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Bioactive marine natural products : analogue synthesis, SAR, and target identification Yan, Luping 2014

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BIOACTIVE MARINE NATURAL PRODUCTS: ANALOGUE SYNTHESIS, SAR, AND TARGET IDENTIFICATION  by Luping Yan  M.Sc., Sichuan University, 2007 B.Sc., Sichuan University, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2014  © Luping Yan, 2014ii  Abstract  3,6,7-trihydroxycoumarin C11 (2.14) was first isolated from the green alga Dasycladus vermicularis in 1983. C11 and 3,7,8-trihydroxycoumarin C21 (2.15), alongside their precursors C12 (2.18) and C22 (2.20), were synthesized for a target-based screen for anti-HCV drugs, where ideal hits eliminate fluorescence signals by inhibiting the proteolytic activity of HCV NS3pro/Pep4A against a synthetic peptide “BS-IQFS”. With C12 and C22 serving as negative controls, C11 and C21 inhibited the NS3pro/Pep4A activity in vitro. The IC50’s of C11 and C21 were 3.07 M and 2.10 M, respectively.  A bioassay-guided fractionation identified sintokamides A – E (3.11 – 3.15) from extracts of the sponge Dysidea sp. in 2008. In a phenotypic screen, the chlorinated dipeptides showed strong to modest inhibition of luciferase activity caused by AR NTD transactivation in LNCaP cells. Larger quantities of sintokamides A and B were isolated from the sponge for further biological study. After developing a practical synthetic route, a comprehensive SAR of the sintokamides was available from the in vitro activities of 29 synthetic analogues/precursors based on a 1,17-dinorsintokamide skeleton. LPY26 [(4R,10R)-3.233] showed the best biological activity in the synthetic analogues prepared to date and it was selected as a drug lead. Mechanism of action study using synthetic probes LPY30 (4.7) and LPY31 (4.8) revealed that the hexachlorinated 1,17-dinorsintokamides covalently bound to the AR, but not to the same AF1 region in the AR NTD as EPI-001 (3.8).  iii  The structure of latonduine A (5.1) isolated from the sponge Stylissa carteri and its total synthesis were published in 2003. Later, latonduine A was shown to be active in a phenotypic screen to find drug leads for the treatment of cystic fibrosis caused by the F508del mutation. Latonduine A could efficiently correct immunofluorescent F508del-CFTR trafficking from the endoplasmic reticulum to the plasma membrane in the engineered cells. Synthetic latonduine A and N-biotinylated latonduine A (5.17) were prepared to support biological studies aimed at identifying its cellular protein target(s). These studies culminated in the finding that latonduine A is an inhibitor of PARP-3 with an EC50 of 400 pM in CFBE41o cells. iv  Preface  Chapter 2 is work done at UBC. All synthetic coumarins (C11, C12, C21, and C22) were synthesized and characterized by the author. The biology of the four synthetic coumarins was completed by Steven McArthur under the supervision of Dr. François Jean in the Department of Microbiology and Immunology at UBC.  Chapter 3 is work done at UBC and the BC Cancer Agency. In this chapter, some copyright content was reused with permissions. Sintokamides A to E were isolated and characterized by Dr. David Williams in the Andersen lab. Preparation of the sintokamide analogues/precursors and their synthetic studies were completed by the author. The biological data of all the synthetic compounds was collected by Nasrin Mawji, Adriana Banuelos, and Iran Tavakoli under the leadership of Dr. Marianne Sadar at the BC Cancer Agency.  Chapter 4 is work done at UBC and the BC Cancer Agency. The biology of the EPI compounds has been published.  Andersen, R. J.; Mawji, N. R.; Wang, J.; Wang, G.; Haile, S.; Myung, J. K.; Watt, K.; Tam, T.; Yang, Y. C.; Banuelos, C. A.; Williams, D. E.; McEwan, I. J.; Wang, Y.; Sadar, M. D., Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer cell 2010, 17 (6), 535–546.   v   Myung, J. K.; Banuelos, C. A.; Fernandez, J. G.; Mawji, N. R.; Wang, J.; Tien, A. H.; Yang, Y. C.; Tavakoli, I.; Haile, S.; Watt, K.; McEwan, I. J.; Plymate, S.; Andersen, R. J.; Sadar, M. D., An androgen receptor N-terminal domain antagonist for treating prostate cancer. The Journal of clinical investigation 2013, 123 (7), 2948–2960. For comparison purposes, some of the biology results in the publications were selected and summarized in this chapter. For the same reason, the biology of sintokamide A described in this chapter was selected and summarized from Iran Tavakoli’s master thesis entitled “Inhibition of castration resistant prostate cancer by sintokamide A: an antagonist of the amino-terminus of the androgen receptor” in 2012.1 The sintokamide A sample used in Tavakoli’s thesis was isolated and characterized by the author. All synthetic analogues and probes were made and characterized by the author. The Click chemistry and biology experiments were performed by Nasrin Mawji, Amy Tien, and Adriana Banuelos under the leadership of Dr. Marianne Sadar at the BC Cancer Agency.  The work in Chapter 5 has been published.  Carlile, G. W.; Keyzers, R. A.; Teske, K. A.; Robert, R.; Williams, D. E.; Linington, R. G.; Gray, C. A.; Centko, R. M.; Yan, L.; Anjos, S. M.; Sampson, H. M.; Zhang, D.; Liao, J.; Hanrahan, J. W.; Andersen, R. J.; Thomas, D. Y., Correction of F508del-CFTR trafficking by the sponge alkaloid latonduine is modulated by interaction with PARP. Chemistry & biology 2012, 19 (10), 1288–1299.2 Latonduines A and B were isolated and characterized by Dr. David Williams in the Andersen lab. The total synthesis of latonduine A was first finished by Dr. Roger Linington. Previous and current co-workers in the Andersen lab: Dr. Robert Keyzers, Dr. Christopher Gray, the author, vi  and Ryan Centko were successively in charge of the chemistry for supporting this collaborative project. Synthetic latonduine A and N-biotinylated latonduine A were constructed and characterized by the author. The author contributed to the experimental section related to the N-biotinylated latonduine A in the publication. All biological data was acquired by the co-authors from the Departments of Biochemistry and Physiology under the supervision of Dr. David Thomas at the McGill University.                 vii  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................. xvii List of Figures ........................................................................................................................... xviii List of Schemes ...........................................................................................................................xxx List of Symbols and Abbreviations ...................................................................................... xxxvi Acknowledgements ................................................................................................................... xliii Dedication .................................................................................................................................. xliv Chapter 1: A Brief Retrospective of Drug Discovery Practices ................................................. 1 1.1 The early days: discovery of medicinal natural products ........................................... 1 1.2 The early 19th century: advances in chemistry of natural products and organic chemistry ................................................................................................................................. 2 1.3 The late 19th century: the emergence of medicinal chemistry .................................... 4 1.3.1 The first half of the 20th century: the golden era of drug discovery ....................... 6 1.3.2 The second half of the 20th century: balancing the safety and efficacy in drug discovery ............................................................................................................................. 8 1.3.3 Since the 1970s: Quest for drugs from the sea ..................................................... 10 1.4 Modern drug discovery: reverse pharmacology and forward pharmacology ........... 12 1.5 Finding new targets: methods for mechanism of action study in forward pharmacology ........................................................................................................................ 15 viii  1.6 Scope of thesis .......................................................................................................... 18 Chapter 2: 3,6,7- and 3,7,8-Trihydroxycoumarins Inhibit HCV NS3pro/Pep4A in vitro ........ 21 2.1 Introduction ............................................................................................................... 21 2.2 Coumarins and coumarins-related anti-HCV studies ............................................... 24 2.3 A new target-based screen for anti-HCV therapy ..................................................... 27 2.4 Syntheses of C12 (2.18), C11 (2.14) and C22 (2.20), C21 (2.15) ............................ 29 2.5 Primary screen and kinetics study............................................................................. 33 2.6 Conclusion ................................................................................................................ 37 Chapter 3: Structure-Activity Relationship Study of Sintokamides ......................................... 38 3.1 Introduction ............................................................................................................... 38 3.1.1 Androgens, normal prostate, and prostate carcinogenesis .................................... 41 3.1.2 Adaptive responses to castration affect the androgen axis in CRPC .................... 45 3.1.3 AR NTD, a promising target for CRPC therapy ................................................... 50 3.2 Sintokamides A to E (3.11 to 3.15) from the sponge Dysidea sp.: collection, isolation and biological activity ............................................................................................ 53 3.2.1 Sintokamide A (3.11) inhibits the AR NTD transactivation in vitro .................... 54 3.2.2 Enrichment of sintokamide A (3.11) and sintokamide B (3.12) ........................... 55 3.3 Syntheses of sintokamide analogues for SAR studies .............................................. 56 3.3.1 Synthesis of NCSTD [(4S,10R)-3.16], the non-chlorinated sintokamide framework ......................................................................................................................... 59 3.3.2 Syntheses of 1,17-dinorsintokamide chlorinated sintokamides ............................ 73 Chapter 4: Mechanism of Action Study of Marine Natural Products Induced Inhibitory Activity of AR NTD Transactivation in Prostate Cancer Cells .............................................. 178 ix  4.1 Introduction ............................................................................................................. 178 4.2 AR activity of EPIs and mechanism of action studies ............................................ 180 4.2.1 AR inhibition of EPI-001 (3.8) is different from hormone therapies in vitro..... 180 4.2.2 EPI-001 (3.8) inhibits the AR NTD via covalent binding to the AF1 protein .... 182 4.2.3 EPI stereoisomers inhibit CRPC xenografts in vivo ........................................... 183 4.3 AR NTD-dependent inhibitory activity of sintokamide A, in vitro and in vivo ..... 184 4.4 Different mechanisms of action of the sintokamides and EPI-001 (3.8) ................ 186 4.5 Click chemistry study based on 1,17-dinorsintokamide analogues ........................ 187 4.5.1 Preparation of the monochlorinated LPY02 (4.5) with an alkyne terminus ....... 188 4.5.2 Preparation of trichlorinated probes with an alkyne terminus ............................ 189 4.5.3 Activities of the probes and results of pull-down assays .................................... 190 4.5.4 Possible mechanistic explanation for the Click chemistry result ........................ 194 4.6 Summary ................................................................................................................. 196 Chapter 5: Synthesis of an N-Biotinylated Probe of Latonduine A for Activity Evaluation and Mechanism of Action Study ................................................................................................... 198 5.1 Background of cystic fibrosis ................................................................................. 198 5.2 Latonduines isolated from Stylissa ......................................................................... 199 5.3 Synthesis of latonduine A ....................................................................................... 201 5.4 Latonduine A corrects F508del-CFTR trafficking in vitro ..................................... 203 5.5 Latonduine A potentiates F508del-CFTR defect in vitro, ex vivo, and in vivo ...... 204 5.6 The mechanism of action study of latonduine A .................................................... 208 5.7 Conclusion .............................................................................................................. 211 Chapter 6: Conclusion............................................................................................................. 213 x  Chapter 7: Experimental ......................................................................................................... 219 7.1 General .................................................................................................................... 219 7.2 Experimental for Chapter 2 ..................................................................................... 220 7.2.1 Preparation of 2.18 (C12) ................................................................................... 220 7.2.2 Preparation of 2.14 (C11) ................................................................................... 221 7.2.3 Preparation of 2.20 (C22) ................................................................................... 222 7.2.4 Preparation of 2.15 (C21) ................................................................................... 223 7.3 Experimental for Chapter 3 ..................................................................................... 224 7.3.1 Preparation of Compound 3.35 ........................................................................... 224 7.3.2 Preparation of Compound 3.37 ........................................................................... 225 7.3.3 C-methylation of compound 3.37 with MeI and KH .......................................... 226 7.3.4 O-methylation of compound 3.37 with Me2SO4 and KH ................................... 227 7.3.5 Preparation of Compound 3.40 ........................................................................... 228 7.3.6 Preparation of Compound 3.32 ........................................................................... 229 7.3.7 Coupling compound 3.32 and compound 3.41 in the presence of MeMgBr ...... 230 7.3.8 Preparation of Compound 3.45 ........................................................................... 231 7.3.9 Preparation of Compound (4S,10R)-3.46............................................................ 232 7.3.10 Preparation of Compound 3.47 ....................................................................... 233 7.3.11 Preparation of (4S,10R)-3.16 (NCSTD) .......................................................... 234 7.3.12 Preparation of Compound (2S,3R,5R)-3.67 .................................................... 235 7.3.13 Preparation of Compound (2S,3R,5R)-3.68 .................................................... 236 7.3.14 Preparation of Compound (R)-3.69................................................................. 236 7.3.15 Preparation of Compound (2R,3S,5S)-3.67..................................................... 237 xi  7.3.16 Preparation of Compound (2R,3S,5S)-3.68..................................................... 238 7.3.17 Preparation of Compound (S)-3.69 ................................................................. 239 7.3.18 Preparation of Compound (2S,3R,5R)-3.71 .................................................... 239 7.3.19 Preparation of Compound (2S,3R,5R)-3.72 .................................................... 240 7.3.20 Preparation of Compound (R)-3.73................................................................. 241 7.3.21 Preparation of Compound (2R,3S,5S)-3.71..................................................... 242 7.3.22 Preparation of Compound (2R,3S,5S)-3.72..................................................... 243 7.3.23 Preparation of Compound (S)-3.73 ................................................................. 243 7.3.24 Preparation of Compound 3.75 ....................................................................... 244 7.3.25 Preparation of Compound 3.76 ....................................................................... 245 7.3.26 Preparation of Compound 3.77 ....................................................................... 246 7.3.27 Preparation of Compound 3.78 ....................................................................... 247 7.3.28 Preparation of Compound 3.79 ....................................................................... 248 7.3.29 Preparation of Compound 3.32 ....................................................................... 249 7.3.30 Preparation of Compound (S)-3.129 ............................................................... 250 7.3.31 Preparation of Compound (S)-3.130 ............................................................... 251 7.3.32 Preparation of Compound (S)-3.131 ............................................................... 252 7.3.33 Preparation of Compound (S)-3.132 ............................................................... 253 7.3.34 Preparation of Compound (S)-3.133 ............................................................... 254 7.3.35 Preparation of Compound (S)-3.124 ............................................................... 255 7.3.36 Preparation of Compound 3.134 ..................................................................... 256 7.3.37 Treatment of 3.134 with trimethyl orthoformate and catalytic sulfuric acid .. 257 7.3.38 Preparation of Compound 3.137 ..................................................................... 257 xii  7.3.39 Preparation of Compound 3.125 ..................................................................... 258 7.3.40 Preparation of Compound (R)-3.129............................................................... 259 7.3.41 Preparation of Compound (R)-3.130............................................................... 260 7.3.42 Preparation of Compound (R)-3.131............................................................... 260 7.3.43 Preparation of Compound (R)-3.132............................................................... 261 7.3.44 Preparation of Compound (R)-3.133............................................................... 262 7.3.45 Preparation of Compound (R)-3.124............................................................... 262 7.3.46 Preparation of Compound 3.138 ..................................................................... 263 7.3.47 Preparation of Compound 3.139 ..................................................................... 264 7.3.48 Preparation of Compound 3.140 ..................................................................... 265 7.3.49 Preparation of Compound 3.126 ..................................................................... 266 7.3.50 Preparation of Compound 3.141 ..................................................................... 267 7.3.51 Treatment of 3.141 with Boc2O and DMAP ................................................... 268 7.3.52 Preparation of Compound 3.144 ..................................................................... 268 7.3.53 Preparation of Compound 3.145 ..................................................................... 269 7.3.54 Preparation of Compound 3.146 ..................................................................... 270 7.3.55 Preparation of 3.60 (LPY00)........................................................................... 271 7.3.56 Preparation of Compound 3.127 ..................................................................... 272 7.3.57 Preparation of Compound 3.128 ..................................................................... 273 7.3.58 Preparation of Compound 3.147 ..................................................................... 274 7.3.59 Preparation of 3.148 (LPY04)......................................................................... 275 7.3.60 Preparation of Compound 3.149 ..................................................................... 276 7.3.61 Preparation of Compound 3.150 ..................................................................... 277 xiii  7.3.62 Preparation of Compound 3.168 ..................................................................... 278 7.3.63 Preparation of Compound 3.169 ..................................................................... 279 7.3.64 Preparation of Compound 3.170 ..................................................................... 280 7.3.65 Preparation of Compound 3.151 ..................................................................... 281 7.3.66 Preparation of Compound 3.180 ..................................................................... 282 7.3.67 Preparation of Compound 3.181 ..................................................................... 283 7.3.68 Preparation of Compound 3.182 ..................................................................... 284 7.3.69 Preparation of Compound 3.183 ..................................................................... 285 7.3.70 Preparation of Compound 3.184 ..................................................................... 286 7.3.71 Preparation of Compound 3.185 ..................................................................... 287 7.3.72 Preparation of Compound 3.186 ..................................................................... 287 7.3.73 Preparation of Compound 3.205 ..................................................................... 288 7.3.74 Preparation of (4R,10R)-3.16 (NCSTD1) ....................................................... 289 7.3.75 Preparation of (4S,10R)-3.16 (NCSTD2) ........................................................ 292 7.3.76 Preparation of Compound (S)-3.206 ............................................................... 293 7.3.77 Preparation of Compound (S)-3.207 ............................................................... 294 7.3.78 Preparation of Compound (S)-3.208 ............................................................... 295 7.3.79 Preparation of Compound (R)-3.206............................................................... 296 7.3.80 Preparation of Compound (R)-3.207............................................................... 296 7.3.81 Preparation of Compound (R)-3.211............................................................... 297 7.3.82 Preparation of Compound (R)-3.73................................................................. 298 7.3.83 Preparation of Compound (4S,10R)-3.209...................................................... 299 7.3.84 Preparation of Compound (4R,10R)-3.212 and (4S,10R)-3.212 ..................... 300 xiv  7.3.85 Preparation of (4R,10R)-3.210 (LPY08) ......................................................... 302 7.3.86 Preparation of (4S,10R)-3.210 (LPY09) ......................................................... 303 7.3.87 Preparation of Compound (S)-3.221 ............................................................... 304 7.3.88 Preparation of Compound (R)-3.221............................................................... 305 7.3.89 Preparation of Compound (S,S)-3.217 ............................................................ 305 7.3.90 Preparation of Compound (R,R)-3.217 ........................................................... 306 7.3.91 Preparation of Compound (S)-3.218 ............................................................... 307 7.3.92 Preparation of Compound 3.223 ..................................................................... 308 7.3.93 Coupling reaction between (S)-3.218 and 3.223 ............................................. 310 7.3.94 Coupling reaction between (S)-3.218 and (R)-3.43 ........................................ 310 7.3.95 Preparation of Compound (R)-3.79................................................................. 311 7.3.96 Preparation of (4S,10R)-3.219 (LPY35) ......................................................... 313 7.3.97 Preparation of (4R,10R)-3.220 (LPY10) ......................................................... 314 7.3.98 Preparation of (4S,10R)-3.220 (LPY11) ......................................................... 315 7.3.99 Preparation of Compound (4S,10R)-3.226...................................................... 316 7.3.100 Preparation of (4R,10R)-3.228 (LPY12) ......................................................... 317 7.3.101 Preparation of (4S,10R)-3.228 (LPY13) ......................................................... 318 7.3.102 Preparation of Compound (4S,10R)-3.229...................................................... 319 7.3.103 Preparation of (4R,10R)-3.231 (LPY17) ......................................................... 320 7.3.104 Preparation of (4S,10R)-3.231 (LPY18) ......................................................... 321 7.3.105 Preparation of Compound (S)-3.79 ................................................................. 322 7.3.106 Preparation of Compound (4S,10S)-3.219 ...................................................... 323 7.3.107 Preparation of (4S,10S)-3.220 (LPY20).......................................................... 324 xv  7.3.108 Preparation of (4R,10S)-3.220 (LPY21) ......................................................... 325 7.3.109 Preparation of (4R,10R)-3.225 (LPY22) ......................................................... 326 7.3.110 Preparation of (4S,10R)-3.225 (LPY23) ......................................................... 327 7.3.111 Preparation of (4R,10R)-3.232 (LPY24) ......................................................... 328 7.3.112 Preparation of (4S,10R)-3.232 (LPY25) ......................................................... 328 7.3.113 Preparation of (4R,10R)-3.233 (LPY26) ......................................................... 329 7.3.114 Preparation of (4S,10R)-3.233 (LPY27) ......................................................... 330 7.3.115 Preparation of (4R,10R)-3.234 (LPY28) ......................................................... 331 7.3.116 Preparation of (4S,10R)-3.234 (LPY29) ......................................................... 332 7.3.117 Preparation of (4R,10R)-3.235 (LPY32) ......................................................... 333 7.3.118 Preparation of (4S,10R)-3.235 (LPY33) ......................................................... 334 7.3.119 Preparation of (4S,10R)-3.219 (LPY34) ......................................................... 335 7.4 Experimental for Chapter 4 ..................................................................................... 336 7.4.1 Preparation of 4.2 (LPY01)................................................................................. 336 7.4.2 Preparation of Compound 4.3 ............................................................................. 337 7.4.3 Preparation of 4.4 (LPY03)................................................................................. 338 7.4.4 Preparation of 4.5 (LPY02)................................................................................. 339 7.4.5 Preparation of 4.6 (LPY16)................................................................................. 340 7.4.6 Preparation of 4.9 (LPY19)................................................................................. 341 7.4.7 Preparation of 4.7 (LPY30)................................................................................. 342 7.4.8 Preparation of 4.8 (LPY31)................................................................................. 343 7.5 Experimental for Chapter 5 ..................................................................................... 344 7.5.1 Preparation of 5.1 (latonduine A) ....................................................................... 344 xvi  7.5.2 Preparation of 5.17 .............................................................................................. 345 Bibliography ...............................................................................................................................347 Appendices ..................................................................................................................................368  xvii  List of Tables  Table 2.1 Results of the primary screen........................................................................................ 33 Table 2.2 C11 inhibition of NS3pro/PepA activity ...................................................................... 37 Table 3.1 New drugs approved for CRPC by the FDA in the last 10 years ................................. 40 Table 3.2 Chlorinated substitution in sintokamides A – E ........................................................... 78 Table 3.3 Possible advantages of 5-protected norvalines over 5-chlorinated norvalines as starting materials to prepare 1,17-dinorsintokamide analogues based on our NCSTD synthesis ........... 102 Table 3.4 Reported trichloromethylation conditions in literature ............................................... 122 Table 3.5 Conditions for trichloromethylation of 3.183 ............................................................. 129 Table 3.6 Preliminary evaluation of the Route Three and the Route Six ................................... 137 Table 5.1 The EC50s of latonduine A against PARPs in CFBE41o− cells ................................. 209  xviii  List of Figures  Figure 1.1 An example of natural product-inspired drug discovery practice: from salicylic acid to aspirin .............................................................................................................................................. 4 Figure 1.2 An example of natural product-inspired drug discovery practice: from cocaine to lidocaine .......................................................................................................................................... 5 Figure 1.3 Three Nobel laureates in the first half of 20th century for their achievements in drug discovery ......................................................................................................................................... 7 Figure 1.4 Birth defects caused by use of thalidomide (1.12) ........................................................ 8 Figure 1.5 The booming of the chemistry of alkaloids from 1950s to 1960s and the structure of taxol (1.13) ...................................................................................................................................... 9 Figure 1.6 Structure of papuamide A (1.14) ................................................................................. 11 Figure 1.7 Primary structure of ziconotide (1.15) ......................................................................... 12 Figure 2.1 Timeline of important advances in the evolution of HCV treatment .......................... 22 Figure 2.2 The TLC of 2.18, 2.20 and 2.14 (DCM/MeOH = 98/2) .............................................. 32 Figure 2.3 Dose-response curves of C11, C12, C21, C22 and C10 .............................................. 36 Figure 3.1 Prostate cancer death and mortality rates in Canada (Adapted with permission of “Mortality, Summary List of Causes”, Statistics Canada, 2009) .................................................. 38 Figure 3.2 The AR gene, protein, and zinc fingers (Reprinted with permission of W.B./SAUNDERS C O. LTD.) ....................................................................................................... 43 Figure 3.3 AR axis and androgen-sensitive transactivation (Reprinted with permission of Nature Publishing Group) ........................................................................................................................ 44 Figure 3.4 Current therapies vs CRPC adaptations affecting the androgen axis .......................... 45 xix  Figure 3.5 Androgen pathways in CRPC ...................................................................................... 47 Figure 3.6 Structures of the FL-AR and AR-V proteins (Reprinted in part with permission of W.B./SAUNDERS C O. LTD.) ....................................................................................................... 49 Figure 3.7 The structures of EPI-001 (3.8), niphatenone A (3.9), and niphatenone B (3.10) ...... 52 Figure 3.8 The structures of sintokamide A – E (3.11 – 3.15) ..................................................... 53 Figure 3.9 Sintokamide A’s inhibitory activity against AR-NTD transactivation in vitro (Reprinted with permission of American Chemical Society) ........................................................ 55 Figure 3.10 (A) Structures of the sintokamides and (B) their in vitro inhibitory activities of R1881 activation of P6.1 luciferase in LNCaP cells..................................................................... 56 Figure 3.11 Structural features of interest in the sintokamide SAR study .................................... 57 Figure 3.12 Treatment of dysidin (3.17) in acidic and basic conditions ....................................... 61 Figure 3.13 Structures of NCSTD (in blue), NCSTD1 (in green) and NCSTD2 (in red) and their 1H NMR spectra comparison ........................................................................................................ 71 Figure 3.14 Bioassay results of NCSTD [(4S,10R)-3.16] on R1881 activation of P6.1luc .......... 73 Figure 3.15 Some examples of highly chlorinated marine natural products ................................ 74 Figure 3.16 Designed structure of LPY00 (3.60), based the structures and activities of NCSTD [(4S,10R)-3.16] and sintokamide E (3.15) .................................................................................... 79 Figure 3.17 Three major structural elements in the simplified 1,17-dinorsintokamide structure . 81 Figure 3.18 Attempts to make chlorinated tetramic acids from 5-chlorinated norvalines............ 95 Figure 3.19 Attempts to make chlorinated active esters from 5-chlorinated norvalines .............. 96 Figure 3.20 Structures of monochlorinated 1-nor-, 17-nor-, and 1,17-dinor-sintokamide analogues..................................................................................................................................... 115 xx  Figure 3.21 Structure comparison of sintokamides E and D, LPY00 (3.60), and LPY04 (3.148)..................................................................................................................................................... 116 Figure 3.22 Bioassay results of LPY00 (3.60) and LPY04 (3.148) on R1881 activation of P6.1luc..................................................................................................................................................... 131 Figure 3.23 Structure comparison of LPY00 (3.60) and sintokamide E (3.15); LPY04 (3.148) and sintokamide D (3.14) .................................................................................................................. 132 Figure 3.24 Synthetic procedure based on Route Two (Part A) ................................................. 133 Figure 3.25 Synthetic procedure based on Route Two (Part B) ................................................. 134 Figure 3.26 Evolution based mechanism of Zakarian’s Ru(II)-catalyzed Cl3C· radical addition to titanium enolate of N-acyl Evans’ chiral auxiliary (Adapted with permission of American Chemical Society) ....................................................................................................................... 140 Figure 3.27 Published syntheses of marine natural products containing chloroleucines by Zakarian and co-workers............................................................................................................. 141 Figure 3.28 Bioassay results of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210] on R1881 activation of P6.1luc ................................................................................................................... 151 Figure 3.29 ORTEP diagram of the Strecker product sulfinimine (S,S)-3.217 .......................... 155 Figure 3.30 Bioassay results of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] on R1881 activation of P6.1luc ................................................................................................................... 159 Figure 3.31 Bioassay results of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231] with LPY11 [(4S,10R)-3.220] on R1881 activation of P6.1luc ................................................................................................................... 164 Figure 3.32 Structures of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231] .......................................................................................... 165 xxi  Figure 3.33 Structures of LPY10 [(4R,10R)-3.220], LPY11 [(4S,10R)-3.220], LPY20 [(4S,10S)-3.220], and LPY21 [(4R,10S)-3.220] .......................................................................................... 166 Figure 3.34 Bioassay results of LPY10 [(4R,10R)-3.220], LPY11 [(4S,10R)-3.220], LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY20 [(4S,10S)-3.220], and LPY21 [(4R,10S)-3.220] with Sint A (3.11) on R1881 activation of P6.1luc ......................................................... 169 Figure 3.35 Structures of LPY22 [(4R,10R)-3.225] and LPY23 [(4S,10R)-3.225], LPY24 [(4R,10R)-3.232], LPY25 [(4S,10R)-3.232], LPY26 [(4R,10R)-3.233], LPY27 [(4S,10R)-3.233], LPY28 [(4R,10R)-3.234], and LPY29 [(4S,10R)-3.234] ............................................................ 171 Figure 3.36 Structures of LPY32 [(4R,10R)-3.235], LPY33 [(4S,10R)-3.235], LPY34 [(4R,10R)-3.219], LPY35 [(4S,10R)-3.219]................................................................................................. 172 Figure 3.37 Bioassay results of different N-acyl 1,17-dinorsintokamide B analogues and key synthetic intermediates on R1881 activation of P6.1luc ............................................................. 173 Figure 4.1 Structures of EPI-001 (3.8) and EPI-002 (4.1) .......................................................... 178 Figure 4.2 A proposed mechanism of action of EPI-001 (3.8) with the AF1 region in the AR NTD ............................................................................................................................................ 183 Figure 4.3 Structures of LPY00 (3.60), LPY04 (3.148) and LPY01 (4.2) and their in vitro activities ...................................................................................................................................... 186 Figure 4.4 Bioassay results of probes LPY02 (4.5) and LPY03 (4.4) on R1881 activation of P6.1luc ........................................................................................................................................ 190 Figure 4.5 Bioassay results of LPY13 [(4S,10R)-3.228] and LPY16 (4.6) with Sint A (3.11) on R1881 activation of P6.1luc ........................................................................................................ 191 Figure 4.6 Bioassay results of LPY30 (4.7) and LPY31 (4.8) with EPI-002 (4.1) and Sint A (3.11) on R1881 activation of P6.1luc ........................................................................................ 192 xxii  Figure 4.7 (A) Western blots using anti Rb/biotin for the cell lysates and the streptavidin pull-down; (B) Western blots using ARN-20 for the cell lysates and the streptavidin pull-down. EPI-053 is a bioactive EPI probe with a terminal alkyne. ................................................................. 193 Figure 4.8 A proposed model incorporating two possible covalent bonds to explain the mechanism of action of LPY10 [(4R,10R)-3.220] ...................................................................... 194 Figure 4.9 LPY10 [(4R,10R)-3.220] may create a covalent bond with the AR via a cyclopropane ring formation/reopening mechanism. ........................................................................................ 195 Figure 5.1. Latonduines (5.1 to 5.6) and structurally related compounds (5.7 to 5.9) isolated from Stylissa ........................................................................................................................................ 200 Figure 5.2. Latonduine A corrects F508del-CFTR trafficking from the ER to cell surface in vitro..................................................................................................................................................... 203 Figure 5.3. Latonduine A potentiates F508del-CFTR in vitro, ex vivo, and in vivo ................... 207 Figure 6.1 The exploration of synthetic approaches to make active 1,17-dinorsintokamide analogues..................................................................................................................................... 215 Figure 6.2 Lead selection based on current SAR understanding of sintokamides’ structural features ........................................................................................................................................ 217 Figure B.1  1H spectrum of 2.18 (C12) recorded in DMSO-d6 at 600 MHz ............................... 372 Figure B.2 1H spectrum of 2.14 (C11) recorded in DMSO-d6 at 300 MHz ............................... 372 Figure B.3 1H spectrum of 2.20 (C22) recorded in DMSO-d6 at 600 MHz ............................... 373 Figure B.4 1H spectrum of 2.15 (C21) recorded in DMSO-d6 at 300 MHz ............................... 373 Figure B.5 1H and 13C NMR spectra of 3.32 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 374 xxiii  Figure B.6 1H and 13C NMR spectra of 3.45 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 375 Figure B.7 1H and 13C NMR spectra of 3.125 recorded in CDCl3 at 400 MHz and 100 MHz, respectively ................................................................................................................................. 376 Figure B.8 1H and 13C NMR spectra of 3.126 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 377 Figure B.9 1H and 13C NMR spectra of 3.144 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 378 Figure B.10 1H and 13C NMR spectra of 3.145 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 379 Figure B.11 1H and 13C NMR spectra of 3.146 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 380 Figure B.12 1H and 13C NMR spectra of 3.60 (LPY00) recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ...................................................................................................................... 381 Figure B.13 1H and 13C NMR spectra of 3.127 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 382 Figure B.14 1H and 13C NMR spectra of 3.128 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 383 Figure B.15 1H and 13C NMR spectra of 3.147 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 384 Figure B.16 1H and 13C NMR spectra of 3.148 (LPY04) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 385 xxiv  Figure B.17 1H and 13C NMR spectra of 3.149 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ................................................................................................................................ 386 Figure B.18 1H and 13C NMR spectra of 3.150 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ................................................................................................................................ 387 Figure B.19 1H and 13C NMR spectra of 3.168 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 388 Figure B.20 1H and 13C NMR spectra of 3.169 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 389 Figure B.21 1H and 13C NMR spectra of 3.170 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 390 Figure B.22 1H and 13C NMR spectra of 3.151 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 391 Figure B.23 1H and 13C NMR spectra of 3.180 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 392 Figure B.24 1H and 13C NMR spectra of 3.181 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ................................................................................................................................ 393 Figure B.25 1H and 13C NMR spectra of 3.182 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 394 Figure B.26 1H and 13C NMR spectra of 3.183 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 395 Figure B.27 1H and 13C NMR spectra of 3.184 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ................................................................................................................................ 396 xxv  Figure B.28 1H and 13C NMR spectra of 3.185 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 397 Figure B.29 1H and 13C NMR spectra of 3.186 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 398 Figure B.30 1H and 13C NMR spectra of 3.205 recorded in CDCl3 at 600 MHz and 150 MHz, respectively. ................................................................................................................................ 399 Figure B.31 1H and 13C NMR spectra of (4R,10R)-3.16 (NCSTD1) recorded in CDCl3 at 600 MHz and 150 MHz, respectively. ............................................................................................... 400 Figure B.32 1H and 13C NMR spectra of (4S,10R)-3.16 (NCSTD2) recorded in CDCl3 at 600 MHz and 150 MHz, respectively. ............................................................................................... 401 Figure B.33 1H and 13C NMR spectra of (S)-3.208 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 402 Figure B.34 1H and 13C NMR spectra of (R)-3.221 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 403 Figure B.35 1H and 13C NMR spectra of (R)-3.73 recorded in CDCl3 at 300 MHz and 75 MHz, respectively. ................................................................................................................................ 404 Figure B.36 1H and 13C NMR spectra of (4S,10R)-3.209 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 405 Figure B.37 1H and 13C NMR spectra of (4R,10R)-3.209 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 406 Figure B.38 1H and 13C NMR spectra of (4S,10R)-3.212 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 407 xxvi  Figure B.39 1H and 13C NMR spectra of (4R,10R)-3.210 (LPY08) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 408 Figure B.40 1H and 13C NMR spectra of (4S,10R)-3.210 (LPY09) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 409 Figure B.41 1H and 13C NMR spectra of (S)-3.218 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 410 Figure B.42 1H and 13C NMR spectra of 3.223 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ................................................................................................................................ 411 Figure B.43 1H and 13C NMR spectra of 7.1 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 412 Figure B.44 1H and 13C NMR spectra of (S)-3.79 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 413 Figure B.45 1H and 13C NMR spectra of (S)-3.218 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. ................................................................................................................................ 414 Figure B.46 1H and 13C NMR spectra of (4S,10R)-3.219 (LPY35) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 415 Figure B.47 1H and 13C NMR spectra of (4R,10R)-3.220 (LPY10) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 416 Figure B.48 1H and 13C NMR spectra of (4S,10R)-3.220 (LPY11) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 417 Figure B.49 1H and 13C NMR spectra of (4S,10R)-3.226 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 418 xxvii  Figure B.50 1H and 13C NMR spectra of (4R,10R)-3.228 (LPY12) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 419 Figure B.51 1H and 13C NMR spectra of (4S,10R)-3.228 (LPY13) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 420 Figure B.52 1H and 13C NMR spectra of (4S,10R)-3.229 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 421 Figure B.53 1H and 13C NMR spectra of (4R,10R)-3.231 (LPY17) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 422 Figure B.54 1H and 13C NMR spectra of (4S,10R)-3.231 (LPY18) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 423 Figure B.55 1H and 13C NMR spectra of (4S,10S)-3.129 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 424 Figure B.56 1H and 13C NMR spectra of (4S,10S)-3.220 (LPY20) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 425 Figure B.57 1H and 13C NMR spectra of (4R,10S)-3.220 (LPY21) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 426 Figure B.58 1H and 13C NMR spectra of (4R,10R)-3.225 (LPY22) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 427 Figure B.59 1H and 13C NMR spectra of (4S,10R)-3.225 (LPY23) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 428 Figure B.60 1H and 13C NMR spectra of (4R,10R)-3.232 (LPY24) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 429 xxviii  Figure B.61 1H and 13C NMR spectra of (4S,10R)-3.232 (LPY25) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 430 Figure B.62 1H and 13C NMR spectra of (4R,10R)-3.233 (LPY26) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 431 Figure B.63 1H and 13C NMR spectra of (4S,10R)-3.233 (LPY27) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 432 Figure B.64 1H and 13C NMR spectra of (4R,10R)-3.234 (LPY28) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 433 Figure B.65 1H and 13C NMR spectra of (4S,10R)-3.234 (LPY29) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ........................................................................................................ 434 Figure B.66 1H and 13C NMR spectra of (4R,10R)-3.235 (LPY32) recorded in methanol-d4 at 600 MHz and 125 MHz, respectively. ............................................................................................... 435 Figure B.67 1H and 13C NMR spectra of (4S,10R)-3.235 (LPY33) recorded in methanol-d4 at 600 MHz and 125 MHz, respectively. ............................................................................................... 436 Figure B.68 1H and 13C NMR spectra of (4R,10R)-3.129 (LPY34) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ............................................................................................... 437 Figure B.69 1H and 13C NMR spectra of 4.4 (LPY03) recorded in C6D6 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 438 Figure B.70 1H and 13C NMR spectra of 4.5 (LPY02) recorded in C6D6 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 439 Figure B.71 1H and 13C NMR spectra of 4.6 (LPY16) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 440 xxix  Figure B.72 1H and 13C NMR spectra of 4.7 (LPY30) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 441 Figure B.73 1H and 13C NMR spectra of 4.8 (LPY31) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 442 Figure B.74 1H and 13C NMR spectra of 4.9 (LPY19) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. ...................................................................................................................... 443 Figure B.75 1H spectrum of 5.17 recorded in DMSO-d6 at 400 MHz ........................................ 444 xxx  List of Schemes  Scheme 1.1 Flowchart of a typical reverse pharmacology-based drug discovery ........................ 12 Scheme 1.2 Flowchart of current forward pharmacology-based drug discovery ......................... 14 Scheme 1.3 A workflow of a typical streptavidin/biotin-based pull-down assay......................... 16 Scheme 1.4 A workflow of a typical Click chemistry-based pull-down assay ............................ 17 Scheme 2.1 The screen design based on the enzymatic property of NS3pro/Pep4A to a BS-IQFS....................................................................................................................................................... 28 Scheme 2.2 The workflow of the target-based screen and the kinetic assay ................................ 29 Scheme 2.3 Syntheses of C11 (2.14) and C21 (2.15) ................................................................... 31 Scheme 3.1 The general synthetic route covering all sintokamide analogues and the structure of LPY26 [(4R,10R)-3.233] identified as the analogue with the best biological activity among the 29 synthetic compounds submitted for biological evaluation....................................................... 58 Scheme 3.2 Structures of a generic sintokamide, NCSTD (3.16, Non-Chlorinated SinTokamiDe), and sintokamide E (3.15). (R1: chloromethyl or methyl; R2: chloromethyl) ................................ 60 Scheme 3.3 Total synthesis of (±)-dysidin (3.17) published by Williard and Laszlo in 1984 ..... 63 Scheme 3.4 Retrosynthetic analysis for NCSTD [(4S,10R)-3.16] ................................................ 64 Scheme 3.5 Synthesis of compound 3.37 ..................................................................................... 65 Scheme 3.6 C-methylation of -keto ester 3.37 with KH and MeI .............................................. 66 Scheme 3.7 O-methylation of -keto ester 3.37 based on Williard and Laszlo’s method ........... 66 Scheme 3.8 Synthesis of compound 3.32 ..................................................................................... 67 Scheme 3.9 Convergent assembly of NCSTD [(4S,10R)-3.16] with Grignard reagent based on Williard and Laszlo’s method ....................................................................................................... 68 xxxi  Scheme 3.10 Synthesis of compound 3.45 ................................................................................... 68 Scheme 3.11 Convergent assembly with n-BuLi and conclusion of the synthesis of NCSTD [(4S,10R)-3.16] ............................................................................................................................. 69 Scheme 3.12 Proposed mechanism for C-4 epimerisation in the presence of a strong base ........ 70 Scheme 3.13 A reasonable biosynthetic pathway for mono-halogenation ................................... 75 Scheme 3.14 A proposed biosynthetic pathway for poly-halogenation ....................................... 75 Scheme 3.15 What are the required chlorination patterns in the leucine moieties for the sintokamides in vitro activities?.................................................................................................... 76 Scheme 3.16 Structure simplification from naturally occurring sintokamides 3.58 to 1,17-dinorsintokamide analogues 3.59.................................................................................................. 77 Scheme 3.17 Retrosynthetic plan for the preparation of chlorinated 1,17-dinorsintokamide analogues 3.59 from 5-chlorinated L-norvalines (3.62) and 5-chlorinated D-norvalines 3.64 based on the synthesis of NCSTD [(4S,10R)-3.16] ................................................................................ 84 Scheme 3.18 Synthesis of N-Boc-5-chloro-D-norvaline [(R)-3.69] .............................................. 85 Scheme 3.19 Synthesis of N-Boc-5-chloro-L-norvaline [(S)-3.69] .............................................. 86 Scheme 3.20 Synthesis of of N-Boc-5,5-dichloro-D-norvaline [(R)-3.73] ................................... 87 Scheme 3.21 Synthesis of N-Boc-5,5-dichloro-L-norvaline [(S)-3.73] ........................................ 87 Scheme 3.22 A recent success in stereoselective trichloromethylation ........................................ 88 Scheme 3.23 Synthesis of N-Boc-5,5,5-trichloronorvaline racemates (3.79) ............................... 89 Scheme 3.24 The 1,4-addition condition worked for methyl acrylate (3.74) could not be used in the case of methyl crotonate (3.80). .............................................................................................. 90 Scheme 3.25 The reported 1,4-addition between methyl crotonate (3.80) and chloroform under electroorganic conditions .............................................................................................................. 90 xxxii  Scheme 3.26 An ideal synthetic route to chlorinated tetramate acids should be milder in conditions and shorter in steps based on the NCSTD synthesis. .................................................. 92 Scheme 3.27 Preparation of N-Boc tetramic acid 3.86 in Jouin’s synthesis of N-protected statine 3.88................................................................................................................................................ 93 Scheme 3.28 Synthesis of methyl tetramate 3.32 via homologation with Meldrum’s acid under Ma and Kraus’ conditions ............................................................................................................. 94 Scheme 3.29 Comparison of (A) the successful synthesis of NCSTD [(4S,10R)-3.16] and (B) the failed synthesis of chlorinated 1,17-dinorsintokamide analogues based on Route One ............... 98 Scheme 3.30 Total synthesis of dysithiazolamide (3.49) (Part A) ............................................... 99 Scheme 3.31 Total synthesis of dysithiazolamide (3.49) (Part B) .............................................. 100 Scheme 3.32 Total synthesis of dysithiazolamide (3.49) (Part C) .............................................. 100 Scheme 3.33 Total synthesis of dysithiazolamide (3.49) (Part D) ............................................. 101 Scheme 3.34 Total synthesis of sintokamide C (3.13) (Part A) .................................................. 103 Scheme 3.35 Total synthesis of sintokamide C (3.13) (Part B) .................................................. 103 Scheme 3.36 Total synthesis of sintokamide C (3.13) (Part C) .................................................. 105 Scheme 3.37 Synthesis of bis-monochlorinated sintokamide 3.123 from the intermediate diol 3.121............................................................................................................................................ 106 Scheme 3.38 Retrosynthetic plan for chlorinated 1,17-dinorsintokamide analogues from 5-silyloxy-norvalines originating from D- and L-glutamic acids ................................................... 108 Scheme 3.39 Synthesis of N-Boc-5-silyloxyl-L-norvaline (S)-3.134 from L-glutamic acid [(S)-3.99] ............................................................................................................................................ 110 Scheme 3.40 Attempt to make methyl tetramate from N-Boc-5-silyloxyl-L-norvaline (S)-3.124..................................................................................................................................................... 111 xxxiii  Scheme 3.41 Synthesis of methyl tetramate 3.125 ..................................................................... 112 Scheme 3.42 Synthesis of N-Boc-5-silyloxyl-D-norvaline (R)-3.124 from D-glutamic acid [(R)-3.99] ............................................................................................................................................ 113 Scheme 3.43 Synthesis of active ester 3.126 from N-Boc-5-silyloxyl-D-norvaline (R)-3.124 ... 114 Scheme 3.44 Attempt to make active ester without benzyl protection of the amino acid .......... 115 Scheme 3.45 Synthesis of LPY00 (3.60), the monochlorinated 17-norsintokamide analogue .. 117 Scheme 3.46 Synthesis of LPY04 (3.148), the bis-monochlorinated 1,17-dinorsintokamide analogue ...................................................................................................................................... 119 Scheme 3.47 Attempt to make 1,17-dinorsintokamide C from diol 3.128 ................................. 120 Scheme 3.48 Synthetic plan to 1,17-dinorsintokamide B analogue based on trichloromethylation of the homologous dihalide 3.152 ............................................................................................... 121 Scheme 3.49 Retrosynthetic plan to bis-trichlorinated 1,17-dinorsintokamide B analogue 3.153 based on Route Two.................................................................................................................... 124 Scheme 3.50 Synthesis of tetramic acid 3.168 from L-aspartic acid [(S)-3.166] ........................ 125 Scheme 3.51 Synthesis of active ester 3.169 from D-aspartic acid [(R)-3.166].......................... 126 Scheme 3.52 Attempt to make sintokamide B analogue 3.153 from diol 3.151 ........................ 128 Scheme 3.53 Synthesis of iodide 3.183 for screen of effective trichloromethylation conditions..................................................................................................................................................... 129 Scheme 3.54 Trichloromethylation of 3.185 .............................................................................. 130 Scheme 3.55 Schemes of Routes Two, Four, and Five .............................................................. 136 Scheme 3.56 Comparison of Routes Three and Six ................................................................... 136 Scheme 3.57 Zakarian’s diastereoselective synthesis of trichloroleucine based on an efficient catalytic radical trichloromethylation ......................................................................................... 139 xxxiv  Scheme 3.58 Zakarian’s total synthesis of sintokamide A ......................................................... 142 Scheme 3.59 Synthesis of non-chlorinated sintokamide analogues NCSTD1 [(4R,10R)-3.16] and NCSTD2 [(4S,10R)-3.16] based on Route Three ....................................................................... 145 Scheme 3.60 Retrosynthetic analysis for LPY09 [(4S,10R)-3.210] in Route Three .................. 148 Scheme 3.61 Synthesis of 5,5-dichloro-L-norvaline methyl ester [(S)-3.208]............................ 149 Scheme 3.62 Synthesis of N-Boc-5,5-dichloro-D-norvaline (R)-3.73 ........................................ 149 Scheme 3.63 Synthesis of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210], the 1,17-dinorsintokamide C analogues .................................................................................................... 150 Scheme 3.64 The 1,17-dinorsintokamide B LPY11 [(4S,10R)-3.220] is the next synthetic target since LPY09 [(4S,10R)-3.210] is inactive. ................................................................................. 153 Scheme 3.65 Retrosynthetic analysis for LPY11 [(4S,10R)-3.220] in Route Three .................. 154 Scheme 3.66 Synthesis of 5,5,5-trichloro-L-norvaline methyl ester [(S)-3.218] ........................ 155 Scheme 3.67 Synthesis of N-propionyl-5,5,5-trichloro-D-norvaline (3.223) ............................. 156 Scheme 3.68 Attempt to make dipeptide 3.224 by coupling compound (S)-3.218 and compound 3.223............................................................................................................................................ 156 Scheme 3.69 Model reaction of making dipeptide by coupling compound (S)-3.218 and Boc-D-leucine [(R)-3.43] ........................................................................................................................ 157 Scheme 3.70 Synthesis of N-Boc-5,5,5-trichloro-D-norvaline [(R)-3.79] .................................. 157 Scheme 3.71 Synthesis of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], the 1,17-dinorsintokamide B analogues .................................................................................................... 158 Scheme 3.72 New synthetic targets LPY13 [(4S,10R)-3.228] and LPY18 [(4S,10R)-3.231] after discovering the in vitro activities of LPY10/11 .......................................................................... 161 xxxv  Scheme 3.73 Synthesis of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], the 17-norsintokamide E analogues ....................................................................................................... 162 Scheme 3.74 Synthesis of LPY17 [(4R,10R)-3.231] and LPY18 [(4S,10R)-3.231], the regioisomeric monotrichlorinated 1-norsintokamide analogues ................................................. 163 Scheme 3.75 Synthesis of N-Boc-5,5,5-trichloro-L-norvaline [(S)-3.79] ................................... 166 Scheme 3.76 Synthesis of 5,5,5-trichloronorvaline methyl esters (3.218) ................................. 167 Scheme 3.77 Synthesis of LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220], two stereoisomers of 1,17-dinorsintokamide B analogues ................................................................ 168 Scheme 4.1 Syntheses of LPY03 (4.4) and LPY02 (4.5) ........................................................... 188 Scheme 4.2 A generic scheme of synthesizing probes from free amine precursors ................... 189 Scheme 5.1. Synthesis of latonduine A (5.1) from 5.10 ............................................................. 202 Scheme 5.2. Synthesis of N-biotinylated-latonduine A (5.17) ................................................... 209  xxxvi  List of Symbols and Abbreviations  # - number $ - dollar(s) (±) - racemic % - per cent   chemical shift 1D- - one dimensional- 2D- - two dimensional- 3HSD - 3-alpha-hydroxysteroid reductase 3CLpro - (SARS coronavirus) 3C-like cysteine protease 3HA - tandem hemagglutinin-epitope tags em - emission wavelength ex - excitation wavelength A.D. - Anno Domini ABC - ATP-binding cassette Abz - aminobenzoyl Ach - acetylcholine AD - antidiuretic (hormone) ADT - androgen-deprivation therapy AF - activation function 1 AIBN - azobisisobutyronitrile ALT - alanine transaminase AMP - adenosine monophosphate AR - androgen receptor ARA70 - androgen receptor-associated protein 70 ARE - androgen response element ARR3 - arrestin 3 AR-V - androgen receptor splice variants xxxvii  ATP - adenosine triphosphate B.C. - Before Christ BAIB - bis(acetoxy)iodobenzene BC - British Columbia BHK - baby hamster kidney Boc - t-butoxycarbonyl BrdU - bromodeoxyuridine BS-IQFS - blue-shifted internally quenched fluorogenic substrate C18 - linear octadecane groups CADD - computer-aided drug design Cbz - carboxybenzyl cDNA - complementary deoxyribonucleic acid CF - cystic fibrosis CFTR - cystic fibrosis transmembrane conductance regulator CL - cytoplasmic loop CRPC - castration-resistant cystic fibrosis CTD - C-terminus domain D- - dexter DAA - direct-acting antiviral agents DBD - DNA binding domain DCC - N,N'-dicyclohexylcarbodiimide DCM - dichloromethane DHEA - dehydroepiandrosterone DHT - dihydrotestosterone DIBAL-H - diisobutylaluminium hydride DIEA - N,N-Diisopropylethylamine DIPEA - N,N-Diisopropylethylamine DMAP - 4-dimethylaminopyridine DMF - dimethylformamide DMP - Dess – Martin periodinane xxxviii  DMS - dimethyl sulfide DMSO - dimethyl sulfoxide DNA - deoxyribonucleic acid Dr. - Doctor E1 - envelope protein 1 EC50 - half maximal effective concentration EDCI - 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EMA - the European Medicines Agency ER - endoplasmic reticulum ERAD - endoplasmic reticulum-associated (protein) degradation EtOAc - ethyl acetate FDA - the Food and Drug Administration FeHeme-BrPO - FeHeme-bromoperoxidase FL-AR - full-length androgen receptor FRET - fluorescence resonance energy transfer g - gram(s) GmbH - Gesellschaft mit beschränkter Haftung GOF - gain of function GR - glucocorticoid receptor h - hour(s) HCV - hepatitis C virus HEK293 - human embryonic kidney 293 HIV - human immunodeficiency virus HMBC - heteronuclear multiple-bond correlation spectroscopy HMPA - hexamethylphosphoramide HOAt - 1-hydroxy-7-azabenzotriazole HOBt - hydroxybenzotriazole HPLC - high-performance liquid chromatography HRESIMS - high resolution electrospray ionisation mass spectroscopy HSP - heat shock proteins xxxix  HTS - high-throughput screening Hz - hertz IC50 - half maximal inhibitory concentration IDP - intrinsically disordered protein IFN - interferon  IGF-1 - insulin-like growth factor 1 IL-6 - interleukin-6 IPCF - isopropenyl chloroformate Isc - short circuit current J - coupling constant K - thousand kb - kilobase Kd - dissociation constant kDa - kilodalton Ki - inhibition constant Km - Michaelis constant L- - laevus LBD - ligand binding domain Leu - leucine LH-RH - luteinizing-hormone-releasing hormone LiHMDS - lithium bis(trimethylsilyl)amide LOF - loss of function M - mole mCRPC - metastatic castration-resistant prostate cancer mg - milligram(s) mg/mL - milligram(s) per milliliter min - minute(s) mL - milliliter(s) mL/min - milliliter(s) per minute M - micromole(s) xl  mmol/L - millimole(s) per liter mRNA - messenger ribonucleic acid MS - mass spectroscopy MSD - membrane spanning domain MW - molecular weight NaHMDS - sodium hexamethyldisilazide NBD - nucleotide binding domain n-BuLi - n-butyllithium NCSTD - non-chlorinated sintokamide NF-B - nuclear factor kappa-B ng - nanogram(s) nm - nanometre(s) nM - nanomole(s) NMR - nuclear magnetic resonance NOE - nuclear overhauser effect NS3 - non-structural (protein) 3 NS3pro - non-structural (protein) 3 protease NTD - N-terminus domain NTPase - nucleoside triphosphatase Nu - nucleophile OAT - organic acid transporters ORTEP - oak ridge thermal ellipsoid plot PARP - poly (adenosine diphosphate -ribose) polymerase PCR - polymerase chain reaction PDE-5 - phosphodiesterase type 5 inhibitor PEG-IFN - pegylated interferon Pep4A - peptide 4A PfpOH - pentafluorophenol PHEX - phosphate regulating endopeptidase homologue pKa - -lg (acid dissociation constant) xli  PM - plasma membrane pM - picomole(s) PR - progesterone receptor PSA - prostate-specific Antigen PTSA - p-toluenesulfonic acid PyBRoP - bromo-tris-pyrrolidinophosphonium hexafluorophosphate QC - quality control R - rectus R&D - research and development RAV - resistance-associated variant RBV - ribavirin RDRP - ribonucleic acid-dependent ribonucleic acid polymerase RFU/s - relative fluorescence units per second RIBA - recombinant immunoblot assay RLU - relative luciferase units RNA - ribonucleic acid RT-PCR - real-time polymerase chain reaction s - second(s) S - sinister SAR - structure-activity relationship SARS - severe acute respiratory syndrome SCUBA - self-contained underwater breathing apparatus SDS-PAGE - sodium dodecyl sulfate-polyacrylamide gel electrophoresis SHBG - sex hormone-binding globulin siRNA - small interfering ribonucleic acid SLCO - solute linked organic (anion carrier) SRD5A - steroid 5 alpha reductase SUMO - small ubiquitin-like modifier SVR - sustained virological response TBAF - tetrabutylammonium fluoride hydrate xlii  TBDPSCl - tert-butyl(chloro)diphenylsilane TEMPO - 2,2,6,6-Tetramethylpiperidine 1-oxyl TEOF - triethyl orthoformate Tf2O - trifluoromethanesulfonic anhydride TFA - trifluoroacetic acid TFAA - trifluoroacetic anhydride TFG - transforming growth factor beta THF - tetrahydrofuran TLC - thin layer chromatography TMS - trimethylsilyl TNF- - tumor necrosis factor alpha tR - retention time UBC - the University of British Columbia UK - the United Kindom UPS - ubiquitin-proteasome system USA - the United States of America V-BrPO - vanadium bromoperoxidase Vmax - maximum reaction rate WGA - wheat germ agglutinin WT - wild type Xq12 - X chromosome 12 YFP - yellow fluorescent protein xliii  Acknowledgements  First of all, I would like to express my sincere gratitude to my supervisor Dr. Raymond Andersen for giving me this opportunity to explore the wonders of the marine natural products and beyond. Thank you Raymond for the encouragement and patience during these years. What I learned from you both in science and personality will definitely be the characteristic parts in my career and life. I would especially thank all the previous and current labmates in the Andersen Lab for your generous help whenever I asked.  Moreover, I would also like to thank my wife, my mom-in-law for taking care of the little Ryan during the writing of this thesis. Your quiet support for my graduation is tremendous and makes me appreciate about what a family I have. I promise that I will spend more time with you and share more responsibility as a dad at home and at work.  At last, I would like to thank my parents. Dear mom and dad, it is still hard at this moment to describe to you what my future looks exactly like but I am trying to be the forever boy you have been proud of ever since we had each other. I wish you happy and healthy.  My life is good because of you all. Thank you! xliv  Dedication         To whom loves and helps Ryan Felix Yan and his siblings  1  Chapter 1: A Brief Retrospective of Drug Discovery Practices  1.1 The early days: discovery of medicinal natural products  The endless pursuit of better welfare and longer life expectancy has made drug discovery a constant activity throughout human history. Besides food and raw materials they harvested from nature, early humankind also tried and recognised nature’s other creations that have curative effects for various diseases. The findings of these natural medicinal sources, in the slang of today’s drug discovery, would be serendipity (pleasant surprise) based on phenotypic correction, which is the observed evidence of improvement. For example, the extract of willow bark (Cortex salicis) has been known to relieve pain and fever since the time of Hippocrates (ca. 400 B.C.). In South America, chewing coca leaf (Folia cocae) has been a tradition for thousands of years. It is believed that the leaves are rich in nutrients and have an analgesic effect. Ancient pharmacopoeias, like the Shen-Nong's Materia Medica (ca. 100 B.C.) and the Galenical Pharmacy (ca. 170 A.D.) represent the repositories of accumulated knowledge about the medicinal usage of natural materials obtained in the early days of human civilization.3       2  1.2 The early 19th century: advances in chemistry of natural products and organic chemistry  With the development of chemistry, biology, and physiology, the isolation of active ingredients from medicinal natural sources has flourished since the early 19th century, alongside emerging pharmacological studies. In 1805, a German pharmacist named Friedrich Sertürner isolated morphine (1.1) from opium (lachryma papaveris). Later on, he tested crystalline morphine on both animals and humans (even on himself!) and proved that the alkaloid was the active ingredient in opium. Morphine was the first-ever alkaloid isolated from a medicinal plant and its discovery initiated research on the chemistry of natural products.4    Morphine was found in Papaver somniferum  Morphine was first isolated in 1805.  Morphine was commercially available in 1827.  Structure elucidation of morphine was done in 1927.  Total synthesis of morphine was finished in 1952.  Morphine is an agonist to the opiate receptor. 1.1, morphine  Mechanism of action studies on bioactive natural products have always been important in pharmacological research. In 1809, François Magendie showed that the spinal cord was the action site of strychnine-containing nux-vomica that caused convulsions in dogs and, in 1842, Claude Bernard discovered that the arrow poison curare could interrupt muscle stimulation by blocking nerve impulses at neuromuscular junctions in frogs.5, 6  3     Strychnine was found in Strychnos nux-vomica.  Strychnine was first isolated in 1818.  The structure was confirmed in 1946.  The total synthesis of strychnine was accomplished by Robert Woodward in 1954.  Strychnine is an antagonist to glycine and acetylcholine (ACh) receptors.  Tubocurarine was found in Chondrodendron tomentosum, the chief constituent of curare.  Tubocurarine was first isolated in 1897.  Tubocurarine in crystalline form was first obtained in 1935.  Tubocurarine is a nicotinic receptor antagonist.  Mechanism of action studies on bioactive natural products have always been important in pharmacological research. In 1809, François Magendie showed that the spinal cord was the action site of strychnine-containing nux-vomica that caused convulsions in dogs and, in 1842, Claude Bernard discovered that the arrow poison curare could interrupt muscle stimulation by blocking nerve impulses at neuromuscular junctions in frogs.5, 6  In parallel, with the advance in organic chemistry, scientists began to find bioactive molecules completely synthesized in chemistry laboratories. In 1540, a German botanist, Valerius Cordus, prepared diethyl ether by distilling ethanol with sulfuric acid.7 Three hundred years later in 1842, ether anesthesia was introduced in surgery by an American surgeon and pharmacist Crawford Long.8 A German chemist, Justus von Liebig, first synthesized chloral hydrate in 1832 by treatment of ethanol with chlorine gas in acidic solution. The sedative effect of chloral hydrate was discovered in 1869.9  4  1.3 The late 19th century: the emergence of medicinal chemistry  The prototype of medicinal chemistry emerged in the late 1800s, when chemists began to use their knowledge in organic chemistry to make better medicinal compounds based on the existing structures of bioactive natural products. Salicylic acid (1.4), the active ingredient in willow bark, was used as an antipyretic analgesic in a clinic in 1875. Acetylsalicylic acid, also known as aspirin (1.5), which was synthesized in a lab in 1853 and marketed in 1899, was shown to have stronger activity and fewer side effects than other salicylates (Figure 1.1).10   Figure 1.1 An example of natural product-inspired drug discovery practice: from salicylic acid to aspirin  The development of local anaesthetics was another successful example from the early days of medicinal chemistry (Figure 1.2). Cocaine (1.6), which was isolated from coca leaves in 1855, was the first used local anaesthetic. However, cocaine’s usage as a local anaesthetic was later restricted due to its strong toxicity. Based on its structure, a less toxic ester named procaine (1.7) was introduced in 1904, and lidocaine (1.8), which replaced ester by amide to achieve longer effect, was synthesized in 1943.10, 11 Today, more than 40 % of drugs used in clinics are natural products, natural product derivatives, or synthetic drugs inspired by natural products.12  5   Figure 1.2 An example of natural product-inspired drug discovery practice: from cocaine to lidocaine                6  1.3.1 The first half of the 20th century: the golden era of drug discovery  The first half of the 20th century was a golden era of new drug discovery and a start of modern medicinal chemistry. With the development of microbiology, the discovery of penicillin (1.9, benzylpenicillin) in 1928 by Sir Alexander Fleming, who was honoured with the Nobel Prize in physiology or medicine in 1945, marked the beginning of a new field of natural products research. Since then, antibiotics have become the principal treatment for bacterial infections, and discovering new antibiotics from microorganisms has been the main task of medicinal microbiologists to fight against the drug resistance developed during antibiotics’ usage in clinics.13 In medicinal chemistry, Paul Ehrlich, the Nobel laureate in physiology or medicine in 1908, proposed the concept of searching for “magic bullets”, referring to molecule-targeting agents, by screening synthetic organic compounds for chemotherapy (the use of chemicals to treat diseases). In 1909, Sahachiro Hata and Paul Ehrlich discovered that arsphenamine (1.10, also called Compound 606), the sixth compound in their sixth collection of synthetic products in activity screening, was the first effective anti-syphilitic treatment.14 Based on a similar philosophy and screening results of thousands of synthetic dyes in mice, Gerhard Domagk discovered an antibacterial prontosil (1.11). Domagk won the Nobel Prize in physiology or medicine in 1939.15 Ehrlich’s theory and its applications, which involved the optimization of a drug lead compound through systematic synthesis of analogues and screening their biological activities, have become the convention for nearly all modern pharmaceutical research.    7     Sir Alexander Fleming shared the Nobel Prize in Medicine in 1945 "for the discovery of penicillin and its curative effect in various infectious diseases"13. Paul Ehrlich shared the Nobel Prize in Medicine in 1908 "in recognition of their work on immunity".14 Gerhard Domagk won the Nobel Prize in Medicine in 1939 "for the discovery of the antibacterial effects of prontosil".15     Figure 1.3 Three Nobel laureates in the first half of 20th century for their achievements in drug discovery          8  1.3.2 The second half of the 20th century: balancing the safety and efficacy in drug discovery  Similar to other disciplines in science and technology, the second half of the 20th century witnessed a dramatic evolution in drug discovery. A longer and stricter regulatory process for preclinical development and clinical trials was imposed on drug discovery practice. Only two years after it got patented, thalidomide (1.12) was marketed over-the-counter in West Germany in 1957 as a “wonder drug” that alleviated morning sickness in pregnant women. Unfortunately, more than 10,000 cases worldwide of severe birth defects were traced back to thalidomide usage (Figure 1.4).16, 17 Such disasters in the history of drug discovery prompted drug administrations around the world to employ tougher policies for the risk and safety assessment of new drugs. Afterward, a typical drug discovery and development cycle took more than one decade.    Figure 1.4 Birth defects caused by use of thalidomide (1.12)  9    Figure 1.5 The booming of the chemistry of alkaloids from 1950s to 1960s and the structure of taxol (1.13)  Advances in chromatographic techniques, together with the popularization of modern spectroscopic methods, revolutionized the efficiency of isolation and the structure elucidation of natural products. For alkaloids alone, the number of new compounds reported in the 1950s was 1,107, exceeding the total number of 950 that was accumulated during the previous century. Moreover, the data even tripled to 3,443 in the 1960s.12 There were two main reasons to explain such progress. First, new isolation principles, such as molecular sieves and reversed phase chromatography, based on a molecule’s intrinsic properties, provided more separation methods for natural product chemists. Second, the mechanization and systemization of traditional isolation maneuvers, for example, the emergence of high-performance liquid chromatography (HPLC), considerably improved the theoretical plate number (a parameter evaluating the separation efficiency), which made the purification of natural products on a microgram scale feasible. On the other hand, in the identification process, it took chemists more than 100 years since it was first isolated to get the correct structure of morphine based on information from chemical degradation studies. Today, thanks to advances in instrumental analysis, especially the 9501107344305001000150020002500300035004000before 1950 1950s 1960sTHE NUMBER OF ALKALOIDS10  ever-improving nuclear magnetic resonance (NMR) spectrometers and mass spectrometers (MS), the routine data collection process for structure elucidation of a natural product sample on 1 mg or an even lower quantity could be completed in hours. The discovery of the tubulin-targeting anticancer drug taxol (1.13), which was initially extracted from the bark of pacific yew (Taxus brevifolia) with a yield of 0.00083 %, represented one of the biggest success stories in the recent development of medicinal natural products into drugs.12  1.3.3 Since the 1970s: Quest for drugs from the sea  Covering 72 % of this planet, the oceans accommodate 50 – 80 % of all life forms on earth, but the marine environment was almost untouched in natural product-based drug discovery before the 1970s. Once they became familiar with skills like snorkelling and scuba diving, chemists in academia, like modern day Christopher Columbuses, began exploring the unknown by collecting interesting marine samples in coastal habitats worldwide, where most macroscopic sea life was found.18, 19 Due to the high biodiversity in the marine environment, bioactive structures with new skeletons were discovered, most of which were unprecedented in natural products from terrestrial sources. For example, the depsipeptide papuamide A (1.14) shown in Figure 1.6, which was isolated from the sponges Theonella collected in Papua New Guinea, exhibited in vitro inhibitory activity against the HIV-1RF infected human T-lymphoblastoid cells with an EC50 of 3.4 ng/mL.20 However, as illustrated in the rest of this thesis, not all bioactive marine natural products have extremely complex structures.  11   Figure 1.6 Structure of papuamide A (1.14)  An exemplary success in marine natural products drug discovery was inspired by the conotoxins secreted by poisonous cone snails.21-27 Conotoxins are neurotoxins, and they are polypeptides featuring disulfide bridges. In the field of neuroscience, the discovery of conotoxins is regarded as the most important discovery in natural products research, because conotoxins are very specific ion channel modulators, which are indispensable tools for routine neurofunctional studies.21, 28 -Conotoxin MVIIA (1.15, ziconotide, PrialtTM), discovered from the venom of the magician's cone (Conus magus), was approved by the FDA in December 2004 for intrathecal treatment of chronic and intractable pain (Figure 1.7).29-31 In clinical anesthesia, ziconotide is 1,000 times more potent than morphine and does not cause morphine-like tolerance because it selectively blocks specific calcium channels on the synapse rather than opiate receptors. Ziconotide is marketed as the primary alternative to morphine (PrialtTM).  12   Figure 1.7 Primary structure of ziconotide (1.15)  1.4 Modern drug discovery: reverse pharmacology and forward pharmacology  Since the 1970s, the traditional phenotype-based drug discovery practice, or “forward pharmacology”, has been challenged by the new molecular target-based research, also called “reverse pharmacology” or “reverse chemical biology”, especially in the pharmaceutical industry.   Scheme 1.1 Flowchart of a typical reverse pharmacology-based drug discovery  The concept of receptor pharmacology has matured since the 1950s in parallel with progress in the field of enzymology. People in drug R&D began to acknowledge that drug molecules could elicit their desired effects by targeting certain important proteins in physiological or pathophysiological pathways. The advent of computational chemistry and structural biology introduced the application of computer-aided drug design (CADD), which could provide disease project selectionmechanism of action study&target identificationassay design&target-based HTSlead optimization13  simulated drug structures complementary to the three-dimensional structural information of known target enzymes or proteins. Compared to “serendipitous” drug discovery, the in silico method to design new drug structures was described as “rational” since it was based on the principles of receptor pharmacology. Development of new synthetic techniques and the introduction of combinatorial chemistry enabled rapid preparation of a large number of structurally related small organic molecules based on rationally designed drug structures, which was also referred as building a chemical library. Expression of purified target proteins and engineered cell lines via molecular biology manipulation provided sustainable biological support for the in vitro screening of the established compound libraries. Active compounds from the screen, also called “hits”, were further subjected to reciprocating SAR study and structural optimization of their pharmacokinetic and physicochemical properties. High-throughput screening (HTS) emerged in concert with the above developments, thanks to the miniaturization of bioassays and robotic automation. Mobilizing all these new technologies in a standardized and integrated protocol redefined and greatly facilitated the whole process of discovering new lead compounds.10  Providing an unprecedented efficiency in generating lead compounds, the combination of target-based drug design, combinatorial chemistry32, 33, and the HTS screen soon took over traditional drug discovery practice in big pharmaceutical companies, beginning in the 1990s.34-37 Most natural product research facilities in the industry were dismantled at that time.38  14   Scheme 1.2 Flowchart of current forward pharmacology-based drug discovery  Recently, a similar revolution has been happening to the phenotype observation-based classical pharmacology.39 Without relying on a predefined molecular target or a known mechanism of action, modern phenotypic screening evenly scrutinizes all possible cellular pathways for “druggable” nodes in a more physiologically related cell-based environment to find hits with therapeutic potential by examination of disease-relevant phenotypic changes. Sometimes, this methodology is referred to as “chemical genetics”, because the cell-based screen, from another perspective, is an investigation of genotype and phenotype relationship via small-molecule perturbation.40  Today, despite the popularity of reverse chemical biology, the phenotypic screening approach still leads the innovation in drug discovery. Between 1999 and 2008, the FDA approved 28 new molecular entities as first-in-class drugs discovered by forward pharmacology, in contrast to 17 drugs from the target-based method.39    disease selection&phenotype studyassay design&phenotypic HTSmechanism of action study&target identificationlead optimization15  1.5 Finding new targets: methods for mechanism of action study in forward pharmacology  Another characteristic of the cell-based forward pharmacology is that the hits from the phenotypic screens could be designed as probes for studying their mechanisms of action in the same cell lines.41 A linker motif, such as a biotin or an alkyne, is attached to the hit compound to form the probe that should maintain a comparable in vitro activity to its parent structure. Afterward, “pull-down” assay is performed using the probe for identification of drug target through probe/protein interaction.42               16   Scheme 1.3 A workflow of a typical streptavidin/biotin-based pull-down assay  Streptavidin, a protein isolated from Streptomyces avidinii, has an exceptionally strong non-covalent binding to biotin with a dissociation constant (Kd) of 1015 M. The specific intermolecular interaction between streptavidin and biotin has been utilized to develop affinity chromatography for purification of the target proteins/enzymes from a complex mixture. As illustrated in Scheme 1.3, the biotinylated probe is incubated with the cell lysates. The ligand moiety in the probe would interact with certain proteins/enzymes in the lysates while the biotin 17  linker builds a connection with streptavidin-bound resins, which is also called “immobilisation”. Then, the resins are washed with gradient buffers. After that, the collected sample is subjected to SDS-PAGE for protein resolution. Specific protein antibodies are widely used for visualisation of the probe-protein adducts in the Western blotting. Modern high-resolution mass spectrometry is also extensively employed for target identification.   Scheme 1.4 A workflow of a typical Click chemistry-based pull-down assay  A probe with a terminal alkyne can be used to investigate whether or not the covalent binding is the mechanism of action between the hit and its target. The protocol is highlighted by a Click chemistry application of Cu (II) catalyzed Huisgen 1,3-dipolar cycloaddition to form a fluorescent triazole between an azide and an alkyne (Scheme 1.4).43 Similarly, the azide can also be designed to attach a linker, such as a biotin, to facilitate purification and the adduct detection 18  in the later stages. SDS-PAGE would break weak noncovalent bonds during the process of electrophoresis.  1.6 Scope of thesis  The significant impacts of natural products have been reevaluated and reenergized in today’s drug discovery practice.44-46 Secondary metabolites not only provide more structural diversity47-49 compared with synthetic derivatives in compound library construction, which is suggested to improve hit rates after the HTS screen, but they also play important roles in drug target identification in forward pharmacology because of their intrinsic property of molecular recognition to biomolecules.50 As a relatively young and unexplored area compared with the terrestrial sources, the oceans hold even greater possibilities in the future for finding bioactive natural products that have potential for the treatment of refractory human diseases. As exemplified in this thesis, three classes of small molecule marine natural products — coumarins C11 (2.14) and C21 (2.15), chlorinated dipeptides sintokamides A – E (3.11 – 3.15), and an alkaloid latonduine A (5.1) — were found to be bioactive from either target-based or phenotypic screening assays in vitro. Their potential as drug leads were further explored as part of collaborations, where productive dialogues and actions were encouraged between marine natural products chemists in the Andersen lab and scientists in three biology laboratories across Canada.  Chapter 2 describes the chemical syntheses of 3,6,7-trihydroxycoumarin (2.14, C11) and 3,7,8-trihydroxycoumarin (2.15, C21), which were identified as hits from a new target-based screen designed to find inhibitors of HCV. As suggested by kinetic data, both compounds elicited their 19  in vitro inhibitory activities through noncompetitive/uncompetitive mechanism, respectively. However, in the following orthogonal assay that was performed in more physiologically related human hepatoma cells, the coumarins exhibited significant toxicities with barely measurable HCV inhibition. Therefore, this project was suspended.  Chapter 3 is the major work in this thesis. Guided by a phenotypic screen in human prostate cancer cells, five active chlorinated dipeptides sintokamides A – E (3.11 – 3.15) were isolated from the marine sponge Dysidea sp.. The screen was designed to detect molecules that could inhibit AR NTD transactivation, which was a newly proposed mechanism for drugs to treat castration-resistant prostate cancer (CRPC). Structurally, the AR NTD belongs to a class of intrinsically disordered proteins (IDPs), which lack higher-order structure of most proteins and, therefore, cannot be applied to rational drug design. In this chapter, an extensive study of structure activity relationships (SARs), which is aimed at elucidating how chemical and stereochemical features in a molecule influence its biological activity, was carried out based on a 1,17-dinorsintokamide skeleton (3.59). 29 analogues/precursors were synthesized from three synthetic routes with different synthetic scopes. Among the natural products and the synthetic analogues, LPY26 [(4R,10R)-3.233] showed superior biological activity and physical stability and has been selected for scale-up synthesis and in vivo evaluation in a mouse xenograft model of CRPC.  Chapter 4, in the beginning, summarizes the studies of mechanism of action of EPI-001 (3.8), the first known AR NTD antagonist that could covalently bind to the AF1 region in the AR NTD. Naturally occurring sintokamide A (3.11) showed many similar in vitro and in vivo properties 20  with EPI-001. However, their mechanisms of action were suggested to be different based on their synergistic effects. A Click chemistry study revealed that the active hexachlorinated 1,17-dinorsintokamide probes LPY30 (4.7) and LPY31 (4.8) exerted their inhibitory activities against AR NTD transactivation through covalent binding to the full-length AR (FL-AR). A plausible mechanism was proposed to explain the unique interaction between the hexachlorinated 1,17-dinorsintokamide analogues and the AR.  Chapter 5 is a part of a ten-year story of how to identify and validate a new drug target from a library of marine natural products, starting with a disease-related phenotypic screen, to find drug leads for treatment of cystic fibrosis resulting from the F508del mutation. The screening assay was designed to visualize the correction of F508del-CFTR transportation by trafficking immunofluorescent mutants onto the cell surface of engineered BHK cells. The screening gave a hit named latonduine A (5.1) from a marine natural products library containing 720 marine extracts. Latonduine A’s activity was further confirmed by a series of in vitro, ex vivo, and in vivo experiments. A mechanism of action study revealed that N-biotinylated latonduine A bound most members of the PARP family, especially PARP-3 (EC50 = 400 pM). The identification of PARP-3 as a target of latonduine A was validated by an siRNA knockdown experiment. 21  Chapter 2: 3,6,7- and 3,7,8-Trihydroxycoumarins Inhibit HCV NS3pro/Pep4A in vitro  2.1 Introduction  Hepatitis C is an infectious liver disease caused by the hepatitis C virus (HCV) through blood to blood contact. When it was first discovered in 1978 that the virus was transmissible from humans to chimpanzees, hepatitis C was called the “non-A, non-B hepatitis”. Hepatitis C got its current name 11 years later in 1989 after the cDNA of HCV had been cloned. Even though patients with chronic HCV infection are often asymptomatic, the viral infection can eventually lead to cirrhosis, liver failure, or hepatocellular carcinoma.51 Today hepatitis C is the No.1 reason for liver transplantation in Canada.52 The World Health Organization estimates that there are roughly 150 million people (~2 % of the world population) living with chronic HCV infection and hepatitis C-related liver diseases cost more than 350,000 lives annually.53 The Public Health Agency of Canada estimates that 242,500 individuals across the country have chronic hepatitis C.54  Hepatitis C is detectable. The development of diagnostic methods for the viral infection was initiated right after the virus’ identification in 1989. Currently, suspected HCV infections are examined by preliminary serum anti-HCV antibody tests followed by confirmatory HCV recombinant immunoblot assays (RIBAs) and HCV RNA polymerase chain reaction (PCR) tests.55 In consideration of possible infections caught before HCV detection became the norm in 22  the early 1990s, the Canadian Liver Foundation recommends that all adults born between 1945 and 1975 undergo the antibody test, which is under the coverage of all provincial health care plans.56 However, there has not yet been a general population HCV screening program existing in Canada.57  Hepatitis C virus has six genotypes and the infections are curable in most cases. The HCV RNA is only cytosolic, so a complete elimination of the virus is possible without touching the nucleus and killing the host cells. Based on this principle, the sustained virological response (SVR) is proposed and widely acknowledged as an important clinical index for hepatitis C treatment. If a patient is HCV RNA free for 24 weeks after the completion of treatment, he/she is thought to be cured and has achieved an SVR24. The FDA and the EMA also accept SVR12 as the conclusion of clinical trials for novel HCV therapies.51   Figure 2.1 Timeline of important advances in the evolution of HCV treatment  1986monotherapyInterferon (IFN)1998dual regimenpegylated IFN/ribavirin (RBV)2011triple regimen for HCV 1telaprevir/boceprevir(NS3/4A DAAs)2013IFN-free all-oral regimen for HCV 2, 3sofosbuvir/RBV(NS5B DAA)23  A timeline of important advances in the evolution of HCV treatment is illustrated in Figure 2.1. The use of interferon (IFN) as a treatment of the “non-A, non-B hepatitis” was reported early in 1986. Since the approval by the FDA in 1998, a dual antiviral regimen of pegylated IFN  (PEG-IFN) plus ribavirin (RBV) replaced IFN monotherapy and the combination became the standard of care for all types of chronic HCV infections in the following ten years. In 2011, two NS3/4A protease inhibitors, telaprevir (INCIVEK®, Vertex) and boceprevir (VICTRELIS®, Schering-Plough/Merck) were approved by the FDA as adjuncts to the dual regimen for treatment of HCV genotype 1 infection. Telaprevir and boceprevir were the first successful examples of direct-acting antiviral agents (DAAs) targeting the viral protein directly. The new triple regimens significantly improved the therapeutic effect compared with the traditional dual regimen.51 In December 2013, the FDA approved the first-in-class NS5B polymerase inhibitor sofosbuvir (Sovaldi®, Gilead) in combination with RBV as the first IFN-free all-oral regimen to treat chronic HCV genotype 2, 3 infections in adults. At the same time, sofosbuvir was also accepted to cure HCV genotype 1 and 4 infections with PEG-IFN and RBV.58 For infections caused by other HCV genotypes, PEG-IFN plus RBV treatment remains the only current therapeutic choice.51  HCV is a positive single-strand RNA virus. The single open reading frame is about 9.6 kb in length, which encodes a polyprotein comprising about 3,000 amino acids. Thereafter, proteolytic cleavage of the polyprotein by cellular and viral proteases generates three structural proteins, six non-structural (NS) proteins, and one small ion channel protein p7. The viral structural proteins consist of a core protein and two envelope proteins (E1 and E2). They are building materials for HCV virion assembly. The viral non-structural (NS) proteins, NS2, NS3, NS4A, NS4B, NS5A, 24  and NS5B, form an enzyme complex, each of which is essential for viral replication and is a possible target for HCV DAA drug discovery. Crystal structures of NS2/NS3, NS3, NS5A, and NS5B have been revealed and are available for rational drug design.51  NS3, NS5A, and NS5B are three non-structural proteins that are the current focal points for HCV DAAs drug discovery. NS3 is a serine protease and NS4A is a cofactor. The NS3/4A protease/cofactor complex is one of the enzymes in charge of cleaving newly synthesized HCV polyprotein into active forms of replicase. The NS3/4A protease inhibitors impede proteolytic activity, diminish required enzymes, and finally prevent HCV replication. NS5B is an RNA-dependent RNA polymerase (RDRP). There are two types of NS5B polymerase inhibitors. The nucleoside inhibitors competitively bind to the NS5B catalytic active site to stop the polyprotein translation early, while the non-nucleoside inhibitors operate noncompetitively to decrease the NS5B enzymatic activity. Even though NS5A is the target for several anti-HCV drugs currently in clinical trials, its exact role in viral replication is not totally understood.51  2.2 Coumarins and coumarins-related anti-HCV studies     2.1  2.2  Coumarin (2.1) itself was first isolated from tonka beans (Dipteryx odorata) in 1820 and coumarin-containing molecules are widely occurring secondary metabolites. So far more than 25  1,300 coumarin-based compounds have been isolated from terrestrial, marine, and microbial sources. Coumarin compounds have a C6/C3 benzo--pyrone core. The 4-phenyl substituted coumarins have a neoflavonoid skeleton (2.2).12    2.3, aflatoxin B1 2.4, armillarisin A     2.5, warfarin 2.6, (+)-Calanolide A  Coumarins are well known for their bioactivities. For example, aflatoxins such as aflatoxin B1 (2.3) are notorious for their hepatic toxicity. Armillarisin A (2.4) can be prescribed to treat acute cholangitis and chronic gastritis. Warfarin (2.5), which has been used as a rodenticide, is a clinically useful anticoagulant acting as a vitamin K antagonist. (+)-Calanolide A (2.6) is a selective inhibitor of HIV reverse transcriptase (IC50 = 0.34 M).59 The biological activities reported for other coumarins include anticancer, antineurodegenerative, antioxidative, and antidepressive activities.60-66   The first laboratory synthesis of coumarin was accomplished by Sir William H. Perkin in 1868 using salicylaldehyde and acetic anhydride (Perkin reaction). Improvements like the Pechmann 26  condensation and the Knoevenagel reaction provide basic methods to make coumarin and its derivatives.     2.7, osthole 2.8, coumestan LQB-34  2.9     2.10, benzimidazole-CH2S-coumarin  2.11, purine-coumarin   Research has already linked coumarins to potential anti-HCV therapies. Osthole (2.7) inhibited caspase-3 (a host cysteine protease) activity, improved the liver histology, and showed better hepatoprotective effects than the currently used glycyrrhizin to prevent anti-Fas antibody induced elevation of plasma ALT in mice.67, 68 Coumestan LQB-34 (2.8) (IC50 = 17 M) and a synthetic coumarin analogue 2.9 (IC50 = 18 M) were novel inhibitors of the NS5B polymerase and they were predicted to interact with the thumb pocket-1 (TP-1).69 It was found that synthetic benzimidazole-CH2S-coumarin (2.10) (IC50 = 3.4 M) and several aromatic equivalents such as imidazopyridine-CH2S-coumarin, purine-CH2S-coumarin, benzoxazole-CH2S-coumarin, and directly linked purine-coumarin (2.11) (IC50 = 3.0 M) conjugates as well as some of their glycosides showed their in vitro activities to inhibit HCV replication with similar IC50’s.70-73 27  2.3 A new target-based screen for anti-HCV therapy     2.12, MC8  2.13, FM16   Esculetin-4-carboxylic acid ethyl ester MC8 (2.12) is a marine natural product based on a coumarin scaffold isolated from the tropical sponge Axinella cf. corrugata by Berlinck and co-workers.74 In 2006, our collaborator Dr. François Jean identified MC8 (2.12) as an inhibitor of the severe acute respiratory syndrome (SARS) coronavirus 3C-like cysteine protease (3CLpro) with an IC50 of 46 M.75 To prove their extrapolation that coumarin derivatives might exhibit inhibitory effects against proteases other than 3CLpro, they tested the ability of MC8 to inhibit HCV NS3/4A. Jean’s results showed that MC8 inhibited NS3/4A with an IC50 of 25.2 M while FM16 (2.13), a structurally related molecule with no anti-3CLpro activity, was unable to inhibit the protease complex. Thus, they designed a target-based screen monitoring fluorescence emission to find better NS3/4A inhibitors based on a library of various coumarin compounds.  A combination of truncated peptides HCV NS3pro and Pep4A has replaced the full length NS3/4A complex in the target-based screening assay. On one hand, NS3 comprises NS3pro, NTPase, and helicase domains from the NTD to the CTD. NS3pro is the serine protease in charge; on the other hand, cofactor NS4A is a protein with 54 amino acids. Pep4A contains residues 21-34 of NS4A and it is sufficient to function as a cofactor. Thus, the NS3pro/Pep4A 28  combination is operational for the target-based screening assay looking for HCV NS3 protease inhibitors.   Scheme 2.1 The screen design based on the enzymatic property of NS3pro/Pep4A to a BS-IQFS  The assay is based on the enzymatic property of NS3pro/Pep4A serine protease to cleave a specially designed synthetic peptide substrate called “blue-shifted internally quenched fluorogenic substrate, (BS-IQFS)” (Scheme 2.1).76 The BS-IQFS consists of a consensus cleavage sequence recognized by NS3pro with an N-terminal fluorophore (an ortho-aminobenzoyl group, Abz) and a C-terminal quenching group [3-nitro-tyrosine, Y(3-NO2)]. Because of their proximity within the molecule, fluorescence resonance energy transfer (FRET) between the fluorophore donor and acceptor moieties results in quenching of the fluorescent energy via non-radiative dipole – dipole coupling, that is, the peptide does not emit florescence at excitation. However, after cleavage at the peptide bond, the donor and acceptor are separated in different molecules. Therefore, fluorescence quenching is eliminated, leading to an increase in fluorescence signal. The effect of inhibition is reciprocal to the intensity of fluorescence.     29   Scheme 2.2 The workflow of the target-based screen and the kinetic assay  Scheme 2.2 illustrates the screening workflow. NS3pro and Pep4A are added to a 96-well plate and then incubated at 30 °C. After 15 min, inhibitors are added and the mixtures are further incubated at 30 °C for another 15 min. After that, substrate BS-IQFS is added. The resulting reaction mixtures are examined for florescence emission at em = 420 nm at the excitation wavelength ex = 320 nm. After collecting kinetic data at 30 °C in 3 h, the reactions are quenched and subjected to separation and component analysis by on-line HPLC-fluorescence spectroscopy.  2.4 Syntheses of C12 (2.18), C11 (2.14) and C22 (2.20), C21 (2.15)     2.14 3,6,7-trihydroxycoumarin (C11)  2.15 3,7,8-trihydroxycoumarin (C21)  After a molecular docking study with the available NS3 crystal structure, 3,6,7-trihydroxycoumarin (2.14, C11) and 3,7,8-trihydroxycoumarin (2.15, C21) were selected as additon of  NS3pro/Pep4A,incubationaddition of inhibitors,incubationaddition of substrate BS-IQFSfluorescence detectionex = 320 nmem = 420 nmkinetic assays,then stop the reactionHPLC30  synthetic targets for testing their HCV inhibitory activities in vitro. C11 is a marine natural product excreted by green algae Cymopolia barbata and Dasycladus vermicularis coping with environmental threats such as extensive UV radiation, raised temperature, abnormal salinity, and physical injuries.77-79 C11 was found to be a vitamin C-comparable antioxidant80, 81 and a potent UV-absorbing substance patented by Merck GmbH in 2007 as a 3-hydroxycoumarin derivative to prevent pigmentation in cosmetic skincare.82 C11 was also reported to have anti-bacterial and microtubule-stabilizing activities. C21, until now, has not been isolated from a natural source. Both C21 and C11, together with several coumarin derivatives with 2 or 3 hydroxyl groups, were patented in 1968 by LIPHA (Lyonnaise Industrielle Pharmaceutique) as agents with therapeutically hypotensive, spasmolytic, and choleretic activities.83              31  A  B  Scheme 2.3 Syntheses of C11 (2.14) and C21 (2.15)  C11 (2.14) and C21 (2.15) were synthesized from 2,4,5- and 2,3,4-trihydroxybenzaldehyde (2.16 and 2.19) in two steps based on literature methodology, respectively (Scheme 2.3).80, 81 In general, the condensation between 2.16 or 2.19 and N-acetylglycine (2.17) in the presence of acetic anhydride and sodium acetate under reflux worked sufficiently in both cases to afford the corresponding 3-acetamido-6,7-diacetoxycoumarin (2.18, C12) and 3-acetamido-7,8-diacetoxycoumarin (2.20, C22) in a yield of 38 % and 54 %, respectively. Hydrolyses of C12 and C22 in a refluxing solution of 3 M HCl and acetic acid gave the final products C11 (2.14) and C21 (2.15) in a yield of 14 % and 24 %, respectively. Sometimes, 3-amino-dihydroxycoumarins 32  (such as 2.21) were isolated as hydrolytic intermediates and complete conversion to C11 and C21 required prolonged reaction time to go to completion.   Figure 2.2 The TLC of 2.18, 2.20 and 2.14 (DCM/MeOH = 98/2)  As shown in Figure 2.2, my experimental observation provides a reasonable explanation to the stepwise hydrolysis results of two unidentified UV-absorbing compounds and C11 isolated from D. vermicularis in 1983.84 On their TLC, an unidentified spot with lower polarity (the top spot) could be hydrolyzed via another unidentified spot with modest polarity (the middle spot) and finally to the most polar C11 (the bottom spot), which was unchanged by extended hydrolysis. In the literature, a prolonged hydrolysis of the crude extract only gave C11 as the major product.     33  2.5 Primary screen and kinetics study  Table 2.1 Results of the primary screen Code$ Name Structure IC50 (M)*C4 esculetin  279.8 MC8 esculetin-4-carboxylic acid ethyl ester  25.2 C2 esculetin-4-acetic acid  >300 C3 4-methylesculetin  >300 C20 nordalbergin  >300 FM373 esculetin-4-carboxylic acid  >300 C5 scoparone  >300 C6 isoscopoletin  >300 C7 scopoletin  >300 34  Code$ Name Structure IC50 (M)*FM16 scopoletin-4-carboxylic acid ethyl ester  >300 FM370 scopoletin-4-carboxylic acid  393 C11 3,6,7-trihydroxycoumarin  3.07 C12 3-acetamido-6,7-diacetoxycoumarin  >300 C13 umbelliferone  >300 C1 umbelliferone-4-carboxylic acid ethyl ester  >300 C14 3-hydroxycoumarin  >300 C15 4-hydroxycoumarin  >300 C8 4-methyldaphnetin  108.8 C10 daphnetin  25.0 35  Code$ Name Structure IC50 (M)*C16 4-methyldaphnetin-3-acetic acid  >300 C17 7-methoxycoumarin  >300 C18 7,8-dimethoxycoumarin  >300 C19 fraxetin  >300 C9 5,7-dihydroxy-4-methylcoumarin  >300 C21 3,7,8-trihydroxycoumarin  2.1 C22 3-acetamido-7,8-diacetoxycoumarin  185 * IC50 values in M were determined by kinetic assay in triplicate. $ Except for C12, C11, C22, and C21, other coumarin compounds in the library were purchased from INDOFINE Chemical Company.  The results of the library screening are shown in Table 2.1. MC8 was confirmed to have an IC50 about 25 M; C10 also had an IC50 of approximately 25 M. The synthetic trihydroxycoumarin molecules, C11 (2.14) and C21 (2.15), exhibited IC50’s of 3.07 M and 2.10 M, respectively. An obvious structure activity relationship is hard to recognize at this time. 36  C12 (2.18) and C22 (2.20), two C11 and C21 acetylated/aminoacetylated precursors, were tested and both have IC50’s > 150 M (Table 2.1 and Figure 2.3). These molecules were then employed as negative controls for C11 and C21, respectively. Interestingly, preliminary results showed that C12 and C22, probably acting as prodrugs, exhibited better pharmacokinetic (PK) properties than C12 and C22 in experiments with cultured human hepatoma cells.   Figure 2.3 Dose-response curves of C11, C12, C21, C22 and C10  Dose-response curves of key compounds (C11, C21, C12, C22 and C10) are shown in Figure 2.3.      37  Table 2.2 C11 inhibition of NS3pro/PepA activity Code [Inhibitor] Vmax (RFU/s) Km (M) Conclusion C11 0 0.4463 51.4 noncompetitive C11 3.3 M 0.2246 51.4  For C11, the inhibition of NS3pro/PepA in Michaelis-Menten experiments revealed an noncompetitive mechanism with a decreased Vmax and a constant Km (Table 2.2). For C21, an uncompetitive mechanism of inhibition was supported by Dixon experiments (Ki = 400 ± 155 nM) with six data sets of triplicates. However, the Michaelis-Menten experiments involving C21 could not be reproduced to give a definite inhibition mechanism.  2.6 Conclusion  This chapter describes an application of a newly developed high-throughput target-based screening assay to find possible drug leads with a coumarin scaffold to inhibit HCV NS3/4A protease activity. The idea was inspired by a coumarin natural product MC8, isolated from the tropical marine sponge Axinella cf. corrugata, which exhibited inhibitory effects on SARS CLpro (IC50 = 46 M). Thanks to the specially designed internal quenched fluorogenic substrate (IQSF), it was the first time that the HTS screen was able to directly detect NS3/4A activity in vitro. Two synthetic coumarins C11 (2.14) and C21 (2.15), along with their acetylated precursors C12 (2.18) and C22 (2.20), were prepared for screening by stepwise syntheses. The bioassay results revealed that 3,6,7-trihydroxycoumarin (C11, IC50 = 3.07 M) and 3,7,8-trihydroxycoumarin (C21, IC50 = 2.10 M) could inhibit HCV NS3pro/Pep4A in vitro. 38  Chapter 3: Structure-Activity Relationship Study of Sintokamides  3.1 Introduction  In Canada, prostatic carcinoma ranks as the most common male cancer and the second largest cause of cancer deaths in men. It is estimated that 1 in 7 Canadian men will have prostate cancer during his lifetime and 1 in 28 will die of it. Every year about 24,000 cases are newly diagnosed and near 4,000 patients die of the disease anually.85   Figure 3.1 Prostate cancer death and mortality rates in Canada (Adapted with permission of “Mortality, Summary List of Causes”, Statistics Canada, 2009)  A malignant tumor in the prostate gland is called prostate cancer. Fortunately, unlike other kinds of cancers, prostate cancer grows slowly.86 Most evidence of prostate cancer under microscopic 010020030040050060070080090010000 - 2 9 3 0 - 3 4 3 5 - 3 9 4 0 - 4 4 4 5 - 4 9 5 0 - 5 4 5 5 - 5 9 6 0 - 6 4 6 5 - 6 9 7 0 - 7 4 7 5 - 7 9 8 0 - 8 4 8 5 - 8 9 9 0 +Number of deaths Age-specific mortality rate per 100,000 population39  examination are clinically unimportant and many patients with prostate cancer usually "die with it, rather than from it".86 Moreover, early detection of the disease has been realized since the FDA approved the prostate-specific antigen (PSA) test to detect prostate cancer for asymptomatic men in 1994. Today, 85 % of diagnosed prostate cancer is in early stages (Stage I and II). Therapeutic options to remove or destroy local cancerous cells, such as surgery and radiation, are curative.87 According to statistics from the Canadian Cancer Society, the current 5-year relative survival rate of prostate cancer in Canada is 96 %.88  However, 20 – 40 % patients are not cured and the cancer recurs and even spreads (Stage III and IV). Androgen-deprivation therapy (ADT), based on a study dated in 1941 by Huggins and Hodges showing the close correlation of prostate tumor growth with androgen concentrations in blood (vide infra), became the key treatment for these patients.89 Radiation and bisphosphonates are also standard treatment options in these stages. Castration therapy initiates favorable therapeutic responses, with a significant regression of the tumor growth and rapid biochemical improvements in medical tests. Unfortunately, the disease will eventually progress and metastasize despite the ADT in 2 – 3 years and this stage of the disease can be defined as castration-resistant prostate cancer (CRPC).87  40   Table 3.1 New drugs approved for CRPC by the FDA in the last 10 years approval date name trade name mechanism indication 5/15/13 Radium-223 chloride xofigo radiopharmaceuticals CRPC with bone metastases 12/10/12 abiraterone acetate Zytiga CYP17A1 inhibitor mCRPC in combination with prednisone 8/31/12 enzalutamide Xtandi AR LBD antagonist mCRPC previously treated with docetaxel 9/16/11 denosumab Prolia monoclonal antibody increasing bone mass in patients 6/17/10 cabazitaxel Jevtana microtubule inhibitor CRPC that is no longer responding to hormone therapy 4/29/10 sipuleucel-T Provenge therapeutic cancer vaccine asymptomatic or minimally symptomatic mCRPC 12/24/08 degarelix Firmagon GnRH receptor antagonist CRPC 5/19/04 docetaxel Taxotere microtubule inhibitor mCRPC in combination with prednisone  41  Current treatment of CRPC is a combination of chemotherapy, hormonal management, and immunotherapy (Table 3.1). For example, the standard dosage schedule for symptomatic patients with metastatic CRPC (mCRPC) is a weekly injection of docetaxel, which can improve overall survival and quality of life. When disease progresses after docetaxel, there are four recently approved therapeutic options with different mechanisms of action: cabazitaxel (2010), abiraterone/prednisone (2012), enzalutamide (2012), and radium-223 chloride (2013).90 All of these agents have shown to improve overall survival after docetaxel failure. However, these therapies can only extend life expectation for 4 – 5 months, and the average life expectation of CRPC is 16 – 18 months from the beginning of progression.91  3.1.1 Androgens, normal prostate, and prostate carcinogenesis  Androgens, acting through the androgen receptor (AR), are required for the development and normal function of the prostate gland. The action of androgens functions through an axis to exert their biological effects by binding to the AR to induce AR transcriptional activity. In short, androgens promote the growth but prevent the death of the prostate epithelia, the primary cell type thought to be transformed in prostate adenocarcinoma. In the normal prostate epithelia, the rate of cell apoptosis is 1 – 2 % per day, which is balanced by a 1 – 2 % rate of proliferation.92  Cancer cells in the prostate also need androgens to grow and survive. Approximately 80 – 90 % of prostate cancers are dependent on androgens at initial diagnosis.92 For those patients, androgen concentration is the key to control the ratio of cells growing to those dying. ADT causes cancer regression, as a result of androgen deprivation, by lowering the rate of cell 42  proliferation while increasing the rate of cell apoptosis. For example, the reduction of serum and prostatic DHT levels by castration resulted in a loss of 70 % of the prostate secretory epithelial cells due to apoptosis in adult male rats.92  3.1.1.1 Androgen receptor: overview  Androgen works via the androgen receptor (AR). The AR belongs to the steroid receptor superfamily and it can bind to various DNA binding sites to activate transcription as a ligand-dependent transcription factor. As shown in Figure 3.2,93 the AR gene on the X-chromosome (Xq12) has 8 exons. The receptor consists of an N-terminal domain (NTD), a central DNA binding domain (DBD), a hinge region, and a C-terminal domain (CTD). The AR DBD is highly homologous with the DBDs of other human steroid receptors such as the glucocorticoid receptor (GR) and the progesterone receptor (PR). The NTD and the CTD contain transactivation domains AF1 and AF2, respectively. However, it is considered that the NTD AF1 is dominant in most AR signalling studies under normal physiological conditions.93  43   Figure 3.2 The AR gene, protein, and zinc fingers (Reprinted with permission of W.B./SAUNDERS C O. LTD.)  The nuclear AR contributes to androgen-axis signalling via transcriptional regulation (Figure 3.3).94 In the nucleus, the ligand-bound AR homodimers recruit various co-factors, bind to androgen-response elements (AREs), and activate transcription in AR-specific genes such as the PSA. Either up- or down-regulation of the AR-specific genes can be achieved on a basis of different ligand concentrations.93  44   Figure 3.3 AR axis and androgen-sensitive transactivation (Reprinted with permission of Nature Publishing Group)        45  3.1.2 Adaptive responses to castration affect the androgen axis in CRPC  The AR transcriptional activity is observed in initially diagnosed prostate cancer cells as well as in the vast majority of cells in mCRPC patients. For example, despite low circulating levels of serum androgens, PSA concentration, which serves as a tumor marker, will eventually rise in most CRPC patients. The androgen signalling axis in CRPC is associated with multiple adaptive mechanisms proposed for the persistent activation of the AR and the development of CRPC (Figure 3.4).93   Figure 3.4 Current therapies vs CRPC adaptations affecting the androgen axis  46  3.1.2.1 Intratumoral synthesis of androgens  CRPC cells can synthesize potent androgens from various steroidal precursors and some CRPC research showed that the conversion of testosterone (3.2) to DHT (3.6) was bypassed and 3.2 was no longer the dominant precursor in DHT synthesis (Figure 3.5).93, 95, 96 Both conventional and adaptive androgen synthesis pathways contribute to intratumoral synthesis of androgens in CRPC cells by increased expression of steroidogenic enzymes involved in both metabolisms.93, 96                 47   Figure 3.5 Androgen pathways in CRPC       48  3.1.2.2 Increased expression of steroid transporters  Organic acid transporters (OATs), the proteins derived from solute carriers (SLCOs) genes, can transport organic molecules across cell membranes. The efficiency of androgen transport affects both efficacy of ADT and transition rate to CRPC afterward. Compared to localized cancers, expression of the transporter genes, such as SLCO2B1 and SLCO1B3, has significantly increased in mCRPC tissues.93, 96, 97  3.1.2.3 Sensitizing the AR to androgens by AR overexpression  Compared to benign tissues and hormone naïve cancers, the AR expression in CRPC has been increased. The elevated AR expression may sensitize the AR transcriptional activity to a low concentration of androgen. It has been demonstrated that an increase in nuclear AR expression in advanced CRPC was correlated with reduced time to prostate-cancer specific mortality.93, 97  3.1.2.4 Promiscuous AR due to adaptive AR mutation  AR gain-of-function (GOF) mutation was observed among patients with ADT, which was more frequently detected in therapeutic regimen of castration/anti-androgen combination. Mutations in the AR LBD could broaden the ligand specificity of the AR, allowing it to be activated by non-androgenic steroids, or anti-androgens (antagonists).93 For example, H874Y mutation was observed among flutamide-treated CRPC patients. The mutation increased AR promiscuity that DHEA, estradiol, progesterone, and hydroxyflutamide in various model systems could trigger 49  AR transcriptional activity. Moreover, AR mutations might alter the binding of co-regulators and other regulatory elements as a result of continued ligand-dependent activation of the AR in CRPC.93, 97  3.1.2.5 AR splice variants with constitutive activity  AR splice variants (AR-Vs) have been recently identified and characterized in CRPC patients (Figure 3.6).93 In some cases of CRPC bone metastases, expression of AR-Vs with relatively low mRNA concentrations could be comparable to that of the FL-AR protein. These truncated receptor variants devoid of the functional LBD displayed constitutive activity, which were capable of regulating target gene expression in the absence of the FL-AR signalling.93, 97   Figure 3.6 Structures of the FL-AR and AR-V proteins (Reprinted in part with permission of W.B./SAUNDERS C O. LTD.)  3.1.2.6 Other proposed mechanisms for CRPC  Several mechanisms were also proposed focused on the role the AR played in CRPC. Over 170 potential co-regulators of the AR have been identified. The relative ratio of co-repressors/co-50  activators recruitment was shown to stabilize the AR, and led to either decreased or increased transcriptional activity by altering ligand specificity and/or sensitivity.93, 96, 97  Other signal transduction pathways, such as those involving TGF, IL-6, and IGF-1, could also modulate the AR activity and provide ligand-independent mechanisms to sustain AR activation. Post-translational modifications of the AR, phosphorylation, SUMOylation, acetylation, and ubiquitylation, could also serve as potential mechanisms effecting AR function, stability, localization, and interactions with other transcriptional factors.93, 97  Finally, the AR axis in CRPC was found to be essentially functional through adaptive mechanisms affecting androgen synthesis, DNA synthesis, and cell cycle progression, as compared to androgen-dependent prostate cancer. The whole network was believed to revive proliferation, motility, and invasion to assist malignant cells in adapting to the decline in androgen because of castration.93, 97  3.1.3 AR NTD, a promising target for CRPC therapy  Since androgens bind to the LBD, all current hormone therapies are focused on the AR CTD, where the LBD is located. However, as shown above, CRPC exhibits vigorous adaptive activity to bypass all the consequences caused by hormone castration to active the AR and exert its transcriptional output.  51  The AR NTD might be the “Achilles’ Heel” in the AR structure for new CRPC therapy.98 First, unlike the AR LBD/DBD, which share a high degree of homology of both domains in other human steroid hormone receptors like the GR and the PR, the AR NTD is unique and has less than 15 % similarity compared with the NTDs in other human steroid hormone receptors. Second, the AF1 in the AR NTD contributes most of the transcriptional activity, which cannot be achieved by the AF2 in the AR CTD. Thirdly, for the AR axis triggered by constitutively-active AR-Vs, the NTD is the only possible target for therapeutic advances because those truncated AR variants keep the full length NTD, while they do not have the LBD at all (Figure 3.6).  Structurally, the AR NTD belongs to the class of intrinsically disordered proteins (IDPs) which lack stable tertiary structure under physiological conditions. Structural flexibility of IDPs facilitates exerting their physiological activity by reversibly binding with multiple proteins via a high-specificity but low-affinity way. In view of drug discovery, small organic molecules would work by disruption of essential protein-protein interaction between the AR NTD and the co-factors that bind to it. However, with intrinsically disordered structure and no available X-ray crystallography information, rational drug design based on the AR NTD cannot be achieved. Till 2008, no drug targeting an IDP has reached clinic trials, nor has a small molecule inhibitor to an NTD of a steroid receptor ever been described.98    52    3.9, niphatenone A  3.8, EPI-001 3.10, niphatenone B  Figure 3.7 The structures of EPI-001 (3.8), niphatenone A (3.9), and niphatenone B (3.10)  With an assay that used LNCaP prostate cancer cells containing an engineered PSA gene with a luciferase reporter, a phenotypic screen of the Andersen marine invertebrate extract library revealed EPI-001 (3.8)99, 100, niphatenones A and B (3.9 and 3.10)101, and sintokamides A – E (3.11 – 3.15)102, as shown in Figure 3.7 and Figure 3.8, which were the first reported small molecule antagonists of the AR NTD, as lead compounds for the development of drugs to treat CRPC. The following sections and Chapter 4 will focus on the story of the sintokamides, including isolation, synthetic plans, and an SAR study as well as work aimed at trying to find their mechanism of action as AR NTD antagonists.      53  3.2 Sintokamides A to E (3.11 to 3.15) from the sponge Dysidea sp.: collection, isolation and biological activity   Figure 3.8 The structures of sintokamide A – E (3.11 – 3.15)  In a continuous effort in the Andersen lab to seek bioactive natural products from marine, terrestrial, and microbial resources around the world, the sponge Dysidea sp. was handpicked while SCUBA diving near Palau Sintok, Karimunjawa archipelago, Indonesia in 2008. Chemical constituents of the MeOH extract of the Dysidea sp. were fractionated under the guidance of a newly developed bioassay for antagonists of the AR NTD (vide infra). After assay-guided fractionation, the five new chlorinated peptides sintokamides A to E (3.11 to 3.15) were revealed as the bioactive components. Their structures were assigned unambiguously by 1D- and 2D-NMR spectroscopy, HR-ESIMS, and X-ray crystallography. Sintokamides were the first reported antagonists of AR NTD transactivation in prostate cancer cells.102     54  3.2.1 Sintokamide A (3.11) inhibits the AR NTD transactivation in vitro  The human prostatic adenocarcinoma cell line LNCaP is androgen-sensitive. Engineered LNCaP cells can stably express luciferase via different mechanisms of AR activation by incorporation of luciferase reporter genes to selected regulatory sequences in different AR domains. This phenotypic screen is designed to measure decreased bioluminescent output resulting from AR transcriptional activity blockage. In LNCaP cells expressing the PSA(6.1)-luciferase reporter, sintokamide A (3.11) at a concentration of 5 g/mL blocked AR transcriptional activity in the presence of a synthetic androgen R1881 (Figure 3.9A). In LNCaP cells expressing AR NTD-Gal4DBD chimera protein/Gal4-luciferase reporter, sintokamide A (3.11) at a concentration of 5 g/mL suppressed forskolin-induced AR NTD transactivation to baseline levels (Figure 3.9B). In unmodified androgen-sensitive LNCaP cells, sintokamide A and bicalutamide (an AR LBD antagonist) showed comparable effects in blocking proliferation caused by androgen treatment through detecting the BrdU incorporation. (Figure 3.9C). However, no significant inhibition of proliferation was observed by sintokamide A (3.11) treatment in androgen-insensitive PC3 human prostate cancer cells (Figure 3.9D). Moreover, sintokamide A’s anti-proliferative activity was not a simple consequence of general cytotoxicity. In LNCaP Cells, sintokamide A at a concentration of 10 g/mL for 48 h did not demonstrate obvious morphological changes. In summary, sintokamide A (3.11) was found to be the first reported compound which could block AR signalling in androgen-sensitive LNCaP cells via inhibition of AR NTD transactivation.102  55   Figure 3.9 Sintokamide A’s inhibitory activity against AR-NTD transactivation in vitro (Reprinted with permission of American Chemical Society)  3.2.2 Enrichment of sintokamide A (3.11) and sintokamide B (3.12)  As the first step in further exploration of the AR NTD antagonistic properties of the sintokamides, larger quantities of the natural products were isolated from the marine sponge to support extensive biological studies (See Chapter 4). ~200 g of frozen sponge material was soaked in 450 mL of methanol overnight at room temperature, and the filtered methanolic extract was dried in vacuo. The extraction process was repeated two more times and the combined methanolic extracts were then partitioned between ethyl acetate (3×100 mL) and water (150 mL). The combined ethyl acetate layers were evaporated to dryness, and the resulting purple oil (2.44 g) was fractionated on silica gel flash chromatography, employing a step gradient from 9:1 hexanes/ethyl acetate to 100 % ethyl acetate. The chromatography concluded with 9:1 56  dichloromethane/methanol elution. Fractions, eluting with hexanes/ethyl acetate 2:1, were combined and dried (480 mg) and then subjected to C18 reversed phase HPLC. With 13:7 acetonitrile/water as eluent at a flow rate of 1.0 mL/min, sintokamide A (3.11) (tR = 45.6 min) and sintokamide B (3.12) (tR = 53.6 min) were collected as amorphous clear solids of 83.6 mg and 10 mg, respectively. Their 1H NMR data were identical with literature values.102  3.3 Syntheses of sintokamide analogues for SAR studies  A B    Figure 3.10 (A) Structures of the sintokamides and (B) their in vitro inhibitory activities of R1881 activation of P6.1 luciferase in LNCaP cells  Having sufficient quantities of the natural products with confirmed structures to carry on all preliminary in vitro and in vivo experiments, a goal was set at the outset of this project of synthesizing sintokamide analogues for SAR studies. Structurally, sintokamides A – E (3.11 – 3.15) are a series of propionylated dipeptides of D-leucine and L-methyl tetramate, which could 0.0%20.0%40.0%60.0%80.0%100.0%120.0%5 μg/mL5 μg/mL5 μg/mL5 μg/mL5 μg/mL5 μg/mL5 μg/mLDMSO BIC sint A sint B sint C sint E CB-0 dysamide A% PSA-LUCIFERASE ACTIVITY57  be derived from homologation of L-leucine (3.29). Most pro-R methyl groups in the sintokamide dipeptides are chlorinated with different degrees of chlorination ( Figure 3.10A). In vitro, the sintokamides exhibited moderate to excellent inhibitory activities of R1881 activated P6.1 luciferase in LNCaP cells ( Figure 3.10B). The different chlorination patterns in the natural products and their relative activities in vitro provided the first sketch of the SAR for this new pharmacophore (vide infra).   Figure 3.11 Structural features of interest in the sintokamide SAR study  Inspiring though it was, the SAR information revealed by the five naturally occurring sintokamides was limited. In order to have a comprehensive SAR understanding, additional structurally related sintokamide analogues needed to be synthesized and tested in the screening bioassay. The aim of our synthetic SAR study was to prepare specially designed, easy-to-make sintokamide precursors/analogues to evaluate every possible AR NTD blocking pharmacophore in the general structure in comparison with the most potent sintokamides A and B as references. 58  Things to consider included the requirements of chlorine atoms and methyl groups in the leucine side chains, the regio- and stereo-preference of chlorination, the optimal N-substitution patterns and other aspects for the AR NTD blocking activity of sintokamides (Figure 3.11). Meanwhile, the design of sintokamide analogues should be practical in terms of asymmetric syntheses since an efficient diastereoselective synthesis of (4S)-5,5,5-trichloroleucine moieties103 in the natural products was unprecedented when this project was started in October 2008. The total syntheses of sintokamides C104 and A, B, E105 were subsequently reported by others in 2010.   Scheme 3.1 The general synthetic route covering all sintokamide analogues and the structure of LPY26 [(4R,10R)-3.233] identified as the analogue with the best biological activity among the 29 synthetic compounds submitted for biological evaluation  59  In this chapter, the synthetic effort (Scheme 3.1) that furnished 29 different compounds (25 analogues and 4 key intermediates) for SAR study and the one general synthetic strategy the author developed to all synthetic sintokamide analogues will be described. Based on the results from the SAR study, we found a synthetic analogue LPY26 [(4R,10R)-3.233] that was practical to make and had superior biological activity and physical stability compared with the natural products and the other synthetic analogues. LPY26 [(4R,10R)-3.233] has been selected for scale-up synthesis and in vivo evaluation in a mouse xenograft model of CRPC.  3.3.1 Synthesis of NCSTD [(4S,10R)-3.16], the non-chlorinated sintokamide framework  3.3.1.1 SAR design and expectation  The AR NTD blocking activities of naturally occurring sintokamides are based on a general dipeptide structure featuring different combinations of chlorination patterns. To investigate the requirement of chlorine atoms, our first synthetic target in this project was compound (4S,10R)-3.16, the Non-Chlorinated SinTokamiDe (NCSTD), which replaces all chlorine atoms with hydrogen atoms in the general structure of the sintokamides (Scheme 3.2).       sintokamides             (4S,10R)-3.16, NCSTD    3.15, sintokamide E  60  Scheme 3.2 Structures of a generic sintokamide, NCSTD (3.16, Non-Chlorinated SinTokamiDe), and sintokamide E (3.15). (R1: chloromethyl or methyl; R2: chloromethyl) Structurally, NCSTD [(4S,10R)-3.16] still keeps several possible pharmacophore elements, such as the dipeptide and the tetramic acid106-109, to stimulate bioactivity. Moreover, NCSTD has a non-chlorinated L-methyl tetramate structure moiety identical to that of sintokamide E (3.15), which showed a strong activity in the AR NTD blocking assay (Figure 9B). The result of the NCSTD bioassay evaluation is expected to answer whether or not chlorine substituents are required for the AR NTD blocking activity.  Synthetically, NCSTD [(4S,10R)-3.16] should be much easier to make compared with the chlorinated natural products. Instead of introducing chiral chloromethyl groups containing leucine side chains without enough literature antecedents and preventing possible dehydrochlorination, the synthesis of NCSTD could be started with commercially available D- and L-leucine derivatives and the synthesis should reasonably tolerate strong acidic and basic conditions.         61   3.3.1.2 Making the N-acyl methyl tetramate: the synthesis of (±)-dysidin (3.17)  Dysidin [(S,S)-3.17], isolated from the sponge Dysidea herbacea, is an N-acyl L-methyl tetramate having a trichloromethyl group at its N-aliphatic terminus.110 Degradation studies in combination with spectroscopic methods were used for its structure elucidation and these studies showed that the amide bond in dysidin was readily cleaved under basic conditions (Figure 3.12). The absolute configuration of dysidin was confirmed by single crystal X-ray diffraction analysis.   Figure 3.12 Treatment of dysidin (3.17) in acidic and basic conditions         62    63  Scheme 3.3 Total synthesis of (±)-dysidin (3.17) published by Williard and Laszlo in 1984 The first total synthesis of (±)-dysidin (3.17) was published by Williard and Laszlo in 1984 (Scheme 3.3).111 The retrosynthetic disconnection was based on the previously described base susceptibility of the N-acyl bond in the natural product. In their synthesis, the O-methyl tetramate moiety 3.20 was prepared from a homologation of racemic N-phthaloylvaline derivatives 3.21 followed by a base-promoted O-methylation and a simultaneous deprotection/cyclisation process (Scheme 3A). The linear trichlorinated O-methyl enol ether moiety 3.28 originated from a radical chain addition of bromotrichloromethane to crotonic acid (3.25), followed by a zinc reduction, a homologation with Meldrum’s acid (3.85) and a base-promoted O-methylation (Scheme 3B). Finally, the convergent synthesis was accomplished by coupling two fragments 3.21 and 3.28 in the presence of a Grignard reagent (Scheme 3C). Much of their work, especially the construction of methyl tetramate and the coupling conditions, could be directly used in our proposed synthesis of NCSTD [(4S,10R)-3.16].           64  3.3.1.3 Retrosynthetic analysis for NCSTD [(4S,10R)-3.16]  My retrosynthetic plan for NCSTD [(4S,10R)-3.16] is outlined in Scheme 3.4. The disconnection begins with breaking the indicated amide bond in the target structure NCSTD. The synthesis is designed in a convergent fashion by N-acylation of the non-chlorinated methyl tetramate 3.32 with the activated amino acid derivative 3.33 prepared from commercially available Boc-D-leucine [(R)-3.43]. Based on the similar protocol described in the total synthesis of (±)-dysidin, the 4-isobutyl L-methyl tetramate moiety 3.32 could readily originate from L-leucine (3.29).   Scheme 3.4 Retrosynthetic analysis for NCSTD [(4S,10R)-3.16]   65  3.3.1.4 Synthesis of NCSTD [(4S,10R)-3.16]  As shown in Scheme 3.5, L-leucine (3.29), N-carbethoxy-phthalimide (3.34) and Na2CO3 were stirred in water to give N-phthaloyl-L-leucine (3.35)112 in quantitative yield. Compound 3.35 was refluxed in SOCl2/DCM to produce the acid chloride 3.36113 in a yield of 91 %. After treatment with 2 equivalents of n-BuLi, 3.36 was homologated with monoethyl malonate (3.22, 79 % from pre-acidified potassium salt114) to give the desired -keto ester 3.37 in a yield of 52 %.   Scheme 3.5 Synthesis of compound 3.37   66   Scheme 3.6 C-methylation of -keto ester 3.37 with KH and MeI  The reactivity of 3.37 was tested with different methylating agents under basic or acidic conditions. As shown in Scheme 3.6, treatment of 3.37 with KH, followed by addition of MeI gave the anticipated C-methylated diastereomeric products (2R,4S)-3.38 and (2S,4S)-3.38 (d.e. = 26 %). The 1H NMR of the crude residue showing quartet splitting patterns at 3.62 and 3.80 (J = 7.2 Hz) confirmed our expectation.   Scheme 3.7 O-methylation of -keto ester 3.37 based on Williard and Laszlo’s method  Because of its acute toxicity, methyl fluorosulfonate (the “magic methyl”) used in Williard and Laszlo’s synthesis is banned in Canada and it was unavailable from any supplier. Instead, KH 67  promoted reaction of 3.37 with dimethyl sulfate generated compound 3.39 in an extremely low yield of 1.2 % (Scheme 3.7). 1H NMR data (5.40, s, 1H; 3.90, s, 3H) indicated 3.39 was the desired O-methylated product.   Scheme 3.8 Synthesis of compound 3.32  Fortuitously, O-methylation under acid catalysis,115-117 which was ineffective in Williard and Laszlo’s synthesis, worked for 3.37 as substrate (Scheme 3.8). In the presence of a catalytic amount of concentrated H2SO4, the -keto ester was treated with trimethyl orthoformate in methanol to give the desired (E)--methoxy methyl acrylate 3.40 in a yield of 70 %. The E configuration in 3.40 was confirmed through 1D-selective NOE and HMBC experiments focusing on the correlation between the olefin proton and the methoxy group. As expected, N-deprotection118 and ring closure took place in one pot by refluxing 3.40 with excess hydrazine hydrate to furnish the methyl tetramate 3.32 in a yield of 55 %.  68   Scheme 3.9 Convergent assembly of NCSTD [(4S,10R)-3.16] with Grignard reagent based on Williard and Laszlo’s method  The Grignard reagent methyl magnesium bromide, which was used in the synthesis of (±)-dysidin, was first chosen for the key coupling reaction (Scheme 3.9). Compound 3.32 was deprotonated by MeMgBr and then treated with N-phthaloyl-D-leucine chloride (3.41) to generate adduct 3.42 in 8 % yield. It was thought that the poor yield could be attributed to the steric hindrance caused by the bulky N-phthaloyl protecting group.   Scheme 3.10 Synthesis of compound 3.45  In order to make a less hindered active ester for coupling rather than acid chloride 3.41, commercially available Boc-D-Leucine [(R)-3.43] was coupled with p-nitrophenol (3.44) in the 69  presence of DCC to give the active ester Boc-D-Leu-ONp (3.45) in a yield of 82 % (Scheme 3.10).119   Scheme 3.11 Convergent assembly with n-BuLi and conclusion of the synthesis of NCSTD [(4S,10R)-3.16]  The synthesis of NCSTD [(4S,10R)-3.16] was completed as illustrated in Scheme 3.11. Deprotonation of 3.32 by n-BuLi in THF at −50 °C created the corresponding lithium amide that was added into the active ester 3.45 to produce adduct (4S,10R)-3.46 in a yield of 47 %. Removal of the Boc in (4S,10R)-3.46 by TFA in 90 % yield followed by N-propionylation gave the final product (4S,10R)-3.16 in a yield of 83 %.  In all, there were 9 steps in this convergent synthesis and the overall yield from L-leucine (3.29) was 6.3 %.  70   Scheme 3.12 Proposed mechanism for C-4 epimerisation in the presence of a strong base  There were concerns about the possible epimerisation at C-4 of the pyrrolidinone substructure in NCSTD [(4S,10R)-3.16] during the coupling reaction (Scheme 3.12). In the presence of a strong base, C-4 could be epimerized in a resonance-like deprotonation/reprotonation process via a stable aromatic pyrrole intermediate 3.48.  71             NCSTD1 (green)  NCSTD2 (red) NCSTD (blue)          Figure 3.13 Structures of NCSTD (in blue), NCSTD1 (in green) and NCSTD2 (in red) and their 1H NMR spectra comparison  72  The doubt was cleared by comparing NCSTD with a pair of structurally confirmed diastereomers NCSTD1 [(4R,10R)-3.46] and NCSTD2 [(4S,10R)-3.46] afforded by another synthetic route in a later stage of this project (Section 3.3.2.7.2). Based on comparison of their Rf values and 1D-NMR data, NCSTD is identical to NCSTD2 which represents the structure of the non-chlorinated sintokamide analogue with (4S,10R) natural configurations.  3.3.1.5 Bioassay results and their SAR implications  The bioactivity of NCSTD [(4S,10R)-3.16] is shown in Figure 3.14. NCSTD [(4S,10R)-3.16] at a concentration of 40 g/mL only exhibited 20 % inhibition of R1881 activation of P6.1 luciferase experiment, compared with 71 % inhibition by sintokamide A (3.11) at a concentration of 5 g/mL and 97 % inhibition by sintokamide B (3.12) at a concentration of 5 g/mL. In short, NCSTD [(4S,10R)-3.16], the non-chlorinated sintokamide, is essentially inactive. Therefore, chlorine atoms are necessary for sintokamide AR NTD blocking activity.  73   Figure 3.14 Bioassay results of NCSTD [(4S,10R)-3.16] on R1881 activation of P6.1luc  3.3.2 Syntheses of 1,17-dinorsintokamide chlorinated sintokamides  3.3.2.1 Chlorinated marine natural products  Seawater is salty. On average, the salinity in the world's oceans is about 3.5 % and the halide counterions are about 0.5 M chloride, 1 mM bromide, and 1 M iodide. Given an environment with such an abundant halogen content, it is not surprising that marine organisms have acclimatised to incorporate halogens into their metabolites (Figure 3.15). It was estimated that most of the 4,000 known natural organohalogens reported before 2004 were identified from marine resources.120 The notable amino acid N-methyl 5,5,5-trichloroleucine is the characteristic halogenated fragment in peptides from sponges in the Dysidea family.121 Many of these halogenated compounds are thought to play defensive roles in keeping predators from eating a 100.0%0.0%80.3%27.2%42.6%30.1%2.9%22.3%0.0%20.0%40.0%60.0%80.0%100.0%120.0%5 μg/mL 40 μg/mL 5 μg/mL 5 μg/mL 5 μg/mL 5 μg/mL 5 μg/mLDMSO BIC NCSTD CB-0 Sint C Sint A Sint B Sint E% PSA-LUCIFERASE ACTIVITY74  particular organism. In many cases, these compounds are also of pharmacological interest due to their biological activities.122     3.49, dysithiazolamine  3.50, dysideapyrrolidone    3.51, dysidenin  3.52, mirabimide E    3.53, barbamide  (S,S)-3.17, dysidin  Figure 3.15 Some examples of highly chlorinated marine natural products  A reasonable biosynthetic pathway for mono-halogenation has been proposed as shown in Scheme 3.13. Haloperoxidases catalyze the oxidation of a halide with the aid of hydrogen peroxide, resulting in the associated electrophilic mono-halogenation of organic substrates. Two types of marine haloperoxidases have been identified: (1) vanadium bromoperoxidase (V-BrPO), a non-heme enzyme, and (2) FeHeme bromoperoxidase (FeHeme-BrPO).122  75   Scheme 3.13 A reasonable biosynthetic pathway for mono-halogenation  On the other hand, the investigation of the origin of N-methyl 5,5,5-trichloroleucine during barbamide biosynthesis uncovered the mechanism for higher degrees of chlorination (Scheme 3.14).123, 124 Attached to a peptidyl carrier protein BarA, L-leucine is trichlorinated on the C-5 pro-R methyl group by tandem action of BarB2 and BarB1, two nonheme Fe(II) halogenases, in the presence of oxygen, chloride, and -ketoglutarate. Briefly, BarB2 is an efficient dihalogenating enzyme but it does not readily catalyze trichlorination. However, this inefficacy is overcome by working cooperatively with a second halogenase, BarB1, which converts dichloroleucine-loaded BarA to the trichloroleucine derivative.   Scheme 3.14 A proposed biosynthetic pathway for poly-halogenation  76  In contrast to the biosynthesis, the availability of synthetic methods for stereoselective trichloromethylation are still highly limited, even though the above chlorinated natural products are attracting increasing attention as target molecules in total synthesis due to their interesting biological activity.121, 125  3.3.2.2 1,17-dinorsintokamide, a simplified skeleton for the chlorination pattern SAR study  Since chlorine atoms are irreplaceable for the AR NTD blocking activity of sintokamides, the next focal point in our SAR study is: what are the required chlorination patterns at the end of each side chain to exert preferred in vitro activity? Mathematically, for 2 chloromethyl groups with 4 possible chlorination degrees, the panorama contains 16 possible combinations, 6 of which have been either just synthesized (the NCSTD) or uncovered from the sponge.   Scheme 3.15 What are the required chlorination patterns in the leucine moieties for the sintokamides in vitro activities?  77   Scheme 3.16 Structure simplification from naturally occurring sintokamides 3.58 to 1,17-dinorsintokamide analogues 3.59  The conventional solution for the SAR study of chlorination patterns would be to prepare all the remaining 10 unknown chlorinated analogues for in vitro testing. However, as I stated before, their preparation would be challenging since at the outset there were few efficient synthetic methods available to prepare (4S)-chloroleucine diastereomers, especially (4S)-5,5,5-trichloroleucines. Therefore, a simplified 1,17-dinorsintokamide skeleton 3.59, as shown in Scheme 3.16, was proposed to serve as a common structure in our further SAR study of chlorination patterns. Such a design of a simplified skeleton would greatly facilitate the synthesis by eliminating the need for diastereoselective preparation of chloroleucines.  The absence of chiral leucine side chains in 3.59 also provided a dedicated chemical scaffold to study the required chlorination patterns for sintokamide bioactivity. As described, the leucine methyl groups alone did not give the in vitro activity to NCSTD [(4S,10R)-3.16] and their role in the naturally occurring sintokamides was so far not clear. The function of the methyl groups in chlorinated leucine moieties could be evaluated later by in vitro comparison of the natural 78  sintokamides with the related chlorinated 1,17-dinorsintokamide analogues having the same chlorination pattern. In addition, such an SAR design might reveal a simpler structure as a drug lead once the required chlorination patterns and the possible need for the leucine methyl groups were figured out.  3.3.2.3 Natural product guided SAR design of chlorinated 1,17-dinorsintokamides  Table 3.2 Chlorinated substitution in sintokamides A – E    Sint A 3.11 Sint B 3.12 Sint C 3.13 Sint D 3.14 Sint E 3.15  # of Cl in R1 2 3 2 1 0  # of Cl in R2 3 3 2 3 3  Careful examination the chlorination patterns in the five naturally occurring sintokamides and their relative in vitro activities reveals some noticeable trends (Table 3.2). First, the chlorinated leucine moieties vary from one sintokamide to the next. Sintokamides A, D and E contain two different chloroleucine moieties in their structures, while sintokamides B and C have only trichloroleucine or dichloroleucine moieties, respectively. This random combination of different chlorination patterns in the sintokamides may be an indication that chlorinated D- and L-leucines and their derivatives were formed before the peptide coupling step in the biosynthesis of sintokamides. Second, in sintokamides A – E (3.11 – 3.15), the D-leucine side chains have more chlorine atoms compared with their L-methyl tetramate counterparts. While the degree of chlorination in the L-methyl tetramate moieties varies from 0 to 3, the D-leucine moieties 79  generally maintain the highest chlorination degree of 3, except for the degree of 2 in sintokamide C (3.13). Third, since in vitro the sintokamides A, B, and E are more active than sintokamide C, it seems that the chlorination degree of D-leucine moieties, rather than the total number of chlorine atoms in the molecule, is more relevant to the activities of the marine natural products. Based on these preliminary observations, it was hypothesized that the in vitro activity of sintokamides is largely determined by the chlorination pattern in their D-leucine moieties, while the degree of chlorination in the L-methyl tetramate moieties may be only supplementary. However, it should be noted that the above SAR gleaned from the structures of the five naturally occurring sintokamides is limited. The definitive SAR of sintokamides should be based on the in vitro activities of all the 16 possible sintokamide structures with different chlorination patterns.       (4S,10R)-3.16, NCSTD  3.15, sintokamide E  3.60, LPY00 inactive  active  ?  Figure 3.16 Designed structure of LPY00 (3.60), based the structures and activities of NCSTD [(4S,10R)-3.16] and sintokamide E (3.15)  Therefore, a selection of chlorinated norsintokamide analogues with or without correspondences in the natural products were prepared for an SAR study of the required chlorination patterns. For example, naturally unprecedented LPY00 (3.60) with the monochlorinated R side chain was made to examine whether only monochlorination rather than trichlorination would be adequate 80  for the full in vitro activity of sintokamide E (3.15) (Figure 3.16). Moreover, based on the partial SAR disclosed by the natural products, the actual number of synthesized compounds was largely reduced from the maximum possibility of 16. By making only four different patterns of the analogues (two patterns contain two diastereomers as their final products), a clear chlorination patterns/activity relationship for chlorinated 1,17-dinorsintokamides was revealed. Besides, a tentative assessment of the role of the chiral methyl groups was realized by direct in vitro comparison of the most potent sintokamide B (3.12) and its 1,17-dinorsintokamide analogues LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220].  3.3.2.4 General synthetic analyses for chlorinated 1,17-dinorsintokamides  Implementation of the carefully designed SAR study requires at least one working synthetic route. The chloromethyl groups, the methyl tetramate ring, and the amide bond are three major structural elements in 3.59, the simplified sintokamide structure (Figure 3.17). The operational route should line up three types of related organic reactions, namely chlorination, cyclisation, and amide coupling, in an optimal order to afford designed molecules for in vitro SAR evaluation. Statistically, there are six different arrangements to put three component reactions in a certain order. 81   Figure 3.17 Three major structural elements in the simplified 1,17-dinorsintokamide structure  Unlike the commercially available L-leucine (3.29) and Boc-D-leucine [(R)-3.43] in our pervious synthesis to NCSTD [(4S,10R)-3.16], L- and D-norvaline derivatives need to be prepared in advance for making 3.59, the 1,17-dinorsintokamide analogues. Either 5-chlorinated norvalines 3.62 and 3.64 or 5-protected norvalines (S)-3.124 and (R)-3.124 could serve as starting materials for the synthesis. If chloromethyl groups were compatible with both cyclisation and amide coupling conditions, 5-chlorinated norvalines would be ideal as the starting materials; otherwise 5-protected norvalines should be chosen if deprotection/chlorination could work in the presence of newly introduced structural features such as the amide bond and the tetramic acid moiety. Furthermore, it might require different synthetic routes to coordinate reaction conditions for different degrees of chlorination.  In this chapter, three synthetic routes were tried and the remaining three synthetic plans were evaluated based on the results from the trials. Both 5-chlorinated norvalines and 5-protected norvalines were prepared as starting materials for the assembly of 1,17-dinorsintokamide analogues 3.59 based on the described synthetic route to NCSTD [(4S,10R)-3.16]. 82  Unfortunately, 5-chlorinated norvalines could not generate the corresponding methyl tetramate and active ester for amide coupling, while the successful syntheses starting with 5-protected norvalines only afforded monochlorinated norsintokamide analogues LPY00 (3.60) and LPY04 (3.148) that were inactive in vitro. Eventually, a third synthetic route that is applicable for all degrees of side chain chlorination was developed. In this route, the methyl tetramate was formed after 5-chlorinated D- and L-norvalines were coupled together. By comparison, the Route Three dwarfed untested three synthetic route arrangements for their usage of 5-protected norvalines as starting materials and their limited chlorination scopes after deprotection (vide infra).  It has to be pointed out that, during the “hard time” in this project, the author was encouraged by the success of two reported total syntheses of sintokamides. I tried adapting their procedures to the syntheses of our targets, found limitations, and improved them. After all, despite published sintokamide natural product synthesis precedents104, 105, the 1,17-dinorsintokamide scaffold is still a distinctive target molecule and our final understanding of the molecule as well as its synthetic plan, after various failures, is novel and comprehensive.         83  3.3.2.5 Route One: chlorination  cyclisation  amide coupling  3.3.2.5.1 Retrosynthetic analysis for Route One  I initiated a synthetic plan to prepare variously chlorinated 1,17-dinorsintokamide analogues based on our previously successful synthesis to NCSTD [(4S,10R)-3.16] (Scheme 3.17). In this plan, starting materials 5-chlorinated L-norvalines 3.62 and 5-chlorinated D-norvalines 3.64 needed to be prepared first and then the stability of the newly introduced chloromethyl groups in the norvaline residues needed to be tested under cyclisation and amide coupling conditions to complete the synthesis. Strong basic conditions were avoided due to possible dehydrochlorination of the 5-chlorinated norvalines. Therefore, a mild homologation/cyclisation condition was proposed (vide infra) to replace previous reactions involving a strong base such as n-BuLi (Scheme 3.5).  84   Scheme 3.17 Retrosynthetic plan for the preparation of chlorinated 1,17-dinorsintokamide analogues 3.59 from 5-chlorinated L-norvalines (3.62) and 5-chlorinated D-norvalines 3.64 based on the synthesis of NCSTD [(4S,10R)-3.16]          85  3.3.2.5.2 Synthesis of N-Boc-5-chloro-D-norvaline [(R)-3.69]  The preparation of the starting materials was initiated with a diastereoselective alkylation reaction that incorporated different chlorinated bromopropanes 3.65 and 3.70 into diphenyl oxazinones (2S,3R)-3.66 and (2R,3S)-3.66 as chiral auxiliaries.126-129   Scheme 3.18 Synthesis of N-Boc-5-chloro-D-norvaline [(R)-3.69]  As shown in Scheme 3.18, 1-bromo-3-chloropropane (3.65) was added dropwise to (2S, 3R)-N-Boc-2,3-diphenylmorpholin-6-one [(2S,3R)-3.66] in the presence of LiHMDS at 78 °C to give oxazinone adduct (2S,3R,5R)-3.67 in a yield of 42 %. The NMR spectra of (2S,3R,5R)-3.67 were recorded at 100 °C to simplify the complexity of the spectra obtained at room temperature due to slow conformational exchange of the Boc group on the NMR time scale. Treatment of (2S,3R,5R)-3.67 with 95 % TFA in H2O, followed by PdCl2 catalyzed hydrogenolysis gave the crude amino acid. The crude residue was directly subjected to a standard N-Boc protection protocol to give N-Boc-5-chloro-D-norvaline [(R)-3.69] in an overall yield of 24 % from the chiral auxiliary. 86  3.3.2.5.3 Synthesis of N-Boc-5-chloro-L-norvaline [(S)-3.69]  Similarly, by addition of 3.65 to the enantiomeric lactone (2R, 3S)-N-Boc-2,3-diphenylmorpholin-6-one [(2R,3S)-3.66], N-Boc-5-chloro-L-norvaline [(S)-3.69] was obtained in 4 steps with an overall yield of 19 % (Scheme 3.19).   Scheme 3.19 Synthesis of N-Boc-5-chloro-L-norvaline [(S)-3.69]  3.3.2.5.4 Synthesis of N-Boc-5,5-dichloro-D-norvaline [(R)-3.73]  Using a similar protocol, the synthesis originally aimed at 5,5,5-tricholoro-norvalines turned out to produce gem-dichlorinated products instead (Scheme 3.20). 3-bromo-1,1,1-trichloropropane (3.70) was added dropwise to (2S,3R)-3.66 in the presence of LiHMDS at 78 °C to give the gem-dichlorinated alkene (2S,3R,5R)-3.71 as intermediate in a yield of 32 %. The double bond in (2S,3R,5R)-3.71 was saturated in a subsequent hydrogenolysis step. Finally, N-Boc-5,5-dichloro-D-norvaline [(R)-3.73] was obtained in four steps with an overall yield of 14 %.  87   Scheme 3.20 Synthesis of of N-Boc-5,5-dichloro-D-norvaline [(R)-3.73]  3.3.2.5.5 Synthesis of N-Boc-5,5-dichloro-L-norvaline [(S)-3.73]  Starting with 3.70 and enantiomeric (2R,3S)-3.66, N-Boc-5,5-dichloro-L-norvaline [(S)-3.73] was obtained in 4 steps with an overall yield of 16 % (Scheme 3.21).   Scheme 3.21 Synthesis of N-Boc-5,5-dichloro-L-norvaline [(S)-3.73]    88  3.3.2.5.6 Synthesis of N-Boc-5,5,5-trichloronorvaline racemates (3.79)  As the inevitable usage of strong base for the coupling reaction causes elimination, any plan to make trichlorinated amino acids must involve milder conditions. Despite nature’s apparently efficient synthesis of trichlorinated leucine residues, all laboratory synthetic attempts have been built on the Kharasch reaction130 and the reported chemical transformations were limited in scope and they proceeded in low yields.125, 131   Scheme 3.22 A recent success in stereoselective trichloromethylation  Recently, A stereoselective radical addition (Scheme 3.22) has been reported,132 which greatly facilitates the synthesis of natural products containing 5,5,5-trichloroleucine building blocks. A short description will be given later in this chapter.  89   Scheme 3.23 Synthesis of N-Boc-5,5,5-trichloronorvaline racemates (3.79)  Our initial approach to the preparation of racemic 5,5,5-trichloro-norvalines involved a conjugate addition followed by a Strecker reaction (Scheme 3.23). Resolution of the racemates was planned in a later stage to give both enantiopure compounds for further reaction steps. Methyl acrylate (3.74) in chloroform was added to a 10 M NaOH solution in the presence of the phase transfer catalyst BnEt3N+Cl− to give an adduct 3.75 in a yield of 81 %.133 Reduction of 3.75 with 1 equivalent DIBAL-H gave the aldehyde 3.76 in a yield of 88 %.133 A Strecker reaction134 was carried out by sequential addition of BnNH2 and TMSCN135, 136 to 3.76 without solvent137-139 to give the cyanide 3.77 in a quantitative yield. Basic hydrolysis140-142 of 3.77 in 3 M NaOH and 30 % H2O2 furnished N-benzyl 5,5,5-trichloronorvalines (3.78). Hydrogenolysis143, 144 of 3.78 followed by N-Boc protection145 gave N-Boc-5,5,5-trichloro-norvalines (3.79). The overall yield starting from methyl acrylate after 6 steps was 27 %.   90   Scheme 3.24 The 1,4-addition condition worked for methyl acrylate (3.74) could not be used in the case of methyl crotonate (3.80).  It was planned to carry out a similar synthesis to racemic N-Boc-5,5,5-trichloroleucines, which could serve as starting materials for the total synthesis of sintokamides. To our surprise, methyl crotonate (3.80) as substrate failed to afford compound 3.81 under the same 1,4-addition conditions (Scheme 3.24). It was found later that Shono and co-workers had successfully performed this conjugate addition by generating the stabilized CCl3 anion under electroorganic conditions and the reported yield was 78 % (Scheme 3.25).146, 147 However, short of such specialised facilities and related expertise, Shono’s reaction conditions were not tested in the lab. For that reason, the conjugate addition approach to trichlorinated leucines was discontinued.  Scheme 3.25 The reported 1,4-addition between methyl crotonate (3.80) and chloroform under electroorganic conditions  91  3.3.2.5.7 An alternative method to make tetramic acids: condensation with Meldrum’s acid  To prepare chlorinated methyl tetramates 3.61 from 5-chlorinated L-norvalines 3.62, the usage of n-BuLi for the homologation of L-leucine chloride (3.36) in the NCSTD synthesis must be replaced with milder conditions to avoid possible dehydrochlorination. In addition, the alternative method needed to be shorter than the previous five-step synthesis, due to the low overall yields of 5-chlorinated norvalines as starting materials.   92   Scheme 3.26 An ideal synthetic route to chlorinated tetramate acids should be milder in conditions and shorter in steps based on the NCSTD synthesis.  In 1987, Jouin and Castro proposed that the tetramic acid derivatives, as precursors to statines, could be easily obtained in two steps in high yield by the treatment of isopropenyl chloroformate (IPCF) /DMAP activated N-protected amino acids with Meldrum’s acid (3.85) (Scheme 3.27).148-150 Even though Jouin claimed that IPCF was the only reagent that would accomplish this transformation and the reaction conditions were stringent, Ma151 and Kraus152 later found that the more affordable and commonly used activating reagent DCC could also be utilized to 93  give tetramic acids in high yield with excellent chiral purity. Their modification to Jouin’s protocol was simple: just stabilizing the intermediate by rapidly diluting the reaction mixture with cold ethyl acetate. In 2006, Tønder and co-workers described that they could also get enantiopure products on gram-scales without chromatography by replacing DCC with EDCI in their synthesis of tetramic acids.153   Scheme 3.27 Preparation of N-Boc tetramic acid 3.86 in Jouin’s synthesis of N-protected statine 3.88  The use of Meldrum’s acid (3.85) would greatly facilitate our construction of the methyl tetramate moiety in our sintokamide analogues. The new protocol was first tested on Boc-L-leucine [(S)-3.43] (Scheme 3.28). The carboxylic acid in (S)-3.43 was activated by DCC/DMAP and then coupled with 3.85. The coupling product 3.89 was unstable, and it was pyrolysed in refluxing ethyl acetate solution for 30 min to give the tetramic acid 3.86 via an intramolecular cyclisation of a highly electrophilic ketene intermediate 3.90. Treatment of 3.86 with trimethyl orthoformate in methanol in the presence of catalytic H2SO4, the same condition used for O-methylation of 3.37 in Scheme 3.8, furnished the vinylogous acid methyl ester 3.32 and 94  simultaneously removed the N-Boc protection in one pot. The overall yield is 40 % from Boc-L-leucine.   Scheme 3.28 Synthesis of methyl tetramate 3.32 via homologation with Meldrum’s acid under Ma and Kraus’ conditions  3.3.2.5.8 Synthesis of chlorinated tetramic acids from 5-chlorinated norvalines  Unfortunately, the cyclisation condition for converting an amino acid to a tetramic acid, which worked for non-chlorinated L-leucine, did not take place when our previous made 5-chlorinated norvalines were used as substrates (Figure 3.18). Replacing DCC with EDCI did not afford the desired products under similar reaction conditions.  95   Figure 3.18 Attempts to make chlorinated tetramic acids from 5-chlorinated norvalines  3.3.2.5.9 Synthesis of active chlorinated esters from 5-chlorinated norvalines  Contrary to the reaction of non-chlorinated leucine, again, the condensation reactions between prepared 5-chlorinated norvalines with p-nitrophenol in the presence of DCC failed to furnish active chlorinated esters for amide coupling (Figure 3.19).  96   Figure 3.19 Attempts to make chlorinated active esters from 5-chlorinated norvalines  Consequently, no amide coupling reactions could be carried out next due to lack of coupling components. Our effort based on Route One, i.e., chlorination  cyclisation  amide coupling, had to be terminated at this point.  3.3.2.5.10 Route One: summary and discussion  Unfortunately, Route One failed to produce 1,17-dinorsintokamide analogues for the SAR study. The synthetic plan was based on our previous successful synthesis of NCSTD [(4S,10R)-3.16] but started with 5-chlorinated norvalines 3.62 and 3.64. For preparation of the starting materials, both enantiomers of N-Boc-5-chloro-norvalines, (R)-3.69 and (S)-3.69, were accessible from a procedure initiated with a diastereoselective reaction by incorporating 1-bromo-3-chloropropane (3.65) into both diphenyl oxazinone chiral auxiliaries, (2S,3R)-3.66 and (2R,3S)-3.66. Our 97  attempts to make enantiopure 5,5,5-trichloro-norvaline derivatives (R)-3.79 and (S)-3.79 starting with 3-bromo-1,1,1-trichloropropane (3.70) using the same protocol, instead gave 5,5-dichloro-norvaline derivatives, (R)-3.73 and (S)-3.73. The dehydrochlorination intermediates were noticed after the alkylation reactions in the presence of LiHMDS, and the alkenes in adducts (2S,3R,5R)-3.71 and (2R,3S,5S)-3.71 were further saturated under the hydrogenolysis conditions used for cleaving the auxiliaries from the products. Ultimately, racemic N-Boc-5,5,5-trichloro-norvalines (3.79) were made from conjugate addition of methyl acrylate with CCl3 anion, followed by Strecker synthesis of the amino acids.  Because chloromethyl groups in the norvalines were found to be sensitive to a strong base, the use of n-BuLi in the preparation of the tetramic acid moiety in Scheme 3.5 needed be replaced with a protocol using milder conditions. A desired method shown in Scheme 3.28 was found that DCC/DMAP activated amino acid could be acylated with Meldrum’s acid (3.85) to generate tetramic acids after decarboxylation. This new reaction proved to be efficient with Boc-L-leucine [(S)-3.43] as a starting material.  Disappointingly, none of the prepared N-Boc-5-chlorinated norvalines could generate related tetramic acids under the new protocol. Besides, the condensation condition that worked for Boc-D-leucine [(R)-3,43] to make active esters 3.45 with p-nitrophenol (3.44), as shown in Scheme 3.10, was inapplicable in all cases of 5-chlorinated norvalines. A tentative explanation was made that the electrophilic chloromethyl groups in the norvalines might be attacked by the nucleophilic Meldrum’s acid and p-nitrophenol used in the reactions.  98  A  B  Scheme 3.29 Comparison of (A) the successful synthesis of NCSTD [(4S,10R)-3.16] and (B) the failed synthesis of chlorinated 1,17-dinorsintokamide analogues based on Route One  To summarize, the success of NCSTD [(4S,10R)-3.16] synthesis could not be repeated by using 5-chlorinated norvalines as starting materials (Scheme 3.29). The lability of chloromethyl groups was not only observed in the presence of a strong base, but also with the nucleophilic reagents irreplaceable in the cyclisation and active ester preparation steps. Furthermore, the possible use 99  of a strong base to deprotonate chlorinated methyl tetramate in the amide coupling step might be another disadvantage of Route One. Based on their optimization of pyrrolidinone related reactions, Tønder and co-workers suggested n-BuLi (pKa = 50) or LiHMDS (pKa = 30) for the amide coupling step.154  3.3.2.6 Route Two: cyclisation  amide coupling  chlorination  In 2006, Jiménez and Rodríguez reported the structure of a new tetrachlorinated dipeptide dysithiazolamide (3.49) from Dysidea sp.155, and the proposed absolute stereochemistry was confirmed by their total synthesis published in 2008 (Scheme 3.30 to Scheme 3.33).156   Scheme 3.30 Total synthesis of dysithiazolamide (3.49) (Part A)  100   Scheme 3.31 Total synthesis of dysithiazolamide (3.49) (Part B)   Scheme 3.32 Total synthesis of dysithiazolamide (3.49) (Part C)  101   Scheme 3.33 Total synthesis of dysithiazolamide (3.49) (Part D)  Structurally, the marine natural product can be regarded as a dipeptide derived from two units of (4S)-5,5-dichloro-L-leucine (3.103). In their synthesis, compound 3.101, originating from L-glutamic acid, was used as a common precursor. The 4S methyl group in 3.101 was introduced earlier by a 1,3-asymmetric induction (Scheme 3.30),157 and the gem-dichlorides were installed by oxidation of the primary alcohols to aldehydes followed by treatment with hydrazine and CuCl2.158-160 To gem-dichlorinate two different synthons, the alcohol in 3.101 was either treated directly (Scheme 3.31), or kept protected as a TBDPS silyl ether until N-methylation, carboxamide preparation,161 and Hantzsch thiazole synthesis were finished (Scheme 3.32). Finally, the convergent synthesis was completed by condensation of the two synthons 3.103 and 3.108 in the presence of the coupling reagent bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP) (Scheme 3.33). What Jiménez and Rodríguez presented in their synthesis of dysithiazolamide (3.49), especially the manipulation of (4S)-5-silyloxy-L-leucine (3.104), stimulated us to reconsider the possibility of preparing chlorinated 1,17-dinorsintokamide analogues based on our NCSTD synthesis, namely, Route Two. 102  Table 3.3 Possible advantages of 5-protected norvalines over 5-chlorinated norvalines as starting materials to prepare 1,17-dinorsintokamide analogues based on our NCSTD synthesis starting materials susceptibility Meldrum’s acid p-nitrophenol strong base leucines stable stable stable 5-chlorinated norvalines observed observed very possible 5-silyloxy-norvalines not likely not likely not likely  As listed in Table 3.3, using 5-silyloxy-norvalines as starting materials based on the synthetic route of NCSTD could overcome all the observed and possible susceptibilities of 5-chlorinated norvalines in the practice of Route One. First, unlike the electrophilic chloromethyl groups, the sterically hindered silyl oxygen is not likely to be attacked by nucleophiles such as Meldrum’s acid and p-nitrophenol. Therefore, it is possible to synthesize tetramic acids and active esters from 5-silyloxy-norvalines. Second, in the course of Route One, there was no chance to test the lability of chloromethyl groups under the amide coupling condition. Even though during the coupling step in the synthesis of (±)-dysidin (Scheme 3.3), the trichloromethyl group was intact in the presence of MeMgBr, Grignard reagents proved to be ineffective in our synthesis of NCSTD, where the deprotonation was accomplished instead by treatment of n-BuLi (pKa = 50). The presence of a side chain protecting group allowed us to pick another suitable base, such as NaH (pKa = 35) or LiHMDS (pKa = 30), which would be strong enough to deprotonate the N-H in the methyl tetramate (pKa = 24), without exposing the lability of chloromethyl groups (pKa chloroform= 15.5).  103   Scheme 3.34 Total synthesis of sintokamide C (3.13) (Part A)   Scheme 3.35 Total synthesis of sintokamide C (3.13) (Part B) 104  The feasibility of our new idea was supported by the total synthesis of sintokamide C (3.13), published by Xu and Ye et al. in early 2010 (Scheme 3.34 to Scheme 3.36).104 The authors believed that the gem-dichlorides in sintokamide C would be susceptible to a strong base, so their convergent synthesis started with preparing a pair of silylated diastereomeric amino acids (2S,4S)-3.110 and (2R,2S)-3.110 for the tetramic acid 3.112 and active ester 3.119. Their assembly of 3.112 was based on Ma and Kraus’ modification (DCC/DMAP/Meldrum’s acid), followed by methylation with diazomethane (Scheme 3.34). On the other hand, the N-fully protected active ester 3.119162 was obtained after a series of functionality manipulations as shown in Scheme 3.35.  105   Scheme 3.36 Total synthesis of sintokamide C (3.13) (Part C)  In their paper, Xu and Ye provided reaction details that served as useful guides for our synthetic efforts (Scheme 3.36). In the presence of protective tert-butyldiphenylsilyl ethers 3.112 and 3.119, amide coupling could take place with either n-BuLi or LiHMDS, but LiHMDS afforded silylated dipeptide 3.120 with a higher yield (70 % versus 30 %). After cleavage of the silyl ethers, the resulting diol 3.121 was oxidized under Parikh – Doering conditions to give bis-aldehyde 3.122 ready for the crucial chlorination. Despite examples in syntheses of dysamide B163 and dysithiazolamide (3.49)156, the authors found that the only dichlorination condition 106  which worked in the case of 3.13 was treatment of compound 3.122 with triphenyl phosphite/Cl2 reagent164. At last, removal of the N-Boc protecting group concluded their total synthesis of sintokamide C.   Scheme 3.37 Synthesis of bis-monochlorinated sintokamide 3.123 from the intermediate diol 3.121.  Likewise, Xu and Ye prepared an unnatural bis-monochlorinated sintokamide 3.123 from the intermediate diol 3.121 (Scheme 3.37).           107  3.3.2.6.1 Retrosynthetic analysis for Route Two  Based on lessons learned in Route One, plus successful stories of making dysithiazolamide and sintokamide C, a revised synthetic plan Route Two was proposed to prepare chlorinated 1,17-dinorsintokamide analogues by manipulation of the alcohol functions in the desilylated intermediate 3.128 into chloromethyl groups in the last step (Scheme 3.38). The rest of the synthetic plan is similar to Route One: 5-silyloxy-norvaline derivatives (S)-3.124 and (R)-3.124, originating from D- and L-glutamic acids [(S)-3.99 and (R)-3.99], served as starting materials to prepare the corresponding methyl tetramate 3.125 and active ester 3.126 components for the amide coupling to give the O-silylated 1,17-dinorsintokamide intermediate 3.127. Accordingly, a flow chart of Route Two could be drawn as “cyclisation  amide coupling  chlorination”.  If all of the desired hydroxyl group manipulations went as anticipated, the intermediate diol 3.128 could serve as a common precursor to 1,17-dinorsintokamide analogues with different degrees of chlorination. However, 3.128 could only lead to analogues with the same degree of chlorination on each side chain without introduction of selective protecting groups.  108   Scheme 3.38 Retrosynthetic plan for chlorinated 1,17-dinorsintokamide analogues from 5-silyloxy-norvalines originating from D- and L-glutamic acids    109  3.3.2.6.2 Synthesis of N-Boc-5-silyloxyl-L-norvaline (S)-3.124  The synthesis of 5-protected L-norvaline (S)-3.124 is shown in Scheme 3.39. Methylation of both carboxylic acids in (S)-3.99 followed by N-Boc protection in one pot yielded N-Boc-glutamate (S)-3.129 in 96 % yield. A second Boc protecting group was introduced quantitatively by treatment of (S)-3.129 with Boc2O and DMAP to afford the N-fully protected glutamate (S)-3.130. Reduction of (S)-3.130 with 3 equivalents DIBAL-H at 40 °C gave the primary alcohol (S)-3.131 in a yield of 46 %. José Padrón et al. suggested that complete protection of the amine in glutamate was required in the reduction reaction for desired yield.165 The protection of the primary alcohol function in (S)-3.131 was finished in a quantitative yield by treatment of (S)-3.131 with TBDPS-Cl and imidazole to give the silyl ether (S)-3.132. Reaction of compound (S)-3.132 with LiBr (conversion rate: 95 %) followed by hydroxylation (conversion rate: 63 %) afforded the proper substrate N-Boc-5-silyloxyl-L-norvaline (S)-3.134 for the next tetramic acid formation. To summarize, (S)-3.124 was obtained in 7 steps and the overall yield from L-glutamic acid [(S)-3.99] was 26 %.  110   Scheme 3.39 Synthesis of N-Boc-5-silyloxyl-L-norvaline (S)-3.134 from L-glutamic acid [(S)-3.99]           111  3.3.2.6.3 Synthesis of methyl tetramate 3.125 from N-Boc-5-silyloxyl-L-norvaline (S)-3.124  The preparation of the methyl tetramate 3.125 started with N-Boc-5-silyloxyl-L-norvaline (S)-3.124 (Scheme 3.40). Transformation of (S)-3.124 using the Ma and Kraus modification gave the tetramic acid 3.134 in a yield of 50 %. However, an O-methylated pyrrolizidinone product 3.136 was obtained upon treatment of compound 3.134 with trimethyl orthoformate under acid catalysis. The structure of 3.136 was based on interpretation of MS data. It was proposed that the 5-exo-tet product was a result of an intramolecular cyclisation after the deprotection of both silyl ether and N-Boc groups in 3.134 under the acidic reaction conditions.   Scheme 3.40 Attempt to make methyl tetramate from N-Boc-5-silyloxyl-L-norvaline (S)-3.124    112  In the literature, the unstable diazomethane was used in the total synthesis of sintokamide C (Scheme 3.34). Its non-explosive alternative trimethylsilyl-diazomethane (TMSCHN2) was found to be relatively safe and it was therefore chosen as the methylating reagent in our synthesis. As shown in Scheme 3.41, treatment of compound 3.134 with TMSCHN2, followed by N-deprotection with TFA, afforded the N-deprotected methyl tetramate 3.125, which was ready for the key coupling reaction.   Scheme 3.41 Synthesis of methyl tetramate 3.125  3.3.2.6.4 Synthesis of N-Boc-5-silyloxyl-D-norvaline (R)-3.124  As illustrated in Scheme 3.42, N-Boc-5-silyloxyl-D-norvaline (R)-3.124 could be obtained in 7 steps with an overall yield from D-glutamic acid [(R)-3.99] of 36 %.  113   Scheme 3.42 Synthesis of N-Boc-5-silyloxyl-D-norvaline (R)-3.124 from D-glutamic acid [(R)-3.99]  3.3.2.6.5 Synthesis of active ester 3.126 from N-Boc-5-silyloxyl-D-norvaline (R)-3.124  Scheme 3.43 shows the preparation of the active ester 3.126 from N-Boc-5-silyloxyl-D-norvaline (R)-3.124. Mild esterification of (R)-3.124 yielded benzyl ester 3.138 in 92 % for the purpose of further functional group manipulation.166, 167 Removal of the N-Boc, followed by N-propionylation gave amide 3.139 in 59 % yield over 2 steps. Reaction of compound 3.139 with Boc2O and DMAP afforded N-fully protected intermediate 3.140, which was subjected to hydrogenolysis, followed by treatment with pentafluorophenol162 and DCC to give 3.126 in 84 % yield over 2 steps. To summarize, there were 6 steps and an overall yield of 28 % from the 5-silyloxylated-D-norvaline (R)-3.124 to compound 3.126, the active ester required for coupling. 114   Scheme 3.43 Synthesis of active ester 3.126 from N-Boc-5-silyloxyl-D-norvaline (R)-3.124  As shown in Scheme 3.44, benzylation of the carboxylic acid in (R)-3.124 was necessary since the model reaction failed to produce N-fully protected active ester in the last step. Besides, deprotonation of 3.45 followed by addition of propionyl chloride did not provide the desired product either.  115   Scheme 3.44 Attempt to make active ester without benzyl protection of the amino acid  3.3.2.6.6 Selection of monochlorinated analogues for the SAR study  In consideration of the economy (the number of chlorine atoms) and the efficiency (the difficulty of polychlorination), monochlorination of the norsintokamide skeleton would be preferred if the resulting analogues were active in vitro. As shown in Figure 3.20, there are 3 different possible structures bearing at least one chloromethyl group.       3.60  3.148  3.143 LPY00  LPY04    Figure 3.20 Structures of monochlorinated 1-nor-, 17-nor-, and 1,17-dinor-sintokamide analogues  116  Monochlorinated LPY00 (3.60) and bis-monochlorinated LPY04 (3.148), were chosen as synthetic targets for the SAR study (Figure 3.21). As discussed before, there is no monochlorination found in the D-leucine side chain among the naturally occurring sintokamides. Therefore, the activities LPY00 (3.60) and LPY04 (3.148), the respective monochlorinated 17-norsintokamide and 1,17-dinorsintokamide analogues of active sintokamide E (3.15) and sintokamide D (3.14), should reveal the capacity of monochlorination to elicit AR NTD inhibitory activity based on the new skeletons.   Figure 3.21 Structure comparison of sintokamides E and D, LPY00 (3.60), and LPY04 (3.148)  3.3.2.6.7 Synthesis of LPY00 (3.60), the monochlorinated 17-norsintokamide analogue  Deprotonation of L-leucine-derived methyl tetramate 3.32 with LiHMDS, followed by addition of the active ester 3.126 gave the adduct 3.144 in a yield of 38 %. The silyl protecting group in 117  3.144 was removed by treatment with HF in pyridine to give alcohol 3.145 in a yield of 73 %. Successive chlorination (76 % yield) and removal of the N-Boc furnished LPY00 (3.60) as our first synthetic chlorinated analogue. To summarize, there are 21 steps (the longest linear steps are 17) in this convergent synthesis and the final yield is 1.8 % based on the longest linear steps.   Scheme 3.45 Synthesis of LPY00 (3.60), the monochlorinated 17-norsintokamide analogue   118  3.3.2.6.8 Synthesis of LPY04 (3.148), the bis-monochlorinated 1,17-dinorsintokamide analogue  Using a similar protocol, bis-monochlorinated analogue LPY04 (3.148) was prepared as illustrated in Scheme 3.46. For the desilylation step, the author tried to replace HF/pyridine with TBAF for easier workup and safety reasons. However, treatment of the bis-silyloxylated intermediate 3.127 with TBAF168 failed to give diol 3.128 for the next step. To summarize, there are 28 steps (the longest linear route is 17 steps) in this convergent synthesis and the final yield is 2.2 % based on the longest linear path.  119   Scheme 3.46 Synthesis of LPY04 (3.148), the bis-monochlorinated 1,17-dinorsintokamide analogue  3.3.2.6.9 Attempt to make 1,17-dinorsintokamide C from diol 3.128  The published procedure used for installation of the gem-dichlorides in the total synthesis of sintokamide C was expected to transform diol 3.128 to sintokamide C analogue LPY09 [(4S,10R)-3.210] for the SAR study (Scheme 3.47). Treatment of 3.128 with Dess – Martin periodinane, instead of the Parikh – Doering oxidation condition used in the literature, gave the desired bis-aldehyde 3.149 in a yield of 50 %. Unfortunately, further reaction of 3.149 with 120  triphenyl phosphite and chlorine gas failed to afford the desired tetrachlorinated product. Instead, five chlorine atoms were detected in 3.150 based on the m/z value and the isotope pattern in the mass spectrum. The additional chlorine atom was tentatively assigned to C-3 on the methyl tetramate ring as shown in the putative structure of 3.150.   Scheme 3.47 Attempt to make 1,17-dinorsintokamide C from diol 3.128  3.3.2.6.10 Attempt to make 1,17-dinorsintokamide B from the diol 3.151  Even though failed to make the 1,17-dinorsintokamide C analogue, the author still explored the potential to prepare 1,17-dinorsintokamide B analogue 3.153 by transforming the alcohol functions in desilylated homologue 3.151 to trichloromethyl groups via a dihalide intermediate 3.152 (Scheme 3.48). Compound 3.151 should be easily halogenated to give 3.152 as a dihalide, 121  and the following trichloromethylation appeared to be straightforward. However, the question was whether the electrophilic alkyl halides could be readily attacked by trichloromethyl anion without interfering with the methyl tetramate in 3.152, which already caused trouble in our efforts to make the tetrachlorinated analogues.   Scheme 3.48 Synthetic plan to 1,17-dinorsintokamide B analogue based on trichloromethylation of the homologous dihalide 3.152 122  Table 3.4 Reported trichloromethylation conditions in literature substrate base carbanion/substrate CHCl3/base temperature & solvent product yield 169 n-BuLi 1.4 1.2 −100 °C to −75 °C in THF/HMPA  68 % 169 n-BuLi 1.3 1.2 −100 °C to −75 °C in THF/HMPA  90 % 169 n-BuLi 1.6 1.2 −100 °C to −75 °C in THF/HMPA  68 % 170 n-BuLi 1.4 1.4 −98 °C to −75 °C in THF/HMPA  71 % 171 NaH 1.0 1.0 0 °C in DMF  57 % 172 t-BuOK 1.0 4.0 −40 °C in DMF  42 %  123  Based on the knowledge learned from the literature and our previous synthesis, it was thought that the proposed synthetic plan to make the sintokamide B analogue was reasonable. Six reaction conditions were reported for alkylation of primary alkyl halides with the trichloromethyl carbanion (Table 3.4).169-172 In most cases, the deprotonation with a base was performed when chloroform was present in excess so that the strongest base left in the resulting reaction system was the CCl3 carbanion (pKa chloroform= 15.5). Since no evidence of epimerisation was observed for the 4-alkyl methyl tetramate even in the presence of LiHMDS and n-BuLi, it was supposed that the basic condition of the alkylation should be safe for our new sintokamide skeleton, and the trichloromethylation of the related dihalide precursor should occur.  124   Scheme 3.49 Retrosynthetic plan to bis-trichlorinated 1,17-dinorsintokamide B analogue 3.153 based on Route Two  Because a successful trichloromethylation would extend the length of each haloalkyl side chain by one carbon unit, our previous retrosynthetic plan of Route Two requires modifications for the new synthetic target (Scheme 3.49). The new synthesis needs to be initiated with L- and D-125  aspartic acids [(S)-3.166 and (R)-3.166], instead of the glutamic acids, to produce the desired precursors for the key trichloromethylation reaction.  3.3.2.6.11 Synthesis of methyl tetramate 3.168 from L-aspartic acid [(S)-3.166]  As shown in Scheme 3.50, the synthesis was carried out starting with L-aspartic acid [(S)-3.166] to generate the methyl tetramate 3.168 over 11 steps in an overall yield of 6.7 %.   Scheme 3.50 Synthesis of tetramic acid 3.168 from L-aspartic acid [(S)-3.166]   126  3.3.2.6.12 Synthesis of active ester 3.169 from D-aspartic acid [(R)-3.166]  Likewise, as shown in Scheme 3.51, the active ester 3.169 was obtained originating from D-aspartic acid [(R)-3.166] over 13 steps in an overall yield of 17 %.   Scheme 3.51 Synthesis of active ester 3.169 from D-aspartic acid [(R)-3.166]  127  3.3.2.6.13 Attempt to make 1,17-dinorsintokamide B (3.153)  Our attempt to make the sintokamide B analogue 3.153 from the diol 3.151 is shown in Scheme 3.52. After coupling and desilylation, diol 3.151 was obtained over two steps in 40 % yield. Treatment of 3.151 with carbon tetrabromide and PPh3 gave the dibromide 3.180 in 55 % yield,173 which was ready for the next key reaction. Reaction of 3.180 with chloroform and NaH in DMF gave compound 3.181. However, instead of the desired linear trichloromethyl group, a cyclopropane ring was detected at C-4 on the methyl tetramate fragment in 3.181.   128   Scheme 3.52 Attempt to make sintokamide B analogue 3.153 from diol 3.151  The N-Boc methyl tetramate ring system in 3.183 was selected as a simplified substrate to screen effective conditions for the desired trichloromethylation (Scheme 3.53). Desilylation of the homologous N-Boc methyl tetramate 3.176, followed by treatment with iodine, PPh3 and imidazole, gave iodide 3.183 in a yield of 83 %.174-176   129   Scheme 3.53 Synthesis of iodide 3.183 for screen of effective trichloromethylation conditions  3.183 was tested with different trichloromethylation conditions, as listed in Table 3.5.  Table 3.5 Conditions for trichloromethylation of 3.183 entry substrate Base substrate/base/CHCl3 temperature product yield 1 3.183 t-BuOK 1.0/2.0/4.0 0 °C N/A N/A 2 3.183 KH 1.0/2.8/4.0 40 °C 3.184 5.0 % 3 3.183 n-BuLi 1.0/2.0/100.0 40 °C 3.184 5.6 % 4 3.183 n-BuLi 1.0/1.0/50.0 40 °C 3.184 5.3 % 5 3.183 n-BuLi 1.0/1.0/50.0 78 °C 3.184 8.7 % 6 3.183 n-BuLi 1.0/1.0/50.0 100 °C 3.184 7.5 %  Unfortunately, compound 3.184, as a result from an intramolecular 3-exo-tet cyclisation, although often in low yields (5 % to 9 %), was always observed in some conditions and no trace of intermolecular SN2 reaction product was detected in the trials.  130  A model reaction was tried to simulate the trichloromethylation at the D-homoserine side chain (Scheme 3.54).177-179 Iodination of (R)-3.173 with iodine and PPh3 gave iodide 3.185 in a yield of 71 %. Trichloromethylation of 3.185 could occur, but with a low yield of 11 %.   Scheme 3.54 Trichloromethylation of 3.185  3.3.2.6.14 Bioassay results and their SAR implications  Compounds LPY00 (3.60) and LPY04 (3.148) were tested in vitro and neither of them was active against R1881 activation of luciferase assays (Figure 3.22). At a concentration of 20 M, LPY00 and LPY04 failed to inhibit the relative luciferase activity and their relative luciferase units (RLUs) were 144 % and 91 % compared with the blank control, respectively.  131   Figure 3.22 Bioassay results of LPY00 (3.60) and LPY04 (3.148) on R1881 activation of P6.1luc  Even though Route Two afforded only two monochlorinated 1,17-dinorsintokamide analogues for the screening and neither had in vitro activity, those inactive analogues with unnatural chlorination patterns still provided valuable SAR information (Figure 3.23). The structural differences between inactive LPY00 (3.60) and active sintokamide E (3.15) showed that only a monochlorinated D-norvaline side chain was not enough to produce the same biological effect as the (4S)-5,5,5-trichloro-D-leucine counterpart in the natural product. Similar information could be drawn from the comparison LPY04 (3.148) and sintokamide D (3.14). Moreover, such a comparison also indicated that the monochlorinated side chain in the methyl L-tetramate moiety could not account for sintokamide D (3.14)’s activity without the participation of either the 17-methyl group as well as the trichlorinated D-leucine moiety at the other terminus. To summarize, monochlorinated 1,17-dinorsintokamide analogues could not exert potent in vitro activity and analogues with higher degrees of chlorination were needed for the next SAR study. Based on 100.0%144.4%91.4%0.0%20.0%40.0%60.0%80.0%100.0%120.0%140.0%160.0%20 μM 20 μMcontrol LPY00 LPY04% PSA-LUCIFERASE ACTIVITY132  LPY00 (3.60) and LPY04 (3.148), the role of the 1- and 17-methyl groups was still not clear because of the negative biological activity results.      3.60 3.15 3.148 3.14 LPY00 sintokamide E LPY04 sintokamide D  Figure 3.23 Structure comparison of LPY00 (3.60) and sintokamide E (3.15); LPY04 (3.148) and sintokamide D (3.14)  3.3.2.6.15 Route Two: summary and discussion  Route Two was our first completed synthetic procedure to chlorinated 1,17-dinorsintokamide analogues (Figure 3.24 and Figure 3.25). Proposed to preclude susceptibilities of pre-installed chloromethyl groups in the presence of strong bases and nucleophiles, Route Two was initiated with 5-silyloxy-norvalines (S)-3.124 and (R)-3.124 and it generated both tetramic acid 3.125 and active ester 3.126 components for amide coupling, neither of which Route One was able to furnish starting with 5-chlorinated-norvalines and using similar reaction conditions (Figure 3.18 and Figure 3.19). The amide coupling reaction in Route Two went smoothly with the aid of LiHMDS. It was initially anticipated that hydroxymethyl groups in the desilylated coupling 133  intermediate 3.128 could be transformed to variously chlorinated methyl groups under selected manipulation conditions (Figure 3.24).   Figure 3.24 Synthetic procedure based on Route Two (Part A)   134   Figure 3.25 Synthetic procedure based on Route Two (Part B)  In practice, I successfully made monochlorinated 1,17-dinorsintokamide analogues LPY00 (3.60) and LPY04 (3.148) by direct chlorination of primary alcohol intermediates 3.145 and 3.128 with CCl4 and PPh3. Unfortunately, polychloromethyl groups could not be introduced into the novel 1,17-dinorsintokamide scaffold under this route. On one hand, Dess – Martin periodinane, rather than the Parikh-Doering activated DMSO oxidant, could oxidise 3.128 to the bis-aldehyde 3.149, but the published conditions for converting 3.149 to the gem-dichlorides found in sintokamide C did not work for the simplified new scaffold. In addition, trichloromethylation of the 4-alkylhalide side chains in the methyl tetramate moiety in 3.180 135  originating from L-aspartic acid was always overwhelmed by formation of a cyclopropane ring via an intramolecular 3-exo-tet mechanism. Due to the above difficulties, I did not try selective protection to synthesize unevenly chlorinated analogues such as 1,17-dinorsintokamide A.  To summarize, Route Two was found to be a viable synthetic route to 1,17-dinorsintokamides with only monochlorinated side chains. By switching starting materials to 5-silyloxy-norvalines (S)-3.124 and (R)-3.124, Route Two overcame all the drawbacks in Route One and generated silylated adducts 3.144 and 3.127 in the presence of LiHMDS. However, after desilylation, only monochlorination of the hydroxymethyl groups occurred to afford LPY00 (3.60) and LPY04 (3.148) for the SAR study; neither gem-dichlorination from related bis-aldehyde 3.149 nor trichloromethylation from brominated homologue 3.180 was achieved to give polychlorinated 1,17-dinorsintokamide analogues. Unlike the branched leucine side chains in the natural products, the methyl tetramates with linear C-4 alkyl-aldehyde and alkyl-halide in the simplified scaffold appeared to be too reactive to the reaction conditions examined in this study. The negative in vitro results observed for LPY00 (3.60) and LPY04 (3.148) called for a new synthetic route to prepare analogues with higher degrees of chlorination for the SAR study.  3.3.2.7 Route Three: chlorination  amide coupling  cyclisation  Previous synthetic routes failed to afford sintokamide analogues that were active in vitro, so a new route to prepare polychlorinated 1,17-dinorsintokamides was required. Based on lessons learned from the synthetic efforts described above, 4 untested synthetic routes were evaluated and tried to find the most reasonable approach to our synthetic goal. 136   Scheme 3.55 Schemes of Routes Two, Four, and Five  In Route Two, the synthetic plan failed to give the desired products in the second last step because the pre-installed pyrrolidinone ring always got involved in the polychlorination of its side chain (Figure 3.25). Therefore, any synthetic plan, such as Route Four or Route Five (Scheme 3.55), in which formation of tetramic acid fragment would be earlier than polychlorination, should be excluded from further consideration.   Scheme 3.56 Comparison of Routes Three and Six  Two similar synthetic routes could be the possible approaches to our desired synthetic analogues (Scheme 3.56). Unlike the routes mentioned above, polychlorination in Route Three and Route 137  Six would not be performed in the presence of the tetramic acid moiety. Route Three and Route Six share a common dipeptide intermediate comprised of 5-chlorinated norvaline derivatives and the chlorinated dipeptide would serve as a substrate for the formation of the final tetramic acid moiety. Route Three generates the chlorinated dipeptide directly from amide coupling of 5-chlorinated norvaline derivatives, while Route Six generates the dipeptide from 5-protected norvaline derivatives. Therefore, in Route Six, additional deprotection and polychlorination are needed to create the polychlorinated intermediate.  Table 3.6 Preliminary evaluation of the Route Three and the Route Six Factors Route Three Route Six # of key steps 2 4 starting materials 5-chlorinated norvalines 5-protected norvalines racemisation possible possible dipeptide chlorinated to be chlorinated formation of chlorinated tetramic acid not achieved not achieved  Preliminary evaluation of the two plausible plans found that neither was perfect based on my knowledge at the time (Table 3.6). Route Three was preferred as it necessitated fewer steps compared with Route Six, if the amide coupling with 5-polychlorinated norvalines was operational; Route Six could be a Plan B in case the direct amide coupling in Route Three failed. However, the efficiency of the subsequent polychlorination of the deprotected dipeptide substrate was the main concern about Route Six. For example, trichloromethylation of 4-iodo-D-homoserine (3.185) only yielded 5,5,5-trichloro-D-norvaline 3.186 in 11 % yield (Scheme 3.54). Moreover, both Route Three and Route Six were challenged by the possible racemisation of the chiral centres in peptide coupling step, and the incompetence of Ma and Kraus modification to 138  finish the assembly of tetramic acid functionality in the presence of 5-chlorinated norvaline derivatives, as I tried in Route One (Figure 3.18). A set of appropriate coupling conditions and a better ring closure protocol would be required for making either route succeed for our purpose.  3.3.2.7.1 An efficient perhaloalkylation and its application in the syntheses of chloroleucine-containing marine natural products  In January 2010, Zakarian and co-workers reported an efficient stereoselective ruthenium (II)-catalyzed radical perhaloalkylation of titanium enolates as an approach to trichlorinated leucine residues (Scheme 3.57).132 After reductive cleavage of the Evans’ auxiliary in 3.188, the resultant polychloromethylated intermediate 3.189 was enantiopure, and it could be transformed via an asymmetric Strecker reaction to polychloroleucine diastereomers such as compound 3.192.180, 181  139   Scheme 3.57 Zakarian’s diastereoselective synthesis of trichloroleucine based on an efficient catalytic radical trichloromethylation            140   Figure 3.26 Evolution based mechanism of Zakarian’s Ru(II)-catalyzed Cl3C· radical addition to titanium enolate of N-acyl Evans’ chiral auxiliary (Adapted with permission of American Chemical Society)  The discovery of the efficient stereoselective trichloromethylation is a result of efforts from generations of organic, inorganic, and theoretical chemists over 65 years (Figure 3.26).132 In 1945, the addition of CHCl3 and CCl4 to olefins in the presence of diacetyl or dibenzoyl peroxide and heat was first observed by Morris Kharasch,130, 182, 183 who set the keynote of such an addition as a free-radical reaction. In 1973, ruthenium(II) complexes like [Ph3P]3Ru(II)Cl2 were introduced to the Kharasch reaction as catalyst to generate a Cl3C· radical, in addition to the radical initiators.184 In 1990, Eguchi found for the first time that TMS enolates could be used as substrates to capture CF3CCl2· radicals generated by a Ru(II) catalyst.185, 186 In 2010, Zakarian and co-workers made the final modification by replacing the TMS enol ether with a titanium 141  enolate187-190 in their Ru(II)-catalyzed radical chloroalkylation of chiral N-acyl oxazolidinone enolates,103, 132 which was inspired by the unconventional biradical character of titanium enolates from -hydroxyl ketones disclosed by Moreira et al. in 2008.191   Figure 3.27 Published syntheses of marine natural products containing chloroleucines by Zakarian and co-workers  With the convenient methodology to diastereoselectively prepare polychlorinated D- and L-leucines, Zakarian and co-workers accomplished the total syntheses of sintokamides A, B and E 142  in November 2010,105 in the context of their synthetic approaches other chloroleucine-derived marine metabolites, such as neodysidenin (3.199),132 dysidenin (3.51),192 dysidin [(S,S)-3.17],192 and barbamide (3.53),192 as shown in Figure 3.27.   Scheme 3.58 Zakarian’s total synthesis of sintokamide A  Synthetically, Zakarian’s successful syntheses of sintokamide A, B and E relied on a procedure similar to Route Three among the six proposed synthetic plans.105 In the paper, they also delivered their solutions to several main concerns during our pre-evaluation of such a synthetic route (Scheme 3.58). In their protecting-group-free synthetic strategy, chloroleucine derivatives 3.200 and 3.201 were subjected to dipeptide amide coupling under selected 1-hydroxy-7-azabenzotriazole (HOAt)193 and EDCI conditions. HOAt is the second generation of triazole peptide coupling reagents after hydroxybenzotriazole (HOBt), and it was accredited “essential” 143  in their syntheses of sintokamides for suppressing racemisation of the stereogenic centres during the amide forming step. Once formed, the single diastereomer 3.202 was hydrolysed, and the resulting carboxylic acid was exposed for the next cyclisation. To complete the tetramic acid formation, IPCF, the exclusive chemical employed by Jouin and Castro in their first preparation of a tetramic acid by incorporation of Meldrum’s acid (3.85) to -amino acids in the aid of DMAP, was reintroduced as an optimal activating reagent rather than DCC or EDCI. Instead of 2 equivalents in the original protocol, the proportion of DMAP was increased to five equivalents. Finally, the total syntheses were concluded by introduction the methyl tetramate via an Mitsunobu reaction.194 In summary, starting with commercially available Evans’ auxiliaries, Zakarian’s total syntheses of sintokamides A, B, E were completed in 14 steps (the longest linear sequence) in a yield of 14 %, 14 %, and 19 %, respectively.             144  3.3.2.7.2 Synthesis of NCSTD1 [(4R,10R)-3.16] and NCSTD2 [(4S,10R)-3.16], the nonchlorinated sintokamide analogues  The feasibility of Zakarian’s method for generating the tetramic acid moiety in 1,17-dinorsintokamide analogues was tested with L-leucine methyl ester hydrochloride (3.204) and N-Boc-D-leucine [(R)-3.43] (Scheme 3.59). A mixture of both commercially available materials was treated with EDCI and HOAt193 to afford dipeptide 3.205 as a single diastereomer in a yield of 91 %. Hydrolysis of compound 3.205 with LiOH gave the carboxylic acid ready for the crucial tetramic acid formation. IPCF/DMAP assisted acylation of the acid with Meldrum’s acid, followed by decarboxylation and vinylogous methylation with TMSCHN2, generated diastereomers (4R,10R)-3.46 and (4S,10R)-3.46. Finally, N-propionylation after removal of the N-Boc protecting group in (4R,10R)-3.46 and (4S,10R)-3.46 accomplished the synthesis of NCSTD1 [(4R,10R)-3.16] and NCSTD2 [(4S,10R)-3.16] in a yield of 37 % and 31 %, respectively.  145   Scheme 3.59 Synthesis of non-chlorinated sintokamide analogues NCSTD1 [(4R,10R)-3.16] and NCSTD2 [(4S,10R)-3.16] based on Route Three        146  3.3.2.7.3 Tetrachlorinated LPY09 [(4S,10R)-3.210] and hexachlorinated LPY11 [(4S,10R)-3.220] are important 1,17-dinorsintokamide analogues for the SAR study    (4S,10R)-3.210 (4S,10R)-3.220 LPY09 LPY10  Tetrachlorinated LPY09 [(4S,10R)-3.210] and hexachlorinated LPY11 [(4S,10R)-3.220] were assigned the highest priority for syntheses and testing in vitro. LPY09 and LPY11 have two identical polychloromethyl groups in their structures, and such an SAR design ensures that the molecules should be active in the bioassay once the threshold value of chlorination degree is reached, regardless of the need for polychlorination in either side chain or the need for synergy between both. In consequence, the inactive compound and its polychloromethyl group should be removed as a point of interest in our future SAR study. Likewise, the two mono-polychlorinated regiomers containing the polychloromethyl groups in the active compound, should be synthesized to determine the preferred location of polychlorination. Moreover, LPY09 [(4S,10R)-3.210] and LPY11 [(4S,10R)-3.220] are the 1,17-dinor-versions of sintokamides C and B, respectively. Comparison of their activities in vitro could help us understand the function of the leucine methyl groups in the AR NTD blocking activity of the naturally occurring sintokamides.  147  In principle, if synthesis permits, the tetrachlorinated LPY09 [(4S,10R)-3.210] should be prepared ahead of hexachlorinated LPY11 [(4S,10R)-3.220] to examine the minimal degree of chlorination required to exert the desired in vitro activity with the 1,17-dinorsintokamide scaffold.  3.3.2.7.4 Retrosynthetic analysis for LPY09 [(4S,10R)-3.210] in Route Three  The retrosynthetic analysis for LPY09 [(4S,10R)-3.210] following Route Three is shown in Scheme 3.60. The target molecule can be regarded as a condensation product between Meldrum’s acid (3.85) and the tetrachlorinated dipeptide (4S,10R)-3.209, which is derived from two units of 5,5-dichloro-norvalines, 3.208 and (R)-3.73. The preparation of the required coupling components could be adapted from the preparation of 5-silyloxy-norvalines in our practice of Route Two (Scheme 3.39 and Scheme 3.42).   148   Scheme 3.60 Retrosynthetic analysis for LPY09 [(4S,10R)-3.210] in Route Three  3.3.2.7.5 Synthesis of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210], the 1,17-dinorsintokamide C analogues  Our synthesis of 5,5-dichloro-L-norvaline methyl ester [(S)-3.208] is illustrated in Scheme 3.61. Reduction of compound (S)-3.130 with 1 equivalent of DIBAL-H gave aldehyde (S)-3.206 in a yield of 59 %. Treatment of (S)-3.206 with (PhO)3P and chlorine gas164 furnished N,N-bis-Boc-gem-dichlorinated L-norvaline [(S)-3.207] in a yield of 83 %. After removal of the N-Boc groups, (S)-3.208 was created in a yield of 80 % and ready for amide formation.  149   Scheme 3.61 Synthesis of 5,5-dichloro-L-norvaline methyl ester [(S)-3.208]  After the successful gem-dichlorination of compound (R)-3.207, N-Boc-5,5-dichloro-D-norvaline [(R)-3.73] was obtained in a yield of 53 % following removal of a N-Boc and hydrolysis of the methyl ester in (R)-3.207 (Scheme 3.62).   Scheme 3.62 Synthesis of N-Boc-5,5-dichloro-D-norvaline (R)-3.73  As shown in Scheme 3.63, starting with the two gem-dichlorinated norvaline derivatives 3.208 and (R)-3.73, the 1,17-dinorsintokamide C analogues LPY09 [(4S,10R)-3.210] and its diastereomer LPY08 [(4R,10R)-3.210], were successfully generated from tetrachlorinated dipeptide (4S,10R)-3.209 in a yield of 18 % and 19 %, based on the application of the synthetic 150  route described earlier in Scheme 3.59 for nonchlorinated sintokamides NCSTD1 [(4R,10R)-3.16] and NCSTD2 [(4S,10R)-3.16].   Scheme 3.63 Synthesis of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210], the 1,17-dinorsintokamide C analogues      151  3.3.2.7.6 Bioassay results and their SAR implications   Figure 3.28 Bioassay results of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210] on R1881 activation of P6.1luc  Figure 3.28 shows the in vitro bioactivity results for LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210]. Both diastereomers inhibited RLU at high concentrations. Their RLUs were 53 % and 66 % compared to that of the control at the concentration of 60 M. However, when LPY08 and LPY09 were tested in parallel with monochlorinated analogues LPY00 (3.60) and LPY04 (3.148) at a concentration of 20 M, all the compounds could be regarded as inactive with a 20 % inhibition rate at most. The direct comparison of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210] with naturally occurring sintokamide C (3.13) to verify the role of methyl groups was not possible due to the paucity of the natural product.  100.0%144.4%91.4%80.1%53.6%86.1%66.2%0.0%20.0%40.0%60.0%80.0%100.0%120.0%140.0%160.0%20 μM 20 μM 20 μM 60 μM 20 μM 60 μMcontrol LPY00 LPY04 LPY08 LPY09% PSA-LUCIFERASE ACTIVITY152    (4R,10R)-3.210 (4S,10R)-3.210 LPY08 LPY09  For SAR information, the inactivity of LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210] suggested that dichlorination in the 1,17-dinorsintokamide skeleton was not sufficient to produce the desired AR NTD inhibition. Therefore, as outlined above, analogues with dichloromethyl groups as their highest degree of chlorination were no longer of interest.  3.3.2.7.7 Synthesis of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], the 1,17-dinorsintokamide B analogues  The next synthetic target was the 1,17-dinorsintokamide B analogue LPY11 [(4S,10R)-3.220] with two trichloromethyl groups in the molecule (Scheme 3.64). The in vitro activities of LPY11 in comparison with naturally occurring sintokamide B (3.12) would support the choice of the 1,17-dinorsintokamide skeleton for SAR study in this project.  153   Scheme 3.64 The 1,17-dinorsintokamide B LPY11 [(4S,10R)-3.220] is the next synthetic target since LPY09 [(4S,10R)-3.210] is inactive.  3.3.2.7.8 Retrosynthetic analysis for LPY11 [(4S,10R)-3.220] in Route Three  As shown in Scheme 3.65, the retrosynthetic analysis for LPY11 [(4S,10R)-3.220] via Route Three is similar to that for LPY09 [(4S,10R)-3.210]. 5,5,5-trichloro-norvaline derivatives could be generated from the asymmetric Strecker reaction using 4,4,4-trichloro-butyraldehyde 3.76 with either enantiomer of Ellman’s sulfinamide as described earlier in Scheme 3.57 for Zakarian’s syntheses of sintokamides A, B and E.  154   Scheme 3.65 Retrosynthetic analysis for LPY11 [(4S,10R)-3.220] in Route Three  3.3.2.7.9 Synthesis of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], the 1,17-dinorsintokamide B analogues  Treatment of 4,4,4-trichloro-butyraldehyde 3.76 with (S)-Ellman’s sulfinamide and anhydrous CuSO4 yielded (S)-sulfinimine (4S)-3.221 in 90 % yield. An enantioselective Strecker reaction of (4S)-3.221 with TMSCN in the presence of a catalytic amount of Sc(OTf)3 furnished a pair of diastereomers. The reaction mixture in DCM automatically recrystallized to afford diastereomerically pure nitrile (S,S)-3.217 in 20 % yield. The absolute configuration of (S,S)-3.217 was determined via single crystal X-ray diffraction analysis. An ORTEP diagram of (S,S)-3.217 is shown in Figure 3.29. Methanolysis of the nitrile in MeOH solution saturated with HCl gas completed the preparation of (S)-3.218 in a yield of 73 % (Scheme 3.66).  155   Scheme 3.66 Synthesis of 5,5,5-trichloro-L-norvaline methyl ester [(S)-3.218]   Figure 3.29 ORTEP diagram of the Strecker product sulfinimine (S,S)-3.217  The synthesis of N-propionyl-5,5,5-trichloro-D-norvaline (3.223) is shown in Scheme 3.67. Diastereomerically pure cyanide (R, R)-3.217 was obtained from recrystallization in a yield of 25 %. Methanolysis of compound (R, R)-3.217, followed by N-propionylation with N-hydroxysuccinimide propionic ester 3.222195 and hydrolysis, gave compound 3.223 in 60 % yield over two steps.  156   Scheme 3.67 Synthesis of N-propionyl-5,5,5-trichloro-D-norvaline (3.223)  Unfortunately, the coupling reaction between compound (S)-3.218 and compound 3.223 in the presence of HOAt and EDCI failed to give the anticipated dipeptide 3.224 (Scheme 3.68). Since N-Boc-5,5-dichloro-D-norvaline [(R)-3.73] in Scheme 3.63 was the effective substrate for tetrachlorodipeptide (4S,10R)-3.209, the N-propionyl group in 3.223 is probably not bulky enough to prevent the possibility of intermolecular dimerization.   Scheme 3.68 Attempt to make dipeptide 3.224 by coupling compound (S)-3.218 and compound 3.223   157   Scheme 3.69 Model reaction of making dipeptide by coupling compound (S)-3.218 and Boc-D-leucine [(R)-3.43]   As shown in Scheme 3.69, our assumption was supported by the result of coupling compound (S)-3.218 with Boc-D-leucine [(R)-3.43]. Under the same conditions, the coupling reaction proceeded smoothly and yielded dipeptide (4S,10R)-3.229 in 87 %.   Scheme 3.70 Synthesis of N-Boc-5,5,5-trichloro-D-norvaline [(R)-3.79]  The synthesis of N-Boc-5,5,5-trichloro-D-norvaline [(R)-3.79] was straightforward (Scheme 3.70). After acidic hydrolysis of (R,R)-3.217 followed by treatment with Boc2O and NaHCO3, (R)-3.79 was obtained in 80 % yield over 2 steps.   158   Scheme 3.71 Synthesis of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], the 1,17-dinorsintokamide B analogues  As shown in Scheme 3.71, amide coupling between compound (S)-3.218 and compound (R)-3.79, followed by tetramic acid formation and three steps of functional group manipulations, accomplished the synthesis of sintokamide B analogue LPY11 [(4S,10R)-3.220] and its diastereomer LPY10 [(4R,10R)-3.220] in a yield of 2.1 % and 1.7 %, respectively.    159  3.3.2.7.10 Bioassay results and their SAR implications   Figure 3.30 Bioassay results of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] on R1881 activation of P6.1luc  Bioassay results of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] are shown in Error! Reference source not found.. Delightfully, both LPY10 and LPY11 showed strong inhibition against R1881 activation of P6.1luc and a clear dose-response relationship could be observed especially for LPY10 [(4R,10R)-3.220] with the unnatural C-4 configuration. Under the same concentration gradient, the synthetic analogues showed better in vitro activities than the naturally occurring sintokamide B (3.12). LPY11 [(4S,10R)-3.220] and sintokamide B at the concentration of 20 M were toxic to cells under microscopic examination.  100.0%53.9%87.0%150.6%19.6%26.8%35.8%50.6%59.0%66.3%0.0%20.0%40.0%60.0%80.0%100.0%120.0%140.0%160.0%10 μM 1.0 μM 0.1 μM 10 μM 1.0 μM 0.1 μM 10 μM 1.0 μM 0.1 μMcontrol Sint B LPY10 LPY11% PSA-LUCIFERASE ACTIVITY160    (4R,10R)-3.220 (4S,10R)-3.220 LPY10 LPY11  The strong inhibition of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] demonstrated that at least for in vitro bioassays, the 1,17-dinorsintokamide skeleton was a reasonable platform for SAR study. Trichloromethylation should be introduced to the 1,17-dinorsintokamide model and the coexistence of the C-1 and C-17 methyl groups in sintokamide B (3.12) might not be crucial for the activity. Moreover, LPY10 [(4R,10R)-3.220] with the unnatural 4R configuration showed the most potent in vitro activity among all compounds tested at that moment with a good dose-response relationship and no observed toxicity under the experimental conditions used in the assay.        161  3.3.2.7.11 Syntheses of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231], the 17-dinorsintokamide E analogues and their 1-dinorsintokamide regioisomers  Encouraged by the bioassay results obtained for LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], 17-nor- and 1-nor-sintokamide monotrichlorinated sintokamide analogues LPY13 [(4S,10R)-3.228] and LPY18 [(4S,10R)-3.231] should be made next to test which trichlorinated side chain is preferred for the in vitro activity of 1,17-dinorsintokamide analogues (Scheme 3.72). The syntheses of LPY13 and LPY18 should be similar to our previous applications of Route Three by incorporating the proper trichlorinated norvalines with commercially available leucine derivatives.   Scheme 3.72 New synthetic targets LPY13 [(4S,10R)-3.228] and LPY18 [(4S,10R)-3.231] after discovering the in vitro activities of LPY10/11   162   Scheme 3.73 Synthesis of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], the 17-norsintokamide E analogues  The synthesis of diastereomeric 17-norsintokamide E analogues is shown in Scheme 3.73. LPY12 [(4R,10R)-3.228] and LPY13 [(4S,10R)-3.228] were obtained under the protocol of Route Three originating from N-Boc-5,5,5-trichloro-D-norvaline [(R)-3.79] and L-leucine methyl ester hydrochloride (3.204).  163   Scheme 3.74 Synthesis of LPY17 [(4R,10R)-3.231] and LPY18 [(4S,10R)-3.231], the regioisomeric monotrichlorinated 1-norsintokamide analogues  The synthesis of LPY17 [(4R,10R)-3.231] and LPY18 [(4S,10R)-3.231] starting with 5,5,5-trichloro-L-norvaline [(S)-3.218] and N-Boc-D-leucine [(R)-3.43] is illustrated in Scheme 3.74. These analogues with unprecedented chlorination patterns were obtained following Route Three in 7 steps.    164  3.3.2.7.12 Bioassay results and their SAR implications   Figure 3.31 Bioassay results of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231] with LPY11 [(4S,10R)-3.220] on R1881 activation of P6.1luc  A clear SAR of side chain-preference to trichloromethylation could be summarized for the 17-norsintokamide skeleton and 1-norsintokamide skeleton by comparison of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231] with LPY11 [(4S,10R)-3.220] in R1881 activation of P6.1luc assay (Figure 3.31). At a concentration of 20 M, LPY12 and LPY13 were at least 30 % more potent compared with LPY17 and LPY18 at inhibiting RLU emission. However, both monotrichlorinated pairs at 20 M only exhibited the degree of RLU inhibition comparable to hexachlorinated LPY11 at a concentration of 10 M. Therefore, for better in vitro activities of 1,17-dinorsintokamide analogues, trichloromethylation at the D-norvaline side chain rather than that in methyl tetramate moiety was preferred. Moreover, the stronger activity observed for hexachlorinated LPY11 over trichlorinated 100.0%50.6% 50.0%55.7%120.0%78.3%0.0%20.0%40.0%60.0%80.0%100.0%120.0%140.0%10 μM 20 μM 20 μM 20 μM 20 μMcontrol LPY11 LPY12 LPY13 LPY17 LPY18% PSA-LUCIFERASE ACTIVITY165  LPY12/13 was thought to be a result of a synergistic effect of the two trichloromethyl groups in LPY11.      (4R,10R)-3.228 (4S,10R)-3.228 (4R,10R)-3.231 (4S,10R)-3.231 LPY12 LPY13 LPY17 LPY18  Figure 3.32 Structures of LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231]  3.3.2.7.13 Synthesis of LPY20 [(4S,10S)-3.220], LPY21 [(4R,10S)-3.220], two stereoisomers of 1,17-dinorsintokamide B analogues  Naturally occurring sintokamides have chlorinated D-leucine as side chains. In this SAR study, two stereogenic centres C-4 and C-10 are present in the 1,17-dinorsintokamide skeleton. Therefore, there are four diastereomers for each of the variously chlorinated analogues. For the most potent hexachlorinated 1,17-dinorsintokamides, LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] are two stereoisomers with the R configuration at C-10. To make a comprehensive understanding of the influence of configuration on the SAR of sintokamide analogues, LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220] with the S configuration at C-10 need to be prepared and tested alongside their stereoisomers LPY10 and LPY11 (Scheme 3.38). 166      (4R,10R)-3.220 (4S,10R)-3.220  (4S,10S)-3.220 (4R,10S)-3.220 LPY10 LPY11 LPY20 LPY21  Figure 3.33 Structures of LPY10 [(4R,10R)-3.220], LPY11 [(4S,10R)-3.220], LPY20 [(4S,10S)-3.220], and LPY21 [(4R,10S)-3.220]  As shown in Scheme 3.75, enantiopure N-Boc-5,5,5-trichloro-L-leucine [(S)-3.79] was prepared from nitrile (S, S)-3.217 over 2 steps in a yield of 68 %.   Scheme 3.75 Synthesis of N-Boc-5,5,5-trichloro-L-norvaline [(S)-3.79]  Since epimerisation was observed during the synthesis of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], the 5,5,5-trichloronorvaline methyl ester coupling components were not required to be enantiopure. Methanolysis of Strecker products 3.217 afforded racemic 5,5,5-trichloronorvaline methyl esters (3.218) in a yield of 83 % (Scheme 3.76).  167   Scheme 3.76 Synthesis of 5,5,5-trichloronorvaline methyl esters (3.218)  The synthesis of LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220] is shown in Scheme 3.77. Starting with the racemate 3.218 and enantiopure (S)-3.79, LPY20 and LPY21 were obtained via another successful execution of Route Three.   168   Scheme 3.77 Synthesis of LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220], two stereoisomers of 1,17-dinorsintokamide B analogues         169  3.3.2.7.14 Bioassay results and their SAR implications   Figure 3.34 Bioassay results of LPY10 [(4R,10R)-3.220], LPY11 [(4S,10R)-3.220], LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY20 [(4S,10S)-3.220], and LPY21 [(4R,10S)-3.220] with Sint A (3.11) on R1881 activation of P6.1luc  Figure 3.34 shows a clear-cut C-10 configurational preference for 1,17-dinorsintokamide in vitro activity. At a concentration of 20 M, the inhibitory activities of LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] were comparable to that of naturally occurring sintokamide A (3.11). When compared with the hexachlorinated LPY10/11, trichlorinated LPY12 [(4R,10R)-3.228] and LPY13 [(4S,10R)-3.228] were less potent in RLU inhibition. However, synthetic analogues LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220] with the S configuration at C-10, even though hexachlorinated, exhibited even weaker effects of RLU inhibition compared to LPY12/13 with the R configuration at C-10. Therefore, the R configuration at C-10 should be maintained in 1,17-dinorsintokamide analogues in order to get the desired in vitro activity. 100.0%51.8% 54.7%35.2%85.5%67.5%97.3%87.3%0.0%20.0%40.0%60.0%80.0%100.0%120.0%20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μMcontrol Sint A LPY10 LPY11 LPY12 LPY13 LPY20 LPY21% PSA-LUCIFERASE ACTIVITY170  3.3.2.7.15 Different N-acyl 1,17-dinorsintokamide B analogues and key synthetic intermediates for SAR study  Free amines LPY24 [(4R,10R)-3.232] and LPY25 [(4S,10R)-3.232], together with three other pairs of N-acyl 1,17-dinorsintokamide B analogues were synthesized and purified for the in vitro assays (Figure 3.35). The amide linkage in the N-propionyl moiety of the sintokamides was thought to be susceptible to cleavage by proteases in vivo, which was consistent with preliminary pharmacokinetic (PK) data that the t1/2 was short in vivo based on a mouse PK analysis with natural sintokamide A. Therefore, LPY26 [(4R,10R)-3.233] and LPY27 [(4S,10R)-3.233] with bulky N-pivaloyl groups, LPY28 [(4R,10R)-3.234] and LPY29 [(4S,10R)-3.234] with protease-resistant N-ethanesulfonyl groups, and the N-Boc protected precursors LPY22 [(4R,10R)-3.225] and LPY23 [(4S,10R)-3.225], were designed to prevent potential proteolytic cleavage and in the case of LPY28 [(4R,10R)-3.234] and LPY29 [(4S,10R)-3.234] improve their solubility in water. Besides, the activity and the metabolic lability of the common synthetic precursors LPY24 [(4R,10R)-3.232] and LPY25 [(4S,10R)-3.232] were of interest. After tedious syntheses and HPLC purifications, these eight analogues were prepared for evaluation in the in vitro assay.       171      (4R,10R)-3.225 LPY22 (4S,10R)-3.225 LPY23 (4R,10R)-3.232 LPY24 (4S,10R)-3.232 LPY25         (4R,10R)-3.233 LPY26 (4S,10R)-3.233 LPY27 (4R,10R)-3.234 LPY28 (4S,10R)-3.234 LPY29  Figure 3.35 Structures of LPY22 [(4R,10R)-3.225] and LPY23 [(4S,10R)-3.225], LPY24 [(4R,10R)-3.232], LPY25 [(4S,10R)-3.232], LPY26 [(4R,10R)-3.233], LPY27 [(4S,10R)-3.233], LPY28 [(4R,10R)-3.234], and LPY29 [(4S,10R)-3.234]  In addition, four other synthetic intermediates were purified for SAR evaluation (Figure 3.36). LPY32 [(4R,10R)-3.235] and LPY33 [(4S,10R)-3.235] are analogues with free tetramic acids and LPY34 [(4R,10R)-3.219] and LPY35 [(4S,10R)-3.219] are hexachlorinated dipeptides. Their in vitro activity could provide information about whether or not the methyl tetramate, the trouble-maker throughout our previous syntheses, was compulsory for the in vitro activity of the 1,17-dinorsintokamide analogues.    172      (4R,10R)-3.235 LPY32 (4S,10R)-3.235 LPY33 (4R,10R)-3.219 LPY34 (4S,10R)-3.219 LPY35  Figure 3.36 Structures of LPY32 [(4R,10R)-3.235], LPY33 [(4S,10R)-3.235], LPY34 [(4R,10R)-3.219], LPY35 [(4S,10R)-3.219]  173  3.3.2.7.16 Bioassay results and their SAR implications   Figure 3.37 Bioassay results of different N-acyl 1,17-dinorsintokamide B analogues and key synthetic intermediates on R1881 activation of P6.1luc  100.0%19.8%31.8%80.0%21.8%25.4%22.5% 22.7% 21.8%63.4%37.2%18.5%89.4%84.8%66.3%59.9%0.0%20.0%40.0%60.0%80.0%100.0%120.0%25 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μM 20 μMcontrol EPI-002 Sint A LPY11 LPY22 LPY23 LPY24 LPY25 LPY26 LPY27 LPY30 LPY31 LPY32 LPY33 LPY34 LPY35% PSA-LUCIFERASE ACTIVITY174  Figure 3.37 reveals the final bioassay results of different N-acyl 1,17-dinorsintokamide B analogues and key synthetic intermediates compared with EPI-002 (4.1) and sintokamide A (3.11) at the concentration of 20 M on R1881 activation of P6.1 luciferase. N-Boc substituted analogues LPY22 [(4R,10R)-3.225] and LPY23 [(4S,10R)-3.225], free amine LPY24 [(4R,10R)-3.232] and LPY25 [(4S,10R)-3.232], and (4R,10R)-N-pivaloylated analogue LPY26 [(4R,10R)-3.233] exhibited good inhibitory activities in accordance with the potent natural product sintokamide A. The activity of (4S,10R)-N-pivaloylated analogue LPY27 [(4S,10R)-3.233] was only 1/3 compared with its diastereomer LPY26 having the unnatural R configuration at C-4.  The pair of N-ethanesulfonylated analogues LPY28 [(4R,10R)-3.234] and LPY29 [(4S,10R)-3.234], which were anticipated to have improved resistance to proteolysis and water solubility, did not show consistent in vitro inhibition in the preliminary parallel bioassays. Their activity study is still undergoing and no confirmed results are available at this moment.  For the bioassay results of the intermediates, LPY32 [(4R,10R)-3.235], LPY33 [(4S,10R)-3.235] and LPY34 [(4R,10R)-3.219], and LPY35 [(4S,10R)-3.219] were all reported showing initial inhibitory activity but their inhibitions were labile. Therefore, methylation of the tetramic acid moiety was required for the observed in vitro inhibition of 1,17-dinorsintokamide analogues. Also, persistent AR NTD inhibitory activity could not be elicited by the hexachlorinated dipeptide structures.   175  3.3.2.8 A summary of SAR results  To sum up, a comprehensive SAR study was done on the 1,17-dinorsintokamide skeleton. Hexachlorinated analogues LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] are the most potent molecules made so far and their activity confirmed our assumption that the polychlorinated leucine derived structures of the natural sintokamides could be simplified to the new 1,17-dinorsintokamide skeleton. However, any lower degrees of chlorination on this skeleton failed to show the desired in vitro activity. The effect of trichloromethylation was more significant on the norvaline side chain rather than on the methyl tetramate moiety, and the potency shown by LPY11 [(4S,10R)-3.220] might be a result of synergy between both trichloromethyl groups in the molecule.  The naturally occurring R configuration at C-10 should be maintained for maximum activity in the active hexachlorinated 1,17-dinorsintokamides, but enhanced activity was observed when the configuration of C-4 was switched to the unnatural R configuration in the methyl tetramate moiety, i.e., LPY10 [(4R,10R)-3.220] exhibited strong, dose-responsive inhibitory activity with no toxicity under the standard assay conditions. As far as N-acylation is concerned, the proteolytic susceptibility of the N-propionyl substitution could be replaced, with both the free amines LPY24 [(4R,10R)-3.232] and LPY25 [(4S,10R)-3.232], the N-Boc analogues LPY22 [(4R,10R)-3.225] and LPY23 [(4S,10R)-3.225], and the (4R,10R)-N-pivaloylated analogue LPY26 [(4R,10R)-3.233] exhibiting similar in vitro activity comparable to sintokamide A. However, the free amine compounds were found to be unstable over time at room temperature. Finally, even though challenging for the synthesis, the methyl tetramate moiety must be a 176  required part of the pharmacophore in the skeleton. Removing either the vinylogous methyl ester or the pyrrolidinone ring led to reduced bioactivity in vitro.  3.3.2.9 A summary of synthetic routes to 1,17-dinorsintokamide analogues  In general, the reaction conditions required for conversion of norvaline derivatives into the 1,17-dinorsintokamide analogues were milder compared with those reported in the literature for conversion of chlorinated leucines into naturally occurring sintokamides. The most challenging reaction in all the synthetic routes was the formation of the tetramic acid moiety. Jouin and Castro’s protocol and its modifications are by far the most practical methods to introduce the tetramic acid substructures to the 1,17-dinorsintokamide analogues.  In this project, the evolution of our synthetic routes was motivated by feedback from in vitro assays. Route One could only generate inactive NCSTD [(4S,10R)-3.16] and failed to form chlorinated coupling components from 5-chlorinated norvaline derivatives. Route Two is a modification of Route One. 5-silyloxyl-norvaline derivatives (S)-3.124 and (R)-3.124 were used instead as starting materials leading to the successful preparation of diol 3.128 as a key intermediate. However, only monochlorinated analogues LPY00 (3.60) and LPY04 (3.148) could be made from 3.128. Dichlorination based on 3.128 and trichloromethylation from the homologous diol 3.151 all failed because of the involvement of the methyl tetramate ring in the reactions. Route Two failed to produce active analogues but suggested a reasonable sequence for installation of key structural features. Finally, Route Three was selected based on careful comparison of available synthetic routes as well as its successful application in a reported total 177  syntheses of sintokamide A, B and E. Most reaction conditions used in the total syntheses were applicable to our preparation of 1,17-dinorsintokamide analogues for in vitro assays.  However, unlike the high yields reported for the total syntheses of the natural products, the low yields of the 1,17-dinorsintokamide analogues available by Route Three might be attributed to the reactivity of the polychloromethyl groups in the 1,17-dinorsintokamide skeleton. However, Route Three currently is the only working synthetic approach to the polychlorinated 1,17-dinorsintokamides, which could handle the lability of polychloromethyl groups, epimerisation in the amide coupling, and the formation of the tetramic acid.  178  Chapter 4: Mechanism of Action Study of Marine Natural Products Induced Inhibitory Activity of AR NTD Transactivation in Prostate Cancer Cells  4.1 Introduction  Natural products, the secondary metabolites originating from the genome of various living organisms, are believed to be biologically active through interfering with the proteome in the natural environment.38, 48 Because of the conservation of protein function in living organisms spanning the range of life form complexity all the way from single cell prokaryotes to highly evolved mammals, it is anticipated that natural products should interact with the human proteome, which contains the majority of new drug targets identified and validated in current drug discovery practice.45 Screening of natural product libraries represents a potentially productive way to find hits with novel skeletons for drug discovery endeavors focusing on IDPs as targets. The absence of X-ray or NMR data to define the binding pockets within IDPs prevents rational drug design for these targets such as the AR NTD.98, 196      3.8, EPI-001   4.1, EPI-002  Figure 4.1 Structures of EPI-001 (3.8) and EPI-002 (4.1) 179  As described before, the AR NTD is an IDP. The collaborative screening program between the Sadar and Andersen laboratories identified several categories of AR NTD antagonists from extracts of sponges with the new hope to treat mCRPC. Among them, EPI-001 (3.8), a mixture of four stereoisomers, exhibited potent in vitro inhibition of AR NTD transactivation in prostate cancer cells. The mechanism of action study of EPI-001 (3.8) has been published. EPI-001 (3.8) was shown to elicit its inhibitory activity by covalently binding to the AF1 region of the AR NTD.99, 100    Sintokamides A – E (3.11 – 3.15), isolated from the sponge Dysidea sp., also showed comparable in vitro and in vivo inhibitory activity against AR NTD transactivation compared with EPI-001 (3.8). However, the sintokamides may have a binding site on the AR NTD different from the AF1 binding site of EPI-001 since a synergistic effect was detected in vitro with a combination of sintokamide A (3.11) and EPI-002 (4.1). Due to the structural complexity of the sintokamides compared to that of EPI analogues, mechanism of action studies have been undertaken using a Click chemistry approach with bioactive hexachlorinated 1,17-dinorsintokamide LPY30 (4.7) and LPY31 (4.8) with an alkyne terminus because of the reduced complexity of synthesizing the probes. LPY30/LPY31 showed evidence for covalent binding to the full length AR (FL-AR), which provided a third example that irreversible chemical binding 180  might be a requisite for a sustained bioactivity exerted by a small molecule drug targeting an IDP. In 2012, it was reported that niphatenones (3.9 and 3.10) from the sponge Niphates digitalis blocked AR transcriptional activity in prostate cancer cells via covalent binding to the AF1 region in the AR NTD.  4.2 AR activity of EPIs and mechanism of action studies  EPI-001 (3.8) demonstrated effective and specific inhibitory activity of AR-dependent transactivation in prostate cancer cells. EPI-001 suppressed LNCaP cell proliferation with an IC50 of 12.63 ± 4.33 M in the presence of 0.1 nM R1881, a potent synthetic androgen. However, no proliferation blocking in response to EPI-001 was detected in PC3 human prostate cancer cells that have no functional AR expression. Moreover, EPI-002 (4.1) failed to inhibit the transcriptional activities of other closely related human steroid receptors such as the GR and the PR. Cell cycle analysis revealed that EPI-001 inhibited the proliferation of LNCaP cells in response to R1881 by reducing cells in S-phase (in which DNA is replicated) with an elevation of the number of cells in the intermediate G1-phase.98-100  4.2.1 AR inhibition of EPI-001 (3.8) is different from hormone therapies in vitro  Antiandrogens, such as bicalutamide and MDV3100, are AR antagonists that competitively bind to the AR LBD. Because of the competitive inhibition, AR inhibitory activities of antiandrogens in vitro would be compromised by increasing the concentration of endogenous or exogenous androgens. For mCRPC models expressing constitutively active AR splice variants with AR 181  LBD deficiency, treatment with antiandrogens is ineffective due to the absence of the drug target in AR-Vs. The use of antiandrogens is also known to cause translocation of cytosolic AR to the nucleus.  Preliminary in vitro bioassays suggested that EPI compounds have an unique mechanism of action with different patterns of AR inhibition compared with antiandrogens. First, the ability of EPI-001 (3.8) to inhibit the AR was not influenced by addition of exogenous androgens. Incubated with R1881 at 50 nM, EPI-001 at a concentration of 25 M still exhibited 80 % of its original AR inhibition. Second, unlike the attenuated activities of bicalutamide or MDV3100 in ARv567es expressing COS-1 cells and ARv567es/FL-AR expressing LNCaP cells, EPI-001 elicited strong inhibitory activity in both systems either with or without the presence of R1881. Third, the antiandrogen induced nuclear translocation of the AR was not observed in EPI-001 (3.8) or EPI-002 (4.1) treated LNCaP cells. Under the experimental conditions, AR-YFP (yellow fluorescent protein) remained cytosolic and there was no florescence observed in the nucleus.  In addition, fluorescence polarization observation also supported that EPI-001 (3.8), unlike R1881 and bicalutamide, did not interact with the AR LBD or compete with existing ligand-binding between a fluoromone and the AR LBD. Moreover, EPI-001 showed no evidence of binding with the GR LBD and the PR LBD, which shared considerable sequence homology with the AR LBD.    182  4.2.2 EPI-001 (3.8) inhibits the AR NTD via covalent binding to the AF1 protein  Besides the LBD, the AR NTD AF1 and AF5 regions can also activate AR transcriptional activity.197 While the AF1 is involved in ligand bound AR transactivation, the AF5 is responsible for AR constitutive activity (without an external stimulus). In absence of androgens, the transactivation of AR NTD can be triggered by addition of forskolin, which elevates cAMP concentration and activates cAMP/PKA pathway. In vitro, EPI-001 (3.8) inhibited the forskolin-induced AR NTD transcriptional activity to baseline level with an IC50 of approximately 6 M.  The structural moiety of a chlorohydrin in the molecule of EPI-001 (3.8) hinted that a possible mechanism of action might be via covalent binding to the AR. This assumption was proven by Click chemistry results that FL-AR could be pulled down from lysates of LNCaP cells that were preincubated with active EPI probes with an alkyne terminus. Further pull-down assays with AR NTD transfected cells, followed with pure recombinant AF1 proteins, confirmed that the mechanism of action of EPI-001 was covalent bound to the AF1 region in the AR NTD. Interestingly, the binding of EPI-001 to an IDP like the AR NTD still required some secondary structural features in the protein, since active EPI probes could not bind to denatured AF1 protein.  Different drug binding sites of AR suggested a possible synergistic inhibition response might be observed when a blend of EPI-001 (3.8) and bicalutamide was tested in vitro. As expected, EPI-001 (5 M) and bicalutamide (5 M) exhibited a 70 % additive inhibition of R1881-induced AR 183  activity in LNCaP cells, while each ingredient at the same concentration alone showed inhibition by 22 % and 27 %, respectively.  A proposed covalent binding mechanism of action of EPI-001 (3.8) is illustrated in Figure 4.2.   Figure 4.2 A proposed mechanism of action of EPI-001 (3.8) with the AF1 region in the AR NTD  4.2.3 EPI stereoisomers inhibit CRPC xenografts in vivo  In vivo, all EPI analogues containing the chlorohydrin moiety exerted significant antitumor activities in the LNCaP CRPC xenograft. Among the analogues, EPI-002 (4.1) was found to be superior due to 60 % of tumor growth regression rate and no significant body weight loss in treated animal, that is, no observed toxicity in vivo under experimental conditions.  184  4.3 AR NTD-dependent inhibitory activity of sintokamide A, in vitro and in vivo  With EPI-001 (3.8) as a model, a new series of similar bioassays were conducted with sintokamide A. The reported activity of sintokamide A (3.11) is related to its AR specificity in prostate cancer cells. Comparable to EPI-001, sintokamide A showed an IC50 of 10.74 ± 2.44 M for inhibition of androgen-dependent proliferation in LNCaP cells transfected with PSA 6.1-luc reporter, but when PC3 cells were exposed to the natural product, no inhibition of proliferation was detected. Moreover, no suppression of GR or PR transactivation was observed in cotransfected LNCaP cells in response to sintokamide A.  Similar to the EPI compounds, sintokamide A’s activity is not related to an AR LBD interaction. First, AR nuclear translocation was also not detected in LNCaP cells treated with sintokamide A (3.11) or EPI-002 (4.1), the most potent stereoisomer component in the stereoisomeric mixture EPI-001 (3.8). After treatment with both AR inhibitors, AR-YFP appeared predominantly in cytoplasm in the cancer cells, compared to nuclear relocalisation upon treatment with R1881, bicalutamide, or MDV3100. Second, there was no indication of AR LBD binding in a fluorescence polarization study with sintokamide A. In the same concentration range of 0.5 nM to 50 M, sintokamide A and EPI-002 could not compete with the recombinant AR LBD/fluoromone binding interaction that was monitored by the assay. Third, while the antiandrogen bicalutamide could inhibit PR transactivation via blocking the PR LBD that is homologous to the AR LBD in the cotransfected LNCaP cells, the AR specificity of sintokamide A suggested that binding to the LBD could not be the reason for the activity of the chlorinated natural product. 185  Based on above observations, sintokamide A’s inhibition of AR transcriptional activity was assumed to be linked to its ability to block the activation of the AR NTD. In support of this assumption, treatment of forskolin activated LNCaP cells with sintokamide A (10 M) suppressed the AR NTD transactivation to baseline level, similar to the result observed with EPI-001 (3.8). Therefore, sintokamide A represents a new class of AR NTD antagonist found in nature.  In LNCaP CRPC xenografts, sintokamide A (3.11) exhibited an effective tumor growth inhibition and no observed toxicity in vivo. Compared with an observed 50 % volume increase in DMSO treated xenografts, tumors treated by intratumoral injection of sintokamide A (30 mg/kg) shrank 20 % in size and appeared less bloody comparing with the control. Treatment with sintokamide A did not change the body weights of the laboratory animals during the in vivo experiment.          186  4.4 Different mechanisms of action of the sintokamides and EPI-001 (3.8)  In Route Two, two monochlorinated sintokamide analogues LPY00 (3.60) and LPY04 (3.148) were prepared for the SAR study. To our surprise, as shown in Figure 4.3, the anticipated simple SN2 replacement of the terminal chlorine atom(s) could not explain the inhibitory activities of sintokamide analogues: 3.60 and 3.148 were all inactive in vitro.         Figure 4.3 Structures of LPY00 (3.60), LPY04 (3.148) and LPY01 (4.2) and their in vitro activities  The bioactive naturally occurring sintokamides do not feature the chlorohydrin of EPI-001 (3.8) in the skeleton as a pharmacophore element, which indicates that the analogues might have a different mechanism of action from the EPI compounds. The deduction was supported by the synergistic therapeutic effect of sintokamide A (3.11) and EPI-002 (4.1). In this study, LNCaP cells were cotransfected with three luciferase reporters: PSA6.1-luc, probasin-luc, and ARR3-luc. Both sintokamide A (3 – 24 M) and EPI-002 (4 – 35 M) independently inhibited the three reporters in the cells in response to R1881. The drug combinations (EPI-002/sintokamide A = 100.0% 97.9%106.1%124.8%0.0%20.0%40.0%60.0%80.0%100.0%120.0%140.0%28 μM 28 μM 25 μMDMSO LPY00 LPY01 LPY04% PSA-LUCIFERASE ACTIVITY187  1.3/1) showed additive effects for three reporters at all experimental concentrations except the highest concentrations where the efficacies seemed saturated. Under the experimental conditions used, no cytotoxicity was detected based on the morphological observation of the LNCaP cells.  4.5 Click chemistry study based on 1,17-dinorsintokamide analogues  An anticipated Click chemistry study of sintokamide A (3.11) was not realized because the conventional installation of an alkyne to the N-propionyl pentachlorinated natural product would inevitably involve the usage of a strong base to deprotonate the remaining N-H, which could cause concomitant elimination of the chlorine atoms.  On the other hand, because of the unique IDP target and the proposed mechanism of action of the EPI compounds, making Click chemistry probes for covalent binding was a constant element of ligand design for the SAR study of the norsintokamide analogues. In Route Two, direct deprotonation of LPY00 (3.60) would cause dehydrochlorination so the preparation of a monochlorinated Click chemistry probe started with treatment of the silylated intermediate 3.144 (Scheme 4.1). In Route Three, free amines could be obtained as intermediates so N-pentynoylation could be performed under mild conditions onto the nitrogen atoms to generate desired probes with a terminal alkyne (Scheme 4.2).    188  4.5.1 Preparation of the monochlorinated LPY02 (4.5) with an alkyne terminus  Monochlorinated probe LPY02 (4.5) and its negative control LPY03 (4.4) were prepared as illustrated in Scheme 4.1. Deprotection of the silyloxylated intermediate 3.144, followed by deprotonation with NaH and treatment with propargyl bromide successfully installed an alkyne into the molecule 4.3 in an overall yield of 53 % over 2 steps.198-200 Following the same procedure used to prepare LPY01 (4.2), desilylation of 4.3 generated alcohol LPY03 (4.4) in 60 % yield, which was monochlorinated to give LPY02 (4.5) in a yield of 95 %.   Scheme 4.1 Syntheses of LPY03 (4.4) and LPY02 (4.5)     189  4.5.2 Preparation of trichlorinated probes with an alkyne terminus  As shown in Scheme 4.2, treatment of the norsintokamide precursors bearing a free amine with 4-pentynoyl chloride in the presence of triethylamine afforded the corresponding N-pentynoyl probes in good yields. A non-chlorinated probe LPY19 (4.9) was also made as a negative reference.     R1 R2 R3 R4 Yield 4.6, LPY16 CH3 H CH3 CCl3 70 % 4.7, LPY30 H H CCl3 CCl3 73 % 4.8, LPY31 H H CCl3 CCl3 70 % 4.9, LPY19 CH3 CH3 CH3 CH3 74 %  Scheme 4.2 A generic scheme of synthesizing probes from free amine precursors        190  4.5.3 Activities of the probes and results of pull-down assays  Monochlorinated LPY02 (4.5) and its primary alcohol precursor LPY03 (4.4) were tested in vitro and neither probes, similar to LPY00 (3.60), showed inhibitory activities against AR NTD transactivation (Figure 4.4). Therefore, no Click chemistry pull-down experiments were performed using these probes.            Figure 4.4 Bioassay results of probes LPY02 (4.5) and LPY03 (4.4) on R1881 activation of P6.1luc  Investigation of covalent binding was initiated with probes LPY16 (4.6) and LPY19 (4.9). Both alkynes were first tested in vitro. As shown in Figure 4.5, the 17-norsintokamide E analogue LPY16 (4.6) elicited inhibitory activity while the non-chlorinated probe LPY19 was inert. Therefore, the active probe LPY16 and the negative control LPY19 were subjected to pull-down experiments with the AR NTD and the AR, respectively. Unfortunately, no readout was obtained from the pull-down assays using LPY16.  100.0%81.3%73.3%0.0%20.0%40.0%60.0%80.0%100.0%120.0%28 μM 28 μMDMSO LPY02 LPY03% PSA-LUCIFERASE ACTIVITY191         Figure 4.5 Bioassay results of LPY13 [(4S,10R)-3.228] and LPY16 (4.6) with Sint A (3.11) on R1881 activation of P6.1luc  Probes LPY30 (4.7) and LPY31 (4.8), based on the structures of our most potent hexachlorinated analogues LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] in vitro, were prepared for pull-down assays with full-length AR. As shown in Figure 4.6, LPY30 and LPY31 exhibited strong in vitro activities. Their potency in vitro were comparable to that of EPI-002 (4.1) and natural occurring sintokamide A.       100.0%28.0% 32.4%37.5%87.6%0.0%20.0%40.0%60.0%80.0%100.0%120.0%10 μM 40 μM 40 μM 40 μMcontrol Sint A LPY13 LPY16 LPY19% PSA-LUCIFERASE ACTIVITY192         Figure 4.6 Bioassay results of LPY30 (4.7) and LPY31 (4.8) with EPI-002 (4.1) and Sint A (3.11) on R1881 activation of P6.1luc  The pull-down assays showed that both active probes LPY30 (4.7) and LPY31 (4.8) covalently bind to the FL-AR with LPY19 (4.9) as the negative control (Figure 4.7). Moreover, the Click chemistry results showed that LPY30 (4.7) bound better than LPY31 (4.8), which could be an explanation of why LPY10 [(4R,10R)-3.220] was superior to LPY11 [(4S,10R)-3.220] in vitro for better dose-response relationship and no observed toxicity at high concentrations. The binding effect of LPY30 (4.7) was similar to that of EPI-053.        100.0%19.8%31.8%37.2%18.5%0.0%20.0%40.0%60.0%80.0%100.0%120.0%25 μM 20 μM 20 μM 20 μMcontrol EPI-002 Sint A LPY30 LPY31% PSA-LUCIFERASE ACTIVITY193  A B                   cell lysates pull-down cell lysates pull-down   Figure 4.7 (A) Western blots using anti Rb/biotin for the cell lysates and the streptavidin pull-down; (B) Western blots using ARN-20 for the cell lysates and the streptavidin pull-down. EPI-053 is a bioactive EPI probe with a terminal alkyne.           194  4.5.4 Possible mechanistic explanation for the Click chemistry result  Two possible covalent binding elements can be identified based on the structure and reactivity of hexachlorinated 1,17-norsintokamide analogues.    covalent bond formation 1 covalent bond formation 2 covalent bond formation 1 + 2    4.11 4.12 4.13  Figure 4.8 A proposed model incorporating two possible covalent bonds to explain the mechanism of action of LPY10 [(4R,10R)-3.220]  First, the trichloromethyl group at the end of either side chain might serve as a good leaving group to facilitate a direct SN2 replacement with a nucleophilic site of an amino acid residue in 195  the binding pocket on the target AR protein to form possible AR/LPY10 adducts 4.11, 4.12 or 4.13 (Figure 4.8). Second, for the possible covalent binding happening to the methyl tetramate structural moiety, the formation of a spirocyclopropane at C-4 observed in Section 3.3.2.6.13 provided another mechanism to create a covalent bond via a nucleophilic ring opening process (Figure 4.9). The reactivity of spirocyclopropane compounds exposed to various nucleophiles has been reported by Kawada et. al.in 1981.201 The actual mechanism of action of the hexachlorinated 1,17-norsintokamides may be involved with a single binding element (replacement of the trichloromethyl group or cyclopropane ring opening) or a combination of the two binding elements.   Figure 4.9 LPY10 [(4R,10R)-3.220] may create a covalent bond with the AR via a cyclopropane ring formation/reopening mechanism.   196  4.6 Summary  This chapter exhibited the strength of research in marine natural products in identification and validation of new drug targets in today’s drug discovery practice, especially for IDPs. Two classes of small molecule based AR NTD antagonists, EPI-001 (3.8) and sintokamides A – E (3.11 – 3.15), were sequentially discovered by the Sadar/Andersen collaborative forward chemical genetics screening program. Both classes of compounds showed comparable in vitro and in vivo activities towards prostate cancer cells. Since the AR NTD is a new promising target for mCRPC, a rigorous investigation of these two classes of inhibitors was performed. First, their activities were proven to be AR specific, since no activity was observed against PC3 prostate cancer cells that are not regulated by the AR and no activity was observed against other human steroid receptors. Second, their inhibition of AR transcriptional activity did not involve competitively binding to the AR LBD, where androgens and antiandrogens work. Third, both compounds inhibited transcription in cells that were deliberately activated by AR NTD transactivation in response to forskolin.  For the mechanism of action study, synergistic effects were detected in vitro with both cocktails EPI-001/bicalutamide and sintokamide A/EPI-002. The former combination suggested EPI-001 (3.8) had a binding site other than the AR LBD, while the latter showed that even targeting the same AR protein, the exact binding sites of both the AR NTD antagonists might be different along the intrinsically disordered protein frame. Later on, Click chemistry results revealed that EPI-001 (3.8) exerted its AR NTD inhibitory activity by covalently binding to the AF1 region in the AR NTD. 197  The monochlorinated norsintokamide analogues LPY00 (3.60) and LPY04 (3.148) failed to show any AR NTD inhibitory activity in in vitro assays. Click chemistry probe LPY16 (4.6) based on the structure of 17-norsintokamide E also failed to pull down the AR or the AR NTD. Finally, based on the structures of the most potent hexachlorinated 1,17-dinorsintokamide analogues LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220], two probes LPY30 (4.7) and LPY31 (4.8) were prepared and showed excellent in vitro activities. Click chemistry demonstrated that both probes could covalently bind to FL-AR, and LPY30 (4.7), which has an R configuration at C-4 in the methyl tetramate moiety, exhibited better binding compared with its stereoisomer LPY31 (4.8). At last, a tentative mechanism of action of LPY10 [(4R,10R)-3.220] was proposed, which could be an explanation for the in vitro activities of the hexachlorinated 1,17-dinorsintokamide analogues observed so far. 198  Chapter 5: Synthesis of an N-Biotinylated Probe of Latonduine A for Activity Evaluation and Mechanism of Action Study  5.1 Background of cystic fibrosis  Cystic Fibrosis (CF) is a life-threatening inherited disease. Having an imbalance of water and minerals in their bodies because of losing large amounts of salt, patients with cystic fibrosis experience health problems in their respiratory, digestive, and reproductive systems. The symptoms of the genetic disorder and their severity change among affected individuals. There is currently no cure.  The incidence of CF varies around the world. Caucasians and Ashkenazi Jews are among the most vulnerable. One in 2,000 – 3,000 newborns in Europe and one in 3,500 children born in North America have CF. Cystic fibrosis is found less common in other continents like Asia and Africa.202 There are 4,000 registered individuals with CF across Canada (2011).203 Approximately 80,000 people suffer from this disease worldwide.204  Cystic fibrosis was once a fatal disease of childhood. In the 1960s, most CF children did not survive long enough to attend kindergarten. As improved therapies have helped to address health issues caused by malnutrition and with better disease management, many CF patients now live well into adulthood and virtually all cystic fibrosis-related deaths are due to lung diseases.205 For 199  CF patients, the “median predicted survival age” in 2011 was 36.8 years in the USA,206 41.5 years in the UK,207 and 48.5 years in Canada.203  5.2 Latonduines isolated from Stylissa  In 2003, latonduines were first published by the Andersen lab for their structural novelty but without any bioactivity.208 Latonduines (5.1 to 5.4), containing a six-membered aminopyrimidine substructure in the place of the five-membered aminoimidazole substructure common to hymenialdisine (5.7) and stevensine (5.8) in the oroidin (5.9) family, were only found in Stylissa.209 Besides latonduine A (5.1), latonduine B (5.2) and its methyl and ethyl esters (5.3 and 5.4) were found from S. carteri collected by SCUBA near Latondu Island, Taka Bonerate National Marine Park, Indonesia in 2003, 3-debromolatonduine A (5.5) and 3-debromolatonduine B methyl ester (5.6) were added to the naturally occurring latonduine family from Indonesian marine sponge Stylissa sp. in 2012.210       200     5.1, latonduine A 5.2, latonduine B 5.3, latonduine B methyl ester       5.4, latonduine B ethyl ester 5.5, 3-debromolatonduine A 5.6, 3-debromolatonduine B  methyl ester    5.7, hymenialdisine 5.8, stevensine 5.9, oroidin  Figure 5.1. Latonduines (5.1 to 5.6) and structurally related compounds (5.7 to 5.9) isolated from Stylissa  In 2007, our collaborator Professor David Thomas at McGill University reported a new high throughput screen for F508del-CFTR trafficking correctors based on an immunofluorescence assay. In short, baby hamster kidney (BHK) cells are engineered to express modified F508del-CFTR which is attached to three tandem hemagglutinin-epitope tags (3HA) in the CL4 region. Once the mutant chloride channels have been correctly transported to the cell surface, their 3HA 201  tags are extracellular and can react with anti-HA antibody conjugated with fluorophore. Thus, their appearance on the cell surface can be detected by fluorometry. The first bioactivity description of the latonduines was discovered by applying such an HTS screen to an Andersen lab natural product library containing 720 marine extracts. In 2012, a detailed depiction of latonduine A’s bioactivity was published in Chemistry & Biology.2 Latonduine A is a strong inhibitor of poly(ADP-ribose) polymerase-3 (PARP-3), which corrects F508del-CFTR trafficking to a therapeutic level.  5.3 Synthesis of latonduine A  Scheme 5.1 shows the published synthetic route to latonduine A.208 4,5-Dibromination of commercially available 2-(trichloroacetyl)pyrrole (5.10) with bromine in acetic acid gave 4,5-dibromo-2-(trichloroacetyl)pyrrole (5.11) in 93 % yield. The trichloromethyl group in 5.11 was replaced with 2-(1,3-dioxolan-2-yl)ethylamine in MeCN to furnish the corresponding amide 5.12 in 81 % yield. Deprotection of the acetal in 5.12, ring closure, and elimination were finished in one pot in neat methanesulfonic acid to give compound 5.13 in 69 % yield. Hydroboration of 5.13 followed by oxidation with H2O2 gave secondary alcohol 5.14 in 84 % yield. Mild oxidation of 5.14 with Dess – Martin periodinane (DMP) afforded the corresponding ketone 5.15 in 98 % yield. Treatment of 5.15 with triethyl orthoformate and TFA gave enol ether 5.16 in 35 % yield. Finally, 5.16 was refluxed with excess guanidine in THF/water to afford latonduine A (5.1) in 85 % yield. The collective yield from 5.10 was 12.7 %.  202   Scheme 5.1. Synthesis of latonduine A (5.1) from 5.10  I prepared more synthetic latonduine A for bioactivity evaluation in July 2008. The synthesis was started with 5.14 (194 mg). DMP was the only effective reagent to oxidase 5.14 to 5.15 with a yield of 50 %. Crude residue of 5.16 was directly used without purification for the final construction of the 2-aminopyrimidine ring. At last, 10 mg of latonduine A (5.1) was obtained and the collective yield was 4.5 %. The NMR and MS data were identical to the literature values.208 203  5.4 Latonduine A corrects F508del-CFTR trafficking in vitro  In wild type CFTR biogenesis, a core glycosylated CFTR is synthesized from the nascent chloride channel in the ER and this “immature” form of CFTR has an MW of 135 – 140 kDa. In the Golgi, the precursor is matured by forming a fully glycosylated protein with an MW of 150 – 160 kDa. On SDS-PAGE/immunoplot, the diffused strips matching the immature and mature CFTR are distinguishable and are designated as “band B” and “band C”, respectively. Therefore, the Western blotting can be used to visualize CFTR trafficking efficiency from the ER to the Golgi.2   Figure 5.2. Latonduine A corrects F508del-CFTR trafficking from the ER to cell surface in vitro 8.0%70.0%100.0%2.0%25.0%100.0%1.2%48.0% 51.0%100.0%0.0%20.0%40.0%60.0%80.0%100.0%120.0%DMSO Latonduine A (10 μM), 24 hDMSO Latonduine A (1 μM), 48 hDMSO Latonduine A (10 μM), 24 hLatonduine B (10 μM), 24 hBHK cells WT CFBE41o- cells WT BHK cells WTBand C expression F508del CFTR rescue rateFrom the ER to the Golgi From the Golgi to cell surface204  Latonduine A was found to correct F508del-CFTR trafficking from the ER to the Golgi significantly. In transfected BHK cells treated with latonduine A (10 M) for 24 h, densitometry measurement of band C of fully glycosylated F508del-CFTR improved to a level of 70 % of that of wild-type CFTR. In the human lung epithelial cell line CFBE41o− cells treated with latonduine A (1 M) for 48 h, SDS-PAGE/Western blot witnessed a 25 % correction compared to the wild-type band C expression.2  Improvement of F508del-CFTR cell surface expression by latonduine A was confirmed under confocal microscopy observation. Transfected BHK cells were marked with wheat germ agglutinin (WGA) and anti-CFTR antibody in sequence after treated with latonduine A (10M) for 24 h. Then the overlapping cell surfaces and CTFR stains were scored under confocal microscopy using a computer-assisted analysis. Again, latonduine A brought 48 % F508del-CFTR appearance on cell surface compared with that of wild-type epithelial cells. These two experiments with three cellular checkpoints of CFTR trafficking showed that latonduine A could substantially rescue misfolded F508del-CFTR from the ER to the cell surface (Figure 5.2).2  5.5 Latonduine A potentiates F508del-CFTR defect in vitro, ex vivo, and in vivo  Only working F508del-CFTR on the epithelial cell surface would produce therapeutic benefit. Latonduine A potentiating F508del-CFTR gating property was verified by in vitro, ex vivo, and in vivo experiments (Figure 5.3).  205  A halide efflux assay was designed to test ion channel’s function in vitro. CFTR is a cAMP regulated chloride channel and functional CFTR secretes halide via activation. The halide secretion generates electric potential difference across the cell surface, which can be recorded by an electrophysiological method. After treatment with latonduine A (1 M) for 24 h, transfected BHK cells were loaded with iodide, washed, and stimulated with forskolin (10 M) and genistein (50 M). It was found that latonduine A generated iodide efflux 42 times as strong as control cells treated with DMSO, which was 51 % compared with the efflux strength of wild-type epithelial cells. In CFBE41o− cells pretreated with latonduine A (10 M) for 24 h, the iodide efflux measured was 31 times the strength of the DMSO control cells.2  Intestinal ileal epithelia pieces from F508del-CFTR homozygous mice were isolated to determine how latonduine A would functionalize the mutant channel ex vivo. After incubation with latonduine A (10 M) for 4 h, the intestinal tissue was setup with an Ussing chamber for measuring short-circuit current (Isc) counterbalancing CFTR channel activity. The latonduine A treated epithelium demonstrated significantly increased Isc over DMSO control. The net Isc gain was 2.5 % of that of wild-type littermates.2  F508del-CFTR homozygous mice were used in a salivary secretion assay to assess how latonduine A would relieve CF manifestation in vivo. The mice were treated with latonduine A (50 mg/kg) by gavage once per day for 2 days and a 75 times increase in the maximum rate of CF mice saliva production was recorded in contrast to DMSO treated mice. The corrected maximum rate of saliva production was 9 % of that of wild-type littermates.2 All the in vitro, ex 206  vivo, and in vivo experimental results established that latonduine A could potentiate F508del-CFTR gating function to a therapeutically beneficial level.  207   Figure 5.3. Latonduine A potentiates F508del-CFTR in vitro, ex vivo, and in vivo  1.23%51.8%100.0%1.23% 38.2%100.0%0.0% 2.5%100.0%0.12%9.0%100.0%0.0%20.0%40.0%60.0%80.0%100.0%120.0%DMSO Latonduine A (10 μM), 24 hWT DMSO Latonduine A (10 μM), 24 hWT DMSO Latonduine A (10 μM), 4 hWT DMSO Latonduine A(50 mg/ kg),i.g.WTBHK cells CFBE41o- cells WT F508del-CFTRhomozygous miceWTlittermatesF508del-CFTRhomozygous miceWTlittermatesHalide efflux Short-circuit current Saliva productionin vitro ex vivo (intestinal ileal epithelia) in vivo (F508del-CFTR homozygousmice)208  5.6 The mechanism of action study of latonduine A  Latonduine A corrects F508del-CFTR only posttranslationally. First, results from real-time polymerase chain reaction (RT-PCR) based on isolated mRNA from CFBE41o− cells pretreated with latonduine A (1 M, 24 h) showed that there was no obvious transcriptional change caused by latonduine A treatment. Second, latonduine A’s effect to correct F508del-CFTR was not compromised by blocked protein translation in the presence of cycloheximide. Moreover, latonduine A is unlikely to work as pharmaceutical chaperone to correct the mutant ion channel: no signs from differential scanning fluorometry supported that latonduine A could bind to CFTR NBD-1 directly. Even at a concentration of 100 M of latonduine A, an upward shift of F508del-CFTR melting point caused by the anticipated direct binding was not observed.2  Latonduine A probably acts as a proteostasis regulator in correction of F508del-CFTR trafficking and its target protein needs to be identified. Structurally, the pyrrole nitrogen in latonduine A is an ideal site to be biotinylated. It is the most acidic proton in the molecule and can be easily coupled with a biotinylated linker in the presence of a weak base. To prove this idea, N-methylated latonduine was prepared and it maintained the biological activity of the parent molecule as an effective F508del-CFTR trafficking corrector in both the Western blotting and halide efflux assays in transfected BHK cells.2 Thus, N-biotinylated latonduine A was selected as the probe to pull down the possible target.2  209   Scheme 5.2. Synthesis of N-biotinylated-latonduine A (5.17)  Scheme 5.2 shows the reaction to make biotinylated latonduine A (5.17). The N-H in the pyrrole of latonduine A (5.1) was deprotonated by potassium carbonate and coupled with a biotin linker in DMF to generate 5.17 in 32 % yield.  Table 5.1 The EC50s of latonduine A against PARPs in CFBE41o− cells  PARP-1 PARP-2 PARP-3 PARP-4 PARP-5a PARP-5b Latonduine A 50 M 20 M 400 pM 9 M 90 M 1 M  With the probe in hand, pull-down assays were repeatedly performed to locate latonduine A’s binding protein or proteins. CFBE41o− cell lysates were pretreated with N-biotinylated latonduine A followed by trypsinization and mass spectrometry. The initial results showed that N-biotinylated latonduine A bound to PARP-1, which was further confirmed by immunoblotting with an anti-PARP-1 antibody and pull-down with the recombinant PARP-1. Moreover, later pull-down assays revealed that N-biotinylated latonduine A also bound to other members in the poly(ADP-ribose) polymerase family such as PARPs 2, 3, 4, 5a, and 5b. Subsequent in vitro experiments showed that latonduine A was a general PARP inhibitor and its inhibitory effect was 210  weak against most of the family proteins except PARP-3. Latonduine A was shown to be a strong PARP-3 inhibitor with an EC50 = 400 pM in CFBE41o− cells.2  Latonduine A’s activity to correct F508del-CFTR trafficking appeared to be linked to its general inhibitory effects against PARPs. Peroxynitrite could activate PARPs and it was found to counteract the F508del-CFTR trafficking correction of latonduine A. When transfected BHK cells were treated with peroxynitrite at different concentrations for 3 h followed by incubation with latonduine A (10 M) for 24 h, the correction effect of latonduine A began to fall at 20Mand could be completely neglected at 100 M. Similarly, the 50 % reduced relative PARP activity in BHK cells pretreated with latonduine A (10 M) for 24 h was compromised by addition of peroxynitrite at 20 M. No band C expression was observed in the Western blot under such a circumstance. However, since there was no preference within peroxynitrite’s activation to specific members in the PARP family, this assay could not tell which PARP was the primary target of latonduine A.2  siRNA knockdown methodology was applied to resolve the above question. Transduced with siRNA for PARPs 1, 3 or 5a, Human Embryonic Kidney 293 (HEK293) cells were treated with latonduine A at different concentrations for 24 h. It was found that a decrease in the EC50 of latonduine A in HEK293 cells was only observed in cells knocked down with PARP-3 siRNA (dropping from 8 nM to 200 pM). Thus, PARP-3 was validated as the specific target in PARP family responsible for latonduine A’s activity to rescue F508del-CFTR.2  211  With a new correction mechanism by inhibiting the PARP-3, latonduine A worked synergically with some investigational drugs having different mechanisms of action currently in clinical trials for F508del-CFTR therapy. Sildenafil is a PDE-5 inhibitor and VX809 corrects CFTR via action on MSD1. Transfected BHK cells were treated with latonduine A (1 M) and sildenafil (1 M) or latonduine A (1 M) and VX809 (100 nM) for 24 h. The latonduine A/sildenafil combination gave 52 % and 95 % increase in surface CFTR signal compared to that given by monotherapy with latonduine A or sildenafil, respectively. In the case of the latonduine A/VX809 combination, the increase was up to 200 % and 75 %, compared to that produced by the individual components latonduine A and VX809 in the combination, respectively.2  5.7 Conclusion  This chapter describes the preparation of latonduine A (5.1) and N-biotinylated latonduine A (5.17) and their application in a series of biological assays and mechanism of action studies. Latonduine A was shown to be dual-acting, which rescued F508del-CFTR trafficking efficiently from the ER to cell surface in vitro and revitalized the mutant channel’s gating defect in vitro, ex vivo, and in vivo. With the knowledge that latonduine A might work posttranslationally and did not directly bind to the CFTR NBD-1, an N-biotinylated latonduine A probe (5.17) was designed and synthesized for pull-down experiments to discover the protein target of latonduine A as a proteostasis regulator. The results showed that latonduine A was a general PARP inhibitor and its inhibitory effect was weak against most of the proteins in this family except PARP-3. In CFBE41o− cells, the EC50 of Latonduine A against PARP-3 was 400 pM. When PARPs were activated by peroxynitrite, the activity of latonduine A was compromised. However, when 212  PARP-3 was knocked down by transducing the corresponding siRNA into HEK293 cells, the EC50 of latonduine A improved from 8 nM to 200 pM. Thus, PARP-3 was validated as the specific target in the PARP family responsible for latonduine A’s activity to correct F508del-CFTR trafficking. Finally, latonduine A’s unique pathway to correct F508del-CFTR was indirectly verified by its additive effects with other F508del-CFTR correctors having different mechanisms of action such as a PDE-5 inhibitor sildenafil and VX809, which corrects CFTR via action on MSD1. 213  Chapter 6: Conclusion  The Andersen lab has traditionally focused efforts on discovering therapeutic agents from the ocean through close collaborations with biologists and pharmacologists. As a part of this tradition, this doctoral thesis has discussed three types of bioactive marine natural products along with their potentials as drug leads.  In Chapter 2, the coumarins C11 (2.14) and C21 (2.15) were found significantly inhibitory against HCV NS3pro/Pep4A in a preliminary target-based screen. Unfortunately, C11 (2.14) and C21 (2.15) were ineffective at blocking HCV infection in a subsequent orthogonal assay using more biologically relevant human hepatoma cell lines. Therefore, this project was terminated in 2010.  Chapter 3, which is the core of this thesis, describes a relatively complete SAR study of the marine natural products sintokamides A – E (3.11 – 3.15) and their inhibitory activities against AR NTD transactivation, a possible new approach for the treatment of CRPC. In total, 29 synthetic precursors/analogues were synthesized for in vitro bioassays and their preparation was based on three synthetic routes with different scopes of chlorination. LPY26 [(4R,10R)-3.233], the N-pivaloylated hexachlorinated 1,17-dinorsintokamide, was identified as the best-in-class compound with superior bioactivity and physical stability. Therefore, LPY26 [(4R,10R)-3.233] has been selected as the lead for further pharmacological studies using a mouse xenograft model of CRPC. A mechanism of action study described in Chapter 4 revealed that LPY30 (4.7) and 214  LPY31 (4.8), the bioactive hexachlorinated 1,17-dinorsintokamide probes with terminal alkynes, could bind covalently to the full length AR.  Chapter 5 builds on an earlier successful forward chemical biology screen that identified a potent F508del-CFTR trafficking corrector latonduine A (5.1) from a library of 720 marine natural products. In vitro, latonduine A treatment facilitated trafficking of the mutant chloride channels through the ER with full glycosylation to the cell surface where they assumed their normal halide channel function. Latonduine A’s ability to correct F508del-CFTR was also confirmed by ex vivo and in vivo experiments in F508del-CFTR homozygous mice. My contribution to the project was to synthesize bioactive probes such as N-biotinylated latonduine A for molecular target identification. Use of this probe showed that latonduine A interacted with proteins in the poly (ADP-ribose) polymerase (PARP) family, especially PARP-3. The marine natural products-inspired forward chemical biology approach provided a novel target for new drug design and a new insight into the molecular pathology of cystic fibrosis resulting from the F508del mutation.215   Figure 6.1 The exploration of synthetic approaches to make active 1,17-dinorsintokamide analogues  216  Our exploration of synthetic approaches to make active 1,17-dinorsintokamide analogues is illustrated in Figure 6.1. Williard and Laszlo’s total synthesis of (±)-dysidin (3.17) in 1984 showed us a successful example of making an N-acyl bond between a methyl tetramate moiety and a trichlorinated carboxylic acid derivative (Scheme 3.3).131 Non-chlorinated sintokamide NCSTD [(4S,10R)-3.16] was thus made as described in section 3.3.1.4 but the molecule was not active in vitro. Our first effective synthetic route to make chlorinated 1,17-dinorsintokamide analogues was similar to Xu and Ye’s total synthesis of sintokamide C (3.13) published in early 2010 (Scheme 3.34, Scheme 3.35, and Scheme 3.36).104 The highlight of their synthesis could be summarized as preparing silylated synthons 3.112 and 3.119 for the key N-acylation step. However, this synthetic strategy showed a limited scope of chlorination for the 1,17-dinorsintokamide skeleton and only generated two inactive monochlorinated analogues LPY00 (3.60) and LPY04 (3.148) (Scheme 3.45 and Scheme 3.46). Later that year, Gu and Zakarian published the total syntheses of sintokamides A, B, and E.105 Their syntheses were different from the convergent protocol: polychlorinated dipeptides were prepared in advance then underwent condensation with Meldrum’s acid (Scheme 3.58). Based on this strategy, our work progressed and eventually furnished tetrachlorinated analogues LPY08 [(4R,10R)-3.210] and LPY09 [(4S,10R)-3.210] (Scheme 3.63) and hexachlorinated analogues LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220] (Scheme 3.71). In vitro experiments showed that the hexachlorinated 1,17-dinorsintokamides LPY10/11 were active. It should be pointed out that our selection of synthetic targets was influenced by the structures of the naturally occurring sintokamides throughout the development of synthetic methodology described above.  217   Figure 6.2 Lead selection based on current SAR understanding of sintokamides’ structural features  Figure 6.2 summarizes the results of the sintokamide SAR study based on the synthetic analogues prepared in this thesis and their inhibitory activities against AR NTD transactivation in vitro. First, the C-1 and C-17 methyl groups were found not necessary to elicit sintokamides’ maximum activities when C-18 and C-19 were both trichlorinated, as indicated by the activities of hexachlorinated 1,17-dinorsintokamides LPY10 [(4R,10R)-3.220] and LPY11 [(4S,10R)-3.220]. The trichloromethyl groups in LPY10/11 showed an additive effect since monotrichlorinated analogues LPY12 [(4R,10R)-3.228], LPY13 [(4S,10R)-3.228], LPY17 [(4R,10R)-3.231], and LPY18 [(4S,10R)-3.231] were inferior to LPY10/11 in vitro. The natural preference of C-19 trichlorination elicited better in vitro activity as observed in LPY12/13 compared to LPY17/18. Second, the methyl tetramate moiety was found to be a required pharmacophore element in the sintokamide structure. Either tetramic acid analogues LPY32 [(4R,10R)-3.235] and LPY33 [(4S,10R)-3.235] or hexachlorinated dipeptide LPY34 [(4R,10R)-218  3.219] and LPY35 [(4S,10R)-3.219] were inactive in vitro. Third, for configuration preferences, the R configuration at C-4 in LPY10 [(4R,10R)-3.220] presented improved dose response and toxicity profiles compared with the natural occurring 4S configuration in LPY11 [(4S,10R)-3.220]. However, C-10 must maintain its R configuration to assure desired in vitro activity, as LPY20 [(4S,10S)-3.220] and LPY21 [(4R,10S)-3.220] were much weaker inhibitors in vitro compared with their 10R diastereomers LPY10/11. Finally, by replacing the N-propionyl group with an N-pivaloyl group, LPY26 [(4R,10R)-3.233] and LPY27 [(4S,10R)-3.233] exhibited enhanced activity in vitro compared with LPY10/11. Putting all the optimal structural requirements together led to LPY26, the (4R,10R)-N-pivaloyl hexachlorinated 1,17-dinorsintokamide, which has been selected for scale-up synthesis and in vivo evaluation in a mouse xenograft model of CRPC.  219  Chapter 7: Experimental  7.1 General  All non-aqueous reactions were carried out in flame-dried glassware and under an argon or nitrogen atmosphere unless otherwise noted. Air and moisture sensitive liquid reagents were manipulated via a dry syringe. Anhydrous tetrahydrofuran (THF) was obtained from distillation over sodium. Other solvents and reagents were used as obtained from commercial sources without further purification. NMR spectra were obtained on Bruker Avance 400 direct, 400 inverse, 300 direct, or Bruker Avance 600 CryoProbe spectrometers at room temperature unless otherwise noted. Flash column chromatography was performed using Silicycle Ultra-Pure silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) plates were aluminum-backed ultrapure silica gel 250 m. Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a Micromass LCT instrument. Optical rotations were measured with a JASCO P-1010 polarimeter at room temperature and 589 nm (sodium D line) in chloroform (g/100 mL). X-ray crystallography was performed by Dr. Brian Patrick at UBC on a Bruker X8 APEX CCD single crystal X-ray diffraction instrument.      220  7.2 Experimental for Chapter 2  7.2.1 Preparation of 2.18 (C12)    2,4,5-trihydroxybenzaldeyde (1.54 g, 9.96 mmol), N-acetylglycine (1.89 g, 16.1 mmol) and NaOAc (1.30 g, 15.8 mmol) were refluxed in Ac2O for 3 h. The resulting mixture was cooled to room temperature. After addition of iced water (10 mL), the reaction mixture was poured into EtOAc (30 mL). The organic extract was washed with saturated NaHCO3 (3×30 mL), H2O (30 mL) and brine (30 mL), dried over anhydrous MgSO4 filtered, and concentrated in vacuo. The crude residue was washed with methanol/H2O solution (60 mL, 1:1) to give 3-acetamido-6,7-diacetoxycoumarin (2.18) (1.22 g, 3.82 mmol) as white solid in a yield of 38 %. 1H NMR (600 MHz, DMSO-d6) : 9.81 (s, 1H), 8.58 (s, 1H), 7.67 (s, 1H), 7.42 (s, 1H), 2.30 (s, 3H), 2.30 (s, 3H), 2.16 (s, 3H); 13C NMR (150 MHz, DMSO-d6) : 169.9, 167.9, 167.6, 156.7, 146.7, 142.0, 138.5, 124.4, 121.8, 121.1, 117.6, 111.1, 23.6, 20.0, 19.9; ESI-MS m/z: 320.0 [M + H]+; ESI-HRMS: m/z calcd for C15H14O7 [M + H]+, 320.0770; found, 320.0765.    221  7.2.2 Preparation of 2.14 (C11)    3-acetamido-6,7-diacetoxycoumarin C12 (1.20 g, 3.77 mmol) was refluxed in 3 M HCl (50 mL) and AcOH (2 mL) for 1 day. After cooled to room temperature, the reaction mixture was extracted with ethyl acetate (3×30 ml). The organic extract was washed with saturated NaHCO3 (3×30 mL), H2O (30 mL) and brine (30 mL), dried over anhydrous MgSO4 filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (3:1) to afford 3,6,7-trihydroxycoumarin (2.14) (100 mg, 0.515 mmol) as black solid in a yield of 14 %. 1H NMR (300 MHz, DMSO-d6) : 9.68 (s, 2H), 9.18 (s, 1H), 6.99 (s, 1H), 6.82 (s, 1H), 6.71 (s, 1H); 13C NMR (75 MHz, DMSO-d6) : 158.9, 146.5, 143.3, 143.1, 138.8, 116.3, 111.9, 110.8, 102.5; ESI-MS m/z: 207.0 [M + Na]+; ESI-HRMS: m/z calcd for C9H6O5Na [M + Na]+, 207.0113; found, 217.0112.        222  7.2.3 Preparation of 2.20 (C22)    2,3,4-trihydroxybenzaldeyde (0.98 g, 6.33 mmol), N-acetylglycine (1.21 g, 10.3 mmol) and NaOAc (0.82 g, 9.96 mmol) were refluxed in Ac2O for 3 h. The resulting mixture was cooled to room temperature. After addition of iced water (10 mL), the reaction mixture was poured into EtOAc (30 mL). The organic extract was washed with saturated NaHCO3 (3×30 mL), H2O (30 mL) and brine (30 mL), dried over anhydrous MgSO4 filtered, and concentrated in vacuo. The crude residue was washed with methanol/H2O solution (60 mL, 1:1) to give 3-acetamido-7,8-diacetoxycoumarin (C22) (1.10 g, 3.44 mmol) as white solid in a yield of 54 %. 1H NMR (600 MHz, DMSO-d6) : 9.83 (s, 1H), 8.64 (s, 1H), 7.66 (dd, J = 7.8, 2.4 Hz, 1H), 7.26 (dd, J = 7.8, 2.4 Hz, 1H), 2.41 (s, 3H), 2.32 (s, 3H), 2.17 (s, 3H); 13C NMR (150 MHz, DMSO-d6) : 170.3, 168.1, 167.4, 156.4, 142.9, 142.3, 129.2, 124.9, 124.4, 122.9, 119.7, 118.6, 23.9, 20.3, 19.9; ESI-MS m/z: 320.0 [M + H]+; ESI-HRMS: m/z calcd for C15H14O7 [M + H]+, 320.0770; found, 320.0772.     223  7.2.4 Preparation of 2.15 (C21)    3-acetamido-7,8-diacetoxycoumarin C12 (0.51 g, 1.60 mmol) was refluxed in 3 M HCl (25 mL) and AcOH (1 mL) for 1 day. After cooled to room temperature, the reaction mixture was extracted with ethyl acetate (3×30 ml). The organic extract was washed with saturated NaHCO3 (3×30 mL), H2O (30 mL) and brine (30 mL), dried over anhydrous MgSO4 filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (3:1) to afford 3,7,8-trihydroxycoumarin (C21) (38 mg, 0.195 mmol) as black solid in a yield of 12 %. 1H NMR (600 MHz, DMSO-d6) : 9.77 (s, 1H), 9.63 (s, 1H), 9.21 (s, 1H), 7.02 (s, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) : 158.7, 146.1, 139.4, 138.4, 132.1, 116.7, 116.2, 113.2, 112.8; ESI-MS m/z: 207.0 [M + Na]+; ESI-HRMS: m/z calcd for C9H6O5Na [M + Na]+, 207.0113; found, 217.0110.     224  7.3 Experimental for Chapter 3  7.3.1 Preparation of Compound 3.35    To a stirred solution of L-leucine 3.29 (5.12 g, 39.0 mmol) and Na2CO3 (4.14 g, 39.0 mmol) in H2O (40 mL) was added N-carbethoxyphathalimide 3.34 (8.55 g, 39.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for additional 2 h. Placed in an ice/water bath, the reaction mixture was added 6 M HCl dropwise until its pH value was adjusted to 0. The aqueous layer was extracted with hexanes (3×100 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (3:1) to afford N-phthalimide-L-leucine 3.35 (10.2 g, 39.0 mmol) as a colourless oil in a quantitative yield. 1H NMR (400 MHz, CDCl3)  11.32 (br. s., 1H), 7.85 (dd, J = 5.4, 3.0 Hz, 2H), 7.73 (dd, J = 5.4, 3.0 Hz, 2H), 4.99 (dd, J =11.5, 4.2 Hz, 1H), 2.36 (ddd, J = 14.3, 10.0, 4.2 Hz, 1H), 1.95 (ddd, J =14.3, 10.0, 4.2 Hz, 1H), 1.37–1.60 (m, 1H), 0.94 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3)  176.0, 167.9, 134.4, 131.9, 123.8, 50.6, 37.2, 25.3, 23.3, 21.2; ESI-MS: m/z 284.2 [M + Na]+. 225  7.3.2 Preparation of Compound 3.37    To a stirred solution of N-phthalimide-L-leucine 3.35 (550 mg, 2.10 mmol) in DCM (4 mL) was added SOCl2 (1.5 mL, 20.5 mmol) at room temperature. The reaction mixture was refluxed for 5 h then it was evaporated in vacuo to afford acid chloride 3.36 (535 mg, 1.91 mmol) as a yellowish oil in a yield of 91 %. The product was used directly without further purification.  To a solution HCl (50 mL, 3 M) was added monoethyl malonate potassium salt (580 mg, 3.41 mmol) at room temperature. The aqueous suspension was extracted with EtOAc (3×50 mL). The combined organic extract was washed with H2O (50 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford a colourless oil 3.22 (450 mg, 3.41 mmol) quantitatively. The crude was used directly without further purification.  To a stirred solution of monoethyl malonate 3.22 (450 mg, 3.41 mmol) in THF (5 mL) at −78 °C was added dropwise n-BuLi (3.7 mL, 7.40 mmol, 2.0 M in hexanes). The resultant white suspension was warmed gradually to −5 °C and then cooled to −78 °C again. The reaction mixture was stirred at −78 °C for 1 h. After addition of acid chloride 3.36 (535 mg, 1.91 mmol) 226  in THF (2 mL) all at once, the reaction mixture was stirred at −78 °C for another 20 min then poured onto a mixture of HCl (7 mL, 1 M) and ether (14 mL). The separated aqueous layer was extracted with ether (2×10 mL). The combined organic extract was washed with saturated NaHCO3 (3×10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (97:3) to afford homologous 1,3-diketone ester 3.37 (331 mg, 0.99 mmol) as a yellowish oil in a yield of 52 %. 1H NMR (300 MHz, CDCl3)  7.88 (dd, J = 5.6, 3.0 Hz, 2H), 7.76 (dd, J = 5.6, 3.0 Hz, 2H), 5.00 (dd, J = 11.3, 4.2 Hz, 1H), 4.14 (q, J = 7.0 Hz, 2H), 3.52 (s, 2H), 2.24 (ddd, J = 14.1, 10.0, 4.1 Hz, 1H), 1.91 (ddd, J = 14.1, 10.0, 4.1 Hz, 1H), 1.37–1.57 (m, 1H), 1.23 (t, J = 7.0 Hz, 3H), 0.95 (d, J = 7.0 Hz, 3H), 0.93 (d, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3)  198.3, 168.0, 166.6, 134.6, 131.9, 123.8, 61.9, 57.7, 46.5, 36.5, 25.3, 23.5, 21.3, 14.2.  7.3.3 C-methylation of compound 3.37 with MeI and KH   To a stirred solution of -keto ester 3.37 (50 mg, 0.15 mmol) in DMF (0.3 mL) was added dropwise KH suspension (6.6 mg, 0.41 mmol) in DMF (1 mL) at room temperature. The reaction 227  mixture was stirred at 5 °C for 20 min then added MeI (0.3 mL, 3.16 mmol) all at once. After addition of saturated NaHCO3 (2 mL), the aqueous layer was extracted with n-pentane (5×10 mL). The combined organic extract was washed with NaOH (2×10 mL, 1 M) and H2O (10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified through a Sep-Pak® Silica (2 g) cartridge eluting with hexanes/acetone (20:1) to afford a mixtures of enol esters (1.0 mg, 0.003 mmol) as a colourless oil in a yield of 1.3 %.  7.3.4 O-methylation of compound 3.37 with Me2SO4 and KH    To a stirred solution of -keto ester 3.37 (80.8 mg, 0.24 mmol) in DMF (0.7 mL) was added dropwise KH suspension (34 mg, 0.85 mmol) in DMF (1 mL) at 5 °C. The reaction mixture was stirred at 5 °C for 20 min then added dimethyl sulfate (0.3 mL, 3.16 mmol) all at once. After addition of saturated NaHCO3 (2 mL), the aqueous layer was extracted with n-pentane (5×10 mL). The combined organic extract was washed with NaOH (2×10 mL, 1 M) and H2O (10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified 228  through a Sep-Pak® Silica (2 g) cartridge eluting with hexanes/acetone (20:1) to afford enol ester 3.39 (1.0 mg, 0.003 mmol) as a colourless oil in a yield of 1.2 %.  7.3.5 Preparation of Compound 3.40    To a stirred solution of -keto ester 3.37 (186 mg, 0.56 mmol) in MeOH (5 mL) were added trimethyl orthoformate (0.25 mL, 2.24mmol) and a drop of conc. H2SO4 at room temperature. The reaction mixture was refluxed overnight. After addition of ether (80 mL), the organic layer was washed with saturated NaHCO3 (3×10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (9:1 → 8:1) to afford desired product E-enol ether methyl ester 3.40 (130 mg, 0.39 mmol) as a yellow oil in a yield of 70 %. 1H NMR (300 MHz, CDCl3)  7.83 (dd, J = 5.4, 2.9 Hz, 2H), 7.71 (dd, J = 5.4, 2.9 Hz, 2H), 6.33 (dd, J = 11.4, 4.8 Hz, 1H), 5.05 (s, 1H), 3.74 (s, 3H), 3.63 (s, 3H), 2.66 (ddd, J = 13.1, 11.4, 3.8 Hz, 1H), 1.67 (ddd, J = 13.1, 11.4, 3.8 Hz, 1H), 1.49–1.60 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H),0.96 (d, J = 6.4 Hz, 3H); 13C NMR (100 229  MHz, CDCl3)  172.3, 168.7, 167.0, 134.0, 132.2, 123.4, 91.2, 56.3, 51.4, 50.2, 38.3, 25.6, 23.4, 21.2. 211 212 212 212 212 212 212 212  7.3.6 Preparation of Compound 3.32    To a stirred solution of enol ether 3.40 (53 mg, 0.16 mmol) in MeOH (5 mL) was added excessive hydrazine hydrate (2 mL) at room temperature. The reaction mixture was refluxed overnight then the solvent was removed in vacuo. After addition of DCM (40 mL) and water (40 mL), the separated aqueous layer was extracted with DCM (2×40 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/MeOH (200:1) to afford methyl tetramate 3.32 (15 mg, 0.088 mmol) as a white solid in a yield of 55 %. 1H NMR (400 MHz, CDCl3)  6.24 (br. s., 1H), 5.00 (d, J = 1.0 Hz, 1H), 4.06 (dd, J = 9.5, 3.4 Hz, 1H), 3.79 (s, 3H), 1.70–1.83 (m, 1H), 1.64 (td, J = 9.2, 4.7 Hz, 1H),1.38 (td, J = 9.2, 4.7 Hz, 1H), 0.97 (d, J = 2.0 Hz, 3H), 0.95 (d, J = 2.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)  179.2, 174.5, 93.3, 58.4, 56.2, 41.6, 25.6, 23.6, 22.0; ESI-MS m/z 170.3 [M+H]+. 211 212 212 212 212 212 212 212   230  7.3.7 Coupling compound 3.32 and compound 3.41 in the presence of MeMgBr    To a stirred solution of mehtyl tetramate 3.32 (10 mg, 0.06 mmol) in THF (2 mL) at 0 °C was added MeMgBr (20 L, 3.0 M in ether). The reaction mixture was stirred at 0 °C for 5 min. After dropwise addition of acid chloride 3.41 (20 mg, 0.07 mmol) in THF (2 mL), the reaction mixture was stirred at room temperature overnight. After addition of saturated NaHCO3 (2 mL), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was washed with brine (10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified through a Sep-Pak® Silica (2 g) cartridge eluting with DCM/MeOH (200:1) to afford coupling product 3.42 (2 mg, 0.005 mmol) as a yellowish oil in a yield of 8 %. ESI-MS m/z: 435.2 [M+Na]+.     231  7.3.8 Preparation of Compound 3.45    To a stirred solution of Boc-D-leucine (R)-3.43 (878.9 mg, 3.80 mmol) and p-nitrophenol 3.44 (581 mg, 4.18 mmol) in THF (20 mL) was added DCC (784.3 mg, 3.80 mmol) at 5 °C. The reaction mixture was stirred at room temperature overnight. After addition of hexanes (50 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (5:1→3:1) to afford active ester 3.45 (1.11 g, 3.125 mmol) as a colourless oil in a yield of 82 %. 1H NMR (400 MHz, CDCl3)  8.28 (d, J = 9.2 Hz, 2H), 7.31 (d, J = 9.2 Hz, 2H), 4.94 (d, J = 6.1 Hz, 1H), 4.52 (br. s., 1H), 1.75–1.86 (m, 2H), 1.61–1.71 (m, 1H), 1.47 (s, 9H), 1.04 (d, J = 2.0 Hz, 3H), 1.02 (d, J = 2.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)  171.6, 155.7, 155.5, 145.6, 125.4, 122.5, 80.6, 52.7, 41.3, 28.5, 25.1, 23.0, 21.9.       232  7.3.9 Preparation of Compound (4S,10R)-3.46    To a stirred solution of methyl tetramate 3.32 (11.2 mg, 0.066 mmol) in THF (2 mL) at −50 °C was added n-BuLi (32 l, 0.066 mmol, 1.60 M in hexanes). The reaction mixture was stirred at −50 °C for 10 min. After dropwise addition of Boc-D-Leu-ONp 3.45 (25.6 mg, 0.073 mmol) in THF (2 mL), the reaction mixture was stirred at −50 °C for another 10 min. The reaction mixture was added AcOH (0.1 mL) then the solvents was evaporated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:1) to afford coupling product (4S,10R)-3.46 (12.0 mg, 0.031 mmol) as a as white powder in a yield of 47 %. 1H NMR (400 MHz, CDCl3)  5.45 (td, J = 2.9, 1.9 Hz, 1H), 5.10 (br. d, J = 8.0 Hz, 1H), 5.04 (s, 3H), 4.58 (t, J = 5.1 Hz, 1H), 1.75–1.88 (m, 6H), 1.46 (s, 9H), 1.32–1.40 (m, 1H), 1.04 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.6 Hz, 6H), 0.89 (d, J = 5.8 Hz, 3H).      233  7.3.10 Preparation of Compound 3.47    Compound (4S,10R)-3.46 (5.0 mg, 0.013 mmol) was dissolved in TFA/DCM (1 mL, 1:3) and stirred at 0 °C for 10 min then poured onto ammonia solution (10 mL, 25 %). The aqueous layer was extracted with DCM (3×10 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified through a Sep-Pak® Silica (2 g) cartridge eluting with DCM/MeOH (98:2) to afford free amine 3.47 (3.3 mg, 0.012 mmol) as a white powder in a yield of 90 %. 1H NMR (400 MHz, CDCl3)  5.05 (s, 1H), 4.60 (t, J = 5.1 Hz, 1H), 4.55 (dd, J = 9.5, 4.2 Hz, 1H), 3.87 (s, 3H), 1.83–1.92 (m, 2H), 1.76 (td, J = 13.3, 6.5 Hz, 1H), 1.54 (ddd, J = 13.4, 9.0, 4.3 Hz, 1H), 1.32 (td, J = 8.9, 4.8 Hz, 1H), 0.98 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 2.6 Hz, 3H), 0.93 (d, J = 2.9 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H); ESI-MS m/z: 283.3 [M+H]+.      234  7.3.11 Preparation of (4S,10R)-3.16 (NCSTD)    To a stirred solution of free amine 3.47 (1.0 mg, 0.0035 mmol) in pyridine (2 mL) was added propionyl anhydride (1.4 l, 0.011 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. After addition of HCl (10 mL, 1 M), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:1) to afford N-propionyl product NCSTD (4S,10R)-3.16 (1.0 mg, 0.0029 mmol) as a colourless oil in a yield of 83 %. 1H NMR (600 MHz, CDCl3) : 6.04 (d, J = 8.8 Hz, 1H), 5.75 (ddd, J = 10.7, 9.0, 2.9 Hz, 1H), 5.05 (s, 1 H), 4.57 (dd, J = 6.2, 4.0 Hz, 1H), 3.87 (s, 3H), 2.25 (q, J = 7.7 Hz, 2H), 1.80–1.83 (m, 3H), 1.75–1.79 (m, 1H), 1.59 (dt, J = 6.9, 3.6 Hz, 1H), 1.40 (dt, J = 6.9, 3.6 Hz, 1H), 1.17 (t, J = 7.5 Hz, 3H), 1.05 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 6H), 0.88 (d, J = 6.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.0, 173.3, 173.1, 169.6, 93.6, 58.9, 58.9, 51.6, 41.7, 39.3, 29.9, 25.2, 24.3, 23.9, 23.8, 22.7, 21.4, 10.0.  235  7.3.12 Preparation of Compound (2S,3R,5R)-3.67    To a stirred solution of (5R, 6S)-4-Boc-5,6-diphenylmorpholin-2-one (2S,3R)-3.66 (300 mg, 0.848 mmol) and 1-bromo-3-chloropropane 3.65 (0.83 mL, 8.39 mmol) in THF/HMPA (33 mL, 10:1) at −78 °C was added lithium bis(trimethylsilyl)amide (1.28 mL, 1.28 mmol, 1.0 M in THF) dropwise. After 2 h, the dry ice/acetone bath was removed. The reaction mixture was stirred at room temperature for additional 2 h then poured onto ice cold water (50 mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified through a Sep-Pak® Silica (5 g) cartridge eluting with hexanes/EtOAc (19:1) to afford chloride (2S,3R,5R)-3.67 (155 mg, 0.360 mmol) as a white powder in a yield of 42 %. 1H NMR (400 MHz, DMSO-d6, 298 K)  6.88–7.38 (m, 10H), 6.49–6.69 (m, 1H), 5.95 (br. s., 1H), 4.72–5.31 (m, 1H), 3.53–3.78 (m, 2H), 1.96–2.58 (m, 4H), 1.41 (br. s., 2H), 1.04 (br. s., 7H); 1H NMR (400 MHz, DMSO-d6, 398 K)  6.94–7.41 (m, 10H), 6.49–6.70 (m, 2H), 6.20 (br. s., 1H), 5.17 (br. s., 1H), 4.81 (br. s., 1H), 3.71–3.77 (m, 2H), 2.15–2.36 (m, 1H), 1.94–2.06 (m, 1H), 1.23 (br. s., 9H); ESI-MS m/z: 452.3 [M+Na]+.   236  7.3.13 Preparation of Compound (2S,3R,5R)-3.68    (2S,3R,5R)-3.67 (131 mg, 0.304 mmol) was dissolved in TFA/H2O (15 mL, 95 %) and stirred at room temperature for 2 h. The solvent was evaporated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1) to afford chloride (2S,3R,5R)-3.68 (85 mg, 0.256 mmol) as a light yellow oil in a yield of 84 %. (2S,3R,5R)-3.67 was directly for the next step used without characterization.  7.3.14 Preparation of Compound (R)-3.69    To a stirred solution of (2S,3R,5R)-3.68 (74 mg, 0.224 mmol) in MeOH/H2O/AcOH (26 mL, 10:2:1) was added PdCl2 (80 mg, 0.451 mmol). The reaction mixture was hydrogenated under atmospheric pressure at room temperature for 12 h. The catalyst was removed through a pad of Celite® with MeOH (20 mL) and the filtrate was concentrated in vacuo to afford amino acid 237  crude (26 mg, 0.169 mmol) as a yellowish oil in a yield of 75 %. The crude residue was used directly for the next step without further purification. ESI-MS m/z: 152.2 [M+H]+.  To a stirred solution of amino acid crude (26 mg, 0.169 mmol), NaHCO3 (28 mg, 0.338 mmol) in H2O/THF (10 mL, 1:1) was added Boc2O (39 mg, 0.178 mmol) at room temperature. The reaction mixture was refluxed overnight then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M NaHSO4 dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2) to afford (R)-3.69 (38 mg, 0.152 mmol) as a colourless oil in a yield of 68 % over two steps. The 1H and 13C NMR spectra of (R)-3.69 recorded in CDCl3 is extremely hard to interpret due to poor purity of the sample and complexities of peaks. The structure was tentatively confirmed with MS data: ESI-MS m/z: 270.2 [M+H2O+H]+.  7.3.15 Preparation of Compound (2R,3S,5S)-3.67    Under a same procedure described in 7.3.12, (2R,3S,5S)-3.67 (155 mg, 0.360 mmol) was prepared from (2R,3S)-3.66 (300 mg, 0.848 mmol) as a white powder in a yield of 40 %. The 238  crude residue was purified through a Sep-Pak® Silica (5 g) cartridge eluting with hexanes/acetone (97:3). 1H NMR (400 MHz, DMSO-d6, 298 K)  7.02–7.30 (m, 10H), 6.51–6.57 (m, 2H), 6.21–6.38 (m, 1H), 5.10–5.28 (m, 1H), 4.68–4.82 (m, 1H), 3.68–3.88 (m, 2H), 2.20 (m, 1H), 1.91–2.02 (m, 1H), 1.41 (br. s., 2H), 1.04 (br. s., 7H); ESI-MS m/z: 452.3 [M+Na]+.  7.3.16 Preparation of Compound (2R,3S,5S)-3.68    Under a similar procedure described in 7.3.13, (2R,3S,5S)-3.68 (85 mg, 0.256 mmol) was prepared from (2R,3S,5S)-3.67 (131 mg, 0.304 mmol) as a light yellow oil in a yield of 80 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1). (2R,3S,5S)-3.68 was directly for the next step used without characterization.        239  7.3.17 Preparation of Compound (S)-3.69    Under a similar procedure described in 7.3.14, (S)-3.69 (38 mg, 0.152 mmol) was prepared from (2R,3S,5S)-3.68 (74 mg, 0.224 mmol) as a colourless oil in a yield of 58 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2). The 1H and 13C NMR spectra of (S)-3.69 recorded in CDCl3 is extremely hard to interpret due to poor purity of the sample and complexities of peaks. The structure was tentatively confirmed with MS data: ESI-MS m/z: 270.2 [M+H2O+H]+.  7.3.18 Preparation of Compound (2S,3R,5R)-3.71    To a stirred solution of (5R, 6S)-4-Boc-5,6-diphenylmorpholin-2-one (2S,3R)-3.66 (300 mg, 0.848 mmol) and 1,1,1-trichloro-3-bromo-propane 3.70 (1.10 mL, 8.56 mmol) in THF/HMPA (33 mL, 10:1) at −78 °C was added lithium bis(trimethylsilyl)amide (1.53 mL, 1.53 mmol, 1.0 M in THF) dropwise. After 2.5 h, the dry ice/acetone bath was removed. The reaction mixture was 240  stirred at room temperature for additional 2.5 h then poured onto ice cold water (50 mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified through a Sep-Pak® Silica (5g) cartridge eluting with hexanes/EtOAc (15:1) to afford gem-dichloride (2S,3R,5R)-3.71 (125 mg, 0.271 mmol) as a white powder in a yield of 32 %. 1H NMR (400 MHz, DMSO-d6, 298 K)  7.20–7.31 (m, 3H), 7.14–7.20 (m, 1H), 7.06–7.12 (m, 2H), 6.98 (d, J = 7.1 Hz, 2H), 6.53–6.62 (m, 2H), 6.14 (t, J = 7.6 Hz, 1H), 5.97 (d, J = 3.0 Hz, 1H), 5.17 (t, J = 7.1 Hz, 1H), 5.02 (d, J = 3.0 Hz, 1H), 2.97–3.05 (m, 2H), 1.45–1.49 (m, 2H), 1.12 (s, 7H); 13C NMR (100 MHz, DMSO-d6)  153.9, 136.5, 134.3, 128.9, 128.4, 128.3, 128.1, 127.9, 127.9, 127.6, 127.4, 126.7, 126.6, 124.3, 124.1, 123.8, 82.3, 81.7, 79.9, 79.4, 61.5, 60.5, 56.4, 55.0, 35.3, 34.8, 28.5, 28.0; 1H NMR (400 MHz, DMSO-d6, 373 K)  7.02–7.31 (m, 8H), 6.58 (d, J = 7.6 Hz, 2H), 6.21–6.30 (m, 2H), 5.17 (br. s., 1H), 4.96 (t, J = 7.3 Hz, 1H), 2.97–3.10 (m, 3H), 1.19 (br. s., 9H).  7.3.19 Preparation of Compound (2S,3R,5R)-3.72    Under a similar procedure described in 7.3.13, (2S,3R,5R)-3.72 (50 mg, 0.138 mmol) was prepared from (2S,3R,5R)-3.71 (82 mg, 0.178 mmol) as a light yellow oil in a yield of 77 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1). 241  1H NMR (400 MHz, DMSO-d6)  7.06–7.30 (m, 10H), 5.84 (t, J = 7.0 Hz, 1H), 4.78 (d, J = 5.1 Hz, 1H), 3.80 (d, J = 5.4 Hz, 1H), 3.01 (t, J = 5.1 Hz, 1H), 2.29–2.45 (m, 2H); 13C NMR (100 MHz, DMSO-d6) 173.8, 143.2, 139.9, 128.5, 127.4, 127.3, 127.2, 126.7, 126.7, 126.5, 119.0, 75.6, 65.6, 65.1, 31.5.  7.3.20 Preparation of Compound (R)-3.73    Under a similar procedure described in 7.3.14, (R)-3.73 (20 mg, 0.069 mmol) was prepared from (2S,3R,5R)-3.72 (43 mg, 0.118 mmol) as a light yellow oil in a yield of 59 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2). The 1H and 13C NMR spectra of (R)-3.73 recorded in CDCl3 is extremely hard to assign due to poor purity of the sample and complexities of peaks. The structure was tentatively confirmed with MS data: ESI-MS m/z: 286.2 [M+H]+.      242  7.3.21 Preparation of Compound (2R,3S,5S)-3.71    Under a same procedure described in 7.3.18, (2R,3S,5S)-3.71 (125 mg, 0.271 mmol) was prepared from (2R,3S)-3.66 (300 mg, 0.848 mmol) as a white powder in a yield of 37 %. The crude residue was purified through a Sep-Pak® Silica (5 g) cartridge eluting with hexanes/EtOAc (19:1). 1H NMR (300 MHz, DMSO-d6)  7.20–7.34 (m, 3H), 7.15–7.20 (m, 1H), 7.06–7.15 (m, 2H), 7.00 (dd, J = 1.1, 7.7 Hz, 2H), 6.53–6.64 (m, 2H), 6.15 (t, J = 7.7 Hz, 1H), 5.99 (d, J = 2.9 Hz, 1H), 5.19 (t, J = 7.2 Hz, 1H), 5.04 (d, J = 3.2 Hz, 1H), 3.03 (t, J = 7.5 Hz, 2H), 1.48 (s, 2H), 1.14 (s, 7H); 13C NMR (75 MHz, DMSO-d6)  153.9, 136.5, 134.3, 128.9, 128.4, 128.3, 128.1, 127.9, 127.9, 127.6, 127.4, 126.7, 126.6, 124.3, 124.1, 123.8, 82.4, 81.7, 79.9, 79.4, 61.5, 60.5, 56.4, 55.0, 35.3, 34.8, 28.5, 28.0.         243  7.3.22 Preparation of Compound (2R,3S,5S)-3.72    Under a similar procedure described in 7.3.13, (2R,3S,5S)-3.72 (50 mg, 0.138 mmol) was prepared from (2R,3S,5S)-3.71 (82 mg, 0.178 mmol) as a light yellow oil in a yield of 75 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1). 1H NMR (400 MHz, DMSO-d6)  7.06–7.30 (m, 10H), 5.84 (t, J = 7.0 Hz, 1H), 4.78 (d, J = 5.1 Hz, 1H), 3.80 (d, J = 5.4 Hz, 1H), 3.01 (t, J = 5.1 Hz, 1H), 2.29–2.45 (m, 2H); 13C NMR (100 MHz, DMSO-d6) 173.8, 143.2, 139.9, 128.5, 127.4, 127.3, 127.2, 126.7, 126.7, 126.5, 119.0, 75.6, 65.6, 65.1, 31.5.  7.3.23 Preparation of Compound (S)-3.73    Under a similar procedure described in 7.3.14, (S)-3.73 (20 mg, 0.069 mmol) was prepared from (2R,3S,5S)-3.72 (43 mg, 0.118 mmol) as a colourless oil in a yield of 59 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2). The 1H and 244  13C NMR spectra of (S)-3.73 recorded in CDCl3 is extremely hard to assign due to poor purity of the sample and complexities of peaks. The structure was tentatively confirmed with MS data: ESI-MS m/z: 286.2 [M+H]+.  7.3.24 Preparation of Compound 3.75     To a vigorously stirred solution of NaOH (10 g, 0.25 mol) in water (10 mL) were successively added BnEt3N+Cl− (0.20 g, 0.878 mmol), methyl acrylate 3.74 (4.0 mL, 44.4 mmol), and chloroform (40 g, 500 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. After addition of DCM (50 mL) and water (50 mL), the separated organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. Vacuum distillation of the crude residue gave methyl 4,4,4-trichlorobutanoate 3.75 (7.39 g, 36.0 mmol) as a colourless liquid in a yield of 81 %. The crude residue could also be purified by silica gel flash chromatography eluting with hexanes/acetone (19:1) to afford 3.75. bp 78 °C–80 °C/1 Torr (lit. bp 80 °C/0.3 Torr); 1H NMR (300 MHz, CDCl3)  3.72 (s, 3H), 2.95–3.13 (m, 2H), 2.68–2.86 (m, 2H) ; 13C NMR (75 MHz, CDCl3)  171.6, 98.7, 52.3, 50.0, 31.4; No EI- or ESI-MS were detected.  245  7.3.25 Preparation of Compound 3.76    To a stirred solution of methyl 4,4,4-trichlorobutanoate 3.75 (1.35 g, 6.57 mmol) in DCM (30 mL) at −78 °C was added DIBAL-H (6.57 mL, 6.57 mmol, 1.0 M in hexanes) at a rate of 1.0 mL/min. The reaction mixture was stirred at −78 °C for 1 h. After successive addition of MeOH (7 mL), HCl (25 mL, 1 M) and Rochelle salt solution (25 mL, 1 M), the reaction mixture was stirred at room temperature for another 1 h. The separated organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (20:1) to afford 4,4,4-trichlorobutanal 3.76 (1.15 g, 5.78 mmol) as a colourless oil in a yield of 88 %. 1H NMR (400 MHz, CDCl3)  9.85 (br. s., 1H), 3.03–3.11 (m, 2H), 2.96–3.02 (m, 2H); 13C NMR (100 MHz, CDCl3)  198.5, 98.9, 47.5, 41.2.       246  7.3.26 Preparation of Compound 3.77    BnNH2 (500 l, 4.56 mmol), TMSCN (680 l, 5.47 mmol) were added successively to aldehyde 3.76 (800 mg, 4.56 mmol) at room temperature. The reaction mixture was stirred for 15 min. After addition of saturated NH4Cl (5 mL), the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (30:1) to afford -aminonitrile 3.77 (1.33 g, 4.56 mmol) as a yellowish solid quantitatively. 1H NMR (400 MHz, CDCl3)  7.28–7.44 (m, 5H), 4.09 (d, J = 12.9 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.59 (t, J = 6.6 Hz, 1H), 2.96–3.07 (m, 1H), 2.79–2.89 (m, 1H), 2.21–2.33 (m, 2H); 13C NMR (100 MHz, CDCl3)  138.0, 128.7, 128.4, 127.8, 119.3, 98.7, 51.5, 51.1, 48.1, 30.5. ESI-MS m/z: 291.0 [M+H]+.       247  7.3.27 Preparation of Compound 3.78    To a stirred solution of NaOH (25 mL, 3 M) and H2O2 (9.0 mL, 30 %) was added -aminonitrile 3.77 (500 mg, 1.71 mmol) at room temperature. The reaction mixture was heated at 70 °C for 7 h and at 110 °C for 1 h and cooled to room temperature. Placed in an ice/water bath, the reaction mixture was added 3 M HCl dropwise until its pH value was adjusted to pH=1. The aqueous suspension was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/MeOH (99:1) to afford 3.78 (478 mg, 1.53 mmol) as a colourless oil in a yield of 90 %. 1H NMR (400 MHz, methanol-d4)  8.19–8.54 (m, 1H), 7.30–7.66 (m, 5H), 4.11 (q, J = 6.9 Hz, 2H), 3.63–3.73 (m, 1H), 3.02–3.09 (m, 1H), 2.91–3.05 (m, 1H), 1.25 (t, J = 7.1 Hz, 2H); 13C NMR (100 MHz, methanol-d4)  173.1, 134.5, 131.6, 130.2, 99.8, 61.7, 44.5, 44.4, 21.2.      248  7.3.28 Preparation of Compound 3.79    To a stirred solution of 3.78 (249 mg, 0.801 mmol) in MeOH/H2O/conc. HCl (35 mL, 3:3:1) was added Pd/C (90 mg, 10 %). The reaction mixture was hydrogenated under atmospheric pressure at room temperature overnight. The catalyst was removed through a pad of Celite® with MeOH (20 mL) and the filtrate was concentrated in vacuo to afford reaction crude (143 mg, 0.561 mmol) as a white powder in a yield of 70 %. The reaction crude was directly used for the next step without further purification.  To a stirred solution of amino acid (143 mg, 0.561 mmol), NaHCO3 (235 mg, 2.80 mmol) in H2O/THF (10 mL, 1:1) was added Boc2O (134 mg, 0.618 mmol) at room temperature. The reaction mixture was refluxed overnight then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M NaHSO4 dropwise until its pH value was adjusted to pH=3. The aqueous suspension was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2) to afford 3.79 (110 mg, 0.343 mmol) as a colourless oil in a yield of 62 %. Due to the large amount of Bn residue, the 1H NMR of 3.79 is hard to interpret. However, the 13C NMR of 3.79 is relatively clean and shows diagnostic peaks for Boc (C 157.4, 80.2, 28.4) and CCl3 ( C 99.4). 249  7.3.29 Preparation of Compound 3.32    To a stirred solution of Boc-L-leucine (S)-3.43 (2.00 g, 8.65 mmol), Meldrum’s acid 3.85 (1.37 g, 9.51 mmol), DMAP (1.48 g, 12.1 mmol) in DCM (10 mL) was added DCC (2.14 g, 10.4 mmol) at 0 °C. The reaction mixture was stirred at room temperature for additional 5 h then poured onto cold EtOAc (50 mL). The white precipitate formed was filtered off. After successive wash with cold KHSO4 solution (30 mL, 5 %), cold water (30 mL) and cold brine (30 mL), the filtrate was dried over anhydrous MgSO4 and concentrated in vacuo. The crude residue was used directly for the next step without further purification.  The yellowish crude residue was refluxed in EtOAc (20 mL) for 1 h then the solvent was removed in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of crude residue in MeOH (5 mL) was added trimethyl orthoformate (3.8 mL, 34.6 mmol) and a drop of conc. H2SO4 at room temperature. The reaction mixture was refluxed overnight then the solvent was removed in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1→1:1) to afford methyl 250  tetramate 3.32 (586 mg, 3.46 mmol) as a colourless oil in a yield of 40 %. 1H NMR (400 MHz, CDCl3)  6.68 (br. s., 1H), 4.99 (s, 1H), 4.05 (dd, J = 3.8, 9.6 Hz, 1H), 3.78 (s, 3H), 1.69–1.86 (m, 1H), 1.55–1.67 (m, 1H), 1.30–1.45 (m, 1H), 0.96 (d, J = 3.4 Hz, 3H), 0.94 (d, J = 3.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)  179.2, 174.7, 93.4, 58.4, 56.3, 41.7, 25.6, 23.7, 22.0.  The attempts to make 3.91 from (S)-3.69, 3.92 from (S)-3.73, and 3.93 from 3.79 were failed based on the above procedure.  The attempts to make 3.94 from (R)-3.69, 3.95 from (R)-3.73, and 3.96 from 3.79 were failed based on the procedure described in 7.3.8 of making 3.45 from (R)-3.43 and 3.44.  7.3.30 Preparation of Compound (S)-3.129    To a stirred solution of L-glutamic acid (S)-3.99 (10.2 g, 69.3 mmol) in methanol (200 mL) at 0 °C was added trimethylsilyl chloride (38.6 mL, 304.1 mmol) at a rate of 1.0 mL/min. The reaction mixture was stirred at room temperature overnight. After successive addition of triethylamine (60 mL, 430.4 mmol) and di-tert-butyldicarbonate (16.6 g, 76.0 mmol), the reaction mixture was stirred at room temperature for additional 2 days then the solvent was evaporated in vacuo. After triturated with ether (3×80 mL), the insoluble was filtered through a 251  pad of Celite® and the combined filtrate was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (8:1→1:1) to afford (S)-3.129 (18.3 g, 66.1 mmol) as a colourless oil in a yield of 96 %. 1H NMR (400 MHz, CDCl3)  5.23 (d, J = 5.5 Hz, 1H), 4.24 (d, J = 4.1 Hz, 1H), 3.65 (s, 3H), 3.59 (s, 3H), 2.25–2.42 (m, 2H), 2.03–2.14 (m, 1H), 1.80–1.93 (m, 1H), 1.35 (s, 9H); 13C NMR (100 MHz,CDCl3)  173.2, 172.7, 155.4, 79.9, 52.9, 52.4, 51.7, 30.1, 28.3, 27.6.  7.3.31 Preparation of Compound (S)-3.130    To a stirred solution of (S)-3.129 (18.2 g, 66.1 mmol) in acetonitrile (100 mL) at room temperature were added Boc2O (31.8 g, 145.7 mmol) in acetonitrile (40 mL) and DMAP (1.62 g, 13.2 mmol). The reaction mixture was stirred at room temperature for 1 day then the solvent was removed in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1→3:1) to afford (S)-3.130 (24.8 g, 66.1 mmol) as a colourless oil in a yield of 100 %. 1H NMR (400 MHz, CDCl3)  4.86 (br. s., 1H), 3.62–3.68 (m, 3H), 3.56–3.62 (m, 3H), 2.28–2.38 (m, 2H), 2.05–2.13 (m, 2H), 1.42 (d, J = 10.1 Hz, 18H); 13C NMR (100 MHz, CDCl3)  173.1, 170.8, 151.9, 83.3, 57.4, 52.2, 51.6, 30.6, 28.0, 25.2.  252  7.3.32 Preparation of Compound (S)-3.131    To a stirred solution of (S)-3.130 (5.31 g, 14.1 mmol) in THF (30 mL) at −40 °C was added DIBAL-H (42.4 mL, 42.4 mmol, 1.0 M in hexanes) at a rate of 1.0 mL/min. The reaction mixture was stirred at −40 °C for 1 h. After successive addition of MeOH (40 mL), HCl (40 mL, 1 M) and Rochelle salt solution (40 mL, 1 M), the reaction mixture was stirred at room temperature for another 2 h. The separated aqueous layer was extracted with DCM (4×40 mL). The combined organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:1) to afford primary alcohol (S)-3.131 (2.26 g, 6.50 mmol) as a colourless oil in a yield of 46 %. 1H NMR (400 MHz, CDCl3)  4.86 (dd, J = 5.5, 8.8 Hz, 1H), 3.69 (s, 3H), 3.61–3.67 (m, 2H), 2.16–2.27 (m, 1H), 1.87–1.97 (m, 1H), 1.56–1.66 (m, 2H), 1.48 (s, 18H); 13C NMR (100 MHz, CDCl3)  171.5, 152.3, 83.3, 62.4, 58.0, 52.3, 29.5, 28.1, 26.6.      253  7.3.33 Preparation of Compound (S)-3.132    To a stirred solution of (S)-3.131 (3.13 g, 9.00 mmol) in DMF (10 mL) at 0 °C were added tert-butyldiphenylsilyl chloride (3.7 mL, 14.2 mmol) and imidazole (2.97 g, 43.6 mmol). The reaction mixture was stirred at room temperature overnight then DMF was removed in vacuo. After addition of HCl (15 mL, 1 M), the aqueous layer was extracted with diethyl ether (3×30 mL). The combined organic extract was washed with water (15 mL) and brine (15 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (12:1) to afford (S)-3.132 (5.27 g, 8.99 mmol) as a colourless oil quantitatively. 1H NMR (300 MHz, CDCl3)  7.69 (d, J = 5.9 Hz, 4H), 7.35–7.47 (m, 6H), 4.90 (dd, J = 5.0, 9.4 Hz, 1H), 3.73 (s, 3H), 3.71 (t, J = 6.3 Hz, 2H), 2.20–2.33 (m, 1H), 1.97–2.08 (m, 1H), 1.60–1.70 (m, 2H), 1.51 (s, 18H), 1.08 (br. s., 9H); 13C NMR (75 MHz, CDCl3)  171.5, 152.2, 135.7, 134.1, 129.8, 127.8, 83.2, 63.6, 58.3, 52.3, 29.6, 28.1, 27.0, 26.7, 19.4.     254  7.3.34 Preparation of Compound (S)-3.133    To a stirred solution of (S)-3.132 (4.40 g, 7.51 mmol) in acetonitrile (100 mL) at room temperature was added lithium bromide (3.04 g, 35.0 mmol). The reaction mixture was stirred 65 °C for 20 h then the solvent was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1) to afford (S)-3.133 (3.47 g, 7.14 mmol) as a colourless oil in a yield of 95 %. 1H NMR (400 MHz, CDCl3)  7.68 (d, J = 6.7 Hz, 4H), 7.35–7.46 (m, 6H), 5.14 (d, J = 6.7 Hz, 1H), 4.34 (br. s., 1H), 3.74 (s, 3H), 3.69 (t, J = 5.8 Hz, 2H), 1.88–2.01 (m, 1H), 1.72–1.83 (m, 1 H), 1.56–1.67 (m, 2H), 1.47 (s, 9H), 1.10 (s, 9H); 13C NMR (100 MHz, CDCl3)  173.6, 155.6, 135.7, 134.0, 129.8, 127.9, 80.0, 63.3, 53.5, 52.4, 31.8, 29.3, 28.5, 27.0, 19.4.        255  7.3.35 Preparation of Compound (S)-3.124    To a stirred solution of (S)-3.133 (1.94 g, 3.99 mmol) in THF (10 mL) at 0 °C was dropwise added lithium hydroxide (8.0 mL, 8.0 mmol, 1 M in H2O) at a rate of 1.0 mL/min. The reaction mixture was stirred at 0 °C for 1 h and at room temperature for 3 h. After THF was removed in vacuo, the reaction mixture was added 1 M HCl dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (5×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/MeOH (98:2) to afford acid (S)-3.124 (1.18 g, 2.50 mmol) as a colourless oil in a yield of 63 %. 1H NMR (400 MHz, CDCl3)  10.51 (br. s., 1H), 7.68 (d, J = 6.7 Hz, 4H), 7.34–7.47 (m, 6H), 6.35, 5.18 (br. s. each, 1H), 4.35, 4.17 (br. s. each, 1H), 3.70 (t, J = 5.6 Hz, 2H), 1.97–2.06 (m, 1H), 1.78–1.87 (m, 1H), 1.61–1.74 (m, 2H), 1.46 (s, 9H), 1.07 (s, 9H); 13C NMR (100 MHz, CDCl3)  178.0 and 177.5, 155.8, 135.7, 133.9, 129.8, 127.9, 81.6 and 80.3, 63.3, 53.6 and 53.5, 29.3, 29.0, 28.5, 28.4, 27.0, 19.4 (31.8s missing).    256  7.3.36 Preparation of Compound 3.134    To a stirred solution of the carboxylic acid (S)-3.124 (1.14 g, 2.42 mmol), Meldrum’s acid 3.85 (348 mg, 2.41 mmol), DMAP (430 mg, 3.52 mmol) in DCM (5 mL) was added DCC (532 mg, 2.58 mmol) at 0 °C. The reaction mixture was stirred at room temperature for additional 5 h then poured onto cold EtOAc (50 mL). The white precipitate formed was filtered off. After successive wash with cold KHSO4 solution (50 mL, 5 %), cold water (50 mL) and cold brine (50 mL), the filtrate was dried over anhydrous MgSO4 and concentrated in vacuo. The yellowish crude residue were used directly for the next step without further purification.  The crude residue was refluxed in EtOAc (15 mL) for 1 h then the solvent was removed in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1) to afford 3.134 (580 mg, 1.17 mmol) as a colourless oil in a yield of 50 %. Due to the complexity of its NMR spectra, the structure of 3.134 was confirmed by mass spectrum data only. The correctness of the structural deduction was further proved by the product structures derived from 3.134. ESI-MS m/z: 518.3 [M+Na]+.   257  7.3.37 Treatment of 3.134 with trimethyl orthoformate and catalytic sulfuric acid    To a stirred solution of  (186 mg, 0.375 mmol) in MeOH (5 mL) were added trimethyl orthoformate (0.17 mL, 1.50 mmol) and a drop of conc. H2SO4 at room temperature. The reaction mixture was refluxed overnight. After addition of ether (80 mL), the organic layer was washed with saturated NaHCO3 (3×10 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (9:1 to 8:1) to afford 3.136. ESI-MS m/z: 176.2 [M+Na]+.  7.3.38 Preparation of Compound 3.137    To a stirred solution of 3.134 (63 mg, 0.127 mmol) in toluene/MeOH (10 mL, 4:1) at room temperature was dropwise added TMSCHN2 (129 l, 0.258 mmol, 2.0 M in hexanes). After addition of AcOH (0.1 mL), the reaction mixture was dried in vacuo. The crude residue was 258  purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1→4:1) to afford 3.137 (65.7 mg, 0.128 mmol) as a colourless oil quantitatively. 1H NMR (400 MHz, CDCl3)  7.64 (d, J = 6.7 Hz, 4H), 7.34–7.46 (m, 6H), 5.07 (s, 1H), 4.51 (dd, J = 2.7, 5.5 Hz, 1H), 3.78 (s, 3H), 3.63 (m, 2H), 2.11–2.25 (m, 1H), 1.88–2.05 (m, 2H), 1.65–1.77 (m, 1H), 1.52 (s, 9H), 1.05 (s, 9H); 13C NMR (100 MHz, CDCl3)  177.6, 169.5, 149.5, 135.7, 134.0, 129.8, 127.8, 94.7, 82.7, 63.6, 59.8, 58.6, 34.1, 28.4, 27.0, 26.3, 25.6, 19.4; ESI-MS m/z: 532.3 [M+Na]+.  7.3.39 Preparation of Compound 3.125    Compound 3.137 (35 mg, 0.0686 mmol) was dissolved in TFA/DCM (1 mL, 25 %) and stirred at 0 °C for 1 h. The solvent was evaporated in vacuo. After addition of saturated NaHCO3 (10 mL), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (7:1→100 % EtOAc) to afford methyl tetramate 3.125 (23.6 mg, 0.057 mmol) as a colourless oil in a yield of 84 %. []20D +14.3° (c 3.0, CHCl3); 1H NMR (400 MHz, CDCl3)  7.66 (d, J = 6.4 Hz, 4H), 7.35–7.47 (m, 6H), 5.75 (br. s., 1H), 5.01 (s, 1H), 4.08 (br. s., 1H), 3.78 (s, 3H), 3.68 (t, J = 5.8 Hz, 2H), 1.86–1.97 (m, 1H), 1.58–1.67 (m, 2H), 1.47–1.58 (m, 1H), 1.06 (s, 9H); 13C NMR (100 259  MHz, CDCl3)  178.4, 174.3, 135.8, 133.9, 129.9, 127.9, 93.9, 63.6, 58.5, 57.3, 28.8, 27.9, 27.1, 19.4; ESI-MS m/z: 432.3 [M+Na]+.  7.3.40 Preparation of Compound (R)-3.129    Under the same procedure described in 7.3.30, (R)-3.129 (12.5 g, 45.4 mmol) was prepared from (R)-3.99 (7.43 g, 50.5 mmol) as a colourless oil in a yield of 90 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (20:3→2:1). 1H NMR (300 MHz, CDCl3)  5.14 (br. s., 1H), 4.31 (d, J = 4.1 Hz, 1H), 3.72 (s, 3H), 3.65 (s, 3H), 2.27–2.50 (m, 2H), 2.07–2.24 (m, 1H), 1.84–2.01 (m, 1H), 1.34–1.48 (m, 9H); 13C NMR (75 MHz, CDCl3)  173.3, 172.8, 155.5, 80.1, 53.0, 52.5, 51.9, 30.2, 28.4, 27.9.         260  7.3.41 Preparation of Compound (R)-3.130    Under a similar procedure described in 7.3.31, (R)-3.130 (7.50 g 19.9 mmol) was prepared from (R)-3.129 (5.80 g, 21.0 mmol) as a colourless oil in a yield of 95 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (12:1). 1H NMR (300 MHz, CDCl3)  4.91 (dd, J = 4.3, 9.4 Hz, 1H), 3.68 (s, 3H), 3.64 (s, 3H), 2.45 (d, J = 8.0 Hz, 1H), 2.31–2.42 (m, 2H), 2.08–2.24 (m, 1H), 1.46 (s, 18H)13C NMR (75 MHz, CDCl3)  173.2, 170.9, 152.0, 83.4, 57.5, 52.3, 51.8, 30.7, 28.1, 25.3.  7.3.42 Preparation of Compound (R)-3.131    Under a similar procedure described in 7.3.32, (R)-3.131 (3.00 g, 8.63 mmol) was prepared from (R)-3.130 (6.20 g, 16.5 mmol) as a colourless oil in a yield of 52 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→1:1). 1H NMR 261  (400 MHz, CDCl3)  4.87 (dd, J = 5.5, 8.5 Hz, 1H), 3.70 (s, 3H), 3.66 (t, J = 6.3 Hz, 2H), 2.15–2.28 (m, 1H), 1.85–1.99 (m, 1H), 1.56–1.69 (m, 2H), 1.49 (s, 18H); 13C NMR (100 MHz, CDCl3)  171.5, 152.3, 83.4, 62.4, 58.0, 52.4, 29.6, 28.2, 26.6.  7.3.43 Preparation of Compound (R)-3.132    Under a similar procedure described in 7.3.33, (R)-3.132 (5.06 g, 0.863 mmol) was prepared from (R)-3.131 (3.00 g, 8.63 mmol) as a colourless oil quantitatively. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (12:1→6:1). 1H NMR (400 MHz, CDCl3)  7.68 (d, J = 6.5 Hz, 4H), 7.34–7.47 (m, 6H), 4.89 (dd, J = 4.8, 9.2 Hz, 1H), 3.73 (s, 3H), 3.71 (t, J = 6.3 Hz, 2H), 2.20–2.33 (m, 1H), 1.95–2.07 (m, 1H), 1.60–1.70 (m, 2H), 1.50 (s, 18H), 1.06 (s, 9H); 13C NMR (100 MHz, CDCl3)  171.5, 152.2, 135.7, 134.1, 129.7, 127.8, 83.2, 63.6, 58.3, 52.3, 29.6, 28.2, 27.1, 26.7, 19.4.      262  7.3.44 Preparation of Compound (R)-3.133    Under a similar procedure described in 7.3.34, (R)-3.133 (4.05 g, 8.34 mmol) was prepared from (R)-3.132 (5.10 g, 8.70 mmol) as a colourless oil in a yield of 96 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1). 1H NMR (400 MHz, CDCl3)  7.66 (d, J = 6.5 Hz, 4H), 7.36–7.47 (m, 6H), 5.10 (d, J = 5.8 Hz, 1H), 4.32 (br. s., 1H), 3.74 (s, 3H), 3.68 (t, J = 5.8 Hz, 2H), 1.88–2.01 (m, 1H), 1.71–1.82 (m, 1H), 1.55–1.65 (m, 2H), 1.45 (s, 9H), 1.06 (s, 9H); 13C NMR (100 MHz, CDCl3)  173.5, 155.6, 135.7, 134.0, 129.8, 127.9, 80.0, 63.3, 53.5, 52.4, 31.8, 29.3, 28.5, 27.1, 19.4.  7.3.45 Preparation of Compound (R)-3.124    Under a similar procedure described in 7.3.35, (R)-3.124 (3.50 g, 7.43 mmol) was prepared from (R)-3.133 (4.24 g, 8.73 mmol) as a colourless oil in a yield of 85 %. The crude residue was purified by silica gel flash chromatography eluting with DCM/MeOH (97:3). 1H NMR (400 263  MHz, CDCl3)  10.24 (br. s., 1H), 7.68 (d, J = 6.5 Hz, 4H), 7.32–7.49 (m, 6H), 6.46, 5.16 (br. s. each, 1H), 4.36, 4.19 (br. s. each, 1H), 3.71 (t, J = 5.1 Hz, 2H), 1.98–2.08 (m, 1H), 1.79–1.90 (m, 1H), 1.61–1.74 (m, 2H), 1.47 (s., 9H), 1.08 (s, 9H); 13C NMR (100 MHz, CDCl3)  177.8, 155.8, 135.7, 133.9, 129.8, 127.9, 80.3, 63.3, 53.5, 29.0, 28.5, 27.1, 19.4 (31.8s missing).  7.3.46 Preparation of Compound 3.138    *To a stirred solution of acid (R)-3.124 (2.0 g, 4.24 mmol), TEA (0.76 mL, 5.46 mmol) and DMAP (52 mg, 0.42 mmol) in DCM (10 mL) at 0 °C was added benzyl chloroformate (0.76 mL, 5.32 mmol). The reaction mixture was stirred at 0 °C for 3 h. After addition of brine (10 mL), the separated aqueous layer was extracted with DCM (3×30 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (15:1→5:1) to afford 3.138 (2.20 g, 3.91 mmol) as a colourless oil in a yield of 92 %. 1H NMR (400 MHz, CDCl3)  7.67 (d, J = 7.0 Hz, 4H), 7.32–7.49 (m, 11H), 5.10–5.27 (m, 3H), 4.39, 4.22 (br. s. each, 1H), 3.67 (t, J = 5.6 Hz, 2H), 1.94–2.05 (m, 1H), 1.73–1.84 (m, 1H), 1.55–1.66 (m, 2H), 1.47 (s, 9H), 1.07 (s, 9H); 13C NMR (100 MHz, CDCl3)  172.9, 155.6, 135.7, 133.9, 129.8, 128.7, 128.5, 128.4, 127.8, 80.0, 67.1, 63.2, 53.6, 29.3, 28.5, 27.0, 19.4 (31.8s missing).  264  7.3.47 Preparation of Compound 3.139    Compound 3.138 (1.76 g, 3.14 mmol) was dissolved in TFA/DCM (30 mL, 25 %) and stirred at 0 °C for 1 h. The solvent was evaporated in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of the crude residue in THF (20 mL) at 0 °C were added TEA (0.56 mL, 4.00 mmol) and propionyl chloride (3.06 mL, 35.0 mmol). The reaction mixture was stirred at 0 °C for 3 h. After addition of saturated NaHCO3 (20 mL), the separated aqueous layer was extracted with EtOAc (3×30 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1) to afford 3.139 (960 mg, 1.85 mmol) as a colourless oil in a yield of 59 %. 1H NMR (400 MHz, CDCl3)  7.64 (d, J = 7.0 Hz, 4H), 7.30–7.47 (m, 11H), 6.02 (d, J = 7.6 Hz, 1H), 5.18 (m, J = 6.4, 31.1 Hz, 2H), 4.70 (td, J = 5.2, 7.8 Hz, 1H), 3.64 (td, J = 2.1, 6.1 Hz, 2H), 2.23 (q, J = 7.6 Hz, 2H), 1.94–2.07 (m, 1H), 1.74–1.85 (m, 1H), 1.46–1.64 (m, 2H), 1.15 (t, J = 7.6 Hz, 3H), 1.04 (s, 9H); 13C NMR (100 MHz, CDCl3)  173.7, 172.8, 135.7, 133.8, 129.8, 128.8, 128.6, 128.4, 127.9, 67.3, 63.2, 52.1, 29.7, 29.2, 28.4, 27.0, 19.4, 9.9 (31.8s missing). 265  7.3.48 Preparation of Compound 3.140    To a stirred solution of 3.139 (513 mg, 0.991 mmol), DIPEA (0.35 mL, 2.00 mmol), DMAP (12 mg, 0.098 mmol) in THF (5 mL) at 0 °C was added Boc2O (432 mg, 1.98 mmol). The reaction mixture was refluxed for 1 h. After addition of brine (5 mL), the separated aqueous layer was extracted with ether (3×20 mL). The combined ethereal extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (30:1→1:1) to afford 3.140 (391 mg, 0.632 mmol) as a colourless oil in a yield of 63 %. 1H NMR (400 MHz, CDCl3)  7.66 (d, J = 6.7 Hz, 4H), 7.30–7.45 (m, 11H), 5.33 (dd, J = 5.2, 9.1 Hz, 1H), 5.10–5.20 (m, J = 1.2, 27.4 Hz, 2H), 3.67 (t, J = 5.9 Hz, 2H), 2.90 (q, J = 7.1 Hz, 2H), 2.25–2.36 (m, 1H), 1.91–2.03 (m, 1H), 1.56–1.66 (m, 1H), 1.47–1.54 (m, 1H), 1.41 (s, 9H), 1.14 (t, J = 7.3 Hz, 3H), 1.05 (s, 9H); 13C NMR (100 MHz, CDCl3)  176.9, 170.8, 152.5, 135.7, 134.1, 129.8, 128.7, 128.4, 128.3, 127.8, 84.0, 67.0, 63.6, 55.9, 31.8, 29.6, 28.0, 27.1, 26.5, 19.4, 9.7.    266  7.3.49 Preparation of Compound 3.126    To a stirred solution of 3.140 (220 mg, 0.356 mmol) in MeOH (10 mL) was added Pd/C (50 mg, 10 %). The reaction mixture was hydrogenated under atmospheric pressure at room temperature for 1.5 h. The catalyst was removed through a pad of Celite® with MeOH (20 mL) and the filtrate was concentrated in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of the crude residue and pentafluorophenol (73.6 mg, 0.399 mmol) in EtOAc (5 mL) was added DCC (88.1 mg, 0.427 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3h. After addition of hexanes (15 ml), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/acetone (30:1) to afford active ester 3.126 (209 mg, 0.302 mmol) as a colourless oil in a yield of 84 %. []20D +4.7° (c 1.27, CHCl3); 1H NMR (400 MHz, CDCl3)  7.67 (d, J = 6.8 Hz, 4H), 7.33–7.49 (m, 6H), 5.61 (dd, J = 5.5, 8.9 Hz, 1H), 3.70 (t, J = 6.0 Hz, 2H), 2.85–3.07 (m, 2H), 2.30–2.43 (m, 1H), 2.01–2.14 (m, 1H), 1.55–1.71 (m, 2H), 1.52 (s, 9H), 1.19 (t, J = 7.3 Hz, 3H), 1.06 (s, 9H); 13C NMR (100 MHz, CDCl3)  176.6, 167.0, 267  152.0, 135.8, 134.0, 129.8, 127.9, 85.2, 63.4, 55.3, 31.7, 29.3, 28.0, 27.1, 26.5, 19.4, 9.6; 19F NMR (282 MHz, CDCl3)  −152.1 (d, J = 18.4 Hz, 2F), −158.2 (t, J = 22.9 Hz, 1F), −162.7 (t, J = 20.6 Hz, 2F).  7.3.50 Preparation of Compound 3.141    Under a similar procedure described in 7.3.47, 3.141 (115 mg, 0.373 mmol) was prepared from 3.45 (350 mg, 0.993 mmol) as a colourless oil in a yield of 38 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (7:1). 1H NMR (400 MHz, CDCl3)  8.25–8.31 (m, 2H), 7.28–7.33 (m, 2H), 5.83 (d, J = 7.31 Hz, 1H), 4.83 (ddd, J = 5.1, 7.7, 8.9 Hz, 1H), 2.31 (q, J = 7.6 Hz, 2H), 1.76–1.89 (m, 2H), 1.65–1.76 (m, 1H), 1.20 (t, J = 7.4 Hz, 3H), 1.04 (t, J = 5.7 Hz, 6H); 13C NMR (100 MHz, CDCl3)  174.2, 171.4, 155.4, 145.8, 125.5, 122.6, 51.3, 41.3, 29.6, 25.3, 23.1, 22.1, 9.8.     268  7.3.51 Treatment of 3.141 with Boc2O and DMAP    Under a similar procedure described in 7.3.48, 3.142 (60 mg, 0.250 mmol) was prepared from 3.141 (85 mg, 0.275 mmol) as a colourless oil in a yield of 91 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (7:1). 1H NMR (300 MHz, CDCl3)  8.20–8.26 (m, 2H), 7.31–7.38 (m, 2H), 1.55 (s, 9H); 13C NMR (75 MHz, CDCl3)  155.9, 150.6, 145.3, 125.3, 122.0, 84.9, 27.7.  7.3.52 Preparation of Compound 3.144    To a stirred solution of methyl tetramate 3.32 (116.6 mg, 0.689 mmol) in THF (4 mL) at −50 °C was dropwise added LiHMDS (0.34 mL, 0.34 mmol, 1.0 M in THF). After dropwise addition of 3.126 (239 mg, 0.344 mmol) in THF (2 mL), the reaction mixture was stirred at −40 °C for 269  additional 3 h. The reaction mixture was added AcOH (0.5 mL) then the solvent was evaporated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1) to afford coupling product 3.144 (88 mg, 0.129 mmol) as a as colourless oil in a yield of 38 %. []20D +45.4° (c 1.1, CHCl3); 1H NMR (400 MHz, CDCl3)  7.64–7.69 (m, 4H), 7.34–7.43 (m, 6H), 5.93 (dd, J = 5.5, 9.4 Hz, 1H), 5.02 (s, 1H), 4.64 (dd, J = 3.2, 6.5 Hz, 1H), 3.85 (s, 3H), 3.69 (t, J = 6.4 Hz, 3H), 2.74–2.92 (m, 2H), 2.09–2.28 (m, 2H), 1.84–1.93 (m, 1H), 1.69–1.80 (m, 3H), 1.55–1.65 (m, 1H), 1.51 (s, 12H), 1.13 (t, J = 7.3 Hz, 3H), 1.04 (s, 9H), 0.90 (d, J = 2.7 Hz, 3H), 0.89 (d, J = 3.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)  180.5, 177.5, 170.0, 169.4, 153.8, 135.8, 134.2, 129.7, 127.8, 93.5, 83.2, 64.0, 59.6, 58.9, 58.8, 39.3, 36.8, 31.7, 30.1, 28.1, 27.1, 24.3, 24.0, 23.0, 19.4, 10.0; ESI-MS m/z: 701.5 [M + Na]+; ESI-HRMS: m/z calcd for C38H54N2O7NaSi [M + Na]+, 701.3598; found, 701.3590.  7.3.53 Preparation of Compound 3.145    To a stirred solution of 3.144 (43 mg, 0.063 mmol) in THF (3 mL) in a Nalgene TM bottle at 0 °C was added HF/pyridine complex (1.0 mL, 20 mmol, ~70 % HF in pyridine) in pyridine (2.5 mL). The reaction mixture was stirred at room temperature for 5 h then cooled to 0 °C again. After 270  carefully neutralizing with saturated NaHCO3, the aqueous layer was extracted with DCM (3×10 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→3:1) to afford 3.145 (20.5 mg, 0.046 mmol) as a colourless oil in a yield of 73 %. []20D +21.3° (c 0.75, CHCl3); 1H NMR (400 MHz, CDCl3)  6.05 (dd, J = 6.3, 8.7 Hz, 1H), 5.03 (s, 1H), 4.66 (dd, J = 3.1, 6.5 Hz, 1H), 3.86 (s, 3H), 3.68–3.76 (m, 1H), 3.61–3.68 (m, 1H), 2.66–2.96 (m, 2H), 2.18–2.32 (m, 2H), 1.79–1.91 (m, 1H), 1.70–1.78 (m, 4H), 1.53 (s, 9H), 1.14 (t, J = 7.3 Hz, 3H), 0.90 (d, J = 4.1 Hz, 3H), 0.88 (d, J = 4.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)  180.9, 177.7, 170.1, 169.9, 153.8, 93.5, 83.5, 61.0, 58.9, 58.8, 58.4, 39.2, 31.7, 29.2, 28.1, 25.9, 24.3, 24.0, 22.9, 10.0; ESI-MS m/z: 463.3 [M + Na]+; ESI-HRMS: m/z calcd for C22H36N2O7Na [M + Na]+, 463.2420; found, 463.2422.  7.3.54 Preparation of Compound 3.146    To a stirred solution of deprotected coupling intermediate 3.145 (20 mg, 0.045 mmol) in carbon tetrachloride (2 mL) at room temperature was added triphenyl phosphine (47.6 mg, 0.18 mmol). The mixture was refluxed overnight. After addition of hexanes (50 mL), the white precipitate 271  formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1) to afford monochlorinated coupling intermediate 3.146 (16 mg, 0.034 mmol) as a white solid in a yield of 76 %. 1H NMR (400 MHz, CDCl3)  5.94 (dd, J = 5.8, 8.9 Hz, 1H), 5.02 (s, 1H), 4.63 (dd, J = 3.2, 6.7 Hz, 1H), 3.85 (s, 3H), 3.49–3.65 (m, 2H), 2.82 (q, J = 7.7 Hz, 2H), 2.16–2.35 (m, 2H), 1.86–2.00 (m, 1H), 1.68–1.86 (m, 4H), 1.54 (s, 9H), 1.14 (t, J = 7.3 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  180.8, 177.6, 169.6, 153.6, 93.4, 83.6, 58.9, 44.9, 39.5, 31.7, 30.0, 28.2, 28.1, 24.4, 24.0, 22.9, 10.0; ESI-MS m/z: 481.3 [M + Na]+; ESI-HRMS: m/z calcd for C22H35N2O6NaCl [M + Na]+, 481.2081; found, 481.2083.  7.3.55 Preparation of 3.60 (LPY00)    Compound 3.146 (13 mg, 0.0283 mmol) was dissolved in TFA/DCM (1 mL, 25 %) and stirred at 0 °C for 1 h. The solvent was evaporated in vacuo. After addition of saturated NaHCO3 (5 mL), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:1) to 272  afford 3.60 (8.9 mg, 0.0247 mmol) as a colourless oil in a yield of 88 %. []20D +53.5° (c 0.28, CHCl3); 1H NMR (400 MHz, CDCl3)  6.25 (d, J = 8.2 Hz, 1H), 5.72 (td, J = 3.2, 8.8 Hz, 1H), 5.07 (s, 1H), 4.64 (dd, J = 3.0, 6.4 Hz, 1H), 3.87 (s, 3H), 3.53–3.69 (m, 2H), 2.26 (q, J = 7.6 Hz, 2H), 1.94–2.13 (m, 2H), 1.69–1.91 (m, 5H), 1.17 (t, J = 7.6 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  181.1, 173.8, 172.0, 169.5, 93.5, 59.0, 58.5, 52.5, 44.7, 39.3, 31.0, 29.9, 29.1, 24.4, 24.0, 22.8, 9.9; ESI-MS m/z: 379.3 [M + H]+; ESI-HRMS: m/z calcd for C17H27N2O4NaCl [M + Na]+, 381.1557; found, 381.1556.  7.3.56 Preparation of Compound 3.127    To a stirred solution of methyl tetramate 3.125 (297.5 mg, 0.726 mmol) in THF (5 mL) at −50 °C was dropwise added LiHMDS (0.54 mL, 0.54 mmol, 1.0 M in THF). After dropwise addition of 3.126 (252 mg, 0.363 mmol) in THF (2.5 mL), the reaction mixture was stirred at −40 °C for additional 3 h. The reaction mixture was added AcOH (0.5 mL) then the solvent was evaporated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (60:1→10:1) to afford coupling product 3.127 (168 mg, 0.182 mmol) as a colourless oil in a yield of 50 %. []20D +46.9° (c 1.66, CHCl3); 1H NMR (400 MHz, CDCl3)  273  7.56–7.76 (m, 8H), 7.31–7.46 (m, 12H), 5.83 (dd, J = 5.6, 9.0 Hz, 1H), 5.03 (s, 1H), 4.68 (dd, J = 2.7, 5.5 Hz, 1H), 3.80 (s, 3H), 3.68 (t, J = 6.5 Hz, 2H), 3.61–3.66 (m, 1H), 3.54–3.61 (m, 1H), 2.81 (q, J = 7.3 Hz, 2H), 2.14–2.29 (m, 2H), 1.97–2.14 (m, 3H), 1.68–1.80 (m, 1H), 1.55–1.66 (m, 2H), 1.49 (s, 9H), 1.13 (t, J = 7.5 Hz, 3H), 1.01–1.05 (m, 18H); 13C NMR (100 MHz, CDCl3)  179.1, 177.5, 170.0, 169.5, 135.6, 153.6, 135.7, 134.2, 134.0, 129.8, 129.7, 127.8, 127.8, 94.0, 83.3, 64.0, 63.5, 59.6, 59.6, 58.8, 31.7, 30.1, 28.1, 27.3, 27.1, 27.0, 25.8, 25.7, 19.4, 10.0; ESI-MS m/z: 941.9 [M + Na]+; ESI-HRMS: m/z calcd for C53H70N2O8Na28Si2 [M + Na]+, 941.4568; found, 941.4578.  7.3.57 Preparation of Compound 3.128    To a stirred solution of 3.127 (166 mg, 0.180 mmol) in THF (5 mL) in a Nalgene TM bottle at 0 °C was added HF/pyridine complex (1 mL, 20 mmol, ~70 % HF in pyridine) in pyridine (1 mL). The reaction mixture was stirred at room temperature for 5 h then cooled to 0 °C again. After carefully neutralizing with saturated NaHCO3, the aqueous layer was extracted with DCM (3×30 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash 274  chromatography eluting with hexanes/EtOAc (5:1→1:4) to afford diol 3.128 (69.1 mg, 0.156 mmol) as a colourless oil in a yield of 86 %. []20D +59.4° (c 0.47, CHCl3); 1H NMR (400 MHz, CDCl3)  5.96 (dd, J = 6.5, 8.1 Hz, 1H), 5.06 (s, 1H), 4.71 (dd, J = 3.0, 5.5 Hz, 1H), 3.86 (s, 3H), 3.64–3.74 (m, 2H), 3.53–3.64 (m, 2H), 2.70–2.88 (m, 2H), 2.19–2.27 (m, 2H), 2.11–2.19 (m, 2H), 1.85–1.97 (m, 2H), 1.62–1.74 (m, 1H), 1.54–1.60 (m, 1H), 1.48–1.54 (m, 9H), 1.13 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  179.6, 177.7, 170.2, 169.8, 153.6, 93.9, 83.7, 62.2, 61.3, 59.2, 59.1, 58.7, 31.8, 29.3, 28.1, 26.2, 25.9, 25.5, 10.0; ESI-MS m/z: 465.3 [M + Na]+; ESI-HRMS: m/z calcd for C21H34N2O8Na [M + Na]+, 465.2213; found, 465.2204.  7.3.58 Preparation of Compound 3.147    To a stirred solution of deprotected coupling intermediate 3.128 (22 mg, 0.497 mmol) in carbon tetrachloride (2 mL) at room temperature was added triphenyl phosphine (52.1 mg, 0.198 mmol). The mixture was refluxed overnight. After addition of hexanes (50 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/EtOAc (300:1) to afford bis-monochlorinated coupling intermediate 3.147 (14.5 mg, 0.030 mmol) as a coulorless oil in a yield of 61 %. []20D 275  +71.4° (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3)  5.83 (t, J = 7.2 Hz, 1 H), 5.07 (s, 1H), 4.65 (dd, J = 3.1, 5.8 Hz, 1H), 3.87 (s, 3H), 3.52–3.65 (m, 2H), 3.50 (td, J = 2.7, 6.5 Hz, 2H), 2.81 (q, J = 7.4 Hz, 2H), 2.25–2.35 (m, 2H), 1.96–2.06 (m, 2H), 1.88–1.96 (m, 1H), 1.82 (m, 1H), 1.63–1.73 (m, 1H), 1.57–1.62 (m, 1H), 1.54 (s, 9H), 1.13 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  179.0, 177.6, 169.8, 169.4, 153.5, 94.1, 83.8, 59.2, 59.1, 58.9, 44.9, 44.6, 31.7, 30.0, 28.2, 28.2, 27.0, 26.3, 10.0; ESI-MS m/z: 501.4 [M + Na]+; ESI-HRMS: m/z calcd for C21H32N2O6Na35Cl2 [M + Na]+, 501.1535; found, 501.1525.  7.3.59 Preparation of 3.148 (LPY04)    Compound 3.147 (14.0 mg, 0.0292 mmol) was dissolved in TFA/DCM (2 mL, 25 %) and stirred at 0 °C for 1 h. The solvent was evaporated in vacuo. After addition of saturated NaHCO3 (2 mL), the aqueous layer was extracted with EtOAc (3×5 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1→1:1) to afford 3.148 (9.4 mg, 0.0247 mmol) as a colourless oil in a yield of 85 %. []20D +66.6° (c 0.3, CHCl3); 1H NMR (600 MHz, CDCl3)  6.23 (d, J = 8.2 Hz, 1H), 5.72 (td, J = 3.6, 8.4 Hz, 1H), 276  5.12 (s, 1H), 4.71 (dd, J = 3.1, 5.6 Hz, 1H), 3.90 (s, 3H), 3.61–3.68 (m, 1H), 3.54–3.61 (m, 1H), 3.51 (t, J = 6.4 Hz, 2H), 2.27 (qd, J = 1.3, 7.6 Hz, 2H), 2.20–2.25 (m, 1H), 2.04–2.10 (m, 1H), 1.96–2.04 (m, 2H), 1.79–1.87 (m, 1H), 1.71–1.78 (m, 1H), 1.56–1.65 (m, 2H), 1.17 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  179.2, 173.9, 172.2, 169.3, 94.2, 59.2, 58.7, 52.6, 44.7, 44.5, 31.0, 29.8, 29.0, 26.5, 26.2, 9.9; ESI-MS m/z: 379.3 [M + H]+; ESI-HRMS: m/z calcd for C16H24N2O4Na35Cl2 [M + Na]+, 401.1011; found, 401.1017.  7.3.60 Preparation of Compound 3.149    To a stirred solution of diol 3.128 (9.4 mg, 0.0212 mmol) in DCM (2 mL) at room temperature was added Dess–Martin periodinane (20 mg, 0.471 mmol). The mixture was stirred at room temperature for 0.5 h. After addition of DCM (10 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/EtOAc (100:1) to afford 3.149 (4.7 mg, 0.106 mmol) as a colourless oil in a yield of 50 %. []20D +85.1° (c 0.24, CHCl3); 1H NMR (600 MHz, CDCl3)  9.79 (s, 1H), 9.73 (s, 1H), 5.67 (dd, J = 5.6, 8.1 Hz, 1H), 5.06 (s, 1H), 4.65 (dd, J = 3.0, 5.6 Hz, 1H), 3.85 (s, 3H), 2.79 (q, J = 7.3 Hz, 3H), 2.62–2.69 (m, 1H), 2.52–2.58 (m, 2H), 2.46–2.51 (m, 277  1H), 2.39–2.45 (m, 1H), 2.28–2.36 (m, 2H), 2.24 (s, 1H), 1.53 (s, 9H), 1.13 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)  201.7, 200.6, 178.8, 177.6, 169.6, 169.3, 153.2, 94.2, 84.1, 59.1, 59.0, 58.9, 41.3, 37.7, 31.8, 28.1, 23.2, 22.5, 9.9; ESI-MS m/z: 461.3 [M + Na]+; ESI-HRMS: m/z calcd for C21H31N2O8 [M + H]+, 439.2080; found, 439.2085.  7.3.61 Preparation of Compound 3.150    To a stirred solution of triphenyl phosphite (6 L, 0.022 mmol) in DCM (1 mL) at −20 °C was carefully bubbled chlorine gas until the solution just became bright yellow. After addition of a few drops of triphenyl phosphite, the solution was discharged. To this almost colourless solution −20 °C were added 3.149 (2.4 mg, 0.005 mmol) in DCM and TEA (6 L, 0.045 mmol). The reaction mixture was stirred at room temperature overnight then refluxed for 1 h. After addition of hexanes (5 ml), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (250:1→99:1) to afford the crude product.  278  Under a similar procedure described in 7.3.38, 3.150 (1.2 mg, 0.0024 mmol) was prepared from the crude residue as a colourless oil in a yield of in a yield of 43 % in two steps. 1H NMR (600 MHz, CDCl3)  6.14 (d, J = 6.66 Hz, 1H), 5.85 (br. s., 1H), 5.78 (br. s., 1H), 5.67 (br. s., 1H), 4.68 (br. s., 1H), 4.37 (br. s., 3H), 2.47 (br. s., 1H), 2.35–2.43 (m, 1H), 2.25–2.34 (m, 3H), 2.18 (br. s., 2H), 1.98–2.11 (m, J = 11.2 Hz, 2H), 1.79–1.90 (m, 1H), 1.12–1.21 (m, 3H); 13C NMR (150 MHz, CDCl3)  174.1, 171.5, 168.3, 165.5, 97.7, 72.8, 72.4, 60.5, 57.4, 52.5, 40.0, 37.0, 29.8, 29.7, 25.3, 9.9; ESI-MS m/z: 505.0 [M + Na]+; ESI-HRMS: m/z calcd for C16H21N2O4Na35Cl5 [M + Ma]+, 502.9842; found, 502.9835.  7.3.62 Preparation of Compound 3.168    Under the same procedure described in 7.3.36, 3.168 (182 mg, 0.460 mmol) was prepared from (S)-3.167 (1.0 g, 2.18 mmol) as a colourless oil in a yield of 21 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (20:3→2:1). []20D +75.2° (c 1.54, CHCl3); 1H NMR (400 MHz, CDCl3)  7.63–7.70 (m, 4H), 7.38–7.49 (m, 6H), 5.01 (s, 1H), 4.20 (dd, J = 3.0, 9.4 Hz, 1H), 3.77 – 3.86 (m, 2H), 3.78 (s, 3H), 2.05 – 2.12 (m, 1H), 1.53–1.68 (m, 1H), 1.08 (s, 9H); 13C NMR (100 MHz, CDCl3)  178.6, 173.9, 135.7, 133.3, 130.2, 128.1, 93.7, 61.8, 58.5, 56.0, 35.1, 27.1, 19.3; ESI-MS m/z: 418.4 [M + Li]+ 279  7.3.63 Preparation of Compound 3.169    Under the same procedure described in 7.3.49, 3.169 (765 mg, 1.12 mmol) was prepared from 3.179 (810 mg, 1.34 mmol) as a colourless oil in a yield of 84 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (30:1). []20D +13.6° (c 0.14, CHCl3); 1H NMR (600 MHz, CDCl3)  7.64–7.70 (m, 4H), 7.36–7.46 (m, 6H), 5.97 (t, J = 6.4 Hz, 1H), 3.78 (td, J = 5.5, 10.8 Hz, 1H), 3.70 (ddd, J = 5.1, 7.4, 10.5 Hz, 1H), 2.87–2.99 (m, 2H), 2.52–2.59 (m, 1H), 2.12 (tdd, J = 6.0, 8.1, 14.0 Hz, 1H), 1.50 (s, 9H), 1.18 (t, J = 7.1 Hz, 3H), 1.07 (s, 9H); 13C NMR (150 MHz, CDCl3)  175.6, 166.7, 151.4, 135.1, 133.0, 132.9, 129.3, 127.3, 127.3, 84.5, 60.0, 52.0, 32.7, 31.2, 27.4, 26.3, 18.7, 8.9.       280  7.3.64 Preparation of Compound 3.170    Under the same procedure described in 7.3.56, 3.170 (235 mg, 0.404 mmol) was prepared from 3.168 (160 mg, 0.404 mmol) and 3.169 (550 mg, 0.808 mmol) as a colourless oil in a yield of 66 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (50:1→10:1). []20D +57.8° (c 3.04, CHCl3); 1H NMR (400 MHz, CDCl3)  6.04 (dd, J = 4.8, 9.7 Hz, 1H), 4.99 (s, 1H), 4.69 (dd, J = 2.7, 5.7 Hz, 1H), 3.75–3.85 (m, 1H), 3.69–3.74 (s, 3H), 3.56–3.68 (m, 3H), 2.74 (dd, J = 7.4, 16.9 Hz, 2H), 2.43–2.54 (m., 1H), 2.19–2.40 (m, 3H), 1.39–1.45 (m, 9H), 1.06 (s, 9H), 1.02 (s, 9H); 13C NMR (100 MHz, CDCl3)  179.8, 177.5, 170.2, 169.3, 153.7, 135.8, 135.8, 135.7, 134.2, 134.0, 133.7, 133.6, 129.9, 129.7, 127.9, 127.8, 93.3, 83.2, 61.8, 59.5, 58.7, 57.8, 57.4, 33.2, 32.1, 31.8, 28.1, 27.0, 27.0, 19.4, 19.3, 9.9; ESI-MS m/z: 913.8 [M + Na]+; ESI-HRMS: m/z calcd for C51H66N2O8Na28Si2 [M + Na]+, 913.4255; found, 501.913.4244.    281  7.3.65 Preparation of Compound 3.151    Under the same procedure described in 7.3.57, 3.151 (60 mg, 0.144 mmol) was prepared from 3.170 (208 mg, 0.233 mmol) as a colourless oil in a yield of 62 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→1:4). []20D +83.8° (c 0.46, CHCl3); 1H NMR (400 MHz, CDCl3)  5.88 (dd, J = 4.6, 9.7 Hz, 1H), 5.04 (s, 1H), 4.71 (t, J = 5.2 Hz, 1H), 3.87 (s, 3H), 3.69–3.78 (m, 1H), 3.65 (t, J = 5.8 Hz, 2H), 3.55–3.62 (m, 1H), 2.74–2.85 (m, 2H), 2.32–2.43 (m, 2H), 2.20–2.32 (m, 1H), 1.91–1.99 (m, 1H), 1.52 (s, 9H), 1.15 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  180.6, 178.3, 170.9, 169.6, 153.3, 93.2, 84.3, 59.4, 59.1, 58.5, 57.8, 57.0, 34.4, 33.4, 31.9, 28.1, 10.0; ESI-MS m/z: 437.3 [M + Na]+; ESI-HRMS: m/z calcd for C19H30N2O8Na [M + Na]+, 437.1900; found, 437.1911.      282  7.3.66 Preparation of Compound 3.180    To a stirred solution of 3.151 (30 mg, 0.072 mmol) in DCM (5 mL) were added CBr4 (73 mg, 0.220 mmol) and PPh3 (58 mg, 0.221 mmol) at room temperature. The reaction mixture was refluxed for 2 h. After addition of hexanes (50 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (8:1) to afford dibromide 3.180 (22 mg, 0.039 mmol) as a colourless oil in a yield of 55 %. []20D +111.1° (c 0.27, CHCl3); 1H NMR (400 MHz, CDCl3)  5.80 (dd, J = 6.4, 7.8 Hz, 1H), 5.07 (s, 1H), 4.65 (dd, J = 3.4, 5.8 Hz, 1H), 3.88 (s, 3H), 3.52 (dt, J = 1.5, 7.0 Hz, 2H), 3.22–3.39 (m, 2H), 2.80 (q, J = 7.1 Hz, 2H), 2.64–2.75 (m, 2H), 2.42–2.57 (m, 2H), 1.55 (s, 9H), 1.12 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)  178.6, 177.7, 169.1, 169.1, 153.1, 94.2, 84.2, 59.1, 59.1, 58.8, 33.5, 33.3, 31.8, 30.2, 28.2, 26.3, 9.8; ESI-MS m/z: 563.0 [M + Na]+; ESI-HRMS: m/z calcd for C19H28N2O6Na79Br2 [M + Na]+, 561.0212; found, 561.0228.    283  7.3.67 Preparation of Compound 3.181    To a stirred solution of NaH (1.0 mg, 0.055 mmol, 60 % in mineral oil) in DMF (5 mL) was added CHCl3 (18 l, 0.222 mmol) at –40 °C. The reaction mixture at that temperature was stirred for 10 min. To the chloroform anion solution was added 3.180 (15 mg, 0.027 mmol) in DMF (1 mL) at –40 °C. The reaction mixture was stirred for additional 2 h. After addition of water (0.5 mL) and EtOAc (15 mL), the organic layer was washed with saturated NaHCO3 (3×5 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo.  The crude residue was dissolved in TFA/DCM (2 mL, 25 %) and stirred at 0 °C for 1 h then was evaporated in vacuo. After addition of saturated NaHCO3 solution (5 mL), the aqueous layer was extracted with EtOAc (3×5 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (30:1) to afford 3.181 (2.1 mg, 0.0061 mmol) as a colourless oil in a yield of 22 %. 1H NMR (600 MHz, CDCl3)  6.30 (d, J = 8.3 Hz, 1H), 5.76 (dt, J = 3.2, 8.6 Hz, 1H), 5.15 (s, 1H), 3.86 (s, 1H), 3.55 (dt, J = 6.3, 9.8 Hz, 1H), 3.41 (dt, J = 5.5, 9.9 Hz, 1H), 2.51 (td, J = 3.7, 9.9 Hz, 1H), 2.26 (q, J = 7.5 Hz, 2H), 2.21–284  2.30 (m, 1H), 2.13–2.20 (m, 1H), 2.02–2.11 (m, 1H), 1.23–1.32 (m, 3H), 1.16 (dt, J = 0.5, 7.5 Hz, 3H); 13C NMR (150 MHz, CDCl3)  179.8, 173.7, 171.2, 170.1, 91.6, 58.8, 53.1, 46.3, 37.2, 29.9, 29.0, 11.2, 11.0, 9.9; ESI-MS m/z: 383.1 [M + Na]+  7.3.68 Preparation of Compound 3.182    Under the same procedure described in 7.3.53, 3.182 (140 mg, 0.544 mmol) was prepared from 3.176 (300 mg, 0.605 mmol) as a colourless oil in a yield of 90 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→2:1). []20D +75.0° (c 1.3, CHCl3); 1H NMR (400 MHz, CDCl3)  4.99 (s, 1H), 4.50 (t, J = 5.1 Hz, 1H), 3.79 (s, 3H), 3.60 (br. s., 2H), 2.68 (br. s., 1H), 2.13–2.25 (m, J = 6.1 Hz, 1H), 1.91–2.04 (m, 1H), 1.48 (s, 9H); 13C NMR (100 MHz, CDCl3)  178.6, 169.3, 150.2, 93.9, 83.1, 58.8, 57.9, 57.7, 33.8, 28.2; ESI-MS m/z: 280.4 [M + Na]+.     285  7.3.69 Preparation of Compound 3.183    To a stirred solution of 3.182 (78 mg, 0.303 mmol) in MeCN (5 mL) were added I2 (123 mg, 0.485 mmol), imidazole (31 mg, 0.454 mmol) and PPh3 (111 mg, 0.424 mmol) at room temperature. The reaction mixture at that temperature was stirred for 2 h. After addition of diethyl ether (50 mL), the organic layer was washed with saturated Na2S2O3 (3×5 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1) to afford iodide 3.183 (104 mg, 0.284 mmol) as a colourless oil in a yield of 94 %. []20D +97.2° (c 0.24, CHCl3); 1H NMR (600 MHz, CDCl3)  5.09 (s, 1H), 4.46 (dd, J = 3.5, 5.1 Hz, 1H), 3.85 (s, 3H), 3.11 (dt, J = 5.1, 9.7 Hz, 1H), 2.95–3.04 (m, 1H), 2.44–2.61 (m, 2H), 1.55 (s, 9H); 13C NMR (150 MHz, CDCl3)  176.6, 168.9, 149.4, 95.0, 83.3, 60.6, 59.0, 34.7, 28.4, –3.4; ESI-MS m/z: 390.2 [M + Na]+, ESI-HRMS: m/z calcd for C12H18NO4NaI [M + Na]+, 390.0178; found, 390.0184.    286  7.3.70 Preparation of Compound 3.184    To a stirred solution of n-BuLi (27 l, 0.0435 mmol, 1.6 M) in THF (5 mL) was added CHCl3 (174 l, 2.17 mmol) at –78 °C. The reaction mixture at that temperature was stirred for 10 min. To the chloroform anion solution were added 3.183 (16 mg, 0.0435 mmol) in THF (1 mL) and HMPA (1 mL). The reaction mixture was stirred at –78 °C for additional 2 h. After addition of water (0.5 mL) and EtOAc (15 mL), the organic layer was washed with saturated NaHCO3 (3×5 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1) to afford 3.184 (0.9 mg, 0.0038 mmol) as a colourless oil in a yield of 8.7 %. 1H NMR (600 MHz, CDCl3)  5.13 (s, 1H), 3.82 (s, 3H), 1.95–1.99 (m, 2H), 1.53 (s, 9H), 1.21–1.24 (m, 2H); 13C NMR (150 MHz, CDCl3)  177.6, 169.7, 149.2, 92.4, 83.0, 58.5, 45.1, 28.4, 11.1; ESI-MS m/z: 262.3 [M + Na]+; ESI-HRMS: m/z calcd for C12H17NO4Na [M + Na]+, 262.1055; found, 262.1051.     287  7.3.71 Preparation of Compound 3.185    Under the same procedure described in 7.3.69, 3.185 (376 mg, 0.848 mmol) was prepared from (R)-3.173 (400 mg, 1.20 mmol) as a colourless oil in a yield of 71 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1). []20D +50.1° (c 0.55, CHCl3) 1H NMR (400 MHz, CDCl3)  4.96 (dd, J = 5.6, 8.3 Hz, 1H), 3.69 (s, 3H), 3.22–3.30 (m, 1H), 3.17 (s, 1H), 2.61–2.72 (m, 1H), 2.30–2.42 (m, 1H), 1.45–1.54 (m, 18H); 13C NMR (100 MHz, CDCl3)  170.5, 152.0, 100.1, 83.6, 58.6, 52.5, 34.7, 28.1, 1.8; ESI-MS m/z: 466.2 [M + Na]+.  7.3.72 Preparation of Compound 3.186    Based on the same condition described in entry 5 in Table 3.5, compound 3.186 (22 mg, 0.0506 mmol) was prepared from 3.185 (209 mg, 0.471 mmol) as a colourless oil in a yield of 11 %. 288  The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1). 1H NMR (400 MHz, CDCl3)  5.51 (dd, J = 7.17, 9.90 Hz, 1H), 4.36–4.48 (m, 1H), 4.09–4.19 (m, J = 6.8 Hz, 1H), 3.51 (s, 3H), 2.71–2.86 (m, 1H), 2.35–2.51 (m, 1H), 1.51 (s, 18H); 13C NMR (100 MHz, CDCl3)  153.6, 108.0, 82.6, 70.3, 60.2, 53.3, 30.4, 28.2; ESI-MS m/z: 456.2 [M + Na]+.  7.3.73 Preparation of Compound 3.205    To a stirred solution of Boc-D-leucine (R)-3.43 (231 mg, 0.99 mmol), L-leucine methyl ester hydrogen chloride 3.204 (181 mg, 1.49 mmol) and HOAt (408 mg, 2.99 mmol) in THF (10 mL) at room temperature was added EDCI (575 mg, 3.0 mmol) and TEA (0.20 ml, 1.5 mmol). The reaction mixture was stirred at room temperature overnight. After addition of HCl (20 mL, 1 M), the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1→4:1) to afford dipeptide 3.205 (327 mg, 0.91 mmol) as a light yellow oil in a yield of 91 %. []20D +35.0° (c 0.55, CHCl3); 1H NMR (600 MHz, CDCl3)  6.62 (br. s., 1H), 4.88 (br. s., 1H), 4.59 (br. s., 1H), 4.15 (br. s., 1H), 3.72 (s, 3H), 1.66–1.72 (m, 2H), 1.61–1.66 (m, 2H), 1.52–1.58 (m, 1H), 1.46–1.52 (m, 1H), 1.45 289  (s, 9H), 0.90–0.96 (m, 12H); 13C NMR (150 MHz, CDCl3)  173.5, 172.5, 155.9, 80.3, 53.2, 52.4, 50.8, 41.6, 41.1, 28.5, 25.0, 23.2, 23.0, 22.1, 22.0; ESI-MS m/z: 365.4 [M + Li]+; ESI-HRMS: m/z calcd for C18H35N2O5 [M + H]+, 359.2546; found, 359.2542.  7.3.74 Preparation of (4R,10R)-3.16 (NCSTD1)    To a stirred solution of dipeptide 3.205 (268 mg, 0.74 mmol) in H2O/THF (8 mL, 1:1) at 0 °C was added LiOH (2.96 mL, 1.48 mmol, 0.5 M in H2O). The reaction mixture was stirred at 0 °C for 1 h then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M HCl dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×20 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of dipeptide carboxylic acid (257 mg, 0.746 mmol), Meldrum’s acid (129 mg, 0.897 mmol), and DMAP (457 mg, 3.74 mmol) in DCM (10 mL) at −10 °C was added IPCC (172 l, 1.5 mmol) in DCM (300 l) was added at a rate of 15 l/min. The reaction mixture was 290  stirred −10 °C for additional 5 h then poured onto KHSO4 solution (30 mL, 5 %). The separated aqueous layer was extracted with EtOAc (3×30 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The yellowish crude residue was refluxed in MeCN (20 mL) for 3 h then the solvent was removed in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of the crude residue in toluene/MeOH (5 mL, 4:1) at room temperature was dropwise added TMSCHN2 (0.38 ml, 0.77 mmol, 2.0 M in hexanes). After addition of AcOH (0.5 mL), the reaction mixture was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:2) to afford (4R,10R)-3.46 (65.6 mg, 0.171 mmol) as a colourless oil in a yield of 23 % and (4S,10R)-3.46 (99.8 mg, 0.261 mmol) as a colourless oil in a yield of 35 %. Both intermediates were used directly for their next steps without characterization.  Compound (4R,10R)-3.46  (52 mg, 0.135 mmol) was dissolved in TFA/DCM (3 mL, 25 %) and stirred at 0 °C for 1 h then was evaporated in vacuo. After addition of saturated NaHCO3 solution (15 mL), the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:2→3:2) to afford free amine (26.4 mg, 0.093 mmol) as a colourless oil in a yield of 68 %. The crude residue was used directly for the next step without further purification.  291  To a stirred solution of the free amine (10 mg, 0.035 mmol) in THF (2 mL) 0 °C was added propionyl chloride (34 l, 0.38 mmol) and TEA (6 l, 0.046 mmol). The reaction mixture was stirred at room temperature for 3 h. After addition of saturated NaHCO3 solution (10 mL), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1→7:1) to afford NCSTD1 (4.4 mg, 0.013 mmol) as a colourless solid in a yield of 37 %. []20D −55.5° (c 0.18, CHCl3); 1H NMR (600 MHz, CDCl3)  6.02 (br. s., 1H), 5.79 (t, J = 8.4 Hz, 1H), 5.05 (s, 1H), 4.63 (dd, J = 3.1, 6.7 Hz, 1H), 3.87 (s, 3H), 2.26 (qd, J = 2.0, 7.5 Hz, 2H), 1.77–1.82 (m, 1H), 1.71–1.76 (m, 3H), 1.67 (ddd, J = 3.3, 10.1, 13.2 Hz, 1H), 1.40–1.46 (m, 1H), 1.16 (t, J = 7.7 Hz, 3H), 1.07 (d, J = 6.1 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  180.9, 173.8, 173.2, 169.5, 93.6, 58.9, 58.5, 51.9, 42.1, 39.3, 29.8, 25.4, 24.4, 24.0, 23.9, 22.8, 21.4, 9.9, ESI-MS m/z: 361.2 [M + Na]+; ESI-HRMS: m/z calcd for C18H31N2O4 [M + H]+, 339.2284; found, 339.2279.         292  7.3.75 Preparation of (4S,10R)-3.16 (NCSTD2)    Obtained in procedure 7.3.74, compound (4S,10R)-3.46 (94 mg, 0.246 mmol) was dissolved in TFA/DCM (3 mL, 25 %) and stirred at 0 °C for 1 h then was evaporated in vacuo. After addition of saturated NaHCO3 solution (15 mL), the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:2→1:2) to afford free amine (48 mg, 0.171 mmol) as a colourless oil in a yield of 70 %. The crude residue was used directly for the next step without further purification.  To a stirred solution of the free amine (30 mg, 0.10 mmol) in THF (5 mL) 0 °C was added propionyl chloride (0.1 mL, 1.14 mmol) and TEA (19 l, 0.136 mmol). The reaction mixture was stirred at room temperature for 3 h. After addition of saturated NaHCO3 solution (15 mL), the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→3:1) to afford NCSTD2 (11 mg, 0.031 mmol) as a colourless oil in a yield of 31 %. []20D +50.0° (c 0.48, 293  CHCl3); 1H NMR (600 MHz, CDCl3)  6.05 (d, J = 8.7 Hz, 1H), 5.75 (t, J = 8.4 Hz, 2H), 5.05 (s, 1H), 4.56 (dd, J = 4.1, 6.1 Hz, 1H), 3.87 (s, 3H), 2.25 (q, J = 7.3 Hz, 2H), 1.75–1.83 (m, 4H), 1.68–1.73 (m, 1H), 1.37–1.43 (m, 1H), 1.16 (t, J = 7.4 Hz, 3H), 1.05 (d, J = 6.1 Hz, 3H), 0.92 (d, J = 6.1 Hz, 6H), 0.88 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.1, 173.5, 173.2, 169.6, 93.6, 58.9, 58.9, 51.6, 41.8, 39.4, 29.9, 25.3, 24.4, 23.9, 23.9, 22.7, 21.5, 10.0; ESI-MS m/z: 361.4 [M + Na]+; ESI-HRMS: m/z calcd for C18H31N2O4 [M + H]+, 339.2284; found, 339.2281.  7.3.76 Preparation of Compound (S)-3.206    To a stirred solution of (S)-3.130 (2.30 g, 6.12 mmol) in THF (20 mL) at −40 °C was added DIBAL-H (6.70 mL, 6.70 mmol, 1M in hexanes) at a rate of 1.0 mL/min. The reaction mixture was stirred at −40 °C for 1 h. After successive addition of MeOH (6 mL), HCl (25 mL, 1 M) and Rochelle salt solution (25 mL, 1 M), the reaction mixture was stirred at room temperature for another 1 h. The separated organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1) to afford 4,4,4-trichlorobutanal (S)-3.206 (1.25 g, 3.61 mmol) as a colourless oil in a yield of 59 %. []20D −43.4° (c 0.23, CHCl3). 1H 294  NMR (600 MHz, CDCl3)  9.76 (s, 1H), 4.88 (dd, J = 5.1, 7.7 Hz, 1H), 3.71 (s, 3H), 2.55–2.64 (m, 1H), 2.45–2.55 (m, 2H), 2.11–2.21 (m, 1H), 1.49 (s, 18H); 13C NMR (150 MHz, CDCl3)  201.1, 170.9, 152.1, 83.6, 57.5, 52.4, 40.7, 28.1, 22.7; ESI-MS m/z: 400.4 [M + CH3OH + Na]+; ESI-HRMS: m/z calcd for C16H28NO7 [M + H]+, 346.1866; found, 346.1872.  7.3.77 Preparation of Compound (S)-3.207    To a stirred solution of triphenyl phosphite (1.73 mL, 6.60 mmol) in DCM (5 mL) at −20 °C was carefully bubbled chlorine gas until the solution just became bright yellow. After addition of a few drops of triphenyl phosphite, the solution was discharged. To this almost colourless solution −20 °C were added (S)-3.206 (1.14 g, 3.30 mmol) in DCM (3 mL) and TEA (1.84 mL, 13.2 mmol). The reaction mixture was stirred at room temperature overnight then refluxed for 6 h. After addition of hexanes (200 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (20:1) to afford gem-dichloride (S)-3.207 (1.10 g, 2.74 mmol) as a colourless oil in a yield of 83 %. []20D −32.1° (c 0.56, CHCl3); 1H NMR (400 MHz, CDCl3)  5.81 (dd, J = 5.0, 6.2 Hz, 1H), 4.87 (dd, J = 5.0, 9.3 Hz, 1H), 3.72 (s, 3H), 2.34–2.44 (m, 1H), 2.26–2.34 (m, 1H), 2.10–2.25 (m, 2H), 1.50 (s, 18H); 13C NMR (100 MHz, CDCl3)  170.8, 295  152.1, 83.7, 73.0, 57.1, 52.5, 40.4, 28.1, 26.5; ESI-MS m/z: 422.3 [M + Na]+; ESI-HRMS: m/z calcd for C16H27NO6Na35Cl2 [M + Na]+, 422.1113; found, 422.1105.  7.3.78 Preparation of Compound (S)-3.208    The (S)-3.207 (1.05 g, 2.62 mmol) was dissolved in TFA/DCM (15 mL, 25 %) and stirred at 0 °C for 1 h then was evaporated in vacuo. After addition of saturated NaHCO3 solution (30 mL), the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (9:1) to afford free amine (S)-3.208 (418 mg, 2.09 mmol) as a colourless oil in a yield of 80 %. 1H NMR (400 MHz, CDCl3)  5.85 (t, J = 5.8 Hz, 1H), 3.74 (s, 3H), 2.24–2.46 (m, 2H), 1.94–2.10 (m, 1H), 1.71–1.89 (m, 1H), 1.45 (br. s., 2H); 13C NMR (100 MHz, CDCl3)  170.8, 73.3, 53.7, 52.4, 40.2, 31.0; ESI-MS m/z: 200.3 [M + H]+; ESI-HRMS: m/z calcd for C6H12NO235Cl2 [M + H]+, 200.0245; found, 200.0241.     296  7.3.79 Preparation of Compound (R)-3.206    Under a similar procedure described in 7.3.76, (R)-3.206 (1.30 g, 3.76 mmol) was prepared from (R)-3.130 (2.83 g, 7.53 mmol) as a colourless oil in a yield of 50 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1). []20D +38.8° (c 0.18, CHCl3); 1H NMR (400 MHz, CDCl3)  9.77 (s, 1H), 4.89 (dd, J = 4.7, 9.3 Hz, 1H), 3.72 (s, 3H), 2.57–2.66 (m, 1H), 2.45–2.56 (m, 2H), 2.10–2.24 (m, 1H), 1.50 (s, 18H); 13C NMR (100 MHz, CDCl3)  201.2, 170.9, 152.2, 83.7, 57.5, 52.5, 40.7, 28.2, 22.7; ESI-MS m/z: 400.3 [M + CH3OH + Na]+; ESI-HRMS: m/z calcd for C16H28NO7 [M + H]+, 346.1866; found, 346.1860.  7.3.80 Preparation of Compound (R)-3.207    Under a similar procedure described in 7.3.77, (R)-3.207 (869 mg, 2.17 mmol) was prepared from (R)-3.206 (1.12 g, 3.24 mmol) as a colourless oil in a yield of 67 %. The crude residue was 297  purified by silica gel flash chromatography eluting with hexanes/EtOAc (20:1). []20D +32.3° (c 0.46, CHCl3); 1H NMR (400 MHz, CDCl3)  5.83 (t, J = 5.6 Hz, 1H), 4.90 (dd, J = 5.2, 9.1 Hz, 1H), 3.74 (s, 3H), 2.36 – 2.47 (m, 1H), 2.28–2.36 (m, 1H), 2.14–2.28 (m, 2H), 1.52 (s, 18H); 13C NMR (100 MHz, CDCl3)  170.8, 152.2, 83.8, 73.0, 57.1, 52.6, 40.4, 28.2, 26.5; ESI-MS m/z: 422.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H27NO6Na35Cl2 [M + Na]+, 422.1113; found, 422.1103.  7.3.81 Preparation of Compound (R)-3.211    To a stirred solution of (R)-3.207 (750 mg, 1.87 mmol) in acetonitrile (50 mL) at room temperature was added lithium bromide (480 mg, 5.52 mmol). The reaction mixture was stirred 65 °C for 12 h then the solvent was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1) to afford (R)-3.207 (428 mg, 1.42 mmol) as a colourless oil in a yield of 76 %. []20D −21.9° (c 0.43, CHCl3); 1H NMR (400 MHz, CDCl3)  5.81 (t, J = 5.6 Hz, 1H), 5.14 (d, J = 7.0 Hz, 1H), 4.34 (br. s., 1H), 3.74 (s, 3H), 2.16–2.34 (m, 2H), 2.05–2.16 (m, 1H), 1.82–1.93 (m, 1H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3)  172.7, 155.5, 80.4, 72.8, 52.7, 52.5, 39.5, 29.3, 28.4; ESI-MS m/z: 322.2 [M + Na]+; ESI-HRMS: m/z calcd for C11H20NO435Cl2 [M + H]+, 300.0769; found, 300.0775. 298  7.3.82 Preparation of Compound (R)-3.73    To a stirred solution of methyl ester (R)-3.211 (287 mg, 0.957 mmol) in H2O/THF (5 mL, 1:1) at 0 °C was added Ba(OH)2 (910 mg, 5.31 mmol). The reaction mixture was stirred at 0 °C for 1 h then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M HCl dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×20 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/EtOAc (98:2) to afford (R)-3.73 (191 mg, 0.667 mmol) as a colourless oil in a yield of 70 %. []20D −15.5° (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3)  11.54 (br. s., 1H), 6.83 and 5.36 (d each, J = 5.7 Hz, 2H), 5.79 (t, J = 5.6 Hz, 1H), 4.34 and 4.13 (br. s. each, 1H), 2.21–2.35 (m, 2H), 2.07–2.18 (m, 1H), 1.83–1.97 (m, 1H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3)  177.4 and 176.4, 157.1 and 155.8, 82.4 and 80.7, 72.7, 53.7 and 52.4, 39.4, 28.3, 20.9; ESI-MS m/z: 284.3 [M + H]+; ESI-HRMS: m/z calcd for C10H17NO4Na35Cl2 [M + Na]+, 308.0432; found,308.0435.    299  7.3.83 Preparation of Compound (4S,10R)-3.209    To a stirred solution of (R)-3.73 (156 mg, 0.545 mmol), 3.208 (109 mg, 0.545 mmol) and HOAt (313 mg, 1.631 mmol) in THF (6 mL) at room temperature was added EDCI (253 mg, 1.635 mmol). The reaction mixture was stirred at room temperature overnight. After addition of HCl (10 mL, 1 M), the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1) to dipeptide (4S,10R)-3.209 (193 mg, 0.412 mmol) as a light yellow oil in a yield of 76 %. []20D +31.2° (c 0.25, CHCl3); 1H NMR (600 MHz, CDCl3)  7.08 (d, J = 5.1 Hz, 1H), 5.82 (dt, J = 5.7, 8.1 Hz, 4H), 5.26 (d, J = 6.1 Hz, 1H), 4.64 (td, J = 4.6, 8.2 Hz, 1H), 4.26 (d, J = 3.6 Hz, 1H), 3.77 (s, 3H), 2.23–2.34 (m, 3H), 2.15–2.22 (m, 2H), 2.09–2.16 (m, 1H), 1.86–1.97 (m, 2H), 1.45 (s, 9H); 13C NMR (150 MHz, CDCl3)  172.1, 171.7, 155.9, 80.8, 72.9, 72.6, 53.4, 53.0, 51.3, 39.6, 39.3, 28.8, 28.7, 28.5; ESI-MS m/z: 491.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H26N2O5Na35Cl4 [M + Na]+, 489.0494; found, 489.0482.   300  7.3.84 Preparation of Compound (4R,10R)-3.212 and (4S,10R)-3.212  To a stirred solution of dipeptide (4S,10R)-3.209 (172 mg, 0.369 mmol) in H2O/THF (2 mL, 1:1) at 0 °C was added LiOH (1.48 mL, 0.739 mmol, 0.5 M in H2O). The reaction mixture was stirred at 0 °C for 1 h then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M HCl dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of crude residue, Meldrum’s acid (63.9 mg, 0.443 mmol), and DMAP (225.7 mg, 1.85 mmol) in DCM (5 mL) at −10 °C was added IPCC (80 l, 0.739 mmol) in DCM (220 l) was added at a rate of 10 l/min. The reaction mixture was stirred −10 °C for additional 5 h then poured onto KHSO4 solution (10 mL, 5 %). The separated aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The yellowish crude residue was refluxed in MeCN (20 mL) for 3 h then the solvent was removed in vacuo. The crude residue was used directly for the next step without further purification.  To a stirred solution of the crude residue in toluene/MeOH (3 mL, 4:1) at room temperature was dropwise added TMSCHN2 (0.18 ml, 0.369 mmol, 2.0 M in hexanes). After addition of AcOH (0.18 mL), the reaction mixture was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1→5:1) to afford (4R,10R)-3.212 (41 mg, 301  0.084 mmol) as a colourless oil in a yield of 23 % and (4S,10R)-3.212 (47 mg, 0.095 mmol) as a colourless oil in a yield of 26 %.    []20D −48.3° (c 0.20, CHCl3); 1H NMR (600 MHz, CDCl3)  5.85 (t, J = 5.6 Hz, 1H), 5.74 (t, J = 5.6 Hz, 1H), 5.32 (t, J = 7.9 Hz, 1H), 5.19 (d, J = 7.2 Hz, 1H), 5.14 (s, 1H), 4.69 (br. s., 1H), 3.91 (s, 3H), 2.40–2.47 (m, 2H), 2.18–2.24 (m, 1H), 2.13–2.17 (m, 1H), 2.04–2.10 (m, 1H), 1.93–2.00 (m, 1H), 1.65–1.74 (m, 2H), 1.45 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.8, 172.6, 169.5, 155.9, 94.5, 80.4, 73.1, 73.0, 59.3, 58.6, 53.6, 40.1, 36.6, 28.7, 28.5, 25.2; ESI-MS m/z: 515.2 [M + Na]+; ESI-HRMS: m/z calcd for C18H27N2O5Na35Cl4 [M + Na]+, 491.0674; found, 491.0665.    []20D +77.9° (c 0.15, CHCl3); 1H NMR (600 MHz, CDCl3)  5.87 (t, J = 5.9 Hz, 1H), 5.75 (t, J = 5.6 Hz, 1H), 5.43 (t, J = 7.9 Hz, 1H), 5.27 (d, J = 8.2 Hz, 1H), 5.14 (s, 1H), 4.74 (br. s., 1H), 302  3.91 (s, 3H), 2.42–2.49 (m, 1H), 2.30–2.40 (m, 2H), 2.10–2.18 (m, 2H), 2.00–2.07 (m, 2H), 1.73–1.80 (m, 1H), 1.44 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.9, 172.2, 169.1, 155.8, 94.3, 80.2, 73.0, 72.6, 59.4, 58.2, 53.8, 40.0, 37.2, 30.1, 28.5, 25.3; ESI-MS m/z: 515.2 [M + Na]+; ESI-HRMS: m/z calcd for C18H27N2O5Na35Cl4 [M + Na]+, 491.0674; found, 491.0667.  7.3.85 Preparation of (4R,10R)-3.210 (LPY08)    Under a similar procedure described in 7.3.74, (4R,10R)-3.210 (7.6 mg, 0.017 mmol) was prepared from (4R,10R)-3.212 (14 mg, 0.028 mmol) as a colourless oil in a yield of 60 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:2). []20D −61.8° (c 0.07, CHCl3); 1H NMR (600 MHz,  CDCl3)  6.19 (d, J = 8.2 Hz, 1H), 5.85 (dd, J = 5.1, 6.7 Hz, 1H), 5.76 (dd, J = 4.4, 6.4 Hz, 1H), 5.57 (td, J = 3.6, 9.0 Hz, 1H), 5.15 (s, 1H), 4.70 (dd, J = 2.6, 5.6 Hz, 1H), 3.92 (s, 3H), 2.35–2.46 (m, 2H), 2.28–2.34 (m, 1H), 2.26 (qd, J = 2.0, 7.5 Hz, 2H), 2.10–2.19 (m, 3H), 1.92–2.00 (m, 1H), 1.73–1.82 (m, 1H), 1.17 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.8, 174.1, 171.9, 169.4, 94.5, 73.1, 73.0, 59.3, 58.6, 52.2, 40.1, 36.6, 29.7, 28.9, 25.2, 9.9; ESI-MS m/z: 491.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H22N2O4Na35Cl4 [M + Na]+, 469.0231; found, 469.0243. 303  7.3.86 Preparation of (4S,10R)-3.210 (LPY09)    Under a similar procedure described in 7.3.74, (4S,10R)-3.210 (11.5 mg, 0.025 mmol) was prepared from (4S,10R)-3.212 (18.1 mg, 0.036 mmol) as a colourless oil in a yield of 70 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→1:1). []20D +76.1° (c 0.06, CHCl3); 1H NMR (600 MHz, CDCl3)  6.26 (d, J = 8.2 Hz, 1H), 5.87 (dd, J = 4.9, 6.4 Hz, 1H), 5.76 (t, J = 5.4 Hz, 1H), 5.73 (td, J = 3.6, 8.7 Hz, 1H), 5.16 (s, 1H), 4.73 (dd, J = 3.1, 5.6 Hz, 1H), 3.92 (s, 3H), 2.41–2.47 (m, 1H), 2.34–2.41 (m, 1H), 2.26 (qd, J = 2.0, 7.5 Hz, 2H), 2.13–2.26 (m, 3H), 2.02–2.07 (m, 2H), 1.79–1.87 (m, 1H), 1.18 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz, CDCl3) 178.9, 174.0, 171.8, 169.0, 94.3, 73.0, 72.6, 59.4, 58.2, 52.2, 39.9, 37.1, 30.0, 29.8, 25.3, 9.9; ESI-MS m/z: 491.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H23N2O435Cl4 [M + H]+, 447.0412; found, 447.0419.     304  7.3.87 Preparation of Compound (S)-3.221    To a stirred solution of aldehyde 3.76 (1.42 g, 8.09 mmol) in DCM (10 mL) were added (S)-(−)-tert-butanesulfinamide (0.98 g, 6.33 mmol) CuSO4 (3.88 g, 24.2 mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 days. The catalyst was removed through a pad of Celite® with DCM (200 mL) and the filtrate was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (100:3) to afford (S)-3.221 (2.0 g, 7.18 mmol) as a colourless oil in a yield of 90 %. []20D +171.9° (c 0.68, CHCl3); 1H NMR (400 MHz, CDCl3)  8.14 (br. s., 1H), 3.03–3.15 (m, 2H), 2.93–3.03 (m, 2H), 1.18 (s, 9H); 13C NMR (100 MHz, CDCl3)  166.0, 98.9, 57.0, 50.2, 33.3, 22.5; ESI-MS m/z: 280.2 [M + H]+; ESI-HRMS: m/z calcd for C8H15NO32S35Cl3 [M + H]+, 277.9940; found, 277.9942.      305  7.3.88 Preparation of Compound (R)-3.221    Under a similar procedure described in 7.3.87, (R)-3.221 (2.92 g, 10.5 mmol) was prepared from 3.76 (2.0 g, 11.3 mmol) as a colourless oil in a yield of 93 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (36:1). []20D −162.3° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3)  8.16 (br. s., 1H), 3.05–3.17 (m, 2H), 2.98–3.04 (m, 2H), 1.19 (s, 9H); 13C NMR (100 MHz, CDCl3)  166.0, 99.0, 57.0, 50.3, 33.3, 22.5; ESI-MS m/z: 280.3 [M + H]+; ESI-HRMS: m/z calcd for C8H15NO32S35Cl3 [M + H]+, 277.9940; found, 277.9938.  7.3.89 Preparation of Compound (S,S)-3.217    To a stirred solution of the sulfinimine (S)-3.221 (1.20 g, 4.31 mmol) in DCM (30 mL) 0 °C were added trimethylsilyl cyanide (1.17 mL, 8.61 mmol) and Sc(OTf)3 (0.32 g, 0.64 mmol). The 306  reaction mixture was stirred at 0 °C for 2 days and at room temperature for another 2 days. The reaction solution was ready to recrystallize to furnish pure diastereomeric cyanide intermediate (S,S)-3.217 (1.13 g, 3.69 mmol) in a yield of 20 % (86 % for both diastereomers). []20D +22.7° (c 0.39, CHCl3); 1H NMR (600 MHz, CDCl3)  4.36 (d, J = 6.7 Hz, 1H), 4.15 (br. s., 1H), 2.92 (t, J = 7.7 Hz, 2H), 2.34–2.47 (m, 2H), 1.28 (s, 9H); 13C NMR (150MHz, CDCl3)  118.5, 98.2, 57.6, 50.6, 45.0, 31.7, 22.7; ESI-MS m/z: 329.3 [M + Na]+; ESI-HRMS: m/z calcd for C9H16N2O32S35Cl3 [M + H]+, 305.0049; found, 305.0051.  7.3.90 Preparation of Compound (R,R)-3.217    Under a similar procedure described in 7.3.89, (R,R)-3.217 (2.27 g, 7.42 mmol) was prepared from (R)-3.129 (2.50 g, 8.97 mmol) as a colourless oil in a yield of 25 % (83 % for both diastereomers). []20D −28.0° (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3)  4.36 (br. s., 1H), 4.16 (br. s., 1H), 2.92 (t, J = 7.6 Hz, 2H), 2.28–2.52 (m, 2H), 1.28 (s, 9H); 13C NMR (100 MHz, CDCl3)  118.5, 98.2, 57.8, 50.6, 45.2, 31.7, 22.8; ESI-MS m/z: 329.3 [M + Na]+; ESI-HRMS: m/z calcd for C9H16N2O32S35Cl3 [M + H]+, 305.0049; found, 305.0056.  307  7.3.91 Preparation of Compound (S)-3.218    HCl gas was generated by dripping HCl (100 mL, conc.) onto anhydrous CaCl2 granule (100 g).   To a stirred solution of (S,S)-3.127 (579 mg, 1.77 mmol) in 10 mL MeOH (10 mL) was bubbled HCl gas for 1 h. The solution was stirred at room temperature overnight. Placed in an ice/water bath, the reaction mixture was added saturated NaHCO3 solution dropwise until its pH value was adjusted to pH=10. The aqueous layer was extracted with EtOAc (3×30 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and evaporated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1) to afford (S)-3.218 (300 mg, 1.28 mmol) as a colourless oil in a yield of 73 %. []20D +7.0° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3)  3.74 (s, 3H), 3.50 (dd, J = 4.9, 8.2 Hz, 1H), 2.84–2.97 (m, 1H), 2.65–2.84 (m, 1H), 2.10–2.30 (m, 1H), 1.87–2.10 (m, 1H), 1.58 (br. s., 2H); 13C NMR (100 MHz, CDCl3)  175.7, 99.7, 53.4, 52.4, 51.7, 31.6; ESI-MS m/z: 234.3 [M + H]+; ESI-HRMS: m/z calcd for C6H11NO235Cl3 [M + H]+, 233.9855; found, 233.9851.     308  7.3.92 Preparation of Compound 3.223    To a stirred solution of N-hydroxysuccinimide (1.0 g, 8.81 mmol) in THF (50 mL) were added propionyl chloride (0.70 mL, 8.0 mmol) and TEA (1.21 mL, 8.81 mmol) at room temperature. The reaction mixture stirred at room temperature overnight. The organic solvent was evaporated in vacuo and extracted with brine. The yellowish oil crystallized upon removal of THF traces under the oil pump vacuum (0.75 g, 54 %). The solid was directly used without purification. 1H NMR (300 MHz, CDCl3)  2.82 (s, 4H), 2.63 (q, J = 7.49 Hz, 2H), 1.26 (t, J = 7.49 Hz, 3H).  Compound (R,R)-3.217 (449 mg, 1.46 mmol) was stirred in HCl saturated methanol solution for 8 h. The solution was stirred at room temperature overnight. Placed in an ice/water bath, the reaction mixture was added saturated NaHCO3 solution dropwise until its pH value was adjusted to pH=10. The aqueous layer was extracted with EtOAc (3×30 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and evaporated in vacuo. The crude residue was directly used without purification.  To a stirred solution of the solid in saturated NaHCO3 solution (15 mL) was added N-hydroxysuccinimide propionic ester (327.5 mg, 1.91 mmol) in THF (15 mL) at room 309  temperature. The reaction mixture was stirred at room temperature overnight. Placed in an ice/water bath, the reaction mixture was added 1M HCl dropwise until its pH value was adjusted to pH=3. The aqueous suspension was extracted with EtOAc (3×10 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1→1:1) to afford the N-propionyl methyl ester (267 mg, 0.876 mmol) as a colourless oil in a yield of 60 %. 1H NMR (600 MHz, CDCl3)  6.08 (d, J = 6.6 Hz, 1H), 4.75 (dt, J = 5.3, 7.8 Hz, 1H), 3.81 (s, 3H), 2.77–2.84 (m, 1H), 2.64–2.71 (m, 1H), 2.36–2.43 (m, 1H), 2.30 (q, J = 7.6 Hz, 2H), 2.08–2.16 (m, 1H), 1.19 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)  173.9, 172.5, 99.2, 53.1, 51.2, 50.8, 30.1, 29.8, 9.9.  Under a similar procedure described in 7.3.35, 3.223 was prepared from the N-propionyl methyl ester as a colourless oil and used without purification for the next step.          310  7.3.93 Coupling reaction between (S)-3.218 and 3.223    Under a similar procedure described in 7.3.73, anticipated product 3.224 was not obtained from coupling between (S)-3.218 (14.7 mg, 0.062 mmol) and 3.223 (17.4 mg, 0.062 mmol).  7.3.94 Coupling reaction between (S)-3.218 and (R)-3.43    Under a similar procedure described in 7.3.73, (4S,10R)-3.229 (57 mg, 0.243 mmol) was prepared from (S)-3.218 (57 mg, 0.243 mmol) and (R)-3.43 (56 mg, 0.243 mmol) as a colourless oil in a yield of 93 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→3:1). Please find data in 7.3.102.  311  7.3.95 Preparation of Compound (R)-3.79  7.3.95.1 Route One    Compound (R,R)-3.217 (449 mg, 1.46 mmol) was refluxed in HCl (10 mL, 6 M) for 0.5 h. The reaction mixture was cooled to room temperature and washed with diethyl ether (2×10 mL). The aqueous layer was evaporated at 50 °C in vacuo and freeze dried overnight. The yellowish solid was directly used without purification.  To a stirred solution of the crude residue (360 mg, 1.17 mmol) in saturated NaHCO3 solution (20 mL) was added Boc2O (254 mg, 1.17 mmol) in THF (20 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. Placed in an ice/water bath, the reaction mixture was added 1M HCl dropwise until its pH value was adjusted to pH=3. The aqueous suspension was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1→1:1) to afford (R)-3.79 (300 mg, 0.935 mmol) as a colourless oil in a yield of 64 %.  312  7.3.95.2 Route Two    Under a similar procedure described in 7.3.91, (R)-3.218 (610 mg, 2.60 mmol) was prepared from (R,R)-3.127 (1.10 g, 3.60 mmol) as a colourless oil in a yield of 72 %.  To a stirred solution of (R)-3.218 (610 mg, 2.60 mmol), NaHCO3 (436 mg, 5.19 mmol) in H2O/THF (10 mL, 1:1) was added Boc2O (573 mg, 2.62 mmol) at room temperature. The reaction mixture was refluxed overnight then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M NaHSO4 dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2) to afford N-Boc methyl ester 7.1 (670 mg, 2.00 mmol) as a colourless oil in a yield of 77 %. 1H NMR (400 MHz, CDCl3)  5.17 (br. s., 1H), 4.40 (br. s., 4H), 3.78 (s, 3H), 2.63–2.90 (m, 2H), 2.34 (t, J = 12.3 Hz, 1H), 2.00–2.17 (m, 1H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3)  172.5, 155.5, 99.2, 80.5, 52.9, 52.4, 51.2, 30.0, 28.5; ESI-MS m/z: 358.2 [M + Na]+; ESI-HRMS: m/z calcd for C11H18NO4NaCl3 [M + Na]+, 356.0203; found, 356.0119.  313  To a stirred solution of 7.1 (540 mg, 1.61 mmol) in H2O/THF (2 mL, 1:1) at 0 °C was added LiOH (6.5 mL, 3.25 mmol, 0.5 M in H2O). The reaction mixture was stirred at 0 °C for 1 h then THF was removed in vacuo. Placed in an ice/water bath, the reaction mixture was added 1 M HCl dropwise until its pH value was adjusted to pH=3. The aqueous layer was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2) to afford (R)-3.79 (505 mg, 1.57 mmol) as a colourless oil in a yield of 98 %. []20D −24.7° (c 4.7, CHCl3); 1H NMR (400 MHz, MeOH-d4) 5.09 (br. s., 2H), 4.19 (dd, J = 4.7, 7.8 Hz, 1H), 2.66–2.91 (m, 2H), 2.21–2.37 (m, 1H), 2.01–2.18 (m, 1H), 1.38 (s, 9H); 13C NMR (100 MHz, MeOH-d4) 174.9, 158.0, 100.6, 80.8, 53.6, 52.6, 50.0, 30.1, 28.8; ESI-MS m/z: 320.3 [M + H]+; ESI-HRMS: m/z calcd for C10H16NO4NaCl3 [M + Na]+, 342.0043; found, 342.0044.  7.3.96 Preparation of (4S,10R)-3.219 (LPY35)    Under a similar procedure described in 7.3.83, (4S,10R)-3.219 (150 mg, 0.279 mmol) was prepared from (S)-3.218 (89 mg, 0.380 mmol) and (R)-3.79 (122 mg, 0.380 mmol) as a colourless oil in a yield of 73 %. The crude residue was purified by silica gel flash 314  chromatography eluting with hexanes/EtOAc (6:1→3:1). []20D +21.9° (c 0.50, CHCl3); 1H NMR (600 MHz, CDCl3)  6.97 (br. s., 1H), 5.14 (d, J = 6.7 Hz, 1H), 4.66–4.78 (m, 1H), 4.30 (d, J = 2.0 Hz, 1H), 3.81 (s, 3H), 2.72–2.89 (m, 3H), 2.63–2.71 (m, 1H), 2.35–2.47 (m, 2H), 2.14–2.21 (m, 1H), 2.07–2.13 (m, 1H), 1.48 (s, 9H); 13C NMR (150 MHz, CDCl3)  171.8, 171.3, 155.9, 99.2, 98.9, 81.2, 53.3, 53.2, 51.3, 51.1, 51.1, 29.6, 29.3, 28.5; ESI-MS m/z: 559.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H24N2O5NaCl6 [M + Na]+, 556.9714; found, 556.9713.   7.3.97 Preparation of (4R,10R)-3.220 (LPY10)    Under a similar procedure described in 7.3.85, (4R,10R)-3.220 (2.4 mg, 0.0047 mmol) was prepared from (4R,10R)-3.219 (150 mg, 0.279 mmol) as a colourless oil in a yield of 1.7 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (7:1→3:2). []20D −28.3° (c 0.53, CHCl3); 1H NMR (600 MHz, CDCl3)  6.22 (d, J = 8.7 Hz, 1H), 5.70 (td, J = 3.6, 9.0 Hz, 1H), 5.19 (s, 1H), 4.75 (dd, J = 3.1, 5.6 Hz, 1H), 3.95 (s, 3H), 2.93–3.01 (m, 1H), 2.75–2.84 (m, 1H), 2.63–2.72 (m, 2H), 2.43–2.51 (m, 1H), 2.33–2.40 (m, 2H), 2.28 (qd, J = 3.3, 7.6 Hz, 2H), 1.91–1.99 (m, 1H), 1.17 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.6, 174.1, 171.7, 169.3, 99.5, 99.2, 94.7, 59.4, 58.3, 51.7, 51.6, 48.4, 30.2, 315  29.8, 26.1, 9.9; ESI-MS m/z: 539.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H21N2O435Cl537Cl [M + H]+, 516.9603; found, 516.9608.  7.3.98 Preparation of (4S,10R)-3.220 (LPY11)    Under a similar procedure described in 7.3.86, (4S,10R)-3.220 (3.0 mg, 0.0058 mmol) was prepared from (4S,10R)-3.219 (150 mg, 0.279 mmol) as a colourless oil in a yield of 2.1 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→1:1). []20D +27.6° (c 0.65, CHCl3); 1H NMR (600 MHz, CDCl3)  6.28 (d, J = 8.2 Hz, 1H), 5.79 (td, J = 3.6, 8.4 Hz, 1H), 5.19 (s, 1H), 4.79 (m, 1H), 3.95 (s, 3H), 2.98 (ddd, J = 4.6, 11.8, 14.3 Hz, 1H), 2.71–2.79 (m, 1H), 2.57–2.63 (m, 1H), 2.54 (dd, J = 6.1, 9.2 Hz, 2H), 2.42–2.49 (m, 1H), 2.34–2.41 (m, 1H), 2.30 (qd, J = 3.8, 7.6 Hz, 2H), 1.99–2.08 (m, 1H), 1.19 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz,  CDCl3)  178.6, 174.0, 171.7, 168.9, 99.3, 98.9, 94.5, 59.5, 57.9, 51.9, 51.4, 48.9, 31.1, 29.9, 26.3, 9.9; ESI-MS m/z: 539.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H21N2O435Cl537Cl [M + H]+, 516.9603; found, 516.9598.  316  7.3.99 Preparation of Compound (4S,10R)-3.226    Under a similar procedure described in 7.3.73, (4S,10R)-3.226 (196 mg, 0.436 mmol) was prepared from 3.204 (140 mg, 0.770 mmol) and (R)-3.79 (165 mg, 0.514 mmol) as a colourless oil in a yield of 85 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:1). []20D +6.4° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3)  6.90 and 6.82 (br. s. each, 1H), 5.35 and 5.25 (d each, J = 5.5 Hz and 7.9 Hz each, 1H), 4.60 (td, J = 4.4, 8.8 Hz, 1H), 4.32 (br. s., 1H), 3.72 (s, 3H), 2.67–2.85 (m, 2H), 2.29–2.41 (m, 1H), 2.01–2.15 (m, 1H), 1.59–1.70 (m, 2H), 1.52–1.59 (m, 1H), 1.45 (s, 9H), 0.87–0.97 (m, 6H); 13C NMR (100 MHz, CDCl3)  173.4, 171.2, 155.9, 99.4, 80.7, 52.6, 51.2, 50.9, 41.5, 41.4, 29.7, 28.5, 25.1, 23.0, 21.9; ESI-MS m/z: 471.3 [M + Na]+; ESI-HRMS: m/z calcd for C17H29N2O5Na35Cl3 [M + Na]+, 469.1040; found, 469.1044.      317  7.3.100 Preparation of (4R,10R)-3.228 (LPY12)    Under a similar procedure described in 7.3.85, (4R,10R)-3.228 (2.5 mg, 0.0058 mmol) was prepared from (4S,10R)-3.226 (100 mg, 0.223 mmol) as a colourless oil. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:2). The yield from (4R,10R)-3.227 is 72 %. []20D −76.0° (c 0.5, CHCl3); 1H NMR (600 MHz, CDCl3)  6.30 (d, J = 8.7 Hz, 1H), 5.74 (td, J = 3.1, 9.0 Hz, 1H), 5.09 (s, 1H), 4.60 (t, J = 5.1 Hz, 1H), 3.89 (s, 3H), 2.98 (ddd, J = 4.6, 11.6, 14.5 Hz, 1H), 2.73–2.80 (m, 1H), 2.32–2.39 (m, 1H), 2.29 (q, J = 7.5 Hz, 2H), 1.88–1.95 (m, 1H), 1.82–1.85 (m, 2H), 1.74–1.80 (m, 1H), 1.18 (t, J = 7.4 Hz, 3H), 0.94 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.3, 173.8, 171.3, 169.8, 99.6, 93.6, 59.0, 59.0, 51.6, 51.5, 39.2, 30.6, 29.9, 24.4, 23.9, 22.7, 10.0; ESI-MS m/z: 451.2 [M + Na]+; ESI-HRMS: m/z calcd for C17H26N2O4Cl3 [M + H]+, 427.0958; found, 427.0961.    318  7.3.101 Preparation of (4S,10R)-3.228 (LPY13)    Under a similar procedure described in 7.3.86, (4S,10R)-3.228 (3.9 mg, 0.00914 mmol) was prepared from (4S,10R)-3.227 (8.5 mg, 0.0228 mmol) as a colourless oil in a yield of 40 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→1:1). []20D +47.5° (c 0.4, CHCl3); 1H NMR (600 MHz, CDCl3)  6.33 (d, J = 8.2 Hz, 1H), 5.77 (td, J = 3.3, 8.6 Hz, 1H), 5.09 (s, 1H), 4.66 (dd, J = 3.1, 6.7 Hz, 1H), 3.89 (s, 3H), 2.99 (ddd, J = 4.4, 12.0, 14.3 Hz, 1H), 2.71–2.78 (m, 1H), 2.46 (tt, J = 4.2, 12.7 Hz, 1H), 2.29 (qd, J = 3.8, 7.6 Hz, 2H), 1.95–2.03 (m, J = 3.8, 9.0, 12.5, 12.5 Hz, 1H), 1.80–1.85 (m, 1H), 1.73–1.80 (m, 2H), 1.18 (t, J = 7.7 Hz, 3H), 0.94 (d, J = 5.6 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.2, 173.9, 171.3, 169.5, 99.5, 93.5, 59.1, 58.6, 51.8, 51.5, 39.4, 31.0, 29.9, 24.5, 24.0, 22.8, 10.0; ESI-MS m/z: 451.2 [M + Na]+; ESI-HRMS: m/z calcd for C17H25N2O4NaCl3 [M + Na]+, 449.0778; found, 449.0775.    319  7.3.102 Preparation of Compound (4S,10R)-3.229    Under a similar procedure described in 7.3.83, (4S,10R)-3.229 (82 mg, 0.183 mmol) was prepared from (S)-3.218 (52 mg, 0.221 mmol) and (R)-3.43 (80 mg, 0.350 mmol) as a colourless oil in a yield of 82 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→3:1). []20D +36.1° (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3)  7.04 and 6.93 (br. s.each, 1H), 5.01 (br. s., 1H), 4.69 (td, J = 4.9, 7.9 Hz, 1H), 4.16 (br. s., 1H), 3.77 (s, 3H), 2.73–2.82 (m, 1H), 2.61–2.71 (m, 1H), 2.32–2.43 (m, 1H), 2.06–2.17 (m, 1H), 1.61–1.72 (m, 2H), 1.47–1.53 (m, 1H), 1.43 (s, 9H), 0.90–0.95 (m, 6H); 13C NMR (100 MHz, CDCl3)  172.9, 171.9, 155.9, 99.1, 80.4, 53.3, 52.9, 51.1, 50.9, 41.0, 29.7, 28.5, 25.0, 23.1, 22.3; ESI-MS m/z: 469.2 [M + Na]+; ESI-HRMS: m/z calcd for C17H30N2O535Cl3 [M + H]+, 447.1220; found, 447.1231.      320  7.3.103 Preparation of (4R,10R)-3.231 (LPY17)    Under a similar procedure described in 7.3.85, (4R,10R)-3.231 (4.0 mg, 0.0093 mmol) was prepared from (4S,10R)-3.229 (79.2 mg, 0.176 mmol) as a colourless oil. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:2). The yield from (4R,10R)-3.230 is 23 %. []20D −25.0° (c 0.4, CHCl3); 1H NMR (600 MHz, CDCl3)  5.97 (d, J = 7.7 Hz, 1H), 5.70 (ddd, J = 3.1, 8.4, 11.0 Hz, 1H), 5.15 (s, 1H), 4.72 (dd, J = 3.1, 5.6 Hz, 1H), 3.92 (s, 3H), 2.76 (ddd, J = 4.4, 11.9, 14.0 Hz, 1H), 2.65–2.72 (m, 1H), 2.43 (ddd, J = 3.8, 11.6, 14.2 Hz, 1H), 2.28–2.35 (m, 1H), 2.24 (qd, J = 4.1, 7.5 Hz, 2H), 1.69–1.76 (m, 1H), 1.60–1.63 (m, 1H), 1.41–1.46 (m, 1H), 1.15 (t, J = 7.7 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.2, 173.8, 173.7, 169.1, 99.4, 94.7, 59.3, 58.3, 51.7, 48.2, 41.0, 29.7, 26.0, 25.2, 23.8, 21.4, 9.9; ESI-MS m/z: 451.3 [M + Na]+; ESI-HRMS: m/z calcd for C17H25N2O4NaCl3 [M + Na]+, 449.0778; found, 449.0780.    321  7.3.104 Preparation of (4S,10R)-3.231 (LPY18)    Under a similar procedure described in 7.3.86, (4S,10R)-3.231 (1.3 mg, 0.003 mmol) was prepared from (4S,10R)-3.230 (8.0 mg, 0.0215 mmol) as a colourless oil in a yield of 14 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→1:1). []20D +60.0° (c 0.33, CHCl3); 1H NMR (600 MHz, CDCl3)  5.96 (br. s., 1H), 5.78 (t, J = 8.4 Hz, 1H), 5.15 (s, 1H), 4.77 (dd, J = 3.3, 5.4 Hz, 1H), 3.92 (s, 3H), 2.55–2.62 (m, 1H), 2.50 (dd, J = 6.7, 9.2 Hz, 2H), 2.30–2.36 (m, 1H), 2.27 (qd, J = 2.3, 7.6 Hz, 2H), 1.71–1.77 (m, 1H), 1.65 (ddd, J = 3.1, 10.2, 13.3 Hz, 1H), 1.44–1.50 (m, 1H), 1.17 (t, J = 7.4 Hz, 3H), 1.07 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.3, 173.9, 173.5, 168.9, 99.0, 94.6, 59.4, 57.8, 51.9, 48.8, 42.1, 29.8, 26.1, 25.4, 23.8, 21.3, 9.9; ESI-MS m/z: 451.3 [M + Na]+; ESI-HRMS: m/z calcd for C17H25N2O4NaCl3 [M + Na]+, 449.0778; found, 449.0781.    322  7.3.105 Preparation of Compound (S)-3.79    Compound (S,S)-3.217 (258 mg, 0.844 mmol) was refluxed in HCl (10 mL, 6 M) for 0.5 h. The reaction mixture was cooled to room temperature and washed with diethyl ether (2×10 mL). The aqueous layer was evaporated at 50 °C in vacuo and freeze dried overnight. The yellowish solid was used without purification.  To a stirred solution of the solid in saturated NaHCO3 solution (20 mL) was added Boc2O (184 mg, 0.844 mmol) in THF (20 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. Placed in an ice/water bath, the reaction mixture was added 1M HCl dropwise until its pH value was adjusted to pH=3. The aqueous suspension was extracted with EtOAc (3×15 mL). The combined organic extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1→1:1) to afford (S)-3.79 (186 mg, 0.575 mmol) as a colourless oil in a yield of 68 %. []20D +26.1° (c 1.8, CHCl3); 1H NMR (400 MHz, MeOH-d4) 5.09 (br. s., 2H), 4.19 (dd, J = 4.7, 7.8 Hz, 1H), 2.66–2.91 (m, 2H), 2.21–2.37 (m, 1H), 2.01–2.18 (m, 1H), 1.38 (s, 9H); 13C NMR (100 MHz, MeOH-d4) 174.9, 158.0, 100.6, 80.8, 53.6, 52.6, 50.0, 30.1, 28.8; 323  ESI-MS m/z: 320.3 [M + H]+; ESI-HRMS: m/z calcd for C10H16NO4NaCl3 [M + Na]+, 342.0043; found, 342.0044.  7.3.106 Preparation of Compound (4S,10S)-3.219    Under a similar procedure described in 7.3.83, (4S,10S)-3.219 (70 mg, 0.130 mmol) was prepared from 3.218 (36 mg, 0.153 mmol) and (S)-3.79 (49 mg, 0.153 mmol) as a colourless oil in a yield of 85 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→3:1). []20D +3.3° (c 1.2, CHCl3); 1H NMR (600 MHz, CDCl3)  6.85 (d, J = 6.1 Hz, 1H), 5.11 (d, J = 7.2 Hz, 1H), 4.72 (td, J = 5.1, 7.9 Hz, 1H), 4.22 (d, J = 6.1 Hz, 1H), 3.81 (s, 3H), 2.74–2.85 (m, 3H), 2.64–2.72 (m, 1H), 2.40–2.47 (m, 1H), 2.32–2.38 (m, 1H), 2.07–2.20 (m, 2H), 1.46 (s, 9H); 13C NMR (150 MHz, CDCl3)  171.7, 171.2, 155.9, 99.2, 99.0, 81.2, 53.4, 53.2, 51.3, 51.2, 51.0, 29.7, 29.1, 28.5; ESI-MS m/z: 559.2 [M + Na]+; ESI-HRMS: m/z calcd for C16H24N2O5NaCl6 [M + Na]+, 556.9714; found, 556.9713.    324  7.3.107 Preparation of (4S,10S)-3.220 (LPY20)     Under a similar procedure described in 7.3.85, (4S,10S)-3.220 (3.8 mg, 0.0073 mmol) was prepared from (4S,10R)-3.219 (60.8 mg, 0.113 mmol) as a colourless oil. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→3:2). The yield from (4S,10S)-3.225 was 60 %. []20D +50.0° (c 0.16, CHCl3); 1H NMR (600 MHz, CDCl3)  6.21 (d, J = 8.2 Hz, 1H), 5.70 (td, J = 3.6, 9.0 Hz, 1H), 5.19 (s, 1H), 4.75 (dd, J = 3.3, 5.4 Hz, 1H), 3.94 (s, 3H), 2.94–3.01 (m, 1H), 2.76–2.82 (m, 1H), 2.64–2.72 (m, 2H), 2.43–2.50 (m, 1H), 2.33–2.40 (m, 2H), 2.28 (qd, J = 3.6, 7.7 Hz, 2H), 1.91–1.99 (m, 1H), 1.17 (t, J = 7.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.6, 174.0, 171.7, 169.3, 99.5, 99.2, 94.7, 59.4, 58.3, 51.7, 51.6, 48.4, 30.2, 29.8, 26.1, 9.9; ESI-MS m/z: 539.1 [M + Na]+; ESI-HRMS: m/z calcd for C16H20N2O4Na35Cl6 [M + Na]+, 536.9453; found, 536.9441.     325  7.3.108 Preparation of (4R,10S)-3.220 (LPY21)    Under a similar procedure described in 7.3.86, (4R,10S)-3.220 (2.2 mg, 0.0042 mmol) was prepared from (4R,10S)-3.225 (8.9 mg, 0.0193 mmol) as a colourless oil in a yield of 22 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2→1:1). []20D −56.0° (c 0.25, CHCl3); 1H NMR (600 MHz, CDCl3)  6.27 (d, J = 8.7 Hz, 1H), 5.79 (td, J = 3.6, 8.7 Hz, 1H), 5.19 (s, 1H), 4.79 (m, 1H), 3.95 (s, 3H), 2.98 (ddd, J = 4.6, 11.8, 14.3 Hz, 1H), 2.75 (ddd, J = 3.6, 12.3, 14.3 Hz, 1H), 2.57–2.63 (m, 1H), 2.54 (dd, J = 6.1, 8.7 Hz, 2H), 2.46 (tt, J = 4.2, 12.7 Hz, 1H), 2.35–2.41 (m, 1H), 2.30 (qd, J = 3.8, 7.6 Hz, 2H), 2.00–2.08 (m, 1H), 1.19 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)  178.6, 174.0, 171.7, 168.9, 99.3, 98.9, 94.5, 59.5, 57.9, 51.9, 51.4, 48.9, 31.1, 29.9, 26.3, 9.9; ESI-MS m/z: 539.1 [M + Na]+; ESI-HRMS: m/z calcd for C16H20N2O4Na35Cl6 [M + Na]+, 536.9452; found, 536.9456.     326  7.3.109 Preparation of (4R,10R)-3.225 (LPY22)    Obtained in 7.3.97, crude (4R,10R)-3.225 was purified through HPLC eluting with MeCN:H2O (3:1) to yield pure compound 2.5 mg. tR = 25.3 min. []20D −14.8° (c 0.27, CHCl3); 1H NMR (600 MHz, CDCl3)  5.37 (dt, J = 3.0, 9.2 Hz, 1H), 5.21 (d, J = 8.1 Hz, 1H), 5.18 (s, 1H), 4.75 (d, J = 2.0 Hz, 1H), 3.94 (s, 3H), 2.93–3.00 (m, 1H), 2.85–2.92 (m, 1H), 2.73 (d, J = 9.2 Hz, 2H), 2.45 (br. s., 1H), 2.28–2.39 (m, 2H), 1.86–1.94 (m, 1H), 1.45 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.5, 172.4, 169.4, 155.9, 99.5, 99.3, 94.7, 80.6, 59.4, 58.3, 53.3, 51.7, 48.4, 29.6, 28.5, 26.0; ESI-MS m/z: 583.2 [M + H]+; ESI-HRMS: m/z calcd for C18H25N2O535Cl537Cl [M + H]+, 560.9865; found, 560.9860.        327  7.3.110 Preparation of (4S,10R)-3.225 (LPY23)    Obtained in 7.3.98, crude (4R,10R)-3.225 was purified through HPLC eluting with MeCN:H2O (7:3) to yield pure compound 4.6 mg. tR = 38.5 min. []20D +45.4° (c 1.3, CHCl3); 1H NMR (600 MHz, CDCl3)  5.48 (dt, J = 3.5, 8.9 Hz, 1H), 5.30 (d, J = 8.7 Hz, 1H), 5.18 (s, 1H), 4.80 (br. s., 1H), 3.94 (s, 3H), 2.95–3.03 (m, 1H), 2.78–2.86 (m, 1H), 2.56–2.63 (m, 1H), 2.54 (dd, J = 5.8, 9.4 Hz, 2H), 2.39–2.44 (m, 1H), 2.33–2.38 (m, 1H), 1.95–2.02 (m, 1H), 1.46 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.6, 172.0, 169.0, 155.8, 99.4, 98.9, 94.5, 80.4, 59.5, 57.8, 53.6, 51.5, 48.9, 30.9, 28.5, 26.3; ESI-MS m/z: 583.2 [M + H]+; ESI-HRMS: m/z calcd for C18H25N2O535Cl537Cl [M + H]+, 560.9865; found, 560.9873.        328  7.3.111 Preparation of (4R,10R)-3.232 (LPY24)    The author did not find a suitable HPLC condition for purifying crude (4R,10R)-3.232. The NMR spectra is hard to assign due to its complexity. ESI-MS m/z: 461.0 [M + H]+; ESI-HRMS: m/z calcd for C13H17N2O3Cl6 [M + H]+, 458.9370; found, 458.9372.  7.3.112 Preparation of (4S,10R)-3.232 (LPY25)    The author did not find a suitable HPLC condition for purifying crude (4S,10R)-3.232. 1H NMR (600 MHz, CDCl3)  5.14 (s, 1H), 4.29 (t, J = 5.0 Hz, 1H), 3.88 (s, 3H), 2.78 (ddd, J = 4.3, 11.4, 14.2 Hz, 1H), 2.54–2.63 (m, 1H), 2.23–2.31 (m, 1H), 2.13–2.22 (m, 1H), 1.75 (br. s., 4H);  ESI-329  MS m/z: 461.0 [M + H]+; ESI-HRMS: m/z calcd for C13H17N2O3Cl6 [M + H]+, 458.9370; found, 458.9381.  7.3.113 Preparation of (4R,10R)-3.233 (LPY26)    Under a similar procedure described in 7.3.85, (4R,10R)-3.233 (2.2 mg, 0.0040 mmol) was prepared from (4R,10R)-3.232 (6 mg, 0.013 mmol) with pivaloyl chloride as a colourless oil in a yield of 31 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (25:2→5:1). []20D −17.3° (c 0.23, CHCl3); 1H NMR (600 MHz, CDCl3)  6.41 (d, J = 7.9 Hz, 1H), 5.60 (dt, J = 3.4, 8.7 Hz, 1H), 5.18 (s, 1H), 4.76 (dd, J = 3.2, 5.1 Hz, 1H), 3.94 (s, 3H), 2.96 (ddd, J = 4.9, 11.4, 14.4 Hz, 1H), 2.76 (ddd, J = 3.5, 11.5, 14.6 Hz, 1H), 2.65–2.73 (m, 2H), 2.43–2.50 (m, 1H), 2.37–2.43 (m, 1H), 2.29–2.37 (m, 1H), 1.94–2.03 (m, 1H), 1.23 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.9, 178.5, 171.8, 169.3, 99.5, 99.2, 94.6, 59.4, 58.3, 51.9, 51.7, 48.4, 39.0, 29.9, 27.7, 26.0; ESI-MS m/z: 567.0 [M + Na]+; ESI-HRMS: m/z calcd for C18H24N2O4NaCl6 [M + Na]+, 564.9765; found, 564.9756.  330  7.3.114 Preparation of (4S,10R)-3.233 (LPY27)    Under a similar procedure described in 7.3.86, (4S,10R)-3.233 (2.4 mg, 0.0044 mmol) was prepared from (4S,10R)-3.232 (6 mg, 0.013 mmol) and pivaloyl chloride as a colourless oil in a yield of 34 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→5:2). []20D +24.2° (c 0.33, CHCl3); 1H NMR (600 MHz, CDCl3)  6.51 (d, J = 7.9 Hz, 1H), 5.75 (dt, J = 3.5, 8.3 Hz, 1H), 5.19 (s, 1H), 4.78–4.80 (m, 1H), 3.94 (s, 3H), 2.97 (ddd, J = 4.7, 11.7, 14.4 Hz, 1H), 2.72 (ddd, J = 3.4, 11.7, 14.7 Hz, 1H), 2.57–2.63 (m, 1H), 2.53–2.56 (m, 2H), 2.47 (tt, J = 4.1, 12.6 Hz, 1H), 2.34–2.41 (m, 1H), 2.01–2.08 (m, 1H), 1.25 (s, 9H); 13C NMR (150 MHz, CDCl3)  178.8, 178.6, 171.8, 168.9, 99.4, 98.9, 94.5, 59.5, 57.9, 51.9, 51.4, 48.9, 39.1, 31.0, 27.7, 26.3; ESI-MS m/z: 567.0 [M + Na]+; ESI-HRMS: m/z calcd for C18H24N2O4NaCl6 [M + Na]+, 564.9765; found, 564.9764.    331  7.3.115 Preparation of (4R,10R)-3.234 (LPY28)    Under a similar procedure described in 7.3.85, (4R,10R)-3.234 (1.3 mg, 0.0023 mmol) was prepared from (4R,10R)-3.232 (5 mg, 0.0108 mmol) and ethanesulfonyl chloride as a colourless oil in a yield of 21 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (25:2→5:1). []20D +14.2° (c 0.21, CHCl3); 1H NMR (600 MHz, CDCl3)  5.22–5.24 (m, 1H), 5.21 (s, 1H), 5.15–5.20 (m, J = 2.8, 9.5 Hz, 1H), 4.78 (dd, J = 3.0, 5.5 Hz, 1H), 3.96 (s, 3H), 3.00–3.07 (m, 2H), 2.92–3.00 (m, 2H), 2.74–2.81 (m, 1H), 2.65–2.71 (m, 1H), 2.44–2.50 (m, 1H), 2.35–2.42 (m, 1H), 2.26–2.32 (m, 1H), 1.87–1.95 (m, 1H), 1.39 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)  179.0, 171.7, 169.6, 99.1, 99.0, 94.6, 59.6, 58.5, 55.7, 51.6, 48.4, 48.0, 30.6, 26.1, 8.4; ESI-MS m/z: 574.9 [M + Na]+; ESI-HRMS: m/z calcd for C15H20N2O5NaSCl6 [M + Na]+, 572.9122; found, 572.9114.      332  7.3.116 Preparation of (4S,10R)-3.234 (LPY29)    Under a similar procedure described in 7.3.86, (4S,10R)-3.234 (1.2 mg, 0.0021 mmol) was prepared from (4S,10R)-3.232 (5 mg, 0.013 mmol) and ethanesulfonyl chloride as a colourless oil in a yield of 20 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→5:2). []20D −13.8° (c 0.36, CHCl3); 1H NMR (600 MHz, CDCl3)  5.31 (br. s., 1H), 5.23 (s, 1H), 4.85–4.89 (m, 1H), 3.98 (s, 3H), 3.01–3.10 (m, 2H), 2.92–3.00 (m, 2H), 2.59–2.65 (m, 1H), 2.57 (dd, J = 5.3, 10.0 Hz, 2H), 2.40–2.44 (m, 1H), 2.35–2.40 (m, J = 4.1 Hz, 1H), 2.07 (br. s., 1H), 1.41 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)  179.0, 171.5, 169.2, 99.0, 98.9, 94.4, 59.6, 58.0, 56.0, 51.3, 48.9, 48.1, 31.5, 26.2, 8.4; ESI-MS m/z: 574.9 [M + Na]+; ESI-HRMS: m/z calcd for C15H20N2O5NaS35Cl537Cl [M + Na]+, 574.9092; found, 574.9084.      333  7.3.117 Preparation of (4R,10R)-3.235 (LPY32)    Obtained in 7.3.97, crude (4R,10R)-3.235 was purified through HPLC eluting with MeCN:H2O (1:1) to yield pure compound 2.6 mg. tR = 47.7 min. []20D −39.3° (c 0.33, CHCl3); 1H NMR (600 MHz, methanol-d4)  5.27 (d, J = 8.8 Hz, 1H), 4.80 (dd, J = 2.9, 5.0 Hz, 1H), 3.33 (br. s., 2H), 2.84–2.95 (m, 2H), 2.69–2.82 (m, 2H), 2.47–2.54 (m, 1H), 2.31–2.38 (m, 1H), 2.22–2.29 (m, 1H), 1.94–2.02 (m, 1H), 1.44 (s, 9H); 13C NMR (150 MHz, methanol-d4)  178.5, 172.3, 171.5, 156.9, 99.3, 99.2, 93.2, 79.3, 58.2, 53.2, 51.7, 27.9, 27.4, 25.1, 22.8; ESI-MS m/z: 568.9 [M + Na]+; ESI-HRMS: m/z calcd for C17H22N2O5NaCl6 [M + Na]+, 566.9558; found, 566.9550.         334  7.3.118 Preparation of (4S,10R)-3.235 (LPY33)    Obtained in 7.3.98, crude (4R,10R)-3.225 was purified through HPLC eluting with MeCN:H2O (1:1) to yield pure compound 4.4 mg. tR = 36.9 min. []20D +49.9° (c 0.44, CHCl3); 1H NMR (600 MHz, methanol-d4)  5.40 (d, J = 5.9 Hz, 1H), 4.86–4.88 (m, 1H), 3.33–3.36 (m, 2H), 2.89–2.97 (m, 1H), 2.83 (m, 1H), 2.62–2.68 (m, 1H), 2.56–2.62 (m, 1H), 2.48–2.54 (m, 1H), 2.36–2.41 (m, 1H), 2.32 (br. s., 1H), 2.04–2.13 (m, 1H), 1.45 (s, 9H); 13C NMR (150 MHz, methanol-d4)  180.1, 173.0, 172.7, 158.3, 100.7, 100.4, 92.6, 80.9, 59.3, 54.9, 52.9, 49.9, 31.3, 28.8, 26.9; ESI-MS m/z: 568.9 [M + Na]+; ESI-HRMS: m/z calcd for C17H22N2O5NaCl6 [M + Na]+, 566.9558; found, 566.9549.        335  7.3.119 Preparation of (4S,10R)-3.219 (LPY34)    Under a similar procedure described in 7.3.83, (4R,10R)-3.219 (411 mg, 0.765 mmol) was prepared from (S)-3.218 (197 mg, 0.842 mmol) and (S)-3.79 (270 mg, 0.842 mmol) as a colourless oil in a yield of 91 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→3:1). []20D +33.3° (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3)  6.96 (d, J = 7.2 Hz, 1H), 5.22 (br. s., 1H), 4.72 (td, J = 5.1, 7.9 Hz, 1H), 4.26 (d, J = 6.1 Hz, 1H), 3.81 (s, 3H), 2.75–2.85 (m, 3H), 2.65–2.72 (m, 1H), 2.38–2.46 (m, 1H), 2.30–2.37 (m, 1H), 2.07–2.20 (m, 2H), 1.50 (s, 9H); 13C NMR (150 MHz, CDCl3)  171.6, 171.3, 155.9, 99.2, 99.0, 81.1, 53.3, 53.1, 51.2, 51.1, 51.1, 29.6, 29.2, 28.5.        336  7.4 Experimental for Chapter 4  7.4.1 Preparation of 4.2 (LPY01)    3.145 (20 mg, 0.0454 mmol) was dissolved in TFA/DCM (1 mL, 25 %) and stirred at 0 °C for 2 h. The solvent was evaporated in vacuo. After addition of saturated NaHCO3 (5 mL), the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:1) to afford 4.2 (9.3 mg, 0.0274 mmol) as a colourless oil in a yield of 60 %. 1H NMR (600 MHz, CDCl3)  6.28 (d, J = 7.1 Hz, 1H), 5.73 – 5.75 (m, 1H), 5.07 (s, 1H), 4.63 – 4.64 (m, 1H), 4.39 – 4.49 (m, 2H), 3.88 (s, 3H), 2.27 (q, J = 7.3 Hz, 3H), 2.00 – 2.10 (m, 1H), 1.90 – 2.00 (m, 1H), 1.78–1.88 (m, 2H), 1.58–1.62 (m, 2H), 1.28–1.32 (m, 1H), 1.17 (t, J = 7.6 Hz, 3H), 0.9.1 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.0, 174.1, 172.5, 169.5, 93.4, 67.5, 59.0, 58.5, 52.3, 39.0, 30.0, 29.8, 24.6, 24.3, 23.9, 22.7, 9.9.  337  7.4.2 Preparation of Compound 4.3    3.114 (16 mg, 0.023 mmol) was dissolved in TFA/DCM (1 mL, 25 %) and stirred at 0 °C for 2 h. The solvent was evaporated in vacuo. The crude residue was used directly without purification.  To a stirred solution of crude residue in THF (2 mL) at 0 °C was added NaH (1.3 mg, 0.054 mmol). After dropwise addition of propargyl bromide (0.1 mL, 0.9 mmol, ~80 % in toluene) in THF (2 mL), the reaction mixture was stirred at room temperature overnight then poured onto a mixture of EtOAc (5 mL) and cold water (5 mL). The separated aqueous layer was extracted with EtOAc (2×5 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (4:1) to afford 4.3 7.8 mg as a yellowish oil in a yield of 53 %. []20D −25.6° (c 0.78, CHCl3); 1H NMR (600 MHz, C6D6)  7.80 (ddd, J = 1.5, 7.4, 12.5 Hz, 4H), 7.21–7.31 (m, 6H), 6.42–6.51 (m, 1H), 6.06 (d, J = 7.1 Hz, 1H), 4.81 (s, 1H), 3.74–3.79 (m, 1H), 3.68–3.74 (m, 1H), 3.45 (dd, J = 2.5, 16.3 Hz, 1H), 2.84 (s, 3H), 2.46–2.53 (m, 1H), 2.42 (dd, J = 4.6, 14.3 Hz, 1H), 2.37 (dd, J = 2.5, 16.9 Hz, 1H), 1.99–2.09 (m, 1H), 1.83 (q, J = 7.5 Hz, 2H), 1.73–1.80 (m, 1H), 1.68 (t, J = 2.3 Hz, 1H), 1.38–1.50 (m, 2H), 1.36 338  (dd, J = 6.6, 14.3 Hz, 1H), 1.19 (s, 9H), 1.02 (t, J = 7.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.69 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, C6D6)  179.2, 173.9, 172.7, 169.9, 136.4, 136.4, 134.9, 134.7, 130.2, 130.2, 95.6, 77.3, 73.1, 69.3, 64.3, 58.0, 53.7, 41.7, 37.1, 31.3, 30.0, 29.9, 27.5, 27.1, 25.0, 24.6, 24.0, 19.9, 14.7, 10.3.  7.4.3 Preparation of 4.4 (LPY03)    To a stirred solution of 4.3 (7.0 mg, 0.011 mmol) in THF (1 mL) in a Nalgene TM bottle at 0 °C was added HF/pyridine complex (0.1 mL, 2.0 mmol, ~70 % HF in pyridine) in pyridine (0.1 mL). The reaction mixture was stirred at room temperature for 5 h then cooled to 0 °C again. After carefully neutralizing with saturated NaHCO3, the aqueous layer was extracted with DCM (3×5 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:3) to afford 4.4 (2.6 mg, 0.0068 mmol) as a colourless oil in a yield of 60 %. []20D −65.3° (c 0.26, CHCl3); 1H NMR (600 MHz, C6D6)  6.36–6.47 (m, J = 1.5 Hz, 1H), 5.98 (d, J = 8.7 Hz, 1H), 4.76 (s, 1H), 3.93–4.04 (m, 1H), 3.72 (td, J = 5.0, 10.8 Hz, 1H), 3.39 (dd, J = 2.8, 16.6 Hz, 1H), 2.84 (s, 3H), 2.39 –2.45 (m, 1H), 339  2.30–2.38 (m, 2H), 1.85 (q, J = 7.3 Hz, 2H), 1.74–1.82 (m, 1H), 1.71 (t, J = 2.5 Hz, 1H), 1.67 – 1.73 (m, 1H), 1.51–1.59 (m, 1H), 1.28–1.38 (m, 2H), 1.02 (t, J = 7.6 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H), 0.64 (d, J = 6.6 Hz, 3H); 13C NMR (150 MHz, C6D6)  179.6, 174.1, 173.0, 170.6, 95.5, 77.1, 73.2, 69.5, 61.1, 58.1, 52.3, 41.7, 30.4, 29.9, 29.3, 27.0, 24.9, 24.5, 24.0, 10.3  7.4.4 Preparation of 4.5 (LPY02)    To a stirred solution of deprotected coupling intermediate 4.4 (2.0 mg, 0.00529 mmol) in carbon tetrachloride (1 mL) at room temperature was added triphenyl phosphine (2.77 mg, 0.01 mmol). The mixture was refluxed overnight. After addition of hexanes (10 mL), the white precipitate formed was filtered off then the filtrate was dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (3:2) to afford monochlorinated coupling intermediate (2.0 mg, 0.005 mmol) as a white solid in a yield of 95 %. []20D +22.2° (c 0.18, CHCl3); 1H NMR (600 MHz, C6D6)  6.38–6.43 (m, 1H), 6.03 (d, J = 7.1 Hz, 1H), 4.78 (s, 1H), 3.38–3.44 (m, 1H), 3.27–3.36 (m, 2H), 2.81 (s, 3H), 2.31–2.38 (m, 2H), 2.25–2.31 (m, 1H), 1.96–2.05 (m, 1H), 1.82–1.87 (m, 1H), 1.80 (q, J = 7.3 Hz, 2H), 1.66–1.74 (m, 1H), 1.63 (t, J = 2.5 Hz, 1H), 1.35–1.42 (m, 1H), 1.29–1.35 (m, 1H), 1.01 (t, J = 7.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 340  3H), 0.66 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, C6D6)  179.3, 173.4, 172.7, 169.9, 95.5, 77.2, 73.0, 69.2, 58.0, 53.0, 45.1, 41.6, 32.1, 29.9, 29.7, 27.0, 25.0, 24.5, 23.9, 10.3; ESI-MS m/z: 419.4 [M + Na]+.  7.4.5 Preparation of 4.6 (LPY16)    Under a similar procedure described in 7.3.85, 4.6 (1.6 mg, 0.0035 mmol) was prepared from free amine precursor (6.0 mg, 0.0161 mmol) and pentynoyl chloride as a colourless oil in a yield of 21 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→2:1); []20D +50.0° (c 0.14, CHCl3); 1H NMR (600 MHz, CDCl3)  6.50 (d, J = 8.1 Hz, 1H), 5.80 (dt, J = 3.3, 8.5 Hz, 1H), 5.10 (s, 1H), 4.66 (dd, J = 3.07, 6.66 Hz, 1H), 3.89 (s, 3H), 2.97–3.04 (m, 1H), 2.75–2.83 (m, 1H), 2.56–2.64 (m, 1H), 2.50–2.55 (m, 1H), 2.44–2.50 (m, 3H), 2.02–2.05 (m, 1H), 1.96–2.02 (m, 1H), 1.80–1.87 (m, 1H), 1.72–1.80 (m, 2H), 0.94 (d, J = 6.1 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)  181.2, 171.0, 170.9, 169.5, 99.5, 93.5, 83.0, 69.8, 59.1, 58.6, 52.0, 51.4, 39.3, 35.6, 31.0, 24.5, 24.0, 22.8, 15.2. ESI-MS m/z: 475.2 [M + Na]+; ESI-HRMS: m/z calcd for C19H25N2O4NaCl3 [M + Na]+, 473.0778; found, 473.0781. 341  7.4.6 Preparation of 4.9 (LPY19)    Under a similar procedure described in 7.3.85, 4.9 (0.9 mg, 0.0024 mmol) was prepared from 3.47 (2.9 mg, 0.0103 mmol) and pentynoyl chloride as a colourless oil in a yield of 23 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→2:1); 1H NMR (600 MHz, CDCl3)  5.77–5.83 (m, 1H), 5.05 (s, 1H), 4.63 (dd, J = 3.2, 6.6 Hz, 1H), 3.87 (s, 3H), 2.50–2.57 (m, 1H), 2.43–2.49 (m, 2H), 1.99 (t, J = 2.6 Hz, 1H), 1.77–1.82 (m, 1H), 1.72–1.77 (m, 2H), 1.65–1.71 (m, 2H), 1.42–1.48 (m, 1H), 1.07 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.89 (d, J = 6.4 Hz, 4H); 13C NMR (150 MHz, CDCl3)  181.0, 172.9, 170.8, 169.5, 93.5, 83.2, 69.4, 58.9, 58.6, 52.1, 42.1, 39.3, 35.6, 25.4, 24.4, 24.0, 23.9, 22.8, 21.4, 15.0; ESI-MS m/z: 385.4 [M + Na]+; ESI-HRMS: m/z calcd for C20H31N2O4 [M + H]+, 363.2284; found, 363.2286.     342  7.4.7 Preparation of 4.7 (LPY30)    Under a similar procedure described in 7.3.85, 4.7 (2.3 mg, 0.0042 mmol) was prepared from (4S,10R)-3.232 (5 mg, 0.0108 mmol) and pentynoyl chloride as a colourless oil in a yield of 39 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (10:1→5:1). []20D −30.0° (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3)  6.41 (d, J = 8.6 Hz, 1H), 5.74 (dt, J = 3.2, 8.9 Hz, 1H), 5.19 (s, 1H), 4.70–4.79 (m, 1H), 3.95 (s, 3H), 2.95–3.02 (m, 1H), 2.81–2.88 (m, 1H), 2.64–2.72 (m, 2H), 2.55–2.63 (m, 1H), 2.51–2.55 (m, 1H), 2.44–2.51 (m, 3H), 2.33–2.42 (m, 2H), 2.03 (t, J = 2.5 Hz, 1H), 1.93–2.00 (m, 1H); 13C NMR (150 MHz, CDCl3)  178.6, 171.4, 171.1, 169.2, 99.5, 99.2, 94.6, 83.0, 69.9, 59.4, 58.4, 51.8, 51.6, 48.5, 35.6, 30.2, 26.2, 15.1; ESI-MS m/z: 562.9 [M + Na]+; ESI-HRMS: m/z calcd for C18H20N2O4Na35Cl537Cl [M + Na]+, 562.9422; found, 562.9424.     343  7.4.8 Preparation of 4.8 (LPY31)    Under a similar procedure described in 7.3.85, 4.8 (2.2 mg, 0.004 mmol) was prepared from (4S,10R)-3.232 (5 mg, 0.0108 mmol) and pentynoyl chloride as a colourless oil in a yield of 37 %. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (5:1→2:1). []20D +25.9° (c 0.27, CHCl3); 1H NMR (600 MHz, CDCl3)  6.47 (d, J = 8.3 Hz, 1H), 5.83 (dt, J = 3.6, 8.4 Hz, 1H), 5.20 (s, 1H), 4.80 (dd, J = 3.7, 4.8 Hz, 1H), 3.95 (s, 3H), 2.97–3.03 (m, 1H), 2.75–2.82 (m, 1H), 2.57–2.64 (m, 2H), 2.52–2.57 (m, 2H), 2.45–2.52 (m, 3H), 2.35–2.42 (m, 1H), 2.04 (t, J = 2.57 Hz, 1H), 2.01–2.09 (m, 1H); 13C NMR (150 MHz, CDCl3)  178.7, 171.4, 171.0, 168.9, 99.3, 98.9, 94.5, 82.9, 69.9, 59.5, 57.9, 52.1, 51.3, 48.9, 35.6, 31.0, 26.3, 15.2; ESI-MS m/z: 562.9 [M + Na]+; ESI-HRMS: m/z calcd for C18H20N2O4Na35Cl537Cl [M + Na]+, 562.9422; found, 562.9427.     344  7.5 Experimental for Chapter 5  7.5.1 Preparation of 5.1 (latonduine A)    To a stirred solution of the alcohol 5.14 (194 mg, 0.598 mmol) in THF (10 mL) was added Dess-Martin periodinane (911 mg, 2.14 mmol). The reaction mixture was stirred at room temperature for 15 min. After filtration of the white solid, the filtrate was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (2:1) to afford ketone 5.15 (97 mg, 0.301 mmol) as a white solid in a yield of 50 %. 1H NMR (400 MHz, acetone-d6)  11.88 (br. s, 1H), 7.14 (br. s, 1H), 3.91 (d, J = 4.8 Hz, 1H), 3.70 (s, 2H); 13C NMR (100 MHz, acetone-d6) 205.9, 164.0, 125.7, 121.1, 106.9, 100.7, 52.1, 41.5.  To a stirred solution of ketone 5.15 (97 mg, 0.301 mmol) in neat trimethyl orthoformate (40 mL) at room temperature was added TFA (0.8 mL). The reaction mixture was refluxed for 18 h. The solvent was evaporated in vacuo. To a redissolved solution of the crude residue in THF/H2O (24 mL, 5/1) were added K2CO3 (70 mg, 0.507 mmol) and guanidine·HCl (50 mg, 0.523 mmol). The reaction mixture was refluxed for 19 h. The cooled reaction mixture was extracted with EtOAc 345  (3×30 mL). The combined organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with hexanes/EtOAc (6:1→100 % EtOAc) to afford latonduine A (5.1) (10 mg, 0.0268 mmol) as a white solid in a yield of 8.9 %. 1H NMR (400 MHz, DMSO-d6)  13.10 (s, 1H), 8.76 (s, 1H), 8.14 (t, J = 5.1 Hz, 1H), 6.88 (br. s, 2H), 3.89 (d, J = 5.1 Hz, 2H).  7.5.2 Preparation of 5.17    To a stirred solution of latonduine A (2 mg, 0.00536 mmol) in DMF (1 mL) were added K2CO3 (9 mg, 0.065 mmol) and N-iodoacetyl-N-biotinylhexylenediamine (6.6 mg, 0.013 mmol). The reaction mixture was stirred at room temperature overnight and then dried in vacuo. The crude residue was purified by silica gel flash chromatography eluting with DCM/methanol (9:1) to afford biotinylated latonduine A 5.17 (1.3 mg, 0.00172 mmol) as a white solid in a yield of 32 %. 1H NMR (400 MHz, DMSO-d6)  8.71 (s, 1H), 8.33 (t, J = 5.4 Hz, 1H), 8.07 (t, J = 5.63 Hz, 1H), 7.72 (t, J = 5.4 Hz, 1H), 6.95 (s, 2H), 6.41 (s, 1H), 6 .35 (s, 1H), 4.25–4.35 (m, 1H), 4.05–4.16 (m, 2H), 3.75–3.79 (m, 1H), 3.03–3.11 (m, 2H), 2.96–3.03 (m, 2H), 2.81 (dd, J = 5.12, 12.4 346  Hz, 1H), 2.59 (s, 1H), 2.55 (s, 1H), 2.03 (t, J = 7.3 Hz, 2H), 1.43–1.54 (m, 3H), 1.32–1.43 (m, 4H), 1.19–1.31 (m, 6H); ESI-MS: m/z: 778.3 [M+Na]+.                      347  Bibliography  1. Tavakoli, I. Inhibition of castration resistant prostate cancer by sintokamide A: An antagonist of the amino-terminus of the androgen receptor. The University of British Columbia, 2012. 2. Carlile, G. W.; Keyzers, R. A.; Teske, K. A.; Robert, R.; Williams, D. E.; Linington, R. G.; Gray, C. A.; Centko, R. 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Pat., 200410066112. 368  Appendices  Appendix A  Crystal Data for (S,S)-3.217  Empirical Formula C9H15N2OSCl3  Formula Weight 305.64 Crystal Colour, Habit colourless, plate Crystal Dimensions 0.03 x 0.18 x 0.25 mm Crystal System monoclinic Lattice Type Primitive Lattice Parameters a = 5.7834(2) Å  b = 9.6140(4) Å  c = 12.3417(4) Å   = 90o   = 102.028(2)o   = 90o  V = 671.15(4) Å3 Space Group P 21  (#4) Z value 2 Dcalc 1.512 g/cm3 F000 316.00 (Mo-K) 8.20 cm-1 369  Intensity Measurements   Diffractometer Bruker APEX DUO  Radiation Mo-K ( = 0.71073 Å) Data Images 2093 exposures @ 2 seconds Detector Position 40.04 mm 2max 60.3o  No. of Reflections Measured Total: 16564  Unique: 3952 (Rint = 0.032; Friedels not merged) Corrections Absorption (Tmin = 0.803, Tmax= 0.976)  Lorentz-polarization 370  Structure Solution and Refinement   Structure Solution Direct Methods (SIR97) Refinement Full-matrix least-squares on F2 Function Minimized  w (Fo2–Fc2)2  Least Squares Weights w=1/(2(Fo2)+(0.0254P) 2+ 0.0861P) Anomalous Dispersion All non-hydrogen atoms No. Observations (I>0.00(I)) 3952 No. Variables 152 Reflection/Parameter Ratio 26.0 Residuals (refined on F2, all data): R1; wR2 0.020; 0.048 Goodness of Fit Indicator 1.04 No. Observations (I>2.00(I)) 3878 Residuals (calculated on F): R1; wR2 0.019; 0.048 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.34 e-/Å3 Minimum peak in Final Diff. Map -0.20 e-/Å3      371            Appendix B  1H NMR and 13C NMR for Selected compounds in Chapters 2, 3, 4, and 5             372   Figure B.1  1H spectrum of 2.18 (C12) recorded in DMSO-d6 at 600 MHz   Figure B.2 1H spectrum of 2.14 (C11) recorded in DMSO-d6 at 300 MHz 373   Figure B.3 1H spectrum of 2.20 (C22) recorded in DMSO-d6 at 600 MHz   Figure B.4 1H spectrum of 2.15 (C21) recorded in DMSO-d6 at 300 MHz 374    Figure B.5 1H and 13C NMR spectra of 3.32 recorded in CDCl3 at 400 MHz and 100 MHz, respectively.  375    Figure B.6 1H and 13C NMR spectra of 3.45 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 376    Figure B.7 1H and 13C NMR spectra of 3.125 recorded in CDCl3 at 400 MHz and 100 MHz, respectively 377    Figure B.8 1H and 13C NMR spectra of 3.126 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 378    Figure B.9 1H and 13C NMR spectra of 3.144 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 379    Figure B.10 1H and 13C NMR spectra of 3.145 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 380    Figure B.11 1H and 13C NMR spectra of 3.146 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 381    Figure B.12 1H and 13C NMR spectra of 3.60 (LPY00) recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 382    Figure B.13 1H and 13C NMR spectra of 3.127 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 383    Figure B.14 1H and 13C NMR spectra of 3.128 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 384    Figure B.15 1H and 13C NMR spectra of 3.147 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 385    Figure B.16 1H and 13C NMR spectra of 3.148 (LPY04) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 386    Figure B.17 1H and 13C NMR spectra of 3.149 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 387    Figure B.18 1H and 13C NMR spectra of 3.150 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 388    Figure B.19 1H and 13C NMR spectra of 3.168 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 389    Figure B.20 1H and 13C NMR spectra of 3.169 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 390    Figure B.21 1H and 13C NMR spectra of 3.170 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 391    Figure B.22 1H and 13C NMR spectra of 3.151 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 392    Figure B.23 1H and 13C NMR spectra of 3.180 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 393     Figure B.24 1H and 13C NMR spectra of 3.181 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 394    Figure B.25 1H and 13C NMR spectra of 3.182 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 395    Figure B.26 1H and 13C NMR spectra of 3.183 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 396    Figure B.27 1H and 13C NMR spectra of 3.184 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 397    Figure B.28 1H and 13C NMR spectra of 3.185 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 398    Figure B.29 1H and 13C NMR spectra of 3.186 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 399    Figure B.30 1H and 13C NMR spectra of 3.205 recorded in CDCl3 at 600 MHz and 150 MHz, respectively. 400    Figure B.31 1H and 13C NMR spectra of (4R,10R)-3.16 (NCSTD1) recorded in CDCl3 at 600 MHz and 150 MHz, respectively. 401    Figure B.32 1H and 13C NMR spectra of (4S,10R)-3.16 (NCSTD2) recorded in CDCl3 at 600 MHz and 150 MHz, respectively. 402    Figure B.33 1H and 13C NMR spectra of (S)-3.208 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 403    Figure B.34 1H and 13C NMR spectra of (R)-3.221 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 404    Figure B.35 1H and 13C NMR spectra of (R)-3.73 recorded in CDCl3 at 300 MHz and 75 MHz, respectively. 405    Figure B.36 1H and 13C NMR spectra of (4S,10R)-3.209 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 406    Figure B.37 1H and 13C NMR spectra of (4R,10R)-3.209 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 407    Figure B.38 1H and 13C NMR spectra of (4S,10R)-3.212 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 408    Figure B.39 1H and 13C NMR spectra of (4R,10R)-3.210 (LPY08) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 409    Figure B.40 1H and 13C NMR spectra of (4S,10R)-3.210 (LPY09) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 410    Figure B.41 1H and 13C NMR spectra of (S)-3.218 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 411    Figure B.42 1H and 13C NMR spectra of 3.223 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 412    Figure B.43 1H and 13C NMR spectra of 7.1 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 413    Figure B.44 1H and 13C NMR spectra of (S)-3.79 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 414    Figure B.45 1H and 13C NMR spectra of (S)-3.218 recorded in CDCl3 at 400 MHz and 100 MHz, respectively. 415    Figure B.46 1H and 13C NMR spectra of (4S,10R)-3.219 (LPY35) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 416    Figure B.47 1H and 13C NMR spectra of (4R,10R)-3.220 (LPY10) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 417    Figure B.48 1H and 13C NMR spectra of (4S,10R)-3.220 (LPY11) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 418    Figure B.49 1H and 13C NMR spectra of (4S,10R)-3.226 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 419    Figure B.50 1H and 13C NMR spectra of (4R,10R)-3.228 (LPY12) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 420    Figure B.51 1H and 13C NMR spectra of (4S,10R)-3.228 (LPY13) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 421    Figure B.52 1H and 13C NMR spectra of (4S,10R)-3.229 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 422    Figure B.53 1H and 13C NMR spectra of (4R,10R)-3.231 (LPY17) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 423    Figure B.54 1H and 13C NMR spectra of (4S,10R)-3.231 (LPY18) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 424    Figure B.55 1H and 13C NMR spectra of (4S,10S)-3.129 recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 425    Figure B.56 1H and 13C NMR spectra of (4S,10S)-3.220 (LPY20) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 426    Figure B.57 1H and 13C NMR spectra of (4R,10S)-3.220 (LPY21) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 427    Figure B.58 1H and 13C NMR spectra of (4R,10R)-3.225 (LPY22) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 428    Figure B.59 1H and 13C NMR spectra of (4S,10R)-3.225 (LPY23) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 429    Figure B.60 1H and 13C NMR spectra of (4R,10R)-3.232 (LPY24) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 430    Figure B.61 1H and 13C NMR spectra of (4S,10R)-3.232 (LPY25) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 431    Figure B.62 1H and 13C NMR spectra of (4R,10R)-3.233 (LPY26) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 432    Figure B.63 1H and 13C NMR spectra of (4S,10R)-3.233 (LPY27) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 433    Figure B.64 1H and 13C NMR spectra of (4R,10R)-3.234 (LPY28) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 434    Figure B.65 1H and 13C NMR spectra of (4S,10R)-3.234 (LPY29) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 435    Figure B.66 1H and 13C NMR spectra of (4R,10R)-3.235 (LPY32) recorded in methanol-d4 at 600 MHz and 125 MHz, respectively. 436    Figure B.67 1H and 13C NMR spectra of (4S,10R)-3.235 (LPY33) recorded in methanol-d4 at 600 MHz and 125 MHz, respectively. 437    Figure B.68 1H and 13C NMR spectra of (4R,10R)-3.129 (LPY34) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 438    Figure B.69 1H and 13C NMR spectra of 4.4 (LPY03) recorded in C6D6 at 600 MHz and 125 MHz, respectively. 439    Figure B.70 1H and 13C NMR spectra of 4.5 (LPY02) recorded in C6D6 at 600 MHz and 125 MHz, respectively. 440    Figure B.71 1H and 13C NMR spectra of 4.6 (LPY16) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 441    Figure B.72 1H and 13C NMR spectra of 4.7 (LPY30) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 442    Figure B.73 1H and 13C NMR spectra of 4.8 (LPY31) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 443    Figure B.74 1H and 13C NMR spectra of 4.9 (LPY19) recorded in CDCl3 at 600 MHz and 125 MHz, respectively. 444   Figure B.75 1H spectrum of 5.17 recorded in DMSO-d6 at 400 MHz            

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