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Ruthenium nitroimidazole complexes as radiosensitizers Chan, Peter Ka-Lin 1988

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RUTHENIUM NITROIMIDAZOLE COMPLEXES AS RADIOSENSITIZERS  by PETER K A - L I N CHAN B.Sc. (Co-op), University of Victoria, 1982 M.Sc, University of British Columbia, 1984  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1988  ©Copyright Peter Ka-Lin Chan, 1988  In  presenting  degree  at  this  the  thesis in  University of  partial  fulfilment  of  British Columbia, I agree  freely available for reference and study. I further copying  of  department  this or  publication of  thesis for by  his  or  her  DE-6G/81)  that the  for  an advanced  Library shall make  it  It  is  granted  by the  understood  that  head of copying  my or  this thesis for financial gain shall not be allowed without my written  CHEmtSTR.V  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  representatives.  requirements  agree that permission for extensive  scholarly purposes may be  permission.  Department of  the  ii ABSTRACT  Local control of tumours by radiotherapy may fail due to the presence of regions of hypoxic cells.  Radiosensitizers, such as nitroimidazoles, enhance killing of  the resistant cells by ionizing radiation.  However, dose limiting side-effects have  prevented the attainment of maximum sensitization.  The successful chemotherapeutic  drug,  and  cz's-diamminedichloroplatinum(II)  (m-DDP),  analogues  show  moderate  radiosensitizing effects, possibly because of binding to DNA. A rationale is then to use the DNA binding property of a metal to carry a sensitizer to the target of radiation damage, DNA, thereby improving the radiosensitizing effect while reducing the toxic side-effects of nitroimidazoles. The complex c/^-RuC^Cdmso)^ was used as a precursor for synthesis of Ru(II)nitroimidazole complexes because of its anti-tumour and DNA binding activities.  A  series of Ru(II) complexes of formulation RuC^dmso^Ljj, where dmso is S-bonded dimethyl sulphoxide, L = a nitroimidazole, and n=l or 2, has been synthesized and characterized, and their toxicities and radiosensitizing abilities examined in vitro. When L = 2-nitroimidazole or a substituted-2-nitroimidazole, n = 2, but the nitroimidazole ligands dissociate in aqueous medium.  With L = the 5-nitroimidazole,  metronidazole, n=2, the sensitizing ability of the six-coordinate cis complex was disappointing with sensitizer enhancement  ratio (SER) of 1.2 in hypoxic Chinese  hamster ovary (CHO) cells. A  series of  4-nitroimidazoles  nitroimidazole (4-N02~Im),  ligands was then studied.  With L = 4-  1-(1' -aziridinyl-2' -propanol)-2-methyl-4-nitroimidazole  (RSU-1170), 2-(l,2-dimethyl-4-nitroimidazolyl)-2-aminoethanol  (RSU-3083), and 1-  methyl-4-nitro-5-phenoxyimidazole (RSU-3100), n=2 and the six coordinate complexes appear to be of all cis geometry.  The NMe-4-N0 -Im ligand (n=l) chelates through  the imidazole-N and the oxygen of N 0  2  2  group as evidenced from spectroscopic data.  Coordination via the  nitrito group is uncommon and other examples involving  nitroimidazole ligands have not been reported. For the l-methyl-5-(2'-thioimidazolyl)4-nitroimidazole (RSU-3159) ligand (n=l), binding to Ru occurs through the thioether and chelation may occur through the imidazole-NCH^. In this series of Ru(II)-4-nitroimidazole complexes studied, RuC^Cdmso^^N0 -Im)2, 5, was the most effective radiosensitizer (SER = 1.6 at 200 ,wM) and is better 2  than the clinically used misonidazole (SER = 1.3 at 200 ^M). sensitize oxic CHO cells.  In addition, 5 did not  Other Ru-N-substituted-4-nitroimidazole complexes gave  SER values of 1.1-1.4 at 100-200 jM.  Complex 5 also produced a dose-dependent  increase in genotoxic activity (as measured by the in vitro induction of chromosome aberrations in CHO cells), which is similar to that of misonidazole but much less than that of c/s-DDP. Two  changes in  ancillary ligands and geometry of complexes  were also  examined: replacement of (i) dmso by tmso (tetramethylene sulphoxide), (ii) CI" by Br". The Ru-nitroimidazole complexes were synthesized from the precursors RuCl2(tmso)  4  and /raHj-RuB^dmso)^  In this series of complexes , only Ru02(tmso)2(4-N02-Im)2,  i l , and RuCl (tmso) (SR-2508), i i , have significantly higher SER values (1.6 and 1.5, 2  2  respectively) than their corresponding nitroimidazole ligands. The tmso complexes of 2N02"Im derivatives were more stable than the dmso series in aqueous solution with respect to the dissociation of the nitroimidazole ligands, which might be due to the improved lipophilicity of tmso complexes.  Complex 18. is suggested to be penta-  coordinated from XPS and ir data. The RuBr2(dmso)2(4-N02-Im)2 was a less effective sensitizer (SER = 1.3 at 200 _#M) than the dichloro analogue which may result from different geometrical structures or different behaviour in aqueous solution chemistry. The enhanced radiosensitizing effect over the corresponding free nitroimidazole ligand observed for complexes 5, J l and 18 may depend on: (a)  the metal's ability to  target the sensitizer to DNA; complex 5 does bind to DNA, dissociation of CI" perhaps  iv  f a c i l i t a t i n g the reaction; (b) the increase i n reduction potential or (c) an increase i n lipophilicity  o f the nitroimidazole  ligand on coordination.  H o w e v e r , the enhanced  radiosensitization does not result f r o m depletion of n o n - p r o t e i n thiols. In the present study, the R u complexes are less toxic than their corresponding n i t r o i m i a z o l e ligands in vitro.  T h e radiosensitization and toxicity o f the complexes 5, 15  and 18 are better than those of the free nitroimidazole ligands and the c l i n i c a l l y used radiosensitizer, misonidazole.  T h e data encourage further investigations of the use of  transition metal complexes as radiosensitizers to combat the h y p o x i c tumour cells.  L i g a n d Structures  /  (3)N  NO,  V  R'  NR(1) <3)N.  NR(1)  NO, R"  = 2-nitroimidazole R = H, OH I  DesmetJiylmisonidazole (De-miso)-  R =CH  3>  R' = H  R" = H : 4-nitroimidazole  R' = H,  2  2  R" = H : N-methy1-4-nitroimidazole  R =CH CH(OH)CH N^] , 2  (4-N0 -Im>  R' = H ,  R" = CH  : RSU-1170  H Etanidazole (SR-2508) OH I  R =CH  I  3<  R' = N-CH -CH -OH,  R" = C H , : RSU-3083  = Hisonidazole ,  NO,  /  CH CH OH 2  CH„  metronidazole  2  R" = H : RSU-3159  V  T A B L E OF CONTENTS PAGE Abstract  ii  Table of Contents  v  List of Tables  xi  List of Figures  xiii  List of Abbreviations  xv  Acknowledgements  xvii  CHAPTER ONE: INTRODUCTION 1.1  Radiation  1  1.2  Radiation and Cell Survival  2  1.3  Radiation and Oxygen Effect  3  1.4  The Proposed Mechanisms for the Oxygen Effect  6  1.5  Cancer and Radiation Therapy  8  1.6  Therapies Which May Overcome the Radioresistance of Hypoxic Cells  10  1.7  General Properties of Radiosensitizers  13  1.8  Radiosensitizing and Hypoxic Toxic Effects of Nitroimidazoles Sulphydryl Binding Compounds as Radiosensitizers Platinum Complexes as Chemotherapeutic  1.9 1.10  14 17  Agents and Radiosensitizers  20  1.11  Metal Complexes as Radiosensitizers  22  1.12  Rationale for Using Metal Radiosensitizer Complexes Metal-Nitroimidazole Complexes as Radiosensitizers  1.13  25 27  vi  T A B L E OF CONTENTS  PAGE 1.14  Ruthenium Complexes as Anticancer Agents  28  1.15  Nature of Sulphoxides as Ligands  29  1.16  Biological Activity of Metal-dimethylsulphoxide Complexes  31  1.17  Short-term in vitro Assays  33  1.18  Objectives  33  CHAPTER TWO: MATERIALS AND METHODS 2.1  Chemicals  35  2.2  Physical Techniques and Methods  3  2.2.1  X-ray Photoelectron Spectroscopy  38  2.2.2  Electrochemical Methods  40  8  (i)  Polarographic Measurements  40  (ii)  Cyclic Voltammetric Measurements  40  2.3  Preparation of N-Methyl-4-nitroimidazole  41  2.4  Preparation of Complexes  41  2.4.1  c/s-RuCl (dmso) , I  41  2.4.2  RuCl (tmso) , 2  42  2.4.3  "RuCl (mpso) ", 3  4  2  2.4.4  //Yz«s-RuBr (dmso) , 4  4  3  2.4.5  RuCl (dmso) (4-N0 -Im) , £  4  3  2.4.6  RuCl (dmso) (NMe-4-N0 -Im), 6  4  4  2.4.7  RuCl (dmso) (RSU-1170) , 7  4  4  2.4.8  RuCl (dmso) (RSU-3083) ,8  4  4  2.4.9  RuCl (dmso) (RSU-3100) , 9  4  5  2  2  2  4  2  2  2  2  2  2  2  2  2  4  2  2  2  2  2  2  2  2  2  2  vii  T A B L E OF CONTENTS PAGE 2.4.10 RuCl (dmso) (RSU-3159), 10  45  2.4.11 RuCl (dmso) (miso) , i i  45  2.4.12 RuCl (dmso) (De-miso) , 12,  46  2.4.13 RuCl (dmso) (2-N0 -Im) , i l  46  2.4.14 RuCl (dmso) (metro) , i4  46  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2.4.15 RuCl (tmso) (4-N0 -Im) , 15  4  7  2.4.16 RuCl (tmso) (NMe-4-N0 -Im) , i6  4  7  2.4.17 RuCl (tmso) (De-miso) ,il  4  7  2  2  2  2  2  2  2  2  2  2  2  2.4.18 RuCl (tmso) (SR-2508), i £  4  8  2.4.19 RuCl (tmso) (CMNI), 19  4  8  2.4.20 RuCl (tmso)(RSU-3159),20  4  8  2  2  2  3  2  2.4.21 RuBr (dmso) (4-N0 -Im) , 21 2  2  2  2  4  2.4.22 RuBr (dmso) (NMe-4-N0 -Im), 22 2  2  2  2.4.23 RuCl (dmso) (l,10-phenanthroline), 23 2  2  2.4.24 RuCl (dmso) (5-Cl-l,10-phenanthroline), 24 2  2  9 4  4  9  9 5  0  2.4.25 RuCl (dmso) (5-NO -l,10-phenanthroline), 25  50  2.4.26 RuCl (dmso) (NMe-Im) , 26  50  2.4.27 RuCl (dmso) (2Me-Im) , 27  51  2.4.28 [Ru(NH ) Cl]Cl , 28  51  2  2  2  2  2  2  2  2  3  5  2  2  2.4.29 [Ru(NH ) (NMe-Im)]Cl , 29 3  5  3  52  2.5  Cell Culture Procedures  52  2.6  Irradiation  53  2.7  Radiosensitization - Cell Survival Experiments  53  2.8  Toxicity Studies - Cell Survival Experiments  56  2.9  Assay for Chromosome Aberrations  59  viii  T A B L E OF CONTENTS PAGE 2.10  Measurement of Non-protein Thiols  60  2.11  Inhibition of Restriction Enzymes  60  2.12  Measurement of Partition Coefficients  62  CHAPTER THREE: CHARACTERIZATION OF COMPLEXES 3.1  XPS Data  66  3.2  Infrared Spectral Data  72  3.2.1 3.2.2 3.2.3 3.2.4 3.3  n  72  Infrared Data of RuCl (tmso) L Complexes (m = 1, 2 or 3; n = 1 or 2)  75  Infrared Data of RuB^dmso^Ljj Complexes (n = 1 or 2)  78  Infrared Data of "RuCl (mpso) "  79  2  m  2  n  2  Nuclear Magnetic Resonance Spectral Data 3.3.1 3.3.2 3.3.3  3.4  Infrared Data of RuCl2(dmso)2L Complexes (n = 1 or 2)  79  Nuclear Magnetic Resonance Data of RuC^dmso^Ljj Complexes (n = 1 or 2) *H nmr Data of RuCl (tmso) L Complexes (m = 1, 2 or 3; n = 1 or 2) 2  m  *H nmr Data of RuBr (dmso) L Complexes (n = 1 or 2) 2  2  80  n  8  7  n  4-Nitroimidazole vs. 5-Nitroimidazole Formulation  9 3 95  CHAPTER FOUR: AQUEOUS SOLUTION CHEMISTRY OF THE RUTHENIUM NITROIMIDAZOLE COMPLEXES 4.1  Solution Stability of the Substituted-2nitroimidazole Complexes  99  ix  T A B L E OF CONTENTS  4.2  Aqueous Solution Chemistry of the R u C l ( R S O ) L Complexes; R SO = dimethylor tetramethylene-sulphoxide; L = 4-N0 -Im (n = 1) or Substituted-4-N0 -Im (n = 1 or 2) 2  2  2  n  2  2  4.3  2  Aqueous Solution Chemistry of the RuBr (dmso) L Complexes (L = 4-N0 -Im, n=2; L = NMe-4-N0 -Im, n=l) 2  2  n  2  2  CHAPTER FIVE: CYTOTOXICITY AND RADIOSENSITIZATION IN VITRO 5.1  Toxicity of the Ru Complexes and Their Nitroimidazole Ligands  5.2  Chromosomal-Damaging Activity of Complex 5  5.3  Radiation Sensitization of Aerobic and Hypoxic CHO Cells 5.3.1  Radiosensitization by the Metal Complexes  CHAPTER SIX: FACTORS R E L A T E D TO RADIOSENSITIZATION 6.1  DNA Binding 6.1.1  Assessment by Inhibition of Restriction Enzymes  6.1.2  DNA Binding by the Ru Complexes  6.2  Partition Coefficient (P)  6.3  Non-protein Thiol Depletion  6.4  Electron Affinities (Reduction Potentials)  6.5  Feasibility in IN VIVO System  X  T A B L E OF CONTENTS PAGE CHAPTER SEVEN: CONCLUSIONS and RECOMMENDATIONS  for F U T U R E WORK  BIBLIOGRAPHY  149  152  APPENDICES I  Preparation of Stock Solutions  161  II  Preparation of Buffers  162  III  Chromatography Through Sephadex G-50  1  6  3  IV  Preparation of Agarose Gel  1  6  4  V  Linearization of pSV2-gpt Plasmid  1  6  5  VI  Preparation of Rat Liver S9 Fraction  1  6  7  VII  Procedures for in vivo Study  1  6  8  xi LIST OF TABLES Table I  Metal compounds ability  Table II  XPS data for Ru(II) compounds and free ligands  Table III  Selected ir spectral data for 4nitroimidazole complexes of Ru and the free nitroimidazole ligands  Table IV  Selected ir spectral data for 2nitroimidazoles, metronidazole and 1,10phenanthrolines, and their Ru complexes  Table V  Selected ir spectral data for RuCl2(tmso) L and RuBr2(dmso) L complexes; m = 1, 2, or 3, n = 1 or 2 m  with  radiosensitization  n  2  n  Table VI  Selected *H nmr spectral data for RuC^dmso^Lp complexes and the free nitroimidazole ligands  Table VII  Selected nmr spectral data for RuCl2(tmso) L complexes and the free nitroimidazole ligands m  Table VIII  n  Selected H nmr and C{ H} chemical shifts for RuBr2(dmso)2(4-N02-Im)2 and RuBr (dmso)2(NMe-4-N0 -Im) ]  13  2  J  2  Table IX  Molar conductivity data for complexes in water at RT  Table X  Plating efficiency of the 4-nitromidazole ligands and their Ru(II) complexes in hypoxic CHO cells (4 h at 37°C)  Table XI  Hypoxic toxicity (PE) of the 2nitroimidazoles and metronidazole and their Ru(II) complexes in CHO cells (4 h at 37°C)  Table XII  Clastogenic activity of RuCUCdmso^^N 0 - I m ) and ds-DDP on CHO cells 2  Table XIII  some Ru  2  Clastogenic activity of m-RuCi2(dmso) and 4-N0 -Im on CHO cells  4  2  Table XIV  The hypoxic SER nitroimidazoles ligands complexes  values of 4and their Ru(II)  xii  LIST OF TABLES PAGE Table X V  The SER values of 2-nitroimidazoles and metronidazole and their Ru(II) complexes  12 7  Table XVI  Relative inhibition of BamHI activity by some Ru complexes, c/5-DDP and transDDP  132  Table XVII  Partition coefficients of nitroimidazole ligands and their ruthenium complexes  136  Table XVIII  NPSH depletion in hypoxia by some Ru(II) complexes  139  Table XIX  Half-wave reduction potentials of Ru(II) compounds and their nitroimidazole ligands  142  xiii LIST OF FIGURES PAGE Figure 1  A typical dose-response survival curve  4  Figure 2  The effect of hypoxia on survival curves  5  Figure 3  The cross-section of a tumour illustrating the oxic and hypoxic regions  9  A scheme showing the direct action mechanism of a radiosensitizer on a target molecule  12  Structural formulae of a nitrofuran, metronidazole and misonidazole  15  Figure 4 Figure 5 Figure 6  The  mechanism  of  oxygen  "inhibition"  of  nitroreduction  18  Figure 7  Proposed mechanism of sulphydryl radioprotection  19  Figure 8  Possible types of DNA interactions with m - D D P  21  Figure 9  Rationale for using metal-radiosensitizer complexes  26  Figure 10  The three resonance structures for the S-O bond of sulphoxides  30  Figure 11  Structural formulae of the 2-nitroimidazole ligands  36  Figure 12  Structural formulae of the 4-nitroimidazole ligands  37  Figure 13  Schematic representation of an anaerobic spectral cell  39  Figure 14  Irradiation set-up  54  Figure 15  The glass vessel for cell suspension toxicity tests  57  Figure 16  Outline of binding assay using plasmid DNA  61  Figure 17  XPS spectra N(ls) of RuCl (dmso)2(4-N0 -Im)2 and 2  2  4-N0 -Im  70  2  Figure 18 Figure 19  nmr spectrum of c/s-RuCl (dmso)4 in CDCl^ nmr spectrum of Ru0 (dmso) (4-N0 -Im) and RuCl (dmso) (NMe-4-N0 -Im) in CDCI3 C{ H] nmr spectra of RuCl (dmso) (4-N0 -Im) and RuClspectra (dmso)of (NMe-4-N0 -Im) and in CDCI3 *H nmr RuCl (tmso) free tmso in CDCK 2  2  Figure 20 Figure 21  81  2  l3  2  2  2  2  2  85  l  2  2  2  2  2  2  4  2  2  88 90  xiv  LIST OF FIGURES PAGE Figure 22  H nmr spectra of RuCl (tmso) (4-N0 -Im) RuCl (tmso) (SR-2508) in C D C I 3 2  2  Figure 23  2  2  and  2  92  2  nmr spectra of RuBr (dmso) (4-N0 -Im) RuBr (dmso) (NMe-4N0 -Im) in C D C I 3 2  2  2  2  2  and  2  9.6  2  Figure 24  pH titration curves  Figure 25  Toxicity of RuCl^(dmso) (4-N0 -Im) and 4-N0 -Im in hypoxic and 0x1c CHO cells  108  Chromosome damaging activity of RuCl (dmso) (4N0 -Im) , misonidazole and 4-N0 -Im in CHO cells in the absence of S9  117  Radiosensitization of CHO cells by RuCl (dmso) (4N 0 - I m ) , 5, or 4-N0 -Im  121  Radiosensitization of CHO cells by RuCl (tmso) (4N 0 - I m ) , 15, or 4-N0 -Im  122  Radiosensitization of CHO cells by RuBr (dmso) (4N 0 - I m ) , 2i, or 4-N0 -Im  123  Radiosensitization of CHO cells by RuCl (dmso) (NMe-4-N0 -Im), 6, or NMe-4-N0 -Im  124  Radiosensitization of CHO cells by RuCWtmsoWSR2508), J8, or SR-2508  126  Inhibition of endonuclease activities of BamHI and EcoRI  130  Titration of BamHI site with RuCl (dmso) (4-N0 Im) , 4-N0 -Im and ds-DDP  131  The E w reduction potentials N 0 - I m ) and 4-N0 -Im  141  Figure 26  2  2  Figure 32 Figure 33  2  2  2  2  2  2  2  2  2  2  2  2  Figure 35  2  2  2  2  Figure 34  2  2  2  Figure 31  2  2  2  2  Figure 30  2  2  2  2  Figure 29  2  2  2  Figure 28  2  2  2  Figure 27  102  2  of RuCl (dmso) (42  2  2  The effect on tumour cells survival of the time interval between RuCl (tmso) (SR-2508) administration and irradiation with 10 Gy of X-rays 2  2  147  XV  LIST OF ABBREVIATIONS  BE  binding energy  as-DDP  czs-diamminedichloroplatinum(II), c-DDP  CMNI  5-chloro-1 -methyl-4-nitroimidazole  CHO  Chinese hamster ovary  De-miso  (2-nitro-1 -imidazolyl)-3-hydroxypropanol (desmethylmisonidazole), Ro-07-9963  dmso  dimethyl sulphoxide  Ej/2  half-wave reduction potential  EDTA  disodium ethylenediaminetetraacetate dihydrate  Ellman's reagent  5,5' -dithiobis-(2-nitrobenzoic acid)  esr  electron spin resonance  FCS  Fetal Calf Serum  Gy  Gray = 100 Rads.  Im  imidazole  ir  infrared  LET  Linear Energy Transfer  MEM  Minimal Essential Medium  metro  l-(2' -hydroxymethyl)-2-methyl-5nitroimidazole (metronidazole) 1 -(2-nitro-1 -imidazolyl)-3-methoxypropanol  miso  (misonidazole), Ro-07-0582 mpso  methylphenyl sulphoxide  NMe-4-N02"Im  N-methyl-4-nitroimidazole  nmr NO->-Im  nuclear magnetic resonance nitroimidazole  xvi LIST OF ABBREVIATIONS  NPSH  non-protein thiols  OER  oxygen enhancement ratio  PBS  phosphate-buffered saline  PE  plating efficiency  RSU-1170  l-(l'-aziridinyl-2'-propanol)-2-methyl4-nitroimidazole  RSU-3083  2-(l,2-dimethyl-4-nitroimidazolyl)2-aminoethanol  RSU-3100  l-methyl-4-nitro-5-phenoxyimidazole  RSU-3159  1 -methyl-5-(2'-thioimidazolyl)4-nitroimidazole  RT  room temperature  S  survival  S9 SER  SH  liver microsomal preparations sensitizer enhancement ratio, usually ratio of radiation doses in hypoxic cells to give 1% survival sulphydryl, thiol  SR-2508  1 -{N-(2-hydroxyethyl)-acetamido}2-nitroimidazole  tmso trans-DDT XPS  tetramethylene sulphoxide fra«5-diamminedichloroplatinum(II) X-ray Photoelectron Spectroscopy  xvii ACKNOWLEDGEMENTS  I am grateful to Professor B.R. James and Dr. K . A . Skov for their guidance, support and encouragement throughout the course of this work. I thank them for their patience and open-minded approach to a major clinical problem from both chemical and biological point of views. I also thank members of the "James' Gang" and the staff of  Medical Biophysics Unit at the B.C. Cancer Research Centre, H. Adomat, I,  Harrison, D. Wurst and Dr. P. Olive, for their support and co-operation.  Early  discussions with Dr. N.P. Farrell on the content and problems of this project were particularly rewarding. I am indebted to my brother, Paul, for helping me on the XPS work and Dr. D.C. Frost for allowing me to use his XPS instrument. The assistance of Mr. P. Borda on micro-analyses, Dr. O.S. Chan on nmr, and the electrical and mechanical services is also gratefully acknowledged. I would like to thank UBC for awarding me a University Graduate Fellowship and the Faculty of Medicine of UBC for awarding Summer Research Studentships during this programme.  The granting agencies, NSERC, MRC and B.C. Cancer  Foundation are also acknowledged for support of this project.  Johnson Matthey Ltd.  provided the loan of RuCl3 3H 0; Dr. T.C. Jenkins is thanked for providing CMNI, -  2  RSU-3100, RSU-1170 and Dr. I.J. Stratford for supplying RSU-3083. Roche Products Ltd. kindly donated Ro-05-9963 (desmethylmisonidazole),  and NCI of the United  States, the SR-2508. I would like to extend a special thanks to my parents for allowing me to achieve what I set out to do. Finally, I dedicate this thesis to my wife, May, for giving me the strength, and going through the ups and downs with me; and also to my new born baby, Emilie, for providing the "Energy after mid-night" to complete this thesis.  xviii  We have not succeeded in answering all your problems. The answers we have found only serve to raise a whole set of new questions. In some ways we feel we are as confused as ever, but we believe we are confused on a higher level and about more important things. from Hickory Plaque Inc. Van Nuys, California, 1972.  1  CHAPTER ONE INTRODUCTION 1.1  Radiation Radiation capable  radiation.  of  ionizing molecules  is  appropriately called ionizing  Ionizing radiation, such as X-rays, causes damage by imparting sufficient  energy to electrons within the irradiated matter to allow them to be released with concomitant ionization of molecules.  This means that the electrons are removed from  atoms, and this can cause chemical bonds to be broken and/or changed: AB ~ v ^ A -  + B- — • P  AB — » A - + B-  [1.1]  £L AR- + BR-  [1.2]  The electrons can also be removed from molecules of water, creating reactive ions which can in turn cause additional damage/ 2H 0 - ^ r ^ c / + ~ 2  e  + Rp —  H 0 3  + r^o"  a  +  + OH-  r^O* — - H- + OHOH- + DNA — * DNA- + r^O  [1.3] [1.4] [1.5] [1.6]  The electrons released by ionization can also impart some of their energy to other electrons, thus creating a "chain reaction" and causing more ionizations, freeing other less energetic electrons.  This process continues until all of the energy available is  expended. These electron reactions can cause "permanent" chemical alterations, or they can terminate in harmless recombination processes/  The most dramatic effects of ionizing radiation on biological systems - mutation and death - are the consequence of the formation of molecular ions which can directly or indirectly cause damage to cellular biomolecules. If the DNA of the cell is damaged, this can result in mutation and chromosomal aberrations which can eventually transform  2  the cell or render the cell unable to divide. However, some of the long-lived chemical changes occurring may be subject to repair by chemical or enzymatic processes.  The term "X-ray dose", as used in this thesis, refers to the absorbed dose, i.e. the energy imparted (absorbed) per unit mass. The Systeme International (SI) unit is the Gray (Gy) which corresponds to an energy absorption of 1 Joule (J) per kilogram (kg) of irradiated material.  Previously, the unit of dose was called the Rad, defined as an  energy absorption of 100 ergs/gram. Since IJ = 10 ergs, the Gray is equivalent to 100 Rads.  1.2  Radiation and Cell Survival In order to study the effects of radiation on biological systems, radiobiologists  have chosen to study various biological endpoints such as DNA strand breaks, cell mutation, loss of cell proliferative capacity (e.g. in bacterial or mammalian cell system) or loss of organ function.'*  An "endpoint" is defined as the effect that is measured at the conclusion of the experiment.  One of the endpoints used in this thesis is the loss of proliferative capacity  or viability of mammalian cells.  For normally dividing cell populations, proliferative  capacity is defined as the capacity of a cell to generate a clone of similar cells, called a colony. In this context, the "lethal" effect of radiation is defined as that which induces loss of proliferative capacity.  Conversely, a "survivor" is defined as a cell which, after  irradiation, has retained its ability to generate a clone of like cells.  Techniques for scoring viable mammalian cells were developed in the mid1950's,^ which allow for dose-response relationships to be obtained for mammalian cells.  Survival curves for mammalian cells, with the surviving fraction plotted  3  logarithmically and the dose linearly, are usually described as having a "shoulder" (Figure 1).  The shoulder region is generally followed by a region of approximately  exponentially decreasing survival.  Therefore, survival curves for mammalian cells at  low doses of radiation show proportionally less killing per unit dose than at higher doses.  There are many different models and theories to describe mammalian cell survival. Most of them are based on some hypothesis as to the mechanism of radiation damage, e.g. the presence of multiple targets per cell, or a single target requiring multiple hits, or a limited cellular repair capacity, could all account for shouldered survival curves (see 5 and references therein).  The use of these expressions allows for  comparisons to be made between parameters describing different cell lines, or between parameters describing different radiation conditions for the same cell lines.  1.3  Radiation and Oxygen Effect The radiosensitizing action of molecular oxygen (dioxygen)-  was observed in  the 1920's,^ but its significance was not appreciated. The effect of oxygen on cellular radiation response is illustrated in Figure 2, which shows the response of Chinese Hamster Ovary (CHO) cells to X-rays, first for irradiation in an air-saturated medium, and secondly for a medium from which all oxygen has been removed by purging with purified nitrogen.  The absence of oxygen has a dramatic radioprotective effect;  the  oxic and hypoxic survival curves are of similar shape, but the dose required to produce a given level of cell kill is very different in the two cases. This difference is measured by the oxygen enhancement ratio (OER), defined as: Dose i n nitrogen OER =  , at equal survival  [1.7]  Dose i n a i r a) The term "oxygen" will be used throughout this thesis to represent molecular 0 (dioxygen).  2  4  F i g u r e 1:  A t y p i c a l dose-response s u r v i v a l curve (taken from r e f . (adapted from r e f . 1).  1).  5  — r CHO  r CELLS  1.0  o  4  <  I  0.1  cc  \  x  0.01  >  >  cc  0.001  3 CO  \  —  0  1  5  2:  2  '  1  1  •  10  15  20  25  D O S E  Figure  o  30  ( G r a y )  The e f f e c t o f hypoxia on s u r v i v a l c u r v e s , o x i c hypoxic c e l l s ( o ) (adapted from r e f . 7).  cells(•),  6  If oxygen is "dose-modifying", that is, if the aerobic and hypoxic survival curves can be superimposed by a change of the dose scale, then the OER will be independent of the value of S chosen for comparison; in practice, S = 0.01 is commonly chosen and is used in this thesis. For irradiation at this dose range the OER for mammalian cells is about 3  7  1.4  The Proposed Mechanisms for the Oxygen Effect The absorption of high energy radiation in matter causes ionizations and  excitations of atoms and molecules (see Section 1.1).  Regardless of the type of incident  radiation or particle, electrons are principally responsible for these effects.  Electron  impact creates free radicals by breaking covalent bonds. This "physical stage" of energy deposition is followed by a "chemical stage" lasting a few microseconds.^ In this latter stage, the free radicals created by the radiation interact with undamaged molecules and with one another.  These reactions can cause "permanent" (or at least, long-lived)  chemical alterations, or they can terminate in harmless recombination processes.  The  biological effect of radiation will be changed by agents that modify the stage of chemical interactions. Presumably, the lethal and mutagenic effects of radiation are due to damage in macromolecules (especially DNA) which is not repaired, or which is misrepaired.  Primary radiation events may be divided into two classes: direct action and indirect action.^  Direct action is the ionization of a macromolecule by a primary  radiation event; indirect action is a secondary reaction caused by a reactive chemical species, which is formed by the absorption of radiation energy in small molecules (such as water). These species may then attack macromolecules.  7  Water is the most common molecule in a living cell, and the action of radiation on water has been studied in detail. The chemistry of water radiolysis was studied by Weiss^ who initially proposed that the process was the production of H and OH" -  radicals, but it is now known that hydrated electrons are the principal reducing species.^  0  These species react with each other to produce even-electron-numbered  molecules: e"  + e" aq aq  OH' + OH" — H  V—  2  2  0  [1.8]  H + 2 0H~ ?  2  [1.9]  2  OH" + e" — O H " aq  [1.10]  However, in the presence of oxygen, other reactions can occur, altering the yields of the primary radiolysis produces, and creating particularly superoxide,^ which can be dangerous when reacting with proteins, lipids, polysaccharides and nucleic acids.^ (aq) H- + 0  2 2  2  — - 0~  +H  t -  +  1  1 2  !  The hydroxyl radical is the most reactive oxidizing species, and thus damaging, of the water radiolysis products.  Indirect damage from this species can then occur either by  H-abstraction or OH' addition, subsequent chemical rearrangement "fixing" the lesion, e.g.: OH- + RH —Uf)  + R-  [1.13]  The process yields products similar to those formed by direct damage: X-ray + RH —»- RH+ + e~ — • R- + H + e" +  Oxygen may act as a radiosensitizer in a number of ways.  [1.14]  Alper^ proposed  that the mechanism involves electron acceptance from target radicals generated by the radiation:  R- + 0  2  —*- R0 - — 2  R +0 +  2  [1.15]  8  These R' radicals have a very short lifetime and may undergo chemical changes; reaction of a product with oxygen to give an organic peroxide is non-reversible and results in biological damage by fixation of a lethal lesion on the target, i.e. DNA. In the absence of oxygen, fewer peroxides are formed and thus more lesions are repaired. A second model of Adams^ suggested that electron affinic compounds, such as oxygen, might act by enhancing the effects of direct damage. After a target molecule has been ionized, the electron was proposed to localize at some electron affinic site on the molecule.  Electron transfer to a sensitizer molecule of greater electron affinity would  reduce the chance of recombination, and increase the chance of decay of the positive target ion to a free radical and subsequent damage fixation which could lead to DNA strand breaks (see Section 1.6).  1.5  Cancer and Radiation Therapy Radiotherapy forms at least part of the treatment for more than half of the new  cancer patients in North America.^-  5  The unlimited cell proliferation associated with  malignant tumour growth often leads to a reduced oxygen concentration within the tumour.  This in turn leads to a relative radioresistance not encountered in normally  healthy tissue.  The full relevance of this physico-chemical effect in therapy was  demonstrated in the study by Gray and co-workers^ who showed that many tumours contain necrotic areas usually separated some distance (150-200 j*m) from the vascular system.  This situation is depicted in Figure 3 and is explained by the fact that the  disordered nature of tumour cell replication and the subsequent requirements of oxygen metabolism produce an oxygen gradient, rendering the cells furthest from the capillaries hypoxic. The clinical relevance is that because of the oxygen effect, these cells, which may represent up to 30% of the total tumour mass will be radioresistant, and upon termination of treatment may become aerobic and tumour growth will recommence.  78  One of the most direct pieces of evidence for the importance of the oxygen effect on  The c r o s s - s e c t i o n of a tumour i l l u s t r a t i n g the o x i c and hypoxic r e g i o n s (oxygen g r a d i e n t ) (taken from r e f . 17). (adapted from r e f . 17).  10  radiotherapy is an analysis by Bush et al}^ of the influence of blood hemoglobin levels on cure rates of patients treated for cervical carcinoma.  Those patients with greater  hemoglobin levels had better cure rates than those with lower levels. Further, Chaplin 20  et al.  have demonstrated that intermittent blood flow can occur in the tumour  vasculature of murine squamous carcinoma, and this loss of flow has been shown to result in acute hypoxia in areas close to blood vessels. As a result of the oxygen effect in tumour cells, the radiation dose which can be delivered to a tumour is limited by the dose which the surrounding healthy tissue can tolerate. Any means of effectively reducing the relative radioresistance of the hypoxic tumour cells will increase the effectiveness of the radiotherapy, and thus should improve the cure rate.  1.6  Therapies Which May Overcome the Radioresistance of Hypoxic Cells Various approaches to the modification of radiotherapy, which may reduce the  radioresistance of hypoxic cells, have been proposed.  These include the use of  hyperbaric oxygen, various forms of radiation, and chemical radiosensitization/-'  The most apparent approach to overcome the hypoxic cell problem is hyperbaric oxygen therapy/  With this technique, patients are irradiated in a pressurized, oxygen-  rich environment.  The rationale for this approach is straightforward: an increase in  blood oxygenation  may reduce  the size of  the  hypoxic fraction in a tumour.  Nonetheless, results with hyperbaric oxygen have not been very encouraging, the reasons probably being two-fold/  First, the increase in blood oxygen-carrying capacity  is far smaller than the increase in oxygen partial pressure.  Secondly, oxygen, because  of rapid depletion by metabolism in the periphery, is unable to penetrate into the hypoxic region of the tumour.  In addition to these problems, hyperbaric oxygen is  11 toxic, and the procedure as a whole is dangerous and difficult to perform on a routine basis.  While this thesis is concerned with X-ray irradiation, there are other forms of ionizing radiation which have been used to overcome the oxygen effect.  The  biological effectiveness of a particular form of radiation such as by X-rays or neutrons is related to its ionization density. This may be expressed in terms of the linear energy transfer (LET) of the radiation. This quantity describes the rate at which the particle deposits energy on travelling through matter. increasing LET;  21  The value of OER  for example, for X-ray irradiation, the OER  neutron irradiation, the OER  decreases with  is about 3 while for  is less than 2. It seems clear that the use of high LET  irradiation in cancer therapy should reduce the limiting effect of tumour hypoxia. Additional benefits may  be if, in addition to giving a lower OER,  the depth-dose  characteristics of the radiation permit better localization of dose in the tumour. However, prior to application, the development of these new radiation modalities have technical difficulties to be overcome. Further, relative cost is higher.  One of the most promising approaches for the control of hypoxic cells is the use of a chemical radiosensitizer, that is, a drug which mimics the radiation-sensitizing properties of oxygen. Adams and Dewey  observed that the compounds known to be  sensitizers shared the property of being efficient electron acceptors as measured by reduction potentials.  In 1969, Adams and Cooke^ suggested that the sensitizer  increases the damage resulting from a direct action mechanism. ionization of the target molecule, e.g. DNA  In this case, direct  (see Figure 4), produces a positive ion and  an electron, the latter migrating to some electron-affinic site on the molecule. If there is a compound of higher electron affinity available, either as a metal complex or as a free molecule, electron transfer from the ionized target molecule to the electron-affinic  12  F i g u r e 4:  A scheme showing t h e d i r e c t a c t i o n mechanism o f a r a d i o s e n s i t i z e r on a t a r g e t m o l e c u l e . Process (a) would l e a d t o DNA s e l f - h e a l i n g , w h i l e process (b) would r e s u l t i n DNA damage  13  molecule could occur. This process (b) would compete with charge recombination (a) in the target molecule which would contribute to self-healing.  Electron-transfer to the  sensitizer would reduce the chance of recombination and favour the decay of the ionized molecule to a free radical (Figure 4).  In support of this mechanism experiments using electron spin resonance have shown directly that a charge transfer complex is formed in irradiated solid mixtures of DNA.  In addition, Adams et al.  have demonstrated rapid one-electron transfer  between nucleotides in solution, implying that intramolecular electron transfer along a polynucleotide should readily occur. radiosensitizers  can  accommodate  This group also demonstrated that known  electrons  that  are  rapidly  and quantitatively  transferred from one-electron adducts of simple pyrimidine and purine bases.  These  results have guided the search for effective radiosensitizers, using a variety of in vitro test systems.  Several compounds were found to be effective in vitro, including stable  nitroxyl free radicals' ** and aromatic nitro compounds/ 2  1.7  7  General Properties of Radiosensitizers For classification as an "effective radiosensitizer", the compound must possess  several properties.  First, the compound must be effective at a clinically achievable  drug concentration, although it does not have to be as effective as oxygen. That is, if a "sensitizer enhancement ratio", SER, is defined as the ratio of radiation doses required to produce a given effect in the absence and presence of the drug, then the SER need not necessarily be as large as the OER. A large SER value is desirable, but an SER as low as 1.3, for example, could still be of significant clinical benefit.  Secondly, the  sensitizer should not increase the radiosensitivity of normal, aerobic cells to the same extent, or the benefits will be nullified.  Thirdly, the sensitizer must be able to  penetrate into the centre of the tumour; this implies ability to cross the barrier  14 presented by the cell membranes of the surrounding tumour tissue.  Generally, this  means that the drug must have lipophilic properties; however, water-solubility is also required for administration.  Fourthly, the sensitizer must be metabolically stable,  otherwise the drug will be consumed before reaching the hypoxic target cells.  Finally,  the toxicity and side-effects of the drug must be kept within a tolerable limit. This last criterion has proven to be the most difficult to satisfy, as it is only assessed in the clinical situation.  1.8  Radiosensitizing and Hypoxic Toxic Effects of Nitroimidazoles In 1973, Foster and Willson^ suggested that available drugs be screened for  structures with radiosensitizing potential.  There were two clinically used, antibacterial  drugs that were a nitrofuran derivative * -  0  and the 5-nitroimidazole drug, l-(2' -  hydroxymethyl)-2-methyl-5-nitroimidazole (flagyl or metronidazole) (Figure 5), which appeared to be good radiosensitizers in vitro. The electron-affinity of these compounds had been measured.  '  Another class of nitroaromatic compounds which showed  more promise was the 2-nitroimidazole derivatives/-*  Of this class, l-(2-nitro-l-  imidazolyl)-3-methoxy-propanol (misonidazole) (Figure 5) has received by far the most attention both in laboratories and in clinics/-* been reported to be 1.6 at 300 ,uM in vitro?*  The SER value for misonidazole has  Within a nitroimidazole series the in vitro  radiosensitizing ability correlated with electron-affinity/^  Anticancer drugs, in many cases, must be delivered systematically (i.e. orally, intravenously), rather than directly at the tumour. Thus, toxic side-effects on normal tissues are frequently encountered. systemic  toxicity be selected.  experience neurotoxicity/-*  It is critical, then, that drugs of outstandingly low For example patients  receiving misonidazole may  Usually, the neuropathic side-effects have been temporary,  and they can be avoided by limiting the total drug dose.  However, such limitations  15  Figure  5:  S t r u c t u r a l formulae misonidazole  of a n i t r o f u r a n , m e t r o n i d a z o l e  and  16  reduce  the  likelihood  of  achieving  effective  radiosensitization.  nitroimidazoles are relatively t o x i c , especially to h y p o x i c c e l l s / ' *  Further,  the  2-  T h i s property might  at first be considered an advantage f o r eradication o f h y p o x i c cells, and indeed many studies are  being directed  at the  interaction  between  toxicity  and  radiosensitizing  properties.  T h e r e was a suggestion that the radiosensitizing a b i l i t y and h y p o x i c and oxic toxicity o f some nitroimidazoles were consequences of their e l e c t r o n - a f f i n i t y ,  i.e. the  ease w i t h w h i c h they c o u l d be reduced to the nitro anion r a d i c a l , R - N O ^ metabolic  reduction  of  aromatic  nitro  compounds  by  mammalian  nitroreductases was first studied by Fouts and B r o d i e i n 1957.^° important observations:  The  systems  via  T h e y made several  enzyme activity was f o u n d i n both the aqueous soluble fraction  and the microsomal f r a c t i o n o f liver; there were large differences i n activity of species; the  enzyme  system  showed  few  rigid  structural  requirements  for  substrates.  F u r t h e r m o r e , the m a m m a l i a n nitroreductase activity was strongly i n h i b i t e d by air.  The  mechanism  of  oxygen  inhibition  of  the  enzymatic  nitroreduction  was  elucidated b y the w o r k of M a s o n and H o l t z m a n , ^ esr spectroscopy being used to demonstrate the f o r m a t i o n of the nitro anion r a d i c a l , d u r i n g the h y p o x i c microsomal reduction of nitrobenzoic a c i d . ^  T h i s showed that the i n i t i a l step i n the  process was a one-electron transfer  reduction  f r o m the enzyme to the nitro c o m p o u n d , and  suggested the possibility of a free radical mechanism i n the oxygen i n h i b i t i o n of the reduction.  In a d d i t i o n , these workers suggested that the i n i t i a l one-electron step i n the  nitroreduction continued i n the presence of oxygen; however, the nitro anion radical was considered to  be  re-oxidized  rapidly  to  the  parent  compound.  Thus  no  net  disappearance o f the nitro c o m p o u n d was observed, and no esr signal f r o m the radical was detected under aerobic conditions.  O x y g e n was reduced to superoxide, resulting i n  17  oxygen depletion and the build-up of superoxide.  The toxic superoxide product was  thought to be rendered harmless by the action of superoxide dismutase and catalase (Figure 6).  However, the actual toxic product from the nitroimidazoles has remained  elusive, and its mode of action (e.g. interaction with DNA, proteins or membranes) unknown.  1.9  Sulphvdrvl Binding Compounds as Radiosensitizers A hydrogen donation model was first proposed in 1954  to explain the reduced  radiosensitivity found under hypoxia in the presence of sulphydryl (SH) compounds. It was postulated that a free radical is produced in the target molecule by radiation, and that subsequent reactions of this free radical lead to permanent biological damage. In the presence of a sulphydryl compound a hydrogen atom is transferred to the target radical before it has time to decompose, and thus the chemical lesion is repaired (Figure 7). The thiol radical RS" then recombines to form the oxidized dithiol species (RSSR).  On the other hand, Loman et al^ have proposed an alternative mechanism of the SH protection that could complement the H-donor model.  During irradiation,  organic free radicals arise in the cell by both direct and indirect mechanisms.  The  model of Loman et al. proposes that these organic free radicals are eliminated by SH compounds through a process of hydrogen donation.  But an essential difference  between these two models is that in the first process, repair of the target molecule has occurred, whereas in the second process, free radical intermediates are eliminated and indirect damage to the target molecule is prevented (Figure 7).  One of the mechanisms proposed to explain the sensitizing effect of certain compounds assumes that the sensitizer acts by binding free, non-protein sulphydryls or thiols (NPSH) groups which normally have a protective effect in irradiated cells/'* One  18  (a)  Reductase R-N0 < / (reduced) j V / \/ +e ( -e  (superoxide)  2  ^ Reductase^ R-NO, (oxidized) (hypoxia)  (reduced products)  L  (b)  H + 0  SOD > h (0 + H 0 )  +  2  2  2  2  CATALASE H  2°2  NET: H + e" + 0 +  Figure 6:  2  >\ 0 + H0 2  —•* H+ + o~  2  3/4 0  2  + \  Mechanism of oxygen 'inhibition' of nitroreduction (a) The i n i t i a l product of nitroreduction i s the n i t r o anion radical. Under hypoxia, this species i s further reduced to unidentified products, including perhaps, toxic and mutagenic species. Under aerobic conditions, the radical anion i s re-oxidized quickly to the parent n i t r o compound. (b) the resulting superoxide i s detoxified by superoxide dismutase (SOD) and catalase.  19  irradiation  irradiation  damage t o target + RSH  + AH + RS-  1 1/2 RSSR  Proposed mechanisms of sulphydryl radioprotection: (a) The hydrogen donation model and (b) the Loman model, AH = organic molecules i n the c e l l .  20  such sulphydryl depleting radiosensitizer is N-ethylmaleimide (NEM).  Experiments  with E. coli have yielded results which are consistent with an SH binding mechanism.^ Another example is the use of buthionine sulphoximine (BSO), which on administration to mice or its incorporation in tissue culture media, results °in rapid depletion of intracellular glutathione concentration by inhibiting glutathione synthesis and enhanced radiosensitivity/^  Nitroimidazoles, such as misonidazole and metronidazole, may  sensitize partly because of removal of endogenous NPSH.  • This appears to be  particularly true under conditions of prolonged incubation such as those used by Hall et  al  48  1.10  Platinum Complexes as Chemotherapeutic Agents and Radiosensitizers In 1969, Rosenberg et al. described several complexes of platinum which have  significant anti-tumour activity/^  Since that time, cis-diamrninedichloroplatinum(II)  (c/s-DDP) has rapidly attained clinical recognition for use alone and in combination with other chemotherapeutic agents/ . 0  Chemists have put considerable effort into a  search for related complexes with improved therapeutic properties/  Platinum complexes bind to DNA,  -5-2  0  - * and this presumably is the means by 5-  which they kill the rapidly dividing tumour cells.  However, the exact nature of the  lethal lesion remains the subject of much research and controversy.  The ris-isomer is  much more effective for killing tumour then the corresponding trans-isomer. One plausible explanation is that c/s-DDP forms a lethal intrastrand crosslink (in addition to the known interstrand crosslink which both isomers can form), which is more difficult to be recognized and requires a longer time to repair this type of damage^ (Figure 8).  The DNA binding action of platinum complexes suggested a rationale for combining the use of the Pt drugs with radiotherapy, and indeed interaction between  Figure 8:  P o s s i b l e types o f DNA  i n t e r a c t i o n s w i t h eis-DDP  (adapted from r e f . 55).  22  radiation and c/s-DDP was reported by Richmond and Powers in 1976, who showed that a concentration (50 JJM) of cis-DDP sensitized the spores of Bacillus megaterium especially under hypoxic conditions/  6  In 1978, Douple and Richmond demonstrated  that radiation sensitization of hypoxic mammalian cells was obtained with the use of c/s-DDP, trans-DDP and ds-dichlorobis(aziridine)platinum(II).  Since this time, many  Pt complexes have been examined in various systems as radiosensitizers/ '^'•^'-'* * 0  >,<  0  Platinum complexes possibly act as radiosensitizers by interfering with the cell's repair mechanisms in some way by binding to DNA. Some groups have been studying sublethal and potentially lethal radiation damage, and find that repair of such damage is inhibited by Pt complexes/^  Other groups have suggested that some Pt complexes,  such as cis-DDP and cis, trans, c;'s-dichlorodihydroxybis(isopropylamine)platinum(IV), show enhanced  radiosensitizing  effects  by  reacting  with  compound; ' however, the reasons are still not yet understood/ 6 2  1.11  the  protecting  NPSH  0  Metal Complexes as Radiosensitizers The early studies on metal salts, chosen for their oxidizing ability, mostly used  bacterial systems and no real mechanistic pointers emerged from these results because no uniform parameter of activity was adopted. However, the examples do demonstrate the potential for study and the need for systematic evaluation of closely related complexes (Table I).  The radiosensitizing effects of copper salts in mammalian cells  have been  demonstrated. Whereas Cu(II) salts had little or no toxicity in normal oxygenated cells, incubation under hypoxic conditions and the reduction to Cu(I), or the introduction directly of  cuprous solutions  in the oxygen-free  medium, resulted in enhanced  sensitivity to radiation, with a dose modifying factor of 1.5 being obtained in the  23  Table I  Metal Compounds with Radiosensitization A b i l i t y  Biological Test Systems  Examples  References  Bacterial  ois -DDP  50, 57  Mammalian  Pt(nitroimidazoles)  70  CuCl.  63  Mammalian  [Fe(CN) N0]2-  64  Bacterial  [Fe(CN) ] 3-  65  Mammalian  64  Bacterial  vitamin B. 12 3+ Co(NH )  Ni  Mammalian  Nidapachol^  69  Mn  Bacterial  MnO,  70  Rh  Mammalian  Metal  Ft  Cu  Fe  Co  Bacterial Mammalian  5  6  3  6  Rh(II) carboxylates  Lapachol i s 2-hydroxy-3-isoprenyl-l,4 - naphthoquinone.  66  71  24  mammalian system.  This example of reduction-enhanced cytotoxicity is of particular  relevance because of the role of copper as an essential trace element and its function in many intracellular redox processes.  The nitroprusside salt, [Fe(CN) NO] , was an effective sensitizer in V-79 cells 5  at 10"  M . This effect was attributed to toxic ligand (CN~) release.  [Fe(CN)g]  Ferricyanide  , is a sensitizer of bacterial cells, the activity being attributed to its thiol  binding capacity "^ and no sensitization of oxic cells by this compound was seen. ^ 6  6  A further apparent example of reduction enhanced cytotoxicity with metal complexes is that of the hexamminecobalt(III) ion, [ C o ( N H ) ] , when reduction to 3+  3  Co(II) also resulted in enhanced sensitivity to radiation.  66  6  Cobalt chelates such as  [Co(2,2'-bipy) ] , however, did not sensitize cells to radiation. 3+  3  67  The more recent  results on the interaction of some inert Co(III) complexes with D N A * may well be 6  relevant to these biological results especially as a 1,10-phenanthroline chelate, such as c/5-[Co( 1,10-phenanthroline)2(NO2)2] +  A nickel chelate of lapachol, 2-hydroxy-3-isoprenyl-l,4-naphthoquinone, has also been studied for its radiosensitizing ability.  69  The chelation is through the 2-OH  and adjacent keto group. The results show that the Ni chelate is a moderate sensitizer (hypoxic CHO cells, SER=1.3 at 100 JJM),  and is better than the Cu or Zn complex.  This Ni chelate also enhances radiation-induced breaks in DNA of hypoxic cells.  69  In vitro sensitization of rhodium complexes containing carboxylates has been 7/ reported.  The Rh carboxylate complexes sensitize in both air and nitrogen, with the  greater effect generally occurring in nitrogen. Radiation chemical experiments show the Rh complexes have substantially low reduction potentials and do not undergo electron  25  transfer reactions, adduct formation, or interaction with radicals derived from DNA bases. The increase in radiation sensitivity of cells treated with Rh(II) carboxylates was attributed to the ability of the Rh compounds to deplete intracellular thiols.  1.12  Rationale for Using Metal-Radiosensitizer Complexes The dose limiting toxic side-effects of nitroimidazoles have prevented the  attainment of maximum sensitization in clinical use. ^ -  A desirable aim is to decrease  the overall concentration of radiosensitizer, while retaining a high drug concentration at the target of ionizing radiation damage, the DNA.  If this could be achieved with good  radiosensitizers such as misonidazole, one might be able to modify or decrease the toxic side-effects which currently limit the use of these drugs.  Studies on DNA as a  radiation target may well lead to the development of new species having radiosensitizing and DNA-binding properties.  In this respect, attachment of a radiosensitizer to  transition metals that are known to bind to DNA may be of value. As a consequence of binding to DNA, the metal could act as a carrier, and increase the local concentration of radiosensitizer at the target and thus decrease the deleterious side-effects;  7,2  further,  the metal itself may exert a radiosensitizing effect (see section 1.11), The hypothesis is that use of a complex (M-S) between a sensitizer (S) and a metal template (M), which binds to DNA, may result in a radiosensitizer with a better therapeutic index than either "parent" drug because of improved radiosensitization - the primary sensitizer S would be located at the target DNA, by virtue of the binding properties of the metal. In addition, the template moiety itself could be contributing to the radiosensitization. Decreased toxicity might be expected because the overall concentration of S would be lowered (Figure 9).  Extensive  structure-activity  relationship  using  free  nitroimidazoles  have  established a correlation between the in vitro radiosensitizing efficiency and the one-  S =  Sensitizer  Molecule  + = M e t a l Complex  Distributed  Throughout  Concentrated  a t DNA  +S  =Metal  Sensitizer  Cell M  Figure  9:  Rationale  for using metal-radiosensltizer  complexes.  27  electron reduction potential of the nitro group.  Metal binding renders this potential  more positive because of the inductive effect resulting from bond formation of the lone-pair nitrogen of the imidazole ring to the metal.  '  In principle, this allows for  "fine-tuning" of the reduction potential of the nitro group, and more  efficient  sensitization.  If  attachment  of  a  metal  to  radiosensitization drastically or increase  a  sensitizer  toxicity,  moiety  does  not  this proposed design  diminish will have  advantages over straightforward combination of the two types of drugs. "* However, a 7  combination of chemical stability, solubility, and pharmacological factors (e.g. partition coefficients, distribution) will also need to be optimized.*'* -  1.13  Metal-Nitroimidazole Complexes as Radiosensitisers Platinum is one candidate for such a carrier because of its known binding 52 53  properties to DNA. '  J  Some Pt complexes are already used as chemotherapeutic  agents'* " " ^- * and some also have moderate radiosensitizing ability." 9  50  5  5-  addition, Pt drugs exhibit their chemotherapeutic  57,60  ' ^' 6  73  In  and radiosensitizing ability at  micromolar levels, whereas much higher, overall cellular concentrations of misonidazole are required to achieve optimum radiosensitization. Initial results on m-tPtC^metronidazole^] (FLAP) were promising with a reported SER of 2.4 and little toxicity, ^ but later studies '*' ""' failed to corroborate 7  7  7  these findings and the complex was at best a weak to moderate radiosensitizer.  A  general problem with the bis(nitroimidazole) complexes is their lack of water solubility. Methods to circumvent this problem include the syntheses of complexes with only one nitroimidazole and also containing an ammine ligand. One example is [PtC^CNH^X^NO2-I1T1)]  which  showed  DNA  binding  property  as  well  as  having  better  28  radiosensitizing enhancement than the bis(nitroimidazole) complexes.  76  Palladium has been used in an attempt to target nitroimidazoles but these 77 complexes generally dissociated in solution.  However, a postirradiation-sensitizing  effect was observed in vivo (KHT sarcoma), possibly because of Pd ions exerting an effect on the repair of potentially lethal damage. 75 Chibber et al.  have been assessing the interaction of Rh(II) species with 7 5 70  radiation, and have used Rh(II) to target misonidazole and analogs.  '  A Rh complex  was better than RSU-1111 (a 2-nitroimidazole ligand) alone, or the corresponding platinum complex (RSU-1113), in sensitizing hypoxic V79 cells. ^ 7  1.14  Ruthenium Complexes as Anticancer Agents The metal, ruthenium, is used in the present study for the development of  metal-based radiosensitizers.  Approaches using ruthenium as antineoplastic agents have  been related to the mechanistic action of the clinically employed c/s-DDP, namely, via on  DNA binding. consequences  In both their initial DNA binding site and in the oncological of  their DNA interactions, ruthenium ammine complexes  [Ru(NH )jH20]^ resemble m-DDP. +  3  such as  Their activity is thought to be exerted, at least  initially, by coordination to guanine N7 sites on cellular nucleic acids.  In vitro biochemical studies demonstrate that Ru(II) and Ru(III) compounds are active in inhibiting DNA synthesis and possess mutagenic activity in the Ames Salmonella and related assays.  It has been suggested that ruthenium complexes with  bipyridine ligands intercalate into DNA prior to covalently residues.  binding to guanine  Preliminary studies of the binding of ruthenium cations to DNA indicate  that advantage can be taken of their inertness to ligand substitution once coordination  29 has been established at a DNA site.  Once coordinated to a nitrogen base, Ru(II) and o  c  Ru(III) usually remain bound to the same base. from the  Additional advantage can be taken  ready availability of both Ru(II) and Ru(III) oxidation states under  physiological conditions,  whereby the drug is inactive in oxic cells as Ru(III), but  which becomes active as a radiosensitizer upon reduction to Ru(II) in hypoxic cells. 1.15  Nature of Sulphoxides as Ligands The sulphoxides, of general formula R2SO, are examples of ambidentate ligands  which may bind to metal ions through either the sulphur or oxygen.  Both atoms have  lone pairs of electron, enabling the sulphoxide to act as a Lewis base.  The three  resonance structures for the S-O bond are shown in Figure 10; structure II is R7  dominant.  Both steric and electronic effects have been shown to dictate the choice of  donor atom and these effects may be summarized** as follows: (a)  Steric Effects: These may be manifested by the sulphoxide, and other ligands in  the coordination sphere.  (i)  effect of sulphoxide ligand Both the [PdCl (dmso)2] and [PdCl (mbso) ] (mbso = methyl-3-methylbutyl 2  2  2  sulphoxide) complexes have both sulphoxides bound through sulphur; however, the op  sterically more demanding mbso ligand results in a trans configuration,  as opposed to  this ds-geometry of the dmso analogue. (ii)  other ligands This is demonstrated in complexes of the type [M(dppe)(dmso)Cl] (where dppe +  =  l,2-bis(diphenylphosphino)ethane,  M=Pd,  Pt).  Bonding through  sulphur is  disfavoured because of interaction of the methyl groups with the phenyl groups of the  30  >s-g  ^s=6=  >s^o.  i  n  i n  gure 10:  The t h r e e resonance s t r u c t u r e s f o r the S-0 bond o f sulphoxides.  31  bulky phosphine ligand, and thus bonding at the sterically-unrestricted oxygen is preferred/  0  (iii) size of central metal ion Within [FeCl (dmso) ] 2  the i  4  same  OJ  is all O-bonded,  O-bonded dmso ligand.  (b)  column  of  the  Periodic Table,  the  complex trans-  whereas c/s-[RuCl (dmso) ] has three S- and one 2  4  OJ  Electronic Effects:  A graphic demonstration of how purely electronic effects  control the mode of coordination of the sulphoxide ligand is demonstrated in dmso adducts of rhodium(II) carboxylates, [Rh (0 CR) (dmso)2]. When R=CH or C H , S2  2  4  coordination of dmso occurs, but when R=CF  3  3  2  5  the highly electronegative C F  3  substituents force preference for O-bonding/''  1.16  Biological Activity of Metal-dimethyl sulphoxide Complexes Intensive research has been carried out to study the mechanism of action of cis-  DDP and to develop suitable analogues with better pharmacological properties by improvement of lipophilicity, lower toxicity and higher water-solubility/^  Metal  complexes with dmso as ligands are generally water-soluble species, and the lipophilicity of dmso and its low toxicity have been of interest in applications of transition metal chemotherapy/'*  The most comprehensively studied complex is m-RuCl (dmso) / ^$$$6 7,9  2  4  I n  a series of studies comparing its biological activity with that of c/s-DDP, the Ru species 97 was shown to produce filamentous growth in E. coli and induce alpha prophage.  The  Ru complex is mutagenic in strain S. typhimurium, carrying the His mutation, specific for base pair substitution mutagens; QO it has in vivo anti-tumour activity at doses of  32  4 0 0 mg/kg, which indicates a high L D ^ Q for this complex.  Other metal-sulphoxide  complexes such as [Ru(dmso) ] , [RhC^dmso^], [RhCl(dmso) ] and [Pt(dmso) ] 2+  2+  6  5  2+  4  have similar behaviour but none of the complexes show more anti-tumour activity than c/5-RuCl2(dmso) ." 4  Apart from the understanding of the mechanism of anti-tumour action of cisDDP, a major interest in the area of nucleic acid-metal ion interactions is the possibility of sequence determination, using specific  binding of metal complexes.  Reactions using [PtCl^dmso)]" with DNA and RNA were found, in which the complex could be used as a differential staining agent for adenine and guanine residues in both polynucleotides/  00  The differential staining is made possible by the binding of two Pt  atoms per adenine residue at both pH 6 . 0 and 7 . 5 , while with guanine at the lower pH, one Pt, and at higher pH, two Pt atoms are b o u n d /  00  An interesting difference emerges between the octahedral Ru(II) and Rh (III) systems and the square planar Pt(II) cases; with adenine, only monodentate binding via N 7 is found for both Ru and Rh systems, whereas with the Pt system, binding can be via N 7  of adenine or guanine/ ^ 0  Hydrogen-bonding between the exocyclic 6 - N H  2  group of adenine and the dmso oxygen atoms or chloride atoms (both H-bond 102  acceptors)  in the octahedral sphere may dictate specificity, these interactions being  minimized in a square-planar complex/ "^ 0  Dimethyl sulphoxide, as a ligand, has been shown to possess many of the properties desirable for metal-based chemotherapeutic agents.  Complexes containing 92 93 103  dmso, rather than ammine, result in more water-soluble, less toxic derivatives.  ' *  In chemical studies, liganded dmso may be displaced by pyrimidine and purine bases,  33  the  products and binding sites being  dictated  by the  nature  of  the starting  complexes/ *' * -  1.17  70-  Short-term In-Vitro Assays Numerous short-term in vitro test systems have been developed to demonstrate  whether or not a chemical is a genotoxic substance.  Some of the genetic damages  employed for this purpose include mutation, DNA damage, chromosome aberration, and gene conversion/ ** 0  These sensitive, rapid, and economical tests for genotoxicity are  also applied to predict the carcinogenic properties of individual compounds ^ or 70  mixtures of chemicals/  06  The usefulness of the tests is based mainly on a fairly good  correlation between results of in vivo carcinogenicity tests on various rodents and in vitro data from genotoxicity assays/  07  Before a metal-based chemical compound can be introduced as an effective radiosensitizer, it would be useful to know whether such a complex exerts a genotoxic effect on normal cells.  The genotoxic effect of some Pt coordination compounds has  been studied by these short-term in vitro assays, which have shown to induce sister chromatid  exchange,  aberrations/ *" 0  777  mutation,  micronuclei  induction,  and  chromosomal  In addition, some Rh(I) and Ru(II) complexes which have anti-  tumor activity have also been shown to raise the number of revertants of S. typhimurium over the background level (i.e. the complexes possess mutagenic activity for bacteria)/  1.18  72  Objectives The purposes of this project were to synthesize and characterize certain Ru-  nitroimidazole complexes with potential as radiosensitizers as adjuncts to radiotherapy, and to study the factors which may contribute to the sensitizing process.  Based on the  literature data available as described above, and expertise available through earlier  3 4  research work in the UBC chemistry department on Ru/sulphoxide systems,  • '  the compounds initially selected for the present work were dihalidebis(sulphoxide) complexes  of  ruthenium(II), containing also known radiosensitizing nitroimidazole  ligands. The potential of such Ru-radiosensitizer complexes as adjuncts to radiation is studied largely in vitro for different sulphoxides and halides complexes in mammalian cells. Certain studies addressing mechanisms of radiosensitization and some preliminary in vivo data are presented.  35  CHAPTER TWO MATERIALS AND METHODS 2.1  Chemicals The 4-nitroimidazole, 2-nitroimidazole, Ellman's reagent  nitrobenzoic  acid)),  aminomethane  tris-(hydroxymethyl)aminomethane  hydrochloride]  (Tris-Cl),  and  (Tris),  disodium  (5,5' -dithiobis-(2[tris-(hydroxymethyl)  ethylenediaminetetraacetate  dihydrate (EDTA) were purchased from Sigma Co. Ltd. (St. Louis, MO), while tetramethylene sulphoxide (tmso), methylphenyl sulphoxide (mpso), N-methylimidazole, 2-methylimidazole, metaphosphoric acid, ds-DDP, 1,10-phenanthroline, 5-NO -l,102  phenanthroline and 5-C1- 1,10-phenanthroline were from Aldrich Chemical Company (Milwaukee, WI).  Metronidazole (metro) was obtained from Poulenc Ltd. (Montreal,  Quebec) and Eagle's Minimal  Essential Medium (MEM),  penicillin, streptomycin  sulphate, kanamycin and fungizone were obtained from Gibco Canada Inc. (Burlington, Ontario).  Fetal calf serum was a Bocknek (Ontario) product. The restriction enzymes  PvuII, BamHI and EcoRI were purchased from Bethesda Research Laboratories Life Technologies Inc. (Gaithersburg, MD). The R u C l 3 H 0 (35% Ru) was received on loan 3  2  from Johnson Matthey Ltd. and was used as provided.  Misonidazole (miso) and  Desmethylmisonidazole (De-miso) (Figure 11) were donated by Roche Products Limited (Welwyn, U.K.).  Of the 4-nitroimidazole ligands, RSU-1170, RSU-3100 and RSU-3159  were kindly supplied by Dr. T.C. Jenkins,^  and RSU-3083 by Dr. I.J. Stratford^  (Figure 12). The compound SR-2508-. was a gift from the National Cancer Institute of the United States.  These chemicals were used without further purification.  common reagents used were at least reagent grade.  All other  Methanol was dried by distilling  over Mg metal turnings for at least four hours before being collected and used. The  b The RSU terminology is used by Drs. T.C. Jenkins and I.J. Stratford (Medical Research Council, Harwell, Didcot, England), the suppliers of these ligands whose structures are given in Figure 12; see for example ref. 114. c SR = Stanford Research.  36  R = H  2-nitroimidazole (2-NO -Im) OH  R = CH -CH-CH ~OH 2  2  Desmethylmisonidazole (De-miso)  0  II R = CH2 -C-N-CH | 2-CH2OH H  = Etanidazole (SR-2508)  OH I  R = CH -CHCHOCH  F i g u r e 11:  = Misonidazole  S t r u c t u r a l formulae o f t h e 2 - n i t r o i m i d a z o l e l i g a n d s .  37  R = H,  R =CH ,  R' = H  R" = H : 4 - n i t r o i m i d a z o l e  R' = H,  R' = H,  R =CH ,  R' = N-CH -CH -OH, 2  R =CH ,  R' = OPh,  R =CH ;-,  R' = \\ u  2  3  3  2  R" = CH  R" = C H  3  3  (NMe-4-NO -Im)  : RSU-1170  : RSU-3083  R" = H : RSU-3100  / ...  f—NH  3  2  R" = H : N - m e t h y l - 4 - n i t r o i m i d a z o l e  R =CH CH(OH)CH N^] , 2  (4-N0 ~Im)  •,  R" = H : RSU-3159  S R =CH , 3  R' =C1,  F i g u r e 12:  R" = H : CMNI  S t r u c t u r a l formulae o f the 4 - n i t r o i m i d a z o l e l i g a n d s .  38  procedures for preparing stock solutions, buffers, rat liver S9 fractions, Sephadex G-50 columns and linearized pSV2-gpt plasmid are given in Appendices I-VI.  2.2  Physical Techniques and Methods Infrared spectra (KI discs or Nujol mulls) were recorded on a Nicolet DX FTIR  spectrometer; *H and C{*H} nmr spectra (in 13  C D C I 3 vs. TMS) were recorded on a  Varian XL-300 or Bruker WH-400 instrument operating in the Fourier transform mode. Conductivity data were measured in water at RT under N  2  with a conductivity cell  connected to a Beckman Serfass Conductance Bridge. The pH-titrations were carried out in aqueous media using a Corning 12 pH-meter and a combination glass-electrode: typically, 25 mL of aqueous HC1 (2 mM), in the absence and presence of added complex (2 mM), were titrated with standard 4 mM NaOH solution; differences between the two titration curves allow for a simple determination of p K values. a  77-5  In each experiment,  about fifteen measurements were made, and care was taken to ensure that each pH reading recorded represented equilibrium conditions.  Carbon dioxide was excluded by  maintaining a flow of nitrogen through the titration cell fitted with a cap.  Optical  spectra were recorded on a Perkin Elmer 552A spectrometer with 1 cm path length quartz cells in air. For recording UV/VIS spectra under anaerobic conditions, one type of anaerobic cell as shown in Figure 13 was employed.  A solid compound was put in  the L tube; a solvent was placed in the 1 cm path length quartz cell, and the two mixed under N . 2  Microanalyses were performed by Mr. P. Borda of the department of  Chemistry, University of British Columbia.  2.2.1  X-rav Photoelectron Spectroscopy The XPS data were taken on a Varian IEE-15 spectrometer at a  background pressure < 10  torr, using MgK radiation (1253.60 eV) at 280 W  and an analyzer pass energy of 100 eV. Resolution was 1.6 eV at full width, half maximum on a gold standard, Au(4f ). 7/2  Each sample was lightly dusted onto  39  V  Figure  13:  i  Schematic r e p r e s e n t a t i o n o f an anaerobic  spectral  40  single-sided 3M scotch tape and introduced into the spectrometer on a rapid insertion probe cooled by liquid N . 2  All binding energies (BE) were calibrated  with reference to the C Is peak (284.0 eV) of graphite. There was no evidence for any sample deterioration during irradiation.  2.2.2  Electrochemical Methods  (i)  Polarographic Measurements The reduction potentials of the compounds were determined using a  Princeton Applied Research Model 364 polarographic analyzer with a dropping mercury electrode.  In a typical experiment, the compounds were dissolved in  0.01 M phosphate-buffered saline (PBS) solution at concentration of 500 JJM solution.  One mL of this solution was then added to the measurement vessel  which contained 9 mL of a 0.01 M PBS solution previously purged with N . The 2  half-wave reduction potential (Ej^ ) of the compound was measured in the 2  vessel at RT, under N , in mV versus a saturated calomel electrode. 2  (ii)  Cyclic Voltammetric Measurements To obtain cyclic voltammograms a standard three compartment H-cell,  fitted with fine frits, was used in conjunction with an E G and G PARC Model 175 Universal Programmer (controlling the range of sweep and the scan speed) linked to a model 173 PAR Potentiostat (controlling the potential of the auxiliary electrode).  The traces were photographed by a Tektronics oscilloscope camera  (C-12) using Polaroid film.  In a typical experiment the compounds were  dissolved in 0.01M PBS solution and the solution purged with argon via a teflon lead for about 10 min. The teflon lead was then raised above the level of the liquid to keep a layer of argon above the solution. The solution was periodically stirred and recharged with argon although before a cyclic voltammogram was  41  run, the agitation of the solution had to be stopped.  The scan speed used was  500 mV/s.  2.3  Preparation of N-methvl-4-nitroimidazole The  compound  N-methyl-4-nitroimidazole  according to a literature procedure.  776  (NMe-4-NC>2-Im)  was prepared  Concentrated HNO^ (25 mL) was added slowly  to N-methylimidazole (10 mL, 0.12 mol) and the reaction mixture cooled to 4°C on an ice-bath, when a vigorous reaction took place.  Concentrated H S 0 2  4  (25 mL) was then  added slowly to the reaction mixture, which was then boiled gently until all nitrous fumes were gone (approximately 4 h). The mixture was then poured into 50 mL of ice and distilled water (1:1) and made basic with concentrated ammonia (approximately 50 mL).  The mixture was then extracted 3 times with CHCI3 (150 mL total), the non-  aqueous layer was collected, and the solvent removed completely by rotovap to give a white solid (3.88 g, 25% yield).  Anal, calcd. for C H N 0 : C 37.76, H 3.93, N 33.04; 4  5  3  2  found: C 37.60, H 3.92, N 33.00.  2.4  Preparation of Complexes The spectroscopic data (UV/VIS, IR, nmr, XPS) for the ligands and complexes  are discussed later in sections 3.1-3.3.  2.4.1  m-RuCl (dmso) . 1 2  (i)  4  The precursor complex ds-RuCl (dmso) , which contains one 2  O-bonded and three S-bonded sulphoxides^ procedure/  6  4  was prepared by a literature  Some RuCl "H 0 (3 g, 11 mmol) was refluxed together with dmso 3  2  (8 mL) for 2 h under N . The resulting dark orange solution was then cooled to 2  RT, and acetone (40 mL) was added slowly to precipitate out a yellow product which was filtered off in air, washed with acetone and diethylether, and dried in  42  vacuo at R T (1.18 g, 63% yield). Anal, calcd. for C o H 0 S C l R u : C 19.83, H 2 4  4.96, Cl 14.65; found: C 19.78, H 4.83, Cl 14.69. A  max  4  4  2  nm(log e), H 0 : 350 2  (2.69), 300 (2.45). (ii)  Another route for making 1 was used, according to that reported  by James et al.  95  Some R u C l j ^ f ^ O (3g, 11 mmol) was added to previously  dried methanol (50 mL), in a two-necked flask equipped with gas-inlet tube, reflux condenser and magnetic stirrer. The solution was refluxed under H  2  (1  atm) overnight (approximately 16 h) to give a deep blue color. To this Ru blue methanolic solution was added 4 mL of dmso. continued for 16 h to yield a red solution.  Refluxing under H  2  was  No reaction was observed when the  refluxing was carried out under N or Ar. The solution was cooled to yield 1.0 g 2  which were filtered off in air. Found: C 19.67, H 5.04,  (59%) of yellow cubes, Cl 14.39.  2.4.2  RuCl (tmso) . 2 2  4  To the Ru blue solution, as mentioned above, was added 3 mL of tmso. Refluxing under H  2  was continued for 6 h yielding an orange solution,  which was cooled to RT when a yellow precipitate formed. Again no reaction was observed when the refluxing was carried out under N  2  or Ar. The solid  was filtered off in air, washed with acetone and dried in vacuo at RT (1.62g, 70% yield).  Anal, calcd. for C , H 0 S C l R u : C 32.61, H 5.48, S 21.74; 6  found: C 32.49, H 5.50, S 21.67. A  2.4.3  max  3 2  4  4  2  nm(log C), H 0 : 343 (2.54), 303 (2.37). 2  "RuCl (mpso) ". 3 2  2  The complex "RuCl (mpso) " was prepared according to a literature 2  procedure.^"*  2  To the blue Ru methanolic solution prepared as described in  section 2.4.1(H) was added mpso (1.3 g, 9.3 mmol), and the solution refluxed  43  overnight under H . The solution turned brownish green and a yellowish orange 2  precipitate was obtained when the solution was cooled to RT. The precipitate was filtered off in air, washed with ethanol and acetone and dried in vacuo at RT (2.9 g, 69% yield).  Anal, calcd. for  C H 0 S C l R u : C 37.16, H 3.57, CI I 4  1 6  2  2  2  15.90; found: C 36.81, H 3.68, CI 15.68.  2.4.4  frans-RuBr (dmso) .4 2  4  Complex 1 (483 mg, 1 mmol) dissolved in C H C 1 (75 mL), and NaBr 2  2  (205 mg, 2 mmol) dissolved in a minimum amount of water (approximately 3 mL), were mixed together, and sufficient methanol (approximately 40 mL) then added to make the two layers miscible. (approximately 16 h) under N  This mixture was refluxed overnight  to give an orange solution, which was roto-  2  evaporated to dryness. The resulting pale orange solid was dissolved in dmso (3 mL) and the solution heated for 1 h. Acetone (approximately 25 mL) was added to precipitate a pale orange solid, which was filtered off in air, washed with acetone/water mixture (2:1) and acetone, and then dried in vacuo at RT (0.34 g, 60% yield). Anal, calcd. for C H 0 S B r R u : C 16.76, H 4.22, S 22.36; found: g  C 17.01, H 4.24, S 22.46. A  2.4.5  m a x  2 4  4  4  2  n m (log C), H 0 : 465 (2.32), 314 (2.17). 2  RuCl (dmsoW4-NQ Im) . 5 2  2  2  Dry methanol (15 mL) was added to RuCl (dmso) (483 mg, 1 mmol) 2  4  and 4-N0 -Im (282 mg, 2.5 mmol), and the mixture refluxed for 4 h under N . 2  2  The resulting brown solution was cooled to RT and diethylether (40 mL) added slowly to precipitate out a brown product, which was filtered off in air, washed with acetone and diethylether, and dried in vacuo at RT (0.36 g, 65% yield). Anal, calcd. for C H p N S C l R u : C 21.66, H 3.25, N 15.16; found: C 22.00, 1 0  l g  6  6  2  2  H 3.06, N 15.38. UV/VIS - see text, section 4.2.  44  RuCl (dmsoWNMe-4-N0 Im). 6  2.4.6  2  2  The complex RuCl (dmso) (483 mg, 1 mmol) was suspended in an 2  4  isopropanol solution (20 mL) containing NMe-4-N0 -Im (254 mg, 2 mmol), and 2  the mixture was refluxed for 4 h under N .  The resulting blue solution was  cooled to RT, when a blue precipitate formed.  This was collected, dissolved in  2  C H C 1 (10 mL), reprecipitated with diethylether (20 mL), filtered off in air and 2  2  dried in vacuo at RT (0.36 g, 8 0 % yield).  Anal, calcd. for C o H 0 N S C l R u : 1 7  C 21.06, H 3.73, N 9.22; found: C 20.99, H 3.93, N 9.25. A  m a x  4  3  2  2  nm(log £ ) , H 0 : 2  303 (4.32), 427 (3.06).  RuCWdmso) (RSU-l 170) . 2  2.4.7  2  The  2  nitroimidazole RSU-1170 (226 mg, 1 mmol) was mixed with  RuCl (dmso) (242 mg, 0.5 mmol) in dry methanol (10 mL), and the mixture 2  4  refluxed for 4 h under N . The resulting green solution was cooled to RT, and 2  diethylether (30 mL) added slowly to precipitate out a green product, which was filtered off in air, washed with acetone and dried in vacuo at RT (0.25 g, 6 5 % yield). Anal, calcd. for C H Q O O N O S C 1 R U : C 33.85, H 5.13, N 14.35; found: C 2 2  33.51, H 5.39, N 13.91.  A  4  2  m a x  n m (log  2  e) H  2  0 : 310 (4.39). ,  RuCl (dmsoWRSU-3083) . 8  2.4.8  2  The  2  compound  RSU-3083  (200 mg,  1 mmol)  was  added  to  RuCl (dmso) ((242 mg, 0.5 mmol) in ethanol (10 mL), and the mixture refluxed 2  4  for 4 h under N->. The resulting red solution was cooled to RT and filtered; slow addition of diethylether (30 mL) yielded a red solid, that was filtered off in air, washed with ethanol and acetone, and dried in vacuo at RT (0.25 g, 7 0 % yield).  45  Anal, calcd. for C H OoN S Cl Ru: C 29.6, H 4.98, N 15.37; found : C 29.34, 16  H 4.64, N 15.66. A  2.4.9  36  max  8  2  2  nm(log £ ), H 0 : 385 (4.40), 450 (4.12). 2  RuCl (dmso) (RSU-3100) . 9 2  2  2  The complex RuCl (dmso)4 (242 mg, 0.5 mmol) was suspended in an 2  isopropanol (10 mL) solution containing RSU-3100 (219 mg, 1 mmol) and the mixture refluxed for 6 h under N .  The resulting brown solution yielded a  2  brown product as in the synthesis of 5. (0.25 g, 65% yield).  Anal, calcd. for  C H O N S C l R u : C 37.58, H 3.92, N 10.96; found C 37.72, H 4.34, N 2 2  3 0  8  6  10.68. A  m a x  2.4.10  2  2  n m (log e), H 0 : 350 (4.13), 396 (4.09). 2  RuCl (dmso) (RSU-3159). 10 2  2  The complex RuCl (dmso) (242 mg, 0.5 mmol) was added to RSU2  4  3159 (225 mg, 1 mmol) in methanol (15 mL), and the mixture refluxed for 3 h under N . 2  The resulting dark green solution was filtered hot, and diethylether  (30 mL) added slowly to the cooled filtrate to precipitate a green solid; this was filtered off in air, washed with acetone and diethylether, and dried in vacuo at RT (0.18 g, 75% yield). Anal, calcd. for Cj j H j g C ^ N ^ C ^ R u : C 23.87, H 3.46, N 12.65; found: C 24.13, H 3.79, N 12.53. A  2.4.11  max  nm(log C ) H 0 : 325 (4.22). 2  RuCl (dmso) (miso) . 11 2  2  2  The complex RuCl (dmso) (483 mg, 1 mmol) was added to miso (402 2  4  mg, 2 mmol) dissolved in toluene (10 mL), and the mixture refluxed for 6 h under N . 2  The resulting blue solution was reduced in volume, and hexanes (30  mL) added slowly to give a blue oily product. liquid N  2  The oily product was cooled in  to yield a blue precipitate, which was filtered in air, washed with  diethylether and dried in vacuo at RT (0.36 g, 50% yield). Anal, calcd. for  46  C H O N S Cl2Ru: 1 8  3 4  1 0  11.86. A  6  m  2.4.12  a  2  x  C 29.63, H 4.69, N 11.51; found: C 29.81, H 4.95, N  nm(log t) EtOH: 325 (3.82), 638 (1.84).  RuClofdmsoWDe-miso^. 1 2 Dry methanol (15 mL) was added to RuCl (dmso) (483 mg, 1 mmol) 2  4  and De-miso (374 mg, 2 mmol), and the solution refluxed for 4 h under N . 2  The resulting blue solution was filtered hot, and the filtrate cooled to RT to give a blue precipitate. This was filtered off in air, washed with diethylether (20 mL) and  dried  in  vacuo at  RT  (0.55  g,  78%  yield).  Anal,  calcd.  for  C , H O N S C l R u : C 27.35, H 4.30, N 11.96; found: C 27.15, H 4.27, N 6  3 0  1 0  12.07. A  2.4.13  6  max  2  2  n m ( l o g e) EtOH: 332 (3.57), 584 (2.01).  RuCl (dmsoW2-NQ -ImU. 13 2  2  The ligand 2-N0 -Im (282 mg, 2.5 mmol) was added to RuCl (dmso) 2  2  4  (483 mg, 1 mmol) in dry methanol (15 mL), and the solution refluxed for 6 h under N . The resulting blue solution was cooled to RT, when slow addition of 2  diethylether (40 mL) gave a blue precipitate, which was filtered off in air, washed with hexanes and diethylether, and dried in vacuo at RT (0.36 g, 65% yield). Anal, calcd. for C H O N S C l R u : C 21.66, H 3.25, N 15.16; found: 1 0  C 21.32, H 3.37, N 15.31. A  2.4.14  ] 8  max  6  6  2  2  n m ( l o g Z ), EtOH: 332 (3.72), 420 (2.34).  RuCl ( dmsoW metroW 14 2  The metronidazole (342 mg, 2 mmol) was mixed with RuCl (dmso) 2  4  (483 mg, 1 mmol) in dry methanol (15 mL), and the solution refluxed for 4 h under N . The resulting brown solution was filtered, and diethylether (40 mL) 2  added slowly to the filtrate to precipitate a brownish yellow solid; this was filtered off in air, washed with acetone and diethylether, and dried in vacuo at  47  RT (0.45 g, 67% yield). Anal, calcd. for C J ^ Q O O N ^ C ^ R U : C 28.66, H 4.51, N 12.53; found: C 28.81, H 4.58, N 12.26. A  2.4.15  max  nm(log Z ), H 0 : 320(3.82). 2  RuCl (tmsoW4-NQ Im) . 15 2  2  2  Dry methanol (15 mL) was added to RuCl (tmso) (588 mg, 1 mmol) 2  4  and 4-N0 -Im (282 mg, 2.5 mmol), and the solution refluxed for 4 h under N . 2  2  The resulting brown solution was roto-evaporated to a reduced volume ( 3 mL) and diethylether/hexane (1:1) mixture (30 mL) added slowly to precipitate out a brown product, which was filtered off  in air, washed with acetone and  diethylether, and dried in vacuo at RT (0.33 g, 55% yield).  Anal, calcd. for  C H 0 N S C l R u : C 27.72, H 3.65, N 13.86; found: C 27.84, H 3.48, N 1 4  2 2  6  6  13.95. A  2.4.16  m a x  2  2  n m (log e), EtOH: 330 (3.88).  RuCl (tmso) (NMe-4-NQ -Im) . 16 2  2  2  2  The complex RuCl (tmso) (588 mg, lmmol) was suspended in a 2  4  methanol solution (20 mL) of NMe-4~N0 -Im(254 mg, 2 mmol), and the mixture 2  refluxed for 6 h under N . 2  The resulting blue solution was filtered hot and  cooled to RT; slow addition of diethylether (30 mL) yielded a blue solid, which was filtered off in air, washed with acetone, and diethylether, and dried in vacuo at R T (0.44 g, 69% yield).  Anal, calcd. for C H 0 N S C l R u : C 30.28, H 1 6  4.13, N 13.25; found: C 30.45, H 4.26, N 13.59.  2 6  A  6  6  2  2  m a x  n m (log C ) , H 0 : 309 2  (3.92), 413 (2.87).  2.4.17  RuCl (tmso) (De-miso) . 17 2  2  2  The De-miso ligand (374 mg, 2 mmol) was added to RuCl (tmso) 2  4  (588 mg, 1 mmol) in dry methanol (10 mL), and the solution refluxed for 6 h under N . 2  The resulting blue solution was cooled to RT and filtered; slow  48  addition of diethylether (30 mL) to the filtrate yielded a blue solid, which was filtered off in air, washed with acetone and dried in vacuo at RT (0.54 g, 72% yield). Anal, calcd. for C 2 H O N S C l 2 R u : 0  34  C 32.22, H 4.84, N 11.29. A  2.4.18  10  m a x  6  C 31.83, H 4.54, N 11.13; found:  2  n m (log C), H 0 : 330 (3.24), 532 (1.93). 2  RuCi2(tmsoWSR-2508). 18 The SR-2508 ligand (428 mg, 2 mmol) was added to RuCl (tmso) 2  4  (588 mg, 1 mmol) in dry methanol (10 mL), and the solution refluxed for 4 h under N . The resulting blue solution was filtered hot and diethylether (30 mL) 2  added slowly to the cooled filtrate to precipitate a blue solid; this was filtered off in air, washed with acetone and diethylether, and dried in vacuo at RT (0.44 g, Anal, calcd. for C H 0 N S C l R u :  74% yield).  1 5  A  found: C 30.38, H 4.73, N 9.04.  2.4.19  2 6  6  4  2  C 30.30, H 4.41, N 9.43;  2  nm(log e), H 0 : 341(3.39), 495 (1.82).  max  2  RuCl (tmso) (CMNI). 19 2  3  The compound CMNI (322 mg, 2 mmol) was mixed with RuCl (tmso) 2  4  (588 mg, 1 mmol) in dry methanol (10 mL), and the solution refluxed for 6 h under N . The resulting green solution was cooled to RT, and diethylether (30 2  mL) was added slowly to precipitate out a green product. This was filtered off, washed with acetone and dried in vacuo at RT (70% yield). C  16 28°5 3 3 H  N  S  C 1  3  R u :  C  2 9  '  7 4  '  H  4  -  3 7  '  N  6  -  5 0  ;  f o u n d :  C  3 0 0 1  »  H  Anal, calcd. for 4  - » 4 2  N  6  -  7 5  -  A j ^ n m (logC), H 0 : 320 (3.71), 381 (2.71). 2  2.4.20  RuCl (tmso)(RSU-3159). 20 2  The complex RuCl (tmso) (588 mg, 1 mmol) was added to RSU-3159 2  4  (450 mg, 2 mmol) in methanol (15 mL), and the mixture refluxed for 6 h under N . 2  The resulting dark green solution was filtered hot, and diethylether (40 mL)  49  added slowly to the cooled filtrate to precipitate a green solid; this was filtered off  in air, washed with acetone and diethylether, and dried in vacuo at RT  (0.31g, 62% yield).  Anal, calcd.for C  {  J H J J O J N ^ C ^ R U :  13.97; found;C 26.33, H 2.93, N 13.74. A  2.4.21  m a x  26.34, H 3.01,  C  N  n m (log £ ) H 0 : 330 (3.89). 2  RuBr (dmsoW4-NQ -Im) . 21 2  2  2  Dry methanol (15 mL) was added to RuBr (dmso) (573 mg, 1 mmol) 2  4  and 4-N0 -Im (282 mg, 2.5 mmol), and the solution refluxed for 4 h under N . 2  The  2  resulting brown solution was cooled to RT and diethylether (40 mL) added  slowly to precipitate out a brown product, which was filtered off in air, washed with acetone and diethylether, and dried in vacuo at RT (0.45 g, 70% yield). Anal, calcd. for C H O N S B r R u : C 18.67, H 2.82, N 13.06; found:C 18.43, 1 0  H 2.71, N 12.78. A  2.4.22  l g  max  6  6  2  2  n m ( l o g £ ), EtOH: 347 (4.17).  RuBr (dmsoWNMe-4-N0 -im). 22 2  The  2  complex RuBr (dmso) (572 mg, 1 mmol) was suspended in a 2  4  methanol solution (15 mL) of NMe-4-N0 -Im (254 mg, 2 mmol), and the 2  mixture refluxed for 4 h under N . 2  RT,  The resulting blue solution was cooled to  when a blue precipitate formed.  This was collected,  washed with  diethylether (20 mL), and dried in vacuo at RT (0.38 g, 70% yield). Anal, calcd. for  C H 0 N S B r R u : C 17.65, H 3.15, N 7.72; found: C 17.72, H 3.31, N 8  7.84.  2.4.23  1 7  A  4  m a x  3  2  2  n m (log £ ) , H 0: 306 (4.28), 440 (3.01). 2  RuCWdmsoW 1.10-phenanthroline). 23 The  complex RuCl (dmso) (483 mg, 1 mmol) was added to 1,102  4  phenanthroline monohydrate (0.396 mg, 2 mmol) in methanol (15 mL), and the solution refluxed for 6 h under N . The resulting brown solution was cooled to 2  50  RT, when a brownish yellow precipitate formed.  This was filtered off in air,  washed with acetone and diethylether, and dried in vacuo at RT (0.40 g, 78% yield). Anal, calcd. for C H 2 O 2 N 2 S C i 2 R u : C 37.78, H 3.94, N 5.51; found: C 16  37.52, H 3.83, N 5.33.  A  2.4.24  2  2  0  m a x  n m (log £ ) , H 0 : 265 (3.98), 332 (2.42). 2  RuCl2(dmso) (5-Cl-1.10-phenanthroline). 24 The 5-C1-1,10-phenanthroline (0.321 g, 1.5 mmol) was mixed with  RuCl (dmso) 2  4  (483 mg, 1 mmol) in dry methanol (15 mL), and the mixture  refluxed for 4 h under N . The resulting brown solution was filtered hot, and a 2  brown precipitate formed when the filtrate was cooled.  This was filtered off in  air, washed with acetone and diethylether, and dried in vacuo at RT (0.38 g, 70% yield). Anal, calcd. for C H 0 2 N S C l 2 R u : C 35.41, H 3.50, N 5.16; found: C 16  35.62, H 3.72, N 5.02. A  2.4.25  max  19  2  2  nm(log e), H 0 : 267(3.84), 328 (2.57). 2  RuCl2(dmsoW5-NO2-1.10-Phenanthroline^. 25 The 5-N0 -1,10-phenanthroline (0.338 mg, 1.5 mmol) was mixed with 2  RuCl2(dmso) (483 mg, 1 mmol) in dry methanol (15 mL), and the solution 4  refluxed for 6 h under N 2 . The resulting brown solution was filtered hot, and a brown precipitate formed when the filtrate was cooled.  This was filtered off in  air, washed with acetone and diethylether, and dried in vacuo at RT (0.42 g, 76% yield).  Anal, calcd. for C H 0 N S C l 2 R u : 1 6  C 34.33, H 3.78, N 7.32.  2.4.26  ^  1 9  m a x  4  3  2  C 34.72, H 3.43, N 7.60; found:  n m (log C ) , H 0 : 267 (4.06) 330 (2.81). 2  RuCUdmsoWNMe-ImK. 26 The  compound  NMe-Im  (0.5  mL, 6.0  mmole)  was  added  to  RuCl2(dmso) (483 mg, 1 mmol) in dry methanol (10 mL), and the solution 4  refluxed for 4 h under N 2 . The resulting yellow solution was cooled to RT and  51  diethylether (30 mL) added slowly to precipitate out a yellow product, which was filtered  off  in air.  This  product was  redissolved  in CH2CI2 (10 mL),  reprecipiated with diethylether (30 mL), filtered off in air, and dried in vacuo at RT (0.36 g, 73% yield). Anal, calcd. for C H 0 N S C l R u : 1 2  N 11.37; found: C 29.37, H 4.62, N 10.98. A  2.4.27  max  2 4  2  4  2  2  C 29.24, H 4.87,  n m ( l o g C), H 0 : 330 (3.67). 2  RuCl (dmso) (2Me-Im) . 27 2  2  2  The compound 2Me-Im (0.164 g, 2 mmol) was added to RuCl (dmso) 2  4  (483 mg, 1 mmol), in dry methanol (15 mL), and the solution refluxed for 4 h under N . The resulting yellow solution was cooled to RT and diethylether (40 2  mL) added slowly to precipitate out a yellow product, which was filtered off in air.  This product was redissolved in C H C 1 2  2  (10 mL), reprecipitated with  diethylether (30 mL), filtered off in air, and dried in vacuo at RT (0.38 g, 78% yield). Anal, calcd. for C , H 0 N S C l R u : 2  C 29.52, H 4.63, N 11.58.  2.4.28  2 4  2  4  2  2  C 29.24, H 4.87, N 11.37; found:  k „ nm (logC), H 0 : 333 (3.82). rn SIX ^ 9  r R u ( N H ) C n C l . 28 3  5  2  The compound [Ru(NH )^Cl]Cl was prepared according to a literature 2  3  /17  procedure.  Some R u C l y t ^ O (3 g, 11 mmol) was dissolved in H 0 (10 mL) 2  in a 200 mL beaker, and hydrazine hydrate (85%, 20 mL) was then added to the well-stirred solution.  The solution was stirred for about 12 h and filtered by  gravity. To the filtered solution, concentrated HC1 was carefully added until the pH was 2 when vigorous gas evolution took place. The solution was boiled, with stirring and became yellow, slowly precipitating the yellow product.  When no  further precipitation was observed, the mixture was cooled to R T , and the crude product collected by filtration.  The product was reprecipitated by heating an  aqueous slurry (3 g in 10 mL H 0 ) to 60°C and concentrated N H was added 2  3  52 dropwise until the yellow complex dissolved to give a wine-red solution, which was filtered hot and cooled in an ice-bath.  To the cooled solution, concentrated  HC1 was added dropwise to reprecipitate the yellow product which was filtered, washed with 6M HC1, water, alcohol and acetone (1.1 g, 39% yield). Anal, calcd. for H N C l R u : 1 5  2.4.29  5  3  H 5.19, N 23.98, Cl 36.35; found: H 5.30, N 24.73, Cl 36.12.  rRu(NH ) (NMe-Im)lCl . 29 3  5  3  The compound [Ru(NH ) (NMe-Im)]Cl was prepared according to a 3  literature procedure.  5  3  The compound NMe-Im (1.0 mL, 12.0 mmol) was added  to 40 mL of 0.05 M HC1. Solid [Ru(NH ) Cl]Cl (145 mg, 0.50 mmol) was 3  5  2  added and the resulting suspension was reduced over zinc amalgam for a period of 4 h, during which time the [Ru(NH )^Cl]Cl2 dissolved completely. 3  A stream  of argon was bubbled through the mixture throughout the course of the reaction. At the end of the reaction period the solution was removed from the zinc, diluted with 60 mL of water and subjected to an air stream for 2 h. The pH of the resulting solution was adjusted to 1-2 with dilute HC1. Ion exchange was carried out using the H Laboratories. product,  +  form of AG50W-X4 resin supplied by Bio-Rad  The column size was approximately 2.5 x 10 cm.  The major  eluted by 3M HC1, was isolated by roto-evaporation to dryness and  reprecipitation from water-ethanol; the product was filtered off in air and dried in vacuo at RT (0.24 g, 65% yield). Anal, calcd. for C H N C l R u : C 12.82, H 4  5.65, N 26.17; found: C 12.94, H 5.78, N 25.87.  A  2 1  m a x  7  3  nm(logE), H 0 : 2  320  (3.42).  2.5  Cell Culture Procedures The in vitro biological experiments reported here were performed on a Chinese  hamster ovary (CHO) cell line grown in tissue culture. The CHO cell line is used widely  53  because it grows rapidly, can be cloned with high plating efficiency, and can be grown in monolayer, suspension, and soft agar cultures.  The CHO cells were grown in  suspension culture in an alpha-medium, supplemented with 10% fetal calf serum (FCS), antibiotics, and bicarbonate buffer (see Appendix I). The flask spinning cultures were grown in an incubator (Incu-cover, Associated Biomedic Systems) at 37°C, with pH regulated to 7.4 by a continuous gassing of the cultures with commercially supplied 5% C0  2  7xl0  in air (Canadian Liquid Air Co. Ltd.). The cell culture was diluted daily to about 4  cells/mL, maintaining asynchronous exponential growth with a doubling time of  about 12 h.  2.6  Irradiation Irradiation was performed using an X-ray source (Picker, 280 kVp, H V L 1.7 mm  Cu or Philips RT 250, 250 kV, 0.5 mm Cu).  Samples were placed in special glass  irradiation vessels, the "arms" of which were taped to the sides of the plexiglass container in which the vessel sat in to prevent it from tilting. Cold water was added to about 600 mL of crushed ice to bring the total volume to 820 mL, which was poured around the irradiation vessel to keep the temperature at 4°C.  A stirrer motor was  suspended above the vessel which contained a teflon coated magnetic bar, while the X ray source was placed below the vessel (Figure 14).  2.7  Radiosensitization-Cell Survival Experiments Radiosensitization experimental procedures were as described by Moore et al.^  except that cells were exposed to drugs for 1 h at 37°C before irradiation at 4°C. Typically, sterile Petri dishes (60x15 mm, Falcon) were prepared the day before the radiosensitization experiment, and 5 mL alpha-medium supplemented with 10% FCS and 10^ "feeder cells" were added. Feeder cells are cells of the same line, sterilized by heavy irradiation, which supply nutrients and growth factors to increase the overall plating  54  F i g u r e 14:  Irradiation set—up The c e l l suspension was p u t i n t o a s p e c i a l g l a s s i r r a d i a t i o n v e s s e l (with a magnetic s t i r bar) which was p l a c e d i n a p l e x i g l a s s c o n t a i n e r c o n t a i n i n g i c e water; a s t i r motor was suspended above the v e s s e l . To o b t a i n a dose r a t e o f 1.6 Gy/min the p l e x i g l a s s c o n t a i n e r was p l a c e d d i r e c t l y on top of the 20 x 20 cm c o l l i m a t o r .  55  efficiency of the cloning procedure, and to overcome the variation in plating efficiency which may be observed when widely differing inocula are used. These Petri dishes were placed in the incubator, and had reached pH and temperature equilibrium by the following day.  An asynchronous population of CHO cells was grown in a spinner flask.  Cells  were then put in the special glass irradiation vessels in a growth medium, with or without the test compounds, in a total volume of 20 mL at approximately 1.25x10^ cells/mL. ppm of 0  The cells were made hypoxic by flowing purified 2  containing less than 5  (Canadian Liquid Air Co. Ltd.), over stirred cell suspensions for 1 h at 37°C  (the time being based on toxicity data, see Section 5.1) prior to the start of irradiation. Aerobic cells were treated identically except that the flow of N  2  was not employed.  Aliquots of 1 mL were withdrawn by auto pipette from the irradiation vessel after the required doses and pipetted in plastic dilution tubes filled with 9 mL of chilled (4°C) alpha-medium (lacking N a H C 0  3  and 10% FCS). Cells were plated from the dilution  tubes into the Petri dishes previously prepared.  The cell concentration in the dilution tubes was determined using a Coulter counter (Coulter Electronics Inc., Hialech, Florida).  The cells were plated from the  dilution tubes so that approximately 100 colonies per Petri dish would emerge after 7 days. After the plating procedure, the dishes were returned to the incubator for 7 days. The medium was then carefully poured out of the Petri dishes and 4 mL of methylene blue stain was added to each dish. After approximately 6 min, the stain was poured off and the Petri dishes rinsed gently in cold running water. counted after the dishes had dried. represent a survivor.  The stained colonies were  A clone of 50 or more cells was assumed to  56  Surviving fractions are defined as plating efficiency (PE) of treated cells divided by PE of controls. The PE is defined as:  PE =  number of colonies  [2.1]  number of c e l l s plated  In general, experiments were repeated at least three times.  Values of log (surviving  fraction) were averaged for each X-ray dose, and standard errors of the mean were calculated. Results are presented as log S vs. dose of X-ray, where  S =  number of colonies number of c e l l s plated  / PE of Controls  [2.2]  Data curves were drawn by using the linear-quadratic fit of the Oi, (3 model/'  2.8  7  Toxicity Studies - Cell Survival Experiments The toxic effect of a compound was measured by exposing cells to the test  compounds for various time intervals, then washing the cells free of the compound with alpha-medium and plating aliquots of the cells into Petri dishes to determine colony Of.  forming ability.  Solutions containing the test compound in medium were prepared  before each experiment in alpha-medium lacking bicarbonate buffer, and sterility was assured by filtering through a Nalgene 0.22 ^m filter unit.  From this stock solution,  19,0 mL was added to a 120 mL wide-mouth, glass centrifuge vessel, this being sealed with a rubber stopper fitted with two syringe needles to permit gas flow, and a stoppered central port for removal of samples (Figure 15). In a typical experiment, such vessels, including controls and various complexes, were set up in a large water-bath, maintained at 37°C and placed on top of a multi-magnetic stirrer unit. apparatus was kept in a 37°C warm room. supplied from a cylinder, and  The entire  The appropriate gas phase ( N  2  or air),  humidified in a glass bubbler filled with sterile  57  •CELL SUSPENSION  MAGNETIC STIR BAR  F i g u r e 15:  The glass v e s s e l f o r c e l l suspension t o x i c i t y t e s t s . N i t r o g e n (5% CO^ was flowed over a s t i r r e d 20 mL suspension of 2.0 x 10 cells/mL contained i n a glass v e s s e l . Samples were o b t a i n e d by removing the s m a l l stopper b r i e f l y and l o w e r i n g a p i p e t down the g l a s s t u b i n g i n t o the suspension. Experiments were performed at 37 c.  58  (autoclaved) water, was admitted through a short tube fitted with a plastic syringe tip. The solutions of test compounds in the medium were pre-gassed before addition of cells for 1 h to achieve temperature and gas phase equilibria.  Cells were harvested from  culture by centrifugation for 8 minutes at 600 rpm using a SORVALL RC-3 centrifuge, and resuspended in compound-free medium at 4x10^ cells/mL, and they remained at this concentration for about 15 min, to ensure metabolic depletion of any remaining 0  2  in the medium. At zero time, 1 mL of cell suspension was added to give a volume of 20 mL, a cell concentration of 2x10^ cells/mL, and a test compound concentration as desired.  Aliquots (1 mL) were removed at zero time (immediately after adding cells)  and at regular intervals thereafter, before being immediately diluted in 9 mL fresh medium at 4°C. The samples were spun to harvest the cells; the supernatant medium was decanted and replaced with fresh medium, and the cells were resuspended by vigorous pipetting and vortexing. Samples of this suspension were then plated into Petri dishes, previously prepared as described in section 2.7, using a micropipette.  Typically, \0 jiL  of cell suspension were plated into a Petri dish. If significant  toxicity were anticipated, larger samples were plated (up to 2 mL); triplicate plates were plated at several inoculum volumes.  Subsequent to the plating procedure, the cell  suspensions were again vortexed and a sample (2 mL) was removed, diluted with PBS, and cell concentration determined with the Coulter counter.  The Petri dishes were then incubated at 37°C in a tray incubator (National Inc.) with 5% C 0 (Canadian Liquid Air Co. Ltd.) flow for 7 days.  The medium was then  carefully decanted and replaced with methylene blue stain.  Finally, the stain was  2  washed off, and the colonies per Petri dish were determined. According to convention, a clone of 50 or more cells was assumed to represent a survivor.  5  59  2.9  Assay for Chromosome Aberrations The method employed was that previously described by Stich et alJ  19  with some  modifications, such as the cells being seeded 2-3 days before the experiment; the cells were exposed to the test compounds for 3 h.  Typically, approximately 140,000 CHO  2 cells were seeded on 22 mm  coverlips in 3.5 cm plastic dishes (Falcon) and kept in 2  mL M E M with 10% FCS, antibiotics (Streptomycin sulphate, 29.6 ^g/mL; penicillin, 125 ^g/mL; kanamycin, 100 ^g/mL, and fungizone, 2.5 ^g/mL) and sodium bicarbonate (1 mg/mL) (10% MEM) at 37°C for 2-3 days.  When cells were 40%-60% confluent, the  tissue culture medium was removed and replaced with 0.5 mL 10% M E M and 0.5 mL of test compounds at various concentrations.  After 3 h incubation at 37°C, the test  compounds and the 10% M E M were removed by suction and the cells washed twice with MEM.  The cells were then incubated for 16 h at 37°C in 2.0 mL 10% M E M . For tests  involving the inclusion of liver microsomal preparations (S9) (see Appendix VI), 0.5 mL aliquots of this activation mixture containing 150 /Ug S9 were added to each Petri dish instead of 0.5 mL 10% M E M .  The S9 was prepared from livers of Aroclor 1254-  pretreated Wistar male rats as described by Ames et al.  120  The activity of the S9  mixture was verified by its capacity to activate the precarcinogen aflatoxin Bl to induce chromosome aberrations in CHO cells.  Aflatoxin Bl without the S9 mixture did not  elevate the frequency of metaphases with chromosome aberrations over that found for non-treated control cell cultures. For an estimation of the frequency of chromosome aberrations, 5 ^g/mL of colchicine was added to the test compounds at 16 h post-exposure and left for 3 h to arrest dividing cells at metaphase.  Cells were then treated with 1% sodium citrate  hypotonic solution for 20 min, this being followed immediately with fixation in ethanol/glacial acetic acid (3:1, V / V ) for 20 min. Air-dried slides were stained with 2% orcein in glacial acetic acid/water (1:1 V / V ) , dehydrated and mounted.  For each  60  sample, 100 metaphases were analyzed for chromatid exchanges and chromatid breaks in each of three replicate cultures.  2.10  Measurement of Non-protein Thiols Measurement of NPSH was carried out essentially as previously described by  Tietze  727  but cells were pre-treated with test compounds before NPSH determination.  Typically, a metaphosphoric acid solution (0.5 mL of 10%) was added to 1 mL of CHO cell suspension (3x10^ cells), which had been pre-treated with the test compounds (200 }iM final concentration) under hypoxia at 37°C for 1 h, in 0.2 M PBS. The cell mixture was sonicated for 10 s and allowed to cool for 10 min in ice, before being centrifuged at 4000 rpm for 10 min. Some PBS (1 mL of 0.5 M), which contained 1.7 g NaOH and 0.2 g EDTA/lOOmL, was added to 1 mL of supernatant, and 0.05 mL of Ellman's reagent was added immediately and the mixture left for 10 min to attain equilibrium at 25°C. Optical absorption of samples were then read at 412 nm on a UV/VIS spectrometer (Aminco DW-2).  2.11  Inhibition of Restriction Enzymes The plasmid, pSV2-gpt, was obtained as described by Skov et al.  growth in E. coli, the DNA (5200 base pairs) was extracted PvuII.  122  After  and linearized using  Figure 16 shows the scheme used: cleavage by BamHI or EcoRI at the defined  sites results in only 2 fragments per enzyme because purified linear plasmid is used. The DNA (20 yL, 20 ,wg/mL) in Tris-Cl (100 mM)/EDTA (1 mM) at pH 8.0, was exposed to the test compound (80 ,wL of desired concentration) for 1 h at 37°C, after which unbound drug was removed using a spin-column of G-50 Sephadex (Pharmacia). Buffer (3.5 uL) (containing 0.33 M Tris (pH 7.9), 0.66 M potassium acetate, 0.10 M magnesium acetate, 0.005 M dithiothreitol and 1 mg/mL bovine serum albumin) and 2 pL  of enzyme were added to the eluted DNA (30 yL).  A solution of EDTA (1 jiL,  0.5  61  pSV2-gpt  I  Pvull  •^XXJOOODOOOOOOOOC^ B  a  m  H  I  /  2.0kb  \ _  EcoRI 3.1kb  X)Ooooa: xxx>ocoooooc :>oooooocccc Doooccc cTAG  OATC  AATT  3.2kb  F i g u r e 16:  <  2.1kb  O u t l i n e o f b i n d i n g assay u s i n g p l a s m i d DNA. B i n d i n g o f complex a t o r near r e s t r i c t i o n s i t e i n h i b i t s r e s t r i c t i o n endonuclease a c t i v i t y on l i n e a r i z e d p l a s m i d ; k b = k i l o b a s e s , number o f bases i n the DNA s t r a n d (taken from r e f . 120).  62  M as the disodium salt) was used to stop enzyme activity after 30 min at 37 C. A tracking dye (7 yL: 0.25% bromophenol blue, 0.25% xylene cyanol, in 30% glycerol water) was added, and the resulting solution (20 piL) was loaded onto a 1% agarose gel, made with E-buffer (0.04 M Tris., 0.005 M sodium acetate, 0.001 M EDTA as the disodium  salt,  pH 7.8  with  glacial  acetic  acid),  and subjected  to horizontal  electrophoresis for 16 h at lV/cm at RT. The slab gel was stained for 15 min with aqueous ethidium bromide solution (500 mL, 1 mg/mL) and photographed under U V light with an MP-4 Polaroid camera. Inhibition of the restriction enzymes BamHI and EcoRI was assessed qualitatively by noting the relative proportions of bands (cleaved vs. uncleaved).  More quantitatively, the proportion of uncleaved DNA was determined  from a densitometric (Kapp and Zonen DD2) scan of the negatives of the recorded pictures.  Analysis was by integration of the scanned peaks, with the fraction in the  uncut band over the total area of the three bands per lane being used to calculate the uncleaved (inhibited) fraction.  2.12  Measurement of Partition Coefficient The experimental procedure for determining the partition coefficients of the Ru  complexes was as outlined by Fujita et al.  124  The analyses of the concentrations of the  partitioned subtances were made using a Perkin Elmer 552A UV/VIS spectrometer. The anhydrous 1-octanol  was used as obtained without further purification.  For the  partitioning, octanol saturated with distilled water and distilled water saturated with octanol were used. The volume ratio of the two phases and the amount of sample were chosen so that, in most cases, the absorbance of a sample from the aqueous layer after partitioning had a value between 0.2 and 0.9 absorbance unit using a 1 cm cell.  Only  the concentration of the sample in the aqueous layer was determined, and that in the octanol was obtained by difference.  The partition coefficient (P) was calculated as the  63  ratio of the concentration of complex in the octanol phase to that in the aqueous phase: Concentration i n octanol P a r t i t i o n Coefficient =  [2.3] Concentration i n water  For each complex, an average of three measurements were made, and care was taken to ensure that each reading recorded represented equilibrium conditions. were done with both aerobic and hypoxic conditions.  Measurements  64  CHAPTER THREE CHARACTERIZATION OF COMPLEXES  Complexes of formulation RuCl (dmso)2L (L=4-N0 -Im, RSU-1170, RSU2  2  2  3083, RSU-3100, 2-N0 -Im, miso, De-miso, metro, NMe-Im and 2Me-Im) are readily 2  synthesized from the precursor complex c/s-RuCl (dmso) , of known structure, ^ 9  2  -96  4  by  substitution of two sulphoxide ligands. The ir and *H nmr data (see sections 3.2.1 and 3.3.1) are consistent with the diamagnetic products (5, 7-9, 11-14. 26 and 27) having a purely cis structure (i.e. cis, cis, cis) with both dmso ligands being S-bonded.  The  syntheses proceed via reaction [3.1]: 0  L  [3.1]  (O or S implies oxygen or sulphur-bonded dmso, respectively).  Complexes 6 and 23-25 of formulation RuCl (dmso) L (L=NMe-4-N0 Im, 2  1,10-phenanthroline,  5-C1-1,10-phenanthroline  2  and 5-N0 -1,10-phenanthroline) 2  2  are  again hexacoordinate. When L=NMe-4-N0 -Im, the L binding is bidentate via an 2  imidazole nitrogen and an oxygen of the N 0  2  group; the phenanthrolines bind in their  common bidentate mode ; the two S-bonded dmso ligands are probably trans. Complex 10. RuCl (dmso) (RSU-3159), has S-bonded dmso ligands and a coordinated thioether 2  2  of RSU-3159, this ligand probably being chelated via the nitrogen of the NMe group (see section 3.3.1).  65  Complexes of formulation RuCl (tmso) L (L=4-N0 -Im, NMe-4-N0 -Im and 2  De-miso)  15-12,  2  2  RuCl (tmso) L (L=SR-2508) 2  2  18,  2  2  RuCl (tmso) (CMNI) 19, and 2  3  RuCl (tmso)(RSU-3159) 20, are synthesized from the precursor complex RuCl (tmso) 2  2  4  of unknown structure, by substitution of two of the all S-bonded tmso ligands (see section 3.1). The ir data of 15-20 indicate that the tmso ligands are S-bonded but the nmr data are very complex (see section 3.3.2) and geometric arrangements of the ligands have not been determined.  Complexes  of  formulation  RuBr (dmsp) (NMe-4-N0 -Im), 2  2  2  22,  RuBr (dmso) (4-N0 -Im) , 2  are  2  synthesized  2  from  21,  2  and  precursor trans-  the  RuBr^dmso)^ - by substitution of two dmso ligands via reaction [3.2]: 2  5  Br  Br  Ru-  -S + 2L (L') -2. S  -S or S-  Ru-  L  Br  Br  L  .Ru  Br  Br  21  22  L /  1  [3.2]  L=4-N0 -Im; L'=NMe-4-N0 -Im; S=S-bonded dmso 2  The  J  2  H nmr data (see section 3.3.3) suggest a purely trans structure with both dmso  ligands being S-bonded for complex 21_.  Complex 22 is also hexacoordinate with L  again binding via an imidazole nitrogen and oxygen of the N 0  2  group, similar to 6,  thus having a trans, cis, cis structure.  The compound activity,  attempted  preparations of  [Ru(NH )^Cl]Cl ,known to 3  on oj ' '  BA  were  2  unsuccessful.  Ru(III)-nitroimidazole bind Only  DNA and the  complexes from the  have  previously  some  anti-tumour  reported  complex,  66 [Ru(NH ) (NMe-Im)]Cl3, 29, 3  5  ' containing the more basic NMe-Im could be made  (see section 2.4.29).  Considerable efforts to grow suitable crystals of any of the nitroimidazole complexes proved frustratingly fruitless.  3.1  XPS Data XPS can be used for determining bonding configurations because the technique  detects changes in the electronic environment of an atom, as evidenced by shifts in the BE of core electrons.  XPS data for N, O, Cl and S atoms within complexes 5, 6, 10,  11, 16, 18, 22, m-RuCl2(dmso) and RuCl2(tmso) , and for some of the free ligands, 4  4  are summarized in Table II. The XPS N Is spectra of 5 and its free 4-N0 -Im ligand 2  are shown in Figure 17.  Coordination of imidazole ligands via the tertiary nitrogen  N(3) is well established for metal ions, including RuQl), ' ' 117  are consistent with this.  126  127  and the XPS data  The XPS data show that there is a shift to higher BE for all  the N atoms present in 5, 6, 15, 16 and 22 compared to the energies within the free ligands, indicating derealization of electrons within the systems. The double intensity peaks centred at 399.6 and 398.9 eV (Figure 17) must each result from the addition of two peaks (considered to be of equal shape) from N(l) and N(3); this allows for a simple deconvolution into the two component peaks as shown. Thus, the deconvolution method assumes the same intensity and shape for each particular type of atom; the error in these particular assignments could be as high as +0.3 eV. In the free ligands 4-NC»2Im and NMe-4-NC>2-Im, the N(3), atoms adjacent to the electron withdrawing NC>2 group are considered to  have a higher BE (399.70 and 399.80 eV, respectively) than  those of N(l) (398.10 and 397.90 eV, respectively).  In the complexes, the coordinated  N(3) might be expected to have an even higher BE than those observed (for 5, 400.20 vs. 399.70 eV; 6, 400.60 vs. 399.80 eV, 15, 400.10 vs. 399.70 eV; 16, 400.40 vs. 399.80  67  Table I I  XPS data f o r Ru(II) compounds and free ligands  Atom  Compound 5, Ru(4-N0 -Im) 2  2  3  Group  BE  Width  -N0  405.80(405.35)  2.6(2.6)  2  0  N(3) N(l)  400.20(399.70)° 2.4(2.6) 399.00(398.10) 2.4(2.5)  -ONO  405.60(404.90)  N(3) N(l)  400.60(399.80) 399.30(397.90)  2.3(2.4)  -N0  2  405.50(405.35)  2.5(2.6)  N(3) N(l)  400.10(399.70) 398.70(398.10)  2.4(2.5) 2.4(2.5)  16, Ru'(NMe-4-N0 -Im)  -N0 N(3) N(l)  405.20(404.90) 400.40(399.80) 398.90(397.90)  2.6(2.4) 2.5(2.4) 2.5(2.4)  18, Ru'(SR-2508)  -N0  2  404.60(404.40)  2.4(2.3)  -ONO  405.30(404.90)  2.5(2.4)  N(3) N(l)  400.60(399.80) 399.00(397.90)  2.4(2.3) 2.5(2.3)  6, Ru(NMe-4-N0 -Im) 2  H>  Ru (4-N0 -Im) 1  2  2  2  22, Ru;MNMe-4-N0-Im) 2  1, cis -RuCl (dmso) ^ 2  2  d  2  -S(0)Me -OSMe  2, RuCl (tmso)^  -S(O)  5, Ra(4-N0 -Im)  N0 -S(0)Me  2  6, Ru(NMe^N0 -Im) 2  3.7 3.7  530.90  3.6  a  531.70(531.70) 532.00  2.9(2.9) 3.6  531.70 (530.70)  2  530.80 532.00  3.8(4.0) 3.8 3.8  9  530.10(530.10) 532.01  3.6(3.6) 3.8  2  -ONO  2  d  d  -S(0)Me 10, Ru(RSU-3159)  N0 -S(0)Me  2.5(2.3) 2.3(2.4)  532.00 533.70  d  2  2  2  d  2  continued  68  continued  Atom  Compound*  Group  3  0 (Is)  15,  Ru'(4-N0 -Im) 2  -N0  2  16, Ru' (NMe^r-N0 -Im) 2  18, Ru'(SR-2508)  19, Ru'(tmso)(CMNI)  2  531.90(531.70)  2  -S(0)  531.20  -N0  531.90(531.70)  2  3.6(3.3) 3.8 3.6(3.8)  531.20  -N0  2  532.00(531.90)  3.6(3.5)  -C=0  531.50(531.45)  3.6  -S(0)  531.20  3.8  -OH  530.40(530.40)  3.4  532.00(531.95)  3.5(3.4)  2  3.8  531.20  -S(0)  531.80(530.70)  -ONO  2  0  -S(0)  -N0  22, Ru"(NMe-4-N0 -Im)  Peak Width  BE°  (3.6M3.5  530.90 -S(0)Me Cl ( 2 P  1,  V2,3/2  )  s ( 2 p  l/2,3/2  cis -RuCl (dmso)^ 2  5, 6 , and 10 1,  ais -RuCl (dmso)^  2,  RuCl (tmso)  2  2  531.70  Cl  197.50  3.0  Cl  197.50-197.60  3.1  SO  166.3  e  2.8  -S(0)  165.10  2.7  )  2  4  5 and 6  -S(0)Me  2  10, Ru(RSU-3159)  -S(0)Me  2  -S 15,  Ru'(4-N0 -Im)  16,  Ru'(NMe-4-N0 -Im)  166.40-166.50  2.9  166.30  2.7  164.20(163.00)  2.7(2.7)  -S(0)  165.10  2.9  -S(0)  165.20  2.9  18, Ru'(SR-2508)  -S(0)  164.70  2.8  20, Ru'(RSU-3159)  -S(0)  165.10  2.8  -S  163.90(163.00)  -S(0)  165.80  22,  2  2  2  Ru"(4-N0 -Im) 2  2  2  2.8(2.7) 2.9  continued  continued  69  Binding energy and peak width given i n eV; estimated maximum error +0.15 eV. Ru, Ru' and Ru" represent RuCl^dmso^, RuC^Ctmso)^ and RuB^Cdmso^ respectively, except 20 has only one tmso ligand, a l l the sulphoxide ligands being S-bondedT Number i n parentheses gives data for corresponding group i n the free ligand; the ranges of BE values for atoms are given i n ref. 128. The deconvolution method assumes the same intensity and shape for each particular type of atom; the error i n these particular assignments could be as high as +0.3 eV. The XPS peaks for sulphurs of the one 0-bonded and three S-bonded ligands are not resolved.  70  71  eV); perhaps a relatively low value results because d , low spin Ru(II) has a quite 117  remarkable  donor ability toward unsaturated N-atom donors.  The greater shift to  higher BE of N(3) of 6 (0.8 eV) compared to those in 5 (0.5 eV), 15 (0.4 eV) and 16 (0.60 eV) is consistent with chelation, with further loss of electron density via an oxygen of N 0 -  Stronger evidence for coordinated nitrito is provided by the doublet  2  seen for the O Is of the N 0  group of £ at 531.70 and 530.80 eV; similarly this O Is  2  doublet is also observed for 20.  This O Is doublet is not seen for 5 and 16 where the  corresponding O Is BE is similar to, or the same as, that of the free ligand at 531.70 eV.  The N Is BE for the N 0  of 18 is similar to that of the free ligand, SR-2508,  2  suggesting no coordination via the N 0  group and a five- coordinate geometry  2  The  531.70 eV and 530.80 eV BE's for O in 6 are assigned to the coordinated and uncoordinated oxygens, respectively.  Chelating ligands utilizing a nitrito moiety are  729 uncommon,  and we are unware of other examples involving nitroimidazole ligands.  On comparison of the N Is and O Is BE's of 4-N0 -Im and NMe-4-N0 -Im, the 2  2  lower values for the latter reflect the electon-donating properties of the methyl group which likely promote the chelation. -* 7  0  The BE's of CI 2 p  1//2  3 / / 2  for complexes 5_, 6  and 10, are 197.5-197.6 eV, which are essentially identical to that of c/.s-RuCI (dmso) . 2  4  Therefore, the possibility of 6 being a chloride-bridged dimer with monodentate imidazole ligands is ruled out. The complex 1, c's-RuCl (dmso) shows two O Is energies (532.0 and 533.7 eV) 2  4  in an intensity ratio of about 3:1, entirely consistent with structural data for the complex/"* (see eq. [4.1]).  However, complex 2, RuCl (tmso) , shows only one O Is 2  (530.90 eV), the value indicating only S-bonded tmso. 2 p  l / 2 3/2  d a t a  a v a n a  cis-RuCl (dmso) 2  4  °l  e  4  The corresponding O Is and S  f ° 5, 6, 10, 15, 16, J_8 and 22, on comparison with those for r  and RuCl (tmso) , reveal the presence of only S-bonded dmso 2  4  ligands in the nitroimidazole complexes.  Previous work using XPS to distinguish O-  and S-bonded dmso ligands has generally been less definitive.  72  The BE for the S 2 p j / 3 / 2  l e v e  2  l  ot  "  t  h  e  thioether sulphur atom of RSU-3159  (163.0 eV) is increased by 1.2 eV and 0.9 eV within complexes 10 (164.2 eV) and 20 (163.9 eV) respectively, strongly implying coordination via this sulphur. Models suggest that chelation of RSU-3159 via the lone pair of N(l), the methylated nitrogen, is plausible.  However, the N Is data for IQ and 20 are insufficiently resolved to give  information about binding to a nitrogen; *H nmr data (see section 3.3.1) provide some support for the chelation.  Further, data for the S 2 p / t  2  3/2  L  E  V  E  L  O  F  T  N  E  T  M  S  0  complexes 2, 1_5_, 16 and J l reveal that the BE for 18 (164.70 eV) is much less than those of the other complexes.  This indicates a weaker interaction between Ru and the S  in 18, and may be consistent with a five- coordinate complex which is trigonal bipyramidal with the tmso ligands in the axial positions, giving a longer bond and weaker interaction with the Ru centre.  3.2  Infrared Spectral Data Infrared spectroscopy is one of the tools available to help elucidate the nature of  the bonding of sulphoxide ligand. The v  for free dmso is seen at 1050 cm'*, ' 95  S Q  131  while coordination through oxygen atom decreases this by enhancing the contribution of resonance structure(I) (Figure 10), because of the withdrawal of electrons from oxygen. This results in a lowering of the SO stretching frequency by approximately 100 cm" ; 1  coordination through the sulphur atom increases the SO stretching frequency by about 70 cm" , by enhancing the contribution of resonance structure(III) (Figure 10).^.^3 1  3.2.1  Infrared Data for RuCl (dmso) L Complexes (n=l or 2) 2  2  n  The ir spectrum of the yellow compound 1 c/s-RuCl (dmso)4, in 2  Nujol is virtually identical to that reported previously  95,96  ' '* and, based on 77  the band assignments, can be used to assign some bands for other complexes  73  described below.  Bands at 1120 and 1091 cm  show the presence of S-bonded  dmso ligands, while a band at 920 cm"' indicates O-coordinated dmso.  Selected  ir  spectral  data  for  the  4-nitroimidazoles  RuCl2(dmso)2L (n=l or 2), are summarized in Table III. n  complexes,  The ir data for all  the complexes £ - J 0 reveal bands in both the 1085-1091 and 1112-1167 c m regions, consistent with S-bonded dmso ligands. known to  be labile relative  to an S-bonded  demonstrated for cis-RuCtydmso)^.  95,96  '  - 1  Oxygen-bonded sulphoxide is one,*  and this has  7  been  Thus, substitution of O-bonded  113  sulphoxide by the nitroimidazole ligand would occur first and leave only Sbonded complex, as revealed by ir data.  All the free 4-nitroimidazole ligands reveal ir bands in the region at 1557-1619 and 1518-1592 cm" , except RSU-3159, which has ir bands at 1641 1  and 1673 cm"', the two values for each ligand are being attributable to the asymmetric (higher value) and symmetric N O 2 stretches. ^  Values within  complex 6, 1543 and 1523 cm" , are both some 20 cm" lower than those of the 1  1  free ligand NMe-4-N02"Im (1565 and 1545 cm" ), presumably reflecting the 1  coordination of the coordinated nitrito group.  Changes in the  coordination of the other nitroimidazoles are smaller. free N M e - 4 - N 0 - I m , attributed to v ^ 2  on  The 2815 cm" band of 1  _ ^ , is found at 2800 cm" with 6, 1  N C H  H  while corresponding bands at 2724, 2730 and 2724 cm" for complexes 8-.10, 1  respectively, are 6-18 cm" lower than that found in the free ligands. 1  These  data provide no strong support for chelation of RSU-3159 via N(l) within complex 10, although the coordination shift is greatest (11 cm" ) for this system. 1  74  Table I I I  Selected i r spectral data for 4-nitroirnidazole complexes of Ru and the free nitroimidazole ligands _1  Cc>mpounds  IR bands (Nujol, cm ) "N-Cr^-H N0„  c  v,SO  _ l e£s-RuCl(drnso)^ s  b  y  Ru-Cl  1120,1091 920  2  5, Ru(4-N0 -Im)  y  1127 1090  1559(1560)asym. 1535(1550)sym.  6, Ra(NMe^-N0 -Im)  1112 1085  1543(1565) 1523(1545)  7, Ru(RSU-1170)  1123 1087  1560(1557) 1522(1518)  8, Ru(RSU-3083)  1167 1086  1627(1619) 1599(1592)  2724(2730)  304 325  9, Ru(RSU-3100)  1137 1091  1614(1611) 1538(1577)  2730(2738)  330 347  10, Ru(RSU-3159)  1125 1085  1684(1673) 1653(1641)  2724(2742)  326 351  2  2  2  2  2  2  328 342 2800(2815)  332 354 325 349  Ru represents RuCl (dmso) ; a l l sulphoxides ligands are-S-bonded. 2  2  Values i n parentheses are for the free nitroimidazole ligands. These bands are tentatively assigned because of uncertainty i n the complex f a r - i r region.  75  The ir corresponding  bands for  complexes  complexes 11-14  reveal v  the 2-nitroimidazoles  are S Q  summarized  in  and metronidazole, and the  Table  IV.  The  ir  data  for  bands i n both the 1078-1094 and 1100-1162 c m  regions, again suggesting S - b o n d e d dmso ligands. bands at about 1500-1548 and 1520-1560 c m "  1  - 1  A l l the complexes have ir  attributable  to symmetric  and  asymmetric N 0 , respectively, the values being slightly lower (by up to 12 c m " 2  than those f o u n d i n the free ligands.  T h e i r data f o r the 1,10-phenanthrolines  and mefhylimidazoles, and  complexes (23.-27) (Table I V ) , reveal bands i n both the 1134 c m "  1  1076-1098 and 1095-  regions, again consistent w i t h S - b o n d e d dmso ligands.  b a n d , attributed to ^ N C H J - H ) * ^ s  o  u  n  d  A 2791 c m "  1  w i t h i n 26, w h i c h is again lower than the  value f o r the free l i g a n d N M e - I m (2804 c m " ) . 1  The of uncertainty 344 c m "  v  R _ci u  b  a n u S  °f  the complexes are d i f f i c u l t to assign because  a n <  i n the m u l t i - b a n d f a r - i r  region.  has been d e f i n i t e l y a s s i g n e d , '  1  9 5  E v e n f o r 1, only one band at  and indeed the presence of only  9 6  one intense R u - C l band i n this region indicated that the product was the isomer. ^ 7  7  trans-  H o w e v e r , this was shown later by structural data to be incorrect,  and the c o m p l e x is i n fact  a cis-isomer. ^  Nevertheless, the structures  9  are  generally thought to contain cr's-chlorides (see section 3.3.1) a n d the two bands listed f o r V ^ _ Q i n Tables III and I V seem appropriate f o r such a geometry. U  3.2.2  I n f r a r e d Data f o r R u C l ( t m s o ) L 2  m  n  C o m p l e x e s ( m = l . 2 or 3: n=l or 2)  Selected i r spectral data f o r the tmso complexes are s u m m a r i z e d i n Table V .  T h e ir bands at 1133 and 1094 c m "  1  f o r c o m p l e x 2 show the presence  76  Table IV  /~1  Selected i r spectral data for 2-nitroimidazoles, metronidazole and 1,10-phenanthrolines, and their Ru complexes  1  IR bands (Nujol, cm  3-  Compounds  y  S0  *N0  b 2  y  )  CH "H 2  yRu-Cl  0  11, Ru(miso)2  1089 1100  1520(1528)asym. 1500(1512)sym.  335 350  12, Ru(De-miso)2  1078 1105  1532(1538) 1515(1527)  328 348  1084 1112  1560(1567) 1548(1559)  336 347  14, Ru(metro)2  1094 1162  1548(1552) 1540(1546)  331 352  23, Ru(phen)  1098 1134  324 338  24, Ru(5-Cl-phen)  1079 1095  329 342  13, Ru(2-N0 -Im) 2  2  25, Ru(5-N0 -phen) 1093 1107 2  326 340  1536(1533) 1515(1518)  26, Ru(NMe-Im)  1076 1114  2791(2804)  330 342  27, Ru(2Me-Im)  1078 1099  2799(2811)  338 351  2  2  Ru represents RuC^dmso^; the sulphoxides ligands are S-bonded. Values i n parentheses are for the free ligands. These bands are tentatively assigned because of uncertainty i n the f a r - i r region.  77  Table V  Selected i r spectral data for RuCl (tmso) L and RuBr (dmso) L complexes; m=l,2 or 3, n=l or 2 9  1  9  m  n  1  9  1 1  IR bands (Nujol, cm ) Compounds  r  3 U  2, RuCl (tmso) 2  S0  D  ^N0  C 2  N-CH -H  y  2  y  Ru-Cl'  1094 1133  4  339  4, trans -RuBr ( dmso )^  1080  15, Ru (4-N0 -Im)  1075 1102  1557(1560)asym. 1537(1550)sym.  16, Ru (NMe^r-N0 -Im)  1088 1115  1554(1565) 1538(1545)  17, Ru'(De-miso)  1078 1114  1534(1538) 1515(1527)  337  18, Ru'(SR-2508)  1050 1093  1532(1538) 1508(1517)  342  19, Ru'(CMNI)  1057 1094  1593(1587) 1574(1568)  336  20, Ru'(RSU-3159)  1079 1106  1681(1673) 1652(1641)  339  1084  1552(1560) 1539(1550)  1073 1092  1542(1565) 1521(1545)  2  1  2  2  1  2  2  2  21, Ru"(4-N0 -Im) 2  2  22, Ru"(NMe^-N0 -Im) 2  348 2809(2815)  343  2806(2815)  Ru' represents RuCl (tmso) , m = 1(20), m = 2(15-18) and m = 3(19); Ru" represents RuBr (dmso) . 2  2  k AH  m  2  bands represent S-bonded sulphoxides.  Values i n parentheses are for the free nitroimidazole ligands. ^ These bands are tentatively assigned because of uncertainty i n the f a r - l r region.  78  of S-bonded tmso ligands, while bands at 1075, 1035, 952 and 875 c m probably due to ( - gy ' ^ v  are  particularly as the XPS data suggest only S-  132 13  T  - 1  in  bonded tmso. The V ^ Q for free tmso is seen at 950 cm"'.''^ The ir spectra of complexes 15-20 reveal sulphur-bonded tmso by characteristic V^Q bands in the 1050-1115 c m  - 1  region. The V Q values assigned to complex 15 are similar to N  those observed for 5j the changes in V ^ Q ^ within 1£ (a six-coordinate compound) on coordination of the  ligand are smaller than the corresponding  differences for the chelated complex 6 and its free ligand. The V Q ^ bands for N  complexes 17 and !§. are in the regions 1508-1515 and 1532-1534 c m , whereas - 1  those v  ^ bands for 19 are at about 1574-1595 c m , but all are similar to - 1  N 0  those of the free ligands. The small change in V J ^ Q ^ between complexes 1_8_ and 12, and their corresponding free SR-2508 and CMNI ligands, respectively, suggests that there is no coordination via the nitrito group (see Section 3.1). Bands due to  V R  U  _ Q  in the far-ir region are again difficult to assign and the  geometric configuration of complexes 11-20 (cis or trans chlorides) cannot be determined from the ir spectrum.  3.2.3  Infrared Data of RuB^dmsoUI^ Complexes (n = 1 or 2) The ir spectrum of the orange product, 4, in Nujol is essentially  identical to that reported previously (prepared from a different method) with a /ra/is-configuration.  725  A strong ir band at 1080 c m  (S-bonded) as previously reported.  - 1  can be assigned as V ^ Q  No band due to O-dmso is observed,  which agrees with data reported previously. ' 95  725  The ir spectra of complexes 21 and 22 possess v  S Q  bands in the 1070-  1090 cm" region, again consistent with S-bonded sulphoxide ligands. The v 1  N Q  ^  values assigned in 2J. are similar to those observed for 5; replacement of CI by  79  Br within 5 thus affects V Q ^ N  very little.  Values within 22, 1542 and 1521  cm" are both approximately 20 cm" lower than the values for the free ligand; 1  1  a similar trend is seen for 6. The data are consistent with coordination to Ru via the nitrito group and the imidazole ring N occurring in 22 as in 6. The RuBr stretch in the far-ir spectrum is difficult to assign, thus making it impossible to determine unambigously the structure of these complexes on the basis of the IR evidence alone.  3.2.4  Infrared Data for "RuCUmpsoW. 3 Compound 3 is very slightly soluble in acetone, CH Cl2 and ethanol. 2  Elemental analysis confirmed the product to be of the stoichiometry shown as demonstrated previously. * The ir spectrum contains a strong band centred at 77-  1128 cm" , assignable to V Q (S-bonded); the free sulphoxide v 1  S  cm" . 1  SQ  is at 1045  Bands are also seen at 940 and 960 cm" but a band due to 1  V<JQ  (O-  bonded) cannot be assigned because the spectrum in the 920-990 cm" region is 1  complicated by the possible presence of the rocking modes of C H * which in 7-  7  3  The low solubility of 2  the free ligand are seen at 987, 972 and 957 cm' .  1 132  (presumably resulting from its polymeric nature *) prevents any chemical 77-  reaction with the nitroimidazole ligands even in a heterogenous phase and, therefore, the chemical properties of this compound were not explored any further.  Nuclear Magnetic Resonance Spectral Data  Nuclear magnetic resonance is one of the most important spectroscopic methods  80  available for structure determination of an unknown molecule.  While infrared  spectroscopy reveals information as to the types of functional groups present in a molecule, nmr gives information about the number of each type of nuclei in such groups. It also gives information regarding the nature of the immediate environment of each of these types of nuclei.  In * H nmr spectroscopy, coordination of sulphoxides to metal centres causes the adjacent proton nuclei to resonate at lower field positions.  Methyl protons of dmso,  other methyl alkyl, and methyl aryl sulphoxides, are deshielded by approximately 1 ppm on coordination to Ru(II) through sulphur and approximately 0.1 ppm on coordination through o x y g e n .  57,96  The ^ Q ' H } nmr spectral data for dmso and imidazole ligands  show that the methyl groups of the sulphoxide are detected at 35-45 ppm, whereas the C resonances of the imidazole ring are at  3.3.1  ppm/ ** -  1 1 0 - 1 2 0  Nuclear Magnetic Resonance Data of RuCWdmso^Ln Complexes (n = 1 or 2 ) The proton nmr spectrum of compound i in CDCI3 was identical to  that reported and discussed previously. "^' "^'^ 9  96  6  Four singlets due to the  methyl protons of the S-bonded dmso ligands occur at 5 3 . 3 3 , 3.44, 3.50 and 3.53 ppm.  The equivalent methyl protons of O-coordinated and and small amounts  of free dmso are observed at 6 2 . 7 3 and 2.60 ppm, respectively (Figure 18). The integration ratio of the downfield singlets to the upfield pair indicates that 1 contains three S-bonded dmso ligands and one O-bonded dmso ligand (eq. The  presence  of  the  four singlets for the  S-bonded  ligands  [3.1]).  is readily  rationalized, because the S-bonded ligands are trans to O-bonded dmso, Sbonded dmso or Cl, and this leads to three different chemical shifts for the methyl resonances of these molecules.  Extra methyl resonances result from the  81  i  i  j  i  i  I~I  I  5  F i g u r e 18:  i  i  i •i 4  -p i  H nmr  i  ;  i  |  i  i  i  i  j  i t i  i  i  i  |  3  spectrum o f c i s - R u C l  i  i  i  i | 2  i  T  J  I  I  I  I  (dmso) , 1, i n CDC1  I 1  I  PPM  .  I  I  I  |  I  I  I  I  j  o  82  formation of isomers from dissolution of the complex,  ,  J  the presence of  some degree of methyl inequivalence could also lead to the complex spectrum observed. - ^ * 96  7  7  5  Table VI lists selected ' H  nmr spectral data for the RuCl (dmso) L 2  2  n  (n = 1 or 2) complexes. Complexes 5-10 show only S-bonded dmso, because no 5 2.7 ppm (the O-bonded region); the  methyl resonance is observed around findings  agree with the ir data.  There is no dissociation of dmso ligand in  CDCI3 solution (Figure 19a), and even in D 0 , complex 5 generates no free 2  dmso .  Oxygen-bonded dmso is known to be labile, thus, as seen in equation  [3.1], substitution of O-bonded dmso and a S-bonded dmso cis to it (necessarily so for the chelating ligand NMe-4-N0 -Im) would lead to the c/5-chloride cis2  sulphoxide geometry for the product.  For complexes 5, 7-2, the presence of  four major, equal intensity resonances, occurring as partly resolved multiplets centred at the shifts listed in Table VI, reveals inequivalent methyl groups for each of the two inequivalent dmso ligands.  Only the low symmetry all cis-  structure of 6-coordinate complexes can give rise to such an nmr pattern. ^' * 7-  7-  6  A model of 5 shows that free rotation about the Ru-S bonds for cis-dmso ligands is restricted because of steric interaction between methyl groups, but this is not a requirement for the magnetic inequivalence of the methyls. "* 7  6  Proton  decoupling experiments did simplify the observed multiplet patterns of the methyls, but these provided no further information on the structures.  For the chelated complex 6_, there are two methyl resonances again of equal intensity (Figure 19b). thus ds-chlorides). * '* 7-  -5,7  6  These are consistent with trans dmso ligands (and Therefore, rearrangement of the dmso ligands has  occurred in this reaction, different from the pathway shown in eq. 3.1. The  83  Table VT  Selected H nmr spectral data f o r RuCl (dmso) L the free nitroimidazole l i g a n d s 9  Compounds^  H-2  C  H-4 (s)  n  N-CH (s)  H-5 (s)  c  complexes and  9  3  C  or 2-CH  1, RuCl (dmso) 2  CH (m)  3  3  3  (dmso) 2.60 2.73 3.33 3.44 3.50 3.53  4  5, Ru(4-N0 -Im) 2  6, Ru(NMe-4-N0 -Im) 2  7, Ru(RSU-1170)  2  8, Ru(RSU-3083)  2  9, Ru(RSU-3100)  JOjRutRsu-sisg)  8.35(8.25) '  7.87(7.80) '  7.96(7.54)  8.78(7.87)  d e  2  d  d  g  1  H,Ru(miso) ^  3.93(3.90)  3.48 3.5^ 3.20 3.25 3.33 3.35  7.91(7.90)  3.60(3.60)  3.37 3.42 3.46 3.52  7.18(7.16)  3.62(3.60)  3.20 3.30 3.39 3.50  7.64(7.92)  3.62(3.49)  3.18 3.20 3.25 3.28  h  2  3.37 3.38 3.40 3.47  d e  7.20(7.19)  7.15(7.11)  3.39 3.48 3.50 3.52  2  7.53(7.45)  7.24(7.19)  3.48 3.53 3.61 3.62  T3,Ri(2-N0 -Im)  8.39(8.37)  8.03(7.99)  3.41 3.46 3.50 3.53  2  12,Ru(De^Tiiso)  2  14,Ru (metro )  k  2  8.05(8.00)  2  2.55(2.45)  3.69 3.77 3.85 3.87  23,Ru(phen)  3.45 3.4^  %,Ru(5-Cl-phen)  3.48 3 . 5 /  25,Ru(5-N0 -phen)  3.52 3.55  2  26,Ru(NMe-Im)  2  27,Ru(2Me-Im)  7.49(7.47)  d  7.13(7.08)  6.91(6.88)  7.11(7.04)  7.05(7.O4)  d  d  2  d  d  3.72(3.70)  3.25 3.31 3.42 3.48  3.76(3.73)  3.25 3.33 3.47 3.51  continued  f  84  .. continued a  2  Oin ppm wrt TMS, i n CDCl^ at room temperature. In each case the integrations are consistent with the assignment.  k Ru represents RuCl^Cdmso^; both sulphoxides ligands are S-bonded. Shift values i n parentheses are for the free nitroimidazole ligands. d  Values are i n good agreement with literature data (ref. 137). The H-2 and H-5 singlets each becomes a doublet when dissolved i n CDC1„ with 57o of trifluoroacetic acid.  ^ The CH^ resonances are singlets. ^ Shift values for ^N-CH ^ HC-CIL multiplets are centred at 54.00 ppm and are the same for tEe free RSU-I170 ligand. anc  2  n  Shift values for HN-CH^t), HO-CH^t) and 2-CH (s) are 53.89, 3.59 and 2.35 ppm,respectively,and are the same for the free RSU-3083 ligand. 3  1  Shift values for 4' and 5' protons are 7.05 and 7.02 ppm, respectively.  j Shift values for the ^N-O^-CH(OH) (m), -CH_ -OCH (m), (HO)CH and 0-CrUs) are at 54.66, 4.42, 4.15 and 3.41 ppm,respectively, and are the same for the free miso ligand. 2  k  3  Shift values for the ^N-O^-CmOH) (m), (HO)CH(m) and (H0)CH (m) are at 54.39, 4.10 and 3.66 ppm, respectively, and are the same for the free De-miso ligand. 2  85  a  >—» •- r - -» ^ - ^ - |  ' • 1  r-^  4  2  PPm  F i g u r e 19:  "Si nmr s p e c t r a o f ( a ) : R u C l ( d m s o ) ( 4 - N 0 ~ I m ) , 2  2  2  2  5_, i n  CDCl.,; and (b) RuCl_(dmso)_(NMe-4-N0 -Im), 6, i n CDCl .  86  presence of four methyl resonances within 10 is again consistent with a chelated 6-coordinate complex with cis arrangements for both chloride and dmso ligands (Table VI).  On comparison with data for the free 4-nitroimidazole ligands (Table VI), the H-2 and H-5 resonances in the complexes are generally shifted slightly to lower field, although the effects are more marked for 6, presumably resulting from the chelation as discussed in section 3.1.  Similarly, the H-4 and H-5  resonances in the complexes 26 and 27 are also shifted slightly downfield on comparison with the free methylimidazole (Table VI), as have been observed in complexes of, for example, Ru(II)  777  and Pt(II).  7iS  The larger downfield shift  for the NMe protons of RSU-3159 on coordination, and a surprising upfield shift of 0.28 ppm for H-2, provide evidence for interaction of the Ru(II) centre with the nitroimidazole ring, presumably via the nitrogen of the NMe. Thus, together with the XPS data (see section 3.1), the nmr data suggest formation of a five-membered chelate ring through the sulphur and nitrogen.  Of the 1,10-phenanthroline complexes, 23-25 show only S-bonded dmso (Table VI). As discussed above, substitution of O-bonded dmso and a Sbonded dmso cis to it, is expected  for the chelating 1,10-phenanthrolines.  There are two methyl resonances again of equal intensity similar to those of complex 6, suggesting rearrangement of the dmso ligands to trans positions, thus giving the /ra/ts-sulphoxides, c/s-chlorides, cis- 1,10-phenanthroline geometric configuration (as for 6 ). The 1,10-phenanthroline complexes show five sets of multiplets in the downfield aromatic region between 57.8-9.2 ppm, and the 5-C1 and 5-NO2 substituents do not markedly affect the position of these resonances, although the total integration on the protons is decreased by one atom.  87  The  C{ H)  nmr spectrum  of  5 shows four  peaks  for the  inequivalent methyls (535.40, 36.63, 44.77, 45.13 ppm) and imidazole carbons at 112.69 (C-2), 116.48 (C-5) and 120.49 (C-4) (Figure 20a). with the  H nmr, two  J  For 6, consistent  C shifts were seen at 38.13 and 45.02 ppm for the  methyls of the two inequivalent dmso ligands; other assigned peaks were 112.41 (C-2), 116.18 (C-5), 119.94 (C-4) and 122.24 (NMe) ppm (Figure 20b).  The *H nmr data for complexes 11-13 (Table VI) again show only Sbonded  dmso  in  CDCl^  solution.  Dissociation  of  the  substituted-2-  nitroimidazole ligands in non-dried CDCI3 or D-,0 solution, as evidenced by UV/VIS spectroscopy and aqueous solution chemistry (see section 4.1), results in the formation of even more complex multiplets for the methyl resonances but no free dmso methyl resonance is observed.  However, when spectra of these two  complexes were recorded in dried CDCI3, relevant *H nmr data could be obtained. Complexes 11-13 have the H-4 and H-5 resonances, and complex 14 has the CH^-2 and H-4 resonances shifted slightly downfield, as compared with the free ligand values, similar to findings for the other complexes 5, 7-10.  The ' H  nmr spectra of complexes 26 and 27 reveal only S-bonded  dmso with four partly resolved multiplets, again indicating inequivalent methyl groups of two inequivalent dmso ligands with the complexes having an all c i s geometric configuration.  3.3.2  - H nmr Data of RuCl2(tmso) L Complexes (m=l. 2 or 3: n = 1 or m  The ' H  n  nmr spectrum of free tmso in CDCI3 reveals three sets of  multiplets centred at 52.60 ( S - C - C H - C H ) , 2.19 (S-CH ) and 1.75 (S-CH ) 2  2  2  2  88  a Solvent Peaks  Ji  l •' i . | 1  IT  '20  T  'l ' ' ' ' | ' ' ' i i i i i i | i i i i | i i i i | i i i i i i i i i | i i i \ | i i i i [ i i i i i 100 80 60 40 20 PPM  S o l v e n t Peaks  111111111111111111111111111111111111111111111111111111111111  120  F i g u r e 20:  100  80  60  13_il C J H } nmr s p e c t r a i n CDC1  40  3  20 PPM  o f (a):' R u C l (dmso) (4-N0 ~Im) , 2  2  2  2  5_, and (b) : R u C l (dmso) (NMe-4-N0 "Im) , 6_. The s o l v e n t peaks e-4-NO -] 2  2  are shown i n the 576-78 ppm r e g i o n .  89  ppm with an integration ratio of 2:1:1, respectively, in agreement with the literature. -*-* The two S - C H resonances are shifted downfield, while the S-C7  2  CH.2-CH2 resonances become shifted to higher field by approximately 0.30 ppm, when the tmso coordinates to Ru(II) (Figure 21, Table VII).  Three sets of  multiplets centred at 5 4.00 (S-CH ), 3.42 (S-CH ) and 2.30 ( S - C - C H - C H ) 2  2  2  ppm are observed, having a relative intensity of 1:1:2, respectively.  2  The large  downfield shift of these S-CH2 multiplets strongly suggests the presence of only S-bonded sulphoxide  (Figure  2l).  8 8  '  1 3 2  -  1 3 3  Indeed,  the  XPS data for  RuCl (tmso) and all the other tmso-containing complexes indicate the presence 2  4  of only S-bonded sulphoxides.  The ' H  nmr spectra for complexes J_5 and 16 in CDCI3 (Table VII)  show five sets of multiplets for the CH2 resonances of tmso (see below), centred at 54.00, 3.65, 3.15, 2.45 and 2.20 ppm, and 5 4.00, 3.51, 3.45, 2.40 and 2.23 ppm, with an integration ratio of 2:1:1:2:2, respectively (Figure 22a).  On  comparison with data for the free imidazole ligands, the H-2 and H-5 resonances in the complexes are shifted to lower field, the effects being more marked for 15 than 16.  The smaller shift in H-5 in J_6 (vs. 6) again supports  chelation via the nitrito group in 6.  The geometric arrangements of ligands  within 15 and 16 cannot be deduced from these spectra.  The ' H  nmr spectrum for complex J_7 in CDCI3 (Table VII) again  shows five sets of multiplets of CH2 resonances centred at 5 3.80, 3.27, 3.12, 2.50 and 2.20 ppm with an integration ratio of 2:1:1:2:2. The ^H nmr data for 18 (Figure 22b, Table VII) also reveal five sets of CH2 multiplets with the same relative intensity as found for 12, centred at 5 3.77, 3.31, 3.22, 2.51 and 2.21. Similarly, ' H  nmr data for 19 and 20 also reveal corresponding patterns centred  90  91  Table VTI  Selected "^H nmr spectral data for RuQ^tmso) L complexes and the free nitroimidazole ligands.  Compounds  H-2 (s)  13  C  H-4 (s) c  H-5 (s)  N-CH (s)  C  3  2, RuCl (tmso), ~ 4  9  9  16, Ru(NMe^-N0o-Im) ~  9  —  Z  17, Ru(De^niso) ~~ ~~  d  18, Ru( SR-2508 ) ~  e  (tmso)  2.30 3.42 4.00  9  15, Ru(4~N0 -Im) ~ ~  Cr^Cm)  8.92(8.25)  8.21(7.80)  7.91(7.54)  8.25(7.87) 3.91(3.90) 2.23 2.40 3.45 3.57 4.00 7.48(7.45) 7.38(7.19)  2.20 2.45 3.15 3.65 4.00  2.20 2.50 3.12 3.27 3.80  7.51(7.45) 7.48(7.24)  2.21 2.51 3.22 3.31 3.77  19, Ru'(CMNI) ~~ ~~  7.01(7.00)  3.56(3.49) 2.00 2.13 3.15 3.35 3.75  20, Ru"(RSU-3159) ~~ ~  7.73(7.92)  2.21 2.51 3.21 3.31 3.75  a  5 i n ppm wrt IMS, i n CDCl^ at room temperature. In each case, the integrations are consistent with the assignment.  k 5i> ^ ^ i " represent RuCl^ttmso^j RuCl (tmso) and RuC^tmso), respectively; a l l sulphoxides ligands are S-Donded. anc  9  3  shift \values given i n parentheses are for the free nitroimidazole Shift ligand. d  6  Shift values for the ^N-O^-CWOH) (m), (HO)CH(m) and (HOJCH^m) are 54.39, 4.10 and 3.66 ppm, respectively, and are the same for the free De-miso. Shift values for the ^N-CH^-CtO) (s), H-N-CH^t) and HD-O^(t) are 55.23, 3.66 and 3.39 ppm, respectively, and are tKe same for the free SR-2508.  93  at §3.75, 3.35, 3.15, 2.13 and 2.00 ppm and 3.75, 3.31, 3.21, 2.51 and 2.21 ppm, respectively. These relative intensities suggest that the two protons of one of the S-CH2  firou  P  s  ° f the coordinated tmso ligand are magnetically inequivalent.  This perhaps results from the fixed orientation of the S=0 when the tmso ligand is coordinated, so that one of the S - C H 2 protons will be in the same "plane" as the O - S - C atoms and the other one will be out of the "plane".  If this  explanation is correct, one would expect to observe a similar effect by the other S-CH_2 groups giving rise to four sets of  S - C H  2  and two sets of  S-C-CH -CH2 2  multiplets with an integration ratio of 1:1:1:1:2:2. However, this is not observed; perhaps the coupling between one set of  protons is smaller and the  S-CH.2  multiplet signal overlaps to give the observed 2:1:1:2:2 integration ratio.  On  comparison with data for the free ligands De-miso and SR-2508, the H-4 and H-5 resonances in the complexes i l and .18 are shifted slightly to lower field at  57.48 and 7.38, and  5 7.51  and 7.48, respectively.  The H spectrum of J l does l  not suggest chelation by the SR-2508 ligand.  3.3.3  - H nmr Data of RuB^fdmsoULp Complexes (n=l or 2) The ' H nmr spectrum of 4 in C D C I 3 is complex at room temperature  (Table VIII), and consists of a complex pattern from 5 3.3 to 3.6 in the S bonded region and a singlet at 52.6 for the free dmso, with relative intensity of the S-bonded to free dmso being 3:1 (Table VIII).  This *H nmr spectrum is  very similar to that observed previously for / r a M s - R u B ^ d m s o ) ^  725  and is  consistent with dissociation of dmso in C D C I 3 solution to give a mixture of five- and six-coordinate species.  725  No O-bonded dmso resonance is observed  in the 2.7 ppm region. In the solid state, /rans-RuB^dmso^ contains all S bonded dmso.  725  94-  Table VIII  Selected H nmr and c{ H } chemical shifts f o r RuBr (dmso) (4-N0 -Im) and RuBr (dmso) (NMe^+-N0 -Im) o  o  o  2  21, Ru"(4-N0 -Im)  2  o  9  CH  N-CH  3  C-5  C-2  4,trans- RuBr (dmso)  o  H-5  H-2  Compounds^  2  o  3  (dmso) 3.35 3.41 3.46 3.57  4  3.59 3.70 45.21 47.22  8.91(8.250° 8.20(7.800° 116.13 112.35 a  a  22, Ru"(NMe-4-N0 -Im)8.03(7.54)° 8.80(7.87)° 3.95(3.90)° 2  108.62  d  112.39  d  119.93  d  3.49 3.53 3.61 3.65 44.91 45.16 47.12 47.58  S i n ppm wrt IMS, i n CDC1 at room temperature.  a  3  k Ru" represents RuBr2(dmso) ; both sulphoxides ligands are S-bonded. 2  c 1 H nmr s h i f t values i n parentheses are for the free nitroimidazole ligand. C| H} values for 21 and 22; C-4 for imidazole carbons of 21 and 22 appear at 119.89 and 116.16 ppm, respectively.  d  13  1  95  The H nmr data for 21 and 22 (Table VIII), like those for 4, show 1  only S-bonded dmso, in agreement  with the ir data (see section 3.2.3). The *H  nmr spectra for these dibromo complexes 21 and 22 are markedly different from those of the corresponding dichloro complexes 5 and 6. The 4-N02-Im complex 2\ has two sets of quartets centred at  5 3.59 and 3.70 ppm, revealing  inequivalent methyl groups for each of the two equivalent dmso ligands (Figure 23a).  These data suggest an all trans configuration for 2L As seen in equation  [3.2], substitution of an S-bonded dmso and another one trans to it would lead to the all trans geometry of the product. For the NMe-4-N0 -Im complex 22, 2  there are four quartets of equal intensity centred at 6 3.65, 3.61, 3.53 and 3.49 ppm,  which  indicate  inequivalent  methyl  groups  for each  of  the two  inequivalent dmso ligands, consistent with a /rans-bromides, c/s-sulphoxides, c/s-NMe-4-N0 -Im configuration (Figure 23b).  This geometric configuration  2  would result from 4 by the substitution of one S-bonded dmso and another dmso cis to it. On comparison with data for the free nitroimidazole ligands, the H-2 and H-5 resonances in the complexes 21 and 22 are shifted to lower field (Table VIII). The effect for H-5 is more marked for the NMe-4-N02-Im complex 22, again presumably resulting from the chelation of the NMe-4-N02"Im via the nitrito group, as observed for 6 (see XPS data, section 3.1). This chelation fits the trans, cis, cis configuration predicted from the ir and *H nmr data of 22. Further, the ^ C ^ H } spectra for complexes  2± and 22 (Table VIII) are  compatible with the corresponding ^H nmr spectra, showing two and four different types of dmso methyl groups respectively.  4-Nitroimidazole vs. 5-Nitroimidazole Formulation  The 4-N0 -Im and 5-N0 -Im ligands are tautomers in solution. 9  9  Laviron  13<  96  I  I  I  I  I  i  I  I {  1  I  4  1  I  Br  I  .,NMe-4-N0 -Im j 2  Z  3 p p m  Br  J.  ~i—i—[—i  i  .  i |  i  i  i  i  i  *  '  i  •  |  •  3  4 p p m  F i g u r e 23:  \ 21,  nmr s p e c t r a i n C D C ^ o f and (b) : RuBr  (dmso) ^  2  ( a ) : RuBr (dmso) (4-NO -Im) , 2  2  (NMe-4-N0 -Im) , 22_. 2  2  97  and Grimison et al.  have shown that in aqueous solution the 4-NC»2-Im is the  predominant form, being approximately 400 times the concentration of the 5-NC»2-Im:  However, complexes with 4-NC>2-Im and 5-N0 -Im as ligands of Pt have been 2  prepared, depending on the experimental conditions: '* 7  white  c/s-PtCi2(5-N02-Im)  complex  2  (as  7  at ambient conditions, the  characterized  by  spectroscopies) was precipitated out initially on treating K P t C l 2  ligand in ethanol; use of large excess of 4(5)-N0 -Im 2  *H nmr 4  and  ir  with 4(5)-N0 -Im 2  ligand and refluxing in  dimethylfuran for 2 h gave the corresponding bright yellow 4-NC>2-Im complex, cisPtCl2(4-N02-Im)2, as confirmed by X-ray crystallography.  In the present study,  complex 5 is considered to contain a 4-N02-Im ligand.  Comparison with the data for metronidazole (a substituted 5-NC>2-Im) reveals that the coordination shift in symmetric VJ^Q^ for this ligand (complex J_4) is 6 c m , - 1  whereas for the 4-NC>2-Im (complexes 5 and 15) and 2-N02~Im (complex 13), the shifts are greater (15, 13 and 11 cm" respectively, Tables III, IV, V). These trends are 1  consistent with a "closer" N 0 i.e. a 4- rather than 5 - N 0  2  2  substituent to the metal in 5 and 15 (similarly for 13).  substituent.  The *H nmr data do not show significant differences between the 4-NC>2-Im (5 and 15) and metronidazole (J4.) complexes, except that the H-2 resonance peak for the  98  4-NC>2-Im complexes is sharp and gives a larger downfield shift compared with the value for the free 4-NC»2-Im ligand.  For the 5-NC>2-Im system (14). the H-4  resonance peak is broad and is slightly downfield shifted compared to data for the free metronidazole ligand.  Similar differences in shapes and shifts of the resonance peaks  are also observed between systems. ^ 7  7  the c/s-PtC^^-NC^-im^  and m - P t C ^ S - N C ^ - I m ^  Use of the NMe-4NC* -Im ligand, which can exist in only this one form, 2  seemed likely to help resolve the tautomer problem; however, this ligand coordinates in a bidentate fashion through the oxygen of the N 0  2  group, resulting in a greater  coordination shifts in the symmetric V Q (~20 cm" ) for this ligand (see section 3.2.1 1  N  and Table III).  99  CHAPTER FOUR AQUEOUS SOLUTION CHEMISTRY OF THE RUTHENIUM NITROIMIDAZOLE COMPLEXES  4.1  Solution Stability of the Substituted-2-nitroimidazole Complexes When the substituted-2-nitroimidazole complexes, JJ_ and 12, are dissolved in  water, PBS, or water/PBS containing a 10-fold excess Cl~ or dmso, under N , the blue 2  colour of the solutions disappears in less than 5 min. The visible bands at 638 and 584 nm for complexes JJ. and 12, respectively, as observed in dry ethanol solution or in the solid state, disappear.  However, if the complexes are dissolved in water containing a  10-fold excess amount of the respective imidazole ligands, the colour of the solutions remains blue and the spectra are unchanged.  These results together with the *H nmr  data in D 0 (see section 3.3.1) indicate that the disappearance of the blue colour is due 2  to the dissociation of the nitroimidazole ligand(s) and that the presence of the following aquation/anation equilibrium exists [4.1]:  R u C l ( d m s o ) ( m i s o ) + H 0 —+• R u C l ( d m s o ) ( H 0 ) ( m i s o ) + x miso [4.1] 2  2  2  2  2  11  2  2  Colourless  Blue  x  y  (x + y = 2)  Further, the aqueous solution of these complexes are non-acidic and non-conducting, and no free CI" is detected using AgNO^; therefore, the possibility of a chloride dissociation is ruled out.  The aquation phenomenon [4.1] is not observed for the tmso  complexes ]_7 and 18, and the colour of aqueous solutions of these remained blue for at least up to 8 h. Complexes 12 and 17 are the same except for the sulphoxide ligands, and therefore the difference in the aquation phenomenon perhaps resulting from the lipophilicity of tmso as a ligand (section 6.3).  In water, complex 13, which contains the  100  unsubstituted 2-N02-Im ligand, does not dissociate the NC^-Im and the colour of aqueous solution remained blue for at least 8 h (see section 3.3.1); presumably the hydrophilic nature of the hydroxy-containing substituents of the miso and De-miso systems favours dissociation of the free ligands in the aqueous media.  4.2  Aqueous Solution Chemistry of the R u C l ( R S 0 ) L Complexes: R2SO = dimethyl- or tetramethvlene-sulphoxide: L = 4-N0 -Im (n=2) or substituted-4NQ -Im (n=l or 2) 2  2  2  n  2  2  On  dissolution  in water  conductivity (100.8 ohm" mol 1  -1  under N , complex  5 instantly gives a molar  2  cm , 2  Table IX) corresponding to a 1:1 electrolyte/'*  2  which results from loss of a single chloride ligand as estimated by A g N 0 titration ; no 3  further dissociation of chloride occurs over 2 days. The UV/VIS spectrum of 5 in the solid state shows a A  max  =345 nm).  m  a  x  identical to that obtained in a solution containing excess CI" (  These data together with those obtained from the XPS (CI 2 p ^  2  3/2)  show that 5 is not the ionic compound, RuCl(dmso) (4-N02-Im)2 Cr. Further, the +  2  resulting aqueous solution is weakly acidic (pH=3.9 for a 2 mM solution of 5), implying partial loss of a proton from coordinated water (-7% dissociation based on the pH 3.9 value, or ~38% dissociation based on an experimentally determined p K value), and a  therefore the molar conductivity corresponding to 1:1 electrolyte must also result partially from this H  +  loss.  Standard pH-titration experiments  yield data that  775  analyze well for a p K value of 4.10 + 0.10 (Figure 24): a  RuCl (R2SO) (4-N0 -Irn) + H 0 — [ R u C l ( H 0 ) (R^O^^-NC^-Im)^ + C l ~ 2  2  2  2  2  2  5, 15 RgSO - dmso, 5 R^SO = tmso, 15  5b, 15b  1!  R  U  C  1  (  0  H  }  (  R  S  i  0  {  }  l  [4  -  21  _l ) + H + Cl~ i L 5c, 15c 4_  N 0  +  m  The p K value seems reasonable for H 0 coordinated at a Ru(II) center. '*-* A 7  a  2  second proton loss measured at higher pH ( p K ' = 8.90 + 0.10) (Figure 24) is attributed a  to  loss of  the  pyrrolic-N  hydrogen to form  coordinated 4-nitroimidazolate; a  101  Table IX  Molar conductivity (A^) for some Ru complexes i n water, at 25°C and 2mM.  Complex  1,  A -1 ohm mol-1cm2 M  3  12.4  c£a-RuCl (dmso) ^ 2  2, RuCl^ltmso)^ 5, Ru(4-N0 -Im) 2  13.7 100.8  2  6, Ru(NMe^+-NO -Im)  15.5  7, Ru(RSU-1170)  2  35.2  h  2  50.1  ?  Ru(RSU-3083)  9, Ru(RSU-3100)  37.4  10, Ru(RSU-3159)  30.7  Ru(miso)  14.1  2  2  12, Ru(De-miso)  16.8  2  Ru(2-N0 -Im) 2  Ru(metro)  27.1  2  22.3  2  15, Ru'(4-N0 -Im) 2  108.2  2  16, Ru'(NMe-4-N0 -Im) 2  Ru'(De-miso)  18.4  2  2  29.1  18, Ru'(SR-2508)  25.4  21, Ru"(4-N0 -Im) 2  118.7  2  22, Ru"(NMe-4-N0 -Im)  20.7  2  Ru, Ru', and Ru" represent RuCl (dmso) , RuCl (tmso) , and RuBr (dmso) respectively. 2  2  2  2  2  102  1 10  1 20  r  1 30  40  Vol of N a O H (mL)  Figure  24:  pH  T i t r a t i o n curves  o b t a i n e d u s i n g 4 mM  NaOH.  103 corresponding value for imidazole itself within Ru(NH3)5(imidazole) is also 8.90. 2+  A UV/VIS spectrum recorded for 5_ at pH 7.0, a pale brown solution ( A  m  a  777  338 nm,  x  log E = 4.63) thus refers to species 5£ minor differences are noted at pH 3.0 (species gb, A___ 343 nm) and at similar acidity in the presence of added CI" (0.1 M) (species I l i a A,  5_, A m  a  x  345 nm). Based on the equilibria shown in equation [4.2], the results suggest 144  that the high CI concentration in the blood ([CI ] ~90 mM)  would prevent the  hydrolysis of complex 5_, and thus species 5_ would dominate in such a medium. However, hydrolysis can presumably occur inside the cells ([CI"] ~1 mM)^^ after penetration through the cell membrane. The main species present under physiological conditions (pH 7.4) in the presence of low Cl~ concentration would be predominantly species 5_c_. The clinically used drug  cis-DDP  has been shown to undergo similar type  of equilibria reactions under physiological conditions (see eq. 4.3).^  The results  1,144  from the studies on complex 1_5_, the tmso analogue, show very similar behaviour in aqueous media (Table IX) giving a  A ^=108.2 ohm" mol"'cm and a pK =4.4, 1  2  a  resulting from the loss of a chloride and the H from the coordinated water. A second +  proton loss measured at higher pH (pK' = 9.01 + 0.10) is again attributed to loss of the a  pyrrolic-N hydrogen to form coordinated 4-nitroimidazolate.  cis-PtCl (NH ) 2  3  cis-[PtCKH 0)(NH ) ] ^ +  2  2  3  -H  2  [FtCNH^Cr^O)^  |J-H pK = 5.6 +  +  a  1^0 cis-[PtCKOH)(NH ) ] 3  2  [Pt(OH)(R 0)(NH ) ] 2  3  2  7.3  (Taken from ref. 50)  [Pt(OH) (NH ) ] 2  3  2  Titration with base of the other complexes 6-10, J4, 16-ii and 23-27 shows no titratable protons are present at least up to pH 11 and, consistent with this, molar  104  conductivity values measured within 10 min of dissolution are lower (15.5-50.1), showing little replacement of Cl" ligand by H 0 . 2  The lack of Cl" dissociation may  result from the electron donating effect of the substituents in the  substituted-4-  nitroimidazoles, and the greater basicity of the phenanthroline ligands (vs. 4-N02"Im). The UV/VIS spectra of all the complexes (see sections on Preparation of Complexes) are dominated by charge transfer bands in the 300-450 nm region, and offer little insight into the coordination geometry.  4.3  Aqueous Solution Chemistry of the RuBr (dmso) L Complexes (L^-NCs-Im. n=2: L=NMe-4-NQ -Im. n=l) 2  2  n  2  On  dissolution in water under N , complex 2 1 , RuBr (dmso) (4-N0 -Im) , 2  instantly gives a molar conductivity of  2  118.7 ohm 'mol  cnr  2  4  2  2  (Table IX), again  corresponding to 1:1 electrolyte/^ which results from loss of a single bromide ligand as evidenced by AgBr formation; no further bromide loss occurs over 2 days under N . 2  As with 5_ and J_5, hydration of 21 occurs giving species 21b. However, unlike the aquo derivatives (5_b and 15b) of 5 and 1 5 , 21b does not deprotonate at the coordinated H 0 2  or coordinated 4-N0 -Im, at least up to pH 11. 2  Thus, the higher molar conductivity  for 21 as compared with 5 and 15 is surprising because there is no H  +  contributions.  Based on these results, the following solution equilibrium [eq. 4.4] is suggested:  RuBr (dmso) (4-N0 -Im) + ^ 0 — 2  2  2  2  [RuBrd^O) (dmso) (4-N0 -Im) ] + Br~ +  2  21  2  21b  2  [4.4]  Thus the more basic bromide system, 21, (compared to the chloride system, 5) has 7  7  coordinated H 0 and imidazole ligands that are at least 10 and 10 2  times, respectively,  less acidic than the corresponding ligands in 5. The lower acidities are consistent with the presence of more basic bromide ligands, but a more important factor almost certainly arises because of differences in the geometrical isomers present.  105  Complex 5 (and 15) has all cis geometry [eq. 4.5] and the chloride replaced is likely to be the one trans to the 4-NC>2-Im (even though dmso has a strong trans effect  ), because only complexes with 4-N02~Im (5 and ]_5), but not N-substituted-4-  NC^-Im, show the dissociation of a chloride (eq. 4.2 and 4.5):  L Ru-  Cl  Ru  -Cl"  Cl  01 ,H  5  5b  Cl  [4.5]  2  For 21, bromide loss must occur according to eq. 4.6:  L Br-  Ru  -Br  Br  -Br"  •Ru  L  L  21  21b  [4.e;  For ligands trans to water, it has been argued that the higher the trans effect, the weaker the metal-oxygen bond and the stronger the oxygen-hydrogen bond, i.e. the weaker the acidity of the coordinated I^O. ^"* However, this suggestion was based on 7  data for just cis and /ra«.s-Pt(NH )2(H20)2^ complexes (cis, p K = 5.6; trans, p K = +  a  3  4.3).  For the data: trans-?t(C H )(U 0) C\2 2  d  2  1  a  (pK = 5); cis-Pt(NH )(H 0)Cl (pK a  3  2  2  a  ~7), the correlation is clearly not valid because in term of trans effects, C2H4 » Cl". Here, the strong TT -acid properties of the C2H4 were invoked to account for stronger  106 acidity of the water within the ethylene complex.  The strong trans effect - weak  acidity argument clearly does not hold for 21b vs. 5t> because in the latter, the water is probably trans to the 4-N02"Im, which is likely to have a strong trans effect judging from its unsaturated nature.  Thus 4-N02"Im is considered to have a stronger trans effect than Br", and yet the coordinated water in 5b is much more acidic than that in 21b.  The strong acidity  could be rationalized in terms of the presence of the strong trans IT -acceptor 4-NC>2Im.  Thus, if the structures of 5 and 2 i are as shown (the evidence being nmr data,  sections 3.3.1 and 3.3.3), then the monoaquo species are probably the isomers shown in 5b and 21b (eq. 4.5 and 4.6).  The discussion on the differences between the aqueous solution chemistry of 21 with 5 (and 15J is clearly very speculative, but at least it mentions the factors that have been used in the literature to account for the acidities of coordinated water.  Species 21b would predominate in aqueous solution and presumably under physiological conditions.  A UV/VIS spectrum recorded for 21 at pH 7.0 ( A  m a x  342  nm, l o g £ = 4.02) thus refers to species 21b: minor differences are noted in the presence of added Br" (0.1 M) (species 21, A _  o v  347 nm, log£= 4.13). Titration of complex 22  with base shows no titratable protons up to pH 11 and the molar conductivity value measured under the same conditions is low (Table IX), showing little replacement of Br" ligand by water.  Clearly, the aqueous solution chemistry of the complexes is germane for an understanding of their biological properties.  Of the complexes synthesized, only 5, 15  and 21 demonstrate high lability according to equations [4.2] and [4.4].  107 CHAPTER FIVE CYTOTOXICITY AND RADIOSENSITIZATION IN VITRO  An important objective in investigating  the sensitizing  properties of new  compounds is to identify a drug that will be superior to misonidazole in the clinic. In addition, these compounds should have beneficial properties, e.g.: the ability to penetrate into the centre of the tumour; metabolic stability and have low side-effects in vivo, but these studies are beyond the scope of the present work. However, the more fundamental beneficial properties of the compounds such as larger SER value in hypoxic cells but not normal oxic ones, and low cytotoxicity, can be assessed in vitro and are discussed below.  5.1  Toxicity of the Ru Complexes and their Nitroimidazole Ligands Various in vitro studies with Chinese hamster cells have been used to study  hypoxic cytotoxicity of nitroimidazoles ^'-^"* * ^ and platinum complexes.- ' ' ' -  6  7  57  60  7  5  The toxicity of the chemicals is measured as plating efficiency (PE) at various times of drug treatment.  In the present experiments, t=0 denotes the time when the cells are  added to the compound dissolved in growth media, immediately removed, washed and plated for survival. The data given represent the average of three resulting PE values. The higher the PE value, the lower the toxicity.  The PE values for CHO cells plotted as a function of incubation time at 37°C in solutions containing 4-N02~Im and the Ru complex 5, under hypoxic and oxic conditions, are shown in Figure 25.  For each point, three aliquots of cell suspension  were processed and plated independently on the same day.  The data show that the  hypoxic toxicity of the complex at 200 juM is significantly less than that of the free ligand at equivalent concentration (400 ^«M). There is no toxicity for either compound at the corresponding concentration in aerobic cells.  Note that there has been no  108  UJ  1  -  01  TIME  F i g u r e 25:  T o x i c i t y o f RuCl  C h o u r s )  2  (dmso) (4-NO -Im) , 5, and 4-NO -Im i n 2 2 2 — 2  h y p o x i c and o x i c CHO c e l l s . Medium c o n t a i n i n g the i n d i c a t e d c o n c e n t r a t i o n o f c h e m i c a l was degassed f o r 1 h a t 37°c b e f o r e c e l l s were added  (t=0). Hypoxic c o n t r o l (•), N  400 pM 4-N0 ~Im  + 200 \iH o f 5^ ( A ) , and 0  2  o f 5 (•).  (T) , N  2  2  2  +  + 200 uM  109  normalization of PE against the drug free control. The hypoxic toxicity data for the other 4-NC>2-Ini complexes 15 and 21 are summarized in Table X. Their toxicities are similar to the value obtained for 5.  The toxicity data obtained when CHO cells are exposed to the various N substituted-4-nitroimidazoles and their Ru complexes (6-K), 16 20 and 22), under hypoxic conditions after 4 h of incubation, are shown in Table X. The hypoxic toxicity of the complexes at lOO^M is again significantly less than that of the corresponding free ligand at equivalent concentration.  In addition, no toxicity is seen in an oxic  environment at these times and concentrations.  The complex concentrations used for  comparison with the ligands NMe-4-N0 -Im and RSU-3159 were 200 2  and 100 MM,  respectively, as the complexes contained only one such ligand per metal. No significant difference in PE is observed between the dmso and tmso complexes (6 vs. 16) or the Cl" and Br" complexes (6 vs. 22).  These data show that the substitution of dmso by tmso  and Cl" by Br" ligands does not alter the toxicity of the corresponding complexes. The hypoxic toxicities of complex 12 and that of its free CMNI ligand were very high, with PE values at 50 JJM of about 0.0007 and 0.0004 respectively, and therefore their radiosensitizing abilities were not studied.  The PE values of CHO cells after 4 h incubation at 37°C with various 2-N02~Im derivatives, metronidazole and their complexes under hypoxic conditions are shown in Table XI. For complexes JJL, 12, J_4, and 18., the hypoxic toxicities of these complexes at 200 MM are similar to those of the free ligands at 400 MM. The hypoxic toxicity data for complexes 13. and 12 are significantly lower than those of their corresponding free ligands at equivalent concentrations. Again, no toxicity is seen in aerobic cells.  110  Table X  Plating efficiency (PE) of the 4-nitroimidazole ligands and their Ru(II) complexes i n hypoxic CHO c e l l s (4 h at 37 C)  PE (cone. |jM) a Complex  „ ,. , Free ligand  r  5, Ru(4-N0 -Im)  , complex  0.052 (400)  0.10  (200)  6, Ra(NMe-4-N0 -Im)  0.49  (200)  0.62  (200)  7, Ru(RSU-1170)  0.037 (100)  0.15  (200)  8, Ru(RSU-3083)  0.036  (100)  0.12  (200)  9, Ru(RSU-3100)  0.0042 (100)  0.12  (200)  io, RutRsu-aisg)^  0.0027 (100)  0.16  (100)  15, Ru"(4-N0 -Im)  0.058 (400)  0.13  (200)  16, Ru'(NMe-4-N0 -Im)  0.49  0.53  (200)  19, Ru'(tmsoMCMNI)  0.0004  20, Ru'(RSU-3159)  0.0027 (100)  0.11  (100)  21, Ru"(4-N0 -Im)  0.058 (400)  0.32  (200)  0.49  0.62  (200)  2  2  b  2  2  2  2  2  2  2  2  b  2  2  22, Ru'"(NMe^+-N0 -Im)  b  2  (200)  0.0007  (50)  (200)  (50)  a Ru, Ru', and Ru" represent RuClo(dmso) , RuCl(tmso)^, and RuBr (dmso) , respectively , except 2(] contains only 4me tmso ligarid. b The concentrations used for the free ligands NMe-4-N0 -Im and RSU-3159 (both presumably bidentate) were 200 JJM and 100 JJM, respectively. 9  z  2  2  L  9  Ill  Table XI  Hypoxic t o x i c i t y (PE) of the 2-nitroimidazoles and metronidazole and their Ru(II) complexes i n CHO c e l l s (4 h at 37°C) a  PE  b  Free ligand (400 pM)  Complex  Complex (200 MM)  11, Ru(miso)2  0.75  0.77  12, Ru(De-miso)2  0.12  0.14  0.0031  0.52  14, Ru(metro)2  0.80  0.79  17, Ru'(De-miso)2  0.13  0.42  18, Ru'(SR-2508)  0.073  0.099  13, Ru(2-N0 -Im) 2  ?  c  Ligand and complex concentrations used were 400 and 200pM, respectively, unless stated otherwise. k Ru and Ru' represent RuC^dmso^ and RuC^tmso^s respectively. C  The concentration used f o r 18 and the ligand SR-2508 were 200 MM, because the complex contains only one such ligand per metal.  112  The hypoxic toxicities of the uncoordinated 1,10-phenanthrolines are very severe, with PE values of about 0.001  at 20 MM after 3 h incubation.  However, the  corresponding Ru complexes (23-25) show drastically reduced hypoxic toxicity with PE values of 0.05 at 200 uM after 3 h incubation.  Only a small degree of toxicities is  observed in aerobic cells for both free 1-10-phenanthroline ligands and the associated Ru complexes.  The  cytotoxicity of nitroimidazole radiosensitizers is believed to result from  metabolic  reduction of  cells."*'*' ^' * 5  7-  5  the  nitro group to derivatives  which cause damage in  This reduction only proceeds under hypoxic conditions or at low oxygen  concentrations and is known to involve the initial addition of a single electron.  The  subsequent addition of electrons proceeds via formation of the nitroso, hydroxylamine and amine derivatives: * 7-  R-N0  2  RN0  T 2  5  —  RN=0 — - R-N-0  — - RNHOH —  RNR^  [5.1]  In the absence of oxygen, a toxic species is produced which interacts with crucial cellular molecules and cell death occurs.*'*'-^ This reduction process is prevented by the -  presence of oxygen which reverses the first stage of nitroreduction, the formation of a radical anion (see section 1.8). toxicity.  This explains why oxic toxicity is not as high as hypoxic  A change in biological interactions with the reduction enzymes, and/or the  depletion of reducing equivalents in hypoxic cells^"*  9,59,7-  *  5  may explain why the Ru  complexes are always less toxic than the free ligands.  5.2  Chromosomal-Damaging Activity of Complex 5 The most dramatic effect on biological systems is DNA damage; this can result in  some genotoxicities such as chromosomal aberrations, * which can eventually transform 70-  113  the cell or render the cell unable to divide, leading to cell death.  Because complex 5,  RuCl2(dmso)2(4-NC>2-Im)2, is a good radiosensitizer, less toxic than the free 4-N02~Im ligand and has been shown to bind to DNA (see section 6.1.1), it was examined for a genotoxic effect on normal cells, as measured by the in vitro induction of chromosome aberrations in CHO cells.  The frequencies of chromosome aberrations in CHO cells exposed for 3 h to complex 5 are shown in Table XII. Complex 5 showed a dose-dependent increase in chromosome damaging activity in CHO cells.  The extent of clastogenic activities is  expressed as the percentage of metaphases plates with at least one chromatid break or one chromatid exchange.  The percentage of metaphases with chromatid aberrations  following exposure to 5 is much less than that elicited by exposure to c/s-DDP. There is a significant increase in chromosomal breakage at 10 ,uM c/s-DDP and the drug becomes toxic, leading to cells death in this experiment after this dose (Table XII).  This  difference in effects of 5 and c/s-DDP might be the result of differential repair of the lesions by the cells.'"'* 5  Several chemicals can be activated by the mixed function oxidases present in the S9 mixture to yield electrophilic species, such as in the case of nitroso species or alkylating agents/^ which bind to DNA, RNA or proteins. ** 7  7  In this study, addition  of the S9 mixture did not enhance the clastogenic activity of 5 and c/s-DDP, indicating that the complexes were acting directly. In a similar study, '** S9 had no effect on the 7  mutagenicity of Pt(II) and Pt(IV) coordination complexes as measured in the Ames Salmonella mutagenic assay. Binding of Pt complexes to DNA is presumably responsible for their genotoxic effects;^ ruthenium complexes have anti-tumor activity*^'^ and 7  also bind to reasonable.  D N A  8  0  '  8  4  ,  1  5  2  and thus the lack of activation by the S9 mixture seems  114  Table XII  Clastogenic a c t i v i t y of RuCl (dmso) (4-N0 -Im) (5) and cis-DDP on CHO c e l l s ' z z Z 9  9  9  9  Average 7 metaphase plates with chromatid aberrations' 0  RuCl (dmso) (4-N0 -Im) o  o  o  Cone. (mM) 10.0  ets-DDP  o  +S9  +S9  Cone. (JJM) b  T  30.0  T  MI  19±1(0.04) 19±2(0.05)  10.0  MI  14±2(0.02) 15±3(0.03)  8.0  61-5(2.54)  8±2(0.02)  4.0  45±4(1.32) 40±5 (1.28)  5±1(0)  2.0  30±5(0.76) 28-2 (0.85)  31-3(0.18)  29±3(0.14)  50.0  6.0  26±2(0.10) 22±2(0.09)  2.0 1.0 0.4  9±2(0.01)  0.2  4±1(0)  T  C  80-10(3.66) 67±5 (2.51)  a. Average "L of three plates - s.d.; the figures i n parentheses show the average number of chromatid exchanges per metaphase. b. T = Toxic, no metaphase was observed. c. MI = Mitotic inhibition: observed.  fewer than 40 diploid metaphases per plate  115  For purposes of comparison, the clastogenic activities of the precursor, cisRuCl2(dmso)4 complex, and the 4-NC*2-Im ligand, were also studied (Table XIII). Similar frequencies of chromatid aberrations are induced by the precursor and by the ligand at the same concentrations. Further, both the precursor and the 4-NC>2-Im ligand produce fewer chromosome aberrations than 5_ at an equivalent molar concentration. The enhanced clastogenic activity of 5, however, appears to result from the combined effect of the metal and the 4-N0 -Im 2  ligand (Tables XII, XIII), suggesting that both  components of 5 are contributing to the chromatid aberrations.  Incubation with  activating enzymes (the S9 mixture) does not increase the extent of damage by the precursor, i or 4-N0 -Im (Table XIII). At these concentrations, dmso ligand does not 2  show any genotoxic effect.  The clastogenic activity of complex 5 is also compared to that of the clinically used nitroimidazole radiosensitizer, miso.  The frequencies of metaphase plates with  chromatid aberrations following exposure to 5, 4-NC»2-Im, or miso, are shown in Figure 26.  Complex 5 and miso produce a similar yield of such metaphases with chromatid  aberrations that is higher than that of 4-N0 -Im at the same concentration. The results 2  indicate that 5 might have some advantage as a radiosensitizer over miso, because the complex produces a similar percent metaphases with chromatid aberrations but yields a higher SER value (see section 5.3).  In addition, 5 is much less genotoxic than m-DDP,  which produces more chromatid multiple-exchanges ^ 7  and which also is used in  conjunction with radiation. '  Despite the lack of understanding of the mechanism of chromosome aberrations, accumulated data have suggested the  role of chromosome  damaging activity in  116 Table XIII  Clastogenic a c t i v i t y of cts-RuCl (dmso) and 4-N0 -Im on CHO c e l l s 9  Z  /  4  0  1  Average "L metaphase plates with chromatid aberrations cis -RuCl (DMSO), 0  Cone  a.  (mM)  4-nitroimidazole  +S9  +S9  10.0  15±2(0.07)  13±2(0.07)  16±3(0.06)  17-3(0.05)  6.0  12±2(0.04)  10±1(0.05)  13-2(0.04)  14±2(0.03)  2.0  7±2(0.02)  6±2(0.03)  10±2(0.02)  11±2(0.01)  1.0  4±1(0.01)  3-1(0.01)  7±2(0)  6±1(0)  0.5  2±1(0)  1-1(0)  4±1(0)  3±1(0)  Average % of three plates - s.d.; the figures i n parentheses show the average number of chromatid exchanges per metaphase.  117  CD R U G )  Figure  26:  C  mM)  Chromosome damaging a c t i v i t y o f 5_ (•), miso ~ °2~ the absence o f S9). 4  N  I m  ( - )  i  n  C  H  O  c  e  l  l  s  (A) and  f o l l o w i n g 3 h exposure ( i n  118 carcinogenesis.  '  Evaluation of the genotoxicity of agents with pharmaceutical  potential is important.  5.3  Radiation Sensitization of Aerobic and Hypoxic CHO Cells In this study, the sensitizing enhancement ratio (SER) value is calculated as the  ratio of X-ray doses for cells in medium with and without the test compound required to produce a survival fraction of 0.01.  The linear-quadratic CY, (3 model' " was used to  fit curves to the data (see section 2.7).  Each data point represents the average of three  individual experiments.  2  5  No radiosensitizing enhancement or protection is observed at  dmso or tmso, or with 200  m-RuCl (dmso) , RuCl (tmso) or trans-  RuB^dmso^ in control experiments.  In these experiments, the chemical is added to  400  2  4  2  4  the cell suspension and incubated for 1 h at 37°C prior to the start of irradiation at 4°C. In no instance was there measurable toxicity from exposure to the compounds under these conditions: the PE values for zero X-ray dose control samples taken immediately prior to irradiation are the same as for cells not exposed to the chemical.  5.3.1  Radiosensitization by the Metal Complexes It is well established that c/s-DDP, trans-DDP, and a variety of other  Pt(II) and Pt(IV), complexes are radiation sensitizers for hypoxic cells, although the detailed mechanisms are not known. •50,57,57,72,76,705 s  o m e  //-arcs-complexes  of Pt are also radiosensitizers and are generally much less cytotoxic than ciscomplexes. ^' ^ * 7  7  -  C/s-dichlorobis(metronidazole)platinum(II) (FLAP)  7,2  was the  first reported metal-sensitizer complex studied but its "initial" activity could not be substantiated by later reports. > $J03 74  7  i  n  contrast, the present study  demonstrates that an enhancement of X-ray damage in hypoxic CHO cells in vitro can indeed be achieved with a range of different Ru(II) nitroimidazole complexes.  119  All of the Ru(II)-nitroimidazole complexes sensitize to a greater extent in hypoxic than oxic cells.  The SER values for the free 4-N0 -Im ligands and 2  respective complexes in hypoxic conditions are summarized in Table XIV. The survival curves obtained with complex 5 and free 4-N0 -Im ligand in CHO cells 2  are shown in Figure 27 (Table XIV). The SER value with 200 MM of 5 is 1.6 in hypoxia, while there is no sensitization of oxic CHO cells to X-ray by this complex. The SER value for 4-N0 -Im at equivalent concentration (400 ^MM) is 2  only 1.2 and again no sensitization of oxic CHO cells is observed.  The oxygen  enhancement ratio (OER) in this experiment is 3.0 which is similar to the value reported by others for CHO cells. '* The SER value for the tmso analogue of 5, 6  complex 15, is also 1.6 at 200 juM in hypoxia (Figure 28 and Table XIV). There is a measurable increase in SER value over ligand alone for the dibromo complex 21 at 200 MM concentration (Figure 29).  Thus, RuBr (dmso) (4-N0 -Im) , 2  2  2  2  complex is not as good a radiosensitizer as the dichloride analogue; this is certainly related to the different isomer forms and aqueous solution behaviour (see sections 4.1 and 4.2).  Figure 30 shows the survival curves obtained when complex 6 or NMe-4-N0 -Im is present during irradiation of hypoxic CHO cells. 2  values obtained for 6 and NMe-4-N0 -Im 2  at 200 pM  The SER  are 1.3 and 1.2,  respectively. The SER values for the other N-substituted-4-N0 -Im ligands and 2  their Ru(II) complexes, 7-10, 16 and 20, are summarized in Table XIV. The SER values for these complexes in hypoxia are between 1.1 and 1.3 and again no radiosensitization of oxic CHO cells by any of these complexes is observed.  In  general, the SER values of the N-substituted-4-N0 -Im complexes are lower 2  than those of 5 and 15 containing unsubstituted 4-N0 -Im ligands. 2  120  Table XIV  The hypoxic SER values of the 4-nitroimidazole ligands and their Ru(II) complexes  SER values  Complex  d  3,  Free ligand (cone. pM)  5, Ru(4-N0 -Im)  1.2 (400)  1.6 (200)  6, Ru(NMe-4-N0 -Im)  1.2 (200)  1.3 (200)  7, Ru(RSU-1170)  2  1.1 (200)  1.3 (100)  1>  Ru(RSU-3083)  ?  1.2 (200)  1.2 (100)  9,  Ra(RSU-3100)  1.2 (200)  1.1 (100)  1.3 (100)  1.3 (100)  1.2 (400)  1.6 (200)  1.2 (200)  1.4 (400)  1.2  1.3  2  2  b  2  10,  2  (RSU-3159)  b  15, Ru'(4-N0 -Im) 2  2  16, Ru' (MMe^N0 -Im)  b  2  19, Ru'(tmso)(CMNI)  C  (50)  1.3 (100)  1.3 (100)  21, Ru"(4-N0 -Im)  1.2 (400)  1.3 (200)  1.2 (200)  1.3 (200)  2  2  22, Ru"(NMe^+-N0 -Im) 2  Ru, Ru', and Ru" represent RuCl (dmso) , RuCl (tmso) , and RuBr (dmso) , respectively, except 2!0 contains only one tmso ligand. 2  b  (50)  20, Ru'(RSU-3159)  b  ci  complex (cone. |jM)  2  2  2  2  2  The concentrations used for comparision with the ligands NMe-4N0 -Im and RSU-3159 (presumably bidentate) were 200 (jM and 100 pM, respectively. 2  The concentrations used f o r this complex and i t s ligand were 50 |JM because of their t o x i c i t y . d  Estimated maximum error + 0.05.  121  i  1  1  T  1  r  I  I  Z  j  i  S  i  18  I  15  D O S E  F i g u r e 27:  25  30  ( G r a y )  Radiosensitization 5_, o r 4-NO  20  (dmso) (4-NO -Im) 2 g 2 2 -Im. The c e l l s were i n c u b a t e d a t 37 C f o r 1 h.  Hypoxic c o n t r o l  o f CHO c e l l s by RuCl  (•), N  5_ ( A ) , o x i c c o n t r o l  2  + 400 pM 4-NO  (•) , and O  -Im  2  (•), N  + 200 pM 5 (•) .  2  + 200 pM  122  i  i  1  1  1  r  Z  DOSE Figure  28:  (Gray)  Radiosensitization 15, o r 4-NO  -Im.  o f CHO  (tmso) (4-NO -Im) 2 2 . 2 The c e l l s were p r e - i n c u b a t e d a t 37 c f o r  1 h. Hypoxic c o n t r o l + 200 pM 15  (•) .  c e l l s by RuCl  (•) , N  + 400 \iM 4-NO 2  -Im 2  ( A ) and N 2  123  IS  D O S E F i g u r e 29:  28  25  38  ( G r a y )  R a d i o s e n s i t i z a t i o n o f CHO c e l l s by RuBr^(dmso)  (4-NO^-Im)^»  21, o r 4-N0 ~Im. The c e l l s were p r e - i n c u b a t e d a t 37°C f o r 2  1 h. Hypoxic c o n t r o l + 200 pM 21 (•).  (•); N  2  + 400 pM 4-N0 ~Im ( A ) and N 2  2  124  T  i  0  5  1  i  _!  Ie  D O S E Figure 30:  1  15  i  r  1  1  20  25  C G r a v  —  i  30  J  >  Radiosensitization of CHO c e l l s by RuCl (dmso) (NMe-4-N0 -Im),  2  2  2  6, or NMe-4-N0 ~Im. The c e l l s were pre-incubated at 37°C for 1 h. Hypoxic control (•), N + 200 M NMe-4-N0 ~Im ( A ) , and 2  2  N  2  + 200 pM 6_ (•) .  M  2  125  The survival curves obtained with SR-2508 and RuC^tmso^SR2508), 18, in CHO cells under hypoxia are shown in Figure 31. The SER values for the ligand SR-2508 (200 jM), and the relevant complex 18 (200 ^M), are 1.3 and 1.5, respectively.  Complex J_8 has the highest SER value of all the 2-  nitroimidazole complexes being studied probably because it is stable in solution with respect to loss of the nitroimidazole ligand unlike the other substituted-2nitroimidazole species.  The free ligand, SR-2508, has been shown to yield a  good SER value (~ 1.6 at 1.5 m M ) . * 7  9  The SER values for the other 2-  nitroimidazole and metronidazole complexes (11-14 and 12) in hypoxia are between 1.2-1.4 (Table XV). There are no significant increases in SER values between the dmso complexes ( H and 12) compared with those for their free nitroimidazole ligands probably because these dissociate from the complexes in solution. As with all the other systems, no oxic sensitization was observed. For the other complexes containing no nitroimidazole ligands, such as 23-29, no radiosensitization effect was observed.  126  D O S E  F i g u r e 31:  ( G r a y )  R a d i o s e n s i t i z a t i o n o f CHO c e l l s by RuCl (tmso)  (SR-2508), 18_,  o r SR-2508. The c e l l s were p r e - i n c u b a t e d a t 37°C f o r 1 h. Hypoxic c o n t r o l 18 (•).  (•) , N  2  + 200 pM SR-2508 ( A ) and N  2  + 200 \iM  127  Table XV  The SER values of 2-rdtroimidazoles, metronidazole, and their Ru(II) complexes  SER values  Complex  11, Ru(miso)2 12, Ru(De-Miso)  2  13, Ru(2-N0 -Im) 2  14, Ru(metro)  2  2  17, Ru'(De-miso)  2  18, Ru'(SR-2508)  b  0  Free ligand (400 pM)  Complex (200 pM)  1.3  1.4  1.3  1.4  1.3  1.4  1.1  1.2  1.3  1.4  1.3  1.5  Ru and Ru' represent RuCl (dmso) and RuCl (tmso) , respectively. 2  2  2  2  k The concentration used for the ligand SR-2508 was 200 MM, because the complex contains only one such ligand per metal. Estimated maximum error + 0.05.  128  CHAPTER SIX FACTORS R E L A T E D TO RADIOSENSITIZATION  Chemicals in general can modify biological response to radiation in a variety of ways/  50  for example: binding to DNA, interaction with radio-protective thiols, and  enhancing radiation damage by electron affinity of the chemicals In most cases where compounds have been shown to radiosensitize, one or several mechanisms have been proposed to explain the observation. The mechanisms of interaction listed below are not necessarily independent of each other.  6.1  DNA Binding With the intent of improving the radiosensitizing properties of radiosensitizers by using DNA-binding metals to carry the sensitizer to the DNA, questions as to whether the desired goal of targeting is achieved, i.e. whether these complexes are in fact binding to DNA, need to be addressed. 6.1.1  Assessment by Inhibition of Restriction Enzymes The method described here for comparing the binding of complexes  to isolated DNA is a variation of that used previously to study cis and transDDP,  7 5 7  and has been modified and expanded by Skov et al}  22  Plasmid was  linearized in order to simplify the interpretation of gel pattern, and titration (inhibition vs. concentration) was used to provide a means of comparing the DNA binding agents.  The assay provides a quick comparison of binding by  various compounds. It is a qualitative assay at present - the numbers obtained are meaningful only when compared with others obtained under the same experimental conditions such as temperature, incubation time and pH.  129  The binding site for cis and trans-DDP has been known for some time to be on G-rich areas of D N A .  5 2 , 7 5 7  Restriction enzymes which cut  DNA in G-rich areas are more inhibited than those with fewer G residues in their recognition sequence.  757  The enzymes BamHI (G-rich) and EcoRI (G-  poor) were chosen to see whether this specificity was affected by attachment of ligands and complexes.  There is no intent to imply that the binding of Ru  complexes occurs specifically at the BamHI site; this location is used only because of its guanine (G) content, which is expected to have a relatively high attraction for platinum m e t a l s . ' ^ ' 80  6.1.2  5  722  '  757  DNA Binding by the Ru Complexes The complexes were assessed qualitatively, i.e., whether there is  noticeable inhibition at 200 uM, after 1 h incubation. Of all the complexes studied, only 5, 23-25, and 28, show inhibition of the endonuclease activity of BamHI, but not of EcoRI, on plasmid pSV2-gpt (Figure 32).  These findings  are assumed to be related to the binding of the complex at or near the G G A T C C sequence, and suggest that these complexes preferentially bind to G rich  regions  similar to  complexes. < >° 0U  0J  those reported previously for other ruthenium  •°° A range of concentrations of 5 was also examined and  compared with data for c/s-DDP as shown in Figure 33. Both 5 and c/s-DDP inhibited BamHI but c/s-DDP also inhibited EcoRI activity. The concentration for 10% inhibition of BamHI by 5 was 27 MM c/s-DDP (12 MM) for 10%).  which was higher than that of  but lower than that for c/s-DDP inhibition of EcoRI (30 MM  Table XVI summarizes the data (concentration to achieve 10%  inhibition) for several of the complexes examined. It should be noted that in control experiments with free ligands, the only inhibition observed in the concentration range under study for the complexes was that for the 4-N02"Im  130  a b c d e f g h i  F i g u r e 32:  j k l m n  I n h i b i t i o n o f endonuclease a c t i v i t y o f BamHI and EcoRI. (a) DNA a l o n e ; (b) DNA + 5_ + EcoRI, (c) DNA + 5 + BamHI; (d) DNA + 15_; (e) DNA + 15 + EcoRI; (f) DNA + 15 + BamHI; (g) DNA + 23_; (h) DNA + _23 + EcoRI; ( i ) DNA + 2_3 + BamHI; (j) DNA + 24 + BamHI; (k) DNA + 5-C1-1,10-phenanthroline; (1) DNA + 5-NO - 1 , 1 0 - p h e n a n t h r o l i n e ; (m) DNA + ais-DDV + BamHI; (n) DNA + BamHI.  131  F i g u r e 33:  T i t r a t i o n o f BamHI s i t e w i t h RuCl 5_, 4-NO -Im, and c-DDP (Cts-DDP).  2  (dmso) (4-NO 2  Table XVT  Relative inhibition of BamHI a c t i v i t y by some Ru complexes,  cis -DDP and trans -DDP  Approximate concentration for 10% inhibition (|jM)  Complex  3  Relative inhibition / i n i t i a l slope,, e.g. Fig. 33 {  ;  cis -DDP  12  0.85  trans -DDP  12  0.85  b  0.06  Ru(4-N0 -Im) , 5  27  0.38  Ru(phen), 23  18  0.49  Ru-(5-Cl-phen), 24  16  0.52  Ru-(5-N0 -phen), 25  18  0.47  b  0.07  cis -RuCl (dmso)^, 1 2  2  2  2  4-N0 -Im 2  Ru represents RuCl (dmso) . 2  2  k 107o inhibition not achieved.  133  (Figure 33).  This suggests that targeting of the ligand via coordination to Ru  (within 5) is occurring and would explain why 5 is a better sensitizer than the free 4-NC>2-Im ligand.  However, the analogous tmso complex 15 which has  aqueous solution behaviour and SER data similar to those of 5, did not show inhibition of activity of restriction enzymes up to the limit of solubility (500 jiM) under the same experimental conditions. This suggests that DNA binding is not the factor giving rise to the improved sensitization for this system. More data are required before any definitive conclusions can be deduced. Within the dichlorobis(dimethyl sulphoxide)-4-nitroimidazole complexes, only 5 binds to DNA, the N-substituted ones do not (i.e. inhibit restriction enzyme activity).  One explanation is the importance of the presence of the pyrrolic  proton of 4-N02-Im, an unsubstituted N-H(l), in enhancing the binding, as observed in the PtCl2(NH )(4-N0 Im) series. 3  76  2  Hydrogen bonding by the N -  H to the phosphate group of the DNA backbone was suggested by van Kralinger et al.  138  and was later shown for some Pt(II) systems.  The lack of  -5-2  DNA binding may also result from the stability of the complexes in solution; in the case of 5, aquation to the neutral hydroxy species may generate an active reagent as demonstrated for the c/s-DDP systems. ^  Further, none of  5  the Ru complexes inhibited EcoRI in this study, a behaviour unlike that of the m - D D P and PtC^NH^XL), L=miso or metro, compounds which also inhibit the activity of this restriction enzyme.  7 ?2  The bromide complex 21, RuBr (dmso) (4-N0 -Im) , does not 2  show any inhibition of enzyme activity.  2  2  2  When 21 is dissolved in aqueous  media, no acidic proton is detected after dissociation of a bromide ion, and the charged species 21b. RuBr(H20)(dmso)2(4-N02-Im)2  +  4.3).  is formed (see section  This might be expected to bind to electron-rich DNA, as shown for  134  [Ru(NH ) H Or 3  5  2  +  by Clarke et alP*  The non-inhibition of activity of  restriction enzymes perhaps results from the way 21 binds to DNA. For the /raws-bromide complex, binding may not be greatly distorting the DNA structure, or may be weak, '* and therefore the restriction enzymes could still 5  cut the plasmid DNA.  General conclusions from a comparison of binding ability of the Ru complexes with enhancement ratio from radiosensitizing experiments cannot be drawn, because other factors may be involved. Complex 5 binds to DNA but 15 and 18 do not according to our assay, even though they all give good SER values. without  Platinum complexes, such as cis and trans-DDP, attachment  of  a  radiosensitizer  ligand,  57  by  radiosensitize even mechanisms  not  understood though presumably related to DNA binding. Conversely, electronaffinic compounds such as nitroimidazoles sensitize without binding to DNA. However, for a series of P t C ^ N ^ X L ) vs. PtCl (L) , L=miso, metro, or 42  N0 -Im, 2  2  the complex which binds to DNA is the better sensitizer  behaviour pattern that 5 appears to follow.  76  a  Thus, it appears that metals may  be used in some cases to target sensitizers to DNA to improve efficacy by their proximity to the target.  6.2  Partition Coefficient (P) In general, the lipophilicity of pharmacological agents (usually measured by  their octanolrwater partition coefficients) has been found to have a major influence on their activity, distribution and toxicity. ^' '* 75  75  For nitroimidazoles, one factor  considered was that their neurotoxicity would be related to their uptake into nervous tissue. Because the amounts of drugs entering neural tissues depend on their ability to cross the blood-brain barriers, this being related to the drugs' lipid solubility, it is  135  expected that drugs of lipophilicity lower than that of miso (P=0.43)  would  penetrate the barriers less efficiently than miso, and hence be less neurotoxic.  Further, the influence of the octanolwater partition coefficient, P, on the radiosensitizing efficiency of a large number of nitroaromatic and nitroheterocyclic compounds has been investigated in vitro. Although Adams et al.  33  showed that P  values over a wide range (0.05-240) were relatively unimportant in determining the radiosensitizing efficiency in mammalian cells, Anderson et alJ^ demonstrated some effect of variation in P using E. coli.  In addition, Brown et al.  156  measured  intracellular uptake of neutral 2-nitroimidazoles with P values in the range 0.01 - 0.43 and found that the rate and extent of uptake correlated with radiosensitization: that is the compound with a higher P value has higher radiosensitizing efficiency.  Butler et  J 57  al.  also found that the partition coefficient  was the most important factor  determining the activity range of 4- and 5-nitroimidazoles administered orally to mice against an infection of Trichomonas foetus. Thus, in the expectation that the degree of lipophilicity influences the distribution and intracellular concentration of the Ru radiosensitizers,  the partition coefficients  of the compounds  synthesized  were  examined, and the results are summarized in Table XVII. Note that at the equilibrium measurement for complexes 5, 1 5 and 2 1 , the hydroxy species 5c and 15c. and the mono aquo species 21b are present in solution (see sections 4.2 and 4.3). The tmso complexes analogues.  have higher P values than the corresponding dmso  Except for the metro ligand and complex 14, 15c has the highest P value  of all the other compounds studied here, and it also has a higher SER value at the same concentration. This observed result for 15c agrees with those previously reported by Brown et al.J  49  whereby the more hydrophilic compounds in a series of 2- and 4-  nitroimidazoles required higher drug concentration to achieve equal radiosensitization  136  Table XVTI  P a r t i t i o n coefficients of nitroimidazole ligands and t h e i r ruthenium complexes  Partition Coefficient, P [ ]Octanol/[ ]Aqueous N0 -Im Ligand  Complex  0.24 (1.2)  0.42 (1.6)  2  5, RuCl [dmso) (4-N0 ~Im) 2  6,  2  2  2  RuCl [dmso) (NMe-4-N0 -Im)  b  a  0.16 (1.2)  0.18  (1.3)  0.22 (1.1)  0.28  (1.3)  8, RuCl (dmso) (RSU-3083)  2  0.38 (1.2)  0.40  (1.2)  9,  2  0.33 (1.2)  0.38  (1.1)  10, RuCl (dmso) (RSU-3159)  0.26 (1.3)  0.29  (1.3)  11, RuCl (dmso) (miso)  0.43 (1.3)  0.12° (1.4)  12, RuCl (dmso)2(De-miso)2  0.13 (1.3)  0.04° (1.4)  14, RuCl (dmso)2(metro)2  0.96 (1.1)  0.77  0.24 (1.2)  0.64 (1.6)  16, RuCl (tmso) (NMe-4-N0 -Im)  0.16 (1.2)  0.20  (1.4)  17, RuCl (tmso) (De-miso)2  0.13 (1.3)  0.22  (1.4)  18, RuCl (tmso) (SR-2508)  0.046(1.3)  0.21  (1.5)  21, RuBr (dmso)2(4-N02~Im)  0.24 (1.2)  0.07 (1.3)  22, RuBr (dmso) (NMe-4-N0 -Im)  0.16 (1.2)  0.14  2  2  2  7, RuCl (dmso) (RSU-1170) 2  2  2  2  2  RuCl (dmso) (RSU-3100) 2  2  2  2  2  2  2  2  2  15, RuCl (tmso)2(4-N0 -Im) 2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  (1.2) b  b  (1.3)  a  Number i n parentheses gives the corresponding SER value from Tables XIV and XV.  b  These values refer to species 5c, RuCKOH) (dmso) (4-N0 -Im) , 15c, RuCl (OH) (tmso) (4-N0 -Im) , ancT~19b, [ RuBr (r^O) (dmso) ^^NO^ImTJ ] . 9  2  c  2  9  9  2  These values were taken i n 4 min after mixing, because of dissociation of nitroimidazole ligands; a l l other values were recorded at equilibrium conditions (± 0.05).  137  level.  F o r the other compounds studied, there is no correlation between S E R and P  values.  T h e free metro ligand has the highest P value (0.96) but only a poor S E R  value of  1.1, whereas S R - 2 5 0 8 has the smallest P value but an S E R value of  s i m i l a r to that observed for miso (P=0.43). values (approximately  1.3  Complexes 5 and 15. have similar S E R  1.6), but the P values f o r 5c and ! 5 c are quite d i f f e r e n t (0.42  vs. 0.64, respectively).  T h u s , the data are i n agreement w i t h A d a m s  suggested that the partition c o e f f i c i e n t is not the dominant  et al '  property  33 34  who  affecting  the  ability o f some nitroimidazoles to act as h y p o x i c cell sensitizers (see also section 1.8); these workers consider electron a f f i n i t y to be the key factor (see section 6.4)  O f the  4 - N 0 - I m complexes, 2J. has the lowest P value (0.07) w h i c h is almost certainly due 2  to the charged species 21b being f o r m e d (equation [4.4]) m a k i n g 19 apparently more hydrophilic.  Complexes H . and 12. have low P values (data measured i n the 600 n m  region) presumably associated w i t h the dissociation of nitroimidazole ligands.  149  B r o w n et al.  suggested that a decrease i n radiosensitizing e f f i c i e n c y  and  c y t o t o x i c i t y of compounds w i t h P values below 0.04 correlated w i t h a decreased ability of the compounds to enter the cells.  In this study, no c o m p o u n d has a P value of less  than 0.04. 6.3  N o n - P r o t e i n T h i o l Depletion T h e s u l p h y d r y l containing compounds have been shown to protect cells f r o m  i r r a d i a t i o n (section 1.9).  M a n y metals b i n d strongly to various sulphur donor ligands  and therefore might be expected to radiosensitize by depleting intracellular t h i o l s , * * ' * "  5  such as glutathione, w h i c h is k n o w n to protect organisms against radiation (e.g. by H atom donation to repair chemically the damaged t a r g e t / "  5 9  see section 1.9).  depletion has been proposed to e x p l a i n c / s - D D P s e n s i t i z a t i o n / '  5 9  Thiol  but this does not  138  appear to be generally accepted.  However, depletion of non-protein thiols (NPSH)  by Rh(II) carboxylates correlated with in vitro radiosensitizing ability.  Nitroimidazoles, such as misonidazole,* ** have been shown to deplete thiols. 6  It has been established that various ortho-substituted-4-nitroimidazoles are much more efficient radiosensitizers than would be predicted from their electron affinity. has been shown that these compounds react readily with t h i o l s , - ' 59  760  ' ' 7  57,7  '  57  59  It  which  reportedly contributes to their abnormally high sensitizing efficiency. In the present work, cells were analyzed for NPSH after 1 h of incubation at 37°C in oxic and hypoxic conditions with test compounds.  Reactions of the  complexes or their free ligands with NPSH were monitored by the extent of formation of an adduct ( A  m  a  x  412 nm) between the SH content and Ellman's reagent.  Of all  the Ru(II) complexes described in this thesis, only complexsJJ. and 12 show some NPSH depletion but this must result partly from the dissociated miso and De-miso ligands, respectively, because the precursor complex m - R u C ^ d m s o ^ deplete NPSH. SR-2508)  and  does not  Of the ligands, the 2-.nitroimidazoles (2-N0 -Im, miso, De-miso and 2  4-N0 -Im 2  depleted  thiol  levels,  nitroimidazoles and metro did not (Table XVIII).  while  the  N-substituted-4-  The results also show that thiol  depletion occurs only under hypoxic and not aerobic conditions.  There have been  reports that hypoxic tissues lose intracellular free thiols by membrane leakage into surrounding fluid. -* 76  However in control experiments, cells incubated under the  experimental conditions did not exhibit thiol loss even after 2 h of hypoxia.  In addition, several other compounds such as ortho-substituted-4- or 5nitroimidazoles are reported to show increased sensitizing efficiency with increasing contact time with cells.  760  For the longer contact time (~2 h), cellular thiols  decreased by one third relative to control values (-30 m i n ) .  760  In the present study, a  139  Table XVTII  NPSH depletion i n hypoxia by some Ru(II) complexes ' 3  7o NPSH depletion Complex  5,  N0 -Im free ligand (400 pM)  0  Ru complex (200 MM)  ?  Ru(4-N0 -Im)  23  0  6, Ru(NMe-4-N0 -Im)  0  0  7, Ru(RSU-1170)  6  1  8, Ru(RSU-3083)  5  1  9, Ru(RSU-3100)  4  1  10, Ru(RSU-3159)  11  2  11, Ru(miso)  15  25  22  20  38  0  0  0  23  2  0  0  22  3  23  1  23  0  0  0  2  2  2  2  2  2  2  12, Ru(De-miso)  2  13, Rj(2-N0 -Im) 2  14, Ru(metro)  2  2  15, Ru*(4-N0 -Im) 2  2  16, Ru'(NMe-4-N0 -Im) 2  17, Ru'(De-miso)  2  2  18, Ru'(SR-2508) 21, Ra"(4-N0 -Im) 2  2  22, Ru"(NMe-4-N0 -Im) 2  No depletion of NPSH was observed under oxic conditions; estimated maximum error Jl 5"L. k No depletion of NPSH was observed for the 1,10-phenanthrolines or their complexes. Ru, Ru', and Ru" represent RuCl (dmso) , RuCl (tmso) , and RuBr (dmso) respectively. 2  2  2  2  2  140  one  hour incubation time  experiments.  was  chosen  to  be relevant  to  the radiosensitizing  The compound, miso, which is known to deplete NPSH,** gave no  further NPSH depletion upon longer incubation times. This result indicated that thiol depletion (to 15 %) which had occurred within the 1 h contact period at 37°C was close to maximum.  Stratford et alJ^  have shown that for some 2- and 4-nitroimidazole systems  4  there is a dependence of thiol reactivity on reduction potential, namely a trend showing an increase in thiol reactivity with increase in reduction potential. However, in the present study, this trend is not followed in that none of the Ru(II) complexes, except IJ. and J_2, react with NPSH, although their reduction potentials (see next section) are higher than those of the free ligands.  Enhancement of radiosensitization  by metallation of the nitroimidazole ligands to Ru does not result from depletion of NPSH.  6.4  Electron Affinities (Reduction Potentials) Various in vitro studies with mammalian cells have shown that hypoxic  cytotoxicity correlates to some extent with electron affinity of the compounds.^"' * 6  The  relevance  of E j y  2  or the one-electron  reduction potential to toxicity by  nitroimidazoles has often been discussed.^" *" 5  59,162  -  164  The half-wave reduction  potentials of the nitroimidazoles and their complexes as measured by polarography (see e.g. Figure 34) are shown in Table XIX. These reduction potential values are in good agreement with values obtained by cyclic voltammetry; the differences between the two values were within +10 mV, except in the case of complex J_0. The compounds are divided into different groups depending on the substituents of the complexes and the positions of the nitro group.  Because free nitroimidazoles showed almost a  hundred times greater toxicity toward hypoxic cell than oxic cells following a one-  141  -685  -400  -600  '  -800  mV  F i g u r e 34;  The 5,  r e d u c t i o n p o t e n t i a l s o f RuCl (dmso)^ (4-NO 2  and 4-NO  -Im  -Im)  142  Table XIX  Half-wave reduction potentials of Ru(II) compounds and their nitroimidazole ligands  (mV)  E  1  Complex  a  5  NG^-Im free ligands  5, Ra(4-N0 -Im) 2  -685 [-690f (1.2)  2  Ru complexes  d  -615  [-620]° (1.6f  e  6, Ru(NMe-4-N0 -Im)  -535 [-545] (1.2)  -518  [-525] (1.3)  7, Ru(RSU-1170)  -410 [-420] (1.1)  -390  [-400] (1.3)  8, Ru(RSU-3083)  -560 [-550] (1.2)  -540  [-535] (1.2)  9, Ru(RSU-3100)  -470 [-460] (1.2)  -455  [-450] (1.1)  10, Ru(RSU-3159)  -370 [-360] (1.3)  -360  [-345] (1.3)  11, Ru'(miso)  -445  (1.3)  -435  (1.4)  -389  (1.3)  -367  (1.4)  -400  (1.3)  -385  -520  (1.1)  -490  (1.2)  -685  (1.2)  -605  (1.6)  16, RJ'(NMe-4-N0 -Im)  -535  (1.2)  -500  (1.4)  20, Ru'(RSU-3159)  -370  (1.3)  -345  (1.3)  17, Ru'(De-miso)  -389  (1.3)  -355  (1.4)  -388  (1.3)  -345  (1.5)  -685  (1.2)  -645  (1.3)  -535  (1.2)  -515  (1.3)  2  2  2  2  2  12, Ru(De-miso)  2  13, Ru(2-N0 -Im) 2  14, Ru(metro)  2  2  15, Ru'(4-N0 -Im) 2  2  2  2  2  18, Ru'(SR-2508) 21, Ifo"(4-N0 -Im) 2  2  22, Ru"(NMe-4-N0 -Im) 2  e  (1.4)  continued ...  continued  143  Values obtained i n PBS w.r.t. SCE; estimated maximum error ± 5 mV. Ru, Ru', and Ru" represent RuCl^ldmso^j RuCL-^tmso^, and RuBr (dmso^j respectively, complexes 5, 15 and 21 are the hydroxy and mono aquo species (see sections 4/2" and" 4.3) The number i n "square brackets" gives Ej. values obtained from cyclic voltammetry. 2  Number i n parentheses gives corresponding SER value. The 4-N0.2-Im and 2-N02-Im complexes give additional Ej. values at -355 mV and -290 mV, respectively, i n the polarographic measurements, but these values were not observed i n c y c l i c voltammetric measurements.  144  electron reduction process,"^"* ' *'"' one assumes that the enhanced hypoxic toxicity in 6 7  the complexes  also results from the reduction of the nitroimidazole within the  complex. An electron is expected to reduce the organic moiety rather than the metal in these Ru complexes, as observed for the c/s-PtCl (metro) , * and PtCl (NH3)(4(5)5  2  N0 -Im) systems. 2  2  The measured half-wave potentials  nitroimidazole ligands are in good agreement  (Ejy  2  with the 31  3 1 C P  potentials for these ligands as reported previously.  2  a  t pH 7) for the free  one-electron  reduction  CQ  The attachment of a  nitroimidazole to the Ru moiety increases the reduction potential with respect to that of the free ligand; that is, the ligand centres are more easily reduced when coordinated to Ru.  This observation is also known to occur upon coordination of  nitroimidazoles to platinum(II).  '  The toxicity of the Ru complexes (Tables X and  XI) toward CHO cells in vitro, compared to that of the respective nitroimidazole ligand, does not follow the trend of increasing toxicity with increasing electron affinity, although such a trend has been observed for unbound nitroheterocyclic compounds."*'*' ' * 7 5  In the present work, the ligands are always more toxic than their  corresponding complexes which are more electron affinic.  This emphasizes that other  factors must also be considered.  Studies with vegetative bacteria, bacterial spores, and mammalian cells have shown that there is a general relationship between the efficiency of radiosensitization and the electron affinity of the compounds, and this has aided considerably the search for new compounds. ^' 3  32  The general relationship between sensitizing efficiency and  electron affinity has been supported by studies on one-electron transfer reactions between known sensitizers, such as the 2-nitroimidazoles, 5-nitrofurans, nitrobenzenes and 4-nitroimidazoles,^'-^"** and the results have suggested that the sensitizing efficiency  increases with increasing electron affinity or reduction potential.  '  Thus, comparison of the reduction potentials (at pH 7.0 in aqueous medium) for  145  accepting a single electron shows that oxygen (the "best" radiosensitizer, SER=3.0) is more electron affinic than the nitroimidazoles: 0 L  In the  2  + e" —»• 0 ",  = "155 m V *  2  + e" — * L ~ ,  present  study,  E  1 / 2  2  = -370 to -685 mV  complexes  5 and 15 have  the  best SER value  (approximately 1.6) and, indeed, the radiosensitizing ability of the 4-nitroimidazole ligands increases with an increase in the reduction potential when the ligand is bound to Ru. For 5 and 1_5, this increase in potential upon metallation is approximately 70 mV which is the largest "coordination shift" among all the systems studied. Complex 18 also shows an increase in the SER value compared to the value for the nitroimidazole free ligand; the increase in reduction potential is -40 mV. The SER values are 1.3 and 1.5 at 200^X1 for SR-2508 ligand and its complex, 18, respectively. The SER values for the other Ru nitroimidazole complexes do not show significant enhancement over those of the free ligands alone at an equivalent molar concentration. This may be a consequence of the relatively small increase in one-electron reduction potential between the free and coordinated ligand, the "coordination shift" being <30 mV (Table XIX). Electron-donor substituents on the nitroimidazole (e.g. the N-CH3, and the amino and thioether substituents of 8 and 10, respectively) lower the reduction potentials. A higher reduction potential will certainly favour thermodynamically abstraction of an electron from irradiated DNA. Surprisingly, 5, and 15 have the lowest electronic affinities ( E j / —610 mV) of all the complexes studied (except one, 2  21) and yet have the highest SER values. affinities  have  been  realized  also  High SER values with low electronic  within  a  series  of  ortho-substituted-4-  nitroimidazoles,"^ but thiol depletion was important in these systems (section 6.3), and is not so in the metal systems. Clearly, factors other than "absolute" electron affinities (higher reduction potentials) also contribute to radiosensitization.  Data on the Ru  146  systems would tend to indicate increased radiosensitizing property with increasing coordination shift of the ligand reduction potential.  6.5  Feasibility in IN VIVO System The in vitro results in the present study have led to a testing of the  radiosensitizing  efficiency  of several of  the  Ru-nitroimidazole complexes in  experimental animal tumours; the experimental work was performed by Ms. D. Ruta under the guidance of Dr. D. Chaplin at the B. C. Cancer Research Centre. The complex RuCl (tmso) (SR-2508) is of particular interest, with a relatively high SER 2  2  value in vitro (1.5 at 200 uM) in view of the presence of only one coordinated nitroimidazole.  Tumour cell survival was determined after a single X-ray dose of 10 Gy delivered to the mice at various times following intraperitoneal injection of RuCl (tmso) (SR-2508), 18, at 300 mg/kg tumour. The results (Figure 35) show that 2  2  the maximum sensitization occurred when complex 18 was administered ~1 h before the start of the irradiation. The interval between drug administration and irradiation, that is required for maximum radiosensitization, is defined as the optimum time. The cell survival at this optimum time with J_8 and X-ray irradiation was 0.04%, whereas the irradiation alone gave a survival of 0.104%. Further, complex 18 alone did not show any toxicity to the mice at 300 mg/kg.  The degree of radiosensitization achieved by a drug may therefore depend on the time-elapsed since dosing.  The importance of the time interval between drug  administration and irradiation for hypoxic cell radiosensitization has been shown by several authors  765  '  766  '  767  who showed that there is an optimum time to irradiate after  drug administration in order to achieve maximum radiosensitization.  If irradiation  147  Hours Before I r r a d i a t i o n  Figure  35:  (h)  The e f f e c t on tumour c e l l s s u r v i v a l on the time i n t e r v a l between R u C l ( t m s o ) (SR-2508) 2  and  (300 mg/kg tumour)  i r r a d i a t i o n w i t h 10 Gy o f X-ray  administration  148  follows too early there may be inadequate time for diffusion of the drug to the hypoxic cells; conversely, if the interval is too long the drug concentration may drop as a result of excretion and/or metabolism.  Further studies will include the use of higher dose of 18 and dose response survival as compared to the free SR-2508 ligand.  In addition, the other 4-N0 -Im  complexes, 5 and 15, which show high SER values in vitro will be examined.  2  149 CHAPTER SEVEN CONCLUSIONS and RECOMMENDATIONS for F U T U R E WORK  Metal complexes can alter the effects of radiation within radiotherapy in many ways.  Several studies suggest that the combination of metal complexes and radiation 70  may  radiosensitize by: (a) enhancement of initial DNA damage,  recovery from radiation damage,  (c) interaction with thiols,  (b) inhibition of  and (d) properties that  71 7 ?  depend  on  electron  affinity.  '  Therefore, a single  metal  complex  could  radiosensitize by several mechanisms. This work was carried out to improve the radiosensitizing ability and reduce the toxicity of nitroimidazoles (currently used clinically), with a view to gaining an understanding of the effects of transition metal (Ru)/nitroimidazole complexes on cellular radiation response.  The data in the present work show that some of the Ru-  complexes have greater sensitizing efficiency, lower toxicity, and a lower reaction rate with NPSH, than the corresponding free nitroimidazole ligand.  One of the better  complexes synthesized, RuCl (dmso)2(4-N02-Im)2, 5, also binds to DNA as assessed by 2  inhibition of restriction enzyme activity.  At present, there is no definite explanation  why 15 (in contrast to 5) does not bind to DNA but steric effects of tmso vs. dmso may play a role. better  The data show that complexes 5 and RuCl2(tmso)2(4-N02-Im)2, i l , are  radiosensitizers  than  the  Ru/nitroimidazole/sulphoxide/halide  free  4-N0 -Im, and than 2  complexes.  When  a range  examined  at  of  other  equivalent  concentrations, they are also better in vitro than clinically used misonidazole.  Replacement of the dmso ligand generally by tmso does not markedly affect the redox properties and radiosensitizing ability of related complexes.  However, such  replacement does affect the stability in aqueous solution of the substituted-2-N02"Im complexes. The substitution of Cl~ by Br" (5 vs. 21) reduces the radiosensitizing ability  150  of the Ru-4-nitroimidazole complexes,  but this may result from the presence of  different forms of geometrical isomers: the all cis geometry for 5 and the transbromide, c/s-sulphoxide, ds-nitroimidazole for 21_; the less electronegative Br" causes a smaller change in "coordination shift" reduction potential and different behaviour in aqueous solution chemistries (equations [4.2] and [4.4]). Complex 5 gives a hydroxy species,  RuCl(OH)(dmso)2(4-N02-Im)2,  while  21  gives  a  mono  aquo  species,  [RuBr(H 0)(dmso)2(4-N0 -Im) ] . +  2  2  2  The better radiosensitizing ability of 5 and 15 compared to other complexes is likely to depend on some or all of the following factors: (a) increased reduction potential of the 4-NC>2-Im ligand when it becomes coordinated, (b) hydration to a species capable of binding to DNA, (c) better lipophilicity, (d) the presence of the pyrrolic proton of imidazole which could be involved in H-bonding that stabilizes a D N A - R u interaction - as demonstrated for Pt(II) systems. ' ' -** 5 2 7  probably not involved.  Thiol depletion is  As regards to (a), a higher reduction potential will certainly  favour thermodynamically abstraction of an electron from irradiation-damaged DNA; electron-donor substituents on the nitroimidazole (e.g. the N - C H 3 , and the amino and thioether substituents of 8 and 10, respectively) lower the reduction potentials.  Enhancement ratios produced by metal complexes as radiosensitizers have often been small using cells in tissue culture.  60  However, a relatively low effect still results  in therapeutic gain in the clinic, as seen for c / s - D D P .  769  It is clearly not possible to  assess the full potential of metal-based radiosensitizers at this stage.  Experimental  investigations must be pursued if the interactions between radiation, metal complexes, and cells are to be optimized to give cell inactivation.  There is a need to gain a more  complete basic understanding of the mechanisms of radiation sensitization so that guidelines can be established for designing and selecting the most effective metal  151  complexes.  For example, the effects of systematic variation of the ligands on a known  radiosensitizing metal complex, or within a given ligand system, variations of the metal on radiosensitizing ability would be instructive. In addition, studies on the mechanisms and factors involved, such as electron affinity, thiol depletion, lipophilicity, DNA binding and repair, must be included. Further studies using Ru(III) as a metal centre should be included to determine if these complexes radiosensitize and have different reduction process(es) as compared with the Ru(II) systems.  The metal-based radiosensitizers must continue to be actively tested in tissue culture and animal tumor systems that are relevant to the selection of metal complexes for application in the radiation therapy of clinical cancer. The individual responses of patients to therapeutic agents are invaluable in the evaluation of cancer treatments. Ideally, for the clinical situation, one would like to find a non-toxic radiosensitizer. The beneficial effects of sensitizing agents must be balanced against detrimental toxic effects.  The data in the present study encourage further investigations of the use of  transition metal complexes as radiosensitizers to combat the hypoxic tumour cells.  152 BIBLIOGRAPHY 1.  B.H.J. Bielski and J.M. Gebicki, in: "Free Radicals in Biology", W.A. Pryor (Ed.), Academic Press, New York, Vol. Ill, pp. 2-48 (1977).  2.  K.H. Chadwick and H.P. Leenhouts, "The Molecular Theory of Radiation Biology", Springer-Verlag, New York, p. 1 (1981).  3.  M.M. Elkind and G.F. 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The solution was sterilized by filtration through a 0.22 micron filter and stored at 4°C.  (c)  Methylene-blue Methylene-blue (2 g) was dissolved in distilled water (1 L) at 37°C. The solution  was allowed to stand for 1 h and filtered.  (d)  Ethidium bromide Ethidium bromide (1 g) was added to distilled water (100 mL). The solution was  stirred for several hours until complete dissolution and was stored in a container wrapped in aluminum foil at 4°C.  162  APPENDIX II  PREPARATION OF BUFFERS  (a)  Electrophoresis buffer (20X) Tris base (387.5 g), sodium acetate (54.4 g) and EDTA (29.8 g) were dissolved in  distilled water (3.8 L). The pH of the mixture was adjusted to 7.8 with approximately 125 mL glacial acetic acid and made up to 4 L with distilled water.  (b)  Loading buffer (6X) Bromophenol blue 0.25% (W/W), xylene cyanol 0.25% (W/W) and glycerol 30%  (W/W) were mixed in distilled water (100 mL) and the solution was stored at 4°C.  (c)  Tris-EDTA (TE) buffer EDTA (1 mM) was mixed with Tris-chloride solution (10 mM) at a 1:1 ratio and  the pH was adjusted to 8.0.  (d)  Tris-acetate (TA) buffer (10X1 The pH of the 10X T A buffer which consisted of: Tris-acetate (0.33 M),  potassium acetate (0.66 M), magnesium acetate (0.10 M) and dithiothreitol (0.005 M), was adjusted to 8.0 and this solution was stored frozen in aliquots at -20°C.  163  APPENDIX III  CHROMATOGRAPHY T H R O U G H SEPHADEX G-50  Sephadex G-50 was used to separate high-molecular weight molecules (e.g. DNA) from smaller molecules (such as salts, chemicals) by gel filtration.  A.  PREPARATION OF SEPHADEX G-50 Sephadex G-50 (Pharmacia, 30 g) was added slowly to T E (250 mL) in a 500 mL  beaker.  The solution was allowed to stand overnight at room temperature, and the  supernatant was decanted and replaced with an equal volume of T E . The mixture was stored at 4 ° C in a screw-capped bottle.  B.  SPIN C O L U M N PROCEDURE 1.  A quick-sep disposable column with sintered disc (5 mL) was packed with Sephadex G-50 (3.5 mL).  2.  The column was spun in a 10 mL polypropylene test tube for 5 min at 1000 rpm and 5 min at 3000 rpm to pack the column and spin out the buffer solution.  3.  The spun-out buffer solution was decanted and an Eppendorf vial with its cap removed was placed in the bottom of the polypropylene test tube. The spun column was then placed back in a test tube.  4.  DNA sample (0.1 mL) was added to the above prepared column, and the column was spun for 5 min at 3000 rpm; the chemicals remained in the column and the DNA was collected into a decapped Eppendorf vial.  164 APPENDIX IV A.  PREPARATION OF AGAROSE G E L The agarose (type I-A, IG) was dissolved in electrophoresis buffer (100 mL).  This agarose solution was poured on a plate with a "comb" in place, whose teeth would provide a gel slot that had 2-3 times the volume of the DNA sample to be loaded. This precaution eliminated the DNA from streaming out of the gel at the top of the slot.  B.  RUNNING G E L 1.  The gel was submerged in IX E-buffer (made from 20X E-buffer).  2.  The digest-dye solution was loaded, so that the slot was only one-half full.  3.  The electrodes were connected, and the gel was electrophoresed for desired time and voltage.  4.  The gel was submerged in an ethidium bromide solution (1:10,000 dilution of 10 mg/mL) and was stained for 15 min.  5.  The gel was carefully placed on a UV-box and the gel was observed with UV-protective glasses and a picture was taken with Polaroid film.  165 APPENDIX V  LINEARIZATION OF pSV2-gpt PLASMID (a)  Tris-acetate buffer (50 yL) and PvuII restriction enzymes (15 )iL) were added to 100 ^g/mL pSV2-gpt (450 »\).  (b)  The above mixture was incubated at 37°C for 3-4 h.  (c)  After 3 h, T E (12j<L), 0.5 M EDTA (1 jAS) and loading buffer (3 ^ L ) were added to the above mixture (3 JJL)-  (d)  An electrophoresis on this mixture and the uncut control was carried out and the DNA bands were compared.  (e)  Once it was ensured that all DNA was linearized, the enzyme activity of PvuII was stopped with 0.5 M EDTA (lO^wL).  (f)  Phenol extraction and ethanol precipitation of the DNA: (1)  The buffered phenol (0.5 mL) was added to the DNA, and the mixture was shaken and centrifuged for 2 min.  (2)  The top aqueous layer was transferred to a fresh Eppendorf vial and the organic phase was discarded. Steps 1 and 2 were repeated several times.  (3)  The residual phenol was extracted with ether (0.5 mL) and the mixture was spun for 10 s; the top ether layer was removed and this procedure was repeated  several times; the mixture was warmed at 37°C for  approximately 5 min to remove residual ether.  166  (4)  NaCl (10  of 5M solution) was then added followed by 95% ethanol (1  mL), and the mixture was chilled at -20°C for 1 h (or overnight) (6)  The mixture was centrifuged for 10 min to spin down the DNA; the supernatant was discarded very carefully, and the DNA was partially dried by an airflow. mL) and stored at 4°C.  The DNA pellet was then redissolved in T E (0.5  167  APPENDIX VI  PREPARATION OF R A T LIVER S9 FRACTION Day 1: Induction of rat liver enzymes for carcinogen activation 1.  Aroclor 1254 was dissolved in corn oil (lg/5mL) by warming up to 37°C (Aroclor is a polychlorinated biphenyl mixture).  2.  The rats were injected with Aroclor (200 g rat - 0.5 mL (IP)).  3.  The rats were given drinking water and Purina Laboratory Chow until day 4.  Day 4: The food was removed.  Day 5: The rats were sacrificed by a blow to the head and then decapitated. 1.  KC1 (0.15 M) was added to the weighed liver (3 mL/g) in a Erlenmyer flask and the liver was homogenized and put into 4 plastic bottles.  2.  The mixture was centrifuged for 10 min at 9000 g, decanted and the supernatant, S9 fraction was saved. The S9 fraction was frozen quickly after distributing into small plastic tubes and was stored at -80°C in a Revco freezer.  168  APPENDIX VII  PROCEDURES for  IN  VIVO  STUDY  The tumour cell line, K H T sarcoma, was maintained in liquid nitrogen, and the tumour transplantation was carried out as described by Chaplin.  770  On attaining a  weight of 450 to 800 mg, the tumours were irradiated in a manner developed from that 171  described by Sheldon and Hill.  The procedure for the preparation of the cell 170  suspensions is described by Chaplin,  and the assay system used for cell survival is the 172  soft agar clonogenic assay of Courtenay.  The relative cell forming unit per gram of  tumour (CFU/g) is calculated as followed: , _ Survival f r a c t i o n o f ^ chemically treated c e l l s J  c e l l y i e l d (chemically ,, . , . c e l l y i e l d (control)  treated)  

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