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N-Thiolated b-lactam antibiotics

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Title:
N-Thiolated b-lactam antibiotics synthesis and structure-activity studies of C3 oxygenated derivatives and attachement to new, functionalized caprolactone monomers and polymers
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Language:
English
Creator:
Leslie, J. Michelle
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Penicillin
Vancomycin
MRSA
Bacillus
Polyester
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: N-Thiolated beta-lactams are a new class of anti-MRSA and anti-Bacillus agents that have recently been reported by our laboratories. From previous studies performed in our laboratories, it is believed that the N-thiolated beta-lactams exert their antimicrobial activity through a unique mode of action that is completely unlike that of classical beta-lactam antibiotics. In the first chapter of this dissertation, a review of previously prepared N-thiolated beta-lactam analogues and their mode of action is presented. In the second chapter, the synthesis of seven different C3-oxygenated derivatives is described. These analogues were tested for antibacterial activity against Staphylococcus aureus, nine different strains of MRSA, and seven different species of Bacillus. The results of the antibacterial testing will be discussed in relation to the differences in the structures of the analogues. In chapter 3, the design and synthesis of two new, functionalized caprolactone monomers are presented. These monomers were subsequently cooligomerized with epsilon-caprolactone, as described in chapter 4. N-thiolated beta-lactams were attached to the functionalized oligomers. These antibiotic containing oligomers were then screened for activity against MSSA, MRSA, and Bacillus. The results of these biological tests and their implications for future experiments are discussed.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
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Includes bibliographical references.
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by J. Michelle Leslie.
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Title from PDF of title page.
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Document formatted into pages; contains 185 pages.
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Includes vita.

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aleph - 001910178
oclc - 173275579
usfldc doi - E14-SFE0001685
usfldc handle - e14.1685
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N -Thiolated -Lactam Antibiotics: Synthesis and St ructure-Activity Studies of C3Oxygenated Derivatives and Attachment to New, Functionalized Caprolactone Monomers and Polymers by J. Michelle Leslie A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Edward Turos, Ph.D. Bill J. Baker, Ph.D. Kirpal S. Bisht, Ph.D. Mohamed Eddaoudi, Ph.D. Date of Approval: July 10, 2006 Keywords: penicillin, vancomycin, MRSA, Bacillus polyester Copyright 2006, J. Michelle Leslie

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Acknowledgements First and foremost, I would like to thank my advisor, Dr. Edward Turos, for allowing me the opportunity to conduct re search in his laboratories. I am especially grateful to him for allo wing me to pursue my own ideas and experiments in the lab. Also, although the numerous group meetings, journal meetings, seminars and colloquia that we were required to attend each week sometimes seemed like cruel and unusual p unishment, I am grateful for them because they helped me to continue learni ng even after my formal coursework was through. Secondly, I would like to thank a ll of the graduate st udents, postdocs and undergraduate students that I worked with over the year s in the Turos group. My labmates (sometimes more like roomma tes) were always there to help with experiments, teaching, presentations, et c., to laugh with and sometimes cry with, to sing badly along to the radio with, and in general to make time spent in the lab much more enjoyable. I would like to thank the members of my committee: Dr. Baker, Dr. Bisht, and Dr Eddaoudi for taking the time out of their schedules to serve on my committee and for always asking insightful questions.

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I would like to acknowledge Sonja Dickey, who ran the biological assays, and Dr. Ted Gauthier, who ran the MALDI spectra. I would like to give a very specia l acknowledgement to my own personal support system: Brian and Jane Leslie (my parents), Nicole and James McQueen (my sister and brother-in-law), Jaenea No rtje, and especially Dave Flanigan. Without the support and encourag ement that they provi ded over the years, it would have been very difficult to complete this work. Finally, I would like to acknowledge Dr. Hilary Jenkins and Dr. Bob Berno, two of my former professors from Saint Ma ry’s University, who started me on this whole adventure when they approached me during my senior year of undergrad and said, “we think it would be a shame if you didn’t go to graduate school”.

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iv Table of Contents List of Tables vi List of Figures viii List of Schemes x List of Abbreviations xii Abstract xiv Chapter One: Introduction 1 1.1 Penicillin and other -lactam antibiotics 1 1.1.1 Mode of action of the -lactam antibiotics 2 1.1.2 Resistance problems 4 1.2 Non-conventionally fused -lactams 4 1.3 Analogues of N -thiolated -lactams 6 1.3.1 First N -thiolated monocyclic -lactam analogues 6 1.3.2 C4 Aryl analogues 7 1.3.3 N-organothio analogues 9 1.3.4 C3 monosubstituted -lactams 11 1.3.5 C3 disubstituted -lactams 12 1.4.0 Mode of action of the N -thiolated -lactam antibacterials 13 1.5.0 Conclusions 16 1.6.0 References 17 Chapter Two: Results of the SAR Studies of N -Thiolated -Lactams and the Synthesis of C3 Hydroxy and Alkoxy Derivatives 19 2.1 Introduction 19 2.2 Results and discussion 20 2.2.1 Synthesis of the C3 hydr oxy and alkoxy derivatives 20 2.2.2 MRSA and Bacillus 26 2.2.3 Kirby-Bauer assay 27 2.2.4 Results of the biological screening against MRSA 28

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v 2.2.5 Results of the biological screening against Bacillus 36 2.3 Conclusions 38 2.4 Experimental 40 2.5 References 48 Chapter Three: Functionaliz ed Caprolactone Monomers: Design, Synthesis and Biological Evaluation 50 3.1 Introduction 50 3.2 Results and discussion 55 3.2.1 Synthesis of a lact one bearing a pendant benzyl ester 55 3.2.2 Synthesis of a lact one bearing an acid-cleavable tert -butyl ester group 60 3.2.3 Attachment of N -thiolated -lactams 63 3.2.4 Results of screening for anti-MRSA activity 66 3.2.5 Results of screening for antiBacillus activity 68 3.3 Conclusions 69 3.4 Experimental 70 3.5 References 76 Chapter Four: Functionalized Caprolactone Cooligomers: Cooligomermerization, Attachment of N -Thiolated -Lactams and Biological Evaluation 78 4.1 Introduction 78 4.1.1 Poly( -caprolactone) 79 4.1.2 Polymerization of -caprolactone 79 4.1.3 Examples of copolymerization of -caprolactone with substituted lactones from the literature 83 4.2 Results and discussion 86 4.2.1 Cooligomerizations of benzyl ester containing lactone with -caprolactone 86 4.2.2 Cooligomerizations of tert -butyl ester containing lactone with -caprolactone 89 4.2.3 Deprotection of the oligomers 90 4.2.4 Attachment of N -thiolated -lactams to the oligomers 92 4.2.5 Results of screening for anti-MRSA and antiBacillus activity 94 4.3 Conclusions 99 4.4 Experimental 101 4.5 References 103 Appendix : Selected spectra 104 About the Author End Page

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vi List of Tables Table 2.1 Average growth inhibition zones for C4 aryl analogues vs. MRSA 30 Table 2.2 Average growth inhibition zones for N -organothio analogues vs. MRSA 31 Table 2.3 Average growth inhibition zones for C3 monosubstituted analogues vs. MRSA 34 Table 2.4 Average growth inhibition zones for C3 disubstituted analogues vs. MRSA 35 Table 2.5 Average growth inhibition zones for C4 aryl analogues vs. Bacillus 37 Table 2.6 Average growth inhibition zones for N -organothio analogues vs. Bacillus 37 Table 2.7 Average growth inhibition zones for C3 monosubstituted analogues vs. Bacillus 38 Table 2.8 Average growth inhibition zones for C3 disubstituted analogues vs. Bacillus 38 Table 3.1 Zones of growth inhibition for lactams and lactam-lactone conjugates against MRSA 67 Table 3.2 Zones of growth inhibition for lactams and lactam-lactone conjugates against Bacillus 68 Table 4.1 Oligomers 147 87

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vii Table 4.2 Oligomers 148 90 Table 4.3 Growth inhibition zones for la ctam-containing oligomers against MRSA 95 Table 4.4 Growth inhibition zones for la ctam-containing oligomers against Bacillus 98

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viii List of Figures Figure 1.1 -lactam family of antibiotics 2 Figure 1.2 N -thiolated bicyclic -lactams 5 Figure 1.3 N -thiolated monocyclic -lactams 7 Figure 1.4 C4 aryl derivatives of N -thiolated -lactams 8 Figure 1.5 N -organothio derivatives of N -thiolated -lactams 10 Figure 1.6 C3 monosubst ituted derivatives of N -thiolated -lactams 12 Figure 1.7 C3 disubstituted derivatives of N -thiolated -lactams 13 Figure 2.1 C3-oxygenated N -thiolated -lactam derivatives 19 Figure 2.2 The Kirby-Bauer test for antibiotic susceptibility 27 Figure 3.1 Poly( -caprolactone) and pr oposed drug-bearing poly( caprolactone) 51 Figure 3.2 5-bromocaprolactone 52 Figure 3.3 Substituted lactones prepared by Hedrick et al. 54 Figure 3.4 Structure of proposed functionalized caprolactone monomer 54 Figure 3.5 Lactone 112 56 Figure 3.6 Lactone 122 60 Figure 3.7 Proposed mechanism of keta l deprotection using molecular iodine in acetone 63

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ix Figure 4.1 Ring-opening polymerization of -caprolactone 79 Figure 4.2 Structure of Sn(Oct)2 79 Figure 4.3 Likely mechanism for the Sn(Oct)2 catalyzed polymerization of -caprolactone 81 Figure 4.4 Intermolecular transesterification 82 Figure 4.5 Intramolecular transesterification 83 Figure 4.6 MALDI spectrum of oligomer 147 88 Figure 4.7 Expanded region 1H NMR spectra for -lactam 73 and -lactam bearing oligomer 150 94 Figure 4.8 Kirby-Bauer disc diffu sion test plate for compounds 131 132 and 151 against MRSA 655 97

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x List of Schemes Scheme 1.1 Formation of cross-links in bacterial cell wall 3 Scheme 1.2 Acylation of acti ve site by penicillin 4 Scheme 1.3 Iodocyclization reaction 5 Scheme 1.4 Reaction of N -thiolated -lactam with 2-mercaptopyridine 14 Scheme 1.5 Transfer of N-organothio substituent to Coenzyme A 15 Scheme 2.1 Synthesis of C3 acetoxy -lactam 84 20 Scheme 2.2 Synthesis of C3 hydroxyl -lactam 73 22 Scheme 2.3 Synthesis of the C3 secondary alkoxy -lactams, 74 75 and 76 23 Scheme 2.4 Synthesis of tertiary alcohol 94 from 87 24 Scheme 2.5 Synthesis of t he C3 tertiary alkoxy -lactams 77 78 and 79 25 Scheme 3.1 In vivo degradation of poly( -caprolactone) 51 Scheme 3.2 Likely outcome of Willia mson ether synthesis of lactone 103 53 Scheme 3.3 Retrosynthesis for substituted lactone 108 55 Scheme 3.4 Synthesis of ylide 115 56 Scheme 3.5 Initial synthesis of lactone 112 58 Scheme 3.6 Alternative synthesis of lactone 112 59 Scheme 3.7 Synthesis of ylide 125 61 Scheme 3.8 Synthesis of lactone 122 61

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xi Scheme 3.9 Deprotection of lactone 112 64 Scheme 3.10 Deprotection of lactone 122 64 Scheme 3.11 Coupling of acid 129 to N -thiolated -lactam 73 65 Scheme 3.12 Coupling of acid 129 to N -thiolated -lactam 131 65 Scheme 4.1 Copolymerization of -caprolactone with 5-bromocaprolac tone 83 Scheme 4.2 Copolymerization of -caprolactone with substituted lactones 84 Scheme 4.3 Cization of -caprolactone with RS-benzylmalonate 85 Scheme 4.4 Cooligom erization of lactone 112 with -caprolactone 86 Scheme 4.5 Cooligom erization of lactone 122 with -caprolactone 89 Scheme 4.6 Deprotection of the be nzyl ester containing oligomer 147 90 Scheme 4.7 Deprotection of the tert -butyl ester cont aining oligomer 148 91 Scheme 4.8 Attachment of N -thiolated -lactams to the carboxylic acid containing oligomer 93

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xii List of Abbreviations alpha AcO acetoxy AcOH acetic acid (aq.) aqueous Ar aryl ATCC American Type Culture Collection beta Bn benzyl Bu butyl bp boiling point br broad (spectral) Bz benzoyl C degrees Celsius 13C NMR carbon-13 nuclear magnetic resonance c concentration (mg/ml) CAN ceric ammonium nitrate cat catalytic CH2Cl2 dichloromethane C6H6 benzene C6H5CH3 toluene delta or chemical shift DMAP 4-dimethylaminopyridine DMSO dimethylsulfoxide Et ethyl Et3N triethylamine EtOAc ethyl acetate eq equivalent(s) g gram(s) (g) gas 1H NMR proton nuclear magnetic resonance h hour(s) Hz hertz IR infrared J coupling-constant(s) KOH potassium hydroxide LiOH lithium hydroxide M molar or moles per liter

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xiii MALDI-TOF ma trix assisted laser desorpti on ionization time-of-flight m CPBA meta -chloroperoxybenzoic acid Me methyl MeCN acetonitrile MeI methyl iodide MeOH methanol mg milligram(s) MHz megahertz MIC minimu m inhibitory concentration min minute(s) mL milliliter(s) mmol millimole(s) mol mole(s) MOM methoxymethyl MRSA methicillin-resistant Staphylococcus aureus MSSA methicillin-susceptible Staphylococcus aureus NaOH sodium hydroxide Pd/C palladium on carbon Ph phenyl ppm parts per million PMP para-methoxyphenyl rt room temperature SAR stru cture activity relationship TBAI tetra-n-butylammonium iodide THF tetrahydrofuran TLC thin layer chromatography g microgram(s) L microliter(s)

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xiv N -Thiolated -Lactam Antibiotics: Synthesis and Structure-Activity Studies of C3-Oxygenated Derivatives and A ttachment to New, Functionalized Caprolactone Monomers and Polymers J. Michelle Leslie ABSTRACT N -Thiolated -lactams are a new class of anti-MRSA and antiBacillus agents that have recently been reported by our laboratories. From previous studies performed in our laborator ies, it is believed that the N -thiolated -lactams exert their antimicrobial activity th rough a unique mode of action that is completely unlike that of classical -lactam antibiotics. In t he first chapter of this dissertation, a review of previously prepared N -thiolated -lactam analogues and their mode of action is presented. In t he second chapter, the synthesis of seven different C3-oxygenated derivatives is de scribed. These analogues were tested for antibacterial activity against Staphylococcus aureus nine different strains of MRSA, and seven different species of Bacillus The results of the antibacterial testing will be discussed in relation to the differences in the structures of the analogues. In chapter 3, the design and synthesis of two new, functionalized caprolactone monomers ar e presented. These monom ers were subsequently cooligomerized with -caprolactone, as described in chapter 4. N -thiolated -

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xv lactams were attached to the functi onalized oligomers. These antibiotic containing oligomers were then screened fo r activity agains t MSSA, MRSA, and Bacillus The results of these biological te sts and their implications for future experiments are discussed.

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1 Chapter 1 Introduction to the N -Thiolated -Lactam Antibiotics 1.1 Penicillin and other -lactam antibiotics For over 60 years, t he penicillins (and other -lactam antibiotics) have been broadly used for the tr eatment and prevention of bacterial infections.1 Following the commercial availability of penicillin in 1940, many other lactam antibiotics were also prepared and tested.2 The general structures of the most common families of -lactam antibiotics are shown in Figure 1.1. The key structural features of penicillin and its analogues such as the penams ( 2 ), penems ( 3 ), carbapenems ( 4 ), cephalosporins ( 5 ), clavulanic acids ( 6 ), nocardicins ( 7 ) and monobactams ( 8 ) are the -lactam ring, and an ionizable ring functionality. The nocardicins, 7 discovered in 19763, were the first monocyclic lactams that exhibited hi gh antibacterial activity.4 The monobactams, 8 were the first -lactam analogues to have a sulfur atom attached directly to the lactam nitrogen.5

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2 Figure 1.1 -lactam family of antibiotics N S O CO2H H N S O CO2H X H N O O CO2H H RHN penams ( 2 ) penems ( 3 ) H HO N O CO2H X H H HO carbapenems ( 4 ) N S O CO2H H H N R O penicillins ( 1 ) N S O RHN H CO2H X cephalosporins ( 5 ) OH clavulanic acids ( 6 ) N O R H N HO2C OH O R noca r dicins ( 7 ) N O SO3 R H N O R monobactams ( 8 ) 1.1.1 Mode of action of the -lactam antibiotics All of the -lactam antibiotics that are shown in Figure 1.1 are water soluble in their salts forms, and attack t he bacteria from t he outside (without entering the cell).1 These antibiotics kill bacteria by targeting the membranebound transpeptidases which are responsible for creating cross-links within the cell wall of the bacteria.2 These crosslinks are formed by the transpeptidases through the replacement of a terminal D-alanine residue on one peptidoglycan strand with the glycine residue from a nei ghbouring peptidoglycan chain. The

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3 cleavage of the D-alanine residue happens w hen an active site serine adds to the amide functionality in a nucleophi lic fashion producing an enzyme-linked peptidoglycan (Scheme1.1). This en zyme-linked peptidogl ycan then undergoes an amidation reaction, which releases the serine for further catalysis. Scheme 1.1 Formation of cro ss-links in bacterial cell wall peptidoglycan H N N H CH3 CH3 O CO2 enzymeserine OH peptidoglycan H N CH3 O O serine-enzyme peptidoglycan H N N H peptidoglycan CH 3 O enzymeserine OH + Penicillin and related -lactams irreversibly block this process by acylating the serine hydroxyl group (Scheme 1.2). T he ionizable ring functionality in these drug molecules is required for binding to the enzyme. The resulting enzyme drugadduct is stable and catalytically inactive. The disruption of these cross-linking proteins leads to structural deformiti es within the cell wall which make the bacteria prone to rupture, which leads to the death of the bacterium.

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4 Scheme 1.2 Acylation of activ e site serine by penicillin enzymeserine OH N S O R CONH CO2H HN S RCONH CO2H O O enzymeserine death of bacterium 1.1.2 Resistance problems Penicillin and the -lactam antibiotics were c onsidered to be “wonder drugs”, and with their discovery, many though t that the war on infectious disease was over. Unfortunately, penicillin-resistant strains of bacter ia began to emerge soon after its introduction.7 The resistant bacteria fought back against the lactam antibiotics by developing enzymes ( -lactamases) which could hydrolyze the -lactam ring, thereby r endering the antibiotic inactive. The advent of resistant bacteria makes the ongoing re search to develop new classes of antibiotics essential. 1.2 Non-conventionally fused -lactams In 1994, Ren et al. first reported the synthesis of non-conventionally fused -lactams.8 In this study, new -lactam antibiotics where the -lactam ring was reorganized so that the lactam nitrogen was directly attached to a sulfur atom were prepared (Figure 1.2). T hese derivatives were called N -thiolated bicyclic lactams and it was hoped that these analogues might provide useful leads in the

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5 search for new antibiotics. Compounds 9 and 10 are penem-monobactam hybrids, and compounds 11 and 12 are clavulanic acid-monobactam hybrids. Figure 1.2 N -thiolated bicyclic -lactams N S H3CO I Ph O H H N S H3CO I Ph O H H O O N S H3CO OAc O H H N S H3CO OAc O H H O O Ph I I Ph ( 9 )( 10 ) ( 11 )( 12 ) These new hybrids were screened for antibacterial activities against a wide variety of common strains of Gra m-positive and Gram-negative bacteria. However, none of these new bicyclic compounds showed any appreciable antimicrobial effects. In the preparat ion of these bicyclic compounds, Ren developed a protocol invo lving an iodocyclization reaction of an unsaturated compound ( 13 ) (Scheme 1.3).9 Scheme 1.3 Iodocyclization reaction N O S CH3 H3CO Ph N S H3CO I Ph O H H N S H3CO I Ph O H H O O mCPBA CH2Cl282% I2, CH2Cl240oC, 80% ( 13 ) ( 9 ) ( 10 )

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6 The intermediate, 13 was also screened for antibacterial activity against the same panel of common bacteria, and it was found to be a powerful growth inhibitor of Staphylococcus aureus including the multi-drug resistant Staphylococcus aureus (MRSA).10 With this discovery, a large structure-activity relationship (SAR) study was undertaken in the Turos laboratory to study these new monocyclic N -thiolated -lactams in more detail. 1.3 Analogues of N -thiolated -lactams 1.3.1 First N -thiolated monocyclic -lactams analogues Compounds that were stru cturally similar to 13 varying only in the spacer unit between the -lactam ring and the aryl substitu ent at the C4 position, were prepared first.11 Compounds where there was no spacer unit ( 18 and 19 ) were selected for further study because their bioac tivities were comparable to that of the C4acetylenic lead compound, 13 and also because they were easily prepared.

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7 Figure 1.3 N -thiolated monocyclic -lactams N O S CH3 H3CO Ph ( 13 ) N O S CH3 H3CO N O S CH3 H3CO ( 14 ) Ph Ph AcO ( 15 ) N O S CH3 H3CO ( 16 ) N O S CH3 H3CO ( 17 ) N O S CH3 H3CO ( 18 ) N O S CH3 H3CO ( 19 ) Ph Ph Cl 1.3.2 C4 Aryl analogues Numerous analogues of 18 where the position and nature of the substituents on the C4 aryl ring wa s varied were prepared (Figure 1.4).11,12

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8 Figure 1.4 C4 Aryl derivatives of N -thiolated -lactams N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO F N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO I N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO Cl Cl Cl MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO Me ( 20 )( 21 )( 22 )( 23 ) ( 24 )( 25 )( 26 )( 27 ) ( 28 )( 29 )( 30 )( 31 ) ( 32 )( 33 )( 34 )( 35 ) N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO N O S CH3 MeO ( 36 )( 37 )( 38 ) ( 39 ) Cl Cl F F I I Cl Cl Cl Cl O2N O OH Ph OBn O2N OAc O BnO Some examples of the C4 aryl anal ogues that were studied include: monohalogenated compounds 20 21 22 23 24 25 26 ,and 27 ; dichlorinated compounds 28 and 29 ; trichlorinated analogue 30 ; nitrated derivatives 33 and 34 ; and compounds that contain electr on-donating substituents such as 31 32 35 36 37 38 and 39

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9 1.3.3 N -organothio analogues The effect of the substituent on the su lfur atom was also studied by Bart Heldreth in our laboratory.13 The length of the substituent chain, degree of branching, and oxidation state of the sulfur was studied. Examples of some of the N -organothio analogues that were prepar ed are shown in Figure 1.5. These analogues include: straight chain sulfenyl compounds 40 41 42 and 43 ; branched sulfenyl compounds 44 45 46 and 47 ; aryl sulfenyl compounds 48 and 49 ; sulfinyl compounds 50 and 53 ; sulfonyl compounds 51 and 54 ; and sulfonic acid 52

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10 Figure 1.5 N -Organothio derivatives of N -thiolated -lactams N O S MeO N O S CH2CH2CH3 MeO N O S (CH2)3CH3 MeO N O S (CH2)7CH3 MeO Cl N O S CH(CH3)2 MeO N O S CH(CH3)CH2CH3 MeO N O S C(CH3)3 MeO N O S Cy MeO Cl N O S Ph MeO N O S Bn MeO N O S Cy MeO N O S Cy MeO Cl Cl Cl Cl N O S MeO Cl ( 40 )( 41 )( 42 ) ( 43 ) ( 44 )( 45 ) ( 46 )( 47 ) ( 48 ) ( 49 )( 50 )( 51 ) ( 52 ) CH2CH3 Cl Cl Cl Cl Cl Cl O O O O O OH N O S CH(CH3)CH2CH3 MeO N O S CH(CH3)CH2CH3 MeO Cl Cl ( 53 )( 54 ) O O O

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11 1.3.4 C3 monosubstituted -lactams It had been previously determined that compounds that were either unsubstituted14 or had a simple alkyl substituent15 at the C3 position were either completely inactive against MRSA, or only weakly active. The compounds that had showed the strongest bioactivity had a methoxy substituent at this position. This suggested that a polar gr oup at the C3 position is r equired for bioactivity. To determine how other substituents at this position affect the bioactivity, a wide variety of compounds that were monos ubstituted at the C3 position were prepared.12 These compounds vary in their polarit ies, lipophilicities, size (steric bulk), hydrogen bonding capabilit ies and stereochemistry. Ex amples of some of the C3 monosubstituted -lactams that were prepared are shown in Figure 1.6. The functional groups incorporated at this position include: azide 55 ; halides 56 and 57 ; amines 58 59 60 and 61 ; ether 62 ; ester 63 ; and sulfonates 64 65 and 66 .12

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12 Figure 1.6 C3 Monosubsti tuted derivatives of N -thiolated -lactams N O S CH3 N3 N O S CH3 I N O S CH3 Cl N O S CH3 N H Cl Cl Cl Cl N O S CH3 BnHN N O S CH3 Et2N N O S CH3 Bu2N Cl Cl Cl N O S CH3 PhO N O S CH3 AcO Cl Cl N O S CH3 MeO2SO N O S CH3 PhO2SO N O S CH3 TolO2SO i( 55 )( 56 )( 57 ) ( 60 ) ( 58 ) ( 59 )( 61 )( 62 ) ( 63 )( 64 )( 65 )( 66 ) 1.3.5 C3 disubstituted -lactams Compounds that were disubstituted at the C3 position were also prepared to continue investigating the role of the C3 substituent and to study the effect of increased steric crowding at this center (Figure 1.7). Compounds 67 68 and 69 have both an alkyl group and an ester group at C3. Compounds 70 71 and 72 contain spirocyclic ethers at the C3 position.

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13 Figure 1.7 C3 Disubsti tuted derivatives of N -thiolated -lactams N O S CH3 N O S CH3 N O S CH3 N O S CH3 Cl Cl Cl N O S CH3 N O S CH3 ( 67 )( 68 )( 69 ) ( 70 ) ( 71 ) ( 72 ) NO2 Ph AcO AcO AcO O O O 1.4 Mode of action of the N -thiolated -lactam antibacterials The initial screening of the N -thiolated -lactams against a variety of common strains of Gram-positive and Gra m-negative bacteria showed that these compounds exhibited narrow s pectrum bioactivity. These lactams showed activity against members of the Staphylococcus genus including S. aureus (and MRSA), S. epidermidis S. simulans S. saprophytcus ; and a few other genera such such as Micrococcus luteus and Neisseria gonorrhoeae .11 A wide variety of other Gram-negative and Gram-positive bacte ria seem to be unaffected by these lactams. The discovery that these -lactams have any antibacterial activity at all was surprising at first, given that they lack an ionizable ring functionality (required by all other known -lactams for binding to th e bacterial transpeptidase). Studies done by Dr. Timothy Long in our laboratory showed that the N thiolated -lactams do not inhibit cell wall bios ynthesis. Bacterial cells that had

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14 been treated with an N -thiolated -lactam were examined by scanning electron microscopy (SEM) and it was observed that there was no change to the cellular morphology. When bacterial cells that had been treated with penicillin were viewed using SEM, drastic alterations in the cell morphology were observed. Gram staining of cells treated with either an N -thiolated -lactam or penicillin showed that there was no change in the thi ckness of the cell wall for those cells treated with the N -thiolated -lactam (staining purple), while the cell walls were significantly thinner (and stained pi nk) when treated with penicillin. These observations showed that the N -thiolated -lactams had a mode of action that was different than classical -lactam antibiotics. Additional studies showed that the N -thiolated -lactams were stable to acidic conditions, mildly basic conditions and radicals. However, it was found that these lactams were not stable to thiols such as glutathione. In the presence of thiols, such as 2-mercaptopyridine, a nucl eophilic attack at the su lfur atom by the thiol is observed to form the mixed disulfide (Scheme 1.4). 17 Scheme 1.4 Reaction of N -thiolated -lactam with 2-mercaptopyridine N O S R Cl H3CO N HS NH O Cl H3CO N S S R + It was found that the N-thio substituent (SR) was required for bioactivity11 and that the addition of thiols, like glut athione, to the lactams reduced their

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15 bioactivity.16,17 These observations led to the hy pothesis that the bioactivity of these N -thiolated -lactams is due to the transfer of the N -organothio group to a thiol target in the cell. It was later f ound that these lactams transfer the thiol group to Coenzyme A (CoA) to form the mixed CoA disulfide (scheme 1.5).17 Scheme 1.5 Transfer of N -organothio substituent to Coenzyme A N O S R Cl H3CO R S S H N O H N O P O O OH O OP O O OO NH OH P O OO N N N H2N CoA Later studies in our laboratory by Dr Kevin Revell have shown that these mixed CoA disulfides inhibi t fatty acid biosynthesis by capping FabH, a key enzyme in type II fatty acid synthesis. Inhi biting fatty acid biosynthesis stops the growth of the bacterium.18

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16 1.5 Conclusions A new class of monocyclic -lactam antibiotics, called the N -thiolated lactams, has been discovered by our labor atories. These lactams exhibit narrow spectrum antibacterial properties and are particularly effective against Staphylococcus aureus including MRSA. Previous work by Tim Long in our laboratory has shown that t hese new antibiotics do not ac t on the bacteria in the same way as the previously known classes of -lactams. Later work by Bart Heldreth and Kevin Revell in our lab has shown that the N -thiolated -lactams enter the bacterial cell and transfer the alkylthio group to coenzyme A to form the mixed CoA disulfide. These mixed CoA disulfides stop the growth of the bacterium by inhibiting FabH, a key en zyme in type II fatty acid synthesis. A large SAR study was undertaken by our group in order to better understand how each of the ring substi tuents contributes to the observed antibacterial properties. The results of t he biological screening of the analogues shown in this chapter, as well as so me new C3 hydroxy and alkoxy analogues, will be discussed in chapter 2.

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17 1.6 References 1. (a) Sweet, R.M. In Cephalosporins and Penicillins, Chemistry and Biology ; Flynn, E.H., Ed.; 1972, p 290. (b) Dunn, G.L. In Comprehensive Heterocylic Chemistry; Lwowski, W. Ed.; Pergamon Press; New York, 1984, Vol. 7, p341-363. 2. Brown, A.G. Pure Appl. Chem 1987 59 475. 3. Hashimoto, M.; Komori, T.; Kamiya, T. J. Am. Chem. Soc 1976 98 3023. 4. For reviews see: (a) Issacs, N.S. Chem. Soc. Rev 1976 76 181. (b) Mukerjee, A.K.; Singh, A.K. Tetrahedron, 1978 34 1731. 5. (a) Imada A.; Kitano, K.; Muroi, M.; Asai, M. Nature, 1981, 291, 590. (b) Sykes, R.B.; Bonner, D. P. Int. Congr. Symp. Se r. –R. Soc. Med 1985 89 3. 6. Waxman, D.J; Strominger, J.L. In Chemistry and Biology of -Lactam Antibiotics Morin, R.B.; Gorman, M. Ed s; Academic Press: NewYork, 1982; Vol. 3, p 209. 7. (a) Chin, G.J., Marx, J. Science 1994 264 359. (b) Antimicrobial Drug Resistance ; Bryan, L.E., Ed.; Academic Press: New York, 1984. (c) Gunda, E.T.; Jaszberenyi, J.C. Prog. Med. Chem 1977 14 181. 8. Ren, X-F.; Turos, E.; J. Org. Chem 1994 59 5858. 9. Ren, X-F.; Konakliev a, M. I.; Turos, E. J. Org. Chem 1995 60 4980. 10. Ren, X-F.; Konaklieva, M.I.; Shi, H .; Dickey, S.; Lim, D.V.; Gonzalez, J.; Turos, E. J. Org. Chem 1998 63 8898.

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18 11. (a) Turos, E.; Long, T.E.; Konaklieva, M.I.; Coates, C.; Shim, J-Y.; Dickey, S.; Lim, D.V.; Cannons, A. Bioorg. Med. Chem. Lett 2002 12 2229. (b) Coates, C.; Long, T.E.; Turos, E.; Dickey, S.; Lim, D.V. Bioorg. Med. Chem 2003 11 193. 12. Turos, E.; Coates, C. Shim, J-Y., Yang, W., Leslie, J.M., Long, T.E., Reddy, G.S.K.; Ortiz, A., Culbreath, M. ; Dickey, S.; Lim, D.V.; Alonso, E.; Gonzalez, J. Bioorg. Med. Chem 2005 13 6289. 13. Heldreth, B.; Long, T.E.; Jang, S.; Reddy, G.S.K.; Turos, E.; Dickey, S.; Lim, D.V. Bioorg. Med. Chem 2006 14 3775. 14. Coates, C., Ph.D. Dissertation, University of South Florida, 2004 15. Konaklieva, M.I., Ph.D. Di ssertation, SUNY at Buffalo, 1997 16. Long, T.E., Ph.D. Dissertation, University of South Florida, 2003 17. Heldreth, B.A., Ph.D. Dissertatio n, University of South Florida, 2004 18. Revell, K.D., Ph.D. Dissertation, University of South Florida, 2006

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19 Chapter 2 Results of the SAR Studies of N -Thiolated -Lactams and the Synthesis of C3 Hydroxy and Alkoxy analogues. 2.1 Introduction This chapter will focus on the results of SAR studies done in the Truos laboratory for the N -thiolated -lactam antibiotics. The re sults of the biological screening of the analogues described in Chapt er one, as well as seven new C3 oxygenated analogues 73 79 (Figure 2.1), will be disc ussed. The synthesis of these seven new analogues wi ll also be described. Figure 2.1 C3-oxygenated -lactam analogues. N O SCH3 HO Cl N O SCH3 O Cl N O SCH3 O Cl N O SCH3 O Cl O N O Cl O SCH3 N O Cl O SCH3 N O Cl O SCH3 O 73 74 75 76 77 78 79

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20 2.2 Results and Discussion 2.2.1 Synthesis of the C3 secondary hydroxy and alkoxy derivatives The -lactam derivatives shown in Figure 2.1 were derived from a common intermediate, the C3 acetoxy -lactam 84 This intermediate was easily prepared as a racemate in two steps as shown in Scheme 2.1. Scheme 2.1 Synthesis of C3-acetoxy -lactam 84 N O O Cl OCH3 O O Cl O O N OCH3 Cl 83 82 84 b Reagents and conditions: (a) neat, 5min; (b) 1.8 eq. 83 3 eq. Et3N, dry CH2Cl2, 12h, 0 o C r t; ( c ) KOH, acetone / MeOH,5min,0o C OCH3 NH2 CHO Cl + a 8081 Upon mixing together p -anisidine ( 80 ) and o -chlorobenzaldehyde ( 81 ) at

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21 room temperature, imine 82 formed as a yellow paste within minutes. The paste was then crystallized from methanol to give pure 82 as bright yellow crystals in nearly quantitative yield. The imine was combined with acid chloride 83 (commercially available or readily prepared in two steps from glycolic acid1) using a typical Staudinger coupling2 to give the -lactam 84 as a white solid with typical yields of 80-90%. The -lactam ( 84 ) was obtained as a racemic mixture of the cis -isomers, whose stereochemistry was a ssigned based on the vicinal coupling constant (J = 5.0 Hz) for the protons at the C3 and C4 positions of the ring3. All of the compounds presented in this chapter are racemic; any stereochemistry that is indicated in the schemes indicates relative stereochemistry, not absolute stereochemistry. The C3 alcohol derivative 73 was prepared in three steps from 84 as shown in Scheme 2.2. The order of protection/deprot ection steps was completed as shown to avoid the intermediate t hat contained both an alcohol and a free amide due to the solubility problem s associated with this compound. The synthesis of -lactam 73 began with the oxidative cleavage of the amide protecting group ( p -methoxyphenyl, or PMP) wit h ceric ammonium nitrate4 (CAN) to give the free amide 85 in modest yields of 60-70%. The free amide was thiolated using N -methylthiophthalimide5 ( 86 ) and a catalytic amount of a tertiary amine base to give 63 as a yellow solid in 70% yield. The hydrolysis of the acetate substituent was accomplished very rapidly with one equivalent of potassium carbonate in methanol to give 73 as a pale yellow solid in 74% yield.

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22 Scheme 2.2 Synthesis of C3 hydroxy -lactam (73). N O O Cl OCH3 O 84 NH O O Cl O 85 N O SCH3 O Cl O 63 N O O SCH3 86 ab Reagents and conditions: (a) 3 eq. CAN, MeCN/H2O, 2h, 0oC; (b) 86 cat. Et3N, dry CH2Cl2, 12h, reflux; (c) K2CO3, MeOH 2min, rt. N O SCH3 HO Cl 73 c The synthesis of the C3 secondary alkoxy derivatives 74 76 also began with lactam 84 (Scheme 2.3). The syntheses of these three C3 secondary alkoxy derivatives began with the hydrol ysis of the acetate group of 84 to give the alcohol 87 This hydrolysis reaction occurs rapidly with potassium hydroxide to give 87 as a white solid in nearly quantitativ e yields. The C3 alkoxy groups were installed via a Williamson ether synthesis using either methoxymethyl (MOM) chloride or allyl bromide and alcohol 87 in the presence of a catalytic amount of tetra -butylammonium iodide (TBAI). The PM P protecting group was then cleaved using CAN. Allyl ether 90 was subjected to catalytic hydrogenation to afford the propyl ether 91 in quantitative yield. All of the compounds were then N -thiolated

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23 with methylthio-transfer reagent 86 in the same manner as described before to give 74 76 in 25, 40, and 35% yield respectively, following column chromatography. The low final yields on t hese reactions are due to the difficulty in separating the final pr oducts from residual reagent 86 Scheme 2.3 Synthesis of the C3 secondary alkoxy -lactams 74, 75 and 76. N O HO Cl OCH3 N O RO Cl OCH3 NH O RO Cl N O RO Cl SCH3 87 88 R = allyl 89 R = MOM b c 90 R = allyl 91 R = propyl 92 R = MOM d 74 R = allyl 75 R = propyl 76 R = MOM Reagents and conditions: (a) KOH, acetone/MeOH, 5min, 0oC (b) NaH, dry CH2Cl2, 15min, rt; R-X, cat. TBAI, 24h, reflux; (c) 3 eq. CAN, MeCN/H2O, 20min, 0 oC;(d) H2(g), cat. 10% Pd/C, MeOH, 12h, rt; (e) 86 cat. Et3N, dry CH2Cl2, 12h, reflux. e N O O Cl OCH3 O 84 a The C3 tertiary ether derivatives were then prepared to explore the effect of steric crowding at this center on bioactivity. All of these compounds were made from the te rtiary alcohol 94 whose synthesis is shown in Scheme 2.4.

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24 Scheme 2.4 Synthesis of tertiary alcohol 94 from 87. N O HO Cl OCH3 N O O Cl OCH3 N O Cl OCH3 HO ab 879394 Reagents and conditions: (a) P2O5, DMSO, 12h, rt; (b) MeMgI, dry Et2O, 4h, -78 oC. Alcohol 87 was subjected to oxidation under Moffatt-Swern conditions6 to give the C3-keto-lactam compound 93 as a light yellow solid in yields of 9095%. Methylmagnesium iodide (gener ated in situ from MeI and Mgo) was then added to 93 in anhydrous diethyl ether to give the tertiary alcohol 94 as a yellow solid in 70-75% yield. The methyl group was added exclusively to the face anti to the C4 aryl substituent, consistent with pr eviously reported data for a similar system by Buynak7, and also determined in our labora tory by Dr. Cristina Coates (for a related com pound) using ROESY-NMR6. The stereochemistry indicated for compounds 87 and 94 in Scheme 2.4 is relative, not absolute. The synthesis of the C3 tertiary alkoxy derivatives 77 78 and 79 was accomplished following an analogous pr ocedure to that used for the C3 secondary alkoxy derivat ives (Scheme 2.5).

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25 Scheme 2.5 Synthesis of the C3 tertiary alkoxy -lactams 77, 78, and 79. N O Cl OCH3 HO N O Cl OCH3 RO NH O Cl RO N O Cl RO SCH3 95 R = allyl 96 R = MOM 97 R = allyl 98 R = propyl 99 R = MOM c 77 R = allyl 78 R = propyl 79 R = MOM Reagents and conditions: (a) NaH, dry CH2Cl2, 15min, rt; R-X, cat. TBAI, 24h, reflux; (b) 3 eq.CAN, MeCN/H2O, 20min, 0 oC ; (c) H2(g), cat. 10% Pd/C, MeOH, 12h, rt; (d) 86 cat. Et3N, dry C H 2 C l 2 12h, r eflux. ab d 94 The synthesis of these three tertiary ether derivatives began with alcohol 94 being treated under Williamson ether synt hesis conditions using either MOM chloride or allyl bromide in the presence of a catalytic amount of TBAI. The yields of these reactions were slightly lo wer than the analogous reactions with the secondary alcohol, presumably due to ster ic crowding at the hydroxy center. The PMP protecting group was then cleaved wi th CAN to give the free amide compounds 97 and 99 in 74 and 95 % yield respectively, after column chromatography. Allyl ether 97 was subjected to catalytic hydrogenation to afford

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26 the propyl ether 98 in quantitative yield. All of the compounds were then N thiolated using N -methylthiophthalimide ( 86 ) as described before. 2.2.2 MRSA and Bacillus The N -thiolated -lactams were individually screened for antibacterial activity against nine different st rains of multi-drug resistant Staphylococcus aureus (MRSA) and also against seven species of Bacillus : Bacillus anthracis, Bacillus globigii, Bacillus thuringenis, Bacillus megat erium, Bacillus coagulans, Bacillus subtilis and Bacillus cereus. MRSA was selected for screening because it is the resistant form of Staphylococcus aureus, the microbe against which the N -thiolated -lactams had previously show n potent growth inhibition.8 For the MRSA screening, penicillin G and vancomyci n (current therapeutic for MRSA) were also screened as a reference. Since Staphylococcus and Bacillus are both members of the same order ( Bacillales ) of bacteria, the N -thiolated -lactams were also screened for antiBacillus activity.9 Recent concerns about the possible use of anthrax (causative agent = Bacillus anthracis ) as a biological weapon has led to increased interest in the developm ent of new anti-anthr ax agents. For the Bacillus screening, the current antiBacillus treatment, ciprofloxacin, was used as a reference.

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27 2.2.3 Kirby-Bauer assay The -lactams that were prepared in this study were individually screened for antimicrobial activity using the Kirby-Bauer method10 of disc or well diffusion (Figure 2.2). Figure 2.2 The Kirby-Bauer test for antibiotic susceptibility Bacterial Growth Cellulose Disc or Well Zone of Growth Inhibition In this assay, the test organism is first inoculated onto Mueller-Hinton agar. Then 20 g of the drug was placed onto the agar plate, eit her on a 6 mm cellulose disc or as a 1mg/mL solution in DMSO that was placed into 6 mm circular holes cut into the agar. The plates were incubated at 37 oC for 24 hours and then the zones of growth inhibition ar ound the disc or well were measured. It has been demonstrated previously in our laboratory that the Kirby-Bauer test results correlate well with the minimum i nhibitory concentration (MIC) values that

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28 were obtained from broth dilution experim ents, and therefore is a reliable way to measure bioactivity within a clos ely related series of analogues11. 2.2.4 Results of biologi cal screening against MRSA The results of the anti-MRSA studies on the C4 aryl analogues that were presented in Chapter 1 (Figure 1.4) are shown in Table 2.1. Also included in Table 2.1 are the zones of growth inhibiti on for the two reference drugs, Penicillin G and Vancomycin.11 Compounds 19 20 and 21 are the ortho meta and para monochlorinated compounds. Compounds 22 23 and 24 are the ortho meta and para monofluorinated compounds. Compounds 25 26 and 27 are the ortho meta and para monoiodinated compounds. For all of these compounds, the activity is independent on which halogen is present on the ring, but is dependent on the position of the substi tution. The compounds possessing ortho substituents displayed the greatest anti-MRSA ac tivity, followed by those with para substituents and then those with meta substituents. Compounds which contained more than one halogen substituent on the C4 aryl ring such as dichlorinated lactams 28 and 29 and trichlorinated analogue 30 were also screened for antiMRSA activity. The zones of inhibition for these polychlorinated compounds were slightly lower than those of the monochlor inated compounds 28 29 and 30 (1724 mm, 16-21 mm and 18-24 mm, respective ly). Nitro substituted compounds 33 and 34 both had lower bioactivities than the monohalogenated compounds 19 27 Again, as was the case with the halogens, the ortho substituted analogue 33 had greater zones of inhibition than the meta substituted analogue (18-23 mm for

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29 33 and 11-22 mm for 34 ). The effect of having electron-donating groups on the C4 aryl ring was also studied. Compounds 31 and 32 which contained small electron-donating groups at the ortho position showed bioactivities that were only slightly less active than the ortho -chloro compound 19 ; the ortho -methoxy compound 31 had zones of inhibition of 23-32 mm while the ortho -methyl compound 32 had zones of inhibition of 20-27 mm. Compounds 36 37 38 and 39 all contained electron donating groups at the para position, and had significantly lower anti-MRSA activity than ortho -substituted compounds 31 and 32 with zones of inhibition of 10-14 mm for para -acetoxy compound 35 10-14 mm for para -acryloxy compound 36 14-19 mm for para -hydroxy compound 37 and 8-12 mm for para -phenyl compound 38 This is again consistent with the previously observed trend that the ortho -substituted compounds have higher bioactivities. Compound 39 disubstituted with benzyl ether groups at both the meta and para positions, displayed activity similar to that of the para -substituted analogues 35 38

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30 Table 2.1 Average growth inhibition zones for C4 aryl derivatives against MRSA12 Pen Van 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 8 16 30 25 26 27 21 25 34 23 27 22 20 24 29 23 18 17 13 13 14 10 13 16 15 29 28 26 28 24 27 29 23 24 21 21 23 32 27 23 22 12 12 19 11 12 10 16 28 22 26 25 18 22 27 21 24 21 19 23 27 23 20 16 11 13 18 10 13 14 16 27 23 25 23 18 21 28 24 24 24 20 22 27 23 22 14 14 12 18 10 13 12 21 27 23 25 2619 25 29 22 25 19 21 19 28 25 22 18 13 13 19 11 14 12 15 25 22 23 26 21 25 28 25 24 20 21 22 27 23 20 18 12 14 19 12 13 19 15 27 19 24 25 18 23 28 23 20 21 19 20 26 22 21 17 12 13 18 9 11 15 15 23 18 18 21 17 20 23 22 17 17 16 18 23 20 18 11 10 10 0 8 10 nd nd 28 26 25 26 20 26 29 24 23 22 19 23 27 23 20 18 12 13 17 10 14 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameter in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound in DMSO solution was used. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purch ased from American Type Culture Collections. Error values are within +/-1 mm. Penicillin G (Pen) and Vancomycin (Van) are included for reference. (nd, not determined). The results of the ant i-MRSA studies on the N -organothio analogues13 that were presented in Chapter 1 (Figure 1.5) are shown in Table 2.2.

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31 Table 2.2 Average growth inhibition zones for N -organothio derivatives against MRSA13 40 41 42 43 44 45 46 47 48 49 50 51 52 33 30 25 13 33 44 10 24 27 28 22 11 0 33 30 24 12 32 41 10 24 24 27 21 10 0 29 28 24 13 31 39 10 23 22 27 29 9 0 29 28 22 12 29 39 10 23 22 27 19 9 0 30 27 23 12 30 39 10 22 23 26 19 8 0 30 27 24 14 31 40 10 23 24 27 20 10 0 31 28 25 13 32 41 10 26 25 29 21 9 0 29 27 24 13 3039 10 24 24 27 19 8 0 31 28 25 14 31 40 10 24 25 28 20 10 0 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA 657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameter in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound in DMSO solution was used. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purchased from American Type Culture Collections. E rror values are within +/-1 mm. Compounds 19 40 41 42 and 43 all contain a linear alkyl chain varying in length from 1-8 carbons as the substituent on the sulfur atom. The N -ethylthio lactam 40 is slightly more active than the lead compound, N -methylthio lactam 19 (zones of inhibition of 29-33 mm for 40 as compared to 23-30 mm for 19 ). Increasing the carbon chain length beyond 2 carbon atoms led to a systematic drop in the bioactivity, with zones of inhibition of 27-30 mm for N -propylthio lactam 41 22-25 mm for N -butylthio lactam 42 and 12-14 mm for N -octylthio lactam 43 Lactams 44 45 46 47 48 and 49 were prepared to study the effect of branching in the N -alkylthio substituent on the anti -MRSA activity. Of these five compounds, N -isopropylthio analogue 44 and the N-sec -butylthio analogue 45

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32 showed excellent bioactiv ities, with zones of inhibition of 29-33 mm for 44 and 39-44 mm for 45 The zones of inhibition of the N-sec -butylthio lactam was found to be ~ 100% larger than those of Vancom ycin, and ~ 200% larger than those of Penicillin G against the nine MRSA strain s! When the degree of branching in the alkylthio side chain was increased farther, as in the N-tert -butylthio lactam 46 the bioactivity dropped dramatically to zones of inhibition of only 10 mm. The N cyclohexylthio lactam 47 the N -phenylthio lactam 48 and the N -benzylthio lactam 49 all had moderate to high bioactivities with zones of inhibition of 22-26 mm, 22-27 mm, and 26-29 mm respec tively. The effect of the oxidatio n state of the sulfur was also examined. It was f ound that the bioactivi ty of the lactams dropped dramatically with incr easing oxidation state of the sulfur: the zones of inhibition for the sulfinyl compound 50 were 19-29 mm, for the sulfonyl compound 51 8-11 mm, and for the sulfonic acid compound 52 0 mm, indicative of an analogue completely devoid of anti-MRSA activity. The results of the anti-MRSA st udies on the C3 monosubstituted analogues that were present ed in Chapter 1 (Figure 1. 6), and the four new C3 monosubstituted analogues described in this chapter, 73 76 are shown in Table 2.3. Compound 19 is the C3 methoxy compound, which is the lead compound for our studies on the effect of the C3 substituent on anti-MRSA activity. Replacement of the methoxy substituent with either an azide group as in compound 55 or an iodine as in compound 56 dropped the zones of growth inhibition down to 19-23 mm and 20-25 mm respectively. A chlorine at the C3 position 57 gave slightly better activity ( 29-31 mm) than the methoxy compound

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33 19 Another interesting thing t hat should be noted about compounds 55 56 and 57 is that they all have a trans relationship of the hydrogens of the -lactam ring. The fact that these compounds show good bi ological activity demonstrates that a cis relationship of the -lactam ring hydrogens is not required for bioactivity. Installation of an amino group at the C3 position ( 58 59 60 and 61 ) eliminated the anti-MRSA activity for t he most part. Only the benzylamine compound 59 had any anti-MRSA activity at a ll, and it was very weak (10-12 mm). The C3 alkoxy analogues ( 62 74 75 and 76 ) all showed greater MRSA activity than Penicillin G, but slightly lower than that of the C3-methoxy lactam 19 The slightly more lipophilic ethers 74 and 75 showed better activity (both 2024 mm) than the more polar compounds 62 and 76 (13-16 mm and 15-19 mm respectively). Acetoxy compound 63 and hydroxy compound 73 also showed slightly lower activities than the C3-methoxy compound 19 (15-24 mm and 17-19 mm respectively). For the C3 sulfonate compounds 64 65 and 66 their activities increased with increased lipophili city, with the methyl< phenyl< p -tolyl.

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34 Table 2.3 Average growth inhibi tion zones for C3 monosubstituted derivatives against MRSA12 55 56 57 58 59 60 61 62 63 64 65 66 73 74 75 76 21 20 30 0 11 0 0 16 18 15 16 21 19 22 23 15 22 23 29 0 12 0 0 15 24 15 17 24 17 24 24 18 22 25 30 0 10 0 0 15 21 18 17 23 17 20 23 19 20 23 30 0 10 0 0 14 23 14 15 20 19 23 22 17 19 23 29 0 10 0 0 16 22 15 16 20 18 23 21 18 21 24 31 0 11 0 0 13 20 15 16 19 16 20 20 18 23 25 30 0 10 0 0 15 21 15 15 20 1920 20 18 22 23 30 0 10 0 0 16 15 14 15 21 18 24 24 16 23 24 29 0 11 0 0 16 18 15 16 21 18 22 23 15 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA 657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameter in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound in DMSO solution was used. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purchased from American Ty pe Culture Collections. Error values are within +/-1 mm. The results for the screening of the C3 disubstituted analogues against MRSA are shown in Table 2.4.

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35 Table 2.4 Average growth inhibition zones for C3 di substituted derivatives against MRSA12 77 78 79 67 68 69 70 71 72 28 28 18 25 28 20 22 17 17 26 26 17 26 27 20 19 18 16 24 24 17 22 26 18 20 15 15 26 26 18 23 26 20 21 16 17 26 26 17 25 28 21 20 17 16 26 25 18 26 29 21 28 15 14 24 25 16 24 26 20 17 16 12 26 25 18 21 24 20 21 14 14 27 29 19 24 27 21 21 18 16 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA 657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameter in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound in DMSO solution was used. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purchased from American Type Culture Collections. Error values are within +/-1 mm. Compounds 77 78 and 79 all had a methyl substituent and an alkoxy group at C3. As was the case for t heir corresponding monosubstituted analogues ( 74 75 and 76 ), the more lipophilic allyl and pr opyl ethers had greater activity (24-28 mm for 77 and 24-29 mm for 78 ) than the more polar methoxymethyl ether 79 (16-18 mm). The activities for all three of these compounds were higher than that of the corresponding monosubsti tuted analogues. For the next three compounds, 67 68 and 69 the ether was replaced with an acetoxy ester, while the alkyl or aryl side chain was varied. Compounds having alkyl side chains, allyl (compound 67 ), and propyl (compound 68 ), both showed larger zones of growth inhibition than the phenyl side chain compound 69 All of these three compounds showed greater activity than compound 63 the C3 monosubstituted acetoxy analogue. The last three disubstituted analogues, 70 71 and 72 all contained a

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36 spirocyclic ether at the C3 position, and slightly lower activity than the nonspirocyclic C3 disubstituted ethers. This slight difference in bioactivity is likely due to the fact that the aryl ring at the C4 position of these compounds is also different. Compounds 70 and 71 have a phenyl at C4 and compound 72 has a para -nitrophenyl at the C4 position. 2.2.5 Results of biolog ical screening against Bacillus10 The results for the screening of the N -thiolated -lactam analogues against the seven strains of Bacillus are shown in Tables 2.5, 2. 6, 2.7, and 2.8. Table 2.5 shows the results of the screening of t he C4 aryl analogues, Table 2.6 shows the results of the screening for the N -organothio analogues and the reference drug ciprofloxacin, Table 2. 7 shows the results for the screening of the C3 monosubstituted analogues, and Table 2.8 s hows the results of the screening of the C3 disubstituted analogues, The trends observed in the screening of these compounds against Bacillus almost exactly parallel the trends that were observed in the screening against MR SA, and therefore do not need to be discussed again in detail. The sim ilar activity trends observed for Bacillus and MRSA suggest that the mode of action of the N -thiolated -lactams against both of these bacterial species is similar.

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37 Table 2.5 Average growth inhibition zones for C4 aryl derivatives against Bacillus Cip 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 39 25 25 20 25 25 20 25 22 22 27 24 20 nd 21 22 10 20 33 18 18 19 18 19 17 17 19 20 22 16 20 14 14 16 10 19 40 19 19 20 19 18 17 17 15 19 20 17 20 13 15 17 0 16 41 16 16 18 17 17 15 15 11 17 21 19 20 10 12 14 0 15 42 2020 15 15 18 16 13 13 22 17 14 22 13 15 17 0 10 41 18 15 10 12 12 10 14 14 18 21 12 18 0 10 17 0 19 33 21 20 0 20 20 18 19 19 23 21 19 20 nd 18 18 17 15 Twenty micrograms of test compound in DMSO solution was used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. Ciprofloxacin (cip) is included for reference. (nd, not determined). Table 2.6 Average growth inhibition zones for N -organothio derivatives against Bacillus 45 52a53 54 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 40 0 10 10 39 0 10 0 30 0 0 9 36 0 0 10 37 0 0 0 39 0 0 nd 30 0 0 nd Twenty micrograms of test compound in DMSO solution was used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. a tested as the tetra n -but y lammoniumsalt. ( nd,notdetermined )

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38 Table 2.7 Average growth inhibi tion zones for C3 monosubstituted derivatives against Bacillus 55 56 57 58 59 60 61 62 64 65 66 73 74 75 76 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 20 22 22 0 10 0 0 20 10 18 19 24 27 27 20 14 24 22 0 9 0 0 18 8 15 12 14 20 24 15 18 15 14 0 9 0 0 11 10 13 11 19 20 19 15 20 13 11 0 7 0 0 12 0 15 11 17 22 20 15 15 11 15 0 0 0 0 18 10 nd 13 17 21 19 15 18 13 14 0 0 0 0 19 10 nd 12 11 23 22 19 21 17 17 0 11 00 22 14 nd 13 19 23 22 19 Twenty micrograms of test compound in DMSO solution was used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. (nd, not determined). Table 2.8 Average growth inhibition zones for C3 disubstituted derivatives against Bacillus 70 77 78 79 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 14 29 28 20 10 25 26 15 9 20 21 16 9 22 22 16 13 20 20 15 0 21 17 13 17 22 21 18 Twenty micrograms of test compound in DMSO solution was used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. (nd, not determined). 2.3 Conclusions In conclusion, it was found that slight ly lipophilic acyloxy or alkoxy groups at the C3 position of the lactam ring lead to larger zones of growth inhibition for both MRSA and Bacillus Also, the stereochemistry at this position does not

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39 seem to be related to the bioactivities of these compounds. At the C4 position, aromatic rings that bear a halogen or a small, electron-donating group at the ortho position of the ring leads to the greatest anti-MRSA and antiBacillus activity. The most dramatic effects on the bioactivity against both MRSA and Bacillus was seen when the N -alkylthio group was varied, with the N-sec butylthio lactam having the st rongest bioactivity of any N -thiolated -lactam analogues prepared in our laboratory to date. All of these observed trends in the bioac tivities lead us to believe that the bioactivity of these N -thiolated -lactams is due to the transfer of the N organothio group to a thiol target in the cell. The role of the C3 and C4 rings substituents is likely to ai d in the transport of the -lactam molecule across the lipid bilayer of the bacterial cell membrane. The parallels in the activity trends against MRSA and Bacillus suggest that the mode of action of the N -thiolated lactams against both of these bacte rial species is similar. This chapter focused on the synthesis of seven C3 oxygenated analogues. From the results of the screening of these seven C3 analogues as well as other C3 analogues, it was dete rmined that as long as the substituent possesses the proper lipophi lic/polar balance, we can introduce different groups at this position and still retain the bioactivity This is important to know in terms of our interest in attaching these N -thiolated -lactams to polymers and oligomers to form new biomaterials. Since such a wide va riety of groups can be tolerated at the C3 position, this position was selected as an ideal site for attachment. Efforts

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40 towards the synthesis of N -thiolated -lactam containing biomaterials will be discussed in the upcoming chapters. 2.4 Experimental All reagents needed for the synthesis of N -thiolated -lactams analogues were purchased from Sigma-Aldric h Company or Fisher Scie ntific and used without further purification unless otherwise stated. Solvents were obtained from Fisher Scientific and used without fu rther purification. Produc ts were purified by flash column chromatography with J. T. Bake r flash chromatography silica gel (200400 mesh). NMR spectra were recorded in CDCl3. 13C NMR spectra were proton decoupled. Synthesis of imine 82 : p -anisidine was recrystallized from water prior to use. O chlorobenzaldehyde (42.23 g, 0.3mol) was added to p -anisidine (37.0 g, 0.3mol) and stirred with a glass rod until a yello w paste formed. The crude product was recrystallized from methanol to yield 67.34g (91%) of im ine as a yellow solid, m.p. 62-63 oC. 1H NMR (250 MHz): 8.95 (1H, s), 8.27-8.23 (1H, m), 7.45-7.36 (3H, m), 7.30 (2H, d, J =8.9Hz), 6.96 (2H, d, J =8.9Hz) 3.86 (3H, s); 13C NMR: (63 MHz) 156.5, 154.6, 144.5, 135.7, 133.3, 131.7, 129.8, 128.3, 127.0, 122.4, 114.8, 114.3, 55.9

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41 Synthesis of -lactam 84 : Imine 82 (5.8 g 23.6 mmol) was dissolved in 100 mL of freshly distilled CH2Cl2 and cooled to 0oC in an ice bath. Triethylamine was added (3. eq., 7.16 g, 70.8 mmol) was added, fo llowed by acetoxy acetyl chloride (1.8 eq., 5.8 g, 42.5 mmol) dissolved in 20 mL of CH2Cl2. The reaction was stirred overnight. The solvent was removed under reduced pressure and the crude material was purified by washing with ice-cold methanol to give 7.1 g (87 %) of lactam 84 as a white solid, m.p. 130-131 oC. 1H NMR (250 MHz): 7.43 ( 1H, d, J =8.9 Hz), 7.32-7.23 (5 H, m), 6.83 (2H, d, J =8.9 Hz), 6.16 (1H, d, J =5.0 Hz), 3.76 (3H, s), 1.76 (3H, s). Synthesis of lactam 85 : Lactam 84 (2.0g, 5.78 mmol) was dissolved in 60 mL of acetonitrile and cooled to 0oC in an ice bath. CAN (9.54g, 11.7 mmol) was dissolved in 15 mL of water and added to t he lactam solution dropwise, over the period of one hour. The reaction mixture wa s then extracted three times with 25 mL of ethyl acetate. The combined ethyl acetate layers were washed with 50 mL of 5% NaHSO3, 50 mL of 5% NaHCO3, and 50 mL of brine, and dried over Na2SO4. The solvent was removed under reduced pressure. The crude product was purified by flash column chromatogr aphy using 4:1 hexanes to ethyl acetate as the eluent to give 0.83 g (60%) of 85 as a light yellow solid. 1H NMR (250 MHz): 7.42-7.23 (4H, m), 6.46 (1 H, br. s), 6.21 (1H, d, J =5.0 Hz), 5.42 (1H, d, J =5.0 Hz), 1.76 (3H, s).

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42 Synthesis of lactam 63 : Lactam 85 (2.0g, 8.35 mmol) was dissolved in 100 mL of CH2Cl2, and 86 (1.70g, 8.35 mmol) was added along with a catalytic amount of triethylamine. The reaction was reflux ed overnight and then the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography using 9:1 hexanes to ethyl acetate as the eluent to give 1.67 g (70%) of 63 as a light yellow solid. 1H NMR (250 MHz): 7.42-7.23 (4H, m), 6.08 (1H, d, J = 5.0Hz), 5.42 (1H, d, J = 5.0Hz), 2.51 (3H, s),1.76 (3H, s). Synthesis of 73 : Lactam 63 (0.20g, 0.7mmol) was dissol ved in 5 mL of methanol and potassium carbonate (0.7mmol as a 1M solution in water) was added. After 1 minute, the reaction mixtur e was extracted three ti mes with 10 mL of ethyl acetate, and then the combi ned ethyl acetate layers were washed with 30mL of brine, dried over Na2SO4, and the solvent removed under reduced pressure to give 73 (0.143g, 78%) as a white solid. 1H NMR (250 MHz): 7.42-7.23 (4H, m), 5.28 (1H, d, J =5.0 Hz), 5.24 (1H, d, J =5.0 Hz), 2.51 (3H, s),1.76 (3H, s). 13C NMR: (63 MHz) 171.7, 133.8, 131.4, 130.0, 129.8 128.3, 127.1, 79.1, 63.9, 21.8. Synthesis of 87 : To a solution of 84 (1.23g, 3.56 mmol) in 30 mL of acetone was added a solution of KOH in methanol at 0 oC. After 5 minutes, the reaction was quenched by adding an equal volume of wate r to precipitate the product out of solution. The product was filtered and dried to give 1.07g (99%) of a white solid, m.p. 183-184 oC. 1H NMR (250 MHz): 7.48 (1H, d, J =7.5 Hz), 7.33-7.22 (5 H,

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43 m), 6.84 (2H, d, J=7. 5 Hz), 5.63 (1H, d, J =5.1 Hz), 5.33 (1H, d, J =5.1 Hz), 3.78 (3H,s), 2.00 (1H, br.s). Synthesis of lactam 88 : To a solution of lactam 87 (1.0 g, 3.3 mmol) in 25 mL of dry CH2Cl2 was added NaH (60% suspension in mineral oil, 0.26 g, 6.6 mmol) and the mixture was stirred for 15 min. Ally l bromide (0.79 g, 6.6 mmol) was then added, along with 5 mg of TBAI. The mi xture was refluxed for 24 h and then quenched with a 5% solution of NH4Cl. The solution was extracted three times with 25 mL of CH2Cl2 and then the combined CH2Cl2 layers were washed with brine, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by fl ash column chromatography using 9:1 hexanes to ethyl acetate as the eluent to give 0.92 g (85%) of 88 as a light yellow solid, m.p. 61-63 oC. 1H NMR (250 MHz): 7.43 (1H, d, J =9.0 Hz), 7.40-7.20 (5H, m), 6.80 (2H, d, J =9.0 Hz), 5.61 (1H, d, J =4.7 Hz), 5.09 (2H, d, J=5.9 Hz), 5.02 (1H, d, J =4.7 Hz), 3.92 (2H, d, J=5.9 Hz), 3.73 (3H, s); 13C NMR: (63 MHz) 163.8, 156.3, 133.2, 133.0, 131.2, 130.3, 129.0, 126. 9, 118.1, 114.3, 82.5, 71.9, 58.9, 55.4. Lactam 89 : off-white solid, m.p. 119-121 oC, 87% yield. 1H NMR (250 MHz): 7.42 (1H, d, J =7.8 Hz), 7.38-7.22 (5H, m), 6.80 (2H, d, J =7.8 Hz), 5.64 (1H, d, J =5.0 Hz), 5.21 (1H, d, J =5.0 Hz), 4.56 (2H, s), 3.74 (3H, s), 3.19 (3H,s); 13C NMR: (63 MHz) 163.9, 156.4, 133.3, 131.5, 130.4 129.5, 126.9, 118.6, 114.4, 96.6, 80.4, 58.9, 55.7, 55.4.

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44 Lactam 90 : Brown semi-solid, 69% yield. 1H NMR (250 MHz): 7.50-7.26 (4H, m), 6.25 (1H br. s.), 5.70-5. 55 (1H, m), 5.26 (1H, d, J =4.6 Hz), 5.11-5.00 (2H, m), 4.97 (1H, d, J =4.6 Hz), 3.93-3.89 (2H, m); 13C NMR: (63 MHz) 168.7, 133.8, 133.2, 129.1, 128.4, 126.9, 117.8, 84.6, 71.8, 56.0. Lactam 91 : 97 % yield 1H NMR (250 MHz): 7.49 (1H, m) 7.46-7.26 (3H, m), 6.30 (1H br. s.), 5.26 (1H, d, J =4.6 Hz), 4.91 (1H, d, J =4.6 Hz), 3.47-3.38 (1H, m); 3.25-3.19 (1H, m), 1.33-1. 24 (2H, m), 0.54 (3H, m) 13C NMR: (63 MHz) 168.6, 133.9, 133.0, 129.2, 129.0, 128.4, 126.8, 85.8, 43.1, 56.1, 22.6, 10.0. Lactam 92 : 64 % yield .1H NMR (250 MHz): 7.48-7.26 (4H, m), 6.26 (1H, br. s), 5.29 (1H, d, J =5.0 Hz), 5.17 (1H, d, J =5.0 Hz), 4.55 (2H, s), 3.21 (3H,s); 13C NMR: (63 MHz) 169.3, 133.3, 129.9, 128.7, 128.5, 127.7, 126.8, 96.1, 84.2, 57.3, 50.2. Lactam 74 : 25% yield. 1H NMR (250 MHz): 7.39-7.26 (4H, m), 5.62-5.51 (1H, m), 5.34 (1H, d, J =4.8 Hz), 5.06-5.03 (2 H, m), 4.99 (1H, d, J =4.8 Hz), 3.93-3.78, (2H, m), 2.45 (3H, s); 13C NMR: (63 MHz) 170.4, 133.7, 132.8, 131.6, 129.5, 129.4, 129.1, 126.7, 118.0, 84.4, 71.6, 62.8, 21.7. Lactam 75 : 40% yield. 1H NMR (250 MHz): 7.34 (1H, m) 7. 27-7.24 (3H, m), 5.31 (1H, d, J =4.8 Hz), 4.91 (1H, d, J =4.8 Hz), 3.38-3.32 (1H, m); 3.09-3.04 (1H,

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45 m), 2.42 (3H, s),1.27-1.19 (2H, m), 0.49 (3H, m) 13C NMR: (63 MHz) 170.5, 134.6, 133.8, 131.6, 129.4, 129.1, 126.6, 85.6, 72.9, 63.0, 22.4, 21.8, 10.0. Lactam 76 : 35% yield. 1H NMR (250 MHz): 7.39-7.26 (4H, m), 5.37 (1H, d, J =5.1 Hz), 5. 19 (1H, d, J =5.1 Hz), 4.51 (2H, s), 3. 14 (3H, s), 2.47 (3H, s) ; 13C NMR: (63 MHz) 170.6, 134.6, 131.8, 129.5, 129.4 128.8, 126.8, 96.3, 82.8, 62.8, 55.7, 21.7. Synthesis of lactam 93 : P2O5 (0.75 g, 2.47 mmol) wa s added to 10 mL of DMSO and allowed to stir for 10 mi nutes. After 10 min, lactam 87 (0.75 g, 2.47 mmol) was added to the solution. After stirring at room temperature overnight, the reaction was quenched with a saturated NaHCO3 solution. The mixture was then extracted three times with 15 mL of ethy l acetate. The combined ethyl acetate layers were washed with water, dried over Na2SO4, and the solvent was removed under reduced pressure to give 0.71 (96 %) of 93 as a yellow solid, m.p. 130-132 oC. 1H NMR (250 MHz): 7.49-7.38 (3H, m), 7.36-7. 16 (4H, m), 6.89-6.82 (2H, m), 6.05 (1H, s), 3.72 (3H, s); 13C NMR: (63 MHz) 189.6, 160.0, 158.0, 133.4, 130.7, 130.6, 129.7, 129. 4, 127.7, 127,5, 119.7, 114.0, 72.0, 55.5. Synthesis of lactam 94 : All glassware was flame-dried under nitrogen atmosphere prior to use. Lactam 93 was dissolved in freshly distilled THF. The flask was flushed with nitrogen and cooled to -78oC using a dry ice/acetone bath. Methylmagnesium iodide (0.35g, 2.5 mmol) (generated in a separate flask from

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46 Mg ribbon, methyl iodide, and a catalyti c amount of iodine) was cooled to -78oC, and then the ketone solution was added to the flask containing the freshly prepared grignard reagent. The r eaction was stirred at -78 oC for 3 hours, and then was allowed to warm to room tem perature. The reaction was quenched with 5% NH4Cl, extracted three time s with ethyl acetate, wa shed with water, dried over Na2SO4 and the solvent was removed under reduced pressure to give 0.39g (74%) of 94 as a light brown solid, m.p. 141-143 oC. 1H NMR (250 MHz): 7.497.38 (1H, m), 7.36-7.16 (5H, m), 6.89-6.82 (2H, m), 5.38 (1H, s), 3.75 (3H, s) 1.83 (3H, s); 13C NMR: (63 MHz) 168.2, 156.5, 133.5, 132.1, 130.5, 129.8, 129.4, 128.1, 127.0, 118.8, 114. 4, 84.0, 66.2, 55.5, 22.0. Lactam 95 : 87% yield. 1H NMR (250 MHz): 7.44-7.41 (1H, m), 7.29-7.16 (5H, m), 6.83-6.80 (2H, m), 5.46-5.35 (1H, m), 5.35 (1H, s), 4.86-4.80 (2H, m) 3.893.87 (2H, m) 3.74 (3H, s) 1.81 (3H, s); 13C NMR: (63 MHz) 166.0, 156.3, 133.6, 133.3, 132.1, 130.6, 129.4, 129. 1, 128.7, 126.6, 118.6, 1 16.0, 114.3, 88.5, 66.9, 65.4, 19.41. Lactam 96 : 80% yield. 1H NMR (250 MHz): 7.45-7.41 (1H, m), 7.29-7.11 (5H, m), 6.83-6.80 (2H, m), 5.26 (1H, s), 4.53 (2H, s), 3.69 (3H, s), 3.04 0 (3H, s), 1.78 (3H, s) ; 13C NMR: (63 MHz) 165.9, 156.5, 133.5, 13 2.4, 130.7, 129.6, 129.2, 128.7, 126.8, 118.7, 114.5, 93.2, 87.4,65.6, 55.7, 55.5, 20.4.

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47 Lactam 97 : 74% yield. 1H NMR (250 MHz): 7.48-7.26 (5H, m), 6.25 (1H, br s), 5.39-5.35 (1H, m), 4.97 (1H, s), 4.86-4. 77 (2H, m) 3.82-3.79 (2H, m), 1.80 (3H, s); 13C NMR: (63 MHz) 170.5, 134.5, 133.7, 133.1, 129.2, 128.8, 127.8, 126.6, 116.0, 90.8, 66.8, 62.6, 19.6 Lactam 98 : 98% yield. 1H NMR (250 MHz): 7.45-7.16 (5H, m), 6.95 (1H, br s), 4.82 (1H, s), 3.21-3.03 (2H, m), 1.64 (3H, s) 1.09-1.01 (2H, m), 0.49 (3H, t); 13C NMR: (63 MHz) 171.40, 134.9, 133.2, 129.3, 128. 8, 128.2, 127.2, 126.6, 90.8, 67.7, 62.8, 22.9, 19.87, 9.99. Lactam 99 : 95% yield. 1H NMR (250 MHz): 7.43-7.32 (2H, m), 7.29-7.24 (2H, m), 6.60 (1H, br s), 4.94 (1H, s), 4.54 (2H, s), 3.06 (3H, s), 1.80 (3H, s) ; 13C NMR: (63 MHz) 170.2, 134.7, 133.1, 129.2, 128.9 127.8, 126.7, 93.0, 90.0, 62.6, 55.6, 20.3. Lactam 77 : 38% yield. 1H NMR (250 MHz): 7.40-7.26 (4H, m), 5.41-5.34 (1H, m), 5.05 (1H, s), 4.84-4.77 (2 H, m) 3.83-3.75 (2H, m), 2.48 (3H, s),1.73 (3H, s); 13C NMR: (63 MHz) 169.7, 133.8, 129.7, 1 29.5, 128.9, 126.9, 116.4, 69.5, 67.1, 21.5, 19.1. Lactam 78 : 54% yield. 1H NMR (250 MHz): 7.36-7.28 (4H, m), 4.82 (1H, s), 3.33-3.29 (1H, m), 3.04-3.01 (1H, m), 2.40 (3H, s), 1.61 (3H, s) 1.23-1.14 (2H,

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48 m), 0.59 (3H, t); 13C NMR: (63 MHz) 173.1, 133.9, 132.7, 129.5, 129.2, 128.7, 126.6, 90.7, 69.4, 67.6, 22.8, 21.2, 19.0, 10.0. 2.5 References 1. Long, T.E., Ph.D. Dissertation, University of South Florida, 2003 2. Bose, A. K.; Anjaneyulu, B.; Bhattacharya, S.K.; Manhas, M.S. Tetrahedron 1967 23 4769. 3. Normal coupling constants for t he vicinal protons on the ring of cis disubstituted -lactams fall in the range of 4. 8-6.0 Hz, while for the trans isomer, the value is typica lly in the range of 1-3 Hz. 4. Kronenthal, D.R.; Han, C.Y.; Taylor, M.K. J. Org. Chem 1982 47 2765. 5. Woulfe, S.R.; Iwagami, H.; Miller, M.J.; Tetrahedron Lett 1985 26 3891. 6. Coates, C., Ph.D. Dissertation, University of South Florida, 2004 7. Buynak, Y.D.; Borate, H.B.; Lamb, G. W.; Khasnis, D.D.; Hustig, C.; Jsum, H.; Siriwardone, U.A.; J. Org. Chem 1993 58, 1325. 8. Ren, X-F.; Konaklieva, M.I.; Shi, H .; Dickey, S.; Lim, D.V.; Gonzalez, J.; Turos, E. J. Org. Chem 1998 63 8898. 9. Turos, E.; Long, T.E.; Heldreth, B .; Leslie, J.M.; Reddy, G.S.K.; Wang, Y.; Coates, C.; Konaklieva, M.; Dickey, S.; Lim, D.V.; Alonso, E.; Gonzalez, J. Bioorg. Med. Chem. Lett 2006 16 2084.

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49 10. NCCLS (National Committee fo r Clinical Laboratory Standards) Methods for Dilution of Antimicrobial Susceptibi lity Tests for Bacteria that Grow Aerobically NCCLS Document M7-A4, Vol. 17. No. 2, 1997 11. Long, T.E.; Turos, E.; Konaklieva, M. ; Blum, A.L.; Amry, A.; Baker, E.A.; Suwandi, L.S.; McCain, M.D.; Rahm an, M.F.; Dickey, S.; Lim, D.V. Bioorg. Med. Chem 2003 11 1859. 12. Turos, E.; Coates, C. Shim, J-Y., Yang, W., Leslie, J.M., Long, T.E., Reddy, G.S.K.; Ortiz, A., Culbreath, M. ; Dickey, S.; Lim, D.V.; Alonso, E.; Gonzalez, J. Bioorg. Med. Chem 2005 13 6289. 13. Heldreth, B.; Long, T.E.; Jang, S.; Reddy, G.S.K.; Turos, E.; Dickey, S.; Lim, D.V. Bioorg. Med. Chem 2006 14 3775.

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50 Chapter 3 Functionalized Caprolactone Monomers: Design, Synthesis, and Biological Evaluation 3.1 Introduction Many MRSA infections occur at the site of an injury, such as a cut in the skin where bacteria can enter the body. It makes sense then to try to find a way to effectively deliver the antibacterial drugs directly at the site of an injury, where water-soluble penicillins may not work we ll. Previous results from Chapter two have shown us that many different alkoxy and hydroxyl groups could be incorporated at the C3 posit ion of the lactam ring of N -thiolated -lactams without diminishing anti-MRSA and antiBacillus activity. For this reas on, this site of the lactam ring was selected as an ideal spot for further modificati on to make new, antibacterial materials. The material that we choos e to try to attach our Nthiolated -lactams to is poly( -caprolactone) ( 100 ) to form a drug-bearing polymer ( 101 ), as shown in Figure 3.1.

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51 Figure 3.1 Poly( -caprolactone) and propo sed drug-bearing poly( caprolactone) O O Drug 101 O O 100 Poly( -caprolactone) is an aliphatic polyester. Aliphatic polyesters are often used as biomaterials because t hey are biodegradable. Materials that biodegrade are attractive for biomedical applications because they do not require a second doctor’s visit or surgery to remove, and as such, have found use for wound closure (staples, sutures), cardiovascu lar applications (stents, grafts), and orthopedic devices (pins, screws, rods, artificial ligaments).1 Poly( -caprolactone) degrades in vivo by the enzymatic hydrolysis of the ester bonds to give 6-hydroxyhexanoic acid, which is then broken down systematically through the citric acid cycle2 (Scheme 3.1). The byproducts of this degradation are therefore non-toxic, and thus poly( -caprolactone) is safe for use in the human body. Scheme 3.1 In vivo degradation of poly( -caprolactone) O O n 101 non-specific esterases HO OH O 102 Citric Acid Cycle

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52 Poly( -caprolactone) has a high permeabilit y to many drugs, and as such has found a lot of use in drug delivery3, including delivery of the anti-MRSA drug, vancomycin, which was encapsulated in t he preformed polymer via emulsification in water.4 Since poly( -caprolactone) lacks func tionality on the polymer backbone, we decided to synthesize a c aprolactone monomer th at bore suitable functionality where a drug molecule coul d be covalently bound. The first thing that we considered was to have a halide on the caprolactone so that we could utilize a Williamson ether synthesis simila r to those described in Chapter 2 to make the C3 alkoxy analogues. A sear ch of the literatur e revealed that 5bromocaprolactone ( 103 ) (Figure 3.2) had been previously synthesized by Trollss et al.5 Figure 3.2 5-bromocaprolactone O O Br 103 Although at first glance this seemed lik e a good monomer to try, we decided against this system because the bromide was at a secondary carbon center, on the nucleophilically-sensitive lactone and ri ng-opening or elimination to give the alkene would be more likely than substituti on under the basic c onditions of the Williamson ether synthesis (Scheme 3.2) It was observed during the syntheses

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53 of the C3 alkoxy lactams 74 79 that the alkylation of lactams 87 and 94 required the use of highly activated alkylating agents. Allyl bromide and methoxymethyl chloride were selected specifically becaus e they were primary, activated, and less likely to undergo base-promot ed elimination reactions. Scheme 3.2 Likely outcome of Williams on ether synthesis of lactone 103 O O Br 103 N O HO S CH3 Cl + 73 NaH, dry CH2Cl2O O 10 4 not O O O O S CH3 Cl 10 5 During the course of the N -thiolated -lactam SAR study (Chapter 2) other compounds that were synthes ized contained acyloxy esters at the C3 position. These acyloxy compounds could be fo rmed easily under relatively neutral conditions. Also, the activity of thes e esterified compounds was found to be comparable to that of the C3 alkoxy derivatives. A further literature result revealed that Hedrick et al. had sy nthesized two caprolactone monomers ( 106 and 107 ) that bore pendant ester groups at the five position of the lactone ring, shown in Figure 3.3.6

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54 Figure 3.3 Substituted lactones prepared by Hedrick et al. O O O BnO O O O BuO t106107 With these two monomers, we were concer ned that it would be di fficult to couple the sterically hindered secondary alcohol of the lactam ring with a carboxylic acid that had branching at the -position. Instead, we pr oposed that introducing an additional methylene spacer in between the caprolactone ring and the ester substituent (as shown in figure 3.4) w ould add more degrees of freedom to the system to allow for an easier coupling with our lactams. Figure 3.4 Proposed lactone. O O CO2R 108 The retrosynthetic analysis for lactone 108 is shown in Scheme 3.3, starting from commercially availabl e 1,4-cyclohexadione monoethyleneacetal ( 111 ). Lactone 108 could be easily formed via a Baeyer-Villiger reaction of the corresponding substituted cyclohexanone 109 Cylohexanone 109 could be

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55 obtained following t he hydrogenation of the Wittig product 110 and deprotection of the acetal. We envisioned that a Wittig reaction between ketone 111 and an ylide derived from an -haloacetate would be a facile way of introducing the required alkyl ester side chain. Scheme 3.3 Retrosynthesis fo r substituted lactone 108 O O CO2R 108 O CO2R O O CO2R O O O 109110111 3.2 Results and discussion 3.2.1 Synthesis of lactone bearing a pendant benzyl ester The first lactone that we decided to synthesize for these studies had a pendant benzyl ester substituent at the 5-position of the lactone ring ( 112 Figure 3.5). This target was selected because it was envisioned that the benzyl ester moiety could be easily deprotected at a suit able point prior to attachment of the hydroxyl lactam.

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56 Figure 3.5 O O CO2Bn 112 The synthesis of the substituted lactone 112 began with phosphorus ylide 115 Although commercially available, ylide 115 could be easily prepared in bulk as shown in Scheme 3.4. Ethyl bromoacetate ( 113 ) was added to a solution of triphenylphosphine in benzene, and stirred overnight at room temperature to form the phosphonium salt 114 as a white solid. The salt was then filtered and washed with benzene to remove any residual tr iphenylphosphine. Phosphonium salt 114 was then dissolved in a solution of 20% sodium hydroxide and stirred at room temperature for five hours to give the ylide 115 This ylide is stabilized by the ester group, and can easily be stored on t he shelf for months before use. Scheme 3.4 Synthesis of ylide 115. Br OEt O (Ph)3P OEt O Br (Ph)3P OEt O a b 113114115 Reagents and conditions: (a) PPh3, C6H6, 12h, rt; (b) 20% NaOH(aq), 5h, rt.

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57 The first route that we used to obtain the lactone 112 is shown in scheme 3.5. First, commercially available 1,4-cyclohexadione monoethyleneacetal ( 111 ) underwent a Wittig olefination with ylide 115 to give the alkene 116 as a clear, colorless liquid in 90-95% yield, after flash column chromatography. Next, the alkene was reduced by catalytic hydrogenation to give 117 also as a clear, colorless liquid in quantita tive yield. Next, both the acetal and the ethyl ester were hydrolyzed by stirring at room temper ature in dilute sulfuric acid solution. Both of the groups could be hydrolyzed to give 118 under these conditions, however, there were a number of problem s involved in this step. At higher concentrations of H2SO4 (5-10% by volume) the yields of the reactions were lower (~50-60%) and there was a lot of dec omposition of the starting material was observed. Lowering the sulfuric acid concentration to 2% eliminated most of the decomposition products and increased t he yields to ~80-90%, but increased the reaction time to ~5 days. Next, the acid 118 was subjected to esterification with benzyl bromide under basic condition s to give the required substituted cyclohexanone 119 as colorless crystals in 88% yield. The Baeyer-Villiger reaction of 119 proceeded very smoothly to give the desired lactone 112 in 9095% yield as a colorless oil initiall y. Drying under vacuum for a few hours transformed the oil into a white, somewhat waxy solid with a low melting point of 59-60 oC.

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58 Scheme 3.5 Initial synthesis of lactone 112 O O O O O O O O O O CO2Bn CO2Et CO2Et O CO2H CO2Bn abc de Reagents and conditions: (a) 2 eq. 115 C6H6, 12h, reflux; (b) 60 psi H2(g), cat. 10% Pd/C, MeOH, 12h; (c) H2SO4(aq), rt; (d) 3 eq. K2CO3, 1.1 eq. BnBr, MeCN, 12h, reflux; (e) 1.5 eq. m CPBA, CHCl3, 3-5h, reflux. 111116117 118119112 Although this synthetic route was successful in producing the desired lactone 112 the yields and reaction condit ions for the hydrolysis were unsatisfactory. We decided to try to av oid the need to hydrolyze both the ester and the acetal in the same step. This could be done by modifying the synthesis shown in Scheme 3.5 to break up the hy drolysis reactions into two separate steps, as shown in Scheme 3.6. First, intermediate 117 (from Scheme 3.5) was subjected to ester hydrolysis under basic conditions using lit hium hydroxide in methanol, followed by acidic work-up and extraction with di ethyl ether to give the free acid 120 as

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59 colorless crystals in 75% yield. Next, t he acid was esterified with benzyl bromide and potassium carbonate to give crude benzyl ester 121 as a clear, colorless oil which was taken on directly to the next st ep without further purif ication. Next, the hydrolysis of the acetal 121 was accomplished by stirring overnight in 70% acetic acid. This hydrolysis went smoothly to give the substituted cyclohexanone 119 in 81 % yield (for the two steps), with none of the decomposition problems that were associated with the sulfuric acid hy drolysis. Finally, the substituted lactone 112 was obtained easily through a Baeyer-Vi lliger reaction as described before. Scheme 3.6 Alternate synthesis of lactone 112. O O O O O CO2Bn CO2Et CO2Bn CO2Bn O O CO2H O O abc d 117 119112 120121 Reagents and conditions: (a) 3 eq. LiOH, MeOH, 12h, rt; 1M HCl; (b) 3 eq. K2CO3, 1.1 eq. BnBr, MeCN, 12h, reflux; (c) 70 % AcOH, 12-24h, rt; (d) 1.5 eq. m CPBA, C H C l 3 3-5h, r eflux.

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60 3.2.2 Synthesis of lactone 122 bearing an acid-cleavable tert -butyl ester group A lactone bearing a pendant tert -butyl ester ( 122 Figure 3.6) was also synthesized to have a protecting group t hat could be easily cleaved later under mild acid conditions. Figure 3.6 O O CO2 tBu 122 The synthesis of the lactone 122 began with the synthesis of the phosphorus ylide derived from tert -butyl bromoacetate ( 123 ). The synthesis of this ylide was accomplished in the same fashion as ylide 115 (Scheme 3.7). First, the tert -butyl bromoacetate was added to a so lution of triphenylphosphine in benzene, which was then stirred overnight at room temperature to give the phosphonium salt 124 as a white solid. The salt was filtered off and washed with benzene to remove any residual tripheny lphosphine. The phosphonium salt 124 was then dissolved in a solution of 20% sodium hydroxide and stirred at room temperature for five hours to give the ylide 125 as a sticky, colorless oil that was used directly in the next scheme (Scheme 3.8).

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61 Scheme 3.7 Synthesis of ylide 125 Br OtBu O (Ph)3P OtBu O Br (Ph)3P OtBu O a b 123124125 Reagents and conditions: (a) PPh3, C6H6, 12h, rt; (b) 20% NaOH(aq), 5h, rt. The synthesis of lactone 122 is shown in Scheme 3.8. The first step was the Wittig olefination using the ylide 125 formed in Scheme 3.7, and the commercially available 111 Scheme 3.8 Synthesis of lactone 122 O O O O O O O O O O CO2 tBu CO2 tBu CO2 tBu ab Reagents and conditions: (a) 2 eq. 125 C6H6, 12h, reflux; (b) H2(g), cat. 10% Pd/C, EtOAc, 24h; (c) 0.1 eq. I2, dry acetone, 1h, rt (d) 1.5 eq. m CPBA, CHCl3, 3h, reflux. d + OtBu O Ph3P + c CO2 tBu 125111126 127128122 This reaction proceeded very smoothly to give the alkene 126 as a clear, colorless liquid in 81% yield. Next, the alkene 126 was subjected to catalytic

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62 hydrogenation using 10% Pd/C as a catal yst. Curiously, even after 24h, the TLC of the reaction mixture still showed two spots. The reaction was stopped at this point, and the two compounds were separat ed via column chromatography. From the column, the expected alkane product, 127 was isolated in 57 % yield. The second product 128 isolated in 19% yield, was the product expected for the next step, where the ketal was depr otected! It seems most likely that there was an acidic contaminant present during the hydrogenation reaction that led to the deprotection of the ketal. For the deprotection of the isolated ketal 127 care had to be taken to select conditions that would not also remove the acid-labile tert butyl ester. A literature search revealed a mild method of removing acetals and ketals using molecular iodine in dry acetone that was published in 2004 by Sun et al.7 The deprotection of ketal 127 following the procedure of Sun proceeded in 1h to give ketone 128 as colorless crystals, m.p. 36-38 oC, in 50% yield. Extending the reaction time beyond 1h di d not improve the yield. Heating the reaction led to the format ion of side products (as observed by the formation of multiple spots on the TLC pl ate). However, in spite of the somewhat low yield, this method of deprotecting the ketal wo rks well for this system because the unreacted ketal can be easily recovered by column chromatography and recycled through the deprotection reaction again. Fo llowing the ketal deprotection, the ketone 128 proceeded smoothly through the Baeye r-Villiger reaction to give the desired lactone 122 as colorless crystals, m.p. 82-83 oC, in 83-97% yield.

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63 The proposed mechanism for the ke tal deprotection using molecular iodine in acetone is shown in Figure 3.7. In this reaction, acetone serves as both the solvent and the substrate. Sun proposed that the deprotecti on proceeds via a substrate exchange mechanism, rather than through a hydrolysis mechanism. A previous report by Firouzabadi et al involving the iodine-catalyzed transthioacetalization of O,O -acetals8 supports this hypothesis. Figure 3.7 Proposed mechanism of ket al deprotection using molecular iodine in acetone7 RO OR O I I O O R R O I I RO OR I2 O 3.2.3 Attachment of N -thiolated -lactams The two new ester-substituted lactones 108 and 122 were deprotected as shown in Schemes 3.9 and 3.10 in order to attach the lactam drug onto the lactone framework. The benzyl ester 112 was deprotected using catalytic hydrogenation to give the free acid 129 as a colorless oil in nearly quantitative yield. The tertbutyl ester was deprotected us ing a equivolume mixture of methylene chloride and trifluoroacetic acid (TFA) to give 129 in 91% yield.

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64 Scheme 3.9 Deprotect ion of lactone 112 O O CO2Bn O O CO2H a 112129 Reagents and conditions: (a) cat. 10% Pd/C, 60 psi H2(g), EtOAc, 12h Scheme 3.10 Deprotect ion of lactone 122 O O CO2 tBu O O CO2H a 122129 Reagents and conditions: (a) 1:1 TFA:CH2Cl2, 1h Following the deprotections to give the free acid 129 the lactone was coupled with N -methylthio lactam 73 using 1-ethyl-3-(3’dimethylaminopropyl)carbodiimide (EDCI) and a catalytic amount of N,N dimethylaminopyridine (DMAP) to gi ve the lactam-lactone conjugate 130 in 36 % yield (Scheme 3.11).

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65 Scheme 3.11 Coupling of acid 129 with N -thiolated -lactam 73 N O O Cl SCH3 O O CO2H O O O N O HO SCH3 Cl a 129130 Reagents and conditions: (a) 1.5 eq. EDCI, cat. DMAP, dry CH2Cl2, 12h, rt 73 + The coupling of the free acid 129 with N-sec -butylthio lactam 131 (synthesized by Dr. Tyler Schertz in our lab) was accomplished using the same reaction conditions as for the coupling of the N -methylthio -lactam 73 to give the desired product, 132 in 31% yield. The yields of these two coupling reactions represent un-optimized yields. Scheme 3.12 Coupling of acid 129 with N -thiolated -lactam 131 N O O Cl SCH(CH3)CH2CH3 O O CO2H O O O N O HO SCH(CH3)CH2CH3 Cl a 129132 Reagents and conditions: (a) 1.5 eq. EDCI, cat. DMAP, dry CH2Cl2, 12h, rt 131 +

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66 The structures of 130 and 132 were confirmed by 1H NMR. The successful attachment of the lactam s to the monomer can be s een by the shift in the 1H NMR spectrum of the two protons of the -lactam ring. In the 1H NMR of 73 these two protons appear as a doublet of doublets at 5.3 and 5.2 ppm, and following the attachment to the acid 129 these two protons appear at 6.1 and 5.4 ppm. In the 1H NMR spectrum of 131 these two protons appear as a multiplet at 5.3 ppm, and following the attachment to 129 these two protons appear as a doublet of doublets at 6.1 and 5.4 ppm. 3.2.4 Results of screeni ng for anti-MRSA activity The new lactam-lactone conjugates, 130 and 132 were screened for antiMRSA activity against the same nine strains of MRSA that were used to test the compounds shown in Chapters 1 and 2. The screening for bioactivity was accomplished using the Kirby-Bauer me thod of well diffusion as described in Chapter 2. The results of the testing are shown in Table 3.1. Also included for comparison are the two commercial dr ugs, penicillin G (Pen) and vancomycin (Van), as well as the -lactams 73 and 131

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67 Table 3.1 Zones of grow th inhibition for lact ams and lactam-lactone conjugates against MRSA Pen Van 73 131 130 132 8 16 19 32 15 23 16 15 17 28 17 21 10 16 17 29 15 22 14 16 19 28 15 21 12 21 18 28 14 22 12 15 16 25 16 21 19 15 19 29 16 21 15 15 18 29 16 21 nd nd 18 30 14 22 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA 657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameters in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound in DMSO solution was used. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laborat ory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purchased from American Type Culture Collections. Error values are within +/-1 mm. Penicillin G (Pen) and Vancomycin (Van) are included for reference. (nd, not determined). The zones of inhibition obtained for all of the compounds in this study were around the same, or higher than, the commercial drugs penicillin G and vancomycin. In both cases, the activity of the -lactams appears to be less for the lactam-lactone conjugates th an for the parent C3 hydroxy -lactams, however, this difference may be due to the molecular weight differences. As was seen in the previous SAR study of the N -organothio -lactams (discussed in Chapter 1), the N -methylthio -lactams ( 73 and 130 ) showed lower anti-MRSA activities than the N sec -butylthio -lactams ( 131 and 132 ). The most significant thing to note from these results is that the anti-MRSA activity of the -lactams is still retained after attachment to the functionalized lactone.

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68 3.2.5 Results of screening for antiBacillus activity The new lactam-lactone conjugates, 130 and 132 were screened for antiBacillus activity against the same seven Bacillus microbes that were used to test the compounds shown in Chapters 1 and 2. The screening for bioactivity was accomplished using the Kirby-Bauer me thod of well diffusion as described in Chapter 2. The results of the testing are shown in Table 3.2. Also included for comparison are the commercial drug, ci profloxacin (Cip), as well as the -lactams 73 and 131 Table 3.2 Zones of grow th inhibition for lact ams and lactam-lactone conjugates against Bacillus Cip 73 131 130 132 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 39 24 25 17 22 33 14 26 15 23 40 19 19 14 17 41 17 24 15 19 42 17 24 15 20 41 11 26 13 22 33 19 19 17 18 Twenty micrograms of test compound in DMSO solution was used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. Ciprofloxacin (Cip) is included as a reference. (nd, not determined). As was the case with the anti-MRSA testing, the N-sec -butylthio -lactams ( 131 and 132 ) showed larger zones of inhibition than the N -methylthio -lactams ( 73 and 130 ). Similar to the results of the SAR studies discussed in Chapters 1 and 2, none of the analogues prepared in this study had bioactivities that surpassed ciprofloxacin. The most significant thing to note from these results is

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69 that the antiBacillus activity of the -lactams is retained a fter attachment to the functionalized lactone. 3.3 Conclusions Lactones bearing a pendant benzyl ester substituent 108 and a pendant tert -butyl ester substituent 122 were both successfully synthesized in five and four steps respectively from commercially available starting materials. Both of these new lactones were easily deprotec ted to give the corresponding lactone bearing a pendant free carboxylic acid 129 The acid 129 was successfully coupled with both the C3 hydroxy N -methylthio -lactam 73 and C3 hydroxy Nsec -butylthio -lactam 131 As was observed previously, the N-sec -butylthio derivatives exhibited great er bioactivities than the N -methylthio deriviatives. When molecular weight differences are take n into account, the bioactivity of the -lactams does not diminish by the attachment to the lactone. Since incorporation of the N -thiolated -lactams onto the caprolactone ring does not adversely affect the bioactivity of the lact ams, it is hoped that attachment to the functionalized poly( -caprolactone) will also not have a detrimental affect on the bioactivity. The efforts towards synthesizing a N -thiolated -lactam containing poly( -caprolactone) are discussed in chapter 4.

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70 3.4 Experimental All reagents and solvents were purchas ed from Sigma-Aldrich Company or Fisher Scientific and used without further purification unless otherwise stated. Products were purified by flash column chromatography with J. T. Baker flash chromatography silica gel (200-400 me sh). NMR spectra were recorded in CDCl3. 13C NMR spectra were proton decoupled. Synthesis of phosphonium salt 114 : Triphenylphosphine (23. 6 g, 90.2 mmol) was dissolved in 100 mL of benzene and ethyl bromoacetate (15.06 g, 90.2 mmol) was added. The solution allowed to stir overnight at room temperature. The phosphium salt was isolated by filtrati on and washed with benzene to remove the residual triphenylphosphine. 1H NMR (250 MHz): 7.88-7.85 (6H, m), 7.75-7.70 (3H, m), 7.67-7.64 (6H, m), 7.32-7.24 (2 H, m), 5.58-5.53 (2 H m), 4.00 (2H, m), 1.04-1.02 (3H, m). Synthesis of ylide 115 : Phosphonium salt 114 was dissolved in 100 mL of 20% sodium hydroxide in water and allowed to stir for 5 hours. The solution was extracted three times with 50 mL of benzene. The combined benzene layers were dried over Na2SO4 and the solvent was removed under reduced pressure to give 30.5 g (97%) or ylide 115 as a white solid. 1H NMR (250 MHz): 7.67-7.24 (15H, m), 5.18 (1H, br s), 3.94 (2 H m), 2.82, 1.30-1.05 (3H, m).

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71 Synthesis of 116 : Commercially available ketone 111 (5.0g, 32.0 mmol) was dissolved in 100 mL of benzene and ylide 115 (2 eq., 22.3g, 64.0 mmol) was added. The reaction was refluxed for 24 hours, the solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography using 9:1 hexanes to ethyl acetate as the eluent to give 6.77 g (94%) of 116 as a colorless liquid. 1H NMR (250 MHz): 5.57 (1H, s), 4.08 (2H, q), 2.92 (2H m), 2.28 (2 H, m), 1.19 (3H, t). Synthesis of 117 : Alkene 116 (6.0g, 26.7 mmol) was dissolved in 40 mL of ethyl acetate. A catalytic amount of 10 % Pd/C was added, and the mixture was shaken under 60 psi of hydrogen gas (Parr hydrogenator) for 24 h. The palladium was removed by filtration through celit e, and the solvent was removed under reduced pressure to give 5.89g (98 %) of alkane 117 as a clear, colorless, liquid. 1H NMR (250 MHz): 3.76 (2H, q), 3.58 (4 H, s), 1.82 (2H, d), 2.28 (2H, m), 1.391.31 (5H, m), 1.28-1.19 (4 H, m), 0.89 (3H, t). Synthesis of 118 : Ester 117 (5.2g, 22.9 mmol) was dissolved in 100 mL of 2% H2SO4 and allowed to stir at room temperature for 5 days (until the TLC showed that the reaction was no longer progressing). The acid was isolated by extracting five times with 50 mL of diethyl ether. The combined ether layers were washed with brine, dried over Na2SO4 and the solvent was removed by evaporating under reduced to pressure to give 2.83 g (81 %) of 118

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72 Synthesis of 119 : Acid 118 (2.83 g, 18.1 mmol) was dissolved in 100 mL of acetonitrile. K2CO3 (3 eq. 7.50g, 54.3 mmol) and ben zyl bromide (1.1 eq. 3.42 g, 19.9 mmol) were added. The reaction was refluxed overnight, and then the base was removed by filtration and the solvent was removed by evaporating under reduced pressure. The crude product was purified by flash column chromatography using first 9:1 and then 4:1 hexanes to ethyl acetate as the eluent to give 3.9 g (88 %) of 119 as a colorless liquid. 1H NMR (250 MHz): 7.3-7.2 (5 H, m), 5.2 (2H, s), 2.44-2.25 (7H, m), 2. 19-2.08 (2H m), 1.52-1.38(2H, m). Synthesis of 112 : Ketone 119 (3.95g, 16.0 mmol) and m CBPA (1.5 eq. 4.16 g, 24.1 mmol) were dissolved in chlorofo rm and the reaction was refluxed for 5 hours (until the TLC showed complete consum ption of the starting material). The chloroform was removed under reduced pressure, and 150 mL of 5% NaHCO3 solution was added to neutralize the reaction. The 5% NaHCO3 solution was then extracted three times with 100 mL of CH2Cl2. the combined CH2Cl2 layers were washed with 5 % NaHCO3, brine, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography to gi ve 3.24 g (77%) of lactone 112 as a colorless soft solid, m.p. 59-60 oC. 1H NMR (250 MHz): 7.35-7.20 (5 H, m), 5.08 (2H, s), 4.38-4.05 (2H, m), 2.71-2.58 (2H m), 2. 23-2.21 (2H, d), 2.192.01 (2H, m) 1.921.18 (5H, m). 13C (63 MHz): 175.2, 171.3, 135.3, 128. 2, 127.9, 127.8, 77.5, 77.0, 76.5, 67.2, 65.9, 40.3, 36.4, 34.5, 32.5, 28.1.

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73 Synthesis of 120 : Ester 117 (6.0 g, 26.5 mmol) was dissolved in 50 mL of methanol and lithium hydroxide (3 eq ., 3.33 g, 79.4 mmol) was added. The mixture was stirred at room temperature until the TLC showed complete conversion to the carboxylate. The r eaction mixture was neut ralized with 1M HCl and extracted five times with 60 mL of di ethyl ether. The combined ether layers were dried over Na2SO4 and the solvent was removed under reduced pressure to give the 4.80 g (90 %) of free acid, 120 Synthesis of 121 : Acid 120 (1.54 g, 7.7 mmol) dissolved in 100 mL of acetonitrile and potassium carbonate (3 eq., 3.19 g, 23.1 mmol) was added, followed by benzyl bromide (1.1 eq., 1.45 g, 8.47 mmol) The mixture was refluxed overnight. The base was removed by filtration and the solvent was removed under reduced pressure. The crude product was taken direct ly to the next step without further purification. 1H NMR (250 MHz): 7.28-7.22 (5H, m), 5.04 (2H, s), 3.76 (4H, S), 2.19 (2H, d), 1.76-1.57 (5 H, m), 1.62-1.42 (2H, m) 1.30-1.10 (2H, m). Compound 122 : colorless crystals, m.p. 82-83 oC, 83 % yield.13C NMR (63 MHz): 175. 6, 171.3, 80.8, 67.8, 42.0, 37.1, 35. 0, 33.0, 28.6, 28.1 Compound 126 : colorless liquid, 81 % yield1H NMR (250 MHz): 5.55 (1H, s), 3.84 (4H, s), 2.88-2.82 (2H, m), 2.30-2.22 (2H, m), 1.78-1 .64 (4H, m), 1.4 (9H, s);

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7413C NMR (63 MHz): 166.0, 158.5, 115.9, 108.0, 79. 6, 64.4, 35.7, 34.9, 34.5, 28.2, 26.7. Compound 127 : colorless liquid, 57 % yield. 1H NMR (250 MHz): 3.69 (4H, s), 1.90-1.85 (2H, d), 1.60-1.40 (5H, m) 1.40-0.90 (4H, m), 1.13 (9H, s); 13C NMR (63 MHz): 172.2, 108.5, 79.9, 64.1, 42.1, 34.1, 33.5, 29.8, 28.1. Compound 128 : Acetal 127 (0.227 g, 0.89 mmol) was dissolved in 3.5 mL of reagent grade acetone (<0.4% water). T hen iodine (0.1 eq. 0.11 g, 0.089 mmol) was added. The reaction was stirred at room temperature for 1 hour, with constant monitoring by TLC. The reaction was stopped when no further change was seen by TLC. The acetone was removed by evaporation, the residue was dissolved in 10 mL of CH2Cl2, and washed with 5 mL of 5% Na2S2O3; 10 mL of water, and 10 mL of brine. The CH2Cl2 layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography using 9:1 hexanes to ethyl acetate as the eluent to give 0.093 g (49%) of 128 as a colorless solid, m.p. 36-38 oC. 1H NMR (250 MHz): 2.35-2.21 (4H, m), 2.15-2.04 (4 H, m), 1.82-2.00 (2H, m), 1.28 (9H, s); 13C NMR (63 MHz): 211.0, 171.6, 80.3, 41.3, 40.5, 33.1, 32.2, 28.0. Synthesis of 130 : Acid 129 (0.025 g, 0.145 mmol), lactam 73 (0.038 g, 0.145 mmol), EDCI (1.2 eq., 0.033 g, 0.174 mmol) and a catalytic amount of DMAP were all dissolved in 2 mL of freshly distilled CH2Cl2, and stirred overnight at

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75 room temperature. The EDCI was re moved by washed with water. The CH2Cl2 was dried over sodium sulfate and the so lvent was removed by evaporation. The crude product was purified by flash column chromatography using 4:1 hexanes:ethyl acetate as the eluent to give 0.0223 g (36%) of 130 1H NMR (250 MHz): 7.37-7.18 (5H, m), 6.08-6.02 (2H, m), 5.46-5.39 (2H, m), 4.32-4.19 (1H, m), 4.12-3.97 (1H, m), 2.45 (3H, s), 2.2-1.1(12H, m); 13C NMR (63 MHz): 170.3, 168.2, 134.4, 130.6, 130.2, 130.0, 129.8, 128.8, 126.8, 126.8, 68.3, 68.1, 62.2, 36.1, 36.0, 30.5, 30.4, 30.3, 28.4, 28.3, 21.9. Compound 132 : 31% yield. 1H NMR (250 MHz): 7.5-7.1 (5H, m), 6.2-6.0 (1H, m), 5.5-5.4 (1H, m), 4.4-4. 2 (1H, m), 4.2-4.0 (1H, m) 3.1-2.9 (1H, m), 2.4-1.5 (10H, m), 1.3-1.1 (5H, m), 1.0-0.9 (3H, m).

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76 3.5 References 1. (a) Kulkarni, R.K.; Pani, K.C.; Neuman, C. ; Leonard, F. Arch. Surg 1966 154 148. (b) Schmitt, E.E.; Polisti na, R.A. U.S. Patent, 3.463.158. 1969 (c) Frazza, E.J.; Schmitt, E.E. J. Biomed. Mater. Res. Symp 1971 1 43. (d) Schneider, A.K. U.S. Patent, 3,636,956, 1972 (e) Brady, J.M.; Cutright, D.E.; Miller, R.A.; Ba ttistone, G.C.; Hunsuck, E.E. Biomed. Mater. Res 1973 7 155. (f) Wasserman, D.; Ve rsfelt, C.C. U.S. Patent 3,839,297, 1974 (g) Gogolewski, S .; Pennings, A.J. Makromol. Chem. Rapid Commun 1983 4 675. (h) Leenslag, J.W. ; Pennings, A.J.; Bos, R.R.M.; Rozenza, F.R.; Boering, G. Biomaterials 1987, 8 311. (i) Vainionp, S.; Rokkanen, O.; Trml, P. Prog. Polym. Sci 1989 14 679. (j) Vert, M. Angew. Makromol. Chem 1989 166 155. (k) Benicewicz, B.C.; Hooper, P.K. J. Bioact. Compat. Polym 1990 5 453. 2. Rutkowska, M.; Dereszewska, A .; Jatrzebska, M.; Janik, H. Macromol. Symp 1998 130 199. 3. (a) Dubernet, C.; Benoit, J.P. ; Couarraze, G.; Duchene, D. Int. J. Pharm. 1987 35 145. (b) Yolles, S.; Eldri dge, J.E.; Woodl and, J.H.R. Polymer News 1971 1 9. (c) Yolles, S.; Polym. Sci. Technol. 1975 8 245. 4. Le Ray, A-M.; Chiffoleau, S.; Ioo ss, P.; Grimandi, G.; Gouyette, A.; Daculsi, G.; Merle, C. Biomaterials 2003 24 443. 5. Detrembleur, M.M.; Mazza, M.; hall eux, O.; Lecomte, P .; Mecerreyes, D.; Hedrick, J.L.; Jrme, R. Macromolecules 2000 33 14.

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77 6. Trollss, M.; Lee, V.Y.; Mecerreyes, D.; Lwenhielm, P.; Mller, M.; Miller, R.D.; Hedrick, J.L. Macromolecules 2000 33 4619. 7. Sun, J.; Dong, Y.; Cao, L. ; Wang, X.; Wang, S.; Hu, Y. J. Org. Chem 2004 69 8932. 8. Firouzabadi, H; Iranpoor, N.; Hazarkhani, H. J. Org. Chem 2001 66 7527.

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78 Chapter 4 Functionalized Caprolactone Cooligomers: Cooligomerization, Attachment of N -Thiolated -Lactams and Biol ogical Evaluation 4.1 Introduction 4.1.1 Poly( -caprolactone) There has been significant research in the past two decades on the use of synthetic polyesters for biomedical purposes such as applications in surgery and medicine. Aliphatic polyesters such as pol y(lactic acid) (PLA), poly(glycolic acid) (PGA) and poly( -caprolactone) (PCL) are often used as biomaterials due to their low toxicity, good biocompatibility, and excellent mechanical properties.1 Recent work in our laboratory has focu sed on the attachment of our N -thiolated -lactam antibiotics to polymers to form new biomaterials.2 This study focuses on covalently binding our lactams to a poly( -caprolactone) backbone formed from the cooligomerization of -caprolactone with the new substituted lactones, 112 and 122 that were described in Chapter 3.

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79 4.1.2 Polymerization of -caprolactone Poly( -caprolactone) is formed from the ring-opening polymerization (ROP) of the -caprolactone monomer 133 (Figure 4.1). Figure 4.1 Ring-opening polymerization of -caprolactone 133 O O O O n -caprolactone monomer 133 poly( -caprolactone) (PCL) 100 ROP There are many catalysts that can be used for this polymerization, but the most commonly used catalyst is tin 2-ethylhexanoate ( 134 ) (Figure 4.2), commonly known as stannous octoate [Sn(Oct)2]. Stannous octoate is used most often because it is inexpensive, non-toxic and highly efficient.3 It is considered safe for use in the making of polymers for biomedi cal applications because it is already approved by the Food and Drug Administ ration (FDA) as a food additive.4 Figure 4.2 Structure of Sn(Oct)2 O O OO 134 Sn

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80 Stannous octoate acts as a catalyst fo r the polymerization reaction, and it is used in conjunction with an active hydrogen compound (either purposely added or as an impurity). The actual pol ymerization mechanism has been much debated3, however, reports in recent years favor the mechanism shown in Figure 4.3.3 The first three steps, A B and C represent initiation steps, D and E are propogation steps, and F is a termination step. In the first step ( A ) of the mechanism, stannous octoate reacts with an active hydrogen compound to form a stannous alkoxide species ( 135 ), thereby liberatin g a molecule of 2ethylhexanoic acid ( 136 ). Next, in the second step of the mechanism, 135 reacts either with another equival ent of alcohol to produc e the stannous dialkoxide initiator ( 137 ) (shown in step B ), or with an equivalent of water to a stannous alcohol derivative ( 138 ) (shown in step C). Of these two possible pathways, the desired pathway is B leading to the formation of the stannous dialkoxide 137 because 137 is a much more efficient initiator than 138 The reaction of 138 with a lactone monomer proceeds via a coordi nation-insertion mechanism shown in step D to generate the first acti vely propagating chain end, 139 This chain can then react with another molecule of the lactone to build onto the chain as shown in step E The chain termination step is shown in step F where 140 can then can then undergo a rapid exchange of the stannous alkoxide monomer for a proton from any hydroxyl compound present (alc ohol, water, chain end). Evidence that supports this mechanism includes the obs ervation that the rate of ROP can be increased by the addition of butanol to distilled Sn(Oct)2,5 and also the direct

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81 observation (by MALDI-TOF) of tin cova lently bound to the end of the polyester chain.6 Figure 4.3 Likely mechanism for the Sn(Oct)2 catalyzed polymerization of -caprolactone3 Sn O O O O 134 + ROH + HO O 136A+ ROH 137 HO O 136B135 + H2O 138 +CROHDO O Sn OR RO O O O RO Sn RO RO O Sn OR RO O SnOR O + 139E+ ROH RO OH O 141 + ROSn OH 137 ROSnOR 135 OctSnOR 135 OctSnOR RO O Sn O 139 O OR O 133 RO O SnOR O 140 2FRO O SnOR O 140 2 OctSnOR ROSnOR +

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82 There are possible side reactions t hat can take place during these polymerization reactions that can lead to lower molecular weight polymers, and broader molecular weight distributions. An intermolecular transesterification reaction, such as the one shown in Figur e 4.4, is one type of side reaction that can occur. Figure 4.4 Intermolecular transesterification RO O SnOR' O + R''O O OR''' O O RO O OR''' O O n n R''O O O SnOR' + Another type of side reaction that c an take place is an intramolecular transesterification reaction that leads to the formation of cyclic oligomers (Figure 4.5). This is known as polymer backbiting. The possibility of interand intramolecular transesterification r eactions increases with higher reaction temperatures and longer reaction times.7

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83 Figure 4.5 Intramolecular tr ansesterification (back-biting) RO O O O O SnOR' O O (CH2)5 O O n n 4.1.3 Examples of copolymerization of -caprolactone with substituted lactones from the literature In recent years, there have been r eports on the copolymerization of caprolactone with caprolactones that cont ain functional groups. Detrembleur et al. reported the copolymerization of -caprolactone with 5-bromocaprolactone ( 103 ) (Scheme 4.1).8 Scheme 4.1 Copolymerization of -caprolactone with 5-bromocaprolactone O O O O Br O O OH O O Br + Al(OiPr)3,PhCH30 oC, 2.5h 133103142 These copolymerization reactions were carried out in toluene solution at 0 oC using aluminum triisopropoxide [Al(OiPr)3] as the catalyst. Copolymers ( 142 )

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84 of the substituted lactone 133 and the unsubstituted -caprolactone 103 were formed successfully in varying ratios with narrow molecular weight distributions. When the content of the substituted lactone 133 was increased beyond 30 mol %, the copolymers were found to exist as viscous liquids. Trollss et al. reported the copolymerization of -caprolactone 103 with lactones that bore pendant ester functionalities (Scheme 4.2).9 Scheme 4.2 Copolymerization of -caprolactone with substituted lactones O O O O RO2C R' O O OH O O CO2R + Sn(Oct)2,R'OH 110 oC, 48h 133106 R=Bn 107 R=tBu 143 R=Bn 144R=tBu These copolymerizations were perfo rmed in bulk (solventless) at 110 oC using Sn(Oct)2 as the catalyst and an alcohol as a initiator. Previously, the same group had reported the observation that s ubstituted lactones generally react slower under these polymerizat ion conditions than unsubstituted -caprolactone 133 .10 In this study, they found that monomers 106 and 107 were particularly slow to react, and that the molecular we ights that they obt ained were lower than what was expected. For the homopol ymerization of the benzyl ester 106 the molecular weights obtained (from size exclusion chromatography, SEC) were 1000 for the reaction using Al(OiPr)3, and 2000 for the reaction using Sn(Oct)2. For the homopolymerization of the tert -butyl ester 107 a molecular weight of

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85 2000 was obtained for the polymerization catalyzed by Al( OiPr )3. For the copolymerization of 20% 107 and 80 % -caprolactone 133 a molecular weight of 13000 (by SEC) was obtained. They obser ved that the copol ymerization of caprolactone with the benzyl ester 106 was not as successful and gave lower than expected molecular weights (the exact va lue was not stated for this system). The copolymerization of -caprolactone with RS-benzyl malolactonate ( 145 ) (Scheme 4.8) was recently reported by He.11 This copolymerization reaction was studied using Sn(Oct)2 as the catalyst. The temperatures, concentration of the catalyst and monomer ratios were varied. Scheme 4.3 Copolymerization of -caprolactone with RS-benzyl malolactonate O O O O O O O + Sn(Oct)2, 90-170 oC, 24h O O CO2Bn C O 2Bn 1331 4 5 1 4 6 This reaction failed at 90 oC, but was successful at 110, 130, 150 and 170 oC. The reaction temperature that was found to give the highest yields and the highest molecular weights was 130 oC. The content of RS-benzyl malolactonate, 145 in the copolymers was observed to be much lower than what was expected, and the molecula r weight of the copolymers 146 decreased as the amount of 145 increased, indicating that 145 reacts much slower than caprolactone 133 Changing the concentration of the catalyst was not found to

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86 have an effect on the molecular weight of the copolymers. A ll of the copolymers that were formed in the study were found to be liquids at temperatures above 50 oC, and the copolymers that c ontained more than 16.2 % of 145 were found to be liquids at room temperature. 4.2 Results and Discussion 4.2.1 Cooligomerizations of benzyl ester containing lactone 112 with caprolactone The lactone bearing a pendant benzyl ester, 112 whose synthesis was described in Chapter 3, was cooligomerized with -caprolactone (Scheme 4.4). The reactions were carried out in bulk at 130 oC using Sn(Oct)2 as the catalyst, following the procedure of He.11 Scheme 4.4 Cooligomerizat ion of lactone 112 with -caprolactone O O O O O O O O O + Sn(Oct)2, 130 oC, 24-48h BnO2C CO2Bn 1331121 4 7 The cooligomerization was attempted wit h 10, 15, 20, 25, 30, and 100 mol % of lactone 112 with a 1/1000 (molar ratio) of the catalyst Sn(Oct)2. The

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87 cooligomerizations usi ng 10, 15, and 20 mol % of 112 were successful. The yields of the copolymerization reactions were extremely low using 25 and 30 mol % of 112 and the products were liquids. Attemp ted isolation of the products from these two reactions was unsuccessful. The attempted homopolymerization of 112 was unsuccessful. This was not surpri sing considering that Trollss had reported for similar monomers ( 106 and 107 ) that homopolymerization did not occur to any appreciable extent.9 Table 4.1 Cooligomers 147 Entry112 Content Feed OligomerbTime MWaA B C 10% 20% 30% 7.6% 13.9% 17.9% 24h 48h 48h ~1000 ~1000 ~1000 a Molecular weights shown represent a crude estimate from the MALDIs p ectrumbfrom1HNM R anal y sis The proportion of the lactone 112 in the cooligomers was 7.6, 13.8, and 17.9 %, slightly lower in than in the feeding dos e (Table 4.1). The molecular weights of the cooligomers were also lo w. The substituted monomer, 112 is a low-melting waxy solid that is very difficult to remove water from. It is likely that the presence of water impurities lowers the molecular weights of the oligomers by forming a less active initiating species ( 138 Figure 4.3). Having a less active initiating species could potentially slow the rate of the oligomerization and increase the likelihood of competing transesterification reactions (Figures 4.4 and 4.5), which would result in lower molecular weight o ligomers. It is also possible that the

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88 Sn(Oct)2 is also coordinating with the car bonyl of the pendant benzyl ester group instead of the carbonyl of the lactone ring. A positive result from these experiments was that exam ination of the MALDI-TOF spectra of the oligomers did confirm the incorporation of lactone 112 into the cooligomers 147 (Figure 4.6). This can be seen by the loss of 262 (the MW of benzyl ester containing lactone 112 ) and the loss of 114 (MW of -caprolactone 133 ) associated with the same set of peaks in the MALDI spectr um. Although the mole cular weight of these oligomers was lower than what was hoped for, this still provided a new material to study t he attachment of the N -thiolated -lactams. This new material could then be checked for anti-MRSA and antiBacillus properties. Figure 4.6 MALDI spectru m of cooligomer 147

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89 4.2.2 Cooligomerizations of tert -butyl ester containi ng lactone 122 with caprolactone The lactone bearing a pendant tert -butyl ester, 122 described in Chapter 3, was cooligomerized with -caprolactone 133 (Scheme 4.5). The reactions were carried out in bulk, at 130 oC, using Sn(Oct)2 as the catalyst. Scheme 4.5 Cooligomerizat ion of lactone 122 with -caprolactone O O O O O O O O O + Sn(Oct)2, MeOH 130 oC, 24-48h CO2 tBu CO2 tBu 133122148 The cooligomerization was attempt ed with 10, 15, and 20 mol % of lactone 122 with a 1/1000 (molar ratio) of the catalyst Sn(Oct)2, and a 1/40 (molar ratio) of methanol was added as an ac tive hydrogen compound. The tert -butyl ester containing monomer 122 was a much more crystalline monomer than the benzyl ester containing monomer 112 and it was hoped that this property would lead to less water impurities in the reaction, and thus lead to higher molecular weight cooligomers ( 148 ). The proportion of the lactone 122 found to be incorporated into the oligomers was 10, 11 and 16% (Table 4.2).

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90 Table 4.2 Oligomers 148 Entry122 Content Feed OligomerbTime MWaA B C 10% 20% 30% 10% 11% 16% 24h 48h 48h ~1000 ~1000 nd a Molecular weights shown represent a crude estimate from the MALDI spectrum after deprotection of the tbutyl ester b from 1H NMR analysis. (nd, not determined) All attempts to analyze these oligomers 148 via MALDI-TOF failed at this stage. However, the oligomers could be analyz ed by MALDI-TOF following the cleavage of the tert -butyl ester group (Section 4.2.3, Scheme 4.7). 4.2.3 Deprotection of the oligomers The deprotection of the oligomers containing the benzyl ester was accomplished by hydrogeno lysis (Scheme 4.6). Scheme 4.6 Deprotection of the benz yl ester containing oligomer 147 O O O O O CO2Bn O O O O O CO2H 10% Pd/C H2, EtOAc 147 149

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91 The hydrogenolysis of the oligom er was carried out in hydrogen atmosphere (under a hydrogenfilled balloon) using 10% Pd/C as the catalyst, and ethyl acetate as the solvent. Ethyl acetate was selected for the solvent because it was capable of dissolving the oligomers both before and after the oligomerization. The reaction was comp lete after stirring overnight under hydrogen atmosphere to give 149 in nearly quantitative yield. The 1H NMR of the hydrogenolyzed product showed the co mplete disappearance of the peaks associated with the benzyl group (at 5.2 and 7.3 ppm). The MALDI spectrum of the hydrogenolyzed product showed the loss of 172 amu, which corresponds to the weight of the substituted monomer afte r the ester protecting group is cleaved. The deprotection of the oligomers containing the tert -butyl ester was accomplished using trifluoroacetic acid (TFA) to give carboxylic acid 149 (Scheme 4.7). Scheme 4.7 Deprotection of the tert -butyl ester contai ning oligomer 148 O O O O O CO2 tBu O O O O O CO2H 1:1 TFA:CH2Cl21h, rt 148 149

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92 The deprotection of the tert -butyl group was accomplished using a solution of 50% TFA in methylene chloride. T he reaction was complete to give deprotected oligomers 149 in quantitative yield after 1 hour of stirring at room temperature. The removal of the tert -butyl ester was confirmed by 1H NMR by the disappearance of the si nglet associated with the tert -butyl group (1.4 ppm). Following the deprotection of the tert -butyl ester groups to give 149 the MALDITOF analysis of the oligomers was attemp ted again. This time, a spectrum was obtained for these oligomers. Unfortunately, the molecular weight of these oligomers was again low (~1000). Howeve r, the MALDI spectrum showed the loss of 172 amu from the chain, confirmi ng that the substituted lactone had been incorporated into the oligomer chain. As was the case with the oligomers 147 (formed from the benzyl ester bearing lactone 112 ) the molecular weights of the oligomers formed from the tert -butyl ester containing lactone 122 were less than what was desired; however, a new, functiona lized oligomer that could be used to test the attachment of our N -thiolated -lactams was obtained. 4.2.4 Attachment of N -thiolated -lactams 73 and 131 to the oligomer The attachment of the N -thiolated -lactams to the oligomer 149 that contained a pendant carboxylic acid gr oup was accomplished using the same method described previously (in Chapter 3) for the atta chment of the N -thiolated -lactams to the lactone monomers (Scheme 4.8).

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93 Scheme 4.8 Attachment of N -thiolated -lactams to the carboxylic acid containing oligomer 149 O O O O n N O HO SR Cl O O O O CO2H n O O N O SR Cl 149 73 R=Me 131 R= s -Bu + 150 R=Me 151 R= sBu a Reagents and conditions: (a) 1.5 eq. EDCI, cat. DMAP, dry CH2Cl2,rt The oligomers containing 7.6% of the carboxylic acid-substituted lactone were selected for this reaction. Coupling of the free acid contai ning oligomers to the N -thiolated -lactams was accomplished using 1.5 equivalents of EDCI and a catalytic amount of DMAP in freshly distilled methylene chloride. The successful attachment of the -lactams to the oligomer c an be seen by the shift in the 1H NMR spectrum of the two protons of the -lactam ring. In the 1H NMR of 73 these two protons appear as a doublet of doublets at 5.3 and 5.2 ppm, and following the attachment to the oligomer 149 these two protons appear at 6.2 and 5.5 ppm (Figure 4.7).

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94 Figure 4.7 Expanded region 1H NMR spectra for -lactam 73 and -lactam bearing cooligomer 150 b) Vicinal protons on the lactam ring of 150 a) Vicinal protons on the lactam ring of 73 In the 1H NMR of 131 these two protons appear as a multiplet at 5.3 ppm, following the attachment to the oligomer 149 these two protons appear as a doublet of doublets at 6.1 and 5.4 ppm. An analogous shift had been observed previously for the coupling of hydroxy lactams 73 and 131 to lactone 129 to give esters 130 and 132 4.2.5 Results of screening for anti-MRSA and antiBacillus activity The new N -thiolated -lactam bearing oligomers 150 and 151 were screened for anti-MRSA activity against the sa me nine strains of MRSA that were used previously for testing the N -thiolated -lactam analogues. The screening for

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95 bioactivity was accomplished using the Kirby-Bauer method of disc diffusion as described in Chapter 2. The oligomers 150 and 151 were tested by disc diffusion rather than well diffusion because they we re completely insoluble in the DMSO solvent that is used for well diffusion. The results of the anti-MRSA testing are shown in Table 4.3. Also included in the Table 4.3 are the zones of growth inhibition for the C3 hydroxy -lactams 73 and 131 the lactam-lactone conjugates 130 and 132 and a control sample of the 7.6% oligomer 147 The control oligomer 147 was included to exclude the possibility that the oligomer itself (or any residual tin initiato r) was inhibiting bacterial growth. Table 4.3 Zones of growth inhibition against MRSA 73 131 130 132 150 151 147a19 32 15 23 9 15 0 17 28 17 21 11 16 0 17 29 15 22 10 15 0 19 28 15 21 10 14 0 18 28 14 22 9 16 0 16 25 16 21 10 15 0 19 29 16 21 11 15 0 18 29 16 21 12 16 0 18 30 14 22 9 15 0 MRSA 652 MRSA 653 MRSA 654 MRSA 655 MRSA 656 MRSA 657 MRSA 658 MRSA 659 MRSA 919 Cmpd # The values indicate the diameters in mm for the zone of growth inhibition after 24 h of incubation at 37 oC. Twenty micrograms of each test compound applied to a cellulose disc as a chloroform solution was used. a 400 micrograms of the control copolymer was applied to the cellulose disc as a chlorform solution. All of the microbes listed are lactamase producing, methicillin-resistant strains of Staphylococcus aureus (MRSA). Those labelled as MRSA 652-659 were obtained from a clinical testing laboratory at Lakeland Regional Medical Center, Lakeland, FL. MRSA 919 is ATCC 43300 and was purchased from American Type C ulture C ollections. E rror valuesarewithin+ / -1mm. All of the compounds which contained the N-sec -butylthio -lactams ( 131 132 and 151 ) showed larger zones of growth inhibition than the compounds which contained the N -methylthio -lactams ( 73 130 and 151 ). This trend is consistent

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96 with what was observed during the N -thiolated -lactam SAR study (Chapter 2). In each series of compounds, the N-sec -butylthio -lactam containing compounds and the N -methylthio -lactam containing compounds, the -lactams ( 73 and 131 ) had greater anti-MRSA activity t han the lactam-lactone conjugates ( 130 and 132 ), which had greater activity than the lactams attached to the cooligomers ( 150 and 151 ) (Figure 4.8) This difference in activity may be due to molecular weight differences and uncerta inty of the loading efficiency of the lactams onto the oligomers. The most important thing to note from these results is the two oligomers ( 150 and 151 ) that have N -thiolated -lactams covalently bound to the oligomer backbone possess an ti-MRSA activity, and that covalent bonding of the -lactams to an oligoester backbone does not interfere with the bioactivity! The control oligomer, 147 showed no zones of growth inhibition in this assay, which confirms that the bioactivity of 150 and 151 is due to the attached -lactams.

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97 Figure 4.8 Kirby-Bauer di sc diffusion test plate for compounds 131, 132, and 151 against MRSA 655 N O O Cl S polyme r O 151 N O HO S Cl 131 N O O Cl S O O O 132 The two new -lactam bearing oligomers, 150 and 151 were also screened for antiBacillus activity against the same seven Bacillus microbes that were examined previously (Table 4.4). Also included in Table 4.4 for comparison are the zones of growth i nhibition for the C3 hydroxy -lactams 73 and 131 and the lactam-lactone conjugates 130 and 132

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98 Table 4.4 Zones of grow th inhibition against Bacillus 73 131 130 132 150 151 B. anthracis Cmpd B. globigii B. thuringensis B. megaterium B. coagulans B. subtilis B. cereus 24 25 17 22 11 22 14 26 15 23 9 23 19 19 14 17 9 14 17 24 15 19 9 19 17 24 15 20 12 20 11 26 13 22 2 22 19 19 17 18 12 18 Twenty micrograms of test compound in DMSO solution wa s used in each case. The values indicate the diameters in mm for the zone of growth inhibition obtained for each compound after 24 h of incubation at 37 oC. Error values are within +/1mm. (nd, not determined). The trend in the bioactivity of these compounds against Bacillus exactly parallels what was seen for MRSA, with the N -sec-butylthio -lactam containing compounds ( 131 132 and 151 ) having larger zones of growth inhibition than the N -methylthio -lactam containing variants ( 73 130 and 150 ). It is exciting to observe that the two -lactam bearing cooligomers ( 150 and 151 ) show antiBacillus activity, indicating that attachment of the lactams to the oligoester backbone did not preclude antibacterial ac tivity of these compounds. From previous studies (described in Ch apters 1 and 2), we know that the N -thiolated lactam antibiotics act by entering the bacteri al cell. Therefore, in order for these new oligomers to possess the same bioac tivity, we must assume that the lactams are entering the cells, however, at this point, we do not know whether or not the lactams are still attached to the oligomers when they do so.

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99 4.3 Conclusions In conclusion, both of the new func tionalized caprolactone monomers ( 112 and 122 ) that were described in Chapter 3 could be cooligomerized with caprolactone to form new functionalized oligomers 147 and 148 While the molecular weights of these new oli gomers were lower than desired, a new material with which to test the attachment of the N -thiolated -lactams was obtained. The deprotection of both 147 and 148 was accomplished under mild conditions to give the oligomer 149 which bore a pendant free carboxylic acid group. An N -methylthio -lactam 73 and an N -sec-butylthio -lactam 131 were both successfully covalently attached to the oligoester backbone, and the resulting -lactam bearing oligomers were screened for anti-MRSA and antiBacillus activity. Both of the new drug-cont aining oligomers were found to inhibit the growth of the a ll nine MRSA and seven Bacillus strains that were tested. This provides the precedent needed for further experiments and to make use of the C3 position of the -lactam ring to bind the antibiotic to a biodegradable oligoester as a means to retain the bioactivity of the lactam. Thus far, the attachment of other antibiotics, such as 6-aminopenicillinic acid and ciprofloxacin has been unsuccessf ul. This is, however, an area which still needs to be explored in detail to dete rmine what other drug molecules can be bound to these oligomers. We believe that they attachment of any drug molecules to the oligomers will need to be accomplished following the oligomerization reaction due to the Lewis ac idity of the oligomerization catalyst.

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100 Based on previous studies of aliphatic polyesters, we believe that these new oligomers will also be biodegradable, but further studies will need to be performed to confirm this. Further experim ents to try to increase the molecular weight of these oligomers should also be explored to increase the scope of potential applications of these new materials. Another alternative to this would be to graft the oligomers to a pre-formed polyester. The experiments described within this dissertation represent the beginning point to an exciting new proj ect that needs to be explor ed further. The successful attachment of the N -thiolated -lactams to a biodegr adable material opens up many new avenues for us to explore. We can envision many biomedical applications for these new materials such as antibacterially -active biodegradable sutures and coatings for implants.

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101 4.4 Experimental -Caprolactone was dried over CaH, di stilled under reduced pressure, and stored over activated 4 molecular sieves before use. Tin (II) ethylhexanoate was purchased from Aldrich and used as rece ived. Toluene was dried over calcium hydride and distilled before use. Anhy drous methanol (<99.9%) was purchased from Acros and used as received. Monomers 112 and 122 were purified by flash column chromatography and dried in an Al bderhalden drying app aratus overnight (P2O5, refluxing acetone). All other r eagents were used as received. NMR spectra were recorded in CDCl3. 13C NMR spectra were proton decoupled. MALDI analysis was performed by Dr. Ted Gauthier in the US F Department of Chemistry Mass Spectrometry Facility, and conducted in positive ion mode using 2,5-dihydroxybenzoic acid (DHB) as the matrix. Representative oligomeriz ation procedure: Lactone 112 ( 0.200g, 0.763 mmol) was weighed into a vial and dried ov ernight using an Albderhalden drying apparatus. An oil bath was preheated to 130 oC. The Albderhalden drying apparatus was opened in a glove bag under a nitrogen atmosphere. Inside the glove bag, previously distilled -caprolactone (0.784g, 6. 87 mmol) was added to the vial containing the substituted lact one. The vial was capped with a cap that contained a teflon septum and then remo ved from the glove bag, sealed with teflon tape and placed in the constant temperature oil bath at 130 oC. After allowing 20 minutes for the monomers to reach the tem perature of the oil bath,

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102 the Sn(Oct)2 initator was added vi a syringe.( 7.63 X 10-3mmol, 76.3 L of a 0.1M solution in freshly distilled toluene). Afte r 24 hours, the vial was removed from the oil bath. The residue was dissolved in a small amount of chloroform and precipitated from an ice-cold solution of 3:2 hexanes:diethyl ether. The solvent was removed from the precipitated c ooligomer and then dried under vacuum overnight to give 0.47g (48 %) of the cooligomer.

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103 4.5 References 1. For review see; Albertsson, A-C.; Varma, I.K. Biomacromolecules 2003 4 1466. 2. (a) Shim, J-Y, Ph.D. Dissertati on, University of South Florida, 2003 (b) Yang, W. Ph.D. Dissertation, Un iversity of South Florida, 2006 3. Storey, R.F.; Sherman, J.W. Macromolecules 2002 35 1504. 4. U.S. Code of Federal Regulations (21CFR) Part 175. 5. Kowlalski, A.; Duda, A.; Penczek, S. Macromol. Rapid. Commun ., 1998 19 567. 6. Kowlalski, A.; Duda, A.; Penczek, S. Macromolecules 2000 33 689. 7. Kowlalski, A.; Duda, A.; Penczek, S. Macromolecules 2000 33 7359. 8. Detrembleur, M.M.; Mazza, M.; Hall eux, O.; Lecomte, P .; Mecerreyes, D.; Hedrick, J.L.; Jrme, R. Macromolecules 2000 33 14. 9. Trollss, M.; Lee, V.Y.; Mecerreyes, D.; Lwenhielm, P.; Mller, M.; Miller, R.D.; Hedrick, J.L. Macromolecules 2000 33 4619. 10. Trollss, M.; Kelly, M.A.; Claesson, H.; Siemens, R.; Hedrick, J.L. Macromolecules 1999 32 4917. 11. He, B.; Chan-Park, M.B.; Macromolecules 2005 38 8227.

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104 Appendix 1 Selected spectra

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105 1H NMR spectrum for 63

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106 1H NMR spectrum for 73

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107 13C NMR spectrum for 73

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1081H NMR spectrum for 74

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10913C NMR spectrum for 74

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1101H NMR spectrum for 75

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11113C NMR spectrum for 75

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1121H NMR spectrum for 76

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11313C NMR spectrum for 76

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1141H NMR spectrum for 77

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11513C NMR spectrum for 77

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1161H NMR spectrum for 78

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11713C NMR spectrum for 78

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1181H NMR spectrum for 79

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11913C NMR spectrum for 79

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1201H NMR spectrum for 82

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12113C NMR spectrum for 82

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1221H NMR spectrum for 84

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1231H NMR spectrum for 85

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1241H NMR spectrum for 87

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12513C NMR spectrum for 87

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1261H NMR spectrum for 88

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12713C NMR spectrum for 88

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1281H NMR spectrum for 89

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12913C NMR spectrum for 89

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1301H NMR spectrum for 90

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13113C NMR spectrum for 90

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1321H NMR spectrum for 91

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13313C NMR spectrum for 91

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1341H NMR spectrum for 92

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1351H NMR spectrum for 93

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13613C NMR spectrum for 94

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1371H NMR spectrum for 95

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13813C NMR spectrum for 95

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1391H NMR spectrum for 96

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14013C NMR spectrum for 96

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1411H NMR spectrum for 97

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14213C NMR spectrum for 97

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1431H NMR spectrum for 98

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14413C NMR spectrum for 98

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1451H NMR spectrum for 99

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14613C NMR spectrum for 99

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1471H NMR spectrum for 112

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14813C NMR spectrum for 112

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1491H NMR spectrum for 114

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1501H NMR spectrum for 115

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1511H NMR spectrum for 116

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1521H NMR spectrum for 117

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1531H NMR spectrum for 118

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1541H NMR spectrum for 119

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1551H NMR spectrum for 121

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1561H NMR spectrum for 122

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15713C NMR spectrum for 122

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1581H NMR spectrum for 126

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15913C NMR spectrum for 126

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1601H NMR spectrum for 127

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16113C NMR spectrum for 127

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1621H NMR spectrum for 128

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16313C NMR spectrum for 128

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1641H NMR spectrum for 129

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1651H NMR spectrum for 130

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16613C NMR spectrum for 130

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1671H NMR spectrum for 131

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1681H NMR spectrum for 132

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1691H NMR spectrum for 147A

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1701H NMR spectrum for 147B

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1711H NMR spectrum for 147C

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1721H NMR spectrum for 148A

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17313C NMR spectrum for 148A

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1741H NMR spectrum for 148B

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1751H NMR spectrum for 148C

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1761H NMR spectrum for 149

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17713C NMR spectrum for 149

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178 1H NMR spectrum for 150

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1791H NMR spectrum for 151

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180 MALDI spectrum for 147a after debenzylation

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181 MALDI spectrum for 147a after debenzylation (expanded region)

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182 MALDI spectrum for 184a after deprotection

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183 MALDI spectrum for 184a after deprotection (expanded region)

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184 MALDI spectrum for 148b after deprotection

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185 MALDI spectrum for 148b after deprotection (expanded region)

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About the Author Michelle Leslie received her Bachelor of Science with a major in Chemistry in May 2000 from Saint Mary’s University, Halifax, Nova Scotia, Canada. Michelle then moved to Tampa, Florida to pursue a Doctorate in Organic Chemistry at the University of S outh Florida. Michelle plans to pursue a career in teaching, and has accepted a position as an Assistant Professor of Chemistry at Florida Southern College in Lakeland, Florida.


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Leslie, J. Michelle.
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N-Thiolated b-lactam antibiotics :
b synthesis and structure-activity studies of C3 oxygenated derivatives and attachement to new, functionalized caprolactone monomers and polymers
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by J. Michelle Leslie.
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[Tampa, Fla] :
University of South Florida,
2006.
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ABSTRACT: N-Thiolated beta-lactams are a new class of anti-MRSA and anti-Bacillus agents that have recently been reported by our laboratories. From previous studies performed in our laboratories, it is believed that the N-thiolated beta-lactams exert their antimicrobial activity through a unique mode of action that is completely unlike that of classical beta-lactam antibiotics. In the first chapter of this dissertation, a review of previously prepared N-thiolated beta-lactam analogues and their mode of action is presented. In the second chapter, the synthesis of seven different C3-oxygenated derivatives is described. These analogues were tested for antibacterial activity against Staphylococcus aureus, nine different strains of MRSA, and seven different species of Bacillus. The results of the antibacterial testing will be discussed in relation to the differences in the structures of the analogues. In chapter 3, the design and synthesis of two new, functionalized caprolactone monomers are presented. These monomers were subsequently cooligomerized with epsilon-caprolactone, as described in chapter 4. N-thiolated beta-lactams were attached to the functionalized oligomers. These antibiotic containing oligomers were then screened for activity against MSSA, MRSA, and Bacillus. The results of these biological tests and their implications for future experiments are discussed.
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Penicillin.
Vancomycin.
MRSA.
Bacillus.
Polyester.
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