Approaches to Ru(II)-Co(III) Dinuclear Hypoxia-Selective Cytotoxins

Approaches to

Ruthenium(II)-Cobalt(III)

Dinuclear Hypoxia-Selective

Cytotoxins

A Dissertation

submitted in partial fulfilment

of the requirements for the degree

of

MChem in Chemistry at the University of

Southampton

by

Alexander Thomas Puttick

4-24890685

School of Chemistry

Southampton

Supervisors: Dr. Simon Gerrard

and

Dr. Jonathan Kitchen

2015

Chemotherapy isn't good for you. So when you feel bad, as I am feeling now, you

think, 'Well that is a good thing because it's supposed to be poison. If it's making the

tumor feel this queasy, then I'm OK with it.'

Christopher Hitchens

Approaches to Co(III)/Ru(II) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick

i

University of Southampton

Faculty of Engineering, Science and Mathematics School of Chemistry

MChem

Approaches to Ruthenium(II)-Cobalt(III) Dinuclear Hypoxia-Selective Cytotoxins

By Alexander Thomas Puttick

Abstract:

Hypoxia, a state of low oxygen, is a common phenotype of solid tumours that

can be exploited with reasonable drug design. This study will outline the potential

application of alkyl bridged ruthenium(II)-cobalt(III) dinuclear complexes as potential

hypoxia-selective prodrugs. Coordination of a nitrogen mustard ligand onto a

kinetically inert cobalt(III) metal centre supresses the alkylating reactivity. Upon bio-

reduction to a labile cobalt(II) species by cellular reductases, the cytotoxic ligand is

released and induces apoptosis in the cell. This reduction is inhibited by molecular O2,

thereby imparting cytotoxic selectivity within the hypoxic regions of tumours. As a

result, various cobalt(III) mustard complexes were synthesised and characterised.

Addition of an extended alkyl chained ruthenium(II) polypyridyl moiety to a mustard

bound cobalt(III) centre allows for greater facilitation into cells, which correlates to

the increase in lipophilicity of the complex. Hydrophobicity can be modulated by

varying the length of the alkyl bridge. Previous work on dinuclear anticancer

complexes has failed to address the emergence of ‘click’ chemistry which provides a

facile, high yielding approach to the synthesis of functionalised 2-pyridyl-1,2,3-

triazole (pyta) bridging systems; molecules that are commonly viewed as surrogates

to traditional bipyridine ligands. Consequently, this study describes the synthesis of a

novel bis-bidentate alkyl linked pyta system that can be utilised to coordinate to both

ruthenium(II) and cobalt(III) metal centres. Although the formation of a stable

ruthenium(II)-cobalt(III) dinuclear system, detailed in the final chapter, was not

achieved, this research outlines significant advances in the field of bioreductive

transition metal complexes as potential hypoxia-selective cytotoxins.

Approaches to Co(III)/Ru(II) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick

i

Table of Contents:

Abstract: ___________________________________________________________ i

Table of Contents: ____________________________________________________ i

Acknowledgements: _________________________________________________ iv

Aims _______________________________________________________________ v

List of Tables _______________________________________________________ vi

List of Figures ______________________________________________________ vi

Chapter 1: __________________________________________________________ 1

Introduction _________________________________________________________ 1

1.1 Introduction ............................................................................................................ 2

1.2 Tumour Hypoxia ........................................................................................................ 2

1.3 Hypoxia Selective Cobalt (III/II) Nitrogen Mustard Complexes .............................. 3 1.3.1 Introduction ............................................................................................................................ 3 1.3.2 Alternative Cobalt (III) Chaperones for Hypoxia-Selectivity ................................................ 5 1.3.3 Copper (II)/(I) Complexes and their Targeted Treatment of Cancerous Cells ....................... 7

1.4 Polypyridyl Ruthenium Complexes ............................................................................. 8

1.6 Click Chelators ............................................................................................................ 12

1.7 Dissertation Overview ................................................................................................. 14

Chapter 2: _________________________________________________________ 16

Synthesis and Characterisation of Some Copper(II) and Cobalt(III)

Complexes PAYLOAD ______________________________________________ 16

2.1 Introduction ................................................................................................................. 17

2.2 Results and Discussion ................................................................................................ 19 2.2.2 Attempts at Copper Complexation ....................................................................................... 19

2.2.2.1 Copper (II) TMEDA Bipy Complex (1) .................................................................... 19 2.2.3 Cobalt Complexation ........................................................................................................... 21

2.2.3.1 Cobalt (III) Ethylene Diamine (EN) bound Complex (3) ........................................ 21 2.2.3.2 Cobalt (III) Tetramethylethylenediamine (TMEDA) bound Complex (4) ............ 21 2.2.3.3 Cobalt (III) N-(2-hydroxyethyl)ethane-1,2-diamine (HEEN) bound Complex (5)23 2.2.3.4 Cobalt (III) N-(2-chloroethyl)ethane-1,2-diamine (CEEN) bound Complex (6):

Synthesis of Cobalt(III) Bound Mustard Agents ................................................................. 26 2.2.3.5 Synthesis of a stable Mustard bound Cobalt(III) Triflate Salt (7) ......................... 28

2.3 Conclusion.................................................................................................................... 30

Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick

ii

Chapter 3: _________________________________________________________ 31

Synthesis and Structural Characterisation of a Polypyridyl Ruthenium(II)

Complex WARHEAD _______________________________________________ 31

3.1 Introduction ................................................................................................................. 32

3.2 Results and Discussion ................................................................................................ 33 3.2.1 Synthesis and characterisation of cis-[Ru(phen)2Cl2] (8) ..................................................... 33

3.3 Conclusion.................................................................................................................... 34

Chapter 4: _________________________________________________________ 35

Synthesis and Characterisation of an Alkyl Bridged 2-pyridyl-1,2,3-triazole

ligand LINKER ____________________________________________________ 35

4.1 Introduction ................................................................................................................. 36 4.1.1 One-Step multi-component CuAAC approach to pyridyl-1,2,3-triazole ligands ................. 37 4.1.2 Two Step CuAAC approach to pyridyl-1,2,3-triazole ligands ............................................. 38 4.1.3 Other CuAAC approaches to pyridyl-1,2,3-triazole ligands ................................................ 38

4.2 Results and Discussion ................................................................................................ 39 4.2.1 Synthesis of an alkyl linked ligand (10) using a One-step CuAAC approach ...................... 39 4.2.2 Synthesis of an alkyl linked ligand (10) using a Two-step CuAAC approach via 1,12-

dibromodododecane ...................................................................................................................... 41 4.2.3 Synthesis of an alkyl linked ligand (10) through a Two-step CuAAC approach via 1,12-

diiododoecane ............................................................................................................................... 43

4.3 Conclusion.................................................................................................................... 44

Chapter 5: Attempts at Heterodinuclear Ruthenium(II)-Cobalt (III)

Complexes _________________________________________________________ 45

5.1 Introduction ................................................................................................................. 46

5.2 Results and Discussion ................................................................................................ 46 5.2.1 Complexation utilising Pyta ‘Click’ Ligand 10 and ruthenium(II) complex 8 .................... 46 5.2.2 Complexation utilising Pyta ‘Click’ Ligand 10 and Cobalt(III) triflate complex 7 ............. 50

5.3 Conclusion.................................................................................................................... 52

Chapter 6: Future Work ____________________________________________ 53

6.1 Uncompleted Experimentation .................................................................................. 54

6.2 Lipophilicity ................................................................................................................. 56

6.3 Electrochemical Studies .............................................................................................. 56

6.4 Understanding Cellular Uptake and Cell death mechanisms ................................. 56

6.5 Controlling Stereochemistry ...................................................................................... 57

6.6 Developing a series of more Stable Cytotoxins ......................................................... 57

6.7 Pyridyl 1,2,3 Triazole Ruthenium(II) Systems ......................................................... 59


iii

Chapter 7: Conclusion _____________________________________________ 60

Appendices _________________________________________________________ 63

Appendix A: Experimental............................................................................................... 64 Chapter 2 ....................................................................................................................................... 64

2.1 [Cu(dmbpy)(TMEDA)2]2BF4 (2) SECTION 2.2.2.1 of main body .............................. 64 2.2 [Co(en)2Cl2]Cl (3) SECTION 2.2.3.1 of main body ....................................................... 65 2.3 [Co(TMEDA)2Cl2]Cl SECTION 2.2.3.2 of main body ................................................. 65 2.4 [Co(HEEN)2(NO2)2]NO3 (5) SECTION 2.2.3.3 of main body ....................................... 66 2.5 [Co(CEEN)2(NO2)(Cl)]Cl (6) SECTION 2.2.3.4 of main body ..................................... 66 2.6 [Co(CEEN)2(OTf)(Cl)]OTf (7) SECTION 2.2.3.5 of main body ................................. 67

Chapter 3 ....................................................................................................................................... 68 3.1 [Ru(phen)2Cl2]2+ (8) SECTION 3.2.1 of main body ....................................................... 68

Chapter 4 ....................................................................................................................................... 69 4.1 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10) through a One step

CuAAC approach SECTION 4.2.1 of main body .................................................................. 69 4.2 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10) through a Two-step

CuAAC approach using 1,12-dibromodododecane SECTION 4.2.2 of main body ............ 70 4.3 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10) through a Two-step

CuAAC approach using 1,12-diiodododecane SECTION 4.2.3 of main body .................... 72 Chapter 5 ....................................................................................................................................... 74

5.1 [Ru(phen)2(pyta)]PF6 Complex (14) ............................................................................ 74 SECTION 5.2.1 of main body .................................................................................................. 74 5.2 [Co(CEEN)2(pyta)]PF6 Complex (16) SECTION 5.2.2 of main body ......................... 75

Appendix B: X-Ray Crystal Data .................................................................................... 76 1.1 Tetranuclear-Copper Complex (2) (SECTION 2.2.2.1) .......................................................... 76 1.2 Trans-[Co(en)2(Cl2)]Cl (3) (SECTION 2.2.3.1) ...................................................................... 77 1.3 Pronated TMEDA and [CoCl4]2-

Cluster (SECTION 2.2.3.2)................................................. 78 1.4 Trans-[Co(HEEN)2(NO2)2]NO3 (5) (SECTION 2.2.3.3) ........................................................ 79

Appendix C: List of Notable Compounds Made ............................................................ 80

Appendix D: Notable Spectroscopic Data ....................................................................... 82 1.0 Complex 5 ESI-MS ................................................................................................................. 82 1.1 Complex 6 13C NMR and ESI-MS .......................................................................................... 82 1.2 Complex 7 1H NMR ................................................................................................................ 83 1.3 Complex 7 19F NMR ............................................................................................................... 84 1.4 Ligand 10 from 1,12-Dibromododecane 1H NMR .................................................................. 84 1.5 Ligand 10 from 1,12-Diiodododecane 1H NMR, 13C NMR, COSY and ESI-MS .................. 85 1.6 Complex 14 1H NMR, UV-vis and ESI-MS ........................................................................... 87 1.7 Complex 16 1H NMR .............................................................................................................. 89 1.8 Complex 8 1H NMR ................................................................................................................ 89

References _________________________________________________________ 91


iv

Acknowledgements:

First and foremost I would like to thank my supervisor Simon for giving me a

fulfilling project, one that has opened my eyes to the intriguing world of scientific

research before I leave university. I have been very blessed to have a supervisor who

would dedicate as much time as he could to helping me progress through my research.

Secondly, I would like to thank Dr. Jonathan Kitchen who has provided some

invaluable pieces of advice for the progression of this project. I am very grateful for

all the time he has given me and hopefully this has been reflected in the quality of

research produced. My gratitude goes to Dr. Tony Keene for helping conceptualise

this project.

I would also like to thank the crystallography team with special thanks going

to Dr. Peter Horton. Without the crystal structures, characterisation of our complexes

would’ve been very difficult.

Finally, I would like to dedicate this project and its future work to anyone who

is suffering from cancer. Trying to tackle one of life’s most unfortunate plights has

been an enchanting journey.


v

Aims 1. To successfully design, synthesise and characterise through appropriate rationale

an alkyl bridged dinuclear ruthenium(II)/cobalt(III) Nitrogen mustard cytotoxin

that exhibits selectivity towards hypoxic cells over healthy cells (Figure 1)

2. To utilise an efficient method to convert a non-toxic cobalt(III) precursor into its

cytotoxic cobalt(III) mustard and apply this principle into the synthesis of our

ruthenium(II)/cobalt(III) complex as late as possible.

3. To synthesise a novel bis-bidentate polypyridyl ligand that is bridged by a

variable alkyl chain using newly discovered ‘click chemistry’.

Figure 1 Proposed Dinuclear Ruthenium(II)/Cobalt(III)) system for Hypoxia Selectivity


vi

List of Tables Table 1: 13C NMR peak comparison between Literature values (left) and observed

(right) for [Co(CEEN)2(NO2)(Cl)]Cl (6) (For full 13C NMR spectrum, see Appendix

D, 1.1) ................................................................................................................................................ 28 Table 21H NMR peak comparison between Literature values (left) and observed

(right) for [Co(CEEN)2(Cl)(OTf)]OTf (7) (For full 1H NMR spectrum, see Appendix

D, 1.2) ................................................................................................................................................ 29 Table 3: Comparison of equivalents used for the one pot synthesis and two step

synthesis ............................................................................................................................................ 41 Table 4 Comparison of Rf values between crude [Ru(phen)2(pyta)] (14) and

[Ru(phen)2Cl2] (8) The eluent used (CH3CN:H2O:NaNO3(sat,aq), 40:4:1 respectively)99

was used to mimic the column conditions that would be used to separate pure

Complex 14 from the obtained crude solid. ............................................................................ 47 Table 5 Comparison of UV-vis absorption peaks for synthesized complex 14 and

reported complex 1596 ................................................................................................................... 49

List of Figures Figure 1 Proposed Dinuclear Ruthenium(II)/Cobalt(III)) system for Hypoxia

Selectivity ............................................................................................................................................ v Figure 2 Nitrogen Mustard Mode of Action, alkylation of N-7 site of guanine9 Once

bound, the second chlorine can be displaced resulting in the formation of interstrand

cross-links. This distortion in the DNA structure forces to the cell to undergo

apoptosis ............................................................................................................................................... 3 Figure 3 Hypoxia Selective [Co(Meacac)2(DCE)]+ complex designed by Denny et al16

Coordination of the nitrogen’s lone pair of electrons suppresses its ability to alkylate

via the intramolecular displacement of the labile chlorine atoms. ...................................... 4 Figure 4 Reductive activation of cobalt (III) complexes as hypoxia selective

cytotoxins24 This reductive mechanism can be undergone in vivo to release a cytotoxic

mustard ligand into cells to facilitate apoptosis. ....................................................................... 4 Figure 5 (Tris(2-methylpyridyl)amine) cobalt(III) complex bound to the anticancer

drug ‘Curcumin’ (shown in red) designed by Hambley et al31. Once bound to the

cobalt(III) centre, the solubility and therefore chemotherapeutic efficacy are increased

................................................................................................................................................................. 6 Figure 6 Structure of a hypoxia-selective copper(II) macrocyclic mustard complex35 . 7

Figure 7 Structure of Cisplatin ....................................................................................................... 8


vii

Figure 8 Structures of NAMI-A (imidazolium trans-

[tetrachloride(imidazole)(dimethylsulfoxide)ruthenate(III)] and KP1019 (indazolium

trans-[tetrachloridobis(1H-indazole)ruthenate(III)]. ............................................................... 9 Figure 9 The chemical structure of Keene et al Rubbn complexes (where n=

2,5,7,10,12 or 16 methylene groups in the alkyl chain51 ..................................................... 10 Figure 10 Confocal Microscopy images of Rubb16. Image (i) shows cellular staining

whilst (ii) shows Mitotracker Green FM staining46 1.5 Ruthenium (II) –

Cobalt (III) Systems ....................................................................................................................... 10

Figure 11 An example of how ruthenium(II)/cobalt(III) complexes have been utilised

for photo-induced ligand release via a polypyridyl bridge62 .............................................. 11 Figure 12 A general schematic of the mode of action for a ruthenium(II)-cobalt(III)

Photoactivated cytotoxin designed by Hartshorn et al 61 .................................................... 11 Figure 13 Fokin and Finn’s proposed catalytic cycle for the azide/alkyne CuAAC

reaction73 ........................................................................................................................................... 13 Figure 14 Fig. General Reaction scheme of Cu(I)-catalysed CuAAC reaction to

generate a bidentate 2-pyridyl-1,2,3-trizole ligand that coordinates to a metal centre

(denoted M). 1,2,3-Triazoles have two possible coordination sites, specifically the 2-

and 3-nitrogen atoms. Introduction of a 2-pyridyl group at the 4-poisition results in

preferred bidentate sites at the 2- triazole nitrogen and 2-pyridyl nitrogen ................... 13 Figure 15 Pt Complex bound to a 1,4 functionalised 1,2,3-triazole ligand that exhibits

anticancer properties71 ................................................................................................................... 14 Figure 16 Representation of how to design a ruthenium(II)-cobalt(III) hypoxia

selective cytotoxin through retrosynthetic analysis displaying the synthetic steps

outlined in each chapter ................................................................................................................ 15 Figure 17 Examples of toxic (DCE, CEEN) and non-toxic (EN, TMEDA, HEEN,

BHEEN, THEEN) ligands which could be used to bind to a cobalt(III) metal centre

when studying Hypoxia-Selective Cytotoxins. ...................................................................... 17 Figure 18 Synthesis of a toxic nitrogen mustard agent (CEEN) from the non-toxic

Hydroxyethylamine equivalent (HEEN) (top) followed by the subsequent formation

of the highly strained aziridinium ion (bottom) ..................................................................... 18 Figure 19 Proposed Reaction Pathway to Complex 1 based on method outlined by

JunJiao et al78 (For full experimental details, see Appendix A: 2.1) ............................... 19 Figure 20 Undesirable Hydroxyl Bridged Tetranuclear Copper Complex 2 (For full X-

ray Data Table, See Appendix B: 1.1) ...................................................................................... 20 Figure 21 General reaction scheme to complex 3 using conditions outlined by Bailar

et al79 (For full experimental details, see Appendix A: 2.2) ............................................. 21 Figure 22 X-ray structure of trans-[Co(en)2(Cl)2]Cl- (For full X-ray data table, see

Appendix B: 1.2) ............................................................................................................................. 21 Figure 23 General Reaction scheme to cobalt(III) complex 2 (For full experimental

details, see Appendix A: 2.3) ...................................................................................................... 22 Figure 24 X-Ray Crystal structure of protonated TMEDA molecules and a

tetrahedrally coordinated [CoCl4]2- species (For full X-ray data table, see Appendix B:

1.3) ...................................................................................................................................................... 22 Figure 25 Synthesis of [Co(HEEN)2(NO2)2]NO3 (5) using conditions outlined by

Downward et al64 (For full experimental details, see Appendix A: 2.4) ........................ 23 Figure 26 Obtained X-ray crystal structure of [Co(HEEN)2(NO2)2]NO3.H2O (5) (For

full X-ray table, see Appendix B: 1.4) ...................................................................................... 24

Figure 27 Hydrogen bonding network of [Co(HEEN)2(NO2)2]NO3.H2O (5) ............... 24


viii

Figure 28 ESI-MS spectrum of the [M]+ peak for [Co(HEEN)2(NO2)2]NO3.H2O (5)

(For full spectrum, see Appendix D, 1.0) ................................................................................ 25 Figure 29 Potential Isomers that could form during the synthesis of

[Co(HEEN)2(NO2)2]NO3.H2O 5 ................................................................................................. 25 Figure 30 Reaction scheme of cobalt(III) complex (5) with thionyl chloride64 (top) to

generate cobalt(III) complex (6) and actual solids that were obtained before and after

reaction(bottom) (For full experimental details, see Appendix A: 2.5) ......................... 26 Figure 31 Fig. Predicted (left) and actual ESI-MS (right) isotope patterns for

[Co(CEEN)2(NO2)Cl]+ (top) and [Co(CEEN)2(NO2)2]+ (bottom). Predicted isotope

patterns acquired from http://www.sisweb.com80 (For Full ESI-MS, see Appendix D

1.1) ...................................................................................................................................................... 27 Figure 32 Reaction scheme of [Co(CEEN)2(NO2)(Cl)]Cl (6) with HOTf to generate

cobalt(III) complex (7) 64,76 (top) and actual solids that were obtained before and after

reaction (bottom) (For full experimental details, see Appendix A: 2.6) ......................... 29 Figure 33 Proposed reaction of a mononuclear cis-[Ru(phen)2Cl2] 8 complex with a

functionalised 2-pyridyl-1,2,3-trizole to form a heteroleptic complex. This step is

required to generate one half of the proposed ruthenium(II)-cobalt(III) hypoxia

selective cytotoxin .......................................................................................................................... 33 Figure 34 Synthesis of cis-[Ru(phen)2Cl2] 8 using methodology outlined by Clercq et

al89 (For full experimental details, see Appendix A: 3.1) .................................................. 33

Figure 35 Predicted (bottom) and measured (top) ESI-MS spectra for cis-

[Ru(phen)2Cl2]2+ (8) . Predicted isotope patterns acquired from

http://www.sisweb.com80 ............................................................................................................. 34 Figure 36 General One Pot CuAAC conditions conducted by Crowley et al68 to

synthesise a library of bis pyta ligands separated by a spacer followed by two

reactions conducted and the yields that were achieved ....................................................... 37 Figure 37 Structure of 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10)

that we aimed to synthesise as a result of utilising Click conditions ............................... 39 Figure 38 Reaction scheme illustrating our one pot methodology (For full

experimental details, see Appendix A: 4.1) ............................................................................ 40 Figure 39 ESI-MS data for purified 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-

yl)dodecane acquired using One-Step synthesis route ....................................................... 40 Figure 40 Reaction scheme to convert 1,12-dibromododecane to 1,12-

diazidododecane using conditions outlined by Dash et al92. STEP ONE (For full

experimental details, see Appendix A: 4.2) ............................................................................ 41 Figure 41 Schematic of CuAAC reaction using larger equivalents of catalytic system

STEP TWO (For full experimental details, see Appendix A: 4.2) ................................... 42 Figure 42 Conversion of 1,12-dibromododecane to 1,12-diiodododecane (For full

experimental details, see Appendix A: 4.3) ............................................................................ 43 Figure 43 Schematic representation of Synthesis of [Ru(phen)2(pyta)]PF6 14 under

microwave conditions (top) and visual observations (bottom). (For full experimental

details, see Appendix A: 5.1) ...................................................................................................... 47 Figure 44 1HNMR Comparison of Ruthenium bound Linker alkyl chain region 14

(top) with free ‘click’ Linker 10 alkyl chain region (bottom). Because the

Ruthenium(II) centre is bound to one side of the ‘Click Ligand’, the alkyl chain

becomes asymmetrical. Each unique hydrogen environment has been highlighted by a

different colour. (For full 1H NMR spectrum of 10 and 14, see Appendix D, 1.5 and

Appendix D, 1.6 respectively) .................................................................................................... 48


ix

Figure 45 Recorded UV-vis absorption data for complex 14 and reported UV-vis

absorption data for complex 15 by Ghosh et al96 (For full spectrum, see Appendix D,

1.6) ...................................................................................................................................................... 49 Figure 46 Flow Injection ESI-MS of crude solid recorded (left) and the predicted

isotope pattern for the suspected dinuclear ruthenium(II) by-product (Right) Predicted

isotope patterns acquired from http://www.sisweb.com80 (For full ESI-MS, See

Appendix D, 1.6) ............................................................................................................................ 50 Figure 47 Schematic representation of Synthesis of [Co(ceen)2(pyta)] 16 (top) and

visual observations (bottom) (For full experimental details, see Appendix A:5.2) ..... 51 Figure 48 Proposed Reaction Pathway to a ruthenium(II)-cobalt(III) cytotoxin using

results from the study .................................................................................................................... 54 Figure 49 Proposed Reaction Pathway to a ruthenium(II)-cobalt(III) cytotoxin using

results from the study .................................................................................................................... 54 Figure 50 Schematic showing how a library of ruthenium(II)/cobalt(III) cytotoxins

can be generated by altering: the length of methylene bridge and using a different

cobalt(III) payload .......................................................................................................................... 55 Figure 51 A group of tridentate ligands that were coordinated to a cobalt(III)centre

and cytotoxicity was tested against a previously reported bidentate (DCE)

cobalt(III)complex28 ....................................................................................................................... 58 Figure 52 Ball and Stick model of triply stranded ruthenium(II)helicates bound by bis

bidentate ‘click’ 2-pyridyl-1,2,3-triazole ligand that exhbit antimicrobial activity121 59 Figure 53 Formation of a Cobalt(III) mustard triflate from a non-toxic hydroxy

analogue ............................................................................................................................................. 61 Figure 54 Schematic displaying the synthetic pathway utilized to generate an alkyl

linked bi-bidentate 2,pyridyl-1,2,3-triazole ligand (10) using a two step copper(I)

catalyzed CuAAC reaction methodology. ............................................................................... 62

Chapter 1 Introduction


1

Chapter 1:

Introduction



2

1.1 Introduction

A Global Cancer statistics study stated that an estimated 14.1 million new

cancer cases and 8.2 million cancer deaths occurred in 2012 worldwide1. The

Development into new cancer treatments provides the medical community with a

spectrum of options when treating a cancerous growth. Researchers additionally aim

to design novel drugs that have decreased negative side effects which are often

associated with cancer treatments.

1.2 Tumour Hypoxia

It is now a well-established fact that a significant proportion of the cells within

solid tumours of both rodents and humans are hypoxic, meaning the cells contain very

low oxygen levels2. The poor vasculature of a rapidly growing tumour results in

inadequate quantities of oxygen and nutrients being able to diffuse into tumour cells,

ultimately leading to the formation of hypoxic (low oxygen) and necrotic (no oxygen)

regions3. Hypoxia plays a crucial role in the progression of cancer. Studies have shown

that tumour hypoxia promotes a resistance to apoptosis4, drives metastatic spread3 and

encourages hypermutation through the inhibition of DNA repair3. Furthermore, tumour

hypoxia has an inherent resistance to both radiotherapy and modern antiproliferative

chemotherapeutics5. Radiotherapeutic resistance can be attributed to what is known as

the ‘oxygen enhancement effect’6. Damage to the cancerous DNA is created by the

direct ionization from the radiation source, usually X-rays, or can be stimulated by

interaction with O2 radicals which are formed by the ionization of water that immerses

the DNA. The DNA strands are broken and those that are not repaired will ultimately

lead to cell death. In the absence of molecular O2, the strand repair is more efficient.

This is because oxygen will react with the broken strands of DNA and form organic

peroxides, which are difficult to repair6. This theory can be substantiated by a study

conducted in 1959 by Deschner et al7 who report oxygenated cells are 2.5-3 times more

radiosensitive than hypoxic cells. In addition, hypoxic cells exhibit resistance to

chemotherapeutics because antiproliferative drugs are often S-phase specific agents

that target the DNA of dividing cells. Due to their lack of oxygen and nutrients,

hypoxic regions are usually out of cycle8.Ultimately, the severity of hypoxia observed

in solid tumours over healthy cells represents an attractive target for the development

of novel therapeutics for selective cancer treatment.



3

1.3 Hypoxia Selective Cobalt (III/II) Nitrogen Mustard

Complexes

1.3.1 Introduction

Due to their redox capabilities, complexes of transition metals have the

potential to be used as hypoxia-selective cytotoxins, but to this date, none have been

selected and developed for clinical use5. Nitrogen mustards are cytotoxic, highly

reactive, small molecules that will bond covalently to the nucleophilic sites of both

large and small biomolecules via a mechanism that involves the intramolecular

displacement of a leaving group, usually chloride, to generate a very strained and

reactive aziridinium cation (Figure 2)9.

Figure 2 Nitrogen Mustard Mode of Action, alkylation of N-7 site of guanine9

Once bound, the second chlorine can be displaced resulting in the formation of interstrand

cross-links. This distortion in the DNA structure forces to the cell to undergo apoptosis

The most frequent site of attachment to DNA is the N-7 site of guanine but

further research has shown other adducts are formed at the O-6 and N-1 sites of

guanine residues10. Additionally, studies have shown that the aziridinium ion can

alkylate histidine and cysteine residues of proteins11. Bifunctional nitrogen mustards

are known to form DNA cross-links12,13. Denny and his group have conducted

comprehensive research into hypoxia selective nitrogen mustard coordinated

cobalt(III) prodrugs which are activated by a reductive mechanism and have shown

significant promise14-21. One of the compounds (Figure 3), exhibited a 30 fold

selectivity towards the hypoxic EMT6 cell line. The cytotoxicity was confirmed to be



4

due to release of the mustard agent, as shown by its effects on cells (UV4) that are

hypersensitive to alkylating agents16.

Figure 3 Hypoxia Selective [Co(Meacac)2(DCE)]+ complex designed by Denny et al16

Coordination of the nitrogen’s lone pair of electrons suppresses its ability to alkylate via the

intramolecular displacement of the labile chlorine atoms.

Octahedral cobalt (III) complexes have a d6 electronic configuration which

renders them kinetically inert. For example, the rate constant for the aquation of

[Co(NH3)6]3+ is equal to 5.8x10-12 s-1, which translates to a half life of 3800 years22.

Comparatively, the rate constant for the aquation of the kinetically labile high spin d7

cobalt (II) species [Co(en)3]2+ is equal to 6.8x102 s-1, a half life of 0.001 s 23. The

reduction from the kinetically inert cobalt (III) complex to a kinetically labile cobalt

(II) species would allow for rapid ligand exchange, thereby releasing the cytotoxic

mustard into the cancerous cell (Figure 4)24.

Figure 4 Reductive activation of cobalt (III) complexes as hypoxia selective cytotoxins24

This reductive mechanism can be undergone in vivo to release a cytotoxic mustard ligand into

cells to facilitate apoptosis.

A therapeutic advantage of using cobalt is that the cobalt (III)/cobalt (II)

reduction potential falls within the range of cellular reductants (-0.2 V to -0.4 V vs.

standard hydrogen electrode)25, so it is reasonable to expect that the one-electron

reduction needed to facilitate mustard release can occur in vivo8. Ahn et al26 found no

relationship linking the expression of cytochrome P450 reductase (the key one-



5

electron reductase in mammalian cells) and the extent of reduction of a particular

cobalt (III) prodrug in vivo. These results suggest that other cellular reductants are

involved such as thiols, NADP(H+) or ascorbate26.

The generally accepted view is that hypoxia selectivity is achieved through the

reoxidation of the labile cobalt (II) species to cobalt (III) by molecular oxygen within

healthy cells before mustard release occurs (Figure 4). Pulse radiolytic studies

conducted by Denny and collaborators have, however, ruled out this redox cycling

mechanism27. Hypoxic selectivity can actually be explained through competition of

the cobalt (III) species with oxygen for the bioreductants28. In oxygenated cells,

oxygen is preferentially reduced, leaving the cobalt (III) species intact.

Nitrogen mustards are ‘Janus-Faced’ cytotoxins. It is well known in the

literature that the cytotoxicity of nitrogen mustards is high (IC50 value of 10-6 M),

meaning they have the potential to eradicate almost all types of tumour cells, whether

they are cycling or non-clycing29. Nevertheless, this comes at the potential cost of

nitrogen mustards exhibiting undesirable cytotoxicity in healthy cells. However, the

alkylating reactivity of the nitrogen mustard agents relies on the availability of the

nitrogen’s lone pair. Coordination onto an inert cobalt (III) centre should mitigate its

reactivity as the lone pair on the nitrogen atom used to form the aziridinium cation is

coordinated to the cobalt (III) centre. Upon reduction of the cobalt (III) centre in a

hypoxic environment, the mustard will be released30. The lone pair of the nitrogen is

no longer coordinated to the metal centre and therefore the drug becomes active

(Figure 4).

1.3.2 Alternative Cobalt (III) Chaperones for Hypoxia-Selectivity

As mentioned in Section 1.3.1, the oxidised cobalt(III) state is kinetically inert

compared to the labile cobalt(II) state. This variation in lability allows cobalt

complexes to be successfully utilised as bioreductive prodrugs. Although this project

will focus on the synthesis of cobalt(III) nitrogen mustard complexes, other cobalt

chaperones have been reported. In each instance, coordination of the organic molecule

to the cobalt centre deactivates its cytotoxic efficacy, either by blocking the active site



6

or in the case of nitrogen mustards, coordinates to the reactive lone pair thereby

yielding an inert prodrug under normal physiological conditions.

Hambley and co-workers recently demonstrated the successful delivery and

release in hypoxic tumour cells of the anticancer drug curcumin using a cobalt(III)

complex (Figure 5)31.

Figure 5 (Tris(2-methylpyridyl)amine) cobalt(III) complex bound to the anticancer drug

‘Curcumin’ (shown in red) designed by Hambley et al31. Once bound to the cobalt(III) centre,

the solubility and therefore chemotherapeutic efficacy are increased

Curcumin had previously suffered in clinical trials due to its low solubility and

short half-life in the blood plasma. Through techniques such as X-ray absorbance near

edge structure spectroscopy (XANES) and fluorescence lifetime imaging it was found

that incorporation of curcumin onto a cobalt(III) species improved stability, solubility

and enhanced both tumour penetration and uptake within hypoxic regions.

As an alternative to enzymatic reduction of cobalt(III) complexes, Ahn and

collaborators have researched reduction in hypoxic regions using clinically relevant

doses of radiation32 . If Radiation-activated prodrugs (RAPS) can be activated only by

radiation and not by endogenous reductases, it is theoretically possible to focus

radiotherapy to confine activation in tumour regions only and therefore avoid toxicity

in areas which are moderately hypoxic, such as the retina33. RAPs could also be used

to target necrotic regions which are hypoxic but lack reductase enzymes32. Other

bidentate ligands, such as ethylenediamine bis-N-(napthyl sulphonamide), could be

used to elicit oxidative DNA cleavage34.



7

1.3.3 Copper (II)/(I) Complexes and their Targeted Treatment of

Cancerous Cells

As well as using cobalt(III), other transition metal centres such as copper have

been reported in the literature as potentially useful hypoxia-selective agents 35-37.

Similar to cobalt, bioreduction of copper(II) to copper(I) by intracellular reductases

forms an unstable copper(I) complex37. This then dissociates to liberate the free ligand

which, depending on its mode of action would damage the cell. Hypoxic selectivity is

achieved through re-oxidation by molecular oxygen in healthy cells. Additionally,

copper can be used to image hypoxic tissue36. For example, several copper

radionuclides are available for positron emission tomography (PET) (60Cu,61Cu, 62Cu

and 64Cu)37. In 2004, Parker et al35 designed a series of copper bound macrocyclic

nitrogen mustard complexes that exhibited both aqueous solubility and hypoxia

selectivity (Figure 6). Previous studies on bioreductive copper(II) complexes found a

lower reduction potential corresponded to increased selectivity for bioreduction37.

Subsequently, the redox potentials can be lowered by increasing the electron-donating

character of the ligand ultimately leading to improved selectivity in hypoxic tissue35.

Figure 6 Structure of a hypoxia-selective copper(II) macrocyclic mustard complex35



8

1.4 Polypyridyl Ruthenium Complexes

Cis-diamminedichloroplatinum(II) (Cisplatin, Figure 7 ) can be considered the

gold standard of metal based chemotherapeutic agents. It interacts with DNA through

covalent binding, halting DNA replication and ultimately induces apoptosis38.

Figure 7 Structure of Cisplatin

Platinum-based chemotherapeutics are essential to many modern cancer

treatments despite their limited solubility39, dose-limiting side effects40 and resistance

to some cancer types41. Ruthenium-based compounds have the potential to be very

promising alternative chemotherapeutic/antimicrobial agents resulting from the wide

variety of complexes that can be formed42. In 2006, a Scifinder database comparison

for the topics ‘’ruthenium anticancer’’ (422 hits) and ‘’platinum anticancer’’ (5773

hits) from 1965-2005 revealed that publications dealing with Platinum based

coordination compounds were still outnumbering those utilising a Ruthenium

centre43. After searching publications from 2005-2015, the topics ‘’ruthenium

anticancer’’ (1481 hits) and ‘’platinum anticancer’’ (5781) showed the rise of

Ruthenium based research but still an overall dominance of Platinum based

publications.

Today, NAMI-A and KP1019 (Figure 8) are the first and only ruthenium(III)

anticancer complexes to reach clinical trials with relatively low toxicity44. It is

postulated that these ruthenium(III) prodrugs are inert until activation within hypoxic

cancerous cells42 and although mechanisms of cytotoxic action are unknown, it is

widely accepted that each complex covalently binds to DNA44.



9

Figure 8 Structures of NAMI-A (imidazolium trans-

[tetrachloride(imidazole)(dimethylsulfoxide)ruthenate(III)] and KP1019 (indazolium trans-

[tetrachloridobis(1H-indazole)ruthenate(III)].

Many polypyridyl ruthenium(II) complexes have been widely reported to

exhibit strong, non-covalent binding to DNA through intercalation, groove binding

and electrostatic interactions45. For example, [Ru(bpy)3]2+ and [Ru(Me4phen)3]

2+

associate electrostatically within the grooves of DNA without disrupting the duplex

despite containing ligands that traditionally intercalate44. Complexes of this type have

the ability to induce photocleavage of DNA45.

Recently, there has been a wealth of research into the anticancer/antimicrobial

properties of dinuclear polypyridyl ruthenium(II) complexes bridged by an alkyl

chained linker46-54. Although a study by Rodger et al54 showed that dinuclear

polypyridyl ruthenium(II) complexes displayed a modest level of cytotoxicity against

cancerous cell lines, it is believed that their cytotoxic properties can be significantly

enhanced by increasing the overall lipophilicity of the complex as observed for a

series of dinuclear ruthenium(II) arene complexes published by Mendoza-Ferri et

al55,47. This finding can be substantiated by Keene et al51 who have shown through

scrupulous testing that increasing the lipophilicity of a flexible alkyl bridge between

two polypyridyl ruthenium(II) centres, denoted Rubbn, does significantly improve

cytotoxic properties (Figure 9). In particular, Rubb12 and Rubb16, where the alkyl

bridge between the two ruthenium(II) centres are 12 and 16 carbons long respectively,

showed significant cytotoxicity to the L1210 murine leukemia cell line46. According

to the research, ΔΔ- Rubb16 exhibited an IC50 value of 5 mM against the L1210 line

making it as cytotoxic as carboplatin, a derivative of cisplatin51.



10

Figure 9 The chemical structure of Keene et al Rubbn complexes (where n= 2,5,7,10,12 or 16

methylene groups in the alkyl chain51

Furthermore, it was reported that the ΔΔ isomers of the ruthenium(II)

complexes had higher cytotoxicity against the leukemia cell line than their ΛΛ

counterpart46.Polypyridyl Ruthenium complexes are excellent candidates for

biological imaging as they exhibit; long-lived, polarized luminescence, large stokes

shifts and oxygen sensitive luminescence which could be used to monitor intra

cellular Oxygen levels56. Keene et al46 exploited this by utilising confocal

microscopy in conjunction with a mitochondrial tracking dye to examine the selective

accumulation of Rubbn complexes in the mitochondria of live cancer cells (Figure 10).

It was also elucidated through Flow cytometric studies that ΔΔ- Rubb16 was taken up

by both L1210 cancer cells and healthy primary B cells to a greater extent than

analogous dinuclear ruthenium(II) complexes with a shorter methylene bridge. More

importantly, there was an overall greater uptake in cancerous cells. This further

provides a corollary between lipophilicity and cellular uptake whilst tentatively

suggesting that the dinuclear complexes selectively accumulate in cancer cells over

healthy cells51.

Figure 10 Confocal Microscopy images of Rubb16. Image (i) shows cellular staining whilst (ii)

shows Mitotracker Green FM staining46



11

1.5 Ruthenium (II) – Cobalt (III) Systems

Introductory work on ruthenium(II)-cobalt(III) bridged systems was

conducted by Taube et al57-60. In these experiments, dinuclear ruthenium(II)-

cobalt(III) systems were bridged by pyridyl and polypyridyl ligands and upon

reduction of ruthenium(III) to ruthenium(II), the rate of electron transfer between

the two metal centres was measured. More recently, Hartshorn et al61-64 have designed

polypyridyl ruthenium(II)-cobalt(III) systems that can be used as photoactivated

cytotoxins (Figure.11).

Figure 11 An example of how ruthenium(II)/cobalt(III) complexes have been utilised for photo-

induced ligand release via a polypyridyl bridge62

In these elegant compounds, photoinduced stimulation by an external light

source transfers an electron from the ruthenium(II) donor (D) to the cobalt(III)

acceptor (A) through the bridging ligand (L), thereby facilitating reduction of the

cobalt centre to cobalt(II) and allowing for the release of the cytotoxic mustard agent

through rapid aquation of the cobalt(II) species61 (Figure 12).

Figure 12 A general schematic of the mode of action for a ruthenium(II)-cobalt(III)

Photoactivated cytotoxin designed by Hartshorn et al 61



12

1.6 Click Chelators

Owing to their kinetically stable bonds, 2,2’-bipyridine derivatives can act as

strong chelating ligands to a wide range of transition metal centres with multiple

applications65. In this respect, ruthenium(II) complexes of bipyridine-type ligands

are particularly interesting as they exhibit predictable photophysical and

electrochemical properties66. Moreover, Richard Keene’s research into dinuclear

ruthenium(II) complexes, which showed selectivity towards leukaemia cells over

healthy cells utilised an alkyl bridged 2,2’-bipyridine moiety as the bridging ligand46.

It was concluded that the additional methylene groups on the bridging ligand

increased lipophilicity of the overall complex, which allowed for greater cellular

accumulation and ultimately enhanced cytotoxicity (Figure 9/ Section 1.4.4).

Therefore, the development of a synthetic strategy for an easy and broad library of

functionalised bipyridine-type ligands is important for the success of this project.

Many modern synthetic routes to obtain functionalised 2,2’-bipyridine ligands are

often: low yielding, require multiple synthetic steps and are marred with meticulous

purification steps65,66.

More recently, a compendium of literature has focused on the copper(I)-

catalysed 1,3-cycloaddtion (CuAAC reaction) of organic azides with terminal alkynes

to synthesise 1,4 functionalised 1,2,3-triazole derivatives as alternative ligands to 2-

2’-bipyridine through ‘Click Conditions’65-71. ‘Click chemistry’ defines a reaction that

meets stringent conditions including: high yielding, generates only inoffensive by-

products, use of a benign solvent that is easily removed and simple product

isolation72. The subsequent purification of products, if required, must be achieved

through non-chromatographic methods, including crystallization and distillation, and

finally the product must be stable under physiological conditions72. Because our

proposed compound is a potential chemotherapeutic, stability in physiological

conditions is crucial. The general process of the CuAAC reaction has been outlined

below (Figure 13).



13

Figure 13 Fokin and Finn’s proposed catalytic cycle for the azide/alkyne CuAAC reaction73

By completely altering the mechanism, the copper(I) catalyst easily

overcomes the otherwise very high activation barrier in the non-catalysed Huisgen

Reaction74. This reaction is particularly useful in the field of coordination chemistry

because the 1,4-functionalised 1,2,3-triazole ligands generated in the CuAAC reaction

are viewed as readily functionalised surrogates for bipyridine and terpyridine ligands

and therefore have the potential to act as N- donor ligands to a variety of metals75

(Figure 14).

Figure 14 Fig. General Reaction scheme of Cu(I)-catalysed CuAAC reaction to generate a

bidentate 2-pyridyl-1,2,3-trizole ligand that coordinates to a metal centre (denoted M).

1,2,3-Triazoles have two possible coordination sites, specifically the 2-and 3-nitrogen atoms.

Introduction of a 2-pyridyl group at the 4-poisition results in preferred bidentate sites at the 2-

triazole nitrogen and 2-pyridyl nitrogen

A number of authors have subsequently examined the complexation of related

1,4 functionalised 1,2,3-triazole ligands with metal centres that exhibit octahedral

geometry such as ruthenium(II), copper(II) and rhenium(I) with positive

results66,67,69,70.Some metal complexes of these ligand types have also been shown to

exhibit some chemotherapeutic properties. For example, research conducted by Kilpin



14

et al70 have demonstrated that ruthenium(II) and osmium(II) n6-arene complexes

bound to related 1,4 functionalised 1,2,3-triazoles ligands have shown excellent

selectivity towards tumour cell lines over non tumour lines whilst Maisonal et al71

have synthesised a platinum based complex bound to a 1,4 functionalised 1,2,3-

triazole ligand that displayed a significant cytotoxicity towards breast cancer cells,

comparable to cisplatin (Figure 15).

Figure 15 Pt Complex bound to a 1,4 functionalised 1,2,3-triazole ligand that exhibits anticancer

properties71

1.7 Dissertation Overview

The purpose of this dissertation is to outline the challenges and approaches

associated with the design and synthesis of an alkyl bridged dinculear ruthenium(II)-

cobalt(III) that under hypoxic conditions, will selectively facilitate the reduction of

the cobalt(III) metal centre into the labile cobalt(II) species causing release of the

cytotoxic mustard agent into the cancerous cell, signalling apoptosis. The synthesis of

the proposed ruthenium(II)-cobalt(III) cytotoxin (Figure 1) can be split into distinct

chapters (Figure 16)

Chapter 2 outlines a method as to how nitrogen mustard ligands (a well-

studied class of DNA alkylators) can coordinate to a cobalt(III) metal centre. This

type of compound can be synthesised from the non-toxic cobalt(III) complex of an

alcohol precursor to a nitrogen mustard agent. The chapter also briefly outlines the

attempt at synthesising copper(II) mustard agents.

Chapter 3 describes the synthesis of a mononuclear polypyridyl ruthenium(II)

species that bears labile chloro ligands. This species can then undergo complexation

reactions with a 2-pyridyl-1,2,3-triazole ligand scaffold. The addition of a lipophilic

ruthenium(II) moiety to our dinuclear species will aid cellular uptake as well bind to

the target DNA.



15

Chapter 4 relates to the design and synthesis of a novel alkyl linked 2-pyridyl-

1,2,3-triazole ligand scaffold that can be used as a bridging linker between the

ruthenium(II) and cobalt(III) metal centres. The variable hydrophobic nature of the

linker will aid the cellular uptake of the drug and therefore increase overall

cytotoxicity.

Chapter 5 encapsulates the findings of previous chapters by coordinating the

polypyridyl ruthenium(II) generated in Chapter 3 with the alkyl chained ‘click’ 2-

pyridyl-1,2,3-triazole linker synthesised in Chapter 4. This chapter also describes the

coordination of a cobalt(III) mustard agent synthesised in Chapter 2 with the alkyl

chained ‘click’ 2-pyridyl-1,2,3-triazole ligand.

Finally, Chapter 6 describes the future prospects of this particular study.

Figure 16 Representation of how to design a ruthenium(II)-cobalt(III) hypoxia selective cytotoxin

through retrosynthetic analysis displaying the synthetic steps outlined in each chapter

Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes


16

Chapter 2:

Synthesis

and

Characterisation

of Some Copper(II)

and

Cobalt(III) Complexes

PAYLOAD



17

2.1 Introduction

One of the major components of this project is the coordination of a cytotoxic

mustard onto a stable redox activated metal centre. This can be achieved by

coordinating the metal centre to non-cytotoxic analogues, such as ethylene diamine

(EN) and N,N,N’,N’-Tetramethylethylenediamine (TMEDA), which can be used to

model the size and coordination of a nitrogen mustard. Conversely, we can coordinate

the nitrogen mustard directly onto the cobalt(III) metal centre. Examples of these

ligands are outlined below (Figure. 17).

Figure 17 Examples of toxic (DCE, CEEN) and non-toxic (EN, TMEDA, HEEN, BHEEN,

THEEN) ligands which could be used to bind to a cobalt(III) metal centre when studying

Hypoxia-Selective Cytotoxins.



18

Research conducted by Hartshorn and his group have found that nitrogen

mustards can be synthesised from the analogous non-toxic hydroxyethylamine by

reaction with thionyl chloride (SOCl2) (Figure 18)76.

Figure 18 Synthesis of a toxic nitrogen mustard agent (CEEN) from the non-toxic

Hydroxyethylamine equivalent (HEEN) (top) followed by the subsequent formation of the highly

strained aziridinium ion (bottom)

Performing this transformation with thionyl chloride before coordinating the

ligand to the cobalt(III) centre may be problematic. This is because the lone pair on

the nitrogen can displace a chlorine atom to give the highly strained aziridinium

cation, as detailed in Section 1.3.1. We can therefore suggest that we will have

competition between decomposition of the mustard agent into the aziridinium cation

and coordination to the cobalt(III) centre. Ultimately, the later the reactive chloro

group is introduced into the complex, the better.

To prevent the undesirable decomposition into the aziridinium ion, a synthetic

strategy has been devised to coordinate the non-toxic alcohol precursor to the

cobalt(III) metal centre, followed by the conversion into the nitrogen mustard with

thionyl chloride76.This particular strategy is however, marred with its own potential

problems. One of which is the coordination of the oxygen atom of the alcohol to the

cobalt(III) centre. Cobalt(III) complexes coordinated to alcohols have been previously

synthesised and crystallised77. One ligand that has been studied in Hartshorn’s group

and subsequently in this study was N-(2-hydroxyethyl)ethane-1,2-diamine HEEN

(Figure 17/18). They concluded that modification of the reaction conditions lead to

the ligand binding in a bidentate manner through the two amine groups, or in a

tridentate fashion, binding through the two amine and hydroxyl group76. In the

remainder of this chapter, I will describe the coordination of various nitrogen based



19

ligands (Figure 17) to a cobalt(III) metal centre as well as attempts at coordination to

a copper(II) metal centre. One of the complexes that was synthesised underwent

conversion to the cobalt(III) mustard complex, detailed in (Figure 18).

2.2 Results and Discussion

2.2.2 Attempts at Copper Complexation

As mentioned in 1.3.3, it is possible to design a Hypoxia selective copper(II)

complex which undergoes bioreduction to a kinetically labile copper(I) species via

intracellular reductases to free the cytotoxic mustard into the cell. However, it was

found that any attempt at synthesising a stable heteroleptic copper(II) complex lead to

the formation of undesirable complexes.

2.2.2.1 Copper (II) TMEDA Bipy Complex (1)

Using Dimethylbipy as a commercially available alternative to our 1,4

functionalised 1,2,3-triazole linker ligand 10 and TMEDA as a bidentate non-toxic

analogue to a mustard agent, we attempted to form a heteroleptic copper(II) complex

1 using a method outlined by JunJiao et al78 who formed an analogously similar

anticancer compound [(Cu(DCA)(phen)) (Figure 19).

Figure 19 Proposed Reaction Pathway to Complex 1 based on method outlined by JunJiao et

al78 (For full experimental details, see Appendix A: 2.1)

However, instead of synthesising the desired copper(II) complex 1, it was

elucidated from SXRD data that the blue crystalline solid was a hydroxyl-bridged



20

tetranuclear copper species 2 (Figure 20). Hydrogens have been omitted for clarity. It

was concluded that the TMEDA ligand was acting as a catalyst through deprotonation

of a water molecule leaving a highly reactive OH- species which was able to form a

multinuclear species with the copper(II) centre. This would suggest that further

reactions would have to be undergone under anhydrous conditions. As we want our

potential chemotherapeutic to be stable in aqueous medium, this is a disadvantage to

using copper. Further reactions utilising the tetradentate THEEN ligand (Figure 17)

and 2,2’-bipyridine (bpy) resulted in products that were extremely viscous that proved

too difficult to precipitate and characterise. Finally, we also found that the copper(II)

complexes were extremely labile under acidic conditions suggesting the

transformation of a hydroxyl nitrogen mustard into its toxic mustard equivalent using

thionyl chloride (Figure 18 (top)) would be very difficult if bound to a copper(II)

centre. Therefore, the series was not carried on further.

Figure 20 Undesirable Hydroxyl Bridged Tetranuclear Copper Complex 2

(For full X-ray Data Table, See Appendix B: 1.1)

These observations along with a comprehensive literature search lead us to

undergoing further complexation reactions using the much more kinetically inert

cobalt(III) centre. A further advantage of using cobalt(III) over copper(II) is the

ability to characterise complexes using NMR. Cobalt(III) is diamagnetic (d6) giving

sharp and distinguishable NMR signals whereas copper(II) is paramagnetic (d9)

leading to a broadening of signals.



21

2.2.3 Cobalt Complexation

2.2.3.1 Cobalt (III) Ethylene Diamine (EN) bound Complex (3)

To help prove that the synthetic pathway to a cobalt(III) complex bound by a

bidentate N-donor ligand was plausible, a test reaction using CoCl2.H2O and ethylene

diamine solution was conducted. The trans-[Co(en)2(Cl2)]Cl 3 was prepared by using

a modification of known methods described by Bailar et al79 (Figure 21)

Figure 21 General reaction scheme to complex 3 using conditions outlined by Bailar et al79

(For full experimental details, see Appendix A: 2.2)

This resulted in a green crystalline solid that SXRD characterised as the

desired Trans-[Co(en)2(Cl2)]Cl complex 3, albeit in a very low yield (29%) (Figure

22).

Figure 22 X-ray structure of trans-[Co(en)2(Cl)2]Cl-

(For full X-ray data table, see Appendix B: 1.2)

2.2.3.2 Cobalt (III) Tetramethylethylenediamine (TMEDA) bound

Complex (4)

Using the reaction conditions that successfully synthesised Trans-

[Co(en)2(Cl2)]Cl 3, the synthesis of Trans –[Co(TMEDA)2(Cl)2]Cl complex 4 was



22

attempted (Figure 23). However, addition of concentrated acid to the reaction mixture

lead to a significant colour change from pink to dark blue. The extracted solid was

analysed by X-ray diffraction to yield a blue crystal structure which instead of

containing the desired complex 4, contained protonated TMEDA molecules and a

tetrahedrally coordinated [CoCl4]2- cluster (Figure 24). This crystal structure can be

attributed to concentrated hydrochloric acid protonating the TMEDA molecules

subsequently causing dissociation of the complex. This finding would help elucidate

the fact that N- donor tertiary amine ligands are only weakly bound to the cobalt(III)

centre. As previously mentioned in 2.2.1.5, it was found that any complexation to a

copper(II) centre using a tertiary N- donor, such as TMEDA, resulted in extremely

insoluble and viscous products which could not be characterised.

Figure 23 General Reaction scheme to cobalt(III) complex 2


It was concluded that further complexation reactions should use ligands

containing primary or secondary N-donor amine functionality. These would

coordinate more strongly to the cobalt(III) centre and therefore improve the overall

stability of our nitrogen coordinated cobalt(III)complex.

Figure 24 X-Ray Crystal structure of protonated TMEDA molecules and a tetrahedrally

coordinated [CoCl4]2- species

(For full X-ray data table, see Appendix B: 1.3)



23

2.2.3.3 Cobalt (III) N-(2-hydroxyethyl)ethane-1,2-diamine (HEEN)

bound Complex (5)

As mentioned in section 2.1, it is required that the N-(2-hydroxyethyl)ethane-

1,2-diamine HEEN ligand (Figure 17) binds to the cobalt(III) metal centre in a

bidentate fashion via the Nitrogen donor atoms. This structure was originally reported

by Hartshorn76 and the synthetic method used was based on a method outlined by

Downward et al64 (Figure 25)

Figure 25 Synthesis of [Co(HEEN)2(NO2)2]NO3 (5) using conditions outlined by Downward et

al64 (For full experimental details, see Appendix A: 2.4)

The reaction yielded orange crystals which were suitable for characterisation

by Single X-ray crystal diffraction. The crystal structure confirmed that the two

HEEN ligands were bound to the cobalt(III) centre through the primary and secondary

amines, in a bidentate manner (Figure 26). The cobalt(III) octahedral centre was also

coordinated to two trans N-bound nitrite ligands. It was predicted by Downward et

al64 that the two nitrite ligands stabilise the complex through inequivalent Hydrogen

bonding interactions. One nitrite ligand will act as a hydrogen bond acceptor to the

secondary amine of the HEEN ligand whilst the other nitrite ligand will act as a

hydrogen acceptor to the primary amine of the HEEN ligand. This observation was

also reported by Hartshorn et al76.



24

Figure 26 Obtained X-ray crystal structure of [Co(HEEN)2(NO2)2]NO3.H2O (5)

(For full X-ray table, see Appendix B: 1.4)

Although there is no direct hydrogen bonding between individual complexes,

a hydrogen bonding network does exist with the mononuclear ‘HEEN’ complexes

being bridged by both water molecules and NO3- counter ions (Figure 27).

Figure 27 Hydrogen bonding network of [Co(HEEN)2(NO2)2]NO3.H2O (5)

Since a single crystal is not always a complete representation of the bulk

product, additional analysis was conducted. Mass spectrometry (ESI-MS) was able to

identify the main ion present in the solid (Figure 28).



25

Figure 28 ESI-MS spectrum of the [M]+ peak for [Co(HEEN)2(NO2)2]NO3.H2O (5)

(For full spectrum, see Appendix D, 1.0)

Even though this reaction achieved a modest yield (59 %), it is still

unexpectedly high due to the large number of isomers that could’ve formed in this

reaction. For example, the ligands could bind through various combinations of donor

atoms (N- and O- donors available) to form tridentate ligands, R and S configurations

are possible as the secondary amine group is a stereogenic centre. Furthermore, the

HEEN ligands could be cis or trans to one another within the trans-bidentate

structure. Some of these theorised isomers can be shown below (Figure 29).

Figure 29 Potential Isomers that could form during the synthesis of

[Co(HEEN)2(NO2)2]NO3.H2O 5

HEEN ligands trans and cis to one another in

trans bidentate complex

HEEN ligands binding in a bidentate or tridentate

fashion



26

2.2.3.4 Cobalt (III) N-(2-chloroethyl)ethane-1,2-diamine (CEEN)

bound Complex (6): Synthesis of Cobalt(III) Bound Mustard Agents

The second aim of this research project was:

‘’To utilise an efficient method to convert a non-toxic cobalt(III)precursor into its

cytotoxic cobalt(III) mustard and apply this principle into the synthesis of our

ruthenium(II)/cobalt(III) complex as late as possible.’’

After examining the literature, a suitable method had been devised by

Downward et al64 who converted the non-toxic hydroxyethyl group from the ‘HEEN’

cobalt(III) complex 5 (section 2.2.3.3) into the toxic chloroethyl group in the

analogous ‘CEEN’ cobalt(III) complex 6. This was achieved through the use of

thionyl chloride. The cobalt(III) complex 5 was stirred in a solution of SOCl2 with a

small amount of DMF to aid dissolution of 5. The reaction mixture was allowed to stir

for 30 minutes and any excess solvent was removed using a vigorous stream of air.

After recrystallization, a pink solid remained which was observably different to the

orange crystals of the cobalt(III) complex 5 (Figure 30).

Figure 30 Reaction scheme of cobalt(III) complex (5) with thionyl chloride64 (top) to generate

cobalt(III) complex (6) and actual solids that were obtained before and after reaction(bottom)




27

The 1H and 13C NMR spectrums showed a considerable number of peaks

which would suggest that the product formed is not pure. ESI-MS data showed the

existence of several products, also noted by Downward et al64. Amongst these

products were: [Co(CEEN)2(NO2)(Cl)]+, [Co(CEEN)2(NO2)2]+

and

[Co(CEEN)(HEEN)(NO2)2]+. As these complexes contain chlorine atoms, we can

expect predictable isotope patterns and this is what we observed (Figure 31). Despite

recrystallization from H2O and EtOH, unwanted by products were still present, albeit

in small traces.

Figure 31 Fig. Predicted (left) and actual ESI-MS (right) isotope patterns for

[Co(CEEN)2(NO2)Cl]+ (top) and [Co(CEEN)2(NO2)2]+ (bottom). Predicted isotope patterns

acquired from http://www.sisweb.com80

(For Full ESI-MS, see Appendix D 1.1)

It’s important to note that the predicted Isotope pattern achieved for

[Co(CEEN)2(NO2)2]+ (bottom pattern) does not match the prediction entirely. This is

most likely due to the fact that this particular compound is in a very small quantity.

Another reason as to why we were not able to acquire pure complex 6 from the

conversion is the possible degradation products from the SOCl2. According to

Earnshaw et al81, SOCl2 can degrade into: sulfuryl chloride, sulphur dichloride,

sulphur dioxide, chlorine and HCl. All of these substituents may have some influence

to the progression of the reaction. The recorded ESI-MS data, colour changes



28

observed in the reaction and 13C NMR data for complex 6 (Table 1) all matched the

data acquired by reported literature sources76,64.

Table 1: 13C NMR peak comparison between Literature values (left) and observed (right) for

[Co(CEEN)2(NO2)(Cl)]Cl (6) (For full 13C NMR spectrum, see Appendix D, 1.1)

13C NMR Peaks Literature64,76δ 13C NMR Peaks Observed δ

52.488 52.56

51.476 51.48

42.705 42.73

40.094 40.13

This lead us to the conclusion that the cobalt(III) mustard complex 6 had been

successfully synthesised and subsequently met this study’s second aim of utilising a

method to convert a non-toxic cobalt(III) complex (5) into its cytotoxic mustard agent

(6).

2.2.3.5 Synthesis of a stable Mustard bound Cobalt(III) Triflate Salt

(7)

For successful coordination of cobalt(III) complex with a 2-pyridyl-,1,2,3-

triazole click chelator (Section 1.6), both reagents must dissolve in a suitable organic

solvent such as acetonitrile. It was found through various testing that the

[Co(CEEN)2(NO2)(Cl)]Cl complex 6 was insoluble in most solvent systems. We can

achieve much greater solubility, however, through formation into the corresponding

trifluromethanesulfonate (triflate) salt. Conversion of cobalt(III) complexes into their

corresponding triflate salt has been previously reported82,83. In general, the reported

procedures treat the cobalt(III) nitrito (NO2-) or chloro (Cl-) complex with HOTf

under a vacuum at a temperature between 60 and 100 oC. It was found, however, by

Downward et al64 that these reaction conditions would not work for

[Co(CEEN)2(NO2)(Cl)]Cl 6. They found that any applied heat to the reaction in

conjunction with the highly exothermic nature of the reaction lead to complete

disassociation of the complex. The reaction was therefore conducted in an ice-salt

bath to mitigate any decomposition of the complex. Conversion of



29

[Co(CEEN)2(NO2)(Cl)]Cl 6 into its corresponding triflate salt 7 was conducted using

conditions outlined by Downward et al64,76. (Figure 32)

Figure 32 Reaction scheme of [Co(CEEN)2(NO2)(Cl)]Cl (6) with HOTf to generate cobalt(III)

complex (7) 64,76 (top) and actual solids that were obtained before and after reaction (bottom)


Although mass spectrometry could not assign the [Co(CEEN)2(Cl)(OTf)]OTf,

complex 7, colour changes and 1H NMR data (Table 2) are analogous to reported

data64,76.

Table 21H NMR peak comparison between Literature values (left) and observed (right) for

[Co(CEEN)2(Cl)(OTf)]OTf (7)

(For full 1H NMR spectrum, see Appendix D, 1.2)

1H NMR Peaks Literature64,76δ 1H NMR Peaks Observed δ

5.723 (br m, 2H) 5.40 (br m, 2H)

5.179 (br s, 1H) 5.18 (br s. 1H)

4.011-3.908 (m, 2H) 4.09-3.86 (m, 2H)

3.122 (m, 3H) 3.11 (m, 3H)

3.006 (m, 2H) 3.00 (m, 2H)

2.715 (m, 1H) 2.73 (m, 1H)



30

Additionally, a 19F NMR of complex 7 was recorded which gave a single peak

at -78.50 ppm (Appendix D, 1.3). Although the single peak was expected for complex

7, it could also suggest a complex with two OTf ligands bound in identical chemical

environments has formed.

2.3 Conclusion

In order to synthesise a cobalt(III) complex that could be used as potential

Hypoxia selective cytotoxins, several ligands which were closely related to reported

cytotoxic mustard agents such as N,N-bis(2chloroethyl)ethane-1,2-diamine (DCE)

(Figure 17) were chosen and their coordination to copper(III)/copper(II) metal centres

were observed. One of the most promising ligands was N-(2-hydroxyethyl)ethane-1,2-

diamine (HEEN), which readily undergoes complexation with a cobalt(III) centre to

form trans-[Co(HEEN)2(NO2)2]NO3 5. This can then be converted into its cytotoxic

mustard agent 6 by treatment with SOCl2 and DMF. Following conversion into

[Co(CEEN)2(Cl)(OTf)]OTf 7, the complex can be coordinated to our 2-pyridyl-1,2,3-

triazole ligand scaffold to form half of our proposed ruthenium(II)-cobalt(III)

cytotoxin (Figure 1)

Chapter 3 Synthesis and structural characterisation of a polypyridyl Ru(II) complex


31

Chapter 3:

Synthesis

and

Structural

Characterisation

of a

Polypyridyl

Ruthenium(II)

Complex

WARHEAD



32

3.1 Introduction

Ruthenium(II) polypyridyl complexes are well-reported DNA groove binders

that exhibit useful spectroscopic properties and low toxicities44. Long before

interactions with DNA were observed, Dwyer et al84 showed that octahedral

[Ru(phen)3]2+ complexes exhibited antibacterial behaviour. They further

demonstrated that the Λ- and Δ- isomers exhibited different activities. Recent

publications have concluded through 2D NMR and SXRD that many ruthenium(II)

polypyridyl systems associate within the minor groove of DNA85,86 . Keene et al49

found that alkyl bridged dinuclear ruthenium(II) complexes (Figure 9) bind within the

minor groove of DNA, noting a significantly higher affinity for non-duplex features

such as bulge and hairpin loop sequences. Due to the flexibility of the species, the

torsional rotation of the alkyl bridged linker allows the second metal center to position

and bind (albeit less strongly) in the minor groove50. It has also been recently

discovered that a polypyridyl cobalt(III) species such as [Co(en)2(phen)]3+ binds to

the minor groove of DNA97 . Therefore, we can postulate that our cobalt(III) mustard

complex 7 may also bind to DNA because a bidentate mustard agent is structurally

analogous to ethylene diamine (en). In conclusion, the addition of a polypyridyl

ruthenium(II) complex into our proposed dinuclear system (Figure 1) will allow for

greater chemotherapeutic efficacy as the complex can bind to DNA. Moreover, the

addition of a luminescent ruthenium(II) centre, means the potential to study cellular

localisation through techniques such as: confocal microscopy88 and wide-field

fluorescence microscopy49 is possible.



33


3.2.1 Synthesis and characterisation of cis-[Ru(phen)2Cl2] (8)

The objective of this section was to synthesise a mononuclear

cis-[Ru(phen)2Cl2] 8 species that upon reaction with a functionalised 2-pyridyl-1,2,3-

trizole ligand (pyta), will displace the labile cis-chloro ligands to form a

[Ru(phen)2(pyta)]2+ type heteroleptic complex (Figure 33). This would therefore

constitute half of the proposed ruthenium(II)/cobalt(III) Hypoxia selective cytotoxin.

The details of the complexation reaction are delineated in Chapter 5.

Figure 33 Proposed reaction of a mononuclear cis-[Ru(phen)2Cl2] 8 complex with a

functionalised 2-pyridyl-1,2,3-trizole to form a heteroleptic complex. This step is required to

generate one half of the proposed ruthenium(II)-cobalt(III) hypoxia selective cytotoxin

In order to obtain the cis monoruthenium complex 8, a revised synthetic procedure

based on a pathway outlined by De Clercq et al89 was used (Figure 34)

Figure 34 Synthesis of cis-[Ru(phen)2Cl2] 8 using methodology outlined by Clercq et al89


Complex 8 was synthesised in a good yield (70. 6%). The 1H NMR spectra for

the recrystallized product showed more peaks than expected (Appendix D 1.8), which

was interpreted to mean that the product was not pure. ESI-MS results were consistent



34

with the formation of several products. Despite various components in the mass

spectrum, it was possible to assign our product 8 as it contained a typical ruthenium

isotope pattern (Figure 35). This data gave us additional confidence in the assignment.

It can be postulated that possibble by-products of this reaction are:

trans-[Ru(phen)2Cl2]2+ and [Ru(phen)3]

3+, both of which will not react with a

functionalised 2-pyridyl-1,2,3-triazole ligand.

d

Figure 35 Predicted (bottom) and measured (top) ESI-MS spectra for cis-[Ru(phen)2Cl2]2+ (8) .

Predicted isotope patterns acquired from http://www.sisweb.com80

3.3 Conclusion From a modification of a reported route by Clerqc et al89, we were able to

successfully synthesise and characterise a cis-[Ru(phen)2Cl2] 8 precursor that could

be used for complexation with our proposed alkyl chained bis 2-pyridyl-1,2,3-triazole

(pyta) ligand 10 (See Chapter 4). To achieve a purer sample of 8 for future reactions,

the reflux can be undergone for 15 hours, as reported by Feiters et al90, to help push

the reaction to completion and minimise the synthesis of unwanted by-products.

Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand


35

Chapter 4:

Synthesis

and

Characterisation

of an

Alkyl Bridged

2-pyridyl-1,2,3-triazole

ligand

LINKER



36

4.1 Introduction

The key component in the design and synthesis of our proposed dinuclear

ruthenium(II)/cobalt(III) cytotoxin is the bridging ligand. The bridging ligand must be

able to bind both metal centres as well as aid the facilitation of the compound into

cells. Traditional N-heterocyclic chelating ligands such as 2,2’-bipyridine (bpy) and

2,2:6’,2’’-terpyridine (tpy) have been widely studied due to predictable coordination

environments and interesting luminescent properties that result from ligand-metal

interactions75. Bipyridine type ligands establish kinetically stable bonds with Metal

centres so the selective synthesis of homoleptic and heteroleptic complexes is

possible66. Similar to Richard Keene’s work on dinuclear ruthenium(II) complexes,

our bridging ligand will contain two bidentate 2,2’-bipyridine type moieties linked by

a polymethylene chain (Figure 9). This alkane functionality is introduced to increase

overall lipophilicity of the complex which will improve cellular uptake to yield a

more potent chemotherapeutic. It has been recognised that hydrophobic compounds

cross the cell membrane more easily than hydrophilic ones47,55. This reflects the fact

that drugs have to cross hydrophobic barriers such as cell membranes to reach their

target.

Modern Synthetic methods for a broad library of functionalised 2-2’-

bipyridines in high yields, however, still remains a challenge. The copper(I)-catalysed

1,3-cycloaddtion (CuAAC) of organic azides with terminal alkynes (Section 1.6)

provides a modular, facile and high yielding method for the generation of readily

functionalised alternatives to (bpy) and (tpy) ligands75.

In this chapter, the synthetic strategies involved in the synthesis of a

polymethylene bridged bis 2-pyridyl-1,2,3-triazole (pyta) ligand through recently

established ‘click conditions’ will be outlined. This will followed by the results

achieved by utilising and modifying these particular methods.



37

4.1.1 One-Step multi-component CuAAC approach to pyridyl-1,2,3-

triazole ligands

Since the discovery of the CuAAC reaction in 2001 by Kolb, Finn and

Sharpless72, many authors have demonstrated a safe, efficient, one pot methodology

where potentially explosive organic azides are generated and subsequently reacted in

situ without the need for isolation91. Crowley et al67,68 exploited some of these

methodologies to provide their own one pot, multicomponent CuAAC method to

rapidly generate a library of more complex alkyl, benzyl, or aryl substituted

polydentate pyridyl-1,2,3-triazole ligands (Figure 36). This method was particularly

interesting as they had successfully generated a bis-bidentate 2-pyridyl-1,2,3-triazole

(pyta) ligand bridged by a six methylene chain in high yields 9.

Figure 36 General One Pot CuAAC conditions conducted by Crowley et al68 to synthesise a

library of bis pyta ligands separated by a spacer followed by two reactions conducted and the

yields that were achieved

For akyl linked systems analogous to our project, reactions conducted at RT

were obtained in a modest yield. This was presumably a result of the substitution of

the halide leaving group by the azide being slow at RT67. They found however that

simply conducting the reaction at 90 OC enabled the desired ligands to be synthesised

in high yields.



38

4.1.2 Two Step CuAAC approach to pyridyl-1,2,3-triazole ligands

By generating the azide substituted spacer prior to the CuAAC reaction, we

eliminate the potential synthesis of highly explosive copper(I) and copper(II) azides

which are plausible by-products using a one pot methodology. Therefore, a literature

search was conducted for a safer two step CuAAC approach which yielded some

excellent methods92,93. In particular, a methodology conceptualised by Dash et al92

utilised a more benign solvent system (tBuOH/H2O, 1:1) than Crowley’s one pot

method (DMF/H2O, 4:1) and allowed the CuAAC reaction to stir for 36 h at room

temperature compared to Crowley’s approach who utilised high temperatures for a

shorter period of time (Figure 35). The use of lower temperatures is desirable for this

reaction as we want to mitigate the explosive nature of organic or copper azides.

Furthermore, conducting the CuAAC reaction under ambient conditions keeps in

concordance with the ‘classical’ click conditions initially outlined by Sharpless et

al72.

4.1.3 Other CuAAC approaches to pyridyl-1,2,3-triazole ligands

Although only one step and two step CuAAC methods were utilised in this

particular project, authors have devised other methods to prepare pyridyl-1,2,3-

triazole derivatives thereby displaying the sheer versatility of these ligand scaffolds.

Schubert et al66 prepared a series of alkyl-substituted pyridyl-1,2,3-triazoles using

microwave assisted conditions. Although reaction times are cut from 24-36 hours to

20 minutes, the potential generation of explosive organic or copper(I)/copper(II)

azides under increased pressure exerted through microwave conditions was a risk we

were not willing to take.



39


4.2.1 Synthesis of an alkyl linked ligand (10) using a One-step

CuAAC approach

Experimental work was initiated with the objective of synthesising a bis

bidentate-2-pyridyl-1,2,3-triazole ligand linked by a twelve carbon polymethylene

chain (Figure 37, 10). The rationale behind this was twofold: to generate a ligand that

contained two N-donor binding sites for ruthenium(II)/cobalt(III) metal centres, as

well as using results from Richard Keene’s work, who found that a dinuclear

ruthenium(II) complex bound by a 12 or 16 carbon chain were the most cytotoxic

towards murine cancer cells46. A 12 carbon chain bridge was chosen simply because

the halide spacer CuAAC precursor (1,12-dibromododecane) was more commercially

available than the 16 carbon chain precursor (1,16-dibromohexadecane).

Figure 37 Structure of 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10) that we

aimed to synthesise as a result of utilising Click conditions

Subsequently, a synthetic route outlined by Crowley et al68 was followed. This

was achieved using 2-ethynylpyridine, NaN3 and 1,12-dibromododeane as the starting

materials. CuSO4.H2O, ascorbic acid and Na2CO3 were used as the catalytic system

(Figure 38). During the workup, a large excess of precipitate in the reaction mixture

was present which was filtered and removed. After extraction with EtOAc, organic

fractions were dried with Na2SO4 and filtered. Filtrate was removed in vacuo to yield

a pale yellow solid. Initial TLC data suggested the filtrate contained a major product

10 as well as unreacted 2-ethynylpyridine and by-products. Possible by-products of

this reaction would be: the mono substituted pyta ligand bound to the 12 carbon

polymethylene bridge along with an unreacted azide functionality on the other end 11

as well as any unreacted 1,12-diazidododecane 12 (Figure 38).



40

Crude material was then purified by column chromatography using a gradient

of (CH3)2CO:CH2Cl2 as the eluent. Product spot was analysed using mass

spectrometry.

Figure 38 Reaction scheme illustrating our one pot methodology


Although after purification there was only enough solid for analysis, mass

spectrometry was conducted on the product spot which showed that the desired

compound 10 had been synthesised (Figure 39). Further analysis was not undergone

as a new synthetic route would be established to improve the poor yield generated

from this method (17 %).

Figure 39 ESI-MS data for purified 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane

acquired using One-Step synthesis route



41

4.2.2 Synthesis of an alkyl linked ligand (10) using a Two-step

CuAAC approach via 1,12-dibromodododecane

Considering the limited success of the One-pot CuAAC approach, it was

decided to synthesise 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane 10 using

a two-step method in which the azide substituted spacer, 1,12-diazidododecane 12,

was generated before the CuAAC reaction. By utilising a Two-step methodology, it is

possible to improve the overall yield as we are eliminating a synthetic step from the

one-step method. Additionally, the overall safety of the reaction is vastly improved as

the synthesis of explosive copper(I) and copper(II) azide is omitted. The initial step in

the synthesis of 10 was to generate 1,12-diazidododecane 12 from 1,12-

dibromododecane via an Sn2 mechanistic pathway based on reaction conditions

outline by Dash et al92 (Figure 40).

Figure 40 Reaction scheme to convert 1,12-dibromododecane to 1,12-diazidododecane using

conditions outlined by Dash et al92. STEP ONE


The final yellow liquid would be used for the subsequent CuAAC reaction.

Although this is not the case, a 100 % reaction conversion from 1,12-

dibromododecane to 1,12-diazidododecane was assumed. Subsequent click conditions

reported by Dash et al92 were followed. However, it was decided to use a much larger

excess of the catalytic system compared to the one pot methodology (Section 4.2.1),

as shown by (Table 3) below.

Table 3: Comparison of equivalents used for the one pot synthesis and two step synthesis

Equivalents used in One pot

methodology Section 4.2.1

Equivalents used in Two step

methodology Section 4.2.2

CuSO4.H2O: 0.4 eqv CuSO4.H2O: 1 eqv

Na2CO3: 0.8 eqv Na2CO3: 2 eqv

Ascorbic Acid: 0.8 eqv Ascorbic Acid: 2 eqv



42

The advantage of using an excess of the catalytic system was to

improve the overall yield of the CuAAC reaction. As reported by Crowley et al67, the

alkyl bridged bis 2-pyridyl-1,2,3-trizole ligand 9 is able to coordinate to copper(II)

metal centres. Once bound, it can no longer undergo further CuAAC reactions and are

therefore limited by the amount of copper(II) utilised in the reaction. Thus, the

reaction of 1,12-diazidododecane with two equivalents of 2-ethynyl pyridine in

tBuOH and H2O (1:1) in the presence of a much larger excess of the catalytic system

produced a colourless solid of 1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane

10 after stirring at RT for 48 hours in a modest yield (51.6 %) (Figure 41).

Figure 41 Schematic of CuAAC reaction using larger equivalents of catalytic system STEP TWO


Compared to previous click reactions, a different workup was utilised

to increase yield of 10. This included: a larger excess of saturated EDTA solution to

remove all copper(II) potentially bound to ligand 10 as well as a different solution to

extract the organic layer (iPrOH:CHCl3, 3:1) compared to (EtOAC) based on the

recommendation of Dr. Steve Goldup of the University of Southampton. After

washing with water, organic fractions were dried with Na2SO4 and filtered. Similar to

the one pot method outlined in Section 4.2.1, TLC analysis confirmed the filtrate

contained a mixture of products. However, the white precipitate that was previously

omitted in previous click reactions contained pure product 10, as confirmed by

1HNMR, 13CNMR, ESI-MS and HSQC (See appendix D, 1.4). Despite insolubility in

various solvent systems, it was found that 10 was soluble in CHCl3: Butanol (1:1) in

the presence of gentle heating. The intrinsic insolubility of 10 can be attributed to the

polar 2-pyridyl-1,2,3-triazole ends coupled by an non-polar alkyl chain. Therefore a

solvent system has to be employed that can dissolve both polar and non-polar

functionalities.



43

4.2.3 Synthesis of an alkyl linked ligand (10) through a Two-step

CuAAC approach via 1,12-diiododoecane

Despite the synthesis of pure ligand 10 using a two-step approach outlined in

Section 4.2.2, a two-step click method using identical conditions was conducted using

1,12-diiododecane 13 as the first step precursor instead of the aforementioned 1,12-

bromododecane. Iodine is a better leaving group than bromine due to its decreased

electronegativity and increased size. Therefore, we can predict step one, the synthesis

of diazidododecane 12, will achieved in a better yield and therefore observe an overall

increase in yield of ligand 10. 1,12-diiodododecane 13 was subsequently generated

using a method outlined by Warnmark et al94 and synthesised in a modest yield (54

%) (Figure 42).

Figure 42 Conversion of 1,12-dibromododecane to 1,12-diiodododecane


Similar to the aforementioned ligand 10 from 1,12-dibromododecane, the

alkyl linked ligand 10 from 1,12-diiodododecane was characterised by ESI-MS, 1H ,

13C, COSY and HSQC NMR. These pyridyl-1,2,3-triazole systems were

characterised by clear [M+H]+ (459.1) and [M+Na]+ (481.3) peaks in the ESI-MS as

well as a diagnostic singlet of the triazole unit (8.27 ppm) (See Appendix D, 1.5). For

final clarification, the analytical data was compared to reported data for a structurally

analogous ligand 9 (Figure 36) that differed only in the length of polymethylene

bridge. Furthermore, ligand 10 was synthesised in a better yield using 1,12-

diiodododecane (83.3 %) as the halide spacer compared to 1,12-dibromododecane

(51.6 %).



44

4.3 Conclusion

From our studies, we have developed a facile, high yielding, multicomponent

method to synthesise a novel and unpublished alkyl linked 2-pyridyl-1,2,3-triazole

ligand 10 from its corresponding halide, sodium azide and alkyne substituents. It was

found that a modification of the two-step CuACC method outlined by Dash et al92

provided a more efficient and safer method to the one pot methodology reported by

Crowley et al68. Furthermore, using 1,12-diiodododecane 13 as the alkyl halide

precursor resulted in a significantly improved yield (83.3 %) of 10 compared to using

1,12-dibromododecane (51.6 %). This alkyl linked ligand 10 can be used as the

bridging linker in our proposed ruthenium(II)/cobalt(III) di nuclear Hypoxia Selective

Cytotoxin (Figure 1). Ultimately, the method used produced pure product 10 and no

separation techniques were required. By simply changing the halide spacer, we can

potentially generate an entire library of functionalised 2-pyridyl-1,2,3-trizole ligand

scaffolds that could be used in future reactions.

Chapter 5 Attempts at Heterodinuclear Ru(II)-Co(III) Complexes


45

.

Chapter 5:

Attempts

at

Heterodinuclear

Ruthenium(II)-Cobalt

(III) Complexes



46

5.1 Introduction

An extensive literature search has shown that the free binding domains of the

2,pyridyl-1,2,3-trizole ligands comparable to 10 (Figure 37, chapter 4) are able to

coordinate to a ruthenium(II) metal centre66, 95,96. To our knowledge, there are no

published papers describing the successful coordination of a cobalt(III) metal centre

to a ‘click’ 2-pyridyl-1,2,3-trizole type ligand but the binding to traditional 2,2’-

Bipyridine or 1,10-phenanthroline analogues has been previously reported87,97,98. We

therefore hypothesise that the ‘click’ ligand 10 will be an ideal bridging linker for our

proposed ruthenium(II)/cobalt(III) di nuclear cytotoxin (Figure 1). Briefly described

in Section 1.5, bridged ruthenium(II)/cobalt(III) complexes reported by Taube et al57-

60 were synthesised for electron transfer studies whilst Hartshorn et al30 have

synthesised bridged ruthenium(II)/cobalt(III) complexes to act as photoactivated

cytotoxins. In this chapter, we will detail the synthesis of two different Metal-Ligand

complexes to elucidate whether our alkyl linked ligand 10 is able to coordinate to

both a ruthenium(II) and cobalt(III) metal centre.


5.2.1 Complexation utilising Pyta ‘Click’ Ligand 10 and

ruthenium(II) complex 8

For our purposes, utilising a method outlined by Gunnlaugsson et al99 seemed

the most useful. Cis-[Ru(phen)2Cl2] 8 (Chapter 3) was reacted with the alkyl linked

ligand 10 (Chapter 4) at 120 o C under microwave conditions using a benign solvent

system (EtOH/H2O, 1:1) to yield a deep red solution with a precipitate. This was then

added to excess NH4PF6 to generate a bright orange emulsion. Filtering the reaction

mixture yielded an orange solid (Figure 43). By altering the molar ratio of Ligand 10

with complex 8 (2:1 respectively), we aimed to coordinate the ruthenium(II) centre to

one side of the alkyl chained ‘click’ ligand 10 to replicate the requirements of the

proposed ruthenium(II)/cobalt(III) cytotoxin (Figure 1).



47

Figure 43 Schematic representation of Synthesis of [Ru(phen)2(pyta)]PF6 14 under microwave

conditions (top) and visual observations (bottom).


The colour changes observed throughout the reaction (Figure 43) were the

same as those reported99,96 which gave us an initial indication that complex 14 had

been successfully synthesised. Subsequent TLC analysis of the crude solid 14 against

the impure cis-[Ru(phen)2Cl2] 8 (Chapter 3) starting material showed that although

there were an expected mixture of products, a new product had been synthesised (Rf:

0.03) whilst other reactants had disappeared (Rf: 0.11, 0.27) (Table 3).

Table 4 Comparison of Rf values between crude [Ru(phen)2(pyta)] (14) and [Ru(phen)2Cl2] (8)

The eluent used (CH3CN:H2O:NaNO3(sat,aq), 40:4:1 respectively)99 was used to mimic the column

conditions that would be used to separate pure Complex 14 from the obtained crude solid.

Rf Values Compound 14 Rf Values Compound 8

0.03 -

- 0.11

0.16 0.16

- 0.27

0.44 0.44



48

Complex 14 was further analysed by one dimensional (1D) (1H, 13C) and two

dimensional (1H-1H COSY, HSQC) NMR, ESI-MS and UV-vis spectroscopy

(Appendix D 1.6). Initial analysis of 1D and 2D NMR data would confirm a mixture

of products and reported column conditions would be required to separate our

proposed compound 14 from by-products99. Due to the fact that the product obtained

was a crude sample, the aromatic peaks could not be assigned. However, a 1H NMR

comparison of the polymethylene bridge region of uncomplexed ligand 10 (Chapter 4)

and complexed ligand 14 (Figure 44) indicates the desired mono substituted complex

has been made.

Figure 44 1HNMR Comparison of Ruthenium bound Linker alkyl chain region 14 (top) with free

‘click’ Linker 10 alkyl chain region (bottom). Because the Ruthenium(II) centre is bound to one

side of the ‘Click Ligand’, the alkyl chain becomes asymmetrical. Each unique hydrogen

environment has been highlighted by a different colour.

(For full 1H NMR spectrum of 10 and 14, see Appendix D, 1.5

and Appendix D, 1.6 respectively)



49

Coordination of a polypyridyl ruthenium(II) centre to one side of the alkyl

linked ligand 10 causes the polymethylene chain to lose its symmetry as the

hydrogens become inequivalent. Each unique hydrogen environment has been

highlighted by a different colour. The alkyl region for complex 14 shows the addition

of new peaks which are equivalent to that of the uncomplexed ‘free’ ligand 10 in

terms of splitting but are shifted to a new ppm value. The presence of these new peaks

suggested an asymmetric complex has formed which would support the argument that

complex 14 has been synthesised, albeit in the presence of at least two by-products

(Table 3).

Additionally, the comparison of the UV-vis data with a reported

[Ru(phen)2(pyta)]2PF6- complex 15 by Ghosh et al96 (Figure 45/ Table 4) provides

further evidence that the desired complex 14 has been synthesised. Reported UV-vis

data of cis-[Ru(phen)2Cl2] 8 (552 nm)100 shows that the UV data from 14 is not due to

any unreacted starting material.

Figure 45 Recorded UV-vis absorption data for complex 14 and reported UV-vis absorption

data for complex 15 by Ghosh et al96

(For full spectrum, see Appendix D, 1.6)

Table 5 Comparison of UV-vis absorption peaks for synthesized complex 14 and reported

complex 1596

14 Observed UV-vis Peaks

(Absorbance)

15 Reported UV-vis Peaks

(Absorbance)96

288 262

400 403

449 445



50

One potential by-product in the synthesis of complex 14 is the coordination of

a polypyridyl ruthenium(II) centre on both ends of the alkyl linked ligand 10 (Figure

46). Although flow injection ESI-MS of the crude solid did not contain the desired

[M]+ peak for complex 14, the presence of this potential by product was suggested

(Figure 46).

Figure 46 Flow Injection ESI-MS of crude solid recorded (left) and the predicted isotope

pattern for the suspected dinuclear ruthenium(II) by-product (Right) Predicted isotope

patterns acquired from http://www.sisweb.com80

(For full ESI-MS, See Appendix D, 1.6)

However, the omission of certain peaks in addition to the lack of a distinct

ruthenium(II) isotope pattern disputes this claim.

5.2.2 Complexation utilising Pyta ‘Click’ Ligand 10 and Cobalt(III)

triflate complex 7

By using the cobalt(III) triflate salt 7 (Chapter 2), it should be possible to

coordinate this complex to one side of the ‘click’ ligand 10 by modulating the molar

ratios of each compound (1: 2 respectively). The rationale behind the synthesis of a

cobalt(III) triflate salt 7 was that it was considerably more soluble in organic solvents.

The cobalt(III) chloro salt 6 was insoluble in most solvents and required significant

heating to dissolve. This is not desirable as we can predict that heating will break the



51

complex apart. As well as this, it has been reported that triflate ligands are weakly

coordinating to a cobalt(III) centre compared to analogous chloro and nitrite ligands49.

We can therefore undergo complexation reactions under benign conditions.

For the complexation of 10 to 7, a method proposed by Hartshorn et al62

seemed the most appropriate (Figure 47). The reaction was conducted in

CHCl3/Butanol (1:1) as this was the only solvent system the ‘click’ ligand 10 would

dissolve in. After stirring at 40 oC for 3 hours, the reaction mixture was added to an

excess of methanolic NH4PF6, filtered and dried to yield a pale orange/yellow solid.

Figure 47 Schematic representation of Synthesis of [Co(ceen)2(pyta)] 16 (top) and visual

observations (bottom)

(For full experimental details, see Appendix A:5.2)

The crude Complex 16 was analysed by one dimensional (1D) (1H, 13C), two

dimensional (1H-1H COSY, HSQC) NMR (Appendix D, 1.7) and (ESI-MS). However

unlike the data for the Ruthenium ligand complex 14, the data for crude 16 was

inconclusive. ESI-MS data showed the presence of free ligand 10 and by products that

could not be characterised whilst 1H NMR displayed a broadening of signals, possible

due to the formation of cobalt(II) paramagnetic compounds. A reason for this may be

due to the intrinsic instability of complex 7 resulting in immediate dissociation under

gentle heating. For future reactions, it may be useful to use more stable cobalt(III)

complexes (Future work, 6.6)



52

5.3 Conclusion

Despite the evidence suggesting that Complex 14 has been synthesised, this

can only be tentatively suggested. This is because only a crude solid was analysed and

therefore gave us inconclusive evidence. The UV-vis spectrum of 14 against a

reported compound was the most promising analytical data. Before any further

analysis of the ruthenium(II) complex 14 is conducted, the product must be separated

using a reported method for analogous compounds99. The synthesis of a stable

cobalt(III)(pyta) heteroleptic type complex was unsuccessful. This is most likely due

to the dissociation of the triflate cobalt(III) complex 7 deriving from its intrinsic

instability. For future reactions, it may be beneficial to use non-toxic analogues to

complex 7 such as [Co(en)2(OTf)2](OTf) for coordination to our alkyl linked ligand

10. The rationale behind this is ethylene diamine (en) will has a similar binding

domain to bidentate nitrogen mustard ligands64. We can therefore increase the scale of

the reaction whilst mitigating any safety hazards associated with using a toxic

mustard ligand. Using a non-toxic mustard ligand is ultimately more suitable to

finding successful reaction conditions. Additionally, the research into more stable

cobalt(III) cytotoxins has been explored in the future work (Future work 6.6). One

such example is using a tridentate nitrogen ligand that due to chelate effects, will be

more stable to substitution and therefore less likely to decompose.

Chapter 6 Future Work


53

Chapter 6:

Future Work



54

6.1 Uncompleted Experimentation Ultimately due to time constraints and synthetic difficulties, we were unable to

successfully synthesise and characterise a stable ruthenium(II)-cobalt(III) cytotoxin

outlined by Aim 1 (Figure 1). We have, however, synthesised the composite

substituents that would make up our proposed dinuclear species. Results from Chapter

5 would tentatively suggest that the alkyl bridging ‘click’ ligand 10 had coordinated

to a polypyridyl ruthenium(II) centre but further reactions and purifications would

have to be completed to clarify this. After finding an appropriate method to synthesise

complex 14, we would then want to coordinate this to cobalt(III) complex 7 to

complete the synthesis of a ruthenium(II)-cobalt(III) hypoxia selective cytotoxin.

Using the information gleaned from the study, a proposed reaction mechanism to

generate a stable ruthenium(II)-cobalt(III) cytotoxin has been outlined (Figure 48)

Figure 48 Proposed Reaction Pathway to a ruthenium(II)-cobalt(III) cytotoxin using results

from the study



55

Once an effective synthetic pathway to a ruthenium(II)/cobalt(III) cytotoxin has been

outlined, a series of complexes could be generated by altering: the length of

methylene bridge between the two complexes and the type of mustard agent/cytotoxin

bound to the cobalt(III) (Figure 50).

Alternative cobalt(III) chaperones have been explored in Section 1.3.2.

After generating a library of cytotoxins, we can employ various techniques to test the

compound’s efficacy as a potential hypoxia-selective cytotoxin. These are briefly

outlined below.

Figure 50 Schematic showing how a library of ruthenium(II)/cobalt(III) cytotoxins can be

generated by altering: the length of methylene bridge and using a different cobalt(III) payload



56

6.2 Lipophilicity

The lipophilicity of our proposed dinuclear cytotoxins could be measured as

the n-octanol/water partition coefficient, log P, by applying the shake flask method101.

UV/vis spectroscopy can then be used to determine the concentration of the

complexes in the organic and aqueous phases.

6.3 Electrochemical Studies

Ruthenium(II)/cobalt(III) dinuclear cytotoxins can exert selectivity towards

hypoxic regions of tumour cells because the cobalt(III)/cobalt(II) redox couple falls

within the range for in vivo bioreduction by endogenous reductases8. Research

conducted by Denny et al28 found that the cobalt(III/cobalt(II) redox couple of a

cobalt(III) complex coordinated to a bidentate mustard agent could be altered

depending on the ancillary ligands present in the complex in addition to the mustard

agent. Electrochemistry is a technique that could be utilised to elucidate the oxidation

and reduction potentials for the metal centres within our proposed dincuclear

complex. The information gleaned from these studies would help indicate whether our

ruthenium(II)-cobalt(III) complexes have the suitable reduction potential for in vivo

reduction within a hypoxic environment.

6.4 Understanding Cellular Uptake and Cell death

mechanisms

As our proposed ruthenium(II)/cobalt(III) dinuclear cytotoxins are potential

chemotherapeutics, an understanding of cellular uptake and localisation is vital to the

progression of these drugs. A study by Pisani et al51 systematically analysed the

uptake mechanism and localisation of a series of dinuclear alkyl bridged, polypyridyl

ruthenium (II) complexes (Figure 9, Section 1.4). Many routes for cellular entry were

examined but ultimately excluded as genuine uptake methods. These included: energy

dependant (requiring ATP) mechanisms, uptake via the assistance of organic cation

transporters (OCTs) and finally the exploitation of the plasma membrane potential (-



57

50 to -70mV102) as a driving force into cells. The research concluded that the cellular

uptake of these complexes was mediated by passive diffusion (energy-independent)

that was assisted by the lipophilicity of the compounds in the cancerous L1210 cell

line51. Once in the cell, it was reported that the dinuclear ΔΔ- Rubb16 complex induced

cell death by apoptosis51. This is in agreement with a study conducted by Chen et al103

who concluded that a dinuclear ruthenium(II) complex induced mitochondria-

mediated apoptosis. By using analogous conditions reported by these publications, it

would be possible to test the cellular uptake and cell death mechanisms of our

ruthenium(II)/cobalt(III) cytotoxins.

6.5 Controlling Stereochemistry

Throughout this study, no attempt at controlling the stereochemistry of the

complexes was taken. Further studies at attempting this may prove useful, as the

relative orientation of the ligands around the metal centre could prove important. It

was initially reported by Dwyer et al84 that Δ- and Λ- [Ru(phen)3]2+ exhibited

different biological activity whilst Keene et al46 noted that the ΔΔ isomer of a series

of dinuclear ruthenium(II) complexes where more cytotoxic to the leukemia cell line

than the analogous ΛΛ isomers. It has also been previously reported that the DNA

interaction with ruthenium(II) complexes depends on the configuration of the

ruthenium metal centre108.

6.6 Developing a series of more Stable Cytotoxins

One synthetic challenge we had during the design of a hypoxic selective

cytotoxin was forming the nitrogen mustard bound cobalt(III) triflate complex 7.

Whilst an experimental procedure devised by Downward and collaborators suggested

we had synthesised the desired triflate complex76,64, the exothermic nature of the

reaction most likely caused a large proportion of the cobalt(III) complex to dissociate

into free ligand and cobalt(II) ions. One way to circumvent the dissociation of the

nitrogen mustard ligand from the cobalt(III) centre is to develop new cytotoxins that

bind to the metal centre more strongly. We postulate that this additional strength

could be achieved through the use of tridentate or even tetradentate ligands. Denny et



58

al28 have reported the coordination of a tridentate mustard N,N-Bis(2-

Chloroethyl)diethylenetriamine (DCD) to a cobalt(III) metal centre agent as well as

non-toxic equivalents including N-(2-aminoethyl)ethane-1,2-diamine (dien) and N,N-

Diethyldiethylenetriamine (DED). The cytotoxicity and hypoxic selectivity of these

complexes was compared against a cobalt(III) complex coordinated to a bidentate

mustard agent (DCE) (Figure 51).

Figure 51 A group of tridentate ligands that were coordinated to a cobalt(III)centre and

cytotoxicity was tested against a previously reported bidentate (DCE) cobalt(III)complex28

Initially, it was shown that the tridentate DCD cobalt(III) complex 1 was

significantly less toxic(IC 50/ µM 750) than the free DCD mustard agent (IC50/µM 50)

against the AA8 cell line, signifying that complexation results in successful

deactivation of the mustard. It was also elucidated through electrochemical studies

that the tridentate mustard complex 1 had a more positive reduction potential (-680

mV) compared to the bidentate mustard complex 2 (-780 mV). Most importantly, the

tridentate mustard complex 1 exhibited less hypoxic selectivity than the bidentate

mustard complex 2. It was further reported that cobalt(III) complexes containing

tridentate mustards were synthesised in low yields due to the coordination of a bulky

tertiary amine moiety onto the metal centre. This finding was observed within our

project when coordinating non-toxic tertiary amine ligands such as TMEDA to a

cobalt(III) centre (See Section 2.2).

We can conclude from this study that although tridentate mustards will be

potentially more stable to convert into their corresponding triflate salts due to chelate

effects, we have accept the potential negative ramifications of reduced hypoxia

selectivity, modification to the reduction potential and lower yielding reaction

pathways.



59

6.7 Pyridyl 1,2,3 Triazole Ruthenium(II) Systems

As mentioned in previous chapters, ruthenium(II) polypyridyl systems have

been used to interact with DNA. Researchers have exploited this to find interesting

applications such as light-switch DNA probes, DNA photocleavers, DNA

luminenscence markers and anticancer drugs104. This interest nucleates from their

high thermodynamic ability in conjunction with their predicatable electrochemical,

photochemical and photophysical properties104. Despite the burgeoning research into

the DNA binding properties of ruthenium(II) polypyridyl systems, the chemical

properties of ruthenium(II) complexes coordinated to a ‘click’ 2-pyridyl-1,2,3-triazole

ligand is seemingly in its infancy. In 2014, Ghosh et al96 synthesised a

[Ru(phen)2(pyta)]2+ complex 15 (Chapter 5) that functioned as a luminescence sensor

for H2PO4-/HP2O7

3- anions whilst in 2015, Crowley et al105 designed a series of di

nuclear ruthenium(II) triply stranded helicates deriving from a bis-bidentate ‘click’ 2-

pyridyl-1,2,3-trizole ligand scaffold that exhibited antimicrobial activity in vitro

against Gram positive and Gram negative microorganisms (Figure 52). Despite a

modest response, they concluded increasing the overall hydrophobicity of the

complex will lead to a more potent antimicrobial agent. The corollary between

hydrophobicity and cytotoxicity was a concept utilised in our study.

Figure 52 Ball and Stick model of triply stranded ruthenium(II)helicates bound by bis

bidentate ‘click’ 2-pyridyl-1,2,3-triazole ligand that exhbit antimicrobial activity121

To our knowledge, no DNA binding studies have been conducted using

ruthenium(II) polypyridyl systems that are coordinated to ‘click’ 2-pyridyl-1,2,3-

triazole ligands so this provides an exciting alternative pathway for the future of this

study.

Chapter 7 Conclusion


60

Chapter 7:

Conclusion



61

The first aim of this project was:

‘ To successfully design, synthesise and characterise through appropriate rationale an

alkyl bridged dinuclear ruthenium(II)/cobalt(III) nitrogen mustard cytotoxin that

exhibits selectivity towards hypoxic cells over healthy cells (Figure 1)’

In order to achieve this proposed dinuclear complex, numerous synthetic

challenges needed to be satisfied. The first of which was the design of a stable

cobalt(III) mustard agent (Chapter 2). The reason for using mustard agents is that

their alkylating reactivity is minimised when bound to a metal centre. Using reported

methods, we were able to successfully show how a non-toxic alcohol precursor bound

to a cobalt(III)centre could be converted to its toxic mustard analogue upon treatment

with SOCl2 thereby completing the second aim of this study. The final step, before

complexation to a functionalised 2-pyridyl-1,2,3-triazole ligand is the formation of

the triflate compound upon reaction with triflic acid (Figure 53).

Figure 53 Formation of a Cobalt(III) mustard triflate from a non-toxic hydroxy analogue

In Chapter 3, we successfully synthesised a ruthenium(II) complex 8 that

could be used for complexation to a 2-pyridyl-1,2,3-triazole ligand. Unfortunately, the

final product was marred with numerous by products but could be improved if the



62

reaction was conducted for a longer duration.

In Chapter 4, the synthesis of a novel and unpublished alkyl linked bis-

bidentate 2,pyridyl-1,2,3-triazole ligand 10 was synthesised and fully characterised

(Figure 54). Because functionalization of these N-chelating ‘Click’ ligand scaffolds is

significantly easier than traditional bipyridine ligands, the properties of the linker can

be easily modulated for a wide range of exciting applications. Additionally, the third

aim of this study has been satisfied and this lipophilic ligand can be used as the

‘linker’ moiety in our proposed ruthenium(II)-cobalt(III) cytotoxin (Figure 1).

Figure 54 Schematic displaying the synthetic pathway utilized to generate an alkyl linked bi-

bidentate 2,pyridyl-1,2,3-triazole ligand (10) using a two step copper(I) catalyzed CuAAC

reaction methodology.

Finally in Chapter 5, we attempted the complexation of the pyta ligand 10

with ruthenium(II) complex 8 as well complexation of 10 with the cobalt(III) mustard

triflate salt 7. Although the resulting ruthenium(II) complex 14 was a crude sample

and therefore difficult to analyse, UV/vis analysis compared with reported data would

suggest coordination had occurred. Additionally, ESI-MS data tentatively suggested

that a di nuclear ruthenium(II) complex has been synthesised but further analysis will

have to be undergone for complete clarification but initial data is promising. The data

for complex 16 was less promising and this was most likely a result of the rapid

decomposition of the cobalt(III) mustard triflate complex 7 under heating.

Ultimately, our primary aim for this project was not achieved but this research

has outlined significant advances in the field of bioreductive transition metal

complexes as potential hypoxia-selective cytotoxins.

Appendices


63

Appendices

Appendices


64

Appendix A: Experimental

Chapter 2

2.1

[Cu(dmbpy)(TMEDA)2]2BF4 (2)

SECTION 2.2.2.1 of main body

Dimethyl bipy (dmbpy) (185.5 mg, 1 mmol) was dissolved in 4 ml ethanol.

This was added to an aqueous solution (5 ml) of Cu(BF4)2.6H2O (1 mmol, 340 mg)

and the reaction was stirred for an hour. TMEDA (1.5 mmol, 0.2399 ml) was added

dropwise to the stirring reaction mixture and stirred for an hour. Ethanol was removed

in vacuo and the solid was recrystallized in DMF/ether into its final blue appearance.

Yield 359 mg. FAILED REACTION

Appendices


65

2.2

[Co(en)2Cl2]Cl (3)


CoCl2.6H2O(2.0 g, 8.40 mmol) was dissolved in 5 ml Water. To this, 7.5 ml of

10 % ethylene diamine (890 mg, 15 mmol) was added and the reaction was stirred for

40 minutes whilst a vigorous stream of air was drawn through the solution. After 40

minutes, 12 ml conc HCl was added and the reaction mixture was stirred. Reaction

mixture was then filtered and washed with 8 ml 6 M HCl, EtOH and Et2O and dried

to yield a crystalline green solid. Yield 610 mg (29 %)

2.3

[Co(TMEDA)2Cl2]Cl


CoCl2.6H2O (2.0 g, 8.40 mmol) was dissolved in 5 ml Water. To this, 7.5 ml

of 10 % TMEDA (1.743 g, 15 mmol) was added and the reaction was stirred for 40

minutes whilst a vigorous stream of air was drawn through the solution. After 40

minutes, 12 ml conc HCl was added and the reaction mixture was stirred. Upon

addition of the acid, solution went from a purple colour to dark blue. Reaction

Appendices


66

mixture was then filtered and the precipitate was washed with MeOH and dried to

yield a dark blue crystalline solid. Yield 700 mg. FAILED REACTION

2.4

[Co(HEEN)2(NO2)2]NO3 (5)


Co(NO3)2.6H2O (11.64 g, 39.9 mmol) and NaNO2 (6 g, 86.95 mmol) were

dissolved in 20 ml H2O and purged with argon. To this was added HEEN ligand (8.3

g, 79.69 mmol) which was dissolved in 10 ml H2O and 3 ml conc HNO3. A vigorous

stream of air was drawn through the solution whilst stirring. The reaction flask was

placed in a freezer overnight. The reaction mixture was then filtered to yield orange

crystals. Yield 10 g (59 %). LRMS (ES+) m/z [M+] 358.8

2.5

[Co(CEEN)2(NO2)(Cl)]Cl (6)


[Co(HEEN)2(NO2)2]NO3.H2O (1.8 g, 4.28 mmol) was placed in 50 ml RBF.

Appendices


67

20 ml SOCl2 was then slowly added whilst stirring. 1 ml of DMF was added dropwise

to facilitate dissolution of the solid. The reaction was noticeably exothermic. After

stirring, the solution was dark pink. A vigorous stream of air was run through the

solution to allow evaporation of SOCl2. The pink slurry was recrystallized in

EtOH/H2O. After cooling and filtration, the final solid was a pink colour. Yield 1.17 g

(66 %) 13C NMR (100 MHz, CDCl3CD3OD 1:1) δ 52.56, 51.48, 42.73, 40.13 LRMS

(ES+) m/z 385 [Co(ceen)2(NO2)(Cl)]+, 396 [Co(NO2)(CEEN)2]+

2.6

[Co(CEEN)2(OTf)(Cl)]OTf (7)


[Co(CEEN)2(NO2)(Cl)]Cl (6) was placed in a 25 ml RBF and cooled using a

salt-ice bath to -20 OC. HOTf was added dropwise whilst stirring until the solid was

covered. The reaction mixture was then stirred under vacuum in an ice salt bath for 45

minutes. Upon completion, the green solution was added dropwise to a stirring

solution of ether (100 ml). The solution was then filtered and dried to yield a green

solid. 1H NMR (500 MHz, CD3CN) δ 5.40 (br m, 2H), 5.18 (br s. 1H), 4.09-3.86 (m,

2H), 3.11 (m, 3H), 3.00 (m, 2H), 2.73 (m, 1H)

Appendices


68

Chapter 3

3.1

[Ru(phen)2Cl2]2+ (8)

SECTION 3.2.1 of main body

RuCl3.H2O (1 g, 2.5 mmol) was added to a dry 100 ml RBF. To this, 1,10

phenanthroline ( 5 mmol, 901.1 mg) was added. 50 ml DMF was added and the

reaction was heated under reflux for 3 hours under a nitrogen atmosphere. The

solution was cooled overnight at 0 oC. 30 ml ethyl acetate was added to the flask to

allow for precipitation. After filtering, the resulting red solid was washed with 30 %

LiCl. Solid was recrystallized in EtOH and filtered and washed with Ice cold EtOH to

produce a final black solid. Yield 940 mg (70.6 %). LRMS (ES+) [M]+ 532

Appendices


69

Chapter 4

4.1

1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (10) through a

One step CuAAC approach


Safety Note: NaN3 is an extremely toxic substance and therefore appropriate

precautions should be taken. Low molecular weight organic azides are potential

explosives, so care is taken at all stages of handling127. Equally, CuI and CuII azides

are highly explosive materials so the precautionary measures for these reactions must

be taken58,59. A standard blast shield was used during ALL one step Click reactions.

Before the CuAAC products are dried, the crude reaction mixture was poured into the

NH4OH/EDTA solution detailed below. Any piece of equipment that had come into

contact with the toxic NaN3 went through a rigorous neutralisation and disposal

process107.

To a stirred solution of 1,12-dibromododecane (1.5 mmol, 0.497 g, 1 eq) in

DMF/H2O (20 ml, 4:1) was added NaN3 (3.18 mmol, 0.209 g, 2.10 eq),

Na2CO3(1.121 mmol, 0.128 g, 0.8 eq), CuSO4.H2O (0.605 mmol, 0.151 g, 0.4 eq) and

ascorbic acid (1.212 mmol, 0.213 g, 0.80 eq). 2-ethynylpyridine (3.105 mmol, 0.320

g, 2.05 eq) was then added to the reaction micture and the resulting mixture was

stirred at 80 OC for 21 hours. The reaction mixture was a bright orange colour upon

addition of all substituents. Upon completion, the reaction mixture was partitioned

between aqueous NH4OH/EDTA solution (200 ml) and EtOAc (200 ml) and the

layers were separated. Organic fractions were further extracted with EtOAc (3 x 150

ml). All the organic fractions were combined and washed with NH4OH/EDTA

solution (200 ml), water (200 ml) and brine (150 ml). Yellow precipitate was filtered

Appendices


70

and the solvent was removed in vacuo to yield a pale yellow solid. The product spot

was then further purified by chromatography (10% acetone in CH2Cl2 then 30%

acetone in CH2Cl2. Yield 120 mg (17 %) 11 LRMS (ES+) 459.1 [M+H]+

4.2


Two-step CuAAC approach using 1,12-dibromodododecane


1,12-diazidododecane 12

To a stirred solution of 1,12-dibromododecane (2.44 mmol, 800 mg, 1 eq) in

DMF (12 ml) was added NaN3 (12.19 mmol, 792 mg, 5 eq) and the reaction was

stirred overnight. Upon completion, the reaction mixture was diluted with water (50

ml) and then extracted with ether (4x 30 ml). Organic layers were washed with brine

and then concentrated. The final compound was a yellow liquid.

1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane 10




are highly explosive materials so the precautionary measures for these reactions must

be taken58,59. Before the CuAAC products are dried, the crude reaction mixture was

poured into the NH4OH/EDTA solution detailed below. Any piece of equipment that

had come into contact with the toxic NaN3 went through a rigorous neutralisation and

disposal process107.

Appendices


71

To a stirred solution of 1,12-diazidodocane (3.17 mmol, 800 mg, 1 eq) in t-

BuOH/H2O (12 ml, 1:1) was added ascorbic acid (6.34 mmol, 1.1166 g, 2 eq),

Na2CO3 (6.34 mmol, 1.256 g, 2 eq), CuSO4.H2O (3.17 mmol, 0.7915 g, 1 eq) and 2-

ethynlpyridine (6.974 mmol, 0.719 g, 2.20 eq). The reaction mixture was then stirred

at RT for 48 hours. Upon completion, the solution was a bright orange colour.

Reaction mixture was then added to saturated NH4OH/EDTA solution (150 ml) and

isopropanol/CH2Cl2 (1:3, 2 x 150 ml) was used to extract the organic fractions.

Organic fractions were then collected and washed with further saturated

NH4OH/EDTA solution (3 x 150 ml) to remove all traces of copper. Organic fractions

were then dried with Na2SO4 and filtered. White Solid from the filtrate contained pure

product and no chromatography was required.

Yield 660 mg (59 %) 1H NMR (400 MHz, CDCl3/CD3OD 1:1) δ 8.53 (d, J= 4, 2H,

Ha), 8.27 (s, 2H, He) 8.09 (d, J= 8, 2H, Hd), 7.87-7.83 (m, 2H, Hc), 7.33-7.30 (m, 2H,

Hb), 4.43 (t, J= 8, 4H, Hf), 1.96-1.93 (m, 4H, Hg), 1.32-1.24 (m, 16H, Hh) 13C NMR

(100 MHz, CDCl3CD3OD 1:1) δ 150.49, 149.82, 148.22, 138.56, 124.10, 123.37,

121.35, 51.40, 30.92, 30.11, 30.04, 29.66, 27.11 LRMS (ES+) 459.1 [M+H]+ , 481

[M+Na]+

Appendices


72

4.3


Two-step CuAAC approach using 1,12-diiodododecane


1,12-diiododecane 13

1,12-dibromododecane (6.124 mmol, 2.0098 g, 1 eq) was added to degassed

acetone (20 ml) and NaI (24.496 mmol, 3.671 g, 4.00 eq). The reaction mixture was

refluxed under a nitrogen atomosphere for 24 h. The solvent was removed in vacuo to

yield a cluster of yellow crystals which were recrystallized in acetone. The final solid

was a white colour. Yield 1.4 g (54 %) LRMS (ES+) m/z [M]+ 422 [M+H]+ 423

[M+2H]+ 211

1,12-diazidododecane 12

To a stirred solution of 1,12-diiodododecane (1.14 mmol, 600 mg, 1 eq) in

DMF (12 ml) was added NaN3 (7.105mmol, 461 mg, 6 eq) and the reaction was

stirred overnight. Upon completion, reaction mixture was diluted with water (50 ml)

and extracted with ether (4x 30 ml). Organic layers were washed with brine and

concentrated. The final compound was a yellow liquid

Appendices


73

1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane 10




are highly explosive materials so extra precautionary measures for these reactions

must be taken58,59. Before the CuAAC products are dried, the crude reaction mixture

was poured into the NH4OH/EDTA solution detailed below. Any piece of equipment

that had come into contact with the toxic NaN3 went through a rigorous neutralisation

and disposal process107

.

To a stirred solution of 1,12-diazidodocane (2.37 mmol, 600 mg, 1 eq) in t-

BuOH/H2O (12 ml, 1:1) was added ascorbic acid (4.74 mmol, 0.8348 g, 2 eq),

Na2CO3 (4.74 mmol, 0.939 g, 2 eq), CuSO4.H2O (2.37 mmol, 0.592 g, 1 eq) and 2-

ethynlpyridine (5.231 mmol, 0.539 g 2.20 eq). The reaction mixture was then stirred

at RT for 48 hours. Upon completion, the solution was a bright orange colour.

Reaction mixture was then added to a saturated NH4OH/EDTA solution (150 ml) and

isopropanol/CH2Cl2 (1:3, 2 x 150 ml) was used to extract the organic fractions.

Organic fractions were then collected and washed with further saturated

NH4OH/EDTA solution (3 x 150 ml) to remove all traces of copper. Organic fractions

were then dried with Na2SO4 and filtered. The white solid from filtrate contained pure

product and no chromatography was required. Yield 435 mg (83.3 %) 1H NMR (400

MHz, CDCl3/CD3OD 1:1) δ 8.46 (d, J= 4, 2H, Ha), 8.21 (s, 2H, He) 8.02 (d, J= 8, 2H,

Hd), 7.82-7.77 (m, 2H, Hc), 7.27-7.24 (m, 2H, Hb), 4.44 (t, J= 8, 4H, Hf), 1.98-1.91

(m, 4H, Hg), 1.32-1.24 (m, 16H, Hh). LRMS (ES+) [M+H]+ 459.1, 481 [M+Na]+

Appendices


74

Chapter 5

5.1

[Ru(phen)2(pyta)]PF6 Complex (14)


1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane (pyta) (183 mg, 0. mmol) was

suspended in 4 ml EtOH/H2O (1:1) in a microwave vial. To this was added solid cis-

[Ru(phen)2Cl2] (106 mg, 0.2 mmol) and the resulting mixture was heated at 120 OC

under microwave irradiation for 45 minutes. The resulting deep red solution with a

small precipitate was added to an aqueous solution of excess NH4PF6 to produce a

bright orange precipitate. Filtering of the precipitate yielded a crude orange solid.

Weight 426 mg.

Appendices


75

5.2

[Co(CEEN)2(pyta)]PF6 Complex (16)


[Co(CEEN)2(NO2)(OTf)]OTf (0.05 mmol, 31.9 mg, 1 eq) and 1,12-bis(pyridine-2-yl)-

1H-1,2,3-triazol-1-yl)dodecane (pyta) (1 mmol, 50 mg, 2 eq) were placed in an

aluminium foil-wrapped 25 ml RBF. 2 ml CHCl3/Butanol (1:1) was added and the

flask was stoppered and allowed to stir for 3 hours at 40 OC to aid dissolution of the

ligand. Upon completion, the solution was pipetted in 4 ml saturated methanolic

NH4PF6 diluted in 60 ml H2O. An orange precipitate began to form. The reaction

mixture was then cooled in ice for 5 minutes and filtered cold and washed with ice

cold EtOH and ether. Final solid was a pale orange colour. Yield 23 mg

Appendices


76

Appendix B: X-Ray Crystal Data

1.1 Tetranuclear-Copper Complex (2) (SECTION 2.2.2.1)

Identification code jk2014_sg2

Empirical formula C23 H25 B2 Cu2 F8 N5 O3

Formula weight 720.18

Temperature 100(2) K

Wavelength 0.71075 Å

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 8.705(4) Å a= 62.79(2)°

b = 13.210(4) Å b= 82.11(3)°

c = 13.606(4) Å g = 79.12(3)°

Volume 1364.1(9) Å3

Z 2

Density (calculated) 1.753 Mg / m3

Absorption coefficient 1.651 mm-1

F(000) 724

Crystal Plate; Blue

Crystal size 0.300 x 0.200 x 0.100 mm3

Theta range for data collection 2.751 - 29.719°

Index ranges -10 <= h <= 12, -17 <= k <= 16, -16 <= l

<= 18

Reflections collected 12910

Independent reflections 6737 [R(int) = 0.0654]

Completeness to theta = 25.242° 99.7 %

Absorption correction Semi-empirical from equivalents

Max. and min. transmission 1.000 and 0.791

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 6737 / 2 / 398

Goodness-of-fit on F2 1.296

Final R indices [F2 > 2sigma(F2)] R1 = 0.0922, wR2 = 0.2251

R indices (all data) R1 = 0.1117, wR2 = 0.2389

Extinction coefficient n/a

Largest diff. peak and hole 1.031 and -1.116 e Å!3

Appendices


77

1.2 Trans-[Co(en)2(Cl2)]Cl (3) (SECTION 2.2.3.1)

Identification code sg4

Empirical formula C2 H10 Cl2 Co0.50 N2 O




Crystal system Monoclinic

Space group P 21/c

Unit cell dimensions a = 10.690(4) Å a= 90°

b = 7.838(3) Å b= 110.880(8)°

c = 9.041(4) Å g = 90°

Volume 707.7(5) Å3

Z 4


Absorption coefficient 1.957 mm!1

F(000) 366

Crystal Prism; Green



Index ranges -13 <= h <= 13, -10 <= k <= 10, -11 <= l

<= 11













Appendices


78

1.3 Pronated TMEDA and [CoCl4]2- Cluster (SECTION 2.2.3.2)

Identification code sg3

Empirical formula C12 H36 Cl8 Co2 N4





Space group P -1


b = 8.2223(14) Å b= 79.317(6)°

c = 13.398(2) Å g = 69.265(5)°

Volume 671.6(2) Å3

Z 1



F(000) 326

Crystal Prism; Blue



Index ranges -8 <= h <= 8, -10 <= k <= 10, -17 <= l <=

17













Appendices


79

1.4 Trans-[Co(HEEN)2(NO2)2]NO3 (5) (SECTION 2.2.3.3)

Identification code 2015srg4_4

Empirical formula C8 H26 Co N7 O10





Space group P -1


b = 9.667(11) Å b= 87.36(7)°

c = 11.579(18) Å g = 62.26(5)°

Volume 870(2) Å3

Z 2



F(000) 460

Crystal Block; Yellow



Index ranges -13 <= h <= 13, -13 <= k <= 13, -15 <= l

<= 16













Appendices


80

Appendix C: List of Notable Compounds Made

Cmpd No./ Section No.

Structure Name

3/2.2.3.1

Trans-[Co(en)2(Cl)2]

5/2.2.3.3

Trans-[Co(HEEN)2(NO2)]NO3-

6/2.2.3.4

Trans-[Co(CEEN)2(NO2)(Cl)]Cl-

7/2.2.3.5

Trans-[Co(CEEN)2(OTf)(Cl)]OTf-

8/3.2.1

Cis-[Ru(phen)2Cl2]

10/4.2.3

1,12-bis(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-

yl)dodecane

12/4.2.2,4.2.3

1,12-diazidododecane

Appendices


81

13/4.2.3

1,12-diiodododecane

14/5.2.1

[Ru(phen)2(pyta)]2+

16/5.2.2

[Co(CEEN)2(pyta)]3+

Appendices


82

Appendix D: Notable Spectroscopic Data

1.0 Complex 5 ESI-MS

1.1 Complex 6 13C NMR and ESI-MS

Appendices


83

1.2 Complex 7 1H NMR

Appendices


84

1.3 Complex 7 19F NMR

1.4 Ligand 10 from 1,12-Dibromododecane 1H NMR

Appendices


85

1.5 Ligand 10 from 1,12-Diiodododecane 1H NMR, 13C NMR, COSY

and ESI-MS

Appendices


86

Appendices


87

1.6 Complex 14 1H NMR, UV-vis and ESI-MS

Appendices


88

Appendices


89



Appendices


90

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