Design & Synthesis of Prostaglandin Ethanolamide Covalent Probes for the Investigation of Novel Physiological Activity and Discovery of a Prostaglandin Intermediate Scaffold for FAAH Inhibition A dissertation presented by Erin Laine Shelnut to The Department of Chemistry and Chemical Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Chemistry Northeastern University Boston, Massachusetts June, 2012 Design and Synthesis of Prostaglandin Ethanolamide Covalent Probes for the Investigation of Novel Physiological Activity and Discovery of a Prostaglandin Intermediate Scaffold for FAAH Inhibition by Erin L. Shelnut ABSTRACT OF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Chemistry in the Graduate School of Northeastern University June, 2012 -2- Abstract Prostaglandin ethanolamides (Prostamides) are an emerging class of endogenous eicosanoids derived from cyclooxygenase (COX) metabolism of the endocannabinoid, anandamide. The chemical structure of the prostamides resembles that of the prostaglandins, with the main distinction attributed to an ethanolamide group in place of the carboxylic acid group. Their biological function has yet to be resolved, however the biosynthetic route for prostamide formation has been well established and their existence in vivo postulated. While the structures and biosynthetic routes are indeed similar, there is growing evidence that prostamides and their prostaglandin counterparts exhibit diverse biological actions. The substitution of ethanolamide for carboxylic acid is sufficient to give the prostamides a diverse biological profile. Indeed, the biosynthetic precursors of prostamides and prostaglandins share this chemical distinction, and are well-known to incite distinct processes. Anandamide is capable of activating the CB1 cannabinoid and TRPV1 vanilloid receptors and serves as their main endogenous substrate. Arachidonic acid, on the other hand, is inactive at both of these receptors and functions mainly as a secondary messenger in cellular signaling and as a precursor to the eicosanoid compounds. In fact, the major pathway of anandamide cannabinergic inactivation is hydrolytic catalysis by the enzyme fatty acid amide hydrolase (FAAH) to arachidonic acid. The disparity between the functions of arachidonic acid and anandamide give credibility to the hypothesis that prostamides might also possess a unique biological purpose. Prostaglandins are known to act at well characterized prostaglandin receptors, named for the specific prostaglandin they bind. Prostamides similarly may interact with distinct, yet to be characterized receptors. Our laboratory attempts to address this prospect by development of novel prostamide analogs for screening against known and orphan receptors and for in vivo inflammation and immunomodulation studies. -3- Chapter 1 provides an overview of the prostamide biosynthetic pathway, including detailed discussion of anandamide release and action and cyclooxygenase structure and function. Additionally, a comprehensive review of current prostamide research covering stability and pharmacology in support of, and in opposition to, the functional distinction between the prostamides and prostaglandins is presented. Conflicting opinions exist as to the uniqueness of the prostamides’ pharmacology, and exploring both sides of the dispute gives a comprehensive review of studies attempting to elucidate prostamides’ physiological targets. Finally, their emerging role in inflammation, neuroinflammation and neuroplasticity is addressed. Most strikingly, evidence suggests that neuroprotective qualities long attributed to the actions of the endocannabinoid, anandamide, are instead the result of its oxidized metabolite, prostamide. Chapter 2 presents the design and synthesis of a set of prostamide analogs to be screened against existing known and orphan receptors and to undergo inflammation and immunomodulation studies. Analogs are designed to incorporate tail moieties that covalently bind a target receptor protein. Modification in head group functionality attempt to address issues of prostamide metabolic stability, and variation of the stereochemistry of the 15R hydroxyl may lead to optimization of a possible hydrogen bond with the active site. The challenges addressed in this extensive synthesis include instability of the cyclopentyl ring hydroxy groups and unachievable chiral reduction of the 15-keto group. The assignment of resulting chirality is designated by thorough Mosher analysis. Using the synthetic route established here, the tail hydroxyl moiety can serve as an entrance to a vast number of future alterations in functionality of both head and tail substituents. The results of biological screening of prostamide analogs on well-established and orphan receptors are presented in Chapter 3. None of the isolated receptors screened showed significant activity toward the prostamides tested. Additionally described within this chapter is the effect of prostamide treatment on an in vivo model of murine inflammatory -4- peritonitis. The single analog tested showed enhanced clearance of pro-inflammatory polymorphonuclear neutrophils and thus exhibited behavior consistent with resolution of inflammation. This study also revealed the inability of the prostaglandin probe to activate the EP prostaglandin receptors. Finally, prostamide’s immunosuppressive effects similar to those of anandamide were demonstrated. Chapter 4 outlines areas of study in which prostamide pharmacological activity can be further explored. Investigations of prostamide action in cells of the central nervous system represent the most promising course of research. Prostamide E2 enhances neuroprotection and suppresses cytokine production, and due to increased production of the precursor anandamide, is expected to be present at relevant concentration in the brain. A study of prostamide activity at EP receptor splice variants may be integral in determining prostamide’s unique pharmacology. Screening prostamide against nuclear receptors such as PPARγ has yet to be completed. Due to the ability of PPARγ to bind prostaglandins and fatty acids, screening on this receptor will determine whether prostamide is an endogenous ligand. Isolation of prostamide-selective receptors from tissues sensitive to prostamide treatment can be achieved by employing radiolabelled or biotinylated, bifunctional prostamide probes. Once isolated, the prostamide-bound proteins can undergo mass spectrometry studies to aid in characterization. Finally, design and synthesis of a subsequent set of prostamide analogs is proposed within this chapter. Chapter 5 introduces a secondary project in which three intermediates of prostamide synthesis were discovered to exhibit inhibition of the FAAH enzyme. The enone and the S and R allylic alcohol compounds inhibited of FAAH in the range of 100 nM-1 µM. Thus, this set of bicyclic lactones represents a promising foundation from which to optimize FAAH inhibition employing SAR study. An initial set of compounds including modifications at the 11-hydroxyl group, at the 20-hydroxyl group, and of the chain length between the 15- hydroxyl and ω tail were designed to assess the structure-activity relationship of the -5- inhibitors to rFAAH. The foundations of a pharmacophore for these bicyclic lactone FAAH inhibitors were roughly established. The 15S-hydroxyl, 20-(1-adamantane carboxylate) compound AM7666, gave the greatest inhibition of the designed analogs, and its large bulk and hydrophobicity suggests that the binding pocket for the lipophilic portion of the inhibitors is considerably large. -6- Acknowledgements Through the enormous challenge of completing this dissertation I realize I owe enormous gratitude to several members of the Department of Chemistry & Chemical Biology and the Center for Drug Discovery. Thank you for providing me this opportunity to learn invaluable research skills and grow as an independent researcher. I am exceeding thankful to Dr Alexandros Makriyannis for his guidance and investment. Thank you for recognizing great potential in me and presenting me your most challenging and interesting research project. Throughout the years you have instilled in me a resilience I will carry always, and I am very grateful for it. I attribute my thanks to all of the members of the Center for Drug Discovery, both past and present, including Dr Lakshmipathi Pandrinathan for his invaluable guidance and training in proper methods of organic synthesis, Dr Kumara Vadivel Subramanian for his continuous assistance in all things lab related, and Jodi Wood for her patience and understanding in training me to perform biological assays. Thank you for sharing your knowledge and investing your time to aid me in my research pursuits and grant me the skills to become a better scientist. I am infinitely grateful to the members of the CDD office, Shawntelle Dillon, Sarah Strassburger, and Brett Greene. Even on your busiest days, you made time to help me with various requests and even gave me emotional support when I needed it most. A number of collaborators have contributed to the research objectives described in this dissertation. Dr Bryan Roth at the University of North Carolina – Chapel Hill screened our compounds on known and orphan receptors through a Psychoactive Drug Screening Program funded by the National Institute of Mental Health. Dr Charles Serhan and members -7- of his group at Harvard Medical