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Synthesis and Biological Evaluation of Dynorphin Analogs, and Caco-2 Permeability of Opioid Macrocyclic Tetrapeptides

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Synthesis and Biological Evaluation of Dynorphin Analogs, and Caco-2 Permeability of Opioid Macrocyclic Tetrapeptides Synthesis and biological evaluation of dynorphin analogs, and Caco-2 permeability of opioid macrocyclic tetrapeptides By C2013 Anand Anant Joshi Submitted to the graduate degree program in department of Medicinal chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Chairperson Dr. Jane V. Aldrich ________________________________ Dr. Michael Rafferty ________________________________ Dr. Teruna J. Siahaan ________________________________ Dr. Blake R. Peterson ________________________________ Dr. Susan M. Lunte Date Defended: 5/17/2013 The Dissertation Committee for Anand Anant Joshi certifies that this is the approved version of the following dissertation: Synthesis and biological evaluation of dynorphin analogs, and Caco-2 permeability of opioid macrocyclic tetrapeptides ________________________________ Chairperson Dr. Jane V. Aldrich Date approved: 6/4/2013 ii Abstract We are interested in the development of potent and highly selective dynorphin (Dyn) analogs targeting kappa opioid receptors (KOR) and studying the pharmacokinetic and physicochemical properties of kappa opioid peptides. In contrast to the extensive structure-activity relationships (SAR) studies on Dyn A, there is minimal SAR information on Dyn B. Therefore we performed an alanine scan of Dyn B amide. The results indicated that Tyr1 and Phe4 residues are critical, while Arg7 is important for maintaining the KOR affinity of Dyn B amide. There is also minimal SAR information for the selective KOR antagonist zyklophin [Nα- 1 5 8 benzylTyr -cyclo(D-Asp ,Dap )]Dyn A(1–11)NH2 which is active in vivo after systemic administration. Hence, we synthesized linear and cyclic zyklophin analogs using solid phase peptide synthesis. To synthesize cyclic analogs the 5-11 linear fragments were selectively deprotected and cyclized, followed by extension of the peptide chain, coupling of the N-terminal N-alkyl amino acids and cleavage from the resin. We modified a key synthetic step to avoid potential racemization of the N-terminal residue. Pharmacological results suggested that the residue in position 5 has a greater influence on the KOR affinity of zyklophin than the residue in position 8. While Phe4 and Arg6 residues in zyklophin were important for maintaining KOR affinity, surprisingly Tyr1 and Arg7 were not important. The natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibited KOR activity after oral administration. Therefore, we are investigating the pharmacokinetic and physicochemical properties of these lead peptides. In Caco-2 cell monolayer permeability studies the D-Trp isomer exhibited 7-fold higher permeability in the apical to basolateral direction than CJ-15,208, and the natural product appeared to be an efflux iii substrate. The permeability of the D-Trp isomer was only 2-fold lower than that of the high permeability compound caffeine. We also examined various agents to solubilize these hydrophobic peptides that are compatible with in vivo studies. The D-Trp isomer had 5- to 40- fold higher solubility than the L-Trp isomer irrespective of the type of solubilizing enhancer used. These studies will help guide the future design of peptidic KOR ligands. iv Acknowledgements This dissertation was possible because of the cooperation, help and valuable guidance of a number of people at and outside University of Kansas, Lawrence. First and foremost I would like to extend my whole-hearted and sincere thank you to Dr. Jane Aldrich, my PhD advisor, for her guidance, sharing of knowledge and directing me on the right path for scientific thinking and research. This thesis would not have been possible without her efforts and guidance. I also want to extend to her my respect for instilling in me the idea of “ownership of your research” which I lacked in my initial period of PhD. I was fortunate for having very helpful and knowledgeable committee members: Dr. Michael Rafferty, Dr. Teruna Siahaan, Dr. Susan Lunte and Dr. Blake Peterson. Their comments and suggestions helped me improve the quality of my dissertation. I would also like to thank my lab members for their continuing support and cooperation. First and foremost I would like to thank Dr. Tatyana Yakovleva for her continuing support, love and unconditional help in research as well as non-research related activities. I would also like to thank Dr. Kshitij Patkar and Dr. Wendy Hartsock who helped and guided me during my initial PhD years and Dr Weijie Fang for providing me with control peptides and standards for biological evaluation. I cannot forget to thank Dr Tanvir Khaliq for his LC-MS/MS method development for the macrocyclic tetrapeptides and Dr Sanjeewa Senadheera for synthesizing the macrocyclic tetrapeptides. I would also like to thank other members in the Aldrich lab including Dr Dmitry Yakovlev, Dr Archana Mukhopadhyay, Solomon Gisemba and Chrstianna Reedy for their help and support during my time in the laboratory. v I would also like to extend my thank you to Dr. Thomas Murray at University of Creighton and his lab members for performing biological evaluation of my peptides and Dr Todd Williams and his laboratory for allowing me to use the LCMS/MS facility in KU Lawrence. The help extended by Norma Henley, Jane Buttenhoff and Karen Montgomery in the administrative formalities over the period of my PhD was worthwhile and my sincere thank you to them. It was also great to have some lifelong and honest friends in Lawrence and they played an important role in keeping me focused on my academic goals. A few to mention who gave their unconditional and self-less support and help include Dr Santosh Thakkar and Dr Himanshu Dande. Some of my other friends who also helped and supported me in time and need include Dr Nikunj Shukla, Dr. Rohita Sinha, Dr. Prashi Jain and Sudhir Raghavan and I would like to thank them as well. Last but not the least my Mom’s and my grandmother’s continuous self-less support always gave me inner strength to advance forward and my wife’s love and support always gave me confidence to complete my academic commitments at KU. This dissertation is dedicated to my family. vi Table of contents Abstract………………………………………………………………………………...............iii Acknowledgements………………………………………………………………….………....v Abbreviations………………………………………………………………………………….xii List of figures………………………………………………………………………………….xviii List of schemes………………………………………………………………………...............xxii List of tables…………………………………………………………………………...............xxiii Chapter 1 - Overview of the research projects................................................................................ 1 1.1 Introduction ...................................................................................................................... 2 Project 1: Synthesis and biological evaluation of alanine substituted analogs of Dyn B amide (chapter 3). ............................................................................................................................... 3 Project 2: Structure-activity relationships of the KOR selective antagonist zyklophin (chapter 4). ............................................................................................................................... 4 Project 3: Caco-2 cell monolayer permeability and solubility analysis of opioid macrocyclic tetrapeptides (chapter 6) .......................................................................................................... 5 1.2 References ........................................................................................................................ 7 Chapter 2 - Literature review – Kappa opioid receptor ligands .................................................... 10 2.1 Introduction .................................................................................................................... 11 2.2 Opioid Receptors ............................................................................................................ 12 2.2.1 Opioid receptor structure ........................................................................................ 14 2.3 Opioids with clinical applications .................................................................................. 19 2.3.1 Clinically used opioids ............................................................................................ 19 2.3.2 Opioids in clinical trials or under clinical development ......................................... 21 2.4 Significance of KOR ...................................................................................................... 23 2.5 Non-Peptidic KOR ligands............................................................................................. 25 2.5.1 KOR agonists .......................................................................................................... 25 2.5.2 KOR antagonists ..................................................................................................... 27 vii 2.6 Overview of peptidic KOR ligands ................................................................................ 31 2.6.1 Peptide agonists ...................................................................................................... 31 2.6.2 Peptide antagonists.................................................................................................. 34 2.7 Dynorphin A analogs ....................................................................................................
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