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N-thiolated β-lactams

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Title:
N-thiolated β-lactams chemistry and biology of a novel class of antimicrobial agents for MRSA
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Book
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English
Creator:
Long, Timothy E ( Timothy Edward )
Publisher:
University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
antibiotic
mode of action
sar
drug resistance
staphylococcus
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: N-Methylthio beta-lactams represent a promising new family of antibacterial agents whose in vitro activity is confined largely to Staphylococcus species, including multidrug-resistant forms of S. aureus. Originally developed in the 1980's for use as synthetic intermediates, N-methylthio beta-lactams have recently been shown in these laboratories to possess intriguing biological properties which are addressed in Chapters I-IV. In terms of the antibacterial activities, the structural features and species specificities exhibited by these compounds are unlike those of any existing family of beta-lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active Beta-lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes. The most active members of this Beta-lactam class appear to be those bearing an aryl (Ar) substituent at C4 of the ring. The synthesis and structure-activity relationship of these analogues is discussed in Chapter III. Moreover, microscopy and 3H pulse-labeling studies, which are described in Chapter IV, demonstrate that N-methylthio beta-lactams appear to be inhibitors of protein biosynthesis.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2003.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Timothy E. Long.
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Includes vita.
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Title from PDF of title page.
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Document formatted into pages; contains 173 pages.

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oclc - 54018516
notis - AJM5946
usfldc doi - E14-SFE0000180
usfldc handle - e14.180
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N -Thiolated -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

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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 lite rature. 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 lactams presented within, would have never explored. In addition, Professors Davi d 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 assist ance and support. First and foremost, my labmates who also initiated this project an d helped in its funding: Cristina M. Coates, Bart A. Heldreth, and Jeung-Yeop Shim. It was truly a wonder ful and pleasurable experi ence 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 a ssistance 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 wh ich 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.

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i 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 ntroduction 1 1.2 -Lactam Antibiotics 1 1.2.1 Cephalosporins 1 1.2.2 Carbapenems 4 1.2.3 N-Thiolated -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 -LACTAMS 2.1 Introduction 10 2.2 N-SO2X -Lactams 10 2.2.1 N -Sulfonic Acid -Lactams 11 2.2.2 N -Chlorosulfonyl -Lactams 15 2.2.3 N -Aryloxysulfonyl and N -Alkoxysulfonyl -Lactams 16 2.3 N-SO2R -Lactams 16 2.4 N-SOR -Lactams 19 2.5 N-SX -Lactams 20 2.6 N-SR -Lactams 22 2.7 Conclusions 29 CHAPTER III: SYNTHESIS AND BIOLOGICAL PROPERTIES OF C4 ARYL SUBSTITUTED N -THIOLATED -LACTAMS 30 3.1 Introduction 30 3.2 Synthesis of C4 Aryl Substituted N -Thiolated -Lactams 30 3.2.1 Synthesis of C -Aryl(imines) 6 31 3.2.2 Synthesis of N -Aryl Protected -Lactams 8 32 by Staudinger Coupling 3.2.3 Dearylation of -Lactams 8 with Ceric Ammonium 34 Nitrate 3.2.4 N -Methylsulfenylation of N -Protio -Lactams 9 35 3.3 The Structure-Activity Profiling of C4 Phenyl Analogues 36

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ii 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 101109 47 3.6 Antibacterial Activity of Heterosubstituted N -Thiolated 48 -Lactams 3.7 Additional N -Thiolated -Lactam Analogues Probed For 51 Biological Activity 3.8 Antifungal Properties of N -Thiolated -Lactams 53 3.9 Antiviral Properties of N -Thiolated -Lactams 54 CHAPTER IV: MODE OF ACTION OF N -THIOLATED -LACTAMS 56 4.1 Introduction 56 4.2 Probing the Modes of Action 57 4.3 N -Thiolated -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 -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 -Lactams as Thiolating Agents 63 4.5.1 Enzyme-Binding Properties of N -Thiolated -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

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iii CHAPTER VI: MATERIALS AND METHODS 77 CHAPTER VII: 1H AND 13C NMR SPECTRA 91 REFERENCES 147 ABOUT THE AUTHOR

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iv 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 -lactam antibiotics. 38 Table 3.03: In vitro susceptibilities of bacteria to N -methylthio -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 101109. 47 Table 3.06: Disc diffusion data for various N -thiolated -lactams. 53 Table 3.07: Cytotoxicity and antiviral activity of N -methylthio -lactams 1 69 and 75 55 in HeLa (cervix carcinoma) cell cultures. Table 3.08: Cytotoxicity and antiviral activity of N -methylthio -lactams 1 69 and 75 55 in HEL (Human Embryonic Lung) cell cultures. Table 3.09: Cytotoxicity and antiviral activity of N -methylthio -lactams 1 69 and 75 in 55 Vero cell cultures. Table 4.01: Important classes of antibiotics and there resp ective intrinsic characteristic s. 56 Table 4.02: Correlation between relative intracellular thiol levels and susceptibility 65 to lactam 68. Table 5.01: Susceptibility comparison of N -methylthio -lactams 67 81, 83 and 97 99. 70

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v LIST OF FIGURES Figure 1.01: Families of clinically important -lactam antibiotics. 1 Figure 1.02: Anti-MRSA cephalosporins in clinical development. 2 Figure 1.03: Anti-MRSA penems in clinical development. 4 Figure 1.04: N -Methylthio -lactams with anti-staphylococcal properties. 5 Figure 1.05: Vancomycin and teicoplanin. 5 Figure 1.06: Oritavancin and dalbavancin. 6 Figure 1.07: Daptomycin and WAP-8294A2. 7 Figure 1.08: Linezolid, VRC-3783, and AZD-2563. 8 Figure 1.09: Anti-MRSA quinolones in clinical development. 8 Figure 1.10: Anti-MRSA coumarin and glycylcycline in clinical development. 9 Figure 2.01: Classes of -lactam antibiotics. 10 Figure 2.02: Chemical classes of N -thiolated -lactams. 10 Figure 2.03: First monobactams discovered by American and Asian scientists. 11 Figure 2.04: Intermediates used to synthesize 3-amino monobactamic acids. 12 Figure 2.05: Synthetic monobactams with enhanced bioactivities. 13 Figure 2.06: Bicyclic monobactam inhibitors of class C -lactamase. 14 Figure 2.07: Bicyclic monobactams 12-21 14 Figure 2.08: Bicyclic and multicyclic N -chlorosulfonyl -lactams 24-27. 15 Figure 2.09: Dimeric N -chlorosulfonyl -lactam 28. 15 Figure 2.10: Deprotection of N -chlorosulfonyl -lactams with thiophenol. 16 Figure 2.11: N -Sulfonyl -lactams inhibitors of porcine pancreatic elastase. 19 Figure 2.12: N -Trifluoromethylsulfinyl -lactam. 20 Figure 2.13: Heterosubstituted N -thiolated -lactams 64-66. 20 Figure 2.14: Examples on N -methylthio monocyclic -lactams. 23 Figure 2.15: N -Trifluoromethylthio -lactam investigated by Merck laboratorie s. 26 Figure 2.16: N-S fused bicyclic -lactams of Pfizer. 27 Figure 2.17: Human leukocyte elastase inhibitor 123. 28 Figure 2.18: Bicyclic N -thiolated -lactam elastase inhibitors 124-129. 28 Figure 3.01: Percent relative activity of penicillin for monosubstituted analogues against 40 methicillin-susceptible Staphylococcus aureus (MSSA). Figure 3.02: Percent relative activity of vancomycin for monosubstituted analogues against 41 methicillin-resistant Staphylococcus aureus (MRSA). Figure 3.03: Equimolar drug loads versus zone of growth inhibition of (a) lactam 69 and 42 (b) penicillin G against MSSA and MRSA. Figure 3.04: Equimolar drug loads versus zone of growth inhibition of lactam against. 42 (a) MSSA and (b) MRSA. Figure 3.05: Growth studies of MRSA with and without blood serum. 43 Figure 3.06: Time-kill studies of 68 against (a) MSSA and (b) MRSA. 44 Figure 3.07: Percent activities of lactams 9699 against (a) MSSA and (b) MRSA. 45 Figure 3.08: Comparison of anti-MSSA activities of lactams 101 109 to penicillin G. 48 Figure 3.09: Anti-MRSA activity comparison of 101 109 48 Figure 3.10: Thiolated bis -lactam dimers 116 and 117. 50 Figure 3.11: Comparison of an ti-MSSA and anti-MRSA activities 116 117, and 119 51 Figure 3.12: Comparision of antifungal activities against C. albicans 54 Figure 3.13: Comparision of antifungal activities against C. tropicalis 54 Figure 4.01: N -Protio -lactam 53 and N -methylthio -lactam 68 58

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vi Figure 4.02: Scanning electron microscopy of S. aureus cultured with (a) no antibiotic, 58 (b) lactam 68, and (c) penicillin G. Figure 4.03: Light microscopy of S. aureus exposed to (a) no antibiotic, (b) lactam 68, and 59 (c) penicillin G. Figure 4.04: Supercoiled DNA treated with lactam 68 at 5-100 M. 61 Figure 4.05: Supercoiled DNA treated with lactam 68 in the presence of thiols. 62 Figure 4.06: Thymidine incorporation in S. aureus. 63 Figure 4.07: Antagonist effect of glutathione (GSH) on the anti-MSSA properties of 64 N -thiolated -lactams. Figure 4.08: Zone diameter of lactam 68 compared to relative thiol levels in select bacteria. 65 Figure 4.09: 1H NMR spectrums of (a) lactam 68 prior to inoculation of S. aureus and 66 (b) lactam 68 following inoculation of S. aureus Figure 4.10: Uridine incorporation in S. aureus 67 Figure 4.11: Isoleucine incorporation in S. aureus 68 Figure 5.01: Chemical structures of penicillin and N -methylthio -lactams. 69 Figure 5.02: Stucture of N -methythio -lactams 1 and 68 71 Figure 5.03: Location of nucleophilic attack on electropositive centers of 3 antibioitics: 72 penicillin, mitomycin C, and N -methylthio -lactam 68. Figure 5.04: Fractionation of cellular components in bacteria. 76

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vii LIST OF SCHEMES Scheme 1.01: Enzyme-catalyzed therapeutic activation mechanism of NB-2001. 4 Scheme 2.01: N -Sulfonation of -lactams with pyridine-sulfur complex. 11 Scheme 2.02: Bases-promoted cyclization for synthesizing monobactams. 12 Scheme 2.03: Acylation of 3-amino monobactamic acids. 12 Scheme 2.04: Mechanism of -lactam hydrolysis by serine transpeptidases. 13 Scheme 2.05: Synthesis of 4-acetoxy N -chlorosulfonyl-azetidin-2-one 23 15 Scheme 2.06: Synthesis of N -aryloxysulfonyl and N -alkoxysulfonyl -lactams. 16 Scheme 2.07: Sulfonylation of N -protio -lactams. 17 Scheme 2.08: Dehydrative ring closure of sulfonamido carboxylic acid 42 17 Scheme 2.09: -Lactam synthesis via olefin-isocyanate cycloaddition. 17 Scheme 2.10: -Lactam synthesis via base-mediated iodocyclization. 18 Scheme 2.11: -Lactam synthesis via Ni-mediated CO in sertion. 18 Scheme 2.12: Proposed mechanism of N -arylsulfonyl -lactams inhibitors of human 18 leukocyte elastase. Scheme 2.13: N-S-Fused bicyclic -lactams. 19 Scheme 2.14: Preparation of N -phthalimidothio -lactams. 20 Scheme 2.15: Preparation of heterosubstituted N -thiolated -lactams via phthalimide 21 exchange. Scheme 2.16: Synthesis of disulfide-linked -lactams. 21 Scheme 2.17: Synthesis of disulfide-linked lactam 72. 22 Scheme 2.18: Preparation of N -sulfenyl -lactams. 22 Scheme 2.19: N -Methylthiolation of -lactams with methanethiosulfonate. 22 Scheme 2.20: Preparation of S -trityl -lactams via enolate-sulfenimine ad dition. 23 Scheme 2.21: Sulfenylation of N -protio -lactams with N -thiophthalimide. 24 Scheme 2.22: Displacement of phthalimide by carbon nucleophile. 24 Scheme 2.23: Decomposition pathways of thiamazins by hydroxide ion. 25 Scheme 2.24: Mechanisms of reactivity of N -thiolated -lactams towards nucleophiles. 26 Scheme 2.25: Cyclization of 110 to the non-penem 111. 26 Scheme 2.26: Preparation of 117 using sulfur bis -phthalimide. 27 Scheme 2.27: Preparation of 117 using N -chlorosuccinimide. 27 Scheme 2.28: Preparation of bicyclic using triphenylphosphine. 28 Scheme 2.29: Preparation of bicyclic lactams via halocy clization. 29 Scheme 2.30: Halocyclization of lactams 85 86 and 132. 29 Scheme 3.01: Iodocyclization of N -methylthio lactam 1 30 Scheme 3.02: General synthetic route to lactam 3 31 Scheme 3.03: Synthesis of C -aryl(imines). 31 Scheme 3.04: Swern oxidation of iodo-benzyl alcohols. 31 Scheme 3.05: Esterification of p -hydroxybenzaldehyde. 32 Scheme 3.06: Synthesis of bis -imine 15 32 Scheme 3.07: Synthesis of N -aryl protected -lactams by Staudinger [2+2] condensation. 32 Scheme 3.08: Ketene-imine cycloaddition. 33 Scheme 3.09: Synthesis of acid chlorides 16 and 18 34 Scheme 3.10: Synthesis of bis -lactam from bis -imine 15. 34 Scheme 3.11: Oxidative dearylation of -lactams 6 35 Scheme 3.12: Synthesis of N -methythio -lactams and methythiomesylate. 35 Scheme 3.13: Synthesis of N -methythio -lactams and N -(methylthio)phthalimide. 36

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viii Scheme 3.14: Synthesis of C4 phenyl analogues 67 81 36 Scheme 3.15: Synthesis of C4 phenolic -lactams from 66 37 Scheme 3.16: Synthesis of multihalogenated N -thiolated -lactams. 45 Scheme 3.17: Synthesis of N -thiophthalimide reagents 94 99 46 Scheme 3.18: Synthesis of N -sulfenyl analogues 101106. 46 Scheme 3.19: Synthesis of N -sulfenyl analogues 107110. 47 Scheme 3.20: Synthesis of heterosubstituted N -thiolated -lactams. 49 Scheme 3.21: Synthesis of N N '-thiobisphthalimide ( 111 ). 49 Scheme 3.22: Reaction of 53 with N N '-thiobisphthalimide ( 111). 49 Scheme 3.23: Synthesis of N -phthalimidothio -lactam 119. 50 Scheme 3.24: Reaction of 118 with morpholine and diisopropylamine. 50 Scheme 3.25: Reaction of 119 with sodium methoxide and isopropanol. 51 Scheme 3.26: Synthesis of 2-thiophene -lactam 127 52 Scheme 3.27: Synthesis of lactams 134, 135, and 137. 52 Scheme 3.28: Synthesis of C3 hydroxy substituted analogue. 53 Scheme 4.01: Possible reactions of N -thiolated -lactams. 57 Scheme 4.02: Acylation of reactivity penicillin-binding proteins (HO-PBP), a serine 57 hydrolase, by pencillin. Scheme 4.03: Methylation of a nucleophilic substrate from the conversion of L-methionine 60 to S -adenosylmethionine. Scheme 4.04: Alkylation of guanosine by leinamycin and mitomycin C. 60 Scheme 4.05: Electrophilic sulfenylation of nucleophiles. 63 Scheme 4.06: Reaction of cysteine and lactam 68. 64 Scheme 4.07: Reaction of Ellman's reagent an d a thiol. 65 Scheme 5.01: Reduction of N -arylated -lactam 90 to azetidine 143 73 Scheme 5.02: Preparation of Lawesson's reagent. 73 Scheme 5.03: Synthesis of thiolactams 144146. 73 Scheme 5.04: Synthesis of 2-methylene azetidines from thiolactams via Eschenmoser 74 sulfide contraction. Scheme 5.05: Unsuccessful synthese s of vinylogous azetidines from N -protio thiolactams. 74 Scheme 5.06: Unsuccessful synthese s of vinylogous azetidines from N -Boc thiolactams. 75 Scheme 5.07: Unsuccessful synt hesis of noncyclic thioamide 156. 75 Scheme 5.08: Proposed synthetic route to [3H]-labeled lactam 68. 75

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ix LIST OF SPECTRA Spectrum 7.01: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 37 91 Spectrum 7.02: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 38 92 Spectrum 7.03: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 39 93 Spectrum 7.04: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 40 94 Spectrum 7.05: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 41 95 Spectrum 7.06: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 42 96 Spectrum 7.07: 1H NMR (250 MHz, CDCl3) of -lactam 43 97 Spectrum 7.08: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 45 98 Spectrum 7.09: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 46 99 Spectrum 7.10: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 47 100 Spectrum 7.11: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 48 101 Spectrum 7.12: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 49 102 Spectrum 7.13: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 50 103 Spectrum 7.14: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 51 104 Spectrum 7.15: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 52 105 Spectrum 7.16: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 53 106 Spectrum 7.17: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 54 107 Spectrum 7.18: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 55 108 Spectrum 7.19: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 56 109 Spectrum 7.20: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 57 110 Spectrum 7.21: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 58 111 Spectrum 7.22: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 60 112 Spectrum 7.23: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 61 113 Spectrum 7.24: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 62 114 Spectrum 7.25: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 63 115 Spectrum 7.26: 1H NMR (250 MHz, CDCl3) of -lactam 64 116 Spectrum 7.27: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 65 117 Spectrum 7.28: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 66 118 Spectrum 7.29: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 70 119 Spectrum 7.30: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 71 120 Spectrum 7.31: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 72 121 Spectrum 7.32: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 73 122 Spectrum 7.33: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 74 123 Spectrum 7.34: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 75 124 Spectrum 7.36: 1H NMR (250 MHz, CDCl3) of -lactam 77 125 Spectrum 7.37: 1H NMR (360 MHz, CDCl3) of -lactam 78 126 Spectrum 7.38: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 81 127 Spectrum 7.39: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 82 128 Spectrum 7.40: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 83 129 Spectrum 7.41: 1H NMR (250 MHz, CDCl3) of -lactam 88 130 Spectrum 7.42: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 89 131 Spectrum 7.43: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 90 132 Spectrum 7.44: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 91 133 Spectrum 7.45: 1H NMR (250 MHz, CDCl3) of -lactam 92 134 Spectrum 7.46: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 94 135

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x Spectrum 7.47: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 95 136 Spectrum 7.48: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 97 137 Spectrum 7.49: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 98 138 Spectrum 7.50: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 99 139 Spectrum 7.51: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 108 140 Spectrum 7.52: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 110 141 Spectrum 7.53: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 117 142 Spectrum 7.54: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 125 143 Spectrum 7.55: 1H NMR (360 MHz, CDCl3) of -lactam 133 144 Spectrum 7.56: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 135 145 Spectrum 7.57: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 137 146

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xi LIST OF ABBREVIATIONS alpha Ar aryl Ac acetyl Aco acetoxy beta Bn benzyl Br broad Bu butyl Boc tert -butyloxycarbonyl bp boiling point br broad (spectral) Bz benzoyl C degrees Celsius 13C carbon-13 c concentration (mg/ml) CAN ceric ammonium nitrate Cbz carbobenzyloxy CH2Cl2 dichloromethane Cl2 chlorine gas cm-1 wave numbers (reciprocal centimeters) CSA camphorsulfonic acid D deuterium (2H) DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC dicyclohexyl carbodiimide DEAD diethyl azodicarboxylate DIBAL diisobutylaluminum hydride DMAP 4-dimethylaminopyridine DMSO dimethylsulfoxide Et ethyl Et3N triethylamine EtOAc ethyl acetate g gram(s) 1H proton 2H deuterium 3H tritium HMDS hexamethyldisilazide hr hour(s) Hnig's base ethyldiisopropylamine Hz hertz IR infrared

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xii J coupling-constant(s) KOH potassium hydroxide LAH lithium aluminum hydride LDA lithium diisopropylamide Lawesson para -methoxyphenylthionophosphine sulphide dimer reagent Me methyl MeCN acetonitrile MeOH methanol mCPBA meta -chloroperoxybenzoic acid mg milligram(s) g microgram(s) mM millimoles per liter mmol millimole(s) mol mole(s) MOM methoxymethyl MRSA methicillin-resistant Staphylococcus aureus MSSA methicillin-susceptible Staphylococcus aureus Ms methanesulfonyl, mesyl N normal NaOH sodium hydroxide OD optical density PCC pyridinium chlorochromate Ph phenyl PhH benzene PhMe toluene ppm parts per million NPhth N -phthalimido PMP para -methoxyphenyl RT room temperature SEM scanning electron microscopy TBAF tetran -butylammonium fluoride TBDPS tert -butyldiphenylsilyl TBS tert -butyldimethylsilyl tBu tert -butoxycarbonyl THF tetrahydrofuran TLC thin layer chromatography Tf trifluoromethylsulfonyl, trifyl (CF3SO2) TFA trifluoroacetic acid TCA tichloroacetic acid Ts para -toluenesulfonyl, tosyl ( p -CH3C6H4SO2)

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N-Thiolated -Lactams: Chemistry and Biology of a Novel Class of Antimicrobial Agents for MRSA Timothy E. Long ABSTRACT N-Methylthio -lactams (1) represent a promising new family of antibacterial agents whose in vitro activity is confined largely to Staphylococcus species, including multidrug-resistant forms of S. aureus. Originally developed in the 1980s for use as synthetic intermediates, N-methylthio -lactams have recently been shown in these laboratories to possess intriguing biological properties which are addressed in Chapters I-IV. In terms of the antibacterial activities, the structural features and species specificities exhibited by these compounds are unlike those of any existing family of -lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active -lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes. The most active members of this -lactam class appear to be those bearing an aryl substituent (Ar) at C 4 of the ring. The synthesis and structure-activity relationship of these analogues is discussed in Chapter III. Moreover, microscopy and 3 H pulse-labeling studies, which are described in Chapter IV, demonstrate that N-methylthio -lactams appear to be inhibitors of protein biosynthesis. N O S C H3 RO Ar H H 1 xiii

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CHAPTER I CLINICAL DEVELOPMENT OF NEW ANTI-MRSA ANTIBIOTICS 1.1 Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant pathogen whose insensitivity to most antibiotics has evolved over the span of five decades. Historically, methicillin, a second-generation semi-synthetic penicillin, was first introduced into hospitals in 1960 in response to the prominence of -lactamase producing staphylococci. However, the bacteria rapidly adapted and in 1961, the first MRSA strain emerged in Cairo, Egypt. 1 As MRSA continues to evolve and becomes more resistant to current antibiotics, the need to find new therapies grows increasingly important. This chapter focuses on the recent progress towards the clinical development of new antimicrobial agents for MRSA. 1.2 -Lactam Antibiotics -Lactams (Figure 1.01) remain the most widely prescribed class of antibiotics despite the continuous rise in resistance that forced the penicillins and cephalosporins to undergo multiple generations of synthetic change. S. aureus resistance to these antibacterials stem primarily from ring cleavage by -lactamase and modification or overproduction of the enzymes involved in cell wall biosynthesis penicillin-binding proteins (PBPs). 2 In recent years, structure-activity relationship (SAR) studies of semisynthetic cephalosporins and penems has identified chemical functionalities that enhance binding to low affinity transpeptidases (ie, PBP2a) and confer resistance to -lactamase. Figure 1.01: Familes of clinically important-lactam antibiotics. NOSCO2HR"R' penicillinsNOSCO2H cephalosporinsNOSCO2H X penamsNOSCO2HR carbapenamsNO monobactamsSO3H RCONH RCONH R' OH OH RCONH R' NO RCONH R' OH CO2H nocardicins 1.2.1 Cephalosporins Traditional cephalosporins are poor substrates for PBP2a and are thus considered ineffective as anti-MRSA agents. During the 1990s, new semisynthetic analogues emerged possessing higher affinity for this low binding transpeptidase combined with improved stability toward -lactamases. The cephalosporins have polar, lipophilic groups at C(3) and C(7) positions of the ring which were discovered to enhance bioactivity and chemical stability. 3 Among these, MC-02479 (RWJ-5442, Essential Therapeutics/RW 1

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Johnson Pharmaceutical Research Institute; Figure 1.02, Table 1.01) and BAL-5788 (BASILEA Pharmaceutica AG) have since advanced to clinical trials in the US and Europe, respectively. Figure 1.02: Anti-MRSA cephalosporins in clinical development. NOSHN R CO 2 OR' CNOHNSClH2N N*SCH2S(CH2)2NH2 NO*CHNH NNSCNOHH2N N*SCH2S(CH2)2NH2 NNSCNOHNH2 MC-02,479 BAL9141 MC-04,546/RWJ-333441R =R'=*** *CH2N+NNHCHOS CP-6679NNSCNOCH2FH2N BMS-247243N+*CH2S C H3CH3NO SCH2*ClClNHOCO2 NNSCNH2NOEt *CH2N+NN(CH2)3NHCH3 +**R =R'= S-3578 CH3 SCH2*ClClO2C N+*CH2SOHNH2 SCH2*ClClO2C N+*CH2SOH2N 12 R =R'= MC-02479 is a broad spectrum cephalosporin with a C(7) 4-pyridinethiol zwitterionic moiety contributing to the lactam's activity and solubility. 4,5 MC-02479 is equipotent to vancomycin against 256 2

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MRSA strains and the binding affinity for PBP2a is reportedly 43-fold greater than imipenem. SAR studies of the 4-pyridinethiol series also led to the discovery of MC-04546 (RWJ-333441, Essential Therapeutics/RW Johnson Pharmaceutical Research Institute; Figure 1.02), an analogue with enhanced serum stability lending to the cephem's above-average pharmacokinetic properties in rats. 6 BAL-5788, the water-soluble prodrug of BAL-9141 (Figure 1.02), is also an efficient substrate for PBP2a and effectively treated MRSA-induced endocarditis in rats. 7 Furthermore, it is stable to class A and C -lactamases, which is attributed to the C(7) hydroxyiminoacetamido side chain. 7 Other anti-MRSA cephems bearing similar C(3) and C(7) substituents have also been reported. Currently, CP-6679 (Meiji Seika Kaisha Ltd; Figure 1.02) is being developed as part of an imidazothiazolium series of cephalosporins. The compound possesses 88-fold greater binding ability to PBP2a than imipenem but displays only modest in vitro activity against MRSA. 8 Another clinical trial candidate is S-3578 (Figure 1.02), a unique lipophilic cephalosporin exhibiting low serum binding (< 25%) while maintaining the therapeutic efficacy of vancomycin in rats with MRSA-induced infections. 9 3 MIC 90 a IC 50 b -lactamase stability ED 50 c (mg/kg) MC-02479 2 0.7 Stable NR MC-04456 1 0.5 Stable 1.4 BAL-9141 2 0.5 Stable 2.4 CP-6679 12.5 5.1 NR 12 S-3578 4 4.42 Stable 5 to 8 BMS-247243 5 0.7 Stable 2.0 to 3.8 NB-2001 0.5 NR Not stable NR Table 1.01: In vitro and in vivo activities of anti-MRSA cephalosporins in clinical evelo p ment. 4-9 d a o a Typical MIC 90 values (minimum inhibition concentration for 90% of MRSA test strains) for vncomycin and ceftriaxone are 2 and > 64 g/ml, respectively; b Typical IC 50 values (concentration t inhibit 50% PBP2a activity) for imipenem are > 100 g/ml; c ED 50 : effective dose in 50% of mice or rats with systemic MRSA infection; NR: not reported or could not be found in the literature. Bristol-Myers Squibb Co is investigating a thiopyridinium series of cephalosporins without a C(7) imino side chain. 10 In lieu of an iminoacetamide group is a dichlorothiophenyl acetamide conferring stability toward A to D class -lactamases. 11 As an early lead compound from this series, BMS-247243 (Bristol-Myers Squibb Co; Figure 1.02) gave rise to other potent anti-MRSA cephalosporins bearing similar functionalities. 11,12 SAR was disclosed in recent publications and new candidates emerged for further pre-clinical evaluation including cephalosporins 1 and 2 (Figure 1.02) whose average PD 50 in mice with systemic MRSA infections is 3.9 and 0.8 mg/kg, respectively. 13 Companies such as Cubist Pharmaceuticals Inc and BASILEA Pharmaceutica AG also boast other anti-MRSA cephalosporins in the late stages of clinical development. CAB-175 (Cubist Pharmaceuticals Inc), 14 an azomethine cephalosporin, is expected to begin human trials during the first half of 2003 for the intravenous treatment of drug-resistant bacteria. CAB-175 demonstrates bactericidal activity against MRSA while maintaining a similar safety profile to ceftriaxone. BAL-2057 (BASILEA Pharmaceutica AG), a member from a new class of narrow spectrum anti-MRSA cephalosporins, is reportedly in the final stages of optimization after displaying consistently high in vivo activity in animal models. 15 However, no additional information on BAL-2057 could be obtained. Using a different chemotherapeutic approach, NewBiotics Inc is utilizing the cephalosporin NB-2001 in an enzyme-catalyzed therapeutic activation (ECTA) method for delivering the bactericide triclosan upon ring hydrolysis of the inactive prodrug (Scheme 1.01). 16 While extensive research on -lactamase-dependent prodrugs has previously been conducted, 17 interest remains in ECTA as a means to circumvent the development of resistance in bacteria by controlled release of the active drug when the drug-resistant pathogen is present.

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Scheme 1.01: Enzyme-catalyzed therapeutic activation mechanism of NB-2001. 16 NOSHNCO2OOClClClOS H2OClOClClO NSOO-NHOSCO2 -Lactamasetriclosan+ NB2001 1.2.2 Carbapenems Although existing carbapenems are not useful drugs for MRSA, this family of antibiotics continues to draw interest because of their chemical stability towards -lactamase. During the 1990s, anti-MRSA carbapenems 18 were being developed by Merck & Co, SmithKline Beecham plc, and Wyeth-Ayerst Research, however, investigations of the naphthosultam penems, 19 trinems, 20 and THF carbapenems, 21 respectively, were discontinued or suspended by 2001. Concurrently, J-111225 (Banyu Pharmaceutical Co Ltd), CS-023 (Sankyo Co Ltd) and CP-6509 (Meiji Seika Kaisha Ltd) are being investigated as ultra-broad spectrum 1-methyl-carbapenems possessing activity against drug-resistant staphylococci. Figure 1.03: Anti-MRSA penems in clinical development. NOHCO2HRCH3 NH*SNHCH3 N*SCH3NOHNH SNNH2CSCH3ONH2 R =J-111225 CS-023*+ CP-5609 O NH H2N O J-111225 (Figure 1.03), a pyrrolidinylthio-1-methylcarbapenem, is a good inhibitor of PBP2a (IC 50 = 2.5 g/ml) which correlates well to its in vitro activity against MRSA (MIC 90 = 4 g/ml). 22 The penem possesses a trans-hydroxyethyl side chain that imparts -lactamase stability and a bulky lipophilic moiety which is understood to enhance PBP2a binding. The effectiveness of J-111225 is comparable to vancomycin in mice with systemic MRSA infections (ED 50 = 5.83 mg/kg) and maintains good stability toward human renal dehydropeptidase I. 23 CS-023 (Figure 1.03) is an analogous broad spectrum pyrrolidinylthio-1-methylcarbapenem being investigated as a parenteral MRSA antibiotic. The drug has a MIC of 8 g/ml against MRSA and an IC 50 of 5.3 g/ml to PBP2a. 24 Another extensively researched penem is CP-5609 (Figure 1.03), a 6,7 disubstituted imidazo[5,1b]thiazole-3-yl-1-methylcarbapenem with excellent affinity for PBP2a and a MIC of 4 g/ml against MRSA. 25 1.2.3 N-Thiolated -Lactams N-Thiolated monocyclic -lactams (Figure 1.04) represent a novel class of synthetic antibacterials whose mechanism of action is not inhibition of cell wall biosynthesis. 26 Additional information on the biological properties of these and related molecules is presented in chapters II-IV. 4

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Figure 1.04: N-Methylthio-lactams with anti-staphylococcal properties. N CH3O Ph O SCH3 H H 3 1.3 Peptide Antibiotics 1.3.1 Second Generation Glycopeptides Glycopeptides 27 (Figure 1.05) embody the most successful family of MRSA antibiotics and remain the drugs of choice for the treatment of hospital-borne infections. Unlike -lactam antibiotics, glycopeptides inhibit cell wall synthesis by forming a complex with the C-terminal D-ala-D-ala of the peptidoglycan crosslinkages. Although the mechanism of action was once considered unbeatable, glycopeptide resistance emerged during the 1980s via enzyme-dependent conversion of the terminal D-alanines to D-lactates. 28 The gene encoding resistance (vanA, vancomycin-resistance) initially evolved in enterococci, however, in June 2002, a S. aureus strain harboring both the mecA (methicillin-resistance) and vanA genes was isolated from a catheter exit site of a diabetic patient in the US. 29 The recent escalation in glycopeptide-resistant bacteria counts has prompted researchers to investigate new semisynthetic analogues of vancomycin while increasing efforts to discover new natural products with enhanced bioactivity or novel modes of action. 27,30 Figure 1.05: Vancomycin and teicoplanin. NONOONOOOHOHNOClONNHOCO2OHOOOHOHOHOi-PrNH2OOClOHNH3OHMeMeNH2Me HHHHH OHOOHOOHOHOHNONOOHNONH3OHOOOHNH O HNOClONNHOCO2OOOH O H OH Cl O OH OH NHAc OH O iPr HHHH vancomycin teicoplanin 5 Oritavancin (InterMune Pharm. Inc; Figure 1.06) is a semisynthetic glycopeptide prepared from a natural product analogue of vancomycin. 31 The peptide displays good in vitro activity against MRSA (Table 1.02) while maintaining the efficacy of vancomycin in rabbits with MRSA-induced endocarditis. 31a After displaying a good safety profile in humans during phase I trials, oritavancin is currently in phase III trials with completion and NDA filing anticipated for January 2004. Another glycopeptide in clinical trials is dalbavancin (Biosearch Italia SpA/Versicor Inc; Figure 1.06, Table 1.02), a semisynthetic glycopeptide being developed as the first once-weekly intravenous treatment for serious infections caused by Gram-positive bacteria. 32,33 Phase I trials established dalbavancin to be well tolerated in healthy individuals and phase II studies during which hospitalized patients with skin and soft tissue infections were administered

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the glycopeptide were reported as positive. 34 Biosearch is also developing ramoplanin (Biosearch Italia SpA/Genome Therapeutics Corp), a lipoglycopeptide antibiotic for use as a topical treatment for nasal carriage of MRSA. 35 However, due to the innate toxicity of ramoplanin, parenteral administration is not possible. Figure 1.06: Oritavancin and dalbavancin. NHOHNOONHOOOHHOHNOClONHNHOHO2C O HOOOHH O OHONH2OOClOHHN C l HOMeMeNH2MeHOMe O NHMe HOOCO2HOHNOOOHOHHONOHNClOONHONHMeHOOOOHHN O HNOClONNHOOHOOH O H HHH oritavancin dalbavancin 1.3.2 Lipopeptides (Daptomycin) Daptomycin (Cubist Pharmaceuticals Inc/Gilead Sciences Inc; Figure 1.07) is a naturally occuring lipopeptide consisting of 13 amino acids and a decanoic acyl side chain. 36 The peptide displays rapid bactericidal activity against MRSA and the VRSA strain isolated in the US. 36a,36b The mechanism of action for daptomycin is not entirely understood although it is known that cellular integrity is disrupted by insertion of the peptide into the plasma membrane. 36c Daptomycin's novel mode(s) of action in bacteria should prevent the occurrence of cross-resistance and studies have shown the incidence of spontaneous-resistance is below detectable levels in S. aureus. 36d 1.3.3 Depsipeptides Wakamoto Pharmaceutical Co Ltd is developing WAP-8294A2 (Figure 4, Table 1.02), a water-soluble depsipeptide antibiotic produced by Lysobactor spp. WAP-8294A2 displays good in vitro activity against MRSA (0.78 g/ml) and VRSA (6.25 g/ml) while maintaining an ED 50 of 0.38 mg/kg in mice with systemic MRSA infections. 37 Shionogi & Co Ltd is also investigating two new depsipetide antibiotics in katanosin and plusbacin A 3 38 The macrocycles inhibit the in vitro growth of MRSA at 0.39 and 1.56 g/ml, respectively, and unlike vancomycin, katanosin and plusbacin A 3 reportedly block cell wall synthesis by inhibiting the formation of lipid intermediates involved in transglycosylation. In vitro studies have demonstrated that the incorporation of [ 14 C]glycine is inhibited at IC 50 values of 2.2 and 2.3 g/ml while synthesis of nascent peptidoglycan is suppressed at IC 50 s of 0.8 and 0.4 g/ml for katanosin and plusbacin A3, respectively. 38 6

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Figure 1.07: Daptomycin and WAP-8294A2. ONHHNO N H2OCO2HNHONHNHNH2(CH2)2i-PrMeNi-PrONHONHOHO(CH2)3i-PrOHNH2 O OHBnNMeONHNHOONH2(CH2)2OOONHOO daptomycin O CH3(CH2)8NHNHNHONNHOOCO2HNONH2MeHONHOOONNHOONHONHNONOHNOOONH2(CH2)3NH2CO2HMeCO2HHOCO2HMeH HHHHH WAP-8294A Table 1.02: Biological activity comparisons of oritavancin, dalbavancin and daptomycin. 31-38 MIC 50 a (g/ml) MIC 90 a (g/ml) MIC w/serum b (g/ml) Protein binding c (%) MBC d (g/ml) Terminal half-life e (h) Oritavancin 0.5 2 1-4 86-90 2 132-356 Dalbavancin 0.13 0.25 2-4 NR 2 166-212 Daptomycin 1 1 NR 90-94 1 8.5 Teicoplanin 0.5 2 2 90 2 > 35 Vancomycin 1 2 1-2 30 2 6 7 a minimum inhibitory concentration for 50 or 90% of MRSA test strains; b 50% bovine serum; c % human plasma protein binding; d minimum bactericidal concentration; e terminal half-life in humans; NR: not reported or data could not be found in the literature. 1.4 Oxazolidinones Oxazolidinones are bacteriostatic antibiotics of Gram-positive bacteria that inhibit protein synthesis by binding to the 30S and 50S ribosomal subunits. 39 Linezolid (Zyvox, Figure 1.08) is the only oxazolidinone in clinical use, although other promising candidates have emerged and advanced to human trials. Currently, Versicor is investigating a thioamide series of oxazolidinones that display good in vitro activity against MRSA (MIC 90 = 1 to 2 g/ml). 40 VRC-3783 (Pharmacia Corp/Versicor Inc; Figure 1.08) and VRC-4104 (Pharmacia Corp/Versicor Inc) are two promising analogues with ED 50 values of 6.2 and 3.8 mg/kg, respectively, in a mouse septicemia model. 40b AstraZeneca plc was developing AZD-2563 (Figure 1.08), an oxazolidinone with a long half-life and good activity against MRSA (MIC = 1 g/ml), but development was discontinued by July 2002 for unknown reasons. 41

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Figure 1.08: Linezolid, VRC-3783, and AZD-2563. MeHNONOOF N O linezolid ( Z y vox ) NOONOONOOHHOFF MeHNSNONHMeOOF VRE-3783AZD-2563 1.5 Quinolones, glycylcyclines and coumarin antibiotics Quinolones are bactericidal agents that act on types II (DNA gyrase, negative supercoiling) and IV topoisomerase (chromosome partitioning) in S. aureus. 42 Several new quinolones, such as DW-116 (Dong Wha Pharmaceutical Industry Co Ltd; Figure 1.09), 43 olamufloxacin (Hokuriku Seiyaku KK; Figure 1.09), 44 and DK-507k (Daiichi Seiyaku Co Ltd, Figure 6) 45 have advanced to clinical trials after demonstrating strong activity against drug-resistant Gram-positive pathogens, such as MRSA (MIC 90 = 0.06, 1.56 and 0.006 g/ml, respectively). These compounds are derived from the popular fluoroquinolone family possessing a C(6) fluorine atom that is known to substantially enhance bioactivity. 42 Des-fluoro(6) quinolones are also being investigated with BMS-284756 (ganefloxacin; Bristol-Myers Squibb Co/Toyama Chemical Co Ltd; Figure 1.09) at the most advanced stage in clinical development for this class 46 Ganefloxacin is completing late phase III trials in the US, Japan and Europe with an anticipated use as a once-daily intravenous treatment for skin and soft tissue pathogens, including MRSA and fluoroquinolone-resistant S. aureus (MIC = 4 g/ml). 46a Non-fluorinated quinolones are also being developed as topical agents for SSTIs. Recently reported is T-3912 (Toyoma Chemical Co Ltd/Ferrer Internacional SA/Maruho KK), a 3-pyridinyl quinolone with potent in vitro activity against drug-resistant staphylococci (MIC 90 = 0.2 g/ml). 47 Figure 1.09: Anti-MRSA quinolones in clinical development. NNCO2 H FOH2NNH2Me NNCO2HFOH2NOMeF olamufloxacin DK-507k HNNOOMeFFCO2H NNNCO2HMeFOF DW-116BMS-284756 8

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Coumarin antibiotics are another class of topoisomerase inhibitors that selectively target DNA gyrase B in S. aureus. 48 RU-79115 (Aventis Pharma AG; Figure 1.10), a coumarin related to novobiocin, displays good activity against multiple drug-resistant forms of S. aureus while maintaining a PD 50 of 1mg/kg in mice with induced systemic infections. 49 Glycylcyclines, although unrelated to the coumarins, are also active against multi-drug resistant strains of S. aureus. Tigecycline (Wyeth Research; Figure 1.06) was the most developed from this class of protein synthesis inhibitors, but a policy change by the US FDA had forced Wyeth to delay the phase III clinical trials. 50 Figure 1.10: Anti-MRSA coumarin and glycylcycline in clinical trials. OOOMeOOOHMeOHMeOMeHNOO NH2ONHOHNNMeMeOHOOHOHONOHMeMe RU-79115tigecycline (GAR-936) 1.6 Conclusions A tremendous worldwide effort to discover new anti-MRSA antibiotics is apparent. -Lactams continue to be the most heavily researched class because of their enduring ability to fight infection while maintaining low toxicity in humans. Semisynthetic vancomycin derivatives have also gained a great deal of attention due to the long-term success of glycopeptides against MRSA. However, future therapies cannot be devised by analogue synthesis of pre-existing antibiotics alone. Compounds with novel modes of action must continue to be researched and developed as S. aureus continues to evolve new mechanisms and new levels of resistance to current antibiotics. In chapter III-V, the synthesis and biological properties of the novel class anti-MRSA antibiotics called "N-methylthio -lactams" is discussed. The structure-activity relationships and investigations into their unknown mode of action will be described in depth for the first time. 9

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CHAPTER II CHEMISTRY AND BIOLOGICAL PROPERTIES OF N-THIOLATED -LACTAMS 2.1 Introduction For over 60 years, the -lactam antibiotics have served as a powerful line of defense against bacterial infections. 51 Following the initial introduction of penicillin during World War II, a variety of other classes of -lactam antibiotics were identified including the cephalosporins, penems, carbapenems, and monobactams (Figure 2.01). 52 One of the landmark discoveries in the -lactam field occurred in 1981, when researchers at the Takeda and Squibb laboratories isolated the first classes of N-thiolated -lactam antibacterial agents from natural sources. 53 The Squibb scientists coined the term "monobactams" in referring to these new monocyclic -lactams. These reports confirmed that -lactam compounds do not necessarily require a conformationally rigid bicyclic ring system to have antibacterial capabilities, or the carboxylic acid moiety found in all other microbially-active -lactams. These -lactam compounds were the first to have a sulfur group attached directly to the lactam nitrogen, in the form of an N-sulfonic acid. In this chapter, a summary of the nearly 5000 -lactam compounds that contain an N-sulfur substituent is provided. The sulfur center in these molecules can bear a diversity of residues and exist in a variety of oxidation states, as classified by the following five structure types (Figure 2.02). The discussion that follows is therefore divided into the following five sections: NOSCO2H penicillinsNOS C O2H cephalosporinsNOSCO2HR penamsNOSCO2HX carbapenamsNO monobactamsSO3H RCONH RCONH X OH OH RCONH R' Figure 2.01: Classes of -lactam antibiotics. 51 Figure 2.02: Chemical classes of N-thiolated -lactams. N O S X O N O S R N O S R O O N O S X N O S R O N-SO2X-LactamsN-SO2R -LactamsN-SOR-LactamsN-SR -LactamsN-SX -LactamsO 2.2 N-SO 2 X -Lactams Compounds within this category include the N-sulfonic acids (X=OH), N-chlorosulfonyl lactams (X=Cl), and N-aryloxysulfonyl or N-alkyloxysulfonyl derivatives (X=OAr or OR). Each of these will be discussed separatedly in this order. 10

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2.2.1 N-Sulfonic Acid -Lactams With over 3600 entries cited in the journal and patent literature, the N-sulfonic acid -lactams represent the largest and most thoroughly studied class of N-thiolated -lactams. The term "monobactam" is now commonly used to refer to structures containing the 2-oxoazetidine-1-sulfonic acid framework. 54 Over the past two decades, a variety of review articles have been written on the discovery and development of the monobactam antibiotics. 55 The first members of this family were described in 1981 in separate disclosures from the Takeda and Squibb laboratories. 55b In February 1981, Takeda reported the isolation of two novel -lactam natural products, sulfazecin and isosulfazecin (Figure 2.03), from Gram-negative Pseudomonas bacteria in soil. Sulfazecin was found to be active against Gram-negative bacteria but weakly active against Gram-positive microbes. Isosulfazecin, on the other hand, exhibited only weak activity against both Gram-negatives and Gram-positives. 53a N NH OCH3 O HN SO3 O CH3 O NH3 O2C N NH OCH3 O HN SO3 O CH3 O NH3 O2C sulfazecin(SQ 26,455)isosulfazecinN SO3 K O NH OCH3 H3C O SQ 26,180 A few months after the Takeda report appeared, Squibb 53b laboratories published their findings on seven closely related -lactams isolated from a diverse range of microbes, including Acetobacterium, Agrobacterium, Chromobacterium, Flexibacterium, and Gluconobacterium species. The compound most frequently found in these screenings, SQ 26,445, turned out to have a structure identical to that of Takeda's sulfazecin. A second lactam, SQ 26,180 seen in Figure 2.03, was also identified. These naturally occurring lactams proved to be highly stable to a wide range of lactamases, but had relatively weak antibacterial activity. These features were attributed to the low affinity the compounds have for the penicillin binding proteins (PBPs) in bacteria. 55b Their selectivity for aerobic Gram-negative microbes warranted further study. Figure 2.03: First monobactams discovered by American and Asian scientists. 55 To be able to carry out detailed structure-activity studies, methods were soon developed to create monobactam analogues by chemically introducing or modifying the side chains on the lactam ring. The most common approach involves the N-thiolation of an N-unsubstituted lactam 1 with pyridine-sulfur trioxide complex (Scheme 2.01). This procedure, developed by the Takeda laboratories, works particularly well for C 4 unsubstituted systems (2, R'=R"=H), giving good yields of the N-sulfonated lactam as its pyridinium salt. 56 The salt can be readily exchanged for other cations (K+) or protonated to give the N-sulfonic acid. Scheme 2.01: N-Sulfonation of -lactams with pyridine-sulfur complex. 56 N O H NH R" R O R' N SO3N O SO3 NH R" R O R' 12CH2Cl2-DMF The N-sulfonic acid moiety can also be transferred onto the ring using the powerful sulfonating agent dimethylformamide-sulfur trioxide (DMF-SO 3 ) followed by aqueous KH 2 PO 4 workup. 57 Treatment of this product with tetrabutylammonium bisulfate and extraction with an organic solvent enables the 11

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monobactam to be obtained as its tetrabutylammonium salt. 58 Alternatively, N-sulfonation can be achieved through a stepwise N-silylation process using trimethylsilyl chloride and triethylamine, followed by treatment of the resulting N-silyl lactam with trimethylsilyl chlorosulfonate ((CH 3 ) 3 SiOSO 2 Cl) at low temperature. 59 The silyl ester group can be hydrolyzed with KH 2 PO 4 buffer to afford the monobactam potassium salt. A different strategy for making monobactams is via base-promoted cyclization of a -methanesulfonyloxyacyl sulfamate 3 in aqueous bicarbonate media, in which the stereochemistry at C 4 arises through an inversion process (Scheme 2.02). 60 Scheme 2.02: Bases-promoted cyclization for synthesizing monobactams. 60 NH O SO3 NH R O N O SO3 NH R" R O R' 32 OMs R' R" aqueousNaHCO3 Monobactam analogues differing only in the acyl side chain can best be obtained by acylation of a 3-amino monobactamic acid 4, as illustrated in Scheme 2.03. The acyl moiety can be introduced using an acyl chloride or an activated ester. 61 Scheme 2.03: Acylation of 3-amino monobactamic acids. 61 N O SO3 H2N R" R' N O SO3 NH R" O R' 45Et3N-DMFPhO O Cl PhO The preparation of the 3-amino monobactamic acid 4 proceeds via monobactam intermediates 6 and 7 (Figure 2.04), neither of which were examined for biological activity. Figure 2.04: Intermediates used to synthesize 3-amino monobactamic acids. N O SO3 N O SO3 N HN BOC BOC CH3 CH3 H3NHN 67 Among the first series of synthetic monobactams studied were those whose side chains resembled the ones on existing -lactam antibiotics (Figure 2.05). Thus, the phenylacetamidyl side chain of penicillin G gave rise to monobactams possessing a C 3 phenylacetamide group (as in SQ 26,324 and SQ 81,427), which were found to have increased activity against Gram-positive bacteria and enhanced affinity for PBP-1, -2, and -3. However, these compounds were still relatively weak compared to the penicillin counterpart. 62 Incorporation of the uriedo moiety of the penicillin, piperacillin led to monobactams having antibacterial activity against a broader spectrum of microbes, including -lactamase-producing strains of Staphylococcus aureus. 55b The most active of the synthetic derivatives came from the attachment of an aminothiazole alkoximino side chain found in cefotaxime, a third generation cephalosporin. One of these derivatives, aztreonam (SQ 26,776), showed high affinity for PBP-3 of Gram-negative bacteria. 55b Structure-activity studies revealed that the aminothiazole oxime is responsible for aztreonams potent 12

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activity against Gram-negative species, while the carboxylic acid on the oxime side chain enhances Pseudomonas activity. Figure 2.05: Synthetic monobactams with enhanced bioactivities. 55b N O SO3 K NH O SQ 26,324N O SO3 K NH O Ph Ph NH N O N O O Et SQ 81,427 N O SO3 Na NH O N SN H3N O CO2H H3C H3C CH3 N O SO3 Na NH O N SN H3N O CO2H O NH2 O aztreonam(SQ 26,776)carumonam(AMA-1080) Like penicillin and the other -lactam antibiotics, the monobactams inhibit bacterial growth by blocking cell wall biosynthesis. The lactams act directly on the penicillin binding proteins (PBP's), a group of membrane-bound serine transpeptidases that catalyze cell wall crosslinking. 61b Upon binding in the active site, the compounds undergo -lactam ring opening by acylating the catalytic serine hydroxyl group as depicted in Scheme 2.04. The PBP, giving a stable enzyme adduct that is catalytically inactive. By disrupting these crosslinking proteins, the -lactams induce morphological deformities in the cell that leads to rupture of the bacterium. The N-sulfonic acid moiety plays a key role by activating the carbonyl of the monobactam ring to nucleophilic attack and ring opening in the enzyme active site. Scheme 2.04: Mechanism of-lactam hydrolysis by serine transpeptidases. O NS O O O [Nu] HO-Ser-Enzyme HNS O O O O O Enzyme-SerThe ability of the monobactams to bind selectively to the bacterial PBP's depends largely on the nature of the C 3 and C 4 side chains on the -lactam ring. The naturally occurring monobactams possess an acylamido side chain and often a methoxy group at C 3 of the ring. The methoxy moiety protects the ring from enzymatic hydrolysis by -lactamases, but also decreases antibacterial activity. Other types of small polar groups at C 3 also reduce antibacterial activity. Monobactams of bacterial origin do not possess C 4 side chains, but synthetic analogues that have alkyl or heteroatom-containing residues at C 4 show significantly enhanced biological activity. 62 Aztreonam (SQ 26,776) and carumonam (AMA 1080; Ro 17-2301), two of the most active agents developed so far, have a methyl and carbamate group, respectively, at C 4 63 Along with enhanced stability to -lactamases, these functionalized monobactams are highly potent against aerobic, Gram-negative bacilli such Pseudomonas aeruginosa, Haemophilus influenzae and members of the Enterobacteriaceae family. Aztreonam is bactericidal in its action 64 and binds selectively to PBP-3 of Gram-negative bacteria, resulting in the elongation of the cell and death through cell lysis. 13

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However, the low affinity aztreonam has for the PBP's of Gram-positive and anaerobic microorganisms allows it to have a narrow spectrum of antimicrobial activity. Most commonly, the drug is used in the treatment of bacteremias, urinary tract infections, pelvic and intra-abdominal infections, and respiratory infections. 65 Since its activity is selective for aerobic Gram-negative bacilli, aztreonam can be combined with other antibacterial agents and be used as an alternative to aminoglycosides or penicillins in penicillin-allergic patients. A variety of bicyclic (bridged) monobactams 8-17 (Figures 2.06 and 2.07) have also been shown to serve as mechanism-based inhibitors of class C -lactamases, some having half-inhibition constants down to 10 M -3 66 The acyl-enzyme adduct formed by these compounds has a half-life toward hydrolysis up to 48 hours. This stability allowed the determination of an X-ray structure for bridged monobactam 8 covalently bound via lactam ring opening to the active site serine of Citrobacter freundii 1203 -lactamase. While none of the inhibitors possess antibacterial activity, synergistic effects were observed for most of the analogues when used in combination with ceftriaxone (a third generation cephalosporin) against -lactamase-producing strains of Enterobacteriaceae. Thus, using a 1:4 ratio of drug:monobactam, the MIC's could be reduced from 128 g/mL (without inhibitor) to as low as 0.25 g/mL for Citrobacter freundii. The compounds are much less effective against either class A or class B -lactamases, and do not have significant synergies against a TEM-3 (class A -lactamase)-producing strain of Escherichia coli or an 18 SH (a class C -lactamase)-producing strain of Pseudomonas aeruginosa. This is probably due to the limited ability of the lactams to permeate the outer membrane of these particular microbes. 67 N O SO3 H2N H H N O SO3 Na N H H Br O N O SO3 Na N H H O O N O SO3 Na N H H HN O R 891011R Figure 2.06: Bicyclic monobactam inhibitors of class C -lactamase. 29 Interestingly, the disubstituted derivatives 12, 13, and 14, and BOC-protected analogue 15 offered no synergistic effect in combination when used in combination with a penicillin. However, lactam 17 (Ro 48-1256) provides a good compromise between -lactamase affinity and membrane permeability. 68 Hoffman-LaRoche AG patented a variety of related bridged monobactam derivatives 18-21 for use in liposome solutions as -lactamase inhibitors. 69 There has also been some interest in the development of monobactams as cysteine proteinase inhibitors. 70 Figure 2.07: Bicyclic monobactams 12-21. 68-70 N O SO3 Na N H H O O N O SO3 Na N H H O O Bn 1213Bn F O NH2 N O SO3 Na N H H O O N O SO3 Na H H 14Bn S NNNN H3C N O O Bu t15 N O SO3H N H H HN O N O SO3H N H H HN O 1617 HN HO ( R o48 1256 ) 14

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N O SO3H NS H H ROSO3 O 18N O SO3H NS H H R O 19N O SO3H NS H H HN O R N O SO3H NS H H NH R 2021 2.2.2 N-Chlorosulfonyl -Lactams N-Chlorosulfonyl -lactams have been exploited primarily as reactive intermediates in synthesis, and have not been studied as bioactive molecules themselves. 71 This is undoubtedly due to their high chemical reactivity and instability in aqueous media. Scheme 2.05 depicts the standard method for preparing N-chlorosulfonyl lactams by a [2+2]-cycloaddition of an olefin with chlorosulfonyl isocyanate 22, 72 as shown for the conversion of vinyl acetate to 4-acetoxy lactam 23. 73 Scheme 2.05: Synthesis of 4-acetoxy N-chlorosulfonyl-azetidin-2-one 23. 72 O CH3 O O=C=NSO2Cl22N O S O CH3 O Cl O O 23 The method has been applied to the synthesis of some bicyclic and multicyclic systems such as 24-27 (Figure 2.08). 74 Figure 2.08: Bicyclic and multicyclic N-chlorosulfonyl -lactams 24-27. 75 N O S Cl O O N O S Cl O O N O S Cl O O N O S Cl O O 24252627 The unusual dimeric compound 28 (Figure 2.09), made by photodimerization, has been employed in the construction of space-separated bis-heterocycles. 75 Figure 2.09: Dimeric N-chlorosulfonyl -lactam 28. 75 N O N O S Cl S Cl O O O O 28 The N-S bond in N-chlorosulfonyl derivatives is readily cleaved by thiol nucleophiles. Thiophenol has been shown to reduce N-chlorosulfonyl -lactams to give N-protio -lactams in the presence of pyridine. This was demonstrated for the deprotection of 29 as seen vide infra. 76 15

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Figure 2.10: Deprotection of N-chlorosulfonyl -lactams with thiophenol. 76 N O S Cl O O Ph Ph SH pyridineN O H Ph Ph 2930 Thus, while providing access to N-unsubstituted -lactams, the inherently high reactivity of N-chlorosulfonyl -lactams would appear to limit their usefulness as potential therapeutic agents per se. 2.2.3 N-Aryloxysulfonyl and N-Alkoxysulfonyl -Lactams Around 30 N-aryloxyand N-alkoxysulfonyl -lactam derivatives have been described in the literature.These -lactams are made by cycloaddition of an olefin with an aryloxyor alkoxysulfonyl isocyanate 31, as illustrated in Scheme 2.06. The method is effective for the synthesis of C 3 unsubstituted adducts such as 33-37 (see Scheme 2.06). Scheme 2.06: Synthesis of N-aryloxysulfonyl and N-alkoxysulfonyl -lactams. 43 OC N S OR O O R' N S OR O O R O 3132 N S O OCH2CCl3 O O Ph N S O OCH2CCl3 O O Ph N S O OR O O Ph 333435 N S O OCH2CCl3 O O 3637O N S O O O O O X Y Starting from the cyclic enol ether, trichloroethoxy derivative 36 and aryloxy (X=H, Y=CH 3 ; X=H, Y=CH 3 O; X=NO 2 Y=H) analogues 37 have also been prepared in this manner. 77 Based on published reports, there have not been any studies or reports on whether these compounds possess biological properties. 2.3 N-SO 2 R -Lactams Numerous articles can be found throughout the literature describing the preparation and use of N-sulfonyl -lactams as intermediates in synthesis. There are several common procedures for synthesizing N-sulfonyl -lactams. The most direct way is by treatment of an N 1 unsubstituted lactam with a sulfonyl chloride in the presence of a base. Two examples are illustrated in Scheme 2.07. 78 16

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Scheme 2.07: Sulfonylation of N-protio -lactams. 78 N H3C CO2CH3 H O ClS Ar O O DBUN H3C CO2CH3 O S Ar O O 3839 N CH3O H O N CH3O O S CH3 O O Ph Ph 1) BuLi, -78oC2) ClSO2CH34041 N-Tosyl -lactams such as 43 have also been formed by dehydrative ring closure of a sulfonamido carboxylic acid 42 in the presence of dicyclohexylcarbodiimide (DCC) and 4-pyrrolidinopyridine (Scheme 2.08). This particular reaction was used in a published carbapenem synthesis. 79 Scheme 2.08: Dehydrative ring closure of sulfonamido carboxylic acid 42. 79 HN S CH3 O OH CH3CH2 OSiPh2tBu O O DCCbaseN S CH3 CH3CH2 OSiPh2tBu O O O 4243 Barrett demonstrated that bicyclic N-sulfonyl derivatives 45-47 could be prepared by olefin-isocyanate cycloaddition as depicted in Scheme 2.09. None of these products were examined for biological activity, however. 77 Scheme 2.09:-Lactam synthesis via olefin-isocyanate cycloaddition. 43 O O=C=NSO2RN O S R O O O 4544 N O S Ar O O N O S A r O O 4746 Biloski employed a different approach for closing the -lactam ring of compound 49 as seen in Scheme 2.10. Here, alkenyl amide 48 underwent iodocyclization in aqueous base to afford the -lactam product. 80 17

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Scheme 2.10: -Lactam synthesis via base-mediated iodocyclization. 80 NH S O O O CH3 I2NaHCO3N S O O O CH3 I 4849 In an isolated report, N-methanesulfonyl lactam 50 was prepared, albeit in poor yield, by a transition metal-mediated CO insertion of N-mesylaziridine as illustrated below (Scheme 2.11). The method was not extended to other sulfonyl substrates or to more complex systems, and would appear to be rather limited in scope. 81 Scheme 2.11: -Lactam synthesis via Ni-mediated CO insertion. 81 N S CH3 O O 1) LiI2) Ni(CO)43) I2CH3 N O S CH3 O O CH3 50 Of the nearly 600 N-sulfonyl -lactams that have been reported, relatively few have been examined for biological properties. This may be related to the fact that the compounds lack the ionic residues needed for binding to bacterial PBP's, and consequently, should be devoid of antibacterial activity. However, N-arylsulfonyl -lactams have been shown to be inhibitors of other serine proteases, including human leukocyte elastase (HLE). 82 The postulated mechanism for elastase inhibition by N-arylsulfonyl -lactams is depicted in Scheme 2.12. 78a The sulfonyl moiety is responsible for activating the -lactam ring towards nucleophilic ring opening by a serine residue, and then after lactam ring opening, for serving as a stable leaving group to create an imine intermediate by loss of arylsulfinic acid. It is proposed that tautomerization of the resulting imine to the enamine enables a second active site nucleophile (perhaps a histidine) to add, giving a crosslinked adduct that is highly resistant to hydrolysis. Attempts to prove the mechanism by trapping of the arylsulfinic acid were inconclusive. Scheme 2.12: Proposed mechanism of N-arylsulfonyl -lactams inhibitors of human leukocyte elastase. 78a N O S Ar O O HLENHSO2Ar Ser-Enzyme O -[HSO2Ar]NH Ser-Enzyme O NH2 Ser-Enzyme O Nu Ser-Enzyme O NH2 Nu However, a high resolution crystal structure of a stable acyl-enzyme adduct formed between N-arylsulfonyl lactam 51 (Figure 2.11) and elastase from porcine pancreas has been reported. 83 The interesting feature found in the structure is that the ester functionality formed between the serine and lactam 18

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moiety does not occupy the oxyanion hole in the active site. This could explain the high resistance this adduct has toward hydrolysis and release. Figure 2.11: N-Sulfonyl -lactams inhibitors of porcine pancreatic elastase. 78,81,83 N O S O O CO2H CH3CH2 CH3 51N O S COPh O O CO2CH2CH3 CH3CH2 52N O S CF3 O O CO2CH3 Ph 53 In addition to the N-arylsulfonyl compounds, N-acylsulfonyl lactam 52 (Figure 2.11) is also an inhibitor of porcine pancreatic elastase. 81 The trifluoromethylsulfonyl lactam 53 (Figure 2.11), however, has no affect on human leukocyte elastase activity. 78a Along with the elastase (serine protease) activity, Merck has shown interest in using N-alkylsulfonyl and N-arylsulfonyl -lactams as anticholesteremic agents through their inhibition of HMG-CoA synthase. 84 Some interesting bicyclic N-sulfonyl -lactams have been reported as well. The N-S-fused bicyclic lactam 55 was prepared by oxidation of sulfenamide 54 with m-chloroperbenzoic acid (Scheme 2.13). 78b-d The compound was devoid of antibacterial activity despite its highly electrophilic -lactam ring. However, the phthalimide analogue 56 (see Scheme 2.13), interestingly enough, was found to possess weak antibacterial activity against Staphylococcus aureus and Vibrio cholerae; the mechanism for this activity was not investigated. Scheme 2.13: N-S-Fused bicyclic -lactams. 78b-d N S O Ph I CH3O H H mCPBACH2Cl2N S O P h I CH3O H H O O 5455 N S O Ph I N H H O O 56O O N S O R H H 57 R=OMe58R = NPhthO O I Ph O O CH3 The highly functionalized bicyclic lactams 57 and 58 (see Scheme 2.13) could likewise be prepared by a similar sulfoxidation of the sulfenamides. These compounds were tested for antimicrobial properties, but did not reveal any activity against a variety of Gram-positive and Gram-negative bacteria. 2.4 N-SOR -Lactams N-Trifluoromethylsulfinyl lactam 59 (Figure 2.12) appears to be the only example of an N-sulfinyl -lactam, being found in a Merck patent on the use of substituted azetidinones as anti-inflammatory and anti-degenerative agents. 85 However, there is no information about whether the compound possesses these biological properties. 19

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Figure 2.12: N-Trifluoromethylsulfinyl -lactam. 85 N O S Ph CF3 O OCH2CH3 O 59 2.5 N-SX -Lactams A variety of structurally interesting molecules have been described in which an S-heteromoiety is attached to a -lactam nitrogen. Typically, these compounds have been isolated as synthetic intermediates or as unexpected reaction products. Miller developed an efficient method for preparing thiophthalimide analogues such as 62 and 63 (see Scheme 2.14). The procedure entails the reaction of the N-protio lactam with sulfur bis-phthalimide 61 in the presence of a catalytic quantity of triethylamine. 86 Scheme 2.14: Preparation of N-phthalimidothio -lactams. 86 N O O H H3C O N O O S 612Et3NN O O S H3C O N O O 6062N O O S H3C O N O O NH O PhO 63 The sulfur linked bis--lactam adduct 64 (Figure 2.13) can also be prepared using this method under certain reaction conditions. Phthalimide lactam 62 is presumably an intermediate of the reaction. 86 Phthalimide lactam 62 can likewise be reacted with other nitrogen nucleophiles such as morpholine to give derivative 65, or with benzyl alcohol in the presence of triethylamine to afford product 66 (Figure 2.13). No biological data was reported for any of these heterosubstituted -lactam derivatives. Figure 2.13: Heterosubstituted N-thiolated -lactams 64-66. 56 N O O O CH3 S N O N O O O CH3 N O O O CH3 S OBn N O O O H3C S 646566 20

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Miller also demonstrated that the phthalimide exchange process could be carried out using bisulfite anion as a means to prepare N-thiosulfuric acid derivatives such as 67 and 68 (see Scheme 2.15). Although only these two examples were shown, the reaction would appear to be general. Thiosulfate lactam 68 was tested for antibacterial properties but found to be inactive, which contrasts sharply with the potent antimicrobial effects of the corresponding monobactam compound (the N-SO 3 H analogue). 87 This confirmed the correlation between the electronegativity of the N-heteroatom of a monocyclic -lactam and biological activity. Nevertheless, this dramatic difference in antibacterial activity in this case may also be due to differences in the way the molecules fit into the enzyme active site. 88 Scheme 2.15: Preparation of heterosubstituted N-thiolated -lactams via phthalimide exchange. 87 N O O S H3C O N O O 62 NaHSO3THF-H2ON O O S H3C O SO3 Na 67N O O S H3C O SO3 NH O PhO 68 There are only two examples of disulfide-linked -lactams of type 69 (see Scheme 2.16), both of which were formed as unexpected reaction products. In the first example, dimeric lactam 71 was obtained by heating S-trityl lactam 70 at 75C in the presence of cupric chloride (Scheme 2.16). 89 The authors make no mention as to whether disulfide 71 was tested for antimicrobial activity. Scheme 2.16: Synthesis of disulfide-linked -lactams. 89 N S S N O O 71CH3 CH3 H3C H3C Ph Ph N S O H3C H3C Ph Ph Ph Ph CuCl2THF-EtOH70N S S N O O 69 Turos reported the formation of disulfide-linked lactam 72 by low temperature lithiation of vinyl iodide 54 as shown in Scheme 2.17. The reaction takes place via an initial metal-halogen exchange, followed by spontaneous ring-opening and dimerization. Compound 72 was screened for antibacterial properties, but found to be inactive against a wide variety of common Gram-positive and Gram-negative microbes. 78c 21

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Scheme 2.17: Synthesis of disulfide-linked lactam 72. 78c N S S N O O BuLi, -78oC Ph Ph CH3O OCH3 THFN S Ph I O CH3O 5472H H 2.6 N-SR -Lactams There have been a number of studies on -lactams having an N-sulfenyl moiety as shown in Scheme 2.18 for structure 74. N-Sulfenyl -lactams can be readily obtained by thiolation of an N 1 unsubstituted lactam 73 with an appropriate sulfenylating agent and base. Scheme 2.18: Preparation of N-sulfenyl -lactams. N O H X-SRbaseN O S R 7374 Shah and Cama reported a procedure for placing a methylthio substituent onto a -lactam nitrogen by treating the N-H precursor 75 with lithium diisopropylamide (LDA) in a mixture of hexamethylphosphoramide (HMPA) in tetrahydrofuran (THF) at low temperature, followed by trapping the amide anion with methyl methanethiolsulfonate (Scheme 2.19). 90 Scheme 2.19: N-Methylthiolation of -lactams with methanethiosulfonate. 90 N O H OCH3 O CH3 OSitBu(CH3)2 L DAHMPA, THFCH3SO2SCH3-78oCN O S C H3 OCH3 O CH3 OSitBu(CH3)2 7576 N O S C H3 OCH3 O CH3 OH N O S C H3 Ph O CH3 OH 7778F F N O S C H3 R O CH3 OSitBu(CH3)2 79 R=OH80 R=PhN O S C H3 R O CH3 O 81 R=OMe82 R=Ph O OCH2CH=CH2 22

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N-Methylthio lactams are stable to acid and base but can be easily deprotected with a thiolate anion. The authors showed a selection of N-methylthio derivatives 76-82 (see Scheme 2.19) that were synthesized, but did not report on their biological properties. Turos has applied this same method to make N-alkylthio lactams 83-90 (Figure 2.14) for use as intermediates in synthesis of bicyclic -lactams. 78b-d For the N-benzylthio and N-phenylthio lactams 89 and 90, respectively, the N-thiolation was done by trapping the amide anion with the sulfenyl chloride (generated from halogenation of the disulfide with chlorine gas). Similarly, researchers at Shionogi research laboratories prepared a series of N-methylthio lactams 91 (Figure 2.14) as intermediates in a carbapenem synthesis (X=F, CN, OH, O 2 CCH 3 OSiMe 2 t Bu, OSi(CH 3 ) 3 or I). None of these lactams appear to have been tested for antibacterial activity. 91 Figure 2.14: Examples on N-methylthio monocyclic-lactams. 78b-d N R Ph O S C H3 H H N CH3O Ph O SR H H ()-83 R=OMe()-84 R=SBnN O R OAc Ph S C H3 H H ()-85 R=OMe()-86 R=NPhth()-89 R=SBn()-90 R=PhN CH3O O S C H3 H H CH3 N CH3O O SCH3 H H CH3 Br CH3 Br ()-87()-88N CH3 TBDMSO R SCH3 O 91 Hart introduced a procedure for preparing S-tritylated lactams via addition of an ester enolate to S-trityl sulfenimines such as 70. This procedure favors formation of the cis -lactam product. While the method works well for S-trityl lactams 70, 94, and 95, attempts to apply the reaction to S-phenyl sulfenimines gave the N-unsubstituted -lactam rather than the desired N-thiolated product (Scheme 2.20). 89 Scheme 2.20: Preparation of S-trityl -lactams via enolate-sulfenimine addition. 89 OCH2CH3 H3C CH3 O 1) LDAN H Ph S Ph Ph Ph 2)9392N O S Ph Ph Ph H3C Ph H3C 70 N O S Ph Ph Ph Ph 94N O S Ph Ph Ph H2N CH3 95H3C CH3 23

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Miller reported a mild N-thiolation procedure that made use of N-thiophthalimides 96 and catalytic triethylamine. The methodology allows a large assortment of organothio groups to be introduced onto the lactam, as illustrated in the formation of ester and carboxylic acid derivatives 97-104 (see Scheme 2.21). 87 Kilburn and colleagues recently used this method to prepare N-phenylthio lactam 105 for studies on radical cyclizations. 92 The authors did not mention if the compound was examined for biological activity. Scheme 2.21: Sulfenylation of N-protio -lactams with N-thiophthalimide. 87,92 N O H N O O SR 96cat Et3N73N O S R 74 N O O S O O CH3 OCH3 N R' O S O OR NH R" O 98 R,R'=Me, R"=OBn99 R=tBu, R'=OAc, R"=CH2OPh100 R=tBu, R'=Me, R"=CH2OPh97N CH3 O S NH BnO O NH C O2 B n OCH3 O 101N CH3 O S O OH NH O N SN H2N OCH3 N R' O S O O K NH O 103 R=OPh, R'=OAcR 104 R=Ph, R'=Me102N O S H3C TBDMSO 105 As described earlier, phthalimide-containing compounds such as 62 can be used to access a range of other S-derivatives. Miller showed that carbon nucleophiles readily displace the phthalimide group from 62 to deliver N-sulfenylated -lactams 106-108 (see Scheme 2.22). 87 Scheme 2.22: Displacement of phthalimide by a carbon nucleophile. 87 N O O S H3C O N O O 62 N CH3 EtO2C O N O O S H3C O 106 CH3 N CO2Et O 24

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N O SCPh3 S CH3 N CO2Et O N O O S H3C O PPh3 CO2E t 107108 The biological studies that have been conducted on the N-sulfenyl -lactams described have led to some surprising, and potentially important, findings. The first studies were those carried out by Miller that compared the synthetic N-SCH 2 CO 2 H lactams (thiamazins) made in his laboratory to their N-OCH 2 CO 2 H counterparts (oxamazins). 87,88,93 The oxamazins are strong antimicrobial agents, but the thiamazins were found to be devoid of antibacterial activity. It was postulated that this remarkable difference in biological properties may be due to the longer N-S bond which prevents the thiamazin binding into the active site of the transpeptidase enzyme. Miller has noted that the carbonyl stretching frequencies of the active oxamazins are consistently 10-20 cm -1 higher than those of the inactive thiamazins, suggesting that there may possibly be an electronic basis for this difference as well. Miller also studied the stability of S-thioacetic acid lactams under basic conditions, and identified three decomposition pathways (Scheme 2.23): (1) hydroxide ion addition to the carbonyl to give the ring-opened carboxylic acid, (2) S N 2 attack at sulfur to cleave the N-S bond, and (3) deprotonation of the acidic carboxyl -proton to provide the NH lactam through loss of the thioaldehyde. Miller determined that approximately 50% of the thiamazin is cleaved at the N-S bond (pathways 2 and 3) at pH 11, and the other 50% by lactam ring opening (pathway 1). 88 The thiamazins are stable below pH 10. Scheme 2.23: Decomposition pathways of thiamazins by hydroxide ion. 88 N S CO2R H O H H ROCNH CH3 OH HN S CO2R H H H ROCNH CH3 NH HO-S CO2R H O H H RCONH CH3 NH S CO2R H O H H ROCNH CH3 O H O +++ H2O attack on carbonylattack on sulfurattack on carbon(1)(2)(3) Recently, Turos has examined N-methylthio lactams 84-91 for antibacterial activity against a variety of common Gram-positive and Gram-negative microorganisms. Curiously, lactam 84 was found to strongly inhibit Staphylococcus aureus. 26a-c The N-methylthio substituent of 84 is required for biological activity, since the N-H and N-SO 2 CH 3 derivatives are totally inactive. The S-benzyl compound 90 also demonstrates appreciable antimicrobial activity against the same microorganisms. The antibacterial activity of these N-alkylthio lactams is surprising, given the total absence of any ionizable ring functionality in the molecules (required for binding to transpeptidases) and the close similarity they have in structure to the inactive thiamazins (cf. Scheme 2.23). This suggests the compounds may be operating through a different mode of action. Further investigations into this novel family of antibacterials is warranted and is the focus of chapters III-V. 25

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Scheme 2.24: Mechanisms of reactivity of N-thiolated -lactams towards nucleophiles. N S CH3 O H MeO H R Nu attack on carbonylattack on sulfurattack on carbon(1)(2)(3)N S CH3 H MeO H R N O H MeO H R N S O H Me O H R Nu O +Nu-SCH3 +Nu-CH3 The N-methylthio -lactams, like the thiamazins, differ from traditional -lactams in that there are at least three sites vunerable to attack by a biological nucleophile. The types of nucleophilic reactions that could in effect provide information on the bacterial mode of action are: (1) addition to the -lactam carbonyl, (2) substitution on the sulfur center, or (3) S N 2 displacement at the N-methylthio carbon atom (Scheme 2.24). Chemical studies and investgations into the intrinsic nataure of N-methylthio -lactams in S. aureus is further detailed in chapter IV Merck laboratories have conducted tests on N-trifluoromethylthio lactam 109 (Figure 2.15) against human leukocyte elastase; however, this lactam was found to have no inhibitory activity (IC 50 = 8 mg/mL). 78a Figure 2.15: N-Trifluoromethylthio-lactam investigated by Merck laboratories. 78a N O S C F3 Ph CO2CH3 109 In addition to the monocyclic systems discussed above, there have been several reports of bicyclic compounds of this class that have the N-S bond at the site of ring fusion. The first example, N-fused -lactam derivative 111, was formed as an unexpected product during attempts to cyclize lactam 111 to the ring-closed penem (Scheme 2.25). The reaction occurs via a rearrangement process. No information was reported about biological testing on this adduct. 94 Scheme 2.25: Cyclization of 110 to the non-penem 111. 94 26 NH O S SCH3 Br O O NO2 N S O SCH3 O O NO2 N S SCH3 O O NO2 O LDACuI-SMe2 110111

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In 1991, the Pfizer laboratories reported the synthesis of some related N-S fused lactam rings represented by structures 112 and 113 in studies on human leukocyte elastase inhibitors (Figure 2.16). 95 Figure 2.16: N-S fused bicyclic -lactams of Pfizer. 95 N O SS N O SS N O SSS N O SSSS 112113114115 Compound 117 was synthesized by cyclizaton of lactam 116 with sulfur bis-phthalimide 61 (Scheme 2.26). The mechanism of the ring closure involved formation of disulfide 118 and trisulfide 119 intermediates (see Scheme 2.26), which could be isolated and independently converted to product 117 using triphenylphosphine. Scheme 2.26: Preparation of 117 using sulfur bis-phthalimide. 95 N O S H H3C OTBDMS OtBu S O 116 61Et3N, THFN O H3C OTBDMS 117SS CO2tB u N O SSS N O SSSS 118119CO2tBu CO2tBu Alternatively, the reaction of 116 to 117 (Scheme 2.27) could be carried out using N-chlorosuccinimide (NCS) proceeding through a sulfenyl halide intermediate 120. 95 Although only one olefin isomer is shown for 117, the reaction in fact gave both geometric isomers which could be separated and independently examined for antibacterial activity. Scheme 2.27: Preparation of 117 using N-chlorosuccinimide. 95 N O S H H3C OTBDMS OtBu S O 116 iPr2EtN CH2Cl2NCSN O S H H3C OTBDMS OtBu SCl O 120 117-HCl The Pfizer group also synthesized the six-membered ring analogue 122 through a similar cyclization starting from monocyclic -lactam 121 (Scheme 2.28). Product 122 was then used to prepare fivemembered ring lactam 117 through a sulfur extrusion process. The conversion of 122 to 117 is thought to proceed through a fascinating, and unexpected, rearrangement process described in the paper. 27

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Scheme 2.28: Preparation of 117 using triphenylphosphine. 95 N O H3C OTBDMS 122R = C O 2 t B uSS S HN O CH3 TBDMSO R PPh3THF117NH O H3C OTBDMS S S HN O CH3 TBDMSO R 61121 R = C O 2 t B uEt3N, THF Both geometric isomers of ester compound 117 were found to be effective inhibitors of human leukocyte elastase (K i = 1.5 M and 4.0 M, respectively). The sodium salt of the carboxylate, lactam 123 (Figure 2.17) was prepared from ester 117 and found to be devoid of antibacterial activity. Thus, to have elastase activity, the ester rather than the anionically-charged carboxylate group is required on these lactams. 95 Figure 2.17: Human leukocyte elastase inhibitor 123. N O H3C OTBDMS 123SS CO2 Na In a subsequent paper, Pfizer studied the elastase inhibitory properties of a series of related bicyclic lactams 124-129 (Figure 2.18), and compared their observed activities to their relative rates of base hydrolysis. Blocking the carbonyl with a bulky side chain as in carbonate 126 increases elastase inhibition while decreasing susceptibility to base hydrolysis. As anticipated, reversing the absolute chirality of the bicyclic ring system, as observed for lactam 127 versus 125, completely destroys the biological activity. The study also indicated that replacement of the ring thioether sulfur atom for a carbon atom as for compounds 128 and 129 slightly increases both the rate of hydrolysis of the lactam ring and the elastase inhibition constant K i The N-S linkage makes the lactam ring more susceptible to base hydrolysis, but does not significantly affect the elastase inhibitory ability. Thus, enhancing the reactivity of the -lactam ring does not increase the compound's potency as an elastase inhibitor, suggesting that the rate determining step in elastase inhibition is not nucleophilic attack on the -lactam carbonyl center (vida supra). 96 Figure 2.18: Bicyclic N-thiolated -lactam elastase inhibitors 124-129. 96 N O H3C OR SS CO2tBu (Z)-124 R=H (E)-125 R=H ( E ) -126 R = C O B nN O H3C OH SS CO2tBu 127N O H3C OR S CO2tBu (Z)-128 R=TBDMS (E)-129 R=H Turos developed a synthesis of structurally related bicyclic lactams 54 and 130 by iodocyclization of unsaturated N-methylthio -lactams 84 and 85 (Scheme 2.29). 78b-d Attempts to construct isopenams 131 by halocyclization of alkenyl sulfenamide 130 were unsuccessful. 28

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Scheme 2.29: Preparation of bicyclic lactams via halocyclization. 78b-d N S O Ph I R H H 54 R=OMe()-84 R=OMe()-85 R=SBnN O SCH3 R H H Ph I2CH2Cl240oC130 R=SBn N S O Br CH3O H H CH3 CH3 N O SCH3 CH3O H H CH3 CH3 ( ) -130131 B r2 However, clavulanic acid-type ring systems 133, 134, and 135 were prepared from monocyclic N-methylthio precursor 85, 86, and 132 (Scheme 2.30). Scheme 2.30: Halocyclization of lactams 85, 86, and 132. 78b-d N O R OAc Ph SCH3 H H ()-85 R=OMe()-86 R=NPhth()-132 R=SBnN S O R H H I Ph O O CH3 I2CH2Cl2()-133 R=OMe()-134 R=NPhth()-135 R = S B n These bicyclic N-S fused compounds have a highly electrophilic -lactam ring, yet are stable over a wide pH range (pH 1 to pH 10). Computational experiments indicate that they have about the same thermodynamic stability as the classical penicillins and penems. Ab initio calculations indicate that the -lactam in these N-S fused systems is slightly more twisted due to a less highly pyramidalized nitrogen center, and have a lower LUMO energy than that of the antibiotics. Despite the structural similarities and higher electrophilicities relative to the known -lactam drugs, lactams 54, 131, 133, 134, and 135 have no antibacterial properties. 76b,76c 2.7 Conclusions The discovery of the monobactams in the early 1980's opened new avenues of investigation in -lactam antibiotics research. This lead to exciting advances and to the introduction of aztreonam as an important clinical agent for control of Gram-negative bacterial infections. The interest in N-thiolated -lactams was further heightened in the mid-1990's with the development of N-arylsulfonyl -lactams as elastase inhibitors. Most recently, the discovery that N-methylthio -lactams have inhibitory activity against S. aureus by a unique mode of action, opens the door to new investigations. As such, the focus of the following chapters will be on the investigations into the chemical and biological properties of N-alkylthio -lactams. 29

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CHAPTER III SYNTHESIS AND BIOLOGICAL PROPERTIES OF C 4 ARYL SUBSTITUTED N-THIOLATED -LACTAMS 3.1 Introduction As alluded to in chapters I and II, N-methylthio -lactam 1, a synthetic precursor to the clavulanic acid-type ring system 2 seen below, was identified as a substance possessing specific biological activity against Staphylococcus aureus. 78b-d Although only 8 analogues of 1 have been reported previously and the antimicrobial screening ad been rather limited, the novelty of this new class of antibiotics warranted their further investigation. This chapter examines for the first time, the structure-activity relationship (SAR) of N-thiolated -lactams antibiotics containing functionalized aromatic residues at the C 4 position. The synthesis and biological evaluation of the lactams against a panel consisting of 33 bacteria species and 10 strains of methicillin-resistant S. aureus (MRSA) is reported. The effect on cell growth and cell survival is also described. The information gathered from the experiments presented in this chapter can in effect be applied towards the understanding of the lactam's mode of action in bacteria. Scheme 3.01: Iodocyclization of N-methylthio lactam 1. N S O P h I R H H 21N O S C H 3 CH3O H H Ph I2CH2Cl240oCN O SR' RO H H Xn 3 3.2 Synthesis of C 4 Aryl Substituted N-Thiolated -Lactams C 4 aryl substituted -lactams (3) were prepared by the series of reactions illustrated in Scheme 3.01. The four stage sequence was initiated by the synthesis of N-(4-methoxyphenyl)imine (6) from aryl aldehyde 4 and p-anisidine. Staudinger coupling of an acid chloride (7) and imine by a formal [2+2] cycloaddition afforded exclusively the cis-(3S,4R)-substituted -lactam 8 as a racemic mixture. Following oxidative cleavage of the p-anisyl residue by aqueous ceric ammonium nitrate, the N-protiolactam 9 was thiolated using readily available sulfenylating reagents to provide the N-thiolated -lactams (3) in four steps. The yields of the reactions were variable depending on the substituents located at C 3 and C 4 positions of the ring and the conditions. The following section details each reaction involved in the synthesis of N-thiolated -lactams. 30

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Scheme 3.02: General synthetic route to lactam 3. N H OCH3 Xn O H Xn 46N O RO H H Xn 8 O C H3 RO O Cl base7p-anisidine5 N O H RO H H Xn CAN9N O SR' RO H H Xn 3 X-SRbase 3.2.1 Synthesis of C-Aryl(imines) 6 N-(4-Methoxyphenyl)imines (6) were prepared from the condensation of aryl aldehyde 4 and p-anisidine (5). Prior to the reaction, the crude p-anisidine was recrystallized in water heated to ~60 o C and dried in vacuo. 97 While most aldehydes were used without further purification, when necessary, the acid contaminant was removed by washing with 10% sodium bicarbonate or distilling at atmospheric pressure. 97 The synthetic protocol was identical for all the monoand multisubstituted C-aryl(imines). The aldehyde and p-anisidine were dissolved in methylene chloride and stirred at room temperature for 1-2 hrs. Approximately 1 mg of camphorsulfonic acid (CSA) was added to the mixture and heating to reflux was sometimes applied to promote the condensing of the starting materials. In most instances, conversion to the imine was completed within 1 hr and could be followed by TLC. Scheme 3.03: Synthesis of C-aryl(imines). N H OCH3 Xn O SO3H cat.CH2Cl2, rt O H Xn + NH2 OCH3 1:1546X = H, F, Cl, Br, I, NO2, CH3, OCH3, O2CR In cases when the aldehydes were not commercially available, the imine precursors could be generated by oxidation of the corresponding benzyl alcohols using oxalyl chloride and DMSO (Scheme 3.04). 98 Aldehydes synthesized by this methodology included 2-, 3-, and 4-iodobenzaldehyde (10-12). Scheme 3.04: Swern oxidation of iodo-benzyl alcohols. I OH (ClCO)2, DMSOCH2Cl2, -78oC then Et3N I H O 10 I=ortho11 I=meta12 I =pa r a 31

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Noncommercial aldeydes bearing an ester functional (13,14) were prepared by the acylation of p-hydroxybenzaldehyde. -Lactams generated from 13 and 14 were used to examine the in vitro performance of N-thiolated -lactams possessing a lipophilic, acyloxy substituent while serving as an intermediate for the preparation of a p-phenol derivative. Scheme 3.05: Esterification of p-hydroxybenzaldehyde. H O HO R Cl O Et3N, CH2Cl213 R=(CH2)4CH314 R=CHCH2 H O O R O Bis-imine 15 was synthesized by the condensation of the dialdehyde glyoxal and p-anisidine, and used to generate bis--lactam analogues. Low solubility in organic solvents, however, limited the usefulness of 15 as an intermediate for making -lactams. Scheme 3.06: Synthesis of bis-imine 15. N H CH3O N H OCH3 15 H O O H p-anisidineCSA, MeOH50oCglyoxal 3.2.2 Synthesis of N-Aryl Protected -Lactams 6 by Staudinger Coupling The most frequently described procedure for preparing monocyclic -lactams is Staudinger coupling. This reaction involves a formal [2+2] cycloaddition of an acid chloride (5) or an activated carboxylic acid 51c to an imine (4, eg, Schiff base), and was the methodology employed to synthesize N-aryl protected -lactams 6 (Scheme 3.05). Scheme 3.07: Synthesis of N-aryl protected -lactams by Staudinger [2+2] condensation. N O RO H H Xn 8 O C H3 RO O Cl + N H OCH3 Xn iPr2EtN, PhMe0oC rt 76 32 The mechanism of -lactam formation in the synthesis of 6 does not entail a direct acylation of the imine with an acid chloride, but rather a ketene cycloaddition. The ketene forms by deprotonation of an activated acid with a Lewis base such as ethyldiisopropylamine. If a base is absent or added after the imine and acid are combined, the ketene does not form and the cycloaddition generally occurs via direct coupling of 4 and 5 resulting in a -lactam with a trans configuration. 51c Table 3.01 categorizes the three types of ketenes that undergo Staudinger coupling. The substituents of 4 and 5 ultimately determine the stereochemical outcome of the reaction. Acid chlorides applied to the synthesis of 6 give rise to exclusively the Bose-Evans ketene which confers the cis conformation when coupled with a diaryl imine (4). 51c This was verified for all C 4 aryl analogues from the chemical shifts in the 1 H NMR spectra.

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Table 3.01: Stereochemical outcome of the Staudinger reaction of ketenes and imines. 51c C O H R Bose-EvansketenesSheehanketenesMooreketenesOR, NHR, N3, Fvinyl, PhthR=Cl, Br, alkyl, Ar, SRS(O)nRproductcistranstransDiaryl IminesproductciscistransAlkyaryl ImineproductcisciscisGlyoxalicIminesproducttranstranstransImidates Imine N-Aryl imines (4) exist primarily in the E-configuration and give rise to cis -lactams. Although thermodynamically less stable, kinetics allows the cis product to be generated via an anionic intermediate which is stabilized by the electron-rich groups of the Bose-Evans ketene. Conversely, if a Z-imine was used in the reaction, a trans -lactam will result depending on the stability of the intermediate. The stereochemical outcome of these reactions is rationalized by the pathway depicted in Scheme 3.08. Orthogonal attack of the imine on the least hindered side of the ketene carbonyl center forms a zwitterionic intermediate which can (1) interconvert via the anionic intermediate, (2) revert back to the imine and ketene, or (3) lead to cis and trans -lactams. Both the -lactam and imine can interconvert as well, but only if the ring opens regenerating the zwitterionic species. Scheme 3.08: Ketene-imine cycloaddition. CN Ar H PMP O H R CN H Ar PMP O H R CN Ar H PMP Nu O H R CNAr H PMP O H R CNH Ar PMP O H R N R Ar H H PMP O N R H H Ar PMP O transcisNu Nu Anionic IntermediateConrotatory Ring ClosureConrotatory Ring Closure Z I m ines E I m ines The ketene precursors (16) were synthesized from the corresponding carboxylic acid and thionyl chloride. This general method was used to make acid chlorides 16 and 18. Although methoxyacetic acid was commercially available, acetoxyacetic acid (17) were prepared from glycolic acid and acetyl chloride in quantitative yield. 33

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Scheme 3.09: Synthesis of acid chlorides 16 and 18. OH O H3CO + SOCl2 Cl O H3CO 160oCthen heat OH O AcO OH O HO 0oCSOCl2 Cl O AcO 0 50oCAcCl1718 The yields of the Staudinger reaction involving acid chlorides 16 and 18 varied greatly depending on the acid chloride, solvent and temperature. Little variation was observed for the different imine derivatives or the type of base. Poor yields did however result when imine 15 was applied toward the syntheses of bis--lactam analogues. Scheme 3.10: Synthesis of bis--lactam from bis-imine 15. N H CH3O N H OCH3 O Cl RO iPr2EtN, PhMerefluxN O N O RO RO O C H3 OCH3 H H H H 15 Staudinger coupling reactions typically require a tertiary amine base such as triethylamine or ethyldiisopropylamine (eg. Hnig's base). The latter was utilized for most reactions but could be replaced by triethylamine with little effect on the isolated yields. A minimum of 3 equivalents of base was needed to enable the reaction to go to completion. Mechanistically, the excess is required to (1) generate the ketene, (2) serve as a proton scavenger, and (3) act as a nucleophile (Nu ) in formation of the anionic intermediate (Scheme 3.06). Toluene became the solvent of choice when yields substantially increased for reactions involving acid chloride 16. Another advantage the nonpolar medium offered was that the ammonium salt of ethyldiisopropylamine/triethylamine and sometimes the -lactam itself were insoluble in toluene at room temperature. After filtration, the product could be cleanly isolated from the precipitated material or triturated from the concentrate of the reaction using diethyl ether or ice-cold methanol. The greatest effect on yield however, was the acid chloride component. Phenoxyacetyl chloride routinely gave the best yields followed by the methoxyand acetoxycounterparts. Of note, the acid chlorides and their respective ketenes were sensitive to heat and decomposed if the temperature was raised too high. Therefore, all reactions were performed at 0 o C and allowed to warm to room temperature. Only in the syntheses of the bis-lactams analogues was reflux required, and generally gave low yields depending on the acid chloride. 3.2.3 Dearylation of -Lactams 6 with Ceric Ammonium Nitrate Kronenthal 99 reported a general method to remove the p-anisyl moiety of protected -lactams using ceric ammonium nitrate (CAN) in aqueous acetonitrile. The same procedure was applied in the synthesis of N-methylthio -lactams by the conversion of N-anisyl lactams 6 to N-protio lactams 9. During the oxidation, the electron-rich aromatic ring permits cleavage by a radical generating species such as CAN. The reaction proceeds through intermediate 19 (Scheme 3.11) which decomposes to the dearylated -lactam and benzoquinone when washed with a 5% sodium bisulfite solution. 34

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Scheme 3.11: Oxidative dearylation of -lactams 6. N O R'O H H Xn N O H R'O H H Xn Ce(NH4)2(NO3)6aq. MeCN0oC69 O C H3 N O R'O H H Xn OH OMe HO 19 Deprotection of the C 4 aryl analogues 6 gave low to moderate yields depending on the solubility of the N-arylated -lactam in acetonitrile. In most cases, the -lactams did not dissolve and heating was required to completely solubilize the starting material. Resuspension of the heat solution in an ice water bath was necessary before the CAN solution could be added otherwise the reaction would fail. Most oxidations were complete after 30 minutes with light stirring followed by aqueous workup of the ethyl acetate extracts with sodium bicarbonate and sodium bisulfite. 3.2.4 N-Methylsulfenylation of -Lactams 9 Two methods were employed to incorporate the N-methylthio substituent onto the N-protio lactam 9. Initially, the procedure reported by Shah and coworkers 90 was applied toward the synthesis of 3 (Scheme 3.12) using methylthiomesylate (20). Deprotonation of 9 with n-butyllithium at low temperature followed by lithium exchange with the methylthio moiety of 20 afforded the N-methylthio-lactams in good yields. Methylthiomesylate was prepared by the oxidation of methyl disulfide with 30% hydrogen peroxide in accordance to an already established literature procedure. 90 Scheme 3.12: Synthesis of N-methythio -lactams and methythiomesylate. N O H R'O H H Xn N O S C H3 R'O H H Xn CH3SSO2CH3 20n-BuLi, THF-78oC93CH3S S CH3 glacial AcOH30% H2O20oC to RTSO O S CH3 CH3 20 Another method of introducing a methythio substituent was reported by Miller using N-methylthiophthalimide (21). 86 The reaction employs a mild base such as triethylamine or Hnig's base under reflux conditions in a low boiling, nonpolar aprotic solvent. The synthesis of 21 was carried out following previously described literature procedures. 100 From methyl disulfide, chlorine gas was used to 35

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generate the methylsulfenyl chloride in situ then cannulated into a flask containing 1 and 1.5 equivalents of phthalimide and triethylamine, respectively. Scheme 3.13: Synthesis of N-methythio -lactams and N-(methyl)thiophthalimide. 86,100 N O H R'O H H Xn N O O SCH3 iPr2EtN, PhH refluxN O SCH3 R'O H H X n 9321 NH O O 1. Cl2, PhH, 0oC, Et3N2. N O O SCH3 CH3S S CH3 21 Reactions involving sulfur reagent 21 was found to be more efficient and reliable than 20 and became the method of choice for the N-methylthiolation of all C 4 aryl -lactams. 3.3 The Structure-Activity Profiling of C 4 Phenyl Analogues 3.3.1 Synthesis and Microbiological Evaluation of 67-81 The SAR studies were initiated with the synthesis and microbial screening of monosubstituted C 4 aryl -lactams 68-81. As a control, an unsubstituted phenyl analogue (67) was included in the series for comparison. A diverse selection of functionalities and locations on the phenyl ring was chosen for the studies including: halogen (68-74), alkyl (75), ether (76), nitro (77,78), nitrile (79), and ester (80,81) substituents. Scheme 3.14 illustrates the synthetic route to compounds 67-81 and the intermediates involved in their preparation. Scheme 3.14: Synthesis of C 4 phenyl analogues 67-81. N O CH3O H H X O C H3 CH3O O Cl N H OCH3 X iPr2EtN, PhMe0oC rt Ce(NH4)2(NO3)6aq. MeCN22 X=H23 X=2-Cl24 X=3-Cl25 X=4-Cl26 X=4-Br27 X=2-I28 X=3-I29 X=4-I30 X=2-CH331 X=2-OCH332 X=2-NO233 X=3-NO234 X=4-CN35 X=4-[O2C(CH2)4CH3]36 X=4-[O2CCHCH2]16()-37 X=H()-38 X=2-Cl()-39 X=3-Cl()-40 X=4-Cl()-41 X=4-Br()-42 X=2-I()-43 X=3-I()-44 X=4-I()-45 X=2-CH3()-46 X=2-OCH3()-47 X=2-NO2()-48 X=3-NO2()-49 X=4-CN()-50 X=4-[O2C(CH2)4CH3]()-51 X=4-[O2CCHCH2]0oC 36

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N O H CH3O H H X N O O SCH3 iPr2EtN, PhHN O SCH3 CH3O H H X 21()-52 X=H()-53 X=2-Cl()-54 X=3-Cl()-55 X=4-Cl()-56 X=4-Br()-57 X=2-I()-58 X=3-I()-59 X=4-I()-60 X=2-CH3()-61 X=2-OCH3()-62 X=2-NO2()-63 X=3-NO2()-64 X=4-CN()-65 X=4-[O2C(CH2)4CH3]()-66 X=4-[O2CCHCH2]()-67 X=H()-68 X=2-Cl()-69 X=3-Cl()-70 X=4-Cl()-71 X=4-Br()-72 X=2-I()-73 X=3-I()-74 X=4-I()-75 X=2-CH3()-76 X=2-OCH3()-77 X=2-NO2()-78 X=3-NO2()-79 X=4-CN()-80 X=4-[O2C(CH2)4CH3]()-81 X=4-[O2CCHCH2]reflux A later addition to this series was a phenolic -lactam 83 which was synthesized by the hydrolysis of the acrylate intermediate 66. Although p-hydroxybenzaldehyde was commercially available, the alcohol residue required protection for the Staudinger coupling and CAN oxidation steps. Hydrolysis of 66 afforded 82 in quantitative yield and subsequent sulfenylation of the -lactam gave the p-phenol analogue in two steps from the acrylate (Scheme 3.15). Scheme 3.15: Synthesis of C 4 phenolic -lactams from 66. N O H CH3O MeOH, 0oCH H O O N O H CH3O H H OH KOH iPr2EtN, PhHN O S C H3 CH3O H H 21OH ()-66()-82reflux()-83 A diverse panel of pathogenic and nonpathogenic bacteria representing 17 genera and 33 species were used in the preliminary screening. Compounds were evaluated in vitro by agar diffusion (Kirby-Bauer) in accordance with the guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS). 101 Initially, the disc variation of the test was applied to assess the susceptibility. Here, a 6 mm cellulose disc impregnated with 20g of the drug was placed on an agar plate inoculated with the test organism. Following incubation, the zones of growth inhibition were measured to determine the potency of the drug after 24 hrs. Another variation which gave consistent results utilizes a well instead of a disc to facilitate drug diffusion. The plates were prepared by cutting 6 mm circular holes into the inoculated medium and applying 20g of the test drug in dimethylsulfoxide (DMSO) to the wells. After incubation, the zone diameters were measured in millimeters to ascertain the relative potency. The results of the susceptibility test are depicted in Table 3.03. The -lactams demonstrated significant inhibitory activity against Bacteroides, Bacillus, Micrococcus, Neisserria, Streptococcus and Staphylococcus species. Peculiarly, these six genera derive from four distinct taxonomic orders defined by their genetic, morphological and metabolic traits (Table 3.02). Although nine genera in toto were found to be susceptible to the lactams, few taxonomic relationships could be used to ascertain the spectrum of activity. The class of bacteria that responded the most to the N-thiolated-lactams was the "Bacilli" which consists of two orders: Bacillales and Lactobacillales. Susceptible members of this taxonomic class included species of Bacillus, Staphylococcus and Streptococcus. Enterococcus Lactococcus and Listeria which are also affliated with the "Bacilli" were mostly insensitive to the drugs, though. The sporadic activity among bacteria species suggests the 37

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compounds may be acting in a different manner from traditional broad spectrum -lactam antibiotics. Chapter IV further examines the metabolic relationships of these microbes and reasons for the randomness in the activities observed for N-thiolated -lactam antibiotics. Table 3.02: Comparison of microbes sensitive to N-thiolated -lactam antibiotics. order genera Gram () morphology respiration activity a Enterobacteriales Salmonella rod aerobic weak Pasteurellales Haemophilus rod aerobic weak Vibrionales Vibrio rod anaerobic weak Bacillales Bacillus + rod aerobic medium Bacteroidales Bacteroides rod anaerobic medium Lactobacillales Streptococcus + cocci anaerobic medium Neisseriales Neisserria cocci aerobic medium Actinomycetales Micrococcus + cocci aerobic strong Bacillales Staphylococcus + cocci anaerobic strong a based on well diffusion data: strong = >20 mm; medium = 15-20 mm; weak = <15 mm. 38

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a Bacteria were ob tained from various commercial mmercial sources. See pg. 87 for more information; b Well diffusio and non co n on Mueller-Hinton ag ar; c Well difussion on Bacteria spp a Table 3.03: In vitro susceptibilities of bacteria to N-methylthio -lactams. 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 83 96 97 98 99 PEN CIP Bacteroides fragalis 15 15 15 0 0 15 0 0 13 0 0 Bacillus anthracis 21 8 7 8 0 0 25 24 20 24 20 22 22 20 18 10 20 14 24 20 37 Bacillus cereus 18 21 20 19 18 19 19 23 18 18 10 15 13 21 19 20 12 33 Bacillus coagulans 14 20 20 20 18 13 15 22 15 13 17 0 10 0 17 14 22 27 40 Bacillus globigii 14 19 18 18 20 17 14 20 15 14 16 0 19 0 22 16 20 30 41 Bacillus megaterium 11 20 16 14 16 15 13 17 12 10 14 0 15 10 21 14 20 41 40 Bacillus subtilis 12 18 15 14 19 14 14 18 10 0 16 0 19 0 21 12 18 41 37 Bacillus thuringensis 15 20 19 17 20 17 17 19 15 13 17 0 16 10 20 17 20 0 33 Enterobactor cloacae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Escherichia coli 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Haemophilus influenzae Klebsiella pneumoniae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Listeria monocytogenes Micrococcus luteus 21 240 31 29 25 24 24 25 21 22 21 25 26 25 26 400 Mycobacterium smegmatis Neisserria gonorrhoeae c 19 16 14 12 12 14 13 12 12 12 12 13 13 10 0 Proteus mirabilis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18 Pseudomonas aeruginosa 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Salmonella typhimurium 8 10 10 8 0 0 0 0 0 0 0 0 0 0 0 0 25 Serratia marcescens 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Staphylococcus aureus 25 26 23 24 23 27 23 24 23 28 17 15 14 16 21 18 18 25 24 23 33 Staphylococcus captitis 19 26 21 21 24 27 19 22 15 18 18 13 0 15 21 19 23 47 Staphylococcus cohnii 17 22 22 22 23 23 20 14 18 17 19 18 17 18 17 19 19 43 Staphylococcus epidermidis 31 29 25 23 30 29 23 28 25 20 18 20 20 20 20 17 20 24 25 23 50 Staphylococcus lentus 12 18 18 18 15 17 18 15 0 10 16 0 0 0 0 0 15 34 Staphylococcus lugdunensis 20 28 28 28 28 28 24 22 19 20 23 19 16 19 22 19 18 47 Staphylocococus saprophyticus 17 20 21 18 19 23 18 18 15 13 15 15 14 15 16 15 22 19 22 30 Staphylococcus simulans 14 16 0 0 18 18 18 16 14 14 12 13 0 15 0 13 21 0 20 13 Staphylococcus warneri 17 25 28 28 24 26 20 22 17 17 18 20 17 13 17 19 20 38 Staphylococcus xylosus 18 28 22 24 26 27 24 24 16 20 22 18 17 18 13 22 22 34 Streptococcus agalactia d 0 11 0 13 15 0 0 0 14 0 11 0 15 0 14 Streptococcus pyrogenes d 14 11 12 14 14 14 0 0 14 0 21 14 16 10 14 Vibrio cholerae 9 10 9 0 0 12 0 0 8 0 0 0 10 0 12 17 Zone of Growth Inhibition (mm) b chocolate agar; d Well diffusion on tryptic soy agar.

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3.3.2 Structure-Activity Relationship of 67-81 and 83 Against MSSA Based on the biological evaluation of lactams 67-81 and 83, it was evident that aromatic substitution is not a prerequisite for antimicrobial activity. Lactam 67 demonstrated equal or greater propensity to inhibit the in vitro growth of bacteria to those containing functionalized phenyl rings (68-81,83). The influence of aromatic substitution on activity appeared to be moderate. An antagonistic effect was observed for analogues possessing highly polar phenyl ring residues against the 33 member panel of bacteria. Compounds containing powerful deactiving or activating groups such as nitro (77,78) and nitrile (79) or acyloxy (80,81) and hydroxy (83), respectively, were about 30% less active than 67. Although the reason for this anomaly is unclear, the ability for a highly polarizable aromatic ring to attenuate bioactivity was evident particularly against species of Bacillus and Staphylococcus. Figure 3.01: Percent relative activity of penicillin for monosubstituted analogues against methicillin-susceptible Staphylococcus aureus (MSSA). No discernable differences in bioactivity could be ascertained for compounds 68-76. These include derivatives that possess weak deactivating aromatic substituents like halogens (68-74) and mild-medium activating groups such as alkyl (75) and alkoxy (76). The bacteria most susceptible were Staphylococcus and Micrococcus luteus. Bacillus spp. was also sensitive to the drugs though not to the same extent. Location and size of the aryl ring substituent had no apparent influence on biological activity. The expectation was that increasing ring bulk would lead to erosion of bioactivity. This however, was not the case for the chloro-, bromo-, and iodo-substituted lactams 68-74. As illustrated in the graph above, the percent activity of penicillin against methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 25923) is nondiscrete for the haloaryl, tolyl, and methoxyphenyl derivatives. Although the zone sizes were slightly larger for ortho-substituted 68, 72, and 76, the minimum inhibitory concentrations (MICs) remained at or about 10 g/ml for MSSA. 3.3.2 Structure-Activity Relationship of 67-81 and 83 Against MRSA Compounds 67-81 and 83 were tested against a panel of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Eight of the ten strains were obtained from Lakeland Regional Medical Center and identified as USF652-659. Prior to screening, each of the Lakeland isolates was assayed for -lactamase activity. Using a previously described acidimetric assay to monitor the hydrolysis of penicillin G, 102 all eight strains was verified to be -lactamase-producing forms of S. aureus. Table 3.04 and Figure 3.02 compare the relative effectiveness of 67-81 and 83 to inhibit the in vitro growth of MRSA. The glycopetide and current treatment of choice for MRSA-type infections, vancomycin, and penicillin were included as controls. Amazingly, the analogues retained their biological activity against forms of S. aureus that were resistant to penicillin. The structure-activity relationship of 67-81 against the multi-drug resistant strains parallel those observed in the microbiological evaluation of 40

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MSSA. It was apparent based on these findings that N-thiolated -lactams are transparent to the destructive forces of -lactamase (eg. penicillinases) which act to neutralize the ability of penicillins and cephalosporins to impede cell wall biosynthesis. In the case of benzylpenicillin, the impact of -lactamase on susceptibility was quite evident for the MRSA. A 50% reduction in zone size was detected against the ten strains for this "activated" -lactam antibiotic. Table 3.04 Kirby-Bauer data for analogues 67-81 and 83 against S. aureus. Strain a 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 83 PEN c VAN d MSSA ATCC 25923 25 26 23 24 26 27 23 24 23 28 17 15 14 16 21 18 33 MRSA ATCC 33591 20 26 26 19 29 28 25 26 17 21 17 20 14 15 20 MRSA ATCC 43300 21 29 25 20 26 30 23 25 19 24 19 17 0 18 20 MRSA USF652 30 30 24 26 25 34 23 27 23 29 18 17 14 17 16 14 8 19 MRSA USF653 30 29 28 27 27 29 23 24 27 32 23 22 16 17 15 19 15 18 MRSA USF654 26 28 22 23 26 27 21 24 23 27 20 16 12 15 20 18 10 19 MRSA USF655 25 27 23 23 29 28 24 24 23 27 22 14 12 15 19 18 14 19 MRSA USF656 28 29 22 25 24 29 22 25 25 28 22 18 13 16 20 19 12 21 MRSA USF657 27 29 22 23 27 28 25 24 23 27 20 18 10 16 21 19 12 18 MRSA USF658 26 27 19 24 20 28 23 20 22 26 21 17 12 14 19 18 19 18 MRSA USF659 24 24 18 18 20 23 22 17 20 23 18 11 12 14 17 0 16 18 Zone of Growth Inhibition (mm) b a USF652-659 were obtained from Lakeland Regional Medical Center, Lakeland, FL. b Kirby-Bauer well diffusion on TSA. c Benzylpenicillin potassium salt (penicillin G) d Vancomycin hydrochloride. Comparison of in vitro susceptibilities of MRSA to N-methylthio -lactams and vancomycin yielded promising results. In the graph below, the mean zone data of the ten MRSA isolates for compounds 67-80 and 83 is depicted as percentage of activity relative to vancomycin. The ability to inhibit the growth of MRSA was greatest for analogues 67-76. The -lactams consistently demonstrated 20-50% and 50-75% greater anti-MRSA activity than vancomycin and penicillin, respectively. Figure 3.02: Percent relative activity of vancomycin for monosubstituted analogues against methicillin-resistant Staphylococcus aureus (MRSA). 41

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3.3.3 Effect of Drug Amount Versus Zone Diameter Subsequent to the screening of compounds 67-81, a series of experiments were conducted to calculate the optimal amount of drug needed for well diffusion measurements. The results of these studies are depicted in graphs of Figures 3.03 and 3.04. Plots 3.03a and b draw a comparison of the relative susceptibilities of MSSA and MRSA to lactam 69 and penicillin, respectively, at equimolar amounts. The first graph reveals a linear relationship between the gs of lactam 69 and the zone diameter generated. Against MSSA (ATCC 25923), the inhibitory activity of 69 rose steadily as the amount increased from 2.8 to 56.2 g/well. The same trend was observed against MRSA (ATCC 43300) indicating that the compound retained full activity against the -lactamase-producing strain at all drug loadings. Conversely, Figure 3.03b illustrates that the potency of penicillin G against the drug-resistant strain was precipitously lower relative to the MSSA isolate. Although the antibiotic was equally effective at all concentrations for MSSA, no anti-MRSA activity was observed until greater than 28 g was applied to the wells. The marked decrease in susceptibility was again a clear indication of the capabilities penicillinases have to neutralize traditional -lactams antibiotics such as penicillin G. Figure 3.03: Equimolar drug loads versus zone of growth inhibition of (a) lactam 69 and (b) penicillin G against MSSA and MRSA. Figure 3.04: Equimolar drug loads versus zone of growth inhibition of lactam against (a) MSSA and (b) MRSA. Figures 3.04a and 3.04b compare the performance of 69 against MSSA and MRSA to penicillin G and vancomycin. The first plot depicts the effectiveness of the -lactam and penicillin G at parallel concentrations against MSSA. It was discovered that compound 69 produced smaller zones than penicillin against the non-resistant S. aureus strain up to 200 mol -3 /well (equivalent to 51.4 g of 69, or 64 g of penicillin G). The ability of penicillin to yield zone sizes >25 mm even at 10 mol -3 was consistent with the lower MIC value of the antibiotic over lactam 69. As predicted, a reversal in activities occurred when the two drugs were screened against MRSA (Figure 3.04b). Lactam 69 demonstrated superior ability to inhibit the in vitro growth of the drug-resistant strain over a 10-200 mol -3 range compared to penicillin. 42

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Vancomycin also displayed less potency than 69 when greater than 75 mol -3 of compound was applied to the well. 3.3.4 Minimum Inhibitory Concentration (MIC) 101 The lack of ionizable groups makes N-thiolated -lactams insoluble in aqueous media, therefore minimum inhibitory concentrations (MIC) proved to be difficult to measure. Some lactams could be solubilized if a 9:1 ratio of aqueous media to dimethyl sulfoxide (DMSO) was utilized. It was reported that S. aureus could sustain logarithmic growth in presence of 8-10% DMSO and this was reconfirmed prior to the MIC determinations. Unfortunately, most of analogues could not be solubilized unless 15-25% DMSO was present in the growth medium. Serial dilutions of TSB media was the initial method of choice to determine MICs though the results were neither consistent nor comparable to the potency levels seen with the raw data from the Kirby-Bauer diffusion evaluation. The agar variation, however, provided dependable results that were not obtainable by broth dilutions of the lactams. The agar media were prepared in 48 well plates and inoculated by applying a 1 l suspension of freshly prepared cultures to the well. Following a 24 hrs incubation period, the plates were examined for growth. The lowest concentration to inhibit the visual growth of bacteria was recorded as the MIC. The MIC values for lactam 68 against MSSA (ATCC 25923) was 10 g/ml. Though penicillin G (<1 g/ml) was superior to 68, the antibiotic did not retain the same effectiveness against the MRSA isolates. The MICs to inhibit 90% of the MRSA strains (MIC 90 ) was >125 g/ml for penicillin G in comparison to 8 g/ml for compound 68. 3.3.5 In Vitro Activity in Blood Serum The ability of a pharmacological agent to retain its antibacterial properties in blood serum is an important attribute if the drug were to be used clinically. Growth studies designed to examine the chemical stability of the N-thiolated -lactams in the presence of serum were performed against MRSA. The graphs in Figure 3.05 depict the efficacy of 68 to inhibit the in vitro growth of S. aureus over 24 hrs in the presence of serum. Bacterial growth was monitored during the course of the study by optical density of the cultures at 630 nm. Figure 3.05: Growth studies of MRSA with and without blood serum. It was apparent based on these studies that the chemical components of blood serum did not affect the lactam's ability to hinder microbial growth. Figure 3.05a and 3.05b are plots of the growth study performed in Mueller-Hinton broth (MHB) in the presence or absence of blood serum. In the antibiotic-free media (DMSO only), a steady progression of cellular growth was observed over 24 hrs for S. aureus. Conversely, bacteria viability was abruptly halted in the presence of lactam 68 at 1x MIC (8 g/ml) after 1 hr. Over 6 hrs, the drug uniformly stunted microbial growth in the serum and serum-free MHB. These data 43

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suggest that the in vitro half-life (t ) of lactam 68 with serum extends beyond 6 hrs. Between 6 and 24 hrs though, both media were equally turbid to the control (DMSO only) suggesting that the t is less than 24 hrs. It was postulated based on these finding that N-thiolated -lactams could be bacteriostatic antibiotics. Intrinsically, the drugs may not be capable of causing cell death but act in a manner that suppresses growth until drug levels were low enough for the bacteria to reproduce. The next section further examines the nature of N-thiolated -lactams to affect the viability of bacteria. 3.3.6 Time-Kill Studies Antibiotics can be categorized by the way they affect the viability of bacteria. Classification as a bacteriostatic or bacteriocidal agent can provide insight on the pharmacokinetic properties such as effective dosage. Additional information on the cellular target of a new antibiotic can be extracted from the intrinsic nature of other known drugs. Apropos those antibiotics that inhibit protein synthesis (ie, chloramphenicol) or secondary metabolites (ie, sulfa drugs) have been historically considered bacteriostatic agents whereas those that disrupt the cell wall (ie, penicillin) or directly damage DNA (ie, metronidazole) are bactericidal. Figure 3.06: Time-kill studies of 68 against (a) MSSA and (b) MRSA. The effect of N-thiolated -lactams on cell survival was examined by monitoring the growth of bacteria at high drug concentrations. Early logarithmic phase cultures of S. aureus were used in the study and growth was measured by viable cell counts. Figures 3.06a and 3.06b depicts cell survival for MSSA and MRSA, respectively, when cultured in the absence or presence of lactam 68 over a 2 hr time frame. In the absence of lactam 68, MSSA and MRSA grew logarithmically; in the presence of 68, bacterial growth was immediately halted. While reproduction ceased at the MIC level of the lactam as well as at 10x MIC, the number of viable cells remains constant throughout the duration of the experiment. Consequently, bacteria growth was clearly inhibited by lactam 68, but little to no decrease in cell population was observed. These data provide substantial evidence that even at high drug concentrations, N-methylthio -lactams are bacteriostatic agents toward staphylococci. 3.4 Multihalogenated Phenyl Analogues and Their Biological Activities 3.4.1 Synthesis of Multihalogenated Phenyl Analogues Follow up studies on the structure-activity relationships of C 4 phenyl analogues were conducted to examine the effect of multisubstitution and lipophilicity on the bioactivity of N-thiolated -lactams. Ergo the multihalogenated analogues 96-99 were prepared (Scheme 3.16) in the same manner that was previously described in Section 3.2. The aldehydes employed to make the pentafluoro(84), dichloro(97,98), and tricholorophenyl (99) N-(methoxyphenyl)imines were obtained from commercial sources. Staudinger coupling followed by anisyl removal and N-thiolation of the protio -lactams 92-95 with N-(methyl)thiophthalimide (21) gave 96-99 for screening. 44

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Scheme 3.16: Synthesis of multihalogenated N-thiolated -lactams. N O CH3O H H Xn O C H3 CH3O O Cl N H OCH3 Xn iPr2EtN, PhMe0oC rt 84 X=F585 X=2,4-Cl86 X=2,6-Cl87 X=2,3,5-Cl15()-88 X=F5()-89 X=2,4-Cl()-90 X=2,6-Cl()-91 X=2,3,5-Cl N O H R'O H H Xn Ce(NH4)2(NO3)6aq. MeCN iPr2EtN, PhHN O S C H3 R'O H H Xn 21reflux0oC()-92 X=F5()-93 X=2,4-Cl()-94 X=2,6-Cl()-95 X=2,3,5-Cl()-96 X=F5()-97 X=2,4-Cl()-98 X=2,6-Cl()-99 X=2,3,5-Cl 3.4.2 Antimicrobial Activity of Multihalogenated Phenyl Analogues Multihalogenated -lactams 96-99 demonstrated comparable bioactivities to the monosubstituted analogues 68-74 (Table 3.02). The spectrum of activity was again narrow which included species of Bacillus, Micrococcus, and Staphylococcus. The multichlorinated lactams 97-99 were equipotent to their monosubstituted counterparts 68-70 against MSSA and MRSA. The diand trisubstituted analogues displayed about 70% the activity of penicillin against MSSA (Figure 3.07a) and 20-40% greater potency against 10 strains of MRSA than vancomycin (Figure 3.07b). Figure 3.07: Percent activities of lactams 96-99 against (a) MSSA and (b) MRSA. Surprisingly, both forms of S. aureus were least susceptible to the pentafluorinated derivative 96. It was postulated before these results that the added hydrophobicity by the fluorine atoms would confer enhanced bioactivity to the -lactams. Lipophilic substances have been known to permeate cell membranes amid greater efficiency thus increasing the intracellular bioavailibilty of the drug. This was clearly not the case for lactam 96. Though the reason for the attenuation of potency is unknown, it is possible that repulsive forces evoked by the electronegativities of the fluorine atoms could decrease recognition and 45

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binding of the lactam to the biological target. However, other explanations can also be put forth. More on the mechanism of action and reactivity of N-thiolated -lactams will be discussed in chapter IV. 3.5 N-Sulfenylated Analogues and Their Biological Activities Based on the data gathered from the microbial screening of 67-81, 83, and 96-99, it was apparent that functionalization of the aryl ring will not improve the in vitro performance of the -lactams. Though few structure-activity relationships could be established from the aryl substituted series, it was believed that modification of the sulfur-bearing substituent could have the greatest impact on biological activity. This section examines the antimicrobial properties of N-thiolated-lactams having different sulfenyl substituents attached to the ring. 3.5.1 Synthesis of N-Sulfenylated Analogues A series of 10 other sulfenyl analogues was synthesized using N-thiolating reagents 94-99. 100 As described in Section 3.2.4, the N-thiophthalimides were prepared via chlorination of the corresponding thiol or disulfide to generate the sulfenyl chloride in situ. The volatile liquid was then canulated into a flash containing phthalimide and triethylamine. The reactions were performed neat for 1 hr in an ice bath (Scheme 3.17). Reagents 94-99 were then recrystallized from methanol and used without further purification. Scheme 3.17: Synthesis of N-thiophthalimide reagents 94-99. 100 NH O O 1. Cl2, PhH, 0oC, Et3N2. N O O S R 94 R=Et95 R=Bu96 R=Bn97 R=Ph98 R=C6H1199 R=C2H4OHRSHorRSSR N-Sulfenylated derivatives of the alkynyllactam 1 were the first to be synthesized and evaluated for antimicrobial activity. Compounds 101-106 were prepared from 100 by base-promoted thiolation using the corresponding N-thiophthalimide (94-99) (Scheme 3.18). Scheme 3.18: Synthesis of N-sulfenyl analogues 101-106. N O H CH3O H H iPr2EtN, PhH94-99reflux()-101 R=Et()-102 R=Bu()-103 R=Bn()-104 R=Ph()-105 R=C6H11()-106 R=C2H4OH N O SR CH3O H H ()-100 46

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Sulfenylated analogues of 67 were also synthesized from the phenyl-substituted lactam 49 using N-thiophthalimides 94-97 (Scheme 3.19). Scheme 3.19: Synthesis of N-sulfenyl analogues 107-110. N O H CH3O H H iPr2EtN, PhHN O SR CH3O H H ()-4921, 94-97reflux()-67 R=Me()-107 R=Et()-108 R=Bu()-109 R=Ph()-110 R=C6H11 3.5.2 Antimicrobial Susceptibilities to Lactams 101-109 A structure-activity profile could be established for -lactams possessing larger sulfur-bearing substituents. The alkynyl and phenyl analogues, 101-106 and 107-109, respectively, were screened against a panel of bacteria that were known to be susceptible to this drug class. In comparison to the N-methylthio -lactams 1 and 67, a 5-40% reduction in the zone diameter was detected for 101-109. The reduced bioactivity was observed against species of Staphylococcus, M. luteus, and N. gonorrhoeae (Table 3.05). It became evident from the data that increasing the sulfur bulk decreased the biological activity of the -lactams. This was again somewhat surprising since the hydrophobicity rose without augmenting the in vitro performance of the lactam. Presumably, the sulfur moiety which is the most labile group on the molecule, has direct involvement in the mode of action. Increasing the size of groups on sulfur could hinder the attack of a biological nucleophile or reduce the binding capabilities of the lactam to its cellular target. Table 3.05: Kirby-Bauer data for analogues 1, 67, and 101-109. Species/Strain a 1 67 101 102 103 104 105 106 107 108 109 PEN c VAN d MSSA ATCC 25923 27 25 23 17 15 12 15 15 21 21 16 33 MRSA USF652 31 30 28 18 15 12 17 18 21 24 18 8 19 MRSA USF653 30 30 27 20 15 10 16 18 25 24 15 15 18 MRSA USF654 28 26 23 16 16 12 14 17 18 20 15 10 19 MRSA USF655 27 25 24 17 17 13 14 15 20 22 16 14 19 MRSA USF656 30 28 24 18 15 12 15 17 20 20 14 12 21 MRSA USF657 28 27 25 16 15 12 14 18 17 20 14 12 18 MRSA USF658 27 26 24 18 18 13 15 18 21 22 17 19 18 MRSA USF659 24 24 22 16 15 13 16 18 20 20 15 16 18 S. epidermidis 30 31 23 18 10 16 14 27 25 0 50 S. saprophyticus 22 22 18 15 10 0 13 16 19 10 30 S. simulans 21 14 15 15 10 8 12 11 18 0 13 M. luteus 23 21 24 23 13 15 17 21 16 16 40 N. gonorrhoeae 13 14 10 8 0 0 0 13 25 0 0 a USF652-659 were obtained from Lakeland Regional Medical Center, Lakeland, FL. b Kirby-Bauer well diffusion on TSA. c Benzylpenicillin potassium salt (penicillin G) dVancomycin hydrochloride. N-Ethylthio lactams (101, 107) and the N-butylthio lactam 108 were the most active members from the series of sulfenyl analogues. Compared to 1 and 67, the compounds were slightly less active against MSSA but more active than the benzyl (103), phenyl (104), and cyclohexyl (105) analogues (vide infra). A linear relationship was established between the bulk of the S-substituent and the observed biological activity. In general, the performance of the -lactams declined as the length of the hydrocarbon Z one ofrowh Inhibition (m) b G t m 47

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chain on sulfur increased. The addition of a heteroatom-containing side chain also had an antagonist effect on the anti-staphylococcal activity of -lactam 106. The reason why the -mercaptoethanol (106) lactam was 30% less active to the mercaptoethane (101) lactam is not yet known. Figure 3.08: Comparison of anti-MSSA activities of lactams 101-109 to penicillin G. The structure-activity relationship observed for lactams 101-109 against MRSA was similar to the profile seen vida supra for the susceptible-form of S. aureus. The size of the S-substituent once again had the greatest impact on biological activity. All compounds appeared stable to -lactamase given that no alteration of the antibacterial properties was detected. Most possessed between 90 and >100% the inhibitory activity than vancomycin though remained less potent than 1 and 67 against eight strains of MRSA (Figure 3.09). Figure 3.09: Anti-MRSA activity comparison of 101-109. 3.6 Antibacterial Activity of Heterosubstituted N-Thiolated -Lactams Attempts to make heterosubstituted N-thiolated -lactams (113) were performed by a substitution reaction involving (2-oxo-1-azetidinyl)-thiophthalimides (112). As described in chapter II, 112 has been 48

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reported to react with nucleophilic substances such as benzyl alcohol, morpholine, and sodium bisulfite to generate 113 in good yields (Scheme 3.20).86 Scheme 3.20: Synthesis of heterosubstituted N-thiolated -lactams. N S O N O O N H O N S O N O O NuN S O Nu N O O S base Nu-112113 2111 Prior to the synthesis of the heterosubstituted N-thiolated -lactams, N,N'-thiobisphthalimide (111) was prepared from the treatment of phthalimide with sulfur monochloride in dry DMF (Scheme 3.21).103 Scheme 3.21: Synthesis of N,N'-thiobisphthalimide (111). N O O S N O O N O O H S2Cl2DMF, rt111 The o-chlorophenyl -lactam 53 and 111 were refluxed in chloroform (Scheme 3.22) and the progress of the reaction was monitored by TLC. Following the disappearance of starting material, the product was purified by silica gel chromatography. Surprisingly, the product of the reaction was not 115 but the dimerized -lactam 116 depicted in Figure 3.10. Scheme 3.22: Reaction of 53 with N,N'-thiobisphthalimide (111). 115N O N O O S CH3O H H H N O CH3O H H S N O O CHCl3, reflux 111()-532Cl Cl not isolated 49

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Figure 3.10: Thiolated bis--lactam dimers 116 and 117. N O CH3O H H S N O O C H3 H H ()-116Cl Cl N O CH3O H H S N O O C H3 H H ()-117Br Br The desired intermediate was generated when the m-bromophenyl -lactam 118 and 111 were reacted without heating (Scheme 3.23). Scheme 3.23: Synthesis of N-phthalimidothio -lactam 119. ()-119N O N O O S CH3O H H H N O CH3O H H S N O O CHCl3, rt 111()-1182Br Br In accordance to the previously described procedure, a 200% mole equivalent of the amine nucleophiles, morpholine and diisopropylamines were reacted with 119 at 025C in attempt to generate the heterosubstituted N-thiolated -lactams 120 and 121 (Scheme 3.24). However, the TLC indicated after 24 hrs that a reaction had not occurred. Heat was next applied and only then was the starting material consumed. Although 119 reacted under reflux, the product that formed was again a -lactam dimer (117). Scheme 3.24: Reaction of 119 with morpholine and diisopropylamine. ()-119 X ON H N HN O CH3O H H S Br N ON O CH3O H H S Br N O 1201210oC reflux CH2Cl20oC reflux CH2Cl2 With the understanding that elevated temperature could not be used for the substitution reactions, other types of nucleophiles were attempted such as sodium methoxide and isopropanol. However, once 50

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again the desired product (122,123) from the displacement of phthalimide was not formed. Either starting material or the N-protio -lactam (118) resulted from the reaction (Scheme 3.25) Scheme 3.25: Reaction of 119 with sodium methoxide and isopropanol. ()-119 X N O CH3O H H S Br O C H3 N O CH3O H H S Br O 122123NaOCH30oC rt CH2Cl2rtiPrOH The heterosubstituted-lactams 116, 117 and 119 were tested for biological activity against MSSA and MRSA. Surprisingly, the compounds were found to possess some antimicrobial properties (see Table 3.05). Although the lactams were less potent than penicillin for MSSA, they were approximately equal to vancomycin in efficacy against MRSA (Figure 3.11). These results contradict previous findings that increasing the molecular bulk on sulfur diminishes the activity of the -lactam (see Section 3.5). To the contrary, 116, 117 and 119 were more effective at eliciting an antimicrobial response than the phenyl (104,109), benzyl (103), and cyclohexyl (105) S-substituted analogues. Though the reason for this remains unclear, it is postulated that the sulfur of the heterosubstituted N-thiolated -lactams is more electrophilic with a second nitrogen bound, making it more reactive, and thus more susceptible, to attack by a biological nucleophile. Figure 3.11: Comparison of anti-MSSA and anti-MRSA activities 116, 117, and 119. 3.7 Additional N-Thiolated -Lactam Analogues Probed For Biological Activity Other N-methylthio -lactam analogues were synthesized and probed for antibacterial activity that did not fit in the series of compounds described in Sections 3.2-3.5. Among these was 127 which contained a 2-thiophene substituent instead a phenyl group at the C 4 position of the ring (Scheme 3.26). The lactam was synthesized by the methodologies previously discussed and tested by well diffusion. The results are depicted in Table 3.05. Analysis of the zone data revealed that a C 4 phenyl ring is not a requirement for antibacterial activity. Lactam 127 was equipotent to the most active N-methylthio -lactams despite lacking a 6-carbon member aryl substituent. These data also suggest that other heterocycles could be placed at C 4 of 51

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the -lactam and still retain biological activity. Synthesis of a 2-furan and 2-pyridine analogue was attempted, though, both decomposed during the CAN reaction. Scheme 3.26: Synthesis of 2-thiophene -lactam 127. N H S OCH3 16iPr2EtN, PhMe 0oCN O CH3O H H O C H3 S aq CANMeCN0oCN O CH3O H H S H 21Et3N, PhHrefluxN O CH3O H H S SCH3 124 ( ) -126 ( ) -125()-127 Alternative substituents at the C 3 position of the -lactam were examined for their influence on the biological properties (Scheme 3.27). Phenoxy (134) and acetoxy (135,137) derivatives were prepared from the corresponding acid chlorides (18,129) and tested by well diffusion. It was apparent from the zone data that the larger substituents at C 3 do not enhance the efficacy of the lactams (Table 3.06). In particularly, the phenoxy analogue of 68 displayed 45% reduced activity with the added molecular bulk. Scheme 3.27: Synthesis of lactams 134, 135, and 137. N H OCH3 iPr2EtN, PhMe 0oCN O RO H H O C H3 aq CANMeCN0oC 21Et3N, PhHreflux23 X=2-Cl()-130 R=Ph, X=2-Cl X X N O RO H H H X N O RO H H SCH3 X RO Cl O 128 X=4-NO218 R=Ac129 R=Ph()-131 R=Ac, X=4-NO2()-132 R=Ph, X=2-Cl ( )-133 R = Ac,X = 4 -NO2()-134 R=Ph, X=2-Cl ( )-135 R = Ac,X = 4 -NO2 21Et3N, PhHreflux ( )-136N O AcO H H H Cl F N O AcO H H S C H3 Cl F ( )-137 With the implication that increasing the size of the C 3 substituent leads to erosion of bioactivity, a substituent smaller that methoxy was incorporated and tested against the same bacteria panel. The group assigned to the position was a hydroxy which was generated by hydrolysis of the acetoxy group in lactam 139 (Scheme 3.28). The zone data revealed that the hydroxy analogue 141 was about 25% less effective than the methoxy derivative 67 at inhibiting the growth of S. aureus (see Tables 3.03, 3.05). 52

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Scheme 3.28: Synthesis of C 3 hydroxy substituted analogue. 18iPr2EtN, PhMe 0oCN O AcO H H O C H3 aq CANMeCN0oCN O AcO H H H 21Et3N, PhHrefluxN O HO H H H 22()-139()-138()-140 aq KOHMeOHN O HO H H SCH3 ()-141 Based on the Kirby-Bauer data, it appeared that the antibacterial properties are influenced by the bulk and polarity of the substituent located at the C 3 position of the -lactam. In this regard, it was hypothesized that MeO AcO > OH > PhO is the order by which the substituents affect the biological activity of N-methylthio -lactams. Table 3.06: Disc diffusion data for various N-thiolated -lactams. Species/Straina 116 117 119 127 134 135 137 141 PENc VANd MSSA ATCC 25923 20 15 18 27 15 15 22 19 33 MRSA USF652 18 21 18 28 18 15 22 15 8 19 MRSA USF653 19 20 20 28 15 11 25 23 15 18 MRSA USF654 19 20 22 27 15 11 20 18 10 19 MRSA USF655 17 19 18 29 14 12 19 20 14 19 MRSA USF656 18 19 19 28 16 14 23 19 12 21 MRSA USF657 19 19 20 26 13 11 21 19 12 18 MRSA USF658 18 16 15 26 15 14 21 18 19 18 MRSA USF659 18 19 20 24 16 11 20 15 16 18 S. epidermidis 28 16 16 26 50 S. saprophyticus 20 14 13 19 30 S. simulans 0 13 0 13 13 M. luteus 15 20 20 20 40 N. gonorrhoeae 11 7 10 15 13 aUSF652-659 were obtained from Lakeland Regional Medical Center, Lakeland, FL. bKirby-Bauer well diffusion on TSA. cBenzylpenicillin potassium salt (penicillin G) dVancomycin hydrochloride. 3.8 Antifungal Properties of N-Thiolated -Lactams N-Thiolated -lactams demonstrated unique antifungal properties against two members of the genus Candida. The compounds were tested by well diffusion on yeast-nitrogen base agar at 50 g quantities. As a control, clotrimazole at the same amount was tested as a gauge for percent activity. The antifungal performance for various N-methylthio -lactams is depicted in the charts below. Against C. albicans and C. tropicalis, a discernable structure-activity profile could be established for the C 4 phenyl substituted analogues. This contrasted the SAR studies for bacteria where few correlations could be detected. The most apparent relationship that could be ascertained for lactams possessing anti-Candida properties was that they all contained a haloaryl residue. The phenyl (67), tolyl (75), and 2-nitrophenyl (77) were completely devoid of activity suggesting that ring substitution has a critical role in drug delivery or in binding to the biological target. The position of the halogen appeared to have little effect on the in vitro perfomance of the lactam. The monosubstituted -lactams were equally effective when the halogen was located ortho, meta, or para, although the m-chorophenyl analogue (70) was oddly found to be inactive against both Candida species (Figures 3.12, 3.11). Z one Growth Inhibition (m) b of m 53

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Figure 3.12: Comparision of antifungal activities against C. albicans. Another effect that was not observed against bacteria was the enhancement of biological activity when the aryl ring contained multiple chlorine atoms. The efficacy of the 97 and 99 to inhibit fungal growth was 15-40% greater than the monosubstituted lactams 68 and 69. Figure 3.13: Comparision of antifungal activities against C. tropicalis. N-Sulfenylated -lactams 101-109 were also screened against the Candida species and as expected, the activity diminished when the size of the S-substituent increased (data not shown). 3.9 Antiviral Properties of N-Thiolated -Lactams In collaboration with Professor Erik De Clercq's laboratory114 at the Rega Institute for Medical Research in Leuven, Belgium, the antiviral properties of lactams 1, 69, and 75 were assessed in preinfected human HeLa (cervix carcinoma), HEL (human embroyonic lung), and Vero (African green monkey kidney) cell cultures. Prior to screening, cytotoxicity measurements were determined for the 3 cell lines and recorded as the minimum cytotoxic concentration to induce microscopic alterations in normal cell morphology (MCC). The antiviral performance of the antibiotics were subsequently assayed and expressed as the minimum concentration to reduce cytopathogenicity by 50% (MIC 50 ). The antiviral activitites of the -lactams against vesicular stomatitis (rhabdovirus), coxsackie (enterovirus), and respiratory syncytial virus (pneumovirus) in HeLa cells is shown in Table 3.07. For comparison, the antivirals brivudin (nucleoside mimic), (S)-DHPA (S-adenosylhomocysteine hydrolase inhibitor), and the ribavarin (reverse transcriptase inhibitor) were included as controls. None of the lactams 54

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demonstrated inhibitory activities against the 3 viruses. Compounds 1, 69, and 75 were ineffective at subtoxic concentrations greater than 80 g/ml in HeLa cells whose MCC value was >400 g/ml. Table 3.07: Cytotoxicity and antiviral activity of N-methylthio -lactams 1, 69, and 75 in HeLa (cervix carcinoma) cell cultures. Compound MIC 50 b MCCa Vesicular Coxsackie Respiratory stomatitis virus Virus B4 syncytial virus 1 400 >80 >80 >80 69 400 >80 >80 >80 75 400 >80 >80 >80 Brivudin >400 >80 >80 >80 (S)-DHPA >400 >80 >80 >80 Ribavirin >400 >80 >80 >80 aminimum cytotoxic concentration (g/ml); bminimum inhibitory concentration (g/ml). Similarly, herpes simplex virus-1 (HSV-1, strain KOS), HSV-1 (strain TKKOS ACVr), HSV-2 (strain G), vaccinia virus, and vesicular stomatitis virus were insensitive to N-methylthio -lactams at concentrations >16 g/ml in HEL cells (Table 3.08). In Vero cells, the compounds were also inactive against parainfluenza type 3 virus, reovirus type 1, Sindbis virus, Coxsackie B4 virus, and Punta Toro virus at MICs >16 g/ml (Table 3.09). Table 3.08: Cytotoxicity and antiviral activity of N-methylthio -lactams 1, 69, and 75 in HEL (Human Embryonic Lung) cell cultures. Compound MIC 50 b MCCa HSV-1 HSV-1 TKHSV-2 Vaccinia Vesicular (KOS) KOS ACVr (G) virus stomatitis virus 1 16 >16 >16 >16 >16 >16 69 16 >16 >16 >16 >16 >16 75 16 >16 >16 >16 >16 >16 Brivudin >400 0.0256 80 >400 3.2 >400 Ribavirin >400 >400 >400 >400 16 240 ACG >400 0.384 9.6 0.384 >400 >400 DHPG >100 0.0038 0.48 0.0064 >100 >100 aminimum cytotoxic concentration (g/ml); bminimum inhibitory concentration (g/ml). Table 3.09: Cytotoxicity and antiviral activity of N-methylthio -lactams 1, 69, and 75 in Vero cell cultures. Compound MIC 50 b MCCa Parainfluenza-3 Reovirus-1 Sindbis Coxsackie Punta Toro virus virus virus B4 virus 1 400 >80 >80 >80 >80 >80 69 400 >80 >80 >80 >80 >80 75 80 >16 >16 >16 >16 >16 Brivudin >400 >400 >400 >400 >400 >400 (S)-DHPA >400 48 48 >400 >400 >400 Ribavirin >400 80 9.6 >400 >400 16 aminimum cytotoxic concentration (g/ml); bminimum inhibitory concentration (g/ml). 55

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56 CHAPTER IV MODE OF ACTION OF N -THIOLATED -LACTAMS 4.1 Introduction Antibiotics can be grouped by at least 3 classification schemes: (1) narrow-spectrum vs. broadspectrum, (2) bacteriostatic vs. b actericidal, and (3) intracellular vs extracellular. In chapter III, N -thiolated -lactams were identified as bacteriostatic agents po ssessing a narrow range of activity which implied that the biological target is internalized. Cytostatic agents usually evoke arrest of intracellular processes involving biosynthetic pathways through enzyme or secondary metabolite modification. Conversely, antibiotics whose function is to weaken the outer periphery of cells (eg, cell wall and plasma membrane) are in most cases, cytocidal ( Table 4.01 ). Based on the attributes of existing antibacterials, N -thiolated lactams were believed to act in a different manner from traditional -lactam antibiotics. The studies presented in this chapter examine the l actam's effect on cellular processes in S. aureus in an effort to define the mechanism of action. Table 4.01: Important classes of antibiotics and ther e respective intrinsic characteristics. representative bacteri activity cellular cell process member(s) ostatic, cidal spectrum target affected I. extracellular a) -lactams penicillins -cidal broad transpeptidase cell wall cephalosporins b) glycopeptides vancomycin, -c idal narrow crosslinkages cell wall teicoplanin in cell wall c) polymyxins polymyxin B -cidal narrow phospholipids plasma membrane d) bacitracin bacitracin A -cidal narrow lipid carrier cell wall II. intracellular a) quinolones Cipro -cidal broad topoisomera se DNA synthesis b) ansamycins Rifadin -cidal broad RNA mRNA synthesis (rifampicin) polymerase c) macrolides erythromycin -ost atic narrow 50S ribosome translocation d) aminoglycosides streptomyc in, -cidal broad 16S rRNA initiation gentamicin e) tetracycline minocylin -o static broad 30S ribosome tRNA binding f) chloramphenicol Chloromycetin -ostatic broad peptidyl tRNA transferase charging g) oxazolidinone Zyvox -ostatic narrow 50S ribosome initiation 4.2 Probing the Modes of Action

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There are at least 3 types of reactions that can occur between N-methylthio-lactams and a biological nucleophile (Nu ) (Scheme 4.01). For the -lactam class of molecules, it is naturally assumed that nucleophilic addition occurs on the carbonyl center of the ring. As in the case of penicillins and cephalosporins, a serine residue within the active site of cell wall peptidases adds to the lactam, disabling the enzyme. Chemical studies of N-thiolated -lactams have shown, however, the ring to be less prone to nucleophilic ring opening such as hydrolysis, therefore other types of reactivity were considered. Scheme 4.01: Possible reactions of N-thiolated -lactams. sulfenylationN O +Nu-SCH3R O A r alkylationN S O +Nu-CH3Ar RO acylationN S CH3 Nu O Ar RO N S CH3 O Ar Nu RO Whereas the least reactive substituents are located at the C 3 (-OR) and C 4 (-Ar) positions of the -lactam, the sulfenyl moiety is the most vulnerable to nucleophilic attack. Alkylation is one manner by which the cellular target might be chemically altered by the S-methyl residue. Most alkylating drugs react with nucleotides to inhibit DNA replication or compromise the integrity of the super helices. Examples of DNA alkylating agents include leinamycin and mitomycin. The S-methyl moiety could as well transfer to the target to induce an inhibitory response. These 3 pathways will be discussed in sections 4.2-4.4 presenting the evidence for or against each reaction type. 4.3 N-Thiolated -Lactams as Acylating Agents Deactivation of the penicillin-binding proteins (eg, transpeptidase), is the only mechanism for which -lactam antibiotics are known to eradicate bacteria. A serine residue in the active site of the PBPs adds to the carbonyl center of penicillins and cephalosporins, forming a stable ester linkage (Scheme 4.02). The addition reaction occurring on the hydroxyl of serine is enhanced by ring strain and a carboxylic acid moiety located in proximity to the -lactam carbonyl. Situated at the -C of the lactam nitrogen, the acid functionality increases the electrophilicity of the carbonyl carbon. 51 Repositioning or eliminating the carboxylate negates the ability of the antibiotics to irreversibly bind to the PBPs. Scheme 4.02: Acylation of penicillin-binding proteins (HO-PBP), a serine hyrdrolase, by penicillin. 51 N S O CO2H RHCONH N S CO2H R H C O N H O PBP-O HO-PBP The lack of an acid functionality in the N-methylthio-lactams suggests that attack on the carbonyl carbon does not occur. Chemical studies of lactam 68 (Figure 4.01) in buffered media verified the ring's stability towards equimolar amounts of potassium hydroxide and serine. In addition, the compound was unreactive to commercial -lactamases (eg, serine hydrolases from B. subtilis) and did not thwart the enzyme's ability to hydrolyze penicillin G to the corresponding -amino acid. However, to prove 57

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unequivocally that the N-thiolated -lactam was not inhibiting the PBPs, light and electron microscopy were used to probe for damage or thinning of the cell wall in S. aureus treated with high concentrations of lactam 68. Figure 4.01: N-Protio -lactam 53 and N-methylthio -lactam 68. N O X CH3O Cl H H ()-53 X=H ( ) -68X=SCH 3 Section 4.3.1 Scanning Electron Microscopy Scanning electron microscopy (SEM) is a technique that can be used to examine cell morphology at >10,000 magnification. Bacteria exposed to antibiotics that disrupt the cell wall (ie, -lactams) or cytoplasmic membrane (ie, polymyxins) can be observed by SEM for physiological damage elicited by the drugs. Cultures of S. aureus inoculated with lactam 68 and penicillin G were inspected by SEM for changes in cell size and appearance in comparison to a culture with no antibiotic. The samples were prepared from Kirby-Bauer diffusion plates by incision of the agar along the outer zones where bacterial growth is inhibited and sublethal doses of the drugs are present. The advantage of preparing the samples from agar cultures as opposed to broth is that a concentration gradient is produced as the antibiotics difuse through the solid media, allowing microscopic examination among a range of exposure amounts. Figure 4.02: Scanning electron microscopy of S. aureus cultured with (a) no antibiotic, (b) lactam 68, and (c) penicillin G. Results of the electron microscopy experiments are depicted in Figure 4.02a-c. The first image portrays the appearance of S. aureus grown with no antibiotic present. In its natural state, staphylococci grow to about 1 m in diameter and reproduce in clusters of spherical-shaped cells. Cultures treated with lactam 68 also resembled S. aureus in its natural state, appearing spherical and uniform with no apparent deformities. When exposed to penicillin G, though, the bacteria were no longer uniform in size and shape (Figure 4.02c). The cocci appeared wrinkled, concaved, and often sheared resulting from deterioration of the cell wall. This was the first substantial evidence that N-thiolated -lactams do not inhibit bacterial transpetidases nor cause rupturing of the cytoplasmic membrane in staphylococcus. Section 4.3.2 Light Microscopy 58

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The inhibition of cell wall biosynthesis in Gram-positive bacteria can be detected by standard light microscopy. Using the Gram-stain technique, bacteria are able to be distinguished by the thickness (eg, crosslinked peptidoglycan content) of their cell walls. The appearances of S. aureus exposed to (a) no antibiotic, (b) lactam 68, and (c) penicillin G after staining is shown below. In its natural state, staphylococcus produces a thick cell wall which retains the crystal-violet stain upon decolorization with 95% ethanol (Figure 4.03a). When the bacterium is treated with a peptidase inhibitor, though, the Gram stain is lost due to thinning of the peptidoglycan shell. The staphylococci appear pink or red under the light microscope as a result. Figure 4.03c reveals the effect of penicillin G on the assimilation of the bacterial cell wall. The vast majority of cells manifested the staining characteristics of Gram-negative bacteria as a consequence of diminished peptidoglycan incorporation. The limited number of staphylococci that did retain the Gram stain in all probability came from the original inoculum before exposure to penicillin G. Mature bacteria with intact cell walls are not affected by -lactam antibiotics and can be Gram stained. Figure 4.03: Light microscopy of S. aureus exposed to (a) no antibiotic, (b) lactam 68, and (c) penicillin G. 59 (a) (b) (c) The effect on cell wall synthesis when S. aureus was treated with lactam 68 is shown in Figure 4.03b. It was quite apparent that all of the bacteria had completely intact cell walls. The staphylococci retained the crystal-violet stain indicating a high level of incorporated peptidoglycan. Based on these findings, it was concluded that N-thiolated -lactams do not inhibit the formation of bacterial cell walls. 4.3.3 Model Membrane Studies While N-thiolated -lactams do not appear to inhibit formation of the cell wall, further proof was needed to establish that the compounds are not extracellular antibiotics. Microscopy experiments revealed that lactam 68 does not cause staphylococcal cells to rupture due to deterioration of the cell wall or plasma membrane. Additional evidence, however, was sought to confirm that the integrity of the plasma membrane was not being altered by the compounds. The lipophilic nature of N-thiolated -lactams might confer "detergent-like" properties enabling incorporation into biological membranes. To determine if lactam 68 can destabilize a phospholipid bilayer, model studies were performed in collaboration with Dr. Pavel Grigoriev 105 on black lipid membranes (eg, poreless membranes) prepared from commercially available long chain fatty acids in a nonpolar medium. Incorporation was monitored by electrical conductance of the lipid bilayers; increased conductivity and subsequent disintegration of the bilayer signified a disturbance in the membrane. For the initial study, conductivity measurements indicated that lactam 68 at concentrations up to 15 g/ml (0.06 M from a DMSO stock solution) did not have an effect on the stability of the bilayer. These findings demonstrate that the lactam did not actively absorb into the interface of the hydrophobic membrane. Poron formation, the cause for the observed changes in conductance, would have resulted if

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absorption had occurred. Lactam 68 was also assayed with a membrane containing model channels (porons) formed by amphotericin B. A change in conductance was observed for this anion-selective, porous bilayer, though, further experiments were needed to confirm these results. In addition, this ongoing collaboration will next examine the effect N-thiolated -lactams possessing longer alkyl side chains for added lipophilicity. 4.4 N-Thiolated -Lactams as Alkylating Agents The ability of lactam 68 to function as a biological alkylator was next examined. Most antibiotics from the family of bioalkylating drugs have nucleic acids as a common cellular target. Hence, investigations of the methylation pathway focused on the interaction of N-thiolated -lactams with supercoiled DNA and their effect on DNA strand replication. Consideration of the alkylation mechanism derived from the in vivo use of methionine in cells to alter the structure and function of proteins, nucleic acids, and phospholipids. Scheme 4.03 depicts the manner in which substrates are methylated by methionine in bacteria. Enzyme-dependent sulfenylation of adenosine with L-methionine generates S-adenosylmethionine which selectively dealkylates in the presence of a methyl transferase and complimentary substrate. Scheme 4.03: Methylation of a nucleophilic substrate from the conversion of L-methionine to S-adenosylmethionine. adenosineSAM synthaseATPNN NN NH2 O OH OH S H3C NH3 O2C Numethyl tranferaseNu-CH3 S -adenosy l m ethionine O2C NH3 CH3S Lm ethionine The clinical application of DNA alkylators is in cancer chemotherapy. Leinamycin 107 and mitomycin C are examples of antitumor drugs which alkylate guanosine residues in helical DNA (Scheme 4.04). Chemical modification of DNA bases can lead to strand breakages with apoptotic consequences. With the possibility that N-thiolated -lactam could be a new member in the drug family of DNA alkylating agents, the anticancer properties of the compounds were investigated by researchers at H. Lee Moffitt Cancer Research Institute. Scheme 4.04: Alkylation of guanosine by leinamycin and mitomycin C. NH SS O O NS CH3 O HO O H3C (CH2)2 HNN NN O H2N dR NH O NS C H3 S O CO2H CH3 OH OH CH3 leinamycin GSH or CSHguanosinedR = deoxyribose; GSH = glutathione; CSH = cysteine. 60

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N O O N CH3 NH2 H3CO H2NCO2 N O O H2N CH3 NH2 mitomycin CNHN N O NH N dR guanosineH2NCO2 4.4.1 Anticancer Properties of Lactam 68 26d N-Methylthio -lactam 68 demonstrated unique anticancer properties against human leukemic and solid tumor cell lines. In Jurkat T (leukemia) cells, the lactam was found to inhibit cellular mitosis and to induce apoptosis through (1) p38 mitogen-activated protein (MAP) kinase activation, (2) mitochondrial cytochrome c release, and (3) caspase activation. Activation of apoptosis was measured by the proficiency of caspase-3 to cleave poly(ADP-ribose) polymerase (PARP) in lysed Jurkat T cell extracts following treatment with lactam 68 (50 M). Apoptotic commitment was observed after 4 hrs of exposure based on the fragmentation of PARP to p38. At this point, the ratio of viable to nonviable cells was 5:1. After 24 hrs, the nonviable population increased to 60% as determined by a trypan blue exclusion assay. A rise in the S-phase population was also detected for Jurkat T cells treated with lactam 68. Pulse-labeling experiments with 3 H-thymidine confirmed that cells stalled in the S-phase had reduced DNA replicating abilities. TUNEL assays further revealed that the attenuation of DNA biosynthesis was due to strand breakages occurring prior to the attainment of S-phase. 4.4.2 DNA Cleavage Experiment Though substantial anticancer properties were demonstrated by lactam 68, it was unknown if the reduced viability of the cancer cells was instigated by chemical modification of DNA. Further experiments were required to verify if N-thiolated -lactams are functional as nucleotide alkylators. A cell-free study was performed to detect whether a plasmid treated with an N-methylthio -lactam could cause strand breakage or reduced torsion of supercoiled DNA. Plasmid pBR322 (0.5g; Sigma Biochemicals) was incubated with lactam 68 at 37C in sodium phosphate buffer (50 mM; pH 7.2) for 24 hrs and analyzed for fragmentation or linearization by agarose gel electrophoresis containing 1% ethidium bromide. Figure 4.04: Supercoiled DNA treated with lactam 68 at 5-100 M. Plasmid pBR322 (0.5g) was incubated with lactam 68 at 37C in sodium phosphate buffer (50 mM, pH 7.2) for 24 hrs and analyzed by agarose gel electrophoresis (ethidium bromide staining). Lane 1: marker. Lane 2: pBR322. Lane 3: pBR322 + DMSO. Lane 4. pBR322 + 5 M 68. Lane 5: pBR322 + 10 M 68. Lane 6: pBR322 + 25 M 68. Lane 7: pBR322 + 50 M 68. Lane 8: pBR322 + 100 M 68. Lane 9: marker. 61 The initial study examined the effect of lactam 68 on DNA integrity at elevated concentrations (5-100 M). Results of this experiment are depicted by the gel in Figure 4.04. In lanes 2 and 3, two bands were observed for the plasmid in the absence of lactam 68. With the supercoiled pBR322 giving rise to the

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denser band, a weak band of relaxed or "nicked" DNA was formed by the electrical current and passage through the gel. Lanes 3-8 contained plasmid samples treated with various concentrations of lactam 68. It was evident that the compound did not cause fragmentation or relaxation of the super helix. The bands were equivalent in location and illumination to those observed for the untreated plasmids (lanes 2,3) thus indicating that chemical modification of the DNA did not occur with 5-100 M of the lactam present. Figure 4.05: Supercoiled DNA treated with lactam 68 in the presence of thiols. Plasmid pBR322 (0.5g) was incubated with lactam 68 at 37C in sodium phosphate buffer (50 mM, pH 7.2) for 24 hrs and analyzed by agarose gel electrophoresis (ethidium bromide staining). Restriction digests conducted with EcoR1 were performed at 37C for 1 hr. Lane 1: pBR322 + 100 M 68. Lane 2: pBR322 + 100 M glutathione. Lane 3: pBR322 + 100 M glutathione + 100 M 68. Lane 4: pBR322 + 100 M DTT. Lane 5: pBR322 + 100 M DTT + 100 M 68. Lane 6: pBR322 + 100 M 2-mercaptoethanol. Lane 7: pBR322 + 100 M 2-mercaptoethanol + 100 M 68. Lane 8: linearized pBR322 (EcoR1digest). Lane 9: pBR322 + EcoR1 + 100 M 68. Lane 10: pBR322 + DMSO. Lane 11: pBR322 + EcoR1 + DMSO. DNA alkylators are often in a prodrug form requiring chemical or enzymatic activation before the nucleotide addition can occur. Leinamycin, for example, is transformed into a potent alkylating antibiotic by a thiol-mediated reaction with cysteine or glutathione. 109 Cells susceptible to leinamycin possess thiol-rich intracellular environments which may also be needed for N-thiolated -lactams to alkylate its cellular target. A second study was performed to examine the stability of plasmid pBR322 (5 M) after treatment of lactam 68 (100 M) with various thiols (100 M) (Figure 4.05). In lane 1, the plasmid following 24 hr exposure to lactam 68 is shown. As expected, the supercoil did not relax or linearized with the antibiotic present. DNA samples loaded in lanes 2-7 were incubated with glutathione (lane 2,3), dithiotreitol (DTT; lane 4,5), or -mercaptoethanol (lane 6,7) as possible activators of 68. Samples with lactam also added, are located in lanes 3,5, and 7. After 24 hrs, the superhelix of the DNA appeared unaltered by the combination of 68 and equimolar amounts of glutathione, DTT, or -mercaptoethanol. The bands of lanes 2-7 were comparable in illumination and location to the antibiotic-free plasmid loading (lane 10). To verify the cleavability of the plasmid, pBR322 was linearize with the endonuclease, EcoR1 (Sigma Biochemicals). Samples containing the digested DNA are located in lanes 8 and 11. A loading comprised of lactam 68 and EcoR1 was applied to lane 9 to establish if the compound could function as an endonuclease inhibitor. Not surprisingly, linearization of the supercoil DNA was unhindered by the N-methylthio -lactam. 4.4.3 Pulse-Labeling Studies of DNA Replication Disruption of the super helix by chemical modifications of nucleotide bases can cause the arrest of DNA replication. To further substantiate that N-thiolated -lactams are not alkylating drugs or affect nucleotide assimilation into double-stranded DNA, pulse-labeling experiments with 3 H-thymidine were performed to monitor DNA replication in bacteria. 110 Protocols of the labeling experiment were as follows: cultures of S. aureus grown to early logarithmic phase (10 4 cfu/ml) were inoculated with 3 H-thymidine (3 Ci/ml) and lactam 68 (20 g/ml in DMSO from a 1 mg/ml stock; 2x MIC), penicillin G (2 g/ml; 2x MIC), or the inhibitor of DNA replication, ciprofloxacin (30 g/ml; 2x MIC). A sample containing only DMSO (20l/ml) was used as a control in the study. Aliquots of 50 l were removed at the appropriate time intervals and precipitated in trichloroacetic acid (TCA). Incorporation was measured by scintillation counts following filtration of the TCA precipitated material on glass fiber filters. 62

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The labeling experiments revealed that lactam 68 has no effect on DNA replication in staphylococcus (Figure 4.06). Figure 4.06a depicts thymidine incorporation as a percent of the control for the three antibiotics. As expected, ciprofloxacin (CIP) was the most potent, inhibiting 65% of thymidine utilization after 30 mins. Conversely, with lactam 68 and penicillin G (PEN) present, 90% of the nucleotide was converted into DNA. The plot in Figure 4.06b further details the rate of thymidine uptake over a period of 1 hr. Again, ciprofloxan caused almost a complete and immediate cessation of DNA replication in S. aureus. Lactam 68 appeared not to affect thymidine incorporation up to 30 mins. Afterwards, the utilization began to temper most likely due to the halt of bacterial reproduction (see Section 3.3.5 for growth studies). Figure 4.06: Thymidine incorporation in S. aureus. 4.5 N-Thiolated -Lactams as Thiolating Agents N-Thiolated -lactams were discovered to have similar sulfenylating properties to the sulfur transfer reagent (20, 21) involved in their preparation. An adjacent electronegative atom enhances the electrophilicity and vulnerability of the sulfurs toward nucleophilic attack (Scheme 4.05). Chemical studies of lactam 68 revealed that the most reactive substances towards 68 are those containing a sulfhydryl group. In buffered media, the S-methyl was rapidly cleaved by cysteine, glutathione (GSH), 1,4-dithio-treitol (DTT), and -mercaptoethanol. Scheme 4.05: Electrophilic sulfenylation of nucleophiles. N S CH3 O RO Ar E l ect r on deficeint N O +Nu-SCH3RO Ar CH3S S O O CH3 N O O S CH3 2021Nu 4.5.1 Enzyme-Binding Properties of N-Thiolated -Lactams Based on the preceding investigations, N-thiolated -lactams appear to inhibit staphylococcal growth by S-methyl transfer to an unidentified protein or metabolite. Chemical studies involving lactam 68 and the 20 L-amino acids comprising proteins revealed that cysteine is the only component of proteins that reacts with the lactam under physiological conditions. The products of the reaction were presumably S-methyl-cystine (142) and N-protio lactam 53 as depicted below. 63

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Scheme 4.06: Reaction of cysteine and lactam 68. NH3 SH O O cysteineN O SCH3 NH3 S O O SCH3 pH 7.2 CH3O Cl +N O CH3O Cl + 6853142 O N O NH3 O SH H O N g l utathioneO O H To evaluate the in vitro effect of cysteine on the anti-staphylococcal properties of N-thiolated -lactams, equimolar amounts of the cysteine-containing tripeptide, glutathione (see Scheme 4.06) and lactam 68 were added to the same well in a Kirby-Bauer diffusion experiment. Following overnight incubation, the plate inoculated with S. aureus was examined for growth inhibition. A zone of inhibition was absent for the well containing lactam 68 and glutathione. The antagonistic effect conferred by the tripeptide could explain the selectivity observed in the activity spectrum of N-methylthio -lactams. To further substantiate the neutralizing effect of glutathione, a plate was prepared with a 1 mg reservoir of the tripeptide located at the center and three surrounding wells containing 20 g of the monochlorophenyl N-methylthio -lactams 68-69 (Figure 4.07). The ability of glutathione to protect the bacteria from the antibiotics was again clearly evident by the presence of concaved zones between the glutathioneand lactam-containing wells. At the dimpled region of the zones, the concentration of glutathione was sufficient to deactivate the lactams allowing proliferation of the staphylococci. The protective role of glutathione likely explains the bacterial selectivity of the antibiotics. Species demonstrating the highest sensitivity to the lactams may have thiol-deficient intracellular environments. To determine if a correlation between the sulfhydryl content and biological activity exists, thiol concentrations in susceptible and nonsusceptible bacteria were measured. Figure 4.07: Antagonist effectof glutathione (GSH) on the anti-MSSA properties of N-thiolated -lactams. 4.5.2 Thiol Determination in Bacteria The concentration of sulfhydryl-containing molecules in bacteria was quantitated with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). 111 The reaction between the reagent and the thiol extracts results in cleavage of the disulfide bond and generation of the fluorophore, 2-nitro-5-thiobenzoate anion (Scheme 4.07). Optical density measurements (OD 412 ) were then taken of the samples and used to calculate the relative amount of thiols contained in the cells. 64

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Scheme 4.07: Reaction of Ellman's reagent and a thiol. 111a S S N O2 O2N COO COO S RSS NO2 O2N COO COO +Ellman's reagent2-Nitro-5-thiobenzoate anion ( f l uo r escence-act i ve)RSH The intracellular sulfhydryl content of eight bacterial species with known susceptibilities to N-methylthio -lactam 68 is depicted in Table 4.02. It was evident from the data that the thiol levels in bacteria were inversely proportional to the sensitivities the organisms displayed toward the antibiotic (Figure 4.08). The greatest concentration was discovered in E. coli which has been recognized in a previous report as a bacterial species maintaining a thiol-rich intracellular environment to guard against oxidants. # N-Thiolated -lactams are entirely devoid of microbiological activity against E. coli and these data suggests that the microbe is protected by its proficiency to produce low molecular weight thiols. 65 A C o (mM) zone (mm) Bacillus anthraces 0.239 0.04 25 Staphylococcus aureus 0.288 0.04 28 Bacillus megaterium 0.312 0.05 21 Bacillus cereus 0.412 0.07 20 Bacillus subtilis 0.475 0.08 19 Bacillus globigii 0.566 0.09 20 Bacillus niger 0.569 0.09 19 Escherichia coli 1.098 0.18 0 Table 4.02:Correlation between relative intracellular thiol levels and susceptibility to lactam 68. Susceptible bacteria were found to have 50% the total thiol content in E. coli. The lowest amounts were found in B. anthraces and S. aureus who also displayed the highest sensitive to lactam 68. Additional species of Bacillus that displayed susceptibilities were also low producers of sulfhydryl compounds, although, the levels were slightly higher than those observed for B. anthraces and S. aureus. The zone sizes were smaller for these bacteria as well, with the implication that the elevated thiol content was responsible for reduced bioactivity. Figure 4.08: Zone diameter of lactam 68 compared to relative thiol levels in select bacteria.

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Though a correlation between sulfhydryl content and susceptibility to N-methylthio -lactams could be established, the selectivity in all likelihood involves other factors. The existence, amount, and accessibility of the biological target(s) are additional factors that would influence the antimicrobial potency of the lactams 4.5.4 1 H NMR Studies NMR studies were conducted to verify that the N-protio lactam is the only byproduct between the interaction of N-methylthio -lactams and staphylococcal cells. The product was recovered by ethyl acetate extraction of an overnight broth culture of S. aureus inoculated with lactam 68. The extracts were combined, concentrated, and analyzed by NMR. The 1 H NMR spectrum of the crude isolated residue revealed the clean formation of the N-protio -lactam 53 as represented by the broad singlet positioned at 6.97 ppm (Figure 4.09b). The loss of the S-methyl substituent was also confirmed by the absence the methyl proton which would give rise to a signal at 2.41 ppm (Figure 4.09a). Although no attempts were made to identify the location of thiotransfer, the evidence from this and prior experiments suggest that the S-methyl is cleaved by a protein or metabolite following entry of the -lactams into the cytoplasm. The N-protio lactam byproduct is subsequently secreted into the extracellular environment where in itself does not possess antibacterial properties. Further studies involving fluorescently-labeled or radiolabeled N-thiolated -lactams are needed, however, to verify this proposed mechanism. Figure 4.09: 1 H NMR spectrums of (a) lactam 68 prior to inoculation of S. aureus and (b) lactam 68 following inoculation of S. aureus. 4.6 Effects of Lactam 68 on Gene Expression 4.6.1 Introduction Gene expression is a biological process targeted by many families of antibiotics including: tetracyclines, macrolides, aminoglycosides, streptogramins, and oxazolidinones (see Table 4.01). In most cases, drugs that affect transcription or translation are bacteriostatic suggesting that one of these processes may be subject to inhibition by N-thiolated -lactams. To test this hypothesis, incorporation of 3 H-uridine and 3 H-isoleucine into RNA and proteins, respectively, was measured in a broth culture of S. aureus treated with lactam 68. 66

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4.6.2 Pulse-Labeling Studies of RNA Assimilation RNA labeling studies 110 with 3 H-uridine were conducted to assess the influence of lactam 68 on transcription in S. aureus. Cultures were grown to early logarithmic phase (10 4 cfu/ml) and inoculated with 3 H-uridine (3 Ci/ml) and lactam 68 (20 g/ml in DMSO from a 1 mg/ml stock; 2x MIC), penicillin G (2 g/ml; 2x MIC), or an inhibitor of RNA synthesis, rifampicin (2 g/ml; 2x MIC). A sample containing only DMSO (20 l/ml) was used as a control in the study. Figure 4.10 compares uridine uptake in cultures treated for 30 mins with the 3 antibiotics. As expected, penicillin G had only a moderate effect on RNA assembly into polynucleotide chains. The slight decrease was probably due to reduced cell counts associated with the bactericidal nature of the antibiotic. Conversely, rifampicin (RIF) clearly impacted the proficiency of S. aureus to transcribe RNA. The nucleotide usage was less than 20% of the control with the culture containing the RNA synthesis inhibitor. Lactam 68 was also found to modulate uridine incorporation though not to the extent of rifampicin. After 30 mins, the nucleotide usage was 50% of the control suggesting that transcription may not be the primary process targeted by the lactam. Frequently, inhibition of a single biosynthetic pathway will hinder subsequent cellular processes by a negative feedback mechanism. These responses are observed in bacteria exposed to translation inhibitors. When protein synthesis stalls, the cell recognizes the accumulation of aminoacyl-tRNA and activates a feedback inhibitor to disable DNA replication, RNA synthesis, and peptidoglycan incorporation into cell walls. This was observed in S. aureus for chloramphenicol which obstructed RNA synthesis after 10 mins of exposure to the peptidyl transferase inhibitor (data not shown). Figure 4.10: Uridine incorporation in S. aureus. 4.6.3 Pulse-Labeling Studies of Protein Synthesis To determine if the primary role of growth inhibition by N-thiolated -lactams is as a transcription inhibitor, the rate of protein synthesis was monitored by the uptake of 3 H-isoleucine. 110 The experiments were conducted in the same manner previously described in Section 4.3.3. An overnight culture of S. aureus was grown to early logarithmic phase (10 4 cfu/ml) and inoculated with 3 H-isoleucine (5 Ci/ml). A larger amount of the radiolabeled amino acid was required because of its lower utility compared to the DNA and RNA precursors. Isoleucine is 1 of 20 amino acids comprising proteins therefore the probability of its incorporation is less than thymidine and uridine. The cultures were then treated with lactam 68 (20 g/ml in DMSO from a 1 mg/ml stock; 2x MIC), penicillin G (2 g/ml; 2x MIC), or the translation inhibitor, chloramphenicol (30 g/ml; 2x MIC). A sample containing only DMSO (20l/ml) was once again used as a control. 67

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Figure 4.11: Isoleucine incorporation in S. aureus. Figures 4.11a and 4.11b compare the relative rates that mRNA was translated in the presence of lactam 68, penicillin G, and chloramphenicol (CHL). Penicillin G and chloramphenicol weakly inhibited protein synthesis in S. aureus after 30 mins compared to lactam 68 (Figures 4.11a) Based on these results, it was clearly evident that the lactam is a more powerful inhibitor of translation than transcription. The plot of Figures 4.11b further elaborates the propensity of the antibiotic to curtail polypeptide formation. For the initial 10 mins of the experiment, a linear progression of isoleucine incorporation was observed for the culture containing lactam 68. Afterwards, a large dissension in protein synthesis was detected. Neither in the DNA or RNA incorporation studies was such a dramatic decline observed. Moreover, cultures treated with the lactam demonstrated a steady increase of thymidine and uridine incorporation that eventually leveled off over time. It was apparent from these results that the primary mode of action of N-thiolated -lactams is to impede protein synthesis while transcription appears to be interrupted as a consequence of the translation blockage. 68

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CHAPTER V DISCUSSION, CONCLUSIONS, AND FUTURE DIRECTIONS 5.1 Discussion and Conclusions 5.1.1 Introduction Over the past 50 years, thousands of -lactam antibiotics have been discovered in nature and through synthetic means. The biological target common to all members in this chemical class of antimicrobials has been the membrane-bound transpeptidases and DD-carboxypeptidases. These enzymes, collectively referred to as penicillin-binding proteins (PBPs), recognize traditional -lactam antibiotics (ie, penicillins and cephalosporins) as the D-ala-D-ala residues in peptidoglycan (Figure 5.01). 55 In misrepresenting the normal substrates of PBPs, these antibacterials bind irreversibly, disabling the enzymes. Chemical structure comparison would suggest that N-methylthio -lactams (3) do not react with the peptidases of bacterial cell walls (Figure 5.01). They lack the D-ala-D-ala backbone and additional binding motifs required for reactivity with the PBPs. Figure 5.01: Chemical structures of penicillin and N-methylthio -lactams. N O NH S CO2 H O R R' N O SCH3 RO Ar penicillin3bonds in bold representtheD-ala-D-alasectionreco g n i zed b y P B Ps.H H Scientists have historically used the -lactams discovered in nature as models to create analogs with enhanced potency and expanded activity spectrums. Seldom have researchers strayed from the concept that -lactams must bind to a peptidase to function as an antibacterial. N-Methylthio -lactams are the first novel design of a -lactam having antimicrobial properties while not possessing PBP-binding capabilities. With an apparent intracellular mode of action, their activity spectrum contrasts to that of traditional -lactam antibiotics amid the inclusion of yeast and cancer. 5.1.2 Narrow vs. Broad Spectrum A narrow range of microbes demonstrated in vitro susceptibility to the inhibitory effects of N-methylthio -lactams. As illustrated in Table 5.01, bacterial species very sensitive to the lactams were represented by 3 genera: Bacillus, Micrococcus, and Staphylococcus. The vast majority of bacteria were not susceptible indicating high selectivity among the 48 species that were screened. The sporadic activity displayed by N-thiolated -lactams is unusual for an antimicrobial agent. Most antibiotics in clinical use possess broad spectrum activities despite being reserved for certain bacterial pathogens. 69

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Table 5.01: Susceptibility comparison of N-methylthio -lactams 67-81, 83, and 97-99. Bacteria Zone of Growth Inhibition (mm) (Species:Strains) Gram () >30 21-30 15-20 <15 0 Bacteriodes sp. (1:1) X X X Bacillus spp. (7:7) + X X X Enterobacter sp. (1:1) X Enterococcus spp. (6:6) + X X Escherichia sp. (1:3) X Fusobacterium spp. (6:16) 112 X Haemophilus sp. (1:3) X Klebsiella sp. (1:1) X Lactococcus sp. (1:1) + X X Listeria sp. (1:2) + X X Micrococcus sp. (1:1) + X X Mycobacterium sp. (1:1) X Neiserria sp. (1:2) X X X Peptostreptococcus sp. (1:1) 112 + X Porphyromonsa (1:2) 112 X Proteus sp. (1:1) X Pseudomonas sp. (1:1) X Salmonella sp. (1:1) X X Serratia sp. (1:1) X Sporobolomyces sp. (1:1) 113 X Staphylococcus spp. (10:21) + X X X X X Streptococcus spp. (2:2) + X X X Vibrio sp. (1:2) X X susceptibilities medium weak none high Broad spectrum antibacterials kill or inhibit a wide range (ie, genera) of Gram-positive and Gram-negative microbes exposed to the drugs. Antibiotics in clinical use that have been classified as broad spectrum include: aminoglycosides (ie, streptomycin, gentamicin), tetracyclines (ie, doxycycline), semi-synthetic penicillins (ie, ampicillin, amoxicillin), 2 nd and 3 rd generation cephalosporins, monobactams (ie, aztreonam), carbapenems (ie, imipenem), macrolides (ie, erythromycin), most quinolones (ie, ciprofloxacin), chloramphenicol, cycloserine, sulfonamides, and many others. Narrow spectrum bacteriocidal or bacteriostatic therapeutics are selective for either Gram-positive or Gram-negative bacteria. Glycopeptides (ie, vancomycin, teicoplanin), natural and semi-synthetic penicillins (ie, penicillin G), and oxazolidinones are used clinically to treat Gram-positive bacterial infections whereas polymyxin (ie, colisitin) and nalidixic acid are mainly effective for Gram-negative bacteria. N-Methylthio -lactams appear to have a limited spectrum of activity similar to the glycopeptides. Although they possess dissimilar modes of action and cytotoxicities, N-methylthio -lactam and glycopeptide antibiotics both inhibit the growth of Gram-positive bacteria deriving from the "Bacilli" class of medically important pathogens. 5.1.3 Bioactivity Spectrum N-Methylthio -lactams demonstrated a unique bioactivity spectrum that was distinct from any pre-existing class of drugs. Inhibitory effects were observed against bacterial, fungal, and cancer cells but lacking for viruses. In bacteria, the most susceptible cell-types to N-methylthio -lactams were Gram-positive. Gram-negative species displayed little or no sensitivities to the lactams with the exception of Neisserria gonorrhoeae which had modest susceptibility towards the antibiotic. Clearly though, the bacterial species most affected by the lactams derived from the genera of Staphylococcus. A broad range of bioactivities was achieved by the drugs against 10 species of staphylococci. MSSA, MRSA, S. epidermidis, 70

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and S. lugdunensis were consistently the most susceptible; S. lentus and S. simulans were consistently the least susceptible. The lactams were also effective against some species of Bacillus, most notably B. anthracis whose pathogenesis causes anthrax disease. The greatest surprise, though, were the bioactivities observed against eukaryotes, particularly yeast and cancer cells. Until the discovery of N-methylthio -lactams, never have antifungal or anticancer properties been observed for a member from the -lactam class of antibiotics. Moreover, their ability to obstruct the viability of eukaryotic pathogens further substantiates that the PBPs are probably not the primary biological target in bacteria. The potential therapeutic use of N-methylthio -lactams for treating mycosis and carcinoma warranted additional screening which are presently ongoing. 5.1.4 Structure-Activity Relationship Few structure-activity relationships were established for the N-thiolated -lactam analogues. In bacteria, changes to the N 1 C 3 and C 4 substituents did not expand the activity spectrum nor enhance the in vitro performance of the lactams against a particular class or species. Increasing polarity or lipophilicity, though, reduced biological activity for bacteria susceptible to the lead compound, lactam 1 (Figure 5.02). Analogues which demonstrated the greatest potency contained a C 4 aryl ring functionalized by a weak activating or deactivating group such as methyl or chlorine, respectively. The monochlorinated lactam 68 (Figure 5.02) gave the best overall activity against bacteria, yeast, and cancer. Analogues with multiple chlorine atoms, however, were the most effective inhibitors of yeast proliferation. Diand trichlorinated lactams demonstrated 15-40% greater activity than 68 and clotrimazole. Figure 5.02: Structure of N-methylthio -lactams 1 and 68. N O S C H3 CH3O Ph ()-1N O S C H3 CH3O H H H H Cl ()-68 5.1.5 Mechanism of Action N-Methylthio -lactams appear to inhibit protein synthesis as their primary mode of action. Substantiating this hypothesis were the results gathered from multiple chemical and biological experiments. Chemical studies demonstrated that the carbonyl carbon and the S-methyl carbon of the thiolated lactams are least vulnerable to nucleophilic attack which was an observation also reported for the thiamazins # (Figure 5.03). As illustrated in Figure 5.04, antibiotics that perform via an acylating (ie, penicillin) or alkylating (ie, mitomycin C) mechanism possess an electropositive center (+) at the sites of reactivity conferred by neighboring electronegative atoms. In the case of N-methylthio -lactams, the highly activated, electron-deficient sulfur center is the most reactive part of the molecule due to the adjacent amide functional (Figure 5.04). For this reason, the ability of the lactams to acylate or alkylate cellular components in bacteria was considered remote. Microscopy and DNA studies further validated the proposed chemical mechanism by demonstrating that the lactams do not inhibit cell wall synthesis through acylation of bacterial PBPs or alkylation of supercoiled DNA thereby causing linearization or disrupting DNA replication. Intracellular transfer of the S-methyl substituent appears to be the means which the viability of bacteria, yeast, and cancer cells is reduced. In bacteria, though, the lactams seem to have a cystostatic effect on growth which implies that the primary process targeted by the compounds is protein synthesis. 71

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Figure 5.03: Reaction of thiamazins at pH 11. 88 N O CH3 RNH S CH2CO2H thiamazinsHN S NH O NH O OH O ++ attack on carbonylattack on sulfurattack on carbonRNH RNH RNH CH3 C H 3 CH3 C H2 C O2H S=CHCO2HHOSCH2CO2Hminor productmajor productsminor productsHO Figure 5.04: Site of nucleophilic attack on electropositive centers of 3 antibiotics: penicillin, mitomycin C, and N-methylthio -lactam 68. N O SCH3 CH3O H H Cl ()-68N O O N CH3 NH2 H3CO H2NCO2 mitomycin CN S O CO2H RHCONH penicillinNu Nu Nu The vast majority of bacteriostatic antibiotics are inhibitors of gene expression including: aminoglycosides, tetracyclines, macrolides, and oxazolidinones. These antibacterials operate by slowing cell proliferation allowing time for the natural defenses of the body to eradicate the infectious bacteria. Reproduction and other biosynthetic processes in bacteria are usually suppressed when gene expression is blocked. This was observed in the incorporation studies with lactam 68 which showed that inhibition of protein synthesis in staphylococci caused a 50% reduction in RNA synthesis. Future studies will be dedicated to determining the precise stage at which protein assimilation is interrupted. 5.2 Future Directions 5.2.1 Structure-Activity Relationship To help define the intracellular target of N-methylthio -lactams, several key investigations were attempted or are, at present, being conducted. Additional SAR studies were performed to determine if the -lactam ring was required for bioactivity. Methylthiomesylate (20) and N-(methylthio)phthalimide (21) (Figure 5.05) were the initial compounds screened but found to be devoid of antistaphylococcal properties. Other molecules analogous to C 4 aryl substituted N-methylthio-lactams were, however, desired to ascertain if the azetidinone was a prerequisite for activity. Figure 5.05: Methylthiomesylate (20) and N-(methylthio)phthalimide (21). N O O SCH3 H3 CS O O S CH3 2021 72

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Preparation of azetidines from N-arylated and N-protio -lactams were the first compounds pursued in these studies. A general method of reducing the carbonyl carbon of -lactams was reported by Ojima 114 using monochlorohydroalane (AlH 2 Cl). As depicted in Scheme 5.01, the metal hydride was generated in situ from the reduction of aluminum trichloride with lithium aluminum hydride. N-Anisyl lactam 90 was then added to the stirring solution of AlH 2 Cl and the mixture was refluxed for 4 hrs. Following aqueous workup, azetidine 143 was isolated as a yellow oil in quantitative yield. Scheme 5.01: Reduction of N-arylated -lactam 90 to azetidine 143. AlCl3dry etherrefluxLiAlH4AlH2Cl dry etherrefluxN CH3O H H Cl Cl O C H3 ()-143N CH3O H H Cl Cl OCH3 ()-90O ()-90 Attempts to deprotect azetidine 143 with cerium ammonium nitrate were unsuccessful. The dearylated azetidine could not be obtained from the reaction as a result of oxidative decomposition. Efforts to reduce the carbonyl carbon of N-protio -lactams with AlH 2 Cl also failed; 1 H NMR spectra of the reaction following workup confirmed the presence of unconverted starting material. Scheme 5.02: Reduction of N-arylated -lactam 90 to azetidine 143. AlH2Cl dry etherrefluxN CH3O H H Cl Cl O C H3 N CH3O H H ()-55O ()-143H N CH3O H H H no product Ce(NH4)2(NO3)6aq. MeCN0oCCl Cl N H CH3O H H Cl Cl no product Despite the failed attempts to make N-methylthio azetidines, additional types of N-containing rings were explored. Azetidine-2-thiones (eg, thiolactams) were successfully prepared from C 4 aryl-substituted -lactams using 2,4-bis(4-methoxyphenyl)-1,3-dithiadiphosphonate-2,4-disulfide (Lawesson's reagent). 115 Lawesson's reagent (143) was synthesized by refluxing anisole and phosphorous pentasulfide in a 10:1 ratio (Scheme 5.03). 116 After 6 hrs, the yellow precipitate was filtered, washed with chloroform/ether, and dried in vacuo at room temperature to prevent polymerization of the reagent. 73

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Scheme 5.03: Preparation of Lawesson's reagent. OCH3 +P4S10 150oC, 6hSPSP CH3O OCH3 S S 143 N-Aryl protected -lactam 40 was then subjected to the thionation conditions using Lawesson's reagent (Scheme 5.04). Compounds 40 and 143 were stirred at approximately 50 o C for 4 hrs followed by silica gel chromatography to give thiolactam 144 as a yellow solid. With the -lactams proving stable to the reaction conditions, N-protio -lactams 53 and 54 were converted to their respective thiolactams 144 and 145. Attempts to prepare N-methylthio thiolactams were, however, unsuccessful. Scheme 5.04: Synthesis of thiolactams 144-146. N S CH3O H H N O CH3O H H ()-40 O C H3 O C H3 Cl Cl ( )-144 PhH, 50oC143 N S H CH3O N S S C H3 CH3O H H H H N O H CH3O H H ()-53 X=2-ClPhH, 50oC143()-145 X=2-ClX X X ()-146 X=3-Cl X ()-54 X=3-Cl Other analogous compounds that were explored included 2-methylene azetidines. Previously, these vinylogous carbamates have been generated from thiolactams either by an Eschenmoser sulfide contraction, 117 thio-Reformasky, 118 or thio-Wittig reaction. 119 Eschenmoser's method has been the most widely used and was first attempted on the N-arylated thiolactam 147 (Scheme 5.05). Ethyl bromoacetate and 147 were stirred at room temperature to generate the thioalkyliminium salt 148 as an intermediate (not isolated). 117c After 1 hr, triphenylphosphine was added to induce the sulfide contraction whereby, eliminating sulfur. However, the vinylgous amide 149 was not obtained, presumably due to ring strain or insufficient nucleophilicity of the thiocarbonyl. Scheme 5.05: Attempted synthesis of 2-methylene azetidines from thiolactam 147 via Eschenmoser sulfide contraction. ()-147N S PMP CH3O N PMP CH3O Ph3P, Et3NCl Cl H H H H N S PMP CH3O Cl H H BrCH2CO2Et dry MeCN, rtdry MeCN, rtno product()-148()-149 O O Et Br O OEt 74

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The thio-Reformansky was then tried with ethyl bromozincacetate on the N-protio thiolactam 146 (Scheme 5.06). 118 Again, the reaction produced unsatisfactory results. Conversion to the vinylogous azetidine was also attempted with ethyl diazoacetate and the stabilized ylide, (carbmethoxymethylene)triphenyl phosphine, but the desired product was not obtained (Scheme 5.06). Scheme 5.06: Unsuccessful syntheses of vinylogous azetidines from N-protio thiolactams. N CH3O H H S H Cl BrZnCH2CO2Etdry ether, refluxN CH3O H H H Cl O OEt ()-146()-150no product N S H CH3O H H Cl 1) N2CHCO2Et2) Ph3P, Et3NN CH3O H H H O OEt Cl ()-145()-151no p r oduct N S H CH3O Ph3P=CHAcPhH, refluxN H CH3O H H H H Cl Cl O ()-152no product()-145 Previous reports 119b have shown that thiolactams require an electron-withdrawing group on the amide nitrogen for the thiocarbonyl to have sufficient reactivity to nucleophilic attack. A common substituent allocated to the nitrogen atom of the thiolactam which can later be removed with hydrofluoric acid is an alkoxycarbonyl. Apropos, N-protio lactam 145 was acylated with di-tert-butylcarbonyl anhydride in the presence of catalytic base to generate N-Boc thiolactam 153 in quantitative yield (Figure 5.07). The thio-Wittig reaction was then attempted on the Boc activated compound, however, only starting material was recovered after 24 hr of reflux in benzene. Scheme 5.07: Unsuccessful attempts to synthesize vinylogous azetidines from N-Boc thiolactams. 75 PPh3=CHAcPhH, refluxN CH3O H H Cl O ()-153no p r oduct()-145N S H CH3O N S CH3O H H H Cl Cl (t-Boc)2ODMAP, MeCNO O ()-154O O

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Synthesis of an open ring S-methyl amide was also attempted via the sulfenylation of amide 155, prepared from the reaction of benzyl amine and methoyxacetyl chloride (Scheme 5.08). The sulfenamide 156, a compound analogous to N-methylthio -lactam 67, could not be obtained from 155 using N-methylthiophthalimide (21). Scheme 5.08: Unsuccessful synthesis of noncyclic thioamide 156. iPr2EtNHPhMe, 0oCN NH2 Cl O CH3O +O H CH3O N O S C H3 CH3O iPr2EtNHPhH, reflux21Benzylamineno p r oduct16155156 5.2.2 Elucidation of the Biological Target Elucidation of the biological target is the primary focus of current investigations. To ascertain the cellular binding site of the lactams, the S-methyl residue will be labeled with tritium which can be tracked inside the bacteria. The proposed synthetic route to the radiolabeled lactam is depicted in Scheme 5.09. Thioacetic acid (157) is alkylated with commercially available [ 3 H]methyl iodide then subsequently cleaved by mesyl chloride (158) under basic conditions to provide [ 3 H]methylthiomesylate (159). Afterwards, the mesylate shall be used to sulfenylate lactam 53 to give the [ 3 H]N-methylthio -lactam for the probing studies. Scheme 5.09: Proposed synthetic route to [ 3 H]-labeled lactam 160. H3C SH O IC(3H)3H3C SC(3H)3 O 1 eq NaOHMeOH1 eq NaOHMeOHClS O O CH3 SS O O CH3 C(3H)3 n-BuLi, THF-78oC53N O CH3O SC(3H)3 Cl H H 157158159160 The [ 3 H]S-methyl will be traced by fractionation of S. aureus into its subcellular components following treatment with 168. The flow diagram in Figure 5.04 depicts the manner by which the fractionation will proceed. 120 A 10 8 cfu/ml suspension of S. aureus in buffer will be treated with the [ 3 H]lactam for 30 mins. The labeled cells are then harvested, lysed, and fractioned by chemical means (Figure 5.04). Radiation levels will next be measured to determine if the S-methyl is bound to a metabolite, RNA, DNA, a protein, or a lipid. Afterwards, the fraction(s) containing the label can be further analyzed to resolve the molecular size of the biological target. 76

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Figure 5.06: Cell fractionation of S. aureus. Staphylococcus aureus cold trichloroacetic acid Small molecules Macromolecules methanol chloroform 77 In conclusion, this thesis has traced the development of the project from a curious and unexpected discovery of an N-methylthio -lactams having antibacterial properties to a potentially valuable class of new antibiotics. The remaining research and the focus of current investigations is to design additional analogues with enhanced potency (eg, lower MICs) and greater stability to glutathione. Furthermore, experiments to identify the cellular target will continue to be conducted to validate the novelty of these antibiotics and bring them to the forefront as a new weapon for the treatment of MRSA. Lipids RNA + DNA + Proteins NaOH RNA DNA + Proteins hot trichloroacetic acid DNA Proteins

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CHAPTER VI MATERIALS AND METHODS 6.1. Synthetic Procedures All reagents needed for the synthesis of N-thiolated -lactams were purchased from Sigma-Aldrich Chemical Company and used without further purification. Solvents were obtained from Fisher Scientific Company. Products purified by flash chromatography used J.T. Baker flash chromatography silica gel (40 m). NMR spectra were recorded in CDCl3. 13C NMR spectra were proton decoupled, but not fluorine decoupled, therefore, some signals in the spectra are split by J C-F-coupling. 6.1.2 Preparation of N-Anisylimines N-Anisylimines (6): Aldehyde (1 mmol), p-methoxyaniline (1 mmol), and 1 mg camphorsulfonic acid (cat.) were dissolved in dichloromethane and stirred at room temperature for 1 hr. Afterwards, an appropriate amount of magnesium sulfate was added and the mixture was filtered. The solvent was then removed in vacuo and purified by silica gel chromatography or recrystallized from methanol. N H OCH3 Xn O SO3H cat.CH2Cl2, rt O H Xn + NH2 OCH3 1:1546X = H, F, Cl, Br, I, NO2, CH3, OCH3, O2CR 6.1.3 Preparation of Acid Chlorides Methoxyacetyl chloride (18): To a 250 ml flame dried round bottom flask containing methoxyacetic acid (31.38 g, 0.35 mol) was added thionyl chloride (41.4 g, 0.35 mol) dropwise while stirring for 0.5 hr at 0C. The ice bath was remove and the mixture reacted for 12 hr at 30C. The pale yellow solution was then distilled to provide pure acid chloride (bp 112-113 C). OH O H3CO + SOCl2 Cl O H3CO 160oCthen heat Acetoxyacetyl chloride (18): To a 250 ml round bottom flask containing glycolic acid (10 g, 0.13 mol) was added acetyl chloride (20 g, 0.26 mol) dropwise at 0C. The ice bath was removed and the resulting slush dissolves within 30 min after the acetyl chloride addition. After 1 hr, a white precipitate forms. Benzene (50 ml) was added and the reaction stirred for an additional 12 hr. The solid was then filtered and washed with benzene and ice cold methanol to give pure product 17 in 80% yield (mp 62-63C). Afterwards, thionyl chloride (31.8 g, 0.24 mol) was added dropwise at 0C to a 250 ml flask containing 78

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acid 17 (24.6 g, 0.24 mol) while stirring. The ice bath was then removed and the resulting slush was stirred overnight at 30C. After 12 hr, the liquid was distilled in vacuo to give the acetoxyacetyl chloride in 60% yield from glycolic acid. OH O AcO OH O HO 0oCSOCl2 Cl O AcO 0 50oCAcCl1718 6.1.4 Preparation of N-4-Anisyl Azetidin-2-ones N-4-Anisyl azetidin-2-ones (6): Imine (1 mmol) and N,N-ethyldiisopropylamine (3 mmol) or triethyl amine (3 mmol) were added to a 250 ml round bottom flask containing dry dichloromethane or toluene (50 ml per g of imine) and the mixture was chilled in an ice bath to 0C. Acid chloride (1.1 mol) in dry toluene (50 ml per g acid) was added dropwise (5 ml per min) to the stirring solution. Following addition of the acid chloride, the flask was removed from the ice bath and the reaction monitored by thin layer chromatography. After 1 hr, the solution was gently heated overnight if imine was still present. The solvent was then removed in vacuo and the crude product was redissolved in ethyl acetate. After dH2O extraction, the organic layer was dried with magnesium sulfate, filtered, and the crude material was concentrated in vacuo. Diethyl ether or methanol was then added and the solution chilled to precipitate the -lactam. When silca gel chromatography was required for purification, separation was achieved with ethyl acetate and hexanes mixtures. N O RO H H Xn ()-6 O C H3 RO O Cl + N H OCH3 Xn iPr2EtN, PhMe0oC rt 54 ()-(3S,4R)-4-(phenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (37): white solid; mp 168-170 C; 1H NMR (250 MHz, CDCl3):7.33-7.16 (m, 5H), 7.19 (d, 2H, J = 9.0 Hz), 6.71 (d, 2H, J = 9.0 Hz), 5.11 (d, 1H, J = 4.8 Hz), 4.74 (d, 1H, J = 4.8 Hz), 3.66 (s, 3H), 3.11 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.7, 156.3, 133.3, 130.5, 128.5, 118.7, 114.3, 84.8, 61.8, 58.4, 55.4. ()-(3S,4R)-4-(2-chlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (38): white solid; mp 183-184 C; IR (neat) 1747 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.43 (d, 1H, J = 7.4 Hz), 7.29-7.19 (m, 5H), 6.80 (d, 2H, J = 9.0 Hz), 5.61 (d, 1H, J = 4.8 Hz), 4.89 (d, 1H, J = 4.8 Hz), 3.73 (s, 3H), 3.27 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.6, 156.4, 133.2, 131.2, 130.3, 129.6, 128.9, 127.0, 118.6, 114.1, 84.9, 59.1, 58.8, 55.4. ()-(3S,4R)-4-(3-chlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (39): white solid; mp 189-191 C; 1H NMR (250 MHz, CDCl3): 7.28-7.21 (m, 4H), 7.17 (d, 2H, J = 8.9 Hz), 6.72 (d, 2H, J = 9.0 Hz), 5.09 (d, 1H, J = 4.7 Hz), 4.74 (d, 1H, J = 4.7 Hz), 3.68 (s, 3H), 3.15 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.5, 156.4, 134.5, 132, 130.3, 129.3, 128.8, 118.7, 114.3, 84.7, 61.1, 58.5, 55.4. ()-(3S,4R)-4-(4-chlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (40): white solid; mp 136-138 C; IR (neat) 1731 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.28-7.21 (app m, 4H), 7.17 (d, 2H, J = 8.9 Hz), 6.72 (d, 2H, J = 9.0 Hz), 5.09 (d, 1H, J = 4.7 Hz), 4.74 (d, 1H, J = 4.7 Hz), 3.68 (s, 3H), 3.15 (s, 3H) 2.39 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.5, 156.4, 134.5, 132.0, 130.3, 129.3, 128.8, 118.7, 114.3, 84.7, 61.1, 58.5, 55.5. 79

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80 ()-(3S,4R)-4-(4-bromophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (41): white solid; mp 154-156 C; 1H NMR (250 MHz, CDCl3): 7.45 (d, 2H, J = 8.4 Hz), 7.23-7.14 (AB m, 4H) 6.73 (d, 2H, J = 9.0 Hz), 5.01 (d, 1H, J = 4.7 Hz), 4.75 (d, 1H, J = 4.8 Hz), 4.79 (d, 2H, J = 4.7 Hz), 3.66 (s, 3H), 3.16 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.5, 156.4, 132.5, 131.8, 130.3, 129.6, 122.7, 118.7, 114.4, 84.7, 61.2, 58.5, 55.4. ()-(3S,4R)-4-(2-iodophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (42): white solid; mp 130132 C; 1H NMR (250 MHz, CDCl3): 7.84 (d, 1H, J = 7.8 Hz), 7.28-7.15 (m, 4H), 7.00 (t, 1H, J = 7.8 Hz), 6.76 (d, 2H, J = 9.0 Hz), 5.40 (d, 1H, J = 4.8 Hz), 4.84 (d, 1H, J = 4.8 Hz), 3.69 (s, 3H), 3.24 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.5, 156.4, 139.4, 135.4, 130.3, 130.1, 128.9, 128.3, 118.7, 114.4, 98.5, 84.8, 66.1, 59.4, 55.4. ()-(3S,4R)-4-(3-iodophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (43): white solid; 1H NMR (250 MHz, CDCl3): 7.67 (s, 1H), 7.62 (d, 1H, J = 8.0 Hz), 7.29 (d, 1H, J = 7.7 Hz), 7.17 (d, 2H, J = 9.0 Hz), 7.04 (t, 1H, J = 7.8 Hz), 6.73 (d, 2H, J = 9.0 Hz), 5.02 (d, 1H, J = 4.8 Hz), 4.73 (d, 1H, J = 4.8 Hz), 3.68 (s, 3H), 3.16 (s, 3H); 13C NMR (63 MHz, CDCl3): 137.8, 136.8, 130.3, 118.7, 114.4, 84.4, 61.0, 55.4. ()-(3S,4R)-4-(2-methylphenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (45): white solid; mp 132-134 C; IR (neat) 1734 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.23-7.08 (m, 6H), 6.75 (d, 2H, J = 8.9 Hz), 5.31 (d, 1H, J = 4.9 Hz), 4.82 (d, 1H, J = 4.9 Hz), 3.69 (s, 3H), 3.20 (s, 3H) 2.39 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.9, 156.3, 135.8, 131.3, 130.7, 130.4, 128.1, 127.1, 126.1, 118.7, 114.3, 84.4, 60.0, 55.4. ()-(3S,4R)-4-(2-methoxyphenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (46): white solid; mp 117-118 C; IR (neat) 1743 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.23 (m, 3H), 7.13 (app d, 1H, J = 7.0 Hz), 6.85 (t, 2H, J = 6.85 Hz), 6.73 (d, 2H, J = 9.0 Hz), 5.52 (d, 1H, J = 4.8 Hz), 4.76 (d, 1H, J = 4.8 Hz), 3.85 (s, 3H), 3.67 (s, 3H), 3.16 (s, 3H); 13C NMR (63 MHz, CDCl3): 164.0, 157.3, 156.2, 130.7, 129.3, 128.2, 121.4, 120.6, 118.7, 114.3, 110.2, 85.8, 58.6, 56.4, 55.4 (d). ()-(3S,4R)-4-(2-nitrophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (47): white solid; mp 143-145 C; 1H NMR (250 MHz, CDCl3): 8.16 (app d, 1H, J = 9.1 Hz), 7.54-7.37 (ABm, 3H), 7.22 (d, 2H, J = 9.0 Hz), 6.78 (d, 2H, J = 9.0 Hz), 5.82 (d, 1H, J = 5.1 Hz), 4.95 (d, 1H, J = 5.1 Hz), 3.71 (s, 3H), 3.30 (s, 3H); 13C NMR (63 MHz, CDCl3): 164.3, 156.6, 148.1, 133.9, 130.6, 130.4, 129.3, 129.0, 125.4, 118.5, 114.5, 85.5, 59.6, 59.5, 55.4. ()-(3S,4R)-4-(3-nitrophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (48): yellow solid; 1H NMR (250 MHz, CDCl3): 8.27 (s, 1H), 8.22 (d, 1H, J = 8.2 Hz), 7.72 (d, 1H, J = 7.7 Hz), 7.56 (d, 1H, J = 7.9 Hz), 7.22 (d, 2H, J = 9.0 Hz) 6.79 (d, 2H, J = 9.0 Hz), 5.30 (d, 1H, J = 4.8 Hz), 4.87 (d, 1H, J = 4.8 Hz), 3.74 (s, 3H), 3.25 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.3, 156.6, 148.3, 136.0, 133.9, 129.9, 129.7, 123.7, 123.7, 123.0, 118.6, 114.5, 84.8, 60.8, 58.7, 55.4. ()-(3S,4R)-4-(4-cyanophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (49): white solid; mp 138-139C; 1H NMR (250 MHz, CDCl3): 7.60 (d, 2H, J = 8.1 Hz), 7.43 (d, 2H, J = 8.1 Hz), 7.13 (d, 2H, J = 8.9 Hz), 6.72 (d, 2H, J = 8.9 Hz), 5.18 (d, 1H, J = 4.6 Hz), 4.79 (d, 2H, J = 4.7 Hz), 3.67 (s, 3H), 3.16 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.1, 156.6, 139.2, 132.3, 130.0, 128.7, 118.6, 118.4, 114.5, 112.5, 85, 61.1, 58 .7, 55.4. ()-(3S,4R)-4-(4-hexanoyloxyphenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (50): white solid; mp 91-93 C; 1H NMR (250 MHz, CDCl3): 7.40 (d, 2H, J = 8.4 Hz), 7.26-7.24 (m, 4H), 7.11 (d, 2H, J = 8.3 Hz), 6.78 (d, 2H, J = 8.4 Hz), 5.17 (d, 1H, J = 4.6 Hz), 4.80 (d, 1H, J = 4.7 Hz), 3.74 (s, 3H), 3.12 (s, 3H), 2.54 (t, 2H, J = 7.5 Hz), 1.74 (app q, 2H), 1.23 (m, 4H), 0.82 (app t, 3H); 13C NMR (63 MHz, CDCl3): 172.1, 163.6, 156.3, 150.9, 130.7, 130.4, 128.9, 121.8, 118.7, 114.3, 84.7, 61.2, 58.4, 55.4, 34.4, 31.2, 24.5, 22.3, 13.9.

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()-(3S,4R)-4-(4-propenoyloxyphenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (51): white solid; mp 93-94 C; 1H NMR (250 MHz, CDCl3): 7.36 (d, 2H, J = 8.5 Hz), 7.20 (d, 2H, J = 7.0 Hz), 7.11 (d, 2H, J = 8.5 Hz), 6.72 (d, 2H, J = 9.0 Hz), 6.53 (d, 1H, J = 17.3 Hz), 6.24 (dd, 1H, J = 17.3, 10.4 Hz), 6.00 (d, 1H, J = 10.3 Hz), 5.13 (d, 1H, J = 4.7 Hz), 4.74 (d, 1H, J = 4.7 Hz), 3.67 (s, 3H), 3.14 (s, 3H); 13C NMR (63 MHz, CDCl3): 164.2, 163.6, 156.4, 150.7, 132.8, 130.9, 130.4, 129.0, 127.7, 121.7, 118.7, 114.3, 84.8, 61.2, 58.5, 55.4. ()-(3S,4R)-4-(2,4-dichlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (89): white solid; mp 124-126 C; 1H NMR (250 MHz, CDCl3): 7.41 (s, 1H), 7.19-7.15 (m, 4H), 6.76 (d, 2H, J = 8.9 Hz), 6.78 (d, 2H, J = 9.0 Hz), 5.52 (d, 1H, J = 4.8 Hz), 4.83 (d, 1H, J = 4.9 Hz), 3.70 (s, 3H), 3.26 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.5, 156.5, 134.7, 133.8, 130.1, 129.9, 129.8, 129.4, 127.4, 118.6, 114.5, 84.5, 59.2, 58.3, 55.4. ()-(3S,4R)-4-(2,6-dichlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (90): crystal; mp 172-174 C; 1H NMR (250 MHz, CDCl3): 7.34 (d, 1H, J = 7.3 Hz), 7.20-7.10 (m, 4H), 6.73 (d, 2H, J = 8.9 Hz), 5.93 (d, 1H, J = 4.9 Hz), 4.87 (d, 1H, J = 4.9 Hz), 3.67 (s, 3H), 3.24 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.2, 156.4, 136.2, 131.1, 130.9, 129.9, 128.6, 117.8, 114.4. ()-(3S,4R)-4-(2,3,5-trichlorophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (91): white solid; IR (neat) 1756 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.46 (app s, 1H), 7.23 (d, 2H, J = 8.9 Hz), 7.15 (app s, 1H), 6.83 (d, 2H, J = 8.9 Hz), 5.57 (d, 1H, J = 4.9 Hz), 4.91 (d, 1H, J = 4.9 Hz), 3.76 (s, 3H), 3.36 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.4, 156.7, 135.2, 134.0, 133.2, 130.0, 129.9, 129.8, 127.0, 118.5, 114.6, 84.9, 59.5, 59.2, 55.4. ()-(3S,4R)-4-(2-thiophenyl)-3-methoxy-1-(4-methoxyphenyl)azetidin-2-one (125): white solid; mp 150-153 C; 1H NMR (250 MHz, CDCl3): 7.27 (m, 3H), 7.12 (d, 1H, J = 2.8 Hz), 6.97 (dd, 1H, J = 4.9, 3.7 Hz), 6.73 (d, 2H, J = 9.0 Hz), 5.40 (d, 1H, J = 4.5 Hz), 4.76 (d, 1H, J = 4.6 Hz), 3.68 (s, 3H), 3.24 (s, 3H); 13C NMR (63 MHz, CDCl3): 163.2, 156.4, 136.5, 130.4, 127.7, 126.8 (d), 125.9, 118.8 (d), 114.3, 84.9, 58.6, 57.7, 55.4. 6.1.5 Dearylation of N-Anisyl Azetidin-2-ones Dearylated azetidin-2-ones (9): To a solution of N-anisyl-lactam 6 (1 mmol) in acetonitrile (25 ml) at 0 C was added 25 ml of an aqueous solution of ammonium cerium (IV) nitrate (3 mmol) over 5 min. The reaction was stirred for 25 min then quenched with 50 ml of water. The solution was extracted 3x with ethyl acetate (25 ml) and the combined organic layers were washed twice with aqueous 5% sodium bicarbonate, once with aqueous 5% sodium bisufite, and once with brine. Follwing treatment with magnesium sulfate and filtration, the solvent was removed in vacuo and the crude material was purified by silica gel chromatography. N O R'O H H Xn N O H R'O H H Xn Ce(NH4)2(NO3)6aq. MeCN0oC69 O C H3 ()-(3S,4R)-4-(phenyl)-3-(methoxy)azetidin-2-one (52): white solid; mp 68-70 C; 1H NMR (250 MHz, CDCl3): 7.31-7.19 (m, 4H), 6.47 (bs, 1H), 4.76 (d, 1H, J = 4.6 Hz), 4.67 (dd, 1H, J = 4.5, 2.8 Hz), 3.11 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.3, 134.2 (d), 129, 128.5, 86.5, 58.2, 57.5. 81

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82 ()-(3S,4R)-4-(2-chlorophenyl)-3-(methoxy)azetidin-2-one (53): white solid; mp 94-95 C; IR (neat) 3275 cm-1 (N-H), 1770 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.47 (d, 1H, J = 6.7 Hz), 7.39-7.27 (m, 3H), 6.97 (bs, 1H), 5.25 (d, 1H, J = 4.5 Hz), 4.83-4.82 (app m, 1H), 3.26 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.5, 133.7, 133.1, 132.9, 129.2, 128.2, 126.9, 86.9, 59.0, 55.9. ()-(3S,4R)-4-(3-chlorophenyl)-3-(methoxy)azetidin-2-one (54): white solid; mp 110-111 C; IR (neat) 3196 cm-1 (N-H), 1762 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.30-7.20 (m, 4H), 6.58 (bs, 1H), 4.76 (d, 1H, J = 4.5 Hz), 4.69-4.67 (app m, 1H), 3.13 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.1, 137.9, 129.6, 128.5, 127.7, 125.8, 86.7, 58.3, 57.6. ()-(3S,4R)-4-(4-chlorophenyl)-3-(methoxy)azetidin-2-one (55): white solid; mp 121-123 C; 1H NMR (250 MHz, CDCl3): 7.36-7.27 (m, 4H), 6.86 (bs, 1H), 4.82 (d, 1H, J = 4.5 Hz), 4.72-4.70 (m, 1H), 3.15 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.1, 134.2, 129.0, 128.5, 86.6, 58.2, 57.5. ()-(3S,4R)-4-(4-bromophenyl)-3-(methoxy)azetidin-2-one (56): white solid; mp 65-67 C; 1H NMR (250 MHz, CDCl3): 7.45 (d, 2H, J = 8.4 Hz), 7.19 (d, 2H, J = 7.9 Hz), 6.39 (bs, 1H), 4.76 (d, 1H, J = 4.5 Hz), 4.70 (app m, 1H), 3.13 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.4, 149.6, 134.7, 131.4, 129.3, 122.3, 116.1, 86.4, 58.2, 57.6. ()-(3S,4R)-4-(2-iodophenyl)-3-(methoxy)azetidin-2-one (57): white solid; mp 133-138 C; IR (neat) 3227 cm-1 (N-H), 1761 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.77 (d, 1H, J = 8.0 Hz), 7.35-7.33 (m, 2H), 6.99 (dd, 1H, J = 8.0, 4.4 Hz), 6.16 (bs, 1H), 5.02 (d, 1H, J = 4.5 Hz), 4.79 (dd, 1H, J = 4.5, 3.4 Hz), 3.23 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.0, 139.2, 137.9, 129.8, 128.2, 98.2, 86.8, 62.7, 59.3. ()-(3S,4R)-4-(3-iodophenyl)-3-(methoxy)azetidin-2-one (58): brown solid; mp 63-65 C; 1H NMR (250 MHz, CDCl3): 7.64-7.60 (m, 2H), 7.28 (d, 1H, J = 7.6 Hz), 7.06 (t, 1H, J = 7.7 Hz), 6.37 (bs, 1H), 4.734.68 (m, 2H), 3.14 (s, 3H); 13C NMR (63 MHz, CDCl3): 167.9, 138.0, 137.4, 136.5, 130.0, 129.9, 94.2, 86.7, 58.4, 57.4. ()-(3S,4R)-4-(2-methylphenyl)-3-(methoxy)azetidin-2-one (60): brown solid; mp 110-111 C; 1H NMR (250 MHz, CDCl3): 7.35-7.32 (m, 1H), 7.19-7.11 (m, 3H), 6.53 (bs, 1H), 4.98 (d, 1H, J = 4.7 Hz), 4.77 (dd, 1H, J = 4.6, 3.1 Hz), 3.19 (s, 3H), 2.26 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.8, 135.6, 134, 130.1, 127.8, 126.1, 126, 86.1, 58.3, 55.7, 19.1. ()-(3S,4R)-4-(2-methoxyphenyl)-3-(methoxy)azetidin-2-one (61): brown solid; mp 81-86 C; 1H NMR (250 MHz, CDCl3): 7.33-7.19 (AB m, 2H), 6.97-6.94 (app t, 1H), 6.82 (d, 1H, J = 8.2 Hz), 6.15 (bs, 1H), 5.17 (d, 1H, J = 4.6 Hz), 4.72 (dd, 1H, J = 4.6, 3.1, Hz), 3.79 (s, 3H), 3.16 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.9, 156.9, 128.9, 127.4, 124.2, 120.5, 109.9, 86.7, 58.6, 55.3, 53.5. ()-(3S,4R)-4-(2-nitrophenyl)-3-(methoxy)azetidin-2-one (62): white solid; mp 153-150 C; 1H NMR (250 MHz, CDCl3): 8.12 (d, 1H, J = 8.1 Hz), 7.64-7.56 (m, 2H), 7.47-7.42 (m, 1H), 7.09 (bs, 1H), 5.50 (d, 1H, J = 4.7 Hz), 4.89-4.86 (m, 1H), 3.26 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.8, 147.7, 134.0, 132.7, 128.7, 125.1, 87.2, 59.5, 56.6. ()-(3S,4R)-4-(3-nitrophenyl)-3-(methoxy)azetidin-2-one (63): white solid; mp 87-90 C; 1H NMR (250 MHz, CDCl3): 8.17-8.14 (m, 2H), 7.67 (d, 1H, J = 7.5 Hz), 7.67 (t, 1H, J = 7.8 Hz), 6.47 (bs, 1H), 4.93 (d, 1H, J = 4.5 Hz), 4.67 (app m, 1H), 3.17 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.0, 148.2, 138.2, 133.7, 129.4, 123.4, 122.6, 86.8, 58.5, 57.3. ()-(3S,4R)-4-(4-cyanophenyl)-3-(methoxy)azetidin-2-one (64): brown oil; 1H NMR (250 MHz, CDCl3): 7.67 (d, 2H, J = 8.2 Hz), 7.50 (d, 2H, J = 8.3 Hz), 6.88 (bs, 1H), 4.94 (d, 1H, J = 4.8 Hz), 4.81 (dd, 1H, J = 4.5, 2.9 Hz), 3.20 (s, 3H).

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()-(3S,4R)-4-(4-hexanoyloxyphenyl)-3-(methoxy)azetidin-2-one (65): white solid; mp 58-60 C; 1H NMR (250 MHz, CDCl3): 7.39 (d, 2H, J = 8.5 Hz), 7.10 (d, 2H, J = 8.5 Hz), 6.33 (bs, 1H), 4.84 (d, 1H, J = 4.5 Hz), 4.76 (dd, 1H, J = 4.5, 2.8 Hz), 3.17 (s, 3H), 2.55 (t, 2H, J = 7.5 Hz), 1.78-1.72 (m, 2H), 1.42-1.36 (m, 4H), 0.93 (t, 3H, J = 6.8 Hz); 13C NMR (63 MHz, CDCl3): 172.7, 171.6, 168.5, 151.1, 133.6, 129.2, 121.9, 87.0, 60.8, 58.5, 57.9, 34.7, 31.6, 31.6, 25.0, 22.7, 21.4, 14.6, 14.3. ()-(3S,4R)-4-(4-propenoyloxyphenyl)-)-3-(methoxy)azetidin-2-one (66): white solid; mp 94-96 C; 1H NMR (250 MHz, CDCl3): 7.33 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 8.4 Hz), 6.55 (d, 1H, J = 17.3 Hz), 6.26 (dd, 1H, J = 17.3, 10.4), 6.00 (d, 1H, J = 10.4 Hz), 4.78 (d, 1H, J = 4.5 Hz), 4.68-4.66 (m, 1H), 3.11 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.0, 164.5, 150.5, 133.2, 132.9, 128.8, 127.7, 121.5, 116.0, 86.6, 58.2, 57.5. ()-(3S,4R)-4-(4-hydroxyphenyl)-3-(methoxy)azetidin-2-one (82): Acrylate -lactam (99 mg, 0.4 mmol) was dissolved in 4 ml of methanol in a 3-neck flask and place in a 0oC ice bath. A potassium hydroxide (44.8 mg, 0.8 mmol) dissolved in 4 ml of a 50% methanol solution was added dropwise over 1 min and allow to stir for an additional 15 min The reaction was quenched with 10 ml of dH2O and the red solution was extracted 3x with 10 ml of ethyl acetate. The organic layer was dried with sodium sulfate then concentrated in vacuo. The product was recrystallized from chloroform giving a brown solid in 84% yield. mp 174-179 C; IR (neat) 3330 cm-1 (O-H), 1729 cm-1 (C=O); 1H NMR (250 MHz, DMSO-d6) 7.38 (bs, 1H), 8.43 (bs, 1H), 7.03 (d, 2H, J = 8.8 Hz), 6.66 (d, 2H, J = 8.5 Hz), 4.60 (d, 1H, J = 4.5 Hz), 4.55 (dd, 1H, J = 4.5, 2.3 Hz), 2.89 (s, 3H); 13C NMR (63 MHz, DMSO-d6): 167.9, 157.4, 129.1, 127.5, 115.2, 86.5, 57.4, 56.4. N O H CH3O MeOH, 0oCH H O O N O H CH3O H H OH KOH()-66()-82 ()-(3S,4R)-4-(2, 3, 4, 5, 6-pentafluorophenyl)-3-(methoxy)azetidin-2-one (92): pale solid; mp 153-155 C; 1H NMR (250 MHz, CDCl3): 6.64 (s, 1H), 5.19 (d, 1H, J = 4.7 Hz), 4.86 (dd, 1H, J = 4.6, 2.0 Hz), 3.42 (s, 3H). ()-(3S,4R)-4-(2,4-dichlorophenyl)-3-(methoxy)azetidin-2-one (93): pale solid; mp 114-116 C; IR (neat) 3196 cm-1 (N-H), 1762 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.39-7.35 (m, 2H), 7.26 (app d, 1H, J = 9.3 Hz), 6.85 (bs, 1H), 4.82-4.78 (app m, 1H), 3.26 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.7, 134.8, 133.9, 132.8, 129.6, 127.7, 87.3, 59.6, 55.9. ()-(3S,4R)-4-(2,3,5-trichlorophenyl)-3-methoxyazetidin-2-one (95): white solid; mp 163-166 C; 1H NMR (250 MHz, CDCl3): 7.39 (s, 1H), 7.30 (s, 1H), 6.82 (bs, 1H), 5.15 (d, 1H, J = 4.6 Hz), 4.82-4.79 (m, 1H), 3.28 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.1, 137.5, 133.8, 133.1, 129.6, 129.5, 126.6, 87.0, 59.5, 56.3. ()-(3S,4R)-4-(2-thiophenyl)-3-methoxyazetidin-2-one (126): white solid; mp 85-88 C; 1H NMR (250 MHz, CDCl3): 7.35 (d, 1H, J = 5.0 Hz), 7.11 (d, 1H, J = 3.0 Hz), 7.05-7.02 (m, 1H), 6.49 (bs, 1H), 5.12 (d, 1H, J = 4.3 Hz), 5.12 (dd, 1H, J = 4.0, 2.8 Hz), 3.28 (s, 3H); 13C NMR (63 MHz, CDCl3): 167.6, 139.2, 126.9, 126.7, 126.3, 86.6, 58.4, 54.0. 83

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6.1.6 Preparation of Sulfur-Transfer Reagents Methyl methanethiolsulfonate (20): Methyl disulfide (14.1 g, 160 mmol) was dissolved in 60 ml of glacial acetic acid and place in an ice water bath. Hydrogen peroxide (34 g of 30% solution) was added slowly to the mixture without stirring over 20 min. The solution was stirred for an additional 30 min at room temperature then slowly warmed to 50C for 2 hr to destroy the excess peroxide. After testing for the presence of peroxide by starch-iodide paper, the glacial acetic acid was removed under vacuum. The residue was treated with 50 ml of sat. sodium bicarbonate and extracted with ethyl acetate. After drying over anhydrous magnesium sulfate, the solvent is removed and the yellow oil distilled. bp 60-70C (0.3 torr); 1H NMR (250 MHz, CDCl3): 2.65 (s, 3H), 3.22 (s, 3H). CH3S S CH3 glacial AcOH30% H2O20oC to RTSO O S CH3 CH3 20 N-(Organothio)phthalimide: In dry benzene containing thiol (1 mmol) or disulfide (1 mmol), chlorine gas (2 mmol in benzene) was added dropwise at 0C while stirring. After 1 hr, the sulfenyl chloride solution was canulated into a flask containing phthalimide (1 mmol) and N,N-ethyldiisopropylamine (1.5 mmol) then stirred for an additional 2 hr. Afterwards, the solid material was filtered, wash with water and n-heptane, and recrystallized from methanol to give N-(organothio)phthalimide in good yields. NH O O 1. Cl2, PhH, 0oC, Et3N2.R"SHorR"SSR" NSR" O O 6.1.7 Preparation of N-Thiolated Azetidin-2-ones N-Thiolated azetidin-2-ones (3): To a solution of 9 (1 mmol) in dry THF at -78 C was added n-butylithium (1.1 mmol). After 30 min, methyl methanethiosulfonate 20 (1 mmol) was added and the mixture was stirred for 12 hr with warming to room temperature. The mixture was poured into 5% aqueous ammonium chloride and extracted 3x with dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated in vacuo. Flash chromatography of the crude material with ethyl acetate/hexanes mixtures gave N-methylthio -lactams 3. N O H R'O H H Xn N O S C H3 R'O H H X n CH3SSO2CH3 20n-BuLi, THF-78oC93 N-Thiolated azetidin-2-ones (3): Lactam 9 (1 mmol) and N-(organothio)phthalimide (1 mmol) were dissolved in dry benzene with a catalytic amount of ethyldiisopropylamine. Following reflux for 1 hr, the mixture was concentrated in vacuo and the residue was purified by silica gel chromatography to give the N-methylthio -lactams 3. 84

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N O H R'O H H Xn N O O SR iPr2EtN, PhH refluxN O SCH3 R'O H H Xn 93 ()-(3S,4R)-4-(phenyl)-3-methoxy-1-(methylthio)azetidin-2-one (67): white solid; mp 51-54 C; 1H NMR (250 MHz, CDCl3): 7.31 (m, 5H), 4.76 (d, 1H, J = 4.9 Hz), 4.72 (d, 1H, J = 4.9 Hz), 3.08 (s, 3H), 2.29 (s, 3H). ()-(3S,4R)-4-(2-chlorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (68) white crystal; mp 71-73 C; IR (neat) 1756 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.35 (d, 1H, J = 7.4 Hz), 7.24 (m, 3H), 5.29 (d, 1H, J = 4.9 Hz), 4.80 (d, 1H, J = 4.9 Hz), 3.16 (s, 3H), 2.40 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.4, 133.8, 131.4, 129.6, 128.9, 126.8, 86.7, 62.7, 58.9, 21.8. ()-(3S,4R)-4-(3-chlorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (69): white crystal; mp 73-75 C; IR (neat) 1740 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.30-7.29 (m, 3H), 7.21 (s, 1H), 4.73 (app s, 2H), 3.14 (s, 3H), 2.33 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.6, 136.1, 134.8, 130.1, 129.5, 129.4, 127.4, 87.0, 66.0, 58.9, 22.6. ()-(3S,4R)-4-(4-chlorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (70): white crystal; mp 62-66 C; IR (neat) 1751 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.33 (d, 2H, J = 8.5 Hz), 7.25 (d, 2H, J = 8.5 Hz), 4.73 (app s, 2H), 3.13 (s, 3H), 2.31 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.2, 134.9, 130.2, 128.6, 86.5, 65.5, 58.4, 22.1. ()-(3S,4R)-4-(4-bromo-phenyl)-3-methoxy-1-(methylthio)azetidin-2-one (71): white solid; mp 80-83 C; IR (neat) 1761 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.48 (d, 2H, J = 8.1 Hz), 7.18 (d, 2H, J = 8.4 Hz), 4.72 (app s, 2H), 3.13 (s, 3H), 2.31 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.6, 133.0, 132.0, 131.0, 123.5, 86.9, 66.0, 58.9, 22.6. ()-(3S,4R)-4-(2-iodophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (72): white crystal; mp 62-65 C; IR (neat) 1756 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.80 (d, 1H, J = 7.8 Hz), 7.30 (t, 1H, J = 7.5 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.00 (t, 1H, J = 7.5 Hz), 5.09 (d, 1H, J = 5.0 Hz), 4.80 (d, 1H, J = 4.8 Hz), 3.82 (s, 3H), 2.41 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.7, 139.9, 136.2, 130.6, 129.3, 128.6, 99.8, 87.1, 70.4, 59.6, 22.2. ()-(3S,4R)-4-(3-iodophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (73): white crystal; mp 97-99 C; IR (neat) 1745 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.65 (m, 2H), 7.27 (d, 1H, J = 7.7 Hz), 7.08 (t, 1H, J = 8.0 Hz), 4.73-4.67 (AB m, 2H), 3.13 (s, 3H), 2.32 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.6, 138.4, 138.2, 136.4, 130.5, 128.5, 94.5, 87, 65.8, 58.3, 22.6. ()-(3S,4R)-4-(4-iodophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (74): white solid; mp 102-105 C; IR (neat) 1766 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.65 (d, 2H, J = 8.3 Hz), 7.03 (d, 2H, J = 8.3 Hz), 4.70 (app s, 2H), 3.10 (s, 3H), 2.29 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.1, 137.5, 133.2, 130.7, 94.9, 86.4, 65.6, 58.4, 22.1. ()-(3S,4R)-4-(2-methylphenyl)-3-methoxy-1-(methylthio)azetidin-2-one (75): white solid; mp 80-81 C; IR (neat) 1745 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.27-7.21 (m, 4H), 5.11 (d, 1H, J = 5.0 Hz), 4.85 (d, 1H, J = 5.0 Hz), 3.21 (s, 3H), 2.45 (s, 3H), 2.37 (s, 3H). 85

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86 ()-(3S,4R)-4-(2-methoxyphenyl)-3-methoxy-1-(methylthio)azetidin-2-one (76): white solid, mp 120123C; 1H NMR (250 MHz, CDCl3): 7.27-7.24 (app m, 1H), 7.19-7.15 (m, 1H), 6.95 (t, 1H, J = 7.5 Hz), 6.86 (d, 1H, J = 8.2 Hz), 5.27 (d, 1H, J = 4.9 Hz), 4.72 (d, 1H, J = 4.9 Hz), 3.80 (s, 3H), 3.12 (s, 3H), 2.37 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.9, 157.7, 129.5, 128.5, 121.5 120.4, 110.3, 86.5, 60.2, 58.5, 55.4, 21.8. ()-(3S,4R)-4-(2-nitrophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (77): yellow solid; 1H NMR (250 MHz, CDCl3): 8.12 (d, 1H, J = 8.2 Hz), 7.66-7.63 (m, 1H), 7.50-7.41 (m, 2H), 5.49 (d, 1H, J = 5.1 Hz), 4.93 (d, 1H, J = 5.2 Hz), 3.23 (s, 3H), 2.43 (s, 3H). ()-(3S,4R)-4-(4-cyanophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (79): yellow solid; mp 88-90 C; 1H NMR (250 MHz, CDCl3): 7.64 (d, 2H, J = 8.0 Hz), 7.40 (d, 2H, J = 8.0 Hz), 4.79 (ABm, 2H), 3.14 (s, 3H), 2.37 (s, 3H). ()-(3S,4R)-4-(4-propenoyloxyphenyl)-3-methoxy-1-(methylthio)azetidin-2-one (81): white solid; mp 87-88 C; 1H NMR (250 MHz, CDCl3): 7.29 (d, 2H, J = 8.4 Hz), 7.08 (d, 2H, J = 8.4 Hz), 6.50 (d, 1H, J = 17.3 Hz), 6.21 (dd, 1H, J = 17.1, 10.3 Hz), 5.92 (d, 1H, J = 10.4), 4.75 (d, 1H, J = 4.8 Hz), 4.69 (d, 1H, J = 4.8 Hz), 3.05 (s, 3H), 2.26 (s, 3H);13C NMR (63 MHz, CDCl3): 170.3, 164.2, 150.9, 132.9, 131.1, 129.9, 127.7, 121.4, 86.5, 65.5, 58.3, 22.0. ()-(3S,4R)-4-(4-hydroxyphenyl)-3-methoxy-1-(methylthio)azetidin-2-one (83): white solid; mp 119123 C; 1H NMR (250 MHz, CDCl3): 7.18 (d, 2H, J = 9.0 Hz), 6.81 (d, 2H, J = 9.0 Hz), 5.73 (bs, 1H), 4.69 (app s, 2H), 3.11 (s, 3H), 2.28 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.9, 168.5, 130.4, 124.9, 115.4, 65.9, 58.3, 22.1. ()-(3S,4R)-4-(2, 3, 4, 5, 6-pentafluorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (96): white solid; mp 724 C; 1H NMR (250 MHz, CDCl3): 5.15 (d, 1H, J = 5.1 Hz), 4.81 (d, 1H, J = 5.1 Hz), 3.35 (s, 3H), 2.35 (s, 3H). ()-(3S,4R)-4-(2,4-dichlorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (97): yellow crystal; mp 102-105 C; IR (neat) 1772 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.37 (s, 1H), 7.28 (d, 1H, J = 8.3 Hz), 7.16 (d, 1H, J = 8.2 Hz), 5.24 (d, 1H, J = 4.6 Hz), 4.79 (d, 1H, J = 4.6 Hz), 3.19 (s, 3H), 2.40 (s, 3H); 13C NMR (63 MHz, CDCl3): 170.5, 135.3, 134.9, 130.6, 130.3, 129.9, 127.7, 87.1, 62.6, 59.4, 22.2. ()-(3S,4R)-4-(2,6-dichlorophenyl)-3-met hoxy-1-(methylthio)azetidin-2-one (98): pale yellow solid; mp 77-80 C; IR (neat) 1771 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.23 (t, 1H, J = 8.1 Hz), 5.72 (d, 1H, J = 5.3 Hz), 4.90 (d, 1H, J = 5.1 Hz), 3.30 (s, 3H), 2.44 (s, 3H); 13C NMR (63 MHz, CDCl3,): 168.3, 131.1, 130.1, 128.8, 88.0, 62.9, 59.2, 21.6. ()-(3S,4R)-4-(2,3,5-trichlorophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (99): yellow crystal; mp 90-94 C; IR (neat) 1761 cm-1 (C=O); 1H NMR (250 MHz, CDCl3): 7.44 (s, 1H), 7.11 (s, 1H), 5.24 (d, 1H, J = 5.0 Hz), 4.83 (d, 1H, J = 5.0 Hz), 3.26 (s, 3H), 2.45 (s, 3H); 13C NMR (63 MHz, CDCl3,): 170.4, 135.8, 134.4, 133.5, 129, 127.6, 87.2, 63.4, 59.7, 22.2. ()-(3S,4R)-4-(phenyl)-3-methoxy-1-(butylthio)azetidin-2-one (108): colorless oil; 1H NMR (250 MHz, CDCl3): 7.34-7.27 (m, 5H), 4.74-4.71 (m, 2H), 3.09 (s, 3H); 13C NMR (63 MHz, CDCl3): 171.0, 133.6, 128.9, 128.8, 128.3, 86.3, 66.9, 58.3, 38.2, 30.8, 21.5, 13.6. ()-(3S,4R)-4-(phenyl)-3-methoxy-1-(phenylthio)azetidin-2-one (109): white solid; mp 58-60 C 1H NMR (250 MHz, CDCl3): 7.37-7.23 (m, 8H), 4.88-4.83 (AB m, 2H), 3.18 (s, 3H). ()-(3S,4R)-4-(phenyl)-3-methoxy-1-(cy clohexylthio)azetidin-2-one (110): oil; 1H NMR (250 MHz, CDCl3): 7.38-7.26 (m, 5H), 4.86-4.82 (m, 2H), 3.02 (s, 3H), 2.89-2.85 (m, 1H), 1.98-1.94 (app m, 1H),

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87 1.75 (app s, 3H), 1.35-1.19 (m, 6H); 13C NMR (63 MHz, CDCl3): 171.3, 133.5, 128.9, 128.8, 128.3, 86.2, 67.6, 58.3, 49.5, 32.2, 30.9, 25.6, 25.4. ()-(3S,4R)-4-(2-thiophenyl)-3-methoxy-1-(methylthio)azetidin-2-one (127): oil; 1H NMR (250 MHz, CDCl3): 7.43 (d, 1H, J = 5.0 Hz), 7.21 (d, 1H, J = 2.9 Hz), 7.06 (dd, 1H, J = 5.0, 2.9 Hz), 5.08 (d, 1H, J = 4.7 Hz), 4.82 (d, 1H, J = 4.8 Hz), 3.32 (s, 3H), 2.30 (s, 3H). ()-(3S,4R)-4-(2,6-dichlorophenyl)-3-phenoxy-1-(methylthio)azetidin-2-one (134): white solid; mp 114116 C; 1H NMR (360 MHz, CDCl3): 7.39 (d, 1H, J = 7.8 Hz), 7.33-7.23 (m, 7H), 6.92 (t, 1H, J = 7.4 Hz), 6.79 (d, 2H, J = 7.9 Hz), 5.59 (app s, 2H), 2.49 (s, 3H); 13C NMR (90 MHz, CDCl3): 167.0, 140.0, 133.6, 133.2, 129.7, 129.5, 128.5, 127.0, 122.6, 119.5, 83.9, 55.6. ()-(3S,4R)-4-(4-nitrophenyl)-3-acetoxy-1-(methylthio)azetidin-2-one (135): white solid; 1H NMR (360 MHz, CDCl3): 8.26 (d, 2H, J = 8.7 Hz), 7.47 (d, 2H, J = 8.7 Hz), 5.96 (d, 1H, J = 5.0 Hz), 5.15 (d, 1H, J = 5.0 Hz), 2.49 (s, 3H), 1.75 (s, 3H); 13C NMR (90 MHz, CDCl3): 182.0, 168.9, 167.8, 148.5, 140.3, 129.7, 123.8, 81.1, 65.1, 22.3, 19.6. ()-(3S,4R)-4-(2-chloro-6-fluorophenyl)-3-acetoxy-1-(methylthio)azetidin-2-one (137): yellow solid; 1787, 1745 cm-1 (C=O); 1H NMR 1H NMR (250 MHz, CDCl3): 7.35 (ddd, 1H J = 8.4, 6.3, 2.1 Hz), 6.91 (t, 2H, J = 8.7 Hz), 5.95 (d, 1H, J = 5.2 Hz), 5.39 (d, 1H, J = 5.2 Hz), 2.38 (s, 3H), 1.81 (s, 3H); 13C NMR (63 MHz, CDCl3): 168.9, 167.9, 162.2 (dd, app JC-F = 250, 6.8 Hz), 131.4 (t, app JC-F = 10.7 Hz), 112.0, 111.7, 108.8 (t, app JC-F = 14 Hz), 77.6, 56.8, 21.7, 19.9. ()-(3S,4R)-4-(phenyl)-3-hydroxy-azetidin-2-one (141): brown oil; 1H NMR (250 MHz, CDCl3): 7.467.26 (m, 5H), 5.17 (d, 1H, J = 5.3 Hz), 4.96 (d, 1H, J = 5.0 Hz), 2.44 (s, 3H). 6.2 Microbiological Test Procedures The following bacteria were used fo r the antimicrobial evaluation of N -thiolated -lactams: Bacillus anthracis (Sterne strain), Bacillus cereus (ATCC 14579), Bacillus coagulans (USF 546), Bacillus globigii (Department of Defense Reagents Program), Bacillus megaterium (ATCC 14581), Bacillus subtilis (19569), Bacillus thuringensis (ATCC 10792), Bacteroides fragalis (obtained from Smith-Kline Laboratory), Candida albicans (clinical isolate), Candida tropicalis (clinical isolate), Enterobacter cloace (environmental isolate, USF510), Enterococcus gallinarium (ATCC 49573), Enterococcus faecalis (ATCC 19433), Enterococcus casseliflavus (ATCC 700327), Enterococcus durans (ATCC 6056), Enterococcus avirum (ATCC 14025), Enterococcus saccharolyticus (ATCC 43076), Escherichia coli (ATCC 23590), Haemophilus influenzae ( USF 561), Klebsiella pneumoniae (USF 512), Lactococcus lactis (ATCC 11454), Listeria monocytogenes (ATCC 19115), Micrococcus luteus (environmental isolate, USF681), Niesserria gonnorheae (obtained from the Tamp a Branch State Laboratory, -lactamase positive, USF 662), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhimurium (obtained from University of South Florida Medical Clinic, USF 515), Serratia marcescens (ATCC 29634), Staphylococcus aureus USF525 (ATCC 25923) Staphylococcus aureus USF652-658 (obtained from Lakeland Regional Medical Center, lactamase positive), Staphylococcus epidermidis (environmental isolate, USF528), Staphylococcus saprophyticus (ATCC 35552), Staphylococcus simulans (ATCC 11631), Staphylococcus capitis (ATCC 35661), Staphylococcus cohnii (ATCC 35662), Staphylococcus lentus (ATCC 700403), Staphylococcus lugdunensis (ATCC 700328), Staphylococcus xylosus (ATCC 29971), Streptococcus pyrogenes Streptococcus agalactiae Vibrio cholerae (biotype E1 Tor Ogawa, cholera toxin positive, CDC E5906), 6.2.1 Antimicrobial Susceptibility Test Culture preparation: From a freezer stock in tryptic soy broth (Difco Laboratories, Detroit, MI) and 20% glycerol, a culture of each organi sm was grown on tryptic soy agar (TSA) plates (Becton-Dickinson Laboratories, Cockeysville, MD) at 37 C for 24 hours. A 108 suspension was then made in sterile phosphate buffered saline (pH 7.2) and swabbed across fresh TSA plates.

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88 Disc method: From each 1mg/ml stock solution in dimethyl sulfoxide (DMSO), sterile 6mm paper discs (Becton-Dickinson Laboratories, Cockey sville, MD) were impregnated with 20 l of the test compounds. At this concentration, the microliter quantity is equiva lent to the micrograms in solution. The discs were allowed to dry in a biohazard safety hood then placed onto the inoculat ed TSA plates. The plates were incubated for 24 hours at 37 C and the antimicrobial susceptibilities were determined by measuring the zones of growth inhibition around each disc. Well method: A 108 standardized cell count suspension was then made in sterile phosphate buffered saline (pH 7.2) and swabbed across fresh TSA plates. Circul ar wells (6 mm in diameter) were cut into the inoculated plates and 20 L of a 1 mg/ml stock solution of the test lactam in dimethylsulfoxide (DMSO) was pipetted into the wells. The plates were incubated for 24 hours at 37 C and the antimicrobial susceptibilities were determined by measuring the zones of growth inhibition around each well. 6.2.2 MIC Calculations Media preparation: The minimum inhibitory concentrations were determined by the agar plate dilution (need reference). The test media were prepared in 24 well plates (Costar 3524, Cambridge, MA) by adding a known concentration of the test drug in DMSO together with a solution of Mueller-Hinton II agar (Becton-Dickinson Laboratories, Cockeysville, MD) for a total volume of 1 ml in each well. Calculations of the overall concentration of antibiotic in the wells were standardized by measuring from a 1mg/ml stock solution of the test drug. At this concentration, the microliter quantity is equivalent to the micrograms in solution. The amount of agar solution added to the wells was determined by subtracting 1000 l from the quantity of test drug in each well to give a combined volume of 1 ml. Following preparation of the well plates, the media were allowed to solidify at room temperature for 24 hours before inoculation. Inoculation: From an 24 hour culture of each organism on tryptic soy agar (TSA) plates (BectonDickinson Laboratories, Cockeysville, MD), the staphylococcal strains we re grown overnight in 5 ml of tryptic soy broth (Difco Labo ratories, Detroit, MI) at 37 C. One microliter of each culture was then applied to the appropriate well of agar and incubated at 37 C overnight. After 24 hr, the MICs were determined by examining the wells for growth. 6.2.3 Growth Studies Overnight cultures of the test strains were grown to logarithmic phase in MHB. An inoculum of 106 cfu/ml was added to fresh MHB and grown for 1 hr at 35C while shaking. The test compounds diluted to 1, 5, or 10 times the MICs in DMSO was app lied to each tube. Viable cell coun ts were determined by plating adequate dilutions of each culture. The plates were incubated and colony counts were taken after 24 hrs. Turbidity measurements were determined by transfer ring 0.2 ml aliquots of cu lture to a 48-well plate (Costar 3524, Cambridge, MA) and optical density readings taken at 630 nm with a Bio-Tek EL800 plate reader. 6.2.4 Metabolism studies To a fresh 106 cfu/ml suspension of S. aureus (ATCC 25923) in 9 ml of sterile saline was added 1 ml of a 400 M solution of lactam 68 in DMSO. After 1 hr, 10 ml of dH2O was added and the solution was extracted 3 times with 5 ml of et hyl acetate. The organic layers were combined, dried with magnesium sulfate, and the solvent was removed under reduc ed pressure. The residue was dissolved in 500 l of CDCl3 and the chemical structure was elucidated by 1H NMR. 6.2.5 Scanning Electron Microscopy (SEM) Experiments Sample preparation: Samples were prepared from sections of agar taken from the disk-diffusion experiment. Sterile 6 mm diameter paper discs (Becton-Dickinson Laboratories, Cockeysville, MD) were

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89 impregnated with 20 g of penicillin-G potassium salt (Sigma Chemical Co, St. Louis, MO) and 68 from their 1 mg/ml stock solutions in DMSO. From a 24-hour culture of Staphylococcus aureus USF525 (ATCC 25923) grown on tryptic soy agar (TSA) plates (B ecton-Dickinson Laboratories, Cockeysville, MD), a 108 suspension was made in sterile phos phate buffered saline (pH 7.2) and swabbed across two separate TSA plates. The discs were placed on the inoculat ed plates and incubated for 24 hours at 37 C. After 24 hours, sections of agar in areas containing the division between the zones of inhibition and lawn of bacteria were cut out and placed into Petri dishes for the SEM preparation. SEM preparation: The agar sections were flooded with 10 ml of a pre-made glutaraldehyde-osmium fixative. After 1 hour, the sections were removed and washed 3 times with 0.1 M sodium cacodylate. The samples were then sequentially submerged in 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and 100% ethanol for periods of 5 minute. Following alcohol tr eatment, the sections were placed into hexamethyl disilazane (HMDS) for chemical drying. The samples were next mounted onto stubs and coated with gold/palladium by a Pelco Model 3 Sputter Coater. Scanning electron microscopy. The morphology of the cells was examined with a Novascan 30 scanning electron microscope. 6.2.6 Light Microscopy Experiment Slide preparation: Sterile 6 mm paper discs (Becton-Dickinson Laboratories, Cockeysville, MD) containing 20 ug of B3o-4, penicillin-G potassium salt (Sigma Chemical Co, St. Louis, MO) and vancomycin hydrochloride (Abbott Laboratory, Chicago, IL) were placed on TSA plates innoculated by S. aureus USF525 (ATCC 25923) from an overnight culture. The plates were incubated at 37 C for 4-5 hours or until the zones of inhibition were visible. Glass 24 x 60mm coverslips (Corning Glass works, Corning, NY) were then gently pressed across the zones to adhere the bacteria. Following heat fixing over a Bunsen burner flame, the coverslips were flooded by Gram crystal violet stain (Becton Dickinson Laboratories, Cockeysville, MD) for one minute. The c overslips were rinsed with water and flooded by Gram iodine (Difco Laboratories, Detroit, MI) for one minute then decolorized by adding 95% ethanol dropwise until the crystal violet no longer flowed off the coverslips. The coverslips were rinsed again with water and counterstained with Gram safranin (Difco Laboratories, Detroit, MI) for one minute. They were then thoroughly rinsed with water, blotted dry, and mounted on glass microscope slides (Fisher Scientific, Pittsburgh, PA). The slides were view with a Nikon LABPHOT Type: 104 bright-field light microscope. 6.2.7 DNA Cleavage Assay To 17 l of sodium phosphate buffer (50 mM, pH 7.4) was added 0.5 g of pBR322 (ICN Biomedicals Inc, Aurora, OH) in 200 l microfuge tubes. 2 L of 2a at 5, 10, 25, 50, and 100 M concentrations in DMSO were added and the samples were vortexed then incubated at 37C. After 24 hrs, 2 l of Blue/Orange 6X Loading Dye (Promega Corp., Madison, WI) were added to 8 l aliquots of the DNA mixtures. The samples were loaded on a 1.2% agarose gel (1.8 g medium EEO agarose; 150 ml TAE; 1 g/ml ethidium bromide) and horizontal electrophoresis was performed at 80 V/10 cm for 2.5 hrs in TAE (40 mM Trisacetate, pH 7.8, 1 mM EDTA). The agarose ge l was visualized and photographed under UV transillumination. The same procedure was used in ex periments conducted with glutathione, dithiothreitol (DTT), and 2-mercaptoethanol. For the enzyme digest samples, 20 l of sterile dH2O, 2 l of 100 M 2a 2 l of restriction enzyme buffer H (90 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, pH 7.5), 1 g of pBR322, and 2 l EcoR1 were added sequentially to a 1.5 ml microfuge tube and incubated in a 37C water bath for 1 hr. 6.2.8 Determination of Thiol Levels in Bacteria Preparation of cell extracts: Cells from a culture grown at 37C in LB broth were harvested by centrifugation (3100 rpm for 5 min) once an OD650 of 1.0 to 1.5 nm was achieved. The liquid media is removed and 10 ml of sterile dH2O is added to the pellet. Following centrifugation (3100 rpm for 5 min),

PAGE 105

90 the water was then removed. One milliliter of warm (60C) 50% aqueous aceton itrile containing 25 mM methanesulfonic acid is added to the tube and vort exed at maximum speed for 1 min. The foamy cell extracts were transferred to a 1.5 ml centrifuge tube and incubated in a 60C water bath for 15 min. The protein and the cellular debris are pelleted by centrifugation for 5 min at 8000 rpms.. The acid supernant fraction is then removed and analyzed by thiol titration with Ellmans reagent. Reagent preparation: 39.6 mg of DTNB is dissolve in 10 ml of 0.5 M potassium phosphate buffer at pH 7.2. Measurement of thiol levels: 0.4 ml of the acid thiol extract is transferred to in a 1 ml cuvette followed by the addition of 0.5 ml of DNTB. The absorbance of the bright yellow solution was measured at 412 nm after 2 mins using dH2O as a blank. Thiol levels (Co) are then calculated by the following equation: Co = [ A412 /13,600] D D = dilution factor A412 = Acuvette1 Acuvette2 where: Acuvette1 = Absorbance of 0.5 ml of DNTB + 0.4 ml of thiol acid extract Acuvette2 = Absorbance of the 0.5 ml of DNTB + 0.4 ml of dH2O. 6.2.9 Macromolecule Synthesis: The effects of lactam 68 on DNA, RNA, and protein synthesis in S. aureus ATCC 25923 was determined by measuring the respective incorporations of [methyl-3H]thymidine, [5-3H]uridine, or L-[4,5-3H]isoleucine (Amersham Life Science). Ciprofloxacin, rifampcin, and chloramphenicol were used as controls for the inhibition of DNA, RNA, and protein synthesis, resp ectively. Radioactive precursors were added to early logarithmic-phase S. aureus (3 Ci of [methyl-3H]thymidine, 3 Ci of [5-3H]uridine, or 5 Ci of L-[4,5-3H]isoleucine) in Luria Broth in the presence or absence of an antibiotic at 2x MIC. To assess the effect on DNA, R NA, and protein synthesis, 50l samples were removed from each reaction tube are the designated time intervals (5, 10, 20, 30, 45, and 60 min) and precipitated in 1 ml of ice cold 10% trichloroacetic acid. After 1 hr, the samples were filtered through glass fiber filters (GF/A; Whatman), washed with 2 ml of ice-cold 5% trichloroacetic acid and 2 ml of ice-cold 95% ethanol, and dried at room temperature overnight. The dried f ilters were placed in 10 ml vials containing 7 ml of counting fluid (Cytoscint, ICN International). Radioactivity was meas ured by liquid scintilation (Beckman Instruments).

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CHAPTER VII 1H and 13C NMR SPECTRA Spectrum 7.01: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 37. N O PMP CH3O H H ()-37 91

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Spectrum 7.02: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 38. N O PMP CH3O H H ( )-38Cl 92

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Spectrum 7.03: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 39. N O PMP CH3O H H ( )-39Cl 93

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Spectrum 7.04: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 40. N O PMP CH3O H H ( )-40Cl 94

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Spectrum 7.05: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 41. N O PMP CH3O H H ( )-41Br 95

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Spectrum 7.06: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 42. N O PMP CH3O H H ( )-42I 96

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Spectrum 7.07: 1H NMR (250 MHz, CDCl3) of -lactam 43. N O PMP CH3O H H ( )-43I 97

PAGE 113

Spectrum 7.08: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 45. N O PMP CH3O H H ( )-45CH3 98

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Spectrum 7.09: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 46. N O PMP CH3O H H ( )-46OCH3 99

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Spectrum 7.10: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 47. N O PMP CH3O H H ( )-47NO2 100

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Spectrum 7.11: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 48. N O PMP CH3O H H ( )-48NO2 101

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Spectrum 7.12: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 49. N O PMP CH3O H H ( )-49CN 102

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Spectrum 7.13: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 50. N O PMP CH3O H H ()-50O O 103

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Spectrum 7.14: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 51. N O PMP CH3O H H ( )-51O O 104

PAGE 120

Spectrum 7.15: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 52. N O H CH3O H H ( )-52 105

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Spectrum 7.16: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 53. N O H CH3O H H ( )-53Cl 106

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Spectrum 7.17: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 54. N O H CH3O H H ( )-54Cl 107

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Spectrum 7.18: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 55. N O H CH3O H H ( )-55Cl 108

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Spectrum 7.19: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 56. N O H CH3O H H ( )-56Br 109

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Spectrum 7.20: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 57. N O H CH3O H H ( )-57I 110

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Spectrum 7.21: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 58. N O H CH3O H H ( )-58I 111

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Spectrum 7.22: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 60. N O H CH3O H H ( )-60CH3 112

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Spectrum 7.23: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 61. N O H CH3O H H ( )-61OCH3 113

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Spectrum 7.24: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 62. N O H CH3O H H ( )-62NO2 114

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Spectrum 7.25: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 63. N O H CH3O H H ( )-63NO2 115

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Spectrum 7.26: 1H NMR (250 MHz, CDCl3) of -lactam 64. N O H CH3O H H ( )-64CN 116

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Spectrum 7.27: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 65. N O H CH3O H H ()-65O O 117

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Spectrum 7.28: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 66. N O H CH3O H H ( )-66O O 118

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Spectrum 7.29: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 70. N O S C H3 CH3O H H ( )-70Cl 119

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Spectrum 2.30: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 71. N O S C H3 CH3O H H ( )-71B r 120

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Spectrum 7.31: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 72. N O S C H3 CH3O H H ( )-72I 121

PAGE 137

Spectrum 7.32: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 73. N O S C H3 CH3O H H ( )-73I 122

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Spectrum 7.33: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 74. N O S C H3 CH3O H H ( )-74I 123

PAGE 139

Spectrum 7.34: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 75. N O S C H3 CH3O H H ( )-75CH3 124

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Spectrum 7.36: 1H NMR (250 MHz, CDCl3) of -lactam 77. N O S C H3 CH3O H H ( )-77NO2 125

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Spectrum 7.37: 1H NMR (360 MHz, CDCl3) of -lactam 78. N O S C H3 CH3O H H ( )-78NO2 126

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Spectrum 7.38: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 81. N O S C H3 CH3O H H ( )-81O O 127

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Spectrum 7.39: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 82. N O H CH3O H H ( )-82OH 128

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Spectrum 7.40: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 83. N O S C H3 CH3O H H ( )-83OH 129

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Spectrum 7.41: 1H NMR (250 MHz, CDCl3) of -lactam 88. N O PMP CH3O H H ( )-88 F5 130

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Spectrum 7.42: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 89. N O PMP CH3O H H ( )-89Cl Cl 131

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Spectrum 7.43: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 90. N O PMP CH3O H H ( )-90Cl Cl 132

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Spectrum 7.44: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 91. N O P M P CH3O H H ()-91Cl Cl Cl 133

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Spectrum 7.45: 1H NMR (250 MHz, CDCl3) of -lactam 92. N O H CH3O H H ( )-92 F5 134

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Spectrum 7.46: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 94. N O H CH3O H H ( )-94Cl Cl 135

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Spectrum 7.47: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 95. N O H CH3O H H ()-95Cl Cl Cl 136

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Spectrum 7.48: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 97. N O S C H3 CH3O H H ( )-97Cl Cl 137

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Spectrum 7.49: 1H NMR (360 MHz, CDCl3) and 13C NMR (90 MHz, CDCl3) of -lactam 98. N O S C H3 CH3O H H ( )-98Cl Cl 138

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Spectrum 7.50: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 99. N O S C H3 CH3O H H ()-95Cl Cl Cl 139

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Spectrum 7.51: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 108. N O S CH3O H H ( )-108 140

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Spectrum 7.52: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 110. N O S CH3O H H ()-110 141

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Spectrum 7.53: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 117. N O CH3O H H S N O O C H3 H H ()-117Br Br 142

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Spectrum 7.54: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3) of -lactam 125. N O PMP CH3O H H ( )-125S 143

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Spectrum 7.55: 1H NMR (360 MHz, CDCl3) of -lactam 133. N O H O H H ( )-133 O NO2 144

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Spectrum 7.56: 1H NMR (360 MHz, CDCl3) and 13C NMR (90 MHz, CDCl3) of -lactam 135. N O S C H3 O H H ( )-135 O NO2 145

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Spectrum 7.57: 1H NMR (250 MHz, CDCl3) and 13C NMR (63 MHz, CDCl3)of -lactam 137. N O S C H3 O H H ( )-137 O Cl F 146

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155 86. Woulfe SR, Iwagami H, Miller MJ: Efficient N -sulfenylation of azetidinones using S -substituted thiophthalimides. Tetrahedron Lett (1985) 26: 3891-3894. 87. Iwagami H, Woulfe SR, Miller MJ: Reations of (2-oxo-1-azetidinyl)-thiophthalimide with nucleophiles. Tetrahedron Lett (1986) 27 : 3095-3098. 88. Boyd DB, Eigenbrot C, Indelicato JM, Miller MJ, Pasini CE, Woulfe SR: Heteroatom-activated blactam antibiotics: considerations of differences in the biological activity of [[3(S)-(acylamino)-2-oxy1-azetidinyl]oxy]acetic acids (Oxamazins) and the correspinding sulfur analogues (Thiamazins). J Med Chem (1987) 30 : 528-536. 89. Burnett DA, Hart DJ, Liu J: -Lactams from esters and sulfenimines: a new route to monobactams. J Org Chem (1986) 51: 1929-1930. 90. Shah NV, Cama LD: Synthesis of a novel carbapenem-potassium (5R,6R)-1,1-difluoro-2-phenyl-6(hydroxyethyl)-carbapen-2-em-3-carboxylate. The use of a new N-protecting group in -lactam synthesis. Heterocycles 1987 25 : 221-227. 91. Sendo Y, Kii M, Sakanoue M, Motokawa K, Kimura Y: Synthesis and antibacterial activity of 1substituted-methyl carbapenems. Chem Pharm Bull (1992) 40 : 2410-2418. 92. Penfold DJ Pike K, Geng A, Anson M, Kitteringham J, Kilburn JD: Radical cyclisations of methylenecyclopropyl azetidinonessynthesis of novel tricyclic -lactams. Tetrahedron Lett (2000) 41 : 10347-10351 93. Woulfe SR, Miller MJ: The synthesis of substituted [[3(S)-(acylamino)-2-oxy-1azetidinyl]thio]acetic acids. J Org Chem (1986) 51 : 3133-3139 94. (a) Oida S, Yoshida A, Hayashi T, Nakayama E, Sato S, Ohki E: 7-Oxo-2-thia-azabicyclo[3.2.0]hept3-ene, a revised structure for penems synthesize d via oxidative addition mediated by copper (I). Tetrahedron Lett (1980) 21 : 619-620; (b) DeNinno F, Linek EV, Christensen BC: Penems. 1. Penem formation via a novel oxidative additi on process mediated by copper (I). J Am Chem Soc (1979) 101 : 2210-2211. 95. Jasys VJ, Kellogg MS, Volkmann RA: Synthesis of novel sulfenamide-containing -lactams. Tetrahedron Lett (1991) 32 : 3771-3774. 96. Faraci WS, Bakker AV, Spencer RW, Williams RA, Jasys VJ, Kellogg MS, Volkmann RA: Inhibition of human leukocyte elastase (HLE) by novel bicyclic -lactams. Bioorg Med Chem Lett (1993) 3 : 22712276. 97. Perrin DD, Armarego WLF, Perrin DR: Purification of Laboratory Chemical. Pergamon Press: New York, USA (1980) 98. Omura K, Swern D: Oxidation of alcohols by "activated" di methyl sulfoxide. a preparative, steric and mechanistic study. Tetrahedron (1978) 34: 1651-1660. 99. Kronenthal DR, Han CY, Taylor MK: Oxidative N -dearylation of 2-azetidinones. p-Anisidine as a source of azetidinone nitrogen. J Org Chem 47: 2765-2768. 100. Behforouz M, Kerwood JE: Alkyl and aryl sulfenamides. J Org Chem (1969) 34: 51-55. 101. NCCLS (National Committee for Laboratory Standards) Methods for Dilution of Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically NCCLS Document M7-A4, Vol. 16, No. 2, 1997.

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ABOUT THE AUTHOR Timothy Long received his bachelor's degree in biology at the Un iversity of South Florida, Tampa, FL. During his senior year, Timothy began research in the synthetic laboratory of Professor Edward Turos. Despite his interest in biology, he greatly enjoyed performing synthesis which ultimately led him to pursue a doctorate in chemistry. Currently, Timothy contin ues to conduct research where his interest lie in solving problems at the interface of biology and chemistry.


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ABSTRACT: N-Methylthio beta-lactams represent a promising new family of antibacterial agents whose in vitro activity is confined largely to Staphylococcus species, including multidrug-resistant forms of S. aureus. Originally developed in the 1980's for use as synthetic intermediates, N-methylthio beta-lactams have recently been shown in these laboratories to possess intriguing biological properties which are addressed in Chapters I-IV. In terms of the antibacterial activities, the structural features and species specificities exhibited by these compounds are unlike those of any existing family of beta-lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active Beta-lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes. The most active members of this Beta-lactam class appear to be those bearing an aryl (Ar) substituent at C4 of the ring. The synthesis and structure-activity relationship of these analogues is discussed in Chapter III. Moreover, microscopy and 3H pulse-labeling studies, which are described in Chapter IV, demonstrate that N-methylthio beta-lactams appear to be inhibitors of protein biosynthesis.
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