potential chemotherapeutic agents lynsey anne …

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.


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.


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.


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.


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


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 .


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.


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


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-


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


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


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.


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.


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.


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.


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.


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


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.


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


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).


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 .


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


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


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,


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'cir̂ mor1), increasing to a steady maximum value after 6 h): 102 f̂ 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.


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.


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


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 ,


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.


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


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.


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.


(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.


(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.


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


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


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


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


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


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.


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


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.


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 ) .


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.


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.


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 ) 2

b

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).


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).


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


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).


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)


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 (-).


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


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


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


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.


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


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


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.


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-


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)°


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).


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 6

a [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.


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.


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


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


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


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


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


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).


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).


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).


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,

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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.

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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.

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Barnes, J. R.; Goodfellow, R. J. Chem. Res., Miniprint 1979, 4301.

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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.


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.


(31) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chern. 1997, 36, 5635.


Chapter 3


(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.


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.


(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.


W O 01/30790 A l , 2001.

(58) Bennett, M . A . ; Smith, A . K . J. Chern. Soc, Dalton Trans. 1974, 233.


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


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.


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.


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.


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


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,


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).


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.


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.


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].


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).


Chapter 4

120

5 60 -to > 50-O 40 -

30 •

20 •

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0 J 1 1 1 . , 1 — r -

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10 -

0 i 1 1 1 — r n 1 — r

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(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 .


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 .


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).


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.


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


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.


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.


(9) Allardyce, C . S.; Dyson, P. J.; El l is , D . J.; Heath, S. L . Chern. Commun. 2001, 1396.


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.



Chapter 4

(13) Yapp, D . T. T.; Rettig, S. J.; James, B . R.; Skov, K . A . Inorg. Chem. 1997, 36, 5635.


(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.


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 ) .

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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).

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Appendix 3

Crys ta l Structure Data

A3.1 Crystal Structure Data for [RuCl(p-cymene)(BESE)]PF6

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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 )


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


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)


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


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)


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


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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