The Synthesis Of Novel Profluorescent Nitroxide Probes …Keddie, D.J. and Bottle S.E. “Synthesis of Profluorescent Isoindoline Nitroxides via Aromatic Functionalisation” ARC Centre

THE SYNTHESIS OF NOVEL PROFLUORESCENT

NITROXIDE PROBES

Submitted by

Daniel Joseph KEDDIE

Bachelor of Applied Science (Honours, Chemistry)

Presented to the School of Physical and Chemical Sciences

In partial fulfilment of the requirements of the degree of

Doctor of Philosophy

January 2008

i

KEY WORDS

Alkenylation, Alkynylation, Buchwald-Hartwig Amination, Cyanation, Fluorescein,

Fluorescence, Fluorophore, Free Radical, Heck Coupling, Hydroxylamine,

Isoindoline, Methoxyamine, Nitroxide, Oxidation, Oxoammonium, Palladium-

Catalysis, Paramagnetic, Profluorescent, Reduction, Redox, Rhodamine, Sonogashira

Coupling, Synthesis, Water Soluble, Xanthene.

ii

ABSTRACT

A number of novel isoindoline nitroxides have been synthesised using a variety of

synthetic techniques. Several carbon-carbon bond forming methodologies, including

the first examples of Heck and Sonogashira coupling applied to the isoindoline

nitroxide class, were utilised to give novel robust aromatic frameworks.

Palladium-catalysed Heck coupling of brominated nitroxides and ester-substituted

olefins generates novel nitroxides possessing extended conjugation. Hydrolysis of

the nitroxide esters gave the corresponding carboxylic acids, which showed enhanced

water solubility.

Sonogashira coupling of an iodo-isoindoline nitroxide gave several novel alkyne-

substituted nitroxides in high yield. Subsequent coupling of a deprotected ethynyl

nitroxide with aromatic iodides gave acetylene-linked nitroxides and an acetylene

linked nitroxide dimer. A butadiyne linked dinitroxide was successfully synthesised

via Eglinton oxidative coupling of two ethynyl nitroxides.

The synthesis of a novel water-soluble dicarboxy nitroxide was achieved by base

hydrolysis of a dinitrile. Functional group interconversion furnished anhydride and

imide substituted nitroxides from the diacid. Subjecting the imide to the Hofmann

rearrangement gave an unexpected brominated amino-carboxy nitroxide. The

dicarboxy nitroxide and the brominated amino-carboxy nitroxide were both shown to

have a protective effect on Ataxia-Telangiectasia cells, indicating a possible role as

antioxidants in the treatment of this disease.

A fluorescein nitroxide was successfully synthesised through the condensation of the

anhydride substituted nitroxide and resorcinol. After limited success using a variety

of other techniques, Buchwald-Hartwig amination was able to furnish a rhodamine

nitroxide, via a triflate-fluorescein nitroxide.

The extended aromatic nitroxides possess suppressed fluorescence and we have

described these systems as profluorescent. The profluorescent nitroxides were found

to have significantly lower quantum yields than the non-radical analogues and

iii

displayed a substantial increase in fluorescence intensity upon radical trapping,

making them useful probes for free radical reactions.

iv

PUBLICATIONS ARISING FROM THIS WORK

Papers

Keddie, D. J.; Johnson, T. E.; Arnold, D. P.; Bottle, S. E. “Synthesis of

Profluorescent Isoindoline Nitroxides via Palladium-Catalysed Heck Alkenylation”

Org. Biomol. Chem. 2005, 3, 2593-2598.

Keddie, D. J.; Bottle, S. E.; Clegg, J. K.; McMurtrie, J. C. “5-[(E)-2-(4-

Methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl” Acta

Cryst. E. 2006, E62(8), o3535-o3536.

Fairfull-Smith, K. E.; Blinco, J. B.; Keddie, D. J.; George, G. A.; Bottle, S. E.; “A

Novel Profluorescent Dinitroxide for Imaging Polypropylene Degradation”

Macromolecules, 2008, 41, 1577-1580.

Keddie, D. J.; Fairfull-Smith, K. E.; Bottle, S. E. “The Palladium-Catalysed Copper-

Free Sonogashira Coupling of Isoindoline Nitroxides: A Convenient Route to Robust

Profluorescent Carbon-Carbon Frameworks”, Org. Biomol. Chem., 2008, accepted

for publication.

Blinco, J. P.; Keddie, D. J.; Wade, T. L.; Barker, P. J.; George, G. A.; Bottle, S. E.

“Profluorescent Nitroxides: Sensors and Stabilisers of Radical-Mediated Oxidative

Damage”, Polym. Degrad. Stab., 2008, accepted for publication.

Lam, M. A.; Pattinson, D. I.; Bottle, S. E.; Keddie, D. J.; Davies, M. J.; “Nitric oxide

and nitroxides can act as efficient scavengers of protein-derived free radicals”, 2008,

submitted for publication.

Morrow, B. J.; Keddie, D. J.; Gueven, N.; Lavin, M. F.; Bottle, S. E. "A Novel

Profluorescent Nitroxide as a Sensitive Probe for the Cellular Redox Environment",

2008, in preparation.

v

Lectures

Keddie, D.J. and Bottle, S.E. “Aromatic Functionalisation of Isoindoline Nitroxides

via Palladium-Catalysis” Brisbane Biological and Organic Chemistry Symposium

(Brisbane, Australia November 2005 – Student Prize)

Keddie, D.J. and Bottle S.E. “Synthesis of Profluorescent Isoindoline Nitroxides via

Aromatic Functionalisation” ARC Centre of Excellence for Free Radical Chemistry

and Biotechnology Winter Carnival (Canberra, Australia June 2006)

Keddie, D.J., Morrow, B.J. and Bottle, S.E. “The Synthesis of Profluorescent

Nitroxides for Monitoring Reactive Oxygen Species (ROS)” Brisbane Biological and

Organic Chemistry Symposium (Brisbane, Australia November 2006)

Keddie, D.J. and Bottle, S.E. “Profluorescent Nitroxides: Stable Radical-Fluorophore

Hybrids for Monitoring Reactive Oxygen Species (ROS) and Cellular Redox Status”

22nd Royal Australian Chemical Institute Organic Conference and the 6th RACI

Conference on Physical Chemistry (OPC) (Adelaide, Australia, January 2007)

Posters

Keddie, D.J., Johnson, T.E., Arnold D.P. and Bottle, S.E. “The Synthesis of

Profluorescent Isoindoline Nitroxides via Heck Alkenylation” XXIX International

Symposium on Macrocyclic Chemistry (XXIX ISMC) and the 20th Organic

Chemistry Conference of the Royal Australian Chemical Institute (20 RACIOC)

(Cairns, Australia, July 2004)

Keddie, D.J., Micallef, A.S., and Bottle, S.E. “Palladium Catalysis in the Synthesis

of Improved Water-Soluble Nitroxide Probes” Connect 2005 Conference - 12th

RACI Convention (Sydney, Australia, July 2005)

Keddie, D.J., Hosokawa, K., and Bottle, S.E. “Synthesis and Evaluation of Novel

Nitroxides as Antioxidants and Probes in Biological Systems” Society of Free

Radical Research (Australasia) Conference (Gold Coast, Australia, December 2005)

vi

TABLE OF CONTENTS

Key Words ............................................................................................................ i

Abstract ...........................................................................................................ii

Publications Arising From This Work ........................................................................ iv

Papers ...................................................................................................................... iv

Lectures .................................................................................................................... v

Posters ...................................................................................................................... v

Table of Contents ........................................................................................................vi

List of Figures ........................................................................................................xiii

List of Tables .........................................................................................................xv

List of Schemes ........................................................................................................xvi

Abbreviations .........................................................................................................xx

Declaration .......................................................................................................xxii

Acknowledgements .................................................................................................xxiii

1. Introduction .......................................................................................1

1.1. Nitroxide Free Radicals.....................................................................1

1.1.1. Nitroxide Stability .............................................................................1

1.1.2. Redox Chemistry of Nitroxides.........................................................2

1.1.3. Radical Trapping ...............................................................................4

1.1.4. Spin Labels/Probes............................................................................4

1.1.5. Profluorescent Nitroxides..................................................................6

1.2. Isoindoline Nitroxides .......................................................................8

1.2.1. Photostability of Isoindoline Nitroxides .........................................10

1.2.2. Drawbacks – Methodologies and Structure ....................................10

1.3. Project Outline.................................................................................11

2. Palladium-Catalysed Heck Coupling of Isoindoline Nitroxides .....12

2.1. Introduction .....................................................................................12

2.2. The Heck Alkenylation Reaction ....................................................12

2.2.1. Heck Reactions Performed on Nitroxides.......................................14

2.3. Results and Discussion....................................................................15

2.3.1. Heck Coupling of 5-bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl

(16) ..................................................................................................15

vii

2.3.2. Heck Coupling of 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-

yloxyl (21)....................................................................................... 20

2.3.3. Synthesis of Methoxyamines (26 and 27)....................................... 21

2.3.4. Fluorescence Data of Vinyl-Substituted Profluorescent Nitroxides22

2.3.5. Water Soluble Profluorescent Nitroxides........................................ 24

2.4. Summary of Results ........................................................................ 25

2.5. Experimental ................................................................................... 25

2.5.1. N-Benzylphthalimide (32) .............................................................. 27

2.5.2. 2-Benzyl-1,1,3,3-tetramethylisoindoline (34)................................. 27

2.5.3. 5-Bromo-1,1,3,3-tetramethylisoindoline (35) ................................. 29

2.5.4. 5,6-Dibromo-1,1,3,3-tetramethylisoindoline (36)........................... 30

2.5.5. 5-Bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl (16).................... 31

2.5.6. 5,6-Dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl (21).............. 32

2.5.7. Methyl 4-vinylbenzoate (20)........................................................... 32

2.5.8. 5-[2-(4-Methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl (18) .................................................................. 33

2.5.9. 5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl (23) ................................................ 34

2.5.10. 5-(2-Methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-2-

yloxyl (17)....................................................................................... 34

2.5.11. 5,6-Bis-(2-methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-

2-yloxyl (22) ................................................................................... 35

2.5.12. 5-[2-(4-Methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-

tetramethyl-isoindoline (26)............................................................ 36

2.5.13. 5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-

tetramethylisoindoline (27) ............................................................. 37

2.5.14. 5-[2-(4-Carboxyphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl (28)....................................................................................... 37

2.5.15. 5,6-Bis-[2-(4-carboxyphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl (29) .................................................................. 38

2.5.16. Fluorescence Quantum Yield Calculations..................................... 38

3. Palladium-Catalysed Sonogashira Coupling of Isoindoline

Nitroxides........................................................................................ 42

3.1. Introduction.....................................................................................42

viii

3.2. The Sonogashira Alkynylation Reaction.........................................42

3.2.1. Sonogashira Reactions Performed on Nitroxides............................46


3.3.1. Sonogashira Coupling of Bromo and Bromo-Nitro Nitroxides ......49

3.3.2. Copper-Free Sonogashira Coupling of Bromo and Bromo-nitro

Nitroxides........................................................................................52

3.3.3. Sonogashira Coupling of the Iodo Nitroxide (59)...........................55

3.3.4. Synthesis of Ethynyl Nitroxide .......................................................58

3.3.5. Profluorescent Nitroxides via Sonogashira Coupling of Ethynyl

Nitroxide..........................................................................................60

3.3.6. Ethyne and 1,3-Butadiyne Linked Nitroxide Dimers......................61

3.3.7. Synthesis of Methoxyamines (“Methyl Traps”)..............................62

3.3.8. Fluorescence Data of Ethynyl Profluorescent Nitroxides...............62

3.4. Summary of Results ........................................................................64

3.5. Experimental ...................................................................................65

3.5.1. 5-Bromo-6-nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl (57)........65

3.5.2. 5-Iodo-1,1,3,3-tetramethylisoindoline (65) .....................................65

3.5.3. 5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (64)........................66

3.5.4. 5-[2-(Trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(55) ..................................................................................................67

3.5.5. Alternate Synthesis of 5-[2-(Trimethylsilyl)ethynyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl (55) ................................................68

3.5.6. 5-Nitro-6-[2-(trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-

2-yloxyl (59)....................................................................................68

3.5.7. 5-(3-Hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl (63) .......................................................................................69

3.5.8. 5-[2-(Phenyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (56)70

3.5.9. 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (70) ..................70

3.5.10. Alternate Synthesis of 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl (70) .......................................................................................71

3.5.11. 5-[2-(1-Naphthyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(72) ..................................................................................................71

3.5.12. 5-[2-(9-Phenanthryl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(74) ..................................................................................................72

ix

3.5.13. 1,2-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]ethyne (76)

......................................................................................................... 72

3.5.14. 1,4-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]-1,3-

butadiyne (75) ................................................................................. 73

3.5.15. Synthesis of methoxyamines (77-84).............................................. 74

3.5.16. General Procedure........................................................................... 74

3.5.17. 5-[2-(Trimethylsilyl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (77) ............................................................................... 74

3.5.18. 5-(3-Hydroxy-3-methyl)butynyl-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (78) ............................................................................... 75

3.5.19. 5-[2-(Phenyl)ethynyl]-2-methoxy-1,1,3,3-tetramethylisoindoline

(79) .................................................................................................. 75

3.5.20. 5-Ethynyl-2-methoxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (80) 76

3.5.21. 5-[2-(1-Naphthyl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (81) ............................................................................... 76

3.5.22. 5-[2-(9-Phenanthryl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (82) ............................................................................... 77

3.5.23. 1,2-Bis-[5,5’-(2-methoxy-1,1,3,3-tetramethylisoindoline)]ethyne

(83) .................................................................................................. 77

3.5.24. 1,4-Bis-[5,5’-(2-methoxy-1,1,3,3-tetramethylisoindoline)]-1,3-

butadiyne (84) ................................................................................. 78

3.5.25. 1-(Phenylethynyl)naphthalene (85)................................................. 78

3.5.26. Fluorescence Quantum Yield Calculations..................................... 79

4. Water Soluble Nitroxides as Antioxidants...................................... 82

4.1. Introduction.....................................................................................82

4.2. Reactive Oxygen Species (ROS) .................................................... 82

4.2.1. Formation of ROS........................................................................... 83

4.2.2. Superoxide Radical (O2•−) ............................................................... 83

4.2.3. Hydrogen Peroxide (H2O2) ............................................................. 83

4.2.4. Hydroxyl Radicals (HO•) ................................................................ 84

4.2.5. Peroxyl Radicals (RO2•) .................................................................. 84

4.2.6. Alkoxyl Radicals (RO•)................................................................... 84

4.3. The Chemistry of Radical Related Cell Damage ............................ 85

4.3.1. Lipid Peroxidation........................................................................... 85

x

4.3.2. Protein Oxidation ............................................................................86

4.3.3. DNA Damage..................................................................................87

4.4. Natural Antioxidants .......................................................................88

4.5. Nitroxides as Antioxidants ..............................................................89

4.5.1. Nitroxide Action on Ataxia-Telangiectasia.....................................90

4.5.2. Isoindoline Nitroxides and A-T.......................................................91


4.6.1. Synthesis of 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl

(102) ................................................................................................94

4.6.2. Synthesis of 5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

(98) ..................................................................................................97

4.6.3. Synthesis of 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-

furano[3,4-f]isoindol-2-yloxyl (100)...............................................98


pyrrolo[3,4-f]isoindol-2-yloxyl (101) .............................................99

4.6.5. The Attempted Synthesis of 5-Amino-6-carboxy-1,1,3,3-

tetramethylisoindolin-2-yloxyl (99) via the Hofmann

Rearrangement ................................................................................99

4.6.6. Syntheses of Methoxyamines (104, 105, 109 and 112) ................102

4.6.7. A-T Cell Survivability Assays of Isoindoline Nitroxides 98 and 108

.......................................................................................................103

4.7. Summary of Results ......................................................................104

4.8. Experimental .................................................................................105

4.8.1. 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl (102)...........105

4.8.2. Alternate Synthesis of 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-2-

yloxyl (102) ...................................................................................106

4.8.3. 5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (98) .........106

4.8.4. 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-furano[3,4-

f]isoindol-2-yloxyl (100)...............................................................107

4.8.5. 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-pyrrolo[3,4-

f]isoindol-2-yloxy (101) ................................................................107

4.8.6. 5-Amino-4-bromo-6-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

(108) ..............................................................................................108

4.8.7. Synthesis of Methoxyamines ........................................................108

xi

4.8.8. General Procedure......................................................................... 109

4.8.9. 5,6-Dicarboxy-2-methoxy-1,1,3,3-tetramethylisoindoline (104) . 109

4.8.10. 2-Methoxy-1,1,3,3-tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-

pyrrolo[3,4-f]isoindole (105) ........................................................ 109

4.8.11. 5-Amino-4-bromo-6-carboxy-2-methoxy-1,1,3,3-

tetramethylisoindoline (109) ......................................................... 110


furano[3,4-f]isoindole (107).......................................................... 110

5. Xanthene-Based Profluorescent Isoindoline Nitroxides ............... 111

5.1. Introduction................................................................................... 111

5.2. Fluorescence Detection of Reactive Oxygen Species (ROS) ....... 111

5.3. Results and Discussion.................................................................. 114

5.3.1. The Synthesis of Nitroxide-Substituted Fluorescein..................... 114

5.3.2. The Synthesis of Methoxyamine-Substituted Fluorescein (130) .. 117

5.3.3. Fluorescence Data of Fluoresceinyl Nitroxide and Methoxyamine

....................................................................................................... 117

5.3.4. Rhodamine Nitroxide Synthesis.................................................... 120

5.3.5. The Attempted Synthesis of Rhodamine B Nitroxide (130) – Direct

Methodologies............................................................................... 121

5.3.6. Synthesis of Rhodamine B Methoxyamine (140) ......................... 123

5.3.7. Attempted Step-Wise Synthesis of Rhodamine B Nitroxide (135)

....................................................................................................... 124

5.3.8. Attempted Acetyl-Protection of the Nitroxide Moiety ................. 126

5.3.9. Buchwald-Hartwig Amination...................................................... 129

5.3.10. Fluorescence Data of Pyrrolidinorhodamine Nitroxide (152) ...... 133

5.4. Summary of Results ...................................................................... 134

5.5. Experimental ................................................................................. 135

5.5.1. 5-Carboxy-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-1,1,3,3-

tetramethylisoindolin-2-yloxyl (127) ............................................ 136

5.5.2. 5-Carboxy-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-methoxy-

1,1,3,3-tetramethylisoindoline (130)............................................. 136

5.5.3. [9-(6-Carboxy-2-methoxy-1,1,3,3-tetramethylisoindolin-5-yl)-6-

diethylamino-xanthen-3-ylidene]-diethyl-ammonium Chloride (140)

....................................................................................................... 137

xii

5.5.4. 6-Acetyl-5,5,7,7-tetramethyl-6,7-dihydro-5H-2-oxa-6-aza-s-

indacene-1,3-dione (144) ..............................................................138

5.5.5. [9-(2-Acetyl-6-carboxy-1,1,3,3-tetramethyl-2,3-dihydro-1H-

isoindol-5-yl)-6-diethylamino-xanthen-3-ylidene]-diethyl-

ammonium Chloride (145) ............................................................139

5.5.6. Bis(trifluoromethanesulfonyl)fluorescein (150) ...........................140

5.5.7. 1-[9-(2-Carboxy-phenyl)-6-pyrrolidin-1-yl-xanthen-3-ylidene]-

pyrrolidinium Chloride (Pyrrolidinorhodamine, 151)...................140

5.5.8. Bis(trifluoromethanesulfonyl)fluoresceinyl Nitroxide (153) ........141

5.5.9. 1-[9-(6-Carboxy-1,1,3,3-tetramethylisoindolin-2-yloxylyl)-6-

pyrrolidin-1-yl-xanthen-3-ylidene]-pyrrolidinium Chloride (152)142

5.5.10. Fluorescence Quantum Yield Calculations ...................................142

6. Conclusions and Future Work.......................................................146

6.1. Conclusions ...................................................................................146

6.2. Future Work ..................................................................................149

7. References .....................................................................................151

Appendices .......................................................................................................162

Appendix 1: Selected HPLC Traces .................................................................162

xiii

LIST OF FIGURES

Figure 1.1: Jablonski Diagram of Electronic Excited States ............................... 6

Figure 2.1: Optimisation of the synthesis of 18 - time varied ........................... 17

Figure 2.2: Optimisation of the synthesis of 18 – Pd catalyst concentration

varied............................................................................................... 17

Figure 2.3: Optimisation of the synthesis of 18 – temperature varied............... 18

Figure 2.4: X-ray crystal structure of 18 (H atoms omitted for clarity) ............ 19

Figure 2.5: UV/Vis and Fluorescence spectra of 18 (- - -) and 26 (―) excited at

330 nm in cyclohexane normalised to 1 µM................................... 22



Figure 2.7: Quantum yield measurements of 18 and 26 at 330 nm in

cyclohexane..................................................................................... 39


cyclohexane..................................................................................... 40

Figure 3.1: X-ray crystal structure of bromo-nitro nitroxide 57 (H atoms

omitted for clarity) .......................................................................... 51

Figure 3.2: X-ray crystal structure of iodo nitroxide 64 (H atoms omitted for

clarity) ............................................................................................. 57

Figure 3.3: UV/Vis and Fluorescence spectra of 72 (---) and 81 (―) excited at




Figure 3.5: Quantum yield measurements of 72, 81 and 85 at 320 nm in

cyclohexane at 480 V...................................................................... 79


cyclohexane at 600 V...................................................................... 80

Figure 4.1: Survivability of A-T cells incubated with CTMIO 66, CTEMPO 95

and CPROXYL 96 .......................................................................... 92

Figure 4.2: Survival of A-T cells incubated with CTMIO 66, α-Tocopherol 92

and Trolox 97.................................................................................. 92

Figure 4.3: The high variability of cyanation reaction ...................................... 96

Figure 4.4: X-ray crystal structure of 98 (H atoms omitted for clarity) ............ 98

xiv

Figure 4.5: X-ray crystal structure of 100 (H atoms omitted for clarity) ..........99

Figure 4.6: Cell survivability of A-T cells treated with 98 and 108................103

Figure 5.1: UV/Vis and Fluorescence spectra of 127 (---) and 130 (―) excited

at 492 nm in 0.1M NaOH normalised to 1 µM .............................118

Figure 5.2: The dependence of fluorescence emission intensity of 127 (―) and

130 (―) on pH ..............................................................................118

Figure 5.3: Fluorescence image of 127 in HeLa cells .....................................120

Figure 5.4: Fluorescence image of 140 in HeLa cells .....................................124

Figure 5.5: UV/Vis and Fluorescence spectra of 152 (---) and 151 (―) excited

at 544 nm in EtOH normalised to 1 µM........................................133

Figure 5.6: Quantum yield measurements of 127 and 130 at 492 nm in 0.1 M

NaOH at 500 V..............................................................................143

Figure 5.7: Quantum yield measurements of 151 and 152 at 544 nm in EtOH at

535 V.............................................................................................144

xv

LIST OF TABLES

Table 2.1: Previously reported yields for mono-substituted Heck products .... 16

Table 2.2: Optimised yields for mono-substituted Heck products................... 19

Table 2.3: Yields for di-substituted Heck products.......................................... 20

Table 2.4: Quantum yields of Heck products................................................... 24

Table 3.1: Traditional Sonogashira couplings of bromo nitroxide and alkynes

......................................................................................................... 50

Table 3.2: Traditional Sonogashira couplings of nitro bromo nitroxide.......... 52

Table 3.3: Copper-free Sonogashira couplings by Li et al. ............................. 53

Table 3.5: Sonogashira coupling of iodo nitroxide 64..................................... 58

Table 3.6: Quantum yields of Sonogashira products ....................................... 64

Table 4.1: Reactive oxygen species ................................................................. 82

Table 4.2: Synthesis of nitroxide dinitrile 102................................................. 95

Table 4.3: Synthesis of dicarboxy nitroxide 98................................................ 97

Table 4.4: Synthesis of methoxyamines 104 and 109.................................... 102

Table 5.1: Quantum yields of fluorescein (127 and 130) and BPEA (131 and

132) compounds. ........................................................................... 120

Table 5.2: Attempted direct syntheses of rhodamine B nitroxide 135........... 122

Table 5.3: Attempted hydrolysis of rhodamine B amide 145........................ 128

Table 5.4: Quantum yields of rhodamine compounds (133, 151 and 152) .... 134

xvi

LIST OF SCHEMES

Scheme 1.1: Delocalisation of nitroxide radical ....................................................1

Scheme 1.2: Disproportionation of phenyl substituted nitroxide..........................2

Scheme 1.3: Disproportionation of nitroxides with hydrogen bound to the α

carbon ................................................................................................2

Scheme 1.4: Reduction of nitroxides .....................................................................3

Scheme 1.5: Oxidation of nitroxides......................................................................3

Scheme 1.6: Bond cleavage of oxoammonium salt ...............................................3

Scheme 1.7: Radical trapping by nitroxide radicals ..............................................4

Scheme 1.8: Naphthalene-based profluorescent nitroxide.....................................7

Scheme 1.9: Structure and numbering of isoindoline nitroxides ...........................8

Scheme 1.10: Stability of TMIO 4 towards α-cleavage.........................................10

Scheme 1.11: Photodegradation of a pyrroline nitroxide 13 .................................10

Scheme 2.1: An example of arylation of alkenes by organomercuries................12

Scheme 2.2: Olefination of iodobenzene by Mizoroki et al. ...............................13

Scheme 2.3: Olefination of aryl iodides by Heck and Nolley .............................13

Scheme 2.4: General outline of the Heck reaction...............................................13

Scheme 2.5: The reaction mechanism of the Heck reaction ................................14

Scheme 2.6: Pyrroline nitroxide Heck coupling by Hideg and co-workers.........15

Scheme 2.7: Synthesis of methoxyamines 26 and 27..........................................22

Scheme 2.8: Synthesis of carboxylic acids 28 and 29 .........................................24

Scheme 3.1: Synthesis of acetylene derivatives by Cassar..................................43

Scheme 3.2: Synthesis of acetylene derivatives by Dieck and Heck...................43

Scheme 3.3: Synthesis of acetylene derivatives by Sonogashira et al.................44

Scheme 3.4: The reaction mechanism of the Pd-catalysed Sonogashira coupling

.........................................................................................................44

Scheme 3.5: The proposed reaction mechanism of the Cu-free Sonogashira

reaction ............................................................................................45

Scheme 3.6: t-Butylphenyl nitroxide Sonogashira coupling by Miura and

Ushitani ...........................................................................................46

Scheme 3.7: t-Butylphenyl and nitronyl nitroxide Sonogashira coupling by Miura

et al. .................................................................................................47

Scheme 3.8: Sonogashira coupling by Romero and Ziessel ................................47

xvii

Scheme 3.9: Nitronyl nitroxide Sonogashira coupling by Romero and Ziessel .. 48

Scheme 3.10: Nitronyl nitroxide Sonogashira couplings by Stroh et al................ 48

Scheme 3.11: Pyrroline nitroxide Sonogashira couplings by Hideg and co-workers

......................................................................................................... 49

Scheme 3.12: Synthesis of bromo-nitro nitroxide 57............................................ 51

Scheme 3.13: Synthesis of iodo amine 65............................................................. 56

Scheme 3.14: Synthesis of iodo nitroxide 64......................................................... 57

Scheme 3.15: Synthesis of ethynyl nitroxide 70 from 55...................................... 59

Scheme 3.16: Synthesis of ethynyl nitroxide 70 from 63...................................... 59

Scheme 3.17: Synthesis of (naphthyl)ethynyl nitroxide 67................................... 60

Scheme 3.18: Synthesis of (phenanthryl)ethynyl nitroxide 74.............................. 61

Scheme 3.19: Synthesis of acetylene dinitroxides 75 and 76................................ 61

Scheme 3.20: Synthesis of butadiyne-linked dinitroxide 75 ................................. 62

Scheme 4.1: Formation of superoxide from one-electron reduction ................... 83

Scheme 4.2: Oxidation of oxyhemoglobin to methemoglobin ............................ 83

Scheme 4.3: Conversion of superoxide to hydrogen peroxide by SOD .............. 83

Scheme 4.4: Formation of hydroxyl radical by the Fenton reaction.................... 84

Scheme 4.5: Production of a peroxyl radical via formation of an alkyl radical .. 84

Scheme 4.6: Formation of an alkoxyl radical from a hydroperoxide .................. 84

Scheme 4.7: Formation of a peroxyl radical from an alkoxyl radical ................. 85

Scheme 4.8: The lipid peroxidation chain reaction ............................................. 86

Scheme 4.9: Termination of peroxyl radicals ...................................................... 86

Scheme 4.10: Formation of oxohistidine 86 by oxidative damage........................ 87

Scheme 4.11: Hydroxylation of tyrosine 88 to form DOPA 89 ............................ 87

Scheme 4.12: Formation of DNA lesions 8-hydroxyguanine 90 and thymine glycol

91..................................................................................................... 88

Scheme 4.13: Dismutation of superoxide .............................................................. 89

Scheme 4.14: Neutralisation of reactive species by antioxidants.......................... 89

Scheme 4.15: The SOD mimicking mechanism of TEMPO 3 .............................. 90

Scheme 4.16: Antioxidant activity of hydroxylamines.......................................... 90

Scheme 4.17: Proposed synthetic route to water soluble nitroxides 98 and 99..... 94

Scheme 4.18: The proposed structure of the phthalocyanine 103 side-product .... 96

Scheme 4.19: Synthesis of anhydride nitroxide 100.............................................. 98

Scheme 4.20: Synthesis of nitroxide imide 101..................................................... 99

xviii

Scheme 4.21: Synthesis of anthranilic acid 107 via the Hofmann rearrangment 100

Scheme 4.22: Synthesis of nitroxides via the Hofmann rearrangement ..............100

Scheme 4.23: Synthesis of dimethylanthranilic acid 106 as described by Godinez

et al. ...............................................................................................101

Scheme 4.24: Speculative mechanism for the formation of amino bromo carboxy

nitroxide 108.................................................................................101

Scheme 4.25: Synthesis of methoxyamine 105...................................................102

Scheme 4.26: Synthesis of methoxyamine 112...................................................103

Scheme 5.1: Fluorescence detection of ROS with dihydrofluoresceins ............111

Scheme 5.2: H2O2 specific probe 118 synthesised by Maeda et al....................112

Scheme 5.3: O2•- specific probe 120 synthesised by Maeda et al. .....................113

Scheme 5.4: Fluorescence detection of singlet oxygen .....................................113

Scheme 5.5: Fluorescence detection of hydroxyl radical ..................................114

Scheme 5.6: Synthesis of fluorescein 125.........................................................115

Scheme 5.7: The two forms of fluorescein ........................................................115

Scheme 5.8: Attempted synthesis of fluoresceinyl nitroxide 127......................116

Scheme 5.9: Synthesis of fluoresceinyl nitroxide 127.......................................116

Scheme 5.10: Synthesis of fluorescein methoxyamine 130.................................117

Scheme 5.11: The two forms of rhodamine B .....................................................121

Scheme 5.12: Synthesis of rhodamine B methoxyamine 140..............................124

Scheme 5.13: Attempted step-wise synthesis of rhodamine nitroxide 135.........125

Scheme 5.14: Synthesis of intermediate 142.......................................................125

Scheme 5.15: Unsuccessful synthesis of rhodamine B nitroxide 135 using PPSE

.......................................................................................................126

Scheme 5.16: Attempted synthesis of acetylated anhydride 143 from AcCl.......126

Scheme 5.17: Synthesis of amide 144 from Ac2O and H2...................................127

Scheme 5.18: Synthesis of rhodamine amide 145...............................................128

Scheme 5.19: Base cleavage of 147 with base reported by Woodroofe et al. .....129

Scheme 5.20: The reaction mechanism of the Buchwald-Hartwig amination.....130

Scheme 5.21: Synthesis of rhodamines 135 and 151 via Pd-catalysed amination

.......................................................................................................131

Scheme 5.22: Synthesis of rhodamine nitroxide 152 via triflate nitroxide 153...132

Scheme 5.23: Attempted synthesis of rhodamine methoxyamine 154................133

Scheme 6.1: Proposed Huisgen 1,3-dipolar cycloaddition of ethynyl nitroxide149

xix

Scheme 6.2: Proposed synthesis of fused xanthene-substituted nitroxides ....... 150

xx

ABBREVIATIONS

Ar aryl

ATR attenuated total reflectance

au arbitrary units

br broad

Bu butyl

calc. calculated

CTMIO 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

d doublet

DABCO 1,4-diazabicyclo[2.2.2]octane

DCTMIO 5,6-dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

dd doublet of doublets

dba dibenzylideneacetone

DCFH2 2’,7’-dichlorodihydrofluorescein

DCFH-DA diacetyl-2’,7’-dichlorodihydrofluorescein

DCM dichloromethane

DMAC N,N-dimethylacetamide

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

EI electron impact

EPR electron paramagnetic resonance

equiv equivalent(s)

ESI electrospray ionisation

Et ethyl

FT fourier transform

GC gas chromatography

h hour(s)

HOMO highest occupied molecular orbital

HPLC high performance liquid chromatography

IR infrared

m multiplet

Me methyl

min minute(s)

xxi

mp melting point

MS mass spectrometry

NMR nuclear magnetic resonance

Ph phenyl

ppm parts per million

PPSE trimethylsilylpolyphosphate

PROXYL 2,2,5,5-tetramethyl-1-pyrrolidinyloxyl

rac-BINAP (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene

ROS reactive oxygen species

RT room temperature

s singlet

t triplet

TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxyl

THF tetrahydrofuran

TMINO 1,1,3-trimethylisoindole-N-oxide

TMIO 1,1,3,3-tetramethylisoindolin-2-yloxyl

TLC thin layer chromatography

UV/vis ultraviolet/visible

xxii

DECLARATION

The work presented in this thesis has not previously been submitted for any diploma

or degree at any higher educational institution. To the best of my knowledge, this

thesis contains no material that has been previously published or written by another

person, except where referenced or cited.

Daniel Joseph Keddie

January 2008

xxiii

ACKNOWLEDGEMENTS

The author would like to sincerely thank:

A/Prof Steven Bottle, my supervisor, for his vast input into this PhD project. Thank

you for having the faith in my abilities to allow me to complete this project over

these past few years; your support has been invaluable.

My two associate supervisors A/Prof Dennis Arnold and Dr Aaron Micallef for their

help, guidance and advice relating to many facets of this project.

Dr John McMurtrie for all his help relating to the X-ray crystallographic analysis of

many of the compounds found within this thesis.

Fellow members of the Bottle research group, both past and present. I make

particular mention to Benjamin Morrow, Dr Kathryn Fairful-Smith, Kazuyuki

Hosokawa and James Blinco who have each had some direct input into aspects of my

project.

The ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, The

Synthesis and Molecular Recognition Research Program, The School of Physical and

Chemical Sciences and The Royal Australian Chemical Institute for financial support

over the period of this project.

Mr Patrick Stevens and the technical staff at The School of Physical and Chemical

Sciences for instrumental assistance throughout the project.

My fellow postgraduate students, both past and present, for friendship, help and

support over the tenure of my postgraduate studies.

My family for all your support over the course of my studies; I dedicate this to you.

Chapter 1 – Introduction

1

1. INTRODUCTION

1.1. Nitroxide Free Radicals

1.1.1. Nitroxide Stability

Nitroxides are stable, persistent free radicals which contain an unpaired electron

delocalised over nitrogen and oxygen atoms (Scheme 1.1);1 this delocalisation

imparts high stability to the nitroxide moiety. The unpaired electron of the nitroxide

gives rise to the intrinsic chemical properties of the compounds and also makes them

paramagnetic.

N

O

R R1

N

O

R R1

Scheme 1.1: Delocalisation of nitroxide radical

The high delocalisation stabilisation (~130 kJ/mol) of the N−O bond prevents

dimerisation of nitroxide radicals.1 The formation of a weak peroxide (NO−ON)

bond in any such dimer would not be able to compensate for the loss of

delocalisation stabilisation for two nitroxide molecules.2

Although the stability of the nitroxide moiety arises from the electronic nature of the

N−O bond, the comparative persistence of nitroxides is inherently dependent upon

the nature of the substituents adjacent to the nitrogen.1

An example of this is the propensity for t-butyl phenyl nitroxide 1 to disproportionate

to give nitrone and amine products (Scheme 1.2 below).1 Delocalisation of spin

density of the unpaired electron onto the aromatic ring increases the chemical

reactivity of the nitroxide. The carbon-centred radical character present on the

aromatic ring allows the molecule to react with another molecule of the nitroxide.

The initially formed dimer rapidly fragments to give products of greater stability than

the original nitroxides.1


2

N

O

N

O

N

O

H

ON

N

O

N

H

+

O

1

N

O

Scheme 1.2: Disproportionation of phenyl substituted nitroxide1

Systems possessing methines or methylenes α to the nitroxide moiety can

disproportionate to give nitrone and hydroxylamine products, as shown in Scheme

1.3.1 Furthermore, radicals that contain hetero-atoms (including oxygen, nitrogen,

phosphorus and sulfur) α to the nitroxide centre also have high reactivity and

inherently short lifetimes.1

N O

C

H

N

O

CN

OH

CH+

CHN

O NO

C

H

2

Scheme 1.3: Disproportionation of nitroxides with hydrogen bound to the α carbon

Bis(t-alkyl) nitroxides, such as PROXYL 2, TEMPO 3 and TMIO 4, are widely

recognised as the most stable nitroxides. This is due to the absence of many viable

degradation mechanisms and destabilising substituents, such as those outlined above.

Their inherent stability allows bis(t-alkyl) nitroxides to be isolated and manipulated

under common laboratory conditions.

N

ON

O

N O

2 3 4

1.1.2. Redox Chemistry of Nitroxides

The redox chemistry of nitroxides plays an important role with respect to their

applications in biological systems. Whilst nitroxides do not generally undergo


3

oxidation or reduction reactions with most organic compounds, they may behave as

either weak oxidants or weak reductants, depending on the redox reactivity of the

substrate present.

Nitroxides may be reduced by a variety of weak reductants in vitro, such as

hydrazine or ascorbic acid, to give hydroxylamines (Scheme 1.4),3 whilst more

powerful reductants may generate secondary amines. Hydroxylamines are quite

reactive and are readily reoxidised to the nitroxide by mild oxidants, such as

[Fe(CN)6]3- and PbO2. They may even be auto-oxidised by atmospheric O2, although

this process may be slow.3 Hydroxylamines are of particular interest, as they are the

major metabolic outcome of nitroxides in vivo.4

N

O

R R1

N

OH

R R1[H]

[O]

Scheme 1.4: Reduction of nitroxides

The oxidation of bis-t-alkyl nitroxides requires relatively strong oxidants,1 such as

Br2 or Lewis acids (e.g. AlCl3), and results in the formation of an oxoammonium salt

(Scheme 1.5).

N

O

R R1

N

O

R R1

[H]

[O]

Scheme 1.5: Oxidation of nitroxides

These species are more reactive than nitroxides, due to their electron-deficiency.2

Secondary reactions occur after formation of the oxoammonium salt, including

reaction with solvents, such as acetone or ethanol, or intramolecular bond scission

(Scheme 1.6).2 Such reactions of the oxoammonium salts can be synthetically

useful,1 selectively and mildly oxidising certain substrates and thereby regenerating

the nitroxide.

N

O

O

ON

OH-H+

Scheme 1.6: Bond cleavage of oxoammonium salt2


4

1.1.3. Radical Trapping

Nitroxides react readily with carbon-centred radicals to form stable alkoxyamines

(Scheme 1.7),1 which underpins their broad use as inhibitors of free radical

processes. Reaction of nitroxides with alkyl radicals is known to occur rapidly, at

close to diffusion-controlled rates.5 Whilst reactions with sulfur-6, 7 and phosphorus-

centred8 radicals, via radical-radical recombination, have also been reported,

nitroxides tend not to react directly with oxygen-centred radicals.

N O

R

R1

R2 N OR2

R

R1

Scheme 1.7: Radical trapping by nitroxide radicals

The field of polymer chemistry has been a significant driver of the development of

applications of nitroxide radical trapping, including the studying of initiation

mechanisms9-12 and nitroxide-mediated “living” free radical polymerisation.13-19

The trapping of radicals in biological systems has received less attention in the

literature. Whilst trapping of alkyl- and aryl-radicals inevitably occurs in these

systems, competition from molecular oxygen limits the prevalence of the trapped

species, instead giving oxygen centred-radicals such as peroxyl-radicals as the major

product. Bio-reduction of the nitroxides also limits trapping ability by lowering the

effective concentration in the system. Due to their low concentration, nitroxide-

trapped species are quite difficult to detect. Therefore, the redox chemistry of

nitroxides has been of greater interest than radical trapping in cellular environments.

1.1.4. Spin Labels/Probes

Nitroxides have found wide use as spin labels and spin probes in many diverse fields

ranging from molecular biology, coordination and analytical chemistry to oil

production and polymer science.1

A nitroxide can be used as a spin label by covalently linking it to a substrate

molecule which occurs naturally in the system being studied, for example a protein

of a biological system. Such spin labelled paramagnetic analogues of the substrate

enable EPR analysis of the system. Spin labelling of a substrate can give information


5

on properties such as viscosity, structure and electric potential of the surrounding

environment.1

Spin labelling of biological molecules, such as nucleic acids, can give insight into

bio-molecular functions within cells. Interactions between DNA and proteins

involved in transcription and translation can be studied using this technique. An

example of a DNA spin label 5 is shown below.20

NO

N

HN

O

R'O OR

O

O

5 Spin labelling and EPR can also give important information about cellular processes

that can lead to improved drug design.

As well as spin labelling, systems can be investigated through the use of spin probes.

A spin probe differs from a spin label in that it is a specifically designed nitroxide

that can give information about a substrate without being covalently bonded to it.1

The measurement of molecular oxygen via EPR oximetry is an example of the use of

nitroxides as spin probes. When in the gas phase, molecular oxygen gives strong

EPR signals, due its paramagnetism. However, when dissolved in liquid, the EPR

signals are so broad that the O2 is undetectable. Interactions with molecular oxygen

in solution cause line broadening of nitroxide EPR spectra. The degree of broadening

can be correlated to the concentration of oxygen.1 This technique is quite powerful as

it has the ability to quantify the concentration of oxygen in cellular tissues, which is

of particular importance in many biological systems. An example of a spin probe

used for oximetry experiments is perdeuterated 15N-TEMPONE 6.21


6

15N

O

ODD

D DCD3D3C

D3C CD3

6

1.1.5. Profluorescent Nitroxides

Nitroxide radicals have long been recognised as effective intermolecular quenchers

of singlet excited states of fluorescent moieties.22-28 The mechanism of the quenching

process has been studied in detail and it is generally considered that the spin state of

the nitroxide moiety facilitates an electron exchange mechanism, resulting in

intersystem crossing to the triplet state or internal conversion to the ground state.27, 29,

30 Normally intersystem crossing (ISC) from a singlet state to a triplet state is ‘spin

forbidden’ (see Figure 1.1 below). Electron exchange between the unpaired electron

of the nitroxide moiety and a fluorophore produces a doublet state for the overall

system. Due to this ISC becomes ‘spin allowed’ because of a conservation of spin

state. This greatly enhances the prevalence of radiation-less decay by internal

conversion, which in turn significantly reduces fluorescence emission from the

system. Whilst the factors outlined above are thought to be of particular significance

to the phenomenon observed, charge transfer, energy transfer and electron exchange

processes have also been suggested to play a part.27, 29

ISC

FP

A

EN

ER

GY

IC

IC A = Photon AbsorptionF = FluorescenceP = PhosphorescenceS = Singlet StateT = Triplet State IC = Internal ConversionISC = Intersystem Crossing

S0

S1

S2

T1

T2

Figure 1.1: Jablonski Diagram of Electronic Excited States


7

Intramolecular fluorescence quenching of nitroxide-fluorophore hybrid molecules

(profluorescent nitroxides) was first demonstrated by Blough and Simpson in 1988.31

In this report, TEMPO was tethered to a range of naphthalene fluorophores via an

ester linkage (see 7 in Scheme 1.8 below). After this initial investigation,

profluorescent nitroxides have gathered greater interest as fluorescent probes.29, 32-48

Ascorbic Acid

N

O

O

7 8O

N

OH

O

O

Scheme 1.8: Naphthalene-based profluorescent nitroxide31

The advantage of these systems arises from the covalent linkage between the

nitroxide and the fluorophore which creates a permanent “collision complex”,

resulting in almost complete quenching of the expected fluorescence output.31

Radical scavenging or redox chemistry involving the nitroxide moiety forms

diamagnetic analogues. These reactions eliminate the fluorescence quenching

process, allowing for the detection of the diamagnetic species by fluorescence

spectroscopy (see 8 in Scheme 1.8 above).

Many examples of profluorescent nitroxides containing relatively labile linkages

such as esters,29, 31, 32, 35, 39-42 amides43-45 and sulfonamides36, 37, 46, 47 or comparatively

labile fluorescamine/amine adducts33, 34, 38, 48 have been the prime focus of this area

of research, presumably due to their relative ease of synthesis. Examples of these are

the ester nitroxide 7 (above), the polypeptide nitroxide44 9, and the sulphonamide

nitroxide37 10 as well as the fluorescamine-nitroxide adduct33 11 (below).

HN CO

N

O

COHN NH C

R

R1

CO

n

9


8

N

O

N

SO

ON

N

O

CO2H

OH

N

O

NMe2

10 11 Unmasking the fluorophore may occur via hydrolysis of a labile covalent tether

between a nitroxide and fluorophore. The fluorescence detected in this instance then

arises from the untethered fluorophore and not from a radical or redox related

product, which may lead to misinterpretation of experimental data. Systems linked

with non-cleavable carbon frameworks are more attractive for use as molecular

probes. With such systems false positives arising from disconnecting the fluorophore

from the nitroxide would be unlikely. Only one example of a profluorescent nitroxide

utilising a robust C-C linkage, published by Kalai et al.,49 had been reported prior to

the study described in this thesis. Further developments in this area have been

reported over the tenure of this project by Kalai et al.,50, 51 Barhate et al.52 and our

research group.53-56

1.2. Isoindoline Nitroxides

The isoindoline nitroxides include all 1,1,3,3-tetraalkylisoindolin-2-yloxyls and their

aromatic-substituted derivatives. The IUPAC numbering system is shown below in

Scheme 1.9.

N O

RR

RR

3

7a1

2

4a

7

6

5

4

X

Scheme 1.9: Structure and numbering of isoindoline nitroxides

The earliest examples of this class of nitroxides were tetraethyl systems, such as

1,1,3,3-tetraethylisoindolin-2-yloxyl 12 (TEIO), synthesised in the late 1960’s and

early 1970’s by Rozantsev and co-workers.57-59 Further work on the tetraethyl

isoindoline nitroxides was performed by the Rassat group,60, 61 in the mid to late

1970’s.


9

N O

12 The complete synthesis of the methyl analogue, 1,1,3,3-tetramethylisoindolin-2-

yloxyl 4 (TMIO), was first published in 1983 by Griffiths et al.62 This nitroxide has

been employed as a radical scavenger to give insight into the initiation of

polymerisation reactions, as reported in several publications originating from the

CSIRO Australia.5, 9, 10, 12 Since these initial reports, the use of TMIO 4 as a radical

trap has become the most extensive area of research involving isoindoline

nitroxides.6-8, 11, 63, 64 Ease of detection, separation and analysis of the radical adducts

gives valuable insight into processes which occur when polymerisation is first

initiated.

N O

4 Several factors make TMIO 4 a useful tool for radical trapping and other studies.

The compound is symmetrical, which leads to less complicated NMR spectra from

the products. The UV chromophore of the aromatic ring facilitates detection of the

radical adducts by standard HPLC systems. The compound is also unreactive

towards alkenes and is inert to free radical attack with the exception of radical

recombination at the nitroxide moiety.5

Further structural analysis of TMIO 4, performed by Busfield et al. in 1986, included

its x-ray crystal structure and the solid state 13C NMR of an alkoxyamine derivative

of TMIO 4.65 These studies confirmed the planarity of the fused ring system. The

complete mass spectral analysis of TMIO 4, performed by Bartley et al., followed

1988.66

Gillies and co-workers have utilised TMIO 4 and a variety of its derivatives as spin

probes in a number of EPR studies.67-76 These investigations showed that isoindoline

nitroxides have much narrower EPR linewidths than those of the TEMPO family of

nitroxides.70


10

1.2.1. Photostability of Isoindoline Nitroxides

The fused aromatic ring present in isoindoline nitroxides makes them more robust

towards α-cleavage from photoexcitation than less rigid systems such as piperidine

and pyrroline nitroxides.77 Recombination of the nitroso group and the carbon

centred radical of the photo-cleaved product is favoured for the isoindoline system

shown in Scheme 1.10.

N O N O

4 Scheme 1.10: Stability of TMIO 4 towards α-cleavage

Pyrroline nitroxides, such as 13, suffer from photodegradation to form NO• and an

alkene,78 presumably because recombination does not occur as readily as it does for

isoindoline nitroxides. This is shown in Scheme 1.11 below.

N

O

+ NO

CONH2CONH2

N

O

rapidslow

CONH2

13 Scheme 1.11: Photodegradation of a pyrroline nitroxide 13

1.2.2. Drawbacks – Methodologies and Structure

Despite their advantages, applications of the isoindoline class of nitroxides have been

restricted by a lack of structural variation. Expansion of the structural diversity of the

isoindoline nitroxides is particularly important with respect to the generation of

profluorescent systems. Prior to this work, few substitution reactions had been

performed directly on the aromatic ring, in the presence of the nitroxide moiety.

Nitration70 and Friedel-Crafts alkylation79 were the only reports in the literature prior

to this current investigation. Further examples, such as Pd-catalysed cyanation,80

have emerged since the beginning of this project, but introduction of other

functionality prior to the formation of the radical moiety is more often favoured.52, 58,

68, 72, 75, 81, 82

hν

hν


11

1.3. Project Outline

The main objective of the research project described in this thesis is the synthesis of

water soluble profluorescent nitroxides. Robust carbon-carbon frameworks, based on

the isoindoline system, that have potential application in biological environments

were of particular interest. Due to the lack of C-C bond forming methodologies in the

presence of nitroxide moieties in the existing literature, the development of several

new synthetic techniques was also explored. Previously, Pd-catalysed Heck coupling

had been used by the author and been shown to be successful.83 Further

developments of Pd-catalysed techniques were of interest during this project, due to

their functional group tolerances. The ability to add the extended conjugation

required for the production of fluorescent products, via methodologies such as Heck

and Sonogashira couplings, added to the attraction of Pd-catalysed techniques.

Fluorescence analysis of the novel profluorescent nitroxides and comparison to

related diamagnetic analogues may give insight into the viability of each compound

as a fluorescent probe.

Chapter 2 – Palladium-Catalysed Heck Coupling of Isoindoline Nitroxides

12

2. PALLADIUM-CATALYSED HECK COUPLING OF

ISOINDOLINE NITROXIDES

2.1. Introduction

The Pd-catalysed Heck reaction is attractive for the synthesis of profluorescent

nitroxides containing non-cleavable carbon-carbon bonds. Heck coupling has the

potential to generate, in one step, the necessary extended conjugation for organic

fluorophores.84 Compounds containing stilbene moieties are achievable via this

methodology. Stilbenes are often used as fluorescent probes because of their high

quantum yields and short excited state lifetimes.85

2.2. The Heck Alkenylation Reaction

In the late 1960s Heck86, 87 discovered the Pd-catalysed arylation and alkylation of

olefins using organomercury, -lead or -tin reagents (see example in Scheme 2.1).

Whilst these reactions led to the desired alkene products, there were also some

drawbacks. It was often problematic to obtain the required organometallics for the

desired transformation, the mixtures were commonly thick slurries of salts and some

of the reagents were highly toxic.88

CH3+

HgOAc CH3Pd(OAc)2

30 psi, MeOH 30 °C, 1 h

Scheme 2.1: An example of arylation of alkenes by organomercuries86

A few years later, when Mizoroki et al.89 and Heck and Nolley88 independently

discovered the palladium-catalysed alkenylation of aryl- and vinyl-halides the

problems arising from the organomercury, -lead or -tin reagents were avoided.

In their original report,89 Mizoroki et al. synthesised substituted styrenes and

stilbenes in moderate to high yields from iodobenzene and olefins (see Scheme 2.2).

Relatively harsh conditions were required, with the reactions undertaken in a

titanium-alloy autoclave at 120 °C employing KOAc and PdCl2 in MeOH.


13

R+

I RPdCl2 KOAc

MeOH 120 °C 2-3 h

26-97 %R = H, alkyl or aryl

Scheme 2.2: Olefination of iodobenzene by Mizoroki et al.89

Heck and Nolley88 determined that using Pd(OAc)2 as the catalyst and (n-Bu)3N as

the solvent and the base at 100 °C gave moderate to high yields of cross-coupled

products of aryl iodides and olefins using more convenient laboratory conditions than

used by Mizoroki (see Scheme 2.3 below).

R'+

I R'Pd(OAc)2(n-Bu)3N

100 °C 1-72 h

37-85 %R = H, OMe, CO2Me, I

R' = H, alkyl or aryl

R R

Scheme 2.3: Olefination of aryl iodides by Heck and Nolley88

After these initial studies88, 89 the reaction was further developed by Heck and co-

workers90-98 to incorporate a variety of functional groups present on both the aryl-

halide and vinyl coupling partners. The Pd-catalysed alkenylation (now commonly

referred to as the Heck reaction) is defined as a vinylic substitution reaction where a

vinyl hydrogen is substituted by an aryl, vinyl or benzyl group (see Scheme 2.4).99

The Heck reaction is an extremely useful synthetic technique as the vinylation of

organic halides cannot be carried out in a single step by any other known method.84

R'R X

RR'Pd (0)

+

Scheme 2.4: General outline of the Heck reaction

The catalytic cycle of the Heck reaction begins with the formation of a Pd(0)

catalyst, usually from Pd(0) complexes such as [Pd(PPh3)4] or [Pd2(dba)3], or by in

situ reduction of Pd(II) salts such as Pd(OAc)2 or PdCl2. This active Pd(0) species

then undergoes oxidative addition of the organic halide, forming a Pd(II) species (see

Scheme 2.5 below). The alkene then coordinates to the Pd(II) complex, after which

the organic fragment undergoes insertion (carbometallation) giving a new Pd(II)

species. This is followed by β-elimination which gives the desired alkene product

and a third Pd(II) species. Reductive elimination of the acid (HX), which is


14

neutralised by a base, regenerates the active Pd(0) catalyst.97 The salt (HX-base)

produced forces the equilibrium of the reversible reductive elimination step towards

the Pd(0) species.97 The Pd(0) catalyst is often stabilised by the addition of

phosphine ligands, although addition of a large excess inhibits the cross coupling

reaction.100

RX

Oxidative Addition

Precursor

[PdII]X

R

Carbometallation

Reductive Elimination

R'

[PdII]LR

R'

beta-Elimination

HX-Base[Pd0]

L

L

L

L

X

LR'

H R

[PdII]X

H

L

L

Scheme 2.5: The reaction mechanism of the Heck reaction99

Alkenes containing electron withdrawing groups are often more effective coupling

partners. Electron rich alkenes commonly form undesirable side-products, such as

regioisomers and diarylated products.99 The stereochemistry of the product is largely

governed by steric factors, whereby the coupling of small molecules may give both

cis and trans isomers, whilst coupling larger groups results in almost quantitative

formation of the trans product.84

2.2.1. Heck Reactions Performed on Nitroxides

To date there have been few reports of Heck coupling in the presence of nitroxide

radicals. The only example in the literature prior to the present investigation was

published in 2002 by Hideg and co-workers101 A brominated pyrroline nitroxide 14

was coupled with ethyl acrylate to give an intermediate diethenyl pyrroline nitroxide,

which gave a substituted isoindoline nitroxide 15 after treatment with MnO2/O2 via a

6π-electrocyclisation (Scheme 2.6).


15

N

O

Br

Pd(OAc)2, PPh3Et3N, DMF100 °C 30 h

CO2Et

CO2Et

N

O

CO2EtEtO2C

N

O

CO2EtEtO2C

MnO2 O2

CHCl3 30 min

14 15 31 %

Scheme 2.6: Pyrroline nitroxide Heck coupling by Hideg and co-workers101

2.3. Results and Discussion

Although Hideg and co-workers101 have successfully demonstrated Heck coupling of

pyrroline nitroxides, the Heck reaction and Pd-catalysis in general have been an

under-utilised technique for the functionalisation of nitroxides. This is possibly due

to a preconceived notion of incompatibility between the nitroxide moiety and the

catalytic system. Initial investigation into the Heck coupling of brominated

isoindoline nitroxides for the synthesis of profluorescent nitroxides was performed

by the author as part of an Honours program.83 These investigations led to the

unoptimised synthesis of nitroxide substituted stilbene and styrene derivatives.

Further synthetic developments related to the Heck coupling of brominated

isoindoline nitroxides are outlined in this chapter. The majority of the results

presented here have been published in the form of a full paper in Org. Biomol.

Chem.,56 with the X-ray crystal structure of 18 (see Figure 2.4) reported in Acta

Cryst. E.102 The synthesis of N-benzylphthalimide 32,103 2-benzyl-1,1,3,3-

tetramethylisoindoline 34,62 5-bromo-1,1,3,3-tetramethylisoindoline 35,75 5-bromo-

1,1,3,3-tetramethylisoindolin-2-yloxyl 16,75 5,6-dibromo-1,1,3,3-tetramethyl-

isoindoline82 36 and 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl82 21 were

achieved by the previously published procedures.

2.3.1. Heck Coupling of 5-bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl

(16)

Initial studies of the Heck coupling of the bromo nitroxide, 5-bromo-1,1,3,3-

tetramethylisoindolin-2-yloxyl 16, by reaction with methyl acrylate 19 or methyl 4-

vinylbenzoate 20 gave two novel compounds. The reaction conditions were not

optimised and the yields (13-34 %) of the coupled products were considered to be


16

low (Table 2.1).83 Due to its low boiling point (80.7 °C),104 a large excess of methyl

acrylate (10 equiv) was used to maximise the available concentration in the reaction

mixture.

N O

BrN O

CO2Me

MeO2C

19

20

R

17 R =

18 R =

MeO2C

MeO2C

16

Pd(OAc)2 (2.5 mol%)PPh3 (5 mol%)

K2CO3 (2 equiv)

DMF100 °C, Ar, 72 h

Table 2.1: Previously reported yields for mono-substituted Heck products83

Entry Alkene Product Yield (%)

1 19

(10 equiv) 17 13

2 20

(1.3 equiv) 18 34

Initial optimisation of the Heck reaction was performed on the stilbene nitroxide 18

rather than the cinnamate nitroxide 17, as it possessed a larger magnitude of relative

fluorescence quenching.

As Heck reactions can be slow, often taking considerable time for full consumption

of the starting material, the reaction time was increased. The reaction was analysed

by HPLC (UV detection at λmax of 18 at 326 nm, sampling at 0, 1, 4, 7, 12, 15 and 21

days). The conversion to the styrylcarboxylate 18 is shown in Figure 2.1 below. The

majority of conversion was complete within ~24 h (ca. 75 %), but the amount of

product detected slowly increased over time, with no bromo starting material able to

be detected after 21 days. Whilst this information was obtained from only one series

of data and therefore was used only as an initial guide, these results did indicate that

complete conversion had not occurred in the initial reaction. Since a reaction time of

over three weeks is impractical, further optimisation was required.


17

0

500

1000

1500

2000

2500

3000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Time/days

Pea

k A

rea

from

HP

LC

Figure 2.1: Optimisation of the synthesis of 18 - time varied

Due to the slow conversion of starting material other factors influencing the yield

needed to be investigated. The reaction yield might be increased by increased

catalyst concentrations, higher reaction temperatures or a combination of both. The

first of these investigated was the catalyst concentration. Again the other conditions

remained the same as the previous conditions (Entry 2, Table 2.1). The amount of

product was determined after 5 and 12 days by HPLC detection. The results of this

investigation are given in Figure 2.2 below, with each group of data representing one

experiment.

0

500

1000

1500

2000

2500

3000

3500

4000

0 2 4 6 8 10 12

Time/days

Pea

k a

rea

fro

m H

PLC

2.5 mol % 5 mol % 10 mol % 20 mol %

Figure 2.2: Optimisation of the synthesis of 18 – Pd catalyst concentration varied

While previous reactions had been performed using 2.5 mol % of Pd catalyst, it can

be seen that the amount of product increases markedly when the catalyst


18

concentration is increased to 5 mol %. Further increases to 10 mol % and 20 mol %

showed decreased catalytic activity and lower yields. From these results it was

determined that 5 mol % of Pd catalyst would provide greater yield than the

previously used amount, 2.5 mol %. Also observable is a decrease in the yields from

5 days to 12 days for the higher catalyst loadings (5 mol% and 10 mol%). This may

be due to some product degradation, which possibly arises from the relatively high

reaction temperature (100 °C) over a prolonged period of time.

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Time/days

Are

a o

f H

PL

C P

eak

80 °C 100 °C 120 °C 140 °C

Figure 2.3: Optimisation of the synthesis of 18 – temperature varied

From the temperature optimisation results shown in Figure 2.3 above (each group of

data represents one experiment), it can be seen that the optimum temperature for the

reaction was 120 °C. Lower yields of the product, which is probably brought about

by degradation, occur at 140 °C, with the amount of product detected being higher at

5 days than 12 days. At 140 °C the reaction mixture rapidly darkens. This colour

change is thought to indicate breakdown of the palladium catalyst and usually takes

3-4 days at 100 °C. At 120 °C the black colour is formed within 1-1.5 days.

However at 120 °C over this time frame, this does not seem to affect the reaction and

a high yield can be obtained. Interestingly, no conversion of the starting material can

be observed by HPLC when the reaction was performed at 80 °C, even after 12 days.

Subsequently, the styryl carboxylate nitroxide 18 was synthesised using the

optimised reaction conditions, 120 °C, with 5 mol % catalyst for three days (three

days was seen as a good compromise between practical time frame and product


19

yield). This gave 19 in a significantly increased (isolated) yield of 85 % (Entry 1,

Table 2.2).

N O

BrN O

R

17 R =

18 R =

MeO2C

MeO2C16

Pd(OAc)2 (5 mol%)PPh3 (10 mol%)K2CO3 (2 equiv)

DMF120 °C, Ar, 72 h

Table 2.2: Optimised yields for mono-substituted Heck products

Entry Alkene Product Yield (%)

1 20

(1.3 equiv) 18 85

2 19

(10 equiv) 17 50

Unequivocal structural determination of 18 was obtained by X-ray crystallography

(data collected by J. K. Clegg, University of Sydney and refined by J. C. McMurtrie,

Queensland University of Technology) and confirmed the trans (E) geometry of the

alkene (Figure 2.4 below).

Figure 2.4: X-ray crystal structure of 18 (H atoms omitted for clarity)

The reaction of the bromo nitroxide 16 with methyl acrylate 19 under the improved

reaction conditions used for the generation of 18 gave the cinnimate nitroxide 17 in


20

an improved yield of 50 % (see Entry 2, Table 2.2). Whilst 17 was isolated in a

moderate yield, this was a marked improvement from the previous isolated yield of

13 %. It is expected that optimisation of the reaction conditions tailored specifically

for the synthesis of 17 would further improve this yield.

2.3.2. Heck Coupling of 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-

yloxyl (21)

The yields of di-substituted Heck products, previously synthesised by the author83

from 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21, were also increased by

optimising catalyst loadings and the temperature. The synthesis of the diacrylate

nitroxide 22 and the distyryl nitroxide 23 had previously been achieved in yields of

32 % and 36 %, respectively (5 % catalyst, 100 °C, Entries 1 and 3, Table 2.3

below).83 Note that the catalyst concentration was doubled from that used in the

mono-substituted reactions to compensate for two coupling reactions per nitroxide

molecule.

N O

BrN O

R

22 R =

23 R =

MeO2C

MeO2C21

Br R

Pd(OAc)2PPh3

K2CO3 (4 equiv)

DMFAr, 72 h

Table 2.3: Yields for di-substituted Heck products83

Entry Alkene Pd(OAc)2

(mol%)

PPh3

(mol%)

Temperature

(°C) Product

Yield

(%)

1 19

(20 equiv) 5 10 100 22 32

2 19

(20 equiv) 10 20 120 22 62

3 20

(2.6 equiv) 5 10 100 23 36

4 20

(2.6 equiv) 10 20 120 23 53


21

The reaction of the dibromo nitroxide 21 with methyl acrylate 19 at 120 °C and with

10 % catalyst concentration gave the diacrylate nitroxide 22 in an improved yield of

62 % (Entry 2, Table 2.3). Similarly, the reaction of the methyl 4-vinylbenzoate 20

with dibromo nitroxide 21 gave the distyryl nitroxide 23 in an improved yield (53 %)

(Entry 4, Table 2.3).

Interestingly, the disubstituted Heck products (22 and 23) are obtained in similar

yields to the mono-substituted products (17 and 18). This may be explained by the

electron density of the aryl halides (16 and 21). Vinylation would be expected to

lower electron density in the ring and therefore promote reactivity for the second

substitution and this may lead to the improvement in yields.

Further evidence of this is the absence of detectable levels of mono-substituted

products, such as 24 or 25, from the di-substitution reactions.

N O

Br

N O

MeO2C

Br

MeO2C

24 25

2.3.3. Synthesis of Methoxyamines (26 and 27)

The methoxyamine analogues 26 and 27 of the styryl-substituted nitroxides 18 and

23 were prepared for further structural characterisation by NMR spectroscopy and as

a fluorescence comparison to the nitroxides. Reaction of nitroxides 18 and 23 with

methyl radicals (formed by Fenton chemistry by reaction of FeSO4·7H2O with H2O2

in DMSO, in which the hydroxyl radicals produced react with the solvent to liberate

methyl radicals; for further details see Section 4.2.4 in Chapter 4) gave the desired

methoxyamine adducts, 26 and 27, in high yields of 77 % and 82 %, respectively

(Scheme 2.7, see Experimental for further details).


22

N O

R

MeO2C

N O

R

MeO2C

MeO2C

18 R = H

23 R =MeO2C

26 R = H 77 %

27 R = 82 %

FeSO4.7H2OH2O2

DMSORT, 30 min

Scheme 2.7: Synthesis of methoxyamines 26 and 27

2.3.4. Fluorescence Data of Vinyl-Substituted Profluorescent Nitroxides

Comparison of the fluorescence spectra of the stilbene nitroxide 18 and its

methoxyamine analogue 26 (Figure 2.5) and the bis-stilbene nitroxide 23 and its

methoxyamine analogue 27 (Figure 2.6) show substantial fluorescence suppression

by the nitroxide moiety.

-0.005

0.005

0.015

0.025

0.035

260 300 340 380

Wavelength/nm

Abso

rbance

0

50

100

150

200

250

340 390 440 490

Wavelength/nm

Inte

nsi

ty/a

u


330 nm in cyclohexane normalised to 1 µM


23

0

0.01

0.02

0.03

0.04

0.05

250 300 350 400

Wavelength/nm

Ab

sorb

an

ce

0

200

400

600

800

1000

340 390 440 490 540 590

Wavelength/nm

Inte

nsi

ty/a

u



The quantum yields of the stilbene nitroxide 18 (ФF = 6 × 10-4) and the bis-

carboxystyryl nitroxide 23 (ФF = 9 × 10-4) were significantly smaller than their

corresponding methoxyamines 26 (ФF = 0.13) and 27 (ФF = 0.71). The quantum

yields were measured in cyclohexane, using anthracene 30 as the standard.

30

The nitroxides 18 and 23 have similar quantum yields to those previously reported

within our research group.54 Interestingly, the bis-system 27 has a much larger

quantum yield than the mono-substituted 26 possibly due to the extended π

conjugation present in 27, which gives the bis-carboxystyryl nitroxide 23 greater

dynamic range when used as a fluorescence probe. As expected, the mono-

carboxystyryl substituted methoxyamine 26 has a maximum emission at a shorter

wavelength (376 nm) than the corresponding di-substituted methoxyamine 27 (418

nm), due to the extended π conjugation of the di-substituted system.

The difference in the fluorescence intensity between the profluorescent nitroxides

(18 and 23) and the diamagnetic methoxyamines (26 and 27) indicates that these

compounds may be useful fluorescent probes for the detection of reactive

radical/redox species.


24

Table 2.4: Quantum yields of Heck products

Compound Quantum Yield (ФF)

18 0.0006

26 0.13

23 0.0009

27 0.71

2.3.5. Water Soluble Profluorescent Nitroxides

For potential application of the profluorescent nitroxides synthesised via Heck

methodology in biological systems, an increase in their water solubility is desired.

Hydrolysis of the esters to carboxylic acids was proposed for this purpose. Reaction

of the mono-methyl ester 18 or di-methyl ester 23 with NaOH in refluxing

THF/water gave the corresponding acids 28 and 29, in high yields of 94 % and 84 %

respectively (Scheme 2.8). Whilst the presence of the carboxylic acid groups did

improve the water solubility of the nitroxides, basic or buffered solutions were

required to completely dissolve the compounds in aqueous media. The fluorescence

spectra of the carboxylic acids 28 and 29 mirrored those of the esters 18 and 23.

N O

R

MeO2C

N O

R

HO2C

MeO2C

18 R = H

23 R =HO2C

28 R = H 94 %

29 R = 84%

NaOH

THF/H2Oreflux, 16 h

Scheme 2.8: Synthesis of carboxylic acids 28 and 29

After its synthesis and characterisation by the author, the carboxy-stilbene nitroxide

28, was supplied to S. Maniam (Australian National University) for use as the axle

(guest) component for synthesis of the first example of a nitroxide-rotaxane 30,105

incorporating α-cyclodextrin 31 as the host.


25

NO

HN

O

O

HOHO

OH

O

OHO

HO OHO

OOH

HO

OH

O

OO

HOOH

HO

O OH

OHHO

O

O

OH

OH

HO

O

=

30 31

2.4. Summary of Results

A series of novel vinyl substituted nitroxides was successfully synthesised via Pd-

catalysed Heck coupling. The nitroxide moiety was found to not adversely affect the

Pd-catalysed coupling reaction. Optimisation of the reaction conditions increased the

yields of the Heck products significantly, with the optimal conditions being 5 mol%

Pd catalyst (Pd(OAc)2/2PPh3) in dry DMF at 120 °C for 3 days. Due to the high

electron density of the bromo nitroxides used in this study, more forcing conditions

than those commonly found in the literature were required to give high yields of

products.

Fluorescence comparison of the mono- 18 and bis- 23 stilbene nitroxides and the

corresponding methoxyamines 26 and 27 showed substantial fluorescence quenching

(~220-fold for 18 and ~780-fold for 23) by the nitroxide moiety. This illustrates the

potential of isoindoline based profluorescent probes for detection of redox/radical

species.

Base hydrolysis of the esters 18 and 23 yielded profluorescent carboxylic acids 29

and 30. These compounds displayed enhanced water solubility, required for potential

applications in the biological field.

2.5. Experimental

All reported synthetic work was performed at the Queensland University of

Technology, Brisbane, Australia.

1H (9.39 Tesla, 400.162 MHz) and 13C (100.051 MHz) NMR spectra were recorded

on a Bruker Avance FT-NMR spectrometer; samples were prepared in CDCl3 (δH=

7.26 ppm, δc=77.0 ppm) unless otherwise stated. Note: due to the paramagnetism of


26

the nitroxide moiety, NMR cannot provide information useful for structural

elucidation of nitroxides.

Infrared spectra were recorded as neat samples using a Nicolet 870 Nexus Fourier

Transform infrared spectrometer equipped with a DTGS TEC detector and an

Attenuated Total Reflectance (ATR) accessory (Nicolet Instrument Corp., Madison,

WI) using a Smart Endurance single reflection ATR accessory equipped with a

composite diamond IRE with a 0.75 mm2 sampling surface and a ZnSe focussing

element. An Optical Path Difference (OPD) velocity of 0.6329 cm s-1 and a gain of 8

were used. Spectra were collected in the spectral range 4000-525 cm-1 with a

minimum of 16 scans, and 4 cm-1 resolution.

Analytical Thin-Layer Chromatography (TLC) was performed using Merck Silica

Gel 60 F254 TLC plates. Preparative column chromatography was performed using

Merck Silica Gel 60 (mesh 230-400).

Low and high resolution mass spectra were recorded at the Australian National

University (ANU) using either a Micromass autospec double focusing magnetic

sector mass spectrometer (EI+ spectra) or a Bruker Apex 3 Fourier transform ion

cyclotron resonance mass spectrometer with a 4.7 T magnet (ESI+ spectra).

Formulations were calculated in the elemental analysis programs of Mass Lynx 4.0

or Micromass Opus 3.6.

Analytical HPLC was performed on a Hewlett Packard 1100 series HPLC, using an

Agilent prep-C18 scalar column (10 µm, 4.6 × 150 mm) at a flow rate of 1mL/min.

Preparatory HPLC was performed on a Hewlett Packard 1200 series HPLC, using an

Agilent prep-C18 column (10 µm, 21.2 × 150 mm) at a flow rate of 15 mL/min.

All UV/Vis spectra were recorded on a single beam Varian Cary 50 UV-Vis

spectrophotometer. Spectrofluorimetry was performed on a Varian Cary Eclipse

fluorescence spectrophotometer equipped with a standard multicell Peltier

thermostatted sample holder. Optimum excitation and emission wavelengths were

obtained by a pre-scan operation. All solutions for UV/Vis and fluorescence analysis

were prepared in cyclohexane and measured using 10 mm quartz fluorescence

cuvettes.


27

All solvents were AR grade unless otherwise stated. Diethyl ether and toluene were

dried and stored over sodium wire. Powdered anhydrous AlCl3 (99.99 %), 4-

vinylbenzoic acid (97 %) and anhydrous dimethylformamide (DMF) (99.8 %) were

purchased from Sigma/Aldrich. Palladium acetate (99 %) was purchased from

Precious Metals Online. All other chemical reagents were of Laboratory Reagent

(LR) quality or better and used as supplied by the manufacturer.

2.5.1. N-Benzylphthalimide (32)103

N

O

O

32

Phthalic anhydride 33 (107 g, 0.722 mol) and benzylamine (120 mL, 1.10 mol, 1.52

equiv) were dissolved in glacial acetic acid (500 mL) and refluxed for 1 hour. The

hot reaction mixture was poured onto ice/water (~1.5 L) with stirring. The resultant

white precipitate, N-benzylphthalimide 32, was isolated by vacuum filtration and

recrystallised from EtOH to give white needles which were dried thoroughly under

high vacuum (168.0 g, 98 %), mp 115–118 °C (lit.,106 116 °C); δH: (CDCl3) 4.85

(2H, s, CH2), 7.23-7.33 (3H, m, 4-H’, 5-H’ and 6-H’), 7.43 (2H, d, J 7.1 Hz, 3-H’

and 7-H’), 7.70 (2H, dd, J 3.2 Hz and 5.4 Hz, 4-H and 7-H), 7.84 (2H, dd, J 3.2 Hz

and 5.4 Hz, 5-H and 6-H); δC: (CDCl3) 41.6 (CH2), 123.3 (C-3’ and C-7’), 127.8 (C-

5’), 128.6 (C-4’ and C-6’), 128.7 (C-4 and C-7), 132.1 (C-3a and C-7a), 134.0 (C-5

and C-6), 136.3 (C-2’), 168.0 (C=O). These data agree with those reported

previously by Hsieh and Cheng.107

2.5.2. 2-Benzyl-1,1,3,3-tetramethylisoindoline (34)62

N

34

Pre-dried magnesium (120 g, 4.94 mol, 11.7 equiv) and several small crystals of

iodine were placed into a flame-dried round-bottomed flask (3 L), equipped with a


28

still-head, a thermometer, two dropping funnels, a mechanical stirrer and two twin

helix condensers connected to a peristaltic pump used to deliver chilled water. The

apparatus was flame-dried under high vacuum, which resulted in a fine coating of

iodine on the magnesium, after which a positive pressure of argon was applied.

Anhydrous Et2O (400 mL) and a small amount of MeI (~5 mL) were added to the

flask to initiate the reaction. After Et2O reflux became evident, the remaining MeI

(155 mL total, 353 g, 2.48 mol, 5.9 equiv) and further Et2O (~ 1 L) were added at a

rate that maintained a constant rate of reaction. When the addition was complete, the

reaction was stirred until all activity subsided. The solution of Grignard reagent was

then concentrated by the distillation of solvent until the reaction mixture reached 80

°C.

The reaction mixture was allowed to cool slightly (to 64 °C) and a solution of N-

benzylphthalimide 32 (100 g, 0.421 mol) in dry toluene (800 mL) was added at such

a rate to maintain a constant temperature (~64 °C). When the addition was complete,

the reaction mixture was heated and solvent was removed by distillation until the

temperature of reaction mixture reached 110 °C. The mixture was refluxed for 3

hours and then concentrated by the removal of solvent (~400 mL).

After allowing the reaction mixture to cool, it was then diluted with hexanes (~1.5 L)

and mixed thoroughly. Upon exposure to atmosphere the reaction mixture became a

dark purple colour. This solution was filtered through Celite and the residue washed

thoroughly with hexanes. Air was bubbled through the purple filtrate overnight,

which was then passed through basic alumina (activity I) to remove highly coloured

purple side-products, yielding a colourless solution. The solvent was removed under

reduced pressure to yield a golden oil which crystallised spontaneously (29.8 g, 1.33

mol, 27 %). Recrystallisation from MeOH gave white crystals of 2-benzyl-1,1,3,3-

tetramethylisoindoline 34 (25.4 g, 1.13 mol, 23 %), mp 57– 60 °C (lit.,62 63−64 °C);

δH: (CDCl3) 1.32 (12H, s, CH3), 4.02 (2H, s, CH2), 7.16 (2H, dd, J 3.2 Hz and 5.6

Hz, 4-H and 7-H), 7.26 (2H, dd, J 3.2 Hz and 5.6 Hz, 5-H and 6-H), 7.24-7.36 (3H,

m, 4-H’, 5-H’, and 6-H’), 7.49 (2H, d,7.4 Hz, 3-H’ and 7-H’); δC: (CDCl3) 28.4

(CH3), 46.4 (CH2), 65.2 (C-1 and C-3), 121.3 (C-4 and C-7), 126.4 (C-5’), 126.7 (C-


29

3’ and C-7’), 127.9 (C-4’ and C-6’), 128.3 (C-5 and C-6), 143.4 (C-2’), 147.8 (C-3a

and C-7a). These data agree with those reported previously by Griffiths et al.62

All Grignard residues and contaminated glassware were deactivated with iso-

propanol, while still wet, to prevent the spontaneous combustion of activated

magnesium and unreacted Grignard reagent.

2.5.3. 5-Bromo-1,1,3,3-tetramethylisoindoline (35)75

N H

Br

35

To a stirred solution of 2-benzyl-1,1,3,3-tetramethylisoindoline 34 (5 g, 18.8 mmol)

in DCM (60 mL), at 0 °C under argon, a solution of liquid Br2 (2.15 mL, 42.0 mmol,

2.2 equiv) in DCM (40 mL) was added, followed by anhydrous AlCl3 (9 g, 67.5

mmol, 3.5 equiv). The reaction mixture was stirred under argon at 0 °C for 1 hour

and then poured onto ice (~150 mL) and stirred for 15 minutes, after which the

solution was basified with NaOH (10 M) and extracted with DCM (3 × 70 mL). The

combined organic phases were washed with brine (2 × 40 mL), dried (Na2SO4) and

the solvent removed under reduced pressure to give a yellow oil.

The residue was taken up in MeOH (~30 mL) and NaHCO3 (~200 mg) was added.

Aqueous H2O2 (30 %) was added until no further effervescence could be detected.

The mixture was treated with H2SO4 (2 M) (75 mL) (caution: effervescence) and

washed with DCM (2 × 50 mL) to remove the benzaldehyde produced. These

combined organic phases were back extracted with H2SO4 (2 M, 3 × 40 mL) to

recover any removed product. The combined acidic phases were washed with DCM

(2 × 40 mL) after which they were cooled in an ice/water bath, basified with NaOH

(10 M) and extracted with DCM (5 × 70 mL). The combined organic phases were

washed with brine, dried (Na2SO4) and the solvent was removed under reduced

pressure to give a golden oil which rapidly crystallised to give a white solid. The

resulting 5-bromo-1,1,3,3-tetramethylisoindoline 35 (3.93 g, 81 %) mp 58−60 °C

(90−95 % purity according to 1H NMR) was used in consequent steps without


30

subsequent purification; δH: (CDCl3) 1.43 (6H, s, CH3), 1.44 (6H, s,CH3), 1.69 (1H,

br, NH), 6.98 (1H,d, J 8.0 Hz, 7-H), 7.23 (1H, d, J 1.8 Hz, 4-H), 7.35 (1H, dd, J 1.8

Hz and 8.0 Hz, 6-H); δC: (CDCl3) 31.7 (CH3), 62.6 (C-1), 62.7 (C-3), 121.4 (C-5),

123.1 (C-6), 124.8 (C-7), 130.2 (C-4), 147.8 (C-7a), 151.2 (C-3a); νmax (ATR-FTIR):

3343 (NH), 3034 (aryl CH), 2957 (alkyl CH), 1457 and 1436 (aryl C-C), 597 (Aryl

C-Br) cm-1. These data agree with those reported previously by Bottle et al.75

2.5.4. 5,6-Dibromo-1,1,3,3-tetramethylisoindoline (36)82

N H

Br

Br

36

To a stirred solution of 2-benzyl-1,1,3,3-tetramethylisoindoline 34 (4 g, 15.1 mmol)

in CCl4 (80 mL) and pyridine (400 µL), under argon at 0 °C, liquid Br2 (12 mL, 234

mmol, 15.5 equiv) was added dropwise. After 15 minutes, anhydrous AlCl3 (12.8 g,

96 mmol, 6.4 equiv) was added. The solution was stirred at 0 °C for a further 4

hours, after which it was poured onto ice/water (~100 mL), basified with NaOH

(10M) and extracted with DCM (4 × 200 mL). The combined organic phases were

washed with water (2 × 150 mL), dried (Na2SO4) and the solvent was removed under

reduced pressure to give a yellow oil.

The residue was dissolved in MeOH (~30 mL) and NaHCO3 (~300 mg) was added.

Aqueous H2O2 (30 %) was added until effervescence ceased. The solution was then

treated with H2SO4 (2 M, ~100 mL; caution effervescence) and the acidic phase

washed with DCM (6 × 150 mL) to remove the benzaldehyde produced. These

combined organic phases were back extracted with H2SO4 (2 M, 3 × 100 mL) to

recover any removed product and combined with the acidic phase, which was then

basified with NaOH (5 M) and extracted with DCM (5 × 150 mL). The combined

organic phases were dried (Na2SO4) and the solvent removed under reduced pressure

to give a golden oil, which solidified to give white crystals of 5,6-dibromo-1,1,3,3-

tetramethylisoindoline 36 (3.5 g, 70 %) mp 74−76 °C (lit.,82 76−77 °C); δH: (CDCl3)

1.42 (12H, s, CH3), 1.72 (1H, br, NH), 7.34 (2H, s, 4-H and 7-H); δC: (CDCl3) 31.8

(CH3), 62.8 (C-1 and C-3), 123.1 (C-5 and C-6), 127.0 (C-4 and C-7), 150.4 (C-3a


31

and C-7a); νmax (ATR-FTIR): 3295 (NH), 3068 (aryl CH), 2963 (alkyl CH), 1465

and 1440 (aryl C-C), 602 (Aryl C-Br) cm-1. These data agree with those reported

previously by Micallef et al.82 The isolated product was used in subsequent steps

without further purification.

2.5.5. 5-Bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl (16)75

N O

Br

16

To a stirred solution of 5-bromo-1,1,3,3-tetramethylisoindoline 35 (~870 mg, 3.43

mmol) (10 % 5,6-dibromo-1,1,3,3-tetramethylisoindoline 36 according to 1H NMR,

~130 mg, 0.381 mmol, giving a total mass of 1 g) in MeOH (9 mL) and MeCN (370

µL) at room temperature, NaHCO3 (370 mg, 4.4 mmol, 1.1 equiv) and

Na2WO4·2H2O (150 mg, 0.45 mmol, 0.12 equiv) were added, followed by H2O2 (30

%, 3.2 mL, 28.3 mmol, 7.3 equiv). After 74 hours, water (~60 mL) was added to the

reaction mixture, which was then extracted with DCM (3 × 60 mL). The combined

organics were washed with H2SO4 (2M, 2 × 50 mL) and brine (2 × 50 mL), dried

(Na2SO4) and the solvent removed under reduced pressure to give a yellow

crystalline solid (0.93 g). The product was triturated with cold n-hexane dissolving

the desired product, 5-bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 16. The

undissolved over-brominated, 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21

(114 mg, 0.33 mmol, 87 %) was recrystallised from MeCN to give yellow needles

(65 mg, 0.19 mmol, 50 %) mp 254−256 °C (decomp.) (lit.,82 258 °C decomp.).

Evaporation of the triturant gave the desired 5-bromo-1,1,3,3-tetramethylisoindolin-

2-yloxyl 16 (851 mg, 3.16 mmol, 92 %) which recrystallised from MeCN to give an

orange solid (696 mg, 2.59 mmol, 75.5 %) mp 107−110 °C (lit.,82 109 °C). νmax

(ATR-FTIR): 3047 (aryl CH), 2973 (alkyl CH), 1480 and 1450 (aryl C-C), 1430

(NO), 630 (aryl C-Br) cm-1. These data agree with those reported previously by

Bottle et al.75


32

2.5.6. 5,6-Dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl (21)82

N O

Br

Br

21

To a stirred solution of 5,6-dibromo-1,1,3,3-tetramethylisoindoline 36 (1.3 g, 3.9

mmol) in MeOH (9 mL) and MeCN (370 µL) at room temperature, NaHCO3 (370

mg, 4.4 mmol, 1.1 equiv) and Na2WO4·2H2O (150 mg, 0.45 mmol, 0.12 equiv) were

added, followed by H2O2 (30 %, 3.2 mL, 28.3 mmol, 7.3 equiv). After 120 hours,

water (~60 mL) was added to the reaction mixture, which was then extracted with

DCM (3 × 60 mL). The combined organics were washed with H2SO4 (2M, 2 × 50

mL) and brine (2 × 50 mL), dried (Na2SO4) and the solvent was removed under

reduced pressure to give a yellow crystalline solid, 5,6-dibromo-1,1,3,3-

tetramethylisoindolin-2-yloxyl 21, (1.19 g, 88 %), which recrystallised from MeCN

to give yellow needles (0.93 g, 68 %) mp 254−256 °C (decomp.) (lit.,82 258 °C); νmax

(ATR-FTIR): 3018 (aryl CH), 2976 (alkyl CH), 1468 and 1444 (aryl C-C), 1429

(NO), 638 (aryl C-Br) cm-1. These data agree with those reported previously by

Micallef et al.82

2.5.7. Methyl 4-vinylbenzoate (20)108

MeO2C

20

A solution of 4-vinylbenzoic acid (1.5 g, 10.1 mmol) in MeOH (150 mL), containing

3 drops of H2SO4, (conc.) was refluxed for 5 hours, after which the solution was

diluted with CHCl3 (150 mL). The solution was washed with saturated NaHCO3

solution (2 × 75 mL) to remove any acidic starting material. The aqueous layer was

extracted with CHCl3 (3 × 100 mL). The combined organics were then washed with

brine (3 × 100 mL), dried (Na2SO4) and the solvent removed under reduced pressure

to give a white solid (1.22 g, 74 %). Methyl 4-vinylbenzoate 20 was purified by

column chromatography (DCM) to give a white solid which was used without further

purification (1.11 g, 68 %) mp 35−38 ° C (lit.,109 35−36 °C); δH: (CDCl3) 3.91 (3H, s,

OCH3), 5.38 (1H, d, J 10.8 Hz, =CH2), 5.86 (1H, d, J 17.6 Hz, =CH2), 6.75 (1H, dd,


33

J 10.8 Hz and 17.6 Hz, =CH-Ar), 7.46 (2H, d, J 8.4 Hz, Ar-H), 8.00 (2H, d, J 8.4 Hz,

Ar-H); δC: (CDCl3) 52.1 (CH3), 116.5 (=CH2), 126.1 (CH), 129.3 (CH), 129.9 (C),

136.0 (CH), 141.9 (C), 166.9 (C=O); νmax (ATR-FTIR): 3094 (=CH2), 2948 (alkyl

CH), 1710 (C=O), 1439 and 1403 (aryl C-C), 1629 (C=C), 1439 and 1403 (aryl C-

C), 1276 (OCH3) cm-1. These data agree with those reported previously by Webb and

Sanders.108

2.5.8. 5-[2-(4-Methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl (18)56

N O

MeO2C

18

5-Bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 18 (100 mg, 0.38 mmol), K2CO3

(100 mg, 0.72 mmol, 1.9 equiv), Pd(OAc)2 (4 mg, 0.018 mmol, 5 mol%) and PPh3 (8

mg, 0.031 mmol, 10 mol%) were placed in a Schlenk vessel, and evacuated and

flushed three times with argon. Methyl 4-vinylbenzoate 20 (80 mg, 0.49 mmol, 1.3

equiv) and anhydrous DMF (5 mL) were then added. The resulting solution was

frozen in liquid N2, evacuated and thawed three times, after which the reaction vessel

was sealed under argon and stirred at 120 °C for 72 hours. Water (~100 mL) was

poured into the brown reaction mixture, which was then extracted with Et2O (3 × 60

mL). The combined organic phases were washed with water (2 × 60 mL), dried

(Na2SO4) and the solvent removed under reduced pressure. The crude product was

purified by column chromatography (70 % EtOAc, 30 % n-hexane). Recrystallisation

from EtOH gave light orange needles of 5-[2-(4-methoxycarbonylphenyl)ethenyl]-

1,1,3,3-tetramethylisoindolin-2-yloxyl 18 (113 mg, 0.323 mmol, 85 %) mp 164−165

°C (found: C, 75.2; H, 6.9; N, 3.9. C22H24NO3 requires C, 75.4; H, 6.9; N, 4.0 %);

νmax (ATR-FTIR): 2976 (alkyl CH), 1715 (C=O), 1604 (C=C), 1492 (aryl C-C), 1434

(NO), 1177 (OCH3) cm-1; +EI MS found M+ 350.17546 (0.46 ppm from calc. mass of

C22H24NO3•); m/z 350 (M+, 30 %), 335 (25), 320 (100), 305 (42).


34

2.5.9. 5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl (23)56

N O

MeO2C

MeO2C

23

5,6-Dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 (100 mg, 0.28 mmol), K2CO3

(154 mg, 1.11 mmol, 4.0 equiv), Pd(OAc)2 (8 mg, 0.035 mmol, 12.5 mol%) and PPh3

(16 mg, 0.061 mmol, 20 mol%) were placed into a Schlenk vessel, which was

evacuated and flushed with argon three times. Methyl 4-vinylbenzoate 20 (120 mg,

0.737 mmol, 2.63 equiv) and anhydrous DMF (5 mL) were then added. The resulting

solution was frozen in liquid N2, evacuated and thawed three times, after which the

reaction vessel was sealed under argon and stirred at 120 °C for 72 hours. Water

(~50 mL) was added to the dark brown solution, followed by extraction with EtO2 (5

× 50 mL). The combined organic phases were washed with water (2 × 50 mL), dried

(Na2SO4) and the solvent removed under reduced pressure. The crude product was

was purified by column chromatography (30 % EtOAc, 70 % n-hexane). Subsequent

recrystallisation from MeCN gave fine light yellow needles of 5,6-bis-[2-(4-

methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl 23 (78 mg,

0.152 mmol, 54 %) mp 212−214 °C (decomp.); νmax (ATR-FTIR): 2973 (alkyl CH),

1708 (C=O), 1602 (C=C), 1484 (aryl C-C), 1432 (NO) cm-1; +EI MS found M+

510.22812 (0.14 ppm from calc. mass of C32H32NO5•); m/z 510 (M+, 15 %), 495 (9),

480 (100), 465 (10), 330 (40) (see HPLC 1, Appendix 1).

2.5.10. 5-(2-Methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-2-

yloxyl (17)56

N O

MeO2C

17


35

5-Bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 16 (100 mg, 0.38 mmol), K2CO3

(100 mg, 0.72 mmol, 1.9 equiv), Pd(OAc)2 (4 mg, 0.018 mmol, 5 mol%) and PPh3 (8

mg, 0.031 mmol, 10 mol%) were placed in a Schlenk vessel, which was evacuated

and flushed three times with argon. Methyl acrylate (0.32 mL, 3.55 mmol, 9.3

equiv) and anhydrous DMF (5 mL) were then added. The resulting solution was



poured into the brown reaction mixture, which was then extracted with DCM (4 × 50

mL). The combined organic phases were washed with water (2 × 50 mL), dried

(Na2SO4) and the solvent was removed under reduced pressure. The crude product

was purified by column chromatography (30 % EtOAc, 70 % n-hexane) and

recrystallised from MeCN to give 5-(2-methoxycarbonylethenyl)-1,1,3,3-

tetramethylisoindolin-2-yloxyl 17 as a yellow solid (52 mg, 0.189 mmol, 50 %) mp

162−164 °C; νmax (ATR-FTIR): 3023 (aryl CH), 2973 (alkyl CH), 1704 (C=O), 1641

(C=C), 1492 (aryl C-C), 1432 (NO), 1164 (OCH3) cm-1; +EI MS found M+

274.14427 (0.18 ppm from calc. mass of C16H20NO3•); m/z 274 (M+, 66 %), 259

(100), 244 (83), 229 (60) (see HPLC 2, Appendix 1).

2.5.11. 5,6-Bis-(2-methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-

2-yloxyl (22)56

N O

MeO2C

MeO2C

22

5,6-Dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 (100 mg, 0.28 mmol), K2CO3

(154 mg, 1.11 mmol, 4.0 equiv), Pd(OAc)2 (8 mg, 0.032 mmol, 10 mol%) and PPh3

(16 mg, 0.062 mmol, 20 mol%) were placed into a Schlenk vessel, which was

evacuated and flushed with argon three times. Methyl acrylate (0.5 mL, 5.55 mmol,

20 equiv) and anhydrous DMF (55 mL) were then added. The resulting solution was



poured into to the brown solution, which was extracted with DCM (3 × 70 mL). The

combined organic phases were washed with water (2 × 50 mL), dried (Na2SO4) and


36

the solvent was removed under reduced pressure. The residue after evaporation was

purified by column chromatography (30 % EtOAc, 70 % n-hexane) and

recrystallised from MeCN giving yellow needles of 5,6-bis-(2-

methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-2-yloxyl 22 (62 mg, 0.173

mmol, 62 %) mp 216−218 °C (decomp.); νmax (ATR-FTIR): 3066 (=CH), 3033 (aryl

CH), 2967 (alkyl CH), 1702 (C=O), 1627 (C=C), 1488 (aryl C-C), 1436 (NO), 1166

(OCH3) cm-1; +EI MS found M+ 358.16511 (0.95 ppm from calc. mass of

C20H24NO5•); m/z 358 (M+, 90 %), 343 (14), 328 (100), 283 (53), 268 (75), 253 (44)

(see HPLC 3, Appendix 1).

2.5.12. 5-[2-(4-Methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-

tetramethyl-isoindoline (26)56

N O

MeO2C

26

A solution of 5-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl 18 (40 mg, 0.114 mmol) and FeSO4·7H2O (64 mg, 0.230 mmol,

2 equiv) in DMSO (4 mL) had H2O2 (30 %, 26 µL) added dropwise. The mixture

was stirred at room temperature for 30 minutes. The resultant solution was poured

onto NaOH (1 M, 30 mL) and extracted with Et2O (3 × 50 mL) and dried (Na2SO4).

Removal of solvent under reduced pressure and subsequent recrystallisation from

EtOH gave colourless crystals of 5-[2-(4-methoxycarbonylphenyl)ethenyl]-2-

methoxy-1,1,3,3-tetramethyl-isoindoline 26 (32.2 mg, 0.088 mmol, 77 %); δH: 1.45

(12H, br s, CH3), 3.80 (3H, s, NOCH3), 3.92 (3H, s, ester OCH3), 7.10 (1H, d, J 16.2

Hz, =CH), 7.11 (1H, d, J 8.1 Hz, 7-H), 7.23 (1H, d, J 16.2 Hz, =CH), 7.27 (1H, d, J

1.3 Hz, 4-H), 7.40 (1H, dd, J 1.3 Hz and 8.1 Hz, 6-H), 7.56 (2H, d, J 8.3 Hz, ArH),

8.02 (2H, d, J 8.3 Hz, ArH); δC: 25.2 and 29.1 (CH3), 52.5 (ester OCH3), 65.9

(NOCH3), 67.8 (C-1 and C-3), 120.0 (=CH), 122.3 (=CH), 126.7 (C-6), 126.8 (C-4),

127.5 (C-7), 129.2 (ArC), 130.4 (ArC), 131.7 (ArC), 136.6 (C-5), 142.3 (ArC), 146.2

(C-7a and C-3a), 167.3 (C=O); νmax (ATR-FTIR): 2978 (alkyl CH3), 1714 (C=O),

1603 (C=C), 1493 and 1435 (aryl C–C), 1178 (OCH3) cm−1; +EIMS found M+


37

365.19948 (1.05 ppm from calc. mass of C23H27NO3); m/z 365 (M+, 5 %), 350 (100),

319 (26), 304 (23).

2.5.13. 5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-

tetramethylisoindoline (27)

N O

MeO2C

MeO2C

27

A solution of 5,6-bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl 23 (20 mg, 0.039 mmol) and FeSO4·7H2O (22 mg, 0.079 mmol,

2 equiv) in DMSO (1.2 mL) had H2O2 (30%, 9 µL) added dropwise. The mixture

was stirred at room temperature for 30 minutes. The resultant solution was poured

onto NaOH (1 M, 20 mL) and extracted with Et2O (3 × 30 mL) and dried (Na2SO4)

and the solvent removed under reduced pressure. Purification by column

chromatography (30 % EtOAc, 70 % n-hexane) gave 5,6-bis-[2-(4-

methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-tetramethylisoindoline 27 (16.6

mg, 0.032 mmol, 82 %); δH: 1.51 (12H, br s, CH3), 3.82 (3H, s, NOCH3), 3.95 (6H,

s, ester OCH3), 7.04 (2H, d, J 16.1 Hz, =CH), 7.33 (2H, s, J 8.2 Hz, 4-H and 7-H),

7.57 (2H, d, J 16.1 Hz, =CH), 7.60 (4H, d, J 8.2 Hz, ArCH), 8.06 (4H, d, J 8.2 Hz,

ArCH); δC: 29.1 (CH3), 52.2 (ester OCH3), 65.6 (NOCH3), 67.1 (C-1 and C-3), 119.9

(=CH), 126.5 (=CH), 129.1 (C-4 and C-7), 129.2 (ArC), 130.1 (ArC), 130.2 (ArC),

135.3 (C-5 and C-6), 141.8 (ArC), 145.9 (C-3a and C-7a), 166.9 (C=O).

2.5.14. 5-[2-(4-Carboxyphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl (28)

N O

HO2C

28


38

5-[2-(4-Methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl 18

(39 mg, 0.111 mmol) was dissolved in THF (10 mL). NaOH (1.25 M, 20 mL) was

added and the mixture refluxed for 16 hours. The resultant solution was washed with

CHCl3 (2 × 20 mL), acidified with HCl (2 M, pH ~0-1) and extracted with CHCl3 (3

× 20 mL). Subsequently, the organic phase was washed with brine (20 mL), dried

(Na2SO4) and the solvent removed under reduced pressure. Recrystallisation from

MeOH gave 5-[2-(4-carboxyphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

28 as a light orange solid (35 mg, 0.104 mmol, 94 %); νmax (ATR-FTIR): 3205 (OH),

2978 (alkyl CH), 2678 and 2553 (OH) 1683 (C=O), 1602 (C=C), 1492 and 1463

(aryl C–C), 1423 (NO) cm−1; +EI MS found M+ 336.15989 (0.24 ppm from calc.

mass of C21H22NO3•): m/z 336 (M+, 39 %), 321 (44), 306 (100), 291 (45).

2.5.15. 5,6-Bis-[2-(4-carboxyphenyl)ethenyl]-1,1,3,3-tetramethyl-

isoindolin-2-yloxyl (29)

N O

HO2C

HO2C

29

5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl 23 (29 mg, 0.057 mmol) was dissolved in THF (10 mL). NaOH (1.25 M, 20

mL) was added and the mixture refluxed for 16 hours. The resultant solution was

washed with CHCl3 (2 × 20 mL), acidified with HCl (2 M, pH ~0-1) and extracted

with CHCl3 (3 × 20 mL). Subsequently, the organic phase was washed with brine (20

mL), dried (Na2SO4) and the solvent removed under reduced pressure.

Recrystallisation from MeOH gave 5,6-bis-[2-(4-carboxyphenyl)ethenyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl 29 (23 mg, 0.048 mmol, 84 %); +EI MS found M+

482.19792 (2.44 ppm from calc. mass of C30H28NO5•): m/z 482 (M+, 20 %), 463 (70),

452 (100), 316 (50).

2.5.16. Fluorescence Quantum Yield Calculations

Fluorescence quantum yield measurements were calculated using cyclohexane as the

solvent and anthracene 30 (ФF = 0.36) as the standard. Stock solutions of Heck


39

products 18 and 23 and their diamagnetic analogues 26 and 27 (approximately 1

mg/100 mL, measured accurately, exact concentrations listed below) were diluted

using analytical glassware to give four solutions of decreasing concentration,

ensuring that the maximum intensity of the UV/Vis did not exceed 0.1 absorbance

units at the fluorescence excitation wavelengths (303 nm or 330 nm). The detector

voltage used for the samples was 600 V for the mono-substituted compounds and

550 V for the di-substituted compounds. Total fluorescence emission was plotted

against UV/Vis absorbance at the excitation wavelength to give a straight line with

gradient (m), which was ratioed against the anthracene 30 standard, giving the

quantum yield (ФF).

y = 349226x + 944.69

R2 = 1

y = 1658.9x + 427.06

R2 = 0.9689

y = 984298x + 200

R2 = 0.9995

0

5000

10000

15000

20000

25000

30000

35000

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

UV-Vis Absorbance

Flu

ores

cenc

e In

tens

ity/a

u

26 18 30 Trendline 26 Trendline 18 Trendline 30

30 26

18

Figure 2.7: Quantum yield measurements of 18 and 26 at 330 nm in cyclohexane


40

y = 380933x + 3219.9

R2 = 1

y = 496.01x + 363.25

R2 = 0.9767

y = 193671x + 82.627

R2 = 0.9948

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14

UV-Vis Absorbance

Flu

ores

cenc

e In

tens

ity/a

u


23

30

27


Anthracene (30)

Stock solution 30 (1.07 mg, 0.00600 mmol, 0.0600 mM). Diluted to give solutions of

9.600, 7.200, 4.800 and 2.400 µM; m330 nm = 984298, m303 nm = 193671.

5-[2-(4-Methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(18)


2.717, 2.038, 1.359 and 0.679 µM; m = 1659; ФF = 0.36(1659/984298) = 0.0006.

5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl (23)


2.728, 2.046, 1.364 and 0.682 µM; m = 496; ФF = 0.36(496/193671) = 0.0009.

5-[2-(4-Methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-



2.824, 2.118, 1.412 and 0.706 µM; m = 349226; ФF = 0.36(349226/984298) = 0.128.


41

5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-2-methoxy-1,1,3,3-



1.584, 1.188, 0.792 and 0.396 µM; m = 380933; ФF = 0.36(380933/193671) = 0.708

Chapter 3 – Palladium-Catalysed Sonogashira Coupling of Isoindoline Nitroxides

42

3. PALLADIUM-CATALYSED SONOGASHIRA COUPLING OF

ISOINDOLINE NITROXIDES

3.1. Introduction

The acetylene (or alkyne) functional group can undergo a broad range of synthetic

reactions, including electrophilic and nucleophilc additions. Acetylenes are able to

undergo stepwise or concerted reaction pathways as well as reactions involving by

heat, light or catalysts.110 In particular, aryl-acetylenes are of significance as they are

common precursors in the synthesis of important natural products, pharmaceuticals

and organic molecular materials.111

Whilst aryl-acetylenes are accessible via several Pd-catalysed cross coupling

methodologies such as Stille (Sn), Suzuki (B) and Negishi (Zn) couplings,111 the

Sonogashira reaction is the most attractive because synthesis of alkynyl-

organometallics is not required prior to the desired coupling reaction.

3.2. The Sonogashira Alkynylation Reaction

Palladium-catalysed alkynylation, commonly referred to as the Sonogashira reaction,

is a convenient and frequently utilised method for the synthesis of aryl- and other

substituted-acetylenes.112 The methodology was pioneered in 1975 by Cassar,113

Dieck and Heck114 and Sonogashira et al.115 The work of Cassar and Dieck and Heck

actually form the first reports of what is now commonly known as the copper-free

Sonogashira reaction, although there have been many developments since these

initial reports.

Cassar showed that aryl- and vinyl-acetylenes could be synthesised in high yield

using catalytic Pd(PPh3)4 in DMF in the presence of a strong base, sodium

methoxide, at reaction temperatures between 40-100 °C (see Scheme 3.1).113 The

NaOMe forms an acetylide anion by deprotonation of the terminal acetylene, which

in turn co-ordinates to the Pd catalyst and undergoes the cross-coupling reaction.


43

XR

R

40-100 °C 3-8 h, N2

+

X= Br, IR = alkyl or aryl

34-97 %

Pd(PPh3)4CH3ONa

DMF

Scheme 3.1: Synthesis of acetylene derivatives by Cassar113

Dieck and Heck114 utilised similar methodology, adapted from the tradional Heck

reaction (Pd-catalysed alkenylation),97 to form aryl-, heterocylic- and vinylic-

acetylenes. Catalytic Pd(OAc)2[PPh3]2 in an amine solvent (Et3N or piperidine) gives

the disubstituted acetylenes in high yield (see Scheme 3.2). In this example the amine

deprotonates the terminal acetylene enabling the coupling reaction to take place. The

fact that piperidine, a secondary amine, enhances the reactivity of the acetylenes

compared with the Et3N reactions has been given as evidence for the formation of a

transient acetylide prior to the coupling taking place.

XR

R

100 °C 0.5-2.5 h, N2

+


53-88 %

Pd(OAc)2PPh3Et3N

Scheme 3.2: Synthesis of acetylene derivatives by Dieck and Heck114

Later the same year, Sonogashira et al.115 described the initial study of what was to

become the traditional Pd-catalysed alkynylation reaction, now commonly referred to

as the Sonogashira reaction. The methodology used was an amalgamation of the

Castro-Stephens reaction,116, 117 a stoichiometric Cu-promoted alkynylation reaction,

and the alkyne adaptation of the Heck reaction.114 Sonogashira et al. used a Cu(I) co-

catalyst (CuI) in the presence of a Pd(0) catalyst (see Scheme 3.3), which allowed the

transformations to be performed at room temperature; much milder conditions than

those used by Cassar or Dieck and Heck.


44

XR

R

rt, 3-6 h, N2

+


80-90 %

Pd(PPh3)2Cl2CuI

Et2NH

XR

R'

R

rt, 3-6 h, N2

+


R'=alkyl, aryl or H

40-99 %

Pd(PPh3)2Cl2CuI

Et2NH

R'

R' R'

Scheme 3.3: Synthesis of acetylene derivatives by Sonogashira et al.115

The reaction mechanism of the Sonogashira reaction is believed to involve firstly the

formation of a Cu-acetylide in situ from the Cu(I) catalyst (see Scheme 3.4 below).

This subsequently undergoes transmetallation, to give a Pd(II) species (cycle B’ in

Scheme 3.4 below), which then reductively eliminates a trace amount of butadiyne

(1:1 stoichiometry with initial Pd(II) source) to give the active Pd(0) catalyst (cycle

A in Scheme 3.4 below). Oxidative addition of RX, commonly an aryl halide, gives a

transient Pd(II) complex. Transmetallation of the copper acetylide and subsequent

reductive elimination give the cross-coupled product and regenerate the Pd(0)

catalyst. Alkylamines, such as triethylamine, are commonly used as the reaction

solvent. In these circumstances they also act as the base, which removes the

hydrogen halide (HX) eliminated.112

Pd

X

XL

L

Pd

L

L

CuC CR'

CuX

C CR'

C CR'

HX-Amine

R'C CH

[Pd0]

[PdII ]X

R

[PdII ]R

C CR'

RC CR'

RX

R'C CCu

CuX

HX-Amine

R'C CH

R'C C CR'C

Oxidative Addition

Transmetallation


Cycle A

Cycle B' Cycle B


Transmetallation

Scheme 3.4: The reaction mechanism of the Pd-catalysed Sonogashira coupling111


45

The Cu(I) salts commonly used in Sonogashira couplings are also known to catalyse

the homocoupling of terminal alkynes in the presence of Pd catalysts.118-121 These are

often observed as unwanted side reactions when coupling less activated aryl

halides.122 Recently, copper-free Sonogashira coupling methodologies have been

gathering greater interest,118, 122-127 in an attempt to eliminate this problem. With the

removal of the Cu (I) co-catalyst, the amine is of increased importance. It performs

multiple roles in these reactions, such as accelerating oxidative insertion as well as

acting as a ligand and a base.128 For these reasons the amine is of critical importance

in the copper-free Sonogashira reaction. Cyclic amines, such as pyrrolidine and

piperidine, perform substantially better than simple alkylamines,129 due to their

enhanced basicity and increased ability to act as a ligand of aryl-Pd(II) complexes.128

[PdII]R

X[PdII]

C CR'

CRR'C RX

HX-Amine

Oxidative Addition


Precursor

L

L

[Pd0]L

L

L

HC CR'

[PdII]

L

R X

R

Deprotonation Complexation

Amine L

HC CR'

L

Scheme 3.5: The proposed reaction mechanism of the Cu-free Sonogashira

reaction128

The mechanism proposed by Tougerti et al.128 (see Scheme 3.5 above) for the

copper-free Sonogashira reaction begins similarly to the traditional reaction, with the

formation of a Pd(0) catalyst from a precursor, which then undergoes oxidative

addition of RX, resulting in a Pd(II) species. Next, the terminal acetylene reversibly

π-bonds to the Pd(II) catalyst to give a second Pd(II) complex. This can then be

deprotonated by the amine (base) to give a third Pd(II) complex in which the


46

acetylene is σ-bonded to the Pd catalyst. Subsequent reductive elimination gives the

cross-coupled product and regenerates the Pd(0) catalyst.

3.2.1. Sonogashira Reactions Performed on Nitroxides

To date, few examples of Sonogashira coupling in the presence of nitroxide radicals

have been documented. The first of these was performed by Miura and Ushitani in

1993,130 where the coupling was used to synthesise a t-butylphenyl nitroxide polymer

39 in high yield (87-94 %). A bis-ethynyl nitroxide 37 was coupled to a diiodo-

aromatic compound 38 as shown in Scheme 3.6 below.

N

PdCl2(PPh3)2 CuI

Et3N/pyridine20 °CO I I

+

NO

n37 38

39

Scheme 3.6: t-Butylphenyl nitroxide Sonogashira coupling by Miura and

Ushitani130

Following on from the initial investigation, the Miura group published another article

in 1996.131 In this case, the Sonogashira couplings were performed on both nitronyl

and t-butylphenyl nitroxides, to give nitroxide polymers incorporating both types of

nitroxide moieties (see Scheme 3.7 below) in high yield (78-83 %). It was proposed

that the polyradicals 41 may have interesting magnetic properties brought about by

through-bond ferromagnetism. Due to stronger through-space antiferromagnetism

this prediction was found to be incorrect.131


47

N

PdCl2(PPh3)2 CuI

Et3N/pyridine20 °C

O

+

NO

n

NN OO

II

NN OO

37 40

41

Scheme 3.7: t-Butylphenyl and nitronyl nitroxide Sonogashira coupling by Miura

et al.131

Romero and Ziessel reported another example of Sonogashira coupling of nitroxides

in 1996,132 where an ethynyl substituted nitronyl nitroxide 43 was synthesised via a

Sonogashira reaction between 42 and (trimethylsilyl)acetylene (see Scheme 3.8

below) to give the (trimethylsilyl)ethynyl nitronyl nitroxide in good yield (63 %).

N

Br

N

N

O

O

Pd(PPh3)4 C6H6/i-Pr2NH

80 °C

N

N

N

O

O

Si

43 63 %

Si

42

Scheme 3.8: Sonogashira coupling by Romero and Ziessel132

Further work from the same group reported the coupling of an ethynyl nitroxide 44

(formed by KF deprotection of 43) with a bromo analogue 42 giving an ethynyl-

bridged nitronyl nitroxide diradical 45 (see Scheme 3.9 below).133


48

N

Br

N

N

O

O

N

N

N

O

O

+

N

NN

O

O

N

NN

O

OPd(PPh3)4

C6H6/i-Pr2NH80 °C

42 44 45 84 %

Scheme 3.9: Nitronyl nitroxide Sonogashira coupling by Romero and Ziessel133

Another example of Sonogashira coupling performed on nitroxide radicals was

published by Stroh et al., wherein the authors described the synthesis a novel nitronyl

nitroxide 46 (see Scheme 3.10 below).134 This work was similar to that previously

reported by Romero and Ziessel except that an m-iodophenyl nitronyl nitroxide 47

was used as the aryl halide rather than a 6-bromopyridinyl nitronyl nitroxide.

I

N

N

O

O

NN

O

OPdCl2(PPh3)2, CuI

TEA, PyRT, 1.5 h

46 59 %

NC

CN

47 Scheme 3.10: Nitronyl nitroxide Sonogashira couplings by Stroh et al.134

As part of a larger study of Pd-catalysis on pyrroline nitroxides, including Heck,

Suzuki and Sonogashira reactions, Hideg and co-workers used Sonogashira

methodology to synthesise novel acetylene-substituted pyrroline nitroxides.101

Couplings were successful when the nitroxide moiety was present on both the vinyl

halide or alkyne coupling partners (see examples in Scheme 3.11 below).


49

N

O N

O

PdCl2(PPh3)2, CuIEt3N, DMF

80 °C 3h

RHN

51 R = H 52 R = SO2Me

36 %50 %

I

NHR

50

N

O

Br O

N

O

O

Ph

PdCl2(PPh3)2, CuIEt3N, DMF80 °C, 3h

49 72 %

Ph

48

Scheme 3.11: Pyrroline nitroxide Sonogashira couplings by Hideg and co-

workers101


Although Sonogashira couplings on the nitroxides outlined above are well

documented, these couplings have been performed on somewhat activated starting

materials. To date very little has been reported on Sonogashira reactions employing

deactivated aromatic halogenated nitroxides, such as the isoindolines, as coupling

partners. This investigation formed the main objective outlined in this chapter. The

synthesis of 5-iodo-1,1,3,3-tetramethylisoindoline 65 and 5-iodo-1,1,3,3-

tetramethylisoindolin-2-yloxyl 64 have been published in the form of a

communication in Macromolecules,55 with the majority of the remaining results

reported in this chapter to be published in the form of a full paper in a forthcoming

article in Org. Biomol. Chem.135

3.3.1. Sonogashira Coupling of Bromo and Bromo-Nitro Nitroxides

After success with Pd-catalysed Heck coupling on brominated isoindoline nitroxides,

initial Sonogashira couplings were attempted on the bromo nitroxide 16, using the

commonly used catalyst system of Pd(PPh3)2Cl2/CuI and triethylamine as the

solvent/base.136 Using these conditions, none of the desired products were obtained


50

when attempting to couple (trimethylsilyl)acetylene 53 or phenylacetylene 54 to the

bromo nitroxide 16 either at room temperature or in refluxing triethylamine (see

Entries 1-4, Table 3.1). The homocoupled alkynes (butadiynes) and bromo nitroxide

starting material 16 were the only compounds isolated from the reaction mixtures.

This can be rationalised by the presence of CuI which is known to disfavour the

Sonogashira cross coupling of less active aryl halides,118 promoting the

homocoupling of the acetylene compounds.

N O

Br

N O

R

55 R = Si

56 R =

Si

54

53

16

Pd(PPh3)2Cl2 (2 mol%)CuI

Et3N, Ar

Table 3.1: Traditional Sonogashira couplings of bromo nitroxide and alkynes

Entry Alkyne CuI (mol%) Conditions Product Yield

(%)

1 53

(1.2 equiv) 0.5 RT, 72 h 55 0

2 54

(2 equiv) 10 RT, 96 h 56 0

3 53

(1.2 equiv) 0.5 90 °C, 72 h 55 0

4 54

(2 equiv) 10 90 °C, 96 h 56 0

In an attempt to reduce the electron density of the nitroxide aryl halide, synthesis of a

bromo-nitro nitroxide 57 was proposed. This was achieved by the adaptation of the

synthesis of 5-nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl 58 originally reported by

Bolton et al.70


51

Direct nitration of bromo-TMIO 16 with H2SO4/HNO3 in glacial acetic acid at 40 °C

for 4 h gave 5-bromo-6-nitro-1,1,3,3-isoindolin-2-yloxyl 57, in excellent yield of 95

% (see Scheme 3.12 below). Recrystallisation from acetonitrile gave orange needles

of 57 of sufficient quality for X-ray crystallographic analysis (data collected by P.

Jensen, University of Sydney and refined by J. C. McMurtrie, Queensland University

of Technology) and whilst there was some disorder in the crystal, whereby the

position of the nitro group and the bromine atom interchanged throughout the lattice,

the data were able to confirm the structure of 57.

N O

Br

O2N

N O

Br

AcOH40 °C, 4 h

HNO3H2SO4

57 95 %16 Scheme 3.12: Synthesis of bromo-nitro nitroxide 57

Figure 3.1: X-ray crystal structure of bromo-nitro nitroxide 57 (H atoms omitted

for clarity)

When the bromo-nitro nitroxide 57 was subjected to the same coupling conditions,

again only the homocoupled acetylenes and starting material were isolated (see

Entries 1 and 2, Table 3.2 below), which suggests the reactivity of the bromo-nitro

nitroxide is not greater than that of the bromo nitroxide.


52

N O

O2N

Br

N O

O2N

Si

5957 Table 3.2: Traditional Sonogashira couplings of nitro bromo nitroxide

Entry Nitroxide Alkyne Catalyst system/Solvent Conditions Product Yield

(%)

1 57 53

(1.2 equiv)

Pd(PPh3)2Cl2 (2 mol%)

CuI (0.5 mol%)/ Et3N RT, Ar, 72 h 59 0

2 57 53

(1.2 equiv)

Pd(PPh3)2Cl2 (2 mol%)

CuI (0.5 mol%)/ Et3N 90 °C, Ar, 72 h 59 0

3.3.2. Copper-Free Sonogashira Coupling of Bromo and Bromo-nitro

Nitroxides

Recently Li et al. published an article which described the use of Pd(OAc)2/DABCO

as an efficient catalyst for copper-free cross coupling of alkynes in the presence of

Cs2CO3 as a base.125 Replacing the inorganic base with additional equivalents of

DABCO, which acts as a ligand and a base, enhanced the efficiency of the cross-

coupling reaction.118 Homocoupling of acetylenes using the same catalytic system,

with the addition of CuI, gave butadiynes in high yield. The inclusion of 2 mol% of

CuI reduced the yield of 60 to 8 %, with approximately 90 % of the homocoupled

product being isolated (Entry 1, Table 3.3 below), compared to 98 % of the desired

cross-coupled product 60 when the reaction is performed without CuI (Entry 2, Table

3.3). This example illustrates the important role CuI plays in these reactions.


53

I

OMe

OMe

54

61 60 Table 3.3: Copper-free Sonogashira couplings by Li et al.118

Entry Catalyst system/Solvent Conditions Yield (%)

1 Pd(OAc)2 (2 mol%)/CuI (2 mol%)

DABCO (3 equiv)/ MeCN RT, air, 24 h 8a

2 Pd(OAc)2 (2 mol%)

DABCO (3 equiv)/MeCN RT, air, 24 h 98

aca. 90 % of diphenylbutadiyne isolated

Due to the inability to couple bromo nitroxides using traditional Sonogashira

techniques, the methodology of Li et al.118 was investigated. The coupling reactions

between (trimethylsilyl)acetylene 53 and the bromo nitroxide 16 or the bromo-nitro

nitroxide 57, using the copper-free conditions of 2.5 mol% Pd(OAc)2, 3 equiv

DABCO in MeCN under air at 50 °C gave trace amounts of both the

(trimethylsilyl)ethynyl nitroxide 55 and the nitro-(trimethylsilyl)ethynyl nitroxide 59,

which were identified by GCMS of the reaction mixture (see Entries 1 and 2, Table

3.4 below), with unreacted starting materials the major component isolated.

Performing the reactions at 80 °C in air did not increase the yields (Entries 3 and 4,

Table 3.4). When the reactions were performed under an argon atmosphere at 80 °C

the yields increased to 5 % and 9 % for 55 and 59 respectively (see Entries 5 and 6,

Table 3.4), again with large amounts of starting material re-isolated from the reaction

mixture. Trace amounts of the desired products were isolated from the coupling

reactions with the alkynes 2-methyl-3-butyn-2-ol 62 and phenylacetylene 54 under

the same conditions (Entries 7 and 8, Table 3.4).

Since the desired products were isolated in low yields from the copper-free reactions,

optimisation of the reaction conditions was undertaken. The reaction mixtures were

analysed via HPLC similar to that described for the Heck optimisation in Chapter 2.

All subsequent optimisation reactions for the Sonogashira cross-couplings were


54

performed on the synthesis of (trimethylsilyl)ethynyl nitroxide 53 from bromo

nitroxide 16.

Increasing catalyst loadings and using Heck type conditions (Entries 9 and 10

respectively) did not improve the yield of the reaction. Additional equivalents of

(trimethylsilyl)acetylene 53 only slightly improved the reaction (Entry 11) whilst

increased reaction temperature, in MeCN or DMF, did not increase the yield of the

desired product (Entries 12-17, Table 3.4), with the DMF reactions failing to produce

any of the product at all. It was concluded, from the results of these attempted

optimisation reactions, that bromo nitroxide 16 and bromo-nitro nitroxide 57 were

not reactive enough to give Sonogashira cross coupled products in acceptable yields

and a new approach was required.

N O

R1

Br

N O

R1

R2

55R1= H, R2 =16R1 = H57R1 = NO2

Si

HO

59R1= NO2, R2 =

63R1= H, R2 =

56R1= H, R2 =

Si

HO

53

62

54

Pd(OAc)2 (2.5 mol%)DABCO (3 equiv)

MeCN72 h

sealed tube

Table 3.4: Copper-free Sonogashira couplings of bromo nitroxides and alkynes

Entry Nitroxide Alkyne Conditions Product Yield (%)

1 16 53

(5 equiv)

50 °C,

air 55 <5

2 57 53

(5 equiv)

50 °C,

air 59 <5

3 16 53

(5 equiv)

80 °C,

air 55 <5


55

4 57 53

(5 equiv)

80 °C,

air 59 <5

5 16 53

(5 equiv)

80 °C,

Ar 59 5

6 57 53

(5 equiv)

80 °C,

Ar 59 9

7 16 62

(5 equiv)

80 °C,

Ar 63 <5

8 16 54

(2 equiv)

80 °C,

Ar 56 <5

9 16 53

(5 equiv)

80 °C,

Ar 55 <5a

10 16 53

(5 equiv)

80 °C,

Ar 55 0a

11 16 53

(20 equiv)

80 °C,

Ar 55 8a

12 16 53

(20 equiv)

100 °C,

Ar 55 <5a

13b 16 53

(20 equiv)

100 °C,

Ar 55 0a

14 16 53

(50 equiv)

100 °C,

Ar 55 <5a

15b 16 53

(50 equiv)

100 °C,

Ar 55 0a

16b 16 53

(20 equiv)

130 °C,

Ar 55 0a

17b 16 53

(50 equiv)

130 °C,

Ar 55 0a

aYield determined by HPLC, product not isolated; bDMF solvent

3.3.3. Sonogashira Coupling of the Iodo Nitroxide (59)

Due to the low reactivity of the brominated isoindoline nitroxides towards

Palladium-catalysed reactions, a limitation originally encountered whilst

investigating Heck coupling,56 the use of an iodo-substituted nitroxide 64 was


56

proposed to improve the viability of the Sonogashira cross-coupling reaction.

Oxidative addition of aryl iodides commonly occurs more efficiently than those of

brominated analogues in Pd-catalysed couplings and in turn leads to increased

yields.137 Acceleration of the reductive elimination step may also play a part due to

the improved stability of iodide (I-) as a leaving group, compared with bromide (Br-).

5-Iodo-1,1,3,3-tetramethylisoindoline 65 was synthesised from 5-bromo-1,1,3,3-

tetramethylisoindoline82 35 by a lithiation procedure similar to that previously

published for the synthesis of 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

(CTMIO) 66.75 Treatment of bromoamine 35 with BuLi (2.6 equiv) followed by

quenching with iodine in dry THF gave the intermediate 2,5-diiodo-1,1,3,3-

tetramethylisoindoline 67 (not isolated), which was subsequently treated with

H2O2/NaHCO3 to remove any N-iodoamine that may have been formed, gave the

desired 5-iodo-1,1,3,3-tetramethylisoindoline 65 in overall yield of 68 % (see

Scheme 3.13 below). This reduction of the N-iodo amine to the secondary amine was

adapted from the previously published synthesis of 5-bromo-1,1,3,3-

tetramethylisoindoline 36 from 2,5-dibromo-1,1,3,3-tetramethylisoindoline 68.82

N I

I

N H

Br 1. BuLi2. I2

THF-78 °C

N H

IH2O2/NaHCO3

MeOH

35 67 65 68 %

Scheme 3.13: Synthesis of iodo amine 65

Subsequent oxidation of the iodo amine 65 with H2O2 in the presence of a

Na2WO4·2H2O catalyst in MeOH/MeCN gave the iodo nitroxide 64 in a yield of 52

% (76 % based on converted starting material) (see Scheme 3.14 below). The

oxidation reaction proceeds slowly presumably due to the limited solubility of the

iodo amine in MeOH. The addition of more solvent to ensure total dissolution of the

starting material increased the reaction rate. Recrystallisation of 64 by slow

evaporation of an Et2O solution gave crystals of 64 of sufficient quality for X-ray

crystallographic analysis (data collected by P. Jensen, University of Sydney and


57

refined by J. C. McMurtrie, Queensland University of Technology) which confirmed

the structure of 64.

N O

I

N H

I

MeOH, MeCNRT, 72 h

H2O2 Na2WO4.2H2O

NaHCO3

64 52 %65 Scheme 3.14: Synthesis of iodo nitroxide 64

Figure 3.2: X-ray crystal structure of iodo nitroxide 64 (H atoms omitted for

clarity)

The coupling of (trimethylsilyl)acetylene 53 with iodo nitroxide 64 under identical

reaction conditions to that of Entry 5 in Table 3.4 above (2.5 mol% Pd(OAc)2, 3

equiv DABCO, MeCN, argon, 80 °C, 24 h), gave the (trimethylsilyl)ethynyl

nitroxide 55 in an excellent yield (92 %, cf. 5 % for BrTMIO) (see Entry 1, Table

3.5), although some difficulty was encountered separating 55 from the iodo starting

material 64 chromatographically, requiring separation by semi-prep HPLC to isolate

pure product 55. This reaction showed a substantial increase in yield by simply

substituting ITMIO 64 for BrTMIO 16 as the aryl halide coupling partner and

illustrates the heightened reactivity of aryl iodides to Pd-catalysis. Since the yield of

55 was deemed sufficient no further optimisation of the reaction was undertaken.

N OO

OH

69

Similarly, reaction of the iodo nitroxide 64 with 2-methyl-3-butyn-2-ol 62 gave the

substituted ethynyl nitroxide 63 in high yield (78 %) (see Entry 2, Table 3.5). The


58

reduced yield is attributed to a competing cyclisation reaction138 which gives a

substituted 3-benzylidene-2,3-dihydrofuran 69 as an undesired side product,

proposed based on GCMS of the reaction mixture and literature precedent,138 in

approximately 10 % yield based on iodo nitroxide starting material.

N O

I

N O

R

55 R = Si

HO63 R =

56 R =

64

Pd(OAc)2 (2.5 mol%)DABCO (3 equiv)

MeCN80 °C, Ar, 24 h

Table 3.5: Sonogashira coupling of iodo nitroxide 64

Entry Alkyne Product Yield (%)

1 53

(5 equiv) 55 92

2 62

(5 equiv) 63 78

3 54

(5 equiv) 56 96

Reaction of phenylacetylene 54 with iodo TMIO 64 under the same coupling

conditions gave the phenylethynyl nitroxide 56 in high yield (96 %) (Entry 3, Table

3.5). The success of these reactions shows the copper-free Sonogashira coupling

methodology is useful for the synthesis of nitroxide functionalised aryl-acetylenes

when the nitroxide moiety is present on the aryl halide fragment.

3.3.4. Synthesis of Ethynyl Nitroxide

After the success of the Sonogashira reaction using iodo TMIO 64, the coupling of

an ethynyl nitroxide as the alkyne coupling partner, was of interest. The synthesis of


59

a nitroxide with terminal alkynyl functionality would facilitate the spin labelling of

fluorescent moieties utilising robust alkyne linkages to give profluorescent

nitroxides. Spin labelling of biological molecules such as pharmaceuticals or proteins

may also be achieved via this synthetic route.

N ON OMeOHRT, 1 h

70 90 %

Si

KOH

55 Scheme 3.15: Synthesis of ethynyl nitroxide 70 from 55

Treatment of the (trimethylsilyl)ethynyl nitroxide 55 with aqueous KOH in MeOH

resulted in the synthesis of 5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 in

high yield (90 %) (see Scheme 3.15 above). Although a high yield of the product was

isolated, separation from the starting material proved troublesome. With the three

related compounds (55, 64 and 70) in this synthetic sequence all having similar

retention on silica gel, purification of the ethynyl product proved quite difficult on

preparative scale, requiring semi-prep HPLC to resolve the components. Due to these

problems an approach utilising a nitroxide with an alternative alkyne protecting

group was undertaken for the synthesis of the ethynyl nitroxide 70 in quantities large

enough to facilitate further coupling reactions.

Deprotection of the nitroxide 63 with KOH in refluxing toluene gave the ethynyl

nitroxide 70 in high yield (88 %) (Scheme 3.16 below).

N ON OToluene

reflux, 1 h

70 88 %

OH

KOH

63 Scheme 3.16: Synthesis of ethynyl nitroxide 70 from 63

The product isolated was identical to that made from the deprotection of the

(trimethylsilyl)ethynyl nitroxide 55. The presence of the polar OH group on the

acetylene starting material facilitated the separation of traces of the protected alkyne


60

63 from the product 70 and allowed 70 to be isolated in larger quantities and in

higher purity than when prepared via the (trimethylsilyl)ethynyl nitroxide 55.

Therefore this was the synthetic route utilised for the synthesis of the ethynyl

nitroxide 70 for the use in subsequent coupling reactions.

3.3.5. Profluorescent Nitroxides via Sonogashira Coupling of Ethynyl

Nitroxide

Profluorescent nitroxides are accessible from the Sonogashira methodology by

coupling the ethynyl nitroxide 70 to aryl halides bearing fluorescent groups such as

polyaromatics. For this study two commercially available polyaromatic iodides,

bearing naphthalene and phenanthrene functionality, were investigated. The

Sonogashira coupling of the ethynyl nitroxide 70 with 1-iodonaphthalene 71 (2

mol% Pd(OAc)2, 3 equiv DABCO, MeCN, argon, 80 °C, 4 h) gave the

(naphthyl)ethynyl nitroxide 72 in high yield (78 %) (see Scheme 3.17). Due to the

electron deficiency of 1-iodonaphthalene 71, the reaction rate was significantly

increased (complete conversion within 4 h) compared with the coupling reactions

performed on the iodo nitroxide 64 (cf. 24 h).

N O

N O

Pd(OAc)2, DABCOMeCN

80 °C, 24h

72 78 %

I

71

70 Scheme 3.17: Synthesis of (naphthyl)ethynyl nitroxide 67

Similar results were obtained using 9-iodophenanthrene 73 as the aryl halide

coupling partner. The reaction gave the (phenanthryl)ethynyl nitroxide 74 in 90 %

yield within 4 h (Scheme 3.18). Although the homocoupled side product, a

butadiyne-linked nitroxide dimer 75 (see Scheme 3.19), could be detected by TLC of

these two reaction mixtures, due to the high reactivity of both the aryl halides 71 and

73, it was formed only in trace amounts and was easily separable from the desired

products.


61

N O

N O

Pd(OAc)2, DABCOMeCN

80 °C, 24h

74 90 %

I

73

70 Scheme 3.18: Synthesis of (phenanthryl)ethynyl nitroxide 74

3.3.6. Ethyne and 1,3-Butadiyne Linked Nitroxide Dimers

To illustrate the versatility of the Sonogashira reaction with respect to nitroxides as

either the aryl halide or alkyne coupling partner, a reaction between iodo nitroxide 64

and the ethynyl nitroxide 70 was undertaken. This reaction gave the desired

acetylene-linked dinitroxide 76, albeit in a moderate yield of 36 % (see Scheme

3.19). The butadiyne-linked nitroxide dimer 75 was observed as a prominent

undesired side product (40 %), resulting from a decrease in reactivity of the aryl

halide 64 (complete conversion 24 h), due to increased electron density of the aryl

halide (compared with 1-iodonaphthalene 71 and 9-iodophenanthrene 73). The

formation of the homocoupled product 75, and the resulting decrease in the

concentration of alkyne 70, may explain the diminished yield of this reaction

compared to those using aryl halides 71 and 73.

NO

N

O

Pd(OAc)2 DABCO MeCN

80 °C, 24h 76 n = 1 36 %

N O

n

70

64

75 n = 2 40 %

N O

I

Scheme 3.19: Synthesis of acetylene dinitroxides 75 and 76

Selective synthesis of the butadiyne nitroxide dimer 75, which was obtained as a side

product from the Sonogashira reaction, was achieved via Eglinton oxidative

coupling. Refluxing the ethynyl nitroxide 70 in pyridine/methanol with Cu(OAc)2

(1.5 equiv)139 gave the butadiyne-linked nitroxide dimer 75 in excellent yield (91 %)

(see Scheme 3.20 below).


62

N O

MeOH, pyridinereflux, 1 h

Cu(OAc)2

N ONO

70 75 91 %

Scheme 3.20: Synthesis of butadiyne-linked dinitroxide 75

3.3.7. Synthesis of Methoxyamines (“Methyl Traps”)

The methoxyamine analogues 77-84 of the ethynyl substituted nitroxides 55, 56, 63,

70, 72 and 74-76 were prepared for further structural characterisation by NMR

spectroscopy. The naphthyl 76 and phenanthryl 77 methoxyamines were also used as

fluorescence controls for their corresponding nitroxides. Reaction of nitroxides 55,

56, 63, 70, 72 and 74-76 with methyl radicals (formed using Fenton chemistry by

reaction of FeSO4·7H2O with H2O2 in DMSO) gave the desired methoxyamine

adducts in moderate to high yields (31-82 %, see Experimental for further details).

3.3.8. Fluorescence Data of Ethynyl Profluorescent Nitroxides

0

0.005

0.01

0.015

0.02

270 320 370

Wavelength/nm

Abs

orba

nce

0

20

40

60

80

100

120

140

160

330 380 430 480

Wavelength/nm

Flu

ore

scen

ce In

ten

sity

/au




63

0

0.01

0.02

0.03

290 340 390

Wavelength/nm

Ab

sorb

ance

0

5

10

15

20

25

330 380 430 480

Wavelength/nmF

luo

resc

enc

e In

ten

sity

/au



Comparison between the fluorescence spectra of naphthyl nitroxide 72 and its

methoxyamine derivative 81 (Figure 3.3) and phenanthryl nitroxide 74 and its

methoxyamine analogue 82 (Figure 3.4) reveals a strong suppression of fluorescence

in both systems.

Quantum yields of 72, 74, 81 and 82 were measured in cyclohexane using anthracene

as a standard and subsequently compared. As expected, the quantum yields of the

naphthyl 81 (ФF = 0.83) and phenanthryl 82 (ФF = 0.26) methoxyamines were

significantly larger than the analogous nitroxides 72 (ФF = 4.0 × 10-3) and 74 (ФF =

4.0 × 10-3). The nitroxides 72 and 74 and the phenanthryl methoxyamine 82 had

similar quantum yields to nitroxides and methoxyamines previously reported by our

research group.54 Interestingly, the naphthyl system 81 had a much larger quantum

yield than initially anticipated, being almost an order of magnitude larger than some

other naphthalene based methoxyamines previously reported.54 Whilst the

unsubstituted parent compound 1-(phenylethynyl)naphthalene126 85 is well known,

its quantum yield was not found in a literature survey. After preparation, via copper-

free Sonogashira coupling analogous to those reported for the ethynyl nitroxides, the


64

quantum yield of 1-(phenylethynyl)naphthalene 85 (ФF = 0.80) was found to be quite

similar to the (naphthyl)ethynyl methoxyamine 81.

Table 3.6: Quantum yields of Sonogashira products


72 0.004

81 0.83

74 0.004

82 0.26

85 0.80


A series of novel acetylene-substituted nitroxides was successfully synthesised via

copper-free Sonogashira coupling. The study indicates that the Sonogashira reaction

can be used for the synthesis of a wide range of isoindoline nitroxides incorporating

many structural moieties. Isoindoline nitroxides can be used as either the aryl halide

or alkyne coupling partner and desired products can be isolated in high yield. Bromo

16 and bromo-nitro 57 substituted isoindoline nitroxides were found to be

incompatible with the reaction, requiring the use of the iodo nitroxide 64 for

successful cross-coupling. This is presumably due to the relatively high electron

density of the aromatic ring, brought about by the dialkyl substituents.

Profluorescent nitroxides with naphthalene 72 and phenanthrene 74 functionality

were synthesised and showed strong fluorescence suppression when compared to

diamagnetic analogues. The nitroxide of the naphthyl system 72 displayed extremely

efficient fluorescence quenching, with a suppression ratio of approximately 200-fold.

Two nitroxide dimers 75 and 76 were also synthesised by Sonogashira and Eglinton

couplings, which may find use in EPR experiments due to the fixed distance between

the two unpaired spins in each dimer. Spin labelling of biologically relevant

molecules, such as pharmaceuticals, by Sonogashira coupling with isoindoline

nitroxides may be pursued in the future.


65

3.5. Experimental

All experimental details were as previously described in Chapter 2 where relevant.

MeCN was dried and stored over molecular sieves (4A). (Trimethylsilyl)acetylene

(98 %), 2-methyl-3-butyn-2-ol (98 %), 1-iodonaphthalene (97 %) and 9-

iodophenanthrene (97 %) were purchased from Sigma/Aldrich.

3.5.1. 5-Bromo-6-nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl (57)

N O

O2N

Br

57

To an ice/water cooled solution of 5-bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl

16 (200 mg, 0.74 mmol) in glacial acetic acid (0.6 mL) was added conc. H2SO4 (1.15

mL), followed by conc. HNO3 (0.3 mL). The resultant solution was heated at 40 °C

for 4 h, after which the reaction was quenched by slow addition of NaOH (5 M, 10

mL) and extracted with CHCl3 (3 × 30 mL). The combined organics were washed

with water (30 mL), dried (Na2SO4) and the solvent removed under reduced pressure.

Recrystallisation from MeCN gave orange needles of 5-bromo-6-nitro-1,1,3,3-

tetramethylisoindolin-2-yloxyl 57 of sufficient quality for analysis by X-ray

crystallography (220 mg, 0.70 mmol, 95 %) mp 246−248 °C (decomp.); νmax (ATR-

FTIR): 3032 (aryl CH), 2979 and 2931 (alkyl CH), 1577 and 1530 (NO2), 1473 and

1463 (aryl C-C), 1430 (NO) cm-1; +EI MS found M+ 313.0187 (0.3 ppm from calc.

mass of C12H14BrN2O3•): m/z 313 (M+, 63 %), 298 (47), 283 (69), 268 (100), 143

(87), 128 (62).

3.5.2. 5-Iodo-1,1,3,3-tetramethylisoindoline (65)

N H

I

65

To a solution of 5-bromo-1,1,3,3-tetramethylisoindoline 35 (7.02 g, 27.6 mmol) in

dry THF (80 mL) at -78 °C (dry ice/acetone) was added BuLi (1.6 M in hexanes, 45


66

mL) dropwise. The resulting mixture was stirred for a further 15 mins, after which a

solution of I2 (21 g, 82.7 mmol, 3 equiv) in dry THF (180 mL) was added dropwise.

The reaction mixture was allowed to warm to room temperature, poured into

ice/water (500 mL) and basified (pH 14) with NaOH (5 M). The aqueous solution

was extracted with DCM (3 × 500 mL) and the combined organics were washed with

brine (500 mL), water (500 mL) and dried (Na2SO4) and the solvent removed under

reduced pressure. The residue was taken up in minimum amount of MeOH (~200

mL) and NaHCO3 (~500 mg) added. The solution was treated with H2O2 (30 %

aqueous solution, ~100 mL), followed by H2SO4 (2 M, ~500 mL, caution:

effervescence). The resulting acid solution was washed with DCM (2 × 500 mL).

The organic washings were back extracted with H2SO4 (2 M, 3 × 500 mL). The

combined acidic aqueous phase was washed with DCM (500 mL), basified (pH 14)

with NaOH (10 M) and extracted with DCM (4 × 500 mL). The DCM extract was

washed with water (500 mL), dried (Na2SO4) and the solvent was removed under

reduced pressure to give 5-iodo-1,1,3,3-tetramethylisoindoline 65 as a colourless oil,

which solidified upon standing (5.61 g, 18.6 mmol, 68 %); δH: (CDCl3) 1.43 (6H, s,

CH3), 1.44 (6H, s,CH3), 1.67 (1H, br, NH), 6.88 (1H, d, J 8.1 Hz, 7-H), 7.44 (1H, d,

J 1.6 Hz, 4-H), 7.56 (1H, dd, J 1.6 Hz and 8.1 Hz, 6-H); δC: (CDCl3) 31.7 (CH3) 31.8

(CH3), 62.6 (alkyl C*), 62.7 (alkyl C*), 92.2 (ArC*), 123.5 (ArCH), 130.8 (ArCH),

136.0 (ArCH), 138.6 (ArCH), 151.4 (ArCH); νmax (ATR-FTIR): 3333 (NH), 3033

(aryl C-H), 2957 and 2916 (alkyl CH), 1439 and 1393 (aryl C-C) cm-1; +EI MS

found M+ 301.03182 (3.10 ppm from calc. mass of C12H16IN): m/z 301(M+, ~1 %),

286 (100), 271 (30), 216 (12). The 5-iodo-1,1,3,3-tetramethylisoindoline 65

synthesised was used subsequently without further purification.

3.5.3. 5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl (64)

N O

I

64

To a solution of 5-iodo-1,1,3,3-tetramethylisoindoline 65 (5.61 g, 18.63 mmol) in

MeOH (70 mL) and MeCN (2 mL) was added Na2WO4·2H2O (810 mg, 2.45 mmol,

13 mol %), NaHCO3 (2.0 g, 23.81 mmol) and H2O2 (30 % aqueous solution, 2.0


67

mL). The resulting solution was stirred at room temperature for 72 h, after which it

was poured onto water (100 mL) and extracted with DCM (3 × 100 mL). The organic

phase was washed with H2SO4 (2 M, 2 × 100 mL), water (100 mL) and dried

(Na2SO4). The solvent was removed under reduced pressure giving 5-iodo-1,1,3,3-

tetramethylisoindolin-2-yloxyl 64 as an orange crystalline solid (3.409 g, 9.64 mmol,

52 %, 76 % based on consumed starting material) mp 132 – 135 °C; νmax (ATR-

FTIR): 3040 (aryl C-H), 2975 and 2926 (alkyl CH), 1465 and 1429 (aryl C-C), 1373

and 1357 (N-O) cm-1. +EI MS found M+ 316.01959 (0.79 ppm from calc. mass of

C12H15INO•): m/z 316 (M+, 75 %), 301 (36), 286 (100), 271 (70), 144 (68), 129 (50).

The acidic washings were basified (pH 14) with NaOH (5 M) and extracted with

DCM (3 × 100 mL). The organic phase was washed with water (2 × 100 mL), dried

(Na2SO4) and the solvent removed to give 5-iodo-1,1,3,3-tetramethylisoindoline 65

(1.79 g, 5.94 mmol) as unreacted starting material. Recrystallisation of 5-iodo-

1,1,3,3-tetramethylisoindolin-2-yloxyl 64 by slow evaporation of Et2O gave crystals

of sufficient quality for analysis by X-ray crystallography.

3.5.4. 5-[2-(Trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl (55)

N O

Si

55

5-Bromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 16 (50 mg, 0.186 mmol), DABCO

(62.5 mg, 0.557 mmol, 3 equiv) and Pd(OAc)2 (1 mg, 2.5 mol%) were dissolved in

dry MeCN (1 mL). (Trimethylsilyl)acetylene 53 (131 µL, 91 mg, 0.927 mmol, 5

equiv) was added and the mixture heated at 80 °C under argon for 24 h. The solvent

was removed under reduced pressure and the residue taken up in CHCl3 (~1 mL).

Purification of the resulting solution by column chromatography (10 % EtOAc, 90 %

n-hexane) gave 5-[2-(trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

55 as a yellow-orange solid (3 mg, 0.010 mmol, 5 %); νmax (ATR-FTIR): 2972 and

2928 (alkyl CH), 2154 (C≡C), 1487 and 1465 (aryl C-C), 1431 (NO) cm-1; +EI MS


68

found M+ 286.16265 (0.25 ppm from calc. mass of C17H24SiNO•): m/z 286 (M+, 12

%), 271 (21), 256 (100), 241 (79), 225 (32).

3.5.5. Alternate Synthesis of 5-[2-(Trimethylsilyl)ethynyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl (55)

N O

Si

55

5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 64 (59 mg, 0.186 mmol), DABCO


dry MeCN (1 mL). (Trimethylsilyl)acetylene 53 (131 µL, 91 mg, 0.927 mmol, 5




n-hexane) gave 5-[2-(trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

55 as a yellow-orange (49 mg, 0.171 mmol, 92 %) (found: C, 70.7; H, 8.5; N, 4.7;

C17H24NOSi• requires C, 71.3; H, 8.4; N, 4.9 %); νmax (ATR-FTIR): 2972 and 2928

(alkyl CH), 2154 (C≡C), 1487 and 1465 (aryl C-C), 1431 (NO) cm-1; +EI MS found

M+ 286.16265 (0.25 ppm from calc. mass of C17H24SiNO•): m/z 286 (M+, 12 %), 271

(21), 256 (100), 241 (79), 225 (32) (see HPLC 4, Appendix 1) .

3.5.6. 5-Nitro-6-[2-(trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-

2-yloxyl (59)

N O

Si

O2N

59

5-Bromo-6-nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl 57 (58 mg, 0.186 mmol),

DABCO (62.5 mg, 0.557 mmol, 3 equiv) and Pd(OAc)2 (1 mg, 2.5 mol%) were

dissolved in dry MeCN (1 mL). (Trimethylsilyl)acetylene 53 (131 µL, 91 mg, 0.927


69

mmol, 5 equiv) was added and the mixture heated at 80 °C under argon for 24 h. The

solvent was removed under reduced pressure and the residue taken up in CHCl3 (~1

mL). Purification of the resulting solution by column chromatography (10 % EtOAc,

90 % n-hexane) gave 5-nitro-[2-(trimethylsilyl)ethynyl]-1,1,3,3-

tetramethylisoindolin-2-yloxyl 59 as a yellow-orange solid (5 mg, 0.016 mmol, 9 %);

νmax (ATR-FTIR): 3045 (aryl CH), 2972 and 2927 (alkyl CH), 2150 (C≡C), 1573 and

1523 (NO2), 1480 and 1467 (aryl C-C), 1431 (NO) cm-1; +EI MS found M+

331.14808 (0.85 ppm from calc. mass of C17H24SiN2O3•): m/z 331 (M+, 100 %), 316

(43), 301 (52), 286 (22).

3.5.7. 5-(3-Hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl (63)

N O

HO

63

5-Iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl 64 (1.18 g, 3.73 mmol), DABCO (1.25

g, 11.14 mmol, 3 equiv) and Pd(OAc)2 (20 mg, 2.5 mol%) were dissolved in dry

MeCN (20 mL). 2-Methyl-3-butyn-2-ol (1.82 mL, 1.60 g, 19 mmol, 5 equiv) was

added and the mixture heated at 80 °C under argon for 24 h. The solvent was

removed under reduced pressure and the residue taken up in CHCl3 (~5 mL).


n-hexane) gave 5-(3-hydroxy-3-methyl)butynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl 63 as a brown-orange oil which solidified upon standing (797 mg, 2.93 mmol,

78 %); (found: C, 74.3; H, 8.3; N, 5.0; C17H22NO2• requires C, 75.0; H, 8.1; N, 5.1

%); νmax (ATR-FTIR): 3387 (OH), 2976 and 2930 (alkyl CH), 2165 (C≡C), 1490 and

1453 (aryl C-C), 1431 (NO) cm-1; +EI MS found M+ 272.1654 (1.3 ppm from calc.

mass of C17H22NO2•): m/z 272 (M+, 100 %), 257 (95), 242 (92), 227 (87).


70

3.5.8. 5-[2-(Phenyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (56)

N O

56



dry MeCN (1 mL). Phenylacetylene (102 µL, 95 mg, 0.930 mmol, 5 equiv) was

added and the mixture heated at 80 °C under argon for 24 h. The solvent was



n-hexane) gave 5-[2-(phenyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl 56 as a

yellow-orange solid (52 mg, 0.179 mmol, 96 %) mp 108−112 °C; (found: C, 82.0; H,

6.9; N, 4.7; C20H20NO• requires C, 82.7; H, 6.9; N, 4.8 %); νmax (ATR-FTIR): 3047

(aryl CH) 2977 and 2928 (alkyl CH), 2214 (C≡C), 1487 and 1465 (aryl C-C), 1431

(NO) cm-1; +EI MS found M+ 290.1542 (1.0 ppm from calc. mass of C20H20NO•):

m/z 290 (M+, 90 %), 275 (75), 260 (100), 245 (50), 215 (40).

3.5.9. 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (70)

N O

70

5-(3-Hydroxy-3-methyl)butynyl-1,1,3,3-tetramethlisoindolin-2-yloxyl 63 (478 mg,

1.76 mmol) was dissolved in dry toluene (320 mL). Solid KOH was added (0.8 g,

14.3 mmol, 8 equiv) and the mixture refluxed for 1 h. The dark brown suspension

was washed with water (3 × 200 mL) and brine (200 mL), dried (Na2SO4) and the

solvent removed under reduced pressure. The residue was taken up in CHCl3 (~ 5

mL) and purified by column chromatography (30 % EtOAc, 70 % n-hexane) to give

5-ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 as a pale yellow crystalline solid

(331 mg, 1.54 mmol, 88 %) mp 126−128 °C; νmax (ATR-FTIR): 3196 (≡CH), 2978


71

and 2929 (alkyl CH), 2097 (C≡C), 1487 and 1464 (aryl C-C), 1428 (NO) cm-1; +EI

MS found M+ 214.1235 (1.5 ppm from calc. mass of C14H16NO•): m/z 214 (M+, 86

%), 199 (90), 184 (100), 169 (84), 152 (53) (see HPLC 5, Appendix 1).

3.5.10. Alternate Synthesis of 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-

yloxyl (70)

N O

70

A solution of 5-[2-(trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl 55

(11.3 mg, 0.039 mmol) was dissolved in degassed MeOH (0.21 mL). Aqueous KOH

was added (5 µl, 0.5 M) and the solution was stirred at room temperature for 1 h. The

reaction mixture was treated with water (5 mL), extracted with Et2O (2 × 5 mL),

dried (Na2SO4) and the solvent was removed under reduced pressure to give the 5-

ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 (8.2 mg, 0.039 mmol, 95 %). The

product was identical to that isolated from the procedure above.

3.5.11. 5-[2-(1-Naphthyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(72)

N O

72

1-Iodonaphthalene 71 (28.3 µL, 49 mg, 0.193 mmol), DABCO (62.5 mg, 0.557

mmol, 3 equiv) and Pd(OAc)2 (1 mg, 2.5 mol%) were dissolved in dry MeCN (1

mL). 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 (50 mg, 0.233 mmol, 1.2




n-hexane) gave 5-[2-(1-naphthyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl 72


72

as an orange solid (51 mg, 0.151 mmol, 78 %); mp 145−148 °C; νmax (ATR-FTIR):

3055 (aryl CH), 2972 and 2924 (alkyl CH), 2211 (C≡C), 1488 and 1460 (aryl C-C),

1430 (NO) cm-1; +EI MS found M+ 340.1701 (0.1 ppm from calc. mass of

C24H22NO•): m/z 340 (M+, 84 %), 325 (60), 310 (100), 295 (32), 265 (35) (see HPLC

6, Appendix 1).

3.5.12. 5-[2-(9-Phenanthryl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-

yloxyl (74)

N O

74

9-Iodophenanthrene 73 (59 mg, 0.194 mmol), DABCO (62.5 mg, 0.557 mmol, 3

equiv) and Pd(OAc)2 (1 mg, 2.5 mol%) were dissolved in dry MeCN (1 mL). 5-

Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 (50 mg, 0.233 mmol, 1.2 equiv)

was added and the mixture heated at 80 °C under argon for 4 h. The solvent was



n-hexane) gave 5-[2-(9-phenanthryl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

74 as an orange solid (52 mg, 0.179 mmol, 96 %); mp 175−178 °C; νmax (ATR-

FTIR): 3070 (aryl CH), 2971 and 2924 (alkyl CH), 2214 (C≡C), 1490 and 1451 (aryl

C-C), 1430 (NO) cm-1; +EI MS found M+ 390.1857 (0.2 ppm from calc. mass of

C28H24NO•): m/z 390 (M+, 72 %), 375 (45), 360 (100), 345 (25) (see HPLC 7,

Appendix 1).

3.5.13. 1,2-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]ethyne (76)

N ONO

76


73



dry MeCN (1 mL). 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 (50 mg,

0.233 mmol, 1.2 equiv) was added and the mixture heated at 80 °C under argon for 4

h. The solvent was removed under reduced pressure and the residue taken up in

CHCl3 (~1 mL). Purification of the resulting solution by column chromatography (10

% EtOAc, 90 % n-hexane) gave the desired 1,2-bis-[5,5'-(1,1,3,3-

tetramethylisoindolin-2-yloxylyl)]ethyne 76 and the homo-coupled 1,4-bis-[5,5'-

(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]-1,3-butadiyne 75. Reversed-phase

preparative HPLC (45 % THF, 55 % H2O) gave 1,2-bis-[5,5’-(1,1,3,3-

tetramethylisoindolin-2-yloxylyl)]ethyne 76 as an yellow crystalline solid (28 mg,

0.070 mmol, 36 %); mp 214−216 °C (decomp.); νmax (ATR-FTIR): 3045 (aryl CH),

2972 and 2928 (alkyl CH), 2207 (C≡C), 1496 and 1466 (aryl C-C), 1433 (NO) cm-1;

+EI MS found M+ 402.2306 (0.3 ppm from calc. mass of C26H30N2O2••): m/z 402

(M+, 95 %), 387 (40), 372 (50), 357 (100), 342 (83) (see HPLC 8, Appendix 1).

3.5.14. 1,4-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]-1,3-butadiyne

(75)139

N ONO

75

5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl 70 (50 mg, 0.233 mmol) and

Cu(OAc)2 (65 mg, 0.358 mmol, 1.5 equiv), were dissolved in MeOH (0.5 mL) and

pyridine (0.5 mL) and the resultant mixture was refluxed for 1 h. H2SO4 (conc.) was

added to the resultant mixture until a suspension formed. This solution was

subsequently extracted with DCM (3 × 20 mL), washed with H2O (20 mL), dried

(Na2SO4) and the solvent removed under reduced pressure to give 1,4-bis-[5,5'-

(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]-1,3-butadiyne 75 as an orange crystalline

solid (45 mg, 0.105 mmol, 91 %) mp 183−185 °C (decomp.); νmax (ATR-FTIR):

3046 (aryl CH), 2975 and 2928 (alkyl CH), 2150 (C≡C), 1487 and 1463 (aryl C-C),

1430 (NO) cm-1; +EI MS found M+ 426.2307 (0.1 ppm from calc. mass of


74

C28H30N2O2••): m/z 426 (M+, 40 %), 411 (28), 396 (35), 381 (100), 366 (33) (see

HPLC 9, Appendix 1).

3.5.15. Synthesis of methoxyamines (77-84)

A general procedure for the synthesis of methoxyamines 77-84 is shown below. For

the synthesis of dimethoxyamines 83 and 84 the amounts of all reagents were

doubled.

3.5.16. General Procedure

To a solution of acetylene-substituted nitroxide (0.077 mmol) and FeSO4·7H2O

(0.154 mmol, 2 equiv) in DMSO (2.6 mL) was added H2O2 (30 %, 18 µL). The

reaction mixture was stirred under argon at room temperature for 1.5 h. NaOH (1 M)

was added and the resulting solution extracted with Et2O. The organic phase was

washed with H2O and dried (Na2SO4). Removal of the solvent under reduced

pressure gave the crude methoxyamine. Subsequent purification was achieved by

column chromatography (see below for specific conditions).

3.5.17. 5-[2-(Trimethylsilyl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (77)

N O

Si

77

Yield: 18.3 mg, mmol, 79 %; Chromatography: 10 % EtOAc, 90 % n-hexane; δH:

0.27 (9H, s, SiCH3) 1.43 (12H, br s, CH3), 3.79 (3H, s, NOCH3), 7.04 (1H, dd, J 0.5

and 7.8 Hz, 7-H), 7.22 (1H, dd, J 0.5 and 1.4 Hz, 4-H), 7.36 (1H, dd, J 1.4 and 7.8

Hz , 6-H); δC: 0.0 (SiCH3) 30.3 (CH3), 65.5 (OCH3), 67.0 (alkyl C*), 67.1 (alkyl C*),

93.3 (C≡C), 105.4 (C≡C), 121.5 (ArC), 121.8 (ArC), 125.2 (ArC), 131.2 (ArC),

145.3 (ArC), 145.9 (ArC).


75

3.5.18. 5-(3-Hydroxy-3-methyl)butynyl-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (78)

N O

HO

78

Yield: 12 mg, 0.042 mmol, 55 %; Chromatography: 30 % EtOAc, 70 % n-hexane;

δH: 1.43 (12H, br s, CH3), 1.64 (6H, s, ≡CCCH3), 2.06 (1H br s, OH), 3.79 (3H, s,

NOCH3), 7.04 (1H, dd, J 0.6 and 7.8 Hz, 7-H), 7.18 (1H, dd, J 0.6 and 1.5 Hz, 4-H),

7.30 (1H, dd, J 1.5 and 7.8 Hz, 6-H); δC: 29.7 (CH3), 31.5 (≡CCCH3), 65.5 (≡CC*)

65.7 (OCH3), 67.0 (alkyl C*), 67.1 (alkyl C*), 82.4 (C≡C), 93.1 (C≡C), 121.4 (ArC),

121.5 (ArC), 124.9 (ArC), 130.8 (ArC), 145.4 (ArC), 145.6 (ArC).

3.5.19. 5-[2-(Phenyl)ethynyl]-2-methoxy-1,1,3,3-tetramethylisoindoline

(79)

N O

79


δH: 1.46 (12H, br s, CH3), 3.81 (3H, s, NOCH3), 7.10 (1H, d, J 7.8 Hz, 7-H), 7.30

(1H, d, J 1.5 Hz, 4-H), 7.36 (3H, m, ArH), 7.43 (1H, dd, J 1.5 and 7.8 Hz , 6-H), 7.55

(2H, m, ArH); δC: 30.3 (CH3), 65.5 (OCH3), 67.1 (alkyl C*), 67.2 (alkyl C*), 88.7

(C≡C), 89.7 (C≡C), 121.6 (ArC), 122.0 (ArC), 123.4 (ArC), 124.8 (ArC), 128.2

(ArC), 128.4 (ArC), 130.8 (ArC), 131.6 (ArC), 145.5 (ArC), 145.6 (ArC).


76

3.5.20. 5-Ethynyl-2-methoxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (80)

N O

80


δH: 1.45 (12H, br s, CH3), 3.07 (1H, s, ≡CH), 3.80 (3H, s, NOCH3), 7.08 (1H, dd, J

0.5 and 7.8 Hz, 7-H), 7.27 (1H, dd, J 0.5 and 1.4 Hz, 4-H), 7.40 (1H, dd, J 1.4 and

7.8 Hz , 6-H); δC: 30.3 (CH3), 65.4 (OCH3), 66.9 (alkyl C*), 67.1 (alkyl C*), 76.5


(ArC), 146.2 (ArC).

3.5.21. 5-[2-(1-Naphthyl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (81)

N O

81


δH: 1.49 (12H, br s, CH3), 3.82 (3H, s, NOCH3), 7.15 (1H, dd, J 0.5 and 7.8 Hz, 7-

H), 7.40 (1H, dd, J 0.5 and 1.5 Hz, 4-H), 7.48 (1H, m, ArH), 7.56 (2H, m, ArH), 7.63

(1H, m, ArH), 7.78 (1H, dd, J 1.5 and 7.8 Hz, 6-H), 7.86 (1H, m, ArH), 7.89 (1H, m,

ArH), 8.46 (1H, m ArH); δC: 30.3 (CH3), 65.5 (OCH3), 67.2 (alkyl C*), 67.3 (alkyl

C*), 86.9 (C≡C), 94.6 (C≡C), 121.0 (ArC), 121.7 (ArC), 122.2 (ArC), 124.8 (ArC),

125.3 (ArC), 126.3 (ArC), 126.4 (ArC), 126.8 (ArC), 128.3 (ArC), 128.7 (ArC),

130.3 (ArC), 130.9 (ArC), 133.2 (ArC), 133.3 (ArC), 145.6 (ArC), 145.8 (ArC).


77

3.5.22. 5-[2-(9-Phenanthryl)ethynyl]-2-methoxy-1,1,3,3-tetramethyl-

isoindoline (82)

N O

82



H), 7.44 (1H, dd, J 0.6 and 1.5 Hz, 4-H), 7.59 (1H, dd, J 1.5 and 7.8 Hz, ArH), 7.63

(1H, m, ArH), 7.70 (1H, m, ArH), 7.74 (2H, m, ArH), 7.90 (1H, dd, J 1.5 and 7.8 Hz,

6-H), 8.11 (1H, s, ArH), 8.58 (1H, m, ArH), 8.70 (1H, m, ArH), 8.74 (1H, m, ArH);

δC: 30.3 (CH3), 65.5 (OCH3), 67.1 (alkyl C*), 67.2 (alkyl C*), 87.1 (C≡C), 94.3

(C≡C), 119.7 (ArC), 121.8 (ArC), 122.1 (ArC), 122.7 (ArC), 122.8 (ArC), 124.9

(ArC), 125.6 (ArC), 127.0 (ArC), 127.1 (ArC), 127.4 (ArC), 128.6 (ArC), 130.1


(ArC), 145.6 (ArC), 145.9 (ArC).

3.5.23. 1,2-Bis-[5,5’-(2-methoxy-1,1,3,3-tetramethylisoindoline)]ethyne

(83)

N ONO

83



H), 7.29 (2H, dd, J 0.6 and 1.5 Hz, 4-H), 7.42 (2H, dd, J 1.5 and 7.8 Hz , 6-H); δC:

30.3 (CH3), 65.5 (OCH3), 67.1 (alkyl C*), 67.2 (alkyl C*), 89.0 (C≡C), 121.6 (ArC),

122.1 (ArC), 124.8 (ArC), 130.8 (ArC), 145.4 (ArC), 145.5 (ArC).


78

3.5.24. 1,4-Bis-[5,5’-(2-methoxy-1,1,3,3-tetramethylisoindoline)]-1,3-

butadiyne (84)

N ONO

84



H), 7.28 (2H, dd, J 0.5 and 1.5 Hz, 4-H), 7.41 (2H, dd, J 1.5 and 7.8 Hz , 6-H); δC:

30.3 (CH3), 65.5 (OCH3), 67.0 (alkyl C*), 67.2 (alkyl C*), 73.4 (C≡C), 81.8 (C≡C),

120.6 (ArC), 121.8 (ArC), 125.8 (ArC), 131.7 (ArC), 145.6 (ArC), 146.7 (ArC).

3.5.25. 1-(Phenylethynyl)naphthalene (85)126

85

1-Iodonaphthalene 71 (57.6 µL, 100 mg, 0.393 mmol), DABCO (130 mg, 1.200

mmol, 3 equiv) and Pd(OAc)2 (2 mg, 2.5 mol%) was dissolved in dry MeCN (1 mL).

Phenylacetylene 54 (51.6 µL, 48 mg, 0.470 mmol, 1.2 equiv) was added and the

mixture heated at 80 °C under argon for 4 h. The solvent was removed under reduced

pressure and the residue taken up in CHCl3 (~1 mL). Purification of the resulting

solution by column chromatography (10 % EtOAc, 90 % n-hexane) gave 1-

(phenylethynyl)naphthalene 85 as a colourless oil (85 mg, 0.373 mmol, 95 %); δH:

7.40-7.73 (8H, m ArH), 7.80-7.92 (3H, m, ArH), 8.49-8.53 (1H, m, ArH); δC: 87.6



(ArC), 131.8 (ArC), 133.3 (ArC), 133.3 (ArC). The NMR data were in agreement

with those previously reported.126


79


Fluorescence quantum yield measurements were calculated using cyclohexane as the

solvent and anthracene (ФF = 0.36) as the standard. Stock solutions of naphthyl and

phenanthryl compounds 72, 74, 81, 82 and 85 (approximately 1 mg/100 mL,

measured accurately, exact concentrations listed below) were diluted using analytical

glassware to give four solutions of decreasing concentration, ensuring that the

UV/Vis maximum of the highest concentration did not exceed 0.1 absorbance units

at the fluorescence excitation wavelength (320 nm). The fluorescence detector

voltage was set at 480 V for the naphthalenes 72, 81 and 85 and 600 V for the

phenanthrenes 74 and 82. Total fluorescence emission was plotted against UV/Vis

absorbance to give a straight line with gradient (m), which was ratioed against the

anthracene standard, giving the quantum yield (ФF).

y = 1896.4x - 0.4857

R2 = 0.9989

y = 361630x + 532.2

R2 = 0.9966

y = 157765x + 43.96

R2 = 0.9945

y = 348354x + 335.35

R2 = 0.9986

0

5000

10000

15000

20000

25000

30000

35000

40000

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

UV-Vis Absorbance

Flu

ores

cenc

e In

tens

ity/a

u

72 81 30 85 Trendline 72 Trendline 81 Trendline 30 Trendline 85

72

81

30

85

Figure 3.5: Quantum yield measurements of 72, 81 and 85 at 320 nm in

cyclohexane at 480 V


80

y = 343353x + 1188.9

R2 = 0.9952

y = 5513.9x + 5.9747

R2 = 0.9861

y = 1338220.6x + 359.4162R2 = 0.9949

0

5000

10000

15000

20000

25000

30000

35000

40000

0 0.02 0.04 0.06 0.08 0.1

UV-Vis Absorbance

Flu

ores

cenc

e In

tens

ity/a

u


30 82

74


at 600 V

Anthracene (30)


12.000, 9.600, 7.200 and 4.800 µM; m480 V = 157765, m600 V = 1338220

5-[2-(1-Naphthyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (72)


5.024, 3.768, 2.512 and 1.256 µM; m = 1896; ФF = 0.36(1896/157765) = 0.004.

5-[2-(9-Phenanthryl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (74)


3.048, 2.540, 2.032 and 1.016 µM; m = 5514; ФF = 0.36(5514/1338220) = 0.004.

5-[2-(1-Naphthyl)ethynyl]-2-methoxy-1,1,3,3-tetramethylisoindoline (81)


4.816, 3.612, 2.408 and 1.204 µM; m = 361630; ФF = 0.36(361630/157765) = 0.825.

5-[2-(9-Phenanthryl)ethynyl]-2-methoxy-1,1,3,3-tetramethylisoindoline (82)


81


4.624, 3.468, 2.312 and 1.156 µM; m = 343353; ФF = 0.36(343353/1338220) =

0.257.

1-(Phenylethynyl)naphthalene (85)


3.784, 2.838, 1.892 and 0.946 µM; m = 348354; ФF = 0.36(348354/157765) = 0.795.

Chapter 4 - Water Soluble Nitroxides as Antioxidants

82

4. WATER SOLUBLE NITROXIDES AS ANTIOXIDANTS

4.1. Introduction

Recent research has highlighted the role of reactive oxygen species (ROS) in

biological systems,140 particularly in the areas of heart disease, neurodegenerative

diseases and cancer research.141 The antioxidant properties of nitroxide free radicals

may play a role in the protection of cellular systems against diseases involving

oxidative stress, such as those described above.

4.2. Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS) is a collective term that includes both oxygen

centred radicals, such as the superoxide radical (O2•−) and the hydroxyl radical (HO•),

and its reactive non-radical derivatives, such as hydrogen peroxide (H2O2) and

singlet oxygen (1O2).142 ROS are able to cause damage to many cellular components,

including lipids, proteins and DNA. Fortunately, the body is able to protect itself

from the oxidative damage inflicted by ROS through antioxidant defence

mechanisms. However, when ROS are produced more rapidly than they can be

removed by the antioxidant defences, the system is deemed to be under “oxidative

stress”.

The relative reactivity of ROS varies greatly, from the extremely reactive hydroxyl

radical (HO•), to relatively stable species, such as the superoxide radical (O2•−) and

hydrogen peroxide (H2O2), which are not highly reactive in aqueous solution. Both

radical and non radical ROS are shown in Table 4.1 below.

Table 4.1: Reactive oxygen species

Radicals Non-radicals

Hydroxyl HO• Hydrogen peroxide H2O2

Superoxide O2•− Hypochlorous acid HOCl

Peroxyl ROO• Ozone O3

Alkoxyl RO• Singlet oxygen 1O2

Hydroperoxyl HOO• Peroxynitrite ONOO-


83

4.2.1. Formation of ROS

There are many mechanisms by which ROS can be formed in vivo. The more

reactive species are commonly thought to be generated from less reactive species.

An example of this is the production of hydroxyl radical (HO•) from reaction of

superoxide radical (O2•−). The mechanisms of production of some key ROS are

examined in more detail below.

4.2.2. Superoxide Radical (O2•−)

The superoxide radical (O2•−) can be formed by the one electron reduction of

molecular oxygen (O2). This process is commonly due to “leakage” from electron

transport chains, especially those in endoplasmic reticulum and mitochondria.143 This

process is shown in Scheme 4.1 below.

O2 e O2+

Scheme 4.1: Formation of superoxide from one-electron reduction

Superoxide can also be formed in enzymatic reactions and by the non-catalytic

oxidation of oxyhemoglobin to form methemoglobin (Scheme 4.2).143

O2O2+Fe2+Hb Fe3+Hb +

Scheme 4.2: Oxidation of oxyhemoglobin to methemoglobin

The formation of superoxide alone does not necessarily produce cell damage.

However, the conversion of superoxide to more reactive species facilitates oxidative

stress to cellular tissues.

4.2.3. Hydrogen Peroxide (H2O2)

Superoxide (O2•−) is converted to hydrogen peroxide (H2O2), via the reaction shown

below in Scheme 4.3, by the enzyme superoxide dismutase (SOD).

H2O2O2 + 2H+ SOD

Scheme 4.3: Conversion of superoxide to hydrogen peroxide by SOD

Although hydrogen peroxide is not a radical nor is it particularly toxic to cells it may

be readily converted to more reactive ROS, such as hydroxyl radical (HO•).


84

4.2.4. Hydroxyl Radicals (HO•)

The hydroxyl radical (HO•) is an extremely reactive oxygen species which can react

with almost every molecule in the living cell, including DNA, lipids, proteins and

carbohydrates.144

Hydroxyl radical (HO•) can be produced from hydrogen peroxide (H2O2) and

superoxide (O2•−) in the presence of transition metals such as Fe2+ or Cu+. An

example of this process includes the Fenton reaction, which is summarised in

Scheme 4.4.

H2O2 HO+ Fe2+ HO + Fe3++

Scheme 4.4: Formation of hydroxyl radical by the Fenton reaction142

4.2.5. Peroxyl Radicals (RO2•)

Peroxyl radicals (RO2•) are formed when an alkyl radical (R•) reacts with O2. The R•

is commonly formed when HO• abstracts a hydrogen atom from a biological

molecule (RH) (Scheme 4.5).

+ H2O

O2 RO2

R +RH HO

R +

Scheme 4.5: Production of a peroxyl radical via formation of an alkyl radical

The reaction between R• and O2 to form RO2• occurs rapidly with rate constants140

often greater than 109 M−1 s−1. Peroxyl radicals are reactive enough to abstract

hydrogen atoms from biological molecules, such as lipids. This reaction forms the

basis of lipid peroxidation, which will be discussed in greater detail later.

4.2.6. Alkoxyl Radicals (RO•)

Alkoxyl radicals (RO•) can be formed by the decomposition of hydroperoxides

(ROOH) (Scheme 4.6) and often undergo reactions to form other radical species,140

such as peroxyl radicals shown in Scheme 4.7 below.

+ROROOH HO

Scheme 4.6: Formation of an alkoxyl radical from a hydroperoxide


85

R O

H H

R OH

H O2O2

Scheme 4.7: Formation of a peroxyl radical from an alkoxyl radical

4.3. The Chemistry of Radical Related Cell Damage

Long term effects from oxidative damage to cellular tissues caused by ROS may lead

to age-related diseases, such as atherosclerosis, Alzheimer’s disease and cancer.

Cellular components affected by ROS, which lead to these diseases, include lipids,

proteins and DNA. A brief explanation of each of these damaging processes is

outlined below.

4.3.1. Lipid Peroxidation

Lipid peroxidation is the radical-initiated autocatalytic process whereby lipids, such

as polyunsaturated fatty acids and phospholipids, undergo degradation by chain

reaction to form hydroperoxides.143 Lipid peroxidation in vivo is a fundamentally

deteriorative reaction that is involved in age-related diseases such as atherosclerosis,

which leads to heart attack and stroke.145

The chain reaction is initiated by hydrogen abstraction from the lipid which forms an

alkyl radical (R•). The R• then reacts with O2 to form a peroxyl radical (RO2•).

Propagation occurs by the RO2• abstracting a hydrogen atom from a nearby lipid,

resulting in lipid hydroperoxides (ROOH) and another alkyl radical (R•), which is

free to continue the chain reaction until termination takes place (Scheme 4.8).


86

OO

Hydrogen

Abstraction

MolecularRearrangement

O2

OOH

Propagation

+

Side reactions leading to further deleterious effects

Scheme 4.8: The lipid peroxidation chain reaction146

Termination, through the disproportionation of two peroxyl radicals, prevents the

two radicals from propagating and forms an alcohol, a ketone and singlet O2 (Scheme

4.9).140

+ 1O2+2R2CHOO R2CR2CHOH O

Scheme 4.9: Termination of peroxyl radicals

Due to the nature of lipid peroxidation, the formation of a hydroxyl radical (HO•),

and its subsequent reaction with lipids, can potentially form an extensive amount of

lipid hydroperoxides before termination takes place. Further reactions, involving the

transient species formed in lipid peroxidation, may take place forming a large variety

of by-products. These by-products, such as singlet O2, hydroxyl radical (HO•) and

alkoxyl radicals (RO•), may react further resulting in extensive damage not only to

the cell membrane but also to many other intra-cellular components.

4.3.2. Protein Oxidation

Amino acid residues of proteins, including those of enzymes and connective tissue,

are highly susceptible to oxidative attack by ROS.147 This can produce oxidised or

cross-linked proteins, carbonyl compounds and formation of disulfide bonds which

results in tertiary structure alterations of the protein that inhibits cellular functions.148,


87

149 Oxidative damage, at an active site of a protein, can induce a progressive loss of a

particular biochemical function within a cell.150

The detection of carbonyl compounds, such as 2-oxohistidine 86 (Scheme 4.10),

from protein oxidation, can be used as a guide to the amount of oxidative cellular

damage.151

N

NH

CO2H

H2N

ROSNH

NH

CO2H

H2N

O

+Other Products

87 86

Scheme 4.10: Formation of oxohistidine 86 by oxidative damage

Hydroxylation of amino acids, such as tyrosine 88 (shown below in Scheme 4.11)

and phenylalanine, can also give information about oxidative damage, although these

species are at much lower concentrations in cells than the overall carbonyl

content.140, 151

R

HO

R

HO OH

HO

88 89

Scheme 4.11: Hydroxylation of tyrosine 88 to form DOPA 89

4.3.3. DNA Damage

Oxidative damage to DNA can occur by many routes including modification of

nucleotide bases, sugars or by forming crosslinks.152 The damage is facilitated by the

production of HO•, from H2O2 and/or O2•−, which once generated can react

immediately with DNA.

Base modification can occur by the addition of HO• to double bonds in both purine

and pyrimidine bases.152 Both single and double strand breaks of DNA can also be

produced by free radical reactions; this is caused by radical reaction at the sugars of

the DNA molecule.149, 152


88

Oxidative modifications to DNA, which are often referred to as DNA lesions, can be

investigated with the aid of biomarkers such as 8-hydroxyguanine 90 and thymine

glycol 91 (Scheme 4.12).153 DNA repair enzymes remove most, but not all, of the

DNA lesions and therefore they accumulate with age.143 The formation of DNA

lesions may lead to mispairing of bases, which can lead to mutational changes that

impair important cellular processes, such as DNA transcription and translation.

HN

N

N

R

O

H2N

HN

N

N

R

O

H2N

OHHO

-H

92 90

HN

N

CH3

O

O

R

HN

NO

O

R

HN

NO

O

R

HN

N

CH3

O

O

R

CH3

OH

H

CH3

H

OH

OHOH

H

93 91

HO

HO

H2O

H2O

-e-

-e-

Scheme 4.12: Formation of DNA lesions 8-hydroxyguanine 90 and thymine glycol

91152

4.4. Natural Antioxidants

Antioxidants, the body’s defence against oxidative stress and ROS, can act by either

preventing the formation of reactive species or neutralising them after their

production.

Due to the vast number of substances that are able prevent oxidative damage, the

definition of an antioxidant is generally a broad one. An antioxidant can be defined

as “any substance which, when present in low concentrations compared with those of

an oxidisable substrate, significantly delays or prevents oxidation of that

substrate”.140


89

The body makes use of two types of antioxidant systems, the first of which are

enzymatic antioxidants. Examples of these are superoxide dismutase (SOD), which

catalyses the conversion of superoxide (O2•−) to hydrogen peroxide (H2O2) and

oxygen (O2) (Scheme 4.13), and glutathione peroxidase (GPX).

O2H2O2+SOD

+2H+2O2

Scheme 4.13: Dismutation of superoxide

Low molecular mass antioxidants make up the other class of natural antioxidants

utilised by the body and are normally derived from the diet. Vitamin E (α-

tocopherol) 92 and Vitamin C (ascorbic acid) 93 are just two examples of these.

O

HO O

HO OH

O

OH

OH

92 93

Low molecular mass antioxidants act by neutralising reactive species present in cells.

This is achieved by transfer of the radical character of the reactive species (R•), such

as hydroxyl radical, to the antioxidant (AH) which results in a less reactive species,

this process shown in Scheme 4.14, below.

AHR + RH A+

Scheme 4.14: Neutralisation of reactive species by antioxidants

The resulting unreactive antioxidant radical (A•) can be converted back to the

antioxidant (AH) by redox cycling processes.

Whilst natural occurring antioxidants can effectively eliminate reactive species,

synthetic antioxidants may be utilised to further reduce oxidative stress levels of

cellular systems.

4.5. Nitroxides as Antioxidants

Whilst nitroxides have been historically used in biological systems as spin

labels/probes and NMR contrast agents,3 recently there has also been considerable

interest in their use as synthetic antioxidants. Nitroxides have been illustrated to


90

reduce levels of oxidative stress in cellular environments154-158 and have been shown

to provide radio-protection towards ionizing radiation.159-162 The redox and, to a

lesser extent, radical trapping properties of nitroxides enable the detoxification of

reactive radical species in vivo.

The redox properties of nitroxides, such as TEMPO 3, enable them to mimic the

function of superoxide dismutase (SOD) by catalysing the conversion of O2•− to

H2O2 and O2.158 This reaction involves the conversion of the nitroxide to an

oxoammonium cation by superoxide. The nitroxide is regenerated by reaction of the

oxoammonium ion with another molecule of superoxide (Scheme 4.15).

O2

H2O2+ 2H+O2

N

O

N

O

-

O2-

3

Scheme 4.15: The SOD mimicking mechanism of TEMPO 3158

Hydroxylamines, produced by bioreduction of nitroxides, are also able to act as

antioxidants in vivo. Their activity is analogous to low molecular mass antioxidants,

described above, where the radical character of the reactive species is transferred

producing a less reactive product (Scheme 4.16).156 The nitroxide formed is in turn

able to confer further antioxidant protection to the cellular environment.

N OH

R

R

N O

R

R

R+ RH+

Scheme 4.16: Antioxidant activity of hydroxylamines156

4.5.1. Nitroxide Action on Ataxia-Telangiectasia

The antioxidant ability of nitroxides has seen their application in many disease states

underpinned by heightened levels of oxidative stress.154-156, 158, 162-166 A study,

reported by Mitchell and co-workers,154 detailed the application of TEMPOL 94 for


91

cancer prevention in a mouse model for the disease Ataxia-Telangiectasia (A-T). A-

T is an autosomal recessive genetic disease which displays, amongst other

symptoms, elevated levels of ROS.167 Patients presenting with the disease are prone

to neurodegeneration, immunodefiency and are predisposed to developing cancer.167

After treatment with the nitroxide TEMPOL 94, the average lifespan of the A-T

diseased mice increased markedly.

N

O

OH

94

4.5.2. Isoindoline Nitroxides and A-T

Recently within our research group, isoindoline nitroxides have been used as

antioxidants to reduce radiation-induced oxidative stress on cells affected with A-T

and thus increase cell survivability.155 To date, the water soluble CTMIO 66 has

proved to be the most effective isoindoline nitroxide, whilst being comparable to

other comercially available carboxy-nitroxides CTEMPO 95 and CPROXYL 96, and

providing better protection than α-tocopherol 92 and its water soluble analogue

Trolox 97.155 Results of the cell survivability assay from the publication by Bottle

and co-workers155 is shown below in Figures 4.1 and 4.2.

N O

HO2CN ON O

HO2C

HO2C

66 95 96

OO

HO HO

C16H33CO2H

92 97


92

Figure 4.1: Survivability of A-T cells incubated with CTMIO 66, CTEMPO 95

and CPROXYL 96155

%Survival vs Time

1

10

100

0 1 2 3 4

Days

% S

urv

ival

Blank Control

CTMIO Control

Vitamin E Control

Trolox Control

Blank A-T

CTMIO A-T

Vitamin E A-T

Trolox A-T

Figure 4.2: Survival of A-T cells incubated with CTMIO 66, α-Tocopherol 92

and Trolox 97155

CTMIO 66 has also been used as a successful antioxidant in a mouse model of A-T.

Purkinje neurons of A-T mutated mice that were treated with CTMIO 66 displayed

increased dendritic growth when compared to untreated A-T mutated mice.168 This

provides further evidence that levels of oxidative stress are reduced when CTMIO 66

is present.

With an aim to improve on the A-T results recently reported by our research

group155, 168 and for potential use on other diseases in which oxidative stress is

%Survival vs Time

1

10

100

0 1 2 3 4 Days

% Survival

Blank ControlCTMIO ControlCTEMPO ControlCPROXYL ControlBlank A-TCTMIO A-TCTEMPO A-TCPROXYL A-T


93

implicated, the synthesis of several nitroxides for use as antioxidants was proposed.

Water solubility is of particular importance for synthetic nitroxides with a view to

potential biological applications.


Due to their biological relevance and ease of preparation, nitroxides containing

carboxylic acid and amine groups are appropriate targets for potential antioxidants.

Two potentially useful compounds, 5,6-dicarboxy-1,1,3,3-tetramethylisoindolin-2-

yloxyl 98 and 5-carboxy-6-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl 99 are

shown below.

N O

HO2C

HO2C

98

N O

H2N

HO2C

99

The dicarboxy substituted nitroxide 98 may be an interesting compound, considering

the effectiveness of the mono substituted CTMIO 66 as an antioxidant in A-T cell

experiments. The carboxy-amino substituted nitroxide 99 is also an appealing target

as the presence of two water solubilising functional groups may further increase

solubility in aqueous media.

The proposed synthetic sequence for the synthesis of the dicarboxy nitroxide 98 and

the aminocarboxy nitroxide 99, as well as intermediate anhydride 100 and imide 101

is shown below (Scheme 4.17).


94

N O

Br

N O

NC

21

Br NC

102

N O

HO2C

HO2C

98

N O

100

O

O

O

Dehydration

N O

101

HN

O

O

N O

H2N

HO2C

Hydrolysis

Cyanation

Hofmann

Imide

Rearrangement

Formation

99

Scheme 4.17: Proposed synthetic route to water soluble nitroxides 98 and 99

4.6.1. Synthesis of 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl

(102)80, 169

The synthesis of the nitroxide dinitrile 102 was initially achieved by the

methodology reported by Micallef and co-workers.80 Heating the dibromo nitroxide

21 with Zn(CN)2 in DMF at 85 °C, in the presence of Pd(PPh3)4 (0.25 equiv), gave

the desired dinitrile 102 in a moderate yield of 35 % (Entry 1 Table 4.2). Whilst this

yield was significantly lower than that reported in the literature,80 this procedure is

known to be highly variable and often gives low yields.170


95

N O

Br

N O

NC

21

Br NC

102

Table 4.2: Synthesis of nitroxide dinitrile 102

Entry Reagents Conditions Yield (%)

1 Zn(CN)2 (2 equiv),

Pd(PPh3)4 (0.25 equiv), DMF 85 °C, Ar, 12 h 35

2

K4[Fe(CN)6]·3H2O (0.4 equiv),

Na2CO3 (2 equiv),

Pd(OAc)2 (5 mol%), DMAC

130 °C, Ar, 72 h >5-72a

a Yields from reaction highly variable

Due to the drawbacks of this methodology, such as the use of a toxic nitrile source

and extremely high Pd-catalyst loadings, another technique originally pioneered at

QUT by A. S. Micallef,169 was undertaken. Heating K4[Fe(CN)6]·3H2O, a non-toxic

cyanide source, with the dibromo nitroxide 21 in the presence of Pd(OAc)2 (5 mol%)

and Na2CO3 in DMAC at 130 °C under argon gave the desired dinitrile 102 in high

yield (72 %) in the first instance, but the procedure proved to be highly variable

(Entry 2 Table 4.2). On some occasions the reaction gave little to no product, whilst

on other occasions it worked successfully, with no apparent alteration to the

methodology. Generally, the outcome of the reaction could be ascertained before

work-up by the colour of the reaction mixture. A dark green-brown solution,

proposed to be coloured by the production of a trace amount of phthalocyanine side

product 103,169 usually indicated a high yield of the dinitrile 102, whilst a light

yellow-brown colour indicated an unsuccessful synthesis, with unreacted starting

material being reisolated. The proposed macrocyclisation, forming the

phthalocyanine 103 from the desired dinitrile 102, is conceivably templated by the

Fe2+ or Pd2+ present in the reaction mixture (Scheme 4.18).


96

N

N

N

N

N

N

N

N M

103 M= Fe2+ or Pd2+

N O

NC

NC

102

N N

NN

O

O

O

O

4Macrocyclisation

Fe2+ or Pd2+

Scheme 4.18: The proposed structure of the phthalocyanine 103 side-product

After performing four simultaneous reactions under identical reaction conditions (see

Figure 4.3 below, note the varying colours of the reaction mixture) it was concluded

that there was a random inherent problem in the reactions, possibly brought about by

the amount of air in the reaction vessels.

Figure 4.3: The high variability of cyanation reaction

Interestingly, when air was completely removed from the reaction via three

freeze/pump/thaw cycles the reaction did not proceed, which suggested some O2 may

be required for the reaction to occur. When the reaction was performed under an

atmosphere of air, again none of the desired product was able to be isolated. Instead

only starting material was isolated along with several degradation products.

Generally, reactions performed under an argon atmosphere without degassing the

reaction mixture prior to heating gave the best yields, usually between 30-50 %

commonly performed on 1-5 g scale of dibromo nitroxide 21. As the inherent


97

problem seemed to be random and attempts to optimise the reaction did not increase

the yields, the reaction was repeated on a large enough scale to give sufficient

amounts of the dinitrile 102 to undertake further reactions. Alternative synthetic

strategies may be able to improve the synthesis of the dinitrile 102 from the dibromo

nitroxide 21 in the future.

4.6.2. Synthesis of 5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

(98)

The synthesis of dicarboxy nitroxide (DCTMIO) 98 was proposed via hydrolysis of

the dinitrile 102. Initial attempts using similar methodology used for the synthesis of

carboxylic acids 28 and 29 from the Heck products (see Chapter 2), using NaOH in

refluxing THF/H2O failed to produced any of the desired di-carboxylic acid 98;

instead only starting material was isolated (Entry 1, Table 4.3).

N O

NC

N O

HO2C

102

NC HO2C

98

Table 4.3: Synthesis of dicarboxy nitroxide 98

Entry Reagents Conditions Yield (%)

1 1.25 M NaOH, THF reflux, 18 h 0

2 9 M H2SO4, AcOH reflux, 6 h 0

3 2.5 M KOH, EtOH Reflux 16 h 99

Subsequently, acid hydrolysis using H2SO4 (9 M) in refluxing AcOH was attempted,

but this method also failed to give any of the dicarboxylic acid 93 (Entry 2, Table

4.3). Finally, base hydrolysis with KOH in EtOH/H2O was utilised to give the

desired diacid nitroxide 98 in near quantitative yield (99 %) (Entry 3, Table 4.3). The

addition of EtOH to the aqueous solution increases the rate of the hydrolysis

reaction.171 Recrystallisation of diacid 98 from H2O gave crystals which were

suitable for X-ray crystallographic analysis (data collected by P. Jensen, University

of Sydney and refined by J. C. McMurtrie, Queensland University of Technology)


98

which confirmed the structure of the diacid (Figure 4.4 below). Further structural

confirmation was achieved by NMR analysis of a corresponding methoxyamine 104

(see Entry 1, Table 4.4), synthesised by the Fenton reaction in DMSO.



furano[3,4-f]isoindol-2-yloxyl (100)

The nitroxide anhydride 100, an intermediate for the proposed synthesis of amino

carboxy nitroxide 99 (as well as xanthene-based profluorescent nitroxides, see

Chapter 5), was synthesised by refluxing DCTMIO 98, in acetic anhydride for 4 h

(Scheme 4.19).

N O

HO2C

N O

98

HO2C

100 100 %

O

O

O

Ac2O

Reflux, 4 h

Scheme 4.19: Synthesis of anhydride nitroxide 100

The reaction was complete when the di-acid 98 was completely dissolved giving an

orange solution. The anhydride 100 crystallised from the cooled reaction mixture

(Ac2O) as bright orange crystals in sufficient quality for X-ray crystallographic

analysis (data collected by P. Jensen, University of Sydney and refined by J. C.

McMurtrie, Queensland University of Technology), which confirmed the planar

anhydride structure (Figure 4.5 below). The anhydride was isolated in quantitative

(100 %) yield after removal of the reaction solvent.


99



pyrrolo[3,4-f]isoindol-2-yloxyl (101)

The nitroxide imide 101 was synthesised by heating the anhydride 100 in the

presence of urea at 140 °C for 25 min.172 After extraction and purification by column

chromatography the nitroxide imide 101 was isolated in high yield (85 %) (Scheme

4.20).

N O N O

100 101 85 %

O HN

O

O

O

O

Urea

140 °C25 min

Scheme 4.20: Synthesis of nitroxide imide 101

The synthesis of phthalimides, like the imide 101, may also be achieved by heating

with conc. NH3 solution until fusion of the reaction mixture,172 but due to the ease of

purification, the procedure using urea as the nitrogen source was preferred. Further

structural confirmation was achieved by NMR analysis of a corresponding

methoxyamine 105 (see Scheme 4.25).

4.6.5. The Attempted Synthesis of 5-Amino-6-carboxy-1,1,3,3-

tetramethylisoindolin-2-yloxyl (99) via the Hofmann

Rearrangement

The synthesis of ortho-aminobenzoic acids (anthranilic acids) from imides via the

Hofmann rearrangement is well known, with many examples being present in the

literature (see Scheme 4.21).173-178


100

106 107

NH2

CO2H

NH

O

O

1. NaOBr

2. HCl

Scheme 4.21: Synthesis of anthranilic acid 107 via the Hofmann rearrangment171

When the nitroxide imide 101 was treated with NaOH/Br2 (NaOBr) using the normal

Hofmann rearrangement conditions,172 none of the desired product 99 was isolated.

Instead the brominated compound 108 was the major product and was isolated in 49

% yield (Scheme 4.22). A scan of the relevant literature failed to reveal any

precedent for the bromination observed.

N O N O

101 99 0 %

H2N

HO2C

HN

O

O

N O

H2N

HO2C

Br

+

108 49 %

80°C 5 min

Br2NaOH

Scheme 4.22: Synthesis of nitroxides via the Hofmann rearrangement

The presence of the bromine can be seen in the mass spectrum the characteristic [M+]

and [M+2+], with an intensity of 1:1 in accordance with isotopic ratio of a mono-

brominated compound, 78 mass units higher than the expected product 99. The

bromine was assigned to the 4-position next to the amine from NMR analysis of the

analogous methoxyamine 109 (see Entry 2, Table 4.4), formed using Fenton

chemistry.

When the Hofmann rearrangement is performed on phthalimides the substrates

commonly do not contain electron-rich aromatic rings. Substituted electron-poor

aromatics174, 176 feature most frequently in the literature. A report by Godinez et al.178

however describes the disubstituted xylene 110, (which is somewhat analogous to the

nitroxide imide 101), undergoing the Hofmann rearrangement to give the substituted

anthranilic acid 111 in 84 % yield (Scheme 4.23). The hydrolysis of the imide and

the Hofmann rearrangement in this case were performed stepwise; the usual

approach is to undertake these reactions in one pot. In this report hypochlorite was


101

used in place of the more commonly used hypobromite, possibly to prevent the

halogenation which was observed in the nitroxide example.

105 106 84 %

NH2

CO2H

NH2

CO2H

O

NaOCl

NaOH

Scheme 4.23: Synthesis of dimethylanthranilic acid 106 as described by Godinez et

al.178

Because of the presence of the t-alkyl substituents, the aromatic ring of the nitroxide

101 may be more electron rich than 110. This may facilitate bromination, by Br2 or

NaOBr, after the Hofmann rearrangement has taken place (Scheme 4.24).

N O

H2N

HO2C

N O

H2N

HO2C

Br

Br+

H

N O

H2N

HO2C

Br

N OHN

O

O

101

108

Scheme 4.24: Speculative mechanism for the formation of amino bromo carboxy

nitroxide 108

After two separate experiments gave the brominated product 108, it was concluded

that the amino carboxy nitroxide 99 could not be synthesised via this methodology.

Due to the presence of water solubilising functionality, the brominated amino

carboxy nitroxide 108 was utilised for the antioxidant investigations envisaged for

the target compound 99.


102

4.6.6. Syntheses of Methoxyamines (104, 105, 109 and 112)

As mentioned above, the methoxyamines 104, 105, 109 and 112, derived from the

nitroxides 98, 101, 108 and 100 respectively, were synthesised in order to give

further structural characterisation of the nitroxides. The dicarboxy 104 and amino

bromo carboxy 109 methoxyamines, synthesised via the Fenton reaction in DMSO,

were isolated in high yields of 95 % and 80 % respectively, as outlined in Table 4.4.

N O

R1

HO2C

N O

R1

HO2C

R2 R2

98or 108 104or 109

FeSO4.7H2OH2O2

DMSORT, 30 min

Table 4.4: Synthesis of methoxyamines 104 and 109

Entry Nitroxide R 1 R2 Product Yield (%)

1 98 CO2H H 104 95

2 108 NH2 Br 109 80

The methoxyamine imide 105 was isolated in a low yield of 38 % (Scheme 4.25).

The yield may be lower than those observed for the acyclic compounds 104 and 109

as the aqueous work up may have led to ring opening reactions and loss of the

desired product 105.

N O N OHN HN

O

O

O

O

FeSO4.7H2OH2O2

DMSORT, 30 min

101 10538 %

Scheme 4.25: Synthesis of methoxyamine 105

The methoxyamine anhydride 112 was synthesised in quantitative yield (100 %) by

dehydration of the diacid methyl trap 104 with Ac2O (Scheme 4.26), similar to the

synthesis of the nitroxide anhydride 100. Aqueous work up was avoided to prevent

ring opening of the anhydride.


103

N O N O

HO2C

HO2C

O

O

O

Reflux4 h

Ac2O

104 112 100 %

Scheme 4.26: Synthesis of methoxyamine 112

4.6.7. A-T Cell Survivability Assays of Isoindoline Nitroxides 98 and 108

The water soluble nitroxides 98 and 108 were subjected to an A-T cell survival assay

(performed by P. Chen, Queensland Institute of Medical Research) to ascertain their

impact as antioxidants for this disease. The cells were cultured and treated with

radiation of 4 Gy as reported previously by our research group.155

Ataxia-Telangiestasia cells treated with the novel nitroxides 98 and 108 were

compared to cells treated with the best performing compound to date, CTMIO 66

(see Figure 4.6). Also included on the graph are cell survivability data of non-

dieseased cells (ND) and untreated Ataxia-Telangiectasia (A-T) cells. The data of

each of the three nitroxides 66, 98 and 108 are the average of two separate cell count

experiments, with each of the experiments performed in triplicate.

1

10

100

0 1 2 3 4

Time/days

% S

urvi

val

ND A-T 98 108 66

Figure 4.6: Cell survivability of A-T cells treated with 98 and 108


104

As can be seen in Figure 4.6 above, each of the carboxy-substituted nitroxides 66, 98

and 108 seem to impart similar protection to the A-T cells. The trend of the data

seems to suggest that CTMIO 66 and DCTMIO 98 may perform slightly better than

brominated amino acid 108, although the uncertainty of the measurements renders

this observation questionable, as the difference between the individual nitroxides is

significantly less than the error of the measurements. Notably however, there is an

observable improvement in survivability for the cells treated with nitroxides 66, 98

and 108 compared with the untreated A-T cells. This suggests that all the nitroxides

are imparting some protective impact on the A-T cells and the carboxylic acid group

may be of particular importance. However, the survivability of the treated cells is

still well below the cell survivability of the non-diseased cells and further

improvement is still required.

It seems that nitroxides which have sufficient solubility in cellular media have the

ability to impart a protective effect on A-T affected cells. This can be seen not only

by the results of the current study but also those previously reported by our group.155

The specificity of a possible structure activity relationship (SAR) for this disease

system is highly speculative as there are distinctive structural differences between

each of the nitroxides tested but no significant difference in activity was shown. For

the design of nitroxides with improved antioxidant efficacy, an assay that limits the

variability in the data observed from individual experiments is required. At present,

under the current methodology, there is not enough certainty in the data to draw any

significant conclusions about the efficacy of the individual nitroxides tested. A

method utilising instrumentation, such as a flow-cytometric method, would greatly

enhance the robustness of the results obtained. That is, of course, if the mechanism of

the protective effect has a relationship specific to the structure of the nitroxides

tested, rather than the cell simply requiring a high enough concentration of nitroxide

to be present, which is also possible.


The dicarboxy nitroxide 98 was succesfully synthesised through base hydrolysis of

the dinitrile 102, in quantitative yield. Although the production of the dinitrile 102

precursor via Pd-catalysed cyanation proved troublesome, sufficient amounts were

able to be synthesised so that further reactions could be performed. Ideally, an


105

improved synthetic technique for the synthesis of this important intermediate is

required. Elaboration of the diacid 98 to the anhydride 100 proved facile and could

easily achieved in quantitative yield. Conversion of the anhydride 100 to the imide

101 was also trouble-free, giving the desired product in high yield. The Hofmann

rearrangement of the imide 101 did not furnish the desired nitroxide anthranilic acid

99, but instead its bromo-substituted analogue 108. This may be due to elevated

electron-density present on the aromatic ring of the nitroxide. However, this

unexpected product 108, along with the diacid 98, was shown to have a protective

antioxidant effect when assessed in an A-T survivability assay. These results suggest

that carboxy-substitution may play an important role in the ability of isoindoline

nitroxides to increase A-T cell survivability, although it is not clear whether this is

due solely to the enhanced water solubility of these nitroxides or also involves other

factors, such as pH for example.

4.8. Experimental

All experimental details were as previously described in Chapters 2 and 3 where

relevant.

Anhydrous dimethylacetamide (DMAC) (99 %) and urea (99 %) were purchased

from Sigma/Aldrich.

4.8.1. 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl (102)80

N O

NC

NC

102

A mixture of 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 (1.0 g, 2.87

mmol), Pd(PPh3)4 (830 mg, 0.72 mmol, 0.25 equiv), Zn(CN)2 (800 mg, 5.87 mmol, 2

equiv) in DMF (5 mL) was degassed via three freeze/pump/thaw cycles and was

heated under argon at 85 °C for 12 hours. The solution was subsequently poured

onto NH3/NH4Cl (250 mL) solution and extracted with CHCl3 (3 × 100 mL). The

organic phase was washed with saturated NaCl solution, dried (Na2SO4) and the

solvent removed under reduced pressure to give crude dinitrile 102. Purification by


106

column chromatography (30 % EtOAc, 70 % n-hexane) gave 5,6-dicyano-1,1,3,3-

tetramethylisoindolin-2-yloxyl 102 (238 mg, 0.99 mmol, 35 %) mp 243-247 °C

(lit.,80 243-248 °C); IR (ATR) νmax: 3039 (alkyl CH), 2979 (aryl CH), 2231 (CN),

1465(aryl C-C), 1432 (NO) cm-1. These data agree with those reported previously by

Barrett et al.80

4.8.2. Alternate Synthesis of 5,6-Dicyano-1,1,3,3-tetramethylisoindolin-

2-yloxyl (102)

N O

NC

NC

102

A mixture of 5,6-dibromo-1,1,3,3-tetramethylisoindolin-2-yloxyl 21 (1.75 g, 5.03

mmol), K4[Fe(CN)6]·3H2O (868 mg, 2.05 mol, 0.4 equiv, pre-ground), Na2CO3 (1.07

g, 10.16 mmol, 2.0 equiv) and Pd(OAc)2 (60 mg, 0.26 mmol, 5 mol%) were heated

in DMAC (11 mL) at 130 °C for 3 days. The solution was subsequently poured onto

water and extracted with CHCl3. The organic phase was washed with saturated NaCl

solution, dried (Na2SO4) and the solvent removed under reduced pressure to give

crude dinitrile 102 and remaining starting material. Purification by column

chromatography (CHCl3) gave 5,6-dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl

102 (869 mg, 3.62 mmol, 72 %) mp 243-247 °C (lit.,80 243-248 °C); IR (ATR) νmax:

3039 (alkyl CH), 2979 (aryl CH), 2231 (CN), 1465(aryl C-C), 1432 (NO) cm-1.

These data agree with those reported previously by Barrett et al.80

4.8.3. 5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (98)

N O

HO2C

HO2C

98

A suspension of 5,6-dicyano-1,1,3,3-tetramethylisoindolin-2-yloxyl 102 (453 mg,

1.89 mmol) in KOH (2.5 M, 7.5 mL) and absolute ethanol (1.6 mL) was heated at

reflux for 16 h. The resultant pale yellow solution was washed with Et2O (2 × 50

mL), acidified with H2SO4 (2 M) and extracted with Et2O (3 × 50 mL). The organic


107

phase was dried (Na2SO4) and the solvent removed under reduced pressure to give

5,6-dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl 98 (521 mg, 1.87 mmol, 99 %)

mp 246-250 °C (decomp.); νmax (ATR-FTIR): 3545 and 3469 (OH), 2975 (alkyl CH),

2906 (aryl CH), 1705 (C=O), 1432 (NO) cm−1; +EI MS found M+ 278.10288 (0.11

ppm from calc. mass of C14H16NO5•): m/z 278 (M+, 5 %), 260 (65), 245 (38), 230

(100), 215 (82), 171 (50). Recrystallisation from water produced crystals that were of

sufficient quality to obtain an x-ray crystal structure.

4.8.4. 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-furano[3,4-

f]isoindol-2-yloxyl (100)

N OO

O

O

100

5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl 98 (610 mg, 2.19 mmol) was

refluxed in Ac2O (10 mL) for 4 h after which a clear orange solution was obtained.

Removal of the solvent under reduced pressure gave 1,1,3,3-tetramethyl-5,7-dioxo-

3,5,6,7-tetrahydro-1H-furano[3,4-f]isoindol-2-yloxyl 100 (570 mg, 2.19 mmol, 100

%) mp 246-250 °C (decomp.); νmax (ATR-FTIR): 2983 (alkyl CH), 2937 (aryl CH),

1845 and 1776 (C=O), 1621 (aryl C-C), 1442 (NO) cm-1; +EI MS found M+

260.09340 (4.30 ppm from calc. mass of C14H14NO4•): m/z 260 (M+, 95 %), 245 (50),

230 (97) 215 (100), 171 (80). Crystals for x-ray analysis were formed by slow

cooling of the Ac2O reaction solution.

4.8.5. 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-pyrrolo[3,4-

f]isoindol-2-yloxy (101)

N OHN

O

O

101

Nitroxide anhydride 100 (100 mg, 0.384 mmol) and urea (35 mg, 0.576 mmol, 1.5

equiv) were stirred at 140 °C for 25 min. The resulting residue was purified by


108

column chromatography (5 % THF, 95 % CHCl3) to give 1,1,3,3-tetramethyl-5,7-

dioxo-3,5,6,7-tetrahydro-1H-pyrrolo[3,4-f]isoindol-2-yloxy 101 as a pale yellow

solid (85 mg, 0.328 mmol, 85 %) mp 235-238 °C (decomp.); νmax (ATR-FTIR): 3168

and 3075 (NH), 2985 (alkyl CH), 2935 (aryl CH), 1766 and 1726 (C=O), 1625 (aryl

C-C), 1432 (NO) cm-1; +EI MS found M+ 259.10828 (0.07 ppm from calc. mass of

C14H15N2O3•): m/z 259 (M+, 68 %), 244 (46), 229 (100), 214 (70), 171 (93) (see

HPLC 10, Appendix 1).

4.8.6. 5-Amino-4-bromo-6-carboxy-1,1,3,3-tetramethylisoindolin-2-

yloxyl (108)

N O

HO2C

H2N

Br

108

To an aqueous solution of NaOH (6.25 M, 0.16 cm3) at 0 °C was added liquid Br2

(11 µL, 34 mg, 0.213 mmol) and the resulting solution cooled back to 0 °C. The

nitroxide imide 101 (46 mg, 0.178 mmol) was added, immediately followed by an

aqueous solution of NaOH (7.25 M, 0.1 cm3). The reaction was heated at 80 °C for 5

min at which stage dissolution occured. The mixture was cooled in ice and conc. HCl

(0.1 cm3) was added to neutralise the base. Precipitation was achieved by addition of

AcOH (0.05 cm3). The resulting suspension was extracted with Et2O (2 × 10 cm3),

washed with H2O (10 cm3) and dried (Na2SO4). The solvent was removed under

reduced pressure to give the brominated amino acid 108 as a yellow crystalline solid

(29 mg, 0.088 mmol, 49 %) mp >300 °C (decomp.); νmax (ATR-FTIR): 3473 and

3361 (NH), 2975 (alkyl CH), 2929 (alkyl CH), 2856 (aryl CH), 1670 (C=O), 1602

and 1552 (aryl C-C), 1436 (NO) cm-1; +EI MS: m/z 327 (M+, 10 %), 312 (20), 297

(10), 282 (100), 236 (45), 219 (55) (see HPLC 11, Appendix 1).

4.8.7. Synthesis of Methoxyamines

A general procedure for the synthesis of methoxyamines 104, 105 and 109 is shown

below.


109

4.8.8. General Procedure

To a solution of nitroxide (98, 101 or 108) and FeSO4·7H2O (2 equiv) in DMSO was

added H2O2 (exact quantities listed below). The reaction mixture was stirred under

argon at room temperature for 1 h. NaOH (1 M) was added and the resulting solution

extracted with Et2O. The organic phase was washed with H2O and dried (Na2SO4).

Removal of the solvent under reduced pressure gave the crude methoxyamine.

Subsequent purification was achieved by column chromatography where applicable

(see below for specific conditions).

4.8.9. 5,6-Dicarboxy-2-methoxy-1,1,3,3-tetramethylisoindoline (104)

N O

HO2C

HO2C

104

5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl 98 (64 mg, 0.230 mmol),

FeSO4·7H2O (128 mg, 0.46 mmol, 2 equiv), DMSO (7 cm3), H2O2 (30 %, 46 µL).

5,6-dicarboxy-2-methoxy-1,1,3,3-tetramethylisoindoline 104 was isolated as a white

crystalline solid (64 mg, 0.218 mmol, 95 %); δH: (MeOH-d4) 1.46 (12H, br s, CH3),

3.78 (3H, s, OCH3), 7.51 (2H, s, 4-H and 7-H); δC: (MeOH-d4) 29.5 (CH3) 64.5

(alkyl C*), 67.0 (OCH3), 121.9 (C4 and C7), 132.3 (C5 and C6), 148.2 (C3a and

C7a), 169.9 (C=O); +EI MS found M+ 293.12636 (0.13 ppm from calc. mass of

C15H19NO5): m/z 293 (M+, ~1 %), 275 (5), 260 (100), 156 (55).


pyrrolo[3,4-f]isoindole (105)

N OHN

O

O

105

Nitroxide imide 101 (30 mg, 0.116 mmol), FeSO4·7H2O (128 mg, 0.244 mmol, 2

equiv), DMSO (4 cm3), H2O2 (30 %, 28 µL). Column chromatography (5 % THF, 95

% CHCl3) gave the methyl trap imide 105 as a white solid (12 mg, 0.044 mmol, 38

%); δH: (DMSO-d6) 1.50 (12H, br s, CH3), 3.80 (3H, s, NOCH3), 7.61 (2H, s, 4-H


110

and 7-H), 7.75 (1H, br s, NH); δC: (DMSO-d6) 30.1 (CH3), 65.6 (OCH3), 67.4 (alkyl

C*), 117.4 (C-4 and C-7), 132.2 (C-5 and C-6), 152.7 (C-3a and C-7a), 167.9 (C=O).

4.8.11. 5-Amino-4-bromo-6-carboxy-2-methoxy-1,1,3,3-


N O

HO2C

H2N

Br

109

5-Amino-4-bromo-6-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl 108 (29 mg,

0.088 mmol), FeSO4·7H2O (128 mg, 0.244 mmol, 2.8 equiv), DMSO (4 cm3), H2O2

(30 %, 28 µL). Column chromatography (20 % EtOH, 80 % EtOH, 0.1 % v/v

AcOH) gave the methoxyamine 109 as white solid (24 mg, 0.070 mmol, 80 %); δH:

(MeOH-d4) 1.40 (12H, br s, CH3), 3.78 (3H, s, NOCH3), 7.64 (1H, s, 7-H); δC:

(MeOH-d4) 29.4 (CH3), 64.7 (OCH3), 65.7 (alkyl C*), 69.8 (alkyl C*), 104.8 (C-7),

111.1 (C-5), 123.6 (C-4), 134.3 (C-3a), 147.9 (C-6), 148.4 (C-7a), 169.3 (C=O).

+ESI MS found m/z 345 (95 %, (M+H)+) 343 (98) 299 (97), 297 (100), 265 (32).


furano[3,4-f]isoindole (107)

N OO

O

O

112

5,6-Dicarboxy-2-methoxy-1,1,3,3-tetramethylisoindoline 104 (64 mg, 0.218 mmol)

was refluxed in Ac2O (1.2 mL) for 4 h after which a clear solution was obtained.

Removal of the solvent under reduced pressure gave the alkoxyamine anhydride 112

as a light yellow solid (60 mg, 0.218 mmol, 100 %); δH: (DMSO-d6) 1.44 (12H, br s,

CH3), 3.71 (3H, s, OCH3), 8.00 (2H, s, 4-H and 7-H); δC: (DMSO-d6) 30.0 (CH3),

65.7 (OCH3), 67.5 (C*), 119.9 (C-4 and C-7), 131.6 (C-5 and C-6), 154.2 (C-3a and

C-7a), 163.5 (C=O); +EI MS found M+ 293.12636 (0.13 ppm from calc. mass of

C15H19NO5): m/z 293 (M+, ~1 %), 275 (5), 260 (100), 156 (55).

Chapter 5 – Xanthene-Based Profluorescent Isoindoline Nitroxides

111

5. XANTHENE-BASED PROFLUORESCENT ISOINDOLINE

NITROXIDES

5.1. Introduction

In recent years fluorescent probes for the detection of reactive oxygen species (ROS)

have attracted increased interest.179, 180 The utility of these probes arises from their

ability to detect elevated levels of ROS in cells, through increased fluorescence

intensity. Traditional probes have been based on reduced xanthenes, such as

dihydrofluoresceins and dihydrorhodamines, which become irreversibly fluorescent

via oxidation by ROS. The fluorescence derived from a redox reversible xanthene-

based profluorescent nitroxide allows the prospect of a real-time measure of cellular

redox status.

5.2. Fluorescence Detection of Reactive Oxygen Species (ROS)

The use of fluorescent probes for the detection and quantification of ROS has the

distinct advantage of greater sensitivity over other non-luminescent techniques, such

as EPR spin trapping. Xanthene-based compounds are commonly employed for this

purpose.

113R = H114R = Cl

OHO OH

CO2H

HR R

OHO O

CO2H

R R

115R = H116R = Cl

ROS

Scheme 5.1: Fluorescence detection of ROS with dihydrofluoresceins

ROS detection by xanthene-based fluorescent probes is commonly performed using

dihydroxanthenes which become fluorescent upon reaction with ROS. Reaction of

reduced fluoresceins, such as dihydrofluorescein181 113 (fluorescin) and the more

commonly used chlorinated analogue, 2’,7’-dichlorodihydrofluorescein 114

(DCFH2) with reactive oxygen species gives the fluorescent analogues 115 and 116

respectively,179 which can be detected spectrofluorimetrically. Dihydrofluoresceins,

such as 113 and 114, and related dihydrorhodamines are known to rapidly auto-


112

oxidize, resulting in a large background fluorescence and problematic analysis of the

fluorescence measurements.179, 182 Whilst the use of freshly prepared solutions can

reduce this effect,182 the problem can be further circumvented by the use of a

protected fluorescein, diacetyl-2’,7’-dichlorodihydrofluorescein 117 (DCFH-DA)

originally synthesised by Brandt and Keston in 1965.182

OAcO OAc

117

Cl ClH

CO2H

The same group concurrently used DCFH-DA 117 (activated with NaOH) for the

detection of minute levels of H2O2 produced by in vitro peroxidase enzymes.183

Since these initial reports by Brandt and Keston, DCFH-DA 117 has been used for

the detection of ROS by a large number of research groups.180, 184, 185

Whilst DCFH-DA 117 and related compounds have found significant utility in the

biological field, they still have some drawbacks.180 In addition to the previously

mentioned auto-oxidation, they also suffer from a lack of selectivity for specific

ROS.180

Recently, Maeda et al. have synthesised sulfonylated fluorescein-based probes which

display specificity for the important ROS hydrogen peroxide186 and superoxide,187 by

non-redox mechanisms. The specificity of the pentafluorophenylsulfonyl fluorescein

118 arises from H2O2 induced cleavage of the sulfonate ester, giving the fluorescent

fluorescein 119 (Scheme 5.2).186

OO O

118 X = H, Cl or F

X X

CO2H

S

O

O

F

F

F

F

F

OO OH

X X

CO2H

H2O2

119 X = H, Cl or F

Scheme 5.2: H2O2 specific probe 118 synthesised by Maeda et al.186


113

Similarly, bis(2,4-dinitrophenylsulfonyl) fluorescein 120 is specific for the detection

of superoxide. Cleavage of the sulfonylated esters of 120 by superoxide gives the

corresponding fluorescein 121 (Scheme 5.3).187

OO O

X X

S

O

O

NO2

O

O

S

O

O

NO2

NO2

O2N

120 X = H, Cl or F

O2

OHO O

X X

121 X = H, Cl or F

CO2H

Scheme 5.3: O2•- specific probe 120 synthesised by Maeda et al.187

Fluorescent probes, specific for the detection of singlet oxygen (1O2), have recently

been synthesised by Nagano and co-workers.188, 189 These xanthene-anthracene based

probes 122 are non-fluorescent due to high energy of the HOMO of the anthracene

moiety in comparison to the xanthene ring. Reaction of these probes with singlet

oxygen (1O2) forms endoperoxides 123, which can be detected by an increase in

fluorescence intensity (Scheme 5.4).

O

R

R

CO2H

OHO O

R

R

CO2H

OHO

1O2

O

O

122 R = Ph or Me 123 R = Ph or Me

Scheme 5.4: Fluorescence detection of singlet oxygen188, 189


114

The Nagano group has also synthesised probes for the detection of highly reactive

oxygen species.190 These aryloxyphenols 124 are O-dearylated by reaction with

hydroxyl radical (•OH) to give fluorescein 115, which can be detected by

spectrofluorimetry, and a quinone product (Scheme 5.5).

O

CO2

OO O

CO2

OHO

124 R = O or NH

OH

OX

115

HX

Scheme 5.5: Fluorescence detection of hydroxyl radical190

Whilst the probes synthesised by the Maeda186, 187 and Nagano188-190 groups show

high specificity for key ROS, the reactions which give rise to the fluorescent species

are irreversible. Therefore they may not give a reversible real-time indication of ROS

present in the cell. A probe involving a reversible transformation which gives rise to

the quenching mechanism would give a more accurate indication of the levels of

ROS, over a given time course. When ROS levels are high, such a probe should

show heightened fluorescence. If the ROS levels are subsequently reduced, by

addition of an antioxidant for example, the fluorescence pertaining to detection of

ROS ideally would then also be reduced.


For the reasons discussed above, the synthesis of xanthene-based profluorescent

nitroxides was highly desirable. The successful synthesis of such systems is

described in this chapter. This represents the first examples of xanthene-based

(fluorescein and rhodamine) profluorescent nitroxides. The redox chemistry of the

nitroxide moiety enables reversible fluorescence detection of ROS and allows a time-

resolved response relating to cellular redox status.

5.3.1. The Synthesis of Nitroxide-Substituted Fluorescein

Xanthene-based fluorescent dyes, such as fluorescein 125, are commonly accessed

via condensation of substituted phthalic anhydrides with ortho-substituted phenols in

the presence of a ZnCl2 catalyst (Scheme 5.6).172


115

O

O

O

OHHO

ZnCl2 180 °C, 2 h

2 equiv

33 125

OHO OH

O

O

126

Scheme 5.6: Synthesis of fluorescein 125

Fluorescein is routinely isolated as a neutral non-fluorescent spirolactone 125 and

exists in this form in acidic environments. Deprotonation of the spirolactone 125 in

basic solution gives the charged, highly fluorescent, quinoidal form 115 of

fluorescein (Scheme 5.7).

OHO OH

125

OHO O

115

CO2O

O

Base

Acid

Scheme 5.7: The two forms of fluorescein

With the nitroxide anhydride 100 in hand (see Chapter 4), the synthesis of a

nitroxide-substituted analogue of fluorescein was envisaged via an analogous

condensation reaction (Scheme 5.8). Heating the nitroxide anhydride 100 with

resorcinol 126, in the presence of ZnCl2, analogous to the synthesis of fluorescein

125, gave the fluoresceinyl nitroxide 127 in trace amounts (<5 %). Decomposition,

presumably due to the incompatibility of the nitroxide moiety to such harsh reaction

conditions, was prevalent leading to various side products which made purification

of the product 127 troublesome.


116

OHHO

ZnCl2180 °C, 2 h

2 equiv

OHO OH

O

O

N OO

O

O NO

127<5 %

126

100

Scheme 5.8: Attempted synthesis of fluoresceinyl nitroxide 127

With the first synthetic approach not particularly successful, a new, less aggressive

route was required for the synthesis of the fluoresceinyl nitroxide 127, in an

acceptable yield. This was achieved by adaptation of the synthesis of 5(6)-

carboxyfluorescein, reported by Burgess and co-workers.191 Reaction of the

anhydride 100, with resorcinol 126 in methanesulfonic acid, at 85 °C for 24 h,

generated the sulfonate ester 128,191 which was not isolated (Scheme 5.9).

N OO

O

O

OHO O

N

O

OHO OH

CO2HN

O

SO3Me

OHHO

MeSO3H85 °C, 24 h

2 equiv

100128

129

OHO OH

O

NO O

127 92 %

CO2

HCl

NaOH

126

Scheme 5.9: Synthesis of fluoresceinyl nitroxide 127


117

Treatment of 128 with NaOH removed the sulfonate, producing the quinoidal

fluorescein anion 129. The product was isolated as the fluorescein nitroxide

spirolactone 127, by precipitation with the addition of HCl. After purification the

fluorescein nitroxide 127 was obtained in a high yield (92 %, see Scheme 5.9).

Isolation and purification proved problematic due to the low solubility of the

spirolactone product 127 in common solvents other than MeOH.

5.3.2. The Synthesis of Methoxyamine-Substituted Fluorescein (130)

For further structural characterisation of the fluoresceinyl nitroxide 127, a

methoxyamine derivative 130 was synthesised, in a yield of 67 %, using the Fenton

reaction (Scheme 5.10). This compound 130 was also used as a fluorescence

comparison with the fluoresceinyl nitroxide 127. The work-up of the methoxyamine

130 mirrored that of the nitroxide 127, whereby isolation of the spirolactone was

facilitated by precipitation by addition of HCl to a basic (NaOH) solution of the

product. Some loss of product may have occurred due to low solubility in solvents

other than MeOH, which makes isolation and purification from unreacted starting

material somewhat troublesome.

OHO OH

O

ONO

OHO OH

O

ONO

FeSO4.7H2OH2O2

DMSORT, 30 min

127 130 67 %

Scheme 5.10: Synthesis of fluorescein methoxyamine 130

5.3.3. Fluorescence Data of Fluoresceinyl Nitroxide and Methoxyamine

The comparison of fluorescence intensity of the fluoresceinyl nitroxide 127 and its

methoxyamine analogue 130 shows fluorescence suppression by the nitroxide radical

(Figure 5.1).


118

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

400 450 500 550 600

Wavelength/nm

Abs

orb

ance

0

50

100

150

200

250

300

350

400

450 550 650

Wavelength/nmIn

ten

sity

/au


492 nm in 0.1M NaOH normalised to 1 µM

Due to the pH dependence of the fluorescence of fluorescein-based compounds 127

and 130 (shown in Figure 5.2) spectra were recorded in 0.1 M NaOH solution, where

fluoresceins are highly fluorescent and the quantum yield of the parent compound

fluorescein is known.192

0

50

100

150

200

250

300

0 7 14

pH

Inte

nsity

/au

Figure 5.2: The dependence of fluorescence emission intensity of 127 (―) and

130 (―) on pH


119

Fluorescein 125 (ФF = 0.93)192 in 0.1 M NaOH was used as a standard for the

calculation of quantum yields of both 127 and 130. Understandably, the quantum

yield of the fluoresceinyl methoxyamine 130 (ФF = 0.93) mirrored that of the

unsubstituted fluorescein 125. Whilst, as expected, the fluoresceinyl nitroxide 127

(ФF = 0.15) had a lower quantum yield than that of the methyl adduct 130, it is

significantly higher than that observed for other profluorescent nitroxides synthesised

during this project. In fact, the fluorescein nitroxide 127 possesses a quantum yield

greater than that of the stilbene 26 and phenanthrene 82 methoxyamines.

This elevated quantum yield of the nitroxide 127 may arise from the large distance

between the nitroxide and the fluorophore combined with inefficient quenching of

the highly fluorescent xanthene moiety by the nitroxide, possibly from a lack of co-

planarity between the fluorophore and nitroxide moieties.

Quantum yield comparison of the fluorescein based compounds 127 and 130 to the

highly fluorescent bis(phenylethynyl)anthracene (BPEA) nitroxide 131 and

methoxyamine 132, synthesised by K. Fairfull-Smith,193 shows that the nitroxide

tethered to the fluorescein quenches approximately 3-4 fold less than the BPEA

nitroxide, whilst the methoxyamines have similar quantum yields (Table 5.1). This

further illustrates the inefficient quenching of the fluorescein nitroxide. The decrease

in dynamic range between the nitroxide and methoxyamine may limit the potential

applications of the fluorescein nitroxide as there may be difficulty in distinguishing

when radical trapping or redox processes have taken place due to an inherently high

baseline fluorescence.

N R

131 R = O132 R = OMe


120

Table 5.1: Quantum yields of fluorescein (127 and 130) and BPEA (131 and

132) compounds.


127 0.15

130 0.93

125 0.93

131 0.04

132 0.95

Confocal microscopy (performed by B. Morrow, Queensland University of

Technology) of the fluoresceinyl nitroxide 127 (Figure 5.3) and methoxyamine 130

showed these compounds were prone to photobleaching.194 The fluorescein

compounds were found to localise in the cellular membrane,194 a property which may

aid in the investigation of lipid peroxidation. Flow cytometry studies, using the

fluoresceinyl nitroxide as a redox probe, have also shown promising results and are

currently being investigated by B. Morrow.194

Figure 5.3: Fluorescence image of 127 in HeLa cells194

5.3.4. Rhodamine Nitroxide Synthesis

After the fluorescein nitroxide had shown interesting fluorescence properties, the

synthesis of a rhodamine B analogue seemed a logical progression, as it may be


121

obtainable via similar synthetic protocols. Rhodamines are known to have longer

fluorescence emission wavelengths and also tend to be more photo-stable than

fluorescein-based compounds.195

OEt2N NEt2

134

OEt2N NEt2

133

CO2HO

O

Base

Acid

Cl

Scheme 5.11: The two forms of rhodamine B

Rhodamines contain basic amino-substituents on the fluorescent xanthene core rather

than acidic phenols, as is the case with fluoresceins. This substitution results in the

rhodamines, such as rhodamine B 133, being in the highly-fluorescent quinoidal

form 133 in acidic solution (which is the commonly isolated form of rhodamine B)

and the non-fluorescent spirolactone form 134 in basic media, which is also known

as rhodamine B base (Scheme 5.11). As these forms of rhodamines in acid/base

solutions are diametrically opposed to the fluoresceins, they can be seen to be

complementary, whereby when rhodamines are fluorescent, fluoresceins are not and

vice versa. However, in buffered solutions and biological systems the fluorescent

quinoidal structure is present for both groups of compounds.

5.3.5. The Attempted Synthesis of Rhodamine B Nitroxide (130) – Direct

Methodologies

After the success of using MeSO3H for the synthesis of the fluorescein nitroxide 127,

its use was again proposed for the synthesis of the analogous rhodamine B nitroxide

135. Unfortunately, heating the nitroxide anhydride 100 and 3-diethylaminophenol

136 in MeSO3H at 85 °C or 140 °C (Entries 1 and 2, Table 5.2) did not produce the

desired product. Only phenolic starting materials, reaction intermediates and

degradation products could be isolated from the reaction mixture.

Repeating the reaction in the more commonly used conc. H2SO4,196 at 140 °C (Entry

3, Table 5.2) also proved unsuccessful, resulting in the production of several dark-

purple/brown phenolic reaction intermediates and degradation products. None of the

desired compound could be isolated from the reaction mixture. More forcing reaction


122

conditions were proposed, as many compounds, proposed to be reaction

intermediates, were isolated from the MeSO3H and conc. H2SO4 reactions.

NEt2HO

2 equiv

OEt2N NEt2

CO2HN OO

O

O

N

O

136Cl

100

135

X

Table 5.2: Attempted direct syntheses of rhodamine B nitroxide 135

Entry Reagents/Conditions Yield (%)

1 MeSO3H,

85 °C, 24 h 0

2 MeSO3H,

140 °C, 24 h 0

3 H2SO4 (conc.),

140 °C, 4 h 0

4 No solvent, 180 °C, 4 h

0

A test reaction, based on the procedure reported by Sansone,197, 198 gave rhodamine B

133 in high yield by heating phthalic anhydride 33 and 3-diethylaminophenol 136 at

180 °C in the absence of a solvent. When these conditions were used for the

synthesis of the nitroxide, none of the desired rhodamine B nitroxide 135 was

isolated. Another product, proposed to be a mixture of two triply-charged rhodamine

B nitrones 137 and 138 (based on high resolution MS), was formed by degradation of

the nitroxide moiety. The nitrones were found to be extremely polar which made

isolation of 137 and 138 in high purity impossible.


123

O NEt2Et2N

N

O

CO2H

O NEt2Et2N

N

O

CO2H

137 138

The formation of the nitrones 137 and 138 as degradation products is conceivable

from the high temperature reaction as the unsubstituted nitrone TMINO 139 is

formed by thermolysis of TMIO 4.199

N O

139

From the attempted syntheses outlined above, it was concluded that the rhodamine B

nitroxide analogue 135 cannot be generated in a single step from the anhydride 95 as

was initially proposed. This is due to instability of the nitroxide functional group

under the rigorous reaction conditions required for the formation of the xanthene

ring.

5.3.6. Synthesis of Rhodamine B Methoxyamine (140)

Due to the problems associated with the direct synthesis of the rhodamine B

nitroxide 135, step-wise syntheses or protection group strategies were proposed.

Before these arduous syntheses were attempted, the rhodamine methyl trap 140 was

prepared to investigate the fluorescence properties of the proposed system. Heating

3-diethylaminophenol 136 and the methoxyamine anhydride 112 (see Chapter 4) at

180 °C gave the desired methyl trap rhodamine 140 in moderate yield (47 %)

(Scheme 5.12).


124

NEt2HO

180 °C, 4 h

2 equiv

OEt2N NEt2

CO2HN OO

O

O

N

O

136

Cl

112

140 47 %

Scheme 5.12: Synthesis of rhodamine B methoxyamine 140

Confocal microscopy (performed by B. Morrow, Queensland University of

Technology) confirmed the photostability of 140 (Figure 5.4).194 As photobleaching

of 140 was negligible, further synthetic methodologies were trialled for the synthesis

of the highly desirable analogous nitroxide 135.

Figure 5.4: Fluorescence image of 140 in HeLa cells194

5.3.7. Attempted Step-Wise Synthesis of Rhodamine B Nitroxide (135)

A step-wise procedure, based on the work of Corrie and co-workers200, 201 was

proposed for the synthesis of the rhodamine nitroxide 135, as both steps in the

synthesis are less harsh than those used for the successful synthesis of the

methoxyamine 140. Unfortunately, heating the nitroxide anhydride 100 and one

equivalent of 3-diethylaminophenol 136 in either refluxing toluene (110 °C) or

xylene (140 °C) did not produce the desired intermediate 141 (Scheme 5.13), with


125

only degradation products isolated from the reaction mixture. Since the first product

of the two-step synthesis could not be achieved in this instance, the step-wise

synthesis of 135 was abandoned.

NEt2HO

O

OHEt2N

CO2H

N OO

O

O

N

O

136

toluene or xylene reflux, 6 h

PPSEDMF

130 °C, 4 h

OEt2N NEt2

CO2H

N

O

Cl

100

141 0 %

135

NEt2HO

136X

Scheme 5.13: Attempted step-wise synthesis of rhodamine nitroxide 135

Interestingly, when a test reaction was performed using phthalic anhydride 33 the

analogous intermediate 142 could be isolated in high yield (85 %) (Scheme 5.14).

NEt2HO

1 equivO

OHEt2N

CO2HO

O

O

136

toluene reflux, 6 h

33142 85 %

Scheme 5.14: Synthesis of intermediate 142

This successful test reaction and those reported by the Corrie group200, 201 employ

phthalic anhydrides with electron-neutral or electron-poor aromatics. The reactivity

of the anhydride group present on the nitroxide anhydride 100 may be significantly

reduced with respect to Friedel-Crafts acylation, due to the high electron density of

the aromatic ring, which would in turn affect the reactivity of the anhydride.


126

NEt2HO

2 equivN OO

O

O

136

PPSEDMF

130 °C, 4 h

OEt2N NEt2

CO2H

N

O

Cl

100

135 0 %

X

Scheme 5.15: Unsuccessful synthesis of rhodamine B nitroxide 135 using PPSE

A one-step synthesis, also based on the work reported by the Corrie group,200 by

heating the nitroxide anhydride 100 and 3-diethylaminophenol 136 in DMF in the

presence of trimethylsilylpolyphosphate (PPSE) (analogous to the second-step in the

proposed method above) proved unsuccessful for the synthesis of 135 (Scheme 5.15).

As with the MeSO3H and H2SO4 reactions discussed above, the reaction did not go to

completion and instead gave products inconsistent with the formation of a rhodamine

analogue. This again may suggest that the anhydride is not reactive enough to acylate

the aromatic ring of the phenol effectively at lower temperatures. For the acylation to

take place effiently, harsh conditions seem to be required, which are incompatible

with the nitroxide moiety. This led to the proposal of protection group strategies.

5.3.8. Attempted Acetyl-Protection of the Nitroxide Moiety

Protection of the nitroxide with an acetyl group was proposed due to its expected

ease of removal via hydrolysis.

N OAcO

O

O

143 0 %

N OO

O

O

AcClpyridine

Pd/C, H2RT, 72 h

100

X

Scheme 5.16: Attempted synthesis of acetylated anhydride 143 from AcCl

Stirring the anhydride nitroxide 100 with acetyl chloride under a hydrogen

atmosphere in the presence of Pd/C and pyridine did not produce the desired product

143, instead only starting material was isolated after oxidation of the intermediate

hydroxylamine (see Scheme 5.16). Due to the low nucleophilicity of the


127

hydroxylamine, more forcing conditions were thought to be required for the

acetylation to take place.

The synthesis of 143 was subsequently attempted through a one-pot procedure

directly from the diacid 98. In this reaction acetic anhydride was utilised to produce

the anhydride and for the attempted acetylation of the nitroxide. After synthesis of

the anhydride by refluxing in acetic anhydride for 4 h, Pd/C catalyst was added to the

reaction which was subsequently heated at 60 °C, under hydrogen for 2h.

Surprisingly, rather than giving the desired product 143 or the unprotected anhydride

nitroxide 100, an amide 144 was produced (Scheme 5.17).

NO

O

O

144 37 %

CH3

O

N O

1. Ac2O, reflux 4 h

2. Pd/C, H2Ac2O, 60 °C, 16 h

98

HO2C

HO2C

Scheme 5.17: Synthesis of amide 144 from Ac2O and H2

Presumably, the nitroxide is reduced by H2 to the secondary amine which

subsequently reacts with the Ac2O to give the amide 144. The heightened

nucleophilicity of the secondary amine, compared with that of an analogous

hydroxylamine may explain the absence of any of the desired product 143.

With the amide in hand, which could potentially be hydrolysed and oxidised to the

nitroxide, the synthesis of a rhodamine B analogue 145 was undertaken. Heating the

amide 144 with 3-diethylaminophenol 136 in a melt at 180 °C gave the rhodamine

amide 145 in high yield of 74 % (Scheme 5.18).


128

NEt2HO

180 °C, 4 h

2 equiv

OEt2N NEt2

CO2HNO

O

O

N

136

Cl

CH3

CH3

O

O

144

145 74 %

Scheme 5.18: Synthesis of rhodamine amide 145

However, basic hydrolysis with KOH in H2O/EtOH, similar to that used for the

synthesis of DCTMIO 98, resulted in the complete destruction of the xanthene

moiety giving non-fluorescent products and none of the desired compound (Entry 1,

Table 5.3).

OEt2N NEt2

CO2H

N

Cl

O

145

OEt2N NEt2

CO2H

N

Cl

146

H

X

Table 5.3: Attempted hydrolysis of rhodamine B amide 145

Entry Reagents Conditions Yield 146 (%)

1 2.5 M KOH, EtOH Reflux, 16 h 0

2 12.5 M H2SO4 Reflux, 16 h 0

Strongly basic conditions have previously been shown by Woodroofe et al.202 to

cleave fluorescein-based compounds, such as 147, to form non-fluorescent


129

intermediates, such as 148 (Scheme 5.19). An analogous process to this may have

occurred during the attempted amide hydrolysis.

OAcO OAc

OHO2C

Cl

O

O

OHAcO

HO2C

Cl

Cl CO2H

147 148 89 %

50 % NaOH

165 °C, 30 min

Scheme 5.19: Base cleavage of 147 with base reported by Woodroofe et al.202

Heating in 70 % H2SO4, a technique often used for the hydrolysis of troublesome

amides,171 failed to produce the desired amine 146 with the starting material

undergoing no observable change (Entry 2, Table 5.3). The hydrolysis of other

isoindoline amides, such as 149, has been shown to be problematic by co-workers in

the research group.193

N

O

HO2C

HO2C

149

Since the acetylated nitroxide could not be synthesised and the amide could not be

hydrolysed, another synthetic strategy was required.

5.3.9. Buchwald-Hartwig Amination

After limited success using protection group strategies, an entirely new approach was

envisioned for the synthesis of the rhodamine nitroxide. As the fluorescein nitroxide

127 already possesses the desired xanthene ring structure, conversion of the

fluorescein 127 to the rhodamine 135 by functional group interconversion was

proposed. Due to their ease of synthesis from phenols and ability to act as leaving

groups in Pd-catalysed Buchwald-Hartwig aminations,203 the synthesis of a triflate

analogue of fluorescein was undertaken to facilitate the conversion.


130

The Pd-catalysed amination of aromatic “halides” (iodides, bromides, chlorides,

sulfonates) was first discovered in 1995 concurrently by the Buchwald204 and

Hartwig205 groups and is commonly referred to as Buchwald-Hartwig amination.

Since these initial studies, the reaction has been further developed by these research

groups to expand the utility of the reaction.206, 207

The proposed catalytic cycle of the Pd-catalysed amination is shown below (Scheme

5.20).206 Oxidative addition of the aryl halide to the Pd(0) catalyst gives an

intermediate Pd(II) complex. Two-step substitution of the amine for the halide gives

another Pd(II) species. Reductive elimination gives the aromatic amine product and

regenerates the Pd(0) catalyst. In some instances, imine side products can be formed

via β-hydride elimination.

ArX

Precursor

[PdII]X

Ar

[PdII]L

[Pd0]L

L

L

L

X

ArNH

R2

HNR1(R2)

R1Base

Base-HX

[PdII]L L

ArN

R2R1

ArNR1(R2)

Oxidative Addition


Halide Elimination

Amine Substitution

beta-Hydride EliminationImine Side

Products

Scheme 5.20: The reaction mechanism of the Buchwald-Hartwig amination206

To test the viability of this synthetic route, test reactions converting fluorescein 125

to rhodamine B 133 via the triflate 150 were undertaken (Scheme 5.21). The

synthesis of the model triflate 150 was achieved by alteration of a synthetic

procedure for the production of sulfonylated fluoresceins reported by Maeda et al.187

Stirring fluorescein 125 and trifluoromethanesulfonyl chloride (triflic chloride) with

Et3N in DCM gave the fluorescein triflate 150 in high yield of 83 % (Scheme 5.21).


131

OEt2N NEt2

CO2HCl

OHO OH

O

O

OO O

O

O

S S

O

OCF3

O

OF3C

ON N

CO2HCl

triflic chloride

Et3N, DCMRT, 6 h

Et2NH, Pd(OAc)2, Cs2CO3, rac-BINAP,

toluene or dioxane100 °C, 16 h

pyrrolidine, Pd(OAc)2, Cs2CO3, rac-BINAP, dioxane

100 °C, 16 h

125 150 83 %

133 <5 %151 79 %

Scheme 5.21: Synthesis of rhodamines 135 and 151 via Pd-catalysed amination

Buchwald-Hartwig amination208 of the triflate 150 with Et2NH, for the synthesis of

133, was unsuccessful in both toluene and dioxane using the conditions previously

reported by Ahman and Buchwald208 for the Pd-catalysed amination of aryl triflates.

When the amine was changed to pyrrolidine, to give the pyrrolidino rhodamine 151,

the yield increased substantially to 79 %, with dioxane as the solvent (Scheme 5.21).

The NMR data of 151 were in agreement to that previously reported by Woodroofe

et al.202 This difference in the yield between the two reactions can be rationalised by

the fact that cyclic amines, like pyrrolidine, often give higher yields than acyclic

amines under Buchwald-Hartwig conditions,209 due to greater accessibility of the

cyclic amine allowing it to react more efficiently.

Using the same conditions described above, the nitroxide triflate 153 was synthesised

and isolated in high yield (85 %), (triflic chloride, Et3N, DCM, RT, 6 h) (Scheme

5.22).


132

ON N

CO2H

N

O

Cl

OHO OH

NO

O

O

OO O

NO

O

O

S S

O

OCF3

O

OF3Ctriflic chloride

Et3N, DCMRT, 6 h

pyrrolidine, Pd(OAc)2, Cs2CO3, rac-BINAP, dioxane

100 °C, 16 h

127 153 85 %

152 24 %

Scheme 5.22: Synthesis of rhodamine nitroxide 152 via triflate nitroxide 153

Subsequent Pd-catalysed amination with pyrrolidine, in the presence of Pd(OAc)2,

rac-BINAP and Cs2CO3, in dioxane at 100 °C under argon, gave the desired

pyrrolidinorhodamine nitroxide 152 in low yield of 24 % (Scheme 5.22). The low

yield may arise from physical losses during work-up due to the small scale of the

reaction. Optimisation of Buchwald-Hartwig amination in the presence of nitroxides

may reveal more favourable conditions for the technique and may be investigated in

the future.

The synthesis of a methoxyamine analogue 154, for further structural

characterisation of the nitroxide 152, proved to be unsuccessful. The nitroxide 152

was found to have low solubility in the dimethylsulfoxide (DMSO) solvent used for

the methyl trap reaction and full conversion proved impossible. A compound that had

TLC retention consistent with that expected for the methyl trap was isolated in trace

quantities (Scheme 5.23). After purification by preparatory TLC this compound was

subjected to 1H NMR analysis. Unfortunately, no conclusive structural data could be

obtained from the NMR due to the small quantity of product isolated. Due to this, the


133

-0.02

0

0.02

0.04

0.06

0.08

0.1

400 500 600 700

Wavelength/nm

Ab

sorb

an

ce

0

500

1000

1500

2000

2500

540 590 640 690

Wavelength/nm

Inte

nsi

ty/a

u

fluorescence of the nitroxide 152 was correlated against the unsubstituted

pyrrolidinorhodamine test compound 151 in place of the methoxyamine 154.

ON N

CO2H

N

O

Cl

152

ON N

CO2H

N

O

Cl

154 <5%

FeSO4.7H2OH2O2

DMSORT, 30 min

Scheme 5.23: Attempted synthesis of rhodamine methoxyamine 154

5.3.10. Fluorescence Data of Pyrrolidinorhodamine Nitroxide (152)

The comparison of fluorescence intensity of the pyrrolidinorhodamine nitroxide 152

and the diamagnetic unsubstituted pyrrolidinorhodamine 151 illustrates the strong

fluorescence suppression of nitroxide radical (Figure 5.5).


544 nm in EtOH normalised to 1 µM


134

Fluorescence spectra were recorded in EtOH and quantum yields of 151 and 152

were calculated using rhodamine B 133 (ФF = 0.71)210 as the standard. Similar to that

of the fluorescein nitroxide 127, the pyrrolidinorhodamine nitroxide 152 (ФF = 0.12)

had a relatively high quantum yield in comparison to profluorescent nitroxides

synthesised via Heck and Sonogashira methodology. The quantum yield of 152 was

found to be substantially lower than that of the diamagnetic analogue 151 (ФF =

0.84), as expected.

Table 5.4: Quantum yields of rhodamine compounds (133, 151 and 152)


152 0.12

151 0.84

133 0.70

The quantum yield of pyrrolidinorhodamine 151 was found to be larger than that of

rhodamine B 133. Increased rigidity of the pyrrolidine ring in comparison to the

diethylamino groups on the xanthene moiety may produce this effect, similar to that

observed for rhodamine 101 155,211 but not to such a marked degree.

ON N

CO2H Cl

155


The first synthesis of two xanthene-based profluorescent nitroxides was successfully

achieved in moderate to low yield. Whilst the fluorescein nitroxide 127 could be

synthesised directly from the nitroxide anhydride 100 via condensation with

resorcinol, the analogous reaction using 3-diethylaminophenol to form a rhodamine

B nitroxide 135 proved unsuccessful, using a number of methodologies. Synthesis of

135 using nitroxide protecting groups also gave limited success. A related


135

pyrrolidinorhodamine nitroxide 152 was eventually able to be synthesised via Pd-

catalysed Buchwald-Hartwig amination of a fluorescein triflate nitroxide 153, which

was initially synthesised from the novel fluorescein nitroxide 127. The synthesis of

the pyrrolidinorhodamine nitroxide 152 is the first example of Buchwald-Hartwig

amination being applied to isoindoline nitroxide systems.

The fluorescein 127 and rhodamine 152 nitroxides have relatively high quantum

yields for profluorescent systems, due to the presence of strongly fluorescent

xanthene fluorophores. In comparison to diamagnetic analogues, the nitroxides

showed strong fluorescence suppression. The fluorescence excitation and emission

wavelengths of the xanthene-based profluorescent nitroxides are ideally suited to

biological applications due to the lack of background signals from the systems under

study, at longer wavelengths. These profluorescent nitroxides may potentially be

applied as probes for the detection of ROS in cells via flow cytometry and

fluorescence microscopy.

5.5. Experimental

All experimental details are as previously described in Chapters 2, 3 and 4 where

relevant.

Resorcinol (98 %), 3-diethylaminophenol (97 %), methanesulfonic acid (99.5 %),

trifluoromethanesulfonyl chloride (99 %), caesium carbonate (99 %), (±)-2,2'-

bis(diphenylphosphino)-1,1'-binaphthalene (rac-BINAP) (98 %) and anhydrous

dioxane (99.8 %) were purchased from Sigma/Aldrich.

All solutions for UV/Vis and fluorescence analysis were prepared in 0.1 M NaOH or

ethanol and measured using 10 mm quartz fluorescence cuvettes.


136

5.5.1. 5-Carboxy-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-1,1,3,3-


NO

O

OHO OH

O

127

Nitroxide anhydride 100 (100 mg, 0.384 mmol) was added to a solution of resorcinol

126 (85 mg, 0.772 mmol, 2 equiv) in MeSO3H (0.78 mL). The mixture was heated at

85 °C for 24 h and subsequently poured onto ice/water. The solution was treated with

NaOH (5 M), to ensure total dissolution of the crude product. The solution was

acidified with dropwise addition of conc. HCl and the product was partitioned into

Et2O (3 × 30 mL) and dissolved by addition of MeOH (~20 mL). The solution was

filtered to remove insoluble material, dried (Na2SO4) and the solvent was removed

under reduced pressure, to give the crude product 127. Purification by column

chromatography (20 % EtOH/80 % EtOAc with 0.1 % v/v AcOH), gave 127 as a

dark orange crystalline solid (158 mg, 92 %) mp > 300 °C (decomp.); νmax (ATR-

FTIR): 3342 (OH), 2977 and 2927 (alkyl CH), 2856 (aryl CH), 1733 (C=O), 1442

(NO) cm-1; +EI MS found M+ 444.1445 (0.5 ppm from calc. mass of C26H22NO6•):

m/z 444 (M+, 100 %), 430 (43), 414 (37), 369 (48) 329 (55) (see HPLC 12,

Appendix 1).

5.5.2. 5-Carboxy-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-methoxy-

1,1,3,3-tetramethylisoindoline (130)

NO

O

OHO OH

O

130

A solution of fluoresceinyl nitroxide 127 (20 mg, 0.045 mmol) in DMSO (1.4 cm3)

had FeSO4·7H2O (25 mg, 0.090 mmol, 2 equiv) and H2O2 (30 % aqueous solution, 9


137

µL) added. The reaction mixture was stirred under argon for 30 min at room

temperature after which it was poured onto NaOH (2 M, 10 cm3) and washed with

Et2O (2 × 10 cm3) and CHCl3 (2 × 10 cm3) to remove DMSO present. The aqueous

phase was acidified with dropwise addition of conc. HCl and partitioned into Et2O

(30 cm3) to remove aqueous salts. MeOH (~5 mL) was added to dissolve the product

and the mixed solvent removed under reduced pressure to give the crude product

130. Purification by column chromatography (20 % EtOH/80 % EtOAc with 0.1 %

v/v AcOH), gave the fluoresceinyl methoxyamine 130 as a dark orange solid (14 mg,

0.030 mmol, 67 %) mp > 300 °C (decomp.); δH: (MeOH-d4); 1.38 (6H, br s, CH3),

1.54 (6H, br s, CH3), 3.78 (3H, s, OCH3), 6.56 (2H, dd, J 2.4 and 8.8 Hz, 2’-H and

7’-H), 6.67 (2H, d, J 2.4 Hz, 4’-H and 5’-H), 6.72 (2H, d, J 8.8 Hz, 1’-H and 8’-H),

7.00 (1H, s, 7-H), 7.78 (1H, s, 4-H); νmax (ATR-FTIR): 3305 (OH), 2962 and 2925

(alkyl CH), 2852 (aryl CH), 1729 (C=O) cm-1; +ESI MS found (M+H)+ 460.174976

(2.3 ppm from calc. mass of C27H26NO6+): +EI MS m/z 459 (M+, 10 %), 444 (100),

369 (30).

5.5.3. [9-(6-Carboxy-2-methoxy-1,1,3,3-tetramethylisoindolin-5-yl)-6-

diethylamino-xanthen-3-ylidene]-diethyl-ammonium Chloride

(140)

N

O

CO2H

OEt2N NEt2

Cl

140

Methoxyamine anhydride 112 (60 mg, 0.218 mmol) and 3-diethylaminophenol 136

(80 mg, 0.484 mmol, 2.2 equiv) were heated at 180 °C for 4 h. The resultant mixture

was treated with dilute NH3 solution. The aqueous solution was then extracted with

benzene, which was back extracted with dilute HCl (2 M). Removal of the aqueous

solvent under reduced pressure gave the crude rhodamine B methoxyamine 140. The

residue was dissolved in EtOH after which slow addition of Et2O was used to

precipitate the inorganic impurities. Purification by column chromatography (20 %


138

EtOH/80 % EtOAc with 1 % v/v Et3N), gave 140 as a dark purple solid (62 mg,

0.102 mmol, 47 %) mp > 300 °C (decomp.); δH: (MeOH-d4); 1.30 (12H, t, J 7.0 Hz,

ethyl CH3), 1.46 (6H, br s, CH3), 1.55 (6H, br s, CH3), 3.67 (8H, q, J 7.0 Hz, ethyl

CH2), 3.81 (3H, s, OCH3), 6.96 (2H, d J 2.2, 4’-H and 5’-H), 7.02 (2H, dd, J 2.2 and

9.2 Hz, 2’-H and 7’-H), 7.11 (1H, s, 7-H), 7.19 (2H, d, J 9.2 Hz, 1’-H and 8’-H),

8.05 (1H, s, 4-H); νmax (ATR-FTIR): 3347 (OH), 2977 and 2931 (alkyl CH), 2856

(aryl CH), 1714 (C=O) cm-1; +ESI MS found M+ 570.331400 (3.1 ppm from calc.

mass of C35H44N3O4+): m/z 570 (M+, 100 %).

5.5.4. 6-Acetyl-5,5,7,7-tetramethyl-6,7-dihydro-5H-2-oxa-6-aza-s-

indacene-1,3-dione (144)

O N

O

O

O

144

5,6-Dicarboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl 98 (100 mg, 0.360 mmol) was

dissolved in Ac2O (10 mL) and heated under argon at reflux for 4 h. The reaction

mixture was cooled, Pd/C (10 %, 10 mg) was added and the mixture was heated

under H2 at 60 °C for 2 h. The solvent was removed under reduced pressure to give

the anhydride amide 144 as a white crystalline solid (37 mg, 0.132 mmol, 37 %) mp

254-256 °C (decomp.); δH: 1.84 and 1.86 (12H, split by restricted rotation of amide,

CH3), 2.35 (3H, s, amide CH3), 7.81 (2H, s, 4-H and 7-H); νmax (ATR-FTIR): 3014

and 2977 (alkyl CH), 2942 (aryl CH), 1845 and 1778 (anhydride C=O), 1635 (amide

C=O) cm-1; +EI MS found M+ 287.1160 (0.8 ppm from calc. mass of C16H17NO4):

m/z 287 (M+, 3 %), 272 (43), 230 (100).


139

5.5.5. [9-(2-Acetyl-6-carboxy-1,1,3,3-tetramethyl-2,3-dihydro-1H-

isoindol-5-yl)-6-diethylamino-xanthen-3-ylidene]-diethyl-

ammonium Chloride (145)

N

CO2H

OEt2N NEt2

Cl

O

145

Anhydride amide 144 (174 mg, 0.605 mmol) and 3-diethylaminophenol 136 (201

mg, 1.21 mmol, 2.0 equiv) were heated at 180 °C for 4 h. The resultant mixture was

treated with dilute NH3 solution. The aqueous solution was then extracted with

benzene, which was back extracted with dilute HCl (2 M). Removal of the aqueous

solvent under reduced pressure gave the crude rhodamine B amide 145. The residue

was dissolved in EtOH after which slow addition of Et2O was used to precipitate the

inorganic impurities. Purification by column chromatography (20 % EtOH/80 %

EtOAc with 1 % v/v Et3N), gave the rhodamine amide 145 as a dark purple solid

(278 mg, 0.450 mmol, 74 %). mp > 300 °C (decomp.); δH: (MeOD-d4); 1.29 (12H, t,

J 7.0 Hz, ethyl CH3), 1.92 (12H, s, CH3), 2.33 and 2.37 (3H, split by restricted

rotation of amide, amide CH3), 3.66 (8H, q, J 7.0 Hz, ethyl CH2), 6.93 (2H, d J 2.2,

4’-H and 5’-H), 7.01 (2H, d, J 9.4 Hz, 1’-H and 8’-H), 7.16 and 7.20 (1H, split by

restricted rotation of amide, 7-H), 7.27 (2H, dd, J 2.2 and 9.4 Hz, 2’-H and 7’-H),

7.96 and 7.98 (1H, split by restricted rotation of amide, 4-H); νmax (ATR-FTIR):

3382 (OH), 2971 and 2929 (alkyl CH), 2888 (aryl CH), 1758 (C=O) cm-1; +ESI MS

found M+ 582.33289 (0.5 ppm from calc. mass of C36H44N3O4+): m/z 582 (M+, 100

%).


140

5.5.6. Bis(trifluoromethanesulfonyl)fluorescein (150)

O

OO O

O

SS

O

OCF3

O

OF3C

150

To a solution of fluorescein 125 (500 mg, 1.5 mmol) in DCM (2 mL), Et3N (0.46

mL, 3.3 mmol, 2.2 equiv) was added. The resulting solution was stirred at 0 °C for 5

min after which trifluoromethanesulfonyl chloride was added (558 mg, 350 µL, 3.3

mmol, 2.2 equiv). The mixture was stirred at room temperature for 6 h, after which

DCM (100 mL) was added and washed with 2 M HCl (2 × 100 mL) and brine (100

mL), dried (Na2SO4) and solvent removed under reduced pressure. The crude product

was purified by column chromatography (98 % DCM/2 % Acetone) to give the

fluorescein triflate 150 as a white crystalline solid (745 mg, 1.25 mmol, 83 %) mp

110 – 113 °C ; δH: (CDCl3); 6.98 (2H, d, J 8.8 Hz, 1’-H and 8’-H), 7.05 (2H, dd, J

2.3 and 8.8 Hz, 2’-H and 7’-H), 7.21 (1H, m, 6-H), 7.32 (2H, d, J 2.3 Hz, 4’-H and

5’-H), 7.71 (1H, m, 5-H), 7.77 (1H, m, 7-H), 8.10 (1H, m, 4-H); δC: (CDCl3); 80.1

(spiro C-1), 110.7 (C-4’ and C-5’), 117.1 (C-2’ and C-7’), 117.7 (C-8a’ and C-8b’),

119.3 (C-5), 120.3 (C-7), 123.8 (C-1’ and C-8’), 125.6 (C-4), 125.7 (C-3), 130.0 (C-

6), 130.7 (C-8), 135.9 (C-3’ and C-6’), 150.2 (CF3), 152.1 (C-4a’ and C-4b’), 168.5

(C=O C-2); νmax (ATR-FTIR): 2971 and 3014 (alkyl CH), 1754 (C=O), 1423 and

1200 (SO3) cm-1.

5.5.7. 1-[9-(2-Carboxy-phenyl)-6-pyrrolidin-1-yl-xanthen-3-ylidene]-

pyrrolidinium Chloride (Pyrrolidinorhodamine, 151)

ON N

CO2H

Cl

151


141

Bis(trifluoromethanesulfonyl)fluorescein 150 (50 mg, 0.084 mmol), Pd(OAc)2 (2

mg, 0.0083 mmol, 10 mol%), rac-BINAP (8 mg, 0.013 mmol, 15 mol%), Cs2CO3

(76 mg, 0.235 mmol, 2.8 equiv) and pyrrolidine (18 mg, 21 µL, 0.252 mmol, 3

equiv) were dissolved in anhydrous dioxane (0.16 mL) and heated under argon at

100 °C for 16 h. The reaction mixture was cooled, HCl (2 M, 15 mL) added and then

extracted with Et2O (2 × 30 mL). The aqueous layer was evaporated under reduced

pressure and the residue taken up in EtOH (~15 mL). Et2O was added to precipitate

inorganic salts, the solution was filtered and solvent removed under vacuum. The

crude product was purfied by preparatory TLC (80 % EtOAc/20 % EtOH with 1 %

v/v Et3N) to give the pyrrolidinorhodamine 151 as a dark purple solid (31 mg, 0.066

mmol, 79 %) mp > 300 °C (decomp.); δH: (DMSO-d6); 1.82 (8H, m, CH2), 3.08 (8H,

m, CH2), 6.81 (2H, d, J 2.1 Hz, 4’-H and 5’-H), 6.92 (2H, dd, J 2.1 and 9.3 Hz, 2’-H

and 7’-H), 7.00 (2H, d, J 9.3 Hz, 1’-H and 8’-H), 7.45 (1H, m, 4-H) 7.80 (1H, m, 7-

H), 7.86 (1H, m, 6-H), 8.10 (1H, m, 4-H). νmax (ATR-FTIR): 3490 (OH), 3095 and

2989 (alkyl CH), 2883 (aryl CH), 1716 (C=O) cm-1. These data are in agreement

with that previously reported by Woodroofe et al.202

5.5.8. Bis(trifluoromethanesulfonyl)fluoresceinyl Nitroxide (153)

NO

O

OO O

O

SS

O

OCF3

O

OF3C

153

To a solution of fluoresceinyl nitroxide 127 (50 mg, 0.112 mmol) in DCM (0.15

mL), Et3N (34 µL, 2.44 mmol, 2.2 equiv) was added. The resulting solution was

stirred at 0 °C for 5 min after which trifluoromethanesulfonyl chloride was added (42

mg, 26 µL, 0.772 mmol, 2 equiv). The mixture was stirred at room temperature for 6

h, after which DCM (20 mL) was added and the combined organic phases were

washed with 2 M HCl (2 × 20 mL) and brine (20 mL), dried (Na2SO4) and solvent

removed under reduced pressure. The crude product was purified by column

chromatography (98 % DCM/2 % Acetone) to give the fluorescein triflate nitroxide


142

153 as a white solid (67.4 mg, 0.095 mmol, 85 %); +ESI MS found M+ 709.050548

(0.8 ppm from calc. mass of C28H21 F6S2NO10•).

5.5.9. 1-[9-(6-Carboxy-1,1,3,3-tetramethylisoindolin-2-yloxylyl)-6-

pyrrolidin-1-yl-xanthen-3-ylidene]-pyrrolidinium Ch loride (152)

N

O

ON N

CO2H

Cl

152

Bis(trifluoromethanesulfonyl)fluorescein nitroxide 153 (35 mg, 0.049 mmol),

Pd(OAc)2 (1.2 mg, 0.005 mmol, 10 mol%), rac-BINAP (4.8 mg, 0.008 mmol, 15

mol%), Cs2CO3 (45 mg, 0.138 mmol, 2.8 equiv) and pyrrolidine (10.7 mg, 12.5 µL,

0.150 mmol, 3 equiv) were dissolved in anhydrous dioxane (0.1 mL) and heated

under argon at 100 °C for 16 h. The reaction mixture was cooled, HCl (2 M, 15 mL)

was added and the combined acidic phases were washed with Et2O (2 × 20 mL). The

aqueous solvent was removed under reduced pressure and the residue taken up in

EtOH (~10 mL). Et2O was added to precipitate inorganic salts, the solution was

filtered and solvent removed under vacuum. The crude product was purfied by

preparatory TLC (80 % EtOAc/20 % EtOH with 1 % v/v Et3N) to give the

pyrrolidinorhodamine nitroxide 152 as a dark purple solid (7 mg, 0.012 mmol, 24 %)

mp > 300 °C (decomp.); νmax (ATR-FTIR): 3421 (OH), 2979 and 2927 (alkyl CH),

2888 (aryl CH), 1743 (C=O) cm-1; +ESI MS found M+ 551.279163 (1.4 ppm from

calc. mass of C34H37N3O4•+) (see HPLC 13, Appendix 1).


Fluorescence quantum yield measurements for fluorescein nitroxide 127 and

methoxyamine 130 were performed in 0.1 M NaOH using fluorescein 125 (ФF =

0.93) as the standard. Quantum yields for rhodamine nitroxide 152 and

pyrrolidinorhodamine 151 were performed in EtOH using rhodamine B 133 (ФF =


143

0.71) as the standard. Stock solutions of compounds 125, 127, 130, 133, 151 and 152

(approximately 1 mg/100 mL, measured accurately, exact concentrations listed

below) were diluted using analytical glassware to give four solutions of decreasing

concentration, ensuring that the UV/Vis maximum of the highest concentration did

not exceed 0.1 absorbance units at the fluorescence excitation wavelength

(fluoresceins 492 nm or rhodamines 544 nm). The fluorescence detector voltage was

set at 500 V for the fluoresceins 127 and 130 and 535 V for the rhodamines 151 and

152. Total fluorescence emission was plotted against UV/Vis absorbance to give a

straight line with gradient (m), which was ratioed against the anthracene standard,

giving the quantum yield (ФF).

y = 395387x + 17161

R2 = 0.997

y = 396487x - 1524.1

R2 = 0.9946

y = 62210x - 25.526

R2 = 0.985

0

5000

10000

15000

20000

25000

30000

35000

40000

-0.02 0 0.02 0.04 0.06 0.08

UV-Vis Absorbance

Flu

ores

cenc

e In

tens

ity/a

u


127

125 130

Figure 5.6: Quantum yield measurements of 127 and 130 at 492 nm in 0.1 M

NaOH at 500 V


144

y = 40400x + 1113.9

R2 = 1

y = 231066.76x - 1323.03

R2 = 0.98

y = 272030x + 766.7

R2 = 0.9831

0

5000

10000

15000

20000

25000

30000

0.005 0.02 0.035 0.05 0.065 0.08 0.095

UV-Vis Absorbance

Flu

orec

ence

Inte

nsity

/au


152

133

151

Figure 5.7: Quantum yield measurements of 151 and 152 at 544 nm in EtOH at

535 V

Fluorescein (125)

Stock solution 125 (0.96 mg, 0.00289 mmol, 0.0289 mM). Diluted to give solutions

of 1.156, 0.867, 0.578 and 0.289 µM; m = 395387.

Fluoresceinyl Nitroxide (127)


of 2.092, 1.569, 1.046 and 0.523 µM; m = 62210; ФF = 0.93(62210/395387) = 0.15.

Fluoresceinyl Methoxyamine (130)


of 2.156, 1.616, 1.077 and 0.539 µM; m =396487; ФF = 0.93(396487/395387) = 0.93.

Rhodamine B (133)


of 0.388, 0.254, 0.169 and 0.085 µM; m = 231066.


145

Pyrrolidinorhodamine Nitroxide (152)


of 7.324, 5.859, 5.127 and 4.394 µM; m = 40400; ФF = 0.71(40400/231066) = 0.12.

Pyrrolidinorhodamine (151)


of 1.516, 1.137, 0.758 and 0.379 µM; m = 272030; ФF = 0.71(272030/231066) =

0.84.

Chapter 6 – Conclusions and Future Work

146

6. CONCLUSIONS AND FUTURE WORK

6.1. Conclusions

Throughout the tenure of this research project, new synthetic strategies, including

Pd-catalysed Heck and Sonogashira couplings, have been developed for the synthesis

of novel isoindoline nitroxides. These new strategies have led to the synthesis and

characterisation of 24 novel isoindoline nitroxides and 18 related precursors and

derivatives.

Palladium-catalysed Heck coupling of brominated nitroxides (16 and 21) yielded

novel nitroxides (17, 18, 22 and 23) bearing extended alkene conjugation. This is the

first example of Heck coupling being performed on the isoindoline class of

nitroxides. After optimisation of the reaction conditions, moderate to high yields of

the nitroxides were isolated. Due to the electron-rich nature of the nitroxide-

substituted functionalised aryl bromides, more forcing reaction conditions than those

commonly found in the literature were required to obtain acceptable yields. The

profluorescent nitroxides (18 and 23) displayed a substantial increase in fluorescence

intensity after radical trapping. The magnitude of the fluorescence quenching

between the nitroxides and the corresponding diamagnetic analogues may be related

to the near co-planarity of the nitroxide moiety and the fluorophore, allowing for

substantial electronic overlap, and the close proximity of the fluorophore to the

nitroxide moiety. Hydrolysis of the methyl esters of the profluorescent nitroxides (18

and 23) furnished the corresponding carboxylic acids (26 and 27), which showed

improved water solubility, albeit in buffered or basic solution.

The first examples of Pd-catalysed Sonogashira couplings were performed on the

isoindoline class of nitroxides during the current investigation. The use of traditional

Sonogashira coupling techniques, which employ a CuI co-catalyst for coupling,

bromo-substituted nitroxides with alkynes, proved to be unsuccessful. Cross-

coupling reactions, between bromo-nitroxides and alkynes, were achieved using

copper-free conditions, although the products were obtained in low yields. Attempts

to optimise these reaction conditions proved unsuccessful. The synthesis of an iodo-

nitroxide (64) and subsequent Sonogashira coupling allowed for the synthesis several


147

alkyne-substituted nitroxides (55, 56 and 63) in high yield. The vast improvement in

yield may be due to accelerated oxidative addition of the aryl iodide, compared to the

analogous aryl bromide. Acceleration of other favoured steps in the catalytic cycle,

such as the reductive elimination due to stability of iodide (I-) as a leaving group,

may also play a part. An ethynyl-nitroxide (70) was successfully synthesised from

the protected acetylene nitroxides (55 and 63). Subsequent Sonogashira coupling

with aryl iodides gave naphthalene and phenanthrene substituted profluorescent

nitroxides (72 and 74). An ethyne-linked dinitroxide (76) was synthesised using

similar synthetic methodology, but was isolated in moderate yield. The reduced

reactivity of the iodo nitroxide (64), in comparison to the polyaromatic iodides, led to

formation of a butadiyne-linked dimer (75) as an undesired side-product, produced

via a competing oxidative homocoupling reaction. The butadiyne-linked dinitroxide

(75) could be selectively synthesised in high yield by Eglinton coupling. The ethyne-

linked profluorescent nitroxides (72 and 74) were found to display strong

fluorescence suppression when compared to diamagnetic analogues, again possibly

due to close proximity of the fluorophore and the nitroxide moiety.

The profluorescent nitroxides synthesised through Heck and Sonogashira couplings

have the potential for use as probes in the study of polymer degradation. Due to their

low polarity, they are most suited for non-polar systems, such as polypropylene. The

magnitude of fluorescence suppression displayed by these compounds will aid in the

detection of radical related degradation processes via fluorescence spectroscopy.

A dicyano-substituted isoindoline nitroxide (102) was successfully synthesised

through Pd-catalysed cyanation of a dibrominated nitroxide (21). After limited

success using the literature procedure, a technique which exploits lower Pd-catalyst

loadings and a non-toxic cyanide source was investigated. Whilst this technique had

the aforementioned advantages, it was found to give variable yields of the desired

dinitrile. Attempts to optimise the reaction to overcome these problems proved

unsuccessful; therefore to obtain sufficient material for further elaboration, the

reaction was required to be repeated several times.

The synthesis of a water-soluble dicarboxy nitroxide (98) was successfully achieved

via base hydrolysis of the dinitrile nitroxide (102). The diacid was subsequently


148

converted to an anhydride (100) by heating in acetic anhydride. The anhydride was

in turn converted to an imide (101) by heating in the presence of urea. Hofmann

rearrangement of the imide resulted in an unexpected brominated amino-carboxy

nitroxide (108). The observed electrophilic aromatic substitution may be facilitated

by the heightened electron density of the aromatic ring, induced by the presence of

the bis-t-alkyl substituents.

The dicarboxy and the brominated amino-carboxy nitroxides were subjected to a cell

survivability assay to ascertain their antioxidant properties with respect to the disease

Ataxia-Telangiectasia. Both compounds were found to have a protective effect

towards A-T cells exposed to radiation, with the data of each of these nitroxides

being within experimental error of the best acting compound to date (CTMIO, 66).

Whilst these two compounds were found to have some activity with respect to

survival of A-T cells, significant improvements are still required for the successful

treatment of this disease.

The first syntheses of profluorescent nitroxides containing the potent xanthene

fluorophore, isoindoline-based or otherwise, were achieved during this research

project. A fluoresceinyl nitroxide (127) was successfully prepared through the

condensation of the nitroxide anhydride (100) and resorcinol. The synthesis of an

analogous rhodamine nitroxide via similar methodologies proved unsuccessful.

Buchwald-Hartwig amination however furnished a pyrrolidinorhodamine nitroxide

(152), via a triflate-functionalised fluorescein (153) synthesised from the

fluoresceinyl nitroxide (127). Both the fluorescein and rhodamine substituted

nitroxides displayed fluorescence suppression when compared with diamagnetic

analogues, although the magnitude of these suppressions were significantly less than

those observed for the nitroxides synthesised through Heck or Sonogashira coupling

methodologies. This could be due to a combination of several factors, which may

include an increased distance between the nitroxide moiety and the xanthene

fluorophore and a lack of co-planarity between the two functionalities. These

compounds may find application as tools to for the investigation of redox status of

cellular systems, due to their water-solubility, long fluorescence excitation and

emission wavelengths and the high quantum yields of related diamagnetic species.


149

6.2. Future Work

Throughout this project several new areas of chemistry have been developed for the

preparation of structurally diverse isoindoline nitroxides. The success of Pd-catalysis

in the synthesis of aromatic functionalised nitroxides suggests that other Pd-catalysed

methodologies, such as Stille, Negishi and Suzuki couplings, may also be successful

in the future. Other transition metals, such as Cu or Zn, may also be utilised for

cross-coupling of nitroxide systems.

Investigation into ROS specificity of the xanthene-based profluorescent nitroxides is

required to enhance their potential for use in biological applications. The specificity

of fluorescent probes allows for the detection and investigation of desired species.

The discovery of which species induce the fluorescence response of these

profluorescent compounds and their subsequent application in cellular systems may

further the understanding of processes involving ROS.

The ethynyl nitroxide may allow spin-labelling of various substrates, either by

Sonogashira couplings or via other synthetic methodologies. Triazole syntheses from

the ethynyl nitroxide and substituted azides via Huisgen 1,3-dipolar cycloaddition,

the premier example of click chemistry, may be utilised to achieve this outcome (see

Scheme 6.1 below). The ability of this reaction to proceed selectively with almost

complete conversion, whilst generally requiring limited purification, makes this

technique particularly attractive.212

N O

N N N

R

N

N

N

N OR

+

NN

N

N O

R

Cu(I)

heat

Scheme 6.1: Proposed Huisgen 1,3-dipolar cycloaddition of ethynyl nitroxide

The xanthene-based nitroxides synthesised during this project possess a low level of

fluorescence quenching. The synthesis of analogues in which the nitroxide


150

functionality is directly connected to the xanthene fluorophore may increase the

magnitude of fluorescence quenching observed.

OX Y

N

O

HO2C

O

OH

X

OH

O

HO Y

N

O

+

X = OH or NR2 Y = OH or NR2

Scheme 6.2: Proposed synthesis of fused xanthene-substituted nitroxides

The synthesis of these compounds may be achieved by the condensation of 4,6-

disubstituted isoindoline nitroxides and the appropriately substituted benzophenones

(see Scheme 6.2). Whilst these compounds are attractive as the magnitude of

fluorescence quenching is expected to increase comparatively to the compounds

synthesised in this thesis, problems may arise during their synthesis. Preparation of

the required 4,6-disubstitued nitroxides may prove difficult, with the ability to

selectively functionalise at the desired positions required. Steric effects may also

hinder the proposed condensation reaction, disfavouring the production of the desired

xanthene compounds. Whilst these issues need to be considered, these compounds

may have enhanced properties and further the potential applications of xanthene-

based profluorescent nitroxides.

Chapter 7 -References

151

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Appendices

162

APPENDICES

Appendix 1: Selected HPLC Traces

HPLC 1: 5,6-Bis-[2-(4-methoxycarbonylphenyl)ethenyl]-1,1,3,3-


HPLC 2: 5-(2-Methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-2-yloxyl

(17)

HPLC 3: 5,6-Bis-(2-methoxycarbonylethenyl)-1,1,3,3-tetramethylisoindolin-2-

yloxyl (22)

HPLC 4: 5-[2-(Trimethylsilyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(55)

HPLC 5: 5-Ethynyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (70)

HPLC 6: 5-[2-(1-Naphthyl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (72)

HPLC 7: 5-[2-(9-Phenanthryl)ethynyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl

(74)

HPLC 8: 1,2-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]ethyne (76)

HPLC 9: 1,4-Bis-[5,5'-(1,1,3,3-tetramethylisoindolin-2-yloxylyl)]-1,3-

butadiyne (75)

HPLC 10: 1,1,3,3-Tetramethyl-5,7-dioxo-3,5,6,7-tetrahydro-1H-pyrrolo[3,4-

f]isoindol-2-yloxy (101)

HPLC 11: 4-Bromo-5-amino-6-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl

(108)

HPLC 12: 5-Carboxy-6-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-1,1,3,3-


HPLC 13: 1-[9-(6-Carboxy-1,1,3,3-tetramethylisoindolin-2-yloxylyl)-6-

pyrrolidin-1-yl-xanthen-3-ylidene]-pyrrolidinium Chloride (152)

Appendices

163

HPLC 1: 23 separated with 50 % THF/50 % H2O

-50

0

50

100

150

200

250

300

0 1 2 3 4 5 6 7 8 9 10 11 12

Time/min

Inte

nsi

ty/a

u


-200

0

200

400

600

800

1000

1200

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Time/min

Inte

nsi

ty/a

u

Appendices

164


-200

0

200

400

600

800

1000

1200

1400

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Time/min

Inte

nsi

ty/a

u


-50

0

50

100

150

200

250

0 2 4 6 8 10 12

Time/min

Inte

nsi

ty/a

u

Appendices

165


-200

0

200

400

600

800

1000

1200

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Time/min

Inte

nsi

ty/a

u


-10

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14 16

Time/min

Inte

nsi

ty/a

u

Appendices

166


-100

0

100

200

300

400

500

600

700

800

900

1000

0 2 4 6 8 10 12

Time/min

Inte

nsi

ty/a

u


-50

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8

Time/min

Inte

nsi

ty/a

u

Appendices

167


-50

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7 8 9

Time/min

Inte

nsi

ty/a

u

HPLC 10: 101 separated with 50 % MeOH/50 % H2O

-50

50

150

250

350

450

550

0 1 2 3 4 5 6 7

Time/min

Inte

nsi

ty/a

u

Appendices

168


-10

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14

Time/min

Inte

nsi

ty/a

u


-20

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6

Time/min

Inte

nsi

ty/a

u

Appendices

169


-1

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6

Time/min

Inte

nsi

ty/a

u

Documents

The Synthesis Of Novel Profluorescent Nitroxide Probes …Keddie, D.J. and Bottle S.E. “Synthesis of Profluorescent Isoindoline Nitroxides via Aromatic Functionalisation” ARC Centre