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The synthesis and characterization of ruthenium disulfoxide complexes and their preliminary in vitro… Huxham, Lynsey Anne 2001

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T H E S Y N T H E S I S A N D C H A R A C T E R I Z A T I O N O F R U T H E N I U M D I S U L F O X I D E C O M P L E X E S A N D T H E I R P R E L I M I N A R Y IN VITRO E X A M I N A T I O N A S P O T E N T I A L C H E M O T H E R A P E U T I C A G E N T S B y L Y N S E Y A N N E H U X H A M B . S c , University of British Columbia, 1998 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Chemistry) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A November 2001 © Lynsey Anne Huxham, 2001 In .presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Date I^QQ. S I / 2 Q O ] DE-6 (2/88) Abstract The anti-cancer activity of the water-soluble complexes cis- and trans-RuCl2(DMSO) 4 has led, in this laboratory, to further investigation of disulfoxide complexes of Ru as potential chemotherapeutic agents. The dithioether precursors and disulfoxides were synthesized in this work following literature procedures. The disulfoxides used were of the formula RS(0)(CH 2 )„S(0)R, where R = Et (BESE) or Ph (BPhSE) for n = 2; and R = Et (BESP) or Pr (BPSP) for n = 3, and the dithioether used was B P T P (l,3-bis(propylthio)propane). These compounds/ligands were reacted with R u precursors to yield complexes characterized mainly by N M R , elemental analysis, IR, and conductivity, while three complexes were also characterized by X-ray crystallography. A mixed sulfoxide/disulfoxide complex, c/s-RuCl2(DMS0)2(BESE), was isolated during this thesis work. The water-soluble complex was characterized by X-ray crystallography, the solution chemistry was studied, and preliminary in vitro tests were performed. The crystal structure shows cis chlorides, one O-bound D M S O , one S-bound D M S O , and S-bound B E S E . The complex does not exhibit significant host toxicity toward C H O (Chinese hamster ovarian) cells below 1.1 m M , and was found to have essentially no anti-cancer activity, at concentrations up to 3 m M , toward human mammary cancer cells. The known complex [RuCl(BESE)(H20)]2(^-Cl)2 ionizes in aqueous solution to generate 2 equiv. of H + and 2 equiv. C l " per mole of complex, and sulfoxide exchange reactions with the in situ formed species (thought to be [Ru(BESE)(0H)(H 20)]2(Ai-Cl) 2) were briefly studied. ii In an attempt to synthesize the previously characterized, dinuclear complex [RuCl 2 (BPTP)] 2 (At-Cl) 2 , frarcs-RuCl 2(BPTP)2 was isolated and characterized by N M R , and elemental analysis. The synthesis of thioether complexes was performed to attempt a subsequent oxidation of the coordinated dithioether while retaining the geometry about the Ru. This thesis work led to the isolation of three Ru disulfoxide-bridged complexes. One such complex, [RuCl 3 (BPhSE)]( / i -BPhSE), was characterized by elemental analysis, IR, and mass spectrometry, and two Ru(p-cymene) complexes with bridging sulfoxides were also isolated. The first, [RuCl 2(p-cymene)] 2(/i-BESE), was characterized by X-ray crystallography and contains S-bound sulfoxide, while the other, [RuCl 2(p-cymene)] 2(/i-BESP) , characterized by N M R , elemental analysis, and IR, appears to be chemically similar to the jU-BESE complex. [RuCl 2 (p-cymene)] 2 (^-BESE) was shown in vitro to exhibit no significant host toxicity below concentrations of 1.1 m M , but did exhibit, in the concentration range 345-360 u,M, anti-cancer activity against human mammary cancer cells. A third p-cymene complex, [RuC10>cymene)(BESE)]PF 6, was characterized by X -ray crystallography and shown to have S-bound sulfoxide. ii i Table of Contents Abstract , i i Table of Contents . iv List of Figures v i i i List of Tables xi List of Abbreviations x i i Acknowledgements xv Chapter 1 IntroductionrRuthenium Sulfoxide Complexes and their Anti-cancer Properties 1 1.1 Introduction 1 1.2 Platinum Chemotherapeutic Agents 2 1.3 Ruthenium Chemotherapeutic Agents 4 1.3.1 Ruthenium Dimethylsulfoxide Complexes 6 1.3.2 D N A Binding of c w - R u C l 2 ( D M S O ) 4 and r ra t t s -RuCl 2 (DMSO) 4 9 1.3.3 Ruthenium Disulfoxide Complexes 11 1.3.4 Ruthenium p-Cymene Complexes 12 1.4 Goals of This Thesis 13 1.4.1 Synthesis of Novel , Water-Soluble Ruthenium Disulfoxide Complexes 13 1.4.2 Preliminary ln Vitro Biological Testing of Ruthenium Disulfoxide Complexes 13 1.4.3 Oxidation of Coordinated Dithioether in Ruthenium Complexes 14 References 15 Chapter 2 General Experimental 19 2.1 Chemicals and Reagents 19 2.2 Physical Techniques and Instrumentation 19 i v 2.2.1 N M R Spectroscopy 19 2.2.2 Infrared and U V - V i s Spectrophotometry 20 2.2.3 Conductivity and Melting Point Measurements 20 2.2.4 Elemental Analyses and Mass Spectral Analyses 21 2.2.5 X-ray Crystallography 21 2.3 Synthesis of Dithioethers 21 2.3.1 3,6-Dithiaoctane [BETE] 21 2.3.2 3,5-Dithiaseptane [ B E T M ] 22 2.4 Synthesis of Disulfoxides 23 2.4.1 l,2-Bis(ethylsulfinyl)ethane(BESE) 23 2.4.2 l,2-Bis(ethylsulfinyl)methane(BESM) 23 2.5 Synthesis of Ruthenium Precursors 24 2.5.1 Ruthenium "Blue" Solution 24 2.5.2 Ruthenium "Red" Solution 24 2.5.3 C w - R u C l 2 ( D M S O ) 4 24 2.5.4 C7s-RuCl 2 (BESE) 2 25 2.5.5 [RuCl(BESE)(H 2 0)] 2 Gu-Cl) 2 25 2.5.6 [RuCl(p-cymene)] 2(Ai-Cl) 2 26 2.5.7 K 3 [ R u C l 6 ] 27 2.6 Synthesis of a Ruthenium M i x e d Sulfoxide Complex 27 2.6.1 C7s -RuCl 2 (DMSO) 2 (BESE) 27 2.7 Synthesis of a Disulfoxide Bridged Ruthenium Complex 29 2.7.1 [ R u C l 3 ( B P h S E ) ] 2 ( ^ - B P h S E ) x H 2 0 ( x = 1,2) 29 2.8 Synthesis of a Ruthenium Dithioether Complex 30 2.8.1 7Vans-RuCl 2 (BPTP) 2 30 2.9 Synthesis of Ruthenium p-Cymene Disulfoxide Complexes 30 2.9.1 [RuCl 2 (p-cymene)] 2 (^-BESE) 30 2.9.2 [RuCl 2(p-cymene)] 2Cu-BESP) 31 2.9.3 [RuCl 2 (p-cymene)(BESE)]PF 6 32 v 2.10 Miscellaneous Reactions 33 2.10.1 Attempted Synthesis of [RuCl(/7-cymene)(BESE)]Cl 33 2.10.2 Reaction of R u C l 2 ( P P h 3 ) 3 with B E S E 33 2.10.3 Reactions of [ R u C l ( B E S E ) ( H 2 0 ) ] 2 ( u - C l ) 2 with B E S P , and with B P S P 34 2.10.4 Reactions of R u C l 3 - 3 H 2 0 with B P S P 34 References 35 Chapter 3 Chemical and Structural Properties of Ruthenium Disulfoxide and Thioether Complexes 36 3.1 Introduction to Sulfoxides 36 3.1.1 Properties of D M S O 36 3.2 Metal-Sulfoxide Bonding 37 3.2.1 Sulfur-Metal Coordination 39 3.2.2 Oxygen-Metal Coordination 40 3.3 Ruthenium M i x e d Sulfoxide/Disulfoxide Complexes 41 3.3.1 C w - R u C l 2 ( D M S O ) 2 ( B E S E ) 44 3.3.2 [RuCl(BESE)(H 2 0)] 2 Gu-Cl) 2 with B E S P , and B P S P 54 3.4 Ruthenium Thioether Complexes 55 3.4.1 Trans-RuCl 2 (BPTP) 2 57 3.5 Ruthenium Sulfoxide-Bridged Complexes 59 3.5.1 [RuCl 3 (BPhSE) ] 2 Cu-BPhSE)x H 2 0 60 3.6 Introduction to Ruthenium/?-Cymene Complexes 61 3.6.1 [RuCl 2 (p-cymene)] 2 Cu-BESE) and [RuCl 2(p-cymene)] 2(jU-BESP) 63 3.6.2 [RuCl(p-cymene)(BESE)]PF 6 70 References 75 vi Chapter 4 Preliminary In Vitro Biological Studies of Water-soluble Ruthenium Sulfoxide Complexes 79 4.1 Introduction 79 4.1.1 The M T T Assay 80 4.1.2 The C H O Toxicity Assay 81 4.2 Experimental Media and Solutions 81 4.2.1 Media 82 4.2.2 Phosphate Buffer Saline Solution (PBS) 82 4.2.3 Solutions of Ruthenium Complexes 83 4.2.4 Methylene Blue Solution 83 4.3 M T T Assay Procedure 83 4.3.1 Cel l Preparation 83 4.3.2 M T T Cel l Plating Procedure and M T T Addition 84 4.4 C H O Toxicity Assay Procedure 87 4.4.1 Cel l Preparation 87 4.4.2 Ce l l Incubation Procedure 87 4.4.3 Cel l Toxicity Assay 88 4.5 M T T Assay Results 89 4.6 C H O Toxicity Assay Results 92 4.7 Toxicity and Anti-cancer Activity of Ruthenium Sulfoxide Complexes 93 4.7.1 C w - R u C l 2 ( D M S O ) 2 ( B E S E ) 93 4.7.2 [RuC] 2(p-cymene)] 2Gu-BESE) 94 References : 96 Appendix 1 Crystal Structure Data for R u C l 2 ( D M S O ) 2 ( B E S E ) 98 Appendix 2 Crystal Structure Data for [RuCl 2 (p-cymene)] 2 (^-BESE) 103 Appendix 3 Crystal Structure Data for [RuCl(p-cymene)(BESE)]PF 6 I l l Appendix 4 Drug Dilution Charts 122 vii List of Figures Figure 1.1 Structure of deoxyguanosine showing the N7 coordination site. Figure 1.2 The aqueous solution chemistry of cz 's-RuCl 2 (DMSO) 4 (A) and z rans -RuCl 2 (DMSO) 4 (B), where S = S-bound D M S O , and O = O-bound D M S O . Figure 1.3 The structures of N A M I and N A M I - A , where S = S-bound D M S O . Figure 1.4 Structures of the diastereomers formed by reaction of c w - R u C l 2 ( D M S O ) 4 and 5 ' G M P ; N represents the N7 of the guanine base and O represents an O-atom of the phosphate group. Figure 1.5 The structure of (1) the two diastereomers formed upon reaction of cis- and ?rans-RuCl 2 (DMSO) 4 with 2 ' -dG, and (2) a third end-product formed upon further reaction with 2 ' -dG. Figure 3.1 The resonance hybrid forms of a sulfoxide, D M S O (R = CH3). Figure 3.2 A molecular structure representation (ORTEP) of c w - R u C l 2 ( D M S O ) 2 ( B E S E ) with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity. Figure 3.3 The proposed aqueous solution behaviour of c w - R u C l 2 ( D M S O ) 2 ( B E S E ) . Figure 3.4 Variation of the ! H - N M R spectra, in D 2 0 , of R u C l 2 ( D M S O ) 2 ( B E S E ) with time; (A) immediately upon dissolving the complex in H 2 0 , and (B) 24 h after dissolving the complex in H 2 0 . Figure 3.5 Variation of conductivity measurements of c w - R u C l 2 ( D M S O ) 2 ( B E S E ) with time. Figure 3.6 A plot of In (AM°° - A M t ) [where A M o ° is the maximum conductivity value and AMt is the conductivity at time (t)] versus time for cis-R u C l 2 ( D M S O ) 2 ( B E S E ) . Figure 3.7 ' H - N M R spectra of R u C l 2 ( B P T P ) 2 in C D C 1 3 , showing an increase in resolution with temperature. Pg. 3 8 9 10 11 37 45 50 51 52 53 58 viii Pg Figure 3.8 The structures of catena-poly{cis-C\2-trans- 60 (CH 3 ) 2 Sn(IV)]( ix-0 ,0 ' -meso-BPSE) and [FtCl 2 (PEt 3 ) ] 2 ( i i -S ,S meso-BPhSE). Figure 3.9 Structures of (1) (R)-2-[(/?)-phenylsulfinyl]propionate, 62 indicating the stereogenic centres (*) and the metal binding sites (-»), and (2) [Ru(ade)(r) 6-/?-cymene)] 4(CF 3S0 3) 4. Figure 3.10 A molecular structure representation (ORTEP) of 64 [RuCl 2(p-cymene)]( /u-BESE) with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity. Figure 3.11 The structure of p-cymene showing the ' H - N M R designations. 66 Figure 3.12 The ' H - N M R spectra for [RuCl 20?-cymene)] 2(,u-BESE) in C D C 1 3 67 at various temperatures from 243 to 313 K , showing that as the temperature decreases the ' H - N M R signals become more resolved. Figure 3.13 The r.t. ' H - N M R spectra in C D C 1 3 for (A) [RuCl 2(p-cymene)] 2( yu-BESE) 68 with one equivalent of B E S E , (B) with 2 equivalents of B E S E , and (C) with excess B E S E . Figure 3.14 The proposed aqueous solution behaviour of 69 [RuCl20p-cymene)] 2( iu-BESE), and the subsequent formation of [RuCl(p-cymene)(BESE)]PF 6 with the addition of NFi4PF6. Figure 3.15 A molecular structure representation (ORTEP) of one 73 conformation of [RuC10>cymene)(BESE)] + with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity. Figure 3.16 A molecular structure representation (ORTEP) of a second 74 conformation of [RuC10j?-cymene)(BESE)]+ with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity. Figure 4.1 The structure of (1) M T T [3-(4,5-dimethylthiazol-2-yl)-2,5- 81 diphenyltetrazolium bromide] (yellow) and (2) the formazan metabolite (purple). ix Figure 4.2 Design and Protocol of the M T T Assay. Figure 4.3 The graphs for two M T T assay trials. Graph (1) shows the cell viability after treatment with c « - R u C l 2 ( D M S O ) 2 ( B E S E ) at drug concentrations from 0.001 to 1.5 m M . Graph (2) shows the same treatment but for concentrations from 0.1 to 3 m M . Figure 4.4 The top graph shows the cell viability after treatment with [RuCl 2(p-cymene)] 2(u.-BESE) at drug concentrations from 0.0001 - 1.5 m M . The inset shows increased detail in the concentration range of the I C 5 0 from 0 .1-1 m M . Figure 4.5 Cellular toxicity results expressed as a percentage of the control for all concentrations tested. The data for cis-R u C l 2 ( D M S O ) 2 ( B E S E ) ( • ) and for RuCl 2 (p - cymene ) ] 2 OBESE) P ) are averages of two experimental conditions (lOuJL and lOOuL). Figure 4.6 Cellular toxicity results expressed as P E for all concentrations tested. The data for a ' s -RuCl 2 (DMSO) 2 (BESE) ( • ) and for RuCl 2(p-cymene)] 2( Ju-BESE) P ) are averages of two experimental conditions (10u,L and lOOuL). Eg 86 90 91 92 93 x List of Tables Table 3.1 Selected Bond Lengths (A) for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) , d s - R u C l 2 ( D M S 0 ) 4 , and c w - R u C l 2 ( B E S E ) 2 Table 3.2 Selected Bond Angles (°) for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) , c w - R u C l 2 ( D M S O ) 4 , and cw-RuCl 2 (BESE) 2 . Table 3.3 The S-O stretching frequency values for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) , cw-RuCl 2 (DMSO) 4 , cis-R u C l 2 ( B E S E ) 2 , and for free D M S O and B E S E . Table 3.4 Selected Bond Lengths (A) and Bond Angles (°) for [RuCl 2(p-cymene)] 2( iu-BESE), [RuCl(p-cymene)(BESE)]PF 6 , and [PtCl 2(PEt 3)] 2(u.-S,S-me5o-BPhSE) Table 4.1 Aliquots of media and ruthenium complex required for 0.1, 0.5, and 1.1 m M experimental drug concentrations. Pg 47 48 49 65 88 xi List of Abbreviations Abbreviation Meaning 2'-dG 2' -deoxyguanosine A M P adenosine monophosphate aq. aqueous b.p. boiling point B B S E 1,2-bis(butylsulfinyl)ethane B C y S E l,2-bis(cyclohexylsulfinyl)ethane B E S E 1,2-bis(ethylsulfinyl)ethane B E S M 1,1 -bis(ethylsulfinyl)methane B E S P 1,3-bis(ethylsulfinyl)propane B M S B 1,4-bis(methylsulfinyl)butane B M S E 1,2-bis(methylsulfinyl)ethane B M S P 1,3-bis(methylsulfinyl)propane BPeSE 1,3-bis(pentylsulfinyl)propane BPhSE 1,2-bis(phenylsulfinyl)ethane B P S E 1,2-bis(propylsulfinyl)ethane B P S P 1,3-bis(propylsulfinyl)propane B B T P 1,3-bis(butylthio)propane B C y T E 1,2-bis(cyclohexylthio)ethane B E T E 1,2-bis(ethylthio)ethane B E T M l,l-bis(ethylthio)methane xi i B E T P l,3-bis(ethylthio)propane BPeTP l,3-bis(pentylthio)propane B P h T E l,2-bis(phenylthio)ethane B P T P l,3-bis(propylthio)propane B T S B 1,2-bis(p-tolysulfinyl)benzene br broad C D circular dichroism C H O Chinese hamster ovary (cell line) C O S Y correlated spectroscopy d(GpG) di(deoxyguanosine)monophosphate (dinucleotide) D M S dimethylsulfide D M S O dimethylsulfoxide coordinated via the O-atom D M S O dimethyl sulfoxide coordinated via the S-atom G M P guanosine monophosphate H E P E S N-2-hydroxylethylpiperazine-N'-2-ethane sulfonic acid I C 5 0 initial concentration where 50 % of the cells die IC20 initial concentration where 20 % of the cells die Im imidazole LSEVIS liquid secondary ionization mass spectrometry metronidazole l-P-hydroxyethyl-2-methyl-5-nitroimidazole m.p. melting point M P S O methylphenylsulfoxide M T T 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide xiii O D optical density O R T E P Oakridge Thermal Ell ipsoid Program PBS phosphate buffered saline solution P E plating efficiency pta l,3,5-triaza-7-phosphatricycol[3.3.1.1]decane r.t. room temperature RB-flask round bottom flask T M S tetramethylenesulfide T M S O tetramethylenesulfoxide A M molar conductivity xiv Acknowledgements I would like to thank firstly my supervisor Dr. Brian James for his support and guidance throughout this thesis work. Many thanks also to Dr . Kirsten Skov, Jenny, and Janet from the B C Cancer Research Centre for their endless help throughout the biological testing and specifically the C H O toxicity assay. I am very grateful to the Biological Services Department and especially Dr. Elena Polishchuk and Mona for their support and help in setting up the C H O toxicity assay. A s well , I would like to thank Jason Sartor, also from the B C C R C , for his efforts in my training during the M T T toxicity assay. Many thanks to the departmental services at U B C namely M r . Peter Borda, the N M R staff, and Dr. Brian Patrick. Thanks also to past and present members of the James group for all their ideas, suggestions, and encouragement. Special thanks to my family and friends for all their love and support. xv Chapter 1 Chapter 1 Introduction: Ruthenium Sulfoxide Complexes and their Anti-cancer Properties 1.1 Introduction Cancer is a progressive disease characterized by abnormal, uncontrolled, and invasive cell growth. Many cell types can be transformed into tumor cells by disruption or disregulation of normal biochemical and cellular processes.1 In the industrialized Western world the most common malignancies, which account for approximately 50% of all cancers, are lung, breast, and colorectal cancer.' If these cancers are detected at an early stage, the chosen treatments are surgery and/or radiation therapy. However, a more advanced, metastatic form of the disease requires chemotherapy (direction of toxic compounds towards malignant cells). Chemotherapy treatment is non-specific, targeting actively dividing cells, and generally only results in prolonged survival for the above mentioned cancers.' Common chemotherapy agents are organic or natural products such as alkylating 2 agents, antibiotics, and alkaloids but, since the discovery of cisplatin (Section 1.2), metal compounds have been used in chemotherapy treatments. Metals as biological agents can participate in biological redox reactions, undergo ligand substitution with biological molecules, or have potential as radioactive isotopes for tumor imaging or therapy.' The development of Ru-based anti-cancer agents and their observed an ti-metastatic activity, provide encouragement for the treatment of resistant tumors as well as prevention and 3,4 reduction of tumor metastases. 1 References on page 15 Chapter 1 1.2 Platinum Chemotherapeutic Agents Since the serendipitous discovery of cell division inhibition by cisplatin [cis-5 diamminedichloroplatinum(II)] in 1965, the complex has become one of the most active and widely used anti-cancer drugs today.6 It is highly effective in the treatment of testicular and 7 ovarian cancers, and is also active against head and neck, lung, cervical, and bladder cancers while breast cancer is recognized as a potential target of cisplatin.6 Cisplatin is administered intravenously and has associated toxicity problems including nephrotoxicity, neurotoxcitiy, 7 nausea, and vomiting at the clinical dosage given. As well, several tumor types exhibit recurrences, after treatment of the initial tumor, due to primary or secondary (acquired) 6 resistance to cisplatin. These problems have led to further investigation of Pt derivatives or second generation Pt drugs for chemotherapy use. New Pt drug developments have considered several characteristics including charge, lipophilicity, stability in the gastric environment, oral bioavailability, and a cis arrangement (to permit intrastrand crosslinking of g DNA) for the design and composition of new drugs. Carboplatin (diammine[l,l-cyclobutanedicarboxylato]-0,0'-platinum(U)), a second generation drug, has achieved a 7 lower toxicity and is routinely clinically used. However, it is only effective in the same range (tumor lines) as cisplatin and does not exhibit activity towards cisplatin resistant 7 tumors. Therefore further work has explored the expansion of the Pt(U) drug line, and of Pt(IV) complexes as potential orally active drugs. Of note, selected trans-Pt complexes as well as di- and tri-nuclear Pt complexes have shown anti-tumor activity, with a different mechanism of action from that of cisplatin, and in particular one tri-nuclear complex has 7,9,10 shown reactivity in both cisplatin sensitive and resistant cell lines. 2 References on page 15 Chapter 1 The mechanism of action of cisplatin, after drug administration, involves diffusion of the complex into cells where loss of chloride occurs in the intracellular environment of low chloride concentration; the complex then becomes biologically active and can bind to D N A 6 The binding of D N A produces interstrand crosslinks (1-5% of lesions) and intrastrand adducts (majority of lesions), the majority of intrastrand adducts being created by the binding of cisplatin to two neighboring deoxyguanosines at the N7 positions (Figure 1.1); these adducts produce local unwinding of the D N A and inhibit replication and transcription.6 It is unclear, however, how this invokes cell death as all cells have mechanisms to repair damaged DNA. Yet it is known that the damage caused by the binding of cisplatin also triggers a cellular response involving activation of some genes, inactivation of other genes, and shifts 6 in cellular metabolism and cell cycle progression, triggering apoptosis. N H 2 Figure 1.1 Structure of deoxyguanosine showing the N7 coordination site. 3 References on page 15 Chapter 1 1.3 Ruthenium Chemotherapeutic Agents Once a cancer has metastasized (spread) to other regions of the body, the method of treatment must be able to access the entire body, and therefore chemotherapy becomes the treatment of choice. However, commonly used chemotherapy drugs, such as cisplatin, which directly interfere with DNA affecting the cell division process are not specific to tumor cells. The exploration of Ru complexes for use as chemotherapeutic agents was initiated in attempts to find less toxic and more specific drugs. Six-coordinate, octahedral Ru agents provide additional coordination sites compared with the square-planar Pt(LT) complexes, and such sites may provide new DNA-binding modes and, depending on the ligands, chirality at the metal centre leading to chiral interactions with the DNA helix.11 As well, Ru(II) and (HI) complexes containing N-ligands are generally substitution inert suggesting they will bind 12 DNA and prevent replication. Ru complexes may utilize different methods of action from those of Pt complexes, and therefore may provide a greater specificity for tumor cells, be active towards different tumor lines than cisplatin, or active toward cisplatin-resistant tumors. Ru agents have shown selectivity for solid tumor metastases and have a lack of 3 significant host toxicity at biologically active doses. The sequence of events thought to 13 occur when these complexes are injected into a living body are as follows (Steps 1-3): 14 1. The Ru moiety binds to transferrin and then is selectively distributed to transferrin-rich receptor tissue. 2. Ru(JJJ) complexes undergo slow exchange reactions until reduction gives the more labile Ru(IJ). 3. Ru exhibits a high DNA-binding affinity. 4 References on page 15 Chapter 1 This proposed behaviour suggests Ru agents may target tumor cells because malignant cells upregulate the expression of transferrin receptors on the cell membrane due 12 to an increased iron requirement. Therefore Ru binding to transferrin provides a method of specificity for ruthenium agents, not available for most of the commonly used anti-cancer 13 agents. The low capacity for exchange of Ru(HI) suggests Ru(nJ) complexes could be used 15 as "prodrugs" of the more active Ru(U) forms. Of note, tumor cells grow more rapidly then normal cells, and as such require increased amounts of glucose and oxygen. 1 5 In fact, the growth is so rapid that the neovascularization process cannot keep pace with the tumor cell growth, and as a result regions of hypoxia form even only at micrometer distances from blood capillaries.' 5 The Ru(m) "prodrugs" wi l l be relatively inert and non-active in the oxic biological environment, but in the hypoxic environment of tumor cells reduction to Ru(II) produces a labile complex that wi l l be active toward D N A . It follows that the high D N A -binding affinity of R u suggests that the drug, once in the intracellular space, wi l l be able to bind D N A . Ruthenium(in) ammine complexes such as cz£-[RuCl2(NH3)4]Cl and JmH[trans-Ru(Im)2Cl4] have shown cytotoxic effects in cell cultures related to D N A - b i n d i n g . 1 6 The 17 mode of DNA-binding is analogous to that of cisplatin (Section 1.2). These multichloro complexes, as well a s / a c - f R u C ^ N H ^ ] , exhibit the best activity for ruthenium complexes , . 16 toward primary tumors. 5 References on page 15 Chapter 1 1.3.1 Ruthenium DimethylsulJvxide Complexes Ru(IJ) and (HI) D M S O complexes exhibit anti-cancer activity at high doses, but are 1 8 relatively non-toxic at high concentrations with L D 5 0 values up to 1 g/Kg. Some of the initial biological studies involved cis- and r r an5 - R u C l 2 ( D M S O ) 4 and suggested that these complexes have anti-cancer properties and specifically anti-metastatic properties. Cis-RuCl2(DMSO)4 is moderately active at very high doses against primary tumors of M C a 1 9 mammary carcinoma and B16 melanoma with survival times moderately increased, while f ra /w-RuCl 2 (DMSO) 4 is active against both primary tumor and metastases formation of B16 1 6 melanoma. Both isomers show no statistically significant effect on primary tumor growth 20 of Lewis lung carcinoma, but do significantly lower lung metastases. Both complexes reduced the number and weight of spontaneous lung metastases by 50 %, with trans-21 R u C l 2 ( D M S O ) 4 being slightly more active at a lower dose. In mice bearing Lewis lung carcinoma, cisplatin is effective at 0.52 mg/(kg-day) compared with zrans-RuCl2(DMSO) 4, 21 which is effective at 37 mg/(kg-day) and cw-RuCl2(DMSO) 4 at 700 mg/(kg-day). In molar terms cw-RuCl2(DMSO) 4 is active at 1238 u M and cisplatin at 1.7 u M showing that a much higher dose is needed to produce an equi-toxic effect using the ruthenium complexes; 20,22 however, this is achieved with reduced host toxicity. The solution activities of c w - R u C l 2 ( D M S O ) 3 ( D M S O ) and r ran^-RuCl 2 (DMSO) 4 are different, thus leading to their different anti-cancer activity. C / s - R u C l 2 ( D M S O ) 4 , once 23 dissolved in H 2 0 , immediately loses the O-bound D M S O which is replaced by H 2 0 . This is followed by slow dissociation of chloride over approximately 10 h to give a 1:1 electrolyte with no further loss of chloride over time (Figure 1.2 (A)); this behaviour is seen at 6 References on page 15 Chapter 1 biological temperatures (37 °C) but a conductivity corresponding to a 1:1 electrolyte is 21 achieved after only 3 h. Alessio et al. have also shown that chloride dissociation occurs at a chloride concentration of 3 m M (intracellular concentration) but is completely inhibited at 21 150 m M (extracellular concentration). This implies that c i s - R u C ^ C D M S O ^ loses D M S O in the extracellular environment with no chloride dissociation, and the resulting neutral species is able to diffuse across the cell membrane where, once inside the cell, it wi l l lose chloride and become active. In contrast, rratts-RuCl2(DMSO)4 on dissolution in H2O immediately releases two S-bound D M S O molecules, and becomes a c/s-diaquo, cis-bis(DMSO), rrans-chloro species (Figure 1.2 (BJ); the release of two S-bound D M S O molecules is thought to occur due to the unfavourable frans-effects between trans S-bound 21 molecules. The neutral trans isomer then slowly loses chloride, over approximately a 24 h 21 period, and after 6 days loses the second chloride. Under biological conditions, the trans isomer loses chloride over more than 6 h and this solution activity is also inhibited at chloride 21 concentrations of 150 m M . 7 References on page 15 Chapter 1 Figure 1.2 The aqueous solution chemistry of c/s-RuCl2(DMSO)4 (A) and trans-R u C l 2 ( D M S O ) 4 (B), where S = S-bound D M S O , and O = O-bound D M S O (adapted from ref. 21). Work in this laboratory has investigated the synthesis of 24,25 RuCl2(sulfoxide)2(nitroimidazole)2 complexes as biological radiosensitizers, while 26 complexes such as Na[rra/M-RuCl4(DMSO)(L)] (L = NH3 or imidazole (lm)) were developed as potential anti-cancer agents. Two complexes, shown in Figure 1.3, Na[trans-RuCl 4 (DMSO)(Im)] ( N A M I ) and ImH[rran5-RuCl 4(DMSO)(Im)] ( N A M I - A ) represent an important development in anti-cancer treatment because of their an ti-metastatic activity 16 which could be vital in eliminating micrometastases following surgery. 8 References on page 15 Chapter 1 Figure 1.3 The structures of N A M I (C = Na) and N A M I - A (C = ImH), where S = S-bound D M S O (adapted from ref. 3). N A M I and N A M I - A are active against M C a mammary carcinoma and Lewis lung 16 carcinoma, while only N A M I is active against B16 melanoma. N A M I does not work by binding to D N A and preventing replication, but instead may act to increase resistance to the 3 formation of metastases. The complex appears to alter the ratio between m R N A s of M M P 2 (a metalloproteinase capable of degrading the extracellular matrix) and TEVIP-2 (the specific 27,28 tissue inhibitor of this enzyme), thereby causing a pronounced increase in extracellular matrix components in and around the tumor, and this hinders the formation of metastases, 16 and blood flow to the tumor. N A M I - A has pharmacological properties and activity similar 16 to those of N A M I ; however, it is more stable and the synthesis is more reproducible. 1.3.2 DNA-binding of cis-RuCl2(DMSO)4 and trans-RuCl2(DMSO)4 29 7Vans-RuCl2(DMSO)4 reacts in vitro and in vivo with D N A , and shows a mechanism 30 of action similar to that of cisplatin even though it has a different geometry. Alessio et al. have shown using N M R spectroscopy and C D that reaction of f n m s - R u C ^ D M S O ^ with 5 ' G M P forms [RuCl (H 2 0) (DMSO) 2 (5 ' -GMP)]" ; two diastereomers with opposite chirality at 31 the Ru are formed upon chelation via the N7 and the a-phosphate group. The N7 site of the 9 References on page 15 Chapter 1 guanine bases is the preferred site of attack on D N A , and studies by Esposito et al. using N M R spectroscopy and molecular modeling have shown reaction between trans-RuCl2(DMSO) 4 and the dinucleotide d(GpG) results in the formation of a 1,2 intrastrand crosslink. 3 2 Reactions of c i 5 - R u C l 2 ( D M S O ) 4 with adenine and cytosine produce Ru 2(ade)3(DMSO) 3Cl4 and R u 2 ( c y t ) 4 ( D M S O ) 2 C l 4 ( C H 3 O H ) 4 , respectively, as shown by 33 elemental and IR analysis. Tian et al. have shown, using and 3 1 P N M R spectroscopy evidence, that under physiological conditions d s - R u C l 2 ( D M S O ) 4 reacts with 5 ' - G M P , coordinating via the N7 and phosphate group, to form predominantly two isomers with opposite chirality at the R u (Figure 1.4); however, the major product from reaction of cis-34 R u C l 2 ( D M S O ) 4 with 5 ' - A M P has only coordinated phosphate. O ii,,, iN . R u , N „nS ' C l C l . R u Figure 1.4 Structures of the diastereomers formed by the reaction of a's-RuCl2(DMSO) 4 and 5 ' G M P ; N represents the N7 of the guanine base and O represents an O-atom of the phosphate group (adapted from ref. 34). A study by Davey et al, using electrospray ionization mass spectrometry and 'Ft N M R spectroscopy, details the reaction products of cz's-RuCl2(DMSO) 4 and trans-RuCl2(DMSO) 4 with 2'-deoxyguanosine (2'-dG). Both complexes react, via different pathways, to give identical end-products, 2 diastereomers with the 2 ' -dG coordinated via the 10 References on page 15 Chapter 1 N7, and a bis adduct with two N7-coordinated 2 ' -dG moieties (Figure 1.5); also, both cis-R u C l 2 ( D M S O ) 4 and trans-RuC\2(DMSO)4 react with 2'-deoxyadenosine (coordinated via N l ) to some degree, while the trans isomer reacts to a small extent with 2'-deoxycytidine and not 35 at all with thymidine. Q H 2 O H 2 S 2'-dG . 2 '-dG ^ R u " ^ R U : $r ^ O H 2 H o O ^ I 'S (1) 2'-dG 2'-dG O H 2 S1 ,„«i'2'-dG ST | ^ 2 ' - d G C l (2) Figure 1.5 The structure of (1) the two diastereomers formed upon reaction of cis- and ?rans-RuCl 2 (DMSO) 4 with 2 ' -dG, and (2) a third end-product formed upon further reaction with 2 ' -dG (adapted from ref. 35). 1.3.3 Ruthenium Disulfoxide Complexes Work in the James group extended the range of Ru(sulfoxide) complexes to include 36,37 disulfoxide complexes, both to examine their in vitro activity and to reduce the number of possible isomers formed during the preparation of RuCl2(sulfoxide)2(nitroimidazole)2 24,25 complexes. Yapp isolated and examined the in vitro toxicity, cell accumulation, and DNA-binding ability of disulfoxide complexes of the formula cz's-RuCl2(L)2 [L = 1,2-bis(ethylsulfinyl)ethane (BESE) , and l,3-bis(methylsulfinyl)propane (BMSP)] , and trans-11 References on page 15 Chapter 1 R u C l 2 ( L ) 2 [ L = l,2-bis(methylsulfinyl)ethane (BMSE) , and l,2-bis(propylsulfinyl)ethane 36,37 (BPSE)]. Further studies by Cheu led to isolation of c w - R u C l 2 ( L ) 2 [L = 1,2-bis(butylsulfinyl)ethane (BBSE) , l,2-bis(pentylsulfinyl)ethane (BPeSE), 1,2-bis(cyclohexylsulfinyl)ethane (BCySE) , and l,3-bis(ethylsulfinyl)propane (BESP)], and 38 r rans-RuCl 2 (L) 2 -H 2 0 (L = B E S E and B P S E ) . Complexes of the formula [RuCl (L)H 2 0] 2 Cu-Cl ) 2 (L = B E S E , B P S E , and B B S E ) and a mixed-valence complex [RuCl(BPSP)] 2 ( / i -Cl) 3 (BPSP = l,3-bis(propylsulfinyl)propane) have been studied similarly in vitro and shown to have low toxicity and to bind D N A to a greater degree than the 38 mononuclear sulfoxide complexes. 1.3.4 Ruthenium p-Cymene Complexes Over the last decade, some reports of Ru(arene) complexes have shown the potential for these complexes as medicinal agents in biological systems. Dale et al. have shown that [(r|6-C6H6)RuCl2(metro)] (metro = metronidazole) damages D N A both under oxic and 39 6 hypoxic conditions, and Vashisht Gopal et al. have shown the ability of [RuCl 2 (r | -40 C 6 H 6 ) ( D M S O ) ] to interfere with the catalytic activity of topoisomerase II. Korn and Sheldrick report that reaction of [RuCl 2(p-cymene)] 2 (^l-Cl 2) with adenine in the presence of A g ( C F 3 S 0 3 ) forms [Ru(ade)(r| 6-/?-cymene)] 4(CF 3S0 3) 4 (Section 3.6; Figure 3.9 (2)), which 41 shows the ability of Ru(p-cymene) complexes to interact with D N A bases. In the last year of this thesis work, it was reported that some RuO>cymene) complexes had been tested for anti-cancer activity, and [Ru(r|6-/?-cymene)Cl2(pta)] (pta = 42 l,3,5-triaza-7-phosphatricycol[3.3.1.1]decane) reveals pH-dependent DNA-binding. The 12 References on page 15 Chapter 1 complex is neutral at physiological p H and is able to diffuse across the cell membrane into cells, while in tissue with reduced pH, such as tumor cells, the pta ligand is protonated and the complex is subsequently unable to leave the cell; the protonated complex induces D N A 42 damage below physiological levels. As well, half-sandwich Ru(JJ) complexes containing N-ligands have been patented for the treatment of cancer, 4 3 and specifically [RuCl(r| 6-/?-cymene)(MA^'-H 2NCH2CH2NH 2)] + has been shown to react with D N A ; this p-cymene-containing cation and 10 other similar Ru(JJ) complexes show activity against a human 44 ovarian cancer cell line (A2780) (Chapter 4; Section 4.7.2). 1.4 Goals of This Thesis 1.4.1 Synthesis of Novel, Water-Soluble Ruthenium Disulfoxide Complexes The biological activity of the water-soluble complexes cis- and rran5 ,-RuCl 2(DMSO) 4 , and of previous water-soluble ruthenium disulfoxide complexes synthesized in this laboratory led to further investigation of Ru(disulfoxide) complexes. One goal of this thesis was to synthesize novel Ru(disulfoxide) complexes, with appreciable aqueous solubility, that could be tested biologically. 1.4.2 Preliminary In Vitro Biological Testing of Ruthenium Disulfoxide Complexes Following reports of cis- and zrans-RuCl2(DMSO)4 anti-metastatic activity, work in the James group has previously looked at the cytotoxicity, cell accumulation, and D N A -binding properties of various Ru(disulfoxide) complexes. A goal of this thesis was to test 13 References on page 15 Chapter 1 novel, water-soluble R u complexes for anti-cancer activity, and toxicity toward Chinese hamster ovarian (CHO) cells. 1.4.3 Oxidation of Coordinated Dithioether in Ruthenium Complexes A further goal of this thesis was to synthesize previously reported R u dithioether complexes of known geometry, and oxidize the coordinated dithioether to the corresponding disulfoxide. This was to determine whether the ligand, after oxidation, would retain its geometry at the R u centre, and to provide a method for obtaining new Ru(disulfoxide) complexes. 14 References on page 15 Chapter 1 References (1) Fricker, S. P. In Metal Compounds in Cancer Therapy (ed. Fricker, S. P.); Chapman and Hal l : London, 1994; p 1. (2) Haiduc, I. Coord. Chern. Rev. 1990, 99, 253. (3) Sava, G . ; Alessio, E . ; Bergamo, E . ; Mestroni, G . Top. Biol. Inorg. Chern. 1999,1, 143. (4) Sava, G . ; Pacor, S.; Bergamo, A . ; Cocchietto, M . ; Mestroni, G . ; Alessio, E . Chemico-Biological Interac. 1995, 95, 109. (5) Rosenberg, B . ; Camp, L . V . ; Krigas, T. Nature 1965, 698. (6) Oldenberg, J.; Los, G . In Drug Resistance in Oncology (ed. Bernal, S. D.); Marcel Dekker: N Y , 1997; p 331. (7) Wong, E . ; Giandomenico, C. M . Chern. Rev. 1999, 99, 2451, and references therein. (8) Kelland, L . R. In Metal Compounds in Cancer Therapy (ed. Fricker, S. P.); Chapman and Hal l : London, 1994; p 41. (9) Farrell, N . In Metal Ions in Biological Systems (eds. Sigel, A . , Sigel, H.); Marcel Dekker: N Y , 1996; V o l . 32; p 603. (10) Farrell, N . ; Qu, Y . ; Bierbach, U . ; Valsecci, M . ; Menta, E . In Cisplatin Chemistry and Biochemistry of a Leading Anti-Cancer Drug (ed. Lippert, B.) ; W i l e y - V C H : Weinheim, 1999; p 479. (11) Clarke, M . J. In Metal Complexes in Cancer Chemotherapy (ed. Keppler, B . K . ) ; V C H : Weinheim, 1993; p 129. 15 References on page 15 Chapter 1 (12) Clarke, M . J.; Galang, R. D . ; Rodriguez, V . M ; Kumar, R.; Pel l , S.; Bryan, D . M . In Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy (ed. Nicol in i , M . ) ; Martinus Nijhoff Publishing: Boston, 1988; p 582. (13) Sava, G . In Metal Compounds in Cancer Therapy (ed. Fricker, S. P.); Chapman and Hall : London, 1994; p 65, and references therein. (14) Messori, L . ; Kratz, F . ; Alessio, E . Metal-Based Drugs 1996, 3, 1. (15) Clarke, M . J. In Metal Ions in Biological Systems (ed. Sigel, H.); Marcel Dekker: N Y , 1980; V o l . 11; p 231. (16) Clarke, M . J.; Zhu, R ; Frasca, D . R. Chern. Rev. 1999, 99, 2511, and references therein. (17) Clarke, M . J. Inorg. Chern. 1980,19, 1103. (18) Farrell, N . In Transition Metal Complexes as Drugs and Chemotherapeutic Agents (eds. Ugo, R., James, B . R.); Kluwer Academic Publishers: Dordrecht, 1989; p 147. (19) Sava, G . ; Zorzet, S.; Giraldi, T.; Mestroni, G . ; Zassinovich, G . Eur. J. Cancer Clin. Oncol. 1984, 20, 841. (20) Sava, G . ; Pacor, S.; Zorzet, S.; Alessio, E . ; Mestroni, G . Pharm. Res. 1989, 21, 617. (21) Alessio, E . ; Mestroni, G . ; Nardin, G . ; Attia, W . M . ; Calligaris, M . ; Sava, G . ; Zorzet, S. Inorg. Chern. 1988, 27, 4099. (22) Sava, G . ; Alessio, E . ; Bergamo, A . ; Mestroni, G . In Metallopharmaceutical I, DNA Interactions (eds. Clarke, M . J., Sadler, P. J.); Springer-Verlag: Berlin, 1999; p 143. (23) Barnes, J. R.; Goodfellow, R. J. J. Chern. Res. 1979, 4301. (24) Chan, P. K . L . ; Chan, P. K . H . ; Frost, D . C ; James, B . R.; Skov, K . A . Can. J. Chern. 1988,66, 117. 16 References on page 15 Chapter 1 (25) Chan, P. K . L . ; James, B . R.; Frost, D . C ; Chan, P. K . FL; Hu , H . - L . ; Skov, K . A . Can. J. Chern. 1989, 67, 508. (26) Alessio, E . ; Balducci, G . ; Lutman, A . ; Mestroni, G . ; Calligaris, M . ; Attia, W . M . Inorg. Chim. Acta 1993, 203, 205. (27) Morgunova, E . ; Tuuttila, A . ; Bergmann, U . ; Isupov, M . ; Lindqvist, Y . ; Schneider, G. ; Tryggvasson, K . Science 1999, 284, 1667. (28) Brown, P. D . ; Whittaker, M . Chern. Rev. 1999, 2735. (29) Alessio, E . ; Attia, W . M . ; Calligaris, M . ; Cauci, S.; Dolzani, L . ; Mestroni, G . ; Monti-Bragadin, C ; Nardin, G . ; Quadrifoglio, F. ; Sava, G . ; Tamaro, M . ; Zorzet, S. In Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy (ed. Nicol in i , M . ) ; Martinus Nijhoff: Boston, 1987; p 617. (30) Mestroni, G . ; Alessio, E . ; Calligaris, M . ; Attia, W . M . ; Quadrifoglio, F. ; Cauci, S.; Sava, G . ; Zorzet, S.; Pacor, S.; Monti-Bragadin, C ; Tamaro, M . ; Dolzani, L . Prog. Clin. Biochem. Med. 1989,10,11. (31) Alessio, E . ; X u , Y . ; Cauci, S.; Mestroni, G. ; Quadrifoglio, F . ; Vigl ino, P.; Marz i l l i , L . J. Am. Chern. Soc. 1989, 111, 7068. (32) Esposito, G . ; Cauci, S.; Fogolsri, F . ; Alessio, E . ; Scocchi, M . ; Quadrifoglio, F . ; Vigl ino, P. Biochemistry 1992, 31, 7094. (33) Khan, B . ; Mehmood, A . / . Inorg. Nucl. Chern. 1978, 40, 1938. (34) Tian, Y . ; Yang, P.; L i , Q.; Guo, M . ; Zhao, M . Polyhedron 1997,16, 1993. (35) Davey, J. M . ; Moerman, K . L . ; Ralph, S. F.; Kanitz, R.; Sheil, M . M . Inorg. Chim. Acta 1998, 281, 10. (36) Yapp, D . T. T. Ph. D . Dissertation, University of British Columbia, Vancouver, 1993. 17 References on page 15 Chapter 1 (37) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chern. 1 9 9 7 , 36, 5635. (38) Cheu, E . L . S. Ph. D . Dissertation, University of British Columbia, Vancouver, 2000. (39) Dale, L . ; Tocher, J. H . ; Dyson, T. M . ; Edwards, D . I.; Tocher, D . A . Anti-Cancer Drug Design 1 9 9 2 , 7, 3. (40) Vashisht Gopal, Y . N . ; Jayaraju, D . ; Kondapi, A . K . Biochemistry 1999, 38, 4382. (41) Korn, S.; Sheldrick, W . Inorg. Chim. Acta 1 9 9 7 , 254, 85. (42) Allardyce, C . S.; Dyson, P. J.; El l is , D . J.; Heath, S. L . Chern. Commun. 2 0 0 1 , 1396. (43) Morris, R. E . ; Sadler, P. J.; Chen, H . ; Jodrell, D . International Publication Number W O 01/30790 A l , 2001. (44) Cummings, J.; A i rd , R. E . ; Morris, R.; Chen, H . ; del Socorro Murdoch, P.; Sadler, P. J.; Smyth, J. F . ; Jodrell, D . I. Clin. Cancer Res. 2 0 0 0 , 6, Supp. (S) Nov, abstract 142, p 4494s. 18 References on page 15 Chapter 2 Chapter 2 General Experimental for Inorganic Syntheses 2.1 Chemicals and Reagents D M S O and alumina (neutral, Brockman activity I) were purchased from Fisher Scientific. Paraformaldehyde, ethanethiol, and 1,2-dibromoethane were purchased from Aldrich, and oc-phellandrene was purchased from Fluka. 3,6-Dithiaoctane (BETE) was purchased from Lancaster Synthesis. R u C l 3 - 3 H 2 0 was donated by either Johnson-Matthey Ltd., or Colonial Metals Inc. l,3-Bis(ethylsulfinyl)propane (BESP), 1,2-bis(phenylsulfinyl)ethane (BPhSE), and 4,8-dithiaunadecane (BPTP) were donated by Elizabeth Cheu (formerly of the James group) and R u C l 2 ( P P h 3 ) 3 was donated by various members of the James group. A l l deuterated solvents ( D 2 0 , CDCI3, and C D 2 C 1 2 ) were purchased from Cambridge Isotope Laboratories Inc. A l l solvents were of reagent grade or better. A l l syntheses were performed in air and all samples and products were stored at r.t. and in air, unless otherwise stated. The biological experimental techniques and materials used are presented in Chapter 4 (Sections 4.2, 4.3, and 4.4). 2.2 Physical Techniques and Instrumentation 2.2.1 NMR Spectroscopy Solution N M R spectra were obtained using a Bruker A C - 2 0 0 E (200.13 M H z ) , or a Bruker A V - 3 0 0 (300.13 M H z for ! H and 121.49 M H z for 3 1 P ) F T - N M R spectrometer. Proton chemical shifts are given as 8, in ppm, with reference to the residual solvent peak as 19 References on page 35 Chapter 2 the internal standard, relative to T M S (5 0.00) [HDO in D 2 0 5 4.63, C H C 1 3 in C D C 1 3 5 7.24, C H 2 C 1 2 in C D 2 C 1 2 5 5.32]. A l l 3 1 P | ' H } N M R chemical shifts are reported relative to 85 % H 3P04(ag); downfield shifts were taken as positive. Trimethylphosphite, [P(OMe) 3 , at 8 141.00 relative to 85 % H 3P0 4(a<?)], was used as an external reference for ^ P l / H } N M R spectra. Variable temperature ' H - N M R spectra and ' H - 1 ! ! C O S Y were all performed using the Bruker A V - 3 0 0 spectrometer. 1 H - N M R chemical shifts are reported as indicated by bs = broad singlet; s = singlet; d = doublet; t = triplet; q = quartet, m = multiplet; sp = septet. 2.2.2 Infrared and UV-Vis Spectrophotometry Infrared spectra were obtained using an A T I Mattson Genesis Series FTIR instrument. Samples were prepared by thoroughly grinding and mixing a solid complex with K B r , and compressing the resulting mixture into a pellet. IR bands are reported in cm" 1. U V - V i s i b l e spectroscopic data were obtained using a Hewlett-Packard H P 8452A Diode Array Spectrophotometer. Wavelength maxima, X m a x , are given in nm, and extinction coefficients are shown after the reported wavelengths as log 8. 2.2.3 Conductivity and Melting Point Measurements Conductivity measurements were obtained using a Thomas Serfass conductivity bridge, and a cell from Yel low Springs Instrument Company. A l l measurements were made at 25 °C, unless otherwise stated, in a thermostatted water-bath, and at 1 0 3 M concentrations of complex. The value of the cell constant was found to be 1.016, and conductivity values are given in Q,'imo\'1cm2. 20 References on page 35 Chapter 2 Melting point measurements were determined using a Fisher-Johns melting point apparatus, and are reported uncorrected as ranges. 2.2.4 Elemental Analyses and Mass Spectral Analyses Elemental analyses were obtained by M r . Peter Borda, of the U B C department, using a Carlo Erba Instruments E A 1108 C H N - O analyzer. Mass spectral analyses were obtained at the U B C facility headed by Dr. G . Eigendorf. LSEvIS and electrospray methods of ionization were used. 2.2.5 X-ray Crystallography A l l X-ray crystallographic data were acquired by Dr. Brian Patrick on a Rigaku/ADSC C C D area detector with graphite monochromated Mo-Koc radiation. 2.3 Synthesis of Dithioethers 2.3.1 3,6-Dithiaoctane [BETE] (MW = 150.31 g/mol) 3,6-Dithiaoctane (although commercially available) was synthesized according to the procedure of Morgan and Ledbury.' Ethanethiol (30 mL, 405 mmol) was added dropwise with constant stirring to a saturated solution of N a O H in M e O H (50 mL) cooled in a dry-ice/acetone bath. The bath was then removed and the solution allowed to slowly warm to room temperature (-22 °C, r.t.) and then heated with stirring for 1 h at 70 °C. This solution was cooled to r.t. and then placed in the dry-ice/acetone bath, before 1,2-dibromoethane (18 mL, 200 mmol) was added dropwise with constant stirring. Again, the mixture was allowed to slowly warm to r.t. and then heated to 70 °C for 1 h. The r.t. solution was then 21 References on page 35 Chapter 2 poured into H 2 0 (100 mL) and the oily, immiscible dithioether layer was collected. The aqueous layer was extracted 3 times with E t 2 0 (40 mL), the organic residues were combined, and the E t 2 0 was removed under vacuum. The product was then dried over MgSCv Yie ld 2.3 g (7 %). 1 H - N M R (CDC1 3 , 200 M H z ) 8 1.25 (t, 6H , CH3, J 7.50 Hz) , 2.58 (q, 4H , C/Y 2CH 3, J 7.50 H z ), 2.72 (s, 4 H , CH2CH2). The ' H N M R data are as expected, and the product was sufficiently pure for further conversion to the corresponding disulfoxide (Section 2.4.1). 2.3.2 3,5-Dithiaseptane [BETM] (MW = 136.12 g/molf 3,5 Dithiaseptane was synthesized following the procedure of Brandsma et al. after 3 substituting ethanethiol for methanethiol. In a RB-flask, ethanethiol (40 mL, 535 mmol) was added slowly and dropwise to a solution of paraformaldehyde (6.68 g, 223 mmol) in 25 % HCI (25 mL). This was stirred for 1 h and in this time the mixture turns from a white cloudy suspension to a clear colourless solution. The solution was then poured into cold water (100 mL). The organic layer was washed with 3 N K O H (25 mL) and dried over M g S 0 4 . Y i e l d 23 g (75 %). 1 H - N M R (CDC1 3 , 300 M H z ) : 8 1.15 (t, 6 H , CH3, J 7.50 Hz), 2.58 (q, 4H , Ctf 2CH 3, J 7.50 H z ), 3.62 (s, 2H, CH2). Anal . Ca lc 'd for C 5Hi 2S 2: C, 44.07; H , 8.88. Found: C, 43.92; H , 8.84 %. ! H N M R data are comparable to those reported in the '2 literature (of note, the literature spectra were taken in CD3CN). 22 References on page 35 Chapter 2 2.4 Synthesis of Disulfoxides 2.4.1 1,2-Bis(ethylsulfinyl)ethane (BESE) (MW = 182.29 g/molf 1,2-Bis(ethylsulfinyl)ethane was synthesized by the acid-catalyzed, D M S O oxidation of the corresponding dithioether following the procedure of H u l l and Bargar. 5 A solution of 3,6-dithiaoctane (5 mL, 33 mmol), D M S O (5.5 mL, 77 mmol) and cone. HC1 (200 uL) was heated with constant stirring at 80 °C for 8 h. The sulfoxide precipitated as thin white, crystals when the reaction mixture was cooled in an ice-bath. Acetone (-20 mL) was then added to precipitate more product and the mixture filtered. The filtrate was heated for a further 4 h and more product was obtained. The crude product was washed with acetone and recrystallized three times from E t O H (50 mL). Y ie ld 780 mg (13 %) 1 H - N M R ( D 2 0 , 300 M H z ) : 5 1.20 (t, 6 H , C H 2 C / / 3 , J 7.50 Hz), 2.88 (m, 4 H , C / / 2 C H 3 ) , 3.15 (m, 4 H , CH2CH2). M .p . 149-150°C. ! H N M R data compare well with those reported in the literature. 2.4.2 1,2-Bis(ethylsulfinyl)methane (BESM) (MW = 168.10 g/mol) Oxidation of l,2-bis(ethylsulfinyl)methane was attempted following the procedure given in Section 2.4.1, but using B E T M (9 mL, 79 mmol), D M S O (10.44 mL, 158 mmol) and cone. HC1 (200 uL). When a white solid formed, the mixture was cooled with ice, and acetone (-20 mL) was added. The solid was filtered, washed three times with acetone and collected. The resulting white powder was purified by sublimation under vacuum at 80 °C. The solid was insoluble in most common solvents and only slightly soluble in D M S O . 23 References on page 35 Chapter 2 Evaluation of this complex using N M R , and mass spectrometry, was unsuccessful and the elemental analysis was inconclusive. 2.5 Synthesis of Ruthenium Precursors 6,7 2.5.1 Ruthenium "Blue" Solution Ruthenium "Blue" solution was synthesized by refluxing R u C l 3 - 3 H 2 0 (500 mg, 1.9 mmol) in dry M e O H (10 mL) under 1 atm H2 for approximately 4 h or until the solution was an intense blue. 8 2.5.2 Ruthenium "Red" Solution R u C l 3 - 3 H 2 0 (1 g, 3.8 mmol) was placed in a RB-flask with 1 N HC1 (6.25 mL) and E t O H (10 mL). The solution was heated under reflux for 2 h in air, and the solvent was then removed by rotary evaporation. The residue was dissolved in 1 N HC1 (~5 mL), the mixture filtered, and the filtrate volume made up to 10.00 m L with I N HC1. 2.5.3 Cis-RuCl2(DMSO)4 (MW = 484.50 g/mol) R u C l 3 - 3 H 2 0 (500 mg, 1.9 mmol) was refluxed (180 °C) in D M S O (10 mL) for 5 min in air. The solution changed from a dark brown to a light yellow and a bright yellow precipitate formed upon cooling. Acetone (30 mL) was then added to further precipitate the product, and the yellow precipitate was filtered off and washed three times with acetone. The filtrate was left overnight to deposit some crystals of the complex. Y i e l d 430 mg 24 References on page 35 Chapter 2 (46 %). 1 H - N M R (CDC1 3 , 200 M H z ) : 8 3.60, 3.57, 3.51, 3.50 (s, D M S O ) , 2.80 (s, D M S O ) , 2.69 (s, free D M S O ) . 1 H - N M R data agree well with those reported in the literature. 2.5.4 Cis-RuCl2(BESE)2 (MW = 536.57 g/mol) Method #1: R u C l 3 - 3 H 2 0 (110 mg, 0.42 mmol) was added to a solution of E t O H (30 mL) and cone. H C I (100 uL), and the solution was refluxed for 5 h in air. B E S E (146 mg, 0.80 mmol) was then added and the solution refluxed for a further 6 h. A yellow solid precipitated as the solution was cooled. This solid was collected, washed with E t O H and dried in vacuo at 70 °C. Y ie ld 155 mg (69 %). ! H N M R ( D 2 0 , 300 M H z ) : 8 1.35, 1.39 (t, 6H, C/7 3), 3.30 - 3.70 (m), 3.75 (s) (8H, -C# 2 S(0 )Cr7 2 CH 5 ) . Signals at 8 1.19 (t, 6H, CH3), 2.85 (m, 4 H , C # 2 C H 3 ) , 3.14 (m, 4 H , CH2CH2) correspond to free B E S E . The ' H 10 N M R data are similar to those reported by Cheu, but there are differences to the data 4 published by Yapp et al. Method #2: Ruthenium "Red" solution (1 mL, 0.38 mmol) and B E S E (146 mg, 0.80 mmol) were added to E t O H (20 mL) in a RB-flask. The solution was refluxed for 1 h. A yellow precipitate formed as the solution was cooled to r.t. Y i e l d 102 mg (48 %). ! H N M R ( D 2 0 , 300 M H z ) : data agree with those reported for Method #1. Anal . Ca lc 'd for C 1 2 H 2 8 C l 2 0 4 S 4 R u : C , 26.86; H , 5.26. Found: C, 27.20; H , 5.23 %. 2.5.5 [RuCl(H20)(BESE)]2(n-Cl)2 (MW = 744.59 g/mol)° R u C l 3 - 3 H 2 0 (500 mg, 1.9 mmol) was added to a solution of E t O H (50 mL) and cone. HCI (500 pi). B E S E (350 mg, 1.9 mmol) was the added and the reaction solution refluxed 25 References on page 35 Chapter 2 for 15 h. The solvent volume was reduced by rotary evaporation to 10 m L and as the solution cooled a yellow precipitate formed. The mixture was filtered, the precipitate washed twice with E t O H , and dried in vacuo at 70 °C. Yie ld 312 mg (44 %). *H N M R ( D 2 0 , 300 M H z ) : 5 1.40 (m, 12H, CH3), 3.30-3.70 (m, 16H, - C t f 2 S ( 0 ) C / 7 2 C H 3 ) . Anal . Ca lc 'd for C 8 H 3 2 C l 4 0 2 S 2 R u 2 : C , 19.35; H , 4.33. Found: C, 18.95; H , 4.24 %. Mass spectrum [electrospray, m/z, matrix: H 2 0 ] : 709 [ M + - C l ] , 690 [ M + - C l - H 2 0 ] , 672 [ M + - 2C1]. ' H 10 N M R data compare well with those reported in the literature. 2.5.6 [RuCl(p-cymene)]2(H-Clh (MW = 612.24 g/mol)1' R u C l 3 - 3 H 2 0 (1 g, 3.8 mmol) was placed under 1 atm N 2 . E t O H (50 mL) was then added via cannula, and the resultant mixture stirred until all the solid was dissolved. cc-Phellandrene (5 mL) was then added via cannula. This solution changed from dark brown to red upon refluxing for 4 h. The solution was then cooled and left overnight to allow full precipitation of the complex. The solvent volume was then reduced under vacuum to 20 mL, and E t 2 0 (-10 mL) was added. The precipitate was filtered off, washed twice with E t 2 0 , and once with M e O H . Y i e l d 790 mg (68 %). ! H N M R (CDC1 3 , 300 M H z ) : 6 1.25 (d, 6H, C H ( C # 3 ) 2 , 7 6 . 9 1 Hz) , 2.13 (s, 3H, C# 3 ) , 2.90 (sp, H , C / / ( C H 3 ) 2 ) , 5.31 (d, 2H, ( C H 3 ) C H C # , J 6.00 Hz), 5.44 (d, 2H , ( C H 3 ) C H C H , J 6.00 Hz). *H N M R ( D 2 0 , 300 M H z ) : 51.19 (d, 6H, C H ( C # 3 ) 2 , 7 6.90 Hz) , 2.06 (s, 3H, CH3), 2.73 (sp, H , C t f (CH 3 ) 2 ) , 5.48 (d, 2H , ( C H 3 ) C H C / / , J 6.00 Hz), 5.70 (d, 2H , ( C H 3 ) C / 7 C H , J 6.00 Hz); small peaks are also seen at 5 1.20 (d, 6H, CH(C# 3 ) 2 ) , 2.09 (s, 3H , CH3), 5.59 (d, 2H, (CH 3 )CHC/7 , J 6.00 Hz) , 5.84 (d, 2H, ( C H 3 ) C # C H , J 6.00 Hz). Anal . Ca lc 'd for C 2 0 H 2 8 C l 4 R u 2 : C , 39.26; H , 4.61. Found: C , 26 References on page 3 5 Chapter 2 39.46; H , 4.79 %. ' H N M R data in C D C 1 3 compare well with those reported in the literature (using the same solvent)." 2.5.7 K3[RuCl6] (MW = 431.08 g/mol)2 R u C l 3 - 3 H 2 0 (1 g, 3.8 mmol) was dissolved in 50 m L dry M e O H , and the solution refluxed under 1 atm H 2 . After 5 h, the yellow-brown solution became green; KC1 (0.09 g, 12 mmol) was added, and the solution refluxed in air. A brown precipitate was filtered off and washed with M e O H . The crude product was recrystallized from 12 M HCI , washed with M e O H and dried in vacuo at r.t. U V - V i s (12 M HCI): 348 (3.49), 314 (3.36), 230 (4.39). 12 The U V - V i s data compare well with those reported in the literature. 2.6 Synthesis of a Ruthenium Mixed Sulfoxide Complex 2.6.1 Cis-RuCl2(DMSO)2(BESE) (MW = 510.53 g/mol) Method #1: [RuCl (BESE)(H 2 0) ] 2 ( / i -C l ) 2 (100 mg, 0.13 mmol) was dissolved in hot H 2 0 (10 mL). D M S O (300 pL , 4.10 mmol) was then added and the yellow orange solution turned pale yellow after refluxing for 3 h. The volume of the solution was reduced to almost dry by rotary evaporation, and acetone (20 mL) was slowly added to precipitate the complex. The precipitate was then filtered off and washed twice with acetone. The volume of the filtrate was reduced to less than 1 m L and acetone was added to precipitate more product. The resulting product was dried in vacuo at 70 °C. Crystals suitable for X-ray analysis were grown by slow evaporation of a 1:1 mixture of an E t O H / C H 2 C l 2 solution of the complex. Yie ld 100 mg (75 %). ! H N M R ( D 2 0 , 300 M H z ) (immediately upon dissolution): 8 1.37 (m, 6H, CH2C//3), 2.60 (s, 6H , free D M S O ) , 3.08, 3.16, 3.17, 3.21, (s, 6H , D M S O ) , 3.4 - 3.8 (m, 27 References on page 35 Chapter 2 8H, - C r 7 2 S ( 0 ) C / / 2 C H 3 ) . ' H N M R ( D 2 0 , 300 M H z ) (after 7 h): 5 1.37 (m, 6H , C H 2 C / / 3 ) , 2.60 (s, 6H, free D M S O ) , 3.08, 3.11, 3.16, 3.17, 3.21, 3.22, (s, 6H , D M S O ) , 3.4 - 3.8 (m, 8H, - C # 2 S ( 0 ) C / 7 2 C H 3 ) . Anal . Ca lc 'd for C,oH 26Cl 20 4S4Ru: C , 23.54; H , 5.14. Found: C, 23.65; H , 5.13 %. IR v s o : 926, 996, 1029, 1101, 1135. Mass Spectrum [LSIMS, m/z, matrix: thioglycerol and H 2 0 ] : 475 [ M + - C l ] , 397 [ M + - C l - D M S O ] . A M ( H 2 0 , increasing to a steady maximum value after 6.5 h): 103 ^" 1 cm 2 mol" 1 ; ( H 2 0 at 37 °C, increasing to a steady maximum value after 2 h): 110 ^ " ' c m ^ o l ' 1 ; ( H 2 0 , 3 m M N a C l (corrected for 3 m M N a C l ; A M : 124 Sr'cir^ mor1), increasing to a steady maximum value after 6 h): 102 f^ cnAnol"1. Method #2: Cz ' s -RuCl 2 (DMSO) 4 (100 mg, 0.21 mmol) was dissolved in H 2 0 (20 mL) in a RB-flask, B E S E (38 mg, 0.21 mmol) was added, and the resultant solution refluxed for 3 h. A precipitate formed as the H 2 0 was removed (total volume remaining, 1 mL) by rotary evaporation; acetone (20 mL) was then added to precipitate more complex, and the mixture was filtered. The isolated light yellow complex was washed twice with acetone and dried in vacuo at 70 °C. Y i e l d 21 mg (10 %). ' H N M R ( D 2 0 , 300 M H z ) : data agree with those reported for Method #1. Of note, using 2 equivalents of B E S E in this method resulted in isolation of c /5 -RuCl 2 (BESE) 2 . Attempts to synthesise d s - R u C l 2 ( D M S O ) 2 ( B E S E ) using cis-R u C l 2 ( B E S E ) 2 in H 2 0 with a stoichiometric amount of D M S O were unsuccessful and resulted in isolation of the starting complex. 28 References on page 35 Chapter 2 2.7 Synthesis of a Disulfoxide Bridged Ruthenium Complex 2.7.1 [RuCl3(BPhSE)]2(p-BPhSE)x H20 (x = 1,2)(MW = 1248.84 g/mol) R u C l 3 - 3 H 2 0 (100 mg, 0.38 mmol) was dissolved in a solution of M e O H (30 mL) and cone. HC1 (100 pJL). The solution was refluxed under 1 atm N 2 for 2 h or until the solution became light orange. At this stage, B P h S E (222 mg, 0.80 mmol) was added and the solution refluxed under N 2 for another 5 h. B P h S E was also refluxed with R u C l 3 - 3 H 2 0 in E t O H according to the procedure reported in Section 2.5.4, and B P h S E was reacted with various ruthenium precursors including, K 3 [ R u C l 6 ] , Ru "Blue" solution, and R u "Red" solution. A l l of the above procedures resulted in the formation of yellow precipitates that were filtered, washed twice with E t O H , and dried overnight in vacuo at 70 °C. *H N M R ( C D 2 C 1 2 , 300 M H z ) (paramagnetic): 8 3.9 (bs, CH2), 7.4 (bm, phenyl groups). Anal . Ca lc 'd for C 4 2 H 4 2 C l 6 0 6 S 6 R u 2 ( -H 2 0): C , 39.78; H , 3.50. Found (for syntheses using R u C l 3 - 3 H 2 0 and K 3 [ R u C l 6]) C , 39.74 - 39.81; H , 3.64 - 3.81%. Anal . Ca lc 'd for C 4 2 H 4 2 C l 6 0 6 S 6 R u 2 (-2H 20): C, 39.22; H , 3.61. Found (for syntheses using Ru "blue" and "red" solutions) C, 39.09 -39.22; H , 3.49 - 3.56 %. IR v s o : 1070, 1082, 1105, 1116. Mass spectrum [LSEvIS, m/z, matrix: thioglycerol]: 1142 [ M + - 3C1], 972 [ M + - BPhSE] . A M (CH 2 C1 2 ) : 0.6 ffWmol"1. The complex dissolved in D M S O to give rrarcs-RuCl 2 (DMSO) 4 as shown by ' H - N M R spectroscopy. Reaction of c / 5 - R u C l 2 ( D M S O ) 4 and B P h S E with 4 h of refluxing in M e O H resulted in isolation of only the free ligand. 29 References on page 35 Chapter 2 2.8 Synthesis of a Ruthenium Dithioether Complex 2.8.1 Trans-RuCl2(BPTP)2 (MW = 556.74 g/mol) R u C l 3 - 3 H 2 0 (100 mg, 0.38 mmol) was placed in M e O H or E t O H (30 mL) and cone. HCI (100 pL) was added. The solution was refluxed under 1 atm N 2 , or air, for 2 h or until the solution became light orange. At this stage, 4,8-dithiaunadecane (BPTP) was added (148 mg, 0.77 mmol) and the solution refluxed under N 2 for another 3 h. The solvent was removed under vacuum, acetone was added (~5 mL), and E t 2 0 (~5 mL) was layered on to the acetone. Crystals formed in the flask over a few days and were determined to be twinned by X-ray crystallography. ! H N M R (CDC1 3 , 300 M H z ) : 8 1.02 (t, 6 H , CH3), 1.67 (m, 4H, C H 2 C / Y 2 C H 3 ) , 2.28 (m, 2H, C H 2 C / Y 2 C H 2 ) , 2.79, 2.90 (bs, 8H, - C / / 2 S C / / 2 C H 2 C H 3 ) . Anal . Calc 'd for C 1 8 H 4 0 C l 2 S 2 R u : C, 38.84; H , 7.24. Found: C, 39.21; H , 7.38 %. U V - V i s (CH 2 C1 2 ) 430 (3.74), 394 (3.78), 246 (4.66). 2.9 Synthesis of Ruthenium p-Cymene Disulfoxide Complexes 2.9.1 [RuCl2(p-cymene)]2(p-BESE) (MW = 794.69 g/mol) [RuCl(p-cymene)] 2( iu-Cl) 2 (200 mg, 0.32 mmol) and B E S E (60 mg, 0.32 mmol) were placed under 1 atm N 2 . C H 2 C 1 2 (20 mL) was then added, via cannula, to the solids and the resultant deep red solution stirred for 30 min. The solvent was reduced in volume to 5 m L and hexanes (10 mL) were added to precipitate the complex. The complex was collected and dried in vacuo at 70 °C. Crystals suitable for X-ray crystallography were grown from slow evaporation of C H 2 C 1 2 from a solution of the complex. Y i e l d 164 mg (64 %). Attempts to 30 References on page 35 Chapter 2 increase the yield by refluxing or adding excess B E S E were unsuccessful. ' H - N M R (CDC1 3 , 300 M H z ) : 5 1.28 (d, p-cymene, CH(Ct f 3 ) 2 ) , 1.36 (t, B E S E , CH3, J 7.50 Hz) , 2.27 (s, p-cymene, CH3), 2.82, 3.21 (bs, B E S E , - C t f 2 S ( 0 ) C t f 2 C H 3 ) , 3.05 (sp, p-cymene, C#(CH 3 ) 2 ) , 5.55 (bs, p-cymene, ( C H 3 ) C H C / / ) , 5.63 (bs, p-cymene, ( C H 3 ) C # C H ) . There are also peaks corresponding to the RuOp-cymene) starting material at 8 1.26 (d, p-cymene, C H ( C / / 3 ) 2 ) , 2.14 (s, p-cymene, CH3), 2.90 (sp, p-cymene, C7/(CH 3 ) 2 ) , 5.31 (d, p-cymene, ( C H 3 ) C H C / 7 , J 5.94 Hz), 5.44 (d, p-cymene, ( C H 3 ) C H C H , J 5.94 Hz) . Anal . Calc 'd . for C 2 6 H 4 2 C l 4 0 2 S 2 R u 2 : C , 39.30; H , 5.33. Found: C, 39.46; H , 5.32 %. IR v s o : 1082, 1110. Mass Spectrum [LSEVIS, m/z, matrix: 3-nitrobenzylalcohol (3 -NBA) ]: 792 [ M + ] . 2.9.2 [RuCl2(p-cymene)]2(H-BESP) (MW = 808.72 g/mol) The procedure used for synthesizing the title complex is that given in Section 2.9.1 but using [RuClfp-cymene)] 2( jU-Cl) 2 (50 mg, 0.081 mmol) and B E S P (15 mg, 0.081 mmol). The volume of the solvent was reduced to 2 m L and E t 2 0 (10 mL) was used to precipitate the complex. The complex was then washed twice with E t 2 0 , collected, and dried in vacuo at 70 °C. Y ie ld 20 mg (30 %). ' H - N M R (CDC1 3 , 300 M H z ) : 5 1.29 (d, p-cymene, CH(C/Y 3 ) 2 ) , 1.34 (t, B E S P , CH3, J 7.50 Hz) , 2.28 (s, p-cymene, CH3), 2.35 (m, B E S P , C t f 2 C H 3 ) , 2.80 (bs, B E S P , C H 2 C / / 2 C H 2 ) , 3.05 (m, B E S P , C t f 2 C H 2 C H 2 ) , 3.08 (sp, p-cymene, C#(CH 3 ) 2 ) , 5.55 (bs, p-cymene, ( C H 3 ) C H C / / ) , 5.63 (bs, p-cymene, ( C H 3 ) C / / C H ) . There are also peaks corresponding to the RuOp-cymene) starting material at 8 1.26 (d, p-cymene, CH(C7/ 3 ) 2 ) , 2.14 (s, p-cymene, Cr7 3 ), 2.90 (sp, p-cymene, C/Y(CH 3 ) 2 ) , 5.31 (d, p-cymene, (CH 3 )CHCr7 , J 5.94 Hz), 5.44 (d, p-cymene, ( C H 3 ) C # C H , J 5.94 Hz), Anal . Ca lc 'd for C 2 7 H44Cl 4 0 2 S 2 Ru 2 : C , 31 References on page 35 Chapter 2 40.11; H , 5.48. Found: C, 39.92; H , 5.57 %. I R v S 0 : 1074, 1082, 1090, 1095,1108,1116, 1122. Mass Spectrum [LSJJvIS, m/z, matrix: thioglycerol ]: 809 [ M + ] . A corresponding reaction of [RuCl(p-cymene)] 2Gu-Cl) 2 and B P h S E was unsuccessful, resulting in no reaction and isolation of starting material. 2.9.3 [RuCl2(p-cymene)(BESE)]PF6(MW = 598.00 g/mol) Method #1: [RuCl(p-cymene)] 2 (^-Cl) 2 (50 mg, 0.081 mmol) and B E S E (30 mg, 0.16 mmol) were dissolved in H 2 0 (20 mL) under 1 atm N 2 or air. The red [RuC10> cymene)] 2 (^-Cl) 2 slowly dissolved with constant stirring to give a yellow solution. Once the [RuCl(p-cymene)] 2( iu-Cl) 2 was completely dissolved, NH4PF6 (26 mg, 0.16 mmol) was added and the H 2 0 was reduced in volume to 5 mL. The solution was then filtered through celite to remove R u metal, left overnight, and yellow needle-like crystals formed. These were filtered off and dried in vacuo at 70 °C. X-ray quality crystals were grown from the complex dissolved in a 1:1 mixture of H 2 0 and M e O H . Y ie ld 30 mg (31 %). 1 H - N M R ( D 2 0 , 300 M H z ) : 5 1.06 (d, 6H , p-cymene CH(C/7 3 ) 2 , J 6.93 Hz) , 1.40 (t, 6H, B E S E C# 3 ) , 2.06 (s, 3H, p-cymene, CH3), 2.70 (sp, H,p-cymene, C/Y(CH 3 ) 2 ) , 3,40 - 3.70 (m, 8H, B E S E -C / Y 2 S ( 0 ) C / / 2 C H 3 ) , 6.28 (s, 4 H p-cymene, CHCH). ' H - N M R (CDC1 3 , 300 M H z ) : 5 1.24 (d, 6H, p-cymene C H ( C / Y 3 ) 2 , J 6.93 Hz) , 1.55 (t, 6H, B E S E C# 3 ) , 2.25 (s, 3H , p-cymene CH3), 2.95 (sp, H,p-cymene, C / / ( C H 3 ) 2 ) , 3.45 (m), 3.75 (m), 3.52 (s) (8H, B E S E , -C /7 2 S(0)C/7 2 CH 3 ) , 6.12 (s, 4 H , p-cymene, CHCH). Anal . Ca lc 'd for C i 6 H 2 8 C 1 0 2 S 2 R u P F 6 : C , 32.14; H , 4.72. Found: C , 32.24; H , 4.77 %. IR v s o : 1072, 1090, 1107, 1119, 1132, 1142. A M (H 2 0) : 75 Q-Wmol"1. 32 References on page 35 Chapter 2 Method #2: [RuCl 2(p-cymene)] 2(,u-BESE) (50 mg, 0.063 mmol) was completely dissolved in H 2 0 (10 mL) and NH4PE6 (21 mg, 0.13 mmol) was added. The solution was concentrated to 5 mL, filtered, left overnight, and yellow needle-like crystals formed. ! H -N M R (CDCI3, 300MHz): data agree with those reported in Method #1. 2.10 Miscellaneous Reactions 2.10.1 Attempted Synthesis of [RuCl(p-cymene)(BESE)]Cl [RuCl(p-cymene)] 2Gu-Cl) 2 (200 mg, 0.33 mmol) and B E S E (118 mg, 0.65,mmol) were dissolved in H 2 0 (20 mL) and the resulting solution stirred for 30 min. The solvent was then removed by rotary evaporation, the residue dissolved in C H 2 C 1 2 , and purified by column chromatography (using 10% M e O H in CH 2 C1 2 ) to remove starting material and R u metal. The isolated solid did not analyze well for [RuClQ9-cymene)(BESE)]Cl. 2.10.2 Reaction of RuCl2(PPh3)3 with BESE R u C l 2 ( P P h 3 ) 3 (200 mg, 0.21 mmol) and B E S E (76 mg, 0.42 mmol) were placed under 1 atm N 2 . C H 2 C 1 2 (20 mL) was added via cannula and an immediate colour change from red to orange was observed. This orange solution was stirred for 10 min, and the solvent volume reduced under vacuum to 2 mL; E t 2 0 was then added to precipitate a beige yellow complex. The mixture was filtered and the precipitate washed twice with E t 2 0 and dried in vacuo at 70 °C. The product was stored under N 2 . *H N M R (CDC1 3 , 300 M H z ) 8 1.20-1.61 (m, B E S E CHj groups), 2.63 - 3.76 (m, B E S E - C # 2 S ( 0 ) C / / 2 C H 3 ) , 7.31- 8.00 (m, phenyl groups). 3 1 P N M R (CDC1 3 , 300 M H z ) : 8-4.18 (s, free PPh 3 ) , 19.17 (s), 19.89 (s). Elemental analysis and ] H , ] H C O S Y data (CDC1 3 , 300 M H z ) were inconclusive. A M 33 References on page 35 Chapter 2 (CH 2C1 2): 1.5 Q^cn^mof" 1 . The solid could not be further purified and is probably a mixture of sulfoxide phosphine complexes and R u C l 2 ( B E S E ) 2 . 2.10.3 Reactions of [RuCl(H20)(BESE)]2(p-Cl)2 with BESP, and with BPSP [RuCl(BESE)(H 2 0)] 2 Gu-Cl)2 (163 mg, 0.22 mmol) and B E S P (83.6 mg, 0.43 mmol) were refluxed in H 2 0 (10 mL) for 4 h. The mixture was then cooled to r.t; after 5 days the solution deposited some crystals that were found to be czs-RuCl2(BESE) 2 by N M R spectroscopy and elemental analysis. *H N M R ( D 2 0 , 300 M H z ) : 5 1.35, 1.39 (t, 6H , C# 3 ) , 3.30 - 3.70 (m), 3.75 (s) (8H, -C / / 2 S(0)C / / 2 CH 5 ) . Signals at 5 1.19 (t, 6H , CH3), 2.85 (m, 4H, C//2CH3), 3.14 (m, 4 H , CH2CH2) correspond to free B E S E . Anal . Ca lc 'd for Ci 2 H 2 8 Cl20 4 S4Ru: C , 26.86; H , 5.26. Found: C, 27.19; H , 5.43 %. Similar results were obtained when [RuCl (BESE) (H 2 0) ] 2 Gu-Cl ) 2 was reacted with B P S P . However, when the dimer alone was refluxed in H 2 0 for 3 h, it was isolated unchanged. 2.10.4 Reactions ofRuCl3-3H20 with BPSP Attempts to synthesize the known dinuclear Ru(n)/(HI) disulfoxide [RuCl(BPSP)] 2(A*-Cl) 3 using Cheu's method 1 0 of refluxing B P S P with R u C l 3 - 3 H 2 0 in E t O H were unsuccessful, and each time an oily orange-red product was isolated. Syntheses attempted under N 2 , and using excess B P S P , were unsuccessful and resulted in isolation of free ligand. Synthesis was also attempted by refluxing cw-RuCl2(DMSO) 4 (200 mg, 0.40 mmol) and B P S P (90 mg, 0.4 mmol) in M e O H (15 mL) under N 2 , but the reaction yielded an oi l , that could not be purified by column chromatography. 34 References on page 35 Chapter 2 References (1) Morgan, G . T.; Ledbury, W . J. Chern. Soc. 1922,121, 2882. (2) Song, H . ; Haltiwanger, R. C ; Dubois, M . R. Organomet. Chern. 1987, 6, 2021. (3) Brandsma, L . ; Vermer, P.; Kooijman, J. G . A . ; Boelens, H . ; Malssen, J. Reel. Trav. Chim. Pays-Bas 1972, 91, 730. (4) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chern. 1997, 36, 5635. (5) Hu l l , M . ; Bargar, T. W . J. Org. Chern. 1975,40, 3152. (6) Rose, D . ; Wilkinson, G . J. Chern. Soc. A. 1970, 1791. (7) Yapp, D . T. T. Ph. D . Dissertation, University of British Columbia, Vancouver, 1993. (8) Lipponer, K . G . ; Vogel , E . ; Keppler, B . K . Metal-Based Drugs 1996, 3, 243. (9) Evans, I. P.; Spencer, A . ; Wilkinson, G . J. Chern. Soc, Dalton Trans. 1973, 204. (10) Cheu, E . L . S. Ph. D . Dissertation, University of British Columbia, Vancouver, 2000. (11) Bennett, M . A . ; Smith, A . K . J. Chern. Soc, Dalton Trans. 1974, 233. (12) James, B . R.; M c M i l l a n , R. S. Inorg. Nucl.Chem. Lett. 1975,11, 837. 35 References on page 35 Chapter 3 Chapter 3 Chemical and Structural Properties of Ruthenium Disulfoxide and Thioether Complexes 3.1 Introduction to Sulfoxides Early on it was recognized that sulfoxides were capable of acting as Lewis bases, and with renewed interest in the 1960s it was established that sulfoxides can act as ambidentate ligands coordinating to specific metals via the oxygen atoms (O-atoms) or sulfur atoms (S-atoms).' The commercial availability and favourable solvent action of D M S O led to further investigation of the coordination chemistry of sulfoxides. Sulfoxides have been used in the solvent extraction of metals during refining processes, and transition metal sulfoxide complexes have various applications as reactive intermediates in preparative coordination chemistry, and in homogeneous catalysis.' Moreover, an interest in the biological activity of sulfoxide complexes has developed over 1,2 the last few decades. 3.1.1 Properties of DMSO Initial work with sulfoxides involved mainly D M S O (b.p. 189 °C) which, because of its resistance towards hydrolysis and thermal decomposition, is an ideal laboratory or industrial solvent. The favourable solvent properties of D M S O result in higher yields and more selectivity in reactions such as alkylation, cyclization, condensation, and ether formation. 3 36 References on page 75 Chapter 3 The structure of D M S O can be considered as the resonance hybrid forms shown in Figure 3.1 (I -HI). Form U is the generally accepted formalization for D M S O ; however, X -ray studies have shown the free molecule is polarized and form I is likely the dominant structure.1 The crystal structures of D M S O have been determined by X-ray diffraction studies at -60 and 5 °C, and show S-0 bond lengths of 1.4714 and 1.531 A 5 , respectively. o 6 These S-O bond lengths of D M S O are shorter than the estimated bond length of 1.66 A for a single bond suggesting the S-O bonds have some double bond character, and the bond lengths are comparable to the 1.54 A found for an S-N double bond. The coordination geometry around the sulfur is a distorted trigonal pyramid with the S-atom sp hybridized. The C - S - 0 bond angles are approximately 107° and the C - S - C bond angle is 98°. 5 In general the C-S-C bond angle is always smaller than the C-S-O angles regardless of the alkyl groups 2 due in part to the repulsion between the S-O double bond and the lone-pair on the S-atom. / S Q •* *• yS=Q •* *• ^ § = 0 R R R I II III Figure 3.1 The resonance hybrid forms of a sulfoxide, D M S O (R = CH3). 3.2 Metal-Sulfoxide Bond ing Sulfoxides are ambidentate ligands binding via either the S- or O-atom to a specific metal. Initial observations with D M S O complexes suggest that sulfoxides bind via the O-1 atom to "hard" first-row transition metals and via the S-atom to "softer" metals, hardness 37 References on page 75 Chapter 3 7 and softness being generally a measure of metal size and orbital diffuseness. A "soft" metal is larger in size and has loosely held outer d-orbital electrons allowing 7i-electron donation to 7 ligands with available empty d-orbitals such as the S-atom. In contrast the O-atom, which is 7 considered a "hard" base, binds to smaller metals with less diffuse orbitals ("hard" metals). "Hard" metals have empty d-orbitals, of low enough energy, available for acceptance of 7 electrons from atoms such as oxygen. Other factors such as steric and electronic effects can g force O-atom bonding to "soft" metals. Steric effects have been implied when comparing, for example, trans-[FeCl 2 (DMSO) 4 ] + , where all D M S O ligands are O-bound, with the complex cis-R u C l 2 ( D M S O ) 3 ( D M S O ) , where one D M S O ligand is O-bound. 8 The larger and softer Ru should allow for all the sulfoxides to bind via the S-atom; however, due to steric constraints, one D M S O binds via the O-atom. Electronic effects exerted by other ligands in mixed ligand complexes can also influence the coordination mode of sulfoxides. This has been observed in a study on linkage isomerism using thiocyanate, with Pd(U) and Pt(U). 9 Thus, in [ M ( N H 3 ) 2 ( S C N ) 2 ] the thiocyanate is S-bound, and in [M(PR 3 ) 2 (NCS) 2 ] it is N-bound. Ligands that are Ti-electron acceptors, such as P R 3 groups, can withdraw electron density from the metal, making it a less 9 diffuse or "harder" metal, which now favours N-bonding to optimize orbital overlap. This is analogous to the S-O bonding situation where variation of the R group in [ R h 2 (0 2 C R)4 ( D M S O ) 2 ] complexes can determine the coordination mode of D M S O . When R = Me or Et, D M S O is coordinated via the S-atom, but when R = C F 3 the high 38 References on page 75 Chapter 3 g electronegativity of the R group causes the D M S O to coordinate via the O-atom: the F-atoms withdraw electron density giving the metal centre a "harder" character. Solvent can also play a role in determining the nature of the sulfoxide species present in solution; for example, in aqueous solutions S-bound sulfoxides are favoured as O-bound sulfoxides are usually readily displaced by H 2 0 . Such solvent effects are seen for both R u C l 2 ( D M S O ) 3 ( D M S O ) , 1 0 and for R u C l 2 ( D M S O ) ( D M S O ) ( B E S E ) synthesized in this work (Section 3.3.1). The trans influence of S-bound sulfoxide can be estimated by observing its effect on the length of a R u - C l bond, and Davies has suggested that the trans influence of D M S O is similar to that of N H 3 . 1 If a metal a-bond such as M - C l is trans to a strong 7t acceptor a shortening of the M - C l bond length is expected. Mercer and Trotter compare mer-[RuCl 3(p-N 2 C 6 H4Me)(PPh 3 ) 2 ] with a mean R u - C l bond length of 2.390(7) A , " which is suggested as typical for an octahedral complex with trans chlorides, with cz 's -RuCl 2 (DMSO) 4 , which has a R u - C l bond length of 2.435(1) A indicating that D M S O has a strong trans influence and is a 12 poor 71-acceptor. 3.2.1 Sulfur-Metal Coordination Studies on coordinated D M S O have shown the molecular geometry of D M S O is essentially unaffected by S- or O-bonding. The S-0 bond order increases when the sulfoxide is coordinated via the S-atom, and this shorter bond length suggests electron density is donated by the O-atom to the S-atom to compensate for the electron withdrawal by the metal. The average S-0 bond lengths for S-bound sulfoxides range from 1.422 -o 2 1.512 A , and the suggested resonance structure of S-bound sulfoxide is closer to that of 39 References on page 75 Chapter 3 form IU (Figure 3.1). The M - S bond length, when compared to the sum of the covalent radii (2.37 A ) of R u (1.33 A ) and S (1.04 A ) , can provide an estimate of the degree of n-back-donation from the metal centre to the S-atom.1 Comparison of data for cis-R u C l 2 ( D M S O ) 4 with a mean Ru-S bond length of 2.268 A, and those for [Ru(NH 3 ) 5 (DMSO)][PF 6 ]2 with a Ru-S bond length of 2.188(3) A, suggests that the shorter bond length results from more 7t-back-donation from the R u to the S-atom.' Infrared spectra show an increase in the stretching frequency (v s o ) from that of free D M S O when the sulfoxide is S-bound, which is consistent with the decrease in S-O bond length. A downfield shift in the ' H - N M R signals of ~1 ppm is expected for S-bound sulfoxide (versus free sulfoxide) due to S-atom coordination through an sp 3 orbital; this withdraws electron density from the C-S bond, and as a result the a-protons are deshielded.1 This trend is also observed for the (3- and y- protons although the extent of the effect is decreased with increasing distance from the S-atom.1 However, this tendency is not always observed as indicated by the ' H - N M R spectrum of d s - R u C l 2 ( T M S O ) 4 which shows the P-protons shifted slightly 13 upfield of free T M S O while the a-protons are shifted downfield of the free ligand. Of note, in general the S-atom wi l l be trans to chloride, or the O-atom when no chloride is 14 available, to avoid n back-bonding competition between trans sulfur ligands. 3.2.2 Oxygen-Metal Coordination As noted above, when sulfoxides coordinate via the O-atom, their structure is generally unaffected and the S-O bond order remains essentially unchanged or decreases I slightly. The corresponding slight increase in the S-O bond length is due to electron 40 References on page 75 Chapter 3 donation from an sp 2 orbital on the O-atom to the metal, which removes electron density from the S-O bond, thus elongating the S-O bond length slightly. The average S-O bond lengths for O-bound sulfoxides range from 1.470 - 1.578 A , and the suggested resonance structure is closer to form I (Figure 3.1). The change in S-0 bond length for O-bound sulfoxide versus free sulfoxide is not as significant as it is for S-bound sulfoxide: the infrared spectrum shows a slight decrease in v s 0 for O-bound sulfoxide corresponding to the increase in S-O bond length upon coordination.' A downfield shift of ~ 0.5 ppm is observed in the ' H - N M R spectra for O-bound sulfoxide, smaller than that seen for S-bound sulfoxide because coordination of the O-atom via an sp 2 orbital does not directly affect the C-S bond, and the a-protons are therefore deshielded to a lesser degree. 3.3 Ruthenium Mixed Sulfoxide/Disulfoxide Complexes The disulfoxides used in this thesis work are of the formula R S ( 0 ) ( C H 2 ) n S ( 0 ) R (n = 2, and R = Et (BESE) , or Ph (BPhSE); or n = 3, and R = Et (BESP), or Pr (BPSP)), of which B E S E (l,2-bis(ethylsulfinyl)ethane) was synthesized and isolated as a mixture of diastereomers (rac-R,R/S,S pairs, and meso-R,S/S,R forms) and recrystallized from E t O H as 15 the meso isomer. O f note, separation of enantiomeric forms of B P h S E (1,2-16,17 bis(phenylsulfinyl)ethane) has been achieved by column chromatography on lactose, while enantiomers of B M S E (l,2-bis(methylsulfinyl)ethane) and B E S E have been 18 synthesized using diacetone-D-glucose and purified by column chromatography on silica. X-ray crystal structures of B M S E , B P S E (l,2-bis(propylsulfinyl)ethane), and B P h S E have suggested that generally the higher melting isomer, obtained from the synthetic procedure 19 involving oxidation of the dithioether with D M S O , is the meso form. 41 References on page 75 Chapter 3 Madan et al. have synthesized various metal disulfoxide complexes of the formulas [ M ( B M S E ) 3 ] ( C 1 0 4 ) 2 ( M = M n 2 + , F e 2 + , C o 2 + , N i 2 + , C u 2 + , Z n 2 + , C d 2 + ) , and M C 1 2 ( B M S E ) ( M = 2+ 2+ 2 0 Pt , Pd ). A s well , complexes with other sulfoxides of the formula [ M ( L ) n ] ( 0 0 4 ) 2 [n = 3 and M = M n 2 + , C o 2 + , N i 2 + , Z n 2 + ;n = 2 and M = C u 2 + ; L = B E S E , B M S P (1,3 bis(methylsulfinyl)propane), and B M S B (1,4 bis(methylsulfinyl)butane)] were later 21 synthesized. IR spectroscopy studies of all these complexes suggest that the sulfoxides are O-bound (shift to a lower v s o ) , except for the P t 2 + and P d 2 + complexes which are S-bound (shift to a higher v s o ) . Musgrave and Kent have synthesized complexes of the formula [M(L) 3 ] (C10 4 ) 2 ( M = C o 2 + , N i 2 + , C u 2 + ; L = meso- and rac-BPhSM {bis(phenylsulfinyl)methane}, and meso- and rac-BPhSE) all with O-bound sulfoxides and 22 the platinum complexes P t ( L ) C l 2 (L = meso- and rac-BPhSE) with S-bound sulfoxides. Khiar et al. have synthesized Fe(HI) complexes with the enantiomerically pure ligands (S,S)-bis(p-tolylsulfinyl)methane and (S,S)-2,2-bis(p-tolylsulfinyl)propane that are O-23 bonded. Tokunoh et al. have synthesized (S,S)-l,2-bis(p-tolylsulfinyl)benzene (BTSB) and the complexes [Rh(BTSB)(COD)](C10 4 ) , f rans-RuCl 2 (BTSB) 2 and P d C l 2 ( B T S B ) containing 24 S-bound sulfoxides. Synthesis and characterization of Rh complexes with chiral sulfoxides such as p-tolyl methyl sulfoxide, and Ru complexes with chiral disulfoxides such as dios, [(2/?3/?)-2,3-0-isopropylidene-2,3-dihydroxy-l,4-bis(methylsulfinyl)butane] were studied as 25,26 catalysts for asymmetric hydrogenation of prochiral olefins. The Khiar group has synthesized and purified, by column chromatography, enantiomerically pure B E S E and B M S E and R u complexes of the formula rran^-RuCl 2 (L) 2 (L = 7?,7?/S,5-BESE and R,R/S,S-4 2 References on page 75 Chapter 3 18 B M S E ) . The B M S E complexes have undergone preliminary biological testing and IC50 18 data for both complexes are presented in Chapter 4 (Section 4.7.1). 27 Complexes such as Na[rrans-RuCl 4 (R 2 SO)(L)] and mer,cw-RuCl 3 (R 2 SO)(L) (L = NH3 or imidazole; R 2 S O = D M S O , and T M S O ) have been synthesized as potential anti-cancer agents (Chapter 1), and some of the initial biological work in this laboratory looked at synthesizing RuCl 2 (R 2 SO) 2 (ni t roimidazole) 2 as potential radiosensitizers, but the structures of the complexes were not definitively resolved because of the number of possible isomers 28,29 formed. The use of sulfoxides was extended to include disulfoxides, both to reduce the number of isomers formed in preparation of the nitroimidazole complexes and to further the range of possible R u sulfoxide anti-tumor complexes. Further work on disulfoxides by Yapp et al. led to the isolation of cw-RuCl 2 (L ) 2 (L = B M S P , and B E S E ) and frans-RuCl 2 (L) 2 (L = B M S E , and B P S E ) , all of which contain S-bound sulfoxide as established by X-ray 30,31 diffraction. A s well , Cheu has isolated cw-RuCl 2 (L ) 2 (L = B B S E , BPeSE , B C y S E , and B E S P ) and r r a n 5 - R u C l 2 ( L ) 2 H 2 0 (L = B E S E and BPSE) , all of which show S-bound sulfoxide by X-ray structure characterization, except for the B P e S E complex which was 32 indicated to be S-bound by IR data. Cheu has also isolated (by using one equivalent of disulfoxide per Ru) and characterized chloride-bridged, dinuclear complexes of the formula [ R u C l ( L ) ( H 2 0 ) ] 2 t > C l ) 2 (L = B E S E , B P S E , and B B S E ) of which only the B E S E complex was structurally characterized, but all complexes indicate S-bound sulfoxide as shown by LR 32 data. A Ru(II)/(III) dinuclear complex [RuCl(BPSP)] 2(jU-Cl) 3 was also synthesized by using two equivalents of disulfoxide, and this was structurally characterized to reveal S-32 bound sulfoxide. Work in this thesis has led to isolation and characterization of the mixed 43 References on page 75 Chapter 3 sulfoxide complex c « - R u C l 2 ( D M S O ) 2 ( B E S E ) with one O-bound D M S O , one S-bound D M S O , and S-bound disulfoxide. 3.3.1 Cis-RuCl2(DMSO)2(BESE) C w - R u C l 2 ( D M S O ) 2 ( B E S E ) , in a 69 % yield, was synthesized from [RuCl (BESE)(H 2 0) ] 2 ( / i -C l ) 2 and D M S O , and was isolated as a pale yellow precipitate that did not need further purification. Crystals suitable for X-ray diffraction were grown by slow evaporation of a solution of the complex in a 1:1 mixture of E t O H / C H 2 C l 2 . The O R T E P diagram (Figure 3.2) shows the structure with cis chlorides, a chelating S-bound meso-BESE (the chirality at S ( l ) is R and at S(2) is 5), one S-bound D M S O , and one O-bound D M S O . Tables 3.1 and 3.2 show various bond lengths and bond angles, respectively, of the title complex, d s - R u C l 2 ( D M S O ) 4 , and m - R u C l 2 ( B E S E ) 2 . The S-O bond lengths for all S-bound sulfoxides fall in the region of 1.470-1.485 A, as expected (Section 3.2.1), showing the S-O bond has some double bond character (cf. 1.531(5) A of free D M S O ) . The S-O bond lengths for O-bound D M S O are 1.529(3) and 1.557(4) A for c « - R u C l 2 ( D M S O ) 2 ( B E S E ) and cis-RuCl 2 (DMSO)4, respectively; these longer bond lengths are expected for O-bound sulfoxide due to electron density donation from the O-atom to the metal (Section 3.2.2). The R u - C l bond lengths are relatively long (Section 3.2) for all three complexes and range from 2.42(1)-2.44(8) A, and suggest that D M S O and B E S E are poor 7i-acceptors. 44 References on page 75 Figure 3.2 A molecular structure representation (ORTEP) of c« -RuCl 2 (DMSO) 2 (BESE) with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity (a stereoview and crystal data are given in Appendix 1 ) . 45 References on page 75 Chapter 3 The mainly S-bound sulfoxide implies a "soft" character of Ru(II). For cw-R u C l 2 ( D M S O ) 2 ( B E S E ) , the Ru-S bond lengths trans to chloride are slightly longer (2.283 and 2.250 A) than that trans to oxygen (2.214 A). The shorter Ru-S bond length suggests more 7i-back donation from Ru to the S-donor when the S-bound sulfoxide is trans to the O-atom. Both cz ' s -RuCl 2 (DMSO) 2 (BESE) and cz 's-RuCl 2 (DMSO) 4 have one O-bound D M S O , perhaps due to steric hindrance, which is trans to an S-atom to avoid 71-back-bonding competition between trans sulfur ligands, as outlined in Section 3.2.1. However, when there are two disulfoxides as with d s - R u C l 2 ( B E S E ) 2 , the sulfoxides are all S-bound. A stereochemical review of R u bis(disulfoxide) complexes suggests this could be due to lower 14 strain energies for S,S-bonding in a disulfoxide with respect to mixed S,0-bonding. The R u - 0 bond lengths, in both c w - R u C l 2 ( D M S O ) 2 ( B E S E ) and c w - R u C l 2 ( D M S O ) 4 , are 2.169(3) and 2.142(3) A, respectively, both significantly shorter than the Ru-S lengths, as expected for a stronger interaction between the O-atom and the Ru than between the S-atom and the Ru. The bond angles in all three complexes show a distorted tetrahedral geometry about the S-atom, with the C - S - C angles being shorter (97.5-100°) in all cases than the C - S - 0 angle (103-109°), because of the S-0 double bond repulsion interaction with the lone-pair on the S-atom (Section 3.1.1). The geometry around the Ru is distorted octahedral with cis angles between 89.1-91.6(9) A, and trans angles between 175.9(5)-179.0(1) A. 46 References on page 75 Chapter 3 Table 3.1 Selected Bond Lengths (A) for d s - R u C l 2 ( D M S O ) 2 ( B E S E ) , cis-R u C l 2 ( D M S O ) 4 , and c w - R u C l 2 ( B E S E ) 2 . Bond d s - R u C l 2 ( D M S O ) 2 ( B E S E ) d s - R u C l 2 ( D M S O ) 4 a d s - R u C l 2 ( B E S E ) 2 b S-O (DMSO) 1.474(4) 1.483, 1.485(5) -S-O (DMSO) 1.529(3) 1.557(4) -S-O (BESE) 1.471, 1.474(3) - 1.470- 1.479(2) S-C (BESE) 1.792(6)- 1.809(5) - 1.796- 1.814(3) S-C (DMSO) 1.762, 1.778(5) 1.783 - 1.808(6) -S-C ( D M S O ) 1.780, 1.782(5) 1.783, 1.793(6) -Ru-Cl 2.428, 2.434(1) 2.435(1) 2.422 - 2.449(8) Ru-S (DMSO) 2.283(l) c 2.276,, 2.277(l) c -2.252(l) d Ru-S (BESE) 2.214(l) d - 2.271 - 2.274(8)c 2.250(l) c 2.297 - 2.308(8)d R u - 0 (DMSO) 2.169(3 ) e 2.142(3) e -a Data taken from ref. 12. b Data taken from ref. 30, 3 1 . c Trans to C l . d Trans to O. e Trans to S. 47 References on page 75 Chapter 3 Table 3.2 Selected Bond Angles (°) for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) , c w - R u C l 2 ( D M S O ) 4 , and c w - R u C l 2 ( B E S E ) 2 . Bond Angle c w - R u C l 2 ( D M S O ) 2 ( B E S E ) c w - R u C l 2 ( D M S O ) 4 a c w - R u C l 2 ( B E S E ) 2b C-S-O ( D M S O ) 105.8, 106.3(2) 106.3(3) - 107.7(4) -C - S - 0 ( D M S O ) 103.1, 105.1(2) 104.6, 104.2(3) -C-S-O (BESE) 106.4 - 108.3(2) - 106 .3 - 109.3(1) C-S-C ( D M S O ) 100.0(3) 9 7 . 5 - 100.1(3) -C-S-C ( D M S O ) 97.5(3) 99.0(4) -C-S-C (BESE) 99.9(2), 100.9(3) - 100 .0- 102.8(1) Ru cis angles 89.1-91.6(9) 85.2-88.8(1) 87.2-92.1(3) Ru trans angles 175.9(5) - 179.0(1) 173.4(5)- 176.1(1) 176 .9- 178.5(3) a Data taken from ref. .2. b Data taken from ref. 30,31. Relevant IR data are given in Table 3.3. The IR of c w - R u C l 2 ( D M S O ) 2 ( B E S E ) shows two bands at a lower frequency than seen for free D M S O (1055 cm" 1), the one of larger intensity at 926 cm" 1 is assigned to D M S O , and the other at 996 cm" 1 is assigned to the methyl rocking frequency (cf. pr = 970 - 1024 cm"1 for cz ' s -RuCl 2 (DMSO) 4 3 3 ) . The bands at 1029 and 1135 cm"1 are assigned to S-bound B E S E , and the band at 1101 cm" 1 is assigned to D M S O . The O-bound sulfoxide shows the characteristic shift to a lower frequency compared with that of free ligand, while S-bound sulfoxide shows a shift to a higher frequency, as expected (Sections 3.2.1 and 3.2.2). 48 References on page 75 Chapter 3 Table 3.3 The S-O stretching frequency values for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) , cis-R u C l 2 ( D M S O ) 4 , d s - R u C l 2 ( B E S E ) 2 , and for free D M S O and B E S E . a Complex Vso (cm"1) Ref. S-bound O-bound c w - R u C l 2 ( D M S O ) 2 ( B E S E ) 1029,1101,1135 926 tw c « - R u C l 2 ( D M S O ) 4 b 1086,1108 927 34 c w - R u C l 2 ( B E S E ) 2 1092, 1122; 1128 - 31,32 B E S E 1019; 1015 - 15,32 D M S O b 1055 - 1 a In K B r , unless stated otherwise. In Nujol. The proposed solution behaviour for c / s -RuCl 2 (DMSO) 2 (BESE) (I) is shown in Figure 3.3. The ' H - N M R spectrum in D 2 0 of (I) immediately upon dissolution shows a singlet in the region of that for free D M S O at 8 2.60 which is due to displacement of the O-bound D M S O by D 2 0 (the addition of D M S O to the N M R sample resulted in an increase of the signal at 8 2.60). A multiplet at 8 1.37 consists of unresolved triplets which correspond to the methyl groups of the B E S E ligand in different environments. The assignments of the triplets are further hindered by the presence of another species (IV) formed by chloride-water exchange (see below). 49 References on page 75 Chapter 3 H 2 O | „„0H 2 1 Figure 3.3 The proposed aqueous solution behaviour of czs -RuCl 2 (DMSO) 2 (BESE) . The multiplet ranging from 8 3.4 - 3.8 represents the C H 2 protons on the backbone and arms of the B E S E ligand, and could not be further assigned. The singlets at 8 3.08, 3.16, 3.17, 3.21 are thought to represent the protons of the S-bound D M S O . The peaks at 8 3.08, and 3.21 are assigned to the methyl groups of the D M S O in (U). The relatively small singlet at 8 3.17 is assigned to the methyl groups of the D M S O in the cationic complex (IV); a singlet is observed because (IV) contains a plane of symmetry. This suggests that a small amount of (IV) is formed immediately upon dissolution of (I) while none of (HI) is formed indicating krv > k m . Of note, there is an unassigned peak (?) slightly downfield of the peak at 8 3.17 which may be due to another isomer in solution. After 24 h, the N M R spectrum changes considerably (Figure 3.4) and two new singlets at 8 3.11 and 3.22 arise along with corresponding decreases of the signals at 8 3.08 50 References on page 75 Chapter 3 and 3.21; these changes are attributed to conversion of (U) into complex (UJ). As well, the singlet at 8 3.17 becomes larger due to further conversion of (U) into (IV). rv ' rv m in I I j I I I I | I I I I | I I I I | I I I I | I I M | 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 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2 6 2.4 2.2 2.0 1.8 1.6 1.4 Figure 3.4 Variation of the 1 H - N M R spectra, in D 2 0 , of R u C l 2 ( D M S O ) 2 ( B E S E ) with time; (A) immediately upon dissolving the complex in H 2 0 , and (B) 24 h after dissolving the complex in H 2 0 . The proposed solution behaviour of d s - R u C l 2 ( D M S O ) 2 ( B E S E ) is analogous to that of 34 c w - R u C l 2 ( D M S O ) 4 in H 2 0 (Chapter 1, Section 1.3.1), and is supported by conductivity data showing the conductivity, in H 2 0 , increases to a steady maximum value of 103 Q" 1 2 1 cm mol" after 6.5 h (Figure 3.5). This suggests that in solution (II) slowly changes from a 35 neutral species to a 1:1 electrolyte system. The conductivity at 37 °C increases to 110 £2" 1 9 1 cm mol" after only 2 h of dissolution of (I) (Figure 3.5). 51 References on page 75 Chapter 3 120 o •o c o • • O 0 0 100 200 300 400 500 600 Time (minutes) Figure 3.5 Variation of the conductivity measurements of c /s -RuCl 2 (DMSO)2 (BESE) with time in H 2 0 at 25 °C ( • ) , in 3 m M NaCI at 25 °C (• ) , and in H 2 0 at 37 °C (-). The conductivity of cz 's-RuCl 2 (DMSO) 2 (BESE) was also followed at 3 m M and 150 m M concentrations of NaCI to show the behaviour of the complex under biological conditions. At 3 m M NaCI (intracellular concentrations), the conductivity increased to a steady maximum value of 102 f X ' c n ^ m o l ' 1 after 6 h, following essentially the same rate as that of the complex in H 2 0 (Figure 3.5). It is thought the dissociation of chloride is inhibited at 150 m M NaCI (extracellular concentrations); however, the conductivity measurements at this concentration are not conclusive because the conductivity of the species present is likely masked by that of NaCI. If chloride dissociation is inhibited, c w - R u C l 2 ( D M S O ) 2 ( B E S E ) could act biologically by diffusing across the cellular membrane, because in an extracellular (150 mM) environment the complex wi l l remain the neutral species (II). Once inside the cell, at a [Cl"] of 3 m M , the dissociation of chloride gives conversion to the cationic complexes (HI) and (IV). Observation of the conductivity over 24 h (for each treatment) 52 References on page 75 Chapter 3 shows the second chloride does not dissociate. Of note, a conductivity study of cis-34 RuCl2(DMSO)4, at a [Cl] of 150 mM, shows complete inhibition of chloride dissociation. The plots of ln(AM D - A M t ) versus time give straight lines, thus showing a first-order dependence for the dissociation of Cl" from cw-[RuCl2(DMSO)(BESE)H20] (Figure 3.6). At 25 °C, k o b s is 7.8 x 10"3 min"1 (ti / 2 is 89 min) and, at 37 °C, k o b s is 3.4 x 10"2 min"1 (t1/2 is 20 min), showing the rate of dissociation is ~ 4.5 times faster at biological temperatures (where k o b s = k m + krv; Figure 3.3). Of note, the conductivity intercepts are slightly different due to the errors involved with the time needed to dissolve the complex and to stabilize the temperature before the first conductivity measurement was taken. Figure 3.6 A plot of In (A M ~ - A M t ) [where A M ° o is the maximum conductivity value and A M t is the conductivity at time (t)] versus time for cz's-RuCl^DMSO^BESE) in H 2 0 at 25 °C (•), and in H 2 0 at 37°C (-). 53 References on page 75 Chapter 3 Cw-RuCi2(DMSO)2(BESE) was tested in vitro for cytotoxicity on Chinese hamster ovarian cells, and for anti-cancer activity on mammalian breast cancer cells, and the results are presented in Chapter 4. Another method for synthesizing cw-RuCl 2(DMSO) 2(BESE), but in lower yield, involves refluxing cw-RuCl2(DMSO)4 for 3 h with 1 equivalent of B E S E . It should also be noted that using 2 equivalents of B E S E in this method results in the isolation of cis-RuCl 2 (BESE) 2 . Attempts to replace the B E S E ligand in c « - R u C l 2 ( B E S E ) 2 using stoichiometric amounts of DMSO were unsuccessful and resulted in isolation of the starting material. 3.3.2 [RuCl(BESE)(H20)]2(lii-Cl)2 with BESP, and with BPSP Refluxing [RuCl (BESE)(H 2 0 ) ] 2 f>Cl ) 2 with 2 equivalents of B E S E results in the 32 isolation of rra«^>-RuCl2(BESE)2 as shown by Cheu. In attempts to synthesize mixed sulfoxide complexes, [RUC1(BESE)(H20)]2(/J-C1)2 was reacted with BESP and with BPSP; however, cw-RuCl 2(BESE) 2 was obtained by crystallization of the reaction product from aqueous solution. An electrospray mass spectrum of [RuCl(BESE)(H20)]2(iu-Cl)2 in aqueous solution was performed to determine if the complex remains dinuclear in solution. Peaks at 709 [M + - Cl], 690 [M + - Cl - H 2 0 ], and 672 [M + - 2C1] suggest that the dimer does not dissociate to monomer. As well, after [RuCl(BESE)(H20)]2(jU-Cl)2 is refluxed in H2O, the complex is isolated unchanged. Previous work has shown that [RuCl(BESE)(H20)]2(AJ-Cl)2 is a 2:1-3:1 electrolyte in H 2 0 , and titration with NaOH showed that two equivalents of base were required for 1 equivalent of [RuCl(BESE)(H20)]2(^-01)2, suggesting that two equivalents of Cl" and two 54 References on page 75 Chapter 3 32 equivalents of H + are liberated on dissolving the dimer in water. It appears the complex in solution perhaps exchanges the terminal chlorides for H2O and becomes [Ru(BESE)(OH)(H 2 0) ] 2 (>Cl ) 2 . That cw-RuCl 2(BESE) 2 is formed on refluxing the dimer with BESP (and with BPSP) is surprising, but clearly the monomer is formed slowly from the hydroxy species. It is not clear, however, why there is no interaction with the added disulfoxides as there is interaction with additional BESE to give frarcs-RuCl 2(BESE) 2. Possibly the BESE complex is thermodynamically more stable than a mixed disulfoxide complex such as RuCl 2(BESE)(BESP). Of note, the 1 H-NMR spectrum in D 2 0 of cis-RuCl 2 (BESE) 2 has signals which correspond to those of the free ligand indicating that one of the BESE ligands dissociates (Section 2.10.3). As well, the singlet seen at 8 3.75, which could only be from a complex with equivalent methylene groups on the backbone of the ligand, suggests that cw-RuCl 2 (BESE) 2 may isomerize in aqueous solution to the trans isomer. 3.4 Ruthenium Thioether Complexes Complexes such as R u X 3 ( L ) 3 (L = DMS or TMS and X = Cl or Br) have been isolated from reactions of ruthenium with acidified DMSO and T M S O . 1 3 ' 3 6 The reactions were performed at higher temperatures (130 - 140 °C) than used for the formation of the anionic complexes rran^- [ (DMSO) 2 H]+ [RuCl 4 (DMSO) 2 ] " and trans-[ (TMSO) 2 H]+ [RuCl 4 (TMSO) 2 ] \ and the formation of the thioether was attributed to redox 36 properties of Ru and sulfoxide. Lidlie et al. proposed the equilibrium shown in eq.l (where R = Bu), so that under highly acidic conditions thioether can form with concurrent oxidation 37 of Ru(II) to Ru(UI). The thioether would then be able to react with Ru(UI) to form 55 References on page 75 Chapter 3 thioether complexes. Conversely, the sulfoxide can be oxidized to sulfone as Ru(ffl) is 37 reduced to Ru(U) as shown in eq. 2. These reactions may play a role in the reduced yields 32 observed in some syntheses of Ru(disulfoxide) complexes because of conversion of sulfoxide to thioether or to sulfone. 2 Ru(U) + R 2 SO + 2 FT i * 2 Ru(UI) + R 2 S + H 2 0 (1) 2 Ru(IJJ) + R 2 SO + H 2 0 • 2 Ru(U) + R 2 S(0) 2 + 2 FT (2) Within the Pt group metals, Lucas et al. have synthesized dithioether complexes of the formula MX2(l,2-bis(benzylthio)ethane), where M = Pd, X = Cl or I, and for M = Pt, X = 38 Cl . Hartley et al. have reported thioether complexes with Pd and Pt of the type ds-[MLX 2 ] and [ML 2](C10 4) 2 (X = Cl , Br, or I; L = RS(CH2)„SR for R = Me or Ph and n = 2 or 3; or L = 39 ds-RSCH = CHSR for R = Me, Ph, or o-C 6 H 6 (SR') 2 (R' = Me or Ph)). As well, they report on [PdLX 2 ] n polymeric complexes (X = Cl and Br; L = PhS(CH2)„SPh for n = 6 or 8) and 39 PdLX 2 and trans-FthCh (X = C l and Br; L = PhS(CH 2)i 2SPh). Other reported thioether complexes are [M(BMTM)X 2 ] (M = Pd or Pt; X = Cl , Br, or I; and B M T M = bis(methylthio)methane), [Rh(BPhTM) 3Cl 3], [Ir(BPhTM) 3Cl 3], and [Ru(BPhTM) 3Cl 3]EtOH 40 where BPhTM = bis(phenylthio)methane. The mononuclear, dithioether Ru complexes zr<ms-RuCl2(BCyTE)2-2 H 2 0 (BCyTE = l,2-bis(cyclohexylthio)ethane) and frans-RuCl2(BPhTE)2 (BPhTE = 1,2-bis(phenylthio)ethane), and dinuclear dithioether complexes of the formula [RuCl2(L)]2(,u-Cl) 2 with L = l,3-bis(ethylthio)propane (BETP), l,3-bis(propylthio)propane (BPTP), 1,3-56 References on page 75 Chapter 3 bis(butylthio)propane (BBTP) , and l,3-bis(pentylthio)propane (BPeTP), have been 3 2 synthesized in this laboratory. The goal of synthesizing these thioether complexes was to determine their geometry, and then oxidize the coordinated thioether using a reported 41 procedure with dimethyl dioxirane to see i f the geometry was retained. This would create a method to synthesize R u complexes that have not otherwise been obtained by direct reaction of ruthenium and sulfoxide. A stereochemical study on Ru(disulfoxide) complexes indicates the cis isomers are more stable than the trans isomers, and therefore the trans complexes may 14 not be obtained by reacting R u precursors with sulfoxide. However, f rans-RuCl 2 (DMSO )4 was shown to be more biologically active, because of its aqueous solution chemistry (Chapter 34 1), than the cis isomer, and the rran.y-Ru(disulfoxide) complexes were shown to accumulate in cells and to bind D N A to a greater degree than the cis isomers (Chapter 4) making the 30,31 trans isomers the preferred products. If isolation of a trans complex cannot be achieved 3 2 by reacting a R u precursor with sulfoxide, as with formation of c /5 , -RuCl 2 (BCySE) 2 , it 32 might be accessible by reacting Ru with the thioether, as with rnms-RuCl 2 (BCyTE) 2 -2 H 2 0 , followed by oxidation of the coordinated thioether. Work in this thesis isolated the thioether complex r ra«5 -RuCl 2 (BPTP) 2 (see below). 3.4.1 Trans-RuCl2(BPTP)2 In an attempt to synthesize the dinuclear complex [RuCl 2 (BPTP)] 2 ( /z-Cl) 2 using the 32 method by Cheu (in air) and under N 2 , r rans-RuCl 2 (BPTP) 2 was isolated. The ' H - N M R in CDCI3 shows a triplet at 8 1.02, which corresponds to the C H 3 groups of B P T P ; one triplet is seen because all the CH3 groups are magnetically equivalent. The multiplet seen at 8 1.67 is 57 References on page 75 Chapter 3 assigned to the CH2 group adjacent to the methyls. The multiplet at 8 2.28 is assigned to the C H 2 protons on the central carbon of the ligand backbone, and the two broad singlets at 8 2.79, and 8 2.90 correspond to the CH2 groups next to the S-atoms. Tentatively, the peaks at 8 2.79 and 2.90 are assigned to the C H 2 protons on the arms of the ligand and those on the backbone, respectively. The ' H peaks become more resolved as the CDCI3 solution of the complex is warmed from 213 to 313 K (Figure 3.8). Elemental analysis also shows that the species isolated is the mononuclear complex and not the previously isolated and structurally 32 characterized [RuCl2 (BPTP)]2 (^-0 )2 . The synthetic work indicates a possible solvent effect because the analyzed crystals of rrarcs-RuCl2(BPTP) 2 (found to be twinned by X-ray crystallography) were grown from acetone/Et 20, whereas crystals of the dinuclear species were grown from CH2CI2. Attempts in this work to isolate crystals from C H 2 C 1 2 were unsuccessful. 3 1 3 K 98 K I . . . . I . 1 . 1 j . . . . I 1 1 . . I 3 . 0 2 . 5 2 . 0 1 . 5 1 . 0 Figure 3.7 1 H - N M R spectra of rran5-RuCl 2 (BPTP) 2 in C D C 1 3 , showing an increase in resolution with temperature. 58 References on page 75 Chapter 3 3.5 Ruthenium Sulfoxide-Bridged Complexes D M S O can bridge Ru centres as shown by the two structures [Ru2(jU-Cl)(^-H)(^-D M S 0 ) C l 2 ( D M S 0 ) 4 ] - 2 C H 2 C 1 2 4 2 and [ R u a G u - C l X ^ - D M S O ^ ^ D M S O M C O y , 4 3 where the bridging D M S O is necessarily both S- and O-bound. The S-0 bond lengths for the bridging D M S O are 1.532(4) and 1.508(5) A, respectively, which are closer to the S-O bond lengths of O-bound than S-bound sulfoxide (Section 3.2.2). The S-O bond lengths for the S-bound D M S O in the complexes range from 1.442 to 1.486 A as expected (Section 3.2.1). A trinuclear complex [Ru 3 (p -MPSO-S ,0 ) 2 Gu-Cl ) 4 (MPSO-S) 4 Cl 2 ] ( M P S O = methyl phenyl 9 44 sulfoxide) with bridging M P S O has an S-O bond length for the ^u-MPSO of 1.507(5) A. Complexes of Sn, Cu , and Pt with bridging disulfoxides have also been synthesized. Carvalho et al. characterized catena-poly{cis-Cl2-tran5-(CH3) 2Sn(rV)](jU-0,0'-meso-BPSE) which has two O-bound B P S E ligands at one Sn centre and each bridges another Sn centre (Figure 3.7). The average S-O bond length is 1.520(3) A, which is consistent with O-bound 45 sulfoxide. Filgueiras et al. have characterized another Sn complex with a bridging disulfoxide, [SnCl-cw-Ph3]2(^-0,0'-rac-l,2-bis(n-propylsulfinyl)ethylene), where the 46 disulfoxide bridges two Sn centres. The structure shows the sulfoxide to be O-bound with an S-O bond length of 1.488(6) A, which is closer to an S-O bond length for S-bound sulfoxide. Geremia et al. have characterized a Cu structure of the formula [Cu(BPSP) 2 (C10 4 ) ] n n + with the Cu-atoms bridged by O-bound B P S P ; the S-O bond length is 5 47 1.533(5) A as expected for O-bound sulfoxide. Two structures with bridging meso-BPhSE have been reported. The first, [SnClPh 3 ] 2 ( i u-BPhSE), was characterized by Zhu et al. and has 19 an S-O bond length of 1.525(4) A , supporting the observed O-bound sulfoxides. The 59 References on page 75 Chapter 3 second is [PtCl 2(PEt 3)] 2(At-S,S meso-BPhSE), shown in Figure 3.7; this has S-bound o 48 sulfoxides and an S-O bond length of 1.475(9) A , consistent with the S-bound sulfoxides. C l + C H 2 S Pr Pr E t 3 P — P t — C l O I II P h — S — C H 2 — C H 2 — S Ph CP J n O C l — P t — P E t 3 Cl (1) (2) Figure 3.8 The structures of catena-poly{cz'i'-Cl2-rran5-(CH 3) 2Sn(IV)]( Ja-0,0'-me50-BPSE) and [PtCl 2 (PEt 3 ) ] 2 ( / i -S,S meso-BPhSE). In this thesis work, the bridging B P h S E complex [RuCl 3 (BPhSE)] 2 ( J u-BPhSE)x H 2 0 was isolated; IR data suggest the sulfoxide is S-bound (Section 3.5.1). As well , two p-cymene complexes with S-bound bridging sulfoxides were synthesized: [RuCl 2 (p-cymene)] 2( )u-BESE) with S-bound sulfoxides has been structurally characterized, and the analogous complex [RuCl 2(p-cymene)] 2( iu-BESP) was also made (Section 3.6.1). 3.5.1 [RuCl3(BPhSE)]2(lA-BPhSE)x H20 [RuCl 3 (BPhSE)] 2 Gu-BPhSE)-x H 2 0 was prepared by various methods (Section 2.7.1). The 1 H - N M R data show a paramagnetic complex, and repeat elemental analyses support the formulation shown with one or two water molecules. The IR stretches 1070, 1082, 1105, and 1116 cm"1 suggest S-bound sulfoxide when compared with the values 32 for free B P h S E (1035, 1089 cm"1), and no bands are seen in the region of O-bound sulfoxide. The mass spectrum shows peaks at 1142 [ M + - 3 C l ] , and 972 [ M + - BPhSE] . The 60 References on page 75 Chapter 3 complex was non-conducting in C H 2 C 1 2 (0.6 i2" 'cm 2 mor 1 ) , 4 9 while the yellow solid dissolved in D M S O to give f rans-RuCl 2 (DMSO) 4 as shown by 1 H - N M R data. 3.6 Introduction to Rutheniump-Cymene Complexes; p-cymene = p-isopropyltoluene Ruthenium arene complexes have shown promising results in catalytic hydrogenation of olefins, ketones, and arenes, and such complexes with ancillary chiral chelating 50 diphosphine ligands have been studied as potential asymmetric hydrogenation catalysts. Mashima et al. have structurally characterized a cationic, dinuclear Ru(IJ) complex triply-bridged by the S-atoms of benzenethiolate ligands ([Ru 2(SPh) 3(p-cymene) 2] +). 5 1 A Ru(p-cymene) complex with a chelating sulfoxide carboxylate ligand, [Ru(p-cymene)(L)Cl], where L = (/?)-2-[(i?)-phenylsulfinyl]propionate that binds as shown in Figure 3.9, has been 52 synthesized and structurally characterized for catalytic use. The S-O bond length of o 1.478(2) A is as expected for the S-bound sulfoxide. 61 References on page 75 Chapter 3 Figure 3.9 Structures of (1) (/?)-2-[(R)-phenylsulfinyl]propionate, indicating the stereogenic centres (*), and the metal binding sites (—>) (adapted from ref. 52), and (2) [Ru(ade)(ri6-/?-cymene)]4(CF3S03)4 (taken from ref. 53). 6 5 3 6 Other arene complexes such as [(r| -CeFf^RuC^metro)] and [RuCl 2 (r | -54 C6H 6 ) (DMSO)] have been synthesized and studied for topoisomerase II activity and D N A damage ability, respectively. Korn and Sheldrick have shown that reaction of [RuCl 2 (p-cymene)] 2 Gu-Cl 2) with adenine in the presence of Ag(CF 3 S03) forms [Ru(ade)(r|6-/7-cymene)] 4(CF 3S03)4 (Figure 3.9) that shows the ability of Ru(p-cymene) complexes to interact with D N A bases. 5 5 Most recently, and after the jU-BESE complex had been synthesized (see below), [Ru(r|6-/?-cymene)Cl2(pta)] has been structurally characterized and 56 shown to exhibit pH-dependent DNA-binding (Chapter 1). A s well , half-sandwich Ru(U) arene complexes containing nitrogen ligands have been patented for the treatment of cancer. 57 In this thesis work, novel [RuCl 2(p-cymene)] 2(/i-disulfoxide) complexes (disulfoxide = B E S E and B E S P ) were synthesized and characterized. The complex [RuCl 2 (p-62 References on page 75 Chapter 3 cymene) ]2( jU-BESE) is the first structurally characterized bridging disulfoxide R u species known. Both disulfoxide bridged complexes are water-soluble, as is the starting material, [RuCl(/7-cymene)] 2 (^-Cl) 2 , which in aqueous solution becomes {[Ru(p-58 cymene)](/ i-Cl) 3 }Cl. A third water-soluble complex [RuCl(p-cymene)(BESE)]PF 6 was also synthesized and structurally characterized. 3.6.1 [RuCl2(p-cymene)]2(/J.-BESE) and [RuCl2(p-cymene)]2(/a-BESP) [RuCl2Gj?-cymene)]2(jU-BESE) was synthesized by reacting [RuCl(p-cymene)] 2( iti-Cl) 2 with B E S E in C H 2 C 1 2 under N 2 . The precipitated red complex needed no purification. IR bands at 1082 and 1110 cm"1 support S-bound sulfoxide. Crystals of the complex suitable for X-ray analysis were grown by slow evaporation of a C H 2 C 1 2 solution of the complex. The O R T E P diagram (Figure 3.10) shows bridging S-bound meso-BESE with 5-chirality at S ( l ) and 7?-chirality at S(2). The r|6-/?-cymene ligands (one at each Ru) are syn with the isopropyl groups pointing in different directions. Selected bond lengths and bond angles of [RuCl 2 (p-cymene)] 2 ( / i-BESE), [RuCl(p-cymene)(BESE)]PF 6 , and [PtCl 2(PEt 3)] 2(A*-S,S meso-BPhSE) are given in Table 3.4. The S-0 bond lengths for the title complex (1.476(2) and 1.483(2) A) are consistent with S-bound sulfoxide, and are similar to the S-0 length of 1.475(9) A seen in [PtCl 2 (PEt 3 )] 2 (^-S,S meso-BPhSE). The Ru-S bond lengths of 2.335(7) and 2.345(7) A are just shorter than the sum of the covalent radii of Ru o 1 and S (2.37 A ) , suggesting very little 7t-back-donation between the Ru- and S-atoms. The sulfoxide has a distorted trigonal pyramid geometry with C-S-C angles of 100.2 and 102.5(1)°, and C-S-O angles of 103.9 - 108.4(1)° 63 References on page 75 Chapter 3 C(7) R U ( I ; C(3) C(4) C(ll) C(5) Cl(2) C(12) C(13) C(10) Figure 3.10 A molecular structure representation (ORTEP) of [RuCl 2(p-cymene)]( iu-BESE) with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity (a stereoview and crystal data are given in Appendix 2). 64 References on page 75 Chapter 3 Table 3.4 Selected Bond Lengths (A) and Bond Angles (°) for [ R u C l ^ - c y m e n e ) ] ^ -B E S E ) , [RuCl(p-cymene)(BESE)]PF 6 , and [PtCl 2 (PEt 3 ) ] 2 i>S,S meso-BPhSE) Bond or Angle [RuCl 2 (p-cymene)] 2 (>BESE} [RuCl(p-cymene)(BESE)]PF 6a [PtCl 2 (PEt 3 ) ] 2 i>S,S meso-BPhSEJ0 S-O 1.476, 1.483(2) 1.461, 1.467(3) 1.475(9) S-C 1.801 - 1.829(3) 1.791 - 1.807(4) 1.78- 1.79(1) R u - C l 2.402, 2.414(7) 2.385(1) -Ru-S 2.335, 2.345 (7) 2.288, 2.302(1) -C - S - 0 103.9- 108.4(1) 107.0- 109.1(2) 107.2-110.4(5) C-S-C 100.2, 102.5(1) 101.0, 103.3(2) 97.9(5) Cl-Ru-S 85.70 - 86.23(3) 87.37, 89.40(4) -Ru-S-0 115.4(1), 114.15(9) 115.3, 118.1(1) -a Data are taken from the structural conformer shown in Figure 3.15 (see below), although the angles and bonds are very similar for both conformers. b The schematic structure is shown in Figure 3.7, and the data are taken from ref. 48. The ' H - N M R spectrum for [RuCl 2 (p-cymene)](jU -BESE), in C D C 1 3 at r.t., gives distinct peaks at 8 1.26 (d), 2.14 (s), 2.90 (sp), 5.31 (d), and 5.44 (d) which correspond to those of the p-cymene precursor material [RuCl(p-cymene)] 2( iu-Cl) 2. There are also downfield shifted peaks, corresponding to the coordinated p-cymene protons of the p - B E S E complex. The doublet at 8 1.28 represents the C H 3 protons (5) of the isopropyl group on the p-cymene, and the septet at 8 3.05 corresponds to the C H proton (4) of the isopropyl group (Figure 3.11). The aromatic protons (3, 2) are seen as broad singlets at 8 5.55 and 5.63, respectively, and the singlet at 8 2.27 is due to the C H 3 group (1) on the p-cymene. 65 References on page 75 Chapter 3 1-5 Figure 3.11 The structure of p-cymene showing the ^ - N M R spectra designations. 1 = methyl group protons (s), 2, 3 = aromatic protons (d), 4 = C H proton of the isopropyl group (sp), 5 = methyl protons of the isopropyl group (d). The peak at 8 1.36 (t) is assigned to the C H 3 groups on the B E S E ligand; however, it is unclear whether this is for free or bridging B E S E because only one triplet is seen; the two 'expected' triplets may be superimposed. The broad peaks at 8 2.82 (bs), and 3.21(bs) are difficult to assign, but likely correspond to the C H 2 protons of bridged or free B E S E . Figure 3.12 shows the variable temperature ' H - N M R spectra for the region 8 2.0 -4.5. Of note, only one triplet at ~ 8 1.36 is seen, at all the temperatures shown, for the C H 3 region of the B E S E suggesting, as mentioned above, that the signals for free and bridging B E S E are superimposed. As the solution is cooled to 243 K the singlet at 8 2.27 for the fx-B E S E complex becomes larger and the peak for the R u precursor diminishes. The peaks in the 8 2.5 - 4.0 region also become more resolved and the doublets seen at 8 3.35 and 4.05 are assigned to the diastereotopic C H 2 protons on the ligand backbone of ^u-BESE. The multiplets at 8 2.75 and 3.62 are assigned to the C H 2 groups on the arms of the ligand. 66 References on page 75 Chapter 3 2 7 3 k 2 8 3 k 3 1 3 K 4 . 0 3 . 5 3 . 0 2 . 5 2 . 0 ppm Figure 3.12 The ' H - N M R spectra for [RuCl 2(p-cymene)] 2(//-BESE) in C D C 1 3 at various temperatures from 243 to 313 K , showing that as the temperature decreases the 1 H - N M R signals become more resolved. Adding one equivalent of B E S E to [RuCl 2(y>cymene)] 2(//-BESE) in C D C 1 3 does not region from 8 2.0 - 6.0 is shown), adding two equivalents of B E S E causes the peaks corresponding to the / ^ - B E S E complex to become more predominant. This is exemplified by the singlet at 8 2.27 and the broad signals at 8 5.55 and 5.63 increasing in intensity and the corresponding signals for the R u precursor decreasing. A t the same time, the peaks corresponding to those o f free B E S E , at 8 1.36 and from 5 2.90 to 3.30, intensify. change the r.t. H - N M R spectrum significantly. However, as shown in Figure 3.13 (only the 67 References on page 75 Chapter 3 JUl 5.5 -i | r-5 . 0 4 • 5 4.0 3.5 3.0 2.5 2.0 ppm Figure 3.13 The r.t. ' H - N M R spectra in C D C 1 3 for (A) [RuCl 2(p-cymene)] 2(//-BESE) with one equivalent of B E S E , (B) with 2 equivalents of B E S E , and (C) with excess B E S E . The data suggest equilibrium behaviour such as in eq. 3 for [RuCl 2 (p-cymene)] 2 0 B E S E ) in C D C 1 3 . The 1 H - N M R experiments indicate that adding B E S E and lowering the temperature favour the left hand side of the reaction. CDC13 [RuCl 2(p-cymene)] 2(//-BESE) ^==* [RuCl(p-cymene)] 20-Cl) 2 + B E S E (3) The [RuCl(p-cymene)] 2(//-Cl) 2 starting material for the synthesis of [RuCl 2 (p-cymene)] 2 (//-BESE) dissolves in water to form the cationic complex {[Ru(p-cymene)]2(//-C1)3}C1. The ' H N M R spectrum in D 2 0 shows a doublet at 8 1.19 and septet at 6 2.73 for the p-cymene protons 5 and 4, respectively. A singlet at 8 2.06 corresponds to the /?-cymene protons 1, and the doublets at 5 5.48 and 5.70 represent the aromatic protons 3 and 2, respectively. There are also small signals shifted slightly downfield within each of the p-cymene proton regions which suggests a small amount of another isomer. The proposed 68 References on page 75 Chapter 3 aqueous solution behaviour for [RuCl 2 (p-cymene)] 2 (//-BESE) is presented in Figure 3.14; the ' H - N M R suggests the complex may dissociate to {[Ru(p-cymene)] 2(>Cl)3}Cl and B E S E before forming the cationic complex [RuCl(j>cymene)(BESE)]Cl. CH2C12, N 2 [RuCl(p-cymene)]2(//-Cl)2 + BESE < > [RuCl2(/7-cymene)]2(//-BESE) H 20 \ / H 20 {[Ru(p-cymene)]2Cu-Cl)3}Cl + BESE — + ci — , / + PF6 N H 4 P F 6 O^S Ru — c i ' w o o=;s— R " — c i o Figure 3.14 The proposed aqueous solution behaviour of [RuCl 2 (p-cymene)] 2 (>-BESE) and the subsequent formation of [RuCl(p-cymene)(BESE)]PF 6 with the addition of N H 4 P F 6 . When [RuCl 2 (p-cymene)] 2 ( / a-BESE) is dissolved in H 2 0 , the ' H N M R shows signals at 8 1.17 (t), 2.84 (m), 3.13 (m) which are those for free B E S E ligand. A s well , observed peaks at 8 1.07 (d), 1.40 (t), 2.06 (s), 2.71 (sp), 3.40-3.65 (m), 6.29 (s) correspond to those of [RuCl(p-cymene)(BESE)]Cl (cf. ' H - N M R data of the characterized [RuCl f> cymene)(BESE)]PF6 complex discussed in Section 3.6.2). Small peaks seen for {[RuOp-cymene)] 2(//-Cl) 3}Cl disappear with time. If [RuCl(p-cymene)] 2(/y-Cl) 2 is placed in water 69 References on page 75 Chapter 3 and then B E S E is added, the 1 H - N M R spectrum shows small peaks for free B E S E which disappear over time, peaks for [RuCl(p-cymene)(BESE)]Cl, and small peaks for {[Ru(p-cymene)]2(//-Cl)3}Cl. The water-soluble p - B E S E complex was tested in vitro for cytotoxicity on Chinese hamster ovarian cells and for anti-cancer activity on mammalian breast cancer cells (Chapter 4). [RuCl2(p-cymene)]2(/^-BESP) was prepared using the same method as for synthesis of [RuCi2(p-cymene)]2(//-BESE). The structure is assumed to be analogous to that of [RuCl 2 (p-cymene)] 2 (/ /-BESE). The ! H N M R spectra for [RuCl2(p-cymene)]2G"-BESP) shows peaks corresponding to the p-cymene protons of the R u precursor, and peaks for the complex at 5 1.29 (d), 1.34 (t), 2.28 (s), 2.80 (bs), 2.35 (m), 3.05 (m), 3.08 (sp), 5.55 (bs), 5.63 (bs); the 'extra' peak at 5 2.35 is due to the 'extra' C H 2 group on the backbone of the ligand. Several IR bands (1074, 1082, 1090, 1095, 1108, 1116, 1122 cm"1) suggest S-bound sulfoxide. A n attempted synthesis of [RuCl2(p-cymene)]2C"-BPhSE) using the same method noted above resulted in isolation of the starting materials. 3.6.2 [RuCl(p-cymene)(BESE)]PF6 [RuCl(p-cymene)(BESE)]PF 6 was synthesized by dissolving [RuCl(p-cymene)]2(//-Cl)2 and B E S E in water (under N2 or air) to give a yellow solution, and then adding NH4PF6, or by dissolving [RuCl2(p-cyrnene)]2(//-BESE) in water to give a yellow solution and adding NH4PF6. Isolation of the product in both cases was by crystallization from a concentrated aqueous solution of complex. X-ray quality crystals were grown from slow evaporation o f a 1:1 H20/MeOH solution of the complex. The O R T E P structures of the 70 References on page 75 Chapter 3 two conformers found in the asymmetric unit are shown in Figures 3.15 and 3.16. The structure in Figure 3.15 shows a cationic complex, with a PF6 counterion (omitted for clarity), with one S-bound meso-BESE (the chirality at S ( l ) is R and at S(2) is S) which is adjacent to the non-isopropyl CH3 group of the r)6-p-cymene. The other conformer (Figure 3.16) shows a similar structure with one maso-BESE (the chirality at S(3) is S and at S(4) is R) in close proximity to the isopropyl group. Selected bond lengths are shown in Table 3.4. The data for both conformers are very similar and therefore only those for the conformer shown in Figure 3.15 are discussed. The S-0 bond lengths of 1.461(3) and 1.467(3) A are consistent with S-bound sulfoxide. The Ru-S bond lengths of 2.288(1) and 2.302(1) A are shorter then those seen in the bridged [RuCl 2 ^-cymene)] 2 ( / / -BESE) complex indicating a higher degree o f 7t-back-bonding between the R u and the S-atom. The C-S-C angles are 101.2 and 103.3(2)°, and the C-S-O angles are 107.0-109.1(2)°, as expected for a distorted trigonal pyramid geometry about the S-atom. The 1 H - N M R spectrum in D 2 0 shows one triplet at 5 1.40 for the CH3 groups o f the B E S E revealing they are equivalent and indicating the molecule is fluctional in solution. A multiplet at 5 3.40-3.70 corresponds to all the C H 2 protons of the B E S E ligand, but cannot be further assigned. The doublet at 8 1.06 and the septet at 8 2.70 correspond to the p-cymene protons 4 and 3, respectively. The singlet at 8 2.06 corresponds to the p-cymene protons 1, and the singlet at 8 6.28 must correspond to the aromatic protons 2, yet it is not clear why a singlet is seen. A singlet was also seen for the aromatic protons of p-cymene complexes of the formula RuX 2 (p-cymene)(A) ( X = C l , A = P B u n 3 , P M e 2 P h , A s M e 2 P h ; and X = B r or I, A 58 = PBu" 3 ) . The IR data show stretching frequencies at 1072, 1090, 1107, 1119, 1132, 1142 cm"1 consistent with the S-bound sulfoxide; the number of IR bands seen must be due to the 71 References on page 75 Chapter 3 presence of two conformers in the solid state. The conductivity of [RuCl(p-cymene)(BESE)]PF 6 in H 2 0 is 75 Q^cir^moT 1 which corresponds to a 1:1 electrolyte in aqueous solution. Attempts to synthesize and isolate the chloride anion form o f the complex were unsuccessful; however, it has been identified in situ (Section 3.6.1). 72 References on page 75 Chapter 3 Figure 3.15 A molecular structure representation (ORTEP) of one conformation of [RuCl(p-cymene)(BESE)] + with 50% probability thermal.ellipsoids shown; H-atoms are omitted for clarity (a stereoview and crystal data are given in Appendix 3). 73 References on page 75 Chapter 3 C(19) Figure 3.16 A molecular structure representation (ORTEP) of a second conformation of [RuCl(p-cymene)(BESE)] + with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity (a stereoview and crystal data are given in Appendix 3). 74 References on page 75 Chapter 3 References Davies, J. A . Adv. Inorg. Chern. Radiochem. 1981, 24, 115. Calligaris, M . ; Carugo, O. Coord. Chern. Rev. 1996,153, 83. Sze, K . FL; Brion, C. E . ; Tronc, M . ; Bodeur, S.; Hitchcock, A . P. Chem. Phys. 1988, 121, 279. Viswamitra, M . A . ; Kannan, K . K . Nature 1966, 209, 1016. Thomas, R.; Shoemaker, C. B . ; Eriks, K . Acta Cryst. 1966, 21, 12. Bennett, M . J.; Cotton, F. A . ; Weaver, D . L . ; Will iams, R. J.; Watson, W . H . Acta. Cryst. 1967, 23, 788. Pearson, R. G . J. Am. Chem. Soc. 1963, 85, 3533. Farrell, N . A C S Symposium Series; ed. Lippard, S. J., 1983; V o l . 209; p 279. Turco, A . ; Pecile, C. Nature 1961,191, 66. Barnes, J. R.; Goodfellow, R. J. Chem. Res., Miniprint 1979, 4301. McArdle , J. V . ; Schultz, A . J.; Corden, B . J.; Eisenberg, R. Inorg. Chem. 1973,12, 1676. Mercer, A . ; Trotter, J. J. Chem. Soc. Dalton Trans 1975, 2480. Yapp, D . T. T.; Jaswal, J. S.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chim. Acta 1990,177, 199. Geremia, S.; Vicentini, L . ; Calligaris, M . Inorg. Chem. 1998, 37, 4094. Hul l , M . ; Bargar, T. W . J. Org. Chem. 1975, 40, 3152. 75 References on page 75 Chapter 3 (16) Taddei, F. Boll. Sci. Fac. Chim. Ind. Bologna 1968, 26, 107; through ref. 45 in Ch. 3 of E . L . S. Cheu's Ph. D . Dissertation (ref. 32). (17) Colonna, S., Personal communication to B . R. James; through ref. 46 in Ch . 3 of E. L . S. Cheu's Ph. D . Dissertation (ref. 32). (18) Araujo, C. S.; Khiar, N . ; Huxham, L . ; James, B . R. Unpublished data, on-going collaboration. 2001. (19) Zhu, F. C ; Shao, P. X . ; Yao, X . K . ; Wang, R. J.; Wang, H . G . Inorg. Chim. Acta 1990,171, 85. (20) Madan, S. K . ; Hu l l , C . M . ; Herman, L . J. Inorg. Chern. 1968, 7, 491. (21) Zipp, A . P.; Madan, S. K . Inorg. Chim. Acta 1977, 22, 49, and references therein. (22) Musgrave, T. R.; Kent, G . D . J. Coord. Chern. 1972, 2, 23. (23) Khiar, N . ; Fernandez, I.; Alcudia, F. Tetrahedron Lett. 1993, 34, 123. (24) Tokunoh, R.; Sodeoka, M . ; Aoe, K . ; Shibasaki, M . Tetrahedron Lett. 1995, 36, 8035. (25) James, B . R.; Morris, R. H . ; Reimer, K . J. Can. J. Chern. 1977, 55, 2353. (26) James, B . R.; M c M i l l a n , R. S. Can. J. Chern. 1977, 55, 3927. (27) Alessio, E . ; Balducci, G . ; Lutman, A . ; Mestroni, G . ; Calligaris, M . ; Attia, W. M . Inorg. Chim. Acta 1993, 203, 205. (28) Chan, P. K . L . ; Chan, P. K . H . ; Frost, D . C ; James, B . R.; Skov, K . A . Can. J. Chern. 1988, 66, 117. (29) Chan, P. K . L . ; James, B . R.; Frost, D . C ; Chan, P. K . H . ; Hu , H . - L . ; Skov, K . A . Can. J. Chern. 1989, 67, 508. (30) Yapp, D . T. T. Ph. D . Dissertation, University of British Columbia, Vancouver, 1993. (31) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chern. 1997, 36, 5635. 76 References on page 75 Chapter 3 (32) Cheu, E . L . S. Ph. D . Dissertation, University of British Columbia, Vancouver, 2000. (33) Evans, I. P.; Spencer, A . ; Wilkinson, G. Chem. Soc., Dalton Trans. 1973, 204. (34) Alessio, E . ; Mestroni, G . ; Nardin, G. ; Attia, W . M . ; Calligaris, M . ; Sava, G. ; Zor'zet, S. Inorg. Chem. 1988, 27, 4099. (35) Huheey, J. E . Inorganic Chemistry (4th ed.): New York, 1993. (36) Jaswal, J. S.; Rettig, S. J.; James, B . R. Can. J. Chem. 1990, 68, 1808. (37) Ledlie, M . A . ; A l l u m , K . G . ; Howell , I. V . ; Pitkethley, C. R. J. Chem. Soc. Perkin I 1976, 1734. (38) Lucas, C. R.; L i u , S.; Newlands, M . J.; Gabe, E . J. Can. J. Chem. 1990, 68, 1357. (39) Hartley, F. R.; Murray, S. G. ; levason, W. ; Soutter, H . E . ; McAul i f fe , C . A . Inorg. Chim. Acta 1979, 35, 265. (40) Murray, S. G . ; Levason, W. ; Tuttlebee, H . E . Inorg. Chim. Acta 1981, 51, 185. (41) Schenk, W . A . ; Frisch, J.; Durr, M . ; Burzlaff, N . ; Stalke, D . ; Fleischer, R.; Adam, W.; Prechtl, F. ; Smerz, A . Inorg. Chem. 1997, 36, 2372. (42) Tanase, T.; A i k o , T.; Yamamoto, Y . Chem. Commun. 1996, 2341. (43) Geremia, S.; Mestroni, S.; Calligaris, M . ; Alessio, E . J. Chem. Soc. Dalton Trans. 1998, 2447. (44) Lessing, S. F.; Lotz, S.; Roos, H . M . ; van Rooyen, P. H . J. Chem. Soc, Dalton Trans 1999, 1499. (45) Carvalho, C. C ; Francisco, R. H . P.; Gambardella, M . T.; Sousa, G . F. ; Filgueiras, C. A . L . Acta Cryst. 1996, C52, 1627. (46) Filgueiras, C. A . L . ; Holland, P. R.; Johnson, B . F. G . ; Raithby, P. R. Acta Cryst. 1982,535, 2684. 77 References on page 75 Chapter 3 (47) Geremia, S.; Calligaris, M . ; Mestroni, S. Inorg. Chim. Acta 1999, 292, 144. (48) Francisco, R. H . P.; Gambardella, M . T. P.; Rodrigues, A . M . D . D . ; de Souza, G . F.; Filgueiras, C. A . L . Acta Cryst. 1995, C51, 604. (49) Geary, W . J. Coord. Chern. Rev. 1971, 7, 81. (50) Fogg, D . E . ; James, B . R. J. Organomet. Chern. 1993, 462, C21-C23, and references therein. (51) Mashima, K . ; Mikami , A . ; Nakamura, A . Chern. Lett. 1992, 1795. (52) Otto, M . ; Parr, J.; Slawin, A . M . Z . Organometallics 1998,17, 4527. (53) Dale, L . ; Tocher, J. H . ; Dyson, T. M . ; Edwards, D . I.; Tocher, D . A . Anti-Cancer Drug Design 1992, 7, 3. (54) Vashisht Gopal, Y . N . ; Jayaraju, D . ; Kondapi, A . K . Biochemistry 1999, 38, 4382. (55) Korn, S.; Sheldrick, W . Inorg. Chim. Acta 1997, 254, 85. (56) Allardyce, C. S.; Dyson, P. J.; El l is , D . J.; Heath, S. L . Chern. Commun. 2001, 1396. (57) Morris, R. E . ; Sadler, P. J.; Chen, H . ; Jodrell, D . International Publication Number W O 01/30790 A l , 2001. (58) Bennett, M . A . ; Smith, A . K . J. Chern. Soc, Dalton Trans. 1974, 233. 78 References on page 75 Chapter 4 Chapter 4 Preliminary In Vitro Biological Studies of Water-soluble Ruthenium Sulfoxide Complexes 4.1 Introduction As outlined in Chapter 1, various Ru(U) and (HI) D M S O complexes exhibit anti-cancer activity but are relatively non-toxic at high concentrations.' Specifically, Cis- and 2-4 frarcs-RuCl 2 (DMSO) 4 as well as certain [RuCl4(DMSO)(Im)] complexes have shown 5,6 appreciable anti-metastatic properties (Section 1.3.1). Several arene complexes such as [RuCl 2(r | 6-C6H 6)(metro)], 7 [RuCl 2 (r] 6 -8 9 C 6 H 6 ) ( D M S O ) ] , and [Ru(r)6-p-cymene)Cl2(pta)] have also been studied for biological activity (Section 1.3.4). As well, half-sandwich Ru(U) arene complexes containing nitrogen 10 ligands have been synthesized for the treatment of cancer, and one of these complexes, [RuCl(r ] 6 -p-cymene)(N,Af ' -H 2 NCH 2 CH 2 NH 2 ) ] + , has shown anti-cancer activity against a n human ovarian cancer cell line (Section 4.7.2). Previous work in this laboratory by Yapp et al. has suggested that trans-RuCl 2 (disulfoxide) 2 complexes accumulate in cells and bind to D N A to a greater degree than 12,13 the cis isomers, while work by Cheu suggests that [RuCl(disulfoxide)(H 20)] 2(/^-Cl) 2 (disulfoxide = B E S E , B P S E , B B S E ) and [RuCl(BPSP)] 2 ( y u-Cl) 3 accumulate in cells, and bind to D N A to a higher degree (-20 times more) than the mononuclear D M S O complexes and 14 -270 times more than the bis(disulfoxide) complexes. A l l the disulfoxide complexes tested exhibited no significant toxicity toward Chinese hamster ovarian (CHO) cells. Two 79 References on page 96 Chapter 4 complexes synthesized in this thesis work, c z ' s - R u C l ^ D M S O ^ B E S E ) and [RuCl 2 (p-cymene)] 2 (jU-BESE), were tested for anti-cancer activity using the M T T assay, and for cytotoxicity using the C H O toxicity assay. 4.1.1 The MTT Assay A need to determine the sensitivity of specific tumors and individualize therapy has led to the development of a number of in vitro assays, and the M T T assay, which measures mitochondrial dehydrogenase activity as a reflection of cell viability, shows considerable promise in the screening and evaluation of potential new anti-cancer agents.1 5 Al ley et al. have noted, in a feasibility study of the M T T assay, that "it appears suitable for initial-stage 16 in vitro drug screening". The assay is quick, taking 4-5 days, and involves addition of M T T to tumor cells incubated with a test complex. The yellow tetrazolium form of M T T is reduced, in active cells, by mitochondrial dehydrogenases to form purple formazan crystals (Figure 4.1). A colourimetric determination is then able to quantify the percentage of viable cells. 80 References on page 96 Chapter 4 (1) Tetrazolium (2) Formazan Figure 4.1 The structure of (1) M T T [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (yellow) and (2) the formazan metabolite (purple). 4.1.2 The CHO Toxicity Assay A cellular toxicity assay was used to test the viability of Chinese hamster ovarian cells after incubation for 2 h with a test complex. The assay measures the colony forming ability of the C H O cells (after 7 days) as a representation of cell viability and corresponding complex toxicity. 4.2 Exper imental M e d i a and Solutions M T T was purchased from Sigma. A l l media and solutions were purchased sterile, or were sterilized through 0.2 urn filters (150 m L flask: 33 mm neck, Corning), unless otherwise stated, and kept under sterile conditions before use. A l l bottles and pipette tips were sterilized in an autoclave. Sterile experiments were performed in laminar flow sterilization hoods, and equipment introduced into the hood was sprayed with 70 % ethanol. 81 References on page 96 Chapter 4 4.2.1 Media For the M T T assay, Dulbecco's Modified Eagle's medium (1 L , Stem Cel l Technologies) in l iquid form was supplemented with 10 % fetal bovine serum before use. Hank's balanced salt solution (Stem Cel l Technologies) was used for washing the cells before trypsinizing. For the C H O toxicity assay, an a-modification of Eagle's minimum essential medium powder (Gibco) was used in all incubation and C H O cell handling procedures. The media were prepared by mixing M E M a-modification powder (1 L pkg), fetal bovine serum 10% (v/v, 100 mL), and 10,000 units penicillin/streptomycin antibiotic (Gibco) in 1 L of deionized water. The solution was then stirred for 2 h at r.t., and split into 2 portions of 500 mL. One portion was buffered with 10 m M H E P E S , the p H adjusted to 7.3 with 4 M (aq.) N a O H , and filtered into a sterile glass bottle to prepare the a +/- medium. To the other portion, N a H C 0 3 (1 g) was added, the p H adjusted to 7.3 with 4 M (aq.) N a O H , and the a+/+ medium filtered. A l l media were stored at 4 °C and warmed to 37 °C for use. 4.2.2 Phosphate Buffer Saline Solution (PBS) The P B S was prepared by dissolving N a C l (4 g), KC1 (100 mg), N a 2 H P 0 4 (575 mg ), and KH2PO4 (100 mg) in deionized water (500 mL). The solution was filtered, stored at 4 °C, and used at 37 °C, unless otherwise stated. 82 References on page 96 Chapter 4 4.2.3 Solutions of Ruthenium Complexes For the M T T assay, the R u complex was dissolved in P B S (1-10 mL, 4 °C), and the solution vortexed and left for 1 h before filtering through a 0.2 u,m needle filter (Nalgen). The stock solution of the complex was then serially diluted with a+/+ medium in 6-well plates (Falcon), according to prepared concentrations (Appendices A4.1 - A4.4), before being added to the 96-well plate (Falcon). For the C H O toxicity assay, the Ru complex was dissolved in P B S (10 mL, 4 °C) to make a 2 m M stock solution. The solutions were vortexed and left for 1 h before filtering through a 0.2 u,m needle filter and adding to the incubation flasks. Three concentrations were chosen based on the amount of ruthenium complex available; 0.1, 0.5, and 1.1 m M (Table 4.1, p.88). 4.2.4 Methylene Blue Solution Methylene blue (200 mg) was dissolved in distilled water (100 mL) and the solution was allowed to stand for 1 h prior to filtration. This solution was used for fixing and staining colonies after incubation to assess colony forming ability. 4.3 M T T Assay Procedure 4.3.1 Cell Preparation The cells used in the M T T assay experiments were obtained from a human mammary cell line M D A - M B - 4 3 5 s . The cell concentrations were determined using a hemacytometer and by diluting 50 U.L of cell suspension in 50 p L of trypan blue solution 0.4 % (Gibco) to 83 References on page 96 Chapter 4 stain dead cells. L ive cells were counted, the counts averaged and multiplied by the standard experimental formula for a hemacytometer (the dilution factor and 104) to obtain the number of cells/mL. The cells (1 x 105) were routinely maintained in T-75 flasks (Falcon) with medium (~ 20 mL) at 37 °C in a NAPCO water-jacketed C 0 2 incubator (Precision Scientific) under an atmosphere of 95 % air/5 % C 0 2 . The cells were trypsinized biweekly with 0.25 % trypsin-EDTA (5 m L , Gibco) at 37 °C for 3 - 4 min (or until the cells were in suspension), counted, and 1 x 105 were plated in a T-75 flask containing a+/+ medium (-20 mL). As a backup, in the event of contamination, 1 x 104 cells were plated in a Tr25 flask (Falcon). To obtain higher cell concentrations for experiments, 1 x 106 cells were plated in a T-75 flask with a+/+ medium (50 mL) and grown for several doubling times, changing the medium every few days. 4.3.2 MTT Cell Plating Procedu re and MTT Addition The general procedure of the M T T assay is outlined in Figure 4.2. For each complex tested, a 96-well plate was prepared by filling the six central wells of column C (control) with 1 x 104 cells in 100 u L of medium (100 p L of cell medium were taken from a cell suspension containing 1 x 105 cells/mL). This procedure was repeated for columns 1-8. Medium (200 pL) was then added to column B to serve as a blank. The outside wells of the plate were filled with sterile H 2 0 (200 pL) , and the plate was incubated for 24 h at 37 °C in a 95 % air/5 % C 0 2 incubator. After 24 h, the prepared stock concentration of Ru complex (Section 4.2.3) was serially diluted with medium, according to a drug dilution chart with concentrations chosen around the IC50 range of the complex (IC50 = concentration of drug at which 50 % of the cells die). The complex was then added to the 6 wells containing cells, 84 References on page 96 Chapter 4 starting with the lowest concentration in column 8, and ending with the highest concentration in column 1. 100 u L of medium were then added to column C and the plate was incubated for 72 h. After 69 h, the M T T (50 p,L of 2.5 mg/mL) was added to each experimental well in the plate, which was incubated for a further 3 h. The wells were then aspirated to remove all liquid, and D M S O (150 |xL) was added to each well to dissolve the formazan crystals. The plates were then vortexed and the proportion of formazan was quantified by absorbance readings at 570 nm using a Dynex Technologies spectrometer (96-well plate reader). 85 References on page 96 Chapter 4 II B C 1 2 3 4 5 6 7 8 Incubate for 24 h A d d Complex 96-Well Plate (10 x 15 cm) IV Incubate for 69 h III B C 1 2 3 4 5 6 7 8 Incubate for 3 h A d d M T T Figure 4.2 Design and Protocol of the M T T Assay. I. I I . III. IV. 100 u.L of medium containing 1 x 10 4 cells was added to the shaded wells of the columns labeled C (control) and 1-8. Next, 200 p L of medium was added to column B (blank). After 24 h, a stock solution of complex, in PBS , was serially diluted with medium and 100 u L of the lowest concentration was added to each shaded well in column 8. This was continued from lowest to highest concentration for columns 7 through 1,respectively, then 100 p i of medium was added to the 6 shaded wells in column C. After 69 h, 50 p L of M T T (2.5 mg/mL) was added to each shaded well (B, C and 1-8). 3 h later, the plate was aspirated, 150 p L of D M S O was added, and the plate was read on a Dynex Technologies spectrometer (96-well plate reader) at 570 nm. 86 References on page 96 Chapter 4 4.4 C H O Toxici ty Assay Procedure 4.4.1 Cell Preparation The cells used in all experiments were obtained from a Chinese hamster ovarian cell line, chosen for its rapid growth and high plating efficiency. The cells (1 x 105) were routinely maintained in T-25 flasks with oc+/+ medium (~ 5 m L ) at 37 °C in a 95 % air/5 % CO2 incubator. The cells were trypsinized three times a week with 0.05% trypsin-EDTA (1 mL, Gibco) at 37°C for 3 - 4 min, counted using a hemacytometer, and 1 x 10 5 cells were plated in a T-25 flask. As a backup, in the event of contamination, 1 x 10 4 cells were plated in a T-25 flask. For higher cell concentrations, 1 x 10 6 cells were plated in T-75 flasks with a+/+ medium (50 mL) and were grown for several doubling times with medium replacement every 2 days. 4.4.2 Cell Incubation procedure Prior to the day of the experiment, 1 x 10 6 cells were plated in each of 4 T-25 flasks with a+/+ medium (5 mL). The cells were incubated for 13-15 h at 37 °C in an incubator with 95 % air/5 % C 0 2 . After incubation, the medium was removed from the cells and aliquots of a+/- medium and complex were added to the flasks according to the outline given in Table 4.1. The cells were then incubated with the complex for 2 h. After incubation, the medium was decanted, the cells washed twice with PBS (2 mL) , and trypsinized with 0.05 % trypsin-EDTA (1 mL) . A s soon as the cells were resuspended ( 3 - 4 min), a+i- medium (9 mL) was added. 87 References on page 96 Chapter 4 Table 4.1 Aliquots of medium and Ru complex required for 0.1, 0.5, and 1.1 m M experimental concentrations. Flask Number 1 (control) 2 3 4 Volume of a +/-medium added (mL) 10 9.5 7.5 4.5 Volume of 2 m M stock complex added (mL) 0 0.5 2.5 5.5 Concentration of complex (mM) 0 0.1 0.5 1.1 4.4.3 Cell Toxicity Assay The toxicities of the complexes towards C H O cells under air were measured by observing the colony-forming ability of cells. Samples (1 mL) were taken from the incubation flasks (after incubation with the complex and resuspension of the cells, Section 4.4.2) and diluted immediately in fresh a+l- medium (9 mL, 4°C). This cell suspension was then counted to determine the number of cells, and aliquots (10, 100, and 1000 pi) were added to polypropylene tubes containing oc+/+ medium (5 mL) . This suspension was poured into Petri dishes (Falcon) that were then incubated for 7 days to allow cell colonies to form. After incubation, the oc+/+ medium was discarded, and the colonies stained with methylene blue solution for ~ 9 min. The stain was then decanted and the dishes rinsed carefully with cold water. The number of colonies per dish was counted and expressed as a plating efficiency (PE) [PE = number of colonies counted/number of cells plated]. 88 References on page 96 Chapter 4 4.5 M T T Assay Results The M T T assay colourimetrically determines the number of cells that are metabolically active after incubation with the test complex for 72 h. The growth inhibitory effect of the added complex is expressed as a percentage of the control processed at the same time [(OD of treated sample/OD of control) x 100]; the percentages were then graphed for each concentration tested in order to create a dosage effect curve, and the curves were then analyzed for the I C 5 0 . C / s - R u C l 2 ( D M S O ) 2 ( B E S E ) was found to have no toxicity toward breast cancer cells at < 1.0 m M and an I C 2 0 > 3 m M (Figure 4.3). However, [RuCl 2 (p-cymene)] 2( Ju-BESE) was found to have an I C 5 0 range of 0.345 - 0.360 m M (345 - 360 uM) (Figure 4.4). 89 References on page 96 Chapter 4 120 5 60 -to > 50-O 40 -30 • 20 • 10 -0 J 1 1 1 . , 1 — r -0.001 0.01 0.1 0.25 0.5 1 1.5 Concentration of RuCI2(DMSO)2(BESE) (mM) (1) 5 5oJ 3 4 0 -30 -2 0 -10 -0 i 1 1 1 — r n 1 — r 0.1 0.5 1 1.51.752 25 3 Ctoncentration of Rua2(DMSO)2(BESE) (mM) (2) Figure 4.3 The graphs for two M T T assay trials. Graph (1) Shows the cell viability after treatment with c « - R u C l 2 ( D M S O ) 2 ( B E S E ) at drug concentrations from 0.001 to 1.5 m M . Graph (2) shows the same treatment but for concentrations from 0.1 to 3 m M . The I C 2 0 is > 3 m M . 90 References on page 96 Chapter 4 Figure 4.4 The top graph (trial 1) shows the cell viability after treatment with [RuCl 2 (p-cymene)] 2(p:-BESE) at drug concentrations from 0.0001 - 1.5 m M . The inset (trial 2) shows increased detail in the concentration range of the I C 5 0 from 0 .1 -1 m M . The I C 5 0 range is 345 - 360 u M . 91 References on page 96 Chapter 4 4.6 C H O Toxici ty Assay Results The preliminary C H O toxicity results, expressed as a percentage of the control, are shown in Figure 4.5 [Percent Viabili ty = (PE of the experimental concentration/PE of the control) x 100]. The results show the percent viabilities of cells treated with cis-R u C l 2 ( D M S O ) 2 ( B E S E ) were 73, 102, and 65 %, and for cells treated with RuC\2(p-cymene)] 2( iu-BESE) the values were 76, 85, and 69 % at concentrations of 0.1, 0.5, and 1.1 m M , respectively. However, the actual P E values are low, ranging from 0.32 to 0.49 for all treatments including the control (Figure 4.6); this suggests that some factor, other than the presence of the complex, is diminishing cell viability. O f note, these data represent two preliminary trials and further experiments are needed to determine definitively the cytotoxicity of these complexes. 1 2 0 0 0 . 1 0 . 5 1.1 C o n c e n t r a t i o n o f C o m p l e x (m M ) Figure 4.5 Cellular toxicity results expressed as a percentage of the control for all concentrations tested. The data for d s - R u C l 2 ( D M S O ) 2 ( B E S E ) ( • ) and for RuCl 2 (p-cymene)] 2( /u-BESE) ( • ) are averages of two experimental conditions (10| iL and 100p:L). 92 References on page 96 Chapter 4 0.6 0 0.1 0.5 1 .1 C o n c e n t r a t i o n of C o m p l e x ( m M ) Figure 4.6 Cellular toxicity results expressed as P E for all concentrations tested. The data for c w - R u C l 2 ( D M S O ) 2 ( B E S E ) ( • ) and for RuCl 2(p-cymene)] 2( iu-BESE) ( • ) are averages of two experimental conditions ( lOuL and lOOuL). 4.7 Toxici ty and Anti-cancer Act iv i ty of Ruthenium Sulfoxide Complexes 4.7.1 Cis-RuCl2(DMSO )2(BESE) The title complex at < 1.0 m M was found to have no toxicity toward breast cancer 17 cells, and an IC 2o > 3 m M (Figure 4.3). In comparison to cisplatin (IC50 = 10 p M ) , the complex is not an effective anti-cancer agent against this breast cancer cell line. However, the similarities in aqueous solution behaviour of this complex (Chapter 3, Section 3.3.1) with that of c / s -RuCl 2 (DMSO )4 indicate the mixed sulfoxide complex may have anti-cancer activity against other types of cancer and/or anti-metastatic activity. O f note, two complexes made by the Khiar group, R,R- and S,S-trans-RuC\2(BMSE)2, were tested using the same M T T assay procedure and cell line as reported in this thesis, and the IC50 ranges were 1.7-18 1.8 m M for both complexes. 93 References on page 96 Chapter 4 The preliminary C H O toxicity results suggest the title complex does not exhibit significant toxicity at or < 1.1 m M . The results indicate percent viabilities for treated cells are 73, 102, and 65 % at concentrations of 0.1, 0.5, and 1.1 m M , respectively. However, as mentioned, the actual P E values are < 0.50 for all treatments including the control and indicates a problem with the experimental design. These experiments involved trypsinizing the cells biweekly and after incubation of the cells with R u complex, unlike the previously 13,14 reported toxicity procedures that used C H O cell cultures maintained in suspension. Trypsin acts by breaking down the adherent cell membrane proteins which cause the cells to stick to the flask wall. If the trypsin is left on the cells too long, or i f the cells have been repeatedly trypsinized, the treatment wi l l affect the cell membrane properties and consequently the cell viability. For further studies, it is recommended that cell cultures in suspension be used. 4.7.2 [RuCl2(p-cymene)]2(/i-BESE) The complex [RuCl20p-cymene)] 2(/i-BESE) was tested using the M T T assay and was found to have an I C 5 0 range of 345 - 360 u M , showing effective anti-cancer activity when 17 compared with the corresponding IC50 of 10 p M found for cisplatin using this cell line. The preliminary C H O toxicity results suggest that the title complex does not exhibit significant toxicity at or < 1.1 m M with percent viability values of 76, 85, and 69 % at concentrations of 0.1, 0.5, and 1.1 m M , respectively. As mentioned in Section 4.7.1 there were problems with the experimental design, and further toxicity experiments should be performed. Of note, the cells in the flask containing [RuClaGp-cymene^Ou-BESE) would not responded to trypsin in the suggested 3 - 4 min incubation time and therefore a longer time 94 References on page 96 Chapter 4 and a higher dose of trypsin were needed to trypsinize the cells. The flask contents at 1.1 m M of complex would not respond to increased doses of trypsin even after 10 min so the cells were removed from the bottom of the flask with a plastic scraper. It has been shown by Geratz et al. that aromatic derivatives of diamidines can inhibit the activity of bovine 19 trypsin, and so it is possible that the aromatic-containing [RuCl2(p-cymene)] 2( Ju-BESE) complex may also inhibit trypsin activity. The in vitro activity of this complex is unknown, and the solution chemistry in a biological environment is uncertain. It is suggested that [RuCl 20>cymene)] 2(,u-BESE) in water gives [RuCl(/?-cymene)(BESE)]Cl (Chapter 3, Section 3.6.1), and this presumably occurs in P B S ; however, at this stage it is unknown whether this charged species enters the cell. Cummings et al. have shown that the similar cationic complex [RuCl(r| 6-/?-cymene)(N, N ' - H 2 N C H 2 C H 2 N H 2 ) ] + , when reacted with D N A , forms a mono adduct with n guanine. This and related complexes exhibit a range of IC50 values from 6 - 200 u M for a human ovarian cancer cell line compared with values of 0.8 u M for cisplatin and 6 p M for carboplatin. 1 1 Therefore, the active species of the bridged [RuCl 2(p-cymene)] 2( iu-BESE) complex may in fact be the [RuCl(p-cymene)(BESE)] + cation. Further research into the activity of this cationic complex should be explored as the isolated species may have increased activity at a lower dose than the parent compound. 95 References on page 96 Chapter 4 References (1) Farrell, N . In Transition Metal Complexes as Drugs and Chemotherapeutic Agents (eds. Ugo, R., James, B . R.); Kluwer Academic Publishers: Dordrecht, 1989; p 147. (2) Alessio, E . ; Mestroni, G . ; Nardin, G . ; Attia, W . M . ; Calligaris, M . ; Sava, G . ; Zorzet, S. Inorg. Chern. 1988, 27, 4099. (3) Sava, G . ; Zorzet, S.; Giraldi , T.; Mestroni, G . ; Zassinovich, G . Eur. J. Cancer Clin. Oncol. 1984, 20, 841. (4) Sava, G . ; Pacor, S.; Zorzet, S.; Alessio, E . ; Mestroni, G . Pharm. Res. 1989, 21, 617. (5) Alessio, E . ; Balducci, G . ; Lutman, A . ; Mestroni, G . ; Calligaris, M . ; Attia, W . M . Inorg. Chim. Acta 1993, 203, 205. (6) Clarke, M . J.; Zhu, F. ; Frasca, D . R. Chern. Rev. 1999, 99, 2511, and references therein. (7) Dale, L . ; Tocher, J. FL; Dyson, T. M . ; Edwards, D . I.; Tocher, D . A . Anti-Cancer Drug Design 1992, 7, 3. (8) Vashisht Gopal, Y . N . ; Jayaraju, D . ; Kondapi, A . K . Biochemistry 1999, 38, 4382. (9) Allardyce, C . S.; Dyson, P. J.; El l is , D . J.; Heath, S. L . Chern. Commun. 2001, 1396. (10) Morris, R. E . ; Sadler, P. J.; Chen, H . ; Jodrell, D . International Publication Number W O 01/30790 A 1,2001. (11) Cummings, J.; A i r d , R. E . ; Morris, R.; Chen, H . ; del Socorro Murdoch, P.; Sadler, P. J.; Smyth, J. F . ; Jodrell, D . I. Clin. Cancer Res. 2000, 6, Supp. (S) Nov, abstract 142, p 4494s. (12) Yapp, D . T. T. Ph. D . Dissertation, University of British Columbia, Vancouver, 1993. 96 References on page 96 Chapter 4 (13) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chem. 1997, 36, 5635. (14) Cheu, E . L . S. Ph. D . Dissertation, University of British Columbia, Vancouver, 2000. (15) Bellamy, W . T. Drugs 1992, 44, 690. (16) Alley, M . C ; Scudiero, D . A . ; Monks, A . ; Hursey, M . L . ; Czerwinski, M . J.; Fine, D . L . ; Abbott, B . J. ; Mayo, J. G . ; Shoemaker, R. H . ; Boyd, M . R. Cancer Res. 1988, 48, 589. (17) Sartor, J. ; Mayer, L . Unpublished data; through collaboration with the BC Cancer Research Centre, 2001. (18) Araujo, C . S.; Khiar, N . ; Huxham, L . ; James, B . R. Unpublished data, on-going collaboration. 2001. (19) Geratz, J. D . ; Cheng, M . C.-F. ; Tidwell , R. R. J. Med. Chem. 1976,19, 634. 97 References on page 96 Appendix 1 Appendix 1 Crystal Structure Data Al. 1 Crystal Structure Data for RuCl2(DMSO)2(BESE) Figure A l . l Stereoview of R u C l 2 ( D M S O ) 2 ( B E S E ) . 98 66 T 01 s O O I || a § c o d iii G s a .2 jj N c. i3 & c o C H D •5 < 3 W _ « to S i 5 £ cj -a O > j ! 6 d ? i: ir. oi a: to Cl § 5 a. 3 o p d K o c5 c E E £ <2 n 2 g " «? • O O N H A Ol II II II II -•0 U ^ E xipnaddy '3. £ 6 E fe 001 1/3 1-H •— oo - - ^ O ! . - ^ „ C 2. w 2. CN co >—< M IM CS i-i i— o —* n CM s CO CM n 00 57 o o O o a U O u O xipnaddy xipnaddy Appendix 2 Crystal Structure Data A2.1 Crystal Structure Data for [RuChtp-cymene)] 2(p.-BESE) Figure A2.1 Stereoview of [RuCl 2Op-cymene)] 2(/i-BESE). 103 SOI M i-t o <— n r-E o < • 10 o OO Ci o CM PI Tf m o co CJ < 0" < a m o —< CM CM CM CM CM CM" to r— 00* cn o u CJ CJ o cj CJ CJ o CJ O CJ X X X X X X X X X X X X o — — i — 10 O I o» oo T in >—' -H t-( io o o oo .-. to -4 l-H O E o < — = CM H « Di oi 2- fr oo o 5 O D 0 5 •7 o O o u O u o O O o o O O xipuaddy 901 — — <M (M CN + + « «-H in oo I C S O I-- ^ r- e-o r> « r- 10 oo , •W - V CO TJ- + s u ' i o en CQ < < ca T m CO to CM CN fN a X K CN ~ o o < o 2 < < CN m CM CQ rt u «! t o " H(20A) a a SS a a a a a a a a H(20A) m y CQ < <: a CJ < o o CN CN n CO •a* CN CN CN Cl CN CN CN X a 3 a a a a a a a xipnaddy LOl °°. ~ x6 t~ o o> o o CO 30 O i— —< t-1 i j . -3* i_i CM CN CM 3 3 ce" 3 . « Ru( 3 cc CM CM m ao CM "S 00 . CM to CM CO to o o u L> o o u o o o C CM 3 £» Q ^7 CM Tf I O C S — ' C l d O C l M 0 4 0 0 O t— G" OD u o o CM" CM* C*4 CM "a" c§ 3 CC "a-CC CO u u " o o U xipnaddy 801 CN — ' C N CM r^" CN — CM —• -— CO C l 0 4 « ri 0? U U O .— w -a1 o o o u r- 00 oo C4 to" CM D O O c o C C O O O ® "H" 2 S a cc CJ CJ ^ CM CM CJ t/) CS CC CM* ^f" CN CM? at rr CN* CN cj cj o CM ' o u cj" o cj CN —I u cj 00 *-i — o u u ' u I i £ S « cc; ct o OO U3 00 Cl 1- c> 00 00 CM t 3 CN CM o i CD o CO CN O CJ u CJ u u CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ o p CM CM CN CN CM CM CN CN p 3 3 3 3 « CC o i CC CC CC CS , a; £ * err CN" ?? U O u CJ CJ ' CJ j xipnaddy 3* is c o u o o U •- ' . — . CN "i5 C ^ . . . . . . ^ - . . . . . H . « O CJ 601 q — to 00 -H m m IO (O to lO m i.O ,-. r r OO 00 U3 LO iO o i o © . oi c i ci c i oi o i oi oi o i 00 ad 06 CO oi c i oi CN CM CM CM o o c o o O o o o o o o o a o o O O 3 U < s* S" 3 O s* CQ S* O LO CD OO 00 Cl o © CM CM CM CO CJ CO T T LO in to to CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM X X X X X X X X X X X X X X X X X • X ' 'x X X X X X rt   - i.O to 00 00 O) © o o CM CM CM CO CO CO iD m m to to 5 CM CM CM - CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM O CJ c j a CJ O CJ CJ CJ CJ CJ CJ CJ a a CJ CJ CJ CJ CJ CJ CJ CJ CJ T o pr CM CM CM CJ X o CJ CM V q to t-> 00 OI m L/5 lO to uO in m LO tn o —. 00 00 m vO "to c i c i co c i 00 Ci d o i oi o i c i Ci c i c i c i c i 00 ob od 00 oi c i an o CM CM f-. CM CM C o o o O O o o o O o o o o c o O < CO CM 3J o o o u o X o 00 Cl lO to q •o m V.*3 Cl m iO LO in in m r- CM t0 to in in in ho CO c i c i oi cc oi oi c i Oi r- c i oi Ci c i c i oi c i oi 0O 00 oo oi o i oi 3 CM CM © © O o o o o o o o o © o o o O . O © o l -CJ Ci CQ Ci _ ffl < < X CQ < X CM ;i2B) X < < £ CM CO* M* LO to to h- 00 Ci ;i2B) X X X X X X X X X X X X X X X X X X X X X X X X x o ' q o o o o o o o o o 0 0 0 0 0 0 0 0 0 0 0 o o 3 ^> s — S" O oo" DO O 5* O? (aoi: - S ^ n s- eg" 13B) CJ a: o C o o Sf sf o O o u O o X in O O O 3= xipuaddy Appendix 3 Appendix 3 Crys ta l Structure Data A3.1 Crystal Structure Data for [RuCl(p-cymene)(BESE)]PF6 Figure A3.1 Stereoview of [RuCl(p-cymene)(BESE)]PF 6 . I l l zu * '3 _ § S fa. "I g = a! -a S S £ 5 E 2 s 1 I 5 a •a o CL o u - E ~ t o & « in II II II a o . u o a S P o 2 SC a O d s & xipaaddy E I •a a 3 5 £11 xipnaddy r-< r - CM CM . - . t~- 1— T-1 < § f-T oo" cT CO c-^ GO CM r-H — — J— S CM cn CM i -£ CJ> 0 — "S o o u u u o o CM CQ CQ CQ CO CM CO u. xipuaddy 911 _^  co o CO T OO CN CO m OO OO CM CM CM CM CM ro Cu CJ O CJ CJ cj CJ CJ O u O O O CJ o co co i - cn CM CO r - h- Ch —' CM tM CM CM CM CM CU u O O a CJ CJ o CJ o CJ cj CM t i c JS g CM CM- o* T? Ch CO CM 5" o CM (O CM CM CM CM CO CO « u. CJ o CJ CJ CJ CJ U o a O CJ CJ CJ c o co OJ CO CO »o Os 5 CM r- 00 i— CM CM CM CM CM a a. o CJ CJ CJ U O u CJ CJ a U CJ o e p 3 « 3 M cc cc K cc C/] Cu & E ^ ^ ^ CM" ^ co us r - T ^ 3 ^ c j w u c j o 5 ? c j c j u o u c j o E c ^ a .— CM H * t f c d c e c s e £ c c ' c d e c - c d : ^ xipnaddy LU CO in 00 CO 00 OO 00 00 o O —i CN CN CM CN CN CN CN CN CN CN CN — — rs CN CN CN O u cj CJ cj CJ CJ CJ O CJ o CJ CJ U CJ CJ CJ o U CJ CJ CJ cj oo -rr CN CM O CJ 66.4(2! 36.5(2] 77.2(2] 36.0(2] 77.5(2] 65.7(2] 115.3(! i05.i(; 108.2(: ii8.i(: 114.7(: o t~ o 116.3C r~ CM C(25) t~ CM O to CM C C(28) oo CM y O o 0(2) C(5) C(5) 0(3) f*< CN >—I o o t-i CN lO o CN CN CN CN CN CN CN CN CN CO CJ CJ CJ CJ c£ o CC o cc O cC CC o s 3 £ H 3 re o u 0 u FT u O y y 3 cn O U 0O o CM" H 2 5' CM y O y y O y y y y y y CM CM • CM y y y E ~ c r ~ ~ _ _ _ _ _ ^ ~ — — ~ - -? ^ sr ff ^ « C J C J C J C J c/i cn - cn cn c"n ./T L O CJ O CJ CJ CJ CJ CJ O O O CJ cTf xipnaddy •811 a CO ST CM tu B CO b. s ca a co 5T u. 5T a ST ca ST OO « ST £T &. O S~ ffl Cu ST b. o ST •o o c o ti. CJ m CJ b, 57 ST . — ' co c^-" ao m CD OO .-I CJ cs ET S -m C J CJ CJ ca ca o C J CJ CO sr u . ti. a co 2- S. ST ST cj ST CO C O :4C) a C J 4B) ST ti. ti. b. — .-. ST ST ST K ST CJ CJ o CJ 6" C O ;3C) o C O fc L i . b. ti. tu m C J b. ffi CO ST ti. CQ ST C71 r~ EV> ST s < CJ CJ o CQ < CJ CJ CQ c a < CJ CJ ca ca o U a a CJ C O •ft C O >— C O CO c o co CJ CJ C N C J u . b . b . b . b . ti- ti. b , b . b . b . b . b . fcu ti. ti. b . ti. ti. b , b . b . b . b . o E 0 . 0. a, ti. c u ti. a. a. Cu CU ti. cu cu CU ft- a, CU cu Cu CU Cu Cu < < •< < < < < < < < < < < < < < < < < < < < CJ C J CJ C M CJ C N co C O C O C O C O CO TP TT b , ti. b . ti. ti. ti. b , ti. ti. ti. b . ti. ti. ti. b . ti. u . U . ti. b . b , ti. b . ti. eo — - — - ,—, ao 5 § i CO Ch CC r-. CO < < U CJ m a < CJ O a a CJ CJ a a CJ m a C N C J C N *c CO C J TT CN r-l C O C O CO C O CO b. b. b. ti. b. ti. b. b. [ L , ti- b. In b. b. ti. tu fcu ti. Su ti. b. ti. fcu ^ B < ' < < < < < < < < < < < : < < < < < < < < < < W ° C l w < J ^ " « C M C M C M C 4 C > l C N c O c O C O e O C O T l « ' T T r T l > xipaaddy 6 CM OS CJ CM — •—• CM "—• u t> u o xipuaddy OZl ^ C S P - i O T P O D a q T P O > t O O r ~ . O a q g o o o o o o o o o o o o o o o o o o o o o o o o CO to CO ao Cft CS CS CO LO to to p- cn Oi 0 CS CN CN cN CN CN CN CN CS 50 CO CO CO CO , CO CO co CO CO CO CO TP TP TP X EE X s* X X CC X X X X X ic X X X 3 3 ' X X X x X X X TP TP tf} UO L.O CO CO CO P- P- r- 00 oo 00 0 . Cft 0 . 0 0 CS - CN CS CS CS 0 0 0 0 O O u 0 0 0 U O O O O 0 0 0 0 0 U 0 0 E 2 5 CJ o CJ CN — K CJ CJ a en CJ C S 5" 0 a K o r -s s I S. rt X s s a o CL-d CJ CN w a cj 0 O O O U U O CO — o" CS CS CS CS CO X X X X x" co ^2. cTf O 0 es 0 CS CN CS CN CS CS CS CS U O r j 0 O 0 0 (19) TP CS CS (38) CS 0 " TP to O 0 X 0 X t c s c s f N c o c o c o T p T f in a U O U O O O O Q U O U CN CO CJ 0 0 a _ CS CO CN CO CJ a CO O in CN CO c. CJ a a CJ a! 0 5 2 CO O u CJ 2 2 s § £ S 2 S a CJ CJ CJ CJ a a a t o CO CO 0 u O 10 ID CJ CJ CM CO CO CJ 0 CJ C S «-f —v —f p- I*. I-l H c f l e o O X O X f f i ' - c r t O w O X m r j U O f f i C S O y o s O O O O xipnaddy f t cs m 00 Ch o O c C-l CS CS CS CS es cs cs CS es cs CO eO CO CO CO CO CO CO CO CJ CJ o o o U O CJ CJ CJ CJ U U U U U CS CS 00 CO CO OS •0 Oi o> CD lO CS CS CS CS CS CO CS TT CS iO CS »-o {§ CJ O D O CJ CJ X U X X O X X o to ao CO Tf CS oo c T P g oi- CS O) oi CO CN oi r— o oi O c i o s s oi O s oi O oi o s CS CO TT Tf Tf TP LO CO Tf (O Tf r- oo © lO o IO iO co to CO iO lO CO CO t.O X X X X X X X X X X X X X X X X X CS CS Tf CS to Cs es r -cs CO CS OO CS cn CS o CO CO CO CO Ci CS CO CS CO Cs CO O CJ O CJ CJ CJ O CJ CJ O CJ CJ U CJ CJ U CJ Tf CO CS CS o • cs CO CS es {--es o CO o CS Oi CS 00 Tf Ch CS OS-es CS CO es TP uO X O O CJ 0? CJ CJ CJ CJ X U O CJ X 3 ~ S. xipuaddy Appendix 4 Appendix 4 A4.1 M T T Drug Dilution Charts: First Trial with R u C l 2 ( D M S O ) 2 ( B E S E ) Table 1 Stock Solution Preparation Compound R u C l 2 ( D M S O ) 2 ( B E S E ) Molecular Weight (g/mol) 510.19 Stock Used (g) 0.005 Diluent PBS Diluent volume (mL) 3 Initial working concentration 3.27E-03 Total working volume 3 Amount per well (pL) 100 Dilution Factor (200 uL total/100 uL drug vol.) 2 Table 2 Serial Dilution Data Final cone. Volume of Working Volume of Diluent Volume remaining for (mM) Solution (mL) (medium, mL) Addit ion to M T T Plate (mL) 1.5 2.75 0.25 1.00 1 2.00 1.00 1.50 0.5 1.50 1.50 1.50 0.25 1.50 1.50 1.80 0.1 1.20 1.80 2.70 0.01 0.30 2.70 2.70 0.001 0.30 2.70 2.70 0.0001 0.30 2.70 3.00 122 Appendix 4 A4.2 M T T Drug Dilution Charts: Second Trial with R u C l 2 ( D M S O ) 2 ( B E S E ) Table 1 Stock Solution Preparation Compound R u C l 2 ( D M S O ) 2 ( B E S E ) Molecular Weight (g/mol) 510.19 Stock Used (g) 0.03 Diluent P B S Diluent volume (mL) 6 Initial working concentration 9.80E-03 Total working volume 8 Amount per well (pL) 100 Dilution Factor (200 u L total/100 uL drug vol.) 2 Table 2 Serial Dilution Data Final cone. Volume of Working Volume of Diluent Volume remaining for (mM) Solution (mL) (medium, mL) Addit ion to M T T Plate (mL) 3 4.90 3.10 1.33 2.5 6.67 1.33 1.60 2 6.40 1.60 1.00 1.75 7.00 1.00 1.14 1.5 6.86 1.14 2.67 1 5.33 2.67 4.00 0.5 4.00 4.00 6.40 0.1 1.60 6.40 8.00 123 Appendix 4 A4.3 M T T Drug Dilution Charts: First Trial with [RuCl 2(p-cyrnene)] 2(//-BESE) Table 1 Stock Solution Preparation Compound fRuCl 2 (p-cymene)l 2 (^-BESE) Molecular Weight (g/mol) 794.69 Stock Used (g) 0.0075 Diluent P B S Diluent volume (mL) 3 Initial working concentration 3.15E-03 Total working volume 3 Amount per well (pL) 100 Dilution Factor (200uL total/ lOOpX drug vol.) 2 Table 2 Serial Dilution Data Final cone. Volume of Working Volume of Diluent Volume remaining for (mM) Solution (mL) (medium, mL) Addition to M T T Plate (mL) 1.5 2.86 0.14 1,00 1 2.00 1.00 1.50 0.5 1.50 1.50 1.50 0.25 1.50 1.50 1.80 0.1 1.20 1.80 2.70 0.01 0.30 2.70 2.70 0.001 0.30 2.70 2.70 0.0001 0.30 2.70 3.00 124 Appendix 4 A4.4 M T T Drug Dilution Charts: Second Trial with [RuCl 2 (p-cymene)] 2 (^-BESE) Table 1 Stock Solution Preparation Compound [RuCl 2(p-cymene)l 2(/^-BESE) Molecular Weight (g/mol) 794.69 Stock Used (g) 0.015 Diluent P B S Diluent volume (mL) 5 Initial working concentration 3.78E-03 Total working volume 4 Amount per well (pL) 100 Dilution Factor (200uL total/ lOOuL drug vol.) 2 Table 2 Serial Dilution Data Final cone. Volume of Working Volume of Diluent Volume remaining for (mM) Solution (mL) (medium, mL) Addition to M T T Plate (mL) 1.5 3.17 0.83 1.33 1 2.67 1.33 1.00 0.75 3.00 1.00 1.33 0.5 2.67 1.33 1.20 0.35 2.80 1.20 1.14 0.25 2.86 1.14 2.40 0.1 1.60 2.40 3.60 0.01 0.40 3.60 4.00 125 

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