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Title:
Design and synthesis of core structural intermediates for novel HIV-1 protease inhibitors & synthesis, biological activity and molecular modeling of novel 20S proteasome inhibitors
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Book
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English
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Avancha, Kiran Kumar Venkata Raja
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Constrained cyclic urea
NPA's
Substituted OPGDA's
Oxaziridine
Apoptosis
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: HIV-1 protease binds to its peptide/protein substrates in extended conformations. Therefore protease inhibitors that are constrained to form extended conformations are likely to produce very active protease inhibitors. This is because they are pre-organized to form favorable interactions with the enzyme environment immediately surrounding the active site. With this hypothesis in mind, we designed a family of structurally related molecules, which contain dipeptide analogs constrained to adopt the extended conformation. Core structural intermediates that are required for the total synthesis of the novel class of HIV-1 protease inhibitors are outlined in Chapter One. Chapter Two discusses the enantioselective synthesis of 2-alkyl-3-nitropropionates (NPA's) that is the part A of the cyclic urea molecule 8, and can also be used as the building block for the synthesis of unnatural beta-amino acids. In conclusion on this project, we were able to successfully achieve the novel ena ntioselective route for the synthesis of NPA's and also obtain the absolute stereochemistry of one of the NPA's by solving the crystal structure. Various routes were explored for the synthesis of the substituted orthogonally protected geminal diamino acids (OPGDA's) and these were discussed in Chapter Three. Chapter Three also discusses the synthesis of a versatile N-Boc transfer reagent and the applications of it in the synthesis of alpha-helix mimics. The outcomes of this project were the efficient synthesis of oxaziridine (104) and the methods that show how we cannot make the "substituted OPGDA's" which can serve as the guidance for future research on them. The proteasome is cellular machinery that is responsible for the breakdown of the complex proteins that are not required by a living cell. The inhibition of its activity in cancerous cells can promote apoptosis. Chapter Four discusses the synthesis of a new class of 20S proteasome inhibitors, their biological testing and lead op timization by molecular modeling, library synthesis and biological evaluation. In short this project achieves our goal for the synthesis of a novel class of 20S proteasome inhibitors that have a potential to act as drug molecules in the future.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
Kiran Kumar Venkata Raja Avancha.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 238 pages.
General Note:
Includes vita.

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Design and Synthesis of Core Structural Intermediates for Novel HIV-1 Protease Inhibitors & Synthesis, Bi ological Activity and Molecu lar Modeling of Novel 20S Proteasome Inhibitors by Kiran Kumar Venkata Raja Avancha 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 McLaughlin, Ph.D. Edward Turos, Ph.D. Abdul Malik, Ph.D. Srikumar Chellappan, Ph.D. Date of Approval: March 31st, 2006 Keywords: Constrained cyclic urea, NPA’s, Substituted OP GDA’s, oxaziridine, apoptosis Copyright 2006, Kiran Kumar Venkata Raja Avancha

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DEDICATION To my mom Prameela and dad Venkata Rao To my love Valli and my brother Arun To my mentor Mark and all my teachers To all my good friends and Well-wishers To the Scientific Community

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ACKNOWLEDGEMENTS First of all I would like to thank my mo m, dad, brother and Valli for their caring and understanding. My dad always encouraged me in obtaining a Ph.D. I can still recollect his words from my childhood Arise Awake. Stop not till the goal is reached. I thank Dr. McLaughlin for being there for me whenever I needed him. He is a great teacher and mentor who not only taught me but also inspired me in achieving this deed. He is a great scientist and I am glad to wo rk for a good person like him. I thank all my committee members Dr. Turos, Dr. Chellappan and Dr. Malik for their suggestions and guidance. I would also like to thank Dr. Wa yne Guida for agreeing to Chair my Defense committee and also for his suggestions and help with Molecular Modeling. I thank my group members current as well as past for th eir constant support and encouragement. I would like to thank Tanaji, who taught me the basics of organic synt hesis and also for his suggestions, directions and for encouraging me in tough times. I thank Stephanie for all her support and her well wishes during tough ti mes both in chemistry and in my life. I also would like to take this opportunity to thank all my friends, well wishers and colleagues in my current lab, previous lab and in other labs: Rao, Mohan, Umut, Jose, Priyesh, Laura, Missy, Mehul, Mingzhou, Jac ob, Phil, Courtney, Sridevi, Santhi, Thushara, Rajesh, Suresh, Raghu, Daita, Raj, DP, Nagaraj, Ravi, Kalyan, and Kiran. I also would like to acknowledge my previous mentors Dr. Somayajulu (Late), Dr. Sankara Bhanu, Sri Sehshagiri Rao, Sri MSN and Dr. Nalini Sasthry.

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I would like to acknowledge the facilities provided to me by the Department of Chemistry at USF and also by the Drug Disc overy Program at H. Lee Moffitt Cancer Center & Research Center. I thank Dr. Said Se bti and his group for th eir collaboration on the biological testing of the novel 20S proteasom e inhibitors. Dr. Kazi in particular at Moffitt helped us in obtaining the biological data on the proteasome inhibitors. I thank Dr. Kenyon Daniel for molecular modeling, Dr Richard Yip for HTS (High-Throughput Screening) and Dr. Harshani Lawrence for tr aining me on the Microw ave synthesizer. I would like to thank Dr. Nick Lawrences Lab in particular for their help and support, especially Dan, Roberta, Simon, Divya, Jayan and Dr. Nick. Dr. Frank Fronczek at LSU helped us in obtaining the crystal structures and also shared his opinion on the crystal structures. Dr. Mike Zaworotko and Dr. Rosa Walsh at Department of Chemistry, USF also helped us in obtaining crystal structures. Dr. Ted Gauthier is very special to me, because of his constant support for my resear ch. He helped me in obtaining Mass spectral data and also invaluable suggestions on various projects. I also would like to acknowledge Dr. Edwin Rivera for the U SF NMR facilities. NIH funding to Dr. McLaughlin is greatly appreciated because of which this research became possible. Last but not the least I would also like to acknow ledge my previous mentor at USF Dr. Bill Baker for helping me in knowing the nuances of the marine natural products research.

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i TABLE OF CONTENTS LIST OF FIGURES iii LIST OF ABBREVIATIONS viii LIST OF SPECTRA x ABSTRACT xiv CHAPTER ONE: HIV PROTEASE INHIBI TORS: INTRODUCTION 1 1.1 Global epidemic: AIDS 1 1.2 HIV-1 Protease: Reason for its inhibition 1 1.3 HIV-1 Protease structure and function 3 1.4 HIV-1 Protease inhibitors approved by FDA 6 1.5 HIV-1 Protease inhibitors with extended conformations 7 1.5.1 Design considerations 9 1.5.2 Retrosynthetic analysis of cyclic urea 8 11 1.6 References 12 CHAPTER TWO: SYNTHESIS OF 2-SU BSTITUTED 3NITROPROPIONATES: PART-A OF CYCLIC UREA 8 15 2.1 Introduction 15 2.2 Results & Discussion 18 2.3 Conclusion 26 2.4 Experimental Data 26 2.4.1 Materials & Methods 26 2.5 References 45 CHAPTER THREE: EFFORT TOWARDS THE SYNTHESIS OF SUBSTITUTED ORTHOGONALLY PROTECTED GEMINAL DIAMINO ACIDS (OPGDAS) & CYCLIC UREA 8 51 3.1 Introduction 51 3.2 Literature reported syntheses and applications of geminal diamino compounds 53 3.2.1 Cushman et al. s synthesis of gem-diamino compounds 58 3.2.2 Reports on orthogonally prot ected geminal diamino acids (OPGDAs) 61

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ii 3.2.3 Our efforts towards the synt hesis of orthogonally protected substituted -diamino acids 64 3.3 Results & Discussion 65 3.3.1 Effort towards the synthesis of cyclic urea 8 73 3.3.2 Electrophilic Amination with Oxaziridines 79 3.3.3 Application of Oxaziridine 104 in -helix mimics synthesis 82 3.3.4 Design considerations in the core structural unit of HIV-1 protease inhibitors synthesis and future direction 85 3.4 Conclusion 86 3.5 Experimental 87 3.6 References 106 CHAPTER FOUR: SYNTHESIS, BIOLOGI CAL ACTIVITY AND MOLECULAR MODELING OF 20S PROTEA SOME INHIBITORS 111 4.1 Introduction 111 4.2 Proteasome structure and ubiquitin-pr oteasome pathway 112 4.3 Proteasome inhibition: A novel approach to cancer therapy 115 4.3.1 Background on the proteasome inhibitors 116 4.3.2 Velcade TM : Only proteasome inhibitor approved by FDA 117 4.3.3 Reason for a new proteasome inhibitor 118 4.4 NSC-12155 lead molecule from HTS core 118 4.4.1 Background on NSC-12155 ( 1,3-Bis-(4-amino-2-methylquinolin-6-yl)-urea 119 4.5 Results & Discussion 120 4.5.1 Synthesis, biological activ ity and molecular modeling of Library 123 4.6 Conclusions and future direction 126 4.7 Experimental 127 4.7.1 20S proteasome inhibition assay 127 4.7.2 Assay for reversible inhibition of NSC-12155 128 4.7.3 Chemistry 129 4.8 References 134 CHAPTER FIVE: APPENDICES 138 Appendix A: Selected 1 H and 13 C NMR spectra 138 Appendix B: X-ray crystallographic data 206 About the Author End Page

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iii LIST OF FIGURES Figure 1.1 Replicative cycle of HIV-1, and the site of action of HIV-1 protease Inhibitors 2 Figure 1.2 Three-dimensional structure of Aspartyl protease from HIV-1 3 Figure 1.3 HIV-1 protease enzyme (a) tunnel view (b ) Flaps in action 4 Figure 1.4 Enzyme reaction mechanism of HIV protease/as partyl protease 5 Figure 1.5 Standard nomenclature of peptide substrates of aspartic proteases 5 Figure 1.6 FDA approved HIV protease inhibitors and their brand names 6 Figure 1.7 Constrained cyclic urea 7 with reported picomolar activity 8 Figure 1.8 (A) Dipeptide bonds in a dipeptide unit; constrained DPU ( D i P eptide U nit); ADPNovel A zaD i P eptide unit 9 Figure 1.9 Novel cyclic urea with all dipeptide bonds constrained 10 Figure 1.10 Retrosynthesis of cyclic urea 8 11 Figure 2.1 -amino acid as the component of bioactive compounds 16 Figure 2.2 Seebachs racemic synthesis of 3-NPA 17 Figure 2.3 Rimkus et al. and Eilitz et al. s enantioselective route for 3-NPA synthesis 17 Figure 2.4 General scheme for the synthesis of 2alkyl-3-nitropropionates 20 Figure 2.5 1 H NMRs for the optically pure and racemic monoacetates and monoalcohols with and without Eu(hfc) 3 21

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iv Figure 2.6 NMR determination of Enantiomeric purity using Eu(hfc) 3 22 Figure 2.7 Lipase PS-30 catalyzed transesterification of substrates 10a-d (Results in tabular form) 23 Figure 2.8 Figure 2.8: Structure of the salt 19, of 2-isobutyl-3-nitropropionic acid ( 16c ) with (1 R ,2R )-(-)-pseudoephedrine ( 18) determined by X-ray structural analysis 25 Figure 3.1 Retro-inverso peptide and its similarity to a normal peptide 52 Figure 3.2 Representative structure of betid amino acids 52 Figure 3.3 Condensation of -acetamino acrylic acid 20 with acetamide 21 53 Figure 3.4 gem -Diamino compounds synthesized by Brenner et al. 54 Figure 3.5 General scheme for the synthe sis of orthogonally protected gem -diamino compounds via modified Curtius reaction 54 Figure 3.6 Enkephalinamide 25 synthesized by Goodman et al. 55 Figure 3.7 gem -Diamino compounds synthesized by Goodman et al. 55 Figure 3.8 Retro-inverso peptide analog of L-Aspartyl-L-phenylalanine ( 31) 56 Figure 3.9 Retro-inverso pe ptide analogs of N -(L-aspartyl)-1,1-diaminoalkane 56 Figure 3.10 Synthesis of orthogonally protected dichloromethane 34 57 Figure 3.11 Synthesis of orthogonally protected -geminal diaminoglycine 37 57 Figure 3.12 Katritzky et al. s scheme for the synthesis of gem-peptides subunit 58 Figure 3.13 Retro-inverso peptide analog 38 of Bombesin 59 Figure 3.14 Failed attempt to synthesize gem -diamino analog of leucine 59 Figure 3.15 Failed attempt via mixed anhydride route 60

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v Figure 3.16 Cushman et al. s successful attempt to synthesize gem -diamino compound 49 via masked amide of 45 61 Figure 3.17 Kohns synthesis of gemdiamino compound 52 61 Figure 3.18 Synthesis of -Fmoc -Boc-aminoglycine analogs 62 Figure 3.19 Synthesis of glycine OPGDA by Davies et al. 62 Figure 3.20 Enantiospecific synthesis of OP GDAs 63 Figure 3.21 Solid-phase peptide synthesis of gem -diamino derivatives 63 Figure 3.22 Antilla et al. s synthesis of N,N -aminals 64 Figure 3.23 Difference between normal OPGDAs and Substituted OPGDAs 64 Figure 3.24 Failed attempt for the enantioselective synthesis of substituted OPGDAs 66 Figure 3.25 Synthesis of 2,4,6-Triios propyl-benzenesulfonyl azide 67 Figure 3.26 Determination of enanti omeric excess of monoacid 63 67 Figure 3.27 Failed attempt to synthesize racemic substituted OPGDA 69 Figure 3.28 Possible mechanism for the ring flipping and elimination of benzyl alcohol 70 Figure 3.29 Failed attempt to synthesize oxazolidine 79 from Oxazolidine 76 70 Figure 3.30 Failed attempt for ring opening of compound 79 71 Figure 3.31 Proposed mechanism for the ring op ening with trimethylsilanolate 71 Figure 3.32 Failed reactions for ri ng opening of oxazolidine 77 72 Figure 3.33 Synthesis of gem -diamino aldehyde 83 73 Figure 3.34 Model Henry reaction on 83 with nitromethane 74 Figure 3.35 Synthesis of amino-alcohol 85 74

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vi Figure 3.36 Failed attempts for cyclization 75 Figure 3.37 Proposed scheme for the synthesis of cyclic urea 93 76 Figure 3.38 Failed rotavap reaction for the synthesis of the 87 77 Figure 3.39 Proposed scheme via selective Boc protection 77 Figure 3.40 Failed attempts of nitroaldol condensation 78 Figure 3.41 Scheme for the synthesis of Oxaziridine 104 80 Figure 3.42 Failed attempt to synthesize imine 69 81 Figure 3.43 Proposed synthesis of substituted OPGDAs via oxaziridine 104 81 Figure 3.44 Failed attempt to make Cbz version of the oxaziridine 104 82 Figure 3.45 Solvent effects on the N-Boc transfer on the secondary amine 83 Figure 3.46 McLaughlin Helix and retrosynthetic analysis of 3-substituted piperazine-2,6-dione re peat units 84 Figure 3.47 Use of Oxaziridine 104 in the key step for the synthesis of the A n subunit 84 Figure 3.48 Synthesis of PheN -Boc-hydrazine in two steps using oxaziridine 104 85 Figure 3.49 Synthesis of DPU 126 as the building block for the HIV-1 protease inhibitors Library 86 Figure 4.1 (a) Crystal structure of the 20S proteasome 6 (b) 26S proteasome complex 112 Figure 4.2 Ubiquitin-proteasome pathway for protein degradation 114 Figure 4.3 Proposed mechanism for the proteolysis catalyzed by the 20S proteasome 114 Figure 4.4 NFB activation pathway 116

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vii Figure 4.5 Structure of the dipeptidyl boronic acid proteasome inhibitor bortezomib 117 Figure 4.6 NSC-12155 the lead molecule 118 Figure 4.7 Sestili et al .s synthesis of 4,6-diam ino-2-methylquinoline ( 129 ) 120 Figure 4.8 Scheme for the synthesis of NSC-12155 121 Figure 4.9 Molecular model of NSC-12155 binding to the chymotrypsin-like subunit of the 20S Proteasome 122 Figure 4.10 Asymmetric urea analogs synthesis 123 Figure 4.11 chymotrypsin-like subunit inhibito ry activity 124 Figure 4.12 Docking of 137, 138 & 139 in the binding pocket 124 Figure 4.13 Comparision of 140 vs. 136 binding orientation in the chymotrypsin-like subunit binding pocket 125 Figure 4.14 Dialysis experiment showing the reversible inhibition of NSC-12155 126 Figure 4.15 Proposed molecules for future syntheses for proteasome inhibition 127

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viii LIST OF ABBREVIATIONS AA = Amino acid Ac = Acetyl ADP = Aza-Dipeptide Unit Boc = tert -Butoxycarbonyl Bn = Benzyl Bt = Benzotriazole BTAH = benzyltrimethyl ammonium hydroxide BTIB/TIB = (bistrifluroacetoxy iodobenzene) o C = degrees Celcius 13 C = carbon 13 Cbz = Benzyloxycarbonyl CDI = Carbonyldiimidazole 18-Crown-6 (18C6) = 1,4,7,10,13,16hexaoxocycloocatadecane Cys = Cysteine DBU = 1,8-Diazabicyclo[5.4.0]unde-7-ene DCC = 1,3-Dicyclohexylcarbodiimide DCM = Dichloromethane DIBAL = Diisobutyl Aluminium hydride DIEA = Diisopropylethylamine DMAP = 4-Dimethylaminopyridine DME or 1,2-DME = Dimethoxyethane DMF = N,N -Dimethylformamide DMSO = Dimethylsulfoxide DPPA = Diphenylphosphoryl azide EDCl = 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide Fmoc = 9-Fluorenylmethoxycarbonyl HATU = O -(7-azabenzotrizol-1yl)-1,1,3,3,tetra methyluronium hexafluorophosphate HOBt = 1-hydroxybenzotriazole NBS = N -Bromosuccinamide NMM = N -Methylmorpholine Oxone = Potassium peroxymonosulfate Ph = Phenyl PTSA/ p-TsOH = p-Toluenesulfonic acid Py = Pyridine TBAF = Tetrabutylammonium fluroide TEA = Triethylamine Tf = Trifluoromethanesulphonyl /Triflate

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ix THF = Tetrahydrofuran TMS = Trimethylsilyl Ts = Tosyl Z or Cbz = Benzylcarbonyl

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x LIST OF SPECTRA Spectrum 5.01 139 Spectrum 5.02 140 Spectrum 5.03 141 Spectrum 5.04 142 Spectrum 5.05 143 Spectrum 5.06 144 Spectrum 5.07 145 Spectrum 5.08 146 Spectrum 5.09 147 Spectrum 5.10 148 Spectrum 5.11 149 Spectrum 5.12 150 Spectrum 5.13 151 Spectrum 5.14 152 Spectrum 5.15 153 Spectrum 5.16 154 Spectrum 5.17 155 Spectrum 5.18 156 Spectrum 5.19 157

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xi Spectrum 5.20 158 Spectrum 5.21 159 Spectrum 5.22 160 Spectrum 5.23 161 Spectrum 5.24 162 Spectrum 5.25 163 Spectrum 5.26 164 Spectrum 5.27 165 Spectrum 5.28 166 Spectrum 5.29 167 Spectrum 5.30 168 Spectrum 5.31 169 Spectrum 5.32 170 Spectrum 5.33 171 Spectrum 5.34 172 Spectrum 5.35 173 Spectrum 5.36 174 Spectrum 5.37 175 Spectrum 5.38 176 Spectrum 5.39 177 Spectrum 5.40 178 Spectrum 5.41 179 Spectrum 5.42 180

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xii Spectrum 5.43 181 Spectrum 5.44 182 Spectrum 5.45 183 Spectrum 5.46 184 Spectrum 5.47 185 Spectrum 5.48 186 Spectrum 5.49 187 Spectrum 5.50 188 Spectrum 5.51 189 Spectrum 5.52 190 Spectrum 5.53 191 Spectrum 5.54 192 Spectrum 5.55 193 Spectrum 5.56 194 Spectrum 5.57 195 Spectrum 5.58 196 Spectrum 5.59 197 Spectrum 5.60 198 Spectrum 5.61 199 Spectrum 5.62 200 Spectrum 5.63 201 Spectrum 5.64 202 Spectrum 5.65 203

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xiii Spectrum 5.66 204 Spectrum 5.67 205

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xiv Design and Synthesis of Core Structural Intermediates for Novel HIV-1 Protease Inhibitors & Synthesis, Biological Activity and Mo lecular Modeling of 20S Proteasome Inhibitors Kiran Kumar Venkata Raja Avancha ABSTRACT HIV-1 protease binds to its peptide/prot ein substrates in extended conformations. Therefore protease inhibitors that are cons trained to form extended conformations are likely to produce very active protease inhibitors This is because they are pre-organized to form favorable interactions with the enzyme environment immediately surrounding the active site. With this hypothesis in mind, we designed a family of structurally related molecules, which contain dipeptide anal ogs constrained to adopt the extended conformation. Core structural intermediates that are required for the total synthesis of the novel class of HIV-1 protease inhibitors ar e outlined in Chapter One. Chapter Two discusses the enantioselective synthe sis of 2-alkyl-3-nitropropionates ( NPAs ) that is the part A of the cyclic urea molecule 8, and can also be used as the building block for the synthesis of unnatural -amino acids. In conclusion on this project, we were able to successfully achieve the novel enantioselective route for the synthesi s of NPAs and also obtain the absolute stereochemistry of one of the NPAs by solving the crystal structure. Various routes were explored for the synthesis of the substituted orthogonally protected geminal diamino acids ( OPGDAs ) and these were discussed in Chapter Three. Chapter Three also discusses the synthesis of a versatile N-Boc transfer reagent and the

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xv applications of it in the synthesis of helix mimics. The outcomes of this project were the efficient synthesis of oxaziridine (104) and the methods that show how we cannot make the substituted OPGDAs which can serve as the guidance for future research on them. The proteasome is cellular machinery that is responsible for the breakdown of the complex proteins that are not required by a living cell. The inhibition of its activity in cancerous cells can promote apoptosis. Chapte r Four discusses the synthesis of a new class of 20S proteasome inhi bitors, their biological testi ng and lead optimization by molecular modeling, library synthesis and bi ological evaluation. In short this project achieves our goal for the synthesis of a novel cl ass of 20S proteasome inhibitors that have a potential to act as drug molecules in the future.

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1 CHAPTER ONE HIV PROTEASE INHIBITORS: INTRODUCTION 1.1 Global epidemic: AIDS Acquired Immunodeficiency Syndrome (AIDS) is an epidemic, which forced the 21 st century human population into the grip of constant fear. Among the top ten global health issues of 2005 published by the World Health Organization (WHO), HIV/AIDS was next only to the avian influenza. It was also ranked number two among the top 10 neglected global health issues of the year 2005. The etiolo gical agent that causes AIDS was previously determined to be a retrov irus, Human Immunodefici ency Virus (HIV). According to the statistics released by WHO, in the year 2005 alone, five million people were newly infected with HIV and more than three million died of HIV/AIDS related issues, among them more than half a million were children. AIDS has killed more than 25 million people since it was first defined in the year 1982 as the manifestation of a deficiency in the human immune system. It is one of the most destructive epidemics in recorded history. 1 1.2 HIV-1 Protease: Reason for its inhibition HIV is a retrovirus with ribonucleic acid (RNA) as the core genetic material. Hence it is also classified under RNA viruses. Since it cannot replicate by itself it has to find a living host cell for its survival. HIV infects the CD4+ lymphocytes, macrophages and dendritic cells. HIV viral replication requires deoxyribonucleic acid (DNA) synthesis

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and viral protein synthesis. The major enzyme in HIV responsible for making the DNA copy of the viral RNA is reverse transcriptase. This DNA copy is then incorporated into the host genome, which is subsequently transcribed into both genomic RNA and mRNA, which are translated into viral proteins. The release of the viral particles results in the manifestation of AIDS. In general, host mRNAs code directly for functional proteins, but in HIV, the RNA is translated into biochemically inert polyproteins. A virus-specific protease then converts these polyproteins into various structural and functional proteins by cleaving at specific sites. HIV-1 Protease is a virus specific protease that binds to the peptide substrates in extended conformations. It is responsible for the maturation of HIV into infectious particles. Because of the absence of this protease in the host cells, HIV protease inhibitor design and synthesis gained tremendous importance over the last decade. The life cycle of the HIV virus and the stage at which HIV protease inhibitors act, is depicted in Figure 1.1. mature extracellular virion nucleus cell membrane cytoplasmAttachment & fusion penetration & uncoating viral RNA cDNA & RNA complex d ou b lest r an d e d unintegrated DNAReversetrancriptase Provirus integration trancription translation Assembly Budding and release Maturation structural proteins regulatory proteins viralmRNAHIV-Protease InhibitorsHIV particle nucleocaspid viral RNA Host Chromosome Provirus X Figure 1.1:Replicative cycle of HIV-1, and the site of action of HIV-1 protease inhibitors 2

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1.3 HIV-1 Protease structure and function The first structure of HIV-1 protease was reported by Navia et al. in 1989. 2 After a decade, more than two hundred crystal structures of various genetic strains of HIV-1 protease and complexes with various enzyme inhibitors and mutant enzymes were deposited in the Protein Data Bank (PDB). HIV-1 protease is a C 2 symmetric homodimer, which consists of two monomers with 99 amino acids in each. Both the monomers are of similar configuration. Each monomer contributes an aspartic acid to form the catalytic site. 3 The preferred cleavage site of the protease enzyme is the Nterminal side of the proline residues, especially between phenylalanine and proline. There are also other general cleavage sites like Tyr/Pro, Leu/Ala, Met/Met, Phe/Tyr, Phe/Leu, and Leu/Phe, which were mentioned in the literature. 4, 5 The design and development of the HIV-1 protease inhibitors became possible because of the X-ray crystal structure of the HIV-1 protease enzyme (Figure 1.2). Figure 1.2:Three-dimensional structure of aspartyl protease from HIV-1 4 The two monomeric chains assemble in such a fashion to form a long tunnel, which is covered by two flexible protein flaps. The flaps open up and the enzyme wraps around the protein chain or the substrate (Figure 1.3). This results in holding the substrate tightly in the tunnel for the cleavage. There is a water molecule in the center of 3

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the tunnel, which is responsible for breaking the protein chain/substrate. This is the active site of the HIV-1 protease. There is no crystal structure of the protein chain bound to the active form of HIV-1 protease because the chain would be cleaved before the crystal structure was solved. The aspartyl residues located in the active site are generally in a tripeptide sequence, Asp-Thr-Gly, which is covered over by two -hairpin structures or flaps. These glycine rich flaps are highly flexible and undergo conformational changes that are localized, so that the substrate and inhibitors can be bound and released. 6 The active site triad is located within the loop that is stabilized by a network of hydrogen bonds formed with the corresponding loop of the other monomer. 7 (a) (b) Figure 1.3:HIV-1 Protease enzyme (a) tunnel view (b) Flaps in action The proposed catalytic mechanism and the transition state intermediate of the amide hydrolysis by an aspartic protease (Figure 1.4) clearly shows that the two aspartic residues participate in general-acid general-base catalysis of the addition of oxygen of water at the carbonyl of the scissile amide. 8 4

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5 OC Asp O HO H HN C R N O CO H O A sp OC Asp O HO H NH C R N O CO HO Asp CO H O Asp HN C R O OH HN OC Asp O + Figure 1.4:Enzyme reaction mechanism of HIV protease/Aspartyl protease According to Schechter and Berger, 9 the standard nomenclature of any peptide substrate complexed with aspartic protease like HIV-1 protease, the active site of the enzyme is designated with subsites S 1 S 2 ,.......S n and S 1 S 2 ,..S n located on both sides of the scissile bond. Where as the amino acid residues of the substrate or the inhibitor are termed P 1 P 2 ,.......P n and P 1 P 2 ,.........P n and counted from the point of cleavage and have the same numbering as the enzyme subsites they occupy (Figure 1.5). P3 NH O P2 HN O P1 NH O P1' HN O P2' NH O P3' S1'S3'S2S3S1S2' Figure 1.5:Standard nomenclature of peptide substrate of aspartic proteases S 1 S 2 .....S n subunits binding specificity for the HIV-1 protease inhibitors is one of the most important features that are needed to be taken in to consideration during the design of the potent inhibitors with less toxic profiles.

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1.4 HIV Protease inhibitors approved by FDA N NH O CONH2 HN O OH N CONH H H (1) Saquinavir[Brand names are INVIRASE and FORTOVASE]N N N OH HN O CONH OH (2) Indinavir[Brand name is CRIXIVAN] HO CH3 O N H OH N S H H (3) Nefinavir[ Brand name is VIRACEPT]O O HN O OH N S NH2 O O (4) Amprenavir[ Brand name is AGENERASE]HNN O HN O OH NH O O (5) Lopinavir[ Brand name is KALETRA]SN N H3C NH O HN O NH O O NS OH (6) Ritonavir[Brand name is NORVIR] Figure 1.6:FDA approved HIV protease inhibitors 10-16 and their brand names There are currently six HIV protease inhibitors that are approved by the FDA. They all act by a similar mechanism. By binding reversibly to the active site of the HIV-1 Protease enzyme, thus resulting in the prevention of cleavage of the viral precursor proteins/polypeptides. All six HIV protease inhibitors currently available in the market are shown in the Figure 1.6. All of the HIV protease inhibitors that have been approved and the ones that are in development are peptidomimetics with non-hydrolysable transition-state mimetics. The cleavage site at the peptide linkage is replaced by 6

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7 transition-state isosteres, such as hydroxyethylamine, which is found in all of the currently approved drugs. The si de chains in the inhibitors can protrude in to the welldefined binding pockets, or subsites, in the ac tive region of the enzyme and thus result in the tight binding interactions. 1.5 HIV-1 Protease Inhibitors with Extended Conformations Since HIV-1 protease binds to their pe ptide/protein substrates in extended conformations, protease inhibitors constrained to form extend ed conformations are likely to produce very active protease inhibitors because they are pre-organized to form favorable interactions with the enzyme environment im mediately surrounding its active environment. We wish to test this hypothesi s, by synthesizing a family of structurally related molecules, which contain dipeptide analogs constrained to adopt the extended conformation. We also planned to use our dipept ide-like units as a central core and attach peripheral groups to the Nand Ctermini that are selected by stru ctural analogy with the most successful currently available HIV-1 protease inhibitors. Due to the synthetic difficulties, the currently approved hydroxyethyl amines and analogs are generally acyclic and therefore are not pre-organized to form the extended conformation. The constrained dipeptide units (DPUs ) we have designed, structurally mi mic natural peptides in extended conformation. There are only a handful of repor ted compounds that ar e pre-organized to adopt the extended conformation before binding and one of these is a 5 pM Ki inhibitor cyclic urea 7 (Figure 1.7 ) 17 which is about 1000 times more active than the approved drugs discussed above. The acyclic flexible hydroxyethyl amine-based drugs strongly bind to the HIV-1 protease due to a favorable combination of specific electrostatic and hydrophobic interactions between the enzyme a nd the inhibitors. But, these flexible

PAGE 27

inhibitors must lose conformational entropy when they bind, whereas, inhibitors that have the same specific electrostatic and hydrophobic interactions and that are pre-organized to adopt the favorable extended conformation can bind with much greater affinity and be much more potent inhibitors. 18 Compound 7 is constrained to adopt an extended-like conformation at the active site of the enzyme and it also has a carbonyl oxygen that replaces the water number 301 found in the crystal structures of the protease structures bound with other flexible HIV-1 protease inhibitors. It is believed that water number 301 plays a key role by helping to stabilize the extended conformation of flexible inhibitors by bridging a hydrogen-bonding network between the amide hydrogens at Ile 50 and Ile 150 and the carbonyl oxygens of the flexible inhibitors. 18 NNN O Ar A r Ph Ph HO 7 Figure 1.7:Constrained cyclic urea 7, with reported picomolar activity There are five peptide backbone bonds in a dipeptide unit. Figure 1.8 illustrates our definition of the peptide backbone bonds of dipeptide unit and two constrained dipeptide units that we have designed. In Figure 1.8, structure A is a canonical dipeptide unit shown in context of a larger peptide. The peptide backbone bonds are shown in bold and these bonds are labeled DP1 DP5. The double bond character of the DP3 bond ensures that either a trans or cis conformation is adopted at that bond; the trans conformation is strongly favored in most peptides. The dipeptide units that we have designed are the Friedinger lactam derivatives DPU (DiPeptide Unit) and the novel ADP 8

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(Aza DiPeptide unit) that are shown below. The unnatural six-membered lactam of DPU constrains the DP2 and DP3 bonds to adopt the extended conformation while maintaining a natural extended strand-like structure on the lower surface. The unnatural six-membered succinylhydrazide ring of ADP constrains the DP2 and DP3 bonds to adopt the extended conformation and the intramolecular hydrogen bond built into ADP restrains the DP4 and DP5 bonds to adopt the extended conformation while maintaining a natural extended strand-like structure on the lower surface. ON N O N HO H H R1 R2 DP2DP3ON NN O N HO H H R1 R2 O H DP2DP3DP4DP5DPUADPONR1 NH O N HO R2 H H DP1DP2DP3DP4DP5H A Figure 1.8:(A) Dipeptide bonds in a dipeptide unit; Constrained DPUD i P eptide U nit; ADP-Novel A za D i P eptide unit 1.5.1 Design Considerations We advance our design strategy further by mimicking the hydroxyethylamine derivative drugs that still comprise constraints on dipeptide bonds, and that are similar to that of the most active inhibitor like cyclic urea 7. The design we considered should also conserve the extended conformation by constraining the DP1 bond as well because of the intramolecular hydrogen bonding built into the cyclic urea 8 (Figure 1.9). This cyclic urea 8 is also similar to hydroxyethylamine derivative where the partially reduced amide bond is a result from the ketone derivative of the ADP. 9

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NN O H H OH N N O H O R1 H H R2 DP1DP4DP3DP2DP58 Figure 1.9:Novel cyclic urea 8 with all the dipeptide bonds constrained The proposed target molecule 8 (Figure 1.9) is worthy of investigating because it is constrained to adopt the backbone conformation that HIV-1 protease binds, but also enables the design and testing of the partially reduced amide bond derivative as a completely novel HIV-1 protease inhibitor. The partially reduced amide bond derivative will be little more flexible than when compared to ADP and may better mimic the tetrahedral proteolysis intermediate like the hydroxyethylamine derivatives. Even the carbonyl oxygen for our urea 8, is similarly oriented as that of the carbonyl oxygen of the urea 7, which was important in the replacement of the water number 301 in the protease binding pocket. We also hypothesize that our constrained cyclic urea 8 with side chains that optimize the binding to the S 1 and S 1 HIV-1 protease binding sites and peripheral groups that optimize binding with the S 3 S 2 and S 2 S 3 HIV-1 protease binding sites respectively, will probably produce the most active HIV-1 protease inhibitors ever prepared. This can also be achieved by attaching the readily available flexible 10

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hydroxyethylamine cores to the cyclic urea 8 that we planned to synthesize. This contributes to optimized binding of our novel HIV-1 protease inhibitors into S 3 S 2 and S 2 S 3 HIV-1 protease binding sites. 1.5.2 Retrosynthetic analysis of cyclic urea 8 The Retrosynthetic analysis of the cyclic urea 8, which is depicted in figure 1.10, clearly indicates that it can be generated via a nitroaldol condensation (Henry reaction) of the 2-subsituted 3-nitropropionate (NPA) A with the aldehyde version of the orthogonally protected geminal diamino compound (OPGDAs) B. The resulting nitro alcohol is subjected to tandem reduction of nitro group and hydrogenolytic cleavage of the Cbz protecting group of B, followed by ring closure should form the proposed six-membered cyclic urea 8. NN O H H OH N N O H O R1 H H R2 12345678 NO2 H OH O R2 5678NH(R) (R')HN COOR'' R1 1234 B ACha p ter-3Cha p ter-2 Figure 1.10:Retrosynthesis of cyclic urea 8 11

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12 Chapter-2 discusses the synthesis of various NPAs and Chapter-3 discusses the efforts towards the synthesis of the substituted OPGDAs which are the precursors for the novel HIV-1 protease inhibitor 8 1.5 References 1) UNAIDS. AIDS epidemic update: December 2005. UNAIDS/05.19E. 2) Navia, M. A.; Fitgerald, P. M.; McKeever, B. M.; Leu, C. T.; Heimbach, J. C.; Herber, W. K.; Sigal, I. S.; Da rke, P. L.; Springer, J. P. Nature 1989, 337 615620. 3) Pearl, L. H.; Taylor, W. R. Nature 1987, 329, 351-354. 4) PDB ID: 2HVP Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach, J. C.; Dixon, R. A.; Scolnick, E. M.; Sigal, I. S. Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 46864690. 5) Flexner, C. The New England Journal of Medicine 1998, 338, 1281. 6) Abdel-Rahman, M. H.; Al-karamany, S. G.; El-Koussi, A. N.; Youssef, F. A.; Kiso, Y. Curr. Med. Chem. 2002, 9, 1905-1922. 7) Martin, J. A.; Redshaw, S.; Thomas, G. J. Progress in Med. Chem. 1995, 32 239. 8) Darke, P. L.; Huff, J. R. Advances in Pharmacology 1994, 25, 399. 9) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157 10) Craig, J. C.; Duncan, I. B.; Hocley, D.; Grief, C.; Roberts, N. A.; Mills, J. S. Antiviral Res. 1991, 16, 295 305.

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13 11) Patick, A. K.; Markowitz, M.; Appelt, K.; Wu, B.; Musicl, L.; Kalish, V.; Kaldor, S.; Reich, S.; Ho, D.; Webber, S. Antimicrob. Agents Chemother. 1996, 40, 292 297. 12) Sham, H. L.; Kempf, D. F.; Molla, A. ; Marsh, K. C.; Kumar, G. N.; Chen, C.M.; Kati, W.; Stewart, K.; Lal, R.; Hsu, A.; Betebenner, D.; Korneyeva, M.; Vasavanonda, S.; McDonald, E.; Saldivar A.; Wideburg, N.; Chen, X.; Niu, P.; Park, C.; Jayanti, V.; Gradowski, B.; Gr anneman, G. R.; Sun, E.; Japour, A. J.; Leonard, J. M.; Plattner, J. J.; Norbeck, D. W. Antimicrob. Agents Chemother. 1998, 42, 3218 3224. 13) Dorsey, B. D.; Levin, R. B.; McDaniel, S. L.; Vacca, J. P.; Darke, P. L.; Zugay, J. P.; Emini, E. A.; Schlief, W. A.; Qu intero, J. C.; Lin, J. H.; Chen, I.-W.; Holloway, M. K.; Fitzgerald, P. M. D.; Axel, M. G.; Ostovic, D.; Anderson, P. S.; Huff, J. R. J. Med. Chem. 1994, 37, 3443 3451. 14) Vacca, J. P.; Dorsey, B. D.; Schlief, W. A.; Levin, R. B.; McDaniel, S. L.; Darke, P. L.; Zugay, J.; Quin tero, J. C.; Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabach, A. J.; Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lin, J.; Chen, I.-W.; Vastas, K.; Ostovic, D.; Anderson, P. S.; Emini, E. A.; Huff, J. R. Proc. Natl. Acad. Sci. 1994, 91, 4096 4100. 15) Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B. G.; Tung, R. D.; Navia, M. A. J. Amer. Chem. Soc. 1995, 117, 1181 1182. 16) Sham, H. L.; Betebenner, D. A.; Chen, X.; Saldivar, A.; Vasavanonda, S.; Kempf, D. J.; Plattner, J. J.; Norbeck, D. W. Bioorg. Med. Chem. Lett. 2002, 12, 1185 1187.

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14 17) Sham, H. L.; Zhao, C.; Stewart, K. D. ; Betebenner, D. A.; Lin, S.; Park, C. H.; Kong, X.-P.; Rosenbrook, W., Jr.; Herri n, T.; Madigan, D.; Vasavanonda, S.; Lyons, N.; Molla, A.; Saldivar, A.; Marsh, K. C.; McDonald, E.; Wideburg, N. E.; Denissen, J. F.; Robins, T.; Kempf, D. F.; Plattner, J. J.; Norbeck, D. W. J. Med. Chem. 1996, 39, 392 397. 18) Bartlett, P.A.; Yusuff, N.; Pyun, H.-J.; Rico, A.C.; Meyer, J.H.; Smith, W.W.; Burger, M.T. "Design of Macrocyclic Pep tidase Inhibitors: The Related Roles of Structure based Approaches and Library Chemistry", in "Medicinal Chemistry into the Millenium", Campbell, M.M., Blagbrough, I.S., Eds., Royal Society of Chemistry, Cambridge (UK), 2001.

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15 CHAPTER TWO SYNTHESIS OF 2-SUBSTITUTED 3-NITROPROPIONATES: PART-A OF CYCLIC UREA 8 2.1 Introduction During the last few years, the preparation of -amino acids that are 1,3difunctionalized have attracted significant attention since they turned out to be components of an array of natural products, such as taxols,1 the dolastatins,2 and also act as antihyperglycemic/antiobes ity compound building blocks.3 Recently they have also emerged as a class of unnatural biopolymers th at offer interesting secondary structures with high potency. Furthermor e, a number of open-chain4 or cyclized5 -amino acids exhibit interesting pharmacological properties a nd act as precursors for stabilized helical peptides with enhanced resist ance to enzymatic degradation.6 Often the substitution of amino acids with -isomers in biologically active peptides increases the activity and enzymatic stability of the resulting peptide.7 Several -amino acids are pharmacologically active and are also the ke y structural components of biologically active terpenes, alkaloids, macrolides and -lactam antibiotics.8 (Figure 2.1) One more reason for the increased development of -amino acid chemistry is the consequence of their ample role in numerous aspects of synthetic organic chemistry, namely their utility as chiral auxiliaries, chiral ligands, chiral building blocks and intermediates in the synthesis of -lactams.9

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16 Figure 2.1:-amino acid as the compon ent of bioactive compounds Although many methods for synthe sizing enantiomerically pure -amino acids have been reported, great effort continue s to be devoted towards more efficient enantioselective methods. Cardillo10 and Konopelski11 used substituted perhydropyrimidin-4-ones, for stereoselectiv e alkylation and subse quent hydrolysis to afford the desired substituted -amino acids. Juaristi and Seebach12 showed the usefulness of chiral deriva tives of 3-aminopropionic acids in order to synthesize branched -amino acids. The syntheses described in the literature using Oppolzer’s sultam,13 the Evans chiral auxiliary,14 or Seebach and Juaristi’s15 chiral pyrimidinone methodology appeared to give highly scalemic -substituted -amino acids. Myers16 has shown (1 R ,2 R )-(-)-pseudoephedrine to be an efficient and inexpensive chiral auxiliary in the stereoselective synthesis of unnatural -amino acids. This methodology has also been extended for the synthesis of chiral -hydroxy acids17 and chiral -substituted acids.18 R NO2 O HO N N N R3 O O R4 R2 R1 H N H H N O H2N R O NH O R COOH R H N NR H2N Peptides Lactam AntibioticsAnxiolytics/antiarrhythmics/ CCK antagonists Antidiabetic/Antiobesity agent Cons t r aine d t u r n m i m e t ic

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17 Further, Nagula and co-workers19 have shown the successful use of the Myers chiral auxiliary for the stereoselective synthesis of -substituted -amino acids. Seebach et al.20 employed methyl 3-nitropropiona tes for the synthesis of 2-alkyl3-nitropropionates in th eir racemic form via -doubly deprotonated species (Figure 2.2 ). Figure 2.2:Seebach’s racemic synthesis of 3-NPA Recently, a few reports were published on the enantioselective synthesis of substituted-nitropropionates using nitroacrylates and dialkylzinc or trialkyl aluminium reagents in the presence of a chiral catalyst (Figure 2.3 ).21 These methods have some inherent limitations, as the yields and enantio control are dependent on the nature of alkyl zinc or aluminum used as well as the ester alkyl group of the nitro acrylate. In addition, these methods are only providing e.e.’s in the range of 5 to 87%, the later being achieved by several optimizations of reaction conditions. Figure 2.3:Rimkus et al. and Eilitz et al.’s enantiomeric route for 3-NPA synthesis H3CO O H NO2 H 2Li + RX -78 0C to -30 0C 70-80% O H3CO NO2 R COOR O2N R2Zn or R3Al, Cu(I) O O P N Ph Ph ROOC R NO2

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18 Thus, there is a need for more robust and versatile enantioselective routes for the synthesis of biologically important -substituted-nitropropionates. We report here a synthetic strategy for the prepar ation of enantiomerically pure -alkyl-nitropropionates starting from inexpensive alkyl malonates. These compounds are very interesting from a synthetic point of view; because of the ease with which they form carbon-carbon bonds in reactions such as Henry and Michael additions.22 The -amino acids can be obtained easily by catalytic hydrogenation of the -nitro acids, which was not the primary focus of this project. In addition, the -NO2 functionality can be transf ormed into an aldehyde or a carboxylic acid.23 Our asymmetric route for the synthesis of potential -alkyl-nitro propionates is novel, highly appl icable, and can be useful fo r the generation of array of nitro propionates by varying the alkyl group at the -carbon. A highly enantioselective s ynthesis of 2-substituted 3nitropropionates is reported using Pseudomonas cepacia lipase (Amano PS-30)-mediate d desymmetrization of the prochiral 2-alkyl-1,3-propanediol as the key reaction step and subsequent transformation of optically pure monoacetates into met hyl 2-alkyl-3-nitropropionates by functional group interconversions. 2.2 Results & Discussion A retrosynthetic analysis of target compounds ( 17a-d ) prompted us to investigate the lipase-catalyzed desymmetrization of the prochiral 1,3-diol intermediate (Refer to scheme in figure 2.4 ). The most striking aspect of this desymmetrization reaction is the enantiocontrol exhibited by the biocatalys t Amano PS-30 Lipase. These prochiral 1,3diols were successfully converted to compounds 11a d in high yields with incorporation of the stereocenter. Our methodology for using this desymmetrization reaction is a very

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19 useful strategy because of th e high yields and the ease with which enantiomerically pure compounds ( 11a d ) were made. This pivotal lipase r eaction is a well known type, indeed, compound 11a was prepared previously in a near identical manner by Itoh et al. and recently by Duhamel and Duhamel, albeit with a different Psedomonas lipase.24 This route also enabled us to achieve stereocenter early in synthetic route and maintain it till the end. The key factor involved in these biocat lysis reactions is the correct choice of the lipases for the desymmetrization of the proc hiral 1,3-diols. But that was successfully achieved because the lipase we selected Amano PS-30 lipase had the ability to undergo various conformational changes in order to accommodate substrates of different sizes.25 This was proved further by the variety of the subsrates selected in this synthetic route. Our synthesis of methyl 2-alkyl-3-nitropr opionates began with the reduction of the substituted diethyl malonates 9a-d into the 2-substituted-1,3-propanediols 10a-d in yields of 70-90% (Step 2 in Figure 2.4 ).26 The transesterification of re sulting diol using lipase from Pseudomonas cepacia (Amano lipase PS-30) and vinyl acetate in THF afforded racemic monoacetates. However, when the diol was treated with neat vinyl acetate in the presence of the lipase, an excellent yield of pure monoacetate 11a was obtained that proved to be of high enantiomeric excess. This modified reactio n condition was succe ssfully applied for making compounds 11b-d in high enantiomeric excess and good yields. The enantiopurity of these monoacetat es was estimated from their 1H NMR spectra acquired in the presence of (+)-Eu(hfc)3 (Figure 2.5 ).

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20 Figure 2.4:General scheme for the synt hesis of 2-alkyl-3-nitropropionates R OTs AcO R OH AcO R NO2O H3CO R NO2O HO R OH HO R I AcO R OEt OEt O O R NO2HO R NO2AcO 9a-d 10a-d 11a-d12a-d 13a-d 14a-d 15a-d 17a-d 16a-d(i) LiAlH4, THF, r.t., (ii) Lipase PS-30, vinyl acetate, r.t., (iii) Acetic anhydride, pyridine, r.t., (iv)Tosylchloride/ TEA; DCM, r.t., (v) NaI, acetone, reflux (vi) AgNO2, Phloroglucinol, DMF, r.t.,(vii) K2CO3/ aq. MeOH, 0 oC (viii) Jone's Oxid., 0 oC to r.t., (ix) CH3I, K2CO3, DMF, r.t.(i) (ii) (iv) (v) (vi) (vii) (viii) (ix) R OH AcO 11a*-d* (racemic) (iii) R OTs AcO 12b* and 12c* (racemic) (iv) ab cd

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21 Figure 2.5:1H NMR’s for the optically pure and racemic monoacetates and monoalcohols with and without Eu(hfc)3 (A) 1H NMR of optically pure 11a with Eu(hfc)3. (B) 1H NMR of racemic 11a* with Eu(hfc)3. (C) 1H NMR of optically pure 12b with Eu(hfc)3. (D) 1H NMR of racemic 12b* with Eu(hfc)3. (E) 1H NMR of optically pure 12c with Eu(hfc)3. (F) 1H NMR of racemic 12c* with Eu(hfc)3. (G) 1H NMR of optically pure 11d with Eu(hfc)3. (H) 1H NMR of racemic 11d* with Eu(hfc)3.

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22 Figure 2.6: NMR Determination of En antiomeric Purity Using Eu(hfc)3 As a control, we checked the effect of the chiral shift re agent on the racemate methyl protons (in 11a* and 11d* prepared by treating the diols with acetic anhydride/DMAP in THF)27 and the tosylate methyl (in 12b* and 12c* prepared by tosylation of the corresp onding racemic monoacetates 11b and 11c *). The appropriate methyl peaks split into two signals of equal in tensity for the racemates in the presence of (+)-Eu(hfc)3 (Figures 2.5 & 2.6 ). The product monoacetates 11a and 11d obtained by lipase catalyzed desymmetrization showed a single singlet for methyl protons in the presence of (+)-Eu(hfc)3, while tosylates 12b and 12c show a single singlet for the tosylate methyl proton (Figure 2.6 ).28 The rationale behind the synthesis and use of 12b* and 12c* for chiral shift NMR experiments is that the racemic monoacetates 11b* and 11c* in the presence of (+)-Eu(hfc)3 even at 1:7 ratio failed to split into two equal intensity signals. The enantiomeric exce ss is reported in the table in figure 2.7 H3CCH3H3C O O CF2CF2CF3 3 Eu O O O S O O CH3 + Racemate Optically Pure H3CCH3H3C O O CF2CF2CF3 3 Eu Racemate Optically Pure + OH O O CH3 (12b*) (12b) (11c*) (11c)

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23 Figure 2.7:Lipase PS-30 catalyzed transe sterification of substrates 10a-d (Results in tabular form). Substrate Product R Time (h)Yield % e.e.a % Config. 10a 10b 10c 10d 11a 11b 11c 11d -CH2C6H5 -CH2C6H11 -CH2CH(CH3)2 -CH2CH2CH2CH3 6 5 12 8 98 90 88 92 >99 >99 >97 >80 b b R b a Determined from 1H NMR spectra in the presence of Eu(hfc)3, by integration of either C H3C=O ( 11a and 11d) or tosylate C H3 ( 12b and 12c ). b No X-ray diffraction quality crystals were obtained for the salts of acids 16a, 16b, and 16d with (1 R ,2 R )-(-)-pseudoephedrine. The enantiomerically enriched monoacetates were successfully converted to their respective tosyl derivatives 12a, 12c, and 12d upon treatment with tosyl chloride, triethyl amine, and DMAP in DCM in excellent yi elds. The similar reaction condition did not work for making compound 12b instead replacement of triethyl amine and DMAP by pyridine yielded 12b in good yield. Direct replacement of tosyl group with nitro function by using NaNO2 or AgNO2 failed to furnish the desired products in acceptable yields. The formation of a silver-halide complex is an important driving for ce for the reaction of alkyl halides with silver nitrite.29 Hence, we decided to conve rt tosyl function into an iodide before treating with AgNO2. The SN2 displacement of the tosylate by treatment with sodium iodide in refluxing acet one afforded the corresponding iodides 13a-d in quantitative yields. Displacemen t of the iodide with NaNO2 in DMF did not afford the

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24 desired product in acceptable yield. However, AgNO2 provided the desired nitro product in <50% yield. The reaction of silver nitrite with alkyl halides is considered as a “pullpush” process, where the pull of silver on the halogen and the push of the NO2 are both crucial in the transition st ate. Interestingly, AgNO2 in the presence of 0.5 to 1.5 equivalents of nitrite scavenge r phloroglucinol in DMF gave the desired nitro products 14a-d in 45-75% yields. An iodide replacemen t by nitrite anion usually gives 40-70% yields of nitro compounds as NO2 is an ambident anion, both the nitrogen atom and oxygen atom can act as the nucleophilic site of reaction, leading to the desired nitro compound (RNO2) and the undesired nitrite (RONO), respectively.30 The nitro acetates 14a-d were hydrolyzed by treatment with potassium carbonate in MeOH-water to give the corresponding nitro alcohols 15a-d in 35-54% yields. The compounds 14b-d were hydrolyzed to afford 15b-d in poor yields without isolation of any side products. This might be due to the polar nature of these intermediates as even after saturating with brine, these -nitroalcohols were difficult to isolate. The resulting nitro alcohols were subjected to Jones oxi dation to afford corresponding carboxylic acids 16a-d in good to excellent yields. In order to measure the absolute configuration, the resulting carboxylic acids were mixed with equimolar amounts of (1 R ,2 R )-(-)pseudoephedrine 18 in a minimum amount of DCM a nd the solvent was allowed to evaporate at room temperature to obtain crystalline samples of the corresponding salts. The results of the representative st ructure determination study of the salt 19, of 2isobutyl-3-nitropropionic acid ( 16c ) with (1 R ,2 R )-(-)-pseudoephedrine ( 18 ) confirmed the ( R )-enantiomer is produced as shown in Figure 2.8 (The detailed crystal structure is reported in Appendix B).

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25 Figure 2.8: Structure of the salt 19, of 2-isobutyl-3-nitroprop ionic acid (16c) with (1 R ,2 R )-(-)-pseudoephedrine (18) determin ed by X-ray structural analysis Unfortunately, we were unable to obtain high quality crystals for the salts of carboxylic acids 16a, 16b and 16d Our attempts to make diastereomeric amides of these acids with optically pure R (+)-methylbenzylamine by standard peptide coupling method resulted in poor qual ity crystals (data not sh own). We hypothesize that compounds 16a, 16b and 16d also have the ( R ) absolute stereochemistry at the chiral center. Our hypothesis is further supported by the analogy between th e side chains in O2N OH O N H CH3 CH3 OH + 16c18

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26 terms of bulk and branching associated with substrates 10a, 10b, and 10d to that of isobutyl side chain in substrate 10c and all of these substrates should assume a similar docking orientation onto the lipase. In addi tion, transesterifica tion of 2-benzyl-1,3propanediol catalyzed by Pseudomonas fluorecens lipase in the presence of vinyl acetate as acyl donor afforded ( R )-3-acetoxy-2-benzyl-1-propano l with enantiomeric excess >94%.31 Finally, esterification of the carboxylic acids 16a-d using methyl iodide and potassium carbonate in DMF le d to the target compounds 17a-d in good to excellent yields. 2.3 Conclusion In conclusion, we came up with a novel and efficient synthetic route for the enantioselective synthesis of NPA’s. This route can be followed for the large scale synthesis of the NPA’s, which can furt her be used for the synthesis of the -amino acids. Also, we were able to obtain the absolu te stereochemistry of the substituted nitropropionic acids due to the solving of the crystal structur e of the isobutyl substituted nitropropionic acid. 2.4 Experimental Data 2.4.1 Materials and Methods: Organic and inorganic reag ents (ACS grade) were obtained from commercial sources and used without further pur ification, unless otherwise noted. Pseudomonas cepacia lipase (Amano PS-30) was obtained from Amano Pharmaceuticals. Thin layer chromatography (TLC) was performed on gla ss plates (Whatman) coated with 0.25 mm thickness of silica gel 60 with fluoresce nt indicator. Column chromatographic purification was performed using silica ge l 60 (#70-230 mesh, Selecto Scientific).

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27 Melting points (uncorrected) were determined using Mel-Temp II, Laboratory Devices, MA, USA. All 1H and 13C NMR spectra were recorded on a Bruker 250 MHz or Varian INOVA 400 MHz spectrometer in CDCl3 or unless otherwise specified and chemical shifts are reported in ppm ( ) relative to internal tetramethylsilane (TMS). Infrared spectra of samples dissolved in DCM were acquired on a Avatar FT IR 320 spectrometer. Gas Chromatography Mass Spectra (LRMS) were recorded using a Shimadzu GC-17A GCMS-QP5000. High resolution mass spectra were obtained on an Agilent LC-MSDTOF. Preparation of the star ting esters (9a-d) Starting esters 9a 9c and 9d were obtained from commerci al sources. While ester 9b was prepared according to the following pro cedure: 60% sodium hydride/mineral oil (0.15 g, 3.75 mmol) was washed under argon flow with hexane (3 x 10 mL). The resulting free flowing powder of sodium hydr ide was suspended in DMF (5 mL) and was treated with diethyl malonate (0.5 g, 3.12 mmol) drop wise. The resulting reaction mixture was stirred for 1h at room temperature and treated with cyclohexylmethyl bromide (0.67 g, 3.8 mmol) slowly. The reaction wa s allowed to stir at room temperature for 12 h. The reaction mixture was quenched wi th water (50 mL), the aqueous phase was extracted with EtOAc (2 x 50 mL), combined or ganic layers were washed with brine (3 x 25 mL), dried over anhydrous Na2SO4, and evaporated under vacuo to afford the crude product. The crude residue wa s purified by column chromatography (3:7 EtOAc/hexane) OEt O OEt O

PAGE 47

28 to afford compound 9b (0.68 g, 85%); a pale yellow oil; Rf = 0.78 (3:7 EtOAc/Hexane); IR (cm-1) 1752; 1H NMR (CDCl3, 400 MHz) 0.88-0.94 (m, 2H), 1.14-1.28 (m, 10H), 1.62-1.73 (m, 5H), 1.77-1.81 (t, J= 7.2 Hz, 2H), 3.42-3.45 (t, J = 7.6 Hz, 1H), 4.164.21 (q, J = 6.8 Hz, 4H); 13C NMR (CDCl3, 62.5 MHz) 13.9, 25.9, 26.2, 32.7, 35.4, 35.9, 49.5, 61.1, 169.7 ppm; MS (GC/MS) m/z 256, 210, 182, 109, 95, 67, 55. A General Procedure for the Preparation of the 2-alkylpropane-1,3-diols (10a-d) The diols 10a-d were prepared according to the literature method25 from their corresponding diesters 9a-d Ethyl 2-benzylmalonate (10 g, 40 mmol), LiAlH4 (6.1 g, 160 mmol), THF (75 mL) gave compound 10a (5.94 g, 90%); a white powder; mp 65-67 0C (lit. mp 66-68); Rf = 0.18 (1:1 EtOAc/hexane); IR (cm-1) 3350; 1H NMR (CDCl3, 400 MHz) 1.97-2.06 (m, 1H), 2.57-2.59 (d, J = 7.6 Hz, 2H), 2.97 (s, 2H), 3.59-3.64 (d, J = 9.6 Hz, 2H), 3.73-3.76 (d, J = 10 Hz, 2H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H); 13C NMR (CDCl3, 100 MHz) 34.4, 44.0, 65.4, 126.3, 128.6, 129.2, 140.0 ppm; MS (GC/MS) m/z 148, 130, 117, 91. 2-Cyclohexylmethylpropane-1,3-diol (10b). 9b (6.21 g, 24.3 mmol), LiAlH4 (2.30 g, 60.6 mmol), gave compound 10b (3.09 g, 75%); as a white powder; mp 64-66 0C; Rf = 0.29 (1:1 EtOAc/hexane); IR (cm-1) 3292, 1275; 1H HO OH H O O H

PAGE 48

29 NMR (CDCl3, 250 MHz) 0.79-0.92 (m, 2H), 1.03-1.08 (t, J = 7.0 Hz, 2H), 1.15-1.31 (m, 4H), 1.68-1.73 (m, 5H), 1.87-1.96 (m, 1H), 2.71 (br s, 2H), 3.57-3.64 (dd, J = 10.4 Hz, 7.5 Hz, 2H), 3.77-3.82 (dd, J = 10.5 Hz, 3.4 Hz, 2H); 13C NMR (CDCl3, 62.5 MHz) 26.2, 26.5, 33.5, 34.8, 35.2, 38.6, 67.0 ppm; MS (GC/MS) m/z 136, 121, 107, 96, 81, 67, 55. 2-Isobutylpropane-1,3-diol (10c). 9c (25.2 g, 112.2 mmol), LiAlH4 (10.6 g, 280.6 mmol), gave compound 10c (14 g, 94%); a colorless oil; Rf = 0.45 (1:1 EtOAc/hexane); IR (cm-1) 3395, 1262; 1H NMR (CDCl3, 250 MHz) 0.83-0.86 (d, J = 6.5 Hz, 6H), 0.98-1.04 (t, J = 7.0 Hz, 2H), 1.54-1.59 (m, 1H), 1.74-1.80 (m, 1H), 3.48-3.55 (dd, J = 10.7 Hz, 7.9 Hz, 2H), 3.66-3.72 (dd, J = 10.7 Hz, 3.7 Hz 2H), 4.13 (br s, 2H); 13C NMR (CDCl3, 62.5 MHz) 22.6, 25.1, 36.7, 39.3, 65.5 ppm; MS (GC/MS) m/z 114, 96, 84, 70, 56. 2-Butylpropane-1,3-diol (10d). 9d (5 g, 23.1 mmol), LiAlH4 (2.2 g, 57.8 mmol), yielded compound 10d (2.2 g, 72%); a colorless oil; Rf = 0.16 (1:4 EtOAc/hexane); IR (cm-1) 3368; 1H NMR (CDCl3, 250 MHz) 0.78-0.83 (t, J = 6.2 Hz, 3H), 1.12-1.22 (m, 6H), 1.63-1.66 (m, 1H), 3.47-3.54 (dd, J = 10.7 Hz, 7.8 Hz, 2H), 3.64-3.70 (dd, J = 10.8 Hz, 4.0 Hz, 2H), 4.77 (br s, 2H); 13C NMR HO OH H O O H

PAGE 49

30 (CDCl3, 62.5 MHz) 13.9, 22.8, 23.8, 27.3, 29.2, 41.8, 65.2 ppm. MS (GC/MS) m/z 114, 96, 84, 71, 57. A General procedure for the preparatio n of optically pure 3-Acetoxy-2-alkyl-1propanol (11a-d) To a mixture of Lipase PS-30 (1 g) and 10a (5 g, 30.12 mmol) was added vinyl acetate (50 mL) and reaction was sti rred at room temperature fo r the time shown in Figure 2.7 and filtered through a Celite pad and washed with ethyl acetate (50 mL). The filtrate was concentrated in vacuo to give 11a (6.25 g, 99%) as a colorless oil: Rf = 0.49 (1:1 EtOAc/hexane); IR (cm-1) 3447, 1729; 1H NMR (CDCl3, 400 MHz) 2.0 (s, 1H), 2.08 (s, 3H), 2.12-2.17 (m, 1H), 2.59-2.72 (m, 2H), 3.48-3.53 (dd, J = 11.2 Hz, 6 Hz, 1H), 3.583.62 (dd, J = 11.6 Hz, 4.8 Hz, 1H) 4.05-4.12 (dd, J = 11.2 Hz, 6.4 Hz, 1H), 4.16-4.20 (dd, J = 11.2 Hz, 4.8 Hz, 1H), 7.17-7-22 (m, 3H), 7.26-7.31 (m, 2H); 13C NMR (CDCl3, 100 MHz) 21.1, 34.5, 42.6, 62.2, 64.2, 126.4, 128.6, 129.2, 139.5, 171.9 ppm; MS (GC/MS) m/z 190, 148, 130, 117, 104, 91.Remaining monoacetates 11b-d were prepared similarly with the following results. The isolated yi eld and the optical purity of the products are listed in table in figure 2.7 A c O O H

PAGE 50

31 3-Acetoxy-2-cyclohexylmethyl-1-propanol (11b) 10b (5.71 g, 33.2 mmol), lipase (2.9 g), vi nyl acetate (50 mL), yielded compound 11b (6.43 g, 90%); a colorless oil; Rf = 0.43 (3:7 EtOAc/hexane); No spectral data for 11b as it was immediately converted to 12b 3-Acetoxy-2-isobutyl-1-propanol (11c). 10c (14.0 g, 106.0 mmol), lipase (7.0 g), viny l acetate (50 mL), yielded compound 11c (13.4 g, 73%); a colorless oil; Rf = 0.49 (3:7 EtOAc/hexane); IR (cm-1) 3474, 1736; 1H NMR (CDCl3, 400 MHz) 0.90-0.91 (d, J = 6.4 Hz, 6H), 1.12-1.21 (m, 2H), 1.60-1.68 (m, 2H), 1.88-1.91 (m, 1H), 1.98 (br s, 1H), 2.08 (s, 3H), 3.46-3.51 (dd, J = 11.2 Hz, 6.4 Hz, 1H), 3.57-3.61 (dd, J = 11.2 Hz, 3.6 Hz, 1H), 4.03-4.08 (dd, J = 11.2 Hz, 6.8 Hz, 1H), 4.19-4.23 (dd, J = 11.2 Hz, 4.4 Hz, 1H); No 13C NMR and mass spectral data for 11c as it was immediately converted to 12c A c O O H A c O O H A cO OH

PAGE 51

32 3-Acetoxy-2-butyl-1-propanol (11d) 10d (1.0 g, 7.57 mmol), lipase (0.25 g), viny l acetate (10 mL), yielded compound 11d (0.66 g, 50%); a colorless oil; Rf = 0.45 (2:5 EtOAc/hexane); IR (cm-1) 3466, 1736; 1H NMR (CDCl3, 250 MHz) 0.87-0.91 (t, J = 6.25 Hz, 3H), 1.15-1.30 (m, 6H), 1.73-1.83 (m, 1H), 2.70 (s, 3H), 3.46-3.63 (dd, J = 16.2 Hz, 6.3 Hz 2H), 4.03-4.10 (dd, J = 11.2 Hz, 6.7 Hz, 1H), 4.17-4.23 (dd, J = 11.2 Hz, 4.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz) 14.1, 21.1, 23.0, 27.7, 29.3, 40.6, 62.8, 64.9, 171.9 ppm; MS (GC/MS) m/z 114, 96, 84, 69, 55. A General Procedure for the preparation of 3-Acetoxy-2-alkyl-1-(4methylbenzenesulfonyloxy)propane (12a-d) To a solution of 11a (6.25 g, 30 mmol), triethyl amine (4.55 g, 45 mmol), DMAP (0.37 g, 3 mmol) in DCM (60 mL) was added to syl chloride (6.3 g, 33 mmol) at 0 0C and stirred at room temperature for 12 h. Reaction mixture was quenched with saturated NH4Cl solution (100 mL). The organic layer was se parated, and the aqueous phase was extracted with DCM (2 x 50 mL). The combined organi c layers were washed with brine (2 x 50 mL), dried over Na2SO4, and evaporated to give the product 12a (10.42 g, 95%) as a white powder. Mp 52-54 0C; Rf = 0.67 (1:1 EtOAc/hexane); IR (cm-1) 1731, 1359, 1171; 1H NMR (CDCl3, 400 MHz) 1.95 (s, 3H), 2.25-2.28 (m, 1H), 2.45 (s, 3H), 2.62-2.65 (dd, J = 7.2 Hz, 4.4 Hz, 2H), 3.90-4.04 (m, 4H), 7.04-7.06 (d, J = 7.6 Hz, 2H), 7.17-7.25 (m, 3H), 7.32-7.34 (d, J = 8 Hz, 2H), 7.75-7.77 (d, J = 8 Hz, 2H); 13C NMR (CDCl3, 100 A c O O T s

PAGE 52

33 MHz) 20.8, 21.8, 34.0, 39.6, 63.2, 69.1, 126.7, 128.2, 128.7, 128.9, 129.8, 132.9, 138.1, 145.1, 170.8 ppm; MS (GC/MS) m/z 148, 130, 117, 104, 91. The remaining acetoxy tosylates 12b-d were prepared similarly, with the following results. 3-Acetoxy-2-cyclohexylmethyl-1-(4-meth ylbenzenesulfonyloxy)propane (12b) 11b (3.11 g, 14.5 mmol), tosyl chloride (3.0 g, 16.0 mmol), pyridine (5.7 g, 72.5 mmol), yielded compound 12b (2.27 g, 43%); a colorless oil; Rf = 0.61 (3:7 EtOAc/hexane); IR (cm-1) 1733, 1237; 1H NMR (CDCl3, 250 MHz) 0.77-0.81 (m, 2H), 1.06-1.19 (m, 6H), 1.55-1.67 (m, 5H), 1.93 (s, 3H), 2.04-2.07 (m, 1H), 2.44 (s, 3H), 3.83-4.05 (m, 4H), 7.347.37 (d, J = 8.1 Hz, 2H), 7.78-7.81 (d, J = 6.6 Hz, 2H); 13C NMR (CDCl3, 62.5 MHz) 20.7, 21.6, 26.1, 26.4, 33.2, 33.2, 34.3, 34.4, 35.0, 63.6, 69.8, 127.9, 129.8, 132.8, 144.8, 170.7 ppm. MS (GC/MS) m/z 213, 155, 136, 124, 96, 67, 55. 3-Acetoxy-2-isobutyl-1-(4-methylbe nzenesulfonyloxy)propane (12c) 11c (13.4 g, 77.4 mmol), tosyl chloride (16.2 g, 85.2 mmol), pyridi ne (30.6 g, 387.3 mmol), yielded compound 12c (16.7 g, 66%); a colorless oil; Rf = 0.60 (3:7 EtOAc/hexane); IR (cm-1) 1732, 1265; 1H NMR (CDCl3, 250 MHz) 0.83-0.86 (d, J = 6.5 Hz, 6H), 1.11-1.18 (m, 2H), 1.52-1.59 (m, 2H ), 1.94 (s, 3H), 2.46 (s, 3H), 3.85-4.05 A c O O T s A c O O T s

PAGE 53

34 (m, 4H), 7.34-7.37 (d, J = 8.0 Hz, 2H), 7.77-7.81 (d, J = 8.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz) 20.9, 21.8, 22.7, 25.1, 35.4, 36.7, 63.7, 70.0, 128.1, 130.0, 145.0, 171.0 ppm. MS (GC/MS) m/z 155, 96, 84, 56. 3-Acetoxy-2-butyl-1-(4-methylbenze nesulfonyloxy)propane (12d) 11d (0.3 g, 1.72 mmol), tosyl chloride ( 0.66 g, 3.44 mmol), DMAP (0.02 g, 0.17 mmol), triethyl amine (0.5 mL, 3.44 mmol), yielded compound 12d (0.52 g, 89%); a pale yellow oil; Rf = 0.36 (1:5 EtOAc/hexane); IR (cm-1) 1732, 1176; 1H NMR (CDCl3, 250 MHz) 0.70-0.75 (t, J = 6.9 Hz, 3H), 1.11-1.22 (m, 6H), 1.82 (s, 3H), 2.33 (s, 3H), 3.76-4.00 (m, 4H), 7.23-7.26 (d, J = 8.0 Hz, 2H), 7.65-7.68 (d, J = 8.2 Hz, 2H); 13C NMR (CDCl3, 62.5 MHz) 13.7, 20.5, 21.4, 22.4, 26.9, 28.5, 37.2, 63.1, 69.5, 127.8, 129.7, 132.5, 144.7, 170.5 ppm. MS (GC/MS) m/z 155, 96, 84, 55. General Procedure for the preparation of 3-Acetoxy-2-alkyl-1-iodopropane (13a-d) To a solution of tosylate 12a (5 g, 13.8 mmol) in acetone (100 mL) was added sodium iodide (4.5 g, 30 mmol) at room temperature. Then the reac tion mixture was refluxed for 19 h. Solvent was evaporated and residue was treated with aqueous saturated NH4Cl solution. The resulting mixt ure was extracted with diethyl ether (3 x 100 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over anhydrous A c O O T s A c O I

PAGE 54

35 Na2SO4 and evaporated to afford the product 13a (4.3 g, 98%) as a yellow oil: Rf = 0.69 (1:4 EtOAc/hexane); IR (cm-1) 1731; 1H NMR (CDCl3, 250 MHz) 1.88-1.95 (m, 1H), 2.0 (s, 3H), 2.59-2.75 (dd, J = 13.0 Hz, 5.8 Hz, 2H), 3.11-3.17 (dd, J = 10.0 Hz, 5.0 Hz 1H), 3.25-3.31 (dd, J = 10.0 Hz, 4.6 Hz, 1H), 3.92-3.95 (dd, J = 11.1 Hz, 7.2 Hz, 1H), 4.09-4.15 (dd, J = 11.0 Hz, 4.8 Hz, 1H), 7.19-7.33 (m, 5H); 13C NMR (CDCl3, 100 MHz) 10.3, 20.9, 37.1, 40.4, 66.4, 126.6, 128.6, 129.0, 138.3, 170.8 ppm; MS (GC/MS) m/z 191, 131, 117, 91, 65; ESI-TOF Calcd for [M+NH4]+ is 336.04550, Found: 336.04528 The remaining iodides 13b-d were prepared similarly, with the following results. 3-Acetoxy-2-cyclohexylmethyl-1-iodopropane (13b) 12b (2.27 g, 6.17 mmol), sodium iodide (2.78 g, 18.51 mmol), yielded compound 13b (1.88 g, 94%); a brown oil; Rf = 0.81 (3:7 EtOAc/hexane); IR (cm-1) 1737, 1268; 1H NMR (CDCl3, 250 MHz) 0.82-0.96 (m, 2H), 1.11-1.30 (m, 6H), 1.60-1.72 (m, 6H), 2.06 (s, 3H), 3.21-3.35 (m 2H), 3.84-3.91 (dd, J = 11.2 Hz, 7.5 Hz, 1H), 4.06-4.12 (dd, J = 11.2 Hz, 4.6 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 11.8, 20.9, 26.1, 26.2, 26.4, 33.2, 33.5, 34.3, 35.3, 39.0, 66.9, 170.8 ppm; MS (GC/MS) m/z 197, 155, 137, 109, 95, 81, 67, 55. A c O I A cO I

PAGE 55

36 3-Acetoxy-2-isobutyl-1-iodopropane (13c) 12c (16.6 g, 50.76 mmol), sodium iodide (22.84 g, 152.29 mmol), yielded compound 13c (12.22 g, 85%); a brown oil; Rf = 0.81 (3:7 EtOAc/hexane); IR (cm-1) 1728, 1262; 1H NMR (CDCl3, 250 MHz) 0.81-0.86 (t, J = 6.3 Hz, 6H), 1.081.18 (m, 2H), 1.52-1.60 (m, 2H), 2.00 (s, 3H), 3.14-3.20 (dd, J = 10.0 Hz, 5.1 Hz, 1H), 3.23-3.29 (dd, J = 10.0 Hz, 4.2 Hz, 1H), 3.76-3.84 (dd, J = 11.1 Hz, 7.6 Hz, 1H), 3.99-4.05 (dd, J = 11.1 Hz, 4.5 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 11.6, 20.8, 22.2, 22.7, 24.7, 35.8, 40.2, 66.7, 170.7 ppm. MS (GC/MS) m/z 157, 115, 97, 81, 69, 55. 3-Acetoxy-2-butyl-1-iodopropane (13d) 12d (0.32 g, 0.94 mmol), Sodium iodide (0.74 g, 4.92 mmol), yielded compound 13d (0.25 g, 93%); a brown oil; Rf = 0.45 (1:10 EtOAc/hexane); IR (cm-1) 1732; 1H NMR (CDCl3, 250 MHz) 0.78-0.84 (t, J = 6.6 Hz, 3H), 1.21-1.32 (m, 6H), 1.45-1.55 (m, 1H), 1.99 (s, 3H), 3.16-3.27 (m 2H), 3.79-3.86 (dd, J = 11.1 Hz, 7.5 Hz, 1H), 3.99-4.06 (dd, J = 11.1 Hz, 4.7 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 11.2, 13.9, 20.8, 22.5, 28.5, 30.8, 38.2, 66.5, 170.7 ppm. MS (GC/MS) m/z 157, 115, 97, 81, 69, 55. General Procedure for the preparation of 3-Acetoxy-2-alkyl-1-nitropropane (14a-d) A c O I A cO NO2

PAGE 56

37 To a solution of iodide 13a (6.22 g, 19.6 mmol) in dry DMF (20 mL) were added phloroglucinol (1.23 g, 9.8 mmol) and AgNO2 (6.0 g, 39.2 mmol) under an argon atmosphere at room temperature and allowe d to stir for 24 h. Reaction mixture was diluted with ethyl acetate a nd filtered through a pad of celite. The filtrate was washed with brine (3 x 25 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. The crude product was purified by column chromat ography (1:4 EtOAc/hexane) to give pure 14a (3.5 g, 75%) as a colorless oil: Rf = 0.46 (1:4 EtOAc/Hexane); IR (cm-1) 1728, 1551, 1175; 1H NMR (CDCl3, 400 MHz) 2.06 (d, J = 2.8 Hz, 3H), 2.32-2.38 (m, 1H), 2.702.75 (dd, J = 13.2 Hz, 7.2 Hz, 2H), 4.04.20 (m, 3H), 4.33-4.46 (ddd, J = 13.2 Hz, 6.4 Hz, 1H), 7.15-7.34 (m, 5H); 13C NMR (CDCl3, 100 MHz) 21.0, 34.6, 39.2, 63.2, 63.7, 76.4, 126.7, 128.8, 129.2, 138.6, 171.1 ppm; MS (GC/MS) m/z 190, 130, 117, 104, 91, 65; ESI-TOF Calcd for [M+NH4]+ is 255.13393, Found: 255.13405 The remaining nitro acetates 14b-d were prepared similarly, with the following results. 3-Acetoxy-2-cyclohexylmethyl-1-nitropropane (14b) 13b (2.5 g, 7.71 mmol), phlorogl ucinol (0.97 g, 7.71 mmol), silver nitrite (4.14 g, 26.98 mmol), yielded compound 14b (1.88 g, 48%); a colorless oil; Rf = 0.70 (3:7 EtOAc/hexane); IR (cm-1) 1733, 1548, 1370; 1H NMR (CDCl3, 250 MHz) 0.80-0.88 (m, 2H), 1.05-1.27 (m, 6H), 1.62-1.66 (m, 5H), 1.99 (s, 3H), 2.65-2.69 (m, 1H), 3.934.14 (m, 3H), 4.29-4.37 (ddd, J = 15.2 Hz, 6.0 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) A cO NO2

PAGE 57

38 20.7, 26.4,32.8, 33.3, 33.4, 35.7, 36.1, 64.2, 76.6, 170.6 ppm; MS (GC/MS) m/z 197, 182, 166; ESI-TOF Calcd for [M+NH4]+ is 261.18088, Found: 261.18063 3-Acetoxy-2-isobutyl-1-nitropropane (14c) 13c (1.1 g, 3.87 mmol), phlorogl ucinol (0.73 g, 5.8 mmol), s ilver nitrite (1.2 g, 7.74 mmol), yielded compound 14c (0.44 g, 56%); a colorless oil; Rf = 0.66 (3:7 EtOAc/hexane); IR (cm-1) 1739, 1549, 1366; 1H NMR (CDCl3, 250 MHz) 0.86-0.91 (t, J = 6.5 Hz, 6H), 1.15-1.27 (m, 2H), 1.60-1.63 (m, 1H), 2.02 (s, 3H), 2.60-2.70 (m, 1H), 3.92-4.15 (m, 3H), 4.32-4.40 (ddd, J = 13.6 Hz, 6 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 20.5, 21.9, 22.6, 24.9, 34.8, 37.5, 64.1, 77.3, 170.5 ppm; MS (GC/MS) m/z 160, 114, 95, 81, 69, 55. 3-Acetoxy-2-butyl-1-nitropropane (14d) 13d (3.55 g, 12.5 mmol), silver nitrite (5.77 g, 37.5 mmol), phlorogl ucinol (0.8 g, 6.25 mmol), yielded compound 14d (1.34 g, 54%); a colorless oil; Rf = 0.39 (1:10 EtOAc/hexane); IR (cm-1) 1735, 1552; 1H NMR (CDCl3, 250 MHz) 0.85-0.90 (t, J = 6.6 Hz, 3H), 1.26-1.36 (m, 6H), 2.03 (s, 3H), 2.50-2.55 (m, 1H), 3.96-4.18 (m, 2H), 4.324.44 (m, 2H); 13C NMR (CDCl3, 62.5 MHz) 13.7, 20.6, 22.6, 28.2, 28.4, 36.9, 64.0, 77.1, 160.9 ppm; MS (GC/MS) m/z 159, 114, 96, 81, 71, 55; ESI-TOF Calcd for [M+NH4]+ is 221.14958, Found: 221.14914 A cO NO2 AcO NO2

PAGE 58

39 General Procedure for the preparation of 3-nitro-2-alkyl-1-propanol (15a-d) To a solution of nitro acetate 14a (2.0 g, 8.44 mmol) in MeOH (180 mL) was added a solution of K2CO3 (3.5 g, 25.3 mmol) in H2O (20 mL) at room temperature. The resulting milky white reaction mixture was stirred at the same temperature for 2 h. the majority of the organic solvent was evaporated and resi due was extracted with EtOAc (3 x 25 mL). Combined organic layers were washed with brine (2 x 25 mL), dried over anhydrous Na2SO4 and evaporated. The crude residue was purified by column chromatography (1:1 EtOAc/hexane) to give the compound 15a (0.74 g, 45%) as a colorl ess oil plus undesired white solid (which did not undergo Jones oxidation) (0.54 g, 33%): Rf = 0.64 (1:1 EtOAc/Hexane); IR (cm-1) 3583, 3409, 1544; 1H NMR (CDCl3, 400 MHz) 1.58 (s, 1H), 2.64-2.79 (m, 3H), 3.58-3.62 (dd, J = 10.8 Hz, 4.8 Hz, 1H), 3.70-3.74 (dd, J = 10.8 Hz, 3.2 Hz, 1H), 4.37-4.41 (dd, J = 12.4 Hz, 5.2 Hz, 1H), 3.52-3.57 (dd, J = 12.4 Hz, 6.4 Hz, 1H), 7.19-7.34 (m, 5H); 13C NMR (CDCl3, 100 MHz) 34.9, 41.8, 62.1, 76.4, 127.0, 128.9, 129.2, 138.0 ppm; MS (GC/MS) m/z 131, 117, 91, 65. The remaining nitro alcohols 15b-d were prepared similarly, with the following results. HO NO2 HO NO2

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40 3-nitro-2-cyclohexylmethyl-1-propanol (15b) 14b (1.31 g, 5.4 mmol), potassi um carbonate (2.24 g, 16.2 mm ol), yielded compound 15b (0.55 g, 51%); A colorless oil; Rf = 0.5 (3:7 EtOAc/Hexane); IR (cm-1) 3440, 1544, 1381; 1H NMR (CDCl3, 250 MHz) 0.83-0.90 (m, 2H), 1.07-1.32 (m, 6H), 1.65-1.70 (m, 5H), 2.38-2.51 (m, 2H), 3.47-3.54 (dd, J = 11.2 Hz, 6.8 Hz, 1H), 3.64-3.70 (dd, J = 11.2 Hz, 4.2 Hz, 1H), 4.30-4.37 (dd, J = 12.0 Hz, 5.9 Hz, 1H), 4.46-4.54 (dd, J = 12.1 Hz, 7.0 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 25.9, 26.0, 26.2, 32.3, 32.9, 34.4, 35.8, 36.9, 62.5, 77.2 ppm; MS (GC/MS) m/z 182, 135, 107, 95, 81, 67, 55. 3-nitro-2-isobutyl-1-propanol (15c) 14c (0.44 g, 2.17 mmol), potassium carbonate (0.9 g, 6.51 mmol), yielded compound 15c (0.15 g, 43%); a colorless oil; Rf = 0.47 (3:7 EtOAc/hexane); IR (cm-1) 3403, 1549; 1H NMR (CDCl3, 250 MHz) 0.92-0.95 (dd, J = 6.5 Hz, 1.7 Hz, 6H), 1.15-1.34 (m, 2H), 1.62-1.73 (m, 2H), 2.45-2.47 (m, 1H), 3.55-3.62 (dd, J = 11.1 Hz, 6.6 Hz, 1H), 3.72-3.78 (dd, J = 11.1 Hz, 4.2 Hz, 1H), 4.37-4.44 (dd, J = 12.1 Hz, 5.8 Hz, 1H), 4.52-4.59 (dd, J = 12.1 Hz, 7.1 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 22.2, 22.6, 25.0, 37.4, 37.7, 62.5, 77.2 ppm; MS (GC/MS) m/z 115, 97, 84, 69, 55. HO NO2 HO NO2

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41 3-nitro-2-butyl-1-propanol (15d) 14d (1.34 g, 6.6 mmol), potassium carbonate (1.37 g, 9.9 mmol), yielded compound 15d (0.36 g, 34%); a colorless oil; Rf = 0.61 (2:5 EtOAc/hexane); IR (cm-1) 3410, 1549; 1H NMR (CDCl3, 250 MHz) 0.86-0.93 (t, J = 2.5 Hz, 3H), 1.28-1.38 (m, 6H), 2.332.37(m, 1H), 3.51-3.58 (dd, J = 11.1 Hz, 6.7 Hz, 1H), 3.66-3.72 (dd, J = 11.1 Hz, 4.3 Hz, 1H), 4.33-4.40 (dd, J = 12.1 Hz, 6.1 Hz, 1H), 4.48-4.55 (dd, J = 12.1 Hz, 7.0 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 11.2, 13.9, 20.8, 22.5, 28.5, 30.8, 38.2, 66.5, 170.7 ppm; MS (GC/MS) m/z 97, 84, 69, 55. General Procedure for the preparation of 3-nitro-2-alkylpropionic acids (16a-d) Jones reagent (2 mL) was added to a solution of nitro alcohol 15a (1.0 g, 5.13 mmol) in acetone (3 mL) at 0 0C. The reaction mixture was stirred for 2 h at same temperature, and then quenched with i -PrOH until the color of the reaction mixture turned to green. After dilution with water and extraction with EtOAc (3 x 25 mL), the combined organic layers were washed with brine (2 x 25 mL), dried over Na2SO4 and the solvent was evaporated under vacuo. The crude product was purif ied by column chromatography (1:1 EtOAc/hexane) to give the carboxylic acid 16a (0.75 g, 70%) as colorless oil. Rf = 0.13 (1:1 EtOAc/hexane); IR (cm-1) 3027, 1714, 1551; 1H NMR (CDCl3, 400 MHz) 2.832.89 (dd, J = 14.4 Hz, 9.2 Hz, 1H), 3.21-3.26 (dd, J = 14.4 Hz, 5.6 Hz, 1H), 3.48-3.55 (m, 1H), 4.35-4.39 (dd, J = 14.8 Hz, 4.4 Hz, 1H), 4.61-4.67 (dd, J = 14.8 Hz, 8.8 Hz, 1H), 7.17-7.19 (d, J = 6.8 Hz, 2H), 7.26-7.36 (m, 3H); 13C NMR (CDCl3, 100 MHz) 35.0, OH O NO2

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42 44.3, 73.7, 127.7, 129.0, 129.3, 136.2, 177.2 ppm; MS (GC/MS) m/z 191, 162, 147, 131, 117, 91, 65; ESI-TOF Calcd for [C10H10O4N]is 208.06153, Found: 208.06120 Remaining nitro acids 16b-d were prepared similarly, with the following results. 3-nitro-2-cyclohexylmethyl propionic acid (16b) 15b (0.55 g, 2.74 mmol), Jones reag ent (5.5 mL), yielded compound 16b (0.39 g, 67%); a white solid; mp 96-98 0C; Rf = 0.57 (1:1 EtOAc/hexane); IR (cm-1) 1722, 1536, 1381; 1H NMR (CDCl3, 400 MHz) 0.89-0.95 (m, 2H), 1.16-1.40 (m, 5H), 1.65-1.81 (m, 6H), 3.32-3.33 (m, 1H), 4.39-4.44 (dd, J = 14.4 Hz, 4.0 Hz, 1H), 4.67-4.73 (dd, J = 14.4 Hz, 9.6 Hz 1H); 13C NMR (CDCl3, 62.5 MHz) 25.8, 26.1, 32.7, 34.7, 36.3, 40.1, 74.8, 178.9 ppm; MS (GC/MS) m/z 168, 121, 107, 95, 81, 67, 55; ESI-TOF Calcd for[C10H16O4N]is 214.10848, Found: 214.10826 3-nitro-2-isobutylpropionic acid (16c) 15c (0.96 g, 5.96 mmol), Jones reagen t (4.5 mL), yielded compound 16c (0.98 g, 95%); a white powder; mp 58-61 0C; Rf = 0.59 (1:1 EtOAc/hexane); IR (cm-1) 1713, 1545, 1373; 1H NMR (CDCl3, 250 MHz) 0.94-0.99 (dd, J = 14.6 Hz, 8.8 Hz, 6H), 1.25-1.44 (m, 2H), 1.62-1.73 (m, 2H), 3.253.33 (m, 1H), 4.39-4.46 (dd, J = 14.6 Hz, 4.3 Hz, 1H), 4.664.76 (dd, J = 14.6 Hz, 9.4 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 21.9, 22.2, 25.5, 37.8, OH O NO2 OH O NO2

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43 40.9, 74.9, 178.7 ppm; MS (GC/MS) m/z 129, 83, 69, 55; ESI-TOF Calcd for [C7H12O4N]is 174.07718, Found: 174.07698 3-nitro-2-butylpropionic acid (16d) 15d (0.1 g, 0.62 mmol), Jones reagen t (1 mL), yielded compound 16d (0.1 g, 93%); a colorless oil; Rf = 0.64 (9:1 EtOAc/hexane); IR (cm-1) 3750, 1713, 1556; 1H NMR (CDCl3, 250 MHz) 0.89 (s, 3H), 1.29-1.39 (m, 6H), 3.20-3.23 (m, 1H), 4.39-4.45 (d, J = 14.6 Hz, 1H), 4.66-4.76 (dd, J = 13.8 Hz, 9.2 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 13.6, 22.3, 28.6, 42.6, 74.5, 178.8 ppm; MS (GC/MS) m/z 129, 83, 73, 55; ESI-TOF Calcd for [C7H12O4N]is 174.07718, Found: 174.07703 General Procedure for the Preparation of Methyl, 3-nitro-2-alky lpropionate (17a-d) To a solution of acid 16a (0.1 g, 0.48 mmol) in dr y DMF (3 mL) at –5 0C was added anhydrous K2CO3 (0.066 g, 0.48 mmol) with stirring und er an argon atmosphere. After stirring for 10 min., methyl iodide (0.075 g, 0.53 mmol) was added and reaction mixture was stirred for another 2 h at same temperatur e. The reaction mixture was diluted with 20 mL of saturated aqueous NaHCO3 solution and extracted with EtOAc (2 x 25 mL). Combined organic layers were washed once with saturated aqueous NaHCO3 solution, then with brine (2 x 25 mL), dried over anhydrous Na2SO4, and evaporated under vacuo OH O NO2 OCH3 O NO2

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44 to afford the pure 17a (0.08 g, 75%) as a light yellow oil: Rf = 0.47 (1:4 EtOAc/hexane); IR (cm-1) 1736, 1551; 1H NMR (CDCl3, 400 MHz) 2.71-2.77 (dd, J = 14.0 Hz, 9.2 Hz, 1H), 3.03-3.08 (dd, J = 14.0 Hz, 6.0 Hz, 1H), 3.34-3.42 (m, 1H), 3.68 (s, 3H), 4.25-4.30 (dd, J = 14.8 Hz, 4.4 Hz, 1H), 4.55-4.60 (dd, J = 14.4 Hz, 9.2 Hz, 1H), 7.05-7.07 (d, J = 7.2 Hz, 2H), 7.18-7.26 (m, 3H); 13C NMR (CDCl3, 100 MHz) 35.3, 44.7, 52.7, 74.2, 127.5, 129.0, 136.6, 172.2 ppm; MS (GC/MS) m/z 178, 117, 105, 91, 65; ESI-TOF Calcd for [M+NH4]+ is 241.11828, Found: 241.11779 The remaining nitro esters 17b-d were prepared similarly, with the following results. Methyl, 3-nitro-2-cyclohexylmethylpropionate (17b) 16b (0.05 g, 0.23 mmol), potassium carbonate (0.037 g, 0.28 mmol), met hyl iodide (0.08 g, 0.56 mmol), yielded compound 17b (0.046 g, 87%); a colorless oil; Rf = 0.80 (3:7 EtOAc/hexane); IR (cm-1) 1739, 1553, 1378; 1H NMR (CDCl3, 400 MHz) 0.86-0.93 (m, 2H), 1.15-1.37 (m, 5H), 1.57-1.79 (m, 6H ), 3.28-3.30 (m, 1H), 3.74 (s, 3H), 4.374.42 (dd, J = 14.0 Hz, 4.4 Hz, 1H), 4.67-4.73 (dd, J = 14.4 Hz, 9.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz) 26.1, 26.4, 33.0, 33.2, 35.2, 36.9, 40.6, 52.5, 75.7, 173.3 ppm; MS (GC/MS) m/z 183, 151, 121, 107, 95, 81, 67, 55; ESI-TOF Calcd for [M+NH4]+ is 247.16523, Found: 247.16470 OCH3 O NO2 OCH3 O NO2

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45 Methyl, 3-nitro-2-isobutylpropionate (17c) 16c (0.05 g, 0.29 mmol), potassium carbonate (0.048 g, 0.35 mmol), Methyl iodide (0.05 g, 0.35 mmol), yielded compound 17c (0.05 g, 93%); a colorless oil; Rf = 0.75 (3:7 EtOAc/hexane); IR (cm-1) 1735, 1560, 1370; 1H NMR (CDCl3, 250 MHz) 0.91-0.96 (dd, J = 11.9 Hz, 6.0 Hz, 6H), 1.29-1.34 (m, 1H ), 1.55-1.65 (m, 3H), 3.22-3.28 (m, 1H), 3.74 (s, 3H), 4.35-4.43 (dd, J = 14.3 Hz, 4.5 Hz, 1H), 4.66-4.75 (dd, J = 14.3 Hz, 9.5 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 21.9, 22.3, 25.6, 38.1, 41.1, 52.2, 75.4, 173.0 ppm; MS (GC/MS) m/z 158, 111, 69, 55; ESI-TOF Calcd for [M+NH4]+ is 207.13393, Found: 207.13390 Methyl, 3-nitro-2-butylpropionate (17d) 16d (0.1 g, 0.57 mmol), methyl iodide (0.12 mL, 2.0 mmol), yielded compound 17d (0.50 g, 47%); a pale yellow oil; Rf = 0.58 (1:5 EtOAc/hexane); IR (cm-1) 1735, 1549; 1H NMR (CDCl3, 250 MHz) 0.85-0.90 (t, J = 6.7 Hz, 3H), 1.221.32 (m, 4H), 1.50-1.71 (m, 2H), 3.12-3.23 (m, 1H), 3.72 (s, 3H), 4.36-4.44 (dd, J = 14.3 Hz, 4.8 Hz, 1H), 4.674.76 (dd, J = 14.3 Hz, 9.4 Hz, 1H); 13C NMR (CDCl3, 62.5 MHz) 13.7, 22.3, 28.6, 28.9, 42.7, 52.3, 75.1, 172.8 ppm; MS (GC/MS) m/z 158, 111, 69, 55; ESI-TOF Calcd for [M+NH4]+ is 207.13393, Found: 207.13389 2.5 References 1) Ojima, I.; Lin, S.; Wang, T. Curr. Med. Chem. 1999 6 927-954. OCH3 O NO2

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46 2) Bates, R. B.; Brusoe, K. G.; Burns, J. J.; Caldera, S.; Cui, Wei.; Gangwar, S.; Gramme, M. R.; McClure, K. J.; Rouen, G. P.; Schadow, H.; Stessman, C. C.; Taylor, S. R.; Vu, V. H.; Yarick, G. V. ; Zhang, J.; Pettit, G. R.; Bontems, R. J. Am. Chem. Soc. 1997 119 2111-2113. 3) (a) Meglasson, M. D.; Wilson, J. M.; Y u, J. H.; Robinson, D. D.; Wyse, B. M.; deSouza, C. J. J. Pharmacol. Exp. Ther. 1993 266 1454-1462. (b) Larsen, S. D.; Connell, M. A.; Cudahy, M. M.; Evans, B. R.; May, P. D.; Meglasson, M. D.; O'Sullivan, T. J.; Schostarez, H. J.; Sih, J. C.; Stevens, F. C.; Tanis, S. P.; Tegley, C. M.; Tucker, J. A.; Vaillancourt, V. A.; Vidmar, T. J.; Watt, W.; Yu, J. H. J. Med. Chem. 2001 44 1217-1230. 4) Tamariz, J. In Enantioselective Synthesis of -Amino Acids ; Juaristi, E., Ed.; Wiley-VCH: New York, 1997; pp. 45-66. 5) Ternansky, R. J.; Morin. Jr., J. M. The Organic Chemistry of -lactams: In Novel Methods for Construction of -Lactam Ring George, G. I., Ed.; Verlag Chemie:New York, 1993; Chapter V, pp. 257-293. 6) (a) Seebach, D.; Overhand, M.; Kuhnle, F.; Florian, N. M.; Martinoni, B. Helv. Chim. Acta 1996 79 913-941. (b) Appella, D. H.; Ch ristianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996 118 13071-13072. (c) Gademann, K.; Ernst, M.; Hoyer, D.; Seebach, D. Angew. Chem. Int. Ed. 1999 38 1223-1226. (d) Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. J. Med. Chem. 2001 44 2460-2468.

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47 7) Spatola, A. F. In Chemistry and Biochemistry of the Amino acids, Peptides and Proteins ; Weinstein, B., Ed.; Marcel Dekke r: New York, 1983; Vol. VII, pp 267357. 8) (a) Colombo, L.; Rassu, G.; Spanu, P. J. Org. Chem. 1991 56 6523-6527. (b) Robl, J. A.; Cimarusti, M. P.; Simpkins, L. M.; Weller, H. N.; Pan, Y. Y.; Malley, M.; DiMarco, J. D. J. Am. Chem. Soc. 1994 116 2348-2355. (c) Kawabata, K.; Inamoto, Y.; Sakane, K.; Iwamoto, T.; Hashimoto, S. J. Antibiot. 1990 43 513518. 9) (a) Hart, D.; Chan, D. Chem. Rev. 1989 89 1447-1465. (b) Palomo, C.; Aizpurua, J. M.; Urchequi, R.; Iturburu, M.; Ochoa, A.; Cuevas, C. J. Org. Chem. 1991 56 2244-2247. 10) Roas, A.; Giuliana, C.; Claudia, T. Heterocycles 1992 34 349-355. 11) Konopelski, J. P.; Chu, K. S.; Negrete, G. R. J. Org. Chem. 1991 56 1355-1357. 12) (a) Juaristi, E.; Quintana, D.; Lamatsch, B.; Seebach, D. J. Org. Chem. 1991 56 2553-2557. (b) Juaristi, E.; Seebach, D. In Enantioselective Synthesis of -Amino Acids ; Juaristi, E., Ed.; WileyVCH: New York, 1997; pp 261-271. 13) Ponsinet, R.; Chassaing, G.; Vaissermann, J.; Lavielle, S. Eur. J. Org. Chem. 2000 1 83-90.

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48 14) (a) Evans, D. A.; Urpi, F.; Sommers, T. C.; Clark, J. S.; Bilodeau, M. T. J. Am. Chem. Soc. 1990 112 8215-8216. (b) Hinternamm, T.; Seebach, D. Helv. Chim. Acta 1998 81 2093-2126. 15) Juaristi, E.; Quintana, D. Tetrahedron: Asymmetry 1992 3 723-726. 16) (a) Myers, A.; Schnider, P.; Kwon, S.; Kung, D. J. Org. Chem. 1999 64 33223327. (b) Myers, A.; Gleason, J.; Yoon, T.; Kung, D. J. Am. Chem. Soc. 1997 119 656-673. (c) Myers, A.; Gleason, J.; Yoon, T. J. Am. Chem. Soc. 1995 117 8488-8489. 17) Roy, R. S.; Imperiali, B. Tetrahedron Lett. 1996 37 2129-2132. 18) Myers, A.; McKinstry, L. J. Org. Chem. 1996 61 2428-2440. 19) Nagula, G.; Huber, V. J.; Lum, C.; Goodman, B. A. Org. Lett. 2000 2 35273529. 20) Seebach, D.; Henning, R.; Lehr, F.; Gonnermann, J. Tetrahedron Lett. 1977 13 1161-1164. 21) (a) Rimkus, A.; Sewald, N. Org. Lett. 2003 5 79-80. (b) Eilitz, U.; Le mann, F.; Seidelmann, O.; Wendisch, V. Tetrahedron:Asymmetry 2003 14 189-191. (c) Eilitz, U.; Le mann, F.; Seidelmann, O.; Wendisch, V. Tetrahedron:Asymmetry 2003 14 3095-3097.

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49 22) (a) Baer, H. H. Adv. Carbohydr. Chem. and Biochem. 1969 24 67-138. (b) Seebach, D.; Colvin, E. W.; Lehr, F.; Weller, T. Chimia 1979 33 1-18. (c) Seebach, D. Angew. Chem. 1979 91 259-278. (d) Seebach, D. Angew. Chem. Int. Ed. Engl. 1979 18 239-258. 23) (a) Matt, C.; Wagner, A.; Mioskowski, C. J. Org. Chem. 1997 62 234-235. (b) Gissot, A.; N’Gouela, S.; Matt, C.; Wagner, A.; Mioskowski, C. J. Org. Chem. 2004 69 8997-9001. 24) (a) Itoh, T.; Chika, J. ; Takagi, Y.; Nishiyama, S. J. Org. Chem. 1993 58 57175723. (b) Monteil, T.; Danvy, D.; Plaquevent, J. C.; Duhamel, L.; Duhamel, P.; Gros, C.; Schwartz, J. C.; Lecomte, J. M. Synth. Commun. 2001 31 211-218. 25) (a) Carr, J. A.; Bisht, K. S. Tetrahedron 2003 59 7713-7724. (b) Xu, C.; Yuan, C. Tetrahedron 2004 60 3883-3892. (c) Sundby, E.; Perk, L.; Anthonsen, T.; Aasen, A. J.; Hansen, T. V. Tetrahedron 2004 60 521-524. 26) Anelli, P. L.; Montanari, F.; Quici, S. J. Org. Chem. 1988 53 5292-5298. 27) Guanti, G.; Narisano, E.; Podgorski, T.; Thea, S.; Williams, A. Tetrahedron 1990 46 7081-7092. 28) The enantiomeric excess was determined from the 1H NMR spectrum recorded in the presence of the (+)-Eu(hfc)3 using the following equation: ee = [(R) – (S)] / [(R) + (S)] x 100%.

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50 29) Kornblum, N.; Smiley, R. A.; Blackwood, R. K.; Iffland, D. C. J. Am. Chem. Soc. 1955 77 6269-6280. 30) Yip, C.; Handerson, S.; Tranmer, G. K.; Tam, W. J. Org. Chem. 2001 66 276286. 31) Atsuumi, S.; Nakano, M.; Koike, Y.; Tanaka, S.; Ohkubo, M.; Yonezawa, T.; Funabashi, H.; Hashimoto, J.; Morishima, H. Tetrahedron Lett. 1990 31 16011604.

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51 CHAPTER THREE EFFORT TOWARDS THE SYNTHESIS OF “SUBSTITUTED” ORTHOGONALLY PROTECTED GEMINAL DIAMINO ACIDS (OPGDA’S) & CYCLIC UREA 8 3.1 Introduction Unnatural amino acids and amino aci d mimics have become increasingly important in the discovery of pharmacological ly active peptides a nd peptidomimetics. Several monomeric building blocks that mimi c the peptide backbone have been proposed and include peptoids,1 azoles,2 2-isoxazolines,3 oligocarbamates, oligosulfones and oligosulfoxides,4 pyrolinones,5 vinylogous backbones,6 -methyl amino acids7, -amino acids,8 and most recently betidamino acids.9,10,11 Biopolymers generated using these unnatural scaffolds, have been found to have physicochemical, chemical, structural, biological, metabolic, absorptive properties that differ from t hose of the parent peptides. The amino acid mimics received considerable at tention due to their important role in the design of conformationally rest ricted peptides with enhanced properties, such as resistance to hydrolysis and enzyme cleavage processes. A number of methods have been devised to stabilize biologica lly active peptides against metabolic degradation and to study their structure – activity relationships. Among these methods is the preparation of partially modified retro-invers o peptide structures (Figure 3.1 ).12-15 A general definition of a retro-isomer of a linear peptide would be the one th at is obtained through formal

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52 reversal of all the peptide bonds in the bac kbone, thus conserving the side chain topology. An absolute retro-isomer can be defined as the one that incorporates amino acids of the opposite chirality while preserving the same resi dues at the N-terminal and C-terminal of the peptide. Synthesis of th e absolute linear retro-isomer has an “end-group” problem,16 which requires the conservation of the C-term inal end group and N-terminal end group. The C-terminal end group problem can be so lved by the replacement of the C-terminal amino acid with an substituted malonic acid. Goodman, Cherov and Willson, the pioneers in the field of synthesis of the retro – inverso peptides realized that the “end-gr oup problem” can be solved by incorporating differentially protected -diamino residues.17 Figure 3.1:Retro-inverso peptide and it s similarity to a normal peptide gem -Diamino compounds also received considerab le attention throughout the literature because they are the basic components in the betidamino acids (Figure 3.2 ). Figure 3.2:Representative structure of betidamino acids. (* indicates a chiral center) Betidamino acids are the novel versatile and c onstrained scaffolds for the drug discovery, that were utilized in the synthesis of the bioactive gonadotro pin-releasing hormone H2N O H N O N H R1 H H R2 R3 H OH O Parent Peptide (all L-amino acids)H2N N H N H R1 H H R2 R3 H OH O O O diamino r esidue D-amino acid malonic acid residue N R1 R2N O R Peptide Peptide O *

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53 (GnRH) analogs.10 “Betidamino” is a term generated from the contraction of “bet a” position with “peptide ” or “amide ”. According to Rivier et al.,10 they are N’monoacylated (optionally, N ’-monoacylated and N-monoor N,N’-dialkylated) aminoglycine derivatives in which each N’-acyl/alkyl group may mimic naturally occurring amino acid side chains or intr oduce novel func tionalities. The use of the gemdiamino compounds for the synt hesis of linear peptides, re tro-isomers as well as our Part-B of the cyclic urea 8 requires two different protecting groups (orthogonal protection), which could be removed selectivel y, so that the two nitrogen atoms can be utilized in different ways. This requiremen t proved to be a synthetic challenge for us. 3.2 Literature Reported Syntheses And Applications Of Geminal Diamino Compounds There were few literature reported procedures for the synthesis of the gemdiamino compounds, but their ap plications were enormous as stated earlier in the introductory section. Bergmann and Zervas18 reported the first method for the stepwise degradation of polypeptides. In their paper they mentioned gem-diamino compounds for the first time. The first reported synthesis of the geminal diamino acid by Shemin and Herbest19,20 included the synthesis of -diacetamino derivative 22, which was made from condensing the -acetamino acrylic acid 20 with acetamide 21 (Figure 3.3).Their synthesis was based on the findings by Zervas et al. Figure 3.3:Condensation of -acetamino acrylic acid 20 with acetamide 21 C NHCOCH3 COOH H2C +CH3CONH2 C NHCOCH3 COOH H3C NHCOCH3 202122

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54 The resulting diacetamino compound 22 is a gem-diamino compound, but it lacks the different protecting groups on the geminal am ines. The next reported synthesis of the gem-diamino is by Greenstein et al.,21,22 where they synthesized the Di(glycylamino)propionic acid hydr ochloride by amination of the Di(chloroacetamino)prop ionic acid. Brenner et al.23 further synthesized the substituted versions of the acid and also reported Cbz protecting groups on the amines (Figure 3.4). Figure 3.4:gem -Diamino compounds synthesized by Brenner et al. A series of reports were published by Goodman et al. on the synthesis of orthogonally protected gem-diamino compounds by Curtiusor Hoffmann type rearrangements of protecte d amino acid derivatives.12,24-26 They used a modified Curtius reaction on the general structure 23, by employing a saturated solution of nitrosoyl chloride in dry THF. The azide that formed was allowed to rearrange to the corresponding isocyanate by heating in toluene at 80 oC. Addition of either tert-butyl alcohol or benzyl alcohol to the reac tion mixture yielded N,N’-diprotected gem-diamino compound with the general formula 24 (Figure 3.5). Figure 3.5:General scheme for the sy nthesis of orthogonally protected gem diamino compounds via modified Curtius reaction. X = -OH, -NH-R'', -NH-CH2-COOH R' =-CH 3 -CH2-COOH COX NHCO H3C NHCO R' R' H N H R H2NHNOC X O NOCl, THFH N H R N3OC X O TolueneH N H R OCN X O OH H N H R HN X O Y 23 24

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55 They used their orthogonally protected gem-diamino compounds in the synthesis of partially modified retr o-inverso-enkephalinamide 25 that was proved to be long-acting analog both in vitro and in vivo (Figure 3.6).24 Their synthesis of novel class of enkephalin analogs was based on the revers al of one or more of the peptide bond residues, thus avoiding the proteolytic degr adation. All the modified structures of the enkephalin analogs they made contain gem-diamino alkyl residues.24 Figure 3.6:Enkephalinamide 25 synthesized by Goodman et al Another report by Goodman et al. came in 1980, where they synthesized the gemdiamino compounds by the similar route they reported earlier, but this time with a variation in the usage of the starting material 26 (Figure 3.7).25 Figure 3.7:gem -Diamino compounds synthesized by Goodman et al. HN3 O N H O N H O N H N H O O NH2 CH3COO H OH H3C H H H S gem -diamino25Z H N C H C C O O PhH2C O OCH2Ph i) LiOH ii)NH2-NH-Boc/DCCZ H N C H C C O O PhH2C O H N H N Boc i) 4N HCl ii) NOClZ H N C H C C O O PhH2C O N3 Z H N C H C N O O PhH2C C O t -BuOHZ H N C H C H N O O PhH2C COOtBu 2627 28 29 30

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56 These gem-diamines were used for the synthesis of the peptide sweeteners, and also to find their effect of modifying the peptide bond on the sweet taste of L-Aspartyl-Lphenylalanine methyl ester and their analogs Goodman’s group was unsuccessful in their attempts to come up with the analogs that are sweeter than L-Aspartyl-L-phenylalanine methyl ester 31 in this attempt (Figure 3.8). Figure 3.8:Retro-inverso peptide analog of L-Aspartyl-L-phenylalanine (31) Their relentless efforts towards synthesis of peptide sweeteners were successful finally, when they synthesized N-(L-aspartyl)-1,1-diaminoalkane-based sweeteners that are up to 1000 times sweeter than sucrose (Figure 3.9).26 Figure 3.9:Retro-inverso peptide analogs of N -(L-aspartyl)-1,1-diaminoalkane Since diamino methane is not a suffici ently stable compound in the monoor unprotonated form to survive the conditions requ ired to use it as a reagent, orthogonally protected diaminomethane was first synthesized by Loudon et al.27,28, where they H3N N O O H O O O gem diamino 31H2N HOOC H N O H N R' O R' = 1000 times 700 times g emd ia m ino

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57 converted the Boc-Glycine 32 to its acyl azide 33 by using bis(p-nitrophenyl )-phosphoryl azide, and the resulting material 33 was rearranged via a modifi ed Curtius reaction in the presence of benzyl alcohol to give th e diprotected methylenediamine derivative 34. Figure 3.10:Synthesis of orthogonally protected dichloromethane 34 Additional interest in the synthesis of ,-diamino compounds was supported, when their possible application as the pep tide carrier systems designed for transporting therapeutically useful compounds into microbial cells was reported.29 Kingsbury et al.29 described a versatile peptide delivery system in which the toxophoric agent was attached to the -carbon of a glycine residue w ithin a peptide chain. Intr acellular cleavage of the peptide by cytoplasmic peptidases results in the formation of an unstable intermediate that decomposes with release of the attached toxophoric group. Bock et al.30 reported an efficient synthesis of -aminoglycine in protected form 37, which complements the existing methodology developed by Goodman’s group (Refer to Figure 3.11). Figure 3.11:Synthesis of orthogonally protected -geminal diaminoglycine 37 BocNHCH2CO2H (O2Np -C6H4O)2PO-N3EtOAc, Et3N BocNHCH2CON3 Benzylalcohol BocNHCH2NH Cbz 32 33 34C6H5CH2OCNHCHCO2H O OH (CH3)2CHSH H2SO4 cat. HOAc C6H5CH2OCNHCHCO2H O SCH(CH3)2 (CH3)3CCO2NH2Hg2+, THF C6H5CH2OCNHCHCO2H O NHCO2C(CH3)3 37 35 36

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58 “gem-peptides” are the peptides containing gem-diamino units in the terminal position of a retro-inverso peptide and th is terminology was introduced by Katritzky et al.31 They reported a convenient and versatile method for the preparation of various simple -substituted monoacyl aminals and al so a novel synthetic route to “gempeptides”. Their synthesis involved Mannich condensation of various amides, aldehydes, and benzotriazole to give adducts of type R1CONH-CH(R2)Bt, which upon reaction with NH3 result in versatile monoacyl aminal structures (R1CONH-CH(R2)NH2 and using protected amino acid amides as the amide component leads to “gem-peptides” (Refer to figure 3.12). Figure 3.12:Katritzkey et al. ’s scheme for the synthesis of “ gem -peptides” subunit 3.2.1 Cushman et al. ’s synthesis of gem -diamino compounds Cushman et al.32 reported the synthesis of end group modified retro-inverso Bombesin Cterminal nonapeptide. Bombesin (GlpGl nArgLeuGlyAsnGlnTrpAlaValGlyHisLeuMetNH2) and structurally related pe ptides display a wide spect rum of biological activity. Cushman et al. followed the approach proposed by Goodman et al.14 for the synthesis of the retro-inverso peptide analog 38 by incorporating the gem-diamino in the termini of Bombesin (Figure 3.13). Their synthesis of the amino terminus analogue was not smooth; they basically had to abandon a couple of schemes before they made their gem-diamino R1CONH2+ CHO R2 + BtH R1COHN H C R2 Bt NH3R1COHN H C R2 NH2 Mannich condensation Bt = benztriazol-1(or 2-)yl

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59 compound. Their schemes will be discussed below in detail, as they closely resemble the approach that we followed in our efforts towards the syntheses of substituted OPGDA’s. Figure 3.13:Retro-inverso peptide analog 38 of Bombesin Their original plan was to construct the ge minal amino amide unit at the end of the chain in the above mentioned re tro-inverso peptide analog 38 of bombesin, by performing a Curtius rearrangement on a peptide containing an asparagine residue. Attempts to rearrange the acyl azide derivative of Z-Lasparagine in TFA or reaction of Z-Lasparagine with diphenyl phosphorazidate (an azi de transfer reagent) gave disappointing results. They concluded that the side chai n amide group of asparagine was interfering with the reaction at the isocyanate stage. Th ey further described some model studies for making orthogonally protected gem-diamino compounds by selecting an amino acid derivative lacking a reactive side chain. They chose Z-L-leucine to conduct their studies, which are outlined in Figures 3.14 & 3.15. Figure 3.14:Failed attempt to synthesize gem -diamino analog of leucine H N H2N O N H O H N O N H O H N O N H O H N O N H O NH2 O H O H2N O NH2 H HN H H H NH N H H H SCH3 gemdiamino CbzHN H OH O i)diphenylphospho r azida t e,TEA ii) Benzene, t -BuOH, refluxCbzHN NHBoc H X 39 40

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60 The reaction of Z-L-leucine 39 with diphenyl phosphorazi date and triethylamine followed by heating at a reflux in benzene containing tert-butyl alcohol also did not lead them to the desired gem-diamino compound 40. They were able to make acyl azide 41 by mixed anhydride method, and were able to achieve the formation of isocyanate 42 via rearrangement. But when this isocyanate 42 was reacted with tert-butyl alcohol in refluxing benzene, it took 15 h before the complete decomposition of the starting material, and they couldn’ t get the desired product 43. In contrast to tert-butyl alcohol, benzyl alcohol reacted readily with the isocyanate 42 to give 44 as the major product (Figure 3.15). Figure 3.15:Failed attempt via mixed anhydride route They concluded that the Curtius rearrangement was not possible if the amino acid has i) a Leucine side chain ii) an amide group like in asparagine. They came up with a new strategy for the construction of the geminal am ino unit of the retro-i nverso peptide analog 38 of Bombesin (Figure 3.16), where they masked the amide group on the asparagine side chain as a nitrile (-CN), and carried out the Curtius rearrangement. They were successful in achieving the gem-diamino compound 49 in moderate yields. CbzHN H OH O CbzHN H N3 O CbzHN N H C O OH CbzHN NHBoc H X39 41 42 43 OH CbzHN O H 44

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61 Figure 3.16:Cushman et al. ’s successful attempt to synthesize gem -diamino compound 49 via masked amide of 45 3.2.2 Reports on orthogonally protected geminal diamino acids (OPGDA’s) Kohn et al.33 reported gem-diamino compounds that showed excellent anticonvulsant activity. In their paper they reported the synt hesis of some of those active compounds by a much simpler scheme depicted in figure 3.17. Figure 3.17:Kohn’s synthesis of gemdiamino compound 52 Rivier et al.’s10 synthesis of betidamino acids (term introduced in the introductory section) involved the solid phase peptide s ynthesis by selectively acylating one of the two amino functionalities of the or thogonally protected aminoglycin e(s) to generate the side chain either prior to or af ter the elongation of the main chain. They used racemic orthogonally protected geminal diaminoglycine as the template for the introduction of BocHN COOH H2NOC DCC/PyridineBocHN COOH NC BocHN CON3 NC BocHN NHCbz NC BocHN NHCbz H2NOC m ixed anhydride method Curtius rearrangement benzyl alcohol reflux basic H2O2in acetone 45 46 47 48 49NHAc EtOOC Br2, CCl4NHAc EtOOC B r Liq. NH3NHAc EtOOC NH2 50 51 52

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62 betidamino acids in Acyline, which is a potent gonadotropinreleasing hormone antagonist. They followed an earlier synthesis of racemic -Fmoc’-Boc-aminoglycine by Qasmi et al.34 (Figure 3.18). Figure 3.18:Synthesis of -Fmoc’ -Boc-aminoglycine analogs Davies et al.35 in their paper reported that they had explored all the literature precedent reports for the synthesis of gem-diamino alkyl units, but on ly two published methods proved fruitful in their hands. The synthe tic route based on the work of Bock et al.30 (Figure 3.11) and synthesis via their adaptation of a procedure by Waki et al.36 (Figure 3.19) were the only synthetic sequences to give significant yields of OPGDA. Figure 3.19:Synthesis of gl ycine OPGDA by Davies et al. Sypniewski et al.11 introduced the first enantiospecific synthesis of (R)-Boc(Fmoc)-aminoglycine and (R)-Boc-(Cbz)-aminoglycine 60. They used (S)-serine (Lserine) 56 as a synthetic template for the enan tiospecific synthesis. Their synthetic FmocNHR2 + (HO)2HCCOOH + HSCH(CH3)2 toluene,PTS A Dean-Stark NR2Fmoc (H3C)2HCS COOH t BuO-CO-NHR1NBS, -30 oC then ambient T NR2Fmoc BocR1N C O O H CbzHN COOEt COOH DCC,HONSu, NH3CbzHN COOEt COONH2 i) TIB, Pyridine t -BuOH ii) baseCbzHN COOH NHBoc 5354 55

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63 strategy took advantage of th e carboxylic acid moiety of 56 to introduce the second amino functionality through car boxylic acid interconversion to a carbamino group via the acyl azide – isocyanate pathway. The hydroxymethyl group was then oxidized to carboxyl functionality (Refer to figure 3.20). Figure 3.20:Enantiospecifi c synthesis of OPGDA’s More recently it was Cantel et al.37 who reported the synthesis of gem-diamino derivatives on solid support. They anchored -aminoacid amide residue by its amine function to a carbamate resin followed by primary amide Hofmann rearrangement to yield gem-diamino residues linked to the resin. Later they acylated the primary amine generated in the previous st ep, with various carboxylic compounds thus to offer a large variety of molecules. Their novel solid-pha se strategy was feasible to synthesize gemdiamino monomeric residues that could not be easily obtain ed in solution phase due to the limited stability of monocarbamate-protected gem-diaminoalkyl derivatives. Their synthetic scheme was outlined below in the figure 3.21. Figure 3.21:Solid-phase peptide synthesis of gem -diamino derivatives H CbzHN HOOC OH DPPA, TEA tBuOH, reflux HN O CbzHN H O (Boc)2O,TE A THF, 0oC BocN O CbzHN H O BTAH, THF -40 oC H CbzHN BocHN OH R S PDC DMF, 25 oC H COOH CbzHN BocHN R 56 57 58 5960 OH Cl-COO-pNP NMM, DCM 0 oC O O O NO2 H2N-CHR1-CO-NH2DIEA, HOBt.H2O, DCM/DMF, rt, 12 h O O N H R1 O NH2 TBIB, pyridine, DMF/H2O O O N H R1 NH2 Fmoc-NH-CHR2-COOH BOP, DIEA O O N H R1 N H O NHFmoc R2

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64 The most recent of the reports on the synthesis of chiral gem-diamino compounds came in 2005 by Antilla et al.38 They synthesized chiral gem-diamino compounds via Brnsted Acid-catalyzed imine amidation (Refer to figure 3.22). Figure 3.22:Antilla et al. ’s synthesis of N,N -aminals 3.2.3 Our efforts towards the synthe sis of orthogonally protected -substituted diamino acids Scores of reports were seen from literat ure so far on the syntheses of compounds of general class-A (Figure 3.23) that are normal OPGDA’s, but none in the class-B. Our efforts that are represented in this chap ter, are towards the synthesis of class-B compounds that we coined as “Substituted OPGDA’s”. These are the building blocks for our designed novel HIV-1 protease inhibitors (Refer to Chapter-1). Figure 3.23:Difference between normal “O PGDA’s” and “Substituted OPGDA’s” YHN NHX COOR H X & Y = common amino protecting groups like -Cbz, -Boc, -Fmoc R = -H or alkyl ClassAYHN NHX COOR R1 X & Y = common amino protecting groups like -Cbz, -Boc, -Fmoc R = -H or alkyl R1= alkyl or arylClassBN O O t -Bu Ar +H2NR Bronsted acid RT, ether Ar N(H)R HN Boc POH O H Catalyst A = Tf2NHCatalyst B = Ph

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65 3.3 Results & Discussion Before I started working on this synthesis of substituted OPGDA’s, Dr. Talele a post-doctoral fellow in our lab had alread y pursued various routes towards their synthesis. The results and rout es explored by Dr. Talele are not mentioned here, but only the ones on which Dr. Talele and I worked or the ones that I worked independently are discussed in this chapter. Our first attempt towards the synthesis of substituted OPGDA’s was in similar lines with that of Goodman’s first synthesis12 of gem-diamino compounds, except that we employed porci ne liver esterase (PLE) for the desymmetrization of the 2-Azido-2methyl-malonic acid diethyl ester 62 to provide us the enantiomeric excess (Figure 3.24 ). Our enantioselective route started with the commercially available 2-met hyl-malonic acid diethyl ester 61. Azido transfer was accomplished successfully by the use of NaH to pull the methine proton of 61 and trisyl azide as the azide transfer r eagent. This reaction was also tried with tosyl azide but the yields of methyl az ido diethylmalonate 62 were poor (23% – 30%). Synthesis of Trisyl azide 68 was carried out by reac ting trisyl chloride 67 with sodium azide in aqueous ethanol, according to the standa rd literature reported procedure (Refer to figure 3.25 ). Desymmetrization of the prochira l methyl azido diethyl malonate 62 was accomplished by selective hydrolysis with P LE according to the literatu re reported procedures. We were able to achieve 84% yield of 2-Az ido-2-methyl-malonic acid monoethyl ester 63 which was then converted to 2-Azido-2azidocarbonyl-propionic acid ethyl ester 64 via the mixed-anhydride method. We tried Curtiu s rearrangement several times on this acyl azide 64 by refluxing in t-BuOH, but we failed to achieve the 2-Azido-2-tertbutoxycarbonylamino-propionic acid ethyl ester 65 Every time we ended up in a product

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66 with the loss of the azide group. We used Rh2(OAc)4 in catalytic amount and refluxed in t-BuOH and this time we were able to achieve 65 in very low yield (28%).This reaction had its own draw back of re producibility. With the compound 65 available in hand, we tried to reduce the azide to amine and follow it by a Cbz protection. But our attempt to reduce the azide with PtO2, EtOH/H2 was unsuccessful. We tried this reaction several times with H2 (balloon) and on hydrogenation apparatus at 25 psi, but in all the cases we failed to synthesize the free amine, instead we observed decomposition. We also tried in situ Cbz protection with PtO2 as the catalyst and H2 (balloon) but we ended up with streak of spots on the TLC, suggesting the possible decomposition of the starting material or the intermediate or the product. Figure 3.24:Failed attempt for the enan tioselective synthesis of substituted OPGDA’s H COOEt H3C COOEt NaH, 68 1,2-DME, reflux 12h, 60% COOEt COOEt N3 H3C PorcineliverEsterase PO4 buffer;CH3CN pH~7.5, r.t.,84% N3 COOH H3C COOEt i) isobutyl chloroformate, NMM,-30oC, 2hr ii)NaN3, H2O, -20oC 3hr, 80% N3 COOEt H3C NHBoc EtOOC NHBoc H3C NHCbz i) PtO2, EtOH/H2ii) Cbz-Cl, Pyridine, THF N3 COOEt H3C CON3 t-BuOH/Rh2(OAc)4reflux, 1hr, 28% X61 62 63 64 65 66 N3 NHBoc H3C O OEt CbzHN NHBoc H3C O OEt i) SnCl2, Dioxane/water ii) Cbz-Cl, pyridineX65 66

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67 Even the reduction of azide with SnCl2, dioxane/water and in situ Cbz protection with Cbz-Cl/pyridine did not yield us the desi red product 2-Benzyloxycarbonylamino-2-tertbutoxycarbonylamino-propionic acid 66 and we had to abandon this route. Figure 3.25:Synthesis of 2,4,6-Tr iiospropyl-benzenesulfonyl azide Desymmetrization of met hyl azido diethylmalonate 62 with PLE yielded monoacid 63 In order to find out th e enantiomeric excess we derivatized the chiral monoacid 63 with a known chiral amine (R)-(+)-1-phenylethylamine 69 and checked for the formation of diasteriomers of 2-Azido-2methyl-(R)-(+)-N-(1-phenyl-ethyl)malonamic acid ethyl ester 70 (Figure 3.26 ). But we were able to see only one spot on the TLC and also one set of peaks on the 1H NMR spectrum of the deri vative, indicating that there is only one diasteriomer (either R,S or R,R) and the enantiomeric excess was determined to be >99% (e.e). We did not determine the absolute stereochemistry of the acid 63 since our aim was to confir m whether we have only a si ngle enantiomer or not at this stage. Figure 3.26:Determination of enan tiomeric excess of monoacid 63 S O O Cl +NaN3Aq. EtOH S O O N3 67 68 N3 H3C O OEt OH O NH2 H3C HATU, HOBT, DCM/DMF DIEA, r.t., 12 h N3 H3C O OEt NH O H3C H H +636970R R, S or R, R

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68 Then we moved on to synthesize subs tituted OPGDA’s via a racemic route and came up with a scheme along similar lines with the synthesis of OPGDA’s by Sypniewski et al.11 Our racemic route shown in figure 3.27, started with a very old literature reported procedure39 for the synthesis of 2, 2-Bis-benzyloxycarbonylaminopropionic acid 73. Pyruvic acid 71 was reacted with phenyl carbamate 72 on a rotavap at 95 oC and under vacuum to pull the water formed during the reaction and thus push the reaction forward for the formation of gem-diamino acid 73 in 80% yield. Compound 73 was activated by making a 2,2-Bis-benzyl oxycarbonylamino-propionic acid 2,5-dioxopyrrolidin-1-yl ester 74 in 90% yield. Ester 74 was reduced to (1Benzyloxycarbonylamino-2-hydroxy-1-methyl-e thyl)-carbamic acid benzyl ester 75 by NaBH4 at 0 oC (70% yield). (4-Methyl-2-oxo-oxazolid in-4-yl)-carbamic acid benzyl ester 76 was obtained from alcohol 75 by the elimination of benzyl alcohol, and subsequent ring closure. We were able to achieve this in two different ways i) using potassium tertbutoxide in THF at -78 oC or ii) by sodium hydride/THF at -78 oC. Using NaH yielded 87% of oxazolidine 76, which is more when compared to 70% produced by KOtBu. Oxazolidine 76 was subjected to Boc protection by using (Boc)2O, DMAP (catalytic amount) and TEA to give 4-Benzyloxycarbo nylamino-4-methyl-2-oxo-oxazolidine-3carboxylic acid tert-butyl ester 77 over 90% yield. We were able to achieve till this step very smoothly with good to moderate yields. But we were unsuccessful in opening the Boc-protected oxazolidine 77 to yield the orthogonally protected (1-tertButoxycarbonylamino-2-hydroxy-1-methyl-e thyl)-carbamic acid benzyl ester 78. When oxazolidine 77 was treated with BTAH (phase tran sfer catalyst) in THF at -78 oC according to the literature reported procedure,11 we ended up in getting the undesired

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69 product (4-Methyl-2-oxo-oxazolidin-4yl)-carbamic acid tert-butyl ester 79 due to the elimination of benzyl alcohol. We tried doing this reaction at different condi tions i) by decreasing the amount of the BTAH used ii ) by changing the solvent from THF to toluene iii) by changing the te mperature conditions (from -78 oC to -40 oC and to room temperature), but still we e nded up with the undesired product 79. A possible mechanism for this ring flipping and elimination of the benzyl alcohol was deduced and was shown in figure 3.28. We concluded that the oxide intermediate during this reaction might be a strong enough nucleophile to attack the carbonyl carbon of the Cbz group and eliminate benzyl alcohol, and thus result in the flipping of the ring. Figure 3.27:Failed attempt to synthesize racemic substituted OPGDA COOH O + O NH2 O NHCbz CbzHN COOH H3C NHCbz CbzHN H3C O O N O O EDC.HCl NHS, DCM 0 oC, 5 h, 90% NaBH4, THF 0oC, 4 h, 70% NHCbz CbzHN H3C OH KOtBu, -78oC 1 h, 70% or NaH, THF, -78oC 1 h, 87% O H N CbzHN H3C O DMAP, Et3N (Boc)2O, THF 0 oC, 5 h, 90% O N CbzHN H3C O Boc BTAH, THF -78 oC, 5 h NHBoc CbzHN H3C OH X95 oC, 1 h, 80%71 72 73 74 75 76 77 78O N CbzHN H3C O Boc O H N BocHN H3C O BTAH, THF -78 oC, 5 h7779

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70 Figure 3.28:-Possible mechanism for the ring flipping and elimination of benzyl alcohol Our observation led us to believe that if we reverse the order of the protecting groups on the oxazolidine ring amine with Cbz and the amine geminal to it with a Boc; we might be able to eliminate the ring flipping. With that intention we tried to deprotect the Cbz from oxazolidine 76 by standard hydrogenation procedure by H2/Pd/C in MeOH. After 3 hr the starting material disappeared on TLC plate, but we did not isolate the free amine. The supposed crude free amine was su bjected to Boc protect ion; we were not successful in obtaining the compound 79. TLC suggested decomposition of the product. We tried to do the in situ Cbz deprotection followed by Bo c protection but the reaction failed again to achieve the compound 79 (Figure 3.29). Figure 3.29:Failed attempt to synthe size oxazolidine 79 from Oxazolidine 76 O H N CbzHN H3C O O H N BocHN H3C O Xi)H2 / Pd,MeOH ii) (Boc)2O, THF, 0oC76 79O N O O O HN Me O O Ph HO O NH O HN Me O O O N O O HN Me O O Ph O OH O N O O HN Me O O Ph O OH

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71 So we changed our plan to open the ring of oxazolidine 79 by a known literature procedure40 using cesium carbonate in MeOH at 0 oC to give the free amine 80. The reaction for ring opening did not yield the free amine 80, but instead the starting material remained unreacted. We increased the temperat ure but this was not of any help because we ended up with multiple spots on TLC suggesting possible decomposition (Figure 3.30). Figure 3.30:-Failed attempt for ring opening of compound 79 We changed our approach to tackle this pr oblem of ring flipping by using a milder base that can form an adduct with the oxide during th e intermediate stage i.e. as soon as it is formed. We proposed a mechanis m for ring opening of oxazolidine 77 with a milder base like trimethylsilanolate. The possible reac tion mechanism was depicted in figure 3.31. Figure 3.31:Proposed mechanism for the ring opening with trimethylsilanolate O N BocHN H3C O XCs2CO3MeOH, 0 oC NHBoc H2N H3C OH H 79 80O N NH H3C O O O O Ph O O Si O N NH H3C O O O O Ph O O Si O N NH H3C O O O O Ph O O Si O N NH H3C O O O O Ph O O Si O NH NH H3C O O O O Ph O O Si H Undesired Product

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72 The reaction mechanism seemed to be reason able for the fact that by protecting the hydroxy group with a trimethylsilanolate reduces the reactivity of the oxide intermediate. This in turn prevents the subsequent att ack of the urethane by the less reactive oxide adduct and prevents elimination of Cbz. We tr ied the reaction that we discussed above by treating oxazolidine 77 with potassium trimethylsilanolate in THF at 0 oC. But the reaction did not occur leaving th e starting material intact. We wanted to push the reaction by refluxing in THF, but this did not he lp us in achieving the desired product 81. Next we used catalytic amount of 18Crown-6 and performed the react ion by refluxing in THF. This time we did push the reaction far enough th at we ended up with the undesired product 79. This is formed due to the una voidable ring flipping a nd we had to abandon this route. We assumed that our proposed mechanism to avoi d ring flipping might be wrong. Instead of breaking the C-N bond if C-O bond breaks in the in termediate, then ring flipping is quite possible (Refer to figure 3.32). Figure 3.32:Failed reactions for ring opening of oxazolidine 77 O N CbzHN H3C Boc O CbzHN NHBoc H3C O KOSi(CH3)3 THF, 0oCXO N CbzHN H3C Boc O CbzHN NHBoc H3C O KOSi(CH3)3 THF, RefluxXO N CbzHN H3C Boc O KOSi(CH3)3 THF, Reflux18Crown-6 O N BocHN H3C H O O O Si(CH3)3 O O Si(CH3)3 77 81 77 81 77 79

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733.3.1 Effort towards the synthesis of cyclic urea 8 Our intention of making the substituted OPGDA’s was to differentiate the two geminal amines in terms of their reactivity so that we can achieve the synthesis of the cyclic urea moiety 8. We asked ourselves a question “Can we make cyclic urea 8 with out OPGDA’s?” To answer that question we wa nted to synthesize an aldehyde from the gemdiamino acid 73 which can undergo a Henry type reaction with our synthesized part-A i.e. substituted nitropropionates (NPA’s) discussed in chapte r 2. The subsequent reduction of the nitro alcohol to an amino alcohol, followed by ring closure potentially can lead us to our desired cyclic urea 8. With that intention we synthesized 2,2-Bisbenzyloxycarbonylamino-propionic acid methyl ester 82 from our previously synthesized gem-diamino acid 73 by standard methylation procedure in 90% yield. We were able to get a decent crystal structure for this compound 82. There were two patterns of crystallization for this molecule and the rela tionship of one patter n to the other in the formation of crystal lattice was reported in Appendix B along with the individual crystal structures of these two patterns. Compound 82 was subjected to reduction with DIBAL in THF at -78 oC to yield the (1-Benzyloxycar bonylamino-1-methyl-2-oxo-ethyl)carbamic acid benzyl ester 83 in 70% yield (Figure 3.33). Figure 3.33:Synthesis of gem-diamino aldehyde 83 NHCbz CbzHN H3C COOH K2CO3, CH3I DMF, 0 oC, 8 h 90% NHCbz CbzHN H3C COOCH3 DIBAL, THF -78oC, 3 h, 70% NHCbz CbzHN H3C CHO 73 82 83

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74 We were able to synthesize the aldehyde 83 also from oxidizing the alcohol 75 with Dess-Martin periodinane according to the literature reported procedure.41 These yields were good when compared to the DIBAL reduction of the ester 82.We did a model Henry reaction on this aldehyde 83 with nitromethane and DBU as the base to deprotonate the nitromethane to generate the carbanion, which in turn attacks the carbonyl carbon of the aldehyde resulti ng (1-Benzyloxycarbonylamino-2-hydroxy-1methyl-3-nitro-propyl)-car bamic acid benzyl ester 84 in 70% yield (Figure 3.34). Figure 3.34:Model Henry reactio n on 83 with nitromethane We failed to reduce this nitro-alcohol 84 to (3-Amino-1-benzyloxycarbonylamino-2hydroxy-1-methyl-propyl)-carbamic acid benzyl ester 85 by hydrogenation in the presence of catalytic amount of PtO2. Instead the reduction of the nitro group was achieved by using 5eq of freshly activated Zi nc in acetic acid/THF solvent system at room temperature. We obtained the amino-alcohol 85 in 70% yield (Figure 3.35). Figure 3.35:Synthesis of amino-alcohol 85 NHCbz CbzHN H3C OH Dess-Martin periodinane DCM, 0oC, 2hr,75% NHCbz CHO CbzHN H3C CH3NO2,DBU THF, 0oC, 3h 70% NHCbz CbzHN H3C NO2 OH 75 83 84NHCbz CbzHN H3C NO2 OH PtO2, A ceticacid H2, r.t NHCbz CbzHN H3C NH2 OH Zn, CH3COOH THF, r.t, 3h, 40%XNHCbz CbzHN H3C NO2 O H NHCbz CbzHN H3C NH2 OH 84 85 84 85

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75 We tried to synthesize the six-membered ring 86, which will be the simplest of our novel HIV-1 protease inhibitor building bl ock. We could theoretically achieve the cyclic urea 86 by eliminating benzyl alcohol from compound 85 and subsequent ring closure to potentially fo rm (5-Hydroxy-4-methyl-2-oxo-hexahydro-pyrimidin-4-yl)carbamic acid benzyl ester 86 (Figure 3.36). Several attempts were made in this direction, various solvents like 1,4-Dioxane, 1,2-DM E, THF and acetone were used at their respective reflux temperatures and we were not able to achieve the desired cyclic urea 86. In almost all the cases thermal decomposition was observed by noting multiple spots on the TLC after the workup or during the reacti on itself. These reactions were closely monitored by checking TLC at various time inte rvals and still we were able to observe the decomposition of the starting material. We tried an in situ nitro reduction and cyclization by treati ng the nitro compound 84 with Zn/CH3COOH and immediately followed with reflux in 1,4-dioxane. This ti me also we observed multiple spots on TLC after 1 hr and these failed attempts forced us to abandon this route from any further exploration. Figure 3.36:Failed attempts for cyclization Our analysis suggests that the difficulty in this ring closure may be due to lack of driving force for the elimination of the benz yl alcohol from one of the Cbz groups and NHCbz CbzHN H3C NH2 OH HNNH CbzHN H3C OH O 1,4-Dioxane, reflux i)Zn/CH3COOH ii) 1,4-Dioxane,reflux 1,2-DME,reflux THF,reflux Acetone,reflux X X X X X 85 86NHCbz CbzHN H3C NO2 O H 84

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76 this could prevent the subsequent ring closur e. We wanted to replace the Cbz protecting group on the gem-diamino with a common protecting gr oup like Boc and try our previous scheme for the ring closure. We were trying to compare the ease of ring closure with the changes in the protecting groups. The proposed scheme for the synthesis of the cyclic urea 93 was depicted in figure 3.37 and this is in similar lines with the failed attempt to synthesize cyclic urea 86. We hoped that this slight modification may generate the desired compound, which was not uncommon in literature where certain reactions work better with a change of the protecting group. Figure 3.37:Proposed scheme for the synthesis of cyclic urea 93 NHBoc COOH BocHN H3C EDC.HCl, NHS NHBoc BocHN H3C O N O O O NaBH4NHBoc BocHN H3C OH Dess-Martin Oxidation NHBoc CHO BocHN H3C CH3NO2 DBU NHBoc BocHN H3C NO2 HO Pd/C, H2NHBoc BocHN H3C NH2 HO HNNH H3C BocHN O OH Cyclization 87 93 88 89 90 91 92NHBoc BocHN H3C NH2 HO NHCbz CbzHN H3C NH2 HO HNNH H3C BocHN O OH HNNH H3C CbzHN O OH 93 86 92 85

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77 So our next step was towards the synthe sis of the 2,2-Bis-tert-butoxycarbonylaminopropionic acid 87 which is exactly similar to that of the gem-diamino compound 73 except that 87 is a di-Boc version of 73. We tried the rotavap reaction (described previously) with t-butyl carbamate 94 and pyruvic acid 71 at 70 oC but we failed to obtain the desired product 87 (Figure 3.38). The starting material t-butyl carbamate remained as the major impurity along with seve ral other uniden tified products. Scheme 3.38:Failed rotavap rea ction for the synthesis of the 87 We proposed another strategy for the synthesis of 93 that is shown in figure 3.39. This avoids using of the di-Boc compound 87 which could not be synthesized previously. Instead we wanted to st art with the di-Cbz compound 75 from the previous scheme. Figure 3.39:Proposed scheme via selective Boc protection O NH2 O + COOH O NHBoc BocHN H3C COOH 70 oC, 4 hrX87 94 71CbzHN NHCbz H3C OH DMAP, TEA (Boc)2O CbzHN N H3C OH Boc Cbz Dess-Martin CbzHN N H3C H Boc Cbz O CH3NO2, DBU CbzHN N H3C Boc Cbz HO NO2 Pd/C, H2H2N HN H3C Boc HO NH2 HNNH H3C BocHN O OH i) Pd/C, H2ii)CDI X 93 75 CDI 95 96 97 98

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78 According to the new strategy, we wanted to selectively perform a Boc protection on one of the Cbz protected amines on the gem-diamino compound 75. This way we could achieve the reduction of the nitro and e limination of Cbz group by the standard hydrogenation procedures. This will not touc h the Boc protected amine. We were not able to do this kind of one pot reduction till now because of th e potential problem of deprotecting both Cbz gr oups (Refer to figure 3.35). This would have resulted in the collapse of the molecule. Even though this st rategy seemed reasonable we couldn’t go much far in the proposed scheme. We tried to selectively protect one of the Cbz protected amines of compound 75 by using 0.5eq of (Boc)2O, 0.5eq of TEA and catalytic amount of DMAP. But we ended up in a compound confir med by NMR to be the Bocprotected alcohol. We could not explor e this route any further. Since we had an availability of a variet y of nitropropionates (from chapter-2), we attempted to perform Henry reaction with the gem-diamino aldehyde 83. We attempted the nitroaldol condensation several times using the isobut yl nitropropionic acid 16c and isobutyl nitropropionate 17c with aldehyde 83 in presence of DBU as the base and THF as the solvent. Various reaction conditions were tried without any success to achieve the desired nitro-alcohol 99 or 100 and we concluded that it might be due to steric effects (Fig 3.40) Figure 3.40:Failed attempts of nitroaldol condensation CbzHN NHCbz H3C CHO + NO2 OH O CbzHN CbzHN H3C OH O2N HO O CbzHN NHCbz H3C CHO + NO2 OCH3 O CbzHN CbzHN H3C OH O2N H3CO O DBU, THF 0 oC to r.tX X16c 17c 83 83 99 100DBU, THF 0 oC to r.t

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793.3.2 Electrophilic Amination with Oxaziridines We realized that all the routes that we had visited so far were almost in similar lines, either the Curtius rearra ngement or the racemic route via oxazolidine ring opening. But still the task for achievi ng the substituted OPGDA’s remained at large. We wanted to approach this problem from a view that is different from that of the literature reported approaches for gem-diamino compounds syntheses. We tried to make a new C-N bond that is geminal to an amine or protected am ine via electrophilic am ination. Electrophilic amination is an important s ynthetic reaction in which el ectron-poor nitrogen carried by the reagent is transferred to a nucleophilic center of the subs trate to form a Nu-N bond in the product. There were several literature reports on electrophil ic amination with oxaziridines.42-49 But the one that specifically caught our attention was N-Boc-3trichloromethyloxaziridine 104 which was reported for the first time by Vidal et al.50 as a versatile and powerful reagent for elec trophilic amination. We synthesized 104 following their procedures (Figure 3.41). We started with t-Butyl carbazate 101 and reacted it with NaNO2 in acetic acid and water at 0 oC for 30 min. The resulting t-butyl azide was immediately reacted with triphenylphos phine to yield N-Boc iminophosphorane 102 in more than 80% yield. We were able to obtain nice crystals of the compound 102 and the crystal structure was reported in the Appendix B. Compound 102 was treated with chloral (a prescription drug in United States), under the reflux conditions of toluene via an Aza-Wittig reaction type to yield imine 103 in 95% yield after a vacuum distillation in a Kugelrohr apparatus. Th e final compound oxaziridine 104 was obtained by oxone oxidation of the imine 103. This was very laborious process where the aqueous phase along with the consumed oxone /K2CO3 in the reaction mixture ha d to be replaced after

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80 every hour. Apparently there was no visualizatio n technique to monitor if this change was really helping or not. We thought if we can make a cha nge in this step, the whole synthesis can become more efficient and le ss time consuming. As mentioned earlier the real challenge in monitoring this reaction is lack of visualization technique since neither the imine nor the oxaziridine show up on th e TLC. We changed various TLC developing agents like PMA, iodine, phenol/sulfuric ac id, ninhydrin etc but none of them worked. We tried to do a model reaction with triflurobenzaldehyde 106 so that we can make an imine 107 that can be monitored on TLC for th e oxidation with oxone, but the reaction between aldehyde 106 and iminophospharane 102 failed (Figure 3.42). Figure 3.41:Scheme for the sy nthesis of Oxaziridine 104 We were able to achieve decent yields of 104 by vacuum distillation of the crude product in a Kugelrohr appara tus instead of the column chromatography performed by Vidal et al. This was only the significant change that we could incorporate in their synthesis. We noticed a potential problem of hydrolysis of imine 103 and the oxaziridine O O NH NH2 i)NaNO2,CH3COOH / H2O(1:2) 00C, 30min ii) Ph3P, diethyl ether, 00C to r.t., 30min O N O P Ph Ph Ph Cl3CCHO, toluene, 1100C, 1hr O N O HC Cl Cl Cl K2CO3, OXONE H2O/CHCl380% Cl3C N O Boc O N H O CH Cl Cl Cl OH 81% 95% 101 102 103 104 105

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81104. If they were left at room temperature unsealed we obtained the hydrolysis product 105, which was confirmed by comp aring with the literature reported melting point. We were able to obtain a good crys tal structure of this mole cule, which was reported in Appendix B. This hydrolysis problem was fu rther avoided by stor ing the oxaziridine 104 in the freezer at -4 oC and in a tightly sealed container. Figure 3.42:Failed attempt to synthesize imine 69 Our aim behind the synthesis of this oxazi ridine was able to use it as an N-Boc transfer reagent on -carbon in an amino acid, so that we can generate the gem-diamino compound. Figure 3.43 outlines our proposed synthesis of the substituted OPGDA’s and the subsequent ring closure to form the cyclic urea 114. Figure 3.43:Proposed synthesis of substituted OPGDA’s via oxaziridine 104 L-phenyl alanine methyl ester 108 was reacted with equimolar benzophenone imine 109 according to the literature reported procedure51,52 to synthesize a benzophenone Schiff O N O P Ph Ph Ph F F F F F CHO O N O H C F F F +F F Toluene, 110oCX102 106 107Ph NH3Cl H3COOC Ph Ph NH DCM, r.t., 24 h 60% Ph N COOCH3 Ph Ph Ph N CHO Ph Ph BocHN CH3NO2,DBU Ph N Ph Ph BocHN NO2 HO H2/Pd CDI HNNH O OH BocHN Ph X104 NaH, THF r.t., 4 h DIBAL Ph N H3COOC Ph Ph BocHN + 108 109 110 111114 113 112

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82 base derivative of pheny l alanine methyl ester 110 in 60% yield. The imine derivative 110 was treated with the oxaziridine 104 in order to transfer the N-Boc on -carbon to that of the imine, but our attemp t failed to synthe size the compound 111. We tried to see if we can make N-Cbz version of the oxazi ridine and use that for the electrophilic amination (Figure 3.44). But our efforts towards the synthesis of compound 116 were futile. We concluded that the intermediate free amine 115 might be highly unstable and not isolable because of its polar nature. We tried in situ Boc deprotection and Cbz protection according to literature procedures, but that was not of much help. We abandoned the idea of making substituted OP GDA’s via electrophilic amination using oxaziridine 104. Figure 3.44:Failed attemp t to make Cbz version of the oxaziridine 104 3.3.3 Application of Oxaziridine 104 in -helix mimics synthesis With an improved synthesis of oxaziridine 104 in terms of overall yield as well as efficiency in purification (by Vacuum dis tillation of the crude product instead of the expensive silica gel chromatography done by Vidal et al.), we were able to perform a multigram scale synthesis of 104. We did a model study on the effect of solvents on the electrophilic amination of diethyl iminodiacetate 117 to synthesize (N'-tertButoxycarbonyl-N-ethoxycarbonylmethyl-hydrazino) -acetic acid ethyl ester 118 and the results were tabulated in the figure 3.45. Cl3C N O Boc TFA/DCM 0oC to R.T Cl3C NH O Cbz-Cl Collidine Cl3C N O Cbz X104 115 116

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83 Entry Solvent Time Temp. % Yield % unreacted 1) CH2Cl2 (5 mL) 12 h -78 oC to r.t.80% 20% 2) THF (5 mL) 12 h -78 oC to r.t. 87% 10% 3) MeOH (5 mL) 12 h -78 oC to r.t.94% not cal. 4) CH3CN (5 mL) 12 h -40 oC to r.t.>99% not cal. 5) EtOAc (5 mL) 12 h -78 oC to r.t.70% 30% 6) 5% MeOH in DCM 12 h -78 oC to r.t.50% 50% Figure 3.45:Solvent effects on the NBoc transfer on the secondary amine Having this data from this model study ha d helped us in achieving a crucial step in the synthesis of -helix mimics, which was another pr oject (currently in progress) in our lab. Dr. McLaughlin designed -helix mimics that have drug-like properties and can be used in preventing certain protein-protein interactions in a cancerous cell and in turn promotes apoptosis. Details regarding this pr oject were discussed elsewhere (refer to Stephanie Weiss’s dissertation work).53 The designed “McLaughlin helix”, a 3substituted piperazine-2,6-dione repeat units and the retro synthetic analysis of this molecule were depicted in Figure 3.46. This helix was built in similar fashion as that of “Hamilton helix”54 but a significant consideration was given to answer the aqueous O O H N O O O O N O O NHBoc Cl3C N O Boc Solvent117 118 104MODEL REACTION

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84 solubility and physiological co mpatibility issues. Some mole cular modeling data was also generated in order to suppor t our idea for mimicking the -helix. Figure 3.46:“McLaughlin Helix” and retro synthetic analysis of 3-substituted piperazine-2,6-dione repeat units The proposed route for the synthesis of An moiety i.e. hydrazine diacid is shown in figure 3.47 (details of the scheme are not discussed here). We wanted to use the oxaziridine 104 as an N-Boc transfer reagent on the secondary amine, which is a key and crucial step for the synthesis of the hydrazino diacid. Figure 3.47:Use of Oxaziridine 104 in th e key step for the synthesis of the An subunit NH NNNHNNHNH O O OH O HO OOO OOO H R1H R2H R3 A1 A2 A3N R O HO H O HO BocHN O O NHBoc Resin An ClH3N R O H3CO H N R O H3CO H O O BocHN N R O HO H O HO BocHN HN R O H3CO H O O O NBoc Cl3C DIEA, BrCH2CO2CH2CH3 CH3CN, r.t., CH3CN, -780CAq. NaOH/EtOH 104An

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85 With the intention stated above we tried our synthesis on phenyl alanine methylester 119 as one of the substituents for the An moiety. We took compound 119 and treated with DIEA and ethylbromoacetate to yield the N-alkylated product 120, which was further treated with the oxaziridine 104 to yield the desired hydrazine 121 in more than 80% yield. This was done in order to prove that oxaziridine works as an excellent N-Boc transfer reagent in our key step and also to standardize the reaction conditions (Figure 3.48). Figure 3.48:Synthesis of Phe-N-Boc-hydrazine in two steps using oxaziridine 104 3.3.4 Design considerations in the core stru ctural unit of HIV-1 protease inhibitors synthesis and Future direction We revised our task to synthesize a much simpler DPU (DiPeptide Unit) subunit 126 compared to that of the much complex cyclic urea 8. The only difference in this change of design is that we don’t need th e substituted OPGDA’s as one of our building blocks, instead we can use an aldehyde derived from N-Boc aspartic acid benzyl ester 122 (Figure 3.49). We activated the acid 122 with NMM and isobutyl chloroformate to H2N Ph O O HCl DIE A ,BrCH2CO2CH2CH3CH3CN, r.t., 18 h, N H Ph O O O O N Ph O O O O NH Boc O N Cl3C Boc CH2Cl2-78oC, 5 h119 120 104 121

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86 form the ester intermediate and subseque ntly reduced the intermediate with NaBH4 to give the primary alcohol 123 in 80% yield. The primary alcohol 123 was subjected to Swern oxidation to yiel d the required aldehyde 124 in 70% yield. We did a model Henry reaction on this alde hyde with nitromethane and DBU as the base to yield the nitroalcohol 125 in 50% yield. The future plan would be to reduce the nitro and deprotect the Cbz group from the carboxylic acid in one pot by hydrogenolysis using Pd as the catalyst. This would yield the intermediate aminoacid. Subsequent ring closure with CDI could potentially yield the DPU compound 126. Figure 3.49:Synthesis of DPU 126 as th e building block for the HIV-1 protease inhibitors Library 3.4 Conclusion In conclusion we can state that various routes for the synthesis of substituted OPGDA’s were explored. Some of them are sim ilar to those reported in the literature and some that we specifically designed to tailor our needs. Unfortunately none of them have BocHN O O OH O i)NMM Isobutylchloroformate ii) NaBH4BocHN O O Ph OH Swern OxidationBocHN CHO O O Ph CH3NO2, TBAF BocHN O O Ph NO2 HO i)H2/Pd MeOH ii) CDINH H BocHN OH O Ph 122 123 124 125 126

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87 yielded our desired subunit (part-B of cyclic urea 8 moiety) substituted OPGDA’s. But all these routes serve as a guideline for the future synthe sis of the substituted OPGDA’s by others. The decent outcome of this project is the efficient synthesis of versatile N-Boc transfer reagent oxaziridine 104, which has a potential applic ation in the synthesis of helix mimics designed by Dr. McLaughlin. 3.5 Experimental Synthesis of 2,4,6-Triisopropylbenzenesulfonyl azide (68) :To suspension of sodium azide (7.0 g, 109 mmol) in absolute EtOH (60 mL), distilled water (~ 50 mL) was added until a clea r solution is seen. 2,4,6-Triisopropyl benzenesulfonyl chloride 67 (10 g, 33 mmol), dissolved in absolute EtOH (150 mL) was added to the above sodium azide solution wh ile stirring. The cloud iness of the reaction mixture was cleared by the addition of a queous EtOH (80%). The reaction mixture was stirred for 24 h at room temperature. Afte r that, EtOH was evaporated, and the aqueous layer was extracted with Et2O (50 mL). The organic layer wa s separated and washed with brine (30 mL), dried over anhydrous Mg2SO4 (3 g) and evaporated in vacuo to afford a pure product 68 as a white solid; Rf = 0.42 (hexane) and mp 42-43 oC (lit. mp 40-42 oC) S O O N3 COOEt COOEt N3 H 3 C

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88Synthesis of 2-Azido-2-methyl-mal onic acid diethyl ester (62):60% NaH in mineral oil (0.25 g, 6.32 mmol ) was washed under argon flow with hexane (3 x 5 mL). The resulting free flowing pow der of NaH was suspended in 1,2-DME (8 mL) and was treated with 2-me thyl-malonic acid diethyl ester 61 (1 g, 5.75 mmol) drop wise. The reaction mixture was heated at 50 oC for 1 h, and a solution of 2,4,6Triisopropyl-benzenesulfonyl azide 68 (1.96 g, 6.32 mmol) in minimum amount of 1,2DME was added in portions. The whole r eaction mixture was brought to reflux and stirred for 12 h. After 12 h starting material was not seen on the TLC plate. The reaction mixture was filtered and the filtrate was subjected to evaporation. The residue was distributed between H2O (20 mL) and EtOAc (30 mL), and the organic layer was subjected to further washing with brin e (2 x 10 mL), drie d over anhydrous MgSO4 and evaporated in vacuo to yield the crude pr oduct. This crude residue was subjected to column chromatography (1:3 EtOAc/hexane ) to give the compound 2-Azido-2-methylmalonic acid diethyl ester 62 (0.72 g, 60%) as a pale yellow oil; Rf = 0.62 (1:1 EtOAc/Hexane); 1H NMR (CDCl3, 250 MHz) 1.23-1.29 (t, J =7.5 Hz, 6H), 1.50 (s, 3H), 4.20-4.29 (q, J = 7.5 Hz, 4H); 13C NMR (CDCl3, 100 MHz) 17.9, 24.3, 66.6, 72.1, 171.7 ppm; ESI-TOF Calcd for [M+H]+ is 216.09788, Found: 216.09786 Synthesis of 2-Azido-2-methyl-mal onic acid monoethyl ester (63):Commercially available Porcine Liver Este rase (Aldrich) (0.89 g, 17,814 units) was weighed and placed immediatel y in a reaction vessel contai ning a solution of 2-Azido-2-N3 COOH H 3 C COOE t

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89 methyl-malonic acid diethyl ester 62 (3.8 g, 17.8 mmol) in phosphate buffer (158 mL) and CH3CN (18 mL) at a pH of 7.5. The reaction mixture was stirred at room temperature for 24 h, and then acidified by adding 1N HC l until the pH was equal to 3.0. EtOAc (100 mL) was added to the reaction mixture and fi ltered through celite. The organic layer was separated from the filtrate and the aqueous la yer was extracted with EtOAc (2 x 100 mL). Combined organic layers were washed with brine (300 mL), dried on anhydrous Na2SO4 and evaporated in vacuo to yield the cr ude compound. The crude product was subjected to column chromatography to yield 2-Azi do-2-methyl-malonic acid monoethyl ester 63 (2.7 g, 84%) as a pale yellow oil; Rf = 0.21 (1:3 EtOAc/hexane); 1H NMR (CDCl3, 250 MHz) 1.25-1.32 (t, J = 7.5 Hz, 3H), 1.58 (s, 3H), 4.22-4.35 (q, J = 7.5 Hz, 2H); 13C NMR (CDCl3, 62.5 MHz) 13.8, 20.3, 63.1, 68.0, 167.3, 172.9 ppm Synthesis of 2-Azido-2-azidocarbonylpropionic acid ethyl ester (64): To a solution of 2-Azido-2-met hyl-malonic acid monoethyl ester 63 (1.1 g, 5.9 mmol) in anhydrous THF (20 mL) at -20 oC, was added NMM (0.6 g, 5.9 mmol) followed by isobutylchloroformate (0.8 g, 5.9 mmol). The reaction mixture was stirred at -20 oC for 2 h, until the starting material disappeared on the TLC. After which it was filtered through celite, and the filtrate was brought to -20 oC and NaN3 (0.96 g, 14.7 mmol) was added while stirring. After 3 h, brine (50 mL) was added to the reaction mi xture and the organic layer was separated and extracted again w ith EtOAc (50 mL). The combined organic layers were subjected to drying on anhydrous Na2SO4 and evaporated in vacuo until N3 COOEt H 3 C CON 3

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90 minimum amount of solvent was left.a A minimum amount of th is product was taken for the characterization and the re st of the material was forw arded to the next step. Rf = 0.65 (1:4 EtOAc/hexane); 1H NMR (CDCl3, 250 MHz) 1.24-1.30 (t, J= 7.5 Hz, 3H), 1.49 (s, 3H), 4.21-4.29 (q, J= 7.5 Hz, 2H); 13C NMR (CDCl3, 62.5 MHz) 14.2, 20.7, 63.5, 69.6, 167.3, 175.3 ppm aNOTE: Complete evaporation of solvent is not recommended because of the highly unstable and explosive nature of acylazides. Synthesis of 2-Azido-2-tert-butoxycarbonylamino-propionic acid ethyl ester (65):To a solution of 2-Azido-2-azi docarbonyl-propionic acid ethyl ester 64 (0.6 g, 2.83 mmol) in t-BuOH (10 mL) catalytic amount of Rh2(OAc)4 (3.0 mg) was added and was refluxed at 80 oC. After 1 h, t-BuOH was evaporated and Et OAc (10 mL) was added to the residue and filtered. The filtrate wa s evaporated in vacuo while the rotavap temperature was maintained at 30 oC. The product obtained was characterized without any further purification. Obtained a colorless oil; Rf = 0.32 (1:4 EtOAc/hexane); 1H NMR (CDCl3, 250 MHz) 1.27-1.32 (t, J= 7.0 Hz, 3H), 1.42 (s, 9H), 1.63 (s, 3H), 4.25-4.30 (m, 2H), 5.72 (br s, 1H); 13C NMR (CDCl3, 62.5 MHz) 14.4, 21.4, 28.4, 60.8, 63.1, 74.1, 154.2, 169.5 ppm N3 H3C O OEt NH O H3C H N3 COOEt H3C NHBoc

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91Synthesis of 2-Azido-2methyl-(R)-(+)-N-(1-phenyl-ethyl)-malonamic acid ethyl ester (70):To a solution of HOBT (0.04 g, 0.30 mmol) HATU (0.12 g, 0.30 mmol), DIEA (47 L, 0.30 mmol) in 5 mL of DCM / DMF solvent sy stem (2:1), was added 2-Azido-2-methylmalonic acid monoethyl ester 63 (0.05 g, 0.27 mmol) and the chiral amine (R)-(+)-1phenylethylamine 69 (62 L, 0.30 mmol) and stirred at room temperature for 12 h. EtOAc (5 mL) was added to the reaction mixt ure and washed with brine (2 x 5 mL) to remove DMF. The organic layer was separated, dried on anhydrous Na2SO4 and evaporated in vacuo to yield th e pure compound 2-Azido-2methyl-(R)-(+)-N-(1-phenylethyl)-malonamic acid ethyl ester 70 (0.055 g, 77%) as a pale yellow oil; Rf = 0.61 (1:4 EtOAc/hexane); 1H NMR (CDCl3, 250 MHz) 1.20-1.26 (t, J = 7.5 Hz, 3H), 1.50-1.53 (d, J = 7.5 Hz, 3H), 1.78 (s, 3H), 4.184.26 (q, J =7.5 Hz, 2H), 5.035.15 (p, J = 7.5 Hz, 1H), 7.30-7.37 (m, 5H); 13C NMR (CDCl3, 62.5 MHz) 14.3, 20.3, 22.0, 49.5, 63.2, 69.2, 126.4, 127.8, 129.0, 129.2, 142.8, 166.2 ppm; ESI-TOF Calcd for [M+H]+ is 291.14517, Found: 291.14518 Synthesis of 2,2-Bis-benzyloxycar bonylamino-propionic acid (73) :To a round bottom flask add benzyl carbamate 72 (15.5 g, 102 mmol) and pyruvic acid 71 (3.9 mL, 56.8 mmol) and rotavap the reactio n mixture. The temperature of the water bath was maintained at 70 oC for 2 h, and then dissolved the reaction mixture in excess ethyl acetate (200 mL). Or ganic layer was washed with cold distilled H2O (2 x 100 mL) in order to get rid of unreacted pyruvic acid. Hexane was added slowly to the organic NHCbz CbzHN COOH H 3 C

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92 layer until a clean white precipitate was obtai ned. Filtered the suspension to obtain 2,2Bis-benzyloxycarbonyl amino-propionic acid 73 (16.8 g, 80%) as a white solid; Rf = 0.4 (1:1 EtOAc/hexane); mp 137-139 oC (lit. mp 139 oC); 1H NMR (CD3OD, 250 MHz) 1.78 (s, 3H), 5.04 (s, 4H), 7.30-7.35 (m, 10H); 13C NMR (CD3OD, 100 MHz) 21.1, 64.6, 65.7, 125.9, 126.2, 126.6, 135.2, 153.6, 171.0 ppm; ESI-TOF Calcd for [M+Na]+ is 395.12136, Found: 395.12112 Synthesis of 2,2-Bis-benzyloxycarbonylamino -propionic acid 2,5-dioxo-pyrrolidin-1yl ester (74) : To a solution of acid 73 (4.23 g, 11.4 mmol) in dry DC M (30 mL) were added N-hydroxy succinamide (1.30 g, 11.4 mmol) and EDCl (2.17 g, 11.4 mmol) while stirring at 0 oC and maintained at 0 oC for 1 h. After which the reac tion mixture was brought to room temperature and further stirred for 5 h, and the washed with H2O (2 x 25 mL). DCM layer was separated, washed with brine (3 x 50 mL), dried over anhydrous MgSO4 and was evaporated in vacuo to give pale yellow o il. The crude product was subjected to column chromatography (1:1 EtOAc/hexane) to give the compound 74 (4.9 g, 92%) as a pale yellow oil; Rf = 0.54; 1H NMR (CDCl3, 250 MHz) 1.91 (s, 3H), 2.71 (s, 4H), 5.00 (s, 4H), 6.39 (br s, 2H), 7.23-7.39 (m, 10H); 13C NMR (CDCl3, 100 MHz) 20.9, 21.2, 25.5, 60.7, 67.4, 128.4, 128.5, 128.7, 128.8, 136.1, 158.1, 172.8, 176.0, 176.5 ppm; ESI-TOF Calcd for [M+Na]+ is 492.13774, Found: 492.13719 NHCbz CbzHN H3C O O N O O

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93 Synthesis of (1-Benzyloxycarbonylamino-2 -hydroxy-1-methyl-ethyl)-carbamic acid benzyl ester (75): To a solution of N-hydroxy succinamide derivative 74 (4.9 g, 10.5 mmol) in anhydrous THF (25 mL), NaBH4 (0.8 g, 21 mmol) was added while stirring at 0 oC. The reaction mixture was stirred for 1 h at 0 oC and then brought to room temperature and further stirred for 24 h, after which it was quenched with 10% KHSO4 solution (20 mL). THF was evaporated and the aqueous layer was extracted with DCM (3 x 100 mL), and the combined organic layers were washed with brine (2 x 100 mL), dried over anhydrous MgSO4 and evaporated in vacuo to yield a pale yellow solid product. The crude product was recrystallised from DCM/hexane to yield pure compound 75 (2.63 g, 71%) as a white solid; Rf = 0.70 (1:1 EtOAc/hexane); mp 102-105 oC; 1H NMR (CDCl3, 400 MHz) 1.57 (s, 3H), 3.73 (s, 2H), 5.04 (s, 4H), 5.17 (br s, 1H), 6.29 (br s, 2H), 7.32-7.36 (m, 10H); 13C NMR (CDCl3, 100 MHz) 25.3, 67.0, 67.3, 68.8, 128.2, 128.4, 128.7, 136.2, 155.8 ppm; ESI-TOF Calcd for [M+Na]+ is 381.14209, Found: 381.14198 Synthesis of (4-Methyl-2-oxo-oxazolidin-4-y l)-carbamic acid benzyl ester (76):Alcohol 75 (2.63 g, 7.35 mmol) from the previous step was dissolved in anhydrous THF (30 mL) and was cooled to -78 oC while stirring under argon atmosphere. To the above solution, KOtBu solution (9.0 mL from stock 1M 50 mL THF, 8.97 mmol) was added in NHCbz CbzHN H 3 C OH O H N CbzHN H3C O

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94 one portion and was allowed to stirred for 10 min after which the reaction was warmed to room temperature and stirred for 1 h. Reaction was quenched with 10% NH4Cl solution (10 mL), THF was evaporated and the aque ous layer was extracted with DCM (3 x 30 mL). The combined organic layers were wash ed with 0.1N HCl (2 x 10 mL), followed by brine wash (2 x 50 mL), dried over anhydrous MgSO4 and evaporated in vacuo to yield the crude product as yellow oil. Recrystalliz ation in DCM/hexane yielded pure compound 76 (1.38 g, 75%) as a white solid; Rf = 0.21 (1:1 EtOAc/hexane); mp 125-128 oC; 1H NMR (CDCl3, 400 MHz) 1.64 (s, 3H), 4.16-4.19 (d, J = 9.6 Hz, 1H), 4.51-4.54 (d, 9.2 Hz, 1H), 5.09 (s, 2H), 7.30-7.36 (m, 5H); 13C NMR (CDCl3, 100 MHz) 26.0, 66.9, 69.3, 75.4, 128.2, 128.3, 128.6, 135.9, 154.9, 158.6 ppm; ESI-TOF Calcd for [M+H]+ is 251.10263, Found: 251.10272 4-Benzyloxycarbonylamino-4-methyl-2-oxo-oxaz olidine-3-carboxylic acid tert-butyl ester (77):Oxazolidine 76 (1.36 g, 5.44 mmol), DMAP (0.013 g, 0.10 mmol), TEA (0.9 mL, 6.53 mmol) were dissolved in anhydrous THF (20 mL) and was cooled down to 0 oC. A solution of tert-butyl chloroformate (1.25 g, 5.71 mmol) in anhydrous THF (5 mL) was added to the above mixture under argon atmo sphere, while stirring over a period of 15 min. The reaction temperature was maintained between 5 oC to 10 oC for 3 h and then the reaction mixture was quenched wi th 0.001 mmol solution of KHSO4 (5 mL). THF was evaporated to leave a white suspension whic h was dissolved in EtOAc (100 mL) and was O N CbzHN H3C O Boc

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95 washed with 10% KHSO4 solution (20 mL), dried over anhydrous Na2SO4 and evaporated in vacuo to yield Boc protected oxazolidine 77 (1.9 g, 99%) as pure white solid; Rf = 0.82 (4:1 EtOAc/hexane); mp 131 – 134 oC; 1H NMR (CDCl3, 400 MHz) 1.50 (s, 9H), 1.77 (s, 3H), 4.11-4.14 (d, J = 9.6 Hz, 1H), 4.67-4.69 (d, J = 8.8 Hz, 1H), 5.09-5.12 (d, J = 10.8 Hz, 2H), 5.69 (br s, 1H), 7.32-7.36 (m, 5H); 13C NMR (CDCl3, 100 MHz) 27.9, 67.1, 71.6, 71.9, 84.5, 128.3, 128.4, 128.5, 128.6, 135.6, 149.1, 151.1, 154.2 ppm; ESI-TOF Calcd for [M+NH4]+ is 368.18161, Found: 368.18129 Synthesis of (4-Methyl-2-oxo-oxazolidin-4-yl )-carbamic acid tertbutyl ester (79): Boc protected Oxazolidine 77 (1.0 g, 2.86 mmol) from the pr evious step was dissolved in anhydrous THF (20 mL) a nd was cooled to -78 oC. Phase transfer catalyst, BTAH (1.3 mL of 40% w/v methanolic solution, 3.15 mmol) was added to the above solution drop wise, while stirring under argon atmosphere and was allowed to stir for 6 h, and then warmed to 40 oC, CH3COOH (1 mL) and distilled H2O (1.5 mL) were added to the reaction mixture and stirred for 30 min, after which the organic solvents were evaporated to leave behind a white residue that was dissolved in EtOAc (100 mL). Organic layer was washed with 10% KHSO4 solution (20 mL) followed by brine (2 x 30 mL), dried over anhydrous Na2SO4 and evaporated in vacuo to yield the deprotected oxazolidine 79 (0.21 g, 23%) as a pure white solid; Rf = 0.26 (1:1 EtOAc/hexane); mp 99 – 102 o C; 1H NMR (CDCl3, 400 MHz) 1.44 (s, 9H), 1.66 (s, 3H), 4.18-4.20 (d, J = 8 Hz, 1H), 4.41-4.43 (d, J = 8.1 Hz, 1H), 5.18 (br s, 1H), 6.15 (br s, 1H); 13C NMR (CDCl3, 100 MHz) 26.8, 28.5, O H N BocHN H3C O

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96 69.3, 75.7, 81.3, 154.4, 158.6 ppm; ESI-TOF Calcd for [M+H]+ is 217.11828, Found: 217.11801 Synthesis of 2,2-Bis-benzyloxycarbonylami no-propionic acid methyl ester (82):To a solution of di-Cbz acid 73 (0.2 g, 0.54 mmol) in dry DMF (1 mL) at -5 oC, powdered anhydrous K2CO3 (0.09 g, 0.65 mmol) was added slowly while stirring. To the above solution CH3I (40 L, 0.65 mmol) was added while stirring, then the reaction was brought to room temperature and stirred fo r 1 h. Reaction mixture was washed with distilled H2O (5 mL) and then extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with brine (2 x 10 mL ), dried over anhydrous sodium sulfate and evaporated in vacuo to yield methyl ester of di-Cbz acid 82 (0.15 g, 75%) as a pure white solid; Rf = 0.72 (1:1 EtOAc/hexane); mp 93 – 96 oC; 1H NMR (CDCl3, 250 MHz) 1.81 (s, 3H), 3.75 (s, 3H), 5.071 (s, 4H), 6.70 (br s, 2H), 7.22-7.32 (m, 10H); 13C NMR (CDCl3, 62.5 MHz) 23.6, 53.5, 66.7, 67.4, 127.9, 128.1, 128.4, 136.0, 154.5, 171.1 ppm; ESI-TOF Calcd for [M+H]+ is 387.15506, Found: 387.15476 Synthesis of (1-Benzyloxycarbonylamino -1-methyl-2-oxo-ethyl)-carbamic acid benzyl ester (83):A solution of the di-Cbz alcohol 75 (0.1 g, 0.28 mmol) in anhydrous DCM (5 mL) was cooled down to -10 oC and then DMP (0.13 g, 0.308 mmo l) was added while stirring. NHCbz COOCH3 CbzHN H3C NHCbz CHO CbzHN H 3 C

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97 After 3 h, the reaction was quenc hed with equal volume of 10% Na2S2O solution (2 mL) and 10% NaHCO3 solution (2 mL). The organic laye r was separated and washed with brine (10 mL), dried over anhydrous MgSO4 and evaporated in vacuo to yield a crude residue. The crude product was subjected to column chromatography (1:1 EtOAc/hexane) to yield the pure product as a white solid (0.05 g, 50%) and also unreacted starting material (0.03 g, 30%); Rf = 0.82 (1:1 EtOAc/hexane); mp 80 – 83 oC; 1H NMR (CDCl3, 400 MHz) 1.63 (s, 3H), 5.07 (s, 4H), 6.31 (br s, 2H), 7.32-7.36 (m, 10H), 9.26 (s, 1H); 13C NMR (CDCl3, 100 MHz) 20.9, 67.3, 70.0, 128.3, 128.5, 128.8, 135.9, 155.2, 192.7 ppm; ESI-TOF Calcd for [M+H]+ is 357.14450, Found: 357.14464 Synthesis of (1-Benzyloxycarbonylamino -2-hydroxy-1-methyl -3-nitro-propyl)carbamic acid benzyl ester (84):Nitromethane (0.2 mL) and DBU (0.4 mL) were stirred at 0 oC for 15 min. After which the aldehyde 83 (0.2 gm, 0.56 mmol) in 1 mL of anhydrous THF was added to the above solution while stirring vigorously at 0 oC. The reaction mixture was stirred for 12 hr at which the temperature was slowly brought to room temperature. Reaction mixture was quenched with 2 mL of saturated NaHCO3 solution. The aqueous layer was extracted with 10 mL of ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and evaporated in vacuo to yield crude pr oduct as yellow oil. The crude product was subjected to column chromatography (2:5 EtOAc/hexane) to obtain the pure compound nitro alcohol 84 as a colorless oil; Rf = 0.36 (1:4 EtOAc/hexane); 1H NMR (CDCl3, 400 NHCbz CbzHN H3C NO2 OH

PAGE 117

98 MHz) 1.25 (s, 3H), 4.22 – 4.24 (m, 1H), 4.55 – 4.61 (dd, J = 14.4 Hz, 6.8 Hz, 1H), 4.70 (br s, 1H), 4.74 – 4.79 (dd, J = 14.4 Hz, 4.4 Hz, 1H), 5.05 (s, 2H), 5.10 (s, 2H), 6.02 (br s, 1H), 7.30 – 7.38 (m, 10H); 13C NMR (CDCl3, 100 MHz) 21.9, 29.7, 42.9, 66.9, 67.2, 73.8, 128.1, 128.2, 128.4, 128.5, 128.6, 135.5, 136.2, 154.7 ppm; ESI-TOF Calcd for [M+Na]+ is 440.14282, Found: 440.14261 Synthesis of N-Boc-Triphenyliminophosphorane (102):To a solution of t-butyl carbazate 101 (47.0 g, 0.35 mol) in CH3COOH (145 mL) and H2O (290 mL) cooled to 0 oC was added NaNO2 (27.0 g) in portions over 15 min. The solution was stirred for 30 min at 0 oC, then extracted with Et2O (3 x 200 mL). The combined organic layers were washed with H2O (300 mL), quickly with sat. aq. NaHCO3 (200 mL), brine (200 mL), and dried over anhydrous Na2SO4. This solution was directly used in the next step. CAUTI ON: To avoid risks of explosi on, did not warm the ethereal solution of the azide and did not concentrat e to dryness! The ether solution of the N-Bocazide was cooled to 0 oC, and PPh3 (93.3 g, 0.35 mol) was a dded in small portions while stirring. Strong evolution of nitrogen occurr ed during the addition of triphenylphosphine. The cooling bath was then removed and the r eaction stirred for 30 min at r.t. The formed white precipitate was filtered, washed with Et2O and dried in vacuo to yield N-BocTriphenyliminophosphorane 102 (109.1 g, 81%); Rf = 0.52 (1:1 EtOAc/hexane); mp 145 148 oC (lit. mpref-2 148 oC); 1H NMR (CDCl3, 400 MHz) 1.37 (s, 9H), 7.44-7.48 (m, O O NH NH2 NaNO2, CH3COOH/H2O 00C, 30min O N O P Ph Ph Ph 81% overall Ph3P,dieth y lether, 00C to r.t.,30min O O N3

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99 6H), 7.53-7.58 (m, 3P), 7.71-7.74 (m, 6H); 13C NMR (CDCl3, 100 MHz) 28.5, 128.6, 128.7, 132.3, 133.2, 133.3 ppm; ESI-TOF Calcd for [M+H]+ is 378.16174, Found: 378.16161 Synthesis of t-Butyl 2,2,2-Trichloro-ethy lidenecarbamate (103):A mixture of anhydrous chlo ral (14.2 mL, 146 mmol) and N-BocTriphenyliminophosphorane 102 (50.0 g, 132.6 mmol) in dry toluene (120 mL) was refluxed for 1 h under Ar. After eva poration of toluene in vacuo, Ph3PO was precipitated by the addition of dry hexane (100 mL) and filt ered off. Evaporation of the filtrate gave crude 103 (34 g), which upon vacuum distillati on in a Kugelrohr apparatus yielded tButyl 2,2,2-Trichloro-ethylidenecarbamate 103 (31.1 g, 95%) as a pure white solid; mp 55 – 58 oC (lit. mp 58 oC); 1H NMR (CDCl3, 400 MHz) 1.57 (s, 9H), 8.08 (s, 1H); 13C NMR (CDCl3, 100 MHz) 27.8, 84.5, 92.9, 159.0, 161.1 ppm; ESI-TOF Calcd for [M+H]+ is 245.98499, Found: 245.98529 Synthesis of 3-Trichloromethyl-oxaziridine2-carboxylic acid tert-butyl ester (104):A solution of oxone (70 g) in chil led water (700 mL) was added at 0 oC to a vigorously stirred mixture of the pure t-Butyl 2,2,2-Trichloro-ethylidenecarbamate 103 (33.0 g, 133.9 mmol) in CHCl3 (350 mL), K2CO3 (55 g) and water (400 mL). After stirring 1 h, the aqueous phase was discarded and replaced by fresh solutions of K2CO3 and oxone. A total of 8 such cycles was pe rformed. The organic phase was (3 x) washed with water, O N O HC Cl Cl Cl Cl 3 C N O Boc

PAGE 119

100 dried on MgSO4 and concentrated in vacuo (bath temp. < 30 oC) to yield the crude 104 (34 g). Kugelrohr vacuum distillation of 104 (20 g) gave the pure oxa ziridine as a stench and colorless oil (15 g, 75%); 1H NMR (CDCl3, 400 MHz) 1.55 (s, 9H), 4.95 (s, 1H); 13C NMR (CDCl3, 100 MHz) 27.7, 81.2, 87.1, 93.8, 158.1 ppm t-Butyl 2,2,2-Trichloro-1-hydroxyethylcarbamate (105): White solid, mp 151 – 154 oC (lit mp 154 oC); 1H NMR (CDCl3, 400 MHz) 1.50 (s, 9H), 5.48 – 5.50 (d, J = 8Hz, 1H), 5.83 – 5.85 (m, 1H); 13C NMR (CDCl3, 100 MHz) 28.4, 81.8, 87.4, 99.0, 154.0 ppm Synthesis of Methyl N-(diphenylmethylene)-L-phenylalaninate (110):L-phenylalanine methyl ester HCl salt 108 (0.1 g, 0.46 mmol) and an equimolar quantity of benzophenone imine 109 (70 L, 0.46 mmol) were stirred at room temperature for 24 h with the exclusion of moisture (CaCl2 tube). The reaction mixture was filtered to remove NH4Cl and evaporated to dryness on a rota ry evaporator. The residue was taken up in 20 mL of ether, filtered, washed with 20 mL of water, dried over anhydrous MgSO4, and evaporated in vacuo to yield a cr ude product. Column chromatography (1:9 EtOAc/hexane) was performed on the crude to yield Methyl N-(diphenylmethylene)-Lphenylalaninate 110 (0.14 g, 88%) as a colorless oil; Rf =0.45 (1:9 EtOAc/hexane); IR (cm-1) 1732, 1617; 1H NMR (CDCl3, 400 MHz) 3.12 – 3.20 (dd, J = 16.0 Hz, 8.0 Hz, O N H O CH Cl Cl Cl OH Ph N COOCH 3 Ph Ph

PAGE 120

101 1H), 3.24 – 3.30 (dd, J = 16.0 Hz, 8.1 Hz, 1H), 4.22 – 4.28 (q, J = 16.1 Hz, 8.0 Hz, 1H), 6.55 (br s, 2H), 6.95 – 7.07 (m, 2H), 7.12 – 7.20 (m, 3H), 7.21 – 7.39 (m, 6H), 7.53 – 7.58 (m, 2H); 13C NMR (CDCl3, 100 MHz) 39.7, 52.2, 67.2, 126.3, 127.5, 127.9, 128.1, 128.3, 128.7, 129.8, 130.2, 136.0, 137.8, 139.3, 170.8, 172.2 ppm Synthesis of (N'-tert-Butoxycarbonyl-N-ethoxycarbonylmethyl-hydrazino)-acetic acid ethyl ester (118): To a solution of diethyl iminodiacetate 117 (0.15 g, 0.53 mmol) in anhydrous DCM (5 mL) at -78 oC was added 3-Trichloromethyl-oxaziridi ne-2-carboxylic acid tert-butyl ester 104(0.15 g, 0.58 mmol) and stirred for 24 h during which it was brought to r.t. The solvent was evaporated and the crude produc t was subjected to column chromatography (1:1 EtOAc/hexane) to yield the pure (N'-tert-Butoxycarbonyl-N-ethoxycarbonylmethylhydrazino)-acetic acid ethyl ester 118 (0.12 g, 80%) as a colorless oil; Rf = 0.66 (1:1 EtOAc/hexane); 1H NMR (CDCl3, 400 MHz) 1.26 – 1.299 (t, J = 6.8 Hz, 3H), 1.44 (s, 9H), 3.81 (s, 4H), 4.16 – 4.21 (q, J = 14.4 Hz, 7.2 Hz, 4H), 6.88 (br s, 1H); 13C NMR (CDCl3, 100 MHz) 14.3, 28.5, 57.4, 61.2, 66.0, 158.1, 170.1 ppm O O N O O NHBoc N H Ph O O O O

PAGE 121

102Synthesis of 2-(Ethoxycarbonylmethyl-amino )-3-phenyl-propionic acid methyl ester (120):To a suspension of Phenylalanine methyl ester HCl salt 119 (0.85 g, 3.94 mmol) in anhydrous CH3CN (15 mL) was added DIEA (2.6 mL, 15.76 mmol) while stirring under argon atmosphere, after the complete addition of the DIEA the suspension becomes clear solution. The reaction mixture was allowed to stir for 15 min, after which ethyl bromoacetate (0.65 mL, 5.91 mmol) was added dr op wise to the above solution, and left stirring at room temperature overnight. Reac tion mixture was quenched with 50% citric acid solution (5 mL) and was extracted with EtOAc (25 mL), separa ted organic layer was washed with brine (2 x 20 mL), dried over anhydrous MgSO4, evaporated in vacuo to yield the crude residue. Column chromatogr aphy (1:4 EtOAc/hexane) was performed to yield 2-(Ethoxycarbonylmethyl-amino)-3phenyl-propionic acid methyl ester 120 (0.78 g, 75%) as a colorless oil; Rf = 0.45 (1:4 EtOAc/hexane); 1H NMR (CDCl3, 400 MHz) 1.20 – 1.24 (t, J = 6.8 Hz, 3H), 2.05 (br s, 1H), 2.93 – 3.01 (ddd, J = 13.6 Hz, 7.2 Hz, 2H), 3.29 – 3.42 (d, J = 16.8 Hz, 2H), 3.57 – 3.61 (t, J = 6.4 Hz, 1H), 3.65 (s, 3H), 4.10 – 4.15 (q, J = 14.8 Hz, 7.2 Hz, 2H), 7.18 – 7.30 (m, 5H); 13C NMR (CDCl3, 100 MHz) 14.3, 39.7, 49.3, 52.0, 61.0, 62.3, 127.0, 128.7, 129.4, 137.1, 171.8, 174.2 ppm; ESI-MS 267.1[M+H]+, 266.1 [M+] N Ph O O O O NH Boc

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103Synthesis of 2-(N'-tert-Butoxycarbonyl-N-ethoxycarbonylmethyl-hydrazino)-3phenyl-propionic acid methyl ester (121):To a solution of 2-(Ethoxy carbonylmethyl-amino)-3-phenyl-pr opionic acid methyl ester 120 (0.05 g, 0.19 mmol) in anhydrous DCM (5 mL) at -78 oC was added 3Trichloromethyl-oxaziridine-2-car boxylic acid tert-butyl ester 104 (0.06 g, 0.23 mmol) and stirred for 24 h during which it was brought to r.t. The solvent was evaporated and the crude product was subjected to column ch romatography (1:1 EtOAc/hexane) to yield the pure 2-(N'-tert-Butoxycarbonyl-N-ethoxycarbonylmethyl-hydrazino)-3-phenylpropionic acid methyl ester 121 (0.05 g, 78%) as a colorless oil; Rf = 0.78 (1:1 EtOAc/hexane); 1H NMR (CDCl3, 400 MHz) 1H NMR (CDCl3, 400 MHz) 1.21 – 1.25 (t, J = 6.8 Hz, 3H), 1.44 (s, 9H), 2.95 – 3.07 (dd, J = 13.2 Hz, 7.0 Hz, 1H), 3.14-3.18 (dd, J = 13.0 Hz, 6.8 Hz, 1H), 3.55 (s, 2H), 3.61 – 3.80 (m, 3H), 4.09 – 4.15 (q, J = 13.2 Hz, 7.6 Hz, 2H), 6.95 (br s, 1H), 7.18 – 7.28 (m, 5H); 13C NMR (CDCl3, 100 MHz) 15.1, 28.5, 36.6, 51.1, 61.2, 69.5, 127.8, 128. 1, 128.9, 137.2, 172. 1 ppm; ESI-MS 403.1 [M+Na]+ Synthesis of 3-tert-Butoxycarbonylamino-4-hydroxy-butyr ic acid benzyl ester (123):To a solution of N-Boc-Laspartic acid -benzyl ester 122 (5.0 g, 15.5 mmol) in anhydrous THF (40 mL) at -20 oC was added NMM (6.8 mL, 62 mmol) followed by isobutyl chloroformate (2.1 mL, 16.3 mmol) and stirred vigorously for 10 min, after which a suspension of NaBH4 (1.0 g, 26.4 mmol) in THF (40 mL) and MeOH (10 mL) was added to the above mixture at -78 oC. The reaction mixture was stirred for 2 h at -78 BocHN O O Ph O H

PAGE 123

104oC, and then quenched with 10% HCl soluti on (20 mL) and further diluted with EtOAc (100 mL), washed with sat. NaHCO3 solution (50 mL) and followed by brine (2 x 50 mL). The organic layers were se parated, dried over anhydrous Na2SO4, evaporated in vacuo to yield the cr ude residue. Column chromatography (1:5 EtOAc/hexane) of the crude residue yielded 3-tert-Butoxycarbonylamino-4-hydroxy-but yric acid benzyl ester 123 (3.95 g, 83%) as a white solid; Rf = 0.20 (1:9 EtOAc/hexane); mp 54 – 57 oC; 1H NMR (CDCl3, 400 MHz) 1.42 (s, 9H), 2.21 (br s, 1H), 2.66 – 2.68 (d, J = 5.6 Hz, 2H), 3.68 – 3.69 (d, J = 4.4 Hz, 2H), 3.98 – 4.01 (m, 1H), 5.12 (s, 2H), 5.21 (br s, 1H), 7.30 – 7.38 (m, 5H); 13C NMR (CDCl3, 100 MHz) 28.5, 36.2, 49.6, 64.6, 66.8, 128.4, 128.5, 128.8, 135.7, 156.0, 171.8 ppm; ESI-TOF Calcd for [M+Na]+ is 332.14684, Found: 332.14690 Synthesis of 3-tert-Butoxycarbonylamino-4-oxo-butyric acid benzyl ester (124):To a solution of oxalyl chloride (2.4 mL, 27.1 mmol) in anhydrous DCM (20 mL) at -78 oC, dry DMSO (4.0 mL) was added and stirred for 10 min. 3-tert-Butoxycarbonylamino4-hydroxy-butyric acid benzyl ester 123 (3.5 g, 11.3 mmol) dissolved in dry DCM (25 mL) was added to the above solution slo wly over a period of 5 min, and stirred vigorously for 15 min at -78 oC. TEA (14 mL, 101.7 mmol) was added to the above reaction mixture slowly and stirred further for 30 min at the same temperature after which the reaction mixture was brought to r.t. and stirred for 2 h. The reaction was quenched with 10% citric acid solution (20 mL), the organic layer was separated washed with distilled water (50 mL), brine (2 x 50 mL), dried over anhydrous MgSO4, evaporated in B o c HN C H O O O Ph

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105 vacuo to obtain crude product. The crude product was purified by recrystallization (DCM/hexane) to yield 3-tert-Butoxycarbonylamino-4-oxo-butyr ic acid benzyl ester 124 (2.4 g, 70%) as a white solid; mp 74 – 77 oC ; 1H NMR (CDCl3, 400 MHz) 1.39 (s, 9H), 2.77 – 2.83 (dd, J = 17.2 Hz, 5.2 Hz, 1H), 2.94 – 2. 98 (dd, J = 17.6 Hz, 5.2 Hz, 1H), 4.28 – 4.31 (m, 1H), 5.05 (s, 2H). 5.48 – 5.56 (d, J = 8.4 Hz, 1H), 7.25 – 7.31 (m, 5H), 9.57 (s, 1H); 13C NMR (CDCl3, 100 MHz) 28.4, 34.7, 56.2, 67.2, 80.8, 128.5, 128.7, 128.8, 135.4, 155.7, 171.3, 199.3 ppm; ESI-TOF Calcd for [M+Na]+ is 330.13119, Found: 330.13091 Synthesis of 3-tert-Butoxycarbonylamino-4-hydroxy-5nitro-pentanoic acid benzyl ester (125):To a solution of 1M TBAF solution in THF (0.46 mL, 0.44 mmol) cooled to 0 oC, 2 mL of dry THF and nitromethane (0.03 mL, 0.5 mmol) were added and stirred for 5 min. After which the aldehyde from the previous step, 3-tert-Butoxycarbonylamino-4-oxobutyric acid benzyl ester 124 (0.1 g, 0.33 mmol) dissolved in 3 mL of dry THF was added to the above solution while stirring at 0 oC and maintained for 20 min. The reaction mixture was poured onto sat. NaHCO3 solution (10 mL) and was extracted with Et2O (3 x 20 mL). The combined organic layers were wa shed with brine (2 x 25 mL), dried over anhydrous MgSO4, evaporated in vacuo to yi eld the crude product. Column chromatography (2:5 EtOAc/hexane) of the crude to yield 3-tert-Butoxycarbonylamino4-hydroxy-5-nitro-pentanoi c acid benzyl ester 125 (0.06 g, 50%) as a colorless oil; Rf = BocHN O O Ph NO2 H O

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106 0.33 (2:5 EtOAc/hexane); 1H NMR (CDCl3, 400 MHz) 1.42 (s, 9H), 2.73 – 2.76 (m, 1H), 4.02 – 4.16 (m, 1H), 4.45 – 4.49 (m, 2H), 4.69 (s, 2H), 5.13 – 5.14 (d, J = 4.8 Hz, 2H), 7.34 – 7.37 (m, 5H); 13C NMR (CDCl3, 100 MHz) 28.4, 37.0, 49.6, 65.4, 67.1, 69.7, 79.0, 127.2, 127.8, 128.6, 128.7, 128.9, 135. 5, 156.0, 171.3 ppm; ESI-TOF Calcd for [M+Na]+ is 391.14757, Found: 391.14727 Synthesis of (3-Amino-1-benzyloxycarbo nylamino-2-hydroxy-1-methyl-propyl)carbamic acid benzyl ester (85):To a solution of nitroalcohol 84 (0.03 g, 0.07 mmol) in 1 mL of anhydrous THF, freshly activated zinc (0.03 g, 0.4 mmol) and glacial acetic acid (0.5 mL) were added at room temperature. The reaction mixture was sti rred vigourously under argon atmosphere for 3 hr, after which the solvents were evaporated The crude product was subjected to column chromatography (9:1 EtOAc/MeOH) to yiled pure amino alcohol 85 (40%) as a colorless oil; Rf = 0.1 (9:1 EtOAc/hexane); 1H NMR (CDCl3, 400 MHz) 1.25 (s, 3 H), 2.00 (br s, 4 H), 2.17 (s, 3H), 4.88 (m, 2H), 4.99 (m, 2H), 5.10 (s, 2H), 7.26 – 7.36 (m, 10H); 13C NMR (CDCl3, 100 MHz) 29.7, 30.9, 66.5, 67.0, 69.1, 127.8, 128.1, 128.2, 128.4, 128.5, 136.1, 154.9 ppm. 3.6 References 1) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 9367 – 9371. NHCbz CbzHN H3C NH2 O H

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107 2) Gordon, T. D.; Singh, J.; Hansen, P. E.; Morgon B. A. Tetrahedron Lett. 1993, 34, 1901 – 1904. 3) Kim, B. H.; Chung, Y. J.; Keum, G.; Kim, J.; Kim, K. Tetrahedron Lett. 1992, 33, 6811 – 6814. 4) Moran, E. J.; Wilson, T. E.; Cho, C. Y.; Cherry, S. R.; Schlutz, P. G. Biopolymers 1995, 37, 213 – 219. 5) Smith, A. B., III; Hirschmann, R.; Pasternak, A.; Akaishi, R.; Guzman, M. C.; Jones, D. R.; Keenan, T. P.; Sprengeler, P. A.; Darke, P. L.; Emini, E. A.; Holloway, M. K.; Schleif, W. A. J. Med. Chem. 1994, 37, 215 – 218. 6) Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.; Schreiber, S. L. J. Am. Chem. Soc. 1992, 114, 6568 – 6870. 7) Kazmierski, W. M.; Urbancz yk-Lipkowska, Z.; Hruby, V. J. J. Org. Chem. 1994, 59, 1789 – 1795. 8) Matthews, J. L.; Braun, C.; Guibourdenche, C.; Overhand, M.; Seebach, D. In Enantioselective Synthesis of -Amino acids; Juaristi, E., Ed.; Wiley-VCH: New York, 1997; pp 105 – 126. 9) Rivier, J. E.; Jiang, G.-C.; Koerber, S. C.; Porter, J.; Craig, A. G.; Hoeger, C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2031 – 2036. 10) Jiang, G.; Miller, C.; Koer ber, S. C.; Porter, J.; Craig, A. G.; Bhattacharjee, S.; Kraft, P.; Burris, T. P.; Campen, C. A.; Rivier, C. L.; Rivier, J. E. J. Med. Chem. 1997, 40, 3739 – 3748. 11) Sypniewski, M.; Penke, B. ; Simon, L.; Rivier, J. J. Org. Chem. 2000, 65, 6595 – 6600.

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108 12) Chorev, M.; Willson, C. G.; Goodman, M. J. Am. Chem. Soc. 1977, 99, 8075 – 8076. 13) Goodman, M.; Chorev, M. Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; Karger: Basel, 1981; p 283. 14) Goodman, M.; Chorev, M. Acc. Chem. Res. 1979, 12, 1 – 7. 15) Pallai, P.; Goodman, M. J. Chem. Soc., Chem. Commun. 1982, 280 – 281. 16) Rudinger, J. “Drug Design”, Vol 2, E. J. Ariens, Ed., Academ ic Press, New York, N.Y., 1971, p 319. 17) Chorev, M.; Goodman, M. Int. J. Pept. Protein Res. 1983, 21, 258 – 260. 18) Bergmann, M.; Zervas, L. J. Biol. Chem. 1935, 2, 341 – 357. 19) Shemin, D.; Herbest, R. M. J. Am. Chem. Soc. 1938, 60, 1954 – 1957. 20) Herbest, R. M. J. Am. Chem. Soc. 1939, 61, 483 – 486. 21) Gonalves, J. M.; Greenstein, P. J. Archives of Biochemistry 1948, 16, 1 – 17. 22) Fu, J.-C.; Levintow, L.; Price, E. V. Greenstein, P. J. Archives of Biochemistry 1950, 28, 440 – 451. 23) Brenner, I. M.; Rufenacht, K. Helv. Chem. Acta 1953, 36, 1832 – 1841. 24) Chorev, M.; Shavitz, R.; Goodman, M.; Minick, S.; Guillemin, R. Science 1979, 204, 1210 – 1212. 25) MacDonald, S. A.; Willson, C. G.; C horev, M.; Vernacchia, F. S.; Goodman, M. J. Med. Chem. 1980, 23, 413 – 420. 26) Fuller, W. D.; Goodman, M.; Verlander, M. S. J. Am. Chem. Soc. 1985, 107, 5821 – 5822. 27) DeBons, E. F.; Loudon, G. M. J. Org. Chem. 1980, 45, 1703 – 1704.

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109 28) Loudon, G. M.; Almond, M. R.; Jacob, J. N.; J. Am. Chem. Soc. 1981, 103, 4508 – 4515. 29) Kingsbury, W. D.; Boehm, C. J.; Mehta, R. J.; Grappel, S. F.; Gilvarg, C. J. Med. Chem. 1984, 27, 1447 – 1451. 30) Bock, M. G.; Dipardo, R. M.; Freidinger, R. M. J. Org. Chem. 1986, 51, 3718 – 3720. 31) Katritzky, A. R.; Urogdi, L.; Mayence, A. J. Org. Chem. 1990, 55, 2206 – 2214. 32) Cushman, M.; Jurayj, J.; Moyer, J. D. J. Org. Chem. 1990, 55, 3186 – 3194. 33) Kohn, H.; Sawhney, K. N.; LeGall, P. ; Robertson, D. W.; Leander, D. J. J. Med. Chem. 1991, 34, 2444 – 2452. 34) Qasmi, D.; Ren, L.; Badet, B. Tetrahedron Lett. 1993, 34, 3861 – 3862. 35) Davies, J. S.; Diddams, M.-S.; Fromentin, R.; Howells, A.; Cotton, R. J. Chem. Soc., Perkin Trans. 2000, 1, 239 – 243. 36) Waki, M.; Kitajima, Y. Izumiya, N. Synthesis 1981, 266 37) Cantel, S.; Boeglin, D.; Rolland, M.; Martinez, J.; Fehrentz, J.-A. Tetrahedron Lett. 2003, 44, 4797 – 4799. 38) Rowland, G. B.; Zhang, H.; Rowland, E. B.; Chennamadhavuni, S.; Wang, Y.; Antilla, J. C. J. Am. Chem. Soc. 2005, 127, 15696 – 15697. 39) Martel, A. E.; Herbst, R. M. J. Org. Chem., 1941, 6, 878-87. 40) Rossi, F. M.; Powers, E. T.; Y oon, R.; Rosenberg, L.; Meinwald, J. Tetrahedron 1996, 52, 10279 – 10286. 41) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277 7287. 42) Andrae, S.; Schmitz, E. Synthesis 1991, 327 – 341.

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110 43) Vidal, J.; Guy, L.; Strin, S.; Collet, A. J. Org. Chem. 1993, 58, 4791-4793. 44) Armstrong, A.; Atkin, M. A.; Swallow, S. Tetrahedron Lett. 2000, 41, 2247 – 2251. 45) Vidal, J.; Damestoy, S.; Guy, L.; Ha nnachi, J.-C.; Aubry, A.; Collet, A. Chem. Eur. J. 1997, 3, 1691 – 1709. 46) Armstrong, A.; Atkin, M. A.; Swallow, S. Tetrahedrom:Asymmetry 2001, 12, 535 – 538. 47) Bulman Page, P. C.; Limousin, C.; Murrell, V. L. J. Org. Chem. 2002, 67, 7787 – 7796. 48) Hannachi, J.-C.; Vidal, J. ; Mulatier, J.-C.; Collet, A. J. Org. Chem. 2004, 69, 2367 – 2373. 49) Greck, C.; Drouillat, B.; Thomassigny, C. Eur. J. Org. Chem. 2004, 1377 – 1385. 50) Vidal, J.; Hannachi, J.-C.; Hourdin, G.; Mulatier, C. J.; Collet, A. Tetrahedron Lett. 1998, 39, 8845 8848. 51) O’Donnell, M. J.; Boniece, J. M.; Earp, S. E. Tetrahedron Lett. 1978, 26412644. 52) O’ Donnell, M. J.; Polt, R. L. J. Org. Chem. 1982, 47, 2663-2666. 53) Stephanie T. Weiss’s do ctoral dissertation work “T he Theoretical Modeling, Design, And Synthesis of Ke y Structural Units for Novel Molecular Clamps and Pro-Apoptotic Alpha Helix Peptidomimetics.” Publication in progress (2006). 54) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001, 123, 5382 – 5383.

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111 CHAPTER FOUR SYNTHESIS, BIOLOGICAL ACTIVITY AND MOLECULAR MODELING OF NOVEL 20S PROTEASOME INHIBITORS 4.1 Introduction Proteins carry out almost al l the lifes essential processes. Cellular proteins are in a dynamic state of turnover, with the relati ve rates of protein synthesis and protein degradation ultimately determining the amount of protein present at any point of time. Breaking down unneeded proteins, a task e qual in importance to synthesizing new proteins, is accomplished by the orderly ac tion of several multiprotein complexes. Usually transcriptional regulation determines the concentrations of specific proteins in cells. But some times the amounts of key en zymes and regulatory proteins, such as cyclins and transcriptional factors, are cont rolled via selective protein degradation. In addition, abnormal proteins arising from the biosynthetic errors or postsynthetic damage must be destroyed to preven t the deleterious consequences of their buildup. Protein degradation is also primarily required to supply amino acids for fresh protein synthesis. 1 There are two major intracellular machines that degrade the damaged proteins or the excess proteins i) Lysosomes and ii) Proteasomes. Lysosomes primarily deal with the extracellular proteins like pl asma proteins and membrane bound proteins that are used in receptor-mediated endocytosis. Protein degrad ation in lysosomes is largely nonselective; selection occurs only during the endocytosis. Proteasomes on the other hand, deal with

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the degradation of the endogenous proteins like transcriptional factors, cyclins and misfolded proteins due to transcriptional errors or encoded by faulty genes. 2-5 Proteasomes are found in eukaryotic as well as in prokaryotic cells. The proteasome is a functionally and structurally sophisticated counterpart to the ribosome. Regulation of protein levels via degradation is an essential cellular mechanism that is both rapid and irreversible. 4.2 Proteasome structure and ubiquitin-proteasome pathway (a) (b) Figure 4.1:(a) Crystal structure of the 20S proteasome 6 (b) 26S proteasome complex 14 112 Proteasomes are large oligomeric structures enclosing a central cavity where proteolysis takes place. The 26S proteasome is 2000-kDa ATP-dependent proteolytic complex. This large structure contains the central 20S (700 kDa) proteasome, in which proteins are degraded, and two 19S complexes, which provide substrate specificity and regulation (Figure 4.1). 6,7 The 20S proteasome is barrel-shaped and is the most abundant particle. It nearly comprises 1% of cellular proteins. The 20S proteasome comprises of four stacked rings that enclose the central cavity or chamber where proteolysis occurs. The two central -rings mainly contain a total of six proteolytic active sites that function together in protein degradation. These sites differ in their substrate specificity and

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113 activity and have been named after enzymes that show similar proteolytic activity or specificity. In case of eukaryot ic cells two of these sites are chymotrypsin-like, two are trypsin-like and two are caspa se-like or post-glutamyl pe ptide hydrolase-like (PGPH). Access of polypeptides/protein s to these specific sites is controlled through a 1.3 nm opening formed by the outer two -rings. The functional 26S pr oteasome consists of the core 20S catalytic complex that is capped at each end by the 19S regulatory subunit or caps. Ubiquitination is the most common mechanism to label a protein for protein degradation in eukaryotes. Ubiquitin is a highly conserved, 76-residue (8.5 kDa) polypeptide and is abundant in the cell. The protein substrate is first conjugated to multiple molecules of ubiquitin in a reaction involving ubiquitin-activating enzyme (E 1 ), ubiquitin carrier protein (E 2 ) and ubiquitin-protein ligase (E 3 ) (Figure 4.2). 8-13 E 1 becomes attached via a thioester bond to the C-terminal glycine residue of the ubiquitin through ATP-driven formation of an ubiquitinadenylate intermediate. Ubiquitin is then transferred from E 1 to an -SH group of E 2 Then E 2 -S~ubiquitin transf ers ubiquitin to free amino groups on proteins selected by E 3 Upon binding a protein substrate, E 3 catalyzes the transfer of ubiquitin from E 2 -S~ubiquitin to free amine groups on the protein, usually a Lys -NH 2 More than one ubiquitin might attach to the protein substrate and the tandem linking of the ubiquitin molecules is catalyzed by isopeptidases. The 19S complexes contain binding sites for ubiquitinated proteins, enzymes then depolymerize the ubiquitin chain and unfold the substrate and facilitates its entry into the 20S proteasome active cavity. Proteins are degr aded by the core partic le in a progressive

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manner, generating peptides of 3-25 amino acids in length and are further hydrolyzed to amino acids by other peptidases. The catalytic mechanism of the 20S proteasome is highly complex and recent studies have revealed the importance of the amino terminal Thr as a nucleophile catalyst. Crews et al. 12 proposed a mechanism for the proteolysis catalyzed by 20S proteasome via Thr-1 and is shown in the figure 4.3. Figure 4.2:Ubiquitin-proteasome pathway for protein degradation 14 Figure 4.3:-Proposed mechanism for the proteolysis catalyzed by the 20S proteasome 12 114

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115 4.3 Proteasome inhibition: A novel approach to cancer therapy Even though the concept of ubiquitin-pro teasome pathway (UPP) as the major proteolytic pathway in the nucleus and the cytosol was discovered more than 20 years ago, the involvement of the proteasome in a poptosis was demonstrated quite recently. Previously, UPP was thought to be merely a disposal system for damaged intracellular proteins. But recent studies concluded that UPP is very much necessary for the regulation of the various essential proteins that gove rn an array of cellular functions. Normal turnover of cell-cycle regulatory proteins is dependent on the proteasome. Adams et al. 15 and An et al. 16 demonstrated that blockade of proteasome function halts cell division. However, malignant cells are even more sensitive to the loss of proteasome activity. The studies comparing normal and malignant cell lines have shown that proteasome inhibition sensitizes malignant cells to a poptosis or programmed cell-death. 17 Apoptotic cell death is fundamentally important in a wide variety of processes during normal development and also considered to be a major factor in anti-cancer therapies. 18, 19 NFB signaling is dependent on the proteas ome activity and recent studies had proven that interference with NFB transcriptional pathway influences tumor cell survival. 13,14,20 In a normal cell, NFB is bound to a specific inhibitor protein I B in the cytoplasm. This makes the NFB inactive and prevents transcription of its target genes (Figure 4.4). In case of external stress on the cell, I B is degraded by the proteasome via UPP and thus allows NFB to translocate to the nucleus. NFB then promotes cell survival by initiating the transcription of genes encoding stress-response enzymes like cyclooxygenase (COX-2), cell-adhesion molecu les, pro-inflammatory cytokines like tumor necrosis factor (TNF) and interleukins (ILs), and anti-apoptotic proteins such as

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Bcl-2 and cIAP1. In case of tumor cells, NF-B is very active and promotes the tumor cell survival by inhibiting apoptosis and reduces the effectiveness of the anticancer therapy. Recent studies by Cusack et al. 21 proved that inhibition of the proteasome blocks the chemotherapy-induced activation of NF-B and in turn increases the apoptosis of the tumor cells in animal models. Figure 4.4:NF-B activation pathway 13 4.3.1 Background on the proteasome inhibitors With the information on the proteasome, UPP and the role of proteasome in apoptosis, a variety of proteasome inhibitors were developed. Proteasome inhibitors were developed as research tools until early 2000, but they grabbed the center stage once their potential application in cancer therapy was recognized by Adams et al. Currently the classes of proteasome inhibitors can be classified as the following: i) Peptide aldehydes 22 ii) Peptide boronates 23 iii) Non-peptide inhibitors like lactacystin 24 iv) epoxyketones 25 v) 116

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Peptide vinyl sulfones 26 vi) New natural product inhibitors like EGCG ((-)-epigallocatechin-3-gallate) 27 and its synthetic analogs 28 gliotoxin 29 etc. Many of these compounds have broad specificity, poor metabolic stability and bind irreversibly to the proteasome. Although the proteasome has multiple active sites, biological experiments proved that the selective inhibition of the Chymotrypsin-like subunit would achieve the required inhibition of the proteasome. 4.3.2 Velcade TM : Only proteasome inhibitor approved by FDA Julian Adams and his co workers at Millennium pharmaceuticals synthesized a dipeptidyl boronic acid bortezomib, which still is the most potent, reversible, and selective proteasome inhibitor (Figure 4.5). 30,31 In 2003 FDA approved this drug under the brand name Velcade TM for the treatment of multiple myeloma. Multiple myeloma is the second most prevalent blood cancer after non-Hodgkins lymphoma. 32 It is a cancer of the plasma cell, an important part of the immune system that produces antibodies to help fight infection and disease. There are approximately 45,000 people in the United States living with multiple myeloma and an estimated 14,600 new cases of multiple myeloma are diagnosed each year. Data showed that Velcade (K i = 0.6 nM) binds to the proteasome with very high affinity and dissociates slowly, conferring stable but reversible proteasome inhibition. 15 NN NH O HN O B OH OH Figure 4.5:Structure of the dipeptidyl boronic acid proteasome inhibitor bortezomib 117

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4.3.3 Reason for a new proteasome inhibitor The search for new proteasome inhibitors still continues in spite of the recent approval of the Velcade because (a) Velcade is highly expensive (3.5 mg = $1000); (b) high-toxic profile for Velcade was observed; (c) adverse effects ranging from cardiac disorders, thrombocytopenia, GI adverse effects, hypotension, peripheral neuropathy and rare cases of acute liver failure; (d) elimination pathways were not fully characterized; (e) studies on pediatric and geriatric patients were not conducted; (f) no formal drug interactions have been conducted; (g) the field of proteasome inhibition for cancer therapy is still juvenile and lot of information is needed. At least some of these reasons do warrant a new class of proteasome inhibitors with a less toxic profile. 4.4 NSC-12155 lead molecule from HTS core High-throughput screening (HTS) Core at the H. Lee Moffitt Cancer Centre & Research Institute have conducted an extensive screening on the 2000 NCI chemical diversity set of compounds, for proteasome inhibition. They have conducted both virtual screening (GLIDE) as well as the enzymatic assays and arrived with a few hit compounds in terms of decent activity. Our journey for a novel proteasome inhibitor started with their screening and identifying potential targets from the NCI diversity set. NSC-12155 was one among the few compounds from their studies which showed IC 50 = 1.2 1.0 M (Figure 4.6). NNHNHONNH2NH2 Figure 4.6:NSC-12155, the lead molecule 118

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119 In order to validate the results generated by the HTS core in terms of virtual screening (GLIDE) as well as the small scale enzymatic assays, we have to take it to level of lead optimization by conducting more biological expe riments. For that we have to have a greater amount of NSC-12155 and thus we ventured into an in-house synthesis of this molecule. 4.4.1 Background on NSC-12155 (1,3-Bis-(4-a mino-2-methyl-quinolin-6-yl)-urea) NSC-12155 is not a new compound as far as synthesis is concerned. The earliest literature report of this molecule came in 1937 and was referred to as surfen back then. Surfen and its analogs were patented by Jensch 33 for their antihelminthic and antibacterial properties. The first synt hesis of surfen was reported by Jensch 34 and it remained as the basic route for all the synt heses of 4,6-diaminoquinolines and its analogs that later followed over the years (Figure 4.8). The antibacterial property and its low toxicity for tissues had prompted many reports for their syntheses. 35-37 Most of these papers also reported the synthesis of asymmetrical ureas, the analogs for studying the structure-activity relationshi p for the antibacterial activ ity. But a report by Hunter and Hill 38 on oncogenic and heparin-neutralizing pr operties of surfen had suddenly dropped the interest of this molecule. But this was not surprising because most of the approved drugs today like cisplatin for the treatment of cancer were reported oncogenic earlier. The revival of interest on 4,6-diaminoquinolines and the substituted asymmetrical urea analogs appeared on the scene because of reported inhibition of C5a receptor binding in human nutrophils. 39 C5a is an anaphylatoxin that is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysi ne. In their study, Lanza 39 and coworkers discover ed the potential of

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NSC-12155 as one of the potential inhibitor of C5a receptor binding. Shinkai et al. 40 reported 4-aminoquinolines as novel nociceptin antagonists with analgesic activity. Panchal et al. 41 reported that NSC-12155 is a very good inhibitor of anthrax lethal factor (LF) and also published a cocyrstal structure of anthrax LF-NSC12155. This report was further supported by Mentecucco et al. 42 Most recently a new synthetic approach for the 4,6-diamino-2-methylquinoline, the monomeric unit of the NSC-12155 was reported by Sestili et al. 43 (Figure 4.7). They also conducted various pharmacological studies on a variety of substituted synthetic analogs of 129 as nociceptin receptor (NOP) antagonists. O2N CN NH2 CH3COCH3SnCl4/Toluenereflux N O2N NH2 N H2N NH2 NiA lMeOH/KOH60oC, 2 h127 128 129 Figure 4.7:Sestili et al.s synthesis of 4,6-diamino-2-methylquinoline 129 Dr. Sebti at Moffitt Cancer Center and his collaborators 44 in 2004 identified 15 compounds from the NCI diversity set of 2000-compound library that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab. NSC-12155 was one among these compounds. 4.5 Results & Discussion The drug discovery team at Moffitt Cancer Center collaborated extensively on the current project that is in discussion. Dr. Sebtis group worked out the biological assays for the 20S proteasome inhibition activity. Dr. Wayne Guidas group supported us with the molecular modeling data. We basically followed the synthesis described by Jensch 33 120

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because of the availability and low cost of the starting materials and the intermediate reagents and we intended to use the intermediates 133 and 134 for making libraries. The synthesis for NSC-12155 is depicted in the figure 4.8. Figure 4.8:Scheme for the synthesis of NSC-12155 H2NHNO NNH2NH2 NHNOOMe MeOOMeO NNHNHONNH2NH2 HNONHOMeO NHNOOH +MeOH, RefluxDowtherm A, 280 0CMe2SO4, toluene, reflux i) AcONH4, 1350Cii) H2SO4, H2O, 1000Ctriphosgene, Et3N, 1,4 -dioxane, reflux 130131132133134135NSC-12155 We started our synthesis by reacting 4-aminoacetanilide 130 with methylacetoacetate 131 in methanol at reflux temperature to yield the methyl 3-(4-acetylaminophenylamino)crotonate 132 in 70% yield. Subsequent cyclization of crotonate 132 at 280 o C yielded 6-acetamide-4-hydroxy-2-methylquinoline 133 in 72% yield. This method of synthesis of quinoline moiety is known as Knorr Quinoline synthesis. Methylation of 133 with dimethylsulfate yielded the 4-methoxyquinoline derivative 134. The methoxy of 134 was converted to amino group with ammonium acetate and the acetyl group was removed by acidic hydrolysis to give 4,6-diamino-2121

PAGE 141

methylquinoline 135 in good yield. Conversion of 135 to NSC-12155 was achieved by triphosgene and TEA. We tested our synthesized molecule of NSC-12155 to compare it with that of the previous result from the enzymatic assay conducted by the HTS Core. The IC 50 value matched with the previous result suggesting that we have a lead and the biological assay results also demonstrated the selectivity of NSC-12155 towards chymotrypsin-like subunit, which is the primary requirement in the proteasome inhibition. The molecular docking of NSC-12155 in the chymotrypsin-like subunit of the 20S proteasome is shown in figure 4.9. The GLIDE molecular modeling software was used to obtain the lowest energy docked model and the model with lowest docking scores are used for our analysis. N CH 3 N H H HN O NH N N H H CH3 H O CH3 Thr1Thr21H N Gly23NSC-12155OO HN H O A B C Figure 4.9:Molecular model of NSC-12155 binding to the chymotrypsin-like subunit of the 20S Proteasome. A: The protein surface of the proteasome is shaded according to the electrostatic potential. Positively charged areas are in light shade and negatively charged areas are shaded dark. For NSC-12155, a wire frame model was shown in the binding pocket of the chymotrypsin-like subunit. B: The hydrogen bonds formed between the NSC-12155 and the protein, via Thr1, Thr21 and Gly23 are shown schematically but not to scale. The H bonds are defined with a minimum donor angle of 122

PAGE 142

90 and minimum acceptor angle of 60 and maximum length of 2.5 C: Surface model of the proteasome with space filling model of NSC-12155. Based on the molecular modeling data we concluded that the pharmacophore of NSC-12155 contains a urea moiety and two aryl groups or one aryl group that can be varied independently. This prompted us to vary the functional units to maximize the chymotrypsin-like activity of the proteasome via focused yet structurally diverse compound libraries. 4.5.1 Synthesis, biological data and molecular modeling of the Library While NSC-12155 is a symmetrical urea, the intermediate 135 can be coupled to various aryl or alkyl isocyanates to build asymmetric urea derivatives. With that intention we designed a small focused library around the urea moiety. By treating the intermediate diamine 135 with the selected isocyanate in acetone with microwave irradiation we were able to achieve the desired asymmetric urea as the sole insoluble product (Figure 4.10). N H3C NH2 NH2 +OC N R A cetoneMicrowave irradiation100 oC, 20 minN H3C NH2 NH NH O R NH NHCH3 CF3 Cl N NH2 NH O N NH2 NH O NHN NH2 NH O NHN NH2 NH O NHN NH2 NH O 136137139138140R = phenyl (136)R = 4-CH3-phenyl (137)R = 4-Cl-Phenyl (138)R = 4-CF3-Phenyl (139)R = 1-naphthyl (140)135 Figure 4.10:Asymmetric urea analogs synthesis 123

PAGE 143

The preliminary results of in vitro chymotrypsin-like activity inhibition were reported in the figure 4.11. The results are very interesting; they all are as active or nearly active as that of the symmetrical urea NSC-12155. CompoundsNSC-12155136137138139140IC50 M)1.2 1.18.4 2.27.4 4.03.6 1.44.3 4.91.4 1.0 Figure 4.11:chymotrypsin-like subunit inhibitory activity Molecular modeling of the compounds 137, 138 and 139 clearly shows that the 4,6-diamino-2-methylquinoline group is critical since it is hydrogen bound to the active site Thr 1 hydroxyl group of the enzyme (Figure 4.12). Since no specific hydrogen binding interactions are predicted for the other 4,6-diamino-2-methylquinoline of the symmetrical urea NSC-12155 (Figure 4.9), our speculation that it can be replaced was proven to be correct both by bioassay as well as molecular docking experiments. NHCH3 CF3 Cl N NH2 NH O NHN NH2 NH O NHN NH2 NH O 137139138 Figure 4.12:Docking of 137, 138 & 139 in the binding pocket 124

PAGE 144

We were surprised to observe the binding orientation of 136 because the phenyl moiety was shown to be in the binding pocket as opposed to the 4,6-diamino-2-methylquinoline group. Even the lowest energy conformation for 136 showed this kind of orientation (Figure 4.13). By comparing the most active compound among our series i.e. 140 with 136 in the binding pocket, the question arises regarding the binding interactions required for inhibition. Because it clearly suggests that compound 136s binding is mainly due to hydrophobic interactions as opposed to the hydrogen bonding in case of the rest of the compounds. Further studies are needed in order to answer this question. NH N NH2 NH O NHN NH2 NH O 136140 Figure 4.13:Comparision of 140 vs. 136 binding orientation in the chymotrypsin-like subunit binding pocket One more study that biologists conducted on NSC-12155 is the Dialysis experiment. This experiment was conducted in order to find whether NSC-12155 is a reversible or irreversible inhibitor. As mentioned in the introductory section a lot of the proteasome inhibitors did not go in to clinical trails because they all irreversibly bind to 125

PAGE 145

the chymotrypsin-like subunit of the 20S proteasome. Experiments proved that our lead molecule NSC-12155 is a reversible inhibitor of 20S proteasome (Figure 4.14). 050001000015000Chymo-like activity DMSO 12155 0 30 60 120 2 40Time (min) Figure 4.14:Dialysis experiment showing the reversible inhibition of NSC-12155 4.6 Conclusion and future direction In conclusion, we accomplished the synthesis of the lead molecule and a small library of molecules that show potential as 20S proteasome inhibitors. There is a lot of potential for NSC-12155 as the lead molecule for the proteasome inhibition. Hence we can make a variety of molecules around the urea and the quinoline moieties (Figure 4.15). Since only one of the two amine protons of the 4-amino group of the 4,6-diamino-2-methylquinolines are specifically hydrogen bound to the Thr21 backbone carbonyl, suggesting that replacement of the 4-amino group with a 4-hydroxy group 141 or with a 4-(N-methylamino) group 142 could retain that interaction. The 6-amino-4-hydroxy-2-methylquinoline derivative 141 needed to test this idea can be prepared from the intermediate 132 in the current synthesis of the 4,6-diaminoquinolines and the intermediate block 142 can be synthesized from 141 via methylation reaction. 126

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N OH NH2 OC N R N OH NH NH O R N OMe NH2 OC N R N OMe NH NH O R AcetonemicrowaveirradiationAcetonemicrowaveirradiationLIBRAR Y 141142132 H2SO4100oC DMS++ Figure 4.15:-Proposed molecules for future syntheses for proteasome inhibition 4.7 Experimental 4.7.1 20S proteasome inhibitory assay Solutions :(a) 50mM Tris, pH 7.6 (b) 20S Proteasome Stock Solution (i) 35ng/l Rabbit 20S Proteasome, Boston Biochem#E-350, 25g/$80.00 (ii) Human 20S Proteasome, Boston Biochem # #-360, 50g/$150.00 (c) Substrate (Chymotrypsin-like; Trypsin-like; or PGPH) (i) CT substrate: Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC), Boston Biochem, #S-280, 5mg/$85.00 20S Proteasome Stock Solution :Dilute 20S proteasome stock (50l) in a total volume of 1428l (add 1378l) of 50mM Tris, pH 7.6, for a final stock concentration of 35ng/l. Aliquot in small volumes and store at -80C. CT substrate (Suc-LLVY-AMC) :127 a) Dilute CT substrate (Suc-LLVY-AMC) 5mg, in 327l of DMSO for a 20mM stock solution. Aliquot in small volumes and store at -20C.

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128 b) Make 2mM working solution of CT s ubstrate (Suc-LLVY-AMC) by diluting 20mM stock 1:10 in DMSO. Equipment :(a) Clear 96-well ELIS A plates ing fluorometer with 355n m excitation and 460nm emission filters (b) Plate-read Protocol :(1) In the wells of 96-well plate, add 96 l of 50mM Tris, pH 7.6, to each well (2) Add 2 l of 35ng/ l 20S proteasome (70ng total) to each well. To the plate blank substrate (20 M final concentration) n/emission filters of 355/460nm 5 mediated proteasome inhibition, 20S his 15 L (225 ng) was used) was wells add 99 l of 50mM Tris, pH 7.6 (3) Add 1 l per well of 100X test sample or vehicle (4) Add 1 l per well of the appropriate (5) Shake plate by hand to mix all reagents (6) Cover plates in foil and in cubate at 37C for 1-4hr (7) Read plates on Fluorometer with excitatio (8) Calculate inhibition ba sed on vehicle control. 4.7.2 Assay for Reversible Inhibition of NSC-12155 To measure the effect of dialysis on NSC-1215 prokaryotic proteasome (9 g in 600 L solution from t incubated with 10 M NSC-12155 or the control solvent DM SO in 50 mM Tris-Hcl, pH 7.5 for 30 min. This was then incubated at 4 o C with dialysis for different time points using a 10,000 MWCO Pierce Slide-A-Lyzer Dialysis Cassette (Rockford, IL) in a rotating bath of 50 mM Tris-Hcl, pH 7.5. Th e proteasomal chymotrypsin-like activity was then assayed as previously described. 45

PAGE 148

129ynthesis of methyl 3-(4-acet solution of 4-aminoacetanilide 130 (20 g, 133.2 mmol) and methyl acetoacetate 131 cooling to 0 oC, ynthesis of 6-acetamide-4-hydrox33):m of methyl 3-(4-acetylaminophenylamino)crotonate 132 (10 g, 40.3 HNONHO 4.7.3 Chemistry MeO S ylaminophenylamino)crotonate (132):A (17.0 g, 146.5 mmol) in methanol (60 mL) was refluxed for 20 h. After the precipitate was collected by filtration to give methyl 3-(4-acetylaminophenylamino)crotonate 132 (23 g, 70%); a colorless solid; mp 134-135 o C; 1 H NMR (CDCl 3 400 MHz) 1.92 (s, 3H), 2.15 (s, 3H), 3.67 (s, 3H), 4.67 (br s, 1H), 6.96-6.98 (d, 2H), 7.44-7.46 (d, 2H), 7.79 (m, 1H), 10.19 (br s, 1H); 13 C NMR (CDCl 3, 100 MHz) 20.4, 24.6, 50.5, 85.5, 120.8, 125.5, 135.4, 135.5, 159.6, 168.7, 171.0 ppm NO HNOH S y-2-methylquinoline (1 The powder for mmol) was added to Dowtherm A (25 mL) while heating at 280 o C. After heating for 10 min, the mixture was cooled room temperature. The resulting precipitate was washed with ethyl acetate (30 mL) and methanol (10 mL) to give 6-acetamide-4-hydroxy-2-methylquinoline 133 (6.3 g, 72%) as a deep yellow solid; 1 H NMR (CD 3 OD, 400 MHz) 2.17 (s, 3H), 2.46 (s, 3H), 4.69 (br s, 1H), 4.98 (br s, 1H), 6.21 (s, 1H), 7.54-7.56 (d, 1H), 7.96-7.99 (dd, 1H), 8.18 (d, 1H); 13 C NMR (CD 3 OD, 100 MHz) 18.7, 22.7, 108.0, 114.5, 118.6, 124.3, 126.0, 135.0, 137.0, 151.8, 171.2, 178.8 ppm

PAGE 149

130ynthesis of 6-acetamide-4-methox34):imethyl sulfate (2.5 mL, 23.6 mmol) was added drop wise to a suspension of 133 in to room temperature, ynthesis of 4,6-diamin mixture of 6-acetamide-4-methoxy-2-methylquinoline 134 (1.3 g, 5.6 mmol) and oC for 4 h. Water (7 mL) and 40% NN HOOMe S y-2-methylquinoline (1 D toluene (20 mL) and the mixture was refluxed for 8 h. After cooling the precipitate was dissolved in water (40 mL) and the solution was alkalinized with 35% aqueous hydroxide solution (3 mL). The resulting precipitate was collected by filtration to give 6-acetamide-4-methoxy-2-methylquinoline 134 (1.5 g, 47%) as a brown solid; 1 H NMR (CD 3 OD, 400 MHz) 2.17 (s, 3H), 2.64 (s, 3H), 4.06 (s, 3H), 6.86 (s, 1H), 7.74-7.78 (m, 2H), 8.48 (s, 1H); 13 C NMR (CD 3 OD, 100 MHz) 22.6, 23.7, 55.3, 101.1, 110.7, 120.1, 123.8, 127.0, 135.8, 145.0, 159.5, 162.9, 170.6 ppm S o-2-methylquinoline (135):NNH2 NH2 A ammonium acetate (6.6 g, 85 mmol) was heated at 135 sulfuric acid (12 mL) were added to the reaction mixture, which was heated at 90 o C for 5 h. After cooling to 0 o C, add charcoal (1 g) and stirred for 5 min. After removal of the charcoal by filtration, the filtrate was alkalinized with 35% aqueous sodium hydroxide while cooling at 0 o C. The resulting precipitate was collected by filtration, washed with water (40 mL) an dried under vacuum at 100 o C to give 4, 6-diamino-2-methylquinoline 135 (0.43g, 45%) as a slightly yellow solid; 1 H NMR (d 6 -DMSO, 400 MHz) 2.28 (s, 3H), 5.02 (br s, 2H), 6.00 (br s, 2H), 6.26 (s, 1H), 6.88 (s, 1H), 6.936.95 (d, 1H), 7.36

PAGE 150

131Synthesis of 1,3-Bis-(4-amino-2-meto a solution of 6-diamino-2-methylquinoline 6 (0.05 g, 0.30 mmol) in 3 mL of 1,4-mmol) was 2-methylquinoline 135 (0.02 g, 0.12 mmol) in dry Acetone dded and the NNHNHNNH2NH2 7.38 (d, 1H); 13 C NMR (d 6 -DMSO, 100 MHz) 25.1, 101.7, 102.7, 119.2, 121.1, 129.5, 142.8, 144.8, 150.0, 153.9 ppm; ESI found 174.1 for [M+H] + O hyl-quinolin-6-yl)-urea (NSC-12155) T dioxane, triethylamine (0.12 mL, 0.9 mmol) and triphosgene (0.015 g, 0.05 added. The reaction was very exothermic and a heavy precipitate was formed. After addition of 3 mL of 1,4-dioxane to dilute the mixture, it was heated under reflux for 12 h. After cooling, the mixture was poured into 5 mL of ice-H 2 O and the product was collected and washed with H 2 O. The material was recrystallised from DMF-MeOH to provide 3-Bis-(4-amino-2-methyl-quinolin-6-yl)-urea NSC-12155 (0.04 g, 75%) as a brown solid; mp 254-256 o C (lit. mp= 255 o C); 1 H NMR (CD 3 OD, 400 MHz) 2.46 (s, 6H), 6.48 (s, 2H), 7.61-7.66 (m, 4H), 8.10 (s, 2H); 13 C NMR (CD 3 OD, 100 MHz) 22.4, 39.2, 102.5, 109.6, 117.4, 123.8, 126.3, 135.4, 143.2, 153.0, 154.2, 156.6 ppm; ESI-TOF, Found: 373.1736 for [M+H] + General Procedure for the microwave assisted synthesis of the Ureas: To a solution of 4, 6-diamino(1 mL) under argon atmosphere the concerned isocyanate (0.12 mmol) was a reaction vessel was sealed. The reaction mixture was placed in the microwave with a previously standardized set of conditions. After the reaction time almost in all the cases a

PAGE 151

precipitate was observed and the precipitate was filtered and dried. The product was characterized with out any further purification. Microwave conditions:132iscover min ynthesis of 1-(4-Amino-2--phenyl-urea (136):. mp= 226.5-N Microwave Type: CEM d Power: 150 W Ramp time: 4:00 Hold time: 20:00 min Temperature: 100 o C Pressure: 220 psi NH2 NH NH O S methyl-quinolin-6-yl)-3 Product was obtained as pale yellow solid (0.005 g); mp 226-228 o C (lit 227.5 o C); 1 H NMR (d 6 DMSO, 400 MHz) 2.37 (s, 3H), 6.39 (br s, 2H), 6.40 (s, 1H), 6.95-9.99 (t, 1H), 7.27-7.30 (t, 2H), 7.47-7.49 (d, 2H), 7.61 (s, 1H), 7.62-7.63 (d, 1H), 7.95-7.96 (d, 1H), 8.65 (br s, 1H), 8.81 (br s, 1H); 13 C NMR (d 6 -DMSO, 100 MHz) 24.5, 102.2, 109.2117.3, 118.0, 121.6, 122.7, 128.5, 128.6, 128.8, 134.4, 139.7, 144.8, 150.6, 152.6, 156.3 ppm; ESI-TOF Calcd for [M+H] + is 293.13969, Found: 293.13976 N H2N NH NH O

PAGE 152

133ynthesis of 1-(4-Amino-2-methyl-quinolin-6-yl)-3-p-tolyl-urea (137):t. mp= 260.4-ynthesis of 1-(4-Amino-(4-chloro-phenyl)-urea (138):6Synthesis of 1-(4-Amino-2-methyl-quinolinifluoromethyl-phenyl)-urea was obtained as pale yellow flakes (0.05 g); mp 265-266 oC; 1H NMR (d6-DMSO, 400 MHz) 2.38 (s, 3H), 6.41 (br s, 3H), 7.62-7.65 (m, 4H), 7.69-7.71 (d, 2H), S Product was obtained as a pale yellow solid (0.005 g); mp 262-263 o C (li 262.4 o C); 1 H NMR (d 6 -DMSO, 400 MHz) 2.24 (s, 3H), 2.37 (s, 3H), 6.38 (br s, 2H), 6.40 (s, 1H), 7.097.10 (d, 2H), 7.36-7.38 (d, 2H), 7.60 (s, 1H), 7.61 (d, 1H), 7.95 (d, 1H), 8.65 (br s, 1H), 8.71 (br s, 1H); 13 C NMR (d 6 DMSO, 100 MHz) 20.2, 24.5, 102.2, 109.3, 117.4, 118.1, 122.7, 128.5, 129.0, 129.2, 130.4, 134.5, 137.1, 144.7, 150.6, 152.6, 156.2 ppm; ESI-TOF Calcd for [M+H] + is 307.15534, Found: 307.15569 N NH2 NH NH O Cl S -2-methyl-quinolin-6-yl)-3 Product was obtained as a brown solid (0.016 g, 44%); mp 264-265 o C; 1 H NMR (d DMSO, 400 MHz) 2.37 (s, 3H), 6.40 (s, 1H), 6.41 (br s, 2H), 7.32-7.34 (d, 2H), 7.51-7.53 (d, 2H), 7.61 (d, 2H), 7.96 (s, 1H), 8.73 (br s, 1H), 8.97 (br s, 1H); 13 C NMR (d 6 -DMSO, 100 MHz) 24.5, 102.2, 109.7, 117.3, 119.5, 122.8, 125.1, 128.5, 134.2, 138.7, 150.6, 152.5, 156.3 ppm; ESI-TOF Calcd for [M+H] + is 327.10054, Found: 327.10054 N CF NH2 NH NH O 3 -6-yl)-3-(4-tr (139):Product

PAGE 153

13422.9, ynthesis of 1-(4-Amino-lin-6-yl)-3-naphthalen-1-yl-urea (140):roduct was obtained as a brown solid (0.05 g); mp 195-196 oC; 1H NMR (d6DMSO, (m, L. Trends Cell Biol. 1998, 8, 397 403 ver, A. Cell 1994, 13 21. Chem. Biol. 1995, 2, 503 508. 434. .; Yasuoka, ID:-1IRU) 7) Coux, O.; Tanaka, K.; Goldberg, A. L. Annu. Rev. Biochem. 1996, 65, 801 847. 8.00 (br s, 1H); 13 C NMR (d 6 -DMSO, 100 MHz) 24.6, 102.3, 109.9, 117.3, 117.6, 1125.9, 128.6, 134.1, 145.0, 150.7, 152.4, 156.5 ppm; ESI-TOF Calcd for [M+H] + is 361.12707, Found: 361.12738 N NH2 NH NH O S 2-methyl-quino P 400 MHz) 2.38 (s, 3H), 6.40 (br s, 2H), 6.42 (s, 1H), 7.47-7.51 (t, 1H), 7.55-7.59 2H), 7.61-7.65 (m, 3H), 7.69 (d, 1H), 7.93-7.95 (d, 1H), 8.02-8.03 (d, 1H), 8.06-8.07 (d, 1H), 8.17-8.19 (d, 1H), 8.96 (br s, 1H), 9.18 (br s, 1H); 13 C NMR (d 6 DMSO, 100 MHz) 24.5, 102.3, 109.3, 117.2, 117.4, 121.3, 122.7, 125.5, 125.7, 125.8, 128.6, 133.6, 134.3, 134.6, 137.8, 142.9, 144.8, 150.6, 153.0, 156.3 ppm; ESI-TOF Calcd for [M+H] + is 343.15534, Found: 343.15551 4.8 References 1) Lee, D. H.; Goldberg, A 2) Ciechano 3) Goldberg, A. L. Science 1995, 268, 522 523. 4) Goldberg, A. L.; Stein, R.; Adams, J. 5) Deshaies, R. J. Trends Cell Biol. 1995, 5, 428 6) Unno, M.; Mizushima, T.; Morimoto, Y.; Tomisugi, Y.; Tanaka, K N.; Tsukihara, T. Structure 2002, 10, 609 618. (PDB

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137 lo, D. N.; J. Med. 40) .; McGrath, C.; Turk, B. E.; Burnett, J.; Aman, M. J.; 42) o, D.; Del Giudice, M. R. Eur. J. Med. Chem. 2004, 39, 1047 1057. unol. 45) 38) Hunter, D. T., Jr.; Hill, J. M. Nature 1961, 191, 1378 1379. 39) Lanza, T. J.; Durette, P. L.; Rolli ns, T.; Siciliano, S.; Cianciaru Kobayashi, S. V.; Caldwell, C. G. ; Springer, M. S.; Hagmann, W. K. Chem. 1992, 35, 252 258. Shinkai, H.; Ito, T.; Iida, T.; Kitao, Y.; Yamada, H.; Uchinda, I. J. Med. Chem. 2000, 43, 4667 4677. 41) Panchal, R. G.; Hermone, A. R.; Ng uyen, T. L.; Wong, T. Y.; Schwarzenbacher, R.; Schmidt, J.; Lane, D Little, S.; Sausville, E. A.; Zaharevitz D. W.; Cantley, L. C.; Liddington, R. C.; Gussio, R.; Bavari, S. Nat. Struct. Mol. Biol. 2004 11, 67 72. Montecucco, C.; Tonello, F.; Zanotti, G. Trends Biochem. Sci. 2004, 29 282 285. 43) Sestili, I.; Borioni, A.; Mustazza, C.; Rodomonte, A.; Turchetto, L.; Sbraccia, M.; Riitan 44) Gasparetto, M.; Gentry, T.; Sebti, S. ; OBryan, E.; Nimmanapalli, R.; Blaskovich, M. A.; Bhalla, K.; Rizzieri, D.; Haaland, P.; Dunne, J.; Smith, C. J. Imm Methods 2004, 292, 59 71. Nam, S.; Smith, D. M.; Dou, Q. P. J. Biol. Chem. 2001, 276, 13322 13330.

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138 CHAPTER 5: APPENDICES Appendix A: Selected 1H and 13C NMR spectra

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Appendix A: (Continued) Spectrum 5.01 1 H NMR (CDCl 3, 400 MHz) of 9b 13 C NMR (CDCl 3, 100 MHz) of 9b 139

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Appendix A: (Continued) Spectrum 5.02 1 H NMR (CDCl 3, 400 MHz) of 10a 13 C NMR (CDCl 3, 100 MHz) of 10a 140

PAGE 160

Appendix A: (Continued) Spectrum 5.03 1 H NMR (CDCl 3, 250 MHz) of 10b 141 13 C NMR (CDCl 3, 62.5 MHz) of 10b

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Appendix A: (Continued) Spectrum 5.04 1 H NMR (CDCl 3, 250 MHz) of 10c 13 C NMR (CDCl 3, 62.5 MHz) of 10c 142

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Appendix A: (Continued) Spectrum 5.05 1 H NMR (CDCl 3, 250 MHz) of 10d 13 C NMR (CDCl 3, 62.5 MHz) of 10d 143

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Appendix A: (Continued) Spectrum 5.06 1 H NMR (CDCl 3, 400 MHz) of 11a 13 C NMR (CDCl 3, 100 MHz) of 11a 144

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Appendix A: (Continued) Spectrum 5.07 1 H NMR (CDCl 3, 400 MHz) of 11c 145

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Appendix A: (Continued) Spectrum 5.08 1 H NMR (CDCl 3, 250 MHz) of 11d 146 13 C NMR (CDCl 3, 100 MHz) of 11d

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Appendix A: (Continued) Spectrum 5.09 1 H NMR (CDCl 3, 400 MHz) of 12a 13 C NMR (CDCl 3, 100 MHz) of 12a 147

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Appendix A: (Continued) Spectrum 5.10 1 H NMR (CDCl 3, 250 MHz) of 12b 148 13 C NMR (CDCl 3, 62.5 MHz) of 12b

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Appendix A: (Continued) Spectrum 5.11 1 H NMR (CDCl 3, 250 MHz) of 12c 149 13 C NMR (CDCl 3, 100 MHz) of 12c

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Appendix A: (Continued) Spectrum 5.12 1 H NMR (CDCl 3, 250 MHz) of 12d 150 13 C NMR (CDCl 3, 62.5 MHz) of 12d

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Appendix A: (Continued) Spectrum 5.13 1 H NMR (CDCl 3, 400 MHz) of 13a 13 C NMR (CDCl 3, 100 MHz) of 13a 151

PAGE 171

Appendix A: (Continued) Spectrum 5.14 1 H NMR (CDCl 3, 250 MHz) of 13b 13 C NMR (CDCl 3, 62.5 MHz) of 13b 152

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Appendix A: (Continued) Spectrum 5.15 1 H NMR (CDCl 3, 250 MHz) of 13c 13 C NMR (CDCl 3, 62.5 MHz) of 13c 153

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Appendix A: (Continued) Spectrum 5.16 1 H NMR (CDCl 3, 250 MHz) of 13d 13 C NMR (CDCl 3, 62.5 MHz) of 13d 154

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Appendix A: (Continued) Spectrum 5.17 1 H NMR (CDCl 3, 400 MHz) of 14a 13 C NMR (CDCl 3, 100 MHz) of 14a 155

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Appendix A: (Continued) Spectrum 5.18 1 H NMR (CDCl 3, 250 MHz) of 14b 13 C NMR (CDCl 3, 62.5 MHz) of 14b 156

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Appendix A: (Continued) Spectrum 5.19 1 H NMR (CDCl 3, 250 MHz) of 14c 157 13 C NMR (CDCl 3, 62.5 MHz) of 14c

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Appendix A: (Continued) Spectrum 5.20 1 H NMR (CDCl 3, 250 MHz) of 14d 13 C NMR (CDCl 3, 62.5 MHz) of 14d 158

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Appendix A: (Continued) Spectrum 5.21 1 H NMR (CDCl 3, 400 MHz) of 15a 13 C NMR (CDCl 3, 100 MHz) of 15a 159

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Appendix A: (Continued) Spectrum 5.22 1 H NMR (CDCl 3, 250 MHz) of 15b 160 13 C NMR (CDCl 3, 62.5 MHz) of 15b

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Appendix A: (Continued) Spectrum 5.23 1 H NMR (CDCl 3, 250 MHz) of 15c 13 C NMR (CDCl 3, 62.5 MHz) of 15c 161

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Appendix A: (Continued) Spectrum 5.24 1 H NMR (CDCl 3, 250 MHz) of 15d 162 13 C NMR (CDCl 3, 62.5 MHz) of 15d

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Appendix A: (Continued) Spectrum 5.25 1 H NMR (CDCl 3, 400 MHz) of 16a 13 C NMR (CDCl 3, 100 MHz) of 16a 163

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Appendix A: (Continued) Spectrum 5.26 1 H NMR (CDCl 3, 400 MHz) of 16b 13 C NMR (CDCl 3, 62.5 MHz) of 16b 164

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Appendix A: (Continued) Spectrum 5.27 1 H NMR (CDCl 3, 250 MHz) of 16c 13 C NMR (CDCl 3, 62.5 MHz) of 16c 165

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Appendix A: (Continued) Spectrum 5.28 1 H NMR (CDCl 3, 250 MHz) of 16d 166 13 C NMR (CDCl 3, 62.5 MHz) of 16d

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Appendix A: (Continued) Spectrum 5.29 1 H NMR (CDCl 3, 400 MHz) of 17a 13 C NMR (CDCl 3, 100 MHz) of 17a 167

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Appendix A: (Continued) Spectrum 5.30 1 H NMR (CDCl 3, 400 MHz) of 17b 13 C NMR (CDCl 3, 100 MHz) of 17b 168

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Appendix A: (Continued) Spectrum 5.31 1 H NMR (CDCl 3, 250 MHz) of 17c 13 C NMR (CDCl 3, 62.5 MHz) of 17c 169

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Appendix A: (Continued) Spectrum 5.32 1 H NMR (CDCl 3, 250 MHz) of 17d 13 C NMR (CDCl 3, 62.5 MHz) of 17d 170

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Appendix A: (Continued) Spectrum 5.33 COOEt COOEt N3 H 3 C 1 H NMR (CDCl 3 250 MHz) of 62 13 C NMR (CDCl 3 62.5 MHz) of 62 171

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Appendix A: (Continued) Spectrum 5.34 N3 COOH H 3 C COOE t 1 H NMR (CDCl 3 250 MHz) of 63 X X 172 13 C NMR (CDCl 3 62.5 MHz) of 63

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Appendix A: (Continued) Spectrum 5.35 N3 COOEt H 3 C CON 3 1 H NMR (CDCl 3 250 MHz) of 64 13 C NMR (CDCl 3 62.5 MHz) of 64 173

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Appendix A: (Continued) Spectrum 5.36 N3 COOEt H3C NHBoc 1 H NMR (CDCl 3 250 MHz) of 65 13 C NMR (CDCl 3 62.5 MHz) of 65 174

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Appendix A: (Continued) Spectrum 5.37 1 H NMR (CDCl 3 250 MHz) of 70 N3 H3C O OEt NH O H3C H X X 13 C NMR (CDCl 3 62.5 MHz) of 70 175

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Appendix A: (Continued) Spectrum 5.38 NHCbz CbzHN COOH H 3 C 1 H NMR (CD 3 OD, 250 MHz) of 73 13 C NMR (CD 3 OD, 100 MHz) of 73 176

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Appendix A: (Continued) Spectrum 5.39 NHCbz CbzHN H3C O O N O O 1 H NMR (CDCl 3 250 MHz) of 74 13 C NMR (CDCl 3 100 MHz) of 74 177

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Appendix A: (Continued) 1 H NMR (CDCl 3 400 MHz) of 75 NHCbz CbzHN H 3 C OH Spectrum 5.40 13 C NMR (CDCl 3 100 MHz) of 75 178

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Appendix A: (Continued) Spectrum 5.41 O HN CbzHN H3C O 1 H NMR (CDCl 3 400 MHz) of 76 13 C NMR (CDCl 3 100 MHz) of 76 179

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Appendix A: (Continued) 1 H NMR (CDCl 3 400 MHz) of 77 O N CbzHN H3C O Boc Spectrum 5.42 13 C NMR (CDCl 3 100 MHz) of 77 180

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Appendix A: (Continued) Spectrum 5.43 O HN BocHN H3C O 1 H NMR (CDCl 3 400 MHz) of 79 13 C NMR (CDCl 3 100 MHz) of 79 181

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Appendix A: (Continued) Spectrum 5.44 NHCbz COOCH3 CbzHN H3C 1 H NMR (CDCl 3 250 MHz) of 82 13 C NMR (CDCl 3 62.5 MHz) of 82 182

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Appendix A: (Continued) Spectrum 5.45 NHCbz CHO CbzHN H 3 C 1 H NMR (CDCl 3 400 MHz) of 83 13 C NMR (CDCl 3 100 MHz) of 83 183

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Appendix A: (Continued) Spectrum 5.46 O N O P Ph Ph Ph 1 H NMR (CDCl 3 400 MHz) of 102 13 C NMR (CDCl 3 100 MHz) of 102 184

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Appendix A: (Continued) Spectrum 5.47 O N O HC Cl Cl Cl 1 H NMR (CDCl 3 400 MHz) of 103 185

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Appendix A: (Continued) Spectrum 5.48 Cl 3 C N O Boc 1 H NMR (CDCl 3 400 MHz) of 104 13 C NMR (CDCl 3 100 MHz) of 104 186

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Appendix A: (Continued) Spectrum 5.49 Ph N COOCH 3 Ph Ph 1 H NMR (CDCl 3 400 MHz) of 110 13 C NMR (CDCl 3 100 MHz) of 110 187

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Appendix A: (Continued) Spectrum 5.50 O O N O O NHBoc 1 H NMR (CDCl 3 400 MHz) of 118 13 C NMR (CDCl 3 100 MHz) of 118 188

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Appendix A: (Continued) NH Ph O O O O Spectrum 5.51 1 H NMR (CDCl 3 400 MHz) of 120 13 C NMR (CDCl 3 100 MHz) of 120 189

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Appendix A: (Continued) N Ph O O O O NH Boc Spectrum 5.52 1 H NMR (CDCl 3 400 MHz) of 121 13 C NMR (CDCl 3 100 MHz) of 121 190

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Appendix A: (Continued) Spectrum 5.53 1 H NMR (CDCl 3 400 MHz) of 123 BocHN O O Ph O H 13 C NMR (CDCl 3 100 MHz) of 123 191

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Appendix A: (Continued) Spectrum 5.54 B o c HN C H O O O Ph 1 H NMR (CDCl 3 400 MHz) of 124 13 C NMR (CDCl 3 100 MHz) of 124 192

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Appendix A: (Continued) BocHN O O Ph NO2 H O Spectrum 5.55 1 H NMR (CDCl 3 400 MHz) of 125 13 C NMR (CDCl 3 100 MHz) of 125 193

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Appendix A: (Continued) Spectrum 5.56 1 H NMR (CDCl 3 400 MHz) of 84 NHCbz CbzHN H3C NO2 OH 13 C NMR (CDCl 3 100 MHz) of 84 194

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Appendix A: (Continued) NHCbz CbzHN H3C NH2 O H Spectrum 5.57 1 H NMR (CDCl 3 400 MHz) of 85 13 C NMR (CDCl 3 100 MHz) of 85 195

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196 Appendix A: (Continued) 13C NMR (CDCl3, 100 MHz) of 132 1H NMR (CDCl3, 400 MHz) of 132 H N O N H O MeO Spectrum 5.58

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197 Appendix A: (Continued) 13C NMR (CD3OD, 100 MHz) of 1331H NMR (CD3OD, 400 MHz) of 133 N H N O OH Spectrum 5.59

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198 Appendix A: (Continued) 13C NMR (CD3OD, 100 MHz) of 1341H NMR (CD3OD, 400 MHz) of 134 N H N O OMe Spectrum 5.60

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199 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1351H NMR (d6-DMSO, 400 MHz) of 135N NH2NH2 Spectrum 5.61

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200 Appendix A: (Continued) 13C NMR (CD3OD, 100 MHz) of NSC-12155 1H NMR (CD3OD, 400 MHz) of NSC-12155 N N H N H O N NH2NH2 Spectrum 5.62

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201 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1361H NMR (d6-DMSO, 400 MHz) of 136N NH2 N H N H O Spectrum 5.63

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202 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1371H NMR (d6-DMSO, 400 MHz) of 137N H2N N H N H O Spectrum 5.64

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203 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1381H NMR (d6-DMSO, 400 MHz) of 138N NH2 N H N H O Cl Spectrum 5.65

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204 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1391H NMR (d6-DMSO, 400 MHz) of 139N NH2 N H N H O CF3 Spectrum 5.66

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205 Appendix A: (Continued) 13C NMR (d6-DMSO, 100 MHz) of 1401H NMR (d6-DMSO, 400 MHz) of 140N NH2 N H N H O Spectrum 5.67

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206 Appendix B: X-ray crystallographic data

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Appendix B: (Continued) Structure of the salt 19, of 2-isobutyl-3-nitropropionic acid (16c) with (1R,2R)-(-)-pseudoephedrine (18) determined by X-ray structural analysis. 207

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Appendix B: (Continued) Molecule-1 Crystal Structure (82) Molecule-2 Crystal Structure (82) Crystal structures of two molecules of 2,2-Bis-benzyloxycarbonylamino-propionic acid methyl ester (82) that are in different orientation. 208

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Appendix B: (Continued) Relationship of Molecule-1 with Molecule-2 in the crystal lattice of 2,2-Bis-benzyloxycarbonylamino-propionic acid methyl ester (82). 209

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210 Appendix B: (Continued) Table 1. Crystal data and structure refinement for 82 Identification code mm011 Empirical formula C20 H22 N2 O6 Formula weight 386.40 Temperature 100(2) K Wavelength 0.71073 Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 14.7237(15) a= 90. b = 16.0439(16) b= 97.260(2). c = 16.9361(17) g = 90. Volume 3968.7(7) 3 Z 8 Density (calculated) 1.293 Mg/m 3 Absorption coefficient 0.096 mm -1 F(000) 1632 Crystal size 0.80 x 0.80 x 0.13 mm 3 Theta range for data collection 1.73 to 22.78. Index ranges -16<=h<=10, -17<=k<=16, -18<=l<=18 Reflections collected 16744 Independent reflections 5356 [R(int) = 0.0663] Completeness to theta = 22.78 99.6 % Absorption correction None Max. and min. transmission 1.000 and 0.802 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 5356 / 0 / 509 Goodness-of-fit on F 2 1.153 Final R indices [I>2sigma(I)] R1 = 0.0689, wR2 = 0.1593 R indices (all data) R1 = 0.0981, wR2 = 0.1824 Largest diff. peak and hole 0.426 and -0.324 e. -3

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211 Appendix B: (Continued) Table 2. Atomic coordinates (x 10 4 ) and equivalent isotropic displacement parameters ( 2 x 10 3 ) for mm011. U(eq) is defined as one th ird of the trace of the orthogonalized U ij tensor. ________________________________________________________________________ x y z U(eq) ________________________________________________________________________ O(1) 2201(2) 3388(2) 2340(2) 26(1) O(2) 2265(2) 2467(2) 3363(2) 24(1) O(3) 970(2) 3483(2) 5265(2) 29(1) O(4) 151(2) 4073(2) 4184(2) 27(1) O(5) -838(2) 3416(2) 2524(2) 25(1) O(6) 7124(2) 3530(2) 1378(2) 22(1) O(7) 6996(2) 3415(2) 39(2) 22(1) O(8) 9343(2) 1811(2) 702(2) 34(1) O(9) 8446(2) 1326(2) -401(2) 30(1) O(10) 10195(2) 3760(2) 1138(2) 33(1) O(11) -1167(2) 2817(2) 3652(2) 28(1) O(12) 10472(2) 3210(2) -21(2) 33(1) N(1) 657(2) 2737(2) 4162(2) 18(1) N(2) 917(2) 3048(2) 2818(2) 21(1) N(3) 8387(2) 3550(2) 788(2) 23(1) N(4) 8706(2) 2678(2) -294(2) 23(1) C(1) 3462(3) 3664(3) 1628(2) 24(1) C(2) -622(3) 3021(3) 3117(2) 24(1) C(3) 335(3) 2627(2) 3326(2) 20(1) C(4) 8772(3) 4173(3) -460(3) 32(1) C(5) 5873(3) 3581(3) 2133(2) 25(1) C(6) 7462(3) 3491(2) 685(2) 18(1) C(7) 1836(3) 2929(2) 2886(2) 20(1) C(8) 559(3) 3484(3) 4502(2) 22(1) C(9) 8924(3) 3445(3) 134(2) 23(1) C(10) 8873(3) 1931(3) 64(3) 26(1) C(11) 6126(3) 3478(3) 1315(2) 25(1) C(12) 3195(3) 3400(3) 2411(3) 32(1)

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212 Appendix B: (Continued) C(13) 267(3) 1692(2) 3144(2) 27(1) C(14) 9930(3) 3471(3) 498(2) 25(1) C(15) 5661(3) 2895(3) 2574(3) 31(1) C(16) 5833(3) 4367(3) 2460(3) 35(1) C(17) 3941(3) 4168(3) 172(3) 38(1) C(18) 2201(3) 3703(3) 6640(3) 32(1) C(19) 5439(3) 2999(4) 3345(3) 48(2) C(20) 3675(3) 3081(3) 1080(3) 31(1) C(21) 3912(3) 3326(3) 349(3) 39(1) C(22) 3498(3) 4495(3) 1437(2) 28(1) C(23) 8557(3) -751(3) -1008(3) 35(1) C(24) 1074(4) 4559(3) 7146(3) 37(1) C(25) 1395(3) 4160(2) 6513(2) 27(1) C(26) 3733(3) 4746(3) 709(3) 33(1) C(27) 8539(3) 481(2) -102(3) 35(1) C(28) 5411(4) 3806(5) 3647(3) 56(2) C(29) 8098(3) -66(3) -758(3) 31(1) C(30) 2346(4) 4022(3) 8027(3) 55(2) C(31) 8158(4) -1237(3) -1635(3) 43(1) C(32) 6849(4) -352(3) -1785(3) 52(2) C(33) 5616(4) 4474(4) 3208(3) 53(2) C(34) 7244(4) 122(3) -1150(3) 48(1) C(35) 7314(4) -1028(3) -2034(3) 45(1) C(36) 877(4) 4259(3) 5691(3) 39(1) C(37) 1551(4) 4487(3) 7908(3) 53(2) C(38) 2664(4) 3643(3) 7394(3) 44(1) C(39) 11451(3) 3225(3) 250(3) 36(1) C(40) -2063(3) 3236(3) 3540(3) 39(1) __________________________________________________________________

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213 Appendix B: (Continued) Table 3. Bond lengths [ ] and angles [] for mm011. _____________________________________________________ O(1)-C(7) 1.347(5) O(1)-C(12) 1.454(5) O(2)-C(7) 1.213(5) O(3)-C(8) 1.356(5) O(3)-C(36) 1.455(5) O(4)-C(8) 1.209(5) O(5)-C(2) 1.196(5) O(6)-C(6) 1.333(5) O(6)-C(11) 1.461(5) O(7)-C(6) 1.222(4) O(8)-C(10) 1.223(5) O(9)-C(10) 1.354(5) O(9)-C(27) 1.447(5) O(10)-C(14) 1.197(5) O(11)-C(2) 1.325(5) O(11)-C(40) 1.471(5) O(12)-C(14) 1.328(5) O(12)-C(39) 1.456(5) N(1)-C(8) 1.345(5) N(1)-C(3) 1.447(5) N(2)-C(7) 1.356(5) N(2)-C(3) 1.455(5) N(3)-C(6) 1.354(5) N(3)-C(9) 1.449(5) N(4)-C(10) 1.351(5) N(4)-C(9) 1.444(5) C(1)-C(22) 1.374(6) C(1)-C(20) 1.382(6) C(1)-C(12) 1.491(6) C(2)-C(3) 1.545(6) C(3)-C(13) 1.532(5) C(4)-C(9) 1.538(6)

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214 Appendix B: (Continued) C(5)-C(16) 1.381(6) C(5)-C(15) 1.387(6) C(5)-C(11) 1.490(6) C(9)-C(14) 1.531(6) C(15)-C(19) 1.396(6) C(16)-C(33) 1.355(7) C(17)-C(26) 1.360(6) C(17)-C(21) 1.386(7) C(18)-C(38) 1.373(6) C(18)-C(25) 1.389(6) C(19)-C(28) 1.394(8) C(20)-C(21) 1.384(7) C(22)-C(26) 1.383(6) C(23)-C(29) 1.384(6) C(23)-C(31) 1.388(6) C(24)-C(25) 1.382(6) C(24)-C(37) 1.394(6) C(25)-C(36) 1.509(6) C(27)-C(29) 1.498(6) C(28)-C(33) 1.361(8) C(29)-C(34) 1.379(6) C(30)-C(38) 1.366(8) C(30)-C(37) 1.381(8) C(31)-C(35) 1.379(7) C(32)-C(35) 1.376(7) C(32)-C(34) 1.385(7) C(7)-O(1)-C(12) 115.8(3) C(8)-O(3)-C(36) 114.5(3) C(6)-O(6)-C(11) 114.6(3) C(10)-O(9)-C(27) 116.9(3) C(2)-O(11)-C(40) 114.2(3) C(14)-O(12)-C(39) 116.1(3) C(8)-N(1)-C(3) 119.2(3) C(7)-N(2)-C(3) 122.7(3)

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215 Appendix B: (Continued) C(6)-N(3)-C(9) 122.1(3) C(10)-N(4)-C(9) 121.0(3) C(22)-C(1)-C(20) 118.7(4) C(22)-C(1)-C(12) 120.4(4) C(20)-C(1)-C(12) 120.9(4) O(5)-C(2)-O(11) 125.8(4) O(5)-C(2)-C(3) 123.1(4) O(11)-C(2)-C(3) 110.9(3) N(1)-C(3)-N(2) 112.2(3) N(1)-C(3)-C(13) 108.7(3) N(2)-C(3)-C(13) 111.4(3) N(1)-C(3)-C(2) 110.5(3) N(2)-C(3)-C(2) 105.3(3) C(13)-C(3)-C(2) 108.7(3) C(16)-C(5)-C(15) 119.0(4) C(16)-C(5)-C(11) 120.2(4) C(15)-C(5)-C(11) 120.8(4) O(7)-C(6)-O(6) 124.3(4) O(7)-C(6)-N(3) 124.3(4) O(6)-C(6)-N(3) 111.4(3) O(2)-C(7)-O(1) 124.9(4) O(2)-C(7)-N(2) 125.0(4) O(1)-C(7)-N(2) 110.2(3) O(4)-C(8)-N(1) 125.5(4) O(4)-C(8)-O(3) 124.2(4) N(1)-C(8)-O(3) 110.3(3) N(4)-C(9)-N(3) 111.9(3) N(4)-C(9)-C(14) 111.6(3) N(3)-C(9)-C(14) 106.4(3) N(4)-C(9)-C(4) 108.3(3) N(3)-C(9)-C(4) 111.3(3) C(14)-C(9)-C(4) 107.2(3) O(8)-C(10)-N(4) 126.0(4) O(8)-C(10)-O(9) 124.8(4)

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216 Appendix B: (Continued) N(4)-C(10)-O(9) 109.3(3) O(6)-C(11)-C(5) 107.1(3) O(1)-C(12)-C(1) 107.8(3) O(10)-C(14)-O(12) 124.5(4) O(10)-C(14)-C(9) 124.3(4) O(12)-C(14)-C(9) 110.9(3) C(5)-C(15)-C(19) 120.3(5) C(33)-C(16)-C(5) 121.2(5) C(26)-C(17)-C(21) 120.3(4) C(38)-C(18)-C(25) 119.6(5) C(28)-C(19)-C(15) 118.4(5) C(1)-C(20)-C(21) 120.8(4) C(20)-C(21)-C(17) 119.3(4) C(1)-C(22)-C(26) 120.9(4) C(29)-C(23)-C(31) 120.3(5) C(25)-C(24)-C(37) 119.8(5) C(24)-C(25)-C(18) 119.7(4) C(24)-C(25)-C(36) 119.0(4) C(18)-C(25)-C(36) 121.3(4) C(17)-C(26)-C(22) 120.0(4) O(9)-C(27)-C(29) 106.1(3) C(33)-C(28)-C(19) 120.8(5) C(34)-C(29)-C(23) 118.5(4) C(34)-C(29)-C(27) 120.8(4) C(23)-C(29)-C(27) 120.6(4) C(38)-C(30)-C(37) 119.6(4) C(35)-C(31)-C(23) 120.4(5) C(35)-C(32)-C(34) 119.6(5) C(16)-C(33)-C(28) 120.3(5) C(29)-C(34)-C(32) 121.4(5) C(32)-C(35)-C(31) 119.7(5) O(3)-C(36)-C(25) 107.5(3) C(30)-C(37)-C(24) 119.9(5) C(30)-C(38)-C(18) 121.4(5)

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217 Appendix B: (Continued) Table 4. Anisotropic displacement parameters ( 2 x 10 3 ) for mm011. The anisotropic displacement factor expone nt takes the form: -2p 2 [ h 2 a* 2 U 11 + ... + 2 h k a* b* U 12 ] ________________________________________________________________________ U 11 U 22 U 33 U 23 U 13 U 12 ________________________________________________________________________ O(1) 12(2) 41(2) 23(2) 10(1) 1(1) 0(1) O(2) 18(2) 32(2) 22(2) 6(1) -3(1) 3(1) O(3) 39(2) 26(2) 18(2) -4(1) -8(1) 11(1) O(4) 29(2) 27(2) 23(2) 0(1) -6(1) 9(1) O(5) 22(2) 36(2) 16(2) 3(1) -2(1) -1(1) O(6) 16(2) 36(2) 15(2) -1(1) 2(1) -2(1) O(7) 19(2) 28(2) 17(2) 2(1) -3(1) -1(1) O(8) 30(2) 41(2) 27(2) 2(1) -7(2) 9(2) O(9) 33(2) 25(2) 32(2) -2(1) -3(2) 4(1) O(10) 25(2) 51(2) 23(2) -4(2) 1(1) -7(2) O(11) 15(2) 42(2) 26(2) 7(1) 3(1) 0(1) O(12) 19(2) 53(2) 27(2) -7(2) 0(1) 2(2) N(1) 21(2) 18(2) 15(2) 3(1) -1(2) 4(2) N(2) 14(2) 30(2) 18(2) 5(2) -2(2) 0(2) N(3) 19(2) 33(2) 16(2) -4(2) -3(2) -5(2) N(4) 23(2) 26(2) 19(2) 0(2) 0(2) 2(2) C(1) 10(2) 37(3) 24(2) -2(2) -2(2) 0(2) C(2) 21(3) 26(2) 23(2) -6(2) 0(2) -5(2) C(3) 20(3) 23(2) 17(2) 2(2) 2(2) -2(2) C(4) 24(3) 32(3) 40(3) 5(2) 2(2) -4(2) C(5) 9(2) 47(3) 17(2) 0(2) -2(2) 1(2) C(6) 18(3) 19(2) 19(2) 0(2) 6(2) 0(2) C(7) 20(3) 20(2) 18(2) -7(2) 1(2) -2(2) C(8) 19(3) 26(3) 19(2) 1(2) 0(2) 2(2) C(9) 19(3) 34(3) 15(2) -7(2) 1(2) -3(2) C(10) 18(3) 34(3) 26(3) -7(2) 5(2) 5(2) C(11) 14(3) 37(3) 24(2) 0(2) -2(2) -2(2) C(12) 17(3) 48(3) 32(3) 6(2) 2(2) -2(2) C(13) 28(3) 27(2) 27(2) -3(2) 1(2) -2(2)

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218 Appendix B: (Continued) C(14) 23(3) 32(3) 20(2) -4(2) 6(2) -5(2) C(15) 15(3) 48(3) 31(3) 3(2) 1(2) 0(2) C(16) 26(3) 46(3) 33(3) -7(2) 5(2) -1(2) C(17) 17(3) 68(4) 28(3) 4(3) 3(2) -2(2) C(18) 30(3) 33(3) 32(3) 7(2) -1(2) 4(2) C(19) 18(3) 97(5) 29(3) 19(3) 1(2) -9(3) C(20) 16(3) 32(3) 43(3) -6(2) -5(2) 0(2) C(21) 17(3) 62(4) 36(3) -19(3) 0(2) 1(2) C(22) 23(3) 35(3) 25(2) -7(2) 2(2) 4(2) C(23) 34(3) 31(3) 40(3) 5(2) 6(2) 4(2) C(24) 51(4) 26(2) 32(3) -8(2) -6(2) 9(2) C(25) 30(3) 21(2) 28(3) 0(2) -4(2) 2(2) C(26) 26(3) 39(3) 33(3) 9(2) 3(2) 2(2) C(27) 40(3) 22(2) 40(3) 3(2) 0(2) 3(2) C(28) 31(3) 115(6) 23(3) -23(3) 10(2) -8(3) C(29) 35(3) 25(2) 35(3) 2(2) 8(2) 4(2) C(30) 73(5) 47(3) 34(3) 8(3) -33(3) -14(3) C(31) 53(4) 30(3) 48(3) -3(2) 16(3) 10(3) C(32) 34(3) 43(3) 74(4) -3(3) -8(3) -3(3) C(33) 41(4) 79(4) 41(3) -27(3) 10(3) -7(3) C(34) 33(3) 40(3) 72(4) -10(3) 4(3) 10(3) C(35) 54(4) 36(3) 46(3) -3(2) 1(3) -6(3) C(36) 53(4) 31(3) 29(3) -8(2) -10(2) 20(2) C(37) 88(5) 43(3) 24(3) -10(2) -11(3) -4(3) C(38) 29(3) 46(3) 51(4) 14(3) -19(3) -5(3) C(39) 19(3) 53(3) 36(3) -1(2) 0(2) 1(2) C(40) 19(3) 64(3) 35(3) 5(2) 6(2) 5(2) ________________________________________________________________

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219 Appendix B: (Continued) Table 5. Hydrogen coordinates ( x 10 4 ) and isotropic displacement parameters ( 2 x 10 3 ) for mm011. ________________________________________________________________________ x y z U(eq) ________________________________________________________________________ H(1A) 915 2320 4445 22 H(2A) 663 3394 2452 25 H(3A) 8670 3655 1266 28 H(4D) 8460 2697 -795 28 H(4A) 9131 4078 -901 49 H(4B) 8968 4695 -190 49 H(4C) 8121 4209 -667 49 H(11A) 5912 2932 1092 31 H(11B) 5842 3923 961 31 H(12A) 3443 2839 2553 39 H(12B) 3443 3796 2832 39 H(13A) 873 1436 3264 41 H(13B) 40 1609 2581 41 H(13C) -157 1432 3473 41 H(15) 5667 2353 2350 38 H(16) 5960 4840 2156 42 H(17) 4107 4343 -326 45 H(18) 2432 3434 6207 38 H(19) 5310 2532 3657 58 H(20) 3659 2504 1206 38 H(21) 4053 2922 -26 46 H(22) 3360 4903 1811 33 H(23) 9148 -888 -749 42 H(24) 529 4881 7063 45 H(26) 3750 5323 583 39 H(27A) 8228 419 380 41 H(27B) 9193 334 34 41 H(28) 5247 3890 4166 67

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220 Appendix B: (Continued) H(30) 2669 3965 8547 66 H(31) 8468 -1717 -1791 51 H(32) 6260 -213 -2048 62 H(33) 5608 5019 3426 64 H(34) 6919 587 -980 58 H(35) 7055 -1348 -2478 55 H(36A) 223 4376 5727 47 H(36B) 1133 4728 5410 47 H(37) 1331 4759 8345 64 H(38) 3217 3332 7477 53 H(39A) 11625 3778 463 54 H(39B) 11790 3101 -198 54 H(39C) 11597 2806 668 54 H(40A) -1974 3841 3569 59 H(40B) -2424 3059 3958 59 H(40C) -2389 3088 3018 59 __________________________________________________________________

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221 Appendix B: (Continued) Table 6. Torsion angles [] for mm011. ________________________________________________________________ C(40)-O(11)-C(2)-O(5) -9.8(6) C(40)-O(11)-C(2)-C(3) 174.2(3) C(8)-N(1)-C(3)-N(2) 72.6(5) C(8)-N(1)-C(3)-C(13) -163.7(4) C(8)-N(1)-C(3)-C(2) -44.5(5) C(7)-N(2)-C(3)-N(1) 52.9(5) C(7)-N(2)-C(3)-C(13) -69.2(4) C(7)-N(2)-C(3)-C(2) 173.2(3) O(5)-C(2)-C(3)-N(1) 138.3(4) O(11)-C(2)-C(3)-N(1) -45.6(4) O(5)-C(2)-C(3)-N(2) 17.0(5) O(11)-C(2)-C(3)-N(2) -166.9(3) O(5)-C(2)-C(3)-C(13) -102.5(4) O(11)-C(2)-C(3)-C(13) 73.6(4) C(11)-O(6)-C(6)-O(7) -0.2(5) C(11)-O(6)-C(6)-N(3) 179.4(3) C(9)-N(3)-C(6)-O(7) -6.2(6) C(9)-N(3)-C(6)-O(6) 174.1(3) C(12)-O(1)-C(7)-O(2) -8.4(5) C(12)-O(1)-C(7)-N(2) 172.5(3) C(3)-N(2)-C(7)-O(2) 0.4(6) C(3)-N(2)-C(7)-O(1) 179.4(3) C(3)-N(1)-C(8)-O(4) 6.3(6) C(3)-N(1)-C(8)-O(3) -174.8(3) C(36)-O(3)-C(8)-O(4) 0.7(6) C(36)-O(3)-C(8)-N(1) -178.3(4) C(10)-N(4)-C(9)-N(3) -64.2(5) C(10)-N(4)-C(9)-C(14) 54.9(5) C(10)-N(4)-C(9)-C(4) 172.7(4) C(6)-N(3)-C(9)-N(4) -52.5(5) C(6)-N(3)-C(9)-C(14) -174.6(3) C(6)-N(3)-C(9)-C(4) 68.9(5)

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222 Appendix B: (Continued) C(9)-N(4)-C(10)-O(8) -13.1(7) C(9)-N(4)-C(10)-O(9) 167.7(4) C(27)-O(9)-C(10)-O(8) 1.8(7) C(27)-O(9)-C(10)-N(4) -179.0(4) C(6)-O(6)-C(11)-C(5) -176.8(3) C(16)-C(5)-C(11)-O(6) 80.5(5) C(15)-C(5)-C(11)-O(6) -100.3(4) C(7)-O(1)-C(12)-C(1) 160.1(3) C(22)-C(1)-C(12)-O(1) 83.6(5) C(20)-C(1)-C(12)-O(1) -96.3(5) C(39)-O(12)-C(14)-O(10) 4.9(6) C(39)-O(12)-C(14)-C(9) 178.5(3) N(4)-C(9)-C(14)-O(10) -141.7(4) N(3)-C(9)-C(14)-O(10) -19.3(6) C(4)-C(9)-C(14)-O(10) 99.9(5) N(4)-C(9)-C(14)-O(12) 44.6(5) N(3)-C(9)-C(14)-O(12) 167.0(3) C(4)-C(9)-C(14)-O(12) -73.8(4) C(16)-C(5)-C(15)-C(19) -1.9(6) C(11)-C(5)-C(15)-C(19) 178.9(4) C(15)-C(5)-C(16)-C(33) 1.8(7) C(11)-C(5)-C(16)-C(33) -179.0(4) C(5)-C(15)-C(19)-C(28) 1.8(7) C(22)-C(1)-C(20)-C(21) -0.6(6) C(12)-C(1)-C(20)-C(21) 179.2(4) C(1)-C(20)-C(21)-C(17) 0.5(6) C(26)-C(17)-C(21)-C(20) -0.4(7) C(20)-C(1)-C(22)-C(26) 0.7(6) C(12)-C(1)-C(22)-C(26) -179.2(4) C(37)-C(24)-C(25)-C(18) -1.2(7) C(37)-C(24)-C(25)-C(36) -178.9(5) C(38)-C(18)-C(25)-C(24) 0.9(7) C(38)-C(18)-C(25)-C(36) 178.5(4) C(21)-C(17)-C(26)-C(22) 0.5(7)

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223 Appendix B: (Continued) C(1)-C(22)-C(26)-C(17) -0.6(7) C(10)-O(9)-C(27)-C(29) -173.9(4) C(15)-C(19)-C(28)-C(33) -1.5(7) C(31)-C(23)-C(29)-C(34) -0.5(7) C(31)-C(23)-C(29)-C(27) -177.7(4) O(9)-C(27)-C(29)-C(34) -47.8(6) O(9)-C(27)-C(29)-C(23) 129.3(4) C(29)-C(23)-C(31)-C(35) 2.2(7) C(5)-C(16)-C(33)-C(28) -1.6(8) C(19)-C(28)-C(33)-C(16) 1.4(8) C(23)-C(29)-C(34)-C(32) -0.7(8) C(27)-C(29)-C(34)-C(32) 176.5(5) C(35)-C(32)-C(34)-C(29) 0.1(8) C(34)-C(32)-C(35)-C(31) 1.6(8) C(23)-C(31)-C(35)-C(32) -2.8(8) C(8)-O(3)-C(36)-C(25) -178.2(4) C(24)-C(25)-C(36)-O(3) -146.9(4) C(18)-C(25)-C(36)-O(3) 35.5(6) C(38)-C(30)-C(37)-C(24) 0.9(8) C(25)-C(24)-C(37)-C(30) 0.3(8) C(37)-C(30)-C(38)-C(18) -1.2(8) C(25)-C(18)-C(38)-C(30) 0.3(7) ___________________________________________________________ Symmetry transformations used to generate equivalent atoms:

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Appendix B: (Continued) Crystal structure of N-Boc-Triphenyliminophosphorane (102). 224

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Appendix B: (Continued) Crystallographic data for compound 102 225

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Appendix B: (Continued) 226

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Appendix B: (Continued) Crystal structure of t-Butyl 2,2,2-Trichloro-1-hydroxy-ethylcarbamate (105). 233

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Appendix B: (Continued) Crystallographic data of t-Butyl 2,2,2-Trichloro-1-hydroxy-ethylcarbamate (105) 234

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Appendix B: (Continued) 235

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About the Author Kiran Avancha received his bachelors in Pharmacy from Osmania University, Hyderabad, India in 2001. He was a register ed pharmacist in India. He started his graduate study in the department of chemistry at University of South Florida in fall 2001. He joined Prof. Mark McLaughlin and the Drug discovery team at H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL in 2003 and continued his research in synthetic organic and medicinal chemistry to receiv e a doctoral degree in Chemistry in spring 2006.