LIST OF FIGURES
Figure 1.1.
Structure of heme 1
1
Figure 1.2.
Depiction of intracellular iron trafficking, storage and utilisation
2
Figure 1.3.
Pathways of cellular damage elicited by UVA and the
consequential generation of ROS and LI release
5
Figure 1.4.
Structure of the chelator EDTA 2, & its metal complex 3
9
Figure 1.5.
Chemical structures of the natural siderophores DFO 4 and
desferrithiocin 5, as well as the more recent synthetic ICs
defirasirox 6 and deferiprone 7.
10
Figure 1.6.
Chemical structure of PIH 8 & its tridentate (2:1) iron complex
12
Figure 1.7.
Chemical structures of the ‘100’ series of iron chelators
13
Figure 1.8.
Chemical structures of the lipophilic aroylhydrazone iron
chelators SIH 10 and NIH 11
14
Figure 1.9.
Chemical structures of the 200 and 300 series of
aroylhydrazone iron chelators
14
Figure 1.10.
Chemical structures of the PKIH series of iron chelators
15
Figure 1.11.
Chemical structure of Triapine® 13 and its iron complex 14
17
Figure 1.12.
The “double-punch” cytotoxic effect of thiosemicarbazone ICs
18
Figure 1.13.
Mechanism of ROS generation, such as the superoxide anion
by redox active thiosemicarbazone-iron complexes
19
Figure 1.14.
Structure of the DpT and BpT iron chelators
19
Figure 1.15.
Structure of the NT class of iron chelators
20
Figure 1.16.
Structure of “pro-chelator” OR10141 18
21
Figure 1.17.
Depiction of a “caged” compound and photorelease of the
bioactive molecule
24
Figure 1.18.
The nitrobenzyl family of PRPGs
26
Figure 1.19.
Structure of the 7-methoxycoumarin-4-ylmethyl ester PRPG
27
Figure 1.20.
Structure of furildioxime 36
30
Figure 1.21.
Structure of NPE-PIH 34 and NPE-SIH 37
30
xvii
Figure 1.22.
Structure of ICRF-159 (Razoxane®) 38
32
Figure 1.23
Structure of aroylhydrazone ICs PIH 8 and SIH 10, along with
their NPE-caged counterparts 34 and 37 respectively.
33
Figure 2.1.
Effects of aryl ring substitution on the photocleavage kinetics of
a nitrobenzyl-caged compound.
42
Figure 2.2.
Examples of coumarinyl caged compounds, depicting various
functional groups through which attachment of the coumarin
moiety can occur
45
Figure 2.3.
Emission spectrum of the Sellas UVA lamp used for decaging
52
Figure 2.4.
UV absorption profiles of the three caged PIH derivatives: NPEPIH 34, NV-PIH 67 and DEACM-PIH 75 at 40 M.
54
Figure 2.5.
HPLC chromatograms of thiosemicarbazone ICs 54a-b, which
were the only two compounds to show any degree of apparent
decomposition.
55
Figure 2.6.
HPLC chromatograms of NPE-SIH 37 and its photoproducts
56
Figure 2.7.
HPLC chromatograms of NPE-PIH 34 and its photoproducts
57
Figure 2.8.
HPLC chromatograms of NPE-NIH 53 and its photoproducts
57
Figure 2.9.
HPLC chromatograms of 55a and its photoproducts
58
Figure 2.10.
HPLC chromatograms of 55b and its photoproducts
59
Figure 2.11.
HPLC chromatograms of 57 and its photoproducts
60
Figure 2.12.
HPLC chromatograms of NV-SIH 66 and its photoproducts
61
Figure 2.13.
HPLC chromatograms of NV-PIH 67 and its photoproducts
61
Figure 2.14.
HPLC chromatograms of DEACM-SIH 78 and its photoproducts
62
Figure 2.15.
HPLC chromatograms of DEACM-PIH 75 and its photoproducts
62
Figure 2.16.
Structure of BrdU 83
64
Figure 2.17.
MTT assay: toxicity of NIH 11 in HaCaT cells after incubation
for 24, 48 or 72 h (n = 3-5)
66
Figure 2.18.
MTT assay: toxicity of NT44mT 54a in HaCaT cells after
incubation for 24, 48 or 72 h (n = 3-5)
67
xviii
Figure 2.19.
MTT assay: toxicity of NT 54b in HaCaT cells after incubation
for 24, 48 or 72 h (n = 3-5)
67
Figure 2.20.
MTT assay: toxicity of 56 in HaCaT cells after incubation for 24,
48 or 72 h (n = 4).
67
Figure 2.21.
Summary of growth inhibitory effects of the parental ICs
measured by MTT assay: NIH 11, NT44mT 54a, NT 54b and
H2PTBH 56.
68
Figure 2.22.
Adjusted results of the colony forming assay with parental ICs
68
Figure 2.23.
MTT assay for the parental, NPE-caged and UVA-irradiated
NPE-caged derivatives of PIH 8 (n = 2-4) and SIH 10 (n = 2) at
100 M in HaCaT cells, 72 h post treatment
69
Figure 2.24.
MTT assay for the parental, NPE-caged and UVA-irradiated
NPE-caged derivatives of NIH 11 at 5 M and 10 M (n = 3)
70
Figure 2.25.
MTT assay for the parental, NPE-caged and UVA-irradiated
NPE-caged derivatives of 54a at 10 M and 20 M (n = 3)
72
Figure 2.26.
MTT assay for the parental, NPE-caged and UVA-irradiated
NPE-caged derivatives of 54b at 5 M and 10 M (n = 3)
73
Figure 2.27.
MTT assay for the parental, NPE-caged and UVA-irradiated
NPE-caged derivatives of 56 at 50 M and 100 M (n = 3)
74
Figure 2.28.
MTT assay for the parental, NV-caged and UVA-irradiated NVcaged derivatives of SIH 10 at 20 M and 40 M (n = 3)
75
Figure 2.29.
MTT assay for the parental, NV-caged and UVA-irradiated NVcaged derivatives of PIH 8 at 100 M (n = 2-3)
76
Figure 2.30.
MTT assay for the parental, DEACM-caged and UVA-irradiated
DEACM-caged derivatives of SIH 10 at 20 M and 40 M
77
Figure 2.31.
BrdU assay: Growth inhibitory effect of aroylhydrazone and
sulfur-containing ICs, along with their NPE-caged and UVAirradiated NPE-caged derivatives 72 h post-treatment (n = 1)
78
Figure 2.32.
MTT assay of HaCaT cells in the absence or presence of UVA
radiation following treatment with NIH 11 or NPE-NIH 53 at 5
M and analysed 72 h post-treatment (n = 1)
80
Figure 2.33.
MTT assay: toxicity of NPK 35 in HaCaT cells after incubation
for 24, 48 or 72 h (n = 3).
83
Figure 2.34.
MTT assay for NPK 35, SIH 10, or a mixture of 35 and 10 at 20
M in HaCaT cells (n = 3)
84
xix
Figure 2.35.
MTT assay for NPK 35, NT 54b or a mixture of 35 and 54b at
10 M in HaCaT cells (n = 3)
84
Figure 3.1.
Structure of OC-NO 93, an example of a “multifunctional” UV
filter for use in sunscreens
88
Figure 3.2.
UV absorption profile of 2-hydroxy- and 2,4-dihydroxycinnamoyl
PIH (120-21) in EtOH at 40 M
100
Figure 3.3.
UV absorption profile of 2-hydroxycinnamoyl-caged SIH 106
and PIH 120 in EtOH at 40 M
101
Figure 3.4.
HPLC chromatograms of hydroxycinnamoyl-caged SIH and PIH
derivatives following exposure to ambient light for 16 h at RT
102
Figure 3.5.
HPLC chromatograms of 2-hydroxycinnamoyl-SIH 106 and its
related photoproducts
103
Figure 3.6.
HPLC chromatograms of 2,4-dihydroxycinnamoyl-SIH 111 and
its related photoproducts
104
Figure 3.7.
HPLC chromatograms of 2-hydroxycinnamoyl-PIH 120 and its
related photoproducts
105
Figure 3.8.
HPLC chromatograms of 2,4-dihydroxycinnamoyl-PIH 121 and
its related photoproducts
106
Figure 3.9.
HPLC chromatograms of 2-hydroxycinnamoyl-SIH 106 and the
extent of decaging at various doses of UVA
107
Figure 3.10.
HPLC chromatograms of 2-hydroxycinnamoyl-PIH 120 and the
extent of decaging at various doses of UVA
108
Figure 3.11.
Extent of uncaging versus UVA dose (kJ/m2) for the 2hydroxycinnamoyl-caged SIH and PIH derivatives 106 and 120
108
Figure 3.12.
MTT assay: toxicity of 2-hydroxycinnamoyl-SIH 106 (+/- UVA)
and SIH 8 in HaCaT cells, 24, 48 or 72 h post-treatment at 20
M (n = 2)
110
Figure 3.13.
MTT assay: toxicity of 2-hydroxycinnamoyl-SIH 106 (+/- UVA)
and SIH 8 in FEK4 cells (20 M), 72 h post-treatment (n = 1)
111
Figure 3.14
Structure of propidium iodide (PI) 136
112
Figure 3.15
Annexin V / PI dual staining assay: proportion of live FEK4 cells
in the absence (‘dark’) or presence of UVA radiation at a dose
of 500 kJ/m2 post-treatment with 8 or 120 (n = 2-13).
113
xx
Figure 4.1.
UV absorption spectra of aminocinnamoyl-caged PIH
derivatives 150a-d in EtOH at a concentration of 40 M
124
Figure 4.2.
UV absorption spectra of 4,5-dimethoxyaminocinnamoyl-caged
CICs 148a and 150a in EtOH at a concentration of 40 M
125
Figure 4.3.
HPLC chromatograms of aminocinnamoyl-CICs following
exposure to ambient light for 16 h
126
Figure 4.4.
HPLC chromatograms of 4,5-dimethoxyaminocinnamoyl-SIH
derivative 148a and its photoproducts
127
Figure 4.5.
HPLC chromatograms of 4,5-methylenedioxyaminocinnamoylSIH derivative 148b and its photoproducts
128
Figure 4.6.
HPLC chromatograms of 4,5-dimethoxyaminocinnamoyl-PIH
derivative 150a and its photoproducts
129
Figure 4.7.
HPLC chromatograms of 4,5-methylenedioxyaminocinnamoylPIH derivative 150b and its photoproducts
129
Figure 4.8.
HPLC chromatograms of aminocinnamoyl-PIH derivative 150c
and its photoproducts
130
Figure 4.9.
HPLC chromatograms of 4-trifluoromethylaminocinnamoyl-PIH
Derivative 150d and its photoproducts
131
Figure 4.10.
HPLC chromatograms of 4,5-dimethoxyaminocinnamoyl-SIH
148a and the extent of decaging at various doses of UVA
132
Figure 4.11.
HPLC chromatograms of 4,5-dimethoxyaminocinnamoyl-PIH
150a and the extent of decaging at various doses of UVA
133
Figure 4.12.
Extent of uncaging versus UVA dose (kJ/m2) for the
aminocinnamoyl-caged SIH and PIH derivatives 148a and 150a
134
Figure 4.13
MTT assay: toxicity of aminocinnamoyl-CIC 148a on HaCaT
cells (20 M) after incubation for 24, 48 or 72 h (n = 2).
135
Figure 4.14.
MTT assay: toxicity of aminocinnamoyl-CIC 148b on HaCaT
cells (20 M) after incubation for 24, 48 or 72 h (n = 2).
138
Figure 4.15.
MTT assay: toxicity of aminocinnamoyl-CICs 148a (A) and 148b 137
(B) on FEK4 cells (20 M). Corresponding treatments with SIH
(IC) are shown for reference (n=1).
Figure 4.16.
Annexin V / PI dual staining assay: photoprotective effect of the
aminocinnamoyl-caged IC 148a (20 M), measured 4 or 24 h
following irradiation of FEK4 cells at a dose of 500 kJ/m2
138
xxi
Figure 4.17.
ROS measurement in FEK4 cells, untreated (control) or treated
with 148a (20 M) and analysed 2 h post UVA-irradiation.
140
Figure 4.18.
Annexin V / PI assay: photoprotective effect of the
aminocinnamoyl-caged IC 148b (20 M), measured 4 h
following irradiation of FEK4 cells at a dose of 500 kJ/m2
141
Figure 4.19.
Annexin V / PI assay: photoprotective effect of the
aminocinnamoyl-caged IC 150c (20 M), measured 4 h
following irradiation of FEK4 cells at a dose of 500 kJ/m2
142
Figure 4.20.
Structure of psoralen, 22a, and carbostyril 140b
142
Figure 4.21.
3-methyl-quinolin-2-one photoproducts 140a-b
143
Figure 4.22.
Backbone of aminocinnamoyl-caged SIH without methyl
substitution on the carbon-carbon double bond
143
Figure 5.1.
Structure of 3-AP (Triapine®, 13) and Dp44mT, 15a
145
Figure 5.2.
Structure of Bp4eT 16d
146
Figure 5.3.
UV absorption spectra of Dp44mT 15a and its iron complex (A)
and the corresponding NPE-caged compound 170a
UV absorbance spectra of the NPE and DEACM-caged
derivatives of Dp4pT 170b and 171.
152
Figure 5.5.
HPLC chromatograms of NPE-Dp4pT 170b and its related
photoproducts
154
Figure 5.6.
HPLC chromatograms of NPE-Dp44mT 170a and its related
photoproducts
155
Figure 5.7.
HPLC chromatograms of DEACM-caged Dp44mT 171 and its
related photoproducts
156
Figure 5.8.
MTT assay: toxicity of Dp44mT 15a on HaCaT cells after
incubation for 24, 48 or 72 h (n = 3-8)
157
Figure 5.9.
MTT assay: toxicity of Dp4pT 15b on HaCaT cells after
incubation for 24, 48 or 72 h (n = 3-8)
158
Figure 5.10.
MTT assay: toxicity of Dp4pT 15b and its NPE-caged analogue
170b (+/- UVA) on HaCaT cells 72 h post-treatment (n = 3).
158
Figure 5.11
MTT assay: toxicity of Dp4pT and its DEACM-caged analogue
159
Figure 5.12
BrdU assay: toxicity of Dp4pT and Dp44mT on HaCaT cells 72
h post-treatment (n = 1)
160
Figure 5.4.
153
xxii
LIST OF SCHEMES
Scheme 1.1.
Overview of the Fenton and Haber-Weiss reactions
4
Scheme 1.2.
Activation of pro-chelator OR10141 18
22
Scheme 1.3.
Activation of boronic ester pro-chelator 21 by H2O2
22
Scheme 1.4.
Psoralen photosensitizers 22a-b and their thymidine adducts
24
Scheme 1.5.
Caged compound ONB-cAMP 24 and its photolysis to release
the active cAMP molecule and nitrosobenzaldehyde
26
Scheme 1.6.
Examples of cinnamoyl-caged compounds and their cyclic
photoproducts
27
Scheme 1.7.
Light activation of Ca2+ chelator 32.
28
Scheme 1.8.
NPE-PIH 34 a “prototype” CIC and its photolysis to yield the
strong iron chelator PIH
29
Scheme 2.1.
Example of the ONB moiety as a protecting group and its
orthogonal photoremoval
35
Scheme 2.2.
One of the first reported NPE-caged compounds and its
photolysis to yield NPK
36
Scheme 2.3.
Mechanism of NPE group photocleavage to produce NPK 35
36
Scheme 2.4.
Photorelease of TsOH after irradiation of 43 at various
wavelengths of light for 60 min
37
Scheme 2.5.
Photolysis of the CIC NPE-PIH 34 to give ‘naked’ PIH 8 and
the NPK photofragment 35
37
Scheme 2.6.
Preparation of parent aroylhydrazone iron chelators 8-11
38
Scheme 2.7.
O-NPE aldehyde synthesis 50-52
39
Scheme 2.8.
Condensation reactions of INH and NPE-caged aldehydes
39
Scheme 2.9.
Tautomerism of pyridoxal, showing its ‘open’ aldehyde and
cyclic furanol 52a or hemiacetal form 52
40
Scheme 2.10.
Preparation of thiobenzhydrazide 56. Also preparation of
sulfur-containing ICs 54a-b and 56 and their NPE-caged
derivatives 55a-b and 57
40
xxiii
Scheme 2.11.
Photocleavage of an NV-caged alcohol to release the free
alcohol and the accompanying nitrosobenzaldehyde
photoproduct
41
Scheme 2.12.
Preparation of nitroveratryl bromide 63 and initial O-alkylation
attempts
43
Scheme 2.13.
Synthesis of NV-caged aroylhydrazone ICs 66 and 67
44
Scheme 2.14.
Mechanism of photocleavage of the coumarin-4-ylmethyl
caging group, exemplified by DEACM
46
Scheme 2.15.
Synthetic route of DEACM-caged PIH and failed O-alkylation
of salicylaldehyde with coumarinyl bromide 73
47
Scheme 2.16.
Mechanism of the intramolecular aldol reaction and the
resulting benzofuranyl derivative which may account for Oalkylation failure
48
Scheme 2.17.
Attempted attachment of the DEACM group to phenolic
oxygen under Mitsunobu conditions
48
Scheme 2.18.
Attempted O-alkylation of salicylaldehyde with DEACM by
formation of carbonate ester 77
49
Scheme 2.19.
Successful base-catalysed formation of benzyl-aryl ether 76
from bromomethyl coumarin 73 and subsequent
aroylhydrazone formation
50
Scheme 2.20.
Non-photochemical synthesis of NPK 35
51
Scheme 2.21.
Enzymatic reduction of the tetrazole MTT 81 to its purple
formazan product 82
64
Scheme 2.22.
Mechanism of NPK 35 reaction with thiolates to give
benzisoxazole 85, as proposed by Corrie et al.
82
Scheme 3.1.
Photolytic mechanism of hydroxycinnamoyl-caged
compounds, resulting in formation of a coumarin and release
of the caged alcohol
86
Scheme 3.2.
Photogenesis of substituted coumarin derivatives 86-88 from
their corresponding hydroxycinnamates
87
Scheme 3.3.
Photogenesis of coumarin derivatives 89-92 from their
corresponding hydroxycinnamates
87
Scheme 3.4.
Initial synthetic route for the preparation of 3,5-dibromohydroxycinnamate 96, and inadvertent formation of coumarin
derivative
88
xxiv
Scheme 3.5.
Preparation of 3,5-dibromo-2,4-dihydroxycinnamoyl-caged
SIH 101
89
Scheme 3.6.
Synthetic routes to the 2-hydroxycinnamoyl-caged SIH
derivative 106
91
Scheme 3.7.
Attempted synthetic route to 2,4-dihydroxycinnamic acid 108
92
Scheme 3.8.
Synthetic route to the 2,4-dihydroxycinnamoyl-caged SIH
derivative 111
92
Scheme 3.9.
Preparation of protected PIH derivative 117
93
Scheme 3.10.
Coupling of TBDPS-PIH 117 to hydroxycinnamic acids and
subsequent desilylation
94
Scheme 3.11.
Formation of insoluble products following treatment of TBAF
with DOWEX ion-exchange resin and calcium carbonate
95
Scheme 3.12.
Preparation of hydroxycinnamoyl ‘caged’ alcohols
96
Scheme 3.13.
Preparation of 2-hydroxycinnamoyl capsaicin derivative 127
97
Scheme 3.14.
Attempted preparation of the 2,4-dihydroxy-5-methoxy
cinnamic acid 131 for subsequent coupling to SIH or PIH
98
Scheme 3.15.
Attempted preparation of the 2,4,5-trihydroxycinnamic acid
derivative 135 for subsequent coupling to SIH or PIH.
99
Scheme 3.16.
Synthetic CIC targets and for the future, which release the
corresponding 7-hydroxycoumarin photoproducts esculetin 91
and scopoletin 92
114
Scheme 4.1.
One of the first reported examples of an aminocinnamate
which is photolytically cleaved to yield carbostyril cyclic
photoproduct 140a
115
Scheme 4.2.
Preparation of 2-aminocinnamic acids 145a-d
116
Scheme 4.3.
Possible intramolecular rearrangement of the acylisourea
intermediate to give the unwanted amide
117
Scheme 4.4.
Coupling of aryl substituted cinnamic acids to either SIH 10 or
silyl-protected PIH 117, and desilylation of the latter.
118
Scheme 4.5.
Initial attempt to prepare carbostyrils 140a-b and by basecatalysed cyclisation of N-propanamide precursors 152a-b
119
xxv
Scheme 4.6.
Preparation of halogenated acrylamide precursor 157
120
Scheme 4.7.
Palladium-catalysed cyclisation of halogenated acrylamide
157 to give the 3-methylquinolin-2-one compound 140c
121
Scheme 4.9.
Preparation of 3-methyl-quinolin-2-ones via Vilsmeier
formylation
123
Scheme 4.10.
Structure of CM-H2DCFDA 166a, and it’s in vitro
transformation to the fluorescent species 166b by intracellular
ROS
139
Scheme 5.1.
S-alkylation of thiosemicarbazide derivative 167 described by
Ouyang et al.
147
Scheme 5.2.
Preparation of DpT iron chelators 15a-b from the
corresponding thiosemicarbazides
147
Scheme 5.3.
S-alkylation of DpT compounds Dp44mT 15a and Dp4pT 15b
with the NPE-caging group
148
Scheme 5.4.
S-alkylation of Dp44mT 15b with the DEACM caging group to
give 171
148
Scheme 5.5.
Attempted synthesis of Bp4eT 16d
149
Scheme 5.6.
Preparation of Bp4eT 16d using revised conditions
149
Scheme 5.7.
Mechanism for the condensation of a thiosemicarbazide and
diarylketone, and its facilitation by an acid catalyst
150
Scheme 5.8.
S-alkylation of thiosemicarbazide 173 with NPE bromide
151
Scheme 5.9.
Preparation of the iron-complex [Fe(Dp44mT)2] ClO4 hydrate
175
151
LIST OF TABLES
Table 1.1.
UK number of diagnoses (incidence) and number of deaths
(mortality) from skin cancer in 2010
7
xxvi