synthesis and characterization of novel platinum … · 2018. 1. 8. · platinum complexes –...

SYNTHESIS AND CHARACTERIZATION OF NOVEL PLATINUM COMPLEXES – THEIR ANTICANCER

BEHAVIOUR

By

JOLANDA MYBURGH

Submitted in fulfilment of the requirements for the degree of

Magister Scientiae at the Nelson Mandela Metropolitan

University

January 2009

Supervisor: Prof JGH du Preez

To my mother..

i

ACKNOWLEDGEMENTS

I wish to express my sincere thanks to:

Prof. J.G.H. du Preez for his motivation, support and guidance.

Dr. Mauritz Wentzel and Malcolm Taylor from Shimoda Biotech and Dr. L. Fourie

from the University of Potchefstroom for the HPLC and mass spectroscopy work.

Debbie du Plessis-Stoman and Anzel Bredenkamp under the supervision of Dr.

M. van de Venter of the Biochemistry department of the NMMU, for the anticancer

testing.

Dr. Eric Hosten of the NMMU for the ICP analysis

Henk Schalekamp for his assistance in the laboratory.

Dr. M. Fernandes of the University of the Witwatersrand for the acquisition of the

crystallographic data.

Dr. B.J.A.M. van Brecht of the NMMU for the analysis of and the contributions

towards the solution and refinement of the crystal structures.

Prof. C.W. Mc Clelland of the NMMU for assisting in the molecular modelling.

Mrs Erica Wagenaar for her expertise in the formatting of this dissertation.

My colleagues, especially Yatish Jaganath, Lukas Oosthuizen, Marissa Louw and

Yolanda Bouwer for their support, guidance, chemical advice and/or assistance

with the experimental work.

My family and friends for all the moral support

God because by the Grace of God I Am What I Am

ii

CONTENTS

Page

ACKNOWLEDGEMENTS ........................................................................................ i LIST OF FIGURES ................................................................................................... vii LIST OF TABLES ..................................................................................................... xiiiii ABBREVIATIONS .................................................................................................... xvi SUMMARY……………………………………………………………………………..xvi

CHAPTER 1: INTRODUCTION

1 HISTORICAL BACKGROUND ................................................................. 1 1.2 PLATINUM COMPOUNDS COMMONLY USED ...................................... 3 1.3 CHEMICAL CHARACTERISTIC OF PLATINUM(II) AND PLATINUM(IV) 5 1.4 IMPORTANT FACTORS REGARDING THE ANTICANCER ACTION OF

PLATINUM COMPLEXES ........................................................................ 8 1.4.1 Kinetics ....................................................................................................... 8 1.4.2 Stereochemistry ......................................................................................... 10 1.4.3 S-N interaction (N7) .................................................................................... 10 1.4.4 Biocompatibility ......................................................................................... 13 1.5 RECENT DEVELOPMENTS IN PLATINUM ANTICANCER RESEARCH .... 14 1.6 OBJECTIVES OF THIS DISSERTATION ................................................. 15

CHAPTER 2: EXPERIMENTAL PROCEDURES

2 INTRODUCTION ....................................................................................... 17 2.1 ANALYSIS TECHNIQUES ........................................................................ 17 2.1.1 Elemental Analysis ..................................................................................... 17 2.1.2 Spectrophotometric Analysis ................................................................... 17 2.1.3 Solubility Studies ....................................................................................... 20 2.1.4 Crystallographic Analysis ......................................................................... 20 2.1.5 Cytotoxicity Studies ................................................................................... 21 2.1.6 Methods applied for the Ionization Studies ............................................. 22 2.2 PREPARATION OF STARTING MATERIALS ......................................... 24

iii

2.2.1 K2PtCl6 ......................................................................................................... 24 2.2.2 K2PtCl4 ......................................................................................................... 24 2.2.3 K2Pt(C2O4)2.2H2O ........................................................................................ 25 2.3 SYNTHESIS OF THE (NS)-DONOR LIGANDS ........................................ 25 2.3.1 1-Methyl-2-methylthioalkylimidazole ........................................................ 25 2.3.1.1 1-Methyl-2-methylthiomethylimidazole(mmtmi) ...................................... 25 2.3.1.2 1-Methyl-2-methylthioethylimidazole (mmtei) .......................................... 27 2.3.1.3 1-Methyl-2-methylthiopropylimidazole (mmtpi) ....................................... 27 2.3.2 1-Butyl-2-methylthiomethylimidazole(bmtmi): ........................................ 28 2.4 SYNTHESIS OF PLATINUM(II) COMPLEXES WITH BIDENTATE (NS)-

LIGANDS .................................................................................................. 28 2.4.1 Synthesis of the (NS)- haloplatinum(II) complexes ................................. 29 2.4.1.1 Preparation of the (NS)-iodoplatinum(II) complexes ............................... 29 2.4.1.2 Preparation of the (NS)-chloroplatinum(II) complexes ........................... 29 2.4.1.3 Preparation of the (NS)-bromoplatinum(II) complexes ........................... 30 2.4.2 Synthesis of the (NS)-oxalatoplatinum(II) complexes ............................. 31 2.5 SYNTHESIS OF PLATINUM(II) COMPLEXES WITH N-DONOR

LIGANDS – MONODENTATE AND BIDENTATE .................................... 32 2.5.1 Synthesis of the aminehalo- and oxalatoplatinum(II) complexes .......... 32 2.5.2 Synthesis of platinum(II) complexes of the type PtL1L2X2 (L1 and L2

are two different monodentate amines) ................................................... 33 2.6 ELEMENTAL ANALYSIS OF PLATINUM(II) COMPLEXES .................... 34 2.7 ANTICANCER ACTION OF THE (NS)-HALOPLATINUM(II) COMPLEXES . 36 2.8 SYNTHESIS OF PLATINUM(IV) COMPLEXES ....................................... 38 2.8.1 Synthesis of Platinum(IV) complexes: …………………………………38

CHAPTER 3: THE (NS)-HALOPLATINUM(II) COMPLEXES

3 INTRODUCTION ....................................................................................... 40 3.1 SYNTHETIC METHODS AND THE IMPLICATIONS THEREOF ............. 42 3.1.1 Introduction ................................................................................................ 42 3.1.2 Method A - Direct ligand exchange from K2PtCl4 .................................... 42 3.1.3 Method B - Silver Method .......................................................................... 44 3.2 (NS)-PLATINUM(II) COMPLEXES - THEIR PHYSICAL PROPERTIES .. 46

iv

3.2.1 Infrared Spectroscopy ............................................................................... 46 3.3 MASS SPECTROMETRY .......................................................................... 50 3.3.1 (NS)-iodoplatinum(II) complexes .............................................................. 50 3.3.2 (NS)-bromo- and chloroplatinum(II) complexes ...................................... 56 3.4 IONIZATION OF THE (NS)-HALOPLATINUM(II) COMPLEXES .............. 61 3.5 ANTICANCER BEHAVIOUR .................................................................... 66 3.6 SOLUBILITY STUDIES ............................................................................ 67 3.7 CONCLUSION .......................................................................................... 67 3.8 APPENDIX ................................................................................................. 70

CHAPTER 4: THE (NS)-DICARBOXYLATOPLATINUM(II) COMPLEXES

4 INTRODUCTION ....................................................................................... 70 4.1 SYNTHETIC METHODS AND THE IMPLICATIONS THEREOF ............. 71 4.2 (NS)-OXALATOPLATINUM(II) COMPLEXES THEIR PHYSICAL

PROPERTIES ........................................................................................... 74 4.2.1 Infrared Spectroscopy ............................................................................... 74 4.2.2 Mass Spectrometry .................................................................................... 75 4.3 IONIZATION STUDIES .............................................................................. 75 4.4 ANTICANCER ACTION ............................................................................ 77 4.4.1 Final comments .......................................................................................... 80 4.5 CONCLUSION ........................................................................................... 82 4.6 APPENDIX ................................................................................................ 82

CHAPTER 5: PLATINUM(II) COMPLEXES WITH N-LIGAND

5 INTRODUCTION ....................................................................................... 86 5.1 NON-LEAVING GROUPS ......................................................................... 88 5.2 AMINEIODOPLATINUM(II) COMPLEXES ............................................... 89 5.3 AMINEBROMOPLATINUM(II) COMPLEXES ........................................... 94 5.4 AMINECHLOROPLATINUM(II) COMPLEXES ......................................... 97 5.5 AMINEOXALATOPLATINUM(II) COMPLEXES ........................................ 102 5.6 INFRARED SPECTROSCOPY ................................................................. 109

v

5.7 COMPARISON OF IONIZATION BEHAVIOUR ....................................... 114 5.8 ANTICANCER BEHAVIOUR .................................................................... 118 5.9 SOLUBILITY STUDIES ............................................................................ 121 5.10 CONCLUSION .......................................................................................... 121

vi

CHAPTER 6: MONONITRO PLATINUM(IV) COMPLEXES

6 INTRODUCTION ....................................................................................... 123 6.1 THE REACTION OF THE (NS)-PLATINUM(II) COMPLEXES WITH NO2 123 6.2 EFFORTS TO SYNTHESIZE PLATINUM(IV) ANALOGUES OF THE

(NS)-OXALATOPLATINUM(II) COMPLEXES .......................................... 124 6.3 EFFORTS TO SYNTHESIZE PLATINUM(IV) ANALOGUES OF THE

AMINEPLATINUM(II) COMPLEXES ........................................................ 126 6.4 MECHANISM OF OXIDATION ................................ ……………………130 6.5 CONCLUSION ..................................................................................... 133

REFERENCES

vii

LIST OF FIGURES

Page

Figure 1:1 Structure of cisplatin ............................................................................ 1

Figure 1:2 Platinum complex of the type PtA2X2Y2 ............................................... 2

Figure 1:3 Structure of carboplatin ........................................................................ 3

Figure 1:4 Structure of oxaliplatin ......................................................................... 4

Figure 1:5 Structures of Lobaplatin (a) and Nedaplatin (b) ................................... 5

Figure 1:6 The dissociative (SN1) and associative (SN2) mechanisms ................. 6

Figure 1:7 Structure of the amino acids methionine (a) and cysteine (b) ............. 8

Figure 1:8 Space filling model (a) and charge distribution values (b) of

adenine. ............................................................................................... 11

Figure 1:9 Space filling model (a) and charge distribution values (b) of guanine .. 11

Figure 1:10 Space filling model(a) and charge distribution values (b) of cysteine .. 12

Figure 2.1 Experimental set up for the synthesis of the platinum(IV) complexes

Figure 3:1 (NS)-Donor Ligands ............................................................................. 41

Figure 3:2 Full Scan Mass Spectra of J13.2 (FAB method) .................................. 43

Figure 3:3a Full Scan Mass Spectra (electronspray method) of J13.2D in

positive ion mode (top) and negative ion mode (bottom) –

(M=monomolecular species and A=auto-ionized species). .................. 43

Figure 3:3b Possible structures of the detected ions - auto-ionized and

monomolecular species ....................................................................... 42

Figure 3:4 Full scan mass spectrum of J13.2 (FAB method) ................................ 45

Figure 3:5 Full scan mass spectrum of J13.2 (electron spray method) in

positive (top) and negative (bottom) ion modes ................................... 46

Figure 3:6 Infrared spectrum of ligand J7 showing the characteristic infrared

stretches .............................................................................................. 47

Figure 3:7 Infrared spectrum of J7.2 showing characteristic stretches of the

(NS)-haloplatinum(II) complexes ......................................................... 47

Figure 3:8 Full Scan mass spectrum of J13.5 (FAB method) ................................ 51

Figure 3:9 Full Scan mass spectra of J13.5 in positive (top) and negative

(bottom) ion modes. Possible structures of detected ions are given

below spectra ....................................................................................... 52

viii


(bottom) ion modes. (M=monomolecular species and A=auto-

ionized) ................................................................................................ 53


(bottom) ion modes. (M=monomolecular species and A=auto-

ionized species.) The possible structures of some of the detected

ions are given below the spectra. ........................................................ 54


(bottom) ion mode. (M=monomolecular species and A=auto-

ionized). Possible structures of some of the detected ions are given

below spectra ....................................................................................... 55

Figure 3:13a Full Scan mass spectra of J7.2 in positive (top) and

negative(bottom) ion modes ............................................................... 56

Figure 3:13b Possible structures of some of the detected ions ................................. 53


(bottom) ion modes. Possible structures of some of the detected

ions are given below spectra ............................................................... 57

Figure 3:15 Full Scan mass spectra of J13.3 in positive (top) and

negative(bottom) ion modes .............................................................. 58

Figure 3:16 Full Scan mass spectra of J5.2 in positive (top) and

negative(bottom) ion modes. Possible structures of detected ions

are given below spectra ....................................................................... 59



below spectra ....................................................................................... 60

Figure 3:18 Full Scan mass spectra of J7.2 showing ionization behaviour in

CH3CN and H2O at room temperature at T= 0 and 80ºC at T = 18 ...... 61

Figure 3:19 Full Scan mass spectra of J5.2 showing ionization behaviour in

CH3CN and H2O at room temperature at T= O and 80ºC at T = 18 ..... 62

Figure 3:20 Effect of the ligand on the ionization rate for the molecular species

[M+NH4] + for (a) the chloro, (b) bromo and (c) iodo species ............... 63

Figure 3:21 Effect of the leaving groups on the ionization rate for the molecular

species [M+NH4] + for (a) J7, (b) J5 and (c) J12. ................................ 64

Figure 3:22: Effect of the leaving groups on the hydrolysis rate for the

ix

monocationic species [ M-X+S]+ for (a) J7, (b) J5 and (c) J12. ........... 65

Figure 4:1 Graph detailing the drop in cisplatin concentration in water (37°C)

over time .............................................................................................. 70

Figure 4:2 Graph indicating the stability in carboplatin concentration in water

(37°C) over time ................................................................................. 70

Figure 4:3 Graph demonstrating the stability in oxaliplatin concentration in

water (37°C) over time ......................................................................... 71

Figure 4:4 Full Scan Mass Spectrum of J7.1 synthesized by the direct method

(method A) in positive and negative ion modes ................................... 72

Figure 4:5 Full Scan Mass Spectrum of J7.1 synthesized by the silver method

(method B) in positive and negative ion modes. Possible structures

of detected ions are given below spectra ......................................... 73

Figure 4:6 LC-UV separation in H2O of J7.1 ......................................................... 74


(method B) in positive mode in CH3CN and H2O at room

temperature at T=O and 80ºC at T= 60Hr. .......................................... 76


(method B) in positive mode in CH3CN and H2O at room

temperature at T=0 and 80ºC at T=60Hr ............................................ 76

Figure 4:9 Anticancer action of J7.1; percentage inhibition as a function of

concentration of J7.1 in μM for HT29 ................................................... 77


concentration of J7.1 in μM for HeLa ................................................... 78


concentration of J7.1 in μM for MCF7 .................................................. 78

Figure 4:12 2-methylthiomethylpyridine .................................................................. 80

Figure 4:13 Full scan mass spectra of J5.1 in positive ion mode (top) and

negative ion modes (bottom) ............................................................... 82

Figure 4:14 Full scan mass spectra of J12.1 synthesised via Method A in


Figure 4:15 Full Scan mass spectra of J12.1 synthesised via Method B in

positive (top) and negative(bottom) ion modes .................................... 83

Figure 4:16 Full scan mass spectra of J13.1 synthesised via Method A in


x

Figure 4:17 Full scan mass spectra of J13.1 synthesised via Method B in


Figure 4:18 Full scan mass spectra of J12.1 synthesized by method b in

positive ion mode in CH3CN and H2O at T=0 and at 80°c at T= 60Hr .. 84

Figure 4:19 Full scan mass spectra of J13.1 synthesized by method B in positive

ion mode in CH3CN and H2O at T=0 and at 80°C at T= 60Hr .............. 85

Figure 5:1 Structure of cisplatin and its hydrolysis products ................................. 86

Figure 5:2 Structure of picoplatin ......................................................................... 87

Figure 5:3 Full scan mass spectrum of JMY13.5 in positive (top spectrum) and

negative (bottom spectrum) ion modes. Possible structures of some

of the detected ions are given below spectra ....................................... 90



of the ions are given below the spectra. ............................................... 91

Figure 5:5 Full scan mass spectra of JMY15.5 in positive (top spectrum) and

negative (bottom spectrum) ion mode. Possible structures of some

of the detected ions are given below spectra. ...................................... 92

Figure 5:6 Full scan mass spectrum of JMY16.5 in positive ion mode. Possible

structures of some of the detected ions are given below spectra ........ 93

Figure 5:7 Full scan mass spectrum of JMY17.5 in positive ion mode and

negative ion mode (M = molecular species and S = symmetrical

species). Possible structures of some of the detected ions are given

below spectra ....................................................................................... 94




Figure 5:9 Full scan mass spectra of JMY14.3 in positive and negative ion

mode. Possible structures of detected ions are given below spectra ... 96

Figure 5:10 Full scan mass spectra of JMY15.3 in CH3CN/H2O solution, positive

(top spectrum) and negative (bottom spectrum) ion mode ................... 97





xi


of the ions are given below spectra ..................................................... 99





structures of detected ions are given below spectrum ......................... 101




Figure 5:16 Full scan mass spectra of JMY14.1 in H2O, positive (top spectrum)

and negative (bottom spectrum) ion modes. Possible structures of

detected ions are given below spectra ................................................. 103





structures of some of the detected ions are given below spectra ........ 105



of the detected ions are given below spectra ................................. 106

Figure 5:20 LC-UV separation of JMY17.1 in 25% CH3CN (JMY17.1 at peak

4.30) ..................................................................................................... 107

Figure 5:21 LC-MS separation of JMY17.1 in 25% CH3CN solution(positive ion

mode) ................................................................................................... 107

Figure 5:22 LC-MS separation of JMY17.1 in 25% CH3CN solution (negative ion

mode) ................................................................................................... 108

Figure 5:23 (a) ball and stick model of JMY15.2, lowest energy conformer. (b) an

alternative ball and stick model for JMY15.2 ........................................ 109

Figure 5:24 Infrared spectrum of JMY13.2 showing the characteristic stretches

of amineplatinum(II) complexes ........................................................... 110

Figure 5:25 Full scan mass spectra of JMY13.5 in H2O/CH3CN solution, showing

ionization behaviour at different time intervals at 37°C ........................ 114


xii




Figure 5:28 (a) Effect of the leaving group on the ionization rate for the

molecular species [M+NH4] + and (b) the monocationic species [M-

X+S] for the chloro, bromo and iodo species; X = Cl, Br or I and S =

CH3CN. ................................................................................................ 116

Figure 5:29 A comparison between the extent of ionization of the (NN)- and

(NS)-complexes for the monocationic species [M-X+S]+ ( X=Cl, Br or

I and S=CH3CN). (a) Comparison between the dichlorocomplexes,

(b) the dibromo complexes. and (c) the diodo complexes .................... 117

Figure 6:1 Structure of CPA-7 ............................................................................... 123

Figure 6:2 Full Scan mass spectra of J12.1.4 in positive (top) and negative


below spectra ....................................................................................... 125

Figure 6:3 Mass spectra of JMY15.1.4 in positive ion mode (top) and negative

ion mode (bottom). Possible structures of some of the detected ions

are given below spectra ....................................................................... 127

Figure 6:4 LC-UV separation of sample JMY15.1.4 in 25% CH3CN ..................... 127

Figure 6:5 Full scan mass spectra of JMY16.1.4 in positive ion mode (top) and

negative ion mode (bottom). Possible structures of some of the


Figure 6:6 Full scan mass spectra of JMY13.1.4 in positive ion mode (top) and

negative ion mode (bottom). Possible structures of some of the


Figure 6:7 Trans-aqua(nitrosyl)tetranitroplatinate(IV) anion .................................. 130

Figure 6:8 Trans-dinitro-trans-dichloro-trans-nitrosylchloroplatinum(IV) ion ......... 131

Figure 6:9

X-ray crystallographic structure of platinum(IV) analogue of

oxaliplatin ............................................................................................. 132

Figure 6:10 Possible stucture of JMY15.1.4 ........................................................... 132

xiii

LIST OF TABLES

Page

Table 2:1 General ICP settings used ................................................................... 18

Table 2:2 Colour and yields of the (NS)- iodoplatinum(II) complexes . ................ 29

Table 2:3 Colour and yields of the (NS)-chloroplatinum(II) complexes prepared

via method A ........................................................................................ 30

Table 2:4 Colour and yields of the (NS)-chloroplatinum(II) complexes prepared

via method B ........................................................................................ 30

Table 2:5 Colour and yields of the (NS)-bromoplatinum(II) complexes

prepared via method B ......................................................................... 31

Table 2:6 Colour and yields of the (NS)-oxalatoplatinum(II) complexes

prepared via method A ......................................................................... 31

Table 2:7 Colour and yields of the (NS)-oxalatoplatinum(II) complexes

prepared via method B ......................................................................... 32

Table 2:8 Colour and yields of the aminehaloplatinum(II) complexes prepared

via method B. ....................................................................................... 32

Table 2:9 Colour and yields of the amine-oxalatoplatinum(II) complexes

prepared via method B. ........................................................................ 33

Table 2:10 Elemental analysis of the (NS)-chloroplatinum(II) complexes via

method A ............................................................................................. 34

Table 2:11 Elemental analysis of the iodoplatinum(II) complexes ............................ 34

Table 2:12 Elemental analysis of the chloroplatinum(II) complexes via method B 35

Table 2:13 Elemental analysis of the bromoplatinum(II) complexes via method

B .......................................................................................................... 35

Table 2:14 Elemental analysis of the oxalatoplatinum(II) complexes via method A ..... 36

Table 2:15 Elemental analysis of the oxalatoplatinum(II) complexes via method B ..... 36

Table 2:16 Cell growth inhibition assay results for (NS)-haloplatinum(II)

complexes on HT29 (colon cancer) cell lines at two different

concentrations in μM. ........................................................................... 36


complexes on Hela (cervical cancer) cell lines at two different

concentrations in μM ............................................................................ 37

xiv


complexes on MCF7 (breast cancer) cell lines at two different


Table 2:19 Chemicals used ................................................................................... 39

Table 3:1 Infrared data for the (NS)-donor ligands .............................................. 48

Table 3:2 Infrared data for complexes of J5 ........................................................ 48

Table 3:3 Infrared data for complexes of J7 ....................................................... 48

Table 3:4 Infrared data for complexes of J12 ..................................................... 49

Table 3:5 Infrared data for complexes of J13 ..................................................... 49

Table 3:6 The ν(C-N), ν(CH2S) def and ν(CH2S) wag shifts of the (NS)-

chloroplatinum(II) complexes ............................................................... 50

Table 3:7 Summary of the major ion clusters (pos ion mode) observed in the

mass spectra of the samples in CH3CN and water and in the

aqueous samples after ≥ 18Hr at 80ºC ................................................ 66

Table 3:8 Solubility of the (NS)-haloplatinum(II) complexes ................................ 67

Table 4:1 Infrared data for the (NS)-oxalatoplatinum(II) complexes .................... 74

Table 4:2 The ν(CH2S) def and ν(CH2S) wag shifts of the (NS)-

oxalatoplatinum(II) complexes ............................................................. 75

Table 4:3 IC50 values obtained for J7.1 and cisplatin ......................................... 79

Table 4:4 Cell growth inhibition assay results for (NS)-oxalatoplatinum(II)

complexes on HT29 (colon cancer) cell lines using two different

complex concentrations. ...................................................................... 79


complexes on HeLa (cervical cancer) cell lines using two different

complex concentrations ....................................................................... 79


complexes on MCF7 (breast cancer) cell lines using two different

complex concentrations ....................................................................... 79

Table 5:1 Infrared data for amine ligands ............................................................ 110

Table 5:2 Infrared data for aminehaloplatinum(II) complexes ............................. 111

Table 5:3 Infrared data of the amine-oxalatoplatinum(II) complexes ................... 112

Table 5:4 The ν(NH2-stretch) ν(NH2-def) and ν(C-N) shifts of the

amineplatinum(II) complexes ............................................................... 113

xv

Table 5:5 Cell growth inhibition assay results for aminehaloplatinum(II)

complexes on the HT29 (colon cancer) cell line at two different

concentrations in μM. ........................................................................... 118


complexes on the HeLa (cervical cancer) cell line at two different



complexes on the MCF7 (breast cancer) cell line at two different

concentrations in µM ............................................................................ 119

Table 5:8 Cell growth inhibition assay results for amine-oxalatoplatinum(II) -

complexes on the MCF7 (breast cancer) cell line at two different

concentrations in μM ............................................................................. 120

Table 5:9 Cell growth inhibition assay results for amine-oxalatoplatinum(II)

complexes on the HT29 (colon cancer) cell line at two different


Table 5:10 Cell growth inhibition assay results for amine-oxalatoplatinum(II)

complexes on the HeLa (cervical cancer) cell line at two different


Table 5:11 Solubility of the amineplatinum(II) complexes ...................................... 121

xvi

ABBREVIATIONS

mmtmi: 1-methyl-2-methylthiomethylimidazole

mmtei: 1-methyl-2-methylthioethylimidazole

mmtpi: 1-methyl-2-methylthiopropylimidazole

bmtmi: 1-butyl-2-methylthiomethylimidazole

xvii

SUMMARY

In this dissertation novel non-leaving groups were employed to synthesize

platinum complexes which can contribute to the understanding or improvement of

anticancer action. These complexes basically consist of (NS)-chelate and

amineplatinum complexes.

Bidentate (NS)-donor ligands were used as non-leaving ligands in the syntheses

of platinum(II) complexes with iodide, chloride, bromide and oxalate anions as

leaving groups. These complexes were synthesized and studied since many

questions regarding the interaction of sulfur donors and platinum still exists.

These relate to thermodynamic and kinetic factors and their influence on

anticancer action. In this dissertation the properties of novel platinum(II)

complexes of a bidentate ligand having an aromatic nitrogen-donor atom in

combination with a thioethereal sulfur atom capable of forming a five membered

ring with platinum(II) were studied. The general structure of the (NS) -ligands

used were N-alkyl-2-methylthioalkyl imidazole. Alkyl groups used were methyl,

ethyl and propyl.

Although amine complexes of platinum have been extensively studied there are

some new aspects of these that are worthwhile investigating. In this dissertation

amines having planar attachments which will be at an angle with the coordination

plane viz. benzylamine and amines having cyclic aliphatic groups namely

cyclopropyl and cyclohexyl were investigated.

Some of the (NS) - and amineplatinum(II) complexes were oxidised to their

mononitroplatinum(IV) analogues . The motivation for the synthesis of these

complexes was the greater kinetic stability of platinum(IV) and recent research

has shown that a specific type of platinum(IV) compound shows suitable

properties as an anticancer agent.

These complexes were characterised by a variety of spectral means (IR, NMR,

mass spectroscopy) as well as elemental analysis, solubility determinations,

thermal analysis (TGA), ionization studies and finally their anticancer behaviour

towards three different cell lines(Hela, MCF 7, Ht29) and in this process they

xviii

were compared to the behaviour of cisplatin as a reference. A few have shown

promising anticancer behaviour.

1

| 1

CHAPTER 1: INTRODUCTION

1 HISTORICAL BACKGROUND

The interest in the pharmacological properties of platinum compounds was

initiated by Rosenberg’s accidental discovery of the inhibition of cell-division by

platinum complexes in 1965.1 It was found that the complexes responsible for the

effects were cis-diamminedichloroplatinum(II) (cis-PtCl2(NH3)2, cisplatin) and cis-

diamminetetrachloroplatinum(IV) (cis-PtCl4(NH3)2). 2,3

H3N

H3N Cl

Cl

Pt

Figure 1:1 Structure of cisplatin

Cisplatin is effective in the treatment of a broad range of solid tumours and has

become one of the most widely used anticancer drugs since 1971 when it entered

clinical trials for the first time.4,5

Although it is commonly used cisplatin has several side effects. Side effects of

cisplatin include cumulative peripheral neuropathy, ototoxicity, hair loss, nausea

and vomiting.

The presence of the chloride anions as leaving

ligands makes cisplatin biologically compatible with the body. In blood and other

body fluids chloride is found in different concentrations.

6 Cisplatin has become the prototype for research and development

of platinum-based antitumour drugs with improved pharmacological properties

and a reduction of negative effects commonly found with cisplatin. The bulk of

these compounds is uncharged cis-configured square planar platinum(II)

complexes with the general formula cis-PtA2X2 where A2 is either two amines or a

diamine as neutral ligands and X2 either a monodentate or bidentate anionic

leaving group such as a halo or a dicarboxylato group. Platinum(IV) complexes of

the form PtA2X2Y2 are also undergoing clinical trials,.7 Y2 being two other

2

| 2

monodentate anionic leaving groups.

A

A X

X

Pt

Y

Y

Figure 1:2 Platinum complex of the type PtA2X2Y2

The only differences between the two types of anticancer platinum complexes are

the higher oxidation state and the two axial leaving groups. Platinum(IV) is less

reactive than platinum(II) and platinum(IV) drugs can possibly be administrated

orally. However these compounds are reduced to platinum(II) in the body

accompanied by the loss of the two axial ligands and thus rather than being seen

as a new type of drug it can be seen as prodrugs.6 Research has shown that

trans-diamminedichloroplatinum(II) (transplatin) is less effective than cisplatin.

Transplatin is kinetically more reactive than cisplatin and more susceptible to

deactivation. 8

A major limitation in the therapeutic success of cisplatin and its derivatives is the

fact that some cancer cells become resistant to the drugs.9,10 Efforts are being

made to overcome resistance. One of the main reasons cells become resistant to

cisplatin is that insufficient amounts of cisplatin reach and bind to the DNA and

this leads to failure of cell death which takes place after binding of platinum to

DNA.6 Reduced platinum accumulation and cytoplasmic detoxification by

glutathione and / or metallothioneins are the most important reasons why

inadequate amounts of platinum reach DNA. Mechanisms within the cell can also

lead to platinum resistance after platinum binding to the DNA has occurred.

These mechanisms include increased DNA repairs of adducts and the ability to

withstand greater levels of DNA damage failure to engage programmed cell death

pathways.8

3

| 3

1.2 PLATINUM COMPOUNDS COMMONLY USED

Since the introduction of cisplatin numerous platinum complexes have been

synthesized and evaluated but only a limited number is used worldwide.9 Most of these

complexes are cisplatin analogues. The most important of these drugs is diammine-

1,1-cyclobutanedicarboxylato-(2)-O,O’-platinum(II) (carboplatin, ([(NH3)2Pt(C4H6O4)]).5

O

OPt

H3N

H3N

O

O

Figure 1:3 Structure of carboplatin

On inspection it can be seen that carboplatin differs from cisplatin by the

replacement of the chloride anions with a dicarboxylic chelate ligand with a

hydrophobic substituent. This derivative of malonic acid is a relatively weak acid.

The weak acidity and the resulting six-membered chelate ring enhance its stability

and thus limit the rate of detoxification and the number of negative side effects.5

Due to the enhanced stability of carboplatin, its ligand exchange reactions occur

at a slower rate than those of cispaltin. Carboplatin is also more readily

transferred across the cell wall due to the presence of the hydrophobic

component.11 The half life of carboplatin is approximately 30h in body fluid. It

resists attack by thiols more readily than cisplatin and the four oxygen atoms

increase its aqueous solubility. Carboplatin is less toxic but also less reactive than

cisplatin and it must be administered in a higher dose.12 A further advantage of

carboplatin is its ability to circumvent the dose limiting nephrotoxicity of

cisplatin.13 It is clinically proven that carboplatin is active against the same range

of tumours than cisplatin but because of its lower toxicity patients tolerate it better

and thus patients who could not continue with cisplatin treatment can continue

with carboplatin treatment.14 The main toxicity of carboplatin is myelosuppression.

A disadvantage of carboplatin is that it also reacts with glutathione to form

inactive platinum-thiol complexes and thus is not active against cisplatin-resistant

tumours.15

4

| 4

More recently trans-R,R-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin)

has gained interest. This can be regarded as the first non-cisplatin analogue.

Oxaliplatin belongs to a generation of drugs that contain different types of chiral

amine molecules6,16

Figure 1:4 Structure of oxaliplatin

Oxaliplatin has shown antitumour activity in colorectal cancer and is used for the

treatment of metastatic colorectal cancer. Oxaliplatin has shown to be effective in

the treatment of cisplatin resistant tumours.6 Oxaliplatin is able to avoid mismatch

repair and replicate bypass. It has reasoned that the 1,2-

diaminocyclohexane(DACH) ligand projects into the major grooves of DNA, which

play an important role in these mechanisms.

17

In addition to oxaliplatin being effective against cisplatin resistant cell lines a

further advantage is that it is less toxic. The decreased toxicity is due to the

oxalato leaving group being ionised more slowly and to a smaller extent than the

chloro leaving groups of cisplatin. Oxaliplatin does not show nephrotoxicity,

myelosuppression and ototoxicity.17,18. However, side effects are present.

Neuropathy and emesis are the toxicities related to oxaliplatin treatment.19 This

chelated diamine ligand has a stereochemical effect. Due to crowding, thiol

interference is less and a larger amount of platinum reaches the DNA thus

circumventing platinum resistance further.20

A few other platinum compounds have achieved regional approval. Lobaplatin

(lactatodiaminomethylcyclobutaneplatinum(II)) has been approved in China for the

treatment of breast cancer21 and Nedaplatin (cis-glycolatodiamineplatinum(II)) in

Japan for the treatment of head and neck, testicular, bladder and ovarian cancer.22

H2NPt

H2N

O

O

O

O

,5

5

| 5

a)

H2N

NH2

Pt

O

O

b)

Pt

H3N

H3N

O

O

O

Figure 1:5 Structures of Lobaplatin(a) and Nedaplatin(b)

1.3 CHEMICAL CHARACTERISTICS OF PLATINUM(II) AND PLATINUM(IV)

Why is platinum effective in the treatment of cancer? Platinum, being a later 5d

transition, metal has the required properties which renders it suitable for

anticancer action. It is highly covalent in its bonding (Platinum(II) is a soft metal

ion indicated by its Marcus softness σ value of +0.33 as compared to K+ with a σ

value of -0.5),23 It forms kinetically stable complexes (ligand exchange rate in

solution is slow) and it prefers bonding to the so-called soft ligands like aliphatic

and aromatic nitrogen-donor ligands and sulfur-donor ligands (e.g. SH- has a σ

value of +0.65; Cl- = -0.09, Br-=+0.17 and I-=+0.50 on a scale where OH- =0)23

Platinum(II) is diamagnetic and its complexes have a square planar symmetry.

Ligand exchange of its complexes can occur via an associative (SN2) or a

dissociative (SN1) mechanism. Ligand exchange of the square planar platinum(II)

complexes occurs via a distorted pentacoordinated species either a trigonal

bipyramidal or square pyramidal intermediate complexes thus by an associative

process. (In very recent literature it was indicated that even the

tetradiaquaplatinum(II) consists of a distorted species with four equal H2O

molecules in the plane and one extended molecule on the apex of a distorted

square pyramid)24

6

| 6

M

R

R

XR

M

R

R

R

M

R

R

R

Y

X

Y

M

R

R

YR

X

Yfast

SN2 Path

SN1 Path

X

5-coordinate intermediate

3-coordinate intermediate

Y = nucleophileX = leaving group

Figure 1:6 The dissociative (SN1) and associative (SN2) mechanisms

Platinum(II) and platinum(IV) mainly differ by the higher covalency and greater

kinetic stability of the latter. Square planar platinum(II) is a 16 electron system

with a vacant low lying orbital which allows for coordination expansion as

indicated by the SN2 mechanism pathway which is normally lower in activation

energy than a SN1 mechanism thus more reactive. In contrast octahedral

platinum(IV) is an 18 electron system in which case only a SN1 mechanism

occurs thus kinetically more stable.

The above behaviour can be used to explain the anticancer action of

dichloroplatinum(II) complexes such as cisplatin. In aqueous solution and

biological fluids (blood and cell fluid) the chloro ligands of platinum(II) complexes

including cisplatin are replaced by aqua ligands e.g. ionization of cisplatin in

blood. The chloride concentration in extracellular fluids is high (100µM in blood)

and lowers the ionization rate whereas in the cell the chloride concentration is low

(4µM) promoting ionization. However chemical equilibrium is not obtained within

the body. 6, 24 See equation 1 and 2 for the stepwise ionization of cisplatin.

7

| 7

Equation one and two:

Pt

Cl

Cl

H3N

H3NPt

Cl

OH2

H3N

H3N

Pt

OH2

OH2

H3N

H3N

2

-Cl +H2O -Cl +H2O

equation 1 equation 2

It is believed that the small amount of aqua species is the actual anticancer agent since it bonds to the aromatic nitrogen of guanine in the DNA strand with two of such atoms thus forming an intrastrand crosslink. This ultimately leads to apoptosis. Deprotonation can occur lowering the reactivity since the hydrolysed species is less reactive.7 See equation 3 -5 for possible deprotonation reactions.

Equation three:

-H

equation 3

Pt

Cl

OH2

H3N

H3N

Pt

Cl

OH

H3N

H3N

Equation four and five:

Pt

OH

OH2

H3N

H3N

H-Pt

OH

OH

H3N

H3N

-H

equation 4 equation 5

Pt

OH2

OH2

H3N

H3N

2

A number of nitrogen (amines) and sulfur-donor ligands are present in the blood

serum and organs. A too high rate of ionization leads to the interaction of such

species with the platinum compound before the platinum reaches the cell. It has

been found that chloride can be directly exchanged for certain sulfur donors such

as the amino acids methionine and cysteine without the need of prior aquation.7

8

| 8

a) NH2

S

O

HO

b)

H2N

SH

O

OH

Figure 1:7 Structure of the amino acids methionine (a) and cysteine (b)

These effects lead to relatively low efficiency of haloplatinum(II) complexes

including cisplatin. Too fast rates of ionization lead to detoxification before these

complexes reach the cell and this results in great toxicity to the rest of the body.

Too slow rates cause inefficiency.24,25

1.4 IMPORTANT FACTORS REGARDING THE ANTICANCER ACTION OF PLATINUM COMPLEXES

In the previous section the relevant basic chemistry of platinum which has a

bearing on anticancer activity was briefly discussed. It is now necessary to

consider all the parameters of the platinum species i.e. combining the platinum

metal properties with the various ligands and their properties thus the total

platinum anticancer agent. These factors are taken into consideration with

specific reference to the design of new anticancer agents having superior action.

The following factors will now be considered namely kinetics, stereochemistry, S-

N interaction (N7) and biocompatibility.

1.4.1 Kinetics

In the previous section the effect of the kinetics of diamminedichloroplatinum(II)

complexes like cisplatin was considered from a basic viewpoint. The importance

of ligand exchange rate was emphasised. Too fast kinetics means high toxicity,

too slow kinetics means inefficient anticancer action. There are a number of

secondary factors of which the trans effect and its variations are very significant.

The trans effect i.e. the capability of a coordinated ligand to labilise a ligand trans to it

in a square-planar platinum complex can in general be applied to ligand exchange in

platinum complexes. The normal order of trans directing is:

9

| 9

CN- ~ CO~ olefins > thioethers ~ H+ ~ arsines ~phosphines > SC(NH2)2 ~ CH3 >

C6H5- ~ NO2- ~ I- ~SCN- > Br- > Cl- > amines ~NH3 ~ OH- ~ H2O.26

i. Coligands can have a great effect on the coordinated ligand in covalent

transition metal chemistry like that of platinum(II) and platinum(IV). E.g.

coligands with high Eneg like N and O cause a high effective nuclear charge

on the platinum leading to a sulfur-donor ligand with a smaller trans effect

than predicted. Such properties are referred to as biphilic. 27

From the above

sequence it appears that sulfur-donor ligands will act differently from Cl- , water and

amines which are the ligands commonly found in the body. Sulfur-donor ligands are

used to demetallate heavy metals and it has been found that cisplatin is removed via

urine bonded to sulfur-donor ligands.27 Considering the above factors it is clear that a

study of the all the factors influencing ligand exchange is necessary when designing

new anticancer agents in an effort to optimise their anticancer properties.

Although the trans effect is important as a general rule there are a number of

other factors also influencing ligand exchange rate.

ii. Sterically hindered systems also influence the nucleophilicity sequence. In

these systems the size and shape of the neucleophile becomes more

important than the order of the sequence. In some cases reversal of

nucleophilicity order occurs. The position of the steric effect is also important,

e.g. a 2-methyl substituted pyridine cis to leaving group has a much bigger

effect than when trans to the leaving group. Stereochemical bulk in a ligand in

the close vicinity of platinum provides an energy barrier for the formation of

the activated intermediate thus negatively influencing the SN2 mechanism and

therefore lowering the ligand exchange rate.7

iii. Lability is also influenced by bond lengths, force constants and stretching

frequencies e.g. the Pt-Cl bond length is 2.21Å trans to NH3 and 2.37Å

trans to a P in Pme3.27

iv. The effect of the cis ligand (cis effect) is very important in Pt(NH3)2Cl2.

When adjacent ligands have weak trans effects the cis effect has a greater

influence in the behaviour of the coordinated ligands. Cl- > NH3 is normal,

but in reality the opposite occurs.27

v. The chelate effect in the case of anions slows down ligand exchange due

10

| 10

to its stabilising effect on the platinum anion bonding.

It should be borne in mind that more than one of the above factors could be

acting simultaneously.

1.4.2 Stereochemistry

i. Crowding nitrogen-donor ligands with alkyl groups does not only slow

down the ligand exchange rate, but it also prevents donation of the large

sulfur atoms present in biological systems which can detoxify the agent by

creating a barrier.16 Crowding also limits organ toxicity.

ii. Stereochemistry also plays a role in the level of replicative bypass of Pt-DNA

adducts and in the repair mechanism. In the case of DACH non-leaving group

it is anticipated that the DACH co-ligand interacts with the major groove of the

DNA assisting in the platination of the aromatic nitrogen of DNA.27

iii. Increased stereochemistry in a non-leaving group results in increased

hydrophobicity which facilitates passive diffusion through the cell wall and

promotes the uptake of anticancer agents.

28

1.4.3 S-N interaction (N7)

The large nucleophilicity of the sulfur-donor ligands give them a trans labilizing

effect as previously discussed. However, this varies from sulfur donor to sulfur

donor. Two types of sulfur-donor ligands exist namely thioethers and sulfoxides

which are known for their σ donor π acceptor action, and secondly thiols and

thiourea and similar compounds that interact via σ donor π donor bonding. It is

known that the stronger σ donor ligands like thiols form more stable complexes

with platinum than the weaker ones depending on π back donation as seen in the

case of thioethers. The σ donor π donor group interacts irreversibiliy with

platinum(II) e.g. the aromatic nitrogen (N7) of guanine cannot replace these. The

former group is thermodynamically less stable and can be replaced by the N7 of

guanine.29

Platinum drugs exert their antitumour effects via damage to DNA. Platinum reacts

with DNA preferentially to the N7 atoms of purine nucleobases. The rate of the

11

| 11

initial binding step and the closure to form the bifunctional adduct are dependent

on the kinetics of the hydrolysis rate. The DNA adducts consist of intrastrand

cross-links involving adjacent bases, most frequently involving adjacent guanine

bases and less frequently adenine adjacent to guanine.

It is of interest that previous ligand exchange studies performed on platinum(II)

anticancer compounds under in vivo conditions i.e. pH7 in the presence of Cl- ions, it

was found that the order of replacement is thiols (glutathione) > GN7 > thioether

(methionine) > AN7..31

A comparative study was done on the two relevant N7 atoms namely adenine and

guanine. Space filling models and charge distribution values of these ligands

were obtained to investigate the possible reasons for the above sequence. (See

figure 1.8 and 1.9).

a) b)

Figure 1:8 Space filling model (a) and charge distribution values (b) of adenine.

a) b)

Figure 1:9 Space filling model (a) and charge distribution values (b) of guanine

From studying the above, the following can be concluded:

i. The N7 atom of guanine is stereochemically less crowded than the N7 atom

of adenine

12

| 12

ii. The negative charges on the two atoms adjacent to adenine N7 atoms are

lower than those of the guanine, thus guanine has a greater potential of

induction by the platinum(II).30

iii. Due to the greater σ basicity of the adenine N7 atom it will get protonated

to a greater extent at pH7 than the guanine N7 atom. The adenine N7 atom

is thus less available for platinum(II).

31

iv. Stronger H-bonding capability of guanine also plays a role in stability of its

platinum-DNA adduct.

There is also a distinct difference between the two types of sulfur atoms. The

thioethers can be regarded as virtually neutral sulfur atoms, but the thiol sulfur

can readily be deprotonated by the very soft 5d transition metal platinum in effect

changing it from a neutral to a monoanionic ligand with enough covalent

character to satisfy the nephelauxetic character of platinum(II).

A space filling model and the charge distribution values of the biothio compound

cysteine was obtained to study the effect of sulfur donation. The large size of the

sulfur atoms immediately becomes clear as well as the slight negative charge

already present before deprotonation

a) . b)

Figure 1:10 Space filling model (a) and charge distribution values (b) of cysteine

If the size of the sulfur atom is compared to that of the aromatic nitrogen-donor

atom then the difference is obvious. Ligand exchange of platinum(II) complexes

occurs via an intermediate distorted pentacoordinated complex and therefore

stereochemical crowding of the platinum(II) square planar complex will have

bigger negative effects on ligand exchange.32 Due to the size difference between

13

| 13

the bulky sulfur atoms and the aromatic nitrogen-donor atom it appears that there

ligand exchange will be influenced more negatively for the sulfur donor than the

nitrogen donor.

Chelated nitrogen-donor ligands are used to control the effect of the sulfur donors

such as thioether (methionine). The usage of chelated nitrogen donors increases

the thermodynamic stability and provides a bigger barrier towards the

replacement by the thioether sulfur atoms.34

However, it should be borne in mind that chemical potential is proportional to

concentration and there is more aromatic nitrogen donors in body fluid and cells

than sulfur-donor atoms and this can influence the situation described above.

1.4.4 Biocompatibility

There are several factors that enhance the biocompatibility of a platinum

compound.

Solubility – In the design of anticancer agents a compromise needs to be reached

between hydrophobicity (which normally lowers the aqueous solubility) and the

aqueous solubility. Too low solubility is to be avoided since enough platinum

compound must be introduced into the body to be effective.5

Membrane transport – Studies have shown that these drugs initially enter the

body through passive diffusion and facilitated diffusion through a gated channel.33

Since the interior of the cell membrane consists of hydrophobic hydrocarbon tails,

increasing the hydrophobicity or lipophilicity of an anticancer agent thus plays a

role in the uptake of these compounds.34, 35

It has been shown that the greater the lipophilic nature of complexes, the more

readily it enters the cells and thus resistance can be circumvented due to

decreased platinum accumulation. Carboxylate, carbonate or carbamate ligands

used in such complexes are suitable ligands since they contain polar and

lipophilic regions. Examples of such complexes are aminedibutylratodichloro

An example of this is JM216

(bis(acetato)ammine-dichloro(cyclohexylamine)platinum(IV), its increased

lipophilicity enhanced its uptake via passive diffusion.35

14

| 14

(cyclohexylamine)platinum(IV) (JM221). NNDP is a lipophilic platinum compound

containing the DACH carrier ligand and lipophilic neodecanoic acid leaving

groups encapsulated in a liposome. The cell uptake for L-NNDP is 8-10 fold

higher compared to cisplatin because of its increased lipophilicity.

I,2-intrastrand crosslinks formed by platinum binding to DNA can create a

hydrophobic notch at the damage site, which can be bound by specific damage

recognition proteins, thus shielding it from NER activity. Thus by using more

hydrophobic platinum complexes, resistance can be prevented and this can lead

to higher antitumour activity.

Biologically active carrier groups – In some cases third generation drugs have a

carrier molecule attached to the normal chloride binding areas, which are

molecules the tumour cells may desire in particular and are more readily taken

into the tumour cells. An example includes the platinum moiety to deoxorubicin

and oestrogen analogues that bind to estrogen receptors. Carrier groups with the

ability to direct platinum to specific organs as well as DNA intercalators that will

localize the platinum in the vicinity of the DNA have been investigated. However,

there have been no clinically significant advances that have been developed from

attaching biologically active groups to platinum compounds.

1.5 RECENT DEVELOPMENTS IN PLATINUM ANTICANCER RESEARCH

Initial studies on the improvement of the anticancer action of platinum complexes

centred around cisplatin and its structure e.g. the synthesis of various

diamineplatinum(II) complexes with different amine non-leaving groups. Extensive

anticancer studies showed that these ‘cisplatin analogues’ have very similar

anticancer behaviour to cisplatin with reference to cell resistance and toxicity.

Some of these properties were modified by using leaving groups that slows down

ligand exchange and thus also slow down detoxification e.g. carboplatin. This was

followed by the so-called second generation anticancer drugs e.g. oxaliplatin. In

the case of oxaliplatin the non-leaving group had a positive effect on the

platination of the N7 of guanine.

15

| 15

The next development in the design of new platinum anticancer drugs was to

produce platinum(IV) compounds which are inert and can thus be taken orally.

The latter two modifications are still being investigated. The effect of variation of

structure and nature of platinum complexes on the anticancer action is still under

investigation. Platinum(IV) proved to be effective against cancer and thus studies

on platinum(IV) compounds are still continued. More recently specific platinum(IV)

compounds appeared in the literature which proved to have promising anticancer

action and Stat-3 behaviour. 36,37

1.6 OBJECTIVES OF THIS DISSERTATION

STAT (signal transduction and translation) proteins migrate during the translation

phase to the cell nucleus where it binds to promoter elements of DNA molecules,

leading to expression of target genes. STAT proteins are normal constituents of

cells, but in STAT-3 activity is frequently up in regulated in many human tumours.

It has been shown that some platinum anticancer agents interact with STAT-3

inhibiting binding to its DNA target. 38

There are still unanswered questions on specific aspects of sulfur-donor ligands.

Targeting of the anticancer cells also became more important in view of the

damage done to the normal cells (thus minimizing the negative side effects).

Some properties of sulfur-donor ligands have been briefly discussed under

kinetics. The dominant role of σ donor π donor atoms which irreversibly bond to

platinum was mentioned as a negative factor. However σ donor π acceptor sulfur

atoms like thioethers have some positive characteristics in so far that the bulky

sulfur atom can protect the platinum through stereochemical crowding although it

can be replaced by the N7 of guanine. The dominant trans directing power of

sulfur can be limited. An example of this is a chelated compound where electron

withdrawal from the sulfur atom can be achieved. In this research this specific

property was studied by using a bidentate ligand having an aromatic nitrogen

linked to a thioethereal sulfur via a methylene group in the form of a chelate

ligand capable of forming a five membered ring. In this case it could be expected

that some sulfur electron density will be drawn through the CH2 connection into

16

| 16

the aromatic system thus weakening the effect.

Platinum(II) complexes of such non-leaving groups having different N1 substituted

imidazole ligands and varying leaving groups (Cl- , Br-, I- and oxalate) were

synthesized, characterized and tested for anticancer action.

Although amine complexes of platinum(II) have been extensively studied some

amine non-leaving groups were selected for specific reasons e.g. having cyclic

substituted groups with the potential to assist in platination and OH substituted

groups for the improvement of aqueous solubility.

The mononitrochloroplatinum(IV) complexes of some of the platinum(II)

analogues especially those that showed significant anticancer action, were

studied.

17

| 17

-CHAPTER 2: EXPERIMENTAL PROCEDURES

2 INTRODUCTION

This chapter will be devoted to discussion of the experimental techniques applied

for both analysis and characterization with special reference to the

instrumentation involved. The instrumentation employed in this research project

will be described here mentioning some of the analytical work done at other

laboratories where instruments were not available in this department.

This information will be followed by the general preparative methods and where

required, alterations of it. This will be sequential to correspond to the later

chapters.

2.1 ANALYSIS TECHNIQUES

2.1.1 Elemental Analysis

Microanalysis for C, H, N, and S was performed at the University of Cape Town.

The samples were analysed by a Thermo Flash 1112 Elemental Analyzer

2.1.2 Spectrophotometric Analysis

Infrared spectroscopy

Infrared spectra were recorded using an Excalibur HE FTS3100

spectrophotometer in the range of 4000-200 cm-1 using the single beam mode.

Solid samples were compressed with dry potassium bromide into discs and

scanned with nitrogen gas as reference. Liquid samples were spread as a thin

film between two sodium chloride plates and scanned using the empty plates as

reference.

18

| 18

NMR spectrometry

The 300.13 MHz 1H-NMR spectra were recorded on a Bruker 300 MHz

spectrometer using SiMe4 as the internal standard. The samples were prepared

using CDCl3 as solvent.

Inductively-coupled plasma emission mass spectroscopy

The platinum content of the samples prepared for the solubility studies were

analysed using a Perkin Elmer SCIEX Elan-6100 ICP-MS Spectrometer. The ICP

settings used are given below in Table 2.1

Table 2:1 General ICP settings used

The isotopes 195Pt and 196Pt were used for optimal analysis. The standards were

prepared in an aqueous medium containing 0.1 M HCl in the range of 5-1000 µg/l.

Samples for analysis were diluted appropriately and prepared in a 0.1 M HCl

matrix.

FAB Mass spectrometry

FAB mass spectra were obtained from the University of Potchefstroom. The

spectra were recorded on a VG70-70E mass spectrometer using a VG2035 data

system. All samples were analysed under positive-ion operating conditions. An

Ion Tech saddle-field using xenon as bombardment gas (8 kV, 1 mA) was

employed to record the spectra. The mass spectrometer was operated at 6 kV

and the magnetic analyser was scanned at 5 s per decade between 500-100 Da.

The matrix used was glycerol or 3-nitrobenzylalcohol.

Setting RF Power 1.4 kW

Coolant gas 15 L/min

Nebulizer gas flow 0.7L/min

Dwell time per AMU 50ms

Sweeps/reading 10

Readings/Replicate 2

Replicate 10

Integration 5.0 s

19

| 19

Electronspray Mass Spectrometry

Electron Spray mass spectra were obtained from Platco laboratories at Shimoda-

Biotech. Measurements were carried out on an LCQ Deca instrument (Finnigan,

San Jose,CA, USA) in either positive or negative ionisation mode. For infusion

studies the samples, in appropriate solvent, were directly infused at a flow rate of

10 μM/min using a 500 μL unimetrics HPLC syringe and the instrument’s own

syringe pump. A 50% CH3CN/10 mM ammonium formate/0.06% formic acid

solution was used as sheath liquid at a flow rate of 150 μL/min. Direct contact

with the analysis solution was allowed by positioning the tip of the fused silica

capillary (i.d.100 μM, o.d. 190 μM) 0.5 to 1.0mm inside the tip of the stainless

steel sprayer capillary. The parameters of the instrument were tuned utilising an

infusion solution of Pt(dach)Cl2 in CH3CN (10 μM at 10 μL/min).

Positive ion mode instrument settings - The instrument parameters were tuned

using the ammonium adduct [M+NH4]+ with the following values: nitrogen sheath

gas flow rate 80 arbitrary units, nitrogen auxiliary gas flow rate 20 arbitrary units,

sprayer voltage 3.5 kV, capillary voltage 46.0 V, tube lens offset 55.0 V, multipole

1 offset -5.75 V, lens voltage -28.0 V, multipole 2 offset -10.00 V, multipole RF

amplitude (V p-p) 400 V, entrance lens -62.0 V and 100 ms maximum injection

time using automatic gain control.

Negative ion mode instrument settings - The parameters of the instrument were

tuned using the formate adduct [M+HCOO]- to the following values: nitrogen

sheath gas flow rate 80 arbitrary units, nitrogen auxiliary gas flow rate 20 arbitrary

units, sprayer voltage 5.0 kV, capillary voltage -12.0 V, tube lens offset 5.0 V,

multipole 1 offset 5.25 V, lens 49 voltage 46.0 V, multipole 2 offset 10.00 V,

multipole RF amplitude (V p-p) 400 V, entrance lens 38.0 V and 100 ms

maximum injection time using automatic gain control.

LC-MS

The LCQ Deca mass spectrometer was connected to a Thermo Surveyor HPLC

system consisting of a quaternary gradient pump and autosampler. For LC-MS

analyses, varying solvents were utilised but in each case, the solvents and

20

| 20

modifiers complied with the overriding requirement of ESI-MS, in that all

components had to be volatile. Thus, no separations could be effected with the

help of phosphates or sulphonic acids and no sodium, potassium or chloride salts

could be used. Instead, volatile buffer systems such as ammonium formate,

ammonium acetate or diethylamine were used.

HPLC-UV

LC separations were performed on a Waters HPLC system consisting of a

WISP717 autosampler, 600E quaternary gradient pump and 484 variable

wavelength UV-Vis detector.

HPLC separation columns used during this study:

• YMC Hydrosphere C18 120 Å 5 μM 250 x 4.6 mm

• Phenomenex Curosil PFP 5 μM 250 x 4.6 mm

2.1.3 Solubility Studies

The solubilities of the complexes were measured by suspending the compound in

2 ml H2O and stirring at 25°C for 30 minutes after which the mixture was

centrifuged for 5 minutes at 5000 rpm's. The supernatant liquor was diluted so

that the platinum concentrations were suitable for ICP-MS analysis. 500 and 1000

times dilutions were sufficient.

2.1.4 Crystallographic Analysis

Full X-ray structure determinations were attempted on some platinum complexes.

The diffraction data was collected at the University of the Witwatersrand using a

Bruker SMART 1K CCD area detector diffractometer with graphite

monochromated MoKα radiation (50 kV, 30 mA). The collection method involved

ω-scans of width 0.3°. Data reduction was carried out using SAINT+38 and

absorption corrections were made using the program SADABS39

The WinGX 1.64.02

.

40 suite of programs was used to solve the crystal structures

using either direct methods or the Pattersen approach. Refinement was done

21

| 21

using SHELXS-9741. Non-hydrogen atoms were first refined isotropically followed

by anisotropic refinement by full matrix least-squares calculation based on F2.

Hydrogen atoms were positioned geometrically and allowed to ride on their

respective parent atoms with one exception. Torsional angles and least squares

planes of selected atoms were calculated using PARST42. Diagrams were

generated using ORTEP43

2.1.5 Cytotoxicity Studies

.

All the complexes prepared in this dissertation were tested for anticancer action

according to known literature methods44,45

Cytotoxicity in cell lines was determined by a modified MTT assay (Sigma). Cells

in exponential growth were trypsinized, counted using a haemocytometer and

diluted to a density of 30 000 cells per ml. Aliquots (200µL) of cell suspensions

were added to each well of a 96-well microtiterplate and incubated for 24 hours.

The novel platinum containing compounds were screened for cytotoxicity using

10 and 100µM concentrations. Cisplatin (10 and 100µM concentrations) served

as a positive control. The spent medium was removed from the 96-well plate and

aliquots (200 µl) of the treatments were added to the wells in quadruplicate. The

plate was incubated in a 37°C-5% CO2 humidified incubator for 48 hours. A

by the Biochemistry department of the

Nelson Mandela Metropolitan University under the supervision of Dr. M. van de

Venter. The compounds were tested on colon (HT29), breast (MCF 7) and

cervical cancer (HeLa) cell lines. Cisplatin was used as reference. A summary of

the general method is given below.

The reduction of tetrazolium salts is now widely accepted as a reliable way to

examine cell proliferation. The yellow tetrazolium salt MTT (3-(4,5-

dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically

active cells, in part by the action of dehydrogenase enzymes. The resulting

intracellular purple formazan can be solubilized and quantified by

spectrophotometric means. The MTT Cell Proliferation Assay measures the cell

proliferation rate and conversely, when metabolic events lead to apoptosis or

necrosis, the reduction in cell viability. The MTT Reagent yields low background

absorbance values in the absence of cells.

22

| 22

5mg/ml stock solution of MTT was prepared in PBSA and used to prepare a

1mg/ml solution of MTT in growth medium, to be applied to wells in 200µL

aliquots. The plate was incubated for 3 hours before the MTT was removed and

replaced by 200µL of DMSO to dissolve MTT formazan. The plates were agitated

on a shaker for 5 minutes, before the absorbance was read at 540nm on a

multiwell scanning spectrophotometer. The values obtained were used to

determine the percentage inhibition of cell growth that these drugs cause.

The treatment solutions (100µM) were made up in pre-warmed (37°C) RPMI

1640 medium containing 10% foetal calf serum. The samples were vortexed for

approximately 1 minute, sonicated for 10 minutes and then briefly vortexed again

before it was ready for use. The 100µM solutions was filtered and sterilized using

0.22µm syringe filter units. A 10x dilution was prepared in order to obtain a 10µM

solution of the treatment. These solutions was applied to cells in 200 µL aliquots

for 48 hours.

All of the complexes synthesized went through a preliminary screening on the cell

lines using the MTT Assay. The results obtained determined which complexes

were used for further experiments.

Dose response curves

Dose response curves were prepared for each of the chosen complexes, as well

as positive controls, in order to obtain IC50 values. The concentrations used was

100, 50, 25, 10, 5 and 1 µM. IC50 values were calculated from the log-dose

response curves using GraphPad Prism 4.

2.1.6 Methods applied for the Ionization Studies

Methods applied for ionization studies on (NS)-platinum(II) complexes- Following

verification of structure by infusion mass spectrometry, the samples were ionized

to determine the individual ionization rates. Two different methods were used as

there was to be found a large difference in the ionization rates of the dichlorides

and the oxalates. The method utilised to monitor the ionization of the dichlorides

consisted of first dissolving the sample in a suitable solvent (600 µM/ CH3OH)

23

| 23

where after an aliquot of the methanolic solution (50 µL) was diluted with water

(450 µL) immediately prior to analysis. The dilution step took an average of 25-

30s. The solution (5 µL) was then injected directly into the mass spectrometer at

10s intervals. A total of 8 to 10 injections were performed.

The oxalates were initially treated as the dichlorides above, but it was found that

no ionization took place during the short time allowed for the fast loop test (ca.

6min). Subsequently fresh samples were suspended in water (ca. 1mM) and the

solutions were infused in a baseline spectrum. The sample was then allowed to

stand at room temperature for 6 hours before a second mass spectrum was

obtained. Finally the sample was placed in an oven at 80ºC for a 60 hour period

prior to final analysis.

Methods applied for ionization studies on (NN)-platinum(II) complexes –

Experiments were performed to evaluate the ionization rates of a set of bidentate

(NN)-dihalideplatinum(II) complexes and compare this with the data obtained for

bidentate (NS)- dihalideplatinum(II) complexes.

Methods - Structural verification was performed by infusing a solution of each

compound in CH3CN (600μM) directly into the mass spectrometer. Unique

molecular ions were obtained demonstrating that the correct structure had been

prepared during synthesis and that the energy imparted by the instrument during

the electronspray process was not excessive. The CH3CN solutions were diluted

in water to 60uM (90% water). This was done to check whether these compounds

underwent rapid ionization as in the case of the analogous (NS)-compounds.

To try and characterize the ionization rate of each individual compound, aqueous

solutions were prepared by diluting the original CH3CN stock with water (1%

CH3CN / 99% H2O) and analysed over time (3Hr). The formation of the ionization

product(s) [M-X+CH3CN]+ and the degradation of the molecular species [M+NH4]+

were quantified, by integration of the peaks produced for each.

Incubation method - The samples were dissolved in CH3CN (600μM). Prior to

analysis (0.5min) an aliquot of the CH3CN solution was diluted with water (37ºC)

to produce a solution of 6uM in 1% CH3CN (99% water). The samples were then

24

| 24

placed in a heated tray (37ºC) and sampled periodically, typically monitoring

transitions between 0.5 and 140min. The difficulty with this technique is that it can be

influenced by instrument drift as the detector is on for the entire period. This implies

that identical samples analysed 2 hours apart may show differences in the ion

intensities and hence the perceived concentration of species. Such differences in ion

intensities will detrimentally affect ionization curves leading to false ionization T1/2 times. The relative abundance of the species will however be reflected correctly.

Therefore plotting the individual ion intensities as a function of the total ion current

(TIC) was considered as a possible solution, the assumption being that all ionization

and degradation products are detected and contribute to the TIC (unsafe

assumption). The best way to compensate for the problem would be through the use

of a suitable internal standard. In the absence of such a compound all steps were

taken to ensure that the analysis time was kept to a minimum

2.2 PREPARATION OF STARTING MATERIALS 2.2.1 K2PtCl6

Pure platinum metal (99.99%) (10 g, 51 mmol) was dissolved in a 1:1 mixture of

aqua regia and distilled water at an elevated temperature. The solution was

concentrated by heating to a silvery paste. The latter was dissolved in

concentrated HCl (100 ml) and again concentrated to a paste. This last step was

repeated. The final paste was dissolved in 1M HCl and made up to 250 ml in a

volumetric flask to act as a stock platinum solution.

Chemically pure potassium chloride (KCl, 0.85 g, 11.4 mmol) was dissolved in 10

ml distilled water and added drop wise to 25 ml stock platinum solution (+/- 1 g,

5.1 mmol) while stirring. An equal volume of 95% ethanol was added to the

platinum mixture and allowed to stand on ice for 45 minutes. The resultant yellow

crystalline K2PtCl6 was filtered and washed twice with 20 ml portions of 50%

ethanol, once with 20 ml 95% ethanol and 3 times with 20 ml portions of ether

where after it was dried in an oven at 50°C. Yield: 95%

2.2.2 K2PtCl4

K2PtCl6 (4.7 g, 9.67 mmol) was suspended in 35 ml distilled water over a 90°C

25

| 25

water bath. A saturated aqueous solution of sulfur dioxide (SO2) was added in

0.6 ml portions over 2-3 minute intervals. The time intervals gradually lengthen

as the reduction nears completion. The next addition can be performed when no

more SO2 is emitted from the platinum(II) solution. Addition of SO2 was stopped

when only a very small portion of solid K2PtCl6 remained in a very red solution.

The platinum solution was filtered while hot to remove the unreacted K2PtCl6 after

which the supernatant was concentrated and allowed to cool. The resultant red

crystalline K2PtCl4 was filtered, washed once with 3 ml acetone and allowed to air

dry in a 50°C oven.

Yield: 65%

2.2.3 K2Pt(C2O4)2.2H2O

K2PtCl4 (2.08 g, 5.0 mmol) and 16 equivalents of K2(C2O4)2 (14.72 g, 79.9 mmol)

were dissolved in 30 ml distilled water at 90°C and stirred for 1 hour maintaining

the temperature at 90°C after which the reaction was allowed to cool for 2 hours

at 5°C. The resultant K2Pt(C2O4)2.2H2O precipitate which formed was filtered and

washed 5 times with 15 ml distilled water, once with 10 ml acetone and allowed to

air dry at 50°C in an oven.

Yield: 95%

2.3 SYNTHESIS OF THE (NS)-DONOR LIGANDS 2.3.1 1-Methyl-2-methylthioalkylimidazole

These ligands were prepared in three consecutive steps. All these compounds

were prepared in the same manner as described below. The difference was that

in the final step the appropriate alkylthiol was used.

2.3.1.1 1-Methyl-2-methylthiomethylimidazole(mmtmi)

Step 1:

A solution of 1-methylimidazole (50 g, 0.61 mol) dissolved in1000 ml of 40 %

formaldehyde was refluxed for 7 days. Sodium hydroxide (60 g, 1.5mol) and 2

26

| 26

litres of water were subsequently added

synthesis and characterization of novel platinum … · 2018. 1. 8. · platinum complexes –...

Documents