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Title:
Design, combinatorial synthesis, and biological evaluation of novel α-helical mimetics based on functionalized piperazines as antagonists of p53/mdm2 interactions
Physical Description:
Book
Language:
English
Creator:
Topper, Melissa
Publisher:
University of South Florida
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Tampa, Fla
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Subjects

Subjects / Keywords:
MDM2
P53
Alpha helix
Protein
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The p53 protein promotes tumor supression upon activation, making it an attractive target in cancer therapies. A reported 50% of all human cancers display aberrant activation of the MDM2 oncoprotein, which directly promotes tumorgenesis by inactivating the transcriptional activity of wild type p53, and is commonly associated with drug, chemo, and radio therapy resistance. Previously reported crystallographic analysis of the p53/MDM2 complex infers that the p53 protein forms a 2.5 turn amphipathic alpha helix whose hydrophobic face interacts within a deep hydrophobic cleft in the N-terminal domain of the globular MDM2. This suggests that the synthesis of small molecular antagonists of p53/MDM2 binding interactions, capable of reactivating wild type p53 function, show a promising therapeutic strategy in pharmaceutical discovery. The use of alpha helix mimics for the disruption of p53/MDM2 binding interactions has been amply documented in the literature; however, these compounds contain hydrophobic scaffolds that limit their usefulness as potential drug candidates. Presented herein is the design, synthesis, and biological evaluation of novel non-peptidic, drug-like, small molecule inhibitors to target p53/MDM2 binding interactions. The mimetics are designed to bind to the N-terminal domain of MDM2 protein leaving p53 unbound and capable of activation. The inhibitor design is based on an alpha helix mimetic scaffold derived from functionalized piperazines, diketopiperazines, and/or pyrimidines. The mimetics are designed to have a higher degree of solubility and facile synthesis yet still maintain the desired spacial arrangements of hydrophobic side chains in the ith, ith+4, and ith+7 positions of a natural alpha helix. The small molecules are designed to act as antagonists of protein/protein interactions, tumor inhibitors, and be potent p53 activators.
Thesis:
Dissertation (PHD)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Melissa Topper.
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Title from PDF of title page.
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Document formatted into pages; contains X pages.

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ABSTRACT: The p53 protein promotes tumor supression upon activation, making it an attractive target in cancer therapies. A reported 50% of all human cancers display aberrant activation of the MDM2 oncoprotein, which directly promotes tumorgenesis by inactivating the transcriptional activity of wild type p53, and is commonly associated with drug, chemo, and radio therapy resistance. Previously reported crystallographic analysis of the p53/MDM2 complex infers that the p53 protein forms a 2.5 turn amphipathic alpha helix whose hydrophobic face interacts within a deep hydrophobic cleft in the N-terminal domain of the globular MDM2. This suggests that the synthesis of small molecular antagonists of p53/MDM2 binding interactions, capable of reactivating wild type p53 function, show a promising therapeutic strategy in pharmaceutical discovery. The use of alpha helix mimics for the disruption of p53/MDM2 binding interactions has been amply documented in the literature; however, these compounds contain hydrophobic scaffolds that limit their usefulness as potential drug candidates. Presented herein is the design, synthesis, and biological evaluation of novel non-peptidic, drug-like, small molecule inhibitors to target p53/MDM2 binding interactions. The mimetics are designed to bind to the N-terminal domain of MDM2 protein leaving p53 unbound and capable of activation. The inhibitor design is based on an alpha helix mimetic scaffold derived from functionalized piperazines, diketopiperazines, and/or pyrimidines. The mimetics are designed to have a higher degree of solubility and facile synthesis yet still maintain the desired spacial arrangements of hydrophobic side chains in the ith, ith+4, and ith+7 positions of a natural alpha helix. The small molecules are designed to act as antagonists of protein/protein interactions, tumor inhibitors, and be potent p53 activators.
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Helical Mimetics Based on Functionalized Piperazines as Antagonists of p53/MDM2 Interactions by Melissa Elizabeth Topper A dissertation submitted in partial fulfillment of 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. Jon Antilla, Ph.D. Wayne C. Guida, Ph.D. John Koomen, Ph.D. Roman Manetsch, Ph.D. Date of Approval: July 8 2010 Keywords: MDM2, p53, helix, apoptosis, protein Copyright 2010, Melissa Elizabeth Topper

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Dedication fail Thank you dad, for making this all possible And for always catching me

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i Table of Contents Table of Contents .............................................................................................................i List of Figures ................................................................................................................ iv List of Schemes .............................................................................................................vii List of Abbreviations ....................................................................................................... x Abstract ....................................................................................................................... xiii CHAPTER ONE: Cancer Formation and its Association with Proteins ............................ 1 1.1 Cancer ............................................................................................................ 1 1.2 Tumor Protein p53 ......................................................................................... 3 1.3 MDM2 ......................................................................................................... 10 1.4 Crystal structure of the p53/MDM2 complex ................................................ 11 -Helix mimetics to disrupt the p53/MDM2 complex ................................... 13 1.6 Fluorescence Polarization Assay .................................................................. 16 1.7 ELISA .......................................................................................................... 17 1.8 Drug Discovery Process ............................................................................... 18 1.9 Goal and Objectives ..................................................................................... 19 1.10 References .................................................................................................. 20 Chapter Two: Design and Synthesis of the Piperazine-2,5-Dione-Piperazine Hybrid Scaffold ....................................................................................... 25 2.1 Introduction .................................................................................................. 25 2.1.1 Terephthalamide Scaffold .............................................................. 25 2.1.2 Rational of the Piperazine-2,5-Dione-Piperazine Hybrid Scaffold ...................................................................................... 26 2.1. 3 Molecular Modeling Studies .......................................................... 28 2.2 Results and Discussion ................................................................................. 31 2.2.1 Retrosynthesis of the Piperazine-2,5-Dione-Piperazine Hybrid Scaffold ...................................................................................... 31 2.2.2 Synthesis of the Piperazine-2,5-Dione Dimers ............................... 33 2.2.3 Synthesis of Trimers 2.13 and 2.14 ................................................ 41 2.2.4 ELISA Assays ............................................................................... 43 2.3 Conclusion ................................................................................................... 46 2.4 Experimental Section ................................................................................... 47 2.4.1 Materials and Methods ................................................................... 47

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ii 2.4.2 Experimental Procedures ............................................................... 48 Chapter Three: Design and Synthesis of the Hybrid 2,5-Piperazine-DionePyrimidine Based Scaffold .................................................................... 87 3.1 Introduction .................................................................................................. 87 3.1.1 Pyrimidines .................................................................................... 87 3.1.2 Hybrid Scaffold Design ................................................................ 89 3.1.3 Molecular Modeling Studies .......................................................... 91 3.2 Results and Discussion ................................................................................. 94 3.2.1 Hybrid Piperazine/Pyrimidine Scaffold, Approach 1 ...................... 94 3.2.2 Hybrid Piperazine/Pyrimidine Scaffold, Approach 2 ...................... 99 3.2.3 Hybrid Piperazine/Pyrimidine Scaffold, Approach 3 .................... 109 3.2.4 Fluorescence Polarization Assays ................................................. 112 3.3 Conclusion ................................................................................................ 112 3.4 Experimental Procedures ............................................................................ 112 3.5 References .................................................................................................. 124 Chapter Four: Design and Synthesis of Functionalized 2,5 and 2,6-Diketopiperazines .................................................................................................. 127 4.1 Introduction ................................................................................................ 127 4.1.1 Piperazine Diones ........................................................................ 127 4.1.2 Design of 3-R-Piperazine-2,5 and 2,6-Dione Scaffold .................. 128 4.2 Results and Discussion for the 3-R-Piperazine-2,6 -Diones ......................... 129 4.2.1 Retrosynthesis of 3-R-piperazine-2,6-Dione Scaffold ................... 129 4.2.2 Synthesis of Key Di-acid Derivatives 4.5a-c ................................ 131 4.2.3 So lid Phase Synthesis .................................................................. 133 4.2.4 Solution Phase Synthesis of 2,6-DKP Monomers ......................... 134 4.2.5 Attempts at Isolation of Cyclic Anhydride 4.6 .............................. 136 4.3 Results and Discussion for the 3-R-Piperazine-2,5-Diones ......................... 137 4.3.1 Retrosynthesis of 3-R-Piperazine-2,5-Dione, Unit A 1 : Approach 1 ............................................................................. 137 4.3.2 Synthesis of Compound 4.19 ........................................................ 139 4.3.3 Attempted Synthesis of the 2,5-DKP Monomer 4.22 .................... 139 4.3.4 Retrosynthesis of 3-R-Piperazine-2,5-Dione, Unit A 1 : Approach 2 ............................................................................. 141 4.3.5 Synthesis of 2,5-DKP Monomer, Route 1 .................................... 142 4.3.6 Synthesis of 2,5-DKP Monomer, Route 2 .................................... 145 4.3.7 Hydrazine Insertion ...................................................................... 146 4.4 Conclusion ................................................................................................ 147 4.5 Experimental Procedures ............................................................................ 148 Chapter Five: Appendicies Appendix A: Selected 1 H and 13 C NMR Spectra ............................................... 167 Appendix B: Selected Mass Spectra ................................................................ 255 Appendix C: X-Ray Crystallographic Data ....................................................... 301

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iii About the Author ................................................................................................ End Page

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iv List of Figures Figure 1.1 Acquired cellular capabilities of cancer .................................................... 2 Figure 1.2 Estimated new cancer cases and deaths in the United States for 2010 .................................................................................................... 3 Figure 1.3 Cellular homeostasis ................................................................................. 4 Figure 1.4 p53 signaling pathway with the p53 protein circled in red ......................... 5 Figure 1.5 N -helix ......................................................................................... 6 Figure 1.6 Cartoon representation of the p53 protein ................................................. 7 Figure 1.7 ...................................... 8 Figure 1.8 Regulation of p53 by MDM2 .................................................................... 9 Figure 1.9 Cancers that commonly display overexpressed MDM2 ........................... 11 Figure 1.10 MDM2 protein bound to the helical domain of the p53 protein ............... 12 Figure 1.11 Simplified image of the three key p53 binding residues .......................... 13 Figure 1.12 H substituent's in the ith, ith +4, and ith +7 positions ................................ .... 14 Figure 1.13 -2 ......................................................... 15 Figure 1.14 Overlay of the Hamilton terphenyl scaffold ............................................ 16 Figure 1.15 Schematic of a fluorescence polarization assay ....................................... 17 Figure 1.16 Schematic of a competitive ELISA assay ................................................ 18 Figure 1.17 Drug discovery protocol ......................................................................... 19 Figure 2.1 Terephthalamide ..................................................................................... 25

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v Figure 2.2 Piperazine-2,5-dione-piperazine hybrid scaffold ..................................... 27 Figure 2.3 -helix representations ........................................................... 27 Figure 2.4 Top glide scoring proposed hybrid scaffold 2.13 ..................................... 28 Figure 2.5 Molecular docking of 2.13 ...................................................................... 29 Figure 2.6 Schematic representations of 2.13 ........................................................... 30 Figure 2.7 Overlay of the p53 protein (transparent light blue) with the proposed hybrid scaffold 2.13 docked in the MDM2 hydrophobic pocket ................................................................................ 30 Figure 2.8 X-ray crystal structures of compounds 2.7e and 2.11g ............................ 41 Figure 2.9 Nutlin-3 and PDI were used as test controls in an ELISA assay to detect binding between immobilized His 6 -p53 and GST-MDM2 ............. 44 Figure 2.10 ELISA results ......................................................................................... 45 Figure 2.11 Piperazine-2,5-dione-piperazine scaffold derivatives .............................. 46 Figure 3.1 Structures of (A) Benzene (B) Pyrimidine .............................................. 87 Figure 3.2 Marketed drugs with pyrimidine sub-units highlighted in blue ................ 88 Figure 3.3 Scaffolds containing piperazine and pyrimidine units circled in blue .................................................................................................... 89 Figure 3.4 Hybrid piperazine/pyrimidne scaffold ..................................................... 89 Figure 3.5 Schematic representations ....................................................................... 90 Figure 3.6 Top scoring hybrid piperazine/pyrimidne scaffold 3.38 ........................... 91 Figure 3.7 Molecular docking of the hybrid piperazine/pyrimidine scaffold ............. 92 Figure 3.8 Hydrophobic interactions of the top scoring hybrid scaffold 3.38 ............ 93 Figure 3.9 Overlay of the p53 protein (transparent light blue) with the proposed hybrid piperazine/pyrimidine scaffold docked to the MDM2 hydrophobic pocket .......................................................... 93

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vi Figure 3.10 Crystal structures for compounds 3.20a, 3.21a, 3.22a, 3.24, and 3.25 ................................................................................................ 106 Figure 4.1 Structures of DKPs ............................................................................... 127 Figure 4.2 2,5 and 2,6 DKP based scaffold ............................................................ 129 Figure 4.3 X-ray crystal structures of compounds 4.27a and 4.27b ........................ 145

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vii List of Schemes Scheme 2.1 Retrosynthesis of the piperazine-2,5-dione-piperazine hybrid scaffold ....................................................................................... 32 Scheme 2.2 Synthesis of compound 2.3a-d and 2.4a-h ............................................... 33 Scheme 2.3 Synthesis of compounds 2.5a-g............................................................... 34 Scheme 2.4 Attempted cyclization of 2.5a ................................................................ 35 Scheme 2.5 Synthesis of compounds 2.6a-g and 2.7a-e .............................................. 36 Scheme 2.6 Synthesis of compounds 2.8a-d and 2.9a-k ............................................. 37 Scheme 2.7 Synthesis of compounds 2.10a-i ............................................................. 38 Scheme 2.8 Synthesis of compounds 2.11a-i and 2.12a-i ........................................... 39 Scheme 2.9 Attempted synthesis of 2.13 .................................................................... 42 Scheme 2.10 Synthesis of trimers 2.13 and 2.14 .......................................................... 43 Scheme 3.1 Retrosynthesis Hybrid 2,5-DKP-Pyrimidine Scaffold, Approach 1 ......... 94 Scheme 3.2 Synthesis of pyrimidine unit A 2 compounds 3.3a-b ................................ 95 Scheme 3.3 Attempted formation of compound 3.4 ................................................... 96 Scheme 3.4 Attempted formation of compound 3.6 ................................................... 97 Scheme 3.5 Attempted indole mediated sulfone displacement ................................... 98 Scheme 3.6 Attempted indole mediated chlorine displacement .................................. 98 Scheme 3.7 Attempted chlorine displacement ............................................................ 99 Scheme 3.8 Retrosynthesis hybrid 2,5-DKP-pyrimidine scaffold, Approach 2 ......... 100 Scheme 3.9 General synthetic method for nitro-containing pyrimidines ................... 101

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viii Scheme 3.10 Proposed synthesis of 3.15, Attempt 1 .................................................. 101 Scheme 3.11 Proposed synthesis 3.15, Attempt 2 ...................................................... 102 Scheme 3.12 Formation of ester 3.17 ......................................................................... 102 Scheme 3.13 Synthesis of 3.14 .................................................................................. 103 Scheme 3.14 Synthesis of 3.21a-b ............................................................................. 104 Scheme 3.15 Synthesis of nitro-based pyrimidines .................................................... 105 Scheme 3.16 Attempted formation of compound 3.26 ............................................... 107 Scheme 3.17 Attempted formation of 3.28 by bromine displacement ......................... 108 Scheme 3.18 Attempted formation of 3.30 by bromine insertion ................................ 108 Scheme 3.19 Attempted amine coupling to compound 3.31 ....................................... 109 Scheme 3.20 Retrosynthesis hybrid piperazine/pyrimidine scaffold, Approach 3 ........................................................................................... 110 Scheme 3.21 Synthesis of N,N -diboc-pseudo guanidinylating agent 3.35 ................... 111 Scheme 3.22 Successful synthesis of hybrid scaffold 3.38 ......................................... 111 Scheme 4.1 Retrosynthesis of the 3-R-piperazine-2,6-dione scaffold ....................... 130 Scheme 4.2 Synthesis of di-acids 4.5a-c .................................................................. 132 Scheme 4.3 Solid phase synthesis of the 3-R-piperazine-2,6-dione scaffold ............. 134 Scheme 4.4 Solution phase synthesis of the 2,6-DKPs ............................................. 135 Scheme 4.5 Attempted cyclic anhydride 4.6 isolation .............................................. 136 Scheme 4.6 Retrosynthesis of 3-R-piperazine-2,5-dione, A 1 : Approach 1 ................ 138 Scheme 4.7 Synthesis of succinimidyl diazoacetate ................................................. 139 Scheme 4.8 Attempted synthesis of 2,5-DKP monomer 4.22 and 4.25 ..................... 140 Scheme 4.9 Retrosynthesis of 3-R-piperazine-2,5-dione, A 1 : Approach 2 ................ 142

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ix Scheme 4.10 Route 1 used to synthesize the 2,5-DKP monomers .............................. 143 Scheme 4.11 Attempts to synthesize 4.26a ................................................................ 144 Scheme 4.12 Route 2 used to synthesize the 2,5-DKP monomer ................................ 146 Scheme 4.13 Attempts at hydrazine formation ........................................................... 147

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x List of Abbreviations Alpha Angstrom Ac 2 O Acetic anhydride AcOH Acetic acid aq. Aqueous Beta BINAP 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl Bn Benzyl Boc tert -Butoxycarbonyl br Broad (spectral) Bu Butyl o C Degree Celsius 13 C NMR Carbon-13 Nuclear Magnetic Resonance CDI N,N' -Carbonyldiimidazole CH 3 CN Acetonitrile Cs 2 CO 3 Cesium carbonate Delta or chemical shift DCC N,N -dicyclohexylcarbodiimide DCM Dichloromethane DIC N,N -diisopropylcarbodiimide DIEA Diisopropylethylamine DKP Diketopiperazine DMAP 4-(dimethylamino)pyridine DMF N,N -Dimethylformamide DMF-DMA N,N -dimethylformamide dimethyl acetal DMSO Dimethylsulfoxide EDC ethyl-( -dimethylamino)propylcarbodiimide-hydrochloride ELISA Enzyme-linked immunosorbent assay Et Ethyl EtOAc Ethyl acetate EtOH Ethanol ESI Electrospray ionization equiv. Equivalent(s) FP Fluorescence polarization g Gram(s) GST Glutathione-S-transferase 1 H NMR Proton Nuclear Magnetic Resonance h Hour(s)

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xi HATU (2 (7 -Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) HBTU O-(Benzotriazol-1-yl)N,N,N',N' -tetramethyluronium hexafluorophosphate HCTU 1H-Benzotriazolium 1-[bis(dimethylamino)methylene]-5chlorohexafluorophosphate (1-),3-oxide HMQC Heteronuclear multiple quantum coherence HOBt 1-hydroxybenzotriazole HPLC High pressure liquid chromatography HR High resolution Hz Hertz IC 50 50% inhibitory concentration J Coupling-constant(s) Ki Inhibitor dissociation constant KOBt Potassium tert -butoxide KOt-amyl Potassium tert -amyloxide Leu Leucine LiOH Lithium hydroxide M Molar or moles per liter mCPBA meta -Chloroperoxybenzoic acid MDM2 Murine double minute 2 Me Methyl MeOH Methanol mg Milligram(s) min Minute(s) mL Milliliter(s) mmol Millimole(s) m.p. Melting point MS Mass spectrum MW Microwave NaH Sodium hydride NaOEt Sodium ethoxide NaOH Sodium hydroxide NMP NMethyl-2-pyrrolidone NOBF 4 Nitrosonium tetrafluoroborate nM Nanomolar ORTEP Oak Ridge thermal ellipsoid plot (crystallography) PCC Pyridinium chlorochromate Pd(OAc) 2 Palladium(II) acetate Pd/C Palladium on carbon Ph Phenyl Phe Phenylalanine ppm Parts per million Pr Propyl PyBrOP Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate RMSD Root mean square deviation

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xii rt Room temperature SAR Structure activity relationship sat. Saturated SPPS Solid-phase peptide synthesis TFA Trifluoroacetic acid THF Tetrahydrofuran TMSCl Trimethylsilyl chloride TLC Thin layer chromatography Trp Trptophan TS Tumor suppressor Microliter(s) Micormolar Val Valine wt Wild type

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xiii -Helical Mimetics Based on Functionalized Piperazines as Antagonists of p53/MDM2 Interactions Melissa Topper Abstract The p53 protein promotes tumor eradication upon activation, making it an attractive target in cancer therapies. A reported 50% of all human cancers display aberrant activation of the MDM2 oncoprotein, which directly promotes tumorgenesis by inactivating the transcriptional activity of wild type p53, and is commonly associated with drug, chemo, and radio therapy resistance. Previously reported crystallographic analysis of the p53/MDM2 complex infers that the p53 protein forms a 2.5 turn amphipathic alpha helix whose hydrophobic face interacts within a deep hydrophobic cleft in the NH 2 -terminal domain of the globular MDM2. This suggests that the synthesis of small molecular antagonists of p53/MDM2 binding interactions, capable of reactivating wild type p53 function, show a promising therapeutic strategy in pharmaceutical discovery. The use of alpha helix mimics for the disruption of p53/MDM2 binding interactions has been amply documented in the literature; however, these compounds contain hydrophobic scaffolds that limit their usefulness as potential drug candidates. Presented is the design, synthesis, and biological evaluation of novel non-peptidic, drug-like, small molecule inhibitors to target p53/MDM2 binding

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xiv interactions. The mimetics are designed to bind to the NH 2 -terminal domain of MDM2 protein leaving p53 unbound and capable of activation. The inhibitor design is based on an alpha helix mimetic scaffold derived from functionalized piperazines, diketopiperazines, and/or pyrimides. The mimetics are designed to have a comparably higher degree of solubility and notably facile synthesis yet still maintain the desired spacial arrangements of hydrophobic side chains in the ith ith+ 4, and ith+ 7 positions of a natural alpha helix. The small molecules are designed to act as antagonists of protein/protein interactions, tumor inhibitors, and potent p53 activators.

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1 Chapter One: Cancer Formation and its Association with Proteins 1 .1 Cancer Cancer is a group of fundamentally genetic diseases that begin within the cell. It is widely agreed upon that the formation of tumors (tumor i genesis) in humans is a complicated, multi step, progressive process. (Busygina and Bale, 2006; Hanahan and Weinberg, 2000; Lengauer et al., 1998) These steps, which display a succession of genetic alterations, are responsible for the progression of normal human cells to abnormal cancerous cells to a conglomerated mass of abnormal cells termed a tumor. (Hanahan and Weinberg, 2000) There are more than 100 different types of cancer and all of those various cancer cells are hallmarked by the same six essential alterations to otherwise norm al cell physiology as shown in Figure 1.1. Cells, which will progress to human malignancies, have the acquired ability to induce and sustain angiogenesis, evade apoptosis, discount anti growth signals, display limitless replication potential, are self s ufficien t in growth signals, and are able to invade surrounding tissue and metastasize. (Genentech, 2008; Hanahan and Weinberg, 2000)

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2 Figure 1 .1: Acquired cellular capabilities of cancer Anyone one of these alterations is attributed to the mutation of a specific gene and a culmination of all six alterations enables the transformation of a normal functioning cell to cancerous cell. The occurrence of gene mutation is a highly inefficient process. The body is designed to maintain pristine genomic order by a complex array actions initiated by tumor suppressor (TS) genes, p roto oncogenes, and DNA repair genes. The TS and proto proliferation, adhesion, and cellular death (apo p tosis). (Busygina and Bale, 2006; Levitt and Hickson, 2002) directly affect cell growth, but are directly involved in the maintenance of g enomic stability by preventing superfluous mutations. (Busygina and Bale, 2006; Hanahan and Weinberg, 2000; Levitt and Hickson, 2002) This is done through the actions of nucleotide ex cision and D NA mismatch repair genes. (Lengauer et al., 1998) Given the

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3 high level of complex it natural maintenance process, it would be highly unlikely that all six aforementioned mutations could or would spontaneously occur in a single normal cell, resulting in cancer development. This poses one of the greatest conun drums in modern medicine as cancer undoubtedly does develop in a substantial portion of the population, causing a world wide hea lth concern (Busygina and Bale, 20 06; Hanahan and Weinberg, 2000) There were an estimated 1,529,560 new cases of cancer reported in the United States alone in 2009, and cancer is the second leading cause of death in the United States (Figure 1.2) (American Cancer Society, 2 010; Heron et al., 2009) This overwhelming statistic would provide sound belief that the genomes of the caretakers, gatekeepers, or possibly both, have been compromised and therefore left susceptible to increased mutability to account for the high inc ident rate of cancer diagnos es (Busygina and Bale, 2006; Hana han and Weinberg, 2000; Lengauer et al., 1998) Figure 1.2 : Estimated new cancer cases and deaths in the United States for 2010 1.2 Tumor Protein p53 P roper cell function is dependent upon a delicate balance between cell proliferation and cell death (apoptosis) (Figure 1.3). An excess of apoptosis can result in embryonic defects and/or lethality, autoimmune diseases, and neurod egenerative diseases. While an excess of cellular proliferation can result in tumorigenesis and metastasis.

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4 Figure 1.3 : Cellular homeostasis There are many signaling pathways within the body whose function is to maintain homeostasis between cellular proliferation and apoptosis. The pathway most commonly regarded as compromised and implicated in tumorigenesis is the p53 signaling pathway. The primary function of this pathway is to induce cell cycle ar rest or apoptos is to irreparabl y damaged cells. (Pogribny et al., 2009) As depicted by Figure 1.4, p53 is the central component of this complex network, directly effec ting ever gene and downstream signal within this pathway d ocumenting th is protein s paramount role in proper cell function.

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5 Figure 1.4 : p53 signaling pathway with the p53 protein circled in red Tu mor suppressor protein 53, more commonly known as p 53, is located at the core of an exceedingly complex network within the living cell and exists within the body helical N terminal do main that binds to MDM2 and MDM X (Laptenko and Prives, 2006) helix, first proposed by Pauling, et al ., and later confirmed in 1951 by Perutz, is the mos t abundant secondary conformation found in naturally occurring proteins (approximately 40%). (Han Yin, 2007; Han ahan and Weinberg, 2000) An helical state has been defined as a state in which the phi and psi display dihedral angles of approximately 60 o twisting repeatedly in the s ame right handed direction. Th e distance separ ating each turn of the helix is 5.4 in a confirmation containing 3.6 amino acids per

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6 turn of the helix, with a rise of 1.5 per amino acid as shown in Figure 1.5 ( Han Yin, 2007; Woster, 2010) Figure 1.5 : helix P53 is a transcription factor that dir ectly effects tumor suppression, and acts as a checkpoint in the cell cycle, inducing cell cycle arrest or apoptosis to irreparable cells. (Sturzbecher and Deppert, 1994) e guardian of the mutations. (Lane, 1992) The p 53 protein regulates expression of over one hundred different targets by acting as a sequence specific transcription factor. (Bai and Zhu, 2006) The p53 protein is a nuclear phosphoprotein with a molecular weight of 53 kDa and is located on the small arm of chromosome 17. (Bai and Zhu, 2006) It consists of 393 amino acids and occurs in the active form as a tetramer composed of four identical subunits

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7 (Figure 1.6) (Bai and Zhu, 2006; Ren et al., 2005) Each monomer contains several structural and functional domains: N terminus Amino terminus domain (residues 1 42), required for transactiva tion activity, contains the MDM2 protein binding site Proline rich region (residues 40 92) contains a series of repeating proline residues that are involved in p53 stability regulated by MDM2 Central Core DNA binding domain (residues 102 292) is the central domain of the p53 protein and is required for sequence specific DNA binding. 90% of p53 mutations are found in this region. C terminus Oligomerization domain (residues 324 355) consists of a beta strand, followed by alpha helix and this domain is necessary for p53 dimerization Carboxy terminus (residues 363 393) is a strongly basic domain that is involved in the downregulation of DNA binding in the central domain and has been connected to the initiation o f cell death Figure 1. 6 : Cartoon r epresentation of the p53 protein

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8 p 53 is functionally activated by a wide array of cellular stress signals such as: DNA damage, UV radiation, hypoxia and chemotherapeutic agents. (Scian et al., 2004) The biological repercuss ion s following p53 activation include: cell cycle arrest, induction of programmed cell death (apoptosis), DNA repair, and cellular senescence (Figure 1.7) (Bai and Zhu, 2006; Braithwaite and Prives, 2006; Laptenko and Prives, 2006; Scian et al., 2004) Two key features of the p 53 protein that a re required for proper function are the ability to mobilize transcriptional co regulators, and the capacity to recognize and bind specific DN A sequences. (Laptenko and Prives, 2006) Figure 1. 7 : R epresentation activation and response In unstressed cells, p53 transcriptional activity is inert and its turnover rate is downregulated via the binding of specific proteins (such as MDM2, COP1, PIRH2 or JNK) that promote p53 degr adation through ubiquitin dependent proteasome pathways. (Alarcon Vargas and Ronai, 2002) The major regulator of p53 is MDM2.

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9 When bound to p53, MDM2 inhib its the phosphorylation of p53 and ultimately results in the degradation and nuclear transport of p53. (Chen et al., 2005) When ce lls receive a genotoxic stress signal, excess p53 is produced which stimulates the production of MDM2. These excess levels of MDM2 lower the p53 levels via a negative feedback reaction and the stress signal induces phosphorylation of serine residues within the activation domain of the p53 protein (Figure 1.8) (Hardcastle, 2007) Figure 1. 8 : Regulation of p53 by MDM2 The phosphorylation of p53 prevents p53 MDM2 binding interactions. (Stur zbecher and Deppert, 1994) Mitogenic signals then activate the p53 by induction of the ADP ribosylation factor (ARF) tumor suppressor which binds to MDM2 and further inhibits the ability of MDM2 to ubiquitinate p53. (Chen et al., 2005) This activation allows for the p53 to induce apoptosis or cell cycle arrest of damaged cells. After these actions are completed, the excess p53 i s quickly dephosphorylated and destroyed by the accumulation of MDM2. Levels of p53 must be strictly regulated for proper cell

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10 function. (Chen et al., 2005; Sturzbecher and Deppert, 1994) Mutations in the p53 gene have been reported in approximately 50% of all malignancies (Bai and Zhu, 2006; Levine et al., 2004; Scian et al., 2004; Sturzbecher and Deppert, 1994) Inactivation of the p53 gene is often associated with increased malignancy, treatment resistance, and decreased survival rates. (O'Brien, 2007) 1.3 MDM2 Levels of p53 must be strictly regulated for proper cell function, and the p redominanat regulator of p53 is cellular MDM2 oncoprotein. (Freedman et al., 1999; Kussie et al., 1996; Lu et al., 2001) When bound to p53, MDM2 prevents phosphorylation and subsequent activation of p53. This binding interaction trigger s the degradation of p53 via the ubiquitin system transport (Alarcon Vargas and Ronai, 2002; Chen et al., 2005; Sturzbecher and Deppert, 1994) A reported 50% of malignancies maintain wild type p53 expression and a large percentage of t hese tumors display compromised p53 function due to an overexpression of the MDM2 protein, as shown in Figure 1.9. (Shangary and Wang, 2009) Over expression of MDM2 and excess p53/MDM2 binding interactions inhib its the p53 ultimately resulting in inhibition of apoptosis. (Freedman et al., 1999) Th is permits tumor prog ression from ben ign, to malignant, to metasta tic (Alarcon Vargas and Ronai, 2002; Chen et al., 20 05; Shangary and Wang, 2009) Due to the paramount role that overexpression of MDM2 plays in the inactivation of wild type p53, th erapeutically

PAGE 27

11 targeting MDM2 for the development of anti tumor agents, to disrupt p53/MDM2 interactions and restore wild type p53 function is an attractive area in cancer research. (Klein and Vassilev, 2004) Figure 1. 9 : Cancers that commonly display overexpressed MDM2 Initial validation of MDM2 as a potential drug candidate have been explored by several different approaches. (Klein and Vassilev, 2004) Microinjection of monoclonal antibodies directed against the p53 binding site on MDM2 resulted in the inhibition of p53/MDM2 binding interactions and subsequent activation and cellu lar accumulation of p53 (B laydes et al., 1997; Bottger et al., 1996) It was reported that inhibition of MDM2 expression by antisense oligonucleotides resulted in activation of p53. (Chen et al., 1998; Chen et al., 1999; Chen et al., 2005; Zhang et al., 2003) 1.4 Crystal structure of the p53/MDM2 complex Previously reported crystallographic analysis of the p53/MDM2 complex (Figure 1 .10 ) infers that there is a de ep hydrophobic cleft in the NH 2 terminal domain of the

PAGE 28

12 globular MDM2 which contains the p53 binding activity. (Bartlett et al., 1990; Chen et al., 2005; Kussie et al., 1996; Yin and Hamilt on, 2005) The p53 peptide forms a 2.5 turn helix whose hydrophobic face buries deep within t he hydrophobic cleft of the MDM2 protein. (Kussie et al., 1996) It has been shown that a triad of hy drophobic and aromatic amino acids (phenalanine 19 (F19), tryptophan 23 (W23), and leucine 26 (L26)) are aligned along one face of the p53 helical peptide in the ith ith+ 4, and ith+ 7 positions, are imperative for binding interactions to occur. (Kussie et al., 1996; Moisan et al., 2008; Yin and Hamilton, 2005) Figure 1. 10 : MDM2 protein bound to the helical domain of the p53 protein P53 (green) with key residues highlighted, F19 (yellow), W23 (pink), and L26 (blue). ( PDB structure 1YCQ ) Studies report that the nature of interactions between p53 and MDM2 are primarily hydrophobic. The binding surface of p53 is dominated by a triad of amino acids

PAGE 29

13 (F19 W23 and L26) that interact with a hydrophobic cleft in the binding domain of the MDM2 protein (Figure 1.10 and Figure 1.11) (Cummings et al., 2006; Kussie et al., 1996; Yin and Hamilton, 2005) This interaction defined the corresponding F19, W23, and L26 pockets necessary for the p53/MDM2 interactions to occur. In this classification, the F19 pocket is defined by amino acid residues R65, Y67, E69, H73, I74, V75, M62, and V93, the W23 pocket is defined by S92, V93, L54, G58, Y60, and V93, and the L26 pocket is defined by Y100, T101, and V53. (Kussie et al., 1996) Figure 1. 11 : Simplified image of the three key p53 binding residues. Labeled Leu, Trp and Phe domains in the MDM2 hydrophobic pocket 1.5 Helix mimetics to disrupt the p53/MDM2 complex Numerous examples of helix mimetics developed to disrupt p53/MDM2 binding inter actions are reported in the literature. (Chen et al., 2005; Cummings et al., 2006; Shangary and Wang, 2009; Yin et al., 2005a) These s mall molecules were designed to helical region of the p53 peptide, containing key residues in the ith ith +4, and ith +7 positions of a natural helix (Figure 1.12 ). (Chen et al., 2005; Cummings and Hamilton, 2010; Kutzki et al., 2002; Yin and Hamilton, 2005) The synthetic targets were designed to bind to the hydrophobic cleft of the MDM2

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14 protein and disrupt p53/MDM2 binding interactions The binding between the synthetic mimic and MDM2 would result in an unbound wild type p53 Leaving p53 capable of phosphorylation and activ ation for apoptosis induction, and disruption of the p53/MDM2 complex inhibit s p53 degradation, making the small molecules antagonists of prote in/ protein interactions, tumor inhibitors, and potent p53 activators (Cummings and Hamilton, 2010) Figure 1. 12 : substituent's in the ith, ith +4, and ith +7 positions Dr. Andrew Hamilton, from Yale University, pioneered the field of using helix mimics to effi ciently d isrupt protein/protein binding interactions (Chen et al., 2005;

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15 Cummings and Hamilton, 2010; Kutzki et al., 2002; Yin et al., 2005b) His most recognizable contribution to the field is the design of the non peptide based terphenyl scaffolds used to disrupt p53 MDM2 binding interactions. Many variation of the terphenyl scaffold were synthesized and found to be biologically active. Figure 1.13 displays a particular terphenyl derivative A 2, that was reported to disrupt the binding protein p rotein interactions of the p53 MDM2 biological system in vitro (182 nM). (Chen et al., 2005; Yin and Hamilton, 2004; Yin and Hamilton, 2005) Analysis conducted by h eteronuclear m ultiple q uantum c oherence ( HMQC) data showed that the side chains pointed in the desired ith, ith +4 and ith +7 helix, as shown in Figure 1.14. (Yin et al., 2005a) Figure 1. 13 : A 2

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16 Figure 1. 14 : Overlay of the Hamilton terphenyl scaffold. T he p53 peptide (green) with the key hydrophobic side chains of F19, W23, and L26 s hown in stick representations. Further evaluation of scaffold A 2 show ed poor potency in ELISA assays of with a reported IC 50 of and the scaffold was not shown to activate p53. It was reasoned that the poor biological activity was due to the scaffolds high degree of hydrophobicity which leads to poor solubility and limits their usefulness as potential drug candidates. Additionally, the overall synthesis was report ed to be lengthy and low yielding (Chen et al., 2005) 1.6 Fluorescence Polarization Assay Fluorescence Polarization is a technique e specially applied to the study of molecular interactions (receptor/ligand studies, protein/protein interactions, DNA/protein interact ions, tyrosine kinase assays, and competitive immun o assays) (Knight et al., 2002; Perrin, 1926) Fluorescence polarization measurements are based on the assessment of the rotational motion of fluorescently labeled macromolecules, as shown in Figure 1.15. (Kinoshita, 2010) The fluorophore is attached to the small binding partner of the

PAGE 33

17 desired int erest and excited by polarized light. Small molecules rotate quickly during the excited stat e, and upon emission, have low polarization values. In contrast, upon excitation, large molecules display little movement the emitted light remains highly polariz ed. A large molecule is formed by the binding of a second molecule to the fluorescently labeled small molecule resulting in slowed tumbling during the lifetime of the excitation and a corresponding ly hi gh polarization value The fluorescence polarization assay reports a direct, n early instantaneous measure of the tracer`s bound/free rat io, and will be used for the specific analysis of the ability of our library of compounds to bind to the NH 2 terminal domain of MDM2. Figure 1. 15 : Schematic of a fluorescence p olarization assay 1.7 ELISA The e nzyme linked immunosorbent assay ( ELISA ) is a plate based assay that is used to determine if a particular peptide, protein, antibody, or hormone is present in a sample and quantify it. (Shurley et al. 2005) An ELISA can also be p erformed as a competitive assay when the targeted an tigen is small and has only one antibody binding

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18 site (Figure 1.16) (GenWay, 2010) In this type of assay, a known antigen and unknown target compete for binding to the capture antibody. There is an inverse relationship between the signal obtained and the concentration of the unknown target in the sample therefore a decrease in signal indicates the presence a n effective inhibitor (Oldreive et al. 2001) Figure 1. 16 : Schematic of a competitive ELISA assay 1.8 Drug Discovery Process Within our lab, a specific drug discovery protocol is followed. The process begins with a strong knowledge base of the desired biological target and/or pathway. Following this, a rational retro synthesis for a library of potential drug targets is propose d. Molecular modeling studies are performed to increase the likelihood for hit generation. Synthesis of the v irtual hits is performed followed by analysis from in vitro biological assays. Optimization of hits is performed un til the molecules display the

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19 desired pharmacological properties necessary for preclinical in vitro analysis. Following successful preclinical trials are clinical trial s and then ultimately the approval of a new drug. This is a lengthy and expensive pr ocess. It was reported that the entire process takes 10 15 years to complete, costs $802 million dollars to develop one new drug, and only one molecule per every 100,000 tested will become a new drug. (DiMa si Joseph et al. 2003) Figure 1. 14 : Drug discovery protocol 1.9 Goal and Objectives In our lab, MDM2 was therapeutically targeted with the goal of restoring wild type p53 activity in cells overexpressing MDM2 by means

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20 helix mimetic based inhibitors We performed the design and synthesis of non peptidic, drug like, small molecule inhibitors to targe t p53/MDM2 binding interaction. The mimetics were designed to bind to the N terminal domain of the MDM2 protein leaving p53 un bound and capable of activation. The molecules were designed to directly mimic ith, ith +4, and ith + 7 positions of an helical The small molecules were designed to act as antagonists of prot ein/protein interactions, tumor inhibitors, and potent p53 activators Our approach toward the development of helix mimetic based inhibitors erphenyl scaffolds, however our design included the addition of polar functional groups to increase drug like properties and potential therapeutic applications. 1.10 References Alarcon Vargas, D., and Ronai, Z. e. (2002). p53 Mdm2 the a ffair that never ends. Carcinogenesis 23 541 547. AmericanCancerSociety (2010). Cancer Facts and Figures 2010. Bai, L., and Zhu, W. G. (2006). p53: structure, function and therapeutic applications. Journal of Cancer Molecules 2 141 153. Bartlett, W. R., Johnson, W. S., Plummer, M. S., and Small, V. R., Jr. (1990). Biomimetic polyene cyclizations. Cationic cyclization of a substrate having an internal acetylenic bond. Synthesis of euphol and tirucallol. Journal of Organic Chemistry 55 2215 2224. Blay des, J. P., Gire, V., Rowson, J. M., and Wynford Thomas, D. (1997). Tolerance of high levels of wild type p53 in transformed epithelial cells dependent on auto regulation by mdm 2. Oncogene 14 1859 1868. Bottger, V., Bottger, A., Howard, S. F., Picksley, S. M., Chene, P., Garcia Echeverria, C., Hochkeppel, H. K., and Lane, D. P. (1996). Identification of novel mdm2 binding peptides by phage display. Oncogene 13 2141 2147.

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21 Braithwaite, A. W., and Prives, C. L. (2006). p53: more research and more questions Cell Death and Differentiation 13 877 880. Busygina, V., and Bale, A. E. (2006). Multiple endocrine neoplasia type 1 (MEN1) as a cancer predisposition syndrome: clues into the mechanisms of MEN1 related carcinogenesis. Yale Journal of Biology and Medic ine 79 105 114. Chen, L., Agrawal, S., Zhou, W., Zhang, R., and Chen, J. (1998). Synergistic activation of p53 by inhibition of MDM2 expression and DNA damage. Proceedings of the National Academy of Sciences of the United States of America 95 195 200. Chen, L., Lu, W., Agrawal, S., Zhou, W., Zhang, R., and Chen, J. (1999). Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression. Molecular Medicine (New York) 5 21 34. Chen, L., Yin, H., Farooqi, B., Sebti, S., Hamilton, A. D., and Chen, J. (2005). p53 alpha Helix mimetics antagonize p53/MDM2 interaction and activate p53. Molecular Cancer Therapeutics 4 1019 1025. Cummings, C. G., and Hamilton, A. D. (2010). Disrupting protein protein interactions with non peptidic, small molecule alpha helix mimetics. Curr Opin Chem Biol 14 341 346. Cummings, M. D., Schubert, C., Parks, D. J., Calvo, R. R., LaFrance, L. V., Lattanze, J., Milkiewicz, K. L., and Lu, T. (2006). Substituted 1,4 benzodiazepine 2,5 diones as alpha helix mim etic antagonists of the HDM2 p53 protein protein interaction. Chemical Biology & Drug Design 67 201 205. DiMasi Joseph, A., Hansen Ronald, W., and Grabowski Henry, G. (2003). The price of innovation: new estimates of drug development costs. Journal of he alth economics 22 151 185. Freedman, D. A., Wu, L., and Levine, A. J. (1999). Functions of the MDM2 oncoprotein. Cellular and Molecular Life Sciences 55 96 107. Genentech. (2008). http://www.biooncology.com/bioonc/research/hallmark/index.m GenWay (20 10). http://www.genwaybio.com/gw_file.php?fid=6056 Han Yin, G. I. L., Andrew D. Hamilton (2007). Alpha Helix Mimetics in Drug Discovery. In Drug Discovery Research: New Frontiers in the Post Genomic Era, (John Wiley & Sons, Inc.), pp. 281 299.

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22 Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell (Cambridge, Massachusetts) 100 57 70. Hardcastle, I. R. (2007). Inhibitors of the MDM2 p53 interaction as anticancer drugs. Drugs of the Future 32 883 896. Heron, M., Hoyert Donna, L., Murp hy Sherry, L., Xu, J., Kochanek Kenneth, D., and Tejada Vera, B. (2009). Deaths: final data for 2006. National vital statistics reports : from the Centers for Disease Control and Prevention, National Center for Health Statistics, National Vital Statistics System 57 1 134. Kinoshita, M. (2010). http://www.glycoforum.gr.jp/science/word/glycotechnology/GT C06E.html Klein, C., and Vassilev, L. T. (2004). Targeting the p53 MDM2 interaction to treat cancer. British Journal of Cancer 91 1415 1419. Knight, S M. G., Umezawa, N., Lee, H. S., Gellman, S. H., and Kay, B. K. (2002). A fluorescence polarization assay for the identification of inhibitors of the p53 DM2 protein protein interaction. Analytical Biochemistry 300 230 236. Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science (Washington, D C) 274 948 953. Kutzki, O., Park, H. S., Ernst, J. T., Orner, B. P., Yin, H., and Hamilton, A. D. (2002). Development of a potent Bcl xL antagonist based on alpha helix mimicry. Journal of the American Chemical Society 124 11838 11839. Lane, D. P. (1992). Cancer. p53, guardian of the genome. Nature 358 15 16. Laptenko, O., and Prives, C. (2006). Transcriptional regulation by p53: one protein, many possibilities. Cell Death and Differentiation 13 951 961. Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998). Genetic instabilities in human cancers. Natu re (London) 396 643 649. Levine, A. J., Finlay, C. A., and Hinds, P. W. (2004). P53 is a tumor suppressor gene. Commentary. Cell (Cambridge, MA, United States) 116 S67 S69. Levitt, N. C., and Hickson, I. D. (2002). Caretaker tumour suppressor genes that defend genome integrity. Trends in Molecular Medicine 8 179 186.

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23 Lu, W., Chen, L., Peng, Y., and Chen, J. (2001). Activation of p53 by roscovitine mediated suppression of MDM2 expression. Oncogene 20 3206 3216. Moisan, L., Odermatt, S., Gombosuren N., Carella, A., and Rebek, J., Jr. (2008). Synthesis of an oxazole pyrrole piperazine scaffold as an alpha helix mimetic. European Journal of Organic Chemistry, 1673 1676. O'Brien, J. (2007). Insights into p53 Tumor Suppressor Gene to Fuel Cancer Stra tegy. In, S.F. University of California, ed. Oldreive, C., Bradley, N., Bruckdorfer, R., and Rice Evans, C. (2001). Lack of influence of dietary nitrate/nitrite on plasma nitrotyrosine levels measured using a competitive inhibition of binding ELISA assay. Free Radical Research 35 377 386. Perrin, F. (1926). Polarization of light of fluorescence, average life of molecules in the excited state. Journal de Physique et le Radium 7 390 401. Pogribny, I. P., Muskhelishvili, L., Tryndyak, V. P., and Beland, F A. (2009). The tumor promoting activity of 2 acetylaminofluorene is associated with disruption of the p53 signaling pathway and the balance between apoptosis and cell proliferation. Toxicology and Applied Pharmacology 235 305 311. Ren, J., Shi, M., Liu R., Yang, Q. H., Johnson, T., Skarnes, W. C., and Du, C. (2005). The Birc6 (Bruce) gene regulates p53 and the mitochondrial pathway of apoptosis and is essential for mouse embryonic development. Proceedings of the National Academy of Sciences of the Unit ed States of America 102 565 570. Scian, M. J., Stagliano, K. E. R., Deb, D., Ellis, M. A., Carchman, E. H., Das, A., Valerie, K., Deb, S. P., and Deb, S. (2004). Tumor derived p53 mutants induce oncogenesis by transactivating growth promoting genes. Oncogene 23 4430 4443. Shangary, S., and Wang, S. (2009). Small molecule inhibitors of the MDM2 p53 protein protein interaction to reactivate p53 function: a novel approach for cancer therapy. Annual Review of Pharmacology and Toxicology 49 223 241. Sh urley, J. F., Legendre, A. M., and Scalarone, G. M. (2005). Blastomyces Dermatitidis Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA. Mycopathologia 160 137 142. Sturzbecher, H. W., and Deppe rt, W. (1994). The tumor suppressor protein p53: Relationship of structure to function (review). Oncology Reports 1 301 307.

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24 Yin, H., and Hamilton, A. D. (2004). Terephthalamide derivatives as mimetics of the helical region of Bak peptide target Bcl xL p rotein. Bioorganic & Medicinal Chemistry Letters 14 1375 1379. Yin, H., and Hamilton, A. D. (2005). Strategies for targeting protein protein interactions with synthetic agents. Angewandte Chemie, International Edition 44 4130 4163. Yin, H., Lee, G. i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005a). Terphenyl based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44 2704 2707. Yin, H., Lee, G. i., Sedey, K. A., Rod riguez, J. M., Wang, H. G., Sebti, S. M., and Hamilton, A. D. (2005b). Terephthalamide Derivatives as Mimetics of Helical Peptides: Disruption of the Bcl xL/Bak Interaction. Journal of the American Chemical Society 127 5463 5468. Zhang, Z., Li, M., Wang, H., Agrawal, S., and Zhang, R. (2003). Antisense therapy targeting MDM2 oncogene in prostate cancer: Effects on proliferation, apoptosis, multiple gene expression, and chemotherapy. Proceedings of the National Academy of Sciences of the United States of A merica 100 11636 11641.

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25 Chapter Two : Design and Synthesis of the Piperazine 2,5 Dione Piperazine Hybrid Scaffold 2.1 Introduction 2.1 .1 Terephthalamide Scaffold Research performed by Dr. Hamilton group at Yale in the development of helical mimetics based on a terephthalamide scaffold (Figure 2 .1) has been extensively investigated by our lab. (Yin et al., 2005) This work was initially interesting due to the small nature of the molecules when compared helix mi mics (Chen et al., 2005; Shangary and Wang, 2009; Volonterio et al., 2007) Figure 2 .1: Terephthalamide ( A ) Generic terephthalamide helical mimetic superimposed on the ith ith +4, and ith +7 position of an ideal helix ( B ) Terephthalamide scaffold shown to display potent inhibition of protein/protein interactions

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26 These mimetics reportedly displayed a higher degree of solubility, notably facile synthesis, when compared to the first generation terphenyl derivates (Yin et al., 2005) yet still maintained the desired spacial arrangements of hydrophobic side chains in the ith ith+ 4 and ith+ 7 positions despite their small size. The terep hthalamide scaffold was reported to be a potent inhibitor of protein/protein interactions (Figure 4.2) with a n IC 50 = 35.0 M for Bcl xL/Bax inhibit ion. Surprisingly, these derivatives were not reported to have activity against p53/MDM2 interactions and the paper reported a difficult linear sy nthesis and poor solubility of the terephthalamides. (Yin et al., 2005) 2 1.2 Rational of the Piperazine 2,5 Dione Piperazine Hybrid Scaffold The Hamiltion terephthalamide scaffold design (Figure 2.1) was used as t he basis for our piperazine 2,5 dione piperazine hybrid scaffold, as shown in Figure 2.2 Our terephthalamide scaffold, with functionalized 2,5 DKPs and piperazine units. It was ant icipated that the reported facile piperazine and DKP synthesis would improve the overall ease of synthesis of the scaffold. The addition of polar functional groups was intended to increase the desired drug like properties of the parent terephthalamide sca ffold while still maintaining the desired spacial arrangement of side chains in the ith ith +3, and ith +7 positions of a natural helix, shown in Figure 2.3 This novel scaffold was designed to be a potent inhibitor of p53/MDM2 binding interactions. Piperazine and piperazinediones are common building blocks featured in many biologically active compounds such as: agonists acting opioid receptors (Alfaro Lopez et al., 1999) inhibitors of MDA MB 231 breast cancer cell proliferation (Ali and

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27 Yar, 2007) and in Gliotoxin (Balibar and Walsh, 2006) There are many documented facile syntheses of substituted piperazines (Dankwardt et al., 1995) (Gordon and Steele, 1995) Figure 2.2: Piperazine 2,5 dione piperazine hybrid scaffold Figure 2.3: Schematic helix representation s ( A ) helix with the i 1, i i + 4, i + 7, and i + 11 residues, ( B ) 2D hybrid piperazine 2,5 dione piperazine scaffold, ( C ) 3D hybrid piperazine 2,5 dione piperazine scaffold.

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28 2.1.3 Molecular Modeling Studies In efforts to validate potential novel inhibitors of p53/MDM2 binding interactions in a virtual screening mode, we conducted computational simulations with S hrodeinder software for variations of the proposed target hybrid scaffold (Figure 2.2 ) docked into the hydrophobic pocket in the N terminal domain of the MDM2 protein derived from the X ray crystal structure of p53 bound to the hydrophobic pocket of MDM2 (PDB 1YCQ) (Kussie et al ., 1996) Computational analysis of the proposed structures were ranked based on the docking GLIDE (Friesner et al., 2004) score and the top scoring structures were then synthesized. Molecular modeling of t he top GLIDE scoring structure of hybrid scaffold 2.13 (shown in Figure 2.5 ) suggested that the hybrid scaffold targeted the same surface area of MDM2 where p53 binds, inserting the side chains into the desired F19, W23, and L26 binding pockets, as shown in Figure 2.5 Figure 2.4 : Top glide scori ng p roposed hybrid scaffold 2.13

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29 Figure 2.5 : Molecular docking of 2.13 Image is displaying the three aromatic side chains of the hybrid scaffold 2.13 occupying the key F19 W23 and L26 positions in the MDM2 hydrophobic pocket ( image by Courtney DuBoulay) Further analysis of the hybrid scaffold 2.13 docked with the p53 protein in transparent light blue (Figure 2. 7 ) displayed numerous hydrophobic interactions with the MDM2 protein, as shown in Figure 2.6 The phenyl gro up in the R 1 position, ith position di s played hydrophobic interactions with Y100 and L54, and the indole group in the R 2 ith +3, position displayed hydrophobic interactions with V93, I99, and F91. The isopropyl group, in the R 3 ith +7, position displayed hydrophobic interactions with Y67. The model also revealed a hydrogen bond between the indolin e NH and L57 which was considered optimal of activity. The hydrophobic interactions of scaffold 2.13 with the MDM2 protein are all reported to be located within the p53 binding pocket. (Kussie et al., 1996)

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30 Figure 2. 6 : Schematic representations of 2.13 ( A ) 2D hybrid piperazine 2,5 dione piperazine scaffold ( B ) Hydrophobic interactions of the top scoring hybrid scaffold 2.13 Figure 2.7 : Overlay of the p53 protein (transparent light blue) with the proposed hybrid scaffold 2.13 docked in the MDM2 hydrophobic pocket ( image by Courtney DuBoulay) The observed hydrophobic interactions at th ese positions were also observed in the binding of the p53 peptide and MDM2 (Kussie et al., 1996) demonstrating that the

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31 hybrid scaffold 2.13 recognizes the MDM2 protein su rface and adopts a similar confo rmation to the p53 peptide. 2.2 Results and Discussion 2.2.1 Retrosynthesis of the Piperazine 2,5 Dione Piperazine Hybrid Scaffold By r etrosynthetic analysis (Scheme 2 .1), it was reasoned that two functional moieties could be introduced to the 2,5 DKP by reacting a n amino acid based orthogonal esters a and b performing selectively hydrolysis d followed by ring closure e and subsequent hydrolysis f to afford the desired dual functionalized 2,5 DKP A 1 2 unit

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32 Scheme 2 .1: Retrosynth esis of the piperazine 2,5 dione piperazine hybrid scaffold

PAGE 49

33 2.2.2 Synthesis of the Piperazine 2,5 Dione Dimers Synthesis of the piperazine 2,5 dione di mers (units A 1 2 Scheme 2.1) began by N acylation of commercially available amino methyl esters 2. 1a d (Scheme 2.2) with bromoacetyl bromide in the presence of a biphasic system of benzene and saturated aqueous sodium bicarbonate to afford the pure products of 2. 3a d in good yields (60 92%) (Maity and Koenig, 2008) Compounds 2. 3a d where were then coupled with an orthogonal amino acid tert butyl esters 2. 2a d in methanol and refluxed overnight, in the presence of TEA, to respectively yield the pure products 2. 4a h again with high purity and in good yields as shown in S c heme 2.2 Entry R 1 Yield 2.3a i Pr 67% 2.3 b i Bu 62% 2.3c Bn 90% 2.3e 4 tert butoxyphenyl 70% Scheme 2.2: Synthesis of compound 2.3a d and 2.4a h Entry R 1 R 2 Yield 2.4a Bn i Pr 60% 2.4b i Bu i Pr 53% 2.4c Bn Bn 64% 2.4d i Bu Bn 65% 2.4e Bn i Bu 82% 2.4f i Pr i Bu 50% 2.4g i Pr s Bu 63% 2.4h Bn s Bu 50%

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34 Sel ective hydrolysis of esters 2.4 was carried out under basic conditions using aqueous 3M sodium hydroxide and methanol at ambient temperatures t o yield 5a g as the pure products shown in Scheme 2.3 Entry R 1 R 2 Yield 2.5a Bn i Pr 90% 2.5b i Bu i Pr 78% 2.5c Bn Bn 42% 2.5d i Bu Bn 40% 2.5e Bn i Bu 56% 2.5f i Pr i Bu 35% 2.5g i Pr s Bu 60% Scheme 2.3: Synt hesis of compounds 2.5a g C yclization of compounds 2. 5a g proved to be the most challenging portion of this synthetic sc heme. As depicted in Scheme 2.4 treatment with TEA or DIEA in MeOH or DMF by traditional and microwave heating did not result in the desired formation of 2 6a Additional attempts using HBTU and HATU with traditional and microwave heating were also unsuccessful at yielding 2. 6a (Montalbetti and Falque, 2005)

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35 Entry Reagents Con ditions Results 1 TEA MeOH, reflux, 24 h No rxn 2 DIEA MeOH, reflux, 24 h No rxn 3 DIEA DMF, 110 C, 5 h No rxn 4 HBTU, DIEA DMF, 110 C, 18 h No rxn 5 HBTU, DIEA DMF, 80 C, 20 min No rxn 6 HATU, DIEA DMF, 80 C, 18 h No rxn Scheme 2.4 : Attempted c yclization of 2. 5a Successful cyclization of 2. 5a was accomplished using pivalic anhydride as a coupling agent, in the presence of TEA under reflux, forming 2,5 DKP 2. 6a g as shown in Scheme 2.4 (Humphrey and Ch amberlin, 1997; Montalbetti and Falque, 2005; Sobolev et al., 1991) Compounds 2. 6a g were subsequently hydrolyzed under acidic conditions with 4N HCl/Dioxane, yielding the target piperazine 2,5 dione dimers 2. 7a e (Scheme 2.5 )

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36 Entry R 1 R 2 Yield 2. 6a Bn i Pr 36% 2. 6b i Bu i Pr 59% 2. 6c Bn Bn 38% 2. 6d i Bu Bn 57% 2. 6e Bn i Bu 54% 2. 6f i Pr i Bu 74% 2. 6g i Pr s Bu 61% Entry R 1 R 2 Yield 2. 7a Bn i Pr 80% 2. 7b i Bu i Pr 59% 2. 7c Bn i Bu 45% 2. 7d i Pr i Bu 9 5 % 2. 7e i Pr s Bu 54% 2. 7f i Bu Bn 85% Scheme 2.5: Synthesis of compounds 2.6a g and 2.7a e The reaction condition s followed for the synthesis of compounds 2.6a g and 2.7a f were used with tert butyl amino a cid esters 2 .2a d as the starting material s as an alternative to amino acid methyl esters 2 .1a d Synthesis of the piperazine 2,5 dione dimers began by N acylatio n of commercially available tert butyl amino acid esters 2.2a d (Scheme 2.6) with bromoac etyl bromide in a biphasic system of benzene and sa turated

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37 aqueous sodium bicarbonate to afford the pure products of 2.8a d in good yields (80 93%) Compounds 2.8a d were then coupled with an orthogonal amino acid methyl esters 2. 1 a f in tert butanol and refluxed overnight, in the presence of TEA, to yiel d the pure products 2. 9 a k in high purity and in good yields (50 80%), as shown in S cheme 2.6 (Maity and Koenig, 2008) Scheme 2.6: Synthesis of compounds 2.8a d and 2.9a k Selective hydrolysis of esters 2.9a k was carried out under acidic conditions with 4N HCl/Dioxane to yield 2.10a i in good yields (50 99%) as the pure produc ts, shown in Scheme 2.7. Entry R 2 R 1 Yield 2.9a i Pr Bn 60% 2.9b Bn Bn 70% 2 .9c Bn i Bu 80% 2.9d Bn s Bu 55% 2.9e i Bu i Pr 70% 2.9f i Pr s Bu 66% 2.9g i Pr 2 methyl 1 H indole 70% 2.9h Bn 2 methyl 1 H indole 60% 2.9i i Bu 2 methyl 1 H indole 64% 2.9k s Bu 2 methyl 1 H indole 50% Entry R 2 Yield 2.8a Bn 83% 2.8b i B u 88% 2.8c i Pr 93% 2.8d s Bu 80%

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38 Entry R 2 R 1 Yield 2. 10a i Pr Bn 99% 2. 10b Bn Bn 90% 2. 10c i Bu Bn 95% 2. 10d Bn s Bu 99% 2. 10e i Bu i Pr 99 % 2. 10f i Pr 2 methyl 1 H indole 90% 2. 10g Bn 2 methyl 1 H indole 92% 2. 10h i B u 2 methyl 1 H indole 99% 2. 10i s Bu 2 methyl 1 H indole 85% Scheme 2.7 : Synthesis of compounds 2.10a i Cyclization of compounds 2.10 was accomplished using pivalic anhydride as the coupling reagent, in the presence TEA under reflux, forming 2,5 DKP s 2 11a i as shown in Scheme 2.7. (Humphrey and Chamberlin, 1997; Montalbetti and Falque, 2005) Compounds 2.11 a i were subsequently hydrolyzed under basic conditions using aqueous 3M lithium hydroxide and CH 3 CN at am bient temperatures to yield 2.12a i as the pure products ( Scheme 2.7 ) (Balakrishnan et al., 2007)

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39 Entry R 2 R 1 Yield 2. 11a i Pr Bn 36 % 2. 11b Bn Bn 40% 2. 11c Bn i Bu 85% 2. 11d i Bu i Pr 75% 2. 11e i Pr 2 methyl 1 H indole 63% 2. 11f Bn 2 methyl 1 H indole 50 % 2. 11g i Bu 2 methyl 1 H indole 52% 2. 11h s Bu 2 methyl 1 H indole 45% 2.11i Bn s Bu 40% Entry R 2 R 1 Yield 2. 12a i Pr Bn 36 % 2. 12b Bn Bn 49% 2. 12c i Pr 2 methyl 1 H indole 59% 2. 12d s Bu 2 methyl 1 H indole 40% 2.12e i Pr s Bu 45% 2.12f i Bu i Pr 40% 2.12g Bn i Bu 55% 2.12h s Bu Bn 50% 2.12i Bn 2 methyl 1 H indole 44% Scheme 2.8 : Synthesis of compounds 2.11a i and 2.12a i In conclusion, r eaction conditions rem ained constant for the synthesis beginning with tert butyl ester with a few minor changes. For the coupling to an orthogonal ester

PAGE 56

40 (step b Scheme 2.6 ) tert butanol was used as the reaction solvent instead of the previou s sly used MeOH in Scheme 4.2. No reaction was observed to occur when MeOH, CH 3 CN, or THF were used as the reaction solvents (data not shown). The basic hydrolysis (step b Scheme 2.7) was performed using 3M LiOH and CH 3 CN, which result ed in shorter reaction t imes and a more facile work u p. The overall reaction conditions and yields were comparable for both the amino acid methyl esters and the amino acid tert butyl esters. X ray crystal structure analysis of compounds 2. 7e and 2. 12c (Figure 4.7) were obtained by Dr. Frank Fronczek at Lo usiana State University The crystal structures verified the s,s sterochemisty of the dimeric units which corresponded to the starting material use of L amino acids.

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41 Figure 2.8: X ray crystal structure s of compound s 2. 7e and 2. 11g 2 .2.3 Synthesis of Trimer s 2.13 and 2 .14 Different synthetic routes were explored for the synthesis of the target trimer 2 .13 before an optimized synthesis was developed ( Scheme 2.8 ) The coupling of 2. 12c with phenyl piperazine was attempte d with EDC /HOBt (Fara et al., 2006; Kitamura et al., 2001) and CDI (Nam et al., 2009; Paul and Anderson, 1960) resulting (in both cases) in very complicated mixture s of pro ducts and side products. The purification of these

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42 mixtures was not successfully accomplished due to the complex composition of the crude material. PyBroP was tried next for its reported use in difficult coupling with N methyl dialky lglycine s with low levels of race mization. (Santagada et al., 2001; Zapf et al., 2005) T hese attempts also resulted in a complicated mixture of products and side products. Entry Reagents Conditions Results 1 EDC, HOBt DCM, rt 24 h Multiple products 2 CDI CHCl 3 rt, 24 Multiple products 3 PyBr oP DIEA DCM, rt, 24 h Multiple products 4 PyBr oP DIEA THF, rt, 24 h Multiple products Scheme 2 .9 : Attempted synthesis of 2 .13 The successful synthesis of 2. 13 and 2. 14 was achieved using standard coupling conditions with EDC and DCM at ambient t emperatures, shown in Scheme 2.9 (Montalbetti and Falque, 2005; Zhang et al., 2002)

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43 Scheme 2 .10 : Synthesis of trimers 2 .13 and 2 .14 2 .2.4 ELISA Assays A competitive ELISA assay to detect binding between immobilized His 6 p53 and free GST MDM2 was performed at the Moffitt Center in the lab of Dr. Jiandong Chen, to det ermine if my compounds could disrupt p53/MDM2 binding interactions (Chen et al., 2005) A previously developed peptide optimized f or binding to MDM2 termed pDI (Phan et al.) and the small molecule Nutlin 3 (Hu et al., 2006; Vassilev et al., 2004) a known in vitro inhibitor of p53/MDM2 binding interac tions, were used as positive controls for the experiment. As expected, Nutlin 3 and PDIW inhibited p53/MDM2 binding with IC 50 of 800 n m (Figure 2.9) (Chen et al., 2005; Hu et al., 2007) In

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44 comparison, my compoun ds were unfortunately unable to disrupt the MDM2 p53 interactions m which is close to its solubility limit in the ELISA assay (Figure 2.10) (Hu et al., 2007) Figure 2.9: Nutlin 3 and PDI were used as test controls in an ELISA assay to detect binding between immobilized His 6 p53 and GST MDM2 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 6 Absorbance at 450 nm Concentration ( mol/L) Nutlin 3 and PDI Controls nutlin PDIW

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45 Figure 2.10: ELISA results: Piperazine 2,5 dione piperazine scaffold derivatives were analyzed for inhibition of p53/MDM2 binding by a competitive ELISA assay at varying concent rations (0 2.6a b 2.6d g 2.7a f 2.11a i 2.12c 2.12e 2.12i and 2.13 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 20 40 60 80 100 120 Absorbance at 450 nm Concentration(umol/L) MDM2 p53 binding ELISA 2.11f 2.11h 2.7f 2.6d 2.11h 2.13 2.11g 2.12e 2.7a 2.12i 2.7b 2.12c 2.6b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 20 40 60 80 100 120 Absorbance at 450 nm Concentration ( mol/L) MDM2 p53 binding ELISA 2.11a 2.6f 2.6e 2.7c 2.6g 2.11e 2.11b 2.11d 2.7d 2.11c 2.11i 2.7e 2.6g

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46 2 .3 Conclusion It was reasoned that the low levels of bio activity were due to the compounds lacking the one of the three key side chains (Figure 2.11, A ) A majority of the compounds tested were the in dimeric form (Figure 2.11, C ) and the two trime r s tested 2.13 and 2.14 contained a hydrogen in the R 3 position (Figure 2.11, B ) as a substitute nt of the desired aromatic or aliphat ic group. Figure 2.11: Piperazine 2,5 dione piperazine scaffold derivatives ( A ) Standard piperazine 2,5 dione piperazine hybrid scaffold ( B ) Trimeric scaffold 2.13 containing aromatic or aliphatic groups in the R 1 2 posi tions and a hydrogen in the R 3 position ( C ) Standard dimeric scaffold I nitially a hydrogen was placed the R 3 position on the scaffold (Figure 2.11, A) to facilitate the synthesis and acquire preliminary biological data. However, the weak potency of the compounds provide a sound rational for the synthesis of a new library of piperazine 2,5 dione piperazine scaffold derivatives containing an aromatic or aliphatic group in the R 3 position. A positive result of the ELISA assay was displayed in the persist ent linearity of the graph through the 100 uM concentration (Figure 2.10) displaying the desired drug like solubility properties of the first generation of piperazine

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47 2,5 dione piperazine scaffold derivatives The addition of side chains to the R 3 positi on should increase the biological activity with the third binding side occupied 2.4 Experimental Section 2.4 .1 Materials and Methods Organic and inorganic reagents and solvents (ACS grade) were purchased from commercially available sources and used with out modification unless otherwise noted. Thin layer chromatography (TLC) was performed on glass plates (EMD) precoated with 0.25 mm thickness of silica gel (60 ) with fluorescent indicator (EMD). Column chromatography was pe rformed using silica gel 60 (S ilicycle). All 1 H, 13 C, and DEPT NMR spectra were obtained using a Varian 400 MHz Mercury plus instrument at ambient temperatures in ch loro form d (CDCl 3 ), unless otherwise specified. Chemical shift values are reported in ppm ( ) relative to internal tetramethylsilane (TMS). Multiplicity is expressed as: s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet. The corresponding coupling constants ( J ) are reported in Hertz (Hz). Automated flash chromatography was performed using a Biotage FlashMaster II system with Biotage silica cartridges. Microwave synthesis was performed in a Biotage Initiator I reactor. High performance liq u id chromatography (HPLC) was performed on a Jasco LC NetII/ADC syst em. High resolution mass spectra were obtained on an Agilent 1100 Series in the ESI TOF mode. Melting points (uncorrected) were obtained using a Melt Temp II Laboratory device. Hydrogenation was performed in a H Cube TM continuous flow hydrogenation reac tor (Thales Technology).

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48 2.4 .2 Experimental Procedures (S) methyl 2 (2 bromoacetamido) 3 methylbutanoate ( 2. 3a) To a suspension of 2.1 a (5.97 mmol) in water (10 mL), cooled in an ice salt bath, was added NaHCO 3 (14.32 m mol) in one portion. A solution of bromoacetyl bromide (5.97 mmol) in benzene (6 mL) was slowly added via addition funnel to the chilled mixture. After completion of the addition, the reaction was brought to ambient temperature and stirred for an additio n 24 h at rt. The aqueous layer was extracted with benzene (3 x 40 mL), the combined organic layers were washed with brine (1 x 100 mL) and dried (Na 2 SO 4 ). Removal of the solvent in vacuo afforded the pure product 2.3 a ( 3.98 mmol, 67 %) as a white gel. 1 H NMR (400 MHz, CDCl 3 J = 8.1 Hz, 1H), 4.46 (dd, J = 8.8, 5.0 Hz, 1H), 3.86 (d, J = 1.4 Hz, 2H), 3.70 (s, 3H), 2.21 2.08 (m, 1H), 0.88 (dd, J = 10.2, 6.9 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 (1C), 52.50 (1C), 31.45 (1C), 29.06 (1C) 19.06 (1C), 17.89 (1C). HRMS (ESI) calc. for C 8 H 14 BrNO 3 (M + H) + 251.0157, found 251.0229. (S) methyl 2 (2 bromoacetamido) 3 (4 (tert butoxy)phenyl)propanoate (2.3e) The product was obtained from 2.1e (3.47 mmol) in a s imilar manner as described for preparation of 2.3a affording the pure compound 4.31c (2.42 mmol, 90%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 6.98 (m, 2H), 6.94 6.90 (m, 2H), 6.81 (d, J =

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49 7.5 Hz, 1H), 4.80 (dt, J = 7.9, 5.9 Hz, 1H), 3.88 3.78 (m, 2H), 3.72 (s, 3H), 3.15 3.05 (m, 2H), 1.32 (s, 9H). 13 C NMR (101 MHz, CDCl 3 (1C), 130.28 (1C), 12 9.94 (2C), 124.58 (2C), 78.75 (1C), 53.95 (1C), 52.70 (1C), 37.27 (1C), 29.05 (3C). HRMS (ESI) calc. for C 16 H 22 BrNO 4 (M + H) + 371.0732, found 371.0808. (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl) amino) 2 oxoethyl) amino) 3 methylbuta noate (2.4a) A solution of 2.2 a (0.802 mmol) in methanol (5 mL) was slowly added via an addition funnel to a solution of compound 2.3 c (0.67 mmol) and TEA (2.01 mmol) in methanol (10 mL) and refluxed for 36 h. The res ulting light yellow solution was cooled to rt, and concentrated under reduced pressure. T he crude residue was dissolv ed in ethyl acetate (100 mL), washed with saturated citric acid (1 x 35 mL), saturated aqueous sodium bicarbonate (2 x 30 mL), brine (2 x 50 mL), and dried over Na 2 SO 4 R emoval of the organic solvent in vacuo afforded the pure compound 2.4 a (1.02 mmol, 60%) as a light yellow oil. 1 H NMR (400 MHz, CD 3 OD) 7.14 (m, 5H), 4.76 (t, J = 6.3 Hz, 1H), J = 165.9, 17.0 Hz, 2H) 3.11 (d, J = 6.9 Hz, 2H), 2.79 (d, J = 5.8 Hz, 1H), 1.85 (dq, J = 13.6, 6.8 Hz, 1H), 1.47 (s, 9H), 0.87 (dd, J = 6.8, 4.3 Hz, 6H). 13 C NMR (101 MHz, CD 3 OD C), 168.11 (1C), 167.61 (1C), 132.15 (1C), 125.48 (2C), 124.90 (2C), 123.43 (1C), 77.81 (1C), 64.04 (1C), 48.77 (1C), 48.51 (1C), 47.36 (1C), 34.25 (1C), 27.69 (1C), 24.40 (3C), 15.63 (1C), 14.18 (1C). HRMS (ESI) calc. for C 21 H 32 N 2 O 5 (M + H) + 392.2311, fo und 392.2144.

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50 (S) methyl 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoate ( 2. 4b) The product was obtained from 2.3 b (1.43 mmol) and 2 .2 a (1.43 mmol) in a similar manner as described for preparation of 2.4 a affording compound 2.4 b (0.89 mmol, 63%) as a clear yellow oil. 1 H NMR (400 MHz, CD 3 4.46 (m, 1H), 3.72 (d, J = 2.6 Hz, 3H), 3.38 (d, J = 16.8 Hz, 1H), 3.05 (d, J = 16.8 Hz, 1H), 2.92 (d, J = 5.8 Hz, 1H), 1.96 (dp, J = 13.5, 6.8 Hz, 1H), 1.72 1.58 (m, 4H), 1.48 (s, 9H), 1.02 0.88 (m, 12H). 13 C NMR (101 MHz, CD 3 (1C), 173.21 (1C), 172.98 (1C), 81.38 (1C), 67.83 (1C), 51.59 (1C), 50.61 (1C), 50.34 (1C), 40.76 (1C), 31.43 (1C), 27.22 (3C), 24.83 (1C), 22.04 (1C), 20.85 (1C), 18.48 (1C), 17.69 (1C). HRMS (ESI) calc. for C 18 H 34 N 2 O 5 (M + H) + 358.2468, found 358.2430. (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 phenylpropanoate (2.4c) The product was obtained from 2.3 c (6.90 mmol) and 2 .2 c (8.03 mmol) in a similar manner as described for pre paration of 2.4 a affording compound 2.4 c (4.32 mmol, 64%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 7.35 7.20 (m, 6H), 7.15 (m, 2H), 7.09 7.03 (m, 2H), 4.73 (m, 1H), 3.71 (s, 3H), 3.34 (m, 2H), 3.05 (dd, J = 13.9, 5.5 Hz, 1H), 2.99 (d, J = 17.4 Hz, 1H), 2.92 2.72 (m, 3H), 2.34 (bs, 1H), 1.39 (s, 9H). 13 C NMR (101 MHz, CDCl 3 173.61 (1C), 172.01 (1C), 171.47 (1C), 137.39 (1C), 136.40 (1C), 129.75 (2C), 129.31 (2C), 128.78 (2C), 128.59 (2C) 127.27 (2C), 126.96 (1C),

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51 82.07 (1C), 63.20 (1C), 52 .84 (1C), 52.49 (1C), 50.79 (1C), 40.00 (1C), 37.68 (1C), 28.22 (3C). HRMS (ESI) calc. for C 25 H 32 N 2 O 5 (M + H) + 440.2311, found 440.2231. (S) methyl 2 (2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino)acetamido) 4 methylpentanoate ( 2. 4d) The product was obtained from 2.3 b (21.55 mmol) and 2 .2 c (25.81 mmol) in a similar manner as described for preparation of 2.4 a affording compound 2.4 d (13 .90 mmol, 64%) as a clear oil. 1 H NMR (400 MHz, CDCl 3 7.31 7.19 (m, 5H), 7.05 (d, J = 8.6 Hz, 1H), 4.45 (m, 1H), 3.69 (s, 3H), 3.43 3.37 (m, 2H), 3.02 (m, 2H), 2.76 (dd, J = 13.7, 8.9 Hz, 1H), 1.54 1.43 (m, 3H), 1.41 (s, 9H), 0.86 (dd, J = 10.4, 6.2 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 173.79 (1C), 173.29 (1C), 171.59 (1C), 137.61 (1C), 129.78 (2C), 128.60 (2C), 126.99 (1C), 82.17 (1C), 63.37 (1C), 52.38 (1C), 50.72 (1C), 50.17 (1C), 40.62 (1C), 40.02 (1C), 28.23 (3C), 24.98 (1C), 23.10 (1C), 21.72 (1C). HRMS (ESI) calc. for C 22 H 34 N 2 O 5 (M + H) + 406. 2468, found 406.2456. (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoate ( 2. 4e) The product was obtained from 2.3 c (3.34 mmol) and 2 .2 b (4.53 mmol) in a similar mann er as described for preparation of 2.4 a affording compound 2.4 e (1.93 mmol, 64%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 8.5 Hz, 1H), 7.31 7.19 (m, 3H), 7.13 7.07 (m, 2H), 4.89 4.83 (m, 1H), 3.69 (s, 3H), 3.38 (d, J = 17.3 Hz, 1H ),

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52 3.11 (t, J = 6.0 Hz, 2H), 3.03 (dd, J = 8.2, 6.3 Hz, 1H), 2.95 (d, J = 17.3 Hz, 1H), 1.66 1.54 (m, 1H), 1.42 (s, 9H), 1.36 1.21 (m, 2H), 0.82 (dd, J = 21.8, 6.6 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 129.42 (2C), 128.82 (2C), 127.36 (1C), 81.67 (1C), 60.89 (1C), 52.71 (1C), 52.44 (1C), 50.94 (1C), 42.96 (1C), 38.09 (1C), 28.27 (3C), 25.01 (1C), 22.99 (1C), 22.27 (1C). HRMS (ESI) calc. for C 22 H 34 N 2 O 5 (M + H) + 406.2467, found 406.2509. (S) tert butyl 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl) amino) 4 methylpentanoate ( 2. 4f) The product was obtained from 2.3 a (3.98 mmol) and 2 .2 b (4.78 mmol) in a similar manner as described for preparation of 2.4 a affording compound 2.4 f (1.95 mmol, 50%) as a white oil. 1 H NMR (400 MHz, CDCl 3 J = 8.5 Hz, 1H), 4.49 (td, J = 8.8, 4.8 Hz, 1H), 3.72 (s, 3H), 3.45 (d, J = 17.2 Hz, 1H), 3.07 (d, J = 5.7 Hz, 1H), 2.99 (d, J = 17.2 Hz, 1H), 2.00 (dt, J = 19.5, 6.5 Hz, 1H), 1.70 1.57 (m, 3H), 1.45 (s, 8H), 1.02 (d, J = 6.8 Hz, 3H), 0.98 0.91 (m, 9H). 13 C NMR (101 MHz, CDCl 3 (1C), 171.15 (1C), 81.87 (1C), 67.40 (1C), 52.01 (1C), 51.42 (1C), 51.13 (1C), 42.04 (1C), 31.73 (1C), 28.23 (3C ), 25.23 (1C), 23.05 (1C), 22.35 (1C), 19.73 (1C), 18.41 (1C). HRMS (ESI) calc. for C 18 H 34 N 2 O 5 (M + H) + 358.3261, found 358.2543.

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53 (2S,3R) tert butyl 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl)amino) 3 methylpentanoate ( 2. 4g) The product was obtained from 2.3 a (9.96 mmol) and 2 .2 d (10.96 mmol) in a similar manner as described for preparation of 2.4 a affording compound 2.4 g (6.24 mmol, 63%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 9.3 Hz, 1H), 4.56 4.51 (m, 1H), 3.71 (s, 3H), 3.52 3.44 (m, 1H), 2.99 2.95 (m, 2H), 2.25 2.12 (m, 1H), 1.91 (bs, 1H), 1.77 1.66 (m, 1H), 1.59 1.47 (m, 1H), 1.47 1.41 (m, 7H), 1.30 1.13 (m, 1H), 0.99 0.85 (m, 12H). 13 C NMR (101 MHz, CDCl 3 171.80 (1C), 81.77 (1C), 67.07 (1C), 56.76 (1C), 52.24 (1C), 51.38 (1C), 38.59 (1C), 31.27 (1C), 28.34 (3C), 25.48 (1C), 19.27 (1C), 17.92 (1C), 16.08 (1C), 11.93 (1C). HRMS (ESI) calc. for C 18 H 34 N 2 O 5 (M + H) + 358. 2468, found 358.2443. (S) 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 3 phenylpropanoic acid ( 2. 5a) To a solution of 2.4 a (0.33 mmol) in methanol (2 mL) was added 3M aq. NaOH (3mL), and t he reacti on was stirred at ambient temperature s for 18 h. S olvent was removed in vacuo the resulting crude oil was redissolved in water (25 mL) and acidified to a pH of 4 with 1N HCl. The resulting acidified solution was extracted with EtOAc (3 x 30 mL), the co mbined organic layers were washed with brine (2 x 25 mL), dried over Na 2 SO 4 and the residual solvent was removed in vacuo to afford the pure compound 2.5 a (0.29 mmol, 90%) as a light yellow oil. 1 H NMR (400 MHz, CDCl 3 7.15 (m, 5H),

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54 7.08 (s, 1H) 4.70 (d, J = 10.0 Hz, 1H), 4.32 (bs, 1H), 3.77 (dd, J = 263.2, 17.5 Hz, 2H), 3.32 (s, 1H), 3.23 (dd, J = 13.9, 4.0 Hz, 1H), 3.06 (dd, J = 13.9, 7.3 Hz, 1H), 2.01 (m, 1H), 1.44 (s, 9H). HRMS (ESI) calc. for C 18 H 34 N 2 O 5 (M + H) + 358.2468, found 358.2443. ( S) 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoic acid (2.5b) The product was obtained from 2.4b (0.89 mmol) in a similar manner as described for preparation of 2.5a affording compo und 2.5b (0.70 mmol, 78%) as a white solid. 1 H NMR (400 MHz, CDCl 3 J = 8.0 Hz, 1H), 4.59 4.53 (m, 1H), 4.09 (dd, J = 123.9, 17.4 Hz, 2H), 3.26 (dd, J = 187.4, 17.4 Hz, 2H), 2.91 (d, J = 5.5 Hz, 1H), 2.04 1.92 (m, 1H), 1.82 1.57 (m, 3H), 1. 47 (s, 9H), 1.04 0.93 (m, 12H). 13 C NMR (101 MHz, CDCl 3 (1C), 50.91 (1C), 42.48 (1C), 41.02 (1C), 31.64 (1C), 28.34 (3C), 25.14 (1C), 23.07 (1C), 22.10 (1C), 19.70 (1C), 18.26 (1C). (S) 2 (2 ((S) 1 tert butoxy 1 oxo 3 phenylpropan 2 ylamino)acetamido) 3 phenylpropanoic acid ( 2. 5c) The product was obtained from 2.4 c (4.32 mmol) in a similar manner as described for preparation of 2.5 a affording compo und 2.5 c (1.80 mmol, 42%) as a light yellow oil. 1 H NMR (400 MHz, CDCl 3 7.28 7.07 (m, 8H), 6.99 (m, 2H), 5.24 (dd, J = 10.9, 5.9 Hz, 1H), 4.04 (m, 1H), 3.64 (dd, J = 111.4, 17.1 Hz, 2H), 3.27 (dd, J = 14.8, 5.9 Hz, 1H), 3.01

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55 (dd, J = 13.9, 3.7 Hz, 1H), 2.84 (dd, J = 14.8, 10.9 Hz, 1H), 2.35 (dd, J = 13.8, 9.5 Hz, 1H), 1. 38 (s, 9H). 13 C NMR (101 MHz, CDCl 3 (1C), 136.35 (1C), 135.46 (1C), 129.62 (2C), 129.23 (2C), 128.94 (2C), 127.68 (1C), 127.36 (1C), 83.03 (1C), 57.47 (1C), 56.75 (1C), 47.10 (1C), 40.16 (1C), 34.66 (1C), 28.21 (3C). H RMS (ESI) calc. for C 24 H 30 N 2 O 5 (M + H) + 426.2155, found 426.2120. (S) 2 (2 ((S) 1 tert butoxy 1 oxo 3 phenylpropan 2 ylamino)acetamido) 4 methylpentanoic acid ( 2. 5d) The product was obtained from 2.4 d (13.90 mmol) in a si milar manner as described for preparation of 2.5 a affording compound 2. 5d (6.19 mmol, 40 %) as a light y ellow gel 1 H NMR (400 MHz, CD 3 OD 7.17 (m, 5H), 5.14 (dd, J = 12.1, 5.0 Hz, 1H), 3.80 (dd, J = 82.1, 17.0 Hz, 2H), 3.38 (dd, J = 14.7, 5.0 Hz, 1H), 3.01 (dd, J = 14.7, 12.1 Hz, 1H), 1.47 (s, 9H), 1.13 (t, J = 7.2 Hz, 2H), 0.94 0.85 (m, 1H), 0.80 (dd, J = 10.1, 6.6 Hz, 6H). 13 C NMR (101 MHz, CD 3 OD (1C), 128.73 (2C), 128.65 (2C) 127.01 (1C), 82.77 (1C), 58.08 (1C), 53.87 (1C), 42.74 (1C), 34.25 (1C), 27.37 (3C), 24.00 (1C), 22.27 (1C), 20.99 (2C). HRMS (ESI) calc. for C 21 H 32 N 2 O 5 (M + H) + 392.2311, found 392.2234.

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56 (S) 2 (2 (((S) 1 (tert butoxy) 4 methyl 1 oxopentan 2 yl)amino)acetamido) 3 phenylpropanoic acid ( 2. 5e) The product was obtained from 2.4 e (1.55 mmol) in a similar manner as desc ribed for preparation of 2.5 a affording compound 2.5 e (1.27 mmol, 82%) as a white solid. 1 H NMR (400 MHz, CD 3 7.16 (m, 5H), 4.68 4.62 (m, 1H), 3.42 (d, J = 16.7 Hz, 1H), 3.26 3.10 (m, 3H), 3.06 3.01 (1H), 1.73 1.60 (3H), 1.47 (s, 9H), 0.89 (dd, J = 14.9, 6.7 Hz, 6H). 13 C NMR (101 MHz, CD 3 (1C), 129.26 (2C), 1 28.27 (2C), 126.60 (1C), 81.95 (1C), 59.99 (1C), 49.09 (1C), 41.61 (1C), 37.54 (1C), 27.10 (3C), 24.83 (1C), 21.67 (2C). HRMS (ESI) calc. for C 21 H 32 N 2 O 5 (M + H) + 392.2311, found 392.2301. (S) 2 (2 (((S) 1 (tert butoxy) 4 methyl 1 oxopentan 2 yl)amino)acet amido) 3 methylbutanoic acid ( 2. 5f) The product was obtained from 2.4 b (1.55 mmol) in a similar manner as described for preparation of 2.5 a affording compound 2.5 b (1.27 mmol, 82%) as a white solid. 1 H NMR (400 MHz, CD 3 O J = 4.8 Hz, 1H), 3.49 (d, J = 16.7 Hz, 1H), 3.34 (d, J = 7.2 Hz, 1H), 3.24 (d, J = 16.7 Hz, 1H), 2.27 2.17 (m, 1H), 1.79 (dp, J = 13.3, 6.7 Hz, 1H), 1.55 (ddd, J = 16.0, 9.9, 6.9 Hz, 2H), 1.48 (s, 9H), 0.96 (td, J = 7.1, 1.2 Hz, 12H). 13 C NMR (101 MHz, CD 3 1C ), 173.57 (1C), 171.42 (1C), 81.93 (1C), 60.28 (1C), 57.82 (1C), 49.17 (1C), 41.78 (1C), 30.86 (1C), 27.10 (3C), 25.01 (1C),

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57 21.79 (1C), 21.64 (1C), 18.49 (1C), 16.74 (1C). HRMS (ESI) calc. for C 17 H 32 N 2 O 5 (M + H) + 344.2311 found 344.2237. (S) 2 (2 (((2S,3R) 1 (tert butoxy) 3 methyl 1 oxopentan 2 yl)amino)acetamido) 3 methylbutanoic acid ( 2 5g) The product was obtained from 2.4 g (6.14 mmol) in a similar manner as described for preparation of 2.5 a affording compound 2.5 g (3.65 mmol, 60 %) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 J = 9.0 Hz, 1H), 6.64 (bs, 1H), 4.47 (dd, J = 9.0, 4.7 Hz, 1H), 3.51 (d, J = 17.2 Hz, 1H), 3.06 (dd, J = 11.3, 5.9 Hz, 2H), 2.29 2.19 (m, 1H), 1.78 1.68 (m, 1H), 1.56 1.49 (m, 1H), 1.45 (s, 9H), 1.25 1.15 (m, 1H), 0.99 0.87 (m, 12H). 13 C NMR (101 MHz, CDCl 3 (1C), 66.87 (1C), 57.38 (1C), 51.06 (1C), 38.40 (1C), 31.00 (1C), 28.33 (3C), 25.67 (1C), 19.39 (1C), 17.83 (1C), 15.89 (1C), 11.90 (1C). HRMS (ESI) calc. for C 17 H 23 N 2 O 5 (M + H) + 34 4.2311, found 344.2384. (S) tert butyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 y l) 3 methylbutanoate 2.6a To a stirring solution of 2.5a ( 1.78 mmol) in anhydrous CHCl 3 (8 mL) under argon, was added DIEA ( 1.95 mmol), and piva lic anhydride ( 1.78 mmol). The solution was heated to a gentle reflux for 24 h. After cooling to ambient temperatures, the reaction was diluted with DI H 2 O (50 mL) and the aqueous portion was extracted with CHCl 3 (3 x 40

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58 mL). The combined organic layers were washed with 10% aq. citric acid (1 x 40 mL), sat. aq. NaHCO 3 (1 x 40 mL), brine (1 x 50 mL), and dried (Na 2 SO 4 ). The residual solvent was removed in vacuo to afford the crude oil which was subsequently purified using flash chromatography on silica g el (30:70 EtOAc /hexanes) affording compound 2.6a (0.64 mmol, 36%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 7.27 (m, 3H), 7.24 7.19 (m, 2H), 4.72 (d, J = 10.0 Hz, 1H), 4.30 4.25 (m, 1H), 3.88 (dd, J = 207.6, 17.4 Hz, 2H), 3.34 (dd, J = 13.9, 3.8 Hz, 1H), 2.95 (dd, J = 13.9, 8.8 Hz, 1H), 2.14 2.03 (m, 1H), 1.45 (s, 9H), 1.01 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.7 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 169.43 (1C), 166.56 (1C), 166.19 (1C), 135.49 (1C), 129.78 (2C), 129.35 (2C), 127.83 (1C), 82.60 (1C), 62.17 (1C), 56.83 (1C), 46.59 (s, 3H), 40.01 (s, 3H), 28.24 (3C), 27.57 (1C), 19.83 (1C), 19.69 (1C ). HRMS (ESI) calc. for C 20 H 28 N 2 O 4 (M + H) + 360.2049, found 360.2022. (S) tert butyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate ( 2. 6b) The product was obtained from 2.5b (0.70 mmol) in a similar manner as described for preparation of 2.6a The crude oil was purified using flash chromatog raphy on silica gel (30:70 EtOAc /hexanes) to afford 2.6b (1.63 mmol, 57%) as a white solid. 1 H NMR (400 MHz, CDCl 3 J = 10.1 Hz, 1H), 4.16 (d, J = 17.2 Hz, 1H), 3.96 (dd, J = 13.1, 4.0 Hz, 1H), 3.87 (d, J = 17.2 Hz, 1H), 2.20 2.08 (m, 1H), 1.80 1.65 (m, 2H), 1.61 1.51 (m, 1H), 1.41 (s, 9H), 0.99 (d, J = 6.6 Hz, 3H), 0.92 (dd, J = 10.7, 6 .3 Hz, 6H), 0.85 (d, J = 6.7 Hz, 3H). 13 C NMR (101 MHz, CDCl 3

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59 (1C), 167.52 (1C), 82.44 (1C), 61.91 (1C), 54.17 (1C), 46.79 (1C), 42.60 (1C), 28.16 (3C), 27.84 (1C), 24.50 (1C), 23.36 (1C), 21.47 (1C), 19.84 (1C), 19.35 (1C). HRMS ( ESI) calc. for C 17 H 30 N 2 O 4 (M + H) + 326.2206 found 326.2186 (S) tert butyl2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate ( 2. 6d) The product was obtained from 2.5d (2.56 mmol) in a similar manner as describ ed for the preparation of 2.6 a affording compound 2.6 d ( 0.41 mmol, 5 9 % ) as a white solid 1 H NMR (400 MHz, CDCl 3 7.11 (m, 5H), 5.30 (dd, J = 12.0, 5.1 Hz, 1H), 3.87 3.83 (m, 1H), 3.82 (d, J = 8.3 Hz, 2H), 3.42 (dd, J = 15.0, 5.1 Hz, 1H), 2.92 (dd, J = 14.9, 12.1 Hz, 1H), 1.46 (s, 9H), 1.40 (d, J = 9.7 Hz, 2H), 1.04 (m, 1H), 0.80 (dd, J = 8.2, 6.7 Hz, 6H) 13 C NMR (101 MHz, CDCl 3 (1C), 128.90 (2C), 128.83 (2C), 127.28 (1C), 82.95 (1C), 57.06 (1C), 54.03 (1C), 47.24 (1C), 42.67 (1C), 34.76 (1C), 28.18 (3C), 24.23 (1C), 23.09 (1C), 21.46 (1C). HRMS (ESI) c alc. for C 21 H 30 N 2 O 4 (M + H) + 374.2206, found 374.2219. (S) tert butyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate ( 2. 6e) The product was obtained from 2.5e (1.27 mmol) in a similar manner as described for preparation of 2.6 a affording compound 2.6e (0.57 mmol, 45%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 7.27 (m, 3H), 7.21 (dd, J = 7.5, 1.6 Hz, 2H), 6.23

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60 (s, 1H), 5.10 (dd, J = 11.1, 5.0 Hz, 1H), 4.34 4.31 (m, 1H), 3.58 (dd, J = 210.4, 17.1 Hz, 2H), 3.25 (dd, J = 13.9, 3.9 Hz, 1H), 3.05 (dd, J = 13.9, 7.5 Hz, 1H), 1.62 (ddd, J = 14.7, 10.0, 5.0 H z, 2H), 1.43 (s, 9H), 1.29 1.14 (m, 1H), 0.91 (dd, J = 8.7, 6.6 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 (2C), 129.21 (2C), 127.74 (1C), 82.64 (1C), 56.80 (1C), 54.52 (1C), 46.04 (1C), 40.20 (1C), 36 .82 (1C), 28.20 (3C), 24.99 (1C), 23.31 (1C), 21.53 (1C). HRMS (ESI) calc. for C 21 H 30 N 2 O 4 (M + H) + 374.2205, found 374.2278. (S) tert butyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate (2.6f) The produc t was obtained from 2.5f (1.24 mmol) in a similar manner as described for preparation of 2.6a The crude oil which was purified using flash chromatog raphy on silica gel (30:70 EtOAc /hexanes) to afford 2.6f (0.57 mmol, 45%) as a white solid. 1 H NMR (400 MH z, CDCl 3 J = 11.0, 5.3 Hz, 1H), 4.05 3.80 (m, 3H), 2.44 2.30 (m, 1H), 1.74 1.62 (m, 4H), 1.44 (s, 9H), 1.03 (d, J = 7.1 Hz, 3H), 0.98 0.89 (m, 9H). 13 C NMR (101 MHz, CDCl 3 82.62 (1C), 61.09 (1C), 54.60 (1C), 46.37 (1C), 36.79 (1C), 32.66 (1C), 28.20 (3C), 25.38 (1C), 23.53 (1C), 21.17 (1C), 19.07 (1C), 16.69 (1C). HRMS (ESI) calc. for C 17 H 30 N 2 O 4 (M + H) + 326.2206, found 326.2214.

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61 (2S,3S) tert butyl 2 ((S) 3 isopropyl 2,5 dio xopiperazin 1 yl) 3 methylpentanoate ( 2. 6g) The product was obtained from 2.5g (3.63 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.6g (2.20 mmol, 61%) as a white powder. 1 H NMR (400 MHz, CDCl 3 J = 9.9 Hz, 1H), 4.15 (d, J = 17.4 Hz, 1H), 3.79 (d, J = 17.6 Hz, 2H), 2.23 (m, 1H), 1.81 (m, 1H), 1.34 (s, 9H), 0.94 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H). 13 C NMR (1 01 MHz, CDCl 3 (1C), 60.53 (1C), 46.59 (1C), 33.49 (1C), 32.72 (1C), 28.10 (3C), 25.81 (1C), 19.03 (1C), 17.01 (1C), 15.87 (1C), 10.86 (1C). HRMS (ESI) calc. for C 17 H 30 N 2 O 4 (M + H) + 326.2205, foun d 326.2097. (S) 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoic acid (2.7c) The product was obtained from 2.6e (1.34 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.7c (0.8 7 mmol, 45%) as a white powder. 1 H NMR (400 MHz, CD 3 7.26 (m,3H), 7.27 7.15 (m, 2H), 5.10 (dd, J = 11.1, 4.8 Hz, 1H), 4.37 (t, J = 4.4 Hz, 1H), 3.75 3.56 (m, 1H), 3.12 (ddd, J = 80.4, 13.8, 4.5 Hz, 2H), 2.92 (d, J = 17.2 Hz, 1H), 1.58 (ddd, J = 14.6, 9.8, 4.8 Hz, 1H), 1.49 1.39 (m, 1H), 1.04 0.93 (m, 1H), 0.88 (dd, J = 21.9, 6.2 Hz, 6H). 13 C NMR (101 MHz, CD 3

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62 172.44 (1C), 167.09 (1C), 166.74 (1C), 135.31 (1C), 130.12 (2C), 128.53 (2C), 127.19 (1C), 56.70 (1C), 53.62 (1C), 45.40 (1C), 39.54 (1C), 36.42 (1C), 26.44 (1C), 24.40 (1C), 22.09 (1C), 20.46 (1C). HRMS (ESI) calc. for C 17 H 22 N 2 O 4 (M + H) + 318.1579, found 318.1664. (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoic acid (2.7d) The product was obtained from 2.6f (2.95 mmol) in a simi lar manner as described for preparation of 2.10a affording compound 2.7d (0.87 mmol, 95%) as a light yellow solid. 1 H NMR (400 MHz, CD 3 J = 11.4, 4.7 Hz, 1H), 3.98 (dd, J = 54.7, 17.3 Hz, 2H), 3.76 (d, J = 5.1 Hz, 1H), 2.25 2.15 (m, 1H), 1.88 1.71 (m, 2H), 1.52 1.45 (m, 1H), 1.03 0.98 (m, 9H), 0.96 (t, J = 6.7 Hz, 9H). 13 C NMR (101 MHz, CD 3 172.49 (1C), 167.69 ( 1C), 167.24 (1C), 61.12 (1C), 54.37 (1C), 46.46 (1C), 36.62 (1C), 33.20 (1C), 25.17 (1C), 22.36 (1C), 20.09 (1C), 17.99 (1C), 16.55 (1C). HRMS (ESI) calc. for C 13 H 22 N 2 O 4 (M + H) + 270.1579, found 270.1666. (2S,3S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl ) 3 methylpentanoic acid ( 2. 7e) The product was obtained from 2.6g (1.84 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.7e (0.99 mmol, 54%) as a white solid. The structural confirmat ion of 2.7e was obtained by single crystal X ray diffraction. 1 H

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63 NMR (400 MHz, CD 3 4.73 (m, 1H), 4.14 (dd, J = 58.8, 17.3 Hz, 2H), 3.78 3.69 (m, 1H), 3.32 (d, J = 15.4 Hz, 1H), 2.26 2.15 (m, 1H), 2.09 1.97 (m, 1H), 1.20 1.09 (m, 1H), 1 .06 0.85 (m, 12H). 13 C NMR (101 MHz, CD 3 167.73 (1C), 167.47 (1C), 61.20 (1C), 60.34 (1C), 46.75 (1C), 33.27 (1C), 32.93 (1C), 25.47 (1C), 18.11 (1C), 16.76 (1C), 15.04 (1C), 9.62 (1C). HRMS (ESI) calc. for C 13 H 22 N 2 O 4 (M + H) + 270.157 9, found 270.1666. (S) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.7f) The product was obtained from 2.6d (1.38 mmol) in a similar manner as described for preparation of 2.10a affording compound 2 .7f (1.17 mmol, 85%) as a white solid. 1 H NMR (400 MHz, CD 3 7.18 (m, 5H), 5.49 (s, 1H), 5.33 (dd, J = 12.4, 4.7 Hz, 1H), 4.00 3.74 (m, 3H), 3.44 (dd, J = 14.8, 4.7 Hz, 1H), 3.08 (dd, J = 14.8, 12.4 Hz, 1H), 1.42 (dd, J = 13.9, 7.3 Hz, 1H), 1.11 (ddd, J = 7.9, 6.4, 1.6 Hz, 2H), 0.79 (dd, J = 9.5, 6.6 Hz, 6H). 13 C NMR (101 MHz, CD 3 (1C), 136.83 (1C), 128.72 (2C), 128.53 (2C ), 126.72 (1C), 57.04 (1C), 53.84 (1C), 42.62 (1C), 34.04 (1C), 23.93 (1C), 21.96 (1C), 20.90 (1C). HRMS (ESI) calc. for C 17 H 22 N 2 O 4 (M + H) + 3 18.1579, found 318.1533.

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64 (S) tert butyl 2 (2 bromoacetamido) 3 phenylpropanoate (2.8a) The product was obtained from 2.2c (7.76 mmol) in a similar manner as described for preparation of 2.3a affording compound 2.8a (6 .45 mmol, 83%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 7.08 (m, 5H), 6.92 (d, J = 7.2 Hz, 1H), 4.77 4.63 (m, 1H), 3.89 3.81 (m, 1H), 3.11 (d, J = 5.9 Hz, 2H), 1.41 (s, 9H). 13 C NMR (101 MHz, CDCl 3 83.05 (1C), 54.15 (1C), 38.06 (1C), 29.05 (1C), 28.17 (3C). HRMS (ESI) calc. for C 15 H 20 BrNO 3 (M + H) + 341.0627 found 307.0563 (S) tert butyl 2 (2 bromoacetamido) 4 methylpentanoate (2.8b) The product was obtained from 2.2b (6.71 mmol) in a similar manner as described for preparation of 2.3a affording compound 2.8b (5.94 mmol, 88%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 7.7 Hz, 1H), 4.49 (td, J = 8.4, 5.2 Hz, 1H), 3.89 (s, 2H), 1.71 1.58 (m, 3H), 1.47 (s, 9H), 0.95 (d, J = 6.0 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 28.21 (3C), 25.17 (1C), 23.04 (1C), 22.36 (1C). HRMS (ESI) calc. for C 12 H 22 BrNO 3 (M + H) + 307.0783, found 307.0675.

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65 (S) tert butyl 2 (2 bromoacetamido) 3 methylbutanoate (2.8c) The product was obtained from 2.2a (5.14 mmol) in a simila r manner as described for preparation of 2.3a affording compound 2.8c (3.98 mmol, 93%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 7.6 Hz, 1H), 4.42 (dd, J = 8.7, 4.4 Hz, 1H), 3.91 (s, 2H), 2.26 2.13 (m, 1H), 1.48 (s, 9H), 0.94 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 28.26 (3C), 19.02 (1C), 17.78 (1C). HRMS (ESI) calc. for C 11 H 20 BrNO 3 (M + H) + 293.0627, found 293.0589. (S) tert butyl 2 (2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino)acetami do) 3 methylbutanoate (2.9a) The product was obtained from 2.2a (0.341 mmol) and 2.1c (0.44 mmol), in the presence of tert butanol (10 mL), in a similar manner as described for pre paration of 2.4a affording compound 2.9a (0.20 mmol, 60%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 ) 7.61 (d, J = 8.9 Hz, 1H), 7.31 7.01 (m, 4H), 4.43 4.33 (m, 1H), 3.63 (m, 3H), 3.51 3.45 (m, 1H), 3.27 (dd, J = 46.0, 16.5 Hz, 1H), 3.06 (d, J = 6.7 Hz, 1H), 2.23 2.03 (m, 1H), 1.44 (s, 9H), 0.95 0.79 (m, 6H). 13 C NMR (101 MHz, CD 3 OD ), 168.11 (1C), 167.61 (1C), 132.15 (1C), 125.48 (2C), 124.90 (2C), 123.43 (1C), 77.81 (1C), 64.04 (1C), 48.77 (1C), 48.51 (1C), 47.36 (1C), 34.25 (1C), 27.69 (1C), 24.40 (3C),

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66 15.63 (1C), 14.18 (1C). HRMS (ESI) calc. for C 21 H 32 N 2 O 5 (M + H) + 392.2311, fou nd 392.2296. (S) methyl 2 ((2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl )amino) 4 methylpentanoate (2.9c ) The product was obtained from 2.2c (3.81 mmol) and 2.1b (4.96 mmol ) in a similar manner as d escribed for preparation of 2.9 a affording compound 2.9 c (3.05 mmol, 80%) as a thick yellow gel 1 H NMR (400 MHz, CDCl 3 J = 8.2 Hz, 1H), 7.30 7.13 (m, 6H), 4.75 4.70 (m, 1H), 3.68 (s, 3H), 3.43 (dd, J = 7.2, 3.3 Hz, 1H), 3.38 (d, J = 17.1 Hz 1H), 3.24 3.19 (m, 1H), 3.15 3.03 (m, 2H), 2.99 (d, J = 17.1 Hz, 1H), 2.06 (s, 2H), 1.64 (dp, J = 13.3, 6.7 Hz, 1H), 1.38 (s, 9H), 0.85 (dd, J = 17.0, 6.6 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 (2C), 128.82 (2C), 127.36 (1C), 81.67 (1C), 60.89 (1C), 52.71 (1C), 52.44 (1C), 50.94 (1C), 42.96 (1C), 38.09 (1C), 28.27 (3C), 25.01 (1C), 22.99 (1C), 22.27 (1C). HRMS (ESI) calc. for C 22 H 34 N 2 O 5 (M + H) + 406.2467 found 406.2540 (2S,3R) methyl 2 ((2 ((( S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 methylpentanoate (2.9d) The product was obtained from 2.2c (2.19 mmol) and 2.1c (2.19 mmol) in a similar manner as described for preparation of 2.9a affording compound 2.9d (1.20 mmol, 55%) as a cloudy yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 8.5 Hz, 1H), 7.32

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67 7.13 (m, 6H), 4.75 (dt, J = 8.5, 6.2 Hz, 1H), 3.70 (s, 3H), 3.41 (d, J = 17.1 Hz, 1H), 3.17 3.01 (m, 3H), 2.95 (d, J = 17.1 Hz, 1 H), 1.73 1.60 (m, 2H), 1.40 (s, 9H), 1.15 1.01 (m, 1H), 0.88 0.83 (m, 6H). 13 C NMR (101 MHz, CDCl 3 170.59 (1C), 136.43 (1C), 129.67 (2C), 128.64 (2C), 127.18 (1C), 82.26 (1C), 66.49 (1C), 53.21 (1C), 51.92 (1C), 51.55 (1 C), 38.43 (1C), 38.35 (1C), 28.17 (3C), 25.30 (1C), 15.95 (1C), 11.81 (1C). HRMS (ESI) calc. for C 22 H 34 N 2 O 5 (M + H) + 406.2467, found 406.2555. (S) tert butyl 2 (2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoate (2.9e) The product was obtained from 2.2b (5.01 mmol) and 2.1a (6.52 mmol) in a similar manner as described for preparation of 2.9a affording compound 2.9 e (3.50 mmol, 70%) as a yellow crystalline solid. 1 H NMR (400 MHz, CDCl 3 J = 8.5 Hz, 1H), 4.53 4.46 (m, 1H), 3.72 (s, 3H), 3.45 (d, J = 17.2 Hz, 1H), 3.07 (d, J = 5.7 Hz, 1H), 2.99 (d, J = 17.2 Hz, 1H), 2.05 1.95 (m, 2H), 1.68 1.59 (m, 3H), 1.45 (s, 9H), 1.02 (d, J = 6.8 Hz, 3H), 0.99 0.92 (m, 9H). 13 C NMR (101 MHz, CDCl 3 172.10 (1C), 171.15 (1C), 81.84 (1C), 67.40 (1C), 52.01 (1C), 51.42 (1C), 51.13 (1C), 42.04 (1C), 31.73 (1C), 28.23 (3C), 25.23 (1C), 23.05 (1C), 22.35 (1C), 19.73 (1C), 18.41 (1C). HRMS (ESI) calc. for C 18 H 34 N 2 O 5 (M + H) + 358.2468, found 358.2422.

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68 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 3 methylbutanoate (2.9g) The product was obtained from 2.2a (0.49 mmol) and 2.1f (0.59 mmol) in a s imilar manner as described for preparation of 2.4a affording compound 2.9g (1.93 mmol, 64%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 9.2 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.18 7.03 (m, 2H), 6.96 (s, 1H), 4.41 (dd, J = 9.2, 4.6 Hz, 1H), 3.61 (s, 3H), 3.39 (d, J = 17.2 Hz, 1H), 3.21 (t, J = 6.7 Hz, 2H), 3.08 (d, J = 17.2 Hz, 1H), 2.17 2.00 (m, 1H), 1.47 (s, 9H), 0.89 (dd, J = 19.9, 6.9 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 (1C), 127.79 (1C), 123.37 (1C), 122.10 (1C), 119.55 (1C), 118.85 (1C), 111.61 (1C), 110.29 (1C), 81.99 (1C), 62.4 3 (1C), 57.35 (1C), 52.12 (1C), 51.26 (1C), 42.87 (1C), 31.42 (1C), 29.51 (1C), 28.27 (3C), 19.27 (1C), 17.81 (1C). HRMS (ESI) calc. for C 23 H 33 N 3 O 5 (M + H) + 431.2420, found 431.2493.

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69 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxop ropan 2 yl)amino) acetamido) 3 phenylpropanoate (2.9h) The product was obtained from 2.2c (1.57 mmol) and 2.1f (1.88 mmol) in a similar manner as described for preparation of 2.4a affording compound 2.9 h (0.94 mmol, 60%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 17.5, 8.1 Hz, 1H), 7.34 7.07 (m, 8H), 6.93 (d, J = 2.1 Hz, 1H), 4.74 4.66 (m, 1H), 3.61 (s, 3H), 3.57 (t, J = 6.1 Hz, 1H), 3.34 (d, J = 17.3 Hz, 1H), 3.19 2.95 (m, 4H), 2.8 7 (dd, J = 13.8, 6.6 Hz, 1H), 1.42 (s, 9H). 13 C NMR (101 MHz, CDCl 3 (1C), 170.71 (1C), 136.61 (1C), 136.51 (1C), 129.63 (2C), 128.65 (2C), 127.77 (1C), 127.16 (1C), 123.45 (1C), 122.19 (1C), 119.63 (1C), 118.95 (1C), 111.66 (1C), 11 0.49 (1C), 82.29 (1C), 62.24 (1C), 54.12 (1C), 53.45 (1C), 52.16 (1C), 51.04 (1C), 42.71 (1C), 38.08 (1C), 29.45 (s, 6H), 28.22 (3C). HRMS (ESI) calc. for C 27 H 33 N 3 O 5 (M + H) + 479.2420, found 479.2493.

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70 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl ) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 4 methylpentanoate (2.9i) The product was obtained from 2.2 b ( 2.21 mmol) and 2.1f ( 2.66 mmol) in a similar manner as described for preparation of 2.4a affording compound 2.9 1 ( 1.42 mmol, 6 4 %) as a clear yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 7.8 Hz, 1H), 7.36 (dd, J = 16.9, 8.4 Hz, 2H), 7.22 7.02 (m, 3H), 4.41 4.38 (m, 1H), 3.66 (s, 3H), 3.38 (d, J = 17.3 Hz, 1H), 3.27 3.10 (m, 2H), 3.08 (d, J = 17.3 Hz, 1H), 1.46 (s, 9H), 1.27 1.19 (m, 2H), 0.96 0.94 (m, 1H) 0.88 (dd, J = 6.3, 2.8 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 123.24 (1C), 122.33 (1C), 119.77 (1C), 119.02 (1C), 111.53 (1C), 111.03 (1C), 81.76 (1C), 62.49 (1C), 52.20 (1C), 51.14 (1C), 50. 97 (1C), 41.44 (1C), 29.54 (1C), 28.25 (3C), 25.13 (1C), 23.06 (1C), 22.15 (1C). HRMS (ESI) calc. for C 24 H 35 N 3 O 5 (M + H) + 445.2577, found 445.2512. (S) 2 (2 ((S) 1 methoxy 1 oxo 3 phenylpropan 2 ylamino) 2 oxoethylamino) 3 methylbutanoic acid ( 2. 10a) Compound 2. 9a (0.92 mmol) was dissolved in 4N HCl/Dioxane (10 mL) and was stirred at ambient temperature for 18 hours. The solvent was removed in vacuo the resulting crude oil was re dissolved in DCM, and evaporated in vacu o (3 x 30 mL). This provided

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71 the pure compound 2. 10a (0.91 mmol, 99%) as a yellow solid. 1 H NMR (400 MHz, CD 3 7.24 (m, 5H), 4.43 4.32 (m, 2H), 3.96 (s, 1H), 3.72 (s, 3H), 3.65 (s, 1H), 3.37 (dd, J = 14.1, 6.0 Hz, 1H), 3.22 (dd, J = 14.0, 8. 0 Hz, 1H), 2.27 2.12 (m, 1H), 1.01 0.93 (m, 6H). 13 C NMR (101 MHz, CD 3 (1C), 133.80 (1C), 129.17 (2C), 128.93 (2C), 127.83 (1C), 66.92 (1C), 60.73 (1C), 57.98 (1C), 52.42 (1C), 35.44 (1C), 30.39 (1C), 18.34 (1C), 16.97 (1C). HRMS (ESI) calc. for C 17 H 24 N 2 O 5 (M H) 336.1652, found 336.1610. (S) 2 (2 ((S) 1 methoxy 4 methyl 1 oxopentan 2 ylamino) 2 oxoethylamino) 3 phenylpropanoic acid ( 2. 10c) The product was obtained from 2.9 c (2. 95 mmol) in a similar manner as described for preparation of 2.1 0a affording compound 2.1 0c (2.94 mmol, 99%) as a light yellow solid. 1 H NMR (400 MHz, CD 3 7.18 (m, 5H), 4.74 (dd, J = 9.4, 4.7 Hz, 1H), 3.95 3.86 (m, 1H), 3.84 (s, 3H), 3.42 (q, J = 7.3 Hz, 1H), 3.25 (d, J = 4.7 Hz, 1H), 2.94 (dd, J = 14.0, 9.5 Hz, 1H), 1.84 1.62 (m, 3H), 0.96 (dd, J = 6.0, 2.3 Hz, 6H). 13 C NMR (101 MHz, CD 3 OD 128.36 (2C), 126.78 (1C), 66.95 (1C), 57.99 (1C), 52.62 (1C), 46.18 (1C), 38.48 (1C), 37.32 (1C), 24.71 (1C), 21.81 (1C), 20.80 (1C). HRMS (ESI) calc. for C 18 H 26 N 2 O 5 (M + H) + 350.1841, f ound 350.1913.

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72 (S) 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoic acid (2.10e) The product was obtained from 2.9e (3.46 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.10e (3.43 mmol, 99%) as a light yellow solid. 1 H NMR (400 MHz, CD 3 4.42 (m, 1H), 4.03 (d, J = 3.9 Hz, 1H), 3.92 (d, J = 6.1 Hz, 1H), 3.86 (s, 3H), 3.65 (s, 2H), 2.46 2.33 (m, 1H), 1.78 1.57 (m, 3H), 1.12 (d, J = 7.0 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H), 0.95 (ddd, J = 12.4, 6.2, 3.9 Hz, 6H). 13 C NMR (101 MH z, CD 3 OD ), 168.31 ( 1C ), 164.96 ( 1C ), 66.94 ( 1C ), 65.44 ( 1C ), 52.45 ( 1C ), 51.11 ( 1C), 40.34 (1C ), 40.21 (1C), 29.49 ( 1C ), 24.79 ( 1C ), 22.07 ( 1C ), 20.58 ( 1C ), 18.03 ( 1C ), 16.38 ( 1C ). (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl )amino)acetamido) 3 methylbutanoic acid (2.10f) The product was obtained from 2.9g (1.23 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.10f (2.57 mmol, 90%) as a light brown solid. 1 H NMR (400 MHz, CD 3 J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.13 (dd, J = 11.1, 4.0 Hz, 1H), 7.09 7.02 (m, 1H), 4.44 (t, J = 6.5 Hz, 1H), 4.37 (d, J = 5.2 Hz, 1H), 4.01 3.90 (m, 2H), 3.70 (s, 3H), 3.50 (qd, J = 15.0, 7.7 Hz, 2H), 3.34 (s, 2H), 2.24 2.17 (m, 1H), 0.96 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, CD 3

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73 (1C), 168.97 (1C), 165.22 (1C), 137.00 (1C), 126.96 (1C), 124.39 (1C), 121.78 (1C), 119.16 (1C), 117.69 (1C), 111.50 (1C), 105.93 (1C), 60.27 (1C), 57.92 (1C), 52.58 (1C), 46.71 (1C), 30 .42 (1C), 25.68 (1C), 18.38 (1C), 16.89 (1C). HRMS (ESI) calc. for C 19 H 25 N 3 O 5 (M + H) + 375.1794, found 375.1855. (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino)acetamido) 3 phenylpropanoic acid (2.10g) The product was obtained from 2.9h (1.04 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.10g (0.94 mmol, 92%) as a light yellow solid. 1 H NMR (400 MHz, CD 3 7.46 (m, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.29 7.11 (m, 8H), 7.09 7.03 (m, 1H), 4.72 (dd, J = 9.2, 4.8 Hz, 1H), 4.23 (t, J = 6.5 Hz, 1H), 3.86 (d, J = 15.9 Hz, 1H), 3.69 (s, 3H), 3.43 (dd, J = 9.9, 6.5 Hz, 2H), 3.34 (s, 3H), 3.24 (dd, J = 14 .0, 4.9 Hz, 1H), 2.91 (dd, J = 14.0, 9.2 Hz, 1H). 13 C NMR (101 MHz, CD 3 172.88 (1C), 169.04 (1C), 164.87 (1C), 136.93 (1C), 129.06 (1C), 128.34 (1C), 126.75 (1C), 124.28 (1C), 121.78 (1C), 119.16 (1C), 117.68 (1C), 111.46 (1C), 105.97 (1C), 60.19 (1 C), 53.94 (1C), 52.50 (1C), 46.68 (1C), 37.25 (1C), 25.76 (1C). HRMS (ESI) calc. for C 23 H 25 N 3 O 5 (M + H) + 423.1794, found 423.1733.

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74 (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino)acetamido) 4 methylpentanoic acid (2.10h) The product was obtained from 2.9i (1.41 mmol) in a similar manner as described for preparation of 2.10a affording compound 2.10h (1.39 mmol, 99%) as a light yellow solid. 1 H NMR (400 MHz, CD 3 J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.18 7.10 (m, 1H), 7.10 7.03 (m, 1H), 4.44 (dd, J = 12.2, 5.6 Hz, 2H), 3.97 3.84 (m, 2H), 3.71 (s, 3H), 3.57 3.43 (m, 2H), 1.73 1.53 (m, 3H), 0.94 (dd, J = 12.6, 6.0 Hz, 6H). 13 C NMR (101 MHz, CD 3 126.94 (1C), 124.36 (1C), 121.81 (1C), 119.18 (1C), 117.66 (1C), 111.49 (1C), 105.93 (1C), 60.26 (1C), 52.57 (1C), 51.10 (1C), 46.65 (1C), 40.36 (1C), 25.66 (1C), 24.82 (1C), 22.11 (1C), 20 .57 (1C). HRMS (ESI) calc. for C 23 H 25 N 3 O 5 (M + H) + 423.1794, found 423.1733. (S) methyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate (2.11a) The product was obtained from 2.10a (0.85 mmol) in a similar m anner as described for preparation of 2.6a affording compound 2.11a (0.31 mmol, 46%) as a white solid. 1 H NMR (400 MHz, CD 3 7.19 (m, 5H), 5.14 (dd, J = 12.1, 4.7 Hz, 1H), 3.97 (dd, J = 17.4, 0.7 Hz, 1H), 3.77 (s, 3H), 3.64 (d, J = 4.9 Hz, 1H), 3.59 (d, J = 17.4 Hz,

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75 1H), 3.42 (dd, J = 14.7, 4.6 Hz, 1H), 3.13 (dd, J = 14.6, 12.2 Hz, 1H), 1.94 1.86 (m, 1H), 0.74 (d, J = 6.9 Hz, 3H), 0.60 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CD 3 170.33 (1C), 167.43 (1C), 166.79 (1C), 136.76 (1C), 128.73 (2C), 128.65 (2C), 126.98 (1C), 60.83 (1C), 58.41 (1C), 51.83 (1C), 33.72 (1C), 33.31 (1C), 17.80 (1C), 15.90 (1C). HRMS (ESI) calc. for C 17 H 22 N 2 O 4 (M + H) + 318.1580, found 318.1502. (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate (2.11b) The product was obtained from 2.10b (0.78 mmol) in a similar manner as described for p reparation of 2.6a affording compound 2.11b ( 0.31 mmol, 40%) as a yellow gel 1 H NMR (400 MHz, CD 3 7.17 (m, 8H), 7.08 7.03 (m, 2H), 4.94 4.89 (m, 1H), 4.18 (t, J = 5.4 Hz, 1H), 3.74 (s, 3H), 3.37 (d, J = 17.1 Hz, 1H), 3.27 3.20 (m, 2H), 3.01 (dd, J = 14.4, 10.5 Hz, 1H), 2.88 (qd, J = 13.8, 5.4 Hz, 2H). 13 C NMR (101 MHz, CD 3 1C), 167.41 (1C), 166.72 (1C), 136.83 (1C), 135.31 (1C), 129.95 (2C), 128.78 (2C), 128.62 (2C), 128.40 (2C), 127.03 (1C), 126.93 (1C), 59.02 (1C), 56.65 (1C), 51.77 (1C), 39.48 (1C), 33.90 (1C). HRMS (ESI) calc. for C 21 H 22 N 2 O 4 (M + H) + 366.1580 found 366 .1525

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76 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate (2.11c) The product was obtained from 2.10c (2.85 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.1 1c (2.42 mmol, 85%) as a yellow powder. 1 H NMR (400 MHz, CDCl 3 7.27 (m, 3H), 7.21 (m, 2H), 5.23 (dd, J = 11.1, 5.0 Hz, 1H), 4.37 4.32 (m, 1H), 3.71 (s, 3H), 3.61 (dd, J = 198.7, 17.2 Hz, 2H), 3.26 (dd, J = 13.9, 3.9 Hz, 1H), 3.05 (dd, J = 13.9 7.6 Hz, 1H), 1.67 (m, 1H), 1.56 1.47 (m, 1H), 1.26 1.14 (m, 1H), 0.91 (dd, J = 8.6, 6.6 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 171.43 (1C), 166.33 (1C), 165.80 (1C), 135.25 (1C), 129.89 (2C), 129.25 (2C), 127.80 (1C), 56.72 (1C), 53.78 (1C), 52.77 (1C) 46.00 (1C), 40.23 (1C), 36.68 (1C), 24.85 (1C), 23.26 (1C), 21.49 (1C). HRMS (ESI) calc. for C 18 H 24 N 2 O 4 (M + H) + 332.1736, found 332.1816. (S) methyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate ( 2. 11d) T he product was obtained from 2.10d (3.46 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.11d (2.58 mmol, 75%) as a white powder. 1 H NMR (400 MHz, CDCl 3 J = 10.6 Hz, 1H), 4.21 (d, J = 17.3 Hz, 1H), 4.01 (d, J = 16.5 Hz, 1H), 3.94 (d, J = 17.3 Hz, 1H), 3.72 (s, 3H), 2.21 (m, 1H), 1.77 (m, 2H), 1.02 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.90

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77 (d, J = 6.7 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ), 166.49 (1C), 61.13 (1C), 54.08 (1C), 52.42 (1C), 46.54 (1C), 42.58 (1C), 27.60 (1C), 24.61 (1C), 23.37 (1C), 21.43 (1C), 19.79 (1C), 19.16 (1C). HRMS (ESI) calc. for C 14 H 24 N 2 O 4 (M + H) + 284.1736, found 284.1828. (S) methyl 3 (1H indol 3 yl) 2 ((S) 3 is opropyl 2,5 dioxopiperazin 1 yl)propanoate (2.11e) The product was obtained from 2.10f (0.83 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.11e (0.52 mmol, 63%) as a yellow crystalline solid. 1 H NMR (400 MHz, CDCl 3 7.52 (m, 2H), 7.28 (t, J = 8.9 Hz, 1H), 7.19 7.04 (m, 3H), 7.00 (d, J = 1.9 Hz, 1H), 5.20 (dd, J = 11.6, 4.7 Hz, 1H), 3.90 (d, J = 17.1 Hz, 1H), 3.76 (s, 3H), 3.69 (d, J = 17.2 Hz, 1H), 3.48 (d, J = 4.6 Hz, 1H), 3.35 3.25 (m, 1 H), 0.97 0.87 (m, 1H), 0.75 (d, J = 7.0 Hz, 3H), 0.48 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 (1C), 127.07 (1C), 122.81 (1C), 122.48 (1C), 119.87 (1C), 118.39 (1C), 111.67 (1C), 110.20 (1C), 60.97 (1 C), 57.60 (1C), 52.82 (1C), 47.98 (1C), 33.09 (1C), 24.38 (1C), 18.77 (1C), 16.03 (1C). HRMS (ESI) calc. for C 19 H 23 N 3 O 4 (M + H) + 357.1688, found 357.1751.

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78 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 (1H indol 3 yl)propanoate (2.11f) The product was obtained from 2.10g ( 1.42 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.11 f (0. 71 mmol, 50 %) as a yellow crystalline solid. 1 H NMR (400 MHz, CD 3 J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.17 7.02 (m, 6H), 6.96 6.91 (m, 2H), 5.11 (dd, J = 10.5, 5.5 Hz, 1H), 4.15 (t, J = 5.2 Hz, 1H), 3.73 (s, 3H), 3.46 (d, J = 17.2 Hz, 1H), 3.37 3.32 (m, 1H), 3.23 3.13 (m, 2H), 2.83 (ddd, J = 40.0, 13.8, 5.3 Hz, 2H). 13 C NMR (101 MHz, CD 3 (1C), 167.29 (1C), 166.79 (1C), 136.97 (1C), 135.17 (1C), 129.87 (2C), 128.30 (2C), 127.22 (1C), 126.96 (1C), 123.09 (1C), 121.52 (1C), 118.85 (1C), 117.82 (1C), 111.35 (1C), 109.27 (1C), 57.90 ( 1C), 56.64 (1C), 51.75 (1C), 39.48 (1C), 23.97 (1C). HRMS (ESI) calc. for C 23 H 23 N 3 O 4 (M + H) + 405.1689, found 405.1588. (S) methyl 3 (1H indol 3 yl) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl)propanoate ( 2. 11g) The produc t was obtained from 2.10h (1.54 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.11g (0.80 mmol, 52%) as yellow crystals. The

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79 structural confirmation of 2.11g was obtained by single crystal X ray diffraction. 1 H NMR (40 0 MHz, CDCl 3 J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.16 (dtd, J = 15.9, 7.1, 1.1 Hz, 2H), 7.04 (d, J = 2.3 Hz, 1H), 6.60 (d, J = 2.2 Hz, 1H), 5.37 (dd, J = 11.6, 5.0 Hz, 1H), 3.89 3.82 (m, 2H), 3.79 (s, 3H), 3.51 (ddd, J = 15.5, 4.9, 1.0 Hz, 1H), 3.29 (dd, J = 15.6, 11.6 Hz, 1H), 1.45 (ddd, J = 12.1, 9.2, 6.5 Hz, 1H), 1.13 1.03 (m, 1H), 0.78 (d, J = 6.6 Hz, 6H). (2S,3S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoate (2.11i) The product was obtained from 2.10d (1.19 mmol) in a similar manner as described for preparation of 2.6a affording compound 2.11i (0.48 mmol, 40%) as a white gel. 1 H NMR (400 MHz, CDCl 3 7.20 (m, 5H), 4.87 (d, J = 10.4 Hz, 1H), 4.35 (s, 1H), 4.07 (d, J = 17.4 Hz, 1H), 3.67 (s, 3H), 3.27 (d, J = 17.4 Hz, 1H), 3.15 (d, J = 4.7 Hz, 2H), 1.80 1.64 (m, 1H), 0.82 (dd, J = 27.1, 5.7 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 1.02 (1C), 166.73 (1C), 166.23 (1C), 135.32 (1C), 130.21 (2C), 129.00 (2C), 127.62 (1C), 59.64 (1C), 56.80 (1C), 52.32 (1C), 45.93 (1C), 39.99 (1C), 33.17 (1C), 25.39 (1C), 15.79 (1C), 10.89 (1C). HRMS (ESI) calc. for C 18 H 24 N 2 O 4 (M + H) + 332.1736, found 3 32.1802.

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80 (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.12a) The product was obtained from 2.11a (0.31 mmol) in a similar manner as described for preparation of 2.12b affording compound 2 .12a (0.09 mmol, 30%) as a yellow solid. 1 H NMR (400 MHz, CD 3 7.28 (m, 3H), 7.26 7.14 (m, 2H), 5.26 (dd, J = 12.4, 4.3 Hz, 1H), 3.90 (dd, J = 40.4, 17.3 Hz, 1H), 3.74 (dd, J = 17.3, 8.7 Hz, 1H), 3.63 (d, J = 4.9 Hz, 1H), 3.48 3.40 (m, 1H), 3.2 4 (dd, J = 21.0, 4.5 Hz, 1H), 3.10 (dd, J = 14.8, 12.5 Hz, 1H), 1.92 1.81 (m, 1H), 0.98 0.85 (m, 1H), 0.83 (d, J = 7.3 Hz, 1H), 0.77 (dd, J = 6.7, 3.4 Hz, 2H), 0.71 (d, J = 7.0 Hz, 2H), 0.62 (d, J = 6.6 Hz, 1H), 0.54 (d, J = 6.8 Hz, 2H). HRMS (ESI) ca lc. for C 16 H 20 N 2 O 4 (M + H) + 304.1423, found 304.1509. (S) 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.12b) To a stirring solution of 2.11b (0.10 mmol) in CH 3 CN (3 mL) was added aqueous LiOH (0.05 mm ol/1 mL H 2 O). The solution was stirred at ambient temperature for 1 8 h The solution was chilled to 0 o C and acidified with 1N HCl (to a pH of 4 5). The aqueous portion was extracted with EtOAc (3 x 35 mL), the combined organic layers were washed with b rine (2 x 40 mL), and dried over Na 2 SO 4 The remaining solvent was removed under reduced pressure to afford the pure compound 2.12b (0.049 mmol, 49%) as a white crystalline solid. 1 H NMR (400 MHz, CD 3 7.17 (m, 8H), 7.08 7.03 (m,

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81 2H), 4.94 4.89 (m, 1H), 4.18 (t, J = 5.4 Hz, 1H), 3.37 (d, J = 17.1 Hz, 1H), 3.27 3.20 (m, 2H), 3.01 (dd, J = 14.4, 10.5 Hz, 1H), 2.88 (qd, J = 13.8, 5.4 Hz, 2H). 13 C NMR (101 MHz, CD 3 C), 166.72 (1C), 136.83 (1C), 135.31 (1C), 129.95 (2C), 128.78 (2C), 128.62 (2C), 128.40 (2C), 127.03 (1C), 126.93 (1C), 59.02 (1C), 56.65 (1C), 39.48 (1C), 33.90 (1C). HRMS (ESI) calc. for C 20 H 20 N 2 O 4 (M + H) + 352.1423 found 352.1495 (S) 3 (1H indol 3 y l) 2 ((S) 3 isopropyl 2,5 dio xopiperazin 1 yl)propanoic acid (2.12c) The product was obtained from 2.11 f (0.45 mmol) in a similar manner as described for preparation of 2.12b affording compound 2.12c (0.27 mmol, 59%) as a n orange solid. 1 H NMR (400 MHz, CD 3 J = 7.9 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.78 6.61 (m, 3H), 4.97 (dd, J = 12.0, 4.0 Hz, 1H), 3.51 (dd, J = 99.6, 17.4 Hz, 2H), 3.23 (d, J = 4.8 Hz, 1H), 3.13 (dd, J = 15.3, 4.5 Hz, 2H), 2.99 (s, 2H), 1.48 1.35 (m, 1H), 0.25 (d, J = 7.0 Hz, 3H), 0.10 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CD 3 167.24 (1C), 167.13 (1C), 136.98 (1C), 127.10 (1C), 123.06 (1C), 121.47 (1C), 118.83 (1C), 117.90 (1C), 111.29 (1C), 109.55 (1C), 60.88 (1C), 33.24 (1C), 24.03 (1C), 17.68 (1C), 15.78 (1C). HRMS (ESI) calc. for C 18 H 21 N 3 O 4 (M + H) + 343.1532, found 343.1588.

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82 (S) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoic acid (2.12f) The product was obtained fro m 2.11g (2.35 mmol) in a similar manner as described for preparation of 2.12b affording compound 2.12f (1.45 mmol, 40%) as a yellow solid. 1 H NMR (400 MHz, CD 3 4.08 (m, 2H), 4.03 3.77 (m, 1H), 2.30 2.12 (m, 1H), 1.89 1.56 (m, 4H), 1.00 0.89 (m, 12H). 13 C NMR (101 MHz, CD 3 (1C), 168.98 (1C), 167.65 (1C), 67.13 (1C), 61.69 (1C), 54.08 (1C), 42.53 (1C), 27.59 (1C), 24.29 (1C), 22.15 (1C), 20.70 (1C), 18.95 (1C), 18.31 (1C). HRMS (ESI) calc. for C 13 H 22 N 2 O 4 (M + H) + 270.1579, found 270.1666. (S) 1 ((S) 3 (1H indol 3 yl) 1 oxo 1 (4 phenylpiperazin 1 yl)propan 2 yl) 3 isopropylpiperazine 2,5 dione ( 2. 14) To a stirring solution of 2.12c (0.29 mmol) in DCM (5 mL), under argon, was added phenyl piperazine (0.35 mmol), EDC (0.35 mmol), and stirred at ambient temperatures for 18 h. The solution concentrated under reduced pressure and the resulting crude resi due was dissolved in ethyl acetate (100 mL), washed with aq. 5% KHSO 4 (1 x 35 mL), saturated aqueous sodium bicarbonate (2 x 30 mL), brine (2 x 50 mL), and dried

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83 over Na 2 SO 4 The residual solvent was removed in vacuo to afford the crude oil which was subs equently purified using flash chromatog raphy on silica gel (60:40 EtOAc /hexanes) affording compound 2.14 (0.09 mmol, 30 %) as a clear oil. 1 H NMR (400 MHz, CDCl 3 7.64 (m, 1H), 7.35 7.30 (m, 1H), 7.25 7.13 (m, 4H), 7.09 (d, J = 2 .3 Hz, 1H), 6.91 6.85 (m, 1H), 6.79 (dd, J = 8.7, 0.9 Hz, 2H), 6.45 (s, 1H), 5.98 (t, J = 8.0 Hz, 1H), 4.51 4.05 (m, 2H), 3.80 (t, J = 3.2 Hz, 1H), 3.74 3.61 (m, 3H), 3.59 3.49 (m, 1H), 3.44 3.33 (m, 1H), 3.21 (dd, J = 14.5, 7.6 Hz, 1H), 3.12 3.0 2 (m, 1H), 2.91 2.82 (m, 2H), 2.60 2.51 (m, 1H), 2.25 2.14 (m, 1H), 0.88 (d, J = 7.0 Hz, 3H), 0.67 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 166.20 (1C), 165.96 (1C), 150.93 (1C), 136.39 (1C), 129.39 (2C), 123.12 (1C), 122.65 (1C), 120.73 (1C), 120.17 (1C), 118.72 (1C), 116.78 (2C), 111.49 (1C), 110.16 (1C), 61.03 (1C), 51.21 (1C), 49.69 (1C), 45.88 (1C), 45.71 (1C), 42.32 (1C) 41.24 (1C), 32.83 (1C), 25.43 (1C), 18.90 (1C), 16.31 (1C). HRMS (ESI) calc. for C 28 H 33 N 5 O 3 (M + H) + 487.2583, found 487.2158. 2.6 References Alfaro Lopez, J., Okayama, T., Hosohata, K., Davis, P., Porreca, F., Yamamura, H. I., and Hruby, V. J. (1999). Exploring the Structure Activity Relationships of [1 (4 tert Butyl 3' hydroxy)benzhydryl 4 benzylpiperazine] (SL 3111), A High Affinity and Selective delta Opioid Receptor Nonpeptide Agonist Ligand. Journal of Medicinal Chemistry 42 5359 5368. Ali, M. A., and Yar, M. S. (2007). Synthesis and antimycobacterial activity of novel 4 [5 (substituted phenyl) 1 phenyl 4,5 dihydro 1H 3 pyrazolyl] 2 methylphenol derivatives. Medicinal Chemistry Research 15 463 470. Balakrishnan, S., Zhao, C ., and Zondlo, N. J. (2007). Convergent and stereospecific synthesis of molecules containing alpha functionalized guanidiniums via alpha guanidino acids. Journal of Organic Chemistry 72 9834 9837.

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84 Balibar, C. J., and Walsh, C. T. (2006). GliP, a Multim odular Nonribosomal Peptide Synthetase in Aspergillus fumigatus, Makes the Diketopiperazine Scaffold of Gliotoxin. Biochemistry 45 15029 15038. Chen, L., Yin, H., Farooqi, B., Sebti, S., Hamilton, A. D., and Chen, J. (2005). p53 alpha Helix mimetics ant agonize p53/MDM2 interaction and activate p53. Molecular Cancer Therapeutics 4 1019 1025. Dankwardt, S. M., Newman, S. R., and Krstenansky, J. L. (1995). Solid phase synthesis of aryl and benzylpiperazines and their application in combinatorial chemistry Tetrahedron Letters 36 4923 4926. Fara, M. A., Diaz Mochon, J. J., and Bradley, M. (2006). Microwave assisted coupling with DIC/HOBt for the synthesis of difficult peptoids and fluorescently labeled peptides a gentle heat goes a long way. Tetrahedron L etters 47 1011 1014. Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., Klicic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Shelley, M., Perry, J. K. et al. (2004). Glide: A new approach for rapid, ac curate docking and scoring. 1. M eth od and assessment of docking accuracy. Journal of Medicinal Chemistry 47 1739 1749. Gordon, D. W., and Steele, J. (1995). Reductive alkylation on a solid phase: synthesis of a piperazinedione combinatorial library. Bioorganic & Medicinal Chemistry Letter s 5 47 50. Hu, B., Gilkes, D. M., and Chen, J. (2007). Efficient p53 Activation and Apoptosis by Simultaneous Disruption of Binding to MDM2 and MDMX. Cancer Research 67 8810 8817. Hu, B., Gilkes, D. M., Farooqi, B., Sebti, S. M., and Chen, J. (2006). M DMX overexpression prevents p53 activation by the MDM2 inhibitor nutlin. Journal of Biological Chemistry 281 33030 33035. Humphrey, J. M., and Chamberlin, A. R. (1997). Chemical Synthesis of Natural Product Peptides: Coupling Methods for the Incorporatio n of Noncoded Amino Acids into Peptides. Chemical Reviews (Washington, D C) 97 2243 2266. Kitamura, S., Fukushi, H., Miyawaki, T., Kawamura, M., Konishi, N., Terashita, Z. i., and Naka, T. (2001). Potent Dibasic GPIIb/IIIa Antagonists with Reduced Prolon gation of Bleeding Time: Synthesis and Pharmacological Evaluation of 2 Oxopiperazine Derivatives. Journal of Medicinal Chemistry 44 2438 2450.

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85 Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science (Washington, D C) 274 948 953. Maity, P., and Koenig, B. (2008). Synthesis and structure of 1,4 dipiperazino benzenes: Chiral terphenyl type peptide helix mimetics. Organic Letters 10 1473 1476. Montalbetti, C. A. G. N., and Falque, V. (2005). Amide bond formation and peptide coupling. Tetrahedron 61 10827 10852. Nam, J., Won, N., Jin, H., Chung, H., and Kim, S. (2009). pH Induced Aggregation of Gold Na noparticles for Photothermal Cancer Therapy. Journal of the American Chemical Society 131 13639 13645. Paul, R., and Anderson, G. W. (1960). N,N' Carbonyldiimidazole, a new peptide forming reagent. Journal of the American Chemical Society 82 4596 4600. Phan, J., Li, Z., Kasprzak, A., Li, B., Sebti, S., Guida, W., Schoenbrunn, E., and Chen, J. Structure based Design of High Affinity Peptides Inhibiting the Interaction of p53 with MDM2 and MDMX. Journal of Biological Chemistry 285 2174 2183. Santagada, V., Fiorino, F., Perissutti, E., Severino, B., De Filippis, V., Vivenzio, B., and Caliendo, G. (2001). Microwave enhanced solution coupling of the alpha ,alpha dialkyl amino acid, Aib. Tetrahedron Letters 42 5171 5173. Shangary, S., and Wang, S. (2009). Small molecule inhibitors of the MDM2 p53 protein protein interaction to reactivate p53 function: a novel approach for cancer therapy. Annual Review of Pharmacology and Toxicology 49 223 241. Sobolev, R. N., Chung, H., Tuan, D. C., Ryakhovskii, V. M., a nd Starostin, G. M. (1991). Evolution of the chemical composition of granitoid complexes in the southeastern Indosinian Massif (southern Vietnam). Tikhookean Geol, 50 58. Vassilev, L. T., Vu, B. T., Gr aves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C. et al. (2004). In Vivo Activation of the p53 Pathway by Small Molecule Antagonists of MDM2. Science (Washington, DC, United States) 303 844 848. Volonteri o, A., Moisan, L., and Rebek, J., Jr. (2007). Synthesis of pyridazine based scaffolds as alpha helix mimetics. Organic Letters 9 3733 3736. Yin, H., Lee, G. i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005). Terph enyl based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44 2704 2707.

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86 Zapf, C. W., Del Valle, J. R., and Goodman, M. (2005). Utilizing the intramolecular Fukuyama Mitsunobu reaction for a flexible synthe sis of novel heterocyclic scaffolds for peptidomimetic drug design. Bioorganic & Medicinal Chemistry Letters 15 4033 4036. Zhang, X., Breslav, M., Grimm, J., Guan, K., Huang, A., Liu, F., Maryanoff, C. A., Palmer, D., Patel, M., Qian, Y. et al. (2002). A New Procedure for Preparation of Carboxylic Acid Hydrazides. Journal of Organic Chemistry 67 9471 9474.

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87 Chapter Three : Design and Synthesis of the Hybrid 2,5 Piperazine Dione Pyrimidine Based Scaffold 3.1 Introduction 3.1.1 Pyrimidines Pyrimidines are six membered rings that display a similar structure to benzene but differing in there containing nitrogen atoms in the 1 and 3 positions of the ring (Figure 3.1). Figure 3.1: Structures of ( A ) Benzene ( B ) Pyrimidine Pyrimidines are an important class of heterocyclic aromatic compounds in the realm of life sciences du e to their unique chemical properties. (Parks et al., 2008) They have electron deficient aromatic systems (Haridas, 2009; Parks et al., 2008) and the pyrimidine structure displays a greater tortional flexibility when compared to similar compounds with a benzyl core, contributing to their high binding affinity. (Haridas, 2009) Pyrimidine analogs are a pt to functionalization and synthesis most commonly involves cyclocondensation reactions with amidine, guanidine or thiourea derivatives. (Parks et al., 2008; Samb et al., 2009) There has been documented synthesis of functionalized pyrimidines by conventional heating solution phase, microwave heating, and solid phase

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88 synthesis.(Kumar et al., 2002; Luo et al., 2002; Samb et al., 2009) Pyrimidine core scaffolds, bearing multiple functionalities, and substituted amino pyrimidines are prevalent in medicinal chemistry and commonly found within marketed drugs, as shown in Figure 3.2. Figure 3.2: Marketed drugs with pyrimidine sub units highlighted in blue Scaffolds, based on a combination of functionalize d piperazines and pyrimidines, have show drug like applications (Figure 3.3) as potent FLT3 tyrosine kinase inhibitors (Figure 3.3, C ) (Gaul et al., 2007) potential antagonists of protein/protein interact ions (Figure 3.3, A ) (Volonterio et al., 2007) and potent do pamine D3 receptor antagonists (Figure 3.3, B ) (Geneste et al., 2006)

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89 Figure 3.3: Scaffolds containing piperazine and pyrimidine units circled in blue ( A ) Rebek based scaffold as antagonists of protein/protein interactions ( B ) H pyrimidin 2 one scaffold as do pamine D3 receptor antagonists ( C ) amino 6 piperazin 1 yl pyrimidine 5 carbaldehyde oximes as FLT3 tyrosine kinase inhi bitors 3.1.2 Hybrid Scaffold Design For this project, we focused on the design, synthesis and biological evaluation of helix mimetics based on a hybrid scaffold comprised of functionalized piperazine or 2,5 DKP, and pyrimidine units, as depicted in Figure 3.4. Figure 3.4: Hybrid piperazine/pyrimidne scaffold This scaffold was designed to mimic the MDM2 binding domain of the p53 peptide, utilizing the same Ha milton design methodology, as previously described in

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90 Chapter 1.5 Our scaffold design replaces the hydrophobic phenyl rings, used in the traditional Hamilton scaffold, with functionalized piperazine and 2,5 pyrimidin e units. This novel design was thought to increase desired drug like properties of the paren t terphenyl scaffold with the addition of polar functional groups while still maintaining the desired spacial arrangement of side chains in the ith ith +4,and ith +7 positions, as shown in Figure 3.5 (Cummings and Hamilton, 2010) Figure 3.5: Schematic representation s ( A ) a natu helix with the i 1, i i + 4, i + 7, and i + 11 residues, ( B ) 2D hybrid piperazine/pyrimidne scaffold, ( C ) 3D hybrid piperazine/pyrimidne scaffold. There are many documented facile syntheses of substituted pyrimidines. (Agarwal et al., 2005; Cherng, 2002; Kumar et al., 2002; Luo et al., 2002) The addition of the facile pyrimidine chemistry coup led with the previously studied synthetic chemistry of substituted piperazines and DKPs (Chapter 2) was hoped to improve the overall ease of synthesis of the scaffold.

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91 3.1.3 Molecular Modeling Studies In efforts to validate potential novel inhibitors of p53/MDM2 binding interactions in a virtual screening mode, we conducted computational simulations wit h Shrodeinger software of variations of the proposed target hybrid scaffold (Figure 3.4) docked into the hydrophobic pocket in the NH 2 terminal domain of the MDM2 protein derived from the X ray crystal structure of p53 bound to the hydrophobic pocket of MD M2 (PDB 1YCQ). (Kussie et al., 1996) Computational analysis of the proposed structures wer e ranked based on the docking GLIDE score and the top scoring structures were then synthesized. Mol ecular modeling of the top GLIDE scoring structure of hybrid scaffold 3.38 (shown in F igure 3.6) suggested that the hybrid scaffold targeted the same surface area of MDM2 where p53 binds, inserting the side chains into the desired F19, W23, and L26 binding pockets, as shown in Figure 3.7. Figure 3.6: Top sc oring hybrid piperazine/pyrimidne scaffold 3.38

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92 Figure 3.7: Molecular docking of the hybrid piperazine/pyrimidine scaffold Displayed are the three aromatic side chains in the key F19, W23, and L26 pockets in the MDM2 hydrophobic grove ( image by Courtney DuBoulay) Further analysis of the hybrid scaffold 3.38 overlayed with the p53 protein (Figure 3.8) displayed numerous hydrophobic interactions with the MDM2 protein, as shown in Figure 3.7. The benzyl group in the R 1 place positions the ith gro up for hydrophobic interactions with Y100, and the benzyl group in the R 2 ith +4, position displayed hydrophobic interactions with V93, L5 4, and V93. The third position benzyl group in the R 3 ith +7, position displayed hydrophobic interactions with M62 and V93. The molecule displayed overall hydrophobic interactions with L54, L57, F55, G58, M62, F91,V93, I99, and I61 which are all reported to be located within the p53 binding pocket. (Kussie et al., 1996)

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93 Figure 3.8: Hydrophobic interactions of the top scoring hybrid scaffold 3.38 Figure 3.9 : Overlay of the p53 protein (transparent light blue) with the proposed hybrid piperazine/pyrimidine scaffold docked to the MDM2 hydrophobic pocket ( image by Courtney DuBoulay) The observed hydrophobic interactions at these positions were also observed in the binding of the p53 peptide and MDM2 (Kussie et al., 1996) demonstrating that hybrid scaffold 3.38 recognizes the target MDM2 protein surface and adopts a similar confirmation to the p53 peptide for its side chains.

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94 3.2 Results and Discussion 3.2.1 Hybrid Piperazine/ Pyrimi dine Scaffold, Approach 1 The key component of this approach, depicted in Scheme 3.1, was the coupling of monomer A 1 (Chapter 2) and monomer A 2 (Scheme 3.2) by sulfur displacement to afford the target hybrid scaffold A 1 A 2 Scheme 3.1: Retrosynthesis of the Hybrid 2,5 DKP Pyrimidine Scaffold, Approach 1 The synthesis 2,5 DKP unit A 1 depicted in Scheme 3.1, is described in detail in Chapter 4, Scheme 4.10 and 4.12. The synthesis of the A 2 pyrimidine unit (Scheme 3.1) b cyanoketones 3.1a c which were sy nthesized in house by other lab members. (Zhou e t al.) Compounds 3.1a c underwent microwave assisted condensation with commercially available 2 methyl 2 thiopseudourea hemisulfate salt in the presence of sodium ethoxide (NaOEt) and methanol to afford pyrimidines 3.2a c as the pure products in high yi elds. (Thomas et al., 2004) Converse ly, no reaction was observed when the reaction was carried out in DMF and yields were

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95 comparable when carried out in ethanol (data not shown). Compounds 3.2a c were subsequently oxidized with meta chloroperoxybenzoic acid (mCPBA), yielding the target A 2 un it, compounds 3.3a b (Thomas et al., 2004) Scheme 3.2: Synthesis of pyrimidine unit A 2 compounds 3.3a b Coupling of piperazine dione 4.28b with pyrimidine 3.3a depicted in Scheme 3.3, was attempted initially with sodium hydride (NaH) in THF or DMF, at various temperatures. The attempt in the aprotic solvent THF resulted in no reaction occurring while attempts using DMF resulted in the formation of undesired elimination product 3.5 Additional reacti ons were performed utilizing alternate bases, solvents, and temperatures in hopes of obtaining the desired substitution product 3.4 (Scheme 3.3). Unfortunately these modifications were not successful and all attempts resulted in no reaction occurring with all starting material still present as shown by TLC and NMR.

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96 Entry Reagents Conditions Results 1 NaH THF, 12 h at rt, 18 h at 40 C No rxn 2 NaH DMF, 100 C, 6 h 3.5 8% 3 NaH DMF, 100 C, 24 h 3.5 10% 4 TEA THF, reflux, 24 h No rxn 5 TEA THF, MW, 120 C, 30 min No rxn 6 DIEA DMF, MW, 70 C, 30 min No rxn 7 DIEA 1 propanol, 105 C, 2 h No rxn 8 DIEA THF, MW, 115 C, 20 min No rxn Scheme 3.3: Attempted formation of compound 3.4 It was reasoned that the amide nitrogen of compound 4.28b was unable to act as an effective nucleophile. Being a better nucleophile than 4.28b compound 4.29b was used in the attempted coupling to compound 3.3a as shown in Scheme 3.4. Again, all attempt s were unsuccessful at forming compound 3.6 and resulted in no reaction (Scheme 3.4).

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97 Entry Reagents Conditions 1 TEA THF, reflux, 24 h 2 TEA THF, MW, 120 C, 30 min 3 DIEA DMF, MW, 70 C, 30 min 4 DIEA 1 propanol, 105 C, 2 h Scheme 3.4: Attempted formation of compound 3.6 Model reactions were performed to assess whether the nucleophilcity of the amide nitrogen, the poor reactivity of the pyrimidine monomer toward nucleophilic aromatic substitution, or a combination of both, were preventing reaction success. As shown in Sch eme 3.5, the indole nitrogen of compound 3.7 was unable to displace the sulfone functional group in pyrimidine 3.3a or the notably more reactive chlorine substituted pyrimidine 3.9 as shown in Scheme 3.6 (Cherng, 2002; Gudmundsson, 2006; Zhang et al., 2006) Further attempts at chlorine displacement with more nucleophilic piperazine 4.29b were also unsuccessful, as shown in Scheme 3.7. (Cushing et al., 1999; Luo et al., 2002)

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98 Entry Reage nts Conditions 1 NaH THF, 0 C for 2 h, reflux for 18 h 2 NaH THF, 0 C for 10 min, reflux for 48 h 3 DIEA DMF, MW, 80 C, 10 min 4 TEA THF, MW, 130 C, 10 min 5 DIEA DMF, MW, 150 C, 40 min Scheme 3.5: Attempted indole mediated sulfone displacement Entry Reagents Conditions 1 DIEA DMF, MW, 150 C, 40 min 2 BINAP, Pd(OAc) 2 Cs 2 CO 3 1,4 dioxane, 120 C, 20 min 3 DIEA NMP, MW, 120 C, 30 min 4 DIEA 2 propanol, MW, 150 C, 3 h 5 DIEA toluene, MW, 100 C, 30 min Scheme 3.6: Attempted indole mediated chlorine displacement

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99 Entry Reagents Conditions 1 TEA toluene, reflux, 18 h 2 DIEA toluene, reflux, 48 h 3 TEA 1,4 dioxane, 80 C, 24 h Scheme 3.7: Attempted chlorine displacement These sets of data verified that neither increasing the nucleophilicity of the reacting amine or the reactivity of the leaving group was able to optimize the coupling reaction. The synthetic approach followed f or the scaffold formation by coupling two different distinct monomers was discontinued and alternate synthetic methodologies were explored for the hybrid piperazine/pyrimidine scaffold formation. 3.2.2 Hybrid Piperazine/ Pyrimidine Scaffold, Approach 2 The next approach at synthesis of the piperazine/pyrimide hybrid scaffold involved the formation of a functionalized amino pyrimidine a followed by bromine displace ment c and intramolecular cyclization to afford the target molecule A 1 A 2 as shown in Sch eme 3.8.

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100 Scheme 3.8: Retrosynthesis hybrid 2,5 DKP pyrimidine scaffold, Approach 2 nitro ketone a that would be used to form the functionalized pyrimidine c upon condensation with an appropriate guanidine b (Scheme 3.8).

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101 Scheme 3.9: General synthetic method for nitro containing pyrimidines nitro ketone (Scheme 3.9, a ), i t was initially thought that coupling commercially available ester 3.13 with nitromethane (CH 3 NO 2 ) in the presence of either potassium tert butoxide (KOBt) or potassium tert amyloxide (KO t ketonitro compound 3.14 (Scheme 3.10). This reaction scheme did not yield the desired product 3.15 and all starting material remained unreacted. (Zhang et al., 2004) Entry Reagents Conditions Results 1 KOBt, CH 3 NO 2 DMSO, 0 o C 10 min, rt 24h No rxn 2 CH 3 CHC(CH 3 ) 2 OK, CH 3 NO 2 THF, 0 o C 10 min, rt 3h No rxn Scheme 3.10: Proposed synthesis of 3.15 Attempt 1 It was anticipated that the subsequent coupling to phenyl ester 3.17 would afford improved results when compared with methyl ester 3.13 t aking into consideration that a phenoxy moiety is a better leaving group than the OMe moiety. (Zhang et al., 2004) The next attempt at the formation of compound 3.15 began with esterification of phenyl acetic acid (compound 17 Scheme 3.11). As shown in Scheme 3.12, the reaction was initially performed in the presence of phenol, N N dicyclohexylcarbodiimide (DCC), 4

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102 (dimethylamino) pyridine (DMAP), and DCM, yieldin g the target ester 3.17, although yields were not optimal and the purification was lengthy and difficult. (Yamazaki et al., 2001) Subsequent treatment with diisopropylcarbodiimide (DIC) did not improve the reaction conditions. Further treatment with ethyl dimethylamino) propylcarbodiimide hydrochloride (EDC) and 1 hydroxybenzotriazole (HOBt) or DMAP did improve conditions and optimal conditions were obtained with microwave assisted coupling in the presence of EDC, DMAP, and DIEA, providing compound 3.17 in good yield, 80%. Scheme 3.11: Proposed synthesis 3.15 Attempt 2 Entry Reagents Conditions Results 1 Phenol, DCC, DMAP DCM, rt, 18h 36% 2 Phenol, DIC, DMAP DCM, rt, 18h 30% 3 Phenol, EDC, HOBt, DIEA DCM, rt, 22h 74% 4 Phenol, EDC, DMAP, DIEA DCM, rt, 22h 70% 5 Phenol, EDC, DMAP, DIEA DCM, MW, 60 o C, 20 min 80% Scheme 3.12: Formation of ester 3.17 Subsequent attempts at the coupling of compound 3.17 with nitromethane to yield compound 3.14 were inefficient, low yielding (17 18%), and difficult to purify, as shown

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103 in Scheme 3.13. (Zhang et al., 2004) These results were not optimal for the synthesis of starting materials, therefore an additional route for the formation of compound 3.15 was explored. Entry Reagents Conditions Results 1 KOBt, CH 3 NO 2 DMSO, rt, 18h 17% 2 KOBt, CH 3 NO 2 DMSO, rt, 48h 18% 3 KOBt, CH 3 NO 2 K 2 CO 3 DMSO, rt, 24h Traces 4 KOBt, CH 3 NO 2 K 2 CO 3 DMSO, rt, 48h Traces Scheme 3.13: S ynthesis of 3.14 nitroketone building blocks is depicted in Scheme 3.14. Aldehydes 3.18a c underwent microwave assisted nitroaldol reactions in the presence of nitromethane and TEA, to afford the pure adducts 3.1 9a c quickly and with modest yields. (Gan et al., 2006; Ma and You, 2007) The alcohols were oxidized to the co rresponding ketones 3.20a b and 3.15 by means of pyridinium chlorochromate (PCC) and subsequent microwave assisted treatment with N,N dimethylformamide dimethyl acetal (DMF nitroketone compounds 3.21a b with mode st yields. (Bartlett et al., 1990)

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104 Scheme 3.14: Synthesis of 3.21a b Pyrimidine formation began with microwave assisted condensation of 3.21a b with guanidine hydrochloride in the presences of potassium carbonate and ethanol afforded pyrimidines 3.22a b as the pure product with high yields (Scheme 3.15). (Radwan and El Sherbiny, 2007) Compound 3.21a was also reacted with benzy l amine hydroch loride under the same microwave assisted conditions affording pyrimidine 3.23 Pyrimidine 3.23 underwent 5% Pd/C catalyzed nitro reduction yielding compound 3.24 in high yields as the pure product. Compound 3.24 subsequently underwent Cu(I) catalyzed bromine insertion, yielding pyrimidine 3.25. (Ueno et al., 2008)

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105 Scheme 3.15: Synthesis of nitro based pyrimidines X ray crystal structures were obtained for compounds 3.20a 3.21a 3.23 3.24 and 3.25 from Dr. Frank Fronczek at Lousiana State University.(Figure 3.10)

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106 Figure 3.10: Crystal structures for compounds 3.20a 3.21a 3.2 2a 3.24 and 3.25 From previous experience with the piperazine 2,5 diones, it was envisioned that compounds 2.3c could be coupled to the 5 amino substituent of pyrimidine 3.24 as shown in Scheme 3.16. Numerous reagents and reaction conditi ons were explored for the synthesis although none were successful. It was reasoned that the 5 amino substituent of pyrimidine 3.24 was not able to react as an efficient nucleophile due to the electron withdrawing effects of the pyrimidine ring.

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107 Entry Reagents Conditions 1 DIEA DMF, 110 C, 24 h 2 DIEA DMF, MW, 150 C, 30 min 3 DIEA Methanol, reflux, 24 h 4 TEA Methanol, reflux, 24 h 5 DIEA Methanol, MW, 100 C, 75 min Scheme 3.16: Attempted formation of compound 3.26 Model reactions were performed to determine whether an alternate synthetic strategy relying on the nucleophilic aromatic substitution of functionalized pyrimidines, for the formation of the hybrid piperazine/pyrimidine scaffold, could be optimized. Bromine displacement of pyrimidine 3.35 with phenyl piperazine 3.27 in the presence of DIEA and 2 ethoxy ethanol failed to provide the desired product compound 3.28 as depicted in Scheme 3.17. Cu(I) catalyzed bromine insertion of pyrimidine 3.29 was unsuc cessful (Scheme 3.18), and the coupling of pyrimidine 3.31 to N Boc phenylalanine 3.32 using a variety of reagents and conditions, as shown in Scheme 3.19, also failed. Unfortunately, these attempts were not successful in generating the desired product. This concluded the use of pyrimidine based bromine displacement as inefficient for the formation of the hybrid piperazine/pyrimidine scaffold and alternative synthetic routes were explored.

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108 Scheme 3.17: Attempted formation of 3.28 by bromine displacement Scheme 3.18: Attempted formation of 3.30 by bromine insertion

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109 Entry Reagents Conditions 1 HOBt, DIC DCM, rt, 24 h 2 DIEA, Isobutyl chloroformate Toluene, 0 C to rt for 1 h, rt, 24 h 3 HOBt, EDC DCM, rt, 4 h, 30 C, 18 h Scheme 3.19: Attempted amine coupling to compound 3.31 3.2.3 Hybrid Piperazine/Pyrimidine Scaffold, Approach 3 Our next approach towards the formation of the hybrd piperazine/pyrimidine scaffold, began with the synthesis of a functionalized piperazine a followed by guanidinylation b and subsequent condensation to afford the target scaffold A 1 A 2 as shown in Sche me 3.20

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110 Scheme 3.20: Retrosynthesis hybrid piperazine/pyrimidine scaffold, Approach 3 This synthetic route, as shown in Schemes 3.21 22, began by reacting previously synthesized piperazine monomeric unit, 2.32b ,with silver triflate in the presence of an N,N diboc pseudo guanidinylating agent 3.35 (synthesis shown in Scheme 3.21) to afford compound 3.36 (Delle Monache et al., 1993; DeMong and William s, 2003) Reactions for this step were also carried out using barium chloride, mercury chloride, and THF as the solvent. (Agarwal et al., 2005; Andrews et al., 1949; Powell et al., 2003) While the desired product, compound 3.35 was synthes ied, the isolation was difficult and the yields were low (20 30%, data not shown). N Boc deprotection of 3.36 was achieved using

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111 standard TFA/DCM conditions, producing compound 3.37 Comp ound 3.37 underwent condensation with compound 3.1b to afford the target scaffold 3.38 Scheme 3.21: Synthesis of N,N diboc pseudo guanidinylating agent 3.35 Scheme 3.22: Successful synthesis of hybrid scaffold 3.38

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112 3.2.4 Fluorescence Polarization Assays Scaffold 3.38 underwent biological testing by means of fluorescence polarization assays to determine if the com pound did disrupt p53/MDM2 or Bcl xL /Bax binding interaction s. An IC 50 > 100 M was obtained for both assays which was not a desirable value for a potential drug candidate. 3.3 Conclusion Although successful, the synthesis of hybrid scaffold 3.38 was lengthy, low yielding, and hard to reproduce. The difficult synthesis coupled with poor biological data encouraged us to put this scaffold synthesis on hold and explore alternative options 3.4 Experimental Procedures 4 tert Butyl 2 methylsulfanyl pyrimidine 5 carbonitrile (3.2a) A mixture of 3.1a (1.11 mmol), sodium ethoxide (1.11 mmol), and 2 methyl 2 thiopseudourea (2.22 mmol) in anhydrous methanol (3 mL) was heated in a Biotage mi crowave at 70 C for 15 minutes. After cooling to ambient temperature, the solution was diluted with 100 mL DI H 2 O, 50 mL of saturated KHSO 4 and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (2 x 30 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel (1:10 EtOAc/hexanes) to afford 3.2a as a light yellow oil (0.68 mmol, 65%). 1 H NMR (400 MHz, CDCl 3 8.61 (s, 1H), 2.586 (s, 3H), 1.487 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ) 178.84 (1C),175.78 (1C), 161.66 (1C), 117.12

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113 (1C), 101.10 (1C), 40.08 (1C), 28.63 (3C), 14.58 (1C). HRMS (ESI) calc. for C 10 H 13 N 3 S (M + H) + 207.0839, found 207.0722. 4 Benzyl 2 methy lsulfanyl pyrimidine 5 carbonitrile (3.2b) This was obtained from 3.1b (0.93 mmol) in a similar manner as described for the preparation of 3.2a The crude product was purified by flash chromatography on silica gel (1:10 EtOAc/hexanes) to afford 3.2b as a light yellow solid (0.84 mmol, 84%). 1 H NMR (400 MHz, CDCl 3 ) 8.60 (s, 1H), 7.42 7.23 (m, 5H), 4.22 (s, 2H), 2.57 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) 176.98 (1C), 171.27 (1C), 159.98 (1C), 135.77 (1C), 129.50 (2C), 129.05 (2C), 127.63 (1C), 115.60 (1C), 102.96 (1C), 29.91 (1C), 14.66 (1C). HRMS (ESI) calc. for C 13 H 11 N 3 S (M + H) + 241.0673, found 241.0747. 2 Methylsulfanyl 4 naphthalen 1 ylmethyl pyrimidine 5 carbonitrile (3.2c) This was obtained from 3.1c (0.378 mmol) in a similar manner as described for the preparation of 3.2a The crude product was purified by flash chromatography on silica gel (1:10 EtOAc/hexanes) to afford 3.2c as a light yellow oil (0.21 mmol, 56%). 1 H NMR (400 MHz CDCl 3 8.62 (s, 1H), 8.26 (d, J = 8.13 Hz, 1H), 7.89 7.78 (m, 2H), 7.55 7.42 (m, 3H), 4. 69 (s, 2H), 2.40 (s, 3H). 13 C NMR (100 MHz, CDCl 3 176.83 (1C), 171.12 (1C), 160.09 (1C), 134.16 (1C), 132.18 (1C), 132.04 (1C), 128.99 (1C),

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114 128.64 (1C), 126.49 (1C), 126.09 (1C), 125.72 (2C), 124.67 (1C), 115.74 (1C), 103.16 (1C), 29.93 (1C), 14.53 (1 C). HRMS (ESI) calc. for C 17 H 13 N 3 S (M + H) + 291.0630, found 291.0902. 4 tert Butyl 2 methanesulfonyl pyrimidine 5 carbonitrile (3.3a) To a stirred and cooled (0 C) solution of compound 3.2a (1.78 mmol) in anhydrous DCM (25 mL) was added m CPBA (7.18 mmol) and the mixture was stirred at 0 C for 4 hours. The mixture was brought to ambient temperatures, treated with 10% Na 2 SO 3 (6 mL), and subsequently filtered. The filtrate was washed with 10 % aqueous NaHCO 3 (2 x 30 mL), brine (2 x 35 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using the Flash Master 3 purification station (3:7 EtOAc/hexanes), affording 3.3a as a w hite solid (1.21 mmol, 68%). 1 H NMR (400 MHz CDCl 3 9 .07 (s, 1H), 3.39 (s, 3H), 1.576 (s, 9H). 13 C NMR (100 MHz CDCl 3 182.67 (1C), 166.15 (1C), 163.61 (1C), 114.84 (1C), 109.63 (1C), 40.60 (1C), 39.17 (1C), 28.63 (3C). HRMS (ESI) calc. for C 10 H 13 N 3 O 2 S (M + H) + 239.0728, found 239.0629. 4 Benzyl 2 methanesulfonyl pyrimidine 5 carbonitrile (3.3b) This was obtained from 3.2b (0.306 mmol) in a similar manner as described for the preparation of 3.3a The crude product was purified by chromatography on silica gel

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115 using the Flash Master 3 purification station (3:7 EtOAc/hexanes), to afford 3.3b as a light yellow solid (0.307 mmol, 42%). 1 H NMR (400 MHz CDCl 3 8.98 (s, 1H), 7.34 7.18 (m, 5H), 4.37 (s, 2H), 3.30 (s, 3H). 13 C NMR (100 MHz, CDCl 3 174.77 (1C), 167.09 (1C), 161.99 (1C), 134.44 (1C), 129.51 (2C), 129.45 (2C), 43.20 (1C), 39.22 (1C). MS (ESI) m/z 274.06 (M + H) + HRMS (ESI) calc. for C 1 3 H 1 1 N 3 O 2 S (M + H) + 273.0572 found 273.0658 phenyl 2 phenylacetate (3.17) To phenyl acetic acid (3.67 mmol), in anhydrous DCM (8 mL), under argon, was added EDC (4.04 mmol) and HOBt (4.04 mmol). The reaction was chilled to 0 o C and phenol (5.51 mmol) in DCM (2 mL) was added via a syringe. The reaction was brought to ambient temperatures and stirred for an additional 22 hours. The reaction mixture was washed with 1 N HCl (1 x 30 mL), brine (2 x 30 mL), dried over Na 2 SO 4 and concentrated in vacuo to afford the cru de oil The crude product was purified by chromatography on silica gel (1:10 EtOAc/hexanes), affording compound 3.17 as a clear oil (2.36 mmol, 78%). 1 H NMR (400 MHz, CDCl 3 7.30 (m, 7H), 7.26 7.20 (m, 1H), 7.11 7.04 (m, 2H), 3.88 (s, 2H). 13 C NMR (101 MHz, CDCl 3 150.97 (1C), 133.71 (1C), 129.62 (2C), 129.55 (2C), 128.96 (2C), 127.58 (1C), 126.09 (1C), 121.68 (2C), 41.68 (1C). MS (ESI) m/z 213.1 (M + H) +

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116 2 nitro 1 phenylethanol (3.19a) A mixture of 3.18a (30 mmol), TEA (45 mmol) in nitromethane (8 mL) was heated in a Biotage microwave at 80 C for 4 minutes. After cooling to ambient temperature, the solution was diluted with DI H 2 O (125 mL), saturated KHSO 4 (20 mL) and subsequently extracted with EtOAc (3 x 35 mL). The combined organic layers were washed with brine (2 x 50 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using t he Flash Master 3 purification station (15:85 EtOAc/hexanes) to afford 3.19a (22.1 mmol, 56%) as a clear oil. 1 H NMR (400 MHz, CDC l 3 7.52 7.24 (m, 5H), 5.36 (dd, J = 9.6, 2.9 Hz, 1H), 4.46 (ddd, J = 16.4, 13.3, 6.3 Hz, 2H), 2.93 (bs, 1H). 13 C NMR (1 01 MHz CDCl 3 138.38 (1H), 132.41 (1C), 129.25 (2C), 129.15 (2C), 126.18 (1C), 81.45 (1C), 71.23 (1C). MS (ESI) m/z 166.1 (M H) 1 nitro 3 phenylpropan 2 ol (3.19b) The product was obtained from 3.18b (8.56 mmol) in a similar manner as described for the preparation of 3.19a The crude product was purified by chromatography on silica gel using the Flash Master 3 purification station (1:1 EtOAc/hexanes) to afford 3.19b (5.30 mmol, 62%) as a clear oil. 1 H NMR (400 MHz, CDCl 3 7.38 7.26 (m, 3H), 7.25 7.20 (m, 2H), 4.60 4.49 (m, 1H), 4.44 4.34 (m, 2H), 2.84 (ddd, J = 28.2, 13.6, 6.1 Hz, 3H). MS (ESI) m/z 180.1 (M H)

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117 4 methyl 1 nitropentan 2 ol (3.19c) T he product was obtained from 3.18c (5.81 mmol) in a similar manner as described for the preparation of 3.19a The crude product was purified by chromatography on silica gel using the Flash Master 3 purification station (1:4 EtOAc/hexanes) to afford 3.19c (4.99 mmol, 86%) as a clear oil. 1 H NMR (400 MHz, CDCl 3 4.30 (m, 3H), 2.87 (bs, 1H), 1.86 1.74 (m, 1H), 1.52 1.42 (m, 1H), 1.25 1.16 (m, 1H), 0.94 0.91 (m, 6H). MS (ESI) m/z 146.1 (M H) 2 nitro 1 phenylethanone (3.20a) To a stirring mixture of 3.19a (6.05 mmol) in DCM (200 mL), was added PCC (15.11 mmol) in one portion, and stirred at ambient temperature for 36 hours. The resulting dark brown solution was filtered over silica and washed with additional DCM (800 mL). The combined organic solutions we re concentrated in vacuo triturated with chilled hexanes, and the precipitate was filtered yielding a crude yellow solid. The resulting yellow crystalline solid was recrystalized in DCM/hexanes (10:1) yielding compound 3.20a (5.39 mmol, 90%) as pale yell ow crystals. The structural confirmation of 3.20a was obtained by single crystal X ray diffraction 1 H NMR (400 MHz, CDCl 3 J = 8.4, 1.2 Hz, 2H), 7.72 7.66 (m, 1H), 7.64 7.48 (m, 2H), 5.91 (s, 2H). 13 C NMR (101 MHz, CDCl 3 135.33 (1C), 129.55 (2C), 128.47 (2C), 127.10 (1C), 81.55 (1C). MS (ESI) m/z 166.2 (M + H) +

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118 4 methyl 1 nitropentan 2 one (3.20b) The product was obtained from 3.19c (5 mmol) in a similar manner as described for t he preparation of 3.20a yielding 3.20b (3.70 mmol, 74%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl 3 J = 6.9 Hz, 2H), 2.25 2.04 (m, 1H), 0.95 (dd, J = 6.6, 0.7 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 49.30 (1C), 24.63 (1C), 22.49 (2C). MS (ESI) m/z 146.2 (M + H) + 1 nitro 3 phenylpropan 2 one (3.15) The product was obtained from 3.19b (8.56 mmol) in a similar manner as described for the preparation of 3.20a yieldin g 3.15 (4.28 mmol, 50%) as a dark yellow oil. 13 C NMR (101 MHz, CD 3 83.39 (1C), 41.58 (1C). MS (ESI) m/z 180.1 (M + H) + (Z) 3 (dimethylamino) 2 nitro 1 phenylprop 2 en 1 one (3.21a) To a mixture of 3.20a (5.41 mmol) in anhydrous toluene (5 mL), was added N,N dimethylformamide dimethylacetal (1.58 mmol) and heated in a Biotage microwave at 70 C for 10 minutes. The excess solvent was removed in vacuo and was co evaporated with MeOH (2 x 30 mL). The resulting orange oil was purified by chromatography on silica gel using the Flash Master 3 purification station (1:1 EtOAc/Hexanes) to afford 3 .21a (47%, 2.52 mmol) as a pale yellow crystals. The structural confirmation of 3.21a was obtained by single crystal X ray diffraction 1 H NMR (400 MHz, CDCl 3 1H), 7.83 (dd, J = 5.2, 3.3 Hz, 2H), 7.58 7.50 (m, 1H), 7.45 (t, J = 7.5 Hz, 2H), 3.37 (s,

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119 3H), 2.78 (s, 3H). 13 C NMR (101 MHz, CDCl 3 (1C), 133.12 (1C), 128.86 (4C), 48.36 (1C), 42.40 (1C). MS (ESI) m/z 221.2 (M + H) + 5 nitro 4 phenylpyrimidin 2 amine (3.22a) To 0.5 2.0 mL microwave vial was added 3.21a (1.4 mmol), guanidine hydrochloride (2.1 mmol), K 2 CO 3 (2.52 mmol), EtOH (1 mL), and heated in a Biotage microwave at 135 C for 30 minutes. The solution was cooled to room temperature, decanted out of the microw ave vial, diluted with DCM (5 mL), and the remaining solution was removed in vacuo The resulting yellow solid was recrystallized in boiling EtOH affording compound 3.22a (71%, 0.99 mmol). The structural confirmation of 3.22a was obtained by single cryst al X ray diffraction 1 H NMR (400 MHz, CDCl 3 7.42 (m, 5H), 5.85 (s, 2H). 13 C NMR (101 MHz, CDCl 3 157.04 (1C), 135.59 (1C), 130.90 (1C), 128.77 (2C), 128.18 (2C). HRMS (ESI) calc. for C 1 0 H 8 N 4 O 2 (M + H) + 216.0647 found 216.0845 5 nitro 2,4 diphenylpyrimidine (3.23) This was obtained from benzamin dine hydrochloride hydrate (1.74 mmol) and compound 3.21a (1.16 mmol) in a similar manner as described for preparation of 3. 22a The crude product was recrystallized in boiling EtOH to afford 3.23 as clear yellow crystals (0.92 mmol, 80%). The structural confirmation of 3.23 was obtained by single crystal X ray

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120 diffracti on 1 H NMR (400 MHz, CDCl 3 8.56 (m, 2H), 7.77 7.72 (m, 2H), 7.62 7.51 (m, 6H). 13 C NMR (101 MHz, CDCl 3 153.85 (1C), 135.92 (1C), 134.55 (1C), 132.66 (2C), 131.61 (2C), 129.62 (2C), 129.09 (2C), 128.86 (2C). MS (ESI) m/z 278.09 (M + H) + HRMS (ESI) calc. for C 1 6 H 1 1 N 3 O 2 (M + H) + 277.0851 found 277.0911 2,4 diphenylpyrimidin 5 amine (3.24) A solution of 3.23 (0.11 mmol) in CH 3 CN (3 mL) underwent hydrogenation in a ThalesNano H Cube Tutor Hydrogenation Reactor, in t he presence of a 5% Pd/C cartridge, at ambient temperatures, 30 bar pressure, and a flow rate of 1 mL/min. This yielded the pure compound 3.24 (0.093 mmol, 85%) as clear crystals. The structural confirmation of 3.24 was obtained by single crystal X ray d iffraction 1 H NMR (400 MHz, CD 3 8.23 (m, 2H), 7.88 7.83 (m, 2H), 7.57 7.34 (m, 6H). 13 C NMR (101 MHz, CD 3 (1C), 138.02 (1C), 137.09 (1C), 129.24 (1C), 128.86 (1C), 128.68 (2C), 128.23 (2C), 128.19 (2C), 126.84 (2C). HRMS (ESI) calc. for C 1 6 H 1 3 N 3 (M + H) + 247.1109 found 247.1173

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121 5 bromo 2,4 diphenylpyri midine (3.25) To an ice bath chilled solution of compound 3.24 (1.14 mmol) in acetic acid (2 mL) was slowly added NaNO 2 (2.27 mmol) dissolved in conc. H 2 SO 4 (0.5 mL) via an addition funnel. The reaction was stirred at 0 o C for 30 min warmed to ambient temperature followed by the addition of Cu(I)Br (2.27 mmol) in 47% aq. HBr (2.3 mL). The solution was subsequently heated to 80 o C for 14 hours, brought to ambient temperature, then chilled to 0 o C. Following cooling, th e reaction mixture was diluted with EtOAc (45 mL), neutralized with solid NaHCO 3 (300 mg) and saturated aqueous NaHCO 3 (10 mL). The aqueous phase was extracted with EtOAc (3 x 35 mL), the combined organic layers were washed with sat. aq. NaHCO 3 (2 x 25 mL ), brine (2 x 50 mL), dried over Na 2 SO 4 and the residual solvent was removed in vacuo The resulting yellow/orange solid was recrystallized in DCM/Hexanes (10:1) yielding compound 3.25 (1.1 mmol, 60%) as clear yellow crystals. The structural confirmation of 3.25 was obtained by single crystal X ray diffraction 1 H NMR (400 MHz, CDCl 3 8.37 (m, 2H), 7.89 7.81 (m, 2H), 7.50 7.36 (m, 6H). 13 C NMR (101 MHz, CDCl 3 160.59 (1C), 137.53 (1C), 136.84 (1C), 131.28 (1C), 130.39 (1C), 129.72 (2C), 128.84 (2C), 128.52 (2C), 128.40 (2C), 116.81 (1C). HRMS (ESI) calc. for C 1 6 H 1 1 Br N 2 (M + H) + 310.0106 found 310.0209

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122 N N' Bis ( tert butoxycarbonyl) S methylisothiourea (3.35) To a stirring mixture of 2 methyl 2 thiopseudourea hemisulfate salt (7.19 mmol) in DCM (14 mL) and saturated aq. NaHCO 3 (12 mL), was added Boc anhydride (14.39 mmol) and stirred at ambient temperatures for 18 hours. The aq ueous layer was extracted with DCM (3 x 40 mL) and the combined organic layers were washed with brine (2 x 50 mL), dried (Na 2 SO 4 ), and concentrated in vacuo to afford the crude oil. The resulting oil was purified by chromatography on silica gel using the Flash Master 3 purification station (5:95 EtOAc/Hexanes) to afford 3.35 (4.27 mmol, 60%) as a white gel 1 H NMR (400 MHz, CDCl 3 13 C NMR (101 MHz, CDCl 3 171.65 (1C), 160.98 (1C), 151.00 (1C), 83.46 (1C), 81. 19 (1C), 28.25 (6C), 14.63 (1C). HRMS (ESI) calc. for C 1 2 H 22 N 2 O 4 S (M + H) + 290.1300 found 290.1382 (S) tert butyl (((tert butoxycarbonyl)amino)(2,4 dibenzylpiperazin 1 yl)methylene)carbamate (3.36) To a solution of 4.33b (0.751 mmol) and 3.35 (0.976 mmol) in anhydrous DMF (4 mL), under argon, was added TEA (2.25 mmol), and stirred at ambient temperatures for 10 minutes. Following this was the addition of silver triflate (1.05 mmol) and the reaction was stirred for an add itional 6 hours at ambient temperatures. The solution was diluted with EtOAc (15 mL) and filtered over a short pad of diatomaceous earth The filtrate of organic solution was washed with brine (2 x 50 mL), dried (Na 2 SO 4 ), and reduced in

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123 vacuo to provide the crude oil which underwent purification by chromatography on silica gel using the Flash Master 3 purification station (20:80 EtOAc/hexanes) to afford 3.36 (0.56 mmol, 78%) as a light yellow oil 1 H NMR (400 MHz, CDCl 3 7.24 (m, 5H), 7.14 (d, J = 6.1 Hz, 3H), 7.07 (s, 2H),3.74 (s, 2H), 3.39 (m, 3H), 2.92 2.82 (m, 2H), 2.65 (d, J = 11.6 Hz, 1H), 2.18 (m, 2H), 2.04 (s, 1H), 1.48 (s, 18H). 13 C NMR (101 MHz, CDCl 3 (2C), 12 8.50 (2C), 128.48 (2C), 127.43 (1C), 126.43 (1C), 79.69 (1C), 63.17 (1C), 62.91 (1C), 60.60 (1C), 53.19 (1C), 53.00 (1C), 36.45 (1C), 31.81 (1C), 28.39 (6C). MS (ESI) m/z 509.31 (M + H) + MS (ESI) m/z 509.3 (M + H) + (S) 4 benzyl 2 (2,4 dibenzylpiperaz in 1 yl)pyrimidine 5 carbonitrile (3.38) To a stirring mixture of 3.37 (0.38 mmol) in EtOH (4 mL) was added 3.1b (0.38 mmol) and TEA (0.77 mmol). The solution was heated to a gentle reflux for 18 hours. After cooling to ambient temperatures, the solution was reduced in vacuo and purified by chromatography on silica gel using the Flash Master 3 purification station (20:80 EtOAc/Hexanes) to afford 3.38 (0.033 mmol, 8%) as a white gel. 1 H NMR (400 MHz, CDCl 3 7.15 (m, 12H), 7.05 6.99 (m, 3H), 4.86 4.78 (m, 1H), 4.59 (dd, J = 29.5, 13.1 Hz, 1H), 4.08 3.95 (m, 2H), 3.49 (d, J = 12.9 Hz, 1H), 3.36 3.23 (m, 2H), 3.19 3.07 (m, 1H), 2.91 (d, J = 11.2 Hz, 1H), 2.73 2.59 (m, 2H), 2.06 (td, J = 12.0, 3.5 Hz, 1H), 1.96 1.89 (m, 1H). 13 C NMR (101 MHz, CDCl 3

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124 (1C), 160.59 (1C), 138.99 (1C), 138.26 (1C), 136.71 (1C), 129.66 (2C), 129.60 (2C), 129.53 (2C), 128.84 (2C), 128.54 (2C), 128.47 (2C) 127.50 (1C), 127.23 (1C), 126.36 (1C), 117.63 (1C), 101.57 (1C), 63.12 (1C), 60.63 (1C), 53.30 (1C), 43.22 (1C), 40.32 (1C), 35.69 (1C), 29.86 (1C). HRMS (ESI) calc. for C 30 H 29 N 5 (M + H) + 459.2423 found 459.2426 3.6 References Agarwal, A., Srivastava, K., Puri, S. K., and Chauhan, P. M. S. (2005). Antimalarial activity and synthesis of new trisubstituted pyrimidines. Bioorganic & Medicinal Chemistry Letters 15 3130 3132. Andrews, K. J. M., Anand, N., Todd, A. R., and Topham, A. (1949). Synthesis of purine nucleosides XXVI. 9 D Glucopyranosidoisoguanine. Journal of the Chemical Society, 2490 2497. Bartlett, W. R., Johnson, W. S., Plummer, M. S., and Small, V. R., Jr. (1990). Biomimetic polyene cyclizations. Cationic cyclization of a substrate having an internal acety lenic bond. Synthesis of euphol and tirucallol. Journal of Organic Chemistry 55 2215 2224. Cherng, Y. J. (2002). Efficient nucleophilic substitution reactions of pyrimidyl and pyrazyl halides with nucleophiles under focused microwave irradiation. Tetrahedron 58 887 890. Cummings, C. G., and Hamilton, A. D. (2010). Disrupting protein protein interactions with non peptidic, small molecule alpha helix mimetics. Curr e n t Opin i o n i n Chem i c a l Biol o g y 14 341 346. Cushing, T. D., Mellon, H. L., Jaen, J. C., Flygare, J A., Miao, S. c., Chen, X., and Powers, J. P. (1999). Preparation of imidazolylpyrimidines and related compounds as antivirals. In, (Application: WO 1 9 9 9 0 8 1 9 : (Tularik Inc., USA).), p. 98 pp. Delle Monache, G., Botta, B., Delle Monache, F., Espinal, R., De Bo nnevaux, S. C., De Luca, C., Botta, M., Corelli, F., and Carmignani, M. (1993). Novel hypotensive agents from Verbesina caracasana. 2. Synthesis and pharmacology of caracasanamide. Journal of Medicinal Chemistry 36 2956 2963.

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125 DeMong, D. E., and Williams, R. M. (2003). Asymmetric Synthesis of (2S,3R) Capreomycidine and the Total Synthesis of Capreomycin IB. Journal of the American Chemical Society 125 8561 8565. Gan, C., Chen, X., Lai, G., and Wang, Z. (2006). Rapid microwave assisted Henry reaction in s olvent free processes. Synlett, 387 390. Gaul, M. D., Xu, G., Kirkpatrick, J., Ott, H., and Baumann, C. A. (2007). 4 Amino 6 piperazin 1 yl pyrimidine 5 carbaldehyde oximes as potent FLT 3 inhibitors. Bioorganic & Medicinal Chemistry Letters 17 4861 4865 Geneste, H., Backfisch, G., Braje, W., Delzer, J., Haupt, A., Hutchins, C. W., King, L. L., Kling, A., Teschendorf, H. J., Unger, L., and Wernet, W. (2006). Synthesis and SAR of highly potent and selective dopamine D3 receptor antagonists: 1H Pyrimidin 2 one derivatives. Bioorganic & Medicinal Chemistry Letters 16 490 494. Gudmundsson, K. (2006). Preparation of carbazoles and related compounds for treatment of dengue fever, yellow fever, west nile virus, and hepatitis C virus infection. In, (Applicati on: WOWO: (Smithkline Beecham Corporation, USA).), p. 59pp. Haridas, V. (2009). From Peptides to Non Peptide Alpha Helix Inducers and Mimetics. European Journal of Organic Chemistry, 5112 5128. Kumar, A., Sinha, S., and Chauhan, P. M. S. (2002). Synthese s of novel antimycobacterial combinatorial libraries of structurally diverse substituted pyrimidines by three component solid phase reactions. Bioorganic & Medicinal Chemistry Letters 12 667 669. Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Mor eau, J., Levine, A. J., and Pavletich, N. P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science (Washington, D C) 274 948 953. Luo, G., Chen, L., and Poindexter, G. S. (2002). Microwave assisted sy nthesis of aminopyrimidines. Tetrahedron Letters 43 5739 5742. Ma, K., and You, J. (2007). Rational design of sterically and electronically easily tunable chiral bisimidazolines and their applications in dual Lewis acid/Broensted base catalysis for highl y enantioselective nitroaldol (Henry) reactions. Chemistry -A European Journal 13 1863 1871. Parks, E. L., Sandford, G., Christopher, J. A., and Miller, D. D. (2008). Perhalogenated pyrimidine scaffolds. Reactions of 5 chloro 2,4,6 trifluoropyrimidine with nitrogen centered nucleophiles. Beilstein Journal of Organic Chemistry 4 No 22

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126 Powell, D. A., Ramsden, P. D., and Batey, R. A. (2003). Phase Transfer Catalyzed Alkylation of Guanidines by Alkyl Halides under Biphasic Conditions: A Conve nient Protocol for the Synthesis of Highly Functionalized Guanidines. Journal of Organic Chemistry 68 2300 2309. Radwan, M. A. A., and El Sherbiny, M. (2007). Synthesis and antitumor activity of indolylpyrimidines: Marine natural product meridianin D ana logues. Bioorganic & Medicinal Chemistry 15 1206 1211. Samb, I., Pellegrini Moise, N., Lamande Langle, S., and Chapleur, Y. (2009). Efficient functionalization of a pyranosido pyrimidine scaffold. Tetrahedron 65 896 902. Thomas, A., Balasubramanian, G. Gharat, L. A., Mohite, J. R., Lingam, V. S. P. R., Lakdawala, A. D., Karunakaran, U., and Verma, R. (2004). Preparation of condensed heterocyclic compounds, in particular indazoles and quinazolines as Phosphodiesterase IV (PDE4) inhibitors for the treatm ent of inflammatory and allergic diso rders. In, (Application: WO 2 0 0 4 1 6 5 9 6 (Glenmark Pharmaceuticals Limited, India).), p. 110 pp. Ueno, A., Kitawaki, T., and Chida, N. (2008). Total Synthesis of (+ ) Murrayazoline. Organic Letters 10 1999 2002. Volonterio, A ., Moisan, L., and Rebek, J., Jr. (2007). Synthesis of pyridazine based scaffolds as alpha helix mimetics. Organic Letters 9 3733 3736. Yamazaki, J., Watanabe, T., and Tanaka, K. (2001). Enantioselective synthesis of allenecarboxylates from phenyl aceta tes through C C bond forming reactions. Tetrahedron: Asymmetry 12 669 675. Zhang, H. Q., Xia, Z., Vasudevan, A., and Djuric, S. W. (2006). Efficient Pd catalyzed synthesis of 2 arylaminopyrimidines via microwave irradiation. Tetrahedron Letters 47 4881 4884. Zhang, N., Tomizawa, M., and Casida, J. E. (2004). alpha Nitro Ketone as an Electrophile and Nucleophile: Synthesis of 3 Substituted 2 Nitromethylenetetrahydrothiophene and tetrahydrofuran as Drosophila Nicotinic Receptor Probes. Journal of Organi c Chemistry 69 876 881. Zhou, M., Topper, M. E., Anderson, L., McLaughlin, J. M., Santiago, D. N., Fronczek, F. R., Guida, W. C., and McLaughlin, M. L. (2010). Design and synthesis of non peptidic alpha helix mimics targeting for MDM2 p53 protein prote in interaction. Abstracts of Papers, 239th ACS National Meeting, San Francisco, CA, United States.

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127 Chapter Four : Design and Synthesis of Functionalized 2,5 and 2,6 Diketopiperazines 4 .1 I ntroduction 4 .1 .1 Piperazine Diones The heterocyclic family of the diketopiperazines (DKP) including 2,6 and 2,5 DKPs has been widely studied and synthesized (Figure 4 1) (Fytas et al., 2008; Maity and Koenig, 2008; Martins and Carvalho, 2007; Oguz et al., 2002; Perrotta et al., 2001; Teixido et al., 2007) T hey have been shown to have important biological activity and a wide spectrum of pharmac eutical applications despite being the smallest cyclic peptide known (Niida et al., 2005) Figure 4 .1: Structures of DKPs ( A ) 2,6 DKP ( B ) 2,5 DKP They are known to display a variety of antitumor (Kanoh et al., 1999; Kanzaki et al., 2000; Nicholson et al., 2006) antifungal (Asano, 2003; Houston et al., 2004) antiviral (Sinha et al., 2004) and antibacterial properties (Abraham, 2005; Sugie et al., 2001) ,to name a few. (Martins and Carvalho, 2007) Di subs transports drugs across the blood brain barrier (Teixido et al., 2007) and several DKPs have significant biological activity in their affinity for the neurokinin 2 receptors. (Nefzi

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128 et al., 1997) Successful synthesis of the 2,5 and 2,6 DKPs, by both solution and solid phase chemistry, has been amply reported in the literature. (Fytas et al., 2008; Maity and Koenig, 2008; Perrotta et al., 2001; Sinha et al., 2004; Teixido et al., 2007) 4.1.2 Design of 3 R Piperazine 2,5 and 2,6 Dione Scaffold Our group initially targeted the use of hydrazine linked functionalized 2,5 and 2,6 helix mimetic scaffolds, as shown in Figure 4.2. MDM2 b inding domain of the p53 peptide, specifically the hydrophobic face of two turns helix, with key aromatic and hydrophobic residues in the ith ith+ 3, and ith+ 7 positions. This novel design was envisioned to increase desired drug like properties of the parent terphenyl scaffold with the addition of polar functional groups. The R and positions of the scaffold can be broadly diversified by altering the functional group and/or stereochemistry of the position by using the desired natural or unnat ural amino acid or amino ester as the starting material. Different starting materials can be used for each of the hydrazine linked monomers to achieve numerous possible combinations.

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129 Figure 4.2: 2,5 and 2,6 DKP based scaffold 4.2 Results and Discussion for the 3 R Piperazine 2,6 Dione Scaffold 4.2.1 Retrosynthesis of 3 R Piperazine 2,6 Dione Scaffold 3 R piperazine 2,6 diones can be prepared by solution or solid phase synthesis. A general retrosynthetic route for the hydrazine bound monomeric unit, A 1 is shown in Scheme 4.1 The amino acid methyl esters a underwent N alkylation b and hydrazine form ation c followed by formation of the cyclic anhydride d Coupling of cyclic anhydride d alanine linker derivative f and subsequent intramolecular cyclization afforded the desired hydrazine bound A 1 unit. Following N Boc deprotection, coupl ing of the A 1 unit with an additional cyclic anhydride d can form the dimeric unit A 1 A 2 which can serve as a building block for the synthesis of the target trimeric scaffold A 1 A 2 A 3 via iteration of these steps.

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130 Scheme 4.1: Retrosynthesis of the 3 R piperazine 2,6 dione scaffold

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131 4.2.2 Synthesis of Key Di acid Derivatives 4.5a c The formation of di acids 4.5a c was performed in a 3 4 step optimized synthesis as depicted in Scheme 2.2. The free amino acids 4.1a c were re acted with thionyl chloride in the presence of methanol yielding methyl ester derivatives 4.2a c as the HCl salts, in high yields. The methyl esters of standard and non standard amino acids were subsequently N alkylated with ethyl bromoacetate in the prese nce of diisopropylethylamine (DIEA), forming 4.3a d, in moderate to high yields, 52 88%. The key step of this synthetic process was the insertion of the N Boc protected hydrazine moiety. This type of reaction had been successfully performed in our lab by means of nitrosation, selective reductions, and subsequent N Boc protection. (Oguz et al., 2002) However, due to the inefficiency of the protocol and the toxici ty of the nitrosamine intermediates, an alternative method using electrophilic amination by means of tert butyl 3 (trichloromethyl) 1,2 oxazirdine 2 carboxylate ( N Boc oxaiziridine, Scheme 2.2) for hydrazine formation was established in our laboratories. T he N Boc oxaiziridine was prepared in our laboratory (Avancha, 2006) according to previously reported literature procedures. (Hannachi et al., 2004; Vidal et al., 1993; Vidal et al., 1998) Use of the N Boc oxaiziridine proved optimal due to its ability to cleanly trans fer the N Boc group to the desired secondary amine position in one step and it was less toxic than previously used agents.

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132 Scheme 4.2: Synthesis of di acids 4.5a c As shown in Scheme 4.2, compounds 4.3a c underwent electrophilic amination, using N boc oxaiziridine, resulting in the formation of the corresponding N Boc protected hydrazines 4.4a c Basic hydrolysis of compounds 4.4a c yielded the desired N Boc protected hydrazine di acids 4.5a c in high yiel ds (75 85%) as the pure products.

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133 4.2.3 Solid Phase Synthesis Dr. Umut Oguz pursued the coupling and cyclization of di acid dervatives 4.5a c by solid phase synthesis (Scheme 2.3) ultimately yielding the trimeric 2,6 DKP scaffold 4.12 which exhibited an IC 50 of 4.7 uM inhibition for Bcl x L /Bax interactions, according to fluorescence polarization assay analysis. The key step of this synthetic route was the formation of the cyclic anhydride 4.6 The synthetic details will not be discussed but analogs prepa red utilizing her novel protocols will be.

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134 Scheme 4.3: Solid phase synthesis of the 3 R piperazine 2,6 dione scaffold 4.2.4 Solution Phase Synthesis of 2,6 DKP Monomers My focus was the solution phase synthesis of the 2,6 DKP (Scheme 4.4) Hydrazine di acids 4.5a b were treated with DIC in the presence o f DCM to generate the cyclic an hydrides 4.6a b in situ Coupling of alanine benzylester hydrochloride with 4.6a afforded 4.13a (Brockunier et al., 2004) The purification of 4.13a was lengthy and

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135 tedious and the yield was poor Compound 4.13a underwent cyclization via activation of the carboxylic acid with acetic anhydride in the presence of sodium acetate, yielding the target 2,6 DKP, 4.14a Scheme 4.4: Solution phase synthesis of the 2,6 DKPs The conditions followed for the synthesis of 4.14a were not optimal, as y ields for the coupling and cyclization steps were low, purification was difficult, and acylation of the amide nitrogen resulted in significant format ion of undesired sideproduct compound 4.15b (and determined by Dr. Oguz). Attempts in the lab to improve this synthesis by using orthogonally protected di esters to generate a mono acid aiming at facilitating the regioselective coupling and improving yiel ds were unsuccessful. (Ande rson, 2009)

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136 4.2.5 Attempts at Isolation of Cyclic Anhydride 4.6 In order to optimize the conditions for the solution phase synthesis of the 2,6 DKP monomers, various reaction conditions were explored in the attempt to isolate and purify the cyclic anhy dride 4.6a b (Scheme 4.5) prior to subsequent coupling (Brockunier et al., 2004; Humphrey and Chamberlin, 1997; Paul and Anderson, 1960) In all cases, the formation of the c yclic anhydrides w ere observed by TLC, however, the isolation of 4.6a b was not accomplished due to the instability of the compounds. Entry Reagents Conditions 4.5b DIC DCM, MW 90 C, 25 min 4.5b DCC THF, MW 110 C, 30 min 4.5b DCC DCM, 0 C to rt, 24 h 4.5b EDC DCM, 0 C to rt, 24 h 4.5b Pivalic Anhydride DCM, rt, 28 h 4.5b Ac 2 O rt to reflux, 12 h 4.5b HATU, DIEA DCM, rt, 24 h 4.5c EDC, HOBt DCM, 60 C, 24 h 4.5c DCC MeCN, rt, 24 h 4.5c CDI THF, rt, 24 h 4.5c Ac 2 O, pyridine DCM, rt, 18 h 4.5c Ac 2 O MW 130 C, 20 min *by L. Anderson Scheme 4.5: Attempted cyclic anhydride 4.6 isolation The cyclization step remained the bottle neck of the synthetic protocol. The low yields and difficult synthesis of the 2,6 DKP based scaffolds were deemed impractical and

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137 inefficient for the generation of focused libraries. Taking all the information int o consideration, the work on the 2,6 DKP based scaffold was concluded and the design of alternative DKP based scaffolds were explored. 4.3 Results and Discussion for the 3 R Piperazine 2,5 Diones 4.3.1 Retrosynthesis of 3 R Piperazine 2,5 Dione, Unit A 1 : Approach 1 The next synthetic str ategy involved the use of the DKP 2,5 dione units as depicted in Scheme 4.6 It was expected that the cyclization of the 2,5 DKP units would be more accessible due to the increased nucleophilicity of the reacting amino gro up. For this approach (Scheme 4.6) we concentrated on synthesizing the monomeric 2,5 DKP unit that did not contain a hydrazine moiety in hopes that the presence of the free amine would facilitate the synthetic process. This approach began by reacting com mercially available amino acids a with succinimidyl diazoacetate (synthesized in house, Scheme 4.7) to form the diazoacetamide derivative b followed by coupling c and ring closure via metal catalyzed N H insertion yielding the target A 1 unit.

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138 Scheme 4.6: Retrosynthesis o f 3 R piperazine 2,5 dione, A 1 : Approach 1

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139 4.3.2 Synthesis of Compound 4.19 Compound 4.19 (Scheme 4.7) was synthesized following literature procedures (Blankley et al., 1969a; Ouihia et al., 1993) and was chosen as a key intermediate because of its reported high stability and easier handling when compared with the moisture sensitive acid chloride analogs. (Blankley et al., 1969b) Scheme 4.7 : Synthesis of succinimidyl diazoacetate 4.3.3 Attempted Synthesis of the 2,5 DKP Monomer 4.22 Depicted in Route 1 of Scheme 4.8, commercially available amino acid methyl esters 4.3a b were treated with 4.19 ( Scheme 4.7), under basic conditions followed by hydrolysis, yielding the diazoacetamide dervatives 4. 20 a b Compound 4.20a was further coupled to commercially available alanine methyl ester hydrochloride yielding compound 4.21 (Scheme 4.8, Route 1). Attempts at ring closure via metal catalyzed N H insertion (compounds 4.22 and 4.25 ) by treatment with Cu(I) catalysis (Ma et al., 2005) failed to provide compound 4.22 (Scheme 4.8, Route 2). Further, re lavent synthetic efforts carried out by our laboratories all proved unsuccessful. (Anderson, 2009)

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140 Scheme 4.8: Attempted synthesis of 2,5 DKP monomer 4.22 and 4.25 The synthesis of 2,5 DKP monomers via approach 1 was not successful. The initial treatment with compound 4.19 and further coupling to commercially available alanine

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141 methyl ester hydrochloride were low yielding and difficult to purify. Further attempts at copper catalyzed ring closure were also unsuccessful on my part as well as additional group members. 4.3.4 Retrosynthesis of 3 R Piperazine 2,5 Dione, Unit A 1 : Approach 2 In the next approach, it was intended to fir st synthesize the 2,5 DKP monomeric unit, then insert the hydrazine moiety as a branching point to extend the scaffold (Scheme 4.9) It was hoped that the chemically simplified monomer unit A 1 would facilitate the synthetic process. It was also advantageous to implore this synthetic method as there were documented syntheses of this nature beginning with commercially available amino acid esters. (Maity and Koenig, 2008) This approach involved the coupling or N acylation of commercially available amino acid methyl esters a hydrolysis b and intramolecular cyclization c followed by hydrazine formation to afford the desired hydrazine linked 2,5 DKP unit, A 1 (Scheme 4.9).

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142 Scheme 4.9: Retrosynthesis of 3 R piperazine 2,5 dione, A 1 : Approach 2 4.3.5 Synthesis of 2,5 DKP Monomer, Route 1 Two different ro utes were investigated to determine an efficient synthesis of the desired 2,5 DKP monomer. Route 1 (Scheme 4.10) began with commercially available esters 4.3a b The se esters underwent microwave assisted coupling reactions with N phenyl glycine in the presence of HBTU and DIEA yielding esters 4.26a b

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143 Scheme 4.10: Route 1 used to synthesize the 2,5 DKP monomers Synthesis of the methylesters 4.26a b was attempted by activation of the carboxylic acid utilizing various reagents (Scheme 4.11). Standard coupling using EDC under basic conditions (Entry 1& 2, Scheme 4.11) yielded a complicated product mixture. The addition of heat to the EDC couplings did not minimize the formation of the undesired side products. Next, the use of an aminium based coupling reagent, 1H Benzotriazolium 1 [bis(dimethylamino)me thylene] 5chloro ,hexafluorophosphate (1 ),3 oxide (HCTU) was investigated (Entry 3, Scheme 4.11) for its reported effects in reducing the rates of racimization in a reaction and high level of stability. Unfortunately HCTU was not a successful reagent in t he clean formation of 4.26a as it too resulted in the formation of numerous side products. Lastly, an alternate aminium based coupling reagent O (Benzotriazol 1 yl) N,N,N',N' tetramethyluronium hexafluorophosphate (HBTU) was used for its reported ability to minimize reaction racimization and higher reactivity when compared with HCTU. As shown in Entry 4 of Scheme 4.11, the use of HBTU did

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144 provide the desired 4.26a compound without the formation of numerous side products although only in trace amounts. Suc cessful synthesis of 4.26a b was determined to be carried out in a Biotage Microwave Reactor at 85 o C for 30 minutes in the presence of HBTU, DIEA, and DMF. (Fara et al., 2006) These reaction conditions showed both good yields (86% of compound 4.26a ) and minimal formation of side products. Entry Entry Conditions Results 1 4.3b EDC, HOBt, DIEA, CH 3 CN, rt, 4h Multiple side products 2 4.3b EDC, DIEA, CH 3 CN, reflux, 24h Multiple side products 3 4.3b HCTU, DIEA, DMF, 80 o C, 24h Multiple side products 4 4.3b HBTU, DIEA, DMF, 80 o C, 24h Traces 5 4.3b HBTU, DIEA, DMF, MW, 85 o C, 30 min 86% Scheme 4.11: Attempts to synthesize 4.26a Esters 4.26a b underwent basic saponification to provide acids 4.27a b in good yields. Synthesis of the diketopiperazines 4.28a b was carried out in the presence of HBTU in a Biotage Reactor. Finally, reduction of 4.28a b wi th lithium aluminum hydride afforded the desired piperazine target monomers 4.29a b Although successful and high yielding, the synthesis of diketopiperazines 4.27a b involved tedious and lengthy purification. Further attempts to employ N aryl and N alkyl glycine as an alternative to N phenyl glycine failed to provide the desired product (data not shown). Efficient examples were limited to the starting materials reported in Scheme 4.10.

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145 X ray crystals structures of compounds 4.27a b (Figure 4.3) we re obtain at Lousiana State University, by Dr. Frank Froncezk, and the crystal structures did verify the s stereochemistry of the compounds. Figure 4.3: X ray crystal structures of compounds 4.27a and 4.27b 4.3.6 Synthes is of 2,5 DKP Monomer, Route 2 Route 2, depicted in Scheme 4.12, was investigated as an alternative synthetic strategy to improve conditions, efficiency, and applicability to a wider range of amino acids. Route 2 began with reactions of esters 4. 3b c, 4.3 0 with bromoacetyl bromide in a biphasic system of aqueous saturated sodium bicarbonate in benzene to yield compounds 4.30a c as the pure product in good yields. (Maity and Koenig, 2008) Esters 4.31a c were subsequently reacted with benzyl amine in methanol to yield the diketopiperazines 4.32a

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146 c as the pure product, which w ere reduced to the corresponding piperazines 4.33a b in the presence of 1M borane/tetrahydrofuran. (Le Bourdonnec et al., 2006) Scheme 4.12: Route 2 used to synthesize the 2,5 DKP monomer Although both Routes 1 and 2 did lead to the desired 2,5 DKP monomer, Route 2 was chosen as the preferred synt hetic protocol based on the higher efficiency, reproducibility, and dexterity of starting material when compared to Route 1. 4.3.7 Hydrazine Insertion A few different options were explored to achieve hydrazine formation of 4.35 (Scheme 4.13) The first was by performing N nitrosation of the amide This was to be achieved by generating NOCL gas in situ using tert butylnitrite and TMS Cl in the presence of pyridine and DCM. (Francom and Robins, 2003) Also nitrosonium

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147 tetrafluoroborate (NOBF 4 ) in the presence of pyridine in acetonitrile was also attempted for N nitrosation. These conditions unfortunately did result in the desired product 4.35 formation. Our efforts at hydrazine insertion to the 2,5 DKP monomer were halted due to the inefficiency of the synthetic route and the high toxicity of the reagents Entry Reagents Conditions 1 TMS Cl, tert butyl nitrite, pyridine DCM, 0 C to rt, 24 h 2 TMS Cl, tert butyl nitrite, pyridine DCM, 0 C to 40 C, 6 h 3 TMS Cl, tert butyl nitrite, pyridine DCM, rt to 40 C, 6 h 4 NOBF 4 pyridine CH 3 CN, 10 C to 0 C, 2 h 5 NOBF 4 pyridine CH 3 CN, 0 C, 2 h Scheme 4.13: Attempts at hydrazine formation 4.4 Conclusion Our efforts in the synthesis of repetitively linked hydrazine bound functionalized 2,5 and 2,6 helix mimetic scaffolds wer e successful in the synthesis of a library of amino di acid derivatives and a variety of functionalized 2,5 and 2,6 DKPs. Numerous approached described were unsuccessful at the desired di and trimeric scaffold formation. The primary difficulty with the described approaches was the low yielding cyclization step or the inability for cyclization to occur. The use of the 2,5 DKP monomeric units did offer promising data with their ease of synthesis and high yields although the insertion of the hydrazine moie ty remains a

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148 challenge. Further evaluation of the functionalized 2,5 DKP units as building block for helix mimetics, provides promising synthetic potential. 4.5 Experimental Procedures Methyl 1 aminocyclohexanecarboxylate hydrochloride (4.2a) To an ice water bath chilled solution of 4.1a (1.41 mmol) in MeOH (10 mL) was slowly added thionyl chloride (4.28 mmol) and brought to ambient temperatures. The solution was stirred for an additional 6 hours at a gentle reflux. The solution was cooled to ambient temperatures, the residual solvent wa s removed in vacuo to afford the pure product 4.2a (1.38 mmol, 95%) as a white solid. 1 H NMR (400 MHz, CD 3 3H), 2.17 2.05 (m, 2H), 1.88 1.46 (m, 8H). 13 C NMR (101 MHz, CD 3 (1C), 59.59 (1C), 52.71 (1C), 31.62 (2C), 24.06 (1C), 20.62 (2C). MS (ESI) m/z 158.56 (M + H) + (S) methyl 2 ((2 ethoxy 2 oxoethyl)amino) 3 methylbutanoate (4.3a) To a solution L valine methyl ester hydrochloride (5.96 mmol) in anhydrous CH 3 CN (20 mL) acetonitrile, under argon, was slowly added diisopropyl ethyl amine (11.93 mmol) and ethyl bromoacetate (11.93 mmol). The reaction mixture was kept under argon at ambient temperatures and stirred for 24 hours. To the mixture was added, DI H 2 O (50 mL) and 10% citric acid (1 0 mL), followed by extraction with ethyl acetate ( 3 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over Na 2 SO 4

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149 and the residual solvent was removed in vacuo to afford the crude product 4.3a as a light yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) afforded compound 4.3a as a clear oil (3.58 mmol, 62%). 1 H NMR (400 MHz, CDCl 3 J = 7.5 Hz, 1H), 4.52 (dd, J = 8.8, 4.8 Hz, 1H), 3.91 (s, 2H), 3.76 (s, 3H), 2.27 2.15 (m, 1H), 0.94 (dd, J = 9.9, 6.9 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 (1C), 19.09 (2C), 17.91 (1C). MS (ESI) m/z 218.1 (M + H) + (S) methyl 2 ((2 ethoxy 2 oxoethyl)amino) 4 met hylpentanoate (4.3b) This was obtained from L leucine methyl ester hydrochloride (0.66 mmol) in a similar manner as described for preparation of 4.3a Purification by flash column chromatography on silica gel (ethyl acetat e/hexane, 1:10) afforded compound 4.3b as a yellow oil (0.46 mmol, 70%). 1 H NMR (400 MHz, CDCl 3 J = 7.1 Hz, 2H), 3.71 (s, 3H), 3.44 3.29 (m, 4H), 2.13 (bs, 1H), 1.74 (dp, J = 13.4, 6.7 Hz, 1H), 1.51 (td, J = 7.1, 2.4 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 6.6, 1.8 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 49.40 (1C), 42.69 (1C), 25.04 (1C), 22.93 (1C), 22.43 (1C), 14.40 (1C). MS (ESI) m/z 345.2 (M H)

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150 methyl 1 ((2 ethoxy 2 oxoethyl)amino)cyclohexanecarboxylate (4.3d) This was obtained from 4.2a (0.84 mmol) in a similar manner as described for preparation of 4.3a Purification by flash column chromatography on silica gel (ethyl a cetate/hexane, 5:95) afforded compound 4.3d as a yellow oil (0.42 mmol, 52%). 1 H NMR (400 MHz, CDCl 3 J = 7.1 Hz, 2H), 3.68 (s, 2H), 3.30 (s, 2H), 2.50 (bs, 1H), 1.95 1.89 (m, 2H), 1.68 1.62 (m, 2H), 1.54 1.48 (m, 2H), 1.44 1.35 (m, 4H), 1.26 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 (1C), 61.11 (1C), 51.99 (1C), 45. 34 (1C), 33.41 (2C), 25.72 (1C), 22.07 (2C), 14.40 (1C). MS (ESI) m/z 242.2 (M H) (S) tert butyl 2 (2 ethoxy 2 oxoethyl) 2 (1 methoxy 3 methyl 1 oxobutan 2 yl)hydrazinecarboxylate (4.4a) A solution 4.3a (3.69 mmol) i n anhydrous MeOH (10 mL), under argon, was chilled to mmol), warmed to ambient temperatures, and stirred for an additional 18 h. The solvent was removed in vacuo and the residual white residue was diluted with DI H 2 O (50 mL). The aqueous solution was extracted with EtOAc (3 x 35 mL), washed with brine (2 x 50 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified using flash chromat ography on silica gel (30:70 EtOAc/hexanes) to afford 4.4a as a clear oil (2.95 mmol, 80%). 1 H NMR (400 MHz, CDCl 3 J = 7.1 Hz, 2H), 3.71 (s, 3H), 3.07 (d, J = 9.7 Hz, 1H), 1.99 (ddt, J = 13.3, 9.9, 6.7 Hz, 1H), 1.44 (s, 9H),

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1 51 1.26 (t, J = 7.1 H z, 3H), 1.12 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). MS (ESI) m/z 281.1 (M + H) + (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymeth yl)hydrazinyl) 3 methylbutanoic acid (4.5a) To a solution of 4.4a (1.51 mmol) in anhydrous MeOH (10 mL), was added 1M NaOH (30 mL) and vigorously stirred for 24 hours. The solvent was removed in vacuo, and to the residue was added DI H 2 O (15 mL), acidified with 15% citric acid (20 mL), and extracted with EtOAc (3 x 30 mL). The combine d organic layers were washed with brine (2 x 30 mL), dried over Na 2 SO 4 and evaporated under reduced pressure to afford the pure compound 4.5a (1.13 mmol, 75%) as a white solid. m.p. = 130 134 o C. 1 H NMR (400 MHz, CD 3 3.60 (m, 2H), 3.30 (dt, J = 3.2, 1.6 Hz, 1H), 3.22 (d, J = 8.1 Hz, 1H), 2.0 1.91 (m, 1H), 1.51 1.41 (m, 9H), 1.10 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CD 3 (1C), 80.84 (1C), 73.53 (1C), 28.75 (1C), 27.4 2 (3C), 19.39 (1C), 18.36 (1C). MS (ESI) m/z 289.1 (M H) (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymethyl)hydrazinyl) 4 methylpentanoic acid (4.5b) This was obtained from 4.4b (1.69 mmol) in a similar manner as described for preparation of 4.5a yielding the pure compound 4.5b (1.27 mmol, 75%). 1 H NMR (400 MHz, CD 3 3.65 (m, 3H), 1.93 (s, 1H), 1.66 1.40 (m, 12H), 0.94 (t, J = 7.3

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152 Hz, 6H). 13 C NMR (101 MHz CD 3 74.86 (1C), 172.38 (1C) 80.81 (1C), 65.43 (1C), 57.82 (1C), 39.05 (1C), 27.37 (1C), 24.54 (1C), 22.19 (1C), 20.98 (1C). MS (ESI) m/z 303.1 (M H) (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymethyl)hydrazinyl) 3 phenylpropanoic acid (4.5c) This was obtained from 4.4c (1.20 mmol) in a similar manner as described for preparation of 4.5a yielding the pure compound 4.5c (0.96 mmol, 80%). 1 H NMR (400 MHz, CD 3 7.16 (m, 5H), 3.90 (t, J = 7.3 Hz, 1H), 3.71 (d, J = 1.6 Hz, 2H), 3.03 (d, J = 7.3 Hz, 2H), 1.46 (s, 9H). 13 C NMR (101 MHz, CD 3 173.46 (1C), 137.54 (1C), 129.05 (2C), 128.21 (2C), 126.41 (2C), 69.31 (1C), 57.17 (1C), 35.90 (1C), 27.50 (2C). MS (ESI) m/z 337.1 (M H) (S) 2 (8 isoprop yl 13,13 dimethyl 3,7,11 trioxo 1 phenyl 2,12 dioxa 6,9,10 triazatetradecan 9 yl)acetic acid (4.13a) To a solution of 4.5a (0.69 mmol) in DCM (5 mL), was added DIC (0.69 mmol), and the solution stirred at ambient temperat ure for 3 hours (or until the disappearance of the starting material 4.5a was observed by TLC confirming the formation of 4.6a ) To the stirring solution of 4.6a alanine benzyl ester hydrochloride (0.69 mmol), triethylamine (1.38 mmol), and stirred at ambient temperature for 18 hours. The solvents were removed in vacuo the resulting residue was diluted with DI H 2 0 (20 mL), acidified with 1N HCl, and extracted with EtOAc (3 x 50 mL). The combined organic

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153 layers were washed with brine ( 2 x 50 mL), dried over Na 2 SO 4 and the solvent was removed in vacuo to give the crude product. The crude product was purified using flash chromatography on silica gel (70:30 EtOAc/hexanes) to afford 4.13a as a light yellow oil (0.21 mmol, 30%). 1 H NMR (400 MHz, CDCl 3 ) 7.41 (m, 5H), 5.17 (s, 2H), 3.3 3.8 (m, 5H), 2.6 (t, 2H), 1.9 (m, 1H), 1.4 (s, 9H), 1.1 (d, 3H), 0.97 (d, 3H). MS (ESI) m/z 452.2 (M + H) + (S) benzyl 3 (4 ((tert butoxycarbonyl)amino) 3 isopropyl 2,6 dioxopiperazin 1 yl)propano ate (4.14a) To a solution of 4.13a (0.22 mmol) in acetic anhydride (10 mL) was added sodium acetate (1.33 mmol). The reaction mixture was refluxed at 75 C for 18 hours then brought to ambient temperatures. To the reaction was added DI H 2 0 (50 mL) and extracted with chloroform (3 x 30 mL). The combined organic layers were washed with 1.2 N NaOH (1 x 60 mL), brine (2 x 50 mL), and dried over Na 2 SO 4. The solvent was removed in vacuo to give the crude product which was purif ied by flash chromatographed on silica gel (30:70 EtOAc/Hexanes) to provide 4.14a (0.04 mmol, 20%) as a clear oil. 1 H NMR (400 MHz, C D C l 3 7.4 (m, 5H), 5.09 (s, 2H), 4.03 4.2 (m, 2H), 3.8 4.0 (dd, 2H), 3.37 (d, 1H), 2 .6 2.7 (m, 2H), 1.9 2.0 (m, 1H), 1.4 (s, 9H), 1.1 (d, 3H), 0.98 (d, 3H). MS (ESI) m/z 451.2 (M + H) +

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154 (E) 2 (2 tosylhydrazono)acetic acid (4.17) A solution of glycolic acid monohydrate (0.22 m) in DI H 2 O (50 mL) was warmed to 60 o C. This warm solution was then subsequently treated with a warm (approximately 60 C) solution of 4.16 (0.22 m) in conc. HCl (10 mL) and heated for an additional 10 minutes. The resulting mixture was brought to ambient temperatures and stirred for an addit ional 12 hours then placed in a refrigerator overnight. The crude product was collected on a filter, washed with cold water, and allowed to dry at ambient temperatures for 2 days. The crude product was recrystallized with boiling benzene and chilled hexan es to afford compound 4.17 ( 0.19 m, 87% ) as white crystals, m.p. 146 152 C The structural confirmation of 4.17 was obtained by single crystal X ray diffraction 1 H J = 8.3 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.17 (s, 1H), 2.36 (s, 3H). 13 138.14 (1C), 136.37 (1C), 130.58 (2C), 127.78 (2C), 21.70 (1C). MS (ESI) m/z 241.2 (M H) (E) 2 (2 tosylhydrazono)acetyl chloride (4.18) To a suspension of 4.17 (0.18 m) in benzene (220 mL) was added thionyl chloride (0.36 m) and heated to reflux for 18 hours. The reaction mixture was cooled to ambient temperatures, filtered through a diatomaceous earth mat on a sintered glas s funnel, and concentrated in vacuo to afford the crude solid The residual solid was tritutrated with

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155 warm benzene to afford acid chloride 4.18 (0.054 m, 30%) as pale yellow crystals, m.p. 102 106 C. 1 H NMR (400 MHz, (D 3 C) 2 J = 8.3 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.10 (s, 1H), 2.36 (s, 3H). 13 C NMR (101 MHz, (D 3 C) 2 138.15 (1C), 136.45 (1C), 130.43 (2C), 127.78 (2C), 21.61 (1C). MS (ESI) m/z 261.6 (M + H) + 2,5 dioxopyrrolidin 1 yl 2 diazoacetate (4.19) To a chilled mixture of Na 2 CO 3 (1.41 mmol) and N hydroxy succinimide (0.85 mmol) in anhydrous DCM (5 mL) was added 4.18 (0.70 mmol) in DCM (3 mL). Reaction was maintained at 0 0 C for 2 hours then stirred at ambient temper atures for an additional 3 hours. The reaction mixture was filtered through a diatomaceous earth mat on a sintered glass funnel, and concentrated in vacuo to afford the crude yellow solid The crude product was recrystallized with boiling DCM and chilled hexanes to afford compound 4.19 ( 0.32 mmol, 45% ) as yellow crystals. The structural confirmation of 4.19 was obtained by single crystal X ray diffraction. (S) methyl 4 methyl 2 (2 (phenylamino)acetamido)pentanoate (4.26a) A mixture of L leucine methyl ester hydrochloride (3.96 mmol), N phenyl glycine (7.94 mmol), HBTU (4.76 mmol), and DIEA (7.94 mmol) in anhydrous DMF (12 mL) was heate d in the Biotage microwave at 85 C for 30 minutes. After cooling to ambient temperature, the solution was diluted with DI H 2 O (100 mL) and saturated KHSO 4 (30

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156 mL). The aqueous solution was extracted with EtOAc (3 x 35 mL), washed with brine (2 x 50 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel performed using the Flash Master 3 purification station (30:70 EtOAC/hexanes) to afford 4.26a as a light yellow oil (3.40 mmol, 86%). 1 H NMR (400 MHz, CDCl 3 ) 7.19 (m, 2H), 7.07 (m, 1H), 6.79 (m,1H), 6.61 (m, 2H), 4.66 (m, 1H), 3.80 (d, J = 4.19 Hz, 2H), 3.67 (s, 3H), 1.65 1.43 (m, 3H), 0.87 (dd, J = 15.07 Hz, 6H). 13C NMR (100 MHz, CDCl 3 ) 173.39 (1C), 170.96 (1C), 147.38 (1C), 129.55 (2C), 119.29 (1C), 113.57 (2C),52.45 (1C), 50.61 (1C), 49.02 (1C), 41.38 (1C), 24.99 (1C), 23.03 (1C), 21.85 (1C). HRMS (ESI) calc. for C 15 H 22 N 2 O 3 (M + H) + 278.1630, found 278.1715. (S) methyl 3 phenyl 2 (2 (phenylamino)acetamido)propanoate (4.26b) This was obtained from L phenylalanine methyl ester hydrochloride (0.66 mmol) in a similar manner as described for preparation of 4.26a The crude product was purified by chromatography on silica gel using a Flash Master 3 purification station (30:70 EtOAc/hexanes) to afford 4.26b as a clear oil (0.95 mmol, 72%). 1 H NMR (400 MHz, CDCl 3 ) 7.27 7.09 (m, 5H), 6.93 (m, 2H), 6.82 (m, 2H), 6.56 (m, 1H), 4.95 (m, 1H), 3.75 (d, J = 8.59 Hz, 2H), 3.70 (s, 3H), 3.06 (dd, J = 6.01 Hz, 2H). 13 C NMR (100 MHz, CDCl 3 ) 171.89 (1C), 170.49 (1C), 147.11 (1C), 135.71 (1C), 129.62 (2C), 129.36 (2C), 128.78 (1C), 127.28 (2C), 119.40 (1C), 113.51 (2C), 52.75 (1C), 52.54 (1C), 48.86 (1C), 38.13 (1C). HRMS (ESI) calc. for C 18 H 20 N 2 O 3 (M + H) + 312.1473, found 312.1603.

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157 (S) 4 methyl 2 (2 (phenylamino)acetamido)pentanoic acid (4.27a) To a solution of 4.26a (0.77 mmol) in methanol (15 mL) was added 3M NaOH (1.92 mmol) and stirred at ambient temperature for 24 hours. The solution was acidified with 6M HCl (4 mL), extracted with EtOAc (3 x 30 mL), washed with brine (2 x 40 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was recrystallized with boiling EtOAc to afford 4.27a (0.58 mmol, 76%) as yellow crystals. The structural confirmation of 4.27a was obtained by single crystal X ray diffraction 1 H NMR (400 MHz, CD 3 O D ) 7.99 (d, J = 8.48 Hz, 1H), 7.16 7.08 (m, 2H), 6.71 6.56 (m, 3H), 4.49 4.47 (m, 1H), 3.77 (q, J = 17.22 H z, 2H), 3.34 (s, 1H), 1.63 1.47 (m, 3H), 0.86 (d, J = 5.79 Hz, 6H). 13 C NMR (100 MHz, CD 3 OD ) 174.55 (1C), 172.87 (1C), 148.06 (1C), 128.90 (2C), 117.94 (1C), 112.93 (2C), 50.61 (1C), 48.69 (1C), 40.46 (1C), 24.71 (1C), 22.20 (1C), 20.54 (1C). HRMS (ESI ) calc. for C 14 H 20 N 2 O 3 (M H) 264.1473, found 264.1401. (S) 3 phenyl 2 (2 (phenylamino)acetamido)propanoic acid (4.27b) This was obtained from 4.26b (0.64 mmol) in a similar manner as described for preparation of 4.27a The crude product was recrystallized with boiling EtOAc to afford 4.27b (0.50 mmol, 77%) as light yellow crystals. The structural confirmation of 4.27b was obtained by single crystal X ray diffraction 1 H NMR (400 MHz, CD 3 OD ) 7.87 (s, 1H), 7.17 7. 00 (m, 7H), 6.70 (m, 1H), 6.51 (m, 2H), 4.76 4.71 (m, 1H), 3.68 (q, J =

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158 17.44 Hz, 2H), 3.07 (ddd, J = 21.65, 6.38 Hz, 2H). 13 C NMR (101 MHz, CD 3 OD ) 173.04 (1C), 172.68 (1C), 148.05 (1C), 136.55 (1C), 129.13 (2C), 128.95 (2C), 128.28 (1C), 126.61 (2C), 1 17.93 (1C), 112.88 (2C), 53.14 (1C), 48.44 (1C), 37.11 (1C). HRMS (ESI) calc. for C 17 H 18 N 2 O 3 (M H) 298.1317, found 298.1267. (S) 3 isobutyl 1 phenylpiperazine 2,5 dione (4.28a) A mixture of 4.27a (0.97 mmol), HBTU (1.16 mmol), and DIEA (1.07 mmol) in anhydrous DMF (2 mL) was heated in the Biotage microwave at 80 C for 25 minutes. After cooling to ambient temperature, the solution was diluted with DI H 2 O (60 mL), extracted with EtOAc (3 x 30 mL), washed with brine (2 x 30 mL), dried (Na 2 SO 4 ), and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using the Flash Master 3 purification station (40:60 EtOAc/hexanes), recrytallized with boiling EtOAc/chi lled hexanes to afford 4.28a as a light yellow crystals (0.96 mmol, 99%). The structural confirmation of 4.28a was obtained by single crystal X ray diffraction. 1 H NMR (400 MHz, CDCl 3 ) 7.43 (m2H), 7.34 7.27 (m, 2H), 7.06 (s, 1H), 4.33 (dd, J = 52.81 Hz, 2H), 4.13 (m, 1H), 1.88 1.73 (m, 3H), 1.00 (dd, J = 14.60 Hz, 6H). 13 C NMR (100 MHz, CDCl 3 ) 166.99 (1C), 166.61 (1C), 140.53 (1C), 129.64 (2C), 127.72 (2C), 125.53 (1C), 54.53 (1C), 52.72 (1C), 42.71 (1C), 24.60 (1C), 23.34 (1C), 21.59 (1C). HRMS (ESI) calc. for C 14 H 18 N 2 O 2 (M + H) + 246.1368, found 246.1441.

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159 (S) 3 benzyl 1 phenylpiperazine 2,5 dione (4.28b) This was obtained from 4.27b (0.16 mmol) in a similar manner as described for preparation of 4.28a The crude product was purified by chromatography on silica gel performed using the Flash Master 3 purifica tion station (30:70 EtOAc/hexanes), affording 4.28b as a light yellow solid (0.13 mmol, 84%). 1 H NMR (400 MHz, CDCl 3 ) 7.44 7.36 (m, 5H), 7.34 7.27 (m, 3H), 7.11 (d, J = 7.40 Hz, 2H), 6.06 6.02 (bs, 1H), 4.46 (m, 1H), 3.85 (d, J = 17.24 Hz, 1H), 3.35 (d, J = 17.20 Hz, 1H), 3.31 3.26 (m, 1H), 3.19 (dd, J = 13.84 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) 166.47 (1C), 165.68 (1C), 140.23 (2C), 135.08 (2C), 130.44 (2C), 129.65 (1C), 129.18 (1C), 128.06 (1C), 127.91 (1C), 125.54 (2C), 57.23 ( 1C), 52.08 (1C), 41.01 (1C). HRMS (ESI) calc. for C 17 H 16 N 2 O 2 (M + H) + 280.1211, found 280.1316. (S) methyl 2 (2 bromoacetamido) 4 methylpentanoate (4.31a) To a suspension of 4.3a (27.52 mmol) in water (10 mL), cooled in an ice salt bath, was added NaHCO 3 (55.63 mmol) in one portion. A solution of bromoacetyl bromide (23.18 mmol) in benzene (8.5 mL) was slowly added via addition funnel to the chilled mixture. After completion of the addition, the reaction was brought to ambient temperature and stirred for an additional 24h at rt. The aqueous layer was extracted with benzene (3 x 50 mL) and the combined organic layers were washed with brine (1 x 100 mL) and dried

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160 (Na 2 SO 4 ). Remova l of the solvent in vacuo afforded the pure product 4.31a (14.37 mmol, 62%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 1 H), 4.62 4.55 (m, 1H), 3.87 (d, J = 1.6 Hz, 2H), 3.73 (s, 3H), 1.71 1.54 (m, 3H), 0.92 (d, J = 6.2 Hz, 6H). 13 C NMR (1 01 MHz, CDCl 3 41.55 (1C), 29.02 (1C s), 25.07 (1C), 23.01 (1C), 22.14 (1C). (S) methyl 2 (2 bromoacetamido) 3 phenylpropanoate (4.31b) The product was obtained from L phenylalanine methyl ester hydrochloride (23.18 mmol) in a similar manner as described for preparation of 4.30a affording the pure compound 4.31b ( 14.02 mmol, 90%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 7.33 7.21 (m, 3H), 7.13 7.08 (m, 2H), 6.91 (d, J = 7.2 Hz, 1H), 4.84 (dt, J = 7.9, 5.8 Hz, 1H), 3.83 (d, J = 2.9 Hz, 2H), 3.73 (s, 3H), 3.20 3.07 (m, 2H). 13 C NMR (101 MHz, CDCl 3 127.57 (1C), 53.96 (1C), 52.75 (1C), 37.91 (1C), 28.82 (1C). MS (ESI) m/z 300.02 (M + H) + (S) methyl 2 (2 bromoacetamido) 3 (4 (tert butoxy)phenyl)propanoate (4.31c) The product was obtained from 4.30 (3.47 mmol) in a similar manner as described for preparation of 4.31a affording the pure compound 4.31c (2.42 mmol, 90%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 6.98 (m, 2H), 6.94 6.90 (m, 2H), 6.81 (d, J = 7.5 Hz, 1H), 4.80 (dt, J = 7.9, 5.9 Hz, 1H), 3.88 3.78 (m, 2H), 3.72 (s, 3H), 3.15 3.05 (m, 2H), 1.32 (s, 9H). 13 C NMR (101 MHz, CDCl 3

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161 (1C), 130.28 (1C), 129.94 (2C), 124.58 (2C), 78.75 (1C), 53.95 (1C), 52.70 (1C), 37.27 (1C), 29.05 (3C). MS (ESI) m/z 372.08 (M + H) + (S) 1 benzyl 3 isobutylpiperazine 2,5 dione (4.32a) A solution of benzylamine (5.56 mmol) in methanol (4.5 mL) was slowly added via an additional funnel to a solution of compound 4.30a (4.64 mmol) and TEA (11.59 mmol) in methanol (10 mL) and refluxed for 36 h. The resulting light yellow solution was cooled to ambient temperatures, concentrated, and the resulting residue was dissolved in ethyl acetate (100 mL). The organic phase was washed with saturated citric acid (1 x 35 mL), saturated aqueous sodium bicarbonate (2 x 30 mL), brine (2 x 50 mL), and dried (Na 2 SO 4 ). Filtration and removal of the organic solvent in vacuo gave a crude pale yellow solid, which was recrystallized from toluene to afford compou nd 4.32a (61%) as a white solid. 1 H NMR (400 MHz, CDCl 3 7.22 (m, 5H), 6.24 (s, 1H), 4.59 (dd, J = 37.3, 14.5 Hz, 2H), 4.09 4.01 (m, 1H), 3.91 3.77 (m, 2H), 1.88 1.61 (m, 3H), 0.97 (dd, J = 12.0, 6.4 Hz, 6H). 13 C NMR (101 MHz, CDCl 3 .67 (1C), 165.87 (1C), 135.41 (1C), 129.20 (2C), 128.57 (2C), 128.39 (1C), 54.10 (1C), 49.98 (1C), 49.05 (1C), 43.23 (1C), 24.53 (1C), 23.40 (1C), 21.44 (1C). HRMS (ESI) calc. for C 18 H 18 N 2 O 2 (M + H) + 294.1368, found 294.1441.

PAGE 178

162 (S) 1,3 dibenzylpiperazine 2,5 dione (4.32b) The product was obtained from 4.31b (3.77 mmol) in a similar manner as described for preparation of 4.32a Yield: 34%, white solid. 1 H NMR (400 MHz, CDCl 3 7.12 (m, 91 0H), 6.18 (bs, 1H), 4.55 4.42 (m, 2H), 4.38 4.29 (m, 1H), 3.54 (d, J = 17.6 Hz, 1H), 3.17 (qd, J = 13.7, 5.3 Hz, 2H), 3.02 (dd, J = 17.6, 0.7 Hz, 1H). MS (ESI) m/z 295.14 (M + H) + (S) 1 benzyl 3 isobutylpiperazine (4.33a) To a solution of 2.31a (1.64 mmol) in anhydrous tetrahydrofuran (10 mL) was added 1M boran tetrahydrofuran solution (9.83 mmol), and the reaction was heated to reflux under argon for 36 h. The mixture was brought to ambient temperature for 10 min then chilled to 0 o C. To the chilled, stirring solution was added methanol (20 mL) and the solution was kept at 0 o C for 1 h. A 4M anhydrous solution of hydrogen chloride in dioxane (5 mL) was subsequently added to the reaction and heated to reflux for 1 h. After cooling to ambient temperature, aqueous ammonium hydroxide solution (10 mL) was added to the mixture, sub sequently stirred for 10 min at rt, and concentrated in vacuo The resulting residue was dissolved in methanol (20 mL) and again concentrated in vacuo This process was repeated 4 times, affording compound 4.32a (53%) as a yellow oil. 1 H NMR (400 MHz, CD Cl 3 7.24 (m, 2H), 6.94 (d, J = 7.9, 2H), 6.86 (t, J = 7.3, 1H), 3.53

PAGE 179

163 (d, J = 10.4, 2H), 3.17 3.10 (m, 1H), 3.02 (td, J = 11.7, 3.0, 1H), 2.97 2.89 (m, 1H), 2.73 (td, J = 11.6, 3.2, 1H), 2.43 2.31 (m, 2H), 1.75 (dt, J = 13.2, 6.6, 1H), 1.38 1.23 (m, 2H), 0.95 (t, J = 6.4, 7H). MS (ESI) m/z 233.20 (M + H) + (S) 1,3 dibenzylpiperazine (4.33b) The product was obtained from 2.31b (1.64 mmol) in a similar manner as described for preparation of 2.32a Yield: 62% clear oil. 1 H NMR (400 MHz, CD 3 7.15 (m, 10H), 3.76 3.44 (m, 4H), 3.32 (dd, J = 17.7, 1H), 3.16 2.84 (m, 4H), 2.47 (t, J = 10.8 Hz, 1H), 2.31 (dd, J = 12.7, 10.1 Hz, 1H). MS (ESI) m/z 267.19 (M + H) + 4.4 References Abraham, W. R. (2005). Controlling pathogenic Gram negative bacteria by interfering with their biofilm formation. Drug Design Reviews -Online 2 13 33. Anderson, L. (2009) Design and synthesis of substituted 1,4 hydrazine linked piperazine 2,5 and 2,6 di ones and 2,5 terpyrimidinylenes as alpha helical mimetics, University of South Florida, Tampa. Asano, N. (2003). Glycosidase inhibitors: update and perspectives on practical use. Glycobiology 13 93R 104R. Avancha, K. K. V. R. (2006) Design and synthesis of core structural intermediates for novel HIV 1 protease inhibitors and synthesis, biological activity, and molecular modeling of novel 20S proteasome inhibitors. University of South Florida, Tampa. Blankley, C. J., Sauter, F. J., and House, H. O. (19 69a). Crotyl diazoacetate. Org Syn 49 22 27. Blankley, C. J., Sauter, F. J., and House, H. O. (1969b). Crotyl diazoacetate. Organic Syntheses 49 Brockunier, L. L., He, J., Colwell, L. F., Habulihaz, B., He, H., Leiting, B., Lyons, K. A., M arsilio, F., Patel, R. A., Teffera, Y. et al. (2004). Substituted piperazines as novel

PAGE 180

164 dipeptidyl peptidase IV inhibitors. Bioorganic & Medicinal Chemistry Letters 14 4763 4766. Fara, M. A., Diaz Mochon, J. J., and Bradley, M. (2006). Microwave assisted coupling with DIC/HOBt for the synthesis of difficult peptoids and fluorescently labeled peptides a gentle heat goes a long way. Tetrahedron Letters 47 1011 1014. Francom, P., and Robins, M. J. (2003). Nucleic Acid Related Compounds. 118. Nonaqueous Diaz otization of Aminopurine Derivatives. Convenient Access to 6 Halo and 2,6 Dihalopurine Nucleosides and 2' Deoxynucleosides with Acyl or Silyl Halides. Journal of Organic Chemistry 68 666 669. Fytas, C., Zoidis, G., and Fytas, G. (2008). A facile and effective synthesis of lipophilic 2,6 diketopiperazine analogues. Tetrahedron 64 6749 6754. Hannachi, J. C., Vidal, J., Mulatier, J. C., and Collet, A. (2004). Electrophilic Amination of Amino Acids with N Boc oxaziridines: Efficient Preparation of N Ort hogonally Diprotected Hydrazino Acids and Piperazic Acid Derivatives. Journal of Organic Chemistry 69 2367 2373. Houston, D. R., Synstad, B., Eijsink, V. G. H., Stark, M. J. R., Eggleston, I. M., and van Aalten, D. M. F. (2004). Structure Based Explorati on of Cyclic Dipeptide Chitinase Inhibitors. Journal of Medicinal Chemistry 47 5713 5720. Humphrey, J. M., and Chamberlin, A. R. (1997). Chemical Synthesis of Natural Product Peptides: Coupling Methods for the Incorporation of Noncoded Amino Acids into P eptides. Chemical Reviews (Washington, D C) 97 2243 2266. Kanoh, K., Kohno, S., Katada, J., Takahashi, J., and Uno, I. (1999). ( ) Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization. Journal of Antibiotics 52 134 141. Kanzaki, H., Imura, D., Nitoda, T., and Kawazu, K. (2000). Enzymatic conversion of cyclic dipeptides to dehydro derivatives that inhibit cell division. Journal of Bioscience and Bioengineering 90 86 89. Le Bourdonnec, B., Goodman, A. J., Graczyk, T. M., Belanger, S., Seida, P. R., DeHaven, R. N., and Dolle, R. E. (2006). Synthesis and Pharmacological Evaluation of Novel Octahydro 1H pyrido[1,2 a]pyrazine as micro Opioid Receptor Antagonists. Journal of Medicinal Chemistry 49 7290 7306. Ma, M., Peng, L., Li, C., Zhang, X., and Wang, J. (2005). Highly Stereoselective [2,3] Sigmatropic Rearrangement of Sulfur Ylide Generated through Cu(I) Carbene and Sulfides. Journal of the American Chemical Society 127 15016 15017.

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165 Maity, P., and Koenig, B. (2008). Synthesis an d structure of 1,4 dipiperazino benzenes: Chiral terphenyl type peptide helix mimetics. Organic Letters 10 1473 1476. Martins, M. B., and Carvalho, I. (2007). Diketopiperazines: biological activity and synthesis. Tetrahedron 63 9923 9932. Nefzi, A., Ost resh, J. M., and Houghten, R. A. (1997). The Current Status of Heterocyclic Combinatorial Libraries. Chemical Reviews (Washington, D C) 97 449 472. Nicholson, B., Lloyd, G. K., Miller Brian, R., Palladino Michael, A., Kiso, Y., Hayashi, Y., and Neuteboom Saskia, T. C. (2006). NPI 2358 is a tubulin depolymerizing agent: in vitro evidence for activity as a tumor vascular disrupting agent. Anti cancer drugs 17 25 31. Niida, A., Oishi, S., Sasaki, Y., Mizumoto, M., Tamamura, H., Fujii, N., and Otaka, A. (20 05). Facile access to (Z) alkene containing diketopiperazine mimetics utilizing organocopper mediated anti SN2' reactions. Tetrahedron Letters 46 4183 4186. Oguz, U., Guilbeau, G. G., and McLaughlin, M. L. (2002). A facile stereospecific synthesis of alp ha hydrazino esters. Tetrahedron Letters 43 2873 2875. Ouihia, A., Rene, L., Guilhem, J., Pascard, C., and Badet, B. (1993). A new diazoacylating reagent: preparation, structure, and use of succinimidyl diazoacetate. Journal of Organic Chemistry 58 164 1 1642. Paul, R., and Anderson, G. W. (1960). N,N' Carbonyldiimidazole, a new peptide forming reagent. Journal of the American Chemical Society 82 4596 4600. Perrotta, E., Altamura, M., Barani, T., Bindi, S., Giannotti, D., Harmat, N. J. S., Nannicini, R., and 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. Sinha, S., Srivastava, R., De Clercq, E., and Singh, R. K. (2004). Synthesis and Anti viral Properties of Arabino and Ribonucleosides of 1,3 Dideazaadenine, 4 Nitro 1,3 dideazaadenine and Diketopiperazine. Nucleosides, Nucleotides & Nucleic Acids 23 1815 1824. Sugie, Y., Hirai, H., Inagaki, T., Ishiguro, M., Kim, Y. J., Kojima, Y., Sakaki bara, T., Sakemi, S., Sugiura, A., Suzuki, Y. et al. (2001). A new antibiotic CJ 17,665 from Aspergillus ochraceus. Journal of Antibiotics 54 911 916. Teixido, M., Zurita, E., Malakoutikhah, M., Tarrago, T., and Giralt, E. (2007). Diketopiperazines as a tool for the study of transport across the Blood Brain Barrier

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166 (BBB) and their potential use as BBB shuttles. Journal of the American Chemical Society 129 11802 11813. Vidal, J., Guy, L., Sterin, S., and Collet, A. (1993). Electrophilic amination: prepar ation and use of N Boc 3 (4 cyanophenyl)oxaziridine, a new reagent that transfers a N Boc group to N and C nucleophiles. Journal of Organic Chemistry 58 4791 4793. Vidal, J., Hannachi, J. C., Hourdin, G., Mulatier, J. C., and Collet, A. (1998). N Boc 3 Trichloromethyloxaziridine: a new, powerful reagent for electrophilic amination. Tetrahedron Letters 39 8845 8848.

PAGE 183

167 Apendix A: Selected 1 H and 13 C NMR Spectra

PAGE 184

168 (S) methyl 2 (2 bromoacetamido) 3 methylbutanoate ( 2. 3a)

PAGE 185

169 (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 methylbutanoate ( 2. 4a)

PAGE 186

170 (S) methyl 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoate ( 2. 4b) (S) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.7f) G63

PAGE 187

171 (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 phenylpropanoate ( 2. 4c)

PAGE 188

172 (S) methyl 2 (2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino)acetamido) 4 methylpentanoate ( 2. 4d)

PAGE 189

173 S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoate ( 2. 4e)

PAGE 190

174 (S) tert butyl 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl) amino) 4 methylpentanoate ( 2.4f)

PAGE 191

175 (2S,3R) tert butyl 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl)amino) 3 methylpentanoate ( 2. 4g)

PAGE 192

176 (S) 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 3 phenylpropanoic acid ( 2. 5a)

PAGE 193

177 (S) 2 (2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoic acid (2.5b)

PAGE 194

178 (S) 2 (2 ((S) 1 tert butoxy 1 oxo 3 phenylpropan 2 ylamino)acetamido) 3 phenylpropanoic acid ( 2. 5c )

PAGE 195

179 (S) 2 (2 ((S) 1 tert butoxy 1 oxo 3 phenylpropan 2 ylamino)acetamido) 4 methyl pentanoic acid ( 2. 5d )

PAGE 196

180 (S) 2 (2 (((S) 1 (tert butoxy) 4 methyl 1 oxopentan 2 yl)amino)acetamido) 3 phenylpropanoic acid ( 2. 5e)

PAGE 197

181 (S) 2 (2 (((S) 1 (tert butoxy) 4 methyl 1 oxopentan 2 yl)amino)acetamido) 3 methylbutanoic acid ( 2. 5 f )

PAGE 198

182 (S) 2 (2 (((2S,3R) 1 (tert butoxy) 3 methyl 1 oxopentan 2 yl)amino)acetamido) 3 methylbutanoic acid (2. 5g)

PAGE 199

183 (S) tert butyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate ( 2.6a )

PAGE 200

184 (S) tert butyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate ( 2. 6b)

PAGE 201

185 (S) tert butyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate ( 2. 6d)

PAGE 202

186 (S) tert butyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate ( 2. 6e)

PAGE 203

187 (S) tert butyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl ) 4 methylpentanoate (2.6f)

PAGE 204

188 (2S,3S) tert butyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoate (2.6g)

PAGE 205

189 (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoic acid (2.7d)

PAGE 206

190 (2S,3S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoic acid ( 2. 7e)

PAGE 207

191 (S) tert butyl 2 (2 bromoacetamido) 3 phenylpropanoate (2. 8a )

PAGE 208

192 (S) tert butyl 2 (2 bromoacetamido) 4 methylpentanoate (2.8b)

PAGE 209

193 (S) tert butyl 2 (2 (((S) 1 methoxy 1 oxo 3 phenyl propan 2 yl)amino)acetamido) 3 methylbutanoate (2.9a)

PAGE 210

194 (S) methyl 2 ((2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl )amino) 4 methylpentanoate (2.9c )

PAGE 211

195 (2S,3R) methyl 2 ((2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 methylpentanoate (2.9d)

PAGE 212

196 (S) tert butyl 2 (2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino)acetamido) 4 methylpentanoate (2.9e)

PAGE 213

197 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 3 meth ylbutanoate (2.9g)

PAGE 214

198 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 3 phenylpropanoate (2.9h)

PAGE 215

199 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 4 methylpentanoate (2.9i)

PAGE 216

200 (S) 2 (2 ((S) 1 methoxy 1 oxo 3 phenylpropan 2 ylamino) 2 oxoethylamino) 3 methylbutanoic acid ( 2. 10a )

PAGE 217

201 (S) 2 (2 ((S) 1 methoxy 4 methyl 1 oxopentan 2 ylamino) 2 oxoethylamino) 3 phenylpropanoic acid ( 2. 10c)

PAGE 218

202 (S) 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoic acid (2.10e)

PAGE 219

203 (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino)acetamido) 3 methylbutanoic acid (2.10f)

PAGE 220

204 (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino)acetamido) 3 phenylpropanoic acid (2.10g)

PAGE 221

205 (S) methyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate (2.11a)

PAGE 222

206 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoate (2.11b)

PAGE 223

207 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate (2.11c)

PAGE 224

208 (S) methyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate ( 2. 11d)

PAGE 225

209 (S) methyl 3 (1H indol 3 yl) 2 ((S) 3 iso propyl 2,5 dioxopiperazin 1 yl)propanoate (2.11e)

PAGE 226

210 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 (1H indol 3 yl)propanoate (2.11f)

PAGE 227

211 (S) methyl 3 (1H indol 3 yl) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl)propanoate ( 2. 11g) (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.12a)

PAGE 228

212 (2S,3S) methyl 2 ((S) 3 benzyl 2,5 dio xopiperazin 1 yl) 3 methylpentanoate (2.11i)

PAGE 229

213 (S) 3 (1H indol 3 yl) 2 ((S) 3 isopropyl 2,5 dioxopipe razin 1 yl)propanoic acid (2.12c )

PAGE 230

214 (S) 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoic acid (2.12f)

PAGE 231

215 (S) 1 ((S) 3 (1H indol 3 yl) 1 oxo 1 (4 phenylpiperazin 1 yl)propan 2 yl) 3 i sopropylpiperazine 2,5 dione ( 2. 14)

PAGE 232

216 4 Benzyl 2 methylsulfanyl pyrimidine 5 carbonitrile (3.2b)

PAGE 233

217 4 tert Butyl 2 methylsulfanyl pyrimidine 5 carbonitrile (3.2a)

PAGE 234

218 2 Methylsulfanyl 4 naphthalen 1 ylmethyl pyrimidine 5 carbonitrile (3.2c)

PAGE 235

219 4 tert Butyl 2 methanesulfonyl pyrimidine 5 carbonitrile (3.3a)

PAGE 236

220 4 Benzyl 2 methanesulfonyl pyrimidine 5 carbonitrile (3.3b)

PAGE 237

221 phenyl 2 phenylacetate (3.17 )

PAGE 238

222 2 nitro 1 phenylethanol (3.19 a)

PAGE 239

223 1 nitro 3 phenylpropan 2 ol (3.19 b) 4 methyl 1 nitropentan 2 ol (3.19 c)

PAGE 240

224 2 nitro 1 phenylethanone (3.20 a)

PAGE 241

225 4 methyl 1 nitropentan 2 one (3.20 b)

PAGE 242

226 1 nitro 3 phenylpropan 2 one (3.21a )

PAGE 243

227 5 nitro 4 phenylpyrimidin 2 amine (3.22a )

PAGE 244

228 5 ni tro 2,4 diphenylpyrimidine (3.23 )

PAGE 245

2 29 2,4 diphenylpyrimidin 5 amine (3.24 )

PAGE 246

230 5 br omo 2,4 diphenylpyrimidine (3.25 )

PAGE 247

231 N N' Bis (tert butoxycarbonyl) S methylisothiourea (3.35 )

PAGE 248

232 (S) tert butyl (((tert butoxycarbonyl)amino)(2,4 dibenzylpiperazi n 1 yl)methylene)carbamate (3.36 )

PAGE 249

233 (S) 4 benzyl 2 (2,4 dibenzylpiperazin 1 yl )pyrimidine 5 carbonitrile (3.38 )

PAGE 250

234 Methyl 1 aminocyclohexanecarboxylate hydrochloride (4.2a)

PAGE 251

235 (S) methyl 2 ((2 ethoxy 2 oxoethyl)amino) 3 methylbutanoate (4.3a)

PAGE 252

236 (S) methyl 2 ((2 ethoxy 2 oxoethyl)amino) 4 methylpentanoate (4.3b)

PAGE 253

237 methyl 1 ((2 ethoxy 2 oxoethyl)amino)cyclohexanecarboxylate (4.3d)

PAGE 254

238 (S) tert butyl 2 (2 ethoxy 2 oxoethyl) 2 (1 methoxy 3 methyl 1 oxobutan 2 yl)h ydrazinecarboxylate (4.4a) (E) 2 (2 tosylhydrazono)acetyl chloride (4.18)

PAGE 255

239 (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymethyl)hydrazinyl) 3 methylbutanoic acid (4.5a)

PAGE 256

240 (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymethyl)hydrazinyl) 4 methylpentanoic acid (4.5b)

PAGE 257

241 (S) 2 (2 (tert butoxycarbonyl) 1 (carboxymethyl)hydrazinyl) 3 phenylpropanoic acid (4.5c)

PAGE 258

242 ( E) 2 (2 tosylhydrazono)acetic acid (4.17)

PAGE 259

243 (S) methyl 4 methyl 2 (2 (phenylamino)acetamido)pentanoate (4.26a)

PAGE 260

244 (S) methyl 3 phenyl 2 (2 (phenylamino)acetamido)pr opanoate (4.26b)

PAGE 261

245 (S) 4 methyl 2 (2 (phenylamino)acetamido)pentanoic acid (4.27a)

PAGE 262

246 (S) 3 phenyl 2 (2 (phenylamino)acetamido)propanoic acid (4.27b)

PAGE 263

247 (S) 3 isobutyl 1 phenylpiperazine 2,5 dione (4.28a)

PAGE 264

248 (S) 3 benzyl 1 phenylpiperazine 2,5 dione (4.28b)

PAGE 265

249 (S) methyl 2 (2 bromoacetamido) 4 methylpentanoate (4.31a)

PAGE 266

250 (S) methyl 2 (2 bromoacetamido) 3 phenylpropanoate (4.31b)

PAGE 267

251 (S) methyl 2 (2 bromoacetamido) 3 (4 (tert butoxy)phenyl)propanoate (4.31c)

PAGE 268

252 (S) 1 benzyl 3 isobutylpiperazine 2,5 dione (4.32a)

PAGE 269

253 (S) 1,3 dibenzylpiperazine 2,5 dione (4.32b) (S) 1 benzyl 3 isobutylpiperazine (4.33a)

PAGE 270

254 (S) 1,3 dibenzylpiperazine ( 4 .33b )

PAGE 271

255 Appendix B: Selected Mass Spectra

PAGE 272

256 (S) methyl 2 (2 bromoacetamido) 3 methylbutanoate (2.3a)

PAGE 273

257 (S) methyl 2 (2 bromoacetamido) 3 (4 (tert butoxy)phenyl)propanoate (2.3e)

PAGE 274

258 (S) tert butyl 2 ((2 (((S) 1 methoxy 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoate (2.4e)

PAGE 275

259 (S) tert butyl 2 ((2 (((S) 1 methoxy 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl) amino) 4 methylpen tanoate (2.4f)

PAGE 276

260 (S) 2 (2 (((2S,3R) 1 (tert butoxy) 3 methyl 1 oxopentan 2 yl)amino)acetamido) 3 methylbutanoic acid (2.5g)

PAGE 277

261 (S) tert butyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate (2.6e)

PAGE 278

262 (2S,3S) tert butyl 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoate (2.6g)

PAGE 279

263 (S) 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoic acid (2.7c)

PAGE 280

264 (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoic acid (2.7d)

PAGE 281

265 (2S,3S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoic acid (2.7e)

PAGE 282

266 (S) tert butyl 2 (2 bromoacetamido) 3 phenylpropanoate (2.8a)

PAGE 283

267 (S) tert butyl 2 (2 bromoacetamido) 4 methylpentanoate (2.8b)

PAGE 284

268 (S) methyl 2 ((2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 4 methylpentanoate (2.9c)

PAGE 285

269 (2S,3R) methyl 2 ((2 (((S) 1 (tert butoxy) 1 oxo 3 phenylpropan 2 yl)amino) 2 oxoethyl)amino) 3 methylpentanoate (2.9d)

PAGE 286

270 (2S,3R) methyl 2 ((2 (((S) 1 (tert butoxy) 3 methyl 1 oxobutan 2 yl)amino) 2 oxoethyl)amino) 3 meth ylpentanoate (2.9f)

PAGE 287

271 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 3 methylbutanoate (2.9g)

PAGE 288

272 (S) tert butyl 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino) acetamido) 3 phenylpropanoate (2.9h)

PAGE 289

273 (S) 2 (2 ((S) 1 methoxy 1 oxo 3 phenylpropan 2 ylamino) 2 oxoethylamino) 3 methylbutanoic acid (2.10a)

PAGE 290

274 (S) 2 (2 ((S) 1 methoxy 4 methyl 1 oxopentan 2 ylamino) 2 oxoethylamino) 3 phenylpropanoic acid (2.10c)

PAGE 291

275 (S) 2 (2 (((S) 3 (1H indol 3 yl) 1 methoxy 1 oxopropan 2 yl)amino)acetamido) 3 methylbutanoic acid (2.10f)

PAGE 292

276 (S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 4 methylpentanoate (2.11c)

PAGE 293

277 (S) methyl 2 ((S) 3 isobutyl 2,5 dioxopiperazin 1 yl) 3 methylbutanoate (2.11d)

PAGE 294

278 (S) methyl 3 (1H indol 3 yl) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl)propanoate (2.11e)

PAGE 295

279 (2S,3S) methyl 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 methylpentanoate (2.11i)

PAGE 296

280 (S) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.12a)

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281 (S) 2 ((S) 3 benzyl 2,5 dioxopiperazin 1 yl) 3 phenylpropanoic acid (2.12b)

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282 (S) 3 (1H indol 3 yl) 2 ((S) 3 isopropyl 2,5 dioxopiperazin 1 yl)propanoic acid (2.12c)

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283 (S) 3 benzyl 1 ((S) 4 methyl 1 oxo 1 (4 phenylpiperazin 1 yl)pentan 2 yl)piperazine 2,5 dione (2.14)

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284 4 Benzyl 2 methylsulfanyl pyrimidine 5 carbonitrile (3.2b)

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285 2 Methylsulfanyl 4 naphthalen 1 ylmethyl pyrimidine 5 carbonitrile (3.2c)

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286 4 tert Butyl 2 methanesulfonyl pyrimidine 5 carbonitrile (3.3a)

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287 4 Benzyl 2 methanesulfonyl pyrimidine 5 car bonitrile (3.3b)

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288 5 nitro 2,4 diphenylpyrimidine (3.23)

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289 2,4 diphenylpyrimidin 5 amine (3.24)

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290 N N' Bis ( tert butoxycarbonyl) S methylisothiourea (3.35)

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291 (S) tert butyl (((tert butoxycarbonyl)amino)(2,4 dibenzylpiperazin 1 yl)methylene)carbamate (3.36)

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292 (S) 4 benzyl 2 (2,4 dibenzylpiperazin 1 yl)pyrimidine 5 carbonitrile (3.38)

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293 (S) methyl 4 methyl 2 (2 (phe nylamino)acetamido)pentanoate (4 .26a)

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294 (S) methyl 3 phenyl 2 (2 (phenylamino)acetamido)propanoate (4.26b)

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295 (S) 4 methyl 2 (2 (phenylamino)acetamido)pentanoic acid (4.27a)

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296 (S) 3 phenyl 2 (2 (phenylamino)acetamido)propanoic acid (4.27b)

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297 (S) 3 isobutyl 1 phenylpiperazine 2,5 dione (4.28a)

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298 (S) 3 benzyl 1 phenylpiperazine 2,5 dione (4.28b)

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299 (S) 1 benzyl 3 isobutylpiperazine 2,5 dione (4.32a)

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300 (S) 1 benzyl 3 isobutylpiperazine (4.33a)

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301 A p p e n d i x C: X R a y C r y s t a l l o g r a p h i c D a t a

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302 ORTEP diagram for compound (2.7e)

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303

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304

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305

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306

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307

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308 ORTEP diagram for compound (2.11g)

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309

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310

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311

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312

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313

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314 ORTEP diagram for compound (3.20a)

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315

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316

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317

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318 ORTEP diagram for compound (3.21a)

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319

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320

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321

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322

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323 ORTEP diagram for compound (3.22a)

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324

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325

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326

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327

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328

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329

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330 ORTEP diagram for compound (3.24)

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331

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332

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333

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334

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335

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336

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337

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338 ORTEP diagram for compound (3.25)

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339

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340

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341

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342

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343

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344

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345 ORTEP diagram for compound (4.17) ORTEP diagram for compound (4 .19 )

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346 ORTEP diagram for compound (4 .28a )

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347 ORTEP diagram for compound (4.27a)

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348

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349

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350

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351

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352

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353 ORTEP diagram for compound (4.27b)

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About the Author Melissa Topper was born in Peoria, Illinois and spent most of her life growing up in Wallkill, NY. She attended the State University of New York at New Paltz where she earned a bachelor degree in December 2003 with a concentration in C hemistry and a minor in Biology. She continued her graduate studies in Chemistry in Augu st 2004 at the University of South Florida. Here she joined the lab of Dr. Mark L. McLaughlin and performed most of her graduate work within the Moffi tt Cancer Research Center. Melissa will receive her doctoral degree in C hemistry with an emphasis in medicinal and synthetic organic chemistry in July 2010.