PHENANTHROPIPERIDINE ALKALOIDS: METHODOLOGY DEVELOPMENT, SYNTHESIS AND BIOLOGICAL EVALUATION BY ©2010 Micah James Niphakis Submitted to the graduate degree program in Medicinal Chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ____________________________________ Chair Committee:____________________________________ ____________________________________ ____________________________________ ____________________________________ Date defended:_______________________ This Dissertation Committee for Micah J. Niphakis certifies that this is the approved version of the following dissertation: PHENANTHROPIPERIDINE ALKALOIDS: METHODOLOGY DEVELOPMENT, SYNTHESIS AND BIOLOGICAL EVALUATION ____________________________________ Chair Committee:____________________________________ ____________________________________ ____________________________________ ____________________________________ Date defended:_______________________ 1 This work is dedicated to my parents, Stavros and Holly. 2 ACKNOWLEDGEMENTS The past five years of research would have been impossible without the gracious support my mentors, coworkers, friends and family. Firstly, I wish to thank Professor Gunda Georg who has been an exemplary doctoral research advisor and has introduced me to the multifaceted nature of Medicinal Chemistry. She has been a constant source of guidance and encouragement in the midst of my blunders and struggles to become an independent scientist and I am immeasurably grateful. I am also greatly indebted to the faculty of the department of Medicinal Chemistry and Chemistry at the University of Kansas who have indelibly shaped my understanding of the molecular world and kindled a sense of wonder that has been the impetus of my own research. Professor Jeffrey Aubé, in particular, has been an outstanding model of an academic scientist and has provided invaluable guidance and support for my scientific career. Professors Gunda Georg, Jeffrey Aubé, Gary Grunewald and Paul Hanson are especially thanked for their strong letters of support which have played a pivotal role in fellowship and post-doctoral applications. I also thank Professors Georg, Aubé, Hanson, Carlson and Dutta for graciously serving as members of my oral examination committee. The majority of my graduate career has been spent at the University of Minnesota (UMN) where I have greatly benefited from the support of the faculty and coworkers in the Institute for Therapeutics Discovery and Development (ITDD). As such, I must thank the department of medicinal chemistry at the UMN for infusing me into their program despite my foreign status. Specifically, I wish to thank Dr. Michael Walters, who has become indispensable to 3 the Georg group, for many enlightening discussions and for guidance in my research. I also thank Dr. Derek Hook, Dr. Jonathan Solberg and Dr. Harry Tian for carrying out cytotoxicity and metabolic studies on short notice. My work has been supported by the service of the National Institute of Health-Psychoactive Drug Screening Program under Director Bryan Roth, M.D., Ph.D. whom I thank. Victor G. Young, Jr. of the University of Minnesota X-Ray Crystallographic Laboratory is thanked for determining the X-ray crystal structure of compound 2.38. Thanks is given to financial supporters: National Institutes of Health (Grants CA90602, GM069663, GM076302 and GM081267), the Kansas Masonic Cancer Research Institute, the University of Minnesota through the Vince and McKnight Endowed Chairs and ACS Division of Medicinal Chemistry (Predoctoral fellowship). The Georg group and my fellow graduate students have been vital for supporting my graduate studies as well. Those who have made a substantial impact include Brandon Turunen and Haibo Ge whom I thank for their foundational work that made much of my research possible. I owe much thanks to Matthew Leighty, Oliver Hutt and Keith Ellis for their mentorship and advice, right my the start of my graduate studies. Also, I am grateful to Andrea Knickerbocker for her extensive and essential role in the Georg group and for her friendship. My parents deserve my utmost thanks. They have made immense sacrifices to provide an education for me and have shown unrivaled support throughout my life. Last, and certainly not least, I wish to thank my wife and best friend, Easten, whose love, patience and support has been my sustenance through the most difficult times of my graduate career. 4 ABSTRACT Phenanthropiperidine Alkaloids: Methodology Development, Synthesis and Biological Evaluation Micah J. Niphakis, Ph. D. The University of Kansas, 2010 The phenanthropiperidines are remarkably potent anti-proliferative alkaloids. Only one member of this class has been tested in the clinic, but the trials were terminated shortly thereafter due to adverse neurological side-effects. This work is directed towards the development of safe phenanthropiperidines for the treatment of cancer. It focuses on synthetic methodologies that facilitate their preparation and biological studies to better understand their neurological effects. The preparation of cyclic enaminones in high enantiomeric purity is made possible through a one-flask, two-step protocol that uses mild Boc-deprotection conditions to suppress racemization. This reaction enables the preparation of monocyclic and bicyclic, six- and seven-membered enaminones, some of which can be used as precursors to phenanthropiperidine alkaloids. Elaboration of the enaminone scaffold was made possible with a direct palladium(II)- catalyzed arylation using aryltrifluoroborates as coupling partners. Combined with the enaminone methodology this approach gives rapid access to enantiomerically pure 3- 5 arylpiperidines. The utility of these scaffolds were demonstrated in the synthesis of two natural products: (+)-ipalbidine and (+)-antofine. A short and high-yielding synthetic route has also been devised to provide tylocrebrine, the only phenanthropiperidine to enter clinical trials. We report the first enantioselective synthesis of tylocrebrine which uses a novel aryl-alkene oxidative coupling reaction that surmounts the historical challenge of obtaining a single phenanthrene regioisomer. A small collection of tylocrebrine analogs were prepared and studied along with tylocrebrine to elucidate the cause of tylocrebrine’s neurological side-effects. Although the causes remain elusive, tylocrebrine and several of its analogs were found to bind to key biogenic amine receptors, disclosing another potential risk factor for their therapeutic use. The phenanthrene substitution pattern, however, had an marked influence on binding and could be used to minimize these off-target interactions. 6 TABLE OF CONTENTS .................................................................................................................List of Figures 12 ...............................................................................................List of Tables and Graphs 15 ..........................................................................................................List of Compounds 18 ..................................................................................................................Abbreviations 26 CHAPTER 1 INTRODUCTION TO PHENANTHROPIPERIDINE ALKALOIDS .....................................................................................................................1.1 Introduction 31 .....................................................................................................................1.2 Background 32 .........1.2.1 Discovery of tylophorine, tylophorinine, cryptopleurine and tylocrebrine 32 .......................................1.2.2 Phenanthropiperidine isolation and structural features 35 ........................................................................................................1.2.4 Biosynthesis 38 ...........................................................................................1.3 Biology of Tylophora indica 41 ....................................................................................................1.3.1 Clinical studies 41 ...........................................................1.3.2 In vitro and in vivo studies with T. Indica 44 .....................................................................1.4 Biology of phenanthropiperidine alkaloids 47 .............................................................................................1.4.1 Anti-cancer activity 48 .........................................................................................1.4.1.1 In vivo studies 48 .........................................................................................1.4.1.2 In vitro studies 50 ...............................................................1.4.1.3 Structure-activity relationships 52 ...........................................................................................1.4.2 Mechanism of action 61 7 .....................................1.4.2.1 Protein, DNA and RNA biosynthesis inhibition 61 ................................................................1.4.2.2 Apoptosis and cell cycle arrest 67 ...........................................................................................1.4.2.3 Angiogenesis 70 ..................................................................................1.4.2.4 Cell differentiation 70 ......................................................................1.4.2.5 NF-κB Signaling pathway 71 ........................................................................................1.4.2.6 DNA and RNA 72 ...........................................................................1.4.2.7 Other molecular targets 73 .................................................................................1.4.3 Anti-inflammatory activity 74 ........................................................................................1.4.4 Miscellaneous activity 75 ..........................................................................1.4.5 Summary of biological activity 76 ...........................................................................................1.5 Obstacles to clinical success 77 ..................................................................1.6 Synthesis of phenanthropiperidine alkaloids 80 ........................................1.6.1 First synthetic efforts towards phenanthropiperidines 81 .......................................................................................1.6.2 Phenanthrene synthesis 83 .............................................................1.6.3. Indolizidine and quinolizidine synthesis 90 ...............................................................1.6.3.1 C3–C4 bond forming reactions 91 ...............................................................1.6.3.2 C2–C3 bond forming reactions 95 ...............................................................1.6.3.3 C4–C5 bond forming reactions 98 ...............................................................1.6.3.4 N–C2 bond forming reactions 102 .......................................................1.6.3.5 N–C8(C9) bond forming reactions 103 ...........................................................1.6.3.6 C5a–C6 bond forming reactions 106 ....................................................................1.6.3.7 Angular OH or Me groups 107 ................................................................................................1.6.4 Closing remarks 108 .......................................................................................................................1.7 Summary 109 8 CHAPTER 2 SYNTHESIS OF SIX- AND SEVEN-MEMBERED CYCLIC ENAMINONES: OPTIMIZATION, SCOPE AND MECHANISTIC STUDIES ...................................................................................................................2.1 Introduction 111 ...................................................................................................................2.2 Background 112 ...................................................................................................2.3 Reaction development 116 ................................2.3.1 Strategy and considerations for enaminone methodology 116 ......................................2.3.2 Synthesis of Weinreb amide and ynone intermediates 119 ..............................................................2.3.3 Optimization of enaminone formation 122 .....................2.3.4 Mitigating stereochemical damage during enaminone formation 124 ..............................................................................................................2.3 Reaction scope 132 ...............................................................................2.3.1 Six-membered enaminones 132 ...........................................................2.3.2 Seven- and five-membered enaminones 136 ...............................................................................2.3.3 Summary of reaction scope 137 ......................................................................................................2.4 Mechanistic insights 138 .......................................................................................................................2.5 Summary 142 CHAPTER 3 PALLADIUM(II)-CATALYZED CROSS-COUPLING OF CYCLIC ENAMINONES AND ORGANOTRIFLUOROBORATES ...................................................................................................................3.1 Introduction 144 .................................................................3.2 Background to Pd-catalyzed cross-coupling 145 ..........................................................................................................3.3 Synthetic strategy 148 .....................................................................3.4 Activation and classical Suzuki approach 150 ..................................................................................................3.5 C–H Functionalization 153 ...................................................................................................3.6 Reaction optimization 161 9 ...............................................................................................3.6.1 Reoxidant screen 161 ..................................................................................3.6.2 Palladium-catalyst screen 165 ...................................................................................................3.6.3 Solvent screen 166 ..................................................................................................3.6.4 Additive screen 167 ...............................................................................................3.7 Scope of trifluoroborates 170 ....................................................................................................3.8 Scope of enaminones 174 ........................................................................3.9 Extensions and directions for the future 176 .......................................................................................................................3.10 Summary 179 CHAPTER 4 SYNTHESIS OF (+)-ANTOFINE AND (+)-IPALBIDINE ...................................................................................................................4.1 Introduction 180 ...................................................................................4.2 Total synthesis of (+)-ipalbidine 182 .........................................................4.2.1 First attempt to synthesize (+)-ipalbidine 182 ....................................................................4.2.2 Second approach to (+)-ipalbidine 185 .......................................................................4.2.3 Third approach to (+)-ipalbidine 188 ......................................................................................4.3 Total synthesis of (+)-antofine 189 .......................................................................................................................4.4 Summary 190 CHAPTER 5 SYNTHESIS AN BIOLOGICAL EVALUATION OF (R)-TYLOCREBRINE AND RELATED ANALOGS ...................................................................................................................5.1 Introduction 192 ..............................................................5.2 Possible mechanisms for neurological effects 193 ...........................................................................5.3 Synthesis of tylocrebrine and analogs 196 .................................................5.4 Biological studies of tylocrebrine and related analogs 207 ...............................................................................................................5.4.1 Cancer 207 10 ................................................................5.4.2 Neurological side-effects hypothesis 209 ...............................................................................5.4.2.1 MPTP-like toxicity 209 ...............................................5.4.2.2 Dopaminergic and other CNS receptors 215 .......................................................................................................................5.5 Summary 221 CHAPTER 6 EXPERIMENTAL DATA ..................................................................................................6.1 Materials and methods 223 .......................................................................................................................6.2 Chapter 2 224 ...........................................................................6.3.1 Preparation of Weinreb amides: 224 ..........................................................................................6.3.2 Preparation of ynones: 241 ........................................................................6.3.3 Preparation of cyclic enaminones 250 .........................................................................................6.3.4 Supplementary studies 264 .......................................................................................................................6.3 Chapter 3 276 .......................................................................6.3.1 Preparation of starting materials 276 ......................................................6.3.2 Pd(II)-catalyzed C3-arylation of enaminones 278 .......................................................................................................................6.4 Chapter 4 292 .......................................................................................................................6.5 Chapter 5 307 ...........................................................6.5.1 Preparation of tylocrebrine and analogs 307 .......................................................................................6.5.2 Biological procedures 330 .........................................................................6.5.2.1 Anti-proliferation assay 330 .............................................................................................6.5.2.2 MOA assay 330 .................................................................................................6.6 X-Ray crystal structure 332 BIBLIOGRAPHY...................................................................................................................... 346 11 LIST OF FIGURES CHAPTER 1 Fig. 1–1. Structure of first discovered phenanthropiperidines. 34 Fig. 1–2. General structural features of the phenanthropiperidines. 36 Fig. 1–3. Under- and over-oxidized scaffolds. 37 Fig. 1–4. Unusual structures of tyloindicines F–I. 38 Fig. 1–5. Structures of several NCI-tested phenanthropiperidines. 49 Fig. 1–6. Scaffolds of phenanthropiperidines and analogs. 55 Fig. 1–7. Structure of PBT-1. 59 Fig. 1–8. Structure of emetine. 64 Fig. 1–9. Structures of tylophorinidine and boehmeriasin A. 69 Fig. 1–10. Antofine’s affinity for DNA structures. 73 Fig. 1–11. Key-step bond formations. 91 CHAPTER 2 Fig. 2–1. Generic structure of the enaminone. 112 Fig. 2–2. Select synthetic transformations of the 6-membered cyclic enaminone. 114 Fig. 2–3. Possible modes of addition for 6-endo-dig cyclization. 117 Fig. 2–4. Potential modes of α- and β-racemization/epimerization. 118 Fig. 2–5. X-ray crystal structure of vinyl iodide 2.38. 128 12 Fig. 2–6. Two potential mechanistic pathways of cyclization. 139 CHAPTER 3 Fig. 3–1. Examples of 3-aryl piperidine medicinal agents. 144 Fig. 3–2. Applications of Pd chemistry. 146 Fig. 3–3. Postulated general cross-coupling catalytic cycle. 147 Fig. 3–4. Postulated Heck-Mizokori catalytic cycle. 148 Fig. 3–5. Plausible mechanisms for Pd-catalyzed C–H functionalization reactions. 154 Fig. 3–6. Directing group-assisted C–H palladation via electrophilic substitution. 155 Fig. 3–7. Reactivity-assisted C–H palladation. 157 Fig. 3–8. C3/C2 Pd-migration of palladated indoles. 159 Fig. 3–9. C3/C2 palladium migration of palladated enaminones. 160 Fig. 3–10. Homocoupling product of trifluoroborate 3.3. 164 Fig. 3–11. Proposed catalytic cycle for direct-arylation of enaminones. 164 Fig. 3–12. Mechanism of Pd(II)-catalyzed olefination. 177 CHAPTER 4 Fig. 4–1. Structures of (+)-ipalbidine and (+)-antofine. 181 13 CHAPTER 5 Fig. 5–1. Promising sites for derivatization. 195 Fig. 5–2. Mechanism for MPTP-induced toxicity. 210 Fig. 5–3. Structure of MPTP, MPP+ and dehydrotylocrebrine. 211 Fig. 5–4. Structurally related molecules of interest. 215 14 LIST OF TABLES AND GRAPHS CHAPTER 1 Table 1–1. Clinical studies with T. indica for the treatment of asthma. 43 Table 1–2. In vivo and in vitro studies with T. indica extracts. 45 Table 1–3. Best NCI in vivo results for L1210 leukemia model. 49 Table 1–4. Growth inhibitory activity for antofine and NCI tested phenanthropiperidines. 51 Table 1–5. Growth inhibitory activity in drug resistance cell lines. 52 Table 1–6. Structure and average GI 50 over select cell lines. 53 Table 1–7. Inhibition of protein, DNA and RNA synthesis. 63 Table 1–8. Apoptosis and cell cycle arrest by phenanthropiperidine alkaloids. 68 Table 1–9. CNS drugs and tylocrebrine’s physicochemical properties. 79 Graph 1–1. Anti-proliferative activity of phenanthropiperidines in fourteen select cell lines. 54 CHAPTER 2 Table 2–1. Preparation of ynones from Weinreb amides. 121 Table 2–2. Optimization of deprotection and cyclization. 123 Table 2–3. Boc-deprotection and β-epimerization. 125 Table 2–4. Substrate scope for the preparation of six-membered enaminones. 134 Table 2–5. Seven-membered enaminone preparation. 137 15 CHAPTER 3 Table 3–1. Suzuki cross-coupling conditions of iodoenaminone. 152 Table 3–2. Screen of reoxidants. 162 Table 3–3. Screen of palladium catalysts. 166 Table 3–4. Screen of solvents. 167 Table 3–5. Screen of additives. 168 Table 3–6. Successfully coupled aryltrifluoroborates. 171 Table 3–7. Aryltrifluoroborates that did not couple. 172 Table 3–8. Successfully coupled of enaminones. 175 Table 3–9. Enaminones that did not couple. 176 CHAPTER 4 Table 4–1. Optimization of dehydration conditions. 187 CHAPTER 5 Table 5–1. Synthesis of biaryl fragment. 202 Table 5–2. Oxidative aryl-alkene coupling optimization. 205 Table 5–3. Final two steps to prepare (±)-tylocrebrine and its analogs. 207 16 Table 5–4. Cytotoxicity of phenanthroindolizidines. 208 Table 5–5. Phenanthropiperidine affinity for monoamine transporters. 213 Table 5–6. Comprehensive CNS target primary screen from NIHM-PDSP. 217 Table 5–7. Affinity for phenanthropiperidine for biogenic amine receptors. 218 Table 5–8. Tylocrebrine’s and dehydrotylocrebrine’s affinity for muscarinic receptors. 220 Graph 5–1. Phenanthropiperidines as a substrate for MAO-A and MAO-B. 212 17 LIST OF COMPOUNDS CHAPTER 2 (S)-tert-Butyl 2-(2-(Methoxy(methyl)amino)-2-oxoethyl)piperidine-1- carboxylate (2.1) 121 (S)-tert-Butyl 2-(2-(Methoxy(methyl)amino)-2-oxoethyl)pyrrolidine-1- carboxylate (2.2) 121 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-(methoxy(methyl)amino)-2- oxoethyl)-pyrrolidine-1-carboxylate (2.3) 121 (2S*,4R*)-tert-Butyl 4-Hydroxy-2-(2-(methoxy(methyl)amino)-2-oxoethyl) pyrrolidine-1-carboxylate (2.4) 121 (2S*,4S*)-tert-Butyl 4-Hydroxy-2-(2-(methoxy(methyl)amino)-2-oxoethyl) pyrrolidine-1-carboxylate (2.5) 121 tert-Butyl 3-(2-(Methoxy(methyl)amino)-2-oxoethyl)morpholine-4-carboxylate (2.6) 121 tert-Butyl (cis)-2-(Methoxy(methyl)carbamoyl)cyclohexyl(methyl)carbamate (2.7) 121 tert-Butyl (trans)-2-(Methoxy(methyl)carbamoyl)cyclohexyl(methyl)carbamate (2.8) 121 (R)-tert-Butyl 3-(Methoxy(methyl)amino)-3-oxo-1-phenylpropylcarbamate (2.9) 121 (S)-tert-Butyl 4-(Methoxy(methyl)amino)-4-oxo-1-phenylbutan-2-ylcarbamate (2.10) 121 18 tert-Butyl 3-(Methoxy(methyl)amino)-3-oxopropylcarbamate (2.11) 121 tert-Butyl Benzyl(3-(methoxy(methyl)amino)-3-oxopropyl)carbamate (2.12) 121 tert-Butyl 3-(Methoxy(methyl)amino)-3-oxopropyl(phenyl)carbamate (2.13) 121 tert-Butyl 2-(3-(Methoxy(methyl)amino)-3-oxopropyl)pyrrolidine-1- carboxylate 121 tert-Butyl 2-(3-(Methoxy(methyl)amino)-3-oxopropyl)piperidine-1-carboxylate 121 tert-Butyl Benzyl ((1 - (2- (methoxy (methyl)amino) 2 - oxoethyl) cyclohexyl) methyl) carbamate 121 tert-Butyl Benzyl(2-(4-chlorophenyl)–4-(methoxy (methyl) amino)-4-oxobutyl) carbamate 121 (S)-tert-Butyl 2-(2-Oxobut-3-yn-1-yl)piperidine-1-carboxylate (2.14) 121 (S)-tert-Butyl 2-(2-Oxopent-3-yn-1-yl)piperidine-1-carboxylate (2.15) 121 (S)-tert-Butyl 2-(2-Oxo-4-phenylbut-3-yn-1-yl)piperidine-1-carboxylate (2.16) 121 (S)-tert-Butyl 2-(2-Oxobut-3-yn-1-yl)pyrrolidine-1-carboxylate (2.17) 121 (S)-tert-Butyl 2-(2-Oxopent-3-yn-1-yl)pyrrolidine-1-carboxylate (2.18) 121 (S)-tert-Butyl 2-(2-Oxo-4-phenylbut-3-yn-1-yl)pyrrolidine-1-carboxylate (2.19) 121 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-oxobut-3-ynyl)pyrrolidine-1- carboxylate (2.20) 121 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-oxopent-3-ynyl)pyrrolidine-1- carboxylate (2.21) 121 19 (2S*,4R*)- tert-Butyl 4-(Benzyloxy)-2-(2-oxo-4-phenylbut-3- ynyl)pyrrolidine-1-carboxylate (2.22) 121 (2S*,4R*)-tert-Butyl 4-Hydroxy-2-(2-oxobut-3-yn-1-yl)pyrrolidine-1- carboxylate (2.23) 121 (2S*,4S*)-tert-Butyl 4-Hydroxy-2-(2-oxobut-3-yn-1-yl)pyrrolidine-1- carboxylate (2.24) 121 tert-Butyl 3-(2-Oxobut-3-ynyl)morpholine-4-carboxylate (2.25) 121 tert-Butyl Methyl((1R*, 2S*)-2-propioloylcyclohexyl)carbamate (2.26) 121 tert-Butyl Methyl((1R*, 2R*)-2-propioloylcyclohexyl)carbamate (2.27) 121 (R)-tert-Butyl 3-Oxo-1-phenylpent-4-ynylcarbamate (2.28) 121 (S)-tert-Butyl (4-Oxo-1-phenylhex-5-yn-2-yl)carbamate (2.29) 121 (S)-tert-Butyl (4-Oxo-1-phenylhept-5-yn-2-yl)carbamate (2.30) 121 tert-Butyl 3-Oxopent-4-ynylcarbamate (2.31) 121 tert-Butyl Benzyl(3-oxopent-4-ynyl)carbamate (2.32) 121 tert-Butyl 3-Oxopent-4-ynyl(phenyl)carbamate (2.33) 121 tert-Butyl 2-(3-Oxopent-4-ynyl)pyrrolidine-1-carboxylate (2.57) 137 tert-Butyl 2-(3-Oxopent-4-ynyl)piperidine-1-carboxylate (2.58) 137 tert-Butyl Benzyl((1-(2-oxobut-3-ynyl)cyclohexyl)methyl)carbamate (2.59) 137 tert-Butyl Benzyl(2-(4-chlorophenyl)-4-oxohex-5-ynyl)carbamate (2.60) 137 (S)-7,8,9,9a-Tetrahydro-1H-quinolizin-2(6H)-one (2.34) 123 (S)-4-Methyl-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (2.46) 134 (S)-4-Phenyl-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (2.47) 145 20 (S)-2,3,8,8a-Tetrahydroindolizin-7(1H)-one (3d) (2.37) 134 (S)-5-Methyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.39) 134 (S)-5-Phenyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.40) 134 (2R*,8aS*)-2-(Benzyloxy)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.41) 134 (2R*,8aS*)-2-(Benzyloxy)-5-methyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (anti-2.42) 134 (2R*,8aR*)-2-(Benzyloxy)-5-methyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (syn-2.42) 134 (2R*,8aS*)-2-(Benzyloxy)-5-phenyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (anti-2.43) 134 (2R*,8aR*)-2-(Benzyloxy)-5-phenyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (syn-2.43) 134 (2R*,8aS*)-2-Hydroxy-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.35) 134 (2S*,8aS*)-2-Hydroxy-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.48) 134 3,4,9,9a-Tetrahydropyrido[2,1-c][1,4]oxazin-8(1H)-one (2.49) 134 (4aS*,8aR*)-1-Methyl-4a,5,6,7,8,8a-hexahydroquinolin-4(1H)-one (2.36) 134 (4aR*,8aR*)-1-Methyl-4a,5,6,7,8,8a-hexahydroquinolin-4(1H)-one (2.50) 134 (R)-2-Phenyl-2,3-dihydropyridin-4(1H)-one (2.51) 134 (S)-2-Benzyl-2,3-dihydropyridin-4(1H)-one (2.52) 134 (S)-2-Benzyl-6-methyl-2,3-dihydropyridin-4(1H)-one (2.53) 134 2,3-Dihydropyridin-4(1H)-one (2.54) 134 1-Benzyl-2,3-dihydropyridin-4(1H)-one (2.55) 134 1-Phenyl-2,3-dihydropyridin-4(1H)-one (2.56) 134 21 (Z)-2,3,9,9a-Tetrahydro-1H-pyrrolo[1,2-a]azepin-7(8H)-one (2.61) 137 (Z)-1,2,3,4,10,10a-Hexahydropyrido[1,2-a]azepin-8(9H)-one (2.62) 137 8-Benzyl-8-azaspiro[5.6]dodec-9-en-11-one (2.63) 137 (Z)-1-Benzyl-6-(4-chlorophenyl)-6,7-dihydro-1H-azepin-4(5H)-one (2.64) 137 (E)-2-(4-Iodo-2-oxobut-3-enyl)pyrrolidinium Iodide (2.38) 128 5-Phenylpent-1-yn-3-one (2.65) 141 (E)-1-Chloro-5-phenylpent-1-en-3-one (2.66) 141 1,1-Dichloro-5-phenylpentan-3-one (2.67) 141 (E)-1-Methoxy-5-phenylpent-1-en-3-one (2.68) 141 CHAPTER 3 (S)-6-Iodo-2,3,8,8a-Tetrahydroindolizin-7(1H)-one (3.1) 151 (S)-6-(4-Methoxyphenyl)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (3.2) 152 4,4'-Dimethoxy-1,1'-biphenyl (3.5) 164 1-Methyl-2,3-dihydropyridin-4(1H)-one (3.18) 175 1-Benzyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (3.19) 175 1-Benzyl-5-(4-methoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.4) 161 1-Benzyl-5-phenyl-2,3-dihydropyridin-4(1H)-one (3.6) 171 1-Benzyl-5-(4-tolyl)-2,3-dihydropyridin-4(1H)-one (3.7) 171 1-Benzyl-5-(2-tolyl)-2,3-dihydropyridin-4(1H)-one (3.8) 171 1-Benzyl-5-(2-methoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.9) 171 22 1-Benzyl-5-(4-(trifluoromethyl)phenyl)-2,3-dihydropyridin-4(1H)-one (3.10) 171 1-Benzyl-5-(4-acetylphenyl)-2,3-dihydropyridin-4(1H)-one (3.11) 171 1-Benzyl-5-(4-bromophenyl)-2,3-dihydropyridin-4(1H)-one (3.12) 171 1-Benzyl-5-(4-chlorophenyl)-2,3-dihydropyridin-4(1H)-one (3.13) 171 1-Benzyl-5-(naphthalen-2-yl)-2,3-dihydropyridin-4(1H)-one (3.14) 171 1-Benzyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.15) 171 1-Benzyl-5-(3-hydroxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.16) 171 1-Benzyl-5-(4-aminobenzyloxycarbonyl)-2,3-dihydropyridin-4(1H)-one (3.17) 171 3-(4-Methoxyphenyl)-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (3.20) 175 (cis)-3-(4-Methoxyphenyl)-1-methyl-4a,5,6,7,8,8a-hexahydroquinolin-4(1H)- one (3.21) 175 ( t r a n s ) - 3 - ( 4 - M e t h o x y p h e n y l ) - 1 - m e t h y l - 4 a , 5 , 6 , 7 , 8 , 8 a - hexahydroquinolin-4(1H)-one (3.22) 175 5-(4-Methoxyphenyl)-1-methyl-2,3-dihydropyridin-4(1H)-one (3.23) 175 1-Benzyl-5-(4-methoxyphenyl)-2-phenyl-2,3-dihydropyridin-4(1H)-one (3.24) 175 (E)-Methyl 3-(1-benzyl-4-oxo-1,4,5,6-tetrahydropyridin-3-yl)acrylate (3.25) 178 CHAPTER 4 23 (S)-tert-Butyl 2-(2-Diazoacetyl)pyrrolidine-1-carboxylate (4.3) 183 (S)-2-(1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)acetic acid (4.4) 183 (S)-tert-Butyl 2-(2-Methoxy-2-oxoethyl)pyrrolidine-1-carboxylate (4.5) 183 (S ) -6- (4-Methoxyphenyl) -1 ,2 ,3 ,5 ,8 ,8a-hexahydroindol iz in-7-yl - trifluoromethanesulfonate (4.6) 185 (S)-6-(4-Methoxyphenyl)-7-methyl-1,2,3,5,8,8a-hexahydroindolizine (4.7) 185 (6S,8aS)-6-(4-Methoxyphenyl)hexahydroindolizin-7(1H)-one (4.8) 186 (6S,7R,8aS)-6-(4-Methoxyphenyl)-7-methyloctahydroindolizin-7-ol (4.9) 186 (6S,8aS)-6-(4-Methoxyphenyl)-7-methyleneoctahydroindolizine (4.10) 186 (+)-(S)-Secoantofine (4.11) 190 (+)-(S)-Ipalbidine (4.1) 181 (+)-(S)-Antofine (4.2) 181 CHAPTER 5 Tylocrebrine (1.4) 34 7 - ( 2 ' , 3 ' , 4 , 5 - Te t r a m e t h o x y - [ 1 , 1 ' - b i p h e n y l ] - 2 - y l ) - 1 , 2 , 3 , 5 , 8 , 8 a - hexahydroindolizine (5.1) 198 2'-Bromo-2,3,4',5'-tetramethoxy-1,1'-biphenyl (5.2) 198 1-Bromo-2-iodo-4,5-dimethoxybenzene (5.3) 198 1,2,3,5,8,8a-Hexahydroindolizin-7-yl Trifluoromethanesulfonate (5.5) 198 24 tert-Butyl 2-Oxopyrrolidine-1-carboxylate (5.6) 198 tert-Butyl 2-Hydroxypyrrolidine-1-carboxylate (5.7) 200 tert-Butyl 2-(2-Ethoxy-2-oxoethyl)pyrrolidine-1-carboxylate (5.8) 200 2-Bromo-3',4,4',5-tetramethoxy-1,1'-biphenyl (5.9) 202 2-Bromo-2',4,5-trimethoxy-1,1'-biphenyl (5.10) 202 2-Bromo-3',4,5-trimethoxy-1,1'-biphenyl (5.11) 202 2-Bromo-4,4',5-trimethoxy-1,1'-biphenyl (5.12) 202 2-Bromo-3',4',5-trimethoxy-1,1'-biphenyl (5.13) 202 2,2'-Dibromo-4,4',5,5'-tetramethoxy-1,1'-biphenyl (5.15) 203 7-(2',4,5-Trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (5.17) 207 7-(3',4,5-Trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (5.18) 207 7-(4,4',5-Trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (5.19) 207 7 - ( 3 ' , 4 , 4 ' , 5 - Te t r a m e t h o x y - [ 1 , 1 ' - b i p h e n y l ] - 2 - y l ) - 1 , 2 , 3 , 5 , 8 , 8 a - hexahydroindolizine (5.20) 207 7-(3',4',5-Trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (5.21) 207 6-Desmethoxytylocrebrine (5.22) 207 6-Desmethoxytylophorine (5.23) 207 Tylophorine (1.1) 34 Deoxypergularinine (5.24) 207 25 ABBREVIATIONS Ac acetyl ACN azobis-cyclohexanecarbonitrile AFP alpha-fetoprotein AIBN azobisisobutyronitrile Ar aryl araC arayinoside cytosine BBB blood-brain barrier Bn benzyl Boc tert-butoxycarbonyl BQ benzoquinone br broad CAN ammonium cerium (IV) nitrate Cbz carbobenzyloxy CDK cyclin-dependent kinase CNS central nervous system Cp cyclopentadienyl CPT camptothecin d doublet d.r. diastereomeric ratio DA dopamine DAT dopamine active transporter dCK deoxycytidine kinase DIAD diisopropyl azodicarboxylate DIBAL-H diisobutylaluminium hydride DMAP 4-dimethylaminopyridine DME dimethoxyethane 26 DMF dimethylformamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DNFB dinitrofluorobenzene dppf 1,1'-bis(diphenylphosphino)ferrocene DTH delayed-type hypersensitivity e.r. enantiomeric ratio EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EtOAc ethyl acetate EtOH ethanol FEV 1 forced expiratory volume in 1 second fod 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate GPCR G-protein coupled receptor h hour HASPO 4,4,5,5-tetramethyl-1,3,2-dioxaphospholane 2-oxide HBD hydrogen bond donor HIF-1 hypoxia-inducible factor I HIR humoral immune response HPLC high performance liquid chromatography HRP horseradish peroxidase HU hydroxyurea i-Pr isopropyl i.p. intraperitoneal i.v. intravenous IR infrared K D dissociation constant LAH lithium aluminum hydride LDA lithium diisopropyl amide 27 LPS lipopolysaccharide m multiplet MAO monoamine oxidase MBC maximum breathing capacity MDR multidrug resistant Me methyl MeOH methanol μW microwave min minutes MPP+ 1-methyl-4-phenylpyridinium MPPP 1-methyl-4-phenyl-4-propionoxypiperidine MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MRP multidrug resistance protein Ms mesyl MW molecular weight NADH nicotinamide adenine dinucleotide NBS N-bromosuccinamide NCI National Cancer Institute NET norepinephrine transporter NMM N-methylmorpholine NMR nuclear magnetic resonance PDSP psychoactive drug screening program PEFR peak expiratory flow rate P-gp P-glycoprotein Ph phenyl PIFA (bis(trifluoroacetoxy)iodo)benzene PK pharmacokinetic PMP p-methoxyphenyl 28 PNS peripheral nervous system PPA polyphosphoric acid PSA polar surface area rac racemic RNA ribonucleic acid RR ribonucleotide reductase rt room temperature s-Bu sec-butyl S-Phos dicyclohexyl(2',6'-dimethoxy-[1,1'-biphenyl]-2-yl)phosphine SAR structure-activity relationship SERT serotonin transporter SN-38 7-ethyl-10-hydroxycamptothecin SRBC sheep red blood cells t triplet t-Bu tert-butyl TBAF tetra-N-butylammonium fluoride TBS tert-butyldimethylsilyl TEA triethylamine Tf trifluoromethanesulfonyl TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THF tetrahydrofuran TMS trimethylsilyl TMV tobacco mosaic virus Topo I topoisomerase I Topo II topoisomerase II TsOH toluenesulfonic acid VEGF vascular endothelial growth factor 29 VC vital capacity VP-16 etoposide XRCC1 X-ray repair cross-complementing gene I 30 CHAPTER 1 INTRODUCTION TO PHENANTHROPIPERIDINE ALKALOIDS 1.1 Introduction With the historical success of pharmacognosy, nature continues to be scoured for new drug leads. The discovery of a medicinally active natural product, however, is only the beginning of the arduous venture of drug development. Nature-derived drug leads often require considerable optimization before they are of any instrumental value and the phenanthropiperidines, the subject of this work, are a case in point. These alkaloids have been pursued as leads for the treatment of cancer and immune-related diseases for over 50 years yet not a single member has emerged for clinical use. Some have abandoned the effort, while others tenaciously pursue next generation phenanthropiperidines with hope that this class is still redeemable. The reasons that undergird both of these positions will be explored in the following pages. Like many bioactive natural products, the phenanthropiperidines were isolated from plants that had been used in folk medicine for untold centuries. 1 The reputed therapeutic value of Tylophora indica (asthmatica) spurred early scientific activity that sought to validate and characterize its biological effects. In spite of periodic lapses of interest over the past six decades, these alkaloids are now receiving more attention than ever. This renewed interest is 31 attributable to recent advances in biotechnology that have just begun to uncover the profound biochemical remodeling induced by these alkaloids in living systems that could be harnessed to treat disease. Herein, we give an extensive review of the literature related to the chemistry and biology of the phenanthropiperidine class. This review differs from previous ones 2-5 in that it is not limited to recent findings, and its scope extends deeper into the pharmacology of these alkaloids. The purpose of this work is to consolidate the widely dispersed literature on this subject to encourage a holistic understanding of what contributes to their potential use as medicinal agents. In this regard, special attention will be given to pharmacologically relevant details in order to comprehend better the poorly understood mechanism of action to assess therapeutic value. Considering that drug or lead accessibility is a major contributor to the success of natural product- and naturally-derived medicinal agents, we will also discuss precedent for the preparation of these alkaloids. Ideally, this compendium will expose impediments to clinical success and will encourage and guide the refinement process in a preclinical environment. 1.2 Background 1.2.1 Discovery of tylophorine, tylophorinine, cryptopleurine and tylocrebrine Folk medicines are a rich source of incipient drugs that are laden with clues for therapeutic use. The phenanthropiperidines owe their discovery to an Indian household remedy, now called T. indica that was brought to the attention of Western scientists in the late 32 19th century. 6 The roots and leaves of this perennial climbing plant have been used for a broad spectrum of medical conditions, and it is listed as an important member of the Indian materia medica. 7 In 1935, with the aim of isolating the active constituents of this natural remedy, Ratnagiriswaran et al. extracted and purified two alkaloids, naming them tylophorine and tylophorinine (Fig. 1–1). 7 The structure of tylophorine was not determined until 1960 after several years of degradation studies and comparison with the synthetically-derived natural product. 8-12 The proposed structure contained an indolizidine heterocycle fused to a symmetrically substituted phenanthrene. 13 The stereochemistry, which had been mistakenly assigned earlier, 14 was unambiguously assigned as R following a total synthesis and comparison of the optical rotation with the isolated alkaloid. 15 Although tylophorine was the first member of its class to be isolated, another phenanthropiperidine had been characterized several years before its structure was determined. 33 In 1948 de la Lande reported the isolation of a highly toxic substance named cryptopleurine that had been extracted from Cryptocarya pleurosperma, a walnut tree indigenous to Austrailia. 16 Seminal pharmacological studies demonstrated that cryptopleurine was lethal in rat, mouse, rabbit, dog and cat models (LD 50 1.5 to 5 mg/kg). It also became apparent that the alkaloid was a potent skin irritant. Upon oral administration, rats developed blisters on the face and feet. Furthermore, topical administration in human volunteers produced vessication on the arm and face that lasted over two weeks. Moreover, gastrointestinal inflammation and ulceration were found in histological studies of rabbits that N MeO OMe MeO OMe N MeO MeO OMe N MeO OMe MeO MeO N OMe MeO MeO OH H H H H (R)-tylophorine (1.1) Source: T. indica Ratnagiriswaran et al. 7 tylophorinine (1.2) Source: T. indica Ratnagiriswaran et al. 7 (R)-cryptopleurine (1.3) Source: C. pleurosperma de la Lande 16 (R)-tylocrebrine (1.4) Source: T. crebriflora Gellert et al. 20 Fig. 1–1. Structure of first discovered phenanthropiperidines. 34 had received oral dosages of the alkaloid. In 1954, the structure of cryptopleurine was determined through X-ray diffraction studies of its methiodide congener making it the first member in its class with a known structure. 17-19 In any discussion of the history of phenanthropiperidines a fourth member of this class, tylocrebrine, is worthy of note. This alkaloid was isolated in 1962 from a North Queensland vine, T. crebriflora which also had vesicant properties. 20 The major alkaloid, tylocrebrine, was found to be a regioisomer of tylophorine. With accumulating evidence that phenanthropiperidine alkaloids have anti-cancer properties, tylocrebrine was tested and found to be considerably more effective than tylophorine, tylophorinine and crytopleurine in the several cancer models. 21 Clinical testing began in 1965, only three years after its isolation. 2 Unfortunately, there were unforeseen problems with the alkaloid and the clinical trials were terminated before tylocrebrine’s therapeutic efficacy could be established. Suffness reported that the studies were terminated due to CNS side-effects. 2 It is of no surprise that medicinal interest in these alkaloids waned in the years to follow. Cryptopleurine was an undiscriminating poison, tylophorine and tylophorinine had only mediocre activity, and worse yet, tylocrebrine, the most promising candidate, was potentially neurotoxic. 1.2.2 Phenanthropiperidine isolation and structural features Since 1965, over 60 phenanthropiperidines and closely related analogs have been isolated and characterized. To date, they have been found primarily in two plant families, the Asclepiadaceae and Moraceae. Five genera (Tylophora, Cynanchum, Vincetoxicum, 35 Pergularia, and Antitoxicum) out of the 300 genera in the Asclepiadacea family are known to contain these alkaloids compared with only one genus (Ficus) from the Moraceae family. 5 These plants are found throughout Asia, Africa, Australia and the Pacific Islands (e.g. Taiwan, Japan and the Philippines). The details of the isolation studies fall outside of the scope of this work. Fortunately, there are several reviews on this subject already. 2, 4 The general structural features of these alkaloids are a bicyclic nitrogenous heterocycle and a polyoxygenated phenanthrene system (Fig. 1–2). The phenanthrene portion is generally substituted with three to five methoxy or hydroxy groups, although methylenedioxy substituents have been isolated as well. 22, 23 A pyrrolidine or a piperidine make up the terminal aliphatic heterocycle (ring E), hence the names phenanthroindolizidine and phenanthroquinolizidine. (4a,4b)-Seco- and dehydro-derivatives (Fig. 1–3)—where ring D is fully aromatized—are frequently isolated along with their phenanthropiperidine parent N 1 2 3 4 5 6 7 8 9 11 12 13 14 N 1 2 3 4 5 6 7 8 9 15 11 12 13 14 N R 1 R 2 R 4 R 5 R 6 R 3 R 7 n R 8 13a 14a 15a 8b 4a 4b 14a 8b 4a 4b A B C D E phenanthroindolizidine phenanthroquinolizidine R 1-6 = H, OH, OMe, -OCH 2 O- R 7 = H, OH R 8 = H, Me, OH n = 1,2 Fig. 1–2. General structural features of the phenanthropiperidines. 36 compounds showing a spectrum of oxidation states that exist within the plant. 3, 4 Oxygenation can also occur as evidenced by indolizidines and quinolizidines bearing a hydroxyl functionality at C14 or C15, respectively. Moreover, an oxygen atom can be introduced at the nitrogen resulting in phenanthroindolizidine N-oxides. 24 Johns et al. isolated a rare phenanthroquinolizidine alkaloid, cryptopleuridine, which possesses a C12- hydroxyl group. 25 To date, this is the only known, natural phenanthropiperidine that has a functionalized ring E system. In 1984, another structural anomaly was observed in the alkaloids of cultivated T. hirsuta. 26, 27 These natural products were essentially identical to several phenanthroindolizidines already isolated except they had a 13a-methyl substitution (R 8 = Me). Recently, Pettit and coworkers isolated and characterized several more of these alkaloids from Hypoëstes verticillaris. 28 Interestingly, in wild populations of T. hirsuta, the principal alkaloidal constituent bears a hydroxyl group at the ring juncture (R 8 = OH) in lieu of an angular methyl group. 29 This labile hemiketal functionality has since been identified in several tyloindicines N R 1 R 2 R 4 R 5 R 6 R 3 n N R 1 R 2 R 4 R 5 R 6 R 3 n Fig. 1–3. Under- and over-oxidized scaffolds. seco-derivative dehydro derivative 37 isolated in 1991 by Ali and coworkers (Fig. 1–4). 30 Besides the rare 13a-hydroxy group that appears in tyloindicine F and G, these anomalous members also have a precariously positioned olefin (15, 15a) that is out of conjugation with ring C. The natural variety that is found within this class has aided an understanding of structure- activity relationships (SARs) and provided swathes of targets for synthetic organic chemists. These will be discussed in detail later on. For our current purposes it is important to point out that practical synthetic routes have provided a sustainable source of many of these alkaloids and, in those cases, total syntheses have supplanted isolation efforts. 1.2.4 Biosynthesis Although the biosynthesis of the phenanthroindolizdines is better understood than that of the phenanthroquinolizidines, it is likely that the pathways are analogous. 31 A representative biosynthetic pathway is outlined in Scheme 1–1. It should be noted that the extent of phenolic methylation of each intermediate is uncertain and is most likely liberally regulated N MeO MeO OMe OH H N MeO MeO OMe OH H OMe N MeO MeO OMe H H HO N MeO MeO OMe H HO OMe Fig. 1–4. Unusual structures of tyloindicines F–I. tyloindicine F tyloindicine G tyloindicine H tyloindicine I 38 39 Scheme 1–1. Proposed biosynthesis of phenanthropiperidines. CO 2 H NH 2 CO 2 H H 2 N ornithine N CO 2 H NH 2 phenylalanine HO H 2 O N O HO N HO N HO HO OH OH OH HO OH HO O [O] N MeO OMe MeO OMe N HO HO HO [H] HO HO N MeO MeO OMe N HO O HO HO N MeO OMe MeO MeO HO methylation methylation tylophorinine tylocrebrine tylophorine methylation and [O] HO O OH HNO HO OH HNO HO OH CO 2 [O] OH H + 1.5 1.6 1.7 1.7 1.8 1.9 1.10 1.12 1.11 1.13 as evidenced by the diversity seen in the natural products, the representative structures in Scheme 1–1 notwithstanding. These pentacyclic alkaloids are thought to be derived from tyrosine, 32 phenylalanine 33 and ornithine. 32 The phenanthroquinolizidine biosynthesis varies only with respect to the latter precursor, which is derived from lysine instead. In the indolizidine class, the pyrrolidine carbons originate from ornithine which decarboxylates, deaminates and cyclizes to pyrroline (1.5). This cyclic imine further reacts with cinnamic acid 1.6 34 —synthesized from phenylalanine—affording a carboxylic acid intermediate (not shown). Following decarboxylation and alcohol oxidation, amine 1.7 undergoes condensation with 3,4-dihydroxyphenylacetaldehyde (1.8) which is derived from tyrosine. 35 The resultant enamine (1.9) cyclizes and upon dehydration yields the seco-derivative 1.10. As already discussed, isolation of the seco-derivatives is relatively facile and gives support to this biosynthetic pathway. The final transformation is thought to proceed through an oxidative coupling and subsequent rearrangement, 34 but no direct evidence has been garnered to support or contradict this hypothesis. Mulchandani et al. suggest that this process is initiated with the formation of dienones 1.11 or 1.12 through ortho- or para-phenolic couplings. The strained spirocycles then undergo a 1,2-alkyl shift to consummate the biosynthetic sequence of tylophorine or tylocrebrine. Another fate for spirocyle 1.11 was also proposed to explain the formation of tylophorinine. In this scenerio, dienone 1.11 is reduced to dienol 1.13 prior to rearrangement resulting in the elimination of hydroxide upon phenanthrene formation. Despite the remaining ambiguities of the latter sequence, this mechanistic hypothesis is plausible when considering its resemblance to the phenolic couplings seen in the biogenesis of morphine. 36 40 Furthermore, this hypothesis has considerable explanatory power, needing minimal revision to explain the biogenetic origins of multiple naturally occurring phenanthropiperidines. 1.3 Biology of Tylophora indica Tylophora extracts have gained considerable attention for the treatment of inflammatory diseases and, to a much lesser extent, cancer. Chitnis et al. investigated the cytotoxic properties of leaf and plant extract of T. indica in mice presenting transplantable leukemia tumors (either L1210 or P388). 37 Notably, mice receiving daily doses of extract survived longer than those treated with a placebo. Since the anti-cancer properties of T. indica have been attributed primarily to specific phenanthropiperidine alkaloids, most investigations have focused on the pharmacology of the purified alkaloids rather than on plant-derived mixtures and hence will be discussed later. On the other hand, the efficacy and mode of action of T. indica for the treatment of immune disorders, such as asthma, have been extensively investigated. 1.3.1 Clinical studies Six clinical studies have been conducted using the extracts of T. indica for the treatment of asthma (Table 1–1). Shivpuri et al. conducted two double-blind clinical investigations of T. indica. 38, 39 In these studies, the leaves of T. indica were administered to over 100 patients with asthma and allergic rhinitis. After a six-day regimen, patients experienced significant 41 symptomatic relief that lasted for several weeks. In a follow-up study, ethanolic extracts were administered to 195 asthmatics. 40 Over 50% experienced moderate to complete symptomatic cessation compared with 31% of patients taking a placebo. Four other clinical investigations have been conducted with either extracts or powdered plant material. 41-43 Two studies showed appreciable symptomatic remission, while the other reported no statistically significant difference between patients receiving the treatment and placebo. In pursuit of a physiological explanation for the apparent symptomatic relief, Gore et al. demonstrated that dried leaf powder had immunosuppressive properties. 44 Interestingly, the immunosuppression, as judged by an eosinophil count, coincided with elevated 17-ketosteroid secretion, suggesting an indirect effect through stimulation of the adrenal cortex. Notably, increased levels of eosinophils in the sputum of asthmatics have a strong correlation with bronchial inflammation. 45 42 An assessment of the methodologies used in five of these clinical trials has recently been reported by Huntley and Ernst. 46 Although positive effects of T. indica in asthmatics are clear, the authors conclude that the clinical relevance of several of these studies was undermined by poorly designed experiments or the omission of important details (e.g. age range of participants, dropouts/withdrawals, etc.). Considering the prevalence of asthma and the appeal of complementary medicine, 47 there is a great need for further characterization of the efficacy and safety of T. indica, which is currently marketed in products such as AsmaAide TM , Ultra Asthmatica and T. Asthmatica Plus. FEV 1 = forced expiratory volume (in 1 second); MBC = maximum breathing capacity; VC = vital capacity; PEFR = peak expiratory flow rate. reference source # of participants study length results Shivpuri 39 leaves 166 6 days 50–100% Symptomatic relief Shivpuri 38 leaves 110 12 weeks 62% vs. 28% (control) Improvement at 6 days; 16% Improvement at 12 weeks Shivpuri 40 EtOH extract 195 12 weeks 50% of participants experienced symptomatic relief vs. 31% of placebo Mathew 43 alkaloid extract 123 12 weeks Improvement in symptomatic relief and FEV 1 Effects peak at 4 weeks Gupta 41 powdered plant 135 3 weeks No significant improvement of symptoms Thiruvengadam 42 dried leaf powder 30 16 days Nocturnal dyspnea symptoms improved Sustained rise in MBC, VC and PEFR Gore 44 dried leaf powder 29 4 weeks Decreased eosinophile count; Increased 17-ketosteroid secretion Table 1–1. Clinical studies with T. indica for the treatment of asthma. 43 1.3.2 In vitro and in vivo studies with T. Indica Preclinical studies have been instrumental in uncovering the intricacies of the anti- asthmatic effects of Tylophora extracts. A summary of these investigations is presented in Table 1–2. Haranath and Shyamalakumari studied the effect of T. indica on in vivo immunosuppression. 48 Aqueous extracts administered intraperitoneally prevented anaphylaxis in guinea-pigs 10 days after its administration and caused leucocytosis and leucopenia—indicative signs of immunosuppression—in dogs upon i.v. administration. Atal et al. later reported the immunomodulating properties of ethanolic extracts of T. indica. 49 Significant inhibition of the humoral immune response (HIR), stimulation of phagocytic function and a significant increase in survival time for a foreign skin graft were reported. Adrenergic effects were noted by Udupa et al. 50 Upon treatment of male albino rats with the extracts of T. indica, the adrenals increased in weight and decreased in cholesterol and vitamin C content indicating direct stimulation of the adrenal cortex. These findings are illuminating considering the use of β 2 -agonists as bronchodilators in asthmatics. 44 With evidence that T. indica can be used for the treatment of pathologies arising from aberrant immune responses (i.e. asthma, anaphylaxis, arthritis, etc.) Ganguly and Sainis sought to further characterize the immunogenic effects of the alkaloids in T. indica. 51 They found that the alkaloid mixture mitigated delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) and contact sensitivity to dinitrofluorobenzene (DNFB), indicating a suppression of T cell-mediated response. Additionally, the alkaloids inhibited mast cell degranulation and suppressed the discharge of pro-inflammatory mediators. Lending further credence to their therapeutic value, prophylactic and remedial benefits were noted in the i.p. = intraperitoneal; i.v. = intravenous; DTH = Delayed-type hypersensitivity; HIR = Humoral immune response; DNFB = dinitrofluorobenzene. Table 1–2. In vivo and in vitro studies with T. indica extracts. reference extract route/dose model result Haranath 48 aqueous i.p. guinea pig prevented anaphylaxis i.v. dog leucocytosis and leucopenia Atal 49 ethanolic oral mouse ↓ DTH ↓ HIR ↑ survival time in skin allograft rejection Udupa 50 multiple oral, i.p. rat stimulation of adrenal cortex Ganguly 51 alkaloidal oral rat ↓ DTH mouse ↓ DNFB-contact sensitivity ↓ IgM response Ganguly 52 alkaloidal > 300 pg/mL T cells ↓ IL-2, ↓ proliferation 19-300 pg/mL macrophage ↑ IL-2, ↑ proliferation 39 ng/mL macrophage ↑ IL-1, macrophage activation 45 sensitized rodent models. Contrary to previous findings, 49 however, the alkaloids did not suppress primary humoral (IgM) immune response to SRBC. The effect of the extracts was also investigated in rodent splenocytes. 52 Concanavalin A, a mitogen, was used to induce splenocyte proliferation as a model for the cellular immune response. Interestingly, a biphasic response was observed where proliferation was inhibited at high concentrations (> 300 pg/mL) and stimulated at lower concentrations (19–300 pg/ mL). Both T cells and macrophages were shown to be perturbed by the alkaloid extracts. As an aside, this result is reminiscent of cryptopleurine’s dual effect on motor nerve sprouting reported by Hoffman in 1952. 53 In addition to T cell mitogenic inhibition, IL-2 secretion was independently inhibited whereas IL-1 production increased. Furthermore, the alkaloids activated macrophages and hence were thought to exert their immunosuppressive activity via production of reactive oxygen and nitrogen species. The molecular basis for T. indica’s anti-inflammatory effects is clearly not straightforward. These preclinical and clinical studies have provided insight into the pharmacologic mechanisms by which T. indica alleviates asthma. The use of raw plant material or plant extracts, however, muddies the water in discussions of the mechanism of action. The chemical composition of any given plant is a complex mixture of biomolecules, and although the most profound effects are often attributed to a primary active constituent, it is conceivable that a few components, if not many, contribute to the overall physiological outcome. A second major challenge arises from the variation among plants of the same species. It is well-known that subtle environmental factors can dramatically change the 46 biochemical landscape within a given plant species, thus, reproducibility may be difficult to attain. 1.4 Biology of phenanthropiperidine alkaloids Investigation of the pure alkaloidal constituents of T. indica has resolved some of the ambiguities that arise from studying complex botanical mixtures. Tylophorine, perhaps due to its relatively high abundance (0.015–0.036%), 7, 30, 54 early discovery, and ease of synthesis, has received the greatest attention in this regard. The physiological effects of tylophorine are not subtle. The earliest isolation attempts report severe contact dermatitis and blistering upon exposure to the alkaline extracts of T. indica. 7 Notably, other plant species containing phenanthropiperidines and, indeed, the alkaloids themselves are notorious for their vesicant properties. 16, 20 These noxious effects, however, have not deterred scientists from exploring their therapeutic potential. With the already established anti-inflammatory, immunosuppressive and cytotoxic properties of the plant extracts it was fitting that the pure alkaloids should be appropriated in the same therapeutic areas. The following section will discuss their biological effects and mechanism of action. 47 1.4.1 Anti-cancer activity 1.4.1.1 In vivo studies The phenanthropiperidines have long been touted as promising anti-cancer agents due to their broad-spectrum and potent cytotoxicity. As seen already, tylophorine, tylocrebrine and cryptopleurine were the first members to enter the limelight. Out of these three alkaloids, cryptopleurine was found to be the most toxic and tylophorine the least. The general toxicity of cryptopleurine was studied in guinea-pig, rat, mouse, rabbit, cat and dog. 16 The alkaloid proved to be lethal at doses as low as 1 mg/kg with little regard for species or the mode of administration. A non-selective toxin is obviously a poor drug. Nevertheless, fatal diseases such as cancer have greater leeway with respect to selectivity if the final outcome with the drug is more desirable than that of the untreated disease. In spite of its potent cytotoxicity, low doses of cryptopleurine (1 mg/kg i.p., twice daily for 8 days) inhibited the growth of Ehrlich ascite tumors in mice without killing the host. 55 Preclinical studies at the NCI (Table 1–3), however, showed that cryptopleurine had only marginal activity in vivo. 2 Tylocrebrine, on the other hand, was a more promising lead and became a clinical candidate soon after its discovery. Still, this alkaloid was found to be inactive in sarcoma 180, adenocarcino 755, B16 melanoma, Lewis lung, P1534 leukemia and Walker 256 carcinosarcoma models. It was tylocrebrine’s structural novelty and superior efficacy to tylophorine and cryptopleurine 21 in lymphoid leukemia L1210 mouse models that justified its entry into clinical trials. 2 In 1966, 48 less than a year after the clinical trials began, the studies were aborted before its therapeutic value could be determined. Suffness reported that this was because of central nervous system (CNS) toxicity evidenced by ataxia and disorientation. 2 Unfortunately, the original clinical data from these studies could not be obtained. compound NSC # dose (mg/kg) life extension (%) (R)-cryptopleurine 19912 1.0 140 (R)-tylocrebrine 60387 20.0 155 (S)-tylophorine 717335 30.0 150 tylophorinine 100055 6.0 130 demethyltylophorinine 94739 15.0 153 Table 1–3. Best NCI in vivo results for L1210 leukemia model. 49 N OMe MeO MeO N OMe MeO MeO OMe N HO MeO OMe OH H H OH H Fig. 1–5. Structures of several NCI-tested phenanthropiperidines. (R)-antofine DCB-3503 demethyltylophorinine 1.4.1.2 In vitro studies Over the next few decades, medicinal interest in tylocrebrine and other phenanthropiperidines waned. Recently, however, there has been a resurgence of activity with regard to these natural products. This can be traced back to a NCI 60 human tumor cell- line screen in the early nineties that included several phenanthropiperidines. To date, eleven naturally occurring members of this class have been tested in this screen. The structures of these can be found in Fig. 1–1, 1–4 and 1–5. Contrary to earlier speculation, many of these alkaloids were found to have potent and broad-spectrum cytotoxicity. Salient members of this class inhibited tumor growth with GI 50 values in the low nanomolar to picomolar range, on par with currently used anticancer drugs. Since the disclosure of this finding, the phenanthropiperidines have been intensely pursued as anti-cancer drug leads. Thus far, a vast majority of these studies have been limited to in vitro anti-proliferative experiments, and the NCI has acquired the majority of the data. A cross-section of the 60-tumor cell line panel screen results is shown in Table 1–4. From this, it should be clear that the alkaloids endowed with activity show little discrimination between cell lines. This is exemplified by the fact that the GI 50 ranges over the 60-tumor cell line panel show moderate selectivity between the most and least susceptible cell lines. To put this in perspective, paclitaxel, under the same assay, has a GI 50 range of 2 to 1000 nM, a 500-fold difference. Although this is not direct evidence, selectivity within the 60-cell line panel may be indicative of selectivity between malignant and normal tissue. 50 A second observation that can be made from the collective data is that these alkaloids retain their potency in drug-sensitive and multidrug-resistant (MDR) cell lines (Table 1–5). 24, 56-58 Antofine and several closely related analogs, for instance, have equipotent growth- inhibitory activity against common and MDR cell lines. 56, 57 Furthermore, in a KB cell line panel containing strains resistant to many of the conventional anti-cancer drugs, tylophorine and its hydroxylated analog, DCB-3503, showed no bias in their anti-proliferative effects. 58 Besides the obvious pertinence to therapeutic utility, this suggests that these alkaloids have a different mechanism of action from drugs that are ineffective in these refractory cancers. To add to this, an earlier evaluation of tylophorine in the NCI’s COMPARE analysis bears further witness to the unique mechanism of action and potentially novel cellular target(s). 59 CEM = leukemia, A549 = non-small cell lung carcinoma, MCF-7 = breast carcinoma, HCT 116 = colon carcinoma, NA = Not applicable, ND = No data. Unless otherwise specified, data were gathered from NCI-DTP Database. a Gao et al. 62 b Fu et al. 63 c Su et al. 56 60-cell line panel GI 50 (nM) individual cell line GI 50 (nM) compound NSC # mean range CEM A549 MCF-7 HCT 116 tylophorine 717335 17.5 10–400 10, 15 a 10 10 10 antofine NA ND ND 5.2 a 10.4, b 0.44 c 12.4 c 9.9 b cryptopleurine 19912 5.1 1.3–50 5.0, 2 a 5.0 5.0 5.0 tylocrebrine 60387 29.5 10–126 25 25 50 25 DCB-3503 716802 29.1 10–160 25 25 16 20 tylophorinine 100055 57.6 10–500 40 63 63 40 demethyltylophorinine 94739 1.13 1.0–16 1.0 1.0 1.0 1.0 tyloindicine F 650393 0.10 0.1–0.1 0.10 0.10 0.10 0.10 tyloindicine G 650394 0.10 0.10–0.16 0.10 0.10 0.10 0.10 tyloindicine H 650395 0.50 0.10–20 0.40 0.63 3.98 0.50 tyloindicine I 650396 4.40 1.6–20.0 3.16 3.98 5.01 2.00 Table 1–4. Growth inhibitory activity for antofine and NCI tested phenanthropiperidines. 51 1.4.1.3 Structure-activity relationships The size of the phenanthropiperidines class has grown significantly since the discovery of tylophorine. There are over sixty members in this class, including seco-analogs and closely related isomers, from which SARs have begun to emerge. For the purpose of this discussion, fifty-one natural and unnatural analogs belonging to eleven scaffolds will be considered. In Table 1–6, the compounds are ordered from the most to the least potent according their average GI 50 activity in up to fourteen of the most commonly used cell lines or, for those that have been tested by the NCI, the mean GI 50 value in the 60-tumor cell line is used. It should be cautioned that compounds in Table 1–6 may not have been tested in the same assay or even in the same cell lines. Furthermore, since internal standards were rarely used, cell line biochemical changes resistance GI 50 (nM) tylophorine DCB-3503 antofine KB (parental) - - 12, 214 a 28 16 a , 1.2 b KB-MDR gp 170 ↑ VP-16, taxol, adriamycin, vincristine 14 26 – KB-7D Topo II ↓, MRP ↑ VP-16, adriamycin, vincristine 12 45 – KB-7D-Rev Topo II ↓ VP-16, adriamycin 11 25 – KB-Ha-R RR ↑, dCK ↓ HU, araC, gemcitabine 25 36 – KB-Ha-Rev dCK ↓ araC, gemcitabine 16 28 – KB-100 Topo I ↓, XRCC1 ↑ CPT, topotecan, SN-38 20 38 – KB-100-Rev Topo I ↓ CPT, topotecan, SN-38 10 41 – KB-VI P-gp ↑ adriamycin, vinblastine, colchicine 173 a – 14 a , 2.3 b a Stærk et al. 57 b Su et al. 56 MRP, multidrug resistance protein; VP-16, etoposide; HU, hydroxyurea; RR, ribonucleotide reductase; dCK, deoxycytidine kinase; Topo II, topoisomerase II; Topo I, topoisomerase I; XRCC1, X-ray repair cross-complementing gene I protein; araC, aravinoside cytosine; CPT, camptothecin; SN-38, 7-ethyl-10-hydroxycamptothecin; P-gp, P-glycoprotein. Table 1–5. Growth inhibitory activity in drug-resistant cell lines (Gao et al.). 58 52 53 entry common name code a scaffold b R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 n Avg GI 50 (nM) c 1 tyloindicine F 43 F H OMe H H OMe OMe H OH d 1 0.1 2 tyloindicine G 44 E OMe OMe H H OMe OMe H OH d 1 0.1 3 – 38 A H OH OMe H OMe OMe S-OH S-H 1 0.2 4 tyloindicine H 45 E H OMe OH H OMe OMe H H d 1 0.5 5 demethyltylophorinine 39 A H OH H H OMe OMe R-OH S-H 1 1.1 6 (±)-cryptopleurine ±-11 A OMe OMe H H OMe H H rac 2 2.0 7 6-demethylantofine 37 A OMe OMe H H OH H H rac 1 4.1 8 tyloindicine I 46 G OMe OMe OH H OMe OMe H H d 1 4.4 9 (R)-cryptopleurine R-11 A OMe OMe H H OMe H H R-H 2 5.1 10 boehmeriasin A 16 A H OMe H H OMe OMe H rac 2 5.2 11 (R)-antofine R-6 A OMe OMe H H OMe H H R-H 1 10.1 12 (±)-antofine ±-6 A OMe OMe H H OMe H H rac 1 14.0 13 – 2 A OMe OMe H H OMe OH H R-H 1 16.0 14 (S)-tylophorine S-1 A OMe OMe H H OMe OMe H S-H 1 17.2 15 – 34 A OMe OMe H H i-PrO H H rac 1 27.8 16 tylocrebrine 18 A OMe OMe H OMe OMe H H rac 1 29.5 17 DCB-3503 22 A OMe OMe H H OMe OMe S-OH S-H 1 29.5 18 – 36 A OMe OH H H OMe H H rac 1 30.3 19 deoxytylophorinidine 27 A H OMe H H OH OMe H R-H 1 35.7 20 – 33 A OMe i-PrO H H OMe H H rac 1 38.7 21 isotylocrebrine 20 A H OMe OMe H OMe OMe H S-H 1 41.0 22 deoxypergularine 19 A H OMe H H OMe OMe H rac 1 64.3 23 demethoxyantofine 7 A OMe OMe H H H H H rac 1 69.5 24 – 35 A OH OMe H H OMe H H rac 1 80.3 25 tylophorine-N-oxide 5 B OMe OMe H H OMe OMe H R-H 1 97.0 26 DCB-3501 21 A OMe OMe H H OMe OMe R-OH S-H 1 108.0 27 (R)-tylophorine R-1 A OMe OMe H H OMe OMe H R-H 1 109.7 28 – 3 A OMe OMe H H OMe OMe H rac 2 117.3 29 – 10 A OMe OMe H H OMe H R-OH R-H 1 130.0 30 antofine-N-oxide 9 B OMe OMe H H OMe H H R-H 1 135.0 31 tylophorinine 28 A H OMe H H OMe OMe R-OH S-H 1 160.1 32 (±) -tylophorine ±-1 A OMe OMe H H OMe OMe H rac 1 254.7 33 (R)-ficuseptine C R-12 A –OCH 2 O– H H OMe H H R-H 1 283.3 34 (±) -ficuseptine C ±-12 A –OCH 2 O– H H OMe H H rac 1 521.4 35 DCB-3506 23 A OMe OH H H OMe OMe S-OH S-H 1 600.0 36 – 32 A i-PrO OMe H H OMe H H rac 1 927.7 37 – 13 H OMe OMe H H OMe H H R-H 1 1064.7 38 tylophorinidine 29 A H OMe H H OH OMe S-OH S-H 1 1832.7 39 dehydroantofine 17 D OMe OMe H H OMe H H rac 1 2360.0 40 (R)-secoantofine 8 C OMe OMe H H OMe H H R-H 1 2535.0 41 tylophoridicine E 24 A H OH H H OMe OMe S-OH S-H 1 3336.7 42 – 30 A H H H H H H H rac 1 3571.2 43 tylophoridicine F 26 B H OMe H H OMe OMe R-OH S-H 1 6378.0 44 tylophoridicine C 25 B H OMe H H OH OMe S-OH S-H 1 8388.3 45 – 31 A H H H H H H H rac 1 9460.0 46 – 15 I OMe OMe H H OMe H H R-H 1 9645.0 47 – 14 I OMe OMe H H OMe H H S-H 1 9685.0 48 – 40 J OMe OMe H H OMe OMe H rac 1 15600.0 49 – 42 K OMe OMe H H OMe OMe H rac 2 42000.0 50 dehydrotylophorine 4 D OMe OMe H H OMe OMe H rac 1 > 50000 51 – 41 K OMe OMe H H OMe OMe H rac 1 > 50000 a Refers to number in Graph 1–1. b See Fig. 1–6. c Average GI 50 in HepG2, Panc-1, CEM, A549, MCF-7, KB, KB-VI, KB-3-1, HCT 116, HT-1080, MDA-MB-231, COLO-205, NUGC-3 and HONE-1. d Unknown stereochemistry Table 1–6. Structure and average GI 50 over select cell lines. 54 N o t e : A l i n e t h a t c o n n e c t s t w o o r m o r e d a t a p o i n t s r e f e r s t o a c o m p a r a t i v e s t u d y b e t w e e n t h e c o m p o u n d s t h a t a r e c o n n e c t e d b y t h a t l i n e . T h e l e t t e r o n e a c h l i n e i n d i c a t e s t h e l i t e r a t u r e r e f e r e n c e w h e r e t h i s d a t a i s r e p o r t e d . C o m p o u n d s t e s t e d i n t h e N C I t u m o r c e l l l i n e p a n e l a r e s h o w n i n r e d . G r a p h 1 – 1 . A n t i - p r o l i f e r a t i v e a c t i v i t y o f p h e n a n t h r o p i p e r i d i n e s i n f o u r t e e n s e l e c t c e l l l i n e s . a . N C I d a t a b a s e b . G a o e t a l . 6 2 c . G a o e t a l . 5 8 d . A b e e t a l . 6 5 e . S t a e r k e t a l . 5 7 f . C h u a n g e t a l . 6 8 g . Y a n g e t a l . 6 4 h . F u e t a l . 6 3 i . S u e t a l . 5 6 j . S t a e r k e t a l . 2 4 k . S y d n e s e t a l . 1 0 4 & B a n w e l l e t a l . 1 0 3 l . G a o e t a l . 6 0 m . H u a n g e t a l . 6 1 n . L u o e t a l . 1 0 5 r e f e r e n c e normalization of these results is virtually impossible. Because of this lack of standardization, a more detailed understanding of the variables specific to each anti-proliferative assay is required to assess the reliability of activity ranking across this class. Graph 1–1 accounts for a number of these variables by incorporating information about each compounds anti-proliferative activity in relation to the specific cell line and assay (i.e. which compound have been tested simultaneously in the same assay). A crude comparison of each compounds activity can be accrued by the range (represented by a blue bar) and mean N R 1 R 2 R 4 R 5 R 6 R 3 R 7 R 8 N R 1 R 2 R 4 R 5 R 6 R 3 n n N R 1 R 2 R 4 R 5 R 6 R 3 R 8 H N R 1 R 2 R 4 R 5 R 6 R 3 R 8 HN R 1 R 2 R 4 R 5 R 6 R 3 R 8 n N R 1 R 2 R 4 R 5 R 6 R 3 R 8 N R 1 R 2 R 4 R 5 R 6 R 3 R 8 n O 4a 4b 4a 4b N R 1 R 2 R 4 R 5 R 6 R 3 O O R 7 A B (N-oxide) C (4a,4b-seco) D E F (4a,4b-seco) G H I J K Fig. 1–6. Scaffolds of phenanthropiperidines and analogs. 55 activity (the center of the blue bar which corresponds to the “Avg. GI 50 ” column in Table 1– 6) of each compound. The innate differences between the cell lines, however, make this type of ranking rather inaccurate. An extreme example of this is seen with reported data for compounds 24 and 25. In HepG2 and Panc-1 cells, compound 25 exhibits more than 50 fold greater activity compared with compound 24, 60 whereas in KB cells compound 24 is at least 25,000 times more active than compound 25. 61 Whether this dramatic difference is a function of the particular cell lines or the different sources of each compound remains unknown, but is clear that simply averaging the GI 50 values obscures this peculiar finding. In order to address this, the activity of the individual cell lines are presented in Graph 1–1 and are distinguished based on the color and shape of each point. Another concern is that even among common cell lines there can be a significant amount of variation between assays. In A549 cells, racemic antofine (±-6) was reported to have a GI 50 value of 25 nM in one report and 0.44 nM in another, over a 50 fold difference. This discrepancy demonstrates the wide variation that can exist between independently acquired data in anti-proliferative assays. In this regard, we have indicated the compounds that have been tested in the same assay by linking them with a line, where the color and stroke (i.e. solid or dashed) of the line refers to the cell line, and the lowercase letter corresponds to the reference for a particular comparative study. In other words, all points that fall along a line of the same style (cell line) and letter (reference) were tested simultaneously. This not only allows the most accurate comparisons between each compound but provides a means to assess the relative activity of compounds that have not been compared in the same assay by finding commonalities between data sets. For instance, (R)-tylophorine (R-1) and (R)- 56 cryptopleurine (R-11), which have not been tested together, can be compared, because both compounds have been tested along with (S)-tylophorine (S-1) in CEM cell lines which can be used as the standard. On the other hand, a comparison of boehmeriasin A (16) with other compounds should be taken with reservation since it has not been tested along with any other members of this class. Nevertheless, by piecing together GI 50 values for particular cell lines that have been reported in the literature, we gain a better, albeit incomplete, understanding of structural features that contribute to the phenanthropiperidine’s anti-cancer activity. The phenanthrene system (rings A, B and C) has been studied most extensively. The seco-analogs are almost universally less active (e.g. Table 1–6, entries 11 and 40). There may, however, be other ways to compensate for this loss in activity as evidenced by tyloindicine F and I (entry 1 and 8) which are extremely cytotoxic in spite of their rings A and C being disconnected. Disrupting the aromaticity of ring B as in DCB-3502 (not shown) can also reduce activity. 58, 62 Furthermore, the location and type of substituents on the phenanthrene has a profound effect on its activity. 57, 61, 63 From Table 1–6, it should be apparent that formulating strict phenanthrene SARs without considering the rest of the scaffold inevitably leads to irreconcilable data. From the fact that when methoxy or hydroxy substituents are removed completely, the naked scaffold is significantly less active (entries 42 and 45), it can be surmised that the presence of these substituents substantially boosts activity. 64 Although this may be due to the requirement of an electron-rich arene, the incorporation of surrogate electron donators, such as amines, has not been investigated. The position of the substituents is also important but data gathered from different sources and cell lines make it difficult to discern precise correlations. Nevertheless, some 57 generalizations can be made. First, the 2,3,6-trisubstituted system seems to be favorable as evidenced by the remarkable potency of antofine and cryptopleurine (entries 6, 9, 11 and 12). A hydroxyl group at C6 can increase potency as in 6-desmethoxyantofine 63 (entry 7 and 12). Depending on the scaffold, C3-hydroxy groups will either increase (entries 5 and 43) 65 or decrease activity 57, 63 (entries 12 and 18). Fu et al. reported a systematic study of antofine’s (entry 12) phenanthrene substituents and found that the C2 position was particularly sensitive to alteration. Replacement of the methoxy group with either a methylenedioxy bridge (entry 34), hydroxy (entry 24) or an isopropoxy group (entry 36) resulted in a significant loss of potency. 62, 66 On the other hand, replacing the methoxy groups at C3 and C6 with hydroxy (entries 7 and 18) or isopropoxy (entries 15 and 20) was well-tolerated. There are several piperidine (ring D) modifications that can be made without losing activity. A 14-hydroxy group (R 7 = OH) seems to be tolerated with only a several-fold loss of activity (entries 14 and 17), 24, 58, 60, 65 but the stereochemistry of C14 does seem to affect the activity (entries 3 and 41; 17 and 26). There are probably multiple factors determining the most active diastereomer. 58, 60, 67 Despite the slightly diminished activity of C14-hydroxyl analogs, demethyltylophorinine (entry 5) is among the most potent members of this class to be tested in the 60-tumor cell line screen (see Table 1–4 also). 67 Interestingly, Gao et al. discovered that a hydroxylated DCB-3503 (entry 17) showed superior activity in vivo compared with its non-hydroxylated parent, tylophorine (entry 14), thus, hydroxylation may ultimately prove to be beneficial. 58, 60 Several groups have constructed N-oxide analogs which, surprisingly, produced only a 2– 10-fold reduction in activity (entries 25 and 27; 11 and 30). 24, 65 Amide analogs, however, are 58 almost completely inactive (entries 48, 49 and 51). 64, 68 Dehydro-analogs, where the piperidine is aromatized to a pyridinium, are also inactive (entries 12 and 39; 32 and 50). 64 It is worth mentioning that several tylophorine-based analogs have been designed that do not contain an intact ring D piperidine (entries 37, 46 and 47). Multiple-bond scissions have been investigated but all permutations of this type have reduced the alkaloid’s anti- proliferative effects. Nevertheless, the ease of preparing these simplified analogs has enabled extensive SAR studies and, through these, the discovery of moderately potent analogs such as PBT-1 (Fig. 1–7) which have IC 50 values of 200–400 nM. 69, 70 Despite their close structural-relatedness, evidence suggests that these unnatural derivatives are mechanistically distinct. 62, 71 There is evidence that the stereochemistry of the bridgehead carbon does contribute to the activity. Only a few labs, however, have tested two enantiomers in the same assay. Gao et al. were able to acquire both enantiomers of tylophorine and found the (S)-isomer (entry 14) to be 3–5-fold more active than the (R)-isomer (entry 27) in HepG2, Panc-1 and CEM cells. 62 Moreover, the anti-proliferative activities of each enantiomer paralleled their inhibitory Fig. 1–7. Structure of PBT-1. O O MeO N OH 59 effects on key transcription factors. In a separate study, (R)-antofine (entry 11) was tested with its racemate (entry 12) in A549, HCT 116 and HT-1080 cells showing the former to be three-fold more active in all three cell lines. 63 From this it may be inferred that, in contrast to tylophorine, the (R)-enantiomer is most active. If this is true, the phenanthrene substituents have differing effects on the activity of each enantiomer and, therefore, the absolute stereochemistry of the eutomer is not uniform across this class. Anecdotal evidence suggests that substitution requirements for C2- and C7- substituents are reversed upon changing the stereochemistry of the indolizidine, but this is still to be borne out in comparative studies. 3 Indeed, there is still a superficial understanding of SARs that, if better understood, could be profitable for the design of improved therapeutic agents. One reason for our poor understanding of SARs is that the available data are disjointed. Over fifty members of this class have been tested in anti-proliferation assays, but most of the results are compiled using specialized cell lines or lack an internal standard resulting in tenuous comparisons between most analytes. Another part of the problem is that SAR studies have been confined to a somewhat homogenous collection of alkaloids. Although the effect of substituent location has been explored, the type of substitution has not strayed beyond hydroxy or alkoxy groups. Surprisingly, even the incorporation of an additional nitrogen atom or halogen has yet to be investigated. There is also a paucity of SAR studies on ring E (Fig. 1–2) other than the observation that indolizidine and quinolidine analogs appear to be comparable in activity. 64, 68 Many of these limitations are understandable in light of the challenge of preparing such analogs. 3 60 1.4.2 Mechanism of action The effect of phenanthropiperidines on living systems has been investigated at the biochemical level. From these studies, several targets have been proposed, but none ratified. Notwithstanding the likelihood that these alkaloids interact with multiple targets, some of the alleged targets are needlessly invoked, only being perturbed at concentrations far above those required to exert the desired effect. Others that are supported by compelling experimental evidence, are unduly overlooked. Accordingly, this discussion will attempt to unify the body of work aimed at identifying the mechanism of action of the phenanthropiperidines with the aim of facilitating a critical analysis of the putative biological targets. 1.4.2.1 Protein, DNA and RNA biosynthesis inhibition The most extensively studied mechanism for the phenanthropiperidine’s anti-cancer activity is protein biosynthesis inhibition. Beginning in 1968, Donaldson et al. reported that tylophorine, tylocrebrine and cryptopleurine inhibited protein synthesis in Ehrlich ascites- tumor cells but did not markedly affect RNA synthesis (entry 1, Table 1–7). 72 Paralleling the order of their anti-proliferative effects, cryptopleurine exhibited the most potent inhibition whereas tylophorine was the least active. (–)-Antofine has recently been shown to inhibit protein biosynthesis with remarkable potency (entry 2). 62 In spite of its potency in eukaryotic systems, cryptopleurine did not inhibit protein synthesis in E. coli (entry 1). 72 It was hypothesized that phenanthropiperidines were exerting their growth inhibitory activity via 61 80S ribosomal inhibition thus explaining their inactivity in prokaryotes that have instead 70S ribosomes. To confirm this hypothesis, a systematic study explored the activity of the three phenenanthropiperidines in S. cerevisiae, a model eukaryote, and E. coli (entry 3). 73 The order of cytostasis was identical to that in the previous studies with cryptopleurine again being the most potent. Ribosomal inhibition studies clearly showed that the alkaloids inhibited protein synthesis with a noticeable preference for yeast cytoplasmic ribosomes over mitochondrial and E. coli ribosomes. Furthermore, cryptopleurine was shown to irreversibly inhibit ribosomal protein synthesis. 62 Delving deeper into the mechanism of inhibition, Huang et al. examined the effects of tylocrebrine in HeLa cells and rabbit reticulocytes, a cell-free model for protein biosynthesis (entry 4). 74 Tylocrebrine, like cryptopleurine, irreversibly inhibited protein biosynthesis. Studies of several phenanthropiperidines corroborate that the inhibition takes place during the translocation step of chain elongation. 74-77 When tylocrebrine was compared with a small collection of emetine-like alkaloids, its ameobocidal activity closely correlated with the activity of compounds that inhibited cell-free protein synthesis to the same extent (entry 5). 78 IC 50 (nM) entry reference model compound PS RS DS 1 Donaldson 72 Ehrlich ascites-tumor cells CP 20 > 100000 - Ehrlich ascites-tumor cells TC 200 > 100000 - Ehrlich ascites-tumor cells TP 1000 > 100000 - E. coli CP > 10000 - - 2 Gao 62 HepG2 cells AF 12 > 50 8 FS 2500 2500 1000 3 Haslam 73 S. cerevisiae; E. coli CP < 1000; > 100000 - - TC 1000; > 100000 - - TP 20000; > 100000 - - 4 Huang 74 HeLa cells TC 50 300 60 reticulocytes TC < 1000 - - 5 Entner 78 reticulocytes; reticulocyte lysate CP 150; 700 - - TC 300; 800 - - 6 Skogerson 76 S. cerevisiae; cry mutant ribosomes CP 180; 18000 - - 7 Gupta 80 CHO cell wild type; Emt R mutant CP 330; 660 - - TC 400; 1800 - - Table 1–7. Inhibition of protein, DNA and RNA synthesis. CP = cryptopleurine, TC = tylocrebrine, TP = tylophorine, AF = antofine, FS = ficuseptine C, PS = Protein synthesis, RS = RNA synthesis, DS = DNA synthesis, Emt R = emetine resistant. 63 The juxtaposition of emetine (Fig. 1–8) and the Tylophora alkaloids is not arbitrary. Emetine’s natural source, ipecacuanha, and T. indica have both been used in traditional medicine as natural emetics. Their similar therapeutic properties even earned T. indica the name “Indian ipecacuanha.” On a chemical level, there are also some similarities that have caused speculation about common binding sites on the ribosome. 79 In 1973, mounting evidence for ribosomal inhibition emerged from the identification of the cry gene that conferred resistance in S. cerevisiae (entry 6). 76 Ribosomes isolated from cry mutants required 100 fold more cryptopleurine to produce the same inhibitory response as parent ribosomes. Interestingly, the normal (uninhibited) activity of 40S ribosomal subunits in cry mutants was impaired revealing the biochemical outcome of the mutation. 76, 77 This finding, however, did not elucidate the ribosomal subunit selectivity of these alkaloids, nor does it identify a specific binding site. Evidence for this was gathered from genetic studies that found the cryptopleurine (Cry R ) and tylocrebrine (Tyl R ) resistant CHO cells bore a remarkably similar cross-resistance profile to emetine-resistant (Emt R ) mutants (entry 7). 80 The fact that Emt R cell’s resistance could be Fig. 1–8. Structure of emetine. MeO MeO N HN H OMe OMe H H 64 traced to a modified 40S ribosomal subunit and that emetine bore structural resemblance to the phenanthropiperidines was adduced as analogical evidence that these protein synthesis inhibitors shared a common binding site. 79, 80 The proposed common pharmacophore of the emetine and cryptopleurine, however, was not predictive of biological activity in synthetic analogs endowed with the alleged essential structural features. 81 Addressing this, evidence for a ribosomal binding site was finally provided by Dolz et al. in 1982. 82, 83 Displacement assays with tritiated cryptopleurine confirmed the existence of a single high-affinity binding site on the 40S and 80S subunits that are shared with tylocrebrine and tylophorine. Emetine and tubulosine, however, only displaced cryptopleurine at very high concentrations, suggesting separate binding locations in spite of their similar effects on translocation. These experiments also showed that non-specific, low-affinity interactions exist on the 40S, 80S and 60S subunits, explaining cryptopleurine’s discrete (peptide bond formation vs. translocation) inhibitory effects at high concentrations. Besides protein synthesis, tylocrebrine-induced inhibition of RNA and DNA synthesis in HeLa cells (entry 4). 74 It should be emphasized that protein and DNA synthetic pathways are interdependent and, consequently, inhibitors of protein synthesis. For example, anisomycin, cycloheximide and emetine, which are potent protein synthesis inhibitors, inhibit DNA synthesis as well. Antofine, a closely related analog, has recently been shown to simultaneously inhibit protein and DNA biosynthesis as well (entry 2). 62 Thus, the effect on DNA synthesis is likely a secondary consequence of tylocrebrine’s and antofine’s ribosomal inhibition. 65 These studies as a whole provide compelling evidence not only for a mechanism of action —namely protein biosynthesis inhibition—but also for the specific molecular target, the 40S ribosome. The question naturally arises: Is this explanation sufficient to explain the pharmacological effects of these alkaloids? This is difficult to answer since protein synthesis is an integral part of a cells normal growth and division and, as such, perturbation of the normal ribosomal function has vast repercussions. To borrow Pestka’s analogy, inhibiting protein synthesis is like sticking a wrench into a very complicated gearbox, “almost anything can happen.” 84 Therefore, it is not unfounded that protein synthesis inhibitors like the phenanthropiperidines are cytostatic to the cells they target. Another important stipulation for anti-cancer therapeutics must also be met. That is, they must exhibit an appropriate level of selectivity for tumor cells. There is accumulating evidence that this can be achieved with protein synthesis inhibitors. 85 First, aberrantly regulated protein synthesis is highly contributive to tumorigenesis 86 and metastatic progression. 87 Second, there is a higher critical threshold for protein synthesis in transformed cells than in normal cells that grow more slowly. Indeed, the rate of cell proliferation positively correlates with the level and rate of translation. Clinical studies have already borne witness to the therapeutic potential of several such inhibitors despite being limited by cardiovascular and other complications 88-90 —hypotension, ventricular tachycardia, atrial irritability, ST-T wave changes and premature ventricular contractions, for example. 90 Still, there is significant interest in developing more selective agents. 85 That multiple phenanthropiperidines potently inhibit protein synthesis and that their anti- proliferative effects correlate with this inhibition evinces the notion that these alkaloids exert 66 their cytotoxic effect, primarily but not exclusively, through protein biosynthesis inhibition. Moreover, as evidenced from studies with tritiated cryptopleurine, one molecular targets of the phenanthropiperidines is the 40S ribosome. 1.4.2.2 Apoptosis and cell cycle arrest Characterizing cellular events associated with anti-cancer lead molecules provides a window into their mechanism of action. Protein biosynthesis inhibitors will, by their nature, abate cell growth, but they are not inherently cytotoxic. Even though cytostatic agents can effectively treat malignancies by subverting the accelerated proliferation in neoplastic growth, cancer cell death or differentiation is required for complete remission. In 2002, it was reported that an alkaloidal extract of T. indica, containing mainly tylophorine, tylophorinine and tylophorinidine, completely inhibited cell growth in K562 (human erythroleukemic) cells at 0.1 µg/mL (entry 1, Table 1–8). 91 Interestingly, when the alkaloidal concentration was increased by ten-fold, the cells underwent apoptosis. Indeed, apoptotic agents engage a strategic battlefront for chemotherapeutic intervention, but the higher concentrations used here to achieve cell death may not be therapeutically practicable. Moreover, since an alkaloidal mixture was used, synergy cannot be ruled out. Lee et al. simplified analysis by using a single alkaloid: antofine (entry 2). 92 Antofine showed potent inhibition of growth and colony formation in A549 and Col2 cancer cells. No evidence of apoptosis or necrosis was observed at these low concentrations suggesting that antofine is primarily cytostatic. In Col2 67 cells, morphological changes and G2/M cell cycle arrest were noted at concentrations as low as 0.1 nM. The Cheng group observed that tylophorine and DCB-3503 did not cause DNA damage as concluded from the fact that p53 was not induced during treatment with either of these alkaloids (entry 3). 58 Both alkaloids (0.03-1 µM) induced S-phase accumulation in KB cells, whereas in HepG2 cells, the cells did not accumulate at any particular phase. Notably, analog DCB-3503 does not induce apoptosis or necrosis at up to 3 µM concentrations, and both tylophorine and DCB-3503 produced complete and sustained growth inhibition in HepG2 cells over 8 days. This persistency of activity was also demonstrated for tylophorine in HONE-1 and NUGC-3 cell lines (entry 4). 93 These long lasting effects are noteworthy considering these alkaloids induce irreversible protein synthesis inhibition. TP = tylophorine, AF = antofine, BA = boehmeriasin A, DCB-3503 = 14-hydroxytylophorine. entry reference cell line compound concentration result at cytostatic concentrations 1 Ganguly 91 K562 T. indica extract 0.1 µg/mL no apoptosis 1 µg/mL apoptosis 2 Lee 92 A549, Col2 AF < 30 nM no apoptosis Col2 AF 0.1 nM G2/M phase arrest; morphological changes 3 Gao 58 KB TP, DCB-3503 0.03-1 µM S phase accumulation HepG2 TP, DCB-3503 0.3-3 µM no DNA damage; sustained growth inhibition 4 Wu 93 HONE-1 NUGC-3 HepG2 TP 2 µM sustained growth inhibition G1 phase arrest 5 Yan 94 MDA-MB-231 BA 19 nM G1 phase arrest, no apoptosis Table 1–8. Apoptosis and cell cycle arrest by phenanthropiperidine alkaloids. 68 In HepG2, HONE-1 and NUGC-3 carcinoma cells, tylophorine induced G1-phase arrest that was linked to a suppression of cyclin A2 expression (entry 4). 93 Furthermore, DCB-3503 and tylophorinidine (Fig. 1–9) were shown to down-regulate cyclin D1. 60 It was noted that cyclin D1 suppression correlated with each compound’s growth inhibitory effects. In breast cancer cell line MDA-MB-231, boehmeriasin A (Fig. 1–9) exerts its potent anti-proliferative effects via G1 cell cycle arrest and not by apoptosis (entry 5). 94 In this study, cyclins D1 and E2 were found to be under-expressed in the treated cells. Given the role of cyclin-dependent kinases (CDKs) in the governance of cell cycle progression, modulators of CDK pathways could potentially correct the faulty regulation that leads to unmitigated cell division in cancer. 95-98 From these data, it is apparent that the archetypal phenanthropiperidines are not apoptotic agents but rather induce cytostasis by overriding a cancer cell’s replicative potential. N MeO HO OMe H OH N MeO MeO OMe tylophorinidine boehmeriasin A Fig. 1–9. Structures of tylophorinidine and boehmeriasin A. 69 1.4.2.3 Angiogenesis Another hallmark of cancer is “sustained angiogenesis”. 99 Highly metabolically active cells, such as those in many types of tumors, require sufficient vasculature to meet their exorbitant aerobic demands. Even slow-growing and anaerobic tumors rely on a blood supply for their basic sustenance. This need is met by actively recruiting blood vessels through the release of growth factors such as VEGF which is triggered by hypoxia. Targeting angiogenesis is, therefore, a highly effective way to suppress tumor growth. Cai et al. investigated cryptopleurine’s and 15R-hydroxycryptopleurine’s effects on hypoxia-inducible factor-1 (HIF-1), 100 a key regulator of a cell’s response to hypoxia and a promising drug target. 101, 102 Both alkaloids potently inhibit HIF-1-mediated gene expression under anaerobic conditions (IC 50 = 8.7 and 48.1 nM, respectively). Furthermore, VEGF expression is also suppressed in a dose-dependent manner. Unfortunately, attempts to determine cryptopleurine’s anti-angiogenic properties in vitro were hampered by its poor solubility. 103 A number of seco-analogs did, however, inhibit angiogenesis in rat aorta blood vessel fragments at low micromolar concentrations. 103, 104 It is still unclear if the anti-angiogenic effects of these alkaloids are independent of their anti-proliferative properties. 1.4.2.4 Cell differentiation When considering the pervasive impact within a cell resulting from protein synthesis inhibition, it is not surprising that the phenanthropiperidines elicit patent phenotypic changes 70 in transformed cells. The possibility that tylophorine induced cell differentiation was investigated through monitoring the expression levels of two tumor biomarkers, alpha- fetoprotein (AFP) and albumin. 58 Treatment with tylophorine (1 µM) resulted in a suppression of AFP expression and an increase in albumin expression which is consistent with cell differentiation. Boehmeriasin A also induced cell differentiation that manifested as morphological changes, lipid droplet accumulation and modulation of gene expression. 94, 105, 106 1.4.2.5 NF-κB Signaling pathway A recent effort has led to the discovery that tylophorine potently inhibits NF-κB– promoted transcription (IC 50 = 30 nM) and, to a lesser extent, that of AP-1 and CRE (IC 50 = 500 nM). 58 Several closely related analogs, including DCB-3503, were also discovered with similar effects. 60 It is unclear if these events are related through overlapping signaling networks but it does provide deeper insight into the root cause of tylophorine’s pharmacological effects. NF-κB–signaling pathways are an integral part of normal cell growth and immunological function. 107-109 A large cohort of cytokines, growth factors and effector enzymes associated with immune responses are strictly regulated by this transcription factor. Furthermore, a growing body of evidence has linked the dysregulation of NF-κB to the onset of cancer and immune-related disorders. 107-109 Thus, the discovery that tylophorine inhibits NF-κB provides a hypothetical link between tylophorine’s seemingly 71 disparate pharmacologies—namely, anti-cancer and anti-inflammatory/immune—and demonstrates therapeutic potential through a validated target. 1.4.2.6 DNA and RNA Other targets for the phenanthropiperidines that have been explored recently are DNA and RNA. Xi et al. prepared a small library of DNA oligonucleotides containing a variety of secondary structures, such as hairpin bends and bulges, and studied antofine’s associative properties. 110 Antofine showed clear preference for duplex oligonucleotides as measured by dissociation from and stability of the DNA-alkaloid complex. Moreover, the alkaloid binds with moderate selectivity for DNA bulges in a 1:1 stoichiometry (Fig. 1–10) and shows slight sequence selectivity. The significance of antofine’s selectivity toward bulged DNA and RNA lies in the fact that these abnormalities are implicated in the etiology of numerous diseases. 111, 112 Docking studies with the bulged structures give reason to conclude that this association is intercalative. It is worth reiterating here that tylophorine does not induce DNA damage as DNA-bulge–selective agent neocarzinostatin does. Tylophorine’s affinity for DNA, however, has yet to be investigated. In addition to DNA binding, antofine has also shown high affinity for the assembly origin of Tobacco Mosaic Virus (TMV) RNA. 113 This RNA motif contains hairpin loop structures with several bulges. Antofine was found to bind to with a K D of 9 nM and a 1:25 stoichiometry. 72 The high affinity of antofine for DNA and RNA structures impels consideration in discussions of its mechanism of action and warrants further characterization in more complex systems such as cells. Furthermore, in view of the homogeneity within this class, these interactions should be determined for other members and correlated with their anti- proliferative activities. Considering the planarity of the phenanthrene system and structural similarity to other DNA intercalators (e.g. ethidium bromide), the question of intercalation is an obvious one and should be addressed. 1.4.2.7 Other molecular targets In addition to the 40S ribosomal subunit and polynucleotides, several other molecular targets have been proposed for the phenanthropiperidines. Thymidylate synthase 114, 115 and dihydrofolate reductase 116 inhibition have been observed at high concentrations (IC 50 > 30 Normal DNA single base  bulge multiple base  bulge K  = 18 nMD   = antofineT A A T G T A A T T G K  = 46 nMD K  = 240 nMD Fig. 1–10. Antofine’s affinity for DNA structures. 73 µM) of tylophorinidine. Since the phenanthropiperidine’s activity is in the low nanomolar range, invoking these targets to explain its anti-cancer activity is seemingly gratuitous. 1.4.3 Anti-inflammatory activity Like anti-cancer properties, the basis for the anti-inflammatory and immunosuppressive effects is not straightforward. The plant extracts were clearly shown to suppress the inflammatory response in cell, animal and human models. With this in mind, the first task was to demonstrate that the pure alkaloids retained the anti-inflammatory effects of T. indica and its extracts. This was borne out in several studies that confirmed that pure tylophorine allayed immunopathological and inflammatory reactions in vitro and in vivo. 117, 118 In pursuit of a novel anti-rheumatic agent, Yin and coworkers identified the promising tylophorine analog, DCB-3503. 119 The compound was chosen based on its inhibitory effects on TNFα-induced NF-κB activity (IC 50 = 100 nM). Although the reason for choosing this analog over tylophorine, in spite of its inferior NF-κB activity, was not disclosed, it is noteworthy that this hydroxylated analog outperformed tylophorine in animal studies, presumably due to improved pharmacokinetics. 58 Mice injected with collagen or lipopolysaccharide (LPS) to induce arthritis were treated with DCB-3503 and closely monitored for signs of efficacy. It was found that the development, onset and severity of LPS- and collagen-induced arthritis were abated with no signs of toxicity. TNFα and IL-1β, two important pro-inflammatory cytokines, were down-regulated at the systemic and local level. Furthermore, immune-cell production of key inflammatory signaling molecules, such 74 as TNFα, IL-12, IL-6, MCP-1, iNOS and COX-2, was significantly impaired, demonstrating DCB-3503’s multifaceted approach to suppressing the arthritic immune response. Indeed, inhibition of NF-κB transcription is a plausible explanation for these extensive biological effects, but this conclusion may be reductionistic. Earlier work in LPS/IFNγ- induced RAW264.7 cells (a murine macrophage cell line), found that tylophorine significantly reduced TNFα, iNOS and COX-2 production at 3-10 µM without causing NF- κB inhibition. 120 AP1 activation, however, was significantly inhibited. This result was accounted for by measuring the levels of upstream signaling factors MEKK1, Akt and c-Jun. Although tylophorine blocked MEKK1 activation, which normally inhibits NF-κB, concurrent Akt activation counteracted its effect. While the relation of these events to the attenuation of inflammatory responses is discernible, the molecular basis for such a response remains elusive. Indeed, the complex network of biochemical processes within this pathway that are constitutively interlaced with feedback loops and other regulatory events makes the search for molecular target an immense challenge. 1.4.4 Miscellaneous activity In addition to their anti-cancer and anti-inflammatory effects, the phenanthropiperidines have been investigated for activity in several other systems. In some of the earliest biological studies with these alkaloids, Barnard reported that cryptopleurine had colchicine-like c- mitotic activity in the root tips of germinating onion seeds. 121 In oyster embryos and yeast, in spite of inhibiting growth, its activity at 10 –6 to 10 –3 M did not seem to resemble that of 75 colchicine. 122 Anti-fungal activity was also observed for cryptopleurine and some closely related analogs. 123 Amoebicidal, 78, 124 insecticidal 125 and anti-feedant 126 properties have also been reported. Anti-viral effects have also been noted. When kidney cells were exposed to crytopleurine and subsequently inoculated with herpes virus hominis, viral growth was stunted. 127 The alkaloid did not, however, have any antiviral activity in coxsackie B-5 or polio-type I viruses. Antofine was also found to exhibit anti-TMV activity in the low micromolar range, presumably due to its affinity for RNA. 128 Following up this discovery, Wang et al. studied tylophorine (racemic, S and R), (±)-antofine and (±)-deoxytylophorine, finding that they also possessed anti-TMV activity. With the goal of improving solubility and stability, a series of tylophorine salt derivatives were prepared. 129 These salinized analogs, besides being more soluble and stable, had improved in vitro and in vivo anti-TMV activity, surpassing that of commercial Ningnanmycin. 1.4.5 Summary of biological activity The prodigious effects of the phenanthropiperidines on living systems are indisputable, but they have yet to be harnessed to treat disease in the clinic. Within the past decade, a there has been a concerted effort to validate their therapeutic potential in both cancer and immune/ inflammatory-related diseases. Although the literature is replete with studies on the phenanthropiperidines, the data extracted from different assays, cell-lines and animal models have led to a fragmented understanding of the mechanism by which these alkaloids modulate 76 immune responses and inhibit the growth of cancer cells. Nevertheless, protein biosynthesis inhibition and modulation of key transcription-factor activity are among the best validated mechanisms of action. This is not to say, however, that there is a single molecular target. Increasing evidence suggests that the phenanthropiperidines perturb multiple macromolecules. For instance, some of the most well-studied members of this class have affinity for the 40S ribosomal subunit, DNA and RNA. Therefore, the identification of specific targets may not be sufficient to explain the extent of their pharmacology. 1.5 Obstacles to clinical success While many phenanthropiperidines exhibit promising activity in vitro, this activity has not translated well into animal models. The NCI has conducted multiple in vivo studies with the salient members in this class: tylophorine, tylocrebrine and tylophorinine. These alkaloids provide up to 150% life-extension in the most responsive tumor model, L1210 leukemia. Unfortunately, other tumor models such as adenocarcinoma 755 and Sarcoma 180 were much less responsive. The basis for this in vitro/in vivo discrepancy is undoubtedly multifactorial and is just beginning to be understood. Cheng and coworkers noticed that despite comparable activities in vitro, tylophorine and DCB-3503 behaved differently in animal studies. 58 Notably, previous assessment in the NCI hollow fiber assay scored DCB-3503 higher than tylophorine (26 vs. 4). Compounds that score above 20 are generally considered for further animal studies. 130 It was posited that this difference was linked to drug delivery or stability. Both compounds were subsequently 77 tested in a HepG2 xenograph murine model using multiple dose regimens. Again, DCB-3503 had superior antitumor activity compared with tylophorine. 58 This idiosyncrasy could result from differing pharmacokinetic profiles, but no extensive PK studies have been reported. Indeed, PK data would be invaluable to understanding why such potent compounds fail to perform in vivo. In addition to the aforementioned toxicity of cryptopleurine, 16 the therapeutic window is another important consideration in this discussion. Some toxicological data can be gathered from the NCI database that reports the mortality in each model system. From this and other in vivo studies it seems that an effective dose can be reached without causing overt toxic effects. Tylophorine has been studied most extensively with respect to its toxicity. One study reported that an oral administration of tylophorine (1.25-2.5 mg/kg/day) over 15 days produced no toxic effects in rats, whereas an oral dose of 35 mg/kg was lethal in 50% of the animals. 131 In another study, rats and hamsters receiving seven oral doses of 5 mg/kg result had a 50 and 80% mortality rate, respectively. 124 Contrasting with these reports, a dose as high as 500 mg/kg of (R)-tylophorine was found to be non-toxic in rats. 117 In addition to acute toxicity, another impediment to the therapeutic potential of these compounds is their neurological effects. It was observed that at high doses, rodent models experienced CNS depression (ptosis, sedation, decreased motor activity and staggered gait). 117 This is noteworthy because of the CNS effects—namely, ataxia and disorientation— experienced by subjects in tylocrebrine’s clinical trials. 2 In light of tylophorine’s similar physicochemical properties, it is clear why this is such a concern. In a recent review, Hitchcock and Pennington show that the degree of passive diffusion across the blood-brain 78 barrier (BBB) correlates with a molecule’s polar surface area (PSA), number of H-bond donors (HBD), cLogP, cLogD and molecular weight. 132 Calculating these properties for tylophorine and tylocrebrine reveals that each value falls well within the suggested parameters for CNS drugs on all accounts (Table 1–9). Of course, if the aim is to reduce CNS-mediated side effects the alkaloids should be endowed with properties less conducive to BBB permeation. Another concern which is intimately related to physicochemical properties is low solubility. The high lipophilicity of these molecules is a likely contributor. Fortunately, mitigating BBB permeation by incorporating polar functional groups will probably improve solubility as well. The planarity of the phenanthrene system is probably another culprit. In a recent review, Loverling et al. show that increasing sp 3 character in drug candidates correlates with improved solubility and hence its clinical success. 133 Whether this strategy can be successfully employed for phenanthropiperidines is still to be realized. If these alkaloids exert their effects through intercalation, relying on sheer planarity, improving PSA = polar surface area, HBD = hydrogen-bond donors, MW = molecular weight. Table 1–9. CNS drugs and tylocrebrine’s physicochemical properties. property top 25 CNS drugs mean values suggested limits tylocrebrine PSA (Å 2 ) 47 < 90 40 HBD 0.8 < 3 0 cLogP 2.8 2-5 4.3 MW 293 < 500 394 79 solubility by increasing saturation may ultimately be counterproductive. Although these impediments to clinical success seem daunting, there are still many “tricks of the trade” that have yet to be explored. 1.6 Synthesis of phenanthropiperidine alkaloids The accessibility of bioactive molecules largely determines the feasibility of drug development. Without an adequate supply, even the most active medicinal agents are rendered useless. Currently, supply is not a limitation for preclinical investigations of the phenanthropiperidines. Compared with many natural products, these alkaloids are structurally simple, and reliable synthetic routes have—for the most part—replaced isolation methods. Spurred by unique structural features and biological properties, organic and medicinal chemists have endeavored to provide a sustainable source of these alkaloids and to give access to novel synthetic analogs with improved therapeutic potential. Indeed, these routes have greatly facilitated lead optimization. With this in mind, the obstacles to clinical success seem less daunting. Synthetic approaches to the phenanthropiperidines have matured since the first efforts. Advancements in transition metal catalysis and heterocycle synthesis have been crucial to this enterprise. There is no shortage of total syntheses for many of the well-known members of this class. Owing to their long history and archetypal structures, tylophorine and cryptopleurine boast over twenty distinct routes. Antofine is another popular target due to its extremely potent cytotoxicity. The number of synthetic undertakings that have been 80 successfully used to prepare these alkaloids far exceeds the scope of this work, so, we will focus only on key sequences within the total syntheses that have led to the construction of the phenanthrene system and indolizidine/quinolizidine scaffolds. Reactions used to prepare parts of these systems (i.e. naphthyl, pyrrolidines, piperidines, etc.) or reactions that elaborate these two substructures will receive minimal attention. Conceivably, improved reaction sequences could be devised by piecing together parts of others. 1.6.1 First synthetic efforts towards phenanthropiperidines Less than five years after the structural elucidation of crytopleurine, Bradsher and Berger reported its synthesis. 134, 135 Slightly delayed because of structural uncertainties, the synthesis of tylophorine followed closely behind. 13 With these methods already established, the synthesis of tylocrebrine could be reported simultaneously with isolation giving strong evidence for its proposed chemical structure. 20 All of these synthetic approaches used the same strategy (Schemes 1–3 and 1–4). First, the phenylacetic acid 1.14 was condensed with NO 2 CHO OMe MeO MeO CO 2 H Ac 2 O MeO OMe X CO 2 H MeO FeSO 4 NH 4 OH (aq.) 1. Amyl nitrite, H 2 SO 4 acetone 2. Cu powder MeO OMe R 2 MeO 3 steps R 1 R 1 R 1 50–72% 14–54% 80% 1.16 (X = NO 2 ) 1.17 (X = NH 2 ) 1.18 (R 2 = CO 2 H) 1.19 (R 1 = H; R 2 = CH 2 Br) 1.20 (R 1 = OMe; R 2 = CH 2 Cl) 1.14 (R 1 = H or OMe) 1.15 Scheme 1–2. Original method to prepare phenanthrene system. 81 nitroveratraldehyde 1.15 (Scheme 1–2). The nitro group was reduced to the amine with ferrous sulfate in preparation for phenanthrene formation (1.16 → 1.17). The biaryl coupling was realized using a Pschorr coupling protocol, involving conversion of amine 1.17 into the corresponding diazonium salt. Subsequent treatment with copper mediated a radical decomposition and bond formation to furnish acid 1.18. The acid was then reduced to an alcohol, and this was displaced by either a bromide or chloride ion. Diverging slightly at this point, cryptopleurine was synthesized via N-alkylation of picolinic aldehyde with the phenanthrene 1.19 and cyclized in PPA (Scheme 1–3). The fully oxidized pentacycle 1.21 was then hydrogenated to (±)-cryptopleurine (1.3) in the final step. Tylophorine (1.1), on the other hand, was prepared through a C-alkylation of pyrrylmagnesium bromide and reduction and then completed using the Bischler-Napieralski protocol from amine 1.22. The synthesis of (±)-tylocrebrine (1.4) was completed in a similar manner. 20 N CHO MeO MeO N H 2 , PtO 2. H 2 , PtO 2 N MgBr MeO OMe MeO OMe HN 1. HCO 2 H 2. POCl 3 3. NaBH 4 OMe 11% (4 steps) 1. 2. PPA 1. 24% (2 steps) 16% (3 steps) Scheme 1–3. Original syntheses of phenanthropiperidines. 1.19 1.20 1.21 1.22 (±)-cryptopleurine (1.3) (±)-tylophorine (1.1) 82 1.6.2 Phenanthrene synthesis The phenanthrene is a relatively rare component of natural products in comparison to the indolizidine. In most cases, this structure has been constructed using robust biaryl coupling reactions. The point in the syntheses at which the full phenanthrene core is realized varies considerably from route to route. There are several things that should be considered when deciding when to incorporate this moiety. In comparison with the indolizidine amine which often requires protection, the phenanthrene system is remarkably stable and is unperturbed in most reaction conditions. Furthermore, compounds containing a phenanthrene moiety are generally stable crystalline solids and hence simplify purification. On the other hand, these stable crystalline lattices can be poorly soluble making late incorporation advantageous. MsO(CH 2 ) 3 CN CpCo(CO) 2 µW, 120 ºC 78% N OMe MeO MeO OMe MeO MeO I PIFA, BF•Et 2 O X X OMe OMe MeO OMe 84% NaBH 4 99% Scheme 1–4. Phenanthrene synthesis via dimerization. 1.23 1.24 (X = I) 1.26 (±)-tylophorine (1.1) 1.25 (X = C≡CH) 3 steps 81% 83 The substitution pattern on the phenanthrene system determines the accessibility of each alkaloid by any given approach. Because of its symmetrical methoxy substituents, tylophorine can be synthesized through dimerization. Deiters and coworkers exploited this symmetry in their synthesis of tylophorine, beginning with a dimerization of 4-iodoveratrole (1.23) and subsequent conversion to the diyne 1.25 (Scheme 1–4). 136 This intermediate was desymmetrized using a [2+2+2]-cyclotrimerization with a judiciously chosen nitrile. Upon cycloaddition, the resulting pyridine spontaneously cyclized by displacing the tethered mesylate and furnished dehydrotylophorine (1.26), which was reduced to the natural product (1.1). In another example, Chelmer and coworkers used a radical-induced dimerization of benzyl bromide 1.27 followed by an oxidative-coupling reaction to prepare symmetrical phenanthrene 1.30 (Scheme 1–5). 137 Alternatively, the benzyl dimer 1.29 can be derived from an alkyne reduction of symmetrical acetylene 1.28 and carried through to phenanthrene 1.30 as before. 138 Further functionalization and desymmetrization were achieved through MeO OMe OMe MeO H 2 , Pd/C OMe MeO MeO OMe OMe MeO Br Cp 2 TiCl 2 81% OMe MeO MeO OMe PIFA BF 3 •Et 2 O R 66–78% 79% 1.27 1.28 1.29 Scheme 1–5. Phenanthrene synthesis via dimerization. 1.30 (X = H) ClSO 3 H 70% 1.32 (X = SO 2 Cl ) 1.31 (X = Br) NBS 98% 84 bromination with NBS 138 or sulfonylation with chlorosulfonic acid to furnish phenanthrene 1.31 and 1.32, respectively. 137 Pearson and Walavalkar demonstrated that the oxidative dimerization of homoveratric acid (1.33) can be used to prepare the necessary precursor (1.34) to (±)-tylophorine (Scheme 1–6). 139 The symmetrical phenanthrene substitution pattern has expedited the latter parts of total syntheses as well. Comins 140, 141 and Padwa 142 have reported the use of Pd-catalyzed bis- arylation reactions to install the requisite aryl groups in septicine, the seco-analog of tylophorine (Scheme 1–7). MeO OMe CO 2 H 1. LDA, –78 ºC 2. I 2 , –78 ºC OMe MeO MeO OMe CO 2 Me CO 2 Me 3. CH 2 N 2 31% (3 steps) 1.33 1.34 Scheme 1–6. Phenanthrene synthesis via dimerization. 10 steps 11% (±)-tylophorine (1.1) 85 Despite the advantages of dimerization, desymmetrization can be challenging. The earliest strategies for phenanthrene construction used a different approach. As shown in Schemes 1–3 and 1–4, the first total syntheses of tylophorine and cryptopleurine used aldol reactions and Pschorr couplings to prepare the phenanthrene. Although the Pschorr reaction has been replaced with more efficient biaryl coupling reactions, the aldol method is still widely used 3, 4 because of its simplicity and amenability to scale up. 143 Notably, the tactic permits substituent variability on either the aldehyde or carboxylate arenes allowing the synthesis of unsymmetrical phenanthrene substitution patterns as in cryptopleurine. The stilbene aldol product is typically converted directly into the corresponding phenanthrene using a series of oxidative biaryl coupling protocols. Iron(III), vanadium(V), thallium(III) N TfO Br OMe MeO ZnBr Pd(PPh 3 ) 4 , THF N OMe MeO MeO OMe N O TfO TfO OMe MeO ZnBr Pd(PPh 3 ) 4 , THF N O H OMe MeO MeO OMe 67% 64% H Scheme 1–7. Bisarylation strategy to synthesize phenanthrene precursor. (R)-septicine 86 and hypervalent iodine reagents have all been successfully used. Thallium works remarkably well for this transformation but is extremely toxic and, hence, is often replaced with less toxic reagents such as iron or PIFA. Photoirradiation can also be used but with poor regioselective control. 144, 145 This oxidative biaryl coupling approach is synthetically attractive due to its atom economy but regioselectivity is a concern. Free rotation around the aryl-alkene bond is responsible for the formation of regioisomers which can make purification extremely difficult. Fortunately, this problem can be mitigated at low temperatures. Another caveat is that the use of oxidative couplings limits the accessibility of phenanthrenes with C4- or C5- substituents. It is true that tylocrebrine, which bears a C5-methoxy group, was among the first phenanthropiperidines to be synthesized, but this approach relied on the isolation of the minor regioisomer (17% vs. 32% of opposite regioisomer) of an unselective Pschorr coupling CO 2 H OMe MeO MeO OMe H 2 N 1. isopentyl nitrite DMF, H 2 SO 4 2. EtOH, reflux CO 2 H OMe MeO MeO OMe CO 2 H OMe MeO MeO + MeO Scheme 1–8. Regioselectivity in synthesis of tylocrebrine. (32%) (17%) 87 protocol to construct the tetramethoxyphenanthrene system (Scheme 1–8). 146 This synthesis provides less than 1% of racemic tylocrebrine over ten steps and required significant effort to separate it from the other regioisomer (recrystallized three times). There is a clear need to surmount this historic challenge. Alternative approaches have been used to form the aryl-aryl bond which show promise for the preparation of phenanthrenes with these difficult substitution patterns. For instance, Kibayashi and coworkers prepared seco-analog 1.35 with a strategically placed bromide to direct C–C bond formation (Scheme 1–9). 147, 148 The phenanthrene could then be formed using either light or Bu 3 SnH/AIBN to initiate the desired biaryl coupling. Since the position of the bromide determines the position of bond formation—this is analogous to the function of the amine in the Pschorr protocol—a single regioisomer (1.36) is obtained. This approach has not been used to synthesize phenanthrenes with C4- or C5-substituents. It is without question that biaryl coupling strategies have dominated phenanthrene synthesis. Fürstner and Kennedy, however, reported an alternative route to four Tylophora alkaloids using a Pt-catalyzed cycloisomerization (Scheme 1–10). 149 This unconventional Br MeO OMe MeO N O H hν (54%) or AIBN, SnBu 3 H (87%) MeO OMe MeO N O H Scheme 1–9. Halogen-directed biaryl coupling. 1.35 1.36 88 approach relied on the assembly of the biaryl system, first via Suzuki cross-coupling. Their judicious use of simple, readily available building blocks (boronic acids and aryl-1,2- dihalides) gives this method the versatility required to access multiple analogs in a divergent manner, a feature that is conducive to lead optimization. Once alkyne 1.37 has been prepared, a Pt(II)-catalyzed cyclization provides phenanthrene 1.38 in up to 97% yield depending on the substrate. Hypothetically, this strategy could give access to the aforementioned phenanthrenes with C4- or C5-substituents, but this has not been reported. Among the reactions discussed, a single example departs from the conventional strategy of piecing together two oxygenated arenes (Scheme 1–11). 150 In this example the tricyclic nucleus arises from a cascade process with well-placed functionalities. The sequence commences with the coupling of alkyne 1.39 and carbenoid 1.40. The newly formed chromium carbene complex 1.41 is ideally situated to the γ-ketone oxygen and furnishes diene 1.42. A strategically placed alkene undergoes a [4+2]-cycloaddition with the pendant furan, and upon ring-opening and dehydration of oxanobernene 1.43, the desired MeO R 1 R 3 R 2 N Boc 56–97% MeO R 1 R 3 R 2 N Boc n n cat. PtCl 2 n = 1,2 Scheme 1–10. PtCl 2 -catalyzed phenanthrene formation. (–)-tylophorine (±)-cryptopleurine (–)-antofine (–)-ficuseptine C 1.37 1.38 89 dihydrophenanthrene system (1.44) is obtained. Using this approach, (±)-antofine could be prepared in 7 steps, but attempts to synthesize (+)-antofine were unsuccessful. 1.6.3. Indolizidine and quinolizidine synthesis With the many creative and elegant approaches to the phenanthrenes discussed, we will now turn to the aliphatic heterocycle. As before, we will speak generally, emphasizing the diversity of approaches to the indolizidine or quinolizidine nuclei rather than dwelling on the intricacies of each. The strategies will be divided based on the position of the bond that is formed to fully assemble the indolizidine or quinolizidine core. Where possible, the heterocycles will be treated as isolated entities and, accordingly, are numbered as such. Unlike the reactions used in preparing the phenanthrene scaffold, the aliphatic core has been MeO MeO O Cr(CO) 5 OMe MeO OMe O N CO 2 Me + 100 ºC 66% Cr(CO) 4 MeO R MeO OMe O MeO R MeO OMe MeO R O H H 2 O MeO OMe MeO R Scheme 1–11. Carbenoid cascade phenanthrene synthesis. 1.39 1.40 1.41 1.42 1.43 1.44 (±)-antofine 90 prepared in a wide variety of ways. The following discussion will be divided based on the location of the bond that is formed in the key step (Fig. 1–11). 1.6.3.1 C3–C4 bond forming reactions The C3–C4 bond is the one point of confluence between the phenanthrene and piperidine structures. These carbons have been successfully conjoined using basic intramolecular aldol condensations. 151-157 Biocatalytic approaches have successfully condensed cylic imines 1.46 and 1.47 which were enzymatically formed from putrescine and cadaverine in situ, with 3-oxopropionic acid 1.45 (Scheme 1–12). 158-160 Alternatively, pyrroline 1.46 can be prepared from 4-aminobutanal diethylacetal and isolated as a stable complex with zinc iodide (not shown). 151, 161 In another example, the N-oxides of imines 1.46 and 1.47 have also proven to be viable precursors to Fig. 1–11. Key-step bond formations. N 2 5 6 7 3 4 n 8 C5a–C6 C4–C5 C3–C4 C2–C3 N–C2 N–C8(C9) 91 the indolizidine 145, 162 and quinolizidine 144 alkaloids. Iida et al. used these nitrones (1.46 and 1.47) in a [1,3]-dipolar cycloaddition reaction yielding isoxazolidine 1.52 or its homologue 1.53. The N-O bond was then reduced, providing β-amino alcohol 1.54. A limitation of these strategies is their lack of stereoselectivity. To address this, Georg and coworkers prepared amine 1.57 in enantiomerically pure from Boc-L-β-homoproline (1.55) in three steps. 152, 154 N O N O Ar reflux 88% (n = 1) O CO 2 H + N catalase + H 2 , Pd/C or Zn/AcOH Ar HNOH n n 85% (n = 1) 79% (n = 2) pH 7.4, 27 ºC, 24 h H 2 N NH 2 n MeO OMe 93% (n = 2) MeO OMe 86% (n = 1) 93% (n = 2) HNO n MeO OMe 1. HN(OMe)Me•HCl EDCI, NMM 2. 4-MeO-C 6 H 4 MgBr 79% NO MeO HO 2 C N Boc R diamine oxidase PhMe n n Scheme 1–12. Preparation of aldol precursors. 1.45 1.49 1.54 1.55 1.56 (R = Boc) 1.481.46 (n =1) 1.47 (n = 2) 1.57 (R = H) 4N HCl or AlCl 3 1.50 (n =1) 1.51 (n = 2) 1.52 (n =1) 1.53 (n = 2) 92 From amine intermediates 1.48, 1.54 and 1.57 there have been two general approaches to the access the natural products. The one that most resembles the biosynthetic pathway begins by treating amine 1.48 with a substituted phenylacetaldehyde (Scheme 1–13). 35, 151, 155, 163-167 The enamine condensation product (1.58) then reacts further to furnish bisarylated indolizidine 1.59. Seco-analog 1.60 is prepared upon reduction with NaBH 4 , situating the intermediate for the final biaryl coupling. This approach is remarkably concise but suffers from low yields (24%). In an alternative approach, amine 1.54 or 1.57 is acylated in preparation for cyclization. 144, 145, 151-153, 162 Diketone 1.61 is then treated with KOH in an alcohol solvent, providing the dehydrated product (1.62) in a single step. N Ar 1 Ar 2 O KOH EtOH or MeOH N Ar 2 Ar 1 O O n n N Ar 2 Ar 1 O n N Ar 2 Ar 1 n NaBH 4 N Ar 2 Ar 1 n OH Ar 2 CH 2 CHO 24% (2 steps) 1. Ar 2 CH 2 COCl; K 2 CO 3 2. CrO 3 67–95% HN(OMe)Me•HCl, EDCI, NMM, Ar 2 CH 2 CO 2 H 52–60% Ar 1 , Ar 2 = MeO MeO MeO or CH 2 Cl 2 58–88% Scheme 1–13. C3–C4 Bond formation via aldol reaction. 1.48 1.58 1.60 1.54 1.62 1.59 1.61 (±)-cryptopleurine (±)-tylophorine (±)-deoxytylophorine (±)-tylophorine (±)-cryptopleurine 1.57 93 A significant limitation was discovered in this approach when the supposedly enantiomerically pure products were found to have undergone partial racemization. 152, 154 Further inquiry into this unexpected result revealed that racemization occurred upon Boc- deprotection (1.56 → 1.57) and during the base-promoted aldol condensation (1.61 → 1.62). 152 It was proposed that a retro-Michael/Michael process was at play that could evidently occur in either acidic or basic conditions (Scheme 1–14). This unfortunate side-reaction has limited the utility of the intramolecular aldol approach. Padwa and coworkers developed a unique Rh(II)-catalyzed [1,3]-dipolar cycloaddition for the preparation of highly-substituted 2(1H)-pyridones that forms the C2–C3 and C5–C5a bonds of the indolizidine simultaneously (Scheme 1–15). 168 Treatment of pyrrolidinone 1.63 with Rh(OAc) 2 generates a cyclic ylide 1.64 in situ, which reacts further with substituted alkenes, providing unstable bridged-bicyclic intermediate 1.65 that spontaneously decomposes to form indolizidine 1.66. This intermediate was then used to complete a formal synthesis of (±)-septicine (see Scheme 1–7). Scheme 1–14. Mechanism of β-racemization in acidic or basic conditions. N Ar 2 Ar 1 O O H N Ar 2 Ar 1 O O H N Ar 2 Ar 1 O O H acid or base 94 1.6.3.2 C2–C3 bond forming reactions The C2–C3 connection was considered by the first chemists to synthesize these natural products. As already discussed this approach relied on the Bischler-Napieralski isoquinoline synthesis in the penultimate step. This reaction, with a few exceptions, 150 has since been replaced with higher yielding and more direct approaches. Very recently Orito and coworkers reported a Pd-catalyzed aromatic carbonylation reaction, which gave access to a variety of benzolactams (1.68, Scheme 1–16). 169 The reactions were generally low yielding unless stoichiometric quantities of palladium were used. N O N 2 PhO 2 S Rh(II) O N O S O OR N O PhO 2 S O RO N O RO HO PhSO 2 H 64% O O Ph R = i-Bu 1.63 1.64 1.65 1.66 (±)-septicine Scheme 1–15. [1,3]-Dipolar cycloaddition cascade to construct the indolizidine core. HN Pd(OAc) 2 , Cu(OAc) 2 N 26–45% R R R R n n n = 1,2 O CO, PhMe Scheme 1–16. C2–C3 Bond formation via Pd-catalyzed carbonylation. 1.67 1.68 95 Reactions of this kind that generate lactams require a subsequent reduction—almost always performed with LAH—to obtain the natural products. A more direct approach, however, has managed to circumvent this final reduction step. This approach, which is now in common usage, relies on the Pictet-Spengler reaction for C2–C3 bond-formation. 63, 137, 143, 149, 170, 62, 171-173 Generally speaking, an amine (1.69) is treated with formaldehyde under acidic conditions to form an electrophilic iminium species (1.70), which reacts further in a 6- endo-trig fashion to afford the phenanthropiperidine 1.71 (Scheme 1–17). Since acidic conditions are used, amines protected with acid-labile protecting groups (e.g. Boc) undergo deprotection in situ. 62, 149 The intramolecular Diels-Alder reaction is another way to construct the C2–C3 bond. Like the Pictet-Spengler approach, this strategy exploits a reactive imine (1.74 and 1.75, Scheme 1–18). Weinreb and coworkers were the first to employ this strategy. The imine dienophile was generated by thermal decomposition of methylol acetate 1.72, which underwent a [4+2]-cycloaddition to form quinolizidine scaffold 1.76. 174, 175 The foregoing protocol could also be used to prepare the indolizidine counterparts. In an example that is N N n n CH 2 O, H + N R R n X 53–97% R R R R Scheme 1–17. Pictet-Spengler approach to conjoin the C2–C3 bond. 1.69 X = H or Boc n = 1,2 1.70 1.71 96 more reminiscent of the Pictet-Spengler approach, Grieco and Parker generated imine 1.75 upon treatment of phenanthrene 1.73 with formaldehyde. 176 After heating the reaction for 10 h at 180 ºC, (±)-cryptopleurine was isolated in excellent yield without the need to reduce an amide. Pearson and Walavalkar have used an intramolecular [1,3]-dipolar cycloaddition reaction to assemble the indolizidine core (Scheme 1–19). 139 This reaction is initiated by an azide- alkene cycloaddition of phenanthrene 1.77 to form a triazoline intermediate 1.78, which upon fragmentation reveals a reactive imine (1.79). The pendant electrophile then alkylates the imine nitrogen to complete the double cyclization. In the final step, reduction of iminium N X [4+2] OMe MeO MeO N X OMe MeO MeO H N OOAc OMe MeO MeO H 2 N CH 2 O 84% 50% 220 ºC, 5 h H 2 O/EtOH 180 ºC, 10 h •HCl Scheme 1–18. Diels-Alder reaction to prepare quinolizidine. 1.72 1.73 1.74 (X = O) 1.75 (X = H 2 ) 1.76 (X = O) (±)-cryptopleurine (X = H 2 ) 97 1.80 with NaBH 4 provides (±)-tylophorine. Kim et al. have recently adopted a similar strategy in the synthesis of (±)-antofine and (±)-cryptopleurine from azide 1.81. 170 1.6.3.3 C4–C5 bond forming reactions The intramolecular Diels-Alder approach is an efficient way to impart complexity in a single reaction. In the aforementioned cycloadditions, however, stereochemical complexity was not addressed. One tactic that has been used to confront the challenge of asymmetric synthesis involves the use of chiral auxiliaries. With previous success in preparing (±)- tylophorine via Diels-Alder chemistry, 177 Ihara et al. next investigated an enantioselective OMe MeO MeO OMe N 3 Cl N Cl N N N Cl N NaBH 4 82% (2 steps) N 2 Cl OMe MeO MeO N 3 1. C 6 H 6 , 135 ºC OMe MeO MeO HN 2. NaBH 3 CN or H 2 , Pd(OH) 2 n n 130 ºC 63-69% C 6 D 6 Scheme 1–19. [1,3]-Dipolar cycloadditions to prepare indolizidine. 1.77 1.78 1.79 1.80 1.81 1.82 (±)-cryptopleurine (n = 2) (±)-tylophorine (±)-antofine (n = 1) 98 route. 178 This approach relies on the synthesis of stilbene amide 1.83 featuring a tethered (E)-unsaturated ester (Scheme 1–20). Using chiral auxiliaries A or B, the desired indolizidine (1.85) could be prepared upon treatment with TBSOTf and TEA. This reaction was deemed an “intramolecular double Michael addition”, but a formal Diels-Alder mechanism has not been ruled out. Although a 4:1 mixture of C3-epimers was obtained, both benzylic stereocenters were destroyed upon oxidation to the phenanthrene system with Tl(CF 3 CO 2 ) 3 , and in a few steps, the first asymmetric synthesis of (R)-tylophorine was completed. Recently, Chemler and coworkers developed a novel Cu(II)-catalyzed enantioselective carboamination reaction that gives rapid access to sulfam (1.86, Scheme 1–21). 137 Upon elimination of sulfur dioxide and Pictet-Spengler reaction, tylophorine could be prepared with an 81% enantiomeric excess. A Pd-catalyzed intramolecular variant of this strategy was MeO OMe OMe MeO O H N R*O 2 C TBSOTf TEA MeO OMe OMe MeO OTBS N R*O 2 C R* = Ph –78 ºC N OMe MeO MeO OMe CO 2 R* O H O O Ph OTBS or A B Scheme 1–20. Diels-Alder strategy to prepare indolizidine. 1.83 1.84 1.85 (R)-tylophorine Chiral auxiliaries: 76–92% 99 recently reported by Herr and coworkers. 138 In this example, phenanthryl bromide 1.31 undergoes oxidative insertion resulting in coordination of the carbamate and pendant alkene. Nucleophilic attack onto the Pd-activated olefin provides pyrrolidine 1.88 and subsequent reductive elimination conjoins the pyrrolidine to the phenanthrene system affording precursor 1.89. This convergent approach was used to construct (±)-tylophorine in only six total steps and with a 35% overall yield. Successful methods to link the C4- and C5-carbons date back to the earliest phenanthropiperidine total syntheses—the first total synthesis of crytopleurine, for instance. 134, 135 This transformation is a Friedel-Crafts–type acylation. The first examples of this strategy treated the amino acid with polyphosphoric acid. 123, 146 By modern standards this OMe MeO MeO OMe S H N OO MnO 2 , PhCF 3 120 ºC, 24 h 64 % N S OMe MeO MeO OMe OO H 2 steps N O N O Cu Ph Ph TfO OTf OMe MeO MeO OMe NHBoc 3 Pd catalyst 67% Br Pd II N Boc OMe MeO MeO OMe N Boc 39% Scheme 1–21. Transition metal-catalyzed C4-C5 construction. 1.86 1.87 1.31 1.88 (R)-tylophorine 2 steps 99% 1.89 (±)-tylophorine 100 reaction provides the product in relatively poor yields (22–35%). When the acid chloride was used instead the yields greatly improved but were highly dependent on the Lewis acid used. 15 Using SnCl 4 , for example, allowed the desired product to be isolated in near quantitative yields, whereas AlCl 3 gave lower yields and caused partial demethylation of the aromatic ethers. The former strategy (i.e. SnCl 4 ) has been reported by several other groups with similar results. 60, 179 The preparation of the Friedel-Crafts starting materials has made this approach quite practical (Scheme 1–22). Akin to the first synthesis of cryptopleurine, simple displacement of the halide of phenanthrene 1.90 with an amino acid (1.91) gives rise to the requisite cyclization substrate (1.92). Thus, the stereochemistry can arise from the chiral pool using amino acids such proline or pipecolic acid. The stereochemical integrity during the Friedel- Crafts reaction is an obvious concern considering the well-known susceptibility of α-amino acid chlorides to racemization. 180 Although the final natural products are reported to be optically active, the broad range of reported optical rotation values for the isolates makes it X HN HO 2 C R R n + N HO 2 C n R R Scheme 1–22. General method to prepare Friedel-Crafts starting material. 1.90 X = Br or Cl 1.91 1.92 101 impossible to deduce the enantiomeric purity within a reasonable margin of error. Analytical chiral resolution will be necessary to settle this. It is worth noting that an intermolecular Friedel-Crafts strategy has also been used that did not result in stereochemical damage (Scheme 1–23). Nordlander and Njoroge used N- trifluoroacetylprolyl chloride (1.93) in the presence of AlCl 3 to acylate symmetrical phananthrene 1.30. 173 Using a lanthanoid chiral shift reagent the enantiomeric purity of the product 1.94 was determined by NMR and found to be greater than 98%. 1.6.3.4 N–C2 bond forming reactions One particular total synthesis effort demonstrates striking ingenuity in constructing the N–C2 bond. Iwao and coworkers reported a directed ortho-metallation strategy in the synthesis of (±)-cryptopleurine and (±)-antofine (Scheme 1–24). 181, 182 The appropriate diethylamide was lithiated with s-BuLi to provide intermediate 1.95 and quenched with an OMe MeO MeO OMe N COCF 3 O Cl AlCl 3 , CH 2 Cl 2 OMe MeO MeO OMe N O F 3 COC e.r. > 98:2 51% + Scheme 1–23. Friedel-Crafts acylation without racemization. 1.30 1.94 1.93 102 aromatic aldehyde. The oxygen of the newly formed alcohol was then translocated upon lactone formation and C-O bond reduction with Zn-Cu. With all the carbons in place, acid 1.97 was converted to the (±)-antofine and acid 1.98 into (±)-cryptopleurine. The penultimate step is the key reaction in this final sequence which forms the N–C2 bond through an acylation of the heterocyclic amine. 1.6.3.5 N–C8(C9) bond forming reactions The piperidine heterocycle (ring D) fused to the phenanthrene is completely conserved within all of these alkaloids whereas the distal heterocycle (ring E) adds a new element of structural diversity. The wealth of reliable C–N bond-forming reactions makes this disconnection an obvious choice. Indolizidine formation via pyridine alkylation has been one approach to realize this bond formation. 183 Using this approach, an inverse electron- MeO OMe MeO O NEt 2 N 2. TsOH MeO OMe MeO O O Ar Li N Bn MeO OMe MeO Ar CO 2 H 1. CH 2 N 2 2. H 2 /Pt 3. LAH 1. H 2 /Pt 2. Δ 3. LAH 1. ArCHO Ar = or KOH Zn-Cu 72–83% 63% (4 steps) 45% (4 steps) Scheme 1–24. Lactamization strategy to form indolizidine and quinolizidine. 1.961.95 1.97 (Ar = 2-pyrrolyl) (±)-antofine (±)-cryptopleurine 1.98 (Ar = 2-pyridinyl) 103 demand cycloaddition using styrenes 1.99 and 1.100 furnished pyran 1.101 which could be converted to the pyridine (1.102) upon treatment with hydroxylamine (Scheme 1–25). Further elaboration provided the tethered primary alcohol which was displaced upon treatment with mesyl chloride. Treatment with NaBH 4 reduced the resultant pyridinium species to the desired indolizidine 1.103. This strategy was applied to indolizidine and quinolizidine formation and was used to prepare, (±)-tylophorine, (±)-antofine and (±)- cryptopleurine. Ar 1 O O Me Ar 2 OMe Yb(fod) 3 99% O Ar 1 Ar 2 OMe O Me N Ar 1 Ar 2 HO 1. MsCl 2. NaBH 4 N Ar 1 Ar 2 N MeO TIPS N O CO 2 R*TIPS Cl N O TIPS NaOMe 91% OMe MeO MeO HN H OH 1. DIAD, PPh 3 OMe MeO MeO N H 2. HCHO, HCl n n 62–86% 71% 4 steps 46% (2 steps) + HH Cbz Scheme 1–25. N-Alkylation to construct ring E. 1.99 1.100 1.101 1.102 1.103 1.104 1.105 1.106 (S)-tylophorine 1.107 (R)-cryptopleurine (±)-tylophorine (±)-antofine (±)-cryptopleurine 104 Achieving similar ends in an asymmetric fashion, Comins et al. prepared enaminone 1.105 from pyridine 1.104 enantioselectively using N-acylpyridinium chemistry. 141 Upon deprotection of the carbamate chiral auxiliary the resultant amine spontaneously cyclized. The enaminone intermediate 1.106 was then used to prepare (S)-tylophorine. Similarly, an intramolecular Mitsunobu reaction of Cbz-aminoalcohol 1.107 was employed in the synthesis of (R)-cryptopleurine by Kim and coworkers. 184 Cascade reactions have a strong reputation for being able to rapidly assemble complex molecules. To construct the indolizidine in tylophorine, Leipa et al. developed a cascade process using hydrogenation conditions (Scheme 1–26). 185 The starting material was easily prepared via Michael-addition of cyanide 1.109 into unsaturated ketone 1.108. Under reductive conditions, cyanide 1.110 was first reduced to primary amine (not shown), which triggered a tandem condensation/reduction/acylation sequence, providing an excellent yield (86%) of bicyclic product 1.112. Preparing heterocycles can often be facilitated through ring expansion reactions and this too has been aptly used in the synthesis of the indolizidine core. Yerxa and coworkers reported an unconventional route to septicine starting from squarate 1.113 (Scheme 1–27). 186 Ar 1 O MeO 2 C Ar 2 CN Ar 1 O MeO 2 C Ar 2 CN H 2 , Pt/C Ar 1 MeO 2 C Ar 2 NH Ar 1 Ar 2 N O 86% K 2 CO 3 61% Scheme 1–26. Hydrogenation cascade reaction to form ring E. 1.108 1.109 1.110 1.111 1.112 Stereochemistry was not reported. 105 The necessary aryl groups were installed sequentially in a 5-step sequence yielding cylcohexadienone 1.117. Desymmetrization takes place upon addition of N-lithiopyrrole into a single carbonyl of diketone 1.114 and trapping the alkoxide with TMS-Cl yields pyrrole 1.115. Upon heating in p-xylene, pyrrole 1.115 is thought to undergo an electrocyclization ring-opening to generate an electrophilic ketene 1.116 that intramolecularly acylates the pyrrole system. Reduction of the resultant seco-analog 1.117 yields (±)-septicine. 1.6.3.6 C5a–C6 bond forming reactions Only one report of a C5a–C6 bond formation reaction exists to construct the indolizidine and quinolizidine system exists. In this example, Chuang et al. formed the indolizidine and O O N Li 2. TMSCl 1. O OTMS N p-xylene N O 86% 88% OMe OMe O O 5 steps O OTMS N O OMe MeO MeO OMe OMe MeO MeO OMe MeO OMe OMe MeO OMe MeO MeO OMe Scheme 1–27. Ring expansion strategy to form ring D. 1.113 1.114 1.115 1.1161.117 106 quinolizidine nucleus by way of a radical cyclization reaction (Scheme 1–28). 56, 68 Alkyl iodides 1.118 (n = 1 and 2) were treated with AIBN as a radical initiator to yield the desired indolizidine and quinolizidine intermediates 1.119. This reaction provides the immediate precursors to the phenanthropiperidine alkaloids. 1.6.3.7 Angular OH or Me groups The rare 13α-methylphenanthroindolizidines, such as the hypoestestatins, are attractive targets because of their unique structures, low abundance and therapeutic potential. 26-28 The methyl group at the ring juncture, however, presents a formidable challenge for synthetic chemists. A singular report discloses a possible route to these alkaloids. 187 Phenanthryl bromide 1.120 was treated with azobis-cyclohexanecarbonitrile (ACN) triggering a 6-endo/5- endo radical cascade cyclization yielding amide 1.121 (Scheme 1–29). To prepare hypoestestatin 1, the amide was reduced with LAH. The NMR spectra, however, did not match with that of the isolated alkaloid. The reason for this discrepancy remains unclear. OMe MeO MeO N I O OMe AIBN, Bu 3 SnH OMe MeO MeO N O OMe n n 87% (n = 1) 80% (n = 2) PhMe Scheme 1–28. Radical cyclization to form the C5a–C6 bond. 1.118 1.119 107 Phenanthroindolizidines bearing 13α-hydroxy groups are also uncommon but considerably more challenging to synthesize. 29, 188 The labile aminal functionality and susceptibility to oxidation have most likely hampered synthetic efforts. Interest in the biological activity of these alkaloids is still of great interest considering the extraordinary anti-proliferative activity of tyloindicine F and G. 1.6.4 Closing remarks The synthetic interest in the phenanthropiperidine alkaloids has given rise to a plethora of novel reactions that accentuates the artistic nature of organic chemistry. We have attempted to present these strategies in a new light, focusing on key transformations rather than on complete reaction sequences. More specifically, we opted to address reactions that result in the consummation of predefined substructures (i.e. the phenanthrene or fused heterocycle core) and by doing so have, unfortunately, excluded some creative methods used to construct their components. Nevertheless, we hope this review will contribute to the development of N O MeO OMe MeO Br N MeO OMe MeO Me O Bu 3 SnH LAH 100% 36% ACN Scheme 1–29. Synthesis of hypoestestatin 1 via cascade radical cyclization. 1.120 1.121 hypoestestatin 1 (NMR did not match) 108 improved reaction sequences to address limitations in yield, stereoselectivity and economy, and to encourage the preparation of novel structures with improved therapeutic utility. 1.7 Summary This review featured a class of alkaloids, the phenanthropiperidines, which have long been known for their medicinal properties but have yet to be refined into a clinically useful drug. These alkaloids show remarkably potent anti-proliferative and anti-inflammatory/ immune activity, providing impetus for their development as therapeutic agents. Their mechanism of action likely involves multiple targets. Evidence suggests that inhibition of protein synthesis through the 40S ribosomal subunit may play a role in their anti-proliferative effects. Moreover, the best-studied phenanthropiperidines perturb NF-κB–mediated transcription and other major signal transduction pathways. There is also evidence that these planar alkaloids intercalate DNA and RNA. Whatever the cause, the upshot is cytostasis and cell differentiation—not death—that leads to long lasting suppression of cell growth. Despite this positive outlook there is still need for refinement. In regard to cancer, there is a marked difference between activity in cell cultures and activity in animal models. Presumably, the alkaloid’s poor PK properties, acute toxicity and low solubility are contributing to this. There is also uncertainty about neurological side effects that could arise as they did in tylocrebrine’s clinical trials. The design of new analogs, therefore, should proceed with the goal of addressing these obstacles. 109 To serve this end, modern synthetic routes have given ready access to the alkaloids and their unnatural congeners, providing a better understanding of SARs and a foundation for designing analogs with improved pharmaceutical and pharmacokinetic profiles. These are formidable challenges, but there is not one here that medicinal chemists have not overcome in the past for already approved drugs. It is hoped that this review will stimulate and guide future investigations into this remarkable class of molecules. 110 CHAPTER 2 SYNTHESIS OF SIX- AND SEVEN-MEMBERED CYCLIC ENAMINONES: OPTIMIZATION, SCOPE AND MECHANISTIC STUDIES 2.1 Introduction The foregoing chapter described a number of obstacles that have thwarted efforts to develop a phenanthropiperidine drug. Challenges of both the biological and chemical nature have emerged and undoubtedly exacerbated one another. In particular, a dearth of novel structures within this class—whether because of synthetic limitations or a failure of synthetic chemists to apply established routes to new targets—has made iterative lead optimization burdensome and left SAR studies in a rather inchoate state. New methodologies that enable the synthesis of well-designed libraries and that diverge from the structurally homogenous collection of already-known phenanthropiperidines would prove a boon to the effort to remedy these alkaloid’s undesirable properties and improve upon desirable ones. Joining this effort, we have developed a cyclization reaction for the synthesis of enantiomerically pure cyclic enaminones and have set precedent for their use in the synthesis of these alkaloids. This chapter will discuss our investigations into the mechanism and scope of this reaction. 111 2.2 Background Enaminones or vinylogous amides (Fig. 2–1) are highly versatile functional groups. As their name suggests they are composed of an alkene, an amine and a ketone. Their reactivity, however, is orthogonal to each of these functional groups in isolation. Remove one of these functional groups and the molecule adopts a completely different nature. For instance, enamines, which lack the carbonyl, are typically prone to hydrolysis or oxidation, 189 whereas enaminones are quite stable and easily isolated. Conversely, enaminones are not as stable as the conventional amide. This, however, makes them more tractable and versatile synthetic intermediates. In addition to their distinct reactivity profile, enaminones have also attracted attention in pharmaceutical development, particularly as anti-convulsants and P-gp modulators. 190-194 Moreover, their stability and favorable physicochemical properties are exemplified by their use as orally active medicinal agents. 193 A combination of the above factors and our interest in anti-cancer natural products encouraged us to develop a synthetic route to previously inaccessible or laboriously synthesized enaminone scaffolds. O N Fig. 2–1. Generic structure of the enaminone. 112 Cyclic enaminones, particularly 6-membered enaminones (2,3-dihydro-4-pyridones), are extraordinarily versatile intermediates for the synthesis of piperidine-containing target molecules. Furthermore, this heterocycle exists in numerous drugs and drug candidates as an indispensable binding element. The piperidine moiety is also prevalent in structural classes of numerous bioactive natural products. 195-199 Considering the ubiquity of biologically active piperidine-containing compounds, practical methodologies for the synthesis of these structures, especially those bearing stereogenic centers, are of great value. The synthetic utility of the enaminone is clear when considering the reactivity of each component (amine, enamine, enone, and alkene) in isolation. The coalescence of these four functional groups in the typical enaminone endows it with four nucleophilic and two electrophilic sites. As depicted in Fig. 2–2, exploiting the unique reactivity of each position on the 6-membered, cyclic enaminone has made possible a plethora of chemoselective transformations (i.e. N-functionalization, 200, 201 O-functionalization, 202-204 C3, 205-208 C4 [1,2- addition], 209-211 C5 212-214 and C6[conjugate addition] functionalization 215-220 and [2+2] cylization, 221 etc.). 113 Many approaches have been developed to construct the 6-membered enaminone core yet only a few are capable of affording non-racemic products (Scheme 2–1). Comins and co- workers have set precedent for the asymmetric synthesis of enaminones employing chiral N- acylpyridinium intermediates. 222 This method has proven to be enormously effective in providing advanced intermediates in the synthesis of numerous natural products. 140, 141, 223-229 More recent efforts have expanded the scope of this chemistry by using an assortment of chiral auxiliaries. 203, 220, 230-236 Another approach, which predates Comins’ method, is the asymmetric hetero-Diels–Alder reaction of imines with Danishefsky's diene. 237-249 In this reaction, the imputed chirality can be derived from various chiral auxiliaries appended to the imine 203, 204, 237-242 or through the use of chiral catalysts. 243-249 A noteworthy corollary of this Fig. 2–2. Select synthetic transformations of the 6-membered cyclic enaminone. 114 classic [4+2] approach has recently been reported by the Rovis group in which alkynes and alkenylisocyanates undergo [2+2+2]-cycloaddition in the presence of a chiral rhodium catalyst. 250-254 Although currently limited to the synthesis of the indolizidine enaminones, this method provides rapid access to these bicyclic molecules with impressive enantioselectivity. Despite the success of these asymmetric approaches, innate limitations, such as ring-size and substituent constraints, warranted an exploration of new avenues for the construction of this useful scaffold. N MeO 1. ClCO 2 R* N O CO 2 R* R TIPS TIPS * N O R 1 R 2 N R 1 R 2 + OMe TMSO catalyst * R 1 , R 2 or catalyst are chiral N O R 1 R 2 R 3 + R 1 R 2 Rh I /L* N C R 3 O N O R 4 R 3 R 2 Boc R 1 1. deprotection 2. cyclization N O R 1 R 4 R 3 R 2 * * * * 2. RMgX N MeO TIPS R CO 2 R* H 3 O + Scheme 2–1. General approaches to access non-racemic 6-membered enaminones. Hetero Diels-Alder Comins Rovis This work 115 In pursuit of this goal, a novel ring-forming reaction of amino acid-derived ynones to yield cyclic enaminones has been developed. 255 Our route accesses the target molecules in high enantiomeric purity by utilizing the chiral pool strategy to take advantage of the chirality of readily available starting materials. A notable feature of this methodology is its ability to generate previously unavailable or circuitously-constructed substrates, namely, bicylic enaminones, 2,5-disubstituted enaminones, and enaminones with α- and β- stereocenters. This concise and operationally facile pathway gives ready access to novel 6- and 7-membered enaminones. 2.3 Reaction development 2.3.1 Strategy and considerations for enaminone methodology As previously said, methods for the construction of non-racemic dihydropyridones (i.e. 6- membered cyclic enaminones) are scarce and the existing few, although highly contributive to this area, have left many structurally simple enaminones out of reach. Thus, we envisioned a complementary route to these molecules using the chiral pool strategy (Scheme 2–2). An amine tethered to an ynone could potentially serve as the requisite precursor to our desired scaffold through an intramolecular Michael reaction. The amino-ynone substrates could conceivably be obtained from β-amino acid precursors. This approach was attractive to us not only because it could furnish pyridones with four distinct appendages and two stereogenic centers, but also because it uses easily accessible chiral starting materials. The 116 strategic use of β-amino acids and their immediate precursors entrusts the chiral pool and well-established asymmetric chemistry 256-263 to provide the desired diversity and complexity of the final products. Despite the numerous examples of intermolecular 1,4-additions of amines to ynones, precedent for an intramolecular reaction as proposed is rare. From the outset, we questioned if suitable amine/ynone orbital overlap could be achieved and were concerned about adverse modes of addition that would yield undesired products. Scheme. 2–2. Retrosynthetic analysis of enaminone construction. 361 N R 1 R 2 R 3 O R 4 N H R 2 R 1 R 3 O R 4 N H OH R 2 R 1 R 3 O enaminone amino-ynone β-amino acid * * * * * * O R X X = nucleophile = R X O R X O R X adds to non-conjugated π-orbital X adds to conjugated π-orbital X O R H + H + Fig. 2–3. Possible modes of addition for 6-endo-dig cyclization. 361 117 Taking Baldwin’s rules for ring closing reactions into account, 264 there are several alternative pathways for this reaction that needed to be considered. Although the proposed 6- endo-dig mode of cyclization appeared to be favored, there remained some uncertainty about our system’s propensity towards the (also favored) 5-exo-dig pathway. We expanded our literature search to reactions of other first-row periodic elements that could undergo 6-endo- dig ring-closures. The few successful examples that we found relied on either metals or π- acids to activate the alkyne for nitrogen addition. 265-273 Deslongchamps and co-workers, however, reported the intramolecular 1,4-addition of β-ketoester nucleophiles into ynones. 274 A new, stereoelectronically-controlled pathway was proposed for this cyclization where, instead of an ordinary addition into the conjugated π-system, the nucleophile adds to the orthogonal π-orbitals to generate a vinylic anion, thereby, circumventing the formation of a highly strained, allenic enolate (Fig. 2–3). This mode of addition was predicted to be applicable for cyclizations of rings smaller than eight atoms 275 and, hence, would be relevant for our system. Fig. 2–4. Potential modes of α- and β-racemization/epimerization. 361 N H O α β NH 3 O + retro-Michael N H O + R 1 R 2 R 1 R 2 N H OR 1 R 2 retro- Mannich R 1 R 2 118 Besides the questions about the mode of addition, concerns arose about racemization and epimerization of the pre-established stereocenters also had to be investigated. Enolization processes leading to loss of α-chiral centers are commonplace and would need to be avoided. We also considered the likelihood of β-amino ketone intermediates to undergo retro-Michael/ retro-Mannich-type processes (Fig. 2–4). 276-278 If this occurred, both the α- and β- stereocenters would be at risk. Worse yet, substrate decomposition could occur if the retro- Michael and Mannich processes were not reversible. With these potential hurdles in mind we ventured to construct the requisite ynone starting materials. 2.3.2 Synthesis of Weinreb amide and ynone intermediates Scheme 2–3. Methods for preparing Weinreb amides 2.1–2.13. 1) TEA, ClCO 2 Et, Et 2 O; then CH 2 N 2 2) 10 mol% Ag(CO 2 CF 3 ) HN(OMe)Me, THF EDCI, NMM N CO 2 H R 2 R 3 Boc N CO 2 H R 2 Boc R 1 R 1 NH 2 1) Methyl acrylate; then Boc 2 O 2) i-PrMgCl, THF HN(OMe)Me•HCl N R 2 Boc O N OMe MeR 1 R 1 HN(OMe)Me•HCl CH 2 Cl 2 N R 2 R 3 Boc R 1 O N OMe Me N Boc O N OMe MeR 1 β-amino acid α-amino acid 1º amine Method A Method B Method C 119 Three routes were devised to obtain β-amino Weinreb amides, the immediate precursors of the desired ynone starting materials. In the cases where the Boc-β-amino acid was commercially available, an EDCI coupling with N,O-dimethylhydroxylammonium chloride in the presence of N-methylmorpholine (NMM) furnished the corresponding amide in a single step (Method A, Scheme 2–3). Alternatively, Boc-α-amino acids were converted to diazoketones which, in the presence of catalytic silver(I) trifluoroacetate, collapsed to the corresponding ketene. The ketene intermediate was trapped in situ with N,O- dimethylhydroxylamine providing the desired Weinreb amides (Method B). Unsubstituted Weinreb amides were synthesized through a one-pot Michael-addition/Boc-protection sequence to afford Boc-β-aminomethylesters (Method C). Treatment of the methylesters with N,O-dimethylhydroxylammonium chloride and isopropylmagnesium chloride (i- PrMgCl) afforded the desired amides. 279 In the event that the Boc-protected amines required N-alkylation, this was accomplished subsequent to amide formation using sodium hydride and an appropriate alkyl halide. In the final step, the desired ynones were obtained from the Weinreb amides through the addition of excess (5 equiv) alkynylmagnesium reagents (Scheme 2–4). These simple steps could be conducted on multi-gram scale with minimal diminution of yield. Scheme 2–4. Method used for preparing ynones from Weinreb amides. N R 2 R 3 Boc O R 1 R 4 XMg THF R 4 N R 2 R 3 Boc R 1 O N OMe Me 2.1–2.13 2.14–2.33 120 121 Table 2–1. Preparation of ynones from Weinreb amides. entry Weinreb amide ynone R method a overall yield b 1 N N O Boc OMe Me 2.1 N O Boc R 2.14 H B 60 2 2.15 Me B 62 3 2.16 Ph B 60 4 N N O Boc Me OMe 2.2 N O Boc R 2.17 H A, B 95, 68 5 2.18 Me A, B 93, 66 6 2.19 Ph A, B 92, 66 7 N N O Boc Me OMe BnO 2.3 N Boc O BnO R 2.20 H B 65 8 2.21 Me B 64 9 2.22 Ph B 60 10 N N O Boc Me OMe R 2.4 N Boc O R 2.23 α-OH B 52 11 2.24 β-OH B c 45 2.5 12 N O N O Boc OMe Me 2.6 N O O Boc 2.25 A 94 13 NMe H H O Boc N OMe Me 2.7 NMe Boc O H H 2.26 cis A 87 14 2.8 2.27 trans A 87 15 O N Ph HN Boc OMe Me 2.9 OPh HN Boc 2.28 A 75 16 O NHN Boc OMe Me Ph 2.10 O HN Ph Boc R 2.29 H B 30 17 2.30 Me B 39 19 O NN Boc OMe Me R 2.11 O N Boc R 2.31 H A 85 20 2.12 2.32 PhCH 2 C 67 21 2.13 2.33 Ph C 46 a See Scheme 2–3. b Isolated multi-step yield from commercially available starting materials. c Stereocenter was inverted from Weinreb amide precursor using the Mitsunobu reaction. Due to our interest in indolizidine and quinolizidine natural products, we chose to synthesize ynones 2.14–2.19 as potential precursors to these important heterocycles (entries 1–6, Table 2–1). The isolated stereogenic center on the pyrrolidine ring of ynones 2.20–2.24 was to be used to facilitate detection of the β-epimerization (entries 7–11). Likewise, cyclohexyl systems 2.26 and 2.27 would allow α-epimerization to be detected (entries 13 and 14). Finally, acyclic ynones 2.28–2.33 were synthesized to obtain monocyclic enaminones (entries 15–21). Although the enaminones to be generated from ynones 2.31–2.33 will be relatively unembellished, we were attracted to these targets because of a surprising lack of general routes to obtain them in spite of their simplicity. 2.3.3 Optimization of enaminone formation Seminal optimization studies for this reaction were conducted by B. J. Turunen who envisioned a protocol in which the Boc-protecting group would be removed to liberate a nucleophilic amine that would, in turn, react with the tethered ynone moiety. Turunen found success in a two-step deprotection/cyclization protocol to provide enaminone 2.34 (Table 2– 2). 255 The use of 4N HCl (entries 4–7, Table 2–2) or TMS-I (entry 8) consistently gave higher yields than when TFA was used to deprotect (entries 1–3), regardless of the cyclization method. We were initially perplexed by this result. The putative ammonium salt intermediates of each method would only differ with respect to their counter ions (Cl - , I - or CF 3 CO 2 - ). Upon closer scrutiny it became clear that HCl and TMS-I were acting bi- functionally. In addition to deprotecting the Boc-group, these reagents also prompted 122 conjugate additions of their halides (Scheme 2–5). Isolation of the deprotected intermediates revealed that, prior to cyclization, the ynone moiety had been converted by HCl and TMS-I to a mixture of vinyl halide I and dihaloketone II. Trifluoroacetic acid (TFA), however, left the ynone intact (III). The addition of chloride or iodide into the ynone prior to cyclization was evidently favoring ring-closure. Another important observation that was made during Turunen’s studies was the apparent dependency of the cyclization on water or MeOH. 255 Regardless of the deprotection method, the enaminone would not form in THF or CH 2 Cl 2 (entries 2, 3, 5 and 6, Table 2–2) unless water was used as a co-solvent (entries 4, 7 and 8). Although these wet solvents were Table 2–2. Optimization of deprotection and cyclization. 255 a Reaction time of cyclization step. b Isolated yield. c Sat. aqueous NaHCO 3 . d Reaction proceeded in a separatory funnel upon work-up. entry 1. deprotection 2. cyclization time a yield b 1 TFA, CH 2 Cl 2 CH 2 Cl 2 , NaHCO 3 c na d 30 2 TFA, CH 2 Cl 2 CH 2 Cl 2 , K 2 CO 3 20 h 0 3 TFA, CH 2 Cl 2 THF, K 2 CO 3 20 h 0 4 TFA, CH 2 Cl 2 CH 2 Cl 2 , H 2 O, K 2 CO 3 5 h 38 5 4N HCl/dioxane CH 2 Cl 2 , K 2 CO 3 20 h 0 6 4N HCl/dioxane THF, K 2 CO 3 20 h 0 7 4N HCl/dioxane CH 2 Cl 2 , H 2 O, K 2 CO 3 1 h 74 8 4N HCl/dioxane THF, H 2 O, K 2 CO 3 1 h 75 9 4N HCl/dioxane MeOH, K 2 CO 3 15 min 87 10 TMS-I, CH 2 Cl 2 MeOH, K 2 CO 3 30 min 95 N Boc O 1. deprotection 2. cyclization N O 2.14 2.34 123 proficient in affecting cyclization, MeOH proved to be the best solvent and was chosen to further explore this reaction (entries 9 and 10). With these optimized conditions we proceeded to establish the scope of this reaction. 2.3.4 Mitigating stereochemical damage during enaminone formation Shortly after we embarked on our study of reaction scope we encountered a formidable challenge. As we had feared, ynones bearing α- or β-stereocenters, when subjected to our one-pot procedure, yielded mixtures of diastereomers or partial racemates. We suspected enolization or β-amino elimination processes were at hand; if not overcome, this would negate the principal advantage of the chiral pool approach. Although both acid- and base- induced epimerizations of this type are possible, we initially focused our attention on the acidic deprotection conditions. Since the most profound stereochemical damage was observed in proline-derived enaminones, hydroxylated ynone 2.23 was employed as our model system to assess the extent of β-epimerization under an array of deprotection conditions (Table 2–3). Scheme 2–5. Fate of ynone upon Boc-deprotection. N O Boc N O HCl or TMS-I N H 2 O X N H 2 O X X + K 2 CO 3 MeOH N H 2 O TFA K 2 CO 3 , MeOH 2.14 I II 2.34 III 124 The previously optimized deprotection conditions had epimerized 15% of the isolated product (entry 1, Table 2–3). In neat and dilute TFA, the yields and diastereomeric ratios of enaminone 2.35 showed no improvement (entries 2 and 3). Slow addition of HCl in ether until all the starting material was consumed also failed to give satisfactory results (entry 4). Positing an acid-induced mode of racemization we attempted to suppress the stereochemical deterioration using basic and neutral conditions (entries 5–7). These too were ineffective. When TMS-I was used to induce Boc-deprotection we observed a marked reduction of epimerization and improvement in yields (entries 8 and 9). It should be noted that ynones have been shown to react with TMS-I at –78 ºC to form β-iodo–allenolates that, upon entry conditions yield % a (d.r.) b 1 4N HCl, dioxane 77 (85:15) 2 TFA (neat) 31 (67:33) 3 TFA/CH 2 Cl 2 (1:1) 18 (88:12) 4 1N HCl, ether 36 (67:33) 5 1. TBSOTf, 2,5-lutidine, CH 2 Cl 2 2. TBAF 0 6 TESOTf, 2,6-lutidine, CH 2 Cl 2 21 (83:17) 7 CAN, MeCN, reflux 0 8 TMS-I (3 equiv), CH 2 Cl 2 , 0 ºC 99 (75:25) 9 TMS-I (1 equiv), CH 2 Cl 2 , –78 to 0 ºC; TMS-I (2 equiv) 60 (94:6) 10 HCO 2 H, rt 0 11 NaI (3 equiv), HCO 2 H, rt 93 (> 95:5) a Isolated yield. b 1 H NMR integration. Table 2–3. Boc-deprotection and β-epimerization. 361 1. conditions 2. K 2 CO 3 , MeOH N Boc O N O HO OH 2.23 2.35 125 warming to 0 ºC, tautomerizes to afford a Danishefsky-type diene (Scheme 2–6). 280 Although this would not directly compromise the integrity of the β-stereocenter, substrates bearing α- stereocenters would be affected. To test this, we subjected enolizable substrate 2.26 to the same conditions, and observed that epimerization was even more profound than when using HCl (Scheme 2–7). In addition to the detrimental stereochemical effects of TMS-I, the necessity of rigorously dried solvents, glassware, and cryogenic temperatures provoked a search for a new, simple protocol that would tolerate epimerizable substrates. Scheme 2–6. Mechanism of α-epimerization with TMS-I. R O TMS-I –78 ºC R OTMS I H 0 ºC R OTMS I R O I H + Scheme 2–7. Epimerization of α-stereocenter using HCl and TMS-I. NBoc O Me 1. a. 4N HCl/dioxane or b. TMS-I, CH 2 Cl 2 2. K 2 CO 3 , MeOH N O Me method, % yield (d.r.) a, 96 (80:20) b, 80 (67:33) H H H H 2.362.26 126 With a wealth of known Boc-deprotection protocols to explore, we were attracted to the use of formic acid to avert epimerization. 281 The simple technique of this method was attractive considering it could accomplish the desired deprotection at ambient temperature and in open-air. Furthermore, we hoped that this relatively weak acid would lessen epimerization. Applying these conditions to ynone 2.23, the Boc-group could be easily removed, yet upon treatment with base, we did not observe any enaminone formation (entry 10, Table 2–3). As shown in earlier experiments, the success of this reaction is not only contingent on the removal of the Boc-group, but also on ynone “activation” with halides (Scheme 2–2). To this end, NaI was added as a nucleophilic halide source and the reaction was repeated (entry 11, Table 2–3). To our delight, the desired enaminone was formed in excellent yield (92%) and without any detectable epimerization. Conceivably, the remote hydroxy group could resist epimerization by directing the re-addition of the eliminated amine to the most favored anti-substituted product. We were encouraged to find that, in the absence of diasteromeric control, enaminones could be obtained in high enantiopurity (Scheme 2–8). When ynone 2.17 was subjected to these deprotection conditions a crystalline solid formed in the reaction media and was determined by X-ray analysis to be the desired deprotected, vinyl iodide intermediate (2.38, Fig. 2–5). Upon treatment of this salt with base, enaminone 2.37 was formed in less than 2 minutes. More importantly, the product was obtained with an enantiomeric ratio of 98.5:1.5 showing that this protocol had effectively mitigated β- epimerization. 127 We next investigated these new conditions on enolizable ynone 2.26 as a model for α- epimerization (Scheme 2–8). The new conditions yielded the desired product (2.36) with a d.r. of 94:6. This was a considerable improvement over the previous method where the d.r. was 80:20. Interestingly, we also saw a time-dependent increase of epimerization indicating that the acid step was indeed responsible, at least in part, for α-epimerization. Enolate formation during the basic step could also potentiate epimerization and was investigated further. 2. K 2 CO 3 , MeOH 1. NaI, HCO 2 H, 6 h N Boc O N O H 98% yield e.r. > 98:2 NBoc O Me 1. NaI, HCO 2 H, time (t) 2. K 2 CO 3 , MeOH N O Me 83% yield, d.r. = 94:6 (t = 6 h) 50% yield, d.r. = 80:20 (t = 24 h) H H H H Scheme 2–8. Racemization of α- and β-stereocenters using mild deprotection conditions. 2.26 2.17 2.36 2.37 Fig. 2–5. X-ray crystal structure of vinyl iodide 2.38. N O I I H H = 2.38 128 Vinyl iodides A, B, and C were chosen as model substrates to examine enolization (Scheme 2–9). These substrates were subjected to the cyclization conditions using deuterated methanol (CD 3 OD) as a solvent and the extent of α-deuteration was determined. Deuterium incorporation at the α-position is indicative of enolate formation revealing another potential source of epimerization. When intermediate A was treated with methanol-d 4 and K 2 CO 3 , enaminone 2.37 was immediately formed and no deuteration was observed. Once formed, this enaminone was resistant to deuterium incorporation for up to 24 h (i.e. to form enaminone 2.37-d 2 ). With the intent of slowing the cyclization we used substituted vinyl iodides B and C. For these cases, the cyclized products 2.39-d 5 and 2.40-d 2 were obtained with complete α-deuteration. Furthermore, as observed before, enaminones 2.39 and 2.40, which were formed in non-deuterated solvent, did not undergo α-deuteration under the prescribed conditions. The point has already been made that enaminones are reluctant to undergo deuteration in the presence of K 2 CO 3 ; however, an anomaly was noted when investigating enaminone 2.39 (Scheme 2–10). This substrate underwent selective and complete γ-deuteration to provide enaminone 2.39-d 3 in 2 hours. Although it is out of the scope of this work, this finding reveals another handle on this versatile scaffold for chemoselective modification. 282, 283 The collective data from the deuterium exchange reactions hinted at another liability in our approach. We feared that the extent of α-deuteration might be diagnostic of pre-cyclized intermediates that would undergo β-amino elimination. Thus, ynones 2.20, 2.21 and 2.22 were subjected to the two-step procedure (Scheme 2–11). Terminal ynone 2.20 cyclized with no observable epimerization whereas the methyl and phenyl substituted ynones (2.21 and 2.22) were obtained as diastereomeric mixtures. 129 In retrospect, these findings are not surprising. The principles of vinylogy would indeed predict an attenuated acidity of the enaminone α-position in relation to that of its ketone progenitor. Thus, α-epimerization or β-amino elimination is precluded by rapid formation of the vinylogous amide. When the rate of cyclization is retarded with sterically encumbered alkynes there is a significant increase in epimerization, thus showing the vulnerability of the enaminone precursors in basic media. Despite the use of mild deprotection conditions, these new findings suggest that base-induced epimerization is predominant when cyclization is slow. N H 2 O I R N O R = H very fast N O D D R = Me, Ph N O H H R N O I R H H D D H H N O D D CD 3 fast R = Me, Ph N O D D Ph or α 24 h slow D K 2 CO 3 CD 3 OD K 2 CO 3 CD 3 OD K 2 CO 3 CD 3 OD K 2 CO 3 CH 3 OD K 2 CO 3 CH 3 OH Scheme 2–9. Deuterium exchange during cyclization. D (R = Me) E (R = Ph) 2.39 (R = Me) 2.40 (R = Ph) 2.37-d 2 2.37 2.39-d 5 2.40-d 2 A (R = H) B (R = Me) C (R = Ph) 130 Suspicions immediately arose concerning the putative role of the acid in β-amino elimination. Perhaps the discrepancies in the stereochemical outcome of each deprotection condition were attributable to the dissimilar haloketone intermediates and their particular reactivities under basic conditions. For instance, a conspicuous difference exists when considering the halides (Cl vs. I). Furthermore, double chloride addition into the ynone occurs in the presence of HCl whereas the diiodo congener is not formed in formic acid and NaI (Scheme 2–5). We were cognizant that these dissimilarities, rather than the strength of the acid used for deprotection, could dictate the extent of epimerization by governing the rate of cyclization. Fortunately, this could be directly investigated. K 2 CO 3 , CD 3 OD N O H H CD 3 2 h N O H H CH 3 > 95% conversion Scheme 2–10. Methyl deuteration of enaminone 2.39. 2.39 2.39-d 3 2. K 2 CO 3 , MeOH 1. NaI, HCO 2 H, 6-24 h N O H R OBn N Boc O BnO R 2.20–2.22 2.41–2.43 Scheme 2–11. Epimerization of β-stereocenters on terminal and internal ynones. R yield d.r. H (2.41) 92% > 95:5 Me (2.42) 94% 2.1:1 Ph (2.43) 88% 1.5:1 131 Experimental evidence suggests that the primary cause of β-epimerization for terminal ynones is the strongly acidic HCl conditions. When ynone 2.20 was subjected to 4N HCl the resultant ammonium salts (2.44 and 2.45) were each generated as 2:1 diastereomeric mixtures (Scheme 2–12). From this we conclude that the acid is directly responsible for inducing amino-elimination. As such, this process can be mitigated when using a weaker acid such as HCO 2 H. It is important to note that the use of HCO 2 H and NaI does not assuage the stereochemical damage in cases where cyclization is slow (i.e. substituted ynones). Thus, this mild protocol is most appropriate for terminal ynones. With these insights we next investigated the reaction scope to validate the utility and generality of this protocol. 2.3 Reaction scope 2.3.1 Six-membered enaminones As can be seen in Table 2–4, a diverse collection of enaminones can be constructed using these developed methods. From this data set we intended to establish a basis for choosing appropriate deprotection conditions. Bicyclic enaminones are formed efficiently, providing a Scheme 2–12. Epimerization during deprotection with HCl. N Boc O BnO NH 2 H BnO O Cl + NH 2 H BnO O Cl Cl 4N HCl dioxane Cl Cl 2.44:2.45 = 1:6 2.44 (d.r. = 2:1) 2.45 (d.r. = 2:1) 2.20 132 facile route to quinolizidine (entries 1–3) and indolizidine (entries 4–11) scaffolds. Synthesis of morpholino-enaminone 2.49 was also feasible (entry 12). Pyrrolidine substrates were particularly susceptible to stereochemical damage; however, this could be mitigated in terminal ynone substrates by using a HCO 2 H and NaI during the deprotection step. Although the reason for this sensitivity has not been directly assessed, we believe this is due, in part, to the alleviation of pyrrolidine ring-strain. Not surprisingly, anti-substitued pyrrolidines were less prone to epimerization than their syn-counterparts (entries 10 and 11), evidencing thermodynamic control of the retro-Michael/Michael process. Installation of aliphatic and aromatic substituents adjacent to the ring-fused nitrogen was also accomplished by employing terminally substituted ynones (entries 5, 6, 8 and 9). As expected, cyclization was relatively slow (15 min to 3 h) in these substrates, but all proceeded in excellent yields. In these cases, however, the HCO 2 H-based deprotection method was ineffective at preventing β-epimerization. Enolizable stereocenters were also a potential liability considering our protocol featured both an acidic and a basic step. As shown before, the use of HCl and TMS-I were destructive when attempting to establish the cis-fused enaminone 2.36. We were pleased to find that α- epimerization could also be suppressed in this case under the milder conditions with HCO 2 H and NaI (entry 13). It should be noted that since the trans-substituted product is expected to be more stable, epimerization is likely to be thermodynamically preferred. With this in mind, it was expected that trans-fused enaminone 2.50 was more resistant to epimerization in both deprotection conditions and could be acquired in high diasteromeric purity (entry 14). We next investigated the synthesis of monocyclic enaminones. These compounds would seemingly be more difficult to form, as they have no conformational constraints facilitating 133 134 entry enaminone R method a yield b e.r. c or d.r. d 1 N H O R 2.34 H A B 87 90 97:3 > 98:2 2 2.46 Me A B 87 80 73:27 73:27 3 2.47 Ph A B 91 85 58:42 69:31 4 N O R H 2.37 H A B 89 96 70:30 98:2 5 2.39 Me A 87 – 6 2.40 Ph A 89 – 7 N O R H BnO 2.41 H A B 94 92 67:33 >95:5 8 2.42 Me A B 87 94 63:37 68:32 9 2.43 Ph A B 85 88 63:37 60:40 10 N O H R 2.35 α-OH A B C 77 93 94 85:15 > 95:5 96:4 11 2.48 β-OH A B C 60 95 70 60:40 92:8 86:14 12 O N O 2.49 A B 80 95 – – 13 N O H H Me 2.36 cis A B C 96 83 80 80:20 94:6 67:33 14 2.50 trans A B 99 82 > 95:5 > 95:5 15 HN O H 2.51 A B 50 50 > 99:1 > 99:1 17 HN O R H 2.52 H A 92 > 95:5 18 2.53 Me A 96 > 95:5 20 N O R 2.54 H A B 70 92 – – 21 2.55 PhCH 2 A B 50 86 – – 22 2.56 Ph A B 50 70 – – a Method A: a) 4N HCl/dioxane, 15 min. b) K 2 CO 3 , MeOH; Method B: a) NaI (3 equiv), formic acid, 6-24 h. b) K 2 CO 3 , MeOH; Method C: a) TMS-I, CH 2 Cl 2 , –78 to 0 ºC. b) K 2 CO 3 , MeOH. b Isolated yield. c Chiral HPLC. d 1 H NMR integration. Table 2–4. Substrate scope for the preparation of six-membered enaminones. ring-closure. In this regard, acyclic β-aminoynones were found to be viable substrates (entries 15–22). Even sterically encumbered, internal ynones undergo cyclization to form monocyclic products (entry 18). In cases where the extruded amine is not tethered to the ynone, as in bicylic substrates (entries 1–12), the retro-Michael process leads to degradation instead of epimerization. Hence, it was observed that several monocyclic products were obtained in lower yields than their bicyclic analogs were. The observation that a subtle ring expansion had profound effects on the extent of β- racemization (entry 1 vs. 4) led us to attempt the construction of phenylglycine-derived enaminone 2.51 (entry 15). We envisioned that this substrate would be particularly susceptible to β-amino elimination. Expecting to see a distinct improvement in yield when formic acid was used instead of HCl, we were surprised that both deprotection methods were equally suited for this sensitive substrate. By conducting this reaction in an NMR tube and carefully monitoring its progression, it became apparent that degradation (i.e. β-amino elimination) occurred upon the addition of methanolic K 2 CO 3 and not during deprotection. The complete retention of stereochemistry suggests that this process was irreversible in contrast to cases where the extruded amine remains tethered to the resultant Michael acceptor (entries 1–12). From these results we suggest alternative methods be used to access enaminones of this type. Fortunately, this is well within the scope of both the Comins’ N- acylpyridinium 230-236 and the hetero Diels-Alder approach. 237-249 In addition to introducing steric bulk, attenuating the nucleophilicity of the amine would seemingly impede an efficient ring-closure. The synthesis of enaminone 2.56 demonstrates that despite the use of a significantly less reactive anilino nitrogen, cyclization still occurs (entry 22), albeit in lower yields. 135 2.3.2 Seven- and five-membered enaminones With these favorable results for constructing 6-membered enaminones, we next explored the feasibility of constructing 5- and 7-membered rings. This method lacks the strict confines of ring size for the alternative routes to cyclic enaminones. Our initial attempts to cyclize α- amino ynones to form 5-membered enaminones were unsuccessful. This is consistent with our hypothesis that this reaction proceeds through an endo-trig mode of cyclization (5-endo- trig is disfavored). The synthesis of 5-membered enaminones remains a limitation of this methodology. Previously reported methods for the construction of cyclic enaminones have not been amenable to the synthesis of 7-membered rings either. Furthermore, to our knowledge, there is no general method for their construction. Without any alteration of our protocol, γ-amino ynones 2.57–2.2.60 rendered four novel 7-membered enaminones (entries 1–4, Table 2–5). Notably, pyrrolo[1,2-a]azepine 2.61 bears resemblance to the core and distinguishing feature of the well-studied Stemona alkaloids. 284, 285 This unique molecule and its piperidino congener 2.62 were both attainable in good yields (entries 1 and 2). Spirocyclic enaminone 2.63 and baclofen-derived enaminone 2.64 were also readily obtained via our deprotection/ cyclization protocol (entries 3 and 4). Although all of these enaminones were constructed in racemic form—hence, their stereochemical liabilities were not investigated—we have no reason to believe that they are susceptible to the stereochemical deterioration seen in β-amino ynones. 136 2.3.3 Summary of reaction scope In summary, we have developed two complementary protocols for synthesizing 6- and 7- membered enaminones. The first method, using HCl, is rapid and able to activate and deprotect internal and terminal ynones in under 15 minutes. It is best suited for substrates without α-stereocenters or those that are not sensitive to acid-induced β-amino elimination. The second method, using formic acid and NaI, although requiring longer reaction times (6-24 h), is ideal for terminal ynones with sensitive α- and β-stereocenters. Both procedures 137 Table 2–5. Seven-membered enaminone preparation. entry ynone a enaminone method b yield c 1 N Boc O 2.57 N O 2.61 A B 60 64 2 N Boc O 2.58 N O 2.62 A B 64 63 3 O N Bn Boc 2.59 N O Bn 2.63 A B 81 84 4 O N Bn Cl Boc 2.60 N O Bn Cl 2.64 A B 66 65 a See Chapter 6 for the synthesis of ynones 2.57-2.60. b Method A: a) 4N HCl/dioxane, 15 min. b) K 2 CO 3 , MeOH; Method B: a) NaI (3 equiv), formic acid, 6-24 h. b) K 2 CO 3 , MeOH; c Isolated yield. are operationally facile and can be carried out in a single vessel. Furthermore, these experimentally simple and environmentally benign conditions are conducive to production of multi-gram quantities of these enaminone scaffolds. 286 2.4 Mechanistic insights During early optimization studies, Turunen stumbled across two notable features of this reaction during optimization that lay the groundwork for our mechanistic studies. Firstly, when the deprotection reaction did not result in vinyl halide intermediate, the yields were significantly lower. Second, the desired enaminone could was not formed unless water or MeOH were used during the cyclization step. By isolating the pre-cyclized intermediates A and B (Fig. 2–6) and verifying a complete consumption of the ynone moiety, the first stipulation (i.e. the need for a halide source while deprotecting the Boc-group) became clear. In sufficiently acidic conditions, halides add into the ynone group forming a vinyl halide that readily undergoes ring-closure to form the desired enaminone. When the ynone remains intact, cyclization is not efficient and intermolecular processes (i.e. polymerization) predominate. Therefore, pre-activation is required for a successful reaction. The discovery that the ynone group was transformed in the presence of halogenic acids demanded a reevaluation of our originally proposed 6-endo-dig mechanism. With direct evidence for the formation of halides A and B, a direct 1,4-addition 6-endo-trig cyclization/ halide elimination would be one plausible mechanism (Fig. 2–6, Pathway A). We were initially impressed, however, by the strong solvent dependence of this reaction. 255 When the 138 reaction was carried out in bulky alcoholic (s-BuOH or i-PrOH) or non-nucleophilic solvents (CH 2 Cl 2 or THF) the reaction was significantly impaired. On the other hand, the use of water or MeOH was highly beneficial and independent of the mode of deprotection. In the first disclosure of this reaction, a mechanism was proposed in which an oxygen nucleophile directly involved in bond-making and bond-breaking. 255 Therein, it was suggested that an addition of MeOH into vinyl chloride C occurred. Then, upon extrusion of the chloride ion the resultant oxonium intermediate E could undergo ring-closure in a 6-exo-trig fashion (Pathway B, Fig. 2–6). Fig. 2–6. Two potential mechanistic pathways of cyclization. HN Cl O Pathway A 6-endo-trig N O H 2 N Cl O Cl + H 2 N Cl O Cl Cl K 2 CO 3 Pathway B HN Cl O RO HN O RO Cl N O OR Cl 6-exo-trig ROH ROH HCl N O HCl H N Boc O 2.14 A B C D E G 2.34 F 139 In an attempt to gain mechanistic insight through detecting transient intermediates, Turunen dissolved the mixture of A and B in methanol-d 4 and monitored the reaction mixture via 1 H NMR after adding a solution of K 2 CO 3 dropwise. Dichloride B was first converted into vinyl chloride A and subsequent additions led to the formation of enaminone 2.34 with no other detectible intermediates. When vinyl iodide analog of A, generated from NaI and formic acid, was subjected to these same conditions the product was also formed with no detectable intermediates. Although Pathway B provided a plausible explanation for the aforementioned solvent effects, dissimilarities in the solubilizing powers of each solvent would still need to be ascertained. Impaired yields would be expected if the protonated amine intermediates (A and B) or the base (K 2 CO 3 ) were poorly soluble. Whether or not a nucleophilic solvent was necessary, however, was difficult to directly assess due the insolubility of the reaction constituents in most organic solvents. To answer this question, we explored alternative organic bases that we expected would allow the reaction to be carried out in non-nucleophilic solvents. Our first success demonstrated that triethylamine (TEA) could be used instead of K 2 CO 3 (Scheme 2–13). As hoped, this base was not only compatible with MeOH but could be used with non-nucleophilic solvents, such as MeCN and DMSO, resulting in the 1. 4 N HCl/dioxane 2. Base: TEA Solvent: MeOH, MeCN or DMSO N Boc O N O Scheme 2–13. Probing cyclization mechanism through model reactions. 2.14 2.34 140 formation of enaminone 2.34. Since these solvents are hygroscopic, adventitious water could potentially facilitate the cyclization in a catalytic manner. To rule out this possibility, we constructed ynone 2.65 to determine the fate of the electrophile in the absence of a tethered nitrogen nucleophile (Scheme 2–14). Following treatment with 4N HCl, which formed a 1:6 mixture of ketones 2.66 and 2.67, the residue was dissolved in MeCN and TEA was added. The NMR spectrum revealed vinyl chloride 2.66 as the sole product. Moreover, upon addition of excess D 2 O, vinyl chloride 2.66 remained intact. Clearly, trace amounts of water were not facilitating the cyclization when the reaction was carried out in MeCN. Delving further we examined the reactivity of intermediates 2.66 and 2.67 in the presence of methanolic K 2 CO 3 . If MeOH facilitated the cyclization through a conjugate addition it would be expected that the isolated vinyl halide 2.66 would be consumed more rapidly than the vinyl chloride A (Fig. 2–6) could form the enaminone. In other words, if the rate of amino cyclization is faster than the rate of addition of MeOH, the former process would Ph O 4N HCl Ph O Cl + Ph O Cl Cl TEA, MeCN >95% yield (NMR) Ph O OMe MeOH K 2 CO 3 2.65 2.66 2.67 2.68 2.66:2.67 = 1:6 33% conversion after 2 h 2.68:2.66 = 1:2 Scheme 2–14. Reactivity of ynone in the absence of an amine. 141 obviate the latter. To test this, a mixture of intermediates 2.66 and 2.67 were reacted in MeOH and K 2 CO 3 (Scheme 2–14). After 2 hours the composition of the crude reaction mixture consisted of a 1:2 mixture of vinyl ether 2.68 and unreacted vinyl chloride 2.66 (33% conversion). Thus, the addition of MeOH to the vinyl chloride is much slower than the cyclization process which occurs in less than 5 minutes. From this data, it is reasonable to suggest that the addition of MeOH into a vinyl chloride should not be invoked in the reaction mechanism. Thus, we suggest that these results are strong evidence for a direct 6-endo-trig mode of cyclization (Pathway A, Fig. 2–6). 2.5 Summary We have developed a practical route to non-racemic 6- and 7-membered enaminones starting from amino acids, providing a complementary approach to preexisting methods. Using this approach, asymmetry can be derived from the rich supply of commercially available amino acids. Moreover, access is gained to enaminones that are substituted at four positions, two of which are stereogenic centers. The key reaction and final step in this sequence is a novel one-flask deprotection/ cyclization reaction of Boc-aminoynones. Two methods were developed to achieve the Boc- deprotection that are suitable for different substrates. The use of 4N HCl in dioxane is most fitting for internal ynones or those without sensitivity to acid-mediated side-reactions. Alternatively, for those substrates that have enolizable stereocenters or are susceptible to acid-promoted β-amino elimination (i.e. retro-Michael) processes, NaI and formic acid are well-suited. The latter conditions provide a mild alternative and work best for terminal 142 ynones. Both protocols are economic and operationally facile, having no need for dry solvents/reagents and can be conducted at room temperature. Furthermore, both methods can be carried out on multi-gram scale with comparable yields. The substrate scope of this reaction was also assessed. It appears that this reaction is general for the construction of 6- and 7-membered enaminones. When the deprotection conditions are judiciously chosen, monocyclic and bicyclic heterocycles can be obtained from amino-ynones in high enantiomeric or diastereomeric purity. Many of the heterocyclic scaffolds reported here, though structurally simple, are unprecedented in the literature. The mechanism of the final cyclization sequence has also been investigated. The success of this reaction relies on the conversion of the ynone moiety into a vinyl halide species and trapping of the protected amine as an ammonium salt following Boc-degredation. This pre- activation, allows for an efficient intramolecular 1,4-addition once the free amine is released upon the addition of base. Thus, this reaction is thought to proceed through a 6-endo-trig ring-closing process. We believe that the asymmetric scaffolds constructed herein will be of great synthetic value for library development and complex molecule synthesis. Specifically, the synthesis of the phenanthropiperidines will be expedited by this direct route. This will be demonstrated in the following chapters. 143 CHAPTER 3 PALLADIUM(II)-CATALYZED CROSS-COUPLING OF CYCLIC ENAMINONES AND ORGANOTRIFLUOROBORATES 3.1 Introduction To expand our method for constructing enantiomerically pure enaminones, we began to investigate practical methods for elaborating this scaffold. Our goal was to install aryl groups at the C3-position, which we envisioned to be an essential transformation en route to the phenanthrene system of the phenanthropiperidine alkaloids. Importantly, this approach, if successful, would be applicable to a broad range of useful 3-arylpiperidine scaffolds besides the phenanthropiperidines. Indeed, many compounds which feature this scaffold are known to have high affinity for an array of validated targets (Fig. 3–1). 198, 287 Fig. 3–1. Examples of 3-aryl piperidine medicinal agents. N OH (R)-preclamol N Me H OH (S)-ipalbidine Nabitan N O O O N anti-emetic/analgesic (CB agonist) non-addictive analgesic D2 agonist 144 Palladium-catalyzed carbon-carbon (C–C) bond formation has been widely used in organic synthesis for arylation reactions, and, therefore, it was an obvious choice for this transformation. Arylation of the C3-position has been accomplished via a two-step sequence involving halogenation and subsequent coupling using a Pd(0)-catalyst, but we realized a more direct arylation protocol that allowed us to avoid preactivation of the enaminone was possible. Herein, we describe our efforts to circumvent the halogenation by taking advantage of the inherent nucleophilicity of the enaminone, which led us to the discovery of a Pd(II)- catalyzed C–H arylation reaction using trifluoroborates. 3.2 Background to Pd-catalyzed cross-coupling Palladium-catalyzed cross-coupling reactions have emerged as an incredibly versatile method for constructing C–C and carbon-heteroatom (C–X) bonds. Applications of cross- coupling in complex natural product syntheses and commercial scale production have demonstrated the utility of this methodology (Fig. 3–2). 288, 289 Materials as far reaching as electron conductive polymers and liquid crystals have relied on cross-couplings for their preparation. 289 Moreover, the routes to numerous drugs, including Eniluracil, Losartan and Rofecoxib, have these coupling reactions as key steps. 288 145 Following the initial reports of cross-coupling reactions by Kumada and Curriu in the 1970s, the number of publications related to this field have grown dramatically. 289 The versatility of these methodologies can be attributed to the tremendous amount of work done to expand the scope of palladium catalysts and coupling partners. The mechanism of the reaction has received much attention and, although the details may be uncertain, is thought to proceed through three general steps (Fig. 3–3): 1. Oxidative addition of an organic electrophile (R–X) into Pd(0) 2. Transmetallation between Pd(II) and an organometallic nucleophile (MR’) 3. Reductive elimination to regenerate Pd(0) and form the C–C σ-bond Highlighted bonds were formed using a Pd-catalyzed coupling reaction. Fig. 3–2. Applications of Pd chemistry. NH NN N N Losartan (Dupont-Merck) Angiotensin II receptor antagonist O O MeO 2 S Rofecoxib (Merck) COX-II inhibitor N OH Cl nBu HN N H O O Eniluracil (Glaxo Smith Kline) Anticancer N N CF 3 CF 3 Me Me CF 3 Insecticide Cl OPh F Pesticide B B ORRO RO OR n Functionalized polymer F F F OC 9 H 19 F OMeC 7 H 15 Liquid crystal 146 Cross-coupling reactions that share this mechanism have been categorized based on the nature of the organometallic nucleophile (M–R’). Among these are the Suzuki-Miyaura (boron-mediated), Migita-Kosugi-Stille (Sn-mediated), Hiyama (Si-mediated), Negishi (Zn- mediated), Kumada (Mg-mediated) and Sonogashira (Cu-mediated) reactions. Each of the organometallic species involved in these reactions exhibit unique reactivity and chemoselectivity profiles allowing reactions to proceed in a broad range of environments— pH and temperature, for example. The Heck-Mizokori reaction is mechanistically distinct from the other cross-coupling reactions and proceeds via carbometallation and subsequent β- hydride elimination, thus, avoiding transmetallation (Scheme 3–4). Fig. 3–3. Postulated general cross-coupling catalytic cycle. Pd 0 Pd II RX Pd II R R' R R' oxidative addition trans- metalation reductive elimination R = sp 3 , sp 2 or sp X = I, Br, Cl, OTf, etc. M = B (Suzuki-Miyaura) Sn (Migita-Kosugi-Stille) Si (Hiyama) Zn (Negishi) Mg (Kumada-Curriu) RX = (Sonogashira) R Cu product R X M R' M X 147 In the past decade, advancements in Pd-catalyzed cross-coupling have focused on expanding the substrate scope of these traditional methods and developing mechanistically distinct coupling reactions. Through the fine-tuning of ligands, coupling partners, transition metals and additive mixtures, some of the typical constraints seen in cross-coupling reactions have been surmounted. The use of well-chosen catalysts and substrates has even allowed the carbon-hydrogen (C–H) bond to substitute as a reactive electrophile or nucleophile in C–C and C–X bond formations. 3.3 Synthetic strategy With such extensive precedent for C–C bond formation using palladium, we laid out our plans to elaborate the enaminone scaffolds discussed in Chapter 2. The Suzuki-Miyaura reaction, which uses organoboron reagents, was chosen to install the aryl moiety based on its Fig. 3–4. Postulated Heck-Mizokori catalytic cycle. Pd 0 Pd II RX oxidative addition carbo- metallation β-hydride elimination product R X R' R' RPd II X H R' R + HX 148 well-known versatility. Boronic acids and esters, the most commonly used coupling partners in this reaction, are significantly more stable than their Mg, Zn or Cu counterparts, yet they are sufficiently reactive under mild conditions. Furthermore, these reagents are non-toxic—a limitation for organostannanes—and hundreds, if not thousands, are commercially available —a practical bonus not shared with organosilanes. The latter advantage was particularly attractive because of our desire to access phenanthrene substituents other than the overly- exploited hydroxy or alkoxy groups. To prepare the natural products, we planned to pre-activate the amino acid-derived enaminone (A) for the Suzuki protocol via halogenation (Scheme 3–1). This would allow a subsequent installation of the arene (Ar 1 ) using an appropriately substituted arylboronic acid (B→C). The final three steps of the synthesis could be completed in a manner that is not completely foreign to the synthesis of these natural products. 140, 141 From aryl enaminone C, the northern arene (Ar 2 ) could be installed using another cross-coupling reaction, only this N R 1 O halogenation R 3 R 2 R 4 N R 1 O R 3 R 2 R 4 X Ar 1 BR 2 Pd(0) N R 1 O R 3 R 2 R 4 Ar 1 N R 1 TfO R 3 R 2 R 4 Ar 1 1,4-reduction; triflation Ar 2 BR 2 Pd(0) N R 1 Ar 2 R 3 R 2 R 4 Ar 1 H [O]-biaryl coupling N R 1 R 3 R 2 R 4 Y Z X = I or Br N R 1 CO 2 H R 3 R 2 Boc Scheme 3–1. Synthetic strategy for preparation of phenanthropiperidines. amino acid A B C D E phenanthropiperidines 149 time pre-activation would generate a triflate (D), which we envisioned happening through a 1,4-reduction and enolate trapping. Triflate D could then react in a cross-coupling reaction to provide seco-analog E. Following the second arylation, the sequence could be completed with an oxidative biaryl coupling reaction furnishing the desired phenanthropiperidine. The proposed sequence has some distinct advantages over previous syntheses. First, the phenanthropiperidines are constructed from three simple building blocks—an amino acid and two boronic acids—that constitute the three extremities of the natural product. These building blocks could be easily interchanged with others from a massive collection of commercially available precursors, giving access to novel permutations of the natural products. As a corollary of this, since the piperidine system (ring D) is derived from amino acids, many of which are available in enantiomerically pure form, asymmetric syntheses will be less daunting. Finally, using various amino acids, unnatural ring E analogs could be prepared and used to probe SARs in this largely unexplored position. 3.4 Activation and classical Suzuki approach In Chapter 2, the reactivity of the enaminone was discussed. From this it is clear why the C3-position is particularly susceptible to electrophilic halogenation. The nitrogen’s nonbonding electrons are conjugated with the π-system explaining this position’s enhanced nucleophilicity. Furthermore, the α, β-unsaturated ketone amplifies the selectivity of this transformation by deactivating the C2-position. Indeed, halogenation of α, β-unsaturated ketones without appended heteroatoms occurs almost exclusively at the α-position. 150 Using enaminone 2.37 as a model substrate, we explored this promising mode of pre- activation. Treatment of the bicyclic system with iodine in the presence of DMAP furnished iodoenaminone 3.1 in near quantitive yield (Scheme 3–2). Bromination was also possible using Br 2 and TEA, but given that C–I bonds are more susceptible to oxidative insertion than are C–Br bonds, iodoenaminone 3.1 was used for the cross-coupling. Previous work in our laboratory had demonstrated that the Suzuki-Miyaura reaction is one possible method for installing the C3-aryl moiety. 213 Under optimized conditions using Pd(PPh 3 ) 4 as a catalyst, the coupling proceeds in 14 h at 100 ºC (entry 1, Table 3–1). Although microwave irradiation could be used to remedy the long reaction times without reduction (or increase) in yield, this demanded a further increase in temperature (entry 2). Observing a considerable amount of degradation under both conditions, we thought it would be profitable to pursue mild alternatives. The necessity of such high temperatures and long reaction times in spite of our use of an iodide electrophile—the most reactive of the halides —is explicable. Oxidative insertion, which is typically the rate-determining step of the Suzuki-Miyaura reaction, proceeds best when the organic electrophile (halide or pseudohalide) is electron-deficient. Since the iodide is located at the most electron-rich position of the vinylogous amide, palladium insertion is less favored. Thus, the nucleophilic N O N O I I 2 , DMAP CH 2 Cl 2 96% H H 2.37 3.1 Scheme 3–2. Iodination of enaminone in preparation for Suzuki reaction. 151 character of C3 is a two-edged sword; it accommodates pre-activation but hinders cross- coupling. Besides increasing the reaction temperature, oxidative insertion may also be promoted through the use of Pd-catalysts with higher reductive potentials. This can be achieved through the use of electron-rich ligands. In this regard, we attempted to compensate for the enaminone’s low reactivity by using the highly-nucleophilic S-Phos ligand. 290 We chose to use conditions which had been successful in the coupling of aryl chlorides—generally unreactive substrates. 291 Using the prescribed organotrifluoroborate coupling partner, we were pleased to discover this reaction proceeded at 50 ºC in 5 h (entry 3). Unfortunately, the yield was not improved over the previous conditions despite using lower temperatures. Moreover, attempts at improving the yield through additive modifications were unsuccessful. Although the best conditions would probably suffice for our purposes, a reconsideration of the enaminone’s innate nucleophilicity led us to pursue a more direct approach through C–H entry Z conditions time yield (%) a 1 b -B(OH) 2 Pd(PPh 3 ) 4 , Ba(OH) 2 , dioxane/H 2 O, 100 ºC 14 h 70 2 b -B(OH) 2 Pd(PPh 3 ) 4 , Ba(OH) 2 , dioxane/H 2 O, 150 ºC, µW 15 min 70 3 c -BF 3 K Pd(OAc) 2 , S-Phos, K 2 CO 3 , MeOH, 50 ºC 5 h 65 N O H N O I H OMe conditions OMe Z + Table 3–1. Suzuki cross-coupling conditions of iodoenaminone. 3.1 3.2 a Isolated yield. b Reaction conditions: iodoenaminone (0.1 M), Pd(PPh 3 ) 4 (20 mol%), boronic acid (1.7 equiv), Ba(OH) 2 (2.0 equiv). c Conditions: iodoenaminone (0.07 M), Pd(OAc) 2 (1.0 mol%), S-Phos (2.0 mol%), trifluoroborate (1.4 equiv), K 2 CO 3 (3.0 equiv). 152 functionalization. A word about this nascent field will explain our rational for using enaminones as C–H donors. 3.5 C–H Functionalization The direct conversion of C–H to C–C bonds is one of the final frontiers of organic synthesis. Recent developments in this field have allowed concise and economical routes to valuable materials and synthetic intermediates. 292-296 The use of stoichiometric transition metal complexes to carry out C–H bond functionalization is well-precedented. However, it was not until the early 1990s that catalytic systems were first reported. Ruthenium-, rhodium- and iridium-based catalyst systems are prevalent in scientific literature for activation of C–H bonds—that is, processes that involve oxidative insertion into a C–H bond. Palladium, on the other hand, has proven to be quite effective for processes involving electrophilic palladation and ultimately C–H functionalization. Despite early examples of C–H functionalization, this field remains largely unexplored due to four major challenges. 297 First, the typical C–H bond, sometimes referred to as the “un-functional group”, 298 is relatively unreactive. Secondly, over-functionalization (chemoselectivity) is often a favorable side-reaction. Third, the ubiquity of C–H bonds has made regioselectivity a significant challenge as organic molecules typically contain many C– H motifs that are difficult to differentiate. Finally, the functionalization of sp 3 carbons allows for the prospect, and hence the challenge, of generating stereogenic centers with high stereoselectivity. 153 From a mechanistic standpoint, the essential feature of Pd-catalyzed C–H functionalization reactions is the generation of a C–Pd bond from a C–H bond. Once this occurs, the fate of the palladium complex parallels that of the classical Pd-catalyzed cross- coupling reactions, and the desired C–C bond is ultimately formed upon reductive elimination (Fig. 3–5) or β-hydride elimination (not shown). The point in the catalytic cycle at which the coupling partner is introduced varies and is dependent on the substrate’s reactivity towards the Pd-catalyst. Coupling partners, such as aryl halides, will undergo oxidative insertion in the presence of Pd(0) prior to palladation of the C–H donor (Fig. 3–5, Pathway A). Alternatively, if an organic nucleophile, such as a boronic acid, is used, transmetallation may occur before (Pathway B) or after (Pathway C) palladation. A key distinction between these catalytic cycles is that Pathway A begins with a Pd(0)-catalyst whereas Pathways B and C begin with a Pd(II)-catalyst. The significance of this lies in the fact that Pd(0) is generated upon reductive elimination—the final step for all pathways— allowing catalytic turnover for only one (Pathway A). Unless an oxidant is present for (OI = oxidative insertion; PA = palladation; RE = reductive elimination; TM = transmetallation) Fig. 3–5. Plausible mechanisms for Pd-catalyzed C–H functionalization reactions. Pd 0 R 2 Pd II X R 2 X R 1 H R 2 Pd II R 1 R 1 R 2 R 1 Pd II R 2 M R 2 Pd II R 1 R 1 R 2 Pd II R 1 H Pd 0 [O] R 2 M R 2 Pd II R 1 H C B A Pathway A Pathway B and C OI PA RE RE TM PA TM PA 154 processes initiated with Pd(II)-mediated palladation or transmetallation (Pathway B and C), the catalytic cycle will not be reiterated. Fortunately, this can be accommodated for by adding an external oxidant which oxidizes Pd(0) into the active Pd(II)-catalyst. Their are two main strategies that have been used to selectively functionalize C–H bonds in Pd-catalyzed cross-couplings. The first strategy is to exploit Lewis basic directing groups to guide palladation. By taking advantage of chelating functionalities, such as acetamides, carboxylates and pyridines, the electrophilic catalyst is guided into close proximity of the C– H bond. There are several plausible mechanisms that can be proposed for palladation, but the exact pathway is highly dependent on the substrate, catalyst, solvent and other additives. 299 The most common and relevant for our purposes is electrophilic substitution (Fig. 3–6). In this process, the electrophilic Pd(II)-complex, which is coordinated to the directing group, reacts with the π-electrons from an appropriately situated olefin resulting in a cationic intermediate. The positive charge is then quenched upon loss of a proton which marks the cleavage of the key C–H bond. When the olefin is embedded within an aromatic system, besides quenching the positive charge, the C–H bond scission is also driven by rearomatization. Once palladation has occurred the palladacycle can be intercepted with a 155 Fig. 3–6. Directing group-assisted C–H palladation via electrophilic substitution. H Z [Pd] H Z [Pd] Z [Pd] variety of coupling partners. This is better demonstrated using case studies. Pathway A (Scheme 3–3) is illustrated well in a reaction reported by Daugulis and Zaitsev. 300 Here, aryl iodides are coupled with anilides in excellent yields. In this reaction the σ-bonded aryl- Pd(II) complex that is formed from oxidative insertion of the active Pd(0)-catalyst into the C–I bond is directed by the acetamide group for palladation. Product formation results from a reductive elimination of the resultant palladacycle. In this example, the ortho methyl group prevents this process from recurring on the opposite side. Pathway B is exemplified in a reaction reported by Shi et al., in which, like before, acetamides are used as directing groups (Scheme 3–3). 301 Unlike before, since the catalytic cycle begins with Pd(II), this reaction required the use of Cu(OTf) 2 and Ag 2 O as oxidants to regenerate the active Pd(II) species necessary for palladation. HN H Pd(OAc) 2 AgOAc CF 3 CO 2 H 90–130 ºC + HN Ac Ac 76% Me I HN Me Pd OMe N H R Pd(OAc) 2 , Cu(OTf) 2 Ag 2 O, PhMe 120 ºC, 24 h + Ar B(OH) 2 N Ar R Ac Ac 20–92% Me Scheme 3–3. Directed Pd-catalyzed C–H functionalization reactions. Daugulis and Zaitsev Shi et al. 156 The second strategy for attaining C–H selectivity is to use the innate reactivity, particularly π-nucleophilicity, of that carbon. 302 If the C–H bond in question is acidic, basic conditions can be used to assist cleavage. 299 There are many substrates, however, that are not acidic and still exhibit sufficient reactivity towards electrophilic palladation. Electron-rich aromatic systems such as phenols, indoles and pyrroles are the commonly used in this type of cross-coupling. 299, 302, 303 Although a directing group is not present, association of the Pd- catalyst and substrate is assisted by the π-nucleophilicity of the active double bond. The mechanism is essentially identical to the previously described directing group-assisted palladation. Following η 2 -coordination of the catalyst, substrate palladation occurs to furnish a stabilized cation which is subsequently quenched upon deprotonation (Fig. 3–7). As before, the cleavage of the C–H bond is driven by rearomatization. Fig. 3–7. Reactivity-assisted C–H palladation. [Pd] [Pd] [Pd] HH H [Pd] H 157 This approach has been applied in numerous settings. 299, 302, 303 Ackermann and Barfüsser have used this strategy in a direct C3-coupling of indoles and aryl bromides (Scheme 3–4). In contrast, Yang et al. reported a C2-selective cross-coupling of indole C–H donors and boronic acids. 304 These two examples represent Pathway A and C (Fig. 3–5), respectively. However, it should not be concluded from this that the regioselectivity of indole arylation is determined by these particular mechanistic pathways. Both C2- and C3-arylations of indoles have been reported for Pd-mediated C–H functionalization reactions. 305 Sames and coworkers give compelling evidence that the regioselectivity of this reaction is dependent on the ease of Pd-migration for specific reaction intermediates (Fig. 3–8). 306 It was proposed that palladation occurs exclusively at the C3-position generating an iminium which can rearrange to stabilize the Pd–C bond through the adjoining nitrogen. The rate of migration, which is influenced by the size of N-substituent and Pd-ligands, determines the regioselectivity of the reaction. In other words, rearomatization precludes Pd-migration and secures the position of C–C bond formation. Scheme 3–4. Direct C2- and C3-arylation of indoles. N H H N H Pd(OAc) 2 , HASPO K 2 CO 3 , dioxane, 95 ºC 81% Br + N H N H Pd(OAc) 2 , AcOH O 2 , rt 83% B(OH) 2 + H Ackermann and Barfüsser Yang et al. 158 The latter of these two strategies (i.e. taking advantage of π-nucleophilicity) inspired us to investigate enaminones in Pd-catalyzed C–H functionalization reactions. Like indoles and pyrroles, the enaminone is endowed with an enamine system which is exceptionally nucleophilic. The major difference between the two substrates is that the enamine in enaminones are embedded within an aromatic systems; hence, C–H bond cleavage would not be driven by rearomatization. Instead, deprotonation would be favored by the acidity of the C3-proton which is flanked by two electron-withdrawing functionalities: a ketone and the transient iminium group (Fig. 3–9). Given the switchable regioselectivity in indole systems, we considered the possibility of a similar C3/C2 palladium migration in the enaminone. We reasoned that this would not be likely based on the instability of the resultant carbocation Fig. 3–8. C3/C2 Pd-migration of palladated indoles. N R H Pd II N R H Pd II N R Pd II N R H Pd II Pd-migration N R H Pd II H + H + H H H H direct palladation 159 (being adjacent to a ketone) and the rate of the highly-favored deprotonation that would compete with the migration. The success of this approach would ultimately depend on the innate nucleophilic character of the enaminone to give access to the same putative transition-metal species as in the two-step process, only in a catalytic and one-step fashion. If this key intermediate was generated in situ, it could presumably be intercepted with a suitable coupling partner. Thus, this strategy uses what was once a liability, namely the nucelophilicity of the enaminone, as an asset, allowing us to circumvent pre-activation of the enaminone. Fig. 3–9. C3/C2 palladium migration of palladated enaminones. Pd-migration H + N O Pd II N O H Pd II N O Pd II H H N O H Pd II H H H N O H Pd II 160 3.6 Reaction optimization 3.6.1 Reoxidant screen Our first goal was to establish the feasibility of this reaction. The first conditions we investigated were inspired by a Pd(II)-catalyzed alkenylation reaction reported by Garg, Caspi and Stoltz. 307 Since our aim was to install arenes instead of olefins like the Stoltz group, we needed a suitable aryl coupling partner. One concern was that the use of an acidic cosolvent, thought to promote palladation by increasing the electrophilicity of the Pd(II) center, 308 would limit the potential of basic or nucleophile-promoted organometallic coupling partners (i.e. organozinc reagents, boronates, silanes…etc). With previous success using organotrifluoroborates—Molander et al. have found to these to be robust equivalents of organoboronic acids 309 —we chose to employ these for our investigations. Initial attempts to couple enaminone 2.55 and trifluoroborate 3.3 using stoichiometric Pd(OAc) 2 were surprisingly successful (Scheme 3–5). With good reason to move forward we next pursued conditions allowing catalytic quantities of palladium. Assuming that this t-BuOH/AcOH/DMSO Pd(OAc) 2 (1 equiv) K 2 CO 3 N O N O OMe H 60 ºC KF 3 B OMe Scheme 3–5. Direct C–H arylation of model enaminone using stoichiometric Pd. 2.55 3.4 3.3 > 50% 161 reaction would proceed through either Pathway B or C (Fig. 3–5), we investigated reoxidants to achieve catalytic turnover. Copper reagents have had longstanding use as palladium oxidants, and so we investigated several Cu(II) reagents for such a purpose (entries 1–3, Table 3–2). Each copper reoxidant proved to be effective in driving the reaction to completion using catalytic quantities of Pd(OAc) 2 . Over two equivalents of each reagent were used since palladium undergoes a two-electron oxidation (Pd 0 → Pd II ) and copper, a single-electron reduction (Cu II → Cu I ). One strategy that has been used to avoid using such high amounts of copper is to carryout the reaction in an oxygen atmosphere. Copper reagents such as Cu(OAc) 2 are known to undergo aerobic oxidation more readily their palladium counterparts and have been exploited in reactions requiring palladium oxidation. In fact, the aerobic oxidation of CuCl to CuCl 2 is among the fastest inorganic reactions—a boon for the development of industrial-scale Wacker processes. 310 Thus, catalytic quantities of the copper reoxidant can be used, because a Reaction conditions unless otherwise specified: enaminone 2.55 (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd(OAc) 2 (0.3 equiv), oxidant, K 2 CO 3 (3 equiv) at 60 °C. b Isolated yield of enaminone 3.4. entry a oxidant (equiv) yield (%) b 1 CuCl 2 (3.0) 66 2 Cu(OTFA) 2 (3.0) 82 3 Cu(OAc) 2 (3.0) 82 4 Cu(OAc) 2 (1.0) / O 2 (1 atm) 40 5 BQ (1.0) 14 6 duroquinone (1.0) 75 Table 3–2. Screen of reoxidants. O O Me Me Me Me duroquinone O O benzoquinone (BQ) 162 the active Cu(II) species is regenerated when it reacts with oxygen. Applying this strategy, the reaction was conducted in the presence of oxygen and substoichiometric quantities of Cu(OAc) 2 (entry 4). Unfortunately, the reaction yield was significantly lower than when 3 equivalents of copper were used. Benzoquinone (BQ) is another widely used palladium oxidant 302 but was deleterious in this reaction (entry 5). Unlike when catalytic amounts of Cu(OAc) 2 was used, this reaction did not simply stop progressing, rather, the starting material was shunted to an undesired byproduct (BQ-adduct C). Under the supposition that enaminone palladation precedes transmetallation, this unexpected product could conceivably arise from enaminone-Pd(II) complex A following the carbometallation and β-hydride elimination of BQ (Scheme 3–6). To prevent this side-reaction, we used duroquinone (entry 6)—a BQ analog with four methyl groups blocking the equivalent reactive positions. This reoxidant provided significantly higher yields than BQ but was not as effective as Cu(OAc) 2 or Cu(OTFA) 2 . Although Cu(OAc) 2 and Cu(OTFA) 2 were comparable as reoxidants, we chose to proceed with the more economic of the two: Cu(OAc) 2. Scheme 3–6. Mechanism for formation of BQ-adduct. N O N O Pd II Pd II O O Bn Bn HPd II N O O O Bn BQ duroquinone A CB 163 A greater concern about the fate of the reoxidant arose when we discovered a significant amount of biphenyl 3.5 was being generated (Fig 3–10). This was accounted for by considering the potential for homocoupling within our proposed mechanistic model (Fig. 3– 11). The catalytic cycle deserves further attention at this point. The mechanism for the palladation of enaminones presumably resembles that of indoles. 296 It is likely initiated by an electrophilic attack of palladium on the enaminone as already OMeMeO Fig. 3–10. Homocoupling product of trifluoroborate 3.3. 3.5 P d 0 KF 3 BX [O] Pd II X Ar PdX 2 reductive elimination Pd II Ar Ar ArAr KF 3 B-Ar N O Bn H N O Bn KF 3 BX KF 3 B-Ar N O Bn reductive elimination P d 0 [O] Pd II X Pd II Ar N O Bn Pd II Ar Ar = 4-methoxyphenyl transmetallation palladation palladation transmetallation Fig. 3–11. Proposed catalytic cycle for direct-arylation of enaminones. 3.4 3.4 3.5 2.55 164 discussed. In the presence of an appropriate organometallic substrate, transmetallation and reductive elimination would provide the desired coupled product. Alternatively, transmetallation could precede enaminone palladation providing an intermediate that would either undergo palladation or another transmetallation. If the latter occurs, reductive elimination would yield homocoupled byproduct 3.5. The upshot of this side-reaction is that both the reoxidant and aryl coupling partner are irreversibly consumed without contributing to product formation. By isolating significant amounts of the homocoupled product, we realized that this process was competing with enaminone palladation and that, if not abated in the future, excess reagents—as we have been using—would continue to be necessary to drive the reaction to completion. With that in mind, keeping the amounts of reoxidant and coupling partner constant, we sought out conditions that would improve yields and would not leave unreacted starting material. 3.6.2 Palladium-catalyst screen First investigating palladium sources, we confirmed that palladium was indeed necessary and that the reaction was not mediated by the copper reagent or trace impurities therein (entry 1, Table 3–3). Pd(OAc) 2 (entry 2) was found to be superior to PdCl 2 and Pd(CF 3 CO 2 ) 2 (entries 3 and 4). It should be noted here that the use of 0.3 equivalents of the catalyst was most effective for complete conversion in this reaction. Considering the limited stability of our enaminone substrate, the higher Pd(II)-catalyst loading allowed us to avoid long reaction times and high temperatures which were otherwise required to drive the reaction to completion. Another reason for using high-catalyst loading was because of an apparent 165 inhibition of the Pd-catalyst. This was confirmed by experiments showing that spiking the reaction mixture with additional palladium, but not with other reagents, could drive arrested reactions to completion. 3.6.3 Solvent screen Although a number of solvents were also investigated, we found that the original solvent mixture gave the best results (Table 3–4, entry 1). Each of the three components were important in this reaction; any one of them in isolation significantly reduced yields (entries 2–5). The presence of DMSO was beneficial and may help to solubilize or stabilize the palladium or copper complexes, but the exact role is unknown (entry 2). The use of AcOH as a solvent enhanced the rate of the reaction and drove it to completion, but the enaminone underwent substantial degradation when it was used as the sole solvent, and less than 10% of the product was isolated (entry 3). The role of the t-BuOH is not entirely clear either; however, we were aware that non-tertiary alcohols were prone to Pd-mediated oxidation a Reaction conditions unless otherwise specified: enaminone 2.55 (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd source (0.3 equiv), Cu(OAc) 2 (3 equiv), K 2 CO 3 (3.0 equiv) at 60 °C. b Isolated yield of enaminone 3.4. entry a Pd catalyst yield (%) b 1 None 0 2 Pd(OAc) 2 82 3 PdCl 2 61 4 Pd(CF 3 CO 2 ) 2 60 Table 3–3. Screen of palladium catalysts. 166 which would result in a rapid depletion of the reoxidant. 311 Nevertheless, t-BuOH was not an effective solvent when used by itself. Realizing that the reaction was extremely slow in the absence of AcOH, we attempted to increase the rate of reaction by raising the reaction temperature. Using several higher-boiling solvents (entries 5–7), we carried out the reaction under acid-free conditions and found that product formation was severely impaired. 3.6.4 Additive screen Our final effort to improve the yield of this reaction was to investigate the role of additives (Table 3–5). Throughout our optimization studies we had chosen to use K 2 CO 3 , but we were still uncertain of its necessity. It soon became clear that K 2 CO 3 was important simply by observing that its absence decreased the reaction yield by almost 20% (entry 2). It is extremely difficult to decipher the role of inorganic additives in complex mixtures, but we posit that the beneficial effects of K 2 CO 3 were distinct from its basic properties. This entry a solvent temperature (ºC) yield (%) b 1 t-BuOH/AcOH/DMSO 60 82 2 t-BuOH/AcOH 60 < 50 3 AcOH 50 < 10 4 t-BuOH 50 < 10 5 DMSO 100 3 6 DMF 100 6 7 PhMe 120 10 Table 3–4. Screen of solvents. a Reaction conditions unless otherwise specified: enaminone 2.55 (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3.0 equiv), K 2 CO 3 (3 equiv) at 60 °C. b Isolated yield of enaminone 3.4. 167 conclusion was borne out of the realization that K 2 CO 3 would decompose in the presence of AcOH, which is in large excess, to generate KOAc, CO 2 and H 2 O. Indeed, upon the addition of the solvent mixture to the solid reagents, we observed bubbling as evidence of CO 2 production. We reasoned from this that the reaction was aided by acetate or potassium ions or both. With this in mind, we replaced K 2 CO 3 with KOAc and found a slight improvement (entry 4). On the other hand, when NaOAc was used instead of K 2 CO 3 , we observed a marked reduction in yield (entry 3). One possible explanation of this is simply that the potassium salt is more soluble than its sodium congener. However the additives work, potassium salts are evidently beneficial. Table 3–5. Screen of additives. entry a additive A (equiv) additive B (equiv) temperature (ºC) yield (%) b 1 K 2 CO 3 (3.0) – 60 82 2 None – 60 63 c 3 NaOAc (3.0) – 60 54 4 KOAc (3.0) – 60 87 5 CF 3 CO 2 K (0.2) – 30 54 6 CF 3 CO 2 K (1.0) – 60 49 7 CF 3 CO 2 K (3.0) – 60 70 8 CF 3 CO 2 Ag (3.0) – 60 41 9 K 2 CO 3 (1.0) CF 3 CO 2 K (1.0) 50 69 10 K 2 CO 3 (2.0) CF 3 CO 2 K (1.0) 50 77 11 K 2 CO 3 (3.0) CF 3 CO 2 K (0.5) 50 70 12 K 2 CO 3 (1.0) CF 3 CO 2 K (0.2) 50 71 13 K 2 CO 3 (2.0) – 60 93 a Reaction conditions unless otherwise specified: enaminone 2.55 (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3.0 equiv), additives A and B (0.2-3 equiv) at 30-60 °C. b Isolated yield of enaminone 3.4. 168 As stated before, we observed that acids enhanced the rate of reaction. This phenomenon reflects the dependence of this reaction on an electrophilic palladation process. The acid presumably enhances the electrophilicity of the palladium complex by assisting anionic ligand dissociation. 308 Another way to enhance the electrophilicity of the Pd-complex is to use ligands that are stable anions, also assisting ligand dissociation. Thus, we investigated the effect of trifluoroacetate salts on the rate and yield of reaction (entries 5–8). Given the apparent benefit of potassium salts, we used potassium trifluoroacetate (CF 3 CO 2 K). Although the yield was not improved, the reaction time was decreased from 8 to 2 h. Quantities as low as 0.2 equivalents conferred complete conversion in less than 3 h at 30 ºC (entry 5). Silver(I) trifluoroacetate (CF 3 CO 2 Ag) did not improve yields at all (entry 8). The lower yields are attributed to the production of multiple side-products since all of the starting material had reacted, but the side-products could be identified. Attempting to maintain the short reaction times but mitigate the side-reactions, we used K 2 CO 3 in conjunction with CF 3 CO 2 K (entries 9–12). Even though the reaction times were improved, the yields did not match those in the absence of CF 3 CO 2 K. Settling on K 2 CO 3 as the only additive to be used, we took one last measure to improve the reaction, relying on the notion that lowering the concentration of the trifluoroborate at any given moment would suppress homocoupling. To do this, the coupling partner was added incrementally over the reaction time. Seeing that this provided the highest yields yet (entry 13), we used these conditions to probe the scope of this reaction by using a diverse set of enaminones and coupling partners. 169 3.7 Scope of trifluoroborates With optimized conditions in hand, we first explored coupling partners with a variety of electronic and steric properties. Electron-rich trifluoroborates underwent rapid (2-6 h) and high-yielding cross-coupling. On the other hand, electron-deficient and sterically- encumbered trifluoroborates (entries 3 and 4, Table 3–6,) required longer reaction times (24 h) and proceeded in relatively low yields. This trend should not be taken too strictly given several unpredictable results we obtained. For instance, it was unexpected that the 2- naphthyl (entry 9) and 3-hydroxyl (entry 11) systems reacted with such high yields. Interestingly, the 3-hydroxyphenyl boronic acid was poorly tolerated in our former Pd(0)- catalyzed approach, which demonstrates that this reaction is not merely a shorter method to arylate enaminones, but that it has a unique and complimentary scope as well. 213 Although electronic considerations are not acutely predictive, the generalization that electron-rich, unhindered trifluoroborates react more rapidly and in higher yields than their electron-poor or hindered counterparts is completely consistent with our findings and sheds new light on the reaction’s mechanism. Both electron-deficient and sterically-hindered coupling partners are less prone to transmetallation. That these properties are sufficient to alter the rate of reaction suggests that transmetallation is—at least in certain circumstances— the rate-determining step. This conclusion does not dismiss the importance of palladation but introduces a competing process that must be considered. 170 Besides giving valuable information about sterics and electronics, this sample set provides a cursory look into functional group compatibility. The phenol moiety is clearly stable under these conditions (entry 11) and the protected aniline group is also well-tolerated (entry 12). More significantly, the scope of this chemistry extends to aryl halides (entry 7 and 8) which react exclusively at the carbon-boron bond. The stability of these entry a Ar (product) yield (%) b entry a Ar (product) yield (%) b 1 3.6 72 7 Br 3.12 73 2 Me 3.7 86 8 Cl 3.13 71 3 Me 3.8 40 9 3.14 96 4 MeO 3.9 27 10 OMe OMe OMe 3.15 83 5 CF 3 3.10 44 11 OH 3.16 97 6 Me O 3.11 42 12 NHCbz 3.17 80 a Reaction conditions: enaminone 2.55 (0.1 M), R-BF 3 K (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3 equiv), K 2 CO 3 (2 equiv) in t-BuOH/AcOH/DMSO (20:5:1) at 60 °C. b Isolated yield. Table 3–6. Successfully coupled aryltrifluoroborates. t-BuOH/AcOH/DMSO Pd(OAc) 2 (0.3 equiv) Cu(OAc) 2 (3 equiv) K 2 CO 3 (2 equiv) 60 ºC, 3-24 h N O H N O Ar Ar—BF 3 K 2.55 3.6–3.17 171 functionalities (i.e. sp 2 halides) under the prescribed conditions reveal the complementarity of this protocol with the classical Suzuki-Miyaura reaction. As discussed already, the classic Suzuki-Miyaura protocol is initiated by a Pd(0)-mediated oxidative insertion process of the organohalide substrate. In the presence of Pd(0), aryl halides would normally react, but apparently, Pd(0), which does not contribute to product formation in this reaction, is oxidized before it can react with the carbon-halide bond. entry a R entry a R entry a R 1 MeO OMe 6 10 Ph 2 O NH 2 7 11 3 N 8 C 5 H 11 12 4 S 9 13 5 S Me 14 Me Table 3–7. Aryltrifluoroborates that did not couple. t-BuOH/AcOH/DMSO Pd(OAc) 2 (0.3 equiv) Cu(OAc) 2 (3 equiv) K 2 CO 3 (2 equiv) 60 ºC, 24 h N O H R—BF 3 K N O R a Reaction conditions: enaminone 2.55 (0.1 M), R-BF 3 K (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3 equiv), K 2 CO 3 (2 equiv) in t-BuOH/AcOH/DMSO (20:5:1) at 60 °C. b Isolated yield. 2.55 not formed 172 The limitations of this reaction were quite clear when we began to investigate more challenging coupling partners (Table 3–7). Fourteen of these organotrifluoroborates were intractable (i.e. no coupled product was observed after 24 h). Knowing that steric congestion can hinder efficient coupling, it was not surprising that 2,6-dimethoxyphenyltrifluoroborate was unable to be coupled (entry 1). Attempts to install the benzamide moiety were also unsuccessful (entry 2). Interestingly, the reaction color change (from blue/green to black) that normally coincides with cross- or homo-coupling was not observed. Although one might suggest that its electron deficiency hampers transmetallation, this does not explain the qualitative difference between equally electron-deficient 4-acetylphenyl group (entry 6, Table 3–6). We postulate instead that the free N-H group is responsible for poisoning the Pd- catalyst. This effect was presumably overridden in the coupling of the N–H-containing anilino group (entry 12, Table 3–6), because the requisite coordinating ability of that nitrogen was sufficiently attenuated by two electron-withdrawing groups: the phenyl and carbamate. Cross-coupling of pyridine and thiophene systems did not furnish the desired product and the starting material remained unreacted (entries 3–5, Table 3–7). Also, attempts to couple alkenyl and alkynyl trifluoroborates were unsuccessful (entries 6–10). This type of organic nucleophile generally undergoes transmetallation more rapidly than their phenyl congeners, and hence, homocoupling would most likely outcompete palladation of the enaminone. This is consistent with the observation that the reaction mixtures turned black almost instantaneously—indicating a coupling of some kind—but the enaminone starting material was essentially unreacted. In contrast, when using sp 3 coupling partners, the reaction mixture remained its original green/blue color, most likely reflecting the less-favored transmetallation process on alkyl centers (entries 11–14). Again, the starting material was intact after 24 h. 173 3.8 Scope of enaminones An initial investigation of enaminone compatibility revealed that monocyclic and bicyclic, unattenuated enaminones were viable substrates in this reaction. Direct arylation of the indolizidine and quinolizidine scaffolds showed the potential of this reaction to be used in the synthesis of the phenanthropiperidine alkaloids (entries 1 and 2, Table 3–8). Interestingly, the reactivity of the two scaffolds were markedly different. Not only did the quinolizidine enaminone give higher yields of product, but it was completely reacted in less than half of the time it took for the indolizidine. One possible explanation of this is that the non-bonding orbital of the lone pair on the nitrogen and olefin p-orbitals are poorly aligned due to the more constrained 5,6-bicyclic system. With less orbital overlap, the contribution of the nitrogen would be less pronounced, resulting in a less nucleophilic enaminone. The ultimate consequence of this is that transmetallation outcompetes palladation and the organoborate and reoxidant are consumed in the production of the homodimer. With reason to be concerned about epimerization of stereocenters (see Chapter 2) we subjected enaminones 2.36 and 2.50 to our optimized protocol (entries 3 and 4). We were pleased that our mildly-acidic conditions were suitable for these substrates, and they reacted with no observable epimerization. 174 Despite the success of this reaction in monocyclic and bicyclic systems there were a several enaminones that did not react (Table 3–9). An N-Boc-protected enaminone was found to be unreactive under our optimized conditions, which we suggest is owing to its 175 a Reaction conditions: enaminone (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3 equiv), K 2 CO 3 (2 equiv) in t-BuOH/AcOH/DMSO (20:5:1) at 60 o C. b Isolated yield. c Starting material was recovered. PMP = 4-methoxyphenyl. entry a C–H donor product time (h) yield (%) b 1 N O H 2.37 N O OMe 3.2 8 70 2 N O H 2.34 N O OMe 3.20 3 98 3 N O Me H H H 2.36 N O Me H H OMe 3.21 7 86 4 N O Me H H H 2.50 N O Me H H OMe 3.22 7 77 5 N O Me H 3.18 N O Me OMe 3.23 6 80 6 N O Bn HPh 3.19 N O Bn OMe Ph 3.24 7 79 Table 3–8. Successfully coupled of enaminones. decreased nucleophilicity (entry 1, Table 3–9). Likewise, adding another degree of unsaturation to the piperidine system is sufficient to suppress the reaction (entries 2–4). In each of these examples, we postulate that palladation is too slow to compete with homocoupling. Another way to impede enaminone palladation is to incorporate steric encumberance. We found that methyl and phenyl substituents could inhibit cross-coupling in the ordinarily reactive quinolizidine system (entries 5 and 6). Notably, the phenylquinolizidine can be coupled under Pd(0)-catalyzed conditions when pre-activated via C3-iodination, 213 showing again that these two methods are complimentary. 3.9 Extensions and directions for the future The success of this reaction seems to rely on a delicate balance between the rates of palladation and transmetallation. This has been a major limitation with respect to both the enaminone and trifluoroborate. The N-alkyl substituted enaminones are on the threshold of entry a C–H donor entry a C–H donor entry a C–H donor 1 N O Boc H 3 N O Bn H 5 N O Me H 2 N O H 4 N O Me Me OH H 6 N O H a Reaction conditions: enaminone (0.1 M), trifluoroborate 3.3 (2-3 equiv), Pd(OAc) 2 (0.3 equiv), Cu(OAc) 2 (3 equiv), K 2 CO 3 (2 equiv) in t-BuOH/AcOH/DMSO (20:5:1) at 60 o C. b Isolated yield. c Starting material was recovered. PMP = 4-methoxyphenyl. Table 3–9. Enaminones that did not couple. 176 reactivity, and when even subtle changes are introduced, such as a methyl group, the desired reaction is overwhelmed by the competing dimerization process. The same effect can result from the use of organotrifluoroborates that are exceedingly prone to transmetallation. On the other hand, alkyl trifluoroborates, which reluctantly transmetallate, fail to react altogether. Such obstacles will be challenging to overcome without approaching the problem from this mechanistic standpoint. One potential solution to this problem is to use coupling partners that are not susceptible to dimerization—those that do not undergo transmetallation, for instance. In this regard, we envisioned a Heck-type modification as an apt extension. Akin to our optimized arylation, this reaction would depend on initial enaminone palladation to generate a σ-bonded Pd- enaminone complex. The subsequent steps would be identical to the classical Heck-Mizokori reaction, involving migratory insertion of an alkene substrate followed by β-hydride elimination (Fig. 3–12). We tested the feasibility of this reaction by replacing our trifluoroborate coupling partner with methylacrylate under our optimized conditions (Scheme 3–7). Even though this olefination was slower than the arylation, we were able to isolate enaminone 3.25 in 62% yield. This is clearly a promising approach to prepare the previously Fig. 3–12. Mechanism of Pd(II)-catalyzed olefination. N O Pd II R N O R Pd II H + + Pd 0 N O R 177 inaccessible C3-alkenyl enaminones. Furthermore, without the competing transmetallation process, enaminones that are less prone to palladation could be still be used. This has been borne out in subsequent studies by Yiyun Yu in our laboratory. The Heck-variation of this reaction is a limited solution to the problem, because only alkenes can be used as coupling partners. If this strategy is to be applied to preparing phenanthropiperidines from less reactive enaminones, aryl groups must also be accommodated. There is much precedent from the literature that suggests arylhalides could be used for this purpose. 299 In a strikingly relevant example reported by Pelletier, Larivée and Charette, cyclic enamines were coupled with aryl iodides using a Pd(0)-catalyst (Scheme 3– 8). 312 This strategy not only minimizes the likelihood of aryl homocoupling but also eliminates the need for a reoxidant. As such, this approach holds great promise to expand the substrate scope of enaminone C–H functionalization reactions. Pd(OAc) 2 , Cu(OAc) 2 K 2 CO 3 , 80 ºC t-BuOH/AcOH/DMSO N O CO 2 Me 62% H N O CO 2 Me Scheme 3–7. Direct C–H olefination of enaminone. 2.55 3.25 178 3.10 Summary In summary, this methodology provides a direct route for the construction of 3- arylpiperidine scaffolds, a privileged structure and prevalent motif in many natural products. Although the substrate scope of this reaction is somewhat limited, this work provides a foundation for future efforts to broaden it. This method is a significant advance over the existing two-step method. Also, this reaction is the first example of C–H functionalization on enaminones (a non-aromatic enamine system) and contributes to the rapid accretion of viable C–H donors. Most importantly, the ultimate goal of this work—to synthesize 3- arylenaminones as intermediates to phenanthropiperidines—has been met. What remains is the utilization of these structures for phenanthropiperidine synthesis. N HR N Ph MeO Ar I PdCl 2 (dppf)•CH 2 Cl 2 NaOAc, Ag 3 PO 4 DMF, 150 ºC N ArR N Ph MeO 32–84% Scheme 3–8. Direct Pd(0)-catalyzed arylation of cyclic enamines by Pelletier et al. 312 179 CHAPTER 4 SYNTHESIS OF (+)-ANTOFINE AND (+)-IPALBIDINE 4.1 Introduction The value of synthetic methodologies is often tentative until they validated through practical applications. Hitherto, this work has described the development of two reactions that have been designed to facilitate the preparation of phenanthropiperidine alkaloids, but now we will turn to how these methods can be utilized. In Chapter 2, we discussed the development of a method that gives ready access to a variety of cyclic enaminones (A, Scheme 4–1), including indolizidine and quinolizidine scaffolds. In Chapter 3, we showed how these scaffolds could be elaborated through the development of a Pd(II)-catalyzed method that directly functionalizes the C3-position of the enaminone and can be used to install the requisite aryl moiety, which constitutes ring C of the phenanthrene system (A → B). As intended, the amino acids and aryl coupling partners are interchangeable building blocks providing a means for preparing numerous permutations of the 3-arylpiperidine scaffold. Herein, we demonstrate that that these intermediates can be used as precursors to (+)-antofine (4.2), a representative phenanthropiperidine, and (+)-ipalbidine (4.1), a structurally-related indolizidine alkaloid (Fig. 4–1). 180 These natural products were chosen not only by virtue of their prototypical structure but also for their remarkable biological profiles. (+)-Ipalbidine (4.1) is a non-addictive analgesic, an oxygen free-radical scavenger, and has demonstrated inhibitory effects on the respiratory burst of leukocytes. 313-315 (–)-Antofine, on the other hand, exhibits low nanomolar cytotoxic activity (IC 50 ) in drug-sensitive and MDR cancer cell lines alike, already being described in Chapter 1. We chose to prepare the unnatural enantiomer, (+)-antofine (4.2), considering that both natural products could be synthesized in a divergent fashion from a late-stage intermediate. Although these natural products have been synthesized before, 142, 158, 316-327, 149, 56, 63, 146, 150, 170-172, 181, 184, 328 our method herein gives access to these molecules in a exceptionally concise and high-yielding manner. N R 1 O R 3 R 2 R 4 Ar 1 BR 2 Pd(0) N R 1 O R 3 R 2 R 4 Ar 1 H N R 1 R 3 R 2 R 4 Y Z N R 1 CO 2 H R 3 R 2 Boc amino acids A B Scheme 4–1. Route to phenanthropiperidines using our developed methods. Chapter 2 Chapter 3 Chapter 4 phenanthropiperidines N Me H HO N H MeO MeO OMe Fig. 4–1. Structures of (+)-ipalbidine and (+)-antofine. (+)-ipalbidine (4.1) (+)-antofine (4.2) 181 4.2 Total synthesis of (+)-ipalbidine Despite being addressed at this point in this work, our efforts to synthesize (+)-ipalbidine predate the full development of our ynone cyclization and direct-arylation protocols that have been already described in depth. In fact, it was not until our total synthesis of (+)-ipalbidine was complete that we discovered that the indolizidine stereocenter had partially racemized. The details of this discovery and our work to suppress what was later discovered to be an acid-mediated process was discussed in Chapter 2. Our seminal work on (+)-ipalbidine also involved pre-activating the enaminone prior to installing the C3-aryl moiety, which was greatly improved upon the discovery and development of a direct-arylation method (see Chapter 3). In order to contrast the old with the new routes, the evolution of our synthetic strategy to (+)-ipalbidine will be discussed in detail. 4.2.1 First attempt to synthesize (+)-ipalbidine Starting with Boc-L-proline, Weinreb amide 2.2 was prepared in three steps (Scheme 4– 2). The amino acid was homologated using a standard Arndt-Eistert reaction, first converting the acid into diazoketone 4.3 and then catalyzing the Wolff rearrangement with CF 3 CO 2 Ag. When using H 2 O as a solvent during the rearrangement, Boc-L-β-homoproline (4.4) was obtained in 49% yield over two steps along with 26% of unreacted starting material. Though the acid could be converted into the desired Weinreb amide 2.2 using a high-yielding EDCI 182 coupling, the difficulty of separating the two homologues following the Arndt-Eistert protocol inspired us to look for improvements. Realizing that the acid functional group was contributing to poor chromatographic separation, we switched solvents during the Wolff rearrangement to MeOH in order to obtain a more tractable methyl ester. We were pleased to find that using MeOH was beneficial, not only because it facilitated purification, but also because it improved the yield. Weinreb amide 2.2 could then be prepared from methyl ester 4.5 using an easily scalable protocol reported by Williams et al. 279 This approach served us well in the preparation of multi-gram quantities of the Weinreb amide, but our constant search for shorter reaction sequences led us to a even shorter route. It occurred to us that if acids and esters could be prepared from the addition of oxygen nucleophiles into ketene intermediate (I, Scheme 4–2), then amides could also be prepared using nitrogen nucleophiles. To test this, we carried out the rearrangement TEA, THF ClCO 2 Et N Boc i-PrMgCl, THF then CH 2 N 2 O N 2 CF 3 CO 2 Ag TEA, H 2 O CF 3 CO 2 Ag TEA, MeOH EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl CF 3 CO 2 Ag, HN(OMe)Me TEA, THF N Boc O Nu N HO 2 C Boc N Boc HO 2 C N Boc O N OMe Me HN(OMe)Me•HCl N Boc MeO 2 C 72% 97% 98% 96% 68% 86% Scheme 4–2. Preparation of Weinreb amide. 4.3 4.4 2.2 4.5 I Boc-L-proline 183 in a non-nucleophilic solvent (THF) and added freshly distilled N,O-dimethylhydroxylamine. The reaction was successful, and the product (2.2) was isolated in 97%. Next, we prepared ynone 2.17 by adding excess ethynylmagnesium bromide to amide 2.2 at 0 ºC (Scheme 4–3). The outcome of this reaction was highly dependent on the quality of the Grignard reagent and on the work-up method. The reaction was typically quenched with a weak acid, since the Boc-protecting group could decompose in strong acids. The acid also served to sequester the nucleophilic N,O-dimethylhyrdroxylamine byproduct as an ammonium salt in order to prevent it from adding into the ynone. 329 Hence, when the reaction was deemed complete, aqueous NaHSO 4 was added and the reaction stirred for 1 h. This method consistently provided the ynone product in greater than 95% yield. From ynone 2.17, enaminone 2.37 was prepared upon stepwise treatment with 4N HCl in dioxane and methanolic K 2 CO 3 , the details of which were discussed in Chapter 2. At the time, unbeknownst to us, the stereocenter had racemized during the deprotection step. This was not obvious at the time, since the product had a high optical rotation value ([α] D = –190) and no standard to compare it to. Furthermore, despite numerous attempts, we were unable to analytically separate the enantiomers using chiral HPLC. N O H 1) 4N HCl, dioxane 2) MeOH, K 2 CO 3 >95% 89% (2 Steps) MgBr THF, 0 ºC; N Boc O N OMe Me N Boc O then NaHSO 4 (aq.) Scheme 4–3. Synthesis of indolizidine enaminone from Weinreb amide. 2.2 2.17 2.37 184 NO H N O H I N O H MeO I 2 , DMAP 96% CH 2 Cl 2 MeO BF 3 K K 2 CO 3 , MeOH S-Phos (2 mol %) Pd(OAc) 2 (1 mol%) 70% L-Selectride THF then Comins' reagent 76% N TfO H MeO Pd(0), MeB(OH) 2 N Me H MeO Suzuki-Miyaura Scheme 4–4. First approach to (+)-ipalbidine. 2.37 3.1 3.2 4.6 4.7 Unaware of its low enantiomeric purity, we subjected enaminone 2.37 to I 2 in the presence of DMAP, to afford iodoenaminone 3.8 (Scheme 4–4). The p-methoxyphenyl group was installed next using a Buchwald modification of the Suzuki-Miyaura reaction (see Chapter 3). 291 To install the methyl substituent in the place of carbonyl group we first investigated a Pd-catalyzed cross-coupling approach. This involved the preparation of triflate 4.6, which could be achieved using L-Selectride to mediate 1,4-reduction and trapping the resulting enolate with Comins’ reagent as an triflate source. We attempted a number of Suzuki-Miyaura protocols to couple the methyl group to the indolizidine scaffold, but they were unsuccessful, providing low yields of difficult-to-purify product. 4.2.2 Second approach to (+)-ipalbidine Turning to our second approach, enaminone 3.2 was once more reduced in a 1,4-fashion with L-Selectride, only this time, without subsequent treatment with Comins’ reagent, and 185 ketone 4.8 was isolated. Methyl lithium was then added to the ketone to furnish tertiary alcohol 4.9. Our initial attempts to dehydrate indolizidine 4.7 were unsuccessful. We had initial concerns that multiple olefin isomers would be obtained and be difficult to separate but hypothesized that the desired isomer would be the most stable—being a tetra-substituted olefin and conjugated with the phenyl group—and that the unwanted isomers could be channeled to the desired product under thermodynamic conditions. This rational also inspired us to prepare indolizidine 4.7 from a methenylation reaction of ketone 4.8 with Tebbe reagent and subsequent isomerization; unfortunately, we could not obtain sufficient quantities of indolizidine 4.10. Returning to tertiary alcohol 4.9, we explored several conventional methods to dehydrate alcohols (Table 4–1). From of the methods tried, neat POCl 3 and SOCl 2 (entries 3 and 6) in pyridine showed promising results with SOCl 2 being the best. Observing that this reaction 186 N O PMP H L-Selectride, THF N HO PMP H H Me N O PMP H H –78 to 0 ºC 90% THF, –78 ºC 75% MeLi N H 2 C PMP H H Tebbe reagent Ti C Cl Al H H Me Me Tebbe reagent N PMP H Me dehydration isomerization Scheme 4–5. Second approach to prepare (+)-ipalbidine. 3.2 4.8 4.9 4.10 4.7 PMP = p-methoxyphenyl was complete in less than 30 minutes and that degradation was causing a significant loss in yield, the reaction was: (1) diluted with THF, (2) run at lower temperatures (–30 to –10 ºC) and (3) run with only a slight excess of SOCl 2 and pyridine (entries 7 and 8). These modifications dramatically improved the outcome of the reaction, providing 88% of the product. From product 4.7, we could prepare (+)-ipalbidine (4.1) by removing the O-methyl group with BBr 3 (Scheme 4–6). It was at this point that we realized that the natural product was not enantiomerically pure. The optical rotation was significantly lower than reported values. Fortunately, ipalbidine’s enantiomers were easily resolved using chiral HPLC, which showed that we had obtained the natural product with an e.r of 70:30. As already discussed, the root cause of entry conditions yield (%) a 1 I 2 , PPh 3 , CH 2 Cl 2 , 16 h NR 2 BF 3 •OEt 2 , CH 2 Cl 2 , 16 h NR 3 POCl 3 , pyridine 30 4 TsOH, AcOH, reflux NR 5 TsOH, PhMe, reflux NR 6 SOCl 2 , pyridine, rt 52 7 SOCl 2 (5 equiv), pyridine (5 equiv), THF, –30 ºC to rt 78 8 SOCl 2 (2.5 equiv), pyridine (5 equiv), THF, –30 to –10 ºC 88 NR = no reaction (i.e. starting material was recovered). a Isolated yield. Table 4–1. Optimization of dehydration conditions. 4.9 4.7 N HO H MeO H Me conditions N H MeO Me 187 racemization was a retro-Michael/Michael process that had occurred during the acidic deprotection step of the ynone cyclization reaction. This was remedied by using HCO 2 H, a much weaker acid, to deprotect the Boc-group, and NaI to activate the ynone prior to cyclization. These conditions virtually eliminated the racemization and the enaminone could be prepared in a high enantiomeric purity. Several other modifications of this third approach, which improve the yields and shorten the entire sequence, deserve further attention. 4.2.3 Third approach to (+)-ipalbidine A third approach to synthesize (+)-ipalbidine was devised to enhance the enaniomeric purity of the final product and also to take advantage of recently developed methods to improve the reaction sequence as a whole (Scheme 4–7). After resynthesizing enaminone 2.37 with our mild cyclization conditions, we used our just-developed Pd(II)-chemistry to bypass the iodination reaction and prepared the intermediate 3.2 in a single step. We were also able to abridge the final sequence by reverting back to triflate intermediate 4.6. Previously, we had had little success installing the methyl group with the Suzuki-Miyaura protocol using this triflate. This was, presumably, due to the steric demand of the a tetra- substituted olefin. To compensate for this, the Negishi reaction, which has an outstanding 75% BBr 3 , CH 2 Cl 2 N H MeO Me N H HO Me –78 to 0 ºC Scheme 4–6. Final reaction to prepare (+)-ipalbidine. 4.7 (+)-ipalbidine (4.1) e.r. = 70:30 188 reputation in alkyl cross-couplings, was used instead. The methyl zincate was prepared immediately beforehand from methylmagnesium bromide and ZnBr 2 (not shown). This reagent was added to triflate 4.6 and Pd(PPh 3 ) 4 , and in 4 h at 60 ºC the product was isolated in 91% yield. (+)-Ipalbidine (4.1) was once again completed upon removal of the O-methyl group, and the enantiomeric ratio was identical to the enaminone it was derived from (e.r. = 98:2). 4.3 Total synthesis of (+)-antofine Our featured route was employed to synthesize (+)-antofine (4.2) as well (Scheme 4–8). Fortuitously, this phenanthropiperidine, which is among the most potent in its class, can be accessed from the same triflate intermediate (4.6) used to prepare (+)-ipalbidine. With previous success using the Negishi protocol, we attempted to install the necessary aryl group using the same method. The zinc reagent was prepared from a lithium-halogen exchange reaction of 4-bromoveratrole and t-BuLi and a subsequent quench with ZnBr 2 . The aryl L-Selectride THF then Comins' reagent 76% 1. Pd(PPh 3 ) 4 , THF MeZnBr, 60 ºC 73% (2 steps) 2. BBr 3 , CH 2 Cl 2 Pd(OAc) 2 (30 mol%) Cu(OAc) 2 (3 equiv) PMP-BF 3 K, K 2 CO 3 t-BuOH/AcOH/ DMSO (20:5:1), 60 o C, 70% N O H N O H MeO N TfO H MeO N O Boc NaI, HCO 2 H; then K 3 CO 3 , MeOH 96% 2.17 2.37 3.2 4.6 Scheme 4–7. Racemization-free approach to (+)-ipalbidine. (+)-ipalbidine (4.1) e.r. = 98:2 189 zincate was then coupled with triflate 4.6 using the same conditions as before to furnish seco- antofine in 96% yield. Finally, a PIFA-mediated biaryl coupling yielded (+)-antofine (4.2), which exhibited optical activity that matched those reported in the literature. 149, 171, 184, 326 4.4 Summary Aided by our new approach to arylated cyclic enaminones, the syntheses of (+)-ipalbidine (4.1) and (+)-antofine (4.2) can be accomplished in a remarkably concise fashion. Moreover, both of the natural product’s were obtained in high yield and enantiomeric purity. By these total syntheses, the utility of two methodologies are exemplified. Specifically, we have shown that cyclic enaminones can be efficiently prepared from the chiral pool and directly functionalized to provide versatile intermediates that can be applied to the synthesis of complex molecules which, in this case, are of medicinal interest. These syntheses can be considered a proof-of-concept. Although they have been validated in a sense, their design was intended to facilitate the production of a library of phenanthropiperidines, not just one. Nevertheless, the two methods, which were developed along the way to these natural products, are a contribution to the rapidly growing arsenal of N TfO H MeO 1. Pd(PPh 3 ) 4 , THF MeO ZnBrMeO 2. PIFA, BF 3 •Et 2 O CH 2 Cl 2 N H MeO MeO OMe 67% (2 steps) Scheme 4–8. Synthesis of (+)-antofine from triflate 4.6. 4.6 (+)-antofine (4.2) 190 organic reactions. The sheer simplicity of the cyclic enaminone and the prevalence of the piperidine and 3-arylpiperidine systems suggest that these reactions will be broadly applicable. Our purposes herein are more focused and, therefore, we will now turn to the preparation of phenanthropiperidine analogs. 191 CHAPTER 5 SYNTHESIS AN BIOLOGICAL EVALUATION OF (R)-TYLOCREBRINE AND RELATED ANALOGS 5.1 Introduction The phenanthropiperidines are being intensely pursued as drug leads, primarily for the treatment of cancer but also for inflammatory conditions, such as arthritis. Although select members of this class are exhibit promising therapeutic potential, there is concern that they may also possess undesired neurological activity. This concern arises from the failed clinical trial of tylocrebrine (1.4), the only member of this class to be tested in humans. Considering the highly-conserved structural features across this class, there is reason to speculate that other phenanthropiperidines are also neurologically active. The molecular basis for tylocrebrine’s side-effects has not been investigated, nor has the neurological effects of other phenanthropiperidine drug leads. Hence, in order to guide our design of phenanthropiperidine analogs, we have begun to study the CNS activity of tylocrebrine and its related analogs. Herein, we report the synthesis and initial biological studies of tylocrebrine and some closely related analogs to better understand the neurological liabilities of these alkaloids. 192 5.2 Possible mechanisms for neurological effects The complete absence of neurological data for the phenanthropiperdines, besides a brief reference to tylocrebrine’s clinical trial, 2 makes our search extraordinarily difficult. Suffness reported that the patients who were administered the alkaloid experienced “disorientation and ataxia” (ataxia being a motor-neurological condition defined as the loss of coordination). 2 Unfortunately, no other data is available. Presumably, more detailed descriptions, such as dosage, mode of administration and alkaloid source, were recorded at the time of the clinical trials, but this data could not be obtained even after correspondence with the NCI. Without this data, we simply concluded that tylocrebrine—in some manner—affects motor centers in the CNS. Although this does not lead us to a molecular target, it does provide a direction for our investigations. Since little is known of the nature of tylocrebrine’s neurological effects or, for that matter, of its pharmacology in general, one cannot rule out the possibility that these two effects are inseparable (i.e. the mechanism of tylocrebrine’s anti-proliferative effects is the same as that of its neurological effects). For this scenario, it would be necessary to characterize the peripheral and central effects in order to assess the feasibility of designing safer phenanthropiperidines. If both peripheral and central effects were equally severe, this would bring the therapeutic potential of these alkaloids into question. On the other hand, if the side- effects were primarily induced by CNS interactions, a promising strategy would be to prevent BBB permeation. Nothing suggests, however, that these alkaloids are exerting their CNS and anti-cancer effects through the same target(s). 193 In the event that the CNS and anti-cancer mechanisms can be differentiated, different strategies could be used. The most obvious one would involve the design of analogs that have greater selectivity for their therapeutic target. The challenge of this approach lies in the difficulty of assessing CNS interactions with adequate throughput. Identifying the specific target(s) responsible for the neurological effects would be highly valuable to this enterprise by providing a means to test synthetic leads in vitro. One advantage of this approach is that it is would not be necessary to identify the therapeutic target given that anti-proliferative cell- based assays are relatively high-throughput already. If the therapeutic target and off-target cannot be differentiated based on binding affinities or functional activity, they can perhaps be differentiated based on location. In other words, if the target(s) responsible for the side- effects is localized within the CNS or is only implicated therein, designing analogs that do not cross the BBB could address this. This latter approach, however, comes with a caveat: namely, that tumors bounded by the BBB will be unaffected. Although this limitation is common to many anti-cancer agents and would not be reason to discontinue development, it is one that, if possible, should be avoided. The treatment of cancers within the CNS is immensely challenging, primarily because of the difficulty of designing active agents that reach tumors within the BBB. 330 With tylocrebrine, however, the challenge is on another front. Tylocrebrine is extremely potent and evidently reaches the CNS already. Thus, the remaining obstacle is to impart neurological selectivity in order to tune-out unwanted side-effects. Although this may also be difficult to achieve, it has yet to be attempted. Designing phenanthropiperidines that do not cross the BBB is more straightforward than designing analogs that do not interact with specific targets. As described in Chapter 1, CNS 194 exposure has been linked to specific physicochemical parameters that can be adjusted to help or hinder BBB-permeation. 132 These parameters are essentially descriptors of lipophilicity (i.e. cLogP, polar surface area, H-bond donors and cLogD). When these properties are taken into account, it not surprising that tylocrebrine, which is highly lipophilic, passes through the BBB. In this regard, this approach should be directed towards increasing the polarity (or hydrophilicity) of the phenanthropiperidines in a manner that does not affect their activity. Incidentally, imparting the physicochemical properties that hinder BBB-permeation will also improve the water solubility, which is another advantage of this approach (concerns about the limited solubility of these alkaloids are discussed in Chapter 1). From what is already known about the SAR of this class, several positions on the phenanthropiperidine scaffold are promising for introducing polar functional groups (Fig. 5–1). The C3-, C6- and C14- positions, for instance, are relatively insensitive to derivatization and could be used to install polarizing groups. Furthermore, ring E (C11–13) is virtually unexplored and may also provide additional points of functionalization. N OMe R 2 R 1 R 3 R 4 14 3 6 E Fig. 5–1. Promising sites for derivatization. 195 The design of non-BBB–penetrating analogs has the luxury being possible without a precise mechanistic understanding of the neurological effects. A more challenging strategy, however, is to design analogs which are neurologically inert yet retain the anti-proliferative effects of the parent alkaloids. We have chosen to pursue this route for the potential it has to treat tumors of the CNS. In order to determine the viability of this approach, we set out to elucidate the mechanism for tylocrebrine’s side-effects to assure that they can be separated from its anti-proliferative effects. Before this search commenced, the alkaloid would need to be obtained. It is crucial that tylocrebrine—not other phenanthropiperidines—be studied since it is the only alkaloid with demonstrated neurological effects in humans. Thus, attaining sufficient quantities of tylocrebrine was our first goal. 5.3 Synthesis of tylocrebrine and analogs To date, the only synthetic strategy that has been used to prepare tylocrebrine relies on the isolation of the minor regioisomer (17%) of an unselective Pschorr coupling protocol, which furnishes the tetramethoxyphenanthrene system (Scheme 1–8). This synthesis provides less than 1% of racemic tylocrebrine over ten steps. 146 Thus, we envisioned the development of a new synthetic route to provide a sustainable source of tylocrebrine and, importantly, to provide it in enantiomerically pure form. In Chapter 4, the synthesis of (+)-antofine was described in which we successfully employed our newly developed methodologies. In relation to previous phenanthropiperidine syntheses (see Chapter 1), our approach was unique except for the final step (i.e. the oxidative biaryl coupling). Although the biaryl coupling method is atom economic and 196 presumably versatile with respect to the aryl substituents, the accessibility of certain phenthrene regioisomers is limited. Free rotation around the aryl-alkene bond makes possible the formation of multiple regioisomers. In our synthesis of antofine, for example, there was a possibility of forming two regioisomers (Scheme 5–1). The major product (IV) of these reactions is predictable when considering the steric interactions that arise from rotation of the arene (I ⇋ II). Hence, the oxidative biaryl coupling approach limits the accessibility of phenanthrenes with C4- or C5-substituents, because bulky substituents in these positions introduce a significant amount of strain. Indeed, the 1,4-strain energy between the C4- and C5-hydrogens in unsubstituted phenanthrene amounts to 5–6 kJ/mol. 331, 332 This synthetic limitation explains the absence of strategies to prepare tylocrebrine (1.4), which bears a C5-methoxy group, despite it being one of the most well-studied members in this class. Considering the shortcomings of the biaryl coupling approach and inaccessibility of certain phenanthrene substitution patterns, it is surprising that these obstacles are largely unconquered. Because our biological studies were dependent on the availability of MeO MeO OMe MeO MeO MeO MeO MeO OMe MeO MeO MeO H [O] [O] major product H 4 5 4 5 Scheme 5–1. Regioisomer formation in biaryl coupling reaction. IIIIII IV 197 tylocrebrine, we had to devise a new strategy allowing construction of C5-substituted phenanthrenes. Our synthetic plan involved three sequential C–C bond forming steps (Scheme 5–2). The key step would be the final ring-closure proceeding through an aryl-alkene oxidative coupling. Unlike, the standard biaryl-coupling approach, this disconnection imposes regioselective control by exploiting the once-problematic methoxy substituent to block coupling at the C5-position. The requisite precusor 5.1, in turn, could be derived from a Negishi reaction of a organozincate prepared from biphenyl bromide 5.2 and triflate 5.5. The biaryl bond (shown in green) could be prepared from a halogen-selective Suzuki reaction from a commercially available boronic acid 5.4 and reported veratrole derivative 5.3. 149 Using our method to prepare 6-membered enaminones, the indolizidine core can be prepared from Boc-L-β-homproline. Alternatively, to access the racemic indolizidine core we N OMe MeO MeO MeO N OMe MeO MeO MeO H H 5 [O] aryl-alkene [O] coupling Negishi coupling OMe MeO MeO MeO Br Suzuki coupling OMe OMe Br OMe OMe B(OH) 2 I + N TfO N Boc HO 2 C N Boc O or Scheme 5–2. Retrosynthetic analysis of tylocrebrine. tylocrebrine (1.4) 5.1 5.2 5.5 5.4 5.3 (R)-4.4 and (S)-4.45.6 198 envisioned starting with N-Boc-pyrrolidinone (5.6). Once the enaminone functionality was realized, the indolizidine enaminone could be converted into triflate 5.5 in preparation for the Negishi protocol. First looking to the indolizidine fragment, we began our synthesis with Boc-D-β- homoproline and Boc-L-β-homoproline, which were to be carried through in parallel (Scheme 5–3). Although each enantiomer of amide 2.2 could be formed directly using an EDCI coupling, the two-step homologation procedure (discussed in Chapter 4) was used as an economic alternative (Boc-D-β-homoproline > $300/g). Nevertheless, the use of large quantities of diazomethane during homologation is still a drawback. CH 2 Cl 2 , 0 ºC HN(OMe)Me•HCl EDCI, NMM N Boc HO 2 C N Boc O N Me MeO N HO 2 C Boc 1. TEA, THF, ClCO 2 Et; then CH 2 N 2 2. CF 3 CO 2 Ag, HN(OMe)Me TEA, THF 95–98% Scheme 5–3. Synthesis of (R) and (S)-Weinreb amide. (R)-4.4 and (S)-4.4 (R)-2.2 and (S)-2.2 Boc-proline 65–70% (2 steps) 199 Considering the amenability of this synthesis to diversification, and thereby library development, we thought that the synthesis of racemic precursors would also be of value, particularly if they could be obtained in large quantities. Thus, an alternative approach was devised for the synthesis of racemic Weinreb amide 2.2 (Scheme 5–4). In this approach, Boc-pyrrolidinone (5.6) was reduced to hemiaminal 5.7 with DIBAL-H and then subjected to Horner-Wadsworth-Emmons conditions in which the putative ring-opened intermediate A spontaneously cyclized to provide the β-proline derivative 5.8. Racemic amide 2.2 could then be directly obtained from ester 5.8 using conditions reported by Williams et al. 279 Although this sequence includes a greater number of steps compared with the enantiospecific synthesis, the payoff is the ease and cost of scale-up. Moreover, it avoids the use of large quantities of hazardous diazomethane which would otherwise be necessary to prepare multi- gram quantities of the desired intermediates. N Boc DIBAL-H THF, –78 ºC NaH, DMF 92% 86% HN(OMe)Me•HCl i-PrMgCl THF, –20 ºC 85% (EtO) 2 P CO 2 Et O NH Boc CO 2 Et O N Boc HO N Boc EtO 2 C N Boc O N Me MeO Scheme 5–4. Synthesis of racemic Weinreb amide 2.2. 5.6 (±)-5.7 A (±)-5.8 (±)-2.2 200 Constructing the indolizidine fragment was our next goal. Grignard addition of ethynylmagnesium bromide to Weinreb amide 2.2 provided the desired ynone 2.17 in excellent yield (Scheme 5–5). Indolizidine formation could be achieved using our one-pot deprotection/cyclization strategy. Either 4N HCl in dioxane or NaI in HCO 2 H can be used for Boc-deprotection. Although the yields of the latter conditions (NaI/HCO 2 H) were generally higher, work-up following the cyclization was easier when using the HCl- deprotection method. It should be recalled, however, that the use of 4N HCl promotes retro- Michael/Michael-type racemization of the β-stereocenter and could not be used for constructing non-racemic indolizidine 2.37. With the indolizidine core assembled, the triflate moiety was installed using a 1,4-reduction and enolate trapping with Comins’ reagent, providing triflate 5.5. N Boc O N Me MeO L-Selectride, THF –78 to 0 ºC; then Comins Reagent –78 to 0 ºC 73% MgBr THF N O N TfO 97% 95% K 2 CO 3 MeOH NaI HCO 2 H N Boc O H 2 N O I I Scheme 5–5. Synthesis of indolizidine triflate 5.5. (R)-2.2, (S)-2.2, (±)-2.2 (R)-2.17, (S)-2.17, (±)-2.17 (R)-2.37, (S)-2.37, (±)-2.37 (R)-5.5, (S)-5.5, (±)-5.5 (R)-2.38, (S)-2.38, (±)-2.38 201 We next turned to the synthesis of the biaryl fragment. Fürstner and Kennedy have previously reported a straightforward route to biarylbromides that were used for the synthesis of several phenanthropiperidine alkaloids. 149 Although biarenes with ortho substituents previously had not been synthesized, the reported conditions showed no impartiality to our hindered boronic acid 5.4 (entry 1, Table 5–1). Notably, this stage of the synthesis is poised for diversification considering the collection of boronic acids available for purchase. Although our primary focus remained on the synthesis of tylocrebrine (1.4), we also synthesized biaryl bromides 5.9–5.13 which bear the substitution pattern of several other prominent members of this class—tylophorine, antofine and deoxypergularinine (entries 2, 4 and 6)—and two unnatural isomers that have never been synthesized before (entries 3 and 5). Our intent was not only to demonstrate the feasibility of diversification at this point in the synthesis, but, also, to explore the scope of this chemistry and its application for the Table 5–1. Synthesis of biaryl fragment. entry R 1 R 2 R 3 R 4 product yield (%) 1 OMe OMe H OMe 5.2 72 2 H OMe OMe OMe 5.9 67 3 OMe H H OMe 5.10 77 4 H OMe H OMe 5.11 68 5 H H OMe OMe 5.12 70 6 H OMe OMe H 5.13 70 I Br OMe R 4 R 1 R 2 B(OH) 2 + (dppf)PdCl 2 (5 mol%) LiCl, Na 2 CO 3 DME, H 2 O, 80 ºC, 16 h R 4 MeO R 1 R 2 Br R 3 R 3 202 construction of C5-unsubstituted analogs—these could potentially form regioisomers in the final aryl-alkene coupling reaction. An important consideration for us was the ease of scale-up and cost efficiency. Admittedly, we did not labor over reaction optimization for the Suzuki protocol, in which we had encountered an number of drawbacks (e.g. challenges in work-up and purification) when preparing multi-gram quantities of product, but realized that a scalable, Pd-free approach to biarylbromide 5.9 was possible (Scheme 5–6). This could be achieved through a PIFA- mediated dimerization of 4-bromoveratrole (5.14). The resultant dibromide 5.15 was desymmetrized upon treatment with one equivalent of n-BuLi and a subsequent MeOH quench. Both steps could be carried out in 20 g batches and, conveniently, without the use of column chromatography providing 13 g of product (80% yield over two steps). Having had previous success with Negishi cross-couplings of sterically-encumbered triflates, we employed analogous conditions to install the biaryl moiety using the appropriate coupling partner. The zincate 5.16 was prepared from biaryl bromide 5.2 via sequential treatment with t-BuLi and ZnBr 2 . The exact conditions as used before were first employed OMe OMe Br PIFA BF 3 •Et 2 O n-BuLi (1.0 equiv) 80% (2 steps) Br OMe OMe OMe CH 2 Cl 2 , –78 ºC THF; then MeOH Br OMe OMe OMe OMe Br MeO Scheme 5–6. Scalable approach to biaryl bromide 5.9. 5.14 (20 g) 5.9 (13 g)5.15 203 for the cross-coupling. Using this protocol, the reaction was complete in 30 min. Because this reaction was so rapid, we reduced the reaction temperature from 60 ºC to room temperature and decreased the catalyst loading to 2 mol%. We were pleased to find that this cross-coupling proceeded in under an hour to provide near quantitative yields of the desired indolizidine 5.1 (Scheme 5–7). Having all the desired carbons in place we could now explore conditions for an oxidative aryl-alkene coupling. To our knowledge there has only been only one report of such a reaction which used (p-BrC 6 H 4 ) 3 N•SbCl 6 . 333 Concerned by this lack of precedent, we undertook a systematic investigation using tylocrebrine precursor 5.1 as a model stubstrate, and reagents that have found success in the related biaryl coupling reaction (Table 5–2). In our first attempt, (p-BrC 6 H 4 ) 3 N•SbCl 6 left the starting material unperturbed (entry 1). Slightly more success was found under photochemical conditions (entry 2) or when using PIFA (entry 3 and 4); however, substantial amounts of degradation were also observed. We were pleased to find that FeCl 3 (entry 5) could induce the desired oxidative coupling with greatly improved yields. The greatest success was found with the use of VOF 3 (entry 7), THF, rt, 1 h Pd(PPh 3 ) 4 (2 mol%) N OMe MeO MeO MeO OMe MeO MeO MeO X + N TfO t-BuLi, THF; then ZnBr 2 Scheme 5–7. Coupling of indolizidine and biaryl fragments. 95% (R)-5.5, (S)-5.5, (±)-5.5 5.16 (X = ZnBr) 5.2 (X = Br) (R)-5.1, (S)-5.1, (±)-5.1 204 which gave the desired product in 62% yield. With the success of this reaction, this method was applied to the both enantiomers of indolizidine 5.1, marking the first asymmetric synthesis of (R)- and (S)-tylocrebrine. The natural products were synthesized in 7 linear steps and in up to 39% overall yield from Boc-D-β-homoproline. In hopes that this methodology would be applicable to more than tyocrebrine, we also attempted the synthesis of various analogs with varying methoxy substitution patterns. Each of the biaryl bromide analogs (5.9–5.13) were coupled with racemic triflate 5.5 using the modified Negishi protocol that was used to prepared indolizidine 5.1 (Table 5–3). The yields conditions N OMe MeO MeO N OMe MeO MeO MeO MeO a NMR yield using MeSiPh 3 as an internal standard. b Recovered starting material. c Isolated yield. entry conditions yield (%) a 1 (p-BrC 6 H 4 ) 3 N•SbCl 6 (1.0 equiv), 2,6-lutidine (0.2 equiv), CH 2 Cl 2 , rt 0 b 2 I 2 (1.0 equiv), PhMe, hν 7 3 PIFA (2.0 equiv), CH 2 Cl 2 :TFA (2:1), 0 ºC to rt 10 4 PIFA (2.0 equiv), BF 3 •Et 2 O, CH 2 Cl 2 , –40 ºC 11 5 FeCl 3 (6.0 equiv), MeNO 2 , CH 2 Cl 2 , rt 44 6 VOCl 3 (1.1 equiv), CH 2 Cl 2 , rt decomp. 7 VOF 3 (4.0 equiv), CH 2 Cl 2 , EtOAc, TFA, TFAA, –78 to –15 ºC, 4 h 70 (62) c Table 5–2. Oxidative aryl-alkene coupling optimization. (±)-5.1 (±)-tylocrebrine (1.4) 205 of these reactions were comparable and, except for compound 5.20, the indolizidine products were isolated in 95% yield or greater. The lower yield for compound 5.20 is probably not an accurate reflection of reactivity but more likely resulted from an experimental oversight. Having obtained these phenanthropiperidine precursors, we next turned to the final oxidative coupling. We were initially concerned that when the C5-methoxy group was not present (R 1 = H, Table 5–3), this would introduce the possibility of producing multiple regioisomeric products. Fortunately, these reactions proceeded in excellent yields, providing phenanthropiperidines 5.23–5.26 with no observable regioisomer formation. Notably, the reaction yields were significantly higher without the C5-methoxy group (R 1 = H) than with it (R 1 = OMe), showing that the C5-substituent is not essential and may even retard the progression of this reaction. Indeed, the oxidative coupling of indolizidines 5.18–5.21 were complete in 1 h—compared with 4 h for tylocrebrine precursor 5.1—and did not require warming above –78 ºC. Conversely, coupling of indolizidine 5.17 was slow and accompanied by numerous side-reactions that left less than 30% of the isolable product. Notwithstanding the poor yields of this reaction, all of the products were isolated in sufficient quantities to begin an investigation of tylocrebrine’s neurological target. Each analog was prepared in only racemic form, whereas racemic, (R)- and (S)-tylocrebrine were synthesized. This route is an improvement over previous routes, being shorter, higher yielding and allowing the synthesis of both enantiomers and previously inaccessible phenanthrene substitution patterns. 206 5.4 Biological studies of tylocrebrine and related analogs 5.4.1 Cancer With six phenanthropiperidine analogs in hand, including tylocrebrine, we began our investigation of their neurological activity in search of “CNS-safe” analogs. The first task was to assess the anti-cancer properties of each compound so that we could eventually determine if the therapeutic effect could be separated from the undesired effects. We chose three human tumor cell lines for our initial studies: MCF-7, COLO-205 and NCI/ADR-RES. N R 4 MeO R 2 N R 4 MeO R 2 R 3 R 3 R 1 R 1 R 4 MeO R 1 R 2 X + R 3 Negishi coupling [O]-coupling X = Br X = ZnBr t-BuLi; ZnBr 2 R 1 R 2 R 3 R 4 Negishi yield (%) a product [O]-Coupling yield (%) a product overall yield (%) b OMe OMe H OMe 95 (±)-5.1 62 c (±)-1.4 27 OMe H H OMe 97 (±)-5.17 28 c (±)-5.22 12 H OMe H OMe 95 (±)-5.18 70 (±)-4.2 30 H H OMe OMe 98 (±)-5.19 85 (±)-5.23 37 H OMe OMe OMe 70 (±)-5.20 68 (±)-1.1 22 H OMe OMe H 98 (±)-5.21 89 (±)-5.24 39 Table 5–3. Final two steps to prepare (±)-tylocrebrine and its analogs. (±)-5.1, 5.17–5.21 Conditions: Negishi coupling, Pd(PPh 3 ) 4 (2 mol%), THF, rt, 1 h; [O]-coupling, VOF 3 , TFA, TFAA, CH 2 Cl 2 , EtOAc, –78 ºC. a Isolated yield. b Yield over 8 steps from N-Boc- pyrrolidinone c Required warming to 0 ºC. (±)-5.5 (±)-1.1, 1.4, 4.2, 5.22–5.24 207 Using the alamarBlue® assay to measure cell proliferation, each analog assessed in the select tumor cell lines. As expected, most of the analogs exhibited potent growth inhibition in each cell line. Unfortunately, exact GI 50 values could not be obtained for several analogs since lowest concentration used resulted in almost complete growth inhibition. For these analogs we have indicated that their GI 50 is less than the lowest concentration used (i.e. 3.1 nM). Although this data does not lend itself to ranking each compound in order of its activity, it does give valuable qualitative data from which new SARs can be gleaned. We have found that tylocrebrine’s racemate exhibited anti-tumor activity which exceeded that of the (S)-enantiomer alone (Table 5–4). Furthermore, of tylocrebrine’s analogs, only one was significantly less cytotoxic than taxol in COLO-205 or MCF-7 cell lines whereas, unlike taxol, each alkaloid retained activity in NCI/ADR-RES, a multi-drug resistant cancer cell line. 6-Desmethoxytylophorine (5.23), an unnatural analog, was much less active than IC 50 (nM) b compound a COLO-205 MCF-7 NCI/ADR-RES Taxol 1.8 1.9 NA (S)-1.4 (S)-tylocrebrine 8.8 105 207 (±)-1.4 (±)-tylocrebrine < 3.1 < 3.1 < 3.1 (±)-5.22 (±)-6-desmethoxytylocrebrine < 3.1 < 3.1 < 3.1 (±)-4.2 (±)-antofine < 3.1 < 3.1 < 3.1 (±)-5.23 (±)-6-desmethoxytylophorine 265 533 1340 (±)-1.1 (±)-tylophorine < 3.1 < 3.1 < 3.1 (±)-5.24 (±)-deoxypergularinine 8.5 5.8 196 Table 5–4. Cytotoxicity of phenanthroindolizidines. a Each phenanthropiperidine is in racemic form unless otherwise specified. b Determined using alamarBlue® assay; COLO-205 = human colorectal adenocarcinoma; MCF-7 = human breast carcinoma; NCI-ADR-RES = drug-resistant human ovarian adenocarcinoma; NA = Not active at the highest concentration tested (293 nM). 208 tylocrebrine (1.4), confirming the importance of the C6-methoxy group, which is present in all of the most active phenanthropiperidines. Corroborating earlier studies, this data shows that removal of either the C2-, C5- or C7-methoxy groups is not detrimental. This information will indeed be valuable for designing novel anti-cancer agents. 5.4.2 Neurological side-effects hypothesis We next began our investigations into the cause of the adverse neurological effects observed in tylocrebrine’s original clinical trial. The absence of clinical data forced us to search for clues elsewhere. The symptoms alluded to by Suffness 2 —who seems to have acquired his data from a primary source—indicate that, at some level, tylocrebrine interferes with normal voluntary movement. Thus, we chose to explore two potential mechanisms for tylocrebrine’s side-effects based on its structural similarity to bioactive molecules known to influence motor function in the CNS: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and dopamine (DA). 5.4.2.1 MPTP-like toxicity In the 1980s, Langston (in collaboration with the NIH) chanced upon a neurotoxin that induced parkinsonism. This substance, later identified as MPTP, affected illicit drug users that had self-injected contaminated batches of MPPP, a synthetic opioid. 334 The neurotoxin was found to have remarkable selectivity for the substantia nigra and DA neurons, thus, mimicking the pathophysiology of Parkinson’s disease. Research over the following decades 209 has shown that MPTP undergoes a fatal sequence of events that leads to DA neuronal cell death (Fig. 5–2). Being highly lipophilic, MPTP passively diffuses across the BBB to enter the CNS. Within glial cells it is oxidized by monoamine oxidase-B (MAO-B) to the active toxin, 1-methyl-4-phenylpyridinium (MPP+, Fig. 5–3)—this makes MPTP, strictly speaking, a protoxin. Following this oxidation, MPP+ is actively transported into DA neurons at the nerve terminal via the dopamine transporter (DAT); hence, the selectivity for dopaminergic neurons. Within the neuron, the active toxin (MPP+) effectively shuts down cellular metabolism in the mitochondria by inhibiting Complex I (NADH dehydrogenase) of the electron transport chain, ultimately resulting in cell death. The basis for investigating tylocrebrine’s similarity to MPTP arose from several commonalities. Firstly, the structure of MPTP (Fig. 5–3) is embedded within tylocrebrine. Second, tylocrebrine readily undergoes oxidation to form MPP+-like dehydrotylocrebrine (Fig. 5–3). Phenanthropiperidines are prone to aerobic oxidation while in solution. The exact mechanism of the oxidation is unknown, but the end product is presumably the dubious Fig. 5–2. Mechanism for MPTP-induced toxicity. 210 pyridinium salt. This raises the question as to the integrity of the phenanthropiperidines in living systems and as to if the side-effects are due to this metabolite rather than the parent compound. Indeed, if oxidation is facile in aerobic conditions, this process may also be implicated in physiological conditions. Another concern is enzymatic oxidation. Having structural similarities to monoamine neurotransmitters and to MPTP, phenanthropiperidines could potentially undergo MAO- mediated oxidation. To explore this hypothesis further, each analog was tested in the MAO Amplex® Red assay (Graph 5–1). This assay measures H 2 O 2 (a byproduct of MAO oxidation) production as an indicator of MAO activity. Using p-tyramine as a standard, MAO-A and MAO-B activities were measured in the presence of the phenanthropiperidines and MPTP. As expected, MPTP was shown to be a substrate for MAO-B and, to a much lesser extent, MAO-A. Conversely, at concentrations up to 100 μM, none of our analogs increased MAO-A or MAO-B activity, showing that oxidation via this mechanism is unlikely. This does not, however, rule out oxidation by other metabolic enzymes or non-enzymatic oxidation. Fig. 5–3. Structure of MPTP, MPP+ and dehydrotylocrebrine. N OMe MeO MeO MeO N Me MPP+ N Me MPTP dehydrotylocrebrine 211 Graph 5–1. Phenanthropiperidines as a substrate for MAO-A and MAO-B. Monoamine oxidase A (MAO-A) Monoamine oxidase B (MAO-B) * Percent activity of the MAO enzyme where the activity induced by p-tyramine (a known substrate) is set at 100%. μ μ 212 In another effort to identify similarities between tylocrebrine and MPTP, we determined if any phenanthropiperidine analogs were subject to active transport by three major neurotransmitter transporters found in the CNS: DAT, NET and SERT. Even if tylocrebrine’s mechanism was different from MPTP, identifying an active transporter would give valuable insight into the region of influence that could aid a physiological explanation when juxtaposed with the tylocrebrine’s side-effects. We also tested dehydrotylocrebrine in this assay, which was prepared from tylocrebrine (1.4), to see if this potential metabolite might exert CNS effects. The affinity of each analog for the transporters was determined at a single concentration (10 μM) in a radioligand displacement assay. It was clear from this assay that none of the phenanthropiperidines tested exhibited marked affinity for the transporters (Table 5–5). % radioligand displacement @ 10 µM b entry compound a DAT NET SERT 1 6-desmethoxytylocrebrine 10 31 4.9 2 6-desmethoxytylophorine 39.7 8 32 3 antofine 26.0 25.7 0 4 deoxypergularinine 25.1 20.4 4.7 5 tylocrebrine 30.3 30.1 0 6 tylophorine 1.6 18.5 8.2 7 dehydrotylocrebrine 19.2 41.2 0 a Each phenanthropiperidine is in racemic form unless otherwise specified. b Determined at the NIHM-PDSP. DAT = dopamine transporter, NET = norepinephrine transporter, SERT = serotonin transporter. Table 5–5. Phenanthropiperidine affinity for monoamine transporters. 213 Further testing is required to unambiguously define tylocrebrine’s (or dehydrotylocrebrine’s) role in the electron transport chain before it can be dismissed as an MPTP-like neurotoxin. Nevertheless, compelling evidence, here and elsewhere, gives reason to conclude tylocrebrine is unique in this respect. Firstly, MPTP is activated via oxidation by MAO-B for which tylocrebrine is not a substrate. Second, MPTP’s active metabolite, MPP+, exploits DAT to reach its cellular target, whereas, dehydrotylocrebrine is not accommodated by this transporter. Thus, if tylocrebrine or an active metabolite exerts its effect in the neurons of the substantia nigra, which is yet to be demonstrated, it does so through a different pathway. Another distinction can be made on the basis of MPTP’s and tyocrebrine’s physiological effects. Administration of MPTP incurs the characteristic parkinsonian rigidity, bradykinesia, tremor and postural instability. These symptoms, as with Parkinson’s disease, result from a damaged substantia nigra which is, among other things, responsible for fine motor control. Strictly speaking, this differs from the ataxia which is allegedly caused by tylocrebrine. Ataxia is defined as the loss of coordination and results from dysfunctional sensory or cerebellar systems. Since ataxia is symptomatically and pathologically distinct from parkinsonism, tylocrebrine’s and MPTP’s motor effects are probably unrelated. Could the known causes of ataxia shed light on the neurological effects associated with the administration of tylocrebrine? Yes, but uncertainty still remains. Their is no evidence to suggest that tylocrebrine’s side-effects are not simply a manifestation of non-specific interactions across the CNS and periphery. Indeed, among its numerous CNS effects, ethanol can induce ataxia. 335 Unfortunately, without the original clinical data, nothing is known about 214 how the ataxic symptoms present, and therefore, we cannot assume that tylocrebrine’s effects are localized or specific. 5.4.2.2 Dopaminergic and other CNS receptors Another concern we had was that tylocrebrine was interfering with neurotransmission by mimicking biogenic amines. This arose out of the realization that tylocrebrine was endowed with the pharmacophore of DA and bore a resemblance to known DA receptor ligands such as dihydrexidine (DHX) and dinoxyline (Fig. 5–4). DA’s pivotal role in the CNS led us to explore the dopaminergic system as the culpable source of tylocrebrine’s neurological side- effects. The dopaminergic system is intimately connected to motor control, cognition, emotion, motivation and many other functions. 336 Furthermore, abnormalities of the dopaminergic system can have devastating consequences which are manifested, for instance, in Parkinson’s disease, schizophrenia, bipolar disorder and drug addiction. Thus, if tylocrebrine is found to modulate the dopaminergic system, there is good reason to attenuate these interactions during lead optimization. NH HO HO H H NH HO HO H O NH 2 HO HO Fig. 5–4. Structurally related molecules of interest. dopamine (DA) dihydrexidine (DHX) full D 1 agonist dinoxyline full D 1 agonist 215 It was not simply the dopaminergic effects, however, that concerned us. There is appreciable overlap between the ligands of DA receptors and other biogenic amine receptors (e.g. adrenergic and serotonergic) in the CNS. 337 Thus, tylocrebrine could possibly exert more extensive effects than those limited to the dopaminergic system. For this reason, we selected a broad panel of CNS targets to investigate rather than focus on a specific receptor. Our collection of phenanthropiperidines was submitted to the National Institutes of Mental Health-Psychoactive Drug Screening Program (NIHM-PDSP) for a comprehensive CNS-target screen (Table 5–6). A primary screen identified compounds which had significant binding (defined as greater than 50% displacement of a radioligand) to receptors at 10 μM. For those “hits”, the K D was determined in a secondary assay. The results from the primary screen confirmed our suspicions about tylocrebrine having DA receptor affinity. Five of the six phenanthropiperidine analogs (excluding dehydrotylocrebrine) bound to the D 1 subtype with moderate selectivity. The uniform activity at D 1 of each tylocrebrine analog could be indicative of a more universal liability for these natural products. These analogs also showed moderate affinity at the D 5 receptor and, to a much lesser extent, the D 2 , D 3 and D 4 subtypes. This selectivity profile is typical for D 1 agonists and antagonists. The D 5 receptor, having a high degree of structural homology with D 1 , is often targeted by D 1 ligands. The D 2 , D 3 and D 4 receptors, on the other hand, all belong to the D 2 -like family and, therefore, achieving selectivity between these three receptors is more difficult than between them and D 1 -like subtypes. The D 1 selectivity exhibited by tylocrebrine is notable, because this receptor is the most highly expressed subtype within the dopaminergic system and widely dispersed in the mammalian forebrain. 336 Modulating this receptor alone could have wide-ranging physiological ramifications. 216 217 T a b l e 5 – 6 . C o m p r e h e n s i v e C N S t a r g e t p r i m a r y s c r e e n f r o m N I H M - P D S P . To better characterize the alkaloids affinity for each receptor, the K D values for each “hit” in the primary screen were determined (Table 5–7). This, however, did not settle the matter. If tylocrebrine had displayed high affinity for the D 1 receptor, this would have forged a promising link to its adverse effects, whereas, if it had weak affinity, it would give good reason to pursue another hypothesis. In reality, tylocrebrine had moderate affinity for the D 1 receptor with a K D of 448 nM, raising new questions about its nature of activity and the details of its clinical trial. To aid interpretation of this result the dosages used in the tylocrebrine’s clinical trials would be extremely valuable. Considering that racemic tylocrebrine was used in these experiments, the affinity of each enantiomer should also be determined before pursuing this route any further. Moreover, the functional effect of binding should be characterized. Although this binding information is useful, a physiological connection cannot be made until a functional assay elucidates tylocrebrine’s efficacy upon K D (nM) b compound a D 1 D 2 D 3 D 4 D 5 5-HT 1D α 1A H2 6-desmethoxytylocrebrine 219 NA NA NA – 1098 2790 885 6-desmethoxytylophorine 89 NA 5078 NA 749 737 105 222 antofine 181 NA 5849 NA 1695 1553 1092 1724 deoxypergularinine 587 NA 7384 NA 5004 2596 1195 2010 tylocrebrine 448 NA NA NA 3644 3123 1784 537 tylophorine 2617 NA NA NA – NA NA 9846 Table 5–7. Affinity for phenanthropiperidine for biogenic amine receptors. a Each phenanthropiperidine is in racemic form unless otherwise specified. b Determined at the NIHM-PDSP. NA = not active (K D is greater than 10 µM). 218 the receptor in question (i.e. whether tylocrebrine is an agonist, antagonist, partial agonist, etc.). The secondary assay also provided some useful information about how structure influences receptor binding. 6-Desmethoxytylophorine had the highest affinity for the D 1 subtype out of all the phenanthropiperidines tested (K D = 89 nM). This compound’s phenanthrene substitution pattern, which renders it much less active in anti-proliferation assays, will therefore be avoided in analog development. Tylophorine, on the other hand, has the least affinity for the D 1 receptor and also exceptional anti-cancer activity. If D 1 - interaction is ever linked to adverse side-effects in the phenanthropiperidines, this information provides a potential strategy to minimize them. Besides showing notable affinity for the D 1 receptor, tylocrebrine and most of the analogs tested also bind to several subtypes within each class (e.g. serotonergic, muscarinic, histaminergic, etc.). Particularly, the 5-HT 1D , α 1A and H 2 receptors were targeted by each analog albeit to a lesser extent than the D 1 receptor. Tylocrebrine had comparable binding to the H 2 receptor and D 1 receptor. The primary functions of the H 2 receptor are in the regulation of gastric acid secretion, smooth and cardiac muscle relaxation and in the immune response. 338 There is little to suggest, however, that H 2 ligands could affect the neurological responses induced by tylocrebrine. Likewise, the α 1A receptors has not been directly linked to motor coordination, but its expression in the ventral and dorsal motor area of rat spinal cord suggests that it may regulate movement in some fashion, 339 as does its α 1B counterpart. 340 Tylocrebrine’s relatively low affinity for this receptor and the 5-HT 1D receptor, which also has no known connection to motor functions, renders these implausible targets. Although 219 these results redirect our search for the mechanism of tylocrebrine’s side-effects, they presage other neurological effects that could potentially hamper lead development. The primary and secondary assays give further confirmation that 6- desmethoxytylophorine and closely related structures could indiscriminately modulate neurotransmission through the biogenic amine receptors. Conversely, tylophorine, which has weak binding to every target that was tested, may be a good lead to base next generation analogs on. It is surprising, however, that this subtle modification—the presence or absence of a single methoxy group—confers such a drastically different CNS receptor profile. Another possible explanation for this anomaly is that tylophorine gives a false negative result by virtue of its insolubility. Indeed, in the process of making DMSO solutions of each alkaloid it was quite apparent that tylophorine was the least soluble. Furthermore, it is significant that tylophorine’s decomposition point (284–285 ºC) is more than 50 ºC above that of the other alkaloids (ranging from 203–230 ºC). Although the decomposition temperature of a chemical entity is not correlated with solubility, as is the melting point, 133 this data demonstrates that the physicochemical properties of tylophorine are significantly different from the other alkaloids, and this difference—rather than actual receptor binding— may be the basis for its anomalous behavior in the PDSP-CNS target screen. K D (nM) b compound a M1 M2 M3 M4 M5 tylocrebrine 2664 4602 NA 3176 NA dehydrotylocrebrine 186 156 339 169 1561 Table 5–8. Tylocrebrine’s and dehydrotylocrebrine’s affinity for muscarinic receptors. a Each phenanthropiperidine is in racemic form unless otherwise specified. b Determined at the NIHM-PDSP. NA = not active (K D is greater than 10 µM). 220 It is also worthy to note that dehydrotylocrebrine exhibits a unique binding profile. Rather than targeting the monoamine receptors, this derivative of tylocrebrine has considerable affinity for the muscarinic receptors (Table 5–6 and 5–8). This could be a concern if dehydrotylocrebrine is generated in vivo. Muscarinic receptors, like dopaminergic systems, are expressed throughout the CNS and PNS, and they are associated with cognition, learning, and memory as well as proper functioning of the heart, gastrointestinal tract, lung bronchioles and urinary bladder. 341, 342 Moreover, these GPCRs are the target of numerous toxins, including its eponymous mushroom toxin muscarine. 343 However, the muscarinic system functions primarily, if not exclusively, in the autonomic nervous system and has not been linked to voluntary motor control or coordination. Therefore, dehydrotylocrebrine’s affinity for these receptors does not explain tylocrebrine’s side-effects, but, as before, discloses potential off-target interactions that could limit the success of phenanthropiperidine-based therapeutics. If oxidation does occur in vivo, preventing it through appropriately designed analogs would be presumably curtail the muscarinic effects. 5.5 Summary Tylocrebrine’s early failure in clinical trials, despite precluding characterization of its efficacy, established a liability for this class of alkaloids and their use as chemotherapeutic agents. Until now, the neurological effects that derailed the clinical trials have not been investigated, despite recent interest in the medicinal properties of the phenanthropiperidines. The lack of practical syntheses for tylocrebrine was a major impediment to these studies. We 221 addressed this by designing a new synthetic approach that accounted for the challenging phenanthrene substitution pattern. Our method provided a simple and high-yielding route to tylocrebrine and provided sufficient quantities to assist characterization of its inimical CNS effects. This route was also used to synthesize a small library of tylocrebrine analogs—some were known natural products, others were unnatural analogs—to aid SAR studies. Herein, we reported preliminary work to understand the CNS effects of tylocrebrine by exploring two hypothetical links between its structure and the nature of its side-effects. Firstly, we investigated the similarities of tylocrebrine to the known neurotoxin MPTP and found that there is unlikely overlap between their biological effects. Second, we investigated tylocrebrine’s and its analogs’ similarity to key biogenic amines in the NIHM-PDSP comprehensive CNS target screen. The screen has uncovered several pitfalls that could be encountered in the development of a phenanthropiperidine drug, especially one that is designed to treat tumors within the CNS. It also uncovered risk factors that contribute to the off-target interactions that will guide lead optimization studies. The mechanism of tylocrebrine-induced ataxia and disorientation is still not understood, making it difficult, without an experimental model, to design “CNS-safe” analogs at this time. One strategy that is feasible, however, involves the preparation of phenanthropiperidines that do not cross the BBB at all. Since this approach does not require a mechanistic understanding of tylocrebrine’s neurological effects, it warrants immediate attention. 222 CHAPTER 6 EXPERIMENTAL DATA 6.1 Materials and methods Unless otherwise specified, all starting materials, reagents and solvents are commercially available and were used without further purification. Flash column chromatography was carried out on silica gel. TLC was conducted on silica gel 250 micron, F 254 plates. 1 H NMR spectra were recorded on a 400 MHz NMR instrument. Chemical shifts are reported in ppm with TMS or CHCl 3 as an internal standard (TMS: 0.00 ppm, CHCl 3 : 7.26 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, b = broad, m = multiplet), integration and coupling constants (Hz). 13 C NMR spectra were recorded on a 100 MHz NMR spectrometer with complete proton decoupling. Chemical shifts are reported in ppm with the solvent as internal standard (CHCl 3 : 77.2 ppm). IR of solids were obtained by dissolving the sample in CHCl 3 and letting the solvent evaporate on a KBr plate. High-resolution mass spectrometry was performed by the University of Minnesota Mass Spectrometry Facility. 223 6.2 Chapter 2 6.3.1 Preparation of Weinreb amides: Method A: Weinreb amide preparation from β-amino acids. Boc N R 3 R 2 R 1 O OH EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl Boc N R 3 R 2 R 1 O N Me OMe The β-amino acid (4.0 mmol, 1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 (100 mL) under an argon atmosphere and cooled to –15 °C. To this solution was added N,O- dimethylhydroxylamine•HCl (4.4 mmol, 1.1 equiv) and N-methylmorpholine (4.4 mmol, 1.1 equiv) followed by EDCI (4.2 mmol, 1.1 equiv). The reaction mixture was then allowed to come to room temperature. After 2 h the reaction was again cooled to 0 °C and quenched by the addition of an ice cold 10% HCl solution (25 mL) and allowed to stir at this temperature for 5 min. The reaction was diluted with water (50 mL) and extracted with CH 2 Cl 2 (x3). The combined organic layers were washed with saturated NaHCO 3 (x1), dried over Na 2 SO 4 , filtered and concentrated. Purification via SiO 2 flash chromatography using EtOAc and hexanes as the eluent afforded pure Weinreb amides. Method B: Weinreb amide preparation from α-amino acids. Boc N CO 2 H R 2 R 1 1) TEA, ClCO 2 Et, Et 2 O; then CH 2 N 2 2) 10 mol% Ag(CO 2 CF 3 ) HN(OMe)Me, THF Boc N R 3 R 2 R 1 O N Me OMe 224 Warning: Large amounts of diazomethane were used for this transformation. Proper care should be taken when handling this highly explosive reagent. All glassware used was free of cracks, scratches or ground-glass joints and a blast shield was used. The N-Boc-amino acid (50.0 mmol, 1.00 equiv) was taken into THF (100 mL) with stirring and cooled to 0 ºC with an ice bath. The reaction solution was treated with TEA (7.66 mL, 55.0 mmol, 1.10 equiv) and allowed to react for 15 min to fully deprotonate the carboxylic acid. With the addition of ethyl chloroformate (5.23 mL, 55.0 mmol, 1.10 equiv), a thick white precipitate formed. Stirring was continued for 15 min then stopped. In a separate flask, an ice-cold ethereal solution of diazomethane was prepared and, without stirring, was carefully decanted into the freshly prepared anhydride reaction flask using a glass funnel. The reaction solution was lightly stirred for 4 seconds then stirring was stopped. The mixture was allowed to warm to room temperature and react overnight. Any additional diazomethane was carefully quenched with 0.5 N acetic acid (100 mL). The drop-wise addition of saturated sodium bicarbonate regulated the solution back to a basic pH 8-9 with gentle stirring. The organic and aqueous layers were separated. The organic phase was washed twice each with saturated sodium bicarbonate and brine then dried over sodium sulfate. The solvent was evaporated under reduced pressure and placed under high vacuum overnight. The diazoketone was taken into THF (250 mL) and cooled to 0 ºC. Foil was used to cover the reaction flask so as to exclude light from the reaction solution. To this was added freshly distilled N,O- dimethylhydroxylamine (9.16 g, 150 mmol, 3.00 equiv). 344 In a separate foil covered flask, silver trifluoroacetate (1.10 g, 5.00 mmol, 0.10 equiv) was dissolved in TEA (130 mL). This solution was added to the diazoketone mixture over 30 min. The reaction temperature was allowed to slowly warm to room temperature and the solution was stirred overnight. To the 225 reaction mixture was added activated charcoal (~2 g) and the reaction mixture was stirred for 5 min and filtered. The filtrate was concentrated and the residue redissolved in EtOAc. To this was added activated charcoal (~2 g) and the process repeated. When the filtrate had been concentrated a second time the residue was purified via SiO 2 flash chromatography using a mixture of EtOAc and hexane as eluent affording the pure Weinreb amide. Method C: Procedure for Weinreb amide preparation from amines. Bn NH 2 1) Methyl acrylate; then Boc 2 O 2) i-PrMgCl, THF HN(OMe)Me•HCl Boc N Bn O N Me OMe A round-bottomed flask was charged with benzylamine (8.00 mL, 73.1 mmol, 3.00 equiv) and cooled to –40 ºC. Methyl acrylate (2.20 mL, 86.1 mmol, 1.00 equiv) was added dropwise over 5 min and the reaction was stirred at this temperature (–40 ºC) for 24 h. Excess benzylamine was distilled off of under reduced pressure. The remaining residue was dissolved in MeOH (50 mL) and di-tert-butyldicarbonate (6.40 g, 29.2 mmol, 1.20 equiv) was added slowly. The reaction mixture was stirred for another 30 min and the solvent was removed in vacuo. The concentrated reaction mixture was redissolved in CH 2 Cl 2 (300 mL) and washed with cold 10% HCl (100 mL x 2) and brine (100 mL x 2). The organic layer was dried with MgSO 4 , concentrated and purified via SiO 2 flash chromatography (25% EtOAc in hexanes) to afford 6.62 g (88%) of the methyl ester as a clear viscous oil. This oil was converted to the corresponding Weinreb amide using the procedure reported by Williams et al. 279 The crude product was purified via SiO 2 flash chromatography (40% EtOAc in 226 hexanes) to give 6.29 g (86%) of the Weinreb amide as a clear viscous oil. See below for spectral data. (S)-tert-Butyl 2-(2-(Methoxy(methyl)amino)-2-oxoethyl)piperidine-1-carboxylate (2.1). The title compound was obtained as a colorless oil using Method B (64% over 2 steps). Spectral data of the title compound was identical to that reported in the literature. 255 (S)-tert-Butyl 2-(2-(Methoxy(methyl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate (2.2). The title compound could be obtained as a colorless oil using either Method A (98%) or Method B (70% over 2 steps). Spectral data of the title compound was identical to that reported in the literature. 152 2.1 N Boc O N Me OMe 2.2 N Boc O N Me OMe 227 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-(methoxy(methyl)amino)-2-oxoethyl)- pyrrolidine-1-carboxylate (2.3). The title compound was obtained as a white solid from Boc-O-benzyl-hydroxyproline using Method B (72% over 2 steps): mp 69.5-70.3 °C; 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.46 (s, 18H), 1.95-2.03 (bm, 2H), 2.30-2.38 (bm, 2H), 2.44-2.54 (bm, 2H), 3.03-3.16 (m, 2H), 3.16 (s, 6H), 3.41-3.56 (m, 3H), 3.67 (s, 6H), 3.67-3.75 (m, 1H), 4.09-4.14 (bm, 2H), 4.27-4.34 (bm, 2H), 4.46-4.54 (m, 4H), 7.26-7.35 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.5, 32.0, 36.4, 36.9, 37.4, 38.1, 51.1, 51.9, 52.9, 61.2, 70.8, 75.7, 76.4, 79.3, 79.7, 127.6, 127.6, 128.4, 138.1, 154.5, 172.1, 172.4; IR (neat) 2974, 1693, 1665, 1397, 1160, 1118 cm -1 ; HRMS (ESI + ) m/e calc’d for [M+H] + C 20 H 31 N 2 O 5 : 379.2233, found 379.2222. 2.3 N Boc O N O Me OMe N Boc HO O N Me OMe H 2 , 10% Pd/C N Boc O O N Me OMe MeOH 2.3 2.4 228 (2S*,4R*)-tert-Butyl 4-Hydroxy-2-(2-(methoxy(methyl)amino)-2-oxoethyl) pyrrolidine-1-carboxylate (2.4). The title compound was obtained as a colorless oil (98%) using the hydrogenation procedure reported by Epperson et al. 345 Spectral data of for this compound was identical to that reported in the literature. 152 2.5 N Boc HO O N Me OMe N Boc HO O N Me OMe i) PPh 3 , DIAD, THF p-NO 2 C 6 H 4 CO 2 H ii) K 2 CO 3 , MeOH 2.4 (2S*,4S*)-tert-Butyl 4-Hydroxy-2-(2-(methoxy(methyl)amino)-2-oxoethyl) pyrrolidine-1-carboxylate (2.5). The title compound was obtained from a Mitsunobu reaction of compound 2.4. Weinreb amine 2.4 (90 mg, 0.32 mmol, 1.0 equiv) was dissolved in THF (10.0 mL) under an argon atmosphere and cooled to 0 °C. Triphenylphosine (164 mg, 0.62 mmol, 2.0 equiv) and p-nitrobenzoic acid (104 mg, 0.62 mmol, 2.0 equiv) were than added sequentially. DIAD (126 mg, 0.62 mmol, 2.0 equiv) was then added slowly and the reaction was allowed to come to room temperature. The reaction was monitored via TLC (100% EtOAc) and judged complete in less than an hour. The reaction mixture was poured into a saturated NaHCO 3 solution and extracted thrice with EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. This crude material was purified via flash chromatography (1 EtOAc/1 hexanes) to provide 134 mg (98%) of a colorless oil: 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.48 (s, 9H), 2.25 (d, J = 14.6 Hz, 1H), 2.43-2.51 (m, 1H), 2.75-2.80 (m, 1H), 3.17 (s, 4H), 3.64 (s, 4H), 3.81-3.86 (m, 1H), 4.40-4.50 (bs, 1H), 5.54-5.57 (m, 1H), 8.21-8.29 (m, 4H); 13 C NMR (125 MHz, CDCl 3 , 50 °C) δ 22.3, 28.9, 229 36.7, 37.5, 52.9, 53.9, 61.6, 75.4, 80.4, 123.9, 131.1, 135.8, 151.2, 154.3, 164.4, 173.3; This p-nitrobenzoyl ester (0.12 g, 0.26 mmol) was dissolved in MeOH (10 mL) and K 2 CO 3 (excess) added. After 5 min the reaction was judged complete by TLC (100% EtOAc) and the reaction solvent was removed under reduced pressure. This residue was then dissolved in a 1:1 CH 2 Cl 2 and water mixture and extracted with CH 2 Cl 2 (x3). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. This crude material was purified via flash chromatography (100% EtOAc) to provide 60 mg (91%) of the desired product: 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.44 (s, 9H), 1.92 (d, J = 14 Hz, 1H), 2.20-2.31 (m, 1H), 2.89-3.11 (m, 3H), 3.15 (s, 3H), 3.38 (d, J = 12 Hz, 1H), 3.55 (bs, 1H), 3.68 (s, 3H), 4.22 (bs, 1H), 4.39 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 , 50 °C) δ 28.9, 32.5, 37.4, 39.8, 54.0, 55.7, 61.5, 70,7, 79.8, 154.7; IR (neat) 3439, 2974, 2935, 1680, 1400, 1172 cm -1 ; HRMS (ES+) m/ e calc’d for [M+H] + C 13 H 25 N 2 O 5 : 289.1763, found 289.1752. tert-Butyl 3-(2-(Methoxy(methyl)amino)-2-oxoethyl)morpholine-4-carboxylate (2.6). The title compound was obtained from N-Boc-3-morpholineacetic acid using Method A as a white solid (99%) after flash chromatography (80% EtOAc/hexanes): mp 71.9-72.7 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 1.46 (s, 9H), 2.54-2.64 (bm, 1H), 2.97-3.17 (bm, 2H), 3.17 (s, 3H), 3.45 (dt, J = 5.8, 2.8 Hz, 1H), 3.58 (dd, J = 11.7, 2.2 Hz, 1H), 3.71 (s, 3H), 3.71-3.88 (m, 3H), 4.41-4.46 (bm, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.4, 31.1, 32.1, 39.2, 48.5, 2.6 N O Boc O N Me OMe 230 61.3, 66.8, 69.1, 80.1, 154.5, 171.9; IR (neat) 2975, 1696, 1663, 1408, 1171, 1106 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 13 H 25 N 2 O 5 : 289.1763, found 289.1770. tert-Butyl (cis)-2-(Methoxy(methyl)carbamoyl)cyclohexyl(methyl)carbamate (2.7). The title compound was obtained from N-Boc-cis-2-aminocyclohexanecarboxylic acid using Method A and a subsequent methylation. The Weinreb amide intermediate (following Method A) was isolated as a colorless oil (95%) after purification via flash chromatography (25% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.16-1.40 (m, 2H), 1.38 (s, 9H), 1.48-1.64 (m, 4H), 1.78-1.88 (m, 1H), 2.05-2.12 (m, 1H), 3.10 (bs, 1H), 3.12 (s, 3H), 3.65 (s, 3H), 3.85 (bs, 1H), 4.98 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 , 50 °C) δ 23.1. 2, 25.9, 28.7, 30.1, 32.7, 41.4, 48.9, 61.6, 79.2, 155.8, 175.2; IR (neat) 3439, 2930, 2860, 1700, 1500, 1168 cm -1 ; HRMS (ES+) m/e calc’d for [M+H] + C 14 H 27 N 2 O 4 : 287.1971, found 287.1971. Methylation Procedure: The Weinreb amide (160 mg, 0.56 mmol, 1.0 equiv) and MeI (170 μL, 2.8 mmol, 5.0 equiv) were dissolved in anhydrous DMF (15 mL) under an argon atmosphere and cooled to 0 °C. To this solution was added 60% NaH (45 mg, 1.1 mmol, 2.0 equiv) in one portion and the reaction was allowed to come to room temperature. It was found crucial to use high quality NaH for this reaction to proceed. After 8 h the reaction was judged complete by TLC (50% EtOAc/hexanes). The reaction was quenched by 2.7 NH O Boc H H NMe N O Boc H H OMe Me OH 1) EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl 2) NaH, MeI, DMF 231 the addition of water and placed in a separatory funnel. This solution was diluted with water and extracted with EtOAc (x3). The combined organic layers were back extracted with water (x2) to remove any residual DMF then dried over Na 2 SO 4 , filtered and concentrated. This crude material was purified via flash chromatography (25% EtOAc/hexanes) to provide 162 mg (92%) of the title compound as a colorless oil: 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.32-1.52 (m, 3H), 1.45 (s, 9H), 1.55-1.66 (m, 1H), 1.78-1.97 (m, 3H), 2.35-2.44 (m,1H), 2.72 (s, 3H), 3.13 (s, 3H), 3.42 (bs, 1H), 3.55 (s, 3H), 4.06 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 , rotameric) δ 20.9, 25.7, 26.0, 28.4, 28.5, 29.1, 29.6, 32.2, 36.4, 37.1, 55.9, 56.9, 61.3, 79.1, 155.9; IR (neat) 3400, 1643, 1145 cm -1 ; HRMS (ES+) m/e calc’d for [M+H]+ C 15 H 29 N 2 O 4 : 301.2127, found 301.2116. tert-Butyl (trans)-2-(Methoxy(methyl)carbamoyl)cyclohexyl(methyl)carbamate (2.8). The title compound was obtained from N-Boc-trans-2-aminocyclohexanecarboxylic acid using Method A and a subsequent methylation. The Weinreb amide intermediate (following Method A) was isolated as a colorless oil (96%) after purification via flash chromatography (25% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.16-1.58 (m, 4H) 1.38 (s, 9H), 1.65-1.82 (bm, 3H), 1.98-2.07 (m, 1H), 2.90 (bs, 1H), 3.15 (s, 3H), 3.42-3.53 (m, 1H), 3.68 (s, 3H), 4.67 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 25. 2, 25.4, 28.7, 29.4, 32.8, 44.9, 2.8 NH O Boc H H NMe N O Boc H H OMe Me OH 1) EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl 2) NaH, MeI, DMF 232 52.6, 61.8, 79.2, 155.4, 175.9; IR (neat) 3303, 2977, 2953, 2856, 1767, 1639, 1178 cm-1; HRMS (ES+) m/e calc’d for [M+H] + C 14 H 27 N 2 O 4 : 287.1971, found 287.1968. Methylation Procedure: The Weinreb amide (70 mg, 0.25 mmol, 1.0 equiv) and MeI (80 μL, 1.3 mmol, 5.0 equiv) were dissolved in anhydrous DMF (20 mL) under an argon atmosphere and cooled to 0 °C. To this solution was added 60% NaH (20 mg, 0.49 mmol, 2.0 equiv) in one portion and the reaction was allowed to come to room temperature. It was found crucial to use high quality NaH for this reaction to proceed. After 8 h the reaction was judged complete by TLC (50% EtOAc/hexanes). The reaction was quenched by the addition of water and placed in a separatory funnel. This solution was diluted with water and extracted with EtOAc (x3). The combined organic layers were back extracted with water (x2) to remove any residual DMF then dried over Na 2 SO 4 , filtered and concentrated. This crude material was purified via flash chromatography (25% EtOAc/hexanes) to provide 73 mg (99%) of the title compound as a colorless oil: 1 H NMR (400 MHz, CDCl3, 50 °C) δ 1.15-1.58 (m, 3H), 1.43 (s, 9H), 1.65-1.89 (bm, 6H), 2.79 (s, 3H), 3.14 (s, 3H), 3.42-3.53 (bs, 1H), 3.69 (s, 3H); 13 C NMR (100 MHz, CDCl3, rotameric) δ 25.0, 25.1, 25.5, 25.6, 28.0, 28.5, 29.2, 29.9, 30.0, 32.3, 42.6, 61.5, 78.8, 79.5, 154.9, 155.8, 175.1, 175.7; IR (neat) 2957, 1680, 1360 cm -1 ; HRMS (ES+) m/e calc’d for [M+H] + C 15 H 29 N 2 O 4 : 301.2127, found 301.2110. 233 (R)-tert-Butyl 3-(Methoxy(methyl)amino)-3-oxo-1-phenylpropylcarbamate (2.9). The title compound was obtained using Method A as a colorless oil (99%) after flash chromatography (70% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 1.41 (s, 9H), 2.83 (dd, J = 15.2, 4.3 Hz, 1H), 3.03-3.10 (m, 1H), 3.10 (s, 3H), 3.50 (s, 3H), 5.11 (bs, 1H), 6.14 (bs, 1H), 7.20-7.26 (m, 1H), 7.29-7.33 (m, 4H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.4, 31.9, 37.8, 51.3, 61.2, 79.3, 126.1, 127.1, 128.5, 142.1, 155.2, 171.8; IR (neat) 2977, 1712, 1655, 1496, 1366, 1170 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 25 N 2 O 4 : 309.1814, found 309.1800; α [ ] D 22 = –12.0 (c 1.00, CHCl 3 ). (S)-tert-Butyl 4-(Methoxy(methyl)amino)-4-oxo-1-phenylbutan-2-ylcarbamate (2.10). The crude material from Method B was purified via flash chromatography (25% EtOAc/ hexanes) to provide the desired product as a colorless oil (54% 2 steps): 1 H NMR (400 MHz, CDCl 3 , 50 °C) δ 1.38 (s, 9H), 2.50-2.62 (bm, 2H), 2.80-2.87 (m, 1H), 2.96-3.02 (m, 1H), 3.13 (s, 3H), 3.49 (s, 3H), 4.14 (bs, 1H), 5.52 (bs, 1H), 7.16-7.30 (m, 5H); 13 C NMR (100 N H O N Boc Me OMe 2.9 2.10 N H O N Boc Me OMe 234 MHz, CDCl 3 , 50 °C) δ 28.8, 32.3, 34.8, 40.7, 49.3, 61.4, 79.3, 126.8, 128.8, 129.7, 138.9, 155.7, 173.0; IR (neat) 3342, 2975, 2934, 1713, 1660, 1496, 1171 cm -1 ; HRMS (ES + ) m/e calc’d for [M+H] + C 17 H 27 N 2 O 4 : 323.1971, found 323.1943; α [ ] D 22 = –21 (c 0.75, CHCl 3 ). tert-Butyl 3-(Methoxy(methyl)amino)-3-oxopropylcarbamate (2.11). The title compound was obtained according to Method A as a colorless oil (95%) after flash chromatography (85% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.43 (s, 9H), 2.63-2.67 (bm, 2H), 3.19 (s, 3H), 3.42 (td, J = 5.8, 5.8 Hz, 2H), 3.68 (s, 3H), 5.31 (bm, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 28.3, 31.9, 32.2, 35.7, 61.1, 61.2, 78.9, 155.9, 173.1; IR (neat) 3349, 2976, 1713, 1660, 1520, 1173 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 10 H 21 N 2 O 4 : 233.1501, found 233.1501. tert-Butyl Benzyl(3-(methoxy(methyl)amino)-3-oxopropyl)carbamate (2.12). The title compound was obtained using Method C as a colorless oil (76% 2 steps) after flash 2.11 N H O N Boc Me OMe 2.12 N Boc O N Me OMe 235 chromatography (70% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.44 (s, 9H), 1.50 (s, 9H), 2.58-2.63 (bm, 2H), 2.67-2,72 (bm, 2H), 3.15 (s, 6H), 3.43-3.48 (bm, 2H), 3.51-3.55 (bm, 2H), 3.63 (bs, 6H), 4.48 (s, 4H), 7.22-7.35 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.4, 31.2, 32.1, 42.8, 43.1, 50.6, 51.6, 61.2, 61.3, 79.8, 127.2, 127.3, 127.8, 128.4, 138.4, 138.8, 155.5, 155.8, 172.5, 172.9; IR (neat) 2974, 1693, 1664, 1413, 1366, 1167 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 17 H 27 N 2 O 4 : 323.1971, found 323.1957. tert-Butyl 3-(Methoxy(methyl)amino)-3-oxopropyl(phenyl)carbamate (2.13). The methyl ester precursor was synthesized from aniline (5.0 mL, 53 mmol, 1.0 equiv) and methyl acrylate (4.7 mL, 53 mmol, 1.0 equiv) as reported by Amore et al. 346 The crude product was redissolved in ethanol (100 mL) and di-tert-butyldicarbonate (12.6 g, 57.8 mmol, 1.10 equiv) was added slowly. The reaction mixure was stirred for 24 h and the solvent was removed in vacuo. The concentrated reaction mixture was redissolved in CH 2 Cl 2 (300 mL) and washed with cold 10% HCl (100 mL x 2) and brine (100 mL x 2). The organic layer was dried with MgSO 4 , concentrated and purified via SiO 2 flash chromatography (25-50% EtOAc in N H O OMe 1) Boc 2 O, EtOH 2) i-PrMgCl, THF HN(OMe)Me•HCl N O N Me OMe Boc NH 2 + CO 2 Me 2.13 Amore et al. 346 236 hexanes) to afford 8.71g (57% for 2 steps) of the methyl ester as a clear viscous oil. This oil was converted to the corresponding Weinreb amide using the procedure reported by Williams et al. 279 The crude product was purified via SiO 2 flash chromatography (40% EtOAc in hexanes) to afford 7.89 g (82%) of the Weinreb amide as a clear viscous oil: 1 H NMR (400 MHz, CDCl 3 ) δ 1.43 (s, 9H), 2.73 (t, J = 7.5, 2H), 3.12 (s, 3H), 3.65 (s, 3H), 3.96 (t, J = 7.5 Hz, 2H), 7.22-7.17 (m, 3H), 7.34-7.31 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 31.1, 32.0, 46.3, 61.3, 80.3, 126.1, 127.0, 128.7, 142.3, 154.5, 172.4; IR (neat) 2976, 1698, 1664, 1456, 1391, 1168 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 25 N 2 O 4 : 309.1814, found 309.1821. tert-Butyl 2-(3-(Methoxy(methyl)amino)-3-oxopropyl)pyrrolidine-1-carboxylate. The methyl ester precursor to the title compound was prepared in 4 steps (88%) from the precedent of Poon et al. 347 The title compound was obtained as a colorless oil using step 2 of Method C: 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.46 (s, 18H), 1.62-1.76 (m, 4H), 1.79-2.00 (bm, 8H), 2.40-2.49 (bm, 4H), 3.18 (s, 6H), 3.29-3.34 (bm, 3H), 3.40-3.47 (bm, 1H), 3.69 (s, 6H), 3.80-3.91 (bm, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 23.0, 23.7, 28.0, 28.5, 29.1, 29.5, 29.7, 30.1, 30.9, 32.2, 46.0, 46.3, 56.7, 56.9, 61.1, 61.2, 78.8 N O Me OMe N Boc CO 2 Me N Boc i-PrMgCl, THF HN(OMe)Me•HCl 237 79.2, 154.8, 174.2; IR (neat) 2972, 1692, 1393, 1367, 1172, 1107 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 25 N 2 O 4 : 287.1971, found 287.1982. \ tert-Butyl 2-(3-(Methoxy(methyl)amino)-3-oxopropyl)piperidine-1-carboxylate. The methyl ester precursor to the title compound was prepared in 4 steps (85%) from the precedent of Poon et al. 347 The title compound was obtained as a colorless oil using step 2 of Method C: 1 H NMR (400 MHz, CDCl 3 ) δ 1.33-1.50 (m, 1H), 1.45 (s, 9H), 1.56-1.73 (m, 6H), 2.05-2.15 (m, 1H), 2.31-2.49 (m, 2H), 2.75-2.81 (m, 1H), 3.17 (s, 3H), 3.66 (s, 3H), 3.99 (bs, 1H), 4.27 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 19.1, 24.6, 25.6, 28.5, 28.9, 29.1, 32.2, 38.5, 50.2, 61.1, 79.1, 155.1, 174.4; IR (neat) 2936, 1687, 1417, 1365, 1273, 1162 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 29 N 2 O 4 : 301.2127, found 301.2132. N O Me OMeN Boc CO 2 Me N Boc i-PrMgCl, THF HN(OMe)Me•HCl 238 NH O i) 5N HCl (aq.), reflux ii) PhCHO, NaOH, MeOH; then NaBH 4 iii) Boc 2 O, NaOH, t-BuOH iv) EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl N Boc O N OMe Me tert-Butyl Benzyl ((1 - (2- (methoxy (methyl)amino) 2 - oxoethyl) cyclohexyl) methyl) carbamate. Spirocyclic lactam (4.0 g, 26 mmol, 1.0 equiv) was dissolved in 5N HCl aq. (100 mL). The solution was heated to reflux for 24 h. The solvent was removed in vacuo leaving an off-white solid. The solid was redissolved in MeOH (60 mL) and to the solution was added solid NaOH (2.6 g, 65 mmol, 2.5 equiv). When everything had dissolved benzaldehyde (3.7 mL, 37 mmol, 1.4 equiv) was added. After 10 min of stirring, the reaction mixture was cooled to 0 ºC and NaBH 4 (1.3 g, 34 mmol, 1.3 equiv) was added and the reaction stirred for 2 h. The solvent was removed under reduced pressure and the residue redissolved in MeOH (50 mL). This was repeated 3 times. The residue was dissolved in t- BuOH (60 mL) and di-tert-butyldicarbonate (7.4 g, 34 mmol, 1.3 equiv) was added slowly. 10% NaOH was added to the reaction mixture until pH = 12. The reaction was stirred for 5 h keeping the pH at 12 by adding 10% NaOH when appropriate. The reaction was quenched by adding 1N NaHSO 4 until the pH reached 2 and EtOAc (150 mL) was added. The product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and purified via SiO 2 flash chromatography using 10 % EtOAc in hexanes (1% AcOH) as eluent to afford 5.3 g (56%) of the carboxylic acid as a clear viscous oil. Method A was used to convert the acid into the Weinreb amide. The title compound was obtained as a colorless oil after flash chromatography (25% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 1.33 (s, 239 9H), 1.33-1.56 (m, 8H), 1.64-1.71 (m, 2H), 2.51 (s, 2H), 3.15 (s, 3H), 3.39 (bs, 1H), 3.48 (bs, 1H), 3.65 (s, 3H), 4.49 (bs, 2H), 7.18-7.23 (m, 3H), 7.27-7.31 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.9, 26.0, 28.3, 32.0, 33.9, 35.1, 39.4, 53.3, 54.5, 61.0, 79.4, 126.7, 127.4, 128.3, 139.5, 157.1, 173.4; IR (neat) 2931, 1695, 1454, 1407, 1248, 1165 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 37 N 2 O 4 : 405.2753, found 405.2738. i) PhCHO, NaOH, MeOH; then NaBH 4 ii) Boc 2 O, NaOH, t-BuOH iii) EDCI, NMM, CH 2 Cl 2 HN(OMe)Me•HCl NH 2 CO 2 H Cl N Boc Cl baclofen O N Me OMe tert-Butyl Benzyl(2-(4-chlorophenyl)–4-(methoxy (methyl) amino)-4-oxobutyl) carbamate. Baclofen (900 mg, 4.2 mmol, 1.0 equiv) was dissolved in MeOH (10 mL) and to the solution was added solid NaOH (190 mg, 4.6 mmol, 1.1 equiv). When everything had dissolved benzaldehyde (0.60 mL, 5.9 mmol, 1.4 equiv) was added. After 10 min of stirring, the reaction mixture was cooled to 0 ºC and NaBH 4 (210 mg, 5.5 mmol, 1.3 equiv) was added and the reaction stirred for 2 h. The solvent was removed under reduced pressure and the residue redissolved in MeOH (10 mL). This was repeated three times. The residue was dissolved in t-BuOH (10 mL) and di-tert-butyldicarbonate (1.2 g, 5.5 mmol, 1.3 equiv) was added slowly. 10% NaOH was added to the reaction mixture until pH = 12. The reaction was stirred for 5 h keeping the pH at 12 by adding 10% NaOH when appropriate. The reaction was quenched by adding 1N NaHSO 4 until the pH of the reaction mixture had reached 2. The product was extracted with EtOAc (3x). The combined organic layers were 240 dried over Na 2 SO 4 and purified via SiO 2 flash chromatography using 15 % EtOAc in hexane (1% AcOH) as eluent to afford 1.2 g (69%) of carboxylic acid as a clear viscous oil. Method A was used to convert the acid into the Weinreb amide. The title compound was obtained as a colorless oil after flash chromatography (35% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.38 (s, 9H), 1.44 (s, 9H), 2.63-2.84 (m, 4H), 3.08 (s, 6H), 3.25-3.34 (m, 2H), 3.37-3.42 (m, 1H), 3.55-3.66 (m, 3H), 3.59 (s, 6H), 4.13 (d, J = 15.6 Hz, 2H), 4.45-4.52 (m, 2H), 7.10-7.31 (m, 18H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 32.1, 35.3, 35.5, 39.2, 39.6, 49.7, 50.1, 50.8, 51.5, 61.2, 79.9, 127.2, 127.9, 128.5, 128.6, 129.3, 132.4, 132.4, 138.0, 138.1, 140.8, 155.8, 172.1, 172.4; IR (neat) 2975, 1693, 1416, 1366, 1165, 701 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 24 H 32 ClN 2 O 4 : 447.2051, found 447.2053. 6.3.2 Preparation of ynones: The Weinreb amide (2.52 mmol, 1.00 equiv) was dissolved in anhydrous THF (40 mL) under an argon atmosphere and cooled to 0 °C. To this reaction vessel, was added dropwise, a solution of ethynyl magnesium bromide reagent (12.6 mmol, 5.00 equiv) in THF and allowed to come to room temperature. After the reaction was judged complete by TLC it was quenched by the addition of an ice cold 10% HCl solution (15 mL) and allowed to stir at this temperature for 5 min. The reaction was diluted with water and extracted with EtOAc (x3). Boc N R 3 R 2 R 1 O N Me OMe R 4 BrMg THF Boc N R 3 R 2 R 1 O R 4 241 The combined organic layers were washed with saturated NaHCO 3 (x1), dried over Na 2 SO 4 , filtered and concentrated. This crude material was purified via flash chromatography using EtOAc in hexanes as eluent to provide pure N-Boc-ynone. N Boc O N Boc O N Boc O N Boc O N Boc O N Boc O N H O Boc N H O Boc Me Me Me NMe NMe Boc O Boc O H H H H N Boc O N Boc O HOHO 2.14 2.15 2.16 2.17 2.18 2.19 2.23 2.24 2.29 2.30 2.26 2.27 Compounds previously reported in Turunen and Georg. 255 242 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-oxobut-3-ynyl)pyrrolidine-1-carboxylate (2.20). The title compound was obtained as a colorless oil (92%) after flash chromatography (20% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.46 (s, 9H), 1.48 (s, 9H), 1.77-1.87 (bm, 2H), 2.35 2.35 (dddd, J = 13.3, 7.7, 3.9, 1.0 Hz, 2H), 2.65-2.73 (bm, 2H), 3.16 (bd, J = 15.3 Hz, 1H), 3.26 (bd, J = 16.9 Hz, 2 H), 3.35-3.42 (m, 3H), 3.56 (bd, J = 11.3 Hz, 1H), 3.78 (bd, J = 11.6 Hz, 1H), 4.06-4.10 (m, 2H), 4.30-4.38 (bm, 2H), 4.44-4.55 (bm, 2H), 4.50 (bd, J = 10.2 Hz, 2H), 7.28-7.36 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.5, 36.9, 38.2, 49.7, 50.9, 51.1, 51.9, 52.2, 70.9, 75.6, 76.1, 78.8, 79.1, 79.7, 80.3, 81.5, 127.6, 127.7, 127.7, 128.5, 137.9, 154.4, 154.5, 185.0, 185.3; IR (neat) 2976, 2091, 1685, 1399, 1367, 1162 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 20 H 26 NO 4 : 344.1862, found 344.1854. 2.20 N Boc O O 243 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-oxopent-3-ynyl)pyrrolidine-1-carboxylate (2.21). The title compound was obtained as a colorless oil (90%) after flash chromatography (20% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.46 (s, 9H), 1.48 (s, 9H), 1.80-1.88 (m, 2H), 2.01 (s, 6H), 2.33 (dddd, J = 13.5, 7.7, 4.1, 1.5 Hz, 2H), 2.60 (dd, J = 15.8, 9.2 Hz, 2H), 3.09 (bd, J = 14.7 Hz, 1H), 3.34-3.44 (m, 3H), 3.55 (bd, J = 10.4 Hz, 1H), 3.75 (bd, J = 11.4 Hz, 1H), 4.09 (p, J = 1.2 Hz, 2H), 4.29-4.37 (m, 2H), 4.46-4.54 (m, 4H), 7.26-7.36 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 4.1, 28.5, 36.8, 38.1, 49.6, 50.9, 51.0, 51.9, 52.5, 70.9, 75.7, 76.2, 79.6, 80.0, 80.4, 127.6, 127.7, 127.7, 128.5, 137.9, 154.4, 185.8, 186.0; IR (neat) 2975, 2219, 1694, 1398, 1366, 1166 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 21 H 28 NO 4 : 358.2018, found 358.2003. 2.21 N Boc O O Me 2.22 N Boc O O 244 (2S*,4R*)-tert-Butyl 4-(Benzyloxy)-2-(2-oxo-4-phenylbut-3-ynyl)pyrrolidine-1- carboxylate (2.22). The title compound was obtained as a yellow oil (85%) after flash chromatography (15% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.46 (s, 9H), 1.50 (s, 9H), 1.87-1.94 (bm, 2H), 2.36-2.42 (bm, 2H), 2.75-2.83 (bm, 2H), 3.22 (bd, J = 14.5 Hz, 1H), 3.41-3.53 (bm, 3H), 3.58 (bd, J = 10.7 Hz, 1H), 3.79 (bd, J = 11.2 Hz, 1H), 4.11 (p, J = 4.1 Hz, 2H), 4.38-4.44 (bm, 2H), 4.46-4.55 (m, 2H), 4.5 (d, J = 10.7 Hz, 2H), 7.27-7.41 (m, 14H), 7.43-7.48 (bm, 2H), 7.57 (d, J = 7.12 Hz, 4H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.5, 36.9, 38.2, 49.6, 50.9, 51.1, 52.0, 52.6, 70.9, 75.7, 76.2, 79.6, 80.2, 88.0, 91.0, 91.4, 119.7, 119.9, 127.6, 127.7, 127.7, 128.4, 128.6, 128.7, 130.7, 130.9, 133.1, 137.9, 154.5, 185.6, 185.9; IR (neat) 2975, 2203, 1692, 1397, 1366, 1160 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 26 H 30 NO 4 : 420.2175, found 420.2157. tert-Butyl 3-(2-Oxobut-3-ynyl)morpholine-4-carboxylate (2.25). The title compound was obtained as a white solid (95%) after flash chromatography (20% EtOAc/hexanes): mp 86.3-86.7 °C 1 H NMR (400 MHz, CDCl 3 ) δ 1.46 (s, 9H), 2.87 (dd, J = 15.8, 5.4 Hz, 1H), 3.08-3.16 (m, 2H), 3.34 (s, 1H), 3.46 (dt, J = 11.8, 2.5 Hz, 1H), 3.59 (dd, J = 11.8, 2.8 Hz, 1H), 3.71-3.90 (m, 3H), 4.43-4.52 (bm, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 39.0, 44.6, N O Boc O 2.25 245 47.9, 66.8, 69.0, 79.0, 80.7, 81.5, 154.3, 184.7; IR (neat) 2977, 2092, 1685, 1408, 1367, 1170 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 13 H 20 NO 4 : 254.1392, found 254.1386. (R)-tert-Butyl 3-Oxo-1-phenylpent-4-ynylcarbamate (2.28). The title compound was obtained as a off-white solid (76%) after flash chromatography (20% EtOAc/hexanes): mp 94.1-95.4 ºC; 1 H NMR (400 MHz, CDCl 3 ) δ 1.42 (s, 9H), 3.08 (dd, J = 16.2, 6.1 Hz, 1H), 3.18-3.24 (bm, 1H), 3.27 (s, 1H), 5.16 (bs, 1H), 5.23 (bs, 1H), 7.25-7.36 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 50.9, 51.3, 79.9, 79.9, 81.1, 126.3, 127.7, 128.8, 140.7, 154.9, 184.3; IR (neat) 3270, 2979, 2094, 1687, 1367, 1169 cm -1 ; HRMS (ESI+) m/e calc’d for [M +H] + C 16 H 20 NO 3 : 274.1443, found 274.1452; α [ ] D 22 = –20 (c 0.61, CHCl 3 ). tert-Butyl 3-Oxopent-4-ynylcarbamate (2.31). The title compound was obtained as a colorless oil (89%) after flash chromatography (25% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 1.43 (s, 9H), 2.86 (t, J = 5.8 Hz, 2H), 3.32 (s, 1H), 3.42 (dt, J = 5.96, 5.96 Hz, 2H) 2.28 N H O Boc N H O Boc 2.31 246 4.97 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.4, 34.9, 45.6, 79.4, 79.5, 81.2, 155.7, 186.1; IR (neat) 3353, 2979, 2093, 1683, 1515, 1172 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 10 H 16 NO 3 : 198.1130, found 198.1121. tert-Butyl Benzyl(3-oxopent-4-ynyl)carbamate (2.32). The title compound was obtained as a colorless oil (89%) after flash chromatography (20% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.45 (s, 9H), 1.50 (s, 9H), 2.75-2.80 (bm, 2H), 2.86-2.91 (bm, 2H), 3.24 (s, 2H), 3.45-3.55 (bm, 4H), 4.45 (bs, 4H), 7.21-7.35 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.4, 41.5, 41.9, 44.3, 44.6, 50.5, 51.6, 79.1, 80.3, 81.3, 127.2, 127.8, 127.3, 128.6, 138.2, 155.4, 185.3; IR (neat) 2977, 2092, 1684, 1415, 1367, 1165 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 17 H 22 NO 3 : 288.1600, found 288.1594. tert-Butyl 3-Oxopent-4-ynyl(phenyl)carbamate (2.33). The title compound was obtained as a colorless oil (99%) after flash chromatography (15% EtOAc/hexanes): 1 H NMR (400 N Boc O 2.32 N Boc O 2.33 247 MHz, CDCl 3 ) δ 1.42 (s, 9H), 2.89 (t, J = 7.2, 2H), 3.24 (s, 1H), 3.99 (t, J = 7.2 Hz, 2H), 7.16-7.23 (m, 3H), 7.32-7.36 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 44.3, 45.1, 79.1, 80.7, 81.2, 126.5, 127.2, 128.9, 141.9, 154.4, 185.0; IR (neat) 2978, 2092, 1694, 1393, 1368, 1163 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 20 NO 3 : 274.1443, found 274.1445. tert-Butyl 2-(3-Oxopent-4-ynyl)pyrrolidine-1-carboxylate (2.57). The title compound was obtained as a colorless oil (90%) after flash chromatography (20% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.47 (s, 9H), 1.58-1.66 (m, 1H), 1.73-2.01 (m, 5H), 2.59-2.68 (m, 2H), 3.25-3.48 (m, 2H), 3.26 (s, 1H), 3.80-3.91 (m, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 23.0, 23.7, 28.5, 30.2, 30.9, 42.4, 42.6, 46.1, 46.5, 78.6, 79.1, 79.5, 81.4, 154.8, 186.6, 186.9; IR (neat) 2974, 2090, 1686, 1396, 1171, 1112 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 14 H 22 NO 3 : 252.1600, found 252.1599. O N Boc 2.57 O N Boc 2.58 248 tert-Butyl 2-(3-Oxopent-4-ynyl)piperidine-1-carboxylate (2.58). The title compound was obtained as a colorless oil (95%) after flash chromatography (20% EtOAc/hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 1.34-1.48 (m, 2H), 1.46 (s, 9H), 1.51-1.75 (m, 5H), 2.06-2.15 (m, 1H), 2,51-2.67 (m, 2H), 2.72 (bdd, J = 13.0, 13.0 Hz, 1H), 3.25 (s, 1H), 3.98 (bs, 1H), 4.25 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 19.0, 23.4, 25.5, 28.4, 29.0, 38.6, 42.3, 49.5, 78.5, 79.5, 81.5, 155.1, 186.6; IR (neat) 2936, 2090, 1683, 1418, 1366, 1162 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 24 NO 3 : 266.1756, found 266.1750. tert-Butyl Benzyl((1-(2-oxobut-3-ynyl)cyclohexyl)methyl)carbamate (2.59). The title compound was obtained as a colorless oil (93%) after flash chromatography (15% EtOAc/ hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 1.26-1.68 (m, 19H), 2.73 (s, 2H), 3.15 (s, 1H), 3.35 (bs, 2H), 4.47 (bs, 2H), 7.16-7.25 (m, 3H), 7.29-7.32 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.7, 25,9, 28.3, 33.9, 40.3, 49.3, 53.2, 54.5, 79.8, 82.8, 126.8, 126.9, 128.4, 139.0, 157.0, 186.5; 13 C NMR (100 MHz, CDCl 3 ) δ 21.7, 25.9, 28.3, 33.9, 40.3, 49.3, 53.2, 54.6, 79.8, 80.3, 82.8, 126.8, 126.9, 128.4, 138.9, 157.0, 186.5; IR (neat) 2930, 2090, 1692, 1409, 1248, 1164 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 32 NO 3 : 370.2382, found 370.2385. 2.59 N Bn O Boc 249 tert-Butyl Benzyl(2-(4-chlorophenyl)-4-oxohex-5-ynyl)carbamate (2.60). The title compound was obtained as a colorless oil (95%) after flash chromatography (15% EtOAc / hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ (1:1 mixture of rotamers) 1.41 (s, 9H), 1.46 (s, 9H), 2.85-3.04 (m, 4H), 3.20 (s, 2H), 3.21 (dd, J = 13.9, 7.7 Hz, 2H), 3.32-3.39 (m, 1H), 3.55-3.70 (m, 3H), 4.07 (bs, 1H), 4.11 (bs, 1H), 4.37-4.46 (m, 2H), 7.05-7.18 (m, 8H), 7.23-7.32 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.3, 38.9, 39.3, 48.6, 48.9, 50.2, 50.8, 51.6, 79.1, 80.3, 81.3, 127.2, 127.3, 127.8, 128.6, 128.8, 129.1, 132.9, 137.9, 139.5, 155.9; IR (neat) 2976, 2092, 1686, 1413, 1366, 1164 cm -1 ; HRMS (ESI+) m/e calc’d for [M+Na] + C 24 H 26 NO 3 ClNa: 434.1493, found 434.1479. 6.3.3 Preparation of cyclic enaminones Boc N R 3 R 2 R 1 O R 4 i) Method A, B or C ii) K 2 CO 3 , MeOH N O R 3 R 2 R 1 R 4 n n n = 1,2 Representative procedures for deprotection: METHOD A: The ynone (0.50 mmol) was dissolved in a 4N HCl-dioxane solution (1.5 mL) and allowed to react for 15 min. After this time the dioxane and excess HCl were 2.60 N Boc BnCl O 250 allowed to evaporate while passing air over the reaction mixture. The remaining solid was placed under vacuum for 15 min and carried on to the cyclization step without further purification. METHOD B: The ynone (0.50 mmol, 1.0 equiv) was dissolved in 98% formic acid (5.0 mL) under a N 2 atmosphere and NaI (230 mg, 1.5 mmol, 3.0 equiv) was added. Note: For terminal ynones the reaction was left stirring for 6 h. When internal ynones were used the reaction was left for 24 h. The solvent was removed by passing N 2 over the reaction mixture. The remaining residue was placed under vacuum for 15 min and was carried on to the cyclization step without further purification. Large Scale Modification: When using greater that 1.0 gram of ynone, the desired deprotected amine can be isolated by pouring the reaction mixture into ether and filtering the precipitate. METHOD C: The ynone (0.50 mmol, 1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 (10 mL) under an argon atmosphere and cooled to –78 °C. A solution of TMS-I ( 0.50 mmol, 1.0 equiv) in anhydrous CH 2 Cl 2 (5 mL) was then added dropwise at this temperature. After 20 min at this temperature the reaction was allowed to warm to 0 °C and additional TMS-I (0.50-2.5 mmol) was added until all starting material was consumed (TLC, 25% EtOAc in hexanes). After 20 min the reaction was judged complete and this mixture was concentrated under reduced pressure and placed under vacuum for 15 min. 251 Representative procedures for cyclization: The deprotected intermediates from Methods A, B and C were dissolved in MeOH (50 mL) and excess K 2 CO 3 (a minimum of 5.0 equiv) was added. The reactions were stirred for various lengths of time depending on the substrate (For enaminones 2.37, 2.41, 2.35, 2.48, 2.36 and 2.50 reactions were stirred for 1 h; for enaminones 2.34, 2.49, 2.52, 2.51, 2.54 and 2.55 reactions were stirred for 3 h; for enaminones 2.46, 2.47, 2.39, 2.40, 2.42, 2.43, 2.53, 2.56, 2.61, 2.62, 2.63 and 2.64 reactions were stirred for 6 h). At this time CH 2 Cl 2 was added, the reaction suction filtered, and the organic solvents concentrated. To the solid residue was added more CH 2 Cl 2 and the precipitates were once again filtered away. This residue was purified via flash chromatography to provide pure enaminone. Large Scale Modification: When the reaction was complete, the MeOH was removed in vacuo and the remaining residue redissolved in brine. The product was extracted with CH 2 Cl 2 (3x). The combined organic layers were dried over Na 2 SO 4 and purified via SiO 2 flash chromatography. Note: Several of the enaminones (e.g. 2.34, 2.37, 2.35, 2.48, etc.) were water soluble and could not be purified by this method. 252 (S)-7,8,9,9a-Tetrahydro-1H-quinolizin-2(6H)-one (2.34). Spectral data of the title compound was identical to that reported in the literature 255 with the exception of optical rotation: Comparison to the reported value, β [ ] D 22 = –146 (c 0.885, CHCl 3 ), indicated an ee of 93% and 99% for Method A ( α [ ] D 22 = –135 (c 0.925, CHCl3)) and Method B ( α [ ] D 22 = –145 (c 1.15, CHCl 3 )), respectively. These enantiomeric ratios were verified via chiral HPLC N N Me O Me O H H H H N O N O N O Me Ph N O N O N O Me Ph N O N O H H H H H H H H HO HO HN O HN O Me H H 2.52 2.352.34 2.46 2.47 2.37 2.40 2.53 2.36 2.50 2.39 2.48 Compounds previously reported by Turunen and Georg. 255 2.34 N O H 253 using a Baker Chiralcel OJ column. Conditions: i-PrOH 2-15% in hexanes, 60 min, 0.5 mL/ min, 30 °C. (–)-Enantiomer: R t = 40.1 min; (+)-Enantiomer: R t = 41.6 min. Method A: ee = 94%. Method B: ee = 96%. (S)-4-Methyl-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (2.46). Spectral data of the title compound was identical to that reported in the literature. 255 The optical rotation, however, has not been previously reported: Enantiomeric ratios were determined via chiral HPLC using a Chiralcel OD column. Conditions: i-PrOH 1-25% in hexanes, 60 min, 1.0 mL/min, 30 °C. (–)-Enantiomer: R t = 36.3 min; (+)-Enantiomer: R t = 39.6 min. Method A: ee = 44%; Method B: ee = 44%. Method A and B α [ ] D 22 = –95.0 (c 0.924, CHCl 3 ). (S)-4-Phenyl-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (2.47). Spectral data of the title compound was identical to that reported in the literature. 255 The optical rotation, however, has not been previously reported: Enantiomeric ratios were determined via chiral 2.46 N O H Me 2.47 N O H 254 HPLC using a Chiralcel AD-H column. Conditions: i-PrOH 1-20% in hexanes, 40 min, 1.0 mL/min, 30 °C. (–)-Enantiomer: R t = 30.4 min; (+)-Enantiomer: R t = 29.0 min. Method A: ee = 16%; Method B: ee = 38%. Method A β [ ] D 22 = –4.2 (c 1.00, CHCl 3 ); Method B α [ ] D 22 = –10.7 (c 1.00, CHCl 3 ). (S)-2,3,8,8a-Tetrahydroindolizin-7(1H)-one (2.37). Spectral data of the title compound was identical to that reported in the literature 255 with the exception of optical rotation. Method A: ee = 40%. Method B: ee = 96%; Method A α [ ] D 22 = –180 (c 1.00, CHCl 3 ); Method B α [ ] D 22 = –727 (c 1.00, CHCl 3 ); Method C α [ ] D 22 = –628 (c 1.00, CHCl 3 ). Enantiomeric ratios were determined via chiral HPLC using a Chiralcel OJ column after converting enaminone 2.37 to aryl derivative 3.2. Conditions for arylenaminone 3.2: i-PrOH 70% in hexanes, 30 min, 1.0 mL/min, 30 °C. (–)-Enantiomer: R t = 20.9 min; (+)-Enantiomer: R t = 11.1 min. 2.37 N O H 2.41 N O H O 255 (2R*, 8aS*)-2-(Benzyloxy)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.41). The title compound was obtained as a colorless oil (Method A, 94%; Method B, 92%) after flash chromatography (100% acetone): 1 H NMR (400 MHz, CDCl 3 ) δ 1.77 (ddd, J = 13.2, 11.2, 4.5 Hz, 1H), 2.35 (dd, J = 16.2, 16.2 Hz, 1H), 2.44-2.51 (m, 2H), 3.60 (d, J = 11.8 Hz, 1H), 3.70 (dd, J = 11.8, 4.9 Hz, 1H), 4.07 (dddd, J = 16.4, 10.9, 5.3, 5.3 Hz, 1H), 4.29 (dd, J = 4.5, 4.5 Hz, 1H), 4.51 (d, J = 11.8 Hz, 1H), 4.56 (d, J = 11.8 Hz, 1H), 4.99 (d, J = 7.1 Hz, 1H), 7.17 (d, J = 7.1 Hz, 1H), 7.29-7.38 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) δ 38.5, 41.4, 55.4, 56.2, 71.0, 77.1, 97.9, 127.6, 128.0, 128.6, 137.5, 149.9, 191.9; IR (neat) 2929, 1631, 1579, 1460, 1306, 1099 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 18 NO 2 : 244.1338, found 244.1332. (2R*,8aS*)-2-(Benzyloxy)-5-methyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (anti-2.42). The title compound was obtained as a colorless oil [Method A, 87% (d.r. 63:37); Method B, 94% (d.r. 68:32)] after flash chromatography (1-10% MeOH/EtOH): 1 H NMR (400 MHz, CDCl 3 ) δ 1.67 (ddd, J = 13.3, 11.4, 4.5 Hz, 1H), 1.92 (s, 3H), 2.22 (dd, J = 16.1, 16.1 Hz, 1H), 2.36-2.43 (m, 2H), 3.33 (d, J = 12.16 Hz, 1H), 3.61 (dd, J = 12.2, 4.7 Hz, 1H), 4.00 (dddd, J = 16.3, 10.7, 5.0, 5.0 Hz, 1H), 4.22 (dd, J = 4.5, 4.5 Hz, 1H), 4.43 (d, J = 11.8 Hz, 1H), 4.51 (d, J = 11.8 Hz, 1H), 4.89 (s, 1H), 7.22-7.31 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) N O H O Me anti-2.42 256 δ 20.4, 38.3, 41.0, 53.5, 56.8, 71.0, 76.3, 98.5, 127.6, 128.0, 128.6, 137.5, 160.8, 191.3; IR (neat) 2928, 1622, 1550, 1497, 1431, 1093 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 20 NO 2 :, 258.1494, found 258.1491. (2R*, 8aR*)-2-(Benzyloxy)-5-methyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (syn-2.42). This compound is the minor diastereomer of the above reaction. The compound was obtained as a colorless oil after flash chromatography (1-10% MeOH/EtOH): 1 H NMR (400 MHz, CDCl 3 ) δ 1.79 (ddd, J = 12.5, 10.0, 7.7 Hz, 1H), 1.89 (s, 3H), 2.33 (d, J = 16.0 Hz, 1H), 2.39 (d, J = 16.0 Hz, 1H), 2.49 (ddd, J = 12.7, 6.4, 6.4 Hz, 1H), 3.41 (dd, J = 11.2, 5.6 Hz, 1H), 3.60 (dd, J = 11.2, 7.0 Hz, 1H), 3.71 (dddd, J = 16.4, 10.0, 6.6, 6.6 Hz, 1H), 4.19 (m, 1H), 4.44 (d, J = 11.8 Hz, 1H), 4.53 (d, J = 11.8 Hz, 1H), 4.87 (s, 1H), 7.23-7.32 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) δ 20.4, 38.3, 41.5, 52.3, 57.4, 71.9, 76.0, 98.9, 127.7, 128.1, 128.6, 137.5, 160.7, 191.3; IR (neat) 2870, 1623, 1551, 1496, 1432, 1125 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 20 NO 2 : 258.1494, found 258.1493. syn-2.42 N O H O Me 257 (2R*,8aS*)-2-(Benzyloxy)-5-phenyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (anti-2.43). The title compound was obtained as a colorless oil [Method A, 85% (d.r. 63:37); Method B, 88% (d.r. 60:40)] after flash chromatography (1-10% MeOH/EtOH): 1 H NMR (400 MHz, CDCl 3 ) δ 1.80 (ddd, J = 13.3, 9.9, 4.7 Hz, 1H), 2.38-2.48 (m, 3H), 3.37 (dd, J = 12.3, 4.8 Hz, 1H), 3.52 (d, J = 12.3 Hz, 1H), 4.12-4.15 (m, 1H), 4.22 (dddd, J = 15.6, 10.3, 5.9, 5.9 Hz, 1H), 4.40 (d, J = 11.9 Hz, 1H), 4.48 (d, J = 11.9 Hz, 1H), 5.03 (s, 1H), 7.21-7.36 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 38.0, 41.4, 54.5, 57.1, 71.1, 76.2, 100.4, 127.5, 127.6, 127.9, 128.6, 128.6, 129.8, 135.9, 137.6, 162.8, 191.8; IR (neat) 2930, 1627, 1531, 1470, 1241, 701 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 21 H 22 NO 2 : 320.1651, found 320.1638. (2R*,8aR*)-2-(Benzyloxy)-5-phenyl-2,3,8,8a-tetrahydroindolizin-7(1H)-one (syn-2.43). This compound is the minor diastereomer of the above reaction. The compound was obtained N O H O anti-2.43 N O H O syn-2.43 258 as a colorless oil after flash chromatography (1-10% MeOH/EtOH): 1 H NMR (400 MHz, CDCl 3 ) δ 1.94-2.00 (m, 1H), 2.34 (dd, J = 16.1, 4.1 Hz, 1H), 2.43 (ddd, J = 13.5, 7.9, 5.6 Hz, 1H), 2.74 (dd, J = 16.2, 16.2 Hz, 1H), 3.32 (dd, J = 11.8, 3.0 Hz, 1H), 3.53 (dd, J = 11.8, 5.2 Hz, 1H), 4.05-4.14 (m, 2H), 4.35 (d, J = 11.9 Hz, 1H), 4.43 (d, J = 11.8 Hz, 1H), 5.08 (s, 1H), 7.18-7.38 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.9, 41.8, 54.9, 57.9, 71.3, 77.3, 100.6, 127.5, 127.8, 127.9, 128.5, 128.6, 130.1, 136.0, 137.5, 162.9, 192.4; IR (neat) 2926, 1626, 1529, 1467, 1242, 700 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 21 H 22 NO 2 : 320.1651, found 320.1640. 3,4,9,9a-Tetrahydropyrido[2,1-c][1,4]oxazin-8(1H)-one (2.49). The title compound was obtained as a white solid (Method A, 80%; Method B, 95%) after flash chromatography (100% acetone): mp 58.1-60.2 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 2.27 (dd, J = 16.3, 14.2 Hz, 1H), 2.33 (dd, J = 16.5, 6.2 Hz, 1H), 3.21-3.29 (m, 2H), 3.39 (dd, J = 10.9, 10.9 Hz, 1H), 3.49-3.57 (m, 1H), 3.65 (ddd, J = 11.5, 10.0, 4.6 Hz, 1H), 3.91-3.98 (m, 2H), 5.06 (d, J = 7.7 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 37.9, 50.2, 55.3, 66.5, 70.7, 100.6, 154.1, 191.1; IR (neat) 2854, 1639, 1590, 1445, 1223, 1121 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 8 H 12 NO 2 : 154.0868, found 154.0859. O N O 2.49 259 HN O H 2.51 (R)-2-Phenyl-2,3-dihydropyridin-4(1H)-one (2.51). The title compound was obtained as a white solid [Method A, 50% (e.r. > 99:1); Method B, 50% (e.r. > 99:1)] after flash chromatography (90% acetone/hexanes): mp 107.7-110.9 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 2.39 (dd, J = 16.3, 5.0 Hz, 1H), 2.62 (dd, J = 16.3, 14.6 Hz, 1H), 4.68 (dd, J = 14.6, 5.0 Hz, 1H), 4.99 (d, J = 7.3 Hz, 1H), 5.93 (bs, 1H), 7.25-7.38 (m, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 44.3, 58.2, 99.0, 126.6, 128.4, 128.9, 139.9, 151.8, 192.4; IR (neat) 3234, 3030, 1620, 1571, 1233, 699 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 11 H 12 NO: 174.0919, found 174.0918; β [ ] D 22 = +233 (c 0.720, CHCl 3 ) [Lit. +332.3 (c 2.42, EtOH)]. 236 Enantiomeric ratios were determined via chiral HPLC using a Chiralcel OD column. Conditions: i-PrOH 1-25% in hexanes, 60 min, 1.0 mL/min, 30 °C. (–)-Enantiomer: R t = 48.0 min; (+)-Enantiomer: R t = 45.2 min. 2,3-Dihydropyridin-4(1H)-one (2.54). The title compound was obtained as a colorless oil (Method A, 70%; Method B, 92%) after flash chromatography (10% EtOH/EtOAc): 1 H NMR (400 MHz, CD 3 OD) δ 2.31 (t, J = 8.1 Hz, 2H), 3.45 (t, J = 8.1 Hz, 2H), 4.82 (d, J = 7.0 Hz, HN O 2.54 260 1H), 7.32 (d, J = 7.0 Hz, 1H) 13 C NMR (100 MHz, CD 3 OD) δ 36.1, 41.9, 99.3, 151.8, 192.8; IR (neat) 3041, 1575, 1409, 1351, 1245, 1174 cm -1 ; HRMS (ESI+) m/e calc’d for [M+Na] + C 5 H 7 NO 2 Na: 120.0425, found 120.0423. 1-Benzyl-2,3-dihydropyridin-4(1H)-one (2.55). The title compound was obtained as an colorless oil (Method A, 50%; Method B, 86%) after flash chromatography (85% EtOAc). Spectral data of the title compound was identical to that reported in the literature. 348 1-Phenyl-2,3-dihydropyridin-4(1H)-one (2.56). The title compound was obtained as an off white solid (Method A, 50%; Method B, 70%) after flash chromatography (100% EtOAc): mp 89.0-90.5 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 2.67 (t, J = 7.6 Hz, 2H), 4.01 (t, J = 7.6 Hz, 2H), 5.24 (d, J = 7.8 Hz, 1H), 7.09-7.18 (m, 3H), 7.37-7.41 (m, 2H), 7.45 (d, J = 7.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 35.9, 47.6, 102.0, 118.2, 124.4, 129.7, 145.1, 149.7, 192.1; IR 2.55 N O N O 2.56 261 (neat) 3057, 1647, 1579, 1495, 1316, 1220 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 11 H 12 NO: 174.0919, found 174.0913. (Z)-2,3,9,9a-Tetrahydro-1H-pyrrolo[1,2-a]azepin-7(8H)-one (2.61). The title compound was obtained as a light yellow solid (Method A, 60%; Method B, 64%) after flash chromatography (100% acetone): mp 106.2-114.4 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 1.29-1.39 (m, 1H), 1.58-1.69 (m, 1H), 2.09-2.34 (m, 4H), 3.00-3.09 (m, 1H), 3.12-3.25 (m, 2H), 3.41-3.50 (bm, 1H), 3.93-4.00 (m, 1H), 4.99 (d, J = 8.4 Hz, 1H), 9.34 (bd, J = 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 28.0, 29.5, 31.4, 34.2, 43.3, 66.8, 95.7, 166.0, 186.8; IR (neat) 2860, 1623, 1583, 1398, 1190, 802 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 9 H 14 NO: 152.1075, found 152.1068. (Z)-1,2,3,4,10,10a-Hexahydropyrido[1,2-a]azepin-8(9H)-one (2.62). The title compound was obtained as a light yellow solid (Method A, 64%; Method B, 63%) after flash chromatography (100% acetone): mp 69.5-70.3 °C 1 H NMR (400 MHz, CDCl 3 ) δ 1.16-1.26 N O 2.61 N O 2.62 262 (m, 1H), 1.38-1.52 (m, 2H), 1.55-1.65 (m, 1H), 1.74-1.81 (m, 1H), 1.87-1.94 (m, 1H), 1.98 (d, J = 13.3 Hz, 1H), 2.20-2.28 (m, 1H), 2.81-2.90 (m, 2H), 3.13-3.21 (bm, 1H), 3.38-3.46 (m, 1H), 3.72 (d, J = 13.0 Hz, 1H), 5.10 (bd, J = 5.7 Hz, 1H), 9.42 (bs, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 23.4, 24.2, 27.8, 28.8, 32.6, 43.7, 62.4, 94.7, 167.6, 187.2; IR (neat) 2940, 1624, 1583, 1394, 1189, 779 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 10 H 16 NO: 166.1232, found 166.1223. 8-Benzyl-8-azaspiro[5.6]dodec-9-en-11-one (2.63). The title compound was obtained as a yellow oil (Method A, 81%; Method B, 84%) after flash chromatography (100% acetone): 1 H NMR (400 MHz, CDCl 3 ) δ 1.36-1.55 (m, 10H), 2.91 (bs, 2H), 3.22 (s, 2H), 4.38 (s, 2H), 5.27 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 6.8 Hz, 2H), 7.28-7.36 (m, 3H), 9.41 (d, J = 6.6 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 23.1, 25.5, 36.0, 39.5, 42.5, 50.1, 63.1, 95.7, 127.5 (2C), 127.9, 128.9, 135.2, 168.0, 187.5; IR (neat) 2925, 1632, 1591, 1552, 1397, 1188 cm -1 ; HRMS (ESI +) m/e calc’d for [M+H] + C 18 H 24 NO: 270.1858, found 270.1852. N O 2.63 263 (Z)-1-Benzyl-6-(4-chlorophenyl)-6,7-dihydro-1H-azepin-4(5H)-one (2.64). The title compound was obtained as a yellow oil (Method A, 66%; Method B, 65%) after flash chromatography (100% acetone): 1 H NMR (400 MHz, CDCl 3 ) δ 3.13-3.22 (m, 1H), 3.44 (dd, J = 10.0, 6.6 Hz, 1H), 3.54-3.66 (m, 2H), 3.80 (dd, J = 10.0, 8.0 Hz, 1H), 4.46 (d, J = 2.8 Hz, 2H), 5.36 (d, J = 7.9 Hz, 1H), 7.11 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 6.64 Hz, 2H), 7.27-7.37 (m, 5H), 9.46 (bd, J = 4.7 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 38.0, 39.5, 50.2, 59.2, 95.7, 127.6, 128.1, 129.0, 133.1, 134.8, 139.7, 166.9, 187.5; IR (neat) 1625, 1590, 1549, 1398, 1190, 702 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 19 ClNO: 312.1155, found 312.1155. 6.3.4 Supplementary studies N H 2 O I I 2.38 (E)-2-(4-Iodo-2-oxobut-3-enyl)pyrrolidinium Iodide (2.38). Ynone 2.17 (0.50 mmol, 1.0 equiv) was dissolved in 98% formic acid (5.0 mL) under a N 2 atmosphere and NaI (230 mg, N O Cl 2.64 264 1.5 mmol, 3.0 equiv) was added. After 6 h, Et 2 O (200 mL) was slowly added to the reaction mixture allowing a precipitate to form. The precipitate was filtered and washed several times with Et 2 O. The filtered precipitate was air dried and used without further purification. To obtain crystals for X-ray analysis, the precipitate was recrystallized from formic acid. See below for X-ray crystal structure report. 1 H NMR (400 MHz, CD 3 OD) δ 1.59-1.69 (m, 1H), 1.82-1.92 (m, 1H), 1.94-2.04 (m, 1H), 1.12-2.20 (m, 1H), 2.97 (dd, J = 18.9, 9.8 Hz, 1H), 3.13-3.24 (m, 3H), 3.76-3.83 (m, 1H), 7.16 (d, J = 15.2 Hz, 1H), 8.17 (d, J = 15.2 Hz, 1H); 13 C NMR (100 MHz, CD 3 OD) 24.6, 31.2, 42.6, 46.6, 56.8, 102.8, 145.2, 196.4; 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 1.49-1.58 (m, 1H), 1.74-1.95 (m, 2H), 2.04-2.12 (m, 1H), 3.04 (dd, J = 18.7 , 9.2 Hz, 1H), 3.04-3.17 (m, 2H), 3.17 (dd, J = 18.7, 4.3 Hz, 1H), 3.71-3.78 (m, 1H), 7.21 (d, J = 15.3 Hz, 1H), 8.26 (d, J = 15.3 Hz, 1H), 8.28 (bs, 1H), 8.85 (bs, 1H); 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ 23.1, 29.7, 41.4, 45.0, 54.3, 104.7, 143.7, 195.4. 5-Phenylpent-1-yn-3-one (2.65). The title compound was prepared using Method A for Weinreb amide formation and subsequent Grignard addition as described above. The product was obtained as a colorless oil (86% over 2 steps) after flash chromatography (5% EtOAc/ hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 2.89-2.93 (m, 2H), 2.96-3.00 (m, 2H), 3.22 (s, 1H), 7.17-7.23 (m, 3H), 7.26-7.30 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 29.5, 46.9, 78.9, 81.3, O 2.65 265 126.3, 128.3, 128.6, 139.9, 186.2; IR (neat) 3029, 2093, 1683, 1454, 1102, 699 cm -1 ; HRMS (EI+) m/e calc’d for [M+H] + C 11 H 11 O: 158.0732, found 158.0722. EXPERIMENT 1: TERMINAL YNONE CYCLIZATION. N O K 2 CO 3 N Boc O N H 2 O I NaI HCO 2 H MeOH-d 4 2.17 2.38 2.37 Vinyl iodide 2.38 was prepared according to Method B. Et 2 O was added to the reaction mixture prior to HCO 2 H evaporation and the precipitate (2.38) was collected via filtration. The precipitate was dried in vacuo for 1 h. Vinyl iodide 2.38 (10 mg, 0.025 mmol) was dissolved in CD 3 OD (1.0 mL) and added (0.60 mL) to an NMR tube. An initial NMR spectrum was recorded. To the NMR tube was added 0.30 mL of a saturated solution of K 2 CO 3 in CD 3 OD and the NMR tube was rigorously agitated. Although an NMR spectrum was recorded immediately, enaminone formation was complete prior to spectrum acquisition (~2 min). 266 EXPERIMENT 1: NMR 267 EXPERIMENT 2: METHYLYNONE CYCLIZATION. Vinyl iodide I was prepared from ynone 2.18 (20 mg, 0.080 mmol) according to Method B. Following evaporation of HCO 2 H, the residue containing vinyl iodide I was dissolved in CD 3 OD (2.0 mL) and added (0.60 mL) to an NMR tube. An initial NMR spectrum was recorded. To the NMR tube was added 0.30 mL of a saturated solution of K 2 CO 3 in CD 3 OD and the NMR tube was rigorously agitated. NMR spectra were recorded at various time increments from 2.0 min to 22.0 min. Deuteration of the α-position was immediate and complete, whereas γ-deuteration was slow, proceeding almost to completion over 22 min, and subsequent to cyclization. N O K 2 CO 3 N Boc O N H 2 O I NaI HCO 2 H MeOH-d 4 Me Me D D CD 3 α γ 2.18 2.39-d 5 I 268 EXPERIMENT 2: NMR 269 EXPERIMENT 3: PHENYLYNONE CYCLIZATION. Vinyl iodide II was prepared from ynone 2.19 (25 mg, 0.080 mmol) according to Method B. Following evaporation of HCO 2 H, the residue containing vinyl iodide II was dissolved in CD 3 OD (2.0 mL) and added (0.60 mL) to an NMR tube. To the NMR tube was added 0.30 mL of a saturated solution of K 2 CO 3 in CD 3 OD and the NMR tube was rigorously agitated. NMR spectra were recorded at various time increments from 2.0 min to 70.0 min. Deuteration of the α-position was rapid and complete. N O K 2 CO 3 N Boc O N H 2 O I NaI HCO 2 H MeOH-d 4 Ph Ph D D Ph α 2.19 II 2.40-d 2 270 EXPERIMENT 3: NMR 271 EXPERIMENT 4: POST-CYCLIZATION DEUTERIUM EXCHANGE. Enaminone 2.37 (10 mg, 0.070 mmol) and 2.39 (11 mg, 0.070 mmol) were dissolved in CD 3 OD (1.0 mL) each and added (0.60 mL of each solution) to separate NMR tubes. An initial NMR spectrum was obtained. To each NMR tube was added 0.30 mL of a saturated solution of K 2 CO 3 in CD 3 OD and the NMR tubes were rigorously agitated. NMR spectra were recorded at various increments over 24 h. Enaminone 2.37 was completely intact at 24 h, whereas, enaminone 2.39 underwent > 95% γ-deuteration after 2 h. N O K 2 CO 3 MeOH-d 4 N O 24 h 2.37 2.37 N O K 2 CO 3 MeOH-d 4 CD 3 γ N O CH 3 γ 2 h 2.39 2.39-d 3 272 EXPERIMENT 4: NMR 273 STUDIES OF YNONE AND VINYL CHLORIDE REACTIVITY IN ABSENCE OF AN INTERNAL NUCLEOPHILE The ynone 2.65 (79 mg, 0.50 mmol) was dissolved in a 4N HCl/dioxane solution (1.5 mL) and allowed to react for 15 min. After this time the dioxane and excess HCl were allowed to evaporate while passing air over the reaction mixture. A 1:6 mixture of 2.66 and 2.67 was obtained. From the residue containing 2.66 and 2.67 10 mg was taken and dissolved in MeCN-d 3 (1.0 mL) and added (0.60 mL) to an NMR tube. After an NMR spectra was obtained for the mixture, one drop of TEA was added and NMR spectra were recorded at various intervals over 2 h. Dichloride 2.67 had completely reacted to form vinyl chloride 2.66 in less than 2 min. After 2 h, a drop of D 2 O was added to the reaction mixture and the NMR tube was vigorously shaken. NMR spectra were recorded at various interval over another 2 h. The vinylchloride moiety remained intact over this period of time. Ph O Ph O Cl + Ph O Cl Cl TEA, MeCN-d 3 ; >95% yield (NMR) Ph O Cl then D 2 O HCl 2.65 2.66 2.67 2.66 274 (E)-1-Chloro-5-phenylpent-1-en-3-one (2.66). The title compound was isolated as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) δ 2.76-2.80 (m, 2H), 2.85-2.90 (m, 2H), 6.45 (d, J = 13.6 Hz, 1H), 7.11 (m, 3H), 7.19-7.23 (m, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 29.6, 42.9, 126.3, 128.3, 128.6, 132.3, 136.8, 140.6, 196.2; IR (neat) 2926, 1682, 1586, 1454, 1169, 942 cm -1 ; HRMS (EI+) m/e calc’d for [M+H] + C 11 H 12 ClO: 194.0498, found 194.0479. 1,1-Dichloro-5-phenylpentan-3-one (2.67). The title compound was isolated as a colorless oil upon evaporation of HCl: 1 H NMR (400 MHz, CDCl 3 ) δ 2.72 (t, J = 7.5 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 3.25 (d, J = 6.4 Hz, 2H), 6.05 (t, J = 6.4 Hz, 1H), 7.10-7.16 (m, 3H), 7.20-7.24 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 29.2, 45.0, 55.5, 67.2, 126.4, 128.3, 128.6, 140.3, 203.4; IR (neat) 2925, 1721, 1454, 1405, 1371, 1092 cm -1 ; HRMS (EI+) m/e calc’d for [M+H] + C 11 H 13 Cl 2 O: 230.0265, found 230.0276. O Cl 2.66 O Cl Cl 2.67 275 The mixture of chlorides 2.66 and 2.67 (10 mg) were dissolved in methanol-d 4 (1.0 mL) and added (0.60 mL) to an NMR tube. An initial NMR spectrum was recorded as a reference. A saturated solution of K 2 CO 3 in methanol-d 4 (0.30 mL) was added to the NMR tube and the tube was vigorously agitated. NMR spectra were recorded at various intervals. After 2 h the spectra showed complete consumption of dichloroketone 2.67 and a 1:2 mixture of ether 2.68 and vinyl chloride 2.66, respectively. NMR spectra for vinyl ether 2.68 matched those found in the literature. 349 6.3 Chapter 3 6.3.1 Preparation of starting materials (S)-6-iodo-2,3,8,8a-tetrahydroindolizin-7(1H)-one (3.1). Enaminone 2.37 (421 mg, 3.07 mmol, 1.00 equiv) and DMAP (751 mg, 6.15 mmol, 2.00 equiv) were dissolved in CH 2 Cl 2 (45 mL). To this flask I 2 (73.8 mL, 3.69 mmol, 1.2 equiv, 0.05M in CH 2 Cl 2 ) was added Ph O Cl + Ph O Cl Cl Ph O Cl MeOH-d 4 K 2 CO 3 + Ph O OMe 2.66 2.67 2.68 2.66 N O I H 3.1 276 dropwise over 20 min and the reaction mixture was stirred for 5 h. The reaction mixture was poured into a saturated solution of Na 2 S 2 O 3 (200 mL) and vigorously stirred for 5 min. The product was extracted with CH 2 Cl 2 (3x), dried over Na 2 SO 4 and purified via SiO 2 flash chromatography (100% EtOAc). The title compound was isolated as a yellow solid (782 mg, 92%). Spectral data matched those found in the literature. 213 Compound 3.18 was prepared from 4-hydroxypyridine using alkylation/reduction methodology developed by Guerry and Neier. 350 NMR spectra matched those found in the literature. 1-Benzyl-2-phenyl-2,3-dihydropyridin-4(1H)-one (3.19) was prepared from 4- hydroxypyridine using N-alkylation 351 and conjugate addition of PhMgCl using methodology reported by Klegraf et al. 230 NMR spectra matched those found in the literature. 351 N O Me 3.18 3.19 N O Bn 277 6.3.2 Pd(II)-catalyzed C3-arylation of enaminones Representative procedure for enaminone arylation: Enaminone (0.20 mmol), Pd(OAc) 2 (14 mg, 0.06 mmol), anhydrous Cu(OAc) 2 powder (110 mg, 0.60 mmol) and granular K 2 CO 3 (55 mg, 0.40 mmol,) 5 were combined in a 20:5:1 mixture of t-BuOH/AcOH/DMSO (2.0 mL) under N 2 6 and stirred for 5 min (Note: Solvents were used without purification or degassing). The reaction mixture was heated to 60 o C and the potassium trifluoroborate (0.40-0.60 mmol) was added slowly over 5 h as a solid. The amount of trifluoroborate needed depends on the rate of addition. Typically 0.20 equivalents of trifluoroborate were added every 30 minutes. The reaction was stirred for an additional h and was monitored by TLC using 100% EtOAc as the mobile phase. The reaction mixture was diluted with EtOAc (8 mL) and excess K 2 CO 3 (~1 g) was added to neutralize the AcOH. After stirring this mixture for 5 min the precipitate was filtered over Celite using EtOAc as the eluent. The filtrate was concentrated and purified by chromatography on SiO 2 (EtOAc/ hexanes). N O R 3 R 2 R 1 R 4 Pd(OAc) 2 Cu(OAc) 2 , K 2 CO 3 t-BuOH/AcOH/DMSO (20:5:1), 60 o C H N O R 3 R 2 R 1 R 4 Ar Ar-BF 3 K 278 1-Benzyl-5-(4-methoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.4). Potassium (4- methoxyphenyl)trifluoroborate was added slowly over 6 h and the reaction was stirred for an additional hour. The title compound was isolated as a yellowish oil. ( 1 H NMR (400 MHz, CDCl 3 ) δ 2.60 (t, 2H, J = 7.8 Hz), 3.44 (t, 2H, J = 7.8 Hz), 3.80 (s, 3H), 4.43 (s, 2H), 6.87 (d, 2H, J = 8.8 Hz), 7.29-7.42 (m, 8H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.4, 46.9, 55.34, 60.2, 111.1, 113.7 (2C), 127.7 (2C), 128.4, 128.8, 129.0 (2C), 129.1 (2C), 135.8, 152.8, 157.8, 188.9; IR (neat) 2835, 1629, 1595, 1298, 1245, 1126 cm -1 ; HRMS (ESI+) m/e calc’d for [M +H] + C 19 H 20 NO 2 : 294.1494, found 294.1482. 1-Benzyl-5-phenyl-2,3-dihydropyridin-4(1H)-one (3.6). Potassium phenyltrifluoroborate was added slowly over 6 h and the reaction was stirred for an additional hour. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.60 (t, 2H, J = 7.8 Hz), 3.45 (t, 2H, J = 7.8 Hz), 4.43 (s, 2H), 7.16 (tt, 1H, J = 1.4, 3.7 Hz), 7.29-7.42 (m, 10H); 3.4 N O OMe N O 3.6 279 13 C NMR (100 MHz, CDCl 3 ) δ 36.3, 46.8, 60.2, 111.2, 125.7, 127.7 (2C), 127.8 (2C), 128.2 (2C), 128.4, 129.1 (2C), 135.7, 136.3, 153.2, 188.6; IR (neat) 3029, 1628, 1595, 1495, 1299, 1127 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 18 H 18 NO: 264.1383, found 264.1388. 1-Benzyl-5-(4-tolyl)-2,3-dihydropyridin-4(1H)-one (3.7). Potassium (4-tolyl)- trifluoroborate was added slowly over 4 h and the reaction was stirred for an additional hour. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.32 (s, 3H), 2.60 (t, 2H, J = 7.8 Hz), 3.44 (t, 2H, J = 7.8 Hz), 4.42 (s, 2H), 7.12 (d, 2H, J = 8.0 Hz), 7.28-7.42 (m, 8H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.1, 36.3, 46.8, 60.2, 111.3, 127.7 (2C), 127.8 (2C), 128.4, 128.9 (2C), 129.1 (2C), 133.3, 135.3, 135.8, 152.9, 188.7; IR (neat) 2919, 1634, 1595, 1382, 1298, 1125 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 20 NO: 278.1539, found 278.1551. 1-Benzyl-5-(2-tolyl)-2,3-dihydropyridin-4(1H ) -one (3.8) . Potassium (2- tolyl)trifluoroborate was added slowly over 12 h and the reaction was stirred for an additional N O Me 3.7 N O Me 3.8 280 12 h. The title compound was isolated as a white solid (mp 94-97 o C). 1 H NMR (400 MHz, CDCl 3 ) δ 2.22 (s, 3H), 2.60 (t, 2H, J = 7.8 Hz), 3.45 (t, 2H, J = 7.8 Hz), 4.37 (s, 2H), 7.07-7.42 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 20.4, 36.0, 47.1, 60.0, 112.2, 125.6, 127.1, 127.7 (2C), 128.4, 129.1 (2C), 130.0, 130.6, 135.8, 136.1, 137.9, 153.9, 188.3; IR (neat) 2960, 1635, 1595, 1322, 1303, 1135 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 20 NO: 278.1539, found 278.1547. 1-Benzyl-5-(2-methoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.9). Potassium (2- methoxyphenyl)trifluoroborate was added slowly over 12 h and the reaction was stirred for an additional 12 h. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.63 (t, 2H, J = 7.8 Hz), 3.46 (t, 2H, J = 7.8 Hz), 3.79 (s, 3H) 4.41 (s, 2H), 6.90 (dd, 1H, J = 1.1, 8.2 Hz), 6.93 (ddd, 1H, J = 1.1, 7.4, 7.4 Hz), 7.19 (ddd, 1H, J = 1.8, 7.4, 8.2 Hz), 7.28-7.42 (m, 7H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.1, 46.9, 55.7, 60.2, 107.2, 111.3, 120.5, 125.0, 127.5, 127.7 (2C), 128.3, 129.0 (2C), 132.0, 135.9, 154.9 157.0, 188.3; IR (neat) cm -1 2960, 1631, 1593, 1302, 1132, 1028; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 20 NO 2 : 294.1494, found 194.1504. 3.9 N O MeO 281 1-Benzyl-5-(4-(trifluoromethyl)phenyl)-2,3-dihydropyridin-4(1H)-one (3.10). Potassium (4-trifluoromethylphenyl)trifluoroborate was added slowly over 12 h and the reaction was stirred for an additional 12 h. The title compound was isolated as a white solid (mp 118-119 o C). NMR (400 MHz, CDCl 3 ) δ 2.61 (t, 2H, J = 7.8 Hz), 3.49 (t, 2H, J = 7.8 Hz), 4.45 (s, 2H), 7.29-7.44 (m, 5H), 7.48 (s, 1H), 7.54 (s, 4H); 19 F NMR (377 MHz, CDCl 3 ) δ -65.9; 13 C NMR (100 MHz, CDCl 3 ) δ 36.2, 46.8, 60.5, 109.8, 124.6 (q, J = 271.7 Hz), 125.2 (q, 2C, J = 3.8 Hz), 127.5 (q, J = 32.3 Hz), 127.6 (2C), 127.8 (2C), 128.7, 129.3 (2C), 135.4, 140.2, 153.5, 188.36; IR (neat) 3032, 1637, 1599, 1327, 1113, 1068 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 19 H 17 F 3 NO: 332.1257, found 332.1259. 1-Benzyl-5-(4-acetylphenyl)-2,3-dihydropyridin-4(1H)-one (3.11). Potassium (4- acetylphenyl)trifluoroborate was added slowly over 12 h and the reaction was stirred for an additional 12 h. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.58 (s, 3H), 2.62 (t, 2H, J = 7.8 Hz), 3.50 (t, 2H, J = 7.8 Hz), 4.50 (s, 2H), 3.10 N O CF 3 3.11 N O O CH 3 282 7.30-7.42 (m, 5H), 7.54 (s, 1H), 7.55 (d, 2H, J = 8.6 Hz), 7.90 (d, 2H, J = 8.6 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 26.5, 36.2, 46.6, 60.5, 109.7, 127.0 (2C), 127.7 (2C), 128.4 (2C), 128.6, 129.2 (2C), 134.1, 135.2, 141.6, 153.5, 188.2, 197.8; IR (neat) 3030, 1672, 1635, 1592, 1271, 1129 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 20 H 20 NO 2 : 306.1489, found 306.1503. 1-Benzyl-5-(4-bromophenyl)-2,3-dihydropyridin-4(1H)-one (3.12). Potassium (4- bromophenyl)trifluoroborate was added slowly over 5 h and the reaction was stirred for an additional hour. The title compound was isolated as a yellowish oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.59 (t, 2H, J = 7.8 Hz), 3.46 (t, 2H, J = 7.8 Hz), 4.45 (s, 2H), 7.27-7.43 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.2, 46.7, 60.3, 109.9, 119.3, 127.6 (2C), 128.5, 129.1 (2C), 129.3 (2C), 131.2 (2C), 135.3, 135.5, 152.9, 188.3; IR (neat) 3029, 1631, 1598, 1378, 1293, 1127 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 18 H 17 BrNO: 342.0488, found 342.0483. 3.12 N O Br 283 1-Benzyl-5-(4-chlorophenyl)-2,3-dihydropyridin-4(1H)-one (3.13). Potassium (4- chlorophenyl)trifluoroborate was added slowly over 7 h and the reaction was stirred for an additional hour. The title compound was isolated as a yellowish oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.59 (t, 2H, J = 7.8 Hz), 3.4 (t, 2H, J = 7.8 Hz), 4.45 (s, 2H), 7.25-7.43 (m, 10H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.2, 46.7, 60.3, 109.9, 127.7 (2C), 128.2 (2C), 128.5, 128.9 (2C), 129.1 (2C), 131.2, 134.8, 135.5, 153.0, 188.4; IR (neat) 3030, 1632, 1599, 1493, 1294, 1128 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 18 H 17 ClNO: 298.0993, found 298.0996. 1-Benzyl-5-(naphthalen-2-yl)-2,3-dihydropyridin-4(1H)-one (3.14). Potassium (2- naphthalene)trifluoroborate was added slowly over 3 h and the reaction was stirred for an additional hour. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.63 (t, 2H, J = 7.8 Hz), 3.46 (t, 2H, J = 7.8 Hz), 4.45 (s, 2H), 7.30-7.43 (m, 7H), 7.52 (s, 1H), 7.59 (dd, 1H, J = 1.9, 8.7 Hz), 7.76-7.80 (m, 3H), 7.83 (d, 1H, J = 1.2 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 36.4, 46.7, 60.3, 111.0, 125.0, 125.5, 125.7, 126.9, 127.4, 127.5, 3.13 N O Cl 3.14 N O 284 127.7 (2C), 127.7, 128.4, 129.1 (2C), 131.9, 133.7, 134.1, 135.7, 153.5, 188.7; IR (neat) 3055, 1636, 1600, 1320, 1297, 1135 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 22 H 20 NO: 314.1539, found 314.1545. 1-Benzyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.15). Potassium (3,4,5-trimethoxyphenyl)trifluoroborate was added slowly over 5 h and the reaction was stirred for an additional hour. The title compound was isolated as a yellowish oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.60 (t, 2H, J = 7.8 Hz), 3.46 (t, 2H, J = 7.8 Hz), 3.83 (s, 3H), 3.86 (s, 6H), 4.45 (s, 2H), 6.64 (s, 2H), 7.29-7.44 (m, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.3, 46.7, 56.1, 60.2, 60.8, 105.3, 111.1, 127.7 (2C), 128.4, 129.1 (2C), 132.1, 135.7, 136.4, 152.9, 153.1, 188.6; IR (neat) 2937, 1630, 1596, 1453, 1328, 1239, 1125 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 21 H 24 NO 4 : 354.1700, found 354.1702. N O OMe OMe OMe 3.15 N O OH 3.16 285 1-Benzyl-5-(3-hydroxyphenyl)-2,3-dihydropyridin-4(1H)-one (3.16). Potassium (3- hydroxyphenyl)trifluoroborate was added slowly over 2 h and the reaction was stirred for an additional h. The title compound was isolated as white solid which later turned pink (mp 145-155 o C). 1 H NMR (400 MHz, CDCl 3 ) δ 2.64 (t, 2H, J = 7.9 Hz), 3.44 (t, 2H, J = 7.9 Hz), 4.45 (s, 2H), 6.69 (ddd, 1H, J = 1.0, 2.5, 8.0 Hz) 6.72 (ddd, 1H, J = 1.0, 1.5, 8.0 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.27-7.42 (m, 5H), 7.46 (dd, 1H, J = 1.5, 2.5 Hz), 7.50 (s, 1H), 8.1 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 36.0, 46.5, 60.5, 110.3, 113.3, 115.8, 117.8, 127.8 (2C), 128.6, 129.3 (2C), 129.4, 135.5, 137.4, 154.5, 156.8, 189.3; IR (neat) 3168, 1594, 1452, 1324, 1303, 1126 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 18 H 18 NO 2 : 280.1332, found 280.1339. 1-Benzyl-5-(4-aminobenzyloxycarbonyl)-2,3-dihydropyridin-4(1H)-one (3.17). Potassium (4-Cbz-aminophenyl)trifluoroborate was added slowly over 4 h and the reaction was stirred for an additional hour. The title compound was isolated as a pale yellow solid (mp 153.5-155 o C). 1 H NMR (400 MHz, CDCl 3 ) δ 2.58 (t, 2H, J = 7.8 Hz), 3.43 (t, 2H, J = 7.8 Hz), 4.43 (s, 2H), 5.19 (s, 2H), 6.71 (s, 1H), 7.30-7.40 (m, 15H); 13 C NMR (100 MHz, (CD 3 ) 2 CO) δ 37.2, 47.3, 60.2, 66.8, 110.3, 118.7, 128.5 (2C), 128.8 (2C), 128.8 (2C), 128.8 (2C), 128.9 (2C), 129.3 (2C), 129.7 (2C), 132.9, 137.2, 137.8, 138.0 153.9, 154.4, 188.4; IR (neat) 3316, 3031, 3.17 N O NHCbz 286 1724, 1593, 1529, 1220 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 26 H 25 N 2 O 3 : 413.1860, found 413.1874. 3-(4-Methoxyphenyl)-7,8,9,9a-tetrahydro-1H-quinolizin-2(6H)-one (3.20). Trifluoroborate 3.3 was added slowly over 2 h and the reaction was stirred for an additional h. 4a was isolated as a pale yellow solid (mp 97-99 o C). 1 H NMR (400 MHz, CDCl 3 ) δ 1.35-1.68 (m, 3H), 1.76-1.89 (m, 3H), 2.45-2.62 (m, 2H), 3.01 (dt, 1H, J = 2.6, 6.3 Hz), 3.32-3.45 (m, 2H), 3.78 (s, 3H), 6.85 (d, 2H, J = 8.8 Hz), 7.05 (s, 1H), 7.29 (d, 2H, J = 8.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 20.7, 23.2, 29.2, 41.4, 50.7, 52.9, 54.8, 109.5, 111.1 (2C), 126.1, 126.6 (2C), 151.3, 155.3, 187.4; IR (neat) 2937, 1636, 1596, 1511, 1243, 1133 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 20 NO 2 : 258.1494, found 258.1488. 6-(4-Methoxyphenyl)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (3.2). Trifluoroborate 3.3 was added slowly over 8 h and the reaction was stirred for an additional hour. The title 3.20 N O OMe 3.2 N O OMe 287 compound was isolated as a pale yellow solid (mp 118-119 o C). Note: See Section 6.4 for a more detailed description of the preparation of enantiomerically pure 3.2. 1 H NMR (400 MHz, CDCl 3 ) δ 1.68-1.76 (m, 1H), 1.92-2.02 (m, 1H), 2.11-2.15 (m, 1H), 2.28-2.33 (m, 1H), 2.49 (t, 1H, J = 16.0 Hz), 2.58 (dd, 1H, J = 5.0, 16.0 Hz), 3.53-3.63 (m, 2H), 3.79-3.87 (m, 1H), 3.79 (s, 3H), 6.85 (d, 2H, J = 8.8 Hz), 7.30 (d, 2H, J = 8.8 Hz), 7.38 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 24.4, 33.1, 42.4, 49.8, 55.4, 57.8, 109.5, 113.5 (2C), 128.8 (2C), 129.6, 148.8, 157.4, 189.0; IR (neat) 2953, 2885, 2833, 1620, 1574, 1510, 1277, 837, 733 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 18 NO 2 : 244.1338, found 244.1327. (cis)-3-(4-Methoxyphenyl)-1-methyl-4a,5,6,7,8,8a-hexahydroquinolin-4(1H)- one (3.21). Trifluoroborate 3.3 was added slowly over 6 h and the reaction was stirred for an additional h. The title compound was isolated as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 1.31-1.54 (m, 4H), 1.60-1.75 (m, 2H), 1.85-1.96 (m, 1H), 2.22-2.33 (m, 1H), 2.73-2.77 (m, 1H), 3.08 (s, 3H), 3.45 (ddd, 1H, J = 3.5, 5.5, 9.0 Hz), 3.78 (s, 3H), 6.84 (d, 2H, J = 8.8 Hz), 7.05 (s, 1H), 7.31 (d, 2H, J = 8.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 22.8, 23.5, 23.7, 24.5, 40.7, 45.7, 55.3, 60.1, 109.2, 113.6 (2C), 128.8 (2C), 129.2, 151.9, 157.5, 190.7; IR (neat) 3.21 N H H O Me OMe 288 2932, 1629, 1599, 1512, 1243, 1035 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 17 H 22 NO 2 : 272.1645, found 272.1652. (trans)-3-(4-Methoxyphenyl)-1-methyl-4a,5,6,7,8,8a-hexahydroquinolin-4(1H)-one (3.22). Trifluoroborate 3.3 was added slowly over 6 h and the reaction was stirred for an additional h. The title compound was isolated as an off-white solid (mp 151-152.5 o C). 1 H NMR (400 MHz, CDCl 3 ) δ 1.08-1.47 (m, 4H), 1.83-1.90 (m, 2H), 2.15-2.25 (m, 2H), 2.46-2.50 (m, 1H), 3.00 (s, 3H), 3.09 (ddd, 1H, J = 3.6, 11.3, 14.8 Hz), 3.78 (s, 3H), 6.84 (d, 2H, J = 8.8 Hz), 7.16 (s, 1H), 7.31 (d, 2H, J = 8.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 24.4, 24.9, 25.0, 30.4, 39.6, 48.7, 55.3, 62.0, 110.9, 113.5 (2C), 128.9, (2C) 129.1, 153.7, 157.7, 191.2; IR (neat) 2919, 1621, 1591, 1513, 1247, 1159 cm -1 ; HRMS (ESI+) m/e calc’d for [M +H] + C 17 H 22 NO 2 : 272.1645, found 272.1651. N H H O Me OMe 3.22 289 5-(4-Methoxyphenyl)-1-methyl-2,3-dihydropyridin-4(1H)-one (3.23). Trifluoroborate 3.3 was added slowly over 5 h and the reaction was stirred for an additional h. The title compound was isolated as a yellowish oil. 1 H NMR (400 MHz, CDCl 3 ) δ 2.62 (t, 2H, J = 7.8 Hz), 3.08 (s, 3H), 3.47 (t, 2H, J = 7.8 Hz), 3.78 (s, 3H), 6.85 (d, 2H, J = 8.8 Hz), 7.13 (s, 1H), 7.28 (d, 2H, J = 8.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 36.3, 43.5, 49.3, 55.5, 110.8, 113.8 (2C), 129.1 (2C), 129.1, 153.8, 157.9, 188.7; IR (neat) 2957, 1626, 1598, 1512, 1299, 1245 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 13 H 16 NO 2 : 218.1181, found 218.1176. 1-Benzyl-5-(4-methoxyphenyl)-2-phenyl-2,3-dihydropyridin-4(1H)-one (3.24). Trifluoroborate 3.3 was added slowly over 6 h and the reaction was stirred for an additional h. The title compound was isolated as a yellowish oil. 1 H NMR (400 MHz, CDCl 3 ) d 2.83 (dd, 1H, J = 7.5, 16.3 Hz), 2.98 (dd, 1H, J = 7.5, 16.3 Hz), 3.80 (s, 3H), 4.19 (d, 1H, J = 15.1 Hz), 4.41 (d, 1H, J = 15.1 Hz), 4.56 (t, 1H, J = 7.5 Hz), 6.89 (d, 2H, J = 8.8 Hz), 7.16-7.38 3.23 N O OMe Me 3.24 N O OMe Bn 290 (m, 12H), 7.49 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) d 44.4, 55.3, 57.5, 61.0, 111.3, 113.8 (2C), 127.1 (2C), 127.8 (2C), 128.3, 128.4, 128.6, 128.9 (2C), 129.0 (2C), 129.1 (2C), 136.0, 138.5, 152.6, 157.9, 187.8; IR (neat) 3030, 1634, 1595, 1512, 1245, 1178 cm -1 ; HRMS (ESI +) m/e calc’d for [M+H] + C 25 H 24 NO 2 : 370.1802, found 370.1811. Oxidative Heck coupling: (E)-Methyl 3-(1-Benzyl-4-oxo-1,4,5,6-tetrahydropyridin-3-yl)acrylate (3.25). Enaminone 2.55 (37 mg, 0.20 mmol), methylacrylate (34 mg, 0.40 mmol), Pd(OAc) 2 (14 mg, 0.06 mmol), Cu(OAc) 2 (110 mg, 0.60 mmol) and K 2 CO 3 (55 mg, 0.40 mmol) combined in a 20:5:1 mixture of t-BuOH/AcOH/DMSO (2.0 mL) and stirred for 5 min. The reaction mixture was heated to 80 o C and stirred until judged complete by TLC. The reaction mixture was diluted with EtOAc (8 mL) and excess K 2 CO 3 (~1 g) was added to neutralize the AcOH. After stirring this mixture for 5 min the precipitate was filtered over Celite using EtOAc as the eluent. The filtrate was concentrated and purified by chromatography on SiO 2 (65% EtOAc/ hexanes). The title compound was isolated as a waxy yellow solid. 1 H NMR (400 MHz, CDCl 3 ) δ 2.53 (t, 2H, J = 7.8 Hz), 3.46 (t, 2H, J = 7.8 Hz), 3.71 (s, 3H), 4.51 (s, 2H), 6.66 (d, 1H, J = 15.6 Hz), 7.22 (d, 1H, J = 15.6 Hz), 7.26-7.28 (m, 2H), 7.36-7.43 (m, 3H), N O Pd(OAc) 2 Cu(OAc) 2 , K 2 CO 3 t-BuOH/AcOH/DMSO (20:5:1), 60 o C N O CO 2 Me CO 2 Me 2.55 3.25 291 7.51 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 35.9, 46.2, 51.1, 60.7, 106.0, 110.9, 127.8 (2C), 128.9, 129.3 (2C), 134.4, 140.4, 156.9, 169.6, 188.7; IR (neat) 2948, 1696, 1653, 1596, 1436, 1224 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 18 NO 3 : 272.1287, found 272.1275. 6.4 Chapter 4 (S)-tert-butyl 2-(2-diazoacetyl)pyrrolidine-1-carboxylate (4.3). Warning: Large amounts of diazomethane were used for this transformation. Proper care should be taken when handling this highly explosive reagent. All glassware used was free of cracks, scratches or ground-glass joints and a blast shield was used. Boc-L-proline (10.8 g, 50.0 mmol, 1.00 equiv) was taken into THF (100 mL) with stirring and cooled to 0 ºC with an ice bath. The reaction solution was treated with TEA (7.66 mL, 55.0 mmol, 1.10 equiv) and allowed to react for 15 min to fully deprotonate the carboxylic acid. With the addition of ethyl chloroformate (5.23 mL, 55.0 mmol, 1.10 equiv), a thick white precipitate formed. Stirring was continued for 15 min then stopped. In a separate flask, an ice-cold ethereal solution of diazomethane was prepared and, without stirring, was carefully decanted into the freshly prepared anhydride reaction flask using a glass funnel. The reaction solution was lightly stirred for 4 seconds then stirring was stopped. The mixture was allowed to warm to room TEA, THF ClCO 2 Et N Boc then CH 2 N 2 O N 2 N HO 2 C Boc 4.3Boc-L-proline 292 temperature and react overnight. Any additional diazomethane was carefully quenched with 0.5 N acetic acid (100 mL). The drop-wise addition of saturated sodium bicarbonate regulated the solution back to a basic pH 8-9 with gentle stirring. The organic and aqueous layers were separated. The organic phase was washed twice each with saturated sodium bicarbonate and brine then dried over sodium sulfate. The solvent was evaporated under reduced pressure. At this point the crude diazoketone could be used for subsequent Wolff rearrangement reactions. Alternatively, the diazoketone could also be purified by chromatography on SiO 2 (15% EtOAc/hexanes) to provide the title compound as a yellow oil (72%). The spectral data matched that in the literature. 352, 353 (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (4.4). Diazoketone 4.3 (8.61 g, 36.0 mmol, 1.00 equiv) was dissolved in 220 mL of a 1:10 mixture of water and THF and cooled to –25 ºC and stirred for 30 minutes. Foil was used to cover the reaction flask so as to exclude light from the reaction solution. In a separate foil covered flask, silver trifluoroacetate (795 mg, 3.60 mmol, 0.10 equiv) was dissolved in TEA (110 mL). This solution was added dropwise to the diazoketone mixture over 30 min. The reaction temperature was allowed to slowly warm to room temperature and the solution was stirred overnight. To the reaction mixture was added activated carbon (~2 g) and the reaction mixture was stirred for 5 min and filtered. The filtrate was concentrated and the residue N Boc O N 2 CF 3 CO 2 Ag, TEA THF/H 2 O N Boc HO 2 C 4.44.3 293 redissolved in EtOAc. To this was added activated carbon (~2 g) and the process repeated. When the filtrate had been concentrated a second time the residue was purified via SiO 2 flash chromatography (30% EtOAc/hexanes) affording the title compound (68%) as a white solid. The spectral data matched that in the literature. 353 (S)-tert-butyl 2-(2-methoxy-2-oxoethyl)pyrrolidine-1-carboxylate (4.5). Diazoketone 4.3 (2.53 g, 10.6 mmol, 1.00 equiv) was dissolved in 45 mL of MeOH and the reaction flask was covered with foil to exclude light from the reaction solution. In a separate foil covered flask, silver trifluoroacetate (240 mg, 1.4 mmol, 0.13 equiv) was dissolved in TEA (30 mL). This solution was added dropwise to the diazoketone mixture over 30 min. The reaction temperature was allowed to slowly warm to room temperature and the solution was stirred overnight. To the reaction mixture was added activated carbon (~1 g) and the reaction mixture was stirred for 5 min and filtered. The filtrate was concentrated and the residue redissolved in EtOAc. To this was added activated carbon (~1 g) and the process repeated. When the filtrate had been concentrated a second time the residue was purified via SiO 2 flash chromatography (15% EtOAc/hexanes) affording the title compound (86%) as a clear oil. The spectral data matched that in the literature. 354 N Boc O N 2 N Boc MeO 2 C CF 3 CO 2 Ag TEA, MeOH 4.3 4.5 294 (S)-tert-Butyl 2-(2-(Methoxy(methyl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate (2.2). Amino acid 4.4 (920 mg, 4.0 mmol, 1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 (100 mL) under an argon atmosphere and cooled to –15 °C. To this solution was added N,O- dimethylhydroxylamine•HCl (430 mg, 4.4 mmol, 1.1 equiv) and N-methylmorpholine (0.49 mL, 4.4 mmol, 1.1 equiv) followed by EDCI (810 mg 4.2 mmol, 1.1 equiv). The reaction mixture was then allowed to come to room temperature. After 2 h the reaction was again cooled to 0 °C and quenched by the addition of an ice cold 10% HCl solution (25 mL) and allowed to stir at this temperature for 5 min. The reaction was diluted with water (50 mL) and extracted with CH 2 Cl 2 (x3). The combined organic layers were washed with saturated NaHCO 3 (x1), dried over Na 2 SO 4 , filtered and concentrated. Purification via SiO 2 flash chromatography (50% EtOAc/hexane) afforded the title compound as a clear oil (98%). See Section 6.2 for spectral data. Methyl ester 4.5 was converted to the Weinreb amide 2.2 using the procedure reported by Williams et al. 279 The crude product was purified via SiO 2 flash chromatography (50% EDCI, NMM, CH 2 Cl 2 N Boc HO 2 C N Boc O N OMe Me HN(OMe)Me•HCl (S)-2.24.4 N Boc O N OMe Me N Boc MeO 2 C i-PrMgCl, THF HN(OMe)Me•HCl 4.5 (S)-2.2 295 EtOAc in hexanes) to give the Weinreb amide (86%) as a clear viscous oil. See Section 6.2 for spectral data. Diazoketone 4.4 (7.37 g, 30.8 mmol, 1.00 equiv)was taken into THF (130 mL) and cooled to 0 ºC. Foil was used to cover the reaction flask so as to exclude light from the reaction solution. To this was added freshly distilled N,O-dimethylhydroxylamine (4.00 g, 92.4 mmol, 3.00 equiv). 344 In a separate foil covered flask, silver trifluoroacetate (1.37 g, 6.20 mmol, 0.20 equiv) was dissolved in TEA (86 mL). This solution was added to the diazoketone mixture over 30 min. The reaction temperature was allowed to slowly warm to room temperature and the solution was stirred overnight. To the reaction mixture was added activated charcoal (~2 g) and the reaction mixture was stirred for 5 min and filtered. The filtrate was concentrated and the residue redissolved in EtOAc. To this was added activated charcoal (~2 g) and the process repeated. When the filtrate had been concentrated a second time the residue was purified via SiO 2 flash chromatography (50% EtOAc/hexanes) affording the pure Weinreb amide as a clear oil (97%). See Section 6.2 for spectral data. CF 3 CO 2 Ag, TEA N Boc O N OMe Me N Boc O N 2 HN(OMe)Me, THF 4.3 (S)-2.2 296 (S)-tert-butyl 2-(2-oxobut-3-yn-1-yl)pyrrolidine-1-carboxylate (2.17). The Weinreb amide (4.66 g, 17.1 mmol, 1.0 equiv) was dissolved in anhydrous THF (200 mL) under nitrogen atmosphere and cooled to 0 °C. To this reaction vessel, was added dropwise, a solution of ethynyl magnesium bromide reagent (171 mL, 85.6 mmol, 5.0 equiv, 0.5 M in THF) and allowed to come to room temperature. After the reaction was judged complete by TLC it was quenched by the addition of an ice cold 10% HCl solution (15 mL) and allowed to stir at this temperature for 5 minutes. The reaction was diluted with water and extracted with EtOAc (x3). The combined organic layers were washed with saturated NaHCO 3 (x1), dried over Na 2 SO 4 , filtered and concentrated. The title compound was obtained as a clear oil (3.94 g, 97% yield) after SiO 2 flash chromatography (20% EtOAc/Hexanes): Spectral data was identical to that reported. 255 (S)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (2.37). Ynone 2.17 (3.74 g, 15.8 mmol, 1.0 equiv) was dissolved in formic acid (50 mL) solution under a N 2 atmosphere and NaI (7.09 g, (S)-2.17(S)-2.2 MgBr THF, 0 ºC; N Boc O N OMe Me N Boc O then NaHSO 4 (aq.) N O H 1. NaI, HCO 2 H 2. MeOH, K 2 CO 3 N Boc O (S)-2.17 (S)-2.37 297 47.3 mmol, 3.0 equiv) was added. The reaction was left stirring for 6 h at room temperature. The solvent was removed by passing N 2 over the reaction mixture. The remaining residue was placed under vacuum for 15 minutes and then dissolved in MeOH (100 mL). A separate flask was charged with 700 mL of MeOH and K 2 CO 3 (10.9 g, 79.0 mmol, 5.0 equiv). To this flask was added the solution of deprotected ynone over 15 minutes. The reaction was stirred for 1 h and the solvent was evaporated. At this time CH 2 Cl 2 was added to redissolve the product (but not the inorganic salts), the slurry was suction filtered, and the filtrate concentrated. To the solid residue was added more CH 2 Cl 2 and the precipitates were once again filtered away. This residue after removal of solvent was purified via SiO 2 flash chromatography to provide the title compound as an off-white solid (2.05 g, 95% yield) after SiO 2 flash chromatography (100% acetone): See Section 6.2 for spectral data. (S)-6-(4-methoxyphenyl)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (3.2). Freshly purified enaminone (750 mg, 5.5 mmol, 1.0 equiv), Pd(OAc) 2 (370 mg, 1.7 mmol, 0.30 equiv), anhydrous Cu(OAc) 2 powder (3.0 g, 17 mmol, 3.0 equiv) and granular K 2 CO 3 (1.5 g, 11 mmol, 2.0 equiv) were combined in a 20:5:1 mixture of degassed t-BuOH/AcOH/DMSO (55 mL) under N 2 and stirred for 5 min (Note: Solvents were used without purification). The reaction mixture was heated to 60 o C and the potassium trifluoroborate (3.5 g, 17 mmol, 3.0 Pd(OAc) 2 (30 mol%) Cu(OAc) 2 (3 equiv) PMP-BF 3 K, K 2 CO 3 t-BuOH/AcOH/ DMSO (20:5:1), 60 o C, 70% N O H N O H MeO (S)-2.37 3.2 298 equiv) was added slowly over 5 h as a solid. Approximately, 0.20 equivalents of trifluoroborate were added every 30 min. The reaction was stirred for an additional hour and was monitored by TLC using 100% EtOAc as the mobile phase. The reaction mixture was diluted with EtOAc and added to a separatory funnel containing brine. The product was extracted with EtOAc (3x). The combined organic layers were washed brine (2x) and sat. NaHCO 3 (aq.) (2x), dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a pale yellow solid (930 mg, 70% yield) after SiO 2 flash chromatography (80% EtOAc/hexanes). The spectral data was identical to that of (±)-3.2 (See Section 6.2). α [ ] D 22 = –97.5 (c 1.00, CHCl 3 ). (6S*,8aS*)-6-(4-methoxyphenyl)hexahydroindolizin-7(1H)-one (4.8). Enaminone 3.2 (120 mg, 0.50 mmol, 1.0 equiv) was dissolved in anhydrous THF (5 mL) under a N 2 atmosphere. The solution was cooled to –78 ºC at which point L-Selectride (0.55 mL, 0.55 mmol, 1.1 equiv, 1.0 M in THF) was added over 15 minutes. After stirring for 1 h, the reaction was slowly warmed to 0 ºC over 1.5 h and stirred at room temperature for an additional 2 h. The reaction was quenched with a saturated solution of NaHCO 3 (aq.) and added to a separatory funnel. The product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound (110 mg, 90% yield) was obtained as a white crystalline solid (mp 118–120 ºC) after SiO 2 flash N O H L-Selectride, THF N O H H –78 to 0 ºC MeO MeO 3.2 4.8 299 chromatography [60% EtOAc/hexanes (1% TEA)]. Spectral data was identical to that reported. 318 α [ ] D 22 = +6.05 (c 1.14, CHCl 3 ). (6S,7R,8aS)-6-(4-methoxyphenyl)-7-methyloctahydroindolizin-7-ol (4.9). Ketone 4.8 (105 mg, 0.43 mmol, 1.00 equiv) taken in anhydrous THF (3.5 mL) in a flame dried RBF and cooled to –78 ºC. Methyl lithium (0.32 mL, 0.52 mmol, 1.2 equiv, 1.6 M in Et 2 O) was added dropwise and stirred for 45 min at –78 ºC. The reaction was quenched with MeOH (1 mL) and allowed to warm to room temperature. The quenched reaction mixture was added to a separatory funnel containing 10% NaOH (aq.) and the product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound (85 mg, 75% yield) was obtained as a white crystalline solid (mp 92–96 ºC) after SiO 2 flash chromatography [50% EtOAc/hexanes (1% TEA)]. 1 H NMR (400 MHz, CDCl 3 ) δ 1.05 (s, 3H), 1.37–1.49 (m, 2H), 1.71–1.78 (m, 1H), 1.81–1.91 (m, 2H), 1.97 (dd, J = 13.2, 2.5 Hz, 1H), 2.23 (dd, J = 17.9, 8.9 Hz, 1H), 2.34–2.39 (m, 1H), 2.67 (dd, J = 11.3, 11.3 Hz, 1H), 2.83 (dd, J = 11.9, 3.8 Hz, 1H), 2.94 (dd, J = 10.6, 3.9 Hz, 1H), 3.07 (dd, J = 8.6, 8.6 Hz, 1H), 3.8 (s, 3H), 6.86 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 29.6, 30.1, 44.3, 51.4, 53.7, 53.7, 55.2, 59.4, 70.4, 113.7, 130.3, THF, –78 ºC MeLi N O H H MeO N HO H H MeO Me 4.8 4.9 300 131.9, 158.6 ; IR (neat) 3416, 2961, 1611, 1512, 1246, 1178, 833 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 24 NO 2 : 262.1807, found 262.1790. α [ ] D 22 = –6.54 (c 1.10, CHCl 3 ). 6-(4-Methoxyphenyl)-7-methyl-1,2,3,5,8,8a-hexahydroindolizine (4.7). Indolizidine 4.9 (62 mg, 0.24 mmol, 1.0 equiv) was dissolved in anhydrous THF (4.0 mL) and cooled to –50 ºC. Pyridine (0.1 mL) followed by SOCl 2 (0.043 mL, 0.59 mmol, 2.5 equiv) in 0.5 mL THF was added dropwise to the reaction mixture. The reaction mixture was kept between –30 and –10 ºC for 1.5 h. The reaction was quenched with 10% NaOH (aq.). The quenched reaction mixture was added to a separatory funnel containing 10% NaOH (aq.) and the product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a colorless oil (51 mg, 88% yield) after SiO 2 flash chromatography [20% EtOAc/hexanes (1% TEA)]. See below for spectral data. N HO H H MeO Me N Me H MeO SOCl 2 , pyridine THF, –50 to –10 ºC 4.9 4.7 301 (S) - 6 - (4-Methoxyphenyl) - 1, 2, 3, 5, 8, 8a – hexahydroindolizin – 7 – yl – trifluoromethanesulfonate (4.6). Enaminone 3.2 (590 mg, 2.4 mmol, 1.0 equiv) was dissolved in anhydrous THF (30 mL) under a N 2 atmosphere. The solution was cooled to – 78 ºC at which point L-Selectride (0.23 mL, 0.23 mmol, 1.0 equiv, 1.0 M in THF) was added over 15 min. After stirring for 1 h, the reaction was slowly warmed to 0 ºC over 2 h. The reaction mixture was once again cooled to –78 ºC and Comins’ reagent (1.1 g, 2.7 mmol, 1.1 equiv) was added all at once. The mixture was stirred for another hour at –78 ºC and then slowly warmed to 0 ºC over 2 h. The reaction was quenched with a saturated solution of NaHCO 3 (aq.) and added to a separatory funnel. The product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a colorless oil (690 mg, 76% yield) after SiO 2 flash chromatography (10% EtOAc/hexanes (1% TEA): 1 H NMR (500 MHz, CDCl 3 ) δ 1.49-1.57 (m, 1H), 1.76-1.84 (m, 1H), 1.87-1.96 (m, 1H), 2.00-2.07 (m, 1H), 2.22 (dd, J = 18.1, 9.2 Hz, 1H), 2.47-2.62 (m, 3H), 3.06 (d, J = 15.8 Hz, 1H), 3.18 (dt, J = 4.4, 2.1 Hz, 1H), 3.72-3.75 (m, 1H), 3.74 (s, 3H), 6.83 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H); 13 C NMR (125 MHz, CDCl 3 ) δ 22.2, 30.5, 35.2, 53.4, 55.3, 55.6, 60.5, 113.9, 118.1 (q, J CF = 320 Hz), 126.6, 129.2, 129.5, 142.0, 159.5 ; IR (neat) 2959, 1610, 1512, 1416, 1209, 1146 cm -1 ; HRMS (ESI +) m/e calc’d for [M+H] + C 16 H 19 F 3 NO 4 S: 378.0987, found 378.0974; α [ ] D 22 = +66.3 (c 1.00, CHCl 3 ). L-Selectride THF then Comins' reagent 76% N O H MeO N TfO H MeO 4.6 3.2 302 (S)-6-(4-Methoxyphenyl)-7-methyl-1,2,3,5,8,8a-hexahydroindolizine (4.7). Preparation of organo zinc reagent. A flame dried RBF under N 2 was charged with anhydrous THF (10.0 mL) and cooled to –78 ºC. Methyl lithium (1.6 mL, 2.5 mmol, 5.0 equiv, 1.6M in Et 2 O) was added and the solution was allowed to sit. A separate RBF was charged with anhydrous ZnBr 2 (590 mg, 2.6 mmol, 5.2 equiv). The ZnBr 2 was dried by heating the RBF under a vacuum with a heat gun for 5 min. When the ZnBr 2 had cooled to room temperature it was dissolved in anhydrous THF (6.0 mL) under N 2 . This ZnBr 2 solution was slowly cannulated into the methyl lithium solution and the resulting solution was stirred at –78 ºC for 5 min and then allowed to warm to room temperature. Triflate 4.6 (190 mg, 0.50 mmol, 1.0 equiv), dissolved in a minimal amount of THF, and Pd(PPh 3 ) 4 (29 mg, 0.025 mmol, 5.0 mol%) were added sequentially to the zinc reagent. If the reaction had not gone to completion after 1 h at room temperature, the reaction mixture was heated to 50 ºC. Upon consumption of the triflate starting material (as judged by TLC) SiO 2 was added to the reaction mixture and the solvent was evaporated to leave a free flowing powder. Following flash chromatography (20% EtOAc/hexanes (1% TEA)) 110 mg (91%) of the title compound was obtained as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) δ 1.46-1.54 (m, 1H), 1.60 (s, 3H), 1.73-1.81 (m, 1H), 1.85-1.94 (m, 1H), 2.00-2.32 (m, 5H), 2.91 (d, J = 15.4 Hz, 1H), 3.22 (dd, J = 3.2, 3.2 Hz, 1H), 3.62 (d, J = 15.4 Hz, 1H), 3.82 (s, 3H), 6.86 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 20.0, Pd(PPh 3 ) 4 , THF MeZnBr, 60 ºC N TfO H MeO N Me H MeO 4.6 4.7 303 21.4, 30.8, 38.5, 54.2, 55.2, 57.8, 60.2, 113.5, 127.9, 130.0, 130.3, 133.8, 158.1; IR (neat) 2907, 1609, 1510, 1244, 1175, 831 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 22 NO: 244.1701, found 244.1688; α [ ] D 22 = +142 (c 1.00, CHCl 3 ). (+)-Secoantofine (4.11). Preparation of organo zinc reagent. A flame dried RBF under N 2 was charged with anhydrous THF (15.0 mL) and cooled to –78 ºC. tert-Butyllithium (6.6 mL, 11 mmol, 11 equiv, 1.7 M in pentane) was added carefully and the solution was allowed to sit for 5 min at –78 ºC. 4-Bromoveratrole (1.2 g, 5.4 mmol, 5.0 equiv) was then added dropwise and the resulting reaction mixture was stirred for 2 h and –78 ºC. A different RBF was charged with anhydrous ZnBr 2 (1.3 g, 5.6 mmol, 5.2 equiv). The ZnBr 2 was dried by heating the RBF under a vacuum with a heat gun for 5 min. When the ZnBr 2 had cooled to room temperature it dissolved in anhydrous THF (12.0 mL) under N 2 . This ZnBr 2 solution was slowly cannulated into the yellow aryllithium reagent solution. The resulting cloudy white solution was stirred at –78 ºC for 5 min and then allowed to warm to room temperature. Upon warming the solution became clear. Triflate 4.6 (400 mg, 1.1 mmol, 1.0 equiv) (dissolved in a minimal amount of THF) and Pd(PPh 3 ) 4 (62 mg, 0.054 mmol, 5.0 mol %) were added sequentially to the zinc reagent. If the reaction had not gone to completion 4.6 (+)-secoantofine (4.11) N TfO H MeO MeO ZnBrMeO N H MeO MeO OMe Pd(PPh 3 ) 4 THF, 60 ºC 304 after 1 h at room temperature, the reaction mixture was heated to 50 ºC. Upon consumption of the triflate starting material (as judged by TLC) SiO 2 was added to the reaction mixture and the solvent was evaporated to leave a free flowing powder. Following flash chromatography [40% EtOAc/hexanes (1% TEA)] 380 mg (96%) of the title compound was obtained as a yellow oil: 1 H NMR (400 MHz, CDCl 3 ) δ 1.51-1.63 (m, 1H), 1.78-2.01 (m, 2H), 2.06-2.14 (m, 1H), 2.25 (dd, J = 9.0, 9.0 Hz, 1H), 2.36-2.45 (m, 2H), 2.69-2.77 (m, 1H), 3.07 (td, J = 16.0, 3.1 Hz, 1H), 3.29 (dt, J = 4.3, 2.0 Hz, 1H), 3.54 (s, 3H), 3.72 (s, 3H), 3.81 (s, 3H), 3.86 (d, J = 15.8 Hz, 1H), 6.47 (d, J = 1.1 Hz, 1H), 6.66-6.69 (m, 4H), 6.97 (d, J = 8.8 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.5, 30.8, 38.6, 54.3, 55.1, 55.5, 55.7, 57.9, 60.4, 110.4, 113.1, 113.4, 120.7, 130.2, 132.6, 132.7, 133.6, 135.1, 147.1, 147.9, 158.0; IR (neat) 2955, 1607, 1511, 1245, 1030, 755 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 366.2064, found 366.2068; α [ ] D 22 = +169 (c 1.00, CHCl 3 ). (+)-Ipalbidine (4.1). O-Methylipalbidine (4.7) (56 mg, 0.23 mmol, 1.0 equiv) was dissolved in CH 2 Cl 2 (1.0 mL) and cooled to –78 ºC under N 2 . To this solution was added BBr 3 (0.23 mL, 0.23 mmol, 1.0 M in CH 2 Cl 2 ). The reaction was allowed to warm to room temperature over night. The reaction was quenched with water (1.0 mL) and then 5.0 mL of a saturated solution of NaHCO 3 (aq.). The product was extracted from the aqueous layer with CH 2 Cl 2 4.7 (+)-ipalbidine (4.1) N Me H MeO N Me H HO BBr 3 , CH 2 Cl 2 –78 ºC to rt 305 (3x). The combined organic layers were dried with Na 2 SO 4 , concentrated and purified via flash chromatography [80% EtOAc/hexane (1% TEA)] to provide 42 mg (80%) of (+)- ipalbidine as a white crystalline solid (mp 122.2-124.6 ºC). 1 H NMR (500 MHz, CDCl 3 ) δ 1.60 (s, 3H), 1.75-1.84 (m, 1H), 1.91-1.99 (m, 1H), 2.06-2.23 (m, 2H), 2.35-2.45 (m, 2H), 2.54-2.61 (m, 1H), 2.79-2.87 (m, 1H), 3.21 (d, J = 14.7 Hz, 1H), 3.45-3.50 (m, 1H), 3.77 (d, J = 15.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 20.0, 21.0, 29.7, 36.0, 53.4, 55.9, 61.0, 115.6, 127.9, 128.6, 129.8, 130.9, 155.7; IR (neat) 2913, 1609, 1513, 1445, 1269, 1169 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 20 NO: 230.1545, found 230.1531; α [ ] D 22 = +202 (c 1.00, CHCl 3 ). [Lit. S-enantiomer: +233.5 (c 1, CHCl 3 ); R-enantiomer: –237 (c 1, CHCl 3 ), –190.5 (c 1, MeOH)]. 326 (+)-Antofine (4.2). Secoantofine (4.11) (48 mg, 0.13 mmol, 1.0 equiv) was dissolved in CH 2 Cl 2 (2.0 mL) and cooled to –78 ºC. To this solution was added PIFA (62 mg, 0.14 mmol, 1.1 equiv) and BF 3 •Et 2 O (16 mg, 0.14 mmol, 1.1 equiv) sequentially. The solution was stirred for 4 h while being monitored by TLC. A solution of PIFA (62 mg in 2.0 mL CH 2 Cl 2 ) was added dropwise to the reaction mixture until the reaction had gone to completion. Upon consumption of starting material the reaction was quenched with 10% NaOH (aq.) and the PIFA, BF 3 •Et 2 O N H MeO MeO OMe CH 2 Cl 2 , –78 ºC N H MeO MeO OMe (+)-antofine (4.2) 4.11 306 mixture was vigorously stirred for 1 h. The product was extracted from the aqueous layer with CH 2 Cl 2 (3x). The combined organic layers were dried with Na 2 SO 4 , concentrated and purified via flash chromatography [70% EtOAc/hexane (1% TEA)] to provide 34 mg (70%) of (+)-antofine as a white crystalline solid: mp 226-227 °C (decomp.) 1 H NMR (400 MHz, CDCl 3 ) δ 1.65-1.75 (m, 1H), 1.80-2.02 (m, 2H), 2.13-2.22 (m, 1H), 2.35-2.46 (m, 2H), 2.79-2.86 (m, 1H), 3.28 (ddd, J = 15.8, 3.7, 1.5 Hz, 1H), 3.38 (dt, J = 4.3, 2.1 Hz, 1H), 3.63 (d, J = 14.9 Hz, 1H), 3.95 (s, 3H), 3.99 (s, 3H), 4.04 (s, 3H), 4.63 (d, J = 14.9 Hz, 1H), 7.13 (dd, J = 9.0, 2.5 Hz, 1H), 7.25 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 2.5 Hz, 1H), 7.85 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.6, 31.3, 33.7, 53.8, 55.0, 55.5, 55.9, 56.0, 60.3, 104.0, 104.7, 114.9, 123.5, 124.1, 124.2, 125.5, 126.7, 127.0, 130.2, 148.4, 149.4, 157.5; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 26 NO 3 : 364.1913, found 364.1909; α [ ] D 22 = +111 (c 1.00, CHCl 3 ). [Lit. R-enantiomer: –113.4 (c 1.23, CHCl 3 ), 149 −125.2 (c 1.27, CHCl 3 ), 184 –108.2 (c 0.71, CHCl 3 ); 171 S-enantiomer: +66 (CHCl 3 ) 355 ]. 6.5 Chapter 5 6.5.1 Preparation of tylocrebrine and analogs tert-butyl 2-(2-ethoxy-2-oxoethyl)pyrrolidine-1-carboxylate (5.7). Pyrrolidinone 5.6 N Boc DIBAL-H THF, –78 ºC O N Boc HO 5.6 5.7 307 (4.21g, 22.7 mmol, 1.0 equiv) was dissolved in THF (65 mL) under a N 2 atmosphere and cooled to –78 ºC. DIBAL-H (32.0 mL, 32.0 mmol, 1.4 equiv, 1.0 M in hexanes) was added slowly (over 15 minutes) and reaction mixture was stirred for 1 h. The reaction was quenched with saturated NH 4 Cl (80 mL) and warmed to room temperature. The quenched reaction was then treated with 10% Na 2 CO 3 (60 mL) and vigorously stirred for 20 minutes. The product was then extracted with CH 2 Cl 2 (3x) and the combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a clear oil (3.92 g, 92% yield) after SiO 2 flash chromatography (30% EtOAc/Hexanes): Spectral data was identical to that reported. 356 tert-butyl 2-(2-ethoxy-2-oxoethyl)pyrrolidine-1-carboxylate (5.8). To a flame-dried flask was added anhydrous DMF (100 mL) and the flask was purged with N 2 . Sodium hydride (1.01 g, 25.2 mmol, 1.2 equiv, 60% in mineral oil) was added to the flask followed by triethylphosphonoacetate (5.0 mL, 25.2 mmol, 1.2 equiv). The mixture was stirred for 1 h at room temperature. To this solution was added hemiaminal 5.7 (3.92 g, 21.0 mmol, 1.0 equiv) in a solution of DMF (100 mL). The reaction was stirred for 12 h at room temperature and quenched with saturated NH 4 Cl (80 mL). The product was then extracted with EtOAc (3x) and washed with brine (2x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a clear oil (4.65 g, 86% yield) NaH, DMF (EtO) 2 P CO 2 Et O N Boc HO N Boc EtO 2 C 5.7 5.8 308 after SiO 2 flash chromatography (25% EtOAc/Hexanes): Spectral data was identical to that reported. 357 tert-butyl 2-(2-(methoxy(methyl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate (2.2). Ester 5.8 (4.65 g, 18.1 mmol, 1.0 equiv) and HN(OMe)Me•HCl (2.65 g, 27.1 mmol, 1.5 equiv) were dissolved in anhydrous THF (55 mL) and cooled to –30 ºC under a N 2 atmosphere. To a flame dried dropping funnel was added i-PrMgCl (27.0 mL, 54.3 mmol, 3.0 equiv, 2.0 M in THF). This Grignard reagent was added dropwise to the reaction mixture over 15 minutes and the reaction was stirred for 30 minutes at –15 ºC. The reaction was quenched with saturated NH 4 Cl (80 mL) and the product was extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a clear oil (4.18 g, 85% yield) after SiO 2 flash chromatography (50% EtOAc/Hexanes): See Section 6.2 for spectral data. 5.8 HN(OMe)Me•HCl i-PrMgCl THF, –20 ºC N Boc EtO 2 C N Boc O N Me MeO (±)-2.2 309 (R)-tert-butyl 2-(2-(methoxy(methyl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate [(R)-2.2]. Boc-D-β-homoproline (4.0 g, 17.4 mmol, 1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 (50 mL) under argon atmosphere and cooled to –0 °C. To this solution was added N,O-dimethylhydroxylamine•HCl (1.87 g, 19.2 mmol, 1.1 equiv) and N-methylmorpholine (2.1 mL, 19.2 mmol, 1.1 equiv) followed by EDCI (3.68 g, 19.2 mmol, 1.05 equiv). The reaction mixture was then allowed to come to room temperature. After 2 hours the reaction was again cooled to 0 °C and quenched by the addition of an ice cold 10% HCl solution (50 mL) and allowed to stir at this temperature for 5 minutes. The reaction was diluted with water (100 mL) and extracted with CH 2 Cl 2 (x3). The combined organic layers were washed with saturated NaHCO 3 (x1), dried over Na 2 SO 4 , filtered and concentrated. The title compound was obtained as a clear oil (4.66 g, 98% yield) after SiO 2 flash chromatography (40% EtOAc/ Hexanes): See Section 6.2 for spectral data. EDCI, NMM, CH 2 Cl 2 N Boc HO 2 C N Boc O N OMe Me HN(OMe)Me•HCl Boc-D-β-homoproline (R)-2.2 310 Compounds 2.17 and 2.37 were prepared according the procedure in Section 6.2 Optical rotations for the R-enantiomers can be found below. (R)-tert-butyl 2-(2-oxobut-3-yn-1-yl)pyrrolidine-1-carboxylate (R-2.17). α [ ] D 22 = +21.3 (c 1.00, CHCl 3 ). (R)-2,3,8,8a-tetrahydroindolizin-7(1H)-one (R-2.37). α [ ] D 22 = +770 (c 1.00, CHCl 3 ). (R)-1,2,3,5,8,8a-hexahydroindolizin-7-yl trifluoromethanesulfonate (R-5.5). Enaminone (R)-2.17 (1.35 g, 9.81 mmol, 1.0 equiv) was dissolved in anhydrous THF (30 mL) under a N 2 atmosphere. The solution was cooled to –78 ºC at which point L-Selectride (10.8 mL, 10.8 mmol, 1.1 equiv, 1.0 M in THF) was added over 15 minutes. After stirring for 1 h, the reaction was slowly warmed to 0 ºC over 2 hours. The reaction mixture was once again cooled to –78 ºC and Comins’ reagent (4.24 g, 10.8 mmol, 1.1 equiv) was added all at once. (R)-2.17, (S)-2.17, (±)-2.17 (R)-2.2, (S)-2.2, (±)-2.2 (R)-2.37, (S)-2.37, (±)-2.37 N Boc O N Me MeO MgBr THF N O 1. NaI, HCO 2 H 2. K 2 CO 3 , MeOH N Boc O L-Selectride, THF –78 to 0 ºC; then Comins Reagent –78 to 0 ºC N O N TfO (R)-2.17, (S)-2.17, (±)-2.17 (R)-5.5, (S)-5.5, (±)-5.5 311 The mixture was stirred for another hour at –78 ºC and then slowly warmed to 0 ºC over 2 hours. The reaction was quenched with a saturated solution of NaHCO 3 (aq.) and added to a separatory funnel. The product was extracted with ethyl acetate (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a colorless oil (2.67 g, 73% yield) after SiO 2 flash chromatography (20% EtOAc/Hexanes (1% TEA): 1 H NMR (400 MHz, CDCl 3 ) δ ; 1.40-1.49 (m, 1H), 1.70-2.01 (m, 3H), 2.14 (dd, J = 17.9, 8.9 Hz, 1H), 2.27-2.46 (m, 3H), 2.82 (d, J = 16.3 Hz, 1H), 3.11 (dt, J = 4.3, 2.3 Hz), 3.51 (dd, 16.3, 4.8 Hz, 1H), 5.72-5.73 (m, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 22.1, 30.4, 35.0, 49.6, 53.3, 60.2, 116.7, 118.5 (q, J = 320 Hz), 147.8; IR (neat) 2794, 1689, 1419, 1210, 1143, 1024, 879 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 9 H 13 F 3 NO 3 S: 272.0568, found 272.0574; (R)-Enantiomer: β [ ] D 22 = –74.5 (c 1.00, CHCl 3 ). (S)-Enantiomer: α [ ] D 22 = +75.2 (c 1.00, CHCl 3 ). SUZUKI COUPLINGS Compounds 5.4, 5.9, 5.10, 5.11, 5.12 and 5.13 were prepared according the procedure shown for compound 5.4. 5.2, 5.9–5.13 I Br OMe R 4 R 1 R 2 B(OH) 2 + (dppf)PdCl 2 (5 mol%) LiCl, Na 2 CO 3 DME, H 2 O, 80 ºC, 16 h R 4 MeO R 1 R 2 Br R 3 R 3 312 2'-bromo-2,3,4',5'-tetramethoxy-1,1'-biphenyl (5.4). DME (60 mL) and Na 2 CO 3 (21.1 mL, 42.3 mmol, 5.0 equiv, 2.0 M aq. solution) were added to a round-bottomed flask and degassed under reduced pressure for 30 minutes. Iodobromoveratrole (2.90 g, 8.45 mmol, 1.0 equiv), arylboronic acid (2.0 g, 11.0 mmol, 1.3 equiv), LiCl (1.07 g, 25.4 mmol, 3.0 equiv) and (dppf)PdCl 2 (310 mg, 0.42 mmol, 0.05 equiv) were added all at once and the reaction mixture was heated to 80 ºC under a N 2 atmosphere. After 16 h the reaction was quenched with a saturated solution of NH 4 Cl (aq.) and added to a separatory funnel. The product was extracted with ethyl acetate (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. The title compound was obtained as a colorless oil (2.15 g, 72% yield) after SiO 2 flash chromatography (20% EtOAc/Hexanes): 1 H NMR (400 MHz, CDCl 3 ) δ 3.63 (s, 3H), 3.83 (s, 3H), 3.91 (s, 3H), 3.91 (s, 3H), 6.82 (dd, J = 7.6, 1.5 Hz, 1H), 6.83 (s, 1H), 6.96 (dd, J = 8.2, 1.5 Hz, 1H), 7.09 (dd, J = 7.9, 7.9 Hz, 1H), 7.12 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 55.8, 56.0, 56.2, 60.7, 111.9, 113.5, 114.2, 115.2, 123.3, 123.4, 131.3, 135.4, 146.7, 147.9, 148.8, 152.8; IR (neat) cm -1 2935, 1579, 1506, 1473, 1262, 1210, 1058, 1007, 941; HRMS (ESI+) m/e calc’d for [M+H] + C 16 H 17 BrO 4 : 353.0388, found 353.0394. OMe MeO MeO MeO Br 5.4 313 2-bromo-3',4,4',5-tetramethoxy-1,1'-biphenyl (5.9) and 2-bromo-3',4',5-trimethoxy-1,1'- biphenyl (5.13). Spectral data was identical to that reported. 149 2-bromo-2',4,5-trimethoxy-1,1'-biphenyl (5.10). 1 H NMR (400 MHz, CDCl 3 ) δ 3.81 (s, 3H), 3.85 (s, 3H), 3.91 (s, 3H), 6.81 (s, 1H), 6.99 (d, J = 8.3 Hz, 1H), 7.03 (ddd, J = 7.4, 7.4, 1.8 Hz, 1H), 7.12 (s, 1H), 7.19 (dd, J = 7.4, 1.8 Hz, 1H), 7.38 (ddd, J = 8.3, 7.4, 1.7 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 55.7, 56.0, 56.2, 111.1, 114.2, 114.2, 115.3, 120.3, 129.3, 130.2, 131.2, 131.7, 148.0, 148.7, 156.7; IR (neat) cm -1 2937, 1597, 1490, 1463, 1245, 1207, 1026, 903, 860; HRMS (ESI+) m/e calc’d for [M+Na] + C 15 H 15 BrO 3 : 345.0102, found 345.0071. OMe MeO MeO Br OMe MeO MeO Br OMe 5.9 5.13 5.10 Br OMe MeO MeO 314 2-bromo-3',4,5-trimethoxy-1,1'-biphenyl (5.11). 1 H NMR (400 MHz, CDCl 3 ) δ 3.85 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 6.84 (s, 1H), 6.90–7.00 (m, 3H), 7.12 (s, 1H), 7.34 (dd, J = 7.9, 7.9 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 55.3, 56.1, 56.2, 112.4, 112.9, 113.8, 115.3, 115.8, 122.0, 129.0, 134.6, 142.4, 148.2, 148.8, 159.1; IR (neat) cm -1 2936, 1602, 1507, 1485, 1464, 1246, 1205, 1028, 932, 857; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 15 BrO 3 : 322.0205, found 325.1938. 2-bromo-4,4',5-trimethoxy-1,1'-biphenyl (5.12). 1 H NMR (400 MHz, CDCl 3 ) δ 3.85 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 6.82 (s, 1H), 6.96 (d, J = 8.7 Hz, 2H), 7.11 (s, 1H), 7.34 (d, J = 8.7 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ) δ 55.3, 56.1, 56.2, 112.7, 113.4, 113.9, 115.7, 5.11 Br OMe MeO MeO 5.12 Br OMe MeO OMe 315 130.6, 133.6, 134.4, 148.2, 148.6, 158.9; IR (neat) cm -1 2937, 1601, 1507, 1486, 1464, 1205, 1028, 932, 857; HRMS (ESI+) m/e calc’d for [M+H] + C 15 H 15 BrO 3 : 322.0205, found 325.1855. 2-bromo-3',4,4',5-tetramethoxy-1,1'-biphenyl (5.9). Dimerization: 4-Bromoveratrole (20.0 g, 92.1 mmol, 1.0 equiv) was dissolved in CH 2 Cl 2 (1.0 L) in a 2 L round-bottomed flask and cooled to –78 ºC. To this solution was added dropwise (over 1 h) a pre-mixed solution of PIFA (21.8 g, 50.7 mmol, 0.55 equiv) and BF 3 •Et 2 O (12.7 mL, 101.4 mmol, 1.1 equiv) in CH 2 Cl 2 (500 mL). The reaction mixture was stirred until the starting material was completely consumed (~ 1 h). The reaction was quenched by adding 10% NaOH (500 mL) and the reaction mixture was vigorously stirred for 1 h. The product was extracted with CH 2 Cl 2 (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo and recrystallized from EtOH. Veratrole dimer 5.15 was obtained as a white crystalline solid (18.8 g, 95% yield). Spectral data was identical to that reported. 358 Dehalogenation: Veratrole dimer 5.15 (10.0 g, 23.0 mmol, 1.0 equiv) was dissolved in anhydrous THF (400 mL) and cooled to –78 ºC. To this solution was added n-BuLi (9.3 mL, OMe MeO Br 1. PIFA, BF 3 •Et 2 O, CH 2 Cl 2 , –78 ºC 2. n-BuLi (1.0 equiv), THF; then MeOH Br OMe MeO MeO OMe 5.9 316 23.0 mmol, 1.0 equiv, 2.5M in hexanes) dropwise via syringe pump (0.62 mL/min). Upon complete addition of n-BuLi the reaction was stirred for another 20 minutes before 2.0 mL of MeOH was added as a proton source. The quenched reaction mixture was stirred for 5 minutes at –78 ºC and then poured into a separatory funnel with 100 mL of H 2 O. The product was extracted with ethyl acetate (3x). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo and recrystallized from MeOH. The title compound was obtained as an off-white crystalline solid (6.79 g, 84% yield). Spectral data was identical to that reported. 149 NEGISHI COUPLINGS Compounds 5.1, 5.17, 5.18, 5.19, 5.20 and 5.21 were prepared according the procedure shown for compound 5.1. THF, rt Pd(PPh 3 ) 4 (2 mol%) N R 4 MeO R 1 R 2 R 4 MeO R 1 R 2 X + N TfO t-BuLi, THF; then ZnBr 2 R 3 R 3 X = Br X = ZnBr 5.5 5.1, 5.17–5.21 317 (R)-7-(2',3',4,5-tetramethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (R-5.1). Representative procedure for preparing zincate solution: A dry RBF containing anhydrous ZnBr 2 (412 mg, 1.83 mmol, 1.65 equiv) was heated with heat gun under vacuum. The flask was then cooled to 25 °C, and anhydrous THF (15 mL) was added. To a separate flask containing 20 mL of THF at –78 °C was added tert-butyllithium (2.06 mL, 3.50 mmol, 3.15 equiv, 1.7 M in THF), and then the biarylbromide 5.4 (590 mg, 1.67 mmol, 1.5 equiv) in a minimal amount of THF (~5 mL) was added dropwise. The mixture was stirred at –78 °C for 2 h, and the solution of ZnBr 2 in THF was transferred via a cannula. The resulting zinc reagent was stirred at -78 °C for 10 min and warmed to 25 °C. Representative procedure for Negishi Coupling: Triflate 5.5 (300 mg, 1.11 mmol, 1.0 equiv) and Pd(PPh 3 ) 4 (26 mg, 0.02 mmol, 0.02 equiv) were added sequentially to the solution of zinc reagent. The resulting yellow mixture was stirred at room temperature until the triflate had been consumed (~1 h). The reaction was quenched with SiO 2 the solvent removed as to provide a free-flowing powder. The SiO 2 was loaded onto a SiO 2 column and purified by flash chromatography (60 % EtOAc/hexanes [1% TEA]). The title compound was obtained as a yellowish foamy solid (415 mg, 95%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.28-1.38 (m, 1H), 1.63-1.73 (m, 1H), 1.75-1.89 (m, 2H), 1.95-2.16 (m, 4H) 2.72 (dd, J = 16.2, 1.5 Hz, (R)-5.1, (S)-5.1 and (±)-5.1 N OMe MeO MeO MeO 318 1H), 3.15 (dt, J = 8.6, 2.1 Hz, 1H), 3.50 (dd, J = 16.2, 4.7 Hz, 1H), 3.59 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 5.53 (m, 1H), 6.82 (dd, J = 7.9, 1.4 Hz, 1H), 6.82 (s, 1H), 6.87 (dd, J = 7.9, 1.4 Hz, 1H), 7.00 (dd, J = 7.9, 7.9 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 30.6, 36.4, 52.7, 54.2, 55.8, 55.9, 55.9, 60.1, 60.4, 111.1, 111.9, 113.8, 123.3, 123.6, 124.7, 127.9, 134.9, 135.7, 137.3, 146.4, 147.1, 147.8, 152.8; IR (neat) 2935, 1515, 1469, 1262, 1008, 751 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 24 H 30 NO 4 : 396.2175, found 396.2163. (R)-Enantiomer: α [ ] D 22 = –62.8 (c 1 .00, CHCl 3 ). 7-(2',4,5-trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (±-5.17). Using the same proportions as above, the title compound was obtained after flash chromatography (60 % EtOAc/hexanes [1% TEA]) as a yellowish foamy solid (467 mg, 97%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.30-1.40 (m, 1H), 1.63-1.73 (m, 2H), 1.76-1.97 (m, 3H), 2.03-2.16 (m, 3H), 2.64 (d, J = 16.2 Hz, 1H), 3.14 (dt, J = 8.6, 2.0 Hz, 1H), 3.46 (ddd, J = 16.2, 4.6, 1.7 Hz, 1H), 3.75 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 5.43-5.44 (m, 1H), 6.81 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 6.95 (dt, J = 7.4, 1.0 Hz, 1H), 7.19 (dd, J = 7.4, 1.7 Hz, 1H), 7.28 (ddd, J = 8.1, 7.4, 1.7 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 30.7, 36.4, 52.8, 54.2, 55.4, 55.8, 55.9, 60.1, 110.5, 111.6, 113.9, 120.2, 124.2, 128.2, 128.5, 130.6, (±)-5.17 N OMe MeO MeO 319 131.5, 135.4, 137.1, 147.2, 147.8, 156.4; IR (neat) 2935, 1515, 1491, 1246, 1028, 753 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 366.2069, found 366.2067. 7-(3',4,5-trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (±-5.18). Using the same proportions as above, the title compound was obtained after flash chromatography (60 % EtOAc/hexanes [1% TEA]) as a yellowish foamy solid (432 mg, 95%). 1 H NMR (400 MHz, CDCl 3 ) δ ;1.24-1.34 (m, 1H), 1.64-1.87 (m, 3H), 1.91-2.00 (m, 3H), 2.11 (dd, J = 18.0, 9.0 Hz, 1H), 2.84 (dd, J = 16.3, 1.8 Hz, 1H), 3.18 (dt, J = 8.6, 2.1 Hz, 1H), 3.59 (dd, J = 16.3, 4.7 Hz, 1H), 3.82 (s, 3H), 3.89 (s, 3H), 3.89 (s, 3H), 5.70-5.71 (m, 1H), 6.81 (s, 1H), 6.82-6.85 (m, 1H), 6.84 (s, 1H), 6.96-7.00 (m, 2H), 7.27 (dd, J = 7.9, 7.9 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 30.6, 36.6, 52.9, 54.2, 55.2, 55.9, 56.0, 60.3, 112.3, 113.0, 113.2, 114.4, 121.5, 125.1, 129.0, 131.8, 134.3, 138.5, 143.1, 147.8 147.9, 159.2; IR (neat) 2956, 1502, 1464, 1238, 1040, 753 cm -1 ; HRMS (ESI+) m/e calc’d for [M +H] + C 23 H 28 NO 3 : 366.2069, found 366.2068. (±)-5.18 N OMe MeO MeO 320 7-(4,4',5-trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (±-5.19). Using the same proportions as above, the title compound was obtained after flash chromatography (60 % EtOAc/hexanes [1% TEA]) as a yellowish foamy solid (444 mg, 98%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.24-1.34 (m, 1H), 1.63-1.73 (m, 2H), 1.75-2.02 (m, 3H), 2.12 (dd, J = 17.9, 9.0 Hz, 1H), 2.84 (dd, J = 16.3, 1.8 Hz, 1H), 3.19 (dt, J = 8.5, 2.0 Hz, 1H), 3.59 (ddd, J = 16.3, 4.7, 1.6 Hz, 1H), 3.84 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 5.69-5.70 (m, 1H), 6.80 (s, 1H), 6.81 (s, 1H), 6.90 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ ; 21.3, 30.6, 36.6, 52.9, 54.3, 55.3, 55.9, 56.0, 60.2, 113.0, 113.2, 113.5, 125.1, 128.5, 129.9, 131.6, 134.2, 138.5, 147.6, 147.8, 158.4; IR (neat) 2956, 1514, 1464, 1249, 1045, 772 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 366.2069, found 366.2074. (±)-5.19 N OMe MeO OMe 321 (R)-7-(3',4,4',5-tetramethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine. (R-5.20) Using the same quantities and proportions as above, the title compound was obtained as a yellowish foamy solid (429 mg, 98%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.23-1.32 (m, 1H), 1.62-1.72 (m, 1H), 1.74-1.85 (m, 2H), 1.87-2.98 (m, 3H), 2.09 (dd, J = 9.0 Hz, 1H), 2.82 (dd, J = 16.3, 1.8 Hz, 1H), 3.17 (dt, J = 8.5, 1.8 Hz, 1H), 3.60 (dd, J = 16.3, 4.6 Hz, 1H), 3.87 (s, 3H), 3.87 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 5.73-5.74 (m, 1H), 6.81 (d, J = 6.0 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.94 (dd, J = 8.2, 1.9 Hz, 1H), 6.98 (d, J = 1.9 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 30.6, 36.6, 53.0, 54.3, 55.8, 55.9, 56.0, 56.0, 60.4, 110.9, 112.3, 113.1, 113.2, 120.9, 124.9, 131.6, 134.1, 134.5, 138.9, 147.7, 147.8, 147.8, 148.2; IR (neat) 2954, 1504, 1464, 1249, 1028, 762 cm -1 ; HRMS (ESI+) m/e calc’d for [M +H] + C 24 H 30 NO 4 : 396.2175, found 396.2177. (R)-Enantiomer: α [ ] D 22 = –138 (c 1 .00, CHCl 3 ). (R)-5.20 and (±)-5.20 N OMe MeO MeO OMe 322 (±)-5.21 N MeO OMe MeO 7-(3',4',5-trimethoxy-[1,1'-biphenyl]-2-yl)-1,2,3,5,8,8a-hexahydroindolizine (5.21). Using the same proportions as above, the title compound was obtained after flash chromatography (60 % EtOAc/hexanes [1% TEA]) as a yellowish foamy solid (485 mg, 98%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.22-1.32 (m, 1H), 1.63-1.98 (m, 6H), 2.09 (dd, J = 17.9, 8.9 Hz, 1H), 2.83 (dd, J = 16.2, 1.6 Hz, 1H), 3.18 (dt, J = 8.5, 1.9 Hz, 1H), 3.61 (ddd, J = 16.2, 4.5, 1.1 Hz, 1H), 3.83 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 5.69-5.70 (m, 1H), 6.82 (dd, J = 8.4, 2.7 Hz, 1H), 6.86 (d, J = 2.7 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.98 (dd, J = 8.2, 2.0 Hz, 1H), 7.01 (d, J = 2.0 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.3, 30.6, 36.5, 53.0, 54.3, 55.3, 55.8, 55.9 60.3, 110.9, 112.1, 112.2, 115.5, 120.9, 124.7, 130.9, 134.4, 134.5, 138.5, 140.4, 148.0, 148.3, 158.6; IR (neat) 2955, 1517, 1464, 1248, 1029, 754 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 366.2069, found 366.2069. 323 ARYL-ALKENE OXIDATIVE COUPLINGS Compounds 1.1, 1.4, 4.2, 5.22, 5.23 and 5.24 were prepared according the procedure shown for compound 1.4. (R)-Tylocrebrine (R-1.4). Indolizidine 5.1 (400 mg, 1.01 mmol, 1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 (100 mL) and cooled to –78 ºC. Two drops of TFAA was then added to this solution. In a separate flask containing VOF 3 (500 mg, 4.04 mmol, 4.0 equiv) was added anhydrous CH 2 Cl 2 (10 mL), anhydrous EtOAc (5 mL), TFA (0.5 mL) and TFAA (2 drops). The VOF 3 solution was then added over 20 minutes to the solution of indolizidine 5.1. The 5.1, 5.17–5.21 VOF 3 , CH 2 Cl 2 , EtOAc N R 4 MeO R 2 N R 4 MeO R 2 TFA, TFAA, -78 ºC R 1 R 1 R 3 R 3 1.1, 1.4, 4.2, 5.22–5.24 tylocrebrine (R)-1.4, (S)-1.4 and (±)-1.4 N OMe MeO MeO MeO 324 reaction mixture was stirred for 1 h at –78 ºC and then slowly warmed over 3 h to ~ –15 ºC or until the starting material had been consumed. To the reaction mixture was added 10% NaOH (50 mL) and the biphasic mixture was vigorously stirred for 1 h at room temperature. The product was extracted with CH 2 Cl 2 . The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 . Filtration and concentration in vacuo gave the crude product. Purification by flash chromatography (80 % EtOAc/hexanes [1% TEA]) provided the title compound as white crystalline solid (247 mg, 62%): mp 227–229 ºC (decomp.) [Lit. mp 218–220 ºC]; 20 1 H NMR (400 MHz, CDCl 3 ) δ 1.73-1.83 (m, 1H), 1.87-1.96 (m, 1H), 1.99-2.10 (m, 1H), 2.19-2.31 (m, 1H), 2.42-2.52 (m, 2H), 2.91 (dd, J = 14.8, 11.3 Hz, 1H), 3.33 (dd, J = 15.8, 3.5 Hz, 1H), 3.46 (t, J = 8.2 Hz, 1H), 3.68 (d, J = 14.7 Hz, 1H), 3.92 (s, 3H), 4.03 (s, 3H), 4.07 (s, 3H), 4.07 (s, 3H), 4.66 (d, J = 14. 8 Hz, 1H), 7.28 (d, J = 9.1 Hz, 1H), 7.33 (s, 1H), 7.65 (d, J = 9.1 Hz, 1H), 9.33 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.6, 31.3, 34.0, 54.2, 55.1, 55.7, 56.5, 60.0, 60.2, 103.5, 109.1, 112.1, 118.8, 123.3, 123.6, 125.8, 126.5, 127.9, 146.3, 147.7, 148.7, 150.6; IR 2936, 1601, 1514, 1467, 1255, 1114, 1033, 913 (neat) cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 24 H 28 NO 4 : 394.2013, found 394.2021. α [ ] D 22 = –105 (c 1.00, CHCl 3 ). [Lit. –45 (c 0.74, CHCl 3 )] 20 325 (R)-Tylophorine (R-1.1). Using the same quantities and proportions as above, the title compound was obtained after flash chromatography (5% MeOH/CH 2 Cl 2 ) as a white crystalline solid (354 mg, 89%). mp 284–285 ºC (decomp.) [Lit. mp 274–276 ºC, 359, 360 275 ºC 11, 14 ] ; 1 H NMR (400 MHz, CDCl 3 ) δ 1.72-1.97 (m, 2H), 1.99-2.10 (m, 1H), 2.20-2.28 (m, 1H), 2.43 (m, 2H), 2.90 (dd, J = 15.6, 10.6 Hz, 1H), 3.35 (dd, J = 15.8, 2.5 Hz, 1H), 3.46 (dt, J = 4.2, 1.6 Hz, 1H), 3.65 (d, J = 14.6 Hz, 1H), 4.05 (s, 3H), 4.05 (s, 3H), 4.11 (s, 6H), 4.61 (d, J = 14.6 Hz, 1H), 7.15 (s, 1H), 7.30 (s, 1H), 7.81 (s, 1H), 7.82 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.6, 31.3, 33.8, 54.1, 55.2, 55.9, 55.9, 56.1, 60.3, 103.2, 103.4, 103.5, 104.0, 123.5, 123.6, 124.4, 125.9, 126.0, 126.3, 148.4, 148.5, 148.7, 148.7; IR (neat) 2960, 1619, 1515, 1248, 1151, 1017, 841 cm -1 ; HRMS (ESI+) m/e calc’d for [M] + C 24 H 24 NO 4 + : 394.2013, found 394.2011. α [ ] D 22 = –99.0 (c 1.00, CHCl 3 ) [Lit. –76.5 (c 0.04, CHC1 3 ), 360 –77.6 (c 0.65, CHCl 3 ) 149 ]. tylophorine (R)-1.1 and (±)-1.1 N OMe MeO MeO OMe 326 (±)-6-Desmethoxytylocrebrine (5.22). Using the same proportions as above, the title compound was obtained after flash chromatography (5% MeOH/CH 2 Cl 2 ) as a white crystalline solid (28 mg, 28%). 1 H NMR (400 MHz, CDCl 3 ) δ 1.72-1.82 (m, 1H), 1.87–1.97 (m, 1H), 1.98-2.10 (m, 1H), 2.42-2.52 (m, 2H), 2.94 (dd, J = 15.8, 10.5 Hz, 1H), 3.38 (dd, J = 16.0, 2.2 Hz, 1H), 3.48 (dt, J = 4.3, 2.0 Hz, 1H), 3.70 (d, J = 14.9 Hz, 1H), 4.07 (s, 3H), 4.09 (s, 3H), 4.12 (s, 3H), 4.68 (d, J = 14.9 Hz, 1H), 7.11 (d, J = 7.7, 1H), 7.36 (s, 1H), 7.48 (dd, J = 8.0, 8.0 Hz, 1H), 7.54 (dd, J = 8.3, 1.1 Hz, 1H); HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 364.1913, found 364.1898. 13 C NMR and IR data could not be obtained, because the sample prepared degraded in CDCl 3 . 6-desmethoxytylocrebrine (±)-5.22 N OMe MeO MeO 327 (±)-Antofine (4.2). Using the same proportions as above, the title compound was obtained after flash chromatography (5% MeOH/CH 2 Cl 2 ) as a white crystalline solid (141 mg, 70%). See Section 6.4 for spectral data. (±)-Desmethoxytylophorine (5.23). Using the same proportions as above, the title compound was obtained after flash chromatography (5% MeOH/CH 2 Cl 2 ) as a white crystalline solid (190 mg, 85%). mp 203–205 ºC (decomp.) 1 H NMR (400 MHz, CDCl 3 ) δ 1.72-1.96 (m, 2H), 1.98-2.09 (m, 1H), 2.20-2.28 (m, 1H), 2.42–2.54 (m, 2H), 2.90 (dd, J = 15.6, 10.5 Hz, 1H), 3.33 (dd, J = 15.8, 2.4 Hz, 1H), 3.46 (dt, J = 8.6, 1.7 Hz, 1H), 3.69 (d, J = 14.8 Hz, 1H), 4.01 (s, 3H), 4.06 (s, 3H), 4.10 (s, 3H), 4.69 (d, J = 14.9 Hz, 1H), 7.20 (dd, J = antofine (±)-4.2 N OMe MeO MeO 6-desmethoxytylophorine (±)-5.23 N OMe MeO OMe 328 9.0, 2.5 Hz, 1H), 7.30 (s, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 2.5 Hz, 1H) 7.91 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.6, 31.3, 33.6, 53.8, 55.0, 55.5, 55.9, 56.0, 60.2, 103.8, 104.0, 104.7, 114.9, 123.5, 124.1, 124.2, 125.5, 126.5, 127.1, 130.2, 148.4, 149.4, 157.5; IR (neat) 2914, 1615, 1512, 1257, 1033, 912, 843 cm -1 ; HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 364.1913, found 364.1909. (±)-Deoxypergularinine (5.24). Using the same proportions as above, the title compound was obtained after flash chromatography (5% MeOH/CH 2 Cl 2 ) as a white crystalline solid (170 mg, 68%). mp 221–222 ºC (decomp.) 1 H NMR (400 MHz, CDCl 3 ) δ 1.71-1.81 (m, 1H), 1.87–1.97 (m, 1H), 1.98-2.09 (m, 1H), 2.20-2.28 (m, 1H), 2.46 (dd, J = 18.0, 9.0 Hz, 1H), 2.43–2.52 (m, 1H), 2.94 (dd, J = 16.0, 10.6 Hz, 1H), 3.41–3.50 (m, 2H), 3.66 (d, J = 14.7 Hz, 1H), 4.02 (s, 3H), 4.06 (s, 3H), 4.10 (s, 3H), 4.61 (d, J = 14.6 Hz, 1H), 7.17 (s, 1H), 7.22 (dd, J = 9.0, 2.5 Hz, 1H), 7.90 (d, J = 2.5 Hz, 1H), 7.92 (s, 1H), 7.95 (d, J = 9.1 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 21.6, 31.3, 33.7, 54.0, 55.2, 55.5, 55.9, 56.0, 60.2, 103.2, 104.0, 104.6, 114.8, 123.3, 125.2, 125.4, 125.6, 125.7, 127.1, 130.4, 148.3, 149.4, 157.6; IR (neat) cm -1 deoxypergularinine (±)-5.24 N MeO OMe MeO 329 2928, 1604, 1528, 1468, 1205, 1165, 1031, 913 HRMS (ESI+) m/e calc’d for [M+H] + C 23 H 28 NO 3 : 364.1913, found 394.1910. 6.5.2 Biological procedures 6.5.2.1 Anti-proliferation assay MCF-7, COLO-205 and NCI-ADR-RES cancer cells were harvested (125 G centrifuge for 5 min) from a exponential-phase maintenance culture. The cells were resuspended in a new culture medium (RPMI Medium 1640) and the cell density was adjusted to 10 5 cells/mL and dispensed in 96-well culture plates at a density 5,000 cells per well (50 µL ). The cells were incubated overnight growth to allow cells to adhere to the wells. The culture medium in each well was replaced with fresh culture medium (50 µL) containing varying concentrations (50, 12.5, 3.125, 0.8, 0.2, 0.05, 0.0125, 0.003 µL) of the test and control (Taxol) compounds. The cultures were grown for an additional 48 h and alamarBlue® (20ul) was added. After 1–2 h, the fluorescence excitation (530 nm) and emission (590 nm) of each well were measured to determine the optical density. (This assay was performed by Dr. Harry Tian at the ITDD) 6.5.2.2 MOA assay The following stock solutions were prepared: MAO-A (5 mg/mL, 75 U/mg), MAO-B (5 mg/ mL, 58 U/mg), p-tyramine (100 mM), Amplex® Red reagent (20 mM), HRP (200 U/mL), 330 reaction buffer. Amplex® Red reagent solution (400 µL) was prepared that consisted of Amplex® Red (50 µL, 20 mM stock solution), p-tyramine (50 µL, 100 mM stock solution) and HRP (2 U/mL). Varying concentrations (100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.0013 µM) of test and control compounds were added wells in a 360-well plate using the ECHO 550 acoustic liquid handler. MAO-A or MAO-B stock solutions (25 µL) were added to the appropriate wells by hand and the plate was incubated at room temperature for 15 min. Amplex® Red solution (25 µL) was added to the appropriate wells by hand and the plate was incubated at room temperature for 30 minutes. The fluorescence excitation (530 nm) and emission (590 nm) were measured for each well. (This assay was performed by Dr. Jonothan Soldberg at the ITDD) 6.5.2.3 Comprehensive CNS target screen and binding assays Receptor binding profiles and K D determinations were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA. For experimental details please refer to the PDSP web site http://pdsp.med.unc.edu. 331 6.6 X-Ray crystal structure REFERENCE NUMBER: 08053a CRYSTAL STRUCTURE REPORT [C8 H13 I N O]+ IReport prepared for: M. Niphakis / Prof. G. Georg March 25, 2008 Victor G. Young, Jr. X-Ray Crystallographic Laboratory Department of Chemistry University of Minnesota 207 Pleasant St. S.E. Minneapolis, MN 55455 DATA COLLECTION A crystal (approximate dimensions 0.45 x 0.20 x 0.05 mm3) was placed onto the tip of a 0.1 mm diameter glass capillary and mounted on a CCD area detector diffractometer for a data collection at 173(2) K. 1 A preliminary set of cell constants was calculated from reflections harvested from three sets of 20 frames. These initial sets of frames were oriented such that orthogonal wedges of reciprocal space were surveyed. This produced initial orientation matrices determined from 122 reflections. The data collection was carried out using MoKα radiation (graphite monochromator) with a frame time of 10 seconds and a detector distance of 4.9 cm. A randomly oriented region of reciprocal space was surveyed to the extent of one sphere and to a resolution of 0.77 Å. Four major sections of frames were collected with 0.30º steps in ω at four different φ settings and a detector position of -28º in 2θ. The intensity data were corrected for absorption and decay (SADABS). 2 Final cell constants were calculated from 2533 strong reflections from the actual data collection after integration (SAINT). 3 Please refer to Table 1 for additional crystal and refinement information. 332 STRUCTURE SOLUTION AND REFINEMENT The structure was solved using Bruker SHELXTL 4 and refined using Bruker SHELXTL. 4 The space group P21 was determined based on systematic absences and intensity statistics. A direct methods solution was calculated which provided most non-hydrogen atoms from the E-map. Full matrix least squares / difference Fourier cycles were performed which located the remaining non-hydrogen atoms. All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were placed in ideal positions and refined as riding atoms with relative isotropic displacement parameters. The final full matrix least squares refinement converged to R1 = 0.0192 and wR2 = 0.0483 (F2, all data). STRUCTURE DESCRIPTION The structure is the one suggested. CHECKCIF determined the P21 structure to be pseudo- centric (83%), but the structure is clearly chiral with the absolute configuration at C4 being S. Data collection and structure solution were conducted at the X-Ray Crystallographic Laboratory, 192C Kolthoff Hall, Department of Chemistry, University of Minnesota. All calculations were performed using Pentium computers using the current SHELXTL suite of programs. 333 References: 1. SMART V5.054, Bruker Analytical X-ray Systems, Madison, WI (2001). 2. An empirical correction for absorption anisotropy, R. Blessing, Acta Cryst. A51, 33-38 (1995). 3. SAINT+ V6.45, Bruker Analytical X-Ray Systems, Madison, WI (2003). 4. SHELXTL V6.14, Bruker Analytical X-Ray Systems, Madison, WI (2000). Some equations of interest: R int = F o 2 − < F o 2 > ∑ / F o 2 ∑ R 1 = F o 2 – F c 2 / F o ∑∑ wR2 = w F o 2 − F c 2 ( ) 2 ⎡ ⎣ ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ / w F o 2 ( ) 2 ⎡ ⎣ ⎤ ⎦ ∑ ⎤ ⎦ ⎥ 1/2 where w = q / σ 2 F o 2 ( ) + a *P ( ) 2 + b *P + d + e*sin(θ)⎡ ⎣ ⎤ ⎦ GooF = S = w F o 2 – F c 2 ( ) 2 ⎡ ⎣ ⎤ ⎦ ∑ / (n – p) ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ 1/2 334 Table 1. Crystal data and structure refinement for 08053a. Identification code 08053a Empirical formula C 8 H 13 I 2 NO Formula weight 392.99 Temperature 173(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2 1 Unit cell dimensions a = 8.7128(13) Å α = 90° b = 7.4839(11) Å β = 96.339(2)° c = 9.3223(13) Å γ = 90° Volume 604.15(15) Å3 Z 2 Density (calculated) 2.160 Mg/m 3 Absorption coefficient 5.168 mm –1 F(000) 364 Crystal color, morphology Colorless, Plate Crystal size 0.45 x 0.20 x 0.05 mm3 Theta range for data collection 2.20 to 27.46° Index ranges –11 ≤ h ≤ 11, –9 ≤ k ≤ 9, 0 ≤ l ≤ 12 Reflections collected 6149 Independent reflections 2704 [R(int) = 0.0252] Observed reflections 2619 Completeness to theta = 27.46° 99.00% Absorption correction Multi-scan Max. and min. transmission 0.7821 and 0.2045 Refinement method Full-matrix least-squares on F2 335 Identification code 08053a Data / restraints / parameters 2704 / 1 / 109 Goodness-of-fit on F 2 1.025 Final R indices [I>2sigma(I)] R1 = 0.0192, wR2 = 0.0479 R indices (all data) R1 = 0.0203, wR2 = 0.0483 Absolute structure parameter 0.00(3) Largest diff. peak and hole 0.570 and –0.438 e.Å –3 Table 2. Atomic coordinates (x10 4 ) and equivalent isotropic displacement parameters (Å 2 x 10 3 ) for 08053a. U eq is defined as one third of the trace of the orthogonalized U ij tensor. x y z U eq N1 6388(3) 3 088(3) 4160(3) 24(1) C1 8059(4) 2548(5) 4568(4) 34(1) C2 8981(4) 4079(6) 4066(4) 43(1) C3 8007(3) 4677(7) 2684(4) 38(1) C4 6372(3) 4679(5) 3141(3) 28(1) C5 5083(3) 4517(6) 1903(3) 27(1) C6 3522(3) 4553(6) 2435(3) 32(1) O1 3376(2) 4524(5) 3716(2) 40(1) C7 2115(3) 4615(7) 1377(3) 35(1) C8 2079(3) 4606(6) -25(3) 32(1) I1 -43(1) 4668(1) -1311(1) 36(1) I2 4346(1) -506(1) 2402(1) 29(1) 336 Table 3. Bond lengths [Å] and angles [°] for 08053a. N(1)-C(4) 1.522(4) N(1)-C(1) 1.519(4) N(1)-H(1A) 0.9200 N(1)-H(1B) 0.9200 C(1)-C(2) 1.503(5) C(1)-H(1C) 0.9900 C(1)-H(1D) 0.9900 C(2)-C(3) 1.530(5) C(2)-H(2A) 0.9900 C(2)-H(2B) 0.9900 C(3)-C(4) 1.532(4) C(3)-H(3A) 0.9900 C(3)-H(3B) 0.9900 C(4)-C(5) 1.524(4) C(4)-H(4A) 1.0000 C(5)-C(6) 1.498(4) C(5)-H(5A) 0.9900 C(5)-H(5B) 0.9900 C(6)-O(1) 1.215(3) C(6)-C(7) 1.487(4) C(7)-C(8) 1.304(4) C(7)-H(7A) 0.9500 C(8)-I(1) 2.090(3) C(8)-H(8A) 0.9500 C(4)-N(1)-C(1) 107.9(2) 337 N(1)-C(4) 1.522(4) C(4)-N(1)-H(1A) 110.1 C(1)-N(1)-H(1A) 110.1 C(4)-N(1)-H(1B) 110.1 C(1)-N(1)-H(1B) 110.1 H(1A)-N(1)-H(1B) 108.4 C(2)-C(1)-N(1) 104.5(3) C(2)-C(1)-H(1C) 110.9 N(1)-C(1)-H(1C) 110.9 C(2)-C(1)-H(1D) 110.9 N(1)-C(1)-H(1D) 110.9 H(1C)-C(1)-H(1D) 108.9 C(1)-C(2)-C(3) 102.7(3) C(1)-C(2)-H(2A) 111.2 C(3)-C(2)-H(2A) 111.2 C(1)-C(2)-H(2B) 111.2 C(3)-C(2)-H(2B) 111.2 H(2A)-C(2)-H(2B) 109.1 C(2)-C(3)-C(4) 102.3(3) C(2)-C(3)-H(3A) 111.3 C(4)-C(3)-H(3A) 111.3 C(2)-C(3)-H(3B) 111.3 C(4)-C(3)-H(3B) 111.3 H(3A)-C(3)-H(3B) 109.2 N(1)-C(4)-C(5) 111.4(3) N(1)-C(4)-C(3) 103.2(3) C(5)-C(4)-C(3) 114.8(3) 338 N(1)-C(4) 1.522(4) N(1)-C(4)-H(4A) 109.1 C(5)-C(4)-H(4A) 109.1 C(3)-C(4)-H(4A) 109.1 C(6)-C(5)-C(4) 111.6(2) C(6)-C(5)-H(5A) 109.3 C(4)-C(5)-H(5A) 109.3 C(6)-C(5)-H(5B) 109.3 C(4)-C(5)-H(5B) 109.3 H(5A)-C(5)-H(5B) 108.0 O(1)-C(6)-C(7) 119.0(3) O(1)-C(6)-C(5) 121.5(3) C(7)-C(6)-C(5) 119.5(3) C(8)-C(7)-C(6) 126.3(3) C(8)-C(7)-H(7A) 116.9 C(6)-C(7)-H(7A) 116.9 C(7)-C(8)-I(1) 119.8(2) C(7)-C(8)-H(8A) 120.1 I(1)-C(8)-H(8A) 120.1 Symmetry transformations used to generate equivalent atoms 339 Table 4. Anisotropic displacement parameters (Å 2 x 10 3 ) for 08053a. The anisotropic displacement factor exponent takes the form: –2π 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 N1 22(1) 27(1) 22(1) -1(1) 4(1) -3(1) C1 21(2) 43(2) 37(2) 6(2) 3(1) 4(1) C2 26(2) 62(3) 39(2) 10(2) 3(2) -8(2) C3 24(1) 48(2) 44(2) 14(2) 7(1) -6(2) C4 25(1) 29(2) 30(1) 5(2) 2(1) 0(2) C5 27(1) 30(2) 25(1) 2(2) 5(1) 5(2) C6 27(1) 37(2) 30(1) -3(2) 2(1) 8(2) O1 32(1) 64(2) 25(1) 2(2) 6(1) 8(2) C7 22(1) 50(2) 32(1) 0(2) 5(1) 4(2) C8 27(1) 32(2) 35(1) 1(2) 1(1) -2(2) I1 32(1) 43(1) 32(1) 1(1) -4(1) 0(1) I2 32(1) 29(1) 25(1) 1(1) 3(1) -6(1) 340 Table 5. Hydrogen coordinates (x 10 4 ) and isotropic displacement parameters (Å 2 x 10 3 ) for 08053a. x y z U eq H1A 5842 2152 3713 28 H1B 5938 3394 4973 28 H1C 8277 2380 5624 41 H1D 8299 1425 4078 41 H2A 10020 3683 3866 51 H2B 9091 5051 4791 51 H3A 8097 3827 1884 46 H3B 8312 5884 2385 46 H4A 6217 5799 3693 34 H5A 5162 5514 1217 33 H5B 5206 3384 1379 33 H7A 1149 4667 1759 42 H8A 3014 4570 -461 38 341 Table 6. Torsion angles [°] for 08053a. C4-N1-C1-C2 12.8(3) N1-C1-C2-C3 -35.3(4) C1-C2-C3-C4 44.7(4) C1-N1-C4-C5 138.4(3) C1-N1-C4-C3 14.7(3) C2-C3-C4-N1 -36.0(4) C2-C3-C4-C5 -157.5(4) N1-C4-C5-C6 63.9(4) C3-C4-C5-C6 -179.3(4) C4-C5-C6-O1 -7.2(6) C4-C5-C6-C7 173.2(4) O1-C6-C7-C8 -178.1(5) C5-C6-C7-C8 1.5(8) C6-C7-C8-I1 179.2(4) Symmetry transformations used to generate equivalent atoms. Table 7. Hydrogen bonds for 08053a [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) N1-H1B...I2#1 0.92 2.62 3.498(2) N1-H1A...I2 0.92 2.61 3.528(3) Symmetry transformations used to generate equivalent atoms: #1 -x+1,y+1/2,-z+1 342 checkCIF/PLATON report No syntax errors found. CIF dictionary Interpreting this report Datablock: 08053a Bond precision: C-C = 0.0044 A Wavelength=0.71073 Cell: a=8.7128(13) b=7.4839(11) c=9.3223(13) alpha=90 beta=96.339(2) gamma=90 Temperature: 173 K Calculated Reported Volume 604.15(15) 604.15(15) Space group P 21 P2(1) Hall group P 2yb ? Moiety formula C 8 H 13 INO, I C 8 H 13 INO, I Sum formula C 8 H 13 I 2 NO C 8 H 13 I 2 NO Mr 392.99 392.99 Dx,g cm -3 2.160 2.160 Z 2 2 Mu (mm -1 ) 5.168 5.168 F000 364.0 364.0 F000’ 362.24 h,k,lmax 11,9,12 11,9,12 N ref 1488( 2761) 2704 Tmin,Tmax 0.301,0.772 0.205,0.782 Tmin’ 0.094 343 Correction method = AbsCorr = MULTI-SCAN Data completeness = 1.82(0.98) Theta(max) = 27.460 R(reflections) = 0.0192( 2619) wR2 (reflections) = 0.0483( 2704) S = 1.025 Npar = 109 The following ALERTS were generated. Each ALERT has the format test-name_ALERT_alert-type_alert-level. Alert level B PLAT111_ALERT_2_B ADDSYM Detects (Pseudo) Centre of Symmetry ..… 83 PerFi Alert level C PLAT850_ALERT_2_C Check Flack Parameter Exact Value 0.00 and su .. 0.03 Alert level G REFLT03_ALERT_4_G Please check that the estimate of the number of Friedel pairs is correct. If it is not, please give the correct count in the _publ_section_exptl_refinement section of the submitted CIF. From the CIF: _diffrn_reflns_theta_max 27.46 From the CIF: _reflns_number_total 2704 Count of symmetry unique reflns 1488 Completeness (_total/calc) 181.72% TEST3: Check Friedels for noncentro structure Estimate of Friedel pairs measured 1216 Fraction of Friedel pairs measured 0.817 Are heavy atom types Z>Si present yes PLAT791_ALERT_1_G Confirm the Absolute Configuration of C4 … S PLAT860_ALERT_3_G Note: Number of Least-Squares Restraints … 1 0 ALERT level A = In general: serious problem 1 ALERT level B = Potentially serious problem 1 ALERT level C = Check and explain 3 ALERT level G = General alerts; check 1 ALERT type 1 CIF construction/syntax error, inconsistent or missing data 2 ALERT type 2 Indicator that the structure model may be wrong or deficient 1 ALERT type 3 Indicator that the structure quality may be low 1 ALERT type 4 Improvement, methodology, query or suggestion 0 ALERT type 5 Informative message, check 344 345 BIBLIOGRAPHY 1. Bhavan, B. 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