Structural Evolution of a Bifunctional Mu-Opioid Receptor (MOR)/Delta-Opioid Receptor (DOR) Peptidomimetic as Nonaddictive Opioid Analgesics by Sean P. Henry A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in the University of Michigan 2019 Doctoral Committee: Professor Henry I. Mosberg, Chair Professor Scott D. Larsen Associate Professor Peter J. H. Scott Professor John. R. Traynor Sean P. Henry [email protected] ORCID iD: 0000-0002-4405-062X © Sean P. Henry 2019 Acknowledgements I would first and foremost like to thank the Lord my God. Without His grace, I would not have been able to enter this PhD program, nor would I have been able to complete the research presented herein. Without His guidance, I would not have been able to find my calling: the synthesis and development of novel pharmaceuticals. For that, I am eternally grateful. To my family back home, Timothy Henry, Brenda Skinner, Kevin Henry, and Joan Henry, thank you all for supporting me and blessing me with all you know. I love you all and hope to give to others what you have given to me. Timothy, you have been a source of inspiration, for both your love of science and for your enduring patience. May you rest in peace. Dr. Henry Mosberg, your guidance and wisdom throughout my time here has thoroughly enriched my understanding of medicine and drug design. Without your efforts, I would not have been able to complete this program and the quality of our work would have suffered. For that, I thank you. I also would like to thank the members of my committee: Dr. Scott Larsen, Dr. Peter Scott, and Dr. John Traynor. Your insights have broadened my horizons and have contributed greatly to my development as a scientist. I would like to thank Dr. Nicholas Griggs, Thomas Fernandez, and Dr. Jess Anand for acquiring the in vitro data presented herein. Without these data, this project would only be an exercise in organic synthesis and the contributions to our understanding of the opioid receptors would be lost. I would also like to thank Bryan Sears, whose studious efforts in acquiring our in vivo data have proven invaluable for the development of these ligands. Finally, I would like Kate Kojiro, whose knowledge in the laboratory have streamlined my work by no small means. ii To Dr. Larisa Yeomans and your husband Matthew Cruz, your kindness and grace have always been warm and welcoming. You have always been there when I needed you, and for that, I count among my dearest of friends. To my coworkers Dr. Tanpreet Kaur, Dr. Deanna Montgomery, Dr. Tony Nastase, and Jay Kim, your levity and companionship have lightened many a rainy day. The completion of this dissertation would have been much less wholesome without you all. Finally, I would like to thank my friends from both GCF and 2:42. Your friendship has contributed to my growth in innumerable ways and I will never forget the good times that we have had together. iii TABLE OF CONTENTS Acknowledgments ii List of Figures vi List of Schemes viii List of Tables ix List of Abbreviations xi Abstract xv Chapter 1: On Opioids 1 1.1 The Opioid Epidemic 1 1.2 The Opioid Receptors and their Effects 2 1.3 Bifunctional MOR/DOR Ligands 4 1.4 From the Opioid Peptides to AAH8 6 1.5 Project Overview 10 Chapter 2: Breaking the THQ Core 14 2.1 Introduction 14 2.2 Results 15 2.3 Discussion and Conclusions 24 2.4 Experimental 29 Chapter 3: Further Derivatization of the Aromatic Core 63 3.1 Introduction 63 3.2 Results 64 3.3 Discussion and Conclusion 73 3.4 Experimental 78 iv Chapter 4: Amine Pendants as MOR Pharmacophores with Improved Stability 100 4.1 Introduction 100 4.2 Results 101 4.3 Discussion and Conclusion 110 4.4 Experimental 114 Chapter 5: Discovery of Aromatic-Amine Pharmacophore 133 5.1 Introduction 133 5.2 Results 135 5.3 Discussion and Conclusions 146 5.4 Experimental 154 Chapter 6: Aromatic-Amine Pharmacophore Enables Removal of Aromatic Core 183 6.1 Introduction 183 6.2 Results 193 6.3 Discussion and Conclusions 183 6.4 Experimental 198 Chapter 7: Aliphatic Heterocycles as Novel Core Elements 216 7.1 Introduction 216 7.2 Results 217 7.3 Discussion and Conclusions 223 7.4 Experimental 226 Chapter 8: Conclusions and Future Directions 246 8.1 Conclusions 246 8.2 Future Directions 251 Appendix 258 References 259 v List of Figures Figure 1: Notable small molecules that exhibit a MOR-agonist/DOR-antagonist profile. 5 Figure 2: General SAR of the opioid peptides. 7 Figure 3: Structural evolution of the endogenous peptide Met-enkephalin into the lead peptidomimetic KSKPP1E. 9 Figure 4: Diversification strategies employed to explore the SAR of KSKPP1E. 10 Figure 5: Normalized metabolic half-life of peptidomimetics that contain a THQ core or similar bicyclic core structure. 11 Figure 6: Known metabolites of KSKPP1E from MLM. 12 Figure 7: Pharmacophore and linker elements of KSKPP1E and our initial monocyclic core series. 15 Figure 8: Molecular modeling of analogue 70 at MOR. 24 Figure 9: Further derivatization of compounds 60 and 70. 64 Figure 10: Proposed CYP450 reaction mechanism on analogues 70, 106, 107, and 114. 77 Figure 11: Design path leading to stable peptidomimetics with basic amine pendants. 101 Figure 12: Antinociceptive activity of analogues 70, 127-128, and 131-134 using the acetic acid stretch assay. 109 Figure 13: Stability ratio of analogues 127-128 and 131-136 against their cLogP. 112 Figure 14: Design of amine pendant analogues with different aromatic core modifications. 135 Figure 15: Molecular docking of superagonist 168 at the three opioid receptors. 145 Figure 16: Antinociceptive activity of 132 and analogues 168, 172, 176, and 180 using the AASA. 146 Figure 17: Literature structures that are close to our discovered aromatic-amine pharmacophore outside of our previously reported peptidomimetic series. 153 Figure 18: Design of analogues possessing greater simplification of core elements. 184 Figure 19: Molecular docking of analogue 193 at the three opioid receptors. 192 vi Figure 20: Antinociceptive activity of the 168 and analogues 193-194, and 197-200 using the AASA. 193 Figure 21: Conversion of aromatic core peptidomimetic to cyclic aliphatic core peptidomimetics. 217 Figure 22: Summary of results from Chapters 2 and 3. 247 Figure 23: Summary of results from Chapters 4 and 5. 248 Figure 24: Summary of results from Chapter 6. 250 Figure 25: Summary of Results from Chapter 7. 251 Figure 26: General design scope upon which further analogues may be developed. 252 Figure 27: Proposed analogues to be synthesized that modify the aromatic-amine pharmacophore. 253 Figure 28: Proposed peptide derivatives of analogues 199 and 200. 253 Figure 29: Proposed analogues that modify the aromatic core. 254 Figure 30: Rigid cyclic core derivatives akin to analogues 196-197. 255 Figure 26: Proposed derivatives of the cyclic aliphatic analogue 229. 256 vii List of Schemes Scheme 1: Synthesis of AAH8. 13 Scheme 2: Synthesis of Substituted Monocyclic Core Analogues 59-74. 16 Scheme 3: Synthesis of Analogues 101-104. 65 Scheme 4: Synthesis of Analogue 105. 66 Scheme 5: Synthesis of Analogues 106-107. 66 Scheme 6: Synthesis of Analogue 114. 67 Scheme 7: Synthesis of Amine or Amide Pendant Analogues Containing an Ethyl Ether. 102 Scheme 8: Synthesis of aromatic core analogues containing amine pendants to produce SAR matrices. 136 Scheme 9: Synthesis of Analogues 193-197. 185 Scheme 10: Synthesis of Peptide Core Analogues 198-200. 186 Scheme 11: Synthesis of Analogues 224-231. 219 viii List of Tables Table 1: The opioid receptors, their location, and their phenotypic effects on activation. 2 Table 2: Binding affinity of the benzylic core compounds at MOR, DOR, and KOR. 19 Table 3: Potency and efficacy of benzylic core compounds at MOR, DOR, and KOR. 20 Table 4: Metabolic stability of benzylic core compounds in MLM. 22 Table 5: Binding affinity of 2nd generation benzylic derivatives at MOR, DOR, and KOR. 68 Table 6: Potency and efficacy of 2nd generation benzylic derivatives at MOR, DOR, and KOR. 69 Table 7: Binding affinity of 2nd generation aromatic derivatives at MOR, DOR, and KOR. 70 Table 8: Potency and efficacy of 2nd generation aromatic derivatives at MOR, DOR, and KOR. 71 Table 9: Metabolic stability of 2nd generation benzylic derivatives in MLM. 72 Table 10: Metabolic stability of 2nd generation aromatic derivatives in MLM. 73 Table 11: Binding affinity of amine or amide pendant analogues at MOR, DOR, and KOR. 104 Table 12: Efficacy and potency of amine or amide pendant analogues at MOR, DOR, and KOR. 106 Table 13: Metabolic stability of amine or amide pendant analogues in MLM. 107 Table 14: Binding affinity matrix (Ki (nM)) of amine analogues of benzylic core structures from Chapter 2 at MOR and DOR. 138 Table 15: Binding affinity (Ki (nM)) matrices of amine analogues of benzylic core structures from Chapter 2 at KOR and their selectivity. 140 Table 16: Potency matrices of amine analogues of previously reported benzylic core structures from Chapter 2 at MOR, DOR, and KOR. 141 Table 17: Efficacy matrices of amine analogues of previously reported benzylic core structures from Chapter 2 at MOR, DOR, and KOR. 143 Table 18: Metabolic stability matrix of amine analogues of previously reported benzylic core structures from Chapter 2. 144 Table 19: Binding affinity of simple core compounds at MOR, DOR, and KOR. 188 Table 20: Efficacy and potency of simple core compounds at MOR, DOR, and KOR.