Marquette University e-Publications@Marquette Dissertations (2009 -) Dissertations, Theses, and Professional Projects Design and Synthesis of Selective Estrogen Receptor β Agonists and Their hP armacology K. L. Iresha Sampathi Perera Marquette University Recommended Citation Perera, K. L. Iresha Sampathi, "Design and Synthesis of Selective Estrogen Receptor β Agonists and Their hP armacology" (2017). Dissertations (2009 -). 735. https://epublications.marquette.edu/dissertations_mu/735 DESIGN AND SYNTHESIS OF SELECTIVE ESTROGEN RECEPTOR β AGONISTS AND THEIR PHARMACOLOGY by K. L. Iresha Sampathi Perera, B.Sc. (Hons), M.Sc. A Dissertation Submitted to the Faculty of the Graduate School, Marquette University, in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Milwaukee, Wisconsin August 2017 ABSTRACT DESIGN AND SYNTHESIS OF SELECTIVE ESTROGEN RECEPTOR β AGONISTS AND THEIR PHARMACOLOGY K. L. Iresha Sampathi Perera, B.Sc. (Hons), M.Sc. Marquette University, 2017 Estrogens (17β-estradiol, E2) have garnered considerable attention in influencing cognitive process in relation to phases of the menstrual cycle, aging and menopausal symptoms. However, hormone replacement therapy can have deleterious effects leading to breast and endometrial cancer, predominantly mediated by estrogen receptor-alpha (ERα) the major isoform present in the mammary gland and uterus. Further evidence supports a dominant role of estrogen receptor-beta (ERβ) for improved cognitive effects such as enhanced hippocampal signaling and memory consolidation via estrogen activated signaling cascades. Creation of the ERβ selective ligands is challenging due to high structural similarity of both receptors. Thus far, several ERβ selective agonists have been developed, however, none of these have made it to clinical use due to their lower selectivity or considerable side effects. The research in this dissertation involved the design of non-steroidal ERβ selective agonists for hippocampal memory consolidation. The step-wise process to achieve the ultimate goal of this research includes: (1) design and synthesis of (4- hydroxyphenyl)cyclohexyl or cycloheptyl derivatives, (2) in vitro biological evaluation of synthesized compounds to identify highly potent and selective candidates, and (3) in vivo biological evaluation of selected candidates for hippocampal memory consolidation. Several (4-hydroxyphenyl)cyclohexyl or cycloheptyl derivatives were synthesized having structural alterations on both aromatic and cyclohexyl/heptyl ring scaffolds. ERβ agonist potency was initially evaluated in TR-FRET ERβ ligand binding assay and compounds having high potency were re-evaluated in functional cell based assays for potency and ERβ vs. ERα selectivity. Two compounds from each series, ISP 163-PK4 and ISP 358-2 were identified as most selective ERβ agonists. Both compounds revealed high metabolic stability, solubility and no cross reactivity towards other nuclear receptors. In vivo efficiency of ISP 358-2 was evaluated in ovariectomized mice (C57BL/6) with object recognition (OR) and object placement (OP) tasks. The results indicate improved memory consolidation at 100 pg/ hemisphere and 0.5 mg/Kg via DH infusion and IP injection respectively. The information learned from this project serves as a foundation for development of other cycloheptyl/hexyl based ERβ agonists or antagonists having acceptable pharmacological profiles. i ACKNOWLEDGMENTS K. L. Iresha Sampathi Perera, B.Sc. (Hons), M.Sc. Since my journey as a PhD candidate is coming to an end, it is time to reminisce the people who stood by me along the way. This is my sincere gratitude to those wonderful souls, who helped in making my story a success. I am ever grateful to my advisor Professor William A. Donaldson for the wonderful opportunity he gave me to join his research group. I can’t thank him enough for his valuable guidance and input during the whole course my research which improved me as a researcher in leaps and bounds. The most special aspect was the faith he showed in me during my research to achieve the results and the kind motivating words whenever I found myself challenged. I haven’t got enough words to thank him for being the motivator and mentor in bringing my research to a high-quality level. I could not have imagined having a better advisor for my Ph.D. study. Thank you again for your amazing support. I would like to thank the rest of my dissertation committee: Prof. Daniel Sem, Prof. James Kincaid and Dr. Christopher Dockendorff for their valuable time and effort in reviewing my dissertation. Further, their insightful comments during the committee meetings incented me to widen my research from various perspectives. Additionally, Dr. Christopher Dockendorff deserves a special mention on granting me access to their laboratory facilities which helped me immensely throughout my research. ii I am also very much thankful to Professor Daniel Sem, Professor Karyn Frick for the fantastic opportunity to work with them on an interesting project. I gained invaluable research experience from the monthly meetings I attended and the insightful suggestions and comments undoubtedly helped me to improve my research work. I also would like to mention Alicia Schulz and Jaekyoon Kim for their support during the collaboration. Thank you for giving me the opportunity to be a part of a fantastic team of researchers. I am also grateful to Department of Chemistry for providing me unrestricted facilities during my stay as a graduate student. I would like to convey my sincere gratitude to Dr. Sergey Lindeman and Dr. Sheng Cai for helping me with X-ray crystallographic and NMR studies. I would like to mention, Dr. Sandra Lukaszewski-Rose for the support she rendered to me during my tenure as a teaching assistant. I will always fondly remember the numerous functions you organized for students and the enjoyment I had. My acknowledgement would be incomplete without a word on Mr. Mark Bartelt. I am utterly grateful to him for all the technical supports on my research work. A very special mention to Prof. Isiah Warner at Louisiana State University for helping me in during my masters. I can’t thank you enough for the support you gave me during my time at Louisiana State University. Your encouraging words made me stronger to believe in myself and pursue my goals. Also, I would like to thank Prof. Chieu D. Tran, who was my first advisor at Marquette for his valuable guidance and help. I would also like to pay gratitude to the Chemistry department at University of Colombo where I took first steps as a researcher during my bachelors. I should specially thank my undergraduate thesis advisor Prof. Ramani Wijesekara for her immense help and guidance. iii I thank all past and present members of Donaldson and Dockendorff groups for their invaluable support. The groups have been a source of friendship as well as good advice. I wish all of you success in all your endeavors. Many thanks to Dr. Ajith Wijenayake and his family who helped me immensely when I moved to Milwaukee and my friends in Milwaukee for supporting me at all times. Further, to all my friends in Sri Lanka, U.S. and all around the world; I appreciate your encouragement and love I received. I love you all. Most importantly, my family was with me at every step of the way in this long journey. My Father Ariyasena Perera and My mother Hema Jayakody were the two pillars of strength whenever I was challenged. Their unconditional love made me stronger to thrive in what I do. I thank to my in-laws, especially my father in-law Mr. Jinadasa Nanayakkara for his encouragement. My brother and his family, my aunties, and my relatives for their lovely support. Finally, my loving husband Muditha Nanayakkara who listened, believed and taught me to be strong and being there for me whenever I needed the most. I love you so much. iv DEDICATION This dissertation is dedicated with love to My parents Hema and Ariyasena Perera My brother Erandha Perera My aunt Buddhi Jayakody and My loving husband Muditha Nanayakkara for supporting, encouraging and believing me in all endeavors…….. v TABLE OF CONTENTS ACKNOWLEDGEMENT....................................................................................................і DEDICATION....................................................................................................................iv LIST OF TABLES............................................................................................................vііi LIST OF FIGURES……………………………………………………………………….ix LIST OF SCHEMES ………………………………………………………………..…...xii LIST OF ABBREVIATIONS AND SYMBOLS..............................................................xiv CHAPTER 1. Background and Goals of Research………………………………………...1 1.1 Discovery of Estrogen Receptors.......................................................................1 1.2 Estrogen Receptors and Human Disease…………………………...……….....2 1.2.1 Estradiol, the Hippocampus and Memory…………………………...5 1.2.2 Estrogen Decrease in Menopause and Hormone Replacement Therapy……………………………………………………………………9 1.3 Estrogen Receptor Structure and Mechanism of Action……………………...11 1.4 Important Interactions within the Ligand Binding Domain…………………..15 1.5 Estrogen Receptor – Agonists, Antagonist and Selective Estrogen Receptor Modulators……………………………………………………………………….17 1.6 Review of ERβ Subtype Selective Ligands……………………………….….23 1.6.1 Design of Non-Steroidal ERβ Selective Agonists………………….24 1.7 Design of 4-Cyclohexyl or Cycloheptyl Phenolic Derivatives as