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Title:
The theoretical modeling, design, and synthesis of key structural units for novel molecular clamps and pro-apoptotic alpha helix peptidomimetics
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Book
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English
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Weiss, Stephanie Tara
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University of South Florida
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Tampa, Fla
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Subjects

Subjects / Keywords:
Hydrazine
Nitrosamine
Substrate-targeted inhibitor
Bax
P53
MDM2
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: This dissertation presents the theory and practice of designing, synthesizing and using peptidomimetics to disrupt protein-protein interactions. Our general strategy is to design and synthesize peptidomimetics that will mimic peptide secondary structures (alpha-helices and beta-sheets). Chapter One is a theoretical examination of the feasibility of using beta-sheet mimics called molecular clamps to inhibit substrate-receptor interactions by blocking the substrate rather than the receptor or enzyme. Several natural and synthetic examples of this approach are given in support of this concept. We also present the results of a kinetic modeling study and a consideration of which types of systems would be the best candidates for a substrate-targeted inhibitor approach. Chapter Two relates a continuation of previous work in our lab to synthesize five novel beta-protected hydrazino amino acids. These hydrazines are essential precursors for synthesizing constrained beta-stran d mimetics. We showed that we could selectively deprotect the alpha-nitrogen of the hydrazines, and we synthesized several novel examples of polar beta-protected hydrazino amino acids. Chapter Three discusses the design and synthesis of small-molecule and peptidomimetic MDM2 inhibitors, including our work on synthesizing a new class of alpha-helix mimics that have improved water solubility compared with previously reported examples of alpha-helix mimics. As with the constrained beta-strand mimics described in Chapter Two, the synthesis of novel hydrazino amino acid precursors is a key step in synthesizing our alpha-helix mimics. One isoleucine hydrazine derivative was synthesized, and progress was made toward synthesizing two other hydrazines from tryptophan. In addition, the synthesis of three potential small-molecule inhibitors of MDM2 is described. Chapter Four describes the use of the GLIDE program to design and evolve an alpha-helix mimic that will interact with the pro-apop totic protein Bax. Progress toward the synthesis of this compound is also reported.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Stephanie Tara Weiss.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 201 pages.
General Note:
Includes vita.

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oclc - 141188497
usfldc doi - E14-SFE0001475
usfldc handle - e14.1475
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The Theoretical Modeling, Design, and Synthesis of Key Structural Units for Novel Molecular Clamps and Pro Apoptotic Alpha Helix Peptidomimetics by Stephanie Tara Weiss A dissertation submitted in partial fulfillment o f the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Mark L. McLaughlin Ph.D. Srikumar Chellappan Ph.D. Abdul Malik, Ph.D. David Merkler, Ph.D. Edward Turos, Ph.D. Date of Approval: March 31, 2006 Keywords: hydrazine, nitrosamine, substrate targeted inhibitor, Bax, p53, MDM2 Copyright 2006 Stephanie T. Weiss

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DEDICATION I dedicate this dissertation to the three people who most profoundly influenced my decision to study chemistry. One is Paul Scudder, my first organic chemistry teacher. The second is Wayne Brouillette, my M.S. advisor. Fina lly, I dedicate it to Mark McLaughlin, my Ph.D. advisor, without whom this dissertation could not have existed.

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ACKNOWLEDGMENTS Many people have helped me in various ways throughout my time in graduate school, and it is impossible for me to mention them all by name, but I hope that they all know how much I appreciate everything that theyve done for me. First, I thank Mark McLaughlin, my Ph.D. advisor, for giving me the opportunity to complete my graduate education. I would also like to thank all of my committee members: Srikumar Chellappan, Abdul Malik, David Merkler, and Edward Turos, as well as Wayne Guida for chairing my defense and proofreading this dissertation. Wayne Brouillette, my M.S. advisor, got me started with chemistry research. I h ave had many great co workers during my time in graduate school, and I would like to thank Umut Oguz, Kiran Avancha, Tanaji Talele, Mohanraja Kumar, Gabriel Garcia, Ben Davis, and all of my other labmates for their support and help. I also would like to r ecognize Emily McIntosh, Scott Haake, and several other undergraduate lab helpers. Ted Gauthier and Edwin Rivera have my utmost appreciation for their help with the MS and NMR instruments, respectively. Shen Shu Sung performed the molecular modeling expe riments described in Chapter Four, and Jiazhi George Sun conducted the biological testing of my compounds against MDM2 and Bax. I would like to thank all of my friends for their support, especially David Rabson for proofreading this dissertation. Final ly, I want to recognize my parents Alicia and Donald Weiss, and my sister Shelly Weiss, for their moral support, because I know it wasnt easy for you to listen to so much chemistry talk!

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i TABLE OF CONTENTS LIST OF FIGURES iii LIST OF SCHEMES vi LIST OF ABBREVIATIONS viii LIST OF NMR SPECTRA x ABSTRACT xiii CHAPTER ONE: GENERAL INTRODUCTION: PROTEINS AND THEIR INTERACTIONS 1 1.1: Proteins, Peptides, and Pep tidomimetics 1 1.2: Molecular Clamps: Background 6 1.3: Molecular Clamps: A Model 11 1.4: References 19 CHAPTER TWO: SYNTHESIS OF POLAR AND NONPOLAR HYDRAZINO AMINO ACIDS 22 2.1: Introduction 22 2.1.1: Hydrazines 22 2.1.2: Synthesis of Hydrazines 24 2.1.3: Use of Hydrazines in Peptidomimetics 30 2.1.4: Use of Hydrazines in -Sheet Mimics 33 2.2: Results and Discussion 35 2.3: Experimental Data 47 2.4: References 69 CHAPTER THREE: SYNTHESIS OF MDM2 INHIBITORS AND KEY STRUCTURAL UNITS 73 3.1: Introduction 73 3.1.1: The Hallmarks of Cancer 73 3.1.2: Apoptosis and MDM2/p53 75 3.1.3: Inhibiting the MDM2/p53 Interaction with Small Molecules 78 3.1.4: Inhibiting MDM2/p53 with Peptides and Peptidomimetics 83

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ii 3.2: Results and Discussion 85 3.3: Experimental Data 92 3.4: References 103 CHAPTER FOUR: DESIGN AND S YNTHESIS OF A BAX PROTEIN -HELIX MIMIC 106 4.1: Introduction 106 4.1.1: Pro-Apoptotic and Anti-Apoptotic Members of the Bcl-2 Family. 106 4.1.2: Mimicking the BH3 Domain of Pro-Apoptotic Proteins. 110 4.2: Results and Discussion 116 4.3: Experimental Data 135 4.4: References 143 CHAPTER FIVE: APPENDIX: PROTON AND CARBON NMR SPECTRA 147 ABOUT THE AUTHOR End Page

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iii LIST OF FIGURES Figure 1.1A: Structure of an Alpha Helix ( 1 ). 2 Figure 1.1B: Structure of a Beta Sheet ( 1 ). 2 Figure 1.2: Leu-Enkephalin, a Natural Pe ptide Ligand of Opiate Receptors, and Morphine, an Opiate Receptor Peptidomimetic ( 4 ). 4 Figure 1.3: Schematic of the Substr ate-Targeted Inhibitor Concept ( 8 ). 7 Figure 1.4: Schematic of the Binding of a Substrate by a MC. 11 Figure 1.5: Dissociation Constants for a Classical System with an Enzyme-Targeted Inhibitor. 12 Figure 1.6: Dissociation Constants for a MC Substr ate-Targeted Inhibitor. 12 Figure 1.7A: Effect of Substrat e Concentration on Relative Enzyme Velocity 14 Figure 1.7B: Effect of MC Concentration on Relative Enzyme Velocity. 15 Figure 1.7C: Effect of Km Value on Relative Enzyme Velocity. 16 Figure 1.7D: Effect of KMC Value on Relative Enzyme Velocity. 17 Figure 2.1: Example of an Azapeptide ( 23 ). 31 Figure 2.2: Examples of Hydrazides ( 26-28 ). 31 Figure 2.3: Examples of N-Amino Amides ( 26,28 ). 32 Figure 2.4: Constraining the Extend ed Conformation of a Peptide to Mimic a Beta Sheet ( 22 ). 34 Figure 2.5: Some Constrai ned Hydrazine-Containing Peptidomimetics ( 31,32 ). 34

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iv Figure 2.6: NMRs of N-Benzyl-(L)-Th reonine Methyl Ester (top) and N-Benzyl-(D)-Threonine Methyl Ester. 41 Figure 3.1: Cellular Pathways that Ca n Be Affected in Cancer Cells ( 1 ). 74 Figure 3.2: The Intrinsic (Mitoc hondrial) and Extrinsic (FAS) Pathways ( 3 ). 76 Figure 3.3: Regulation of p53 by MDM2 ( 7 ). 77 Figure 3.4: Small Molecule Inhibitors of MDM2 ( 13 ). 80 Figure 3.5: Structure of syc-7, a Non-Peptide Inhibitor of HDM2 ( 19 ). 82 Figure 3.6: Design, Model, and Structures of Hamilton’s Terphenyl Helices ( 21,22 ). 84 Figure 3.7: Structure and Model of the McLaugh lin Piperazine Helix. 85 Figure 3.8: Synthesis of Mc Laughlin Piperazine Dione -Helix Mimics. 86 Figure 3.9: MDM2 Small-Molecule Inhibitor Lead Compound NSC-131734. 90 Figure 4.1: The Bcl-2 Fa mily of Proteins ( 2 ). 107 Figure 4.2: Theorized Mech anism of Action of Bax ( 6 ). 109 Figure 4.3: Structures of Some Small-Mol ecule Bcl-2 Family Inhibitors ( 8, 9 ). 111 Figure 4.4: Structures of Two Pr o-Apoptotic Natural Products ( 12,13 ). 111 Figure 4.5: Chemical and Stereo X-Ra y Structure of an Oligoamide ( 17 ). 113 Figure 4.6: Terphenyl Derivatives with Binding Affinity for Bcl-xL ( 21 ). 114 Figure 4.7: Terephthalamide Derivatives with Binding Affinity for Bcl-xL ( 23 ). 115 Figure 4.8: Structure of the Pro-Apopto tic Bax Protein. 117 Figure 4.9: Structures of Some NCI Compounds Virtually Screened to Fit into the Hydrophobic Pocket of Bax. 118

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v Figure 4.10: Some Iterations to Obtain Compound 24 from NSC 109816. 119 Figure 4.11: Mimicking the Alpha Helix of Bax. 120 Figure 4.12: Compound 24 Docked to Bax in Lieu of the Helix. 121

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vi LIST OF SCHEMES Scheme 2.1: Reduction of a Hydrazone to a Hydrazine ( 7 ). 24 Scheme 2.2: Strecker-Like Synt hesis of a Hydrazino Acid ( 6 ). 24 Scheme 2.3: Reaction of Hydrazine with an -Halo Acid ( 8, 9 ). 25 Scheme 2.4: Synthesis of a Hydrazine via Rearrangement of a Hydantoic Acid ( 10 ). 26 Scheme 2.5: Attempt to Synthesize a Hydrazine Using a Hydroxylamine Derivative ( 10 ). 27 Scheme 2.6: Synthesis of a Hydrazine Via N-Amination ( 10 ). 27 Scheme 2.7: Synthesis of a Hydrazine from a Nosyloxy Ester ( 12 ). 28 Scheme 2.8: Hydrazine Formation Via Asymmetric Electrophilic Amination ( 13,14 ). 28 Scheme 2.9: Synthesis of H ydrazines Using Oxaziridines ( 17, 20 ). 29 Scheme 2.10: Synthesis of Hydrazines from Amino Acids Via Nitrosamines ( 21 ). 30 Scheme 2.11: Procedure for the S ynthesis of Five Nonpolar Amino Acids ( 21,22 ). 36 Scheme 2.12: Synthesis of the Ala (6a), Lys (6b) Ser (6c), Thr (6d), and Tyr (6e) Boc-Protected Hydrazines from the Free Amines (1a-e). 37 Scheme 2.13: Unsuccessful TwoStep Method for Synthesizing Compound 2a 38 Scheme 2.14: Epimerization of Thr Due to 2-St ep Benzylation Procedure. 40 Scheme 2.15: Unsuccessful Attempt to Reduce Lys Nitrosamine 4b. 42

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vii Scheme 2.16: Procedure for Synthesizing Co mpound 5b (Lys). 43 Scheme 2.17: Synthesis of Ser, Thr, and Tyr Methyl Esters (1c-e). 44 Scheme 2.18: Procedure for Synthesizing Co mpound 1b (Lys). 45 Scheme 2.19: Deprotection of Nonpolar Ami no Acid Hydrazines. 46 Scheme 3.1: Synthesis of Alkylated Hydrazi no Amino Diacids. 87 Scheme 3.2: Synthesis of Tryptophan Methyl Esters 14b and 14c. 88 Scheme 3.3: Results of the Hydrolysis of 16a with NaOH versus LiOH. 90 Scheme 3.4: Synthesis of MDM2 Inhibitors 23a-c. 91 Scheme 4.1: Retrosynthesis of Compound 24. 122 Scheme 4.2: Synthesis of Indole 25. 123 Scheme 4.3: Failed Attempts to S ynthesize 31 via Reductive Amination of 29. 124 Scheme 4.4: Successful Synthesis of 31 via N, N-Dialkylation. 125 Scheme 4.5: Unsuccessful Attempts to Oxidize 29 and 31 to the Carboxylic Acids. 126 Scheme 4.6: Unsuccessful Attempt to N,N-Dialkylate a Benzoic Ester. 128 Scheme 4.7: Unsuccessful Attempt to Perform Reductive Amination on 38. 129 Scheme 4.8: Synthesis of Succinimide 41 from Aniline 29. 130 Scheme 4.9: Side Product of the Succinimide Reaction. 131 Scheme 4.10: Proposed Synthesis of 44 from 41. 133 Scheme 4.11: Alternative Proposed Route to Compound 26. 134

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viii LIST OF ABBREVIATIONS M: Micromolar AcOAc: Acetic Anhydride AcOH: Acetic Acid ADP: Azadipeptide Unit Ala: Alanine ANT: Adenine Nucleotide Transporter Bcl-2: B-Cell Lymphoma 2 BH3: Bcl-2 Homology Region 3 Boc: tert -Butoxycarbonyl Cbz: Carboxybenzyl conc.: Concentrated Dap: Diaminopimelic Acid DBAD: Ditert -butyl Azodicarboxylate DCM: Dichloromethane DIEA: Diisopropylethylamine DMF: N,N-Dimethylformamide DMSO: Dimethylsulfoxide DP: Dipeptide DPU: Dipeptide Unit E: Enzyme ESI: Electrospray Ionization HDM2: Human Double Minute 2 HR: High Resolution IC50: 50% Inhibitory Concentration Ile: Isoleucine Ki: Inhibitor-Enzyme Dissociation Constant Km: Enzyme-Substrate Dissociation Constant KMC: Inhibitor-Substrate Dissociation Constant LiOH: Lithium Hydroxide Leu: Leucine Lys: Lysine MC: Molecular Clamp MDM2: Mouse Double Minute 2 MeOH: Methanol MIM: Mitochondrial Inner Membrane mM: Millimolar MOM: Mitochondrial Outer Membrane

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ix MP: Melting Point MS: Mass Spectrum NBS: N-Bromosuccinimide nM: Nanomolar NMR: Nuclear Magnetic Resonance Spectrum P: Product PBP: Penicillin Binding Protein Phe: Phenylalanine pM: Picomolar PTP: Permeability Transition Pore p-TSOH: p-Toluenesulfonic Acid Rf: Retardation Factor S: Substrate Ser: Serine TBAB: Tetrabutylammonium Bromide TEA: Triethylamine TFA: Trifluoroacetic Acid THF: Tetrahydrofuran Thr: Threonine TLC: Thin Layer Chromatography Trp: Tryptophan Tyr: Tyrosine Val: Valine VDAC: Voltage-Dependent Anion Channel

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x LIST OF NMR SPECTRA Spectrum 5.1: 148 Spectrum 5.2: 149 Spectrum 5.3: 150 Spectrum 5.4: 151 Spectrum 5.5: 152 Spectrum 5.6: 153 Spectrum 5.7: 154 Spectrum 5.8: 155 Spectrum 5.9: 156 Spectrum 5.10: 157 Spectrum 5.11: 158 Spectrum 5.12: 159 Spectrum 5.13: 160 Spectrum 5.14: 161 Spectrum 5.15: 162 Spectrum 5.16: 163 Spectrum 5.17: 164 Spectrum 5.18: 165 Spectrum 5.19: 166

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xi Spectrum 5.20: 167 Spectrum 5.21: 168 Spectrum 5.22: 169 Spectrum 5.23: 170 Spectrum 5.24: 171 Spectrum 5.25: 172 Spectrum 5.26: 173 Spectrum 5.27: 174 Spectrum 5.28: 175 Spectrum 5.29: 176 Spectrum 5.30: 177 Spectrum 5.31: 178 Spectrum 5.32: 179 Spectrum 5.33: 180 Spectrum 5.34: 181 Spectrum 5.35: 182 Spectrum 5.36: 183 Spectrum 5.37: 184 Spectrum 5.38: 185 Spectrum 5.39: 186 Spectrum 5.40: 187 Spectrum 5.41: 188

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xii Spectrum 5.42: 189 Spectrum 5.43: 190 Spectrum 5.44: 191 Spectrum 5.45: 192 Spectrum 5.46: 193 Spectrum 5.47: 194 Spectrum 5.48: 195 Spectrum 5.49: 196 Spectrum 5.50: 197 Spectrum 5.51: 198 Spectrum 5.52: 199 Spectrum 5.53: 200 Spectrum 5.54: 201

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xiii The Theoretical Modeling, Design, and Synthe sis of Key Structural Units for Novel Molecular Clamps and Pro-Apoptoti c Alpha Helix Peptidomimetics Stephanie Tara Weiss ABSTRACT This dissertation presents the theory a nd practice of design ing, synthesizing and using peptidomimetics to disrupt protein-protei n interactions. Our general strategy is to design and synthesize peptidomimetics that wi ll mimic peptide secondary structures ( helices and -sheets). Chapter One is a theoretical examination of the feasibility of using -sheet mimics called molecular clamps to inhibit substrate-receptor interactions by blocking the substrate rather than the recepto r or enzyme. Several natural and synthetic examples of this approach are given in suppor t of this concept. We also present the results of a kinetic modeling study and a cons ideration of which types of systems would be the best candidates for a substrate-target ed inhibitor approach. Chapter Two relates a continuation of previous work in our lab to synthesize five novel -protected hydrazino amino acids. These hydrazines are essentia l precursors for synthe sizing constrained strand mimetics. We showed that we could selectively deprotect the -nitrogen of the hydrazines, and we synthesized se veral novel examples of polar -protected hydrazino amino acids. Chapter Three discusses the de sign and synthesis of small-molecule and peptidomimetic MDM2 inhibitors, including our work on synthesizing a new class of helix mimics that have improved water solub ility compared with previously reported

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xiv examples of -helix mimics. As with the constrained -strand mimics described in Chapter Two, the synthesis of novel hydrazino amino acid precursors is a key step in synthesizing our -helix mimics. One isoleucine hydr azine derivative wa s synthesized, and progress was made toward synthesizing two other hydrazines from tryptophan. In addition, the synthesis of three potential small-molecule inhibitors of MDM2 is described. Chapter Four describes the use of the GLIDE program to design and evolve an -helix mimic that will intera ct with the pro-apoptotic pr otein Bax. Progress toward the synthesis of this compound is also reported.

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1 CHAPTER ONE GENERAL INTRODUCTION: PROTEI NS AND THEIR INTERACTIONS 1.1 Proteins, Peptides, and Peptidomimetics Proteins and peptides ar e biopolymers comprised of monomers called amino acids. There are twenty natu rally occurring amino acids, and they are linked together by amide bonds to form linear chains. Shorter ch ains of less than 40 amino acids are called peptides, while longer chains are ca lled polypeptides or proteins ( 1 ). The linear amino acid sequence of a protei n is called its primary structure. A protein’s primary structure is ultimately res ponsible for the structure and function of that protein. However, proteins are also organi zed on additional levels based on the local and global folding of the polypeptide chain. Lo cal folding gives rise to the protein’s secondary structures and occurs due to hydrogen bonding among amino acids. Two common secondary structures are the -helix and the -sheet ( 1 ). An -helix is a spiral shape that o ccurs due to hydrogen bonding between the carbonyl group of one amino acid and the am ine group of another amino acid located four positions up the chain. The hydrogen bonds ar e oriented parallel to the axis of the helix, while the side chains of the amino aci ds point out approximately perpendicularly. See Figure 1.1A below ( 1 ). Beta sheets resemble a pleated piece of paper, and are also held together by hydrogen bonds between th e carbonyl and amino groups of different amino acids. The hydrogen bonds lie within the plan e of the pleats, while the amino acid

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2 side chains are oriented perpendicular to the peptide backbone both above and below the plane of the pleats. See Figure 1.1B ( 1 ). Figure 1.1A: Structure of an Alpha Helix ( 1 ). Hydrogen bonds in an helix are parallel to the helical axis, while the amino acid side chains are oriented perpendicularly. Figure 1.1B: Structure of a Beta Sheet ( 1 ). The peptide backbone forms the pleated sheet, while the amino acid side chains stick out above and below it. Proteins also have a global folding conf ormation that comprises their tertiary structure. The -helices, -sheets, and other local foldin g patterns interact with one another to form the overall shape of the protein ( 1 ). Although infinite possible protein conformations are possible, each protein must be folded correctly in order to be capable of performing its biological function. This function often entails in teraction with other proteins ( 2 ). The importance of protein-protein interactions has only recently become appreciated, along with the possibi lities for interfering with pr otein-protein interactions in order to probe biological system s and develop new therapeutics ( 3 ). There are several ways in which protein-pr otein interactions mi ght be prevented. One obvious possibility is to screen for or design a small organic molecule inhibitor. Small molecule inhibitors are often effectiv e in cases where the binding residues of the

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3 protein are clustered together or ther e is a known natural product inhibitor ( 3 ). However, since the size of a protein is very large compared with a typical natural product or synthetic organic inhibitor, it may be difficu lt to block the entire protein surface by using small molecules. There is also the possibi lity that there may be no binding pocket on the surface of a flat protein to which a small or ganic inhibitor could bind; the surface of many proteins is flat ( 3 ). A second possibility is to use inhibitors that are actual peptid es with the same sequence as the portions of the protein that interact with ot her proteins. Such peptides serve as competitive inhibitors of the proteins whose binding sites they resemble. This strategy was successfully used to inhibit severa l examples of protein-protein interactions, including HIV-1 protease dimerization, herp es simplex virus ribonucleotide reductase dimerization, and p53/HDM2 interactions ( 3 ). However, this stra tegy tends not to work well in cases where amino acid residues fr om noncontiguous parts of the protein’s primary sequence must come together to fo rm an epitope that is then capable of interacting with another protein ( 3 ). In addition, peptides te nd to have limitations in terms of poor bioavailability and meta bolic stability in living organisms ( 3 ), as well as poor pharmacokinetics and potential side e ffects due to a lack of selectivity ( 4 ). However, the peptide-based strategy is not limited merely to making an exact copy of the binding peptide portion of the protein of interest. The chemical and biological properties of inhibitory peptides can be modified by incorporating non-natural amino acids into their sequence, or by even dispensing with the peptide’s amide-based backbone altogether ( 3 ). Such modifications give rise to yet another possible source of inhibitors called peptidomimetics.

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4 Peptidomimetics have peptide-like structur es and activities. However, since they do not have actual peptide bonds, the pharmacoki netic properties of peptidomimetics are modified. This allows peptidomimetics to interact with receptors and enzymes analogously to their parent pe ptides while avoiding many of the problems that arise from using actual peptides ( 4 ). One of the best-known exam ples of a peptidomimetic is morphine, an opiate receptor agonist ( 4 ). Unlike most peptide-pe ptidomimetic pairs, the peptidomimetic morphine was known long befo re the natural enkepha lin peptide ligands of the opiate receptors were discovered ( 4 ). The structures of Leu-enkephalin and morphine are shown in Figure 1.2. OH NH2NH O NH O NH O NH O COOH Leu-enkephalinOH O OH N morphine Figure 1.2: Leu-Enkephalin, a Natural Pept ide Ligand of Opiate Receptors, and Morphine, an Opiate Receptor Peptidomimetic ( 4 ). The analgesic activity of morphine has been known for over 2000 years, but the natural peptid e enkephalin ligands were not discovered until the 1970s. Unlike morphine, most peptidomimetics are purposefully designed based on the structure of their parent peptides ( 4 ). Farmer’s Rules descri be a systematic method for converting a peptide into a peptidomimetic ( 4 ). These rules include using the simplest possible active portion of the peptide (t he pharmacophore), maintaining the steric parameters of the parent peptide, beginni ng with a flexible molecule and then progressively constraining it through severa l rounds of structureactivity relationship

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5 studies, and mimicking the three-dimensional sh ape of the parent pe ptide, particularly with regard to -helices and -sheets ( 4 ). This dissertation presents the theory a nd practice of design ing, synthesizing and using peptidomimetics to disrupt protein-protein interactions. In general, our strategy for doing this is to design and synthesize pepti domimetics that will mimic peptide secondary structures ( -helices and -sheets). The remaining por tion of Chapter One is a theoretical examination of the feasibility of using -sheet mimics called molecular clamps to inhibit substrate-receptor interactio ns by blocking the substrate rather than the receptor or enzyme. Several natural and synthe tic examples of this approach are given in support of this concept, and a kinetic mode ling study is presented. Chapter Two relates a continuation of previous work in our lab to synthesize -protected hydrazino amino acids. These hydrazines are essential precursors for synthesizing constrained -strand mimetics. Chapter Three summarizes the cu rrent literature concer ning the design and synthesis of -helix mimics, to block the interac tion between p53 and MDM2. It also describes our work on synt hesizing a new class of -helix mimics that have improved water-solubility compared with previously reported examples of -helix mimics. As with the constrained -strand mimics described in Chapter Two, the synthesis of hydrazino amino acid precursors is a key step in synthesizing our -helix mimics. Chapter Four describes the use of molecular modeling and docking software to design an -helix mimic that will interact with the pro-apoptotic protein Bax. Also, the ongoing synthesis of one compound that showed promis e in the computer screening is reported.

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6 1.2 Molecular Clamps: Background The canonical strategy for blocking en zyme-substrate or receptor-ligand interactions is to screen fo r or design a small molecule or peptide that can bind to the enzyme or receptor in such a way as to inhib it that enzyme or receptor. This inhibition can either be competitive, where it is target ed to the active site, or noncompetitive, where it is targeted to a separate allosteric site. Both types of inhibition share the same general strategy, however, which is to prevent the enzyme or receptor from conducting its normal activity. This strategy has lead to multiple successes in drug discovery and continues to do so, as exemplified by recent reports of reti noic acid analogs that inhibit retinoic acid receptors ( 5 ) and inhibitors of cyc lin-dependent kinases ( 6 ). However, this strategy is not suitable in all cases, because many enzymes catalyze reactions on more than one substrate. Blocking such enzymes leads to the undesired effect of blocking other pathways that ma y not be involved in the mechanism whose modulation is desired. One recent example of the potential bad outcome due to the inhibition of promiscuous enzymes is farnes yltransferase and gera nylgeranyltransferase inhibitors intended to prev ent prenylation of K-Ras ( 7) Inhibition of K-Ras prenylation is desirable for cancer therapy; however, us e of farnesyltransferase-specific inhibitors will not prevent prenylation of K-Ras by geranylgeranyltransferase, and use of dualpurpose prenylation inhibitors is toxic to the point of complete lethality ( 7 ). One possible way to solve this problem is to take the complementary approach, which entails having the inhib itor bind to the substrate instead of to the enzyme or receptor. Rather than having the inhibitor compete with the substrate for the enzyme’s active or allosteric site, the inhibitor competes with the enzyme to bind the substrate,

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7 analogous to the way antibodies recognize and neutralize their antigens ( 8 ). Thus, the substrate binds to the inhibito r, and this prevents that pa rticular substrate from being acted upon by the enzyme. However, the enzyme is left free to continue to perform its functions on all ot her substrates ( 8 ). Figure 1.3 is a schematic comparing the effects on a dual-substrate enzyme of using no inhibitor (top panel), a traditional enzyme-targeted inhibitor (middle panel), or a substrat e-targeted inhibitor (bottom panel). Figure 1.3: Schematic of the Substrate-Targeted Inhibitor Concept ( 8 ). The top panel shows two substrates (squi ggles) interacting with the same enzyme (block) in the absence of inhibitor. The middle panel show s an inhibitor (triangle) that targets the enzyme, thereby blocking all functions of that enzyme. The bottom panel shows an inhibitor (boomerang) that targets one substrat e (left squiggle), leaving the enzyme free to continue catalyzing its reaction with the second substrate (right squiggle).

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8 There is good reason to believe that this approach of inhibiting the substrates rather than the enzymes or receptors would be successful based on the prevalence of both natural and synthetic examples in the literatu re. Some of the natural precedents include not only antibodies, but also antibiotics in the vancomycin family. The vancomycin group antibiotics are cell wall synthesis inhibi tors in bacteria, but their mechanism of action is quite different from that of the peni cillins, which also inhibit cell wall growth ( 9 ). The penicillins accomplish this task by binding to and inhibiting the penicillinbinding proteins (PBPs), which are a group of enzymes required by the bacterium to synthesize its cell wall ( 10 ). Thus, the penicillins are examples of enzyme-targeted inhibitors, as shown in the middle panel of Figure 3. The vancomycin group antibiotics, in contrast, complex with the peptidoglycan precursors needed to synthesize the bacterial cell wall, but do not affect the PBP enzymes ( 9 ). Thus, they function analogously to the substrate-targeted inhibitor por trayed in the bottom panel of Figure 3. The fundamentally different strategies for inhibiting cell wall synthesis by the penicillins versus the vancomycins explains why bacteria that have developed resistance to the penicillins may still be susceptible to the vancomycins, and vice versa ( 9 ). The use of antibodies themselves in th is context is an obvious approach, and several examples of antibodi es are in clinical use ( 11 ). While therapeutic use of antibodies has multiple advantages, including hi gh specificity, long half-life, and lack of resistance, antibodies also have serious disadvantages in th at they can be immunogenic or toxic, and they have limited ability to cro ss the blood-brain barrier or penetrate solid tumors ( 11 12 ). Some of these problems may be solved through the use of human recombinant antibodies rather than murine ones ( 11 12 ). In addition, several approaches

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9 using small molecules rather than antibodies have been published ( 8 ). However, small molecules have problems of their own, one of wh ich is that they have fewer interactions with the substrate as compared to polypeptides ( 8 ). Thus, several techniques have been developed that attempt to remedy this by mi micking antibody-antigen interactions and creating a multivalent inhibito r capable of recognizing multiple epitopes on the target substrate protein ( 8 ). One such technique involves using double synthetic peptides linked together to form “molecular forceps” that were shown to be specific for the carboxy terminus of hRas ( 8,13) These forceps consisted of two tetrap eptides connected by either a flexible or a rigid linker ( 13 ). The peptides on each arm of the forceps were identical, increasing the collective binding affinity of the conglomerate for th e substrate analogous to an antibody’s two identical epitope-binding sites ( 8 ). Large numbers of potential forceps were synthesized and screened using combin atorial chemistry procedures previously developed ( 13 ). In addition, active forceps were found to contain multiple Lys residues, either as part of the linker or as part of the forceps sequence ( 13 ). Testing for binding of other Ras sequences showed that the molecula r forceps were specific for the particular hRas sequence; other substrates continued to be farnesylated in the presence of the forceps ( 13 ). It was found that binding resoluti on as sensitive as a single amino acid could be obtained using molecular forc eps, precluding the need for complex macromolecular structures ( 13 ). However, the IC50 values for the best molecular forceps were on the order of 100 M, and improvement in their affinity is necessary before they could be used in a therapeutic context ( 13 ). The authors also were concerned that their forceps might not be capable of penetrating cell membranes ( 13 ).

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10 A second group used a similar approach where multiple identical pentapeptides were attached to library beads, which were then used to screen for a dopamine D2 receptor target sequence that was attached to magnetic beads ( 8,14) This approach had the additional advantage that the bound inhi bitor-substrate complexes could be easily isolated using a magnet to pull them to th e side of the tube, while unbound ligand beads would simply settle to the bottom ( 8 ). This was the first example where small peptide ligands were found that bind to a small peptide target molecule ( 14 ). Binding affinity was better for this bead-to-bead technique, with the best Kd values on the order of 100 nM ( 14 ). Molecular clamps (MCs) function slightly differently than these previously reported strategies. They prevent substrates from being posttranslationally modified by binding only to the specific substrate sequence upon which an enzyme acts, thus preventing the active site of the enzyme from be ing able to interact with and catalyze that substrate. Each MC consists of 12 ami no acid residues connected by a peptidomimetic linker. Functioning as a –turn mimic, the MC itself undergoes a change in conformation from a random coil when unbound, to form a small, stabile –sheet upon binding the target sequence. MCs can be designed to ha ve affinity for any known substrate site of posttranslational modification. The approach has the added advantage of only requiring knowledge of the enzyme’s activity and the s ubstrate sequence that the enzyme modifies, but not the structure of the enzyme or its ac tive site. A schematic of the mechanism of MC activity is shown in Figure 1.4. The remainder of this chapter describes the applications and considerations n eeded to use the MC strategy.

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11 ksub 1k s u b -1molecular clamp ksub 2ksub -2 substrate sequence A B C Figure 1.4: Schematic of the Bi nding of a Substrate by a MC The substrate protein has multiple sequences of amino acids, but only one sequence that is specific for the MC ( A ). Binding of the MC to the substrate to form a three-stranded -sheet occurs in a twostep process, where first one side of the clamp binds ( B ), followed by rapid binding of the second side ( C ). 1.3 Molecular Clamps: A Model Classical enzyme kinetics systems are comprised of an enzyme (E) and a substrate (S). The enzyme and substrate react to form an enzyme-substrate complex (ES). The ES complex can either dissociate back to enzy me and substrate, or it can go on to form the product (P). Km, the Michaelis-Menton constant, describes the propensity for ES complex dissociation to occur. If an enzy me-targeted inhibitor (I) is present in the system, it will react with the enzyme to form an enzyme-inhibitor (EI) complex, thereby tying up some of the enzyme and giving rise to an EI dissociation constant called Ki. All of these relationships are shown in Figure 1.5. The Michaelis-Menton equation describes the rate (v) at which an enzymatic reaction will occur for a given concentration of substrate: Vmax[S] Km + [S] v = The reaction rate also depends on the maximum rate at which the enzyme can catalyze the reaction (Vmax), as well as on Km.

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12KiE + S E + P + I E S E I Km Figure 1.5: Dissociation Constants for a Cla ssical System with an Enzyme-Targeted Inhibitor. Ki is the dissociation constant for the enzyme-inhibitor complex, and Km is the dissociation constant for the enzyme-substrate complex. See the text for the definitions of the remaining variables. However, the presence of a substrate-targeted inhibito r like the MC complicates this scenario considerably. This is because the enzyme can only act upon the free substrate ([S]free), but the MC would sequester some of the substrate and make it unavailable for binding. The enzyme binds to the remaini ng free substrate similarly to the previous scenario. However, rather th an binding to the enzyme like the classical inhibitor does, the MC binds directly to th e substrate instead, giving rise to a new dissociation constant (KMC). These relationships are shown in Figure 1.6. KMCE + S E + P + MC E S MC S Km Figure 1.6: Dissociation Constants for a MC Substrate-Targeted Inhibitor. Km is the dissociation constant for the enzyme-substrate complex, and KMC is the dissociation constant for the inhibitor-substrate complex. See the text for the definitions of the remaining variables.

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13 The total amount of substrate is equal to the sum of the free substrate and the MCsubstrate complex: [S]total = [S]free + [S•MC] The concentration of free substrate depends on the concentration of MC, as well as on the binding affinity of the MC for the substrate: [S]free = [{([MC]total [S]total + KMC)2 + 4KMC[S]total}0.5 ([MC]total [S]total + KMC)] Since only the free substrate is availabl e to be catalyzed by the enzyme, [S]free must be substituted for [S] in the Michaelis-Menton equation: Vmax[S] Km + [S]freev = free Substituting the formula for [S]free into the Michaelis-Menton equation gives a revised version of the Michaelis-Menton equation that can be used to study the kinetics of a system comprised of an enzyme, a substr ate, and a MC. This equation was then manipulated to study the effects of the MC on enzyme kinetics. There are four potential variables that can be studied usi ng this model: the concentration of substrate ([S]), the concen tration of MC inhibitor ([MC]), the binding constant of the enzyme for the substrate (Km), and the binding constant of the MC for the substrate (KMC). Changing each of these variables affects the expected outcome of the model in predictable and logica l ways. In each graph, the reac tion velocity was plotted as the percentage of the actual velocity compared to the maximum velocity. If the concentration of substrate is incr eased beyond a certain point, it is expected that the ability of the MC to sequester the substrate would be overpowered; this conclusion is supported by the model. See Fi gure 1.7A. When all other variables are

PAGE 31

14 held constant, a high enough concentration of substrate (> 1 mM) will allow the enzyme to reach its maximum velocity, a ssuming that the enzyme has a Km in the low micromolar range and the MC is al so present in a low micromolar concentration. The model suggests that once the substrate and MC are present in approximately a 1:1 ratio, the usefulness of the MC begins to diminish. This is true even in a scenario like th e one shown in Figure 1.7A, where the MC has a much higher binding a ffinity for the substrate than the enzyme does. In contrast, if the [MC] is at least an order of magnitude greater than the total [S], then the MC will be effective at removing most or all of the free substrate from the system and inhibiting formation of the ES complex. Effect of Substrate Concentration on Relative Enzyme Velocity0 10 20 30 40 50 60 70 80 90 100 1E-15 1E-13 1E-11 0.00000000 1 0.0000001 0.00001 0.001 0.1 Total [S]% Vmax Figure 1.7A: Effect of Substrate Concentr ation on Relative Enzyme Velocity. A sufficient concentration of substrate will overpower the MC. [MC] = 5 M, Km = 30 M, KMC = 50 nM.

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15 Effect of Molecular Clamp Concentration on Relative Enzyme Velocity0 10 20 30 40 50 60 70 80 90 100 0.00E+001.00E-062.00E-063.00E-064.00E-065.00E-066.00E-06 [MC]% Vmax Figure 1.7B: Effect of Molecular Clamp Concentration on Relative Enzyme Velocity. A sufficient concentration of MC will sequester the substrate and prevent catalysis. [S] = 1 nM, Km = 30 M, KMC = 50 nM. Increasing the concentrati on of MC was predicted to decrease the relative enzyme velocity, and this was indeed found to be the case. If the s ubstrate/MC ratio is approximately 1:1, inhibition is relatively lo w, and the enzyme reaction velocity is relatively close to normal. It is not until the [MC] gets ab out three orders of magnitude higher than [S] that catalysis is essentially blocked. Again, this scenario assumes that the MC has a higher affinity for the substrate than the enzyme does. The activity of an enzyme that binds tightly to its substrates is considerably less affected by the presence of the MC compared with an enzyme that does not, assuming that excessive concentration of substrate is not present. This can clearly be seen by comparing the velocities of se veral enzymes with different Km values while holding the

PAGE 33

16 concentrations of substrate and MC and the KMC constant. If the Km of the enzyme were sub-nanomolar, the MC would likely not be able to effectively compete with it, as noted previously, unless its own bi nding constant was also sub-nanomolar. See Figure 1.7C. Effect of Km Value on Relative Enzyme Velocity0 10 20 30 40 50 60 70 80 90 100 1E-15 1E-13 1E-11 1E-09 0.0000001 0.00001 0.001 0.1 Km% Vman Figure 1.7C: Effect of Km Value on Relative Enzyme Velocity. As the enzyme’s affinity for the substrate increases, it is able to out-compete the MC for the substrate. [MC] = 5 M, [S] = 1 nM, KMC = 50 nM Finally, the binding affinity of the MC al so plays an important role, with the effectiveness of the inhibition increasing as the affinity of the MC for the substrate increased when all other factors were held constant. A molecular clamp present at high nM concentrations with a KMC of 1 mM was not able to a ffect the rate of substrate catalysis by the enzyme. Similarly, the MC with a KMC of 1 M had only a minimal effect. However, when the KMC dropped to 1 nM, catalysis was dramatically decreased

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17 even at these low concentr ations of inhibitor; a KMC of 1 pM essentially prevented catalysis altogether, as shown in Figure 1.7D. These results were also in line with our expectations. Effect of KMC Value on Relative Enzyme Velocity0 10 20 30 40 50 60 70 80 90 100 1.00E-121.00E-111.00E-101.00E-091.00E-081.00E-071.00E-061.00E-051.00E-041.00E-03 KMC (M)% vmax Figure 1.7D: Effect of KMC Value on Relative Enzyme Velocity. As the MC’s binding affinity improves, the MC becomes more ef fective at sequestering the substrate and preventing catalysis. [S] = 1 nM, Km = 30 M, [MC] = 95.9 nM. Based on these model results, it is reas onable to conclude that MCs would be most useful in systems where the concentration of the substrate is low, the affinity of the enzyme for the substrate is low (high Km), and the affinity of the MC for the substrate is high (low KMC). It is also beneficial to raise th e concentration of MC as high as is feasible within pharmacokinetic and toxicologi cal limits. Consideration must be given to the concentration of the substrate that is naturally present in cells in order to select the

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18 substrate candidates likeliest to be successfully inhibited using MCs. If the substrate to be inhibited is normally present at low concen trations, then the amount of MC needed to inhibit it will also be low, and within an ach ievable range. However, if the substrate is present in large amounts in cells, then the co ncentration of MC needed to inhibit that substrate may be prohibitively high or toxic. Thus, substrates present in large amounts are not likely to be good candi dates for inhibition by MCs. Since MCs are essentially peptides w ith a small peptidomimetic region, one possible problem that might limit their use is membrane impermeability. Proteins and peptides are not generally able to penetrate cell membranes. However, it is also known that there are various techniques that can be us ed to allow peptides to enter cells, and that certain peptide sequences are themselves r eadily transferred across cell membranes along with any proteins attached to them ( 12 16,17 ). The most important feature of these peptides appears to be the inclusion of multiple basic re sidues in them, particularly arginine ( 16,17) Such guanidine-containing sequences could easily be incorporated into MCs as well, facilitating their uptake into cells. Successful penetration of fusion proteins has already been demonstrated with a number of other peptide examples ( 17 ). Membrane permeability can also be increased through palmitoylation, myristoylation, or other addition of long-chain hydr ophobic groups to the peptide (18 ). There is also the issue of peptide stability to proteases. As with the issue of cell membrane penetration, chemical modifica tion of the MC by conjugating it with a polymer is one possibility ( 19 ). Such conjugation has the added advantage of reducing the immunogenicity of the peptide ( 19 ). It is also possible to increase the peptidomimetic character of the MCs as needed if degradation of its peptid e bonds is a major problem.

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19 Finally, the biochemistry and pharmaco logy of MCs are not known. Since the intracellular MC-bound substrate will not necessari ly be degraded, such substrates could function as undesired inhi bitors of other metabolic pathwa ys. It is also unknown whether MCs will be toxic to the kidneys or other organs where they might accumulate. These questions require answers before the MC strategy could be us ed in a clinical scenario. In conclusion, we have considered the pros and cons of using MCs as drugs, as well as established some guidelines to de termine which enzymes would be the best candidates for the use of MCs. We also pe rformed some virtual kinetics studies on a hypothetical system where we varied the MC concentration, the s ubstrate concentration, the enzyme-substrate dissociation constant, a nd the MC-substrate dissociation constant to see how each of these changes would affect th e usefulness of the MC. Future work on this project would involve designing and s ynthesizing some actual MCs that target a carefully selected substrate, as well as performing biol ogical tests to increase our knowledge of the behavior of MC s with respect to th eir ability to penetrate cells and their pharmacological properties. 1.3 References 1. Garrett, R. H. and Grisham, C. M. Biochemistry, 2nd Ed. ; Saunders College Publishing: Fort Worth, 1999. 2. Branden, C. and Tooze, J. Introduction to Protein Structure, 2nd Ed. ; Garland Publishing: New York City, 1999. 3. Thorsten, B. (2003) Modulati on of Protein-Protein Inter actions with Small Organic Molecules, Angewandte Chemie, Int. Ed. 42, 2462-2481. 4. Luthman, K. and Hacksell, U. Peptides and Peptidomimetics. In Textbook of Drug Design and Discovery, 3rd Ed. ; Krogsgaard-Larsen, P., Liljefors, T. and Madsen, U., Ed.; Taylor and Franci s: New York, 2002; pp. 459-485.

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20 5. Patel, J. B.; Huynh, C. K.; Handratta, V. D.; Gediya, L. K.; Brodie, A. M. H.; Goloubeva, O. G.; Clement, O. O.; Nanne, I. P.; Soprano, D. R.; and Njar, V. C. O. (2004) Novel Retinoic Acid Metabolism Bl ocking Agents Endowed with Multiple Biological Activities Are Efficient Growth Inhibitors of Human Breast and Prostate Cancer Cells in Vitro and a Human Breast Tumor Xenograft in Nude Mice, J. Med. Chem. ASAP article 6. Markwalder, J. A.; Arnone, M. R.; Benfield, P. A.; Boisclair, M.; Burton, C. R.; Chang, C. H.; Cox, S. S.; Czerniak, P. M.; Dean, C. L.; Doleniak, D.; Grafstrom, R.; Harrison, B. A.; Kaltenbach, R. F.; Nugiel, D. A.; Rossi, K. A.; Sherk, S. R.; Sisk, L. M.; Stouten, P.; Trainor, G. L.; Worland, P. ; and Seitz, S. P. (2004) Synthesis and Biological Evaluation of 1-Aryl-4, 5-dihydro-1 H -pyrazolo[3,4d ]pyrimidin-4-one Inhibitors of Cyclin-Dependent Kinases. J. Med. Chem., 47 5894-5911. 7. deSolms, S. J.; Ciccarone, T. M.; MacTough, S. C.; Shaw, A. W.; Buser, C. A.; EllisHutchings, M.; Fernandes, C.; Hamilton, K. A. ; Huber, H. E.; Kohl, N. E.; Lobell, R. E.; Robinson, R. G.,; Tsou, N. N.; Walsh, E. S.; Graham, S. L.; Beese, L. S.; and Taylor, J. S. (2003) Dual Protein Farnes yltransferase-Geranylgeranyltransferase I Inhibitors as Potential Can cer Chemotherapeutic Agents, J. Med. Chem. 46 29732984. 8. Kodadek, T. (2002) Inhibition of Pr oteolysis and Other Posttranslational Modifications with Substr ate-Targeted Inhibitors, Biopolymers 66 134-140. 9. Barna, J. C. J. and Williams, D. H. (1984) The Structure and Mode of Action of Glycopeptide Antibiotics of the Vancomycin Group, Annu. Rev. Microbiol., 38, 339357. 10. Waxman, D. J. and Strominger, J. L. (1983) Penicillin-Binding Proteins and the Mechanism of Action of -Lactam Antibiotics, Ann. Rev. Biochem., 52, 825-869. 11. Roskos, L. K.; Davis, C. G.; and Schwa b, G. M. (2004) The Clinical Pharmacology of Therapeutic Monoclonal Antibodies, Drug Development Research, 61 108-120. 12. Sanz, L.; Blanco, B.; and Alverez-Vallina, L. (2004) Antibodies and Gene Therapy: Teaching Old “Magic Bullets” New Tricks, Trends in Immunology, 25 85-91. 13. Dong, D. L.; Liu, R.; Sherlock, R.; Wigler, M. H.; and Nestler, H. P. (1999) Molecular forceps from combinatorial librari es prevent the farnes ylation of Ras by binding to its carboxyl terminus, Chemistry & Biology, 6 133-141. 14. Sasaki, S.; Takagi, M.; Tana ka, Y.; and Maeda, M. (1996) A New Application of a Peptide Library to Identify Selective Interaction Between Small Peptides in an Attempt to Develop Recognition Molecules Toward Protein Surfaces, Tetrahedron Letters, 37 85-88.

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21 15. Segel, I.H., Enzyme Kinetics John Wiley: New York, 1975. 16. Lundberg, P. and Langel, U. (2003) A br ief introduction to cellpenetrating peptides, J. Molecular Recognition, 16 227-233. 17. Tung, C. H. and Weissleder, R. (2003) Arginine containing peptides as delivery vectors, Advanced Drug Delivery Reviews, 55 281-294. 18. Martin, M. L. and Busconi, L. (2000) Membrane localization of a rice calciumdependent protein kinase (CDP K) is mediated by myrist oylation and palmitoylation, The Plant Journal, 24 429-435. 19. Torchilin, V. P. and Lukyanov, A. N. (2003) Peptide and protei n drug delivery to and into tumors: challenges and solutions, Drug Discovery Today, 8 259-286.

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22 CHAPTER TWO SYNTHESIS OF POLAR AND NONP OLAR HYDRAZINO AMINO ACIDS 2.1 Introduction 2.1.1 Hydrazines Hydrazines are compounds that contain a nitrogen-nitrogen single bond. The first hydrazine synthesis to be reported in the ch emical literature was for phenylhydrazine in 1875 by Emil Fischer ( 1 ). Ordinary hydrazine was synt hesized about a decade later by Theodor Curtius ( 1 ). The physical, chemical, and bi ological properties of hydrazine and its derivatives are well documented. Thes e compounds are surprisingly common in nature, as well as from synthetic sources such as agriculture, industr y, military and space vehicle fuels, and even medicinal drugs ( 1 ). The use of over thirty hydrazi nes as drugs is especially interesting in light of the fact that the majority of hydraz ines studied thus far are known to be toxic, teratogenic, and carcinogenic ( 1,2 ). However, it is not unprecedented for antineoplastic agents to also be carcinogens. Mustard gas, alkyla ting compounds, cyclophosphamide, and several other antineoplastic agents are also known to both i nduce and inhibit cancer ( 2 ). The exact mechanism of action of the antineopl astic hydrazines is not known, but they are postulated to be cytotoxic, interfere with glycolysis and gluc oneogenesis, chelate essential trace metals, inhibit mitosis, and/or degrade DNA ( 2 ).

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23 Most hydrazine-containing drugs are hetero cyclic compounds, and they exhibit a wide range of activities ( 1 ). A sample of hydrazine-con taining drugs includes several anti-hypertensives (pheniprazin e, hydralazine), anti-depress ants (nialamide, marplan), cancer chemotherapeutics (pro carbazine and proresid, along with hydrazine itself), Parkinson’s disease chemothera peutics (carbidopa), anti-vira l drugs (methiazone), antituberculotics (isoniazid, phtiv azid), and antibiotics (sulfa phenazole, sulfamethizole), among others ( 1,2 ). Hydrazine derivatives have also been te sted as inhibitors of enzymes that metabolize -amino acids ( 3,4 ). Some of these compounds have been shown to be potent, specific inhibitors of su ch enzymes in bacteria, giving rise to a class of hydrazines with antimicrobial activities. One well-studi ed example is a class of diaminopimelic acid (DAP) analogs. These compounds were foun d to inhibit the enzyme meso-DAP Ddehydrogenase in B. sphaericus and the enzyme LL-DAP epimerase in E. coli thereby preventing these bacteria from synthesizing the amino acid lysine and interfering with their cell wall synthesis ( 4 ). More recently, a series of hydrazine-containi ng peptides were reported that inhibit the enzyme DAP aminotransferase in E. coli ( 5 ). The authors were able to observe good in vivo antimicrobial activity from some of th eir compounds when they coupled their hydrazines to normal amino acids to make short peptides. They speculated that the presence of the natural amino acids would a llow their compounds to be transported into the cells as peptide prodrugs that would th en be cleaved intracellularly via a peptide transporter. However, they found that some of the transportable pept ides were not active,

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24 which suggests that the regular amino acids may actually play a role in the inhibitory properties of the active compounds ( 5 ). 2.1.2 Synthesis of Hydrazines There are several methods to synthesize hyd razines that have been reported in the literature. Before 1970, there were basica lly three methods that had been reported: reduction of the hydrazone of an -keto acid, functionalization of a carbonyl compound (analogous to the Strecker synthesis) and reaction of hydrazine with an -halo acid ( 6 ). Reduction of hydrazones was ach ieved using sodium and mercury to give the free hydrazine ( 7 ). This procedure is shown in Scheme 2.1. The Strecker-like synthesis, shown in Scheme 2.2, involves the use of hydr azine and potassium cyanide to give the cyanohydrazine. This can then be hydrolyzed to the hydrazino acid ( 6 ). N H O OH O N H OH O NHNH2 Na(Hg) NH2NH2N H NNH2O O NH2NH3 Scheme 2.1: Reduction of a Hydrazone to a Hydrazine ( 7 ). This procedure gives the racemate of the hydrazine. O HO O NH2NH2KCN O HO NHNH2CN O HO NHNH2COOH H3O Scheme 2.2: Strecker-Like Synt hesis of a Hydrazino Acid ( 6 ). This procedure also gives the racemate of the hydrazine.

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25 These two methods could not be used to form optically pure isomers; only the reaction of hydrazine with an -halo acid procedure was useful in synthesizing hydrazino acids ( 6 ). This method in volves reacting the -hydrazino acid with hydrazine via a bimolecular nucleophilic substitution reaction. Since the -carbon is already functionalized in this case, an opti cally active product can be obtained ( 8, 9 ). Two examples of this reaction are shown in Scheme 2.3. N N COOH Cl H NH2NH2N N COOH H NHNH2ZHN COOH Br NH2NH2ZHN COOH NHNH2 Ref. 8 Ref. 9 Scheme 2.3: Reaction of Hydrazine with an -Halo Acid (8,9). This procedure can be used to obtain an optically active hydrazine. Z denot es the Cbz protecting group. Subsequently, it was discovered that hyda ntoic acids could be rearranged into hydrazines using sodium hypochlorite ( 10 ). Substituted hydroxylamines had been used previously to form nitrogen-nitrogen bonds, but since excess amine was required, the purification of the products was difficult ( 10 ). The synthesis route involved reaction of the amino acid with potassium cyanate to form the hydantoic acid. The hydantoic acid

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26 was then reacted with sodium hypo chlorite to form the hydrazine ( 10 ). However, the yield of the rearrangement reaction wa s relatively low, between 20-30% ( 11 ). More recently, it was found that using potassium hypochlorite instead of sodium hypochlorite could raise the yield of this method to around 70% ( 11 ). The synthesis of hydrazines via rearrangement of hydantoic acids is depicted in Scheme 2.4. O HO NH2COOH KCNO O HO NH COOHNH2O NaOCl O HO NHNH2COOH Scheme 2.4: Synthesis of a Hydrazine vi a Rearrangement of a Hydantoic Acid (10). This procedure gives an optically pure product. These authors had also attempted to make hydrazines via two other methods ( 10 ). One procedure that th ey tried was to reac t an amino acid with hydroxylamine-O-sulfonic acid, which theoretically should have given the hydrazino acid directly ( 10 ). However, they were not able to separate the hydrazine product from the amine starting material; this was a major problem because the parent amine had to be used in excess ( 10 ), as shown in Scheme 2.5. They also tried N-aminating an optically active ace tylated amino cyanide ( 10 ). This approach was successful, givi ng the optically active acetylated hydrazino cyanide, which could then be hyd rolyzed to the hydrazino acid ( 10 ). This reaction is shown in Scheme 2.6. Another group found that they were able to synthesize opt ically active hydrazines from 2-(((4-nitrobenzene)sulfonyl)oxy) (nosylo xy) esters via a bimolecular nucleophilic substitution mechanism ( 12 ). The nosyloxy compounds were made by reacting 2-

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27 hydroxy esters with nosyloxy chlo ride, followed with Boc-prot ected hydrazine, to give the Boc-protected hydrazine ( 12 ). This procedure is shown in Scheme 2.7. NH2OSO3Na O HO NH2COOH XO HO NHNH2COOH Scheme 2.5: Attempt to Synthesize a Hy drazine Using a Hydroxylamine Derivative (10). The hydrazine product was obtained, but it could not be readily purified from the parent amine. O HO NH CNO NaHO HO N CNO ClNH2 O HO NNH2CNO HClO HO NHNH2COOH Scheme 2.6: Synthesis of a Hydrazine Via N-Amination (10). This method gives an optically active product. Several groups successfully synthesized optically active hydrazines via asymmetric electrophilic amination ( 13-16 ). This is a complementary approach to several of the previous procedures describe d where the hydrazine or its synthon served as the nucleophile ( 14 ). In contrast, asymmetric elec trophilic amination uses the carbon skeleton as the nucleophile and the nitrog en-containing compound as the electrophile

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28 ( 13-16 ). The electroph ilic hydrazine synthon used was ditert -butyl azodicarboxylate (DBAD) ( 13,14 ); this particular azodicarboxylate wa s chosen because it was found that a bulkier alkyl group improves the dias teroselectivity of the reaction ( 15 ). In addition, DBAD is stable and commercially available, and the tert -butoxy group can be removed from the hydrazine by conditions that will not affect the N-N hydrazine bond ( 16 ). Some examples of hydrazine formation via electr ophilic amination are shown in Scheme 2.8. CO2CH3OH NsCl TEA CO2CH3ONs BocNHNH2MeCN CO2CH3NHNHBoc Scheme 2.7: Synthesis of a Hydrazine from a Nosyloxy Ester (12). The nosyloxy esters are made from chiral hydroxy esters. PhO NMe2OSiMe3R TiCl4O O N O O N DCM PhO NMe2O R N N H Boc Boc Ref. 13N O O O R Bn N O O O R Bn BocNH-BocN O O N O O N LiNR21. 2. Ref. 14 Scheme 2.8: Hydrazine Formation Via Asymmetric Electrophilic Amination (13, 14). This procedure is complementary to previo us procedures that used hydrazine as the nucleophile.

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29 A few groups have also synthesized hydrazines by reacting amino acids with oxaziridines ( 17-20 ). The oxaziridines themselves ar e produced by reacting a Schiff base with a peracid like oxone ( 18-20 ), or by electrophilic aminat ion of a carbonyl compound ( 17 ). Although N-amination is needed to produce hydrazines from oxaziridines, oxaziridines are well-known aminating agents th at can also be used to attach nitrogen atoms to nucleophilic carbon atoms, sulfur atoms, oxygen atoms, and even phosphorus atoms ( 17,19 ). In addition, the synthesis of oxaziri dines with protecti ng groups that are amenable to peptide synthetic methods made this methodology useful for the synthesis of peptides and peptidomimetics ( 18 ). Oxaziridines have been synthesized with Boc ( 1820 ), Cbz ( 19 ), Fmoc ( 19 ), and other protecting groups on them. Some examples of protected hydrazines synthesized by reacting an amine with an oxaziridine are shown in Scheme 2.9. NH MeO MeO NH O 1. 2. 2N HCl N MeO MeO NH2 HCl Ref. 17 O NH O N Cl3CBoc O N NHBoc Ref. 20 Scheme 2.9: Synthesis of Hydr azines Using Oxaziridines (17,20). Several stable oxaziridines like the second example shown have been synthesized.

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30 Recently, we published a method to synt hesize hydrazines from amino acids by the reduction of nitrosamines ( 21,22 ). The amino acid is first converted to a nitrosamine, and this is then reduced using Zn/conc. HCl at –78 C. The free hydrazine is reactive and has a tendency to self-condens e, so we protected the free -nitrogen using Boc anhydride ( 21,22 ). This procedure was very successful for the synthesis of the Boc-protected hydrazines of several nonpolar amino acids ( 21,22 ). N O O H ONO N O O N O N O O NH2 Zn/HCl -78 C Scheme 2.10: Synthesis of Hydrazines from Amino Acids Via Nitrosamines (21). After formation of the hydrazine, it was i mmediately reacted with Boc anhydride to protect it as the stable Boc derivative. 2.1.3 Use of Hydrazines in Peptidomimetics Hydrazines are the key structural un its of both azapeptide and hydrazinopeptide peptidomimetics. Azapeptides are isosteres of peptides wh ere the alpha carbon has been replaced with a nitrogen atom. The first re ported azapeptide, an analog of the peptide hormone angiotensin II, is shown in Figure 2.1 ( 23 ). The authors found that this compound was about 100-fold less active than angiotensin II, but its duration of activity was twice as long ( 23 ). Another azapeptide with an unnatural N-cyclohexyl substituent was found to be an excellent inhibitor of HIV protease ( 24). More recently, azapeptides were studied as anti-cancer agents ( 25 ).

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31H2N N N NN N N N OH O O OO OO O HOOC H H H HH HN HNNH2HO NNH Figure 2.1: Example of an Azapeptide (23). This is the first reported example in the literature of an azapeptide isostere. The azapeptide portion of the molecule is boxed. H2N N N N N N N N N NH2O O O O OO O O H H H H H H H H NH2O HO S N Boc N N N OO H H H N N N OH O HO H HO Ref. 27 Ref. 26 Ref. 28 Figure 2.2: Examples of Hydrazides (26-28). In a hydrazide, the hydrazine is coupled to the rest of the peptoid by its -nitrogen. The hydrazines in each molecule are boxed.

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32 Azapeptides are easier to synthesize in comparison with hydrazinopeptides. One reason is that the chiral alpha carbon has been replaced with an achiral nitrogen atom. In addition, there are fewer problems with regiosel ectivity in coupling the nitrogens to make an azapeptide ( 26 ). However, hydrazinopeptides where the normal -amino acid is replaced with an -hydrazino amino acid, more clos ely mimic the three-dimensional structure of real peptides. There are two gr oups of hydrazinopeptides that are classified based on how the hydrazine subunits are coupled to the rest of the molecule ( 26 ). Hydrazides are coupled by the -nitrogen, whereas N-amino amides are coupled by the -nitrogen ( 26 ). Some examples of hydrazides and N-amino amides are shown in Figures 2.2 and 2.3, respectively ( 26-29 ). N Boc N O NH2N O H N N O HO NH2 OH O Ref. 26 Ref. 28 Figure 2.3: Examples of N-Amino Amides (26, 28). In N-amino amides, the hydrazine is coupled to the rest of the peptoid by its -nitrogen. The hydrazines in each molecule are boxed.

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332.1.4 Use of Hydrazines in -Sheet Mimics Proteins are ubiquitous in biology, and th ere is consequently a great deal of interest in interfering with their interac tions as well as mimicking their secondary structures. The use of peptidomimetics is al so considered to be a way to overcome the disadvantages inherent in using peptides as dr ugs, such as poor bi oavailability, enzymatic degradation, antigenicity, a nd high cost of production ( 29 ). The hydrazines described in this chapter can be used as structural s ubunits for conformationally restricted peptide secondary structures like -helices and -sheets. This section briefly describes the construction of -sheet mimics, while -helix mimics are described in Chapter Three. Peptide ligands must adopt a specific confor mation in order to interact with their receptors. There is a tremendous loss of entr opy that occurs when a flexible ligand in solution is forced to interact in a single c onformation with a protein such as a receptor ( 30 ). In general, processes where a large num ber of torsional motions become restricted upon binding are unfavorable due to entropic factors, even if they are enthalpically favored ( 30 ). Thus, it would be advantageous to conformationally rest rict such peptide ligands in order to reduce entropic losses, as this would be expected to increase their binding affinity. To this end, our group has been worki ng on the construction of two units that mimic the extended conformation of a peptide by constraining the peptide as part of a six-membered ring ( 22 ). The first diagram in Figure 2.4 shows a regular peptide that is free to rotate around its singl e backbone bonds. There are five such dipeptide (DP) bonds, numbered DP1 to DP5. Forming a sixmembered dipeptide unit (DPU) ring as in the middle diagram constrains two of thes e DP bonds and prevents any rotation around

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34 them. Beyond this, using a hydrazine as in the right-hand azadipeptide (ADP) structure forms two six-membered rings; now there are four constrained DP bonds. We are currently working on the synthe sis and testing of constrai ned ADP-type peptidomimetics using our amino hydrazines. Figure 2.4: Constraining the Extended Conf ormation of a Peptide to Mimic a Beta Sheet. ( 22 ) The first figure shows a regular peptide in an exte nded conformation. All of the dipeptide (DP) bonds are bolded. The middle figure s hows a partially constrained dipeptide unit (DPU), where a six-membered ring prevents the peptide from rotating around DP bonds 2 and 3. The figure on the right shows a completely constrained azadipeptide (ADP) unit, where the peptide is prevented from rotating around DP bonds 2, 3, 4, and 5. N N O N H O HO O H2N HO N O H H N O H NHBn O HO Ref. 31N N Bzl CO2CH3NO2O2N Ref. 32 Figure 2.5: Some Constrained Hydraz ine-Containing Peptidomimetics (31, 32). The ring size can be six-membered, as in the second example, or much larger. There are some examples in the literatu re where hydrazines have been used to construct conformationally restricted peptidomimetics. One is a mimic of the CD4 loop O N N O N HO H H R1 R2 DP2DP3O N N N O N HO H H R1 R2 O H DP2DP3DP4 DP5DPU ADPO N R1 N H O N HO R2 H H DP1DP2DP3DP4 DP5H A

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35 region that prevents the HIV glycopr otein gp120 from binding to CD4 on human lymphocytes ( 31 ). Another group synthesized a si x-membered ring with a hydrazine ( 32 ). These two structures are shown in Figure 2.5. 2.2 Results and Discussion This work builds on our group’s previous work to synthesize the hydrazines of five nonpolar amino acids: valine, isoleuci ne, leucine, methionine, and phenylalanine ( 21,22 ). Initially, the synthesis of compounds 6a-6e, shown in Scheme 2.12, was attempted using our previ ously developed methodology ( 21,22 ), shown in Scheme 2.11. However, we ran into trouble at several key points during the synthe sis, such that two new procedures were necessary. One cha nge was to use a single-step reductive amination rather than a twostep procedure to benzylate the amino groups. Second, the successful synthesis of Lys compound 6b required the use of oxaziridine 10 rather than the nitrosamine reduction pathway used for th e other four amino acids reported here. Our original procedure begins with the pr otection of the methyl ester of the amino acid as the benzyl amino ester. This wa s done via a two-step process that involved forming the imine using benzaldehyde and th e amino acid methyl ester, isolating the imine, and then reducing it to the benzyl amine using sodium borohydride. The final compounds were made by converting each benzyl amine to the corresponding nitrosamine using tert -butylnitrite, reducing th e nitrosamine with zinc and concentrated HCl at –78 C, and protecting the -nitrogen of the hydrazin e by reacting it with Boc anhydride neat ( 21,22 ). The original procedure is depicted in Scheme 2.11.

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36H2N O O R HCl O H TEAMgSO4THF 1. 2. NaBH4MeOH N O O R H N O O R NH O O ONO 1. 2. DCM Zn/HClMeOH -78 C Boc2O S R = 3. Scheme 2.11: Procedure for the Synthesis of Five Protected Nonpolar Amino Acids (21, 22). This work was the starting basis for th e synthesis of the f our polar amino acid and alanine hydrazines descri bed in this chapter. The general synthetic procedure used to synthesize the analogous alanine, lysine, serine, threonine, and tyrosi ne hydrazines is shown in Scheme 2.12. This synthesis begins with the reductive amination of the free amine 1a-e using benzaldehyde, triethylamine, and sodium cy anoborohydride to give compounds 2a-e. The benzylprotected amines were then treated with tert -butyl nitrite to give nitrosamine compounds 3a-e in high yield and purity. The nitrosamin es were reduced using Zn/conc. HCl to give the reactive intermediate hydrazines 4a-e, which were immediately reacted with Boc anhydride to give the stab le, Boc-protected hydrazines 5a-e. Finally, the benzyl protecting group was removed from the -nitrogen via hydrogenolysis to give compounds 6a-e. Lysine compound 5b could not be obtained by this method, and was synthesized by an alternative procedure as discussed below.

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37 ONO DCM 94100% N O N R O O 3a-e conc. HCl MeOH Zn N O H R O 2a-e4090% N O H H R O 1a-eN O NH2R O 4a-e MeOHO H Et3N NaBH3CN 67100% 38100% (0% for Lys) N O N R O H Boc 5a-eN O H N R O Boc H 6a-e H2Pd/C MeOH Boc2O CH3CN a b c d eR = CH3 N O O O O O Boc = O O Scheme 2.12: Synthesis of the Ala (6a), Lys (6b) Ser (6c), Thr (6d), and Tyr (6e) BocProtected Hydrazines from the Free Amines (1a-e). Compound 1a was commercially available, while compounds 1b-1e were synthesized as described below. Compound 5b could not be obtained by this procedure.

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38 .1a 2aNH O OCH3 H2N HCl O OCH3 2. NaBH490-95 % 1.H O X Scheme 2.13: Unsuccessful Two-Step Method for Synthesizing Compound 2a. Unlike the other nonpolar amino acids published previously, the Ala compound 2a could not be obtained using this two-step method. The two-step benzylation method shown in Scheme 2.11 was successfully used for all five nonpolar amino acids attempted previously. Therefore, we began our synthesis of the new hydrazine examples by attempting to protect the corresponding free amines with a benzyl group us ing the two-step process ( 21,22 ). However, we ran into some difficulties with the synthesis of se veral of the new benzyl amino acids. One problem was that we were unable to obtain the alanine benzyl amine 2a using the twostep procedure, even after multiple attempts as depicted in Sche me 2.13. Instead, only benzyl alcohol was isolated as evidenced by TLC and NMR. Amino acid imine stability studies have s hown that bulkier side chain amino acids like Leu and Ile provide greater protection against imine hydrol ysis when compared with smaller groups ( 33 ). This would explain why the two-step procedure worked for the larger nonpolar amino acids but not for Ala. However, since re duction of the imine apparently occurs faster than hydrolysis dur ing the one-step reducti on, we were able to successfully obtain 2a by that route. Further conversion of 2a to the protected hydrazine 6a was performed by the usual procedures.

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39 Second, we had trouble benzyl-protecti ng the polar amino acids due to the racemization of the alpha carbon, as depicted in Scheme 2.14. Initia lly, we did not know about this problem because in most cas es, racemization would produce a pair of enantiomers, which would not be detectable. However, when the imine of threonine was reduced to the benzyl amine, we were clearly able to detect two stereoisomers. This is due to the presence of a second chiral center in the side chain of threonine that does not exist in any of the other four amino acids. Thus, when the alpha carbon of threonine racemized, we could clearly detect two diastereomers by TLC, and they could be separated by column chromatography. An examination of the NMRs of the two diastereomers shows th at they are very similar, but the groups of protons have slightly different coupling constants. This can be seen in Figure 2.6. The aromatic protons in 2d, the (L)-isomer, show up as a multiplet ranging from 7.22-7.38 ppm. In contrast, th e aromatic protons of the (D)-isomer 13b are more closely overlapping, with the multiple t for the aromatic protons ranging from 7.247.35 ppm. The benzyl CH2 protons appear as a multip let ranging from 3.92-3.99 ppm for 2d, versus 3.82-3.94 ppm for 13b. The alpha proton shows up as a doublet at 3.11 ppm with a coupling constant of 3.5 Hz for 2d, while the same doublet shows up at 3.25 ppm and has a coupling constant of 6.0 Hz for 13b. Finally, the methyl group on the side chain of threonine shows up 1.24 ppm for 2d with a coupling constant of 6.2 Hz, versus 1.19 ppm for 13b, with the same coupling constant. It is clear that these two spectra are of the same compound, with very slight differe nces due to the spatial orientation of the groups that are attached to the tw o chiral centers in each molecule.

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40N O H HOO 1d O H NaBH4THF MeOHN O OO H 13a 13bN O HOO 18%2d36%N O HOO Scheme 2.14: Epimerization of Thr Du e to 2-Step Benzylation Procedure. The (L) and (D) isomers 2d and 13 were formed in a 2:1 ratio du e to the acidity of the imine intermediate’s alpha proton. Our nitrosamine reduction procedure has se veral advantages, in that the starting materials are all cheap and readily available, and that it can be used to synthesize hydrazino amino acids from the corresponding am ino acids without a lo ss of chirality at the -carbon. However, there are also some problems: the tert -butylnitrite reagent is toxic and carcinogenic, the highly acidic condi tions limit the types of side chain groups and protecting groups that can be present, and there is a tendency to over-reduce the nitrosamine back to th e parent amine rather than stoppi ng at the hydrazine. We had the most trouble with the synthesis of the benzyl lysine hydrazine 5b. Although we were able to selectively protect, depr otect, and react each of the tw o amino groups of lysine, as well as form the nitrosamine 3b, we were not able to reduce 3b to the hydrazine 4b cleanly, as depicted in Scheme 2.15. Rath er, we obtained a multitude of decomposition products, as evidenced by TLC and proton NMR.

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41 Figure 2.6: NMRs of N-Benzyl-(L)-Threoni ne Methyl Ester (top) and N-Benzyl-(D)Threonine Methyl Ester. The two diastereomers could clearly be differentiated by proton NMR, as well as by TLC. N-Benzyl -(L)-Threonine ppm (t1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 7.357 7.311 7.282 7.260 7.233 4.017 4.003 3.992 3.977 3.968 3.953 3.943 3.923 3.718 3.630 3.577 3.183 3.168 3.131 2.173 1.254 1.229 1.131 1.079 2.1 0 3.0 0 1.1 3 1.0 4 0.9 3 4.71 9.2 0 3.4 0 N O HOO N-Benzyl -(D)-Threonine ppm (t1) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 7.348 7.285 7.275 7.266 7.255 7.232 7.329 7.316 7.309 3.941 3.887 3.854 3.830 3.805 3.670 3.616 3.258 3.234 3.728 2.049 1.202 1.177 1.126 1.0 0 4.4 0 1.2 4 1.1 5 2.8 2 1.2 6 1.1 9 3.4 5 8.7 0 N O HOO

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423b XZn/HCl MeOHN O O N O NOO 4bN O O N O NH2O Scheme 2.15: Unsuccessful Attemp t to Reduce Lys Nitrosamine 3b. Unlike the other amino acids we have reported, compound 3b gave a complex mixture of products upon repeated attempts to reduce it using Zn/HCl. This problem was solved by reacting lysine benzyl amine 2b with oxaziridine 10, as shown in Scheme 2.16, to directly fo rm the desired benzyl lysine hydrazine 5b in good yield. Oxaziridine 10 was synthesized in our lab fo llowing a literature procedure ( 20,34 ), and the yield and ease of using it to synt hesize a Boc-protected hydrazine were an improvement over our own nitrosamine method. 10 also has the advantage of being much less carcinogenic and volatile compared with tert -butylnitrite. However, the procedure to synthesize 10 is highly laborious and time-co nsuming, and it requires the use of chloral, which is a controlled substanc e in the United States that is not readily available for purchase fro m commercial sources. Unlike the nonpolar amino acids, the polar amino acids were not readily available as the hydrochloride salts of the methyl esters. Thus, we obtained compounds 11c–e as the free acids with th eir amino groups protected by a Cbz. 11 was treated with potassium carbonate and methyl iodide to give methyl ester 12, which was then deprotected on the amine by hydrogenolysis to give starting material 1c–1e. This series of protection and deprotection steps is de picted in Scheme 2.17.

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43Boc = O O XPd/C H2AcOH MeOH40% MeOHO H Et3N NaBH3CN1bN O H HO N O O H CH3CN67%N O HO N O O 2b10N O Boc Cl Cl Cl 5bN ONOH Boc N O O N OHNOH Boc N O O 6b Scheme 2.16: Procedure for Synthesizing Compound 6b (Lys). The nitrosamine of Lys 5b could not be reduced to hydrazine 6b using Zn/HCl, but compound 6b was successfully synthesized using the Boc-protected oxaziridine 10 according to literature procedure ( 20,34 ). The final hydrogenation step resulted in multiple products. The lysine starting material 1b was also formed from the free acid, but a few additional steps were necessary in order to selectively protect the alpha and epsilon nitrogens. We began with compound 11b, which was orthogonally protected by a Boc group on the alpha nitrogen and a Cbz group on the epsilon nitrogen. This was reacted with methyl iodide and potassium carbonate to form methyl ester 12b as before. The Cbz

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44 group was then removed from the epsil on nitrogen by hydrogenolysis, and compound 7 was immediately reacted with phthalic anhydride 8 to form the lysine phthalimide 9 in excellent yield. Finally, the alpha nitrogen of 9 was deprotected using a 1:1 mixture of TFA and DCM to give starting material 1b. This procedure is shown in Scheme 2.18. Cbz = O O 12c-eN OH Z H R O 11c-eN O Z H R O K2CO3CH3I DMF 91-100% H2/Pd 50 psi MeOH 92-100%N O H H R O 1c-eR = c d eO O O Scheme 2.17: Synthesis of Ser, Thr, and Tyr Methyl Esters (1c-e). The three tert butyl ether-protected alcohol side chain amino esters were synthesized from the commercially available Cbz-pr otected acids in high yield.

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45 Boc = O O Cbz = O O N O H HO N O O H 1b9N O Boc HO N O O 7N O Boc HO N HH H Pd/C H2AcOH MeOH12bN O Boc HO N HCbz K2CO3CH3I DMF11bN OHBoc HO N HCbz TFA DCM Et3NO O 100%O O O 8 Scheme 2.18: Procedure for Synthesizing Compound 1b (Lys). The amino group on the side chain of Lys was protected as a pht halimide in order to selectively form the hydrazine on the alpha amino group.

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46Boc = O O 53100% N O N R O H Boc 5f-iN O H N R O Boc H 6f-i H2Pd/C MeOH iS f g hR = Scheme 2.19: Deprotection of Nonpolar Amino Acid Hydrazines. Compounds 5f – 5i were synthesized as described previously by our group ( 21, 22 ). Hydrogenolysis of the benzylated hydrazines gave compounds 6f 6i. Finally, all of the benzyl-p rotected hydrazines that were synthesized previously by our group were deprotected via hydrogenol ysis. Previously, only the phenylalanine example had been attempted and published ( 21 ). The procedure for synthesizing 6f–6i was analogous to that described for depr otection of the polar amino acid hydrazines above, and is shown in Scheme 2.19. It was difficult to deprotect the methionine benzyl hydrazine 5i due to the sulfur poisoning the cataly st. We attempted to overcome this problem by adding several drops of acetic aci d, as well as by adding excess Pd/C. The

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47 use of excess Pd/C was more helpful than adding acetic acid, but even with this change to the procedure, the yield was stil l significantly lower than it was for the other amino acids. The final lysine compound 6b could not be obtained; repeated attempts to hydrogenate benzyl-protected hydrazine 5b gave either starting material or multiple decomposition products as evidenced by TLC and NMR. In conclusion, we have successfully deprotected the nonpolar hydrazino amino acids that were published by our group previously ( 21 ). We have also synthesized the hydrazine derivatives of five additional amino acids using modified procedures. Future work on this project will entail continued e fforts to successfully deprotect the lysine benzyl hydrazine 5b, as well as the synthesis of the hydrazino amino acid derivatives of the remaining ten natural amino acids. 2.3 Experimental Data N O OO H 13a Experimental Procedure to Synthesize Methyl tert-Butoxy-N-Benzylidene-(R)Threoninate (13a). A 100 mL Schlenk flask was placed in an ice bath with a stir bar and flushed with argon. The methyl amino acid 1d (2.55 mmol) was dissolved in the flask in a few mL of anhydrous THF, and this mixture was stirred for ten minutes at 0 C. Then benzaldehyde (5.10 mmol) and TEA (2.55 mmol) were added, along with a small scoop of MgSO4 to keep the reaction dry. The reac tion was allowed to come to room

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48 temperature and stirred overnight. The MgSO4 was then removed by filtering the reaction mixture through a powde r funnel and rinsing the MgSO4 several times with THF. The crude product 13a was obtained by evaporating the THF under reduced pressure, and was carried on to the next step without purification due to the instability of the imine to column chromatography. Rf = 0.56 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, MeOD) 2.17 (s, 9H), 2.26 (d, J = 7.6 Hz, 1H), 2.39 (d, J = 6.3 Hz, 1H), 2.56 (quin, J = 6.3 Hz), 2.75 (quin, J = 6.2 Hz, 1H), 3.33 (s, 3H), 5.83-5.91 (m, 3H), 6.22-6.25 (m, 2H), 6.67 (s, 1H), 6.75 (s, 1H); 13C NMR (75 MHz, CDCl3) 19.48, 20.44, 28.54, 28.72, 52.21, 69.26, 75.42, 80.13, 80.90, 129.44, 132.10, 136.60, 166.58, 166.77, 172.68 (The peak intensities were approximately th e same for the both isomers, so the signals reported are for both isomers); ES I-MS (0.1% AcOH in MeOH) 278.1 (M+H)+. Experimental Procedure to Synthesize (S)and (R)-Methyl tert-Butoxy-N-BenzylThreoninate (2d and 13b). Compound 13a (2.55 mmol) was dissolved in several mL of dry MeOH, flushed with argon, and stirred at room temperature. Sodium borohydride (10.20 mmol) was added slowly in three portio ns over the course of ten minutes due to vigorous bubbling in the reaction. The reactio n was stirred at room temperature for two hours, and then worked up with 1M NaOH. This was extracted th ree times with ethyl acetate, and the three organic layers were co mbined and washed with brine, dried over MgSO4, and evaporated under reduced pressu re to give the benzyl amines 13b and 2d. The crude products were chromatographed in 9: 1 hexanes/ethyl acetate to give the pure products in a 2:1 (S):(R) ratio in an overall yield of 54.5%.

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49N O HOO 2d Methyl tert-Butoxy-N-Benzyl-(S)-Threoninate (2d). Rf = 0.56 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.12 (s, 9H), 1.24 (d, J = 6.2 Hz, 3H), 2.23 (broad, 1H), 3.12 (d, J = 3.5 Hz, 1H), 3.57-3.62 (m, 1H), 3.70 (s, 3H), 3.92-3.99 (m, 2H), 7.22-7.38 (m, 5H); 13C NMR (75 MHz, CDCl3) 20.57, 28.29, 51.52, 52.08, 65.87, 68.50, 73.65, 126.85, 128.20, 128.27, 140.09, 174.17; ESI-MS (0.1% AcOH in MeOH) 280.1 (M+H)+. 13bN O HOO Methyl tert-Butoxy-N-Benzyl-(R)-Threoninate (13b). Rf = 0.44 (3:1 hexanes/ EtOAc); 1H NMR (250 MHz, CDCl3) 1.13 (s, 9H), 1.19 (d, J = 6.2 Hz, 3H), 2.36 (broad, 1H), 3.24 (d, J = 5.8 Hz, 1H), 3.64-3.67 (m, 1H), 3.73 (s, 3H), 3.84 (quin, J = 5.2 Hz, 1H), 3.90-3.94 (m, 1H), 7.24-7.26 (m, 1H), 7.31-7.35 (m, 4H); 13C NMR (100 MHz, CDCl3) 19.83, 28.56, 51.79, 52.45, 66.38, 69.02, 74.31, 127.22, 128.50, 128.55, 139.80, 174.47; ESI-MS (0.1% AcOH in MeOH) 280.1 (M+H)+.

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50Experimental Procedure for Reductive Amination (2a-e). The commercially available HCl salt of the alanine methyl ester 1a (10.7 mmol) or the previously synthesized methyl ester 1b-e was dissolved in dry MeOH (50 mL), and cooled to 0 C. TEA (10.7 mmol) was added, followed by ben zaldehyde (21.5 mmol). The reaction was allowed to come to room temperatur e and stirred for 20 minutes. Sodium cyanoborohydride (43.0 mmol) was then added to the reaction, which was stirred overnight. The reaction mixture was quenche d with 1M NaOH (30 mL), saturated with NaCl, and extracted three times with ethyl a cetate. Then, the combined ethyl acetate layers were extracted with satura ted NaCl solution, dried over MgSO4, and evaporated under reduced pressure. The pure product 2a-e was isolated by column chromatography (4:1 hexanes:ethyl acetate) in 40-90% yield. 2aNH O OCH3 Methyl N-Benzyl-(S)-Alaninate (2a). Rf = 0.31 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.69 (d, J =7.2 Hz, 3H), 3.69-3.67 (m, 1H), 3.78 (s, 3H), 4.16 (d, J =12.8 Hz, 1H), 4.25 (d, J =12.8 Hz, 1H), 7.40-7.35 (m, 3H), 7.64-7.62 (m, 2H); 13C NMR (100 MHz, CDCl3) 15.08, 48.92, 53.13, 53.30, 129.16, 129.61, 129.74, 130.86, 169.06; ESIMS (0.1% AcOH in MeOH) 194.1 (M+H)+.

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51 N O Bnz HO N O O O H NaBH3CN MeOH TEAN O H HO N O O H 9N O Boc HO N O O 1b 2b TFA DCM Methyl N’-Phthalyl-N-Benzyl-(S)-Lysinate (2b) via Methyl N’-Phthalyl-(S)Lysinate (1b). Boc-protected amine 9 (0.383 mmol) was dissolved in a large excess of 1:1 DCM/TFA. The reaction mixture was s tirred under argon at room temperature for ten minutes, and then the DCM/TFA was remo ved under reduced pressure to give crude 1b as the trifluoroacetate salt. 1b was immediately carried on as described above to give 2b in 66.7% yield. Rf = 0.13 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.361.44 (m, 2H), 1.60-1.65 (m, 4H), 1.89 (broad, 1H), 3.22 (t, J = 6.8 Hz, 1H), 3.56-3.70 (m, 6H), 3.73-3.78 (m, 1H), 7.18-7.26 (m, 5H), 7.66 (dd, J = 5.4 Hz, 2.8 Hz, 2H), 7.79 (dd, J = 5.4 Hz, 2.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) 23.29, 28.47, 33.15, 37.89, 51.90, 52.32, 60.62, 123.36, 123.38, 127.23, 128.42, 128.55, 128.57, 132.33, 134.08, 139.98, 168.55, 176.02; HR ESI-MS (0.1% AcOH in MeOH) 381.18056 (M + H)+ N O HOO 2c Methyl tert-Butoxy-N-Benzyl-(S)-Serinate (2c). Rf = 0.43 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.15 (s, 9H), 2.23 (broad, 1H ), 3.43-3.47 (m, 1H), 3.58-3.62

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52 (m, 2H), 3.70-3.75 (m, 1H), 3.73 (s, 3H), 3.89-3.95 (m, 1H), 7.24-7.37 (m, 5H); 13C NMR (75 MHz, CDCl3) 27.31, 51.65, 51.93, 61.00, 63.11, 73.14, 126.96, 128.25, 128.31, 139.76, 173.82; HR ESI-MS (0.1% AcOH in MeOH) 266.17505 (M+H)+. N O HOO 2d Methyl tert-Butoxy-N-Benzyl-(S)-Threoninate (2d). Rf = 0.61 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.13 (s, 9H), 1.24 (d, J = 6.2 Hz, 3H), 2.16 (broad, 1H), 3.12 (d, J = 3.6 Hz, 1H), 3.58-3.63 (m, 1H), 3.72 (s, 3H), 3.92-3.99 (m, 2H), 7.23-7.38 (m, 5H); 13C NMR (75 MHz, CDCl3) 20.51, 28.31, 51.49, 52.13, 66.01, 68.53, 73.66, 126.83, 128.19, 128.26, 140.13, 174.19; ESI-MS (0.1% AcOH in MeOH) 280.2 (M+H)+. N O HOO 2e Methyl tert-Butoxy-N-Benzyl-(S)-Tyrosinate (2e). Rf = 0.46 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.35 (s, 9H), 1.96 (broad, 1H), 2.93 (d, J = 7.0 Hz, 2 H), 3.52 (t, J = 7.0 Hz, 1H), 3.61-3.66 (m, 1H), 3.63 (s 3H), 4.70 (s, 2H), 6.92 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.20-7.39 (m, 5H); 13C NMR (75 MHz, CDCl3) 28.80, 39.10, 51.64, 51.93, 62.09, 65.23, 78.36, 124.21, 126.95, 127.02, 127.58, 128.09, 128.33,

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53 128.52, 129.59, 132.17, 139.46, 153.94, 175.17; HR ESI-MS (0.1% AcOH in MeOH) 342.20687 (M+H)+. Experimental Procedure for Ni trosamine Formation (3a-e). The benzyl protected amine 2 (1.14 mmol) was dissolved in DCM (10 mL), cooled to 0 C, and tertbutylnitrite (1.25 mmol) in DCM was added drop-wise from an addition funnel. The reaction was refluxed for 4 hours, cooled to RT, and stirred overnight. The solvent was evaporated under reduced pre ssure to give nitrosamine 3 as yellow oil in a 100 % yield. Nitrosamines were typically pure and did not require chromatography. N O NO O 3a Methyl N-Benzyl-N-Nitroso-(S)-Alaninate (3a). Rf = 0.33 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.69 (d, J = 7.2 Hz, 3H), 2.48 (d, J = 6.8 Hz, 3H), 3.57 (s, 3H), 3.67 (s, 3H), 4.54 (q, J = 7.6 Hz, 1H), 4.72 (d, J = 15.2 Hz, 1H), 4.94 (d, J = 15.2 Hz, 1H), 5.21 (q, J = 7.6 Hz, 1H), 5.39 (s, 2H), 7.08-7.09 (m, 2H), 7.27-7.30 (m, 3H), 7.37-7.39 (m, 5H); 13C NMR (100 MHz, CDCl3) 13.14, 16.47, 46.64, 52.35, 52.72, 52.96, 55.42, 59.96, 127.78, 127.98, 128.40, 128.61, 128.73, 128.91, 134.32, 134.61, 168.94, 170.87 (The peak intensities were approx imately the same for the both isomers, so the signals reported are for both isomers); ESI-MS (0.1% AcOH in MeOH) 223.1 (M+H)+.

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54N O NO N O OO 3b Methyl N’-Phthalyl-N-Benzyl-N-Nitroso-(S)-Lysinate (3b). Rf = 0.09, 0.15 (3:1 hexanes/EtOAc; two nitrosamine stereoisomers); 1H NMR (400 MHz, CDCl3) 1.171.29 (m, 2H), 1.44-1.60 (m, 2H), 2.09-2.24 (m, 2H ), 3.46 (s, 3H), 3.47-3.62 (m, 2H), 4.64-4.74 (m, 1H), 4.90-5.03 (m, 1H), 5.28-5. 38 (m, 1H), 7.03-7.10 (m, 1H), 7.20-7.42 (m, 4H), 7.71-7.73 (m, 2H), 7.82-7.83 (m, 2H); 13C NMR (100 MHz, CDCl3) 23.42, 28.02, 28.14, 29.50, 37.48, 46.94, 52.38, 52.80, 55.66, 56.15, 64.65, 123.37, 128.06, 128.16, 128.67, 128.80, 128.85, 128.94, 129.01, 129.84, 132.21, 134.16, 134.20, 134.48, 168.44, 170.40; (The spectrum is complex as with all of the nitrosamin es; several of the peaks split for both proton and carbon NMRs. No MS was taken of this compound due to its toxicity and carcinogenicity.) N O NO OO 3c Methyl tert-Butoxy-N-Benzyl-N-Nitroso-(S)-Serinate (3c). Rf = 0.43, 0.48 (3:1 hexanes/ EtOAc; two nitrosamine stereoisomers); 1H NMR (250 MHz, CDCl3) 1.05 (s,

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55 9H), 1.10 (s, 9H), 3.58 (s, 3H), 3.71 (s, 3H), 3.73-3.77 (m, 2H), 3.92 (dd, J = 4.1 Hz, 9.7 Hz, 1H), 4.00-4.07 (m, 1H), 4.45-4.50 (m, 1H ), 4.75-4.81 (m, 1H), 5.05-5.11 (m, 1H), 5.20-5.26 (m, 1H), 5.34-5.39 (m, 1H), 5.74-5. 80 (m, 1H), 7.09-7.11 (m, 1H), 7.26-7.28 (m, 2H), 7.36-7.40 (m, 2H); 13C NMR (75 MHz, CDCl3) 26.98, 27.19, 47.91, 52.08, 52.50, 56.87, 58.65, 60.84, 65.07, 73.80, 127.27, 127.64, 128.32, 128.65, 134.71, 168.88 (The peak intensities were approximately th e same for the both isomers, so the signals reported are for both isomers. No MS was take n of this compound due to its toxicity and carcinogenicity.) N O NO OO 3d Methyl tert-Butoxy-N-Benzyl-N-Nitroso-(S)-Threoninate (3d). Rf = 0.37, 0.44 (3:1 hexanes/EtOAc; two nitrosamine stereoisomers); 1H NMR (250 MHz, CDCl3) 1.15 (s, 9H), 1.16 (d, J = 6.2 Hz, 3H), 1.20 (s, 9H), 3.50 (s, 3H ), 3.64 (s, 3H), 4.35-4.36 (m, 1H), 4.53-4.58 (m, 1H), 4.75-4.81 (m, 1H), 5.14-5. 25 (m, 2H), 5.79-5.85 (m, 1H), 7.17-7.47 (m, 5H); 13C NMR (75 MHz, CDCl3) 20.88, 22.01, 28.55, 28.69, 49.52, 51.84, 52.27, 57.53, 61.59, 66.05, 68.16, 70.41, 74.81, 127.13, 128.21, 128.40, 129.31, 134.73, 135.04, 168.82 (The peak intensities were approximately in a 2:1 ratio for the two isomers, so the signals are reported for both isomers. No MS was taken of this compound due to its toxicity and carcinogenicity.)

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56N O NOO O 3e Methyl tert-Butoxy-N-Benzyl-N-Nitroso-(S)-Tyrosinate (3e). Rf = 0.45 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.32 (s, 9H), 1.35 (s, 9H), 3.01-3.06 (m, 1H), 3.24-3.29 (m, 2H), 3.40-3.49 (m, 2H), 3.51 (s, 3H), 3.62 (s, 3H), 4.15-4.23 (m, 3H), 4.52-4.82 (m, 4H), 4.80-4.84 (m, 1H), 5.40-5.46 (m, 1H), 6.85 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 7.16-7.22 (m, 5H), 7.30-7.36 (m, 5H); 13C NMR (75 MHz, CDCl3) 28.78, 32.63, 35.86, 47.60, 52.22, 52.70, 56.98, 59.60, 65.11, 65.37, 78.58, 124.29, 124.61, 126.90, 127.45, 127.78, 128.38, 128.60, 128.68, 129.10, 129.54, 130.74, 131.61, 133.28, 133.50, 154.32, 167.59, 169.80 (The peak intens ities were approximately the same for the both isomers, so the signals re ported are for both isomers. No MS was taken of this compound due to its toxicity and carcinogenicity.) Experimental Procedure for Boc-Protected -Hydrazino Esters via Hydrazine Formation (5a-e). Zinc was activated by washing with 10% HCl, dI water, methanol, and diethyl ether. The nitrosamine 3 (1.60 mmol) was dissolved in dry MeOH (15 mL) under argon in a 3-necked flask. A powder a ddition funnel was filled with activated Zn (12.8 mmol) and fitted to one neck, while th e other two necks were sealed with rubber septa. The reaction system was then cooled to -78 C, and activated Zn was slowly added to the stirred suspension, followed by addition of conc. HCl (12.8 mmol) via

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57 syringe. The reaction was then stirred under argon at -78 C for 4 hours. To work up the reaction, the excess Zn was filtered while th e reaction mixture was still cold, and the filtrate was treated with cold 6N KOH until it was strongly basic (pH 12-13). It was then extracted with 3 equal portions of cold ethyl acetate. The combined ethyl acetate layers were dried over Na2SO4, and evaporated under reduced pressure from a RT water bath, to give the hydrazine. The neat oil will oligomerize, so the hydrazines were immediately protected using Boc anhydride wi th no characterization. Melted tert -Boc2O (4.32 mmol) was added directly to 4 (3.92 mmol) and they were stirre d together for 30 min at room temperature neat. After 30 min, a very sma ll amount of acetonitrile was added to prevent the reaction from solidifying, and the reac tion mixture was stirred overnight. The acetonitrile was removed under reduced pressure at RT, and the product 5 was isolated by column chromatography (4:1 hexanes:ethyl acetate) in 38-100% yield. N O NO H Boc 5a Methyl N-Amino-N-Benzyl-N’-tert-butoxycarbonyl-(S)-Alaninate (5a). Rf = 0.45 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.32-1.44 (m, 12H), 3.62-3.64 (m, 1H), 3.71 (s, 3H), 3.95-4.05 (m, 2H), 6.60 (b road, 1H), 7.24-7.30 (m, 3H), 7.31-7.40 (m, 2H); 13C NMR (100 MHz, CDCl3) 16.21, 28.19, 51.55, 60.08, 60.65, 79.52, 127.41, 128.20, 129.27, 136.89, 155.37, 175.50; HR ESI-MS ( 0.1% AcOH in MeOH) 331.18090 (M + H)+

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58N O Bnz O N O ON HBoc 5b Methyl N’-Phthalyl-N-Amino-N-Benzyl-N’’-tert-Butoxycarbonyl-(S)-Lysinate (5b). Rf = 0.29 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.33 (s, 9H), 1.56-1.60 (m, 2H), 1.70-1.78 (m, 4H), 3.35-3.39 (m, 1H), 3.63-3.65 (m, 2H), 3.88-3.91 (m, 1H), 3.98-4.00 (m, 1H), 6.70 (broad, 1H), 7.20-7.37 (m, 5H), 7.70 (d, J = 2.8 Hz, 2H), 7.82 (d, J = 2.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) 23.59, 28.33, 29.86, 37.96, 51.81, 61.32, 64.23, 79.94, 95.90, 123.37, 127.74, 128.46, 129.65, 130.09, 132.41, 134.05, 136.89, 155.37, 168.62, 174.38; HR ESI-MS (0.1% AcOH in MeOH) 496.24433 (M + H)+ N O NO H BocO 5c Methyl tert-Butoxy-N-Amino-N-Benzyl-N’-tert-Butoxycarbonyl-(S)-Serinate (5c). Rf = 0.40 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.12 (s, 9H), 1.36 (s, 9H), 3.65-3.81 (m, 3H), 3.73 (s, 3H), 3.94-4.04 (m, 2H), 6.69 (broad, 1H), 7.23-7.31 (m, 3H), 7.39-7.41 (m, 2H); 13C NMR (100 MHz, CDCl3) 27.25, 27.37, 27.47, 27.54, 28.47, 51.68, 61.24, 61.88, 65.33, 65.71, 73.47, 79.80, 127.65, 128.39, 128.59, 129.29, 129.60, 137.00, 172.49; HR ESI-MS (0.1% AcOH in MeOH) 403.22016 (M + Na)+

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59N O NO H BocO 5d Methyl tert -ButoxyN -Amino -N -BenzylN ’tert -Butoxycarbonyl-( S )-Threoninate (5d). Rf = 0.54 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.10 (s, 9H), 1.27 (d, J = 6.3 Hz), 1.38 (s, 9H), 3.44-3.45 (m, 1H), 3.72 (s, 3H), 3.92-3.98 (m, 1H), 4.17-4.22 (m, 2H), 6.94 (broad, 1H), 7.22-7.42 (m, 5H); 13C NMR (100 MHz, CDCl3) 20.24, 28.32, 51.30, 61.62, 68.63, 69.93, 73.74, 79.10, 127.25, 128.09, 129.17, 137.49, 155.05, 172.37; HR ESI-MS (0.1% AcOH in MeOH) 395.25477 (M + H)+ N O NOO Boc H 5e Methyl tert -ButoxyN -Amino -N -BenzylN ’tert -Butoxycarbonyl-( S )-Tyrosinate (5e). Rf = 0.46 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.34 (s, 9H), 1.38 (s, 1H), 2.89-3.01 (m, 1H), 3.06-3.12 (m, 1H), 3.62 (s, 3H), 3.67-3.71 (m, 1H), 3.92-3.99 (m, 2H), 6.64 (broad, 1H), 6.90 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.22-7.29 (m, 5H); 13C NMR (75 MHz, CDCl3) 28.18, 28.80, 35.55, 51.37, 66.73, 78.13, 79.76, 107.88, 123.93, 127.41, 128.08, 129.30, 129.66, 132.73, 136.47, 147.68, 151.93, 152.90, 153.81, 173.38; HR ESI-MS (0.1% AcOH in MeOH) 479.25147 (M + Na)+

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60 General Procedure for Hydrogenolysis of Boc-Protected -Hydrazino Esters (6ai). Compound 5 (1.32 mmol) was dissolved in MeOH (8 mL) and 10% Pd/C (0.280 g) was added. The solution was hydrogenated at 45 psi H2 in a Paar Hydrogenator for 3 hours. The reaction mixture wa s filtered through a Celite cake, which was rinsed with MeOH. Evaporation of the MeOH yielded 6 in 67-100% yield. N O H NO Boc H 6a Methyl N-Amino-N’-tert-Butoxycarbonyl-(S)-Alaninate (6a). Rf = 0.15 (3:1 hexanes/ EtOAc); 1H NMR (400 MHz, CDCl3) 1.33 (d, J = 7.2 Hz, 3H), 1.45 (s, 9H), 3.71-3.75 (m, 4H), 4.17 (broad, 1H), 6.34 (broad, 1H); 13C NMR (100 MHz, CDCl3) 16.00, 28.30, 52.11, 58.47, 80.68, 156.36, 174.30; HR ESI-MS (0.1% AcOH in MeOH) 241.11595 (M + Na)+ N O H NO Boc HO 6c Methyl tert-Butoxy-N-Amino -N’-tert-Butoxycarbonyl-(S)-Serinate (6c). Rf = 0.36 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.14 (s, 9H), 1.44 (s, 9H), 2.69

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61 (broad, 1H), 3.68-3.73 (m, 2H), 3.75 (s, 3H ), 6.39 (broad, 1H), 6.64 (broad, 1H); 13C NMR (100 MHz, CDCl3) 27.45, 28.52, 44.23, 51.66, 52.21, 61.47, 63.88, 68.01, 73.51, 73.62, 80.60, 156.25, 172.19; HR ESI-MS (0.1% AcOH in MeOH) 313.17306 (M + Na)+ N O H NO Boc HO 6d Methyl tert-Butoxy-N-AminoN’-tert-Butoxycarbonyl-(S)-Threoninate (6d). Rf = 0.20 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.10 (s, 9H), 1.30 (d, J = 6.2 Hz, 3H), 1.42 (s, 9H), 3.43 (broad, 1H), 3.72 (s, 3H), 3.98-4.02 (m, 1H), 6.22 (broad, 1H); 13C NMR (75 MHz, CDCl3) 20.64, 28.26, 51.85, 67.49, 69.38, 73.87, 80.12, 155.93, 172.84; HR ESI-MS (0.1% AcOH in MeOH) 327.18926 (M + Na)+ N OHNOO Boc H 6e Methyl tert-Butoxy-N-Amino -N’-tert-Butoxycarbonyl-(S)-Tyrosinate (6e). MP = 48-49 C; Rf = 0.39 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.32 (s, 9H), 1.41 (s, 9H), 2.93-3.04 (m, 2H), 3.67 (s, 3H), 3.91 (t, J = 6.6 Hz, 1H), 4.18 (broad, 1H), 6.18 (broad, 1H), 6.91 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H); 13C NMR (75 MHz,

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62 CDCl3) 28.22, 28.77, 36.42, 51.93, 64.25, 80.60, 124.23, 129.53, 131.17, 154.18, 156.08, 173.15; HR ESI-MS (0.1% AcOH in MeOH) 389.20463 (M + Na)+ N O H NO Boc H 6f Methyl N-Amino-N’-tert-Butoxycarbonyl-(S)-Valinate (6f). Rf = 0.28 (3:1 hexanes/ EtOAc); 1H NMR (250 MHz, CDCl3) 0.97-1.02 (m, 6H), 1.44 (s, 9H), 2.01-2.06 (m, 1 H), 3.48 (d, J = 4.4 Hz, 1 H), 3.75 (s, 3H), 4.17 (b road, 1H), 6.19 (broad, 1H); 13C NMR (75 MHz, CDCl3) 18.93, 19.40, 28.69, 30.34, 52.30, 69.74, 81.09, 156.70, 173.95; HR ESI-MS (0.1% AcOH in MeOH) 269.14784 (M + Na)+ N O H NO Boc H 6g Methyl N-Amino-N’-tert-Butoxycarbonyl-(S)-Isoleucinate (6g). Rf = 0.37 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 0.76-0.86 (m, 6H), 1.13-1.18 (m, 1H), 1.32 (s, 9H), 1.37-1.44 (m, 1H), 1.66-1.68 (m, 1H), 3.41 (d, J = 4.9 Hz, 1H), 3.62 (s, 3H), 4.09 (broad, 1H), 6.48 (broad, 1H); 13C NMR (75 MHz, CDCl3) 11.86, 12.09, 15.18, 15.77, 25.95, 26.43, 28.52, 34.86, 36.97, 51.90, 52.05, 67.80, 68.46, 80.52, 156.73, 173.80, 174.20; HR ESI-MS (0.1% AcOH in MeOH) 283.16317 (M + Na)+

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63 N O H NO Boc H 6h Methyl N-Amino-N’-tert-Butoxycarbonyl-(S)-Leucinate (6h). Rf = 0.40 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 0.85 (d, J = 7.3 Hz, 6H), 1.35 (s, 9H), 1.38-1.44 (m, 1H), 1.69-1.75 (m, 1H), 3.57 (m, 1H ), 3.64 (s, 3H), 4.08 (broad, 1H), 6.48 (broad, 1H); 13C NMR (75 MHz, CDCl3) 22.44, 23.24, 25.22, 28.64, 39.99, 52.36, 62.26, 80.79, 156.64, 174.95; HR ESI-MS (0.1% AcOH in MeOH) 283.16326 (M + Na)+ N O H NO Boc HS 6i Methyl N-Amino-N’-tert-Butoxycarbonyl-(S)-Methioninate (6i). Rf = 0.14 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.44 (s, 9H), 2.00-2.08 (m, 2H), 2.09 (s, 3H), 2.65 (t, J = 7.8 Hz, 2H,) 3.68-3.72 (m, 1H), 3.75 (s, 3H), 4.25 (broad, 1H), 6.25 (broad, 1H); 13C NMR (125 MHz, CDCl3) 15.64, 28.49, 30.06, 30.58, 52.45, 62.42, 80.93, 156.51, 173.79; HR ESI-MS (0.1% AcOH in MeOH) 301.12002 (M + Na)+

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649N O Boc HO N O O Experimental Procedure to Synthesize Methyl N’-Phthalyl-N-tert-Butoxycarbonyl(S)-Lysinate (9). Compound 12b (2.54 mmol) was dissolved in ~10 mL methanol, to which was added a few drops of glacial AcOH and 0.1 g 10% Pd/C. The mixture was shaken under H2 at 50 psi for one hour, filtered through a Celite cak e to remove the Pd/C, and evaporated under reduced pre ssure to give the free amine 7. Deprotected amine 7 (2.54 mmol) was immediately dissolved in 20 mL dry dioxane in a three-necked flask. Phthalic anhydride 8 (2.80 mmol) and TEA (2.80 mmol ) were added, the reaction was fitted with a condenser and drying tube, and it was then refluxed overnight. The reaction was cooled after 16 hours and diluted with EtOAc. Then th e organic layer was washed with dI water (3x) and brine (2x), dried over MgSO4, and evaporated under reduced pressure. The pure product 9 was isolated in 100% yield with no purification needed. Rf = 0.15 (3:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 1.43-1.44 (m, 11H), 1.651.72 (m, 3H), 1.82-1.85 (m, 1H), 3.66-3.70 (m, 2H), 3.73 (s, 3H), 4.28-4.29 (m, 1H), 5.08 (broad, 1H), 7.72 (dd, J = 5.4 Hz, 3.2 Hz, 2H), 7.84 (dd, J = 5.2 Hz, 3.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) 22.75, 28.28, 28.48, 32.34, 37.66, 52.44, 53.42, 67.25, 80.01, 123.39, 132.28, 134.07, 155.56, 168.57, 173.41; HR ESI-MS (0.1% AcOH in MeOH) 413.16832 (M + Na)+

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65Experimental Procedure for Methyl Esterification (12b-12e). The commercially available amino acid 11b-11e (3.49 mmol) was dissolved in dry DMF (20 mL) and cooled to 0 C. Anhydrous potassium carbonate ( 3.84 mmol) was added, and the reaction was flushed with argon and stirred at 0 C for 10 minutes. Met hyl iodide (6.98 mmol) was then added drop-wise via syringe, and th e reaction was allowed to warm up to room temperature. After stirring for 4.5 hours, th e reaction was diluted with ethyl acetate (50 mL) and washed twice with saturated sodi um bicarbonate solution. The organic layer was then washed once with saturated NaCl solution, dried over MgSO4, and evaporated under reduced pressure. The pure product 12c-12e was isolated by fi ltering through a small pad of silica using 3:1 hexa nes:ethyl acetate in 91-100% yield. 12bN O Boc HO N HCbz Methyl N-tert-Butoxycarbonyl-N’-Carboxybenzyl-(S)-Lysinate (12b). Rf = 0.29 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 1.36-1.41 (m, 2H), 1.43 (s, 9H), 1.49-1.56 (m, 2H), 1.60-1.64 (m, 1H), 1.76-1.83 (m, 1H), 3.16-3.21 (m, 2H), 3.73 (s, 3H), 4.24-4.29 (m, 1H), 4.87 (broad, 1H), 5.09 (s, 2H), 5.12 (broad, 1H), 7.31-7.36 (m, 5H); 13C NMR (100 MHz, CDCl3) 14.41, 22.61, 28.52, 29.59, 30.54, 32.58, 40.84, 52.49, 53.38, 66.85, 80.14, 128.31, 128.34, 128.72, 136.83, 156.70, 171.36, 173.48; ESIMS (0.1% AcOH in MeOH) 417.1 (M + Na)+

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66N O Z HOO 12c Methyl tert-Butoxy-N-Carboxybenzyl-(S)-Serinate (12c). Rf = 0.35 (3:1 hexanes/ EtOAc); 1H NMR (250 MHz, CDCl3) 1.12 (s, 9H), 3.57 (dd, J = 3.1 Hz, 9.0 Hz, 1H), 3.74 (s, 3H), 3.81 (dd, J = 2.9 Hz, 9.1 Hz, 1H), 4.45-4.49 (m, 1H), 5.13 (s, 2H), 5.68 (d, J = 8.8 Hz, 1H), 7.31-7.37 (m, 5H); 13C NMR (75 MHz, CDCl3) 27.21, 52.33, 54.59, 61.94, 66.94, 73.38, 128.12, 128.49, 136.25, 156.11, 171.11; HR ESI-MS (0.1% AcOH in MeOH) 310.16486 (M + H)+ N O Z HOO 12d Methyl tert-Butoxy-N-Carboxybenzyl-(S)-Threoninate (12d). Rf = 0.48 (3:1 hexanes/EtOAc); 1H NMR (250 MHz, CDCl3) 1.10 (s, 9 H), 1.22 (d, J = 6.3 Hz, 3H), 3.72 (s, 3H), 4.22-4.26 (m, 2H), 5.14 (s, 2H), 5.60 (d, J = 9.6 Hz, 1H), 7.32-7.41 (m, 5H); 13C NMR (75 MHz, CDCl3) 21.00, 28.24, 52.20, 59.80, 67.01, 67.28, 74.00, 128.13, 128.52, 136.25, 156.76, 171.61; HR ESI-MS (0.1% AcOH in MeOH) 346.16278 (M + Na)+

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6712eN O Z HOO Methyl tert-Butoxy-N-Carboxybenzyl-(S)-Tyrosinate (12e). Rf = 0.36 (3:1 hexanes/ EtOAc); 1H NMR (250 MHz, CDCl3) 1.33 (s, 9H), 3.07 (d, J = 5.9 Hz, 2H), 3.70 (s, 3H), 4.64 (q, J = 8.1 Hz, 1H), 5.10 (s, 2H), 5.28 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 7.32-7.35 (m, 5H); 13C NMR (75 MHz, CDCl3) 28.79, 37.56, 52.26, 54.83, 66.94, 78.43, 124.25, 128.11, 128.19, 128.52, 129.67, 130.43, 136.16, 154.39, 155.58, 172.02; HR ESI-MS (0.1% AcOH in MeOH) 408.17881 (M + Na)+ Experimental Procedure for Cbz Deprotection (1c-1e). The methyl ester 12c-12e (3.04 mmol) was dissolved in MeOH (10 mL), and the mixture was slightly acidified by adding 10 drops of glacial acetic acid. Th en 10% Pd/C (0.4 g) was added, and the reaction was placed under a H2 environment at 50 psi on a Paar hydrogenator. After shaking for 1 hour, the product was filtered th rough a small pad of Celite and evaporated under reduced pressure. No purification was necessary to obtain the pure product 1c-1e in 92-100% yield.

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68N O H HOO 1c Methyl tert-Butoxy-(S)-Serinate (1c). Rf = 0.10 (100% EtOAc); 1H NMR (250 MHz, CDCl3) 0.96 (s, 9H), 3.41-3.48 (m, 3H), 3.53 (s, 3H), 3.75 (broad, 2H); 13C NMR (75 MHz, CDCl3) 27.09, 51.81, 54.47, 63.00, 72.91, 173.67; HR ESI-MS (0.1% AcOH in MeOH) 176.12745 (M+H)+. N O H HOO 1d Methyl tert-Butoxy-(S)-Threoninate (1d). Rf = 0.16 (100% EtOAc); 1H NMR (250 MHz, CDCl3) 1.11 (s, 9H), 1.22 (d, J = 6.2 Hz, 3H), 3.32-3.33 (m, 1H), 3.53 (broad, 2H), 3.70 (s, 3H), 3.99-4.04 (m, 1H); 13C NMR (75 MHz, CDCl3) 20.82, 28.36, 51.93, 60.08, 68.02, 73.72, 174.58; HR ESI-MS (0.1% AcOH in MeOH) 190.14360 (M+H)+. 1eN O H HOO

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69Methyl tert-Butoxy-(S)-Tyrosinate (1e). Rf = 0.26 (100% EtOAc); 1H NMR (250 MHz, CDCl3) 1.32 (s, 9H), 2.33 (broad, 2H), 2.80-2.88 (m, 1H), 3.04 (dd, J = 5.3 Hz, 13.6 Hz, 1H), 3.70 (s, 3H), 3.72-3.75 (m, 1H), 6.92 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H); 13C NMR (75 MHz, CDCl3) 28.77, 40.25, 51.96, 55.63, 78.37, 124.27, 129.64, 131.75, 154.16, 175.29; HR ESI-MS (0.1% AcOH in MeOH) 252.15986 (M+H)+. 2.4: References 1. Toth, B. (2000) A Review of the Natu ral Occurrence, Synthe tic Production, and Use of Carcinogenic Hydrazines and Related Chemicals, In Vivo 14, 299-319. 2. Toth, B. (1996) A Review of the Antin eoplastic Action of Ce rtain Hydrazines and Hydrazine-Containing Natural Products, In Vivo 10, 65-96. 3. Scaman, C. H., Palcic, M. M., McPhalen, C., Gore, M. P., Lam, L. K. P., Vederas, J. C. (1991) Inhibition of Cytoplasmic Aspa rtate Aminotransferase from Porcine Heart by R and S Isomers of Aminooxysu ccinate and Hydrazinosuccinate, Journal of Biological Chemistry 266, 5525-5533. 4. Lister, L. K. P., Arnold, L. D., Kalantar T. H., Kelland, J. G., Lane-Bell, P. M., Palcic, M. M., Pickard, M. A., Vederas, J. C. (1988) Analogs of Diaminopimelic Acid as Inhibitors of meso-D iaminopimelate Dehydrogenase and LLDiaminopimelate Epimerase, Journal of Biological Chemistry 263, 11814-11819. 5. Cox, R. J., Jenkins, H., Schouten J. A., Stentiford, R. A., Wareing, K. J. (2000) Synthesis and in vitro enzyme activity of peptide derivatives of bacterial cell wall biosynthesis inhibitors, Journal of the Chemical Soc iety, Perkin Transactions 1 2023-2036. 6. Kerady, S., Ly, M. G., Pines, S. H., Sletzinger, M. ( 1971) Synthesis of Dand L(3,4-Dihydroxybenzyl)-hydrazinopropionic Acid via Resolution, Journal of Organic Chemistry 36, 1946-1948. 7. Glamkowski, E. J., Gal, G ., Sletzinger, M., Porter, C. C., Watson, L. S. (1967) A New Class of Potent Decarboxylase Inhibitors. -(3-Indolyl)-hydrazinopropionic Acids, Journal of Medicinal Chemistry 10, 852-855. 8. Sletzinger, M., Firestone, R. A., Reinhol d, D. F., Rooney, C. S. (1968) The Hydrazino Analog of Histidine, Journal of Medicinal Chemistry 11, 261-263.

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70 9. Sawayama, T., Kinugasa, H., Nishimura, H. (1976) Syntheses of Ornithine Decarboxylase Inhibitors: Dand DL-Hydrazinoornithine, Chemical and Pharmaceutical Bulletin 24, 326-329. 10. Kerady, S., Ly, M. G., Pines, S. H., Sl etzinger, M. (1971) Synthesis of L-(3,4Dihydroxybenzyl)-hydrazinopropionic Acid from Op tically Active Precursors by N-Homologization, Journal of Organic Chemistry 36, 1949-1951. 11. Viret, J., Gabard, J., Collet, A. (1987) Practical Synthesi s of Optically Active Hydrazino Acids from -Amino Acids, Tetrahedron 43, 891-894. 12. Hoffman, R. B., Kim, H. O. (1990) The pr eparation of 2-Hydrazinyl Esters in High Optical Purity from 2-Sulfonyloxy Esters, Tetrahedron Letters 31, 2953-2956. 13. Gennari, C., Colombo, L., Bertolini, G. (1986) Asymmetric Electrophilic Amination: Synthesis of -Amino and -Hydrazino Acids with High Optical Purity, Journal of the American Chemical Society 108, 6394-6395. 14. Evans, D. A., Britton, T. C., Dorow, R. L ., Dellaria, J. F. (1986) Stereoselective Amination of Chiral Enolates. A New Appr oach to the Asymmetric Synthesis of aHydrazino and a-Amino Acid Derivatives, Journal of the American Chemical Society 108, 6395-6397. 15. Trimble, L. A., Vederas, J. C. (1986) Amination of Chiral Enolates by Dialkyl Azodiformates. Synthesis of -Hydrazino Acids and -Amino Acids Journal of the American Chemical Society 108, 6397-6399. 16. Evans, D. A., Britton, T. C., Dorow, R. L., Dellaria, J. F. (1988) The Asymmetric Synthesis of -Amino and -Hydrazino Acid Derivatives via the Stereoselective Amination of Chiral Enolates with Azodicarboxylate Esters, Tetrahedron 44, 55255540. 17. Andrae, S., Schmitz, E. (1991) Electr ophilic Aminations with Oxaziridines, Synthesis 327-341. 18. Vidal, J., Guy, L., Sterin, S., Collet, A. (1993) Electrophilic Amination: Preparation and Use of N-Boc-3-(4-cyanophenyl)oxaziridi ne, a New Reagent that Transfers a NBoc Group to Nand C-Nucleophiles, Journal of Organic Chemistry 58, 4791-4793. 19. Vidal, J., Damestoy, S., Guy, L., Hannachi, J. C., Aubry, A., Collet, A. (1997) NAlkyloxycarbonyl-3-aryloxaziridines: Their Prep aration, Structure, and Utilization as Electrophilic Amination Reagents, Chemistry: A European Journal 3, 1691-1709.

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71 20. Vidal, J., Hannachi, J. C., Hourdin, G., Mula tier, J. C., Collet, A. (1998) N-Boc-3trichloromethyloxaziridine: a new powerfu l reagent for electrophilic amination, Tetrahedron Letters 39, 8845-8848. 21. Oguz, U., Guilbeau, G., McLaughlin, L. (2002) A Facile Stereospecific Synthesis of -Hydrazino Esters, Tetrahedron Letters 43 2873-2875. 22. Oguz, U. (2003) Design and Synthesis of Constrained Dipeptide Units for Use as Sheet Promoters, Dissertation, Louisiana State University. 23. Hess, H. J., Moreland, W. T., Laubach, G. D. (1963) N-[2-Is opropyl-3-(L-aspartylL-arinyl)-carbazoyl]-L-tyrosyl-L-valyl-Lhistidyl-L-prolyl-L-phenylalanine, an Isostere of Bovine Angiotensin II, Journal of the American Chemical Society 85, 4040-4041. 24. Fasler, A., Bold, G., Capraro, H. G., Cozen s, R., Mestan, J., Ponc ioni, B., Rosel, J., Tintelnot-Blomley, M., Lang, M. (1996) Aza-Peptide Analogs as Potent Human Immunodeficiency Virus Type-1 Protease In hibitors with Oral Bioavailability, J. Med. Chem. 39, 3203-3216. 25. Bouget, K., Aubin, S., Delcro s, J. G., Arlot-Bonemains, Y., Baudy-Floc’h, M. (2003) Hydrazino-Aza and N-Azapeptoids with Ther apeutic Potential as Anticancer Agents, Bioorganic & Medicinal Chemistry 11, 4881-4889. 26. Lecoq, A., Marraud, M., Aubry, A. ( 1991) Hydrazino and N-Amino Peptides. Chemical and Structural Aspects, Tetrahedron Letters 32, 2765-2768. 27. Niedrich, H. (1967) Synthese des Ele doisin-(4-11)-Octapepti des Lys-Asp(NH2)-AlaPhe-Ile-Gly-Leu-Met-NH2 und seines Hete rologen mit Hydrazinoessigsaure statt Glycin, Chemische Berichte 100, 3273-3282. 28. Killian, J. A., Van Cleve, M. D., Shayo, Y. F., Hecht, S. M. (1998) RibosomeMediated Incorporation of Hydrazinophenylalanine into Modi fied Peptide and Protein Analogs, Journal of the American Chemical Society 120, 3032-3042. 29. Guy, L., Vidal, J., Collet, A. (1998) Design and Synthesis of Hydrazinopeptides and Their Evaluation as Human Leukocyte Elastase Inhibitors J. Med. Chem. 41, 48334843. 30. Chen, S., Chrusciel, R. A., Nakanishi, H., Raktabutr, A., Johnson, M. E., Sato, A., Weiner, D., Hoxie, J., Saragovi, H. U., Gr eene, M. I., Kahn, M. (1992) Design and synthesis of a CD4 -turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes, Proc. Natl. Acad. Sci. 89, 5872-5876.

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72 31. Mammen, M., Shakhnovich, E. I., Whitesides, G. M. (1998) Using a Convenient, Quantitative Model for Torsional Entropy to Establish Qualitative Trends for Molecular Processes That Rest rict Conformational Freedom, Journal of Organic Chemistry 63, 3168-3175. 32. Hannachi, J. C., Vidal, J., Mulatier, J. C., Collet, A., (2004) Electrophilic Amination of Amino Acids with N-Bocoxaziridines: Efficient Pr eparation of N-Orthogonally Diprotected Hydrazino Acids and Piperazic Acid Derivatives, J. Org. Chem 69, 2367-2373. 33. Vazquez, M. A., Munoz, F., Donoso, J. (1990) Influence of the Side Chain on the Stability of Schiff-Bases Formed Between Pyridoxal 5’-Phosphate and Amino Acids, International Journal of Chemical Kinetics 22, 905-914. 34. Avancha, K. (2006) Design and Synthesis of Novel HIV-1 Protease Inhibitors and Synthesis and Biological Activ ity of Novel 20S Proteasom e Inhibitors, Dissertation, University of South Florida.

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73 CHAPTER THREE SYNTHESIS OF MDM2 INHIBITORS AND KEY STRUCTURAL UNITS 3.1 Introduction 3.1.1 The Hallmarks of Cancer Cancer is fundamentally a genetic disease ( 1 ). Our current best understanding suggests that cancer arises from multiple genetic transformations, and that the development of normal cells into can cerous cells is a multi-step process ( 1 ). While no one genetic transformation is likely to be sufficient to cause cancer, a combination of several such transformations has been experime ntally and clinically observed to lead to tumorigenesis ( 1 ). In contrast to normal cells, whic h have tightly controlled cell growth cycles, cancer cells have some defect in the regulation of their cell cycles that allows them to proliferate in an uncontrolled manner ( 1 ). Although there are literally dozens of know n types of cancers in various tissues, cancer cells tend to have seve ral common characteristics that relate to their abnormal proliferative ability. These include self-suffi ciency in producing growth signals, lack of response to anti-growth signals, evasion of programmed cell death (a poptosis), no limit to cell reproduction, the inducti on of new blood vessels to support tumor growth (angiogenesis), and the ability to in vade other tissues (metastasis) ( 1 ). These six defining characteristics are thought to be present in mo st if not all cancers; there may be additional common factors such as senescence as well ( 1 ).

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74 Figure 3.1: Cellular Pathways that Ca n Be Affected in Cancer Cells ( 1 ). These pathways are involved in cellular growth a nd death. Bax and p53 are both depicted on the right side of the drawing as compone nts of the intrinsic apoptotic pathway. There are several pathways in cells that are involved with cellular growth and death ( 1 ). An examination of Figure 3.1 emph asizes how complex and redundant these pathways are, as well as their inter-relatedne ss. MDM2, which is an inhibitor of the proapoptotic tumor suppressor gene p53, and Ba x, which is a promoter of apoptosis, can both be located at the right-han d side of the figure. These two proteins and many others involved with apoptosis are poten tial targets for disrupting prot ein-protein interactions in

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75 order to promote apoptosis. This chapter will describe the design and synthesis of three potential small-molecule inhibitors of MDM 2, as well as three key structural components needed to synthesi ze inhibitory MDM2 -helix mimics. Chapter 4 will focus on the design and synthesis of an -helix mimic that is intended to stabilize the active form of the Bax protein. 3.1.2 Apoptosis and MDM2/p53 The cells in a multicellular organism ex ist in a precarious balance between life and programmed cell death, or apoptosis. A poptosis is the process by which unwanted cells that have genetic abnormalities, physical damage, or parasitic infection can be removed from the body ( 2 3 ). In normal cells, the deci sion to undergo apoptosis is governed by the relative levels of pro-apopto tic and anti-apoptotic proteins. Abnormal expression of many of these prot eins has been implicated in the transformation of normal cells into cancer cells, and in fact is a common charac teristic of cancer cells ( 1,3 ). Apoptosis is a controlled process that is often contrasted to necrosis, where cells die off in an uncontrolled manner ( 2 ). However, it is probably more correct to think of necrosis and apoptosis as two extremes on a continuum of types of cell death ( 2 ). Apoptosis can be recognized based on specifi c morphological features, including nuclear fragmentation, chromatin conde nsation, cell shrinkage, and “blebbing” of the cell membrane ( 4 ). Traditionally, apoptosis was considered as only being effected by a family of proteases called caspases, but there are also non-caspase dependent forms of controlled cell death known ( 2 ). The caspases are activated by two primary pathways, which are illustrated in Figure 3.2. The intrinsic pathway involves the mitochondria, while the

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76 extrinsic pathway involves si gnaling via death receptors ( 3,4 ). Intrinsic pathway apoptosis occurs due to the release of cytochrome c fr om the mitochondria following depolarization of the mitochondrial proton gradient ( 3,4 ). The intrinsic pathway is discussed further in the Introduc tion section of Chapter Four. Figure 3.2: The Intrinsic (Mitochondria l) and Extrinsic (FAS) Pathways ( 3 ). Both p53/MDM2 and Bax (discussed in Chapter 4) ar e members of the intrinsic pathway. The members of this pathway function by either promoting or inhibiting cytochrome c release from the mitochondria. One method by which cells become cancerous is due to the abnormal expression of mutated oncogenes, which c ode for proteins that affect the cell cycle, or tumor suppression genes, which bl ock cell cycle progression ( 1 ). p53 is a tumor suppressor

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77 protein that detects damage to a cell’s DNA and either prevents the cell from dividing until after the DNA has been repaired, or ca uses the cell to unde rgo apoptosis if the damage to the DNA is severe enough ( 1,3,5 ). p53 is regulated by MDM2 or HDM2, which are oncogene proteins in mice and humans respectively ( 3 ). The remainder of this Introduction will use the te rm MDM2 to refer to both proteins. MDM2 physically inhibits p53, and it also promotes the ubiquitination of p53; p53 ubiquitination leads to the degradation of p53 by the proteasome ( 3,5,6 ). In normal cells, p53 is unstable and has a short half-life due to MD M2-mediated degradation ( 6 ). Prevention of the MDM2-p53 interaction leng thens p53’s half-life considerably, from minutes to hours ( 6 ). Figure 3.3: Regulation of p53 by MDM2 ( 7 ) This drawing show s the auto-regulatory feedback loop between p53 and MDM2. Figure 3.3 is a schematic showing the regulation of p53 by MDM2 ( 7 ). MDM2 and p53 are tied together in an auto-regulat ory feedback loop, where p53 stimulates the expression of MDM2, and MDM2 inhibits p53 ( 7 ). However, the pathway is very

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78 complex and involves multiple other proteins. For example, one protein called ARF is responsible for inhibiting MDM2 ( 7 ). There are also multiple splice variants of MDM2, as well as related proteins with similar functions such as MDMX ( 7 ). MDMX and MDM2 are known to interact with each other, and this interac tion is also important in the regulation of p53 ( 8 ). Since apoptosis pathways are abnormal in many cancer cells and are not abnormal in regular cells, it is theoretically possible to develop pro-apoptotic drugs that selectively target cancer cells ( 1,3 ). For example, it is estimated that up to half of human cancers have a disrupted or mutated p53 pathway, maki ng p53 and its protein-protein interactions an important target of anti-cancer chemotherapy ( 3 ). Although p53 itself can be under-expressed due to mutation, its turnover can also be increased due to abnorma lly high levels of MDM2 ( 3,4 ). For this reas on, inhibitors of MDM2 would be expected to restore the ap optotic pathway in cancer cells that overexpress this protein ( 7 ). This assumes that the cells st ill have normal expression of p53, which is typically the case ( 7 ). There are certain types of cancers that are more likely to have higher levels of MDM2 over-e xpression, including so ft tissue tumors, osteosarcomas, and gliomas ( 8 ). 3.1.3 Inhibiting the MDM2/p53 Interaction with Small Molecules MDM2 is an attractive target for several reasons ( 9 ). One of them is that the p53 regulatory function of MDM2 has been well characterized at the biological level ( 9 ). Another reason is that the crystal structure of the MDM2/p53 bindi ng complex has been known for the past ten years ( 9 ). (The crystal structure of the MDM2/p53 complex is shown in panel A of Figure 3.6 below.) A nd finally, experiments with site-directed

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79 mutagenesis and peptide inhibitors have established a clearly-defined pharmacophore, such that the binding requirements of any anti-MDM2 drug are well understood ( 9 ). Several compounds have been reported that inhibit the p53-MDM 2 interaction; in general, they mimic p53 as opposed to MDM2 ( 6 ). This is because MDM2 (but not p53) has a well-defined bind ing site, and p53 (but not MDM2) ha s an interface comprised of a single, short stretch of ami no acids arranged in an alpha helix secondary structure ( 6,7 ). The MDM2/p53 interaction has al so been studied using mole cular modeling in order to analyze the movements of both proteins when they are bound together versus unbound ( 10 ). It was found that the conformation of MDM2 changes very little upon association with the p53 helix, suggesti ng that a good inhibitor of MDM2 can be developed ( 10 ). The first reported p53 inhibitors were phage -display peptide libraries that showed greater affinity for MDM2 than even p53 itself did ( 6 ). The use of unnatural amino acids and conformational restrictions improved pep tide potency even more, and also provided the proof of concept that in hibitors of MDM2 could bloc k its interaction with p53 ( 6 ). Unfortunately, peptides do not make good drugs for many reasons, including their lack of oral bioavailability and their susceptibility to proteases ( 11 ). Thus, several groups are working to make small-molecule inhibitors that can block MDM2/p53 interactions that would hopefully show improved drug-like prop erties in comparison to peptides. Until quite recently, it was believed that using small molecules to inhibit proteinprotein interactions would be difficu lt at best and impossible at worst ( 12 ). However, there have now been several examples of small-molecule protein-protein inhibitors published. These compounds can either act direc tly such that they inhibit the proteins from interacting, or indirectly at an allosteric site ( 12 ). Although many protein-protein

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80 interactions are difficult to disrupt using sma ll-molecule inhibitors due to the lack of a well-defined binding pocket, the p53/MDM2 interaction is particularly attractive in this regard because the interface be tween the two molecules is relatively small, and it is known that p53 does bury itself into a pocket on MDM2 ( 7 ). Figure 3.4: Small Molecule Inhibitors of MDM2 ( 13 ). Panel A is an example of a chalcone, and Panel B is a boron ic chalcone. Chlorofusin is shown in Panel C. Panel D shows nutlin 2, the best currently known small-molecule inhibitor of MDM2. The first small-molecule inhibitors of MDM2 to be identified were chalcones, which are pictured in panel A of Figure 3.4 ( 13 ). These compounds are weak MDM2

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81 inhibitors with IC50 values in the high micromolar range ( 13 ). Chalcones were known to have anti-tumor properties, and NMR studies showed that they bind to a portion of the p53-binding site on MDM2 ( 14 ). In addition, biochemical experiments established that chalcones can disrupt MD M2/p53 interactions ( 14 ). Boron-containing chalcone derivatives have also been described ( 13 ); an example is shown in panel B of Figure 3.4. Another MDM2 inhibitor is chlorofusin, a cyclic peptide derivative that was isolated and characterized from a microorganism associated with a Fusarium genus fungus ( 13,15 ). The structure of chlorofusin is s hown in Panel C of Figure 3.4. It is comprised of a cyclic peptide with a non-peptide chromophore attached to it ( 15 ). An examination of this structure shows that it is too large, complex, and peptide-like to be a good drug candidate. The cyclic peptide por tion of chlorofusin was synthesized and tested alone; it was found not to in hibit the p53-MDM2 interaction ( 16 ). This suggests that the chromophore portion of the molecule may be needed for the compound’s activity, which is also in the micromolar range ( 13 ). Recently, the nutlins, a class of potent, orally bioavailable small-molecule inhibitors of the MDM2/p53 interaction were reported ( 17 ). The nutlins were found to bind MDM2 in the p53 pocket, activate p53 in tumor cells, and induce apoptosis ( 17 ). They are active at a submicromolar level ( 17 ). The structure of nutlin-2 is shown in panel D of Figure 3.4. Nutlin-2’s crystal st ructure with MDM2 showed that nutlin-2 closely mimics the binding properties of p53 ( 17 ). In particular, th ree binding residues of p53 (Phe, Trp, and Leu) were all mimicked by nu tlin-2, in contrast to previous inhibitors like the chalcones that only replaced a portion of the p53 alpha helix ( 13 ).

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82 Biological tests with another compound, nutlin -3, showed that nutlin-3 selectively induces the p53 pathway, promoting cell cycle arre st and apoptosis in vivo as well as in vitro ( 18 ). Cancer cells with over-expressed or even normal levels of MDM2 both showed sensitivity to nutlin-3, but only if p53 expression was normal ( 18 ). Fortunately, it appears that most tumors that over-expre ss MDM2 do not also have mutations of p53 as well ( 18 ). It seems reasonable that tumor cel ls that over-express MDM2 would most likely have intact p53 signa ling, because further attenua tion of p53 would not be necessary for them to avoid undergoing apoptosis ( 18 ). OH O O O NH HN O Figure 3.5: Structure of syc-7, a No n-Peptide Inhibitor of HDM2 ( 19 ). This compound and some of its deri vatives were able to inhi bit HDM2/p53 interactions and induce apoptosis in some tumor cell lines. Finally, there have also been a series of small-molecule inhibitors of the HDM2/p53 association reported ( 19 ). These compounds, called syc compounds, were conceived using computer-aided design, and then synthesized ( 19 ). One compound in particular, syc-7, showed an ability to inhi bit HDM2-p53 interactions and also could be seen to activate the p53 pathway and induce apoptosis in four different tumor cell lines ( 19 ). However, the concentrations of syc-7 needed to i nduce apoptosis were

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83 prohibitively high for a drug, in the low millimolar range ( 19 ). The structure of syc-7 is shown in Figure 3.5. 3.1.4 Inhibiting MDM2/p53 with Pe ptides and Peptidomimetics As mentioned previously, the first inhib itors of the MDM2/ p53 interaction were peptides and modified peptides obtained by screening phage libraries, reported in 1996 ( 6,20 ). These researchers originally used peptides to establish the p53 pharmacophore, and they discovered that the hexapeptide Thr-Phe-Ser-Asp-Leu-Trp was the consensus sequence ( 20 ). However, this peptide had fairly low binding affinity that was in the micromolar range ( 20 ). Somewhat better results we re obtained by screening a phage display peptide library, which improve d the binding affinity by 28-fold ( 20 ). Starting with an octapeptide lead, the binding affin ity of this peptide for HDM2 was improved even more to the low nanomolar range by replacing non-pharmacophore amino acids with -disubstituted amino acids ( 20 ). It is known that the use of disubstituted amino acids can stabilize the -helix secondary structure of a short peptide ( 20 ). More recently, Andy Hamilton’s group at Yale has made a variety of -helix-like peptidomimetics based on a terphenyl scaffold ( 21,22 ). The substituents on the benzene rings in these structures are too close together to allow the benzene rings to be coplanar, which gives the molecule a twisted or helical shape. Placing substituents at the ortho positions on each benzene ring orients these substituents so that they mimic the orientation of the amino acid si de chains on an alpha helix ( 21,22 ). The relationship between Hamilton’s helices and the p53 -helix can be seen in Figure 3.6. Panel A shows the crystal structure of p53 interacting with MDM2. The orientation of the side

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84 chains at the i i +4, and i +7 positions can be seen in p53 versus a terphenyl helix in Panel B. (These are the essential Phe, Trp, and Leu residues identified previously.) Figure 3.6: Design, Model, and Structures of Hamilton’s Terphenyl Helices ( 21 22 ). Panel A shows the x-ray crystal structure of p53 (backbone) complexed with MDM2 (space-filling). Pane l B shows how the physi cal topography of an -helix is mimicked by the Hamilton helices. Several examples of Hamilton helices that were screened against MDM2 are shown in Panel C. Hamilton’s group discovered that the best inhibition occurred with the use of an isobutyl or benzyl group on the fi rst phenyl ring, mimicking Phe ( 22 ). In addition, it was helpful to have an extra methyl side chain on the second phenyl ri ng, since this made the compound more rigid and better able to induce activation of p53 ( 22 ). Fortuitously, it was found that several of the terphenyl comp ounds were membrane-permeable, and they could induce p53 activation and accumulation in tumor cells ( 22 ). Several examples of

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85 p53-like terphenyl helices are s hown in Panel C. Other te rphenyl helices have been developed as mimics of the Bak BH3 domain; this work will be discussed further in Chapter Four. 3.2 Results and Discussion Although Hamilton’s helices seem like a promising idea for p53 mimics, they do have the disadvantage of being nonpolar and th erefore not very water-soluble. They are also difficult to synthesize. Accordingly, we have desi gned a new set of p53 helices based on hydrazino subunits that can be assembled into a set of piperazine dione rings, as shown in the top panel of Figures 3.7. As with Hamilton’s terphenyl helices, the McLaughlin compounds are also predicted to form a twisted shape based on molecular models. The model is shown in the bottom panel of Figure 3.7. Figure 3.7: Structure and Model of the McLaughlin Piperazine Helix. Not only does this helix twist like the Hamilton Helix doe s, it has the additional advantage of being more polar and water-soluble. The building blocks of the helix can be synthesized from ordinary natural or unnatural amino acids, allo wing for a great divers ity of side chains.

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86 The chemistry to make piperazine dione s is well known, and there are several examples in the medicinal chemistry literature ( 23 ). These compounds can be made from ethylene diamine tetraacetic acid tetraethyl ester scaffolds ( 23 ). Members of several classes of drugs contain pipe razine diones, including topoiso merase inhibitors, tricyclic anti-depressants, and antihistamines ( 23 ). In addition, thes e compounds can be made using solid-phase synthesis, as well as in solution ( 24 ). As far as we are aware, there are currently no examples in the literature of hydrazino pipera zine diones from amino acids. X O N BocHN R1 H O X RN N O O R1 NHBoc H 1) 95% TFA 2) TEA 3) couple X O N BocHN R2 H O X 4) 95% TFA 5) TEA 6) couple ResinN N O O R1 N H N O O NHBoc R2 H RN N O O R1 N H N O O N R2 H N O O R3 H NHBoc repeat NNH O O R1 N H NH O O N R2 H NH O O R3 H NH A1A2Resin O O NHBoc HO O O OH O 10) 95% TFA 11) TEA 12) CO2Bn X O 13) HBr, AcOH for A3 Figure 3.8: Synthesis of McLaughlin Piperazine Dione -Helix Mimics. The hydrazino units will be incorporated into pi perazine dione peptidomimetics via solidphase synthesis. X represents an activated carboxylic acid; R is an amino acid side chain. The McLaughlin helices have the advant age of being amenable to solid-phase peptide synthesis, thus facil itating their constructi on. They also have the advantage that multiple natural and unnatural amino acid si de chains can be inserted into the peptidomimetic. Figure 3.8 shows how the hydr azino subunits can be combined into the piperazine dione compound. Compounds 17a – c and other alkylated hydrazino amino

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87 acids can be incorporated into the piperazine dione alpha helix mimics described above. This is currently being done in our lab via solid-phase sy nthesis through a series of deprotection, activation, and coupli ng steps. The synthesis of hydrazines 17a-c is discussed below. O Br O O N O OO R H O N O O O R N O O H HO N OH OO R N O O H H2N O O R ClHLiOH CH3OH DIEA CH3CN CH3OH O NCl3CO O 1015a-c 14a-c 16a-c 17a-c a bcR = N H N Scheme 3.1: Synthesis of Alky lated Hydrazino Amino Diacids. N-amination of the three alkylated amino acids with oxaziridine 10 ( 25, 26) was the key step in this synthesis. Hydrolysis of hydrazine 16a gave product 17a The synthesis of compounds 16b-c and 17b-c was not completed.

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8818 NaH MeI DMF H2/Pd MeOH AcOH K2CO3CH3I DMF 91-100%19a 19b 14c 14bCbz = O O H2/Pd MeOH AcOHN OHCbz HO NH N O Cbz HO NH N O H HO N N O H HO NH N O Cbz HO N Scheme 3.2: Synthesis of Trypto phan Methyl Esters 14b and 14c. Both compounds were prepared via a series of protection and deprotection steps from commercially available Cbz-tryptophan. Each of these steps proceeds with approximately quantitative yields. Hydrazino amino acid derivative 17a was synthesized to be used as a subunit in the construction of the McLaughlin helices. It was synthesized using the procedure depicted in Scheme 3.1. The methyl ester of the amino acid 14a was first reacted with

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89 ethyl bromoacetate in the presence of diis opropylethylamine to form secondary amine 15a This compound was then reacted with oxaziridine 10 ( 25, 26 ) to give the Bocprotected hydrazine 16a Oxaziridine 10 here is the same co mpound that was used previously to synthesize compound 5b as described in Chapter Two. The final step in the synthesis was to hydrolyze both esters using lithium hydroxide to give 17a The synthesis of two other hydr azino amino acid derivatives ( 17b-c ) was partially completed. The isoleucine methyl ester 14a was commercially availa ble as the hydrochloride salt, but the two tryptophan methyl esters 14b and 14c had to be synthesized from commercially available compound 18 as shown in Scheme 3.2. Compound 18 is the Cbzprotected amino acid tryptophan. The first step was to make the methyl ester 19a of 18 using potassium carbonate an d methyl iodide. Compound 19a was then split into two portions. One portion was hydrogenated to directly form compound 14b which was then used to form the hydrazine diacid 17b as shown in Scheme 3.1. The other portion was Nalkylated on the indole nitrogen to give 19b and compound 19b was then also hydrogenated to form the alkyl ated indole tryptophan ester 14c Finally, compound 14c was converted into hydrazine diacid 17c also shown in Scheme 3.1. Initially, our synthetic procedure called fo r using sodium hydr oxide to hydrolyze the esters rather than lithium hydroxide. This procedur e worked fine for some of the first amino acids tried, which included phenylalanine and leucin e. However, the hindered amino acids valine and isoleu cine did not hydrolyze to the diacid with NaOH. Instead, treatment of isoleucine hydrazine diester 16a with NaOH consistently yielded monoacid 20 instead of the desired diacid 17a We tried refluxing 16a for 48 hours using NaOH, but we still could not obtain the diacid. Ho wever, we finally were able to obtain 17a

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90 when we used lithium hydroxide. S ubsequent attempts at hydrolysis of 16a with NaOH were finally successful, but doing this required harsh conditions that might lead to the racemization of the alpha carbon. However, we did not observe any such racemization. O N O O O N O O H HO N OH OO N O O H LiOH CH3OH 16a 17aO N O O O N O O H HO N O O O N O O H 16a 20NaOH CH3OH Scheme 3.3: Results of the Hydrol ysis of 16a with NaOH versus LiOH. Unlike the less hindered amino acids, use of NaOH to hydr olyze the Ile diester led to the monoacid product 20 rather than the desired diacid 17a Compound 17a was ultimately obtained by hydrolysis of 16a with LiOH. N OH O O N H NSC-131734 Gly-465 Figure 3.9: MDM2 Small-Molecule I nhibitor Lead Compound NSC-131734. Our analogs 23 attempted to mimic the quinoline and anthracene systems of this compound, and all three compounds could be sy nthesized easily in a single step.

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91N H Cl NaH DMFN N H Cl NaH DMFN N H Cl NaH DMFN 21a 21b 21c 22 22 22 23a 23b 23c Scheme 3.4: Synthesis of MDM2 Inhibitors 23a-c. All three compounds were synthesized in a single step from 1-(chlorom ethyl)naphthalene and the appropriate indole or carbazole derivative. We have also been working on the desi gn and synthesis of a small library of small-molecule inhibitors of MDM2. One compound that showed modest MDM2 inhibitory activity was NCI compound NSC131734, shown in Figure 3.9. Three mimics 23 of NSC-131734 were easily synthesized usi ng similar chemistry as previously described for the tryptophan alkylation. Th is procedure is shown in Scheme 3.4. 2,3dimethylindole ( 21a ), carbazole ( 21b ), and 1,2,3,4-tetrahydracarbazole ( 21c ) were each treated with sodium hydride and th en 1-(chloromethyl)-naphthalene ( 22 ) to give Nalkylated products 23

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92 All three of these compounds were tested as inhibitors of MDM2 and were found to be inactive. This may be due to th eir hydrophobicity, as a ll three compounds are insoluble in water and are significantly less polar than the model NCI compound. It may also be due to the molecules being too sma ll to adequately cover the entire p53 binding pocket on MDM2. In conclusion, we successfully carried out the synthesis of is oleucine hydrazino amino acid derivative 17a We also made progress toward the synthesis of two tryptophan hydrazino acid derivatives 17b-c Finally, we synthesized three potential small-molecule inhibitors ( 23a-c ) of MDM2 that were modeled after a screened NCI compound. Further work on this project wi ll entail completion of the syntheses of 17b-c assembly of the hydrazino amino acid deriva tives into piperazine diones, and further biological testing of both th e piperazine diones and the small-molecule inhibitors 23 3.3 Experimental Data Experimental Procedure for N-Alkylation. The free amine 14 (5.50 mmol) was dissolved in acetonitrile (10 mL). Diis opropylethylamine ( 11.01 mmol) and ethyl bromoacetate (16.51 mmol) were added via syri nge. The reaction was stirred overnight in an argon atmosphere at room temperature. The resulting product was washed with 5% citric acid, the organic layer was separate d, and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine (1 x 20 mL), dried with magnesium sulfate and eva porated under reduced pressure, yielding a yellow oil. The crude product was purified by column chromatography (4:1 hexane/EtOAc) to give pure 15 a colorless oil.

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93O N O OO H 15a Methyl N-(2-Ethoxy-2-Oxoethyl)-( S )-Isoleucinate (15a). Rf = 0.62 (3:1 hexanes /EtOAc); 1H NMR (400 MHz, CDCl3) 0.82-0.86 (m, 6 H), 1.12-1.22 (m, 4 H), 1.451.51 (m, 1 H), 1.63-1.69 (m, 1 H), 1.96 (s, 1 H), 3.08 (d, J = 6.0 Hz, 1 H), 3.24 (d, J = 17.2 Hz, 1 H), 3.34 (d, J = 17.2 Hz, 1 H), 3.65 (s, 3 H), 4.11 (q, J = 7.2 Hz, 2 H); 13C NMR (100 MHz, CDCl3) 11.62, 14.33, 15.72 25.63, 38.48, 49.94, 51.65, 60.90, 65.76, 171.96, 174.83 ppm; MS (GC/MS) m/z 232, 199, 172, 158, 146, 130, 114, 100, 86, 69, 56. 15bN O O HNH O O Methyl N-(2-Ethoxy-2-Oxoethyl)-( S )-Tryptophanate (15b). Rf = 0.73 (100% EtOAc), Rf = 0.04 (3:1 hexanes /EtOAc); 1H NMR (400 MHz, CDCl3) 1.19 (t, J = 6.8 Hz, 3H), 2.37 (broad, 1H), 3.14-3.19 (m, 1H), 3.26 (dd, J = 6.0 Hz, 14.4 Hz, 1H), 3.323.36 (m, 1H), 3.45-3.49 (m, 1H), 3.66 (s, 3H), 3.72 (t, J = 6.0 Hz, 1H), 4.10 (q, J = 6.8 Hz, 2H), 7.10-7.13 (m, 2H), 7.16-7.20 (m, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 8.27 (broad, 1H); 13C NMR (100 MHz, CDCl3) 14.32, 29.33, 49.45, 52.17, 61.11, 61.38, 110.94, 111.48, 118.87, 119.68, 122.31, 123.33, 127.60, 136.48, 171.84, 174.59; HR ESI-MS (0.1% AcOH in MeOH) 327.13188 (M + Na)+

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9415cN O O HN O O Methyl N’-Methyl-N-(2-Ethoxy-2-Oxoethyl)-( S )-Tryptophanate (15c). Rf = 0.81 (100% EtOAc), Rf = 0.12 (3:1 hexanes /EtOAc); 1H NMR (400 MHz, CDCl3) 1.22 (t, J = 7.2 Hz, 3H), 2.17 (broad, 1H), 3.14 (dd, J = 7.2 Hz, 14.8 Hz, 1H), 3.24 (dd, J = 5.6 Hz, 14.4 Hz, 1H), 3.31-3.36 (m, 1H), 3.44-3.3.50 (m, 1H), 3.66 (s, 3H), 3.70 (t, J = 5.6 Hz, 1H), 3.75 (s, 3H), 4.11 (q, J = 7.6 Hz, 2H), 6.98 (s, 1H), 7.09-7.13 (m, 1H), 7.20-7.24 (m, 1H), 7.26-7.30 (m, 2H), 7.59 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) 14.30, 28.70, 32.96, 49.03, 52.40, 61.24, 61.44, 108.66, 109.57, 118.91, 119.30, 122.00, 127.98, 128.27, 137.27, 170.92, 172.76; HR ESI-MS (0.1% AcOH in MeOH) 341.14739 (M + Na)+ Experimental Procedure for Hydrazine Formation (16a-c). Compound 15 (5.75 mmol) was dissolved in methanol (12 mL) in a round bottom flask and cooled to –78 C under an argon atmosphere. Oxaziridine 10 (6.55 mmol, Ref. 25,26 ) was dissolved in methanol (10 mL) and added to the reaction mixture slowly via syringe, and the reaction was allowed to warm to room temperature and stirred overnight. The reaction was then washed with water, the organic layer was se parated, and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine (1 x 25 mL), dried with magnesium sulfate a nd evaporated under reduced pressure. The

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95 crude product was purified by column chromat ography (9:1 hexane/EtOAc) to give pure 16 a colorless oil. O N O OO NHO O 16a Methyl N”tert -Butoxycarbonyl-N-(2-Ethoxy-2-Oxoethyl)-( S )-Isoleucinate (16a). Rf = 0.53 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 0.83-0.94 (m, 6 H), 1.16-1.22 (m, 1H), 1.23 (t, 3 H), 1.44 (s, 9 H), 1.74-1.78 (m, 1 H), 1.95-2.01 (m, 1H), 3.16-3.21 (m, 1H), 3.57-3.61 (m, 1 H), 3.69-3.76 (m, 4 H), 4.15 (q, J = 7.2, 2H), 7.21 (broad, 1H); 13C NMR (100 MHz, CDCl3) 11.03, 11.67, 14.33, 14.37, 15.47, 15.74, 25.71, 28.18, 28.41, 28.47, 34.91, 38.47, 49.92, 51.47, 51.75, 56. 55, 60.93, 61.03, 65.78, 71.99, 80.15, 170.52, 171.91, 172.89, 174.76 ppm; MS (GC/MS) m/z 346, 287, 273, 246, 231, 217, 189, 173, 157, 145, 129, 113, 101, 87, 69, 57; HR ESI-MS (0.1% AcOH in MeOH) 369.19977 (M + Na)+ 20HO N O OO NHO O

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96 Procedure for N”tert -Butoxycarbonyl-N-(2 -Ethoxy-2-Oxoethyl)-( S )-Isoleucinate Synthesis (20) Compound 16a (0.75 mmol) was dissolved in methanol (3 mL), and 1.87 mL of a 1.0 solution of sodium hydroxide was a dded to it by syringe The reaction was stirred for four hours at RT. The resulting prod uct was washed with potassium bisulfate. The organic layer was separated and the aqueous layer was extracted w ith ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (1 x 15 mL), dried with magnesium sulfate and dried und er reduced pressure, yielding 20 (0.21 g, 79%), a colorless oil; Rf = 0.69 (9:1 DCM/MeOH); 1H NMR (400 MHz, CDCl3) 0.80-0.86 (m, 6 H), 1.14-1.19 (m, 1 H), 1.40 (s, 9 H), 1.73-1.76 (m, 1 H), 1.77-1.89 (m, 1H), 3.16 (d, J = 9.6 Hz, 1 H), 3.45-3.54 (m, 2 H), 3.71 (s, 3 H), 7.21 (broad, 1H); 13C NMR (100 MHz, CDCl3) 11.14, 15.70, 26.26, 28.36, 28.41, 35 .50, 52.12, 72.74, 82.78, 158.00, 171.17 ppm; ESI-MS (0.1% AcOH in MeOH) 317.1 (M H)Procedure for Diacid Synthesis. Compound 16a (0.45 g, 1.3 mmol) was dissolved in methanol (13 mL). 0.5 M lithium hydroxide (9.29 mL) was added, the flask was heated to 50 C and stirred for 24 hours. The resu lting product was wash ed with potassium bisulfate. The organic layer was separate d and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (1 x 15 mL), dried with magnesium sulfate and dried under reduced pressure. The crude product was recrystallized (DCM/hexane) yielding 17a (0.17g, 45%), a white solid.

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9717aHO N OH OO NHO O N”tert -Butoxycarbonyl-N-Carboxymethyl-( S )-Isoleucinate (17a) MP = 98-100 C; Rf = 0.13 (9:1 DCM/MeOH); 1H NMR (400 MHz, CD3OD) 0.89-0.95 (m, 6 H), 1.171.30 (m, 1 H), 1.46 (s, 9 H), 1.71 -1.87 (m, 2 H), 3.34-3.36 (m, 1 H), 3.64-3.69 (m, 2 H); 13C NMR (100 MHz, CD3OD) 9.87, 10.41, 10.60, 14.55, 14.86, 15.38, 25.12, 25.61, 26.03, 27.40, 34.38, 35.11, 35.45, 53.63, 57.82, 70.56, 71.82, 72.19, 80.83, 157.12, 172.26, 174.07, 174.18, 174.74 ppm; ESI-MS (0.1% AcOH in MeOH) 303.1 (M H)19aN O Cbz HO NH Experimental Procedure for Methyl Es terification of N-Carboxybenzyl-( S )Tryptophanate to Methyl N-Carboxybenzyl-( S )-Tryptophanate (19a). The commercially available Cbzprotected Trp 18 (1.48 mmol) was dissolved in dry DMF (20 mL) and cooled to 0 C. Anhydrous potassium car bonate (1.63 mmol) was added, and the reaction was flushed w ith argon and stirred at 0 C for 10 minutes. Methyl iodide

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98 (2.96 mmol) was then added drop-wise via syri nge, and the reaction was allowed to warm up to room temperature. After stirring for 4.5 hours, the reaction was diluted with ethyl acetate (50 mL) and washed twice with sa turated sodium bicarbonate solution. The organic layer was then washed once with saturated NaCl solution, dried over MgSO4, and evaporated under reduced pr essure. The pure product 19a was isolated by filtering through a small pad of silica using 3:1 hexanes:ethyl acetate in 100% yield. Rf = 0.59 (1:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 3.31 (d, J = 5.6 Hz, 2H), 3.68 (s, 3H), 4.71-4.75 (m, 1H), 5.07-5.15 (m, 2H), 5.42 (d, J = 8 Hz, 1H), 6.92 (s, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.32-7.35 (m, 6H), 7.53 (d, J = 8.0 Hz, 1H), 8.42 (broad, 1H); 13C NMR (100 MHz, CD3OD) 28.18, 52.61, 54.81, 67.19, 109.87, 111.59, 118.78, 119.84, 122.39, 123.24, 127.76, 128.36, 128.41, 128.77, 136.44, 136.55, 156.12, 172.74; ESI-MS (0.1% AcOH in MeOH) 353.1 (M + H)+ 19bN O Cbz HO N Experimental Procedure to Form Methyl N’-Methyl-N-Carboxybenzyl-( S )Tryptophanate (19b). The Trp methyl ester 19a (1.48 mmol) was dissolved in 25 mL anhydrous DMF and the reaction mixture was cooled to 0 C. Unwashed 60% sodium hydride was added slowly, and the reaction was stirred for fifteen minutes until all effervescence had ceased. Then methyl i odide (1.63 mmol) was added, and the reaction

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99 was allowed to warm to room temperature an d stirred for three hou rs. It was quenched with water, forming an oily white precipitate that could not be filtered. The reaction mixture was acidified with 10% KHSO4 to pH = 3, and the aqueous layer was extracted three times with DCM. The combined organi c layer was then extracted twice with dI water and once with brine, dried over MgSO4, and evaporated under reduced pressure to give the pale yellow oil 19b in 77.8% yield. Rf = 0.67 (1:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) 3.33 (d, J = 5.6 Hz, 2H), 3.71 (s, 3H), 3.72 (s, 3H), 4.73-4.75 (m, 1H), 5.10-5.18 (m, 2H), 5.40 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 7.10-7.14 (m, 1H), 7.227.37 (m, 7H), 7.54 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CD3OD) 28.04, 32.94, 52.59, 54.81, 67.15, 108.45, 109.59, 118.98, 119.44, 122.05, 127.79, 128.20, 128.30, 128.41, 128.78, 136.64, 137.19, 156.07, 172.69; ESI-MS (0.1% AcOH in MeOH) 367.1 (M + H)+ Experimental Procedure to Remove the Cbz Protecting Group (14b-c). The methylated tryptophan derivatives (0.426 mm ol) were dissolved in a few mL of methanol/THF, to which was added 0.1 g 10% Pd/C and a couple of drops of glacial acetic acid. This mixture was then placed in a hydrogen atmosphere at approximately 50 psi and shaken for one hour, after which the Pd/C was removed by filtering the dissolved product through a Celite bed. The product was obtained as a white solid in 100% yield.

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10014bHN O O HNH Methyl ( S )-Tryptophanate (14b). MP = 101-103 C; Rf = 0.08 (100 % EtOAc); 1H NMR (400 MHz, CD3OD) 3.59 (s, 3H), 4.00-4.03 (m, 1H), 6.89-6.91 (m, 1H), 6.956.97 (m, 1H), 7.01 (s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 8.0 Hz); 13C NMR (100 MHz, CD3OD) 27.93, 52.60, 54.26, 107.63, 111.90, 118.22, 119.49, 122.13, 124.64, 127.63, 137.55, 171.75; HR ESI-MS 219.11305 (0.1% AcOH in MeOH) (M + H)+ 14cHN O O HN Methyl N’-Methyl -( S )-Tryptophanate (14c). MP = 105-106 C; Rf = 0.15 (100% EtOAc); 1H NMR (400 MHz, CDCl3) 3.07-3.13 (m, 1H), 3.29 (dd, J = 4.4 Hz, 14.4Hz, 1H), 3.85-3.88 (m, 1H), 3.73 (s, 3H), 3.76 (s, 3H ), 4.96 (broad, 2H), 6.94 (s, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.21-7.26 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) 30.27, 32.93, 52.35, 54.75, 109 .27, 109.55, 119.03, 119.32, 122.02, 128.05, 128.14, 137.30, 175.48; HR ESI-MS ( 0.1% AcOH in MeOH) 233.12831 (M + H)+

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101 Procedure to Synthesize MDM2 Inhibitors. 60% sodium hydride (0.897 mmol) was washed two times with dry DMF to remove the oil, and then suspended in approximately ten mL dry DMF. The reaction was cooled to 0 C, and then the indole or carbazole derivative (0.598 mmol) was added slowly. Th e reaction mixture was then stirred for fifteen minutes at 0 C, af ter which 1-chloromethylnaphtha lene (0.658 mmol) was added dropwise. The reaction was allowed to warm up to room temperature and stirred at room temperature for five hours. Ethyl acetate was th en used to dilute the reaction, and it was washed three times with dI water and once wi th brine and dried over magnesium sulfate. Evaporation under reduced pressure yielded th e crude product, which was recrystallized in ethyl acetate mixed with a few drops of hexanes to give the pure product in 66-91% yield. N 23a 1-(1’-Methylnaphthyl)-2,3-Dimethylindole (23a). MP = 150-152 C; Rf = 0.77 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 2.29 (s, 3H), 2.38 (s, 3H), 5.76 (s, 2H), 6.37 (d, J = 6.8 Hz, 1H), 7.11-7.17 (m, 3H), 7.21-7.26 (m, 2H), 7.59-7.63 (m, 3H), 7.74 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) 9.24, 10.21, 44.41, 107.40, 109.10, 118.32, 119.20, 121.10, 122.42, 122.91, 126.06, 126.11, 126.62, 127.77, 129.02, 129.27, 130.53, 132.94, 133.39, 133.79, 136.76; HR ESI-MS (0.1% AcOH in CH3CN) 286.1590 (M + H)+.

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102N 23b 9-(1’-Methylnaphthyl)-Carbazole (23b). MP = 146-148 C; Rf = 0.73 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 5.94 (s, 2H), 6.68 (d, J = 7.2 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1 H), 7.30-7.32 (m, 2H), 7.34-7.38 (m, 2H), 7.43-7.48 (m, 2H), 7.647.71 (m, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 8.8 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) 44.57, 109.31, 119.68, 120.78, 122.61, 123.29, 123.44, 125.98, 126.25, 126.28, 126.80, 128.11, 129.41, 130.94, 131.94, 134.04, 141.18; HR ESI-MS (0.1% AcOH in CH3CN) 308.1429 (M + H)+. N 23c 9-(1’-Methylnaphthyl)-1,2,3,4Tetrahydrocarbazole. MP = 162-164 C; Rf = 0.76 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 1.92-1.93 (m, 4H), 2.62 (s, 2H), 2.86 (s, 2H), 5.73 (s, 2H), 6.45 (d, J = 6.4 Hz, 1H), 7.10-7.17 (m, 3H), 7.23-7.27 (m, 1H), 7.59-7.65 (m, 3H), 7.75 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) 21.45, 22.16, 23.46, 23.52, 44.08, 109.25, 110.33, 118.10, 119.22, 121.10, 122.46, 123.02, 126.04, 126.09, 126.61, 127.76, 127.82, 129.26, 130.58, 133.47, 133.79, 136.15, 136.99; HR ESI-MS (0.1% AcOH in CH3CN) 312.1743 (M + H)+.

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103 3.4 References 1. Hanahan, D., Weinberg, R. A. (2000) The Hallmarks of Cancer, Cell 100, 57-70. 2. Lockshin, R. A., Zakeri, Z. (2002) Caspase-Independent Cell Deaths, Current Opinion in Cell Biology 14, 727-733. 3. Fesik, S. W. (2005) Promoting apoptosis as a strategy for cancer drug discovery. Nature Reviews Cancer, 5(11), 876-885. 4. Senderowicz, A. M. (2004) Targeting cell cycle and apoptosis for the treatment of human malignancies, Current Opinion in Cell Biology 16, 670-678. 5. Shmueli, A., Oren, M. (2004) Regulati on of p53 by Mdm2: Fate is in the numbers Molecular Cell 13(1), 4-5. 6. Moll, U. M., Petrenko, O. (2003) The MDM2-p53 Interaction, Molecular Cancer Research 1, 1001-1008. 7. Chene, P. (2003) Inhibiting the p53-MDM2 Interaction: An Important Target for Cancer Therapy, Nature Reviews: Cancer 3, 102-109. 8. Levav-Cohen, Y, Haupt, S., Haupt, Y., (2005) MDM2 in growth signaling and cancer, Growth Function 23, 183-192. 9. Chene, P. (2004) Inhibition of the p53MDM2 Interaction: Targeting a ProteinProtein Interface, Molecular Cancer Research 2, 20-26. 10. Barrett, C. P., Hall, B. A., Noble, M. E. M. (2004) Dynamite: a simple way to gain insight into protein motions. Biological Crystallography 60 (1), 2280-2287. 11. Guy, L., Vidal, J., Collet, A. (1998) Design and Synthesis of Hydrazinopeptides and Their Evaluation as Human Le ukocyte Elastase Inhibitors Journal of Medicinal Chemistry 41, 4833-4843. 12. Pagliaro, L., Felding, J., Audouze, K., Niel sen, S. J., Terry, R. B., Krog-Jenson, C., Butcher, S. (2004) Emerging classes of protein-protein intera ction inhibitors and new tools for their development, Current Opinions in Chemical Biology 8, 442-449. 13. Klein, C., Vassilev, L. T. (2004) Targ eting the p53-MDM2 interaction to treat cancer, British Journal of Cancer 91, 1415-1419.

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104 14. Stoll, R., Renner, C., Hansen, S., Palme, S., Klein, C., Belli ng, A., Zeslawski, W., Kamionka, M., Rehm, T., Muhlhahn, P., Sc humacher, R., Hesse, F., Kaluza, B., Voelter, W., Engh, R. A., Holak, T. A. (2001) Chalcone Derivatives Antagonize Interactions between the Hu man Oncoprotein MDM2 and p53, Biochemistry 40, 336-344. 15. Duncan, S. J., Gruschow, S., Williams, D. H., McNicholas, C., Purewal, R., Hajek, M., Gerlitz, M., Martin, S., Wrigley, S. K., M oore, M. (2001) Isolation and Structure Elucidation of Chlorofusin, a N ovel p53-MDM2 Antagonist from a Fusarium sp., Journal of the American Chemical Society 123, 554-560. 16. Malkinson, J. P., Zloh, M., Kadom, M., Errington, R., Smith, P. J., Searcey, M. (2003) Solid Phase Synthesis of the Cyclic Peptide Portion of Chlorofusin, an Inhibitor of p53-MDM2 Interactions, Organic Letters 5, 5051-5054. 17. Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D., Podl aski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C., Fo touhi, N., Liu, E. A. (2004) In Vivo Activation of the p53 Pathway by Sma ll-Molecule Antagonists of MDM2, Science 303, 844-848. 18. Tovar, C., Rosinski, J., Filipovic, Z., Hi ggins, B., Kolinsky, K., Hilton, H., Zhao, X., Vu, B. T., Qing, W., Packman, K., Myklebost, O., Heimbrook, D. C., Vassilev, L. T. (2006) Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: Implications for therapy, Proceedings of the National Academy of Sciences 103, 1888-1893. 19. Zhao, J., Wang, M., Chen, J., Luo, A., Wa ng, X., Wu, M., Yin, D., Liu, Z. (2002) The initial evaluation of non-peptide sma ll-molecule HDM2 inhibitors based on p53HDM2 complex structure, Cancer Letters 183, 69-77. 20. Garcia-Echeverria, C., Chene, P., Blommers, M. J. J., Furet, P. (2000) Discovery of Potent Antagonists of the Interaction be tween Human Double Minute 2 and Tumor Suppressor p53, Journal of Medicinal Chemistry 43, 3205-3208. 21. Yin, H., Lee, G., Park, H. S ., Payne, G. A., Rodriguez, J ., Sebti, S. M., Hamilton, A. D. (2005) Terphenyl-Based Helical Mimetics That Di srupt the p53/HDM2 Interaction, Angewandte Chemie International Edition, 44, 2704-2707. 22. Chen, L., Yin, H., Farooqi, B., Sebti, S ., Hamilton, A. D., Chen, J. (2005) p53 Helix mimetics antagonize p53/MDM2 interaction and activate p53, Molecular Cancer Therapeutics 4, 1019-1025. 23. Insaf, S. S., Witiak, D. T. ( 2000) Synthesis of All Distinct -Methyl-Substituted Isomers of Amino Bis(2,2’-Ethanoic Acid ) Diethyl Ester and Ethylene Diamine Tetraacetic Acid Tetraethyl Ester Scaffolds, Tetrahedron 56, 2359-2367.

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105 24. Perotta, E., Altamura, M., Barani, T., Bindi, S., Giannotti, Harmat, N. J. S., Nannicini, R., Maggi, C. A. (2001) 2,6-Diketopiperazines from Amino Acids, from Solution-Phase to Solid-Phase Organic Synthesis, Journal of Combinatorial Chemistry 3, 453-460. 25. Vidal, J., Hannachi, J. C., Hourdin, G., Mula tier, J. C., Collet, A. (1998) N-Boc-3trichloromethyloxaziridine: a new powerfu l reagent for electrophilic amination, Tetrahedron Letters 39, 8845-8848. 26. Avancha, K. (2006) Design and Synthesis of Novel HIV-1 Protease Inhibitors and Synthesis and Biological Activ ity of Novel 20S Proteasom e Inhibitors, Dissertation, University of South Florida.

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106 CHAPTER FOUR DESIGN AND SYNTHESIS OF A BAX PROTEIN HELIX MIMIC 4.1 Introduction 4.1.1 Pro-Apoptotic and Anti-Apoptotic Members of the Bcl-2 Family Apoptosis was discussed in the Introducti on section of Chapter Three regarding the MDM2-p53 interaction. As shown in Fi gure 3.2, there are two apoptotic pathways: the extrinsic (FAS) pathway, which invol ves death receptors; and the intrinsic (mitochondrial) pathway, which involves cytoch rome c release from the mitochondria as a result of action by the Bcl-2 family of proteins ( 1 ). This chapter will discuss some of the Bcl-2 family members, as well as ideas for manipulating them in order to promote apoptosis in cancer cells. The Bcl-2 family of proteins is compri sed of three groups of pro-apoptotic and anti-apoptotic members ( 2,3 ), several of which are shown in Figure 4.1. The top panel shows the Bcl-2 group, which includes Bcl-xL and Bcl-2; these protei ns are anti-apoptotic ( 2 ). They share a sequence homology to Bcl-2 in four regions that are designated Bcl-2 homology one through four (BH1-BH4), as shown in Figure 4.1 ( 2 ). The pro-apoptotic Bax group is shown in the middle panel; thes e proteins have three Bcl-2 homologous regions numbered from BH1-BH3 ( 2 ). The Bik group members, shown at the bottom of Figure 4.1, are another set of pro-apoptotic pr oteins that only share homology in the BH3 region ( 2 ).

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107 Figure 4.1: The Bcl-2 Family of Proteins ( 2 ). The Bcl-2 group proteins are antiapoptotic, while the Bax and Bik groups are both pro-apoptotic. It is the interplay of various Bcl-2 family members that determines the mitochondrial response, and ul timately the cell’s fate ( 3,4 ). The Bcl-2 family proteins can form heterodimers between pro-apoptotic and anti-apoptotic memb ers; this inhibits the biological activity of their partners ( 2-4 ). Heterodimerizati on occurs via the BH3 region of a pro-apoptotic prot ein being inserted into a hydrophobic cleft comprised of the BH1, BH2, and BH3 regions of an anti-apoptotic protein ( 2 ). The BH1, BH2, and BH4 regions are all necessary for anti-apoptotic activity, but BH3 alone is sufficient for proapoptotic activity ( 2 ). Some of the proapoptotic family members like Bax and Bak can also form homo-oligomers that allow for outer mitochondrial membrane permeabilization in response to a loss of the mitochondrial proton gradient ( 3 ).

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108 Originally, it was thought that any of the pro-apoptotic proteins could pair up with any of the anti-apoptotic ones ( 4 ). However, it is now known that several of the Bcl-2 family members have specificities for certain partners and not for others ( 4 ). It appears that many of the BH3-only pro-apoptotic fa mily members interact with certain antiapoptotic partners, and that once these anti-a poptotic proteins are neutralized, the proapoptotic Bax group of proteins are free to permeabilize the mitochondria and other organelles ( 4 ). There are, however, some BH3-only pro-apoptotic protei ns that can pair with any of the anti-apoptotic proteins; one consequence of this differential specificity is that the BH3-only proteins with more specificity are weaker cell killers in comparison to the broader-spectrum BH3-only proteins ( 4 ). It has been suggested that the more specific binding properties of some prot eins may be due to their great er rigidity, while the more general binding properties of other proteins occur because they are more flexible ( 4 ). Several members of the Bcl-2 family can form channels in the mitochondrial membrane, including Bax, Bcl-2, and Bcl-xL ( 5 ). The pro-apopto tic Bax presumably forms channels to permit the escape of cytoch rome c from the mitochondria, but it is not known what the purpose is of the channels fo rmed by the anti-apoptotic Bcl-2 proteins ( 5 ). Presumably, their channel-forming activ ity relates to promo ting cell survival ( 5 ). It is not precisely known how pro-apoptotic family memb ers like Bax are able to cause mitochondrial rupture and cytochrome c re lease, but some of th e details have been worked out. One thing that is known is that the ability of Bax to form channels in the mitochondria is pH-dependent ( 5 ). A proposed mechanism for the Bax channel-forming process is shown in Figure 4.2 ( 6 ). There are several steps that are necessary before apoptosis can occur.

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109 Under normal, non-apoptotic conditions, a mitochondrion is characterized by a proton gradient across its inner membrane. The cytochrome c molecules are located in the intermembrane space on the mitochondrial i nner membrane (MIM). Bax is soluble in the cytoplasm of the cell. A protein called the permeability transition pore (PTP), which spans both the MIM and the mitochondrial outer membrane (MOM) in its active form, is inactive under normal conditions. This scenar io is depicted in Panel A of Figure 4.2 ( 6 ). Figure 4.2: Theorized Mechanism of Action of Bax ( 6 ). Panel A shows the normal mitochondrial membrane, with Bax solubilized in the cytoplasm and a proton gradient across the mitochondrial membrane. In Panel B, the proton gradient has been dissipated, which causes a conformational change in Bax and allows it to insert its 9th alpha helix into the mitochondrial membrane (Panel C). The Bax helices form channels that allow the cytochromes to escape into the cytopl asm (Panel D), thereby killing the cell.

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110 The first thing that must happen in order for Bax-induced apoptosis to occur is that the mitochondrial proton grad ient must be lost. There ar e two proteins that comprise the PTP, called the voltage-dependent ani on channel (VDAC) and the adenine nucleotide transporter (ANT). VDAC, which is in th e MOM, and ANT, which is in the MIM, associate to form the PTP, which spans both membranes ( 2 ). The PTP opens, allowing protons to move down their electrochemical gr adient and enter the mitochondrial matrix. The proton gradient is thus dissipated ( 6 ), as shown in Panel B of Figure 4.2. The loss of the mitochondrial proton gradie nt causes a conformational change in Bax. Individual Bax proteins begi n to insert their C-terminal -helices into the MOM ( 6 ). This is shown in Panel C of Figure 4.2. As the Bax proteins continue to insert into the mitochondrion, they begin to oligomerize and form channels in the MOM. These channels allow the cytochrome c proteins in the intermembrane space to pass through the MOM into the cytoplasm, where they recruit the caspases that ultimately cause cellular death ( 6 ). Panel D depicts the Bax oligomer channels allowing the cytochrome c molecules to escape from the m itochondrion into the cytoplasm. 4.1.2 Mimicking the BH3 Domain of Pro-Apoptotic Proteins To date, there have been several exampl es of small molecules, natural products, antisense nucleotides, peptides, and peptidomim etics that inhibit the Bcl-2 family protein interactions reported in the literature ( 7-23 ). Generally, these co mpounds interfere with the interactions between the pro-apoptotic and anti-apoptotic family members. For example, the Bcl-2 antisense nucleotid e ODN 2009 was found to induce apoptosis in leukemia cells, and to work synergistically with the cy totoxic compound chlorambucil ( 7 ). Several small molecules have been reporte d that inhibit the inte ractions between pro-

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111 apoptotic BH3 domains and Bcl-xL ( 8 ) and Bcl-2 ( 9-11 ). These compounds were also found to promote apoptosis in tumor cells; their structures ar e shown in Figure 4.3. S N O S HOOC Br BH3I-1 (Ref. 8)Br OH Cl O NH Cl S O O Cl BH3I-2 (Ref. 8)ONH2O O O N Br HA14-1 (Ref. 9) Figure 4.3: Structures of Some SmallMolecule Bcl-2 Family Inhibitors ( 8, 9 ). The first two compounds inhibit Bcl-xL, and the third compound inhibits Bcl-2. Alisol B acetate (Ref. 13) N H H O OMeO NH O O O O O O 2-Methoxyantimycin A (Ref. 12) Figure 4.4: Structures of Two Pr o-Apoptotic Natural Products ( 12, 13 ). These compounds are two natural products that were found to promote apoptosis. There are also natural produc ts that have been reporte d to promote apoptosis ( 1213 ). One is a methoxy derivative of antimycin A, shown on the right si de of Figure 4.4. This compound competes with the Bak BH 3 peptide to bind to Bcl-2 and Bcl-xL ( 12 ).

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112 Another pro-apoptotic compound that was recently reported is alisol B acetate, shown on the left side of Figure 4.4. This com pound was found to induce Bax up-regulation and the insertion of Bax into the nuclear membrane ( 13 ). Alisol B acetate does not affect the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL ( 13 ), suggesting that its mechanism of action (up-regulation of Bax) is different than that of most of the previous compounds discussed, which function by inhibi ting the anti-apoptotic proteins. It is known that the interface between the anti-apoptotic proteins and the proapoptotic BH3 helices is not flat; in fact, the BH3 helices bind in a groove on their antiapoptotic partners ( 14 ). This fact makes the normally difficult process of finding a smallmolecule inhibitor of a protei n-protein interaction seem more hopeful. However, all of the small-molecule and natura l product apoptosis promoters described thus far are active in the low micromolar concentration range, wh ich is considerably higher in comparison to the natural BH3 helix ligands ( 14 ). In addition, it is know n that the BH3 domain is unstructured in solution, but it takes on a helical conformation when it binds to its antiapoptotic partner ( 14 ). Thus, it is reasonabl e to propose that rigid, -helix-mimicking compounds should have higher affinity and po ssibly higher specificit y than the natural ligands do, since their binding woul d not result in as large of an entropic penalty due to the loss of conformational degrees of freedom ( 14 ). One group reported a protein grafting met hod where the active BH3 amino acid side chains were strategically pl aced on a rigid helical scaffold ( 15 ). This design was then followed by a peptide selection proce ss using phage display libraries where the peptides that were the most stable an d had the best binding were selected ( 15 ). The best proteins were found to have dissociation consta nts in the nanomolar ra nge that were even

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113 superior to the BH3 helix itself ( 15 ). These authors also reported that the BH3-binding pockets on Bcl-2 versus Bcl-xL are different enough to ma ke it possible to develop inhibitors specific to on e protein over the other ( 14,15 ). More recently, a hydrocarbon stapling stra tegy was reported where BH3 peptides that had been chemically constrained into th e helical binding shape were used to inhibit Bcl-2 ( 16 ). These compounds were found to also be resistant to prot eases and permeable to cell membranes, as well as having improved affinity for Bcl-2 in comparison with the native BH3 peptide ( 16 ). As with the grafted peptid es, these compounds were able to activate apoptosis in cancer cells ( 16 ). Finally, it is also possible to inhi bit the anti-apoptoti c proteins using peptidomimetics that are similar to the BH3 peptides, but that have entirely different scaffolds ( 17-23 ). Several scaffolds have been re ported, including oligoamide foldamers ( 17 ), terphenyl derivatives ( 18-21 ), and terephthalamide derivatives ( 21-22 ). These compounds are shown in Figures 4.5, 4.6, and 4.7 respectively. Figure 4.5: Chemical and Stereo X-Ray Structure of an Oligoamide ( 17 ). This compound is flat, but the isopropyl ether side chains twist out of the plane at a 45 angle.

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114 The oligoamide compounds were shown to have a flat backbone, but their side chains twist out of the plane at a 45 angle, as shown in Figure 4.5 ( 17 ). These compounds were found to have a low micromolar affinity for Bcl-xL, which is about an order of magnitude worse than the binding a ffinity of the native BH3 peptide ligand ( 17 ). Attempts to lengthen the backbone to four uni ts were unsuccessful due to the insolubility of the compound, and hydrolyzing the ester to improve solubility did not improve the affinity of the longer peptidomimetic ( 17 ). Figure 4.6: Terphenyl Derivatives wi th Binding Affinity for Bcl-xL ( 21 ). Panel A shows a schematic of how the terphenyl mimi cs the side chain orientation on the BH3 helix. The crystal structure of th e terphenyl is overlaid with an -helix in Panel B. The use of a terphenyl backbone was even more successful ( 18-21 ). These socalled “proteomimetics” have several adva ntages over the molecules previously

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115 described, including their modular synthesis from subunits, their ability to proj ect the side chains in the same orientation as the nativ e BH3 peptide, and their twisted shape that mimics an -helix ( 18 ). The phenyl rings in the backbone are able to ro tate around the single bonds between them, but the bulky side ch ains prevent them from being able to lie flat ( 19 ). These compounds had binding constants for Bcl-xL in the high nanomolar range ( 20 ). In the minimum energy conformati on, the ortho substitution pattern on the terphenyl backbone mimics the i i +4, and i +7 side chains on the BH3 helix; these are the residues that are important for BH3 to be able to interact with Bcl-xL ( 21 ). The structure of these compounds is shown in Figure 4.6. Figure 4.7: Terephthalamide Derivatives with Binding Affinity for Bcl-xL ( 23 ). Panel A shows the general structure of the te rephthalamide derivatives. The overlap of a terephthalamide with an -helix is shown in Panel B, wh ile Panel C shows the overlap of a terphenyl compound with a terephthalamide. The authors note that the terphenyl de rivatives were too hydrophobic to be watersoluble and that they were difficult to synthesize ( 22 ). Thus, they developed a new

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116 proteomimetic based on a tere phthalamide scaffold, which is shown in Figure 4.7. Basically, the two terminal phe nyl rings of the terphenyl we re replaced with carboxamide groups, which also have a restricted geometry due to the planar amide bonds they contain ( 22 ). This compound maintained the side ch ain geometry of the terphenyl compounds, while increasing the hydrophi licity of the backbone ( 22 ). The terephthalamides were tested against Bcl-xL and found to be high nanomolar inhi bitors, similar to their terphenyl predecessors ( 23 ). All of the peptidomimetics described thus far function by affecting the pairings of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins. These compounds affect the apoptotic pathway upstream of the Bax family members, which form the membrane channels that release cytochrome c from the mitochondria. A search of the literature did not turn up any compounds that di rectly affect Bax itse lf with the possible exception of alisol B. The remainder of this chapter will discuss the effort to design and synthesize an -helix mimic that could bind to Bax itself, thus forcing Bax to adopt its active, channel-forming conformation and ultimately inducing apoptosis. 4.2 Results and Discussion The Bax protein is comprised of nine -helices ( 24 ). The first eight helices resemble the anti-apoptotic protein Bcl-xL, while the ninth C-terminal helix is involved with Bax dimerization and mitochondrial pore formation ( 24 ). When Bax is in its inactive conformation, the ninth helix is tuck ed into a hydrophobic pocket formed by the rest of the protein ( 24 ). Early in the apoptosis proc ess, helix 9 inserts itself into mitochondrial membranes and forms the channels that permit cytochro me c to escape into the cytoplasm ( 24 ). The structure of Bax is shown in Figure 4.8.

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117 It is known that disruption of the inactiv e structure of Bax by detergents will cause oligomer formation ( 24 ). Presumably, these oligomers would lead to membrane penetration, cytochrome release, and apopt osis. We therefore propose a novel compound that is hypothesized to disrupt the inac tive conformation of Bax, leaving the hydrophobic helix 9 free to insert into the mitochondrial membranes. Figure 4.8: Structure of the Pro-Apoptotic Bax Protein. The apoptosis-inducing helix 9 can be seen at the top of the pict ure, horizontal and nearest to the viewer.

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118 We began by virtually screening a library of 1981 NCI compounds (~2500 3D models) in an effort to fit them into th e hydrophobic binding site of helix 9 using the GLIDE automated docking program. Since the binding site of the helix is relatively large, we obtained compounds that docked into the C-terminal, N-terminal, and middle portions of the Bax molecule. A sample of the compounds that we re found to dock to Bax is shown in Figure 4.9. An examination of these molecules shows that they have several characteristics in comm on, including that they tend to be relatively symmetrical, contain aromatic rings, and unsurprisi ngly tend to be relatively hydrophobic. NN O H HO NSC109816 N N OOH O HO Cl Cl NSC210627 N NN NN N Cl Cl NSC343227 N N N N OH NSC231643 Figure 4.9: Structures of Some NCI Compo unds Virtually Screened to Fit into the Hydrophobic Pocket of Bax. The hit compounds tend to be relatively symmetrical, nonpolar, and aromatic.

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119 We then began a series of iterations to build up these small molecules into larger molecules that could occupy the entire helix 9 binding site. This required several rounds of design and screening. Although we initially built upon all four of the compounds shown in Figure 4.9, we discovered that the li gands with the best docking scores were derivatives of compound NSC109816. We also found that the best compound 24 contained an additional central carbonyl, and that molecular symmetry was apparently helpful. Some of the intermediate compounds that evolved between the starting compound NSC109816 and the final compound 24 are shown in Figure 4.10. Figure 4.10: Some Iterations to Obtain Compound 24 from NSC 109816 Each successive group of compounds was virtually sc reened, and the results were used to design the next set. Eventuall y, this process re sulted in compound 24 the synthesis of which is discussed below. Based on the GLIDE docking simulations, compound 24 was predicted to have a similar shape and binding orientation in compar ison to helix 9, and to fit into the same NN O H HO NSC109816O H O H CH3O NH N N NH NH NH O O O NH N NH O N N H H N N O O N N N N 24

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120 hydrophobic binding pocket. Although the com pound structure appears to be flat, the model predicts that 24 will exist in a twisted conf ormation, making its shape more analogous to an -helix. The structure of 24 its orientation according to the GLIDE model, and helix 9 are all shown in Figure 4.11. O N N H H N N O O N N N N 24 Figure 4.11: Mimicking the Alpha Helix of Bax. The bottom panel shows the alpha helix from the Bax protein. Analogously, target compound 24 twists like an alpha helix, shown in the middle panel. The chemical structure of 24 is shown in the top panel. A superimposition of compound 24 with the Bax protein minus helix 9 is shown in Figure 4.12. This diagram shows that 24 is large enough to occupy most of the

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121 hydrophobic helix 9 binding pocket. It also shows the twisted conformation of compound 24. Figure 4.12: Compound 24 Docked to Bax in Lieu of the Helix. Computer simulations predict that 24 will bind Bax in the same orientation and place where helix 9 normally folds.

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122 The remainder of this discussion presents our progress toward the synthesis of compound 24 Scheme 4.1 shows the retrosynthesis of 24 Since 24 is a symmetrical compound, only one half of the molecule mu st be synthesized, and the two identical pieces can then be joined together as a urea to make the entire compound. Each half of 24 was further broken down into an indole portion 25 and a benzoic acid portion 26 These two pieces are theoretically accessi ble from available starting materials 27 and 29 O N N H H N N O O N N N N 24 N NH2HO O 225 262N NH2 Scheme 4.1: Retrosynthesis of Compound 24. 24 is a dimer requiring two pieces: indole derivate 25 and benzoic acid derivative 26 We began with the synthesis of 25 because it is the simpler piece of the molecule; these reactions are shown in Scheme 4.2. The first step was the N-alkylation of 5nitromethylindole using sodium hydride and methyl iodide. This reac tion is basically the

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123 same as in our previous syntheses of Nmethyl tryptophan and the small molecule MDM2 inhibitors described in Chapter Three. The next step would be to reduce the nitro group to an amine via hydrogenation. This is a well-known literature procedure ( 25-27 ), but we have not yet performed it on compound 28 due to potential difficulties with storing it. 25N NH2 28N NO2 27N H NO2 NaH CH3I DMF H2/Pd/C MeOH 100% AcOH Scheme 4.2: Synthesis of Indole 25. Compound 25 could readily be synthesized from commercially available nitroindole 27 We did not complete the final step of the synthesis while we were attempting the synthesis of 26 We then turned our attention to synthesizing 26 the other half of the molecule. Our initial plan was to begin with 2-methyl-5-nitroaniline 29 and reductively aminate it using 2,5-dimethoxytetrahydrofuran 30 to form tertiary aniline 31 Compound 30 is a diacetal that we planned to open by adding acid, after whic h we would proceed with our normal reductive amination procedures as in Chapter Two. We would then oxidize the

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124 methyl group up to the carboxylic acid, and redu ce the nitro to the amine. Unfortunately, several attempts at conducting a reductive amin ation were all unsuccessful, as shown in Scheme 4.3. We initially tried using acetic aci d, a weak acid. When that reaction failed, we attempted to use p-toluenesulfonic acid. This reaction also failed, as did a third attempt using 2N HCl. NH2NO2 29O O O XN NO2 AcOH 1. MeOH 2. NaBH3CN30 31 NH2NO2 29O O O XN NO2 1. MeOH 2. NaBH3CN30 31p-TsOH NH2NO2 29O O O XN NO2 1. MeOH 2. NaBH3CN30 312N HCl Scheme 4.3: Failed Attempts to Synthe size 31 via Reductive Amination of 29. Several acids were tried, including acetic, ca talytic p-toluenesulfonic, stoichiometric ptoluenesulfonic, and hydrochloric acids.

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125 We next attempted to synthesize compound 31 via an N,N-dialkylation using 1,4dibromobutane. Our first attempt used DIEA in toluene following a literature procedure ( 28 ), which was not successful in our hands. This failure is probably due to the high electron-withdrawing capacity of th e nitro group on the benzene ring of 29 which greatly reduces the nucleophilicity of the aniline n itrogen. The reported literature examples either had electron-donating groups or weaker electron-withdrawing gr oups attached to the benzene ring, but no exampl es of aniline dialkylation with a nitrobenzene were reported ( 28 ). However, when we attempted the reaction again using potassium carbonate and the phase tr ansfer catalyst TBAB ( 29 ), we were able to successfully obtain 31 These reactions are both shown in Scheme 4.4. NH2NO2 29 XN NO2 31Br Br DIEA NH2NO2 29N NO2 31Br Br K2CO3TBAB 59.3% Scheme 4.4: Successful Synthesis of 31 via N, N-Dialkylation. The first attempt to synthesize 31 gave only starting materials, but addition of TBAB, a phase transfer catalyst, and use of potassium carbonate gave 31 as a bright orange solid.

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126N NO2 31 XN NO2HO O 32KMnO4H2O34 29NH2NO2 X33KMnO4H2O NH2NO2HO O Cl KMnO4H2O Cl OOH 3571.0% Scheme 4.5: Unsuccessful Attempts to Ox idize 29 and 31 to the Carboxylic Acids. Regardless of whether the amine was tert iary or primary, the methyl groups in 29 and 31 could not be oxidized up to the corresponding acids in the presence of the amine. However, the oxidation reaction did proceed normally on toluene derivative 34 which lacks the amino group, to give acid 35 Having obtained the tertiary aniline 31 we next attempted to oxidize the methyl group up to carboxylic acid 32 using potassium permanganate. This reaction is also a

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127 common, well-known lite rature procedure ( 30, 31 ). However, we were not able to successfully obtain product 32 after several attempts at oxidizing 31 Attempting the procedure on the initial starting material primary aniline 29 was also unsuccessful. We were, however, able to successf ully oxidize p-chlorotoluene 34 to the corresponding benzoic acid. This suggested that our reagents were fine, and that somehow the presence of the aniline group was problematic. We were furthermore able to rule out the nitro group as being the problem because one lite rature procedure reported the successful oxidation of a nitrotoluene to th e corresponding nitr obenzoic acid ( 29 ). These reactions are shown in Scheme 4.5. Since we were unable to oxidize the methyl group in the presence of the amine, it was necessary to find an alternative route to synthesize 32 We found another literature procedure where the methyl group had been ox idized in the presence of an acetylated amine ( 32 ). Thus, our next strategy was to acetylate the aniline first, and then oxidize the methyl to a carboxylic acid. This could be pr otected as an ester, after which the aniline could be deacetylated and then re acted with 1,4-dibromobutane to give 32 We were able to successfully synthesize the acetylated aniline 36 using acetic anhydride and acetic acid ( 33,34 ), and oxidation of the me thyl group with potassium permanganate gave benzoic acid 37 We then protected 37 as the methyl ester using methyl iodide to give 38 and hydrolyzed the amide back to the free aniline 39a using HCl ( 35 ). However, when we attempted to perform the dialkylation reaction on 39a to give tertiary aniline 39b we found that no reaction would occur, even in the presence of TBAB. Again, this is likely to be due to the presence of an additional electronwithdrawing group on the benzene ring. Befo re the methyl group was oxidized, it was

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128 mildly activating. However, once it was oxidized to the acid or ester, it became deactivating. This series of r eactions is shown in Scheme 4.6. X29NH2NO2 AcOAc AcOH N NO2H O 3663.6% KMnO4H2O MgSO4N NO2H O O OH 3775.5% K2CO3CH3I 100% DMF N NO2H O O O 38NH2NO2O O 39a conc. HCl 77.8% EtOH N NO2O O 39bBr Br K2CO3TBAB Scheme 4.6: Unsuccessful Attempt to N,N-Dialkylate a Benzoic Ester. Acetylation of the amine allowed oxidation of the methyl group with KMnO4, but N,N-dialkylation of the ester was not successful due to the extremely low nucleophilicity of the aniline nitrogen.

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129 At this point, we attempted to perfor m another reductive amination of starting compound 29 using 2.5 M sulfuric acid and s odium borohydride in THF and methanol, following a literature procedure ( 36 ). These reaction condition s were powerful enough to allow the reductive amination of 29 to give compound 31 in good yield. However, when we attempted to repeat this procedure on aniline 38 there was no reaction. Again, this is likely to be due to th e low nucleophilicity of 38 which possesses two powerful electronwithdrawing groups attached to the benzene ring in contrast to the weakly electrondonating methyl group that is present on 29 The procedure for reductive amination using sulfuric acid is shown in Scheme 4.7. X29NH2NO2 NH2NO2O O 38 N NO2O O 39 O O O 30 THF/MeOH 2.5M H2SO4NaBH4N NO2 31O O O 30THF/MeOH 2.5M H2SO4NaBH459.5% Scheme 4.7: Unsuccessful Attempt to Perform Reductive Amination on 38. Although several earlier attempts to do reduc tive amination had failed, the reaction was finally performed successfully on 29 using 2.5M H2SO4. However, the reaction failed when it was attempted on compound 38

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130 X29NH2NO2 15.2% O O O 40 N NO2O O 41dioxane TEA29NH2NO2 Cl Cl O O TEA N NO2O O 41 42reflux DCM29NH2NO2 O O O 40 N NO2O O 41AcOH 30.3% Scheme 4.8: Synthesis of Succinimide 41 from Aniline 29. Using succinyl chloride was messy and led to multiple products, but a small amount of product 41 could be obtained. Succinic anhydride gave the produc t if refluxed, along with a side product that was initially thought to be the incomplete cyclization produc t. This side product could not be cyclized using peptide coupling agen ts, and it was later determined to be the acetylated aniline 36 Our next strategy was to attempt to synthesize the succinimide 41 which would be followed by oxidation of the methyl group to the carboxylic acid a nd then reduction of

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131 the imide and nitro groups. We predicted that the succinimide, like the acetylated aniline, should not interfere with the permanganate oxidation of the me thyl group into the carboxylic acid. Our first attempt to synthe size the succinimide by refluxing succinic anhydride in dioxane was not successful. We then attempted to synthesize 41 using succinyl chloride; this reaction did produce a small amount of product, but the yield was poor and the reaction was messy and difficult to purify. Finally, we followed a literature procedure ( 37 ) to make 41 from succinic anhydride by re fluxing it in acetic acid. This reaction turned out to be the most successful and all of these proc edures are shown in Scheme 4.8. reflux29NH2NO2 O O O 40 N NO2O O 41AcOH 30.3% 36NH NO2O ~30% Scheme 4.9: Side Product of the Succinimide Reaction. We concluded by NMR and TLC that the other major product of this reaction was acetanilide 36.

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132 However, even the second succinic anhydr ide procedure was still relatively lowyielding, and TLC showed the presence of a s econd major spot present in about the same proportion as the desired product 41. NMR and TLC tests allowed us to conclude that this side product was actually 36, the acetylated aniline that we had already synthesized previously. This is not ultimately surprising, since the succinimide-forming conditions are similar to the acetylating conditions ( 33,34 ). The literature yield for this reaction was also relatively low at 36% ( 37 ). The reaction and both products formed are depicted in Scheme 4.9. At this point, we had obta ined sufficient quantities of 41 to allow us to continue with the synthesis. The separation of 41 from 36 is relatively diffi cult since the two products run fairly close together on TLC, but we were able to separate them by chromatographing them in dichloromethane. 41 has a higher Rf compared with 36, and by using 100% DCM as the eluent it is possible to collect 41 while 36 remains on the silica. 36 can be isolated by adding a small pe rcentage of methanol to the DCM. 41 was then oxidized to carboxylic acid 42. This reaction proceeded successfully as we expected based on our previous experience with oxidizing the acetylated aniline. The next step in the synthesis is the reduction of the acid, imide, and nitro groups using lithium aluminum hydride to the primar y alcohol, tertiary amine, and primary amine, respectively. Then, the alcohol will be oxidized back to the carboxylic acid using Jones reagent to give 26, which can then be combined with 25 to give 44, the symmetrical half of 24. These reactions are shown in Scheme 4.10. Finally, the free amines on 44 can be connected together as a urea to give target molecule 24, as discussed previously.

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133N NH2HO 26 N NO2O O 41KMnO4H2O N NO2O O O HO 43 25N H2N 44N NH2O N N H THF LiAlH4Jones reagent N NH2HO O 26a63.6% Scheme 4.10: Proposed Synthesis of 44 from 41. Compound 44 would then be used to synthesize target compound 24. The synthesis of 43 was successfully completed. Should this series of reactions not be successful, we propose to attempt an alternative route where we would form the pyrrolidine either by dialkylating the aniline ring of 29 using 1,4-dibromobutane or by reductive amination. We would then convert

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134 the methyl group to the primary alcohol usi ng wet NBS, followed by oxidation of alcohol 45 to acid 46 with Jones reagent. 46 could then be hydrogenate d to give intermediate 26, as shown in Scheme 4.11. The first reaction in this procedure to form 31 has already been successfully accomplished as described previously. NO2NH2 29 Br Br NO2N NBS CHCl3H2O NO2N HO Jones reagent NO2N HO O H2/Pd/C NH2N HO O 31 45 46 26 Scheme 4.11: Alternative Proposed Route to Compound 26. This route will be attempted next if the proposed route in Scheme 4.10 is unsuccessful.

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135 In conclusion, we have successfully us ed GLIDE docking simulations to virtually screen a library of NCI compounds, and we developed target compound 24 based on multiple screening iterations. We then made significant progress on the synthesis of 24. Further work will entail completing the synthe sis described above, as well as performing biological testing on 24. Experimental Data: 28N NO2 Procedure for Synthesis of NMethyl-5-Nitroindole (28). Sodium hydride (0.925 mmol) was washed twice with dry DMF, and then suspended in a third aliquot of dry DMF. The reaction mixture was cooled to 0 C, and 5-nitroindole (0.671 mmol) was added slowly. The reaction was then warmed up to room temperature and stirred for 15 minutes under Ar, after which methyl i odide (0.678 mmol) was added dropwise via syringe. The reaction was stirre d for thirty minutes at RT, and TLC then showed that it was complete. Ethyl acetate was added to th e reaction, and it was then washed with dI water. The water was back-extracted with ethyl acetate, and the combined ethyl acetate layers were washed once with water and twice with brine, dried over MgSO4, and evaporated under reduced pressure to give 28, a yellow solid, in 100% yield. MP = 160162 C; Rf = 0.32 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 3.85 (s, 3H), 6.66 (d, J = 2.8 Hz, 1H), 7.20 (d, J = 2.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 9.2 Hz,

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136 1H), 8.56 (s, 1H); 13C NMR (100 MHz, CDCl3) 33.43, 103.95, 109.22, 117.33, 118.25, 127.74, 132.18, 139.55, 141.67; HR ESI-MS (0.1% AcOH in CH3CN) 177.06553 (M + H)+; (The compound begins to decompose above 100 C.) N NO2 31 Procedure for Dialkylation Synthesis of 2-Pyrollidino-4-Nitrotoluene (31). 1methyl-5-nitroaniline (0.657 mmol) was dissolved in 5 mL toluene, and 1,4dibromobutane (0.657 mmol), potassium car bonate (0.657 mmol), and TBAB (0.066 mmol) were added. The reaction was then re fluxed overnight and worked up by washing it with 2N HCl and back-extra cting the aqueous layer with DCM. The DCM was backextracted with 2N HCl three times, and then all of the aqueous layers were combined and basified with solid NaOH. The aqueous laye r was extracted five times with DCM, and these five organic layers were combined, dr ied over magnesium sulfate, and evaporated under reduced pressure. Column chromatogr aphy using 9:1 hexanes/ethyl acetate gave 31, an orange solid, in 59.3% yield. MP = 73-74 C; Rf = 0.68 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 1.96-2.00 (m, 4H), 2.40 (s, 3H ), 3.27-3.29 (m, 4H), 7.18 (d, J = 8.0 Hz, 1H), 7.62-7.64 (m, 2H); 13C NMR (100 MHz, CDCl3) 21.48, 25.48, 109.98, 114.58, 132.22, 135.52, 147.16, 150.35; ESI-MS (0.1% AcOH in CH3CN) 207.1 (M + H)+.

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137N NO2 31 Procedure for Reductive Amination Synthesi s of 2-Pyrollidino-4Nitrotoluene (31). 2,5-dimethoxytetrahydrofuran (2.32 mmol) was di ssolved in THF, and reacted with 2.5 M sulfuric acid (4.50 mmol). The reaction mixture was cooled to 0 C and stirred for 15 minutes. In a separate flask, 1-methyl-5-nit roaniline (1.77 mmol) was dissolved in a 1:1 methanol/THF mixture and cooled to 0 C. The acidic acetal solution was then placed into an addition funnel, and this solution was added to the aniline reaction mixture dropwise. The combined reaction mixture wa s stirred at 0 C for 15 minutes, and then sodium borohydride was added from a powder addition funnel over twenty minutes. The reaction was then warmed up to room temperat ure and stirred for an hour, after which it was quenched with NaOH and water, and extr acted with DCM. Chromatography in 9:1 hexanes/ethyl acetate gave 31 as a bright orange powdery solid in 59.5% yield. MP = 7475 C; Rf = 0.63 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 1.96-1.99 (m, 4H), 2.40 (s, 3H), 3.27-3.30 (m, 4H), 7.17 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) 21.48, 25.48, 51.30, 109.99, 114.58, 132.22, 135.52, 150.35; ESI-MS (0.1% AcOH in CH3CN) 207.1 (M + H)+.

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138N NO2H O 36 Procedure for Synthesis of N-Acet yl-2-Amino-4-Nitrotoluene (36). Aniline 29 (0.0329 mol) was dissolved in 5 mL acetic acid, to which was added 8 mL acetic anhydride. The flask was placed in a water ba th at 45 C, causing it to solidify. 25 mL chloroform was added, and the reaction was stir red at 45 C for 10 minutes, until the SM had dissolved to form a reddish-brown so lution. The chloroform was removed under reduced pressure, and the product was recrystal lized from a 1:1 solution of ethanol:water to give white, flowery crystals in a 68.6% yield. MP = 149-150.5 C; Rf = 0.56 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 2.27 (s, 3H), 2.37 (s, 3H), 7.07 (broad, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 8.78 (s, 1H); 13C NMR (100 MHz, CDCl3) 18.12, 24.31, 118.05, 119.78, 130.97, 136.35, 136.48, 146.76, 168.57; ESI-MS (0.1% AcOH in CH3CN) 193.0 (M H)-. N NO2H O O OH 37 Procedure for Synthesis of N-Acetyl -2-Amino-4-Nitrobenzoic Acid (37). Acetyl aniline 36 (0.476 mmol) was suspended in 10 mL dI water, to which magnesium sulfate

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139 (0.297 mmol) was added. The reaction flask wa s fitted with a condenser, and heated to 80 C, after which potassium permanganate (1.19 mmol) was added. The reaction was stirred at 85 C for one hour, then cooled to RT. Small por tions of sodium bisulfite and concentrated sulfuric acid were added until the solid manganese dioxide byproduct dissolved and the product 37 precipitated out of solution as a pale peachy yellow powder in 75.5 % yield. MP = 197-198 C; Rf = 0.22 (100% EtOAc); 1H NMR (400 MHz, CD3OD) 2.24 (s, 3H), 7.90-7.93 (m, 1H), 8.23-8.26 (m, 1H), 9.44 (s, 1H); 13C NMR (100 MHz, CD3OD) 23.84, 114.55, 116.60, 119.97, 131.13, 132.69, 142.10, 169.53; ESI-MS (0.1% AcOH in CH3CN) 223.0 (M H)-. N NO2H O O O 38 Procedure for Synthesis of Methyl N-A cetyl-2-Amino-4-Nitrobenzoate (38). Benzoic acid 37 (0.416 mmol) was dissolved in 5 mL DMF, and the reaction was cooled to 0 C. Potassium carbonate (0.458 mmol) was added, and the reaction was stirred for ten minutes at 0 C before adding methyl iodide (0.833 mmol). Th en the reaction was allowed to warm to RT and stirred for four hours at RT. The reac tion was diluted with ethyl acetate and washed with saturated sodium bicarbonate so lution, dI water, and brine solution to give 37 as a yellow oil in 76.9% yield. Rf = 0.31 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 2.29 (s, 3H), 4.00 (s, 3H), 7.87 (dd, J = 2.4 Hz, 8.8 Hz, 1H),

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140 8.19 (d, J = 8.8 Hz, 1H), 9.59 (d, J = 2.0 Hz, 1H); 11.12 (broad, 1H); 13C NMR (100 MHz, CDCl3) 25.45, 53.12, 115.26, 116.50, 119.05, 131.94, 142.40, 151.26, 167.36, 169.28. NH2NO2O O 39a Procedure for Synthesis of Methyl 2-Amino-4-Nitrobenzoate (39a). Acetyl aniline 38 (0.323 mmol) was suspended in 7 mL absolu te ethanol, to which was added 1.4 mL concentrated HCl. The reaction was refluxe d for 18 hours at 79 C, after which it was soluble in ethanol. The ethanol was rem oved under reduced pressure, and the crude product was suspended in dI water and basi fied with 1M NaOH until the pH was 6 to give a gelatinous yellow solid in 77.8 % yield. Rf = 0.00 (3:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 3.92 (s, 3H), 6.06 (broad, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.51 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) 52.42, 110.30, 111.35, 115.16, 133.05, 150.91, 151.58, 167.50; ESI-MS (0.1% AcOH in CH3CN) 197.0 (M + H)+. N NO2O O 41

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141Procedure for Succinyl Chloride Synthesi s of 2-Succinimido-4-Nitrotoluene (41). Aniline 29 (3.29 mmol) was dissolved in 25 mL DC M, to which was added succinyl chloride (3.61 mmol) and TEA (6.57 mmol). Th e reaction was stirred at RT for one hour, after which the solid byproduct formed was filtered off, and the supernatant was chromatographed on silica in 2:1 hexanes/EtOAc to give 41 as pale yellow crystals in a 15.2% yield. MP = 214-217 C; Rf = 0.29 (1:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 2.27 (s, 3H), 2.99 (s, 4H), 7.51 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 2.4 Hz, 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) 18.49, 28.90, 124.11, 124.48, 129.06, 131.14, 132.18, 144.08, 175.56. Procedure for Succinic Anhydride Synthesi s of 2-Succinimido-4-Nitrotoluene (41) and Acetylated Side Product (36). Aniline 29 (3.29 mmol) and succinic anhydride (6.57 mmol) were dissolved in glacial acetic acid, and the reaction was refluxed for two hours. It was then cooled to RT, and extract ed with saturated bica rbonate solution. The crude product was chromatographed on silica in 1:1 hexanes/EtOAc to give succinimide product 41 as pale yellow crystals in 30.3% yi eld, and side product acetyl aniline 36 as a white powdery solid in 30% yield. N NO2O O 41

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1422-Succinimido-4-Nitrotoluene (41). MP = 215-218 C; Rf = 0.30 (9:1 DCM/MeOH), Rf = 0.33 (1:1 hexane/EtOAc); 1H NMR (400 MHz, CDCl3) 2.27 (s, 3H), 2.99 (s, 4H), 7.52 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 2.4 Hz, 8.4 Hz); 13C NMR (100 MHz, CDCl3) 18.47, 28.90, 124.11, 124.47, 132.17, 144.10, 146.99, 175.56. N NO2H O 36 N-Acetyl-2-Amino-4-Nitrotoluene (36). MP = 149-150 C; Rf = 0.12 (9:1 DCM /MeOH); 1H NMR (400 MHz, CDCl3) 2.26 (s, 3H), 2.37 (s, 3H), 7.16 (broad, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 8.75 (s, 1H); 13C NMR (100 MHz, CDCl3) 18.33, 24.57, 118.20, 120.00, 131.18, 136.26, 136.71, 147.10, 168.58; ESI-MS (0.1% AcOH in CH3CN) 193.0 (M H)-. N NO2O O O HO 43 2-Succinimido-4-Nitrobenzoic Acid (43). MP = 145-146 C; Rf = 0.00 (100% DCM); 1H NMR (400 MHz, DMSO) 2.56-2.59 (m, 2H), 2.67-2.70 (m, 2H), 7.95 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 9.30 (d, J = 2.4 Hz, 1H), 11.22 (broad, 1H);

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14313C NMR (100 MHz, DMSO) 29.25, 32.78, 114.97, 117.52, 124.54, 133.31, 141.79, 150.82, 168.77, 171.74, 174.19. 4.3 References 1. Fesik, S. W. (2005) Promoting apoptosis as a strategy for cancer drug discovery. Nature Reviews Cancer, 5(11), 876-885. 2. Tsujimoto, Y., Shimizu, S. (2000) Bcl-2 family: Life-or-death switch, FEBS Letters 466, 6-10. 3. Breckenridge, D. G., Xue, D. (2004) Regulation of mitochondrial membrane permeabilization by Bcl-2 family proteins and caspases, Current Opinion in Cell Biology 16, 647-652. 4. Chen, L., Willis, S. N., Wei, A., Smith, B. J., Fletcher, J. I., Hinds, M. G., Colman, P. M., Day, C. L., Adams, J. M., Huang, D., C ., S. (2005) Differential Targeting of Pro-Survival Bcl-2 Proteins by Their BH 3-Only Ligands Allows Complementary Apoptotic Function, Molecular Cell 17, 393-403. 5. Antonsson, B., Conti, F., Ciavatta, A. M ., Montessuit, S., Lewi s, S., Martinou, I., Bernasconi, L., Bernard, A., Mermod, J. J., Mazzei, G., Maundrell, K., Gambale, F., Sadoul, R., Martinou, J. C. (1997) Inhi bition of Bax Channel-Forming Activity by Bcl-2, Science 277, 370-372. 6. de Giorgi, F., Lartigue, L., Bauer, M. K. A., Schubert, A., Grimm, S., Hanson, G. T., Remington, J. S., Youle, R. J., Ichas, F. (2002) The permeability transition pore signals apoptosis by directing Bax tr anslocation and multimerization, The FASEB Journal 16, 607-610. 7. Pepper, C., Hooper, K., Thomas, A., Hoy, T., Bentley, P. (2001) Bcl-2 Antisense Oligonucleotides Enhance the Cytotoxic ity of Chlorambucil in B-Cell Chronic Lymphocytic Leukaemia Cells, Leukemia and Lymphoma 42, 491-498. 8. Degterev, A., Lugovskoy, A., Cardone, M., Mulley, B., Wagner, G., Mitchison, T., Yuan, J. (2001) Identification of small-mo lecule inhibitors of interaction between the BH3 domain and Bcl-xL, Nature Cell Biology 3, 173-182. 9. Wang, J. L., Liu, D., Zhang, Z. J., Shan, S., Han, X., Srinivasula, S. M., Croce, C. M., Alnermri, E. S. Huang, Z. (2000) Struct ure-based discovery of an organic compound that binds Bcl-2 protein and i nduces apoptosis of tumor cells, Proceedings of the National Academy of Science 97, 7124-7129.

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144 10. Chen, J., Freeman, A., Liu, J., Dai, Q., Lee, R. M. (2002) The Apoptotic Effect of HA14-1, a Bcl-2-interacting Small Molecular Compound, Requires Bax Translocation and is Enhanced by PK11195, Molecular Cancer Therapeutics 1, 981987. 11. Sinicrope, F. A., Penington, R. C., Tang, X. M. (2004) Tumor Necrosis FactorRelated Apoptosis-Inducing Li gand-Induced Apoptosis Is Inhibited by Bcl-2 but Restored by the Small Molecule Bcl-2 I nhibitor, HA14-1, in Human Colon Cancer Cells, Clinical Cancer Research 10, 8284-8292. 12. Tzung, S. P., Kim, K. M., Basanez, G., Giedt, C., D., Simon, J., Zimmerberg, J., Zhang, K. Y. J., Hockenbery, D., M. (2001) Antimycin A mimics a cell-deathinducing Bcl-2 homology domain 3, Nature Cell Biology 3, 183-191. 13. Huang, Y. T., Huang, D. M., Chueh, S. C., Te ng, C. M., Guh, J. H. (2006) Alisol B acetate, a triterpene from Alismatis rhizoma induces Bax nuclear translocation and apoptosis in human hormone-resist ant prostate cancer PC-3 cells, Cancer Letters 231, 270-278. 14. Rutledge, S. E., Chin, J. W., Schepartz, A. (2002) A view to a kill: ligands for Bcl-2 family proteins, Current Opinion in Chemical Biology 6, 479-485. 15. Chin, J. W., Schepartz, A. (2001) Design and Evolution of a Miniature Bcl-2 Binding Protein, Angewandte Chemie International Edition 40, 3806-3809. 16. Walensky, L. D., Kung, A. L, Escher, I., Malia, T. J., Barbuto, S., Wright, R. D., Wagner, G., Verdine, G. L., Korsmeyer, S. J. (2004) Activation of Apoptosis in Vivo by a Hydrocarbon-Stapled BH3 Helix, Science 305, 1466-1470. 17. Ernst, J. T., Becerril, J., Park, H. S., Yin, H. Hamilton, A. D. (2003) Design and Application of an -Helix-Mimetic Scaffold Based on an Oligoamide-Foldamer Strategy: Antagonism of th e Bak BH3/Bcl-xL Complex, Angewandte Chemie International Edition 42, 535-539. 18. Orner, B. P., Ernst, J. T., Hamilton, A. D. (2001) Toward Proteomimetics: Terphenyl Derivatives as St ructural and Functional Mimics of Extended Regions of an -Helix, Journal of the American Chemical Society 123, 5382-5383. 19. Ernst, J. T., Kutzki, O., Debnath, A. K. Jiang, S., Lu, H., Hamilton, A. (2002) Design of a Protein Surface Antagonist Based on -Helix Mimicry: Inhibition of gp41 Assembly and Viral Fusion, Angewandte Chemie International Edition 41, 278-281.

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145 20. Kutzki, O., Park, H. S., Ernst, J. T., Orne r, B. P., Yin, H., Hamilton, A. D. (2002) Development of a Potent Bcl-xL Antagonist Based on -Helix Mimicry, Journal of the American Chemical Society 124, 11838-11839. 21. Yin, H., Lee, G., Sedey, K. A., Kutzki, O., Park, H. S., Ornder, B. P., Ernst, J. T., Wang, H. G., Sebti, S. M., Hamilton, A. D. (2005) Terphenyl-Based Bak BH3 Helical Proteomimetics as Low-Mol ecular-Weight Antagonists of Bcl-xL, J. Am. Chem. Soc., 127, 10191-10196. 22. Yin, H., Hamilton, A. D. (2004) Terephtha lamide derivatives as mimetics of the helical region of Bak peptide target Bcl-xL protein, Bioorganic and Medicinal Chemistry Letters 14, 1375-1379. 23. Yin, H., Lee, G., Sedey, K. A., Rodriguez, J., Wang, H. G., Sebti, S. M., Hamilton, A. D. (2005) Terephthalamide Derivatives as Mimetics of Helical Peptides: Disruption of the Bcl-xL/Bak Interaction. J. Am. Chem. Soc., 127, 5463-5468. 24. Suzuki, M., Youle, R. J., Tjandra, N. (2000) Structure of Bax: Coregulation of Dimer Formation and Intracellular Localization, Cell 103, 645-654. 25. Matassa, V. G., Maduskuie, T. P., Shapiro, H. S., Hesp, B., Snyder, D. W., Aharony, D., Krell, R. D., Keith, R. A. (1990) Journal of Medicinal Chemistry 33, 17811790. 26. Tietze, L. F., Haunert, F., Fe urstein, T., Herzig, T. ( 2003) A Concise and Efficient Synthesis of seco -Duocarmycin SA, European Journal of Organic Chemistry 562566. 27. Baraldi, P. G., Romagnoli, R., Beria, I., Cozzi, P., Geroni, C., Mongelli, N., Bianchi, N., Mischiati, C., Gambari, R. (2000) Synthesis and Antitumor Activity of New Benzoheterocyclic Derivatives of Distamycin A, Journal of Medicinal Chemistry 43, 2675-2684. 28. Verboom, W., Lammerink. B. H. M., Egberin k. J. M., Reinhoudt, D. N., Harkema, S. (1985) Synthesis of Mitomy cin C Analogues. 2. Introduction of a Leaving Group at C-1 and Oxidation of the Aromatic Ring in 2,3,9,9a-Tetrahydro-1 H -pyrrolo[1,2a]indoles, Journal of Organic Chemistry 50, 3797-3806. 29. Huang, Y. L., Lin, C. F., Lee, Y. J., Li, W. W., Chao, T. C., Bach erikov, V. A., Chen, K. T., Chen, C. M., Su, T. L. ( 2003) Non-classical Antifolates, 5-(NPhenylpyrrolidin-3-yl)-2 ,4,6-triaminopyrimidines and 2,4-Diamino-6(5H)oxopyrimidines, Synthesis and Antitumor Studies, Bioorganic and Medicinal Chemistry 11, 145-157.

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146 30. Leonard, N. J., Boyd, S. N. (1946) Cinno lines. I. Synthesis of Aminoacetophenones and Aminopropiophenones, Journal of Organic Chemistry 11, 405-418. 31. Adams, R., Gold, M. H. (1940) The Synthesis of 1,3Dephenyldihydroisobenzofurans, 1,3Diphenylisobenzofurans, and oDibenzoylbenzenes from the Diene A ddition Products to Dibenzoylethylene, Journal of the American Chemical Society 62, 56-61. 32. Rogister, F., Laeckmann, D., Plasman, P. O., Van Eylen, F., Ghyoot, M., Maggetto, C., Liegeois, J. F., Geczy, J., Herchuelz, A ., Delarge, J., Masereel, B. (2001) Novel inhibitors of the sodium-calcium excha nger: benzene ring analogues of N-guanidino substituted amiloride derivatives, European Journal of Medicinal Chemistry 36, 597614. 33. Glinsukon, T., Weisburger, E. K., Benjamin, T ., Roller, P. P. (1975) Preparation and Spectra of Some Acetyl Deri vatives of 2,4-Toluenediamine, Journal of Chemical and Engineering Data 20, 207-209. 34. DeRuiter, J., Swearingen, B. E., Wandrekar, V., Mayfield, C. A. (1989) Synthesis and in Vitro Aldose Reductase Inhibitory Activity of Compounds Containing an NAcylglycine Moiety, Journal of Medicinal Chemistry 32, 1033-1038. 35. Zhou, Z. L., Kher, S. M., Cai, S. X., Whitte more, E. R., Espitia, S. A., Hawkinson, J. E., Tran, M., Woodward, R. M., Weber, E., Keana, J. F. W. (2003) Synthesis and SAR of Novel Diand Tris ubstituted 1,4-Dihyd roquinoxaline-2,3-dion es Related to Licostinel (Acea 1021) as NMDA/Glycine Site Antagonists, Bioorganic and Medicinal Chemistry 11, 1769-1780. 36. Verardo, G., Dolce, A., T oniutti, N. (1999) Reductiv e One Batch Synthesis of NSubstituted Pyrrolidines from Primary Amines and 2,5-Demethoxytetrahydrofuran, Synthesis 1, 74-79. 37. Marshalkin, V. N., Brovko, D. A., Samet, A. V., Semenov, V. V. (2001) Reaction of Polynitrotoluenes with Phthalic Anhydride, Russian Journal of Organic Chemistry 37, 1244-1248.

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147 CHAPTER FIVE APPENDIX: PROTON AND CARBON NMR SPECTRA

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148 Spectrum 5.1 N O H H O O 1 c

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149 Spectrum 5.2 N O H H O O 1 d

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150 Spectrum 5.3 1 e N O H H O O

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151 Spectrum 5.4 2 a N H O O C H 3

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152 Spectrum 5.5 N O H O N O O 2 b

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153 Spectrum 5.6 N O H O O 2 c

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154 Spectrum 5.7 N O H O O 2 d

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155 Spectrum 5.8 N O H O O 2 e

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156 Spectrum 5.9 N O N O O 3 a

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157 Spectrum 5.10 N O N O N O O O 3 b

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158 Spectrum 5.11 N O N O O O 3 c

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159 Spectrum 5.12 N O N O O O 3 d

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160 Spectrum 5.13 N O N O O O 3 e

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161 Spectrum 5.14 N O N O H B o c 5 a

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162 Spectrum 5.15 N O B n z O N O O N H B o c 5 b

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163 Spectrum 5.16 N O N O H B o c O 5 c

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164 Spectrum 5.17 N O N O H B o c O 5 d

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165 Spectrum 5.18 N O N O O B o c H 5 e

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166 Spectrum 5.19 N O H N O B o c H 6 a

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167 Spectrum 5.20 N O H N O B o c H O 6 c

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168 Spectrum 5.21 N O H N O B o c H O 6 d

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169 Spectrum 5.22 N O H N O O B o c H 6 e

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170 Spectrum 5.23 N O H N O B o c H 6 f

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171 Spectrum 5.24 N O H N O B o c H 6 g

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172 Spectrum 5.25 N O H N O B o c H 6 h

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173 Spectrum 5.26 N O H N O B o c H S 6 i

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174 Spectrum 5.27 9 N O B o c H O N O O

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175 Spectrum 5.28 1 2 b N O B o c H O N H C b z

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176 Spectrum 5.29 N O Z H O O 1 2 c

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177 Spectrum 5.30 N O Z H O O 1 2 d

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178 Spectrum 5.31 1 2 e N O Z H O O

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179 Spectrum 5.32 N O O O H 1 3 a

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180 Spectrum 5.33 1 3 b N O H O O

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181 Spectrum 5.34 1 4 b H N O O H N H

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182 Spectrum 5.35 1 4 c H N O O H N

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183 Spectrum 5.36 O N O O O H 1 5 a

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184 Spectrum 5.37 1 5 b N O O H N H O O

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185 Spectrum 5.38 1 5 c N O O H N O O

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186 Spectrum 5.39 O N O O O N H O O 1 6 a

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187 Spectrum 5.40 1 7 a H O N O H O O N H O O

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188 Spectrum 5.41 1 9 a N O C b z H O N H

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189 Spectrum 5.42 1 9 b N O C b z H O N

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190 Spectrum 5.43 2 0 H O N O O O N H O O

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191 Spectrum 5.44 N 2 3 a

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192 Spectrum 5.45 N 2 3 b

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1 93 Spectrum 5.46 N 2 3 c

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194 Spectrum 5.47 2 8 N N O 2

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195 Spectrum 5.48 N N O 2 3 1

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196 Spectrum 5.49 N N O 2 H O 3 6

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197 Spectrum 5.50 N N O 2 H O O O H 3 7

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198 Spectrum 5.51 N N O 2 H O O O 3 8

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199 Spectrum 5.52 N H 2 N O 2 O O 3 9 a

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200 Spectrum 5.53 N N O 2 O O 4 1

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201 Spectrum 5.54 N N O 2 O O O H O 4 3

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ABOUT THE AUTHOR Stephanie Tara Weiss was born in Detroit, Michigan and grew up in Plantation, Florida. After graduating from South Plantation High School in 1993, she attended New College of Florida, where she majored in natural sciences an d Spanish. She earned her B.A. there in 1997, and then went to the University of Alabama at Birmingham, where in 2001 she earned her M.S. in pharmaceutical design and medicinal chemistry under the supervision of Dr. Wayne Brouillette. In the spring of 20 02, she began her graduate studies at the University of South Florida, where she joined the lab of Professor Mark L. McLaughlin at the Moffitt Cancer Center and Research Institute. She will receive her Ph.D. in organic chemistry with a focus on peptidomim etics in May 2006.