Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2015 Design, Synthesis, and Biological Evaluation of Folate Targeted Photosensitizers for Photodynamic Therapy Laura Jean Donahue Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Chemistry Commons Recommended Citation Donahue, Laura Jean, "Design, Synthesis, and Biological Evaluation of Folate Targeted Photosensitizers for Photodynamic Therapy" (2015). Dissertations. 1940. https://ecommons.luc.edu/luc_diss/1940 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2015 Laura Jean Donahue LOYOLA UNIVERSITY CHICAGO DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF FOLATE TARGETED PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN CHEMISTRY BY LAURA J. DONAHUE CHICAGO, IL AUGUST 2015 Copyright by Laura J. Donahue, 2015 All rights reserved. ACKNOWLEDGMENTS I would like to acknowledge my research advisor, Dr. David Crumrine, for all of his guidance, patience, and support throughout my years of graduate work. I would also like to thank the members of my dissertation committee, Dr. James Babler, Dr. Ken Olsen, Dr. Martina Schmeling, and Dr. Stefan Kanzok for their continued guidance, support, and participation as members of my committee. I would like to thank other graduate students at Loyola who have been of assistance to me, in particular, Jeff Trautmann, Matthew Reichert, Brian Leverson, Cory Reidl, and RoJenia Jones. In addition, I would like to thank other faculty and staff at Loyola who have been very helpful, including Dr. Robert Polak, Dr. Rodney Dale, Dr. Matthew Bartucci, Dr. David French (dec.), Dr. Jessica Eisenberg, Dr. Paul Chiarelli, Doug Steinman, Peidong Zhao, Denise Hall, Carol Grimm, and Stacey Lind. I would like to thank the many undergraduate students, especially Sana Hira, who worked with cell cultures and cell killing studies, Munira Munshi, who worked on the oxidation studies of the phenothiazine trimer, and all of the others who worked with me in the research group, as their assistance was invaluable. I am also grateful for the financial assistance from Loyola University in the form of teaching assistantships and a 3rd year teaching fellowship, without which this research would not have been possible. iii Finally, I would like to thank my family and my most beloved friend, John Sahagian, for their support of me throughout this entire endeavor. iv TABLE OF CONTENTS ACKNOWLEDGMENTS iii LIST OF SCHEMES vii LIST OF FIGURES viii LIST OF TABLES xiii LIST OF ABBREVIATIONS xv ABSTRACT xxii CHAPTER ONE: CANCER AND PHOTODYNAMIC THERAPY 1 Introduction 1 Photodynamic Therapy 2 Mechanisms of Photodynamic Therapy 3 Role of Oxygen in Photodynamic Therapy 9 Importance of Light Dosimetry and Types of Light Sources for PDT 12 Characteristics of a Good Photosensitizer 14 Historical Uses of Phototherapy 15 First Generation Photosensitizers 18 Second Generation Photosensitizers 20 Third Generation Photosensitizers 25 Folate Targeted Cancer Therapy 32 Folate Receptor Expression Patterns 33 Current Uses of PDT for Non-cancerous Conditions 34 Vascular Targeting in Photodynamic Therapy 46 Inflammation and Immune Responses to PDT 51 Photobleaching and Quenching of Singlet Oxygen in PDT 57 Buchwald-Hartwig Chemistry for Pd-Catalyzed Cross Coupling Reactions Forming C-N Bonds 59 Pd-Catalyzed Cross Coupling Reactions Forming C-C Bonds 64 Ullmann Conditions for Catalysis of Cross Coupling Reactions Forming C-N Bonds 66 HeLa Cells to Test Cancer Cell Killing Potential 68 CHAPTER TWO: PURPOSE OF THE RESEARCH 70 v CHAPTER THREE: EXPERIMENTAL RESEARCH & DEVELOPMENT 74 Experimental Description of Research 74 Experimental Materials 96 Chemical Materials 96 Biological Materials 97 Experimental Procedures 97 Synthesis of N-benzylphenothiazine 97 Synthesis of N-benzyl-3,7-dibromophenothiazine 98 Attempted Ullmann Synthesis of N-benzyl-3,7- diphenothiazinylphenothiazine 99 Buchwald-Hartwig Synthesis of N-benzyl-3,7- diphenothiazinylphenothiazine 100 Procedure for Deprotection of N-benzylphenothiazine 101 Synthesis of Acetyl Phenothiazine Using Acetyl Chloride 102 Synthesis of Acetyl Phenothiazine Using Acetic Acid NHS Ester 103 Synthesis of Methyl 4-N-methylenephenothiazinylbenzoate 103 Synthesis of Methyl (3,7-dibromo)-4-N- methylenephenothiazinylbenzoate 104 Synthesis of Methyl (3,7-diphenothiazinyl)-4-N- methylenephenothiazinylbenzoate 105 Synthesis of N-Octadecylbenzamide 106 Ullmann Synthesis of Methyl 4-phenothiazinylbenzoate 107 Synthesis of Chlorin e6–PEG–Folate Conjugate 108 CHAPTER FOUR: EVALUATION OF A CHLORIN e6–PEG–FOLATE CONJUGATE AS A PHOTODYNAMIC THERAPY AGENT 110 Determination of the Phototoxicity of the Chlorin e6–PEG–FA Conjugate 110 Results and Discussion 113 Cytotoxicity / Phototoxicity Assays 116 Conclusions 125 CHAPTER FIVE: CONCLUSION 129 APPENDIX A: LIST OF FIGURES 133 APPENDIX B: LIST OF TABLES 211 REFERENCE LIST 241 VITA 284 vi LIST OF SCHEMES Scheme 1. Synthesis of N-benzylphenothiazine 74 Scheme 2. Synthesis of N-benzyl-3,7-dibromophenothiazine 75 74 Scheme 3. Ullmann Reaction Attempt to Produce Phenothiazine Trimer 76 Scheme 4. Buchwald-Hartwig Reaction to Produce Phenothiazine Trimer 76 Scheme 5. Synthetic Approach to N-benzyl-3,7-diiodophenothiazine 77 Scheme 6. Buchwald-Hartwig Synthesis of Phenothiazine Trimer 79 Scheme 7. Model Deprotection of N-benzylphenothiazine 81 Scheme 8. Model Acetylation of Phenothiazine using Acetyl Chloride 82 Scheme 9. Model Acetylation of Phenothiazine using Acetic Acid NHS Ester 84 Scheme 10. Phenothiazine Protection using methyl 4- (bromomethyl)benzoate 85 Scheme 11. Bromination of methyl 4-N- methylenephenothiazinylbenzoate 85 Scheme 12. Synthesis of methyl (3,7-diphenothiazinyl)-4-N- methylenephenothiazinylbenzoate 86 Scheme 13. Model Reaction Synthesis of N-octadecylbenzamide 86 Scheme 14. Ullmann Synthesis of methyl 4-N-phenothiazinylbenzoate 88 Scheme 15. Synthesis of Chlorin e6–PEG–Folate 89 vii LIST OF FIGURES Figure 1. Type 1 and 2 Mechanisms of Forming ROS 4 Figure 2. Structure of 8-oxo-7,8-dihydroguanosine 6 Figure 3. Buchwald-Hartwig Cross Coupling Reaction 63 Figure 4. Structure of Losartan 66 Figure 5. Endocytosis 71 Figure 6. Cross-linked Hemoglobin 72 Figure 7. Other Heterocyclic Compounds to Replace Bromine 75 Figure 8. Structures of XPhos Ligand and Pd Catalyst 79 Figure 9. Structure of PEG linker with COOH group 83 Figure 10. Structure of PEG linker with NHS ester group 83 Figure 11. Mechanism of Aminolysis Using TBD Catalyst 87 Figure 12. Structure of NH2–PEG–FA 88 Figure 13. Mechanism of Amide Coupling of Chlorin e6 to NH2–PEG–FA (Note – RCOOH indicates the carboxylic acid group at C-17 of Chlorin e6) 90 Figure 14. UV-VIS Spectrum of Ce6–PEG–FA in PBS 91 Figure 15. Reduction of Resazurin to Resorufin 111 Figure 16. Cytotoxicity / Phototoxicity of Ce6–PEG–FA 112 Figure 17. Chlorin e6–PEG–Folate Structure and FR Interactions 114 Figure 18. Structural Interactions of Folate with the Folate Receptor 115 viii Figure 19. Average Fluorescence Counts for Cells Only Control: 4 Expts. 116 Figure 20. Cytotoxicity Average % Viability for Cells Control, Ce6 Controls, and CPF: 4 Expts. 117 Figure 21. Average % Viability for Ce6 Control: 4 Expts. 118 Figure 22. Average % Viability for Ce6–PEG–FA: 4 Expts. 120 Figure 23. Average % Viability for Ce6 / CPF 10 µM: 4 Expts. 121 Figure 24. Average % Viability for All Compounds: 4 Expts. 123 Figure 25. Average % Viability for Ce6 / CPF 5 µM: 4 Expts. 124 Figure 26. Diagram of N-benzyl-3,7-diphenothiazinylphenothiazine Showing Delocalization of a Lone Electron 131 Figure 27. UV-VIS Spectrum of N-benzyl- 3,7-diphenothiazinylphenothiazine 134 Figure 28. UV-VIS Spectrum of methyl (3,7-diphenothiazinyl)-4-N-methylenephenothiazinylbenzoate 135 Figure 29. UV-VIS Spectrum of Ullmann methyl 4-N-phenothiazinylbenzoate 136 Figure 30. UV-VIS Spectrum of Folic Acid Dihydrate Standard 137 Figure 31. UV-VIS Spectrum of Folic Acid Dihydrate Standard 138 Figure 32. UV-VIS Spectrum of Folic Acid Dihydrate Standard 139 Figure 33. UV-VIS Spectrum of Folic Acid Dihydrate Standard 140 Figure 34. UV-VIS Spectrum of Ce6 Standard 141 Figure 35. UV-VIS Spectrum of Ce6 Standard 142 Figure 36. UV-VIS Spectrum of Ce6 Standard 143 Figure 37. UV-VIS Spectrum of Ce6 Standard 144 ix Figure 38. IR Spectrum of N-benzyl-3,7-diphenothiazinylphenothiazine 145 Figure 39. IR Spectrum of methyl (3,7-diphenothiazinyl)-4-N- methylenephenothiazinylbenzoate 146 Figure 40. Mass Spectrum of N-benzyl-3,7- diphenothiazinylphenothiazine 147 Figure 41. Mass Spectrum of methyl 4-N- methylenephenothiazinylbenzoate 148 Figure 42. Mass Spectrum of methyl (3,7-diphenothiazinyl)-4-N- methylenephenothiazinylbenzoate 149 Figure 43. Mass Spectrum of Chlorin e6 150 Figure 44. Mass Spectrum of Ce6–PEG–FA 151 Figure 45. Mass Spectrum of Ce6–PEG–FA 152 Figure 46. 1H NMR of N-benzylphenothiazine 153 Figure 47. 1H NMR Expansion of N-benzylphenothiazine 154 Figure 48. 13C NMR Expansion of N-benzylphenothiazine 156 Figure 49. 1H NMR of N-benzyl-3,7-dibromophenothiazine 157 Figure 50. 1H NMR Expansion of N-benzyl-3,7-dibromophenothiazine