DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL SELECTIVE CANNABINOID RECEPTOR 2 (CB2) LIGANDS WITH THERAPEUTIC POTENTIALS by Abdulrahman A. Almehizia B.Sc. in Pharmaceutical Sciences, King Saud University, 2007 Submitted to the Graduate Faculty of The School of Pharmacy in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2015 UNIVERSITY OF PITTSBURGH SCHOOL OF PHARMACY This dissertation was presented by Abdulrahman A. Almehizia It was defended on March 17th , 2015 and approved by Dr. Paul Schiff, Professor, Pharmaceutical Sciences Dr. Regis Vollmer, Professor, Pharmaceutical Sciences Dr. Paul Floreancig, Professor, Chemistry Dr. LiRong Wang, Research Assistant Professor, Pharmaceutical Sciences Thesis /Dissertation Advisor: Dr. Xiang-Qun (Sean) Xie, Professor, Pharmaceutical Sciences ii Copyright © by Abdulrahman A. Almehizia 2015 iii DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL SELECTIVE CANNABINOID RECEPTOR 2 (CB2) LIGANDS WITH THERAPEUTIC POTENTIALS Abdulrahman A. Almehizia, PhD University of Pittsburgh, 2015 Cannabinoid receptors 1 and 2 (CB1 and CB2) belong to the rhodopsin-like family of the G-Protein Coupled Receptors (GPCRs). CB1 receptor is highly expressed in the central nervous system, while CB2 receptor is expressed mainly in the immune cells and the periphery. Targeting the CB2 receptor is believed to avoid the psychoactive side effects associated with CB1 receptor. CB2 receptor has been shown to be involved in several physiological functions as well as diseases, such as pain, multiple sclerosis, osteoporosis, and cancer, demonstrating the importance of the CB2 receptor as a therapeutic target. In the present study, we employed chemistry design and discovery to identify novel CB2 ligands, carried out in-vitro functional studies, and evaluated the therapeutic potentials. Several chemical scaffolds were discovered and evaluated. The di-amide scaffold was discovered utilizing pharmacophore drug discovery and molecular docking studies. Several derivatives of the di-amide scaffold demonstrated potent and highly selective CB2 inverse agonists as well as potent osteoclast (OCL) inhibition capabilities. The di-amide derivatives suffered from weak anti-multiple myeloma (MM) properties and poor pharmacokinetic properties. A new scaffold was identified utilizing scaffold-hopping and molecular docking studies. However, the 2- (sulfonylamino)-2-phenylacetamide scaffold demonstrated weak CB2 binding affinity. Due to the iv limitation of the two previous scaffolds, virtual screening and structure-based drug design were utilized for scaffold-hopping in order to identify highly potent CB2 specific ligands with new scaffolds. A new lead compound was identified and structure activity relationship (SAR) studies were conducted on the scaffold 4-(aminomethyl)-N,N-diethylaniline. Several novel compounds were discovered with high potency and selectivity towards the CB2 receptor. Cellular function assays were applied to characterize the functionality (agonist and inverse agonist) of these compounds. Overall, therapeutic studies showed that inverse agonism is essential for the OCL inhibition effects while anti-MM experiments showed that CB2 agonists are more effective than inverse agonists. v TABLE OF CONTENTS LIST OF TABLES ...................................................................................................................... XI LIST OF FIGURES ................................................................................................................. XIII LIST OF SCHEMES ............................................................................................................ XVIII ACKNOWLEDGMENTS ....................................................................................................... XIX ABBREVIATIONS .................................................................................................................. XXI 1.0 INTRODUCTION ........................................................................................................ 1 1.1 THE ENDOCANNABINOID SYSTEM AND THE CANNABINOID RECEPTORS ........................................................................................................................ 1 1.1.1 THE G-PROTEIN COUPLED RECEPTORS SUPERFAMILY ............... 1 1.1.2 THE ENDOCANNABINOID SYSTEM ........................................................ 4 1.1.3 CANNABINOID RECEPTORS 1 (CB1) ...................................................... 9 1.1.4 CANNABINOID RECEPTORS 2 (CB2) .................................................... 10 1.1.5 ORPHAN RECEPTORS .............................................................................. 11 1.1.6 CANNABINOID RECEPTORS SIGNALING ........................................... 12 1.2 CANNABINOID RECEPTOR LIGANDS ...................................................... 19 1.3 THERAPEUTIC SIGNIFICANCE OF CANNABINOIDS........................... 30 1.3.1 CANNABINOIDS AND TUMOR GROWTH ............................................ 31 1.3.2 CANNABINOIDS AND BONE HOMEOSTASIS ..................................... 43 2.0 RECENT DEVELOPMENT OF CANNABINOID RECEPTOR 2 LIGANDS ... 47 2.1 TETRAHYDROCANNABIOID DERIVATIVES .......................................... 48 2.2 PURINE DERIVATIVES ................................................................................. 50 vi 2.3 PYRIDINE DERIVATIVES ............................................................................. 52 2.4 PYRIMIDINE CARBOXAMIDES DERIVATIVES ..................................... 56 2.5 DIHYDROQUINOLINE-3-CARBOXAMIDES AND 1,8- NAPHTHYRIDINONE ...................................................................................................... 58 2.6 INDOLE-BASED DERIVATIVES .................................................................. 72 2.7 BENZIMIDAZOLE DERIVATIVES .............................................................. 74 2.8 IMIDAZOLES ................................................................................................... 78 2.9 THIOPHENE AMIDE DERIVATIVES.......................................................... 81 2.10 SULFONAMIDE DERIVATIVES................................................................... 83 2.11 CONCLUSION .................................................................................................. 86 3.0 GENERAL METHODS AND MATERIAL ............................................................ 88 3.1 COMPUTER MODELING .............................................................................. 88 3.2 CHEMISTRY ..................................................................................................... 88 3.3 CANNABINOID RECEPTORS MEMBRANE PROTEIN PREPARATIONS ............................................................................................................................. 89 3.4 RADIO-LIGAND COMPETITION BINDING ASSAYS ............................. 90 3.4.1 Saturation Binding Assay ............................................................................. 90 3.4.2 Radio-Ligand Competition Binding Assay ................................................. 92 3.5 CYCLIC ADENOSINE MONOPHOSPHATE (CAMP) ASSAY ................ 94 3.6 CELL-VIABILITY ASSAY .............................................................................. 96 3.7 OSTEOCLAST FORMATION ASSAY .......................................................... 97 3.8 STATISTICAL ANALYSIS ............................................................................. 98 vii 4.0 DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATIONS OF DI-AMIDE DERIVATIVES AS SELECTIVE CB 2 INVERSE AGONISTS ........................................... 99 4.1 INTRODUCTION ............................................................................................. 99 4.1.1 Discovery of Cannabinoid Receptor 2 (CB2) Ligands ............................... 99 4.1.2 Scaffold Hopping and Lead Optimization................................................. 103 4.2 RESULTS AND DISCUSSION ...................................................................... 107 4.2.1 Chemistry Synthesis .................................................................................... 107 4.2.2 Structure Activity Relationship Analysis .................................................. 109 4.2.3 In-Vitro Functional Assay ........................................................................... 120 4.2.4 CB2 Inverse Agonist as Osteoclast Inhibitors ........................................... 122 4.2.5 Multiple Myeloma Anti-Proliferation Studies .......................................... 124 4.2.6 Stability and Pharmacokinetic Studies ...................................................... 126 4.3 CONCLUSION ................................................................................................ 128 4.4 EXPERIMENTAL ........................................................................................... 130 4.4.1 Pharmacophore Modeling and Virtual Screening .................................... 130 4.4.2 Chemistry ..................................................................................................... 130 5.0 DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF SUBSTITUTED 2-(SULFONYLAMINO)-2-PHENYLACETAMIDE DERIVATIVES .. 148 5.1 INTRODUCTION ........................................................................................... 148 5.2 RESULTS AND DISCUSSION ...................................................................... 150 5.2.1 Chemistry Synthesis ...................................................................................