New-Generation Bioinorganic Complexes 9783110348903, 9783110348804

Table of contents :
Contents
Preface
List of contributing authors
1. Noncovalent interactions in biocomplexes
1.1 Introduction
1.2 Noncovalent interactions in metal complexes
1.2.1 Some historical backgrounds
1.2.2 Types of interactions in and around metal complexes
1.2.3 Characterisation of interactions
1.2.4 Interactions within complex molecules
1.2.4.1 Through-metal ligand?ligand interactions
1.2.4.2 Through-space ligand?ligand and metal?ligand interactions
1.2.5 Detection and evaluation of ligand?ligand interactions in solution
1.2.5.1 Stability constants
1.2.5.2 Spectral data
1.2.6 Ligand?ligand interactions in ternary metal complexes involving amino acids
1.2.6.1 Stability enhancement
1.2.6.2 Structure and selectivity
1.3 Structural and functional characterisation of noncovalent interactions in chemistry and biology
1.3.1 Association of oppositely charged ions in Cu(II)?arginine complexes
1.3.2 Interactions between metal complexes and surrounding groups
1.3.2.1 Adduct formation and its effect
1.3.2.2 Protein?small molecule interactions
1.3.2.3 Interactions involving coordinated ligands at the metal site of proteins
1.3.3 Close contact between the metal center and the side chain groups
1.3.3.1 Metal?aromatic ring interactions
1.3.3.2 Interactions involving a hydrogen atom
1.4 Concluding remarks
1.5 Abbreviations
Acknowledgments
References
2. Photo-sensitive complexes based on azobenzene
2.1 Azobenzene
2.2 UV-sensitive complexes
2.2.1 Azobenzene with nickel (Ni)
2.2.2 Azobenzene with platinum (Pt) and palladium (Pd)
2.2.3 Azobenzene with cobalt (Co)
2.2.4 Azobenzene with manganese (Mn), rhenium (Re) and ruthenium (Ru)
2.2.5 Azobenzene with ferro (Fe) and zinc (Zn)
2.2.6 Azobenzene with Copper (Cu)
Acknowledgment
References
3. Complexes of biogenic amines in their role in living systems
3.1 Introduction
3.2 Polyamines in living systems
3.3 Polyamines as tumour markers
3.4 Weak interactions of polyamines
3.5 Complex formation of bioamines
3.6 Application of polyamine complexes in medicine
References
4. Synthetic aspects, crystal structures and biological activities of d- and f-metal salen-type complexes
4.1 Synthesis and crystal structure of d-metal salen-type complexes
4.2 Synthesis and crystal structure of metal salen-type complexes
4.3 Synthesis and crystal structure of the heteronuclear salen-type complexes of d- and f-metal ion
4.4 Biological activities of d- and f-metal salen-type complexes
4.5 Conclusions
References
5. Biocomplexes in radiochemistry
5.1 Introduction
5.2 Bone-seeking complexes
5.2.1 Diagnostic bone-seeking radiopharmaceuticals
5.2.2 Development of novel diagnostic bone-seeking technetium complexes
5.2.3 Development of novel diagnostic bone-seeking gallium complexes for PET
5.2.4 Bone-seeking radiopharmaceuticals for palliative therapy of bone metastases
5.2.5 Development of novel bone-seeking complexes for palliation therapy of bone metastases
5.3 Radio-complexes for imaging of apoptosis
5.3.1 Technetium-labeled annexin A5
5.3.2 99mTc-4, 5-bis(thioacetamido)pentanoyl-annexin A5 (99mTc-BTAP-annexin A5)
5.3.3 99mTc-HYNIC-annexin A5
5.3.4 99mTc-labeled annexin A5 constructed with histidine residues
5.3.5 99mTc-C3(BHam)2-annexin A5
5.3.6 68Ga-labeled annexin A5
5.3.7 Summary of radiolabeled annexin A5 for imaging of apoptosis
5.4 Summary
References
6. Peptides and biocomplexes in anticancer therapy
6.1 General introduction on cancer
6.1.1 Cancer and metastasis origins and global burden
6.1.2 Genomic instability and the other side of the Darwinian coin
6.1.3 The hallmarks of cancer
6.1.4 Molecular basis of the metastatic cascade
6.2 General used therapies of cancer
6.2.1 Therapy against cancer and metastasis
6.3 Biocomplexes in cancer therapy
6.3.1 Photodynamic therapy (PDT)
6.3.2 Metal complexes as platforms for cancer therapy
6.3.2.1 Platinum-based cancer therapy: a start of a new phase
6.3.2.2 Zinc in cancer therapy
6.3.2.3 Copper in cancer therapy
6.3.2.4 Peptide therapeutics
6.3.2.5 Peptides in cancer therapy
6.3.2.6 TAT-RasGAP317–326 as a dual sensitizer and anti-metastatic tool
6.4 Conclusions
Acknowledgment
References
7. Developments in platinum anticancer drug
7.1 Discovery of anticancer activity of cisplatin
7.2 Platinum complexes
7.2.1 Platinum (II) complexes
7.2.2 Platinum (IV) complexes
7.3 Anticancer drug delivery
References
Index

Citation preview

Jastrząb, Tylkowski (Eds.) New-Generation Bioinorganic Complexes

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New-Generation Bioinorganic ­Complexes Edited by Renata Jastrząb and Bartosz Tylkowski

DE GRUYTER

Editors Dr. habil. Renata Jastrząb Adam Mickiewicz University Faculty of Chemistry Umultowska 89b 61-614 Poznan, Poland [email protected] Dr. Bartosz Tylkowski Rovira i Virgili University Department of Chemical Engineering Av. Paisos Catalans 26 43007 Tarragona, Spain [email protected]

ISBN 978-3-11-034880-4 e-ISBN (PDF) 978-3-11-034890-3 e-ISBN (EPUB) 978-3-11-038644-8 Set-ISBN 978-3-11-034891-0 Library of Congress Cataloging-in-Publication Data A CIP catalog record for this book has been applied for at the Library of Congress. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2016 Walter de Gruyter GmbH, Berlin/Boston Cover image: Wolfgang Filser/gettyimages Typesetting: Compuscript Ltd., Shannon, Ireland Printing and binding: CPI books GmbH, Leck ♾ Printed on acid-free paper Printed in Germany www.degruyter.com

Preface Bioinorganic chemistry is an essential part of life. As one of the most dynamic fields in contemporary science, bioinorganic chemistry lies at a natural juncture between chemistry, biology, and medicine. This rapidly expanding field probes fascinating questions about the uses of metal ions in nature. Whenever nature has a difficult task to perform it can be expected that a metal ion or a cluster of such ions is employed. Metal ions are indispensable for the integrity of biological structures and the catalysis of life processes. The book aims to review the art of the next generation of bioinogranic complexes and to provide the readers with a comprehensive and in-depth understanding of recent developments and innovative applications in this field. Chapter 1 describes noncovalent interactions which perform essential roles in biological systems such as molecular recognition, protein stabilization, specificity, and efficiency of enzymatic reactions. Chapter 2 provides an overview on photosensitive complexes based on azobeznene compounds and their applications. Chapters 3 details complexes of biogenic amines in the aspect of their role in living systems, while Chapter 4 emphasizes synthetic aspects, crystal structures, and biological activities of d- and f-metal salen-type complexes, respectively. Knowledge about metal ions’ interaction with proteins, nucleic acids, and other organic moieties is very important for the development of new pharmaceuticals and medicine. The topics detailed in Chapters 5–7 provide current developments and applications of bio-complexes extremely useful in radiochemistry and anticancer therapy. We would like to express our gratitude to the contributing authors in making this project a success, and to Dr. Ria Fritz and Ms. Gesa Plauschenat of ­DeGruyter Publisher, Germany as well as Prof. Henryk Koroniak and Prof. Maciej Kubicki, deans of Faculty of Chemistry of Adam Mickiewicz University, Poznan, Poland, for their assistance and encouragement in this venture. Moreover we would like to ­gratefully acknowledge a financial support from European Community’s Seventh Framework Programme (FP/2007–2013) under which Dr. Tylkowski co-edits this book as a part of outriching activity of his Individual Outgoing Marie Curie Grant agreement no. 328794. Renata Jastrząb and Bartosz Tylkowski

Contents Preface v List of contributing authors

xi

Osamu Yamauchi 1 Noncovalent interactions in biocomplexes 1 1.1 Introduction 1 1.2 Noncovalent interactions in metal complexes 2 1.2.1 Some historical backgrounds 2 1.2.2 Types of interactions in and around metal complexes 3 1.2.3 Characterisation of interactions 5 1.2.4 Interactions within complex molecules 5 1.2.4.1 Through-metal ligand‒ligand interactions 5 1.2.4.2 Through-space ligand‒ligand and metal‒ligand interactions 1.2.5 Detection and evaluation of ligand‒ligand interactions in solution 7 1.2.5.1 Stability constants 7 1.2.5.2 Spectral data 8 1.2.6 Ligand‒ligand interactions in ternary metal complexes involving amino acids 11 1.2.6.1 Stability enhancement 11 1.2.6.2 Structure and selectivity 14 1.3 Structural and functional characterisation of noncovalent interactions in chemistry and biology 19 1.3.1 Association of oppositely charged ions in Cu(II)‒arginine complexes 19 Interactions between metal complexes and 1.3.2 surrounding groups 20 1.3.2.1 Adduct formation and its effect 20 1.3.2.2 Protein‒small molecule interactions 21 1.3.2.3 Interactions involving coordinated ligands at the metal site of proteins 24 1.3.3 Close contact between the metal center and the side chain groups 25 1.3.3.1 Metal‒aromatic ring interactions 25 1.3.3.2 Interactions involving a hydrogen atom 28 1.4 Concluding remarks 30 1.5 Abbreviations 30 Acknowledgments 32 References 32

6

viii 

 Contents

Bartosz Tylkowski, Renata Jastrząb and Monika Skrobańska 2 Photo-sensitive complexes based on azobenzene 41 2.1 Azobenzene 41 2.2 UV-sensitive complexes 44 2.2.1 Azobenzene with nickel (Ni) 44 2.2.2 Azobenzene with platinum (Pt) and palladium (Pd) 46 2.2.3 Azobenzene with cobalt (Co) 49 2.2.4 Azobenzene with manganese (Mn), rhenium (Re) and ruthenium (Ru) 54 2.2.5 Azobenzene with ferro (Fe) and zinc (Zn) 55 2.2.6 Azobenzene with Copper (Cu) 61 Acknowledgment 64 References 64 Renata Jastrząb, Lechosław Łomozik and Bartosz Tylkowski 3 Complexes of biogenic amines in their role in living systems 3.1 Introduction 69 3.2 Polyamines in living systems 70 3.3 Polyamines as tumour markers 74 3.4 Weak interactions of polyamines 77 3.5 Complex formation of bioamines 81 3.6 Application of polyamine complexes in medicine 88 References 93

69

Małgorzata T. Kaczmarek 4 Synthetic aspects, crystal structures and biological activities of d- and f-metal salen-type complexes 107 4.1 Synthesis and crystal structure of d-metal salen-type complexes 108 Synthesis and crystal structure of metal salen-type 4.2 complexes 110 Synthesis and crystal structure of the heteronuclear salen-type 4.3 complexes of d- and f-metal ion 113 Biological activities of d- and f-metal salen-type complexes 4.4 115 4.5 Conclusions 117 References 118 Kazuma Ogawa 5 Biocomplexes in radiochemistry 121 5.1 Introduction 121 5.2 Bone-seeking complexes 121 5.2.1 Diagnostic bone-seeking radiopharmaceuticals

121

Contents 

5.2.2

 ix

Development of novel diagnostic bone-seeking technetium complexes 123 5.2.3 Development of novel diagnostic bone-seeking gallium complexes for PET 126 5.2.4 Bone-seeking radiopharmaceuticals for palliative therapy of bone metastases 128 5.2.5 Development of novel bone-seeking complexes for palliation therapy of bone metastases 130 5.3 Radio-complexes for imaging of apoptosis 132 5.3.1 Technetium-labeled annexin A5 132 99mTc-4, 5-bis(thioacetamido)pentanoyl-annexin A5 5.3.2 (99mTc-BTAP-annexin A5) 132 99mTc-HYNIC-annexin A5 5.3.3 133 99mTc-labeled annexin A5 constructed with histidine residues 5.3.4 134 99m 5.3.5 Tc-C3(BHam)2-annexin A5 134 68Ga-labeled annexin A5 5.3.6 135 5.3.7 Summary of radiolabeled annexin A5 for imaging of apoptosis 137 5.4 Summary 137 References 137 Hadi Khalil, Mathieu Heulot and David Barras 6 Peptides and biocomplexes in anticancer therapy 143 6.1 General introduction on cancer 143 6.1.1 Cancer and metastasis origins and global burden 143 6.1.2 Genomic instability and the other side of the Darwinian coin 143 6.1.3 The hallmarks of cancer 145 6.1.4 Molecular basis of the metastatic cascade 147 6.2 General used therapies of cancer 149 6.2.1 Therapy against cancer and metastasis 149 Biocomplexes in cancer therapy 6.3 151 Photodynamic therapy (PDT) 6.3.1 151 Metal complexes as platforms for cancer therapy 6.3.2 152 Platinum-based cancer therapy: a start of a new phase 6.3.2.1 152 Zinc in cancer therapy 6.3.2.2 153 Copper in cancer therapy 6.3.2.3 153 Peptide therapeutics 6.3.2.4 154 Peptides in cancer therapy 6.3.2.5 154 6.3.2.6 TAT-RasGAP317–326 as a dual sensitizer and anti-metastatic tool 156 6.4 Conclusions 157 Acknowledgment 157 References 158

x 

 Contents

Bartosz Tylkowski, Renata Jastrząb and Akira Odani 7 Developments in platinum anticancer drug 160 7.1 Discovery of anticancer activity of cisplatin 160 7.2 Platinum complexes 162 7.2.1 Platinum (II) complexes 162 7.2.2 Platinum (IV) complexes 166 7.3 Anticancer drug delivery 169 References 171 Index

174

List of Contributing Authors Chapter 1 Osamu Yamauchi Nagoya University 1-14-3 Meguri, Hirakata 573-1171, Osaka, Japan Chapter 2 Bartosz Tylkowski Universitat Rovira i Virgili Departament de Enginyeria Química Av. Països Catalans 26 43007, Tarragona, Spain Renata Jastrząb Adam Mickiewicz University Faculty of Chemistry Umultowska 89b 61-614 Poznan, Poland Monika Skrobańska Adam Mickiewicz University Faculty of Chemistry Umultowska 89b 61-614 Poznan, Poland Chapter 3 Bartosz Tylkowski See Chapter 2 Renata Jastrząb See Chapter 2 Lechosław Łomozik Adam Mickiewicz University Faculty of Chemistry Umultowska 89b 61-614 Poznan, Poland Chapter 4 Małgorzata T. Kaczmarek Faculty of Chemistry Adam Mickiewicz University Umultowska 89b, 61-614 Poznań, Poland

Chapter 5 Kazuma Ogawa Graduate School of Medical Sciences Kanazawa University Kanazawa, 920-1192 Japan Chapter 6 Hadi Khalil Cincinnati children’s hospital medical center 240 Albert Sabin Way 45229-3039, Cincinnati Ohio, USA Mathieu Heulot Department of Physiology University of Lausanne, 1005 Lausanne Switzerland David Barras Swiss Institute of Bioinformatics 1015 Lausanne Switzerland Chapter 7 Bartosz Tylkowski See Chapter 2 Renata Jastrząb See Chapter 2 Akira Odani Kanazawa University Department of Clinical and Analytical Sciences Kakuma-machi, 920-1192 Kanazawa, Japan

Osamu Yamauchi

1 Noncovalent interactions in biocomplexes 1.1 Introduction Noncovalent interactions perform essential roles in biological systems such as molecular recognition, protein stabilisation, and specificity and efficiency of enzymatic reactions [1–4]. They are formed and cleaved instantaneously and are dependent on such factors as the properties of the interacting groups or atoms, the distances between them, and the media in which they are present. The interactions, which are often called weak interactions, are also important for metal complex systems involving nucleotides and DNA [5, 6] and supramolecular architecture [7, 8]. Essential transition metal ions such as copper and zinc are bound to proteins mainly by the side chain groups of the amino acid residues such as the histidine (His) imidazole, cysteine (Cys) thiol, and tyrosine (Tyr) phenol moieties. The ligands forming the metal site may be in contact with the amino acid residues forming the molecular environment through weak interactions, and therefore the metal centre is under conditions which are different from bulk water. Such interactions protect the metal centre from the attack of solvent molecules and have subtle effects on the properties of the metal ion. The function of metalloproteins therefore depends on the active site structure and the noncovalent interactions with the molecular environment. As seen in cytochrome c peroxidase [9, 10] and type 1 copper sites [11], interactions between the coordinating groups such as the imidazole and thiolate moieties and the protein side chain groups surrounding the metal site can influence the structure and electron density and thus the reactivity of the metal centre. However, various metal ions and complexes are known to be enzyme inhibitors [12], and new steps toward metallodrugs [13–16] and functional complexes considering the second coordination sphere [17] have been made, which indicate that noncovalent interactions and structural fitness are important for the activity. DNA is well known as the target of the anticancer drugs such as cisplatin and its analogues and metallo-intercalators, the latter of which bind with DNA by noncovalent interactions, especially aromatic ring stacking and electrostatic interactions. Studies have been carried out for developing effective and specific metallo-intercalators and metallo-insertors and clarifying their binding modes [5, 18]. Zinc finger proteins are a class of proteins that bind with Zn(II) to form finger structures with basic, polar, and aromatic amino acids such as arginine (Arg) and His at the finger domains, whose noncovalent interactions with DNA have attracted much attention [19, 20]. In view of the importance of noncovalent interactions in biological systems, this chapter is intended to give a perspective of noncovalent interactions in and around

2 

 1 Noncovalent interactions in biocomplexes

the metal centre and their relevance to the metal site of proteins, focusing on ligand‒ ligand interactions in metal‒amino acid and related complexes and interactions involving metal complexes and proteins.

1.2 Noncovalent interactions in metal complexes In the past 50 years there has been growing interest in noncovalent interactions in metal complexes of biological ligands. This section will give an overview of the backgrounds and basic findings related to metal‒ligand systems. 1.2.1 Some historical backgrounds An early indication of intramolecular ligand–ligand interactions was provided by the solution studies on ternary (mixed ligand) complexes of Cu(II) etc. Preferential formation of ternary complexes depending on certain combinations of ligands has been shown by Sigel and his collaborators by evaluation of the stability enhancement relative to the complexes without such a ligand set [21–23]. Studies have been reported for the intramolecular ligand‒ligand stacking interactions in ternary Cu(II) complexes with aromatic diimines (DA) and nucleotides such as Adenosine 5'-monophosphate (AMP) [22, 24–26], and complexes containing amino acids with aromatic, aliphatic, charged, or polar side chains capable of various interactions have been studied [27–29]. Metal transport in biological systems has been an important and interesting subject from the view point of bioinorganic chemistry. Most of the Cu ions (ca. 95 %) in blood serum are bound to ceruloplasmin and are not exchangeable, and the rest are present mainly as Cu(II)‒serum albumin and to a smaller extent as mixed amino acid complexes containing His, both of which are considered to be involved in copper transport [30, 31]. A ternary complex containing His and threonine (Thr), Cu(His)(Thr), was detected in human blood serum [32], while the tracer studies using 64Cu showed that the amino acids Thr, glutamine (Gln), and asparagine (Asn) effectively formed ternary complexes with His, Cu(His)(L) (L = Thr, Gln, and Asn) [33]. The structure of Cu(His)(Thr) was established by X-ray analysis to have His bound to Cu(II) through the amine and imidazole nitrogens with the carboxylate oxygen at an axial position [34]. Later the structure of Cu(His)(Asn) (Fig. 1.1(a)) was revealed to have the same coordination structure as that of Cu(His)(Thr) [35]. Preferential formation of certain ternary amino acid Cu(II) complexes was also indicated by computer simulation of Cu(II)-amino acid systems in solution. Some discrepancies between the tracer experiment and computer simulation regarding the preferred formation of the above mentioned mixed amino acid complexes have been carefully reinvestigated, and the conclusions from both approaches are now in satisfactory agreement [36].

1.2 Noncovalent interactions in metal complexes 

NH2

O

O

O

C

O

O

–

NH2 N

2+ Cu

N

O

H2N C 2+ Cu

NH2 O–

O

N H

N H (a)

O

–

NH2

NH2 O–

 3

(b)

Fig. 1.1: Structures of Cu(His)(Asn) (a) and its possible conformational isomer with ligand‒ligand interaction (b) [35, 37].

Since the side chains of all the above amino acids L have a polar group, the structure of Cu(His)(Asn) suggested the possibility of hydrogen bonding between the axially coordinated His carboxylate oxygen and the amide group of Asn. They are located on the same side of the coordination plane, and the amide NH2 moiety may then approach the coordinated oxygen atom by rotation around the Cb-Camide bond and a slight deformation of the chelate ring (Fig. 1.1(b)) [37]. The situation is considered to be similar for Thr and Gln in place of Asn, and formation of His-containing ternary Cu(II) complexes in blood plasma as shown by tracer studies and isolation of Cu(His) (Thr) may be due to such intramolecular hydrogen bonding. The structures of metalloenzymes were first reported for a zinc-containing enzyme carboxypeptidase A (CPA) and its complex with a substrate model peptide glycyltyrosine (GlyTyr) by Lipscomb and his collaborators [38, 39]. The active site structure of the CPA‒GlyTyr complex revealed the coordination of GlyTyr to Zn(II) to form a complex, and in addition it showed weak interactions between the carboxylate and phenol moieties of GlyTyr and the guanidinium group of an arginine (Arg) residue (Arg145) and a hydrophobic pocket of the enzyme, respectively (Fig. 1.2(a)) [38]. This enzyme-substrate complex is considered as a ternary Zn(II) complex, and these interactions can be regarded as ligand‒ligand interactions, which may be partly mimicked by ternary amino acid complexes as shown in Fig. 1.2(b) [28]. These and other features of the enzyme‒substrate complex prompted studies on ligand‒ligand interactions and ligand reactivities in ternary metal complexes containing amino acids [40–43]. 1.2.2 Types of interactions in and around metal complexes Noncovalent interactions are bonding interactions that are not covalent and with rather long interatomic distances (2‒5 Å) and energies usually less than 1/10 of covalent bonds ( ion‒dipole > dipole‒dipole > dipole‒quadrupole [44]. In metal complexes there are metal‒ligand coordinate covalent bonds forming the coordination structure, and in addition there can be various weak interactions between the ligands and between the metal ion and ligands. Metal‒ligand systems may involve the following interactions in addition to the metal‒ligand coordinate bonds: (i) Through-metal ligand‒ligand interactions (ii) Through-space ligand‒ligand interactions (a) Hydrogen bonds and electrostatic interactions (b) Aromatic ring stacking interactions (c) CH‒p and other interactions (iii) Metal‒aromatic and metal‒alkyl interactions (iv) Intermolecular interactions between a metal complex and neighbouring molecules Interactions (i)–(iii) as well as factors such as the statistical factor and neutralisation of charges are considered to contribute to mixed ligand metal complex formation. Complexes with interacting groups may undergo intermolecular interactions (iv) with neighboring molecules to form molecular adducts, where selective binding may lead to molecular recognition.

1.2.4 Interactions within complex molecules 1.2.4.1 Through-metal ligand–ligand interactions Through-metal ligand–ligand interactions are regarded as electronic interactions between ligands mediated by the central metal ion. These interactions have been concluded for ternary Cu(II) complexes containing DA (= 2,2ʹ-bipyridine (bpy), 1,10 phenanthroline (phen), etc.) and a negatively charged oxygen ligand such as catecholate (cat), Cu(DA)(cat), where the combination of an electron-deficient DA

6 

 1 Noncovalent interactions in biocomplexes

with an electron-rich oxygen ligand, cat, in the Cu(II) coordination plane is favoured [21–23] (cf. 2.5.1.). The stabilising effect of such a ligand combination is explained by electron donation from ligands such as –O‾ and Cl‾ with filled p orbitals to the metal ion (p-donation) and from the metal ion to ligands such as pyridine and CN‾, which have empty p* orbitals at a relatively low energy level (p-back donation). The former ligands are called p-donors or p-bases and the latter are called p-acceptors or p-acids. Tanaka presented an equation for estimating the stability constants of ternary Ni(II) and Cu(II) complexes of nitrogen- and/or oxygen-donor ligands from mechanistic considerations by introducing the ligand interaction terms, dij, which were calculated from the reported stability constants and allow for the effect of the donor atom Xi of a ligand on the donor atom Yj of the other ligand [47, 48]. The dij values may be considered to reflect the through-metal ligand-ligand interactions, and the equation gave estimates of the stability constants for mixed ligand complexes such as His-containing ternary Cu(II) complexes, which were in excellent agreement with the experimental values [49].

1.2.4.2 Through-space ligand‒ligand and metal‒ligand interactions In ternary (mixed ligand) complexes there can be steric repulsion between the coordinated ligands due to bulky side chain groups, but here we will consider attractive ligand‒ligand interactions, such as hydrogen bonds, electrostatic interactions, and interactions involving aromatic rings, which favour ternary complex formation. Most L-a-amino acids are effective biological N,O-donor ligands, and those with a metal binding side chain group, especially His, Cys, methionine (Met), aspartate (Asp), glutamate (Glu), and Tyr, are important metal binding sites in proteins. a-Amino acids with the side chain group (X) at neutral pH as shown below are capable of interaction and thus of interest for their possible interactions in and around metal complexes: (i) Negatively charged group: Asp, Glu (X = ‒COO‾ ) (ii) Positively charged groups: Arg (X = ‒NHC(NH2)2+); lysine (Lys) (X = ‒NH3+) (iii) Polar groups: serine (Ser), Thr (X = ‒OH); Asn, Gln (X = ‒CONH2) (iv) Aromatic rings: phenylalanine (Phe) (X = phenyl); Tyr (X = p-hydroxyphenyl); tryptophan (Trp) (X = 3-indolyl); His (X = 4(5)-imidazolyl) At higher pH or at the metal site, some X groups and Cys (X = ‒SH) dissociate to give a negative charge and may be involved in noncovalent interactions and/or metal binding. In addition, the derivatives such as phosphotyrosine (PTyr) and phosphoserine (PSer) (X = ‒OPO3‾) may be involved in hydrogen bonds or electrostatic interactions, and biological amino acids such as cysteic acid (CySO3H; X = ‒SO3‾), ornithine (Orn; X = ‒NH3+), and citrulline (Cit; X = ‒CONH2) also have interacting side chains.

1.2 Noncovalent interactions in metal complexes 

 7

Bulky alkyl side chain groups may be involved in CH‒p and hydrophobic interactions in and around the metal centre [27, 43, 50]. 1.2.5 Detection and evaluation of ligand‒ligand interactions in solution Preferential formation of ternary complexes due to ligand‒ligand interactions may be concluded on the basis of the information most commonly from stability constants and spectral data, while X-ray crystal structure analysis provides detailed information on the mode and strength of interactions in the solid state and serves as a basis for their existence. 1.2.5.1 Stability constants The existence of intramolecular ligand‒ligand interactions has been concluded for various complexes in solution by stability constant measurements. The stability enhancement of ternary complexes can be evaluated by using the values such as log Km, Dlog K, and log K calculated from the relevant stability constants [22, 29, 51]. Here the constants will be expressed as the stepwise stability constants (K values) for 1:1 complexes and as the overall stability constants (b values) for complexes with more than one ligand. The equilibrium constant, Km, is defined as follows (charges are omitted for simplicity) [22, 51]:

MA2 + MB2  

Km

2MAB

​ A​ ​​ + log ​b M​ ​ B​ ​​ ) log Km = 2log b MAB – (log ​b M​ 2

2

(1.2)

where M is a metal ion and A and B are bidentate ligands such as a-amino acids. The statistical value of log Km is 0.6 for square-planar complexes, and therefore the log Km value greater than 0.6 indicates that the ternary complex MAB is favoured over the binary complexes MA2 and MB2. The Dlog K value corresponds to the logarithm of the equilibrium constant Kʹ for eq. (1.3) and indicates the preference for binding of A to MB or binding of B to MA rather than binding of A or B to the solvated metal ion M [22, 51]:

MA + MB  

Kʹ

MAB + M

log Kʹ = log b MAB – (log KMA + log KMB) = ∆log K

(1.3)

However, the constant K is defined for the following equilibrium [29]:

MAʹB + MABʹ  

K

MAB + MAʹBʹ

log K = log b MAB + log b MAʹBʹ – (log b MAʹB + log b MABʹ) 

(1.4)

where A and B are ligands with an interacting group and Aʹ and Bʹ are corresponding ligands without it. This is an equilibrium showing preferred formation

8 

 1 Noncovalent interactions in biocomplexes

of complex MAB with ligand‒ligand interactions, with MAʹBʹ serving as the standard. While the log Km and Dlog K values indicate the preferential formation of ternary complexes due to various factors, the log K value reflects the stability increase of MAB relative to MA’B’ mainly due to ligand‒ligand interactions [29, 40]. Stabilisation of ternary complexes by through-metal ligand‒ligand interactions has been known for complexes with DA and a phenolate ligand such as cat (L) as seen in 1.2.4.1. The log Km and Dlog K values for Cu(DA)(L) with DA = bpy or phen and L = cat or oxalate are greater than those for Cu(en)(L) and Cu(DA)(en) (en = ethylenediamine) due to the above mentioned electron flow, which does not occur with en [21–23].

1.2.5.2 Spectral data NMR spectra provide information on ligand‒ligand interactions. The interactions expected for ternary M‒amino acid complexes such as those containing an acidic and a basic amino acid [28] and those containing His and a polar amino acid [37] can affect the side chain conformation of amino acids, which may be studied by 1H NMR spectral measurements of Pd(II) and low-spin Ni(II) complexes with a coordination structure similar to that of Cu(II) complexes. Changes in the side chain conformations of coordinated amino acids due to the intramolecular interactions in complexes can be detected by the changes in the populations (P) of their staggered rotamers (Fig. 1.3(a)) calculated from the coupling constants, J, of the 1H NMR spectra according to the following equations [52, 53]:

( JAC − Jg) PI = ______ ​     ​ 



( JAB − Jg) PII = ______ ​     ​ 



 ( Jt + Jg) − ( JAB + JAC)    ​      PIII = ​  _________________  ( Jt − Jg) 

( Jt − Jg)

( Jt − Jg)

(1.5)

where JAB and JAC are coupling constants between the protons shown in Fig. 1.3(a) and Jt = 13.56 Hz and Jg = 2,60 Hz [52]. The population of rotamer III (PIII), which enables the intramolecular interactions in complexes as shown for Pd(Lys)(CySO3H) in Fig. 1.3(b)), increases with the temperature decrease and the solvent polarity decrease as compared with rotamers I and II, indicating the effect of the electrostatic side chain interactions [54]. Use of rotamer populations for determining the side chain conformations has been reported, for example, for metal‒peptide systems such as Ni(II)‒TyrGlyGly and Pd(II)‒AlaTyr (Gly = glycine; Ala = alanine), which exhibited the PIII increase due to the metal ion‒side chain aromatic ring interaction [55–57].

1.2 Noncovalent interactions in metal complexes 

NH2

NH2

HB

HC

R

HA

COO–

HA

(a)

 9

NH2 HB

HC

R

COO–

HA

COO–

R

HC

HB

I

II

III HB

–

L-CySO3H Pd

NH2 HC

SO3

HA

COO–

–

HA

OOC (CH2)4

2+

H3N+

HC NH2 L-Lys

HB (b) Fig. 1.3: (a) Staggered rotamers of a-amino acids. (b) Structure of [Pd(L-CySO3H)(L-Lys)] showing that rotamers III of the amino acids are necessary for intramolecular interactions [54].

[Pt(phen)(en)]–NMP [Pt(phen)(en)]–(NMP)2 l = 01 and 0.2 M (NaCl)

Δδn/ppm

40

NAD0.1 GMP0.2 AMP0.1 AMP

30

0.2 AMP0.2 GMP0.1 NAD0.1 GMP0.1 GMP0.2 AMP0.1 IMP0.1 IMP0.1

20 10

0

10

20 30 –ΣΔHn°/kJ mol–1

40

Fig. 1.4: 195Pt NMR downfield shifts (Ddn/ppm) plotted against enthalpy changes (−SDHn°/kJ mol‒1; n = 1 (●) and 2 (○)). The ionic strength (I) is indicated for each nucleotide as a suffix [61].

Aromatic ring stacking in complexes may be detected by the upfield shifts of the proton NMR signals due to the ring current effect [58]. The upfield shifts have been used for evaluating the stacking interactions in ternary complexes involving DA (= bpy, phen, etc.) and nucleotides, amino acids, or peptides [27, 59, 60]. The adduct

10 

 1 Noncovalent interactions in biocomplexes

formation between Pt(II) complexes such as Pt(phen)(en) and nucleotides such as AMP caused upfield shifts of the 1H NMR signals and downfield shifts of the 195Pt NMR signals, Ddn, which have been shown to increase with the increase of the enthalpy changes, −SDHnº (n = 1, 2 ), for the 1:1 and 1:2 adduct formations (Fig. 1.4) [61]. The downfield shifts indicate that the electron density of Pt(II) decreases with the increasing stability of the stacked form probably due to delocalisation of the electrons over the stacked structure. Absorption and CD spectra are important sources of information on ligand‒ ligand interactions. Stacking interactions give rise to charge transfer (CT) bands in the near ultraviolet region as observed, for example, for ternary Cu(II)‒bpy‒ nucleotide systems involving bpy‒purine base stacking [62]. CT bands were also observed for Cu(II)‒DA‒amino acid complexes; a Trp-containing complex Cu(bpy) (Trp), which has an average bpy···indole distance of 3.67 Å, exhibited a broad band centred at 320 nm in the difference spectrum [63], and similarly Cu(phen)(XPhe) (XPhe = Phe, Tyr, or p-aminophenylalanine (NH2Phe)) and related complexes gave weak peaks assigned to CT at 320–400 nm [64, 65]. Optically active ligands such as L-a-amino acids and their peptides coordinated to transition metal ions give CD bands in the d-d region due to the vicinal effect of the asymmetric carbon. The CD magnitude (De) of the Cu(II) complexes of oligopeptides are known to be an additive function of the magnitudes due to the component amino acid residues [66]. In the absence of ligand‒ligand interactions the CD magnitudes of ternary amino acid‒Cu(II) complexes have also been found to be an additive function of the contributions from each amino acid. The magnitude for M(A)(B) (M = Cu(II), Pd(II); A and B = amino acids), Decalcd, can be estimated from the magnitudes DeCu(A)2 and DeCu(B)2 observed for the binary complexes M(A)2 and M(B)2, respectively, by the following equation [28]:



Decalcd = 1_2​  ​   (De MA + De MB ) 2

2

(1.6)

When there exist interactions between the side chains of A and B in M(A)(B), the observed magnitude, De, deviates from the additivity, i.e. De/Decalcd ≠ 1, due to increased asymmetry. The magnitude anomaly was first observed for ternary complexes containing an acidic and a basic amino acid such as Cu(edma)(Arg) (edma = ethylenediamine-N-monoacetate) and Cu(Asp)(Arg) and the corresponding Pd(II) complexes (Tab. 1.2) [28, 67, 68] and has been assigned to the intramolecular electrostatic ligand‒ligand interactions between the oppositely charged side chains as shown in Fig. 1.2(b). For the complexes with stacking interaction such as Cu(phen)(AA) (AA = aromatic amino acids), the CD magnitude has been found to be much larger than that for Cu(en)(AA) and depend on the aromatic ring of AA in the order Phe < TyrO < Tyr < Trp (TyrO = Tyr with the deprotonated phenol moiety) [29]. Various other methods such as ESR and resonance Raman spectroscopies are also useful for dectection of structural changes due to interactions.

1.2 Noncovalent interactions in metal complexes 

 11

Tab. 1.2: CD spectral magnitude anomaly observed for ternary metal(II)-L-a-amino acid systemsa [54, 67, 68, 71]. System

pH

λmax

Cu(Ala)(Arg) Cu(Ala)(Asp) Cu(Val)(Arg) Cu(Val)(Glu) Cu(Asp)(Arg) Cu(Asp)(Lys) Cu(Glu)(Arg) Cu(Glu)(Lys)

9.4 9.3 7.2 7.4 7.2 7.2 7.3 7.3

600

Cu(Ala)(PTyr) Cu(Ala)(Pser) Cu(PTyr)(Arg) Cu(PSer)(Arg) Cu(PTyr)(Lys)

10.0 7.5 10.0 7.8 9.7

603

Pd(Asp)(Arg) Pd(Asp)(Lys) Pd(Ala)(CySO3H) Pd(CySO3H)(Lys) Pd(His)(Ala) Pd(His)(Val) Pd(His)(Ser) Pd(His)(Thr) Pd(His)(Asn)

Dε

Dε/Dεcalcd

−0.10 −0.06 −0.19 −0.19 −0.09 −0.08 −0.14 −0.13

1.0

−0.177 −0.128 −0.411 −0.214 −0.382

1.15

630 585 616 591

0.96b 2.54 1.57 2.27

6.4 6.4 6.6 6.4

327 328 304 304

0.41 0.41 0.32 0.20

0.86 0.70 1.00 0.87

7.1 7.0 6.9 7.1 6.8

324

−0.10 −0.47 −0.11 −0.29 −0.12

0.93

630 590 600 630 640 600 600

319 321 315 307

1.0 1.0 1.0 1.4 1.2 1.4 1.2

0.96 0.58 0.90 0.64

aThe bI

ionic strength (I) of the solutions was not adjusted (I = var.). = 0.1 M (NaClO4).

1.2.6 Ligand‒ligand interactions in ternary metal complexes involving amino acids The formations and properties of the complexes with intramolecular ligand‒ligand interactions have been studied by various methods. Stability increase due to throughmetal ligand‒ligand interactions as observed for Cu(bpy)(cat) etc. was briefly mentioned in 1.2.5.1. Here we will see some typical examples of through-space noncovalent interactions in amino acid-containing complexes.

1.2.6.1 Stability enhancement In ternary complexes such as Cu(DA)(AA) the side chain group of aromatic amino acids (AA = Phe, Tyr, Trp) can be involved in intramolecular stacking interactions with coordinated DA (DA = bpy, phen, histamine (hista), etc.), and the observed

12 

 1 Noncovalent interactions in biocomplexes

stability enhancement has indicated the existence of such interactions. Tab. 1.3 shows a comparison of the log K values calculated for Cu(DA)(AA) by eq. (1.7) based on eq. (1.4),

log K = log βCu(DA)(AA) + log βCu(en)(Ala) − (log βCu(en)(AA) + log βCu(DA)(Ala))

(1.7)

which revealed the stability sequences due to DA and the side chain groups of AA as follows: DA: phen > bpy > hista Side chain aromatic group of AA: I

HO N H

I

≈ HO

> I

(HTrp)

(I2Tyr) >

> HO (Tyr)

N CH3 (MTrp) >

(Phe)

>

N H

≈

–

O I

(Trp)

O

>

–

(TyrO)

2–

(I2TyrO)

O3PO (PTyr)

Scheme 1

The sequences (Scheme 1) clearly show that the stabilisation due to ligand‒ ligand stacking interactions depends on the size and electron density of the aromatic rings of DA and AA involved. The enhanced stability observed for I2Tyr (= 3,5-diiodotyrosine) and I2TyrO (I2Tyr deprotonated from the phenol moiety) suggests the stabilising effect of the interaction between the iodine atom and the aromatic ring [69, 70]. In contrast to this, phosphorylation of Tyr to PTyr reduced the log K values by ca. 1 log unit for DA = bpy or phen (Tab. 1.3), which indicates that the stacking interaction is virtually lost [71]. The dependence of the complex stabilisation on the Hammett s values (sp) of various p-substituents of Phe for the complexes Cu(DA)(XPhe) (DA = 4,4′-disubstituted-2,2 ′-bipyridines; XPhe = Phe, NH2Phe, Tyr, NO2Phe, FPhe) indicated that the stability enhancement due to stacking is larger for the systems with a larger electron density difference between the stacked rings [70]. For example, for DA = (NEt2)2bpy with two diethylamino groups, the ternary complex with NO2Phe containing an electron-deficient ring showed a larger log K value than that for the complex with NH2Phe with an electron-rich ring, while for DA = (COOEt)2bpy with two electron-attracting ester substituents the log K value was larger for NH2Phe than for NO2Phe.

1.2 Noncovalent interactions in metal complexes 

 13

Tab. 1.3: Structure dependence of log K values for Cu(DA)(AA) calculated according to eq. (1.7) (25 °C; I = 0.1 M (KNO3)) [29, 69, 71, 158]. DA

AA Val

  Phe

   Tyr

TyrO

PTyr

I2Tyr

I2TyrO

 Trp

MTryp

HTrp

bpy 0.02 phen 0.08 hista 0.06

0.60 0.64 0.26

0.90 1.05 0.51

0.25

−0.14 −0.02 −0.15

1.88 2.18 0.84

1.20 1.38 0.28

1.19 1.39 0.60

1.86 0.83

1.80 2.22 0.87

0.11

A small stability difference was previously reported for the D- and L-His-containing ternary Cu(II) complexes with basic amino acids (B) such as L-Lys and L-Arg with a protonated side chain at neutral pH. In Cu(D/L-His)(L-B), L-B is considered to be coordinated as seen for L-Asn in Fig. 1.1(a), and thus its side chain can interact with the axially bound carboxylate oxygen of L-His but not of D-His, resulting in stabilisation of Cu(L-His)(L-B); this stability difference disappeared upon deprotonation from the side chain of B, indicating that the interaction is electrostatic [72, 73]. The difference in the equilibrium constants due to the interactions in ternary complexes with an acidic amino acid (A) and B, Cu(A)(B), was detected in the deprotonation process of the protonated side chain group of B, whose pKa value was 0.57–0.87 log units higher for Cu(A)(B) as compared with Cu(Ala)(B), which is devoid of the interacting group [74]. The result indicates that the proton of the side chain group of B is necessary for the interaction with A as shown in Fig. 1.2(b). For the ternary Cu(II) complexes with A = PTyr, PSer, etc. and B = Lys, Arg, etc., the log K values calculated from eq. (1.4) with Aʹ = Bʹ = Ala were found to be larger at I = 0.1 M (KNO3) than at 1 M (KNO3), which supports that the stabilising effect is due to the intramolecular electrostatic interactions involving the phosphorylated phenol moiety [71]. As compared with the low stability of stacking in Cu(DA)(PTyr) described above, the phosphoester moiety is effectively involved in electrostatic interactions with Arg and Lys. The result may suggest a possible conversion of the Tyr phenol moiety from stacking to electrostatic interactions upon protein phosphorylation [29]. Stabilisation due to noncovalent interactions may occur in binary complexes when ligands have a set of side chain groups as in peptides. The reactions of Cu(II) with a pentapeptide, NSFRY (= AsnSerPheArgTyr-NH2), which is a fragment from the 28-peptide atrial natriuretic factor, and its analogs have been studied by pH titrations and spectroscopic methods, and this particular peptide was revealed to form an exceptionally stable 4N-donor complex with Cu(II) compared with the analogs [75, 76]. From comparative studies of the stability constants, the stabilisation has been attributed to various interactions between the side chain groups; the Asn side chain is considered to interact with the amino group to lower its pKa and make it react easily with Cu(II) and further form a fence with Phe. In the 4N-donor complex, hydrogen bonding between the polar atoms of Asn and Tyr is considered to be

14 

 1 Noncovalent interactions in biocomplexes

formed. An interesting stabilising effect comes from the Arg and Tyr residues, which is explained to be due to formation of an additional fence over the one formed by Asn and Phe [75–79]. The results suggest that non-coordinating side chain groups located close together in the coordination sphere interact with each other and cover the space around the metal centre, thus protecting it from the attack of solvent molecules, etc. The observations suggest a situation that is comparable to that at the metal site in proteins.

1.2.6.2 Structure and selectivity As described in the foregoing sections, noncovalent ligand‒ligand interactions in complexes M(DA)(AA) and M(A)(B) (M = Cu(II), Pd(II)) have been concluded from O C O O

N N edma

Cu

N

O L–Arg

N

N

C

C

N N N

C

O N

O O

L–Arg

O

N

N Cu

N edma

Fig. 1.5: Schematic presentation of intermolecular guanidinium‒carboxylate interactions in [Cu(edma)(L-Arg)]+ in the solid state [80].

L-Ala (a)

S

AQS

O

3.40 Å

2.80 Å

O

N

N

O

Pt

AQS phen

Pt(II) complex

N (b)

Fig. 1.6: [Pt(phen)(L-Ala)]···AQS adduct. (a) Molecular structure and (b) stacking between the adducts [81].

1.2 Noncovalent interactions in metal complexes 

 15

GMP 3.29 Å

N

N N 2.93 Å P

O 2.83 Å N

Pt N

O

N

O

[Pt(bpm)(L-Arg)]2+ 2.79 Å N Fig. 1.7: Structure of [Pt(bpm)(Arg)]2+···GMP2− adduct [82].

solution and spectral studies. The X-ray structures of the complexes isolated as crystals have provided the details of the interactions, although the mode of interactions in the solid state may not always be the same as that in solution. Crystal growth requires interactions between molecules, and intramolecular interactions may be converted to intermolecular interactions to form a polymeric chain. The electrostatic ligand‒ ligand interactions concluded for Cu(edma)(Arg) in solution [28] were found to be intermolecular in the solid state as shown in Fig. 1.5 [80]. While stacking within complexes such as M(DA)(AA) often remains localised, association of complexes or adducts with a stacked structure can occur, forming an infinite pile of stacked rings. In the crystal structure of [Pt(phen)(L-Ala)](AQS) (AQS = anthraquinone-2-sulfonate), [Pt(phen)(L-Ala)]+ stacks with AQS‾ (Fig. 1.6(a)), and this adduct unit stacks with the other units, resulting in an alternate pile of the coordinated phen and AQS rings (Fig. 1.6(b)) [81]. A similar solid state structure was disclosed, e.g. for [Pt(bpm)(L-Ala)](IA) (bpm = 2,2ʹ-bipyrimidine; IA = indole-3-acetate) [82]. In contrast, the adduct of [Pt(bpm)(L-Arg)]2+ with a nucleotide (GMP2‾ ) exhibited an interesting structure (Fig. 1.7), which is formed as a discrete unit by intramolecular interactions, a stacking interaction between coordinated bpm and the guanine ring of GMP and hydrogen bonds between the guanidinium and amino groups of Arg and the phosphate oxygen atoms of GMP [82]. This structural unit is connected to the neighbouring units through unique guanine‒guanine hydrogen bonds with the distances of 2.82‒2.86 Å. A number of structures with p‒p stacking interactions have been reported for ternary complexes containing an aromatic amino acid (AA) or its peptide and DA, such as Cu(phen)(Trp) [83], Cu(bpy)(Trp) [63], Cu((CONH2)2bpy)(Phe) (Fig. 1.8(a)) [70], Cu(hista)(I2TyrO) (hista = histamine) [84], Cu(phen)(TyrGlyH-1) (Fig. 1.8(b)) [85], and Pd(bpy)(TyrGlyH-1) [85], where the aromatic side chain of AA is involved in intramolecular stacking with coordinated DA. Figure 1.8(b) shows that stacking takes place between

16 

 1 Noncovalent interactions in biocomplexes

3.74 Å L-Phe

3.24 Å 3.17 Å O Cu N

(a)

N

N (CONH2)2bpy

Cu

N

N

N

O

N

(b)

Fig. 1.8: Structures of Cu(CONH2)2bpy)(L-Phe) (a) [70] and Cu(phen)(L-TyrGlyH-1) (b) [85].

the Tyr phenol ring and phen bound perpendicular to the Cu(II) plane, which is different from the parallel stacking in Pd(bpy)(TyrGlyH-1) on the Pd(II) plane. The interaction in M(DA)(AA) usually remains localised in the complex molecule with the shortest atomic distances of 3.0–3.5 Å. The stability sequence due to intramolecular stacking of Phe shows that Cu(DA)(Phe) is rather weakly stabilised by stacking compared with Tyr and Trp (cf. 1.2.6.1. and Tab. 1.3). Probably because of this, the side chain of Phe in [Cu(phen)(Phe)Cl] has been found to be both in the stacked structure and the extended structure without stacking [64], and other examples such as [Cu(bpy)(Phe)(H2O)]+ [64] and [Cu(phen)(Phe)]ClO4·H2O [86] were also without intramolecular stacking. Stacking interactions in proteins are well known to contribute to the stability of proteins [3], where the modes of stacking are often edge-to-face and offset or parallel-displaced to avoid p‒p repulsion [87, 88] and possibly for steric reasons. In metal complexes with aromatic nitrogen donors such as M(DA)(AA), stacking is usually offset, showing limited overlapping due to the steric requirements of the coordination structure and the ligand side chain length. However, the distortion of the coordination plane observed in X-ray structures suggests that the aromatic rings involved in stacking tend to be close to each other to be in a parallel position. This may reflect the weakening of p‒p repulsion due to the electron density decrease of coordinated aromatic nitrogen heterocycles, which are already with a low p-electron density [89]. The stacking in M(DA)(AA) may be expressed as MLp‒L’p interactions shown in Fig. 1.9, where ML and L’ denote a metal-bound aromatic ligand and a pendent aromatic ring, respectively [70]. The stacking also implies M‒L’p or d‒p interactions and other interactions when there are ring substituents X and Y. Preference for stacking partners is seen from the structures of the Cu(II) and Pd(II) complexes of tridentate ligands containing a pyridine and a phenolate moiety as donor groups and a side chain indole ring as shown in Fig. 1.10, where the electron-rich indole ring stacks with the pyridine ring but not with the electron-rich phenolate ring [90]. It is interesting

1.2 Noncovalent interactions in metal complexes 

 17

to note in this connection that a Trp residue was found to be in contact with the Cubound Tyr residue by stacking at the active site of a copper enzyme galactose oxidase; the coordinated Tyr272 phenolate ring with a thioether bridge stacks with the Trp290 indole ring, where the indole ring is considered to play important roles in stabilising the electron-deficient phenoxyl radical formed in the course of the reaction and in the functioning of the enzyme [91, 92]. L’

X

M–L’π

MLπ–L’π M–N

Y L

N N

Cl

Fig. 1.9: p‒p stacking between a metal-coordinated aromatic nitrogen ligand L and a pendent aromatic ring L′ [70]. X and Y are ring substituents.

O

Pd N

Fig. 1.10: Stacking of the indole ring in a Pd(II) complex involving a pyridine and a phenolate moiety as coordinating groups [90]. O 3.49 Å O

N (a)

Cu

3.45 Å

N O

N N

N

Cu

N

N

N

(b)

Fig. 1.11: Structures of Cu(hista)(L-Phe) (a) and Cu(hista)(L-Try) (b) [93].

18 

 1 Noncovalent interactions in biocomplexes

Phe 12

His 90 N His 37

Cu

N S

Cys 87 S Met 95

Fig. 1.12: Stacking of coordinated imidazole with Phe in plastocyanin from fern (PDB code: 1KDJ) [94].

The histidine imidazole ring is an important metal binding site in metalloproteins and can be involved in stacking with other aromatic rings. The X-ray analysis of ternary complexes, [Cu(hista)(AA)(ClO4)] (hista = histamine; AA = Phe, Tyr), revealed intramolecular stacking interactions shown in Fig. 1.11 [93]. From the log K values listed in Tab. 1.3, the hista-containing ternary complexes are less stabilised by stacking than those containing bpy or phen probably due to the smaller ring size and higher electron density. The structures show that the shortest distances between the stacked rings are 3.45‒3.49 Å, which are within the normal range. Although ring overlapping is limited and the dihedral angles between the stacked rings are rather large (38.1‒38.5°) in the model complexes, the results suggested the possibility of His‒Phe and His‒Tyr stacking interactions at the metal sites in proteins. Later the coordinated His‒Phe stacking has been actually detected at the Cu site of a plastocyanin from fern (Dryopteris crassirhizoma) between coordinated His90 and nearby Phe12 (Fig. 1.12), which is the first observation of stacking interactions involving a coordinated His residue [94]. While steric hindrance in ternary complex formation is well known as a source of stereoselectivity of ligands, noncovalent interactions are important for chiral and molecular recognition in systems involving complexes and other molecules [4, 43]. Selective incorporation of the L-enantiomer of DL-His via formation and isolation of Cu(His)(L-Asn or L-Cit) is in line with the intramolecular hydrogen bonds inferred from the syntheses and structures of the ternary Cu(II) complexes containing His and an amino acid with a polar side chain (Fig. 1.1(b)) [37, 95] (cf. 1.2.1 and 1.2.6.1). Stereoselectivities or chiral recognitions upon complex formation with His- or histacontaining ligands have been reported for complexes, such as Cu(II)‒His‒amino acid [96] and Cu(II)‒cyclo-HisHis‒amino acid complexes [97] by stacking, and for Cu(II)‒ hista-functionalised b-cyclodextrin‒amino acid complexes by hydrophobic interactions [98]. Differences in the steric requirements for D- and L-Ala in a chiral Co(III) complex [99] and in the rate of complex formation by a chiral Co(III) complex of a leucine-containing ligand with D- and L-Phe due to p‒p stacking [100] and stereoselective binding of a-amino acids by a chiral cyclen‒Co(III) complex [101] serve as further examples of enantioselectivity arising from ligand‒ligand interactions.

1.3 Structural and functional characterisation 

 19

1.3 Structural and functional characterisation of noncovalent interactions in chemistry and biology Noncovalent interactions have influences on the properties of complexes, which is seen from the structures in the solid state and the behaviour in solution. Molecular recognition and stereoselectivity are functions typically expected for noncovalent interactions in metal complexes, but various other contributions to structures and functions of complexes have been reported. In this section, examples showing the effects of intra- and intermolecular interactions in systems involving metal ions will be presented, and their relevance to biological systems will be considered.

1.3.1 Association of oppositely charged ions in Cu(II)‒arginine complexes The guanidinium group of Arg has three NH/NH2 moieties positively charged and is known to be involved in the hydrogen bonding called a salt bridge with the carboxylate group of Asp and Glu, which is effective for protein structure stabilisation [1, 3] and substrate binding by enzymes such as in CPA [38, 39] and Cu,Zn-SOD [102]. It is also known to interact with aromatic rings such as indole to undergo cation‒p interactions (cf. 1.3.3.). The binary Cu(II) complex of Arg, [Cu(Arg)2](NO3)2, in the solid state has two Arg molecules coordinated in the cis configuration due to the hydrogen bonds between the amino groups and a nitrate ion (Fig. 1.13(a)) [80]. When the nitrate ions are replaced by a dianion, X, with two hydrogen bond acceptors, the [Cu(Arg)2]2+ core unit

N

X2-(A1,A2: C or N) A1

N N O

O O O O

N Cu

O

O

O

N

N

O

Arg side chain

H2 N

O

O

N N

O

O N

A2

O O

Arg side chain

H2 N

Cu O O [Cu(L-Arg)2]X

O

Double helical structure

N (a)

(b)

(c)

Fig. 1.13: (a) Structure of [Cu(L-Arg)2](NO3)2 (A) [80]; (b) formation of a double helical structure by association of [Cu(L-Arg)2]2+ with a dicarboxylate by guanidinium-carboxylate interactions; (c) space-filling model of [Cu(L-Arg)2](mbc) [103, 104].

20 

 1 Noncovalent interactions in biocomplexes

self-organises with X to give supramolecular structures depending on the structure of X; with X = isophthalate (m-benzenedicarboxylate (mbc)) and pyridine-2,6- and -3,5-dicarboxylates (2,6- and 3,5-pdc, respectively) having the acceptors of the hydrogen bonds with the Arg guanidinium groups and the coordinated amino groups in suitable positions, [Cu(Arg)2]2+ forms a double-helical structure reflecting the chirality of Arg (Figs. 1.13(b) and 1.13(c)) [103], and a single-helical structure is formed with X = SO42− [104]. However, dianions such as terephthalate and benzene-1,3-disulfonate bind with [Cu(Arg)2]2+ having the NH2 groups coordinated in trans positions to give a tape structure, which then associates to form a sheet structure [104]. The Cu(II)-L-Arg system with a phosphate ion as a counter ion has been reported to give a doubly phosphate-bridged dimer, which is then bound to neighbouring dimers by the guanidinium-carboxylate hydrogen bonds to give a layer [105]. A dipeptide complex, Cu(ArgGlyH-1), was found to form hydrogen bonds between the guanidinium group and the Cu(II)-bound b-carboxylate group of a neighbouring complex molecule, resulting in a zigzag chain [106]. These examples suggest that owing to the guanidinium group, Arg-containing metal systems can be prototypes for molecular recognition and constituents or synthons of supramolecules [107].

1.3.2 Interactions between metal complexes and surrounding groups Intermolecular interactions between metal complexes and non-coordinated molecules or ions may form adducts, which could be regarded as second-sphere coordination. Such interactions may influence the structures and functions of the complexes and/or the surroundings. The metal site of proteins is in the molecular environment produced by the proteins, and the interactions between them are essential for the activity. For these reasons, there is a growing interest in the effects of the microenvironment of metal complexes in chemical and biological systems [17].

1.3.2.1 Adduct formation and its effect Pt(II) complexes such as Pt(phen)(Arg) bind with IA, AQS (Fig. 1.6(a)), GMP (Fig. 1.7), and other aromatic molecules by stacking and hydrogen bonding [81, 82]. FMN (riboflavin 5’-phosphate) is known as the prosthetic group of redox carrier proteins flavodoxins; in the flavodoxin from Anabaena, FMN is noncovalently bound to the protein through its isoalloxazine ring sandwiched between the Tyr phenol and Trp indole rings in addition to hydrogen bonds, and the molecular environment is known to control the semiquinone/hydroquinone redox potential of FMN [108, 109]. FMN was reported to form ternary Cu(II) complexes with DA = bpy or phen, where it is bound to Cu(II) through the phosphate moiety and stacking with DA [110]. The Pt(II) complexes were found to interact with FMN to form 1:1 adducts as seen for AQS with a similar

1.3 Structural and functional characterisation 

 21

three consecutive ring system (Fig. 1.6(a)), and the stability constants (log K values) were determined to be 2.83~3.42 by 1H NMR spectra [81]. Upon adduct formation the redox potential (E1/2) for the two-electron redox processes of FMN exhibited anodic shifts due to the electron density decrease, the shift differences indicating the effects of both stacking and hydrogen bonding. Complex molecules can interact with the other molecules in the second coordination sphere. For example, crown ethers have been shown to surround Ru-ammine complexes in place of solvent molecules [111]; 18-crown-6 and other crown ethers form adducts with the Ru complexes by hydrogen bonds between the oxygen atoms and the coordinated ammine ligands and cause a cathodic shift of the Ru redox potential, which indicates that the electron density of the Ru centre increases due to binding with the oxygen donors. The factors affecting the adduct formation have been studied [112]. In view of the functions of polyamines such as putrescine and spermidine in genetic information transfer processes, Lomozik and collaborators have investigated the complex formations in metal-ligand systems involving polyamines and nucleosides or nucleotides and interactions between metal complexes and noncoordinated molecules or groups. In ternary systems involving amino acids, nucleotides etc., coordinated polyamines have some protonated amine nitrogens, which bind with the other ligand by hydrogen bonds [113, 114] (for details, see Chapter 1.3.3.2).

1.3.2.2 Protein‒small molecule interactions Interactions of metal complexes with biological macromolecules are of current interest in view of the activities of metallodrugs [13–18]. Biological processes of enzyme catalysis and electron transfer require interactions between the enzyme and its substrate and between the electron donor and the acceptor, respectively. Structural studies have been performed, for example, for the enzyme‒substrate model complex of CPA (cf. 2.1) and the electron transfer complexes such as cytochrome c‒cytochrome c peroxidase [9] and amicyanin‒methylamine dehydrogenase‒cytochrome c551i [115], where various interactions, notably electrostatic interactions between basic and acidic amino acid residues and hydrophobic interactions, have been observed within the complexes. Plastocyanin (PC) is a mobile electron transfer protein involved in photosynthesis and accepts an electron from cytochrome f (cyt f) of photosystem II and transfers it to photosystem I [116]. Higher plant and green algae PCs have consecutive acidic amino acid residues at the solvent-accessible site near the Tyr residue (negative patch), while cyt f has a Lys residue-rich site exposed to solvents (positive patch) [117]. These oppositely charged sites are known to be involved in the recognition process. Subtle effects of the interaction between the proteins on the structural and electrochemical properties of Silene pratensis PC [118] have been studied by using positively charged Lys peptides such as tetralysine in place of cyt f at neutral pH (Fig. 1.14) [119, 120]. The absorption spectral changes of PC at around 600 nm caused by pentalysine indicated that the Cu site structure or the Cu‒S(Cys) bond was altered, and from the

22 

 1 Noncovalent interactions in biocomplexes

Cu site NH3+

+ H3N + H3N Tyr83

Negative patch

+ H3N + H 3N

CH C O HN CH O C NH CH C O HN CH COO–

Lysine peptide

Fig. 1.14: Structure of Silene pratensis plastocyanin (PDB code: 1BYO) [118] and schematic presentation of interaction with a lysine peptide [119]. Modified from ref [120].

difference resonance Raman spectrum in the 200–600 cm‒1 region (excitation wavelength, 591.0 nm), several bands at 375–475 cm‒1 related with the Cu‒S bond were found to be slightly shifted to lower frequencies, indicating that the Cu‒S(Cys) bond was weakened by addition of pentalysine. The resonance Raman spectral changes of PC caused by lysine peptides were the same as those by cyt c, which is positively charged, and the fact that the peptides, especially tetra- and pentalysine peptides, competitively inhibited the electron transfer from reduced cyt c to oxidised PC indicates that lysine peptides serve as the PC interacting site models of cyt c, cyt f, etc. The PC‒lysine peptide association constants were found to increase with the peptide length, dilysine < trilysine < tetralysine < pentalysine, showing that PC binds more strongly with longer peptides or peptides with more positive charges. Interestingly, as the Cu‒S(Cys) bond became longer due to the PC‒lysine peptide interaction, the redox potential shifted to a higher value, facilitating the electron transfer from the redox partner to PC. Recently proteins have become important targets for metal complexes and metalbased pharmaceuticals, both for therapeutic and diagnostic purposes [16,  121]. The specificity of enzymes depends on the structural fitness of the substrate to the active site, where the substrate is bound to the enzyme by noncovalent interactions. Transition metal ions have the possibilities of forming diverse structures by complex formation and may fit into the protein’s crucial site, serving as metallodrugs. Interesting studies have been reported by Meggers and collaborators on fitting the structures of inert complexes to the protein kinase active site by mimicking a natural product staurosporine (Fig. 1.15(a)) known as an effective inhibitor of the enzyme [122]. They developed pyridocarbazole ligands resembling staurosporine

1.3 Structural and functional characterisation 

H N

N

O

H N

O

O

NH2

N

O

N

N O

Ru

C

NH2 N

Cl

O

 23

NH (a)

(b)

Fig. 1.15: Structures of staurosporine (a) and an octasporine (b) [122].

and synthesised mixed ligand complexes, which were named as octasporines (Fig. 1.15(b)), and tailored them to fit into the ATP binding site of protein kinases, where the pyridocarbazole moiety occupies the adenine binding site. The inhibitory activities of the complexes thus prepared were studied, and the Ru complex shown in Fig. 1.15(b), for example, has been found to be a selective inhibitor of the a-form of glycogen synthase kinase 3 [123]. The structure of a protein kinase, human 8-oxo-dGPTase, with a bound Ru complex is shown in Fig. 1.16(a), where the complex (Fig. 1.16(b)) interacts with the Lys, Asn, and Asp residues of the kinase by hydrogen bonds in a hydrophobic environment with Phe, Trp, etc. [124]. The studies indicate the importance of steric fitness of the complex to the active site and noncovalent interactions between them. The approach toward site specific binding of small complexes to proteins will be important for the elucidation of the active site structures and functions and developing new metallodrugs and other functional complexes that can pinpoint the target.

H2N Lys 23 Phe 27

Asn 33

CH3

Asp 120 O

N Asp 119 Leu 9

(a)

N

O

Trp 123

C

Ru

N

O

O O

(b)

Fig. 1.16: Structures of protein kinase‒inhibitor complex (PDB code: 3WHW) (a) and the inhibitor (b) [124].

24 

 1 Noncovalent interactions in biocomplexes

1.3.2.3 Interactions involving coordinated ligands at the metal site of proteins Functions of metalloproteins depend on the coordination structure and properties of the central metal ion, donor atoms, and the effect of the molecular environment. Hydrogen bonds and stacking interactions of the donor groups with the secondsphere groups such as the peptide -NHCO- and the side chain groups of amino acid residues can affect the electron density of the coordinating groups and thus the redox properties of the central metal ion. We will see some examples of noncovalent interactions in blue copper proteins and iron-sulfur proteins and their models. Blue copper proteins such as PC [116] (cf. 1.3.2.2.) have a unique Cu coordination structure with two His imidazole nitrogens and Cys thiolate and Met thioether sulfurs. As shown in Fig. 1.12, PC from fern has a Phe residue stacked with the coordinated imidazole and exhibits a higher redox potential than the other PCs from higher plants [94]. Further studies have been reported on the spectroscopic and electrochemical effects of mutation of Achromobacter cycloclastes pseudoazurin at Met16 located close to coordinated His81 to aromatic and aliphatic amino acids [125, 126]. Mutation to Tyr, Trp, and Phe caused shifts of the Cu‒S(Cys) stretching modes to a higher frequency region, indicating that a structural perturbation has occurred to make the Cu‒S(Cys) bond stronger. Comparison of the CysS-to-Cu(II) CT bands and ESR spectra indicated a trigonal disposition of the Cu site. The redox potentials were found to be higher for the mutants with the aromatic amino acids than the wild type and the mutants with aliphatic amino acids [125, 126].

Phe 114

Gly 45

Gly 45 Cu

His 117 NH

His 117

His 46

Met 121 Cys 112

Thr 113

Cu

Mutation

Met 121 NH

NH Pro 114

Asn 47

N

Asn 47 Cys 112

Thr 113

+297 mv (a)

His 46

(b)

+211 mv

Fig. 1.17: Shift of the redox potential (midpoint potential Em) due to loss of a N‒H···S hydrogen bond at the copper site in a blue copper protein azurin. (a) Pseudomonas aeruginosa azurin (PDB code: 4AZU) [127]; (b) Phe114Pro mutant of azurin (PDB code: 2GHZ) [128]. Hydrogen bonds are indicated by broken lines. Reproduced from ref [129] by the courtesy of Asakura Publishing, Tokyo, Japan.

1.3 Structural and functional characterisation 

 25

Asn 112

His 114

Cys 111 His 16

Fe His 41

His 47

IIe 113

Fig. 1.18: NH···S hydrogen bonding in a non-heme iron enzyme superoxide reductase (SOR) from Pyrococcus furiosus (PDB code: 1DQI) [132]. Hydrogen bonds are indicated by broken lines. Reproduced from ref. [129] by the courtesy of Asakura Publishing, Tokyo, Japan.

At the metal centre of proteins, hydrogen bonds between the donor groups and the peptide bonds and/or side chain groups of the protein are known to modulate the redox activities of the metal centre. The Cu sites of a blue copper protein azurin and its mutant are shown in Fig. 1.17 [127, 128]. Wild type azurin has two hydrogen bonds involving coordinated Cys thiolate sulfur and two peptide groups [127], but upon mutation of Phe114 to Pro114 one of the NH moieties was lost, leaving a hydrogen bond with the peptide NH of Asn47 only [128]. This caused a redox potential lowering from 297 mV to 211 mV. A similar shift has been reported for amicyanin, where a hydrogen bond added at the Cu site by mutation caused an anodic shift of the redox potential [130]. Hydrogen bonds around the metal centre have also been known for iron-sulfur [131] and nonheme-iron proteins such as shown in Fig. 1.18 for superoxide reductase (SOR) [132]. Recent detailed ENDOR and DFT studies on a heme enzyme cd1 nitrite reductase indicated dynamic formation of hydrogen bonds between the reduction product nitric oxide bound to the heme and the distal His and Tyr residues [133]. Among the approaches to the iron-sulfur proteins, the iron-sulfur clusters were synthesised by using a Cys-containing peptide ligand incorporating the characteristic CysGlyAla sequence of the metal site and related ligands [134]. Fe4S4(ZCysGlyAlaOMe)4]2‒, which is capable of N‒H···S hydrogen bonding when bound through –S‒, exhibited a higher redox potential than [Fe4S4(ZCysGlyOMe)4]2‒ where hydrogen bonding is not possible. The result was supported by further studies [135, 136].

1.3.3 Close contact between the metal centre and the side chain groups 1.3.3.1 Metal‒aromatic ring interactions Aromatic rings are known to contribute to the protein structure stabilisation [1, 3, 137] and molecular recognition [138–140] and are often located close to the coordination sphere. This is well known for Cu(II) complexes, where the side chain aromatic ring of amino acids and peptides have been shown to be bent over the Cu(II) coordination plane. As shown earlier (cf. 1.2.6.2), the aromatic rings of AA and DA in Cu(DA)(AA)

26 

 1 Noncovalent interactions in biocomplexes

undergo stacking interactions within the complex molecule, but at the same time the side chain aromatic ring is located close above the Cu(II) centre to be within the sum of the van der Waals radii, suggesting electronic interactions between them. Aromatic rings tend to occupy a space above the Cu(II) coordination plane even in the absence of aromatic‒aromatic stacking. In the solid state structure of Cu(II)‒GlyTrp complex [141], deprotonated GlyTrp (GlyTrpH-1) coordinates to Cu(II) as a tridentate ligand in the planar positions with the side chain indole ring of Trp bent over Cu(II) with a rather large dihedral angle of 50°, which is probably due to the planarity of the dipeptide coordination with a deprotonated peptide nitrogen; on the other side of the Cu(II) plane, however, there is the indole ring from a neighbouring molecule at the distance of 3.12 Å and with an angle of 13°. The aromatic ring‒metal ion interaction was also proposed by NMR studies of the Tyr-containing dipeptide complexes of Pd(II) [56] and tripeptide complexes of Ni(II) and Pd(II) [55], and similar Cu(II)aromatic ring contact was established by X-ray analysis of complexes such as Cu(LTyr)2 [142]. These observations indicate that aromatic rings have a tendency to be close to the metal centre possibly due to electrostatic, electronic, and/or hydrophobic interactions. The intramolecular stacking shown in the earlier sections is considered to involve both metal-coordinated ligand‒ligand p‒p (or MLp‒L’p) and metal d‒L’p interactions (Fig. 1.9). The presence of the electronic interaction between the metal ion and the aromatic ring is suggested by the 195Pt NMR shift upon adduct formation with AMP, etc. (Fig. 1.4) considered to be due to delocalisation of the d electrons, and the relatively large enthalpy changes, −SDHo, indicate that the adduct is formed as a result of bonding interactions rather than the entropy effect [61].

Trp 44 5 4 2.86 Å Met 49

NH 2.67 Å Cu Met 47

2.270(5) Å

His 36

(a)

2.228(5) Å Cu N N

N

(b)

Fig. 1.19: (a) Cu(I)‒p interaction in a copper chaperone CusF (PDB code: 2VB2) [148]. Reproduced from ref. [129] by the courtesy of Asakura Publishing, Tokyo, Japan. (b) Cu (I)‒indole h2-bonding with a 3N-donor ligand [154].

1.3 Structural and functional characterisation 

 27

Cation–p interactions are of current interest, and indeed they are well recognised in protein structures, ion channels, and enzyme reactions, where the interactions of alkali metal ions, the Arg guanidinium group, and the trimethylammounium group of acetylcholine have been shown to interact with the aromatic rings of Trp, etc. [143–147]. A striking example of transition metal‒aromatic ring interactions in proteins has been reported for a copper chaperone CusF, where the Cu(I) ion bound with two Met sulfurs and a His imidazole nitrogen interacts with a Trp indole ring located close to the Cu site (Fig. 1.19(a)) [148]. This is the first example of the interaction of the Trp indole ring with a metal ion in biological systems. Figure 1.19(a) shows that the indole C(4)-C(5) moiety of Trp44 is in contact with Cu(I) with rather long distances of 2.67 and 2.86 Å, and the interaction has been assigned to a cation‒p interaction [148, 149]. Subsequent studies indicated that the Cu(I) binding affinity (log K) of CusF is 14.3 ± 0.1 [150] and that the Cu(I)-Trp44 interaction is important for stabilising the complex and protecting Cu(I) from oxidation by water [151, 152].

2.336 Å N

2.211 Å Cu N N Fig. 1.20: d‒p interaction in Cu(I) complexes [155].

On the other hand, Cu(I) is known to form p-type bonds with alkenes [153]. The reaction of Cu(I) with a 3N-donor ligand containing a pendent indole ring has been shown to give a Cu(I) complex, whose X-ray structure revealed that Cu(I) bound to three nitrogen atoms forms an h2-bond with the C(2)‒C(3) moiety as shown in Fig. 1.19(b) [154]. The bond distances between Cu(I) and the carbon atoms (2.228 and 2.270 Å) are longer than those of the Cu(I)‒alkene complexes (1.943–2.028 Å), indicating that the bonds are rather weak; when acetonitrile is added to the solution of the complex dissolved in CH2Cl2, the carbon donors are replaced by acetonitrile with concomitant changes of the Cu(I)-to-indole CT band at 308 nm. In this connection structures and quantitative evaluation of similar h2-bond formation between Cu(I) and a side chain phenyl ring have been reported for ligands containing a p-substituted phenyl ring in place of the indole ring such as shown in Fig. 1.20 [155], where the Cu‒C bond distances were 2.336 and 2.211 Å and comparable with those for the indole ring. These Cu(I)-arene interactions were concluded to consist mainly of the interaction between the Cu(I) dz2 orbital and the phenyl ring p orbital. The cation‒p interactions involving transition metal ions are considered to be somewhat different from those of alkali metal ions. A theoretical study on the Cu(I)‒benzene systems indicated that the electrostatic interaction

28 

 1 Noncovalent interactions in biocomplexes

is not important and that Cu(I) forms an h6 cation‒p complex with the decrease of the Cu(I) 3d electron density as a result of a 3d→p* electron flow, i.e. back donation from Cu(I) d to p* of the aromatic ring [156]. The study also showed that Cu(I) tends to form h2 complexes in the presence of counterions. As shown in Fig. 1.19, the distances for the Cu-indole interaction in CusF are longer than those for the Cu(I)‒indole complex, which suggests that the interaction is a more electrostatic cation‒p interaction rather than d‒p interactions. Thus, depending on the metal ions involved and the distances between the interacting groups, cation‒p interactions may lie somewhere between covalent h-type bonds and electrostatic interactions. A spectroscopic study on Cu(II)‒Trp interactions using GlyAsnHisTrp-NH2 showed the UV and CD spectral changes which were ascribed to the cation‒p interaction, and the most stable structure obtained by molecular mechanics calculations indicated that the Cu(II) ion bound by four nitrogens of the peptide is located above the pyrrole moiety of the indole ring with a distance of 3.85 Å, which supported that the interaction is a cation‒p interaction [157]. The pyrrole NH moiety of the indole ring has a very weak acidity which may be compared with the phenol ring [158], and the metal binding ability of indole by the nitrogen or by the carbon atoms has been reviewed recently [159]. These results suggest further possibilities of Trp‒metal ion interactions in biological systems, for example, in prion‒Cu(II) binding.

1.3.3.2 Interactions involving a hydrogen atom A hydrogen atom from a metal-coordinated ligand has attracted attention due to its ability to interact with an aromatic ring in the second coordination sphere. The active site of metalloproteins is often associated with aromatic rings from aromatic amino acid residues such as Trp and Phe. The hydrogen atom bound to a ligand may gain a positive charge from the metal ion and undergo an effective cation‒p type interaction. By detailed surveys of the crystallographic data of the Protein Data Bank (PDB), Zaric and collaborators revealed the existence of such interactions in metalloproteins and called them “metal ligand aromatic cation‒p (MLACp)” interactions [160]. MLACp interactions will be stronger when the number of bonds from the coordinating atom to the hydrogen atom is smaller. In metalloproteins the distances between the aromatic centroid and the nearest non-hydrogen ligand atom were found to be 3.09—4.41 Å, and the energies of interactions were calculated to be 4—120 kJ/mol [161]. Interestingly the structure of the alcohol dehydrogenase‒ethanol complex indicated the MLACp interaction of the CH2 moiety of ethanol bound to Zn(II) with a Phe residue, which may suggest a possible pathway for the hydride transfer to NAD [160, 161]. Close proximity of an alkyl side chain at the type 1 copper site of laccases and domain 2 of ceruloplasmin may be interesting in view of the influence of the molecular environment on the metal site of proteins. The Cu site of these proteins is formed by

1.3 Structural and functional characterisation 

 29

Leu 329

His 276

CH3 3.71Å Cu

N

N S

Cys 319 Fig. 1.21: Cu···alkyl close contact at the type I Cu site of ceruloplasmin (PDB code: 2J5W) [162]. Reproduced from ref. [129] by the courtesy of Asakura Publishing, Tokyo, Japan.

His 324 (4) (5)

*

(a) (1)

(2) (b)

(3)

5 3

1

*

N

2 4

1

Pd

N

N 3

2 1 Chemical shift/ppm

0

N

Cl [PdCl(Mbu–L)]+

N

3 2

5 4

N

Mbu–L

Fig. 1.22: Behaviour of the branched alkyl side chain in a Pd(II) complex [163]. (a) 1H NMR spectra: (a) ligand Mbu-L; (b) Pd(II)‒Mbu-L complex. (b) Structures of Mbu-L and its Pd(II) complex.

two His imidazoles and a Cys thiolate, and a branched alkyl group of Leu occupies the position above the trigonal plane with the Cu···C distance of 3.71 Å for ceruloplasmin (Fig. 1.21) [162]. It has a very high redox potential as compared with that of the blue copper proteins and oxidases with a 2N2S-donor distorted tetrahedral structure. Although the alkyl side chain is not considered as a ligand to the Cu ion, the rather short Cu···alkyl distance suggests certain noncovalent interactions which could affect the properties of the central ion. As structural models for the active site, Cu(II) and Pd(II) complexes of 3N-donor ligands with a branched alkyl side chain have been synthesised and revealed to have a structure as shown for a Pd(II) complex in Fig. 1.22, where the methyl group (C(4)) is located above the coordination plane with the metal‒C(4) distances of 3.30–3.35 Å [163]. The 1H NMR spectra indicated that the conformation with the methyl group above the Pd(II) plane is maintained in solution, and the downfield shifts of the signals were larger in solvents with a lower dielectric constant. Considering that the ligand side chain is flexible and can be extended outward in less

30 

 1 Noncovalent interactions in biocomplexes

polar solvents, these observations indicate that the metal‒alkyl contact is considered to be a weak bonding interaction of electrostatic nature. A similar Cu‒alkyl contact has been reported for the Cu(II) complex of an ephedrine derivative [164]. The M‒H‒C angles observed for these complexes are greater than 100°, and the interactions may be classified as hydrogen bonds and not agostic bonds [165]. An interesting property probably arising from the interaction is that the Cu redox potentials of the complexes with the Cu‒alkyl contact were more positive than those of the complexes without the contact, which suggests that the electron density is decreased due to the Cu···H‒C interaction [163]. These observations suggest that the Cu sites of laccase, etc., with an alkyl side chain in close proximity, may also have a decreased electron density.

1.4 Concluding remarks Noncovalent interactions have long been recognised in chemistry, but in the past they seem to have been mostly behind the scenes in part due to the difficulty of detection and evaluation. With deeper insights into noncovalent interactions around the metal centre in the field of bioinorganic chemistry, their importance is now well recognised, and without the knowledge of the interactions, it is impossible to fully understand and control chemical and biological reactions. The chemistry of biocomplexes supported by information on noncovalent interactions will play essential roles in elucidation of biological systems involving metal ions.

1.5 Abbreviations A AA Ala AlaTyr AMP AQS Arg ArgAspH˗1 Asn Asp B bpm bpy cat CD Cit

amino acid; bidentate ligand; acidic amino acid aromatic amino acid alanine alanyltyrosine adenosine 5′-monophosphate anthraquinone-2-sulfonate arginine arginylaspartate deprotonated from the peptide NH asparagine aspartate amino acid; bidentate ligand; basic amino acid 2,2′-bipyrimidine 2,2′-bipyridine catechol circular dichroism citrulline

1.5 Abbreviations 

carboxypeptidase A CPA CT charge transfer Cu,Zn-superoxide dismutase Cu,Zn-SOD cyclo-HisHis cyclo-L-histidyl-L-histidine cysteine Cys cysteinylglycylalanine CysGlyAla CySO3H cysteate cyt c cytochrome c cyt f cytochrome f DA aromatic diimine Dba 2,4-diaminobutyrate DFT density functional theory edma ethylenediamine-N-monoacetate en ethylenediamine ENDOR electron-nuclear double resonance ESR electron spin resonance FMN flavin mononucleotide Gln glutamine Glu glutamate GlyAsnHisTrp-NH2 glycylasparaginylhistidyltryptophanamide GlyTrp glycyltryptophan GlyTrpH˗1 GlyTrp deprotonated from the peptide NH GlyTyr glycyltyrosine guanosine 5′-monophosphate GMP His histidine hista histamine HTrp 5-hydroxytryptophan IA indole-3-acetate IMP inosine 5′-monophosphate I2Tyr 3,5-diiodotyrosine I2TyrO I2Tyr with the deprotonated phenol moiety J coupling constant Leu leucine Lys lysine M metal ion mbc m-benzenedicarboxylate Met methionine MTrp N-methyltryptophan NAD nicotinamide adenine dinucleotide NMP nucleotide 5′-monophosphate nuclear magnetic resonance NMR NSFRY asparaginylserylphenylalanylarginyltyrosinamide

 31

32 

 1 Noncovalent interactions in biocomplexes

Orn PC 2,6-pdc,3,5-pdc Phe phen PSer PTyr Ser SOR Thr Trp Tyr TyrGlyGly TyrGlyH˗1 TyrO X XPhe ZCysGlyAlaOMe ZCysGlyOMe

ornithine plastocyanin pyridine-2,6-, pyridine-3,5-dicarboxylates phenylalanine 1,10-phenanthroline phosphoserine phosphotyrosine serine superoxide reductase threonine tryptophan tyrosine tyrosylglycylglycine tyrosylglycine deprotonated from the peptide NH Tyr with the deprotonated phenol moiety side chain group; dianion (X2˗) p-X substituted phenylalanine benzyloxycarbonylcysteinylglycylalanine methyl ester benzyloxycarbonylcysteinylglycinamide

Acknowledgments The author thanks Professor Tatsuo Yajima, Kansai University, and Professor Yuichi Shimazaki, Ibaraki University for assistance with preparation of the figures. Some of the figures were made available by the courtesy of Asakura Publishing Co., Tokyo, Japan, to which the author’s thanks are due.

References [1] Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Molecular biology of the cell. New York, Garland Publishing 1994;89–138. [2] Frieden R. Non-covalent interactions: Key to biological flexibility and specificity. J Chem Educ 1975;52:754–61. [3] Burley SK, Petsko GA. Weakly polar interactions in proteins. Adv Protein Chem 1988;39:125–89. [4] Nishio M. CH/p hydrogen bonds in organic reactions. Tetrahedron 2005;61:6923–50. [5] Zeglis BM, Pierre VC, Barton JK. Metallo-intercalators and metallo-insertors. Chem Commun 2007;4565–79. [6] Terrόn A, Fiol JJ, García-Raso A, Barcelό-Oliver M, Moreno V. Biological recognition patterns implicated by the formation and stability of ternary metal ion complexes of low-molecular-weight formed with amino acid/peptides and nucleobases/nucleosides. Coord Chem Rev 2007;251:1973–86. [7] Lehn J-M. Supramolecular chemistry: Concepts and perspectives. Weinheim, VCH, 1995. [8] Desiraju GR. Supramolecular synthons in crystal engineering a new organic synthesis. Angew Chem Int Ed Engl 1995;34:2311–27.

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[150] Bagchi P, Morgan MT, Bacsa J, Fahrni CJ. Robust affinity standards for Cu(I) biochemistry. J Am Chem Soc 2013;135:18549–59. [151] Loftin IR, Blackburn NJ, McEvoy MM. Tryptophan Cu(I)‒p interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF. J Biol Inorg Chem 2009;14:905–12. [152] Chakravorty DK, Wang B, Ucisik MN, Merz KM, Jr. Insight into the cation‒p interaction at the metal binding site of the copper metallochaperone CusF. J Am Chem Soc 2011;133:19330–3. [153] Thompson JS, Harlow RL, Whitney JF. Copper(I)‒olefin complexes. Support for the proposed role of copper in the ethylene effect in plants. J Am Chem Soc 1983;105:3522–7. [154] Shimazaki Y, Yokoyama H, Yamauchi O. Copper(I) complexes with a proximal aromatic ring: Novel copper‒indole bonding. Angew Chem Int Ed 1999;38:2401–3. [155] Osako T, Tachi Y, Doe M, et al. Quantitative evaluation of d‒p interaction in copper(I) complexes and control of copper(I)‒dioxygen reactivity. Chem Eur J 2004;10:237–46. [156] Zhang SL, Liu L, Fu Y, Guo QX. Cation‒p interactions of Cu+. J Mol Struc: THEOCHEM 2005;757: 37–46. [157] Yorita H, Otomo K, Hiramatsu H, Toyama A, Miura T, Takeuchi H. Evidence for the cation‒p interaction between Cu2+ and tryptophan. J Am Chem Soc 2008;130:15266–87. [158] Shimazaki Y, Yajima T, Yamauchi O. Properties of the indole ring in metal complexes. A comparison with the phenol ring. J Inorg BIochem 2015;148:105–15. [159] Shimazaki Y, Yajima T, Takani M, Yamauchi O. Metal complexes involving indole rings: structures and effects of metal‒indole interactions. Coord Chem Rev 2009;253:479–92. [160] Zarić SD, Popovoć DM, Knapp EW. Metal ligand aromatic cation‒p interactions in metalloproteins: ligands coordinated to metal interact with aromatic residues. Chem Eur J 2000;6, 3935–42. [161] Zarić SD. Metal ligand aromatic cation‒p interactions. Eur J Inorg Chem 2003;2197–209. [162] Lindley PF, Card G, Zaitseva I, Zaitsev V, Reinhammer B, Selin-Lindgren E, Yoshida K. An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity. J Biol Inorg Chem 1997;2:454–63. [163] Yamauchi O, Yajima T, Fujii R, et al. CH···metal(II) axial interaction in planar complexes (metal = Cu, Pd) and implications for possible environmental effects of alkyl groups at biological copper sites. J Inorg Biochem 2008;102:1218–26. [164] Castro M, Cruz J, López-Sandoval H, Barba-Behrens N. On the CH···Cu agostic interaction: chiral copper(II) compounds with ephedrine and pseudoephedrine derivatives. Chem Commun 2005:3779–81. [165] Thakur TS, Desiraju GR. Misassigned C‒H···Cu agostic interaction in a copper(II) ephedrine derivative is actually a weak, multicentered hydrogen bond. Chem Commun 2006:552–4.

Bartosz Tylkowski, Renata Jastrząb and Monika Skrobańska

2 Photo-sensitive complexes based on azobenzene In the last few decades, the development of photo-responsive materials has become an intensive area of research. These substances are intended for the production of “smart chemical systems”, whose properties – and eventually functionality – are controlled by changes in the environment (light irradiation) [1]. These systems have already been implemented in a wide range of modern materials and devices for daily applications such as sunglass lenses, memory devices, photochromic inks, etc. [2]. Light offers unparalleled opportunities as a non-invasive regulatory element for biological applications. First, it shows a great degree of orthogonality toward most elements of chemical and biochemical systems. In contrast to chemicals, which are used for regulating biological processes, photons do not cause contamination of the studied object and have low or negligible toxicity. Second, light can be delivered with very high spatial and temporal precision, which is of paramount importance for controlling the action of bioactive compounds. Finally, light can be regulated in a qualitative and quantitative manner by adjusting wavelength and intensity [3]. Despite the importance and versatility of transition metal complexes, smart photoresponsive examples remain rather unexplored in comparison with the large number of well-known light-triggered organic switches. In principle, photo-responsive metal complexes can be obtained by incorporation of organic photochromic units in the structure of their ligands. These photo-sensitive ligands, rather than acting as conventional spectators that tune the properties of their complexes, transform them into dynamic smart entities able to offer a functional response to an external stimulus. This chapter is divided into two parts. In the first part we discuss the photoisomerisation processes of azobenzene molecules as organic photochromic units, while in the second part we describe selected examples of complexes containing these UVsensitive moieties in their structures.

2.1 Azobenzene Azobenzene (Fig. 2.1) was described for the first time in 1834 [4],  and one century later, in 1937, G. S. Hartley published a study of the influence of light on the configuration of its N N double bonds [5]. Azobenzene-based chromophores are versatile molecules and have received much attention in both fundamental and applied research. The strong electronic absorption maximum can be tailored by ring substitution to fall anywhere from ultraviolet to red-visible regions, allowing chemical fine-tuning of colour. This, combined with the fact that these azo groups are relatively robust and chemically stable, has prompted extensive study of azobenzene-based structures as dyes and colorants. The

42 

H

 2 Photo-sensitive complexes based on azobenzene

N

C Fig. 2.1: The structure of azobenzene.

rigid mesogenic shape of the molecule is well suited for spontaneous organisation into liquid-crystalline (LC) phases, and hence polymers doped or functionalised with azobenzene-based chromophores (azo polymers) are common as LC media. With appropriate electron donor/acceptor ring substitution, the π electron delocalisation of the extended aromatic structure can yield high optical nonlinearity, and azo chromophores have been extensively studied for nonlinear optical applications as well. One of the most interesting properties of these chromophores, however, and the subject of this chapter, is the readily induced and reversible isomerisation about the azo bond between trans and cis geometric isomers and the geometric changes that result when azo chromophores are incorporated into complexes. For purpose of classification of geometrical photoswitching, the nature and behaviour of an azobenzene based chromophore can be well described by four variables: –– electronic absorbance maximum, λmax –– dipole moment, µ –– shape, which can be roughly quantified by the aspect ratio ra –– effective occupied volume, OV. Each of these variables can be controlled synthetically with the introduction of appropriate ring substituents, or in the case of ra also by linking together additional phenyl rings with azo bonds to form dis- and tris-azobenzene dyes. For a wide range of λmax values displayed by various azo chromophores (and hence a wide range of colours and properties) a useful classification scheme was introduced by Rau. Azo aromatic chromophores can be considered to belong to one of three spectral types based on the energetic ordering of their (π*, n) and (π*, π) electronic states as that of azobenzene type, aminoazobenzene type, or pseudo-stilbene type. Azobenzene-type molecules display a low-intensity π* ← n absorption band in the visible region and a high-intensity π* ← π band in the UV one. Ortho- or parasubstitution with an electron-donating group (such as an –NH2 amino) leads to the aminoazo­benzene type where the π* ← n and π* ← π bands are very close or overlapped in violet or near-visible UV due to an increase in the π orbital energy and a decrease in the energy of the π* orbital. This effect is enhanced with the 4 and 4′ position substitution of electron donor and electron acceptor (push/pull) substituents (such as an amino and –NO2 nitro group, respectively), which shifts the π* ← π transition

2.1 Azobenzene 

 43

band toward the red (past that of the π* ← n) to assume a reverse order, and places the molecule in the pseudo-stilbene spectral class. Key to some of the most interesting applications of azobenzene complexes is the readily induced and reversible isomerisation about the azo bond between trans (E) and cis (Z) geometrical isomers, which can be interconverted by light and heat (Fig. 2.2). 5.5 Å

UV light blue light or heat

9Å

8Å

8Å

Fig. 2.2: Trans (left) and cis (right) geometric isomers of azobenzene.

The trans-isomer can be photoisomerised to the cis-isomer, which can be converted back to the trans form again either photochemically or thermally. As shown in Fig. 2.2, the geometrical change associated with trans- to cis-isomerisation of azobenzenes is significant, and can be used to destroy or rearrange the order in a wide variety of organised media. The conversion from trans- to cis-azobenzene decreases the distance between the 4 and 4′ ring positions from 9.0 to 5.5 Å [6], increases the average freevolume requirement [7], and can produce a substantial change in many of the observable properties of azo-containing systems. The isomerisation is completely reversible, and free from side reactions. This applies to azobenzenes in solution, liquid crystals, sol-gel systems, monolayer films, dispersed in polymers, and bound in polymers, though with marked differences in kinetics and quantum yields. It is important to note that the composition of the photostationary state, the equilibrium state of the three conversion processes under irradiation, is unique to each system and can depend on irradiation intensity, temperature, quantum yields, free volume, and substituents. The trans form of azobenzene is more stable with a difference in ground-state energies of cis and trans of 50 kJ/mol. Unlike the planar trans form, cis-azobenzene assumes a geometry with the phenyl rings twisted at right angles to the C–N N–C plane. For purposes of azobenzene isomerisation, the nature of the light that is used to induce isomerisation can be defined simply by three properties: –– wavelength –– intensity –– polarisation.

44 

 2 Photo-sensitive complexes based on azobenzene

The irradiation used in most experimental work with azo polymers can be generated by low-power gas lasers (such as Ar+ or HeNe) or solid-state (such as YAG or GaAs) lasers, as they produce light that is of more than sufficient intensity and of the appropriate frequency. The wavelengths of light produced by these readily available lasers can address usual λmax values of a wide range of substituted azo chromophores, from the UV (Ar+ line at 350 nm), through blue (Ar+ line at 488 nm) and green (Ar+, YAG, HeNe lines at 514, 532, 545 nm), to the red (HeNe, GaAs lines at 633, 675 nm). The rates and extent of isomerisation of azobenzenes depend on the irradiation intensity, the quantum yields for the two processes (Φtrans and Φcis), and the rate constant k, which governs the thermal relaxation from the cis back to the trans form. The net effect of these competing processes can be summarised by the cis concentration [cis] in the photostationary state (PSS), representing the extent of isomeric conversion achieved under irradiation. In general, k for the spectral classes of azobenzene, aminoazobenzene, and pseudo-stilbene is observed to be of the order of hours, minutes, and seconds, respectively, and the isomeric ratio in the PSS is observed to be predominantly cis, near unity, and predominantly trans, respectively, under usual irradiation levels. In the dark, [cis] usually lies below the limit of detection, and the system can be considered to be composed of 100% trans molecules. The mechanism of isomerisation of azobenzene is still unclear. Early suggestions of a rotation about the –N N– double bond axis were in disagreement with later work suggesting an inversion mechanism through a linear transition state, though further work with substituent dependencies of thermal relaxation again suggested a rotation mechanism. The range of results suggests that there may not be one general mechanism, but a competition between the two, depending on the spectral class of the chromophore and the local environment [8–12]. A combination of these remarkable photoswitching properties of azobenzene with the electrochemical, magnetic, catalytic, or biological properties of the metal complexes could give rise to multifunctional molecules. In many of these metal species, the ligand is chelated to the metal through one of the nitrogen atoms and either a second nitrogen donor substituent or a carbon atom, so that the photoisomerisation of the azo group is not possible [13]. Thanks to the light-induced interconversion, the systems incorporating azobenzenes can be used as photoswitches, effecting rapid and reversible control over a variety of chemical, mechanical, electronic, and optical properties [14]. According to Professor Han [15], light-responsive metal complexes are considered potential candidates for applications in sensors, molecular motors, optical data storage, and medicine.

2.2 UV-sensitive complexes 2.2.1 Azobenzene with nickel (Ni) Azobenzene–metal complexes are considered potential candidates for applications in medicine, especially in magnetic resonance imaging (MRI). MRI is one of

2.2 UV-sensitive complexes 

 45

the most important noninvasive tools in diagnostic medicine. As opposed to other deep tissue imaging modalities such as computer tomography (CT) or positron emission spectroscopy (PET), no ionising radiation is used in MRI examinations, and no radiation damage is induced. To date, >200 million doses of MRI contrast agents (CAs) have been administered to patients worldwide [16].  Commercially available 7 CAs are mainly gadolinium(III) chelate complexes [17]. With a spin of S = 2 , these molecules are highly paramagnetic and decrease the NMR relaxation time of surrounding water protons (or other NMR-active nuclei), which in turn leads to signal enhancement in MRI. The majority of clinically used Gd(III) chelates are strongly hydrophilic; therefore, after intravenous injection, the complexes stay mainly in the blood circuit, leading to high contrast of blood vessels. Since the MRI signal enhancement correlates with the concentration of CAs, they primarily increase anatomical contrast. Further physiological information could be obtained by using responsive or “smart” CAs whose relaxivity (capability of reducing the relaxation time of surrounding nuclei) is controlled by metabolic parameters. The design of responsive CAs reporting on parameters such as temperature, pH, or biochemical markers is a subject of intensive research because they are potentially capable of visualising the site of a disease in a magnetic resonance image. Research in this field started in the mid-1990s. Most of the approaches since then have been based on Gd(III) complexes whose relaxivity is controlled by controlling water coordination to the Gd3+ ion, which is the most efficient relaxation mechanism. A number of CAs have been developed that respond to proteins and enzymes, carbohydrates, pH values, and ions like Ca2+, Zn2+, Cu+/2+, and K+ [18]. One of the first studies on photo-sensitive CAs published by Tu et al. [19] concerning functionalisation of Gd(III) chelates with photochromic spiropyrans gave rise to relaxivity changes of ~20%. Whereas a Gd complex in the “off” state, with a completely filled coordination sphere blocking water access, still exhibits a residual relaxivity by outer-sphere relaxation (through-space magnetic dipole interaction), a diamagnetic transition metal complex with  S  = 0 is completely MRI silent. Thus, spin state switching offers the potential to achieve a higher efficiency in relaxivity control. Contrast switching is very important in interventional radiology (catheter-based surgery under imaging control). The change in contrast so far has been obtained by administering additional CAs each time it has been required. After multiple injections, the CAs accumulate in the bloodstream to a level at which they are harmful, and the contrast change is gradually lost. The advantage of light-sensitive CAs is that they need to be administered only once and the contrast can be switched rapidly via an optical fiber. A breakthrough discovery in this field has been made by Prof. Herges from the Otto-Diels-Institut für Organische Chemie, Christian-Albrechts-Universität, Germany. His group has developed a highly efficient, light-responsive molecular magnetic switch using azobenzene-Ni complexes, as shown in Fig. 2.3 [20]. As presented in Fig. 2.3, green (500 nm) and violet-blue (435 nm) light were used to switch the relaxation time of solvent protons in a 3 mM solution by a factor of >2,

46 

 2 Photo-sensitive complexes based on azobenzene

430 nm

500 nm N

N=N N

=

N N

N N NiNN R

R N Ni N

N

N R

R

H2O 430 nm

500 nm

R

Fig. 2.3: Azobenzene-Ni complexes for magnetic resonance imaging. Reprinted with permission from [20]. Copyright (2015) American Chemical Society.

and the relaxivity (R1) of the contrast agent changes by a factor up to 6.7. The change in ­contrast was clearly visible in a clinical MRI scanner. Contrast control was based on a cascade of events that includes photoisomerisation of an azo ligand, coordination change at Ni2+, spin switch, and MRI contrast change. The system was optimised in such a way that each step was close to quantitative. No side reaction or fatigue was detected after >100,000 switching cycles. The metastable cis form (contrast “on” state) has a half-life of >1 year at room temperature. Prof. Herges’s light-driven coordinationinduced spin state switch approach has the potential to provide the basis for the development of a number of interesting applications, including the design of temperature- or pH-responsive contrast agents for MRI. The latter would be useful to detect tumours because they exhibit a higher temperature and a lower pH than surrounding tissue. 2.2.2 Azobenzene with platinum (Pt) and palladium (Pd) As mentioned in the introduction section, extensive studies have led to general categorisation of azobenzene derivatives into three types, namely azobenzene (AB), aminoazobenzene (aAB), and pseudostilbene types (pAB), on the basis of the relative energetic order of their n−π* and π−π* transition bands. The materials of pABand aAB-type, obtained through attachment of electron-accepting and/or – donating moieties to the parent AB, display the desired red-shifted photoexcitation energies for isomerisation in comparison to the AB-type materials. However, both aAB and pAB exhibit significantly increased rates of thermal cis to trans isomerisation, which may limit their use in applications that require longer lived cis isomers [21]. Even though the AB family as a whole offers a wide span of photoexcitation energies and cis thermal stabilities, there is still a major challenge to develop new synthetic approaches that would allow forward and back isomerisation of AB with visible light and thermal stabilities of the corresponding cis isomers within a wide

2.2 UV-sensitive complexes 

 47

range of time scales. Among several synthetic strategies employed to control the photochemical properties given above, azo-conjugated transition-metal complexes have been shown to provide new advanced molecular functions via combinations of the remarkable photoswitching properties of AB and the magnetic, electrochemical, or coordination properties of the transition metal complexes [22–24]. For this reason the research group of Prof. Mirkin from the Department of Chemistry and International Institute for Nanotechnology, Northwestern University in Evanston, USA has been particularly interested in exploiting the photochemistry of AB moieties in the context of the weak-link approach (WLA), a coordination-chemistry-based method for the construction of allosterically controlled supramolecular complexes. This approach allows modulation of the structure and properties of functional moieties embedded into the supramolecular architecture via coordination chemistry at distal sites, and it has been extensively employed in catalytic switches, chemical sensors, and signal amplification applications.  Prof. Mirkin and his group envisioned that AB could bestow WLA systems with multiple new functionalities, since its photoisomerisation can allow the toggling of properties intrinsic to the behaviour of catalytic switches, such as reactive cavity sizes and substrate binding affinities. Furthermore, they hypothesised that AB could provide signaling for coordination changes that directly relate to the activity of a WLA system through the modulation of AB’s electronics upon changes in coordination to the structural regulatory centers. Recently, the authors published a paper that presented results concerning the synthesis and study of a series of d8 transition-metal (Pt(II) and Pd(II)) coordination complexes incorporating phosphinefunctionalised aminoazobenzene derivatives as hemilabile phosphino−amine (P, N) ligands as model WLA photoresponsive constructs (Fig. 2.4) [25]. The group of Prof. Mirkin has found that the optical and photochemical properties of these complexes are highly influenced by various tunable parameters in WLA systems, which include the type of metal, coordination mode, type of ancillary ligand, solvent, and outer-sphere counteranions. The authors conclude that in dichloromethane, reversible chelation and partial displacement of the P, N coordinating moieties allow the toggling between aminoazobenzene- or pseudostilbene- and azobenzene-type derivatives. The reversible switching between electronic states of azobenzene can be controlled through either addition or extraction of chloride counterions and is readily visualised in the separation between π−π* and n−π* bands in the complexes’ electronic spectra. In acetonitrile solution, the WLA variables inherent to semiopen complexes have a significant impact on the half-lives of the corresponding cis isomers, allowing tuning of their half-lives from 20 to 21000 s, while maintaining photoisomerisation behaviours with visible light. Therefore, one can significantly increase the thermal stability of a cis-aminoazobenzene derivative to the extent that single crystals for X-ray diffraction analysis can be grown for the first time, uncovering an unprecedented edge-to-face arrangement of the phenyl rings in the cis isomer. Overall, the azobenzene-functionalised model complexes shed light on the design parameters relevant for photocontrolled WLA molecular switches, as well

48 

 2 Photo-sensitive complexes based on azobenzene

N

N

Ph2P

N

+

R1

Cl Ph2P

M

Cl S–R2

1, R1 = H 2, R1 = CN

CH2Cl2 or NaBF4, CH2Cl2

R1

N

+ X– N

N

Ph2P Ph2P

AgBF4, CH2Cl2

M

Cl S–R2

3, R1 = H, R2 = CH3, M = Pt, X– = Cl– 4, R1 = H, R2 = CH3, M = Pt, X– = BF4– 5, R1 = H, R2 = CH3, M = Pd, X– = BF4– 6, R1 = H, R2 = Ph, M = Pt, X– = BF4– 7, R1 = CN, R2 = CH3, M = Pt, X– = Cl– 8, R1 = CN, R2 = CH3, M = Pt, X– = BF4–

N(n-Bu)4Cl, CH2Cl2

+ 2 2BF4– Ph2P Ph2P

M

Cl S–R2

N

N

N

9, R1 = H, R2 = CH3, M = Pt 10, R1 = H, R2 = Ph, M = Pt 11, R1 = H, R2 = CH3, M = Pd 12, R1 = H, R2 = CH3, M = Pt

Fig. 2.4: Synthetic Routes to Aminoazobenzene-Conjugated WLA Complexes, Reprinted with permission from [25]. Copyright (2013) American Chemical Society.

as offer new ways of tuning the properties of azobenzene-based, photoresponsive materials. Palladium complexes with azobenzene moieties as photo-sensitive ligands have also been a subject of study of Prof. Han from Nagoya University in Japan. Azobenzene-based palladium(II) complexes have been extensively studied because of their widespread applications in catalysis, organic and organometallic synthesis, optoelectronic devices, and in the design of a new class of metallomesogens. On reaction with readily available palladium, platinum, and a variety of other transition metals, azobenzene frequently undergoes cyclometalation at the ortho carbon of the phenyl ring along with the lone pair of a nitrogen of the azo group. Recent investigation has suggested that cyclopalladated azobenzene complexes derive their novel liquid crystalline, electrochemical, photoconducting, and fluorescence characteristics from interactions between transition

2.2 UV-sensitive complexes 

 49

metal ions and azobenzenes. However, once a cyclopalladation reaction occurs between a central metal ion and photoisomerisable azobenzene ligands, azobenzene becomes a rigid trans-blocked ligand and does not show reversible conformation changes in response to light wavelength. One way to overcome this drawback is to obstruct facile cyclopalladation at the ortho carbon of the phenyl ring and to synthesise stable mononuclear palladium complexes through only one N:/Pd s-bond [15]. For this reason, Prof. Han and his group has decided to design a strategy for stable light-sensitisation of palladium complexes, and to describe the important determinants of stabilising/destabilising azobenzene-based palladium complexes capable of undergoing repeated light-triggered conformation changes. Experimental results presented by the authors [15] suggest that unusually distorted trans-azobenzene is hardly influenced by the complexation reaction which requires considerable distortion of the azobenzene unit, thus stabilising mononuclear palladium complexes. In nonpolar solvents, these complexes underwent repeated conformation changes under alternating UV and visible light irradiation. However, in polar solvents, the UV-triggered conformation change was accompanied by facile light-assisted breaking of the N:/Pd bond. Even dark incubation in polar organic solvents caused the dissociation of azobenzene ligands from the complexes. The same authors have discovered that the breaking rate of the N:/Pd bond increased in the order of benzene z dichloromethane < acetone < DMF, with more polar solvents inducing faster dissociation. The results obtained clearly show that the solvent polarity effect on the stability of azobenzene-based complexes can be interpreted in terms of the degree of polarisation of the metal–ligand bond formed as a consequence of interactions between the palladium ion as a soft acid and azobenzene nitrogen as a hard base.

2.2.3 Azobenzene with cobalt (Co) The group of Prof. Nishihara from the University of Tokyo, Japan, has investigated the photochemical properties of azobenzene-bound bipyridine and terpyridine metal complexes [26, 27]. Generated results show that tris(bipyridine)cobalt complexes exhibit a number of interesting behaviours, including: (1) a simple electronic structure exhibiting only weak ð-ð* absorption bands in the UV region and very weak d-d* absorption in the visible region, which could simplify photochemical investigations; and (2) the reversible redox behaviour of cobalt bipyridine complexes, which yields electron-related phenomena of interest, including light induced electron transfer. The same authors have found that the Co-(III) complexes [Co(pAB)3](BF4)3 {pAB) 4-[4-(4-tolylazo)-phenyl]-2,2ʹ-bipyridine and [Co(mAB)3](BF4)3 mAB)4-[3″-(4‴tolylazo)phenyl]-2,2ʹ-bipyridine significantly prevent formation of the cis isomer by UV light irradiation, whereas the corresponding Co(II) complexes do not [28].

50 

 2 Photo-sensitive complexes based on azobenzene

­ ccording to the investigators, the achieved result could be applied to realise the A reversible trans-cis photoisomerisation of azobenzene moieties using a single UV light source and the CoIII/CoII redox reaction (Fig. 2.5).

CoII

N=N

N=N CoII

Single light source (UV light)

Reduction

CoIII

hv

Oxidation

N=N

N=N hv

CoIII

Fig. 2.5: Reversible isomerisation using a single light source and redox reaction. Reprinted with permission from [28]. Copyright (2005) American Chemical Society.

Furthermore the group of Prof. Nishihara has investigated photoisomerisation properties of tris(bipyridine)cobalt complexes containing six or three azoben­ zene moieties (Fig. 2.6) [29], namely, [CoII(dmAB)3](BF4)2 dmAB) 4,4ʹ-bis[3″-(4‴tolylazo)phenyl]-2,2ʹ-bipyridine, [CoIII(dmAB)3](BF4)3, [CoII-(mAB)3](BF4)2 mAB) 4-[3″-(4‴-tolylazo)phenyl]-2,2ʹ-bipyridine, and [CoIII(dmAB)3](BF4)3, derived from the effect of gathering azobenzenes in one molecule and the effect of the cobalt(II) or cobalt(III) ion using UV-vis absorption spectroscopy, femtosecond transient spectroscopy, and 1H NMR spectroscopy. Under this study, in the photostationary state of these four complexes, nearly 50% of the trans-azobenzene moieties of the Co(II) complexes were converted to the cis isomer, and nearly 10% of the trans-azobenzene moieties of the Co(III) complexes isomerised to the cis isomer, implying that the cis isomer ratio in the photostationary state upon irradiation at 365 nm is controlled not by the number of azobenzene moieties in one molecule, but rather by the oxidation state of the cobalt ions. The femto­second transient absorption spectra of the ligands and the complexes suggested that the photoexcited states of the azobenzene moieties in the Co(III) complexes were strongly deactivated by electron transfer from the azobenzene moiety to the cobalt center to form an azobenzene radical cation and a Co(II) center. The cooperation among the photochemical structural changes of six azobenzene moieties in [CoII(dmAB)3](BF4)2 was investigated with 1H NMR spectroscopy. The time-course change in the 1H NMR signals of the methyl protons indicated that each azobenzene

2.2 UV-sensitive complexes 

 51

n+ N N

N N N N

N N

N mAB

N

N Co N N N

N N

N N N

NN

N

dmAB [Co(pAB)3]n+ (n = 2,3) N N N N

N

N

N Co N

n+

N N

N

N N

N

N Co N

N N

N N

NN

[Co(mAB)3]n+ (n = 2,3)

N N

n+

N N

N N

NN

[Co(dmAB)3]n+ (n = 2,3)

Fig. 2.6: Azobenzene-bound tris(bipyridine) cobalt complexes. Reprinted with permission from [29]. Copyright (2005) American Chemical Society.

moiety in [CoII(dmAB)3](BF4)2 isomerised to a cis isomer with a random probability of 50% and without interactions between the azobenzene moieties as shown in Fig. 2.7. Very interesting results have been presented by Wang et al. [30]. They showed that by using trimethylphosphine-supported organocobalt compounds in low oxidation states, they were able to activate the C−H bond with an azo N atom and phenoxy O atom as anchoring groups through cyclometalatio and obtain the aniline derivatives, shown in Fig. 2.8, as a result of the N N cleavage. New dimeric η2-diyne complexes of cobalt, linked through an azobenzene ligand, presented in Fig. 2.9, have been a subject of investigation of Moreno and co-workers [13]. The authors synthesised cobalt-azobenzene complexes via c­ oupling reactions of 4,4′-diiodoazobenzene with excess trimethylsilylacetylene (TMSA)

52 

 2 Photo-sensitive complexes based on azobenzene

n+

n+ N N

N N

N N N Co N

N N

Co(II)

N N N Co N

N N N N

Co(III)

N N

N N

N N

N N

N N

N N

N N

N N

N N

Under UV light

Fig. 2.7: Photoisomerisation of cobalt complexes investigated by Yamaguchi et al. [29]. Reprinted with permission from [29]. Copyright (2005) American Chemical Society.

OH Me

O

N N 1, 2

+ Co(PMe3)3Cl R

Me

N N

PMe3 Cl Co PMe3

R R = Me Br 1 2 3 4

NH2 • R 3, 4 (3)

Fig. 2.8: Reactions of 2-(4′-R-phenylazo)-4-methylphenols (R = Me (1), Br (2)) with Co(PMe3)3Cl afford two organocobalt(III) complexes, Co(PMe3)2Cl(Me(C6H3O∩N NC6H3R)·(H2NC6H4R)) (R = Me (3), Br (4)), whereas the reactions of 1 and 2 with Co(PMe3)4 and Co(PMe3)4Me afford the dinuclear complex [Co2(PMe3)4(MeC6H3O∩NH)2] with the cleavage of N N bond. Reprinted with permission from [30]. Copyright (2008) American Chemical Society.

and 1-decyne, respectively, under Sonogashira coupling conditions. Under this study, para-Alkynyl azobenzene derivatives (R-Azo-R) and their organometallic cobalt complexes linked to the azobenzene unit by a π-conjugated carbon bridge were synthesised in satisfactory yields by direct reaction between Co2(CO)8 and the organic ligands. Complexes containing Co2(CO)4(L-L) have been obtained by substitution reaction of carbonyl ligands, in the presence of Me3NO. Figure 2.10 shows a polarising optical photomicrograph of the complex obtained by Moreno et al. at 37 °C on cooling from the isotropic liquid. The results obtained clarified the scientific knowledge about the role of   transsubstituents, and the mesomorphic behaviour of several para-alkynyl azobenzene derivatives (R-Azo-R) and their organometallic cobalt complexes linked to the azobenzene unit by a π-conjugated carbon bridge.

2.2 UV-sensitive complexes 

 53

Fig. 2.9: Dimeric η2-diyne complexes of cobalt linked through an azobenzene ligand. Reprinted with permission from [13]. Copyright (2015) American Chemical Society.

Fig. 2.10: Polarising optical photomicrograph (20×) of the complex obtained by the at 37 °C on cooling from the isotropic liquid. Reprinted with permission from [13]. Copyright (2015) American Chemical Society.

Research groups of Prof. Itoh from Japan [31] and Prof. Shumelyuk from Ukraine [32] have performed intensive studies in order to understand the influence of cobaltazobenzene complexes on the composite films behaviour. In particular, the group of Prof. Itoh has investigated polarised spectra of hybrid materials of chiral Schiff base cobalt(II), nickel(II), copper(II), and zinc(II) complexes and photochromic azobenzenes in polymethylmethacrylate (PMMA) cast films, while the group of Prof. Shumelyuk has recorded holograms in films of a 4-methacroyloxy-(4-carboxy-3-hydroxy)-2chloroazobenzene polycomplex with cobalt, for parallel and orthogonal orientation of the light beam polarisation. Prof. Itoh’s group studied a series of hybrid materials in the aspect of photo-tuning of optical anisotropy and conformational changes caused by the so-called Weigert’s effect (merely polarised light induces optical anisotropy in azo-compounds) accompanying cis–trans photoisomerisation, by means of alternate irradiation of UV or visible polarised light. In order to confirm certain intermolecular interactions, these authors used polarised light irradiation and polarised absorption spectroscopy to generate or observe molecular arrangement. This approach was one of several attempts to design multi-input and multi-output digital logic circuits by using conventional organic–inorganic hybrid materials and spatial information.

54 

 2 Photo-sensitive complexes based on azobenzene

The results have provided evidence that a dichroism of polarised spectra could be observed for the cis-form of azobenzene but not for the trans-form of azobenzene. The group of Prof. Shumelyuk, based on the results obtained, hypothesised that the information-related characteristics of the recording medium can be controlled by external electric or magnetic fields, owing to the presence of magnetic metal ions in the composition of the medium.

2.2.4 Azobenzene with manganese (Mn), rhenium (Re) and ruthenium (Ru) Although the toxicity of carbon monoxide is well established (it is often referred to as the silent killer) [33], the salutary effects of this diatomic molecule have only recently been recognised [34, 35]. CO is endogenously produced through the catabolism of heme by the enzyme heme oxygenase (HO) [36]. Surprisingly, at low concentrations, CO imparts significant anti-inflammatory and antiapoptotic effects in mammalian physiology through various pathways [34]. CO also provides protection from myocardial infraction [37] and has been employed during pretreatment in procedures of organ transplantation and preservation [38]. Despite such beneficial roles of CO in different therapeutic settings, difficulties in handling this toxic gaseous molecule loom as a major concern in CO therapy [39]. To evade such impediments, researchers have undertaken initiatives to synthesise various transition metal carbonyl complexes for use as pro-drugs, which eventually deliver CO to biological targets [40]. Many of these carbonyl complexes, commonly known as CORMs (carbon monoxide releasing molecules), undergo solvent-assisted release of CO and thus serve as agents for controlled CO delivery. However, such CORMs often suffer from poor solubility in biological systems, poor stability under aerobic conditions, or short half-lives. These drawbacks called for an alternative trigger for CO release from CORMs in addition to achieve better stability in biological milieu. Because further control of such CO delivery through light-triggering can be achieved with photoactive metal carbonyl complexes (photoCORMs), over the past few years much effort has been directed to isolating such complexes. Typical metal carbonyl complexes release CO when exposed to UV light, a fact that often deters their use in biological systems. A significant contribution to photoCORMs has been brought by a research group of Prof. Mascharak from the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, United States. The group has focused its investigation on identifying the design principles that could lead to photoCORMs that would release CO upon illumination with low-power (5−15 mW/cm2) visible and near-IR light. Prof. Mascharak [41] and his co-workers have used Mn(I) center to ensure overall stability of the carbonyl complexes as shown in Fig. 2.11. Prof. Mascharak [41] and his co-workers have hypothesised that transfer of electron density from the electron-rich metal centers to π* MOs of the ligand frame

2.2 UV-sensitive complexes 

 55

14000

CO OC

Mn

OC

N= N

Br

N

ε (M-1 cm-1)

12000 10000 8000 6000 4000 2000 0 300

400

600 500 Wavelength (nm)

700

Fig. 2.11: Azobenzene−Mn complex studied in [41]. Reprinted with permission from [41]. Copyright (2014) American Chemical Society.

via strong metal-to-ligand charge transfer (MLCT) transitions in the visible/near-IR region would weaken metal−CO back-bonding and promote rapid CO photorelease. This expectation has been realised in a series of carbonyl complexes derived from a variety of designed ligands in combination with a smart choice of ligand/ coligand. Several principles have emerged from their systematic approach to the design of principal ligands and the choice of auxiliary ligands (in addition to the number of CO) in synthesising these photoCORMs. In each case, density functional theory (DFT) and time-dependent DFT (TDDFT) study afforded insight into the dependence of the CO photorelease from a particular photoCORM on the light wavelength. Results of these theoretical studies indicate that extended conjugation in the principal ligand frames as well as the nature of donor groups lower the energy of the lowest unoccupied MOs (LUMOs), while auxiliary ligands like PPh3 and Br− modulate the energy of the occupied orbitals depending on their strong σ- or π-donating abilities. As a consequence, the ligand/coligand combination dictates the energy of MLCT bands of the resulting carbonyl complexes. The rate of CO photorelease can be altered further by proper disposition of the coligands in the coordination sphere to initiate transeffect or alter the extent of π back-bonding in the metal−CO bonds. Addition of more CO ligands blue shift the MLCT bands, while intersystem crossing impedes labilisation of metal−CO bonds in several Re(I) and Ru(II) carbonyl complexes. These authors anticipate that their design principles will provide help in the future design of photoCORMs that could eventually find use in clinical studies.

2.2.5 Azobenzene with ferro (Fe) and zinc (Zn) In recent years, a large variety of synthetic molecular machines have been published [42]. The design of artificial molecular machines often takes inspiration from the macroscopic world [43]. The huge challenge has been to construct a single molecule that shows

56 

 2 Photo-sensitive complexes based on azobenzene

mechanical motion caused by external stimulation resembling the movement of its macroscopic analogue. These attempts have yielded analogues of rotors [44], gears [45], (4) clutches [46], shuttles [47], ratchets [48], elevators [49], and muscles [50]. Angular motion of the cyclopentadienyl rings about the metal center in ferrocene has been used for developing molecular machines [51]. Aida and co-workers have reported chiral molecular scissors (Fig. 2.12) that performed a light-induced open−close motion [52].

hν hν’ Closed

Open

Fig. 2.12: Molecular structures of trans-1 (left) and cis-1 (right) azobenzene isomers, optimised with DFT calculation (B3LYP/3-21G*), and schematic representation of its open−close motion induced by photoisomerisation of the azobenzene unit scissors. Reprinted with permission from [52]. Copyright (2003) American Chemical Society.

N N

Fe

(a)

(b)

(c)

Fig. 2.13: Photo-sensitive complex synthesised by the authors of [52]. Reprinted with permission from [52]. Copyright (2003) American Chemical Society.

Figure 2.13 shows the photo-sensitive complex synthesised by Aida and co-workers­. The complex consists of two phenyl groups (a) as the blade moieties, a ferrocene unit (b) as the pivot part, and two phenylene groups (c) as the handle parts, which are linked together by an azobenzene unit. In its native state, the azobenzene adopts a trans-configuration which keeps the blades in a “closed” state. Upon irradiation with UV light (λ = 350 nm), the trans-azobenzene is converted to the cis-isomer, thereby inducing an angular motion of the ferrocene unit, which opens the blade moieties. Subsequent irradiation with visible light (λ > 400 nm) results in the cis- to trans-isomerisation, which closes the blades. Later investigation has established that, upon changing the oxidation state of the ferrocene, the reversible open−close motion of molecular scissors could be actuated only by UV light [53]. Subsequently, the Aida group devised a signal transmission system (Fig.  2.14) consisting of three different movable components: a chiral “scissoring” unit (3*; red),

2.2 UV-sensitive complexes 

Ar

ZnPshort Ar

Ar

Ar N

H N

N H

N Zn

N

N HN N H N

N N Zn N

N

N N N Zn N

Zn N

N

Ar

N S S

Ar

N N

N Ar

Ar N

N

N

Ar

Fe

ZnPlong

N

Ar

 57

N

Ar Ar

3*

2

1Open

Fig. 2.14: Schematic illustration of the expected ternary complex of 1open, 2, and 3* (Ar: 3, 5-dioctyloxyphenyl). Reprinted with permission from [54]. Copyright (2008) American Chemical Society.

an intermediate “bridging” unit (2; blue/purple), and a photochromic “signalling” unit (1open; green) [54]. These components were mechanically interconnected through coordinative interactions. Signaling unit 1open is a pyridine-appended­dithienylethene (DTE) derivative, and can undergo switching between open and closed forms through irradiation. Scissoring component 3* involves a chiral tetrasubstituted ferrocene core bearing two pyridyl groups, capable of c­ oordinating to the zinc porphyrin handles of bridging module 2. Bridging module 3* is a biaryl derivative bearing two sets of zinc porphyrin handles. Upon irradiation, 1open undergoes an opening or closing motion. As the bridging module is coordinated to the signaling unit, the opening/closing motion induced an angular motion in the bridging unit. This rotary motion is translated into a scissoring motion of 3*. As in the previous examples, the scissoring motion was monitored through CD measurements. Subsequently, Aida and co-workers attached zinc porphyrin units to the blades, which provided a binding site for guest molecules wherein bidentate

58 

 2 Photo-sensitive complexes based on azobenzene

ligands were found to coordinate in a 1:1 fashion with a high binding affinity [55]. Reversible photoisomerisation of the azobenzene strap in response to irradiation with UV and visible light induced a scissor- or pedal-like conformational change of the zinc porphyrins, which was translated into a twisting motion of the rotary guest repeatedly in clockwise and counterclockwise directions. The rotary motion of the bound guest was monitored through circular dichroism (CD) spectroscopy. In isolation, the guest is not optically (CD) active because of the free rotation about the C−C bond connecting the two bicyclic rings. However, upon binding to the host, the guest loses this freedom and becomes optically active. Upon irradiation with UV light, the CD intensity decreased, which suggested that the transto-cis isomerisation of the azobenzene induced a conformational change (twist) in the guest. Viau and co-workers have studied a photo-sensitive star-shaped polymer containing complexes based on azobenzene molecules [56]. First, the authors designed a new type of 4,4′-bis- (styryl)-2,2′-bipyridine functionalised by a dialkylamino-azobenzene group. This ligand allowed them to prepare photoisomerisable octupolar tris(bipyridyl)zinc(II) complexes and the corresponding star-shaped polymer by atom transfer radical polymerisation (ATRP) of methyl methacrylate (MMA) showed in Fig. 2.15. Authors reported the photoisomerisation properties of such new metallochromophores and presented for the first time the macroscopic molecular orientation of the corresponding doped and star-shaped non-linearly oriented polymer films [56]. The results indicated a possibility to generate a new class of nonlinear quadratic materials capable of answering the request for stability and efficiency in conjunction with the symmetry control. Arguably, catalysis is among the most attractive functions of photoswitch. However, reversible photomodulation of catalytic activity has thus far been poorly explored, and the few reported cases [57] suffer from a lack of generality and low on/off-ratios, i.e. small changes in activity. In search of a more general photoswitchable catalyst, Peters and co-workers [58] have targeted photocontrol over accessibility to metalloporphyrins, which are ubiquitous in nature since they perform important binding, activation, and catalytic processes. Their design involved a direct incorporation of an azobenzene unit into the framework of a meso-tetraphenylporphyrin in such a way that two o-phenylazo substituents could occupy the space above and below the plane of the metalloporphyrin, leading to an effective shielding of the catalytically active metal center as shown in Fig. 2.16. Peters and co-workers expected that, upon irradiation, the large structural reorganisation accompanying the trans−cis isomerisation of the azobenzenes would open the pocket and dramatically increase access to the axial positions of the metal center, thereby enabling binding and catalysis to occur. They reported the

 59

2.2 UV-sensitive complexes 

X

N X N

2’,2Trisphat

N N N N

N N

N

X N N

N

N

Zn

N

N

N N N N

N

N X 2a, X=H X 2b, X=OH (i) 2c, X=OC(O)C(Me)2Br (ii) 2d, X=OC(O)C(Me)2[CH2CMe(CO2Me)]nBr

N N

N

X

Fig. 2.15: Structures of zinc complexes 2a−da a  conditions: (i) BrC(Me)2C(O)Br, pyridine, THF, rt, 77%; (ii) MMA/CuBr/iPrN C−Py, (600/6/12), MeOH, 60 °C, 85%. Reprinted with permission from [56]. Copyright (2004) American Chemical Society.

Substrate

Substrate

hν hν’, ∆

Product Fig. 2.16: Concept of photomodulating axial accessibility and therefore catalytic activity in azobenzene-substituted metalloporphyrins. Reprinted with permission from [58]. Copyright (2006) American Chemical Society.

60 

 2 Photo-sensitive complexes based on azobenzene

H3C N Ar

N N Ar

Zn

Ar

N

N

N N N N

Fig. 2.17: Azobenzene–zinc investigated by Peters and co-workers [58]. Reprinted with permission from [57]. Copyright (2006) American Chemical Society.

e­ fficient synthesis as well as structural and photophysical characterisation of novel ­bisazobenzene-substituted porphyrin complexes with zinc (Fig. 2.17). Marchi and co-workers [59] have also concentrated their work on the Zinc com­ plexes; however they decided to investigate a photo-sensitive dendrimer. Dendrimers [60,  61], a class of multibranched molecules that can – by design – exhibit a high degree of order and complexity, are ideal scaffolds to organise multiple units into a nano-object. Dendrimers containing photochromic [62, 63] or luminescent units [64] or acting as ligands of metal ions [21, 65] have been extensively investigated in the past decade; however Marchi et al. have reported the first example of dendrimers (Fig.  2.18) containing photochromic (azobenzene), luminescent (naphthalene), and metal coordinating units and thus performing three different functions: light-harvesting, metal coordination and photoswitching. Because of their proximity, the various

UV

Energy transfer Zn2+

Zn2+

Zn2+ Isomerization

Fig. 2.18: Photo-sensitive dendrimer designed by [59]. Reprinted with permission from [59]. Copyright (2012) American Chemical Society.

2.2 UV-sensitive complexes 

 61

functional groups of the dendrimers interact: the azobenzene unit enables to control the distance between the two cyclam moieties upon light stimulation. 2.2.6 Azobenzene with copper (Cu) A special challenge is construction of molecular machines showing unidirectional, multi-state switching cycles. The problem in the design of such systems is that the path of leaving the starting state must differ from the path returning back to the starting point, as otherwise no mechanical work is performed during one cycle. Therefore, a system is needed that, on the one hand, is flexible enough to allow a complex motion sequence and, on the other hand, is so rigid that the movements are controlled in a single direction. The targeted unidirectional nanomachine should pass through a cycle of four states. Such a cycle (I → II → III → IV → I), together with the structure, concept, and principle of the nanomachine activity, is depicted in Fig. 2.19. The system consists of a scaffold, which must be chiral to control the direction of motion, two arms, and two types of hinge (blue and red circles in Fig. 2.19). This system can be compared to the arm movements of a human breaststroke swimmer, the scaffold corresponds to the human torso and the arms represent human arms. The blue hinge corresponds to the shoulder joints, and the red hinges represent the elbow joints. In state I, both arms are stretched forward. During the transition from state I to state II, a rotation of the stretched arms around the blue hinge takes place. This motion can also be considered to be the power stroke of a breaststroke swimmer. It is of the utmost importance that this motion is directed (unidirectional); this means that in our case the right arm in Fig. 2.19 must rotate only clockwise, whereas the left arm must move counterclockwise. The second stroke is the folding of the two arms

I

IV

II

III

Fig. 2.19: Structure, concept, and principle of the nanomachine. The nanomachine consists of a torso and two arms. The arms can be unidirectionally rotated around the blue hinge. The stretching/ folding of the two arms takes place around the two red hinges. Sequence of motions: I → II (directed motion of the forward-stretched arms from the front to the side; power stroke), II → III (folding of the arms), III → IV (rotation of the folded arms), and III → IV (stretching of the arms). Reprinted with permission from [66]. Copyright (2015) American Chemical Society.

62 

 2 Photo-sensitive complexes based on azobenzene

around the two red hinges (II  →  III). In the third stroke, a rotation of the folded arms around the blue hinge takes places (III → IV) whereby the direction occurs in exactly the inverse fashion to that in the first step. The fourth stroke is the stretching of the arms, which leads back to starting state I and completes the swimming cycle (IV → I). Haberhauer and co-workers [66] have designed molecular machines that demonstrate unidirectional, four-state switching cycles that bear similar characteristics to the arm movements of a human breaststroke swimmer. The photo-sensitive machines are based on a peptidic, macrocyclic scaffold that controls the direction of motion. The arms performing the swimming movements are rotated around one hinge and are stretched and folded by another hinge. The first hinge is a meta-substituted azobenzene attached to the chiral scaffold and can be switched by light. The arms are bipyridine units that are either directly attached or connected via rigid spacers to the azobenzene unit. The bipyridine units are stimulated by Cu2+ ions. The movements of these machines are triggered by alternating addition of chemicals and irradiation with light. The limiting factor for the repeatability of the cycles is the dilution effect caused by the addition of solutions, which worsens the recording of the spectra. The rate-limiting factor is the time to record the spectra for each cycle. According to Haberhauer and co-workers their design is a promising small step to an artificial nano-swimmer that is propelled in solution by an external stimuli. Very fascinating results concerning the assembly of artificial molecular machines that allow precisely controlled motion that responds to light and electrons  have been presented by Kume et al. [67]. These authors have shown that UV/bluecontrolled ­repetitive motion of azobenzene moieties in 6,6′-bis(4″-tolylazo)-4,4′-bis (4-­tertbutylphenyl)-2,2′-bipyridine (Fig. 2.20) causes reciprocal CuI translocation between two coordination environments, resulting in pumping of the redox potential of CuI, as presented in Fig. 2.21. Therefore, UV/blue light information can be successfully

+ But

N

N N N

Bu

t

N

N

N

Cu

N

Bu

t

N(31)

N N N

N(41)

N

t

Cu(1)

N(11)

N(21)

Bu

[Cu(oAB)2]+ Fig. 2.20: Azobenzene–Cu complex obtained by Kume et al. [67]. Reprinted with permission from [67]. Copyright (2005) American Chemical Society.

2.2 UV-sensitive complexes 

Isomerization

UV light CuI

Ligand exchange

Ligand exchange

+

= 2,2’–bipyridine

+

+

CuI

Blue light

+

CuI Chemical potential

= Cis2–oAB

Isomerization

= Trans2–oAB

 63

CuI

π–stacking CuII/I redox potential Fig. 2.21: Schematic representation of energy migration in the artificial molecular machines designed by Kume et al. [67]. Reprinted with permission from [67]. Copyright (2005) American Chemical Society.

transformed into an electrode potential change and positive/negative current response, which is closely related to natural visual transduction both functionally and mechanically. This result demonstrates a new strategy for artificial molecular machine assembly, i.e. forming a path with multistep chemical reactions between input/output couples of choice. The research group of Prof. Hirota from the Nara Institute of Science and Technology, Japan, has published very interesting results about photocontrol of the cooperation of metal ions for DNA cleavage [68]. Over the last decade, DNA cleavage by metal complexes has been actively studied to design artificial metallonucleases. For this purpose, many dinuclear metal complexes have been shown to be effective, in which the metal ion centers could exhibit cooperation for DNA cleavage [68–70]. Peptide and amino acid metal complexes have also been shown to be effective for DNA cleavage [71]. Photoresponsive molecules are of enormous interest in the aspect of control of the structure and function of biomolecules. The helical structure and spatial orientation of DNA-binding peptides have been photocontrolled by modification of the peptides with an azobenzene cross-linker, which was photoisomerised between the trans and cis forms [68, 72–74]. The research group of Prof. Hirota has envisaged that the cooperation of the metal ion centers in DNA cleavage could be photocontrolled by linking two metal complexes with an azobenzene derivative, in which the distance between the metal ion centers could be modulated by changes in their spatial orientation by photoisomerisation of the azobenzene linker [68]. Therefore, Prof. Hirota and coworkers linked CysGly (cystylglycine) dipeptides by an azobenzene derivative,  trans-4,4′-bisbromomethylazobenzene, and obtained a photoisomerisable peptide (1) as a ligand and its dicopper complex (1-2Cu2+; Fig. 2.22). The copper complex 1-2Cu2+ was reactive for DNA cleavage in the

64 

 2 Photo-sensitive complexes based on azobenzene

O

S H2N

12 Å

O

N=N

OH2

O

Cu N

Cu H2O

O

O

S

S

O N O

1–trans–2Cu2+

8Å

S

355 nm 430.6 nm

NH2

O

N=N

N

O

O Cu

NH2 H2N

H2O

Cu

H2O

N O

O

1–cis–2Cu2+

Fig. 2.22: Photoconversion between the trans and cis forms of 1-2Cu2+, in which the azobenzene chromophore links Cu(II)-bound CysGly peptides. Reprinted with permission from [68]. Copyright (2008) American Chemical Society.

cis form (1-cis-2Cu2+), in which the copper(II) ion centers are oriented close to each other. However, the reactivity of  1-2Cu2+  was negligible in the trans form (1-trans2Cu2+), in which the copper(II) ions are far away from each other. These results demonstrate the photocontrol of the spatial orientation of the copper(II)-bound dipeptides linked by an azobenzene derivative for DNA cleavage.

Acknowledgment Financial support from European Community's Seventh Framework Programme (FP/2007-2013) under the Individual Outgoing Marie Curie Grant agreement no. 328794 is gratefully acknowledged by Dr. Bartosz Tylkowski.

References   [1] Tylkowski B, Pregowska M, Jamowska E, Garcia-Valls R, Giamberini M. Preparation of a new lightly cross-linked liquid crystalline polyamide by interfacial polymerization. Application to the obtainment of microcapsules with photo-triggered release. European Polymer Journal 2009;45:1420–32. [2] Perez-Miqueo J, Telleria A, Munoz-Olasagasti M, Altube A, Garcia-Lecina E, de Cozar A, Freixa Z. Azobenzene-functionalized iridium(iii) triscyclometalated complexes. Dalton Transactions 2015;44:2075–91. [3] Velema WA, Szymanski W, Feringa BL. Photopharmacology: Beyond Proof of Principle. Journal of the American Chemical Society 2014;136:2178–91. [4] Mitscherlich, E. Ueber das Stickstoffbenzid. Annalen der Pharmacie 1834;12:311–34. [5] Hartley GS. Cis form of azobenzene. Nature 1937;140:281–2. [6] Sato M, Kinoshita T, Takizawa A, Tsujita Y. Photoinduced conformational transition of polypeptides containing azobenzenesulfonate in the side chains. Macromolecules 1988;21:1612–6.

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 2 Photo-sensitive complexes based on azobenzene

[23] Samanta S, Ghosh P, Goswami S. Recent advances on the chemistry of transition metal complexes of 2-(arylazo)pyridines and its arylamino derivatives. Dalton transactions (Cambridge, England : 2003) 2012;41:2213–26. [24] Kume S, Nishihara H. Photochrome-coupled metal complexes: molecular processing of photon stimuli. Dalton Transactions (Cambridge, England : 2003), 2008:3260–71. [25] Park JS, Lifschitz AM, Young RM, Mendez-Arroyo J, Wasielewski MR, Stern CL, Mirkin CA. Modulation of electronics and thermal stabilities of photochromic phosphino– aminoazobenzene derivatives in weak-link approach coordination complexes. Journal of the American Chemical Society 2013;135:16988–96. [26] Yutaka T, Mori I, Kurihara M, Mizutani J, Tamai N, Kawai T, Irie M, Nishihara H. Photoluminescence switching of azobenzene-conjugated Pt(II) terpyridine complexes by trans-cis photoisomerization. Inorganic Chemistry 2002;41:7143–50. [27] Yutaka T, Kurihara M, Kubo K, Nishihara H. Novel photoisomerization behavior of Rh binuclear complexes involving an azobenzene-bridged bis(terpyridine) ligand. Strong effects of counterion and solvent and the induction of redox potential shift. Inorganic Chemistry 2000;39:3438–9. [28] Kume S, Kurihara M, Nishihara H. Reversible trans-cis photoisomerization of azobenzeneattached bipyridine ligands coordinated to cobalt using a single UV light source and the Co(iii)/Co(ii) redox change. Chemical Communications 2001:1656–7. [29] Yamaguchi K, Kume S, Namiki K, Murata M, Tamai N, Nishihara H. UV−Vis, NMR, and time-resolved spectroscopy analysis of photoisomerization behavior of three- and six-azobenzene-bound tris(bipyridine)cobalt complexes. Inorganic Chemistry 2005;44:9056–67. [30] Wang A, Sun H, Li X. N-assisted carbon−hydrogen bond activation by cobalt(i) complexes. Organometallics 2008;27:5434–7. [31] Akitsu T, Itoh T. Polarized spectroscopy of hybrid materials of chiral Schiff base cobalt(II), nickel(II), copper(II), and zinc(II) complexes and photochromic azobenzenes in PMMA films. Polyhedron 2010;29:477–87. [32] Davidenko NA, Davidenko II, Pavlov VA, Popenaka AN, Savchenko IA, Shumelyuk AN. Recording medium based on a polymer azobenzene complex with cobalt for polarization holography. Theoretical and Experimental Chemistry 2009;45:54–7. [33] Chance B, Erecinska M, Wagner M. Mitochondrial responses to carbon monoxide toxicity. Annals of the New York Academy of Sciences 1970;174:193–204. [34] Motterlini R, Otterbein LE. The therapeutic potential of carbon monoxide. Nature reviews. Drug Discovery 2010;9:728–43. [35] Heinemann SH, Hoshi T, Westerhausen M, Schiller A. Carbon monoxide--physiology, detection and controlled release. Chemical Communications (Cambridge, England) 2014;50:3644–60. [36] Kikuchi G, Yoshida T, Noguchi M. Heme oxygenase and heme degradation. Biochemical and Biophysical Research Communications 2005;338:558–67. [37] Stein AB, Bolli R, Dawn B, Sanganalmath SK, Zhu Y, Wang OL, Guo Y, Motterlini R, Xuan YT. Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium. Journal of Molecular and Cellular Cardiology 2012;52:228–36. [38] Kohmoto J, Nakao A, Stolz DB, Kaizu T, Tsung A, Ikeda A, Shimizu H, Takahashi T, Tomiyama K, Sugimoto R, Choi AM, Billiar TR, Murase N, McCurry KR. Carbon monoxide protects rat lung transplants from ischemia-reperfusion injury via a mechanism involving p38 MAPK pathway. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2007;7:2279–90. [39] Alberto R, Motterlini R. Chemistry and biological activities of CO-releasing molecules (CORMs) and transition metal complexes. Dalton Transactions (Cambridge, England : 2003) 2007:1651–60. [40] Romao CC, Blattler WA, Seixas JD, Bernardes GJ. Developing drug molecules for therapy with carbon monoxide. Chemical Society Reviews 2012;41:3571–83.

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[41] Chakraborty I, Carrington SJ, Mascharak PK. Design strategies to improve the sensitivity of photoactive metal carbonyl complexes (photoCORMs) to visible light and their potential as CO-donors to biological targets. Accounts of Chemical Research 2014;47:2603–11. [42] Wegner HA. Molecular switches. Second Edition. Edited by Ben L. Feringa and Wesley R. Browne. Angewandte Chemie International Edition 2012;51:2281. [43] Balzani VV, Credi A, Raymo FM, Stoddart JF. Artificial molecular Machines. Angewandte Chemie International Edition 2000;39:3348–91. [44] Kottas GS, Clarke LI, Horinek D, Michl J. Artificial molecular rotors. Chemical Reviews 2005;105:1281–376. [45] Manzano C, Soe WH, Wong HS, Ample F, Gourdon A, Chandrasekhar N, Joachim C. Step-by-step rotation of a molecule-gear mounted on an atomic-scale axis. Nature Materials 2009;8:576–9. [46] Setaka W, Nirengi T, Kabuto C, Kira M. Introduction of clutch function into a molecular gear system by silane-silicate interconversion. Journal of the American Chemical Society 2008;130:15762–3. [47] Brouwer AM, Frochot C, Gatti FG, Leigh DA, Mottier L, Paolucci F, Roffia S, Wurpel GW. Photoinduction of fast, reversible translational motion in a hydrogen-bonded molecular shuttle. Science 2001;291:2124–8. [48] Mahadevan L, Matsudaira P. Motility powered by supramolecular springs and ratchets. Science 2000;288:95–100. [49] Badjic JD, Balzani V, Credi A, Silvi S, Stoddart JF. A molecular elevator. Science 2004;303:1845–9. [50] Bruns CJ, Stoddart JF. Rotaxane-based molecular muscles. Accounts of Chemical Research 2014;47:2186–99. [51] Kinbara K, Muraoka T, Aida T. Chiral ferrocenes as novel rotary modules for molecular machines. Organic & Biomolecular Chemistry 2008;6:1871–6. [52] Muraoka T, Kinbara K, Kobayashi Y, Aida T. Light-driven open−close motion of chiral molecular scissors. Journal of the American Chemical Society 2003;125:5612–3. [53] Muraoka T, Kinbara K, Aida T. Reversible operation of chiral molecular scissors by redox and UV light. Chemical Communications 2007:1441–3. [54] Kai H, Nara S, Kinbara K, Aida T. Toward long-distance mechanical communication: Studies on a ternary complex interconnected by a bridging rotary module. Journal of the American Chemical Society 2008;130:6725–7. [55] Muraoka T, Kinbara K, Aida T. Mechanical twisting of a guest by a photoresponsive host. Nature 2006;440:512–5. [56] Viau L, Bidault S, Maury O, Brasselet S, Ledoux I, Zyss J, Ishow E, Nakatani K, Le Bozec H. All-optical orientation of photoisomerizable octupolar zinc(II) complexes in polymer films. Journal of the American Chemical Society 2004;126:8386–7. [57] Cacciapaglia R, Di Stefano S, Mandolini L. The bis-barium complex of a butterfly crown ether as a phototunable supramolecular catalyst. Journal of the American Chemical Society 2003;125:2224–7. [58] Peters MV, Goddard R, Hecht S. Synthesis and characterization of azobenzene-confined porphyrins. The Journal of Organic Chemistry 2006;71:7846–9. [59] Marchi E, Baroncini M, Bergamini G, Van Heyst J, Vögtle F, Ceroni P. Photoswitchable metal coordinating tweezers operated by light-harvesting dendrimers. Journal of the American Chemical Society 2012;134:15277–80. [60] Schmittel M, Mahata K. Diversity and complexity through reversible multiple orthogonal interactions in multicomponent assemblies. Angewandte Chemie International Edition 2008;47:5284–6. [61] Franc G, Kakkar AK. “Click” methodologies: Efficient, simple and greener routes to design dendrimers. Chemical Society Reviews 2010;39:1536–44.

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[62] Deloncle R, Caminade A-M. Stimuli-responsive dendritic structures: The case of light-driven azobenzene-containing dendrimers and dendrons. Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2010;11:25–45. [63] Nguyen TT, Turp D, Wang D, Nolscher B, Laquai F, Mullen K. A fluorescent, shape-persistent dendritic host with photoswitchable guest encapsulation and intramolecular energy transfer. Journal of the American Chemical Society 2011;133:11194–204. [64] Kuroda DG, Singh CP, Peng Z, Kleiman VD. Mapping excited-state dynamics by coherent control of a dendrimer’s photoemission efficiency. Science 2009;326:263–7. [65] Branchi B, Ceroni P, Balzani V, Klarner FG, Vogtle F. A light-harvesting antenna resulting from the self-assembly of five luminescent components: A dendrimer, two clips, and two lanthanide ions. Chemistry (Weinheim an der Bergstrasse, Germany) 2010;16:6048–55. [66] Haberhauer G, Burkhart C, Woitschetzki S, Wölper C. Light and chemically driven molecular machines showing a unidirectional four-state switching cycle. The Journal of Organic Chemistry 2015;80:1887–95. [67] Kume S, Murata M, Ozeki T, Nishihara H. Reversible photoelectronic signal conversion based on photoisomerization-controlled coordination change of azobenzene-bipyridine ligands to copper. Journal of the American Chemical Society 2005;127:490–1. [68] Prakash H, Shodai A, Yasui H, Sakurai H, Hirota S. Photocontrol of spatial orientation and DNA cleavage activity of copper(II)-bound dipeptides linked by an azobenzene derivative. Inorganic Chemistry 2008;47:5045–7. [69] Mancin F, Scrimin P, Tecilla P, Tonellato U. Artificial metallonucleases. Chemical Communications (Cambridge, England), 2005:2540–8. [70] van den Berg TA, Feringa BL, Roelfes G. Double strand DNA cleavage with a binuclear iron complex. Chemical Communications (Cambridge, England), 2007:180–2. [71] Copeland KD, Fitzsimons MP, Houser RP, Barton JK. DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators. Biochemistry 2002;41:343–56. [72] Guerrero L, Smart OS, Woolley GA, Allemann RK. Photocontrol of DNA binding specificity of a miniature engrailed homeodomain. Journal of the American Chemical Society 2005;127:15624–9. [73] Hirota S, Fujimoto Y, Choi J, Baden N, Katagiri N, Akiyama M, Hulsker R, Ubbink M, Okajima T, Takabe T, Funasaki N, Watanabe Y, Terazima M. Conformational changes during apoplastocyanin folding observed by photocleavable modification and transient grating. Journal of the American Chemical Society 2006;128:7551–8. [74] Jousselme B, Blanchard P, Gallego-Planas N, Levillain E, Delaunay J, Allain M, Richomme P, Roncali J. Photomechanical control of the electronic properties of linear pi-conjugated systems. Chemistry (Weinheim an der Bergstrasse, Germany) 2003;9:5297–306.

Renata Jastrząb, Lechosław Łomozik and Bartosz Tylkowski

3 Complexes of biogenic amines in their role in living systems 3.1 Introduction

Although polyamines (PA) belong to relatively simple aliphatic substances, their role in life processes of animals and plants is of key importance [1–5]. The group of the most important amines, called biogenic ones includes: Spermine (Spm): Spermidine (Spd): Putrescine (Put):

H2N(CH2)3NH(CH2)4NH(CH2)3NH2 H2N(CH2)3NH(CH2)4NH2 H2N(CH2)4NH2.

Of secondary importance are homologues of biogenic amines, occurring in lower contents in living organisms [2, 6–8]: H2N(CH2)3NH2 1,3-diaminopropan: Cadaverine: H2N(CH2)5NH2 Homospermidine: H2N(CH2)4NH(CH2)4NH2 Norspermine (3,3,3-tet): H2N(CH2)3NH(CH2)3NH(CH2)3NH2 Thermospermine: H2N(CH2)3NH(CH2)4NH(CH2)4NH2 Caldopentamine: H2N(CH2)3NH(CH2)3NH(CH2)3NH(CH2)3NH2. The first polyamine discovered in a living organism was tetramine, a spermine crystallised out of sperm in 1678 by Van Leewenkeuk [9]. Putrescine was discovered in the end of the 19th century in microbes and then triamine: spermidine was discovered in the beginning of the 20th century [2]. Later studies have shown that in animal cells spermidine and spermine occur at elevated levels, while in prokaryotes spermidine and putrescine contents are dominant. Putrescine, spermidine, 1,3-diaminopropan, homospermidine, norspermidine, and norspermine have been found in many gramnegative bacteria and algae [7, 10, 11]. Total concentration of PA in living organisms is on the order of millimols, however, the concentration of free polyamines is much lower. A low level of free amines follows from the fact that they are involved in noncovalent interactions with biomolecules occurring in living organisms such as nucleic acids, proteins, or phospholipids. High concentrations of non-bonded polyamines have been detected first of all in young molecules in the process of growth, in particular in rapidly proliferating cancer cells [6, 12]. Elevated levels of free polyamines have been observed, e.g. in breast, colon, lung, prostate, and skin tumours, accompanied by

70 

 3 Complexes of biogenic amines in their role in living systems

changed levels of enzymes responsible for biosynthesis and catabolism of polyamines. Because of the increased level of free polyamines and a tendency of their interaction with nucleic acids and other bioligands, these compounds have become objects of intense study [1, 13–19]. There is no doubt that the regulation of biosynthesis of polyamines and catabolism is one of the most important pathways in the search strategy for chemoprevention and chemotherapeutic drugs [14, 15, 20–38]. The present state of knowledge of these processes, their significance in biological systems, and their application in medicine are presented in subsequent sections of this chapter.

3.2 Polyamines in living systems Polyamines occur in practically all living organisms, although the level of their concentration depends on the type of species, type of tissue, and first of all on the age of the cells [10, 14, 39–45]. The organ in which the highest concentration of polyamines (2 mM spermidine and 4 mM spermine) has been detected, is the pancreas [10]. High levels of polyamines (spermine and spermidine) have also been detected in the kidneys, spleen, liver, lungs, and in semen [7, 20]. Spermidine and its analogues are also present in high levels in the central nervous system, and that is why spermidine is also called neuridine. It should be pointed out that spermidine is not uniformly distributed in the entire nervous system and occurs in high concentration in the white matter of the brain. Also, an untypical increase in the concentration of spermidine and spermine in the brain has been noted with increasing age of the organism [39, 40]. A similar situation as in the nervous system (different concentration of amine in the same tissue) has been noted in the skin. Measurements with liquid chromatography and fluorescence methods have proved much higher concentrations of spermidine and spermine in the epidermis than in the dermis of the skin. Regular measurements of changes in the PA level in the skin are necessary to control the condition of patients with dermatological diseases [46]. Small amounts of biogenic amines have been found in blood tissues, e.g. 0.01 mM in erythrocytes, and in urine (putrescine and spermidine) [41], in which an increase in their concentration is a symptom of disease. Increasing concentration of polyamines is also observed in people with cystic fibrosis or mucoviscidosis, most probably spermidine and the products of its metabolic decomposition play an important role in the pathogenesis of dysfunction of cell membranes [47]. Another interesting fact is that polyamines, strongly basic substances, show a high affinity to protons. This affinity increases with an increasing methylene chain of the amine. In physiological conditions, at pH close to 7, biogenic amines are fully protonated. Amines with a methylene chain shorter than three methylene groups in physiological conditions can be partly protonated, which consequently show a lower affinity to polyanions than biogenic amines [48].

3.2 Polyamines in living systems 

 71

From a pharmacological point of view, polyamines are toxic [15], but at appropriate concentrations play various positive roles in living organisms, Fig. 3.1. These compounds interact with entire cells, cellular organelles, and nucleic acids, and participate in many metabolic reactions [5, 49]. Polyamines have been found to catalyse and control biosynthesis of nucleic acids and are directly responsible for macromolecular synthesis taking place upon cell growth (synthesis of DNA is stimulated by spermidine) [44]. Strongly basic polyamines show high affinity to anionic compounds and thus show the ability to bind to nucleic acids. When interacting with phosphate groups of polynucleotides, they prevent denaturation and stabilise the structure of the nucleic acid [6, 10, 50, 51]. Polyamines show a tendency towards initiation of t-RNA methylation in vitro, prevent enzymatic degradation and radiation-induced damage to ribosomes, and influence synthesis of nucleic acids and proteins [10, 41]. Moreover they affect the degree of DNA packing in a bacteriophage [52, 53]. Biogenic polyamines also influence the functioning of biological membranes (spermine stabilises membrane activity in bacteria), and their effect depends first of all on the positive charge of the amine chain (the greater the charge the better the stabilisation). A very important consequence of attachment of PA to double-layered membranes is protection against lipid peroxidation. Biogenic polyamines also modulate the synthesis of triacylglycerols necessary for membrane construction DNA stabilization Modulation of chromatin structure

Receptor – ligand interactions

Transcription

Functioning of ion channels

Translation

BIOAMINES

Migration

ATP binding

mRNA stabilization

Membrane stability

Signal transduction Cell growth and proliferation

Fig. 3.1: Role of biogenic amines in living organisms.

72 

 3 Complexes of biogenic amines in their role in living systems

[24, 26, 27, 43, 49]. Spermidine is also known to exert an antimutagenic effect in bacteria [44]. Some polyamines are precursors of amino acids that are formed by oxidative deamination of amines. One of those amino acids (derivatives of amines) is putreanine, which is a natural component of the brains of vertebrates [42]. The pathway of polyamine biosynthesis has been for the first time defined on the basis of investigation of microorganisms. The later developed scheme of polyamine biosynthesis for mammals proved almost identical, Fig. 3.2. In bacteria (e.g. in Escherichia Coli) and in plants, putrescine is synthesised along two parallel pathways: 1) from ornithine as a result of its decarboxylation in the presence of ornithine decarboxylase 2) from arginine in the process of decarboxylation to agmatine, which is then hydrolysed with involvement of agmatine ureohydrolase directly or through the intermediate product N-carbamyl-1, 4-diaminobutane to putrescine and urea. Catabolism of polyamines in animals has been discovered as a two-stage process controlled by the enzymes spermidine/spermine N1-acetyltransferase (SSAT) [54–59]. Thanks to the activity of these enzymes, acetylated polyamines become substrates for further conversion of spermidine to putrescine. SSAT catalyses the formation of N1-acetylspermine or N1-acetylspermidine by a transfer of acetyl group from acetylcoenzyme A to the N1 position of spermidine or spermine. In the second stage of this pathway, peroxisomal flavin adenine dinucleotide (FAD) is formed, and its level is directly related to the activity of N1-acetylpolyamine oxidase (APAO) [60–65]. APAO is the enzyme that catalyses the cleavage of acetylated polyamines and is responsible for formation of spermidine or spermine, 3-aceto-aminopropanal and H2O2 [62, 63, 66–68]. Alternatively, spermine can be oxidised back directly to spermidine with involvement of spermine oxidase (SMO) the enzyme dependent on Flavin adenine dinucleotide (FAD) [67–69]. Induction of SMO has been reported to accompany the inflammatory conditions related to the colon and lung cancer, which suggests that SMO may be an attractive target for chemoprevention strategies [70–74]. A side effect of the oxidation reaction is generation of toxic hydrogen peroxide (H2O2). The molecules of H2O2 are known to play an essential role in DNA damage, which leads, among other things, to catabolism of polyamines. Moreover, a significant reduction of the level of intracellular polyamines, which accompany the process of catabolism, is inhibited by an increase in the level of inhibitors of this process [42]. The best recognised transport of polyamines in animals suggests that in the first stage PAs enter the cell through as yet unidentified membrane transporter/ carrier. This process is powered by the membrane potential and then by rapid accumulation of polyamines into polyamine-sequestering vesicles driven by a vacuolar − ATPase pH gradient and proton exchange. This system of transportation

3.2 Polyamines in living systems 

NH2 COOH H2N

C NH

COOH

HN

NH2

L-Ornithine

CH3 S

NH2

L-Arginine

–CO2

1

C

–CO2

+ ATP, Mg2+

NH2 N

N

NH

N

HN

NH2

L-Methionine

NH2 3

N

– PP, P1 4 COOH

+ CH(CHOH)2CHCH2S

NH2

NH2

CH3

O

S – Adenosyl methionine

Aganatine 2 –Urea

+ Mg2+

NH2 H 2N

COOH

N

N

NH2

N

Putrescine

N

–CO2 5

+ CH(CHOH)2CHCH2S O

NH2

CH3

Decarboxylated S – Adenosyl methionine

6 NH2 H2N

H2 N

N

NH2

Spermidine 7

Spermine

N

CH(CHOH)2CHCH2SCH3 O

Decarboxylated S – Adenosyl methionine

NH

N

N

NH

NH

Methyl Adenosyl

NH2

Fig. 3.2: The pathway of polyamine synthesis in living organisms. The enzymes involved are 1 – arginine decarboxylase, 2 – agmatine ureohydrolase, 3 – ornithine decarboxylase, 4 – S-adenosylmethionine synthase, 5 – S-adenosylmethionine decarboxylase, 6 – spermidine synthase, and 7 – spermine synthase.

 73

74 

 3 Complexes of biogenic amines in their role in living systems

permits explanation of the seemingly high total concentration of intracellular polyamines, although the actual concentration of free PAs is believed to be relatively low [75–78]. Another model of polyamines transportation assumes that spermine is bonded to heparin sulphate and glypican 1 (GPC1), which act as agents transporting amine inside the cell [72, 75, 76]. The enzymes and the amines synthesised (especially putrescine) are the direct target of the efforts undertaken to inhibit the synthesis of polyamines in the diseases in which the elevated level of polyamines is observed, particularly in cancer.

3.3 Polyamines as tumour markers Neoplastic tumours, along with circulatory system diseases, are the leading causes of death in developed countries [79]. It has been recently established that increased incidence of inflammatory conditions and increased level of synthesis of polyamines are related to the development of intraepithelial neoplasia, which is a risk factor for cancer development. There is ample literature on the relation between the increased level of polyamines concentration and neoplastic diseases, which suggests a relation between induced biosynthesis of PAs and neoplasia [1, 72, 80]. Metabolism of polyamines is an integral component of the mechanisms of carcinogenesis in epithelium tissues. Development of neoplastic disease is a multistage process that involves specific transformations leading to progressive change from healthy normal cells to cancer cells. If the range of inflammatory conditions is irregular, the cell response leads to chronic inflammatory conditions in which the inflammatory focuses are dominated by macrophages and other inflammatory cells. This leads to generation of increased amount of growth agents and cytokines as well as reactive species of oxygen and nitrogen responsible for DNA damage [81, 82]. In persons with neoplastic diseases a considerable increase in the concentration of polyamines in blood and urine is observed; however, no specific values characteristic of the disease could be established because the level of polyamines depends on activity and degree of development of the disease [10, 15, 41, 83–85]. In many types of cancer, e.g. in persons with cancer of the stomach, pancreas, and breast, the concentration of polyamines is practically unchanged (only in 20% and 25% of patients is an increase in the concentration of spermidine and spermine observed, respectively), while an increased level of PAs is observed in about 50% of persons with malignant cancer of the lungs. In persons with cancer of the genital tract, prostate, or bladder, an increased level of PAs is noted in about 90% of patients [20, 45, 74]. Measurements of the concentrations of spermine and spermidine in urine and blood often facilitate diagnosis and evaluation of health

3.3 Polyamines as tumour markers 

 75

conditions and can be used for monitoring the progress of therapy. If the therapy is effective, a significant decrease in the level of polyamines is observed starting from the first week of chemotherapy [15, 45, 86]. However, it has been established that analogues of bio­genic amines (spermidine and spermine) inhibit the growth of tumours in model systems and show an antimalarial effect. These types of derivatives are mainly obtained by the Mitsunobu reaction (between alcohol and activated amine, leading to a chiral derivative of polyamine) [87]. In general, cells have well-developed mechanisms regulating the correct intracellular level of PAs, and deregulation of this metabolism (biosynthesis and catabolism) and transport is of key importance for cell growth. An increased level of polyamines (as a consequence of their increased synthesis) is noted as a result of inflammatory conditions or increased proliferation following from rapid growth of cancer cells (PA have pleiotropic effects on cell physiology) [88]. In the 1960s, Russell and Snyder for the first time noted a high level of concentration of ornithine decarboxylase (ODC) accompanying the occurrence of cancer [89]. High activity of ODC and a high level of PAs have been observed to accompany an increase in familial adenomatous polyposis in cases of genetic predispositions for colon cancer related to mutation of adenomatous polyposis coli [90]. Pioneer works aimed at checking correlations between a high level of polyamines and the occurrence of skin cancer have shown that an elevated level of ODC is necessary and often sufficient for triggering cancer in mice [73]. It has been also established that the level of ODC increases in persons with human non-melanoma skin cancer (NMSC) [91]. Induction of ODC and an increase in the level of PAs level have been correlated with breast cancer [16, 92] and prostate cancer [17]. Also other enzymes such as spermidine synthase and spermine synthase are closely related to carcinogenesis. The level of enzymes involved in catabolism is also related to carcinogenesis, e.g. increased activity of SMO is observed in inflammation conditions connected to neoplastic diseases such as infection and the ensuing cancer of alimentary tract (Heliobacter pylori) [93]. Infection of gastric epithelial cells provoked by H. pylori also leads to deregulation of SMO expression, leading to an increased level of DNA damage and apoptosis. Removal of the source of inflammation has been observed to be correlated with a reduction in SMO expression [94]. A decrease in the level of SMO can bring a reduction in the inflammatory condition caused by H. pylori and consequently inhibition of gastric cancer development. An elevated level of SMO expression, relative to its level in healthy prostate tissues, has been also found in tissues from patients with prostatic intraepithelial neoplasia and prostate cancer [95] and ulcerative colitis, the presence of which is related to a high risk of colon cancer [96]. Introduction to the organism of appropriate inhibitors of biosynthesis, e.g. those that inhibit the activity of ornithine decarboxylase, reduces the level of biogenic amines in the organism, Fig. 3.3. Of particular importance is α-difluormethylornithine (DFMO), obtained in 1978, Fig. 3.4.

76 

 3 Complexes of biogenic amines in their role in living systems

NH2

H2N

Ornithine DFMO

COOH MDL 72527

ODC

SAM486A

CO2 O

H2N Putrescine

CH3 Ado

AdoMetDC

S+

NH2

O

H

APAO

CH3 Ado

S+

COOH NH2

Spermidine synthase

O

NH2

H2N

Ado―S―CH3 SAM486A

Ado

AdoMetDC

S+

H2N

N H Spermidine

MDL 72527 Exported from cell

NH2 O

APAO

CH3 Ado

CH3

N H

N H

SSAT

MTA

CH3

CH3 +H2O2

N H

H

S+

H2O2

O N H

CH3 +H2O2

COOH NH2

Spermidine synthase

NH2

SMO

Ado―S―CH3

H N

O N H

N H

CH3

SSAT

MTA

H2N

H2N

H N Spermine

N H

NH2

Enzyme Inhibitor

Fig. 3.3: Targets in the polyamine metabolic pathway [72].

Significant inhibition of cell replication after the introduction of DFMO (or similar inhibitors) suggests a potential therapeutic strategy for the treatment of neoplastic diseases. Interference into the metabolism of polyamines through the use of inhibitors of biosynthesis or catabolism seems promising in the treatment of inflammatory conditions bearing substantial risk of cancer development. Clinical trials using DFMO, a selective inhibitor of polyamines synthesis, have shown that after a year of DFMO application the size of enlarged prostates and the amount of serum prostatespecific antigen were reduced by half in persons genetically predisposed to prostate cancer. Similarly, the 3-year application of a combination of DFMO and sulindac, a non-steroid anti-inflammatory drug (NSAID), to persons with stage 1 colon cancer led to a 70% reduction in the amount of cancer cells [1, 97, 98].

3.4 Weak interactions of polyamines 

NH

NH2

H2 N

H2N

COOH

F2HC

N

N H

N

NH2 N

NH

H N

NH2

N

N H3C

HO OH

R

N

NH2 NH

N N

N

O

N H

N

CGP 39937, 3 NH2

N

N

H2N

NH

MGBG, 2

DFMO, 1

H 2N

CH3

 77

O

S+

HO OH

H N

NH2

R = H, AdoDATO, 5 R = aminopropyl, AdoDATAD, 6

AbeAdo, 4

N H

H N MDL 72527, 7

N H

N H

N H

N H

MDL 27695, 8 N H

N H

N H

N H

BENSpm, 9 N H

N H

BESpm, 10

H N

N H

H N

H N

H N

N H

BEHSpm, 11 N H

H N

N H

H N

N H

BE–4X4, 12 Fig. 3.4: Structures of the classical inhibitors of polyamine biosynthesis [99].

3.4 Weak interactions of polyamines The most common types of weak noncovalent interactions in biological systems are illustrated in Fig. 3.5.

78 

C O

 3 Complexes of biogenic amines in their role in living systems

H

N

C

O – O

Hydrogen bonding

+ H3N

H3C

CH3 CH CH2

H3C

CH3 CH CH2

Electrostatic interaction Hydrophobic interaction

Stacking

Fig. 3.5: Types of weak interactions.

In the systems of polycations of biogenic amines with other bioligands, the dominant types of interactions are ion–ion or ion–dipole, with involvement of protonated PAs and biomolecules that contain atoms or a group of atoms of negative charge or high electron density [8]. The main factor determining the possibility of occurrence of weak interactions is high basicity of polyamines. In physiological environments, polyamines occur in protonated form [2], and −NHx+ groups are potential positive centres of interactions of electrostatic type with other biomolecules such as nucleosides, nucleotides, amino acids, proteins, nucleic acids, or phospholipids [86]. Protonation of primary amine groups in the PA molecule is more exothermic than protonation of secondary groups. Differences in the subsequent ΔH values decrease with increasing length of the chain, which is a consequence of changes in the transmission of inducing effect depending on the number of methylene groups. If the chain is shorter than three methylene groups, at least one pK value is less than seven, and the molecule is partly protonated in the physiological medium [99–101]. This explains a low activity of short polyamines in noncovalent interactions in biological systems [48]. Reliable information on the formation of molecular complexes as a result of noncovalent interactions with involvement of protonated polyamines have been published about 40 years ago [102, 103]. As a result of interactions of this type, electron density at the reaction centre changes, which leads to a shift in NMR signals and permits identification of the sites of interactions. On the basis of NMR studies, the protonated −NHx+ groups of spermidine and spermine have been found to react with deprotonated phosphate groups and high electron density fragments of AMP, ADP or ATP (5’-adeninomono-, di- and triphosphate, respectively) [102, 104]. According to the polyelectrolytic Manning theory, the main factor determining the mode of interactions is the charge of reagents [105, 106]. However, this approach does not explain the specificity of certain reactions. Analysis of the character of weak interactions of polyamines has revealed that their structure influences the character of the reaction and formation of adducts with other biomolecules. PAs cannot be approximated as a point charge, as has been assumed in analysis of the reaction of biomolecules with metal ions, e.g. magnesium(II) [107]. Spatial matching of polyamine to other bioligands determines the stability of adducts formed. According to the calorimetric studies of polyamine interaction with DNA, much better agreement between theory and experiment is obtained when taking into account the structural

3.4 Weak interactions of polyamines 

 79

factors [108]. Particular improvement in this agreement is obtained for the biogenic amines spermidine and spermine. For biogenic amines the effectiveness of interactions related to structural factors increases in the order Put < Spd < Spm, which corresponds not only to the number of amine groups but also to the length of these PA molecules. As the formation of molecular complexes is accompanied by a shift of the acid-base equilibrium in the reversible process: HxPA + HyNuc

(Nuc)Hx + y ˗ n(PA) + nH+ PA = polyamine; Nuc = nucleoside,

the liberation of hydrogen cations permits determination of thermodynamic stability of adducts by a potentiometric method with computer analysis of experimental data. Such determinations were performed for a series of binary and ternary systems of polyamines with nucleosides, nucleotides, and amino acids [8, 109–115]. A scheme of the adduct forming in the system of AMP/(3,3-tri), 3,3-tri NH2(CH2)3NH(CH2)3NH2 is presented in Fig. 3.6 [110]. Interestingly, according to NMR results, the longer polyamine, spermine, in the system with the same nucleotide, interacts only with endocyclic nitrogen atoms from AMP (at different spatial arrangement), see Fig. 3.6 [111].

NH2

N

Cu

N

(PO3)3 O H H

OH

O

+ NH2

NH2 NH2+ + NH2 N

N

+ NH3

+ NH3

N

O H

H H OH

AMP/3,3–tri system

N

PO32–

H

O

OH

+ NH2

NH2 N

+ NH3

N

H H OH

AMP/Spm system

Fig. 3.6: Tentative mode of interactions (noncovalent interaction).

Molecular complexes form in the pH range in which amine is protonated and act as a positive centre of interaction, while the nucleoside (nucleotide, amino acid) is deprotonated and acts as a negative centre of interaction. Dissociation of polyamine leads to adduct decomposition (Fig. 3.7). In comparison to the system with cytidine (Cyd) the pH range of adduct formation in the solution with uridine (Urd) is significantly shifted towards higher pH values, which is a consequence of greater basicity of the endocyclic nitrogen atom from the second nucleoside (log KHCyd  =  4.5, log KHUrd = 9.2) and a different pH range of its dissociation (Fig. 3.7a, b) [8, 114, 115].

80 

 3 Complexes of biogenic amines in their role in living systems

100 [%] 80

100 [%] 80

100 [%] 80

60

60

60

40

40

40

3

20 0

(a)

1 4

6

8

2 10 pH

1 2

20 0

(b)

4

6

8

3

10 pH

2

0

3

1

20 4

6

8

10 pH

(c)

Fig. 3.7: Distribution diagram for (a): Urd/Spm, (b): Cyd/Spm, (c): CMP/Spm systems; (a): 1-(Urd)H3(Spm), 2 – (Urd)H2(Spm), 3 – (Urd)H(Spm); (b): 1 – (Cyd)H4(Spm), 2 – (Cyd)H3(Spm), 3 – (Cyd)H2(Spm); (c): 1 – (CMP)H5(Spm), 2 – (CMP)H4(Spm), 3 – (CMP)H3(Spm).

The presence of an additional reaction centre, such as a phosphate group from CMP, causes a shift in the pH range of adduct formation towards higher acidity (Fig. 3.7c). Dissociation of the first proton from the nucleotide takes place at a lower pH than that of the dissociation of nucleoside. Of importance is also the ionic composition of the solution. The presence of Na+ or K+ cations weakens the effectiveness of the interactions. The observed formation of complexes of polyamines with inorganic ions and the salt effect changing the acid-base character of the ligands [116–118] supports the thesis that formation of adducts of polyamines with other bioligands is of ion–ion and ion–dipole nature. In general, the thermodynamically stable molecular complexes have been found to form only when at least two centres of interaction between the ligands are present [107]. The NMR results have also revealed differences in the types of interactions between different polyamines and ATP molecule. It has been suggested that spermine in contrast to spermidine, prefers pyrimidine base to triphosphate group [119, 120]. Other information provided by NMR results is that in the systems of PA with amino acids the inversion effect takes place. The amine groups from PA can be positive or negative reaction centres depending on the degree of amine protonation [112, 113, 121]. The above discussed type of interactions and noncovalent interactions in the system with metal ions is essential for molecular recognition and self-assembly of biomolecules in living organisms [122, 123]. In the systems with nucleic acids, putrescine polycation at a low concentration locates in and interacts with minor and major DNA grooves, while spermidine and spermine locate only in the major groove. In contrast to putrescine the interaction of spermidine when at high concentrations is much stronger, and this amine is located in major grooves, as indicated by Infrared spectroscopy (IR) results. The authors of [124] suggest the presence of hydrophobic interactions and, apart from them, the presence of electrostatic interactions between polyamines and DNA phosphate groups. The results obtained for the system of DNA and spermine also point to the occurrence of electrostatic interactions [125, 126]. The interactions of spermine polycation in the system with DNA and metal ions have been studied by X-ray diffraction method. The polyamine

3.5 Complex formation of bioamines 

 81

joins in the crystal lattice the duplexes of nucleic acid through a system of hydrogen bonds between phosphate groups and nitrogen atoms of the bases [127]. Significant differences have been noted between the interaction of biogenic amines and their analogues. The former bind to DNA through major and minor grooves and phosphate groups, while e.g. 1,11-diamine-4,8-diazaundecane (analogue of spermine shorter by one –CH2 group) binds mainly through the N7 atom from guanine and phosphate groups. Moreover, DNA complexes with analogues of biogenic amines are thermodynamically weaker than those with spermine, spermidine, or putrescine. Bonding of amines with DNA leads to partial transformation of B-DNA to A-DNA, which has not been observed for biogenic amines [128]. The study of polyamine interactions with dendrimers has revealed that biogenic amines make more stable complexes than their synthetic analogues and the amine affinity to dendrimers depends on the charge of the polycation [129]. The reactions of a series of polyamines with phytic acid (myo-inositolhexakisphosphate acid, IP) were investigated by potentiometric measurements and 31P NMR. As a result of multifunctional noncovalent interactions, stable complexes of the type (IP)Hn(PA) of the molar ratio IP:PA 1:1 and of a different number of protons, are formed in the system. Nonselective interactions in the adducts take place between the positive protonated –NHx+ groups from polyamines and negative centres of deprotonated phosphate groups from IP [130, 131]. Biogenic amines have been also found to show antioxidative activity. Spermine is a stronger antioxidant than spermidine, which is suggested to be related to the higher ability of Spm for metal chelation and damage of radicals [132]. The role of polyamines in processes taking place in living organisms has stimulated further studies of this group of ligands. The application of complexes in medicine is described in another part of this chapter. Recently, a number of review papers have been published on the significance of PA in living systems, describing their role as necessary components of biological systems and their negative influence on the health [133–136].

3.5 Complex formation of bioamines The complexing properties of aliphatic polyamines depend on the number and nature of –NH2 groups and the length of methylene chains in the molecules. Literature data on the coordination compounds, mainly in binary systems, have been reviewed by D.A. House [137]. One of the most important and earliest studied ligands is ethylene diamine (en), which in metal-free systems assumes trans or cis (gauche) conformation, the transformation energy is low, close to 4 kJ/mol. Bonding with metal ions usually leads to formation of a five-membered ring. Such species can assume enantiomeric conformations λ (left-handed helicity) or δ (right-handed helicity). The energy barrier of λDδ inversion is low and equal to about 20 kJ/mol [138]. Although en is a typical chelating ligand, its complexes with monofunctional coordination or the complexes in which it is a bridging ligand are also known [139–143]. The number of

82 

 3 Complexes of biogenic amines in their role in living systems

stereochemical conformations in bis(en) complexes is greater. If the two ligands are trans isomers there are λλ, δδ, and λδ possible conformations. The first two are energetically equivalent, while λδ is less stable by about 4 kJ/mol [137]. The simplest triamine, diethylenetriamine (dien), forms six-membered rings with metal ions, and the compounds prefer chair-type coordination, although, particularly in systems with alkyl derivatives, the compounds making twist conformation are also known [139]. Linear triamines react with formation of meridional or facial conformation, while tripodal-type amines, RC(CH2NH2)3, where R = H, Me, Et, occur only in the facial form, both in solution [144, 145] and in solid state [146, 147]. The length of a polyamine chain has a significant effect on the character of interaction with metal ions. In solid state complexes, of ML2 type made by Cd(II) with dien, 2,3-tri and 3,3-tri, all six donor nitrogen atoms are involved in the coordination. In the complex with dien, the cadmium ion is sandwiched between two ligands of crystallographic symmetry C2, in contrast to the complexes with 2,3-tri and 3,3-tri, in which deformed octahedron was detected to be formed, Fig. 3.8 [148].

C3 C4

N2

C3ʹ C1

N3

C2

C2ʹ C1ʹ

N1 Cd1

N1A C2A

N3A N2A C1A

C4A

C4ʹ N2ʹ

N1 C1 C2

C3

N1ʹ

C6 N1

N3 N2

C5 C4

Cd(2,3–tri)2

C1ʹ C2ʹA

C5

N2

C1

N3ʹ

C3A

Cd(dien)2

C2

C5ʹ

N1ʹ Cd1

C4

C3

C2ʹB C3ʹ

Cd1

N3

N2ʹ C5ʹA C4ʹ

N3ʹ

C6ʹ C5ʹB

Cd(3,3–tri)2

Fig. 3.8: Structures of complexes in Cd(II)/polyamine systems.

Results of the equilibrium studies of metal/amine systems have been presented in a number of papers [8, 137, 142, 149–151], and also of anhydrous systems [152]. Kinetic studies of formation of complexes with polyamines and their analogues have been reported in [153–156]. In the formulae of the complexes the charge values have been omitted, unless necessary for understanding of the text. The literature on metal complexes with biogenic amines: putrescine, spermidine, and spermine is rather poor relative to that on metal complexes with other amines, especially in binary systems. Only in the end of the last century was the formation of copper(II) complexes with putrescine finally confirmed, after many years of controversies in this matter [157]. The influence of the size of the ring on processes of chelation has been observed [158]. A larger ring of Put than shorter

3.5 Complex formation of bioamines 

 83

diamines lead to downfield shift in 59Co NMR signal because of a smaller overlap of metal-ligand σ-bond [159, 160]. In a series of platinum complexes of biogenic amines and their analogues, a clear correlation between the rate of ring-closing and the size of the ring has been found [161]. The differences in reactivity of particular ligands are related rather to enthalpy than entropy of activation. In some platinum complexes amines act as a bridging ligand [162]. Results of the studies of bis(platinum) reactions have proved that dimers are kinetically more active than their monomer analogues [163]. The differences in the spectra of ternary complexes Co(NH3)5[NH2(CH2)nNH2] (n = 2, 8, 10) and Co(NH3)5[NH2(CH2)nNH3] are assigned to the formation of the charge-transfer bands, which is related to the formation of intramolecular hydrogen bonds between free amino groups and protons from coordinated amino groups [140]. After many attempts, 1,ω-diamine copper(II) complex with putrescine was successfully isolated in solid phase [164]. In the solid complex [Co(PA)3]Br3 the chair conformation of a seven-membered ring with the {N6} coordination geometry, trigonally elongated [165–167]. The seven-membered ring is stabilised by the metal ion, although in solution the conformation can change as a result of solvation effect and ion pairs formation [158]. In the systems with amines of long chains, the tendency to formation of monomeric forms and bridging structures dominates over the formation of chelate complexes [168–170]. The series of bis and tris complexes: [Ni(Put)3]Cl2·H2O, [Ni(Put)2(H2O)2]Br2, [Ni(Put)2](NCS)2, and many products of their pyrolysis are characterised by the symmetry Oh [171]. Also halogen and pseudo-halogen complexes of Zn(II), Cd(II), and Hg(II) with 1,3-diaminopropane, putrescine, and cadaverine have been isolated. Diamines behave as bridging or chelating ligands [137]. Investigation of copper(II) complexes with linear triamines has shown that the most stable complexes form five- and six-ring sequency [99, 100, 172, 173]. In the series of tetramines, the value of log K increases in the order: Spm < 3,3,3-tet < 2,2,2-tet < 3,2,3-tet < 2,3,2-tet and depends on the tension in the ring and on the ability to assume the chair or twist-chair conformation. It does not explain the low stability of spermine complex, although it correlates with the observation that Spm often behaves as two independent fragments of NH2(CH2)3NH separated by a long methylene chain, which is supported by formation of {N2} type complex with Cu(II) and Spm [111]. A similar behaviour has been reported to occur in the system with Spm analogue 4,9-dioxa-1,12-dodecanediamine. This mode of coordination permits noncovalent interactions with the protonated amine groups from Spm. The heat of formation of protonated complexes of spermidine with copper is similar to that for Cu(tn)2 complex, but much lower than the heat of formation of complexes with five-membered rings, which indicates the formation of six-membered ring in Cu(HSpd) species [174, 175]. As follows from analysis of thermodynamic data, in Cu(Spd) the six-membered ring is fused into the seven-membered ring with involvement of three donor nitrogen atoms in the coordination. The enthalpy of Cu(Spd) formation is by 12.5 kJ/mol higher than that of Cu(HSpd) formation. Stability of particular copper(II) complexes forming in the systems with

84 

 3 Complexes of biogenic amines in their role in living systems

triamines and tetramines is determined by the enthalpy and entropy, respectively [176]. Computer analysis of the potentiometric data in combination with spectroscopic analysis permits the choice of a coordination model, including the stoichiometric composition of the complexes and their thermodynamic stability. Determination of the solution structure in binary systems of metal/polyamine is relatively easy and boils down to finding the number of donor nitrogen atoms from polyamine in the internal coordination zone. Low stability of the seven-membered ring in the systems with putrescine explains the presence of monofunctional coordination in the complex and formation of the species Cu(HPut) at pH close to 7 [157]. In the systems of copper(II) ions with longer biogenic amines the metalation usually begins at pH close to 5 with formation of the six-membered species Cu(HSpd) and Cu(H2Spm). The thermodynamically less preferred seven-membered ring is stabilised by the presence of one or two six-membered rings in the Spd and Spm complexes, respectively. The formation of the three-ring chelate corresponds to a high increase in the stability constant of Cu(Spm) complex (log K = 14.70) relative to that of Cu(Spd) (log K = 11.7). Although the absorption bands in electron spectra can differ for different systems, the value λmax = 564 nm suggests the coordination with {N4} chromophore. Decrease in the ligand field strength results in a shift of the absorption energy towards weaker fields, λmax = 626 nm in Cu(Spd), {N3} coordination but λmax = 655 nm in Cu(HSpd), {N2} coordination [13, 177, 178]. For monofunctional coordination, as e.g. in Cu(HPut), λmax = 705 nm [157]. Analysis of these results points to the equatorial geometry of species in {N4} coordination. In the electron spectrum of the complex with {N5} coordination, a red shift appears as a result of localisation of the fifth nitrogen atom in the axial position [142, 177, 179]. The geometry of the complexes is supported by the studies of Cu(II), Zn(II) and Co(II) reactions with longchain polyamines [180, 181]. The above conclusions on the mode of coordination in solution correlate with the results of a study of the complexes in solid state. The isolated [Cu(Spd)]X2 (X = Cl,Br,I) assume the structure of a square pyramid with one halogen atom at the axial position [182]. The stability of triamine complexes with Hg(II) increases in the series en < tn < Put, which can explain the tendency of this metal ion to linear coordination. The tension in seven-membered ring is relatively the lowest [183]. In the series of solid state complexes of rhodium with tetramines, the shortest ligand 2,2,2-tet makes only cis-α isomer, while 2,3,2-tet makes cis-α and trans isomers, and the longer ones 3,2,3-tet, 3,3,3-tet, and Spm assume a trans configuration [184]. These differences are related to different tensions in particular rings. The size of the ring also determines the kinetic properties of the complexes. In an acidic medium, [Cu(Spd)]Cl3 immediately undergoes hydrolysis and shows increased lability of the coordinated chlorine ion with respect to the shorter amines 2,3-tri and 3,3-tri. The change in the rate of Co(PA)(H2O)n complex reduction to Co(II) increases in the order: Spd < 3,3-tri < dien, which corresponds to the changes in the activation energy [185].

3.5 Complex formation of bioamines 

 85

Binary systems were studied mainly in the 1970s and 1980s. Although many problems in bioorganic and bioinorganic chemistry have not been solved yet, the simple structure of amines soon diminished the interest in this group of compounds [186]. At present the research work has been concentrated on more complex, multicomponent model systems that are better approximations of the in vivo systems. Much attention has been paid to the interactions and processes related to the transfer of genetic information, mainly to the reactions of polyamines with nucleic acids in the model systems with DNA or RNA fragments (nucleosides, nucleotides) and metal ions [8, 179, 187, 188]. As mentioned above, the interactions of protonated amines stabilise DNA (or RNA) mainly as a result of charge neutralisation. Because of their coordinating abilities, transition metal ions should be treated as factors interfering with the system of noncovalent bonds between the negative fragments of nucleic acid and the polyamine that is a positive centre. The potential centres of the reaction of polyamine with a nucleotide simultaneously make the metalation sites (Fig. 3.9). In ternary complexes of metal/PA/PNuc (PNuc = nucleotides), besides the phosphate residues, the donor atoms N(3) from pyrimidine nucleosides and N(1) and N(7) atoms from purine nucleosides are the preferred sites of metal binding with fragments of nucleic acids.

NH2―(CH2)3―NH―(CH2)4―NH2

NH2 N 8

7

5

9

4

N R

CH2 H

4ʹ

H

3ʹ

OH

O

H

3

1

N

2

N

5ʹ

O

6

1ʹ

METAL

H OH 2ʹ

Fig. 3.9: A scheme of interactions in metal/nucleotide/polyamine systems, R = phosphate groups (red arrows indicate potential sites of interactions).

Analysis of the mode of coordination in the systems containing purine nucleosides or nucleotides shows with no doubt that simultaneous bonding of the metal with N(1) and N(7) is impossible for steric reasons. However, in a series of metal/nucleotide and metal/nucleotide/polyamine systems, the coordination dichotomy with monofunctional metalation and formation of a mixture of isomers in which the ions Cu(II), Ni(II), Co(II), Zn(II), Cd(II), Hg(II) were bonded either to N(1) or to N(7) atoms [178, 189–193].

86 

 3 Complexes of biogenic amines in their role in living systems

The presence of a phosphate group in nucleotides significantly changes the mode of interaction of this ligand with respect to that of nucleoside. For instance, in the system Cu(II)/Nuc/Spm the complexes with {N5} type coordination are formed, in which the metal ion binds four nitrogen atoms from the polyamine in equatorial position and the nitrogen atom from Nuc in the axial position (Nuc = Ado or Cyd). In the system Cu(II)/PNuc/Spm, (PNuc = AMP or CMP), metalation is realised only via the phosphate group, while spermine is engaged in noncovalent interactions with nitrogen atoms of high density from a purine base, Fig. 3.10 [111, 194].

NH NH2

Cu

NH NH2 N N

HO H H

OH

O

N N

N

(O3P)

H

Cu(Ado)(Spm)

O

H H

OH

+ NH2

NH2

N

Cu

O H OH

+ NH2

+ NH3

NH2

N N

+ NH3

NH2 NH NH2

Cu

N

NH

(O3P)

NH2 O H

H H OH

Cu(AMP)H4(Spm)

H

OH

N O

N N

H H OH

Cu(AMP)(3,3,3–tet)

Fig. 3.10: Tentative mode of coordination.

Solution structures presented in Fig. 3.10 are just a few examples from many illustrating that even small differences in the length of polyamines exert significant effects on the character of interactions determining the specificity of the reaction. Of course, also the nature of the metal influences the character of interactions in ternary systems. The effect of metal is best illustrated by the differences between Co(II) and Ni(II). The nickel (II) ions show low effectiveness of binding to phosphate groups from the nucleotide, while cobalt (II) binds both the endocyclic nitrogen atoms and coordinates with involvement of the phosphate groups [189]. In many ternary systems, not all donor atoms from the polyamine are engaged in the metal ions binding in MLL �Hx species (L = polyamine, L � = nucleotide) that permits the appearance of noncovalent interactions with the protonated –NHx+ groups from the polyamine, as illustrated in Fig. 3.11. Such an intramolecular interaction has a stabilising effect on the complex [8, 110, 111, 195]. Also intramolecular interactions between ligands in the inner coordination sphere of the species metal/nucleotide and the polyamine located in the external sphere lead to the formation of molecular complex of ML····L � type [196, 197]. The mode of interaction is significantly dependent on the concentration of polyamine [198].

3.5 Complex formation of bioamines 

NH2

NH2 NH2+ + NH2

N

Cu

N

N

(PO3)3 O H H

O

+ NH3

N

H

Fig. 3.11: Tentative mode of coordination in Cu(ATP)H3(Spm).

H OH

OH

+ H2N

+ H3N

N

H H

OH

O

+ H2N

+ NH3

NH2 N

O3P2– O

 87

N

N

H2N

N

Cu

N (O3P)

O

H H OH

(AMP)H3(3,3–tri)

H H

OH

O

+ NH3

NH2 N N

H H OH

Cu(AMP)H2(3,3–tri)

Fig. 3.12: Tentative mode of interaction.

The formation of ML····L � type complexes has been also observed in the systems of metal/polyamine/amino acid [112, 113]. The influence of copper ions on the copper srtructure is also illustrated on Fig. 3.12. The introduction of Cu(II) ions to the binary system eliminates the phosphate groups from noncovalent interactions [110]. However, polyamine can be treated as an agent interfering into the metal-nucleotide interactions. In the system Cu(II)/CMP, Cu(II) ions bind the phosphate group from the nucleotide and the endocylic N(3) atom. The polyamine introduced into the system blocks N(3) atom as a result of the noncovalent interaction, while the copper ion coordinates to the donor nitrogen atom from putrescine and to the phosphate group from CMP [110]. Moreover, uridine introduced to the ternary system of Cu(II)/ ATP/3,3,3-tet is involved in noncovalent interactions with the purine base from the nucleotide, while the presence of metal ions and the polyamine changes the specific mode of interactions of complementary bases (according to Watson-Crick), observed in the metal-free system, Fig. 3.13 [199].

88 

 3 Complexes of biogenic amines in their role in living systems

OH

H

OH

H HO

O

HN

H

H

NH Cu

Space of the effective weak interaction

N

H2N (PO3)3 O

O NH O

Space of the effective weak interaction

NH2

N

H2N

N

N

O

H

OH

H N

H

H

OH

Fig. 3.13: Tentative mode of interaction in Cu(ATP)(3,3,3-tet)H(Urd).

3.6 Application of polyamine complexes in medicine The majority of currently used therapeutic drugs are organic compounds, while much less attention has been paid to the effect of inorganic compounds. Metals in coordination or organometallic compounds offer great chemical diversity and thus larger therapeutic use [4]. Inorganic compounds have been shown to be particularly effective in therapy for malignant forms of neoplasms, as their activity is based on the specific interaction with DNA, which leads to cell damage and eventually cell death [200–206]. The coordination compounds of platinum have been used for chemotherapy since 1978, when cisplatin (cis-diamminedichloroplatinum (II)) was first introduced in the USA as a component of chemotherapeutic treatment [207]. Although thousands of mononuclear analogues of cisplatin have been synthesised and tested as potential anticancer drugs, only two compounds, carboplatin and oxaliplatin, have been used in clinical treatment of neoplastic diseases, while other analogues were therapeutically inactive [200, 207–223]. It is known now that the antineoplastic properties of platinum complexes are related to their selective reaction with DNA and possible involvement in the formation of bridging systems with donor nitrogen atoms N(7) and N(1) [210, 224], which affects the processes of replication and transcription. The very promising results of the studies on mononuclear complexes of platinum have aroused interest in the polynuclear platinum complexes containing two or three metallic centres. The polynuclear compounds may be more cytotoxic because they cause considerable irreparable damage to DNA. From among them, the polynuclear platinum complexes containing bridging polyamines make a new class of compounds potentially more effective in antineoplastic diseases

3.6 Application of polyamine complexes in medicine 

H N

H2 N Pt

Cl

NH

NH2

NH2 Pt

H 2N

Cl H 2N

Pt

H N

N H

H2N

BBR 3464 34

NH2

H N

N H

H N

N H

Cl Pt

H3N

N H

H N

N H 36

S

Cl

S

Ru

Cl

N H

S

37

S = DMSO CO

OC

NH

Re OC

Cl

H N

HN

N H

N OC

Re

OC

N H 39

N

Cl NH2

NH2

(HCl)4 NH2

NH2

N H 38

Pt

Cl

(HCl)4

H N

N H

Pt

NH2

H N

N H

NH2

H2N

BBR 3610 35 H3N

 89

(CF3COOH)8 H N

NH2

(CF3COOH)4

Cl CO

Fig. 3.14: Polyamine–transition metal complexes with antitumour activity [99].

therapy than the hitherto used ones, Fig. 3.14 [211, 215, 217, 224]. The introduction of biogenic amines to the first generation platinum complexes has an essential influence on their cytotoxic properties [217].

90 

 3 Complexes of biogenic amines in their role in living systems

The cytotoxic effect of these compounds is higher than that of cisplatin [209, 215, 225, 226]. Dimers of this new class of complexes show a wider range of activity than their monomer analogues because of the possibility of intra- and inter-strand cross-links formation [211, 215]. Small differences in the structure of polyamines were found to lead to considerable changes in the cytotoxic properties of their complexes with platinum. Besides the correlation between the structure and activity, the cytotoxic effectiveness of biomolecules also depends on the charge, flexibility, and noncovalent interactions [206, 215]. One such trinuclear Pt(II) compounds, BBR3464, successfully passed clinical trials, Fig. 3.15 [227]. Also, cytotoxicity of the new complexes of polyamines with platinum (IV) has been tested [224, 228, 229]. Therapeutic possibilities of platinum (IV) complexes are similar to those of platinum (II) because, as has been suggested, in vivo the Pt(IV) ions are reduced. In the search for anticancer drugs, the better solubility of platinum (IV) compounds should be taken into account [216]. Many polyfunctional chelates containing di, tri, or tetramines as linkers have been the subject of intense studies [225, 230–236]. Intense studies of anticancer therapeutic drugs of a wide pharmacological spectrum of activity, high therapeutic profile, and low toxicity have shown that polynuclear platinum complexes are very promising candidates for replacement of cisplatin and carboplatin in therapeutic applications in which the latter are ineffective. Biogenic amines, putrescine, spermidine, and spermine and their N-alkylated correspondents are used in particular as bridging ligands in the complexes of Pt(II) and Pd(II) [212, 215, 216, 233, 237–251]. The presence of connectivities of this type permits the occurrence of untypical mechanisms of interaction with DNA such as “long-distance” inter- and intrastrand crosslinks inside the DNA helix [252], which contribute to improvement in the anticancer activity shown by some of the complexes, e.g. they give a positive response in the treatment of cancers usually resistant to cisplatin [253, 254]. Besides the complexes with platinum, also those with palladium (II) have been tested for cytotoxic activity. The value of ID50 of palladium (II) complexes with putrescine and spermidine is better than for cis-DDP, but much worse for the complexes with spermine, which corresponds to the fact that the latter is not able to produce conformational changes in DNA [212, 213]. Higher anticancer activity of the compounds in which Pd(II) has replaced Pt(II) as the central atom, has been already described in literature [255–257]. A number of trinuclear complexes of polyamines with Pt(II) and Pd(II) have been synthesised and tested for the structure-activity relationship with regard to their potential cytotoxic properties [258]. Complexes of palladium have been found to reduce the cell activity of the ornithine decarboxylase enzyme more then complexes of platinum [259]. The already recognised functions of polyamines in the processes taking place in living organisms include not only the effect on cell growth and differentiation or cell death, but also the protection of nucleic acids against damage caused by reactive oxidative species (ROS) generated by different substances and also by transition metal ions Cu(II) and Fe(II) [260–262].

3.6 Application of polyamine complexes in medicine 

 91

Moreover, the influence of BBR3464 complex on the shortening of cell lifetimes was much lower than that produced by cisplatin [253]. The application of polynuclear compounds has brought promising therapeutic effects in the treatment of melanoma, pancreatic, lung, and ovarian cancers [263–265].

4+

(a)

(b) 8+

Pt C N

Cl H

Fig. 3.15: Polyamine-bridged polynuclear Pt(II) complexes: BBR3464 (Triplatin) [254] (a), Pt2Spm (Spm = spermine) [240] (b), and TriplatinNC [267] (c) [247].

The cytotoxic effect on the cell lines of human cancer (HSC-3) has been also checked for the dinuclear complex of Pd(II) spermine (Spm) chelate, (PdCl2)2(Spm) and compared with the effect of the earlier described Pt(II) analogues [215]. The results confirmed higher cytotoxic effect of the compounds with Pd(II) than those with Pt(II) [266]. It is known that Pd(II) complexes are usually very labile and their deactivation by cis-trans isomerisation is unlikely. Spermine makes extremely strong chelate bonds with Pd(II) because of a high chelating effect [240]. Spm, while strongly bonded to Pd(II), imposes the cis coordination on the labile ligands such as e.g. Cl−, and prevents such deactivation. Deactivation as a result of interaction with the cell components other than DNA, thanks to the high lability of Pd(II) complexes, is possible. For the complexes with Pd(II), the range of ligands undergoing exchange is much wider than for the analogous species with Pt(II). It is important that inside the cell,

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 3 Complexes of biogenic amines in their role in living systems

N

HN

(CH2)4

Pd Cl

Cl

Cl

Cl Pd N

NH Fig. 3.16: Tentetive mode of coordination in (PdCl2)2(Spm).

Fig. 3.16, (PdCl2)2(Spm) undergoes a fast hydrolysis that permits the appearance of hydrated species (e.g. thiols) that interact with cell components other than DNA more strongly and earlier than with DNA. Although the rate of water exchange is 106 times higher for Pd(H2O)42+ than Pt(H2O)42+ [240, 250], for other systems the Pd(II) and Pt(II) complexes may show the same rate of ligand exchange [268–270]. The effect of polyamine analogues, N1,N11-bis(ethyl)norspermine (BENSpm) and N1-cyclo-propylmethyl-N11-ethylnorspermine (CPENSpm), as well as the synthesised dinuclear complexes Pd2(BENSpm), Pt2(CPENSpm), and Pd2(Spm) on the heathy cells of breast epithelial MCF-10A and the breast cancer cell lines JIMT-1 and L56BR-C1, has been studied to evaluate their activities. It has been established that palladination of BENSpm increases cytotoxicity, while platination of CPENSpm leads to its reduction. Moreover, Pd2(BENSpm) was the most effective compound damaging DNA and reducing the population of bacteria colony. However, Pt2(CPENSpm) and Pd2(Spm) show lower toxicity in all tests. Pd2(Spm) efficiently reduces the level of cell glutathione, which probably causes their metabolic deactivation by linking thiols to their endogenic parts. The formation of many cross-link bonds connectivities with DNA leads to distortion of the DNA particle, thus the key biological processes such as DNA replication and transcription are inhibited, which causes inappropriate synthesis of proteins and inhibition of cell proliferation [271, 272]. The problems related to side effects such as neurotoxicity, nephrotoxicity, and development of an acquired resistance to cisplatin are the main factors limiting the effectiveness of therapy with this drug [271, 272]. Another group of profound interest in experimental cancer research is that of polyamine analogues [273]. Platinum-based therapeutic drugs, such as cisplatin, are very effective but show undesirable side effects, and their effectiveness is limited to a few types of cancer cells. Therefore, intense research work on development of new drugs of a wider range of activity and lower toxicity is continued. The effectiveness of cisplatin and its analogues (II generation drugs) as anticancer drugs make them the basis for designing alternative drugs showing other modes of activity and less acute side effects [274–277]. The medical use of such metals as Ag, Au, and Cu is well known. Au(III) complexes with porphyrin,

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dithiocarbamate, and polyamine show in vitro high stability constants and much effort has been made to evaluate the anticancer effects of such complexes [278–280]. As yet, the main group of inorganic anticancer drugs includes the compounds containing platinum (II) and platinum (IV), palladium (II), gold (I) and gold (III), ruthenium (II) and ruthenium (III), bismuth (III), rhenium (I), and copper (II) as well as gallium (III) and tin (IV). Many of them show in vitro much higher cytotoxicity than cisplatin [281].

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[225] Farrell N. Nonclassical platinum antitumor agents: perspectives for design and development of new drugs complementary to cisplatin. Cancer Invest 1993;11:578–89. [226] Jansen BAJ, Van der Zwan J, Reedijk J, Den Dulk H, Brouwer JA. A tetranuclear platinum compound designed to overcome cisplatin resistance. Eur J Inorg Chem 1999;9:1429–33. [227] Calvert AH, Thomas H, Colombo N, Gore M, Earl H, Sena L, Camboni G, Liati P, Sessa C. Phase II clinical study of BBR 3464, a novel, bifunctional platinum analogue, in patients with advanced ovarian cancer. Eur J Cancer 2001;37:260–7. [228] Souzu H. Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase. Biochem Biphys Acta 1986;861:353–60. [229] Alvarez-Valdes A, Perez JM, Lopez-Solera I, Lannegrand R, Continente JM, Amo-Ochoa P, Camazon MJ, Solans X, Font-Bardia M, Navarro-Ranninger C. Preparation and characterization of platinum(II) and (IV) complexes of 1,3-diaminepropane and 1,4-diaminebutane:  Circumvention of cisplatin resistance and DNA interstrand cross-link formation in CH1cisR ovarian tumor cells. J Med Chem 2002;45:1835–44. [230] Nishioka K. Polyamines in Cancer: Basic Mechanisms and Clinical Approaches, Springer, Berlin, Germany, 1966. [231] Qu Y, Scarsdale NJ, Tran MC, Farrell NP. Cooperative effects in long-range 1,4 DNA-DNA interstrand cross-links formed by polynuclear platinum complexes: An unexpected syn orientation of adenine bases outside the binding sites. J Biol Inorg Chem 2003;8:19–28. [232] Chvalova K, Kasparkova J, Farrell N, Brabec V. Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes. FEBS J 2006;273:3467–78. [233] Monti E, Gariboldi M, Maiocchi A. Cytotoxicity of cisplatinum(II) conjugate models. The effect of chelating arms and leaving groups on cytotoxicity: A quantitative structure-activity relationship approach. J Med Chem 2005;48:857–66. [234] Costa Couri MR, Vieira de Almeida M, Soares Fontes AP. Synthesis of polyamines from ethylenediamine and their platinum(II) complexes, potential antitumor agents. Eur J Inorg Chem 2006;9:1868–74. [235] Liu Q, Qu Y, van Antwerpen R, Farrell N. Mechanism of the membrane interaction of polynuclear platinum anticancer agents. Implications for cellular uptake. Biochem 2006;45:4248–56. [236] Williams JW, Qu Y, Bulluss GH, Alvorado E, Farrell NP. Dinuclear platinum complexes with biological relevance based on the 1,2-diaminocyclohexane carrier ligand. Inorg Chem 2007;46:5820–2. [237] Komeda S, Moulaei T, Chikuma M. The phosphate clamp: A small and independent motif for nucleic acid backbone recognition. Nucleic Acids Res 2011;39:325–36. [238] Fiuza SM, Amado AM, Oliveira PJ, Sardão VA, Batista de Carvalho LAE, Marques MPM. Pt(II) vs Pd(II) polyamine complexes as new anticancer drugs: A structureactivity study. Lett Drug Des Discov 2006;3:149–51. [239] Marques MPM, Girão T, Pedroso de Lima MC, Gameiro A, Pereira E, Garcia P. Cytotoxic effects of metal complexes of biogenic polyamines. I. Platinum(II) spermidine compounds: Prediction of their antitumour activity. BBA-Mol Cell Res 2002;1589:63–70. [240] Soares AS, Fiuza SM, Gonçalves MJ, Batista de Carvalho LAE, Marques MPM, Urbano AM. Effect of the metal center on the antitumor activity of the analogous dinuclear spermine chelates (PdCl2)2(Spermine) and (PtCl2)2(Spermine). Lett Drug Des Discov 2007;4:460–3. [241] Navarro-Ranninger C, Perez JM, Zamora F, Gonzalez VM, Masaguer JR, Alonso C. Palladium(II) compounds of putrescine and spermine. Synthesis, characterization, and DNA-binding and antitumor properties. J Inorg Bioch 1993;52:37–49. [242] Navarro M, Peña NP, Colmenares I, González T, Arsenak M, Taylor P. Synthesis and characterization of new palladium-clotrimazole and palladium-chloroquine complexes showing cytotoxicity for tumor cell lines in vitro. J Inorg Biochem 2006;100:152–7.

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Małgorzata T. Kaczmarek

4 Synthetic aspects, crystal structures and biological activities of d- and f-metal salen-type complexes Salen-type Schiff bases derived from salicylaldehyde and diamines have numerous applications, such as fluorgenic agents, pesticides, herbicidal agents [1], and ­ion-selective electrodes for the determination of anions in analytical samples [2]. The  interest in Schiff base ligands has grown recently because of their antitumor, antibacterial, antivirus, and antifungal activity improved by coordinating ligands to a metal ion [3, 4]. Transition metal complexes with salen-type ligands have applications in heterogeneous and homogenous catalysis [5–8], diagnostic pharmaceuticals and laser technology [9]. Cobalt Schiff base complexes are investigated as a models for Cobalamine (B12) coenzymes classified as oxygen carriers. They have been applied as a catalyst for the preparative oxygenation of phenols and amines [10]. The interest in salen-type complexes have been also increased because of their magnetic and optical properties [11]. Requirements of metal ions determine the structure of metal complexes. Lanthanide ions show high coordination numbers as well as variable and flexible coordination environments, therefore they have great potential in the synthesis of novel crystal structures [12–13]. One of the main factors contributing to the structural changes experienced by lanthanide ions is the well-known lanthanide contraction. The lanthanide contraction is associated with the systematic decrease in ionic radius that takes place when increasing the atomic number throughout the series of lanthanides. Its result is a decrease in the coordination number of the metal ions; the coordination number for the light lanthanides is 10 or 9, while it is 9 or 8 (sometimes 7) for the heavy ones [14–17]. Besides the metal ions’ requirements, the structure of ligands is very important for the development of new coordination compounds. Flexible salen-type ligands may provide more potential for the construction of unique frameworks because of their freedom of conformation, and they can show different coordination modes with metal ions in the construction of supramolecular frameworks [18]. For example, it has been found that a flexible salen-type ligand has been used to obtain infinite one- and two-dimensional coordination polymers [19–20]. However, when a rigid salen-type ligand was used, finite monomeric complexes were obtained [21]. The self-assembly process is another factor determining the structure of the d- and f-electron compounds. This process, which leads to organisation of simple molecules in more complicated structures, involves noncovalent interactions, such as electrostatic, π–π interactions, Van der Waals, donor-acceptor,­ and hydrogen bonding [22]. Among the supramolecular interactions, ­ hydrogen bonding is one the most effective instruments that organise building blocks into supramolecular structures. The number of hydrogen bonds and arrangement of the donor and acceptor groups determines the strength of hydrogen bonded c­ omplexes.

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The structures of salen-type complexes are influenced by a variety of factors such as the type of metal ions, metal ionic radius, the nature of counter ions, and pH of the environment.

4.1 Synthesis and crystal structure of d-metal salen-type complexes One of the methods for preparation of salen-type Schiff base complexes is a metalpromoted one-step (template) condensation reaction. The method has been used for many years for the synthesis of Schiff base macrocyclic compounds. A metal ion is used as a template to induce orientation of the reacting groups of linear substrates in the required conformation for the ring to close [23]. This method has been found very effective for obtaining salen-type complexes. The template reaction of salicylaldehyde with 4-methyl-1,3-phenylenediamine in ethanol in the presence of zinc chloride provided Zn(II) salen-type complex (1) containing N,N ′-bis(salicylidene)-4-methyl-1, 3-phenylenediamine (Fig. 4.1). CH3 N OH

N OH

Fig. 4.1: N,N′-bis(salicylidene)-4-methyl1,3-phenylenediamine [24].

In the crystal structure of the complex, Zn(II) ion is four-coordinated and bonded to neutral salen ligands and two chlorides. Generally, salen-type ligands are fourcoordinated, but in this case only oxygen donor atoms are involved in the coordination (Fig. 4.2) [24]. Another method for the preparation of salen-type Schiff base complexes is direct synthesis. The first step of direct synthesis is the preparation of an organic ligand, while the second step is the reaction of the ligand and a metal ion. This method was used to prepare of the homometalic salen-type coordination polymer of Zn(II) (2) or Co(II) (3) ions with 3-formyl-4-hydroxybenzoic acid. The crystal structure of (2) reveals that this compound 3D framework formed of tetranuclear [Zn4(µ4-O)(carboxylate)8] units. One Zn(II) ion is located at the center of the salen ligand and is coordinated with two nitrogen and two oxygen atoms from the ligand and the coordination resembles distorted square-planar geometry, while the other Zn(II) ion exhibits distorted tetragonal pyramid geometry. The crystal structure of (3) reveals that this compound adopts a 1D chain structure constructed of Co(II) ions and a ligand derived from the hydrolysis to one imine group of ligand. Co(II) ion shows octahedral geometry [25].

 109

4.1 Synthesis and crystal structure of d-metal salen-type complexes  

C35B C35A C36A

C37B C34A C33A

C37A C38A

034B

C41A C36B

C34A

C32A

N31A

C4A

C5A

N31B C38B

C32B

C2B

C1A

C2A

Cl2

N11A

Zn1

C12A C13A C18A

O14A C14A C15A

C17A

C16A

C4B C5B

C3B

C6A

C3A

C41B

C6B C1B N11B

O14B C14B

C12B C13B C18B

Cl1

C17B

C15B C16B

Fig. 4.2: Crystal structure of (1) [24].

Other examples of salen-type complexes are compounds of Ni(II) (4) and Co(III) (5) with the pentadentate H2L Schiff base ligand – 2,2′-{(methylimono)bis[propane-3, 1-diylnitrilomethylylidene]} diphenolate. The complexes were obtained in the reactions of nickel(II) perchlorate or cobalt(III) perchlorate with the pentadentate ligand in the presence of azide ion and acetonitrile. In the presence of a Ni(II) ion a binuclear compound containing m1,3-N3 bridge was obtained, while in the presence of a Co(II) ion, a mononuclear octahedral complex was produced. In both complexes metal ions are six-coordinated and adopt a distorted octahedral geometry. In (4) the main structural unit can be described as a binuclear species comprising two asymmetric [Ni(LH)]+ units, connected through a m1,3-N3 bridge [26]. The new one-dimensional manganese(III) complex (MnL(bix)]ClO4·2H2O (6) (H2L = N,N′-bis(salicylidene)phenylenediamine; bix = 1,4-bis-(imidazol-1-ylmethyl) benzene) has been synthesized. Crystal structure has revealed that Mn(III) ion is coordinated by the tetradentate ligand L2- in the equatorial plane, while 1,4-bis-(imidazol1-ylmethyl)benzene ligand acts as a bridge. The Mn(III) ion is distorted octahedral coordinated by N2O2 donors of one deprotonated ligand L2- and two N donors from two different bix ligands. 1,4-bis-(imidazol1-ylmethyl)benzene ligands function as a bridge linking monomeric [Mn(L)2]+ units into a 1D chain [27].

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 4 Synthetic aspects, crystal structures and biological activities

New five complexes of Cr(III) [CrL1(en)]Br0.3Cl0.7 (7), [CrL1(pr)]Cl (8), [CrL2(en)]ClO4 (9), [CrL2(pr)]Cl (10) and [CrL1(m-OMe)]2 (11) with ligands: N,N′-bis(2-hydroxybenzyl­)-1, 2-ethanediamine (H2L1) and N,N′-bis(2-hydroxybenzyl)-1,3-diaminepropane (H2L2) have been obtained and characterized. All the complexes could be isolated as single crystals by a solvent (MeOH) evaporation process except (8). The solid-state structures of the mononuclear and dinuclear complexes (7), (9), (10), and (11) were determined by X-ray crystallographic studies. (7), (9), and (10) crystallize as a mononuclear species with the Cr(III) ion chelated by L(1)2- – tetradentate and 1,2-ethanediamine­(en) bidentate ligands, L(2)2- – tetradentate and 1,2-ethanediamine ligands and by L(2)2- – tetradentate and 1,3-propanediamine (pr) bidentate ligands, respectively. (11) crystallizes as a dinuclear species, each chromium metal center is ­chelated by L(1)2- – tetradentate ligand and connected to one another via two cismethoxy­bridging groups. The metals’ center Cr(III) adopts a slightly distorted octa. The hydrogenation of tetradentate Schiff bases increases ligand hedral geometry­ flexibility. The combination of polydentate ligands (L(1)2-, L(2)2-, 1,2-ethanediamine and 1,3-propanediamine) enhances the stability of Cr(III) complexes system by chelate effect. The difference of stability of these complexes is mainly due to the size of the chelate rings changing from five-membered for 1,2-ethanediamine to six-membered for 1,3-propanediamine [28].

4.2 Synthesis and crystal structure of metal salen-type complexes The one-pot metal-promoted reaction between salicylaldehyde and 4-methyl-1, 3-phenylenediamine in the presence of lanthanum(III) or gadolinium(III) nitrate gave new salen-type complexes [Ln2(H2L)4(NO3)3] (La (12) and Gd (13)) containing N,N′-bis(salicylidene)-4-methyl-1,3-phenylenediamine (H2L) (Fig. 4.1). Crystal structure analysis has revealed that the complex exists as a centrosymmetric binuclear compound at the 2:4 metal-ligand ratio. Ln(III) ions are nine-coordinated, and the ­geometry around the central ions can be described as a distorted tricapped trigonal prism. Two ligands acting as bidentate chelators bridge the lanthanide ions, while the third ligand appears to be monodentate involving only one oxygen atom in ­coordination, the nitrogen atoms remain uncoordinated. The second oxygen atom of this ligand does not take part in any intermolecular interactions. Additionally, each metal ion is coordinated by three bidentate nitrate counterions (Fig. 4.3) [29]. The result of the one pot template (metal-promoted) reaction of salicylaldehyde and putrescine (1,4-butanediamine) in the presence of lanthanum(III) ion was [La(H2L)2(NO3)3] (14), where H2L = N,N′-bis(salicylidene)-1,4-(butanediamine). The single-crystal X-ray analysis has revealed that complex (14) crystallized as an infinite [La2(H2L)4(NO3)6]∞ polymeric structure. The La(III) ions are ten-coordinated, with the

4.2 Synthesis and crystal structure of metal salen-type complexes  

C38a

O34b

C41b C4b C5b C6b

C2b C1b

C12b C18b

N31b O23 C3b

C13b

N11b

O31

O14b

N3 O33

C14b C15b

C17b

N2

O22

C41a

C35b C34b

C36b

O12

O21

O14a C16a

C36a

C33a

N31a

C34a

C35a

O34a

C3a

C12a C13a C18a

O11 Gd1

C37a

C1a

N11a O13

N1

C4a C2a

C33b C38b C6a C32b

C15a O32

C5a

C37b

C32a

 111

C14a C17a

C16b

Fig. 4.3: Crystal structure of (13) [29].

coordination polyhedron, which resembles a distorted bicapped dodecahedron, and only oxygen atoms are involved in the coordination [30]. Also new lanthanide salen-type complexes of the formula of [Ln(H2L)(NO3)3] (H2L), where  Ln = La(III) (15), Nd(III) (16), Eu(III) (17), Gd(III) (18), Ho(III) (19), Er(III) (20), Tb(III) (21), Yb(III) (22) and H2L = N,N′-bis(salicylidene)-4-methyl-1,3phenylenediamine­ (Fig.  4.1) were obtained in a one pot template (metal-promoted) synthesis. All the complexes are isostructural. The Ln(III) ions are in the ninecoordinate environment with a coordination polyhedron that can be described as a tricapped trigonal prism. The three neutral ligands are coordinated with the metal ion, while the fourth ­molecular ligand is built into the crystal structure, but it is not bonded to the lanthanide ion (Fig. 4.4). Interestingly, in all ligand molecules only one oxygen and none of the ­nitrogen atoms are involved in the coordination.

Fig. 4.4: Crystal structure of the representative (16) [21].

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 4 Synthetic aspects, crystal structures and biological activities

The additional H2L ligand molecule acts as a guest in the salicylaldimine complex host and stabilizes the overall self-assembled supramolecular network [21]. The gadolinium(III) nitrate complex [Gd(H2L)(NO3)3(EtOH)]MeOH (23), where H2L = N,N′-bis(5-methylsalicylidene)-4-methyl-1,3-phenylenediamine (Fig. 4.5), was obtained in one pot template (metal-promoted) synthesis, too. CH3 H3C

N OH

CH3

N OH

Fig. 4.5: N,N′-bis(5-methylsalicylidene)-4-methyl-1, 3-phenylenediamine H2L.

Single-crystal X-ray diffraction analysis has shown that the central Gd(III) ion is ninecoordinated, and as in the example above, the coordination resembles a distorted tricapped trigonal prism. Interestingly, only oxygen atoms are involved in the coordination (one oxygen from each of the ligands, six oxygen atoms from three nitrate groups, and one from the coordinated ethanol molecule). In both ligand molecules, only one oxygen and none of the nitrogen atoms are involved in coordination. The second OH group is involved in intramolecular O-H···N hydrogen bonds. In the crystal structure there is an uncoordinated solvent (methanol) molecule. It plays an important role in the crystal packing by hydrogen bonding, as it connects the complex molecules into infinite chains (Fig. 4.6) [31].

Fig. 4.6: Fragment of the hydrogen-bonded chain of (23) [31].

It was found that the products of template metal-promoted reactions between 5-methylsalicylaldehyde and 4-methyl-1,3-phenylenediamine in the presence of lanthanide metal ions: La, Nd, Sm, Tb, Ho, and Yb were identified as three polymorphic forms of the ligand H2L, with different numbers of symmetry-independent molecules: (24) for ligand H2L and Sm, Tb, Ho, and Yb reactions, with one, (25) for La with three, and (26) for Nd with four molecules in the asymmetric part of the unit cell. The perspective view of one of the molecules of (24) is shown in Fig. 4.7 [32].

 113

4.3 Synthesis and crystal structure of the heteronuclear salen-type complexes 

C41 C5 C6

C4 C3

C1 C2

C12 C18 C171 C17

N11 C13

C14 C16

O34 C34 N31

C35

C33

C36

C32 C38

C37

O14

C371

C15

Fig. 4.7: A perspective view of the molecule H2L as determined in the structure of (24) [32].

The salen-type ligands have been proven to be particularly suitable for the synthesis of 4f SMMs (single–molecule magnets). In the reaction of hexadentate salen ligand N,N′-bis(3-methoxysalicylidene)cyclohexane-1,2-diamine (H2L) with LnCl3·6H2O (Ln = Tb(III), Dy(III), Ho(III)) the tetranuclear complexes of the formulas [Dy4(L)2(HL)2Cl2(m3OH)2]2Cl2(OH)2·3C2H5OH·H2O (27) and [Ln4(L)2(HL)2Cl2(m3-OH)2]2Cl2·5C2H5OH·4CH4Cl2 (Ln = Tb(III) (28), Ho(III) (29)) were obtained. X-ray crystallographic analysis reveals that all complexes are isostructural, and four Ln(III) ions with eight oxygen atoms form distorted defective dicubane {Ln4O8} cores. In each of two units Ln4(L)2(HL)2 two Ln(III) ions (Ln1(III) and Ln2(III)) are linked by two m3-OH in different coordination modes. The Ln1(III) ion is eight-coordinated, adopting a distorted bicapped triangular prism, while Ln2(III) ion is nine-coordinated, forming a distorted monocapped square antiprism [33].

4.3 Synthesis and crystal structure of the heteronuclear salen-type complexes of d- and f-metal ion Heterometalic polynuclear complexes (d-4f) containing d-block and lanthanide (4f) ions are currently of interest as potential new optical, electrical, and magnetic materials. The potential applications of the polynuclear Schiff base complexes are as contrast agents for medical resonance imaging, luminescent stains for fluoroimmunoassays, catalysts for the selective cleavage for RNA and DNA, and tunable photonic light-converting.

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 4 Synthetic aspects, crystal structures and biological activities

The precursor Zn-Schiff base complexes were obtained from the simple salen-type Schiff base ligands: N,N′-bis(salicylidene)ethylene-1,2-diamine or N,N′bis(salicylidene)phenylene-1,2-diamine (H2L1 or H2L2, respectively). Then, reaction of the respective precursors [ZnL1(py)] or [ZnL2(py)] with Ln(NO3)3·6H2O (Ln = Nd, Yb, Er or Gd) resulted in the formation of heterotrinuclear complexes (Zn2Ln). The eight complexes [ZnL1(py)Ln(NO3)3] (Ln = Nd (30), Yb (31), Er (32), Gd (33)), and [ZnL2(py) Ln(NO3)3] (Ln = Nd (34), Yb (35), Er (36), Gd (37)) were obtained and characterised by analytical and spectroscopic methods. The crystal structures of both neodymium complexes (30 and 34) were resolved. In both compounds, the four phenoxo oxygen atoms of two [ZnL1(py)] or [ZnL2(py)] components of (30) or (34), respectively, coordinate to one Nd(III) ion resulting in the formation of trinuclear complexes. Each Zn(II) ion has a five-coordinate environment and a distorted square pyramidal geometry. The two Nd(III) ions in (30) or (34) are nine- and ten-coordinated, respectively. Apart from phenoxo oxygen atoms, they complete their coordination environment with five or six oxygen atoms from nitrate ions, in (30) and (34), respectively [34]. Two salen-type heteronuclear Cu–Gd complexes, of the formulas [(GdCuL1Cl3 (CH3OH)2] (38) and [Gd2Cu4L24Cl2(OH)](PF6)3, (39) where (H2L1 = N,N′-bis(2-hydroxy-3methoxybenzylidene)-1,2-diaminocyclohexane and H2L2  =  N,N′-bis(2-hydroxy-3methoxybenzylidene)-1,3-diaminopropane) have been isolated. Their crystal s­ tructure analysis reveals that the molecular unit of (38) comprises one Gd(III) ion, one Cu(II) ion, one ligand, three chloride ions, and two methanol molecules. The Gd(III) ion is eight-coordinated to four O atoms from the ligand, two chloride ions and two O atoms from the methanol molecules. The Cu(II) ion is five-coordinated to two N, two O atoms of the ligand, and one chloride ion to give a pyramidal geometry. The Gd(III) and Cu(II) ions are bridged up by the two phenolate atoms. The X-ray analysis reveals that (39) features an unusual hexanuclear Cu–Gd structure. In the asymmetric unit of the complex, the Gd(III) ion is nine-coordinated to eight oxygen atoms from two ligands and one hydroxyl O atom in a mono-capped pseudo-square anti-prismatic geometry. The Cu(II) ion is five-coordinated to two N atoms and two O atoms of the ligand and one chloride ion in a pyramidal environment. The Gd(III) and Cu(II) ions are bridged by two phenolate O atoms [35]. The heterotri- and heterodinuclear complexes of the general formulae [Cu2Ln(L)2(NO3)(H2O)2](NO3)2·3H2O (where Ln = Ce (40), Pr (41), Nd (42), and La (43)), and [CuLn(L)(NO3)2(H2O)3MeOH]NO3·MeOH (where Ln = Dy (44) and Er (45)), involving the salen-type ligands H2L = N,N′-bis(5-bromo-3-methoxysalicylidene) propylene-1,3-diamine were obtained. In the crystal structure of trinuclear complexes, Cu(II) ions occupy the N2O2 cavity. The coordination polyhedron has distorted square pyramidal geometry with a water molecule in the apical position, while in the crystal structure of the dinuclear complexes, the coordination environment around the copper(II) ion is distorted octahedral. The Cu2+ ion is held within the inner N2O2 compartment of the Schiff base ligand. The apical vertices

4.4 Biological activities of d- and f-metal salen-type complexes 

 115

of the deformed octahedron are occupied by one methanol atom and one nitrate oxygen atom. In the trinuclear complexes, Ln(III) ions are ten-coordinated into a structure of geometry resembling three face- and one edge-capped trigonal bipyramid, while in the dinuclear complexes the lanthanide(III) cations are nine-coordinated by one bidentate nitrate ion, three water oxygen atoms, and four oxygen atoms of the salen-type ligand [36].

4.4 Biological activities of d- and f-metal salen-type complexes In bioinorganic chemistry, the interest in the Schiff base salen-type complexes derives from their ability to provide synthetic models for metal-containing sites in metalloproteins and to contribute to developments in medicinal chemistry. Thus, Schiff base salen-type ligands and their complexes have a variety of applications in biological, clinical, and analytical fields [37]. Metal ions have a great influence on biological processes and have been a subject of interest in medical applications. Indeed, the mechanisms of action of these metal ions are complicated, but they are believed to involve covalent bonding to the hetero-atoms of the heterocyclic residues of biomolecules, such as proteins, enzymes, and nucleic acids. Salen-type complexes can be used as anticancer agents, therefore it is very interesting and important to study different types of coordination compounds with especially designed biologically active ligands. Mn(II), Cu(II), and Zn(II) salen-type Schiff base complexes were prepared by condensation of 2-hydroxy-1-naphthaldehyde with either 4-nitrobenzene-1,2-diamine (Mn(II) (46), Cu(II) (47), and Zn(II) (48)) or 4-methylbenzene-1,2-diamine (Mn(II) (49), Cu(II) (50), and Zn(II) (51)). The cytotoxic activity of the compounds was evaluated on a human liver cancer cell line (HepG2) by MTT colorimetric assay. (46), (49), and (50) showed more potent antiproliferative activity with IC50 (concentration of test substance to achieve 50% inhibition) 1.24, 2.22, and 3.56 μg/ ml, respectively. The results were comparable with the effect of a standard drug 5-fluorouracil (IC50 4.6 μg/ml). Interestingly, the complexes exhibited better antiproliferative effect than the ligands, as their IC50 values were found to be significantly low [38]. The salen-type zinc complexes have been shown to be active as anti-tumor and anti-HIV or ­anti-bacterial agents. The zinc(II) complexes containing N,N′-bis (salicylidene)-4-methyl-1,3-phenylenediamine (H2L) ([Zn(HL)Cl(H2O)2]·C2H5OH (52) and [Zn(H2L)2Cl(NO3)(H2O)]·CH3OH) (53) and the ligand were tested for antimicrobial activity against Staphylococcus aureus in a minimum inhibitory concentration (MIC) experiment. MIC (μg/mL), which is defined as the lowest concentration of the sample that inhibits bacterial growth, was determined in sterile plates compared to the drugfree control wells. The efficiencies of the complexes and the ligand were compared with the activity of a standard antibiotic – c­ hloramphenicol. The results revealed an MIC value of 50 μg mL−3 for (52), 500 μg mL−3 for (53), and 100 μg mL−3 for the

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 4 Synthetic aspects, crystal structures and biological activities

free ligand. The activities of the complex and ligand could not reach the effective­ ness of the conventional antibacterial agent, chloramphenicol, with an MIC value of 5 μg mL−3 [3]. The interactions of the complexes of Cu(II) (54), Ni(II) (55), and Zn(II) (56) with the ligand H2L diethyl-2,2 (propane-1,3-diylbis((2-hydroxy-3-methoxy benzyl)azanediyl))diacetate, and calf thymus DNA were tested. The interactions were ­investigated by UV-Vis absorption titration, ethidium bromide displacement assay, cyclic ­voltammetry methods, and agarose gel electrophoresis. The compounds (54–56) were found to bind to calf thymus DNA through the intercalation mode. It was evidenced by a slight bathochromic shift of intraligand (π–π*) transition in the region 270–280 nm in the range of 1.6–1.7 nm. Furthermore, (54) was found most effective to promote cleavage of pUC19 DNA from the super coiled form to the nicked form in the presence of H2O2. Incubation of DNA with (54) leads to its conversion to forms II and III. In the absence of H2O2, the compounds (54–56) did not show any effect towards the cleavage of DNA. The antibacterial activities of (54–56) were tested against Gram-positive­ bacteria, Streptococcus pyogenes and Staphylococcus aureus, and Gram-negative­ bacteria, Escherichia coli, Klebsiella mobilis, Aeromonas aquariorum, and Serratia ­marcescens, by the diffusion method. Standard antibiotics, ampicillin and amoxicillin, were used as controls. It was found that the metal complexes were more effective than the ligand or metal salts but less active than the controls against all the bacteria tested. (54) showed higher antibacterial activity against Streptococcus ­pyogenes and ­Escherichia Coli than the other metal complexes, while (55) showed higher activity against Klebsiella mobilis and Aeromonas aquariorum, and (56) was found to show moderate activity towards the bacteria tested [39]. Two salen-type Schiff base complexes of cobalt(III) of the formulas [Co(L)(L1)(NCS)] ClO4 (57), where L  =  propane-1,2-diamine, L1  =  2-[N-(2-aminopropyl)ethanimidoyl] phenolate and [Co(L2)(N3)]2·4H2O (58), where L2 = 2,2′-[propane-1,2-diylbis(nitriloeth-1yl-1-ylidene)]diphenolate ion, have been tested in vitro to assess their growth inhibitory activity against two Gram-positive bacteria (Staphylococcus aureus MTCC 2940 Bacillus subtilis MTCC 441) and Gram-negative bacteria (Pseudomonas aeruginosa MTCC 2453 and Escherichia coli MTCC). Antibacterial activities of the compounds were evaluated by measuring the inhibition zone diameters (IZD, the area of media in which bacteria are unable to grow). The results of the antibacterial activities indicate that both complexes exhibit broad spectrum antibacterial activity against all the four chosen reference bacteria. The IZD (cm) data show that the ligand L is active against Streptococcus aureus and Escherichia Coli but is inactive against Bacillus subtilis and Pseudomonas aeruginosa. The Schiff base H2L2 was found to be mildly active against Bacillus subtilis and Escherichia coli but inactive against the other two bacteria. The Escherichia coli bacteria were found to be the most sensitive to all the compounds. The majority of the compounds tested show mild to moderate antibacterial activities that increase with dose. However, the activities are much lower than those of the commercial antibiotic (Gattifloxacin) at similar concentrations [40].

4.5 Conclusions 

 117

4.5 Conclusions This chapter describes methods for the syntheses of d- and f-metal salen-type complexes, characterisation of their crystal structures and biological activities. Schiff base salen-type ligands are able to coordinate many different metals and to stabilise them in various oxidation states, enabling the use of salen-type ligands to obtain compounds with numerous potential applications. The Schiff base salen-type ligands can be used to obtain mono- and polynuclear complexes and as well as supramolecular and coordination polymers. The Schiff base salen-type complexes can be obtained in two ways. One of the methods for the synthesis of salen-type Schiff base complexes is the metal-promoted one-step (template) condensation reaction. The method has been used for many years for the synthesis of Schiff base macrocyclic compounds. A metal ion is used as a template to induce the orientation of the reacting groups of linear substrates in the required conformation for the ring to close. The template condensation reactions were successfully used for the preparation of Schiff base salen-type complexes. Another method proposed for the synthesis of salen-type Schiff base complexes is direct synthesis. The first step of direct synthesis is the preparation of the organic ligand, and the second step is the reaction of the ligand and the metal ion. The stoichiometry and structures of these complexes depend on the Schiff base ligands employed in their syntheses as well as metal ionic radius, counter ions, and reaction conditions. Generally, Schiff-base salen-type ligands are four-coordinated, but sometimes only oxygen donor atom is involved in the coordination. Interestingly, it was confirmed that Schiff bases could act as neutral undeprotonated ligands in which only oxygen atom (and not the nitrogen atoms) are involved in the coordination. Jones and co-workers have shown four different bonding modes for salen-type complexes with ytterbium: I monodentate bonding to one Yb3+ ion; II tetradentate bonding to one Yb3+ ion; III bidentate bonding to two Yb3+ ions; and IV pentadentate bonding to two Yb3+ ions [18]. The results of antibacterial screening of the salen-type ligand and complexes have revealed their mild to moderate bactericidal activities. In addition to the synthetic and structural investigations, this study helps to evaluate the potentiality and effectiveness of new Schiff base complexes as antibacterial agents. Metal complexes and ligands have been tested against gram positive (e.g. Salmonella typhi, Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis) and gram negative (e.g. Escherichia coli, Aeromonas aquariorum, Serratia marcescens and Pseudomonas aeruginosa) bacteria using methods: Minimum Inhibition Concentration (MIC, μg/mL), Minimal Bactericidal Concentration (MBC, mg/mL), Inhibition Zone Diameters (IZD - Kirby Bauer Method) as a qualitative assay using a standard antibiotics e.g. Kanamycine, Chloramphenicol, Ampicillin, Amoxicillin and Gattifloxacin as reference standards. The antibacterial activity tests indicate that the complexes exhibit higher antimicrobial activities against the Gram-negative

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and Gram-positive bacteria than the free ligand. In general, the antibacterial results show that the majority of the metal complexes were more active than their respective Schiff bases salen-type ligands [41].

References [1] Wang H, Zhao P, Shao D, Zhang J, Zhu Y. Synthesis, characterization and spectra studies on Zn(II) and Cu(II) complexes with thiocarbamide ligand containg Schiff base group. Struct Chem 2009;20:995–1003. [2] Naeimi H, Safari J, Heidarnezhad A. Synthesis of Schiff base ligands derived from condensation of salicylaldehyde derivatives and synthetic diamine. Dyes Pigm 2007;73:251–3. [3] Kaczmarek MT, Jastrząb R, Hołderna-Kędzia E, Radecka-Paryzek W. Self-assembled synthesis, characterization and antimicrobial activity of zinc(II) salicylaldimine complexes. Inorg Chim Acta 2009;362:3127–33. [4] Fleck M, Karmakar D, Ghosh M, Ghosh A, Saha R, Bandyopadhyay D. Synthetic aspects, crystal structure and antibacterial activity of two new Schiff base cobalt(III) complexes. Polyhedron 2012;34:157–62. [5] Cozzi PG. Metal–Salen Schiff base complexes in catalysis: practical aspects. Chem Soc Rev 2004;33:410–21. [6] Rajabi F. A heterogeneous cobalt(II) Salen complex as an efficient and reusable catalyst for acetylation of alcohols and phenols. Tetrahedron Lett 2009;50:395–7. [7] Kleij AW. Nonsymmetrical salen ligands and their complexes: synthesis and applications, Eur J Inorg Chem 2009;193–205. [8] Lima LF, Corraza ML, Cardoza-Filho L, Màrquez-Alvarez H, Antunes OAC. Oxidation of limonene catalyzed by metal(Salen) complexes. Brazilian J Chem Eng 2006;23:83–92. [9] Papadopoulos C, Kantiranis N, Vecchio S, Lalia-Kantouri M. Lanthanide complexes of 3-methoxy-salicylaldehyde thermal and kinetic investigation by simultaneous TG/DTG–DTA coupled with MS. J Therm Anal Calorim 2010;99:931–8. [10] Kinaf AH, Mahmood WAK, Dinari M, Azarian MH, Khafri FZ. Novel nanohybrids of cobalt(III) Schiff base complexes and clay: Synthesis and structural determinations. Spectrochim Acta, Part A, 2014;127:422–8. [11] Layek M, Ghosh M, Sain S, Fleck M, Muthiah PT, Jenniefer SJ, Ribas J, Bandyopadhyay D. Synthesis, crystal structure and magnetic properties of nickel(II) and cobalt(III) complexes of a pentadentate Schiff base. J Mol Struct 2013;1036:422–6. [12] Łyszczek R, Mazur L. Polynuclear complexes constructed by lanthanides and pyridine3,5-dicarboxylate ligand: Structures, thermal and luminescent properties. Polyhedron 2012;41:7–19. [13] Wang L, Ni L, Yao J. Synthesis, structures and fluorescent properties of two novel lanthanide [Ln=Ce(III), Pr(III)] coordination polymers based on 1,3-benzenedicarboxylate and 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline ligands. Solid State Sci 2012;14:1361–6. [14] Radecka-Paryzek W, Pospieszna-Markiewicz I, Kubicki M. Self-assembly two-dimersional salicylaldimine lanthanum(III) nitrate coordination polymer. Inorg Chim Acta 2007;360:488–96. [15] Aguilà D, Barrios LA, Velasco V, Arnedo L, Aliaga-Alcalde N, Menelaou M, Teat SJ, Roubeau O, Luis F, Aromi G. Lanthanide contraction within a series of asymmetric dinuclear [Ln2] complexes. Chem Eur J 2013;19:5881–91. [16] Janicki R, Starynowicz P, Mondry A. Lanthanide carbonates. Eur J Inorg Chem 2011;3601–16.

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[17] Wang H, Wen R-M, Hu TL. Two series of lantahnide metal-organic frameworks constructed from crown-ether-like secondary building units. Eur J Inorg Chem 2014;1185–91. [18] Yang X, Jones RA, Huang S. Luminescent 4f and d-4f polynuclear complexes and coordination polymers with flexible salen-type ligands. Coord Chem Rev 2014;273–4:63–75. [19] Radecka-Paryzek W, Pospieszna-Markiewicz I, Kubicki M. Self-assembly as a route to one-dimersional lanthanum(III) salicylaldimine coordination polymer. J Rare Earth 2010;28:51–5. [20] Radecka-Paryzek W, Pospieszna-Markiewicz I, Kubicki M. Self-assembled two-dimersional salicylaldimine lanthanum(III) nitrate coordination polymer. Inorg Chim Acta 2006;366: 488–96. [21] Kaczmarek MT, Kubicki M, Mondry A, Janicki R, Radecka-Paryzek W. Self-assembly ­salicylaldimines with a unique coordination mode. Eur J Inorg Chem 2010;2193–200. [22] Lin JCY, Huang CJ, Lee YT, Lee KM, Lin IJB. Carboxylic acid functionalized imidazolium salats: sequential formation of ionic, zwitterionic, acid-zwitterionic and lithium salt-zwitterionic liquid crystal. J Mater Chem 2011;21:8110–21. [23] Radecka-Paryzek W, Patroniak V, Lisowski J. Metal complexes of polyaza and polyoxaaza Schiff base macrocycles. Chem Rev 2005;249:2156–75. [24] Kaczmarek MT, Kubicki M, Radecka-Paryzek W. Crystal structure and spectral characterization of rare example of a salen-type zinc complex with neutral monodentate oxygen donor ligands coordination. Monatsh Chem 2006;137:997–1003. [25] Hao LN, Lu Y, He ZZ, Lui ZJ, Wang E. Two new homometallic coordination polymers based on a carboxylate-functionalized salen ligand. Inorg Chem Commun 2015;55:88–91. [26] Layek M, Ghosh M, Sain S, Fleck M, Muthiah PT, Jenniefer SJ, Ribas J, Bandyopadhyay D. Synthesis, crystal structure and magnetic properties of Nickel(II) and cobalt(III) complexes of a pentadentate Schiff base. J Mol Struct 2013;1036:422–6. [27] Zhang CM, Gao XF, Zhu M, Li YG, Wang QL, Li LC. Synthesis, crystal structure and magnetic characterization of two Mn(III) chains with Schiff-base ligands. J Mol Struct 2013;1033:8–13. [28] Lui B, Chai J, Feng S, Yang B. Structure, photochemistry and magnetic properties of ­tetrahydrogenated Schiff base chromium(III) complexes. Spectrochim Acta, Part A 2015;140:437–43. [29] Kaczmarek MT, Pospieszna-Markiewicz I, Kubicki M, Radecka-Paryzek W. Novel lanthanide complexes with unusual coordination mode. Inorg Chem Communn 2004;7:1247–9. [30] Pospieszna-Markiewicz I, Kaczmarek MT, Kubicki M, Radecka-Paryzek W. Self-assembly in lanthanum(III) and calcium(II) complexes of salicyldiamine derived from putrescine. J Alloys Compd 2008;451:403–5. [31] Kaczmarek MT, Jastrząb R, Kubicki M, Gierszewski M, Sikorski M. Suplamolecular polymer of Schiff base gadolinium complex: Synthesis, crystal structure and spectroscopic properties. Inorg Chim Acta 2015;430:108–13. [32] Kaczmarek MT, Kubicki M. The trimorphic structure of N,N′-bis(5-methyl-salicylidene)-4-methyl1,3-phenylenediamine. Acta Crystallogr. Section B 2014;B70:792–800. [33] Luan f, Liu T, Yan P, Zou X, Li X, Li G. Single-molecular magnet of a tetranuclear dysprosium complex disturbed by a salen-type ligand and chloride counterions. Inorg Chem 2015;54:3485–90. [34] Bi W, Wei T, Lu X, Hui Y, Song J, Zhao S, Wong WK, Jones RA. Hetero-trinuclear near-infrared (NIR) luminescent Zn2Ln complexes from Salen-type Schiff-base ligands. New J Chem 2009;33:2326–34. [35] Xu L, Zhang Q, Hou G, Chen P, Li G, Pajerowski DM, Dennis CL. Syntheses, structures, and magnetic properties of salen type Cu–Gd dimer and hexamer complexes with strong ferromagnetic interactions. Polyhedron 2013;52:91–95.

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[36] Cristóvão B, Miroslaw B, Kłak J. N,N′-bis(5-bromo-2-hydroxy-3-methoxybenzylidene)-1,3diaminopropane Cu–4f–Cu and Cu–4f complexes: Synthesis, crystal structures and magnetic properties. Polyhedron 2012;34:121–8. [37] Sarı N, Pişkin N, Öğütcü H, Kurna N. Spectroscopic characterization of novel d-amino acid-Schiff bases and their Cr(III) and Ni(II) complexes as antimicrobial agents. Med Chem Res 2013;22:580–7. [38] Farag AM, Guan TS, Osman H, Majid AMSA, Iqbal A, Ahamed MBK. Synthesis of metal(II) [M = Cu, Mn, Zn] Schiff base complexes and their Pro-apoptotic activity in liver tumor cells via caspase activation. Med Chem Res 2013;22:4727–36. [39] Jeslin Kanaga Inba, Annaraj B, Thalamuthu S, Neelakantan MA. Cu(II), Ni(II), and Zn(II) Complexes of Salan-Type Ligand Containing Ester Groups: Synthesis, Characterization, ­Electrochemical Properties, and In Vitro Biological Activities. Bioinorganic Chemistry and Applications 2013;1–11. [40] Fleck M, Karmakar D, Ghosh M, Ghosh A, Saha R, Bandyopadhyay D. Synthetic aspects, crystal structure and antibacterial activity of two new Schiff base cobalt(III) complexes. Polyherdon 2012;34:157–62. [41] Salehi M, Amirnasr M, Meghdadi S, Mereiter K, Bijanzadeh HR, Khaleghian A. Synthesis, characterization, and X-ray crystal structure of cobalt(III) complexes with a N2O2-donor Schiff base and ancillary ligands. Spectral, antibacterial activity, and electrochemical studies. Polyhedron 2014;90–7.

Kazuma Ogawa

5 Biocomplexes in radiochemistry 5.1 Introduction Compounds are labeled with radionuclides and are used in nuclear medicine. These compounds are generally injected intravenously, after which they accumulate in target tissues, decay, and emit radiation. If the radiation produced is in the form of very high frequency electromagnetic waves, such as gamma rays or X-rays, it is highly penetrating and can be detected in patients by gamma scintigraphy, single photon emission computed tomography (SPECT), or positron emission tomography (PET). Therefore, images can be obtained that show the distribution of the radiopharmaceuticals within the body. The purpose of the images acquired in nuclear medicine is to provide functional information as opposed to anatomical information provided by other techniques such as X-ray, computed tomography (CT), and magnetic resonance imaging (MRI). Although the spatial resolutions of SPECT and PET are much lower than those of CT and MRI, SPECT and PET still provide useful information, such as imaging of processes at the molecular and cellular level and quantitative data useful in clinical assessments. If a radionuclide emits radiation which has relatively low penetration and loses its high energy within a short distance, such as beta and alpha radiation, it is suitable for use in internal radionuclide therapy for cancer. The best known internal radionuclide therapy is radioimmunotherapy. In this type of therapy, antibodies to the molecules which are over expressed on the surface of cancer cells are used as carriers of radionuclides for delivery to the tumours. For this purpose, radiolabeled antibodies are injected into cancer patients. In clinical use, two successful radiolabeled antibodies, 90Y-ibritumomab tiuxetan (Zevalin®) and 131I-tositumomab (Bexxar®), which are beta particle-emitter-labeled anti-CD20 monoclonal antibodies, have been approved by the US Food and Drug Administration (FDA) for the treatment of nonHodgkin lymphoma. In this chapter, I present some radiometal complexes used for diagnosis and therapy and discuss my recent research, focusing on radiolabeled compounds used for bone disorders and apoptosis.

5.2 Bone-seeking complexes 5.2.1 Diagnostic bone-seeking radiopharmaceuticals Bisphosphonates are synthetic pyrophosphate analogs that are stable in vivo because of their P-C-P central structure, rather than the P-O-P configuration of

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OH O P OH O P OH

O P OH H H O P OH

OH

OH

O

(a)

OH

OH

(b)

O P OH OH H 3C O P OH OH (c)

O HO P HO HO HO (d)

P

N O

OH O P OH N

OH P OH

OH O P OH H H O P OH OH

O (e)

Fig. 5.1: Chemical structures of bisphosphonates analogs (a) pyrophosphate, (b) MDP, (c) HMDP, (d) EDTMP, and (e) HEDP.

­ yrophosphates, which affords greater resistance to phosphatase hydrolysis p (Fig.  5.1a). Because bisphosphonates inhibit osteoclast-mediated bone resorption and bone turnover, they have been used in the treatment of skeletal disorders, such as osteoporosis, metastatic bone cancer, and Paget’s disease [1, 2]. ­Bisphosphonates have a high affinity to bones, especially to hydroxyapatite, which is a mineral present in bones. Bisphosphonates also have been used as carriers of radioisotopes to bones. For many years, two 99mTc-bisphosphonate complexes, 99mTc-methylenediphosphonate (99mTc-MDP, Fig. 5.1b) and 99mTchydroxymethylenediphosphonate (99mTc-HMDP, Fig. 5.1c), have been clinically used in nuclear medicine for diagnosis of bone disorders, such as metastatic bone cancer [3–5], because their high sensitivity can detect bone disorders before the occurrence of anatomical changes. Bone metastases are classified as osteolytic, osteosclerotic, or mixed types that reflect osteolytic or osteosclerotic changes caused by the highly activated osteoclasts or osteoblasts that occur in bone metastases. In addition, technetium-99m (99mTc) is one of the most important radionuclides in nuclear medicine. 99mTc has frequently been clinically used because (1) it has adequate physical half-life (T1/2 = 6.01 h) for clinical use, (2) the gamma ray energy it emits (141 keV) is appropriate for SPECT imaging, and (3) it can be produced from the radionuclide generator 99Mo/99mTc, which enables generation of 99mTc on demand. The 99mTc-bisphosphonate complex accumulates in bones because of its high affinity for hydroxyapatite in the bisphosphonate structure. It is assumed that 99mTc-MDP forms a bidentate – bidentate bridge with hydroxyapatite, whereas 99mTc-HMDP must form a bidentate – tridentate bridge because of the presence of an additional hydroxyl group on the central carbon of the C-P-C structure in HMDP and is expected to enhance the hydroxyapatite affinity of the 99mTc complex [6, 7]. The uptake mechanisms of 99mTc-bisphoshonate complexes in bone metastases have not been completely elucidated. One of the factors related to the higher tracer uptake at the metastatic sites is increased vascularity and regional distribution of blood flow associated with the disease. However, regional bone blood flow alone

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does not explain the increased uptake of 99mTc-bisphoshonate [8]. Other factors are also related to the binding and interaction between the complexes and the bones. It is known that 99mTc-bisphoshonate complexes accumulate at sites of new bone formation or calcification [9, 10]. Kanishi has reported that the accumulation mechanisms might involve both adsorption onto the surface of hydroxyapatite in the bone and incorporation into the crystalline structure of hydroxyapatite [11]. The crystalline structure of hydroxyapatite in newly formed bone is amorphous and has a greater surface area than that in normal bone [12]. Bisphosphonate compounds show significantly higher in vitro adsorption onto amorphous calcium phosphate than onto crystalline forms [8]. 99mTc is supplied from the 99Mo/99mTc generator as 99mTcO −. The oxidation state 4 99m of Tc in 99mTcO4− is +7. Bisphosphonate compounds form multiple complexes with reduced 99mTc. By using high-performance liquid chromatography (HPLC), the relative composition of 99mTc-bisphosphonate complexes in a reaction mixture has been found to vary with pH, 99Tc carrier, and oxygen concentrations [13]. Wilson et al. have assumed that 99mTc-bisphosphonate complexes would be a mixture of monomers, oxobridged dimers, and oligomeric clusters with various technetium-oxo core configurations, oxidation states, and ligand coordination numbers [14]. These 99mTc-bisphosphonate complex species have different biodistribution properties in rats. Pinkerton et al. has reported that the smallest, low charge, mononuclear 99mTc-bisphosphonate complex has the greatest uptake in bone lesions and the highest lesion-to-muscle and lesionto-normal bone ratios in experiments using each isolated complex by HPLC [15]. Although these studies were performed over a quarter century ago, the exact structures and mechanisms of action of 99mTc-bisphosphonate complexes remain unclear.

5.2.2 Development of novel diagnostic bone-seeking technetium complexes Although 99mTc-MDP and 99mTc-HMDP are most frequently used as bone scintigraphy agents, their chemical and pharmaceutical properties have not been optimised. As mentioned above, these complexes are not well-defined single-chemical species but rather mixtures of short-chain and long-chain oligomers [13]. In 99mTc-bisphosphonate complexes, the phosphonate groups are used both as ligands for complexation and as carriers of the radionuclide to bone [16], which could decrease the inherent affinity of bisphosphonate for bone. To develop superior bone-seeking radiopharmaceuticals, a more logical design strategy based on conjugation of a stable 99mTc complex with a carrier like bisphosphonate has been proposed by our research group and other groups. I will now discuss some studies that used this drug design, which allowed the ligand and carrier function to operate independently and effectively. Verbeke et al. have designed and evaluated a 99mTc-L,L-ethylenedicysteine (EC) complex, a renal tracer agent known to have rapid renal excretion, conjugated to

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HOOC

 5 Biocomplexes in radiochemistry

HN O

O

N

Tc S

S

O

OH O P OH NH O P OH OH

O

N O

N

Tc S

(a)

N

O

(b)

OH O P OH OH O P OH OH

O N H

O O H O HO

Tc N HN H

O

N

OC (d)

Tc CO

OC (f)

Tc CO

N H N N

CO

OC (e)

N H N

O

NH2

O

N N

N H

OH O P OH NH O P OH OH

O

N

N

OH O P OH OH O P OH OH

O

O

(c)

N N

CH3

N

CO

NH2

Tc CO

CO

OH O P OH OH O P OH OH OC

NH2

N

OH O P OH OH O P OH OH

OC

N

N

Tc CO

N

OH O P OH OH O P OH OH

(g)

Fig. 5.2: Chemical structures of 99mTc-complex-conjugated bisphosphonate compounds (a) 99mTc-EC-AMDP, (b) 99mTc-MAG3-HBP, (c) 99mTc-HYNIC-HBP, (d) [99mTc(CO)3(PzNN-BP)], (e) [99mTc(CO)3(PzNN-ALN)], (f) [99mTc(CO)3(PzNN-PAM)], and (g) 99mTc(CO)3-DPA-alendronate.

bisphosphonate (99mTc-EC-AMDP, Fig. 5.2a) [17]. 99mTc-EC-AMDP showed a faster blood clearance and a higher bone/blood ratio, which is an index signal/noise (S/N) ratio, relative to those of 99mTc-MDP in animal experiments.

5.2 Bone-seeking complexes 

 125

Our research group developed stable 99mTc-complex-conjugated bisphosphonate compounds: 99mTc-mercaptoacetylglycylglycylglycine (MAG3)-conjugated bisphosphonate (99mTc-MAG3-HBP, Fig. 5.2b) and 99mTc-6-hydrazinonicotinic acid (HYNIC), with tricine and 3-acetylpyridine as co-ligands conjugated to bisphosphonate (99mTc-HYNIC-HBP, Fig. 5.2c) [18]. In hydroxyapatite-binding experiments in vitro, the binding affinities of 99mTc-complex-conjugated bisphosphonate compounds to hydroxyapatite were higher than that of 99mTc-HMDP. In animal experiments, 99mTc-complex-conjugated bisphosphonate compounds showed higher accumulation in bone than did 99mTc-HMDP, which reflected the findings of hydroxyapatite binding experiments in vitro. However, the blood clearance of 99mTc-MAG3-HBP was delayed because its protein-binding rate in blood was high. Thus, the bone/blood ratio of 99mTc-MAG3-HBP was lower than that of 99mTc-HMDP. The blood clearance of 99mTc-HYNIC-HBP was similar to that of 99mTc-HMDP. The bone/blood ratio of 99mTc-HYNIC-HBP was higher than that of 99mTc-HMDP. Palma et al. developed a 99mTc-tricarbonyl complex, which is anchored by a pyrazolyl (Pz)-containing ligand, conjugated to the bisphosphonate compounds ([99mTc(CO) 3(PzNN-BP)], [99mTc(CO) 3(PzNN-ALN)], and [99mTc(CO)3(PzNN-PAM)], Figs. 5.2d–2f) [19, 20]. The ligands N2S2 and N3S have classically been some of the most useful ligands for complexation of 99mTc complexes. The 99mTc complexes have a (TcO)3+ core with technetium in its +5 oxidation state. In contrast, the 99mTc-tricarbonyl complex has a [Tc(CO) ]+ core with technetium in its +1 oxida3 tion state. 99mTc-tricarbonyl complexes, which are compact and kinetically inert, could be formed from the [Tc(CO)3]+ core with suitable ligands [21]. Palma et al. have confirmed the structures of 99mTc-tricarbonyl-complex-conjugated bisphosphonate compounds by using reversed-phase HPLC analyses. The analyses showed that the compounds had retention times identical to those of the corresponding nonradioactive rhenium (Re) complexes, which revealed the structural analogies because nonradioactive technetium does not exist (99Tc is also a radionuclide). In animal experiments [99mTc(CO)3(PzNN-BP)] showed moderate bone uptake, but the uptake was lower than that of 99mTc-MDP. In contrast, the bone accumulations of [99mTc(CO)3(PzNN-ALN)] and [99mTc(CO)3(PzNN-PAM)] were high and comparable to that of 99mTc-MDP. At 4 h after injection of tracers, [99mTc(CO)3(PzNN-ALN)] and [99mTc(CO)3(PzNN-PAM)] showed higher bone/blood and bone/muscle ratios because they had faster clearance than that of 99mTc-MDP. The differences in bone accumulation among the 99mTc-tricarbonyl complex-conjugated bisphosphonate compounds could be derived from the existence of a hydroxyl group at the central carbon of the P-C-P structure in bisphosphonate compounds because some authors have described that bisphosphonate compounds containing the hydroxyl group have higher affinity for bone [22–24]. A 99mTc-tricarbonyl complex-conjugated bisphosphonate that has a structure similar to that of [99mTc(CO)3(PzNN-ALN)] but with dipicolylamine (DPA) was developed by de Rosales et al. and used as a ligand for complexation [99mTc(CO)3-DPA-alendronate,

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Fig. 5.2g] [25]. 99mTc(CO)3-DPA-alendronate showed higher affinity for hydroxyapatite than did 99mTc-MDP in in vitro experiments. In animal experiments, the bone accumulation of 99mTc(CO)3-DPA-alendronate was as high as that of 99mTc-MDP. As mentioned above, some 99mTc-complex-conjugated bisphosphonate compounds have shown superior biodistribution of radioactivity as bone imaging agents relative to that of the existing bone scintigraphic agents, 99mTc-complex, 99mTc-MDP, and 99mTcHMDP. Consequently, the strategy and concept of 99mTc-complex-conjugated bisphosphonates could be promising for the diagnosis of bone disorders, such as bone metastases.

5.2.3 Development of novel diagnostic bone-seeking gallium complexes for PET Presently, 68Ga has attracted much attention as a radionuclide for PET because its radiophysical properties are useful clinically, particularly as a 68Ge/68Ga generatorproduced radionuclide with a half-life (T1/2) of 68 min [26]. Its production does not require an expensive cyclotron, and 68Ga can be produced on demand in a hospital. Indeed, because the half-life of the parent nuclide 68Ge is long (T1/2 = 270.8 d), the lifespan of the 68Ge/68Ga generator also must be long. The above-mentioned concept of a stable complex-conjugated bisphosphonate could also be applicable to 68Ga complexes. To develop superior PET tracers for diagnosis of bone disorders, some kinds of radiogallium complex-conjugated carriers for delivery to bone have been reported. We developed a 67Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) complex-conjugated bisphosphonate (67Ga-DOTA-Bn-SCN-HBP, Fig. 5.3a) because DOTA forms a stable complex with gallium [27]. Although the aim was to develop a superior bone-seeking 68Ga-labeled agent for PET, 67Ga was used in the initial basic study because of its longer half-life (T1/2 = 78 h). In animal experiments, 67Ga-DOTA-Bn-SCN-HBP accumulated rapidly and highly in bone but was rarely observed in tissues other than bone. Consequently, the bone/blood ratio of 67Ga-DOTA-BnSCN-HBP was comparable to that of 99mTc-HMDP. Fellner et al. have reported a human study of 68Ga-DOTA-conjugated bisphosphonate (68Ga-BPAMD, Fig. 5.3b) [28]. 68Ga-BPAMD showed high uptake in osteoblastic metastatic lesions in a prostate cancer patient. The maximum standardised uptake values (SUVmax) were 77.1 and 62.1 in the 10th thoracic and L2 vertebra, respectively, for 68Ga-BPAMD; the respective values were 39.1 and 39.2 for 18F-fluoride, which is a typical bone imaging agent for PET. Basic experiments on 68Ga-BPAMD using µ-PET and a bone metastasis rat model have been also reported [29]. 68Ga-BPAMD showed higher accumulation in metastatic bone lesions than in healthy bone of the same animal (contrast factor = 3.97 ± 1.82). Furthermore, Suzuki et al. have developed 68Ga-1,4,7-triazacyclononane-1,4, 7-triacetic acid (NOTA)-conjugated bisphosphonate (68Ga-NOTA-BP, Fig. 5.3c) [30].

5.2 Bone-seeking complexes 

O

O

O

N N

HO

OH

N Ga

OH

S

N

N H

O

O

O

O

O

O P OH OH O P OH

N H

OH

(a)

HO

N N

O

O

N Ga

OH O P OH NH

N

O

O

(b)

O P OH OH

OH

O

O

N

O Ga N

O

N O

O (c)

O

 127

O

H N

OH O P OH OH O P OH OH

O

N N

HO (d)

O

O

N H

N Ga

O

N

O

OH n

O O

Fig. 5.3: Chemical structures of 67/68Ga complex-conjugated bisphosphonate compounds (a) 67Ga-DOTA-Bn-SCN-HBP, (b) 68Ga-BPAMD, (c) 68Ga-NOTA-BP, and (d) 67Ga-DOTA-(Asp)n.

Triazamacrocyclic ligands may be more suitable for gallium complexation because of their high conformational and size selectivity. Actually, it has been reported that NOTA forms highly stable chelates and allows faster incorporation of gallium at lower temperatures than DOTA [31]. In animal experiments using Wistar rats, 68Ga-NOTA-BP showed faster clearance and a higher bone/blood ratio than did 99mTc-MDP and 18F-fluoride. Moreover, in a PET study using a mouse model of bone metastasis, 68Ga-NOTA-BP highly accumulated in osteolytic lesions in the tibia. Next, we investigated acidic amino acids as carriers of radionuclides to bone. Several major noncollagenous bone proteins, such as osteopontin and bone sialoprotein, contain repeating sequences of acidic amino acids, such as Asp or Glu, in their structures [32–34]. It has been reported that polyglutamic and polyaspartic acids have high affinities for hydroxyapatite and could be used as carriers for drug delivery to bones [35–37]. Recently, we reported 67Ga-DOTA-conjugated L-Asp peptides [67Ga-DOTA(Asp)n, Fig. 5.3d] with varying peptide lengths (n = 2, 5, 8, 11, or 14) [38]. The binding affinities for hydroxyapatite of 67Ga-DOTA-(Asp)n depended on their peptide lengths; longer peptides had higher affinities for hydroxyapatite. In biodistribution experiments

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in normal mice, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed selectively high accumulation in bones (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively). Although the bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, which suggested that bisphosphonates have higher affinities for bone than do polyaspartic acids, 67Ga-DOTA-(Asp)n showed faster blood clearance than did 67Ga-DOTA-Bn-SCN-HBP. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp) and 67Ga-DOTA-(Asp) were comparable to that of 67Ga-DOTA-Bn11 14 SCN-HBP. These results suggest that not only bisphosphonate molecules but also acidic amino acid peptide sequences could be useful as carriers of radionuclides to bones. Moreover, the use of radiometal complex-conjugated carrier molecules for delivery to bones could be a useful approach for the development of 68Ga PET tracers for bone disorders such as bone metastases.

5.2.4 Bone-seeking radiopharmaceuticals for palliative therapy of bone metastases Gamma ray emitting radionuclide- and positron emitting radionuclide-labeled boneseeking agents are used for the diagnosis of bone disorders such as bone metastases. Bones are one of the most common organs affected by metastatic cancer because of the presence of numerous growth factors in them [39, 40]. Especially, certain cancers, such as breast cancer, lung cancer, and prostate cancer, have a tendency to metastasize to bones. Most cancer patients in late phases of bone metastases suffer severe pain, but the pathophysiology is not well understood, and multiple mechanisms have been postulated. Control of metastatic bone pain is very important for improving patients’ quality of life [41–43]. As palliative treatments, nonsteroidal anti-inflammatory drugs (NSAIDs) are the first option for most patients, followed by progression to opioids as the intensity of pain rises by using a three-step “ladder” proposed by the WHO for cancer pain relief. However, it is difficult to control pain in patients with bone metastases. After the initial standard palliative treatment, about half of these patients continue to suffer from substantial bone pain [44]. Moreover, although efforts are made to decrease the side effects of drugs used for pain palliation, these drugs can produce unwanted side effects, such as gastrointestinal ulceration, neutropenia, enhanced bleeding, and disruptions in renal function in the case of NSAIDs, and nausea, sedation, and constipation in the case of opioids. Localised radiation therapy from an external source is an effective method for palliation of metastatic bone pain [45]. Localised radiation therapy decreases metastatic bone pain in many patients. However, because most patients with bone metastases have multiple metastatic lesion sites, treating patients, bone metastases with multiple metastatic lesions with localised radiation therapy is not easy. In that case, internal radiotherapy using bone-seeking compounds labeled with high-energy beta or alpha particle-emitter

5.2 Bone-seeking complexes 

 129

radionuclides has been shown to be an effective alternative that has fewer side effects than those associated with other treatments [46]. Pain palliation is achieved from beta or alpha particles emitted from radionuclides, but the mechanism of this pain palliation has not been elucidated. Because beta or alpha particles cause damage to tumour cells, palliation effects might occur because of a reduction of mechanical pressure. However, the palliative effects are usually observed before the tumour mass is reduced. Because it is known that lymphocytes, which are radiation-sensitive cells, secrete many kinds of cytokines related to pain, palliation effects could be derived from not only reduction of tumour cells but also damage to lymphocytes in the tumour tissue. I will next discuss the approved radiopharmaceuticals and recent research related to radiolabeled compounds for palliative therapy. 89SrCl2 (Metastron®) was the first radiopharmaceutical approved by the FDA for the palliation of metastatic bone pain. Strontium (Sr) and calcium (Ca) are alkaline earth metals that are members of family IIA on the periodic table. It is known that the characteristics of Sr and Ca are similar and that Sr accumulates at sites of high osteoblastic activity through incorporation into mineralizing collagen during new bone formation [47]. 89Sr has a long physical half-life of 50.5 d and emits high-energy beta particles, with a maximum energy of 1.46 MeV, and 0.01% gamma rays, with an energy of 910 keV. The usual dose of 89SrCl2 is 148 MBq (4 mCi) or 1.5–2.2 MBq/kg body weight. It has been reported that there is no dose-dependence for pain relief [48]. There is a lot of data on the palliative effects of 89SrCl2 for breast and prostate cancer patients with metastatic bone pain. Review articles have reported pain relief rates ranging from 57% to 92% [49–51]. Palliation effects are usually observed within 6 weeks after intravenous injection of 89SrCl2, and the mean duration of pain relief is approximately 6 months [52]. Samarium-153 (153Sm) emits beta particles with maximum energies of 0.81 MeV (20%), 0.71 MeV (49%), and 0.64 MeV (30%) and a 28% abundance of gamma rays with an energy of 103 keV and can be used for imaging, unlike 89Sr. 153Sm has a physical half-life of 46.3 h. 153Sm-EDTMP (Quadramet®), which has also been approved by the FDA, is a complex of 153Sm with ethylenediaminetetramethylene phosphonic acid (EDTMP: lexidronam, Fig. 5.1d), which is a tetraphosphonate chelator with high affinity for bone. Because the biodistribution of 153Sm-EDTMP is similar to that of 99mTc-MDP, the dosimetry of 153Sm-EDTMP could be predicted using 99mTcMDP bone scintigraphy [53]. The blood clearance of 153Sm-EDTMP is very rapid, and the compound is excreted via the kidney into the urine [54]. The blood clearance is applicable to a biexponential model with an estimated half-life of 5.5 ± 1.1 and 65.4 ± 9.6 min [55]. About 50% of the injected dose is excreted into the urine at 6–7 h after injection. Most of the remaining dose is accumulated in bones. There is little accumulation in soft tissue, such as the liver, and less than 1% of the injected dose remains in the blood. The usual dose of 153Sm-EDTMP is 37 MBq/kg (1 mCi/kg). Review articles have reported pain relief rates ranging from 62% to 84% [49–51].

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 5 Biocomplexes in radiochemistry

When 89SrCl2- and 153Sm-EDTMP-treated groups of prostate cancer patients with bone metastases were compared, no statistical differences were observed in response rates and side effects [56]. Radium-223 chloride (223RaCl2), in which 223Ra is the alpha particle-emitting radionuclide, was approved by the FDA and European Medicines Agency (EMA) in 2013. Radium (Ra) also is an alkaline earth metal and a member of family IIA in the periodic table, as are Ca and Sr. Among Ra isotopes, 223Ra has a suitable half-life of 11.4 d for therapy and decays through a chain of daughter nuclides with an emitted total energy of approximately 28 MeV, with most of the energy released as alpha particles. In a phase III randomised trial (Alpharadin in Symptomatic Prostate Cancer Patients: ALSYMPCA), surprisingly, 223RaCl2 significantly improved overall survival in castration-resistant prostate cancer patients with bone metastases [57, 58]. Moreover, 223RaCl is associated with a low incidence of myelosuppression, which is assumed 2 to be the major side effect and could be the dose-limiting factor. 223RaCl2 is the first alpha particle-emitting radiopharmaceutical approved for clinical use and has been demonstrated to be an effective therapy for bone metastases; therefore, it is currently attracting much attention.

5.2.5 Development of novel bone-seeking complexes for palliation therapy of bone metastases Rhenium has chemical properties similar to those of technetium because both elements are members of family VIIA in the periodic table. Of the rhenium isotopes, there are two radionuclides, 186Re and 188Re, that are useful for radionuclide therapy [59]. Both rhenium radionuclides emit not only high-energy beta particles for therapy but also gamma rays for imaging: 186Re (T1/2 = 3.7 d, β−max = 1.07 MeV, γ = 137 keV) and 188Re (T1/2 = 17.0 h, β−max = 2.12 MeV, γ = 155 keV). Furthermore, because 188Re is a daughter nuclide of 188W (T1/2 = 60 d), 188Re is obtained from an in-house aluminabased 188W/188Re generator, similar to a 99Mo/99mTc generator [60]. In the case of the use of rhenium radionuclides for bone-seeking radiopharmaceuticals in a manner similar to that of 99mTc-MDP and 99mTc-HMDP, it has been reported that rhenium forms complexes with some bisphosphonate derivatives as ligands. 186/188Re-1-hydroxyethylidene-1, 1-diphosphonate (186/188Re-HEDP), which is a 186/188Re-complex with HEDP (Fig. 5.1e), has been evaluated in clinical research [61–63]. Although the chemical characteristics of rhenium and technetium are similar, rhenium is more easily oxidised than is technetium [64], and the stability of 186Re-HEDP is lower than that of 99mTc-bisphosphonate complexes [65]. Some studies have shown that gastric accumulation of radioactivity was observed after injection of 186Re-HEDP in patients with bone metastases [66, 67]. It is known that 186/188ReO4− accumulates in the stomach, as does 99mTcO4−, and it is thought that this occurs because of the in vivo instability of 186Re-HEDP [68]. Moreover, similar to the phosphonate

5.2 Bone-seeking complexes 

OH

O N

H N

N

O

O

Re S

O

OH

N

O P OH

(a)

O

H N

N O

Re

OH

S

OH

O

P OH

S

O

P OH

O

P OH

OH OH

S

(b) O O

N

O

N OC

Re S

N

O

O (c)

 131

N H

OH O P O P OH

OH

OC

OH

OH O P OH

N

N

Re CO

N

OH O P OH OH

OH (d)

Fig. 5.4: Chemical structures of 186/188Re complex-conjugated bisphosphonate compounds (a) 186Re-MAMA-BP, (b) 186Re-MAMA-HBP, (c) 186Re-MAG3-HBP, and (d) 188Re(CO)3-DPA-alendronate.

groups in 99mTc-MDP and 99mTc-HMDP, those in 186/188Re-HEDP are used as both a ligand for complexation and a carrier to bone, which may reduce the inherent affinity of HEDP to bones. To develop superior 186/188Re-labeled bone-seeking radiopharmaceuticals, I assumed that the above-mentioned concept of a stable complex-­ conjugated bisphosphonate would be more useful. Therefore, our research group designed and evaluated 186Re-monoaminemonoamidedithiol (MAMA)- and 186Re-mercaptoacetylglycylglycylglycine (MAG3)-conjugated bisphosphonate compounds (186Re-MAMA-BP, 186Re-MAMA-HBP, 186Re-MAG3-HBP; Figs. 5.4a–c) and reported our findings [24, 69–72]. When 186Re-complexes were incubated in buffered solution at 37°C, the Re-complex-conjugated bisphosphonate compounds, 186Re-MAMA-HBP, 186Re-MAMA-BP, and 186Re-MAG3-HBP, were more stable than 186Re-HEDP, as expected. In biodistribution experiments in mice, little gastric accumulation of radioactivity was observed after injection of 186Re-MAMA-HBP, 186Re-MAMA-BP, and 186Re-MAG3-HBP. The drug design of Re-complex-conjugated bisphosphonates led to better stability in vitro and in vivo. Among these 186Re-­complexconjugated bisphosphonate compounds, 186Re-MAG3-HBP showed the most favourable biodistribution characteristics as a bone-seeking agent, such as high and selective bone accumulation. These favourable biodistribution characteristics could be related to high hydrophilicity (log P value:, −2.68 ± 0.01) and the introduction of a hydroxyl group to the central carbon of the bisphosphonate P-C-P structure. The therapeutic potential of 186Re-MAG3-HBP for palliation of metastatic bone pain in a rat model of bone metastasis was evaluated. Compared with untreated control group

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rats, 186Re-MAG3-HBP-treated rats showed significant palliation effects, as assessed by the hind paw withdrawal response to stimulation with von Frey filaments [73], and the palliation effects of 186Re-MAG3-HBP tended to be higher than those of 186ReHEDP. Although 186Re-HEDP did not inhibit tumour growth, 186Re-MAG3-HBP significantly inhibited tumour growth. Because one of the 99mTc-complex-conjugated bisphosphonate compounds, 99mTc(CO) -DPA-alendronate, has been introduced above, the same ligand was used 3 to design and evaluate 188Re(CO)3-DPA-alendronate by the same group (Fig. 5.4d) [74]. 188Re(CO) -DPA-alendronate also showed higher in vitro stability than did 188Re-HEDP, 3 which oxidised to 188ReO4− (up to 75%) after incubation in PBS for 48 h at 37°C. In imaging experiments, 188Re(CO)3-DPA-alendronate showed superior biodistribution of radioactivity than did 188Re-HEDP, i.e. 188Re(CO)3-DPA-alendronate highly accumulated in metabolically active bone, such as joints with low soft-tissue uptake. These results indicate that the concept of stable 186Re-complex-conjugated bisphosphonates could be very useful and that novel 186Re-complex-conjugated bisphosphonate complexes could be attractive candidates as palliative agents in metastatic bone pain.

5.3 Radio-complexes for imaging of apoptosis It is known that apoptosis is associated with maintaining homeostasis, most diseases, and responses to therapy. Therefore, imaging of apoptotic cells could help with elucidation of disease mechanisms and with early detection of therapeutic effects. Next, I will discuss radiocomplexes for imaging of apoptosis, their drug design, preclinical studies, and applications in medicine.

5.3.1 Technetium-labeled annexin A5 Phosphatidylserine (PS) exists on the intracellular face of the cell membranes of normal cells. When apoptosis occurs, the lipid distribution of the plasma membrane changes so that PS is exposed to the outside of the cell membrane. Therefore, PS could be a target for imaging of apoptosis, and the typical compound of PS-targeted carriers is annexin A5, which is a 36-kDa human protein with a nanomolar affinity for membrane-bound PS [75–77]. For imaging of apoptosis, many researchers have developed and evaluated radiolabeled annexin A5 compounds.

5.3.2 99mTc-4, 5-bis(thioacetamido)pentanoyl-annexin A5 (99mTc-BTAP-annexin A5) As mentioned above, 99mTc is an ideal radionuclide for clinical use because of its physical properties. However, because most proteins and polypeptides do not possess chelation sites for formation of technetium complexes, a ligand for complexation with

5.3 Radio-complexes for imaging of apoptosis  

 133

technetium should be introduced into proteins and polypeptides. Tetradentate chelators, such as N3S and N2S2 coordination molecules, could form stable square pyramidal 99mTc complexes with a [Tc=O]3+ core in which technetium is in the oxidation state +V.

O

O Annexin A5

NH

O

(a) NH Annexin A5

(c)

HN

N O N Tc S S

S

O

Annexin A5

NH

O

N

H O N H N N Tc O O O

(b)

O N O

N H

OH OH OH OH OH

H N O

N ON Tc N N O O

Fig. 5.5: Structures of (a) 99mTc-BTAP-annexin A5, (b) 99mTc-HYNIC-annexin A5, and (c) 99mTc-C3(BHam)2-annexin A5.

Among the 99mTc-labeled annexin A5 compounds for imaging of apoptosis, 99mTc-4, 5-bis(thioacetamido)pentanoyl (BTAP)-annexin A5 (Fig. 5.5a), which has a N2S2 ligand for complexation with 99mTc, was reported to be the first 99mTc-labeled annexin A5 compound. 99mTc-BTAP-annexin A5 was used as an imaging agent for transplant rejection of cardiac transplantation by Narula et al. When 99mTc-BTAPannexin A5 was injected into cardiac allograft recipients, 99mTc-BTAP-annexin A5 accumulated in the hearts of some recipients who showed at least moderate transplant rejection. These results suggested that 99mTc-labeled annexin A5 was useful for noninvasive imaging of apoptosis [78]. However, N2S2 ligands, such as BTAP, should require harsh conditions for preparation of 99mTc complexes because the protecting groups for thiol must be deprotected just before radiolabeling. Thus, 99mTc-BTAPannexin A5 was prepared by a preformed-chelate approach. The preformed-chelate approach means that the conjugation between the 99mTc-BTAP complex and annexin A5 is performed after the complexation of 99mTc with the BTAP ligand. The preformedchelate approach is complicated and has low radiochemical yields because multiple steps and purification are needed. Easy 99mTc labeling of annexin A5 is required for routine clinical use.

5.3.3 99mTc-HYNIC-annexin A5 In 1998, Blankenberg et al. reported 99mTc-HYNIC-annexin A5 (Fig. 5.5b), for imaging of apoptosis [79]. HYNIC is one of the most familiar ligands for labeling of peptides and proteins with 99mTc. HYNIC forms a mixed ligand complex with 99mTc and the proper

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coligands. In the complex, it has been reported that HYNIC works as a monodentate or bidentate ligand [80]. Several coligands, such as glucoheptonate, tricine, ethylene diamine diacetic acid (EDDA), and ternary ligand systems containing tricine and watersoluble phosphines or tricine and imine-N-containing heterocycles have been reported. Among these coligands, (tricine)2 has been frequently used for 99mTc labeling of proteins because the 99mTc-(HYNIC)(tricine)2 complex can be prepared under mild conditions with high radiochemical yields in a short reaction time; specifically, 99mTc-(HYNIC) (tricine)2 has been obtained without any purification after a one-step reaction [81, 82]. Presently, 99mTc-HYNIC-annexin A5 with (tricine)2 as coligands is the gold standard among agents used for imaging of apoptosis in nuclear medicine. Because easy labeling has helped researchers with imaging of apoptosis in studies using 99mTc-HYNIC-annexin A5, many of these studies have been conducted in the clinic and as basic research [83]. However, 99mTc-HYNIC-annexin A5 has some disadvantages because of the instability of the 99mTc-(HYNIC)(tricine)2 complex; in particular, 99mTc-HYNIC-annexin A5 shows high uptake and long retention in nontarget tissues, such as the kidney and liver [84]. In basic research, accumulation of 99mTc-HYNIC-annexin A5 in apoptosis-induced Jrukat T-cell lymphoblasts has shown a correlation with the percentage of FITClabeled annexin A5 labeled cells (r2 = 0.922) [85]. Moreover, many researchers have demonstrated that 99mTc-HYNIC-annexin A5 has high affinity for apoptotic cells and can enable visualisation of apoptosis in various animal models [84, 86–88]. In a clinical study in 2007, 99mTc-HYNIC-annexin A5 imaging before and after initiation of platinum anticancer agent-based chemotherapy in non-small-cell lung cancer patients was reported. All patients who showed increased uptake of 99mTc-HYNIC-annexin A5 in tumours achieved complete or partial responses. The uptake of 99mTc-HYNIC-annexin A5 by tumours correlated with treatment outcome (r2 = 0.86; P = 0.0001) [89].

5.3.4 99mTc-labeled annexin A5 constructed with histidine residues In 2002, preparation of mutant annexin A5 with N-terminal extensions constructed with three or six histidine residues for 99mTc labeling was reported [90]. A mutant annexin A5 with six histidine residues in the N-terminal showed higher radiochemical yield, radiochemical purity, and bioactivity than did the mutant annexin A5 with three histidine residues in the N-terminal. This study demonstrated that it was possible to successfully construct annexin A5 with histidine residues in the N-terminal to form specific chelation sites for the 99mTc-carbonyl complex without altering its high affinity.

5.3.5 99mTc-C3(BHam)2-annexin A5 A novel 99mTc-labeled annexin A5, 99mTc-C3(BHam)2-annexin A5, which has a bis(hydroxamamide) derivative as a bifunctional chelating agent to achieve low

5.3 Radio-complexes for imaging of apoptosis  

 135

uptake and retention in nontarget tissues, was recently reported by my research group (Fig. 5.5c) [91]. It has been reported that a bis(hydroxamamide) derivative, N, N’-trimethylenedibenzohydroxamide ligand [C3(BHam)2 ] forms a stable 99mTc complex over a wide pH range under mild reaction conditions within short reaction times [92]. The radiochemical yield is very high even at ligand concentrations as low as 2.5 × 10 ˗6 M. In mice, the radioactivity in liver was not residual after intravenous injection of 99mTc-C3(BHam)2-IgG [93], which indicated that the radiometabolite of 99mTc-C3(BHam)2-IgG could be not residual in metabolic organs. In the case of annexin A5, I assumed that there would be less residual radioactivity in metabolic organs after injection of 99mTc-C3(BHam)2annexin A5 and therefore, designed 99mTc-C3(BHam)2-annexin A5. The bioactivity of 99mTc-C3(BHam)2-annexin A5 was comparable to that of 99mTcHYNIC-annexin A5. In mouse biodistribution, the uptake by the kidney was lower for 99mTc-C3(BHam)2-annexin A5 than for 99mTc-HYNIC-annexin A5. Moreover, the radioactivity of metabolic organs, such as the liver and kidney, after injection of 99mTc-C (BHam) -annexin A5 gradually decreased, whereas there was residual 3 2 radioactivity in metabolic organs after the injection of 99mTc-HYNIC-annexin A5. In therapeutic experiments, tumour growth in mice treated with 5-fluorouracil (5-FU) was significantly inhibited. Accumulation of 99mTc-C3(BHam)2-annexin A5 in tumours significantly increased at 24 h after 5-FU treatment. The accumulation of 99mTc-C3(BHam)2-annexin A5 correlated positively with the counts of terminal dUTP nick-end labeling (TUNEL)-positive cells. Moreover, the intratumoral accumulations of 99mTc-C3(BHam)2-annexin A5 by autoradiography significantly correlated with TUNEL-staining positive cells in corresponding sections (r = 0.716, P < 0.001, Fig. 5.6).

5.3.6 68Ga-labeled annexin A5 A protein labeling system using 68Ga and a sulfhydryl-derivatised chelator, 2, 2′-(7-(1-carboxy-4-(2-mercaptoethylamino)-4-oxobutyl)-1, 4,7-triazonane-1, 4-diyl)diacetic acid (NODA-GA-T), was reported in 2011 [94]. In that study, 68Ga-labeled annexin A5 was prepared within only 15 min and accumulated in the apoptotic area of myocardial infarctions in a PET study using an animal model. In the same year, site-specific 68Ga-labeled annexin A5 compounds, which are variants of annexin A5 containing a single cysteine residue at a position of 2 or 165 (Cys2-annexin A5 and Cys165-annexin A5, respectively), have been reported [95]. 68Ga-Cys2-annexin A5 and 68Ga-Cys165-annexin A5 were prepared within approximately 55 min with a 25% radiochemical yield (43% if corrected for decay). Both 68Ga-labeled annexin A5 compounds preserved bioactivity and showed high in vitro stability. The uptakes of 68Ga-Cys2-annexin A5 and 68Ga-Cys165-annexin A5 by tumours were not high but were significantly increased by the treatment of cyclophosphamide and radiation therapy in a tumour model.

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(a)

(b)

(c)

1 mm

(x 10–6) 40

Radioactivity in grid (%dose)

35 30 25 20 15 10 5 0 (d)

r = 0.716, P < 0.001 0

100 150 50 The number of TUNEL positive cells per grid

200

Fig. 5.6: Representative (a) autoradiographic images and (b, c) TUNEL-staining images for adjacent tumour sections from mice treated with 5-FU. (d) Correlation between the number of TUNEL-positive cells in each grid (0.45 mm × 0.55 mm) of a tumoural section and 99mTc-C3(BHam)2-annexin A5 accumulation (%dose) determined by autoradiography in each corresponding grid of an adjacent section from mice treated with 5-FU.

Radiolabeled annexin A5 seems to require some time before imaging of apoptosis after injection of radiotracers can be performed. Accordingly, although 68Ga is a promising radionuclide for PET imaging, the half-life of 68Ga may be too short if annexin A5 is used as a carrier to apoptotic cells.

5.4 Summary 

 137

5.3.7 Summary of radiolabeled annexin A5 for imaging of apoptosis Quantitative imaging agents for apoptosis are useful tools because imaging enables early determination of therapeutic effects on diseases, even before anatomical changes occur at the lesion site. Selection of an appropriate therapy by using imaging data from an individual patient may be possible. Although some radiolabeled-annexin A5 compounds are in use, their biodistributions are not necessarily ideal. I hope that novel apoptosis imaging agents using other carriers to apoptotic cells that enable superior imaging to that currently available will be developed in future.

5.4 Summary Radiometal complexes can be used as radiopharmaceuticals because some show ideal biodistribution as diagnostic or therapeutic agents in nuclear medicine. One of the advantages of radiometal complexes is the high radiochemical yield. Radiolabeled compounds with high radiochemical yields do not need purification after radiolabeling. For example, in the case of generator-produced radionuclides, such as 99mTc and 68Ga, radiolabeling and on-demand administration to patients just after eluting radionuclides from generators in the hospital is useful clinically, especially in emergency cases. Two categories of nuclear medicine applications, bone metastases and apoptosis, have been reviewed here, and radiometal complexes have shown great utility in these fields. Development of new radiometal complexes for nuclear medicine is anticipated in the future.

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Hadi Khalil, Mathieu Heulot and David Barras

6 Peptides and biocomplexes in anticancer therapy 6.1 General introduction on cancer 6.1.1 Cancer and metastasis origins and global burden Hippocrates (460 B.C.) first established the word carcinos from the Greek (which means crab) to point at what we now know as a tumour. This term referred to the morphology of the tumour Hippocrates saw, the irregular shape and the hanging blood vessels reminded him of crab claws and limbs. This animal symbol was preserved, and the term “cancer” was employed afterwards by Aurelius Cornelius Celsus (from the Latin “Canker”, which also means crab). The first cases of cancer were reported on papyrus by Egyptians (1600 B.C.). Joseph Claude Anthelme Récamier, a French gynaecologist was the first to coin the word “metastasis” in 1829 as being a spread cancer found in the bloodstream. Sixty years after, Stephen Paget strengthened knowledge by proposing the “seed and soil” theory stating that “the distribution of the secondary growth is not a matter of chance” but the cancer cell’s (the seed) ability to invade an organ (the soil) strongly depends on the properties of this organ [1] (reviewed in [2]). Without describing any molecular aspect, he however pointed out one of the most important features of metastases. These notions were deepened later by James Ewing, in 1928, in his “Treatise on tumours”. Nowadays, cancer is the leading cause of death in economically developed countries and has emerged to be the second one in developing countries [3]. The increasing population ageing and lifestyles imposed by the emerging consumption society like sedentary behaviour, inadequate nutrition, and smoking mainly account for this curse. In 2008, global cancer statistics estimated 12.7 million new cancer cases and 7.6 million cancer-associated deaths. Lung cancer so far is the deadliest cancer in males representing 22.5% of cancer deaths, and the second deadliest cancer in females after breast cancer (13.7%). (Fig. 6.1).

6.1.2 Genomic instability and the other side of the Darwinian coin Mutations in the genome affecting the function of proteins that regulate homeo­ stasis of the cell account for cancer development. Every form of cancer displays high genomic instability. Genomic instability refers to the high rate by which changes rise in chromosome structures and numbers, and therefore in genomic functions [4]. Genomic instability favours the continuous acquisition of DNA aberrations which, as a result of the selection pressure, prompt the cancer cells to adapt, resist, and become

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Female Breast 458,400 Lung & bronchus 427,400 Colon & rectum 288,100 Cervix Uteri 275,100 Stomach 273,600 Liver 217,600 Ovary 140,200 Oesophagus 130,700 Pancreas 127,900 Leukaemia 113,800 All sites but skin 3,345,800

Male Lung & bronchus 951,000 Liver 478,300 Stomach 464,400 Colon & rectum 320,600 Oesophagus 276,100 Prostate 258,400 Leukaemia 143,700 Pancreas 138,100 Urinary bladder 112,300 Non-Hodgkin lymphoma 109,500 All sites but skin 4,225,700

Fig. 6.1: Estimation of cancer-related deaths in 2008.

continuously more aggressive. Cancer is therefore the result of Darwinian natural selection, where the fittest cell would have the opportunity to grow and divide. Paradoxically, the other side of the coin is the eventual death of the “host” and therefore the cancer extinction. How genomic instability arises is still a subject of intense debate. There are two main opposing theories concerning this matter. The mutator hypothesis supports that an original early mutation renders the precancerous cell genetically unstable (e.g. by disabling “caretaker” genes affecting the DNA repair machinery) and leads to an increase in the rate of spontaneous mutations [5]. This allows the cancer cell to transform, and according to the selective pressure (e.g. anticancer therapy, hypoxia, etc.) to adapt to or escape an unfavourable environment. This theory however predicts that mutations affecting the fitness of a cell are also occurring that would lead to cancer extinction, obviously this does not reflect the reality. The oncogene-induced DNA replication stress model states that mutations in sporadic cancer can arise and affect oncogenes or tumour suppressor genes (by opposition to “caretaker” genes). In the absence of a functional tumour suppressor called p53, which prompts the cell to undergo senescence or alternatively apoptosis when DNA is not intact, these oncogenic mutations would lead to the collapse of the DNA replication forks and ultimately to DNA strand breaks and genomic instability [4]. High throughput genetic data obtained on breast and colon cancer favour the oncogenic stress model

6.1 General introduction on cancer 

 145

as the majority of the DNA mutations that were found lie within non-caretaker genes [6, 7]. Certain non-caretaker genes appear almost always mutated in the analysed samples like those encoding for K-Ras (Kirsten rat sarcoma viral oncogene homolog), APC (adenomatous polyposis coli), and PI3K (phosphoinositide 3-kinase). In contrast, mathematical predictions shift the balance in favour of the mutator hypothesis [5]. It is not strange that the reality is more complex and surely imbricates elements of these two models.

6.1.3 The hallmarks of cancer Neoplastic diseases are highly complex and heterogeneous. However, these diseases have shared common traits. Douglas Hanahan and Robert Weinberg published in 2000 “The hallmarks of cancer”, a report of principles that rationalise the complexity displayed by malignancies [8]. Six distinctive and complementary hallmarks that eventually favour tumour growth and metastatic dissemination have been proposed at this time and updated 10 years after [9]. They are briefly explained here (Fig. 6.2).

Evading growth suppressors

Enabling replicative immortality

Resisting cell death Hallmark of cancer

Activating invasion and metastasis

Inducing angiogenesis

Sustaining proliferative signaling Fig. 6.2: The hallmarks of cancer.

The accumulation of mutations due to genomic instability favours the appearance of the following hallmarks: (i) Sustained proliferative signalling. Cancer cells have the ability to maintain constant proliferation. In contrast, normal cells control their proliferation which confers on them the maintenance of homeostasis and tissue architecture. Cancer cells reprogram the release of growth factors and the expression of their associated receptors in order to increase their proliferation rate. Every pathway that controls positively or negatively the mitogenic activity is a target of choice for carcinogenesis-inducing mutations. Cancer cells may even also reprogram and induce neighbouring normal cells to emit growth factors [10].

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 6 Peptides and biocomplexes in anticancer therapy

(ii) Evasion from growth suppressors. Sustained constant proliferation occurs through the first-mentioned hallmark but cannot be completed unless the negative regulators of proliferation are neutralised. Some proteins, the so-called tumour suppressors, are responsible to sense and further decide if the cell should undergo division, senescence, or apoptosis. Rb (retinoblastoma protein) [11] and p53 (commonly called the “guardian of the genome”) [12] are such proteins and are unsurprisingly common targets of inactivating mutations. (iii) Resistance to cell death. Normal cells that undergo replication failure or those that encounter excessive stress normally undergo a form of cell suicide called apoptosis. The regulation of apoptosis is finely tuned at physiological levels, in particular by showing an equilibrated balance between anti- and pro-apoptotic protein activities [13]. Apoptosis can be triggered by extrinsic factors (for example by engagement of the Fas receptor) or from within the cell (intrinsic mitochondrial cascade), typically after DNA damage. Both situations lead to the eventual activation of the proteases called caspases, which cleave various proteins essential for cell homeostasis maintenance, and therefore leads to progressive cell disassembly. Genomic instability allows the cancer cells to generate an unbalanced anti-apoptotic signature by activating anti-apoptotic signalling and repressing the pro-apoptotic ones [13]. The diversity of strategies that cancer cells use to counteract apoptosis reflects the multiplicity of signalling leading to cell death. Other characterised kinds of cell death like autophagy [14] and necrosis [15] are also bypassed by cancer cells. (iv) Enabled replicative immortality. Normal cells are only able to pass through a limited number of division cycles, after which they enter into a viable but nonreplicative senescence phase. They alternatively enter in a phase called crisis and die by apoptosis. This fate is mainly attributed to the telomere (chromosome extremities) shortening [16]. Telomeres of dividing cells shorten at every cell cycle, which is thought to eradicate their protective effect on chromosomal ends. The telomerase is a specialised DNA polymerase that adds DNA segments to the telomeres. In normal cells, telomerase is almost always absent, while more than 90% of the reported immortalised cells display high levels of telomerase [16]. Acquisition of the enhanced telomerase activity confers a strategy for unlimited replication of cancer cells. (v) Induction of angiogenesis. The fast growth and division of cancer cells necessitates constant support in nutrients and oxygen. These latter are supplied by the blood. The generation of new vessels is called angiogenesis and is a physiological process during embryogenesis and female reproductive cycling. It is however mainly switched off at adult stages and physiological conditions. To alleviate the tumour needs of nutrients and oxygen, tumour cells have evolved strategies to create their own vasculature [17]. The angiogenic switch triggered by cancer cells is heterogeneous but almost always results in production of a released factor called vascular growth factor-A (VEGF) that prompts endothelial cells to from a neovasculature [18].

6.1 General introduction on cancer 

 147

(vi) Activated invasion and metastasis. The ability of cancer cells to disseminate through the body is undoubtedly their most harmful property, which almost always leads to death. It is also the least understood hallmark of all. In addition to providing the nutrients and oxygen, the newly created vessels offer to cancer cells the possibility to disseminate across the body. Only a few cancer cells will have the ability to complete the long metastatic journey. Succeeding in initiating metastasis involves several critical steps discussed in detail below. (vii) Other biological processes. In addition to the six hallmarks set out in this section, studies conducted over the last decades identified other biological processes as emerging hallmarks of cancer. Among those is the ability of cancer cells to escape from immune surveillance and the reprogramming of energy metabolism, a process that is also called the “Warburg effect”. In addition to the fact that the cancer cells can escape from immune surveillance, recent data indicate that immune cells could also provide growth, survival, and pro-angiogenic factors to the tumour cells (reviewed in [19]). To fuel their increasing need of energy, the cancer cells are prone to reprogram their energy metabolism by opting for an aerobic glycolytic metabolism, which is a mechanism allowing to produce lactate starting from glucose in presence of oxygen [20]. This glycolytic switch acidifies the tumour microenvironment which results in several selective advantages for the cancer cells (reviewed in [20]). Another important observation is the increasing consideration for the protective role of the stroma associated with cancer cells.

6.1.4 Molecular basis of the metastatic cascade The metastatic cascade is highly complex and involves sequential multistep processes [21]; the consequence of this is that only a few pre-metastatic cells will survive until the end of the process to eventually form metastases [22]. There are still debates on which mode is utilised by the cell to form a metastasis [23]. The linear progression model states that all the mutations that are necessary to disseminate are acquired within the primary tumour while the parallel progression model favours the hypothesis that mutations are acquired sequentially in a spatial manner, and therefore that cells which leave the primary tumour are not fully metastatic [24]. Regardless of the dissemination mode, metastatic cells should be able to undergo a physiological process called epithelial-mesenchymal transition (EMT). EMT is a reversible reprogramming allowing epithelial cells to dedifferentiate and acquire de-adhesive and pro-migratory properties [25]. Concretely, pre-metastatic cells have first to detach from the primary tumour mass (Fig. 6.3). This phenomenon is now widely accepted to be a consequence of the loss of the E-cadherin adhesion receptors that mediate cellto-cell interactions [26]. Modulation of the interaction with the extracellular environment is also a crucial event and is mainly achieved by activating or repressing a set of adhesion receptors called integrins. The activation of proteases such as the matrix

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 6 Peptides and biocomplexes in anticancer therapy

Primary tumor Proliferation Local invasion Stoma

sation Intrava

essel Blood v

ation emin s s i D

n tio asa v a r n tio Ext iza n o l Co

Proliferation

Micromentastasic Macroscopic Metastasic Fig. 6.3: The multistep metastatic cascade.

metalloproteinases (MMPs) permits cancer cells to find a path through the extracellular matrix (ECM), which is a network of proteoglycans, polysaccharides, and fibers mainly constituted by collagen, fibronectin, laminin, and vitronectin [27]. This is however not sufficient for the invasion process. In order to weave a path, the cells must increase their motility machinery by modulating the cytoskeleton (discussed in detail below). Once the pre-metastatic cell has reached the lymphatic or hematogenous systems, it has to cross the endothelial barrier, a process called intravasation [28]. The dissemination is then driven by the flow of the lymph or blood, but cells that are not able to survive in an anchorage-independent fashion will die at this step. Additionally, surviving cells should escape from the immune system surveillance. The succeeding ones are then helped by platelets and leukocytes to attach to the vessels in a selectindependent manner and will then cross the endothelial barrier back, a process called extravasation [28]. Finally, cells have to invade the distant organ parenchyma, which is often different from the primary tumour microenvironment in terms of ECM components [28]. At this moment, the metastatic cells reverse the mesenchymal phenotype in order to acquire a proliferative and structured state back; this program is called mesenchymal-epithelial transition (MET) and allows the cells to colonise and form micrometastasis and then eventually macrometastases [29]. Despite the impressive obstacle-associated journey to form a metastasis, the metastatic cell proteome only slightly differs from the primary tumour cell as exemplified

6.2 Generally used therapies for cancer  

 149

in the breast-to-bone malignant dissemination [30]. Overexpressed proteins involved in this breast-to-bone dissemination signature are proteins involved in bone-specific functions like bone homing and degradation, but also proteins involved in more general aspects of dissemination like angiogenesis (FGF5 and CTGF), extracellular matrix degradation (MMP-1 and proteoglycan-1). Metastatic markers that target the motility machinery were also found; for example RhoC (Ras homologous C), a small GTPase involved in actin cytoskeleton remodelling [31]. Any failure to complete one single step of the metastatic cascade would lead to the arrest of the entire process. This therefore raises hope for finding drugs that prevent the metastatic cascade by targeting isolated and specific features of metastatic cells.

6.2 Generally used therapies for cancer 6.2.1 Therapy against cancer and metastasis Genomic instability makes cancer a moving target. The most commonly used treatment against cancer remains surgery when applicable, in addition to radiotherapy and systemic therapies including chemo-, immune-, and hormonal therapy. Surgery aims to physically remove the tumour mass; it is however not feasible in every organ as well as in many invasive cancer, the shape of which is not well delimitated. Radiotherapy uses ionizing radiation to kill malignant cells. Ionizing radiation induces DNA simple and double strand breaks that lead the cell to undergo apoptosis or mitotic catastrophe. Fifty percent of the cancer-diagnosed patients are treated with radiotherapy with possible adjuvants of anticancer drugs. Chemotherapies are organised in several groups: 1) The alkylating agents that cause DNA cross-linking and strand break, leading to replicative failure and apoptosis. 2) The antimetabolites are analogues of nucleic acids that inhibit enzymes involved in DNA synthesis. 3) The alkaloids that bind and disrupt the microtubule cytoskeleton, this latter being crucial for the mitotic spindle and therefore for the cell cycle. 4) The antibiotics that also cross-link the DNA. 5) The inhibitors of topoisomerase, an enzyme required during DNA replication. Immunotherapy generally aims to target or treat tumours with immuno-enhancers. These latter prompt the immune system to attack the tumour cells. Hormonal therapy is also suited for treating certain cancers that strictly depend on the presence of a hormone. In this case, the therapy aims to block the hormone-induced signalling. Side-effects and resistance to most of these therapies remain however a major concern. The genomic instability favours heterogeneity and therefore the possibility to render a few cells resistant to the drug used, which is the main issue in cancer therapy. Nowadays, plenty of small molecule inhibitors have come out and appear promising

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 6 Peptides and biocomplexes in anticancer therapy

EGFR inhibitors

Aerobic glycolysis inhibitors

Proapoptotic BH3 mimetics

PARP inhibitors

Cyclin-dependent kinase inhibitors Sustaining proliferative signaling

Deregulating cellular energetics Resisting cell death Genome instability & mutation

Evading growth supperssors Avoiding immune destruction Therapeutic targeting of the hallmarks of cancer

Inducing angiogenesis

Enabling replicative immortality

Tumor promoting inflamation Activating invasion & metastasis

Inhibitors of VEGF signaling

Immune actuvating Antu-CTLA4 mAb

Telomerase inhibitors

Selective antiinflammatory drugs

Inhibitors of HGF/c-Met

Fig. 6.4: Therapeutic targeting of the hallmarks of cancer.

as well as target more specifically some hallmarks of cancer (Fig. 6.4). An attractive strategy to reduce resistance to therapy is to prevent cells from having the possibility to use an “emergency exit” by creating a cocktail of drugs that target different hallmarks of the cancer cells (Fig. 6.4). The emergence of personalised therapy will help to define the Achilles heel of each tumour and therefore to use the appropriate cocktail of drugs. As mentioned earlier, patients die from metastases. Unfortunately, only a few drugs target the metastatic cascade per se [32]. The others are rather hampering the proliferation of pre-established metastases. An illustration of this is the B-Raf inhibitors for malignant melanoma treatment. B-Raf, an effector of Ras, was often found mutated and thereby constitutively activated in malignant melanoma [33]. B-Raf inhibitors development substantially helped in treating these cancers, they however target the proliferating capacity of the metastasis and not their dissemination capacities. Drugs that are used to counteract the metastatic cascade are mainly ­anti-angiogenic agents, inhibitors of matrix metalloproteinases (MMPs) and those that target the tumour microenvironment of the secondary site [34]. An a ­ nti-angiogenic agent such as Bevacizumab, a monoclonal antibody directed against VEGF, is now used for treating metastatic colorectal cancer. Such drugs prevent vessel development and have the double effect of depriving the cells of nutrients and of a transport mean. An example of targeting the secondary site microenvironment is provided by Denosumab, an antibody targeted against RANKL (receptor activator of nuclear factor kappa-B (NF-kB) ligand). The release of RANKL leads to bone resorption, which is a facilitating event during bone invasion. Neutralizing RANKL with Denosumab is used

6.3 Biocomplexes in cancer therapy 

 151

against osteoporosis and for bone metastasis prevention. The invasion process is also the subject of intense therapeutic development. Melanomas and glioblastomas are currently treated with inhibitors of aV-integrin, an adhesion protein involved in cellECM interaction. aV-integrin inhibitors comprise neutralising antibodies (such as etaracizumab) or cyclic peptides derived from the RGD tripeptide integrin ligand (such as cilengitide). Anti-MMPs were also developed and are currently under clinical trials. They prevent invading cells from digesting the extracellular matrix. A novel class of inhibitors, the inhibitors of c-MET, a tyrosine kinase that controls drug resistance, stemness, invasion, and angiogenesis, seem promising as they simultaneously target several cancer hallmarks at the same time. Despite all that, cancer therapy still lacks effective anti-metastasis drugs.

6.3 Biocomplexes in cancer therapy 6.3.1 Photodynamic therapy (PDT) Newly emerged therapies of cancer include the combination of multiple facets of approaches; one of the promising applications is photodynamic therapy (PDT). This type of therapy has emerged as an alternative approach to chemotherapy and radiotherapy for cancer treatment. Such therapy is based on a photosensitizer (PS), which is perhaps the most critical component of PDT, and this newly introduced therapy is a continuously interesting area of intense scientific research. Classically, the photosensitizer’s traditional use was achieved by molecules like porphyrins, which have dominated the field of photosensitizer therapy (Fig. 6.5). However, photosensitizer agents, major disadvantages are low water solubility, poor light absorption, and reduced selectivity for targets. In order to overcome these disadvantages, experts in the field have introduced polysilsesquioxane (PSilQ) nanoparticles that are crosslinked homopolymers formed by the condensation of functionalised trialkoxysilanes or bis(trialkoxysilanes). PSilQ particles provide an interesting R

R N OC OC

H

N Re

O

N

O

R=

H

N CO

Photoinduced anticancer activity

1

2

3

N

N OC

Re

N

CO 4 Luminescent imaging probe

OC

Fig. 6.5: Photodynamic therapy in targeting cancer. Non toxic luminescent probe of Rhenium that can be activated by a specific wavelength in order to induce an anti-cancer compound.

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 6 Peptides and biocomplexes in anticancer therapy

platform for developing PS nanocarriers. Tackling the major problems of introducing the therapeutic components, the PSilQ nanoparticles provide the reliability to carry a large payload of PS molecules; their surface and composition can be tailored to be more target-specific based on the load. In addition to their small size, nanoparticles can penetrate deep into tissues and be readily internalised by cells. The authors describe the PSilQ nanoparticles with a high payload of photosensitizers that were synthesised, characterised, and applied in vitro. The network of this nanomaterial is formed by “protoporphyrin IX (PpIX) molecules chemically connected via a redox-responsive linker”. Under reducing environment such as the one found in cancer cells, the nanoparticles can be degraded to efficiently release single photosensitizers in the cytoplasm. In fact, recent advances have shown that the phototoxicity of this porphyrin-based PSilQ nanomaterial was successfully demonstrated in vitro using human cervical (HeLa) cancer cells. Future research will build on this finding in order to improve the platform so that the nanoparticles hopefully can be further functionalised with other components including for example polyethylene glycol (PEG) and target-specific ligands to improve its biocompatibility and target specificity.

6.3.2 Metal complexes as platforms for cancer therapy Metals are essential cellular components that play a major role in the function of several indispensable biochemical processes for living organisms, mainly being enzymes cofactors. Metals are endowed with unique characteristics that include redox activity, variable coordination modes, and reactivity towards organic substrates. Metals are tightly regulated under normal conditions due to their high reactivity, and therefore aberrant metal ion concentrations are associated with various pathological disorders, including cancer. For the above-mentioned reasons, coordination complexes, either as drugs or prodrugs, become very attractive probes as potential anticancer agents. After the discovery of cisplatin, cis-[PtII(NH3)2Cl2], which led to the interest in platinum(II) and other metal-containing complexes as potential novel anticancer drugs. However the interests in this field are concerned with uptake, toxicity, and resistance to metallodrugs.

6.3.2.1 Platinum-based cancer therapy: a start of a new phase Platinum-based compounds anticancer therapy is based on ligand exchange kinetics. For example, a platinum-ligand bond exhibits similar thermodynamic durability (less than 100 kJ/mol), which is actually much weaker than typical coordination bonds, such as C-C, C-N, or C-O single and double bonds (between 250 and 500 kJ/mol). The ligand exchange behaviour is rather slow. Such a slow exchange behaviour gives a high kinetic stability and allows much slower ligand exchange reactions, rendering the reaction period rather longer on the order of minutes to days. Additionally, in the

6.3 Biocomplexes in cancer therapy 

 153

case of Pt(II) compounds, ligands are mainly oriented in the trans position and are more rapidly substituted than those in the cis- position. The next chapter of this book is focused on the platinum-based drugs.

6.3.2.2 Zinc in cancer therapy Zinc is a cofactor of many cellular enzymes and another indispensable trace element that plays a critical role in a wide range of cellular processes including cell proliferation, differentiation, and defense against free radicals. Zinc acts as a key structural component in many proteins and enzymes, including transcription factors, and cellular signaling proteins. The effects of the well-defined complexes of pyrrolidine diothiocarbamate complexes containing zinc, Zn(PyDTC)2, and copper, Cu(PyDTC)2, were investigated as potential proteasome inhibitors. Interestingly, these complexes were found to specifically target and inhibit the chymotrypsin-like activity of cellular 26S. In additon this effect does not seem to be specific to zinc only since the authors demonstrated that both Cu(EtDTC)2 and Zn(EtDTC)2 displayed a much higher potency to inhibiting the 26S proteasome in intact breast cancer cells. Another target metal that is being foreseen in cancer therapy is copper, which is enriched in various human cancer tissues and is an essential cofactor for tumour angiogenesis processes. Under normal biological conditions, copper exists in both (Cu+) and (Cu2+), and it serves as a cofactor in redox reactions, such as the mitochondrial electron transport chain. Copper was found to be a critical angiogenic effector by stimulating the proliferation and migration of endothelial cells. Therefore copper could be implicated in anti­tumour development, by an anti-angiogenic therapy using copper chelators. Compounds such as diethyldithiocarbamate (DDTC) and pyrrolidine dithiocarbamate (PDTC) were capable of binding copper and able to form complexes that potently inhibit the proteasomal chymotrypsin-like activity that suppresses proliferation and induces apoptotic cell death in cultured human prostate cancer cells [35].

6.3.2.3 Copper in cancer therapy It is known that copper plays a role in the growth and progression of malignancy has been the subject of intense investigation. It was found that copper levels are altered in tumours in mice and humans; in addition, copper is found in serum and tissue found in various human tumours including breast, prostate, colon, lung, and brain. ­Disulfiram (tetraethylthiuram disulfide, DSF) is a clinically employed drug that when mixed with CuCl2 undergoes a dramatic colour change, which indicates that a c­ hemical reaction between DSF and copper has occurred. Therefore employing the biology of a tumour where higher levels of copper compared to normal tissue are found, very innovative strategies are being developed of targeting elevated copper levels with DSF, and the authors of this work hypothesised that the r­ esultant

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 6 Peptides and biocomplexes in anticancer therapy

­ SF-copper complex could possess potent tumour-specific killing activity by selecD tively inhibiting the proteasome in tumour cells [35].

6.3.2.4 Peptide therapeutics The field of peptide therapeutics has grown rapidly in recent years. In 2012, the number of peptides that reached market approval and clinical trials has never been higher [36]. Twelve peptides were approved in 2012 compared to ~1.3 per year in the beginning of the 2000s. Virtually all biological fields are concerned by peptide development, with oncology and metabolism domains being at the forefront of this research. Peptides have suffered a lot from their low biodelivery, bioavailability, and short half-lives [37, 38]. In addition, they are often rapidly cleared from the circulation by hepatic and renal routes. Still there are solutions to overcome these limitations. The proteolytic cleavage of peptides can be significantly improved first by replacing the oral administration by in situ injections, which already prevent the negative effects of gastrointestinal proteases. Second, the usage of unnatural amino acids (D-configuration), peptide cyclisation or the replacement of peptide bonds by isosteric bonds alo prevents proteases from cleaving these peptides in [37]. The short half-life of peptides is not always a bad aspect as compared to small molecules that stably toxically accumulate within organs, the peptide accumulation is often only transient and thus doses can be more easily controlled. The short size of peptides gives them the potential to penetrate the cells better than recombinant proteins or antibodies. In addition, coupling these peptides to cell-penetration peptides (CPPs) and organ targeting sequences further improves their delivery and specificity. More generally, it is widely accepted that peptides display more specificity towards their targets as they are often mimicking natural proteins, which in addition limits the side-effects associated to non-specific targets [39]. In cancer therapy, peptide efficacy was approved particularly due to their efficient induction of apoptosis or antagonizing integrins for example. This field is currently extensively studied and much more remains to be discovered.

6.3.2.5 Peptides in cancer therapy Until recently most research efforts aimed at developing anti-cancer tools were mainly concentrated on small molecules and their biology. The current usage of smaller compounds and alternative compounds are now being increasingly assessed for their potential anti-cancer properties, including peptides and peptide-derivatives. The previous limitation of peptide usage was the fact that they do not optimally cross cell membranes. This point was alleviated with the characterisation of cell-­permeable sequences. Most anti-cancer peptidic compounds induce

6.3 Biocomplexes in cancer therapy 

 155

apoptosis of tumour cells. This is achieved by modulating various pathways that might shift the balance between anti-apoptotic and pro-apoptotic proteins. The biology of these modulating peptides is principally based on altering the activity of Bcl-2 family members that control the release of death factors from the house of power in the cells (the mitochondria) or by inhibiting negative regulators of caspases, the proteases that mediate the execution of protein cleavage and mainly induce the apoptotic response in cells besides other newly discovered non apoptotic functions. In parallel, the shift in the pro-and anti-apoptotic balance could be achieved by inhibiting the inhibitors. Researchers have identified Smac inhibitors that target the inhibitors of apoptosis proteins (IAPs), the family known to inhibit caspases and therefore induces cell death and ultimately vanishes the cancer colony. Similarly, a tumour suppressor and oncogenic sensor called p53 inhibits cell cycle for repair or induces apoptosis. The p53 protein counteracts cancer development by its apoptosis-inducing capacity. Unsurprisingly, p53 and p53 regulators are often mutated in many human cancers, and one of the major goals in cancer therapy is to identify a way to allow the restoration of a functional p53 apoptosis signaling response in cancer cells. For example, a 15 amino acid long peptide derived from a carboxyterminal domain of p53 was shown to negatively regulate its own transcriptional activity, and it was shown that this peptide can activate latent forms of p53 when injected into cells, therefore reverting many of the inactivated p53 mutant back into activation phase. This finding shows the promising facet of peptides as re-activation of the p53 pathway would lead to tight control in the cell cycle again and therefore a rapid control on cancerous cells. ­Pro-apoptotic ­anti-tumour peptides will soon be tested for their efficacy in patients with cancers. There is a long list of pro-apoptotic peptides (Tab. 6.1) [40]. Most of these peptides were tested and verified in animal models. Some of these peptides have been shown to inhibit the growth of tumours in mouse models. Tab. 6.1: List of peptides targeting the apoptotic signalling pathway. Adapted from Barras et al. 2011. Cancer specificity

In Vivo validation

Amino-Acid sequence (Without CPP and CST)

Pen

N/A

No

poly-Arg

N/A

No

Yes

No

MGQVGRQLA IIGDDINRRY MGQVGRQLA IIGDDINRRY MGQVGRQLA IIGDDINRRY

Name

Origin

CPP

Ant-BH3 (Ant-Bak) R8-Bak

Bak Bak

LHRH-BH3

Bak

CST

LHRH

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 6 Peptides and biocomplexes in anticancer therapy

Tab. 6.1 (continued) Cancer specificity

In Vivo validation

Amino-Acid sequence (Without CPP and CST)

N/A

No

Pen

N/A

No

Bax

poly-Arg

N/A

No

p3Bax

Bax

TAT

N/A

No

TAT-DV3-BH3 TATRasGAP317–326 cpm-1285

PUMA p120RasGAP

TAT TAT

Yes Yes

Yes Yes

KLSECLKRIG DELDS STKKLSECLKRIG DELDSNM STKKLSECLKRIG DELDSNM MDGSGEQLGSG GPTSSE­QIMKTG AFLLQGFIQ LRRMADDLN WMWVTNLRTD

Bad

CH3(CH2)8CO

Yes

Yes

ANTBH3BAD

Bad

Pen

No

No

Ant-Bad

Bad

Pen

No

No

R8-Bad

Bad

poly-Arg

No

No

SAHBA

Bid

N/A

Yes

TAT-Bim

Bim

N/A

Yes

peptide 2

IP3R

N/A

No

None given None given

Smac Smac

Yes N/A

Yes Both

None given None given Shepherdin RI-TATp53C′

Smac Smac Survivin p53

N/A N/A Yes Yes

No No Yes Yes

Name

Origin

None given

Bax

Ant-Bax

Bax

R8-Bax

CPP

CST

DV3

TAT

TAT Pen/ poly-Arg

Pen TAT

DV3

KNLWAAQRYG RELRRMSD E­FEGSFKGL NLWAAQRYG RELRRMSDEFVD NLWAAQRYG RELRRMSDEFVD NLWAAQRYG RELRRMSDEFVD EDIIRNIARHLA *VGD*NLDRSIW EIWIAQELRRIG DEFNAYYAR NVYTEIKCNSLLP LDDIVRV AVPIAQK Variation of AVPI AKPF dimer AVP*IAQKSE KHSSGCAFL KKHRSTSQGK KSKLHSSHARSG

6.3.2.6 TAT-RasGAP317–326 as a dual sensitizer and anti-metastatic tool TAT-RasGAP317–326 is a cell-permeable peptide that contains a ten amino acid sequence derived from the p120 GTPase-activating protein (RasGAP). This peptide has been shown to increase the sensitivity of tumour cells specifically toward several anticancer ­treatments both in vitro and in vivo. Indeed, chemotherapeutic agents such as

6.4 Conclusions 

 157

cisplatin, doxorubicin, adriamycin, etc. have been shown to be more efficient in the presence of TAT-RasGAP317–326. Moreover, this sensitising effect was also observed using radiotherapy and photodynamic therapy. Importantly, this peptide alone does not induce apoptosis [41–44]. The RasGAP-derived peptide was also shown to be an inhibitor of cell migration and invasion [45]. Furthermore, among the ten amino acids from RasGAP, only two tryptophan residues were shown to be crucial for both sensitisation to apoptosis and inhibition of cell migration properties, making the WxW motif a potential candidate for anticancer drug design [46].

6.4 Conclusions There is no doubt about the critical role that metals play in the functioning and maintenance of life, highlighted by playing major roles of cofactors of enzymes in biological reactions. The clinical success of cancer treatment would be made much more efficient by the merging of one or more of the anti-cancer treatments we discussed in this chapter. The great success of cisplatin provided a proof that for investigating metals, and their role in defined biological processes might be essential in addition to their coordination in complexes as potential anticancer agents. Design strategies of novel platinum for example has led to the finding of different generations of the compound that is still being under intense investigation to address shortcomings of previous generation of platinum compounds. These findings, including what has been reported mainly by targeting the ubiquitin-proteasome pathway with metal based compounds (copper-, zinc- and gold-containing complexes), is an emerging concept in developmental therapeutics and would represent a significant progress in the developing novel anticancer drugs. Although bio-complexes are not leading the pharmaceutical market in contrast to small molecules, this chapter has highlighted that they remain very attractive. Metals display potent anti-cancer activities, and their costs is very low. Anticancer peptides, on the contrary, might be more cost-effective but generally display more specificities towards their biological target, which is not negligible when a major current goal is to decrease drug side effects. Nevertheless, their development remains very slow. One active field of research is the translation of these bio-complexes into small molecules bearing the same activities and being more attractive in terms of industrial development. Smac mimetics represent a good example of this trend since several clinical trials are now ongoing.

Acknowledgment Dr. Khalil as a corresponding author of this chapter would like to thank the Swiss ­National Science Foundation “SNSF” for a financial support of the project P300P3_158486 from 01.05.2015 to 31.10.2016 during which this work was completed.

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 6 Peptides and biocomplexes in anticancer therapy

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Bartosz Tylkowski, Renata Jastrząb and Akira Odani

7 Developments in platinum anticancer drugs Platinum compounds represent one of the great success stories of metals in medicine. Following the unexpected discovery of the anticancer activity of cisplatin (Fig. 7.1) in 1965 by Prof. Rosenberg [1], a large number of its variants have been prepared and tested for their ability to kill cancer cells and inhibit tumor growth. Although cisplatin has been in use for over four decades, new and more effective platinum-based therapeutics are finally on the horizon. A wide introduction to anticancer studies is given by the authors of the previous chapter. This chapter aims at providing the readers with a comprehensive and in-depth understanding of recent developments of platinum anticancer drugs and to review the state of the art. The chapter is divided into two parts. In the first part we present a historical aspect of platinum and its complexes, while in the second part we give an overview of developments in the field of platinum anticancer agents.

H3N

Cl Pt

H3N

Cl

Fig. 7.1: Cisplatin.

7.1 Discovery of anticancer activity of cisplatin Platinum  is a  chemical element  with  the symbol  Pt. Its name is derived from the Spanish term  platina, which is literally translated into “little silver”. The platinum coordination complexes are a group of antineoplastic agents that are usually classified as alkylating agents. Early in the 19th century, the study of the compounds of platinum began to interest a number of chemists. The existence of ammonium chloroplatinate was of course well known as the source of the pure metal on heating, and other similar triple salts were soon recognised. One of the first to undertake investigation of a wide range of Pt compounds was Edmund Davy. He discovered that a spiral of platinum wire in the vicinity of an ignited wick in the lamp would catalyse the continued oxidation of coal gas (methane) with sufficient vigor to glow white hot after the wick was extinguished. At least, that is how one would now describe his experiment; but Davy’s central concern at the time was with the nature of combustion and safety in mines. He recognised that the addition of the platinum spiral was a useful modification to the miner’s lamp, but of the phenomenon he had observed, he simply remarked that it was “more like magic than anything I have seen . . . it depends upon a perfectly new principle in combustion”. In 1812 he published a long paper in

7.1 Discovery of anticancer activity of cisplatin 

 161

The Philosophical Magazine “On Some combination of Platina”, in which he reported his findings on its reactions with phosphorus, oxygen, chlorine, and ammonia, concluding his paper with a rather prophetic comment: Platina appears to be characterised no less by its valuable properties than by its disposition to form particular triple compounds; and there can be little doubt that a more expensive acquaintance with the combination of this metal will ass considerably to the number of such substances. It would be an interesting inquiry, whether the same laws which seem to govern the formation of binary compounds extend likewise to those of ternary compounds. For an investigation of this kind no metal seems so well adopted as platina.

The constitution and structure of the first coordination compounds remained unknown for many years, and they were customarily named after their discoverers. Cisplatin was first described in 1845 as Peyrone’s salt, and in 1893 its chemical structure was elucidated. The first platinum ammine was reported by Magnus, and it is still called Magnus’ Green Salt, while its chlorinated derivatives discovered by James Gros are called Gros’ compounds [2]. The great success story of platinum compounds was initiated by accident exactly 50 years ago in the laboratory of physicist-turned-biophysicist Barnett Rosenberg at the Michigan State University, East Lansing, Michigan, United States. Rosenberg was interested in applying electromagnetic radiation to bacterial and mammalian cells to investigate whether electric or magnetic dipole fields might be involved in cell division. Inadvertently, in the early experiments using Escherichia coli, a set of platinum electrodes (considered to be inert) was included in the growth chamber. When the field was turned on, the bacteria appeared as very long filaments (300 times the usual length) rather than as the normal short rods. This effect was shown not to be due to the electric field but, rather, to electrolysis products arising from the platinum electrodes. Detailed chemical analysis identified two active complexes – the neutral cis-isomer [Pt(II) (NH3)2Cl2], which went on to be cisplatin, and a platinum(IV) analogue, cis-diamminetetrachloro-platinum(IV) – as the causative molecules of this intriguing biological effect. The  trans  isomer was much less active. These findings were published in 1965. In 1968, following further tests against various bacteria,  cis-diamminedichloroplatinum(II) (cisplatin) was administered intraperitoneally to mice bearing a standard murine transplantable tumour of the day, sarcoma-180, at the non-lethal dose of 8 mg/kg, and was shown to cause marked tumour regression. Since 1978 when the American Food and Drug Administration (FDA) approved it as an anticancer compound, cisplatin has had a profound and far-reaching impact on the field of chemotherapy, substantially increasing survival for many cancer patients. Most notably, long-term survival rates of testicular cancer patients improved from less than 10% to greater than 90% following the introduction of cisplatin to the treatment regimens [3]. Apart from its wonderful clinical value, cisplatin, one of the few approved transition-metal-based drugs, inspired generations of inorganic chemists

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 7 Developments in platinum anticancer drugs

to pursue applications of their research in the medical sciences. With confirmatory  in vivo  tests performed at the Chester Beatty Institute in London, United Kingdom, cisplatin was taken on by the US National Cancer Institute (NCI) for clinical testing. The first patients were treated in 1971 – a remarkably short time, in modern terms, from the original “bench” discovery [1, 4].

7.2 Platinum complexes 7.2.1 Platinum(II) complexes Following the initial reports of the anticancer activity of cisplatin, inorganic chemists began preparing a variety of platinum complexes with different ligands and testing their antineoplastic effects. The collective result of these many separate studies was the emergence of a set of rules governing molecular structure that appeared to be required in order for a platinum complex to show activity. Traditional structure– activity relationships specified that the active platinum complex should: –– have square-planar geometry –– be charge neutral –– contain two cis amine ligands –– have two cis anionic ligands. Moreover the anionic ligands could not bind the platinum too tightly, or activity would be reduced. If these ligands were too labile, however, the compounds exhibited prohibitively high levels of toxicity. Furthermore, the two ammine ligands or two anionic ligands could be replaced by a chelating diamine or chelating dicarboxylate, respectively [5]. While cisplatin is among the most effective anticancer agents in the armory of drugs available to cancer clinicians, this broad-spectrum cytotoxic is not without its drawbacks. Side effects include: –– nephrotoxicity –– nausea –– vomiting –– loss of sensation in the extremities. The success of cisplatin in the clinic led to a period in which many thousands of platinum-containing compounds were screened for their activity, with around 30 entering clinical trials. Figure. 7.2 provides chemical structures of platinum-based anticancer drugs in clinical use worldwide. The first of these follow-up agents to enter worldwide clinical use was carboplatin. Carboplatin contains the cis-Pt(NH3)2 active fragment of cisplatin, but the two chloride leaving groups are replaced by a bidentate dicarboxylate. In line with this

7.2 Platinum complexes 

H3N H3N

Pt

O

O

NH2 O Pt NH O

O

2

O

Carboplatin NH2 NH2 Lobaplatin

Pt

CH3

O

O

O

H3N H3N

Pt

O

O O

Nedaplatin

Oxaliplatin O

O

 163

H3C

O

NH2

H3C

O

NH2

Heptaplatin

Pt

O

O

O O

Fig. 7.2: Chemical structures of platinum-based anticancer drugs in clinical use worldwide.

simple change of leaving groups, the biological mechanism of action of carboplatin appears to be entirely analogous to that of cisplatin and the DNA adducts to be the same. The spectrum of cancers that can be treated is also identical in vivo. The change of leaving group does reduce the activity of the agent somewhat, however. While it is as effective in ovarian cancer, it is less potent against testicular, head, and neck cancers. Correspondingly, the side effects are less severe. Cisplatin has tended to remain the agent of choice, with carboplatin used when there is a clinical need to minimize the platinum drug side effects because of other medical conditions. In 2004, another platinum drug, oxaliplatin has become the third to achieve worldwide clinical approval. The clinical advantage of oxaliplatin is that it has a different spectrum of activity: in particular, it is effective against colorectal cancer, a disease not treatable using cisplatin or carboplatin. Moreover, oxaliplatin is active against some cisplatin-resistant cancers. The predominant DNA adducts formed by oxaliplatin are 1,2-intrastrand GG adducts analogous to those formed by cisplatin. However, in oxaliplatin the amines are incorporated into a (trans)-R,R-1,2-diaminocyclohexane (DACH) framework and the adducts are thus not cis-Pt(NH3)2 adducts but Pt(DACH) DNA adducts. The precise biological reasons for the difference in the spectrum of activity and the ability of this agent to circumvent some cisplatin resistance mechanisms remain to be fully elucidated, but seems to hinge on the DACH ligand. The alternate diamine ligands used in lobaplatin and heptaplatin do not confer these same effects. Lobaplatin is a platinum(II) complexes containing diastereometric mixture of 1,2-bis(aminomethyl)cyclobutane stable ligand and lactic acid as the leaving group. The two diastereoisomers can be separated in plasma ultrafiltrate samples from cancer patients by highperformance liquid chromatography linked to an ultraviolet detector after solidphase extraction. Lobaplatin was developed by ASTA Pharma AG (Frankfurt, Germany) that subsequently discontinued development of the drug, undertaken then by Zentaris AG. In 2003, Zentaris AG and Hainan Tianwang International Pharmaceutical signed a contract for the manufacture and marketing of ­lobaplatin

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 7 Developments in platinum anticancer drugs

in China. Lobaplatin has been approved in China for the treatment of chronic myelogenous leukaemia (CML) and inoperable, metastatic breast and small-cell lung cancer. Lobaplatin has also completed phase II clinical trials in the US, Australia, EU, Brazil, and South Africa for the treatment of various cancers, including breast, oesophageal, lung, and ovarian cancers as well as CML. The main toxicity of lobaplatin is thrombocytopenia, and its dose should be corrected according to renal function. Heptaplatin has been approved for the treatment of gastric cancers in South Korea. Preclinical studies suggested that this drug might have a greater antitumour activity and a lower toxicity than cisplatin. The molecular mechanisms of heptaplatin in cisplatin-resistant cancer cell lines and the involvement of metallothionein were investigated. Since 1978 the development of the platinum anticancer complexes has not been only a great interest of research & development (R & D) platforms based at pharmaceutical corporations that have provided the above-mentioned drugs but also of small/medium size research groups located at universities and public institutions around the world that have performed fundamental studies. Very interesting investigations have been reported by Odani et al. [6]. The authors studied intermolecular noncovalent interactions of several platinum(II) complexes Pt(L)(en) (L = bpy, phen, nphen, Me4phen1) with various purine and pyrimidine bases of NMP (= AMP, GMP, CMP, UMP) in solution and in the solid state with a view to revealing the presence and mode of intermolecular associations between them. The research group of Prof. Natile [7] has extended the investigation to the synthesis and biological evaluation of cisplatinum(II) complexes with ligands containing a carbobicyclic framework and two methylamino substituents in 1,2 positions in order to have a 1,4-diaminobutane-like structure (Fig. 7.3). This type of molecule was designed to slightly increase the flexibility and the steric hindrance of the diamine carrier ligand with respect to the corresponding complexes with DACH ligands. Moreover, the stereochemistry and lipophilicity of the new ligands were modulated by the scientists to investigate how they can influence the interaction with DNA, as the target molecule, and the pharmacokinetics of the compounds. By using a two- or three-step synthetic pathway, the authors were able to synthesize four compounds which showed better antiproliferative activity than cisplatin (up to 4.28 times more active than the clinically relevant drug). Elerman and co-workers have focused their research on the synthesis and characterization of cisplatin analogues containing substituted benzimidazole ligands [8].

X

NH2 Cl Pt NH2

X = O, CH2 or CH2CH2

Cl

Fig. 7.3: Basic structure of the platinum compounds studied by Natali et al. Reprinted with permission from [7]. Copyright 2008 American Chemical Society.

7.2 Platinum complexes 

 165

The authors decided to use the benzimidazole ligands because they have four main features that could be important in the interaction of their platinum(II) complexes with DNA: 1. The benzimidazole nucleus is found in a variety of naturally occurring compounds such as vitamin B12 and its derivatives, and it is structurally similar to purine bases. 2. The substituents at position 1 and/or position 2 of the benzimidazole ligands can induce notable changes in the electronic, steric, and hydrophobic properties of the compounds. 3. The nonplanar benzimidazole ligands are flexible and bulky enough to affect the kinetics and cytotoxicities of the corresponding platinum(II) complexes. 4. The benzimidazole ligands having acetoxy, hydroxyl, and/or free N1−H moiety, which would have hydrogen-bond donor and/or acceptor properties, could facilitate novel types of lesions with cellular DNA, and might exhibit sequence selectivity. Moreover the authors showed that synthesised Pt(II) complexes were less cytotoxic than cisplatin and were comparable to carboplatin. Furthermore, reported results of the plasmid DNA interaction and the restriction studies suggest that changing the chemical structure of the benzimidazole ligands may modulate DNA binding mode and the sequence selectivity. Recently, Lippard and Wilson synthesised five cis-diammineplatinum(II) complexes bearing different β-diketonates, shown in Fig. 7.4 [9]. Theses particular ligands were selected to systematically modify the electronic properties and hydrophobicity of the

H3C

H3C

CH3 O

O

O

(SO4)0.5

Pt

O Pt

NH3

H3N

H3N

(1)

O

(NO3)

Pt H3N

(NO3)

NH3

(3)

O

O

(NO3)

Pt H3N

NH3

O Pt

H3N

(2)

O

O

(SO4)0.5

NH3

CF3

(4)

CH3

CF3

NH3

(5)

Fig. 7.4: Chemical structures of cis-diammineplatinum(II) complexes bearing different β-diketonates. Reprinted with permission from [9]. Copyright 2012 American Chemical Society.

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resulting platinum complexes through the use of trifluoromethyl and phenyl groups, respectively. The authors discovered that both of these properties have direct effects on the overall cytotoxicity of the resulting complexes in cancer cells. The primary O,O′ bidentate chelating mode of these ligands is analogous to that of the CBDCA ligand of carboplatin, thus warranting consideration of their use as leading group ligands for novel platinum anticancer drug candidates. Although β-diketonates have been used as supporting ligands for titanium(IV) [10] and organometallic ruthenium(II) anticancer agents [11, 12], they were also used as ligands by the research group of Prof. Marsigliante from the University of Salento, Italy. The authors designed and synthesised platinum(II) complexes containing acetylacetonate (acac) in the coordination sphere of the metal, such as: [PtCl(O,O′-acac)(DMSO)] with only one oxygen-bonded (O,O′acac) acac, [Pt(O,O′-acac)(g-acac)(DMSO)] containing both O,O′-acac and an s-bonded (g-carbon bonded) acac, their dimethylsulfide (DMS) analogs having the same key structures [13]. The authors reported that the higher and selective cytotoxicity of  [Pt(O,O′-acac)(g -acac)(DMS)] toward cancer cells in both immortalised cell lines and in breast cancer cells in primary cultures, stimulated a pre-clinical study so as to evaluate its therapeutic potential in vivo. During the study, the efficacy of [Pt(O,O′acac)(g -acac)(DMS)] was assessed using a xenograft model of breast cancer developed by injection of MCF-7 cells in the flank of BALB/c nude mice. Treatment of solid tumour-bearing mice with  [Pt(O,O′-acac)(g -acac)(DMS)]  induced up to 50% reduction of tumour mass compared with an average 10% inhibition recorded in cisplatintreated animals. Thus, chemotherapy with  [Pt(O,O′-acac)(g -acac)(DMS)]  was much more effective than cisplatin. Marsigliante end co-workers also demonstrated enhanced  in vivo  pharmacokinetics, biodistribution, and tolerability of  [Pt(O,O′-acac) (g -acac)(DMS)] when compared with cisplatin administered in Wistar rats. Pharmacokinetics studies with  [Pt(O,O′-acac)(g -acac)(DMS)]  revealed prolonged Pt persistence in systemic blood circulation and decreased nefrotoxicity and hepatotoxicity, major target sites of cisplatin toxicity. Overall, according to the authors [Pt(O,O′-acac) (g -acac)(DMS)] turned out to be extremely promising in terms of greater in vivo anticancer activity, reduced nephrotoxicity, and acute toxicity compared with cisplatin. Moreover, the research carried out by Lord and co-workers [14] with β-diketonate hafnium complex, showed that β-diketonate ligands have a significant effect on the cytotoxic potential of the complexes, and that these group IV complexes warrant further evaluation as novel metal-containing anticancer agents.

7.2.2 Platinum(IV) complexes The anticancer activity of platinum(IV) complexes such as  cis-[PtCl4(NH3)2] has been known since the discovery of cisplatin (cis-[PtCl2(NH3)2]) in 1965. Although substant­ially fewer platinum(IV) complexes have been studied as potential anticancer agents than have platinum(II) complexes [15] and [16], some have shown

7.2 Platinum complexes 

 167

sufficient promise to enter clinical trials. Iproplatin (CHIP, JM9,  cis,trans,cis[PtCl2(OH)2(isopropylamine)2]) was selected from a range of platinum(IV) complexes synthesised by Tobe and co-workers for its high solubility [15].  Iproplatin was sufficiently well tolerated to enter phase II and III clinical trials [17], but was ultimately found to be less active than cisplatin and so it has not entered widespread clinical use.  Tetraplatin (ormaplatin, [PtCl4(d,l-cyclohexane-1,2-diamine)]) showed great promise in preclinical studies but caused severe neurotoxicity in treated patients, and the trials were subsequently abandoned at the phase I level [18]. JM216 (satraplatin, cis,trans-[PtCl2(OAc)2(NH3)cyclohexylamine] is a rationally designed drug that was recently in phase III trials, but trials were abandoned due to variability in drug uptake [19]. That none of the platinum(IV) compounds trialled clinically have revealed significantly greater activity in humans than that of cisplatin is particularly disappointing in light of the report by Kelland et al. that analogues of JM216 are up to 840-fold more active than cisplatin in in vitro assays [19]. The high activity was ascribed to high cellular uptake, but in vivo reduction alters the pharmacological properties and thus the effectiveness of the drug. However, platinum(IV) complexes have enormous potential as anticancer agents in terms of both high activity and low toxicity, but this potential has not been realised by the drugs investigated to date, probably because they are reduced too readily in the bloodstream. The potential advantages of platinum(IV) complexes that remain in the higher oxidation state in the bloodstream are that their lower reactivity would diminish the loss of active drug and lower the incidence of unwanted side reactions that lead to toxic side effects. In addition, the higher lipophilicity of some platinum(IV) complexes would be retained, leading to potential improvements in cellular uptake. It is widely believed that reduction to platinum(II) is essential for the anticancer activity of platinum(IV) complexes to be effected [20–22]. If this is the case, then the ease with which a platinum(IV) complex is reduced can be expected to influence its biological activity. There are a number of factors to consider when assessing the possible effects of administering the platinum(IV) analogue of a platinum(II) complex. The kinetic inertness of platinum(IV) complexes means that there is increased opportunity for the complex to arrive at the cellular target intact. Modifying the axial ligands of platinum(IV) complexes alters the solubility of the complex (lipophilic vs. hydrophilic) and thus its ability to enter tumour cells before being reduced to yield the active platinum(II) drug. Preventing side reactions by administering platinum(IV) complexes has the potential to lead to a drug that has reduced toxicity and high activity. The reduction potentials of diam(m)ine platinum(IV) complexes are dependent on the nature of the axial and equatorial ligands, but the axial ligands generally exert stronger influence [23]. Reduction occurs most readily when the axial ligands are chloro, while the least readily when they are hydroxo, and is intermediate when they are carboxylato [24]. These results have been confirmed by Choi et al. who also showed

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 7 Developments in platinum anticancer drugs

that the reduction rates correlate with reduction potentials [25]. The development of new synthetic methodologies for accessing the platinum(IV) manifold can expand the range of complexes showing novel properties [25]. Recently, Farrer et al. [26] have published information on the synthesis, characterisation, and biological activity of a series of six “cis” photo-active dia(m)mine Pt(IV) diazido complexes, with the general formula cis,trans,cis-[Pt(N3)2(OH)2(NH3)(X)] (X = propylamine, or butylamine, or pentylamine, or pyridine, or 2-methylpyridine, or 3-methylpyridine), presented in Fig. 7.5.

H3N Amine

OH Pt OH

N3 N3

vs

H3N N3

OH Pt OH

N3 Amine

Fig 7.5: Photo-active dia(m)mine Pt(IV) diazido complexes synthesised by [26]. Reprinted with permission from [26]. Copyright 2010 American Chemical Society.

The authors reported that inactive drug precursors (prodrugs) can be activated by light, following administration, to create a reactive species specifically where it is required, for example, at the tumour site. Light-triggered drug release has been investigated for other therapeutic benefits too, for example, for drugs such as ibuprofen [27]. Photoactivation of a drug provides a degree of spatial and temporal control over drug dosage, and this strategy is being investigated for a number of metal-based anticancer drugs [28]. Photoactive drugs are currently used in the clinic in the modality of photodynamic therapy (PDT), which is an extremely effective treatment for a number of cancers including those of the skin, lung, brain, and esophagus [29, 30]. The selectivity of PDT that results from the cytotoxic species being produced only where it is required has advantages over other forms of therapy including surgery and chemoand radiotherapy, in that it can spare the normal tissue and structure of the organ and can be repeated as often as required. A limitation of PDT is that the cytotoxic mechanism requires oxygen, which can be problematic since tumours are often hypoxic [31]. Photochemotherapy, which would be less dependent on oxygen for the cytotoxic effect, is therefore desirable [32]. The same research group, in a prior study has shown that photoactivation of nontoxic Pt(IV)-azido complexes such as  trans,trans,trans[Pt(N3)2(OH)2(NH3)(py)] can generate potent, cytotoxic species (up to 90× more cytotoxic following irradiation than treatment with cisplatin) capable of causing cell death in a number of cell lines by a mechanism distinct from that of cisplatin [33]. Pt(IV) complexes such as these demonstrate good aqueous solubility in comparison to their Pt(II) counterparts and are much less biologically reactive – a general feature of Pt(IV) complexes [34] – which minimises the potential for side reactions (and thus the cytotoxicity) of the prodrug on the way to its cellular target.

7.3 Anticancer drug delivery 

 169

7.3 Anticancer drug delivery A major challenge for improving anticancer treatments is to direct the therapeutic agent specifically to the tumour cells or tumour blood vessels, thus enhancing the efficacy of the treatment and decreasing undesirable side effects [35, 36]. Therefore, application of targeted drug delivery system (TDDSs) is significant for reducing the undesirable side effects of anticancer drugs in healthy cells/organs, thereby improving the therapeutic efficacies [35]. Different strategies have been designed to achieve this goal. Platinum drugs can be passively targeted to solid tumours through the enhanced permeability and retention effect  [37]. Alternatively, platinum drugs can be actively targeted to both solid tumours and leukaemia through the use of aptamers,  peptides, antibodies,  or cancer-related substrates (such as folate). Research group of Prof. Wheate has tethered the active component of the anticancer drug oxaliplatin to a gold nanoparticle (NPs), for improved drug delivery [38]. During this investigation, the platinum molecule was the active component of oxaliplatin: [Pt(1R,2R-diaminocyclohexane]2+  ([Pt(dach]). The authors decided to use used gold nanoparticles because they are well known as an ideal drug-delivery scaffold due to their nontoxic and nonimmunogenic behavior. Gold nanoparticles can be readily functionalised with multiple targeting molecules and have so far shown excellent potential for the delivery of other nonplatinum-based drugs and a platinum(IV) complex.  Furthermore, they are easy to synthesise, and their high surface area increases drug density on the surface, allowing higher concentrations of a drug or indeed multiple drugs to be simultaneously loaded onto a single nanoparticle. The unique physiochemical properties of gold NPs can also trigger drug release at remote sites, creating a multipronged approach to destroying cancer cells [39, 40]. Prof. Wheate and co-workers functionalised the naked gold nanoparticles with a thiolated poly(ethylene glycol) (PEG) monolayer capped with a carboxylate group. The compound [Pt(1R,2R-diaminocyclohexane) (H2O)2]2NO3  was added to the PEG surface to yield a supramolecular complex with 280 (±20) drug molecules per nanoparticle. The authors reported that the platinum-tethered nanoparticles demonstrated the cytotoxicity as good as, or significantly better, than oxaliplatin alone towards all cell lines studied and an unusual ability to penetrate the nucleus in the lung cancer cells. It is important to note that several gold nanoparticle-based drugs are currently under development by CytImmune, with their lead drug, Aurimune, in clinical trials. The size of the gold nanoparticles can also be controlled. Typically, gold nanoparticles ranging from 13 to 60 nm can be easily made by a simple reduction of gold salts in water [41, 42] or by starting with seed particles of 13 to 20 nm followed by a second reduction step involving more gold salt [43–45].  Very fascinating studies have been performed by Kumar and coworkers [46]. Neuropilin-1 (Nrp-1) receptor was selectively targeted to enhance delivery of a therapeutic drug such as platinum(IV). By selecting this receptor the authors achieved

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 7 Developments in platinum anticancer drugs

higher cytotoxic effects in prostate cancer cells. It is well known that during the development of metastatic tumours, cells can upregulate certain cell-surface receptors molecules and secreted factors, as well as express several oncogenic proteins primarily involved in embryonic development. However, cancer cells often have similar characteristics to those of surrounding healthy tissue, making the treatment more challenging. In such cases, ligands can be designed to have high specificity and affinity to receptors that are overexpressed on cancerous cells. Since the targeting ligands are generally present on the outermost sector of the nanoparticle delivery system,  glutathione-stabilised gold (Au@GSH) nanoparticles were designed by the authors with a targeting moiety on the surface with the aim of selectively increasing cellular binding and internalisation via receptor-mediated endocytosis. Nrp-1 plays a significant role during angiogenesis and vascular permeability. Nrp-1 is a transmembrane glycoprotein that binds peptides with a C-terminal amino acid motif R/KXXR/K, called the CendR motif (found in semaphorin 3A, VEGF-A165, and iRG). Binding of the CendR peptide to Nrp-1 mediates cell internalisation and tissue penetration. Nrp-1 is expressed by a large variety of tumours, including osteosarcoma, melanoma, lung cancer, brain tumor, colon cancer, pancreatic cancer, prostate cancer, breast cancer, myeloid leukaemia, salivary adenoid cystic carcinoma, infantile hemangioma, ovarian neoplasm, and bladder cancer. Nrp-1 has high affinity toward its cognate ligands, thereby enabling nanoparticles to penetrate into tumour cells and tissues. Kumar and co-workers assumed that introduction of a targeting peptide specific for Nrp-1 receptor may be a more effective way to increase the efficacy of tumour penetration by platinum(IV) drugs. The authors combined the properties of gold NPs stabilised with glutathione (Au@GSH) with a platinum(IV) drug and the targeting CendR peptide ligand Cys-Arg-Gly-Asp-Lys (CRGDK) into a single platform to create an effective TDDS for prostate cancer treatment, as shown in Fig. 7.6.

Au@GSH Pt(IV) CRGDK Neuropilin

Lysosome

Nucleus

Fig 7.6: Anticancer single platform designed by Kumar et al. Reprinted with permission from [46]. Copyright 2014 American Chemical Society.

References 

 171

In the absence of targeting ligands, nanoparticles interact nonspecifically with cell membranes, which are insufficient to achieve an optimal effect of a drug at the disease site.  Kumar et al. have shown that small Au@GSH NPs (5.2 nm) functionalised on the surface with platinum(IV) and CRGDK have effective targeting activity and demonstrate potent cytotoxicity against prostate cancer cells that overexpress Nrp-1 receptors. As mentioned above, the possibility to control anticancer drug activity and release on demand is very attractive in cancer therapy. The photoactivated platinum(IV) prodrug is stable in the dark and can be activated by UV light. Very recently, Dai and co-workers [47] have developed a multifunctional drug delivery system combining upconversion luminescence/magnetic resonance/computer tomography trimodality imaging and NIR-activated platinum pro-drug delivery. The authors used the core–shell structured upconversion nanoparticles NaYF4:Yb3+/Tm3+@NaGdF4/Yb3+ to convert the absorbed NIR light into UV to activate the  trans-platinum(IV) prodrug,  trans,trans,trans-[Pt(N3)2(NH3)(py)(O2CCH2CH2COOH)2], as shown in Fig. 7.7. Compared with the direct use of UV, the NIR offers a longer tissue penetration depth and is less harmful to health. Meanwhile, the upconverted nanoparticles can effectively deliver the platinum(IV) pro-drugs into the cells by endocytosis. The results obtained showed that the mice treated with pro-drug-conjugated nanoparticles under near-infrared (NIR) irradiation demonstrated better inhibition of tumour growth than those under direct UV irradiation. According to the authors, this multifunctional nanocomposite could be used as multimodality bioimaging contrast agent and a transducer by converting NIR light into UV for control of drug activity in practical cancer therapy.

UCL

NIR

UV

Diagnosis

CT

NIR

Therapy

Tumor

MRI

Fig. 7.7: Multifunctional nanocomposite developed by Dai et al. Reprinted with permission from [47]. Copyright 2013 American Chemical Society.

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Index 1,2-bis(aminomethyl)cyclobutane 163 α-difluormethylornithine 75 Achromobacter cycloclastes 24 Aeromonas aquariorum 116, 117 amicyanin 21, 25 aminoazobenzene 42, 44, 46, 47 Anabaena 20 angiogenesis 170 anticancer 160, 166 anticancer activity 160, 166, 167 anticancer agents 166, 167 anticancer drug 169, 171 anticancer treatments 169 antiproliferative activity 164 antitumour activity 164 azobenzene 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 56, 58, 59, 60, 62, 63, 64 Bacillus subtilis 116, 117 benzimidazole ligands 164, 165 biogenic amines 69, 70, 71, 72, 75, 78, 79, 81, 82, 84, 89 Biogenic polyamines 71 biosynthesis 70, 71, 72, 74, 75, 76, 77 bis(trialkoxysilanes) 151 bisphosphonate compounds 124, 125, 126, 127, 131, 132 bisphosphonate molecules 128 Bisphosphonates 121 bladder cancer 170 brain tumor 170 breast cancer 166, 170 Cadaverine 69 Caldopentamine 69 cancer 160, 163, 164, 166 cancer cells 166, 170 cancer therapy 171 Carboplatin 162, 163, 165 carcinos 143 catalyse 160 ceruloplasmin 2, 28 chelating diamine 162 chelating dicarboxylate 162 chemotherapy 161, 166 chronic myelogenous leukaemia 164

cilengitide 151 cis-diamminedichloroplatinum(II) 161 cis-diamminetetrachloro-platinum(IV) 161 cisplatin 1, 160, 161, 162, 163, 164, 165, 166, 167, 168 cisplatin analogues 164 cisplatinum(II) complexes 164 Cobalamine 107 collagen 148 colon cancer 170 complexes 161 computer tomography 45 coordination compounds 161 coordination number 107 cysteine 1, 31 cytochrome c peroxidase 1, 21 cytochrome f 21, 31 cytotoxic 165, 166, 168, 170 cytotoxicity 166, 169, 171 diethyldithiocarbamate 153 difluormethylornithine 76 dilysine 22 dipole moment 42 Disulfiram 153 epithelial-mesenchymal transition 147 Escherichia coli 72, 116, 117, 161 esophagus 168 etaracizumab 151 fibronectin 148 Gamma ray 128 gold nanoparticle 169 hepatotoxicity 166 heptaplatin 164 herbicidal agents 107 histidine 1, 18, 31 homeostasis 132 Homospermidine 69 hydrophilic 167 imidazole 1, 2, 18, 24, 27 infantile hemangioma 170 inoperable 164

Index 

intravasation 148 Iproplatin 167 Klebsiella mobilis 116 laminin 148 Lanthanide 107 leukaemia 169 lipophilic 167 lipophilicity 164, 167 lobaplatin 163, 164 liquid chromatography 70 liquid-crystalline 42 lung cancer 170 mammalian cells 161 melanoma 170 metallodrugs 1, 21, 22, 23 metallomesogens 48 metallonucleases 63 metalloporphyrins 58 metalloproteinases 148, 150 metalloproteins 1, 18, 24, 28 metallothionein 164 metastatic breast 164 Mutations 143 myeloid leukaemia 170 nefrotoxicity 166 Neoplastic diseases 145 nephrotoxicity 166 neurotoxicity 167 noncovalent interactions 1, 2, 3, 4, 6, 11, 13, 18, 19, 22, 24, 29, 30 Norspermine 69 nucleic acids 69, 71, 78, 80, 85, 90 oesophageal 164 oncogene 144 organometallic 48, 52 organometallic ruthenium(II) anticancer agents 166 ormaplatin 167 osteosarcoma 170 ovarian neoplasm 170 oxaliplatin 163, 169 pancreatic cancer 170 pentalysine 21 pesticides 107

 175

phenol 1, 3, 10, 12, 13, 16, 20, 28, 31, 32 phenolate moiety 16 phospholipids 69, 78 photoactivated platinum(IV) pro-drug 171 Photoactive drugs 168 Photochemotherapy 168 photochromic units 41 photodynamic therapy 168 photoexcitation 46 photoisomerization 41, 44, 46, 47, 50, 53, 58, 63 photorelease 55 photo-responsive materials 41 photo-sensitive complex 56 photosensitizer 151 photosystem 21 plastocyanin 18, 21, 32 platina 160, 161 platinum 160, 162 platinum ammine 161 platinum anticancer 160 platinum anticancer complexes 164 platinum-based anticancer drugs 162 platinum complex 162, 166 platinum compounds 160, 161 platinum drug 163, 169 platinum(II) complex 165, 166, 167 platinum(IV) analogue 167 platinum(IV) complex 166, 167, 169 platinum(IV) compounds 167 platinum(IV) drug 170 platinum(IV) pro-drugs 171 polyamine 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 90, 92, 93 Polysilsesquioxane 151 prodrugs 168 prostate cancer 170, 171 proteins 69, 71, 78, 92, 127, 132, 133 proteoglycans 148 Pseudomonas aeruginosa 116, 117 pseudostilbene 46, 47 Putrescine 69 pyridocarbazole moiety 23 pyrrolidine dithiocarbamate 153 radiogallium complex 126 radioimmunotherapy 121 Radiolabeled annexin 136 radiometal complexes 121, 137

176 

 Index

radionuclides 121, 122, 127, 128, 129, 130, 137 Radiopharmaceuticals 121, 128 radiotherapy 168 salicylaldehyde 107, 108, 110 salivary adenoid cystic carcinoma 170 Schiff bases 107, 110, 117, 118 Serratia marcescens 116, 117 Silene pratensis 21 spermidine 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 82, 90 spermine 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 86, 90, 91 Staphylococcus aureus 115, 116, 117 staurosporine 22 Streptococcus pyogenes 116, 117 supramolecular architecture 1

targeted drug delivery system 169 tetralysine 21 Tetraplatin 167 Thermospermine 69 thiol 1 thiolated poly(ethylene glycol) 169 thrombocytopenia 164 transition-metal-based drugs 161 (trans)-R,R-1,2-diaminocyclohexane 163 trialkoxysilanes 151 trilysine 22 trimethylsilylacetylene 51 tumor 143, 144, 145, 146, 147, 148, 149, 150, 153, 155, 156, 160 tumour cells 167, 169 tyrosine 1, 32 vitronectin 148