Application of the Principle of Hard and Soft Acids and Bases to Mechanisms of Bioinorganic Reactions -Monograph

Livre de Lyon Academic Works of Livre de Lyon

Science and Mathematical Science

2019

Application of The Principle of Hard and Soft Acids and Bases to Mechanisms of Bioinorganic Reactions -Monograph-

Tanja V. Soldatović Novi Pazar State University, Serbia, [email protected]

Follow this and additional works at: https://academicworks.livredelyon.com/sci_math

Part of the Inorganic Chemistry Commons, Materials Chemistry Commons, Medicinal-Pharmaceutical Chemistry Commons, and the Organic Chemistry Commons

Recommended Citation Soldatović, Tanja V., "Application of The Principle of Hard and Soft Acids and Bases to Mechanisms of Bioinorganic Reactions -Monograph-" (2019). Science and Mathematical Science. 5. https://academicworks.livredelyon.com/sci_math/5

This Book is brought to you for free and open access by Livre de Lyon, an international publisher specializing in academic books and journals. Browse more titles on Academic Works of Livre de Lyon, hosted on Digital Commons, an Elsevier platform. For more information, please contact [email protected]. Ć I V O T H

A P

D A

L R

O G

S O

. N

V O

A M J N A T

M O N O G R A P H TANJA V. SOLDATOVIĆ

c n e e d i t e t p e u t y e o l a b b h i a

r I t i t

e t . a n v o s c o t

a m c n h e

e b h e d

l r l c l m i i a e i p h

f w

o h

l w s

c i . d e i s h s v n h t e d e a

l g w

s d m i f r s f e o

o l e p r

y f h u y r

w t t c o s i o r r s t i c l n a g o u k

e l

m s h s e e e w c e v r h r

r e

c a h

n d e

e g c f e i i h s t o t h n

c r y a e p e

r l l g h n e r e e t i

v o s h o

g j

o n l

i l h n c i i o o p r s i t i w a

e b

d r

d f h i t g n o i s p n o

n a e n d n r o d o e o g i c u

g o s m t ,

a n s n d s

g i o a e e n t h p r m t h e x

t o

e s n d

i p r I n h o e o t r t f a

Tanja V. Soldatović

APPLICATION OF THE PRINCIPLE OF HARD AND SOFT ACIDS AND BASES TO MECHANISMS OF BIOINORGANIC REACTIONS

-MONOGRAPH-

Lyon 2019

Author • Tanja V. Soldatović Editor in Chief • Caroline Justet Reviewers Prof. em. Dr. Dr. h. c. mult. Rudi van Eldik Prof. Dr. Basam M. Alzoubi Prof. Dr. Mohamed M. Shoukry

Cover Design • Betul Akyar First Edition• © May 2019

ISBN: 978-2-490773-02-2

© copyright All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by an means, electronic, mechanical, photocopying, recording, or otherwise, without the publisher’s permission.

Publisher Livre de Lyon Adress: 37 rue marietton, 69009, Lyon France Phone: +330605595613 website: http://www.livredelyon.com e-mail: [email protected]

Dr. Tanja V. Soldatović

Associate Professor Deaprtment of Chemical-Technological Sciences, State University of Novi Pazar

APPLICATION OF THE PRINCIPLE OF HARD AND SOFT ACIDS AND BASES TO MECHANISMS OF BIOINORGANIC REACTIONS

Reviewers:

Prof. em. Dr. Dr. h. c. mult. Rudi van Eldik

Emeritus Professor of Inorganic Chemistry, University of Erlangen-Nuremberg, Germany Professor of Inorganic Chemistry, Jagiellonian University, Krakow, Poland Professor of Inorganic Chemistry, N. Copernicus University, Torun, Poland

Prof. Dr. Basam M. Alzoubi

Professor of Physical Chemistry, AlBalqa Applied University, AlSalt, Jordan Professor of Physical Chemistry Al-Huson University College, Alhuson, Jordan

Prof. Dr. Mohamed M. Shoukry

Professor of Inorganic Chemistry. Department of Chemistry. Faculty of Science, Cairo University, Egypt

Published by Livre de Lyon

Dr. Tanja Soldatović

anja Soldatović is associate professor at Department of Chemical-Technological Sciences, State University of TNovi Pazar. Her research field is bioinorganic and medicinal inorganic chemistry (studies related to the biological activity and medical application of transition metal anti-tumour complexes). Currently, her research is focused on zinc and copper complexes. Dr. Tanja Soldatović is author and coauthor of more of 30 peer reviewed articles and two books. She participated in the realization of national scientific research project and international DAAD project. She was a research fellow at University of Sassari, Department of Chemistry, Sassari-Italy and University of Erlangen- Nürnberg, Inorganic Chemistry, Department of Chemistry and Pharmacy, Erlangen-Germany. Dr. Tanja Soldatović is member of professional societies: Serbian Chemical Society, Association for Cancer Research (Serbia) and European Association of Cancer Research.

PREFACE

It is well known that metal ions play an important role in the biological and biomedical processes. Many processes such as breathing, metabolism, photosynthesis, growth, reproduction, muscle contraction cannot be imagined without the presence of some metal ions. Bioinorganic chemistry, among other thing, is focused on mechanism of biological processes in the organism that occur during applications of essential and non-essential element’s compounds in medicine. Many metal-based coordination compounds are widely used in medicine for the treatment of many diseases, including various cancers, Alzheimer's disease, diabetes, rheumatoid arthritis, etc.

In this monograph, the influence of HSAB principle on preference of central metal ions to biologically relevant molecule has been discussed. Taking into account the author’s scientific work on investigations the kinetics and mechanism of interaction between transition metal-based compounds with potential antitumor activity, such as Pt(II), Pd(II) (soft acids) and Zn(II) and Cu(II) (intermediate acids) and biomolecules, a review of the scientific results in this field has been made. Author for more than two decades, investigate substitution processes of monofunctional, bifunctional and dinuclear complexes of platinum(II), palladium(II), lately, zinc(II) and copper(II) with biologically relevant nucleophiles.

In this monograph selected results from this field have been reported, considering the high velocity of developments. I hope this monograph will help the researchers and will be catalytic for the students to join research groups which are motivated and engaged in discovery of new knowledges which contribute to expansion of bioinorganic chemistry fields.

Novi Pazar, Author May 2019.

Abbreviations:

D - dissociative mechanisms A - associative mechanisms I - interchange mechanisms k - second-order rate constant H° - standard enthalpy of reaction H - enthalpy of activation S - entropy of activation G - Gibbs energy of activation V - volume of activation - acid dissociation constant K a - pseudo-first order rate constant kobs k - solvolysis rate constants or second-order rate constant 1 - second-order rate constant for direct nucleophilic k2 substitution - anation rate constant k-1 cisplatin - cis-diamminedichloridoplatinum(II) - cis-diamminedichloridoplatinum(II) cis-DDP CDDP - cis-diamminedichloridoplatinum(II) transplatin - trans-diamminedichloridoplatinum(II) - 1,1-cyclobutanedicarboxylate CBDC carboplatin - cis-diammine(1,1- cyclobutanedicarboxylate)platinum(II) nedaplatin - cis-diammineglicolatoplatinum(II) - 2,2-bis-(aminomethyl)-1,3-propanediol zeniplatin (1,1-cyclobutanedicarboxylato)platinum(II) enloplatin - tetrahydro-4H-pyran-4,4-dimetanamine (1,1-cyclobutanedicarboxylato)platinum(II) - 2-methyl-1,4-butanediamine CI-973 (1,1-cyclobutanedicarboxylato)platinum(II) BBR3464 - triplatin tetranitrate - 1,2-diaminocyclohexane dach DNA - deoxyribonucleic acid 5’-GMP - guanosine-5'-monophosphate - adenosine-5'-monophosphatesfat 5’-AMP - guanosine GUO INO - inosine 5’-IMP - inosine-5'-monophosphate - thiol RSH - S-methyl-thioether R-SCH3 DDTC - diethyldithiocarbamate AMP - adenosine- monophosphate ADP - adenosine- diphosphate

ATP - adenosine- triphosphate 1,2-d(GpG) - 1,2-guanylyl(3’- 5’)guanosine 1,2-d(ApG) - 1,2-adenosyl(3’- 5’)guanosine 1,3-d(GpG) - 1,3-guanylyl(3’- 5’)guanosine SDS - sodium dodecyl sulfate Tu - thiourea DL-asp - DL-aspartic acid L-met - L-methionine L-cis - L-cysteine gly - glycine GSH - glutathione (-glutamyl-cysteinyl-glycine) GSMe - S-methyl-glutatihione en - 1,2-diamnoethane or ethylenediamine DMSO - dimethylsulfoxide bipy - bipyiridine dien - diethylentriamine (1,5-diamino-3-azapentane) kaq - hydrolysis rate constants terpy - 2,2′:6′,2′′- terpyridine Hepes buffer - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid PBS buffer - phosphate buffer A - absorption λ - wavelength (nm) t - time (s) UV-Vis - ultraviolet and visible spectroscopy NMR - nuclear magnetic resonance spectroscopy EPR - electron paramagnetic resonance MS - mass spectrometry DFT - density functional theory HPLC - high-performance liquid chromatography MALDI-TOF MS - matrix assisted laser desorption/ionization-time of flight mass spectrometry HPLC-MS - high-performance liquid chromatography-mass spectrometry

CONTENTS

INTRODUCTION 9

1. TRANSITION METAL ION CHEMISTRY 13 13 1.1. Lewis acid character 1.2. Mechanisms of substitution reactions 16 1.3. Characteristics of the various mechanisms 19 1.4. Bioinorganic reaction mechanism 20

2. PLATINUM-BASED ANTICANCER AGENTS 23 2.1. Mechanism of interactions of anticancer platinum drugas 25 with biomolecules 2.2. Interactions of model monofunctional platinum(II) and 30 palladium(II) complexes with sulfur- and nitrogen donor biomolecules 2.3. Interactions of model bifunctional platinum(II) complexes 42 with sulfur and nitrogen donor biomolecules 2.4. Interactions of dinuclear platinum(II) complexes with sulfur 62 and nitrogen donor biomolecules

3. BIOINORGANIC INTERACTIONS OF ZINC(II) 79 AND COPPER(II) COMPLEXES IN CORRELATION

WITH HSAB PRINCIPLE

3.1. Interactions of zinc(II) complexes with biomolecules in 82 correlation with HSAB principle

3.2. Cytotoxic activity of zinc(II) complexes 98 3.2. Interactions of copper(II) complexes with biomolecules in 101 correlation with HSAB principle

CONCLUSION 109

REFERENCES 113

INTRODUCTION

Bioinorganic chemistry is an interdisciplinary field, examines the role of essential micro- and macro-elements in biological systems. Many processes in living systems such as metabolism, respiration, photosynthesis, growth, reproduction, muscle contraction, etc., cannot take place without the presence of some metal ions. Bioinorganic chemistry connects inorganic chemistry with organic chemistry in addition to synthesize the new complex compounds of transition metal ions, which are used as therapeutic and diagnostic agents. This field includes studies of kinetic and thermodynamic interactions between transition metal ions coordination compounds and biomolecules such as enzymes, nucleic acids, proteins, peptides, amino acids and others. Also, bioinorganic chemistry connects inorganic chemistry with biology and medicine during investigation in vitro and in vivo potential appropriate therapeutic agents, as well as, in examination of transport of these compounds through the cell membrane and elimination of them from biological systems using appropriate ligand by forming the stable complex compounds.

Our desire to understand bioinorganic processes after administration of potential antitumor metal-based drugs in cell leads us to investigation of ligand-substitution reactions and factors that have impact and control the binding of small molecules. This is the most fundamental type of chemical reaction that can occur when a metal complex is dissolved in solution in the presence of other nucleophiles. Ligand substitution and acid-base properties of central metal ions are very important for understanding the mechanism of interactions between metal ions (Lewis acids) and various biomolecules (Lewis bases) with different donor atoms. General chemical properties of

9 metals that are very important to consider in research of bionorganic mechanisms are:

 Charge. Metal ions are positively charged in aqueous solution, and its charge depends on the coordination environment.  Interactions with ligands. Metal ions bind to ligands via donor atoms, the interactions could be either strong or labile and selective. The thermodynamic and kinetic properties of metal-ligand interactions influence ligand exchange reactions.  Structure and bonding. Transition metal complex compounds are different geometrical structures. The bond lengths, bond angles, and number of coordination sites can vary depending on the metal and its oxidation state.  Lewis acid character. The terms „hard“ and „soft“ acids arise from a polarizabilities of the metal ions. Metal ions with high electron affinity can significantly polarize groups that are coordinated to them, facilitating hydrolysis reactions.  d-block elements. For the transition metals, the number of electrons in the d-shell orbitals has influence on electronic and magnetic properties to transition metal complexes.  Redox activity. Various numbers of electrons in the d-shell is the ability for many transition metals to undergo one-electron oxidation and reduction reactions.

Generally the reactivity of metallic centers in biology and medicine depends of their Lewis acid or redox-active characters. Today, many inorganic compounds are widely used in medicine to treat many diseases. It is known that some complexes of platinum(II), mainly cis-diamminedichloridoplatinum(II), cis-[PtCl2(NH3)2], and more recently

10 complexes of platinum(IV) and polynuclear platinum(II) complexes are used in chemotherapy as anticancer drugs. Interaction of platinum complexes with DNA are responsible for their antitumor activity. However, complexes of platinum(II) as „soft“ acid have high affinity to biomolecules such as amino acids, peptides, proteins, enzymes, etc. that have sulfur donor atoms „soft“ base (L-cysteine, L-methionine, glutathione, metallothionein, etc.) Interactions of platinum complexes with sulfur-containing biomolecules are responsible for their toxic effects (nephrotoxicity, ototoxicity, neurotoxicity, etc.). In addition, these biomolecules deactivated complexes of platinum(II) and cause resistance during treatment with chemotherapeutics.

In monograph we discuss the connection between principle of hard and soft acids and bases (HSAB) and interactions of some transition metal complex compounds of ions with various biomolecules. This HSAB principle is qualitatively useful to predict the preference of the metal for the ligand and to predict the stability of M-L bonds. Hard–hard or soft–soft bonds of acid and base contribute to stabilization and strength of the bonds between donor and acceptor. These factors also include the charges and sizes of the cation and donor atom, their electronegativities and the orbitals overlap between them.

12 1. TRANSITION METAL ION CHEMISTRY

1.1. Lewis acid character

A Lewis acid is an electron acceptor, and a Lewis base is an electron donor. In a coordination complex, the central metal ions act as a Lewis acid and are coordinated (bonded) by one or more molecules or ions (ligands) which act as Lewis bases. Formed coordinated bonds between central atom or ion with ligands have covalent character and are known under name coordinate covalent bond or simple coordinate bond. Atoms in the ligands that are directly bonded to the central atom or ion are donor atoms. If we consider the acceptor properties of metal ions towards ligands (i.e. Lewis acid–Lewis base interactions), two classes of metal ion can be identiﬁed, although the distinction between them is not clear-cut. This two classes are „hard“ acids or class (a) cations and „soft“ acids or class (b) cations. Similar patterns were found for other donor atoms: ligands with O- and N-donors form more stable complexes with class (a) cations, while those with S- and P-donors form more stable complexes with class (b) cations.

The terms „hard“ and „soft“ acids arise from a description of the polarizabilities of the metal ions. Hard acids are typically either small monocations with a relatively high charge density or are highly charged, again with a high charge density. These ions are not very polarizable and show a preference for donor atoms that are also not very polarizable, e.g. F. Such ligands are called hard bases. Soft acids tend to be large monocations with a low charge density, e.g. Pt2+, and are very polarizable. Soft metal ions prefer to form coordinate bonds with donor atoms that are also highly polarizable, e.g. I. Such ligands are called soft bases. Pearson’s classiﬁcation

13 of hard and soft acids comes from a consideration of a series of donor atoms placed in order of electronegativity:

F > O > N > Cl > Br > C ~ I ~ S > Se > P > As > Sb

A hard acid is one that forms the most stable complexes with ligands containing donor atoms from the left side of the series. The reverse is true for a soft acid. This classiﬁcation is listed in Table 1

Table 1 Selected hard and soft metal centers (Lewis acids) and ligands (Lewis bases) and those that exhibit intermediate behavior.

Hard (acids) Intermediate (acids) Soft (acids) Li+, Na+, K+, Rb+, Be2+, Mg2+, Pb2+, Fe2+, Co2+, Zero oxidation state metal Ca2+, Sr2+, Sn2+, Mn2+, Al3+, Ni2+, Cu2+, Zn2+, centers, Tl+, Cu+, Ag+, Au+, 3+ 3+ 3+ 3+ 3+ 2+ 3+ 3+ 2+ 2+ 2+ 2+ 2+ Ga , In , Sc , Cr , Fe , Os , Ru , Rh , [Hg2] , Hg , Cd , Pd , Pt , Co3+, Y3+, Th4+, Pu4+, Ti4+, Ir2+ Ru2+ Tl3+ 4+ 2+ + Zr , [VO] , [VO2] Hard (bases) Intermediate Soft (bases) (bases) ------F , Cl , H2O, ROH, R2O, [OH] Br , [N3] , py, I , H , R , [CN] (C-bound), CO - - 2- - , [RO] , [RCO2] , [CO3] , [SCN] (N-bound), (C-bound), RNC, RSH, R2S, - 3- 2- - - - [NO3] , [PO4] , [SO4] , ArNH2, [NO2] , [RS] , [SCN] (S-bound), R3P, - 2- 2- [ClO4] , [ox] , NH3, RNH2 [SO3] R3As, R3Sb, alkenes, arene

The implications and applications of the HSAB principle are possibility to predict thermodynamically stable M-L bonds. For example:

 Fe(III) belongs to a class of hard acids, prefers the hard bases e.g. O. Thus, it is understandable why concentration of Fe(III) ions in the body is controlled by OH-, O2- and RO- species. In ferritin protein that stores iron and releases it in a controlled fashion Fe(III) ions are bound by the phenolate group -OPh

14  Pt(II) a soft acid prefers soft bases S-donor instead of N-donor ligands. Antitumor activity of platinum(II)-based drugs is explained by the assumption that the firstly react with S-donor biomolecules, that is kinetically more favorable and then comes to formation thermodynamically more stable Pt-DNA adducts

Ligands with hard N- or O-donor atoms form more stable complexes with s- and p-block metal cations (e.g. Na+, Mg2+, Al3+), early d-block metal cations (e.g. Cr3+, Fe3+) and f-block metal ions (e.g. Ce3+, Th4+). On the other hand, ligands with soft P- or S-donors have a preference for heavier p-block metal ions (e.g. Tl+) and later d-block metal ions (e.g. Cu+, Ag+).

Complex formation involves ligand substitution. In aqueous solution ligands substitute H2O

z+ z+ [M(H2O)6] + L [M(H2O)5L] + H2O (1)

z+ If we suppose that M is a hard acid then bond with hard H2O ligands is a favourable hard–hard interaction. If L is a soft base, ligand substitution will not be favourable. If L is a hard base, there are several competing interactions to consider. Aquated L possesses hard–hard L–OH2 interactions z+ z+ or aquated M possesses hard–hard M –OH2 interactions or the product complex will possess hard–hard Mz+–L interactions. Overall, it is observed that such reactions lead to only moderately stable complexes, and values of H° for complex formation are close to zero.

If we consider the case where Mz+ in Eq.1 is a soft acid and L is a soft base.

The competing interactions will be aquated L possesses soft–hard L–OH2 z+ z+ interactions or aquated M possesses soft–hard M –OH2 interactions or the product complex will possess soft–soft Mz+–L interactions. In this case,

15 experimental data indicate that stable complexes are formed with values of H° for complex formation being large and negative. Although successful, the HSAB principle initially lacked a satisfactory quantitative basis. Today is possible to use DFT theory to derive electronic chemical potential values (electronic chemical potential) and chemical hardness values [1].

1.2. Mechanisms of substitution reactions

Complex compounds could be involved in a number of substitution reactions such as: ligand exchange, solvent exchange, complexation or anation reactions, solvolysis, acid and base hydrolysis, inter- and intramolecular isomerisation, racemization, metal ion exchange, etc. [2]. Substitution reactions of complexes are divided on electrophilic (SE) or nucleophilic (SN) depending on the replacement of either central metal ion or ligand. If the metal ion is substituted during the reaction, i.e. electrophile, the reactions are electrophilic substitution (Eq. 2), otherwise if a ligand is replaced that is nucleophilic substitution reaction (Eq. 3) [3,4].

(2) [MLn] + M' [M'Ln] + M

(3) [MLn] + X [MLn-1X] + L

Ligand substitution reactions in metal complexes can occur in two ways, either by a combination of solvolysis and substitution by ligand or simple exchange in which there is a replacement of one ligand by another without the direct inclusion of solvent. The direct substitution is more relevant for

16 square-planar complexes with regard to octahedral complexes. For other complex geometries both routes are used [5].

Nucleophilic substitution reactions, according to Langford and Gray, are carried out in three different mechanisms: dissociative (D), associative (A) or interchange mechanism (I) (Figure 1) [3].

interchange mechanism (I)

[MLn-1X] [MLn-1X]L +L [MLn]X

[MLn] associative mechanism (A)

[MLnX] [MLn-1] +L dissociative mechanism (D)

X+

[ML X] [ML X] n-1 +L n-1

Figure 1. Schematic representation of the mechanisms for substitution reactions.

In the dissociative mechanism (D) the first step of the reaction is dissociation of the one ligand L from the inner coordination sphere, whereby an intermediate with a decreased coordination number forms. In the next step, the entering ligand X binds to the central metal ion. Since the first step of the reaction is slower, it determines the overall rate of the substitution reaction. In the associative mechanism (A), in the first step, the entering ligand X

17 binds to the central metal ion, forming an intermediate with an increased coordination number, and then, in the second step, the leaving ligand L leaves the coordination sphere of the complex. The formation of an intermediate with an increased coordination number is slower and it determines the rates of this substitution process. When an intermediate cannot be detected by kinetic, stereochemical, or product distribution studies, the so-called interchange mechanisms (I) are invoked. Associative interchange mechanisms (IA) have rates dependent on the nature of the entering group, whereas dissociative interchange (ID) mechanisms do not. If the process of breaking the bond between the central metal ion and the outgoing ligand L has a greater impact on the rate of reaction, the mechanism is ID, and if the forming a new bond between the central metal ion and the entering ligand X has a greater impact on the chemical reaction rate, the mechanism is marked with IA [3,4].

As was mentioned before, the factors affecting metal ion lability include size, charge, electron configuration, coordination number Lewis acid characters. The associative mechanism is well known and preferred for four-coordinated square-planar complexes. Dissociative mechanisms are more common for six-coordinated octahedral complexes. Five-coordinated complexes could react in both mechanisms [5]. The study of kinetics and mechanism of the bioinorganic reactions of transition metal complexes expanded with the development of experimental techniques such as spectroscopic techniques (UV-Vis, NMR, Mössbauer, IR, Raman, EPR spectroscopy, MS), rapid cryogenic X-ray structure determinations of reactive intermediates, matrix isolation of reactive intermediates, fast kinetic techniques, low-temperature kinetics, high-pressure kinetic and thermodynamic techniques to construct volume profiles as compared to energy profiles, and theoretical methods to analyze and predict reaction mechanisms. The main aims of study are

18 determination of rates of substitution processes, investigation of the influence of different parameters (change of reactant concentration, pH, temperature and pressure change, introduction catalyst, etc.), investigation of interactions between potential antitumor metal-based drugs and biologically relevant molecules [2-5].

1.3. Characteristics of the various mechanisms

Determination of the mechanism of the nucleophilic substitution reaction is carried out on in relation to values of the thermodynamic parameters that characterize the studied process. One of the parameters by which the substitution mechanism can be preliminarily determined in a very simple way is the rate of the chemical reaction rate. Based on the equations characterized by the processes of dissociative, associative and interchange mechanism (Figure 1), it can be seen that the process of substitution by the dissociative mechanism is the first-order reaction, and by the associative mechanism the second-order reaction [3,4].

A more reliable criterion for determining the mechanism is the value of the entropy of activation S. Since activation entropy is a measure of the disorder of a system, and based on the knowledge that an intermediate with greater or minor disorder is formed in different mechanisms, this parameter enable to determine the substitution mechanism. In dissociative mechanism, as mentioned before, when an intermediate with decreased coordination number forms the system disorder increases and S has a positive value. In associative mechanism, the formation an intermediate with increased

19 coordination number the system order decrease and S has a negative value. In the case of I the mechanism S is approximately equal to zero.

The most reliable criterion for determining the mechanism is the value of the activation volume [6,7]. Taking into account the type of intermediate in different mechanisms, the increase in pressure will accelerate the reactions occurring in the A mechanism and slow down the reactions of the D  mechanism. Therefore, the negative value V indicates the A or IA  mechanism, and the positive value V indicates the D or ID substitution mechanism. In the case of a change mechanism, the pressure does not significantly affect the rate of substitution.

1.4. Bioinorganic reaction mechanism

Under the classification of bioinorganic reactions we consider the interactions of metal ions with bio-ligands under physiological conditions. Ligand affinity and possible coordination geometries of the metal center are important bioinorganic principles. Metal-ligand bonds are closely related to the HSAB nature of metals and their preferred ligands. Many factors could affect metal–ligand complex formation including formation of competing equilibria–solubility products, complexation, and/or acid–base equilibrium constants–sometimes referred to as „metal ion speciation” all affect complex formation. Ion size and charge, preferred metal coordination geometry, and ligand chelation effects all affect metal uptake. In biological systems, as in all others, metal ions exist in an inner coordination sphere with ligands binding directly to the metal.

In order for the coordination complexes to be approved as metal drugs it is necessary to detailed examination of the fundamental aqueous chemistry of the proposed drug, including its pharmacokinetics, the metabolic processes in

20 blood and intracellularly, and the effects of the drug on the target of choice. The interactions between metal ions and biomolecules are very important for medical application of drugs. The bioionorganic reactions mechanism includes investigation of all processes which occur during applications of metal-based drugs. Thus, the determination of mechanism helps to clarify what will happen after administrations of the drugs and helps to improve medical characteristics of them.

22 2. PLATINUM-BASED ANTICANCER AGENTS

Cisplatin or cis-diamminedichloridoplatinum(II), cis-DDP is one of the most famous antineoplastic drugs, which has wide application in the treatment of various cancers [8]. Regardless of the fact that this complex compound is the most effective antitumor agent, it also has varios negative effects. For this reason, the research in this area is focused on synthesis of the new compounds of platinum(II) with higher selectivity towards biomolecules but less toxicity.

Cisplatin is square-planar geometry, its analog trans- diamminedichloridoplatinum(II) hasn’t shown any antitumor activity (Figure 2) [9].

H3N Cl H3N Cl

Pt Pt

H3N Cl Cl NH3

cisplatincisplati n a t rtransplatinansplatina

Figure 2. Isomers cisplatin and transplatin

The results of a large number of studies indicate that antitumor activity of platinum(II) complexes is linked with their structures. among the most widely used drugs for the treatment of cancer. Thanks to the successful and widespread use of cisplatin a large number of analogous compounds were synthesized. All these compounds have a several common characteristics:

1. Bifunctional complex compounds with cis-geometry.

23 2. The general formula of these compounds is cis-[PtA2X2] where A2 are two inert monodentate nitrogen donor ligands or one inert bidentate nitrogen donor ligand, while with X2 are two labile monodentate or one labile bidentate ligand.

3. The oxidation state of platinum in the complexes is +2 or +4.

4. Nitrogen-donor ligands have to contain at least one NH bond.

On Figure 3 are presented some of platinum complexes that are in the medicinal use worldwide.

O O H2 H3N O N O N O O Pt Pt Pt N H3N O N O CH3 O O H2

KCarboplatinarboplatina NK 121 LobaplatinLobaplatin a

O H2 H2 N O O N O H N O O R 3 Pt Pt Pt R O O N O N H3N O H2 H2 O

OOxaliplatinksaliplatina L-NDDP NNedaplatinedaplatina

O CH3 H2 OH H2 Cl iPr N Cl N Cl H3N Cl Pt Pt Pt N Cl iPr N Cl N Cl H2 O H2 OH H2 Cl CH3 O

JM-216 IIproplatinproplatina OOrmaplatinrmaplatina

Figure 3. Platinum antitumor complexes with adopted commercial names.

24 The second generation of platinum(II) antitumor complexes are carboplatin, oxaliplatin, nonplatinum, zenithplatin, enloplatin, CI-973 and others. Instead of labile chloride ligands they contain bidentate ligands such as 1,1-cyclobutanedicarboxylate, glycolate, and complexes with 1,2-diaminocyclohexane as an inert ligand, while the labile ligands are sulfates, malonates and other ligands [10]. The second generation complexes based on the cisplatin structure were developed in attempts to improve toxicity and/or expand the range of useful anticancer activity. The third generation of platinum antitumor complexes are octahedral platinum(IV) coordination compounds with general formula cis-[PtA2X2Y2], where two labile monodentate or one labile bidentate ligand is labelled as

Y2. The platinum(IV) drugs are orally administered to patients. In the presence of various biomolecules such as cysteine or ascorbic acid, the redaction to Pt(II) occurred by leaving the axial ligands Y2. Also, this group includes new complexes with a trans-geometric structure, polynuclear platinum complexes (BBR3464), and complexes containing a ligand with an asymmetric carbon atom [11].

2.1. Mechanism of interactions of anticancer platinum drugas with biomolecules

The mechanism of the antitumor effects of platinum complexes consists in their binding to DNA molecules, thereby preventing replication and transcription of DNA, the process of uncontrolled cell growth [12-14]. From the moment of injection of the drugs in the body to their binding to DNA molecules, a large number of secondary processes happen that are responsible for the occurrence of toxic effects [12,13]. The mechanism of the interactions between platinum(II) complexes with biomolecules is in

25 correlation with hard-soft acid-base principle. Antitumor activity of these complexes takes a few steps.

The first step of administration of Pt(II) drugs is hydrolysis. Hydrolysis of Pt(II) drugs in the body occurs as a result of a different concentration of chloride ions in and out of the cell. The high concentration of Cl- ion in the extracellular fluid (104 mM) suppresses the hydrolysis process, while in the intracellular low concentration of about 4 mM, it is suitable for the hydrolytic reactions of platinum(II) antitumor drugs [15,16]. The resulting aqua-chloride complex is more reactive than the dichlorido complex, which is one of the essential characteristics of this compound, and equilibrium depends on the pH and concentration of chloride (Figure 4).

+ 2+ Cl Cl OH2

H2O H2O H3N Pt Cl - H3N Pt OH2 H3N Pt OH2 Cl Cl-

NH3 NH3 NH3

H+ pKa = 6,85 H+ pKa = 5,93

+ Cl OH2

H2O H3N Pt OH H3N Pt OH Cl-

NH3 NH3

H+ pKa = 7,87

H3N Pt OH

NH3

Figure 4. Hydrolysis of cisplatin.

26 The antitumor platinum(II) agents must not be either too reactive or too inert, since in both cases their toxicity is increasing [10]. On the other hand, the essential characteristic of these compounds must be selectivity towards certain biomolecules [12]. High affinities for the platinum complexes show the biomolecules that contain sulfur, as the thiols and the thioethers, as soft Lewis acid platinum(II) drugs form very stable bonds with sulfur donor biomolecules e.g. soft bases. The resulting compounds are responsible for negative side effects during treatment (such as vomiting, resistance, nephrotoxicity, ototoxicity, neurotoxicity, cardiotoxicity, etc.).

As was mention, the antitumor activity of platinum(II) is the result of binding to the genetic DNA found in the nucleus. Also, there is a possibility of binding to a mitochondrial DNA inherited exclusively by a mother. Interactions with mitochondrial DNA are less responsive for antitumor activity of platinum drugs . After hydrolysis, binding of the complex to DNA occurs primarily through the N7 guanine atoms, while the binding of N7 and N1 adenine and N3 cytosine is less represented. Since the DNA molecule in its complementary spiral structures contains a different sequence of purine and pyrimidine bases, it was found that 60-65% represented the coordination of the 1,2-(GpG) complex, i.e. the bond between the two guanosine-5'- monophosphate molecules on the opposite chains of DNA. About 20-25% is represented coordination by a bond type 1,2-(ApG), i.e. bond to adenosine- 5'-monophosphate and a guanosine-5'-monophosphate disposed on opposite DNA chains. Other ways of coordination (monophunctional binding of the complex, type 1,3-(GpG) coordination via guanosine located on the same chain of DNA molecules, etc.) are less represented. The bond between platinum and N-bounded biomolecules (intermediate bases) is thermodynamically very stable [10-21].

27 From the moment of administration of drugs into the organism, most often intravenously, through the hydrolysis process, to the DNA molecule coordination, platinum-based anticancer agents can react with other biomolecules, and such interactions are, as noted above, responsible for the appearance of toxic effects. Thus, platinum(II) possesses high affinity to the sulfur and in the blood plasma itself reacts immediately with albumin or other biomolecules that contain sulfur (proteins or peptides in which L-cysteine or L-methionine). Considering that the concentration of thiol, including L-cysteine and glutathione, in intracellular fluid is about 10 mM, it is presumed that the platinum(II) base antitumor reagents first react with sulfur donor nucleophiles, which is kinetically favoured and after that form thermodynamically more stable Pt-DNA compounds. On Figure 5 schematic are presented intercell processes during application of platinum-based antitumor drugs.

28 Substitution

of S-CH3 by N7

CH3 Substitution mitochondria of N7 by GSH Pt S R hydrolysis Substitution RNA

of S-CH3 by SH R-S-CH3 HS-R Pt(II)-SR Pt(II)-complex cytoplasm [Cl-] ~ 4 mM

ddtc extracellular [Cl-] ~ 104 mM excretion uptake

Figure 5. Intercell processes during application of platinum-based antitumor agents.

Sulfur interactions with donors are very important, but the characteristics of the compounds formed are fundamentally different depending on whether sulfur comes from the thiol or thioether molecules. If sulfur is from the thioether molecule, the resulting Pt-S(thioether) bond may be interrupted in the presence the DNA molecule, i.e. the N7 atom from the guanosine-5'- monophosphate may substitute the molecule of thioether from the resulting complex (Figure 5). Also, the thiol molecule may substitute the thioether from compound. However, the bond between the platinum complex and the molecule containing the thiol group is not favorable, the compounds are extremely stable and non-selective. The Pt-S(thioether) products are

29 "platinum reservoirs" in the organism, they are suitable intermediates in platinum complex(II) and DNA molecule reactions, while Pt-S(thiol) compounds completely deactivate complexes forming the compounds responsible for toxic effects. After the treatment it is necessary to remove platinum from the organism, or to terminate a very stable Pt-S(thiol) bond, using sulfur containing nucleophiles, such as diethyldithiocarbamate (ddtc), thiourea, thiosulfate or glutathione (Figure 5).

2.2. Interactions of model monofunctional platinum(II) and palladium(II) complexes with sulfur- and nitrogen donor biomolecules

Monofunctional platinum(II) complexes contins stabile tridentate ligand such as dien (diethylentriamine or 1,5-diamino-3-azapentane) or terpy (2,2′:6′,2″- terpyridine) while the fourth coordination place is occupied with labile ligand, mostly chlorido ligand. These complexes represent a good model for investigations of platinum(II) interactions with various biomolecules which contains sulfur and nitrogens. The structures of complexes disable the bifunctional coordination to DNA, because of that, they do not exhibit antitumor properties, but simplifies investigation of substitution reactions of these complexes.

The monofunctional [PtCl(terpy)]+ and related complexes of the general type 2+ - [Pt(terpy)X] (X = H2O, Cl , etc.) is extensively studied. Terpy ligand affects on nucleophilic substitution reactions which are controlled by strong π-acceptor ability of the tridentate chelate 2,2′:6′,2′′-terpyridine. The electronic communication between three pyridine rings causes a decrease in

30 electronic density on the platinum center due additional formation of π-back bond and makes it more electrophilic and more reactive. The obtained values for lengths of chemical bonds between the platinum(II) and three nitrogen donor atoms of terpyridine system show that the shortest connection is to the secondary nitrogen atom.

Considering that platinum as soft acid prefers soft bases such are sulfur- coordinated biomolecules we have studied kinetics for the complex formation of the [PtCl(terpy)]+ with 5’-GMP in the presence and absence of GSH at pH ca. 6, with a concentration ratio [PtCl(terpy)]+ : GSH : 5’-GMP of 1:2:10 [22]. The second order rate constants, obtained from linear least- squares analysis of the kinetic data, clearly point to a kinetic preference of [PtCl(terpy)]+ toward the GSH at pH ca. 6. Nitrogen donor guanosine-5’- monophospahte is also a very good nucleophile for Pt(II) complexes, but at neutral pH cannot compete with GSH. The second-order rate constant for GSH is 102 times higher then for 5’-GMP [22]. The progress of the reaction of [PtCl(terpy)]+ with these biomolecules over extended periods of time was monitored with HPLC technique, which allows aliquots separated from the reaction mixture at programmed times to be analyzed. The studied reactions were carried out in water, without any buffer, since buffer ions (e.g. phosphate) are potential ligands for Pt(II). The pH of each solution was checked over the reaction time, and was shown to be kept between 4.5 and 5.5. Prior to the study of the reaction between [PtCl(terpy)]+ and glutathione and 5’-GMP, we first investigated the reactions between platinum(II) complex with each of the nucleophiles. The products formed were isolated by reversed-phase HPLC and characterized by MALDI-TOF mass spectrometry. As was expected, the products obtained corresponded to the adducts [Pt(terpy)(GS)]+ and [Pt(terpy)(N7-GMP)]+ (m/z 734,2 and 789,8, respectively).

31 The competition reactions between [PtCl(terpy)]+, of mixtures of GSH and 5’-GMP in molar 1:1:12, respectively, has shown that that [PtCl(terpy)]+ reacted faster with glutathione than with 5’-GMP, but this did not prevent a small amount (< 16%) of [Pt(terpy)(N7-GMP)]+ from being formed at beginning of the process by reaction of [PtCl(terpy)]+ (Figure 6) [22].

5’-GMP

Pt-GSH

Pt-GMP

0 10 t(min)

Figure 6. HPLC profile of an aliquot of the reaction mixture [PtCl(terpy)]+:glutathione:5’-GMP 1:1:12 after 1 week reaction time[22].

The relative proportion of this adduct remained virtually constant throughout the reaction process, which indicates that once formed it remains unaltered. The possibility that [Pt(terpy)(GS)]+ reacted with the excess of 5’-GMP

32 present in the reaction mixture was not observed, unless glutathione did not replace 5’-GMP from formed [Pt(terpy)(N7-GMP)]+. The formed adducts were also characterized by mass spectrometric analysis of the products isolated from the reaction mixture by HPLC (Figure 7) [22].

terpy cation+

+ [Pt(terpy)(GS)]

+ [PtCl(terpy)]

Figure 7. MALDI-TOF mass spectrum of the adduct [Pt(terpy)(GS)]+ isolated from the reaction mixture [PtCl(terpy)]+:glutathione:5’-GMP 1:1:12[22].

33 According HSAB principle the platinum prefers sulfur donor biomolecules as soft base, but with nitrogen donors biomolecules (intermediate) builds thermodynamically very stables complexes.

The other the most studied monofunctional complex is [PtCl(dien)]+ and his aqua analog in different reaction conditions. The monofunctional complexes provide insight into the initial complexation reactions of cisplatin, since the reactions of the complex with sulfur containing amino acids and peptides, are often complicated by the formation of polymeric complexes [13], and are frequently accompanied by the release of coordinated ammonia [23].

In view that interactions of anticancer platinum-based drugs with sulfur thioether biomolecules are more favorable we have investigated the competitive reactions of [PtCl(dien)]+ (10 mM) with L-methionine and 5’-GMP in a molar ratio: [PtCl(dien)]+:L-methionine:5’-GMP = 1:1:3 [24,25]. In the initial stage of the reactions (<40 h), 1H NMR peak for the free L-methionine ( 2.142 ppm) decrease in the intensity, and new peak of the [Pt(dien)(S-meth)]2+ appeared in the spectrum ( 2.544 ppm), whereas a little of the 5’-GMP reacted. In the later stages (72 h), the peaks for the bounded L-methionine and free 5’-GMP ( 8.208 ppm) decreased in intensity, whereas those for free L-methionine increased in intensity, as did those assignable to bound 5’-GMP in [Pt(dien)(N7-GMP)]2+ ( 8.624 ppm) as shown in Figure 8 [24] .

1 t=840 h

t=384 h

t= 264 h 3 t= 168 h 4 1 t= 120 h

2 t= 72 h

Figure 8. 1H NMR spectra of the reactions of [PtCl(dien)]+ (10 mM) with mixture of L-methionine and 5’-GMP in the ratio 1:1:3 (where 1 is the signal for the [Pt(dien)(S-meth)]2+, 2 is the signal for the [Pt(dien)(N7- GMP)]2+, 3 is the signal for the free L-methionine and 4 is the signal for the free 5’-GMP[24].

In separated experiments we confirmed that the reactions of [PtCl(dien)]+ with L-methionine is relatively fast, and the complex [Pt(dien)(N7-GMP)]2+ can be formed from [Pt(dien)(S-meth)]2+ in direct displacement of coordinated L-methionine by 5’-GMP. Moreover, the 5’-GMP proton signals of the end product are identical to those belonging to [Pt(dien)(N7-GMP)]2+ formed by direct reactions of 5’-GMP and [PtCl(dien)]+ complex [24].

[Pt(dien)(S-Met)]2+ 40

20 % adduct %

10 [Pt(dien)(N7-GMP)]2+

0 0 200 400 600 800 1000 1200 1400 t(h)

Figure 9. Observed product formation during the competition reaction of [PtCl(dien)]+ with L-methionine and 5’-GMP in molar ratio [PtCl(dien)]+: L-methionine:5’-GMP = 1:1:3[24].

As could be seen, initially there is rapid formation of [Pt(dien)(S-meth)]2+ followed by displacement of L-methionine by 5’-GMP (Figure 9). In the later stages the concentration of [Pt(dien)(N7-GMP )]2+ is predominant [24]. The spectra of the starting complex, [PtCl(dien)]+ and the final reaction product is presented on the Figure 10 [25].

1.0

0.8

0.6 A

0.4 [Pt(dien)(N7-GMP)]2+

+ 0.2 [PtCl(dien)]

0.0 200 250 300 350 400 Wavelenght l(nm)

Figure 10. Electronic spectra for the starting complex, [PtCl(dien)]+, and for the final product, [Pt(dien)(N7-GMP)]2+[25].

In these experiments we confirmed that mechanism of interactions of platinum(II) complexes as soft acid with soft sulfur bases originating from thiols and thioethers differs in presence of an excess intermediate nitrogen bases (e.g. DNA constituent), depending on biomolecules that will react.

Substitution reactions of the [PtCl(dien)]+ complex with biomolecules in the presence and absence of micelles, sodium dodecyl sulfate (SDS), in aqueous

0.10, 0.05 and 0.01 M NaClO4 at pH 2.5 were also investigated [26]. The presence of anionic micelles induced the acceleration of complex formation. The largest effect of micelles has been observed in the case of L-methionine. On the other hand, an increase in the ionic strength in the presence of

37 micelles induced a decrease in the rate constants (Figure 11). The negative entropies of activation support the operation of an associative complex- formation mechanism.

0.2

0.1 a)

0 I1/2

) c) 0 0 0.1 0.2 0.3 0.4

-0.1 b) log(k/k

-0.2 c)

-0.3

-0.4 b)

Figure 11. The effect of the ionic strength in the presence (open symbols) and in the absence (solid symbols) of 0.01 M SDS on the reaction rate between [PtCl(dien)]+ and a) L-methionine, b) GSH and c) 5’-GMP at pH = 2.5[26].

These studies are important from biochemical aspects, could serve as models of ligand exchange reactions on the surface of biomembrane or at the interface of a globular protein.

38 Monofunctional palladium(II) complexes have almost identical properties as Pt(II), they react about 103 - 105 times faster than the corresponding platinum complexes(II). They do not show antitumor activity, one of the reasons is high reactivity. Complexes Pd(II) in vivo are involved in hydrolytic pathway and form different hydroxyl complexes faster than react with sulfur-binding biomolecules. High reactivity also leads to rapid cis- trans isomerization, while in the case of Pt(II) compounds it is most often kinetically inhibited.

The kinetics and mechanism of the complex-formation reactions of 2+ [Pd(AEP)(H2O)] , where AEP stands for 1-(2-aminoethyl)piperazine, with biologically relevant ligands were studied as a function of selected nucleophiles and pH. The reactivity of the ligands follows the sequence L-methionine > guanosine-5’-monophosphate > glycine> inosine > glutathione [27]. The substitution reactions with glutathione showed two reaction steps in which the first step involves coordination through nitrogen and depends on the nucleophile concentration, whereas the second step involves intra-molecular isomerization from N- to S-bonded glutathione and does not depend on the nucleophile concentration (Figure 12).

39 4.0 a) 4.0 b)

3.5 3.5

3.0 3.0

2.5 2.5

2.0 2.0

Absorbance Absorbance 1.5 1.5

1.0 1.0 0.5 0.5

0.0 0.0 200 250 300 350 400 450 500 550 600 200 300 400 500 600 Wavelenght (nm) Wavelenght (nm)

Figure 12. Rapid-scan spectra recorded for the reaction of 2+ [Pd(AEP)(H2O)] with glutathione at time intervals of 1 s after mixing at pH = 2.5 (a) and pH = 4.75 (b). The increase and subsequent decrease in absorbance indicates the presence of two subsequent reaction steps[27].

Figure 13 summarizes the experimental data for the two observed reaction steps. Whereas the first step clearly obeys rate law kobs = k1[Nu] + k-1 for both pH values, the second reaction step is independent of the entering nucleophile concentration and can be accounted for in terms of an intramolecular isomerization process from N- to S-bonded GSH.

0.35 0.6 400 nm pH= 2.5 400 nm pH = 4.75 the first step the first step 0.30 the second step 0.5 the second step

0.25 0.4

0.20

-1 -1

s 0.3

/s obsd/

0.15 obsd k k 0.2 0.10

0.1 0.05

0.00 0.0 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.000 0.001 0.002 0.003 0.004 0.005 0.006 [GSH]/M [GSH]/M

Figure 13. Pseudo-first order rate constants as a function of nucleophile concentration for the first and second steps of the reaction between 2+ o [27] [Pd(AEP)(H2O)] and glutathione at different pH and 25 C .

The first reaction step (k1) is somewhat faster at pH 4.75 than at pH 2.5, whereas the intercept (k–1) is larger at pH 2.5 than at pH 4.75. The latter trend can be ascribed to protonation of the N-bonded GSH complex in more acidic solution that will accelerate the reverse aquation reaction. The second -1 reaction step is faster at pH 4.75 than at pH 2.5, viz. k2 = 0.075  0.002 s and 0.030  0.001 s-1, respectively, which can be ascribed to a faster isomerization from N- to S-bounded GSH with increasing pH as a result of the deprotonation of the –SH group [27].

This study demonstrated an exceptionally low reactivity for glutathione. Under our experimental conditions the SH group of GSH is protonated and prevent direct coordination through sulfur. Increasing steric hindrance of the AEP chelate in the complex is also expected to slow down the ligand- substitution reactions for GSH. The first coordination step involves binding of the N-donor, and the second reaction step involves isomerization to

41 S-bonded GSH, which is independent of the entering nucleophile concentration [27].

The slower rate for the complex-formation reactions with glutathione could be more favorable since the drug resistance of such anti-tumor complexes through the interaction of sulfur-containing proteins will be less efficient both in blood plasma and inside the tumor cells.

2.3. Interactions of model bifunctional platinum(II) complexes with sulfur and nitrogen donor biomolecules

Over the last decades many bifunctional platinum drugs have been developed in an attempt to improve the toxicity profile and particularly to design a drug that is able to overcome resistance. More information about interactions were obtained from a number of studies implemented in vitro, among which are investigation of the substitution reactions of bifunctional platinum complexes with various biomolecules at different conditions. Nitrogen donor biomolecules have a high affinity for platinum compounds it is known that interactions with DNA are responsible for the antitumor effect of platinum drugs. On other hand many studies indicate a kinetic preference for, e.g., thioethers over nitrogen donors, but from a theoretical point of view (HSAB principle) sulfur donors win the competition for the soft metal Pt(II) center. Sufficient amounts of the Pt(II) drug reach and react with the DNA target, indicating that the conditions under which the reactions proceed are of significant importance for the chosen pathway.

A set of three oxaliplatin derivatives containing 1,2-trans-R,R- diaminocyclohexane (dach) as a inert bidentate ligand and different chelating

42 leaving groups X–Y, viz., [Pt(dach)(O,O-cyclobutane-1,1-dicarboxylate)] or Pt(dach)(CBDCA), [Pt(dach)(N,O-glycine)]+ or Pt(dach)(gly), and [Pt(dach)(N,S-methionine)]+, or Pt(dach)(L-met), where L-met is L-methionine, were synthesized and the crystal structure of Pt(dach)(gly) was determined by X-ray diffraction [28]. The effect of the leaving group on the reactivity of the resulting Pt(II) complexes was studied for the nucleophiles thiourea, glutathione (GSH) and L-methionine (L-met) under pseudo-first-order [28].

Figure 14 shows the structures of investigated complexes.

+ + - - ClO4 ClO4

H2N NH2 H2N NH2 H2N NH2 Pt Pt Pt Me

O O H2N O H2N S

  O O O -OOC    

 Pt(cbdca) Pt(gly) Pt(L-met)

Figure 14. Structures of the Pt(dach) derivatives with different chelating leaving groups X–Y: O-O (CBDCA2–), N–O (glycine–) and S–N (L-met)[28].

Crystallization of [Pt(dach)(gly)]ClO4 from aqueous ethanol furnished the compound as single crystals, the monoclinic unit cell of which was found to contain two crystallographically independent cations and anions connected

43 by multiple NH·······O hydrogen bonds (Figure 15). In the two cations, the amino nitrogen and carboxylate oxygen atoms define the coordination environments around the central metal ions which deviate only slightly from planarity, the dihedral angles spanned by the pairs of planes Pt1–N1– O1/Pt1–N2–N3 and Pt2–N3–O4/Pt2– N5–N6 being 16.8(8)º and 2.2(4)º, respectively. The cyclohexane rings of the dach ligands adopt the expected chair conformation with the NH2 groups in equatorial positions. The platinum glycinate chelates are puckered, with the donor groups deviating only little from the two least-squares coordination planes and the connecting carbon atoms above these planes. The Pt-glycinato-O and Pt-glycinato-N bond lengths are 2.004(11) and 2.043(14) Å in cation 1, and 2.029(9) and 2.068(14) Å in cation 2.

Figure 15. Perspective view of the two crystallographically independent

[Pt(dach)(gly)]ClO4 ion pairs (carbon-bonded hydrogen atoms omitted for clarity)[28].

44 The lengths of the Pt-NH2 bonds between the two central metals and the dach ligands vary from 1.991(12) to 2.084(12) Å, which are at the lower and upper limits of the range of d(Pt-dach-NH2) separations measured for several closely related [Pt(dach)(dicarboxylato)] complexes (2.01–2.05 Å) [29,30], respectively.

1,2-Diaminocyclohexane contains two asymmetric carbon centers and therefore exists in three isomeric forms, among which the enantiomeric 1,2-trans-R,R form of the ligand, i.e., dach, was chosen for synthesis. The stereochemistry remains during the synthesis and no enantiomers are formed – during the process. The presence of ClO4 as a counterion was found to stabilize the crystal structure through the formation of hydrogen bonds. A view along the b-axis is presented by way of example in Figure 16 and shows the formation of a fishbone pattern within the crystal structure, due to the accumulation of the hydrophobic cyclohexane residues and the hydrophilic glycinato residues.

Figure 16. Crystal packing of [Pt(dach)(gly)]ClO4 with molecules arranged in a fishbone pattern[28].

45 The substitution kinetics of the coordinated chelate were investigated spectrophotometrically by following the change in absorbance at a suitable wavelength (at pH of 7.4 at 37.5 ºC in Hepes buffer) as a function of time for the nucleophiles, thiourea, L-met and GSH while NMR techniques were applied to study the reactions with 5’-GMP. Proposed reaction pathways for the substitution reactions of Pt(dach) derivatives with a series of nucleophiles are given on Figure 17.

k2 H N NH X + Nu, - X-Y 2 2 Nu: tu, GSH Pt + Y Nu Nu

k1 H N NH H N NH 2 2 Nu 2 2 Pt Pt X Y k-1 X Nu k2 -Nu H N NH X - X-Y 2 2 Y Pt + Nu: L-met Y Nu

Figure 17. The substitution process of Pt(dach)(X-Y) complexes with nucleophiles (X-Y: O-O (CBDCA2-), N-O (gly), S-N (L-met) Nu: Thiourea (Tu), glutatione (GSH), L-methionine (L-met), guanosine-5’-monophosphate (5'- GMP)[28].

46 The nature of the chelate, being O–O (CBDCA2–), N–O (glycine) or S–N (L-met) was shown to play an important role in the kinetic and mechanistic behavior of the Pt(II) complexes. Pt(dach)(CBDCA) exhibits a higher reactivity towards the sulfur donor L-met than Pt(dach)(gly), whereas the order is the opposite for the nitrogen donor 5’- GMP and the sulfur donors thiourea and GSH in the first reaction step.

At pH 7.4 the reaction occurs between the zwitterionic form of + L-methionine (pKCOOH = 2.28 [31], 2.13 [32], pKNH3 = 9.2 [32]) and the complex. The dependence of the absorbance on reaction time is shown on Figure 18 for various nucleophile concentrations by way of example for the reaction of L-met with Pt(dach)(CBDCA).

0.40 10 mM L-Met 20 mM 0.39 30 mM 40 mM 0.38 50 mM

0.37

0.36 absorbancenm] [265 0.35

0.34 0 2000 4000 6000 8000 10000 12000 time [s]

Figure 18. Time traces obtained for the reaction of 1 mM Pt(dach)(CBDCA) with L-met. T = 37.5 °C, pH 7.4 (N-2-hydroxyethylpiperazine-N′-2- ethanesulfonic acid, Hepes), l = 265 nm[28].

47 The rise and fall of the absorbance at 265 nm as a function of time is characteristic for a reaction that involves an intermediate B in the overall process during which A is transformed into C as shown in Equation 4, the time dependence of which is given by Eq. 5 [29]:

k 1 k 2 A B C (4)

k [ A] o b s 1 0 - k o b s 1 t - k o b s 2 t (5) [ B] t = e - e ( k - k ) o b s 2 o b s 1

In Eq. 4, B represents the 1:1 complex with both the entering and the leaving chelates, resulting from the nucleophilic attack of the sulfur donor of the thioether group in L-met accompanied by dechelation of Y in X–Y in the first step of the reaction. Subsequently a six-membered ring is formed by the nitrogen donor of the amine group of L-met under liberation of the leaving group X–Y, resulting in C. With regards to the acid dissociation constants of free L-met ring closure involves deprotonation of the amine group of the amino acid. This will occur readily at physiological pH since the pKa values of L-met decrease significantly upon metal ion coordination. Also, it can be notice that the shape of the kinetics traces in Figure 18 depends so strongly on the selected nucleophile concentration. The larger the excess of L-met, the higher the absorbance maximum achieved and the better the subsequent absorbance decrease was observed. At L-met concentrations below 30 mM, species B represents a reactive intermediate with kobs2 > kobs1 and B is rapidly transformed to product C. At concentrations higher than 30 mM, is opposite:

48 kobs1 > kobs2. Therefore, the lifetime of the intermediate B depends on the nucleophile concentration used in the experiment.

The Pt–N bond was always found to be very strong, especially for the reaction with 5’-GMP, in which the 1:1 reaction product [Pt(dach)(N- gly)(N7-GMP)] is very stable and hardly reacts with another molecule of 5’-GMP to form the 1:2 product (Figure 19).

The reactions with L-met were also investigated by 1H NMR method and have alreday demonstrated that the Pt–N bond in Pt(dach)(gly) during the second step does not break as easily as the Pt–O bond in Pt(dach)(CBDCA), such that we observed a contribution from a back reaction, the rechelation of glycine. Furthermore, the second-order rate constant for second reaction step

(k2) was found to be significantly smaller compared to k2 for Pt(dach)(CBDCA). This is consistent with the results obtained for the reaction of Pt(dach)(gly) with 5’-GMP. A total peak area of only 7% for the 1:2 reaction product stagnated at this value even after months (see the 1H NMR spectrum in Figure 19). The 1:1 complex, [Pt(dach)(gly)(N7-GMP)], is a very stable intermediate and exists to an extent of 93%. The equilibrium constant was calculated from the peak area of the intermediate, the final product and free 5’-GMP, and was found to be very small, viz., K= 0.07 M-1.

This clearly points to a very unfavorable equilibrium k2/k–2 with respect to the formation of the 1:2 product.

49 (1:1)

H2N NH2 H2N NH2 -CH2 -CH2 Pt + 5‘GMP2- Pt

H2N O H2N 5'-GMP

H O O O

H3, H4 H5, H8 H5‘/H8‘ H6/H7

3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0  (ppm)

Figure 19. 400-MHz 1H NMR spectra of a solution of Pt(dach)(gly) (9.18 mM) with 5’-GMP (18.3 mM) in D2O at pH 7.4 and 37.5 °C recorded as a function of time. The predominant reaction product is the stable 1:1 intermediate with 93% of the total integral. The peak that could be assigned to the liberated -CH2 of glycine after completion of the reaction as well as that of the 1:2 reaction product (7%) appear at the same chemical shift [28] (shoulder at 3.55) as for the -CH2 in Pt(dach)(gly) .

The result of an attempt to shift the equilibrium towards the right side of Eq. 5 by increasing the temperature is shown in Figure 20. The temperature was steadily increased from 25 to 80 °C and then decreased again to 25 °C. The spectral changes observed at 80 °C persist even after decreasing the

50 temperature again down to 25 °C, indicating a very slow equilibration process at 25 °C. The equilibrium did not shift to the product side, but rather to the side of the reactants, viz., the Pt(dach)(gly) complex and free 5’-GMP. This can be seen in both the increase in the signals of the H1’ protons of liberated 5’-GMP and -CH protons of chelated glycine and the decrease in the signals of the H1’ protons and the -CH protons of glycine of the 1:1 intermediate.

1:1 5‘GMPfree -CHchelate -CHopen 1:2

T = 25°C

-CHfree T = 80°C

T = 25°C 6.08 6.00 5.92 5.84 3.70 3.60 3.50 3.40 3.30 3.20 3.10  (ppm)

Figure 20. 400-MHz 1H NMR spectra of Pt(dach)(gly) (9.18 mM) with

5’-GMP (18.3 mM) in D2O at pH 7.4 after completion of the reaction. The temperature was consecutively increased from 25 to 80 °C and decreased again to 25 °C to shift the equilibrium of the reaction to the 1:2 reaction product. The spectral changes observed at 80 °C persist even after decreasing the temperature again down to 25 °C, indicating a very slow equilibration

51 process. The equilibrium was shifted to the side of the reactants, as can be seen by the increase in the signals of the H1’ protons of liberated 5’-GMP (left) and the chelated glycine -CH protons (right) and the decrease in the signals of the 1:1 product of the H1’ protons and the ring-opened glycine -CH protons[28].

By contrast, the liberation of H2CBDCA in Pt(dach)(CBDCA) in the second reaction step was faster than the rate-determining first reaction step and could not be analyzed under the selected experimental conditions. The 1H NMR spectral changes for 5 mM Pt(dach)(CBDCA) and 5’-GMP (10 mM) in D2O are recorded as a function of reaction time (Figure 20). Signals for the Pt(dach)(CBDCA) complex, the reaction product

[Pt(dach)(N7-5’-GMP)2] and the liberated H2CBDCA were assigned, but the signals for the -CH2 and -CH2 protons of the reaction intermediate seem to overlap with the signals of the liberated acid. The intermediate is highly reactive and does not exceed 2% of the total amount of 5’-GMP. Thus, the signals are very small and tend to overlap with those of the reactants in the spectral range selected in Figure 21.

52 H, H3/H4 H, H5, H8 H H5‘/H8‘ H6/H7 time (h)

245

3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0  (ppm)

Figure 21. 400-MHz 1H NMR spectra of a solution of 5 mM

Pt(dach)(CBDCA) and 5’-GMP (10 mM) in D2O at pH 7.4 and 37.5 °C recorded as a function of time. The spectral changes can be attributed to the reactants and the 1:2 final product and the liberated acid H2CBDCA (H and H). No peaks in this area could be assigned to the highly reactive intermediate[28].

Evaluation of the integrals for the reactant and the product peaks resulted in similar time dependencies. This observation, together with the high reactivity of the intermediate, implies that the second step, the liberation of the chelate, is fast compared with the first step, the ring-opening of the chelate, which is the rate-determining step in this reaction. However, for the H8 proton in

53 5’-GMP, a peak of 2% of the total integral for all H8 5’-GMP signals was obtained for the intermediate at 8.4878 ppm, which is presented in the 1H NMR spectrum of the purine protons after a reaction time of 8 h 55 min. Because B is such a highly reactive intermediate, it was impossible to analyze the second reaction step for Pt(dach)(CBDCA) to assign k2. The mechanism of all substitution reactions is associative as supported by the large and negative values of ΔS#.

The impact of chloride concentration on the kinetics and mechanism of ligand substitution reactions of bifunctional [PtCl2(SMC)] complex with biologically relevant ligands at pH 2.5 and 7.2 was investigated. All reactions were studied as a function of chloride and nucleophile concentrations. The set of nucleophiles (L-methionine, glutathione, inosine, inosine-5’-monophosphate, guanosine-5’-monophosphate) was selected because of their difference in nucleophilicity, steric hindrance, binding properties and biological relevance. Due to the strong trans effect of the sulfur donor of S-methyl-L-cysteine in the coordination sphere of

[PtCl2(SMC)], hydrolysis of coordinated chloride trans to sulfur occurs in the first reaction step. Furthermore, this also causes significant labilization of coordinated chloride in the absence of a stronger nucleophile such that an equilibrium mixture of chloro and aqua complexes are expected to exist in equilibrium in aqueous solution. The pre-equilibrium results in two parallel substitution routes during which either chloride or water can be substituted by the entering nucleophile (see Scheme 1) [30].

Cl S S Cl K Pt + H O 2 Pt + Cl- N Cl N H O 2

+ H2O, k-b + Nu, ka - + Nu, kb + Cl , k-a S Cl

N Nu

Scheme 1. The proposed mechanism for the reaction of the [PtCl2(SMC)] complex with the selected nucleophiles in the presence of various concentrations of chloride at pH 2.5 and 7.2[30].

The presence of a pre-equilibrium suggests that the observed kinetics for the first reaction step should depend strongly on the concentration of chloride ions in solution. The expression for the observed first-order rate constant is given in Eq. 6 according to which a characteristic [Cl-] dependence is expected for both the intercept and the slope of plots of kobs versus [Nu]. The reverse k-b path was not included in Eq. 6 since in most cases no significant intercept was observed in the absence of added chloride [30].

k [Cl-] + k K a b - k o b s d = N u + k-a [Cl ] (6) K + [Cl-]

55 The results show that the second order rate constants, ka and kb, show throughout the same trend in that ka << kb, i.e. the chloro-aqua complex is significantly more reactive than the dichlorido complex as expected. In addition, the values of the equilibrium constant K are, within the quoted error limits, all of the same order of magnitude (~10-3 M), which is in line with all expectations since the pre-equilibrium constant should be independent of the - studied nucleophile. At high [Cl ] the value of the slope (i.e. ka) is almost zero, indicating that addition of excess chloride almost completely suppresses the displacement of chloride by the entering nucleophile. The values of both ka and kb follow the order L-met > GSH ~ INO > 5’-GMP [30]. According to hard-soft acid base theory soft S-donor biomolecules took advantage before N-donors.

To account for the kinetic data obtained for the back reaction it suggested that the chlorido-nucleophile complex rapidly form an ion-pair with the excess chloride ion (KIP), followed by a rate-determining ligand exchange step (kc) (see Scheme 2).

56 Cl Cl S K S + IP + + Cl- . Cl- Pt Pt

N Nu N Nu

S Cl

Pt + Nu

N Cl

Scheme 2. An ion-pair formation mechanism for the back reaction of the

[PtCl2(SMC)] complex with the selected nucleophiles in the presence of various concentrations of chloride at pH 2.5 and 7.2[30].

The rapid formation of ion-pair leads leads to a modification of Eqn. 6 to 7.

- - ka[Cl ] + kbK kcKIP [Cl ] k = obsd Nu + - K + [ C l - ] 1 + K I P [ C l ] (7)

The data showed that the ion-pair formation constants are of the same order of magnitude for all the data at pH 2.5 where the coordinated nucleophiles are either neutral or exist as zwitterions. The only value that seemed to be

57 significantly smaller is the value obtained for L-methionine as nucleophile at pH 7.2. Under these conditions uncoordinated L-met is in the zwitter-ionic form which will on coordination become an anion due to the lowering of the pKa2 value. This will in turn lead to significantly weaker ion-pair formation, viz. [Pt+ - Nu-]° and result in a four times lower ion-pair formation constant [30].

At physiological pH = 7.2, L-methionine is more reactive than at pH = 2.5 while 5’-GMP is less. A possible explanation for the lower reactivity is the fact that at pH 7.2 the 5’-monophosphate residue of the nucleotide (pKa ≈ 6) can bind to the metal center trough oxygen, which can lead to additional complications in the mechanism of complex-formation at higher pH [30].

The pKa1 and pKa2 values of the coordinated water molecules in 2+ [Pt(SMC)(H2O)2] were determined and are 3.49 and 8.80, respectively

[31]. pKa1 corresponds to deprotonation of the coordinated water molecule trans to the coordinated amine group, whereas pKa2 has a significantly higher value due to the strong trans labilization effect of sulfur on the coordinated water molecule.

1H NMR spectroscopy was used to investigate the substitution reactions of

[PtCl2(SMC)] with 5’-GMP in aqueous solution at pD = 2.91 and 298.2 K. According to the strong trans effect of the coordinated sulfur of S-methyl-L- cysteine the first step of this reaction was hydrolysis of two non-equivalent chlorides in [PtCl2(SMC)], and the second was coordination of guanosine-5’- monophosphate through the N7 atom [31]. At low pH was established that chloride trans to sulfur is much more labile than the chloride trans to N [20].

Coordination by Pt trough N and S seems to have little effects on pKa1, nor does the difference in charge between cationic free amino acid, neutral

58 [PtCl2(SMC)] and cationic cis- and trans-(O,S)-[PtCl(H2O)(SMC)] complexes [31].

At the previous stage in the reaction we have observed two singlets at δ = 2.685 ppm (the signal designated as 1 on the spectrum) and δ = 2.657 ppm (signal 2) and both are from S–CH3 (Figure 22). These pairs of peaks have been assigned of the S–CH3 group in [PtCl2(SMC)] and cis-(O,S)-

[PtCl(H2O)(SMC)] complexes and are decreased in intensity. At a later stage in the reaction, the two new peaks appear with increasing in intensity at δ = 2.599 ppm (signal 3) and δ = 2.505 ppm (signal 4) that indicated coordinated 5’-GMP (Figure 22). The multiplicity of the signals 1 and 2 during the time, t = 8341–14765 s, corresponds to equilibrium between the aqua complex cis-(O,S)-[PtCl(H2O)(SMC)] and [PtCl2(SMC)] [31]

1 Figure 22. H NMR spectra of the reactions of [PtCl2(SMC)] (10 mM) with

5’-GMP in the ratio 1:2 (where 1 is the signal for the [PtCl2(SMC)], 2 is the signal for the cis-(O,S)-[PtCl(H2O)(SMC)], 3 is the signal for the cis-(S,N7)-[PtCl(5’-GMP)(SMC)] complex and 4 is the signal trans-(S,N7)- [PtCl(5’-GMP)(SMC)]+)[31].

The integral of the singlet of the H8 proton and the doublet of the H1’ proton of 5’-GMP was unavailable to measure because there was multiplicity of a different product signals at the same shifts (Figure 23).

1 Figure 23. H NMR spectra of the reactions of [PtCl2(SMC)] (10 mM) with 5’-GMP in the ratio 1:2 (where with circle is assigned the signal of free 5’-GMP and with square are assigned signals of coordinated 5’-GMP)[31].

61 As was mentioned, the first reaction step was hydrolysis whereas the product we obtained the trans-(O,S)-[PtCl(H2O)(SMC)] complex which is very reactive and the peak of S–CH3 group in this complex did not appear in the

NMR spectra. Instead, the singlet of the S–CH3 group of the complex with coordinated 5’-GMP (δ = 2.505 ppm) in the trans position of the sulfur atom in S-methyl-L-cysteine was recognized. The other product of hydrolysis was the cis-(O,S)-[PtCl(H2O)(SMC)] complex with the peak of the S–CH3 group shifted by δ = 2.657 ppm. This complex reacts with 5’-GMP and a new singlet of S–CH3 at δ = 2.599 ppm appeared (coordinated 5’-GMP) (Figure 21) [31].

The values of rate constant showed faster substitution of coordinated H2O in the trans position to the S donor atom of S-methyl-L-cysteine, whereas the slower reaction was assigned to the displacement of the cis coordinated aqua molecule. This happens due to the strong trans labilization effect of coordinated sulfur. The results indicate that N-bonding ligands, such as 5’-GMP, have a high affinity for the platinum(II) complex. Thermodynamically Pt-N bonds are more stable while Pt-S are kinetically favourable [31].

2.4. Interactions of dinuclear platinum(II) complexes with sulfur- and nitrogen donor biomolecules

The third generation anti-tumor complexes that are tested in pre-clinical trials represent orally active Pt(IV) complexes, sterically hindered Pt(II) complexes, polynuclear Pt(II) complexes and sulfur-containing platinum complexes [10-12]. These polynuclear Pt(II) complexes consist of either two or three platinum centers that are linked through a flexible bridge such as an

62 aliphatic chain, or a rigid bridge that consist for instance azole molecules. The reasons for increasing interest in multinuclear complexes is their ability to form DNA adducts that differ significantly from those formed by cisplatin and related complexes, which results in a completely different anti-tumour behavior [32].

Polynuclear (dinuclear and trinuclear) bifunctional DNA binding agents are amongst the best studied of these nonclassical structures. The class as a whole represents a second, distinctly new structural group of platinum-based anticancer agents. The first example of this class to advance to clinical trials was BBR3464 [33]. The polynuclear platinum compounds are in opposite to mononuclear platinum complexes because the predominant DNA lesions are long-range inter- and intra-strand cross-links where the sites of platination may be separated by up to four base pairs. The consequent structural and conformational changes in DNA are also distinct. Due to the charged nature of polynuclear platinum complexes, ranging from 2+ to 4+, binding to DNA occurs significantly more rapidly than for cisplatin. The binding of polyamine-bridged dinuclear compounds is even faster than BBR3464, suggesting strong preassociation or electrostatic binding prior to covalent attachment.

Some of trans azole-bridged dinuclear platinum(II) complexes are highly effective in vitro in cisplatin-resistant cell line, as well as in several tumour cell line [34,35]. As a main structural feature the azole-bridged complexes possess the leaving hydroxo group, an appropriate Pt∙∙∙Pt distance, and some flexibility to provide the 1,2-intrastrand cross-links with a minimal distortion of the DNA.

As part of our research we have synthesized dinuclear Pt(II) complexes started from transplatin using various type of bridged ligands (Figure 24).

63 The novel dinuclear Pt(II) complexes are [{trans-Pt(NH3)2Cl}2(- pyrazine)](ClO4)2 (Pt1), [{trans-Pt(NH3)2Cl}2(-4,4′- bipyridyl)](ClO4)2·DMF (Pt2), and [{trans-Pt(NH3)2Cl}2(-1,2-bis(4- pyridyl)ethane)](ClO4)2 (Pt3).

N H 3 N H3

Cl Cl Pt N N Pt C l O 4 2 N H N H 3 3

[{trans - Pt(NH3 ) 2 Cl}2 ( - pyrazine)](ClO4 ) 2, Pt1

N H3 N H3

C l N N Pt C l ClO * dmf Pt 4 2

N H NH 3 3

[{tra ns - Pt(NH3 )2 Cl}2 ( - 4,4’- bipyridyl)](ClO4 ) 2 · DMF , Pt2

N H 3 N H 3 N Pt Cl

C l O 4 2 C l Pt N N H 3

N H 3

[{trans - Pt(NH3 ) 2 Cl} 2 ( - 1,2 - bis(4 -pyridyl)ethane)](ClO 4 ) 2 , Pt3

Figure 24. Structures of the investigated dinuclear platinum(II) complexes[36].

64 Тhе pKa constants of diacqua complexes were determined UV-Vis spectrophotometrically at 25 °C in the presence of 0.01 M NaClO4. Spectral changes after each addition of standard NaOH solution are shown in the Figures 25.

2.4 0.40

0.35 2.0 0.30

0.25

1.6 Absorbance 0.20

1.2 0.15

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Absorbance pH 0.8

0.4

0.0 200 220 240 260 280 300 320 340 Wavelength, nm

Figure 25. UV-Vis spectra of the diaqua Pt3 complex recorded as a function of pH in the range 2 to 10 at I = 0.01 M (NaClO4) and 25 °C. Inset: titration curve at 280 nm[36].

The titration data for the complexes were fitted to Eq. 8 for measuring in two pKa values.

65 b  a c  b y  a   (8)  x  pK   x  pK  1 2,718  a1  1 2,718  a2   m   n 

In this equation x refers to pH, and y represents the absorbance value. The parameter a represents the value of the absorbance at the beginning of the titration, b represents the absorbance during the titration and c represents the absorbance at the end of the titration. The parameters m and n are used to optimize the titration curve.

The aqua ligands coordinated to each platinum center can exhibit different pKa values depending on the distance between the two Pt(II) centers. The conjugated -electron system in some of the bridging ligands supports electronic communication between the two platinum centers and contributes to the pKa values [37].

The platinum centers in the studied complexes are thermodynamically and kinetically independent of each other, and for all the complexes two pKa values (Table 2) were observed. A shorter distance between the two Pt(II) centers results in the addition of the charges on the platinum centers by which each platinum center becomes more electrophilic and thus more acidic, which leads to lower pKa values than that found for 2+ charged platinum centers of mononuclear complexes. The pKa1 value of the diaqua Pt1 complex is lower than for the Pt2 and Pt3 complexes, and could be associated with the -acceptor ability of the pyrazine ligand in Pt1. Following deprotonation of the first coordinated water in all the dinuclear Pt(II) complexes, the overall charge of the complex decreases from 4+ to 3+ and the electrophilicity and acidity of the second platinum center also

66 decreases. These effects account for the smaller difference between the pKa1 and pKa2 values (see Table 2) [36].

Table 2. Summary of pKa values for the deprotonation steps of the investigated diaqua complexes[36].

pKa1 pKa2 Pt1 3.94 ± 0.07 5.0 ± 0.5 Pt2 4.6 ± 0.2 5.69 ± 0.05 Pt3 4.58 ± 0.06 5.5 ± 0.3 Pena 3.93 ± 0.03 5.69 ± 0.03 Hepa 4.07 ± 0.02 5.27 ± 0.06 Deca 4.03 ± 0.04 5.06 ± 0.06 a Ref. [37b]

In Table 2 are summarized pKa values of dinuclear platinum(II) complexes 4+ type [Pt2(N,N,N′,N′-tetrakis(2-pyridylmethyl)diamine)(H2O)2] . Based on the literature data it can be asserted that on the pKa value the distance between the two metal centers has a large influence [37b]. The electrophilicity of the dinuclear complexes is higher when Pt–Pt distance is smaller, which results in lower pKa values [36,37].

The substitution reactions of all the studied dinuclear Pt(II) complexes proceeded in two subsequent reactions steps that both depended on the nucleophile concentration. All kinetic traces gave an excellent fit to a double exponential function, typical for a two step reaction. The so-obtained pseudo-first-order rate constants, kobs1 and kobs2, calculated from the kinetic traces were plotted against the concentration of the entering nucleophiles. A

67 linear dependence on the nucleophile concentration was observed for all the studied reactions (see Figures 26 and 27). The linear fits passing through the origin indicated that possible parallel or backward reactions are insignificant or absent, whereas the observed small intercepts for the dichlorido complexes were ascribed to the back reaction with the excess chloride present in solution to prevent the spontaneous hydrolysis of the dichlorido complexes (see Figures 27). The substitution process was characterized by two rate constants k1, which represents the second-order rate constant for the first step of substitution, and k2 for the second step of substitution [36].

68 Pt1

6 12 2 -1 -1 First step 10 kobsd2/s Second step kobsd1/s GSH 10 Tu

4 8 5'-GMP 6

GSH 2 4

5'-GMP 2

3 10 [L]/M 103 [L]/M 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Pt2

36 10 3 -1 4 -1 10 kobsd1/s First step 10 kobsd2/s Second step 32 Tu GSH 28 8

24 5'-GMP 6 20 GSH 16 4 12

8 5'-GMP 2 4 103 [L]/M 103 [L]/M 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Pt3

16 6 3 -1 4 -1 Second step 10 kobsd1/s First step 10 kobsd2/s GSH Tu

12 4 5'-GMP

8 GSH 2 4 5'-GMP

103 [L]/M 103 [L]/M 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Figure 26. Pseudo-first order rate constants as a function of nucleophile concentration for the first and second substitution reactions of the diaqua Pt1, Pt2 and Pt3 complexes by Tu, GSH, 5’-GMP at pH = 2.5, 0.01 M o [36] NaClO4 and 37 C .

69 Pt1

10 3 -1 12 10 kobsd1/s 4 -1 First step Tu 10 kobsd2/s Second step 10 GSH 8

8 6 GSH

4 4

2 2 5'-GMP 5'-GMP 103 [L]/M 103 [L]/M 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Pt2

8 6 3 -1 Tu 4 -1 10 kobsd1/s First step 10 kobsd2/s Second step GSH

6 4 GSH

2 2 5'-GMP 5'-GMP 3 3 10 [L]/M 0 10 [L]/M 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Pt3

6 4 3 -1 4 -1 10 kobsd1/s First step 10 kobsd2/s Second step Tu

3 4

GSH 2 GSH

2 1 5'-GMP 5'-GMP 3 3 10 [L]/M 0 10 [L]/M 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Figure 27. Pseudo-first order rate constants as a function of nucleophile concentration for the first and second substitution reactions of the dichlorido Pt1, Pt2 and Pt3 complexes by Tu, GSH, 5’-GMP at pH=7.2, 25 mM Heppes, 20 mM NaCl and 37 oC[36].

As was mention under selected experimental conditions (pH 2.5), the substitution of the aqua ligands in the complexes involved two reaction steps

70 determined by the second-order rate constants k1 and k2 as shown on Scheme 3 [36].

In the case of thiourea as the nucleophile the first reaction turned out to be very fast, whereas the second reaction was extremely slow, and both steps showed a dependence on the thiourea concentration. The fast step was assigned to the substitution of both labile aqua ligands, whereas the second step was ascribed to the displacement of the bridging ligand as a result of the strong trans effect of coordinated thiourea [36].

NH3 NH3

X Pt L Pt X

NH3 NH3 Nu

k1 X

NH3 NH3

Nu Pt L Pt X

NH3 NH3 Nu

k2 X

NH3 NH3

Nu Pt L Pt Nu

NH3 NH3

- X = H2O, Cl Nu = Tu, GSH, 5’-GMP L = pyrazine, 4, 4’-bipyridyl, 1, 2-bis(4-pyridyl)ethane

Scheme 3. Proposed reaction pathways for the reaction of multinuclear Pt(II) complexes with a series of nucleophiles[36]

71 Nucleophile thiourea as an soft base S-donor nucleophile have shown the strongest effect followed by glutathione and guanosine-5′-monophosphate. Due to the observed decomposition of the dinuclear bridged complex during the subsequent slow reaction with thiourea, this process was not studied in any further detail.

The order of reactivity of the diacqua complexes are Pt1 > Pt2 > Pt3. The higher lability of the diacqua Pt1 complex can be ascribed to the decrease in electron density on the platinum center caused by the  acceptor ability of the pyrazine ligand, by which the metal centers in Pt1 become more electrophilic and favour the rapid binding of the entering nucleophile. The Pt2 and Pt3 react significantly slower because the nature of the bridging ligand is such that less -back bonding can occur to increase the electrophilicity of the metal centers. This was in agreement with other published results for the reactions of related dinuclear Pt(II) complexes with thiourea [37,38].

In the case of the substitution reactions with glutathione, we observed a lower reactivity than with thiourea. The kinetic traces showed two reaction steps. Over time the first reaction step with GSH for all the dinuclear platinum(II) complexes was complete, and it was possible to study the second substitution step (see Figure 26). If we take into account the pKa values of glutathione, viz. pKa1 = 2.05, pKa2 = 3.40, pKa3 = 8.79 and pKa4 = 9.49 [39], the SH group will be partially protonated under our experimental conditions (pH 2.5). Coordination primarily took place through the sulfur donor and the reactions go to completion. In the reactions with glutathione the first step could be described as a substitution of the first aqua molecule by glutathione, while the second substitution step is substitution of the second aqua molecule. The slower reaction for the second step is caused by

72 the steric effect after the coordination of the first GSH, and by the charge on the complex. After coordination of the first glutathione the overall charge of the complexes is reduced from 4+ to 3+, the electrophilicity on the Pt(II) center decreased, which causes a slower second substitution step [36].

The interaction of glutathione with the dinuclear platinum(II) complexes has biological importance because glutathione is the most prevalent intracellular thiol. However, the role of glutathione appears to be dual: glutathione both activates and deactivates platinum drug cisplatin [40]. Higher effectiveness of cisplatin has also been demonstrated by co-administering cisplatin and glutathione in patients. For now it is not clear whether the increase in effectiveness is due to the reduced toxicity or due to the modification of the platinum drug by binding to the metal.

The substitution reactions with guanosine-5′-monophosphate were over during the observed reaction time and kinetic traces also showed two substitution steps. Under our experimental conditions (pH 2.5) only the N7 position of 5′-GMP will bind to the dinuclear Pt(II) complexes, since at this pH the N1 position is protonated (pKa = 8.88). The first step of the substitution reaction could be interpreted as substitution of the first water molecule by the nucleophile, whereas the second step is substitution of the other water molecule by 5′-GMP. The steric hindrance may not significantly differ for the first step as free rotation around the Pt–N azine bonds is present within both of them. However, for the second step effects of steric hindrance should be considered, as caused by the binding of the first nucleophile. Moreover, the charge of the complex may also play an important role because after the coordination of the first 5′-GMP2- the charge of the complex is 2+ and the second substitution reaction is much slower. Furthermore, the substitution process is slow in comparison with S-donor

73 ligands. It is well know, that Pt(soft acid)–N(intermediate base) complexes are thermodynamically more stable products.

The reactions of the dichlorido dinuclear platinum(II) complexes were studied at pH 7.2 in 25 mM Hepes buffer in the presence of 20 mM NaCl at 37 °C. The substitution processes also showed two reaction steps determined by two second-rate constants k1 and k2 (Scheme 3). Only in the case of thiourea was it not possible to study the second reaction step because labilization of the bridging ligand as a result of the strong trans-effect of Tu. The order of reactivity of the nucleophiles for all complexes is Tu > GSH > 5′-GMP. Dichlorido complexes are less reactive than their diaqua analogues, partly because of the stronger Pt–Cl bond and the lower electrophilicity of the platinum center of the chlorido complexes arising from the lower overall charge of 1+. The order of reactivity of the complexes is Pt1 > Pt2 > Pt3 [36].

1H NMR spectroscopy was used to investigate the substitution reactions with 5’-GMP in aqueous solution at pD= 6.70 and 22 °C. The substitution kinetics was studied under second-order conditions with an initial molar ratio of 1:2 for dichlorido Pt2 complex:5′-GMP [36]. Considering that the initial concentrations of 5′-GMP and the complex, ca0 and cb0 respectively, can be expressed by ca0 ≠ cb0 for the first reaction step, the Eq. 9 was applied for the determination of k1. The concentration of the 1:1 product, i.e. singly substituted complex, is represented by x [29]. The concentration x is calculated considering area of the signals for free 5’-GMP and coordinated 5’-GMP.

1 cb0(ca0 - x) k 1 t = ln (9) ca0 - cb0 ca0(cb0 - x)

The integral of of doublet of the H1’ proton of 5′-GMP used to measure intensity of the signal. The intensity of signal for free 5’-GMP at 5.95 ppm decreases during the reactions whereas at the same time a signal for coordinated 5’-GMP appears around 6.12 ppm and increases.

A plot of the right hand side of Eq. 9 versus reaction time results in a straight line passing through the origin (Figure 28). The value of k1 was obtained from the slope, when is one 5′-GMP molecule coordinated. The second reaction step it does not to go completion during the selected reaction time and we didn’t consider.

300

250

-x) 0

(b 200

0 -x)/(a

0 150

)*ln(a 0

-b 100

0 (1/a 50

0 0 5000 10000 15000 20000 25000 30000 35000 40000 t(s)

Figure 28. Second order plot for the reaction of Pt2 (10 mM) complex with 5’-GMP (20 mM) (in molar ratio complex:ligand = 1:2).The y axis represents the right-hand side term of Eq. 9. at pD = 6.7 and 22 °C[27].

75 The value of k1 obtained for substitution of chloride by 5’-GMP investigated 1 -2 -1 -1 by H NMR spectroscopy (k1 = (0.71 ± 0.03)·10 M s ) was smaller then -2 - the value obtained from UV-Vis spectrophotometry (k1 = (6.1 ± 0.4)·10 M 1s-1) [36]. The main reason is the temperature effect the reaction between dichlorido Pt2 complex and 5’-GMP investigated by 1H NMR spectroscopy. This reaction was performed at 22 °C while all reactions on UV-Vis were performed at 37 °C. Increasing the temperature in 15 degrees the second- order rate constant increased around 9 times. The obtained k1 value are in agreement with literature data for the similar dinuclear platinum(II) complexes [37c].

The cytotoxic activity of these dinuclear platinum(II) and some platinum(IV) complexes such as [PtCl4(bipy)] and [PtCl4(dach)], toward TOV21G (ovarian cancer cell line) and HCT-116 (colon cancer cell line) human carcinoma cell line have been investigated [41]. The cytotoxic capacity toward TOV21G, HCT116 tumor human cell lines and human MSC, normally dividing cells, was compared (Figure 29).

All the complexes displayed a dose-dependent and time dependent cytotoxicity toward the tested cell lines, but the highest cytotoxic effect was shown toward TOV21G cells (Figure 29). The complex [PtCl4(dach)] at lower concentrations induced significantly higher cytotoxic effect toward TOV21G cells than the other four complexes. HCT116 cells were more resistant to cytotoxic effects of the selected complexes. Again, [PtCl4(dach)] was the most efficient and exerted very similar activity toward HCT116 cells as cisplatin [41]. The complexes Pt2 and Pt3 displayed cytotoxicity toward

MSC similar to cisplatin, but the other three complexes Pt1, [PtCl4(dach)] and [PtCl4(bipy)] were more toxic.

Figure 29. Cytotoxic activity of tested complexes measured by MTT test (Mean ± SE)[41].

77 The dinuclear azine-bridged Pt(II) complexes, for example, [{cis-

Pt(NH3)2Cl}2(-pyrazine)](NO3)2, [{cis-Pt(NH3)2Cl}2(-pyridazine)](NO3)2 show significant cytotoxicity for the ovarian carcinoma cell line and a lower cytotoxicity toward colon cancer cell line, compared to cisplatin [42].

A chiral ligand, 2-(((1R,2R)-2-aminocyclohexyl)amino)acetic acid (HL), was designed and synthesized to prepare a series of dinuclear Pt(II) complexes with dicarboxylates or sulfate as bridges. All compounds showed antitumor activity to HCT116 very close to the activity of oxaliplatin and better than our the dinuclear Pt(II) complexes [43]. Also, dinuclear Pt(II) complex,

{[cis-Pt{NH3)2Cl]2(l-4,40-methylendianiline)}(NO3)2, is highly cytotoxic against the murine leukemia (P-388) and the human non-small-cell lung cancer (A-549) cell lines, and it is more cytotoxic than cisplatin at most concentrations tested [44].

78 3. BIOINORGANIC INTERACTIONS OF ZINC(II) AND COPPER(II) COMPLEXES IN CORRELATION WITH HSAB PRINCIPLE

Transition metal compounds play crucial roles in bioinorganic reactionts as cofactors in metalloproteins, they act mainly as a Lewis acid. Two essential metal ions, namely zinc and copper ions, modulate enzymes activities, catalytic and regulatory functions, oxidative-reductive processes, etc. [45,46]. The electronic properties of Zn(II), such as intermediate Lewis acidity, redox inertness and flexible coordination geometry, render it a suitable cofactor in several proteins that perform essential biological functions. On other hand the chemistry of copper is dominated by the +2 oxidation state, e.g. copper(II) complex ions but in many biological processes turned to +1 as in Cu/Zn-superoxide dismutase (Figure 30), during catalyzed dismutation of superoxides .

Figure 30. The active site of Cu/Zn-superoxide dismutase.

79 Zinc(II) ions are known to be cytotoxic/tumor suppressor agents in several cancers [47]. Cellular accumulation of zinc exhibits tumor-suppressor effects that are incompatible with the process and activities of prostate malignancy, thus, the concentration of zinc existing in the normal prostate epithelial cells is cytotoxic in the malignant cells [48]. The most studied metalloproteins in which zinc serves a structural role belong to the zinc-finger family, which is involved in control of nucleic acid replication, transcription and repair [49]. In zinc-finger proteins, zinc is tetrahedrally coordinate to histidines and/or cysteines, the coordination of aspartic acid and glutamic acid residues to the metal, also has been found in metalloenzymes (see Figure 31) [50].

Figure 31. Active site in zinc-finger protein.

Zinc(II) complexes have shown potential utilization as radioprotective agents [51], antibacterial or antimicrobial agents [52], antidiabetic insulin-mimetic [53] and tumor photosensitizers [54]. They are kinetically labile, the mechanism of antitumor activity of zinc(II) coordination compounds could be connected with: (i) fast interconversion among its four-, five-, and six- coordinate states; (ii) preference of the variable coordination geometries (tetrahedral, five-coordinate, octahedral) that zinc(II) is able to adopt, toward diverse donor site of relevant nucleophiles [55,56]. Knowledge of the

80 mechanism of interactions between zinc(II) complexes and biomolecules or other relevant ligands is essential for understanding the cellular biology of delivery of complexes to DNA and proteins.

On the other hand, copper(II) controls cancer development. It serves as a limiting factor for multiple aspects of tumour progression, growth, angiogenesis and metastasis [57-59]. Copper(II) complexes offer various potential advantages as antimicrobial, antiviral, anti-inflammatory, antitumor agents, enzyme inhibitor, chemical nucleases, and they are also beneficial against several diseases like copper rheumatoid and gastric ulcers [60, 61]. It has been shown recently that Zn(II) and Cu(II) complexes of imidazole terpyridine (itpy) have potential applications in chemotherapy [62]. Changing the ligand environment toward the specific target is a possible way of tuning the selectivity of a drug molecule. The nature of the ligands plays an important role in the binding of a metal complex to a biomolecule such as DNA or protein [62,63]. Research efforts have shown that the cytotoxic activity of the Cu(II) complexes is highly dependent on the organic ligand frame around the metal. The donor set and ligand scaffold can influence important factors such as the lipophilic/hydrophilic nature of the compound, the favored oxidation state of the metal center, as well as the observed reactivity of the complex.

Design of novel non-platinum DNA and protein targeting metal-based anticancer agents with potential in vitro toxicity have gained importance in recent years [46]. The non-platinum antitumor complexes could be alternatives to platinum-based drugs due to their better characteristics and less negative side effects, especially because some transition metal ions are essential cellular components involved in several biochemical processes. Investigation of mechanism of the interaction zinc(II) and copper(II) ions

81 with biomolecules and other relevant ligands provided useful information for the future design of potential zinc- and copper-based anticancer drugs. Different mechanism of interactions with selected biomolecules compared to platinum-based drugs has been observed, that could be discussed in terms of hard soft acid base principle.

3.1. Interactions of zinc(II) complexes with biomolecules in correlation with HSAB principle

Knowledge of the mechanism of interactions between zinc(II) complexes and biomolecules gives valuable information for the synthesis of new drugs. Zinc(II) complexes are kinetically labile, variable coordination geometries such as tetrahedral, five-coordinate, octahedral, that zinc(II) is able to adopt in solution have preference toward diverse donor site of relevant nucleophiles [55,56]. In our studies, we have shown interconversion of the four-coordinated tetrahedral [ZnCl2(en)] complex to five- and six- 2- coordinated octahedral [ZnCl4(en)] anion in the presence of various chloride concentration [64,65]. The excess of chloride had no affect on coordination geometry of complex [ZnCl2(terpy)] [64]. Strong π-acceptor ability of the tridentate chelate 2,2′:6′,2”-terpyridine stabilizes the square- pyramidal geometry [66].

Mole ratio method was used for determination of metal-ligand stoichiometry. This method is an alternative to the method of continuous variations (Job’s method) in which the amount of one reactant, usually the molar concentration of metal, is held constant, while the amount of the other reactant is varied [67,68]. In order to determine metal-ligand stoichiometry between [ZnCl2(en)] complex and chloride at pH 7.2, the absorbance

82 changes over the wavelength range 200 to 400 nm for different molar ratio

[Cl-]/[ZnCl2(en)] were recorded (see Figure 32) [65]. The differences between spectra of [ZnCl2(en)] complex and spectra of the solutions with various molar ratio [Cl-]/[ZnCl2(en)] only can be noticed in intensity of the absorbance, and maximum has been shifted due to coordination of the chloride from 213 nm to 227 nm. The existence of mixed Cl-H2O complexes was not observed [65].

Figure 32. Absorbance changes for the reactions between [ZnCl2(en)] complexes (0.001 M) and chloride in different molar ratio at pH 7.2 (0.025 M Hepes buffer) and 295 K[65].

The absorbance was monitored at a wavelength where the metal–ligand complex absorbs. At selected wavelength 234 nm we have observed two

83 equivalence points, corresponding to step-wise formation of 1:1 and 1:2 complexes (see Figure 33).

Figure 33. Stoichiometry of chloride-[ZnCl2(en)]complexes (Mole-ratio method)[65].

- 2- Three complex species the [ZnCl2(en)], [ZnCl3(en)] and [ZnCl4(en)] complexes absorb at the selected wavelength. The coordination number of Zn(II) is changed from 4 up to 6, according to this the geometry of formed complexes undergoes to change [65].

Metal-ligand stoichiometry between [ZnCl2(en)] and [ZnCl2(terpy)] complexes and imidazole at pH 7.2 in the presence of an excess of chlorides also was determined [64]. The absorbance changes over the wavelength

84 range 200 to 600 nm for different molar ratio [imidazole]/[Zn(II) complex] were recorded. Different stoichiometry of zinc-imidazole complexes we observed which indicated formation of different complex species. According to our research in the presence of chlorides different complex species are formed [65] In the presence of 0.001 M NaCl at selected wavelength 218 nm we observed one equivalence point, corresponding to formation of 1:1 complexes specie [ZnCl2(en)(imidazole)] (see Figure 34).

1.2 0.5 eq 1.0 eq 0.38 1.5 eq 2.0 eq 1.0 2.5 eq 0.36 3.0 eq 3.5 eq 0.34

0.8 0.32

0.30 0.6 0.28

Absorbance 0.4 0.26 Absorbance 1:1.38 0.24 0.2 0.22

0.20 0.0 200 250 300 350 400 450 500 550 600 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wavelength (nm) Molar ratio

Figure 34. Titrations of [ZnCl2(en)] with imidazole as monitored by UV-

Vis spectra. Left: [ZnCl2(en)]-imidazole, Right: Cross-section of UV-Vis spectra at 218 nm in presence of 0.001 M NaCl[64].

Otherwise, in the presence of 0.010 M NaCl at selected wavelength 205 nm we observed two equivalence points, corresponding to step-wise formation of 1:1 and 1:2 complexes and we assumed that octahedral complex

[ZnCl2(en)(imidazole)2] is formed rapidly (Figure 35) [64].

1.4 0.5 eq 1.0 eq 1.5 eq 1.2 2.0 eq 2.5 eq 1.4 3.0 eq 3.5 eq 4.0 eq 1.0 4.5 eq 1.2 5.0 eq

0.8 1.0

0.6 0.8 Absorbance

1 : 2.3

0.4 0.6 Absorbance

0.4 0.2 1 : 1.1

0.2 0.0 200 250 300 350 400 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Wavelength (nm) Molar ratio

Figure 35. Titrations of [ZnCl2(en)] with imidiazole as monitored by UV-

Vis spectra. Left: [ZnCl2(en)]-imidiazole, Right: Cross-section of UV-Vis spectra at 212 nm in presence of 0.010 M NaCl[64].

Five-coordinated complex [ZnCl2(terpy)] is very stable, in the presence of 0.001 M NaCl at selected wavelength 350 nm we observed one equivalence point. This result implied a 1:2.15 zinc:imidazole stoichiometry that probably corresponds to formation of the five-coordinate specie

[Zn(terpy)(imidazole)2] in which chlorides are substituted by imidazole (Figure 36) [64].

86 0.5 eq 3.0 1.0 eq 0.14 1.5 eq 2.0 eq 2.5 2.5 eq 0.12 3.0 eq 3.5 eq 0.10 2.0 4.0 eq

0.08 1.5

0.06 Absorbance Absorbance 1.0 1 : 2.15 0.04

0.5 0.02

0.0 0.00 200 225 250 275 300 325 350 375 400 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Wavelength (nm) Molar ratio

Figure 36. Titrations of [ZnCl2(terpy)] with imidiazole as monitored by

UV-Vis spectra. Left: [ZnCl2(terpy)]-imidiazole, Right: Cross-section of UV-Vis spectra at 350 nm in presence of 0.001 M NaCl[64].

In the presence of 0.010 M NaCl we did not obtain the valuable results. The reason could be competition of coordination between chlorides and imidazole, chlorides were suppressed the substitution [64].

The mechanism and kinetics of substitution reactions between these two complexes and bio-ligand have been investigated in the presence and absence of chloride [64,65,68]. All substitutions proceed in two consecutive reactions steps, both are depending on the nucleophile concentration. All kinetic traces gave an excellent ﬁt to a double exponential function, typical for a two step reaction. The obtained pseudo-ﬁrst-order rate constants, kobs1 and kobs2, were calculated from the kinetic traces were plotted against the concentration of the entering nucleophiles. A linear dependence on the nucleophile concentration was observed for all studied reactions.

Observed pseudo-first-order rate constants, kobs1 and kobs2, depend on the entering nucleophile (Nu) concentration as given in Eqn. 10 and 11.

87 kobs1 = k1[Nu] + k-1 (10)

kobs2 = k2[Nu] + k-2 (11)

Linear fits passing through the origin for some reactions in present study, indicate that possible parallel reactions are insignificant or absent, i.e. k-1 and k-2 are negligible, and Eqn. 10 and 11 simplify to kobs1 = k1[Nu] and kobs2 = k2[Nu]. Thus, in the present systems, direct nucleophilic substitution is the major observed reaction pathway under the selected conditions.

The first step of the substitution reactions of [ZnCl2(en)] with biomolecules such as inosine-5′-monophosphate (5′-IMP), guanosine-5′-monophosphate (5′-GMP), L-methionine (L-met), glutathione (GSH) and DL-aspartic acid (DL-asp), in the presence of 0.010 M NaCl at pH, 7.2 could be interpreted as substitution of the axial chlorido ligands in cis position toward bidentate coordinated ethylenediamine by the nucleophiles, whereas the second step is substitution of the equatorial chlorido ligand. As was mentioned before in the 2- presence of excess of chloride the step-wise formation of [ZnCl4(en)] complex firstly occured, after that substitution reactions took place [65]. Due to a large negative inductive effects of amino groups in ethylenediamine the basicity of N-donors atoms increase and the interactions between Zn(II) and

–NH2 groups are stronger. According to this, both chlorido ligands in axial position are kinetically labile and equal for parallel substitution routes. A linear dependence on the nucleophile concentration was observed for all the studied reactions (see Figure 37), and only in the case of the DNA constituent (5’-IMP and 5’-GMP) a small intercept has been observed [65].

1.6 0.045 first step first step 1.4 second step 0.040 second step 0.035 1.2

0.030 1.0

0.025 -1

0.8 -1

/s /s

0.020

obsd

obsd k k 0.6 0.015

0.4 0.010 0.2 0.005

0.0 0.000 0.000 0.001 0.002 0.003 0.004 0.005 0.000 0.001 0.002 0.003 0.004 0.005 [5'-IMP]/M [5'-GMP]/M

0.24 0.32 0.12 first step 0.22 first step second step first step 0.20 0.28 second step second step 0.10 0.18 0.24 0.16 0.08 0.14 0.20

-1 0.12

/s -1

-1 0.16 0.06

/s /s

obsd 0.10

obsd

obsd k 0.08 k 0.12 0.04 0.06 0.08 0.04 0.02 0.02 0.04

0.00 0.000 0.001 0.002 0.003 0.004 0.005 0.00 0.00 0.000 0.001 0.002 0.003 0.004 0.005 0.000 0.001 0.002 0.003 0.004 0.005 [L-Met]/M [GSH]/M [DL-Asp]/M

Figure 37. Pseudo-ﬁrst order rate constants plotted as a function of nucleophile concentration for the first and second substitution reactions of the [ZnCl2(en)] complex by 5’-IMP, 5’-GMP, L-met, GSH and DL-asp at pH 7.2 (0.025 M Hepes buffer) in the addition of 0.010 M NaCl at 295 K[65].

The first reaction step is accompanied by dissociation of one chloride ligand in equatorial position and five-coordinate complex have been obtained. The dissociation of ligand in six-coordinate complex of zinc(II) and formation of

89 a five-coordinate complex happens with little energy loss. On the other side, four-coordinate complexes can add a fifth ligand with little energetic barrier [46,69,70].

The second reaction step could be interpreted as substitution of the last chloride ligand. The order of reactivity of the investigated nucleophiles for the first reaction step is 5’-IMP > GSH > L-met > DL-asp > 5’-GMP, while for the second reaction step is GSH > L-met > 5’-IMP > DL-asp > 5’-GMP [65].

Zinc(II) as borderline hardness Lewis acid displays high affinity for nitrogen and oxygen donor atoms as well as for sulfur, depending on coordination number [71]. It is therefore found to be bound to histidines, glutamates or aspartates, and cysteines [46]. The square-pyramidal structure of Zn(II) in biological systems prefers O-carboxylate, carbonyl and N-imidazole donor bioligands. The versatility of coordination of Zn(II) and DNA has been founded in zinc finger proteins as part of nucleic acid polymerases and hydrolyses [71]. With variable coordination geometries (tetrahedral, five- coordinate, octahedral) that zinc(II) is able to adopt, its balance in donor site preference (N,O) may account for the lowest reactivity of 5’-GMP. The steric hindrance also could be reason of the similar reactivity of 5’-GMP for both reaction steps [72,73].

The kinetics of ligand substitution reactions of [ZnCl2(en)] and

[ZnCl2(terpy)] complexes and biologically relevant nitrogen nucleophiles such as imidazole, 1,2,3-triazole and L-histidine were investigated at pH 7.2 as a function of nucleophile concentration in the presence of 0.001 M and

0.010 M NaCl [64]. In the presence of 0.001 M NaCl [ZnCl2(en)] undergoes - to changes and five-coordinate complex specie [ZnCl3(en)] is formed, while 2- in the presence of 0.010 M NaCl octahedral complex anion [ZnCl4(en)] is

90 present and all substitution processes of these complex species should be considered [64]. In the presence of 0.001 M NaCl the geometry of five- coordinate complex species after substitution remains. The order of reactivity of investigated nucleophiles for the first reaction step is L-histidine > 1,2,3- triazole > imidazole while for the second is imidazole > L-histidine > 1,2,3- triazole [64]. In the presence of 0.010 M NaCl octahedral complex anion 2- [ZnCl4(en)] is formed rapidly [65]. The order of reactivity of the investigated nucleophiles for the first reaction step is 1,2,3-triazole > imidazole > L-histidine, while for the second reaction step is L-histidine > imidazole > 1,2,3-triazole [64]. The different order of reactivity of relevant nitrogen nucleophiles could be consequence of different geometrical structure of [ZnCl2(en)] complex caused by different chloride concentration.

The structure of [ZnCl2(terpy)] complex remains unchanged in the presence of chloride, the substitution proceed in two reaction steps, the booth chloride are substituted by nucleophiles. The rates of nucleophilic substitution reactions are controlled by strong π-acceptor ability of the tridentate chelate 2,2′:6′,2”-terpyridine and by the excess of chloride present in solution [64]. Different chloride concentration affects only on the rate of substitution. The substitution reactions in the presence of the 0.001 M NaCl are approximately two or three times faster, the reason could be competition between relevant nitrogen nucleophiles and chloride. The order of investigated nucleophiles in the presence of different chloride concentration is the same for the first step: L-histidine > 1,2,3-triazole > imidazole. The second order of reactivity in the presence of 0.001 M NaCl is L-histidine > imidazole > 1,2,3-triazole, while in 0.010 M NaCl the nucleophiles follows the order: L-histidine > 1,2,3- triazole > imidazole. We observed the similar order of magnitude for the second rate constants, for the both substitution processes of [ZnCl2(terpy)] complex by L-histidine in the presence of 0.001 M NaCl. This suggests that

91 there are two parallel reaction paths. The steric hindrance also could be reason of diverse reactivity of nucleophiles for second reaction steps [72,73]. The second-order rate constants in the presence of various chloride concentrations are summarized in Tables 3 and 4.

Table 3. Second-order rate constants for the first and second substitution reactions between zinc(II) complexes and imidazole, 1,2,3-triazole, L-histidine at pH 7.2 (0.025 M Hepes buffer) in the addition of 0.001 M NaCl at 295 K[64].

[ZnCl2(en)] 1mM imidazole 1,2,3- triazole L-histidine 295 -1 -1 k1 /M s 233 ± 15 301 ± 10 355 ± 20

2 295 - -1 -1 10 k-1 [Cl ]/M s - 0.31± 0.03 -

295 -1 -1 k2 /M s 82 ± 8 15.9 ± 0.3 49 ± 3

2 295 - -1 - 10 k-2 [Cl ]/M s 0.20 ± 0.01 0.057 ± 0.009

[ZnCl2(terpy)] 1mM imidazole 1,2,3- triazole L-histidine

295 -1 -1 k1 /M s 317 ± 18 401 ± 18 471 ± 23

2 295 - -1 -1 10 k-1 [Cl ]/M s 0.46 ± 0.06 - 0.441 ± 0.076

295 -1 -1 k2 /M s 84 ± 9 17 ± 1 118 ± 7

2 295 - -1 - 10 k-2 [Cl ]/M s - 0.039 ± 0.004 0.238± 0.020

92 Table 4. Second-order rate constants for the first and second substitution reactions between zinc(II) complexes and imidazole, 1,2,3-triazole, L- histidine at pH 7.2 (0.025 M Hepes buffer) in the addition of 0.010 M NaCl at 295 K[64].

[ZnCl2(en)] 10mM imidazole 1,2,3- triazole L-histidine

295 -1 -1 k1 /M s 202 ± 21 311 ± 18 195 ± 13

2 295 - -1 -1 10 k-1 [Cl ]/M s - 0.22 ± 0.06 -

295 -1 -1 k2 /M s 12 ± 1 7 ± 1 49 ± 5

2 295 - -1 - 10 k-2 [Cl ]/M s 0.024 ± 0.004 0.033 ± 0.003 0.051 ± 0.014

[ZnCl2(terpy)] 10mM imidazole 1,2,3- triazole L-histidine

295 -1 -1 k1 /M s 91 ± 1 199 ± 14 268 ±14

2 295 - -1 -1 10 k-1 [Cl ]/M s - 0.18 ± 0.05 0.72 ± 0.05

295 -1 -1 k2 /M s 12 ± 2 20 ± 2 33 ± 3

2 295 - -1 - 10 k-2 [Cl ]/M s 0.024 ± 0.006 0.028 ± 0.007 0.083± 0.009

The mechanism of interactions between the tetrahedral [ZnCl2(en)] and square- pyramidal [ZnCl2(terpy)] complexes with DNA constituent guanosine-5’-monophospate has been investigated at pH around 4.5 to prevent spontaneous hydrolysis. The 1H NMR spectra of the products of the substitution reaction were recorded for several weeks. Different pathways in the substitution reaction have been observed. The geometry of the complexes, e.g. tetrahedral or square-pyramidal, had great impact on rate of reactions. DFT calculations (B3LYP(CPCM)/-6-311+G**) have been used in combination with NMR spectroscopic data to solve the structures of formed complexes [66].

93 1 H-NMR data of reaction between [ZnCl2(en)] and 5’-GMP (Figure 38) exhibited upfield shift of all signals due coordination, after 48 hours.

Figure 38. NMR spectra of the reaction between [ZnCl2(en)] and 5’-GMP at

295 K, pD 4.5 in D2O: a) spectrum at initial time; b) spectrum obtained after 48 h[66].

In initial stage of the reaction, the singlet of H8 and the doublet of the H1’ proton from 5’-GMP appeared at 9.02 and 6.1 ppm, respectively, while the singlet of NH2 from coordinated ethylenediamine in [ZnCl2(en)] appeared at 5.14 ppm. After 48 h, upfield shifts of H8 (δ = 0.77 ppm) and H1’ δ = 0.11 ppm) signals were observed (Figure 38). These changes in the chemical shifts suggest that N7 nitrogen atom is a coordination site. The singlet of

NH2 at δ 5.14 ppm decreased in intensity while a new singlet appeared at δ 4.97 ppm after 48 h. After monitoring of the reaction during several weeks,

94 no further changes in spectrum were noticed. pD of the solution dropped slightly from 4.5 to 4.0, implying that substitution reached completion. The formed complex is very stable. The rate of substitution from this complex is lower than from [ZnCl2(terpy)], which could be a consequence of the changes in coordination sphere of tetrahedral zinc(II) complexes at the beginning of the reaction. It is proposed that an equilibrium between tetrahedral and square-pyramidal structure of Zn(II) complexes exists in solution and after substitution the geometry converts to tetrahedral. This could be the reason of lower reaction rate [66].

The mechanism of interaction between [ZnCl2(terpy)] and 5’-GMP also has been investigated under the same reaction conditions. The reaction reached completion for less than one minute, which was visible in NMR tube. The color of solution after addition of 5’-GMP in molar ratio 1:1 turns in white. 1H NMR spectra were recorded after 24 h, 48 h and during several weeks, but changes in spectrum have not been observed (Figure 39).

Figure 39. NMR spectrum of the reaction between [ZnCl2(terpy)] and [66] 5’-GMP at 295 K, pD 4.5 in D2O after 1 minute .

95 The singlet of H8 proton of coordinated 5’-GMP appeared at 8.70 ppm, while doublet of the H1’ proton was at 5.97 ppm. Corresponding to terpy ligand in spectrum six protons pattern have been observed. Doublet of the 6,6′′ appeared at 8.95 ppm, multiplet formed by covered signals of 3′,5′ protons, from middle pyridine, and 4,4′′ protons appeared in the range 8.49- 8.42 ppm, triplets of 4′ proton from middle pyridine ring and 5,5′′ protons were at 8.19 and 7.71 ppm, respectively. Doublet of 3,3′′ protons seems to be at the same position as H8 proton of 5’-GMP (the signal was broadened) (Figure 39) [66]. The protons in the aromatic region of the spectra all correspond to signals from known terpy ligand coordinated to metal ions as is expected [74].

Complex [ZnCl2(terpy)] reacted immediately with DNA constituent, the rate of nucleophilic substitution reaction is controlled by strong π-acceptor ability of the tridentate chelate 2,2′:6′,2”-terpyridine [66]. The electronic communication between three pyridine rings causes a decrease in electronic density on the zinc center due additional formation of π-back bonding and makes it more electrophilic and more reactive. According to results from our research [ZnCl2(terpy)] is very stable in solution makes hydrates very easy, .... but the Zn OH2 hydration bond is rather weak [64,75].

X-ray diffraction crystallography provides structural information in the solid state, while NMR spectroscopy as well as DFT-calculations (B3LYP/6- 31G*) are useful in structural elucidation in solution. Combination of these two methods was applied to the Zn-GMP-complexes in order to confirm whether the zinc(II) complexes with 5’-GMP ligand adopt square-pyramidal, trigonal-bipyramidal or tetrahedral geometries. The experimental 1H NMR data ﬁt with the DFT (B3LYP/6-31G*) calculated structures for

96 [ZnCl(en)(N7-GMP)] and [ZnCl(terpy)(N7-GMP)] complexes better than for [ZnCl2(en)(N7-GMP)] [66].

In order to confirm the geometry around the coordinated center, structural index τ [76] was calculated for both [ZnCl(terpy)(N7-GMP)] and [ZnCl(en)(N7-GMP)] complexes. For five-coordinated Zn(II) complex the structural index τ5 = (β – α )/60° (α and β are the two largest angles around the central atom) [76], which represents the relative amount of trigonality

(square-pyramid, τ5 = 0; trigonal-bipyramid, τ5 = 1) is 0.32. The coordination geometry around zinc ion could be best described as somewhat between square-pyramidal and trigonal-bipyramidal, more like leaned toward distorted square-pyramidal geometry. The tetrahedral geometry of

[ZnCl(en)(N7-GMP)] was confirmed by structural indexes τ4 [76c] and τ′4

[76d]. Both indexes have similar values but τ′4 better differentiates the examined structures. The structural index τ4 is 0.91 (square-planar, τ4 = 0; tetrahedral, τ4 = 1; τ4 = (360°- (α +β ))/(360°-2θ); where α and β are the two −1 1 largest angles around the central atom and θ = cos (− ⁄3) ≈ 109.5° is a tetrahedral angle), while τ′4 is 0.90 (τ′4 = (β – α )/(360°-θ) + (180°- β)/(180°-θ)). Thus on the basis of calculated structural index, it could be concluded that [ZnCl(en)(N7-GMP)] adopt a tetrahedral geometry with minor distortion [76].

Determined activation parameters for the substitution processes between

[ZnCl2(terpy)] and L-methionine at pH 7.38 support an associative mechanism A or Ia [75]. Chelate formation and pre-equilibrium were obtained in substitution process between [ZnCl2(terpy)] complex and glutathione [75]. At pH 7.38 GSH is deprotonated [39], formation of five- membered chelate ring is possible via O-carboxylate and N-ammine group from -glutamyl residue [77]. The rise and fall of the absorbance at 281 nm

97 as a function of time is characteristic for a reaction that involves an intermediate in the overall process during which reactant is transformed into product [75].

Generally, great influence on substitution processes of zinc(II) complexes with biomolecules has the hard-soft nature of the metal and bio-ligands, the versatility of coordination of the biomolecules and different geometrical structures of complex that can adopt in solution.

3.2. Cytotoxic activity of zinc(II) complexes

The cytotoxic activity of zinc(II) complexes were tested on human cancer cell lines (MDA-MB-231 and HCT-116) and normal human lung fibroblast (MRC-5) as healthy control cell line. Both zinc(II) complexes reduced viability of all tested cells (Figure 40). [ZnCl2(en)] showed strong, dose- dependent and time-dependent decrease of MDA-MB-231, HCT-116 and

MRC-5 cell viabilities. All tested doses with [ZnCl2(terpy)] significantly decreased the percentage of viable cells (Figure 40), without dose dependence. Time dependence was present in treatments of MDA-MB-231 and HCT-116 cells, and not on MRC-5 cell line [66].

Generally, tasted complexes significantly reduced MDA-MB-231, HCT-116 and MRC-5 cell viabilities. Significant cytotoxic and anticancer effect of

[ZnCl2(en)] complex hasn’t shown, while [ZnCl2(terpy)] was significantly cytotoxic on MDA-MB-231 after 72 h, and HCT-116 after 24 h (IC50 values in Table 5) [66]. The better cytotoxicity of [ZnCl2(terpy)] complex is correlated with its square-pyramidal structure. The planar terpyridine ligand could serve as an intercalator.

Table 5. Cytotoxic effects - IC50 values (μM) of zinc(II) complexes on HCT- 116, MDA-MB-231 and MRC-5 cell lines after 24 and 72 h exposure. Results are calculated upon percentages of viable cells[66].

complex IC50 MDA-МB-231 HCT-116 MRC-5 24 h 72 h 24 h 72 h 24 h 72 h Zn(en) 196.6 154.5 160.2 149.7 72.1 87.7 Zn(terpy) x 23.0 10 x x 94.0 x – undefined

It can be concluded that the nature of chelate ligands, terpy versus en, has an important role in cytotoxic activity. These results indicate that square- pyramidal complexes of zinc(II) with planar chelate ligand will have better anticancer properties compared to tetrahedral zinc(II) complexes which are susceptible to changes in coordination geometry in solution.

Figure 40. The effect of [ZnCl2(en)] (left) and [ZnCl2(terpy)] (right) complexes on MDA-MB-23, HCT-116 and MRC-5 cells viability after 24 and 72 h of exposure. All values are mean, n=6; percentages of viable cells [66].

100 3.3. Interactions of copper(II) complexes with biomolecules in correlation with HSAB principle

Clear understanding of complex formation reactions between copper(II) complexes and bio-relevant nucleophiles is still largely missing. Substitution behavior of Cu-complex compounds at physiological conditions is very complex due to the rather high molecular mobility and distortions of complex local symmetry and different geometrical structures. Adopted geometry of complex compounds conditionals different preferences toward bio-ligands. Thus, the square-pyramidal structure of Cu(II) in biological systems prefers O-carboxylate or carbonyl and N-imidazole donor bio- ligand, while tetrahedral prefers S-thiolate or thioether, N-imidazole [45]. As a result of the d9 electronic conﬁguration of copper(II) ions, an elongation of the axial-bound solvent molecules can be observed. Due to such distortion, the axial water molecules are weakly bound to the central atom and therefore can be more easily substituted [78]. The strong ligand field forces the metal ion into a different geometry and removes the dynamic Jahn-Teller effect a labilizing effect [78d]. The bulk of five-coordinate {Cu(terpy)(bipy)} and {Cu(terpy)(phen)} (terpy=2,2′:6′,2′′- terpyridine or derivative, bipy= 2,2′- bipyridine or derivative, phen=1,10-phenanthroline or derivative) complexes exhibiting ostensibly square-pyramidal geometries and also shows an additional interaction in the remaining axial site leading to six-coordinated products [78c].

Recently, we have investigated by different methods (UV-Vis, EPR, HPLC- MS, mole-ratio, etc.) the kinetics and mechanism of the reactions between square-planar [CuCl2(en)] and a square-pyramidal [CuCl2(terpy)] with bio-nucleophiles (inosine-5’-monophosphate (5’-IMP), guanosine-5’-

101 monophosphate (5’-GMP), L-methionine (L-met), glutathione (GSH) and DL-aspartic acid (DL-asp) as a function of entering nucleophile concentrations and temperature at pH 7.4 [79]. The main goals were: (i) to investigate the ligand-substitution reactions between the copper(II) complexes and biologically important nucleophiles under physiological conditions; (ii) to investigate the changes in the coordination geometry around Cu(II).

In the presence of an excess of chloride, the square-planar complex

[CuCl2(en)] exists as a pseudo octahedral complex with two axially and weakly-bound solvent ligands, these ligands are rapidly replaced/substituted 2- by chloride ions to form [CuCl4(en)] as a pre-equilibrium intermediate, while Geometry of [CuCl2(terpy)] complex have not changed under the existing experimental conditions [79]. The order of reactivity of the 2- investigated bio-relevant nucleophiles toward [CuCl4(en)] complex was:

GSH > 5’-GMP > 5’-IMP > DL-asp > L-met, while toward [CuCl2(terpy)] the order of reactivity is: DL-asp > L-met > GSH > 5’-GMP > 5’-IMP, for the first reaction step. Different order of reactivity of biomolecules toward

[CuCl2(en)] and [CuCl2(terpy)] complexes could be explained by different geometrical structures of complexes (octahedral and square-pyramidal, respectively) in the presence of chloride and their different preferences toward donor atoms of biomolecules.

Mass spectrum of [CuCl2(terpy)] in Hepes buffer has shown two new signals at m/z = 477.150 and m/z = 521.00, assigned to [CuCl(terpy)]+−Hepes fragments of coordinated Hepes buffer. These signals also appear in mass spectra of ligand-substitution reactions between [CuCl2(terpy)] and biomolecules in molar ratio 1:1 and 1:2 (see Figure 41). If we take into account molar concentrations of investigated complex (0.0001 M), ligands

102 (0.0001 or 0.0002 M) and Hepes buffer (0.025 M), it is understandable why the signals corresponding to the fragments of free and coordinated Hepes molecule are dominant in the mass spectra. Fragmentation occurs during ionization. In the process of ionization, chlorido ligands are substituted with Hepes fragments in the coordination sphere of the complex (see Figure 40) [79].

103

O N N N 6 7 1 NH 8 Cu N N Cl 9 3 N O N N N N N N Fragmentation NaO P O 5' Cu O O 1' ONa H H - CH3 Cu O O S O H H N 6 N N OH OH 7 1NH O- N 8 O Cl C10H11N4Na2O8P 9 3 C31H38CuN9O8P Exact Mass: 392.01 O N N Exact Mass: 758.19 C15H11Cl2CuN3 N 6 N Exact Mass: 365.96 Mol. Wt.: 392.17 5' 7 1 NH Mol. Wt.: 759.21 HO P O 8 Mol. Wt.: 367.72 O H C 1' 9 3 3 OH H H C H CuN O PS2- O N N H H 33 40 9 12 HO OH OH Exact Mass: 880.16 HO P O 5' N Mol. Wt.: 881.31 O 1' OH H H C8H18N2O4S Exact Mass: 238.1 H H OH OH O N Mol. Wt.: 238.3 HO S

O Cl N N

tation Cu Cl agmen N N Fr CH2 C25H31ClCuN5 N N Exact Mass: 499.16 Cu O Mol. Wt.: 500.55 S O- N N O N CH2

N F ra O- gm en tat ion Cl C23H27ClCuN5O4S Exact Mass: 567.08 N N Mol. Wt.: 568.56 C22H27ClCuN5O Cu CH3 Exact Mass: 475.12 Mol. Wt.: 476.48 N N

N OH Inten. 100

90 302,000

499,050 105,150 80

60 HEPES fragments

50 737,050

30 239,100

261,000 477,100

20 146,050

10 759,050 618,150

0 100 200 300 400 500 600 700 m/z

Figure 41. Mass spectra for the reaction between [CuCl2(terpy)] and 5’-IMP in molar ratio 1:1 at pH 7.4 (0.010 M NaCl 0.025 M Hepes buffer)[79].

104 The EPR spectrum of [CuCl2(en)] complex is a sum of two axially symmetric signals with rather close g values (line 1: gǁ =2.240 and g=

2.053, line 2: gǁ =2.207 and g= 2.047 at 300 K) and the ratio of the signals intensities decreases from 7.3 at 300 K to 5.6 at 110 K. Both signals are characterized by gǁ > g> 2.040 indicating that the Cu(II) center is in the dx2- y2 ground state, which is typical for a square-planar geometry [80]. The presence of two signals in the spectra is due to specific crystal packing caused by hydrogen bonding which gives rise to the disorder in the ethylenediamine ring [81]. In the case of [CuCl2(terpy)] complex a rhombic type spectrum with g3 > g2 > g1 (g3=2.187, g2=2.135 g1=2.074 at 300 K) was observed. For such systems the ratio of (g3 – g2)/(g2 – g1)=R is useful to distinguish the complex geometry. The five-coordinated [CuCl2(terpy)] complex shows value of R less than 1 (0.84 at 300 K and 0.88 at 110 K). It means that the ground state is predominantly dx2-y2 which is typical for a square-pyramidal geometry [80]. In solutions two factors affecting the EPR spectra can be observed: (i) mobility of the complexes varies the degree of spectral anisotropy, (ii) the structure of Cu(II) complexes might change be distorted [79].

The EPR spectrum of [CuCl2(en)] complex in water shows 6 lines: quartet with hyperfine splitting constant about 6.8 mT (67.4×10−4 cm−1) and two isotropic lines. Thus, a mixture of various conformations with rather complicated conformational dynamics is observed in the aqueous solution. In

0.010 M NaCl 0.025 M Hepes buffer the EPR spectrum of [CuCl2(en)]- complex consists of a quartet and an isotropic line. The EPR parameters of these spectral features are practically the same as ones in the distilled water. The spectrum does not exhibit any noticeable changes indicating the possible coordination of buffer to [CuCl2(en)] complex or a complex geometry distortion due to presence of buffer molecules. These results are in a good

105 agreement with the mass spectroscopy data. Likely the presence of buffer molecules in the solution (concentration is approximately 365 Hepes molecules per one Cu-complex) just slightly affects the complex mobility and conformational dynamics [79].

The addition of ligands can affect the EPR spectra in several ways: (i) ligand molecule might form a stable complex with [CuCl2(en)] or cause a distortion of existing complex, (ii) affect the solution conformational dynamics and complex mobility (this case a some kind of competition between ligand and Hepes molecules might be observed). The EPR spectra demonstrate that all ligands interact with [CuCl2(en)] resulting in the Aǁ constant changes and g shifts as well as the appearance of new isotropic lines. The [CuCl2(en)] exists as distorted octahedral where its axial positions are filled by weakly bonded water molecules which can be easily occupied by ligand molecules. According to EPR data L-met forms the most stable complex with

[CuCl2(en)] among the ligands that were considered (Figure 42).

106

Figure 42. Left: EPR spectrum of 0.0001 M [CuCl2(en)] complex solution in 0.010 M NaCl 0.025 M Hepes buffer, pH 7.4, at 27 °C. Right: EPR spectrum of 0.0001 M equimolar [CuCl2(en)]−L-met solution in 0.0010 M NaCl 0.025 M Hepes buffer, pH 7.4, at 27 °C [79].

The EPR spectrum of [CuCl2(terpy)] in water shows 6 lines: an axial type quartet due to hyperfine interaction between an electron spin and nuclear spin of the Cu and two isotropic lines without any resolved hyperfine structure. Such quartet is typical for square-pyramidal or octahedral stereochemistries. Since the value of Aǁ constant (7.5 mT or 74.8×10−4 cm−1) is rather low, the square-based pyramid should be the preferential geometry for the complex [79]. The EPR parameters of the axial type quartet of [CuCl2(terpy)] in 0.010 M NaCl 0.025 M Hepes buffer or any

[CuCl2(terpy)]−ligand solution are practically the same as ones in the distilled water. It means that [CuCl2(terpy)] complex is very stable and there are no significant changes in its square-pyramidal geometry in the presence of buffer or ligands molecules. Nevertheless, the EPR parameters of isotropic

107 lines are affected by addition of buffer or nucleophiles. In particular, the rather weak lines with g values about 2.136-2.139 might be attributed to

[CuCl2(terpy)]-Hepes complexation which observed in the mass spectroscopy measurements. Addition of ligands molecules also causes small but rather well pronounced changes in the spectra (see Figure 43). It means the possibility of ligand substitution reactions involving [CuCl2(terpy)] complex.

Figure 43. Left: EPR spectrum of 0.0001 M [CuCl2(terpy)] complex solution in 0.010 M NaCl 0.025 M Hepes buffer, pH 7.4, at 27 °C. Right: EPR spectrum of 0.0001 M 1:2 copper-complex:ligand [CuCl2(terpy)]−GSH solution in 0.010 NaCl 0.025 M Hepes buffer, pH 7.4, at 27 °C [79].

108

CONCLUSION

Ligand preference and possible coordination geometries of the metal center are important bioinorganic principles. Metal ligand preference is closely related to the hard–soft acid–base nature of metals and their preferred ligands. In biological systems where Ca(II) ions (hard acid) are frequently found coordinated with carboxylate (hard base), Fe(III) (hard acid) with either carboxylate or phenoxide (hard base), Cu(II) (intermediate acid) with the nitrogens of the imidazole ring (intermediate base) of histidine residues and cadmium (soft acid) with the sulfhydryl groups (soft bases) of proteins. Metallothioneins, a group of small proteins whose structures contain about 30 per cent cysteine, are believed to protect cells by complexing with soft toxic metals such as mercury(II) and lead(II).

The terms hard and soft refer to the availability and mobility of the electrons possessed by the acid or base. Soft species have electrons that are easily removed (relatively mobile) whilst hard species have electrons that are firmly held (not very mobile). Softness is associated with a low charge density while hardness is related to a high charge density. As is known, hard acid will have a high positive charge density and a small size whereas a soft acid would have a low positive charge density and a large size. A large number of species have been classified as hard and soft acids and bases using these definitions but, as expected with all such definitions, a number of borderline cases are also known.

The theory of hard and soft acids and bases (HSAB) has proven to be a useful tool in predicting the outcome of bionorganic substitution reactions.

109 According to this principle, electrophile such as complex compounds of transition metal ions react preferentially with donor atoms of biologically relevant nucleophiles of similar hardness or softness. The concept of hard and soft acids and bases can be used to predict the relative strengths of bonds in complexes. A bond formed between two atoms with the same degree of electron mobility would be stable, having an almost even distribution of electrons. However, a bond formed between atoms with widely differing degrees of electron mobility would be less stable. Consequently, strong bonds are formed between hard acids and hard bases, soft acids and soft bases or borderline acids with borderline. In aqueous solution, the stability of complexes formed by soft acids and Lewis bases has been found to be in decreasing order of stability: S > C > I > Br > Cl > F. The bonds between borderline Lewis acids and bases are generally formed between either oxygen, nitrogen or sulfur donor atoms depending of the coordination number i.e. geometrical structures of initial complexes in solution. The stability of the these complexes depends of steric hindrance and electronic factors. For example, the electronic configuration of Zn2+ is 3d10 with two electrons per orbital. In coordination compounds, there is no ligand field stabilization energy, and the coordination number is determined by a balance between bonding energies and repulsions among the ligands. Therefore, zinc complexes are kinetically labile.

Thus, platinum(II) and palladium(II) belong to soft acid and prefer soft bases. Kinetically are preferred reactions with sulfur donor biomolecules but more termodinamically stable are Pt-N or Pt-N products. On other hand zinc(II) and copper(II) are borderline hard/soft ions and readily complexes with ligands containing a range of donor atoms, e.g. hard O-, intermediate N- and soft S-donors according to coordination numbers. The extensive investigations were performed in order to define the mechanism of

110 interaction of potential antitumor complexes with biomolecules. HSAB principle contributes to the better understanding of interactions between complexes of selected metal ions and biomolecules and helps to future design of less toxic potential metal-based anticancer drugs. We hope that future researches in combination with theoretical knowledge will help in bionorganic/medicinal inorganic chemistry to to predict and clarify the mechanism of processes in cell level.

Non scholae, sed vitae discimus

111

112 REFERENCES

[1] Housecroft CE, Sharpe AG. Inorganic chemistry. 4th ed. Harlow, England; New York: Pearson; 2012. 1213 p. ISBN-13: 978-0273742753

[2] Katakis D, Gordon G. Mechanism of inorganic reactions. Wiley: New York; 1987. 416 p. ISBN-13: 978-0471842583

[3] Tobe ML, Burgess J, editors. Inorganic reaction mechanism. Longman: England; 1999, 688 p. ISBN-13: 978-0582236776

[4] Langford CH, Gray HB. Ligand substitution processes. 2nd ed. Benjamin: New York; 1974, 103p. ISBN: Unknown

[5] Wilkins RG. Kinetics and mechanism of reactions of transition metal complexes. 2nd thoroughly revised edition. Wiley-VCH Verlag GmbH; 2002. 465 p. ISBN: 3-527-28389-7

[6] Kotowski M, van Eldik R. in Inorganic High Pressure Chemistry, Kinetics and Mechanism, Ed., R. van Eldik, Elsevier, Amsterdam, 1986, ch. 1 and 3. ISBN: 978-0128128343

[7] van Eldik R, Asano T, Le Noble WJ. Activation and reaction volumes in solution. Chemical Reviwes. 1989:89(3):549-688.DOI:10.1021/cr00093a005

[8] a) Lippard SJ. New chemistry of an oldmolecule: cis-[PtCl2(NH3)2]. Science.1982:218:1075-1082. DOI: 10.1126/science.6890712 b) Todd RC, Lippard SJ. Structure of duplex DNA containing the cisplatin 1,2- 2+ {Pt(NH3)2} -d(GpG) cross-link at 1.77 A resolution. Journal of Inorganic Biochemistry. 2010:104(9):902-908. DOI: 10.1016/j.jinorgbio.2010.04.005;

[9] a) Rosenberg B, van Camp L, Krigas T. Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature. 1965:205:698-699. DOI:10.1038/205698a0 b) Rosenberg B, van Camp L,

113 Trosko JE, Mansour VH. Platinum compounds: a new class of potent antitumour agents. Nature. 1969:222:385-386. DOI: 10.1038/222385a0 c) Rosenberg B, van Camp L. The successful regression of large solid sarcoma 180 tumors by platinum compounds. Cancer Research. 1970:30:1799-1802 d) Rosenberg B, van Camp L, Grimley EB, Thomson AJ. The inhibition of growth or cell division in Escherichia coli by different ionic species of platinum(IV) complexes. Journal of Biological Chemistry 1967: 242:1347-1352.

[10] Jakupec MA, Galanski M, Keppler BK. Tumour-inhibiting platinum complexes state of the art and future perspectives. Reviews of physiology, biochemistry and pharmacology. 2003;146:1-53. DOI: 10.1007/s10254-002- 0001-x.

[11] Farrell N. Metal complexes as drugs and chemotherapeutic agents. In McCleverty JA, Meyer TJ, editors. Comprehensive coordination chemistry II. 2nd ed. Elsevier Ltd.; 2003. p.809-840. ISBN: 9780080437484

[12] Lippert B, editor. Cisplatin, chemistry and biochemistry of leading antitumor drugs. Wiley-VCH: Zuruch. 1999. 563 p. ISBN 3906390209

[13] Reedijk J. Why does cisplatin reach guanine-N7 with competing S- donor ligands available in the cell? Chemical Reviews. 1999; 99: 2499-2510. DOI: 10.1021/cr980422f

[14] Fuertes M A, Alonso C, Pérez JM. Biochemical modulation of cisplatin mechanisms of action: Enhancement of antitumor activity and circumvention of drug resistance. Chemical Reviews. 2003;103(3):645-662. DOI: 10.1021/cr020010d

[15] Martin RB. Platinum complexes: Hydrolysis and binding to N(7) and N(1) of purines. In: Lippert B, editor. Cisplatin, chemistry and biochemistry

114 of leading antitumor drugs. Wiley-VCH: Zurich. 1999. p. 183-206. ISBN 3906390209

[16] Legendre F, Chottard JC. Kinetics and selectivity of DNA‐platination. In: Lippert B, editor. Cisplatin, chemistry and biochemistry of leading antitumor drugs. Wiley-VCH: Zurich.1999. p. 223-245. ISBN 3906390209

[17] Rau T, van Eldik R. Mechanistic insight from kinetic studies on the interaction of model palladium(II) complexes with nucleic acid components. In: Sigel A, Sigel H, editors. Metal Ions in Biological Systems. Volume 31. Marcel Dekker Inc., New York; 1996. p. 339-378. ISBN 0-8247-9688-8

[19] Reedijk J. Improved understanding in platinum antitumour chemistry. Chemical Communications. 1996:801-806. DOI:10.1039/CC9960000801

[20] Hegmans A, Berners-Price SJ, Davies MS, Thomas DS, Humphreys AS, Farrell N. Long range 1,4 and 1,6-interstrand cross-links formed by a trinuclear platinum complex. Minor groove preassociation affects kinetics and mechanism of cross-link formation as well as adduct structure. Journal of the American Chemical Society. 2004:126(7):2166- 2180. DOI: 10.1021/ja036105u

[21] Kozelka J, Legendre F, Reeder F, Chottard JC. Kinetics aspects of interactions between DNA and platinum complexes. Coordination Chemistry Reviews, 1999:190-192:61-68. DOI: 10.1016/S0010-8545(99)00065-X

[22] Bugarčić ŽD, Soldatović T, Jelić R, Algueró B, Grandas A. Equilibrium, kinetic and HPLC study of the reactions between platinum(II) complexes and DNA constituents in the presence and absence of glutathione. Dalton Transactions. 2004:3869-3877. DOI:10.1039/B411168K

115 [23] Norman RE. Ranford JD, Sadler PJ. Studies of platinum(II) methionine complexes: metabolites of cisplatin Inorganic Chemistry. 1992:31(5):877– 888. DOI: 10.1021/ic00031a033

[24] Soldatović T, Bugarčić ŽD. Study of the reactions between platinum(II) complexes and L- Methionine in the presence and absence of 5'-GMP. Journal of Inorganic Biochemistry 2005:99(7):1472-1479. DOI: 10.1016/j.jinorgbio.2005.04.005

[25] Soldatović T, Čanović P, Čanović D, Bugarčić ŽD. Competitive reactions of L-Methionine and 5’-GMP towards platinum(II) complexes Medicinal Chemistry. 2005:1(6):547-561. DOI: 10.2174/157340605774598117

[26] Soldatović T, Vasić V, Bugarčić ŽD. Effects of micelles on the complex formation of [PtCl(dien)]+ with biologically relevant ligands. Bulletin of the Chemical Society of Japan. 2006:79(12):1889-1893. DOI: 10.1246/bcsj.79.1889

[27] Soldatović T, Shoukry M, Puchta R, Bugarčić ŽD, van Eldik R. Equilibrium and kinetic studies of the reactions between aqua(1-(2- aminoethyl)piperazine)palladium(II) and biologically relevant nucleophiles. European Journal of Inorganic Chemistry. 2009:15:2261–2270. DOI: 10.1002/ejic.200801229

[28] Summa N, Soldatović T, Dahlenburg L, Bugarčić ŽD, van Eldik R. The impact of different chelating leaving groups on the substitution kinetics of mononuclear PtII(dach)(X-Y) complexes. Journal of Biological Inorganic Chemistry. 2007:12(4):461-475. DOI: 10.1007/s00775-006-0200-z

[29] Connors KA.Chemical kinetics: The study of reaction rates in solution. VCH, Weinheim: New York. 1990. 480 p. ISBN 9781560810063

116 [30] Soldatović Т, Bugarčić ŽD, van Eldik R. Influence of the chloride concentration on ligand substitution reactions of [Pt(SMC)Cl2] with biologically relevant nucleophiles. Dalton Transactions 2009: 4526-4531. DOI: 10.1039/B822718G

[31] Soldatović T, Čanović P, Nikolić R, Jelić R, Bugarčić ŽD. Equilibrium and 1H NMR kinetic study of the reactions of dichlorido [S-Methyl-L- Cysteine(N,S)]platinum(II) complex with some relevant biomolecules. Journal of Solution Chemistry. 2009:38:57-71. DOI: 10.1007/s10953-008- 9346-3

[32] Kasparova J, Marini V, Najajreh Z, Gibson D, Brabec V. DNA binding mode of the cis and trans geometries of new antitumor nonclassical platinum complexes containing piperidine, piperazine or 4-picoline ligand in cell-free media. Relations to their activity in cancer cell lines. Biochemistry. 2003:42:6321-6332. DOI: 10.1021/bi0342315

[33] Qu Y, Appleton TG, Hoeschele JD, Farrell N. Cisplatin as synthon. Synthesis and characterization of triplatinum complexes containing three cis-

Pt(amine)2 units linked in a linear fashion. Inorganic Chemistry. 1993:32:2591-2593. DOI: 10.1021/ic00063a063

[34] Komeda S, Lutz M, Spek AL, Chikuma M, Reedijk J. New antitumor- active azole-bridged dinuclear platinum(II) complexes: synthesis, characterization, crystal structures, and cytotoxic studies. Inorganic Chemistry. 2000:39(19):4230-4236. DOI: 10.1021/ic000273v

[35] Komeda S, Lutz M, Spek AL, Yamanaka Y, Sato T, Chikuma M, Reedijk J. Journal of American Chemical Society. 2002:124(17):4738-4746. DOI: 10.1021/ja0168559

117 [36] Soldatović T, Jovanović S, Bugarčić ŽD, van Eldik R. Substitution behaviour of novel dinuclear Pt(II) complexes with bio-relevant nucleophiles. Dalton Transactions. 2012:41:876-884. DOI:10.1039/C1DT11313E

[37] a) Hofmann A, van Eldik R. Thermodynamic and kinetic study of the interaction between the Pt(II) centres in [Pt2(N,N,N′,N′-tetrakis(2- 4+ pyridylmethyl)diamine)(H2O)2] . Influence of the bridging ligand. Dalton Transactions. 2003:2979-2985. DOI:10.1039/B305174A b) Ertürk H, Hofmann A, Puchta R, van Eldik R. Influence of the bridging ligand on the substitution behaviour of dinuclear Pt(II) complexes. An experimental and theoretical approach. Dalton Transactions. 2007, 2295-2301. DOI:10.1039/B700770C c) Ertürk H, Maigut J, Puchta R, van Eldik R. Substitution behaviour of amine-bridged dinuclear Pt(II) complexes with bio-relevant nucleophiles. Dalton Transactions 2008:2759-2766. DOI: 10.1039/B718177A d) Ertürk H, Puchta R, van Eldik R. Synthesis, characterization, thermodynamic and kinetic properties of a new series of dinuclear PtII complexes. European Journal of Inorganic Chemistry. 2009:1331-1338. DOI: 10.1002/ejic.200801135

[38] Mambanda A, Jaganyi D, Hochreutther S, van Eldik R. Tuning the reactivity of chelated dinuclear Pt(II) complexes through a flexible diamine linker. A detailed kinetic and mechanistic study. Dalton Transactions. 2010:39:3595-3608. DOI:10.1039/B921687A

[39] Smith RM, Martel AE, Critical Stability Constants Vol. 6, 2nd Suppl., Plenum Press: New York, 1989. 640 p. ISBN 978-1-4615-6766-0

[40] Valckova E, Dudones LP, Bose RN. HPLC determination of binding of cisplatin to DNA in the presence of biological thiols: implications of

118 dominant platinum-thiol binding to its anticancer action. Pharmaceutical Research. 2002:19:124-131. DOI: 10.1023/A:1014268729658

[41] Arsenijević M, Milovanović M, Volarević V, Čanović D, Arsenijević N, Soldatović T, Jovanović S, Bugarčić ŽD. Cytotoxic properties of platinum(IV) and dinuclear platinum(II) complexes and their ligand substitution reactions with guanosine-5′-monophosphate. Transition Metal Chemistry. 2012: 37:481–488. DOI: 10.1007/s11243-012-9613

[42] Komeda S, Kalayda GV, Lutz M, Spek AL, Yamanaka Y, Sato T, Chikuma M, Reedijk J. New Isomeric Azine-Bridged Dinuclear Platinum(II) Complexes Circumvent Cross-Resistance to Cisplatin. Journal of Medicinal Chemistry. 2003:46(7):1210–1219. DOI: 10.1021/jm020004+

[43] Boulikas T, Pantos A. Designing platinum compounds in cancer: structures and mechanisms. Cancer Therapy. 2007:5:537–583

[44] Damin F, Xiaoliang Y, Xiaoyong W, Shouchun Z, Jiafei M, Jian D, Liping L, Zijian G. A dinuclear monofunctional platinum(II) complex with an aromatic linker shows low reactivity towards glutathione but high DNA binding ability and antitumor activity. Journal of Biological Inorganic Chemistry 2007:12:655–665. DOI: 10.1007/s00775-007-0214-1

[45] Roat-Malone RM. Bioinorganic chemistry: A short course. Hoboken, NJ: John Wiley & Sons Inc.; 2002. 348 p. ISBN 0-471-15976-X

[46] Bertini I, Gray HB, Stiefel EI, Valentine JS. Biological inorganic chemistry. Structure and reactivity. Sausalito, CA: University Science Books; 2007. 739 p. ISBN-13: 978-1891389436

[47] Costello LC, Franklin RB. Cytotoxic/tumor suppressor role of zinc for the treatment of cancer: an enigma and an opportunity. Expert Review of Anticancer Therapy. 2012:12(1):121-128. DOI: 10.1586/era.11.190

119 [48] Costello LC, Franklin RB. The clinical relevance of the metabolism of prostate cancer; zinc and tumor suppression: connecting the dots. Molecular Cancer. 2006:5:1-17. DOI: 10.1186/1476-4598-5-17

[49] Berg JM, Godwin HA. Lessons from zinc-binding peptides. Annual Review of Biophysics and Biomolecular Structure. 1997;26:57-371. DOI: 10.1146/annurev.biophys.26.1.357

[50] Jernigan R, Raghunathan G, Bahar I. Characterization of interactions and metal-ion binding sites in proteins. Current Opinion in Structural Biology. 1994;4:256-263. DOI: 10.1016/S0959-440X(94)90317-4

[51] Emami S, Hosseinimehr SJ, Taghdisi SM, Akhlaghpoor S. Kojic acid and its manganese and zinc complexes as potential radioprotective agents. Bioorganic & Medicinal Chemistry Letters. 2007:17(1):45-48. DOI: 10.1016/j.bmcl.2006.09.097 [52] a) Chohan ZH, Arif M, Sarfraz M. Applied Organometallic Chemistry. Metal‐based antibacterial and antifungal amino acid derived Schiff bases: their synthesis, characterization and in vitro biological activity. 2007:21:294- 302. DOI: 10.1002/aoc.1200 b) Singh VP, Katiyar A. Synthesis, structural studies and bio-activity of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes with p-amino acetophenone salicyloyl hydrazone. Journal of Coordination Chemistry. 2008:61(20):3200-3212. DOI: 10.1080/00958970802017646 [53] a) Nakayama A, Hiromura M, Adachi Y, Sakurai H. Molecular mechanism of antidiabetic zinc–allixin complexes: regulations of glucose utilization and lipid metabolism. Journal of Biological Inorganic Chemistry.2008:13(5):675-684. DOI: 10.1007/s00775- 008-0352-0 b) Sakurai H, Yoshikawa Y, Yasui H. Current state for the

120 development of metallopharmaceutics and anti-diabetic metal complexes. Chemical Society Reviews. 2008:37:2383-2392. DOI: 10.1039/b710347f [54] Huang Q, Pan Z, Wang P, Chen Z, Zhang X, Xu H. Zinc(II) and copper(II) complexes of β-substituted hydroxylporphyrins as tumor photosensitizers. Bioorganic & Medicinal Chemistry Letters. 2006:16:3030. DOI: 10.1016/j.bmcl.2005.02.094 [55] Bertini I, Luchinat C, Rosi M, Sgamellotti A, Tarantelli F. Tarantelli F. pKa of zinc-bound water and nucleophilicity of hydroxo-containing species. Ab initio calculations on models for zinc enzymes. Inorganic Chemistry. 1990:29:1460-1463. DOI: 10.1021/ic00333a004

[56] Williams RJP. Bio-inorganic chemistry: its conceptual evolution. Coordination Chemistry Reviews. 1990:100:573. DOI: 10.1016/0010- 8545(90)85020-S

[57] Jany, T, Moreth A, Gruschka C, Sischka A, Spiering A, Dieding M, Wang Y, Samo SH, Stammler A, Bogge H, von Mollard GF, Anselmetti D,

Glaser T. Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone. Inorganic Chemistry. 2015;54:2679–2690. DOI: 10.1021/ic5028465

[58] Chen HH, Kuo MT. Overcoming platinum drug resistance with copper- lowering agents. Anticancer Research. 2013;33:4157–4161. PMID: 24122978

[59] Ohrvik H, Thiele DJJ. The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy. Journal of Trace Elements in Medicine and Biology. 2014;31:178–182. DOI: 10.1016/j.jtemb.2014.03.006.

[60] P.J. Sadler. Inorganic Chemistry and drug design. Advances in Inorganic Chemistry. 1991;36:1-48. DOI: 10.1016/S0898-8838(08)60035-5

121 [61] Fricker SP. Metal based drugs: from serendipity to design. Dalton Transactions. 2007;43: 4903-4917. DOI: 10.1039/b705551j

[62] Manikandamathavan VM, Weyhermüller T, Parameswari RP, Sathishkumar M, Subramaniana V, Nair BU. DNA/protein interaction and cytotoxic activity of imidazole terpyridine derived Cu(II)/Zn(II) metal complexes. Dalton Transactions. 2014;43:13018-13031. DOI: 10.1039/C4DT01378F

[63] Manikandamathavan VM, Rajapandian V, Freddy AJ, Weyhermuller T, Subramanian V, Nair BU. Effect of coordinated ligands on antiproliferative activity and DNA cleavage property of three mononuclear Cu(II)-terpyridine complexes. European Journal of Medicinal Chemistry. 2012;57: 449-458. DOI: 10.1016/j.ejmech.2012.06.039

[64] Selimović E, Soldatović T. Impact of chloride concentration on ligand substitution reactions of zinc(II) complexes with biologically relevant nitrogen nucleophiles. Progress in Reaction Kinetics and Mechanism. 2018:43(3-4): 244-253. DOI: 10.3184/146867818X15319903829164

[65] Soldatović T, Selimović E. Kinetic studies of the reactions between dichlorido(1,2-diaminoethane)zinc(II) and biologically relevant nucleophiles in the presence of chloride. Progress in Reaction Kinetics and Mechanism 2018:43(1):53-61. DOI: 10.3184/146867818X15066862094897

[66] Soldatović T, Selimović E, Šmit B, Ašanin D, Planojević N, Marković S, Puchta R, Alzoubi B. Interactions of zinc(II) complexes with 5’-GMP and their cytotoxic activity. Journal of Coordination Chemistry. 2019. In press DOI: 10.1080/00958972.2019.1569229.

122 [67] Yoe JH, Jones AL. Colorimetric determination of iron with disodium- 1,2-dihydroxybenzene-3,5-disulfonate. Ind. Eng. Chem. Anal. Ed.(Analytical Chemistry). 1944:16(2):111-115. DOI: 10.1021/i560126a015

[68] Chriswell CD, Schilt AA. New and improved techniques for applying the mole ratio method to the identification of weak complexes in solution. Analytical Chemistry. 1975:47(9):1623-1629. DOI: 10.1021/ac60359a021

[69] Bock CW, Katz AK, Glusker JPJ. Hydration of zinc ions: A comparison with magnesium and beryllium ions. Journal of American Chemical Society. 1995:117(13): 3754-3765. DOI: 10.1021/ja00118a012

[70] Basolo F, Pearson RG. Mechanisms of Inorganic Reactions. 2nd ed, Wiley: New York, N.Y.1967. 715 p. ISBN: 978-0471055457

[71] Sigel H, Massoud SS, Corfu NA. Comparison of the extent of macrochelate formation in complexes of divalent metal ions with guanosine (GMP2-), Inosine (IMP2-), and adenosine 5'-monophosphate (AMP2-). The crucial role of N-7 basicity in metal ion-nucleic base recognition. Journal of American Chemical Society. 1994:116(7):2958-2971. DOI: 10.1021/ja00086a028

[72] Zhang J, Yang W, Piquemal J.-P. Ren P. Modeling structural coordination and ligand binding in zinc proteins with a polarizable potential. Journal of Chemical Theory and Computation. 2012:8(4):1314−1324. DOI: 10.1021/ct200812y

[73] Orpen A, Brammer L, Allen FH, Kennard O, Watson DG, Taylor R. Tables of bond lengths determined by X-Ray and neutron Diffraction. Part 1. Bond lengths in organic compounds. Journal of the Chemical Society, Perkin Transactions 2. 1989: S1-S83. DOI: 10.1039/p298700000s1

123 [74] a) Cummings SD. Platinum complexes of terpyridine: Interaction and reactivity with biomolecules. Coordination Chemistry Reviews. 2009:253(9-10):1495-1516. DOI: 10.1016/j.ccr.2008.12.009 b) Manikandamathavan VM, Weyhermüller T, Parameswari RP, Sathishkumar M, Subramaniana V, Balachandran UN. DNA/protein interaction and cytotoxic activity of imidazole terpyridine derived Cu(II)/Zn(II) metal complexes. Dalton Transactions. 2014:43: 13018-13031. DOI: 10.1039/C4DT01378F c) Ziessel R, Grosshenny V, Hissler M, Stroh C. cis-[Ru(2,2′:6′,2′′-terpyridine)(DMSO)Cl2]: Useful precursor for the snthesis of heteroleptic terpyridine complexes under mild conditions. Inorganic Chemistry. 2004:43:4262-4271. DOI: 10.1021/ic049822d

[75] Selimović E, Jeremić S, Ličina B, Soldatović T. Kinetics, DFT study and antibacterial activity of zinc(II) and copper(II) terpyridine complexes. Journal of the Mexican Chemical Society. 2018:62:1-21

[76] a) Addison AW, Rao TN, Reedijk J, van Rijn J. Verschoor GC. Synthesis, structure, and spectroscopic properties of copper(II) compounds containing nitrogen–sulphur donor ligands; the crystal and molecular structure of aqua[1,7-bis(N-methylbenzimidazol-2′-yl)-2,6- dithiaheptane]copper(II) perchlorate. Journal of the Chemical Society, Dalton Transactions. 1984:7:1349-1356. DOI: 10.1039/DT9840001349 b) Uhlenbrock S, Wagner R, Krebs B. Syntheses and characterization of novel tri- and hexa-nuclear zinc complexes with biomimetic chelate ligands. Journal of the Chemical Society, Dalton Transactions. 1996:18:3731-3736. DOI: 10.1039/DT9960003731 c) Yang L, Powell DR, Houser RP. Structural variation in copper(I) complexes with pyridylmethylamide ligands: structural analysis with a new four-coordinate geometry index, tau4. Dalton Transactions. 2007:9:955-964. DOI: 10.1039/b617136b

124 [77] Wiseman DA, Sharma S, Black SM. Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator.Biometals 2010:23(1):19-30. DOI: 10.1007/s10534-009-9263-y

[78] a) Shaban SY, Heinemann FW, van Eldik R. New trigonal‐bipyramidal II [Cu (pytBuN3)Cl2] complex: Synthesis, structure and ligand substitution behaviour. European Journal of Inorganic Chemistry.2009:3111-3118. DOI: 10.1002/ejic.200900302 b) Hugh Powell D, Merbach AE, Fabian I, Schindler S, van Eldik R. Evidence for a chelate-induced changeover in the substitution mechanism of aquated copper (II). Volume profile analyses of water exchange and complex-formation reactions. Inorganic Chemistry. 1994:33(20):4468-4473. DOI: 10.1021/ic00098a011 c) Constable EC, Housecroft CE, Price JR, Zampese JA. When five are six: the myth of five- coordinate copper(II) in supramolecular chemistry. CrystEngComm, 2010:12:3163-3171. DOI: 10.1039/C0CE00019A

[79] Selimović E, Komolkin AV, Egorov AV, Soldatović T. Substitution behavior of square-planar and square-pyramidal Cu(II) complexes with bio- relevant nucleophiles. Journal of Coordination Chemistry. 2018;71(7):1003- 1019. DOI: 10.1080/00958972.2018.1456656

[80] Harvey DA, Lock CJ. Dichloro(ethylenediamine)copper(II). Acta Crystallographica Section C. 1986:C42:799-801. DOI: 10.1107/S0108270186094490

[81] Hathaway BJ, Billing DE. The electronic properties and stereochemistry of mono-nuclear complexes of the copper(II) ion. Coordination Chemistry Reviews. 1970:5(2):143-207. DOI: 10.1016/S0010-8545(00)80135-6

125

126

127

128

129

130

131