Synthesis of Insecticidal Mono- and Diacylhydrazines for Disruption of K+ Voltage-Gated Channels, Elucidation of Regiochemistry, and Description of Conformational Isomerism by NMR Spectroscopy and Computation Joseph Shelby Clements II Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemistry Paul R. Carlier, Chair David G. I. Kingston James M. Tanko Gordon T. Yee May 2, 2017 Blacksburg, VA Keywords: Hydrazine, acylation, α-effect, regioselectivity, hydrazide, voltage- gated, (E)-/(Z)-, 19F NMR, single electron transfer (SET), rotational barrier, N-N bond rotation, rotational isomerism, helicity, transition state, dihedral angle Copyright 2017, Joseph S. Clements II Synthesis of Insecticidal Mono- and Diacylhydrazines for Disruption of K+ Voltage-Gated Channels, Elucidation of Regiochemistry, and Description of Conformational Isomerism by NMR Spectroscopy and Computation Joseph Shelby Clements II ABSTRACT Based on the success of diacyl-tert-butylhydrazines RH-5849 and RH-1266 in controlling agricultural crop pests, we endeavored to synthesize our own diacylbenzyl- and arylhydrazine derivatives for use against the malaria vector Anopheles gambiae. In the process of producing a library of compounds for assay against An. gambiae, it became clear that employing regioselective acylation techniques (in molecules that feature two nucleophilic, acyclic nitrogen atoms α to one another) would be imperative. Synthesis of the library derivatives proceeded rapidly and after topical assay, we found three compounds that were more toxic than the RH- series leads. One of the three displayed an LD50 value of half that of RH-1266, though patch clamp assay concluded that toxicity was not necessarily linked to inhibition of mosquito K+ channel Kv2.1. The acylation of monoarylhydrazines appears simple, but its regioselectivity is poorly understood when assumed as a function of basicity correlating to nucleophilic strength. We determined the ratio of the rate constants for distal to proximal N-acylation using 19F NMR spectroscopic analysis of reactions of 4-fluorophenylhydrazine with limiting (0.2 equiv) acylating agent in the presence of various bases. Acid anhydrides gave consistent preference for distal acylation. The selectivity of acylation by acyl chlorides when using pyridine gives strong distal preference, whereas use of triethylamine or aqueous base in conjunction with aroyl chlorides showed a moderate preference for proximal acylation. This observation yielded a convenient one-step method to synthesize proximal aroylarylhydrazines in yields comparable or superior to that provided by the standard three-step literature approach. Combined with NMR evidence of the distal nitrogen as the unambigiously stronger base of the two nitrogens, we propose a single electron transfer mechanism that predicts the regiochemistry of arylhydrazines toward acylating agents better than the nucleophilicity model based on pKa values. While synthesizing the acylhydrazine library for assay against An. gambiae, NMR spectroscopy revealed rotational isomerisms of two types: chiral helicity (M)/(P) and acyl (E)/(Z)-isomerism due to hindered rotation. Variable temperature NMR allowed the measurement of N-N bond rotational barriers, as well as estimate the barrier of (E)/(Z) interconversion. We obtained the X-ray crystal structures of four diacylhydrazines to test this hypothesis and revealed both the twist conformation around the N-N bond axis and (E)/(Z)- isomerism around the proximal acyl group. Computation (which agreed with the crystal structures) allowed us to estimate which (E)/(Z)-isomers were most likely being observed in solution at room temperature by NMR spectroscopy. In addition, we were able to calculate transition structures corresponding to N-N bond rotational barriers of (E,Z)- and (Z,Z)-isomers of model molecules and rationalize the difference in coalescence temperatures between (E,Z)- and (Z,Z)-isomers. GENERAL AUDIENCE ABSTRACT Herein we present the work of both synthesizing and characterizing the mosquitocidal and chemical properties of acylhydrazines. Part of the challenge of working with hydrazines comes in part from deceptive comparisons to amines and ammonia; hydrazine is as different from ammonia as hydrogen peroxide is from water. We were successful in identifying effective synthetic techniques to obtain our desired acylhydrazines reliably and managed to discover compounds that were better at eliminating Anopheles gambiae (the african malaria mosquito vector) than lead compounds from previous researchers. In the process of making the library of compounds for mosquito testing, we explored hydrazine reactivity toward acylating agents in a direct and deeper way than previous work, as well as their dynamic structural features. We employed a battery of techniques, including NMR, X-ray crystallography, and computational molecular modeling to understand these molecules and possibly contribute insight into their biochemical efficacy. Acknowledgements Firstly, I would like to extend my gratitude to my PhD advisor Dr. Paul R. Carlier. More than once, we would sit down for a 30 minute meeting about some results from the past week and 2 hours later we would have come up with a new idea to test. My work with Paul Carlier has always been mutual collaboration with emphasis on discovery and clarity. He has shown receptiveness to my ideas and independent thinking; all the while, not being afraid to tell me when I’m wrong...and sometimes vice versa. As we transition from the relationship of the student and the teacher to becoming colleagues, I will still look toward Dr. Carlier for his professional advice and counsel. It has been a pleasure to work with him over these past several years. I would also like to thank my advisory committee Drs. Kingston, Tanko, and Yee for their suggestions, patience, and interest in my work and my professional success. Secondly, I would like to thank my fellow graduate students, post-doctoral colleagues, collaborators, and analytical staff for their willingness to work with me to achieve success in my research work. I’d like to thank Drs. Qiao-Hong Chen and Ming Ma for their indispensable and practical guidance at the very beginning of my graduate career in organic synthesis. I’d like to thank Drs. Dinesh Nath and Zhong-Ke Yao for working alongside me on acylhydrazine syntheses at the beginning of that project, as well as my former pupil Michael Black. I thank Dr. Eric Nybo for our long friendship and conversations on our research which has proved inspiring time after time. I extend my gratitude to Dr. Gary Richoux, Dr. Astha Verma, Dr. Eugene Camerino, Dr. Neeraj Patwardhan, Sha Ding, and Lixuan Liu for your time and inclination to discuss my work and offer your thoughts. Maryam Ghavami deserves special thanks for contributing her synthetic prowess to my project on regioselective proximal acylation of aryl hydrazines. I am grateful to Dr. Carla Slebodnick for her expertise and advice toward obtaining v the vital X-ray crystal structures for my research. My sincere gratitude goes to Dr. Narasimhamurthy Shanaiah and Ken Knott for their guidance and hours of time in front of the 600 MHz NMR, helping me learn how to do variable temperature measurements properly. I’d like to thank Bill Bebout and Dr. Mehdi Ashraf-Khorassani for their hard work, troubleshooting, and patience with me and my compounds. I’d like to thank our collaborators Drs. Jeff Bloomquist, Fan Tong, Rafique Islam, and Baonan Sun at the University of Florida Department of Entomology and Nematology for their diligent work in biological assay of my compound library for mosquito toxicity as well as K+ channel inhibition. Finally, those outside of academia have contributed greatly to my success and they too deserve recognition for their part this accomplishment. My parents, Markus and Sharon Clements deserve thanks for their love, their moral and material support, and allowing me to pursue science and encouraging it at a young age. I thank my sister Kristi Wilkinson for her support and protectiveness of her little brother in his vulnerable young years. My Mother and Father in-law, David and Peggy Heimerdinger supported me when I needed it and I will remember it always. Lastly, I would like to thank my wife Rachel Kathryn Heimerdinger most of all. You were there for me when I was at my weakest in mind and body; it was your love, work, patience, courage, and fortitude that have seen me through to this moment and I am forever grateful. vi Dedication I dedicate this work to Rachel Kathryn Heimerdinger and all those who would pursue and facilitate scientific endeavor for the furtherance of our human civilization. vii Chapter 1. Introduction to Hydrazine Chemistry: Nucleophilicity, Synthesis, Conformational Chirality and Isomerism .................................................................................. 1 1.1 Introduction ........................................................................................................................... 1 1.2 Insecticidal action of diacylhydrazines ................................................................................. 5 1.3 The α-effect ........................................................................................................................... 9 1.4 Direct alkylation of phenylhydrazine by alkylhalides. ....................................................... 17 1.5 Acylation