MACROMOLECULAR DERIVATIVES OF METHOTREXATE AND FERROCENE AS POTENTIAL PRODRUGS IN CANCER CHEMOTHERAPY Ilunga Mufula A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science. Supervisor: Professor E.W. Neuse Johannesburg, 2010 ii DECLARATION I declare that this dissertation is my own, unaided work. It is submitted for the degree of Master of Science, in the University of the Witwatersrand, Johannesburg, South Africa. It has not been submitted before for any degree or examination in any other University. ________________________ Ilunga Mufula ________________day of __________________, 2010 iii ABSTRACT Cancerous diseases present a formidable health problem worldwide. While the chemotherapy of cancer, in conjunction with other treatment modalities, has reached a significant level of maturity, efficacious use of such agents is still restricted by numerous pharmacological deficiencies, such as poor solubility, short serum circulation lifetimes, and low bioavailability resulting from lack of affinity to cancer tissue and inadequate mechanisms of cell entry. More critically still, most drugs suffer from toxic side effects and a risk of drug resistance. In an attempt to enhance the therapeutic effectiveness of carcinostatic drugs, the concept of anchoring bioactive agents to polymeric carriers has proved to be a promising approach to overcome these deficiencies and was the main aim of this project. Water-soluble, biodegradable macromolecular carriers used were polyaspartam ides, prepared by an aminolytic ring-opening process of polysuccinimide; polyam ides obtained by ester-amine base-catalyzed polyaddition; and polyam idoam ines prepared by Michael- type addition polymerization. The drug-anchoring potential of carrier polymers was demonstrated by the coupling of methotrexate (MTX), ferrocene and platinum drug models. MTX was linked to carrier via polymer attached amine by N-acylation of linear amine- functionalized polyaspartamide carriers with the acid group from methotrexate. Acylation was brought about by mediation of HBTU coupling agent. The resulting MTX content of the conjugates was in the range of 10-19% by mass. In the present dissertation, series of water-soluble ferrocene conjugates were synthesized as for MTX by N-acylation of linear amine-functionalized polyaspartamide carriers with 4-ferrocenylbutanoic acid. Acylation was brought about again by mediation of HBTU coupling agent. The resulting iron content of the conjugates was in the range of 6-13% by mass. iv Polymer-attached dihydroxylato-type ligands were used to anchor the platinum drug to the polymeric carriers. The platinum content of the conjugates was in the range of 6-8% by mass. A member of selected conjugates was submitted to the Department of Immunology, University of Pretoria, and to the School of Pharmacy, University of California, Los Angeles, CA, for biomedical activity assessment. In order to demonstrate the multidrug-binding capacity of the polyaspartamide-type carriers, ferrocene was co-conjugated to selected polymeric conjugates containing MTX or folic acid. The latter was used to ensure target-specific drug delivery. v DEDICATION This dissertation is dedicated to my father for his prayers and constant support throughout my studies. vi ACKNOWLEDGEMENTS I would like to express my sincere gratitude to the following people and organizations for making this work possible: ? The master of time and circumstances, the Lord God Almighty, for the gift of life. ? Professor E.W. Neuse, my supervisor, for his valuable assistance, tireless guidance, patience, helpfulness, confidence and understanding throughout this project. It was his faith in me that made me come so far. ? The Foundation for Research Development, NRF, (2007-2008) and Bradlow Postgraduate Awards (2006-2009) for their financial assistance. ? Mr Richard Mampa of the School of Chemistry for countless NMR spectra he scanned for me, and his kindness. ? All my colleagues in Polymer Research Laboratory. Their warm friendship, encouragement and sense of humor made this experience worthwhile. ? My father for being so supportive and trusting God for me to come this far. ? My wife Isabelle Mufula for her love, prayers, courage and support in all the ways. ? My children Gedeon, Deogracias, Salem and Benedicta Mufula for their prayers, love, courage and support. ? My brothers and sisters for their support and guidance. ? Last but not least, all of those who pray for me. Your prayers were not worthless. vii TABLE OF CONTENTS Declaration ii Abstract iii Dedication v Acknowledgements vi Table of contents vii List of Figures x List of Schemes xi List of Tables xiii List of Abbreviations xv Chapter 1: Introduction 1 1.1 What is cancer 1 1.2 Cancer problems 1 1.3 Aims of the project 3 Chapter 2: Literature review 2.1 Causes of cancer 5 2.1.1 Carcinogens 5 2.1.2 Age 5 2.1.3 Genetic make up 6 2.1.4 The immune system 6 2.1.5 Diet 6 2.1.6 Day to day environment 7 2.1.7 Viruses 7 2.1.8 The many or other causes of cancer 7 2.2 Cancer treatments 7 2.2.1 Surgery 7 2.2.2 Radiation therapy 8 viii 2.2.3 Targeted therapies 9 2.2.4 Immunotherapy 9 2.2.5 Chemotherapeutic treatment of cancer 10 2.2.5.1 Drug carriers 14 2.2.5.2 Polymer as drug carrier 14 2.2.5.2.1 Natural polymers as drug carriers 15 2.2.5.2.2 Synthetic polymers as drug carriers 15 2.3 The polymer-drug Anchoring strategy 15 2.4 The bioactive agents 19 2.4.1 Methotrexate 19 2.4.2 Ferrocene 22 2.4.3 Platinum compounds 24 Chapter 3: Results and discussion 3.1 Synthesis of carrier polymers 26 3.1.1 Polyaspartamides 28 3.1.1.1 Poly-DL-succinimide 29 3.1.1.2 Poly?, ?-DL-aspartamides 29 3.1.2 Other polyamides 49 3.1.3 Polyamidoamines 54 3.1.3.1 Polyaddition reaction of methylenebisacrylamide with primary monoamines and primary diamines 55 3.2 Polymer-drug conjugation 61 3.2.1 Polymer-Methotrexate conjugation 61 3.2.1.1 Polymer-Folic acid conjugation 61 3.2.1.2 Polymer-MTX conjugation 61 3.2.2 Polymer-Ferrocene conjugation 71 3.2.3 Polymer-Platinum conjugation 76 3.2.4 Polymer-multidrug conjugation 80 3.3 Biomedical testing 85 ix Chapter 4: Experimental 4.1 General procedure 86 4.2 Reagents, Reactants and Solvents 86 4.3 Preparation of polymeric carriers 4.3.1 Poly-DL-Succinimide (PSI) 2 87 4.3.2 Synthesis of poly-?,?-DL-aspartamides 87 4.3.3 Other polyamides 98 4.3.4 Polyamidoamines 99 4.4 Preparation of polymeric conjugates 103 4.4.1 Polymer-folic acid conjugates 103 4.4.2 Polymer-Methotrexate conjugates 108 4.4.3 Polymer-Ferrocene conjugates 109 4.4.3.1 Synthesis of ferrocenylbutanoic acid 109 4.4.3.2 Preparation of polymer-ferrocene conjugates 109 4.4.4 Polymer-platinum conjugates 114 4.4.4.1 Preparation of DACH-Pt 114 4.4.4.2 Polyamides-platinum anchoring 114 4.4.5 Polymer multidrug conjugates 115 Chapter 5: Summary and Conclusion 120 References 123 Appendix 128 x LIST OF FIGURES Figure2.1: General structure of a polymer carrier as proposed by Ringsdorf 17 Figure2.2: Structure of Methotrexate 21 Figure2.3: Mechanism of action of Methotrexate 22 Figure2.4: Cisplatin and other platinum analogues of clinical application 25 Figure 3.1: Structure of polyamide-type carrier 28 Figure 3.2: Structures showing resemblance of folic acid and MTX 66 xi LIST OF SCHEMES Scheme2.1: Polymer-drug conjugate as proposed by our group 18 Scheme2.2: Reactions of ferrocene complex in biological environment 24 Scheme 1: Polycondensation of DL-aspartic acid 29 Scheme 2: Synthesis of Poly-?, ?-DL-aspartamide 30 Scheme 3: Copolymer, Poly-?, ?-DL-aspartamide 30 Scheme 4: Synthesis of Copolyaspartamides 4a to 4e 33 Scheme 5: Synthesis of Copolyaspartamides 5a to 5c 36 Scheme 6: Synthesis of Copolyaspartamides 6a to 6c 40 Scheme 7: Synthesis of Copolyaspartamide 7a to 7c 44 Scheme 8: Synthesis of Copolyaspartamides 8a to 8b 47 Scheme 9: Synthesis of Copolyamide 9a 51 Scheme 10: Synthesis of Copolyamide 9b 51 Scheme 11: Synthesis of Copolyamide 9c 52 Scheme 12: Synthesis of Copolyamide 9d 52 Scheme 13: Synthesis of Polyamidoamines 10-14 57 Scheme 14: Reaction for the synthesis of 4a(90)-FA, 4b(90)-FA, 5a(90)-FA, 5a(95)- FA, 4c(90)-FA and 4d(90)-FA 62 Scheme 15: Reaction for the synthesis of 6a-FA, 6b-FA, 6c-FA, 7a-FA, 7c-FA, 8a-FA and 8b-FA 63 Scheme 16: Reaction for the synthesis of 4a(90)-MTX, 4b(90)-MTX, 5a(90)-MTX, 5b(95)-MTX 67 Scheme 17: Reaction for the preparation of 4-ferrocenenylbutanoic acid 72 Scheme 18: Reaction for the synthesis of 4a(90)-Fc to 4d(90)-Fc 72 Scheme 19: Reaction for the synthesis of 6a-Fc to 8b-Fc 73 Scheme 20: Reaction for the synthesis of dihydroxylato platinum conjugate 9a-Pt 77 xii Scheme 21: Reaction for the synthesis of dihydroxylato platinum conjugate 9b-Pt 77 Scheme 22: Preparation of platination agent DACH-Pt aq 78 Scheme 23: Preparation of polyaspartamide MTX/Fc co-conjugates 81 Scheme 24 Preparation of polyaspartamide FA/Fc co-conjugates 82 xiii LIST OF TABLES Table 3.1: Summary of experimental data of Polyaspartamide carriers 4a to 4e 34 Table 3.2: 1H NMR data for Polyaspartamide carriers 4a to 4e 35 Table 3.3 Summary of experimental data of Polyaspartamide carriers 5a to 5c 38 Table 3.4: 1H NMR data for Polyaspartamide carriers 5a to 5c 39 Table 3.5 Summary of experimental data of Polyaspartamide carriers 6a to 6c 42 Table 3.6: 1H NMR data for Polyaspartamide carriers 6a to 6c 43 Table 3.7 Summary of experimental data of Polyaspartamide carriers 7a to 7c 45 Table 3.8: 1H NMR data for Polyaspartamide carriers 7a to 7c 46 Table 3.9 Summary of experimental data of Polyaspartamide carriers 8a to 8b 48 Table 3.10: 1H NMR data for Polyaspartamide carriers 8a and 8b 49 Table 3.11: Synthesis of Polyamides 9a-9d 53 Table 3.12: 1H NMR data for Polyamides 9a-9d 53 Table 3.13: Preparative data of Polyamidoamines 10 to 14 58 Table 3.14: 1H NMR data for Polyamidoamines 1a to 10a 59 Table 3.15: Summary of experimental data for folic acid conjugates (4a(90)-FA to 8b- FA) 64 Table 3.16: 1H NMR data and viscometric results for folic acid conjugates (4a(90)-FA to 8b-FA) 65 Table 3.17: Summary of experimental data for MTX conjugates (4a(90)-MTX to 5b(95)-MTX) 69 Table 3.18: 1H NMR data and viscometric results for MTX conjugates (4a(90)-MTX to 8b-MTX) 70 Table 3.19: Summary of experimental data for the conjugates 4a(90)-Fc to 8b-Fc 74 Table 3.20: 1H NMR data and viscometric results for the conjugates 4a(90)-Fc to 8b- Fc 75 xiv Table 3.21: Summary of experimental conditions and analytical data for the dihydroxylato platinum conjugates 9a-Pt and 9b-Pt 79 Table 3.22: Summary of experimental data of Polyaspartamide co-conjugates 83 Table 3.23: 1H NMR data and viscometric results for co-conjugates 84 xv LIST OF ABBREVIATIONS AEE 2-(2-Aminoethoxy)ethanol AEM 4-(2-aminoethyl)morpholine APM 4-(3-aminopropyl)morpholine aq aqueous d day(s) DACH 1,2-diaminocyclohexane DCC N,N?-dicyclohexylcarbodiimide DEEA 2-(diethylamino)ethylamine DEP 3-(diethylamino)-1-propylamine DET diethylenetriamine Detart diethyl L-tartrate DMEA 2-(dimethylamino)ethylamine DMF N,N-dimethylformamide DMP 3-(dimethylamino)-1-propylamine DMSO dimethyl sulphoxide DNA deoxyribonucleic acid EA ethanolamine EDDA 2,2-(ethylenedioxy)-diethylamine FA folic acid Fc ferrocenyl ?inh inherent viscosity HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium fluorophosphates MBA methylenebisacrylamide MTX methotrexate Net3 triethylamine NMR nuclear magnetic resonance PDA 1,3-propylenediamine ppm parts per million PSI poly(D,L-succinimide) xvi RNA ribonucleic acid RT room temperature TRIA 4,7,10-trioxa-1,13-tridecanediamine SOLUMIX: mixture of (% w/w): Toluene (42.96%), m-xylene (14.39%), p-xylene (6.72%), o-xylene (6.39%), ethylbenzene (6.08%), heptane and isomers (10.01%), n- hexane (2.26%), hexane, mixture of isomers (1.60%), pentane (0.16%), isopentane and 2- methylbutane (0.12%).