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

Für meine Eltern

Ein gerader Weg führt immer nur ans Ziel.

- André Gide

Content

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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.

Abbreviations

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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

Abbreviations

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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

Abbreviations

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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

Abbreviations

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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

1 Introduction

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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.

1 Introduction

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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.

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.

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

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

1 Introduction

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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

1 Introduction

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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.

1 Introduction

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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

1 Introduction

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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

1 Introduction

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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.

1 Introduction

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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

1 Introduction

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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-

1 Introduction

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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

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.

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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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

1 Introduction

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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,

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

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.

1 Introduction

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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

1 Introduction

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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

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

1 Introduction

Page | 32

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

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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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

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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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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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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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