-
Oligo-Aminoferrocenes for Cancer Treatment
Oligo-Aminoferrocene in der Krebstherapie
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Gina Lorrain Zeh
aus Bad Reichenhall
-
Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-
Universität Erlangen-Nürnberg.
Tag der mündlichen Prüfung: 10.01.2020
Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer
Gutachter/in Prof. Dr. Andriy Mokhir
Prof. Dr. Svetlana Tsogoeva
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Für meine Eltern
Ein gerader Weg führt immer nur ans Ziel.
- André Gide
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Content
Page | i
CONTENT
1
INTRODUCTION..........................................................................................................................9
1.1 CANCER: DEVELOPMENT, RISK FACTORS AND CANCER CELL
PROLIFERATION ...... 10
1.1.1 The Cell Cycle: Understanding Cell Proliferation and
Growth .......................................... 10
1.1.2 Hallmarks of Cancer: Identification of Cancer Targets
...................................................... 13
1.2 CANCER TREATMENT: FROM CHEMOTHERAPY TO SPECIFIC TARGET
ASSESSMENT
.........................................................................................................................................................
16
1.2.1 Chemotherapy: Small Drugs for Cancer Treatment
........................................................... 17
1.2.2 Targeted Therapy: History, Tumor Targets and Treatment
................................................ 21
1.2.3 Reactive Oxygen Species
..................................................................................................
24
1.2.3.1 Antioxidants
..................................................................................................................
28
1.2.3.2 ROS in Cancer Treatment
..............................................................................................
30
1.3 FERROCENES, AMINOFERROCENES AND OLIGO-(AMINO)FERROCENES FOR
CANCER
TREATMENT
..................................................................................................................................
37
1.3.1 Bioisosteres: Ferrocene-Analogues of Organic Drugs
....................................................... 38
1.3.2 Ferrocene
Bio-Conjugates.................................................................................................
40
1.3.3 Multi-(Hetero)metallic Ferrocene-Derived Drugs
..............................................................
41
1.3.4 Ferrocenes as Drug Carrier
...............................................................................................
43
1.3.5 Oligomers of Ferrocene as Drugs
......................................................................................
44
2 MOTIVATION AND AIM OF THE WORK
.............................................................................
49
3 THE DENDRITIC APPROACH: RESULTS AND DISCUSSION
........................................... 50
3.1 CONCEPT: DENDRITIC RELEASE OF ACTIVE DRUGS
....................................................... 50
3.2 OVER ALL SYNTHETIC ROUTE TO CARBAMATE-DENDRON 6 AND PRODRUG
A ....... 54
3.2.1 Synthesis of Link Building Block 4
..................................................................................
55
3.2.2 Variations of Link Synthesis “a” and “b”
..........................................................................
59
3.2.3 Synthesis of Trigger-Link Molecule 6
...............................................................................
62
3.3 OVERALL SYNTHESIS ROUTE TO ETHER-BRIDGED DENDRON 13
................................ 69
3.3.1 Synthesis of Link Building Block 8
..................................................................................
71
3.3.2 Synthesis of Dendritic Prodrug
13.....................................................................................
72
3.4 SUMMARY FOR DENDRITIC APPROACH
............................................................................
76
4 THE OLIGOMERIC APPROACH: CONCEPT, RESULTS AND DISCUSSION
................... 77
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4.1 CONCEPT: TUNING THE OXIDATION POTENTIAL OF AMINOFERROCENES
................ 77
4.2 MONOMER-SERIES
.................................................................................................................
82
4.2.1 Synthesis of Ethyl-Monomer 15 and DM-Monomer 16
..................................................... 82
4.2.2 Experiments in Cell-Free Settings
.....................................................................................
85
4.2.3 Electrochemical Analysis of Monomer-Series
...................................................................
92
4.2.4 Experiments in Cell Culture
............................................................................................
103
4.2.4.1 Cell Viability Assay (MTT Assay)
...............................................................................
103
4.2.4.2 Oxidative Stress Assay on BL-2 Cell Line
...................................................................
107
4.2.5 Conclusion Monomer-Series
...........................................................................................
109
4.3 DIMER-SERIES
.......................................................................................................................
113
4.3.1 Synthesis of Propyl-Dimer 18 and Hexyl-Dimer 19
........................................................ 114
4.3.2 Experiments in Cell-Free Settings
...................................................................................
117
4.3.3 Electrochemical Analysis of Dimer-Series
......................................................................
124
4.3.4 Experiments in Cell Culture
............................................................................................
134
4.3.4.1 Cell Viability Assay (MTT Assay)
...............................................................................
134
4.3.4.2 Oxidative Stress Assay on BL-2 Cell Line
...................................................................
137
4.3.5 Conclusion Dimer-Series
................................................................................................
138
4.4 TRIMER 20
..............................................................................................................................
142
4.4.1 Experiments in Cell-Free Settings
...................................................................................
144
4.4.2 Electrochemical Analysis of Trimer 20
...........................................................................
147
4.4.3 Experiments in Cell Culture
............................................................................................
151
4.4.4 Conclusion Trimer 20
.....................................................................................................
153
4.5 TETRAMER 21
........................................................................................................................
155
4.5.1 Experiments in Cell-Free Settings
...................................................................................
162
4.5.2 Electrochemical Analysis of Tetramer 21
........................................................................
167
4.5.3 Experiments in Cell Culture
............................................................................................
171
4.5.4 Conclusion Tetramer
21..................................................................................................
174
4.6 COMPARISON OF ALL ANALYZED OLIGO-AMINOFERROCENES
................................. 177
5 COOPERATION PROJECTS
..................................................................................................
185
5.1 FLUORESCENCE MICROSCOPY MEASUREMENT OF IN-CELL-SYNTHESIS
OF
ARTEMISININ-DERIVATIVES
....................................................................................................
185
5.2 CELL VIABILITY, CELL PERMEABILITY AND OXIDATIVE STRESS
MEASUREMENTS
OF
CLATHROCHELATES............................................................................................................
189
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6 SUMMARY AND OUTLOOK
..................................................................................................
199
7
ZUSAMMENFASSUNG............................................................................................................
202
8 EXPERIMENTAL SECTION
...................................................................................................
205
8.1 GENERAL COMMENTS AND INSTRUMENTS
....................................................................
205
8.2 THE DENDRITIC APPROACH - SYNTHESES
......................................................................
207
8.2.1 Synthesis of Carbamate-Dendron
....................................................................................
207
8.2.1.1 Synthesis of Link-Building Block 4
.............................................................................
208
8.2.1.1.1 Synthesis of 2,4-Dimethyl-1-nitrobenzene 1
..............................................................
209
8.2.1.1.2 Synthesis of 4-Nitroisophthalic Acid 2
......................................................................
210
8.2.1.1.3 Synthesis of (4-Nitro-1,3-Phenylene)-Dimethanol 3
.................................................. 211
8.2.1.1.4 Syntheses of Compounds 3a and 3b
..........................................................................
213
8.2.1.1.5 Synthesis of Compound 4a
........................................................................................
215
8.2.1.1.7 Synthesis of Compound 4b
.......................................................................................
216
8.2.1.2 Activation of Trigger 5
................................................................................................
217
8.2.1.2.1 Synthesis of Carbonate 5a
.........................................................................................
217
8.2.1.2.2 Synthesis of Chloramate 5b
.......................................................................................
218
8.2.1.3 Synthesis of Compound 6
............................................................................................
219
8.2.2 Synthesis of Ether-Dendron 13
.......................................................................................
221
8.2.2.1 Synthesis of Building Block ‘Link’ L2
.........................................................................
221
8.2.2.1.1 Synthesis of Compound 7
.........................................................................................
222
8.2.2.1.2 Synthesis of Compound 8
.........................................................................................
223
8.2.2.2 Synthesis of Trigger 9
..................................................................................................
224
8.2.2.3 Synthesis of TBS-Dendron 10
......................................................................................
225
8.2.2.4 Synthesis of Unprotected Dendron 11
..........................................................................
226
8.2.2.5 Synthesis of Precursor 1-Azidocarbonyl ferrocene 12
.................................................. 227
8.2.2.6 Synthesis of Ether-Aminoferrocene-Dendron 13
.......................................................... 228
8.3 THE OLIGOMERIC APPROACH - SYNTHESES
...................................................................
229
8.3.1 MONOMER-SERIES
............................................................................................................
229
8.3.1.1 Synthesis of Ethyl-Monomer 15
...................................................................................
229
8.3.1.2 Synthesis of DM-Monomer 16
.....................................................................................
231
8.3.2 DIMER-SERIES
....................................................................................................................
232
8.3.2.1 Synthesis of Propyl-Dimer 18
......................................................................................
232
8.3.2.2 Synthesis of Hexyl-Dimer 19
.......................................................................................
233
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8.3.3 SYNTHESIS OF TRIMER 20
................................................................................................
234
8.3.4 SYNTHESIS OF TETRAMER 21
..........................................................................................
235
8.3.5 SYNTHESIS OF ISOCYANURIC TRIMER 22
.....................................................................
238
8.4 MEASUREMENTS IN CELL-FREE SETTINGS
.....................................................................
240
8.4.1 Determination of the n-Octanol-Water-Distribution
Coefficient ...................................... 240
8.4.2 Determination of ROS Generation in vitro
......................................................................
241
8.4.3 Determination of Turbidity of Aqueous Solutions of Drugs
............................................. 242
8.4.4 Determination of Direct Reduction of MTT by Drugs
..................................................... 242
8.5 MEASUREMENTS IN CELL CULTURE (IN VITRO)
.............................................................
244
8.5.1 Cell Lines, Instruments, Cultivation Protocols
................................................................
244
8.5.2 Measurement of Cell Viability – MTT-Assay
.................................................................
247
8.5.3 Estimation of Cell Permeability for Clathrochelates
CC1-CC3 ........................................ 248
8.5.4 Estimation of Oxidative Stress (BL-2)
............................................................................
248
8.5.5 Visualization of Cellular Distribution of AC98, CM486, AC
and the Reaction of AC and
CM486 Using Fluorescence Microscopy (A2780)
...................................................................
249
9 APPENDIX A - REFERENCES
................................................................................................
251
10 APPENDIX B – SPECTRA (NMR, UV/VIS, MS)
..................................................................
257
10.1 OVERVIEW OF SYNTHESIZED MOLECULES
..................................................................
257
10.2 NUCLEAR MAGNETIC RESONANCE (NMR) SPECTRA
.................................................. 257
10.3 MASS SPECTRUM ANALYSIS
............................................................................................
270
11 APPENDIX C – CELL-FREE EXPERIMENTAL SPECTRA
.............................................. 271
12 APPENDIX D: CELL EXPERIMENTAL SPECTRA
........................................................... 277
12.1 CELL VIABILITY ASSAY (MTT)
........................................................................................
277
12.2 OXIDATIVE STRESS ASSAY
..............................................................................................
282
13 APPENDIX E – X-RAY CRYSTALLOGRAPHIC DATA
.................................................... 283
DANKSAGUNG
...................................................... FEHLER!
TEXTMARKE NICHT DEFINIERT.
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Abbreviations
Page | v
ABBREVIATIONS
8-HQ 8-hydroxyquinoline
A absorbance
a.u. arbitrary units
A2780 human breast carcinoma cell line
abs. absolute
Ac acetyl
aq. aqueous
ATP adenosine triphosphate
BL-2 human Burkitt lymphoma cell line
bs broad singlet
CAC citric acid cycle (Krebs cycle)
Cas carbon anhydrase
CC column chromatography on SiO2-gel (unless otherwise
stated)
CM chloromethyl-
conc. concentrated
δ chemical shift
d doublet
DCF 2’,7’-dichlorofluorescein
DCFH 2’,7’-dichlorodihydrofluorescein
DCFH-DA 2’,7’-dichlorodihydrofluorescein diacetate
DCM dichloromethane
DIPEA N,N-diisopropylethylamine
DMAP N,N-dimethylpyridine-4-amine
DMEM Dulbecco’s Modified Eagle’s Medium
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
DPV differential pulse voltammetry
DSMZ German collection of microorganisms and cell cultures
(Deutsche Sammlung
von Mikroorganismen und Zellkulturen)
DU145 human prostate cancer cell line
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Abbreviations
Page | vi
E emission
EDTA N,N,N’,N’-ethylenediamine tetra acetic acid
EI electron ionization
EMEM Eagle’s Minimum Essential Medium
eq. equivalents
ESI electrospray ionization
EtOAc ethyl acetate
EtOH ethanol
FBS fetal bovine serum
Fc ferrocene
Fc+ ferrocenium
GF growth factor
GM01379 human lung fibroblast cell line
GS growth signal
GSH glutathione
GSSG glutathione disulfide
HeLa human cervical cancer cell line
HPLC high potential/pressure liquid chromatography
Hz hertz
IC50 inhibiting concentration 50 %
ihexane usually a mixture of n-/cyclo-/iso-hexane with
iso-derivative > 50 %
J Joule
λ wavelength
λem emission wavelength
λex excitation wavelength
µ micro
m meta
M molar (mol/L)
m multiplet
m/z mass to charge ratio
MALDI matrix-assisted laser desorption/ionization
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Abbreviations
Page | vii
MCF-7 human cervix adenocarcinoma cell line
MCT monocarboxylate transporter
MeOH methanol
MHz megahertz
MOPS 3-N-(morpholino)propane sulfonic acid
MS mass spectrometry
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
NADP nicotinamide adenine dinucleotide phosphate
NHDF normal human dermal fibroblast cell line
NHE Na+-H+-exchanger protein
NMR nuclear magnetic resonance
NP normal phase
o ortho
Opti-MEM minimal essential medium
p para
PBS phosphate buffered saline (10 mM, pH 7.4, 154 mM NaCl)
PI propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)
propyl]-6-
phenylphenanthridinium diiodide)
ppm parts per million
R residue
Rf retention factor
ROS reactive oxygen species
rpm revolutions per minute
RPMI 1640 Roswell Park Memorial Institute medium
RP-TLC reverse phase thin layer chromatography (C18)
RT room temperature (22 °C – 25 °C)
s singlet
sat. saturated
t tertiary
t triplet
TBS tert-butyl dimethyl silane
https://en.wikipedia.org/wiki/Di-https://en.wikipedia.org/wiki/Methylhttps://en.wikipedia.org/wiki/Thiazolehttps://en.wikipedia.org/wiki/Phenyl
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Abbreviations
Page | viii
TEA triethylamine
TES triethyl silane
THF tetrahydrofuran
TLC thin layer chromatography (SiO2)
TMS trimethyl silane
UV ultra violet
VEGF vascular endothelial growth factor
Vis visible
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1 Introduction
Page | 9
1 Introduction
With an average lifespan of 80 years, the median chance to
develop at least one tumorous tissue during
human lifetime is about 6:1000 (per population) with increased
likelihood1. According to the World
Cancer Report, published by the World Health Organization (WHO),
about 14.1 million people are
expected to develop cancer annually2. About half as much cancer
patients are estimated to die of cancer
every year (2018: 9.6 million), rating malign tumors the fourth
most common cause of death in Germany
(most common one: cardiovascular diseases).3,4
The alarming fear of cancer lies in its likewise appearance in
under- and over nutrient countries. On the
one hand, overweight, bad physical condition and sugar-rich
diets lead to an increased risk of cancer
development, on the other hand, unbalanced diet, stress and
physical overexertion were found to
correlate with cancer growth.4
Despite the numerous types of cancer, some of them occur
frequently in humans. The most common (in
terms of new diagnoses/year in 2018) cancers are lung and breast
cancer (about 2.09 million
diagnoses/year), followed by colorectal (1.8 million
diagnoses/year), prostate, skin and stomach cancer
(each about 1 million diagnoses/year). Excellent preventive
medical checkup programs facilitate the
early treatment of tumors and lead to fewer cancer-related
deaths. Nevertheless, for some types of
cancer, e.g. lung cancer, the number of deaths per year is
almost as high as the number of new diagnoses
per year. These statistics emphasize the importance of new
cancer treatment methods by highly efficient
tumor-specific cytotoxic drugs.4
The present thesis is meant to elucidate the cancer-specific
toxicity of new redox-active drugs. First, a
scientific basis will be established by the explanation of
cancer cell proliferation and tumor
development. After a short resume of the most common cancer
treatment methods, focus will be set on
chemotherapy and subsequently targeted (chemo)therapy. In a
short overview, the history of iron-
containing drugs will be highlighted. Afterwards, the
challenging synthetical accesses to new anti-
cancer drugs, based on aminoferrocenes, and preliminary results
in cell culture assays will be presented.
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1 Introduction
Page | 10
1.1 Cancer: Development, Risk Factors and Cancer Cell
Proliferation
To understand the transformation from a normal, healthy cell to
a tumor cell, one should take a closer
look at the life cycle of cells. Cells – with some exceptions –
are naturally in their quiescent state,
meaning, they do not grow and proliferate. If cell growth,
differentiation or proliferation is necessary,
mitogenic growth signals (GS) alter cells into their active
proliferative state. The usually diffusible,
ubiquitous signal molecules either pass the cell membrane by
transmembrane receptors or bind to
extracellular receptors for signal transfer. This process goes
in both directions: Cells can send GS to
other cells, or receive GS that bind to receptors and alter the
metabolic processes within the cell. Fact
is, until now no cell was found that can proliferate without
external or internal trigger as GS.5
1.1.1 The Cell Cycle: Understanding Cell Proliferation and
Growth
Upon activation by GS, the cell exits its quiescent state (G0)
and enters the cell cycle to proliferate. The
cell cycle can be divided in two major phases, interphase, where
the cell division is prepared, and mitotic
phase, where the cell physically divides into two equivalent
cells. In the first step of interphase (G1,
Figure 1), the cell duplicates all its cellular content except
the contents with genetic information (DNA
in chromosomes). Physically, the cell grows to approximately
double its size. The molecular building
blocks produced in this step will be needed later. In S phase,
DNA in the nucleus as well as centrosomes
(separate DNA during M phase) is doubled. During the second gap
phase (G2), the cell forms proteins
and organelles in preparation for mitosis. Now, entering M
phase, DNA is organized in chromosomes.
The mitotic spindle reorganizes chromosomes and divides them
into two identically sets, the genetic
basis for two new cells. The M phase ends with cytokinesis,
where the cytoplasm divides into two new
cells. Slowly or not proliferating cells can exit cell cycle by
resting cell division. This state without
proliferation is called G0 phase. G0 phase is for some
cell-types, e.g. neurons, a permanent state. Most
other cell-types can re-enter cell cycle at any time.
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1 Introduction
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Figure 1: Cell cycle of a normal, healthy, eukaryotic cell. G0 –
cell cycle arrest; G1 – duplication of
all cellular contents except chromosomes; S – duplication of DNA
(chromosomes); G2 – double check
for errors in DNA replication and repairs; M – mitosis: cell
division and cytokinesis.
A tumor, also called neoplasm, evolves from normal, healthy
cells that divide independently, continued
constantly and with high division rates (Figure 2). Usually,
spontaneous mutations in healthy tissue are
detected and repaired by repair mechanisms of the cell. In rare
cases (mutation rate for epithelial cells ~
10-7 per cell division6), a mutated cell is either not detected
or divides faster than the repair mechanism
can identify and repair the mutation. The mutated cell passes on
an advantage of selectivity as it grows
faster than the surrounding normal cells. The cell proliferates,
and a tissue of mutated cells grows.
Spontaneously, a second mutation occurs on one of the mutated
cells, leading to a double mutated cell
with two selectivity markers, for example rapid growth and
optimized adaption to the
microenvironment. The double mutated cell is selected,
proliferates and a tissue consisting of double
mutated cells can grow. The consequent mutation- and
selection-steps proceed, and a tumor tissue can
evolve, resulting in cancer cells with multiple mutations and
selective growth advantage.6 A cancer cell
of a malign tumor typically exhibits about six different
mutations.
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1 Introduction
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Figure 2: Schematic representation of multiple tissue mutations,
resulting in malign tumor. After
every mutation step a selection step for the cell with the
fastest growth or best adaption to environment
follows, allowing proliferation of mutated cells despite
numerous cellular control systems.6
Not all tumors are lethal and therefore must be treated. Benign
tumors, e.g. common skin wart, stay
within their local boundaries and do not integrate into adjacent
tissue. Usually, if the benign tumor is
small and does not grow further, these tumor tissues do not need
to be treated or removed. In contrast,
malign tumors, colloquially called cancer, must be treated as
soon as possible after detection. In the
early stage of tumor growth, the mutant cells stay benign and
remain within the normal tissue boundaries
of a tissue. As the tumor grows, it becomes malignant and
invades adjoining tissues in the need of
nutrients and space. There, the mutant cells destroy or repress
healthy, normal cells. Additionally, cancer
cells integrate into blood vessels and lymph vessels and spread
into other tissues or organs. These
offshoots of the main cancer tissue are called metastases.
Within the last years, a third category was
found, so called semi malign tumors. This small group of cancer
cells integrates into adjacent tissue but
does not form metastases and because of that is readily
accessible for invasive treatment. Tumor cells
in general easily adapt to new conditions like varying pH,
oxygen-concentration or metabolic support.
That, together with their ability to metastasize and invade,
makes it difficult for the immune system as
well as scientists to find, identify and defeat malign
tumors.6,7
Tumors can be further divided in cancer types that differ in the
cellular origin of tumors. The most
common one is carcinoma, a tumor that originates from epithelial
tissue, for example from skin, mucous
membrane or glandular tissue. Approximately 90 % of malign human
cancers are derived from mutated
epithelial cells.6 Sarcomas origin from connective or supporting
tissue, for example, from fat tissue,
muscles, bones or fibrous tissue. They are solid and thereby
easy to treat by surgery, but rare tumors in
humans. Leukemias and lymphomas are derived from blood-forming
and immune cells6. The last one,
blastomas, are embryonic tumors that develop during tissue- and
organ progression7. Out of these types
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1 Introduction
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of tumors, carcinomas are the favorite subject for cancer
research due to their abundancy and challenging
treatment.6,7
The number of diagnosed cancers was found to correlate with the
age of patients and certain risk factors
were identified that increase the chance of tumor development.
Genetical mutations, as shown above,
lead to cancer emerge. These mutations can be induced by
pre-existing illness, but in most cases, the
ability to develop a tumor due to genetical encoding is
bequeathed (e.g. breast cancer). During the last
years, environmental risk factors played a growing role in
cancer development. Chemicals or radiation
lead to mutations or deletions in the genetic material and
increase the risk of non-specific cancer
development. While excessive smoking is associated with an
increased risk for lung cancer
development, most environmental risk factors induce a variety of
cancer types. In general, obesity,
sedentary and stress weaken the body’s immune system and lead to
an increased likelihood of mutations
in genetic material or fail of the repair of genetic mutations.
Excellent preventive medical checkup
programs, e.g. annual mammography to detect breast cancer, help
detecting tumor evolution in the very
early states of carcinogenesis and facilitate complete and
permanent success of treatment.8
1.1.2 Hallmarks of Cancer: Identification of Cancer Targets
Tumorigenesis in mammalians is a multistep process involving the
production of oncogenes and an
increase in importance of these due to a repression of tumor
suppressor genes. Despite the fact that
almost every cancer is unique and perfectly adapted to its
surrounding, there are six acquired capabilities
that all cancer cells have in common and that define a cell as a
cancer cell, as proposed by D. Hanahan
and R. A. Weinberg in 2000.5
1) Self-sufficiency in growth signals
In contrast to normal cells, cancer cells don’t necessarily need
GS to grow and proliferate. Instead of
remaining in the quiescent cell state, three mechanisms evolved
allowing cancer cells independence
from external growth signaling. First, tumor cells generate
their own GS, the so-called oncogenes.
Oncogenes control the induction of apoptosis by sensing lethal
mutations. In tumorous cells, oncogenes
were found to trigger cancer development by inducing
proliferation and cell survival instead of
apoptosis. Oncogenes imitate GS so that cancer cells are
completely independent in cell growth and
proliferation from external stimuli. Second, the uptake of GS
through the cell membrane is altered by
over-expression of receptors that transduce GS. The result is
hyper-sensitivity to GS that normally would
not initiate cell proliferation. Additionally, cancer cells can
promote the expression of certain GS
receptors, for example those that help cell growth. Third, once,
the GS are in the cell, the downstream
cytoplasmic circuitry is adapted to maintain growth factors (GF)
as long as possible in cancer cell
signaling cascades. The elucidation of cell signaling pathways
showed that maintaining GF besides
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1 Introduction
Page | 14
proliferation led to suppression of death factors, release of
survival factors and ultimately changes in
gene expression that are clearly favorable for the cancer
cell.5,9
2) Insensitivity to anti-growth signals
Cellular quiescence and tissue homeostasis are balanced by
proliferative and antiproliferative signals.
Antiproliferative signals block cell proliferation by two
mechanisms. Either cells are forced into G0 state
until GS initiate cell proliferation again or they differentiate
into a specific cellular subgroup. Cancer
cells were found to monitor their environment during cell cycle
and block functions of cell cycle clock
sensors that initiate cell cycle arrest. The disturbance of
these sensor proteins leads to an insensitivity
towards anti-growth signals. Acquired insensitivity is gained by
tumor cells by various other
mechanisms, including a number of enzymes and proteins, that are
not well understood yet. By evolution
of insensitivity to anti-growth signals, cancer cells are
hypothetically able to replicate limitless, enabling
a fast tissue growth.5,9
3) Evading apoptosis
In case of abnormal cell mutations that cannot be repaired, the
cell will undergo a programmed cell
death by disruption of cellular membranes, brake-down of nuclear
and cytoplasmic skeletons, ejection
of the cytosol and degradation of all cellular compartments. The
whole process of apoptosis takes a
maximum of 120 min and, together with the uptake of the
remaining building blocks by surrounding
cells, is finished after 24 h.5,9
To evade apoptosis, genes, that induce programmed cell death,
must be suppressed by cancer cells.
Tumor suppressor proteins, e.g. p53, control apoptosis.
Therefore, the suppression of p53 leads to
abnormal proliferation of tumor cells. About 50 % of human
cancers evidenced a mutation of p53 gene,
leading to a repression of DNA damage sensors. This mutation
also leads to other tumor-specific
abnormalities like hypoxia (low oxygen supply of tissue) and
hyperexpression of certain genes. A
revocation of apoptosis was also observed by overexpression of
oncogenes. These alterations lead to
cancer cell immortality.5,9
4) Limitless replicative potential
The first three hallmarks are strongly dependent on cell-to-cell
signaling by GS. But an interference in
GS signaling in tumor cells was proven to not necessarily
promote cancer cell proliferation. Mammalian
cells bear an intrinsic, independent from other cells program
that regulates proliferation. After cutting
all extracellular signaling pathways, the fourth step in
transformation to a cancer cell lies in disruption
of the own proliferation-regulating signaling. Cell senescence,
a stop in replication after a certain
number of cell cycles, is disabled by inhibition of tumor
suppressor genes (e.g. p53). Cells with inhibited
tumor suppressor genes multiply further, until they abruptly die
(called crisis state). The erosion of
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1 Introduction
Page | 15
telomeres, the ends of DNA, was found to play a major role in
entering crisis state. To elude crisis,
cancer cells increase expression of telomerase enzyme and
maintain telomeres. This simple adaption,
together with up-to-date not well circumvention of cell
senescence, is a key event in enabling replicative
immortality.5,9
5) Sustained angiogenesis
As mentioned before, oxygen supply is of major importance in
cell growth and proliferation. Blood
vessels that ensure oxygen saturation of surrounding tissue are
carefully grown during the development
of organs. Angiogenesis, the act of growing new blood vessels,
is regulated by cellular signals, e.g.
vascular endothelial growth factor (VEGF) that binds to
transmembrane tyrosine kinase receptors and
thereby transduce the signal into the cell. Possible
interference-mechanisms include inhibition of
tyrosine kinase receptors, interference in signal transduction
in the cell and in the intracellular space.
Angiogenesis in general is a multi-step process that includes
numerous proteins, receptors and signal
cascades, offering a number of therapeutic targets for cancer
treatment.5,9
6) Tissue invasion and metastasis
After cutting off signaling to other cells, controlling own
signaling cascades for limitless proliferation
and ensuring nutrient supply, the last step in malign tissue
growth is metastasis. As mentioned before,
the invasion of other organs and tissues is a tumor-innate
ability and cause about 90 % of human cancer
deaths10. Invasion of adjacent tissues provides more space for
tumor growth, and a primary excellent
supply with nutrients. Cells that are capable of invading
adjacent tissue have altered proteins for cell-
to-cell interactions and cell-to-matrix interactions. Latter
ones are exemplified by E-cadherin, a protein
that binds neighboring cells. Via this bridge, GS as well as
antigrowth signals are transmitted.
Elimination of E-cadherin represents a key step in metastasis.
The second key step is the enhanced
expression of proteases that are needed to degrade cellular
matrices. The ability of malign cancer cells
to metastasize challenges tumor treatment and leads, even after
successful treatment of carcinomas,
often to resurrection of cancer tissues.5,9
These so-called hallmarks of cancer are continuously extended by
additional characteristic markers for
malign cancer, as, for example, abnormal metabolism (anaerobic
glycolysis instead of citric acid cycle
in favor of a fast construction of building blocks for
proliferation, summarized as Warburg Effect)11,
reversed pH-milieu (plasma deprotonation leads to an
intercellular alkaline pH)12, genomic instability9,
resistance against attacks of the immune system9 and a higher
risk of cancer development during chronic
inflammation9. All these factors belong to a cascade of
signaling that primarily is designed to undergo
planned cell death, apoptosis, but the signal cascade in cancer
cells is altered or interfered at several
stages leading to abnormal cell growth and proliferation.
Conversely, every cancer hallmark that is
properly identified and characterized, opens up new targets for
successful cancer treatment.
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1.2 Cancer Treatment: From Chemotherapy to Specific Target
Assessment
Cancer treatment is strongly dependent on cancer type and
origin. Conventional cancer treatment
includes surgery, radiation therapy, immunotherapy, chemotherapy
and targeted therapy. Newer therapy
methods are hormone therapy, stem cell transplant and precision
therapy. Hormone therapy is strongly
connected with breast cancer. Breast cancer is one of the first
tumors that were found to be hormone-
dependent (hormone: estrogen) and estrogen-down-regulators, for
example tamoxifen, a selective
estrogen receptor modulator (SERM), lead to an improvement of 11
% in the 10-year survival rate in
estrogen-positive (ER+) cancer patients. Focusing on
conventional therapy methods, solid tumors that
do not metastasize and are locally accessible, as, for example,
skin tumor, can easily be treated by local
surgery. Surgery is, compared to other treatment methods, the
only one with a cure rate of nearly 100 %
because all tumor cells are seized and killed13. This treatment
is restricted to solid, non-metastasized
tumors and can’t be used for diffuse tumors like blood cancers
(leukemia). It is the most invasive method
to treat cancer, but as the whole cancer is cut out of the body,
the risk of relapse is low. For cancer types
that spread over the body, immunotherapy is used to potentialize
the body’s own immune system to
defeat cancer. Radiation therapy uses doses of radiation to
destroy cancer cells and shrink tumors. With
about 45 % of new cancers receiving radiation therapy, it is
mainly used for prostate, neck, breast, cervix
and thyroid cancer because of their good accessibility13. As the
side-effects include destruction of
surrounding tissue, radiation therapy is often used in
combination with other cancer treatment methods
and thereby in smaller doses. The most prominent method to treat
cancer is chemotherapy. Small
molecules are introduced into the tumorous tissue and exploited
to kill rapidly dividing cells. Drugs for
chemotherapy can be administered orally, intravenously or by
several other methods, making
chemotherapy the least invasive method of cancer treatment. Side
effects include the slow-down and
killing of healthy, normal rapidly dividing cells, fatigue and
loss of facial hair. Although the side effects
are severe, chemotherapy can be applied for all cancer types and
the rates of successful treatment are
incomparably high. The newest and last conventional cancer
therapy is targeted therapy. As mentioned
before, cancer cells are identified by several hallmarks of
cancer. Targeted therapy utilizes drugs that
target these hallmarks, leading to less destruction of
surrounding tissue and thereby fewer side effects.14
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Figure 3: Methods of cancer treatment.
Lately, the combination of several drugs or therapy methods
improved cancer treatment rates. This
approach is considered as precision therapy or individual
therapy, where for each patient an individual
and personalized treatment is applied. Advantages of precision
therapy are fewer side effects and better
treatment results due to cancer-type specific treatment. Most
often, hormone- or immunotherapy is
combined with drugs known from targeted therapy. Thereby, new
drugs for targeted therapy gained
mounting interest during the last decades. The next chapter will
summarize the basic demands of
chemical molecules that are used for cancer treatment as the
concept of targeted therapy was established
on chemotherapy.
1.2.1 Chemotherapy: Small Drugs for Cancer Treatment
For chemotherapy, usually small drugs are used for cytostatic
treatment of malign cancers. These
chemotherapeutic agents are designed to inhibit mitosis by
DNA-damaging or -interference and thereby
stop fast proliferating cells from dividing. Often, they are
administered in single doses or multiple doses
of maximum tolerated doses (MTD), where no severe toxicity
towards the whole organism is observed.
For curative chemotherapy, a combination of drugs is used to
kill cancers.13 The following chapter
highlights milestones of chemotherapeutic agents and most recent
drugs for chemotherapy.
During first world war, the use of mustard gas (also Lost, I,
Figure 4) induced a loss of lymphocytes
and bone marrow depression in victims of mustard gas. The
treatment of lymphatic tumors with nitrogen
mustard-derivative of Lost (mustine, II, Figure 4) was tested in
1942 and resulted in an outstanding
tumor suppression of lymphatic tumor tissue in mice and in
patients with Non-Hodgkin-Lymphoma.
Although no complete or permanent cancer treatment was induced
by mustine II, it was the first time a
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small molecule was successfully used in cancer treatment and the
basis for chemotherapy was built. In
the following years, the mechanism of action of mustine was
clarified. The molecule binds covalently
to guanine-bases of DNA and thereby crosslinks DNA-strands
intermolecular. This leads to a standstill
of DNA-replication and to cell cycle arrest. Mustine II reacts
with itself in a SN2-intramolecular reaction
and forms the aziridinium-derivative II-1. The aziridinium is
attacked by guanine of DNA-double strand
to form II-2. The intramolecular SN2-reaction to the aziridinium
occurs a second time and intermediate
II-3 is attacked by another guanine-base. The resulting
guanine-mustine-dimer II-4 either crosslinks
two DNA molecules or the two strands within a DNA, disenabling
DNA unzipping and replication. The
effectiveness of mustine II originates from its ability to
affect selectively rapidly dividing cell due to
their fast DNA-replication.15
Figure 4: Structures of mustard gas and anti-cancer drug mustine
(sold as Mustargen®) and
mechanism of action of mustine.
With the rational development of methotrexate (III, Figure 5), a
chemotherapeutic for the treatment of
acute lymphatic leukemia (ALL), J. C. Wright was able to treat
for the first time not only leukemia, but
also solid tumors. Up to that date, the only method of treatment
of solid tumors was a surgery. J. C.
Wright observed cytotoxic effects of methotrexate III on breast
cancer cells. It took some more years to
elucidate the mechanism of action of methotrexate III. The drug
inhibits in competition with folic acid
IV reversible the enzyme dihydrofolate reductase. The enzyme
catalyzes the formation of active
tetrahydrofolate out of dihydrofolate. The product is essential
for the synthesis of thymidine, one of four
bases for DNA synthesis. Methotrexate III silences
thymidine-synthesis and thereby DNA-synthesis.
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This leads ultimately to a cell cycle arrest and explains why
folic-acid-antagonist methotrexate
selectively shows cytotoxicity on cells that divide rapidly:
These cells are consuming building blocks as
nucleosides much faster than non-cancerous cells.
Figure 5: Structures of anti-cancer drug methotrexate III and
its bio-antagonist folic acid IV.
Labeled uracil, an RNA-specific nucleoside, was introduced into
rats that suffered from liver tumor
(Figure 6, V). The artificial nucleoside-derivative was observed
to accumulate in the tumorous tissue of
the rats due to the rapid consumption of nucleosides in cancer
cells and the strong need of these building
blocks. Analogous structures of nucleosides are used during
biosynthesis of DNA and RNA but cannot
be transcribed during DNA replication. The, in terms of cell
proliferation and replication, useless DNA
strands lead to a stop of replication and thereby cancer cell
death. C. Heidelberger and his group
developed, encouraged by the accumulation of artificial
nucleosides in tumorous tissue, fluorinated
pyrimidines. The lead compound, 5-fluorouracil, is still in use
for the treatment of solid tumors as
prostate cancer (Figure 6, VI). Mechanistic studies revealed an
additional inhibition of thymidylate-
synthase, the enzyme, that catalyzes the synthesis of thymidine,
another nucleoside for DNA-synthesis.
Unfortunately, 5-fluorouracil VI is also introduced into
non-cancerous cells and serves as nucleoside-
derivative for DNA- and RNA-synthesis, leading to severe side
effects.
Figure 6: Structures of RNA-base uracil its synthetic analogous
5-fluorouracil.
A great break-through – maybe the biggest break-through at all –
in using chemotherapeutics for cancer
treatment was marked by the rather unintended discovery of
cisplatin. B. Rosenberg observed that
bacteria died after taking an electrostatic field on them.
Analysis of the active toxic species revealed
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organo-platinum-derivatives that were released from the
platinum-electrodes upon electrolysis. Further
analysis helped the development of platinum-derived cytostatic
drugs based on flagship-compounds
cisplatin, oxaliplatin and carboplatin (Figure 7).16
Figure 7: Structures of cisplatin, oxaliplatin and
carboplatin.
Platinum-based cytotoxic drugs diffuse passively or by active
transport via copper-transport enzymes
through the cell membrane. The chloride-concentration in the
cell is significantly lower (~ 4 mM) than
outside the cell membrane (~ 100 mM). Inside the cell,
chloride-ligands of cisplatin VII are substituted
by water molecules (Figure 8). The mono- (VII-1) or
di-aqua-species of cisplatin (VII-2) tends to bind
to guanine N7-position of DNA (VII-3), forming inter- and
intra-strand crosslinks (VII-4) that stop DNA
replication and thereby proliferation of cells. As mentioned for
other chemotherapeutics before, the fast
cell division of cancer cells triggers a successful treatment
with cisplatin VII and its analogues VIII and
IX as the DNA replication takes place more often than in
non-cancerous cells. Cancer treatment with
cisplatin usually requires administration of several small doses
of the drug. A growing issue in the
treatment of malign tumors with platinum-based cytostatic drugs
is the resistance of several cancer cell
lines against cisplatin and its derivatives that leads to a high
risk of recurrence of cancer cells that are
insensitive to platinum-derived anti-cancer drugs.16,17
Figure 8: The mechanism of action of DNA-binding of
cisplatin.16
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Besides a high risk of recurrence of cancer cells after
successful treatment, the main drawback of
chemotherapy is its low selectivity. As the therapy exploits
cancer cells’ rapid proliferation and cell
division, certain non-tumorous, but rapidly growing cells are
also attacked by chemotherapeutic agents.
These types of cells include hair follicles, which leads to hair
loss, blood cells, which leads to anemia,
and cells of the gastrointestinal system, which causes nausea,
vomiting, anorexia and diarrhea. Newly
synthesized drugs for chemotherapy focus on better selectivity
towards cancer cells and higher activity
in killing cancer cells to overcome the drawbacks of
chemotherapy.
1.2.2 Targeted Therapy: History, Tumor Targets and Treatment
Most chemotherapeutic drugs address DNA replication and cell
cycle (platin-derivatives, nucleobase
mimicrys). In targeted therapy, cancer drugs are designed to
address a specific molecular target that was
found to play a critical role in tumor signaling pathways and
growth and – in most cases – repair these
altered networks. To find and identify appropriate molecular
targets, the understanding of molecular
changes based on transformation of a cell to a tumor cell is
crucial. Evolving from the afore-mentioned
hallmarks of cancer, new therapeutic strategies promise an
elaborate concept of tumor therapy. Two
main approaches emerged for targeted therapy: therapeutic
monoclonal antibodies (mAbs) and small
molecule agents. Both therapeutic approaches address the growth
factor and signaling pathways. Latter
ones have multiple target applications, are metabolically
stable, are easily absorbed after oral
administration and show selectivity towards cancer
cells.18,13
Many small molecules for targeted therapy are designed to stay
inactive until they reach their target
where they selectively get activated. This logical approach is
referred to as prodrug concept. The inactive
drug is harmless to biological systems until it gets activated.
Activation usually occurs upon external
stimuli in cancer cells, e.g. changes in pH, enzymes or photo
activation. The active compound either
releases the drug moiety/moieties or is a drug itself.19
The following chapters focus on small-molecule tumor targets and
gives relevant examples for each
target.
Blood Supply/Angiogenesis: Bevacizumab
A target, all cancer cells have in common, is angiogenesis. The
ability of cancer cells to grow blood
vessels to ensure a sufficient oxygen saturation in tumor tissue
is directly linked to cancer cell growth
and spread. As targets, various GF were identified since 1971
that can be used for target-specific cancer
treatment. The best-characterized GF in terms of understatement
of signaling pathways is vascular
endothelial growth factor (VEGF). In 2004, a VEGF-addressing
anti-cancer drug, bevacizumab
(Avastin®), was finally FDA approved for the treatment of
colorectal cancer in combination with 5-
fluorouracil. Whereas many anti-angiogenic drugs inhibit other
targets, e.g. GF receptors, as well, until
now bevacizumab is the only drug that’s activity is clearly and
solely attributable to its anti-
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angiogenesis. By now, many drugs that were intentionally
designed to address another target in cancer
cells were found to target blood supply as well. Therefore, the
design of solely anti-angiogenic drugs
was overwhelmed by the design of multi-target drugs that usually
additionally target angiogenesis.13
Tyrosine Kinase Inhibitors: Gleevec™
Kinases are enzymes that get activated by GF and induce
phosphorylation and dephosphorylation of
proteins, proving kinase enzyme class as one of the most widely
spread and ubiquitous necessary
enzyme class in cells. Mutations of kinases where the activity
of the enzyme is blocked or silenced lead
to a variety of diseases where tumor progression is only one
example. In chronic myeloid leukemia
(CML), a fusion protein of a kinase, namely BCR-ABL, is
responsible for pathogenesis. With
conventional cancer therapies, less than 20 % of CML patients
were cured. By screening a data base of
natural products for their kinase-inhibition activity, a series
of similar structures was found to interact
with ABL tyrosine kinase and chosen as lead compound.
Optimization of the lead compound led to the
small compound imatinib-mesylate (traded as Gleevec™, Figure 9,
X). Imatinib-mesylate inhibits BCR-
ABL and – as shown later – other kinases with outstanding
efficiency. The inhibitor achieves 90 %
complete remission of CML in clinical trials and was proven to
not harm non-cancerous cells.13,20,21
Figure 9: Structure of tyrosine kinase inhibitor
imatinib-mesylate.
Over-Expressed Enzymes: Capecitabine
Besides designing new potential drugs for targeted therapy,
well-known and -understood
chemotherapeutics that show appropriate cytotoxicity but low
selectivity towards cancer cells were
tuned by introduction of a cancer cell target-specific moiety.
5-Fluorouracil VI, for example, causes
severe side effects due to no selectivity in cancer treatment.
On basis of 5-fluorouracil VI, the drug
capecitabine XI was developed. It is activated in three steps
via enzymes, that are overexpressed in
tumors. Carboxylic esterases cut the carbamate-residue of
capecitabine to yield XI-1. A second enzyme
class, cytidine deaminases, convert the amine moiety into a
carbonyl group XI-2 before thymidine
phosphorylases release 5-fluorouracil VI, the active drug. The
first enzymatic step takes place in liver,
from where XI-1 integrates to surrounding tissue. The second
enzymatic step occurs specifically in liver,
plasma and tumor tissue as these cells overexpress cytidine
deaminases. The last enzymatic step
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1 Introduction
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provides best selectivity, as thymidine phosphorylases are up to
10 times higher expressed in solid tumor
tissue than in healthy, normal tissue.22
Figure 10: Release of 5-fluorouracil from capecitabine in a
three-step enzymatic cleavage.
Tumor Hypoxia: Indolequinone 5-Fluorouracil
As mentioned before, cancer cells need for survival a sufficient
supply with oxygen. As tumor tissue
growths faster than normal cells, certain regions within the
tumor tissue will suffer from persistent lower
supply with nutrients, including oxygen. Small drug molecules
were developed to address the resulting
hypoxic regions of tumor tissues as they are hardly treatable
with conventional cancer therapy.23
An example for a drug that is selectively activated at anaerobic
conditions is an indolequinone prodrug
derivative of 5-fluorouracil (Figure 11, XII).
Figure 11: Mechanism of degradation of indolequinone prodrug
derivative of 5-fluorouracil XII
to release active cytotoxic drug VI.
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1 Introduction
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The drug shows almost no cytotoxicity against tumor cells under
aerobic conditions, but excellent
cytotoxicity under hypoxic conditions with an IC50 of 150 nM.
Compound XII is activated under
hypoxic conditions to give hydroquinone XII-1. Further
degradation releases iminium cation
hydroquinone XII-2 and the active cytotoxic drug VI. The
50-times lower IC50-value of XII compared
to its parent compound VI was found to be caused by the iminium
cation XII-2 that exhibited strong
cytotoxic effects against various cancer cell lines.24
Reactive Oxygen and Nitrogen Species (ROS and RNS)
The elevated basal level of reactive oxygen (ROS) and nitrogen
(RNS) species provides a ubiquitous
target for cancer specific treatment. ROS and RNS are small but
extraordinary important molecules in
signal transduction and involved in numerous enzymatic
metabolisms. Therefore, by addressing
abundant ROS and RNS in cancer cells, a variety of cell
metabolic pathways are affected. The drugs
discussed in this work exploit the increased level of ROS in
cancer cells. The following chapter starts
with an introduction into ROS generation and detoxification,
notes the most important anti-oxidants that
are biosynthesized in cells and finally discusses, how ROS can
be used to selectively treat cancer.
1.2.3 Reactive Oxygen Species
Reactive oxygen species (abbr. ROS) are small, highly reactive
molecules that are found in humans,
animals and plants25. ROS are peroxides, anionic or radical
species of oxygen and are therefore
inherently high reactive. Best known for their abundant need in
biological systems are superoxide radical
anions (O2‾•), hydrogen peroxide (H2O2), singlet oxygen (
1O2) and the hydroxyl radical (HO•). These
small molecules are crucial counterparts in signal transduction,
cell differentiation and -proliferation, as
well as in pathogen resistance and therefore are produced
ubiquitously in the human body. High
intracellular concentrations of ROS lead to damage of
macromolecules like lipids, proteins and DNA
and finally to oxidative stress and apoptosis.26
As displayed in Figure 12, the generation of ROS originates from
a single-electron transfer to molecular
oxygen. Oxygen, in its triplet ground state, displays two
unpaired electrons that occur in different
orbitals and possess parallel spin. Therefore, an additional
electron must be antiparallel (except of the
formation of singlet oxygen 1O2, where with energy one electron
with parallel spin is forced to turn its
spin around to give a pair of antiparallel electrons). After
addition of one antiparallel electron, the
resulting superoxide ion can further accept one electron to give
the peroxide and after addition of one
more electron to give the O23-ion. The last one degrades under
consumption of two H+ to water and the
oxene ion. The ion can be further oxidized to the oxide ion.
Second-grade ROS include perhydroxyl
radical (HO2•), hydrogen peroxide (H2O2), and the hydroxyl
radical (HO
•).25
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1 Introduction
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Figure 12: General formation of ROS starting from O2.25
ROS are found in the environment as pollutants, tobacco smoke,
iron salts and radiation26. ROS have
been divided into two major categories, according to their rate
of reactivity and damage against organic
molecules/biomolecules (Table 1). Slow reacting ROS usually do
not have radicals or only have radicals
that are well stabilized and sterically hindered within the
molecule. As they react slowly with
surrounding molecules, their concentration in cells is,
depending on the rate of formation, usually high.
Fast reacting ROS are usually formed in situ and rapidly
consumed up in solution. They react with
almost any surrounding molecule, as exploited in several assays
for ROS detection, and are thus
considered as strong oxidative, highly cytotoxic agents that can
be used for cancer treatment. In
mammalian cells, slow reacting ROS are usually predominant as
they are less harmful for DNA and cell
organelles. Although, when needed, these slow reacting ROS are
transformed into fast oxidizing, highly
toxic ROS that support the immune system to defeat
pathogens.27
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Table 1: Reactive oxygen species (ROS): Reactivity and damage on
cell compartments27
Name Formula Reactivity
(M-1s-1)
Damage against
Slo
w r
eact
ing
RO
S
Hydrogen peroxide H2O2 0.9
2 x 10-2 28
Variety of
biomolecules
Thiols (as cysteine):
oxidation to sulfonic
acid and thereby
inactivation
Peroxyl HOO• < 5 x 101 28 Biomolecules
Fas
t re
acti
ng R
OS
Hypochlorous acid HOCl 3-4 x 107 28 Biomolecules
Superoxide anion O2•‾
3 x 103 - 107
< 0.3 28
Fe-S-cluster-proteins:
Fe2+-release
Hydroxyl radical29 HO• 4-7 x 109 28 DNA (irreversible):
oxidative
damage/mutations
Proteins/Thiols:
oxidation to
sulfoxides and
thereby inactivation
Organic peroxy
radicals/alkoxy
radical30,31
RO2
RO•
1-9 x 1010 Lipids: oxidation +
peroxidation
The generation of fast reacting, highly toxic ROS out of slow
reacting ROS involves iron- or copper-
metal ions in cells. The Fenton-Haber-Weiss-reaction summarizes
redox-reactions that lead to fast
reacting ROS in cells (Figure 13). In aqueous systems, Fe2+ and
oxygen are in equilibrium with Fe3+ and
superoxide. Fe3+ can catalyze the reaction of hydrogen peroxide
to HO2• and a proton. H2O2 can also be
reduced to hydroxy radical and hydroxide anion. The latter
reaction is called Fenton-reaction. In total,
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1 Introduction
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two molecules of slow reacting ROS hydrogen peroxide and one
molecule of non-toxic oxygen are
transformed into highly toxic superoxide, hydroxy radical and
HO2•.
Figure 13: Combination of Fenton- and Haber-Weiss-reaction. Fe2+
and Fe3+ catalyze the generation
of fast reacting ROS out of slow reacting ones.
ROS can be taken up by cells from the extracellular space, e.g.
via signal transduction, but the major
source of ROS is the metabolism of cells themselves. Cells can
produce ROS via various mechanisms.
In biological systems, enzymes play major roles in the
generation of ROS. Enzymes consist of a metal
core center for catalysis and peptides that build the enzyme
structure. The metal center can transfer
electrons to surrounding molecules, provided that the molecules
are small enough to enter the active site
of the enzyme (Figure 14). Oxygen is converted into superoxide
at the electron transport chain (ETC)
of mitochondrial respiratory chain or NADPH oxidase (NOX).
Superoxide can react with NO• to give
ONOO-. More likely, superoxide is converted into hydrogen
peroxide (H2O2) by superoxide dismutase
(SOD). The slow reacting and thereby less harmful H2O2 can react
with Cl-, for example, to give HOCl
catalyzed by myeloperoxidase (MPO). If iron is present, hydrogen
peroxide can take up one electron
from Fe(II) to disproportionate to hydroxy radical and hydroxy
anion. The former one can abstract one
proton from other alcohols, resulting in oxygen-centered
radicals. After uptake of one more electron and
a proton, water is formed, and the detoxification process
completed.26,27
Figure 14: General scheme for ROS production including cellular
enzymes. ETC: Electron
Transport Chain; NOX: NADPH oxidase; SOD: Superoxide dismutase;
MPO: Myeloperoxidase;
The first cellular compartment identified to play an important
role in maintaining redox homeostasis in
cancer cells was mitochondria. In 1966, mitochondria was found
to be the main producer of reactive
oxygen species, respectively superoxide anion (O2•-). The
mitochondrial respiratory chain, in particular
the electron transport chain (ETC), unintentionally produces a
small amount of superoxide and hydrogen
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1 Introduction
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peroxide (0.1 – 2 % of all electrons passing through ETC) due to
a lack of full reduction of molecular
oxygen to molecular water (compare Figure 15). Even though ROS
are only by-products of the ETC, ist
high efficiency makes the mitochondria the biggest producer of
reactive oxygen species in cells. Another
well-understood source of ROS is ubiquitous NADPH oxidase (NOX).
First mentioned in 1990, it is
now known that there are seven enzymes, all belonging to
NADPH-oxidase-family, that use NADPH as
substrate to produce superoxide out of molecular oxygen in a
one-electron-transfer-reaction. The
activation of the enzyme is strongly dependent on the ligands,
often also referred to as co-factors: growth
factors (GF), chemokines or tumor necrosis factors can bind to
the enzyme and thereby regulate its
activity.26,27
Figure 15: Electron transport chain (ETC) within the
mitochondrial respiratory chain.
There are numerous enzymes more in cells that produce ROS.
Mammalian targets of rapamycin (mTOR), p53 and BCL-2 were
found
to increase the ROS-level in cells. Any kind of oxidase or
oxygenase that
is redox-active (i.e. xanthine oxidase (XO), lipoxygenases,
cyclooxygenases) unintentionally produce ROS due to a lack
for
electrons. Even the heme-center of CypP450 (Figure 16) produces
ROS
due to its ability to transfer electrons from the iron-atom in
heme to
surrounding molecules.26,27
As signaling molecules, ROS play an important role in cell
proliferation,
growth and survival. To ensure cell growth and proliferation but
no
toxification with ROS, each cell balances oxidants and
antioxidants
carefully to maintain redox-homeostasis, an equilibrium between
production and scavenge of ROS
(Figure 14)27,25. Any perturbance in homeostasis in- or
decreases the ROS-level of cells, for example, as
environmental changes can lead to over-expression of enzymes,
that produce ROS. Perturbances in the
redox homeostasis usually lead to the so-called oxidative burst
that will be further explained in chapter
1.2.3.2. To prevent oxidative burst, cells evolved a
detoxification mechanism for ROS including
enzymes that use ROS as substrates to generate water molecules
and antioxidants that react with free
radicals to prevent cells from oxidative damage and cell
death.
1.2.3.1 Antioxidants
A slightly increased level of ROS leads to cell proliferation
and cell growth whereas a significant
increase in ROS-concentration in cells results in apoptosis
and/or necrosis of the cell. It is crucial for
Figure 16: Heme b.
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cell survival to carefully balance ROS generation and ROS
degradation depending on cell status, micro-
and macroenvironment.
With the target to maintain a low basal level of ROS but still
proliferate, cells evolved antioxidants like
ascorbates, glutathione (GSH), tocopherols, flavonoids and other
small molecules for nonenzymatic
ROS-scavenging as well as numerous enzymes for detoxification
like superoxide-dismutases (SOD),
catalases (CAT) and peroxidases (Px). The ubiquitous redox
homeostasis system in cells is displayed in
Figure 17.
Figure 17: Oxidant and antioxidant system and their role in
reactive oxygen species (ROS)
generation and degradation. ETC = electron transport chain; NOX
= NADPH oxidase; SOD =
superoxide dismutase; MPO = myeloperoxidase; GPO =
glycerol-3-phosphate oxidase; Cat = catalases;
AA = amino acids.
Glutathione (GSH, Figure 18, XIV) plays a key role in
antioxidant response to an increased level of
ROS. It donates electrons of its thiol-sidechain to ROS and
reacts afterwards with another molecule of
glutathione to from glutathione-disulfide (GSSG, XV). The
process of building a disulfide-bridge is
catalyzed by the enzyme glutathione peroxidase. GSSG can be
converted back to the active scavenger
by the enzyme glutathione reductase under consumption of
NADH.27
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Figure 18: Structures of antioxidant glutathione (GSH, XIV) and
its oxidized counterpart GSSG
(XV).
The before described prooxidative and antioxidative systems
establish a broad and diverse platform for
treatment methods. The perturbance of ROS homeostasis in cancer
cells is an excellent tool for inducing
selective oxidative stress in cancer cells as the homeostasis in
cancer cells is already altered. The
following chapter focusses on exploiting the elevated ROS level
in cancer cells by induction of small
molecules as drugs.
1.2.3.2 ROS in Cancer Treatment
Perturbances of redox homeostasis in cells lead to oxidative
stress. Primarily described as an imbalance
between antioxidants and prooxidants in a biological system in
favor of the prooxidants by Helmut Sies
in 1985, until now, the concept of oxidative stress is still not
well defined or described in literature
although its use is wide spread. The hypothesis of oxidative
stress tries to summarize a number of sub-
molecular to enzymatically flow-down mechanisms that can be
affected even by small, redox-active
molecules. To formulate a concept that describes both, the
small, immediate impact of a redox active
drug on the oxidative system of a cell as well as the secondary,
big impacts on the biosynthesis, immune
response and cell signaling of the cell, is almost
impossible.32
Several attempts address the categorization of oxidative stress
regarding physiological damage,
nitrosative stress or toxic oxidative stress. In accordance with
the categorization one must consider
intensity of damage, going from a basal oxidative stress level
to highly intensive to apoptotic oxidative
stress. Nevertheless, all descriptions of oxidative stress share
an elevated oxidative environment that
leads to alterations in cell metabolism and ultimately to cell
death. In accordance with the scientific
point of view, oxidative stress is usually caused by a
chemotherapeutic (pro)drug that is introduced into
the cell. The drug either inhibits the antioxidative system of
the cell or is redox-active itself. In the
former case, the drug disturbs the detoxification from ROS and
other reactive species and thereby
indirectly acts as prooxidant. In the latter case, the drug acts
as prooxidant (in elaborated cases, the drug
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1 Introduction
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is designed to do both in parallel). By altering the
redox-balance of a cell, homeostasis is disrupted and
the amount of ROS in cell increases, leading to apoptosis,
necrosis or combined cell death.26,32
The up to ten-fold increased basal level of ROS in cancer cells
marks a potent target for selective cancer
therapy33. Cancer cells tend to react more sensitive to
additional, induced oxidative stress than non-
cancer cells (Figure 19). Healthy cells can easily handle a
slightly increased level of ROS due to their
reserve antioxidant capacity. Thereby, in non-cancer cells,
redox homeostasis is maintained. In contrast,
in cancer cells, the additional amount of ROS leads to a
transgression of the toxic threshold and thereby
results in cancer cell death.26
Figure 19: Concept of cancer redox biology. The artificial ROS
increase leads to cancer cell specific
cell death.
Several chemotherapeutics target the elevated level of ROS in
cancer cells. To understand design goals
for effective ROS-modulating anti-cancer drugs, landmarks of
ROS-targeting chemotherapeutics and
their mechanisms of action are highlighted in the following
section.
An example for a therapeutic, prooxidative drug is the group of
bleomycins (Figure 20, XVI). First
isolated in 1966 from bacteria by H. Umezawa and colleagues,
bleomycins were soon found to be perfect
suitable not only for treatment bacterial infections, but also
for combinational therapy of testicular
cancer and lymphoma. One molecule of the bleomycin-family,
Bleonoxane, is already in clinical use.
In combination with cisplatin and etoposide, 90 % of testicular
cancers are cured with Bleonoxane.34
Bleomycins, representative of Bleonoxane, are administered in
their inactive form. To be activated, a
reduced transition metal as Fe(II) or Cu(I) and oxygen is
needed. Bleonoxane forms a complex with the
metal, where one metal ligand is oxygen. The transition metal
can donate one electron to oxygen,
resulting in a highly toxic superoxide anion (O2•‾). The active
intermediate is formed by consumption
0
20
40
60
80
100
120
Normal Conditions ROS increaseRO
S l
evel
(arb
itra
ry l
inea
r sc
ale
)
Normal Cells Cancer Cells
Threshold of cell death
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of an electron and a proton, resulting in bleomycin-Fe(III)-OOH.
This active intermediate can cleave
DNA and RNA as well as proteins by generating highly reactive
hydroxyl radicals, which ubiquitously
react with every molecule in their immediate vicinity. The
active complex binds to DNA/RNA by its
bithiazole-tail and the nitrogen-atoms of pyrimidine. ROS
generated in direct surrounding of DNA/RNA
induce strand brakes. Unfortunately, bleomycins were found to
induce dose-dependent pneumonitis that
affects half of the number of patients treated with; 3 % of them
actually die from pneumonitis.34
Figure 20: Generalized structure of bleomycin with structural
units.
In contrast, NO-donating aspirin (NO-ASA, Figure 21, XVII) works
by suppressing the antioxidative
system of a cell and thereby, indirectly, enhancing the
ROS-level in cells. Nitrogen monoxide donating
para-acetylsalicylic acid XVII is cleaved enzymatically by
cellular esterases into acetylsalicylic acid
(ASA, XVII-1) and nitrogen monoxide-p-quinone (XVII-2).
NO-donating unit XVII-2 provides a
precursor of p-quinone methide (XVIII) that disproportions
further by 1,6-elimination to p-quinone
methide and nitrate. p-Quinone methide XVIII can react with
nucleophiles like antioxidant glutathione
(GSH) and bind them irreversible. The cytotoxic effect of NO-ASA
XVII is caused by the perturbance
of the antioxidative system by p-quinone methide as
GSH-scavenger and not, as assumed before, by the
release of NO3- and acetylsalicylic acid XVII-135. Thereby, the
redox homeostasis is disturbed, the level
of ROS increases and the cell finally undergoes apoptosis.
Unfortunately, NO-ASA XVII does not
selectively alter the ROS-amount of cancer cells, but of all
cells. The risk of the affection of healthy,
non-cancer cells is increased and can lead to severe side
effects as well as secondary tumors.36,37
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1 Introduction
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Figure 21: Mechanism of action of ROS-enhancing drug NO-ASA.
Glutathione is the basis of the anti-oxidant system of cells and
therefore a gladly used target for redox
interference. Enzymes like peroxidases, peroxiredoxins and thiol
reductases consume reduced GSH to
maintain redox homeostasis and cell biosynthesis. GSH-scavenging
as with NO-ASA XVII or inhibition
of GSH-synthesis/reduction leads to a depletion of GSH and
oxidative stress. Buthionine sulfoximine
(BSO, Figure 22, XIX), for example, causes cancer cell death by
inhibition of an enzyme for GSH-
synthesis. The main drawback of targeting GSH and thereby the
antioxidative system is drug resistance.
Especially tumor tissue in the late disease stage is
well-adapted to oxidative stress and provides a well-
established antioxidative system. Consequently, these cancer
cells inherit resistance mechanisms against
many anti-cancer drugs (e.g. cisplatin VII) and a need of new
and efficient treatment methods ignited
the search for new potential anti-cancer drugs.26
On the search for new redox-active agents, drugs that are
already known to generate ROS in other
treatment methods were tested for their efficiency in cancer
therapy. Growing interest in natural products
for cancer treatment arouse in the course of highly potent
tubulin-targeting paclitaxel and its
derivatives38. Most recently, artemisinin-based drugs were
tested for their cytotoxic efficiency (Figure
22, XX). In 2015, T. Youyou was awarded the Nobel prize for
physiology and medicine for her research
on artemisinin as anti-malarial. In parallel, first
investigations on the cytotoxicity of artemisinin were
done by Y. Yuthavong and C. R. Chitambar. Artemisinin, a natural
product derived from artemisia
annua, is a sesquiterpene bridged by a peroxide-bridge. The
peroxide can degrade under catalysis of
iron, releasing oxygen-centered, highly reactive and therefore
toxic radicals. Erythrocytes and
plasmodia, the main target for malaria-treatment, accumulate
iron ions. As soon as artemisinin
accumulates in plasmodia, the plasmodium gets destroyed by an
increase in oxidative stress. The
mechanism of action is proposed as described, but still not
fully understood nor clarified.39
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1 Introduction
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Figure 22: Structures of cytotoxic drug BSO (XIX),
anti-malarial- and anti-cancer-drugs
artemisinin XX and artesunate XXI and a representative of an
artemisinin-quinazoline-hybrid
XXII.
The main drawback of artemisinin as drug is the lack of good,
high-yielding synthetic access to the final
product. Semi-synthetic derivatives of artemisinin, namely
artesunate (Figure 22, XXI), are easier to
access and active derivatives of artemisinin that are believed
to act in the same manner as the parent
compound. Artesunate XXI was tested against 55 cell lines with
the result that it showed excellent
cytotoxicity against leukemia (50 % of maximal inhibition of
cell proliferation (GI50) = 1.11 ± 0.56 µM)
and colon cancer (GI50 = 2.13 ± 0.74 µM) cell lines40. Besides
an elevated level of intrinsic oxidative
stress due to ROS-generation of artesunate XXI, the cytotoxic
activity was linked to an additional
mechanism of action that includes the down-regulation of hBUB3,
a mitotic spindle assembly
checkpoint gene that modulates anaphase initiation. This
combined mechanism ultimately leads to cell
cycle arrest and/or apoptosis.40
Although the dual mechanism of action of artesunate XXI was not
rationally designed but rather
unintentional, the combination of two or more mechanisms of
action unambiguously potentializes a
cancer drug’s efficiency. In aim to exploit not only the
elevated level of oxidative stress in cancer cells
but additionally overcome drug resistances, the combination of
several drugs or prodrugs within one
molecule experienced a rise of interest in the last years.
Hybrids of artemisinin and multi-use-drug
quinazoline (Figure 22, XXII) were found to inhibit leukemia
cancer cell growth in the low micromolar
range and exhibited micro- to nanomolar activity against
bacteria and viruses41-43. The combination of
ROS-generating and GSH-depleting drugs is of particular appeal
as these agents address the same target,
disturbance of redox homeostasis, from two sides.
The cytotoxicity of chemotherapeutic, electrophilic alkylating
agents like nitrogen mustards II or
cisplatin VII and its derivatives was reported to partially rely
on the irreversible reaction with N-atoms
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1 Introduction
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of DNA or S-atom of glutathione XIV35. Clathrochelates, caged
structures that combine a cage of
electrophilic, alkylating agents that scavenge GSH XIV, and a
Fe2+-core that produces ROS in a Fenton-
Haber-Weiss-type reaction upon release, were designed and
analyzed by Y. Z. Voloshin et al. (Figure
23, XXIII)44-46. The hexachloro-substituted Fe-clathrochelate
XXIII inhibited HL-60 cancer cell growth
by 50 % at a concentration of 6.5 ± 4.6 µM. The negative
control, a fully alkylthiolated clathrochelate
that could not react with GSH XIV, was found to be non-toxic in
cancer cells (IC50 > 50 µM). This result
led to the assumption that ROS-generation of clathrochelate
XXIII was possible due to partial
substitution with nucleophiles as GSH as the fully substituted
derivative (XXIII-1) did not generate
ROS in vitro.45
Figure 23: Reaction of clathrochelate XXIII with GSH XIV to form
XXIII-1.
The main drawback of cancer redox biology and its drugs is the
administration of an already active
compound. The compound affects non-cancerous cells in the same
way as cancerous cells, leading to
side effects, necrosis and – in the worst case – evolution of a
new tumor tissue. To achieve selectivity
towards cancer cells and suppress side effects by eradication of
non-cancerous cells, an elegant method
for designing new ROS-targeting anti-cancer drugs includes a
ROS-specific activatable unit. The
inactive prodrug is introduced into all cell, no matter if
cancerous or not, but is selectively activated by
the elevated ROS-level in cancer cells. After cleavage of the
trigger moiety, the active drug is released
and affects the cancer cell whereas the non-cancerous tissue
remains unaffected (Figure 24).
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1 Introduction
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Figure 24: Cancer specific redox biology concept.26,27,
modified.
One of the first examples for a ROS-activated cancer drug is a
nitrogen mustard-based prodrug (Figure
25, XXIV). The ROS-sensitive trigger moiety is a boronic ester
that gets cleaved by hydrogen peroxide.
Phenolic intermediate XXIV-1 further degrades and releases one
molecule of p-quinone methide
(XVIII) and DNA-crosslinking active drug nitrogen mustard (II).
Compound II forms in solution highly
reactive aziridium-ion XXIV-2 that reacts with guanine bases of
DNA, resulting in irreversible DNA-
interstrand-crosslinks (XXIV-3). The formation of DNA duplex
interstrand cross-links was observed in
vitro exclusively in the presence of hydrogen peroxide. In cell
experiments, X. Peng et al. proved the
successful treatment of leukemia, lung and renal cancer cell
lines and no cytotoxicity of nitrogen-
mustard prodrug against non-cancerous human
lymphocytes.27,47-49
Threshold of cell death
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1 Introduction
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Figure 25: Activation of nitrogen mustard prodrug XXIV by
hydrogen peroxide.
ROS-sensitive triggers were proven to be versatile, promising
inducers of selectivity for anti-cancer
drugs. Since the first use of hydrogen peroxide-sensitive
trigger boronic acid for cancer treatment in
2011, several attempts were made to connect the selectivity
marker to already existing and/or new
developed potential prodrugs. For further reading on
ROS-triggered prodrug concepts, the reviews of
M. H. Clausen et al.50 and X. Peng/V. Gandhi49 are highly
recommended.
1.3 Ferrocenes, Aminoferrocenes and Oligo-(Amino)Ferrocenes for
Cancer Treatment
Ferrocene, a small sandwich-complex consisting of two
cyclopentadienyl-ligands (cp‾) coordinating
ionically an iron(II) center, was discovered in the early 1950
by Kealy, P. L. Pauson and Miller
independently (Figure 26, XXV)51. Its incompatible stable
nature, easy handling and non-toxicity made
ferrocene XXV a major subject for chemical, physical and
biological investigations52. In 1975, the
redox-switchable properties of ferrocene XXV were already
well-known and -analyzed by Raman and
IR-spectrometry, electronic absorption and paramagnetic
resonance measurements53. The first clinical
use of ferrocene XXV is traced back to the treatment of iron
deficiency anemia in USSR around 1970
with ferrocerone, a ferrocene derivative (Figure 26, XXVII)54.
Ferrocerone XXVII is thought to be not
only the first, but also the only ferrocenyl drug ever in
medicinal use. It can be administered orally due
to the lipophilicity of ferrocenyl groups, and is still used as
drug. Eight years later, first investigations
on antigen-bearing ferrocene derivatives and their use as
anti-cancer-agents were published by S. Brynes
et al.55. The antigen was designed to bind to nucleic acids and
thereby cause an antigenic response, but
the analyzed molecules showed no cytotoxicity in mice with
lymphotic leukemia.
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1 Introduction
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Figure 26: Structures of ferrocene, ferrocenium and
ferrocerone.
It took a few more years until the first investigations on
ferrocene/ferrocenium-salts as anti-tumor-agents
were performed by P. Köpf-Maier, H. Köpf and E. W. Neuse in
198456,57. The investigation of several
metallocenes regarding their activity in cancer cell lines
(metallocene dihalides of early transition
metals58,59) failed to give the desired results, until medium to
late transition metal sandwich complexes
were synthesized and analyzed. The results opened a new field of
application for the iron(II)-
metallocene: It’s charged counterpart ferrocenium XXVI was found
to be an active cancer drug against
Ehrlich ascites tumor (EAT) in CF1-mice (whereas ferrocene
itself did not show any tumor suppressive
character; Figure 26). Until 1990 it was thought that only
already-oxidized ferrocenium XXVI can act
as tumor drug or drug in general. E. W. Neuse and F. Kanzawa
showed that ferrocene XXV and its
positively charged counterpart exist in equilibrium in cellular
compartments, introducing ferrocene-
derivatives as prodrugs for cancer treatment60. Almost thirty
years ago, the proposed mechanism of
action for ferrocenium XXVI depicted the coordination of the
iron(III)-core atom of ferrocenium XXVI
to DNA and an intercalation-caused stop of replication61. DNA
cleavage in vitro was proven to be
generated by hydroxyl radicals through Fenton mechanism,
catalyzed by ferrocenium XXVI
compounds, in 199762. Still it remained to be clarified whether
ferrocenium intercalates into DNA and
hydroxyl radicals are genera