcd for C 1 2 H 9 N 6 0 6 Ru-2H 2 0: C, 30.63; H, 2.77; N, 17.87. Found: C, 30.87; H, 2.58; N, 17.47. 127 5.6.12 Ru(Im-C0 2)(Im-C0 2H) 2Cl 2EtOHH 20 (33) Im-C0 2 H (0.084 g, 0.75 mmol) was added to a brown solution of RuCl 3 3H 2 0 (0.065 g, 0.25 mmol) in EtOH (10 mL). The resulting solution was refluxed for 4 h, when it had become yellow. The solvent was reduced in volume to ~2 mL and 15 mL of Et 2 0 was added to afford a yellow precipitate that was collected, washed with Et 2 0 (3x5 mL), and dried under vacuum at 78 °C for 24 h. Yield: 0.11 g (76 %). *H NMR (CD 3OD): 8 3.45 (q, HOCH 2 CH 3 ) , 1.12 (t, H 0 C H 2 C / / 3 ) , -4.49, -7.20, -10.16 (s, H(5)-Im), -14.83, -16.75, -23.92 (br s, H(2)-Im). IR (KBr pellet): v 3467 (O-H, br s), 3217 (O-H, m), 3149 (N-H, m), 1718 (C=O a s y m , s), 1706 (C=O a s y m , s), 1570 (C=O a s y m , s), 1384 (C=O s y m, m), 1350 (C=O s y m, m), 1313 (C=O s y m, m), 1188 (m), 1089 (m). ESI-MS: 508 (M+), 472 ( M + - CI), 396 ( M + - Im-C02H), 360 (M + - Im-C02H - CI). A M (MeOH) = 19 Q ' W m o l " 1 . Anal. Calcd for C^HnNeOeChRuEtOHHzO: C, 29.42; H, 3.33; N, 14.71. Found: C, 29.36; H, 3.17; N, 14.44. 5.7 References (1) Deacon, G.; Phillips, R. Coord. Chem. Rev. 1980, 33, 227. (2) Nakamoto, K.; McCarthy, P. J. Spectroscopy and Structure of Metal Chelate Compounds; John Wiley & Sons, Inc.: New York, 1968. (3) Ellis, R.; Quilligan, J.; Williams, N.; Yandell, J. Aust. J. Chem. 1989, 42, 1. (4) Barral, M . ; Jimenez-Aparicio, R.; Royer, E.; Saucedo, M . ; Urbanos, F.; Gutierrez-Puebla, E.; Ruiz-Valero, C. J. Chem. Soc. Dalton Trans. 1991, 1609. (5) Ghatak, N.; Chakravarty, J.; Bhattacharya, S. Polyhedron 1995,14, 3591. (6) Kingry, K.; Royer, A.; Vincent, J. J. Inorg. Biochem. 1998, 72, 79. (7) Cahtterjee, M. ; Maji, M . ; Ghosh, S.; Mak, T. J. Chem. Soc, Dalton Trans. 1998, 3641. (8) Chakov, N.; Collins, R.; Vincent, J. Polyhedron 1999,18, 2891. (9) Stearns, D. M . ; Armstrong, W. H. Inorg. Chem. 1992, 31, 5178. (10) Li , W.; Olmstead, M . M. ; Miggins, D.; Fish, R. H. Inorg. Chem. 1996, 35, 51. (11) Speetjens, J. K.; Collins, R. A.; Vincent, J. B.; Woski, S. A. Chem. Res. Toxicol. 1999,12, 483. 128 (12) Anderson, R. A.; Bryden, N. A.; Polansky, M . M . J. Am. Coll. Nutr. 1997, 16, 273. (13) Stephenson, T.; Wilkinson, G. / . Inorg. Nucl. Chem. 1966, 28, 2285. (14) Martin, F. S. J. Chem. Soc. 1952, 2682. (15) Geary, W. J. Coord. Chem. Rev. 1971, 7, 81. (16) Huheey, J. E . Inorganic Chemistry: Principles of Structure and Reactivity, 3rd ed.; Harper Collins Publishers: New York, 1983. (17) Boskovic, C ; Folting, K.; Christou, G. Polyhedron 2000,19, 2111. (18) Sengupta, P.; Dinda, R.; Ghosh, S.; Sheldrick, W. Polyhedron 2001, 20, 3349. (19) Tolstyakov, V. V.; Pevzner, M . S. Russ. J. Org. Chem. 1997, 33, 1803. (20) Bagal, L. I.; Pevzner, M . S.; Frolov, A. N.; Sheludyakova, N. I. Khim. Geterotsikl. Soedin. [English Translation] 1970, 2, 240. (21) Cipens, G. Metody Poluch. Khim. Reaktivov Prep. 1966,14, 9. (22) McMurry, J. Organic Chemistry, 3rd ed.; Brooks/Cole Publishing Co.: Pacific Grove, 1992. (23) Davis, D. P.; Kirk, K. L.; Cohen, L. A. J Heterocylic Chem. 1982,19, 253. (24) Novikov, S. S.; Khmel'nitskii, L . I . ; Lebedev, O. V.; Sevast'yanova, V. V.; Epishina, L. V. Khim. Geterotsikl. Soedin. [English Translation] 1970, 6, 465. (25) Pavia, D. L.; Lampman, G. M. ; Kriz, G. S. Introduction to Spectroscopy; Harcourt Brace College Publishers: Orlando, 1996. (26) Nagao, H.; Hirano, T.; Tsuboya, N.; Shiota, S.; Mukaida, M . ; Oi, T.; Yamasaki, M . Inorg. Chem. 2002, 41, 6267. (27) Hirano, T.; Oi, T.; Nagao, H.; Morokuma, K. Inorg. Chem. 2003, 42, 6575. (28) Serb, B.; Zangrando, E.; Gianferrara, T.; Yellowlees, L. ; Alessio, E. Coord. Chem. Rev. 2003, 245, 73. (29) Kavadias, G.; Luh, B.; Saintonge, R. Can. J. Chem. 1982, 60, 723. (30) Vinogradova, N. B.; Kromov-Borisov, N. V. Metody Poluch. Khim. Reactivov. Prep. 1966,14, 40. 129 Chapter 6 Antiproliferatory Activity of Ruthenium Complexes Using the MTT Assay 6.1 Introduction For preliminary drug screening, tumour cell lines are used to test the antiproliferatory activity of compounds in vitro. Compounds then showing activity can be tested in vivo to determine if the in vivo and in vitro properties correlate. The MTT assay is a colourimetric assay commonly used as a screening process to examine the antiproliferatory activity of compounds over large concentration ranges.1 The assay quantifies the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to formazan by mitochondrial dehydrogenase (Figure 6.1). In viable cells (Figure 6.2), M T T (yellow) is reduced to formazan (purple) which precipitates onto the surface of the wells. Dissolving the precipitate in DMSO gives a purple solution (Figure 6.3, details of the procedure are given in Section 6.3, as well as in a recent publication by our group2). The amount of formazan in each well is then measured spectrophotometrically to determine the number of viable cells in each well. The absorbance values at determined at each concentration are then divided by the absorbance values of the control and plotted vs. concentration. From these graphs, I C 5 0 values can be Figure 6.1 Mitochondrial reduction of M T T to Formazan. 130 Figure 6.2 Pictures of cells on Day 5 of M T T assay before adding M T T in a control experiment with no complex (left), and in an experiment incubated for 69 h in 2 m M Ru(ma) 3 (right). Figure 6.3 Outline of M T T assay from Day 1 - Day 5; see Section 6.3. The outer rows of wells are filled with water, and the inner wells with cells increasing in concentration of complex from right to left. The leftmost two columns are the blank (farthest left) and control (second from left). The intensity of the purple colour is proportional to the number of viable cells present (highest for the control and low concentration samples). determined, the concentration at which the number of viable cells is 50 % that of the control. 3 ' 4 Low IC50 values with respect, in particular, to that of cisplatin (40 u M for the M D A - M B - 4 3 5 S cell line under the reaction conditions described in this Section 6.3, Table 6.4), are desirable and suggest that the compound is having an antiproliferatory effect at low concentrations.5 Complexes exhibiting high IC50 values may also have useful biological applications, particularly in the case of EF5 complexes (Table 6.3), which may be specific to hypoxic cells. 6 There is a need to develop useful imaging agents for hypoxia that should specifically target hypoxic cells while, at the same time, be 131 free of any cytotoxic effects that might also harm healthy cells (i.e. exhibit high IC50 values).7 The results of the M T T assay have been found generally to correlate well with in vivo studies as well as with other in vitro studies such as the dye exclusion assay.8 Measurements of formazan absorption in MTT tests have been shown to correlate very well with the number of counted cells both using a Coulter counter3 and using microscopic counting techniques.9'10 Like all assays, however, results from the MTT assay vary between cell lines and, within a given cell line, results may vary from week to week (Section 6.4.2, ± 5 %) although this variation can often be difficult to distinguish from the natural distribution of the data.11 For these reasons the M T T assay is only a preliminary assay. Positive results at this stage then lead to more detailed testing in vitro using more cell lines as well as in vivo studies. Experience in the development of NAMI-A (Figure 1.8, Section 1.5.3.1) has led to a set of suggested guidelines for the testing of Ru complexes for anti-cancer properties. The Callerio Foundation, the group responsible for the development of NAMI-A, lists the M T T assay as the first step in a screening process recommended for determining whether a Ru complex may exhibit any antiproliferatory activity toward a given cancer cell line.5 6.2 General Experimental Procedures 6.2.1 Experimental Media, Solutions, and Materials Leibovitz's L-15 medium with L-glutamine (L-15), Dubelco's modified essential medium (DMEM), fetal bovine serum (FBS), zinc bovine insulin, penicillin/streptomycin antibiotic, phosphate buffered saline solution 7.4 (PBS), and trypsin-EDTA (0.25 % trypsin in ImM Na 4(EDTA)) were purchased from Gibco. 96-Well plates, T-25 and T-75 flasks were purchased from Falcon. MTT was purchased from Aldrich. The growth medium for the MDA-MB-435S cell line consisted of 500 mL L-15, 50 mL FBS, and 5.0 mg of insulin. The growth medium for the MDA435/LCC6-WT cell line consisted of 500 mL D M E M , 50 mL FBS, and 5,000 units of penicillin/streptomycin antibiotic. PBS, MTT and the growth medium were stored at 4 °C, while the trypsin-EDTA and FBS were stored at -20 °C. FBS was filter-sterilized through 0.1 um filters before use. 132 6.2.2 Cell Preparation Human breast cancer cells (MDA-MB-425S) were purchased from the American Type Tissue Culture Collection (ATCC), while MDA435/LCC6-WT cells 1 2 1 3 were kindly donated by Dr. K Skov of the BC Cancer Research Center. The MDA-MB-435S cells were maintained in L-15 medium in T-75 tissue culture flasks at 37 °C under air. The cells were transferred every 3-4 days to new flasks at a dilution factor of approximately 8. Before transfer, the medium was removed from the flask, and the cells were rinsed for 20 s with 2 mL trypsin-EDTA, which was then removed and 2 mL of fresh trypsin-EDTA were added to the flask. After 3-4 min the cells were observed to detach from the flask surface, and 8 mL of medium were added. The cells were then counted under a microscope using a hemacytometer (Hausser Scientific, 0.100 mm deep). 2 x 106 cells (typically in 2-3 mL of medium) were then transferred to a T-75 flask, and the suspension diluted to 20 mL with medium to yield a final density of 1 x 105 cells/mL. For the MDA435/LCC6-WT cells, the cell preparation was the same as for the MDA-MB-435S cells using D M E M medium in place of L-15 medium (the preparation of the media is described in Section 6.2.1). 6.2.3 Preparation of Ru Complexes The Ru complexes were weighed into glass vials and dissolved in PBS to make 1.5 mL of 4 mM stock solutions for each complex. These solutions were then sonicated and heated to 37 °C to ensure the complex was completely dissolved, and then filtered through 0.2 pm filters to remove any remaining particulates. From this stock solution, subsequent dilutions yielded the following final concentrations: 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 mM (Section 6.3.2). Follow-up experiments for several compounds focused on a lower concentration range, and thus stock solutions of 0.400 mM were used instead and the dilutions were adjusted to test concentrations as low as 500 nM. For the complexes containing EF5 (14), 2N02lm (15) and 3N02tri (17), stock solutions of 4 mM could not be prepared because of poor complex solubilities in PBS. Instead 2 mM stock solutions of 15 and 17 were prepared in 20 % DMSO in PBS, while a 2 mM solution of 14 was prepared in 5 % DMSO in PBS. These three solutions were then heated to 50 °C 133 to ensure complete solubility of the complexes, filter sterilized while hot, and then cooled to room temperature before use. 6.2.4 Preparation of Ligand Precursor Solutions Hma, HEma, DMSO and N-heterocyclic compounds (see Table 6.2) that acted as ligand precursors were tested over a concentration range from 5 to 0.02 mM. They were weighed into glass vials and dissolved in PBS to yield 1.5 mL of 10 mM stock solutions for each compound. 6.3 M T T Assay Procedure 6.3.1 Day 1 - Plating Cells On day one of the M T T assay, cells were plated into 96-well plates. The medium was removed from a T-75 flask containing the cells, and 2 mL of trypsin-EDTA were then added. The flask was gently agitated to rinse the cells. The-trypsin-EDTA was removed and an additional 2 mL were added to the flask which was then sealed for 3-4 min. After 3 min the cells were examined under a microscope to determine whether they were still adhering to the plastic flask or whether they had become detached. If the cells were still adhering, the flask was again gently shaken to assist in the removal of the cells from the flask wall. L-15 medium (8 mL) was then added to the flask. This suspension was thoroughly mixed by pipetting the mixture "up and down" several times. A 50 uL aliquot of the suspension were then removed and plated onto a hemacytometer to determine the cell count. Then the dilution factor needed to make a stock suspension of 1 x 105 cells/mL was calculated (5.4 mL of this suspension are required per plate), and 100 uL of this suspension were then added to each of the wells in columns 3-11 in rows B-G (Figure 6.4). Medium (100 uL) was added to the wells in column 2 rows B-G (blank). Water (200 pL) was then added to all the remaining wells to help prevent evaporation of the medium from the experimental wells. The plates were then placed into the incubator for 24 h. 134 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Figure 6.4 Experimental 96-well plate set-up showing the control column (dark grey), the blank (white), the wells with water (light grey) and those containing the complex solutions (speckled). 6.3.2 Day 2 - Addition of Ru Complexes and Ligand Precursors The 96-well plates were removed from the incubator after 24 h. Stock solutions of Ru complexes were prepared as described in Section 6.2.3. Dilutions of such stock solutions were then prepared in 700 u L tubes using P B S (except for 14, 15, and 17, see below), in order to yield eight 650 u L solutions spanning, when using a 4 stock solution, from 4 m M to 0.02 m M (Table 6.1). For those experiments in which a 0.4 m M stock solution was used, the concentrations of the corresponding diluted solutions were one order of magnitude less; in some cases different dilutions were used in order to test concentrations below 2 u M . The diluted solutions for all experiments were mixed using a multi-channel pipette and then 100 p L of each solution was pipetted into the wells in rows B - G and columns 2-11 (Figure 6.4). Only PBS (or P B S / D M S O in some cases, as discussed later below) was added to columns 2 and 3, as these two columns were the blank and control, respectively. Solutions of 14, 15, and 17 were prepared as D M S O / P B S solutions and were thus subsequently diluted with the same solvent mixture used for their preparation (20 % D M S O for 15 and 17, and 5 % D M S O for 14) to ensure that any D M S O effect would be 135 minimized. In Section 3.3.3, it was observed that both 15 and 17 in DMSO dissociated ligands; however, the resulting solutions of these complexes in DMSO:PBS (1:1) were still tested using the M T T assay. The DMSO:PBS solvent mixtures were added to the control and blank to ensure that any DMSO effect would manifest itself in the control as well. The stock solutions of these three complexes were only 2 mM (see Section 6.2.3), and therefore the final concentrations of the diluted samples were half those shown in Table 6.1, which shows the final concentrations for samples diluted from a 4 mM stock solution. Table 6.1 Dilution table for the addition of complexes using a 4 mM stock solution.3 Solutions of 650 U.L were made for each concentration of Ru complex, as well as those for the blank and the control. Column 2 3 4 5 6 7 8 9 10 11 Concentration of complexes (uM) 0 0 4000 2000 1000 400 200 100 40 20 Vol. PBS (uL) 650 650 0 325 487.5 585 617.5 633.75 643.5 646.75 Vol. stock solution CMT 1 0 0 650 325 162.5 65 32.5 16.25 6.5 3.25 a For some complexes different stock solution concentrations or different dilution factors were used to perform the assay (see Sections 6.2.3 and 6.2.4). For the ligand precursors, 10 mM stock solutions were prepared in PBS, the samples then being diluted in PBS to final concentrations spanning 10 to 50 uM. The dilutions used were as for the Ru complexes (Table 6.1), with the final concentrations being 2.5 times greater than those shown, as 10 mM stock solutions were used instead of 4 mM ones. 6.3.3 Day 5 - Addition of M T T and Plate Reading A modified version of a published procedure was used for the addition of M T T . 1 4 After incubation of the cell/complex mixture for 69 h, 50 uL of an M T T solution (2.5 mg/mL in PBS) were added to each of the wells in rows B-G, columns 2-11. The plates were then incubated for an additional 3 h, at which time the medium was removed from all the wells to leave purple formazan crystals. DMSO (150 |iL) was then added to each 136 of the columns in rows B - G , columns 2-11, and the plates were then scanned at 570 nm on a Spectra Max 190 El isa plate reader (Molecular Devices Co.) with a shake time set to 10 s before reading the plates (Figure 6.5). Values for the blank absorbance were averaged and automatically subtracted from those read for each well using Softmax Pro v2.2.1 software. 6.3.4 Data Analysis For each compound, six replicates were performed at each of eight different concentrations. The absorbance of each well at 570 nm was determined. The values obtained were normalized by subtracting the average absorbance value for the 6 blank replicates from the value for each experimental and control well . A t each concentration the average absorbance over the 6 replicates was determined and divided by the average value for the 6 control replicates to determine the percent cell viability at each concentration. Values presented in this chapter are the IC50 values determined by graphing the percent viability versus concentration, and for the complexes are the average value as determined from two or more experiments. For the free ligands, the IC50 values reported are those from a single experiment. With the M D A - M B - 4 3 5 S cell line, IC50 values were found to be reproducible within experimental uncertainty from week to week. Error bars are included on each graph and represent 2 standard deviations or a 95 % confidence limit. Figure 6.5 Image of plate after M T T treatment. The control is clearly seen as a purple column on the left, and the purple colours on the right clearly show the increase in cell viability at lower concentration. 137 6.4 Results and Discussion 6.4.1 Ligand Precursors Table 6.2 shows the I C 5 0 values for the ligand precursors used to form the Ru complexes that were tested. The only one of these compounds that showed significant activity was 2N02Im, exhibiting an IC50 value of 50 uM. Only two other compounds exhibited I C 5 0 values below 500 uM, EF5 and 3N02tri, both N-heterocycles containing a Table 6.2 I C 5 0 values for the ligand precursors against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h. Compound3 IC 5 0 (uM) (±10 %) Hma 1000 HEma 900 Im >5000 2MeIm >5000 4MeIm >5000 lMelm >5000 H 2Me 2biim 650 H 2biim 800 2N0 2Im 50 EF5 350 3N02tri 400 metro >5000 Hpic 950 Im-C0 2 H 4000 H C A N T 1000 DMSO >5000b a See page xxii for compound abbreviations. b Ref. 2. 138 nitro group. The lack of any significant activity of ma, Ema and most of the imidazoles (IC50 values are much higher than those observed for complexes containing these compounds as ligands, see Section 6.4.2) suggests that the lower IC50 values for the complexes are not a result of dissociated ligand inside the cell. 6.4.2 Ru Maltolato and Imidazole Complexes In total, 23 complexes were screened using the M T T assay (Tables 6.3-6.6). Because of some variability in the assay from week to week resulting from changes in cell morphology over time, Ru(ma)3 (1) and [Ru(ma)2(2MeLm)2]CF3S03 (10) were used as standards to ensure consistency from week to week. These complexes were chosen as they exhibited activity in different concentration ranges (IC 5 0 value for 1 is 140 uM and for 10 is 5 uM), and showed similar variability from week to week (e.g. if the IC50 value for 1 went up 5 %, so did the IC 5 0 value for 10; in general, weekly variability was < 5 % and thus values non-normalized values still fell within experimental error limits); in contrast, the activity of cisplatin, did not vary. Cisplatin was also tested and used as a control to help compare the results with those of other groups, who have used different cell lines but have used cisplatin as a standard.15 The tests with the free ligands (Table 6.2) suggest that the intact complexes in solution likely give rise to the activity and not free ligand present as a result of ligand dissociation over the incubation period. Table 6.3 shows the IC50 values for the maltolato complexes that were tested. Only 3 complexes with nitro-substituted ligands were tested because of poor solubilities in PBS. In fact, at higher complex concentrations, the 570 nm absorbances for the complexes with EF5 (14), 2N0 2Im (15) and 3N02tri (17) increased as a result of metal-containing species precipitating from the reaction medium. This was confirmed by examining the cells under a microscope on day 5 of the M T T assay, before the addition of MTT. Under the microscope, blue solids for 15 and 17, and a brown solid for 14, were observed at concentrations of 500 uM and higher. Against the MDA-MB-435S cell line, [Ru(ma)2(2MeIm)2]CF3S03 (10) showed the lowest IC50 value of all the maltolato complexes tested (5 uM) (Figure 6.6); [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) was also highly active (15 uM). The next most 139 Table 6.3 I C 5 0 values for Ru-maltolato complexes against MDA-MB-435S cells in L-15 medium and against MDA435/LCC6-WT cells in D M E M after incubation at 37 °C for 69 h.a Complex I C 5 0 (MM) MDA-MB-435S IC 5 0 (pM) M D A43 5/LCC6-WT rrflwj-[Ru(ma)2(2MeIm)2]CF3S03 (10) 5 5 rra^-[Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) 15 80 [Ru(ma)2(Im)2]CF3S03 (7) 70 >2000 rran5-[Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) 80 >2000 rrarcs-[Ru(ma)2(metro)2]CF3S03 (4) 80 >2000 trans-[Ru(ma)2(EtOH)2]CF3S03 (3) 300 -fra^-[Ru(ma)2(EF5)2]CF3S03-EtOH (14) >500 -[Ru(ma)2(2N02Im)2]CF3S03 (15) 25 90 [Ru(ma)2(3N02tri)2]CF3S03 (17) >500 -a Error limits are ± 10 %. 1 5 0 b 1 0 0 Concentration (mM) Figure 6.6 Cell viability vs. concentration for [Ru(ma)2(2MeIm)2]CF3S03 (10), with error bars representing 95% confidence limit. 140 active imidazole complexes were the corresponding Im (7) and lMelm (8) species with IC 5 0 values of 70 and 80 uM, respectively. These complexes (6, 7, 8, and 10) showed no signs of ligand dissociation or reactivity in the PBS/medium solutions as judged by T L C analysis of the complex solutions after 5 days. This suggests that the intact complexes are responsible for the observed activity, and not a new complex resulting from partial ligand dissociation or displacement in the medium. [Ru(ma)2(2N02lm)2]CF3S03 (15) exhibited an IC 5 0 value of 25 uM, half that of free 2N02lm (Tables 6.2 and 6.3), the activity likely resulting from the presence of free 2N02Im, as T L C analysis of 15 dissolved in PBS:DMSO (1:1) showed a large band for this compound. The 25 uM IC50 value for 15 perhaps indicates that all of the 2N02Im is dissociating, thus exhibiting an IC50 value half that observed for the free ligand (25 uM complex is equiv. to 50 uM free ligand). If the 2N02Im is only partially dissociating in solution, the activity observed may be attributed to remaining undissociated Ru complex. The PBS/medium solution of the bis(EtOH) complex, 3, changed colour from red to green over the 3 day incubation, likely as a result of displacement of the EtOH ligands; EtOH is readily displaced by water at room temperature but the aqua complex is also red in colour (Section 3.2.3). The green colour suggests components of the PBS/medium solution are coordinating to the Ru. Some of the complexes listed in Table 6.3 were also tested against another cell line, MDA435/LCC6 - W T , 1 6 and the IC50 values differed from those observed against the MDA-MB-435S cell line for most of the complexes; 4, 7, and 8 exhibited IC50 values > 2 mM against the MDA435/LCC6-WT cell line, compared to values of 70 - 80 uM against the MDA-MB-435S cell line. Only the IC50 value for 10 was the same against both cell lines (5 uM). Ru(ma)3 (1) (Figure 6.7) and Ru(Ema)3 (2) (Figure 6.8) gave I C 5 0 values of 140 and 90 uM, respectively, against the MDA-MB-435S cell line (Table 6.4); the values agree with those reported earlier against this cell line under similar conditions (150 and 80 uM). 1 7 The increase in cell proliferation observed at lower concentration for 2 (at < 50 uM, Figure 6.8) was reproducible, suggesting that 2 has an advantageous effect on 141 cell proliferation at such concentrations as well as a deleterious one at higher concentrations. 150 1 t, 100 0 I ••— — : - 1 • • ^ • ; 0.01 0.1 1 10 Concentration (mM) Figure 6.7 Cell viability vs. concentration for Ru(ma)3 (1), with error bars representing 95% confidence limit. Figure 6.8 Cell viability vs. concentration for Ru(Ema)3 (2), with error bars representing 95% confidence limit. 142 Table 6.4 IC50 values for cisplatin,3 and three Ru complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h.b Ru(ma)3 (1) and Ru(Ema)3 (2) are precursors for the complexes in Tables 6.3 and 6.5, and [RuCl(p-cymene)]2(p-Cl)2is a precursor for biologically active Ru sulfoxide complexes. Complex I C 5 0 (pM) Ru(ma)3 (1) 140 Ru(Ema)3 (2) 90 Cisplatin 40 [RuCl(p-cymene)]2(p-Cl)2 350 a Error limits are ± 5 % for cisplatin. b Error limits are ± 10 % for the Ru complexes. To determine if this trend of greater activity for Ema complexes (vs. ma species) was more general, Ema-4MeIm and -2MeIm derivatives were synthesized because 6 and 10 showed the lowest I C 5 0 values of the ma complexes (Table 6.3). Again, the Ema systems exhibited lower I C 5 0 values (Table 6.5): indeed, [Ru(Ema)2(2MeIm)2]CF3S03 has an IC 5 0 value of 500 nM (Figure 6.9), an order of magnitude lower than that of any other complex tested. It is unclear why the Ema derivatives exhibit IC50 values lower than those of the corresponding ma complexes, but this finding will be discussed further with other results given in Chapter 7. Table 6.5 I C 5 0 values for ethylmaltolato complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h.a Complex I C 5 0 (pM) [Ru(Ema)2(2MeIm)2]CF3S03 (13) 0.5 [Ru(Ema)2(4MeIm)2]CF3S03 (12) 5 a Error limits are ± 10 %. 143 150 b 100 • IS > Concentration (mM) Figure 6.9 Cell viability vs. concentration for [Ru(Ema)2(2MeIm)2]CF3S03 (13), with error bars representing 95% confidence limit. Cisplatin was tested several times against the MDA-MB-435S cell line in order to provide a "known standard," and had an IC50 value of 40 u M (Figure 6.10, Table 6.4), while the value against the human ovarian cell line A2780 is reported as 0.5 u M , 1 5 eighty times lower. IC50 values are also reported for RuX(p-cymene)(N-N) complexes, where X = CI or I and N-N = ethylenediamine or (CH 3 CN) 2 , against the A2780 cell line, some exhibiting IC5o values as low as 8 u M . 1 5 Previously in our group, Huxham et al. found that [RuCl(p-cymene)]2(|i-BESE) exhibited an IC 5 0 value of 350 | i M against the MDA-MB-435S breast cancer cell line.18 As no one has reported on the biological activity of the precursor for these Ru-p-cymene complexes, [Ru(p-cymene)Cl2]2, this dimer was tested against the MDA-MB-435S breast cancer cell line and its IC50 was also found to be 350 uM (Table 6.4). Per Ru atom, the IC 5 0 values of both the starting dimer and [RuCl(p-cymene)]2(u-BESE) are 700 | iM; thus the presence of the chelated sulfoxide is not necessary to produce the observed activity for this complex.18 144 150 ^ 100 Concentration (mM) Figure 6.10 Cell viability vs. concentration for cisplatin, with error bars representing 95% confidence limit. This experiment was done over a shortened concentration range to produce more points near the IC50 value and improve the accuracy of the assay. 6.4.3 Ru 4,4'-Biimidazole Complexes Complexes of 4,4'-biimidazoles were also tested using the M T T assay, including maltolato biimidazole complexes (Table 6.6). After a 2MeIm derivative was shown to exhibit the lowest IC50 value of a series of Ru-ma complexes (Table 6.3), 2,2'-dimethyl-4,4'-biimidazole (Me2biim) was synthesized (Section 4.8) as well as 4,4'-biimidazole (biim) (Section 4.8), and a Ru ma complex of each was examined, as well as other complexes containing either of these two ligands. The I C 5 0 value for [Ru(ma)2(Me2biim)]CF3S03-2H20 (25) (Figure 6.11) is 15 pM, the same as for 6 (Table 6.3), while [Ru(ma)2(biim)]CF3S03-2H20 (24) (Figure 6.12) exhibited an IC50 value of 50 pM. These two complexes exhibit activities different from those of the analogous bis(Im) (7, IC 5 0 = 70 pM) and bis(2MeIm) (10, IC 5 0 = 5 pM) complexes (Table 6.3). Two major differences that may account for this are the 145 Table 6.6 I C 5 0 values3 for biim and Me2biim complexes against MDA-MB-435S cells in L-15 medium after incubation at 37 °C for 69 h; data for new Ru complexes containing picolinic acid (Hpic) or imidazole-4-carboxylic acid (Im-C02H) against the same cell line under the same conditions are also shown. Complex I C 5 0 (MM) [Ru(ma)2(biim)]CF3S03-2H20 (24) 50 [Ru(ma)2(Me2biim)]CF3S03-2H20 (25) 15 [Ru(biim)3][CF3S03]2 (26) 18 [Ru(Me 2biim) 3][CF 3S0 3] 2 (27) 36 RuCl2(DMSO)2(biim) (20) 800 RuCl2(DMSO)2(Me2biim) (22) 400 [Ru2(pic)4(EtOH)Cl]-H20 (31) 100 Ru(Im-C0 2 )(Im-C0 2 H) 2 Cl 2 H 2 OEtOH (33) 400 3 Error limits are ± 10 %. geometric arrangement of the ligands (cis-ma for 24 and 25 vs. trans-ma for 7 and 10) and the Ru(m/n) reduction potential in MeCN vs. SCE (-101 and -35 mV for 24 and 25, respectively (Table 4.1), and -705 and -844 mV for 7 and 10, respectively (Table 3.4)). Clarke et al. have suggested that the reduction of Ru(H[) to Ru(U) may play an important role in the activation of Ru complexes inside the cell. 1 9' 2 0 The low reduction potentials of 7 and 10 perhaps indicate that such a reduction is not relevant for these two complexes. 24 and 25, however, have much more positive reduction potentials and might be reduced inside the cell. The cis geometry of the biimidazole complexes 24 and 25 compared to the trans geometry of all the complexes listed in Table 6.3 (Section 6.4.2) may also play an important role. The orientation of ligands can be important for their biological activity as in the case of cisplatin: the trans-isomer (rrans-[PtCl2(NH3)2]) is completely inactive because it is unable to form the necessary intrastrand DNA crosslinks to prevent cell division. 24 and 25 appear to remain intact throughout the experiment (as judged by TLC), at least outside the cells. From the experiments conducted, it could not be 146 150 o J , ; , 1 0.0001 0.001 0.01 0.1 1 Concentration (mM) Figure 6.11 Cell viability vs. concentration for [Ru(ma)2(Me2biim)]CF3S03-2H20 (25), with error bars representing 95% confidence limit. 0.0001 0.001 0.01 0.1 1 Concentration (mM) Figure 6.12 Cell viability vs. concentration for [Ru(ma)2(biim)]CF3S03-2H20 (24), with error bars representing 95% confidence limit. 147 determined if an intra-cellular process might lead to any ligand dissociation. Even in the absence of vacant sites for binding components of intra-cellular molecules, the geometry of the ligands can still play a role in how the complexes interact with protein active sites: a transport protein, for example, may recognize only one of the cis- or trans-isomers. Some Ru(U) complexes with biimidazoles were also tested (Table 6.6). The mixed biimidazole/DMSO complexes showed considerably less activity against this cell line than the Ru(UI) complexes tested. Our group has reported on the activity of Ru(IT)-maltolato sulfoxide complexes2 and Ru(U)-acetylacetonato sulfoxide complexes22 against the MDA-MB-435S cell line (data taken from Wu's M.Sc. thesis),17 the lowest IC 5 0 value being 190 pM for Ru(Ema)2(DMSO)2. [Ru(Me 2biim)3][CF 3S0 3] 2 (26) and [Ru(biim)3][CF3S03]2 (27) exhibit IC50 values of 36 and 18 pM, respectively (Figures 6.13 and 6.14), which indicates significant antiproliferatory activity in the absence of the ma ligand, these complexes are almost certainly acting through a different mechanism than that of the Ru(m) ma complexes. No M T T studies have been reported using [Ru(Im)6][CF3S03]2 or other similar homoleptic imidazole complexes, so the effect of the 150 Concentration (mM) Figure 6.13 Cell viability vs. concentration for [Ru(Me 2biim) 3][CF 3S0 3] 2 (27), with error bars representing 95% confidence limit. 148 bidentate versus the corresponding monodentate ligands cannot be compared. Previous work in our group showed that incubation of SCCVII cells with [Ru(Im)6][CF3S03]2 for 3 h did lead to some Ru uptake into the cells.6 Further study of these Ru(JJ) systems is needed to arrive at a better understanding of their behaviour, and to determine what effect the biimidazoles produce with respect to complexes with monodentate imidazoles. 150 T , Concentration (mM) Figure 6.14 Cell viability vs. concentration for [Ru(biim)3][CF3S03]2 (26), with error bars representing 95% confidence limit. 6.4.4 Ru Carboxylate complexes A Ru(ni) complex with imidazole-4-carboxylic acid (32), and a mixed valence Ru(ITJ/]I) bimetallic complex with picolinic acid (31) (Figure 6.15) were also tested using the MTT assay. Both exhibited higher IC50 values than did the imidazole maltolato complexes (Table 6.6); the IC50 value per Ru atom for the mixed valence species would be 200 uM. Further study of these two complexes is still warranted as they do show activity toward this cell line. 149 150 Concentration (mM) Figure 6.15 Cell viability vs. concentration for [Ru2(pic)4(EtOH)Cl]-H20 (31), with error bars representing 95% confidence limit. 6.5 Summary Direct comparison of the M T T assay data obtained here with those from other groups is difficult as results likely vary depending on the cell line used. However, by comparison with data for cisplatin, it is clear that a number of the Ru complexes show significant antiproliferatory activity and should be studied further to determine how they behave inside the cell. IC50 values reported for other Ru(III) complexes, as determined by MTT assays, include those for NAMI (Section 1.5.3.2) against the human colon cancer cell lines SW707 (195 pM) and SW948 (220 pM), 3 and that reported for a complex written as [H][Ru(l,2-dimethylimidazole)Cl4] against SKW-3 cells(> 400 pM), a human T-lymphoma cell line.4 Ideally, the best way to gauge the activity of the new Ru complexes would be to test them simultaneously with NAMI-A, which is currently undergoing phase I clinical trials (Section 1.5.3.2), or other standards on the same cell line, and compare their activities. Performing these tests on the same cell lines and at the same time under the 150 same conditions would lead to a more comprehensive understanding of the effectiveness of the various complexes, and perhaps of their different levels of activity in different tumour models. Performing M T T assays on other cell lines, particularly cisplatin resistant cell lines, would also help determine the effectiveness of these new Ru complexes to arrest cell proliferation. 6.6 References (1) Alley, M . C ; Scudiero, D. A.; Monks, A.; Hursey, M . L.; Czerwinski, M . J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M . R. Cancer Res. 1988, 48, 589. (2) Wu, A.; Kennedy, D.; Patrick, B. O.; James, B. R. Inorg. Chem. 2003, 42, 7579. (3) Keppler, B. K.; Galeano, A.; Berger, M . R. Drug Res. 1992, 42, 821. (4) Nikolova, A.; Ivanov, D.; Buyukliev, R.; Konstantinov, S.; Karaivanova, M . Drug Res. 2001, 57, 758. (5) Sava, G.; Bergamo, A. Int. J. Oncol. 2000,17, 353. (6) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents, Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. (7) Hodgkiss, R. J. Anti-Cancer Drug Des. 1998,13, 687. (8) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 936. (9) Loosdrecht, A. A. V. d.; Beelen, R. H. J.; Ossenkoppele, G. J.; Broekhoven, M . G.; Langenhuijsen, M . M . A. C. J. Immunol. Methods 1994,174, 311. (10) Loosdrecht, A. A. V. d.; Nennie, E.; Ossenkoppele, G. J.; Beelen, R. H. J.; Langenhuijsen, M . M . A. C. / . Immunol. Methods 1991,141, 15. (11) Bellamy, W. T. Drugs 1992, 44, 690. (12) Brinkley, B. R.; Beall, P. T.; Wible, L. J.; Mace, M . L. ; Turner, D. S.; Cailleau, R. M . Cancer Res. 1980, 40, 3118. (13) Siciliano, M . J.; Barker, P. E.; Cailleau, R. Cancer Res. 1979, 39, 919. (14) Mosmann, T. J. Immunol. Methods 1983, 65, 55. 151 (15) Morris, R. E.; Arid, R. E.; Murdoch, P. S.; Chen, FL; Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler, P. J. J. Med. Chem. 2001, 44, 3616. (16) Leonessa, F.; Green, D.; Licht, T.; Wright, A.; Wingate-Legette, K.; Lippman, J.; Gottesman, M . M . ; Clarke, R. Br. J. Cancer 1996, 73, 154. (17) Wu, A. Synthesis and Characterization of Ruthenium Maltolato, Sulfoxide, and Nitroimidazole Complexes as Potential Anticancer Agents, M . Sc. Dissertation, University of British Columbia: Vancouver, 2002. (18) Huxham, L. A.; Cheu, E. L. S.; Patrick, B. O.; James, B. R. Inorg. Chim. Acta 2003, 352, 238. (19) Clarke, M . J. Coord. Chem. Rev. 2002, 232, 69. (20) Clarke, M . J.; Zhu, F.; Frasca, D. R. Chem. Rev. 1999, 99, 2511. (21) Lippard, S. J.; Berg, J. M . Principles of Bioinorganic Chemistry; University Science Books: Mill Valley, 1994. (22) Wu, A.; Kennedy, D.; Patrick, B. O.; James, B. R. Inorg. Chem. Commun. 2003, 6, 996. 152 Chapter 7 In Vitro Toxicity, Ru-Uptake and Ru-DNA Binding Studies of Ru Complexes, and Antibody Recognition Studies of a New Nitrotriazole in Chinese Hamster Ovarian (CHO) Cells 7.1 Introduction A persistent problem in radiation therapy of cancerous tumours is that many tumours contain hypoxic regions that are resistant to radiation treatment. Rapid growth of tumours can lead to inadequate vasculature development and thus poor transport of oxygen to the tumour cells.1 Typically an oxygen-gradient will be established with the innermost part of tumour being lowest in oxygen concentration (see Chapter 1, Figure 1.1); these oxygen depleted regions in which the cells are still viable, are called hypoxic regions, and their resistance to many treatments makes it important to identify these regions before a treatment is prescribed.1 Nitroimidazoles have been shown to accumulate in hypoxic cells, the nitro group being reduced inside the cell and the resulting reduced species then reacting with macromolecules to form adducts. Quantification of the nitroimidazole content inside cells can help identify regions of hypoxia.1 Through incorporation of radioactive atoms into the nitroimidazole, the localization of the radioactive species can be quantified using SPECT, PET, or MRI imaging.1 Antigenic properties of the resulting adducts can also be used to quantify the levels of drug uptake by removing a tissue or cell sample and treating it with monoclonal antibodies. The concentration of bound antibodies can then be determined using fluorescence spectroscopy.3'4 There are literature examples of Ru complexes binding to DNA bases,5"7 but in order to propose a mechanism of action involving DNA binding (as with cisplatin), one must first determine if the complex is in fact reaching the DNA in vitro and binding to it. It has also been suggested that some Ru complexes may act through extracellular mechanisms.8 If so, then one would not expect to find correlation between the cellular uptake of Ru and the biological activity of the complex. The results of Ru-uptake and 153 DNA binding assays, discussed in this chapter, give information not only about hypoxia selectivity, but also about how and where the complexes might be interacting inside the cells. Furthermore, a lower plating efficiency of the cells after their incubation with a given complex for 3 h, an arbitrarily chosen time, compared with that of the control, indicates that the complex is having a cytotoxic or antiproliferatory effect on the cells over this period. 7.2 General Experimental 7.2.1 Materials, Media, and Prepared Solutions Eagle's minimum essential medium powder (cc-modification), and penicillin/streptomycin antibiotic were purchased from Gibco. Sterile tissue culture dishes (5 cm diameter), T-25 and T-75 flasks, and 15 and 50 mL Conical tubes were purchased from Falcon. Methylene blue was purchased from Aldrich. Other materials used in this chapter are listed in Section 6.2.1 (p 132). The media were prepared by the staff in the biological services laboratory in the UBC chemistry department, a-l-Medium was prepared by adding one packet of a-medium powder and 10,000 units of penicillin/streptomycin antibiotic to 1 L of doubly distilled (dd) H 2 0 , and stirring the resulting solution for 2 h at r.t. a+I- Medium was prepared by adding 10 % heat-inactivated fetal bovine serum (FBS) to a-l- medium buffered with 10 mM HEPES. a+/+ medium was prepared by adding 10 % FBS and Na(HC0 3) (2 g/L) to a-l- medium and then adjusting the pH to 7.30 with 2 M NaOH. The media were then filter- sterilized and stored at 4 °C, while the trypsin-EDTA and FBS were stored at -20 °C. Methylene blue solution was prepared by dissolving the solid (Aldrich) (200 mg) in 100 mL of ddH 20; the resulting solution was filtered through a 0.1 um filter to remove any undissolved solid and stored at 4 °C. The following solutions were prepared exclusively for the monoclonal antibody binding assay (Section 7.3.4) with the assistance of staff at the BC Cancer Research Centre (BCCRC): PBS* was prepared by adding 0.25 % (v/v) 0.1 M Thimerosal (Aldrich) and 0.25 % (v/v) 1.0 M NaN 3 to the standard PBS solution. PBS" was prepared by adding 0.3 % Tween 20 (Aldrich) (v/v) to a PBS* solution. Ab Carrier, available 154 from BCCRC, consisted of 1.5 % bovine serum albumin (0.15 g) in PBS" (10 mL); as the bovine serum albumin is partially insoluble, the suspension was stirred at 37 °C for 1 h prior to use. Blocking solution was also available from BCCRC and consisted of 20 % skim milk and 5 % mouse serum added to the Ab Carrier mixture. Paraformaldehyde (pF) was prepared by boiling a solution of 100 pL of 1 M NaOH in 60 mL ddH 20, and then adding this to a slurry of 4 g pF in 30 mL ddH 20. The resulting colourless solution was cooled to 0 °C, when 10 mL of lOx PBS (available at B C C R C and having ten times the ionic strength of normal PBS) were added. This mixture was then filtered through a 0.22 pm sterile filter and to the filtrate was added 100 pL of 1 M HCl. pF was stored at 0 °C. The monoclonal antibody (MoAb), ELK3-51, was donated by Dr. C Koch (University of Pennsylvania) to BCCRC. Atomic absorption spectroscopy (AAS) was performed by Celator Technologies on a Varian SpectrAA-300 Zeeman Atomic Absorption Spectrometer controlled by a Compaq Deskpro 386s computer. The instrument was calibrated using stock solutions of Ru from Aldrich, and a Ru hollow cathode lamp at a 10 mA current ( X m a x = 349.9). A re-slope calibration was performed after every fifth sample to ensure that there was no significant shift in the background reference. 7.2.2 Cell Preparation Chinese Hamster Ovarian (CHO) cells were donated by Dr. K. Skov at the BCCRC (Figure 7.1). After the cell culture was grown for three days, the cells were passaged and a frozen stock of cells was prepared (ten 1 mL vials containing 1 x 106 cells in medium with 5 % DMSO). The cells were maintained in T-75 tissue culture flasks in a 5 % C 0 2 /air incubator from Forma Scientific in a+/+ media at 37 °C. The cells were transferred every 3-4 days using the procedure described in Section 6.2.2 for the MDA-MB-435S cell line, but using a+/+ medium instead of L-15 medium. 155 Figure 7.1 C H O cells in a+/+ medium (~106 cells/mL). 7.2.3 Stock Solutions of Ru Complexes The Ru complexes were weighed into glass vials and dissolved in PBS to a concentration of 1.0 m M ; 3.0 m L of stock solution were prepared for each complex. These solutions were then sonicated and heated to 37 °C to ensure the complex was completely dissolved, and then filter-sterilized through 0.2 urn filters, to remove an remaining particulates. 7.3 Hypoxia Selectivity Assays 7.3.1 Incubation of Complexes and Plating Efficiency The hypoxia assays were performed at U B C in an innova 4300 incubator shaker (from New Brunswick Scientific) under a flow of N 2 or air, according to established protocols. 9 Centrifugation was performed on a Dynac Centrifuge, a+l- medium (8 mL) and Ru complex solution (1 mL) were added to 50 m L Falcon tubes. In most cases, two tubes were prepared for each complex, one for incubation under air, the other under N 2 ; however, some complex solutions were only tested under an air atmosphere (see Table 7.2, Section 7.4.2). The tubes were then placed in the incubator, attached to a gas line, shaken (150 rpm) at 37 °C for 1 h (Figure 7.2), and then removed from the incubator and 156 placed in a sterile hood. To each tube was then added 1 mL of a prepared cell suspension containing 3 x 106 cells/mL in a+/- medium. The tubes were then placed back in the incubator, reattached to the gas lines, and shaken (150 rpm) at 37 °C for 3 h. The suspensions were then transferred to 15 mL Falcon tubes and centrifuged (8 min at 800 rpm). The supernatant was removed, and the cells were resuspended in 5 mL of PBS to rinse them of any remaining Ru. The cell suspensions were centrifuged again (8 min at 800 rpm) and the supernatant removed. The pellets were then resuspended in 2 mL of PBS and the cell density of each suspension was determined by counting the cells under a microscope using a hemacytometer. A 10 uL aliquot of each cell suspension was then added to 5 mL of a+/- medium and the mixture vortexed to ensure that the diluted cell suspensions were homogeneous; 50 uL of each of these was then added to 5 mL of cc+/+ medium in a 5 cm tissue culture dish. This was repeated 2 more times giving a total of 3 dishes for each sample. The tissue culture dishes were then placed in the 5 % C 0 2 incubator for 7 days to allow colonies to form. gas in gas out Figure 7.2 Conical tube set-up used for CHO experiments (gas = air or N 2). The remaining 2 mL suspension in PBS and 5 mL suspension in a+/- medium were combined for each sample and centrifuged (8 min at 800 rpm). The cells in each flask were then resuspended in 2 mL of PBS. For those samples for which the DNA binding assay was performed, the suspensions were then divided in half, with 1 mL of the 157 cell suspension being used for the Ru-uptake assay (Section 7.3.2), and the other half for the D N A binding determination (Section 7.3.3). For those samples for which only the Ru-uptake assay was performed, the entire 2 m L suspension was used for the procedure. Alternately, for the Ru-free compounds, EF5 and triF5, only the monoclonal antibody binding assay was performed, the 2mL suspension being used to carry out the procedure described in Section 7.3.4. After 7 days, the medium was removed from each dish and methylene blue solution was added (0.40 mL). The dishes were swirled to ensure the dye covered the entire surface of the dish and, after 10 min, the dishes were rinsed with water and the remaining colonies (Figure 7.3) were counted to determine the plating efficiency (PE), defined as the number of colonies counted for each sample divided by the number of cells plated 7 days earlier. Figure 7.3 Colony of C H O cells after 7 days, following staining with methylene blue. 7.3.2 Ru-Uptake Determination The final cell suspensions allotted for Ru-uptake determination (1 or 2 mL, see Section 7.3.1) were transferred to a fresh 15 m L Falcon tube and then centrifuged (10 min at 800 rpm). The supernatant was then removed and the tubes were placed with lids open in the shaker incubator (37 °C, 150 rpm) for 16 h to dry the cell pellets. Cone. H N 0 3 (100 pL) was then added to each tube, which was then closed and left at r.t. for 24 158 h in order that the cell pellet be digested. Then, 250 pL of ddFJ^O was added to dilute the acid, and the samples were then sent for A AS to determine the Ru content of each sample. 7.3.3 DNA Isolation The DNA isolation was performed using a Wizard Genomic D N A Purification Kit from Promega. The 1 mL aliquots of the cell suspensions (see Section 7.3.1) were transferred to 1.5 mL microcentrifuge tubes and pelleted using centrifugation (15,000 x g for 10 s). The supernatant was then removed from each tube, leaving the cell pellet in -50 pL of residual medium. PBS (200 pL) was then added to each pellet, and these mixtures were vortexed at high speed for 30 s to resuspend the cells. The cell suspensions were then centrifuged again (15,000 x g for 10 s). The supernatant was again discarded from each tube leaving the cell pellets in approximately 20 pL of liquid. Nuclei Lysis Solution (600 pL) (from the Kit) was then added to each cell pellet, and the mixtures gently "pipetted" until no visible clumps of cells remained. RNase (3 pL) Solution (from the Kit) was then added to each tube, and the samples were mixed. The cell mixtures were then incubated for 30 min at 37 °C to allow the cells to lyse. The tubes were then removed from the incubator and cooled to r.t. Protein Precipitation Solution (200 pL) (from the Kit) was then added to each tube, and the resulting mixtures were vortexed at high speed for 20 s and then cooled in an ice-bath for 5 min. The tubes were then centrifuged (15,000 x g for 4 min) causing the precipitated protein to form a white pellet at the bottom of each tube. The supernatant containing the DNA was then carefully removed and transferred to a clean 1.5 mL microcentrifuge tube containing 600 pL of isopropanol. The resulting solutions were then carefully mixed by inverting the tubes until white, thread-like strands of DNA became visible. The tubes were then centrifuged (15,000 x g for 1 min) to pellet the DNA. The supernatant was then removed and 600 pL of 70 % aq. EtOH was added. The tube was inverted several times to wash the DNA. The tubes were again centrifuged at 15,000 x g for 1 min to pellet the DNA and the EtOH was aspirated with a pipette leaving only the DNA pellet. The tubes were then left open and inverted over a paper towel to air dry the pellet for 30 min. Finally, 100 pL of DNA Rehydration Solution (from the Kit) was added to each tube and the 159 sealed tubes were then heated at 65 °C for 1 h. The tubes containing the rehydrated DNA were then stored at 4 °C until the amount of isolated DNA from each sample was determined by UV-Vis spectroscopy (Section 7.3.3.1) and the amount of associated Ru by AAS . The amount of DNA in each sample was determined by measuring the optical density, and then employing Equation 7.1. The absorption of a DNA suspension was recorded at both 260 and 280 nm on a Perkin Elmer Lambda 2 UV/VIS spectrometer in a 1 cm optical cell. The ratio of the absorptions at 260 and 280 nm provides a check on the DNA purity: values > 1.7 are considered to be good,10 and for all the samples, this ratio was between 1.71 and 1.85. The rehydrated DNA samples were diluted from 30 to 800 pL, a dilution factor of 26.67, before the absorbances at 260 and 280 nm were recorded. A (OD) x D x 50 (ng mL"1 OD"1) x (0.100 mL) = pg DNA (Eq. 7.1) A = absorption at 260 nm, D = dilution factor, 0.100 mL is the total sample volume and 50 is a constant. 7.3.4 Monoclonal Antibody Binding Assay This assay was performed at the BCCRC with the assistance of Haibo Zhou. The two compounds tested, EF5 and triF5, were incubated with CHO cells using the procedure described in Section 7.3.1 for the incubation of complexes. The 2 mL cell suspensions from the EF5 and triF5 experiments were then centrifuged (8 min at 800 rpm) and the supernatant, removed from each using a 1000 pL micropipettor, was discarded. pF (3 mL) was then added dropwise to each tube as it was vortexed at medium speed. The tubes were then inverted continuously for 1 h at 4 °C. After 1 h, the cells were centrifuged (8 min at 800 rpm) and washed twice with PBS* (by addition of 5 mL of PBS*, centrifugation for 8 min at 800 rpm and then removal of the supernatant). After the second wash, the cells were resuspended in 1 mL PBS* and then transferred to Eppendorf tubes. These were then centrifuged (8 min at 800 rpm), their supernatant removed, and 100 pL of blocking solution was added to each tube (blocking solution prevents the antibody from binding molecules other than the intended target inside the cell). The tubes were then inverted for 5 h at 4 °C. Then 1 mL of PBS* was added to each Eppendorf tube, and these were shaken gently to mix the suspensions. The mixtures 160 were then centrifuged (8 min at 1200 rpm) and the supernatant removed; 100 uL of a MoAb solution was then added (the ELK3-51 antibody was conjugated to a Cy3 fluorescent label11 (Figure 7.4) to make detection of the antibody possible, and the MoAb solution contained a 20:1 mixture of Ab Carrier.MoAb). The resulting suspensions were then covered with Al foil and rotated overnight at 4 °C (12 h). (Note: all work with the MoAb was done in a dark-room as the intensity of the fluorescence of the MoAb decreases if the antibody is exposed to light). The samples were then centrifuged (8 min COaH Figure 7.4 H3C. ? H 3 C H = C H (CH2)4S03 C0 2H (CH 2) 4S0 3K Structure of the fluorescent dye, Cy3, used to label the ELK3-51 MoAb. at 1200 rpm) and the supernatant containing the MoAb solution was removed. PBS" was then added to each tube and the resulting mixtures gently vortexed to resuspend the pellet. The tubes were then rotated for 40 min at 4 °C, centrifuged (8 min at 1200 rpm), and the supernatant discarded. This procedure was repeated two more times. After the last wash with PBS", the samples were resuspended in 400 uL of PBS". These suspensions were diluted with 2 mL of 1 % pF and then submitted for analysis at BCCRC by flow cytometry. The final suspensions were stored at 4 °C for 1-2 weeks before the analyses were performed. The concentration of MoAb adducts and thus the concentration of EF5 or triF5 in a given sample was measured by a technician at BCCRC on a Coulter Epics Elite fluorescence-activated cell-sorter, with a Coherent Inova 90 Ar laser. Detection of the MoAb adducts is facilitated by the Cy3 label on the MoAb. The sample is passed through the laser using an ultrasonic vibrator to split the sample into droplets; only drops 161 containing a single cell were analyzed. The laser was set to 510 nm for the excitation wavelength, and the emission was detected between 580-590 nm. For each sample, the fluorescence of 10,000 cells was measured. 7.4 Results and Discussion 7.4.1 Plating Efficiency of C H O Cells The toxicity of several compounds (Table 7.1) was investigated under hypoxic (under N 2) and oxic (under air) conditions by determining the plating efficiency (PE) of the cells after a 3 h incubation period in media usually containing 100 uM of the compound; [Ru(ma)2(EF5)2]CF3S03-EtOH (14) was tested at 48 uM because of a solubility limitation. The procedure for this experiment is outlined in Section 7.3.1. The plating efficiency was determined by counting the colonies for of the 3 replicates for each sample, and then dividing by the number of cells plated 7 days earlier after the 3 h incubation with the complex. The PE data (Table 7.1) are the average of two experiments, each having a plating efficiency determined by averaging the number of colonies in the 3 replicates. For the controls, the values are the average of six experiments, as a control was performed each time a complex or ligand was tested, and only 5 complexes could be tested at once. The. error in the PE of the controls is only ± 4 % because there are more experiments within the average value, and the variation of the PE values for the controls was less than those for the complexes. Several of the complexes do exhibit a small cytotoxic effect; however, there is no significant hypoxia selectivity observed for the PE of any of the complexes. The two nitro-heterocycles, EF5 and triF5, showed no toxicity at all. Repeating these experiments might be valuable as a greater number of trials would decrease the error limits. The experiments could also be lengthened or monitored over time to determine how the amount of complex in the cells varies with time. The use of a Coulter counter (not available in the UBC chemistry department) might help reduce the errors as counting cells on the hemacytometer was not very accurate for this particular assay. Clumping of cells was a major problem, both in the counting of cells, and the fact 162 that cell clumps form large cell masses (Figure 7.5) and not individual countable colonies. A B C Figure 7.5 Large cell colony formations resulting from clumped cells (A and B) compared with a colony grown from a single cell over the same time period, C. Our group has previously synthesized and tested similar Ru-acac-imidazole and -nitroimidazole complexes in SCCVII cells, and also found no hypoxia selective toxicity.12 There may be several reasons why the PEs of complexes active in the MTT assay (with IC50 values well below 100 uM, see Section 6.4) do not show any significant toxicity in this PE assay. A different cell line was used here than that for the M T T assay (CHO vs. MDA-MB-435S) because of past experience at BCCRC in which CHO cells were known to provide a good model for testing hypoxia. This PE assay also differs in that the incubation is only 3 h compared with 69 h for the M T T assay, and the complexes may not accumulate quickly enough to have a significant effect on the cells. The cells are also plated in media in which no complex is present. If the complexes are able to diffuse out through the cell membrane, then an equilibrium between complex inside and outside the cell may be important; removing the extracellular complex may drive the complex outside of the cell. This sort of mechanism is not unreasonable, as there is no evidence to suggest that the complex decomposes inside the cell, and no intracellular targets have yet been established to suggest that, once the complex is taken up, it is 'trapped' inside the cell. 163 Table 7.1 Plating efficiency3 of CHO cells after a 3 h incubation period under both aerobic and anaerobic conditions with several Ru(IU) complexes at 100 pM. Compound PE-oxic PE-hypoxic Control 69b 66b Ru(ma)3 (1) 42 35 Ru(Ema)3 (2) 41 32 [Ru(ma)2(metro)2]CF3S03 (4)... 36 48 [Ru(ma)2(2MeIm)2]CF3S03 (10) 72 58 [Ru(ma)2(4MeIm)2]CF3S03-CH2Cl2 (6) 50 48 [Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2 (8) 48 62 [Ru(ma)2(EF5)2]CF3S03-EtOH (14) 72c 65c [Ru(Ema)2(2MeIm)2]CF3S03 (13) 43 45 [Ru(Ema)2(4MeIm)2]CF3S03 (12) 35 35 Ru(Im-C0 2 )(Im-C0 2 H) 2 Cl 2 EtOHH 2 0 (32) 32 39 EF5 68 66 triF5 65 62 3 Error limits are ± 12 %. b The error for these values is only ±4%. c The final concentration of [Ru(ma) 2(EF5) 2]CF 3S0 3EtOH was 48 pM due to poor solubility. 7.4.2 Ru-Uptake The uptake of Ru into CHO cells was determined in order to help understand how the Ru(HI) complexes that showed low IC50 values (Chapter 6) might be acting in vitro. o Sava and Bergamo have suggested that some Ru complexes may bind extracellular components in order to produce cytotoxic or antiproliferatory effects, while others may act with intracellular components such as proteins or DNA to produce a biological effect. In the former case, one would expect low levels of Ru to be taken up into the cells. In the latter case, the level of activity might be dependent on the amount of complex in the cell. 164 The data (Table 7.2) show that the complexes containing the Ema ligands (2, 12 and 13) are taken up more readily into the cells than the corresponding ma complexes (1, 6, and 10), and the former group also exhibit lower IC50 values in MDA-MB-435S cells than the latter group (Section 6.4.2). This suggests a link between the Ru-uptake and antiproliferatory activity for these complexes. Conversely, the Ru-uptake for [Ru(ma)2(2MeIm)2]CF3S03 (10) and [Ru(ma) 2(lMeIm) 2]CF 3S0 3-CH 2Cl 2 (8) under both oxic and hypoxic conditions are exactly the same for each complex, whereas the corresponding IC50 values are 5 and 70 pM, respectively (Section 6.4.2). These findings imply that the nature of the imidazole ligand is important for the biological effect; as the free imidazoles (2MeIm and lMelm) alone produce no significant cytotoxic effect (Section 6.4.1), at least one of the imidazole ligands must remain bound in vitro in order for the Ru complexes to produce their biological effects. CHO cells incubated with [Ru(ma)2(EF5)2]CF3S03-EtOH (14) showed the highest level of Ru-uptake, suggesting that this complex is taken up more readily than analogous imidazole- and nitroimidazole-maltolato complexes; 14, however, exhibits no significant antiproliferatory activity (Section 6.4.2), nor does this complex accumulate selectively in hypoxic cells as does free EF5 (Section 7.4.4). Indeed, none of the complexes tested under both oxic and hypoxic conditions shows any hypoxia selectivity (Table 7.2). The Ru-uptake values reported for 14 (Table 7.2) are for a 3 h incubation at 48 pM as determined by AAS after the complex solution had been filtered. Other workers have noted a linear correlation between Ru-uptake vs. concentration at concentrations below 100 pM in M E M (minimum essential medium),13 suggesting that at 100 pM, the values listed in Table 7.2 for 14 would be 169 and 152 ng Ru/106 cells under oxic and hypoxic conditions, respectively. For the biimidazole complexes, the Ru-uptake values for [Ru(ma)2(biim)]CF3S03-2H20 (24) and [Ru(ma)2(Mebiim)]CF3S03-2H20 (25) are very similar, as are the values for [Ru(biim)3][CF3S03]2 (26) and [Ru(Mebiim)3][CF3S03]2 (27). Nevertheless, the complexes with perhaps higher uptake (25 and 26) do have lower IC50 values (15 and 10 pM, respectively) than do 24 and 27 (50 and 25 pM, respectively). Further experiments that might help determine if these differences are real or not will be noted in Chapter 8. 165 Table 7.2 Uptake of R u by C H O a cells after a 3 h incubation period under both aerobic and anaerobic conditions with several R u complexes at 100 u M . Compound Oxic - Ru Hypoxic - Ru I C 5 0 values0 (ng/106 cells) (ng/106 cells) (uM) Control 1.0 0.5 -Ru(ma) 3 (1) 15.3 16.7 140 Ru(Ema) 3 (2) 42.3 36.5 90 [Ru(ma)2(metro) 2]CF 3S0 3 (4) 14.9 15.2 80 [Ru(ma) 2 (2MeIm) 2 ]CF 3 S0 3 (10) 14.3 16.5 5 [Ru(ma) 2 (4MeIm) 2 ]CF 3 S0 3 -CH 2 Cl 2 (6) 27.0 21.0 15 [ R u ( m a ) 2 ( l M e I m ) 2 ] C F 3 S 0 3 - C H 2 C l 2 (8) 14.1 17.2 80 [Ru(ma) 2 (Im) 2 ]CF 3 S0 3 (7) 16.7 19.6 70 [Ru(ma) 2 (EF5) 2 ]CF 3 S0 3 -EtOH (14) 81.3 b 72.8 b >500 [Ru(ma)( lMeIm) 4 ]CF 3 S0 3 (9) 13.3 - -[Ru(ma) 2 (b i im)]CF 3 S0 3 -2H 2 0 (24) 12.7 - 50 [Ru(ma) 2 (Me 2 bi im)]CF 3 S0 3 -2H 2 0 (25) 18.0 - 15 [Ru(b i im) 3 ] [CF 3 S0 3 ] 2 (26) 26.0 - 18 [Ru(Me 2 b i im) 3 ] [CF 3 S0 3 ] 2 (27) 20.0 - 36 R u ( I m - C 0 2 ) ( I m - C 0 2 H ) 2 C l 2 E t O H H 2 0 (32) 15.4 24.3 400 [Ru(Ema) 2 (2MeIm) 2 ]CF 3 S0 3 (13) [Ru(Ema) 2 (4MeIm) 2 ]CF 3 S0 3 (12) 67.2 82.0 0.5 79.5 94.1 5 a Error limits are ± 5 ng/106 cells. b The final concentration of [ R u ( m a ) 2 ( E F 5 ) 2 ] C F 3 S 0 3 E t O H was 48 u M due to poor solubility. Data from Tables 6.3-6.6 (p. 138, 141, and 144) using the M D A - M B - 4 3 5 S cell line. Recently, Frausin et al. have examined the Ru-uptake of N A M I - A in K B cells (human nasopharynx cancer cells), and concluded that the complex enters the cell both by passive diffusion across the membrane and by active transport.1 4 Over 4 h, at both 4 and 37 °C, the complex only accumulated in the cells at the higher temperature, the Ru-uptake increasing when the experiment was performed in PBS instead of M E M ; this was thought 166 to result from competition for transport sites in the membrane with other components present in M E M . When the concentration was varied between 10 and 1000 pM, the percent of Ru taken into the cell remained constant in M E M , while in PBS, the percent of Ru-uptake from the total present in solution decreased as the concentration increased.14 Ru-uptake data have also been reported for the dimeric Ru complexes [RuCl4(DMSO)]2(p-L), where L = pyrazine, pyrimidine, 4,4'-bipyridine as well as other I n substituted pyridine ligands; these were also tested against KB cells, and uptake values were similar to those found for NAMI-A in the same cell. Further experiments examining the Ru-uptake of the complexes discussed in this section might help to understand how the complexes act in vitro. Use of the MDA-MB-435S cell line used for the M T T assay (Chapter 6) instead of CHO cells would allow a more direct comparison to be made between the Ru-uptake values (Table 7.2) and IC50 values (Section 6.4.1, Table 6.6). 7.4.3 Ru-DNA Binding DNA was isolated from cells after a 3 h incubation with a selected Ru(ffl) complex in order to determine if it was interacting with DNA. With the exception of [Ru(ma)2(EF5)2]CF3S03-EtOH (14), none of the other complexes tested (1, 2, 4, 6, 8,10) exhibited any DNA binding (Table 7.3). 14 exhibits some hypoxia selectivity for binding DNA: 5.9 and 10.5 ng Ru/mg DNA at 48 pM for oxic and hypoxic conditions, respectively. Previous studies in our group with [Ru(acac)2(EF5)2]CF3S03 in SCCVII cells12 showed that, after a 3 h incubation, the amount of bound Ru was 9.6 and 11.1 ng Ru/mg DNA for oxic and hypoxic conditions, respectively. These values are calculated here for 48 pM complex solutions, assuming that there is a linear correlation between Ru-DNA binding and Ru concentration, as poor solubility of [Ru(acac)2(EF5)2]CF3S03 limited its testing to a concentration of 22 p M . 1 2 Such an assumption has been made for the Ru-uptake of NAMI-A at low concentrations.13 This complex also exhibited a 4-fold increase in fluorescence over that of free EF5 in the MoAb assay,12 suggesting that the complex facilitates the accumulation of EF5 inside the cell to a greater concentration 167 Table 7.3 Amount of Ru a associated with DNA isolated from CHO cells after a 3 h incubation period under both aerobic and anaerobic conditions with several Ru(UI) complexes at 100 pM. Compound Oxic Hypoxic Ru (ng/mg DNA) Ru (ng/mg DNA) (± 3 ng/mg) (± 3 ng/mg) Control 0 0 1, 2, 4, 6, 8,10 0 0 ; [Ru(ma)2(EF5)2]CF3S03-.EtOH (14) . 5.9" Error limits are ± 3 ng/mg, A A detection limit ~ 1 ng/mg DNA. B The final concentration of 14 was 48 pM due to poor solubility. than when EF5 acts alone. The higher level of Ru-DNA binding for [Ru(acac)2(EF5)2]CF3S03 suggests that, in the cell, the EF5 is being released from the complex and replaced by donor ligands of DNA. The same may be true for 14, and thus further study, in particular the MoAb binding assay, is needed on this complex. The DNA binding assay should also be repeated using the MDA-MB-435S cell line so that a more accurate correlation can be made between the DNA-binding results and the IC 5 0 values from the M T T assay (Chapter 6). For the other complexes, their method of action inside the cell does not include DNA binding; thus further experiments are needed to locate other potential targets for these complexes in vitro. In Section 7.4.5, a preliminary study will be discussed with L-cysteine potential intracellular target. 7.4.4 The Monoclonal Antibody Binding Assay This assay was performed using only EF5 (Section 3.6.2) and the corresponding fluorinated nitrotriazole derivative, triF5 (Section 3.6.3). For triF5, detection of the MoAb adducts was carried out using flow cytometry and, as in the case of earlier work on EF5, 1 2 triF5 accumulates preferentially in hypoxic cells (Figure 7.4). However, the mean fluorescence for the triF5 MoAb adducts was considerably less than that observed 168 for the EF5 M o A b adducts: at 100 p M , the signal for EF5 adducts are up to 7 times greater than that for triF5 adducts. More important is that the ratio of the hypoxic:oxic signals for triF5 is much less than that of E F 5 ; in a search for new hypoxia imaging agents, this ratio determines the effectiveness of the compound to detect specifically hypoxic cells with low levels of background fluorescence in normal tissue. 500 0 200 400 600 800 1000 1200 cone, compound (pM) Figure 7.6 Median fluorescence intensity of C H O cells incubated with EF5 or triF5 under N 2 or air and then treated with the ELK3-51 /Cy3 antibody. 7.4.5 Reaction of [Ru(ma)2(2MeIm)2] C F 3 S 0 3 (10) with L-cysteine A n in situ, r.t. reaction of [Ru(ma) 2 (2MeIm) 2 ]CF 3 S0 3 (10, Section 3.7.9) in D 2 0 (~ 10"3 M ) under air with 1 equivalent of L-cysteine (Figure 7.7) was followed by ' H N M R spectroscopy. After 24 h, the initial intensities of the proton resonances for 10 were unchanged, and there were no new paramagnetic or diamagnetic signals, showing that 10 was still intact. The resonances for L-cysteine (5 3.92 (-CH-), 3.08 (-CH2-)) had decreased in intensity by ~ 50 %, and new resonances for L-cystine (8 4.13 (-CH-), 3.32 169 (-C//2-)) were present in about equal intensity as those for L-cysteine. In a control with only L-cysteine in D 2 0 , only trace amounts of L-cystine were present after 24 h. 10 is apparently catalyzing the aerial solution oxidation of L-cysteine. O , N H , O S H Figure 7.7 Molecular structure of L-cysteine The 'H NMR spectrum 10 in D 2 0 in air remained unchanged for up to 48 h. For the L-cysteine sample at this time, the resonances for L-cystine were more intense than at 24 h, but still only ~ 10 % of the L-cysteine had been oxidized. In the spectrum of the 1:1, 10:L-cysteine system, there were no longer any resonances for L-cysteine present; and the only paramagnetic signals observed were those for 10, although a trace of 2-methylimidazole was now present in solution. No signals could be seen for either free or diamagnetically bound maltolato, showing that none of the initial Ru(HJ) had been reduced to Ru(U). At the concentrations used, the signals for 10 are weak and difficult to resolve (minimum 150 scans, Section 3.3.2) and it is possible that trace amounts of other Ru(IU) species might be present. The integration of the 2-methylimidazole signals with respect to those of L-cystine suggests that ~ 0.05 equivalents of 2-methylimdiazole have dissociated, leaving 95 % of intact 10. From this it can be concluded that the complex is catalytically oxidizing the cysteine to cystine in air. Repeating the reaction under Ar could give evidence for a reduced species. T L C analysis after 48 h of solutions of 10, and 10 with L-cysteine, revealed the same band as a sample of 10 which had just been dissolved. All three samples also gave a brown spot (Rf = 0) that was less intense for the freshly prepared sample. No new bands for reduced species or newly formed complexes were observed. 170 Glutathione (a tripeptide containing a cysteine) can reduce certain Ru(IH) species in situ,]5 while reaction with glutathione is also the primary means by which cisplatin is deactivated in vivo}6 The stability of 10 in the presence of L-cysteine suggests that this complex is not being reduced in situ to a biologically active Ru(EI) species, and an intact or only partially substituted Ru(UI) species is likely responsible for the antiproliferatory activity observed and not a species from which both imidazole ligands have been dissociated. Further experiments monitoring the reaction of glutathione with 10 and other Ru(irj) complexes listed in Table 7.2 are needed to help determine more definitively the fate of the complex inside cells. 7.5 References (1) Hodgkiss, R. J. Anti-Cancer Drug Des. 1998,13, 687. (2) Wardman, P. Environ. Health Perspect. 1985, 64, 309. (3) Matthews, J.; Adomat, H.; Farrell, N.; King, P.; Koch, C ; Lord, E.; Palcic, B.; Poulin, N.; Sangulin, J.; Skov, K. Br. J. Cancer 1996, 74, S200. (4) Koch, C. J.; Evans, S. M. ; Lord, E. M . Br. J. Cancer 1995, 72, 869. (5) McNamara, M . ; Clarke, M . J. Inorg. Chim. Acta 1992,195, 175. (6) Hotze, A. C. G.; Broekhuisen, M . E. T.; Velders, A. H.; Kooijman, H.; Spek, A. L.; Haasnoot, J. G.; Reedijk, J. J. Chem. Soc, Dalton Trans. 2002, 2809. (7) Hotze, A. C. G.; Broekhuisen, M . E. T.; Velders, A. H ; Van der Schilden, K.; Haasnoot, J. G.; Reeijk, J. Eur. J. Inorg. Chem. 2002, 369. (8) Sava, G.; Bergamo, A. Int. J. Oncol. 2000,17, 353. (9) Moore, B. A.; Palcic, B.; Skarsgard, L. D. Radiat. Res. 1976, 67, 459. (10) Ausubel, F. M . ; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman, J. G.; Smith, J. A.; Struhl, K. Short Protocols in Molecular Biology 2nd ed.; John Wiley and Sons: New York, 1992. 171 (11) Southwick, P. L.; Ernst, L. A.; Tauriello, E. W.; Parker, S. R.; Mujumdar, R. B.; Mujumdar, S. R.; Clever, H. A.; Waggoner, A. S. Cytometry 1990, 11, 418. (12) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents, Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. (13) Bergamo, A.; Stocco, G.; Gava, B.; Cocchietto, M . ; Alessio, E.; Serb, B.; Iengo, E. ; Sava, G. J. Pharmacol. Exp. Ther. 2003, 305, 725. (14) Frausin, F.; Cocchietto, M. ; Bergamo, A.; Scarcia, V.; Furlani, A.; Sava, G. Cancer Chemother. Pharmacol. 2002, 50, 405. (15) Clarke, M . J. Coord. Chem. Rev. 2002, 232, 69. (16) Hall, M . D.; Hambley, T. W.. Coord. Chem. Rev. 2002, 232, 49. 172 Chapter 8 Conclusions, and Direction of Future Work 8.1 Conclusions Several new Ru(UI)-ma imidazole and -ma nitroimidazole complexes have been synthesized and tested for biological activity; selected, corresponding Ema complexes were also synthesized and tested (Chapter 3). The 'H NMR spectra of the paramagnetic Ru(ITJ)-ma imidazole complexes were used to determine their purity and molecular structure. In the 'H NMR spectra of 6, 8 and 10, single resonances for the ma-Me protons were assigned to the trans-ma, trans-imidazole isomer, and X-ray crystallography confirmed such a structure for 8 and 10. Similarly, in the ' H NMR spectra of the Ema complexes 11 - 13, single resonances for the -CH2- groups were also assigned to trans-isomers, which was demonstrated for 11 by X-ray analysis. Ru(ITJ)-ma complexes of nitroimidazoles in which the N(l) proton was not substituted (EF5 and metro are both 1-substituted nitroimidazoles) were not soluble (without decomposition) in any solvent tested and were not obtained in high purity. Ema complexes were taken up more readily into CHO cells than their ma analogues (Chapter 7), and this higher uptake corresponded with lower observed IC50 values (Chapter 6). Within the series of Ru-ma complexes, [Ru(ma)2(2MeIm)2]CF3S03 (10) exhibited the lowest I C 5 0 value (5 pM), while the corresponding Ema complex, [Ru(Ema)2(2MeIm)2]CF3S03 (13), exhibited the lowest I C 5 0 value of all complexes tested, 500 nM. Differences in IC50 values that resulted from changes in the imidazole ligand did not correlate with changes in Ru-uptake. [Ru(ma)2(EF5)2]CF3S03-EtOH (14) exhibited the highest Ru-uptake of all the complexes tested (Chapter 7), and was the only complex to exhibit DNA binding, which increased by ~ 50 % under hypoxic conditions. Despite its high uptake and DNA binding values, this complex did not exhibit any noticeable antiproliferatory activity up to a concentration of 500 pM (Chapter 6). 173 Of several new complexes synthesized using 4,4'-biimidazole ligands (Chapter 4), four Ru(II) and two Ru(IJJ) species exhibited low IC 5 0 values and warrant further investigation (Chapter 6). The biimidazole complexes vary in charge, oxidation state, and ancillary ligands, making it unclear what biological mechanism is operating, although more than one mechanism is likely involved judging by the similar activity of the different complexes. The findings suggest that known Ru complexes of other bidentate N-donor ligands such as 2,2'-bipyridine or 2,2'-biimidazole might also exhibit similar activity. A triazole derivative of EF5 (triF5) was developed and tested for hypoxic selectivity, but was less specific for hypoxia than EF5; triF5 may utilize different metabolic pathways than EF5 or may be less able to bind the fluorescent antibodies used for detection. Finally, some new Ru carboxylate complexes were synthesized and tested for biological activity (Chapter 5). In particular, a new bimetallic complex with picolinate (31) and a monomeric complex with Im-C0 2 H (33) were studied. Preliminary reactions with a 3-nitro-l,2,4-triazole-5-carboxylic acid were also performed. 8.2 New Complexes to Synthesize and Test Ru(UI)-ma complexes with nitroimidazole and nitrotriazole ligands containing an N(l)-H group were not soluble in any solvent except DMSO in which they decomposed. Methylation at the N(l) position of 2N02lm, 4N0 2Im or 3N02tri might provide ligands that form stable Ru-ma complexes with higher solubilities, allowing for easier characterization and biological testing. The high activity (low IC50 values) of several of the complexes containing biim and Me2biim suggests that they should be studied in greater detail. Similar complexes with other N-donor ligands are known, but have not been tested for antiproliferatory activity or other biological activity. In particular, [Ru(2,2'-biimidazole)3][PF6],1,2 [Ru(imidazole)6][CF3S03]23 and [Ru(2,2'-bipyridine)3][PF6]21,2 could be tested alongside [Ru(4,4'-biimidazole)3][CF3S03]2 (26) to determine the effect of (a) changing from a monodentate imidazole to a chelating biimidazole, (b) changing the connectivity of the 174 biimidazole, and (c) changing the ring structure from imidazole to pyridine. Results from such comparisons could then give insight into the effect of the imidazole group, and could lead to new complexes with maltolato and other ancillary ligands (e.g. [Ru(ma)2(2,2'-biimidazole)]CF3S03). As noted in Chapter 7, complexes of Ema tend to be taken up more readily into cells than the corresponding ma complexes, and so the synthesis of the Ema derivative of [Ru(ma)2(EF5)2]CF3S03-EtOH (14) might lead to improved uptake in cells and higher levels of DNA binding. New ma-complexes could be synthesized using some of the carboxylate ligands discussed (Chapter 5), for example, [Ru(ma) 2(HCANT) 2]CF 3S0 3 or Ru(ma)2(CANT) containing nitro-N-heterocycles. Similar-type complexes may also be possible with Hpic and Im-CC^H ligands. The incorporation of the nitro and carboxylate groups within the N-heterocyclic ligand may significantly affect the biological properties. Isopropylmaltol and n-butylmaltol are known4 and the corresponding tris-(maltolato)complexes with Ru could be prepared and tested. Pyridinones, in which the O in the ma ring has been substituted by a N-R group, might also be useful ligands to try. A member of our group has synthesized Ru(3-hydroxy-l,2-dimethyl-4-pyronate)3, although attempts to incorporate some ancillary imidazole ligands has yet to yield positive results.5 8.3 New Directions for Hypoxia Markers The high level of cellular uptake and selective DNA binding of [Ru(ma)2(EF5)2]CF3S03-EtOH (14) suggest that this complex is worth further investigation as a hypoxic marker. Previously in our group, Baird reported on the activity of [Ru(acac)2(EF5)2]CF3S03 that showed a degree of Ru uptake into SCCVII cells similar to that found for 14 in CHO cells, but exhibited no selectivity toward hypoxic cells in the DNA binding assay.6 This complex did, however, show a 75-fold increase in fluorescence in hypoxic cells over that observed in normal cells in the monoclonal antibody binding assay, and levels of fluorescence that were 4 times greater than those 175 for the free ligand alone (free EF5 itself also showed a 75-fold selectivity for hypoxic cells)6 From these results it can be inferred that [Ru(acac)2(EF5)2]CF3S03 is taken up by all cells equally, and once inside the cell the EF5 is released; the EF5 is then able to diffuse back out of the normal cells. This would account for the non-specific DNA binding and cellular uptake values for Ru, and the selective accumulation for EF5 in hypoxic cells. The complex may be taken up by cells more readily than the free ligand, thus resulting in the 4-fold increase in fluorescence. Further tests with [Ru(acac)2(EF5)2]CF3S03 were not pursued because of its poor water-solubility. [Ru(ma)2(EF5)2]CF3S03-EtOH (14) is more water-soluble than the acac analogue and, unlike [Ru(acac)2(EF5)2]CF3S03 j shows selectivity for DNA binding in hypoxic cells. Results from the antibody binding assay with 14 might show that it is a viable candidate for further testing as a hypoxia imaging agent. The ELK3-51 antibody used to quantify EF5 was supplied by BCCRC; however, to continue this work, a new source for this antibody is needed. 8.4 Future Biotests The higher cellular uptake of Ema complexes may mean that they can more readily diffuse across cell membranes. Lipophilicity studies would help determine if the complexes are entering the cell through passive diffusion or via an activated transport mechanism. The synthesis of complexes with isopropyl and n-butyl derivatives of ma would be useful in determining the effect of the ma-substituents on cellular uptake. The Ru-uptake and Ru-DNA binding assays should be repeated using MDA-MB-435S cells. The CHO cells were used to mimic a hypoxic environment, but as only 14 exhibited any hypoxia selectivity, the other complexes should be tested on the same cell line used for the M T T assay in order to make more accurate correlations between cell uptake and antiproliferatory activity. In addition, complexes could be tested against other cancer cell lines to see if the activity is specific for the MDA-MB-435S cell line or if the effect of the complexes is more general. In particular, tumour cell lines resistant to cisplatin should be used to determine if these Ru-ma complexes are active against such tumour models in vitro. 176 As only 14 interacted with DNA in vitro, other tests are needed to determine what type of interaction the complexes experience inside the cell and how the complexes enter the cell. The latter can be tested by performing the Ru-uptake assay (see Section 7.3.2) using PBS in place of growth medium. For some other Ru complexes,7 the uptake of Ru has been found higher in PBS because components in the growth medium compete with the complex for active transport sites. In PBS, this competition for transport proteins is removed and the concentration of complex inside the cell increases. No increase in Ru-uptake in PBS might suggest that the complex is entering the cell via passive diffusion. To determine the ultimate fate of the complexes inside the cell, it would be helpful to first run a gel on a protein extraction from control MDA-MB-435S cells, and then on a protein extraction of cells that have been incubated with [Ru(Ema)2(2MeIm)2]CF3S03 (13, lowest IC50 value, Section 6.4.2). Differences in the protein expression between the two extractions may indicate how and where inside the cell the complex is interacting. Proteins that are either over- or under-expressed in the cells incubated with 13 could then be isolated from the gel, and identified by comparison of their amino acid composition with on-line library data. Gradient centrifugation could then be used to separate and isolate various components of the cell in which the identified proteins are known to be present. Another experiment that could be performed to determine the cellular distribution of Ru is a micro-SRFXE study (Synchrotron Radiation-Induced X-ray Emission). This experiment can be used to detect the cellular distribution of elements, down to ppm levels.8 This technique has not been reported for detecting Ru, but has been employed to detect Pt, Zn, K, Cu, and Ca distributions inside cells.8'9 8.5 References (1) Goulle, V.; Thummel, R. Inorg. Chem. 1990, 29, 1767. (2) Rillema, D. P.; Sahai, R.; Matthews, P.; Edwards, A. K.; Shaver, R. J.; Morgan, L. Inorg. Chem. 1990, 29, 167. (3) Bastos, C. M. ; Gordon, K. A.; Ocain, T. D. Bioorg. Med. Chem. Lett. 1998, 8, 147. 177 (4) Thompson, K. H.; Liboiron, B. D.; Sun, Y.; Bellman, K. D. D.; Setyawati, I. A.; Patrick, B. O.; Karunaratne, V.; Rawji, G.; Wheeler, J.; Sutton, K.; Bhanot, S.; Cassidy, C.; Mcneill, J. H.; Yuen, V. G.; Orvig, C. J. Biol. Inorg. Chem. 2003, 8, 66. (5) Wu, A. 2003, Unpublished Results. (6) Baird, I. R. Fluorinated Nitroimidazoles and Their Complexes: Potential Hypoxia-Imaging Agents, Ph. D. Dissertation, University of British Columbia: Vancouver, 1999. (7) Frausin, F.; Cocchietto, M. ; Bergamo, A.; Scarcia, V.; Furlani, A.; Sava, G. Cancer Chemother. Pharmacol. 2002, 50, 405. (8) Hall, M . D.; Hambley, T. W. Coord. Chem. Rev. 2002, 232, 49. (9) Hall, M . D.; Dillon, C. T.; Zhang, M. ; Beale, P.; Cai, Z.; Lai, B.; Stampfl, A. P. J.; Hambley, T. W. J. Biol. Inorg. Chem. 2003. 178 mer-Ru(ma)3 (1) Appendix A l Experimental Details for the X-ray Crystallographic Study of m£?r-Ru(ma)3 (1) E m p i r i c a l f o r m u l a Formula w e i g h t Temperature Wavelength C r y s t a l system, space group U n i t c e l l d i m e n s i o n s Volume Z, C a l c u l a t e d d e n s i t y A b s o r p t i o n c o e f f i c i e n t F(000) T h e t a range f o r d a t a c o l l e c t i o n L i m i t i n g i n d i c e s R e f l e c t i o n s c o l l e c t e d / u n i q u e Completeness t o t h e t a = 27.93 Refinement method Data / r e s t r a i n t s / p a r a m e t e r s G o o d n e s s - o f - f i t on F / v2 F i n a l R i n d i c e s [ I > 2 s i g m a ( I ) ] R i n d i c e s ( a l l d a t a) L a r g e s t d i f f . peak and h o l e C i s H i s 0 9 Ru 476.37 173(2) K 0.71069 A t r i c l i n i c , Pbca a = 17.017(3) A a l p h a = 90 deg. b = 11.6860(8) A b e t a = 90 deg. c = 18.6414(14) A gamma = 90 deg. 3707.0(7) A"3 8, 1.707 Mg/m~3 0.895 mm A-l 1912 3.04 t o 27.93 deg. -15<=h<=19, -9<=k<=13, -22<=1<=19 3820 / 3820 [ R ( i n t ) = 0.0000] 85.9 % F u l l - m a t r i x l e a s t - s q u a r e s on F A2 3820 / 0 / 291 0.716 R l = 0.0380, wR2 = 0.0889 R l = 0.0879, wR2 = 0.1042 0.478 and -0.663 e.A A-3 179 Appendix Al Table A 1.1 Atomic Coordinates (x 1 0 4 ) and U(eq) mer-Ru(ma)3 ( 1 ) X y z U(eq) R u ( l ) 1313(1) 2225(1) 2180(1) 42 (1) 0 (1 ) 617 (2) 1430 (3) 2882 (2) 58 (1) 0 (2 ) 463 (2) 3462 (3) 2239(2) 51(1) 0 (3 ) - 1 2 0 5 ( 2 ) 2475(3) 3643(2) 71(1) 0 (4 ) 1986 (2) 2913 (3) 2940 (2) 64 (1) 0 (5 ) 2165(2) 977(2) 2197 (2) 48 (1) 0 (6 ) 3843 (2) 1901(4) 3619 (2) 77 (1) 0 (7 ) 1915 (2) 3098 (3) 1416(2) , 64(1) 0(8) 755(2) 1562(4) 1298(2) 67 (1) C ( l ) -7 (3) 2093(4) 3042 (2) 47 (1) C(2) - 5 6 9 ( 4 ) 1787 (4) 3524 (3) 60 (1) C(3) - 1 2 7 2 ( 4 ) 3484(5) 3290 (3) 75(2) C(4) -744 (3) 3840(4) 2827 (3) 61(1) C(5) - 7 9 ( 3 ) 3164(4) 2680(3) 46 (1) C(6) - 5 7 4 ( 4 ) 706(5) 3943(3) 91(2) C(7) 2621 (3) 2261(4) 3047(2) 51 (1) C(8) 3207(4) 2571(5) 3515(3) 69(2) C(9) 3905(4) 914(6) 3248(3) 73 (2) C (10) 3364(3) 549(4) 2781(3) 60 (1) C ( l l ) 2698(3) 1218(4) 2652 (3) 45(1) C(12) 3232(5) 3660(6) 3931 (4) 111 (3) C(13A) 971(8) 1925(11) 740(4) 49 (4) C(14A) 645 (7) 1575 (10) 93 (6) 81(5) 0(9A) 911(8) 2045(11) -548 (4) 140(6) C(15A) • 1502(8) 2866(10) - 5 4 0 ( 4 ) 101 (8) C(16A) 1828 (7) 3216 (10) 107(5) 76 (5) C(17A) 1562(7) 2746(11) 748(4) 52 (4) C(18A) 63(16) 817(17) - 1 3 ( 1 0 ) 208 (12) C(13B) 1730 (6) 3008(9) 861 (3) 45(4) C(14B) 2061(4) 3621(7) 298 (3) 84 (6) 0 (9B) 1774(5) 3483(10) - 3 9 5 ( 3 ) 108(4) C(15B) 1156 (7) 2734 (12) - 5 2 5 ( 4 ) 138(12) C(16B) 825 (7) 2121(11) 39(5) 109 (7) C(17B) 1112 (7) 2259 (10) 731(5) 70(6) C(18B) 2651(6) 4381 (10) 257(5) 100 (5) 180 Appendix Al OTer-Ru(ma)3 (1) Table A1.2 Bond Lengths (A) Ru(l)-0(4) 1 . 990(3) Ru(l)-0(1) 1 .993 (3) Ru(l)-0(7) 2 .030(3) Ru(l)-0(2) 2 .049 (3) Ru(1)-0(8) 2 .052 (3) Ru(l)-0(5) 2 .056 (3) 0( 1 ) - C ( l ) 1 .347(5) 0(2)-C(5) 1 .284(5) 0(3)-C(3) 1 .355(7) 0(3)-C (2) 1 .366 (6) 0(4)-C(7) 1 .337(6) 0 ( 5 ) - C ( l l ) 1 273(5) 0(6)-C(9) 1 349(7) 0(6)-C(8) 1 351(7) 0(7)-C(17A) 1 443(8) 0(8) -C(13A) 1 181(7) C(l)- C ( 2 ) 1 361(6) C(l)-C(5) 1 427(7) C(2)-C(6) 1 485(7) C(3)-C(4) 1 313(7) C(3)-H(3) 0 9500 C(4)-C(5) 1 408(7) C(4)-H(4) 0 9500 C (6) -H(6A) 0 9800 C(6)-H(6B) 0 9800 C(6)-H(6C) 0 9800 C(7)-C(8) 1 373 (7) C ( 7 ) - C ( l l ) 1 430(7) C (8)-C(12) 1 491(8) C (9) -C(10) 1 338(8) C(9)-H(9) 0 9500 C (10)-C(11) 1 397(7) C(10)-H(10) 0 9500 C(12)-H(12A) 0 9800 C(12)-H(12B) 0 9800 C(12)-H(12C) 0 9800 C(13A)-C(14A) 1 3900 C(13A)-C(17A) 1 3900 C(14A)-0(9A) 1' 3900 C(14A)-C(18A) 1 34(2) 0(9A)-C(15A) 1 3900 C(15A)-C(16A) 1 3900 C(15A)-H(15A) 0 9500 C(16A)-C(17A) 1 3900 C(16A)-H(16A) 0 9500 C(18A)-H(18A) 0 9800 C(18A)-H(18B) 0 9800 C(18A)-H(18C) 0 9800 C(13B)-C(14B) 1 3900 C(13B)-C(17B) 1 3900 C(14B)-C(18B) • 1 3433 C(14B)-0(9B) 1 3900 181 Appendix Al mer-Ru(ma)3 (1) Table A1.2 Bond Lengths (A) (contd.) 0 ( 9 B ) - C ( 1 5 B ) 1 3 9 0 0 C ( 1 5 B ) - C ( 1 6 B ) 1 3 9 0 0 C ( 1 5 B ) - H ( 1 5 B ) 0 9 5 0 0 C ( 1 6 B ) - C ( 1 7 B ) 1 3 9 0 0 C ( 1 6 B ) - H ( 1 6 B ) 0 9 5 0 0 C ( 1 8 B ) - H ( 1 8 D ) 0 9 8 0 0 C ( 1 8 B ) - H ( 1 8 E ) 0 9 8 0 0 C ( 1 8 B ) - H ( 1 8 F ) 0 9 8 0 0 182 Appendix Al m e r - R u ( m a ) 3 (1) Table A1.3 Bond Angles (°) 0 ( 4 ) - R u ( l ) - 0 ( 1 ) 93 . 61 (15 ) 0 ( 4 ) - R u ( l ) - 0 ( 7 ) 90 . 3 9 ( 1 6 ) 0 ( 1 ) - R u ( l ) - 0 ( 7 ) 173 . 8 4 ( 1 5 ) 0 ( 4 ) - R u ( l ) - 0 ( 2 ) 94 . 8 0 ( 1 4 ) 0 ( 1 ) - R u ( l ) - 0 ( 2 ) 82 .78 (13) 0 ( 7 ) - R u ( l ) - 0 ( 2 ) 92 . 2 2 ( 1 4 ) 0 ( 4 ) - R u ( l ) - 0 ( 8 ) 171 . 6 2 ( 1 6 ) 0 ( 1 ) - R u ( l ) - 0 ( 8 ) 94 . 3 0 ( 1 6 ) 0 ( 7 ) - R u ( l ) - 0 ( 8 ) 81 . 9 7 ( 1 6 ) 0 ( 2 ) - R u ( l ) - 0 ( 8 ) 88 . 9 6 ( 1 4 ) 0 ( 4 ) - R u ( l ) - 0 ( 5 ) 82 . 5 6 ( 1 3 ) 0 ( 1 ) - R u ( l ) - 0 ( 5 ) 94 . 4 8 ( 1 3 ) 0 ( 7 ) - R u ( l ) - 0 ( 5 ) 90 . 6 8 ( 1 3 ) 0 ( 2 ) - R u ( l ) - 0 ( 5 ) 176 . 0 9 ( 1 2 ) 0 ( 8 ) - R u ( l ) - 0 ( 5 ) 94 . 0 5 ( 1 4 ) C ( l ) - 0 ( 1 ) - R u ( l ) 110 . 2 ( 3 ) C ( 5 ) - 0 ( 2 ) - R u ( l ) 110 4 (3) C ( 3 ) - 0 ( 3 ) - C ( 2 ) 120 0(4) C ( 7 ) - 0 ( 4 ) - R u ( l ) 109 9(3) C ( l l ) - 0 ( 5 ) - R u ( l ) 110 8(3) C ( 9 ) - 0 ( 6 ) - C ( 8 ) 119 0(5) C ( 1 7 A ) - 0 ( 7 ) - R u ( l ) 104 6(4) C ( 1 3 A ) - 0 ( 8 ) - R u ( 1 ) 115 2 (6) 0 ( 1 ) - C ( l ) - C ( 2 ) 123 4 (5) 0 ( 1 ) - C ( l ) - C ( 5 ) 117 8(4) C (2) - C (1) -C (5) 118 8(5) C ( 1 ) - C ( 2 ) - 0 ( 3 ) 120 7(5) C (1) - C ( 2 ) - C ( 6 ) 125 1(5) 0 ( 3 ) - C ( 2 ) - C ( 6 ) 114 3(5) C ( 4 ) - C ( 3 ) - 0 ( 3 ) 122 6(6) C ( 4 ) - C ( 3 ) - H ( 3 ) 118 7 0 ( 3 ) - C ( 3 ) - H ( 3 ) 118 7 C ( 3 ) - C ( 4 ) - C ( 5 ) 119 9(5) C ( 3 ) - C ( 4 ) - H ( 4 ) 120 0 C ( 5 ) - C ( 4 ) - H ( 4 ) 120 0 0 ( 2 ) - C ( 5 ) - C ( 4 ) 123 3(5) 0 ( 2 ) - C ( 5 ) - C ( l ) 118 6(4) C ( 4 ) - C ( 5 ) - C ( l ) 118 1(5) 0 ( 4 ) - C ( 7 ) - C ( 8 ) 122 1(5) 0 ( 4 ) - C ( 7 ) - C ( l l ) 118 9(4) C ( 8 ) - C ( 7 ) - C ( l l ) 119 0(5) 0 ( 6 ) - C ( 8 ) - C ( 7 ) 121 4(5) 0 ( 6 ) - C ( 8 ) - C ( 1 2 ) 113 4 (5) C ( 7 ) - C ( 8 ) - C (12) 125 2 (6) C ( 1 0 ) - C ( 9 ) - 0 ( 6 ) •123 5(5) C ( 1 0 ) - C ( 9 ) - H ( 9 ) 118 2 0 ( 6 ) - C ( 9 ) - H ( 9 ) 118 2 C ( 9 ) - C ( 1 0 ) - C ( l l ) 119 5(5) C ( 9 ) - C ( 1 0 ) - H ( 1 0 ) 120 3 C ( l l ) - C ( 1 0 ) - H ( 1 0 ) 120 3 0 ( 5 ) - C ( l l ) - C ( 1 0 ) 124 7 (5) 0 ( 5 ) - C ( l l ) - C ( 7 ) 117 7 (4) C ( 1 0 ) - C ( 1 1 ) - C ( 7 ) 117 6(5) 183 Appendix Al mer-Ru(ma)3 (1) Table A1.3 Bond Angles (°) (contd.) 0 ( 8 ) - C ( 1 3 A ) - C ( 1 4 A ) 122 .3 0 ( 8 ) - C ( 1 3 A ) - C ( 1 7 A ) 117 6 c (14A) -C(13A) -C(17A) 120 0 0 (9A) - C ( 1 4 A ) - C(13A) 120 0 0 (9A) - C ( 1 4 A ) - C(18A) 112 0 c (13A) -C(14A) -C(18A) 128 0 c (14A) - 0 ( 9 A ) - C(15A) 120 0 c (16A) -C(15A) -0(9A) 120 0 c (16A) -C(15A) -H ( 1 5 A ) 120 0 0 (9A) - C ( 1 5 A ) - H (15A) 120 0 c 15A) -C(16A) -C(17A) 120 0 c 15A) -C(16A) -H ( 1 6 A ) 120 0 c 17A) -C(16A) -H ( 1 6 A ) 120 0 c 16A) -C(17A) -C(13A) 120 0 c 16A) -C(17A) -0(7) 119 5 c 13A) -C(17A) -0(7) 120 5 c 14A) -C(18A) -H ( 1 8 A ) 109 6 c 14A) -C(18A) - H ( 1 8 B ) 109 4 H 18A) -C(18A) - H ( 1 8 B ) 109 5 c 14A) -C(18A) -H ( 1 8 C ) 109 5 H 18A) -C(18A) -H ( 1 8 C ) 109 5 H 1 8 B ) -C(18A) -H ( 1 8 C ) 109 5 c 1 4 B ) - C ( 1 3 B ) - C ( 1 7 B ) 120 0 c 1 8 B ) - C ( 1 4 B ) - C ( 1 3 B ) 133 3 c 18B) -C(14B) -0(9B) 106 7 c 13B) -C(14B) -0(9B) 120 0 c 15B) - 0 ( 9 B ) - C(14B) 120 0 0 9B) - C ( 1 5 B ) - C(16B) 120 0 0 9B) - C ( 1 5 B ) - H (15B) 120 0 c 16B) -C(15B) -H ( 1 5 B ) 120 0 c 17B) -C(16B) -C(15B) 120 0 c 17B) -C(16B) -H ( 1 6 B ) 120 0 c 15B) -C(16B) -H ( 1 6 B ) 120 0 c 16B) -C(17B) -C(13B) 120 0 c 14B) -C(18B) -H (18D) 109 5 c 14B) -C(18B) -H ( 1 8 E ) 109 5 H 18D) -C(18B) -H ( 1 8 E ) 109 5 c 14B) -C(18B) - H ( 1 8 F ) 109 5 H 18D) -C(18B) - H ( 1 8 F ) 109 5 H 18E) -C(18B) -H ( 1 8 F ) 109 5 184 Appendix Al fran^-/^u(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) Appendix A2 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) E m p i r i c a l f o r m u l a F o r m u l a w e i g h t T e m p e r a t u r e W a v e l e n g t h C22 H24 C12 F3 N4 09 Ru S 749.48 173(2) K 0.71069 A C r y s t a l sy s t em, space g r o u p U n i t c e l l d i m e n s i o n s Volume Z, C a l c u l a t e d d e n s i t y A b s o r p t i o n c o e f f i c i e n t F (000) C r y s t a l s i z e T h e t a range f o r d a t a c o l l e c t i o n Index r a n g e s R e f l e c t i o n s c o l l e c t e d / u n i q u e C o m p l e t e n e s s to 2 t h e t a = 27 .09 Ref inement method D a t a / r e s t r a i n t s / p a r a m e t e r s G o o d n e s s - o f - f i t on F^2 F i n a l R i n d i c e s [ I>2s igma(I ) ] R i n d i c e s ( a l l d a t a ) L a r g e s t d i f f . peak and h o l e t r i c l i n i c , PI (#2) a = 9 .1784(8) A a l p h a = 64 .082(14) d e g . b = 12 .9918(3) A b e t a = 78 .771(19) d e g . c = 13 .6855(3) A gamma = 85 .95(2) deg . 1439.48(13) A~3 2, 1.729 Mg/m A 3 0.878 mrrr-1 754 0.20 x 0.10 x 0.02 mm 2.26 to 27 .09 d e g . - l l < = h < = l l , -16<=k<=15, -17<=1<=16 .5707 / 5707 [ R ( i n t ) = 0.0000] 90 .1% F u l l - m a t r i x l e a s t - s q u a r e s on F A 2 5707 / 69 / 421 0.981 R l = 0 .0595 , wR2 = 0.1345 R l = 0 .1147 , wR2 = 0.1563 1.049 and -0 .802 e . A ^ - 3 185 Appendix A2 rran5-/Ru(ma)2(lMeIm)2]CF3S03-CH2C]2 (8) Table A2.1 Atomic Coordinates (x 104) and U(eq) X y z U(eq) occ R u ( l ) 5000 0 5000 19 (1) Ru(2) 10000 0 10000 18 (1) S ( l ) 9026 (5) 4518 (3) 3007 (3) 26 (1) 0 75(1) F ( l ) 10758 (7) 4699 (5) 4233 (5) 57 (2) 0 75 (1) F(2) 11914 (7) 4882 (7) 2637 (5) 85 (3) 0 75(1) F(3) 11150 (15) 3212 (6) 3913 (6) 76 (3) 0 75(1) 0(1) 5196 (5) 1665 (4) 3926 (3) 24 (1) 0(2) 3911 (5) 669 (4) 6062 (3) 25 (1) 0(3) 4180 (5) 4123 (4) 4473 (4) 32 (1) 0(4) 8197 (5) -159 (4) 9440 (3) 23 (1) 0(5) 10494 (5) 1328 (4) 8437 (3) 22 (1) 0(6) 6831 (5) 1922 (4) 7050 (4) 33 (1) 0(7) 7969 (7) 4017 (6) 4012 (4) 41 (2) 0 75(1) 0(8) 8934 (8) 5736 (4) 2420 (5) 53 (3) 0 75(1) 0(9) 9201 (10) 3918 (6) 2330 (6) 66 (3) 0 75(1) N ( l ) 6937 (5) 94 (4) 5503 (4) 20 (1) N(2) 8785 (7) -383 (6) 642 6 (6) 42 (2) N(3) 8868 (6) . 1176 (4) 10499 4) 21 (1) N(4) 8113 (7) 2796 (5) 10536 (5) 31 (1) C ( l ) 3987 (7) 1754 (6) 5584 5) 26 (2) C (2) 3435 (7) 2474 (6) 6123 6) 26 (2) C (3) 3548 8) 3602 (6) 5551 6) 31 (2) C (4) 4711 7) 3486 (6) 3905 6) 27 (2) C(5) 4672 7) 2322 5) 4450 5) 22 (1) C (6) 5298 8) 4162 6) 2728 5) 35 2) C(7) 7919 8) 997 7) 5072 6) 36 (2) C (8) 9067 8) 709 7) 5658 7) 43 2) C(9) 7484 8) -740 7) 6324 6) 34 (2) C (10) 9727 10) -1048 9) 7208 7) 60 3) C (11) 9351 7) 1546 6) 7972 5) 23 1) C (12) 9212 8) 2532 6) 6994 5) 29 2) C (13) 7948 8) 2666 6) 6586 5) 34 2) C (14) 6880 8) 965 6) 7995 5) 28 2) C (15) 8097 8) 752 5) 8496 5) 24 2) C (16) 5539 8) 211 6) 8396 6) 36 2) C (17) 7808 7) 957 6) 11418 5) 25 1) C (18) 7342 8) 1938 6) 11456 6) 32 2) C (19) 9017 7) 2311 5) 9972 5) 24 1) C (20) 7956 11) 4024 6) 10208 7) 48 2) C (21) 10784 9) 4318 6) 3454 5) 53 3) 0 75(1) C (22) 6780 7) 7270 2) 538 15) 55 3) 0 85(1) C l ( l ) 4844 3) 7263 2) 926 3) 59 1) 0 85(1) CI (2) 7311 6) 6875 4) -551 3) 95 2) 0 85(1) S (IB) 9132 16) 4364 12) 2980 11) 26 1) 0 25(1) 0(7B) 8370 3) 4541 19) 3909 14) 41 2) 0 25(1) 0(8B) 9260 3) 5354 15) 1943 13) 53 3) 0 25(1) 0(9B) 8730 3) 3332 14) 2959 19) 66 3) 0 25(1) 186 Appendix A2 fran5-APvu(ma)2(lMeIm)2]CF3S03-CH2C]2 (8) Table A2.1 Atomic Coordinates (x 104) and U(eq) (contd.) C(21B) 10960(17) 4050(18) 3315(17) 53 (3) 0 25(1) F(1B) 11890 (2) 3805 (17) 2559(14) 57(2) 0 25 (1) F(2B) 11600(2) 4910(2) 3380(2) 85(3) 0 25(1) F(3B) 10920(5) 3130 (2) 4292(17) 76 (3) 0 25(1) C(22B) 6989 (19) 7130(13) 460(8) 55 (3) 0 15(1) C I ( I B ) 5460 (2) 7304(14) 1370(14) 59 (1) 0 15(1) CI(2B) 6440(4) 6790 (2) -510(2) 95(2) 0 15(1) 187 Appendix A2 rran5-/Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) Table A 2 . 2 Bond Lengths (A) R u ( l ) - 0 ( 1 ) 2 012(4) R u ( l ) - 0 ( 1 ) # 1 2 012(4) R u ( 1 ) - N ( 1 ) 2 057(5) R u ( l ) - N ( l ) # 1 2 057 (5) R u ( l ) - 0 ( 2 ) 2 . 072(4) R u ( l ) - 0 ( 2 ) # 1 2 072(4) Ru (2 ) -0 (4 )#2 2 012(4) R u ( 2 ) - 0 ( 4 ) 2 012(4) R u ( 2 ) - 0 ( 5 ) 2 065(4) Ru (2 ) -0 (5 )#2 2 065(4) Ru (2 ) -N(3)#2 2 072(5) R u ( 2 ) - N ( 3 ) 2 072(5) S ( l ) - 0 ( 7 ) 1 429(4) S ( 1 ) - 0 ( 9 ) 1 431(4) S ( 1 ) - 0 ( 8 ) 1 432(4) S ( 1 ) - C ( 2 1 ) 1 797(8) F ( l ) - C ( 2 1 ) 1 354(4) F ( 2 ) - C ( 2 1 ) 1 342 (5) F ( 3 ) - C ( 2 1 ) 1 341(5) 0 ( 1 ) - C ( 5 ) 1 348(8) 0 ( 2 ) - C ( l ) 1 269(8) 0 ( 3 ) - C ( 3 ) 1 349(8) 0 ( 3 ) - C ( 4 ) 1 373(8) 0 ( 4 ) - C ( 1 5 ) 1 332(7) 0 ( 5 ) - C ( l l ) 1 283 (7) 0 ( 6 ) - C ( 1 3 ) 1 319(8) 0 ( 6 ) - C ( 1 4 ) 1 352(7) N ( l ) - C ( 9 ) 1 329(8) N ( l ) - C (7) 1 369(8) N ( 2 ) - C ( 8 ) 1 351 (10) N ( 2 ) - C ( 9 ) 1 364(9) N ( 2 ) - C ( 1 0 ) 1 444(9) N ( 3 ) - C ( 1 9 ) 1 331(8) N ( 3 ) - C ( 1 7 ) 1 364(8) N ( 4 ) - C ( 1 9 ) 1 335(8) N ( 4 ) - C ( 1 8 ) 1 366(8) N ( 4 ) - C ( 2 0 ) 1 461(9) C ( l ) - C ( 5 ) 1 425(9) C ( l ) - C ( 2 ) 1 438(9) C ( 2 ) - C ( 3 ) 1 326(9) C ( 2 ) - H ( 2 ) • 0 9500 C ( 3 ) - H ( 3 ) 0 9500 C ( 4 ) - C ( 5 ) 1 363(9) C ( 4 ) - C ( 6 ) 1 .466 (9) C ( 6 ) - H ( 6 A ) 0 9800 C ( 6 ) - H ( 6 B ) 0 . 9800 C ( 6 ) - H ( 6 C ) 0 . 9800 C ( 7 ) - C ( 8 ) 1 .380(10) C ( 7 ) - H ( 7 ) 0 . 9500 C ( 8 ) - H ( 8 ) 0 . 9500 C ( 9 ) - H ( 9 ) 0 . 9500 C ( 1 0 ) - H ( 1 0 A ) 0 . 9800 C ( 1 0 ) - H ( 1 0 B ) 0 . 9800 188 Appendix A2 rran5-/Ru(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) Table A2.2 Bond Lengths (A ) (contd.) C ( 1 0 ) - H ( 1 0 C ) 0 9800 C ( l l ) - C ( 1 2 ) 1 412(8) C ( 1 1 ) - C ( 1 5 ) 1 450.(9) C ( 1 2 ) - C ( 1 3 ) 1 351(9) C ( 1 2 ) - H ( 1 2 ) 0 9500 ' C ( 1 3 ) - H ( 1 3 ) 0 9500 C ( 1 4 ) - C ( 1 5 ) 1 372(9) C ( 1 4 ) - C ( 1 6 ) 1 487(9) C ( 1 6 ) - H ( 1 6 A ) 0 9800 C ( 1 6 ) - H ( 1 6 B ) 0 9800 C ( 1 6 ) - H ( 1 6 C ) 0 9800 C ( 1 7 ) - C ( 1 8 ) 1 334(9) C ( 1 7 ) - H ( 1 7 ) 0 9500 C ( 1 8 ) - H ( 1 8 ) 0 9500 C ( 1 9 ) - H ( 1 9 ) 0 9500 C ( 2 0 ) - H ( 2 0 A ) 0 9800 C ( 2 0 ) - H ( 2 0 B ) 0 9800 C ( 2 0 ) - H ( 2 0 C ) 0 9800 C ( 2 2 ) - C 1 ( 2 ) 1 751(6) C ( 2 2 ) - C I ( 1 ) 1 752(6) C ( 2 2 ) - H ( 2 2 A ) 0 9900 C ( 2 2 ) - H ( 2 2 B ) 0 9900 S ( 1 B ) - 0 ( 9 B ) 1 429(5) S ( 1 B ) - 0 ( 7 B ) 1 430(5) S ( I B ) - 0 ( 8 B ) 1 430(5) S ( 1 B ) - C ( 2 1 B ) 1 797(9) C ( 2 1 B ) - F ( 3 B ) 1 343 (5) C ( 2 1 B ) - F ( 2 B ) 1 343(5) C ( 2 1 B ) - F ( 1 B ) 1 .343(5) C ( 2 2 B ) - C I ( 2 B ) 1 .751 (7) C ( 2 2 B ) - C I ( I B ) 1 .752 (7) C ( 2 2 B ) - H ( 2 2 C ) 0 . 9900 C ( 2 2 B ) - H ( 2 2 D ) 0 . 9900 189 Appendix A2 rran5-/Tlu(ma)2(lMeIm)2]CF3S03-CH2Cl2 (8) Table A2.3 Bond Angles (°) 0 1 ) - R u ( l ) -0(1)#1 180 0 0 1 ) - R u ( l ) - N ( l ) 90 94(19) 0 1)#1-Ru( 1 ) - N ( l ) 89 06(19) 0 1 ) - R u ( 1 ) - N ( l ) # 1 89 06(19) 0 1)#1-Ru( 1 ) - N ( l ) # 1 90 94(19) N 1 ) - R u ( l ) - N ( l ) # 1 180 0 0 1 ) - R u ( l ) -0 (2) 82 04(17) 0 1)#1-Ru( 1 ) -0 (2 ) 97 96(17) N 1 ) - R u ( l ) -0 (2) 88 15(19) N 1)#1-Ru( 1 ) -0 (2 ) 91 85(19) 0 1 ) - R u ( l ) -0(2)#1 97 96(17) 0 1)#1-Ru( 1 ) -0(2)#1 82 04(17) N 1 ) - R u ( l ) -0(2)#1 91 85(19) N 1)#1-Ru( 1 ) -0(2)#1 88 15(19) 0 2 ) - R u ( l ) -0(2)#1 180 0 0 4)#2-Ru( 2 ) - 0 ( 4 ) 179 999(1) 0 4)#2-Ru( 2 ) - 0 ( 5 ) 97 77(16) 0 4 ) - R u ( 2 ) -0 (5) 82 23(16) 0 4)#2-Ru( 2) -0(5)#2 82 23(16) 0 4 ) - R u ( 2 ) -0(5)#2 97 77(16) 0 5 ) - R u ( 2 ) -0(5)#2 180 000(1) 0 4)#2-Ru( 2)-N(3)#2 88 92(18) 0 4 ) - R u ( 2 ) -N(3)#2 91 08(18) 0 5 ) - R u ( 2 ) -N(3)#2 92 77(19) 0 5)#2-Ru( 2)-N(3)#2 87 23(19) 0 4)#2-Ru( 2 ) -N(3 ) 91 08(18) 0 4 ) - R u ( 2 ) -N(3) 88 92(18) 0 5 ) - R u ( 2 ) -N(3) 87 23(19) 0 5)#2-Ru( 2 ) -N(3 ) 92 77(19) N 3)#2-Ru( 2 ) -N(3 ) 180 000(1) 0 7) - S ( l ) - 0(9) 114 9(3) 0 7 ) - S ( 1 ) - 0(8) 114 5(3) 0 9 ) - S ( 1 ) - 0(8) 114 7(3) 0 7 ) - S ( 1 ) - C(21) 104 1(4) 0 9 ) - S ( 1 ) - C (21) 102 9(4) 0 8 ) - S ( 1 ) - C (21) 103 6(4) c 5 ) - 0 ( 1 ) - R u ( l ) 110 2 (4) c 1 ) - 0 ( 2 ) - R u ( l ) 110 1(4) c 3)-0(3} - C(4) 120 3(5) c 15) -0 (4 ) -Ru(2) 109 9(4) c 11) -0 (5 ) -Ru(2) 109 7 (4) c 13) -0 (6 ) -C(14) 121 1(5) c 9 ) - N ( l ) - C(7) 106 4(6) c 9 ) - N ( l ) - R u ( l ) 125 6(5) c 7 ) - N ( l ) - R u ( l ) 128 0(5) c ( 8 ) - N ( 2 ) - C(9) 108 5 C.6) c (8)-N(2)- C(10) 125 0(7) c (9) -N(2)- C (10) 126 5(8) c (19) -N(3) -C(17) 105 8(5) c (19) -N(3) -Ru(2) 126 7 (4) c (17) -N(3) -Ru(2) 127 6(4) c (19) -N(4) -C(18) 107 6(6) 190 Appendix A2 rran^-/T<u(ma)2(lMeIm) 2]CF3S03-CH2Cl2 (8) Table A2.3 Bond Angles (°) (contd.) c 19) - N ( 4 ) - C ( 2 0 ) 125 9 ( 6 ) c 18) - N ( 4 ) - C ( 2 0 ) 126 5 ( 6 ) 0 2) - C ( 1 ) - C ( 5 ) 1 1 9 7 ( 6 ) 0 2) - C ( l ) - C ( 2 ) 123 9 ( 6 ) c 5) - C ( 1 ) - C ( 2 ) 116 4 ( 6 ) c 3) - C ( 2 ) - C ( l ) 1 1 9 5 ( 6 ) c 3) - C ( 2 ) - H ( 2 ) 1 2 0 2 c 1) -C ( 2 ) - H ( 2 ) 1 2 0 2 c 2) -C ( 3 ) - 0 ( 3 ) 123 1 ( 7 ) c 2) -C ( 3 ) - H ( 3 ) 118 4 0 3) - C ( 3 ) - H ( 3 ) 118 4 c 5) - C ( 4 ) - 0 ( 3 ) 1 1 9 7 ( 6 ) c 5) - C ( 4 ) - C ( 6 ) 1 2 5 7 ( 6 ) 0 3) - C ( 4 ) - C ( 6 ) 1 1 4 6 ( 6 ) 0 1) - C ( 5 ) - C ( 4 ) 1 2 1 6 ( 6 ) 0 1) - C ( 5 ) - C ( 1 ) 117 5 ( 6 ) c 4) - C (5) - C ( 1 ) 120 8 ( 6 ) N 1) - C ( 7 ) - C ( 8 ) 109 4 ( 7 ) N 1) - C ( 7 ) - H ( 7 ) 125 3 c 8) - C ( 7 ) - H ( 7 ) 125 3 N 2) - C (8) - C ( 7 ) 105 8 ( 6 ) N 2) - C ( 8 ) - H ( 8 ) 127 1 C 7) - C ( 8 ) - H ( 8 ) 127 1 N 1) - C ( 9 ) - N ( 2 ) 1 0 9 8 (7) N 1) - C ( 9 ) - H ( 9 ) 1 2 5 1 N 2) - C ( 9 ) - H ( 9 ) 1 2 5 1 0 5) - C ( l l ) - C ( 1 2 ) 124 2 ( 6 ) 0 5) - C ( 1 1 ) - C ( 1 5 ) 118 4 ( 6 ) C 12) - C ( 1 1 ) - C ( 1 5 ) 117 4 ( 6 ) C 13) - C ( 1 2 ) - C ( l l ) 118 6 ( 6 ) C 13) - C ( 1 2 ) - H ( 1 2 ) 120 7 C 11) - C ( 1 2 ) - H ( 1 2 ) 1 2 0 7 0 6) - C ( 1 3 ) - C ( 1 2 ) 123 5 ( 6 ) 0 6) - C ( 1 3 ) - H ( 1 3 ) 118 2 C 12) - C ( 1 3 ) - H ( 1 3 ) 118 2 0 6) - C ( 1 4 ) - C ( 1 5 ) 120 4 ( 6 ) 0 6) - C ( 1 4 ) - C ( 1 6 ) 114 1 ( 6 ) C 15) - C ( 1 4 ) - C ( 1 6 ) 1 2 5 5 ( 6 ) 0 4) - C ( 1 5 ) - C ( 1 4 ) 123 3 ( 6 ) 0 4) - C ( 1 5 ) - C ( l l ) 117 8 ( 6 ) C 14) - C ( 1 5 ) - C ( 1 1 ) 118 9 ( 6 ) C 18) - C ( 1 7 ) - N ( 3 ) 109 9 ( 6 ) c 18) - C ( 1 7 ) - H ( 1 7 ) 125 0 N (3) - C ( 1 7 ) - H ( 1 7 ) 1 2 5 0 C 17) - C ( 1 8 ) - N ( 4 ) 106 5 ( 6 ) C (17) - C ( 1 8 ) - H ( 1 8 ) 126 8 N (4) - C ( 1 8 ) - H ( 1 8 ) 126 8 N ( 3 ) - C ( 1 9 ) - N ( 4 ) 1 1 0 2 ( 6 ) N (3) - C ( 1 9 ) - H ( 1 9 ) 124 9 N ( 4 ) - C ( 1 9 ) - H ( 1 9 ) 124 9 F (3) - C ( 2 1 ) - F ( 2 ) 107 4 ( 5 ) F (3) - C ( 2 1 ) - F ( l ) 106 1 ( 5 ) F (2) - C ( 2 1 ) - F ( l ) 106 0 ( 5 ) 191 Appendix A2 Table A2.3 Bond Angles (°) (contd.) rra«5-/T<u(ma)2(lMeIm)2]CF3S03-CH2Cl2(8) F ( 3 ) - C ( 2 1 ) - S ( l ) 112 2(7) F ( 2 ) - C ( 2 1 ) - S ( l ) 113 4(6) F ( l ) - C ( 2 1 ) - S ( 1 ) 111 3(6) C 1 ( 2 ) - C ( 2 2 ) - C 1 ( 1 ) 111 8(6) C I ( 2 ) - C ( 2 2 ) - H ( 2 2 A ) 109 3 C 1 ( 1 ) - C ( 2 2 ) - H ( 2 2 A ) 109 3 CI ( 2 ) - C ( 2 2 ) - H ( 2 2 B ) 109 3 C 1 ( 1 ) - C ( 2 2 ) - H ( 2 2 B ) 109 3 H ( 2 2 A ) - C ( 2 2 ) - H ( 2 2 B ) 107 9 0 ( 9 B ) - S ( I B ) - 0 ( 7 B ) 114 9(5) 0 ( 9 B ) - S ( I B ) - 0 ( 8 B ) 114 8 (4) 0 ( 7 B ) - S ( I B ) - 0 ( 8 B ) 114 8(5) 0 ( 9 B ) - S ( I B ) - C ( 2 1 B ) 102 2 (15) 0 ( 7 B ) - S ( I B ) - C ( 2 1 B ) 100 5 (13) 0 ( 8 B ) - S ( I B ) - C ( 2 1 B ) 107 3 (14) F ( 3 B ) - C ( 2 1 B ) - F ( 2 B ) 106 8(6) F ( 3 B ) - C ( 2 1 B ) - F ( 1 B ) 106 9(6) F ( 2 B ) - C ( 2 1 B ) - F ( 1 B ) 106 8(6) F ( 3 B ) - C ( 2 1 B ) - S ( 1 B ) 111 (2) F ( 2 B ) - C ( 2 1 B ) - S ( I B ) 113 8(16) F ( 1 B ) - C ( 2 1 B ) - S ( 1 B ) 111 4(16) C I ( 2 B ) - C ( 2 2 B ) - C I ( I B ) 111 8(7) C I ( 2 B ) - C ( 2 2 B ) - H ( 2 2 C ) 109 2 C I ( I B ) - C ( 2 2 B ) - H ( 2 2 C ) 109 2 C I ( 2 B ) - C ( 2 2 B ) - H ( 2 2 D ) 109 2 C I ( I B ) - C ( 2 2 B ) - H ( 2 2 D ) 109 2 H ( 2 2 C ) - C ( 2 2 B ) - H ( 2 2 D ) 107 9 192 Appendix A2 rrans-[Ru(ma)2(2MeIm)2]CF3S03 (10) Appendix A 3 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(ma)2(2MeIm)2]CF3S03 (10) E m p i r i c a l f o r m u l a F o r m u l a w e i g h t T e m p e r a t u r e W a v e l e n g t h C r y s t a l s y s t e m , space group U n i t c e l l d i m e n s i o n s Volume Z, C a l c u l a t e d d e n s i t y A b s o r p t i o n c o e f f i c i e n t F (000) C r y s t a l s i z e T h e t a range f o r d a t a c o l l e c t i o n L i m i t i n g i n d i c e s R e f l e c t i o n s c o l l e c t e d / u n i q u e A b s o r p t i o n c o r r e c t i o n Max. and m i n . t r a n s m i s s i o n Re f inement method Data / r e s t r a i n t s / p a r a m e t e r s G o o d n e s s - o f - f i t on F A 2 F i n a l R i n d i c e s [ I>2s igma(I ) ] R i n d i c e s ( a l l data ) L a r g e s t d i f f . peak and h o l e C21 H22 F3 N4 09 S Ru 664.55 173.2 K 0.7107 A T r i c l i n i c , P -1 a = 8 .9512(6) A a l p h a = 94 .908(5) deg . b = 9 .2949(7) A b e t a = 93 .453(5) deg . c = 15 .817(1) A gamma = 93 .059(4) deg . 1306 .5(2) A"3 2, 1.689 Mg/m A 3 0.758 m m A - l 670.00 0.20 x 0.15 x 0.15 mm 2.20 to 27 .86 d e g . -9<=h<=ll , - l l < = k < = l l , -18<=1<=19 11677 / 5269 [ R ( i n t ) = 0.04146] S e m i - e m p i r i c a l f rom e q u i v a l e n t s 1.000 and 0.8142 F u l l - m a t r i x l e a s t - s q u a r e s on F"2 5269 / 0 / 363 0 . 951 R l = 0 .032 , wR2 = 0.040 R l = 0 .0603 , wR2 = 0.0896 0.78 and - 0 . 8 3 193 Appendix A3 trans- [Ru(ma)2(2MeIm)2] CF 3 S 0 3 (10) Table A3.1 Atomic Coordinates (x 104) and U(eq) X y z U(eq) R u ( l ) 0 0 10000 18(1) Ru(2) 0 0 5000 22 (1) S ( l ) 4642(1) -5654(1) 7488 1) 32 (1) F ( l ) 4116(3) -5039 (3) 9080 1) 49 (1) F (2) 2821(3) -6863 (3) 8472 2) 60(1) F(3) 2271 (3) -4732(3) 8209 2) 79(1) 0(1) 1420 (2) 76 (2) 9069 1) 22 (1) 0(2) 1689 (2) -1099(2) 10555 1) 22(1) 0(3) 4980 (2) -1557 (2) 8936 1) 28(1) 0(4) -1508(2) -1640 (3) 5101 1) 30(1) 0(5) 1351(2) -1242 (2) 5705 1) 28 (1) 0(6) -968(4) -4971 (3) 6049 2) 66(1) 0(7) 3753(3) -6381 (3) 6790 2) 47 (1) 0(8) 5118(4) -4179(3) 7411 (2) 61(1) 0(9) 5824 (3) -6447(4) 7847 (2) 62 (1) N ( l ) -897 (2) -1979 (3) 9428 2) 21(1) N(2) -2018 (3) -3709 (3) 8576 (2) 30(1) N(3) 651 (3) -1038 (3) 3892 (2) 24(1) N(4) 1931(3) -1996 (3) 2883 (2) ' 33 (1) C ( l ) 2623 (3) -659 (3) 9255 2) 21(1) C (2) 2755 (3) -1239(3) 10056 2) 20(1) C(3) 4085 (3) -1929(3) 10275 (2) 25 (1) C (4) 5134 (3) -2042(4) 9713 (2) 30(1) C (5) 3723 (3) -870(3) 8700 (2) 25 (1) C (6) 3724(4) -422(4) 7826 (2) 37(1) C (7) -986 (3) -3226(4) 9844 (2) 26(1) C (8) -1699(4) -4304(4) 9325 (2) 32 (1) C(9) -1537 (3) -2311(3) 8650 (2) 24(1) C (10) -1682(4) -1349(4) 7949 (2) 37 (1) C (11) -863(4) -2664(4) 5515 (2) 28(1) C (12) 656(4) -2424(4) 5825 (2) 30(1) C (13) 1310(5) -3566(4) 6245 (3) 48 (1) C (14) 514(5) -4754(5) 6321 (3) 68(2) C (15) -1656(4) -3926(4) 5643 (3) 44(1) C (16) -3251(4) -4292(5) 5380 (3) 54(1) C (17) -333(4) -1540(4) 3211 (2) 29 (1) C (18) 452(4) -2128(4) 2584 (2) 38(1) C (19) 2025(4) -1337(4) 3671 (2) 29(1) C (20) 3465(4) -992(5) 4185 (3) 48(1) C (21) 3396(4) -5551(4) 8355 (2) 35(1) H(3) 4230 -2314 10830 30 H(4) 6059 ' -2500 9872 36 H(6A) 2692 -297 7614 44 H(6B) 4161 -1166 7457 44 H(6C) 4318 495 7829 44 H(7) -591 -3313 10428 31 H(8) -1937 -5299 9454 38 H(10C) -682 -1034 7792 45 H(10A) -2222 -502 8138 45 194 Appendix A3 rranHRu(ma)2(2MeIm)2]CF3S03 (10) Table A3.1 Atomic Coordinates (x 104) and U(eq) (contd.) H (10B) -2238 -1878 7455 45 H(13) 2355 -3452 6475 57 H(14) 1002 -5536 6588 81 H (16C) -3781 -4572 5870 65 H (16A) -3330 -5098 4934 65 H(16B) -3700 -3449 5160 65 H(17) -1424 -1478 3189 35 H(18) 49 -2563 2025 46 H (20B) 4271 -1478 3904 58 H (20C) 3381 -1326 4752 58 H (20A) 3693 56 4237 58 H(21) -2460(40) -4110(40) 8220(20) 31(9) H(22) 2630(50) -2340(50) 2630(30) 60(10) 195 Appendix A3 ?ra/25-[Ru(ma)2(2MeIm)2]CF3S03 (10) Table A3.2 Bond Lengths ( A ) R u ( l ) - 0 ( 1 ) 2 006(2) R u ( l ) - 0 ( 1 ) # 1 2 006(2) R u ( l ) - 0 ( 2 ) 2 063 (2) R u ( l ) - 0 ( 2 ) # 1 2 063 (2) R u ( 1 ) - N ( 1 ) 2 078 (2) R u ( l ) - N ( l ) # 1 2 078 (2) R u ( 2 ) - 0 ( 4 ) 2 005 (2) Ru(2 ) -0 (4 )#2 2 005(2) R u ( 2 ) - 0 ( 5 ) 2 061(2) Ru(2 ) -0 (5 )#2 2 061(2) R u ( 2 ) - N ( 3 ) 2 058(2) Ru(2) -N(3)#2 2 058(2) S ( l ) - 0 ( 7 ) 1 421 (2) S ( l ) - 0 ( 8 ) 1 431 (3) S ( l ) - 0 ( 9 ) 1 438 (3) S ( l ) - C ( 2 1 ) 1 818(4) F ( 1 ) - C ( 2 1 ) 1 320(4) F ( 2 ) - C ( 2 1 ) 1 330(4) F(3) -C(21 ) 1 316(4) 0 ( 1 ) - C ( l ) 1 338 (3) 0 ( 2 ) - C ( 2 ) 1 279 (3) 0 ( 3 ) - C ( 4 ) 1 346(4) 0 ( 3 ) - C ( 5 ) 1 371(4) 0 ( 4 ) - C ( 1 1 ) 1 337(4) 0 ( 5 ) - C ( 1 2 ) 1 268(4) 0 ( 6 ) - C ( 1 4 ) 1 369(5) 0 ( 6 ) - C ( 1 5 ) 1 364(5) N ( l ) - C ( 7 ) 1 383(4) N ( l ) - C ( 9 ) 1 329(4) N ( 2 ) - C ( 8 ) 1 371(5) N ( 2 ) - C ( 9 ) 1 340(4) N ( 2 ) - H ( 2 1 ) 0 72 (3) N ( 3 ) - C ( 1 7 ) 1 382(4) N ( 3 ) - C ( 1 9 ) 1 335(4) N ( 4 ) - C ( 1 8 ) 1 374(4) N ( 4 ) - C ( 1 9 ) 1 337(4) N ( 4 ) - H ( 2 2 ) 0 82 (4) C ( 1 ) - C ( 2 ) 1 419(4) C ( 1 ) - C ( 5 ) 1 369(4) C ( 2 ) - C ( 3 ) 1 421(4) C(3) - C ( 4 ) 1 334(4) C ( 3 ) - H ( 3 ) 0 98 C ( 4 ) - H ( 4 ) 0 98 C ( 5 ) - C ( 6 ) 1 477(5) C (6) -H(6A) 0 98 C ( 6 ) - H ( 6 B ) 0 98 C ( 6 ) - H ( 6 C ) 0 98 C ( 7 ) - C (8) 1 346(5) C ( 7 ) - H ( 7 ) 0 98 C ( 8 ) - H ( 8 ) 0 98 C (9) -C(10) 1 487(5) C ( 1 0 ) - H ( 1 0 C ) 0 98 196 Appendix A3 Table A3.2 Bond Lengths (A) (contd.) frarcHRu(ma)2(2MeIm)2]CF3S03 (10) C (10) -H(10A) 0 98 C(10) -H(10B) 0 98 C ( l l ) -C(12 ) 1 417(4) C (11) -C(15 ) 1 375(5) C(12) -C(13 ) 1 433(5) C(13) -C(14) 1 300(6) C(13) -H(13) 0 98 C(14) -H(14) 0 98 C(15) -C(16) 1 476(5) C(16) -H(16C) 0 98 C(16) -H(16A) 0 98 C(16) -H(16B) 0 98 C(17) -C(18 ) 1 344(5) C(17) -H(17) 0 98 C (18) -H(18) 0 98 C (19) -C(20 ) 1 486(5) C (20) -H(20B) 0 98 C(20) -H(20C) 0 98 C (20) -H(20A) 0 98 197 Appendix A3 rranHRu(ma)2(2MeIm)2]CF3S03 (10) Table A 3 . 3 Bond Angles (°) O l - R u l - 0 2 82 .32(7) O l - R u l - N l 88 .88(9) 0 1 - R u l - 0 1 _ a 180 .00 0 1 - R u l - 0 2 _ a 97 .68(7) 0 1 - R u l - N l _ a 91 .12(9) 0 2 - R u l - N l 87 .87(8) 0 1 _ a - R u l - 0 2 97 .68(7) 0 2 - R u l - 0 2 _ a 180.00 0 2 - R u l - N l _ a 92 .13(8) 0 1 _ a - R u l - N l 91 .12(9) 0 2 _ a - R u l - N l 92 .13(8) N l - R u l - N l _ a 180.00 0 1 _ a - R u l - 0 2 _ a 82 .32(7) 0 1 _ a - R u l - N l _ a 88 .88(9) 0 2 _ a - R u l - N l _ a 87 .87(8) 04-Ru2-N3 89 .74(9) 0 4 - R u 2 - 0 4 _ b 180.00 0 4 - R u 2 - 0 5 _ b 97 .81(8) 04 - Ru2-N3_b 90 .26(9) 0 5 - Ru2-N3 90 .41(9) 04_b-Ru2-05 97 .81(8) 0 5 - R u 2 - 0 5 _ b 180.00 0 5 - R u 2 - N 3 _ b 89 .59(9 ) 04_b-Ru2-N3 90 .26(9) 05_b-Ru2-N3 89 .59(9) N3-Ru2-N3_b 180.00 0 4 _ b - R u 2 - 0 5 _ b 82 .19(8) 0 4 _ b - R u 2 - N 3 _ b 89 .74(9) C 2 - C 1 - C 5 119 .2(3) 0 2 - C 2 - C 1 118 .9(2) 0 2 - C 2 - C 3 122 .8(3) C 1 - C 2 - C 3 118 .3(3) C 9 - C 1 0 - H 1 0 A 109.47 H10B-C10-H10C 109.48 H10A-C10-H10C 109.50 0 4 - C 1 1 - C 1 2 118 .8 (3 ) C 1 2 - C 1 1 - C 1 5 120 .4(3) 04 - C11-C15 120 .8(3) C 1 1 - C 1 2 - C 1 3 116 .8(3) 0 5 - C12-C13 124 .4(3) 0 5 - C12-C11 118 .8(3) N 1 - C 9 - C 1 0 127 .5(3) N1-C9-N2 108 .6(3) C 2 - C 3 - H 3 120.58 C 4 - C 3 - H 3 120.55 C 1 1 - C 1 5 - C 1 6 125 .9(4) 06 - C15-C11 119 .9(3) N3-C17-C18 108 .8(3) N 4 - C 1 8 - C 1 7 • 106 .3 (3 ) N 4 - C 1 9 - C 2 0 123 .5(3) N 3 - C 1 9 - C 2 0 127 .4(3) N3-C19-N4 109 .1(3) C12-C13-H13 120.07 C14-C13-H13 120.07 C13-C14-H14 118.20 06 -C14-H14 118.27 C 1 5 - C 1 6 - H 1 6 A 109.42 C 1 5 - C 1 6 - H 1 6 C 109.52 198 Appendix A3 rran5-[Ru(ma)2(2MeIrn)2]CF3S03 (10) Table A3.3 Bond Angles (°) (contd.) C19-C20-H20B 109 45 C 1 9 - C 2 0 - H 2 0 C 109 51 H20A-C20-H20B 109 44 H20A-C20-H20C 109 47 S 1 - C 2 1 - F 1 111 6(3) S 1 - C 2 1 - F 2 110 3(3) S 1 - C 2 1 - F 3 112 3 (2) F 1 - C 2 1 - F 2 106 7(3) F 1 - C 2 1 - F 3 108 1(3) F 2 - C 2 1 - F 3 107 6(3) 05_b-Ru2-N3_b 90 41(9) 04 -Ru2-05 82 19 (8) 0 9 - S 1 - C 2 1 100 75 (.17) 0 8 - S 1 - 0 9 112 0(2) 0 8 - S 1 - C 2 1 104 11(18) 0 7 - S 1 - C 2 1 104 53(17) 0 7 - S 1 - 0 8 116 70(18) 0 7 - S 1 - 0 9 116 25(19) R u l - O l - C l 109 94(16) R u l - 0 2 - C 2 110 10(16) C 4 - 0 3 - C 5 120 2 (2) R u 2 - 0 4 - C l l 109 63(19) Ru2-05-C12 110 43(19) C 1 4 - 0 6 - C 1 5 119 4(3) C 7 - N 1 - C 9 106 8(3) R u l - N l - C 9 129 7 (2) R u l - N l - C 7 123 5(2) C 8 - N 2 - C 9 109 8(3) C8-N2-H21 122(3) C9-N2-H21 128(3) Ru2-N3-C19 129 3 (2) C17-N3-C19 106 9(3) Ru2-N3-C17 123 7 (2) C18-N4-C19 108 8(3) C18-N4-H22 125 (3) C19-N4-H22 126 (3) 0 1 - C 1 - C 5 122 2(3) 0 1 - C 1 - C 2 118 5(2) H10A-C10-H10B 109 48 C 9 - C 1 0 - H 1 0 C 109 44 C 9 - C 1 0 - H 1 0 B 109 45 C 2 - C 3 - C 4 118 9(3) 0 3 - C 4 - C 3 123 0(3) 0 3 - C 5 - C 1 120 3 (3) C 1 - C 5 - C 6 126 4(3) 0 3 - C 5 - C 6 113 3 (2) N 1 - C 7 - C 8 109 6(3) N 2 - C 8 - C 7 105 .1(3) N 2 - C 9 - C 1 0 123 .8(3) 0 5 - C 1 2 - C 1 1 118 .8(3) C 1 2 - C 1 3 - C 1 4 119 9(4) 0 6 - C 1 4 - C 1 3 123 .5(4) 0 6 - C 1 5 - C 1 6 114 .2(3) 0 3 - C 4 - H 4 118 .47 C 3 - C 4 - H 4 118 . 54 H 6 A - C 6 - H 6 C 109 .45 C 5 - C 6 - H 6 A 109 .38 H6B-C6-H6C 109 . 54 . H 6 A - C 6 - H 6 B 109 .55 199 Appendix A3 rranHRu(ma)2(2MeIm)2]CF3S03 (10) Table A3.3 Bond Angles (°) (contd.) C 5 - C 6 - H 6 C 109 45 C 5 - C 6 - H 6 B 109 46 N1-C7-H7 125 22 C 8 - C 7 - H 7 125 16 C 7 - C 8 - H 8 127 47 N2-C8-H8 127 40 H16A-C16-H16B 109 44 C15-C16-H16B 109 47 H16B-C16-H16C 109 45 H16A-C16-H16C 109 53 N3-C17-H17 125 57 C18-C17-H17 125 62 N4-C18-H18 126 87 C17-C18-H18 126 80 H20B-C20-H20C 109 50 C 1 9 - C 2 0 - H 2 0 A 109 46 200 Appendix A 3 rrans-[Ru(Ema)2(metro)2]CF3S03 (11) Appendix A4 Experimental Details for the X-ray Crystallographic Study of trans-[Ru(Ema)2(metro)2]CF3S03 (11) E m p i r i c a l f o r m u l a F o r m u l a w e i g h t T e m p e r a t u r e W a v e l e n g t h C r y s t a l sys tem, space g r o u p U n i t c e l l d i m e n s i o n s Volume z , C a l c u l a t e d d e n s i t y A b s o r p t i o n c o e f f i c i e n t F(000) C r y s t a l s i z e T h e t a range f o r d a t a c o l l e c t i o n Index r a n g e s R e f l e c t i o n s c o l l e c t e d / u n i q u e C o m p l e t e n e s s t o 2 t h e t a = 27 .87 A b s o r p t i o n c o r r e c t i o n Max. and m i n . t r a n s m i s s i o n Ref inement method Data / r e s t r a i n t s / p a r a m e t e r s G o o d n e s s - o f - f i t on F A 2 F i n a l R i n d i c e s [ I>2s igma(I ) ] R i n d i c e s ( a l l da ta ) L a r g e s t d i f f . peak and h o l e C28 H34 F3 N6 016 Ru S 900.74 173(2) K 0.71069 A T r i c l i n i c , P - l a = 11.0867(12) A a l p h a = 105 .636(4) deg . b = 12 . 5113(14) A b e t a = 97 .737(3) deg . c = 13.8897(15) A gamma = 99 .838(4) d e g . 1794.6(3) A"3 2, 1.667 M g / m ^ 0.591 m m A - l 918 0.25 x 0.20 x 0.15 mm 2 .66 t o 27 .87 d e g . -12<=h<=14, -16<=k<=14, -17<=1<=15 7380 / 7380 [ R ( i n t ) = 0.0000] 86.2% S e m i - e m p i r i c a l f rom e q u i v a l e n t s 1.0000 and 0.7654 F u l l - m a t r i x l e a s t - s q u a r e s on F A 2 7380 / 37 / 545 0 . 969 R l = 0 .0501 , wR2 = 0.1190 R l = 0 .0855, wR2 = 0.1322 1.155 and -0 .963 e . A A - 3 201 Appendix A4 rranHRu(Ema)2(metro)2]CF3S03 (H) Table A4.1 Atomic Coordinates (x 104) and U(eq) X y z U(eq) R u ( l ) 0 0 0 16(1) Ru(2) 0 0 5000 21(1) 0(1) 1772 3) 234 2) 816 2) 24(1) 0(2) 953 3) 628 2) -931 2) 25(1) 0(3) 4252 3) 1826 3) -497 3) 37(1) 0(4) 562 3) 3497 3) 3807 2) 40 (1) 0(5) -365 3) 4673 2) 3271 2) 38(1) 0(6) -3001 4) 3428 3) 369 3) 41 (1) 0(7) 933 3) 79 2) 3827 2) 30(1) 0(8) 1358 3) 1372 2) 5750 2) 27 (1) 0(9) 3256 3) 3162 3) 4631 3) 44 (1) 0(10) -2814 3) 1258 3) 1833 2) 44 (1) 0(11) -3177 4) 2832 3) 2737 3) 44 (1) 0(12) -3952 5) 2798 4) 5640 4) 80(2) 0(13) -4835 4) 696 3) 3094 3) 58 (1) N ( l ) -114 3) 1627 2) 788 2) 19 (1) N(2) -467 3) 3364 2) 1241 3) 22(1) N(3) 58 3) 3800 3) 3119 3) 27(1) N(4) -1020 3) 1080 3) 4540 3) 23 (1) N(5) -1928 3) 2512 3) 4506 3) 21(1) N(6) -2741 4) 1977 3) 2648 3) 30(1) C ( l ) 2590 (4) 690 (3) 392 (3) 25(1) C(2) 2179 (4) 950 (3) -511 3) 26(1) C(3) 3022 (5) 1557 (4) -908 (4) 32(1) C(4) 4659 (5) 1515 (4) 318 (4) 41 (1) C (5) 3907 (4) 972 (4) 782 (4) 33(1) C(6) 2705 (6) 2029 (5) -1747 (4) 50(1) C(7) -462 (4) 2458 (3) 440 (3) 23(1) C(8) -82 (4) 3085 (3) 2110 (3) 22 (1) C(9) 127 (4) 2028 (3) 1825 (3) 20(1) C(10) -742 (5) 2395 (4) -641 (3) 36(1) C ( l l ) -832 (4) 4415 (3) 1126 (4) 30(1) C (12) -2205 (5) 4364 (4) 1170 (4) 36(1) C(13) 1727 (4) 1028 (4) 4063 (3) 29(1) C(14) 1924 (4) 1756 (3) . 5078 (3) 26(1) C(15) 2674 (4) 2820 (4) 5333 (4) 36(1) C(16) 3118 (5) 2447 (5) ' 3687 (5) 49 (1) C(17) 2411 (5) 1396 (4) 3368 (4) 39(1) C(18) 2930 (6) 3694 (4) 6324 (4) 51(2) C (19) -1275 (4) 2035 (3) 5114 (3) 22(1) C (20) -2081 (4) 1807 (3) 3520 (3) 23 (1) C(21) -1509 (4) 942 (3) 3550 (3) 23 (1) C(22) -883 (5) 2515 (4) 6237 (3) 34 (1) C(23) -2319 (4) 3601 (3) 4878 (3) 26(1) C (24) -3676 (5) 3411 (4) 4957 (4) 43 (1) C(25) -4232 (6) -506 (6) 4029 (6) 68 (2) C (26) -5054 (5) -227 (4) 3241 (4) 42 (1) C(27) -6167 (6) -1106 (5) 2626 (6) 72(2) S(1B) -2670 (4) 5263 (5) 8491 (3) 24 (1) 202 Appendix A4 Table A4.1 rranHRu(Ema)2(metro)2]CF3S03 (H) Atomic Coordinates (x 104) and U(eq) (contd.) (x 104) 0(14B) -2128 (18) 6309 (8) 9217 (8) 73 (6) 0(15B) -2277 (15) 4335 (8) 8714 (9) 32 (4) 0(16B) -2676 (18) 5259 (11) 7482 (6) 53 (5) C(28B) -4271 (9) 5087 (11) 8590 (10) 41 (5) F(1B) -4889 (18) 5736 (16) 8253 (17) 77 (6) F(2B) -4365 (16) 5306 (13) 9533 (10) 59 (4) F(3B) -4900 (15) 4068 (11) 8129 (12) 70 (5) S(1A) -2885 (2) 4690 (2) 8165 (3) 72 (1) 0(14A) -2102 (7) 4904 (11) 9111 (5) 228 (9) 0(15A) -2266 (5) 4828 (5) 7379 (4) 73 (2) 0(16A) -3889 (7) 3760 (5) 7905 (7) 246 (9) C(28A) -3649 (5) 5850 (5) 8411 (5) 64 (3) F(1A) -2847 (6) 6796 (4) 8595 (7) 138 (4) F(2A) -4467 (7) 5789 (7) 7634 (8) 140 (4) F(3A) -4296 (7) 5878 (7) 9119 (7) 142 (4) 0(6B) -2280 (3) 5560 (2) 1270 (2) 66 (9) 203 Appendix A4 rranHRu(Ema)2(metro)2]CF3S03 (H) Table A4.2 Bond Lengths (A ) R u ( l ) - 0 ( 2 ) # 1 1. 998 3) R u ( l ) - 0 ( 2 ) 1. 998 3) R u ( l ) - 0 ( 1 ) 2 . 051 3) R u ( l ) - 0 ( 1 ) # 1 2 . 051 3) R u ( l ) - N ( l ) # 1 2 . 080 3) R u ( 1 ) - N ( 1 ) 2 . 080 3) R u(2 ) -0 (8 )#2 2 . 007 3) R u ( 2 ) - 0 ( 8 ) 2 . 007 3) Ru(2 ) -0 (7 )#2 2 . 060 3) R u ( 2 ) - 0 ( 7 ) 2 . 060 3) R u ( 2 ) - N ( 4 ) 2 . 075 3) Ru(2) -N(4)#2 2 075 3) 0 ( 1 ) - C ( 1 ) 1 280 5) 0 ( 2 ) - C ( 2 ) 1 350 5) 0 ( 3 ) - C ( 4 ) 1 340 6) 0 ( 3 ) - C (3) 1 353 6) 0 ( 4 ) - N ( 3 ) 1 221 5) 0 ( 5 ) - N ( 3 ) 1 240 4) 0 ( 6 ) - C ( 1 2 ) 1 438 6) 0 ( 7 ) - C ( 1 3 ) 1 283 5) 0 ( 8 ) - C ( 1 4 ) 1 339 5) 0 ( 9 ) - C ( 1 6 ) 1 345 7) 0 ( 9 ) - C ( 1 5 ) 1 360 6) 0 ( 1 0 ) - N ( 6 ) 1 225 5) 0 ( 1 1 ) - N ( 6 ) 1 231 4) 0 ( 1 2 ) - C ( 2 4 ) 1 404 7) 0 ( 1 3 ) - C ( 2 6 ) 1 216 6) N ( l ) - C ( 7 ) 1 350 5) N ( l ) - C ( 9 ) 1 363 (5) N ( 2 ) - C ( 7 ) 1 358 (5) N ( 2 ) - C ( 8 ) 1 378 (5) N ( 2 ) - C ( 1 1 ) 1 481 (5) N ( 3 ) - C ( 8 ) 1 417 (5) N ( 4 ) - C ( 1 9 ) 1 345 (5) N ( 4 ) - C ( 2 1 ) 1 362 (5) N ( 5 ) - C ( 1 9 ) 1 360 (5) N ( 5 ) - C ( 2 0) 1 384 (5) N ( 5 ) - C ( 2 3 ) 1 481 (5) N ( 6 ) - C ( 2 0 ) 1 414 (5) C ( l ) - C ( 2 ) 1 416 (6) C ( 1 ) - C ( 5 ) 1 434 (6) C ( 2 ) - C ( 3 ) 1 369 (6) C ( 3 ) - C ( 6 ) 1 471 (7) C (4) - C ( 5 ) 1 327 (7) C ( 7 ) - C ( 1 0 ) 1 .469 (6) C ( 8 ) - C ( 9 ) 1 .344 (5) C ( ' l l ) -C(12) 1 .523 (6) C ( 1 3 ) - C ( 1 4 ) 1 .422 (6) C (13) - C (17) 1 .426 (7) C ( 1 4 ) - C ( 1 5 ) 1 .370 (6) C ( 1 5 ) - C ( 1 8 ) 1 .466 (7) C ( 1 6 ) - C ( 1 7 ) 1 .334 (8) C ( 1 9 ) - C ( 2 2 ) 1 .485 (6) 204 Appendix A4 Table A4.2 Bond Lengths (A ) (contd.) rranHRu(Ema)2(metro)2]CF3S03 (H) C (20 ) -C(21) 1 352(5) C ( 2 3 ) - C ( 2 4 ) 1 505(6) C ( 2 5 ) - C ( 2 6 ) 1 483(8) C ( 2 6 ) - C ( 2 7 ) 1 486(8) S ( 1 B ) - 0 ( 1 4 B ) 1 396(5) S ( I B ) - 0 ( 1 6 B ) 1 399(5) S ( 1 B ) - 0 ( 1 5 B ) 1 402(5) S ( I B ) - C ( 2 8 B ) 1 779(8) C ( 2 8 B ) - F ( 2 B ) 1 286(6) C ( 2 8 B ) - F ( 1 B ) 1 287(6) C ( 2 8 B ) - F ( 3 B ) 1 287(6) S ( 1 A ) - 0 ( 1 6 A ) 1 395(5) S ( 1 A ) - 0 ( 1 5 A ) 1 399(4) S ( 1 A ) - 0 ( 1 4 A ) 1 406(5) S ( 1 A ) - C ( 2 8 A ) 1 781(7) C ( 2 8 A ) - F ( 2 A ) 1 289(5) C ( 2 8 A ) - F ( 3 A ) 1 290(5) C ( 2 8 A ) - F ( 1 A ) 1 293(5) 205 Appendix A4 ?rattHRu(Ema)2(metro)2]CF3S03 (H) Table A4.3 Bond Angles (°) 0(2)#1 -Ru( D - 0 ( 2 ) 180 . 0 0 (2 )#1 -Ru( 1 ) -0 (1 ) 97 . 86 (12) 0 ( 2 ) - R u ( l ) -0(1) 82 . 14(12) 0 (2 )#1 -Ru( 1) -0 (1)#1 82 . 14(12) 0 ( 2 ) - R u ( l ) -0(1)#1 97 . 86(12) 0 ( 1 ) - R u ( l ) -0(1)#1 180. 0 0 (2 )#1 -Ru( 1 ) - N ( l ) # 1 90 . 01(12) 0 ( 2 ) - R u ( l ) - N ( l ) # 1 89 . 99(12) 0 ( 1 ) - R u ( l ) - N ( l ) # 1 89 . 12(11) 0 (1 )#1 -Ru( 1 ) - N ( l ) # 1 90 . 88(11) 0 (2 )#1 -Ru( 1 ) - N ( l ) 89 99 (12) 0 ( 2 ) - R u ( l ) - N ( l ) 90 01(12) 0 ( 1 ) - R u ( l ) - N ( l ) 90 88(11) 0 (1 )#1 -Ru( 1 ) - N ( l ) 89 12(11) N ( l ) # 1 - R u ( 1 ) - N ( l ) 180 0 0 (8 )#2 -Ru( 2 ) - 0 ( 8 ) 180 0 0 (8 )#2 -Ru( 2)-0(7)#2 81 48(12) 0 ( 8 ) - R u ( 2 ) -0(7)#2 98 52(12) 0 (8 )#2 -Ru( 2 ) - 0 ( 7 ) 98 52(12) 0 ( 8 ) - R u ( 2 ) -0(7) 81 48(12) 0 (7 )#2 -Ru( 2 ) - 0 ( 7 ) 180 0 0 (8 )#2 -Ru( 2) -N(4 ) 91 92(12) 0 ( 8 ) - R u ( 2 ) -N(4) 88 07(12) 0 (7 )#2 -Ru( 2) -N(4 ) 93 18(13) 0 ( 7 ) - R u ( 2 ) -N(4) 86 82(13) 0 (8 )#2 -Ru( 2) -N(4)#2 88 07(12) 0 ( 8 ) - R u ( 2 ) -N(4)#2 91 93(12) 0 (7 )#2 -Ru( 2)-N(4)#2 86 82(13) 0 ( 7 ) - R u ( 2 ) -N(4)#2 93 18(13) N ( 4 ) - R u ( 2 ) -N(4)#2 179 999(1) C ( l ) - 0 ( 1 ) - R u ( l ) 110 7(3) C ( 2 ) - 0 ( 2 ) - R u ( l ) 109 7(3) C ( 4 ) - 0 ( 3 ) - C(3) 120 1(4) C ( 1 3 ) - 0 ( 7 ) -Ru (2) 110 6 (3) C ( 1 4 ) - 0 ( 8 ) -Ru (2) 109 3(2) C ( 1 6 ) - 0 ( 9 ) -C(15) 120 1(4) C ( 7 ) - N ( l ) - C,(9) . 107 0(3) C ( 7 ) - N ( l ) - R u ( l ) 130 .1(3) C ( 9 ) - N ( l ) - R u ( l ) 122 8(3) C ( 7 ) - N ( 2 ) - C(8) 106 .6(3) C ( 7 ) - N ( 2 ) - C ( l l ) 123 .4(4) C ( 8 ) - N ( 2 ) - C (11) 130 .0(3) 0 ( 4 ) - N ( 3 ) - 0(5) 123 .0(3) 0 ( 4 ) - N ( 3 ) - C(8) 117 .5(3) 0 ( 5 ) - N ( 3 ) - C(8) 119 .4(4) C ( 1 9 ) - N ( 4 ) -C(21) 107 .9(3) C ( 1 9 ) - N ( 4 ) -Ru (2) 130 .6(3) C ( 2 1 ) - N ( 4 ) -Ru (2) 122 .0(3) C ( 1 9 ) - N ( 5 ) -C(23) 124 • 2(3) C ( 2 0 ) - N ( 5 ) - C (23) 129 .5(3) 0 ( 1 0 ) - N ( 6 ) -0(11) .123 • 8(4) 0 ( 1 0 ) - N ( 6 ) -C(20) 116 .6(3) 206 Appendix A4 Table A4.3 Bond Angles (°) (contd.) franHRu(Ema)2(metro)2]CF3S03 (11) 0 ( 1 1 ) - N ( 6 ) - C ( 2 0 ) 119 . 6(4) 0 ( 1 ) - C ( 1 ) - C ( 2 ) 1.18 . 6(4) 0 ( 1 ) - C ( l ) - C ( 5 ) 124 . 2 (4) C ( 2 ) - C ( 1 ) - C ( 5 ) 117 . 3 (4) 0 ( 2 ) - C ( 2 ) - C ( 3 ) 122 . 1(4) 0 ( 2 ) - C ( 2 ) - C ( l ) 118 . 5(4) C ( 3 ) - C ( 2 ) - C ( l ) 119 . 3(4) 0 ( 3 ) - C ( 3 ) - C ( 2 ) 120 . 8(4) 0 ( 3 ) - C ( 3 ) - C ( 6 ) 113 . 9(4) C ( 2 ) - C ( 3 ) - C ( 6 ) 125 . 2(5) C ( 5 ) - C ( 4 ) - 0 ( 3 ) 123 . 3(5) C ( 4 ) - C ( 5 ) - C ( l ) 118 . 8(5) N ( l ) - C ( 7 ) - N ( 2 ) 109 . 5(4) N ( l ) - C ( 7 ) - C ( 1 0 ) 124 6(3) N ( 2 ) - C ( 7 ) - C ( 1 0 ) 125 9(4) C ( 9 ) - C ( 8 ) - N ( 2 ) 108 0(3) C(9) - C ( 8 ) - N ( 3 ) 126 8 (4) N ( 2 ) - C ( 8 ) - N ( 3 ) 125 2 (3) C(8) - C ( 9 ) - N ( l ) 108 9 (4) N ( 2 ) - C ( 1 1 ) - C ( 1 2 ) 111 0(3) 0 ( 6 ) - C ( 1 2 ) - C ( 1 1 ) 111 9(4) 0 ( 7 ) - C ( 1 3 ) - C ( 1 4 ) 117 6(4) 0 ( 7 ) - C ( 1 3 ) - C ( 1 7 ) 124 0(4) C ( 1 4 ) - C ( 1 3 ) - C ( 1 7 ) 118 4(4) 0 ( 8 ) - C ( 1 4 ) - C ( 1 5 ) 122 4(4) 0 ( 8 ) - C ( 1 4 ) - C ( 1 3 ) 118 3(4) C ( 1 5 ) - C ( 1 4 ) - C ( 1 3 ) 119 3(4) 0 ( 9 ) - C ( 1 5 ) - C ( 1 4 ) 120 3 (4) 0 ( 9 ) - C ( 1 5 ) - C ( 1 8 ) 113 2 (4) C ( 1 4 ) - C ( 1 5 ) - C ( 1 8 ) 126 5(5) C ( 1 7 ) - C ( 1 6 ) - 0 ( 9 ) 124 0(5) C ( 1 6 ) - C ( 1 7 ) - C (13). 117 7(5) N ( 4 ) - C ( 1 9 ) - N ( 5 ) 109 5(3) N ( 4 ) - C ( 1 9 ) - C ( 2 2 ) 125 5(4) N ( 5 ) - C ( 1 9 ) - C ( 2 2 ) 125 0(3) C ( 2 1 ) - C ( 2 0 ) - N ( 5 ) 108 2 (3) C ( 2 1 ) - C ( 2 0 ) - N ( 6 ) 127 0 (4) N ( 5 ) - C ( 2 0 ) - N ( 6 ) 124 8(3) C ( 2 0 ) - C ( 2 1 ) - N ( 4 ) 108 .2(3) N ( 5 ) - C ( 2 3 ) - C ( 2 4 ) 111 .6(3) 0 ( 1 2 ) - C ( 2 4 ) - C ( 2 3 ) 112 .7 (4) 0 ( 1 3 ) - C ( 2 6 ) - C ( 2 5 ) 121 .5(5) 0 ( 1 3 ) - C ( 2 6 ) - C ( 2 7 ) 120 • 1(5) C ( 2 5 ) - C ( 2 6 ) - C ( 2 7 ) 118 .4(5) 0(14B) -S (IB.) -0(16B) 114 .6 (5) 0 ( 1 4 B ) - S ( I B ) - 0 ( 1 5 B ) • 114 .2 (5) 0 ( 1 6 B ) - S ( 1 B ) - 0 ( 1 5 B ) 113 .6(5) 0 ( 1 4 B ) - S ( I B ) - C ( 2 8 B ) 102 .9(10) 0 ( 1 6 B ) - S ( I B ) - C ( 2 8 B ) 104 .0(10) 0 ( 1 5 B ) - S ( 1 B ) - C ( 2 8 B ) 105 .9(9) F ( 2 B ) - C ( 2 8 B ) - F ( 1 B ) 104 .4(15) F ( 2 B ) - C ( 2 8 B ) - F ( 3 B ) 107 .5(14) F ( 1 B ) - C ( 2 8 B ) - F ( 3 B ) 105 .2(14) 207 Appendix A4 rran5-[Ru(Ema)2(metro)2]CF3S03 (11) Table A4.3 Bond Angles (°) (contd.) F ( 2 B ) - C(28B) -S(1B) 109 6 (11) F ( 1 B ) - C(28B) -S(1B) 116 9 (13) F ( 3 B ) - C(28B) -S(1B) 112 7 (11) 0(16A) -S (1A) -0(15A) 116 0 (3) 0<16A) -S (1A) -0(14A) 115 8 (3) 0(15A) -S (1A) -0(14A) 114 8 (3) 0(16A) -S (1A) -C(28A) 101 7 (4) 0(15A) - S ( 1 A ) -C(28A) 103 7 (4) 0(14A) - S ( 1 A ) -C(28A) 101 6 (6) F ( 2 A ) - C(28A) - F ( 3 A ) 102 9 (8) F ( 2 A ) - C(28A) - F ( 1 A ) 106 3 (7) F ( 3 A ) - C(28A) - F ( 1 A ) 111 6 (7) F ( 2 A ) - C(28A) -S(1A) 111 8 (5) F ( 3 A ) - C(28A) -S(1A) 114 0 (5) F ( 1 A ) - C(28A) -S(1A) 109 9 (5) 208 Appendix A4 l,r-Bis(triphenylmethyl)-4,4'-biimidazole Appendix A5 Experimental Details for the X-ray Crystallographic Study of l,l'-Bis(triphenylmethyl)-4,4'-biimidazole Empirical formula Formula weight Crystal system Space group Unit cell dimensions Volume Z Density (calculated) F(000) Diffractometer Mo K a Radiation Linear absorptions coefficient Data collection range Index ranges Temperature Programs Crystal dimension Transmission coefficient T M a x / T M i n Reflections collected Independent reflections Refinement (on F 2 ) Reflections observed No. of parameters refined Largest peak/hole GOF=S=[Z[w(F02 - F c 2)] 21 (n-p)]1 /2 R=ZTF 0 CT - aFCT | SCTFOO-wR 2=[I[w(F 0 2 - F c 2) 2] | S[w(F 0 2)] 2]' C 2 2 . 5 H 1 8 N 2 CI M = 351.84 g mol"1 monoclinic C 2/c (No. 15) a = 14.7825(4) A a = 90.000 ° b = 13:3779(2) A (3 = 93.2370(10)° c = 18.3481(4) A Y = 90.000° 3622.71(14) A 3 Z = 8 p= 1.290 gem"3 1472 Four circle diffractometer Siemens Smart X = 0.71073 A \x (Mo Ka) 0.218 mm"1 4.10< 29<45 -21 < h < 20, -17 < k < 19, -24 < 1 < 26 293 K SHELX86, SHELX93 0.60x0.53x0.14 mm 3 0.9766 / 0.5171 13577 4158 [R ( i n t ) = 0.0850] Full matrix least squares n = 4146 p = 299 0.467 and -0.824 e.A"3 1.028 0.0719 [F0>4a(F0)], 0.1437 (all data) 0.1714 [Fo>4a(F0)], 0.2071 (all data) 209 Appendix A 5 1,1 '-Bis(triphenyImethyl)-4,4'-biimidazole Table A5.1 Atomic Coordinates (x 104) and U(eq) Atom x/a y/b z/c U, C(l) 0.3200(5) 0.0119(4) 0.6032(2) 0.0053(1 N(l) 0.3134(1) 0.4566(2) 0.0296(1) 0.0036(1 N(2) 0.4522(2) 0.4127(2) 0.0678(1) 0.0045(1 C(l) 0.4571(2) 0.4828(2) 0.0125(1) 0.0038(1 C(2) 0.3722(2) 0.5090(2) -0.0114(2) 0.0042(1 C(3) 0.3663(2) 0.4004(2) 0.0760(2) 0.0041(1 C(4) . 0.2137(2) 0.4739(2) 0.0265(1) 0.0033(1 C(5) 0.1668(2) 0.3915(2) 0.0695(2) 0.0036(1 C(6) 0.1064(2) 0.3239(2) 0.0363(2) 0.0042(1 C(7) 0.0607(3) 0.2554(3) 0.0765(2) 0.0058(1 C(8) 0.0752(3) 0.2518(3) 0.1509(2) 0.0065(1 C(9) 0.1352(3) 0.3168(3) 0.1850(2) 0.0060(1 C(10) 0.1794(2) 0.3874(3) 0.1451(2) 0.0047(1 C(ll) 0.1822(2) 0.4706(2) -0.0552(1) 0.0035(1 C(12) 0.1197(2) 0.5361(2) - 0.0864(2) 0.0040(1 C(13) 0.0898(2) 0.5258(3) -0.1594(2) 0.0052(1 C(14) 0.1222(2) 0.4507(3) -0.2013(2) 0.0056(1 C(15) 0.1858(2) 0.3863(3) -0.1711(2) 0.0056(1 C(16) 0.2156(2) 0.3952(3) -0.0987(2) 0.0048(1 C(17) 0.1941(2) 0.5749(2) 0.0632(1) 0.0037(1 C(18) 0.1054(2) 0.5981(3) 0.0780(2) 0.0046(1 C(19) 0.0839(3) 0.6869(3) 0.1110(2) 0.0058(1 C(20) 0.1509(3) 0.7535(3) 0.1302(2) 0.0066(1 C(21) 0.2388(3) 0.7314(3) 0.1181(2) 0.0070(1 C(22) 0.2608(2) 0.6429(3) 0.0846(2) 0.0054(1 C(23) 0.0 -0.0597(5) 0.2500 0.0080(2 Cl(l) 0.0981(1) 0.0092(1) 0.2445(1) 0.0126(1 210 Appendix A 5 1 J'-Bis(triphenylmethyl)-4,4'-biirnidazole Table A5.2 Bond Lengths (A ) bond length bond length N(l)-C(3) 1.351(3) C(ll)-C(12) 1.374(4) N(l)-C(2) 1.374(4) C(ll)-C(16) 1.394(4) N(l)-C(4) 1.491(3) C(12)-C(13) 1.393(4) N(2)-C(3) 1.297(4) C(13)-C(14) 1.367(5) N(2)-C(l) 1.387(4) C(14)-C(15) 1.369(5) C(l)-C(2) 1.353(4) C(15)-C(16) 1.381(4) C(l)-C(l)#l 1.448(5) C(17)-C(22) 1.382(4) C(4)-C(17) 1.544(4) C(17)-C(18) 1.390(4) C(4)-C(5) 1.542(4) C(18)-C(19) 1.378(5) C(4)-C(ll) 1.545(4) C(19)-C(20) 1.364(6) C(5)-C(6) 1.387(4) C(20)-C(21) 1.363(6) C(5)-C(10) 1.391(4) C(21)-C(22) 1.381(5) C(6)-C(7) 1.378(5) C(7)-C(8) 1.371(5) C(23)-C1(1)#2 1.726(4) C(8)-C(9) 1.369(6) C(23)-C1(1) 1.726(4) C(9)-C(10) 1.381(5) 211 Appendix A5 l,r-Bis(triphenyImethyl)-4,4'-biimidazole Table A5.3 Bond Angles (°) (contd.) Atom angle Atom angle C(3)-N(l)-C(2) 105.6(2) C(12)-C(ll)-C(16) 118.3(3) C(3)-N(l)-C(4) 130.3(2) C(12)-C(ll)-C(4) 123.1(3) C(2)-N(l)-C(4) 123.7(2) C(16)-C(ll)-C(4) 118.6(2) C(3)-N(2)-C(l) 105.2(2) C(ll)-C(12)-C(13) 120.5(3) C(2)-C(l)-N(2) 109.1(3) C(14)-C(13)-C(12) 120.7(3) C(2)-C(l)-C(l)#l 128.8(4) C(13)-C(14)-C(15) 119.4(3) N(2)-C(l)-C(l)#l 122.1(3) C(14)-C(15)-C(16) 120.5(3) C(l)-C(2)-N(l) 107.0(3) C(15)-C(16)-C(ll) 120.7(3) N(2)-C(3)-N(l) 113.1(3) C(22)-C(17)-C(18) 117.5(3) N(l)-C(4)-C(17) 109.1(2) C(22)-C(17)-C(4) 123.5(3) N(l)-C(4)-C(5) 110.0(2) C(18)-C(17)-C(4) 118.9(3) C(17)-C(4)-C(5) 107.6(2) C(19)-C(18)-C(17) 121.5(3) N(l)-C(4)-C(ll) 106.0(2) C(20)-C(19)-C(18) 119.7(4) C(17)-C(4)-C(ll) 113.2(2) C(21)-C(20)-C(19) 120.1(4) C(5)-C(4)-C(ll) 110.9(2) C(20)-C(21)-C(22) 120.6(4) C(6)-C(5)-C(10) 117.3(3) C(21)-C(22)-C(17) 120.6(4) C(6)-C(5)-C(4) 122.6(2) C(10)-C(5)-C(4) 120.0(3) C1(1)#2-C(23)-C1(1) 115.4(4) C(7)-C(6)-C(5) 121.5(3) C(8)-C(7)-C(6) 120.1(4) C(9)-C(8)-C(7) 119.6(4) C(8)-C(9)-C(10) 120.4(4) C(9)-C(10)-C(5) 121.0(4) 212 Appendix A5 

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