Novel Cannabidiol and Anandamide Analogs Thesis Presented by Marsha Rebecca D’Souza to The Bouvé Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Medicinal Chemistry NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS March 23rd, 2012 Signature page 1 Northeastern University Bouvé Graduate School of Health Sciences Thesis title: Novel Cannabidiol and Anandamide Analogs Author: Marsha Rebecca D’Souza Program: Medicinal Chemistry Approval for thesis requirement of the Doctor of Philosophy in Medicinal Chemistry Thesis Committee (Chairman) ________________________ Date __________ ________________________ Date __________ ________________________ Date __________ ________________________ Date __________ ________________________ Date __________ Director of the Graduate School _________________________ Date __________ Dean _________________________ Date __________ Copy Deposited in Library _________________________ Date __________ Signature page 2 Northeastern University Bouve' Graduate School of Health Sciences Thesis title: Novel Cannabidiol and Anandamide Analogs Author: Marsha D’Souza Program: Medicinal Chemistry Approval for thesis requirements of the Doctor of Philosophy in Medicinal Chemistry Thesis Committee (Chairman) ________________________ Date ________ ________________________ Date ________ ________________________ Date ________ ________________________ Date ________ ________________________ Date ________ Director of the Graduate School ________________________ Date ________ i Abstract Part I Delta-9-tetrahydrocannabinol (Δ9-THC) and (-)-cannabidiol (CBD) are the major constituents of Cannabis sativa (marijuana). (-)-CBD shares many of Δ9-THC’s therapeutic properties without inducing negative psychotropic effects. These include potential medicinal uses for anti-inflammation, neuroprotection, anxiolytic, anti-nausea and anti-cancer that are all of great therapeutic importance. The clinical potential of (-)- CBD has been realized with the recent approval of Sativex® in Canada, a drug consisting of a 1:1 mixture of Δ9-THC and (-)-CBD for relief of neuropathic and cancer-related pain. Nonetheless, the levorotatory (-)-CBD natural product binds with low affinity to the two principal cannabinoid (CB) G protein-coupled receptors, CB1R and CB2R, whereas the synthetic dextrorotatory (+)-CBD enantiomer binds to both with high (nanomolar) affinity. However, little is known regarding (+)-CBD ligand-binding and functional domains at these receptors, and structure-activity relationship (SAR) data around (+)- CBD is sparse. In this dissertation, a number of high-affinity (+)-CBD analogs have been synthesized in order to explore the SAR. The SAR focused on the side-chain, northern- end, and phenolic hydroxyl pharmacophores of the (+)-CBD prototype. In vitro leads were selected based on their high binding affinity, selectivity as CB2R agonists or CB1R partial agonists, drug-like physicochemical properties, and modulation of in vitro pharmacological activity (cAMP, β-arrestin assays). These leads were profiled in a panel of rodent paradigms for in vivo cannabinergic activity (hypothermia, catalepsy and tail- flick tests). AM9200 was demonstrated to have a longer duration of action as compared to its metabolite, AM9201, whereas (+)-CBD analogs AM9217 and AM9248 were ii (weak) partial agonists active in vivo. AM9252 and AM 9222 showed potent analgesic and hypothermic effects in mice and rats, suggesting agonist activity at both CB1R and CB2R. AM 9252 was also shown to have analgesic effects comparable to synthetic Δ9- THC analog (AM 4054) in non-human primates. Also, in order to obtain structural information regarding the binding site of (+)-CBD analogs with these membrane-bound proteins, pharmacologically active (+)-CBD analogs designed as covalent probes to wild- type and mutant CB1R and CB2R are being profiled to help characterize their binding site(s). Part II Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are the two key endocannabinoids that act at CB1R and CB2R to modulate physiological and pathological processes including nociception, inflammation, neuroprotection, feeding behavior, anxiety, memory, and cell proliferation. They are produced “on demand,” are rapidly inactivated by enzymatic hydrolysis, and serve as substrates for oxidative metabolism by cyclooxygenases and lipoxygenases, making it difficult to study directly their in vivo physiology and pharmacology. For the development of novel endocannabinoid templates with potential resistance to hydrolytic and oxidative metabolism, we targeted the methylation of the bis-allylic carbons of the arachidonoyl skeleton. Towards this end, the synthesis and preliminary biological data for the (13S)- methyl-anandamide analog were recently disclosed from our laboratory. This compound was found to have the highest CB1 binding affinity among anandamide analogs to date. Based on this discovery, this dissertation reports the total synthesis of the (10S)- and (10R)-methyl-counterparts. The synthetic approach used was stereospecific, efficient, and iii provided the chiral analogs without the need for resolution. Biological testing showed that (10S)- and (10R)-methyl anandamide analogs bound to CB1 and CB2 with moderate affinity. To explore the binding motifs of the novel (13S)-methyl-substituted arachidonoyl template, the respective tail-modified covalent probes (at C-20) were synthesized and profiled. The covalent probes bound to the CB1R and CB2R with low nanomolar affinity and are currently being tested for their ability to covalently label the CB receptors. iv Acknowledgements I am very excited to have successfully completed my doctorate. This journey has been possible mainly because of my Lord and Savior Jesus, who has been by my side through all my trials and successes. There is nothing that I can do without him. I would like to convey my deepest appreciation to my committee chair, Professor Alexandros Makriyannis, for his support and encouragement throughout this process. The experience and knowledge that I gained from his lab research has motivated me to continue my career in this area. I would also like to express gratitude to my mentor Dr. Spyros Nikas, who has not only taught me excellent chemistry skills, but also trained me to think like a scientist. He has always been there to help me in practicing for presentations, proof reading my thesis and helping me with some of my questions from the classes I took. Dr. David Janero has helped me immensely in proof reading my thesis and has given me excellent suggestions for its improvement. I am also grateful to Dr. Robert Hanson for serving on my thesis committee and providing me suggestions for my thesis. I really appreciate Dr. Andreas Goutopoulos, who took time form his busy schedule to make it for all my committee meetings. He also gave me an industrial perspective on my project and good suggestions for improving my thesis research. I want to thank my lab mates (Megan, Erin, Sherrica, Shukla, Rishi, Shama, Kiran, Kyle and Heidi) who have made this a fun experience. I am very thankful to Dr. Kumar Vadivel for his selfless help. He has always been ever ready to help me in discussing chemistry mechanisms and fixing my presentations. I am grateful to Dr. Jessica Garcia who proofread my chemistry and results section and gave me good input to improve my thesis and presentations. I would like to thank Dr. David Finnegan and Shwantelle Dillon v for proof reading my experimental and introduction. I am truly indebted and thankful to Sarah Strassburg, Shawntelle Dillon and Brett for their help in scheduling meetings. I also want to thank my collaborators, Dr. Jodi Wood, Han Zhou, Dr. Aneetha Halikhedkar, Pusheng Fan, Othman Benchama, SriKrishnan Mallipeddi, Yan Peng, Girija Rajarshi, Shivagi Joshi, Dr. Jarbe, Dr. Dustin Smith, Dr. Aaron Lichtman and Dr. Carol Paronis who have provided me with in vitro and in vivo data. Last but not the least I want to thank my parents for their unconditional support, love and prayers. It is my dad who first encouraged me to get into science and I am really proud that I can call myself a scientist. My mom has never ceased to pray for my smooth sailing through these five years. My brother Helius and my little sisters Malissa and Moira have always been there to advice me in tough times. Words cannot express how thankful I am to my boyfriend Elliott Eno, who has always been there for me during these five years, in good times and in bad times. vi TABLE OF CONTENTS PAGE ABSTRACT .......................................................................................................................... ii ACKNOWLEDGMENT ........................................................................................................... v LIST OF TABLES ................................................................................................................. ix LIST OF FIGURES ................................................................................................................ xi LIST OF SCHEMES ............................................................................................................. xiv LIST OF ABBREVIATIONS .................................................................................................. xvi CHAPTER 1 THE CANNABINOID REALM BACKGROUND ................................................................................ 1 SIGNIFICANCE .............................................................................