METAL-PEPTIDE BIOCONJUGATES FOR TARGETED ANTI- THERAPY

Dissertation

submitted to the Faculty of Chemistry and Biochemistry at the Ruhr-University Bochum, Germany to obtain the degree Doctor of Natural Sciences

presented by Dariusz Śmiłowicz, M. Sc.

Bochum, January 2020

This work has been carried out between December 2015 and January 2020 under the supervision of Prof. Dr. Nils Metzler-Nolte at the Chair of Inorganic Chemistry I – Bioinorganic Chemistry at the Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum.

Date of oral examination: 08.05.2020 1st Referee: Prof. Dr. Nils Metzler-Nolte 2nd Referee: Prof. Dr. Gilles Gasser

2 Acknowledgements

First of all, I would like to express my sincere gratitude to Prof. Dr. Nils Metzler-Nolte for giving me the opportunity to carry out my Ph.D. thesis under his supervision. In my opinion the most important is to work and learn from intimidating person and outstanding scientist. I was lucky to have both. Nils is a lively, enthusiastic, and energetic person, and is always in a good mood, eager to discuss any issue. This is particularly amazing. Moreover, I am also very grateful to Nils for his scientific advices, knowledge, many insightful discussions and suggestions, and for giving me the freedom to pursue various projects.

I would like to gratefully acknowledge Prof. Dr. Gilles Gasser for being the second referee of my thesis.

In particular I‘d like to thank Dr. Reece Miller for many scientific and life-related discussions. He belongs to the group of interesting people, which you may be lucky to encounter on your way.

Dr. Jack Slootweg, my supervisor in the lab during my first practical time in this group, I also thank for his guidance and a highly inspiring time.

To Dr. Matthew Reback, Sugina Thavalingam and Pina Eichert, I would like to say massive thanks for always advising on chemistry matters, the interesting discussions, countless advices, valuable time and for always being supportive.

All the members of the AC1 group: Isabelle Daubit, Liudmila Janzen, Milena John, Nicole Lorenz, Daniel Obitz, Dr. Evgenia Olshvang, Dr. Sreedhar Kumar Vellas and Dr. Joan Soldevila-Barreda, I thank for the constant support and for their remarkable willingness to help.

I would also like to thank the former Ph.D. students in this group, Dr. Martin Strack, Dr. Daniel Siegmund, Dr. Kathrin Klein, Dr. Anna Cordes and Dr. Yvonne Gothe. They have been helpful in providing advice many times during my research. And a special thanks to Nicole Ray, Dr. Christiane Klare, Carsten Lodwig, Marvin Heller, Annegret Knüfer and Dr. Klaus Merz. I could always ask them for advice and opinions on lab and on life related issues.

And last but not least, I would like to especially thank my mom, sisters, and brothers. I know I can always rely on my family when times are rough.

3

―Celui qui passe à coté de la plus belle histoire de sa vie n'aura que l'âge de ses regrets et tous les soupirs du monde ne sauraient bercer son âme.‖

- Yasmina Khadra, Ce que le jour doit à la nuit

―The man who let the love of his life pass him by will end up alone with his regrets and all the sighs in the world won't soothe his soul.‖

- Yasmina Khadra, What the day owes the night

4

Content

1 Introduction...... 9

1.1 Metals in anti-cancer therapy...... 9

1.2 Cell penetrating peptides...... 19

1.3 Cell targeting peptides...... 22

1.4 Metal-peptide bioconjugates...... 25

2 Objective...... 29

3 Results...... 30

3.1 Synthesis of monofunctional (IV) carboxylate precursors for use in Pt(IV)–peptide bioconjugates...... 30

3.1.1 Abstract...... 30

3.1.2 Introduction...... 31

3.1.3 Results and discussion...... 33

3.1.4 Conclusion...... 44

3.1.5 Experimental section...... 45

3.2 Bioconjugates of Co(III) Complexes with Schiff Base Ligands and Cell Penetrating Peptides: Solid Phase Synthesis, Characterization and Antiproliferative Activity...... 51

3.2.1 Abstract...... 51

3.2.2 Introduction...... 52

3.2.3 Results and discussion...... 55

3.2.4 Conclusion...... 65

3.2.5 Experimental section...... 66

5 3.3 Bioconjugation of cyclometalated gold(III) lipoic acid fragment to linear and cyclic breast cancer targeting peptides...... 71

3.3.1 Abstract...... 71

3.3.2 Introduction...... 72

3.3.3 Results and discussion...... 76

3.3.4 Conclusion...... 87

3.3.5 Experimental section...... 88

4 Summary...... 93

5 Literature...... 95

6 List of contributions...... 121

7 Appendix (Supporting Information)...... 122

7.1 Appendix for chapter 3.1...... 122

7.2 Appendix for chapter 3.2...... 134

7.3 Appendix for chapter 3.3...... 145

6

List of abbreviations

AA amino acid

ACN acetonitrile

Boc tert.-butyloxycarbonyl

Carboplatin [(diammine)(1,1-cyclobutanedicarboxylato)platinum(II)]

Cisplatin cis-[PtCl2(NH3)2]) CBDC cyclobutane-1,1-dicarboxylate CDDP cis-[diamminedichloridoplatinum(II)] () DACH diaminocyclohexane DCM dichloromethane

DIPEA N,N-diisopropylethylamine

DMEM Dulbecco‘s Modified Eagle‘s Medium

DMF dimethylformamide

DMSO dimethyl sulfoxide

EDTA ethylenediaminetetraacetic acid

ESI electrospray ionisation

Et2O diethyl ether

FDA Food and Drug Administration

Fmoc fluorenylmethoxycarbonyl

Heptaplatin [(Propanedioato)(2-(1-methylethyl)-1,3-dioxolane-4,5- dimethanamine)platinum(II)] HOBt 1-hydroxybenzotriazole

HPLC high performance liquid chromatography

IC50 half maximal inhibitory concentration

IR infrared lobaplatin [(2-Hydroxypropanoato)(1,2-cyclobutanedimethanamine)platinum(II)]

7 m/z mass to charge ratio

MALDI matrix assisted laser desorption/ionization

MeOH methanol

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [diammine(hydroxyacetato)platinum(II)] NMR nuclear magnetic resonance ox oxalate [1(R),2(R)-cyclohexane-1,2-diammine](oxalato)platinum(II)

PBS phosphate buffered saline

SPPS solid-phase peptide synthesis

TBTU O-(benzotriazol-1-yl)-N,N,N‘,N‘-tetramethyluronium tetrafluoroborate

TFA trifluoroacetic acid

TIS triisopropylsilane

For amino acids 1-letter codes as abbreviations are used. D-amino acids are shown in lower- case letters.

8

1 Introduction

1.1 Metals in anti-cancer therapy

In medicinal bioinorganic chemistry, we can observe a clear shift from organic molecules to metal-containing complexes.1 Such choice is not surprising since metals possess advantages over conventional carbon-based compounds, such as partially filled d orbitals, a wide range of coordination numbers and geometries, accessible redox states, thermodynamic and kinetic characteristics, and the intrinsic properties of the cationic metal ion itself. 2 Different coordination numbers and modes provide a wide range of geometries, which eventually influence the pharmacological properties of metal complexes. 3 Furthermore, metal-based complexes exhibit one more major advantage over purely organic, carbon-based compounds, namely the ability to undergo ligand exchange reactions.4 This characteristic provides the capacity to interact reversibly as well as irreversibly with various biological molecules. 5

Of course such a shift and the wide use of metal complexes as anti-cancer agents certainly started from the serendipitous discovery of cisplatin‘s properties. 6 Cisplatin is an internationally approved drug for the treatment of ovarian, cervical, head and neck, non-small cell lung carcinoma and testicular .7 Moreover, it is commonly used in combination treatment with , , 5- and . 8 Cisplatin

[PtCl2(NH3)2], a platinum(II) inorganic complex, is comprised of two ammonia moieties as carrier ligands and two chloride atoms as leaving groups. In living cells, cisplatin can be taken up by either passive or active transport (Figure 1).9 The high concentration of chloride ions in blood plasma, around 100 mM, prevents cisplatin from early hydrolysis. After cellular uptake, cisplatin‘s chloride ligands are replaced by water molecules, due to low intracellular chloride concentrations (4-23 mM). This step is crucial, since only activated cationic species can bind 10 2+ to DNA. These [Pt(H2O)2(NH3)2] species bind covalently to nitrogen atoms of guanine or adenine from DNA and form mainly 1,2- and 1,3-intrastrand cross-links. The adducts between cisplatin and DNA cause the inhibition and finally of cells. 11 Along with high activity and an impressive cure rate, cisplatin is unfortunately also responsible for

9 variety of severe side effects, such as neuro-, hepato- and nephrotoxicity. 12 The use of cisplatin is also highly hampered by inherent as well as acquired resistance.13

Figure 1. The cytotoxic pathway for cisplatin.7

The discovery of the anti-proliferative effect of cisplatin and especially the above mentioned limitations of it triggered the interest in research on other platinum(II) complexes. 14 This led to the second generation platinum-based anti-cancer agent ([Pt(CBDC)(NH3)2]), which was developed and approved by the FDA in 1989 (Figure 2). 15 The chelating dicarboxylate ligand increases the stability due to the formation of a six-membered ring. Carboplatin is used effectively in the treatment of ovarian carcinoma, lung, and head and neck

10 cancers, since it overcomes side effects, such as ototoxicity and nephrotoxicity compared to cisplatin. Among third generation anti-cancer platinum drugs, oxaliplatin ([Pt(ox)(DACH)]), possessing chelating carrier as well as leaving groups and received approval by the FDA in 2002 (Figure 2).16 Oxaliplatin is clinically used for the treatment of colorectal cancer, which is resistant to cisplatin. Further platinum(II) dicarboxylates, namely nedaplatin, lobaplatin and heptaplatin (Figure 2) have been approved for clinical use in Japan, China, and South Korea, respectively.17 However, despite the improved aqueous solubility, higher stability and slightly diminished side effects of carboplatin and oxaliplatin compared to cisplatin, all these anti- cancer drugs exhibit the same mechanism of action, possess a similar spectrum of activity and lead to intrinsic and acquired resistance.18,19 Another, different approach focuses on the synthesis of complexes, which can bind selectively to DNA through non-covalent interactions. Triplatin tetranitrate BBR3464 (TriplatinNC) is an unusual trinuclear platinum complex with an overall charge of +4 (Figure 2).20 This complex interacts with DNA through hydrogen bonds between phosphate and amine groups. 21 TriplatinNC even reached the phase II in clinical trials on lung cancer. However, because of undesired side effects, such as neutropenia and diarrhoea, further studies on triplatinNC were abandoned.22

Figure 2. Structures of platinum(II) anti-cancer drugs.

11 To reduce the side-effects of platinum(II) drugs, a prodrug strategy became attractive since anti-cancer complexes synthesized in prodrug form are inert under normal physiological conditions.23 Significantly, in different environment such prodrugs become more labile and can be converted to active drugs. Such environments can be provided by redox status, pH, the presence of enzymes or even local light application. The hypoxic microenvironment in most solid tumours is characterised by a poor oxygen supply, relatively high amounts of reducing agents and reductase enzymes.24 As mentioned in the beginning d-transition metals possess a variety of oxidation states. Platinum possesses two available oxidation states: II and IV and platinum(IV) complexes are suitable as potential anti-cancer prodrugs.117

Figure 3. Structures of platinum(IV) prodrugs.

12 Since six-coordinated platinum(IV) complexes possess octahedral geometry, the two additional ligands on the octahedral metal centre can be used not only to improve the stability, and lipophilicity, but also the pharmacokinetic properties and even cytotoxic activity. 25 The latter can be achieved by introducing in axial positions bioactive ligands, which are able to provide antiproliferative activity by themselves. After a two-electron reduction of platinum(IV) inside the cells to a square planar platinum(II) complex, two axial ligands are detached and after release, they can further display their own roles. 26 These bioactive ligands can target specific types of cancer cells or even specific enzymes. Simultaneously, platinum(IV) is reduced to platinum(II), activated and then reacts with DNA in the classical way.27 The platinum(IV) prodrug strategy provides higher resistance to deactivation by thiol moieties in blood stream and higher selectivity for hypoxic tumour regions through an activation-by-reduction mechanism. For these reasons, numerous platinum(IV) prodrugs with bioactive axial ligands were synthesized.117

The most prominent example of platinum(IV) prodrugs is a platinum(IV) complex with two estrogen molecules in axial positions (Figure 3).28,29 The intercellular reduction leads to the release of one equivalent of cisplatin and two equivalents of estrodiol. Treatment of estrogen receptor (ER)-positive breast cancer cells with such prodrugs has overcome cisplatin resistance. Also, platinum(IV) complexes with two 2,2-dichloroacetic acid (DCA) as axial ligands were prepared (mitaplatin) (Figure 3). 30 As reported, DCA inhibits the enzyme pyruvate dehydrogenase kinase. Since cancer cells rely on cytosolic aerobic glycolysis and healthy ones on mitochondrial oxidative phosphorylation, DCA can target tumour cells leaving normal cells unaffected by shifting cellular metabolism from glycolysis to glucose oxidation. 31 The mitaplatin prodrug displays a dual mode of action after reduction, DCA disrupts mitochondrial function, while active species of cisplatin cause damage to DNA in the nucleus. Unfortunately, unlike different kinetically inert platinum(IV) prodrugs, mitaplatin was found to undergo hydrolytic degradation. 32 Another platinum(IV) prodrug (ethacraplatin) consists of ethacrynic acid (Figure 3), which is known to inhibit glutathione S-transferase (GST).33 This enzyme, responsible for detoxification of xenobiotics, including cisplatin, was found to be over-expressed in many cisplatin-resistant tumors. After reduction ethacrynic acid inhibits GST, prevents platinum drug detoxification and finally improves cisplatin potency.34 Another interesting strategy involves the photo-activation of platinum(IV) prodrugs. 35 This type of activation can provide higher selectivity for cancer cells over healthy cells.36 The most prominent platinum(IV) prodrug possesses dihydroxy ligands in the trans positions and two

13 azide ligands (Figure 3), either positioned cis or trans.37 This complex appeared to be stable and inactive in the dark. Upon irradiation, azido platinum(IV) prodrugs caused growth inhibition of human bladder cancer cells and simultaneously reduced toxicity to human skin cells.38

Unfortunately, the use of the platinum(IV) prodrug strategy did not significantly improve the cytotoxic potential of cisplatin. One of the most prominent examples is (JM216), an orally administered, octhahedral platinum(IV) prodrug, which has reached clinical trials, but in III phase has shown similar activity to cisplatin on hormone refractory human prostate cancer and further clinical investigation were forsaken.39 Similar activity does not surprise, because the mechanism of action, after reduction to platinum(II) moiety, is the same as that for other platinum-based drugs.118

To overcome such limitations two different strategies can be undertaken. Either toxic metal complexes with a different target can be involved or non-toxic metals can be used, which act as a carrier system for enzyme inactivation. To address the shortcomings of platinum drugs, new strategies involving gold(III) and cobalt(III) have been explored, since both metals can be used as prodrugs and activated by reduction inside the cells. 40,41

Since d8 gold(III) is isoelectronic and adopts the same square-planar geometry like platinum(II) complexes, gold(III) appeared as a natural candidate for next generation anti- cancer drugs. As it turned out, unlike platinum(II) complexes, square planar gold(III) analogues were relatively unstable under physiological conditions, undergo easy reduction to metallic gold and finally were also light-sensitive.42 In order to overcome these obstacles, firstly multidentate ligands such as bipyridyl, phenathroline, ethylenediamine, diethylenetriamine, porphyrins, cyclam, terpyridine were involved. Such complexes like + [AuCl2(phen)] exhibit a square planar arrangement of the gold(III) center (Figure 4). Two coordination positions of the square planar environment are occupied by two nitrogen donors and the remaining positions by two chloride or hydroxide groups.43

14

Figure 4. Structures of gold(III) complexes.

Organogold(III) compounds, bearing the bipyridyl motif, were developed as second- generation gold(III) compounds with improved stability (Figure 4).44 Such complexes possess the following donors to the square planar gold(III) center: one nitrogen atom of the bipyridyl moiety, one carbon atom of the phenyl group, and one chloride anion. The next interesting group of gold(III) complexes consists of triphenylphosphine ligands (Figure 4). 45 The exploration of these encouraging complexes was however abandoned, since triphenylphosphines appeared to be highly toxic to normal cells. In order to provide high lipophilicity to the gold complexes, preferentially N-heterocyclic carbenes (NHCs) were then introduced (Figure 4). 46 A variety of structurally different organometallic gold(III) compounds with 5-ring NHCs were synthesized with a lower systemic toxicity. Moreover, NHCs are better water and air-stable and therefore easier to handle than triphenylphosphines. Most of the gold complexes are firstly synthesized as gold(I) complexes and then subsequently treated with oxidation reagents such as Cl2, Br2 or I2 resulting in conversion to the corresponding organometallic Au(III)-N-heterocyclic carbene complexes. 47 Recently, gold(III) dithiocarbamates have come into the limelight, due to their wide range of potential applications.48 Chelating dithiocarbamate ligands cause a strong stabilisation of the gold(III) center, which prevents gold(III) complexes from undergoing hydrolysis. The dithiocarbamate gold(III) [AuBr2(DMDT)] complex (Figure 4), appeared to be more cytotoxic than cisplatin

15 and able to significantly overcome both intrinsic and acquired resistance. Dithiocarbamates derived from amino acids have received great interest, since the presence of three functional groups amino, dithiocarbamate, carboxylate, which can potentially bond to wide variety of biomolecules.49

Various biological investigations of the mechanism of action of gold(III) complexes showed that their cytotoxic mechanism of action is distinctively different from that of platinum complexes. 50 Thorough investigation as shown that, platinum-based drugs target specific sequences on DNA which finally leads to apoptosis. Surprisingly, DNA seems not to be a primary target for cytotoxic gold(III) compounds. Classical square planar gold(III) complexes exhibited significantly different and weaker interactions with DNA than those of their platinum analogues. Further investigations were directed to different targets, namely proteins to monitor binding properties. Highly cytotoxic gold(III) complexes appeared to be inhibitors of the enzyme thioredoxin reductase (TrxR). TrxR is a well-known seleno-containing enzyme responsible for cell protection against oxidative stress. Inhibition of thioredoxin reductase triggers mitochondrial cytochrome c release, which finally activates the apoptotic cascade. Consequently, a mitochondrial pathway was assigned to gold(III) complexes as the proapoptotic mechanism related to selective inhibition of thioredoxin reductase.

Moreover, due to the high binding affinity of gold ions to thiols, gold(III) drugs are able also to strongly inhibit thiol-containing enzymes, such as cysteine protease cathepsins and glutathione reductase.51 Another study on gold(III) dithiocarbamate showed the proteasome as a molecular target for gold(III) complexes. 52 Such complexes inhibit the chymotrypsin-like activity of a purified rabbit 20S proteasome and the 26S proteasome in highly metastatic MDAMB- 231 breast cancer cells.53

Cobalt also came in the research spotlight as a bioreductive prodrug candidate, since it has two accessible oxidation states: kinetically inert, low-spin 3d6 configuration cobalt(III) and labile high-spin 3d7 cobalt(II).54 Since cobalt is an essential, relatively non-toxic trace element in the form of cobalamin, cobalt(III) complexes are thought to exhibit lower systemic toxicity than platinum based drugs.55 Cobalt(III) complexes were involved as prodrugs for selective delivery of anti-cancer agents to the hypoxic regions of a tumour. Numerous inert cobalt(III) chaperones consisting of quinoline, nitrogen mustards, curcumin and marimastat (the matrix metalloproteinase inhibitor) have been synthesized.159 Immediately, when cobalt(III)

16 complexes reach the hypoxic environment, they are reduced to cobalt(II) and the active molecules are released through ligand exchange.56

Figure 5. Different modes of action of cobalt(III) prodrugs.

However, in contrast to DNA-targeting platinum(II) drugs, DNA is not the primary target for Co(III) complexes.57 Similarly to gold(III) complexes the mechanism of action seems to rely on the interactions with specific enzymes. The mechanism of action is highly dependent on the nature of complexes and their ligands. 58 Some ligands can be exchanged through interaction with biomolecules (1), bioactive ligands can also bind directly to enzymes (2) and some complexes are activated by bioreduction to release active cobalt(II) species and bioactive ligands (3).59 Besides the various targets for bioactive ligands, which depend on their nature, for cobalt complexes histidine-rich enzymes are considered to be the effective

17 target apart from the structure of metal complexes. 60 The axial ligands are substituted by the imidazole moieties of histidine residues at the active site of enzymes, causing enzymatic deactivation. Cobalt complexes appeared to inactivate HSV-1 serine protease, zinc finger motifs, thermolysin, α-thrombin and MMP-2 enzymes.183 Besides well-known Co(III) complexes possessing polydentate N-donor ligands (pyridine rings, Schiff bases), new classes with sulphur-containing ligands (dithiocarbamates, bis(thiosemicarbazones)) (Figure 6) emerged with promising in vitro anti-proliferative activity.61, 62

Figure 6. Cobalt(III) prodrugs with sulphur-containing ligands.

As discussed in the previous paragraphs, all metal complexes are not specific - they do not selectively target cancer cells over normal ones. Different approaches including a peptide- based drug delivery system, which could make platinum(IV), gold(III) and cobalt(III) complexes specific, is required. However, the real challenge is how to link such metal complexes to peptides. This issue will be discussed in following chapters.

18 1.2 CELL-PENETRATING PEPTIDES (CPPs)

To improve the transport of anti-cancer drugs through plasma membranes, cell-penetrating peptides (CPPs) came in the spotlight. The adventure of such vectors started from observations that histones and polylysine increased the uptake of albumin. 63,64 CPPs became deeply explored after the discovery that a natural polycationic protein (RKKRRQRRR), the trans-acting activator of transcription (Tat) of the human immunodeficiency virus (HIV-1), facilitates the penetration through membranes.65 Cell penetrating peptides (CPPs) are mostly cationic peptides consisting of typically up to 30 amino acids. 66 CPPs possess the ability to cross the hydrophobic cellular membrane (Figure 7). So far it is stated that CPPs can influence intracellular delivery using both endocytic and non-endocytic pathways. The mechanism of internalization is dictated by the type of CPPs, their concentration, types of cells as well as of the nature of molecular cargo.67

Figure 7. Mechanisms of CPPs uptake across the cellular membrane.65

19 CPPs are utilized as delivery systems for small organic molecules, imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, oligonucleotide/DNA/RNA, nanoparticles, nucleotides, other peptides as well as for proteins. 68 Three main groups of CPPs can be distinguished, namely: cationic, amphipathic and hydrophobic. The most numerous are the polycationic ones.69 Natural protein motifs constitute a rich source for CPP derivatives. Heparin-binding proteins, DNA and RNA-binding proteins, homeoproteins, signal peptides, antimicrobial peptides and viral proteins are the major classes of CPPs derived from natural proteins.70

The well-known penetratin (RQIKIWFQNRRMKWKK), a DNA binding protein, derives from third the helix of the homeodomain of Drosophila Antennapedia. 71 Since penetratin is able to rapidly internalized across the blood-brain barrier (BBB), it was involved in the treatment of neurodegenerative Alzheimer's disease, Parkinson's disease and Huntington's disease. Another CPP derived from natural proteins, lactoferricin (KCFQWQRNMRKVRGPPVSCIKR), an antimicrobial peptide, is a derivative from human lactoferrin (hLF), an 80-kDa iron-binding glycoprotein.72 Lactoferricin plays a dual role, in that it efficiently crosses the cellular membrane in mammalian cells and simultaneously exhibits antimicrobial activity.

Nevertheless the most common and most frequently investigated CPPs are those based on designed CPPs. This group is represented by leading arginine-rich cell-penetrating peptides.73 Cellular uptake studies revealed that an octaarginine (R8) is the minimal sequence, which provides increased cellular uptake. On the second hand, polylysine, also positively charged CPPs, shows a much lower uptake than polyarginine. 74 Arginine-rich cell-penetrating peptides were further modified to optimize the interaction with membranes and thus the cellular uptake of bioactive molecules with low membrane permeability. Li et al. have synthesized polyarginine with protoporphyrin (PpIX) for tumour-targeting . 75 The results showed that the PpIX conjugate was effectively accumulated at the tumour site by fluorescence enhancement. Takayama et al. have introduced a new term describing an additional sequence on N-terminus of CPPs in order to improve the interaction with membranes, namely penetration-accelerating sequence (Pas).212 Introduction of small hydrophobic sequences (FFLIPKG) enhanced cellular uptake and subsequently cytosolic translocation. A polyarginine peptide was also modified by adding cysteine-histidine rich 76 moieties to the N- and C-terminus (C-H5-R9-H5-C). Such modified CPPs enhanced uptake of quantum dots in comparison to unmodified polyarginine (R9). Also to well-known CPPs:

20 octaarginine and penetratin, a phenylalanine was coupled. 77 Such hydrophobicity distal provided higher accumulation of bovine serum albumin and quantum dot cargoes.

Table 1. CPPs used for anti-cancer drug delivery

Name Sequence Cargo Lit. R8 RRRRRRRR Taxol [78] TATp-Cys CYGRKKRRQRRR Paclitaxel [81] R7 RRRRRRR sulfate [78] TAT GRKKRRQRRRPQ Doxorubicin [81] R8 RRRRRRRR Doxorubicin [78] Penetratin RQIKIWFQNRRMKWKK Doxorubicin [80]

Due to their high affinity to cell membranes, CPPs were used as vectors to deliver molecular cargo. Pure organic chemotherapeutics, such as taxol, paclitaxel, vincristine sulfate and doxorubicin have been conjugated to CPPs (Table 1).78 The conjugation of such anti-cancer agents with CPPs is perceived to increase the cellular uptake of chemotherapeutics and therefore reduce the systematic toxicity. 79 Doxorubicin, a chemotherapeutic agent used in treatment of breast cancer, bladder cancer and lymphoma, was conjugated to penetratin.80 Such bioconjugates showed similar activity, but exhibited diminished side effects compared to free doxorubicin. The bioconjugate consisting of doxorubicin coupled to TAT peptide facilitated the delivery of doxorubicin in multidrug resistant MCF-7/ADR breast cancer cells.81

21 1.3 CELL-TARGETING PEPTIDES (CTPs)

Although CPPs are widely used as a delivery system for anti-cancer cargos, they possess a wide range of drawbacks. Those drawbacks range from low stability to metabolic degradation, penetration dependence on cell type over binding to extracellular matrix (ECM) components, to lack of correlation between in vitro and in vivo delivery.82 However a major drawback is the absence of specific cellular delivery. A new approach involving cell targeting peptides (CTPs) has been offered. CTPs like RGD and NGR are relatively small peptides (Table 2), which exhibit high affinity and specificity to a cell or tissue target.83 CTPs have shown advantages since they display a dual role, firstly increase intracellular delivery of cytotoxic agents across biological membranes, secondly improve the therapeutic efficacy. These peptides are characterized by lower immunogenicity and toxicity. Nonetheless, the issue of paramount importance is their capacity to delivery therapeutic agents cell-type specifically. Such extraordinary activity is correlated with the binding ability of CTPs to cell surface receptors.84

Table 2. Cell targeting peptides and their receptors.

Peptide Sequence Receptor Target cells Lit.

RGD RGD αvβ3 integrin breast carcinoma [87] iRGD CRGDKGPDC integrin αvβ3/αvβ5, angiogenic endothelial [88] NRP-1/2 cells, prostate NGR CNGRC aminopeptidase N tumor neovasculature [83] iNGR CRNGRGPDC aminopeptidase tumor endothelial cells [83] N,NRP-1/2 LTVSPWY LTVSPWY erbB2 breast carcinoma [96]

Cell surface receptors are receptors that are embedded in the plasma membrane of cells (Figure 8).90 They take part in cell signalling by binding to extracellular molecules. The latter can be represented by hormones, cytokines, neurotransmitters, growth factors, cell adhesion molecules and finally CTPs.85 All these extracellular molecules react with the receptor, cause cascading chemical changes in the metabolism and eventually in activity of a cell. RGD peptides were the first tumour-homing peptides that were discovered.86 Such cell specific

22 CTPs are internalized by endocytosis. RGD peptides show high specificity and strong affinity to surface receptors on tumor endothelium. Those receptor are from the integrin family, transmembrane heterodimeric glycoprotein cell surface receptors. Among them, αvβ3 and αvβ5 integrin are exclusively over-expressed on the membrane surface of many tumor cells.87 Integrins consist of two non-covalently bound transmembrane α and β subunits. The αvβ3 integrin receptor takes part in angiogenesis of solid tumors, cell migration, invasion and also metastatic activity.88 Integrin αvβ3 is highly expressed on activated endothelial cells and new- born vessels, but simultaneously is absent in resting endothelial cells and most normal organ systems. This phenomena is used in targeted drug delivery systems for cancer therapy.89

Figure 8. Mechanism of uptake of RGD-containing CTPs.90

One of the limitations of using CTPs is a high susceptibility to proteolytic degradation. 91 To overcome this drawback and increase affinity of CTPs to their receptor two strategies have been involved, namely cyclization and incorporation of D-forms of amino acids. 92 The common way to obtain the cyclic form of CTPs is inserting cysteines at the N- and C-terminal ends. Linear peptides can be converted to a cyclic form through the formation of a disulfide

23 bridge. Such cyclization causes a constrained conformation, which enhances the affinity of the interaction between the peptide and the target receptor in comparison to the linear form. The linear RGD peptides is converted in cyclic form either by disulfide bridge or by amide bond through N-terminal α-amine and the C-terminal α-carboxyl groups. In order to provide high stability, flexibility and reactivity of the native cyclic tri-peptide two additional amino acids have been inserted into the RGD sequence. The best results were obtained by incorporating phenylalanine (F) and lysine (K). Furthermore, such sequence was modified by substitutions of phenylalanine by its D-form giving the peptide c-RGDfK, where f stands for a D-amino acid. 93

Similarly to CPPs also CTPs were linked to organic anti-cancer molecules (Table 3).94,95 Studies revealed that attaching pro-apoptotic alpha-tocopheryl succinate (α-TOS) to the LTVSPWY peptide reduces the high-level expression of erbB2 in breast cancer in transgenic mice.96 Such results incited the extended research on delivery of organic anticancer drugs by CTPs. A cyclic RGD derivative possessing the CDCRGDCFC sequence was conjugated to doxorubicin. 97 Such conjugate with CTPs showed improved cytotoxicity in comparison to parent doxorubicin in treatment in nude mice having MDA-MB-435 breast carcinoma. The cyclic RGD peptide obtained through head to tail amide cyclization and possessing D-form of phenylalanine (RGDfK) was conjugated to . 98 Such conjugate was better internalized and accumulated into cancer cells compared to the free form of the drug.

Table 3. Cell targeting peptides as drug delivery systems for anti-cancer drugs.

Peptide Anti-cancer drug Target model Lit.

RGD camptothecin PC3 prostatic carcinoma, A498 renal [98] carcinoma, A2780 ovarian carcinoma

RGD nab-paclitaxel, doxorubicin, 22Rv1 human prostate cancer and BT474 [94] trastuzumab human breast cancer xenografts

RGDfK , camptothecin αvβ3 integrin receptor [95] iRGD Pancreatic adenocarcinoma models [95] iRGD doxorubicin HepG2 and Hu-7 human hepatocellular [97] carcinoma xenografts

NGR doxorubicin vascular endothelium [97]

24

1.4 METAL-PEPTIDE BIOCONJUGATES

The promising results of coupling organic anti-cancer agents to CPPs and CTPs, outlined in the previous section, have incited deep investigations of such bioconjugates. As a consequence, these studies initiated the research on coupling metal complexes to such peptides. However, such a strategy can be more challenging, because of redox activity of d- transition metals.

For example, in the case of platinum(IV) prodrugs, photoactivatable Pt(IV) complexes were coupled to well-known cyclic RGD-containing peptides. 99 The conjugation of such platinum(IV) complexes was achieved via the formation of an amide bond with the peptides (Figure 9). Platinum(IV) derivatives were synthesized with succinic acid in the axial position to a provide a carboxylic acid as a functional group to enable peptide coupling. The synthesis of the linear KRGDF peptide with a polyethyleneglycol spacer was carried out using a solid- phase peptide synthesis (SPPS) on 2-chlorotrityl chloride resin, followed by cleavage under mild acidic conditions and cyclization of the side-chain protected peptide. After final cleavage, removal of all protecting groups and purification, the coupling between platinum(IV) prodrugs and cyclic RGD-containing peptides was carried out in solution through amide bond formation.

Figure 9. Synthesis of platinum(IV)-peptide bioconjugate.

However, the first approach can lead to undesired side-reactions since coupling of the activated platinum(IV) complex takes place with the fully deprotected peptide bearing many functional groups. The most classical way to obtain platinum(IV)-peptide bioconjugate is represented by the synthesis of oxaliplatin-TAT peptide bioconjugates (Figure 10).134 The platinum(IV) derivate of oxaliplatin was prepared by incorporation of succinic acid in axial group. The TAT-peptide fragment (YGRKKRRQRRR) was prepared by solid-phase peptide synthesis. To avoid undesired effects, the coupling with the platinum complex was

25 accomplished on the resin with the fully protected peptide and finally the cleavage and deprotection gave the desired platinum(IV)-TAT bioconjugate.

Figure 10. Synthesis of platinum(IV)-peptide bioconjugate by SPPS.

The most prominent examples of cobalt-peptide bioconjugates are those based on cobaltocenium, organometallic cobalt(III) complexes.100 Since cobaltocenium carboxylic acid is highly stable, the conjugation of metal complexes to a nuclear localization peptide sequence (KPKKKRKV) through amide bond formation was reported to be successful (Figure 11).101 At first, resin-bound peptide was synthesized according to SPPS, then cobaltocenium carboxylic acid was coupled through amide bond formation and finally the whole bioconjugate was cleaved from the resin.

Figure 11. Synthesis of cobalt(III)-peptide bioconjugate by SPPS.

Furthermore, a cobalt(III) complex was coordinated to the short antimicrobial peptide analogue (RWRW-OBn) (Figure 12).102,103 To the resin-bound peptide, a polydentate ligand, tris(2-pyridylmethyl)amine (tpa), was appended by SPPS procedure. Following this, the linker-peptide was detached from the resin and metalation with Co(III) salt was carried out in solution.

26

Figure 12. Synthesis of cobalt(III)-peptide bioconjugate.

Another example of a cobalt-peptide bioconjugate involves a cobalt carbonyl coupled to the somatostatin-derived cyclic octapeptide octreotate (FCFWKTCT) (Figure 13). 104 Firstly cyclic peptide was synthesized on solid support followed by coupling of an alkyne linker, namely 4-Pentynoic acid, which then was finally cleavaged from the resin. To such a modified, unprotected cyclic peptide with an alkyne group on the N-terminus, dicobalt hexacarbonyl was coupled in solution, due to the selective binding of the cobalt carbonyl unit to the alkyne function.

Figure 13. Synthesis of cobalt-peptide bioconjugate.

Surprisingly, among gold-peptide bioconjugates, there is a lack of a synthetic strategy for gold(III) complexes linked to peptides. Only reliable procedures for bioconjugates consisting of gold(I) complexes have been reported (Figure 14).105 As an example, in order to obtain gold(I)-azide bioconjugates, azide-alkyne cycloaddition (AAC) was used. Firstly, a tetrapeptide with a mitochondria-targeting sequence (FRFK) was synthesized on the resin using a modified phenylalanine residue with an alkyne functional group. In the next step, a non-catalyzed [3+2] cycloaddition reaction between the gold(I) azide complex and the alkynyl peptide was carried out on the resin. After accomplished on-resin cycloaddition, the bioconjugate was cleaved from the resin.

27

Figure 14. Synthesis of gold (I)-peptide bioconjugate.

To mononuclear gold(III) complexes only short L-histidine-containing dipeptides were coordinated (Figure 15).106 Firstly, L-histidine was coupled in the solution with other amino acids like glycine, alanine and leucine to obtain dipeptides with good chelating capacity for an Au(III) ion. After purification, the coordination of those dipeptides to gold(III) was performed in solution. Crystallographic studies confirmed that those L-histidine-containing peptides coordinate to the Au(III) ion through the N3 imidazole nitrogen, the deprotonated nitrogen of the amide bond and the nitrogen of the N-terminal amino group.

Figure 15. Synthesis of gold(III)-peptide bioconjugate.

The intensive search for metal-peptide bioconjugates has been ongoing ever since. However, the most challenging aspect is the selective synthesis of such bioconjugates. Metal complexes with appropriate functional groups suitable for SPPS have to be designed. In the case of literature-known symmetrical platinum(IV)-peptide bioconjugates a problem is caused by the mixture of mono- and bi-functional products. The reliable synthesis of mono-functional, unsymmetrical platinum(IV) complexes with cell penetrating peptides using SPPS has to be elaborated. The same obstacles are observed for cobalt(III) complexes since the octahedral geometry favours bi-functional complexes with equal axial groups making the selective synthesis of bioconjugates tedious. For gold(III) peptide bioconjugates an entirely new strategy has to be established.

28

2 OBJECTIVE

To synthesize metal-peptide bioconjugates the different nature of metal complexes and peptides has to be taken into account during the design step. Since metal complexes differ in terms of stability, the bioconjugation techniques have to be tailored accordingly. Therefore, the chemistry of different metal complexes will be adjusted to provide a selective synthesis of mono-functional metal-peptide bioconjugates.

Spurred by a continuous lack of synthetic approaches of metal-peptide bioconjugates for drug delivery, three projects covering the preparation of N-terminal modified peptides and corresponding metal-peptide conjugates will be presented. The challenge to combine prodrug strategy with CPP and CTPs to create efficient drug delivery systems for platinum(IV), cobalt(III) and gold(III) complexes will be undertaken. CPPs consisting of polyarginine and polyphenylalanine sequences and CTPs consisting of linear and cyclic RGD sequences will be synthesized. Unsymmetrical platinum(IV) and cobalt(III) complexes possessing functional carboxylic groups will be synthesized and the coupling with different CPPs will be carried out using SPPS procedure. Organogold(III) complexes will be linked to breast cell targeting peptides in the solution using the di-thiol moiety.

All metal complexes, CPPs, CTPs and prodrug-peptide bioconjugates will be characterized by analytical HPLC and mass spectrometry (ESI-MS, MALDI-TOF). Additionally, metal- peptide bioconjugates will be screened for their in vitro cytotoxicity by MTT assay against different human tumor cell lines and normal human cells. The results will be compared to their relative parent metal complexes and unmodified peptides as control experiments.

29

3 RESULTS

3.1 SYNTHESIS OF MONOFUNCTIONAL PLATINUM(IV) CARBOXYLATE PRECURSORS FOR USE IN PT(IV)-PEPTIDE BIOCONJUGATES

Dariusz Śmiƚowicz, Nils Metzler-Nolte*

Inorganic Chemistry I – Bioinorganic Chemistry Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany E-mail: [email protected]

Keywords: Bioconjugates, Medicinal Inorganic Chemistry, Peptides, Platinum Compounds,

3.1.1 Abstract

Herein we present platinum(IV) bioconjugates with polyarginine peptides as prospective prodrug delivery systems. Asymmetrical platinum(IV) complexes 3 were obtained via oxidation of parent platinum(II) complexes 2 with N-bromosuccinimide (NBS) in the presence of succinic anhydride. The combination of these two oxidation reagents furnishes the platinum(IV) environment with two different axial ligands, one of which bears a free carboxylic acid. All platinum(II) and (IV) compounds were characterized by FT-IR, ESI-MS, HPLC, 1H-, 13C- and 195Pt-NMR. Standard solid-phase peptide chemistry was used for the synthesis of polyarginine (R9) peptides. Coupling of N-terminal peptides with the platinum complexes afforded peptide monoconjugates, which were purified by semi-preparative HPLC and characterized by analytical HPLC and ESI-MS. Platinum(IV)-peptide bioconjugates as well as platinum(II) and platinum(IV) complexes were tested as cytotoxic agents against two different human cancer cell lines (MCF-7, HepG2) and normal human fibroblasts cell lines (GM5657T). Preliminary in vitro data showed that all platinum(IV) complexes exhibit lower activity than their platinum(II) precursors towards most cell lines. Interestingly, in the case of

30 HepG2 cells, the Pt(IV)-(R)9-G-A-L bioconjugate (4a) showed even higher activity compared to the non-targeting platinum(IV) parent compound.

3.1.2 Introduction

Since the discovery of the toxicity of cisplatin by Barnett Rosenberg in 1965 and its admission as a drug by The Food and Drug Administration in 1978, cisplatin is routinely used for the treatment of cancer.107,108 Unfortunately, it has well-known acute toxic side effects, intrinsic or acquired drug resistance, and poor aqueous solubility.109,110,111 Surprisingly, since the discovery of cisplatin, only two other inorganic platinum-based complexes, namely carboplatin and oxaliplatin have gained worldwide approval as anticancer agents (Figure 16).112,113,114

Figure 16. Structures of platinum based anticancer agents.

Regardless of the success of other current anticancer complexes, these compounds either possess severe side-effects or undergo inactivation in the blood stream before reaching the cancer cells. 115,116 In order to overcome these drawbacks, platinum(IV)-based drugs were developed as a possible improvement in . 117 Inside the cells, platinum(IV)- based prodrugs can be reduced to platinum(II) species, for example by ascorbic acid and glutathione, and can then interact with the cellular DNA to initiate apoptosis, in a fashion identical to their related Pt(II) species 118 Such platinum(IV)-based prodrugs are less reactive,because the platinum centre is coordinatively saturated, hence they exhibit higher stability in the blood stream. It has been suggested that thisleads to lower side effects and paves the way for oral uptake. 119 Additionally, the axial ligands can be varied to change the lipophilicity and the redox potential.120,121,122

31

Figure 17. Examples of symmetrical platinum(IV) complexes.

A number of platinum(IV)-based drugs were developed and tested in vitro and in vivo, most prominently among them iproplatin and satraplatin (Figure 17).123,114 The most successful complex, satraplatin, has undergone Phase III clinical trials against human prostate cancer. Satraplatin reduces the risk of prostate cancer progression by 40%. Unfortunately, satraplatin did not obtain FDA approval as it failed to show a convincing patient benefit in terms of overall survival.124,125 According to these drawbacks, there is a need to develop new, efficient and selective drug delivery systems which are able to fully eliminate or at least drastically reduce the side-effects of chemotherapy. 126 , 127 , 128 One possibility to enhance the cellular uptake and thereby potentially cytotoxicity is coupling of platinum complexes with a delivery vector in the form of a macromolecule. 129,130This approach may provide higher stabilization of the drug and longer residence time of the drug in the body leading to more drug efficiently reaching its biological target. In the literature, there are known Pt(IV) bioconjugates, which can target specifically genomic DNA or mitochondria. As one example, Lippard and co-workers obtained a platinum(IV) complex conjugated with the vitamin E analogue α-tocopherol succinate (α-TOS), which displays an activity similar to cisplatin in cancer cells and causes damage to DNA and mitochondria simultaneously.131Another promising method is to usecell- penetrating peptides (CCPs), which can pass the cell membrane because of their positive charge. The use of other metals, such as osmium agents with CPPs, has been investigated by Sadler and co-workers132which showed that attaching CPPs to an organometallic osmium(II) anticancer complex, [(η6-biphenyl)Os(picolinate)Cl], improved cytotoxicity, cellular uptake and DNA binding. These data and our previous results 133,134 revealed the cytotoxic potency of metal-peptide bioconjugates, especially platinum bioconjugates, and the need for further biological investigation. Unfortunately, there is no reliable and straightforward synthesis strategy for platinum(IV)-peptide bioconjugates. This prompted us to develop a reliable synthesis route

32 for such bioconjugates, starting from platinum(II) through oxidation to platinum(IV) complexes, and coupling to cell-penetrating peptides. To exemplify the relative ease of synthesis of such bioconjugates, the asymmetrical platinum(IV) complexes were to be coupled to a cell penetrating peptide by solid phase peptide synthesis. The monofunctional platinum(IV) complex (Figure 18 (I)) was our target complex to be conjugated to peptides to obtain platinum(IV) bioconjugates as exemplified in Figure 18 (II). Different peptides were chosen possessing polyarginine and polyglutamic residues. While the ones with polyarginine sequences should be readily taken up by cells and cause acute cytotoxicity, sequences with a ―neutralizing‖ additional polyglutamic part should not.

Figure 18. Synthesis strategy for platinum(IV) prodrugs bioconjugates.

3.1.3 Results and discussion

Synthesis and characterisation of platinum(II) complexes: The reaction of the diamine 1R,

2R-DACH and K2PtCl4 in a 1:1 molar ratio in water afforded the complex [PtCl2((1R, 2R)- DACH)],which upon treatment with the silver salts of 1,1-cyclobutanedicarboxylic acid (CBDC) afforded complex 2b (Scheme 1b). Complex 2a was a special case, as it could not be prepared directly from K2PtCl4 without obtaining a mixture of cis- and trans-platinum(II) 135 complexes. For this reason K2PtCl4 was firstly converted to the iodide derivative since iodide has a higher trans effect. Then, the reaction between [PtI2(NH3)2] and Ag2CBDC in water in 1 : 1 molar ratio gave complex 2a (Scheme 1a). The platinum(II)complexes were characterized by ESI-MS, FT-IR,1H-, 13C-, and 195Pt-NMR (Figs. S2-S6, ESI†).

33

Scheme 1. Synthesis pathway of platinum(II) complexes

Synthesis and characterisation of platinum(IV) complexes: The axial ligands in octahedral platinum(IV) complexes can play an important role in the drug targeting and delivery (DTD) strategy because of their capacity for functionalization with a variety of small-molecule or macromolecule moieties. Pt(IV) derivatives with only one dicarboxylic acid in the axial positions are expected to be a suitable platform for this purpose because one carboxylic group is axially linked to the platinum centre while the second carboxylate group remains free and is available for further reactions, thus yielding exclusively monofunctional platinum(IV) bioconjugates. For this reason, both platinum(II) complexes 2 were oxidized to their platinum(IV) analogues via asymmetrical oxidation, as shown in Scheme 2. Complexes 3a and 3b were synthesized by using a procedure similar to that reported in literature for the oxidative chlorination of carboplatin and oxaliplatin derivatives. 136 Through oxidation via N- bromosuccinimide (NBS) in the presence of succinic anhydride, platinum(IV) prodrugs were obtained with bromide and succinate ligands in the axial positions. The reactions were carried out under mild conditions, and the final crude products were precipitated by an acetone/diethyl ether mixture. The oxidative bromination of the platinum(II) complexes, in which NBS acts as a source of bromine, was carried out in a mixture of acetone/water (1:1). The choice of solvents was dictated by the limited aqueous solubility ofsuccinic anhydride and limited solubility of platinum(II) complexes in pure acetone, respectively. Notably, no coordination of solvents to the metal centre was observed. According to our results, addition of only one equivalent of NBS was sufficient to oxidize platinum(II) species to platinum(IV), as well as to provide a bromide ligand to one of the axial positions.We strongly aimed to

34 synthesize unsymmetrical platinum(IV) prodrugs with one carboxylate scaffold, because such complexes provide only monoconjugate products. Symmetrical platinum(IV) complexes bearing two carboxylate groups in axial positions would be able to react with two peptides. According to this strategy, two types of products, namely monoconjugate and diconjugate could be expected, which could cause obstacles during separation of such bioconjugates. The presence of a bromide ligand in the second axial position was dictated by our choice of NBS as oxidating reagent. The resulting compounds are highly soluble and stable in DMF, which makes them convenient intermediates for further functionalization with peptides by solid phase peptide synthesis.137,138 Compounds 3a and 3b were comprehensively characterized by FT-IR, ESI-MS, HPLC,13C- and 195Pt-NMR.

Scheme 2. Synthesis of platinum(IV) complexes.

Characterization of platinum(II) and platinum(IV) complexes: The IR spectra of (2a) and (2b) show a typical pattern for dicarboxylate ligands bound in a bidentate fashion to a metal centre (Fig.S4, in the ESI†). The ν(C=O) band disappears at 1705 cm-1 in the free - -1 cyclobutane-1,1-dicarboxylic acid, and the carboxylate band νs(OCO ) appears at 1346 cm -1 - (2a) and 1348 cm (2b). Additionally, the νas(OCO ) stretching frequency, characteristic for a coordinated carboxyl group, appears at 1638 cm-1 (2a) and 1620 cm-1 (2b). The mode of - carboxylate coordination to a metal can be determined based on the Δ values (Δ=νas(OCO ) - - -1 -1 νs(OCO )). A calculated Δ value of 263 cm (2a) and 272 cm (2b) compared to Δ' value of disodium cyclobutane-1,1-dicarboxylate (240 cm-1) suggests a monodentate coordination mode for each carboxylate group. The IR spectra of both platinum(IV) complexes showed

35 frequencies clearly associated with the characteristic functional group of desired ligands. The IR spectral data of important functional groups of the transition metal complexes are - - presented in Table 4. The characteristic bands of the νas(OCO ) and νs(OCO ) group from 1,1- cyclobutanedicarboxylate ligand appeared at 1617 cm-1and at 1340 cm-1 for complex 3a. These frequencies were shifted towards the lower wave numbers in the spectra of platinum(IV) complexes compared to those of platinum(II) complexes. This is indicative of the oxidation of platinum(II) to platinum(IV). The very broad absorption bands of the - - νas(OCO ) and νs(OCO ) group can be explained by the fact that they consist also of absorption bands of one coordinated carboxylic group from succinic acid. The IR spectra of 3a and 3b show not only a typical pattern for dicarboxylate ligands, but also the characteristic band for free carboxylic acid groups from the succinic acid ligand at 1701 cm-1 and 1700 cm- 1, respectively (Fig.S7, in the ESI†).

Table 4. IR data for platinum complexes.

IR [cm-1]

- - νas(OCO ) νsym(OCO ) ν(COOH) (2a)cis-[Pt(CBDC)(NH3)2] 1638 1346 - (2b)cis-[Pt(CBDC)(DACH)] 1620 1348 - (3a) cis,trans,cis-[Pt(CBDC)(succ)(Br)(NH3)2] 1617 1340 1701 (3b) cis,trans,cis-[Pt(CBDC)(succ)(Br)(DACH)] 1614 1343 1700

Electrospray ionization mass spectrometry (ESI-MS) was applied to investigate the mass of the desired compounds (Fig.S3 in the ESI†). In the full-scan mass spectra, the molecular ion peak [M+H]+ (m/z = 393 and m/z = 451) were found and assigned to complexes 2a and 2b, respectively. The ESI-MS data support the proposed monomeric structures of platinum(II).The m/z values are consistent with the expected isotopic mass distribution pattern of platinum species. Mass spectra of the platinum(IV) compounds 3a and 3b are shown in Figure 4. Platinum(IV) complexes give a molecular ion peak at m/z=566 and 646 for 3a and 3b respectively, which could be assigned in both cases to the [M-H]- peak. The mass spectrum of complex 3b displayed a peak with high intensity corresponding to the [M+Na-H]- species at m/z=673. Moreover, the mass spectrum of complex 3b displayed other indicative signals, one of moderate intensity at m/z=528 corresponding to the [M-succ]- species and a second at m/z=448 corresponding to the [M-succ-Br]- species. This feature confirms the

36 fragmentation pathways which were initiated by the loss of ligands in the axial positions: firstly succinic acid and finally the bromide ligand. In the case of complex 3a, fragmentation was accomplished by loss of the succinic acid ligand, leading to a signal at m/z=450. The full- scan mass spectra are available in the ESI†(Fig.S9).

Figure 19. ESI-MS spectra (neg. ion detection mode) of platinum(IV) complexes: 3a (left) and 3b (right).

The purity and identity of the platinum(II) and platinum(IV) complexes was further confirmed by RP-HPLC (Table 5). Complexes with R,R-cyclohexane-1,3-diamine ligandderived from oxaliplatin possess higher lipophilicity than those with NH3 ligands. After oxidation, octahedral platinum(IV) complexes possesseven further increased lipophilicity in comparison to their parent compounds. The data show that the retention time is remarkably affected by the presence of coordinated axial ligands after oxidation. (Figs.S5 and S8, in the ESI†).

Table 5. RP-HPLC retention times for metal complexes.

t [min] Compounds R

(2a)cis-[Pt(CBDC)(NH3)2] 4.92 (2b)cis-[Pt(CBDC)(DACH)] 8.95 (3a)cis,trans,cis-[Pt(CBDC)(succ)(Br)(NH3)2] 13.18 (3b)cis,trans,cis-[Pt(CBDC)(succ)(Br)(DACH)] 14.48

The coordination sphere of platinum complexes can unequivocally be determined based on 195 their Pt resonances. Signals in the range between -2000 and -1600 ppm (vs. aq. K2PtCl4)

37 are characteristic for dicarboxylate platinum(II) complexes (Fig.S6, in the ESI†). Platinum(II) complexes 2a and 2b exhibit one signal at -1705 and -1906 ppm, respectively (Table 6). The high field shift of these signals is attributed to a [PtN2O2] ligand environment. The observed single resonance confirms the presence of one platinum(II) species only. Oxidation from platinum(II) precursors to platinum(IV) prodrugs was investigated also via 195Pt-NMR. The spectra showed only one peak, which confirms the oxidation of the platinum(II) precursor to the one species of platinum(IV) complex (Fig.S10, in the ESI†). For an accurate classification, the spectra were compared with literature.14,15 In general, the range between 900 and 1100 ppm is characteristic for octahedral dicarboxylate platinum(IV) complexes. Complexes 3a and 3b exhibit one signal at 1090 and 1043 ppm, respectively (Table 6). The additional axial ligands lead to deshielding of the metal complexes, which causes the signal from platinum(IV) species to be shifted downfield by about 3000 ppm in comparison to the parent platinum(II) complexes.139,140

Table 6. Measured 195 Pt NMR shifts of synthesized platinum complexes.

Compounds 195Pt NMR [ppm]

(2a) cis-[Pt(CBDC)(NH3)2] - 1705 (2b)cis-[Pt(CBDC)(DACH)] - 1906 (3a)cis,trans,cis-[Pt(CBDC)(succ)(Br)(NH3)2] 1090 (3b)cis,trans,cis-[Pt(CBDC)(succ)(Br)(DACH)] 1043

Synthesis and characterisation of peptides. In order to explore the reaction scope for conjugation of the asymmetrical platinum(IV) complexes with biomolecules, cell penetrating peptides were synthesized. The synthesis route of the peptides reported in this article is shown in the ESI† (Fig.S11). Solid-phase peptide synthesis (SPPS) Fmoc strategy was employed to synthesise two sequences of CPP‘s.141,142 Peptide P1, which consists of only a polyarginine sequence, was synthesized manually. Peptide P2, which consists of an additional polyglutamic sequence to neutralize the positive charge of polyarginine, was assembled on an automated, microwave-assisted synthesizer (Figure 20). 143 ,144 Deprotection of temporary Fmoc protecting groups was performed with 20% piperidine in DMF. The coupling was performed with an excess of the Fmoc-amino acid (4 eq.), activated with 2-(1H-benzotriazole- 1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) in the presence of N-

38 hydroxybenzotriazole (BtOH, 4 eq.) and an excess of diisopropylethylamine (DIPEA, 6 eq.).145,146 SPPS consists of repeated cycles of N-terminal protecting group removal on the last amino acid, followed by coupling of the next incoming amino acid. Through repetitions of a reaction cycle of peptide coupling, washing, deprotection and washing the desired peptideswere obtained.

P1

H Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ala Leu

OH

P2

H Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ala Leu

Gly HO Glu Glu Glu Glu Glu Glu Glu Glu Glu Pro Leu

Figure 20. Structures of peptide P1(up) and peptide P2 (down).

After completion of peptide synthesis and before the bioconjugation reaction with platinum prodrugs the successful synthesis of peptides P1 and P2 was established by HPLC and ESI- MS. For this purpose, 10 mg of the resin from each peptide was treated with 20% of piperidine in DMF in order to remove the Fmoc group from the N-terminus. Then, after washing and drying the peptides were cleaved from the resin by treatment with a mixture of

TFA/TIS/H2O (95%/2.5%/2.5%). After cleavage from the resin the peptides were lyophilized. Both crude peptides were purified by reverse-phase semi-preparative HPLC. Finally, the peptides were characterized by HPLC and the mass was identified by ESI-MS measurements. The purity of the peptides was confirmed by analytical HPLC (Figs.S14 and S17, in the ESI†). ESI-MS spectra confirm the molecular mass [M+H]+ for peptide P1 at m/z = 1664 and for peptide P2 at m/z= 3096. Besides the signals of the peptides multiply charged species were

39 formed, in the case of P1 (M+2H)2+ at m/z = 832, (M+3H)3+ at m/z = 555, (M+4H)4+ at m/z= 416, and for P2 (M+2H)2+ at m/z= 1548 and (M+3H)3+ at m/z= 1033 (Figs.S13 and S16 in the ESI†).

Figure 21. ESI-MS spectrum of P1(left) and MALDI-MS spectrum of 4a (right).

Synthesis and characterisation of bioconjugates. The coupling of Pt complexes 3a and 3b with the Fmoc-deprotected peptides was performed with the fully protected peptide on the resin in order to avoid the undesired side reactions (Figure 22). The carboxylate group in the axial position provided a suitable functionality for coupling by SPPS. The metal complex was dissolved in DMF and subsequently the conjugation reaction with the peptide was carried out. Coupling was performed with an excess of the platinum(IV) complex (5 eq.), activated with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) in the presence of N-hydroxybenzotriazole (BtOH, 4 eq.) and an excess of diisopropylethylamine (DIPEA, 6 eq.). After 24 h, excess of reagents was removed by filtration, the resin was washed with DMF, DCM, Et2O and dried under vacuum. Cleavage from the resin and deprotection of the pbf side-chain protecting groups of arginine and the tert-butyl side-chain protecting groups of glutamic acid was achieved with 95% TFA. The obtained metal-peptide bioconjugates were purified by preparative HPLC to obtain the desired products. All metal- peptide bioconjugates were characterized by analytical HPLC, ESI-MS and MALDI-MS.

40

Figure 22. Synthesis of platinum(IV)-peptide bioconjugate (4a)

41 Analysis by RP-HPLC showed main peaks (Fig.S19, in the ESI†), which were isolated and characterized by ESI mass spectrometry. After purification by semi-preparative HPLC and lyophilization, the conjugates 4a (overall yield 27%) and 4b (overall yield 12%) were obtained as white solids. ESI-MS analysis of the conjugates showed m/z values that were + consistent with that of the charged species ([M+NH4] for bioconjugate 4a and doubly charged species ([M + H]2+ for bioconjugate 4b, also showing the expected isotopic mass distribution patterns of platinum (Fig.S20, in the ESI†).

Table 7. RP-HPLC retention times for peptides and bioconjugates.

Compounds tR [min]

(P1) (R)9-G-A-L 10.38 (P2)(R)9-G-A-L-G-L-P-(E)9 15.03 (4a)Pt(IV)-(R)9-G-A-L 14.03 (4b)Pt(IV)-(R)9-G-A-L-G-L-P-(E)9 20.05

Cytotoxicity. The anti-proliferative activity of platinum complexes and platinum-peptide bioconjugates was investigated against human liver cancer cells (HepG2), human breast cancer cells (MCF-7) and normal human fibroblast cells (GM5657T) by the MTT assay. For control purposes, the IC50 of cisplatin was also determined using the same assay. All of the results are expressed as IC50 values (half-maximal inhibitory concentration) and are summarized in Table 8. The dose-response curves are shown in the ESI† (Fig.S21). Firstly, we investigated the cytotoxic activity of platinum complexes after oxidation. For complex 3a moderate activity was observed. HepG2 liver cancer cells were shown to be the most sensitive (43.0±1.5 µM) to 3a. Generally, the platinum(IV) complex 3a was found to be less active than its parent platinum(II) complex towards all cell lines. It is worth noting that 147 such high IC50 values are typical for platinum(IV) prodrugs. The lower antiproliferative activity of platinum(IV) complexes in comparison to their parent platinum(II) compounds is frequently correlated in the literature to the nature of the axial ligands, which influence their lipophilicity, as well as on the rate of reduction. The easier the reduction, the faster is aquation, the faster the coordination to DNA and consequently the higher the activity.148,149,150. Secondly, we investigated the influence of cell penetrating peptides attached to platinum(IV) complexes on in vitro cytotoxicity to determine whether this can lead to increased activity.

42 The bioconjugate 4a with the polyarginine sequence showed only moderate activity towards MCF7 (85.0±3.3 µM) and GM5657T (71.7±1.3 µM). Towards HepG2 liver cancer cells, 4a exhibits slightly higher activity (37.3±1.6 µM) than the parent platinum(IV) complex (43.0±1.5 µM). In contrast, under the same conditions, bioconjugate 4b showed no growth inhibition against all tested cell lines. This fact is especially interesting in the case of normal human fibroblasts GM5657T. The platinum(IV) peptide bioconjugate 4a consisting of only the polyarginine sequence is active towards human fibroblasts. In bioconjugate 4b, which possesses an additional polyglutamic sequence in the peptide structure, the cationic charge is neutralized and hence there is much lower cell uptake (Figure 23). This bioconjugate completely loses activity of cell-growth inhibition against healthy cells. The difference in toxicities between 4a and 4b apparently can thus be attributed to their differing peptide sequences that strongly impact cellular uptake.

Figure 23. Mechanism of action of platinum(IV) prodrug bioconjugates.

43 Table 8. Cytotoxicity dataas determined by the MTT assay after 48 h of incubation.

a IC50 [µM] Cell lines Cisplatin 2a 3a 4a 4b MCF7 25.3±2.3 33.8±0.6 65.3±0.9 85.0±3.3 ˃250 HepG2 13.3±1.4 21.1±0.5 43.0±1.5 37.3±1.6 ˃250 GM5657T 9.0±2.2 14.0±1.1 46.5±1.3 71.7±1.3 ˃250

a The IC50 values are averages of three independent determinations. All compounds were dissolved in DMSO (final concentration 0.5 %).

3.1.4 Conclusion

Transition metal-based chemotherapeutic drugs coupled with macromolecules have attracted considerable attention in recent years as they offer the possibility of targeted delivery. In this paper we demonstrate a reliable method for making platinum(IV)-peptide bioconjugates. Asymmetrical platinum(IV) prodrugs were obtained though oxidative bromination with N- bromosuccinimide (NBS), which was used as a brominating and oxidizing agent. Cell penetrating peptides were synthesized by solid phase peptide synthesis (SPPS). Consequently, the SPPS reactions of resin-bound peptides and the Pt complexes with one free carboxylate group afforded monoconjugated platinum-peptide species, which were purified by RP-HPLC and characterized by ESI-MS. Platinum(IV) complexes coupled to peptides were found to be stable even under strongly acidic cleavage conditions. All compounds were screened for their cytotoxicity against selected cancer cell lines. Platinum(II) and platinum(IV) precursors show promising cytotoxicity in the lower micromolar range. The bioconjugate 4b, which consists of polyarginine and polyglutamic acid, exhibits no activity on cell proliferation of any tested cell lines. Bioconjugate 4a, which consists of a CPP-polyarginine peptide sequence only, was found to be considerably active towards HepG2 liver cancer cells. As synthesized, our compounds inevitably still have one bromide ligand opposite the carboxylate/CPP attachment point. It was previously shown by Hambley, Gibson, Osella, and others that there is significant scope for optimization of the antiproliferative activity by installing amide or acetate ligands at this position rather than halides,148, 151, 152and these findings will certainly guide optimization of our peptide bioconjugates in the future as well. Nevertheless, the results reported here indicate that already this family of platinum(IV) complexes and their

44 bioconjugates with polyarginine-CPPs possesses interesting antiproliferative properties. This result encourages the design of such peptide delivery systems, which in the future could provide selectivity to active anticancer platinum moieties.

3.1.5 Experimental Section

General experimental conditions. Unless otherwise noted, all the reactions were carried out under normal atmospheric conditions without protection from light. All chemicals which were not synthesised were purchased from Sigma-Aldrich, J.T. Baker Chemical Company, AppliChem GmbH, Carl Roth GmbH & Co. KG, Acros Organics, Fisher Scientific GmbH, IRIS Biotech GmbH, Alfa Aesar, Novabiochem or VWR International GmbH and used without further purification.

Instrumentation and analytical measurements. Solid phase peptide synthesis was carried out on a CEM Microwave Peptide Synthesizer Liberty. Analytical and semi-preparative HPLC were performed on a Knauer Smartline System by using reversed-phase Reprosil-Pur C18 columns (analytical HPLC: 1.5 μm material, 250 x 4.6 mm column, flow 1 ml/min; semi- preparative HPLC: 5 μm material, 250 x 10 mm column, flow 2.5 ml/min) purchased from Dr.Maisch GmbH. Buffer A (water/acetonitrile/TFA, 95:5:0.1, v/v/v) and Buffer B (acetonitrile/water/TFA, 95:5:0.1, v/v/v) were used as gradients over 40 minutes. Retention times were recorded at 214 nm. NMR spectra were obtained at room temperature on Bruker DPX 200 MHz and Bruker DRX 400 MHz spectrometers. They were measured at 200 MHz as well as 400 MHz for 1H and 62.860 MHz for 13C and 53.747 MHz for 195Pt with DMSO- 1 13 d6 as solvent. The reference standard was tetramethylsilane for H NMR as well as C NMR 195 and Na2PtCl6 for Pt NMR. Chemical shifts were reported in parts per million (ppm) and referenced to solvent resonances. ESI-MS spectra were measured on a Bruker Daltonics Esquire 6000 and IR measurements were performed on a Bruker Tensor 27 FT-IR using ATR (600 – 4000 cm-1). MALDI-TOF mass spectrometry was performed on a Bruker Daltonics Autoflex using α-Cyano-4-hydroxycinnamic acid as matrix. UV-Vis absorption spectra were recorded on a Spectrophotometer Microplate Reader (Berthold Detection System).

HPLC analysis and purification. HPLC analysis and purifications were carried out using C18 analytical (Varian Dynamax, 4.6 mm x 250 mm) and C18 semi-preparative (Varian Dynamax, 21.4 mm x 250 mm) columns on a customised Varian Prostar instrument. Linear gradient, 5–95% MeCN in 18 min, eluents: H2O and MeCN both containing 0.1% (v/v) TFA.

45 Analytical (flow rate: 1.0 mL min-1) and preparative (flow rate: 4.0 mL min_1) runs were performed with a linear gradient of A (95% millipores water, 5% MeCN, 0.1% TFA (v/v/v)) and B (5% millipores water, 95% MeCN, 0.1% TFA (v/v/v)). Analytical runs: t = 0 min: 0% B; t = 18 min: 100% B; t = 26 min: 0% B; t = 30 min: 0% B. Preparative runs: t = 0 min: 0% B; t = 45 min: 50% B; t = 55 min: 100% B; t = 65 min: 0% B. All samples were filtered before injection using a 0.22 mm syringe filter. Spectra were recorded at 254 nm and ambient temperature, retention times (tR/[min]) were noted in each case.

Solid-phase peptide synthesis. The resin bound Fmoc-polyarginine was synthesized on an automated peptide synthesizer using standard protocols (amino acid coupling: TBTU in DMF (0.5 M), HOBt in DMF (0.5 M), DIPEA in NMP (2 M) and amino acids in DMF (0.2 M); arginine coupling: 25 min, 75 1C, 0 W followed by 5 min, 75 1C, 25 W; standard amino acid coupling: 5 min, 75 1C, 24 W. For deprotection: 20% piperidine in DMF: initial deprotection: 0.5 min, 75 1C, 30 W followed by deprotection: 3 min, 75 1C, 50 W). Afterwards, aliquots of 100 mg peptide containing resin was transferred into a filter-containing syringe for further derivatization. For the synthesis of all compounds, the N-terminal Fmoc group was deprotected and coupled with carboxylic group from platinum(IV) prodrugs. After each step the resin was washed 5 times using 2 mL DMF.

Deprotection of Fmoc. The deprotection was performed twice by treating the Fmoc protected peptide with 2 mL of 20% piperidine in DMF (each 10 min).

Cleavage. The resin was washed with DMF and DCM, shrunk with Et2O and dried under vacuum for 30 min. Finally, cleavage of the bioconjugate from the resin was performed using TFA/water/triisopropylsilane (TIS) (2 mL, 95 : 2.5 : 2.5) or TFA/phenol/TIS (2 mL, 85 : 10 : 5) for 6 h at room temperature. The resin was filtered and washed with 0.5 mL TFA. Addition of cold diethyl ether yielded a precipitate, which was washed repeatedly with diethyl ether.

The crude product was dissolved in MeCN/H2O (1:1), filtered, lyophilized and afterwards purified and analyzed by RP-HPLC, and finally characterized with ESI-MS.

Platinum(IV) coupling. After Fmoc-deprotection, platinum(IV) prodrugs were coupled to the free N-terminus using platinum(IV) complexes, 1-hydroxybenzotriazole (HOBt), 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TBTU), DIPEA, (2 : 4 : 4 : 6 equiv.) in DMF for 24 hours.

46 Synthesis of disilvercyclobutane-carboxylate: Cyclobutane-1,1-dicarboxylic acid (374.5 mg, 2.6 mmol) was dissolved in 15 mL of dist. water. Sodium hydroxide (207.8 mg, 5.2 mmol) was dissolved separately in 5 mL of dist. water and was added dropwise to the solution. Under the exclusion of light, silver nitrate (882.5 mg, 5.2 mmol) was added and the mixture was stirred for 1 h. The white precipitation was filtered and washed with each 5 mL of water, ethanol and diethyl ether. Finally, the product was dried under vacuum. Yield: 762.6 -1 - mg (82.0%). IR (ATR, cm ) 2935 (w), 1542 (m), 1499 (s, 휈 asym(OCO )), 1396 (m), 1346 - (m,휈 sym.(OCO )). Elemental analysis: calculated: C: 20.14%, H: 1.69%, observed: C: 19.80%, H: 1.69%

Synthesis of cis-[Pt(I2)(NH3)2] (1a): Potassium tetrachloroplatinate(II) (500 mg, 1.2 mmol) was dissolved in 15 mL of water and then potassium iodide (1595 mg, 9.6 mmol) was added. The mixture was stirred for 30 min to obtain a brown solution with the intermediate product potassium tetraiodoplatinate(II). Then 33% ammonia solution (1.54 mL, 26.6 mmol) was added. After 2 hours yellow solid precipitated. This solid was washed with 5 mL of water, ethanol and diethyl ether and afterwards dried under vacuum to obtain 1a. Yield: 498.9 mg -1 (85.8%). IR (ATR, cm ) 3267(m,휈 sym.(NH3)), 3194 (m), 1288 (s), 1259 (s) 736 (s).

Synthesis of cis-[Pt(Cl2)(DACH)] (1b): Potassium tetrachloroplatinate(II) (500 mg, 1.2 mmol) was dissolved in 15 mL of water and solid 1,2-diaminocyclohexane (137.5 mg, 1.2 mmol) was added. The mixture was stirred for 24 h, and the resulting yellow precipitate was filtered and washed with each 5 mL of water, ethanol and diethyl ether to obtain 1b. Yield: 411.5 mg (89.3%). IR (ATR, cm-1) 3272 (m), 3182 (m), 2934 (m), 2863 (w) 1563 - 1 (s,휈 asym(OCO )). H NMR (400 MHz, DMSO-d6, δ): 6.40-4.90 (m, 4H, -NH2), 2.35 (s, 1H, -

CH), 2.09 (s, 1H, -CH), 1.90 (m, 2H, -CH2) 1.46 (dd, 2H, -CH2) 1.26 (m, 2H, -CH2) 0.99 (dt, 13 2H, -CH2). C NMR (62.86 MHz, DMSO-d6, δ): 61.5 (1Ct), 61.3 (1Ct), 31.6 (1Cs), 31.4

(1Cs), 23.9 (1Cs), 23.7 (1Cs).

Synthesis of [Pt(CBDC)(NH3)2] (2a): 1a (498.9 mg, 1.0 mmol) was suspended in 10 mL of water and 10 mL of ethanol. Disilvercyclobutane-carboxylate (368.6 mg, 1.0 mmol) was added and the mixture was stirred for 48 hin the dark. Then the mixture was centrifuged and the supernatant was filtrated. The solvents were removed by slow evaporation. The obtained solid was washed with acetone and dried under vacuum to obtain 2a with a yield of 183.7 mg -1 - (48.0%). IR (ATR, cm ) 3262(m,휈 sym.(NH3)), 2925 (w), 1638 (s), 1694 (s,휈 asym.(OCO )), 1376 - - (s, 휈 sym.(OCO )). 1346 (s). MS (ESI-MS, negative ion detection mode, m/z): 370 [M-H] , 265

47 - 1 [M-(CBDC)+(H2O)2] . H NMR (400 MHz, DMSO-d6, δ): 4.09 (s, 6H, -NH3), 2.67 (t, 4H, - 13 CH2), 1.64 (qi, 2H, -CH2). C NMR (62.86 MHz, DMSO-d6, δ): 177.6 (2Cq, C=O), 55.6 195 (1Cq), 30.4 (2Cs), 15.0 (1Cs). Pt NMR (53.75 MHz, DMSO-d6, δ ppm): -1705.

Synthesis of [Pt(CBDC)(DACH)] (2b): 1b (396.52 mg, 1.0 mmol) was dissolved in 10 mL of water and 10 mL of ethanolic disilvercyclobutane-carboxylate (372.16 mg, 1.0 mmol) was added and the mixture was stirred for 48 h. Then the mixture was centrifuged and the supernatant was filtrated. The solution was slowly evaporated to dryness. The obtained solid was washed with acetone and dried under vacuum to obtain 2b with a yield of 147.98 mg -1 - - (31.4%). IR (ATR, cm ) 3409 (w), 2936 (w), 1620 (s,휈 asym(OCO )), 1348 (s, 휈 sym.(OCO )). MS (ESI-MS, neg. ion detection mode, m/z) 474 [M+Na-H]-, 451 [M-H]- 338 [M-DACH-H]- 1 . H NMR (400 MHz, DMSO-d6, δ): 5.86 (d, 2H, -NH2), 5.14 (t, 2H, -NH2), 2.68 (dq, 4H, -

CH2), 2.27 (t, 1H, -CH) 2.05 (t, 1H, -CH) 1.81 (d, 2H, -CH2) 1.65 (qi, 2H, -CH2) 2.44 (d, 2H, 13 -CH2) 1.21 (d, 2H, -CH2) 1.03 (m, 2H, -CH2). C NMR (62.86 MHz, DMSO-d6, δ): 177.3 195 (2Cq, C=O), 62.0 (2Ct), 55.5 (1Cq), 31.5 (2Cs), 30.2 (2Cs), 24.0 (2Cs), 15.0 (1Cs). Pt NMR

(53.75 MHz, DMSO-d6, δ ppm): -1906.

Synthesis of cis,trans,cis-[Pt(CBDC)(succ)(Br)(NH3)2] (3a): 2a (50 mg, 0. 13 mmol) was suspended in 5 mL of water and 10 mL of acetone and then succinic anhydride (26.96 mg, 0.27 mmol) was added. After dissolving the N-bromosuccinimide (23.98 mg, 0.13 mmol) was added and the mixture was stirred for 5 h under the exclusion of light. The solution was evaporated, a yellow oil was washed with acetone and evaporated to dryness. This procedure was repeated until a yellowish solid was obtained. The final product was dried under vacuum -1 to obtain 3a with a yield of 53.34 mg (69.8%). IR (ATR, cm ) 3203 (w,휈 sym.(NH3)), 1702 (w) - - 1620(s, 휈 asym(OCO )), 1422 (w) 1339(s, 휈 sym.(OCO )). MS (ESI-MS, negative ion - - 13 detectionmode, m/z) 590 [M+Na-H] , 567 [M-H] . C NMR (DMSO-d6, 125.7 MHz): δ 14.8

(OCO-C-CH2- CH2), 28.6 (OCO-CH2-CH2-COOH), 29.3 (OCO-C-CH2-CH2), 30.1(OCO-C-

CH2-CH2), 30.8 (OCO-CH2-CH2-COOH), 53.9(OCO-C-CH2-CH2), 172.5 (OCO-CH2-CH2-

COOH), 174.6 (OCO- C-CH2-CH2), 175.3 (OCO-C-CH2-CH2), 178.2 (OCO-CH2-CH2- 195 COOH) ppm. Pt NMR (53.75 MHz, DMSO-d6, δ ppm): 1090.

Synthesis ofcis,trans,cis-[Pt(CBDC)(succ)(Br)(DACH)](3b): 2b (50 mg, 0.11 mmol) was suspended in 5 mL of water and 10 mL of acetone and succinic anhydride (22.17 mg, 0.22 mmol) was added. After dissolving all starting materials, the N-bromosuccinimide (19.72 mg, 0.11 mmol) was added and the mixture was stirred for 5 h under the exclusion of

48 light. The solution was evaporated, a yellow oil was washed with acetone and evaporated again. This procedure was repeated until a yellowish solid was obtained. This product was dried under vacuum to obtain 3b with a yield of 31.11 mg (43.4%). IR (ATR, cm-1) 3143 (w), - - 2944 (w), 1709 (w) 1629 (s, 휈 asym(OCO )), 1447 (w) 1342 (s, 휈 sym.(OCO )). MS (ESI-MS, - - 13 negative mode, m/z) 647 [M-H] , 360 [M-(succ)-(CBDC)-Br+(OH)3] . C NMR (DMSO-d6,

125.7 MHz): δ15.5 (OCO-C-CH2- CH2), 23.4 (NH2-CH-CH2-CH2), 23.5 (NH2-CH-CH2-

CH2),29.6 (OCO-CH2-CH2-COOH), 30.6 (NH2-CH-CH2-CH2), 30.7 (OCO-C-CH2-CH2), 30.9

(NH2-CH-CH2-CH2), 31.6(OCO-C-CH2-CH2), 31.8 (OCO-CH2-CH2-COOH), 55.7(OCO-C-

CH2-CH2), 61.3 (NH2-CH-CH2-CH2), 61.4 (NH2- CH-CH2-CH2), 173.7 (OCO-CH2-CH2-

COOH), 176.2 (OCO- C-CH2-CH2), 176.3 (OCO-C-CH2-CH2), 180.0 (OCO-CH2-CH2- 195 COOH) ppm. Pt NMR (53.75 MHz, DMSO-d6, δ ppm): 1043.

Synthesis of peptide(R)9 −G−A−L (P1) and(R)9 −G−A−L−G−L−P−(E)9 (P2): The peptides were synthesized by solid phase peptide synthesis (SPPS). The Wang-resin connected with the first protected amino acid was swelling in DCM and DMF. After this the general synthesis cycle was involved. In the first step the amino acid connected with the resin was deprotected with 20% piperidine in DMF, which results detaching of Fmoc group. The resin with free amine group on N-terminus chain was washed and then the coupling with the next amino acid containing free carboxylic group was performed. For making amide bond the common coupling reagent were used: TBTU, HOBt. After complete synthesis deprotection of Fmoc group was carried out and finally cleavage of resin and all protecting group in acidic + conditions (TFA/TIS/H2O). MS (ESI-MS, positive mode, m/z) P1: 1666.0 [M] , P2:1548.8 [M]2+.

Synthesis of cis,trans,cis-[Pt(CBDC)(succ)(Br)(NH3)2]-(R)9-G-A-L (4a1), cis,trans,cis-

[Pt(CBDC)(succ)(Br)(NH3)2]-(R)9-G-A-L-G-L-P-(E)9 (4a2), bioconjugates: The bioconjugation reaction was carried out between platinum(IV) prodrugs with cell penetrating peptides on the resin. Two types of peptides were involved: P1 (R)9−G−A−L and P2 (R)9−G−A−L−G−L−P−(E)9. The synthesis of these compounds is described in detail for 4a as an example. Peptide P1 bounded to Wang-resin (88.69 mg, 0.057 mmol) was washed three times with 2 mL of DCM and three times with 2 mL of DMF for one minute each. Then deprotection of Fmoc group was performed by treatment of the peptide on the resin with 2 mL of 20% piperidine in DMF twice for ten minutes each. In the next step the coupling mixture of compound 3a (70.80 mg, 0.125 mmol), PyBOP (59.06 mg, 0.114 mmol),

49 DIPEA (79.07 µl, 0.454 mmol) in 2.5 mL DMF was prepared and loaded into a syringe. The reaction was carried out for 24 hours. The resin with bioconjugate was washed three times with 1 mL of each DMF, DCM and diethyl ether and then dried under vacuum. Afterwards a cleavage mixture of phenol (32.53 mg, 0.346 mmol), TFA (1.425 mL), and H2O (50 µl) was prepared and cleavage process was performed for 6 h. The platinum(IV) bioconjugate was precipitated from reaction mixture by n-hexane/diethyl ether (1:1) mixture. Finally, the product was dissolved in ACN/H2O, frozen and lyophilized. The bioconjugate was purified by semi-preparative HPLC. Yield: 4a1 15 mg (11,9%).

Cell culture. General procedure. Cells were grown in RPMI 1640 with 1% sodium pyruvate, 1% L-glutamine, 100units per mL Pen Strep, 10% fetal bovine serum. The cells were maintained at 37°C in a humidified incubator under an atmosphere containing 5% CO2.

Cytotoxicity experiments: Dulbecco‘s Modified Eagle‘s Medium (DMEM), containing 10% fetal calf serum, 1% penicillin and streptomycin, was used as growth medium. MCF-7, HepG2 and GM5657T cells were detached from the wells with trypsin and EDTA, harvested by centrifugation and resuspended again in cell culture medium. The assays were carried out on 96 well plates with 6000 cells per well for all cell lines: MCF-7, HepG2 and GM5657T.

After 24 h of incubation at 37.8°C and 10 % CO2, the cells were treated with the compounds (with final DMSO concentrations of 0.5 %) with a final volume of 200 mL per well. For a negative control, one series of cells was left untreated. The cells were incubated for 48 h followed by adding 50 mL MTT (2.5 mg mL-1). After an incubation time of 2h, the medium was removed and 200 mL of DMSO were added. The formazan crystals were dissolved and the absorption was measured at 550 nm, using a reference wavelength of 620 nm. Each test was repeated in quadruplicates in three independent experiments for each cell line.

50 3.2 Bioconjugates of Co(III) Complexes with Schiff Base Ligands and Cell Penetrating Peptides: Solid Phase Synthesis, Characterization and Antiproliferative Activity

Dariusz Śmiłowicz, NilsMetzler-Nolte* Inorganic Chemistry I – Bioinorganic Chemistry Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany E-mail: [email protected]; Fax: +49 234 321 4378

Keywords: Bioconjugates, Cell Targeting Peptides, Cobalt(III), Medicinal Inorganic Chemistry, Schiff base ligands, Solid Phase Peptide Synthesis

3.2.1 Abstract

In this work we synthesized a chelating Schiff base by a single condensation of salicylaldehyde with 3,4-diamino benzoic acid (5). This ligand was used further for complexation to CoCl2·6H2O under nitrogen. In the next step, three six-coordinate Co(III) complexes were synthesized by coordinating this complex with imidazole (6), 2- methyimidazole (7) and N-Boc-L-histidine methyl ester (8) in axial positions with simultaneous oxidation of Co(II) to Co(III) under ambient environment. All Co(III) complexes were characterized by multinuclear NMR spectroscopy (1H, 13C and 59Co NMR), FT-IR, mass spectrometry and HPLC. The Co(III) complexes were conjugated to three different cell penetrating peptides: FFFF (P3), RRRRRRRRRGAL (P4) and FFFFRRRRRRRRRGAL (P5). Standard solid-phase peptide chemistry was used for the synthesis of cell penetrating peptides. Coupling of N-terminal peptides with the cobalt complexes, possessing a carboxylic group on the tetradentate Schiff base ligand, afforded Co(III)-peptide bioconjugates, which were purified by semi-preparative HPLC and characterized by analytical HPLC and mass spectrometry. The antiproliferative activity of the synthesized compounds was studied against different human tumor cell lines: lung cancer A549, liver cancer HepG2 and normal human fibroblasts GM5657T, in comparison with the activity of cisplatin as a reference drug. The bioconjugate 25 containing the Co complex 8 and the combined phenylalanine and polyarginine cell penetrating sequence P5 shows better activity against the liver cancer line HepG2 than the parent Co(III) complex 8.

51

3.2.2 Introduction

Cobalt is an essential trace element in the human body where it exists exclusively in the form of vitamin B12 (cobalamin), which is a cofactor for a number of enzymes, like isomerases, methyl transferases or dehalogenases. 153 Further, cobalamin takes part in creating neurotransmitters and stimulates the formation of erythrocytes in bone marrow. 154 Deficiency of cobalt is strongly related to the vitamin B12 level and causes anaemia and thyroid hypofunction.155, 156 Recently, other cobalt compounds have gained extended attention in the field of medicinal inorganic chemistry, especially in the search for new anticancer drug candidates. 157 The severe side effects of platinum-based anticancer drugs have forced researchers towards finding metal-based anticancer drugs as alternatives. 158 From a chemical and physical point of view cobalt, similar to platinum, adopts a wide variety of coordination numbers, geometries, oxidation states, and ligand binding affinities, which makes cobalt chemistry thoroughly investigated, not only but also in the field of new anticancer agents. 159, 160 Interestingly, despite the fact that cobalt(III) complexes possess the same electron configuration (d6) as platinum(IV) prodrugs, cobalt(III) complexes exhibit a different mode of action from platinum-based anticancer drugs. 161 The very inert oxidised cobalt(III) state is reduced to more labile cobalt(II), facilitating the exchange of axial ligands. 162,163,164 Binding of the complexes to histidine residues in or nearby the active sites of proteins causes irreversible inhibition of activity.165,166 Thus, Co(III) complexes inhibit histidine-containing proteins and enzymes including zinc finger transcription factors (TFs) and metalloendopeptidases. Protein inhibition occurs through a dissociative exchange of labile axial ligands for the imidazole nitrogens of histidine residues, as drawn schematically in Figure 24.167,168

Figure 24. Cobalt binding to histidine residues through dissociative axial ligand exchange.

52 Moreover, cobalt is generally less toxic to humans than platinum, which creates a basis for the investigation of cobalt-containing compounds as less toxic alternatives to platinum-based anticancer drugs.169,170 The simple Co3+ ion is unstable in water, but can be stabilized to hamper reduction to Co2+ by coordination to chelating N,O or N,S donor ligands. 171 Cobalt(III) complexes derived from these donor ligands find applications as antibacterial and antiviral agents.172 One of the most promising classes of Co(III) complexes containing N, O donor ligands are those based on chelating Schiff bases. 173 So far, among all Co(III) complexes, clinical trials have been reached only by a cobalt(III) Schiff base complex containing bis(acetylacetone)ethylenediimine (acacen), with two axially coordinated 2- 174 methylimidazole rings [Co(III)(acacen)(2-mimd)2] (Figure 25 A). The drug formulation was developed as Doxovir™ by the Redox Pharmaceutical Corporation. In 2013, Doxovir successfully completed phase II clinical trials for the treatment of Herpes Simplex Virus Type 1 (HSV-1) labialis infections and phase I clinical trials for the treatment of two viral eye infections, the major causes of blindness (ophthalmic herpetic keratitis and adenoviral conjunctivitis). 175 The promising antiviral activity of Doxovir is attributed to the direct interaction of the Co(III) Schiff base complex with its molecular target, the herpes virus maturational protease, a serine protease containing large amounts of histidine.

Figure 25. Examples of cobalt(III) inorganic drug candidates.

Another promising group of cobalt(III) complexes are those with nitrogen mustards ligands, which are potent cytotoxins, utilizing the lone pair on the amine nitrogen to initiate a reaction sequence, in which DNA is cross-linked by double alkylation.176,177 ,178 The Co(III) complex + [Co(Meacac)2(DCE)] (Meacac = Methlyacetylacetonate, DCE=N,N-bis(2-

53 chloroethyl)ethylenediamine) (Figure 25 B) had 20-times greater activity against cancer cells under hypoxic than oxic conditions, due to bioreduction of the starting Co(III) complex to Co(II) in the hypoxic regions of solid tumours and subsequent release of the cytotoxic free from the substitutionally more labile Co(II) ion. 179 Along the same lines, the Hambley group has exploited bioreductive prodrugs of chaperone cobalt(III) complexes for the delivery of cytotoxic ligands to hypoxic solid tumours. 180 Firstly, Hambley et al. presented a bioreductively activated carrier system for the delivery and release of curcumin. The dichlorido precursor complex, [CoCl2(tpa)]ClO4, (tpa = tris-(2- pyridylmethyl)amine), exhibited no toxicity up to a concentration of 200 µM against the colorectal cancer cell line DLD-1, whereas the Co(III) complex with coordinated curcumin 181 possesses an IC50 value of 39 ± 4 µM. Secondly, Hambley et al. have prepared a series of cobalt(III) complexes with the tripodal, ancillary tpa ligand as chaperones for delivery of derivatives of hydroxamic acid, which inactivates enzymes by binding to catalytic zinc ions through the hydroxamic acid moiety. The [Co(tpa)(c343ha)]ClO4 complex with a fluorescent hydroxamic acid ligand (c343ha) showed enhanced antiproliferative activity against DLD-1 182 colon cancer cells (31 ± 2 µM) in comparison to the free c343haH2 ligand (113 ± 8 µM). Later, Hambley and co-workers extended the concept of Co(III) prodrugs to selectively deliver inhibitors of matrix metalloproteinases (MMP) enzymes, which are involved in the process of tumour metastasis. The Co(III) carrier system consists of the MMP inhibitor marimastat with a tetradentate tpa carrier ligand (Figure 25 C). In vivo antimetastatic activity tested against Balb/c mice with 4T1.2 tumour implants of C showed a higher level of tumour- growth inhibition than free marimastat. 183 Meade et al. have synthesized Co(III) Schiff base- DNA conjugates targeting C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway, which regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. Such Co(III)-DNA bioconjugates resulted in a targeted inhibitor of the single C2H2 zinc finger transcription factor Cubitus Interruptus (Ci). 184,185 ,186 In the literature there are also examples of Co(III) conjugates with and pepleomycin, glycopeptide antitumor antibiotics, which are used in the treatment of Hodgkin‘s lymphoma, carcinomas of the skin, head and neck.187,188 Such Co-glycopeptide conjugates bind to the DNA and under UV or visible light irradiation result in DNA cleavage. 189,190 ,191 ,192 Despite the fact that cobalt(III) complexes with small molecular bioactive ligands as described above were extensively studied as antiviral, antimicrobial and anticancer agents, the development of cobalt(III) bioconjugates with larger biomolecules and suitable drug delivery systems for them is still in its infancy. Our research is devoted to the design of peptide

54 delivery systems for various anticancer drugs. 193,194 ,195 In the past, we have synthesized a dicobalt hexacarbonyl alkyne compound linked to the neurotensin peptide hormone. The cobalt-peptide bioconjugates showed moderate cytotoxicity against HeLa cervical cancer cells (26.4 ± 5.8 µM).196 Thus, despite their demonstrated versatility cobalt derivatives, especially cobalt-peptide bioconjugates have not been further studied as anticancer agents. This fact and our previous work on bioconjugation techniques for inorganic pharmaceuticals,197 ,198 ,199 ,200 prompted us to investigate cell penetrating peptides as a drug delivery system for promising Co(III) anti-cancer candidates, as an alternative to platinum-based therapy.

3.2.3 Results and discussion

Synthesis and characterisation of cobalt(III) complexes

The key idea of this study is to synthesize a Schiff base ligand containing a functional group, suitable for coupling with peptides. Such covalent linking to peptides via the ligand has not been achieved before. The literature-known ligand H2salophen 5 was synthesized by a single condensation of salicylaldehyde with 3,4-diamino benzoic acid (Scheme 3).201,202 The ligand was fully characterized by FT-IR, ESI-MS, HPLC, 1H- and 13C-NMR (ESI†).

Scheme 3. Synthesis of Schiff base ligand 5.

In the next step, the complexation reaction of Schiff base ligand 5 to cobalt(II) was carried out under nitrogen atmosphere (Scheme 4). The ligand 5 was dissolved in absolute MeOH and subsequently CoCl2·6H2O was added. The formation of the expected cobalt(II) complex of ligand 5 in situ was confirmed by electrospray mass spectrometry(see ESI†). In the full-scan mass spectra, the molecular ion peak [M+H]+ (m/z = 417) was found and assigned to cobalt(II) complexes with ligand 5. 3 hours were obligatory to obtain the cobalt(II) complex in situ. To the in situ formed cobalt(II) complex bearing the Schiff base ligand 5, three

55 different types of N-donors were added as the axial ligands (2 eq.), namely imidazol, 2- methyimidazol and N-Boc-L-histidine methyl ester. The imidazole-based ligands were chosen because of the previously noted remarkable activity of cobalt(III) Schiff base complexes with coordinated 2-methylimidazole rings.174 In the case of N-Boc-L-histidine methyl ester, both functional groups (amino and carboxylic group) were protected to avoid any side reactions during future coupling with peptides by solid phase peptide synthesis. After adding the N- donors, the reaction mixture was opened to air to allow oxidation of cobalt(II) to cobalt(III) (Scheme 4).

Scheme 4. Synthesis of cobalt(III) complexes.

All cobalt(III) complexes were characterized by FT-IR, mass spectrometry, HPLC and multinuclear NMR spectroscopy (1H, 13C and 59Co NMR). The IR spectra of the cobalt(III) complexes 6 – 8 were compared to those of the free ligands. The IR spectral bands with their

56 assignment observed in the region 4000-400 cm-1 are listed in Table 9. The medium and broad absorption peaks in the range 3102-3356 cm-1 were assigned to ν(O–H) stretching frequencies showing the existence of a carboxylic acid. The sharp bands indicative of NH vibrations are located at 2900-2999 cm-1. IR spectra show that the vibration bands of the ν(C=N) imino groups were shifted to 1600–1675 cm-1. These shifts by 15 cm-1 compared to the free ligand 5 indicate that the nitrogen atoms of the imino groups coordinate to the cobalt 203 -1 centre. The sharps bands of the νas(COO) stretching frequencies at 1502–1633 cm and -1 νsym(COO) at 1289–1370 cm indicate the existence of carboxylate salts. All IR spectra are reproduced in the ESI†.

Table 9. Principal IR bands, assignments, and59Co NMR chemical shifts.

Compound IR assignment 59Co NMR - - ν(O-H) ν(N-H) ν(C=N) νas(COO ) νs(COO ) δ (ppm) ν1/2 (Hz) 5 3344 - 1670 1633 1340 - - 6 3230 2988 1680 1537 1324 4550 5200 7 3223 2999 1688 1566 1354 4670 2900 8 3220 2987 1695 1588 1365 4812 2400

Electrospray ionization mass spectrometry (ESI-MS) was applied to confirm the mass of the desired compounds (Figure 26a). In the full-scan mass spectra, the molecular ion peaks [M+H]+ were found and assigned to all complexes. The m/z values are consistent with the proposed constitution of the compounds. The mass spectra of cobalt(III) complexes displayed two additional peaks with moderate intensity corresponding to the [M-L]+ and [M-2L]+ species. This feature confirms the expected primary fragmentation pathway, which would be characterized by the gradual loss of one and the second ligand in the axial positions. The full- scan mass spectra of complexes 6 - 8 are available in the ESI†. To fully accomplish characterization of the Co(III) complexes also their 59Co NMR spectra were measured. Representative spectra are shown in Figure 26, and chemical shifts (δ) and 59 line widths (ν1/2) are summarized in Table 9. The remaining Co NMR spectra are available in the ESI†. All spectra showed only one peak, which confirms the oxidation of the cobalt(II) precursor to only one cobalt(III) complex. The 59Co NMR data show that the nature of the different axial ligands affect the amount of electron density at the Co nucleus. 204 The signal is shifted downfield, when the axial ligand is altered from imidazole (6) to 2-methylimidazole (7) and to N-Boc-L-histidine methyl ester (8).

57

59 Figure 26. a) ESI-MS spectrum of complex 8; b) Co NMR spectra of complex 8 in dmso-d6.

Synthesis and characterisation of peptides

To enhance the cellular uptake and hence potentially the cytotoxicity of cobalt(III) Schiff base complexes we decided to utilize cell penetrating peptides (CPPs). CPPs are usually rich in positively charged amino acids, such as arginine (R) and lysine (K). 205 Oligoarginine / arginine-rich cell-penetrating peptides have been successfully used as vectors for the intracellular delivery of small anticancer drugs without specific receptors. 206 It has been also reported that the attachment of a short peptide segment, namely hydrophobic sequences of phenylalanine residues (F) results in enhanced translocation through cell membranes of arginine-rich CPPs, due to stronger interaction with membranes. 207,208 Therefore, we prepared two peptides containing an arginine-rich sequence, one with an additional FFFF segment at the N-terminus. A third peptide, consisting only of phenylalanine residues, was design as a control peptide for cytotoxicity experiments. Solid-phase peptide synthesis (SPPS) with Fmoc strategy was employed to synthesize those CPPs (Scheme 5).209 Peptide P3, which consists of only a tetraphenylalanine sequence, was synthesized manually. The longer peptides P4 and P5, were assembled on an automated, microwave-assisted synthesizer. Deprotection of temporary Fmoc protecting groups was performed with 20% piperidine in DMF. The coupling was performed with an excess of the Fmoc-amino acid (4 eq.), activated with 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) in the presence of N-hydroxybenzotriazole (HOBt, 4 eq.) and an excess of diisopropylethylamine (DIEA, 6 eq.). SPPS consists of repeated cycles of N-terminal protecting group removal on the last amino acid, followed by coupling of the next incoming amino acid. Through

58 repetitions of a reaction cycle of peptide coupling, washing, deprotection and washing the desired peptides were obtained.

Scheme 5. Synthesis of peptides P3, P4 and P5 by SPPS.

After completion of peptide synthesis and before the bioconjugation reaction with Co(III) complexes 6 – 8 the successful synthesis of peptides P3, P4 and P5 was established by HPLC and ESI-MS. For this purpose, 10 mg of the resin from each peptide was treated with 20% of piperidine in DMF in order to remove the Fmoc group from the N-terminus. Then, after washing and drying the peptides were cleaved from the resin by treatment with a mixture of

TFA/phenol/H2O (95%/2.5%/2.5%). After cleavage from the resin the peptides were

59 lyophilized. The crude peptides were purified by reverse-phase semi-preparative HPLC. Finally, the peptides were characterized by HPLC and ESI-MS measurements. The purity of the peptides was confirmed by analytical HPLC (ESI†). ESI-MS spectra confirm the molecular mass [M+H]+ for peptide P3 at m/z = 606, for peptide P4 at m/z = 1664 and for peptide P5 at m/z = 2253. Besides the signals of molecular masses, multiply charged species were formed, in the case of peptide P4 (M+2H)2+ at m/z = 832, (M+3H)3+ at m/z = 555, (M+4H)4+ at m/z = 416, and for peptide P5 (M+2H)2+ at m/z = 1127 and (M+3H)3+ at m/z = 752 (ESI†).

Table 10. Characterization of peptides and bioconjugates after purification by semi- preparative HPLC.

+ Compound Yield [%] m/zexptl m/zcalcd[M+H] tR [min] P3 44 606.40 606.28 6.12 P4 38 1664.03 1664.06 9.30 P5 41 2252.87 2252.34 9.80 17 12 1140.02 1139.09 7.19 18 25 1168.08 1167.12 10.30 19 23 1342.55 1341.46 11.50 20 18 2198.42 2197.09 7.30 21 32 2226.00 2225.12 10.72 22 17 2400.09 2399.24 12.10 23 36 2785.40 2785.43 8.20 24 33 2813.80 2813.46 12.60 25 22 2969.90 2969.52 13.25

Synthesis and characterisation of bioconjugates

In the last synthesis step, the peptides were coupled to our Co(III) complexes which contain a free carboxylic acid group for bioconjugation.

60

Scheme 6. Synthesis of Co(III)-peptide bioconjugates.

61 The coupling of Co(III) complexes 6, 7 and 8 with the Fmoc-deprotected peptides P3, P4, P5 was performed with the fully side-chain protected peptides on the resin in order to avoid any undesired side reactions (Scheme 6). The carboxylic acid group on the Schiff base ligand provided a suitable functionality for coupling by SPPS. The metal complex was dissolved in DMF and coupling was performed with an excess of the cobalt complexes (4 eq.), activated with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) in the presence of N-hydroxybenzotriazole (HOBt, 4 eq.) and an excess of diisopropylethylamine (DIEA, 8 eq.). After 24 h, an excess of reagents was removed by filtration, the resin was washed with DMF, DCM, Et2O, and then dried under vacuum. Cleavage from the resin and deprotection of the pbf side-chain protecting groups of arginine, for peptides P4 and P5, was achieved with 95% TFA. The obtained metal-peptide bioconjugates were purified by preparative HPLC to obtain the desired products. All metal- peptide bioconjugates were characterized by analytical HPLC, ESI-MS and MALDI-MS. Analysis by RP-HPLC showed main peaks (ESI†), which were isolated and characterized by ESI mass spectrometry. After purification by semi-preparative HPLC and lyophilization, all bioconjugates were obtained as pale brown solids. ESI-MS analysis of the conjugates showed m/z values that were consistent with that of the charged species [M+H]+, [M+Na]+ and doubly charged species [M + 2H]2+, also showing the expected isotopic mass distribution patterns (ESI†). The observed m/z values are listed in Table 10.

62

Figure 27. Structures of Co(III)-peptide bioconjugates.

63 Cytotoxicity Our next objective was to evaluate the anti-proliferative activity of Co(III) complexes and cobalt-peptide bioconjugates against human liver cancer cells (HepG2), human lung cancer cells (A549) and normal human fibroblast cells (GM5657T) by the MTT assay. The choice of cell lines was guided by our previous studies on platinum, gold and metallocene 195,196, 197,210 bioconjugates. For comparison, the IC50value of cisplatin was also determined using the same assay, and identical conditions. We utilized DMSO as the solvent for coherence, so that all compounds are treated in an absolutely identical fashion. While we are aware of the effect of DMSO on cisplatin activity reported by Hall et al., 211 we did not observe a similar influence of DMSO on ligand exchange in our Co complexes by 59Co NMR.

Cytotoxicity results are expressed as IC50 values (half-maximal inhibitory concentration) and are summarized in Table 11. Dose-response curves are shown in the ESI†. Firstly, we investigated the cytotoxic activity of all cobalt complexes. As indicated in Table 11 all Co(III) complexes display moderate in vitro activity. HepG2 liver cancer cells appeared to be the most sensitive to cobalt complexes among all testes cell lines. Complex 8 was the most active towards HepG2 human liver cancer cells (13.2±0.8 µM) comparable with cisplatin (13.3±1.4 µM). The GM5657T non-cancerous human fibroblast cells were the most sensitive (22.0±0.9 µM) to complex 4.

Table 11. Cytotoxicity data of metal complexes, metal-free peptides and Co-peptide conjugates determined by the MTT assays after 48 h of incubation.

a Cell lines IC50 [µM]

A549 HepG2 GM5657T

Cisplatin 25.3±2.3 13.3±1.4 9.0±2.2 6 43.8±1.6 28.7±0.9 22.0±0.9 7 65.3±0.9 43.0±1.5 46.5±1.3 8 32.0±2.2 13.2±0.8 51.2±1.1 P3 >250 >250 >250 P4 >250 >250 >250 P5 >250 >250 >250 19 64.3±2.8 49.3±1.9 72.4±3.3 22 35.8±1.1 20.0±0.9 61.0±3.1

64 25 25.3±1.9 9.0±1.2 36.4±1.6 a The IC50 values are averages of three independent determinations. All compounds were dissolved in DMSO (final concentration 0.5 %).

Secondly, we investigated the influence of cell penetrating peptides attached to the cobalt(III) complexes on in vitro cytotoxicity (Table 11). Since complex 8 appeared to be the most potent cytotoxic agent among all cobalt complexes, only bioconjugates containing this complex (19, 22, 25) were investigated by the MTT assay. Bioconjugate 19 with the short phenylalanine sequence showed only moderate activity towards all cancer cells, lower than complex 8. Bioconjugate 22 consisting of an polyarginine domain showed activity similar to the parent Co(III) complex. Finally, bioconjugate 25 possessing the combined polyarginine sequence and a short uptake-accelerating tetra-phenylalanine sequence on the N-terminus shows slightly improved activity in comparison to complex 8 against all tested cells. Similar observations were made by Takayama et al., who observed that the attachment of a small hydrophobic peptide segment to arginine rich CPPs improves cellular uptake.212 Also Sadler et al. described the improved cytotoxic activity of osmium(II) complexes after conjugation to polyarginine peptides. 213 Since cell penetrating peptides without any particular selectivity were used in this work cancer cells as well as normal human fibroblast cells were about equally affected.

In a control experiment we investigated the anti-proliferative impact of the cell penetrating peptides alone, i.e. without cobalt(III) Schiff base complexes attached. Together, the metal- free peptides P3, P4 and P5 were examined by MTT assays using the same cell lines and conditions as for the parent bioconjugates (Table 11). These peptides exhibit no in vitro activity at concentrations of up to 250 μM. These results are in agreement with observations made by Gross et al.195, where similar peptides with only a slight C-terminal modification appeared to be inactive as well.

3.2.4 Conclusion

Cobalt complexes emerge as a promising alternative to classical platinum-based anticancer drugs. Here we present the synthesis of Co(III) complexes with salen-type Schiff base ligand as chelating donor and three different imidazole derivatives containing N-donors as axial

65 ligands. Notably, a free carboxylic group on the salen-type ligand provides a handle for the synthesis of covalent bioconjugates. By reaction with this carboxylate group, cobalt(III)- bioconjugates with cell penetrating peptides have been synthesized and characterized for the first time. First, the cell penetrating peptides were synthesized by solid phase peptide synthesis (SPPS). Then, an SPPS reactions scheme of resin-bound peptides and the Co(III) complexes with one free carboxylate group afforded monoconjugated cobalt-peptide species, which were purified by RP-HPLC and characterized by ESI-MS. All Co complexes and selected Co-peptide bioconjugates were screened for their cytotoxicity against selected cancer cell lines. The Co(III) complexes alone show promising cytotoxicity in the mid-to-low micromolar range. Among all bioconjugates, the bioconjugate which consists of a CPP- polyarginine peptide sequence with four addition phenylalanine residues (25), was found to be most active against HepG2 liver cancer cells. It is noteworthy that the peptide sequences chosen here are not know for any particular intra-cellular targeting other than uptake through lysosomes. On the other hand, adding a peptide amplifies the molecular weight of the (small) Co complex significantly, and this might potentially alter its propensity to interact with the intracellular target. On the other hand, the fact that we do see acceptable bioactivity of the Co- peptide conjugates in our work indicates that at least a fraction of the Co complex will reach the intracellular target and exert its activity. Further investigations as to what this target would be shall be carried out and the results reported subsequently.

3.2.5 Experimental section

Materials and reagents

All reagents and chemicals were purchased from commercial sources and used without further purification. Salicylaldehyde, 3,4-diamino benzoic acid and CoCl2·6H2O were purchased from Merck. All Fmoc-protected amino acids were purchased from Iris Biotech GmbH. All the materials and organic solvents including absolute methanol, ethanol, dimethyl sulfoxide and dimethylformamide were commercially obtained from Merck, Aldrich or Fluka. Unless otherwise noted, all manipulations were performed using standard Schlenk techniques under nitrogen atmosphere. For the biological experiments, Dulbecco‘s Modified Eagle‘s Medium (DMEM), fetal bovine serum, penicillin/streptomycin mixture, trypsin/EDTA, and phosphate- buffered saline (PBS) were purchased from Sigma-Aldrich. Compounds were dissolved in DMSO and diluted with the tissue culture medium before use.

66 General Methods: Solvents were dried according to standard procedures and stored over molecular sieves (4 Å). Solid-supported reactions were performed in 5 mL plastic syringes with a porous polypropylene disc as filter. HPLC was performed by using two buffer systems

(buffer A: H2O/MeCN/TFA, 95:5:0.1, v/v/v; buffer B: MeCN/H2O/TFA, 95:5:0.1, v/v/v) as the mobile phase. Preparative HPLC runs were performed by using a Dr. Maisch reprosil C18 reversed-phase column (250 × 20 mm) at a flow rate of 10 mL/min with a linear gradient of buffer B (100 % in 40 min) from 100 % buffer A with a total run time of 60 min. Analytical HPLC runs were performed by using a Knauer Eurospher-II C18 reversed-phase column (250 × 4.6 mm) at a flow rate of 1.0 mL/min with a linear gradient of buffer B (100 % in 20 min) from 100 % buffer A with a total run time of 40 min. FT-IR spectra were recorded with ATR technique on a Spectrum 65 PerkinElmer instrument. NMR spectra were recorded at room temperature with a Bruker Avance 300 Digital (1H at 300 MHz) spectrometer. 1H and 13C

NMR spectra were referenced using the residual solvent chemical shift (DMSO-d6). Mass spectra were recorded with a Bruker Esquire 6000 (ESI-MS) spectrometer. UV-Vis absorption spectra were recorded on a Spectrophotometer Microplate Reader (Berthold Detection System).

Synthesis of the ligand

(5): Salicylaldehyde (1.60 g, 0.13 mmol) was mixed with ethanol (10.0 mL) and simultaneously 3,4-diamino benzoic acid (1.00 g, 0,01 mmol) with the same solvent (10.0 mL). This solution of 3,4-diamino benzoic acid was added dropwise to the salicylaldehyde solution and the reaction mixture was stirred overnight at room temperature. After 24h, the orange precipitation was collected and washed three times with Et2O. The product was dissolved and purified by preparative HPLC using 0.01% TFA as the aqueous mobile phase and acetonitrile as the organic mobile phase with a linear gradient from 10% to 90% of the organic mobile phase over 20 min. The final product was dried by rotary evaporation, followed by lyophilization, to give a pale yellow oil. Yield: (2.09 g, 52%). Anal. Calc. for

C21H16N2O4: C, 69.99; H, 4.48; N, 7.77. Found: C, 70.11; H, 5.03; N, 7.51. MS (ESI-MS, pos. mode, m/z): obsv.: 361.00 [M+H]+; calcd: 361.11 [M+H]+, obsv.: 383.00 [M+Na]+; calcd: 383.10 [M+Na]+; obsv.: 255.40 [M-L]+; calcd: 254.07 [M-L]+. 1H NMR (300 MHz, DMSO- d6) δ: 8.94 (s, 2H), 8.13 (d, 2H), 7.80 (ddd, 2H), 7.71 (m, 4H), 7.65 (d, 2H), 7.35 (d, 2H), 7.32

(s, 2H), 6.93 (t, 4H). 13C NMR (126 MHz, DMSO-d6) δ: 163.6, 161.1, 160.0, 144.6, 132.4, 132.1, 130.1, 127.2, 123.6, 123.5, 121.4, 120.5, 117.8.

67 Synthesis of Co(III) complexes

(6): To a methanolic suspension (5 mL) of ligand 5 (40 mg, 0.0989 mmol), CoCl2·6H2O complex (51.7 μL, 0.297 mmol) was added directly under nitrogen atmosphere at room temperature. After 2 hours, 2 eq. of imidazol (12.54 mg, 0.0989 mmol) was added to the reaction. The solution was opened to air and the reaction was stirred for 12 h to produce a brown solution. The solution was concentrated. Addition of diethyl ether precipitated a brown solid. The product was filtered and washed with cold diethyl ether. Yield: (0.657 g, 49%).

Anal. Calc. for C27H22ClCoN6O4: C, 55.07; H, 3.77; N, 14.27. Found: C, 55.42; H, 3.83; N, 14.49. MS (ESI-MS, pos. mode, m/z): obsv.: 564.80 [M+Na]+; calcd: 574.09 [M+Na]+, obsv.: 485.90 [M-L]+; calcd: 484.06 [M-L]+, obsv.: 485.90 [M-2L]+; calcd: 417.03 [M-2L]+. 1H

NMR (300 MHz, DMSO-d6) δ: 8.94 (s, 2H), 8.13 (d, 2H), 7.80 (ddd, 2H), 7.71 (m, 4H), 7.65

(d, 2H), 7.35 (d, 2H), 7.32 (s, 2H), 6.93 (t, 4H). 13C NMR (126 MHz, DMSO-d6) δ: 163.6, 161.1, 160.0, 144.6, 132.4, 132.1, 130.1, 127.2, 123.6, 123.5, 121.4, 120.5, 117.8.

(7): To a solution of the ligand 5 (0.50 g, 2.45 mmol) in MeOH (5.0 mL) was added directly solid CoCl2·6H2O complex (0.24 g, 1.025 mmol) under nitrogen atmosphere at room temperature with continuous stirring. The mixture was stirred at room temperature for 2 h. After that, 2 eq. of 2-methylimidazol (12.54 mg, 0.0989 mmol) was added to the reaction. The reaction was subjected to stirring for 18 h in an open-air environment to produce a bright brown precipitation. Finally, the precipitate was filtered, washed with Et2O (5.0 mL) and dried. Yield: (0.657 g, 49%). Anal. Calc. for C29H26ClCoN6O4: C, 56.46; H, 4.25; N, 13.62. Found: C, 56.86; H, 4.33; N, 14.01. MS (ESI-MS, pos. mode, m/z): obsv.: 582.00 [M+H]+; calcd: 581.48 [M+H]+, obsv.: 499.80 [M-L]+; calcd: 498.70.10 [M-L]+, obsv.: 416.90 [M- + + 2L] ; calcd: 417.03 [M-2L] . 1H NMR (300 MHz, DMSO-d6) δ: 8.94 (s, 2H), 8.13 (d, 2H), 7.80 (ddd, 2H), 7.71 (m, 4H), 7.65 (d, 2H), 7.35 (d, 2H), 7.32 (s, 2H), 6.93 (t, 4H). 13C NMR

(126 MHz, DMSO-d6) δ: 163.6, 161.1, 160.0, 144.6, 132.4, 132.1, 130.1, 127.2, 123.6, 123.5, 121.4, 120.5, 117.8.

(8): The ligand 5 (0.31 g, 1.5 mmol) was suspended in MeOH (10 mL) and to the suspension was added immediately solid CoCl2·6H2O complex (0.24 g, 1.025 mmol) under nitrogen atmosphere at room temperature with continuous stirring. The reaction was carried out for 3 h, and then 2 eq. of N-Boc-L-histidine methyl ester (12.54 mg, 0.0989 mmol) was added to the reaction. The solution was opened to air and an immediate colour change to deep brown was observed. After stirring at room temperature for 24 h, the resulting solution was allowed

68 to evaporate resulting in the precipitation of a deep brown solid, which was collected by filtration and washed with Et2O. Yield: (0.657 g, 49%). Anal. Calc. for C45H52ClCoN8O12: C, 54.52; H, 5.29; N, 11.30. Found: C, 54.92; H, 5.37; N, 11.46. MS (ESI-MS, pos. mode, m/z): obsv.: 458.88 [M+H]+; calcd: 458.11 [M+H]+; obsv.: 480.82 [M+Na]+; calcd: 481.10 [M+Na]+; (ESI-MS, neg. mode, m/z): obsv.: 456.85 [M-H]-; calcd: 457.10 [M-H]-; obsv.: - - 478.73 [M+Na-H] ; calcd: 480.09 [M+Na-H] . 1H NMR (300 MHz, DMSO-d6) δ: 8.94 (s, 2H), 8.13 (d, 2H), 7.80 (ddd, 2H), 7.71 (m, 4H), 7.65 (d, 2H), 7.35 (d, 2H), 7.32 (s, 2H), 6.93

(t, 4H). 13C NMR (126 MHz, DMSO-d6) δ: 163.6, 161.1, 160.0, 144.6, 132.4, 132.1, 130.1, 127.2, 123.6, 123.5, 121.4, 120.5, 117.8.

Peptide Synthesis: The FFFF peptide (P3) was synthesized manually by means of the Fmoc protocol on an Fmoc-Phe-Wang resin (0.64 mmol/g). This peptide was synthesized on a 0.25 mmol scale. Each synthetic cycle consisted of the following steps: Fmoc removal, washing, coupling, washing. Fmoc removal: The resin (357 mg, 0.250 mmol) was treated with a 20 % solution of piperidine in DMF (3.0 mL; 2 × 10 min). The solution was removed and the resin washed with DMF (3.0 mL; 3 × 1 min) and CH2Cl2 (3.0 mL; 3 × 1 min). The presence of free α-amino functionalities was checked by the bromophenol blue (BPB) test (blue-green beads). Coupling of standard amino acids: A mixture of Fmoc-Xxx-OH (1.00 mmol, 4 equiv.), 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1.00 mmol, 4 eq.), N-hydroxybenzotriazole (HOBt, 1.00 mmol, 4 eq.) and DIEA (1.5 mmol, 6 equiv.) in DMF (3.0 mL) was added to the resin and the suspension mixed by shaking at room temperature for 45 min. Reagents and solvents were removed by filtration, and the resin was subsequently washed with DMF (3.0 mL; 3 × 1 min) and CH2Cl2 (3.0 mL; 3 × 1 min). Completion of the coupling(absence of free α-amino functionalities) was verified either by the Kaiser test or the

BPB test (colorless beads in both cases). Then, the resin was washed with CH2Cl2 (3.0 mL; 3

× 1 min), Et2O (3.0 mL; 3 × 1 min), and finally was dried under vacuum. The resin bound polyarginine peptides RRRRRRRRRGAL (P4) and FFFFRRRRRRRRRGAL (P5) were synthesized on a CEMLibertyBlueTM automated microwave peptide synthesizer using standard protocols (amino acid coupling: TBTU in DMF (0.5 M), HOBt in DMF (0.5 M), DIEA in NMP (2 M) and amino acids in DMF (0.2 M); arginine coupling: 25 min, 75 1C, 0 W followed by 5 min, 75 1C, 25 W; standard amino acid coupling: 5 min, 75 1C, 24 W. For deprotection: 20% piperidine in DMF: initial deprotection: 0.5 min, 75 1C, 30 W followed by deprotection: 3 min, 75 1C, 50 W). Afterwards, aliquots of 100 mg of peptide-containing resin were transferred into a filter-containing syringe for further derivatization.

69 Deprotection and Cleavage. The Fmoc deprotection was performed twice by treating the Fmoc protected peptide with 2 mL of 20% piperidine in DMF (each time 10 min). For the synthesis of all compounds, the N-terminal Fmoc group was deprotected, and after each step the resin was washed 5 times using 2 mL of DMF. TFA cleavage: The resin was swirled in a mixture of TFA/phenol/H2O (2.0 mL; 95:2.5:2.5, v/v/v) at room temperature for 1 h. Then the resin was removed and the residual TFA solution transferred to a 50 mL falcon tube and an ice-cold mixture of Et2O/hexane (20 mL; 1:1 v/v) was added to precipitate the peptide. The supernatant was removed after centrifugation, and the peptide pellets were washed twice with

Et2O/hexane. The crude peptides were dissolved in MeCN/H2O (1:1, v/v) and lyophilized. Crude peptides were purified by preparative HPLC, and the pure fractions were pooled and lyophilized. Finally, the peptides were analyzed by analytical HPLC and characterized by ESI-MS.

Cell culture. General procedure. Cells were grown in RPMI 1640 with 1% sodium pyruvate, 1% L-glutamine, 100 units per mL Pen Strep, 10% fetal bovine serum. The cells were maintained at 37°C in a humidified incubator under an atmosphere containing 5% CO 2.

Cytotoxicity experiments: Dulbecco‘s Modified Eagle‘s Medium (DMEM), containing 10% fetal calf serum, 1% penicillin and streptomycin, was used as growth medium. A549, HepG2 and GM5657T cells were detached from the wells with trypsin and EDTA, harvested by centrifugation and re-suspended again in cell culture medium. The assays were carried out on 96 well plates with 6000 cells per well for all cell lines: human lung cancer (A549), human liver cancer (HepG2) and normal human fibroblast (GM5657T). After 24 h of incubation at

37.8 °C and 10 % CO2, the cells were treated with the compounds (with final DMSO concentrations of 0.5 %) with a final volume of 200 mL per well. For a negative control, one series of cells was left untreated. The cells were incubated for 48 h followed by adding 50 mL MTT (2.5 mg mL-1). After an incubation time of 2 h, the medium was removed and 200 mL of DMSO were added. The formazan crystals were dissolved and the absorption was measured at 550 nm, using a reference wavelength of 620 nm. Each test was repeated in quadruplicates in three independent experiments for each cell line.

70 3.3 BIOCONJUGATION OF CYCLOMETALATED GOLD(III) LIPOIC ACID FRAGMENTS TO LINEAR AND CYCLIC BREAST CANCER TARGETING PEPTIDES

Dariusz Śmiłowicz, Jack C. Slootweg, Nils Metzler-Nolte*

Inorganic Chemistry I – Bioinorganic Chemistry Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany E-mail: [email protected]

Keywords: Bioconjugates, Breast Cancer, Cell Targeting Peptides, Drug Targeting, Gold(III), Medicinal Inorganic Chemistry, Precision Medicine, Solid Phase Peptide Synthesis (SPPS)

3.3.1 Abstract

Cell targeting peptides (CTPs) are increasingly used in the field of cancer research due to their high affinity and specificity to cell or tissue targets. In the search for novel metal-based drug candidates, our research group is particularly focused on bioconjugates by utilizing peptides to increase the selectivity of cytotoxic organometallic compounds. Motivated by the relatively high cytotoxic activity of gold complexes, such as Auranofin (approved to treat rheumatoid arthritis), for the treatment of various diseases, we anticipated that gold peptide bioconjugates would present interesting candidates for novel breast cancer therapies. For this, we investigate the use of the natural compound lipoic acid (Lpa) as a bioconjugation handle to link Au complexes in the oxidation state +III to peptides using the di-thiol moiety. Using this strategy, we have synthesized Au(III) complex bioconjugates linked to the linear LTVSPWY peptide and two cyclic DfKRG and KTTHWGFTLG tumor-targeting peptides. Solid phase peptide synthesis (SPPS) was used to prepare the peptides, with lipoic acid introduced N-terminally as a conjugation handle. After peptide cleavage, the metal complex was introduced in solution by first reducing the internal disulfide bond, followed by reaction with Au(ppy)Cl2 (26, ppy: 2-phenyl-pyridine), to yield the Au(III)–Lpa–peptide bioconjugates. The new bioconjugates were successfully synthesized, purified by preparative HPLC and characterized by ESI-MS.

71 Au(III)-peptide bioconjugates were tested as cytotoxic agents against two different human breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human fibroblasts cells (GM5657T) and compared to cisplatin, the parent Au(III) dichloride complex, and metal-free peptides. These in vitro data show that the Au(III)-peptide bioconjugate 30, possessing the cyclic integrin-targeting RGD-derived peptide sequence in the structure, exhibits improved activity compared to the parent gold(III) compound Au(ppy)Cl2 (26) as well as to cisplatin or the metal-free peptide. Moreover, the excellent targeting properties of 30 are supported by the fact that a Au(III)-peptide conjugate with the exact same peptide sequence, but in a linear rather than the cyclic form of 30 exhibits 10 times lower cytotoxic activity.

3.3.2 Introduction

Breast cancer is one of the most commonly occurring and deadly types of cancer. In order to overcome this major public health problem worldwide, numerous approaches have been developed and implemented. All approaches can be placed into one of three categories: radiotherapy, chemotherapy or hormone therapy. 214 Current chemotherapy treatments for breast cancer include the use of (i.e. doxorubicin) or (i.e. paclitaxel) (Figure 28). In most cases of adjuvant or neoadjuvant treatment, chemotherapy is more effective, when combinations of drugs are used. 215 Hormone therapy is recommended for women with hormone receptor-positive (ER-positive and/or PR-positive) breast cancers. 216,217 Among the drugs used in hormone therapy, the clinically approved drugs, tamoxifen and fareston, act by blocking the estrogen receptors of breast cancer cells (and are therefore called anti-estrogens).218

Figure 28. Structures of tamoxifen, doxorubicin and paclitaxel, all used in the treatment of breast cancer.

72 Due to intensive screening and successful, optimized conventional treatments, the mortality rate among women with breast cancer has been reduced. However, the reduction of toxic side effects, treatment of triple-negative breast cancer patients, drug resistance and lack of selectivity still remain significant challenges in breast cancer treatment. To overcome these obstacles, several other therapeutic strategies have attracted attention recently. Hanson et al. synthesized bioconjugates consisting of a steroidal anti-estrogenand the potent cytotoxin doxorubicin for targeting estrogen receptor-positive breast cancer cells.219 The bioconjugate, evaluated for selective uptake and cytotoxicity in ER(+)-MCF-7 and ER(-)-MDA-MB-231 breast cancer cells, was 70-fold more potent than doxorubicin in inhibition of cell proliferation and promoting cell death in ER(+)-MCF-7 human breast cancer. Peneva et al. synthesized the cytotoxic drug doxorubicin coupled to the tumor-targeting peptide vector octreotide via a disulfide-intercalating cross-linking reagent. 220 The obtained bioconjugate was tested against MCF-7 breast cancer cells, which display a high expression of the somatostatin receptor subtype 2 that strongly binds the somatostatin analog octreotide. The cytotoxic effects of the mentioned bioconjugate was much stronger (27 ± 2.5 µM) compared to the octreotide peptide alone (>150 µM). In order to provide a selective treatment of breast cancer bone metastases, Caliceti et al. designed a novel polymer bioconjugate bearing paclitaxel and alendronate (a bisphosphonate) attached to the pullulan polysaccharide backbone.221 Paclitaxel, the drug of choice for treatment of breast cancers, was attached to the polymer through a cathepsin K-cleavable peptide (GGPNle). The choice of Cathepsin K was dictated by the fact that it is overexpressed by breast cancer cells and secreted to the bone lacunae, where it is involved in osteoclast resorption. According to studies performed by using human MDA-MB-231-BM (bone metastases-originated clone), murine 4T1 breast cancer cells, murine K7M2, and human SAOS-2 osteosarcoma cells, paclitaxel was rapidly released from the bioconjugate by Cathepsin K and the bioconjugate exerted an enhanced antiproliferative activity compared to the conjugate without the bisphosphonate. Another extensively studied approach concerns the use of nanoparticles in the treatment of breast cancer. 222,223 , 224 ,225 , 226 Govindaraju et al. synthesized biogenic gold nanoparticles (AuNPs) and investigated their anticancer efficacy in the treatment of breast cancer cells.227A cytotoxic effect of gold nanoparticles on MDA-MB-231 cells was observed at 4 µg/mL (IC50 value) and on MCF-7 cells at 6 µg/mL. Also, no significant toxicity was observed in normal HMEC cells in the range of 10–80 µg/mL. Moreover, Banu et al. conjugated doxorubicin through folate to polymeric gold nanoparticles for the targeted treatment of breast cancers, simultaneously supported by adjunctive laser photothermal therapy. 228 The cytotoxicity assays

73 showed that in the case of MDA-MB-231 cells, the IC50 value for AuNPs bioconjugated to doxorubicin after laser photothermal therapy, was as low as 0.008 µM, while the treatment of the cells with only doxorubicin gives an activity at 0.18 µM. In MCF-7 cells, doxorubicin in the AuNP-conjugated form was active at 0.11 µM, which makes it slightly more active than doxorubicin alone (0.20 µM). Improved therapeutic efficacy for doxorubicin AuNPs compared to that of free doxorubicin treatment in MDA-MB-231 breast cancer cells was explained by overexpression of surface folate receptors (αHFR), compared to MCF-7 breast cancer cells that express low levels of the folate receptor. The main aim of the aforementioned research was to develop breast cancer specific bioconjugates, containing a targeting drug delivery system as a one component, which can provide selectivity, and a commonly used anti-breast cancer therapeutic agent as a second component. In contrast to using purely organic drugs like doxorubicin or paclitaxel, our research interest is directed at using molecular metal compounds, especially gold in the oxidation state +III, which are isoelectronic and often isostructural to platinum(II) anticancer drugs. However, Au(III) complexes exhibit a cytotoxic mechanism of action different from that of cisplatin, targeting the mitochondrial membrane and ultimately leading to mitochondria-induced apoptosis. 229 , 230 , 231 Among the numerous gold complexes investigated,232,233especially organometallic Au(III) complexes display noteworthy anticancer activity against a variety of cancer cells.234,235 Among them, also a few were highlighted as active compounds against breast cancer cells. 236 Messori et al. have designed and synthesized dmb dmb the organogold(III) compound [Au(bipy -H)(2,6-xylidine-H)][PF6] (AuXyl), where bipy = 6-(1,1-dimethylbenzyl)-2,2‘-bipyridine), and have investigated its cytotoxic properties inter alia on MCF-7 breast cancer cells. This complex AuXyl exhibited similar activity (5.2 ± 0.4 µM) to cisplatin (5.3 ± 0.9 µM).237 Dinda et al. have synthesized a Au(III) organometallic complex supported by two N-heterocyclic carbene (NHC) ligands. The cytotoxicity of this complex, tested against MCF-7 breast cancer cells, was also improved (6.2 ± 1.4 µM) in comparison to cisplatin (9.4 ± 1.0 µM).238,239 Our group is especially focused on exploring metal-peptide bioconjugates as potential agents in the cancer treatment. 240 , 241 , 242 , 243 , 244 Surprisingly, there is a lack of Au(III)-peptide bioconjugates in the literature, designed to increase the activity or specificity towards cancer cells. Intrigued by the lack of such bioconjugates and encouraged by the high activity of parent organometallic Au(III) compounds, we designed a synthesis pathway to bioconjugates of gold complexes and cell targeting peptides (CTPs). As the organometallic Au(III) starting material, we chose [AuCl2(ppy)] ((ppy = 2-phenyl-pyridine). We selected three different

74 peptides targeting breast cancer cells, one linear peptide with the LTVXPWX sequence, and two cyclic peptides possessing DfKRG and KTTHWGFTLG sequences. The linear LTVSPWY cell-binding peptide was found to facilitate the uptake of antisense oligonucleotides into the breast cancer cell line SKBR3 through targeting the HER2 receptor.245 Cyclic peptides containing the HWGF sequence are selective inhibitors of MMP- 2 and MMP-9, which are responsible for tumour growth, angiogenesis and metastasis. The CTTHWGFTLC cyclic peptide was found to specifically inhibit the activity of these enzymes, thereby preventing the growth of tumours in mice and improving survival of mice bearing human tumours. 246 Cyclic peptides with the RGD motif (DfKRG in this work) are well- known to bind to the αvβ3 integrin receptor, which plays a role in angiogenesis of solid tumors, cell migration, invasion and also metastatic activity.247 For linking the cytotoxic gold complex and the cell targeting peptides together we utilized lipoic acid (Lpa) as a bioconjugation handle. This linker possess a free carboxylic acid group suitable for covalent binding to amino groups during solid phase peptide synthesis and two thiol groups able to coordinate to the Au(III) centre in a chelating fashion (Scheme 7). α-Lipoic acid is synthesized de novo in mitochondria as a cofactor of multi-enzyme complexes such as pyruvate dehydrogenase.248 Moreover, lipoic acid has itself been investigated as an apoptosis inducing agent in various cancer cell lines.249 It suppressed thyroid cancer cell proliferation and tumor growth through activation of AMPK and subsequent down-regulation of the mTOR-S6 signaling pathway.250 α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation. 251Also, α-lipoic acid induces apoptosis in MCF-7 human breast cancer cells via changes of the ratio of the apoptotic-related proteins Bax/Bcl-2. 252 In the literature, there is one example of a lipoic acid-peptide conjugate, consisting of the pentapeptide KTTKS, with potency of inhibition of melanin synthesis and tyrosinase activity in B16F10 melanoma cells. 253

75

Scheme 7. Synthesis strategy towardsAu(III)-peptide bioconjugates.

3.3.3 Results and Discussion

Synthesis and characterisation of linear LTVXPWX peptide: The synthesis of the linear peptide was performed manually on a Wang solid support according to standard Fmoc-based SPPS procedures. 254 , 255 , 256 , 257 Deprotection of temporary Fmoc protecting groups was performed with 20% piperidine in DMF. The coupling was performed with an excess of the Fmoc-protected amino acid (4 eq.), activated with 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) in the presence of N- hydroxybenzotriazole (HOBt, 4 eq.) and an excess of diisopropylethylamine (DIEA, 6 eq.).258,259After peptide chain assembly, the Fmoc group was removed and lipoic acid was attached for the incorporation of the gold moiety (Scheme 8). The lipoic acid (4eq.) was dissolved in DMF, activated with TBTU (4eq.) and HOBt (4eq.) and mixed with the resin overnight. After completion of the peptide synthesis and before the bioconjugation reaction with the gold(III) complex, the successful synthesis was established by HPLC and ESI-MS.

For this purpose, 10 mg of the resin with peptide was treated with a mixture of TFA/H2O (95%/5%). After cleavage from the resin the peptide was lyophilized. The crude peptide was

76 characterized by HPLC and mass spectrometry (ESI-MS). The purity of the peptide was confirmed by analytical HPLC (Fig.S2, in the ESI†). The ESI-MS spectrum confirms the molecular mass [M+H]+ for the peptide at m/z = 1053. Besides this signal also peaks corresponding to [M+Na]+ at m/z = 1075 and [M+K]+ at m/z = 1091 were observed (Fig.S3, in the ESI†).

Scheme 8. Synthesis of the linear LTVSPWYpeptide (27).

Synthesis and characterisation of cyclic DfKRG and KTTHWGFTLG peptides: In order to synthesize cyclic peptides, the methodology had to be changed. Firstly, we decided to carry out a ‗head to tail cyclization‘ in solution with the combination. This kind of cyclization entails the following requirements: 1) N-terminus and C-terminus have to be deprotected selectively before cyclization, 2) cyclization can be carried out only for protected linear

77 peptides, the orthogonally protected groups from the side chain may be removed only after cyclization. This clearly demanded that lipoic acid cannot be coupled to the N-terminus, like in the case of the linear peptide 27. We chose a lysine to accommodate the lipoic acid as part of the peptide sequence through a side chain. In this synthesis route, firstly the lipoic acid was activated by using TBTU and DIEA to yield the active ester in situ (Scheme 9). Secondly, the side chain of commercial Fmoc-Lys(Boc)-OH was selectively deprotected with TFA in DCM and the deprotected Fmoc-Lys(H)-OH was added in slight excess, in order to react the free amino group of the side-chain with the lipoic acid active ester. This two-step one-pot coupling procedure favoured the formation of the desired product 28. After purification by silica gel column chromatography (2 % MeOH in CH2Cl2) the product was obtained as a yellow solid in 83 % yield. Compound 28 was unambiguously characterized (data are available in the ESI†, Fig.S6) and used as a building block for the peptide synthesis.

Scheme 9. Synthesis of Fmoc-Lys(Lpa)-OH (28).

In order to have the termini available for cyclization, while side-chain protection is intact, the 2-chlorotrityl chloride resin was chosen. This type of resin is known to be cleaved at mild acid conditions, which ensures that the side chain groups are still protected. In principle, the respective linear sequence can be synthesized starting from any amino acid since after cyclization the same peptide is always obtained. In practise, we chose to start the sequence from glycine as this amino acid does not have a chiral center no precautions against racemization were required even after long storage of the resin. 260 For both peptides, firstly Fmoc-Gly-OH was loaded onto the 2-chlorotrityl chloride resin with a loading of 1.55 mmol/g. The DfKRG and KTTHWGFTLG peptides, conjugated with lipoic acid through side chain of lysine, were synthesized by standard solid-phase peptide synthesis (SPPS) using Fmoc strategy. For side-chain protection the 2,2,4,6,7- pentamethyl dihydrobenzofuran-5-

78 sulfonyl (Pbf) group for arginine, tert.-butyloxycarbonyl (Boc) group for tryptophan, trityl (Trt) group for histidine and the tert.-butyl (tBu) group for aspartic acid and threonine were used. Peptides couplings were carried out with TBTU and HOBt as coupling reagents in DMF (Scheme 10). After accomplishing the synthesis, the N-terminus was selectively deprotected on solid-phase using a solution of 20% piperdine in DMF. In the next step, the peptides were cleaved from the resin with a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM to give the C-terminus free acid peptide, but with intact protecting groups on all side chains, which enables head-to-tail cyclization. Further, optimized conditions for peptide cyclization reactions using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

(PyBOP) as coupling reagent and DIEA as base in CH2Cl2 were adopted for making the desired cyclic peptides in solution. After removing the solvent, finally the protecting groups were removed with a mixture of TFA/H2O (95%/5%). After cleavage the peptides were lyophilized. Both crude peptides were purified by reverse-phase semi-preparative HPLC. The purity of the peptides was confirmed by analytical HPLC (Figs.S8 and S11, in the ESI†). ESI- MS spectra confirm the molecular mass [M+H]+ for peptide 29 at m/z = 792 and for peptide 30 at m/z= 1319 (Figs. S9 and S12, ESI†). Peptides 29 and 30 were isolated in 49% and 51% yield, respectively.

79

Scheme 10. Synthesis of cyclic peptides (29) and (30).

Synthesis and characterisation of Au(III)-bioconjugates: The coupling of gold complex 26 with peptides 27, 29, and 30 was performed with the fully unprotected peptides in solution according to the general strategy outlined in Scheme 11. The corresponding peptide was first dissolved in a mixture of ACN/H2O. The thiol groups in the lipoic acid structure provide a suitable functionality for coupling peptides with the Au(III) moiety. The reducing agent tris(2- carboxyethyl)phosphine (TCEP) was used to reduce the lipoic acid to the dithiol form.261 Subsequently, coupling was performed by adding the Au complex 26 in a slight excess (1.5 eq.). The bioconjugation reaction was monitored by analytical HPLC (Figure 29). Different times of conversion were observed for different peptides. For the linear peptide with LTVXPWX sequence, in 4 hours complete conversion was observed. For the two cyclic peptides possessing DfKRG and KTTHWGFTLG sequences, 6 and 12 hours of reaction time were necessary for complete conversion, respectively. After these times, the reactions were

80 quenched and the samples were lyophilized. All crude products were purified by reverse- phase semi-preparative HPLC and characterized by analytical HPLC and ESI-MS. The conjugate 31 (overall yield 19%) was obtained as a pink solid and the conjugates 32 (overall yield 36%) and 33 (overall yield 27%) were obtained as white solids. ESI-MS analysis of the conjugates showed m/z values that were consistent with that of the charged species [M+H]+ for bioconjugate 31 at m/z = 1404, for bioconjugate 32 at m/z = 1142 and for bioconjugate 33 at m/z = 1668, respectively. Experimental isotope patterns corresponded to the calculated ones (Figs.S15, S18 and S21, in the ESI†).

absorbancenM) 214 (at

10 20 30 time [min]

Figure 29. HPLC traces of 27 before conjugation (black) and the crude reaction mixture of 31 after conjugation with the metal complex 26 (blue).

81

Scheme 11. Synthesis of Au(III)-peptide bioconjugates.

82

Figure 30. Structures of Au(III)-peptide bioconjugates 31, 32 and 33.

Cytotoxicity: With pure peptides and Au-peptide conjugates in hand, the cytotoxic activity was investigated. The MTT assay was performed to determine the cell viability of two breast cancer cell lines, MCF-7 and MDA-MB-231, after treatment with the plain gold compound

[AuCl2(ppy)] (26) as well as the Au(III)-peptide bioconjugates. The stability of gold(III) complexes in DMSO was reported in literature. 262 For control purposes, the cytotoxicity of cisplatin was also determined against both cell lines using the same assay and conditions. All of the results are expressed as IC50values (half-maximal inhibitory concentration) after 48 h of incubation and are summarized in Figure 31. The dose-response curves are shown in the ESI†

(Fig. S22 and Table S1). The IC50values for cisplatin against MCF-7 and MDA-MB-231 were found to be 8.9±2.3 and 22.4±2.5 µM, respectively, which is consistent with the literature 263,264 data. The gold complex [AuCl2(ppy)] exhibits higher activity against the MCF-7 cell line (IC50=44.3±3.6 µM) than against MDA-MB-231 (IC50=77.9±5.1 µM), however it is less active than cisplatin, in the case of both tested breast cancer cell lines. Also, the Au(III) complex 26 possesses lower activity than literature known Au(III) dithiocarbamate complexes against breast cancer cells. 265,266 In comparison, all Au(III)-peptide bioconjugates revealed enhanced cytotoxic activity in comparison to 26. The bioconjugate 31 consisting of the linear peptide sequence LTVSPWY showed doubly increased activity (IC50 = 24.8±2.9 µM) against the MCF-7 cell line, but still lower activity than cisplatin (IC50=8.9±2.3 µM). Similar observations were also reported for gold nanoparticles (AuNPs) loaded with an -lipoic acid- peptide conjugate (Lpa-WKRAKLAK). 267 Simultaneously, good activity for the same

83 bioconjugate 31 is observed against MDA-MB-231 cell line. This bioconjugate was 5 times more active (IC50=12.5±0.5 µM) than the parent Au(III) complex 26 and twice as active as cisplatin (IC50=22.4±2.5 µM) against this cell line. These cytotoxicity data confirm the potency of increased cellular uptake of the LTVSPWY peptide sequence and targeting capacity to breast cancer. 268 , 269 , 270 Moreover, it was seen that bioconjugate 32, which possesses the cyclic RGD peptide sequence, was able to hamper the cell growth against both breast cancer cells more effectively than the Au(III) complexes alone and even cisplatin with

IC50 values of 6.3±1.3 and 9.7±1.6 µM, respectively. For bioconjugate 33, more moderate activity was observed against both MCF-7 and MDA-MB-231 breast cancer cells. This bioconjugate, consisting of the cyclic peptide with KTTHWGFTLG sequence, exhibits slightly improved activity in comparison to the parent Au(III) complex. Such moderate activity of bioconjugate 33 in comparison to the parent gold compound can be explained by the fact that the MMP enzymes, which such sequence should target, are expressed in very low basic level in MCF-7 as well as in MDA-MB-231 cell line.271 An over-expression can be obtained in such cell lines only after treatment with specific activators.272,273,274 (Figure 31). However, as observed, the linear peptide (LTVSPWY) as well as cyclic peptide (DfKRG) provide higher activity against breast cancer cells, namely MCF-7 and MDA-MB-231. Unfortunately, they also show higher activity against normal cells (GM5657T). Such a lack of specificity might suggest that receptors existing in cancer cells are not the only targets for our peptide conjugates.275

84

90 77,9 80

70

60 50,8 50,1 50 44,3

µM] 40,2 [ 36,3 50 40

MCF7 IC 30 24,8 MDA-MB-231 22,4 20 12,5 GM5657T 10,2 11,5 9,7 8,9 7,2 10 6,3

0

Figure 31. Cytotoxicity data determined by the MTT assay after 48 h of incubation. The IC50 values are averages of three independent determinations. All compounds were dissolved in DMSO (final concentration 0.5 %).

Encouraged by the promising activity of cyclic gold-peptide bioconjugate 32 we have synthesized a linear peptide with the DfKRG sequence (34) and its Au(III) bioconjugate 35 (see Experimental Section) in order to investigate the influence of the peptide structure (cyclic or linear) on the cytotoxicity. Also, the cyclic peptide 29 with the DfKRG sequence and the lipoic acid linker, but devoid of a toxic metal complex was prepared. Together, the metal-free DfKRG peptides 29 and 34 (cyclic and linear), and the linear Au(III)-DfKRG bioconjugate 35 were examined by the MTT assay as well (Figure 32). Interestingly, bioconjugate 35 showed

85 ten times lower activity than its cyclic analogue, and was also less active than the parent Au(III) complex 26 against all tested cell lines (Figure 33). Such cytotoxic activity can be easily explained by the fact that the linear sequence possesses no biologically active RGD moiety, which is formed only after cyclization in the structure of cyclic bioconjugate 32. On the other hand, improved activity of the Au(III)-DfKRG linear bioconjugate 35 in comparison to non-active peptide 34 confirms the impact of the Au(III) moiety on the antiproliferative activity. The cyclic peptide 29 exhibits by itself moderate in vitro activity, which parallels 276 observations made by Massaguer et al. Minor differences in IC50 values of cyclic RGD peptides across different cancer cells are caused by different levels of integrin expression between non-metastatic breast cancer MCF7- and metastatic breast cancer MBA-MB-231 cells.277

Figure 32. Structures of compounds 29, 34 and 35.

86

300

250 250 250 250

200 174,2 MCF7

151,4 µM] [ 150 MDA-MB-231 129,3 50 110,5 106,7

IC GM5657T 100 80,6

50

0 29 34 35

Figure 33. Cytotoxicity data determined by the MTT assay after 48 h of incubation. The IC50 values are averages of three independent determinations. All compounds were dissolved in DMSO (final concentration 0.5 %).

3.3.4 Conclusions Herein, to the best of our knowledge, we report the first synthesis of Au(III)-peptide bioconjugates. Lipoic acid provides a suitable scaffold for gold coordination, in which the aliphatic chain terminated with a carboxylic acid group takes part in SPPS and the di-thiol moiety in coordination to the gold complex. We have synthesized conjugates composed of the potent cytotoxic organometallic Au(III) complex with three different cell targeting peptides (CTPs), namely one linear peptide LTVSPWY and two cyclic peptides: DfKRG and KTTHWGFTLG. For the linear bioconjugate 31, the gold moiety with lipoic acid was attached to the N-terminus, while for the cyclic bioconjugates 32 and 33, the Au(III) complex

87 was introduced on the side chain of a lysine moiety. All new compounds were evaluated against two breast cancer cell lines, namely MCF-7 and MDA-MB-231, and normal human fibroblast cell lines (GM5657T). These peptide-drug-conjugates showed potential for improving the cytotoxic efficacy of chemotherapeutic drugs. All Au(III)-peptide bioconjugates exhibit increased cytotoxicity in comparison to the non-targeting Au(III) complex and the metal-free peptides alone. The cyclic bioconjugate 31, possessing an integrin-binding RGD-derived sequence, appeared to be the most potent derivative with remarkable in vitro activity against both cancer cell lines. In a structure-activity relationship (SAR) sense, the critical features for highly active RGD-derived Au-peptide bioconjugates were identified. This result encourages us to investigate in the future the mechanism responsible for such activity of Au(III)-peptide bioconjugates. Our findings suggest that such cell targeting peptides conjugated to organometallic gold moieties might be a promising lead for targeted breast cancer treatment.

3.3.5 Experimental Section

General Methods: Chemicals were obtained from commercial suppliers and used without further purification. All Fmoc-protected amino acids were purchased from Iris Biotech GmbH. Pure L amino acids were used throughout (and denoted by capital letters in the one- letter shorthand notation), except for phenylalanine in the RGD-derived sequence, for which the D enantiomer was used and denoted by a small ―f‖. Solvents were dried according to standard procedures and stored over activated molecular sieves (4 Å). Solid-supported reactions were performed in 5 mL plastic syringes with a porous polypropylene disc as filter.

HPLC was performed by using two buffer systems (buffer A: H2O/MeCN/TFA, 95:5:0.1, v/v/v; buffer B: MeCN/H2O/TFA, 95:5:0.1, v/v/v) as the mobile phase. Preparative HPLC runs were performed by using a Dr. Maisch Reprosil C18 reversed-phase column (250 × 20 mm) at a flow rate of 10 mL/min with a linear gradient of buffer B (100 % in 40 min) from 100 % buffer A with a total run time of 60 min. Analytical HPLC runs were performed by using a Knauer Eurospher-II C18 reversed-phase column (250 × 4.6 mm) at a flow rate of 1.0 mL/min with a linear gradient of buffer B (100 % in 20 min) from 100 % buffer A with a total run time of 40 min. Mass spectra were recorded with a Bruker Esquire 6000 (ESI-MS) spectrometer. UV-Vis absorption spectra were recorded on a Spectrophotometer Microplate Reader (Berthold Detection System).

88

Synthesis of Fmoc-Lys(Lpa)-OH: Fmoc-Lys(Boc)-OH (5.00 mmol, 2.34 g) was dissolved in

10 mL of the mixture of TFA/CH2Cl2 (1:1). The solution was stirred for 30 min and the mixture was concentrated in vacuo. The residue was washed two times with CHCl3 (5 mL) ad evaporated. Simultaneously, -lipoic acid (Lpa-OH, 5.00 mmol, 1.03 g) was dissolved in

CH2Cl2 (10 mL). After the addition of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 5.00 mmol, 1.61 g) and diisopropyl-ethylamine (DIEA, 10.00 mmol, 1.74 mL), the solution darkened, and it was stirred for 45 min. Then, Fmoc-Lys(H)- OH (5.00 mmol, 2.34 g) was added to the solution, stirring was continued for another 3 h, and the reaction was monitored by TLC on silica (10 % MeOH/CH2Cl2). The mixture was concentrated in vacuo and extracted with EtOAc (100 mL). The organic layer was washed with aq. 1 M HCl (3 × 60 mL) and brine (2 × 60 mL) and dried with NaSO4. EtOAc was removed in vacuo, and after purification by silica gel column chromatography (3 % MeOH in

CH2Cl2→ 5 % MeOH in CH2Cl2) the product was obtained as a yellow solid (2.31 g, 4.1 mmol, 81 %). Rf = 0.24 (CH2Cl2/MeOH, 90:10). MS (ESI+): calcd. for C29H36N2O5S2 556.7; found 556.9 [M]+, 578.9 [M + Na]+, 594.8 [M + K]+.

Linear Peptides Synthesis: The LTVSPWY (27) and DfKRG (34) peptides were synthesized manually by means of the Fmoc/tBu protocol on an Fmoc-Tyr(tBu)-Wang resin (0.70 mmol/g), similar to a procedure described for other metal-peptide conjugates.278 The peptides were synthesized on a 0.25 mmol scale. Each synthetic cycle consisted of the following steps. Fmoc removal: The resin (357 mg, 0.250 mmol) was treated with a 20 % solution of piperidine in DMF (3.0 mL; 2 × 10 min). The solution was removed and the resin washed with DMF (3.0 mL; 3 × 1 min) and CH2Cl2 (3.0 mL; 3 × 1 min). The presence of free α- amino functionalities was checked by the bromophenol blue (BPB) test (blue-green beads). Coupling of standard amino acids: A mixture of Fmoc-Xxx-OH (1.00 mmol, 4 equiv.), TBTU (1.00 mmol, 4 eq.), N-hydroxybenzotriazole (HOBt, 1.00 mmol, 4 eq.) and DIEA (1.5 mmol, 6 equiv.) in DMF (3.0 mL) was added to the resin and the suspension mixed by shaking at room temperature for 45 min. Coupling of Lpa-OH was achieved by using the following conditions: Lpa-OH (1.00 mmol, 4 equiv.), TBTU (1.00 mmol, 4 equiv.), HOBt (1.00 mmol, 4 eq.) and DIEA (1.5 mmol, 4 equiv.) in DMF (3.0 mL) were added to the resin, and the suspension was mixed by shaking at room temperature for 60 min. Reagents and solvents were removed by filtration, and the resin was subsequently washed with DMF (3.0 mL; 3 × 1 min) and CH2Cl2 (3.0 mL; 3 × 1 min). Completion of the coupling(absence of free α-amino

89 functionalities) was verified either by the Kaiser test or the BPB test (colorless beads in both cases). Then, the resin was washed with CH2Cl2 (3.0 mL; 3 × 1 min), then Et2O (3.0 mL; 3 × 1 min), and the resin was dried in vacuo. TFA cleavage: The resin was swirled in a mixture of TFA/triisopropylsilane/phenol (2.0 mL; 95:2.5:2.5, v/v/v) at room temperature for 3 h. Then the resin was removed and the residual TFA solution transferred to a 50 mL falcon tube and diluted with ice-cold Et2O/hexane (20 mL; 1:1 v/v) to precipitate the peptide. The supernatant was removed after centrifugation, and the peptides pellets were washed twice with

Et2O/hexane. The crude peptides were dissolved in MeCN/H2O (1:1, v/v) and lyophilized. Crude peptides were purified by preparative HPLC, and the pure peptide fractions were pooled and lyophilized. Finally, the peptides were analyzed by analytical HPLC and characterized by ESI-MS.

Cyclic Peptides Synthesis: The DfKRG (29) and KTTHWGFTLG (30) peptides were synthesized manually by means of the Fmoc/tBu protocol on an Fmoc-Gly-2-Chlorotrityl resin (1.55 mmol/g). Both the peptides were synthesized on a 0.25 mmol scale. Each synthetic cycle consisted of the Fmoc removal and coupling of standard amino acid. The resin (161 mg, 0.250 mmol) was treated with a 20 % solution of piperidine in DMF (3.0 mL; 2 × 10 min).

The solution was removed and the resin washed with DMF (3.0 mL; 3 × 1 min) and CH2Cl2 (3.0 mL; 3 × 1 min). The presence of free α-amino functionalities was checked by the bromophenol blue (BPB) test (blue-green beads). A mixture of Fmoc-Xxx-OH (1.00 mmol, 4 equiv.), TBTU ( 1.00 mmol, 4 eq.), HOBt (1.00 mmol, 4 eq.) and DIEA (1.5 mmol, 6 equiv.) in DMF (3.0 mL) was added to the resin and the suspension mixed by shaking at room temperature for 45 min. Coupling of Fmoc-Lys(Lpa)-OH (28) was achieved by using the following conditions: Fmoc-Lys(Lpa)-OH (28) (1.00 mmol, 4 equiv.), TBTU (1.00 mmol, 4 equiv.), HOBt (1.00 mmol, 4 eq.) and DIEA (1.5 mmol, 4 equiv.) in DMF (3.0 mL) wereadded to the resin, and the suspension was mixed by shaking at room temperature for 60 min. Reagents and solvents were removed by filtration, and the resin was subsequently washed with DMF (3.0 mL; 3 × 2 min) and CH2Cl2 (3.0 mL; 3 × 2 min). Completion of the coupling (absence of free α-amino functionalities) was verified either by the Kaiser test or the BPB test (colorless beads in both cases). When chain assembly was completed, the fully protected peptide on the resin was dried in vacuo. The final deprotection was performed twice by treating the Fmoc protected peptide with 2 mL of 20% piperidine in DMF (each 10 min).

Next, prior to the cleavage peptide from the resin, the resin was washed with CH2Cl2 (3.0 mL;

3 × 2 min), then Et2O (3.0 mL; 3 × 2 min) and the resin was dried in vacuo. In the next step,

90 freshly prepared mixture of HFIP/DCM (20:80) was added (3 mL) to the resin. After 1 h of cleavage the filtrate was collected and concentrated. Cyclisation in solution was carried out by using the following conditions: peptide (1.00 mmol, 1 equiv.), PyBOP (1.20 mmol, 1.2 equiv.) in 10 mL of CH2Cl2 for 45 minutes. The final cleavage of the protecting group from cyclic RGDfK and KTTHWGFTLG peptides was performed in a mixture of TFA/triisopropylsilane/phenol (2.0 mL; 95:2.5:2.5, v/v/v) at room temperature for 3 h. Then, the TFA solution transferred to a 50 mL falcon tube and diluted with ice-cold Et2O/hexane (20 mL; 1:1 v/v) to precipitate the peptide. The supernatant was removed after centrifugation, and the peptide pellet was washed twice with Et2O/hexane. The crude peptide was dissolved in MeCN/H2O (1:1, v/v) and lyophilized. Crude peptides were purified by preparative HPLC, and the pure peptide fractions were pooled and lyophilized. Finally, the peptides were analyzed by analytical HPLC and characterized by ESI-MS.

Cell culture, general procedures: Cells were grown in RPMI 1640 with 1% sodium pyruvate, 1% L-glutamine, 100 units per mL Pen Strep, 10% fetal bovine serum. The cells were maintained at 37°C in a humidified incubator under an atmosphere containing 5% CO 2.

Cytotoxicity experiments: Dulbecco‘s Modified Eagle‘s Medium (DMEM), containing 10% fetal calf serum, 1% penicillin and streptomycin, was used as growth medium. MCF-7 and MDA-MB-231 cells were detached from the wells with trypsin and EDTA, harvested by centrifugation and re-suspended again in cell culture medium. The assays were carried out on 96 well plates with 6000 cells per well for all cell lines: MCF-7 and MDA-MB-231. After

24 h of incubation at 37.8 °C and 10 % CO2, the cells were treated with the compounds (with final DMSO concentrations of 0.5 %) with a final volume of 200 mL per well. For a negative control, one series of cells was left untreated. For a positive control cisplatin was used. Stock solutions and dilutions of cisplatin were prepared in exactly the same way as described above for our compounds in order to keep all parameters as similar and comparable as possible, despite the fact that the stability and activity of cisplatin in DMSO/water mixtures has been 279 critically evaluated. Thus, the absolute IC50 values of cisplatin in this paper are probably too high and should be taken with appropriate care. The cells were incubated for 48 h followed by adding 50 mL MTT (2.5 mg mL-1). After an incubation time of 2 h, the medium was removed and 200 mL of DMSO were added. The formazan crystals were dissolved and the absorption was measured at 550 nm, using a reference wavelength of 620 nm. Each test was repeated in quadruplicates in three independent experiments for each cell line.

91

Acknowledgements. Dr. Jan Dittrich is kindly acknowledged for providing Au(ppy)Cl2. This research was partly funded by the Federal Ministry of Education and Research (BMBF, project ―KATMETHAN‖).

92 4. Summary

The aim of this thesis was to introduce and optimize novel synthetic protocols for the synthesis of metal-peptide bioconjugates. Platinum(IV) and cobalt(III) complexes were linked to cell penetrating peptides through amide bonds on the resin and finally cleaved to their corresponding metal-peptide bioconjugates. Such a strategy required the preparation of platinum(IV) and cobalt(III) precursors with incorporated functional carboxylic acid group. On the contrary, gold(III) complexes were coupled to breast cancer cell targeting peptides in solution. The natural compound lipoic acid was utilized as a bioconjugation handle to link gold(III) complexes to CTPs using the di-thiol moiety. All metal complexes, peptides and metal-peptide bioconjugates were thoroughly characterized by FT-IR, mass spectrometry (ESI-MS, MALDI), HPLC and multinuclear NMR spectroscopy (1H, 13C, 59Co and 195Pt NMR).

Furthermore, the anti-proliferative activity of metal complexes, peptides and their metal- peptide bioconjugates against human liver cancer cells (HepG2), human lung cancer cells (A549), human breast cancer cells (MCF-7 and MDA-MB-231), and normal human fibroblast cells (GM5657T) were evaluated by the MTT assay. Bioconjugate 4a, which consists of platinum(IV) complex and a CPP-polyarginine peptide sequence, was found to be considerably active towards HepG2 liver cancer cells. Among all cobalt(III)-peptide bioconjugates, the bioconjugate, which consists of a CPP-polyarginine peptide sequence with four addition phenylalanine residues (25), was found to be slightly more active against HepG2 liver cancer cells (9.0±1.2 µM) in comparison to the parent cobalt(III) complex 8 (13.2±0.8 µM) as well as to cisplatin (13.3±1.4 µM). Among the family of gold(III)-peptide bioconjugates, the cyclic bioconjugate 31, possessing organometallic gold(III) complex

([AuCl2(ppy)]) and an integrin-binding RGD-derived sequence, appeared to be the most potent derivative with remarkable in vitro activity against both breast cancer cell lines (MCF- 7 and MDA-MB-231).

To overcome the lack of selectivity we have coupled platinum(IV) complexes to activatable cell penetrating peptides (ACPPs) with the enzyme-cleavable linker (PLGLAG) between the polycationic and neutralizing polyanionic domains. The cytotoxic experiment has shown that bioconjugate 4b, which consists of an additional polyglutamic sequence to neutralize the positive charge of polyarginine, showed no growth inhibition against cancer as well as normal cells. Whereas, the platinum(IV) peptide bioconjugate 4a consisting of only the polyarginine

93 sequence was more active towards HepG2 liver cancer cells (37.3±1.6 µM) than the parent platinum(IV) complex (43.0±1.5 µM).

Future investigations would be necessary to facilitate the delivery of metal-peptide bioconjugates selectively to cancer cells over healthy cells. Biological experiments involving a wide range of cancer cells, which over-express MMMP-2 enzymes, have to be carried out. Moreover, the activation of over-expressed MMP-2 enzymes has to be elucidated and controlled.

94

5. Literature

1 Haas, K. L.; Franz, K. J., Application of metal coordination chemistry to explore and manipulate cell biology. Chemical reviews 2009, 109 (10), 4921-4960. 2 Cohen, S. M., New approaches for medicinal applications of bioinorganic chemistry. Current opinion in chemical biology 2007, 11 (2), 115-120. 3 Selvaganapathy, M.; Raman, N., Pharmacological activity of a few transition metal complexes: A short review. Journal of Chemical Biology & Therapeutics 2016, 1 (02), 2572. 4 Ndagi, U.; Mhlongo, N.; Soliman, M. E., Metal complexes in cancer therapy–an update from drug design perspective. Drug design, development and therapy 2017, 11, 599. 5 Frezza, M.; Hindo, S.; Chen, D.; Davenport, A.; Schmitt, S.; Tomco, D.; Ping Dou, Q., Novel metals and metal complexes as platforms for cancer therapy. Current pharmaceutical design 2010, 16 (16), 1813-1825. 6 Carter, S., Cisplatin—past, present and future. In Platinum Coordination Complexes in Cancer Chemotherapy, Springer: 1984; pp 359-376. 7 Desoize, B.; Madoulet, C., Particular aspects of platinum compounds used at present in cancer treatment. Critical reviews in oncology/hematology 2002, 42 (3), 317-325. 8 Boulikas, T.; Vougiouka, M., Recent clinical trials using cisplatin, carboplatin and their combination chemotherapy drugs. Oncology reports 2004, 11 (3), 559-595. 9 Johnstone, T. C.; Wilson, J. J.; Lippard, S. J., Monofunctional and higher-valent platinum anticancer agents. Inorganic chemistry 2013, 52 (21), 12234-12249.

10 Siddik, Z. H., Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003, 22 (47), 7265.

11 Dasari, S.; Tchounwou, P. B., Cisplatin in cancer therapy: molecular mechanisms of action. European journal of pharmacology 2014, 740, 364-378.

12 Hamers, F.; Gispen, W.; Neijt, J., Neurotoxic side-effects of cisplatin. European Journal of Cancer and Clinical Oncology 1991, 27 (3), 372-376. 13 Galluzzi, L.; Senovilla, L.; Vitale, I.; Michels, J.; Martins, I.; Kepp, O.; Castedo, M.; Kroemer, G., Molecular mechanisms of cisplatin resistance. Oncogene 2012, 31 (15), 1869.

95

14 Abu-Surrah, A. S.; Kettunen, M., Platinum group antitumor chemistry: design and development of new anticancer drugs complementary to cisplatin. Current medicinal chemistry 2006, 13 (11), 1337-1357. 15 van der Vijgh, W. J., Clinical pharmacokinetics of carboplatin. Clinical pharmacokinetics 1991, 21 (4), 242-261. 16 Raymond, E.; Faivre, S.; Woynarowski, J. M.; Chaney, S. G. In Oxaliplatin: mechanism of action and antineoplastic activity, Seminars in oncology, 1998; pp 4-12.

17 Kang, X.; Xiao, H.-H.; Song, H.-Q.; Jing, X.-B.; Yan, L.-S.; Qi, R.-G., Advances in drug delivery system for platinum agents based combination therapy. Cancer biology & medicine 2015, 12 (4), 362. 18 Stewart, D. J., Mechanisms of resistance to cisplatin and carboplatin. Critical reviews in oncology/hematology 2007, 63 (1), 12-31. 19 Extra, J.-M.; Marty, M.; Brienza, S.; Misset, J.-L. In Pharmacokinetics and safety profile of oxaliplatin, Seminars in oncology, 1998; pp 13-22. 20 Kabolizadeh, P.; Kipping, R.; Menon, V.; Ryan, J.; Peterson, E.; Povirk, L.; Farrell, N., A non-covalent binding platinum drug, TriplatinNC, induces a G1 phase arrest and p53-independent mechanisms of apoptosis. AACR: 2010. 21 Rosa, N. M.; Ferreira, F. H. d. C.; Farrell, N. P.; Costa, L. A. S., TriplatinNC and Biomolecules: building models based on non-covalent interactions. Frontiers in chemistry 2019, 7.

22 Kipping, R.; Kabolizadeh, P.; Frezard, F.; Ryan, J.; Farrell, N., Anticancer drug potential of ―noncovalent‖ platinum compounds. The biology of the phosphate clamp. AACR: 2007.

23 Testa, B., Prodrug research: futile or fertile? Biochemical pharmacology 2004, 68 (11), 2097-2106. 24 Groot, F. M. d.; Damen, E. W.; Scheeren, H. W., Anticancer prodrugs for application in monotherapy targeting hypoxia, tumor-associated enzymes, and receptors. Current medicinal chemistry 2001, 8 (9), 1093-1122. 25 Hall, M. D.; Mellor, H. R.; Callaghan, R.; Hambley, T. W., Basis for design and development of platinum (IV) anticancer complexes. Journal of medicinal chemistry 2007, 50 (15), 3403-3411.

96

26 Choi, S.; Filotto, C.; Bisanzo, M.; Delaney, S.; Lagasee, D.; Whitworth, J. L.; Jusko, A.; Li, C.; Wood, N. A.; Willingham, J., Reduction and anticancer activity of platinum (IV) complexes. Inorganic chemistry 1998, 37 (10), 2500-2504. 27 Mi, Q.; Shu, S.; Yang, C.; Gao, C.; Zhang, X.; Luo, X.; Bao, C.; Zhang, X.; Niu, J., Current status for oral platinum (IV) anticancer drug development. Radiation Oncology 2018, 7, 231- 247. 28 Barnes, K. R.; Kutikov, A.; Lippard, S. J., Synthesis, characterization, and cytoto xicity of a series of estrogen-tethered platinum (IV) complexes. Chemistry & biology 2004, 11 (4), 557- 564. 29 Descôteaux, C.; Leblanc, V.; Bélanger, G.; Parent, S.; Asselin, É.; Bérubé, G., Improved synthesis of unique estradiol-linked platinum (II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta. Steroids 2008, 73 (11), 1077-1089. 30 Ferretti, V.; Bergamini, P.; Marvelli, L.; Hushcha, Y.; Gemmo, C.; Gambari, R.; Lampronti, I., Synthesis and characterization of Pt complexes containing dichloroacetate (DCA), designed for dual anticancer action. Inorganica Chimica Acta 2018, 470, 119-127.

31 Sutendra, G.; Michelakis, E. D., Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology. Frontiers in oncology 2013, 3, 38. 32 Dhar, S.; Lippard, S. J., Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate. Proceedings of the National Academy of Sciences 2009, 106 (52), 22199- 22204. 33 Li, S.; Li, C.; Jin, S.; Liu, J.; Xue, X.; Eltahan, A. S.; Sun, J.; Tan, J.; Dong, J.; Liang, X.-J., Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs) with ethacraplatin micelles in vitro and in vivo. Biomaterials 2017, 144, 119-129.

34 Song, H.; Xiao, H.; Zhang, Y.; Cai, H.; Wang, R.; Zheng, Y.; Huang, Y.; Li, Y.; Xie, Z.; Liu, T., Multifunctional Pt (IV) pro-drug and its micellar platform: to kill two birds with one stone. Journal of Materials Chemistry B 2013, 1 (6), 762-772.

35 Mackay, F. S.; Woods, J. A.; Moseley, H.; Ferguson, J.; Dawson, A.; Parsons, S.; Sadler, P. J., A photoactivated trans‐diammine platinum complex as cytotoxic as cisplatin. Chemistry–A European Journal 2006, 12 (11), 3155-3161. 36 Imberti, C.; Zhang, P.; Huang, H.; Sadler, P. J., New designs for phototherapeutic transition metal complexes. Angewandte Chemie International Edition 2020, 59 (1), 61-73.

97

37 Farrer, N. J.; Woods, J. A.; Munk, V. P.; Mackay, F. S.; Sadler, P. J., Photocytotoxic trans- diam (m) ine platinum (IV) diazido complexes more potent than their cis isomers. Chemical research in toxicology 2009, 23 (2), 413-421. 38 Kasparkova, J.; Kostrhunova, H.; Novakova, O.; Křikavová, R.; Vančo, J.; Trávníček, Z.; Brabec, V., A photoactivatable platinum (IV) complex targeting genomic DNA and histone deacetylases. Angewandte Chemie International Edition 2015, 54 (48), 14478-14482.

39 Kelland, L. R.; Abel, G.; McKeage, M. J.; Jones, M.; Goddard, P. M.; Valenti, M.; Murrer, B. A.; Harrap, K. R., Preclinical antitumor evaluation of bis-acetato-ammine-dichloro- cyclohexylamine platinum (IV): an orally active platinum drug. Cancer research 1993, 53 (11), 2581-2586. 40 Milacic, V.; Fregona, D.; Dou, Q. P., Gold complexes as prospective metal-based anticancer drugs. Histology and histopathology 2008. 41 Messori, L.; Scaletti, F.; Massai, L.; Cinellu, M. A.; Gabbiani, C.; Vergara, A.; Merlino, A., The mode of action of anticancer gold-based drugs: a structural perspective. Chemical Communications 2013, 49 (86), 10100-10102.

42 Shaw, C. F., Gold-based therapeutic agents. Chemical reviews 1999, 99 (9), 2589-2600.

43 Casini, A.; Diawara, M. C.; Scopelliti, R.; Zakeeruddin, S. M.; Grätzel, M.; Dyson, P. J., Synthesis, characterisation and biological properties of gold (III) compounds with modified bipyridine and bipyridylamine ligands. Dalton Transactions 2010, 39 (9), 2239-2245.

44 Coronnello, M.; Mini, E.; Caciagli, B.; Cinellu, M. A.; Bindoli, A.; Gabbiani, C.; Messori, L., Mechanisms of cytotoxicity of selected organogold (III) compounds. Journal of medicinal chemistry 2005, 48 (21), 6761-6765. 45 Staples, R.; Grant, T.; Fackler, J.; Elduque, A., Structure of trichloro (triphenylphosphine) gold (III),[AuCl3 {P (C6H5) 3}]. Acta Crystallographica Section C: Crystal Structure Communications 1994, 50 (1), 39-40. 46 Lin, I. J.; Vasam, C. S., Review of gold (I) N-heterocyclic carbenes. Canadian journal of chemistry 2005, 83 (6-7), 812-825.

47 Pažický, M.; Loos, A.; Ferreira, M. J.; Serra, D.; Vinokurov , N.; Rominger , F.; Jäkel, C.; Hashmi, A. S. K.; Limbach, M., Synthesis, reactivity, and electrochemical studies of gold (I) and gold (III) complexes supported by N-heterocyclic carbenes and their application in catalysis. Organometallics 2010, 29 (20), 4448-4458.

98

48 Ronconi, L.; Aldinucci, D.; Ping Dou, Q.; Fregona, D., Latest insights into the anticancer activity of gold (III)-dithiocarbamato complexes. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 2010, 10 (4), 283-292. 49 Ronconi, L.; Marzano, C.; Zanello, P.; Corsini, M.; Miolo, G.; Macca, C.; Trevisan, A.; Fregona, D., Gold (III) dithiocarbamate derivatives for the treatment of cancer: solution chemistry, DNA binding, and hemolytic properties. Journal of medicinal chemistry 2006, 49 (5), 1648-1657. 50 Casini, A.; Messori, L., Molecular mechanisms and proposed targets for selected anticancer gold compounds. Current topics in medicinal chemistry 2011, 11 (21), 2647-2660.

51 Zou, T.; Lum, C. T.; Lok, C.-N.; Zhang, J.-J.; Che, C.-M., Chemical biology of anticancer gold (III) and gold (I) complexes. Chemical Society Reviews 2015, 44 (24), 8786-8801. 52 Milacic, V.; Dou, Q. P., The tumor proteasome as a novel target for gold (III) complexes: implications for breast cancer therapy. Coordination Chemistry Reviews 2009, 253 (11-12), 1649-1660.

53 Milacic, V.; Chen, D.; Ronconi, L.; Landis-Piwowar, K. R.; Fregona, D.; Dou, Q. P., A novel anticancer gold (III) dithiocarbamate compound inhibits the activity of a purified 20S proteasome and 26S proteasome in human breast cancer cell cultures and xenografts. Cancer research 2006, 66 (21), 10478-10486. 54 Bustamante, F. L.; Metello, J. M.; de Castro, F. A.; Pinheiro, C. B.; Pereira, M. D.; Lanznaster, M., Lawsone dimerization in cobalt (III) complexes toward the design of new prototypes of bioreductive prodrugs. Inorganic chemistry 2013, 52 (3), 1167-1169. 55 Eussen, S. J.; de Groot, L. C.; Clarke, R.; Schneede, J.; Ueland, P. M.; Hoefnagels, W. H.; van Staveren, W. A., Oral cyanocobalamin supplementation in older people with vitamin B12 deficiency: a dose-finding trial. Archives of Internal Medicine 2005, 165 (10), 1167-1172. 56 Ott, I.; Gust, R., Non platinum metal complexes as anti‐cancer drugs. Archiv der Pharmazie: An International Journal Pharmaceutical and Medicinal Chemistry 2007, 340 (3), 117-126. 57 M‘bitsi-Ibouily, G.; Marimuthu, T.; Kumar, P.; du Toit, L.; Choonara, Y.; Kondiah, P.; Pillay, V., Outlook on the Application of Metal-Liganded Bioactives for Stimuli-Responsive Release. Molecules 2017, 22 (12), 2065. 58 Souza, E. T.; Castro, L. C.; Castro, F. A. V.; do Canto Visentin, L.; Pinheiro, C. B.; Pereira, M. D.; de Paula Machado, S.; Scarpellini, M., Synthesis, characterization and biological

99 activities of mononuclear Co (III) complexes as potential bioreductively activated prodrugs. Journal of inorganic biochemistry 2009, 103 (10), 1355-1365.

59 Bustamante, F. L.; Miranda, F. S.; Castro, F. A.; Resende, J. A.; Pereira, M. D.; Lanznaster, M., A study on the properties and reactivity of naphthoquinone–cobalt (III) prototypes for bioreductive prodrugs. Journal of inorganic biochemistry 2014, 132, 37-44.

60 Guise, C. P.; Mowday, A. M.; Ashoorzadeh, A.; Yuan, R.; Lin, W.-H.; Wu, D.-H.; Smaill, J. B.; Patterson, A. V.; Ding, K., Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia. Chinese journal of cancer 2014, 33 (2), 80.

61 West, D. X.; Mokijewski, B. L.; Gebremedhin, H.; Romack, T. J., Nuclear magnetic resonance spectral study of 2-acetylpyridine 4 N-alkylthiosemicarbazones and their cobalt (III) complexes. Transition Metal Chemistry 1992, 17 (5), 384. 62 Beebe, S. J.; Celestine, M. J.; Bullock, J. L.; Sandhaus, S.; Arca, J. F.; Cropek, D. M.; Ludvig, T. A.; Foster, S. R.; Clark, J. S.; Beckford, F. A., Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt (III) complex with a thiosemicarbazone ligand. Journal of inorganic biochemistry 2020, 203, 110907. 63 Shen, W.-C.; Ryser, H., Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins. Proceedings of the National Academy of Sciences 1978, 75 (4), 1872-1876. 64 Ryser, H. J.-P.; Hancock, R., Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 1965, 150 (3695), 501-503. 65 Layek, B.; Lipp, L.; Singh, J., Cell penetrating peptide conjugated chitosan for enhanced delivery of nucleic acid. International journal of molecular sciences 2015, 16 (12), 28912- 28930. 66 Schmidt, N.; Mishra, A.; Lai, G. H.; Wong, G. C., Arginine‐rich cell‐penetrating peptides. FEBS letters 2010, 584 (9), 1806-1813. 67 Maiolo, J. R.; Ferrer, M.; Ottinger, E. A., Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides. Biochimica et Biophysica Acta (BBA)- Biomembranes 2005, 1712 (2), 161-172. 68 Bolhassani, A., Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2011, 1816 (2), 232-246.

100

69 Copolovici, D. M.; Langel, K.; Eriste, E.; Langel, U., Cell-penetrating peptides: design, synthesis, and applications. ACS nano 2014, 8 (3), 1972-1994. 70 Milletti, F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discovery Today 2012, 17 (15-16), 850-860. 71 Pietersz, G. A.; Li, W.; Apostolopoulos, V., A 16-mer peptide (RQIKIWFQNRRMKWKK) from antennapedia preferentially targets the Class I pathway. Vaccine 2001, 19 (11-12), 1397- 1405. 72 Hartwig, B.; Alcalde, J. T.; Windhab, N.; Panzano, M. d. P. A.; Carrera, M. J. V., Human lactoferrin derived peptide for use as an antigen masking agent. Google Patents: 2017. 73 Melikov, K.; Chernomordik, L., Arginine-rich cell penetrating peptides: from endosomal uptake to nuclear delivery. Cellular and Molecular Life Sciences CMLS 2005, 62 (23), 2739- 2749. 74 Robison, A. D.; Sun, S.; Poyton, M. F.; Johnson, G. A.; Pellois, J.-P.; Jungwirth, P.; Vazdar, M.; Cremer, P. S., Polyarginine interacts more strongly and cooperatively than polylysine with phospholipid bilayers. The Journal of Physical Chemistry B 2016, 120 (35), 9287-9296. 75 Li, S.-Y.; Cheng, H.; Qiu, W.-X.; Liu, L.-H.; Chen, S.; Hu, Y.; Xie, B.-R.; Li, B.; Zhang, X.-Z., Protease-activable cell-penetrating peptide–protoporphyrin conjugate for targeted photodynamic therapy in vivo. ACS applied materials & interfaces 2015, 7 (51), 28319- 28329. 76 Liu, B. R.; Huang, Y.-w.; Winiarz, J. G.; Chiang, H.-J.; Lee, H.-J., Intracellular delivery of quantum dots mediated by a histidine-and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials 2011, 32 (13), 3520-3537. 77 Sayers, E. J.; Cleal, K.; Eissa, N. G.; Watson, P.; Jones, A. T., Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides. Journal of controlled release 2014, 195, 55-62. 78 Farkhani, S. M.; Valizadeh, A.; Karami, H.; Mohammadi, S.; Sohrabi, N.; Badrzadeh, F., Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules. Peptides 2014, 57, 78-94. 79 Sugawara, K.; Kadoya, T.; Kuramitz, H., Monitoring of the interaction between U937 cells and electroactive daunomycin with an arginine-rich peptide. Bioelectrochemistry 2015, 105, 95-102.

101

80 Nakase, I.; Konishi, Y.; Ueda, M.; Saji, H.; Futaki, S., Accumulation of arginine-rich cell- penetrating peptides in tumors and the potential for anticancer drug delivery in vivo. Journal of controlled release 2012, 159 (2), 181-188.

81 Borrelli, A.; Tornesello, A.; Tornesello, M.; Buonaguro, F., Cell penetrating peptides as molecular carriers for anti-cancer agents. Molecules 2018, 23 (2), 295.

82 Juliano, R. L.; Alam, R.; Dixit, V.; Kang, H. M., Cell‐targeting and cell‐penetrating peptides for delivery of therapeutic and imaging agents. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2009, 1 (3), 324-335. 83 Barry, M. A.; Dower, W. J.; Johnston, S. A., Toward cell–targeting gene therapy vectors: Selection of cell–binding peptides from random peptide–presenting phage libraries. Nature medicine 1996, 2 (3), 299. 84 Cluzel, C.; Saltel, F.; Lussi, J.; Paulhe, F.; Imhof, B. A.; Wehrle-Haller, B., The mechanisms and dynamics of αvβ3 integrin clustering in living cells. J Cell Biol 2005, 171 (2), 383-392. 85 Lodish, H.; Berk, A.; Zipursky, S. L.; Matsudaira, P.; Baltimore, D.; Darnell, J., Molecular cell biology 4th edition. National Center for Biotechnology Information, Bookshelf 2000. 86 Horton, M. A., The αvβ3 integrin ―vitronectin receptor‖. The international journal of biochemistry & cell biology 1997, 29 (5), 721-725. 87 Soldi, R.; Mitola, S.; Strasly, M.; Defilippi, P.; Tarone, G.; Bussolino, F., Role of αvβ3 integrin in the activation of vascular endothelial growth factor receptor‐2. The EMBO journal 1999, 18 (4), 882-892. 88 Weis, S. M.; Cheresh, D. A., αV integrins in angiogenesis and cancer. Cold Spring Harbor perspectives in medicine 2011, 1 (1), a006478.

89 Cai, W.; Chen, X., Anti-angiogenic cancer therapy based on integrin αvβ3 antagonism. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti- Cancer Agents) 2006, 6 (5), 407-428.

90 Vrettos, E. I.; Mező, G.; Tzakos, A. G., On the design principles of peptide–drug conjugates for targeted drug delivery to the malignant tumor site. Beilstein journal of organic chemistry 2018, 14 (1), 930-954.

102

91 Bogdanowich‐Knipp, S. J.; Chakrabarti, S.; Siahaan, T. J.; Williams, T. D.; Dillman, R. K., Solution stability of linear vs. cyclic RGD peptides. The Journal of peptide research 1999, 53 (5), 530-541. 92 Vivès, E.; Schmidt, J.; Pèlegrin, A., Cell-penetrating and cell-targeting peptides in drug delivery. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2008, 1786 (2), 126-138. 93 Gentilucci, L.; De Marco, R.; Cerisoli, L., Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Current pharmaceutical design 2010, 16 (28), 3185-3203. 94 Dal Pozzo, A.; Ni, M.-H.; Esposito, E.; Dallavalle, S.; Musso, L.; Bargiotti, A.; Pisano, C.; Vesci, L.; Bucci, F.; Castorina, M., Novel tumor-targeted RGD peptide–camptothecin conjugates: Synthesis and biological evaluation. Bioorganic & medicinal chemistry 2010, 18 (1), 64-72. 95 Hamdan, F.; Bigdeli, Z.; Balalaie, S.; Sewald, N.; Michalek, C., Efficient synthesis of novel RGD based peptides and the conjugation of the pyrazine moiety to their N-terminus. New Journal of Chemistry 2019, 43 (6), 2702-2709. 96 Palao-Suay, R.; Aguilar, M. R.; Parra-Ruiz, F. J.; Martín-Saldaña, S.; Rohner, N. A.; Thomas, S. N.; San Román, J., Multifunctional decoration of alpha-tocopheryl succinate- based NP for cancer treatment: effect of TPP and LTVSPWY peptide. Journal of Materials Science: Materials in Medicine 2017, 28 (10), 152. 97 Burkhart, D. J.; Kalet, B. T.; Coleman, M. P.; Post, G. C.; Koch, T. H., Doxorubicin- formaldehyde conjugates targeting αvβ3 integrin. Molecular cancer therapeutics 2004, 3 (12), 1593-1604.

98 Mousavizadeh, A.; Jabbari, A.; Akrami, M.; Bardania, H., Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review. Colloids and Surfaces B: Biointerfaces 2017, 158, 507-517. 99 Gandioso, A.; Shaili, E.; Massaguer, A.; Artigas, G.; González-Cantó, A.; Woods, J. A.; Sadler, P. J.; Marchán, V., An integrin-targeted photoactivatable Pt (IV) complex as a selective anticancer pro-drug: synthesis and photoactivation studies. Chemical Communications 2015, 51 (44), 9169-9172. 100 Noor, F.; Wüstholz, A.; Kinscherf, R.; Metzler‐Nolte, N., A cobaltocenium–peptide bioconjugate shows enhanced cellular uptake and directed nuclear delivery. Angewandte Chemie International Edition 2005, 44 (16), 2429-2432.

103

101 Noor, F.; Kinscherf, R.; Bonaterra, G. A.; Walczak, S.; Wölfl, S.; Metzler‐Nolte, N., Enhanced cellular uptake and cytotoxicity studies of organometallic bioconjugates of the NLS peptide in Hep G2 cells. ChemBioChem 2009, 10 (3), 493-502. 102 Allam, A.; Maigre , L.; Alimi , M.; Alves de Sousa , R.; Hessani, A.; Galardon , E.; Pagès, J.- M.; Artaud, I., New peptides with metal binding abilities and their use as drug carriers. Bioconjugate chemistry 2014, 25 (10), 1811-1819.

103 Pagès, J.-M.; Kascàkovà, S.; Maigre , L.; Allam , A.; Alimi , M.; Chevalier , J.; Galardon , E.; Réfrégiers , M.; Artaud, I., New peptide-based antimicrobials for tackling drug resistance in bacteria: single-cell fluorescence imaging. ACS medicinal chemistry letters 2013, 4 (6), 556- 559. 104 Gross, A.; Habig, D.; Metzler‐Nolte, N., Synthesis and Structure–Activity Relationship Study of Organometallic Bioconjugates of the Cyclic Octapeptide Octreotate. ChemBioChem 2013, 14 (18), 2472-2479. 105 Köster, S. D.; Alborzinia, H.; Can, S.; Kitanovic, I.; Wölfl, S.; Rubbiani, R.; Ott, I.; Riesterer, P.; Prokop, A.; Merz, K., A spontaneous gold (I)-azide alkyne cycloaddition reaction yields gold-peptide bioconjugates which overcome cisplatin resistance in a p53- mutant cancer cell line. Chemical Science 2012, 3 (6), 2062-2072. 106 Warżajtis, B.; Glišić, B. Đ.; Savić, N. D.; Pavic, A.; Vojnovic, S.; Veselinović, A.; Nikodinovic-Runic, J.; Rychlewska, U.; Djuran, M. I., Mononuclear gold (iii) complexes with l-histidine-containing dipeptides: tuning the structural and biological properties by variation of the N-terminal amino acid and counter anion. Dalton Transactions 2017, 46 (8), 2594- 2608. 107Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V. H., Platinum compounds: a new class of potent antitumour agents. nature 1969, 222 (5191), 385-386. 108Rosenberg, B.; Van Camp, L.; Krigas, T., Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. nature 1965, 205 (4972), 698-699. 109Pinzani, V.; Bressolle, F.; Haug, I. J.; Galtier, M.; Blayac, J. P.; Balmès, P., Cisplatin- induced renal toxicity and toxicity-modulating strategies: a review. Cancer chemotherapy and pharmacology 1994, 35 (1), 1-9. 110 O'Dwyer, P. J.; Stevenson, J. P.; Johnson, S. W., Clinical pharmacokinetics and administration of established platinum drugs. Drugs 2000, 59 (4), 19-27.

104

111Reedijk, J., Why does cisplatin reach guanine-N7 with competing S-donor ligands available in the cell? Chemical reviews 1999, 99 (9), 2499-2510.

112Wexselblatt, E.; Gibson, D., What do we know about the reduction of Pt (IV) pro-drugs? Journal of inorganic biochemistry 2012, 117, 220-229. 113van Rijt, S. H.; Sadler, P. J., Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs. Drug Discovery Today 2009, 14 (23-24), 1089-1097. 114Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R., The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Transactions 2010, 39 (35), 8113-8127. 115 Dilruba, S.; Kalayda, G. V., Platinum-based drugs: past, present and future. Cancer chemotherapy and pharmacology 2016, 77 (6), 1103-1124. 116Gibson, D., The mechanism of action of platinum anticancer agents—what do we really know about it? Dalton Transactions 2009, (48), 10681-10689. 117Kenny, R. G.; Chuah, S. W.; Crawford, A.; Marmion, C. J., Platinum (IV) Prodrugs–A Step Closer to Ehrlich's Vision? European Journal of Inorganic Chemistry 2017, 2017 (12), 1596- 1612. 118Graf, N.; Lippard, S. J., Redox activation of metal-based prodrugs as a strategy for drug delivery. Advanced drug delivery reviews 2012, 64 (11), 993-1004.

119Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J., The next generation of platinum drugs: targeted Pt (II) agents, nanoparticle delivery, and Pt (IV) prodrugs. Chemical reviews 2016, 116 (5), 3436-3486. 120Wang, D.; Lippard, S. J., Cellular processing of platinum anticancer drugs. Nature reviews Drug discovery 2005, 4 (4), 307. 121Abu Ammar, A.; Raveendran, R.; Gibson, D.; Nassar, T.; Benita, S., A lipophilic Pt (IV) oxaliplatin derivative enhances antitumor activity. Journal of medicinal chemistry 2016, 59 (19), 9035-9046.

122Theiner, S.; Varbanov, H. P.; Galanski, M.; Egger, A. E.; Berger, W.; Heffeter, P.; Keppler, B. K., Comparative in vitro and in vivo pharmacological investigation of platinum (IV) complexes as novel anticancer drug candidates for oral application. JBIC Journal of Biological Inorganic Chemistry 2015, 20 (1), 89-99. 123Hall, M. D.; Hambley, T. W., Platinum (IV) antitumour compounds: their bioinorganic chemistry. Coordination Chemistry Reviews 2002, 232 (1-2), 49-67.

105

124Göschl, S.; Varbanov, H. P.; Theiner, S.; Jakupec, M. A.; Galanski, M.; Keppler, B. K., The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum (IV) prodrugs. Journal of inorganic biochemistry 2016, 160, 264- 274. 125 Sternberg, C. N.; Petrylak, D. P.; Sartor, O.; Witjes, J. A.; Demkow, T.; Ferrero, J.-M.; Eymard, J.-C.; Falcon, S.; Calabrò, F.; James, N., Multinational, double-blind, phase III study of prednisone and either satraplatin or placebo in patients with castrate-refractory prostate cancer progressing after prior chemotherapy: the SPARC trial. Journal of clinical oncology 2009, 27 (32), 5431. 126 Renfrew, A. K., Transition metal complexes with bioactive ligands: mechanisms for selective ligand release and applications for drug delivery. Metallomics 2014, 6 (8), 1324- 1335. 127Butler, J. S.; Sadler, P. J., Targeted delivery of platinum-based anticancer complexes. Current opinion in chemical biology 2013, 17 (2), 175-188. 128Parker, J. P.; Ude, Z.; Marmion, C. J., Exploiting developments in nanotechnology for the preferential delivery of platinum-based anti-cancer agents to tumours: targeting some of the hallmarks of cancer. Metallomics 2016, 8 (1), 43-60. 129Basu, U.; Banik, B.; Wen, R.; Pathak, R. K.; Dhar, S., The Platin-X series: activation, targeting, and delivery. Dalton Transactions 2016, 45 (33), 12992-13004. 130Zheng, Y.-R.; Suntharalingam, K.; Johnstone, T. C.; Lippard, S. J., Encapsulation of Pt (IV) prodrugs within a Pt (II) cage for drug delivery. Chemical Science 2015, 6 (2), 1189- 1193. 131Suntharalingam, K.; Song, Y.; Lippard, S. J., Conjugation of vitamin E analog α-TOS to Pt (IV) complexes for dual-targeting anticancer therapy. Chemical Communications 2014, 50 (19), 2465-2468. 132Rijt, S. H. v.; Kostrhunova, H.; Brabec, V.; Sadler, P. J., Functionalization of osmium arene anticancer complexes with (poly) arginine: effect on cellular uptake, internalization, and cytotoxicity. Bioconjugate chemistry 2011, 22 (2), 218-226.

133Gaviglio, L.; Gross, A.; Metzler-Nolte, N.; Ravera, M., Synthesis and in vitro cytotoxicity of cis, cis, trans-diamminedichloridodisuccinatoplatinum (iv)–peptide bioconjugates. Metallomics 2012, 4 (3), 260-266.

106

134Abramkin, S.; Valiahdi, S. M.; Jakupec, M. A.; Galanski, M.; Metzler-Nolte, N.; Keppler, B. K., Solid-phase synthesis of oxaliplatin–TAT peptide bioconjugates. Dalton Transactions 2012, 41 (10), 3001-3005. 135Wilson, J. J.; Lippard, S. J., Synthetic methods for the preparation of platinum anticancer complexes. Chemical reviews 2013, 114 (8), 4470-4495. 136Ravera, M.; Gabano, E.; Pelosi, G.; Fregonese, F.; Tinello, S.; Osella, D., A new entry to asymmetric platinum (IV) complexes via oxidative chlorination. Inorganic chemistry 2014, 53 (17), 9326-9335. 137 Slootweg, J. C.; Albada, H. B.; Siegmund, D.; Metzler-Nolte, N., Efficient reagent-saving method for the N-terminal labeling of bioactive peptides with organometallic carboxylic acids by solid-phase synthesis. Organometallics 2016, 35 (18), 3192-3196. 138 Slootweg, J. C.; Prochnow, P.; Bobersky, S.; Bandow, J. E.; Metzler‐Nolte, N., Exploring Structure–Activity Relationships in Synthetic Antimicrobial Peptides (synAMPs) by a Ferrocene Scan. European Journal of Inorganic Chemistry 2017, 2017 (2), 360-367. 139Still, B. M.; Kumar, P. A.; Aldrich-Wright, J. R.; Price, W. S., 195Pt NMR—theory and application. Chemical Society Reviews 2007, 36 (4), 665-686. 140Tsipis, A. C.; Karapetsas, I. N., Accurate prediction of 195 Pt NMR chemical shifts for a series of Pt (ii) and Pt (iv) antitumor agents by a non-relativistic DFT computational protocol. Dalton Transactions 2014, 43 (14), 5409-5426. 141Merrifield, R. B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society 1963, 85 (14), 2149-2154.

142Dirscherl, G.; Knape, R.; Hanson, P.; König, B., Solid-phase synthesis of metal-complex containing peptides. Tetrahedron 2007, 63 (23), 4918-4928. 143N‘Dongo, H. W. P.; Ott, I.; Gust, R.; Schatzschneider, U., Microwave-assisted solid-phase synthesis, cellular uptake, and cytotoxicity studies of cymantrene–peptide bioconjugates. Journal of Organometallic Chemistry 2009, 694 (6), 823-827. 144Gross, A.; Alborzinia, H.; Piantavigna, S.; Martin, L. L.; Wölfl, S.; Metzler-Nolte, N., Vesicular disruption of lysosomal targeting organometallic polyarginine bioconjugates. Metallomics 2015, 7 (2), 371-384. 145 Albada, B.; Metzler-Nolte, N., Highly potent antibacterial organometallic peptide conjugates. Accounts of chemical research 2017, 50 (10), 2510-2518.

107

146Albada, B.; Metzler-Nolte, N., Organometallic–peptide bioconjugates: synthetic strategies and medicinal applications. Chemical reviews 2016, 116 (19), 11797-11839. 147Cubo, L.; Hambley, T. W.; Miguel, P. J. S.; Carnero, A.; Navarro-Ranninger, C.; Quiroga, A. G., The preparation and characterization of trans-platinum (IV) complexes with unusually high cytotoxicity. Dalton Transactions 2011, 40 (2), 344-347. 148Ravera, M.; Gabano, E.; Zanellato, I.; Fregonese, F.; Pelosi, G.; Platts, J. A.; Osella, D., Antiproliferative activity of a series of cisplatin-based Pt (IV)-acetylamido/carboxylato prodrugs. Dalton Transactions 2016, 45 (12), 5300-5309. 149Zanellato, I.; Bonarrigo, I.; Colangelo, D.; Gabano, E.; Ravera, M.; Alessio, M.; Osella, D., Biological activity of a series of cisplatin-based aliphatic bis (carboxylato) Pt (IV) prodrugs: how long the organic chain should be? Journal of inorganic biochemistry 2014, 140, 219-227. 150Gibson, D., Platinum (IV) anticancer prodrugs–hypotheses and facts. Dalton Transactions 2016, 45 (33), 12983-12991.

151 Zhang, J. Z.; Bonnitcha, P.; Wexselblatt, E.; Klein, A. V.; Najajreh, Y.; Gibson, D.; Hambley, T. W., Facile Preparation of Mono‐, Di‐and Mixed‐Carboxylato Platinum (IV) Complexes for Versatile Anticancer Prodrug Design. Chemistry–A European Journal 2013, 19 (5), 1672-1676. 152Pelosi, G.; Ravera, M.; Gabano, E.; Fregonese, F.; Osella, D., Unprecedented one-pot synthesis of an unsymmetrical cisplatin-based Pt (iv)–acetamidato complex. Chemical Communications 2015, 51 (38), 8051-8053. 153 Kobayashi, M.; Shimizu, S., Cobalt proteins. European Journal of Biochemistry 1999, 261 (1), 1-9. 154 Simonsen, L. O.; Harbak, H.; Bennekou, P., Cobalt metabolism and toxicology—a brief update. Science of the Total Environment 2012, 432, 210-215. 155 Czarnek, K.; Terpiłowska, S.; Siwicki, A. K., Selected aspects of the action of cobalt ions in the human body. Central-European journal of immunology 2015, 40 (2), 236. 156 Yamada, K., Cobalt: its role in health and disease. In Interrelations between Essential Metal Ions and Human Diseases, Springer: 2013; pp 295-320. 157 Law, B. Y. K.; Qu, Y. Q.; Mok, S. W. F.; Liu, H.; Zeng, W.; Han, Y.; Gordillo-Martinez, F.; Chan, W.-K.; Wong, K. M.-C.; Wong, V. K. W., New perspectives of cobalt tris (bipyridine) system: anti-cancer effect and its collateral sensitivity towards multidrug-resistant (MDR) cancers. Oncotarget 2017, 8 (33), 55003.

108

158 Franz, K. J.; Metzler-Nolte, N., Introduction: Metals in Medicine. ACS Publications: 2019. 159 Heffern, M. C.; Yamamoto, N.; Holbrook, R. J.; Eckermann, A. L.; Meade, T. J., Cobalt derivatives as promising therapeutic agents. Current opinion in chemical biology 2013, 17 (2), 189-196.

160 Wang, X.; Wang, X.; Jin, S.; Muhammad, N.; Guo, Z., Stimuli-responsive therapeutic metallodrugs. Chemical reviews 2018, 119 (2), 1138-1192. 161 Zhang, P.; Sadler, P. J., Redox‐active metal complexes for anticancer therapy. European Journal of Inorganic Chemistry 2017, 2017 (12), 1541-1548. 162 Graf, N.; Lippard, S. J., Redox activation of metal-based prodrugs as a strategy for drug delivery. Advanced drug delivery reviews 2012, 64 (11), 993-1004. 163 Renfrew, A. K., Transition metal complexes with bioactive ligands: mechanisms for selective ligand release and applications for drug delivery. Metallomics 2014, 6 (8), 1324- 1335. 164 de Souza, I. C. A.; Faro, L. V.; Pinheiro, C. B.; Gonzaga, D. T. G.; da Silva, F. d. C.; Ferreira, V. F.; da Silva Miranda, F.; Scarpellini, M.; Lanznaster, M., Investigation of cobalt (III)-triazole systems as prototypes for hypoxia-activated drug delivery. Dalton Transactions 2016, 45 (35), 13671-13674. 165 Sonnay, M.; Fox, T.; Blacque, O.; Zelder, F., Modulating the cobalt redox potential through imidazole hydrogen bonding interactions in a supramolecular biomimetic protein- cofactor model. Chemical Science 2016, 7 (6), 3836-3842. 166 Wegner, S. V.; Spatz, J. P., Cobalt (III) as a Stable and Inert Mediator Ion between NTA and His6‐Tagged Proteins. Angewandte Chemie International Edition 2013, 52 (29), 7593- 7596. 167 Heffern, M. C.; Reichova, V.; Coomes, J. L.; Harney, A. S.; Bajema, E. A.; Meade, T. J., Tuning cobalt (III) Schiff base complexes as activated protein inhibitors. Inorganic chemistry 2015, 54 (18), 9066-9074.

168 Lee, J.-Y., Cobalt (III) complexes as novel matrix metalloproteinase-9 inhibitors. Bulletin of the Korean Chemical Society 2012, 33 (8), 2762-2764. 169 Egorova, K. S.; Ananikov, V. P., Toxicity of metal compounds: knowledge and myths. Organometallics 2017, 36 (21), 4071-4090.

109

170 Renfrew, A. K.; O'Neill, E. S.; Hambley, T. W.; New, E. J., Harnessing the properties of cobalt coordination complexes for biological application. Coordination Chemistry Reviews 2018, 375, 221-233. 171 Munteanu, C. R.; Suntharalingam, K., Advances in cobalt complexes as anticancer agents. Dalton Transactions 2015, 44 (31), 13796-13808. 172 Ghosh, A. K.; Mitra, M.; Fathima, A.; Yadav, H.; Choudhury, A. R.; Nair, B. U.; Ghosh, R., Antibacterial and catecholase activities of Co (III) and Ni (II) Schiff base complexes. Polyhedron 2016, 107, 1-8. 173 Böttcher, A.; Takeuchi, T.; Hardcastle, K. I.; Meade, T. J.; Gray, H. B.; Cwikel, D.; Kapon, M.; Dori, Z., Spectroscopy and electrochemistry of cobalt (III) Schiff base complexes. Inorganic chemistry 1997, 36 (12), 2498-2504. 174 Hall, M. D.; Failes, T. W.; Yamamoto, N.; Hambley, T. W., Bioreductive activation and drug chaperoning in cobalt pharmaceuticals. Dalton Transactions 2007, (36), 3983-3990. 175 Chang, E. L.; Simmers, C.; Knight, D. A., Cobalt complexes as antiviral and antibacterial agents. Pharmaceuticals 2010, 3 (6), 1711-1728. 176 Ware, D. C.; Palmer, B. D.; Wilson, W. R.; Denny, W. A., Hypoxia-selective antitumor agents. 7. Metal complexes of aliphatic mustards as a new class of hypoxia-selective cytotoxins. Synthesis and evaluation of cobalt (III) complexes of bidentate mustards. Journal of medicinal chemistry 1993, 36 (13), 1839-1846. 177 Downward, A. M.; Polson, M. I.; Kerr, W. R.; Kariyawasam, J.; Hartshorn, R. M., Synthesis of a nitrogen mustard ligand on a cobalt (III) metal centre. Polyhedron 2013, 52, 617-622. 178 Craig, P. R.; Brothers, P. J.; Clark, G. R.; Wilson, W. R.; Denny, W. A.; Ware, D. C., Anionic carbonato and oxalato cobalt (III) nitrogen mustard complexes. Dalton Transactions 2004, (4), 611-618. 179 Ware, D. C.; Brothers, P. J.; Clark, G. R.; Denny, W. A.; Palmer, B. D.; Wilson, W. R., Synthesis, structures and hypoxia-selective cytotoxicity of cobalt (III) complexes containing tridentate amine and nitrogen mustard ligands. Journal of the Chemical Society, Dalton Transactions 2000, (6), 925-932. 180 Yamamoto, N.; Renfrew, A. K.; Kim, B. J.; Bryce, N. S.; Hambley, T. W., Dual targeting of hypoxic and acidic tumor environments with a cobalt (III) chaperone complex. Journal of medicinal chemistry 2012, 55 (24), 11013-11021.

110

181 Renfrew, A. K.; Bryce, N. S.; Hambley, T. W., Delivery and release of curcumin by a hypoxia-activated cobalt chaperone: a XANES and FLIM study. Chemical Science 2013, 4 (9), 3731-3739. 182 Green, B. P.; Renfrew, A. K.; Glenister, A.; Turner, P.; Hambley, T. W., The influence of the ancillary ligand on the potential of cobalt (III) complexes to act as chaperones for hydroxamic acid-based drugs. Dalton Transactions 2017, 46 (45), 15897-15907. 183 Failes, T. W.; Cullinane, C.; Diakos, C. I.; Yamamoto, N.; Lyons, J. G.; Hambley, T. W., Studies of a cobalt (III) complex of the MMP inhibitor marimastat: a potential hypoxia‐activated prodrug. Chemistry–A European Journal 2007, 13 (10), 2974-2982. 184 Hurtado, R. R.; Harney, A. S.; Heffern, M. C.; Holbrook, R. J.; Holmgren, R. A.; Meade, T. J., Specific inhibition of the transcription factor Ci by a cobalt (III) Schiff base–DNA conjugate. Molecular pharmaceutics 2012, 9 (2), 325-333. 185 Louie, A.; Meade, T., A cobalt complex that selectively disrupts the structure and function of zinc fingers. Proceedings of the National Academy of Sciences 1998, 95 (12), 6663-6668. 186 Heffern, M. C.; Kurutz, J. W.; Meade, T. J., Spectroscopic elucidation of the inhibitory mechanism of Cys2His2 zinc finger transcription factors by cobalt (III) Schiff base complexes. Chemistry–A European Journal 2013, 19 (50), 17043-17053. 187 Goodwin, K. D.; Lewis, M. A.; Long, E. C.; Georgiadis, M. M., Crystal structure of DNA- bound Co (III)· bleomycin B2: Insights on intercalation and minor groove binding. Proceedings of the National Academy of Sciences 2008, 105 (13), 5052-5056. 188 Murray, V.; Chen, J.; Chung, L., The interaction of the metallo-glycopeptide anti-tumour drug bleomycin with DNA. International journal of molecular sciences 2018, 19 (5), 1372. 189 Dabrowiak, J.; Tsukayama, M., Cobalt (III) complex of pseudotetrapeptide A of bleomycin. Journal of the American Chemical Society 1981, 103 (25), 7543-7550. 190 Caceres-Cortes, J.; Sugiyama, H.; Ikudome, K.; Saito, I.; Wang, A. H.-J., Interactions of Deglycosylated Cobalt (III)− Pepleomycin (Green Form) with DNA Based on NMR Structural Studies. Biochemistry 1997, 36 (33), 9995-10005. 191 Fedeles, F.; Zimmer, M., Conformational/configurational analysis of all the binding geometries of cobalt (III) bleomycin. Inorganic chemistry 2001, 40 (7), 1557-1561. 192 Subramanian, R.; Meares, C. F., Photosensitization of cobalt bleomycin. Journal of the American Chemical Society 1986, 108 (20), 6427-6429. 193 Battistin, F.; Siegmund, D.; Balducci, G.; Alessio, E.; Metzler-Nolte, N., Ru (ii)-Peptide bioconjugates with the cppH linker (cppH= 2-(2′-pyridyl) pyrimidine-4-carboxylic acid):

111 synthesis, structural characterization, and different stereochemical features between organic and aqueous solvents. Dalton Transactions 2019, 48 (2), 400-414. 194 Albada, B.; Metzler-Nolte, N., Organometallic–peptide bioconjugates: synthetic strategies and medicinal applications. Chemical reviews 2016, 116 (19), 11797-11839. 195 Gross, A.; Alborzinia, H.; Piantavigna, S.; Martin, L. L.; Wölfl, S.; Metzler-Nolte, N., Vesicular disruption of lysosomal targeting organometallic polyarginine bioconjugates. Metallomics 2015, 7 (2), 371-384. 196 Gross, A.; Neukamm, M.; Metzler-Nolte, N., Synthesis and cytotoxicity of a bimetallic ruthenocene dicobalt-hexacarbonyl alkyne peptide bioconjugate. Dalton Transactions 2011, 40 (6), 1382-1386. 197 Śmiłowicz, D.; Metzler-Nolte, N., Synthesis of monofunctional platinum (iv) carboxylate precursors for use in Pt (iv)–peptide bioconjugates. Dalton Transactions 2018, 47 (43), 15465-15476. 198 Albada, B.; Metzler-Nolte, N., Highly potent antibacterial organometallic peptide conjugates. Accounts of chemical research 2017, 50 (10), 2510-2518. 199 Slootweg, J. C.; Prochnow, P.; Bobersky, S.; Bandow, J. E.; Metzler‐Nolte, N., Exploring Structure–Activity Relationships in Synthetic Antimicrobial Peptides (synAMPs) by a Ferrocene Scan. European Journal of Inorganic Chemistry 2017, 2017 (2), 360-367. 200 Slootweg, J. C.; Albada, H. B.; Siegmund, D.; Metzler-Nolte, N., Efficient reagent-saving method for the N-terminal labeling of bioactive peptides with organometallic carboxylic acids by solid-phase synthesis. Organometallics 2016, 35 (18), 3192-3196. 201 Mohammadi, K.; Niad, M.; Irandoost, A., Synthesis, spectral, thermal and thermodynamic studies of oxovanadium (IV) complexes of Schiff bases derived from 3, 4-diaminobenzoic acid with salicylaldehyde derivatives. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2013, 107, 145-150. 202 Oliveri, V.; Vecchio, G., A novel artificial superoxide dismutase: Non-covalent conjugation of albumin with a MnIII salophen type complex. European journal of medicinal chemistry 2011, 46 (3), 961-965. 203 Jing, C.; Wang, C.; Yan, K.; Zhao, K.; Sheng, G.; Qu, D.; Niu, F.; Zhu, H.; You, Z., Synthesis, structures and urease inhibitory activity of cobalt (III) complexes with Schiff bases. Bioorganic & medicinal chemistry 2016, 24 (2), 270-276.

112

204 King, A. P.; Gellineau, H. A.; Ahn, J.-E.; MacMillan, S. N.; Wilson, J. J., Bis (thiosemicarbazone) complexes of Cobalt (III). Synthesis, characterization, and anticancer potential. Inorganic chemistry 2017, 56 (11), 6609-6623. 205 Guo, Z.; Peng, H.; Kang, J.; Sun, D., Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications. Biomedical reports 2016, 4 (5), 528-534. 206 He, X.; Lin, M.; Guo, J.; Qu, Z.; Xu, F., Experimental and simulation studies of polyarginines across the membrane of giant unilamellar vesicles. RSC Advances 2016, 6 (36), 30454-30459. 207 Sayers, E. J.; Cleal, K.; Eissa, N. G.; Watson, P.; Jones, A. T., Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides. Journal of controlled release 2014, 195, 55-62. 208 Kalafatovic, D.; Giralt, E., Cell-penetrating peptides: Design strategies beyond primary structure and amphipathicity. Molecules 2017, 22 (11), 1929. 209 Malishev, R.; Nandi, S.; Smilowicz, D.; Bakavayev, S.; Engel, S.; Bujanover, N.; Gazit, R.; Metzler-Nolte, N.; Jelinek, R., Interactions between BIM protein and beta-amyloid may reveal a crucial missing link between Alzheimer's disease and neuronal cell death. ACS chemical neuroscience 2019. 210 Śmiłowicz , D.; Slootweg, J. C.; Metzler-Nolte, N., Bioconjugation of Cyclometalated Gold (III) Lipoic Acid Fragments to Linear and Cyclic Breast Cancer Targeting Peptides. Molecular pharmaceutics 2019. 211 Hall, M. D.; Telma, K. A.; Chang, K.-E.; Lee, T. D.; Madigan, J. P.; Lloyd, J. R.; Goldlust, I. S.; Hoeschele, J. D.; Gottesman, M. M., Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes. Cancer research 2014, 74 (14), 3913- 3922. 212 Takayama, K.; Hirose, H.; Tanaka, G.; Pujals, S.; Katayama, S.; Nakase, I.; Futaki, S., Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery. Molecular pharmaceutics 2012, 9 (5), 1222-1230. 213 Rijt, S. H. v.; Kostrhunova, H.; Brabec, V.; Sadler, P. J., Functionalization of osmium arene anticancer complexes with (poly) arginine: effect on cellular uptake, internalization, and cytotoxicity. Bioconjugate chemistry 2011, 22 (2), 218-226. 214Siegel, R.; DeSantis, C.; Virgo, K.; Stein, K.; Mariotto, A.; Smith, T.; Cooper, D.; Gansler, T., Lerro, C.; Fedewa, S.;Lin, C.; Leach, C.; Cannady, R.S.; Cho, H.; Scoppa, S.; Hachey, M.;

113

Kirch, R.; Jemal, A.; Ward, E.Cancer Treatment and Survivorship Statistics.CA: CANCER J. CLIN. 2012,62, 220-241.

215Becker, S. A historic and scientific review of breast cancer: The next global healthcare challenge. Int. J. Gynecol. Obstet. 2015, 131, 36-39. 216 Lukong, K.E. Understanding breast cancer-The long and winding road. BBA Clinical.2017, 7, 64–77. 217Gabbiani, C.; Pratesi, A.; Marchetti, L.; Casini, A.; Leoni, P.; Pillozzi, S.,Crociani O.; Bartoli G.; Messori, L. Potent in vitro antiproliferative properties for a triplatinum cluster toward triple negative breast cancer cells. J. Inorg. Biochem. 2016, 163, 318-322.

218 Ali, S.; Buluwela, L.; Coombes, R.C. Antiestrogens and Their Therapeutic Applications in Breast Cancer and Other Diseases.Annu. Rev. Med.2011, 62, 217-232. 219 Dao, K.L.; Sawant, R.R.; Hendricks,J.A.; Ronga,V.; Torchilin,V.P.; Hanson,R. N. Design, Synthesis, and Initial Biological Evaluation of a Steroidal Anti-Estrogen-Doxorubicin Bioconjugate for Targeting Estrogen Receptor-Positive Breast Cancer Cells.Bioconjug.Chem.2012, 23, 785−795. 220Lelle, M.; Kaloyanova, S.; Freidel, C.; Theodoropoulou, M.; Musheev, M.; Niehrs, C.; Stalla, G.; Peneva, K. Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin-Peptide Conjugate.Mol. Pharm. 2015, 12, 4290−4300. 221 Bonzi, G.; Salmaso, S.; Scomparin, A.; Eldar-Boock, A.; Satchi-Fainaro, R.; Caliceti,P. Novel Pullulan Bioconjugate for Selective Breast Cancer Bone Metastases Treatment. Bioconjug.Chem.2015, 26, 489-501. 222 Lee, J.; Chatterjee, D.K.; Hyuk Lee, M.; Krishnan, S. Gold nanoparticles in breast cancer treatment: Promise and potential pitfalls. Cancer Lett. 2014, 347, 46-53. 223Liu, R.; Hu, C.; Yang, Y.; Zhang, J.; Gao, H. Theranostic nanoparticles with tumor-specific enzyme-triggered size reduction and drug release to perform photothermal therapy for breast cancer treatment, Acta Pharm. Sin. B. 2019, 9 (2), 410-420. 224 Her, S.; Jaffray, D.A.; Allen, C. Gold nanoparticles for applications in cancer radiotherapy: Mechanisms and recent advancements. Adv. Drug Deliv. Rev.2017, 109, 84-101. 225 Bryce, N.S.; Pham, B.T.; Fong, N.W.; Jain, N.; Pan, E.H.; Whan, R.M.; Hambley, T.W.; Hawkett, B.S. The composition and end-group functionality of sterically stabilized nanoparticles enhances the effectiveness of co-administered cytotoxins. Biomater. Sci.2013,1(12), 1260-1272.

114

226 Ruggiero, E.; Alonso-de Castro, S.; Habtemariam, A.; Salassa, L. Upconverting nanoparticles for the near infrared photoactivation of transition metal complexes: new opportunities and challenges in medicinal inorganic photochemistry. Dalton Trans. 2016, 45(33), 13012-13020.

227 Uma Suganyaa, K.S.; Govindarajua, K.; Ganesh Kumara, V.; Prabhub, D.; Arulvasub, C.; Stalin Dhasa, T.; Karthicka, V.; Changmai N. Anti-proliferative effect of biogenic gold nanoparticles against breastcancer cell lines (MDA-MB-231 & MCF-7). Appl. Surf. Sci. 2016,371, 415–424. 228 Banu, H.; Kaur Sethi, D.; Edgar, A.; Sheriff, A.; Rayees, N.; Renuka, N.; Faheem, S.M.; Premkumar, K.; Vasanthakumar, G. Doxorubicin loaded polymeric gold nanoparticles targeted to human folate receptor upon laser photothermal therapy potentiates chemotherapy in breast cancer cell lines. J. Photochem. Photobiol. B, Biol.2015, 149, 116-128. 229 Nardon, C.;Boscutti, G.; Fregona, D. Beyond platinums: gold complexes as anticancer agents. Anticancer Res. 2014, 34(1), 487-492.

230Laskay, Ü.A.; Garino, C.; Tsybin, Y.O.; Salassa, L.; Casini, A. Gold finger formation studied by high-resolution mass spectrometry and in silico methods. Chem. Comm. 2015, 51(9), 1612-1615.

231Gamberi, T.; Magherini, F.; Fiaschi, T.; Landini, I.; Massai, L.; Valocchia, E.; Bianchi, L.; Bini, L.; Gabbiani, C.; Nobili, S.; Mini, E.; Messori L.; Modesti, A. Proteomic analysis of the cytotoxic effects induced by the organogold(III) complex Aubipyc in cisplatin-resistant A2780 ovarian cancer cells: further evidence for the glycolytic pathway implication. Mol. BioSyst. 2015, 11(6), 1653-1667.

232 Nardon, C.; Schmitt, S.M.; Yang, H.; Zuo, J.; Fregona, D.; Ping Dou, Q. Gold(III)- Dithiocarbamato Peptidomimetics in the Forefront of the Targeted Anticancer Therapy: Preclinical Studies against Human Breast Neoplasia. PLOS ONE. 2014, 9, 84248-84258. 233Altaf, M.; Monim-ul-Mehboob, M.; Kawde, A.N.; Corona, G.; Larcher, R.; Ogasawara, M.; Casagrande, N.; Celegato, M.; Borghese, C.; Siddik, Z.H.; Aldinucci, D.; Isab, A.A. New bipyridine gold(III) dithiocarbamate-containing complexes exerted a potent anticancer activity against cisplatin-resistant cancer cells independent of p53 status. Oncotarget2017, 8, 490-505.

115

234 Sivaram, H.; Tan,J.; Vinh Huynh, H. Syntheses, Characterizations, and a Preliminary Comparative Cytotoxicity Study of Gold(I) and Gold(III) Complexes Bearing Benzimidazole- and Pyrazole-Derived N-Heterocyclic Carbenes. Organomet. 2012, 31, 5875−5883. 235 Lemke, J.; Pinto, A.; Niehoff, P.; Vasylyeva, V.; Metzler-Nolte, N. Synthesis, structural characterisation and anti-proliferative activity of NHC gold amino acid and peptide conjugates. Dalton Trans.2009, 7063, 7063-7070. 236Altaf, M.; Casagrande, N.; Mariotto, E.; Baig, N.; Kawde, A.N.; Corona, G.; Larcher, R.; Borghese, C.; Pavan, C.; Seliman, A.A.; Aldinucci, D.; Isab A.A. Potent In Vitro and In Vivo Anticancer Activity of New Bipyridine and Bipyrimidine Gold(III) Dithiocarbamate Derivatives. Cancers2019, 11, 474:1-14. 237 Messori, L.; Marcon, G.; Cinellu, M.A.; Coronnello, M.; Mini, E.; Gabbiania C.; Oriolia, P. Solution chemistry and cytotoxic properties of novel organogold(III) compounds. Bioorg. Med. Chem. 2004, 12, 6039-6043. 238Kumar Rana, B.; Nandy, A.; Bertolasi,V.; Bielawski, C.W.;Das Saha,K.; Dinda, J. Novel Gold(I)-and Gold(III)-N-Heterocyclic Carbene Complexes: Synthesis and Evaluation of Their Anticancer Properties. Organomet. 2014, 33, 2544-2548. 239 Dinda, J.; Samanta, T.; Nandy, A.; Saha, K.D.; Seth, S.K.; Chattopadhyay, S.K.; Bielawski, C.W. N-heterocyclic carbene supported Au (I) and Au (III) complexes: A comparison of cytotoxicities. New. J. Chem.2014, 38(3), 1218-1224.

240 Muñoz-Osses, M.; Godoy, F.; Fierro, A.; Gómeza, A.; Metzler-Nolte, N. New organometallic imines of rhenium(I) as potential ligands of GSK-3β: synthesis, characterization and biological studies. Dalton Trans. 2018, 47, 1233-1242. 241König, M.; Siegmund, D.;Raszeja,L.J.; Prokop, A.; Metzler-Nolte, N. Resistance-breaking profiling and gene expression analysis on an organometallic ReI-phenanthridine complex reveal parallel activation of two apoptotic pathways.MedChemComm. 2018,9, 173-180. 242 Graf, M.; Gothe, Y.;Siegmund, D.; Metzler-Nolte,N.; Suenkel, K. Synthesis and characterization of cyclometallated rhodium(III) and iridium(III) compounds with antiproliferative activities in the nanomolar range.Inorg. Chim. Acta. 2018, 471, 265-271. 243 Gothe, Y.; Romero-Canelon, I.; Marzo, T.;Sadler, P.J.; Messori,L.; Metzler-Nolte, N. Synthesis and Mode of Action Studies on Iridium(I)-NHC Anticancer Drug Candidates. Eur. J. Inorg. Chem. 2018, 20-21, 2461-2470. 244 Gasser, G.; Ott, I.; Metzler-Nolte, N. Organometallic Anticancer Compounds. J. Med. Chem.2011, 54, 3–25.

116

245 Shadidi, M.; Sioud, M. Selective targeting of cancer cells using synthetic peptides. Drug Resist. Updat.2003, 6, 363-371. 246Koivunen, E.; Arap, W.; Valtanen, H.; Rainisalo, A.;Medina, O.P.; Heikkilä, P.; Kantor, C.; Gahmberg, C.G.; Salo, T.; Konttinen, Y.T.; Sorsa, T.; Ruoslahti, E.; Pasqualini, R.Tumor targeting with a selective gelatinase inhibitor, Nat.Biotechnol. 1999, 17, 768-774.

247Vivès, E.; Schmidt, J.; Pèlegrin, A. Cell-penetrating and cell-targeting peptides in drug delivery.Biochim. Biophys. Acta. 2008, 1786, 126-138. 248Schonauer, M.S.; Kastaniotis, A.J.; Samuli Kursu,V.A.; Hiltunen,J.K.; Dieckmann, C.L. Lipoic Acid Synthesis and Attachment in Yeast Mitochondria. J. Biol. Chem. 2009, 284, 23234-23242. 249Dörsam, B.; Fahrer, J. The disulfide compound α-lipoic acid and its derivatives: A novel class of anticancer agents targeting mitochondria.Cancer Lett.2016, 371, 12-19. 250Jeon, M.J.; Kim, W.G.; Lim, S.; Choi, H.J.; Sim,S.; Kim, T.Y.; Shong, Y.K.; Kim,W.B. Alpha lipoic acid inhibits proliferation and epithelial mesenchymal transition of thyroid cancer cells. Mol. Cell. Endocrinol. 2016, 419,113-123. 251Yang, L.; Wen, Y.; Lv, G.; Lin, Y.; Tang, J.; Lu, J.; Zhang, M.; Liu, W.; Sun, X. α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation.Biochem. Biophys. Res. Commun. 2017, 494, 325-331 252 Dozio, E.; Ruscica, M.; Passafaro, L.; Dogliotti, G.; Steffani, L., Pagani, A.; Demartini, G.; Esposti, D.; Fraschini, F.; Magni, P. The natural antioxidant alpha-lipoic acid induces p27Kip1-dependent cell cycle arrest and apoptosis in MCF-7 human breast cancer cells.Eur. J. Pharmacol. 2010, 641, 29-34. 253 Lu, C.; Mi Kim, B.; Lee, D.; Hee Lee, M.; Hwa Kim, J.; Pyo, H.B.; Chai, K.Y. Synthesis of lipoic acidpeptide conjugates and their effect on collagen and melanogenesis. Eur. J. Med. Chem. 2013, 69, 449-454. 254 Albada, B.; Metzler-Nolte, N. Highly Potent Antibacterial Organometallic Peptide Conjugates. Acc. Chem. Res. 2017, 50, 2510-2518.

255 Albada, B.; Metzler-Nolte N. Organometallic-Peptide Bioconjugates: Synthetic Strategies and Medicinal Applications. Chem. Rev. 2016, 116, 11797–11839.

256 Chantson, J.Y.; Falzacappa, M.V.V.; Crovella, S.; Metzler-Nolte N. Solid-Phase Synthesis, Characterization, and Antibacterial Activities of Metallocene-Peptide Bioconjugates. ChemMedChem.2006, 1, 1268-1274.

117

257 Di Marco, L.; Zhang, J.Z.; Doan, J.; Kim, B.J.; Yamamoto, N.;Bryce, N.S.; Hambley, T.W. Modulating the Cellular Uptake of Fluorescently Tagged Substrates of Prostate-Specific Antigen before and after Enzymatic Activation. Bioconjug. Chem.2018, 30(1), 124-133.

258 Lemke, J.; Pinto, A.; Niehoff, P.; Vasylyeva, V.; Metzler-Nolte, N. Synthesis, structural characterisation and anti-proliferative activity of NHC gold amino acid and peptide conjugates. Dalton Trans.2009, 7063-7070. 259Caddy, J.; Hoffmanns, U.; Metzler-Nolte, N. Introduction of phosphine-gold(I) precursors into a Cys-modified Enkephalin neuropeptide as part of solid phase peptide synthesis. Z. Naturforsch. B 2007, 62B, 460-466. 260 Davies, J.S. The Cyclization of Peptides and Depsipeptides. J. Peptide Sci.2003, 9, 471- 501. 261 Gorąca, A.; Huk-Kolega, H.; Piechota, A.; Kleniewska, P.; Ciejka, E.; Skibska B. Lipoic acid-biological activity and therapeutic potential. Pharmacol Rep. 2011, 63, 849-858. 262Czerwińska, K.; Golec, M.; Skonieczna, M.; Palion-Gazda, J.; Zygadło, D.; Szlapa-Kula, A.; Krompiec, S.; Machura, B.; Szurko, A. Cytotoxic gold(III) complexes incorporating a 2,2′:6′,2′′-terpyridine ligand framework - the impact of the substituent in the 4′-position of a terpy ring. Dalton Trans. 2017, 46, 3381-3392.

263 Prabhakaran, P.; Hassiotou, F.; Blancafort, P.; Filgueira, L. Cisplatin induces differentiation of breast cancer cells. Front Oncol. 2013, 3, 134-144. 264Suberu, J.O.; Romero-Caneln, I.; Sullivan, N.; Lapkin, A.A.; Barker, G.C. Comparative Cytotoxicity of Artemisinin and Cisplatin and Their Interactions with Chlorogenic Acids in MCF7 Breast Cancer Cells. Chem. Med. Chem. 2014, 9, 2791 – 2797. 265Milacic, V.; Chen, D.; Ronconi, L.; Landis-Piwowar, K.R.; Fregona, D.; Dou, QP. A Novel Anticancer Gold(III) Dithiocarbamate Compound Inhibits the Activity of a Purified 20S Proteasome and 26S Proteasome in Human Breast Cancer Cell Cultures and Xenografts. Cancer Res. 2006, 66, 10478-10486 266 Nardon, C.; Schmitt, S.M., Yang, H.; Zuo, J.; Fregona, D.; Dou, Q.P. Gold(III)- Dithiocarbamato Peptidomimetics in the Forefront of the Targeted Anticancer Therapy: Preclinical Studies against Human Breast Neoplasia, PLOS ONE2014, 9, e84248. 267Akrami, M.; Balalaie S.;Hosseinkhani S.;Alipour M.; Salehi F.; Bahador A.;Haririan I. Tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms. Sci. Rep. 2016, 6, 31030.

118

268 Khodadust, F.; Ahmadpour, S.; Aligholikhamseh, N.; Abedi, S.M.; Hosseinimehr, S.J. An 99m improved Tc-HYNIC-(Ser)3-LTVSPWY peptide with EDDA/tricine as co-ligands for targeting and imaging of HER2 overexpression tumor. Eur. J. Med. Chem.2018, 144, 767- 773. 269 Gong, C.; Pan, D.; Qiu, F.; Sun, P.; Zhang Y.H. Selective DNA Delivery to Tumor Cells Using an Oligoarginine-LTVSPWY Peptide. PLOS ONE. 2014, 9 (10), 110632-110640. 270Jie, L.Y.; Cai, L.L.; Wang, L.J.; Ying, X.Y.; Yu, R.S.; Zhang, M.M.; Du, Y.Z. Actively- targeted LTVSPWY peptide-modified magnetic nanoparticles for tumor imaging, Int. J. Nanomedicine. 2012, 7, 3981–3989.

271 Singer, C.F.; Kronsteiner, N.; Marton, E.; Kubista, M.; Cullen, K.J.; Hirtenlehner, K.; Seifert, M.; Kubista, E. MMP-2 and MMP-9 expression in breast cancer-derived human fibroblasts is differentially regulated by stromal-epithelial interactions. Breast Cancer Res. Treat. 2002, 72, 69-77. 272 Roomi, M.W.; Monterrey, J.C.; Kalinovsky,T.; Rath, M.; Niedzwiecki, A. Patterns of MMP-2 and MMP-9 expression in human cancer cell lines.Oncol. Rep. 2009, 21, 1323-1333. 273 Li, H.; Qiu, Z.; Li,F.; Wang, C. The relationship between MMP-2 and MMP-9 expression levels with breast cancer incidence and prognosis.Oncol. Lett. 2017, 14, 5865-5870. 274 Pellikainen, J.M.; Ropponen, K.M.; Kataja, V.V.; Kellokoski, J.K.; Eskelinen, M.J.; Kosma, V.M. Expression of Matrix Metalloproteinase (MMP)-2 and MMP-9 in Breast Cancer with a Special Reference to Activator Protein-2, HER2, and Prognosis. Clin. Cancer Res. 2004, 10, 7621-7628. 275 Kapp, TG.; Rechenmacher, F.; Neubauer, S.; Maltsev, O.V.; Cavalcanti-Adam, E.A., Zarka, R.; Reuning, U.; Notni, J.; Wester, H.J.; Mas-Moruno, C.; Spatz, J.; Geiger B.; Kessler, H. A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins, Sci. Rep. 2017, 7, 39805.

276Massaguer, A.; González-Cantó, A.; Escribano, E.; Barrabés, S.; Artigas, G.; Morenoc, V.; Marchán, V. Integrin-targeted delivery into cancer cells of a Pt(IV) pro-drug through conjugation to RGD-containing peptides. Dalton Trans. 2015, 44, 202-213.

277 Taherian, A.; Li, X.; Liu Y.; Haas, T. A. Differences in integrin expression and signaling within human breast cancer cells. BMC Cancer. 2011, 11, 293-308.

119

278Kirin, S. I.; Noor, F.; Metzler-Nolte, N.; Mier, W. Manual Solid Phase Peptide Synthesis. J. Chem. Educ.2007, 84, 108-111. 279Hall, M.D.; Telma, K.A.; Chang, K.E.; Lee, T.D.; Madigan, J.P.; Lloyd, J.R.; Goldlust, I.S.; Hoeschele J.D.; Gottesman, M.M. Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes, Cancer Res.2014, 14, 3913-22.

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6. List of contributions

Śmiłowicz, D.; Metzler-Nolte, N., Synthesis of monofunctional platinum (iv) carboxylate precursors for use in Pt (iv)–peptide bioconjugates. Dalton Transactions 2018, 47 (43), 15465-15476.

Author Contribution: I contributed to the concept of the research, developed the methodology, carried out investigations and data analysis and I wrote the original draft of manuscript.

Śmiłowicz , D.; Slootweg, J. C.; Metzler-Nolte, N., Bioconjugation of Cyclometalated Gold (III) Lipoic Acid Fragments to Linear and Cyclic Breast Cancer Targeting Peptides. Molecular pharmaceutics 2019.

Author Contribution: I developed the methodology, carried out investigations and data analysis and I wrote the original draft of manuscript.

Śmiłowicz, D.; Metzler-Nolte, N., Bioconjugates of Co(III) Complexes with Schiff Base Ligands and Cell Penetrating Peptides: Solid Phase Synthesis, Characterization and Antiproliferative Activity. Journal of Inorganic Biochemistry 2019, under revision

Author Contribution: I contributed to the concept of the research, developed the methodology, carried out investigations and data analysis and I wrote the original draft of manuscript.

121

Electronic Supporting Information

for

SYNTHESIS OF MONOFUNCTIONAL PLATINUM(IV) CARBOXYLATE PRECURSORS FOR USE IN PT(IV)-PEPTIDE BIOCONJUGATES.

by

Dariusz Śmiłowicz†, Nils Metzler-Nolte*, †

† Chair of Inorganic Chemistry I – Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Bochum, Germany

Contents

1. Characterisation of platinum(II) complexes p.123-125 2. Characterisation of platinum(IV) complexes p.126-127 3. Characterisation of cell penetrating peptides p.128-131 4. Characterisation of platinum(IV)-peptide bioconjugates p.131-133

122

Figure S1. 1H NMR spectrum of dicarboxylate platinum(II) complex (2a)

Figure S2. 13C NMR spectrum of dicarboxylate platinum(II) complex (2a)

123

Figure S3. ESI-MS spectrum of dicarboxylate platinum(II) complex (2a)

102

100

98

96

94

92

90 transmittance [%] transmittance 88

86

84

4000 3500 3000 2500 2000 1500 1000 500 wavenumber [cm-1]

Figure S4. FT-IR spectrum of dicarboxylate platinum(II) complex (2a)

124

tR=7.27

5 10 15 20 time [min]

Figure S5. HPLC chromatogram at 214 nm of dicarboxylate platinum(II) complex (2a)

Figure S6. 195Pt NMR spectrum of dicarboxylate platinum(II) complex (2a)

125

1,00

0,95

0,90

0,85

transmittance [%] transmittance 0,80

0,75

0,70 4000 3500 3000 2500 2000 1500 1000 500 wavenumber [cm-1]

Figure S7. FT-IR spectrum of dicarboxylate platinum(IV) complex (3a)

tR=14.03

10 20 time [min]

Figure S8. HPLC chromatogram at 214 nm of dicarboxylate platinum(IV) complex (3a)

126

Figure S9. ESI-MS spectrum of dicarboxylate platinum(IV) complex (3a)

Figure S10. 195Pt NMR spectrum of dicarboxylate platinum(II) complex (2a)

127

Figure S11. General synthesis of P1 peptide

128

Figure S12. Structure of polyarginine peptide (P1)

1664.02 [M+H]+

1000 1500 2000 2500 m/z

Figure S13. ESI-MS spectrum of polyarginine peptide (P1)

129

tR=10.38

10 20 time [min]

Figure S14. HPLC chromatogram at 214 nm of polyarginine peptide (P1)

Figure S15. Structure of polyarginine-polyglutamic peptide (P2)

130

1548.75 [M]2+

1000 1500 2000 m/z

Figure S16. ESI-MS spectrum of polyarginine-polyglutamic peptide (P2)

tR=15.03

5 10 15 20 time [min]

Figure S17. HPLC chromatogram at 214 nm of polyarginine-polyglutamic peptide (P2)

131

Figure S18. Structure of platinum(IV)-peptide bioconjugate (4a)

tR=14.03

10 20 time [min]

Figure S19. HPLC chromatogram at 214 nm of platinum(IV)-peptide bioconjugate (4a)

132

Figure S20. MALDI spectrum of platinum(IV)-peptide bioconjugate (4a)

Figure S21. The dose-response curve for the determination of the IC50 value of 4a on HepG2 cells.

133

Electronic Supporting Information

for Bioconjugates of Co(III) Complexes with Schiff Base Ligands and Cell Penetrating Peptides: Solid Phase Synthesis, Characterization and Antiproliferative Activity

by

Dariusz Śmiłowicz†, Nils Metzler-Nolte*, †

† Chair of Inorganic Chemistry I – Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Bochum, Germany

Contents

1. Characterisation of Schiff base ligand p.135-136 2. Characterisation of Co(III) complexes p.136-139 3. Characterisation of cell penetrating peptides p.139-142 4. Characterisation of Co(III)-peptide bioconjugates p.142-144 5. Cytotoxicity data determined by MTT p.144

134

135

136

137

138

139

140

141

142

143

Figure S20. The dose-response curve for the determination of the IC50 value of complex 8 on A549 cells.

144

Electronic Supporting Information

for BIOCONJUGATION OF CYCLOMETALATED GOLD(III) LIPOIC ACID FRAGMENTS TO LINEAR AND CYCLIC BREAST CANCER TARGETING PEPTIDES

by

Dariusz Śmiłowicz†, Jack C. Slootweg†, Nils Metzler-Nolte*, †

† Chair of Inorganic Chemistry I – Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Bochum, Germany

Contents

1. Characterisation of linear LTVXPWX peptide p.146-147 2. Characterisation of Fmoc-Lys(Lpa)-OH p.147-148 3. Characterisation of cyclic RGDFK peptide p.149-150 4. Characterisation of cyclic KTTHWGFTLG peptide p.150-151 5. Characterisation of gold(III)- LTVXPWX bioconjugate p.152-153 6. Characterisation of gold(III)- RGDFK bioconjugate p.153-154 7. Characterisation of gold(III)- KTTHWGFTLG bioconjugate p.155-156 8. Cytotoxicity data determined by MTT p.156-157

145

Figure S1. Structure of linear LTVXPWX peptide (27).

146

Figure S3. ESI-MS spectrum of linear LTVXPWX peptide (27).

Figure S4. Structure of Fmoc-Lys(Lpa)-OH (28).

147

Figure S6. ESI-MS spectrum of Fmoc-Lys(Lpa)-OH (28).

148

Figure S7. Structure of cyclic RGDFK peptide (29).

149

Figure S9. ESI-MS spectrum of cyclic RGDFK peptide (29).

Figure S10. Structure of cyclic KTTHWGFTLG peptide (30).

150

Figure S12. ESI-MS spectrum of cyclic KTTHWGFTLG peptide (30).

151

Figure S13. Structure of gold(III)- LTVXPWX bioconjugate (31).

152

Figure S15. ESI-MS spectrum of gold(III)- LTVXPWX bioconjugate (31).

Figure S16. Structure of gold(III)- RGDFK bioconjugate (32).

153

Figure S18. ESI-MS spectrum of gold(III)- RGDFK bioconjugate (32).

154

Figure S19. Structure of gold(III)- KTTHWGFTLG bioconjugate (33).

155

Figure S21. ESI-MS spectrum of gold(III)- KTTHWGFTLG bioconjugate (33).

Figure S22. The dose-response curve for the determination of the IC50 value of bioconjugate 32 on MDA-MB-231 cells.

156

Table S1. Cytotoxicity data determined by MTT after 48 h of incubation.

Cell lines a IC50 [µM] MCF7 MDA-MB-231 GM5657T CDDP 8.9±2.3 22.4±2.5 10.2±2.1 26 44.3±3.6 77.9±5.1 50.8±4.1 30 24.8±2.9 12.5±0.5 11.5±0.8 31 6.3±1.3 9.7±1.6 7.2±1.3 32 36.3±4.3 50.1±4.6 40.2±3.2 29 80.6±12.6 106.7±15.1 110.5±6.1 34 >250 >250 >250 35 129±5.3 174.2±6.5 151.3±4.1 a The IC50 values are averages of three independent determinations. All compounds were dissolved in DMSO (final concentration 0.5 %).

157