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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
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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.
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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Chapter 1:
Introduction
Chapter 1 Introduction
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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.
Chapter 1 Introduction
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Chapter 1 Introduction
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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-
Chapter 1 Introduction
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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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.
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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
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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.
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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.
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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
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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
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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).
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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
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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.
Chapter 1 Introduction
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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Chapter 2:
Synthesis
and
Characterisation
of Some Copper(II)
and
Cobalt(III) Complexes
PAYLOAD
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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.
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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
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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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.
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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
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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
(For full experimental details, see Appendix A: 2.3)
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)
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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.
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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).
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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)
(For full experimental details, see Appendix A: 2.5)
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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)
(For full experimental details, see Appendix A: 2.6)
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)
Chapter 2 Synthesis and Characterisation of some Cu(II) and Co(III) complexes
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
31
Chapter 3:
Synthesis
and
Structural
Characterisation
of a
Polypyridyl
Ruthenium(II)
Complex
WARHEAD
Chapter 3 Synthesis and structural characterisation of a polypyridyl Ru(II) complex
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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.
Chapter 3 Synthesis and structural characterisation of a polypyridyl Ru(II) complex
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
33
3.2 Results and Discussion
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
(For full experimental details, see Appendix A: 3.1)
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
Chapter 3 Synthesis and structural characterisation of a polypyridyl Ru(II) complex
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
35
Chapter 4:
Synthesis
and
Characterisation
of an
Alkyl Bridged
2-pyridyl-1,2,3-triazole
ligand
LINKER
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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.
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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.
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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.
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
39
4.2 Results and Discussion
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).
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
(For full experimental details, see Appendix A: 4.1)
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
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
(For full experimental details, see Appendix A: 4.2)
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
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
(For full experimental details, see Appendix A: 4.2)
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.
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
(For full experimental details, see Appendix A: 4.3)
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 %).
Chapter 4 Synthesis and characterisation of an alkyl bridged 2-pyridy-1,2,3-triazole ligand
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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.
Chapter 5:
Attempts
at
Heterodinuclear
Ruthenium(II)-Cobalt
(III) Complexes
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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 Results and Discussion
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).
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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)
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
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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)
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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
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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
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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)
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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
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Chapter 6:
Future Work
Chapter 6 Future Work
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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
Chapter 6 Future Work
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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
Chapter 6 Future Work
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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 (-
Chapter 6 Future Work
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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
Chapter 6 Future Work
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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.
Chapter 6 Future Work
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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
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Chapter 7:
Conclusion
Chapter 7 Conclusion
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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
Chapter 7 Conclusion
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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
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Appendices
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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2.2
[Co(en)2Cl2]Cl (3)
SECTION 2.2.3.1 of main body
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
SECTION 2.2.3.2 of main body
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
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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)
SECTION 2.2.3.3 of main body
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)
SECTION 2.2.3.4 of main body
[Co(HEEN)2(NO2)2]NO3.H2O (1.8 g, 4.28 mmol) was placed in 50 ml RBF.
Appendices
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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)
SECTION 2.2.3.5 of main body
[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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
SECTION 4.2.1 of main body
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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
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
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. 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.
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
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
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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1,12-bis(pyridine-2-yl)-1H-1,2,3-triazol-1-yl)dodecane 10
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 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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
74
Chapter 5
5.1
[Ru(phen)2(pyta)]PF6 Complex (14)
SECTION 5.2.1 of main body
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.
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5.2
[Co(CEEN)2(pyta)]PF6 Complex (16)
SECTION 5.2.2 of main body
[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
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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
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Formula weight 178.48
Temperature 170(2) K
Wavelength 0.71073 Å
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
Density (calculated) 1.675 Mg / m3
Absorption coefficient 1.957 mm!1
F(000) 366
Crystal Prism; Green
Crystal size 0.140 x 0.140 x 0.140 mm3
Theta range for data collection 3.304 - 27.476°
Index ranges -13 <= h <= 13, -10 <= k <= 10, -11 <= l
<= 11
Reflections collected 5970
Independent reflections 1616 [R(int) = 0.0342]
Completeness to theta = 25.242° 99.6 %
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 1.000 and 0.809
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 1616 / 0 / 94
Goodness-of-fit on F2 0.738
Final R indices [F2 > 2sigma(F2)] R1 = 0.0261, wR2 = 0.0856
R indices (all data) R1 = 0.0319, wR2 = 0.0925
Extinction coefficient n/a
Largest diff. peak and hole 0.424 and -0.261 e Å!3
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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1.3 Pronated TMEDA and [CoCl4]2- Cluster (SECTION 2.2.3.2)
Identification code sg3
Empirical formula C12 H36 Cl8 Co2 N4
Formula weight 637.91
Temperature 170(2) K
Wavelength 0.71073 Å
Crystal system Triclinic
Space group P -1
Unit cell dimensions a = 6.8666(12) Å a= 72.366(5)°
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
Density (calculated) 1.577 Mg / m3
Absorption coefficient 2.037 mm!1
F(000) 326
Crystal Prism; Blue
Crystal size 0.360 x 0.320 x 0.090 mm3
Theta range for data collection 3.184 - 27.480°
Index ranges -8 <= h <= 8, -10 <= k <= 10, -17 <= l <=
17
Reflections collected 7086
Independent reflections 3069 [R(int) = 0.0267]
Completeness to theta = 25.242° 99.8 %
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 1.000 and 0.759
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 3069 / 0 / 126
Goodness-of-fit on F2 0.844
Final R indices [F2 > 2sigma(F2)] R1 = 0.0303, wR2 = 0.0976
R indices (all data) R1 = 0.0406, wR2 = 0.1126
Extinction coefficient n/a
Largest diff. peak and hole 0.550 and -0.523 e Å!3
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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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
Formula weight 439.29
Temperature 170(2) K
Wavelength 0.71075 Å
Crystal system Triclinic
Space group P -1
Unit cell dimensions a = 9.444(14) Å a= 69.83(6)°
b = 9.667(11) Å b= 87.36(7)°
c = 11.579(18) Å g = 62.26(5)°
Volume 870(2) Å3
Z 2
Density (calculated) 1.677 Mg / m3
Absorption coefficient 1.053 mm!1
F(000) 460
Crystal Block; Yellow
Crystal size 0.140 x 0.130 x 0.100 mm3
Theta range for data collection 1.892 - 32.543°
Index ranges -13 <= h <= 13, -13 <= k <= 13, -15 <= l
<= 16
Reflections collected 11415
Independent reflections 5690 [R(int) = 0.0753]
Completeness to theta = 25.242° 99.8 %
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 1.000 and 0.811
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 5690 / 1 / 255
Goodness-of-fit on F2 1.008
Final R indices [F2 > 2sigma(F2)] R1 = 0.0703, wR2 = 0.1781
R indices (all data) R1 = 0.1172, wR2 = 0.2117
Extinction coefficient n/a
Largest diff. peak and hole 0.495 and -0.953 e Å!3
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
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
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
82
Appendix D: Notable Spectroscopic Data
1.0 Complex 5 ESI-MS
1.1 Complex 6 13C NMR and ESI-MS
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
83
1.2 Complex 7 1H NMR
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
84
1.3 Complex 7 19F NMR
1.4 Ligand 10 from 1,12-Dibromododecane 1H NMR
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
85
1.5 Ligand 10 from 1,12-Diiodododecane 1H NMR, 13C NMR, COSY
and ESI-MS
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
86
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
87
1.6 Complex 14 1H NMR, UV-vis and ESI-MS
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Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
88
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
89
1.7 Complex 16 1H NMR
1.8 Complex 8 1H NMR
Appendices
Approaches to Ru(II)/Co(III) Dinuclear Hypoxia Selective Cytotoxins Alexander Puttick
90
References
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91
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