Design and Synthesis of Cyclic Analogs of the Kappa Opioid Receptor Antagonist Arodyn
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Design and synthesis of cyclic analogs of the kappa opioid receptor antagonist arodyn By © 2018 Solomon Aguta Gisemba Submitted to the graduate degree program in Medicinal Chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Chair: Dr. Blake Peterson Co-Chair: Dr. Jane Aldrich Dr. Michael Rafferty Dr. Teruna Siahaan Dr. Thomas Tolbert Date Defended: 18 April 2018 The dissertation committee for Solomon Aguta Gisemba certifies that this is the approved version of the following dissertation: Design and synthesis of cyclic analogs of the kappa opioid receptor antagonist arodyn Chair: Dr. Blake Peterson Co-Chair: Dr. Jane Aldrich Date Approved: 10 June 2018 ii Abstract Opioid receptors are important therapeutic targets for mood disorders and pain. Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and 1,2,3 4 8 depression. Arodyn (Ac[Phe ,Arg ,D-Ala ]Dyn A(1-11)-NH2), an acetylated dynorphin A (Dyn A) analog, has demonstrated potent and selective KOR antagonism, but can be rapidly metabolized by proteases. Cyclization of arodyn could enhance metabolic stability and potentially stabilize the bioactive conformation to give potent and selective analogs. Accordingly, novel cyclization strategies utilizing ring closing metathesis (RCM) were pursued. However, side reactions involving olefin isomerization of O-allyl groups limited the scope of the RCM reactions, and their use to explore structure-activity relationships of aromatic residues. Here we developed synthetic methodology in a model dipeptide study to facilitate RCM involving Tyr(All) residues. Optimized conditions that included microwave heating and the use of isomerization suppressants were applied to the synthesis of cyclic arodyn analogs. Initial pharmacological data indicates the constraints involving aromatic residues were generally well tolerated at KOR with most of the analogs exhibiting affinities within 3- to 4-fold that of arodyn. RCM was also used in the synthesis of head to side chain cyclized arodyn analogs. Attempted cyclizations involving Tyr(All) residues proceeded in low yields, in contrast to cyclizations involving AllGly residues. However, ring contraction products as a result of olefin isomerization were also observed during the latter cyclizations. The resulting head to side chain cyclized arodyn analogs exhibited a 5-fold decrease in KOR affinity compared to arodyn. We further explored synthesis of arodyn analogs cyclized in both the N-terminal and C- terminal segments resulting in bicyclic arodyn analogs. Here, we present the synthesis of two initial bicyclic peptide KOR ligands with different topologies. The RCM-based bicyclic arodyn iii analog exhibited KOR affinity within 3-fold that of arodyn, whereas the lactam-based bicyclic analog displayed a substantial loss in affinity for KOR. There are currently no reports of bicyclic opioid peptide ligands and such bicyclic arodyn analogs could be useful as pharmacological tools. iv Acknowledgements This dissertation is the product of years of work and generous support from more individuals than I could possibly list here. Even so, I wish to express my gratitude to a number of individuals for their kind support. First, I would like to thank my advisor Dr. Jane Aldrich for her mentorship, patience, and support during the course of my PhD. I was very fortunate to have Dr. Aldrich as a mentor; I have gained a wide variety of skill sets and learned a great deal in research and scientific thinking. In addition, I would like to mention my helpful and knowledgeable committee members whose valuable comments improved the quality of my dissertation. My sincere gratitude to Dr. Michael Rafferty, Dr. Blake Peterson, Dr. Teruna Siahaan, and Dr. Thomas Tolbert. I would also like to thank current and former members of the Aldrich lab for their continuing support and cooperation over the years. I thank Dr. Dmitry Yakovlev and Dr. Tatyana Yakovleva for their helpful discussions and always being there to ensure the lab is running; I was privileged to have their kind support. I cannot forget to thank Dr. Archana Mukhopadhayay for helpful discussions and training in cell culture and biology experiments. I also thank Dr. Michael Ferracane for helpful discussions and performing molecular modeling and docking studies. I would also like to thank Dr. Sanjeewa Senadheera and Dr. Anand Joshi for their help and training at the beginning of my graduate studies. I also thank other members of the Aldrich lab including Christianna Reedy, Dr. Tanvir Khaliq, Dr. Laura Hanold, Grant Simpson, Robyn Pescatore, Jeremy Coleman, and Jan-Louis Cosme. It has been a pleasure to work with all of you. I would also like to thank Dr. Thomas Murray and his lab members at the University of Creighton for performing pharmacological evaluation of my peptides. I was also fortunate to work with knowledgeable staff members; I thank Dr. Justin Douglas at the University of Kansas NMR v lab and Jim Rocca in the McKnight Brain Institute at the National High Magnetic Field Laboratory’s AMRIS facility at the University of Florida for helpful discussions and training in NMR experiments. Finally, I wish to thank my family: my parents John and Grace, my siblings Chris, Claudia, and Jeremy as well as my fiancée Tiffany for their unwavering commitment and overwhelming support. Words cannot express how thankful I am. Your words of encouragement have been a pillar of strength. This dissertation is dedicated to my family. vi Table of contents Abstract………………………………………………………………………………..................iii Acknowledgements………………………………………………………………….…………...v Abbreviations…………………………………………………………………………………….xii List of figures………………………………………………………………………………….....xv List of schemes………………………………………………………………………..................xx List of tables…………………………………………………………………………..................xxi Chapter 1 - Research overview ........................................................................................................1 1.1 Background and significance .................................................................................................2 1.2 Research projects and hypotheses ..........................................................................................3 1.3 References ..............................................................................................................................3 Chapter 2 - Literature review: opioid receptors ...............................................................................8 2.1 History and background of opioids ........................................................................................9 2.2 Opioid receptors ...................................................................................................................10 2.2.1 Clinically used opioids .............................................................................................12 2.2.2 Opioid receptor signaling .........................................................................................14 2.2.3 Ligands used for in vitro studies of opioid receptors ...............................................15 2.2.4 Opioid receptor structures .........................................................................................16 2.2.5 Ligand-directed signaling .........................................................................................22 2.3 Significance of the KOR ......................................................................................................24 2.3.1 KOR selective agonists .............................................................................................26 vii 2.3.2 KOR selective antagonists ........................................................................................29 2.3.2.1 Non-peptidic KOR selective antagonists ...........................................................29 2.3.2.2 Peptidic KOR selective antagonists ...................................................................32 2.4 Conclusions ...........................................................................................................................36 2.5 References .............................................................................................................................36 Chapter 3 - Literature review: challenges and opportunities for ring closing metathesis in peptide cyclization ......................................................................................................................................56 3.1 Introduction ..........................................................................................................................57 3.1.1 Solution phase and solid phase cyclization of peptides ............................................60 3.2 Ring closing metathesis (RCM) of peptides ........................................................................62 3.2.1 Polycyclic ligands and RCM ....................................................................................67 3.2.2 RCM and opioid peptides .........................................................................................72 3.2.2.1 RCM of KOR antagonists ..........................................................................75 3.3 Conclusion ............................................................................................................................78 3.4 References ............................................................................................................................78