University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 11-18-2003 N-Thiolated β-Lactams: Chemistry and Biology of a Novel Class of Antimicrobial Agents for MRSA Timothy E. Long University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Long, Timothy E., "N-Thiolated β-Lactams: Chemistry and Biology of a Novel Class of Antimicrobial Agents for MRSA" (2003). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/1420 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. N-Thiolated β-Lactams: Chemistry and Biology of a Novel Class of Antimicrobial Agents for MRSA by Timothy E. Long A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Science University of South Florida Major Professor: Edward Turos, Ph.D Daniel V. Lim, Ph.D. David Merkler, Ph.D. Gregory Baker, Ph.D. Date of Approval: November 18, 2003 Keywords: Staphylococcus, Antibiotic, Mode of Action, SAR, Drug-Resistance © Copyright 2003, Timothy E. Long ACKNOWLEDGEMENTS The completion of this research would not have been possible without the assistance and guidance of many people with in the Departments of Chemistry and Biology at the University of South Florida. First and foremost, I am grateful to my research advisor, Professor Edward Turos, who has taught me the art of organic synthesis and the importance of reading the literature. Throughout the years, Professor Turos has maintained an environment rich in intellectual thought for which to mature in as a scientist. I am also grateful to him for granting me the freedom to conduct investigations that were of interest despite the occasional failed experiments. The phenomenal experience of working in Dr. Turos' laboratory will serve me well in the future endeavors for which I will always be indepted. During my studies at the University of South Florida, I was obliged to receive direction from an outstanding committee. Firstly, Professor Daniel V. Lim who has guided me throughout these investigations. Without his assistance, several key experiments pertaining to the biology of N-thiolated β- lactams presented within, would have never explored. In addition, Professors David J. Merkler and Gregory R. Baker have been tremendous advisors and have made my research experience over recent years an enjoyable one. While in Professor Turos' laboratory, I had the privilege to work with a number of outstanding scientists. I am grateful to all my current and former colleagues for their assistance and support. First and foremost, my labmates who also initiated this project and helped in its funding: Cristina M. Coates, Bart A. Heldreth, and Jeung-Yeop Shim. It was truly a wonderful and pleasurable experience working with them over the years. In later years, several other remarkable individuals who joined the lab and had the same influence included: Sampath Abeylath, Marcie Culbreath, Dr. Seyoung Jang, J. Michelle Leslie, Dr. Suresh Reddy, and Yang (Helen) Wang. Finally, I would to thank the talented undergraduate researchers whom I was honored to assist in their experiments: Alex Ortiz, Jaenea Polk, Arturo Torres, and Brenda K. Yantzer. I would also like to acknowledge the technical assistance of Sonja Dickey. Her efforts made all the biological studies possible and I am very much indepted to her. Moreover, the microscopy studies were made possible with the aid of Betty Loraamm. She provided invaluable assistance helping use the electron microscope and taking the photographs. Finally, I want to thank my parents, Stephen and Janice Long. They have provided everything needed to succeed in my life, for which I am deeply grateful. Without their love and support, I would never have been able to accomplish any of this work. This is as well extended to members of my immediate family including my brothers, Chris and Dan, and my grandparents. TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF SCHEMES vii LIST OF SPECTRA ix LIST OF ABBREVIATIONS xi ABSTRACT xiii CHAPTER 1: CLINICAL DEVELOPMENT OF NEW ANTI-MRSA 1 ANTIBIOTICS 1.1 Ιntroduction 1 1.2 β-Lactam Antibiotics 1 1.2.1 Cephalosporins 1 1.2.2 Carbapenems 4 1.2.3 N-Thiolated β-Lactams 4 1.3 Peptide Antibiotics 5 1.3.1 Second Generation Glycopeptides 5 1.3.2 Lipopeptides (Daptomycin) 6 1.3.3 Depsipeptides 6 1.4 Oxazolidinones 7 1.5 Quinolones, Glycylcyclines and Coumarin Antibiotics 8 1.6 Conclusions 9 CHAPTER II: CHEMISTRY AND BIOLOGICAL PROPERTIES OF N-THIOLATED 10 β-LACTAMS 2.1 Introduction 10 2.2 N-SO2X β-Lactams 10 2.2.1 N-Sulfonic Acid β-Lactams 11 2.2.2 N-Chlorosulfonyl β-Lactams 15 2.2.3 N-Aryloxysulfonyl and N-Alkoxysulfonyl β-Lactams 16 2.3 N-SO2R β-Lactams 16 2.4 N-SOR β-Lactams 19 2.5 N-SX β-Lactams 20 2.6 N-SR β-Lactams 22 2.7 Conclusions 29 CHAPTER III: SYNTHESIS AND BIOLOGICAL PROPERTIES OF C4 ARYL SUBSTITUTED N-THIOLATED β-LACTAMS 30 3.1 Introduction 30 3.2 Synthesis of C4 Aryl Substituted N-Thiolated β-Lactams 30 3.2.1 Synthesis of C-Aryl(imines) 6 31 3.2.2 Synthesis of N-Aryl Protected β-Lactams 8 32 by Staudinger Coupling 3.2.3 Dearylation of β-Lactams 8 with Ceric Ammonium 34 Nitrate 3.2.4 N-Methylsulfenylation of N-Protio β-Lactams 9 35 3.3 The Structure-Activity Profiling of C4 Phenyl Analogues 36 i 3.3.1 Synthesis and Microbiological Evaluation of 67-81 36 3.3.2 Structure-Activity Relationship of 67-81 and 83 40 Against MSSA 3.3.2 Structure-Activity Relationship of 67-81 and 83 40 Against MRSA 3.3.3 Effect of Drug Amount Versus Zone Diameter 41 3.3.4 Minimum Inhibitory Concentration (MIC) 43 3.3.5 In Vitro Activity in Blood Serum 43 3.3.6 Time-Kill Studies 44 3.4 Multihalogenated Phenyl Analogues and Their Biological 44 Activities 3.4.1 Synthesis of Multihalogenated Phenyl Analogues 44 3.4.2 Antimicrobial Activity of Multihalogenated Phenyl 45 Analogues 3.5 N-Sulfenylated Analogues and Their Biological Activities 46 3.5.1 Synthesis of N-Sulfenylated Analogues 46 3.5.2 Antimicrobial Susceptibilities to Lactams 101-109 47 3.6 Antibacterial Activity of Heterosubstituted N-Thiolated 48 β-Lactams 3.7 Additional N-Thiolated β-Lactam Analogues Probed For 51 Biological Activity 3.8 Antifungal Properties of N-Thiolated β-Lactams 53 3.9 Antiviral Properties of N-Thiolated β-Lactams 54 CHAPTER IV: MODE OF ACTION OF N-THIOLATED β-LACTAMS 56 4.1 Introduction 56 4.2 Probing the Modes of Action 57 4.3 N-Thiolated β-Lactams as Acylating Agents 57 4.3.1 Scanning Electron Microscopy 58 4.3.2 Light Microscopy 59 4.3.3 Model Membrane Studies 59 4.4 N-Thiolated β-Lactams as Alkylating Agents 60 4.4.1 Anticancer Properties of Lactam 68 61 4.4.2 DNA Cleavage Studies 61 4.4.3 Pulse-Labeling Studies of DNA Replication 62 4.5 N-Thiolated β-Lactams as Thiolating Agents 63 4.5.1 Enzyme-Binding Properties of N-Thiolated β-Lactams 63 4.5.2 Thiol Determination in Bacteria 64 4.5.4 1H NMR Studies 66 4.6 Effects of Lactam 68 on Gene Expression 66 4.6.1 Introduction 66 4.6.2 Pulse-Labeling Studies of RNA Assimilation 67 4.6.3 Pulse-Labeling Studies of Protein Synthesis 67 CHAPTER V: DISCUSSSION, CONCLUSIONS, AND FUTURE DIRECTIONS 69 5.1 Discussion and Conclusions 69 5.1.1 Introduction 69 5.1.2 Narrow vs Broad Spectrum 69 5.1.3 Bioactivity Spectrum 70 5.1.4 Structure-Activity Relationship 71 5.1.5 Mechanism of Action 71 5.2 Future Directions 72 5.2.1 Structure-Activity Relationship 72 5.2.2 Elucidation of the Biological Target 75 ii CHAPTER VI: MATERIALS AND METHODS 77 CHAPTER VII: 1H AND 13C NMR SPECTRA 91 REFERENCES 147 ABOUT THE AUTHOR iii LIST OF TABLES Table 1.01: In vitro and in vivo activities of anti-MRSA cephalosporins in clinical 3 development. Table 1.02: Biological activity comparisons of oritavancin, dalbavancin and daptomycin. 7 Table 3.01: Stereochemical outcome of the Staudinger reaction of ketenes and imines. 33 Table 3.02: Comparison of microbes sensitive to N-thiolated β-lactam antibiotics. 38 Table 3.03: In vitro susceptibilities of bacteria to N-methylthio β-lactams. 39 Table 3.04: Kirby-Bauer data for analogues 67-81 and 83 against S. aureus. 41 Table 3.05: Kirby-Bauer data for analogues 1, 67, and 101-109. 47 Table 3.06: Disc diffusion data for various N-thiolated β-lactams. 53 Table 3.07: Cytotoxicity and antiviral activity of N-methylthio β-lactams 1, 69, and 75 55 in HeLa (cervix carcinoma) cell cultures. Table 3.08: Cytotoxicity and antiviral activity of N-methylthio β-lactams 1, 69, and 75 55 in HEL (Human Embryonic Lung) cell cultures. Table 3.09: Cytotoxicity and antiviral activity of N-methylthio β-lactams 1, 69, and 75 in 55 Vero cell cultures. Table 4.01: Important classes of antibiotics and there respective intrinsic characteristics. 56 Table 4.02: Correlation between relative intracellular thiol levels and susceptibility 65 to lactam 68. Table 5.01: Susceptibility comparison of N-methylthio β-lactams 67-81, 83, and 97-99.