Synthesis and Characterization of Novel Ru(Ii) Dipyrrin
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SYNTHESIS AND CHARACTERIZATION OF NOVEL RU(II) DIPYRRIN COMPLEXES FOR USE AS PHOTODYNAMIC THERAPY AGENTS IN CANCER TREATMENTS Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Chemistry By Max Tsao Dayton, Ohio August 2019 SYNTHESIS AND CHARACTERIZATION OF NOVEL RU(II) DIPYRRIN COMPLEXES FOR USE AS PHOTODYNAMIC THERAPY AGENTS IN CANCER TREATMENTS Name: Tsao, Max APPROVED BY: Shawn Swavey, Ph.D. Committee Chair Mark Masthay, Ph.D. Faculty Advisor Angela Mammana, Ph.D. Faculty Advisor ii ABSTRACT SYNTHESIS AND CHARACTERIZATION OF NOVEL RU(II) DIPYRRIN COMPLEXES FOR USE AS PHOTODYNAMIC THERAPY AGENTS IN CANCER TREATMENTS Name: Tsao, Max University of Dayton Advisor: Dr. Shawn Swavey Cancer remains a significant obstacle in modern healthcare, and efforts are needed to discover and advance new treatment methods and compounds. Accordingly, two novel dipyrrin compounds, consisting of a heteroleptic monometallic ruthenium(II) complex and a heteroleptic trimetallic ruthenium(II) complex, were synthesized and characterized for use as photosensitizers in photodynamic therapy of cancer. In the monometallic complex, a π to π* transition is observed originating from the bipyridyl groups along with overlapping MLCT from Ru(dπ) to bpy(π*) and ligand centered transition occurring at a range between 520 nm and 600 nm. The trimetallic complex contains an expected π to π* dipyrrin transition at 294 nm and Ru(dπ) to bpy(π*) MLCT transitions at 355 nm and 502 nm. An intense transition occurring at 578 nm from overlapping dipyrrin π to π* and Ru(dπ) to dipyrrin(π*) is also observed. These transitions were predominantly analyzed through electrochemical and spectroelectrochemical experiments. Performance of the complexes was initially tested through irradiation in the 600 to 850 nm photodynamic therapy window while in the presence of plasmid DNA, with both complexes showing the ability to cause photo-damage to the DNA backbone. However, in the absence of iii oxygen, the trimetallic Ru(II) complex also generated photo-induced DNA damage, which is suggestive of a photo-oxidative Type I process. Cytotoxicity of the complexes up to 50 µM concentration towards A549 lung cancer cells was negligible in the absence of light. The trimetallic complex demonstrated significantly greater photo-cytotoxicity compared to the monometallic analog upon irradiation of the cells with a 420 nm low power light source. A dose dependent response curve results in an IC50 value of 92 µM for complex B. iv ACKNOWLEDGMENTS I’d like to thank everyone who has been a part of my life and given support me over the years. v TABLE OF CONTENTS ABSTRACT …………………………………………………………..........................….iii ACKNOWLEDGMENTS ……………………………………………………………..….v LIST OF FIGURES ..…………………………………………………………………....viii LIST OF ABBREVIATIONS ……………………………………………………………..x CHAPTER 1 INTRODUCTION ………………………………………………...…..……1 Cancer and Conventional Treatments………………………………….………….1 Photodynamic Therapy....................……………………………...…..……….…..4 Current PDT Agents……….………………...……………………..…….………..7 Ruthenium Complexes……………………………………………..…….………..9 CHAPTER 2 EXPERIMENTAL…… ……………………………..……….………..…..12 Materials and Methods……………………………………………….…………..12 Synthesis of Ruthenium Complexes A and B……………………..….………….12 Complex A………........................................................…….……………12 Complex B…………………………………………………….…………13 Electronic Spectroscopy……………………………………………….…………14 Electrochemistry…………………….……………………………….…………...14 DNA Photo-Cleavage Studies…….……………………...………….....…………15 A549 Cell Culture and Photo-Irradiation…………………….…..……….……....15 A549 Viability and Reactive Oxygen Species Assessments……............………..16 CHAPTER 3 RESULTS AND DISCUSSION …….………………………...……….….17 Synthesis……………………………………………………………….………...17 vi Electronic Spectroscopy……………………….……………………………...….19 Electrochemistry.…………………………………………………………………21 DNA Photo-Cleavage Studies…….................…………………………...……….23 Singlet Oxygen Studies………………………………………………...…….......27 A549 Cell Studies…………….…………………………………...………....…...29 Cellular Reactive Oxygen Species....……..………………………..………...…...31 CHAPTER 4 CONCLUSIONS …………………………………..…………………..….35 REFERENCES …………………………………………………………………….…….37 vii LIST OF FIGURES Figure 1. Cost increases of chemotherapeutic drugs after release to market ......................3 Figure 2. Jablonski diagram of type I and II pathways for photodynamic therapy .............5 Figure 3. Behavior and propagation of light through tissues ……………………………..6 Figure 4. Structure of Photofrin, in which n = 1-8 …………………………………..……8 Figure 5. Historical progression of Ru anticancer compounds …………...………….…..10 Figure 6. Structures of the heteroleptic (A) and supramolecular (B) ruthenium(II) complexes ……....…………………………………………………….………….11 Figure 7. Synthesis of the heteroleptic Ru(II) complex A ……………………….………17 Figure 8. Synthesis of the Ru(II) supramolecular complex B ………………..…….……18 Figure 9. Electronic spectra of N-methylated dipyrrin, complex A, and complex B in acetonitrile at room temperature …….......………………....…19 Figure 10. Cyclic voltammetry of N-methylated dipyrrin, complex A, and complex B in acetonitrile with Bu4NPF6 as supporting electrolyte ..........……….21 Figure 11. A) Spectroscopic changes associated with bulk electrolysis of an acetonitrile solution of complex B at V = 1.10 vs Ag/AgCl. B) Spectrum of complex B before electrolysis and after reduction to original state …...…...…22 Figure 12. Gel electrophoresis of circular plasmid DNA pUC18 in the absence (lanes C) and presence (lanes 1–9) of complex A (A, top) and complex B (B, bottom) at a 5:1 base pair to complex ratio at 5 min intervals with 550 nm filter .............................................................................................................….24 Figure 13. Gel electrophoresis of circular plasmid DNA (pUC18) in the viii absence (lanes C) and presence (lanes 1–9) of complex A (A, top) and B (B, bottom) at a 5:1 base pair to complex ratio at 10 min intervals with 420 nm filter in absence of oxygen …………………………………..….….26 Figure 14. Absorption spectra of DPBF acetonitrile solutions in the presence 2+ of complexes A (A, left), B (B, middle), and [Ru(bpy)3] (C, right) after irradiation with a 550 nm long band pass filter for A/B or a 420 nm long band pass filter in the case of C …………………………………...……………..28 Figure 15. Determination of A549 viability following a 24 h exposure to varying concentrations from 1 to 50 μM of either complex A (A, left) or complex B (B, right) without photo-stimulation …………………….………..30 Figure 16. Examination of A549 viability following concurrent complex exposure and irradiation with a 420 nm light source for 15 min, with complex A (A, left) and complex B (B, right) at varying concentrations from 1 to 50 μM .…………………………...………………………………….…31 Figure 17. Quantification of A549 stress, as assessed through ROS activation following photo-stimulated exposure to complex A (A, left) and complex B (B, right) at varying concentrations from 1 μM to 50 μM …………....………….32 Figure 18. Quantification of the IC50 value for complex B following irradiation was performed using a high dose-dependent cytotoxicity analysis …..………….34 ix LIST OF ABBREVIATIONS ANOVA analysis of variance BODIPY boron dipyrromethene bpy 2,2’-bipyridine Bu4NPF6 tetrabutylammonium hexafluorophosphate DI deionized DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPBF 1,3-diphenylisobenzofuran E1/2 half-wave potential ΔE potential difference between anodic and cathodic peaks Epa potential at peak anodic current Epc potential at peak cathodic current Et3N trimethylamine FDA Food and Drug Administration h-cell half cell HR-ES-MS high resolution-electrospray ionization-mass spectroscopy IC50 half maximal inhibitory concentration ILCT intra-ligand charge transfer ISC intersystem crossing IR infrared LLCT ligand to ligand charge transfer x MLCT metal to ligand charge transfer NAMI sodium (Na) imidazole (Mi) anticancer agent NH4PF6 ammonium hexafluorophosphate NSCLC non-small cell lung cancer pH log10 of 1/hydrogen ion activity PD-1 programmed death 1 protein PD-L1 programmed death ligand 1 protein PDT photodynamic therapy PF6 hexafluorophosphate PS photosensitizer pUC18 plasmid University of California 18 DNA ROS reactive oxygen species RPMI Roswell Park Memorial Institute Tris buffer tris(hydroxymethyl)aminomethane UV ultraviolet UV-vis ultraviolet-visible ε molar absorptivity π pi bonding orbital π* pi antibonding orbital xi CHAPTER 1 INTRODUCTION Cancer and Conventional Treatments Cancer continues to be one of the primary challenges for modern medicine, with an expected 1.7 million new cases and 610 thousand deaths in the US in 2018 alone [1]. Significant financial resources and manpower have been dedicated to tackling this issue, with an estimated 1.3 billion dollars needed to bring a drug to market [2]. Progress in cancer agents has remained shockingly low, with only 8% of compounds that enter phase I eventually reaching the market and only 50% of compounds that reach phase III successfully complete the trials [3]. Cancerous cells have undergone significant DNA mutations, resulting in cells that aggressively proliferate, evade the immune system, and spread to multiple parts of the body [4], all of which contribute to obstacles in treatment and necessitate patient specific plans. Current conventional treatment options typically consist of surgery, radiation therapy, chemotherapy, and more recently, immunotherapy,