Small Molecule Inhibitors of Protein-Protein Interactions by Kareem Khoury Bachelor of Science, University of Pittsburgh, 2008 Submitted to the Graduate Faculty of School of Pharmacy in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2013 UNIVERSITY OF PITTSBURGH SCHOOL OF PHARMACY This dissertation was presented by Kareem Khoury It was defended on December 5th, 2013 and approved by Barry I. Gold, Professor, Department of Pharmaceutical Sciences Xiang-Qun Xie, Professor, Department of Pharmaceutical Sciences Song Li, Professor, Department of Pharmaceutical Sciences Carlos Camacho, Department of Structural Biology Dissertation Advisor: Alexander Doemling, Professor, Department of Pharmaceutical Sciences ii Copyright © by Kareem Khoury 2013 iii Discovery of Small Molecule Inhibitors for Protein-Protein Interactions Kareem Khoury, PhD University of Pittsburgh, 2013 Protein-protein interactions (PPIs) constitute a rising class of targets for the next generation of therapeutic intervention. Though they play a fundamental role in many biological processes and almost all pathological conditions including cancer, diabetes, and autoimmune diseases, , PPIs remain underrepresented in drug discovery. Small molecules are the ideal candidates for PPI inhibitors due to low production cost and better absorption, distribution, metabolism, and excretion (ADME) properties compared to biological agents. Discovering small molecule inhibitors of PPIs has proven to be difficult because of the relatively large contact areas between proteins. Herein are described novel approaches to the chemical synthesis as well as a screening tool which will facilitate the discovery of small molecule inhibitors of PPIs. Currently, molecules derived from multicomponent reactions (MCRs) are rarely distributed in general screening libraries. The exceptions are special scaffolds, e.g. dihydropyrimidines, whose class includes the blockbuster Ca antagonist, Nifedipine. Interestingly, however, MCR molecules have been frequently described as inhibitors of PPIs. Examples include p53/mdm21,2, Bcl23, HIV-1/gp414, CCR55 and oxytocin antagonists6. These findings support the notion that MCR molecules are especially suitable for mimicking peptides. We have developed a virtual library of scaffolds derived from a diverse set of MCRs which are easily chemically accessible. iv In addition, our lab has set forth to discover novel MCRs to add to our virtual library. One such scaffold described in this dissertation is derived from the Ugi-4-component-5- cenetered reaction. A novel amidation of this classic scaffold adds a true four-component reaction to our MCR database. The scope and limitations of this reaction are described in detail allowing for an accurate representation of compounds with a high probability of being synthesized. Various cyclizations of this scaffold are also explored and described in detail. A receptor-based drug discovery approach can be applied when an accurate three- dimensional (3D) structure of a specific PPI complex is available. A novel, complementary and transformative docking approach for the rational design of small molecule inhibitors was developed for our virtual library based on the “anchor” concept. Applying our method, we efficiently discovered several new scaffolds of inhibitors of the p53/MDM2 interaction with lower micromolar affinity binding to MDM2, which can serve as starting point for medicinal chemistry optimization. In summary, the methods and tools described in this dissertation are important contributions to the fields of medicinal chemistry and structure-based drug discovery because they combine structural insights and ligand design to expedite the discovery of novel small molecule inhibitors of PPIs. v TABLE OF CONTENTS LIST OF SCHEMES ............................................................................................................... XIII PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 PROTEIN-PROTEIN INTERACTIONS AS DRUG TARGETS ................... 1 1.2 THE P53/MDM2 AXIS ....................................................................................... 3 1.2.1 p53/MDM2 Biology.......................................................................................... 4 1.2.2 p53/MDM2 interactions .................................................................................. 8 1.2.3 Small molecule-MDM2 co-crystals .............................................................. 10 1.2.4 MDMX-co crystal .......................................................................................... 21 1.2.5 Clinical Trials ................................................................................................. 24 2.0 ANCHORQUERY: AN EASILY ACCESSIBLE VIRTUAL SCREENING LIBRARY/TOOL ....................................................................................................................... 26 2.1 COMPOUND SCREENING LIBRARY GENERATION ............................. 33 2.1.1 Isocyanide Based MCRs ................................................................................ 34 2.1.1.1 Ugi Reaction......................................................................................... 34 2.1.1.2 Tetrazole Reaction .............................................................................. 35 2.1.1.3 Hydantoine Reaction........................................................................... 37 2.1.1.4 Imidazole .............................................................................................. 38 vi 2.1.1.5 Diketopiperazine ................................................................................. 39 2.1.1.6 Praziquantel (Ugi/Pictet-Spengler) .................................................... 40 2.1.1.7 Ugi 4-component-5-centered reaction ............................................... 42 2.1.1.8 Ugi-deprotective Cyclization - Benzimidazole .................................. 43 2.1.1.9 Ugi-deprotective Cyclization - Dihydrobenzodiazepine-acetamide 45 2.1.1.10 Ugi-deprotective Cyclization - Tetrahydrobenzodiazepine - carboxamide ....................................................................................................... 46 2.1.1.11 Ugi- deprotective Cyclization - Dihydrobenzodiazepine- carboxamide ....................................................................................................... 47 2.1.1.12 Ugi- deprotective Cyclization - Tetrazolyl- dihydrobenzodiazepine….................................................................................. 49 2.1.1.13 Groebke/Blackburn/Bienayme ........................................................ 50 2.1.1.14 Thienodiazepine ................................................................................ 51 2.1.1.15 Orru .................................................................................................... 53 2.1.1.16 Schöllkopf reaction ........................................................................... 54 2.1.1.17 Thiazole Amidation ........................................................................... 56 2.1.2 Non-Isocyanide Based Multicomponent Reactions .................................... 58 2.1.2.1 Döbner .................................................................................................. 58 2.1.2.2 Gewald.................................................................................................. 59 2.1.2.3 Isoquinoline Amidation ...................................................................... 61 2.1.2.4 Reaction mapping and smarts codes ................................................. 62 2.1.3 Principal Moment of Inertia ......................................................................... 68 2.2 ANCHORQUERY IMPLEMENTATION ...................................................... 70 vii 3.0 DISCOVERY OF NEW MULTICOMPONENT REACTIONS FOR VIRTUAL SCREENING ............................................................................................................................... 74 3.1 IMINODICARBOXAMIDES BY VARIATION OF THE UGI-4- COMPONENT-5-CENTERED REACTION .................................................................. 75 3.1.1 Materials and Methods ................................................................................. 85 3.2 CYCLIZATION SCAFFOLDS BY VARIATION OF THE UGI-4- COMPONENT-5-CENTERED REACTION ................................................................ 105 3.2.1 Isoindolinone ................................................................................................ 107 3.2.2 Dioxopyrrolidines ........................................................................................ 110 3.2.3 Pictet-Spengler ............................................................................................. 113 3.2.4 Materials and Methods ............................................................................... 121 4.0 UGI-4-COMPONENT-5-CENTERED REACTION FOR USE IN P53/MDM2 155 4.1 ANCHORQUERY SEARCH AGAINST P53/MDM2 ................................. 155 4.2 SYNTHESIS AND IN VITRO ACTIVITY OF SELECTED COMPOUNDS 158 4.2.1 Materials and Methods ............................................................................... 164 4.3 CO-CRYSTAL AND CELL BASED SCREENING .................................... 181 4.3.1 Materials and Methods ..............................................................................