Cannabinoid Receptor

Conformationally Constrained Analogs of Org27569 as Allosteric Modulators of CB1 Cannabinoid Receptor Master’s Thesis Research By Siddhi Honavar Advisor: Dr. Ganesh Thakur, PhD Department of Pharmaceutical Sciences Northeastern University August 2015 1 Acknowledgements I would like to express my sincere gratitude to my advisor Dr. Ganesh Thakur for the continuous support during my thesis project. I thank him for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. Besides my advisor, I would like to extend a special thanks to Dr. Ganesh Chaturbhuj who has not only been an excellent guide and mentor for my thesis project, but he has also been an inspiration for me to work hard. His continuous encouragement has driven me to being a better researcher. I want to thank him especially for being there to help even across the seas, and being patient and resolving my abundant queries. I would also like to thank Abhijit Kulkarni for training me in my initial days and being a support through the entire research journey. My sincere thanks also goes to Dr. Sumantha Garai, Dr. Pushkar Kulkarni, and Dr. Gopalkrushna Waghule, who have been very helpful in guiding me in the lab and shared their experiences and knowledge which have helped me, grow as a researcher. I thank my fellow lab mates Sharvik, Brinda, Ninad and Prisca for being so understanding and kind, and for all the fun we have had. I would also like to extend thank you to Robert Laprairie and Dr. Eileen Denovan-Wright from Dalhousie University for characterization of compounds and helping with the biological data. Last but not the least I would like to thank my parents for their belief in me and being supportive throughout my MS, and also my friends and roommates in Boston who have been with me through all good and bad times. 2 CONTENTS: 1. Abbreviations………………………………………………………………………………………………...3 2. List of Figures………………………………………………………………………………………..…….….4 3. List of Tables……………………………………………………………………………………….…………..5 4. Abstract.………………………………………………………………………………………………………....6 5. Specific aims……………………………………………………………………………………………………7 6. Background and Significance……………………………………………………………………………8 7. Research Plan………………………………………………………………………………………………..18 8. Chemistry and Schemes………………………………………………………………..………………22 9. Biological Assay Data…………………………………………………………………………………....28 9.1 Data Analysis………………………………………………………………………………..…………33 10. Conclusion……………………………………………………………………………………………..……..34 Experimental Section……………………………………………………………………………..……..35 11. References…………………………………………………………………………………………………….44 3 Abbreviations: CB - Cannabinoid GPCR – G-Protein Coupled Receptor Org – Organon 2-AG – 2-arachidonolyglycerol MAPK – Mitogen activated protein kinase JNK – c-Jun N- terminal kinase FAAH – Fatty acid amide hydrolase ∆9-THC - ∆9- tetrahydrocannabinol PAM – Positive allosteric modulator NAM – Negative allosteric modulator DMF – Dimethylformamide DCM – Dichloromethane TEA – Triethylamine DPPA – Diphenylphosphoryl azide THF - tetrahydrofuran quant – quantitative 4 List of Figures: Figure A: The CB1 receptor signaling Figure B: Structures of cannabinoid antagonists Figure C: Examples of endocannabinoids Figure D: Cannabinoid receptor agonists Figure E: Allosteric and orthosteric ligands Figure F: Negative allosteric modulators at cannabinoid receptor Figure G: Structures of Organon compounds Figure H: Summary of SAR studies of the Org scaffold Figure I: Constrained analog approach for Org27569 Figure J: Structures of GAT700 and GAT701 Figure K: Core structure of constrained analog series Graph A: Data for GAT700 and GAT701 for Modulation of CB1 and Gαq dependent PLCβ3 phosphorylation. Graph B: Data for GAT700 and GAT701 for pPLCβ3 Graph C: Data for cAMP inhibition by GAT700 and GAT701 5 Graph D: Data of GAT700 and GAT701for ERK1/2 phosphorylation List of Tables: Table 1: Structures of proposed compounds 6 Abstract: The Cannabinoid receptors have become the focus of research due to their importance as targets for treating a number of disorders. These receptors which are a part of the G-protein coupled receptor (GPCR) superfamily are of two subtypes, CB1 receptors which are present abundantly in the brain and in the peripheral and adipose tissues and CB2 receptors which are predominantly found in the immune cells. The cannabinoid receptors were always known to show its function through the orthosteric ligand binding, but the discovery of allosteric site on the CB1 receptors, has opened up a whole new horizon for research. Three of Organon analogs displayed a paradoxical allosterism at the CB1 receptors, wherein they were negative allosteric modulators (NAM) of function but positive allosteric modulators (PAM) of binding of orthosteric ligand at the CB1 receptor. The SAR around these three molecules has not been explored as much, and as all three almost shared the same phamacophoric properties, Org27569, the most potent analog of these, was selected as the lead compound. Org27569 has shown hypophagic effect independent of the presence of the CB1 receptor, and hence pointing towards the possibility of it‟s off target binding which is a significant limitation in its further development as a drug. Exploring the SAR around Org27569 would give a better insight into the molecule‟s structural requirements for allosteric modulation at CB1 receptor. The conformational restriction approach is adopted as a tool for molecular modification and design of the analogs. This projects aims at synthesizing conformationally constrained analogs of Org27569 as GAT700 and GAT701, to explore the receptor binding and functional selectivity of the allosteric modulators at the CB1 cannabinoid receptor. 7 Specific Aims: 1. Rational design of constrained analogs: The project aims at synthesizing analogs of Org27569 that have a restricted orientation, which would give us more control over the stereochemistry of the compound, and which might increase its selectivity for CB1 receptor and pacify the off target binding, giving us more control over the receptor function. 2. The structural characterization of these molecules. 3. Biological testing of analogs, to study the effect on CB1 receptor: The analogs were tested in different functional assays to measure their functional potencies and efficacies, which would help in expanding the SAR study for this chemotype. 8 1. Background & Significance 1.1 Endocannabinoid system: The endocannabinoid system encompasses two of the G-protein coupled receptors (GPCRs), CB1 and CB2, the endogenous cannabinoid receptor ligands, along with the enzymes and proteins that are involved in their synthesis and inactivation1-4. The ones identified so far are derivatives of long chain polyunsaturated fatty acids, the ones that have been researched the most; these include anandamide (N-arachidonoylethanolamine), and 2-arachinonolyglycerol (2-AG). Experiments have also shown endocannabinoids to act as retrograde synaptic messengers5-7. 8,9 The CB1 receptors are found in both central and peripheral nervous system , but in abundance in the central nervous system, and most prominently at the neuronal terminals where they play a 2,3 pivotal role in modulation of the neurotransmitter release and the CB2 receptors are mainly occurring in the immune cells and are involved in cytokine release and immune cells migration10. 11 Figure A: The CB1 receptor signaling 9 Similar to many GPCRs, multiplicity of signal transduction is demonstrated by the CB1 receptors. Though it shows preferential coupling with Gi/o type proteins, there is possible 12 13 4 interaction with Gs and Gq type proteins in some conditions . CB1 receptor activation is responsible for the drop of the cAMP levels in cells due to its inhibitory action on adenylyl- cyclase. Along with this CB1 receptor also modulates the mitogen-activated protein kinases activation 14, which comprises of extracellular signal regulated kinase-1 and -2, p38 MAPK, p42/p44 MAPK, and c-Jun N-terminal kinase (JNK) 14. Moreover they can inhibit N- and P/Q 2+ + type voltage-gated Ca channels and activates the A-type, leading to inward rectification of K 15 channels . CB1 receptors have a complicated signaling network which brings up the presence of receptor functions that are controlled by modulatory mechanisms. 1.2 Therapeutic potential of the cannabinoid receptors: The wide distribution of the cannabinoid receptors in different tissues accounts for the psychotropic and peripheral effects of THC. CB1 receptors are predominant in the neuronal circuit (CNS) and are also present in the periphery, whereas CB2 receptors are restricted to the immune cells. CB1 receptors are involved in neuromodulatory actions which cause the regulation of cardiovascular, garstrointestinal16 and cardiovascular functions17 along with regulation of pain 18 perception . CB1 receptors are also involved in regulation of the hormones in males and females 1,19 and in turn controlling the reproductive functions . Vast evidences show that the CB1 receptor is an important target for the treatment of a number of disorders like obesity20,21, pain22, inflammation23, osteoporosis24, cancer25, gastrointestinal disorders, psychosis, and schizophrenia3. 10 1.3 Cannabinoid receptor ligands: CB1 and CB2 receptors, which have been identified, are primarily located in the central nervous system and on the immune cells respectively. The discovery of these receptors, and the importance of their role in the body as neuromodulators and immunomodulators have driven

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