Chapter 1: Synthesis of Sidechain Functionalized Polyamines and Study of Their RNA-Binding Properties

NORTHWESTERN UNIVERSITY Part 1: Synthesis of Side-Chain Functionalized Polyamines and Study of their RNA-Binding Properties Part 2: Synthesis and Evaluation of Heterocycle-Based Selective Inhibitors of Neuronal Nitric Oxide Synthase with Improved Bioavailability A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree DOCTOR OF PHILOSOPHY Field of Chemistry By Graham R. Lawton EVANSTON, ILLINOIS JUNE 2007 2 © Copyright by Graham R. Lawton 2007 All Rights Reserved 3 ABSTRACT Part 1: Synthesis of Side-Chain Functionalized Polyamines and Study of their RNA-Binding Properties Part 2: Synthesis and Evaluation of Heterocycle-Based Selective Inhibitors of Neuronal Nitric Oxide Synthase with Improved Bioavailability Graham R. Lawton Part 1. The complex folded structures associated with RNA allow for specific protein-RNA interactions and also create binding sites for small molecules. Developing organic molecules that can bind RNA with high affinity and specificity is a challenge that must be overcome for RNA to be considered a viable drug target. Polyamines with different side chains were synthesized to test for binding affinity and specificity to TAR and RRE of HIV. Binding interactions between polyamines and RNAs were examined using fluorescence assays and two foot-printing assays based on terbium-induced cleavage and magnesium-catalyzed cleavage at high pH. Binding constants and specificity were highly dependent on the side chains of the polyamines, demonstrating that this class of molecules is a promising starting point for the development of highly selective RNA binding ligands. Part 2. The overproduction of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS) has been implicated in a variety of neurological diseases, including Parkinson’s and neuronal damage due to hypoxic conditions such as stroke. Inhibition of nNOS could have therapeutic benefit, but must be achieved without inhibition of the endothelial isoform (eNOS), as that would 4 lead to hypertension. Using computer modeling of drug-like fragments in the crystal structures of nNOS and eNOS, a class of highly selective and potent nNOS inhibitors was discovered. Modification of the structures of these lead compounds to optimize for pharmacological properties without sacrificing potency and selectivity was carried out. Replacing a 2- aminopyridine group in the leads with aminothiazoles resulted in a dramatic loss in potency. The replacement of secondary amines with ether and amide linkages generally reduced potency; however, in one case an ether-containing compound was as potent and selective as the best lead compound. Brain uptake studies in mice proved that the modification did have a beneficial effect on the amount of compound that crosses the blood brain barrier. The neutralization of other secondary amines in the molecule with amides and carbamates was carried out to investigate whether a prodrug approach could further increase brain uptake. In all cases, however, the capping of the secondary amine had no beneficial effect on brain uptake. 5 ACKNOWLEDEMENTS I would like to express my deepest gratitude to all those who helped me throughout my graduate career. I have been extremely lucky in my colleagues, mentors and collaborators without whom this work would not have been possible. I am grateful to Professor Richard B. Silverman for taking me into his group in 2004 and giving me the chance to carry out interesting chemistry on an exciting project. I appreciate the freedom he gave me to design my own syntheses, explore new avenues in the project, and develop into an independent researcher. I wish to thank all of the members of the Silverman group, past and present, for creating an enjoyable atmosphere in which to carry out research and for their assistance and ideas. I especially need to thank Dr. Haitao Ji for the use of his molecular modeling diagrams and for his assistance with my syntheses, Michael Clift for the work he did on ether formation reactions, and Ian Pulford for help with the aminoimidazole synthesis. I would like to thank Dr. Daniel H. Appella for his enthusiasm, encouragement and guidance on the polyamine project. I want to thank all of the members of the Appella lab from 2002-2004, in particular Dr. Michael C. Myers, who gave me many practical tips on organic synthesis. I wish to thank Professor Olke Uhlenbeck for allowing me to carry out the RNA binding assays in his laboratory, providing essential equipment and supplies, and for offering advice and encouragement. I am grateful to all the members of his laboratory who went out of their way to help me with my project, especially Dr. Richard P. Fahlman and Dr. Fedor (Ted) V. Karginov, whose ideas and critiques were invaluable in the design of my RNA assays, interpretation of the results and preparation of the corresponding manuscript. 6 My warmest thanks go to Professor D. Martin Watterson for providing me with a place to carry out research essential to my project. In addition, he offered helpful advice and taught me a great deal about pharmacology and drug discovery. I apologize to his group members for any inconvenience I may have caused them while conducting research in their lab, and am grateful to them all for their assistance, especially Dr. Hantamalala Ralay Ranaivo, who carried out the majority of the animal administrations, and Laura K. Wing, who helped me design and interpret the in vitro metabolism assays. I am grateful to Professor Michael J. Avram and Lynn Luong for carrying out the LCMS analysis of my biological samples. They were both extremely helpful and accommodating and I enjoyed working with them and learning from them. My deepest thanks go to all those who have offered me emotional support and have encouraged me and made me the person I am today. I want to thank my Mum and Dad and all my family for their love and encouragement over the years, and for supporting my decision to come to Chicago. I want to thank my wife’s family, particularly Howard and Beverly, for providing me with a loving support network here in the U.S. I want to thank Dr. Benjamin Brooks for igniting my interest in chemistry, all my friends, Ozzie and Kenny, and all others who have been a part of my life over the last 5 years. Finally, and most importantly, I need to thank my beloved wife, Victoria Lawton, for her love, friendship and support, for helping to keep me sane, and for being the best companion a man could want. 7 TABLE OF CONTENTS Abstract...................................................................................................................................... 3 Acknowledgements.................................................................................................................... 5 Table of Contents....................................................................................................................... 7 List of Figures............................................................................................................................ 10 List of Schemes.......................................................................................................................... 15 List of Tables ............................................................................................................................. 17 Chapter 1: Synthesis of Sidechain Functionalized Polyamines and Study of their RNA-Binding Properties................................................................... 18 1.1 RNA as a drug target ........................................................................................................... 19 1.2 Antibiotics that bind ribosomal RNA .................................................................... 22 1.3 Binding of small molecules to other RNAs........................................................... 23 1.4 Polyamines as RNA-binding small molecules....................................................... 29 1.5 Analysis of RNA binding ...................................................................................... 30 1.6 Synthesis ................................................................................................................ 34 a) Reduction of resin-bound tripeptides using borane ........................................... 34 b) Reductive amination.......................................................................................... 37 1.7 Fluorescence-based assay ...................................................................................... 44 1.8 Footprinting assays ................................................................................................ 48 1.9 Test for epimerization during reductive amination................................................ 53 1.10 Synthesis of alternative aldehydes....................................................................... 57 a) Fmoc protected γ-amino aldehydes ................................................................... 57 b) Trityl protection of glutamine and citrullene sidechains................................... 59 1.11 Summary.............................................................................................................. 61 1.12 Experimental procedures ..................................................................................... 62 8 Chapter 2: Synthesis and Evaluation of 2- and 4-Aminothiazole-based Inhibitors of Neuronal Nitric Oxide Synthase ......................................................... 89 2.1 Biological role of nitric oxide...............................................................................

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