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View Is Primarily on Addressing the Issues of Non-Permanently Charged Reactivators and the Development of Treatments for Aged Ache

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View Is Primarily on Addressing the Issues of Non-Permanently Charged Reactivators and the Development of Treatments for Aged Ache Design, Synthesis, and Evaluation of Therapeutics for the Treatment of Organophosphorus Poisoning by Nerve Agents and Pesticides Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Andrew Joseph Franjesevic Graduate Program in Chemistry The Ohio State University 2019 Dissertation Committee Professor Christopher M. Hadad, Advisor Professor Thomas J. Magliery Professor David Nagib Professor Jonathan R. Parquette Copyrighted by Andrew Joseph Franjesevic 2019 2 Abstract Organophosphorus (OP) compounds, both pesticides and nerve agents, are some of the most lethal compounds known to man. Although highly regulated for both military and agricultural use in Western societies, these compounds have been implicated in hundreds of thousands of deaths annually, whether by accidental or intentional exposure through agricultural or terrorist uses. OP compounds inhibit the function of the enzyme acetylcholinesterase (AChE), and AChE is responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh), and it is extremely well evolved for the task. Inhibition of AChE rapidly leads to accumulation of ACh in the synaptic junctions, resulting in a cholinergic crisis which, without intervention, leads to death. Approximately 70-80 years of research in the development, treatment, and understanding of OP compounds has resulted in only a handful of effective (and approved) therapeutics for the treatment of OP exposure. The search for more effective therapeutics is limited by at least three major problems: (1) there are no broad scope reactivators of OP-inhibited AChE; (2) current therapeutics are permanently positively charged and cannot cross the blood-brain barrier efficiently; and (3) current therapeutics are ineffective at treating the aged, or dealkylated, form of AChE that forms following inhibition of of AChE by various OPs. iii Herein we report initial computational investigations, the synthesis, and then the biological evaluation of a new class of therapeutic quinone methide precursors (QMPs) for the purpose of treating OP exposure. We have thus far been able to demonstrate that our compounds bind with AChE, reactivate OP-inhibited AChE, and resurrect (restore) native function of the aged-AChE. These compounds are non-oxime structures and address at least two of the three major problems with current therapeutics. The testing of a structure-activity library of 40 substituted QMPs for the resurrection of methylphosphonate-aged AChE yielded great success. In total, 20 of the 40 compounds showed resurrection of the methylphosphonate-aged AChE, with the best compound achieving ~14.5% recovery in 24 hours at a pH of 7.5. Further testing of the top seven compounds revealed that there is a significant dependence of resurrection on the medium’s pH and the ratio of [QMP]:[AChE] in solution. Interestingly, at varying concentrations of [QMP]:[AChE], one compound is shown to be the most effective, recovering 54.9% and 62.6% of the original native activity at pH 7.5 after 24 hours when the [QMP]:[AChE] ratio is increased by 5 and 10 fold, respectively. Members of this structure-activity relationship library were even more effective with organophosphate pesticides, and many of these compounds recovered >50% in 24 hours. With methyl paraoxon as the pesticide, our QMPs could recover >20% in 1 hr, and with an effective concentration (EC50) of about 100 µM. The vast improvement in therapeutic efficiency over the course of this research demonstrates the potential of these compounds for the treatment of OP exposure and warrants significant continued investigation. iv Acknowledgments Absolutely none of this would have been possible without the continued support of my family and friends. There were points in times when I no longer wished to continue pursuit of my doctorate degree but the continued support of my immediate family and Professor Christopher Hadad gave me the motivation and strength to continue forward. At times this program left me distraught but the love and support of my mother, father, and brother never waivered. I would like to personally thank Chris for his continued guidance both in my chemistry endeavors as well as personal experiences during the course of this program. His belief in my abilities as both a chemist and mentor drove me to continue my career in organic chemistry and the sciences. I also wish to thank Dr. Christopher Callam for his mentorship in regards to both chemistry as well as graduate school and career pursuits. To Dr. Qinggeng (Albert) Zhuang and Dr. William (Bill) Coldren, thanks for making lab interactive. I have greatly enjoyed our various conversations whether chemistry related or not. To Dr. Thomas (Tom) Corrigan for his assistance in helping me develop my synthetic skills in the time we managed to work with one another. And to all the current and future group members, I share such a passion for the research conducted in the Hadad lab. Each year the group has grown closer and has proven to be more fun. I wish the best in your future research and I’m excited for the progress to come. v Vita 2014 ………………………………………………...B.S. Chemistry, Wittenberg University 2014 to present ……………………………………..Graduate Research Associate, Department of Chemistry and Biochemistry, The Ohio State University Publications Franjesevic, A. J.; Sillart, S. B.; Beck, J. B.; Vyas, S.; Callam, C. S.; Hadad, C. M. Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase. Chem. Eur. J. 2019, 25, 5337-5371. Zhuang, Q.; Franjesevic, A. J.; Corrigan, T. S.; Coldren, W. H.; Dicken, R.; Sillart, S.; DeYong, A.; Yoshino, N.; Smith, J.; Fabry, S.; Fitzpatrick, K.; Blanton, T. G.; Joseph, J.; Yoder, R. J.; McElroy, C. A.; Dogan Ekici, O.; Callam, C. S.; Hadad, C. M. Demonstration of In Vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents. J. Med. Chem. 2018, 61, (16), 7034–7042. vi Yoder, R. J.; Zhuang, Q.; Beck, J. M.; Franjesevic, A.; Blanton, T. G.; Sillart, S.; Secor, T.; Guerra, L.; Brown, J. D.; Reid, C.; McElroy, C. A.; Dogan Ekici, O.; Callam, C. S.; Hadad, C. M. Study of para-Quinone Methide Precursors toward the Realkylation of Aged Acetylcholinesterase. ACS Med. Chem. Lett. 2017, 8, (6), 622–627. Fields of Study Major Field: Chemistry vii Table of Contents Abstract .............................................................................................................................. iii Acknowledgments ............................................................................................................... v Vita ..................................................................................................................................... vi List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1. INTRODUCTION TO ORGANOPHOSPHORUS COMPOUNDS AND THEIR INHIBITION AND AGING OF ACETYLCHOLINESTERASE ........................ 1 1.1 Historical Development of Organophosphorus Pesticides and Nerve Agents .......... 1 1.2 Recent Uses of Organophophorus Pesticides and Nerve Agents for Terrorism. ...... 7 1.3 Acetylcholinesterase Structure and Function in the Native, Inhibited, and Aged States. .............................................................................................................................. 9 1.4 Butyrylcholinesterase Structure and Function in the Native, Inhibited, and Aged States. ............................................................................................................................ 19 1.5 Reactivation of Acetylcholinesterase. ..................................................................... 24 1.6 Recent Results from Efforts to Develop Broad Scope Reactivators of Acetylcholinesterase. .................................................................................................... 25 1.7 Developing Non-Permanently Charged Oxime Reactivators of OP-Inhibited Acetylcholinesterase. .................................................................................................... 43 1.8 Developing Non-Oxime Based Reactivators of Inhibited Acetylcholinesterase. ... 54 1.9 Difficulties with the Evaluation of Developed Reactivators. ................................. 62 1.10 Reactivation of Butyrylcholinesterase. ................................................................. 64 1.11 Recent Results for the Reactivation of Butyrylcholinesterase. ............................. 66 1.12 Resurrection of Aged Acetylcholinesterase. ......................................................... 77 1.13 Early Attempts at the Realkylation of Aged Acetylcholinesterase. ...................... 78 1.14. Quinone Methides as Alkylating Agents of Model Nucleophiles. ...................... 89 1.15 Mannich Bases for the Alkylation of Aged Acetylcholinesterase. ....................... 95 1.16 Problems with Realkylation of Aged AChE. ...................................................... 101 1.17 Perspective and Future Work. ............................................................................. 103 viii Chapter 2. COMPUTATIONAL INSIGHTS INTO THE ALKYLATION REACTIONS OF PYRIDINE AND PYRIDINIUM QUINONE METHIDE PRECURSORS: AN EFFORT TO REVERSE THE EFFECTS OF ACETYLCHOLINESTERASE FOLLOWING AGING ..................................................................................................
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