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Studies on antibacterial activities of n-thiolated beta-lactams and their polymeric nanoparticles against mrsa

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Studies on antibacterial activities of n-thiolated beta-lactams and their polymeric nanoparticles against mrsa
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English
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Shim, Jeung-Yeop
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University of South Florida
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Subjects / Keywords:
Drug resistant
Antibiotics
Biopolymers
MRSA
Beta-lactams
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: Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hospitals and in care centers, and has brought on the need to develop new drugs for MRSA. This thesis centers on studies of N-thiolated beta-lactams, a new family of potent antibacterial compounds that selectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Chapter 1 describes MRSA in more detail. Chapter 2 outlines experiments on the effect of a fatty ester group (CO2R) on the C4-phenyl ring of N-methylthio fO-lactams, expecting that attachment of long chain ester moieties might increase the hydrophobicity, and thus enhance the drugs ability to penetrate through the cell membrane. However, the results indicate that antibacterial activity drops off rapidly when more than seven carbon atoms are in the chain.These results led to the idea about examining a fO-lactam conjugated polymer as a possible pro-drug delivery method, which is the focus of Chapter 3. To synthesize the initial drug-polymer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N-methylthio fO-lactam. This method has advantages over the conventional emulsion polymerization methods because a solid co-monomer (fO-lactam drug) can be utilized. SEM studies show that these polymeric nanoparticles have a microspherical morphology with nano-sizes of 40-150 nm. The N-thiolated fO-lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancomycin.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2003.
Bibliography:
Includes bibliographical references.
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by Jeung-Yeop Shim.
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Title from PDF of title page.
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Document formatted into pages; contains 145 pages.
General Note:
Includes vita.

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oclc - 62782950
usfldc doi - E14-SFE0000630
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ABSTRACT: Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hospitals and in care centers, and has brought on the need to develop new drugs for MRSA. This thesis centers on studies of N-thiolated beta-lactams, a new family of potent antibacterial compounds that selectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Chapter 1 describes MRSA in more detail. Chapter 2 outlines experiments on the effect of a fatty ester group (CO2R) on the C4-phenyl ring of N-methylthio fO-lactams, expecting that attachment of long chain ester moieties might increase the hydrophobicity, and thus enhance the drugs ability to penetrate through the cell membrane. However, the results indicate that antibacterial activity drops off rapidly when more than seven carbon atoms are in the chain.These results led to the idea about examining a fO-lactam conjugated polymer as a possible pro-drug delivery method, which is the focus of Chapter 3. To synthesize the initial drug-polymer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N-methylthio fO-lactam. This method has advantages over the conventional emulsion polymerization methods because a solid co-monomer (fO-lactam drug) can be utilized. SEM studies show that these polymeric nanoparticles have a microspherical morphology with nano-sizes of 40-150 nm. The N-thiolated fO-lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancomycin.
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Studies on Antibacterial Activities of N -Thiolated -Lactams and Their Polymeric Nanoparticles Against MRSA by Jeung-Yeop Shim 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 Julie P. Harmon, Ph..D. Bill J. Baker, Ph.D. Kirpal S. Bisht, Ph.D. Michael W. Fountain, Ph.D. Date of Approval: November 21, 2003 Keywords: Antibiotics, Biopolymers, MRSA, -Lactams, Drug-Resistance Copyright 2003, Jeung-Yeop Shim

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ACKNOWLEDGEMENT I would like to thank with all my h eart Professor Edward Turos, my major professor for all of his help, guidance, and knowledge. He has given me nume rous opportunities in order to experience a wonderful career in multi-science areas. He has provided a tremendous learning environment to thrive over th e years. I am also grateful for his warm heart and his patience. He is my teacher as well as my be st friend. With my sincerest respect, I would like to express my deepest thanks to my teacher, Dr. Edward Turos for sharing of all. I would also like to thank my committee members, Professor Julie Harmon, Professor Bill J. Baker, and Professor Kirpal Bisht for their guidance while at USF. Also I would like to acknowledge Dr. Michael Fount ain for opening my eyes to the world of drug delivery and business. Also, I would like to express my appreciation to Dr. ChangKiu Lee for encouraging my breakable mind to further pursue in science. His inspiration, guidance, and encouragement are beyond my ability to describe. While in Dr. Turos lab, I met and wo rked with a wonderful gr oup of individuals: Dr. Seyoung Jang, Dr. Suresh Reddy, Bart Heldre th, Cristina Coates, Timothy Long, J. Michelle Leslie, Sampath Abeylath, Hele n Wang, Marci Culbreath, Kerriann Greenhalgh, Casey Cosner. They all gave me warm heart and good friendship.

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Last, I want to express my sincer est appreciation to my wife. Without her invested and scarified time, efforts, love, patience, I would not have completed this goal. Her continued encouragement led me to complete this success. I want to say to my wife I love you forever. Also, I want to say my two sons, David and Da niel, I love you.

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv LIST OF SCHEMES viii ABBREVIATIONS ix ABSTRACT x CHAPTER 1. METHIC ILLIN-RESISTANT Staphylococcus aureus (MRSA) 1.1. Introduction 1 1.2. Cell Wall Biosynthesis and Its In hibition by Penicillins in Gram-Positive Bacteria 3 1.3. Methicillin-Resistance 6 1.4. Trends in Resistance and Prospect for New Therapies 8 References 10 CHAPTER 2. INFLUENCE OF FATTY ES TER SIDE CHAINS ON THE ANTIBACTERIAL ACTIVITY OF N -THIOLATED -LACTAMS 2.1. Introduction 11 2.2. Synthesis 14 2.3. Biological Activity 20 2.4. Discussion 25 2.5. Conclusion 29 2.6. Experimental 30 References 52 CHAPTER 3. POLYMERIC NANOPA RTICLES CONTAINING AN NMETHYLTHIO -LACTAM 3.1. Introduction 53 3.2. Conventional Microemulsion Polymerization 56 3.3. Synthesis of C3-Acryloyl N-Methylthio -Lactam 59 3.4. Main Components for Microemulsion Polymerization 61 3.4.1. Choice of Drug Monomer 61 3.4.2. Choice of Co-monomer 62 3.4.3. Choice of Surfactant 63 3.4.4. Choice of Radical Initiator 65

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ii 3.4.5. Choice of Aqueous Media 66 3.5. The Preparation of Polymeric Na noparticles Using Microemulsion Polymerization 66 3.6. Characterization 68 3.6.1. Scanning Electron Microscopy (SEM) 68 3.6.2. Coalescing Process 77 3.6.3. Determination of Solid Content (%) 78 3.6.4. 1 H NMR Spectra Analysis 79 3.7. Biological Activity Against MRSA 81 3.7.1. Initial Testing 81 3.7.2. Antibacterial Testing of Homo Po ly(ethyl acrylate) Nanoparticles (Without N-Methylthio -Lactam) 82 3.7.3. Antibacterial Testing of Lactam-containing Nanoparticles Against MRSA 83 3.8. Antifungal Testing of Nanoparticle Emulsions 90 3.9. Discussion 92 3.10. Conclusions 99 3.11. Experimental 100 References 105 CHAPTER 4. PREPARATION OF FLUORESCENCE ACTIVE NANOPARTICLES AND FUTURE APPLICATIONS 4.1. Introduction 108 4.2. Preparation of an Acrylated Fl uorescence Monomer 109 4.3. The Preparation of Fluor escence-Active Polymeric Nanoparticles in an Aqueous Emulsion 112 4.4. Characterization of Fluoresence-Active Emulsified Nanoparticles 117 4.4.1. Scanning Electron Microscopy (SEM) 117 4.4.2. 1 H NMR Spectra Analysis 119 4.5. Discussion 121 4.6. Experimental 125 References 128 CHAPTER 5. CONCLUSIONS AND FU TURE DIRECTIONS 129 ABOUT THE AUTHOR End Page

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iii LIST OF TABLES Table II-1. Zones of Inhibition for Compound 7s and 13s 22 Table II-2. Zones of Inhibition for Compound 15s 23 Table II-3. Zones of Inhibition for Compound 20s 24 Table III-1. Formulation of Microemu lsion Polymerization 71 Table III-2. Zones of Inhibition Obtained Fr om Agar Well Diffusion Experiments 87 Table III-3. Zones of Inhibition Obtained Fr om Agar Well Diffusion Experiments 94 Table IV-1. Formulation of Microemulsion Poly merization 118 Table IV-2. Formulation of Microemulsion Poly merization 119 Table IV-3. Formulation of Microemuls ion Polymerization 120

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iv LIST OF FIGURES Figure 1-1. Chemical Structure of Peni cillin G and Cephalosporin C 2 Figure 1-2. Gram-positive Bacteria Cell Wall 3 Figure 1-3. Chemical St ructure of NAG-NAM 4 Figure 1-4. Bacteria Cell Wall Synthesis of Gram-positive Bacteria 4 Figure 1-5. Structure of Pe ptidogylcan 5 Figure 1-6. Inhibition of GTPase by Penicillin 6 Figure 1-7. Hydrol ysis of Penicillin by -Lactamase 7 Figure 1-8. Proportion of S. aureus Nocosomial Infections Resistant to Oxacillin (MRSA) Among Intensive Ca re Unit Patients, 1989-2001 9 Figure 2-1. Effect of Increasing R Chain Length on Antibacterial Activity Against MRSA for -Lactams 25 Figure 2-2. Comparison of Bi oactivities for Methoxy and Acetoxy -Lactams 26 Figure 2-3. Comparison of Bioactivities for trans and cis -Lactams 27 Figure 2-4. Comparison of Bioactivities for ortho and para Isomers 28 Figure 2.7. Comparison of Bioact ivities for MRSA 28 Figure 3-1. Comparison of Conventional a nd Controlled Release Profile 54 Figure 3-2. Schematic Representation of an Emulsion Polymerization System 58 Figure 3-3. 1 H NMR Spectra of C 3 -Acryloyl -Methylthio -Lactam 61 Figure 3-4. Structure of Dr ug Monomer 62 Figure 3-5. -Lactam Polymeric Emulsion 67

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v Figure 3-6. SEM Picture fo r Homo(ethyl acrylate) Polyme ric Nanoparticles 69 Figure 3-7. SEM Pictur e for 20:1 Copolymeric Nanoparticles 70 Figure 3-8. SEM Pictur e for 13:1 Copolymeric Nanoparticles 71 Figure 3-9. SEM Pictur e for 10:1 Copolymeric Nanoparticles 72 Figure 3-10. SEM Picture for 7:1 Copol ymeric Nanoparticles 73 Figure 3-11. SEM Picture for 5:1 Copol ymeric Nanoparticles 74 Figure 3-12. SEM Picture for 2.5:1 Copol ymeric Nanoparticles 75 Figure 3-13. Particle Size Distribution of -Lactam Copolymeric Nanoparticles 76 Figure 3-14. Representation of the Coalescing Process 77 Figure 3-15. Determination of Solid Content (%) 78 Figure 3-16. 1 H NMR Spectra, the Molar and Solid Content of -Lactam and Ethyl Acrylate Copolymers 80 Figure 3-17. Initial Antibacterial Testi ng of Nanoparticles and Polymer Films Against MRSA 652 85 Figure 3-18. Antibacterial Testing of Homo Poly(ethyl acrylate) Nanoparticles Against MRSA 83 Figure 3-19. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 652 85 Figure 3-20. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 653 85 Figure 3-21. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 654 86 Figure 3-22. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 655 86 Figure 3-23. Antibacterial Testing of Drug-embedde d Nanoparticles Against 87 MRSA 656 Figure 3-24. Antibacterial Testing of Drug-embedded Nanoparticles Against

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vi MRSA 657 87 Figure 3-25. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 658 88 Figure 3-26. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 659 88 Figure 3-27. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 919 89 Figure 3-28. Antibacterial Testing of Drug-embedded Nanoparticles Against MRSA 920 89 Figure 3-29. Antibacterial Testing of Drug-embedded Nanoparticles Against S. aureus 849 90 Figure 3-30. Comparison of Antib acterial Activities of N-Thiolated -LactamContaining Emulsified Nanoparticles 93 Figure 3-31. Bioactivity of Polymeric Nanoparticles As a Function of Disk Loading Amounts (MRSA 653) 95 Figure 3-32. Bioactivity of Polymeric Nanoparticles As a Function of Disk Loading Amounts for S. aureus 849 96 Figure 3-33. Bioactivity of 7:1 Copo lymeric Nanoparticles As a Function of Decreasing Disk Loading Amounts 97 Figure 3-34. Comparison of Antibacterial Activities Against MRSA 653 98 Figure 3-35. Diagram of Endocytosis Process 99 Figure 4-1. 1 H NMR Spectra of (a) Dansyl, (b ) Naphthyl, and (c) Anthracenyl Acrylates 111 Figure 4-2. SEM Image for -Lactam Fluorescence-active Emulsified Nanoparticles with Particle Size (60-120 nm) 117 Figure 4-3. SEM Image for Naphthyl Fluorescence-active Emulsified Nanoparticles with Particle Size (30-60 nm) 118 Figure 4-4. SEM Image for Anthrace nyl Fluorescence-active Emulsified Nanoparticles with Particle Size (60-120 nm) 118

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vii Figure 4-5. 1 H NMR Spectra of (a) Dansyl, (b ) Naphthyl, and (c) Anthracenyl Fluorescence-active Copolymers 120 Figure 4-6. Comparison of the Non Fluorescence-active -Lactam and Fluorescence-Active Naphthyl and Anthracenyl Emulsified Nanoparticles and Their Corre sponding Thin Films Upon UV Irradiation 124

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viii LIST OF SCHEMES Scheme II-1 16 Scheme II-2 17 Scheme II-3 18 Scheme II-4 19 Scheme III-1 60 Scheme III-2 67 Scheme IV-1 110 Scheme IV-2 114 Scheme IV-3 115 Scheme IV-4 116

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ix ABBREVIATIONS MRSA Methicillin-resistant Staphylococcus aureus NAG N -Acetylglucosamine NAM N -Acetylmuramic acid DAP Diaminopimelate GTPase Glycopeptide transpeptidase MIC Minimum inhibitory concentration VISA Vancomycin-intermediate Staphylococcus aureus VRSA Vancomycin-resistant Staphylococcus aureus DCC Dicyclohexyl carbodiimide DMAP 4-Dimethylaminopyridine ATCC American type culture collection PEG Polyethylene glycol CMC Critical micelle concentration SEM Scanning Electron Microscopy EDC 1-ethyl-3-(3-dimet hylaminopropyl) carbodimide DIPEA Diisopropylethylamine

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x Studies on Antibacterial Activities of N -Thiolated -Lactams and Their Polymeric Nanoparticles Against MRSA Jeung-Yeop Shim Abstract Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hos pitals and in care centers, and has brought on the need to develop new drugs for MRSA This thesis centers on studies of N -thiolated lactams, a new family of potent antibacteri al compounds that selectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Chapter 1 describes MRSA in more detail. Chapter 2 outlines experiments on the effect of a fatty ester group (CO 2 R) on the C 4 -phenyl ring of N -methylthio -lactams, expecting that attachment of long chain ester moieties might in crease the hydrophobicity, and thus enhance the drugs ability to penetrate through the cell membrane. However, the results indicate that antibacterial activity drops off rapidly when more than seven carbon atoms are in the chain. These results led to the idea about examining a -lactam conjugated polymer as a possible pro-drug delivery method, which is the focus of Chapter 3. To synthesize the initial drug-poly mer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic

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xi polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N methylthio -lactam. This method has advantag es over the conventional emulsion polymerization methods because a solid co-monomer ( -lactam drug) can be utilized. SEM studies show that these pol ymeric nanoparticles ha ve a microspherical morphology with nano-sizes of 40-150 nm. The N -thiolated -lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancom ycin. Although at this time the relationship between particle size and activity is not clear and the mode of action is unknown, the N thiolated -lactam containing nanoparticles dramatically enhance bioactivity, possibly due to increased bioavailability of the antibiotic via endocytosis. In chapter 4, Fluorescence-active emulsified nanoparticles containing naphthyl or anthracenyl side chains were also su ccessfully prepared by microemulsion polymerization for possible use in fluorescence st udies to determine if the drug enters the cell of MRSA through endocytosis, and where possible bioaccumulation site are located.

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1 Chapter One Methicillin-resistant Staphylococcus aureus (MRSA) 1.1 Introduction 1,2,3,4 Penicillin was the first antibiotic chemotherapeutic agen t discovered and it was proven to be effective against specific bact eria when administered in the human body without destroying the body's own cells. In 1928, Sir Alexander Fleming, prof essor of bacteriology at St. Mary's Hospital in London, was culturing Staphylococcus aureus. He noticed zones of growth inhibition where mold spores were growing. He named the mold Penicillium rubrum. It was determined that a secretion of the mold was effective against Gram-positive bacteria. In 1940 Lord Howard Florey and Sir Ernst Chai n successfully isolated the antimicrobial agent. It was determined to be an inhibitor of cell wall synthesis in gram-positive bacterial. Amidst the need for antibacterial agents in WW II, penicillin was isolated, purified and injected into experimental an imals, where it was found to not only cure infections but also to possess low toxi city. This opened the age of antibiotic chemotherapy and promoted an intense search for similar antimicrobial agents of low toxicity that might prove useful in the treatment of infectious disease. Some time later, another mold was found which produced a bact eria-killing chemical, and the structure of the molecule was determined to be very simila r to the penicillin mo lecule; this chemical

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and its cousins were called "cephalosporins" after the Cephalous mold from which they were derived (Fig. 1-1). Chemical changes have been made to the molecules over the years to improve their bacteria-fighting abilities and to help them overcome biochemical breakdown and "immunity" of resistant bacteria. N S O CH3 CH3 H HN O OH O N S O CH3 O OH O HN HO H O O H2N O Penicillin GCephalosporin C Fig. 1-1 Chemical structure of penicillin G and cephalosporin C The isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several other antibiotics were in clinical usage. The bacterial cell has a cell wall similar to the outer layer of plant cells, but which is missing in human and animal cells. This wall must grow along with the cell, or the growing cell will eventually become too big for the wall and burst and die. Penicillins and cephalosporins kill bacteria by messing up the wall-building system. Since human cell do not have cell walls, and plants have a different wall-building system, neither human, nor animals, nor plants are affected by the penicillins. Some bacteria have changed the structure of their walls to prevent penicillin from entering, or have come up with ways to break down the penicillin structure. In the 1940's and 1950's, most bacteria could be killed by penicillin. Now, because no longer penicillins and cephalosporins have 2

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been used so often there are many bacteria that no longer are killed by penicillin. Antibiotic resistance is a growing problem. 1.2 Cell Wall Biosynthesis and Its Inhibition by Penicillins in Gram-positive Bacteria The Gram-positive bacteria possess a thick cell wall composed of a cellulose-like polymer covalently interbound via short peptide units into layers (Fig. 1-2). This peptidoglycan substance is also found in Gram-negative cells, but is thinner and less fortified. Gram negative organisms also possess an outer membrane composed of lipoproteins, lipopolysaccharides, and phospholipids, which provide a natural barrier to the antimicrobial effects of penicillin. 5 The polysaccharide portion of the peptidoglycan structure (fig. 1-5) is made of repeating units of N-acetylglucosamine (NAG) linked to N-acetylmuramic acid (NAG-NAM) (Fig. 1-3). The peptide varies, but begins with L-Ala and ends with D-Ala. In the middle is a dibasic amino acid, diaminopimelate (DAP). The terminal Ala residues are used for glycan linkage; the DAP for interpeptide linkages. Different species of Gram-positive bacteria possess different peptide units in the peptidoglycan cell wall. 4,5 3 peptidoglycan plasmamembrane Fig. 1-2 Gram-positive bacteria cell wall 5

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O O O OH HO HN O O O HO OH O HN O n Fig. 1-3 Chemical structure of NAG-NAM 1,2,5 NAG 4 NAM NAG NHCHCH3 C NH CHCH3 COO O O B H NHCHCH3 C O O Ser NAGNAG NAM NAGNAGNH2 D-Ala D-Ala NHCHCH3 C O NAGNAG NAM NAGNAGNH GPTase D-Ala D-Ala D-Ala NAM NAM Fig. 1-4 Cell wall biosynthesis of gram-positive bacteria 1 The NAM part of the NAG-NAM monomer group provides the point of attachment for the peptide. The L-Ala residue is attached to the NAM. DAP provides a linkage to the

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D-Ala residue on an adjacent peptide, which continues to link distally with another layer of NAG-NAM (Fig. 1-4). OOHO HO NH O O HO NH O O HO NH O O HO NH O OOH HO NH O O O O O HN NH HN HN NH HN HN O O O O O O O O O O O O HO HN NH O O O NH NH HN NH O O O O O O O OOOH OH HN O O OH HN O OO OH HN O O OH HN O OHO OH HN O O O O O HN NH HN HN NH HN HN O O O O O O O O O O O O HO HN NH O O O NH NH HN NH O O O O O O HN NH HN HN NH HN HN O O O O O O O O O O O O HO HN NH O O O NH NH HN NH O O O O O O O O Fig. 1-5 Structure of peptidoglycan 5 The transpeptide linkages are catalyzed by the enzyme glycopeptide transpeptidase (GPTase). A ser residue in the active site of GPTase is the target of penicillin attack. The anionic Ser residue attacks the carbonyl carbon of the -lactam ring since the

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conformation resembles the transition state of the normal GPTase substrate. This forms an indefinitely stable structure; thereby rendering the enzyme inactive. Without an active site, glycoprotein transpeptidation can not occur. Without transpeptidation, Gram-positive cell walls can not be crosslinked. Therefore, those bacteria are easily killed (Fig. 1-6). 4,5 O N S O NH O R O O B H O HNS NH R O O O O GPTase Fig. 1-6 Inhibition of GTPase by penicillin 1.3 Methicillin-Resistance As early as the 1940s, bacteria began to develop resistance to penicillin. -Lactamases (or penicillinases) are enzymes produced by a bacterium which render penicillin useless by hydrolyzing the -lactam ring. The -lactamases catalytically disrupt the amide bond of penicillin, and other -lactams, via the formation of a serine-ester-linked acyl enzyme derivative. -Lactams are structural analogs of the peptidyl-D-alanyl-D-alanine terminal moiety of peptidoglycan cell wall precursors. Following -lactam binding, the -lactam ring is opened by nucleophilic attack by the hydroxyl group of a serine residue. Acylation of the enzyme generates an acyl enzyme intermediate, 6

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which undergoes subsequent hydrolysis to release the penicilloyl moiety and regenerating the active enzyme (Fig. 1-7). 6,7,8,9 N S O HN H H O R O OH -lactamase Nu H HNS NH R O O -lactamase Nu O OH H2O H H HNS NH R O O HO O OH H H -lactamase Nu + Fig. 1-7 Hydrolysis of penicillin by -lactamase Strains of Staphylococcus aureus are resistant to methicillin, cloxacillin, flucloxacillin and to all the other -lactam antibiotics, including all penicillins and cephalosporins. because of a change in the cell wall proteins. MRSA also has the ability to pick up other resistance mechanisms and may become resistant to all other anti-staphylococcal drugs. With the widespread emergence of MRSA, glycopeptide antibiotics such as vancomycin or teicoplanin have been more frequently used in the clinical practice. Popular use of these agents has led to the onset of glycopeptide resistance at the end of the 20th century. In 1997, an isolate of S. aureus with reduced susceptibility to vancomycin (MIC 8 g/mL) was first reported from Japan. 10 Until 2002, a total of 24 cases of Vancomycin-Intermediate Staphylococcus aureus (VISA) infections were reported from 11 countries in the world. In 2002, two strains of VRSA with high-level resistance to vancomycin (MIC 32 g/mL) with van A gene from 7

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8 enterococci were reported in the U.S. 11,12 It is anticipated that gl ycopeptide resistance will only continue to get worse within a few years. 1.4 Trends in Resistance and Prospect for New Therapies Natural selection is the driving for ce for the appearance of drug resistant strains. Once one strain of bacteria has become re sistant, the resistance can be transferred between strains by several mechanisms invol ving exchange of genetic material. These mechanisms include conjugation, trans duction, and transformation. The more an antibiotic is used, the greater the selective pre ssure of bacteria to become resistant, and the faster resistance spreads. The most eviden t example is penicillin. When penicillin was first discovered, it was believed to be a miracl e drug and was used at any sign of infection. The emergence of resistant strains was al most immediate. MRSA is now the most challenging bacterial pathogen that currently affects patients in hospital and in the community. Infection caused by this impor tant nosocomial bacterium has become a serious national and global problem. Fig. 1-8 13 indicates the development of MRSA in the recent years. Therefore, by studying th e mechanisms by which strains become resistant, a better understanding of how to st op or slow down the emergence of resistant strains is possible. For example, for strains that produce -lactamases, a -lactamase inhibitor can be added in addition to the -lactam drug to render the bacteria sensitive to the antibiotic. Development of drugs with a new mechanism of action are needed. Alternatively, new ways to deliver the antibiot ic to the cell may help overcome resistance and minimize further expose of bacteria to the drug.

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In next chapter, studies on a new family of potent antibacterial compounds, N-thiolated -lactams, that selectively inhibit the growth of Staphylococcus species are described. 0102030405060 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 Year Fig. 1-8 Proportion of S. aureus Nosocomial Infections Resistant to Oxacillin Source: National Nocosomial Infections Surveillance (NNIS) 9

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10 References 1. Sweet, R. M., In Cephalosporins and Penicillins, Chemistry and Biology; Flynn, E. H., Ed.; 1972. 2. Morin, R.B. and Gorman, M., Chemistry and Biology of -Lactam Antibiotics; Academic Express: New York, 1982, Volumes 1-3. 3. Kukacs, F. and Ohno, M., Recent Progress in the Chemical Synthesis of Antibiotics; Springer-Verlag: Berlin-Heidelberg, 1990. 4. Silverman, R.B. The Organic Chemistry of Drug Design and Dr ug Action; Academic Press: IL, 1 st Ed., 1992. 5. Lim, D., Microbiology; Kendal l/Hunt Publishing Company: Iowa, 3 rd Ed., 2003. 6. Alkema, W.B.L. et al ., Charecterisation of the -lact am binding site of penicillin acylase of Escherichia coli by structural and site-dir ected mutagenesis studies, Protein Engineering 2000 13, 857. 7. Lamotte-Brasseur, J. et al ., Streptomyces albus G serine -lactamase, Biochem. J. 1992, 82, 189. 8. Lamotte-Brasseur, J. et al., Mechanism of acyl transfer by the class A serine lactamase of Streptomyces albus G. Biochem. J. 1991, 279, 213. 9. Zawadzke, L.E. et al., An engineered Staphylococcus aureus PC1 serine -lactamase that hydrolyses third-ge neration cephalosporins, Protein Engineering 1995 12, 1275. 10. Hiramatsu K, Hanaki H, Ino T, et al Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 1997, 40, 135. 11. Centers for Disease Control and Prevention. Staphylococcus aureus resistant to vancomycin-United States, 2002. Morb Mort Weekly Report. 2002, 51, 565. 12. Centers for Disease Control and Prevention. Public health dispatch : vancomycinresistant Staphylococcus aureus Pennsylvania, 2002. Morb Mort Weekly Report. 2002, 51, 902. 13. http://www.cdc.gov/ncidod/hip/ARESIST/mrsa.htm

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Chapter Two Influence of Fatty Ester Side Chains on the Antibacterial Activity of N-Methylthio -Lactams 2.1 Introduction N-Thiolated -lactams are a new family of potent antibacterial compounds that selectively inhibit the growth of Staphylococcus species. Members of this family of antibiotics show enhanced activity towards Staphylococcus microbes, including methicillin-resistant Staphylococcus aureus (MRSA), over other common bacterial genera. 1 The initial lead compound I-A developed earlier in the Turos laboratory led to further investigations on the structure-activity studies patterns of these new antibacterial agents. 2 Previous work found that certain lipophilic and electron withdrawing functionalities on the ring may be preferred. 11 I-A NOSOCH3 CH3 1234 The analogs of N-methylthio -lactams, II-A, II-B, II-C, II-D, show potent activities against MRSA. 3 These compounds are lipophilic and have the electron withdrawing functionality on the C 4 -phenyl group.

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NOSOCH3CH3CN NOSOCH3CH3 O2N NOSOCH3CH3Cl NOSOCH3CH3CO2Me II-AII-BII-CII-D C 4 -Saturated/unsaturated side chain aryl analogs, III-A, III-B, III-C also have moderate antibacterial activities against MRSA. 3 These compounds are lipophilic and even have no electron withdrawing substituent at C 4 NOSOCH3CH3 NOSOCH3CH3 NOSOCH3CH3 III-AIII-BIII-C C 4 -Fluoro aryl analogs, IV-A, IV-B, IV-C, IV-D, also have strong antibacterial activities against MRSA. 4 12

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NOSOCH3CH3F NOSOCH3CH3F NOSOCH3CH3F F F F NOSOCH3CH3F F F F F IV-BIV-CIV-DIV-A One of these compounds, ester analog II-B, has an ester group (CO 2 Me) on the C 4 -phenyl ring of the lactam. This compound was found to be a reasonably potent analog among those we examined. The electron withdrawing ester group on the C 4 -phenyl group as well as the long alkyl chain of the ester group can possibly increase the hydrophobicity of N-methylthio -lactam and thus may increase bioavailability by enhancing penetration through the cell membrane. Therefore, it is a good candidate for systematic investigation of the structure-activity relationship for N-methylthio -lactams. In this study, the synthesis and evaluation of additional fatty ester analogs was carried out to address whether increasing the length or the degree of branching, and thus the hydrophobic character of the ester side chain, may alter the antibacterial properties. 13

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2.2 Synthesis A series of N-thiolated -lactams 1, having a C 4 -fatty ester on the phenyl group was prepared. NOSR'OCH3 CO2R1 Scheme II-1 summarizes the synthesis of the first of these analogs, 3-methoxysubstituted C 4 -benzoate alkyl esters 7. 4-Carboxybenzaldehyde 2 was coupled with the appropriate alcohol using dicyclohexylcarbodiimide/dimethylaminopyridine (DCC/DMAP), followed by reaction with p-anisidine, to give alkyl ester imines 4. Staudinger coupling 5-8 of methoxyacetyl chloride with the imine 4 gave N-aryl protected -lactam alkyl ester 5. Dearylation of -lactam 5 with ceric ammonium nitrate and methylthiolation with N-(methylthio)phthalimide affords C 3 -methoxy N-methylthio -lactam ester analogs 7. 2 Ortho-substituted -lactams 13 are also good candidates for the study of biological activity. These compounds were prepared as shown in Scheme II-2. Even though ortho substituted methyl, ethyl, propyl and pentyl ester analogs were tried under the usual Staudinger coupling conditions, only ortho ethyl ester, gave results. In this case, cis and trans adducts were obtained. These adducts were taken on to the N-methylthio -lactams 13, which independently were tested for antibacterial activity by the Kirby-Bauer method of disc diffusion on agar plates. 14

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15 Scheme II-3 summarizes the synthesis of C 3 -acetoxy N-methylthio -lactams 15. Staudinger coupling of acetoxyace tyl chloride with imine 4 gave C 3 -acetoxy N -aryl protected -lactam 14. In some cases, both cis and trans adducts could be obtained from the Staudinger reaction. Dearylation of -lactam 14 with ceric ammonium nitrate, followed by methylthiolation with N-(methylthio)phthalimide affords C 3 -acetoxy N methylthio -lactams 15. C 3 -Alkyl carbonate and methyl dithiocarbonate moieties also could be introduced to examine the effect of differ ent functionalities and chain br anching on bioactivity of the lactam. Scheme II-4 summarizes the synthesis of C 3 -alkylcarbonate N-methylthio lactam analogs 20. The C 3 -hydroxy -lactam 17 could be easily obtained by cleavage of C 3 -acetoxy -lactam 16 with methanolic KOH. The re sulting free hydroxyl compound 17 was reacted with alkyl orthoc hloroformate to give the C 3 -alkyl carbonate -lactams 18. Dearylation and methylthiola tion provided N-methylthio -lactams 20.

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Scheme II-1 O H OH O N OR O MeO N O MeO OR O OMe N O S MeO OR O CH3 4574a CH3 984b CH2CH3 984c (CH2)4CH3 674d (CH2)6CH3 894e (CH2)9CH3 91Rcis-5a CH3 78cis-5b CH2CH3 18cis-5c (CH2)4CH3 17cis-5d (CH2)6CH3 19trans-5d (CH2)6CH3 18cis-5e (CH2)9CH3 63RRcis-7a CH3 66cis-7b CH2CH3 70cis-7c (CH2)4CH3 79cis-7d (CH2)6CH3 70trans-7d (CH2)6CH3 68cis-7e (CH2)9CH3 81H ab Conditions: (a) ROH, DCC, DMAP, dry acetone, reflux; in case of 3a: MeOH, SOCl2, 0oC to rt. (b) p-anisidine, Et3N, CH2Cl2. (c) MeOCH2COCl, Et3N, CH2Cl2. ( d ) ( NH 4 ) 2 Ce ( NO 3 ) 6 MeCN-H 2 O. ( e ) N( meth y lthio ) phthalimide,Et 3 N,CH 2 Cl 2 NH O MeO OR O 6Rcis-6a CH3 65cis-6b CH2CH3 72cis-6c (CH2)4CH3 19cis-6d (CH2)6CH3 89trans-6d (CH2)6CH3 88cis-6e (CH2)9CH3 91 O H OR O cde233a CH3 993b CH2CH3 433c (CH2)4CH3 503d (CH2)6CH3 453e (CH2)9CH3 36RYield(%)Yield(%)Yield(%)Yield(%)Yield(%) 16

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N OMe O O N O O OMe O O CH2CH3 H3C N O S O O O CH2CH3 H3C CH3 N O S O O O CH2CH3 H3C CH3 +Scheme II-2CH2CH3 81011cis-13trans-13cis : trans (1:1) mixtureO OH O Conditions: (a) EtI, K2CO3,dry acetone, reflux. (b) p-anisidine, Et3N, CH2Cl2. (c) MeOCH2COCl, Et3N, CH2Cl2. (d) (NH4)2Ce(NO3)6, MeCN-H2O;(e) N-(methylthio)phthalimide, Et3N, CH2Cl2. O O CH2CH3 9O H NH O O O O CH2CH3 H3C 12cis : trans (1:1) mixture abcde18%60%36%36%72% 17

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Scheme II-3 N OR O MeO N O AcO OR O OMe N O SMe AcO OR O 41415cis-14a CH3trans-14a CH3cis-14b CH2CH3trans-14c (CH2)4CH3trans-14d (CH2)6CH3cis-14e (CH2)9CH3trans-14e (CH2)9CH3Rcis-15a CH3 8trans-15a CH3 10cis-15b CH2CH3 12trans-15c (CH2)4CH3 5trans-15d (CH2)6CH3 2cis-15e (CH2)9CH3 9trans-15e (CH2)9CH3 12R ab:Conditions: (a) AcOCH2COCl, Et3N, CH2Cl2. (b) (i) (NH4)2Ce(NO3)6, MeCN-H2O; (ii) N-(methylthio)phthalimide, Et3N, CH2Cl2. Yield(%)2543124057Yield(%) 18

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Scheme II-4N O OMe N O O 171818a Me, X=O 5518b Et, X=O 3918c CMe3, X=O 98 18d Ph, X=O 2118e Me, X=S 15R Conditions: (a) KOH, MeOH, 0oC. (b) NaH, CH2Cl2, RT then ROCOCl; in case of 14e: CS2, NaH, THF, RT then MeI. (c) (NH4)2Ce(NO3)6, MeCN-H2O. ( d ) N( meth y lthio ) phthalimide,Et 3 N,CH2Cl2. HO X X N O OMe 16 AcO aN O SMe O 2020a Me, X=O 520b Et, X=O 3520c CMe3, X=O 120d Ph, X=O 5320e Me, X=S 1R X X N O H O 1919a Me, X=O 6319b Et, X=O 219c CMe3, X=O 7219d Ph, X=O 7819e Me, X=S 1R X X OMe R R R bcdYield(%)Yield(%)Yield(%) 19

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20 2.3 Biological Activity Fatty ester N-methylthio -lactams 7, 13, 15 and 20 were tested for antibacterial activity by the Kirby-Bauer method of disc diffusion on agar plat es. Mr. Timothy Long and Ms. Sonja Dickey performed these assays Table 1, 2 and 3 displays a plot of the microbiological data for these 20 -lactams.

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Table II-1. Zo n es of i n h i b i t io n o b t ai n edfro m agarwelldiffusio n e x peri m e n t s usi n g 20 gof t h e t es t co m pou n d.T h evaluescorrespond to the diameters in mm for the zone of growth inhibition appearing around the well after 24 hours. Penicillin G (Pen G) was used as a reference antibiotic. Staphylococcus aureus and-lactamase-producing strains of methicillin-resistant Staphylococcus aureus were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL (labeled MRSA USF652-659) or from ATCC sources. ("nt" indicates "not tested") Microorganisms cis-7a cis-7b cis-7c cis-7d trans-7d cis-7e cis-13 trans-13 Pen GMRSA USF652 12 17 17 11 9 0 18 18 8MRSA USF653 14 14 13 8 8 0 21 23 15MRSA USF654 8 9 11 10 9 0 19 20 10MRSA USF655 14 12 13 9 9 0 20 22 14MRSA USF656 11 11 14 10 8 0 20 22 12 MRSA USF657 12 14 14 9 9 0 19 21 12MRSA USF658 9 11 12 9 8 0 21 23 19MRSA USF659 13 13 12 10 9 0 18 19 16S. aureus ATCC 25923 13 13 13 11 10 0 16 18 33S. epidermidis 12 16 12 9 8 0 nt nt 50 22

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Table II-2. Zo n es of i n h i b itio n o b tai n edfro m agarwelldiffusio n e x peri m e n tsusi n g20 goft h etestco m pou n d.The values correspond to the diameters in mm for the zone of growth inhibition appearing around the well after24 hours. Penicillin G (Pen G) was used as a reference antibiotic. Staphylococcus aureus and -lactamase-producing strains of methicillin-resistant Staphylococcus aureus were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL (labeled MRSAUSF652-659) or from ATCC sources. Microorganisms cis-15a trans-15a cis-15b trans-15c trans-15d cis-15e trans-15e Pen GMRSA USF652 15 18 14 15 9 0 0 8 MRSA USF653 12 15 12 11 8 0 0 15MRSA USF654 12 16 11 11 9 0 0 10MRSA USF655 13 16 12 13 8 0 0 14MRSA USF656 14 17 14 14 9 0 0 12 MRSA USF657 11 15 11 12 8 0 0 12MRSA USF658 14 15 13 11 8 0 0 19MRSA USF659 13 18 13 13 9 0 0 16S. aureus ATCC 25923 14 17 13 12 9 0 0 33S. epidermidis 14 15 12 11 8 0 0 50 23

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Table II-3 Zo n esofi n h i b i t io n o b t ai n edfro m agarwelldiffusio n e x peri m e n t susi n g20 gof the test compound. The values correspond to the diameters in mm for the zone of growth inhibition appearing around the well after 24 hours. Penicillin G (Pen G) was used as a reference antibiotic. Staphylococcus aureus and -lactamase-producing strains of methicillin-resistant Staphylococcus aureus were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL (labeled MRSAUSF652-659) or from ATCC sources. Microorganisms 20a 20b 20c 20d 20e Pen GMRSA USF652 23 21 18 13 15 8 MRSA USF653 24 20 20 9 15 15MRSA USF654 21 18 18 8 14 10MRSA USF655 23 21 19 9 16 14MRSA USF656 22 18 18 10 15 12 MRSA USF657 20 17 15 9 14 12MRSA USF658 21 18 16 10 16 19MRSA USF659 15 17 13 8 17 16S. aureus ATCC 25923 23 20 20 9 13 33 24

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2.4 Discussion It is apparent that -lactams cis-7a, cis-7b, and cis-7c are about equal in potency, suggesting that alkyl chain length of the benzoate ester moiety may not influence in vitro antimicrobial activity. However, activity drops dramatically for derivatives having seven carbons (cis-7d and trans-7d) or more in the ester chain, as for the inactive decyl ester cis-7e. Fig. 1 displays the observed structure-activity relationship for -lactams 7. 25 R = CH3CH3CH2CH3(CH2)4CH3(CH2)6CH3(CH2)6CH3(CH2)9 (Tested as racemate)MRSA MRSAMRSAMRSAMRSAMRSASize ofZone(mm)NOSOCH3OOCH3 R MRSA7 trans cis Fig. 2-1 Effect of increasing R chain length on antibacterial activity against MRSA for -lactams 5. In addition to the fatty ester residues on the C 4 aryl ring, as in the above series of compounds, the substitution of the C 3 methoxy group for an acetoxy group also can provides a good opportunity to study of two series of compounds for biological activity.

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Fig. 2 displays the structure-activity relationship for -lactams 7 and 15. It is clear that -lactams 7 and 15 have about equal biological activity, suggesting that C 3 methoxy and acetoxy moieties may exert equal influence over antimicrobial activity. However, for both -lactams 7 and 15, activity drops dramatically for derivatives having seven carbons or more in the ester chain, as for the inactive decyl ester analogs. Size ofZone(mm) 26 R = CH3CH3CH2CH3(CH2)4CH3(CH2)6CH3(CH2)9 transNOSOO O RCH3 CH3 O NOSOORCH3 OCH3 MRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSA715 Fig. 2-2 Comparison of bioactivities for methoxy and acetoxy -lactams 7 & 15. It is also interesting to study side-by-side the cis and trans isomers of C 3 -acetoxy N-methylthio -lactams 15. Fig. 3 shows that there is not much difference between the cis and trans analogs, and once again, activity drops dramatically for derivatives having seven carbons or more in the ester chain. In the case of methyl ester analogs, the trans isomer was found to be about 10% more active than the cis analogs.

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R = CH3CH3CH2CH3(CH2)4CH3(CH2)6CH3(CH2)9R Size ofZone(mm)NOSOOOCH3 CH3 O NOSOOOCH3 R CH3 O MRSAMRSAMRSAMRSAMRSAMRSAtranscis Fig. 2-3 Comparison of bioactivities for trans and cis -lactams 15 Fig. 4 displays the comparison of bioactivities for ortho and para ethyl ester analogs. It is apparent that ortho analogs have about 40% more potent activity than that of the para analogs, so bioactivities depend on the position of the ester substituent on the phenyl ring. The reason for more potent activity of the ortho analogs is not clear yet. The other hand, C 3 -alkyl carbonate and methyl dithiocarbonate analogs 20 are good candidates for examining the effect of different functionalities and chain branching on bioactivity at C 3 Fig 5. shows the comparison of activities as increasing the bulkiness of the alkyl group. Upon increasing the size of alkyl group, the activity gradually drops up to 50% compared to the methyl and phenyl analogs. It is also apparent that the methyl carbonate analog has about 25% more activity than that of methyl dithiocarbonate. 27

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Size ofZone(mm)NOSOCH3 CH3 O O O CH3 NOSOCH3 O O CH3 CH3 NOSOCH3 CH3 O O CH3 NOSOCH3 CH3 O O CH3 MRSAMRSAMRSAMRSA Fig. 2-4 Comparison of bioactivities for ortho and para isomers 0510152025 N O S O O O N O S O S S 28 R CH3 CH3 CH3CH3CH2C(CH3)3C6H5 CH3 MRSAMRSAMRSAMRSAMRSASize ofZone(mm)20a20b20c20d20e Fig. 2-5 Comparison of bioactivities for MRSA

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29 2.5 Conclusion In general, the antibacterial properties of the fatty ester N-methylthio -lactams is loosely associated with lipophilicity within the C 4 side chain. Activity drops off rapidly when more than seven carbon atoms are in the ester. However, the cis and trans stereochemistry and methoxy/ acetoxy substitution at C 3 seem to exert no significant effect. Ortho -substituted C 4 -aryl analogs have more poten t activity than that of the para analogs. Therefore, as these studies show, th ere is an optimal chain length for the fatty ester groups, with activity dropping off rapidly when more than seven carbon atoms are in the chain. These results gave us the idea about developing a -lactam prodrug system and a -lactam conjugated polymer drug delivery system. The inactive form of -lactam conjugated in the long cacrbon chain like the polymer can be changed to the active form after biological or enzymatical cleava ge from polymer. In the next chaper, -lactam conjugated polymeric nanoparticles are going to be described as a possible new drug delivery system.

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30 2.6 Experimental All reagents were purchased fr om Sigma-Aldrich Chemical Company and used without further purification. So lvents were obtained from Fi sher Scientific Company. Thin layer chromatography (TLC) was carried out using EM Reagent plates with a fluorescence indicator (SiO 2 -60, F-254). Products were pur ified by flash chromatography using J.T. Baker flash chromatography silica gel (40 m). NMR spectra were recorded in CDCl 3 unless otherwise noted. 13 C NMR spectra were prot on broad-band decoupled. Procedure for the Synthesis of 4-( Methoxycarbonyl)benzaldehyde (3a) To a stirred milky solution of 4-carboxyben zaldehyde (1.00 g, 6.7 mmol) in dry methanol (20 ml) in an ice-water bath was added dropwise thionyl chloride (0.54 ml, 7.3 mmol). After 30 min, the mixture was allowed to warm to room temperature and stirred for an additional 8 h. The mixture was diluted with CH 2 Cl 2 (20 ml), followed by evaporation under reduced pressure to 1.08 g (99%) of aldehyde 3a as an off-white solid. mp 44-45 o C. 1 H NMR (250 MHz) 10.11 (s, 1H), 8.21 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 3.97 (s, 3H). 13 C NMR (63 MHz) 191.6, 165.4, 139.0, 135.3, 130.0, 129.4, 61.5.

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31 Procedure for the Synthesis of 4-(A lkyloxycarbonyl)benzaldehydes 3b-3e To a stirred milky solution of 4-car boxybenzaldehyde (1g, 6.66 mmol) and DCC (1.51g, 7.33 mmol) in dry CH 2 Cl 2 (20 ml) at rt was added dry et hanol (1 ml). The resultant mixture was refluxed for 8 h. The solvent was removed under reduced pr essure to yield a white solid. The crude material was purified by flash column chromatography on silica gel (1:9 EtOAc:hexanes) to afford 0.51 g (43%) of aldehyde 3b as a colorless semi-solid. 1 H NMR (250 MHz) 9.97 (s, 1H), 8.06 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 4.29 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 191.6, 165.3, 139.0, 135.2, 130.0, 129.3, 61.4, 14.1. 4-(Pentoxycarbonyl)benzaldehyde (3c) Colorless semi-solid, 50%. 1 H NMR (250 MHz) 10.06 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 4.31 (t, J = 6.7 Hz, 2H), 1.73 (m, 2H), 1.36 (m, 6H), 0.90 (m, 3H) 13 C NMR (63 MHz) 191.6, 165.5, 139.0, 135.4, 130.1, 129.4, 65.7, 28.3, 28.1, 22.3, 13.9. 4-(Heptyloxycarbonyl)benzaldehyde (3d) Colorless semi-solid, 45%. 1 H NMR (250 MHz) 10.05 (s, 1H), 8.14 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 4.30 (t, J = 6.7 Hz, 2H), 1.74 (m, 2H), 1.26-1.38 (m, 8H), 0.84 (m, 3H). 13 C NMR (63 MHz) 191.6, 165.5, 139.0, 135.4, 130.0, 129.4, 65.7, 31.6, 28.9, 28.6, 25.9, 22.5, 14.0. 4-(Decyloxycarbonyl)benzaldehyde (3e) Colorless semi-solid, 36%. 1 H NMR (250 MHz) 10.08 (s, 1H), 8.17 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.3 Hz, 2H), 4.33 (t, J = 6.7 Hz, 2H), 1.76 (m, 2H), 1.24-1.41 (m, 14H), 0.84

PAGE 45

32 (m, 3H). 13 C NMR (63 MHz) 191.6, 165.5, 139.0, 135.4, 130.1, 129.4, 65.7, 31.8, 29.5 (double), 29.2 (double), 26.0, 22.6, 14.1. Procedure for the Synthesis of N -(4-Methoxyphenyl)-Substituted Imines 4. To a solution of p-anisidine (9.45 g, 77.0 mm ol) in 50 ml of CH 2 Cl 2 was added benzaldehyde (10.50 g, 64.0 mmol) and a catalytic amount of camphorsulfonic acid. The resultant mixture was stirred until TLC indicated the disappearance of starting materials. The solvent was removed under reduced pressu re, and the crude material was purified by recrystallization from metha nol to yield 17.02 g (98%) of 4a as a yellow solid. mp 157159 C. 1 H NMR (250 MHz) 8.54 (s, 1H), 8.14 (d, J = 7.9 Hz, 2H), 7.97 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 3.96 (s, 3H), 3.85 (s, 3H). 13 C NMR (63 MHz) 166.3, 158.7, 156.9, 144.2, 140.2, 132.3, 129.9, 128.4, 122.4, 114.4, 55.4, 52.3. An analogous procedure was used to prepare lactams 4b-e 4-(Ethoxycarbonyl)benzaldehyde N -(4-methoxyphenyl)imine ( 4b ). Yellow solid, mp 104-107 C, 98%. 1 H NMR (250 MHz) 8.51 (s, 1H), 8.11 (d, J = 8.1 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 166.2, 158.7, 156.9, 144.2, 140.2, 132.3, 129.9, 128.3, 122.4, 114.4, 61.2, 55.5, 14.3. 4-(Pentoxycarbonyl)benzaldehyde N -(4-methoxyphenyl)imine ( 4c ). Yellow solid, mp 68-70C, 67%. 1 H NMR (250 MHz) 8.54 (s, 1H), 8.13 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 4.34 (t, J = 6.7 Hz, 2H), 3.84 (s, 3H), 1.80 (m, 2H), 1.42 (m, 4H), 0.94 (m, 3H). 13 C NMR (63

PAGE 46

33 MHz) 166.2, 158.7, 156.9, 144.2, 140.2, 132. 3, 129.9, 128.3, 122.4, 114.4, 65.4, 55.5, 28.4, 28.2, 22.4, 14.0. 4-(Heptyloxycarbonyl)benzaldehyde N -(4-methoxyphenyl)imine ( 4d ). Yellow solid, mp 59-60 C, 89%. 1 H NMR (250 MHz) 8.54 (s, 1H), 8.13 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.9 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 4.34 (t, J = 6.7 Hz, 2H), 3.84 (s, 3H), 1.80 (m, 2H), 1.32-1.44 (m, 8H), 0.90 (m, 3H), 13 C NMR (63 MHz) 166.2, 158.7, 156.8, 144.2, 140.2, 132. 3, 129.9, 128.3, 122.4, 114.4, 65.4, 55.5, 31.7, 28.9, 28.7, 26.0, 22.6, 14.1. 4-(Decyloxycarbonyl)benzaldehyde N -(4-methoxyphenyl)imine ( 4e ). Yellow solid, mp 40-41C, 91%. 1 H NMR (250 MHz) 8.54 (s, 1H), 8.13 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 4.34 (t, J = 6.7 Hz, 2H), 3.85 (s, 3H), 1.79 (m, 2H), 1.27-1.44 (m, 14H), 0.88 (m, 3H), 13 C NMR (63 MHz) 166.2, 158.7, 156.9, 144.2, 140.2, 132.3, 129.9, 128.3, 122.4, 114.4, 82.6, 65.4, 63.7, 60.8, 55.3, 52.3, 28.4, 28.1, 22.3, 20.4, 13.9. Procedure for the Formation of N -(4-Methoxyphenyl)-substituted -Lactams 5. To a stirred solution of imine 4 (6.94 g, 25.8 mmol) and triethylamine (10.8 ml, 77.4 mmol) was added dropwise over 10 minutes a solution of methoxyacetyl chloride (2.83 ml, 37.7 mmol) in CH 2 Cl 2 The resultant mixture was stirre d at rt until TLC indicated the disappearance of starting material. The so lvent was removed under reduced pressure, and the crude material was purified by washing wi th ice-cold methanol to afford 6.89 g (78%) of lactam cis5a as a white solid. mp 157-159 C. 1 H NMR (250 MHz) 8.05 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 5.22

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34 (d, J = 4.7 Hz, 1H), 4.84 (d, J = 4.7 Hz, 1H), 3.91 (s, 3H), 3.74 (s, 3H), 3.20 (s, 3H). 13 C NMR (63 MHz) 166.7, 163.4, 156.5, 138.7, 130.4, 130.3, 129.8, 128.0, 118.7, 114.4, 84.9, 61.4, 58.5, 55.4, 52.2. An analogous procedure was used to prepare lactams 5b-e cis4-(4-Ethoxycarbonylphenyl)-3-methoxyN -(4-methoxyphenyl)-2-azetidinone ( cis 5b) White solid, mp 104-107 C, 18%. 1 H NMR (250 MHz) 8.05 (d, J = 8.2 Hz, 2H). 7.46 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 5.22 (d, J = 4.8 Hz, 1H), 4.85 (d, J = 4.8 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.73 (s, 3H ), 3.19 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H), 13 C NMR (63 MHz) 166.1, 163.4, 156.4, 138.6, 130.8, 130.3, 129.7, 127.9, 118.6, 114.3, 84.9, 61.4, 61.1, 58.5, 55.4, 14.3. cis3-MethoxyN -(4-methoxyphenyl)-4-(4-pentoxycar bonylphenyl)-2-azetidinone ( cis -5c) White solid, mp 68-70 C, 17%. 1 H NMR (250 MHz) 8.08 (d, J = 8.3 Hz, 2H). 7.49 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 5.22 (d, J = 4.7 Hz, 1H), 4.88 (d, J = 4.7 Hz, 1H), 4.34 (t, J = 6.4 Hz, 2H), 3.78 (s, 3H), 3.52 (s, 3H), 1.79 (quintet, J = 6.9 Hz, 2H), 1.43 (m, 4H), 0.94 (t, J = 6.9 Hz, 3H), 13 C NMR (63 MHz) 166.2, 163.4, 156.4, 138.6, 130.8, 130.3, 129.8, 127.9, 118.7, 114.4, 85.0, 65.2, 61.4, 58.6, 55.4, 28.4, 28.2, 22.3, 14.0. cis -4-(4-Heptyloxycarbonylphenyl)-3-methoxyN -(4-methoxyphenyl)-2-azetidinone ( cis -5d)

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35 White solid, mp 51-53C, 19%. 1 H NMR (250 MHz) 8.06 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.9 Hz, 2H), 6.79 (d, J = 8.9 Hz, 2H), 5.23 (d, J = 4.7 Hz, 1H), 4.85 (d, J = 4.7 Hz, 1H), 4.31 (t, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.21 (s, 3H), 1.75 (m, 2H), 1.30-1.38 (m, 8H), 0.89 (m, 3H). 13 C NMR (63 MHz) 166.2, 163.4, 156.4, 138.6, 130.8, 130.3, 129.8, 127.9, 118.7, 114.4, 85.0, 65.2, 61.4, 58.5, 55.4, 31.7, 28.9, 28.7, 26.0, 22.6, 14.0. trans-4-(4-Heptyloxycarbonylphenyl) 3-methoxyN -(4-methoxyphenyl)-2azetidinone ( trans-5d) White solid, mp 55-57C, 18%. 1 H NMR (250 MHz) 8.09 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 6.80 (d, J = 8.9 Hz, 2H), 4.97 (app s, 1H), 4.43 (app s, 1H), 4.34 (t, J = 6.4 Hz, 2H), 3.75 (s, 3H), 3.60 (s, 3H), 1.78 (quintet, J = 6.5 Hz, 2H), 1.22-1.38 (m, 8H), 0.90 (m, 3H). 13 C NMR (63 MHz) 166.0, 163.1, 156.5, 141.3, 131.0, 130.5, 130.2, 126.0, 118.8, 114.4, 91.2, 65.3, 62.8, 58.2, 55.4, 31.7, 29.0, 28.7, 26.0, 22.6, 14.1. cis4-(4-Decyloxycarbony lphenyl)-3-methoxyN -(4-methoxyphenyl)-2-azetidinone ( cis -5e) White solid, mp 95-96C, 63%. 1 H NMR (250 MHz) 8.05 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 5.22 (d, J = 4.7 Hz, 1H), 4.85 (d, J = 4.7 Hz, 1H), 4.30 (t, J = 6.4 Hz, 2H), 3.74 (s, 3H), 3.21 (s, 3H), 1.75 (m, 2H), 1.26-1.42 (m, 14H), 0.89 (m, 3H). 13 C NMR (63 MHz) 166.2, 163.4, 156.5, 138.6, 130.9, 130.3, 129.8, 127.9, 118.7, 114.4, 85.0, 65.3, 61.4, 58.6, 55.4, 31.8, 29.5 (double), 29.3 (double), 28.7, 26.0, 22.6, 14.1.

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36 Procedure for the N -Dearylation of N -(4-Methoxylphenyl)-substituted -Lactams 6. To a solution of cis5a (1.00 g, 2.93 mmol) in 15 ml of ac etonitrile in ice-water bath, was added ceric ammonium nitr ate (4.82 g, 8.79 mmol) in 15 ml of water. The resultant mixture was stirred for 5 minutes, and 20 ml of water was added. The solution was extracted (3 x 25 ml) with Et OAc. The combined organic layers were washed with 75 ml of 5% NaHSO 3 5% NaCO 3 and dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown oil, which after purification by flash column chromatography on silica gel (1:2 EtOAc:hexanes) afforded 0.45 g (65% yield) of lactam cis -6a as a brown oil. 1 H NMR (250 MHz) 8.03 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 6.40 (bs, 1H), 4.89 (d, J = 4.5 Hz, 1H), 4.77 (dd, J = 4.5, 2.7 Hz, 1H), 3.90 (s, 3H), 3.15 (s, 3H). An analogous procedure was used to prepare lactams 6b-e cis4-(4-Ethoxycarbonylphenyl)-3 -methoxy-2-azetidinone ( cis -6b) Brown oil, 72%. 1 H NMR (250 MHz) 8.08 (d, J = 8.2 Hz, 2H). 7.47 (d, J = 8.2 Hz, 2H), 6.70 (bs, 1H), 4.93 (d, J = 4.5 Hz, 1H), 4.81 (dd, J = 4.5, 2.9 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 3.19 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). cis3-Methoxy-4-(4-pentoxycarbonylphenyl)-2-azetidinone ( cis-6c ). Brown oil, 19%. 1 H NMR (250 MHz) 8.09 (d, J = 8.2 Hz, 2H). 7.48 (d, J = 8.2 Hz, 2H), 6.36 (bs, 1H), 4.95 (d, J = 4.7 Hz, 1H), 4.83 (dd, J = 4.7, 2.8 Hz, 1H), 4.35 (t, J = 6.7 Hz, 2H), 3.22 (s, 3H), 1.79 (quintet, J = 6.7 Hz, 2H), 1.43 (m, 4H), 0.94 (t, J = 6.9 Hz, 3H). cis -4-(4-Heptyloxycarbonylphenyl)-3-methoxy-2-azetidinone ( cis-6d )

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37 Brown oil, 89%. 1 H NMR (250 MHz) 8.06 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 6.38 (bs, 1H), 4.92 (d, J = 4.5 Hz, 1H), 4.80 (dd, J = 4.5, 2.8 Hz, 1H), 4.31 (t, J = 6.7 Hz, 2H), 3.18 (s, 3H), 1.78 (quintet, J = 6.5 Hz, 2H), 1.22-1.38 (m, 8H), 0.89 (m, 3H). trans-4-(4-Heptyloxycarbonylpheny l)-3-methoxy-2-azetidinone ( trans6d ). Brown oil, 88%. 1 H NMR (250 MHz) 8.11 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 6.35 (bs, 1H), 4.64 (app s, 1H), 4.43 (app s, 1H), 4.34 (t, J = 6.4 Hz, 2H), 3.60 (s, 3H), 1.78 (quintet, J = 6.5 Hz, 2H), 1.22-1.38 (m, 8H), 0.90 (m, 3H). cis -4-(4-Decyloxycarbonylphenyl)-3-methoxy-2-azetidinone ( cis -6e). Brown oil, 91%. 1 H NMR (250 MHz) 8.02 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 6.89 (bs, 1H), 4.89 (d, J = 4.5 Hz, 1H), 4.77 (dd, J = 4.5, 2.5 Hz, 1H), 4.28 (t, J = 6.6 Hz, 2H), 3.13 (s, 3H), 1.75 (quintet, J = 7.1 Hz, 2H), 1.26-1.42 (m, 14H), 0.89 (t, J = 6.7 Hz, 3H). Procedure for the N -Methylthiolation of Lactams 7. To a solution of cis6a (455 mg, 1.91 mmol) in 10 ml of dry CH 2 Cl 2 was added N (methylthio)phthalimide (410 mg, 2.10 mmol ) and 0.25 ml of triethylamine. The resultant mixture was refluxed overnight. The solvent was removed under reduced pressure to yield a brown solid. The brown solid was redissolved in CH 2 Cl 2 and washed with 1% aqueous NaOH. The organi c layer was dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown oil, which after purification by column chromatography on silic a gel (1:4 EtOAc:hexanes) yielded 0.356 g (66%) of cis7a as a white solid. mp 118-120 C. 1 H NMR (250 MHz) 8.03 (d, J =

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38 8.1 Hz, 2H). 7.45 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.9 Hz 2H), 3.18 (s, 3H), 5.22 (d, J = 4.7 Hz, 1H), 4.84 (d, J = 4.7 Hz, 1H), 3.90 (s, 3H), 3.72 (s, 3H). 13 C NMR (63 MHz) 170.2, 167.0, 138.7, 130.7, 129.6, 12 8.8, 86.7, 65.8, 58.5, 52.2, 22.1. An analogous procedure was used to prepare lactams 7b-e cis -4-(4-Ethoxycarbonylphenyl)-3-methoxy -N -methylthio-2-azetidinone ( cis -7b) White solid, mp 113-114 C, 70%. 1 H NMR (250 MHz) 8.03 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 5.22 (d, J = 4.8 Hz, 1H), 4.84 (d, J = 4.8 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.73 (s, 3H), 3.19 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 170.1, 166.2, 138.6, 131.0, 129.5, 128.8, 86.7, 65.8, 61.1, 58.4, 22.1, 14.3. cis3-MethoxyN -methylthio-4-(4-pentoxycarbo nylphenyl)-2-azetidinone ( cis -7c) White solid, mp 84-85 C, 79%. 1 H NMR (250 MHz) 8.07 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 4.88 (d, J = 4.8 Hz, 1H), 4.82 (d, J = 4.8 Hz, 1H), 4.32 (t, J = 6.5 Hz, 2H), 3.17 (s, 3H), 2.39 (s, 3H), 1.77 (m, 2H), 1.41 (m, 4H), 0.93 (t, J = 6.5 Hz, 3H). 13 C NMR (63 MHz) 170.2, 166.3, 138.6, 131.1, 129.5, 128.8, 86.8, 66.0, 65.4, 58.4, 28.3, 28.2, 22.1, 22.0, 13.9. cis4-(4-Heptyloxycarbonylphenyl)-3-methoxyN -methylthio-2-azetidinone ( cis -7d ). White solid, mp 51-52 C, 70%. 1 H NMR (250 MHz) 8.08 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 4.88 (d, J = 4.9 Hz, 1H), 4.82 (d, J = 4.9 Hz, 1H), 4.33 (t, J = 6.6 Hz, 2H), 3.18 (s, 3H), 2.39 (s, 3H), 1.75 (m, 2H), 1.25-1.42 (m, 8H), 0.90 (m, 3H). 13 C NMR (63 MHz) 170.1, 166.1, 138.5, 131.3, 129.5, 128.8, 87.0, 65.4, 65.0, 58.4, 31.7, 29.1, 28.9, 26.0, 23.0, 22.6, 14.1.

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39 trans4-(4-Heptyloxycarbonylphenyl)-3-methoxyN -methylthio-2-azetidinone ( trans-7d ). White solid, 53-55 C, 68%. 1 H NMR (250 MHz) 8.09 (d, J = 7.9 Hz, 2H). 7.38 (d, J = 7.9 Hz, 2H), 4.67 (app s, 1H), 4.44 (app s, 1H), 4.33 (t, J = 6.5 Hz, 2H), 3.53 (s, 3H), 2.43 (s, 3H), 1.77 (m, 2H), 1.25-1.41 (m, 8H), 0.89 (m, 3H). 13 C NMR (63 MHz) 170.1, 166.0, 140.9, 131.2, 130.3, 126.8, 91.7, 66.2, 65.4, 58.4, 31.7, 29.7, 28.9, 26.0, 22.6, 21.8, 14.0. cis4-(4-Decyloxycarbony lphenyl)-3-methoxyN -methylthio-2-azetidinone ( cis -7e ). White solid, 45-46 C, 81%. 1 H NMR (250 MHz) 8.06 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 4.88 (d, J = 5.0 Hz, 1H), 4.82 (d, J = 5.0 Hz, 1H), 4.31 (t, J = 6.7 Hz, 2H), 3.15 (s, 3H), 2.38 (s, 3H), 1.76 (m, 2H), 1.26-1.42 (m, 14H), 0.87 (m, 3H), 13 C NMR (63 MHz) 170.1, 166.2, 138.6, 131.0, 129.5, 128.8, 86.7, 65.8, 65.3, 58.5, 31.8, 29.5, 29.3, 28.7, 26.0, 22.7, 22.1, 14.0. Procedure for the Synthesis of 2-Ethoxycarbonylbenzaldehyde (9) To a stirred solution of 2-carboxybenzaldehyde 8 (5.00 g, 33.3 mmol) in dry acetone (20 ml) was added K 2 CO 3 (4.60g, 40.0 mmol) and the solution was stirred for 1h. To this was added dropwise a solution of ethyl iodide (3.2 ml, 40 mmol) in dry acetone (1 ml), and the mixture was refluxed for overnight. The solvent was removed under reduced pressure to yield a brown semi-solid. The brown solid was redissolved in EtOAc, and washed with water. The organic layer was dried over anhydrous MgSO 4 The crude material was purified by column chromatography on silica gel (1:9 EtOAc:hexanes) to yield 1.07 g (18%) of aldehyde 9 as a colorless semi-solid. 1 H NMR (250 MHz) 10.58

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40 (s, 1H), 7.88-7.96 (m, 2H), 7.53-7.63 (m, 2H), 4.41 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 192.2, 136.9, 132.9, 132.4, 132.2, 130.7, 130.3, 128.3, 62.0, 14.2. Procedure for the Synthesis of N -(4-Methoxyphenyl)-4-ethoxycarbonyl phenylimine (10). To a solution of p-anisidine (0.55 g, 4.5 mmol) in 10 ml of CH 2 Cl 2 was added aldehyde 9 (0.80 g, 4.5 mmol) and a catalytic amount of camphorsulfonic acid. The resultant mixture was stirred at rt until TLC indicated the disappearance of both starting materials. The solvent was removed under reduced pressu re, and the crude material was purified by recrystallization from methanol to yield 0.76 g (60%) of imine 9 as a yellow solid. mp 52-53 C. 1 H NMR (250 MHz) 9.23 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.58 (t, J = 7.1 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 4.38 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H),1.38 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 166.9, 158.3, 157.6, 144.8, 137.3, 132.1, 130.7, 130.3, 129.9, 128.1, 122.5, 114.2, 61.3, 55.4, 14.2. Synthesis of 4-(2-Ethoxycarbonylphenyl)-3-methoxyN -(4-methoxyphenyl)-2azetidinone ( cis and trans adduct) To a stirred solution of imine 4 (0.34 g, 1.21 mmol) and triethylamine (0.51 ml, 3.63 mmol) was added dropwise over 10 minutes a solution of methoxyacetyl chloride (0.22 ml, 2.42 mmol) in CH 2 Cl 2 The resultant mixture was refluxed for overnight. The solvent was removed under reduced pressure to yiel d a brown oil. The crude material was

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41 purified by flash column chromatography on s ilica gel (1:4 EtOAc:hexanes) to afford 0.18 g (36%) of lactam 11, Brown semisolid (cis:trans = 1:1 mixture), 1 H NMR (250 MHz) 1.40 (t, J=7.1 Hz, 3H), 3.19 (s, 3H), 3.73 (s, 3H), 4.37 (q, J=7.1 Hz, 2H), 4.84 (d, J=4.8 Hz, 1H,), 5.22 (d, J=4.8 Hz, 1H,), 6.77 (d, J=8.9 Hz 2H), 7.22 (d, J=8.9 Hz, 2H), 7.45 (d, J=8.1 Hz, 2H), 8.03 (d, J=8.1 Hz, 2H). 13 C NMR (63 MHz) 14.3, 22.1, 58.4, 61.1, 65.8, 86.7, 128.8, 129.5, 131.0, 138.6, 166.2, 170.1. Procedure for synthesis of 3-Methoxy-4 -(2-ethyloxycarbonyl)phenyl-2-azetidinone ( 12) To a solution of 10 (0.18 g, 0.52 mmol) in 1 ml of CH 3 CN in an ice-water bath was added a solution of ceric ammonium nitrate (0.86 g, 1.57 mmol) in 1 ml of water. The resultant mixture was stirred for 5 min, and 2 mL of water was added. The solution was extracted (3 x 5 ml) with EtOAc. The combined organic layers we re washed with 5% NaHSO 3 5% NaHCO 3 and dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown oil, which was purified by flash column chromatography on silica gel (1:2 EtOAc:hexanes) to afford 47 mg (36%) of lactam 11 as a brown semi-solid (1:1 cis:trans mixture). 1 H NMR (250 MHz) 8.08 (d, J = 7.5 Hz, 1H). 8.02 (dd, J = 7.5, 1.5 Hz, 2H), 7.41 (m, 8H), 7.28 (m, 8H), 6.78 (two d, J = 9.0 Hz, 6H), 6.01 (d, J = 5.1 Hz, 1H), 5.98 (s, 2H), 4.95 (d, J = 5.1 Hz, 1H), 4.43 (q, J = 7.1 Hz, 4H), 3.71 (s, 6H), 3.59 (s, 6H), 1.42 (two t, J = 7.1 Hz, 6H), 13 C NMR (63 MHz) 166.9, 166.6, 164.6, 163.9, 156.3, 137.8, 136.0, 132.8, 132.4, 131.2, 131.0, 130.7, 130.3, 129.4, 129.2, 128.1, 127.8, 126.1, 118.9, 118.6, 114.3, 91.5, 85.6, 61.5, 61.2, 60.3, 59.1, 58.9, 57.9, 55.3, 30.9, 14.3. Procedure for the Synthesis of N-Methylthiolation of -Lactams 13.

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42 To a solution of 12 (47 mg, 0.19 mmol) in 2 ml of dry CH 2 Cl 2 was added N(methylthio)phthalimide (55 mg, 0.28 mmol) and 3-5 drops of triethylamine. The resultant mixture was refl uxed overnight. The solven t was removed under reduced pressure to yield a brown solid. The brown solid was redissolved in methylene chloride, and washed with 1% sodium hydroxide. The organic laye r was dried over magnesium sulphate. The solvent was removed under redu ced pressure to yield a brown semi-solid. The crude material was purified by column chromatography on silica gel using a gradient elution (1:19, 1:9 and 1:4 EtOAc:hex anes) to yield 23 mg (35%) of cis -lactam 13a and 24 mg (37%) of trans -lactam 13b as white solids (total 72% yield). cis -4-(2-Ethoxycarbonylphenyl)-3-methoxy -N -methylthio-2-azetidinone (13a). White solid, mp 95-97 C, 35%. 1 H NMR (250 MHz) 8.06(m, 1H). 7.61(m, 1H), 7.43 (m, 2H), 5.72 (d, J = 5.1 Hz, 1H), 4.95 (d, J = 5.1 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.23 (s, 3H), 2.48 (s, 3H), 1.43 (t, J = 7.1 Hz 3H). 13 C NMR (63 MHz) 171.3, 166.7, 136.1, 132.4, 130.9, 129.8, 127.9, 87.6, 63.8, 61.2, 58.9, 29.7, 21.5, 14.3. trans-4-(2-Ethoxycarbonylphenyl)-3-methoxy -N -methylthio-2-azetidinone (13b) White solid, mp 75-76 C, 37%. 1 H NMR (250 MHz) 8.01 (m, 1H), 7.58 (m, 1H), 7.43(m, 1H), 7.29 (m, 1H), 5.71 (d, J = 1.9 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 4.40 (d, J = 1.9 Hz, 1H), 3.56 (s, 3H), 2.46 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 171.1, 166.8, 137.6, 132.6, 131.1, 130.3, 128.2, 126.3, 92.3, 62.0, 61.5, 58.2, 21.6, 14.2. Procedure for the Synthesis of 3-AcetoxyN -(4-Methoxyphenyl)-substituted Lactams 14.

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43 To a stirred solution of imine 4a (0.50 g, 1.9 mmol) and triethylamine (0.56 g, 5.6 mmol) was added dropwise over 10 min a solution of acetoxyacetyl chloride (0.24 ml, 2.2 mmol ) in dry CH 2 Cl 2 (10 ml). The resultant mixture was stir red at rt for overnight. The solvent was removed under reduced pressure, and th e crude material was purified by column chromatography on silica gel by gradient elutio n (1:9 then 1:4 EtOAc: hexanes) to yield 175 mg (25%) of lactam 14a (1:1 cis : trans mixture) as a brown semi-solid. 1 H NMR (250 MHz) 8.00 (two d, J = 8.9 Hz, 4H), 7.38 (two d, J = 8.9 Hz, 4H), 7.20 (two d, J = 8.9 Hz, 4H), 6.77 (two d, J = 8.9 Hz, 4H), 5.94 (d, J = 4.8 Hz, 1H), 5.37 (d, J = 4.8 Hz, 1H), 5.32 (s, 1H, trans isomer), 4.94 (s, 1H, trans isomer), 3.89 (s, 6H), 3.72 (s, 3H), 3.71 (s, 3H), 2.17(s, 3H), 1.67 (s, 3H). An analogous procedure was used to prepare -lactams 14b-e 3-Acetoxy-4-(4-ethoxycarbonylphenyl)N -(4-methoxyphenyl)-2-azetidinone (14b) Brown semi-solid, 20:1 cis:trans mixture, 43%. Data for cis isomer: 1 H NMR (250 MHz) 8.02 (d, J = 7.9 Hz, 2H). 7.37 (d, J = 7.9 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 5.96 (d, J = 4.5 Hz, 1H), 5.38 (d, J = 4.5 Hz, 1H), 4.36 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H), 1.69 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H). 13 C NMR (63 MHz) 169.1, 166.0, 161.0, 156.7, 137.4, 130.9, 130.0, 129.7, 127.9, 118.7, 114.4, 76.3, 61.2, 61.1, 55.4, 19.9, 14.3. 3-AcetoxyN -(4-methoxyphenyl)-4-(4-pentoxycarbonylphenyl)-2-azetidinone (14c) Brown semi-solid, 1:5 cis:trans mixture, 12%. Data for the trans isomer: 1 H NMR (250 MHz) 8.03 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.9 Hz, 2H), 6.76 (d, J = 8.9 Hz, 2H), 5.31 (s, 1H), 4.95 (s, 1H), 4.27 (t, J = 6.5 Hz, 2H), 3.70 (s, 3H), 2.17 (s, 3H), 1.72 (m, 2H), 1.37 (m, 4H), 0.89 (m, 3H). 13 C NMR (63 MHz) 169.7, 165.9,

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44 160.7, 156.6, 140.0, 131.1, 130.3, 129.9, 126.4, 118.8, 114.4, 82.3, 65.2, 63.3, 55.3, 28.3, 28.1, 20.4, 13.9. 3-AcetoxyN -(4-methoxyphenyl)-4-(4-heptyloxycarbonylphenyl)-2-azetidinone (14d) Brown semi-solid, 1:2 cis:trans mixture, 40%. 1 H NMR (250 MHz) 8.04 (two d, J = 8.6 Hz, 3H), 7.42 (two d, J = 8.6 Hz, 3H), 7.20 (two d, J = 8.7 Hz, 3H), 6.79 (two d, J = 8.7 Hz, 3H), 5.97 (d, J = 4.6 Hz, 0.5H cis isomer), 5.39 (d, J = 4.6 Hz, 0.5H, cis isomer), 5.33 (s, 1H, trans isomer), 4.95 (s, 1H, trans isomer), 4.30 (t, J = 6.5 Hz, 3H), 3.73 (s, 4.5H), 2.19 (s, 3H), 1.74 (m, 3H), 1.69 (s, 1.5 H), 1.25-1.37 (m, 12H), 0.88 (m, 4.5H). 3-Acetoxy-4-(4-decyloxycarbonylphenyl)N -(4-methoxyphenyl)-2-azetidinone (14e) Brown semi-solid, 1:1 cis:trans mixture, 57%. 1 H NMR (250 MHz) 8.04 (two d, J = 8.5 Hz, 4H). 7.40 (two d, J = 8.5 Hz, 4H), 7.22 (two d, J = 8.9 Hz, 4H), 6.79 (two d, J = 8.9 Hz, 4H), 5.96 (d, J = 4.8 Hz, 1H, cis isomer), 5.39 (d, J = 4.8 Hz, 1H, cis isomer), 5.33 (s, 1H, trans isomer), 4.96 (s, 1H, trans isomer), 4.38 (m, 4H), 3.74 (s, 3H), 3.73 (s, 3H), 2.19 (s, 6H), 1.73 (m, 4H), 1.69 (s, 6 H), 1.26-1.69 (m, 28H), 0.87 (m, 6H). Procedure for the Synthesis of N -Methylthio-substituted Lactams 15. To a solution of 14a (100 mg, 0.27 mmol) in 2.5 ml of CH 3 CN in an ice-water bath was added ceric ammonium nitrat e (0.450 g, 0.81 mmol) in 2.5 ml of water. The resultant mixture was stirred for 5 min, and 5 ml of water was added. The solution was extracted (3x5 ml) with EtOAc. The combined organi c layers were washed with 5% NaHSO 3 5% NaHCO 3 and dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a crude brown oil, which was disso lved in 10 ml of dry CH 2 Cl 2 and N (methylthio)phthalimide (52 mg, 0.268 mmol) and 3-5 drops of triethylamine were added.

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45 The resultant mixture was refluxed for overnight. The solvent was removed under reduced pressure to yield a brown solid. The brown solid was redissolved in CH 2 Cl 2 and washed with 1% NaOH. The organic layer was dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown semi-solid, which was purified by column chromatography on silica gel with gradient el ution (1:9 then 1:4 EtOAc:hexanes) to yield 23.6 mg (18%) of a brown semi-solid containing lactams as a mixture of cis:trans isomers. The cis and trans isomers were separated by recrystallization in methanol to give cis -15a (8 mg) and trans15a (11 mg) in combined 18% yield. cis3-Acetoxy-4-(4-methoxycarbonylphenyl)N -methylthio-2-azetidinone ( cis -15a ). White solid, mp 81-83 C, 8%. 1 H NMR (250 MHz) 8.05 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 5.92 (d, J = 5.0 Hz, 1H), 5.08 (d, J = 5.0 Hz, 1H), 3.93 (s, 3H), 2.45 (s, 3H), 1.69 (s, 3H). 13 C NMR (63 MHz) 168.7, 168.1, 166.5, 137.6, 130.8, 129.5, 128.6, 78.1, 65.4, 52.2, 22.1, 19.8. trans3-Acetoxy-4-(4-methoxycarbonylphenyl)N -methylthio-2-azetidinone ( trans 15a). White solid, mp 66-68 C, 10%. 1 H NMR (250 MHz) 8.09(d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 5.38 (d, J = 1.5 Hz, 1H), 4.72 (d, J = 1.5 Hz, 1H), 3.92 (s, 3H), 2.43 (s, 3H), 2.16 (s, 3H). 13 C NMR (63 MHz) 169.4, 168.0, 166.4, 140.0, 131.0, 130.2, 127.1, 82.7, 66.0, 52.3, 21.8, 20.4. An analogous procedure was used to prepare lactams 15b-e cis3-Acetoxy-4-(4-ethoxycarbonylphenyl)N -methylthio-2-azetidinone ( cis 15b ). White solid, mp 95-97 C, 12%. 1 H NMR (250 MHz) 8.06 (d, J = 8.3 Hz, 2H), 7.35 (d,

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46 J = 8.3 Hz, 2H), 5.93 (d, J = 5.0 Hz, 1H), 5.07 (d, J = 5.0 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 2.45 (s, 3H), 1.71 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 14.3, 20.0, 22.1, 61.2, 65.4, 78.1, 128.6, 129.5, 131.1, 138.0, 166.9, 168.6, 169.2. 3-AcetoxyN -methylthio-4-(4-pentyloxycarbonylphenyl)-2-azetidinone ( trans 15c ). Brown oil, 1:7 cis:trans mixture, 5%. Data for trans isomer: 1 H NMR (250 MHz) 8.11(d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 5.39 (d, J = 1.9 Hz, 1H), 4.72 (d, J = 1.9 Hz, 1H), 4.33 (t, J = 6.6 Hz, 2H), 2.45 (s, 3H), 2.18 (s, 3H), 1.78 (m, 2H), 1.42 (m, 4H), 0.91 (m, 3H). 13 C NMR (63 MHz) 7.8, 20.6, 23.7, 24.3, 28.1, 28.4, 65.4, 66.1, 82.7, 127.1, 130.2, 131.2, 139.9, 166.4, 167.9, 169.3. trans3-Acetoxy-4-(4-heptyloxycarbonylphenyl)N -methylthio-2-azetidinone ( trans 15d ). Brown oil, 2%. 1 H NMR (250 MHz) 8.05 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 5.35 (app s, 1H), 4.70 (app s, 1H), 4.28 (t, J = 6.6 Hz, 2H), 2.40 (s, 3H), 2.12 (s, 3H), 1.72 (m, 2H), 1.21-1.36 (m, 8H), 0.84 (m, 3H). 13 C NMR (63 MHz) 169.4, 168.0, 165.9, 139.8, 131.3, 130.1, 127.1, 82.7, 65.9, 65.3, 31.6, 28.9, 28.6, 25.9, 22.1, 21.7, 20.3, 14.0. cis3-Acetoxy-4-(4-decyl oxycarbonylphenyl)N -methylthio-2-azetidinone ( cis -15e) Brown oil, 9%. 1 H NMR (250 MHz) 8.06 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 5.93 (d, J = 5.0 Hz, 1H), 5.08 (d, J = 5.0 Hz 1H), 4.33 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H), 1.77 (m, 2H), 1.71 (s, 3H), 1.28-1.43 (m, 14H), 0.88 (t, J = 6.2 Hz, 3H). 13 C NMR (63 MHz) 168.9, 168.3, 166.6, 137.7, 131.1, 129.5, 128.6, 78.1, 65.4, 31.9, 29.5, 29.3, 28.6, 26.0, 22.1, 19.9, 14.1. trans3-Acetoxy-4-(4-decyloxycarbonylphenyl)N -methylthio-2-azetidinone ( trans 15e ).

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47 Brown oil, 12%. 1 H NMR (250 MHz) 8.07 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 5.37 (d, J = 2.0 Hz, 1H), 4.71 (d, J = 2.0 Hz, 1H), 4.30 (t, J = 6.6 Hz, 2H), 2.42 (s, 3H), 2.14 (s, 3H), 1.74 (m, 2H), 1.24-1.40 (m, 14H), 0.85 (m, 3H). 13 C NMR (63 MHz) 169.4, 168.0, 165.9, 139.8, 131.3, 130.2, 127.1, 82.7, 66.0, 65.3, 31.8, 29.5, 29.2, 28.6, 26.0, 22.6, 21.8, 20.3, 14.1. Procedure for the Synthesis of 3-AcetoxyN -(4-methoxyphenyl)-4-phenyl-2azetidinone (16). To a stirred solution of N -(4-methoxyphenyl)imine (5.31 g, 25.2 mmol) and triethylamine (7.64 g, 75.5 mmol) was added a solution of acetoxyacetyl chloride (5.15 g, 37.7 mmol) in methylene chloride dropwise over 10 minutes The resultant mixture was stirred at rt until TLC indicated the disappearance of star ting material. The solvent was removed under reduced pressure, and the crude materi al was purified by washing with ice-cold methanol to give 6.89 g (89%) of 16 as white solid, mp 153-155 o C. 1 H NMR (250 MHz) 7.34-7.30 (t, 5H), 7.28 (d, J = 8.9 Hz, 2H) 6.79 (d, J = 8.9 Hz, 2H), 5.92 (d, J = 4.8 Hz, 1H), 5.33 (d, J = 4.8 Hz, 1H), 3.74 (s, 3H), 1.66 (s, 3H). 13 C NMR (63 MHz) 169.1, 161.2, 156.5, 132.2, 130.2, 128.7, 128.4, 127.8, 118.7, 61.3, 55.3, 19.7. Procedure for the Synthesis of 3-HydroxyN -(4-methoxyphenyl)-4-phenyl-2azetidinone (17). To a solution of -lactam 15 (21.2 g, 68.3 mmol) in 500 ml of acetone was added KOH (3.83 g, 68.3 mmol) in 50 ml of methanol. The resultant mixture was stirred for 5 minutes, and 300 ml of water was added. Th e product was precipitated and isolated by filtration to yield 18.0 g (98%) of 17 as a white solid, mp 199-202 o C. 1 H NMR (250

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48 MHz) 7.44-7.28 (m, 7H), 6.83-6.79 (q, 2H), 5.29 (d, J = 5.2 Hz, 1H), 5.90 (d, J = 5.2 Hz, 1H), 3.76 (s, 3H), 2.10 (bs, 1H). 13 C NMR (63 MHz, DMSO-d 6 ), 166.7, 155.9, 135.2, 131.1, 128.5, 128.4, 128.1, 114.8, 77.2, 62.2, 55.5. Procedure for the Synthesis of 3-(methoxycarbonyl)oxyN -(4-methoxyphenyl)-4phenyl-2-azetidinone ( 18a ). To a solution of -lactam 17 (500 mg, 1.86 mmol) in 5 ml of freshly distilled CH 2 Cl 2 was added NaH (60% suspension in mineral o il, 74.4 mg, 1.86 mmol), and the mixture was stirred for 15 min. Methyl chloroformate (0.17 ml, 2.23 mmol) was then added, and the resultant mixture was stirred for 5 h or until TLC indicated the disappearance of starting material. The reaction was quenched with a 5% solution of NH 4 Cl and extracted (3x20 ml) with CH 2 Cl 2 The combined organic layers were dried over anhydrous MgSO 4 and purified with column chromatography on silica gel (1:4, EtOAc:hexanes) to give 0.35 g (55%) of 17a as a white solid, mp 123-124 C. 1 H NMR (250 MHz) 7.35 (s, 5H), 7.28 (d, J = 8.9 Hz, 2H) 6.81 (d, J = 8.9 Hz, 2H), 5.86 (d, J = 4.8 Hz, 1H), 5.36 (d, J = 4.8 Hz, 1H), 3.75 (s, 3H), 3.55 (s, 3H). 13 C NMR (63 MHz) 160.2, 156.6, 154.0, 131.8, 130.2, 129.0, 128.6, 127.9, 118.8, 114.4, 78.5, 61.3, 55.4, 55.3. An analogous procedure was used to prepare lactams 18b-e. 3-(Ethoxycarbonyl)oxyN -(4-methoxyphenyl)-4-phenyl-2-azetidinone ( 18b ). White solid, mp 105-106 C, 39%. 1 H NMR (250 MHz) 7.35 (s, 5H), 7.28 (d, J = 9.0 Hz, 2H) 6.81 (d, J = 9.0 Hz, 2H), 5.83 (d, J = 4.8 Hz, 1H), 5.36 (d, J = 4.8 Hz, 1H), 3.96 (q, J = 7.1 Hz, 2H), 3.75 (s, 3H), 1.03 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 160.2,

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49 156.6, 154.0, 131.8, 130.2, 128.9, 128.6, 128.0, 118.8, 114.4, 78.4, 64.7, 61.4, 55.4, 13.9. 3-(1,1-Dimethylethoxy)carbonyloxyN -(4-methoxyphenyl)-4-phenyl-2-azetidinone ( 18c ). White solid, mp 169-170 C 98 %. 1 H NMR (250 MHz) 7.35 (s, 5H), 7.28 (d, J = 8.9 Hz, 2H) 6.80 (d, J = 8.9 Hz, 2H), 5.74 (d, J = 4.7 Hz, 1H), 5.33 (d, J = 4.7 Hz, 1H), 3.75 (s, 3H), 1.20 (s, 9H). 13 C NMR (63 MHz) 160.2, 156.6, 154.0, 131.8, 130.2, 129.0, 128.6, 127.9, 118.8, 114.4, 78.5, 61.3, 55.4, 55.3. 3-PhenoxycarbonyloxyN -(4-methoxyphenyl)-4-phenyl-2-azetidinone ( 18d ). White solid, mp 154-155 C, 21%. 1 H NMR (250 MHz) 7.42 (s, 5H), 7.34-7.19 (m, 5H), 6.83 (d, J = 8.9 Hz, 2H) 6.73 (d, J = 8.9 Hz, 2H), 5.89 (d, J = 4.8 Hz, 1H), 5.40 (d, J = 4.8 Hz, 1H), 3.75 (s, 3H). 13 C NMR (63 MHz) 159.8, 156.7, 152.0, 150.5, 131.7, 130.1, 129.5, 129.1, 128.7, 128.2, 126.3, 120.9, 120.7, 118.9, 114.4, 78.9, 61.3, 55.4. 3-(methoxydithiocarbonyl)oxyN -(4-methoxyphenyl)-4-phenyl-2-azetidinone ( 18e) White solid, mp 125-126 C 15%. 1 H NMR (250 MHz) 7.32 (s, 5H), 7.29 (d, J = 9.0 Hz, 2H) 6.81 (d, J = 9.0 Hz, 2H), 6.67 (d, J = 4.8 Hz, 1H), 5.41 (d, J = 4.8 Hz, 1H), 3.75 (s, 3H), 2.28 (s, 3H). 13 C NMR (63 MHz) 160.6, 156.6, 131.9, 130.1, 128.9, 128.4, 128.2, 118.9, 114.7, 81.7, 61.7, 55.5, 18.9. Procedure for the 3-(Ethoxycarbonyl)oxy-4-phenyl-2-azetidinone (19a). To a solution of 18a (330 mg, 1.01 mmol) in 5 ml of CH 3 CN was added ceric ammonium nitrate (1.66 g, 3.03 mmol, 3 eq.) in 5 ml of water. The result ant mixture was stirred for 5 min, and 10 ml of water was added. The so lution was extracted ( 3x10 ml) with EtOAc. The combined organic layers were washed with 5% NaHSO 3 5% NaHCO 3 and dried

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50 over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown oil. The crude material was purified by flash column chromatography on silica gel (1:2 EtOAc:hexanes) to give 150 mg (63%) of 19a as a brown semi-solid. 1 H NMR (250 MHz) 7.37-7.28 (m, 5H), 6.90 (bs, 1H), 5.75 (dd, J = 4.7, 2.7 Hz, 1H), 5.03 (d, J = 4.7 Hz, 1H), 3.52 (s, 3H). An analogous procedure was used to prepare lactams 19b-e. 3-(Ethoxycarbonyl)oxy-4-phe nyl-2-azetidinone (19b). Brown semi-solid, 2%. 1 H NMR (250 MHz) 7.35-7.31 (m, 5H), 7.02 (bs, 1H), 5.73 (dd, J = 4.7, 2.7 Hz, 1H), 5.01 (d, J = 4.7 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H), 1.22 (t, 3H). 3-(1,1-Dimethylethoxy)carbonyloxy4-phenyl-2-azetidinone (19c). Brown semi-solid, 72%. 1 H NMR (250 MHz) 7.35 (s, 5H), 6.67 (bs, 1H), 5.65 (dd, J = 4.5, 2.8 Hz, 1H), 5.02 (d, J = 4.5 Hz, 1H), 1.19 (s, 9H). 3-Phenoxycarbonyloxy-4-phenyl-2-azetidinone (19d) Brown semi-solid, 78%. 1 H NMR (250 MHz) 7.43 (s, 5H), 7.30-7.20 (m, 3H), 7.17 (bs, 1H), 6.72 (m, 2H), 5.78 (dd, J = 4.5, 2.8 Hz, 1H), 5.05 (d, J = 4.5 Hz, 1H). 3-(methoxycarbonyl)oxy-4-phenyl-2-azetidinone (19e). Brown semi-solid, 1%. 1 H NMR (250 MHz) 7.33 (s, 5H), 6.61 (dd, J = 4.7, 2.5 Hz, 1H), 6.53 (bs, 1H), 5.03 (d, J = 4.7 Hz, 1H), 2.31 (s, 3H). Procedure for Synthesis of N -Methylthio-3-methoxycarb oxy-4-phenyl-2-azetidinone (20a). To a solution of 19a (200 mg, 0.90 mmol) in 5 ml of dry CH 2 Cl 2 was added N (methylthio)phthalimide (260 mg, 1.35 mmol) and 3-5 drops of triethylamine. The resultant mixture was refl uxed overnight. The solven t was removed under reduced

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51 pressure to yield a brown solid. The brown solid was redissolved in methylene chloride, and washed with 1% sodium hydroxide. The organic laye r was dried over magnesium sulphate. The solvent was removed under redu ced pressure to yiel d a brown oil. The crude material was purified by column chromatography (1:4 ethyl acetate: hexanes) to yield 11.8 mg (5%) of 20a as a white solid. mp 83-84 C. 1 H NMR (250 MHz) 7.297.41 (m, 5H), 5.83 (d, J = 5.0 Hz, 1H), 5.03 (d, J = 5.0 Hz, 1H), 3.54 (s, 3H), 2.43 (s, 3H). 13 C NMR (63 MHz) 167.3, 153.6, 131.9, 129.2, 128.7, 128.5, 80.1, 65.7, 55.4, 22.1. 3-(Ethoxycarbonyl)oxyN -methylthio-4-phenyl-2-azetidinone (20b). White solid, mp 71-72 C, 35%. 1 H NMR (250 MHz) 7.30-7.40 (m, 5H), 5.80 (d, J = 4.9 Hz, 1H), 5.02 (d, J = 4.9 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.01 (t, J = 7.1 Hz, 3H). 13 C NMR (63 MHz) 167.4, 153.7, 132.0, 129.1, 128.8, 128.4, 80.1, 65.8, 64.8, 22.1, 3-(1,1-Dimethylethoxy)carbonyloxyN -methylthio-4-phenyl-2-azetidinone (20c). White solid, mp 81-83 C, 1%. 1 H NMR (250 MHz) 7.30-7.39 (m, 5H), 5.69 (d, J = 4.8 Hz, 1H), 5.01 (d, J = 4.8 Hz, 1H), 2.44 (s, 3H), 1.16 (s, 9H). 13 C NMR (63 MHz) 167.5, 151.1, 132.1, 128.9, 128.3, 128.4, 83.3, 79.8, 66.0, 27.2, 22.2. N -Methylthio-3-phenoxycarbonyloxy-4phenyl-2-azetidinone (20d). White solid, mp 117-118 C, 53%. 1 H NMR (250 MHz) 7.18-7.49 (m, 8H), 6.69 (m, 2H), 5.87 (d, J = 4.9 Hz, 1H), 5.09 (d, J = 4.9 Hz, 1H), 2.48 (s, 3H). 13 C NMR (63 MHz) 166.8, 151.7, 150.4, 131.8, 129.4, 128.9, 128.6, 126.4, 120.6, 80.5, 66.7, 22.2. N -Methylthio-3-methoxydithiocarboxy4-phenyl-2-azetidinone (20e).

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52 White solid, mp 77-78 C, 1%. 1 H NMR (250 MHz) 7.28-7.37 (m, 5H), 6.61 (d, J = 4.9 Hz, 1H), 5.09 (d, J = 4.9 Hz, 1H), 2.45 (s, 3H), 2.31 (s, 3H). References 1. T. E. Long and E. Turos, Curr. Med. Chem. Anti-Infective Agents 2002, 1, 251. 2. (a) X. F. Ren, M. I. Konaklieva, H. Shi, S. Dickey, D. V. Lim, J. Gonzalez, E. Turos, J. Org. Chem 1998, 63, 8898. (b) E. Turos, M. I. Konaklieva, X. F. Ren, H. Shi, J. Gonzalez, S. Dickey, D. V. Lim, Tetrahedron 2000, 56, 5571. 3. E. Turos, T. E. Long, M. I. Konaklieva, C. Coates, J.-Y. Shim, S. Dickey, D. V. Lim, A. Cannons, Bioorg. Med. Chem. Lett. 2002, 12, 2229. 4. T. E. Long, E. Turos, M. I. Konaklieva, A. L. Blum, A. Amry, E. A. Baker, L. S. Suwandi, M. D. McCain, M. F. Rahman, S. Dickey, D. V. Lim, Bioorg. Med. Chem. 2002, 11, 1859. 5. Gomez-Gallego, M.; Mancheno, M. J.; Sierre, M. A. Tetrahedron 2000, 56, 5743. 6. Delpiccolo, C. M. L.; Mata, E. G. Tetrahedron: Asymmetry 2002, 13, 905. 7. Banik, I.; Becker, F. F.; Banik, B. K. J. Med. Chem. 2003, 46, 12. 8. Banik, I.; Hackfeld, L.; Banik, B. K. Heterocycles 2003, 59, 505.

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53 Chapter Three Polymeric Nanoparticles Co ntaining an N-Methylthio -Lactam 3.1 Introduction In recent years, there has been a ra pid growth in the area of drug discovery. This has been facilitated by novel technologies su ch as combinatorial chemistry and highthroughput screening. 1,2,3 These novel approaches have led to numerous drug candidates within very shor t periods of time compared with the conventional drug discovery method. Many of these analogs show promising biological activity in vitro and in vivo. However, most of them do not progress to commercialization because of the problems of unwanted cytotoxicity (no targeting and no controlled release), very poor water solubility and, consequently, low bioavailability. These problems are closely related to optimal drug delivery systems in the human body which is why research on drug delivery systems has been and remains a hot topic in drug discovery. 4,5 Fig. 3-1 shows a hypothetical comp arison of the concentr ation of drug found in the plasma as a function of time following conventional drug release versus controlled drug release by oral drug administration As can be seen in Fig. 3-1, the conventional oral drug administration does not provide ideal pharmacokinetic profiles, especially for drugs which di splay high toxicity and/or na rrow therapeutic periods of

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time. For such drugs, if they can be conjugated to an optimal drug delivery system, the ideal pharmacokinetic profile can be achieved wherein the drug concentration reachs therapeutic levels without exceeding the maximum tolerable dose, and maintains these concentrations for extended periods of time until the desired therapeutic effect is reached. Therefore, optimizing drug delivery is a good answer for treatments that involve highly cytotoxic drugs such as certain anticancer agents, so that toxic side effects and damage to healthy tissues are minimized. 0123456780102030405060 conventional release profile controlled release profile minimum effective concentration maximum tolerable concentration Fig. 3-1 6 Comparison of conventional and controlled release profile In recent years, significant effort has been devoted to developing micro or nano-size microsphere technology for drug delivery. 7,8,9 These microspheres offer a suitable means of delivering small molecular weight drugs, as well as biomacromolecules such as proteins, peptides or genes by either sustained or targeted 54

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55 delivery to the tissue of interest. This technology focuses on formulating therapeutic agents in biocompatible or biodegra dable polymeric nanocomposites such as nanoparticles or nanocapsules. Since th ese systems are often polymeric and submicron in size, they have multiple advantages in drug delivery. These systems in general can be used to provide cellular or tissue delivery of drugs, to improve oral bioavailability, to sustain drug/gene effect in target tissue, to enhance the water solubility of drugs for intravascular de livery, and to improve the stability of therapeutic agents against enzymatic de gradation (nucleases and proteases). 7 However, the majority of current technologi es are focusing on controlled or sustained release systems via drug encapsulation me thods using biodegradable polymeric nanoparticles, even though the nanometer-si ze ranges of these delivery systems offer certain distinct advantages for drug delivery. Therefore, if nanoparticles can penetrate deep into tissues and through endocytos is are taken up efficiently by the cells 10 this allows efficient delivery of therapeutic agents to target sites in the body. Also, by modulating polymer characteri stics, one can control the release of the drug from nanoparticles to achieve a desired therapeu tic level in target tissue for optimal therapeutic effects. Further, nanoparticles can be delivered to target sites either by localized delivery using an enhanced permeation and rete ntion (EPR) effect 11, 12 or by targeted delivery through c onjugation to a biospecific ligan d which could direct them to the target tissue or organ. 7 In this research, microemulsion polymerization was done in aqueous solution to form hydrophilic polymeric nanoparticles c ontaining a highly wa ter-insoluble solid antibiotic, an N -methylthio -lactam. N -Methylthio -lactams are a new family of

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56 potent antibacterial co mpounds that selectively inhibi t the growth of methicillinresistant Staphylococcus aureus (MRSA). These drugs tend to be insoluble in water, and it was hoped that this method could enhance water solubility and bioavailability of these drugs. In addition, the antibacteri al activity of these resulting polymeric nanoparticles was investigated against MRSA strains. The synthesis of N -thiolated -lactam acrylic monomers for radical polymerization, and the conjugation of thes e acrylic monomers into a polymeric nanoparticle using a novel microemulsi on polymerization method, are described herein. We also describe the characteri zation of the polymer, its antibacterial properties, and the relationship that may exist between antibacterial activity, % drug content, and particle size. Finally, the preparation and th e characterization of a fluorescence-active nanoparticle is reported in chapter four for di agnostic purposes such as elucidating the biological mode of action, acting as a bi ological sensor a nd biological imaging. 3.2 Conventional Microemulsion Polymerization Microemulsion polymerizatio n was first reported in the early 1980s. This process can produce transparent or translucen t polymeric microlatexes with particle diameters of 50nm or higher. Over 45% of al l industrial polymers have been prepared by using emulsion polymerization. The main advantages of this process are: a) water is used as the sole solvent, b) the vi scosity of emulsion is relatively low and independent of the polymer molecular wei ght, and c) high molecular weight polymers with a narrow weight distribution can be produced in a controlled environment. 13, 14

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57 Therefore, this emulsion polymerization is bringing about the renaissance in the industrial polymer area. A conventional emulsion polymeriza tion consists of disper sing a water insoluble acrylic monomer phase, which is the low visc osity liquid phase, in an aqueous phase with the aid of surfactants. Polymerizati on is then brought a bout by free radical initiation in the water phase. The polymer fo rmed is stabilized within the emulsion by absorption onto surfactants to form a protective colloid form as the polymerization proceeds. The resultant products are homoge neous and relatively stable emulsified polymeric microspheres. The surfactant is a hydrocarbon chain with one end being hydrophobic and the other hydrophili c. If the concentratio n of surfactant is high enough, the hydrophobic ends of several su rfactants form aggregates known as micelles. The surfactant serves as a stab ilizer for the polymer particles and the monomer droplets. Therefore, the hydrophobic ends will att ach to the particles while the hydrophilic ends will remain in the water phase. The charges on these surfactants form what is known as an electrical double layer which prevents the particles from coagulating. In other words, the emulsifier serves to keep the particles suspended in the water. The micelles can also be the lo cation of particle nucleation. The monomer is present in the reaction mixture in the form of large droplets. These droplets act as reservoirs of the monomer. The monomer in the droplets can diffuse through the water phase and into the micelles. When the initiator is added to the reaction mixture it dissociates into two radicals in the pres ence of heat. The initiator radicals are very reactive towards any monomer in the wate r phase. The monomer in the water phase continues to add to the radical until the chain grows long enough such that it is no

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longer soluble in water. The oligomeric radical chain (multiple monomeric units) is now hydrophobic enough to enter a polymer particle or to enter a micelle to nucleate a new particle. Thus, the conventional emulsion polymerization will occur in three stages. The first stage involves the nucleation (birth) of polymer particles. This can occur by either micellar or homogeneous nucleation. The second stage involves the growth of the particles until the monomer droplets disappear. The third and final stage begins with the disappearance of the monomer droplets and continues until the end of the reaction (Fig. 3-2). 15, 16 RR 2 R defusion of monomersmonomeremulsion droplet continuousaqueous phasesurfactantinitiatoremulsified polymeric particle Fig. 3-2 Schematic representation of an emulsion polymerization system 15 Conventional emulsion polymerization methods are difficult to use for the preparation of polymeric microspheres for drug delivery systems, since most drugs are either partially or completely insoluble in water, and as such cannot form the 58

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59 liquid droplet in the emulsion polymerization process. The acrylic part on the original chemical structure of the drug has to be introduced synthetically for free radical polymerization without losing activity. Th erefore, the challenge is to prepare polymeric microspheres for drug de livery systems using a new emulsion polymerization method which can overcome these problems. 3.3 Synthesis of C 3 -Acryloyl N -Methylthio -Lactam as a Key Monomer The acrylation of N -methylthio -lactams is required for microemulsion polymerization using free radicals. Acryla tion can be accomplished at either the C 3 or the C 4 position of the -lactam. In this study, the C 3 -acrylation of the -lactam was studied. The synthesis of C 3 -acryloyl N -methylthio -lactam is summarized in Scheme III-1. The key step is to introduce the acryloyl group at the C 3 -position, which can then participate in the radical copolymerization with the other acrylic comonomer. 2-Chlorobenzaldehyde ( 21) was coupled with p-anisidine, to give imines 22. Staudinger coupling of acetoxyacetyl chloride with imine 22 gave C 3 -acetoxy N -aryl protected -lactam 23. Hydrolysis of acetoxy group under basic conditions gave the C 3 -free hydroxyl -lactam 24. Acrylation of free hydroxyl -lactam 24 with acryloyl chloride gave C 3 -acryloyl N -aryl protected -lactam 25. Dearylation of -lactam 25 with ceric ammonium nitrate gave N-dearylated -lactam 26, followed by methylthiolation with N-methylthiophthalimide affords C 3 -acryloyl N -methylthio

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lactam 27 which is a white solid, mp 92-93 o C The 1 H NMR spectra of C 3 -acryloyl N-methylthio -lactam 27 is displayed in Fig. 3-3. N O O H3C O OCH3 N O HO OCH3 N O O O OCH3 N O H O O N O S O O KOH/0oCacetone-MeOHNaH/CH2Cl2/RT Cl O CANCH3CH-H2O0oC N O O S CH3 CH2Cl2/RT96%72%76%Cl Cl Cl Cl Cl Cl O H N Cl OCH3 OCH3 NH2 CSA/CH2Cl2O Cl O CH3 O Et3N/CH2Cl289%86%88%mp: 92-93 oC CH3 21222324252627Scheme III-1 60

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61 9.08.58.07.57.06.56.05.55.04.54.03.53.02.52.01.51.00.50.0 4.043.092.041.05 N O H H O S CH3 Cl O H H H abc6.36.26.16.05.95.85.75.65.55.4 2.041.051.000.97 cdeabcdce Fig. 3-3 1 H NMR Spectra of C 3 -acryloyl N-methylthio -lactam 3.4 Main Components for Microemulsion Polymerization The microemulsion polymerization used to prepare the polymeric nanoparticles consists of four major components: 1) acrylic drug monomer, 2) acrylic co-monomer, 3) surfactant, and 4) deionized water. The selection and requirement of each of these components are described below. 3.4.1 Choice of Drug Monomer The drug monomer needs to contain three essential parts; the first one is the drug itself, the second one is the acrylic moiety for radical polymerization and the third one is the linker that connects the drug with the acrylic moiety such that it can break down biologically or enzymatically. The general features are shown in Fig. 3-4.

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R O O linker drug Fig. 3-4 Structure of drug monomer A wide variety of drugs (antibacterial, antiviral, antifungal and anticancer agents, etc.) can potentially be used in this method, including those which have little or no water solubility as well as those that have good water solubility. A variety of acrylic monomers can conceivably be synthetically coupled with a drug containing a functional group such as a carboxylic acid, an amine or an alcohol. Examples of such monomers include acryloyl chloride, methacryloyl chloride, acrylic acid, maleic acid, itaconic acid, crotonic acid, N-methylol acrylamide, acrylonitrile, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-hydroxyethyl methacrylate Modified acrylamide, modified methacrylamide, (PEG) modified acrylate, amino acid oligomeric acrylate etc. also can be possibly used for specific controlled release system since the amino acid oligomer which is active for a specific enzyme can be selectively cleaved under certain biological conditions. 3.4.2 Choice of Co-monomer 62 Commercial acrylic or vinyl monomers can be used as the co-monomers. The choice of co-monomers is entirely dependent on the chemical and physical properties of the drug monomer. It is a critical that the commercial or synthetically modified acrylic monomers are able to play two roles in the polymerization process: (1) as the co-monomer for the radical emulsion polymerization and (2) as the solvent for

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63 dissolving the synthetically modified drug monomers in order to make homogeneous liquid phase. Therefore, it is very important to find a specific acrylic co-monomer that matches a specific drug monomer, and th is is accomplished by trial and error. Fortunately, there are many acrylic monom ers available commercially and/or synthetically: acrylonitrile, acrylic acid, maleic acid, methyl acrylate, ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, methoxyethyl acrylat e, dimethylamino acrylate, methacrylic acid, methyl methacrylate, et hyl methacrylate, butyl methacrylate, isobutyl methacrylate, 2-ethyl hexyl meth acrylate, lauryl methacrylate, stearic methacrylate, dimethyl amino methacry late, allyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2hydroxyethyl methacrylate, modified acrylamide, modified methacrylamide, glycidyl acrylate, styrene, vinyl acetate, vinyl toluene, and all synthetically modified acrylics can be used. 3.4.3 Choice of Surfactant The surfactant that is used in emulsion pol ymerizations has two ends of different solubility. One end, termed the tail, is a long hydrocarbon that is soluble in nonpolar, organic compounds. The other end, the head, is often a hydrophilic functional sodium or potassium salt, which is water soluble. The water soluble salt can be the salt of a carboxylic acid or sulfonic acid. The technical term for the chemical display of "dual personalities" is amphipathic

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O S O O O Na nonpolar tailwater soluble headSodium Lauryl Sulfate The selection of emulsifiers (or the surfactants) is very important for a successful formulation as it can control many of the properties of emulsion polymers. There is a critical concentration below which an emulsifier will not form micelles. The minimum level required for micelle formation is known as the critical micelle concentration (CMC). Emulsifiers are classified according to the ionic type of the hydrophilic group, ionic or non-ionic. Ionic emulsifiers generally have a lower CMC than non-ionic emulsifiers and they provide low particle size emulsions. However, there is a problem with long term storage of these compounds. In the case of non-ionic emulsifiers, they need higher CMC level because of their low water solubility so that it leads to the formation of small aggregates or grainy emulsions. However, once the particles are formed they are very stable in aqueous systems. As a result of these advantages and disadvantages, ionic/non-ionic emulsifier mixtures can be employed in emulsion polymerization. Therefore, the factors for selecting the emulsifiers are totally dependent on the chemical or physical properties of the applied drug monomer and co-monomer, radical initiator and aqueous system. As the choice of surfactants, anionic, cationic and nonionic surfactants such as lauryl alcohol (+6EO), nonyl phenol (+10EO, +15EO, +30EO), sodium lauryl sulphate, sodium lauryl sulphate (+2EO, +4EO), sodium dodecylbenzenesulphonate, 64

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sodium dioctyl sulphosuccinate, polyvinyl alcohol, polyol, unsaturated and saturated fatty acid sodium and potassium salts, and synthetically modified PEG surfactants can, in principle, be used. 3.4.4 Choice of Radical Initiator The initiator must be water soluble and the free radicals have to be generated thermally or by use of an oxidation-reduction (or redox) couple. The major initiators used in emulsion polymerization are persulphates. SO O S O O O O O O KK Potassium Persulfate Even though the initiating efficiency and the half life of persulphates vary, ammonium persulphate is preferred in practice because of its better solubility. Hydroperoxides are often used particularly as a post reaction initiator to kill the unreacted monomers after emulsion polymerization. The rate of free radical generation increases with temperature, and it is normal to employ reaction temperatures of 60-90 o C when using thermal generation techniques. However, when redox couples (thiosulphates, metabissulphites and hydrosulphides) are employed, the rate of free radical generation is increased to that provided by thermal generation at the same temperature. Therefore, when using redox couples, reaction temperatures can be as low as 30 o C. Possible free-radical initiators for use in the initiating step of 65

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66 polymerizations include: peroxides; persul phates; alkyl hydroper oxides; persulphates with sodium, ammonium and potassium; thiosulphates; metabisulphites; and hydrosulphides. 3.4.5 Choice of Aqueous Media The aqueous media is the major part of the emulsion polymer ization. The ions or metals in the aqueous media can act as a radical scavenger. Therefore the aqueous media used has to be either de-ionized water or nano-pure water for biological purposes. It is sometimes necessary to use a buffer solution depending on the surfactant and the par ticle stabilization. 3.5 The Preparation of Polymeric Nanoparticles Using Microemulsion Polymerization Polymeric nanoparticles of N-methylthio -lactam were prepared by a novel microemulsion polymerization technique described in Scheme III-2 The key point of this method was the complete dissolution of the solid dr ug monomer in a commercial acrylic monomer at elevated temperature, followed by its dispersion into an aqueous media containing a surfactant fo r the stabilization of the suspension. Therefore, it is very critical to get a homogeneous liquid mixture. In the first example studied, C 3 acryloyl N-methylthio -lactam 21 and ethyl acrylate were combined at 70 o C. The mixture was then dispersed into an aqueous media containing a surfactant, sodium lauryl sulphate, to allow the formation of droplets with vigorous stirring until a milky suspension was formed. The emulsion radical polymerization then was launched by

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adding the initiator, potassium persulfate, at 70 o C to afford the polymeric nanoparticles as a milky colloidal suspension (Fig. 3-5). 67 ON Cl S O H3C N O SCH3 Fig. 3-5 -lactam polymeric emulsion Other anaologs were prepared by emulsion polymerization by varying the ratios of monomers and reaction conditions. The formulations of polymerization are summarized in Table 3-1. O O Cl O O CH2CH3 O + ON Cl S O H3C O mipo croemulsionlymerization O O CH2CH3 Scheme III-2

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entry mole ratio monomers(mg) surfactant (mg) initiator(mg) water (ml) rxn. temp.(oC) EA1 : -lactam1 -lactam1 EA1 lauryl sulphate1 persulphate1 1 EA homo 0 1000 20 5.0 5.0 60 22 20 : 1 100 700 16 4.0 4.0 70 32 13 : 1 100 456 12 3.0 2.8 70 42 10 : 1 100 350 9 2.5 2.3 70 52 7 : 1 100 245 7 1.8 1.8 70 62 5 : 1 100 175 6 1.5 1.4 70 72 2.5 : 1 100 88 3 0.8 0.7 70 1 EA:ethyl acrylate, -lactam: C3-acryloyl N-methylthio -lactam, lauryl sulphate: sodium salt, persulphate: potassium salt 2 The scale was varied several times based on volume. Table III-1 Formulation of MicroemulsionPolymerization 3.6 Characterization of Emulsion 3.6.1 Scanning Electron Microscopy (SEM) The morphology and the particle size of the emulsified particles were examined by Scanning Electron Microscopy (SEM). The sample of nanoparticles was prepared on the silicon wafer by air blowing and then was coated with gold sputter under high vacuum. The gold-coated nanoparticles were then observed by SEM (Hitach S800). The SEM images of the copolymeric nanoparticles for the -lactam and ethyl acrylate are shown in Fig. 3-6 through Fig. 3-12. The SEM image of the polymeric nanoparticles for homo poly (ethyl acrylate): Fig. 3-6 shows the polymeric nanoparticles deformed by the electron beam of SEM because of the low Tg, -23 o C of homo poly(ethyl acrylate). The SEM images 68

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of the copolymeric nanoparticles, (20:1 through 2.5:1 for ethyl acrylate: -lactam) show that the particles have microspherical morphology with the particle size distribution of 40 nm to 150 nm in diameter. It is very interesting that the 7:1 copolymeric nanoparticles have the smallest particle size distribution (40-80 nm), while the 2.5:1 copolymeric nanoparticles had a uniform particle size of 70 nm. The particle size distribution of the copolymeric nanoparticles for the -lactam and ethyl acrylate, determined by SEM analyses, are summarized in Fig. 3-13. Fig. 3-6 SEM picture for homo ethyl acrylate polymeric nanoparticles 69

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(a) (b) Fig. 3-7 SEM pictures for 20:1 copolymeric nanoparticles which has particle size, 60-150 nm; (a) and (b) 70

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(a) (b) Fig. 3-8 SEM pictures for 13:1 copolymeric nanoparticles which has particle size, 60-150 nm; (a) and (b) 71

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(a) (b) Fig. 3-9 SEM pictures for 10:1 copolymeric nanoparticles which has particle size, 100-130 nm; (a) and (b) 72

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(a) (b) Fig. 3-10 SEM pictures for 7:1 copolymeric nanoparticles which has particle size, 40-80 nm; (a) and (b) 73

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(a) (b) Fig. 3-11 SEM pictures for 5:1 copolymeric nanoparticles which has particle size, 130-150 nm; (a) and (b) 74

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(a) (b) Fig 3-12. SEM pictures for 2.5:1 copolymeric nanoparticles which has particle size, 70nm; (a) and (b) 75

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501001502002.5:15:17:110:113:120:1homo particleSize (nm)-lactamcopolymericnanoparticles(ethyl acrylate: -lactamratio) Fi g 3-13 Particle size distribution of -lactam co p ol y meric nano p articles 76

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3.6.2 Coalescing Process 15,16 For further characterization of -lactam copolymeric nanoparticles, thin films were prepared by a coalescing process. The polymeric particles experience an irreversible structural change during film formation. The particles, upon evaporation of water, come into contact, fuse and form a uniform film through a process called coalescense. In general it is assumed that the film formation can be separated into the following three stages: Stage 1: Water evaporates slowly and thus polymer particles become concentrated. Stage 2: The particles deform to form a dense closed packing. Stage 3: The fully coalesced particles produce a uniform film. The coalescing process is summarized in Fig. 3-14. 77 substratesubstratesubstrate random distributionof particles close-packingof particles dry film of fullycoalesced particles emulsified polymericnanoparticles dry film of fullycoalesced particles Fig. 3-14 Representation of the coalescing process 15

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3.6.3 Determination of Solid Content (%) The measurement of the non-volatile solid (-lactam) content in polymer is critical for determining the amount of the drug in the polymeric emulsion. The solid content is defined as the weight percent of the non-volatile solid among total weight of the emulsion. The weight percent of polymer in the emulsion can be calculated quantitatively after film formation via the coalescence process. The calculation method is described in Fig. 3-15. All solid content measurements were done in triplicate and the average value is reported. Al foilonlyAl foil+samplesolutionAl foil+dry polymer ABC1.A B = The weight of sample solution2.A C= The weight of dry polymerSolid Content (%) = (A B) / (A C) x 100 Fig. 3-15 Determination of solid content (%) 78

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79 3.6.4 1 H NMR Spectra Analysis 1 H NMR spectroscopy is a very powerful tool to analyze chemical structure of organic molecules. It can also be used to determine the mole ratio of each of the monomeric units, ( -lactam and ethyl acrylate) in c opolymers. Fig. 3-16 shows the cumulated 1 H NMR spectra for the dry films obta ined by coalescing the nanoparticle emulsions of homopoly(ethyl acrylate) and six different copolymers of the -lactam and ethyl acrylate. Signals at (d) 2.6, (b ) 5.6 and (c) 6.1 ppm ar e assigned to S-CH 3 C 3 -H and C 4 -H on -lactam respectively. The signal at (a) 4.0 ppm is assigned to the methylene proton of ethyl acrylate. The olef in protons of acrylate in the range of 5.66.1 ppm do not show in the spectrum. That indicates that all of the monomeric lactam acrylate and ethyl acrylate was convert ed to polymeric particles. In addition, the composition of the polymeric nanoparticles can be determined by 1 H NMR spectroscopy as mentione d. The mole ratio of -lactam and ethyl acrylate in the copolymer was determined from the peak integration of the methylene proton (a ) of ethyl acrylate and a proton of C 3 ( b ) or C 4 ( c ) on the -lactam respectively. Fig. 3-16 also shows the solid content of each of the polymers. The -lactam loading amount on the each polymers can be calculated qua ntitatively based on these parameters, based on the mole ratio of each monomers, th e loading volume and the solid content.

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ON Cl S O H3C O O O CH2CH3 H H abcdadbc Fig. 3-16 1 H NMR Spectra, the mole ratio and the solid content of -lactam and ethyl acrylate copolymers 80

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81 3.7 Biological Activity Against MRSA 3.7.1 Initial Testing The N-methylthio -lactam-conjugated polymeric nanoparticles and their coalesced plastic films were tested for antibacterial acti vity using the Kirby-Bauer method of disc diffusion on agar plates. The 7:1 (ethyl acrylate : lactam) copolymeric nanoparticles and their plastic film as well as the homo poly(ethyl acrylate) nanoparticles and their plastic film, were also tested against a st rains of methicillinresistant Staphylococcus aureus (MRSA). Fig. 3-17 shows the results of this testing. It is obvious that the plas tic films of the 7:1 copolym er and the homo polymer, as well as the homo poly(ethyl acrylate) nanopa rticles have no activity against MRSA. However, the 7:1 copolymeric nanoparticles surprisingly have strong activity against MRSA. That means the film polymers, whethe r they contain the dr ug or not, have no activity, and the nanoparticles that do not bear a drug, also have no activity. This initial result indicates that further research should be focused on nanoparticles having the attached -lactam drug.

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7:1 acrylate:lactam nanoparticles 7:1 acrylate:lactam dry film* (with drug) (with drug) homo polyacrylate homo polyacrylate nanoparticles dry film* (without drug) (without drug) *Dry film was dissolved in DMSO for testing. Fig. 3-17 Initial antibacterial testing of nanoparticles and polymer films against MRSA 652 3.7.2 Antibacterial Testing of Homo Poly(ethyl acrylate) Nanoparticles (Without N-Methylthio -Lactam) The antibacterial testing of homo poly(ethyl acrylate) nanoparticles was performed as a control experiment. A sample of the nanoparticle suspension was tested against MRSA on an agar plate, with increasing disk loading amounts from 20 l to 100l. No activity was observed even at 100 l of disk loading (Fig. 3-18). This result is consistent with that of the initial testing showing that the polymeric nanoparticle which has no drug, has no activity. Therefore, the focus next was to the test the antibacterial activity of the nanoparticles that do contain the N-methylthio -lactam drug. 82

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Fig. 3-18 Antibacterial testing of homo poly(ethyl acrylate) nanoparticle against MRSA 3.7.3 Antibacterial Testing of Lactam-containing Nanoparticles Against MRSA Nanoparticles containing the N-methylthio -lactam were evaluated for antibacterial activity against ten MRSA strains by Kirby-Bauer of disc diffusion on agar plates. Six different samples of the nanoparticle emulsions were tested, varying in the relative amounts of drug to ethyl acrylate used to prepare the particles (20:1 to 2.5:1). Table 3-2 displays the observed zones of inhibition for the nanoparticles. It is very interesting that all samples containing the N-methylthio -lactam are active against the MRSA strains as well as the non-resistant strain of S. aureus. In addition, the strongest bioactivity was observed for the 7:1 (acrylate:lactam) nanoparticles, which have the smallest particle size distribution, 40-80 nm. Therefore, these facts 83

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indicate that research should be focused on the biological mode of action of these conjugated nanoparticles, and the relationship of particle size to biological activity. The images of the agar plates used to test against MRSA and S. aureus are displayed in Fig. 3-19 through Fig. 3-29. strains homo EA 20 : 1 15 : 1 10 : 1 7 : 1 5 : 1 2.5 : 1MRSA 652 0 14 11 18 23 16 17 653 0 15 13 24 28 24 24 654 0 14 12 15 23 17 16 655 0 0 11 18 22 17 15 656 0 12 15 18 23 17 18 657 0 12 12 18 24 17 16 658 0 0 12 17 24 18 16 659 0 0 14 16 22 17 17 919 0 0 11 17 22 17 17 920 0 0 12 16 23 18 16 S. aureus 849 0 13 16 18 24 20 19 nanoparticles having monomer ratio of EA* : -lactam TableIII-2.Zonesofinhibitionobtainedfromagarwelldiffusione x perimentsusing20 loftheemulsifiedsuspensionofthetestnanoparticles.Thevaluescorrespondtothediameters in mm for the zone of growth inhibition appearing around the well after 24 hours. Staphylococcusaureusand-lactamase-producingstrainsofmethicillin-resistantStaphylococcusaureus(labeledMRSAUSF652-659)wereobtainedfromaclinicaltestinglaboratory at Lakeland Regional Medical Center, Lakeland, FL or from ATCC sources.* eth y l acr y late (EA) 84

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homo7:15:120:12.5:1 Fig. 3-19 Antibacterial testing of drug-embedded nanoparticles against MRSA 652 (ratios of ethyl acrylate : lactam indicated) homo2.5:15:120:17:1 85 Fig. 3-20 Antibacterial testing of drug-embedded nanoparticles against MRSA 653 (ratios of ethyl acrylate : lactam indicated)

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2.5:1homo5:120:17:1 Fig. 3-21 Antibacterial testing of drug-embedded nanoparticles against MRSA 654 (ratios of ethyl acrylate : lactam indicated) 2.5:1homo7:15:120:1 Fig. 3-22 Antibacterial testing of drug-embedded nanoparticles against MRSA 655 (ratios of ethyl acrylate : lactam indicated) 86

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2.5:1homo7:15:120:1 Fig. 3-23 Antibacterial testing of drug-embedded nanoparticles against MRSA 656 (ratios of ethyl acrylate : lactam indicated) 2.5:1homo7:15:120:1 Fig. 3-24 Antibacterial testing of drug-embedded nanoparticles against MRSA 657 (ratios of ethyl acrylate : lactam indicated) 87

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homo7:15:120:12.5:1 Fig. 3-25 Antibacterial testing of drug-embedded nanoparticles against MRSA 658 (ratios of ethyl acrylate : lactam indicated) homo7:15:120:12.5:1 88 Fig. 3-26 Antibacterial testing of drug-embedded nanoparticles against MRSA 659 (ratios of ethyl acrylate : lactam indicated)

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homo7:15:120:12.5:1 Fig. 3-27 Antibacterial testing of drug-embedded nanoparticles against MRSA 919 (ratios of ethyl acrylate : lactam indicated) Fig. 3-28 Antibacterial testing of drug-embedded nanoparticles against MRSA 920 (ratios of ethyl acrylate : lactam indicated) 89

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homo7:15:120:12.5:1 Fig. 3-29 Antibacterial testing of drug-embedded nanoparticles against S. aureus 849 (ratios of ethyl acrylate : lactam indicated) 3.8 Antifungal Testing of Nanoparticle Emulsions In the previous section the 7:1 (acrylate:drug) nanoparticles showed the most promising antibacterial activity against MRSA strains. Thus, it is very meaningful to extend the testing of these emulsions to fungus, because N-methylthio -lactams have previously been found (Marci Culbreath, unpublished results) to have activity against fungal strains. The antifungal testing of these nanoparticles was performed by Kirby-Bauer disc diffusion on agar plates against eight genera of fungi. Table 3-4 displays the zones of inhibition observed nanoparticles. It is very interesting that nanoparticles are very active against all of the fungal strains, with antifungal activity of the N-thiolated lactam (1 g) in the nanoparticles being similar to that of the standard, 90

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clotrimazole (50 g). That means the 7:1 (acrylate:lactam) nanoparticles are fifty times more potent than clotrimazole. This indicates that drug containing nanoparticles are promising leads to new antifungal agents as well as an antibacterial antibiotics. 26 26 2020 24 22 2019 20 16 18 1421 23 20 21 3724 27 29 27 2731 32 32 32 2519 20 22 20 3222 23 23 23 24C. albicansC. tropicalisC. glabrataC. kefyrC. kruseiC. lusitaniaeC. parapsilosisC. utilisaveragestandardclotrimazole( 50 g)1st2nd3rdnanoparticlesfungalstrains* Ta b leIII-4.Zonesofinhi b i t iono b t ainedfro m agarwelldiffusionex p eri m en t susing20mlof7:1(ethylacrylate:lactam)nanoparticleemulsion.Thiscorresponds to 1 mg of active drug in the particle. The values correspond to the diametersinmmforthezoneofgrowthinhibitionappearingaroundthewellafter 48 hours. Fungi were chosen on the basis of their potential pathogenicity. C. albicans and C. tropicalis were donated by Dr. Ray Widen from the University of South Florida, School of Medicine. C. glabrata (ATCC 15126), C. krusei (ATCC 14243), C. keyfr (ATCC 20409), C. parapsilosis (ATCC 22019), C. lusitaniae (ATCC 34449) and C. utilis (ATCC 29950) were obtained commercially. 91

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92 3.9 Discussion Nanoparticles comprised of poly(ethyl acrylate) were made by a novel microemulsion polymerization procedure. This method has the advantage over conventional emulsion polymerization me thods because a solid co-monomer ( lactam drug) is utilized. This method may open a new area in drug delivery and help to solve the main problems in drug discovery: unwanted cytotoxicity, water insolubility and low bioavail ability of drug. The core feature of this method, as mentioned in section 3.5, is to make a homogeneous solu tion of monomeric substances at elevated temperature (60 o C or 70 o C) and to disperse this mixture in aqueous media containing a suitable surfac tant (to stabilize the nanoparticles). Radical polymerization within these pre-form ed particles then ensues to make the nanospherical polymers. A water-insoluble solid antibiotic, N-methylthio -lactam, was synthetically converted to a C 3 -acryloyl -lactam derivative and its hom ogenized into a liquid state with ethyl acrylate as a co-monomer was generated at 70 o C. The N-methylthio lactam-containing nanoparticles were then prepared by free radical microemulsion polymerization after dispersing in an aqueous phase. Thus, it was demonstrated that the N-methylthio -lactam could be incorpoated as a monomer to generate a new Nmethylthio -lactam-containing nanoparticle. It is likely that this new method can be applied to many other water-insoluble solid drugs (or those having high vi scosity). The N-methylthio -lactam-containing nanopart icles were subjected to antibacterial testing against various MRSA st rains. Fig. 3-30 displays a comparison of

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homo 20:1 13:1 10:1 7:1 5:1 2.5:15101520253035 Pen G Vancomycin monomer 0.41 g (20 l) 0.00 g (20 l) 0.50 g (20 l) 0.66 g (20l) 1.02 g(20 l) 0.91 g(20 l) 1.76 g (20 l) 20 g 20 g 20 g acrylateand-lactamemulsified copolymers S. aureus849 standardsMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSA0 homo 20:1 13:1 10:1 7:1 5:1 2.5:15101520253035 Pen G Vancomycin monomer 0.41 g (20 l) 0.00 g (20 l) 0.50 g (20 l) 0.66 g (20l) 1.02 g(20 l) 0.91 g(20 l) 1.76 g (20 l) 20 g 20 g 20 g acrylateand-lactamemulsified copolymers S. aureus849 standardsMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSAMRSA0 Fig. 3-30 Comparison of antibacterial activities of N-thiolated -lactam-containing emulsified nanoparticles. 93

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94 antibacterial activities for each of the differe nt emulsified nanoparticles to penicillin G (Pen. G), vancomycin, and C 3 -acryloyl -lactam 27 as standards. It also shows the actual amounts of drug contained within 20 l of the nanoparticle emulsion tested. It is apparent that all N-methylthio -lactam containing nanoparticles are active against MRSA strains as well as the non-resistant strain, S. aureus 849. Their activity trend appears to increase gra dually as the portion of drug ( -lactam) increase from 20:1 (ethyl acrylate:antibiotic)to the 7:1 and reachs the maximum at the 7:1. However, the bioactivity decreased for par ticles having an ethyl acrylate:lactam ratio of 5:1, and the activities of the 5:1 and 2.5:1 nanoparticles are similar even though the portion of drug ( -lactam) is increased. Therefore, the result indicates that the antibacterial performance of N-methylthio -lactam containing nanoparticles is enhanced dramatically over that of the free antibiotic, and the 7:1 (ethyl acrylate:lactam) nanoparticle s show the best activity. The comparison of antibacterial activity of the N-methylthio -lactam containing emulsified nanoparticles and the standards: penicillin G (Pen. G), vancomycin, and C 3 -acryloyl -lactam 27 against MRSA, shows that the emulsified nanoparticles have similar and/or better activi ties at very low drug amount compared with those of standards. However, the activ ity of 7:1 (ethyl acrylate:la ctam) nanoparticles are better even at low drug amount (0.91 g) compared with that of vancomycin (20 g). It is likely that the activity of th e 7:1 nanoparticles is over 20 times more than that of vancomycin.

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Fig. 3-31 and Fig. 3-32 display a comparison of the antibacterial activities of the 2.5:1, 5:1, 7:1 and 10:1 (ethyl acrylate:lactam) nanoparticles against MRSA and non-resistant S. aureus with decreasing sample loading volume (20 l to 12 l). The amount of sample was proportional to the bioactivity. However, the N-methylthio -lactam containing nanoparticles still possess good activities even at 12 l of loading volume. Most notably the 7:1 copolymeric nanoparticles still show the strongest activity. 20 l 18 l 16 l 14 l 12 l zone of inhibition (mm)0510152025307:15:110:12.5:1 disk loading amount (l of emulsion) Fig. 3-31 Bioactivity of polymeric nanoparticles as a function of disk loading amounts (MRSA 653) 95

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20 l 18 l 16 l 14 l 12 l zone of inhibition (mm)25201510507:110:15:12.5:1 disk loading amount (l of emulsion) Fig. 3-32 Bioactivity of polymeric nanoparticles as a function of disk loading amounts for S. aureus 849 Fig. 3-33 displays antibacterial activity of the 7:1 nanoparticles as the sample loading volume is decreased from 20 l to 2 l against MRSA and S. aureus. Surprisingly, the activity of the 7:1 nanoparticles for MRSA maintained a strong level of potency (over 20 mm zone of inhibition) until the volume was reduced to 6 l (0.27 g) and even at 2 l (0.09 g, 14 mm in the zone of inhibition). In addition, against S. aureus the particles also show a good level of antibiotic activity until the loading amount is reduced to 6 l (0.27 g, 16 mm zone of inhibition). The relative antibacterial activities for 7:1 (ethyl acrylate:lactam) nanoparticles, Pen G, vancomycin, and C 3 -acryloyl N-methylthio -lactam 27 were also evaluated at 96

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1 g since 20 l of volume for the 7:1 nanoparticles contains approximately 1 g of -lactam. zone of inhibition (mm) disk loading amount (l of emulsion)0510152025300.91 g (20 l) 0.82 g (18 l) 0.73 g (16 l) 0.64 g (14 l) 0.55 g (12 l) 0.46 g (10 l) 0.36 g (8 l) 0.27 g (6 l) 0.18 g (4 l) 0.09 g (2 l) Fig. 3-33 Bioactivity of 7:1 copolymeric nanoparticles as a function of decreasing disk loading amounts Fig. 3-33 shows a comparison of relative antibacterial activities against MRSA. It is obvious that Pen G and C 3 -acryloyl N-methylthio -lactam have no activity and vancomycin is very weak at 1 g of drug amount level. However, the 7:1 (ethyl acrylate:lactam) nanoparticles show very strong activity. Therefore, it is a natural question to ask why the N-methylthio -lactamcontaining nanoparticles have such enhanced antibacterial activities even at very low concentrations. 97

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7:1 -lactamcopolymericnanoparticles(1 g of lactam) Penicillin G1 g Vancomycin1 gN O S O O Cl CH3 N O S O O Cl CH3 homopolymericnanoparticles(0 g of lactam)+ 1 g1 g Fig. 3-34 Comparison of antibacterial activities against MRSA 653 Though the mode of antibacterial action of these nanoparticles is unknown, one possible answer is that N-methylthio -lactam-containing nanoparticles have nano-size diameters (40 nm) and can easily penetrate into the bacteria cell through a process called endocytosis, and thus, the bioavailability of the antibiotic can be dramatically increased. Fig. 3-33 shows an example of the endocytosis process for a polymer drug. To study this proposed mechanism, the relationship between particle size and bioactivity, as well as the preparation of fluorescence probe containing nanoparticles, are the subject of our current investigations. 98

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drug polymeric nanoparticles adsorption endocytosis cell membranelysosomalhydrolysis release drugs Fi g 3-35 Pro p osed mechanism of dru g deliver y b y endoc y tosis 13 3-10 Conclusions N-Methylthio -lactam containing nanoparticles were prepared by a novel nanoemulsion polymerization technique with ethyl acrylate as a co-monomer. This method has advantages over the conventional emulsion polymerization methods because a solid co-monomer (-lactam drug) can be utilized. SEM studies show that the polymeric nanoparticles have a microspherical morphology with particle sizes of 40-150 nm, and the 7:1 copolymeric nanoparticles have the smallest particle sizes (40-80 nm). The N-thiolated -lactam containing 99

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100 nanoparticles display potent anti-MRSA activity at low drug amount compared with penicillin G and vancomycin. The strongest bioactivity was observed in the 7:1 (ethyl acrylate:lactam) nanoparticles, which correlates with it having the smallest particle size (40-80 nm). A lthough at this time, the relations hip between particle size and activity is not clear and the mode of action is unknown, the N -thiolated -lactam containing nanoparticles dramatically enhance bioactivity, possibly due to increased bioavailability of the antibiotic via endocyt osis. Further examination of this proposed mechanism is the subject of our current investigations. 3.11 Experimental All reagents were purchased fr om Sigma-Aldrich Chemical Company and used without further purification. Solvents were obtained from Fisher Scientific Company. Thin layer chromatography (TLC) was carried out using EM Reagent plates with a fluorescence indicator (SiO 2 -60, F-254). Products we re purified by flash chromatography using J.T. Baker flash chromatography silica gel (40 m). NMR spectra were recorded in CDCl 3 unless otherwise noted. 13 C NMR spectra were proton broad-band decoupled. Procedure for the Synthesis of N -(4-Methoxyphenyl)-(2-chlorophenyl)imine (22). To a solution of p-anisidine (9.64 g, 78 mmol) in 25 ml of CH 2 Cl 2 was added 2chlorobenzaldehyde 21 (10.50 g, 64 mmol) and a catal ytic amount of camphorsulfonic acid. The resultant mixture was stir red until TLC indicated the disappearance

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101 of starting materials. The solvent was re moved under reduced pressure, and the crude material was purified by recr ystallization from methanol to yield 15.56 g (89%) of 22 as a yellow solid. mp 56-57 C. 1 H NMR (250 MHz) 8.95 (s, 1H), 8.25 (m, 1H), 7.43-7.35 (m, 3H), 7.29 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H). 13 C NMR (63 MHz) 158.6, 154.6, 144.5, 135.7, 133.4, 131.7, 129.8, 128.3, 127.0, 122.5, 114.3, 55.4. Procedure for the Synthesis of 3-Acetoxy-N -(4-methoxyphenyl)-4-(2-chlorophenyl)2-azetidinone (23). To a stirred solution of N -(4-methoxyphenyl)-(2-chlorophenyl)imine 22 (17.00 g, 69.15 mmol) and triethylamine (26.8 g, 36 ml, 207.6 mmol) was added a solution of acetoxyacetyl chloride (9.76 g, 7.69 ml, 90.0 mmo l) in methylene chloride (30 ml) dropwise over 10 minutes. The resultant mixture was stirred at rt until TLC indicated the disappearance of starting material. The so lvent was removed under reduced pressure, and the crude material was purified by washi ng with ice-cold methanol to give 19.48 g (86%) of 23 as white solid, mp 130-132 o C. 1 H NMR (250 MHz) 7.43 (d, J = 7.8 Hz), 7.29-7.24 (m, 5H), 6.83 (d, J = 8.3 Hz, 2H), 6.16 (d, J = 4.6 Hz, 1H), 5.78 (d, J = 4.6 Hz, 1H), 3.76 (s, 3H), 1.76 (s, 3H). 13 C NMR (63 MHz) 168.7, 161.4, 156.7, 133.8, 130.2, 130.0, 129.8, 128.7, 126.8, 118.6, 114.5, 75.4, 58.2, 55.4, 19.9. Procedure for the Synthesis of 3-HydroxyN -(4-methoxyphenyl)-4-(2-chloro phenyl)-2-azetidinone (24). To a solution of -lactam 23 (8.00 g, 23.1 mmol) in 50 ml of acetone was added KOH (1.30 g, 23.1 mmol) in 20 ml of methanol at 0 o C. The resultant mixture was stirred for 5

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102 minutes, and 50 ml of water was added. The product was precipitate d and isolated by filtration to yiel d 6.8 g (96%) of 24 as a white solid, mp 178-180 o C. 1 H NMR (250 MHz) 7.48 (d, J = 7.3 Hz, 1H), 7.34-7.24 (m, 5H), 6.85 (d, J = 9.0 Hz, 2H), 5.63 (d, J = 5.1 Hz, 1H), 5.34 (d, J = 5.1 Hz, 1H), 3.78 (s, 3H), 1.74 (bs, 1H). 13 C NMR (63 MHz, DMSO-d 6 ), 166.6, 156.1, 133.1, 132.9, 131.0, 129.8, 129.6, 128.9, 127.4, 118.6, 115.0, 77.2, 60.0, 55.7. Procedure for the Synthesis of 3-acryloyl-N -(4-methoxyphenyl)-4-(2-chlorophenyl)2-azetidinone ( 25). To a solution of C 3 -hydroxy -lactam 24 (5.80 g, 19.1 mmol) in 30 ml of freshly distilled CH 2 Cl 2 was added NaH (60% suspension in mineral oil, 0.83 g, 21.0 mmol), and the mixture was stirred for 15 min at room temperature. Acryloyl chloride (2.59 g, 28.64 mmol) was then added dropwise and the resultant mixture was stirred until TLC indicated the disappearance of starting material. The reaction was quenched with a 5% solution of NH 4 Cl and extracted (3x20 ml) with CH 2 Cl 2 The combined organic layers were dried over anhydrous MgSO 4 and purified with column chromatography on silica gel (1:4, EtOAc:hexanes) to give 4.92 g (72 %) of 25 as a white solid, mp 99-100 C. 1 H NMR (250 MHz) 7.33 (d, J = 7.9 Hz, 1H), 7.23-7.10 (m, 5H), 6.75 (d, J = 8.9 Hz, 2H), 6.17 (d, J = 5.0 Hz, 1H), 5.98 (dd, J = 16.9, 1.00 Hz, 1H), 5.74 (dd, J = 16.9, 10.4 Hz, 1H), 5.69 (d, J = 5.0 Hz, 1H), 5.59 (d, J = 10.4, 1.0 Hz, 1H), 3.66 (s, 3H). 13 C NMR (63 MHz) 163.6, 161.2, 156.6, 133.7, 132.3, 130.3, 130.1, 129.8, 128.5, 126.8, 126.5, 118.6, 114.4, 75.3, 61.3, 58.2, 55.3.

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103 Procedure for the Synt hesis of 3-acryloyl-N -methylthio-4-(2-chlorophenyl)-2azetidinone (27). To a solution of 25 (4.00 g, 11.2 mmol) in 40 ml of CH 3 CN in an ice-water bath was added ceric ammonium nitrat e (18.39 g, 33.54 mmol) in 40 ml of water. The resultant mixture was stirred for 5 min, and 20 ml of water was added. The solution was extracted (3x5 ml) with EtOAc. The combined organic layers were washed with 5% NaHSO 3 5% NaHCO 3 and dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield 2.14 g (76 %) of 26 as a crude brown oil. Without futher purification, compound 26 (2.00 g, 8.0 mmol) was dissolved in 30 ml of dry CH 2 Cl 2 and N -(methylthio)phthalimide (2.30 g, 11.9 mmol) and 3-5 drops of triethylamine were added. The resultant mixture was refluxed for overnight. The solvent was removed under reduced pressure to yield a brown solid The brown solid was redissolved in CH 2 Cl 2 and washed with 1% NaOH. The organic layer was dried over anhydrous MgSO 4 The solvent was removed under reduced pressure to yield a brown semi-solid, which was purified by column chromatography on silica gel with gradient elution (1:9 then 1:4 EtOA c:hexanes) to yield 2.00 g (88%) of 27 as a white solid. 1 H NMR (250 MHz) 7.35-7.26 (m, 4H), 6.20 (d, J = 5.1 Hz, 1H), 6.06 (dd, J = 16.7, 1.9 Hz, 1H), 5.78 (dd, J = 16.7, 10.4 Hz, 1H), 5.68 (dd, J = 10.4, 1.9 Hz, 1H), 5.54 (d, J = 10.4 Hz, 1H), 2.51 (s, 3H). 13 C NMR (63 MHz) 168.3, 163.4, 156.6, 134.3, 132.6, 129.8, 128.6, 126.6, 126.3, 62.1, 21.9. Procedure for the synthesis of 2.5:1 (e thyl acrylate:lactam) nanoparticles containing N-methylthio -lactam

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104 The mixture of 3-acryloyl N -methylthio -lactam 27 (500 mg, white solid, mp 92-93 o C) and ethyl acrylate (440 mg) was warmed to 70 o C with slow stirring under a nitrogen atmosphere. Stirring was continued until the mixture was completely mixed to give a homogeneous liquid phase. De ionized water (7.94 ml) containing dodecyl sulfate, sodium salt (Acros 15 mg) was added with vigor ous stirring and the mixture was stirred for one hour to give a milky pre-emulsion state. A solution of potassium persulfate (Sigma, 4 mg) di ssolved in deionized water ( 0.3 ml) was added under a nitrogen atmosphere and the mixture was stirred rapidly at 70 o C for 6 hr. A solution of potassium persulfate (1 mg) dissolved in deionized water (0.1 ml) was added to the emulsion and rapid stirring was continue d for 1hr, to give the N-methylthio -lactam containing nanoparticles as a milky emulsion. Other analogs were prepared similarly based on the formulation described in Table 32. Process for the preparation of samples for SEM An aliquot (0.1 ml) of the emulsified su spension of the nanoparticles was diluted 20,000 to 30,000 times with deionized wate r (2000 ml3000 ml). One drop of the diluted emulsion was carefully applied to the surface of a silicon wafer and evaporated under a gentle stream of air. The spot of emulsion on the wafer was marked with a pen and coated with gold sputter under high vacuum. The gold coated nanoparticles were observed by SEM. Note: it is really important to keep the s ilicon wafer clean from any dust. Therefore, the best way is to prepare the sample in a clean room.

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105 Process for the preparation of thin film on the glass. Samples of the nanoparticle emulsions were converted to thin f ilms by coalescence as described in section 3.6.1. A rectangular ca rdboard frame was fixed on glass and the emulsion was poured into the frame. The emulsi on was left to dry for 48 hours to give a transparent thin film. References 1. Lebl, M. Parallel Personal Comments on Classical Papers in Combinatorial Chemistry. J. Comb. Chem 1999, 1, 3. 2. a) Hudson, D. Matrix Assisted Synthetic Transformations: A Mosaic of Diverse Contributions. I. The Pattern Emerges. J. Comb. Chem 1999, 1, 333. b) Hudson, D. Matrix Assisted Synthetic Tran sformations: A Mosaic of Diverse Contributions. II. The Pattern is Completed. J. Comb. Chem 1999, 1, 403. 3. Rao, V.S.V.Vadlamudi; Thomas, P. C. High Throughput Screening: Revolution in Drug Discovery Research. Asian Chemistry Letters 2001, 5, 61. 4. Greenwald, R.B.; Choe, Y.H.; McGuire, J.; Conover, C.D. Effective drug delivery by PEGylated drug conjugates. Advanced Drug Delivery Reviews 2003, 55, 217. 5. a) Langer, R. Drug delivery and targeting. Nature 1998, 392 5. b) Garnett, M. C. Targeted Drug Conjugates: Principles and Progress. Advanced Drug Delivery Reviews 2001 53, 171. 6. Internet Web Site Paper; Polymeric Drug Deliver, A Brief Review; http://www.drugdel.com/polymer.htm

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106 7. Moghimi, S.M.; Hunter, A.C.; Murray, J.C.; Long-Circulating and Target Specific Nanoparticles: Theory to Practice. Pharmacol. Rev. 2001, 53, 283. 8. a) Langer, R. New Methods for Drug Delivery. Science 1990, 249, 1527. b) Langer, R. Perspectives: Dr ug Delivery-Drugs on Target. Science, 2001 293, 58. 9. a) Lambert G.; Fattal E.; Couvreur P. Nanoparticulate Systems for the Delivery of Antisense Oligonucleotides. Advanced Drug Delivery Reviews 2000, 47, 99. b) Kawaguchi, H. Functional Polymer Microspheres. Prog. Polym. Sci 2000, 25, 1171. 10. a) Vinagradov,S.V.; Bronich,T.K.; Kabanov, A.V. Nanosized Cationic Hydrogels for Drug Delivery: Preparati on, Properties and Interactions with Cells, Adv. Drug Del. Rev. 2002, 54, 223. b) Okamoto, C.T. Endocytosis and Transcytosis. Advanced Drug Delivery Reviews 1998, 29, 215. 11. a) C. Song, V. Labhasetwar, X. Cu i, T. Underwood, R.J. Levy, Arterial Uptake of Biodegradable Nanoparticles for Intravascular Local Drug Delivery: Results with an Acute Dog Model, J. Control. Release 1998 54, 201. b) Rihova, B.; Jelinkova, M.; Stroha lm, J.; Subr, V.; Plocova, D.; Hovorka, O.; Novak, M.; Plundrova, D.; Germano, Y.; Ulbrich, K. Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules-II. Anti-cancer Activity of Antibody or (Fab)2-Targeted Conjugates and Combined Therapy with Immunomodulators. J. Control. Release 2000 64, 241.

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107 12. Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapies in Cancer Chemotherapy: Mechanism of Tumouritropic Accumulation of Proteins and Antitumour Agent SMANCS, Cancer Res 1986, 6, 6387. 13. a) Harkins, W.D. A General Theory of the Mechanism of Emulsion Polymerization J. Am. Chem. Soc. 1947, 69, 1428. b) Blackley, D.C. Emulsion Polymerization, Applied Science. London, 1975. c) Stoffer, J.O.; Bone, T. J. Polym. Sci. Polym. Chem. Ed. 1980, 18, 2641. d) Stoffer, J.O.; Bone, T. J. Dispersion Sci. Technol. 1980, 1, 37. 14. a) Atik, S.S.; Thomas, J.K. Polymerized Microemulsions. J. Am. Chem. Soc. 1981, 103, 4279. b) Atik, S.S.; Thomas, J.K. Photochemistry in Polymerized Microemulsion Systems J. Am. Chem. Soc. 1982 104, 5868. c) Atik, S.S.; Thomas, J.K. Photoinduced Reactions in Polymerized Microemulsions J. Am. Chem. Soc. 1983, 105, 4515. 15. Wicks, Z.W. Jr; Jones, F.N.; Pappas S.P. Organic Coatings: Science and Technology. John Wiley & Sons, Inc., 1992, Vol. I, p64. 16. Barbour, M.; Clarke, J.; Fone, D.; Hoggan, A.; James, R.; Jones, P.; Lam, P.; Langham, C.; OHara, K.; Oldring, P. ; Raynor, G.; Royston, I.; Tuck, N.; Usher, R. Waterborne & Solvent Based Acrylics and Their End User Applications. SITA Technology Limited, 1996, Vol. 1, p103. 17. Ouchi, T.; Ohya, Y. Macromolecular Prodrugs. Prog. Polym. Sci 1995, 20, 211.

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108 Chapter Four Preparation of Fluorescence Active Nanoparticles 4.1 Introduction Fluorescence has proven to be a vers atile tool for numerous applications. This powerful technique enables one to study molecula r interactions in life science It is promoting the phenomenal sensitivity for the life scientist working on biological analysis. But the fluorescence technology offers much more than mere signal-gathering capabilities. New developments in instrumentation, software probes, and applications have resulted in a burst of popularity sinc e it was observed over 150 years ago. As the theory of fluorescence became better understood, a more power ful set of applications emerged and afforded the detailed information about complex molecules and their reaction pathways. 1,2,3 The preparation of N-methylthio -lactam containing fl uorescence-active nanoparticles are the subject of our current investigations for studying the biological mechanism of the nanoparticles. However, th ere are no reports to prepare fluorescenceactive polymeric nanopart icles. Therefore, it may ope n a new area in biological diagnostics if fluorescence-active nanoparticles are successfully prepared and applied appropriately to detection of specific biologi cal molecules, acting as a water dispersed biological sensor or biol ogical imaging agent.

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109 4.2 Preparation of an Acrylated Fluorescence Monomer The acrylation of fluorescent molecules to serve as free radica l acceptors is required for microemulsion polymerization. Three co mpounds were selected for these studies: dansyl chloride 1 (5-dimethylamino-1-naphthalenesulfonamide, 29) 1-naphthoic acid 1 ( 30) and 9-anthracenecarboxylic acid 1 ( 31) They are all fluorescence active. 2-Hydroxy ethyl acrylate was used for acrylation of each co mpound since the terminal hydroxyl group is easily coupled with the carboxylic acid group of each substance, and the resultant diester linker can be easily hydrolyzed in a biol ogical environment. Dansyl chloride ( 29) was reacted with 2-hydroxyethyl acrylate using amine base, to give compound 32 with 56% yield. Both 1-naphthoic acid ( 30) and 9-anthracenecarboxylic acid ( 31) was also coupled with 2-hydroxy acrylate under EDC/DMAP conditions, to give compound 32 and 33 with 82% and 21% yield, respectively. The synthesis and 1 H NMR spectra are displayed in Scheme IV-1 and Fig. 4-1, respectively.

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O O OH O OH O O O O EDC / DMAP / CH2Cl2 82 %33+ O O OH S N Cl O O CH3 H3C DIPEA0oC to RT S N O O O CH3 H3C O O +RT56% O O OH O OH O O O O EDC / DMAP / CH2Cl2 21 %+RT 341)2)3)Scheme I V -128293031282832 110

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N CH3 H3C S O O O O O 987654321 0 6.494.262.141.951.801.031.000.93 (a) O O O O 987654321 0 3.982.981.011.011.011.000.970.97 (b) O O O O 987654321 0 4.013.912.031.011.000.980.91 (c) Fig. 4-1 1 H NMR spectra of (a) dansyl, (b) naphthyl, and (c) anthracenyl acrylates 111

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112 4.3 The Preparation of Fluorescence-Active Polymeric Nanoparticles in an Aqueous Emulsion The microemulsion polymerization described in section was performed with the fluorescent acrylates in an attempt to prepare fluorescence-active nanoparticles. -Lactam containing fluorescence-active emul sified nanoparticles were successfully prepared with C 3 -acryloyl N-methylthio -lactam 27, naphthyl acrylate 32 and ethyl acrylate at 70 o C. After making a homogeneous solu tion of these three monomeric substances at 70 o C, this mixture was dispersed in aqueous media containing the surfactant, sodium lauryl sulf ate. Radical polymerization within this pre-formed particle mixture then was performed with an init iator (potassium persulfate) to give the nanospherical polymers. Its synthesis and form ulation are described in Scheme IV-2 and Table IV-1 respectively. Dansyl acrylate 31 could not be used in the emulsion polymerization, because it underwent the hydrolysis under the above conditions. The naphthyl containg fluorescen ce-active emulsified nanoparticles (without the lactam drug) were prepared with naphthyl acrylate 32 and ethyl acrylate in aqueous phase. A homogeneous solution of mono meric substances could be made at room temperature and this mixture was dispersed in aqueous media with the aid of sodium lauryl sulphate at 60 o C. Its synthesis and formulation are disp layed in Scheme IV-3 and Table IV-2 respectively. Finally, anthracenyl fluorescence-activ e emulsified nanoparticles were also prepared with anthracenyl acrylate 33, styrene and butyl acrylate in aqueous phase at 70 o C. A homogeneous solution of monomeric substances could be ma de with styrene and butyl

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113 acrylate at 70 o C and this mixture was dispersed in aqueous media contai ning a surfactant, sodium lauryl sulfate, to give the anthra cenyl fluorescence-active nanoparticles as a milky emulsion. However, a homogeneous solu tion of monomeric substances could not be obtained with either ethyl acrylate and butyl acrylate sin ce they could not dissolve the anthracenyl acrylate 34 at 70 o C. Its synthesis and the formulation are also displayed in Scheme IV-4 and Table IV-3 respectively.

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O O O O O CH2CH3 N O S CH3 O Cl O O ++ microemulsion polymerization O O CH2 O O N Cl O S CH3 O O O O 4.510.6:: O O O O N O S CH3 O Cl O O O CH2CH3 CH3 Scheme I V -2 Table I V -1 FormulationofMicroemulsionPolymerization for Fluorescence-Active -Lactam Copolymeric Nanoparticlescomponentsamountethyl acrylate (mg)-lactam acrylate (27) (mg)naphthyl acrylate (33) (mg) surfactant1 (mg)initiator1 (mg)deionized water (ml)temperature (oC)1701005071.82.070 1 surfac t an t : sodiumlaurysulfa t e;ini t ia t or:po t assiumpersulfa t e 114

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O O O O O CH2CH3 O O O O O + microemulsion polymerization O O O O O O O O CH2 CH3 CH2 CH3 Scheme I V -38:1 Table I V -2 FormulationofMicroemulsionPolymerization for Fluorescence-Active Naphthyl Copolymeric Nanoparticlescomponentsamountethyl acrylate (mg)naphthyl acrylate (33) (mg) surfactant1 (mg)initiator1 (mg)deionized water (ml)temperature (oC)30010082.03.060 1 surfac t an t : sodiumlaurysulfa t e;ini t ia t or:po t assiumpersulfa t e 115

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O O O O O CH2 O ++ microemulsion polymerization O O CH2 O O O O O O O O O O CH2 SchemeI V -4 H3CH2CH2CH2C CH2 CH2 CH3 CH2 CH3 Table I V -3 FormulationofMicroemulsionPolymerization for Fluorescence-Active Anthracenyl Copolymeric Nanoparticlescomponentsamountbutyl acrylate (mg)styrene (mg)anthracenyl acrylate (34) (mg) surfactant1 (mg)initiator1 (mg)deionized water (ml)temperature (oC)70030010205.05.070 1 surfac t an t : sodiumlaurysulfa t e;ini t ia t or:po t assiumpersulfa t e 116

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4.4 Characterization of Fluorescence Active Emulsified Nanoparticles 4.4.1 Scanning Electron Microscopy (SEM) The morphology and the size of the emulsified particles were examined by Scanning Electron Microscopy (SEM). The sample of nanoparticles was prepared on a silicon wafer by evaporation of water under a gentle stream of air, and then coated with gold sputter under high vaccum. The gold-coated nanoparticles were then observed by SEM. The SEM images of -lactam, naphthyl, and anthracenyl fluorescence-active nanoparticles are displayed in Fig. 4-2, 4-3, and 4-4 respectively. The images of nanoparticles show that the particles have microspherical morphology and a particle size distribution of about 30-120 nm. Fig. 4-2 and 4-4 also show that some of particles are fused by the coalescence due to not enough dilution of the samples when they are prepared. Fig. 4-2 SEM image for -lactam fluorescence-active emulsified nanoparticles with particles size (60-120 nm) 117

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Fig. 4-3 SEM image for naphthalyl fluorescence-active emulsified nanoparticles with particle size (30-60 nm) Fig. 4-4 SEM image for anthracenyl fluorescence-active emulsified nanoparticles with particle size (60-120 nm) 118

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119 4.4.2 1 H NMR Spectra Analysis 1 H NMR Spectroscopy is a very useful tool to analyze the chemical structure of organic molecules. It can also be used to determine the mole ratio of each of the monomeric units, in copolymers. Fig. 4-5 shows 1 H NMR spectra for the dry films obtained by coalescing the nanoparticle emulsions of naphthyl (a), -lactam (b), and anthracenyl (c) copolymer. The olefin protons of the acryla te moiety in the range of 5.66.1 ppm do not appear in the spectrum, indicati ng that all acrylic monomers participated in polymerization. In addition, each m onomer composition in the polymer was determined by the peak integration in the 1 H NMR spectrum. For instance, the comparison of the peak integration for C 3 or C 4 proton of -lactam, methylene protons of ethyl acrylate, and one of arom atic protons in naphthyl group in (b) spectra gave the mole ratio of each monomer in the copolymer.

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O O O O O O CH2 CH3 (a) O O O O N O S H3C O Cl O O O CH2CH3 10987654321 9.096.703.112.661.041.000.790.730.66 0 (b) O O O O O O CH2CH2CH2CH3 10987654321 40.1221.4419.039.440.76 0 (c) Fig. 4-5 1 H NMR spectra for (a) naphthyl, (b) -lactam and (c) anthracenyl fluorescence-active copolymer 120

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121 4.5 Discussion Fluorescence-active emulsified nanopa rticles were successfu lly prepared by a novel microemulsion polymerization described in chapter three. The fluorescence-active nanoparticles were made for the first time and may open opportunitie s to construct new biological diagnostic systems to detect a cert ain microbe, cell, or biomolecule. This will require further development. Acrylation with naphthyl carboxylic acid 30 and anthracenyl carboxylic acid 31 was performed with 2-hydroxy ethyl acrylate using EDC/DMAP condition to give acrylated compound 33 and 34 which are all fluorescence-active. The -lactam containing fluorescence active emul sified nanoparticles were prepared with the monomer combination of C 3 -acryloyl N-methylthio -lactam 27, naphthalyl acrylate 33, and ethyl acrylate. The homogeneous solution of monomeric substances was formed at 70 o C, followed by dispersion in water phase with aid of surfactants. The mole ratio of each monomer in the polymer is 1 : 4.5 : 0.6, and the particle size distribution is approximately 60-120 nm. These nanoparticle s were prepared for possible use in identifying the mechanism, of how the drug enters the cell of MRSA (chapter 3). The fluorescence-active polymeric nanopa rticles without drug are also expected to be very important materials as leads to bio-dia gnostics because of easy and fast detection of the fluorescence probe. First, naphthyl-c ontaning fluorescence-active emulsified nanoparticles were prepared with ethyl acry late as a co-monomer using microemulsion polymerization. A water-insoluble hi gh viscous oil, naphthyl acrylate 33, was pre-mixed with ethyl acrylate at room temperature to give a homogeneous liquid. Following this, emulsion polymerization was performed at 60 o C to give naphthyl fluorescence-active

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122 emulsified nanoparticles as a milky emulsi on. The mole ratio of ethyl acrylate and naphthyl acrylate in the polymer is 8:1 and th e particle size distribut ion is approximately 30-60 nm. Anthracenyl fluorescence-active emul sified nanoparticles wh ich have a different fluorescent emission were also prepared with butyl acrylate and styrene as co-monomers using microemulsion polymerization. The mole ratio of each monomer was not determined with 1 H NMR spectra analysis since the p eak integration of anthracenyl group is too small to detect. The particle size distribution is approximately 60-120 nm. Even though the emulsion polymerization was attemp ted with the monomer combination of ethyl acrylate-anthracenyl acrylate and th e butyl acrylate-anthracenyl acrylate respectively, the anthracenyl emulsified na noparticles were not prepared since neither monomer combinations could form a homogeneous liquid phase at 70 o C. Thus, these experiments failed. The fluorescent emission colors fo r a naphthyl and an anthracenyl emulsions and their corresponding thin films formed by coalescence were compared with non fluorescent -lactam emulsion and its thin film u pon UV irradiation. The image (a) and (a) in Fig. 4-6 displays the fluorescent emission colors for the non fluorescent -lactam nanoparticles as well as that of naphthyl, and anthracenyl system upon UV irradiation. The non-fluorescent -lactam emulsion shows no color change while both the naphthyl and anthracenyl nanoparticle emulsions emit a blue and a bright bl ue-green fluorescent color respectively. The image (b) and (b) in Fig. 4-6 also displays the fluorescent emission colors for the thin films of co rresponding samples upon UV irradiation. Each sample shows the same fluorescent emissi on colors as in the emulsion. Therefore,

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123 naththyl and anthracenyl emulsified nanopart icles and their corresponding thin films are all fluorescence-active and emit a blue a nd a bright blue-green fluorescent color respectively. As an on-going study, the naphthyl and anthracenyl copolymeric emulsified nanoparticles are going to be prepared by using microemu lsion polymerization. If the copolymeric nanoparticles emit the totally di fferent color in fluorescent emission, that means new fluorescence-active nanoparticles can be created with simple microemulsion copolymerization of two different fluorescent probes. Therefore, this research can potentially bring significant advantages and applications in the bio-diagnostics area.

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(a) UV Lamp Off (b) UV Lamp Off (a) UV Lamp On (b) UV Lamp On la cta m na p hth y l anthracen y l 124 la cta m na p hth y l anthracen y l Fig. 4-6 Comparison of the non fluorescence-active -lactam and the fluorescence-active naphthalyl and anthracenyl emulsified nanoparticles and their corresponding thin films upon UV Irradiation

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125 4.6 Experimental All reagents were purchased from Sigma-Aldrich Chemical Company and used without further purification. Solvents were obtained fr om Fisher Scientific Company. Thin layer chromatography (TLC) was carried out using EM Reagent plates with a fluorescence indicator (SiO 2 -60, F-254). Products were purified by flash chromatography using J.T. Baker flash chromatography silica gel (40 m). NMR spectra were recorded in CDCl 3 unless otherwise noted. 13 C NMR spectra were prot on broad-band decoupled. Procedurefor the synthesis of 2-(5-dimethylami no-naphthalene-1-sulfonyloxy)-ethyl acrylate (32). To a solution of 2hydroxyethyl acrylate 28 (25.8 mg, 0.2 mmol) in 2 ml of freshly distilled CH 2 Cl 2 was added diisopropylethylamine (DIPEA, 0.35 ml, 0.2 mmol) dropwise at 0 o C. Dansyl chloride (50.0 m g, 0.19 mmol) was then added dropwise and the resultant mixture was stirred at room temperature until TLC indicated the disa ppearance of starting material. The reaction was quenched with a 5% solution of NH 4 Cl and extracted (3x20 ml) with CH 2 Cl 2 The combined organic layers were dried over anhydrous MgSO 4 and purified with column chromatography on silica gel (1:4, EtOAc:hexanes) to give 37.5 mg (56 %) of 32 as a yellow semi solid. 1 H NMR (250 MHz) 8.61 (d, J = 8.6 Hz, 1H), 8.26 (t, J = 6.3 Hz, 2H), 7.56 (m, 2H), 7.20 (d, J = 7.7 Hz, 1H), 6.19 (dd, J = 16.5, 2.5 Hz, 1H), 5.80 (dd, J = 16.5, 10.3 Hz, 1H), 5.71 (dd, J = 16.5, 10.3 Hz, 1H), 4.25 (m, 4H), 2.88 (s, 6H). 13 C NMR (63 MHz) 158.0, 151.7, 131.8, 131.5, 130.9, 130.6, 129.8, 128.8, 127.3, 123.0, 119.4, 115.6, 67.8, 61.5, 45.4.

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126 Procedure for the synthesis of naphthal ene-1-carbonyloxy-2-ethyl acrylate (33). To a solution of 2-hydroxyethyl acrylate 28 (1.01 g, 8.7 mmol) and 1-naphthoic acid 30 (1.00 g, 5.8 mmol) in 10 ml of freshly distilled CH 2 Cl 2 was added EDC (1.60 g, 8.7 mmol) and DMAP (cat. amount) at room temper ature. The resultant mixture was stirred at room temperature until TLC indicated th e disappearance of starting material. The reaction was quenched with a 5% solution of NH 4 Cl and the mixture was washed with water. After extraction with EtOAc (3x20 ml ), the organic layers were dried over anhydrous MgSO 4 and purified with column ch romatography on silica gel (1:4, EtOAc:hexanes) to give 1.29 g (82 %) of 33 as a colorless oil. 1 H NMR (250 MHz) 8.91 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 7.1 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.57 (m, 3H), 6.49 (d, J = 17.3 Hz, 1H), 6.19 (dd, J = 17.3, 10.4 Hz, 1H), 5.71 (d, J = 10.4 Hz, 1H), 4.66 (m, 2H), 4.58 (m, 2H). 13 C NMR (63 MHz) 167.2, 165.9, 133.8, 133.6, 131.5, 131.3, 130.5, 128.6, 128.0, 127.8, 126.6, 126.2, 125.7, 124.5, 62.7, 62.3. Procedure for the synthesis of anthracen e-9-carbonyloxy-2-ethyl acrylate (34). To a solution of 2-hydroxyethyl acrylate 28 (0.78 g, 6.8 mmol) and 1-naphthoic acid 30 (1.00 g, 5.8 mmol) in 10 ml of freshly distilled CH 2 Cl 2 was added EDC (0.86 g, 4.5 mmol) and DMAP (cat. amount) at room temper ature. The resultant mixture was stirred at room temperature until TLC indicated th e disappearance of starting material. The reaction was quenched with a 5% solution of NH 4 Cl and the mixture was washed with water. After extraction with EtOAc (3x20 ml ), the organic layers were dried over

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127 anhydrous MgSO 4 and purified with column ch romatography on silica gel (1:4, EtOAc:hexanes) to give 0.30 g (21 %) of 34 as a yellow solid, mp 66-68 o C. 1 H NMR (250 MHz) 8.54 (s, 1H), 8.06 (dd, J = 14.7, 8.0 Hz, 4H), 7.52 (m, 4H), 6.21 (d, J = 17.2 Hz, 1H), 6.21 (dd, J = 17.2, 10.5 Hz, 1H), 5.91 (d, J = 10.5 Hz, 1H), 4.89 (m, 2H), 4.62 (m, 2H). 13 C NMR (63 MHz) 169.3, 165.3, 131.7, 130.9, 129.7, 128.7, 127.9, 127.1, 125.5, 124.9, 63.2, 62.3. Procedure for the synthesis of fluores cence-active emulsified nanoparticles The mixture of 3-acryloyl N -methylthio -lactam 27 (100 mg, white solid, mp 92-93 o C), naphthyl acrylate 33 (50 mg), and ethyl acrylate (170 mg) was warmed to 70 o C with slow stirring under a nitrogen atmosphere. St irring was continued unt il the mixture was completely mixed to give a homogeneous liquid phase. Deionized water (1.7 ml) containing dodecyl sulfate, sodium salt (A cros, 7 mg) was added with vigorous stirring and the mixture was stirred for one hour to gi ve a milky pre-emulsion state. A solution of potassium persulfate (Sigma, 1.8 mg) dissolved in deionized water ( 0.3 ml) was added under a nitrogen atmosphere and the mi xture was stirred rapidly at 70 o C for 6 hr. A solution of potassium persulfate (0.5 mg) dissolved in deionized water (0.1 ml) was added to the emulsion and rapid stirring was co ntinued for 1hr, to give the N-methylthio -lactam containing fluorescence-active emulsi fied nanoparticles as a milky emulsion. Other analogs were prepared similarly based on the formulation described in Table IV-2 and Table IV-3 respectively.

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128 Process for the preparation of thin film on the glass. Samples of the nanoparticle emulsions were converted to thin f ilms by coalescence as described in previous chapter. A rectangular cardboard frame was fixed on glass and the emulsion was poured into the frame. The emulsi on was left to dry for 48 hours to give a transparent thin film. References 1. Du, H.; Fuh, R. A.; Li, J.; Corkan, A.; Lindsey, J. S. PhotochemCAD: A computer-aided design and research tool in photochemistry. Photochemistry and Photobiology 1998, 68, 141. 2. Rhys Williams, A. T. Clinical Chem istry Using Fluorescence Spectroscopya Review of Current Techniques. Perkin-Elmer Ltd (1976). 3. Rhys Williams, A. T. Fluorescence Deri vatisation in Liquid Chromatography Perkin-Elmer Ltd. (1984).

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129 Chapter Five Conclusions and Future Directions Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hospita ls and in care centers. Infections caused by this bacterium has become a serious nationa l and global problem. The need to develop new drugs for MRSA is the force driving this research project. N -Thiolated -lactams are a new family of pot ent antibacterial compounds that selectively inhibit the growth of Staphylococcus species including methicillin-resistant Staphylococcus aureus (MRSA), over other common bact erial genera. Recent efforts within this laboratory have been on understa nding possible structureactivity profiles of this previously unstudied family of antibiotics. In chapter 2 of this thesis, results were discussed on the eff ect of a fatty ester group (CO 2 R) on the C 4 -phenyl ring of N -methylthio -lactams. The initial expectation was that attachment of long chain ester moieties might increase the hydr ophobicity, and thus enhance the drugs ability to penetrate thr ough the cell membrane. However, the result indicate that there is an optimal chain length for the fa tty ester groups, with antibacterial activity dropping off rapidly when more than seven carbon atoms are in the chain. These results led to the idea about examining a -lactam conjugated polym er as a possible drug

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130 delivery method, with the polymer essentially serving as a surrogate for an extremely large, lipophilic ester side ch ain on the lactam ring. The ques tion being asked is whether this would enhance, or de stroy, biological activity. To synthesize the initial drug-poly mer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N methylthio -lactam. This method has advantag es over the conventional emulsion polymerization methods because a solid co-monomer ( -lactam drug) can be utilized. SEM studies show that these pol ymeric nanoparticles ha ve a microspherical morphology with nano-sizes of 40-150 nm. The N -thiolated -lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancom ycin. Although at this time the relationship between particle size and activity is not clear and the mode of action is unknown, the N thiolated -lactam containing nanoparticles dramatically enhance bioactivity, possibly due to increased bioavailability of the antibiotic via endocytosis. Therefore, the preparation and the biological testing of N -thiolated -lactam containing nanoparticles of differe nt particle size distributions have to be performed to ascertain the relationship between particle size and activity. In vivo and cytotoxicity testing are also necessary for furthe r progress towards commercialization. Fluorescence-active emulsified nanopart icles containing naphthy l or anthracenyl side chains were also successfully prepared by microemulsion polymeri zation for possible use in fluorescence studies to determine if th e drug enters the cell of MRSA through endocytosis.

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131 It will be necessary to further develop these and possibly other related fluorescenceactive emulsified nanoparticles into more inte lligent forms which are able to selectively bind to certain biological targets. The emulsi fied nanoparticles can be converted to dry films that are readily solubl e in organic solvents, but wh ich do not retain biological activity. Future work in this laboratory is likely to focus on development of drugcontaining thin film polymers for a variet y of biomedical and research related applications.

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ABOUT THE AUTHOR Jeung-Yeop Shim currently serves as the Founder a nd Principle Inves tigator of the NanoDDS Biotechnology Corpor ation. Dr. Shim has focused the past 10 years on creating drug discovery as well as a novel drug delivery system by using nonotechnology. He has successfully patented innovative drug delivery system and diagnostic products for marketplaces. He has demonstrated leadersh ip in new business formation having created and managed new ventures, NanoDDS Biotechnology Corporation. He served as a group leader and an executive manager in R&D center at the DPI Co. in Korea and developed several new concepts of polymer coating systems such as waterborne type-thin layer coating system by using nanotechnology. Dr. Shim received his Honors BS and MS from Kangwon National University in Korea, and Ph.D. from University of South Florida. End Page