UNIVERSITY OF CALIFORNIA SAN DIEGO Conversion of Metal Chelators to Selective and Potent Inhibitors of New Delhi Metallo-beta-lactamase A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry by Allie Yingyao Chen Committee in charge: Professor Seth M. Cohen, Chair Professor Michael D. Burkart, Co-Chair Professor Bradley S. Moore Professor Joseph M. O’Connor Professor Faik Akif Tezcan 2020 The Dissertation of Allie Yingyao Chen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ Co-Chair _____________________________________________________________ Chair University of California San Diego 2020 iii DEDICATION To my family - thank you & I love you. iv TABLE OF CONTENTS Signature Page………………………………………………………………………………….iii Dedication……………………………………………………………………………………….iv Table of Contents…………………………………………………………………………..v List of Symbols and Abbreviations……………………………………….……………..ix List of Figures……………………………………………………………….……………..xiii List of Schemes………………………………………………………………..…………...xvi List of Tables…………………………………………………..…………………………..xviii Acknowledgements………………………………………..……………..……….…………xx Vita…………………………………………………..…………………………………………xxiii Abstract of the Dissertation………………………………………………………..…..xxiv Chapter 1. Introduction…………………………………………………..…………..……...1 1.1 Targeting Metalloenzymes for Therapeutic Intervention……………………...………2 1.2 Fragment-Based Drug Discovery Approach for the Development of Metalloenzyme Inhibitors………………………………………………………………………………………....7 1.3 Introduction to New Delhi Metallo-β-lactamase-1……………………….……………11 1.4 Outlook……………………………………………………………………….…………….16 1.5 Acknowledgements……………………………………………………………………..17 1.6 References……....……………….………………………………………………………..18 Chapter 2. Dipicolinic Acid Derivatives as Inhibitors of NDM-1……..……………………23 2.1 Introduction………….……………………………………………………………………..24 2.2 Results and Discussion……………………………………………………………….….26 v 2.2.1 MBP Library Screen and Selection of DPA as Lead Fragment.……..……...……..26 2.2.2 Design and Synthesis of DPA Derivatives (Sublibrary 1 – Sublibrary 3).………...30 2.2.3 In Vitro Activity of DPA Derivatives as Inhibitors of NDM-1…………………...…..35 2.2.4 Investigation of Inhibitor Selectivity……...…………………………………………...43 2.2.5 Determination of Mode of Inhibition…..................…………………………………...45 2.2.6 Microdilution Broth Minimum Inhibitory Concentrations (MICs)……………….…...57 2.3 Conclusion…………………………………………………………………………….60 2.4 Experimental………………………………………………………………….…………62 2.5 Acknowledgements…………………………………………………………………87 2.6 References………………………………………………………………………………..88 Chapter 3. Investigation of DPA Isosteres for the Inhibition of MBLs…………………...93 3.1 Introduction…………………….…………………………………………………………..94 3.2 Results and Discussion……………….………………………………………………….96 3.2.1 DPA Isostere Design and Synthesis (Sublibrary 4)…………………………...…….96 3.2.2 Evaluation of DPA Isosteres Against MBLs.……………………….……...............100 3.2.3 DPA Isostere pKa Analysis …………….….…………............................................102 3.2.4 Mechanism of Isostere Inhibition………………....……………………...................104 3.2.5 Derivatization of Isostere 49 (Sublibrary 5)……………………………………….108 3.3 Discussion………….………...................…………...................………….................115 3.4 Conclusion…………...................…………...................…………….…….................118 3.5 Experimental...........…………...................…………………….………………………119 3.6 Acknowledgements...………….......................…………..............................141 vi 3.7 References………….......................…………..............................................142 Chapter 4. Iminodiacetic Acid as a Metal-binding Pharmacophore for New Delhi Metallo- β-lactamase Inhibitor Development……………………………………………………...145 4.1 Introduction………........................................................................................146 4.2 Results and Discussion………................................................................................148 4.2.1 IDA MBP Design and Verification…………………………………........................148 4.2.2 IDA Derivative Synthesis and Inhibitory Activity (Sublibrary 6 – Sublibrary 7)….153 4.2.3 Thermal Shift Assay………………………………...........................................157 4.2.4. Native State Electrospray Ionization Mass Spectrometry……………………..…159 4.3 Conclusion………........................................................................................165 4.4 Experimental……….....................................................................................166 4.5 Acknowledgements………………...........................................................................188 4.6 References………........................................................................................189 Chapter 5. Alternative DPA Libraries and the Future of NDM-1 Inhibitor Development………………………………………………………………………………….192 5.1 Introduction..................................................................................................193 5.2. meta-Substituted DPA Derivatives (Sublibrary 8)……………….......……………...195 5.2.1. Synthesis and Evaluation of Sublibrary 8………………………...........................197 5.2.2 Discussion……………………………………………………………………….……..200 5.2.3. Experimental…………………………………………………………………………..201 5.3 Tetrazole DPA Derivatives (Sublibrary 9)……………………………….…………….213 5.3.1 Synthesis and Evaluation of Sublibrary 9…………………...................................214 vii 5.3.2 Discussion……………………………………………...........................................216 5.3.3 Experimental.............................................................................................217 5.4 Alternative Isosteres for Inhibitor Development (Sublibrary 10).............................220 5.4.1 Synthesis and Evaluation of Sublibrary 10…………..........................................222 5.4.2. Discussion.........................................................................................225 5.4.3. Experimental.........................................................................................226 5.5 Synthesis and Evaluation of Aspergillomarasmine A.............................................231 5.5.1. Synthesis and Evaluation of AMA…………………………………………………233 5.5.2 Discussion............................................................................................235 5.5.3 Experimental………………………………………………………………………...…236 5.6 Outlook and Perspective on Future NDM-1 Inhibitor Development……………….242 5.7 References..................................................................................................246 viii LIST OF SYMBOLS AND ABBREVIATIONS AMA Aspergillomarasmine A ATP Adenosine Triphosphate BBL B β-Lactamase BLA Biologic License Application Bn Benzyl C Celsius CDC Centers for Disease Control and Prevention CHAPS 3-((3-Cholamidopropyl) Dimethylammonio)-1-propanesulfonate CuAAC Copper-catalyzed Azide-alkyne Cycloaddition DCC N,N′-Dicyclohexylcarbodiimide DCU Dicyclohexylurea DMF N-N’-Dimethylformamide DMSO Dimethyl Sulfoxide DPA Dipicolinic Acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA Ethylenediaminetetraacetic Acid EPR Electron Paramagnetic Resonance ESBL Extended Spectrum β-Lactamases EtOAc Ethyl Acetate FBDD Fragment-based Drug Discovery FDA Food and Drug Administration ix GHz Gigahertz Glo1 Glyoxalase 1 h Hour hCAII Human Carbonic Anhydrase II HDAC Histone Deacetylase HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid HOBT Hydroxybenzotriazole HPLC High-performance Liquid Chromatography HTS High-throughput Screening IDA Iminodiacetic acid IMP IMiPenemase K Kelvin kHz Kilohertz LasB Pseudomonas Aeruginosa Elastase LE Ligand Efficiency LLE Lipophilic Ligand Efficiency MBI Metal-binding Isostere MBL Metallo-β-Lactamase MBP Metal-binding Pharmacophore MeOH Methanol MHz Megahertz MIC Minimum Inhibitory Concentration x min Minute mL Milliliter μM Micromolar mM Millimolar mmol Millimoles MMP Matrix Metalloproteinases MOE Molecular Operating Environment MS Mass Spectrometry μs Microsecond ms Miliseconds mW Milliwatts NDM New Delhi Metallo-β-lactamase ng Nanogram nm Nanometer nM Nanomolar NMEs New Molecular Entities NMR Nuclear Magnetic Resonance NSA ([1,1'-Biphenyl]-4-ylsulfonyl)-D-phenylalanine PBP Penicillin-binding Protein PDB Protein Data Bank PK Pharmacokinetic pM Picomolar xi ppm Parts Per Million RO3 Rule of Three s Second SAR Structure-activity Relationship SBL Serine-β-Lactamase TEA Triethyl Amine TFA Trifluoroacetic Acid THF Tetrahydrofuran TLC Thin Layer Chromatography VIM Verona Integron-encoded Metallo-β-lactamase X-Phos 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl xii LIST OF FIGURES Figure 1-1. FDA-approved metalloenzyme inhibitors between 2013 and 2017 with MBP motifs shown in red…………………………………………………………..…………………6 Figure 1-2. Representative compounds in the MBP fragment library……...……………10 Figure 1-3. Top: NDM-1 hydrolysis of β-lactam drug; Bottom: Structure of NDM-1 with the di-Zn(II) active site, L3, L5, and L10 labeled (adapted from PDB 4EYB). Figure generated via Pymol..……………………………………………………………………..13 Figure 1-4. Examples of current NDM-1 inhibitors reported in literature………….…….15 Figure 2-1. Z’ Factor determination using the substrate chromacef. Absorbance values for positive () controls and negative () controls. Solid lines are the mean values for each set, dashed