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Biologically active secondary metabolites isolated from marine and terrestrial sources Cinel, Bruno 2001

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BIOLOGICALLY ACTIVE SECONDARY METABOLITES ISOLATED FROM MARINE AND TERRESTRIAL SOURCES by  BRUNO CINEL B . S c , Simon Fraser University, 1994  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Chemistry  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A October 2001 © Bruno Cinel, 2001  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his or her  representatives.  It is understood  that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  DE-6  (2/88)  OCT  T  /I  /  2.0^  I  11  ABSTRACT A series of new and known secondary metabolites were isolated from marine and terrestrial sources guided by two newly developed, cell-based assays. antimitotic properties  of a crude extract  from the  Investigations into the  Caribbean octocoral Erythropodium  caribaeorum resulted in the isolation and identification of the known antimitotic agent eleutherobin (61) and six novel structural analogues, 69-74. E. caribaeorum proved to be a new and abundant source of eleutherobin (61), whose pre-clinical development had been impeded by its scarcity, and the structural variations of the new diterpenoids offered key insights into proposed pharmacophore models for microtubule-stabilizing compounds.  In addition, single  crystal X-ray diffraction analysis and N O E difference experiments provided the first solid-state and solution conformations for eleutherobin (61) and may aid in the development of new models for microtubule stabilization.  61  R =Ac;R  69  Rj = A c ; R = H ; R = H ; A  70  R, = H ; R = H ; R = Me; A  71  Rj = H ; R = A c ; R = M e ; A ' ' (E)  1  OH  = H ; R = Me;A '' '(£) 2  2  3  3  2  (E)  2 , 3  3  2  (E)  2 , 3  3  2  2  3  73  R=  74  R = Ac  I  W ^  O  H  OAc  3  ,2',3'  72 R = A c ; R = H ; R = M e ; A ^ (Z) {  2  3  The crude extract from a marine sponge, Stylissa flabelliformis, exhibited potent activity in a new bioassay for G 2 cell cycle checkpoint inhibitors. Bioassay-guided fractionation of this active  extract  resulted  in  the  isolation  and  identification  of  the  natural  product  iii debromohymenialdisine (91) and three related alkaloids (85, 94, 103).  These compounds were  the first G 2 checkpoint inhibitors to be found by a rational screen and were structurally distinct from previously reported G 2 checkpoint inhibitors. In addition, debromohymenialdisine  (91)  was found to specifically act on the protein kinases C h k l and Chk2, thus providing a new biochemical tool to probe the molecular basis of G 2 checkpoint inhibition.  91  85  103  94  A s a result of a large-scale screen of natural extracts for G 2 checkpoint inhibitors, a series of a-pyrones from the Taiwanese tree Cryptocarya concinna were found to exhibit potent inhibitory activity. Bioassay-guided purification resulted in the isolation and identification of the natural product cryptofolione (130)  and three related polyketide lactones (148,  131,  138).  Synthetic modifications on these metabolites yielded four additional analogues and led to mode of action and structure-activity relationship studies. The G 2 checkpoint inhibitors isolated from C. concinna were structurally different from previously known inhibitors and appear to act by a novel mechanism of action.  131  138  iv  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  viii  List of Figures  ix  List of Schemes List of Abbreviations  Acknowledgements  xvi xvii  xxi  CHAPTER 1: General Introduction 1.1. Natural Products Chemistry  1  1.2. Marine Natural Products Chemistry  4  1.3. Novel Anticancer Screens  11  1.3.1. The Cell Cycle  11  1.3.2. Antimitotic Agents  14  1.3.3. The Antimitotic Bioassay  16  1.3.4. Cell Cycle Checkpoints  19  1.3.5. The G2 Checkpoint Inhibition Bioassay  21  1.3.6. Biochemical Tools and Chemical Genetics  24  1.4. Research Summary  27  References  29  CHAPTER 2: Antimitotic Diterpenoids from the Gorgonian Erythropodium caribaeorum 2.1. Introduction  36  2.1.1. Introduction to Coelenterates and Gorgonians  36  2.1.2. Review of Diterpenoids from Octocorals  38  V  2.1.3. Review of Known Metabolites from Erythrop odium caribaeorum 2.1.4. Review of Microtubule-Stabilizing Antimitotic Agents 2.2. Results and Discussion  43 46 50  2.2.1. Isolation of Antimitotic Compounds  50  2.2.2. Structure Elucidation of Antimitotic Diterpenoids  54  2.2.2.1. Desmethyleleutherobin  55  2.2.2.2. Eleutherobin  67  2.2.2.3. Desacetyleleutherobin  72  2.2.2.4. Isoeleutherobin A  77  2.2.2.5. Z-Eleutherobin  81  2.2.2.6. Caribaeoside  85  2.2.2.7. Caribaeolin  91  2.2.2.8. Diacetyleleutherobin  96  2.2.2.9. Known Compounds  102  2.2.3. Biological Activity  104  2.2.4. Solid-State and Solution Conformations  110  2.2.5. Additional Results  123  2.2.6. Future Studies  127  2.3. Conclusions  128  References  129  CHAPTER 3: G2 Cell Cycle Checkpoint Inhibitors from Stylissa flabelliformis 3.1. Introduction  135  3.1.1. Introduction to Marine Sponges  135  3.1.2. Review of Oroidin Alkaloids from Marine Sponges  138  vi  3.1.3. Review of G2 Checkpoint Inhibitors 3.2. Results and Discussion  143 145  3.2.1. Isolation of G2 Checkpoint Inhibitors  145  3.2.2. Structure Elucidation of G2 Checkpoint Inhibitors  148  3.2.2.1. Debromohymenialdisine  148  3.2.2.2. Hymenialdisine  152  3.2.2.3. Z-Debromoaxinohydantoin  155  3.2.2.4. Aldisin  158  3.2.3. Biological Activity  161  3.3. Conclusions  165  References  166  CHAPTER 4: G2 Cell Cycle Checkpoint Inhibitors from Cryptocarya concinna 4.1. Introduction  170  4.1.1. Review of Plant-Derived Compounds  170  4.1.2. Review of Cryptocarya Phytochemistry  173  4.2. Results and Discussion  177  4.2.1. Isolation of G2 Checkpoint Inhibitors  177  4.2.2. Structure Elucidation of G2 Checkpoint Inhibitors  179  4.2.2.1. ^-Cryptofolione  180  4.2.2.2. Z-Cryptofolione  185  4.2.2.3. Cryptofolione ketone  189  4.2.2.4. Compound 138  193  4.2.3. Synthetic Derivatives of ZJ-Cryptofolione 4.2.3.1. Cryptofolione acetonide  197 197  vii  4.2.3.2. Diacetylcryptofolione  '  201  4.2.3.3. p-mercaptoethanol adduct  205  4.2.3.4. Compound 152  209  4.2.4. Biological Activity  213  4.3. Conclusions  215  References  216  EXPERIMENTAL APPENDIX  218  225  viii  LIST OF TABLES Page  Table 2.1. N M R data for desmethyleleutherobin (69) recorded in D M S O - J 6 .  61  Table 2.2. N M R data for eleutherobin (61) recorded in DMSO-c/6.  69  Table 2.3. N M R data for desacetyleleutherobin (70) recorded in DMSO-c/6.  74  Table 2.4. N M R data for isoeleutherobin A (71) recorded in D M S O - J 6 .  79  Table 2.5. N M R data for Z-eleutherobin (72) recorded in DMSO-t/6.  83  Table 2.6. N M R data for caribaeoside (73) recorded in D M S O - J 6 .  ' 88  Table 2.7. N M R data for caribaeolin (74) recorded in D M S O - J 6 .  93  Table 2.8. N M R data for diacetyleleutherobin (75) recorded in DMSO-c/6.  98  Table 2.9. Summary of antimitotic activity of the compounds isolated from E. caribaeorum. Table 2.10.  107  Difference N O E and R O E S Y data for eleutherobin (61) in C D C 1 andDMSO-e?6.  3  112  Table 3.1. G 2 checkpoint inhibition by debromohymenialdisine (91) and related compounds.  162  Table 4.1. Summary o f a-pyrones isolated from various Cryptocarya species.  175  Table 4.2. N M R data for £-cryptofolione (130) recorded in C D C 1 .  181  Table 4.3. N M R data for Z-cryptofolione (148) recorded in C D C 1 .  186  3  3  Table 4.4. N M R data for cryptofolione ketone (131) recorded in C D C 1 .  190  Table 4.5. N M R data for compound 138 recorded in C D C 1 .  194  3  3  Table 4.6. N M R data for cryptofolione acetonide (149) recorded in C D C 1 .  198  Table 4.7. N M R data for diacetylcryptofolione (150) recorded in C D C 1 .  202  3  3  Table 4.8. N M R data for P-mercaptoethanol adduct (151) recorded in C D C 1 .  206  Table 4.9. N M R data for compound 152 recorded in C D C 1 .  210  3  3  ix  LIST OF FIGURES Page  Figure 1.1. Distribution of marine natural products reported in 1999 according to the phylum of the source organism.  10  Figure 1.2. A n overview of the eukaryotic cell cycle.  12  Figure 1.3. Fluorescent micrographs depicting cells in various stages of mitosis.  13  Figure 1.4. Microtubule structure.  15  Figure 1.5. The antimitotic bioassay.  18  Figure 1.6. The series of signal transduction pathways in mammalian cell cycle checkpoints.  19  Figure 1.7. Rationale for the use of G2 checkpoint inhibitors in cancer therapy.  22  Figure 1.8. The G2 cell cycle checkpoint inhibitor bioassay.  24  Figure 1.9. The relationship between genetics and chemical genetics in exploring protein function.  26  Figure 1.10. Small molecule-based studies of the cell cycle and cell cycle checkpoints.  27  Figure 2.1. Structural classes of gorgonian di terpenoids.  41  Figure 2.2. Erythropodium caribaeorum (photo by P. Humann). Figure 2.3. Antimitotic activity (reported as Absorbance o5nm) of each fraction collected  43  4  from liquid chromatography.  53  Figure 2.4. Selected H M B C and C O S Y correlations for substructure A of compound 69.  56  Figure 2.5. Selected H M B C and C O S Y correlations for substructure B of compound 69.  57  Figure 2.6. Selected H M B C and C O S Y correlations for substructure C of compound 69.  59  Figure 2.7. Selected H M B C and C O S Y correlations for substructure D of compound 69.  60  Figure 2.8. ' H N M R spectrum of desmethyleleutherobin (69) recorded in DMSO-c/6 at 500 M H z . Figure 2.9. C N M R spectrum of desmethyleleutherobin (69) recorded in DMSO-J6 at 100 M H z . '•  62  I 3  63  X  Figure 2.10. C O S Y spectrum of desmethyleleutherobin (69) recorded in D M S O - J 6 at 500 M H z .  64  Figure 2.11. H M Q C spectrum of desmethyleleutherobin (69) recorded in D M S O - J 6 at 500 M H z .  65  Figure 2.12. H M B C spectrum of desmethyleleutherobin (69) recorded in D M S O - J 6 at 500 M H z .  66  Figure 2.13. Selected H M B C and C O S Y correlations for the cyclic ketal subfragment of compound 61.  68  Figure 2.14. ' H N M R spectrum of eleutherobin (61) recorded in D M S O - J 6 at 500 M H z .  70  Figure 2.15.  71  l 3  C N M R spectrum of eleutherobin  (61) recorded in D M S O - J 6 at 100 M H z .  Figure 2.16. Selected C O S Y correlations for the arabinose subunit of compound 70. Figure 2.17. ' H N M R spectrum of desacetyleleutherobin (70) recorded in DMSO-<i6  73 75  at 500 M H z .  Figure 2.18.  (70) recorded in D M S O - J 6  76  Figure 2.19. Selected C O S Y correlations for the arabinose subunit of compound 71.  78  C N M R spectrum of desacetyleleutherobin at 100 M H z .  1 3  Figure 2.20. ' H N M R spectrum of isoeleutherobin A (71) recorded in D M S O - J 6 at 500 M H z .  80  Figure 2.21. Selected H M B C and C O S Y correlations for the yV-methylurocanic acid ester subunit of compound 72.  82  Figure 2.22. ' H N M R spectrum of Z-eleutherobin (72) recorded in D M S O - d 6 at 500 M H z .  84  Figure 2.23. Selected H M B C and C O S Y correlations for the cyclohexene ring subunit of compound 73.  86  Figure 2.24. Selected R O E S Y correlations for the diterpene core of compound 73.  87  Figure 2.25. ' H N M R spectrum of caribaeoside (73) recorded in DMSO-c/6 at 500 M H z .  89  Figure 2.26. Expanded R O E S Y spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  90  Figure 2.27. Selected H M B C correlations around C-15 of compound 74.  92  Figure 2.28. Selected R O E S Y correlations for the diterpene core of compound 74.  92  xi  Figure 2.29. ' H N M R spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  94  Figure 2.30. Expanded R O E S Y spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  95  Figure 2.31. Selected C O S Y and H M B C correlations for the arabinose subunit of compound 75.  97  Figure 2.32. ' H N M R spectrum of diacetyleleutherobin (75) from the acetylation of eleutherobin (61). Recorded in D M S O - ( i 6 at 500 M H z .  99  Figure 2.33. ' H N M R spectrum of diacetyleleutherobin (75) from the acetylation of desacetyleleutherobin (70). Recorded in D M S O - J 6 at 500 M H z .  100  Figure 2.34. *H N M R spectrum of diacetyleleutherobin (75) from the acetylation of isoeleutherobin A (71). Recorded in D M S O - J 6 at 500 M H z ,  101  Figure 2.35. Immunofluorescence photograph of A549 cells after exposure to eleutherobin (61) at a concentration of 60 ng/ml.  105  Figure 2.36. Antimitotic activity of the compounds isolated from E. caribaeorum.  106  Figure 2.37. Overlay of nonataxel (cyan) with a) paclitaxel b) epothilone B c) eleutherobin and d) discodermolide, all in yellow.  109  Figure 2.38. Areas of common overlap among microtubule-stabilizing compounds.  109  Figure 2.39. O R T E P drawing of eleutherobin (61).  111  Figure 2.40. Torsional angles around the cyclohexene ring of eleutherobin (61).  113  Figure 2.41. N O E difference experiments of eleutherobin (61) recorded in C D C 1 Figure 2.42.  at 400 M H z . N O E difference experiments of eleutherobin  3  114  (61) recorded in C D C 1  3  at 400 M H z .  115  Figure 2.43. R O E S Y spectrum of eleutherobin (61) recorded in C D C 1 at 500 M H z .  116  Figure 2.44. R O E S Y spectrum of eleutherobin (61) recorded in DMSO-rf6 at 500 M H z .  117  Figure 2.45. Comparison of the conformations of eleutherobin (61).  119  3  Figure 2.46. Proposed common overlap for paclitaxel (4), epothilone B (53), and eleutherobin (61).  120  Figure 2.47. Proposed common overlap and pharmacophore for paclitaxel (4), epothilone B (53), and sarcodictyin A (55).  121  xii  Figure 2.48. Superposition of nine solution conformations of eleutherobin (61) in DMSO-c/6.  123  Figure 2.49. Taxonomy of the antimitotic extracts from the N C I Open Repository relative to E. caribaeorum.  124  Figure 2.50. M a p of the geographic collection locations of sarcodictyin/eleutherobinproducing organisms.  125  Figure 3.1. Stylissa flabelliformis (photo by P.L. Colin).  145  Figure 3.2. ' H N M R spectrum of debromohymenialdisine (91)-HC1 recorded in Figure 3.3.  D M S O - ^ 6 at 400 M H z .  150  C N M R spectrum of debromohymenialdisine (91)-HC1 recorded in D M S O - J 6 at 100 M H z .  151  1 3  Figure 3.4. *H N M R spectrum of hymenialdisine (85)-HCl recorded i n D M S O - d 6 Figure 3.5.  at 500 M H z .  153  C N M R spectrum of hymenialdisine (85)-HCl recorded in D M S O - J 6 at 100 M H z .  154  1 3  Figure 3.6. ' H N M R spectrum of Z-debromoaxinohydantoin (94) recorded in D M S O - J 6 at 400 M H z .  Figure 3.7.  1 3  156  C N M R spectrum of Z-debromoaxinohydantoin (94) recorded in  D M S O - J 6 at 100 M H z .  157  Figure 3.8. ' H N M R spectrum of aldisin (103) recorded in D M S O - ^ 6 at 400 M H z .  159  Figure 3.9.  160  I 3  C N M R spectrum of aldisin (103) recorded in D M S O - t / 6 at 100 M H z .  Figure 3.10. Inhibition of the G 2 checkpoint by debromohymenialdisine (91).  162  Figure 3.11. Inhibition of protein kinase C h k l by debromohymenialdisine (91).  164  Figure 3.12. Inhibition of protein kinase Chk2 by debromohymenialdisine (91).  164  Figure 4.1. Characteristic conformations and C N M R chemical shift values for synl 3  and £M/M,3-diols.  182  Figure 4.2. ' H N M R spectrum of £-cryptofolione (130) recorded in C D C 1 at 400 M H z .  183  Figure 4.3.  184  3  1 3  C N M R spectrum of ^-cryptofolione (130) recorded in C D C 1 at 100 M H z . 3  Figure 4.4. ' H N M R spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z .  187  Figure 4.5.  188  3  1 3  C N M R spectrum of Z-cryptofolione (148) recorded in C D C 1 at 100 M H z . 3  xiii  Figure 4.6. ' H N M R spectrum of cryptofolione ketone (131) recorded in C D C 1  3  at 500 M H z .  Figure 4.7.  L ,  191  C N M R spectrum of cryptofolione ketone (131) recorded in C D C 1  3  at 100 M H z .  192  Figure 4.8. ' H N M R spectrum of compound 138 recorded in C D C 1 at 500 M H z .  195  Figure 4.9.  19.6  3  1 3  C N M R spectrum of compound 138 recorded in C D C 1 at 100 M H z . 3  Figure 4.10. ' H N M R spectrum of cryptofolione acetonide (149) recorded in C D C 1  3  at 400 M H z .  Figure 4.11.  199  C N M R spectrum of cryptofolione acetonide (149) recorded in C D C 1 at 100 M H z .  1 3  Figure 4.12. ' H N M R spectrum of diacetylcryptofolione (150) recorded in C D C 1  3  200  3  at 500 M H z .  Figure 4.13.  1 3  203  C N M R spectrum of diacetylcryptofolione (150) recorded in CDCI3  at 100 M H z .  204  Figure 4.14. Selected C O S Y correlations in the (3-mercaptoethanol adduct (151). Figure 4.15. ' H N M R spectrum of p-mercaptoethanol adduct (151) recorded in C D C 1  206 3  at 500 M H z .  Figure 4.16.  I 3  207  C N M R spectrum of (3-mercaptoethanol adduct (151) recorded in C D C 1  at 100 M H z .  3  "  208  Figure 4.17. ' H N M R spectrum of compound 152 recorded in C D C 1 at 500 M H z .  211  Figure 4.18.  1 3  C N M R spectrum of compound 152 recorded in C D C 1 at 100 M H z .  212  Figure 4.19.  Inhibition of the'G2 checkpoint by natural and semi-synthetic a-pyrones.  214  Figure A.l. C O S Y spectrum of eleutherobin (61) recorded in D M S O - J 6 at 500 M H z .  225  Figure A.2. H M Q C spectrum of eleutherobin (61) recorded in D M S O - d 6 at 500 M H z .  226  Figure A.3. H M B C spectrum of eleutherobin (61) recorded in D M S O - t / 6 at 500 M H z .  227  3  3  Figure A.4. C O S Y spectrum of desacetyleleutherobin (70) recorded in D M S O - J 6 at 500 M H z .  228  Figure A.5. H M Q C spectrum of desacetyleleutherobin (70) recorded in D M S O - ^ 6 at 500 M H z .  229  xiv  Figure A.6. H M B C spectrum of desacetyleleutherobin (70) recorded in D M S O - d 6 at 500 M H z .  230  Figure A.7. C O S Y spectrum of isoeleutherobin A (71) recorded in D M S O - d 6 at 500 M H z .  231  Figure A.8. H M Q C spectrum of isoeleutherobin A (71) recorded in D M S O - J 6 at 500 M H z .  232  Figure A.9. H M B C spectrum of isoeleutherobin A (71) recorded in DMSO-rf6 at 500 M H z .  233  Figure A.10. C O S Y spectrum of Z-eleutherobin (72) recorded in D M S O - J 6 ! at 500 M H z .  234  Figure A.11. H M Q C spectrum of Z-eleutherobin (72) recorded in D M S O - ^ 6 at 500 M H z .  235  Figure A.12. H M B C spectrum of Z-eleutherobin (72) recorded in DMSO-<i6 at 500 M H z .  236  Figure A.13. C O S Y spectrum of caribaeoside (73) recorded in D M S O - ^ 6 at 500 M H z .  237  Figure A.14. H M Q C spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  238  Figure A.15. H M B C spectrum of caribaeoside (73) recorded in DMSO-c/6 at 500 M H z .  239  Figure A.16. R O E S Y spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  240  Figure A.17-. C O S Y spectrum of caribaeolin (74) recorded in D M S O - d 6 at 500 M H z .  241  Figure A.18. H M Q C spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  242  Figure A.19. H M B C spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  243  Figure A.20. R O E S Y spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  244  Figure A.21. C O S Y spectrum of debromohymenialdisine (91)-HCl recorded in DMSO-ci6 at 500 M H z .  245  Figure A.22. H M Q C spectrum of debromohymenialdisine (91)-HC1 recorded in D M S O - J 6 at 500 M H z .  246  Figure A.23. H M B C spectrum of debromohymenialdisine (91)-HC1 recorded in DMSO-</6 at 500 M H z .  247  Figure A.24. C O S Y spectrum of hymenialdisine (85)-HCl recorded in DMSO-c/6 at 500 M H z .  248  XV  Figure A.25. H M Q C spectrum of hymenialdisine (85)-HCl recorded in DMSO-c/6 at 500 M H z .  249  Figure A.26. H M B C spectrum of hymenialdisine (85)-HCl recorded in DMSO-c/6 at 500 M H z .  250  Figure A.27. C O S Y spectrum of £-cryptofolione (130) recorded in C D C 1 at 500 M H z .  251  Figure A.28. H M Q C spectrum of £-cryptofolione (130) recorded in C D C 1 at 500 M H z .  252  Figure A.29. H M B C spectrum of ^-cryptofolione (130) recorded in C D C 1 at 500 M H z .  253  Figure A.30. C O S Y spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z .  254  Figure A.31. H M Q C spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z .  255  Figure A.32. H M B C spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z .  256  3  3  3  3  3  3  Figure A.33. C O S Y spectrum of cryptofolione ketone (131) recorded in C D C 1  3  at 500 M H z .  257  Figure A.34. H M Q C spectrum of cryptofolione ketone (131) recorded in C D C 1  3  at 500 M H z .  258  Figure A.35. H M B C spectrum of cryptofolione ketone (131) recorded in C D C 1  3  at 500 M H z .  259  Figure A.36. C O S Y spectrum of compound 138 recorded in C D C 1 at 500 M H z .  260  Figure A.37. H M Q C spectrum of compound 138 recorded in C D C 1 at 500 M H z .  261  Figure A.38. H M B C spectrum of compound 138 recorded in C D C 1 at 500 M H z .  262  3  3  3  xvi  LIST OF SCHEMES Page  Scheme 2.1. Formation of geranylgeranyl pyrophosphate.  39  Scheme 2.2. Proposed biosynthetic relationship between cembranoid diterpenes.  40  Scheme 2.3. Isolation procedure for antimitotic diterpenoids from E. caribaeorum.  52  Scheme 2.4. Taxonomic scheme showing the related chemistry isolated from octocorals.  125  Scheme 3.1. Biogenetic precursors of odiline/stevensine from the sponge T. mprchella.  139  Scheme 3.2. Representative structural groups of oroidin alkaloids.  140  Scheme 3.3. The six cyclization modes of the oroidin skeleton.  141  Scheme 3.4. Isolation procedure for G 2 checkpoint inhibitors from S. flabelliformis. Scheme 4.1. Isolation procedure for G 2 checkpoint inhibitors from C. concinna.  146 178  xvii  LIST OF ABBREVIATIONS A  angstrom  [a]d °  specific rotation at wavelength of sodium D line at 20 °C  Ac  acetyl  ATM  ataxia telangiectasia mutated kinase  ATR  ataxia telangiectasia mutated-related kinase  ATP  adenosine triphosphate  ax  axial  B.C.  British Columbia  br  broad  c  concentration (g/100 ml)  CD  circular dichroism  CDCI3  deuterated chloroform  Cdk  cyclin-dependent kinase  CHCI3  chloroform  2  CH C1 2  2  dichloromethane  CI  chemical ionization  COSY  correlation spectroscopy  °  degrees  5  chemical shift in parts per million  d  doublet  ID  one-dimensional  2D  two-dimensional  DBH  debromohymenialdisine  xvni DCM  dichloromethane  DMSO  dimethyl sulfoxide  DMSO-J6  deuterated dimethyl sulfoxide  DNA-PK  DNA-activated protein kinase  e  molar absorptivity coefficient  EI  electron impact  ELICA  Enzyme Linked Immuno-Cytochemical Assay  ELISA  Enzyme Linked Immuno-Sorbent Assay  Et  ethyl  EtOAc  ethyl acetate  eq  equatorial  g  gram  GTP  guanosine triphosphate  Gy  grays  HMBC  heteronuclear multiple bond multiple quantum coherence  HMQC  heteronuclear multiple quantum coherence  HPLC  high performance liquid chromatography  HRCIMS  high resolution chemical ionization mass spectrometry  HRELMS  high resolution electron impact mass spectrometry  HRFABMS  high resolution fast atom bombardment mass spectrometry  HRP  horseradish peroxidase  Hz  hertz  i  signal due to impurity  IC50  Inhibitory Concentration resulting in 50% maximal response  ('Pr) NH 2  diisopropyl amine  XIX  J  scalar coupling constant  LRFABMS  low resolution fast atom bombardment mass spectrometry  m  multiplet  M  molar  M  +  molecular ion  Me  methyl  MeOH  methanol  MHz  megahertz  ml  milliliter  m/z  mass to charge ratio  uM  micromolar  N  normal  NCI  National Cancer Institute  nm  nanometers  NMR  Nuclear Magnetic Resonance  NOE  Nuclear Overhauser Effect  ORTEP  Oak Ridge Thermal Ellipsoid Plot  Ph  phenyl  ppm  parts per million  q  quartet  ROESY  rotating frame Overhauser effect spectroscopy  s  singlet or signal due to solvent  SCUBA  self-contained underwater breathing apparatus  SD  standard deviation  sp.  species  t  triplet  TFA  trifluoroacetic acid  THF  tetrahydrofuran  UBC  University of British Columbia  US  United States  UV  ultraviolet  w  signal due to water  x  times  xxi  ACKNOWLEDGEMENTS I wish to express my deepest appreciation to my research supervisor, Professor Raymond J. Andersen for the many years of endless enthusiasm, brilliant insight, constant encouragement and support. I will be forever grateful for the opportunity to learn and work under his esteemed guidance. Many heartfelt thanks are extended to Dr. David E . Williams and Dr. M i c h e l Roberge for all their invaluable instruction and patient assistance over the past years.  I have learned  much and benefited enormously from their efforts. M y appreciation to all the members of the Andersen and Roberge research groups, especially John Coleman, Lynette L i m , Darko Curman, Cristina Biggs, and Rebecca Osborne who assisted on the biological testing and other aspects of this thesis research.  Thanks also to Michael LeBlanc and Robert Britton for collection of the  marine specimens used in this research, and the staff of the N M R , M S , and X-ray facilities at U B C for their assistance in collecting data. T o my friends for their kindness and encouragement throughout the years: Jason, Erica, Patti, and especially Kyle, Susie, and above all, Carine with all my love. Finally, I wish to acknowledge the incalculable and unwavering support of my parents, Ivo and Bianca Cinel, for everything. Words fail to describe my profound gratitude for all you have done for me...  CHAPTER 1 1. General Introduction Cancer remains one of the leading causes of death worldwide, and the limitations of conventional therapies  coupled with an ageing population w i l l continue to impact the  pervasiveness of this disease well into the new millennium.  This threat, along with the  emergence of resistance mechanisms to current therapeutic agents, has continued to drive the search for novel therapies.  Recent breakthroughs in molecular biology have identified  important genetic differences between normal and cancerous cells, yielding new insights into the molecular basis of this disease.  B y identifying new chemical entities with specific  antimitotic or cell cycle checkpoint inhibitory activities, the intricate machinery of a cancerous cell may be better understood and new treatments or cures developed. The goal of this thesis was to use two recently developed cell-based assays to identify antimitotic compounds and G 2 checkpoint inhibitors from marine and terrestrial sources. These screens facilitated the isolation and structure determination of novel antimitotic diterpenoids from a Caribbean octocoral, alkaloid G 2 checkpoint inhibitors from a marine sponge, and polyketide G2 checkpoint inhibitors from a Taiwanese tree.  Further investigations on these  biologically active secondary metabolites and their semi-synthetic analogues provided key insights into the structural features required for activity and helped to identify molecular targets and possibly novel mechanisms of action.  1.1. Natural Products Chemistry From the ancient Mesopotamian and Chinese civilizations to the present day, plant extracts have been widely used as traditional medicinal agents.  1  A t the dawn of the new  millennium, natural sources continue to provide important compounds as possible new  2 therapeutic agents or biological tools in the fight against life-threatening diseases such as cancer. Cancer is currently the leading cause of death in Canada and is set to emerge as the number one killer in the United States.  2,3  This ominous news comes nearly three decades after a  "war on cancer" was declared in the U S and it highlights the urgent need for improved medical treatments.  The chemotherapeutic approach represents one well-established and still attractive  approach for treating this serious disease. '  4 5  New chemical entities offer the promise of future  drugs with increased specificity, fewer side effects, novel modes of action to counter resistance, and potential to act as essential biological tools to probe the cellular mechanisms of this disease. Historically, natural products have been proven to be the foremost source for new leads in drug discovery and development. Indeed, some of the earliest active compounds isolated as pure entities are still in widespread use today.  Morphine (1) was isolated in 1806 from an  extract of Papaver somniferum and became the first commercially-pure medicinal product some twenty years later.  6  A t present, there are still no alternatives to morphine for patients suffering  from the severe pain that is common in the late stages of cancer.  The discovery that the  analgesic and antipyretic properties of the bark of the willow tree were due to salicylic acid led to the development of the natural product derivative, aspirin (2).  Over one hundred years after  its initial introduction, aspirin remains the most successful drug ever developed.  6  The  serendipitous discovery of penicillin by Fleming in 1928 (as penicillin G , 3) revolutionized 7  drug discovery by revealing the potential of soil microorganisms to be a rich new source of biopharmaceuticals. Cultures of microbes have since provided a seemingly unlimited array of unique and complex structures with a broad variety of biological activities. Production of drugs via microbial fermentation removes the barriers presented by large-scale harvesting of wild specimens and difficult or costly syntheses that plague the development of some terrestrial plant agents.  3  Despite the obstacles involved in large-scale production of phytogenic drugs, plant metabolites have continued to be invaluable as therapeutics.  Paclitaxel, or Taxol® (4), was  originally isolated in the late 1960's from the Pacific Y e w tree, Taxus brevifolia, after an extensive screen of over 110,000 plant extracts for cytotoxic agents.  8  Its novel mechanism of  action and the urgent need for improved anticancer chemotherapeutics led to its present day use in the treatment of ovarian and metastatic breast cancers.  The traditional Chinese medicinal  plant qinghao, or Artemisia annua, has been used for centuries in the treatment of fever and malaria.  9  Isolation of the active agent, artemisinin (5), provided a therapeutic treatment for  quinine-resistant forms of malaria.  10  Both paclitaxel (4) and artemisinin (5) have spawned  clinically useful semi-synthetic analogues, including docetaxel (6) and artemether (7), which have improved medicinal properties and decreased side effects.  4  5  4  HQ  O O'  NH  O  O  OH  H  :o OH;  O  H  o  6  H OMe  7  1.2. Marine Natural Products Chemistry In addition to terrestrial plants and soil microorganisms, a third resource of natural product chemical diversity is the marine environment. The total inhabitable space in the world's oceans greatly exceeds the narrow land surface zone and provides a vast array of species biodiversity throughout its wide range of habitats.  O f the 28 principle animal phyla, 26 are  represented in the marine biota and eight of these are exclusively aquatic in nature.  In  particular, many marine invertebrates exhibit soft bodied, sessile, slow moving, slow growing, or brightly coloured characteristics that leave them seemingly unprotected from predation. However, these organisms may chemically defend themselves by use of a highly complex arsenal of toxic secondary metabolites. Greater access to the marine environment came with the advent of S C U B A in 1943, opening the door to nearly 40 years of investigation into marine natural products. During this time, research in the secondary metabolites from marine organisms flowed along three parallel tracks: marine chemical ecology, marine toxins, and marine biopharmaceuticals." In contrast to primary metabolites such as amino acids and nucleotides, secondary metabolites are not ubiquitous in nature, are nonessential to the daily existence of an organism, and are often  species specific. antifeedant,  12  However, these compounds are thought to play key ecological roles as  antifouling,  13  antiovergrowth, or antimicrobial agents and as chemical cues to 14  mating, settling, or metamorphosis. '  13 15  The biological roles of most marine invertebrate natural  products remain unknown, but such biodiversity is an attractive lure for modern drug discovery. When  marine  plants  and microorganisms  are also included, marine  organisms  present  themselves as a vast and largely unexplored source of species and chemical diversity. Some of the largest and most impressive marine metabolites have emerged from the study of marine toxins related to algal blooms and the associated seafood poisonings.  The  polyether "ladder-like" structure common to a majority of these toxins was first revealed by the study of the dinoflagellate metabolite brevetoxin B (8).  The brevetoxins, produced by the "red  tide" dinoflagellate Gymnodinium breve, comprise a series of related neurotoxins that interfere with sodium voltage channels and are responsible for massive marine life  fatalities. '  16 17  Maitotoxin (9), isolated from the dinoflagellate Gambierdiscus toxicus and involved in ciguatera seafood poisoning, represents the largest and possibly most lethal non-biopolymer identified to 18  date.  These very complex compounds exhibiting such potently harmful biological activities  stand in contrast to the many marine metabolites showing promising therapeutic effects.  Much  of the research  in marine natural products  chemotherapeutics or pharmaceutical agents. 1950's  when  Bergmann  spongothymidine (11.)  et  al.  isolated  has focused on finding  new  The first success in this area came early in the two  ara-nucleosides,  spongouridine  from the Caribbean sponge Cryptotethya crypta}  9  (10)  and  These compounds  were the first nucleosides found to possess an arabinose sugar moiety and they served as "lead structures" for the development of many modified nucleosides with antiviral and antitumour properties.  1  The synthetic compound A r a - A , or arabinosyladenine (12), has been used since the  1970's as an antiviral agent to treat Herpes encephalitis and was later found to occur naturally 20 in the Mediterranean gorgonian Eunicella cavolini.  Fifteen years after Bergmann's discovery  of 10 and 11, the cytosine arabinoside A r a - C (13) 21  was developed into a clinical anticancer  agent  and it currently remains one of the most active agents available for treatment of acute  non-lymphocytic leukemia.  5  OH  OH  10 R = H  11 R = C H  3  12  OH  13  7 Many compounds more recently isolated from marine organisms have progressed to clinical trials for evaluation as new chemotherapeutics.  Bryostatin 1 (14),  one of a series of  potently cytotoxic macrolides isolated from the bryozoan Bugula neritina, is currently in phase I and phase U clinical trials for the treatment of melanoma.  22  A n insufficient supply of 14 from  harvesting w i l d bryozoan specimens led to the successful implementation of an aquaculture program capable of producing enough material for testing. '  With recent studies suggesting a  microbial symbiont as the true source of bryostatin 1, advances in the genetic manipulation of biosynthetic genes may forever eliminate future supply problems.  24  Investigations of the  shallow water tunicate Ecteinascidia turbinata by two independent research groups led to the simultaneous discovery of a series of tetrahydroisoquinoline alkaloids, including ecteinascidin 743 (15).  252 6  This compound, which acts as a guanine-specific D N A alkylating agent, is  currently in clinical trials as an antitumour agent. ecteinascidins and the safracins  27  The similarity in structure between the  isolated from Streptomyces  lavendulae " 27  28  suggest these  metabolites may all be microbial in origin and thus may eliminate the need to harvest wild specimens or undertake expensive chemical syntheses to obtain sufficient quantities for future use.  14  15  8 Chemical syntheses were required to confirm the structure of dolastatin 10 (16),  a potent  antimitotic agent originally isolated from approximately two tonnes of the Indian Ocean sea hare Dolabella auricularia  This compound contains a series of novel amino acids and  29  exhibits a complex mechanism of action involving inhibition of tubulin assembly during cell division.  225  Promising antineoplastic activity against all experimental cancers tested so far has  led to the clinical trials of dolastatin 10 and related analogues.  In contrast to dolastatin 10's  inhibition of tubulin polymerization, the Caribbean sponge metabolite discodermolide (17)  30  interferes with cell division by stabilizing microtubules against depolymerization.  31  Although  this mechanism of action is similar to that of paclitaxel (4), discodermolide was also found to inhibit the growth of paclitaxel-resistant ovarian and colon cancer cells. sponge natural product, the unusual sesterterpenoid dysidiolide (18), inhibitor of the protein phosphatase C d c 2 5 A .  3;>  Another marine  32  was found to be a possible  Inhibition of C d c 2 5 A phosphatase, an enzyme  known to be overexpressed in a number of tumour cell lines, should arrest the cell cycle at the G 2 / M transition just prior to cell division.  Compounds such as dysidiolide (18)  could  potentially exploit the genetic differences between normal and cancerous cells and lead to new cancer chemotherapeutics.  17  18  9 While most of the efforts in finding new biopharmaceuticals from marine natural products have focused on anticancer compounds, there have been key discoveries in other areas as  well.  In particular, the  Pseudopterogorgia  elisabethae,  pseudopterosins  isolated from  the  Caribbean gorgonian,  exhibit potent anti-inflammatory and analgesic activities. ' 34  33  The diterpene pentose glycoside pseudopterosin C (19) mediates inflammation by acting on both lipooxygenase and phospholipase A? and has found commercial use as an active ingredient in a cosmetic, anti-ageing skin cream. sponge Petrosia contignata, stimulated cells,  36  A compound isolated from the Pap'ua New Guinean  has been found to inhibit the release of histamine from anti-Ige  thus showing promise as an anti-asthmatic treatment.  This discovery of  contignasterol (20) and its biological activity has led to the clinical development of an orallyactive synthetic analogue that appears to lack the toxic side-effects associated with current steroidal anti-asthma drugs.  OH  Investigations of the secondary metabolites produced by marine organisms  have  revealed thousands of unique terpenoid, alkaloid, and polyketide chemical structures that have no terrestrial counterparts.  37  A s shown in Figure 1.1, the two most prolific marine sources of  new compounds reported in the recent literature are the sponges (phylum Porifera) and the corals (phylum Coelenterata).  Organisms from these two phyla remain a rich source of  10 structurally diverse organic molecules and form the basis of two of the chemical investigations presented in this thesis.  Figure 1.1. Distribution of marine natural products reported in 1999 according to the phylum of the source organism. 37  Although interest in natural products as a source of chemical structural diversity occasionally  becomes  overshadowed  by  other  combinatorial chemistry and computer-based  approaches  to  drug  discovery such  as  molecular modeling, statistics show natural  products or synthetic analogues account for over 60% of recently approved drugs or late stage clinical trial drug candidates for cancer and infections diseases.  Synthetic combinatorial  libraries often contain closely related structural analogues, whereas a collection of biological extracts from widely divergent taxonomic groups, ecological niches, and geographic regions possess a much higher degree of molecular diversity. In addition, only a small percentage of the world's plant and animal species have been systematically evaluated for their medicinal value  39  and traditional screens for biological activity have focused on cytotoxicity or antimicrobial activity. While current drugs are estimated to target only 417 different enzymes or receptors, it has been suggested that the study of human genomics will improve drug therapy by providing  11 an estimated 10,000 potential new drug targets.  6  The recent development of novel mechanism-  based bioassays searching for specific inhibitors of molecular targets or biological processes demands a reassessment of previously examined natural extracts along with investigations into new collections.  1.3. Novel Anticancer Screens The term "cancer" refers to a broad group of more than 100 different forms of disease that are characterized by uncontrolled proliferative growth and the spread of aberrant cells from their site of origin. Most of the major cancers, or solid tumours, occur in localized tissues and arise from a common ancestral cell that has lost its ability to control cellular growth and reproduction as a result of mutations.  Advances in genetics and molecular biology have  identified many genetic differences between cancerous and normal c e l l s , " 40  43  differences that  can be exploited to selectively target and control malignant cells.  1.3.1. The Cell Cycle  4445  The growth and reproduction of eukaryotic cells is determined by a precisely timed sequence of events known as the cell cycle.  A normal functioning cell progresses through  various stages of development and ultimately divides to form two genetically identical daughter cells.  A n y alteration or mutation in the complex cellular machinery responsible for these  functions may result in uncontrolled cell proliferation, altered phenotype, or cell death.  These  genetic changes and cell cycle pathways may be exploited by anticancer agents that act by interfering with some aspect of the cell cycle. The cell cycle can be divided into two main stages, interphase and M phase (see Figure 1.2). Interphase represents the stage of cellular growth and replication of D N A and consists of three separate phases beginning with the G l or G a p l phase.  During G l , the cell grows and  produces enzymes necessary for the duplication of its genetic material.  The S phase that  12 follows is responsible for synthesizing an exact copy of the cell's D N A . After completion of the S phase, the cell enters the second gap phase, or G2. At this time, there is an increase in protein synthesis in preparation for cell division. Interphase  Figure 1.2. A n overview of the eukaryotic cell cycle.  The M phase of the cell cycle consists of mitosis, the distribution of a complete set of chromosomes to each daughter nucleus, and cytokinesis, the formation of two genetically identical cells. Mitosis itself can be divided into four sequential stages: prophase, metaphase, anaphase and telophase.  Although these divisions have distinct morphological characteristics,  the transition from one stage to the next is smooth and continuous (see Figure 1.3, A - F ) . The first stage of mitosis, prophase, is characterized by the condensation of the sister chromatids of each chromosome, the migration of the centrioles to opposite ends of the cell, and the early assembly of microtubules into the mitotic spindle. nuclear envelope disperses  A t the onset of metaphase, the  and the spindle microtubules begin to attach to the  center  13 (kinetochore) of each chromosome.  Towards the end of metaphase, the mitotic spindle is  completely formed and each chromosome is aligned along the spindle equator. Anaphase marks the separation of the two sister chromatids of each chromosome as the microtubules slowly pull the now independent daughter chromosomes towards opposite poles. The last stage of mitosis, telophase, concludes with the decondensation of the chromosomes and the formation of a new nuclear envelope to surround each complete set of D N A .  Cytokinesis results in the final  division of the cytoplasm and creation of two daughter cells.  C  B  / D  E  F  •••••  •  Figure 1.3. Fluorescent micrographs depicting cells in various stages of mitosis. In these examples, the microtubules (red) were stained using indirect immunofluorescent methods, while the chromosomes (green) were stained with the DNA probe Hoechst 33342. (A) A late prophase cell in which the two centrioles and their associated microtubules have separated to opposite sides of the nucleus. (B) A early metaphase cell that contains one monooriented chromosome (white arrow). (C) A metaphase cell in which all the chromosomes are aligned on the spindle equator. (D) A cell just entering anaphase in which the chromatids are disjoining. (E) A late anaphase cell in which the two groups of chromosomes are at the spindle poles. (F) A telophase cell in which the two groups of chromosomes are reforming nuclei and in which cytokinesis (between the white arrowheads) is almost complete.  14  1.3.2. Antimitotic Agents The uncontrolled cellular proliferation characteristic of cancerous tumours may be inhibited by compounds acting as antimitotic agents. Indeed, several natural products that act by arresting cells in mitosis have become clinically important anticancer drugs.  The Vinca  alkaloids vincristine (21) and vinblastine (22) were originally isolated from the periwinkle plant and have been used for over three decades in the chemotherapy of neoplastic diseases, such as childhood leukemia and Hodgkin's disease.  47  They, along with the taxanes paclitaxel (4) and  docetaxel (6), cause mitotic arrest by interfering with the assembly or disassembly of a- and Ptubulin into microtubules.  48  R  21 22  C0 Me 2  R = CHO R = Me  As shown earlier, microtubules play a fundamental role throughout the process of cellular division, most importantly in attaching to and segregating  the  chromosomes.  Microtubules are largely composed of the protein tubulin, a dimer comprised of a and (3 subunits, that joins in a head-to-tail fashion in the presence of GTP to form a long protein fiber called a protofilament. Typically 12 or 13 of these protofilaments group together to ultimately form the microtubule's hollow, pipe-like structure (see Figure 1.4).  Rapid assembly and  disassembly of tubulin dimers occurs at the ends of the microtubules resulting in a continuous, dynamic state of elongation and contraction.  It is this dynamic polymerization and  depolymerization of the microtubules that is disrupted by antimitotic agents.  15  There are two main mechanisms by which most antimitotic compounds act on tubulin and microtubules. A t high concentrations most antimitotic agents, including the Vinca alkaloids and dolastatin 10 (16),  ' inhibit microtubule assembly and cause complete depolymerization.  48 47  A second group of antimitotic compounds, such as the taxanes and discodermolide  (17), '  49 47  stabilizes the microtubules against depolymerization and leads to abnormal microtubule arrays or bundles.  Neither depolymerization nor bundling is observed at low concentrations but the  dynamics of tubulin addition and removal from the mitotic spindle microtubules are sufficiently affected so as to inhibit normal function. Thus, the microtubules are prevented from attaching to and segregating the chromosomes during mitosis and the cells arrest at this stage of cell division.  50,51  Prolonged arrest eventually leads to cell death, either in mitosis or after an  eventual escape from mitotic arrest. The current antimitotic chemotherapeutics are far from ideal. They display numerous unwanted side effects, including myelosuppression and neurotoxicity, and many cancers now demonstrate multi-drug resistance to these therapies. '  48 49  Resistance mechanisms can arise from  overexpression of the P-glycoprotein drug efflux pump, from tubulin isotypes exhibiting weaker drug binding, and from mutational or post-translational alterations in the structure of tubulin.  16 Screening natural extracts may provide new chemical entities with increased specificity, fewer unwanted side effects, and antineoplastic activity against drug-resistant cancers. The antimitotic agents currently available, including those in pre-clinical studies or clinical trials, were discovered either by screening for cytotoxicity, by exhibiting cytotoxicity patterns against multiple cell lines similar to other antimitotics, or by serendipity. " The determination of their mode of action was secondary  to their isolation and characterization as cancer-killing  compounds. In response to the lack of a rational drug discovery screen for antimitotic agents, a rapid and reliable cell-based assay was recently developed in the laboratory of Michel Roberge.  33  1.3.3. The Antimitotic Bioassay  53  Roberge's antimitotic bioassay was found to be suitable for drug discovery and for quantitative determination of antimitotic activity. In addition, this cell-based system provides many advantages over receptor or enzyme-based screens.  These advantages include the  elimination of many non-specific false positives associated with cell-free screens due to the need for agents to cross the cell membrane to demonstrate activity.  54  In a recent study, over  90% of compounds identified from in vitro target-based assays either failed to cross the plasma membrane or were degraded rapidly, resulting in no biological activity.  55  In Roberge's assay, exposure to antimitotic compounds, either in pure form or as crude natural extracts, causes rapidly cycling human breast carcinoma cells to arrest in mitosis. The arrested cells are detected by an Enzyme-Linked Immuno-Sorbent Assay ( E L I S A ) using the monoclonal antibody T G - 3 .  5 6  The T G - 3 monoclonal antibody is highly specific for mitotic cells  and flow cytometry studies show a >50-fold more intense T G - 3 immunofluorescence in mitotic cells than in interphase cells. nucleolin,  58  57  The antibody specifically recognizes^ a phosphoepitope of  found abundantly in cells arrested in mitosis but only present in low levels in  17 cycling cells. ELISA  5 9  This method detects phosphorylated nucleolin and thus mitotic cells by an  using 96-well microtitre plates and the addition of an HRP-conjugated secondary  antibody. The colorimetric determination of H R P activity provides a quantitative determination of antimitotic activity. The antimitotic bioassay is shown schematically in Figure 1.5. Human breast carcinoma M C F - 7 cells were cultured as monolayers, seeded on 96-well microtitre plates, and allowed to grow overnight.  Pure compounds or crude natural extracts were then added to the cells and  incubated for 16-20 hours. A t this stage, the presence of an antimitotic agent would cause the cells to arrest in mitosis and the phosphorylation of nucleolin to occur. After incubation with the extracts, the cell culture medium was carefully withdrawn from the plate without removing any cells. The remaining attached cells were lysed by buffer and transferred to 96-well protein binding E L I S A plates. Addition of the T G - 3 monoclonal antibody was followed by incubation for 16-20 hours, during which time any phosphorylated nucleolin would bind to the antibody. Introduction of the secondary antibody, HRP-conjugated goat antimouse I g M , and subsequent overnight incubation would attach the secondary antibody to any TG-3/protein complexes present. After a final rinse and addition of hydrogen peroxide, the H R P activity was determined by measuring the absorbance at 405 nm. A positive colorimetric result would indicate the cells had been arrested in mitosis due to the addition of an antimitotic sample. Improvements to the bioassay resulted from the substitution of the lengthy and labourintensive E L I S A procedure with a simpler, shorter protocol. The E L I C A , or Enzyme-Linked Immuno-Cytochemical Assay, involves fixing the cells on the 96-well microtitre plates with formaldehyde and permeabilizing the cells with methanol and detergents. These modifications, along with the subsequent simultaneous addition of the T G - 3 primary antibody and the H R P conjugated secondary antibody, reduce the total time and steps by half and eliminate the transfer to new plates.  The suitability of both the E L I S A and E L I C A procedures for quantifying  18 antimitotic activity was tested by using the known antimitotic paclitaxel (4) and confirmed by microscopy. Human breast carcinoma MCF-7 cells seeded on 96-well plates grown overnight  incubate 16-20 hours  No antimitotic agent present cells continue cycling nucleolin un-phosphorylated  Antimitotic agent present cells arrested in mitosis nucleolin phosphorylated  cell culture medium removed cells lysed and transferred to ELISA plates TG-3 antibody added incubate for 16-20 hours  No binding of TG-3 antibody  TG-3 antibody binds nucleolin-P HRP-conjugated antibody added  •  Complex of two antibodies and nucleolin-P  No complex incubate overnight rinse and add hydrogen peroxide NEGATIVE SIGNAL NO ANTIMITOTIC PRESENT IN E X T R A C T  POSITIVE SIGNAL ANTIMITOTIC AGENT PRESENT IN E X T R A C T  Figure 1.5. The antimitotic bioassay.  The antimitotic screen developed by Roberge et al. provided a new rational assay for natural products capable of arresting cells in mitosis. In total, over 24,000 extracts of marine microorganisms, marine invertebrates,  and terrestrial plants were screened for antimitotic  activity, with 119 extracts testing positive.  The bioassay-guided investigation of one of the  19 active extracts, originating from a Caribbean octocoral, is the subject of Chapter Two of this thesis.  1.3.4. Cell Cycle Checkpoints  As a cell replicates and divides, it is vital that the genetic information encoded in its D N A is duplicated and passed on accurately to the two new daughter cells.  Any errors or  deficiencies in these processes could lead to mutations and the manifestation of proliferative disorders such as cancer or even cell death.  In response to D N A damage, a series of signal  transduction pathways called checkpoints are activated which delay cell cycle progression and allow for D N A repair. " 60  62  Checkpoints arrest cells in G l phase to prevent the replication of  damaged D N A and in G2 phase to prevent the segregation of damaged chromosomes during mitosis.  While many investigations have focused on genetic studies in yeast, the complex  mechanisms in mammalian cell cycle checkpoints have only recently become better understood (see Figure 1.6).  ft  .V //•• //•  Apoptosis ?'?  Figure 1.6. The series of signal transduction pathways in mammalian cell cycle checkpoints. (Biochemically well-defined responses are shown in red).  20 The first D N A damage checkpoint occurs prior to the S phase of the cell cycle and exists to prevent replication of defective genetic material. The G l cell cycle checkpoint is regulated by three important gene products: A T M , p53 and p21, whose levels and activities strictly control entry into S phase. " 64  66  In response to D N A damage, the ataxia telangiectasia mutated  ( A T M ) kinase is thought to contribute to p53 activation by phosphorylation at the serine-15 res i d u e . ' 67  68  Recently, A T M was also found to activate the kinase Chk2, which in turn  phosphorylates p53 at serine-20 (see inset Figure 1.6).  69  In undamaged cells that are dividing  normally, p53 is highly unstable and rapidly degraded. After D N A damage induced by ionizing radiation, the two phosphorylation events by A T M and Chk2 lead to significant stabilization of p53 and subsequent increase in its transcription of p21. '65  70  Cells deficient in p53 function are  unable to arrest in G l in response to y-irradiation and this transcriptional activation of the cyclin-dependent kinase (Cdk) inhibitor p21 is linked to the ability of p53 to induce G l arrest. The elevated levels of p21 lead to the binding to and inactivation of a variety of cyclin/Cdk complexes, such as cyclin E / C d k 2 ,  71  thus preventing entry into S phase and the replication of  genetic material. In addition to activating the G l cell cycle checkpoint, D N A damage also initiates D N A repair processes, G 2 checkpoint pathways and, depending on the severity of the genetic damage, apoptosis.  According to the current understanding of the G 2 checkpoint signaling pathways,  D N A damage activates members of the phosphoinositide-3 kinase family including A T M , ATM-related kinase ( A T R ) , and DNA-activated protein kinase ( D N A - P K ) .  7 2  The A T M -  dependent signaling pathway involves activation of the downstream protein kinases C h k l and Chk2. ' " 64  73  77  These kinases directly mediate the phosphorylation of serine-216 on Cdc25c  phosphatase, resulting in an increased binding affinity for the 14-3-3 family of proteins (see Figure 1.6). ' " 76 78  81  The 14-3-3 proteins, present in larger amounts due to p53 transcription,  inactivate the Cdc25c phosphatase and promote its export from the nucleus and sequestration in  21  the cytoplasm. The inactivation of Cdc25c results in the inhibition of the cyclin B/Cdc2 kinase complex responsible for the G 2 / M cell cycle transition, leading to G 2 arrest. also  phosphorylate  phosphorylation.  83,84  and  activate  Weel,  a  kinase  that  catalyses  C h k l and Chk2  82  Cdc2  inhibitory  Since it is necessary for Cdc2 to be dephosphorylated for entry into  mitosis, this provides a second mechanism by which the ATM-dependent C h k proteins arrest cells in G 2 as a result of D N A damage.  1.3.5.  The G 2 Checkpoint Inhibition Bioassay  85,86  In response to D N A damage, cells can activate biochemical control pathways called checkpoints to halt progression of the cell cycle or to trigger cell death.  When these tightly  regulated responses break down, D N A replication and cell division may occur regardless of the cellular damage and the resulting genetic changes may lead to malignancy.  However, this  failure of cell cycle arrest responses in malignant or cancerous cells can also be exploited therapeutically.  87  Over half of all human cancers completely lack a G l checkpoint as a result of a mutation in the p53 tumour suppressor gene.  This fact has important clinical implications since agents  that disrupt the G 2 cell cycle checkpoint are found to selectively potentiate the cytotoxic effects of DNA-damaging agents on cancer cells that lack p 5 3 _ - ' - ' 42  85  89  90  91  j  n  combination with D N A -  damaging treatments, inhibition of the G 2 checkpoint in tumour cells lacking p53 function would facilitate mitotic entry and lead to cell death (see Figure 1.7a). Normal cells exposed to D N A damage and a G 2 checkpoint inhibitor would still arrest at the functioning G l checkpoint to repair damaged D N A (Figure 1.7b), leading to an increased survival over mutant p53- tumour cells. For this reason, components of the G 2 checkpoint are particularly attractive targets for novel antineoplastics, and their inhibition may increase the sensitivity of tumour cells to standard chemotherapy and radiation. "  22  A. p53- tumour cells G2 checkpoint  Gl checkpoint mutated  inhibited, y  M  G2  Gl  Cell Death B. p53+ normal cells G2 checkpoint  Gl checkpoint  inhibited p \ |  Gl  s  M  G2  Cells Arrested  Cell Death  Figure 1.7. Rationale for the use of G 2 checkpoint inhibitors in cancer therapy. A. In response to D N A damaging agents, tumour cells will not arrest at cell checkpoints, leading to cell death. B. Under the same conditions, normal cells will arrest in G l and repair their D N A .  The few G 2 checkpoint inhibitors identified to date include caffeine (23) and 1substituted caffeine analogues, 2-aminopurine and 6-dimethylaminopurine, staurosporine and 7hydroxystaurosporine ( U C N - 0 1 , 24), SB-218078, and isogranulatimide (25).  86  Several of these  compounds, including caffeine (23), U C N - O l (24), and isogranulatimide (25), have shown the ability to enhance the cytotoxicity of DNA-damaging agents. ' 85  Caffeine and its analogues  93  demonstrate in vitro inhibition of the A T M and A T R kinases, ' 62  pharmacological effects prevent clinical use. ' 95  96  however their numerous  94  Although U C N - 0 1 displays broad protein  kinase activity, it is currently in phase I clinical trials and has been shown to increase the cytotoxic effect of DNA-damaging agents in breast cancer c e l l s . ' 89  97  Because this is a new  23 approach to anticancer treatment, there is a need for a rational screen to identify better G2 checkpoint inhibitors that possess greater specificity and reduced unwanted cellular effects.  23  24  25  In response to the need for an assay for G 2 checkpoint inhibition, a cell-based screen was developed in the laboratory of M i c h e l Roberge and used to screen thousands of natural extracts from marine and terrestrial sources (see Figure 1 . 8 ) .  8586  In this assay, human breast  cancer cells lacking p53 function ( M C F - 7 p53-) were cultured as monolayers, seeded on 96-well microtitre plates, and allowed to grow for 24 hours.  The cells were then exposed to D N A  damage in the form o f 6.5 G y o f y-irradiation. Pure compounds or crude natural extracts were then added to the cells 16 hours after irradiation along with the antimitotic agent, nocodazole. At this point, cells in the presence of a G 2 checkpoint inhibitor w i l l not arrest in G 2 as a result of the induced D N A damage but will enter and arrest in mitosis.  The cells, extracts, and  nocodazole were incubated together for 8 hours after which time the cell culture medium was carefully withdrawn from the plate without removing any cells.  The remaining steps of the  bioassay involve detection of the presence of phosphorylated nucleolin using the T G - 3 antibody and HRP-conjugated secondary antibody (as was seen earlier in section 1.3.3). A positive signal from this E L I S A protocol would indicate a G 2 cell cycle checkpoint inhibitor was present in the extract, thus allowing the cells to overcome the barrier to entering mitosis.  24  Human breast carcinoma M C F - 7 p53- cells seeded on 96-well plates incubate for 24 hours  DNA damage  6.5 Gy of y-irradiation incubate for 16 hours  Extracts and Nocodazole added incubate for 8 hours  G2 checkpoint inhibitor present cells enter mitosis and arrested nucleolin phosphorylated  No G2 checkpoint inhibitor present cells arrested in G2 nucleolin un-phosphorylated  cell culture medium removed cells lysed and transferred to ELISA plates T G - 3 antibody added  incubate for 16-20 hours  HRP-conjugated antibody added  t No complex  Complex of two antibodies and nucleolin-P incubate overnight rinse and add hydrogen peroxide POSITIVE SIGNAL G2 C H E C K P O I N T I N H I B I T O R P R E S E N T IN E X T R A C T  Figure 1.8.  NEGATIVE SIGNAL N O G2 C H E C K P O I N T I N H I B I T O R P R E S E N T IN E X T R A C T  The G 2 cell cycle checkpoint inhibitor bioassay.  1.3.6. Biochemical Tools and Chemical Genetics Not only can G 2 checkpoint inhibitors provide potential chemotherapeutic benefits, they are also invaluable as biochemical tools to investigate the machinery of cell cycle checkpoints.  25 Since most of our understanding of cell cycle checkpoint pathways has come from genetic studies on yeast signaling pathways, finding agents that prevent the G2 arrest in mammalian cells would provide useful information on their components and gene targets.  The use of  natural products and natural product-like compounds to decipher and control the cellular and 98  physiological roles of proteins is referred to as "chemical genetics". The traditional genetic approach for determining the cellular function of a protein requires indirectly altering its function by an inactivating or activating genetic mutation. This method is extremely powerful for identifying the roles of genes that encode proteins of interest. However, limitations arise in using the classical genetic approach in mammals because of their slow reproductive rates, large physical size, and more complex genome." In addition, indirect genetic mutations do not allow for the instantaneous or conditional alteration of protein function and investigations into essential genes render organisms nonviable.  100  A complementary approach to classical genetics involves the use of exogenous ligands to alter the function of a single gene product or protein.  The chemical genetics approach  involves small molecules capable of either inactivating or activating proteins directly.  For  instance, the first antimitotic agent to be characterized, colchicine, inactivates the protein function of tubulin whereas steroid hormones, such as dexamethasone, activate transcription in nuclear hormone receptors (see Figure 1.9).  Thus, this approach emulates the logic of a  98  classical genetic screen but differs in that it relies upon small molecules, rather than mutations, to conditionally modulate biological processes.'  01  This direct method allows for "genetic-like"  screens in cells and tissues of higher organisms and complements other indirect genetic screening approaches.  100  26  inactivating mutation in gene C \  activating mutation in gene C  genetic  / cell division, differentiation,  chemical genetic f  Y  W o o ,  a  PP™  . X  ^ A=:0  a c h  Q x^- ^  colchicine  cell death, etc. OH  dexamethasone  Figure 1.9. The relationship between genetics and chemical genetics in exploring protein function. 98  One of the fundamental requirements of chemical genetics is the existence of a diverse library of ligands to bind very specific molecular targets within an organism.  The chemical  diversity necessary for this approach relies, at present, on compounds primarily from natural sources since most of the large combinatorial libraries synthesized so far contain simple 102  compounds with few or no stereocenters.  Indeed, compounds rivaling the structural  complexity, high potency, and target specificity of natural products are not available using existing combinatorial synthetic methods.  100  T o date,  biological screens of natural and  synthetic libraries have identified small molecule ligands for many proteins (including kinases,  103  phosphatases,  104  proteases,  105  and various receptors  cellular processes (such as blocking mitotic progression, ' 101  arrest, ' 110  85  and inducing apoptosis "). 1  109  10  -  l 0 1  ' ) and agents that act on m  inducing or suppressing cell-cycle  Small molecule-based studies of the cell cycle and cell  cycle checkpoints have been used to gain new insights into the circuitry of cell cycle progression, including the effects of discodermolide," lactacystin," and rapamycin" (see 2  3  4  Figure 1.10). Biologically active small molecules identified by rational screens may thus be  27 used as molecular probes to elucidate cell cycle pathways and protein functions, such as for the D N A damage checkpoint and its mediator c h k l .  1 1 5  '  7 3  Figure 1.10. Small molecule-based studies of the cell cycle and cell cycle checkpoints.  The checkpoint inhibitor screen developed by Roberge et al. provided the first rational assay for natural products capable of overcoming the G 2 checkpoint. These natural agents can be investigated as potentiators of D N A damage or used as molecular probes to further elucidate the G 2 cell cycle checkpoint. In total, over 30,000 extracts of marine microorganisms, marine invertebrates, and terrestrial plants were screened for checkpoint inhibitors.  116  The bioassay-  guided investigation of two of these active extracts, originating from a marine sponge and a Taiwanese tree, is the subject of Chapters Three and Four of this thesis, respectively.  1.4. Research Summary A series of new and known secondary metabolites were isolated from marine and terrestrial sources guided by two newly developed, cell-based assays. The crude extract from a Caribbean octocoral Erythropodium caribaeorum caused cells to arrest in mitosis and to display morphological characteristics similar to the microtubule-stabilizing compound, paclitaxel (4).  28 Bioassay-guided fractionation  resulted in the isolation and identification of the known  antimitotic agent eleutherobin and six novel structural analogues. E. caribaeorum proved to be a new and abundant source of eleutherobin, whose pre-clinical development had been impeded by its scarcity, and the structural variations of the new diterpenoids offered key insights into proposed pharmacophore models for microtubule-stabilizing compounds.  In addition, single  crystal X-ray diffraction analysis and N O E difference experiments provided the first solid state and solution conformations for eleutherobin. These results advance the current understanding of this structural class of marine diterpenoids and may facilitate the development of new models for microtubule stabilization. The crude extract from a marine sponge, Stylissa flabelliformis, exhibited potent activity in a new bioassay searching for G 2 cell cycle checkpoint inhibitors.  Bioassay-guided  fractionation of this active extract resulted in the isolation and identification of the natural product debromohymenialdisine and three related alkaloids. These compounds were the first G 2 checkpoint inhibitors to be found by a rational screen and were structurally distinct from previously reported G 2 checkpoint inhibitors. In addition, debromohymenialdisine was found to specifically act on the protein kinases C h k l and Chk2, thus blocking two branches of the checkpoint pathway downstream of A T M .  The discovery of these biologically active natural  products validated the power of the new bioassay and contributed to the understanding of the G2 checkpoint pathway by providing new biochemical tools to probe the molecular basis of inhibition. A s a result of a large-scale screen of natural extracts for G 2 checkpoint inhibitors, a series o f a-pyrones from the Taiwanese tree Cryptocarya concinna were found to exhibit potent inhibitory activity. Bioassay-guided purification resulted in the isolation and identification of the natural product cryptofolione and three related polyketide lactones. Synthetic modifications on these metabolites yielded four additional analogues and led to mode of action and structure-  29 activity relationship studies.  The G 2 checkpoint inhibitors isolated from C. concinna were  structurally distinct from previously known inhibitors and appear to act by a novel mechanism of action.  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Natl. Acad. Sci. U.S.A. 1996, 93, 2850. b) Keith, C . T . ; Schreiber, S.L. Science 1995, 270, 50.  1 1 6  Roberge, M . , personal communication.  36  CHAPTER 2 2.1. Antimitotic Diterpenoids from the Gorgonian Erythropodium caribaeorum In searching for biologically active natural products from marine sources, the crude extract from a Caribbean octocoral, Erythropodium caribaeorum, was found to contain a series of new diterpenoids.  Specimens of E. caribaeorum were collected off the coast of Dominica  and the methanolic crude extract exhibited promising activity in the antimitotic bioassay.  In  addition, cells arrested in mitosis by this extract displayed morphological characteristics similar to the microtubule bundling seen with paclitaxel. Bioassay-guided fractionation of the active extracts yielded six new antimitotic diterpenoids and the known antimitotic compound, eleutherobin. A s well as identifying an important new source for eleutherobin, this discovery led to structure-activity relationships offering key insights into pharmacophore models for microtubule-stabilization and the first solid-state and solution conformational studies on this class of promising anticancer agents.  2.1.1. Introduction to Coelenterates and Gorgonians The animal phylum Coelenterata, also known as Cnidaria, represents a diverse group of marine invertebrates that includes hydras, jellyfish, sea anemones, and corals. With more than 10,000 known species, the coelenterates  are arguably the most common and conspicuous  invertebrates present in shallow tropical waters, reaching levels of diversity and importance unequalled by any other phylum.  1  Coral reefs are responsible for some of our earth's most  fascinating, complex, and varied ecosystems by hosting an incredible multitude of animal and plant life.  Consisting of four main classes, the coelenterates all exhibit radial symmetry, a  single opening (coelenteron) for ingesting and expelling food, and tentacles armed with stinging structures (nematocysts) for capturing prey and defense.  T w o basic body types are found in  37 coelenterates, a sessile polyp form that lives attached to a substrate and an unattached, freeswimming medusa form.  While most members of the Hydrozoa, Scyphozoa, and Cubozoa  classes display alternate polyp and medusa stages, the remaining Anthozoa class exists only as solitary polyps or colonial polyps. Chemically, the Coelenterate phylum is second only to the sponges as the most prolific marine source for natural products reported in the recent literature (as seen in Figure 1.1), with compounds arising almost exclusively from the class Anthozoa (corals, sea fans, sea anemones, and zoanthids).  2  The anthozoans represent the largest and most diverse class within the Coelenterata and can conveniently be divided into two subclasses depending on their polyp morphology. The Hexacorallia usually display six unbranched tentacles  or multiples thereof,  whereas  the  Octocorallia always exhibit eight tentacles that bear tiny pinnate (feather-like) projections or side branches.  The colonial octocorals, in turn, can be divided into four primary groups: the  stoloniferans, alcyonaceans (soft corals), penatulaceans (sea pens), and gorgonians.  Easily  recognized as "sea fans" or "sea whips", the gorgonians are prominent and eye-catching members of the reef fauna of the West Indies and represent an estimated 38% of the known fauna.  However, they are often mistaken for plants because of their permanent attachment to a  substrate, bushy colony form, and passive mode of existence (their branches move only because of currents or wave action).  4  Many shallow water species of gorgonians and octocorals also  play host to scores of symbiotic unicellular algae, or zooxanthellae, present in their surface tissues and responsible for the relatively dull brownish appearance of the host organism.  5  L i k e most octocorals, gorgonians are filter feeders that inhabit areas where currents flow and are represented by about 1 7 0 species in the Caribbean region. While most of these are restricted to depths greater than 5 0 meters or are not found in reef habitats, the remaining shallow  water gorgonians are among the most common and abundant  species.  4  The  morphological differences in the arrangement and shape of the polyps along with the branching  38 of the stem anchoring the polyp to the substrate can often be used to distinguish the genus and occasionally species of gorgonian.  However, positive identification requires the microscopic  examination of the location, pattern, shape, and size of the skeletal spicules embedded in the polyp's and colony's common tissue.  6  The calcareous spicules are very small structures (0.05-  1.00 mm) of various shapes that form the internal skeleton of gorgonians and may also function to deter predators.  In addition, the rich chemistry often associated with these organisms is 5 7  thought to contain toxic compounds that play a key role as feeding deterrents. '  2.1.2. Review of Diterpenoids from Octocorals The natural products  isolated from coelenterates  dominated by the diterpenoid class of compounds.  2  and, specifically,  octocorals is  Biosynthetically, the large and structurally  diverse family of diterpenoids arises from the linking of four isoprene (C5) units in a head-to-tail fashion to form a 20-carbon structural framework.  The assembly of the diterpene skeleton  begins with the creation of two biochemical isoprene units, dimethylallyl  pyrophosphate  ( D M A P P ) and isopentenyl pyrophosphate (IPP), from acetate precursors through a mevalonic acid intermediate. The enzymatic formation of geranyl pyrophosphate from D M A P P and IPP is thought to involve the generation of an allylic cation, electrophilic addition yielding a tertiary cation, and the subsequent stereospecific loss of a proton, HR (see Scheme 2.1).  8  This process is  repeated two more times, ultimately forming the C20 linear geranylgeranyl pyrophosphate (GGPP).  39 a) formation ol allylic cation  c)  b) electrophilic addition giving tertiary cation  "OPP  C\  OPP  DMAPP  HR  IPP  OPP H  geranyl p y r o p h o s p h a t e  S  c)  a) allylic cation b) electrophilic addition  OPP  stereospecilic loss of proton  stereospecilic loss of proton  OPP (GPP)  IPP a) allylic cation b) electrophilic addition  OPP farnesyl p y r o p h o s p h a t e  OPP IPP  (FPP)  c)  stereospecilic loss of proton  OPP geranylgeranyl pyrophosphate  (GGPP)  Scheme 2.1. Formation of geranylgeranyl pyrophosphate.  The linear G G P P can undergo a variety of oxidation, reduction, and cyclization reactions to produce the multitude of diterpenoids isolated from octocorals.  In a recent review of the  secondary metabolites reported from Caribbean gorgonians, diterpenoids were found to account for close to 65% of the compounds isolated over a 37 year period, with one third of these belonging to the cembranoid structural class.  9  The formation of the cembranoid diterpene  framework involves a cyclization between carbons 14 and 1 of G G P P (as shown in Scheme 2.2). One  of the simplest members of this class is cembrene A (26), isolated from marine soft corals  and a variety of terrestrial sources. " 10  12  The carbon skeletons of two related classes of bicyclic  diterpenes, the briaranes and the eunicellanes (or cladiellanes), are thought to arise from additional cyclizations of the cembrane ring system, as shown in Scheme 2 . 2 .  13  Briarein A (27)  40 and eunicellin (28) were the first known members of these diterpene structural classes and were isolated from the gorgonians Briareum abestinum  and Eunicella stricta,^  14  respectively.  28 Scheme 2.2. Proposed biosynthetic relationship between cembranoid diterpenes.  In total, over twelve different structural classes of diterpenes have been reported from gorgonians (see Figure 2.1), with each class displaying an impressive number of members possessing unique substitution patterns and functionalities.  In addition to the cembranes,  briaranes, and eunicellanes, the most common carbon skeletons include the asbestinins (related to the eunicellins by a 1,2-methyl migration), rearranged,  13  the pseudopteranes and cubitanes (both  12-membered, monocyclic rings containing two isopropyl groups), the related  dolabellane/dolastanes,  and  dilophol/elemene  classes.  9  Gorgonians  of  the  genus  41 Pseudopterogorgia have proven to be especially prolific in terms of diterpene structural classes, producing  secondary  elisapterane,  19  from  the  amphilectane,  16  serrulatane,  17  elisabethane,  18  and gersolane structural classes, just to name a few.  asbestinin  dolastane  serrulatane  metabolites 20  pseudopterane  cubitane  dilophol  dolabellane  elemene (fuscol)  elisabethane  elisapterane  amphilectane  gersolane  Figure 2.1. Structural classes of gorgonian diterpenoids.  There has been considerable interest in the secondary metabolites of gorgonians because of the many biological and pharmaceutical activities associated with these compounds. Investigations of Briareum excavatum have led to the isolation of 40 briarane diterpenoids, the 21 22  excavatolides A - Z and briaexcavatolides A - N ,  '  some of which exhibit significant cytotoxic  activity against a variety of cancer cell lines (i.e., excavatolide M (29)).  Pinnatin A (30), a  polycyclic diterpenoid lactone belonging to the rare gersolane class," was recently found to show significant differential antitumour activity in the National Cancer Institute's 60 cell-line panel.  20  In addition to cytotoxicity, anti-inflammatory properties have been reported from  42 numerous briarane diterpenes and diterpene glycosides isolated from gorgonians. diterpene  glycosides  include  the  amphilectane-containing  pseudopterosins,  25  These  24  such  as  26  pseudopterosin A (31), the elemene-containing fuscosides, calyculaglycoside B (32).  27  Furthermore, gorgonian secondary metabolites continue to attract 28  interest because of the antiviral, antifouling  32  and the dilophol-containing  29  antibacterial,  30  ^I  insecticidal,  immunomodulatory,  properties associated with some of these compounds.  and  Numerous ecological  33  studies  indicate the diterpenoids produced by gorgonians can act as potent feeding deterrents  to reduce predation by fish (i.e., furanocembranolide 3 3 ) .  34  O  32  33  While diterpenoids comprise the majority of secondary metabolites reported from gorgonians  and  octocorals, there are  also frequent  reports  of novel  sesquiterpenoids,  acetogenins, prostanoids, and highly functionalized steroids isolated from these organisms.  2  Although over forty years have passed since Burkholder and Burkholder highlighted the occurrence of antibiotic substances from a Caribbean gorgonian,  35  only a small percentage of  the many species of gorgonians from this region have been systematically scrutinized to date.  9  Even species that have been extensively studied in the past continue to excite researchers with  43  novel compounds and structures, often with new biological activities, that were previously overlooked.  2.1.3.  Review of Known Metabolites from Erythropodium caribaeorum The gorgonian Erythropodium  caribaeorum  (Duchassaing & Michelotti,  1860) is  endemic to the Caribbean region and is classified as "uncommon", typically found from the southern tip of Florida, east to the lesser Antilles, and into the western Caribbean.  3  Its genus is  taxonomically monospecific and is described morphologically as an encrusting octocoral, with a tan upper surface colouration and reddish underside. The colonies are quite bushy but grow in flat, vertical planes with distinctive, contrasting light yellow-brown polyps and dark brown rind.  6  Colony sizes vary from 10 centimeters to 1 meter and usually inhabit depths easily  accessible by S C U B A , from 1 to 30 meters. Figure 2.2 shows the typical appearance of this gorgonian with its fine, hair-like polyps extended and retracted.  Figure 2.2. Erythropodium  caribaeorum (photo by P. Humann).  6  44 The chemistry of E. caribaeorum has been extensively investigated by many research groups over the past 17 years. The first report dates back to 1968 when Schmidt and Ciereszko 36  isolated choline sulphate (34) and scyllitol (35) from this gorgonian coral.  A further 16 years  passed before Fenical et al. reported the structures of two new chlorinated diterpenoids. erythrolides A (36) and B (37),  from a sample collected in the waters off B e l i z e .  7  The authors  suggest erythrolide A arises from a photochemical di-7r-methane rearrangement of erythrolide B , the first observation of such a rearrangement in a natural product.  Later that same year, the  Fenical and Djerassi groups published a study investigating whether the terpenes isolated from gorgonians  are produced by the organisms themselves  or by their associated symbiotic  37  zooxanthellae.  Most octocorals inhabiting the Caribbean Sea host symbiotic algae known as  zooxanthellae which provide a large portion of the nutrient requirements of the octocoral through photosynthesis and carbon transfer.  7  Since the true origin of secondary metabolites  isolated from gorgonians was in doubt, the authors used  1 3  C / C isotope ratio mass spectrometry 1 2  to compare compounds known to be of algal origin, such as the sterol gorgosterol (38), and the 38  newly identified diterpenoids, the erythrolides. Their data suggests the sterols isolated from E. caribaeorum are produced by the zooxanthellae, a result consistent with data from other gorgonians, whereas the diterpenes are true gorgonian metabolites. OH  36  37  38  45 A few years later, erythrolides A and B along with seven additional erythrolides, C-I  (39-45), were isolated from E. caribaeorum collected in the U . S . Virgin Islands and Jamaica/  9  A l l of these erythrolides, with the exception of erythrolide H (44), are chlorinated at the C-6 position. While erythrolides C (39), D (40), and H (44) all possess epoxide functionalities, the remaining diterpenes (41-43, 45) comprised a new group of ether-cyclized briaranes featuring an unprecedented C-2 to C-8 ether link. While in the process of conducting feeding-deterrent studies on the compounds from this gorgonian, Fenical and Pawlik reported the presence of a new spirobicyclic sesquiterpene,  named erythrodiene (46).  40  Results o f the feeding studies  showed, however, that the erythrolides, particularly erythrolides B (37) and D (40), were responsible for the defensive properties of the gorgonian towards a collection of predatory reef fishes.  O  44  46  46 Subsequent studies of E. caribaeorum collected off the waters of Tobago and Jamaica led to the isolation of erythrolide J (47), erythrolide K (48), and the three acetate analogues 41  42  of the erythrolides E (49), H (50), and I (51), respectively.  43  Until our present work, the  secondary metabolites isolated from the gorgonian Erythropodium caribaeorum had offered insight into their ecological roles as feeding deterrents and provided challenges to synthetic chemistry but were devoid of any pharmaceutical interest or potential.  49  44  51  R = Ac  O 50  R=  ^ ^ ^ ° Y ^  O 2.1.4. Review of Microtubule-Stabilizing Antimitotic Agents Compounds capable o f binding to tubulin and stabilizing the microtubule assembly during cell division provide one important mechanism to halt the uncontrolled cellular proliferation associated with cancerous tumours. Arresting these cells in mitosis can eventually lead to apoptosis, or cell death, and presents a clinically important approach to the treatment of  47 slow-growing solid tumours usually occurring in adults, such as those targeting the lungs, colon, prostrate, and brain. Paclitaxel (4), originally isolated from the bark of the Pacific Yew tree, was the first chemical entity found to exhibit this unique mechanism of action. The subsequent clinical success of paclitaxel (4) and the related docetaxel (6) has generated considerable interest in identifying additional chemotypes that stabilize microtubules.  Four additional structural  classes of microtubule-stabilizing compounds that have been identified include the epothilones A and B , discodermolide, laulimalide, and the sarcodictyin/eleutherobin classes.  4  6  Epothilones A (52) and B (53) were initially isolated from the soil bacteria Sorangium cellulosum and were the first compounds identified after paclitaxel (4) to exhibit microtubulestabilizing properties.  45  In contrast to paclitaxel, the epothilones can be easily produced in large  quantities through bacterial fermentations and are equally active against multi-drug resistant human cancer cells overexpressing the P-glycoprotein efflux pump  4 6  The recent isolation of  the biosynthetic gene cluster responsible for production of the epothilones has revealed another promising avenue of investigation and possible new source of analogues via combinatorial biosynthesis.  47  The next two classes of microtubule-stabilizing agents were discovered as  secondary metabolites from marine sponges. Discodermolide (17) was originally isolated from the Caribbean sponge Discodermia dissoluta as an immunosuppressant and was later screened for antimitotic activity on the basis of a computer-assisted structure analysis that compared it to  48 other tubulin-interacting drugs  4 8  Laulimalide (54),  however, was identified from the sponge  Cacospongia mycofijiensis in a mechanism-based screening program specifically aimed at the discovery of new antimicrotubule agents.  49  54 The fourth structural class of antimitotic compounds with microtubule-stabilizing properties is represented by the soft coral metabolites eleutherobin and the sarcodictyins. Pietra et al. originally isolated sarcodictyins A (55)  and B (56)  from a Mediterranean stolonifer  Sarcodictyon roseum and later reported four other sarcodictyins, C to F (57-60) from the same source. ' 50  51  There was, however, no mention of any biological activity associated with these  new eunicellin-type diterpenoids. It was eight years after the discovery of the sarcodictyins that Fenical et al. filed a Patent application describing the isolation of eleutherobin (61) cytotoxic properties.  52  and its  The authors reported the isolation of eleutherobin from a rare  alcyonacean, identified as an Eleutherobia species, collected in the waters of Western Australia and showed eleutherobin (61) to be a potent cancer cell growth inhibitor against a diverse panel of tumour tissue cell lines. The mechanism of action was still undetermined at this point and  49 shortly thereafter, Kashman et al. isolated sarcodictyin A (55) (63)  from the South African soft coral Eleutherobia  biological activity. application  by  sarcodictyins,  54  and eleuthosides A (62) and B  aurea, again with no mention of any  This flurry of activity spanning 1995 and 1997 included a Patent  53  Pietra  et  al.  describing  the  microtubule-stabilizing properties  of  the  followed soon after by a journal article by Fenical et al. reporting the isolation  and paclitaxel-like activity of eleutherobin (61),  55  eleutherobin (61)  by Nicolaou's group.  syntheses of eleutherobin (61)  56  and the subsequent total synthesis of  Despite all this research interest and further total  and the sarcodictyins, " 57  59  the pre-clinical development of  eleutherobin (61) as an antimitotic agent had been impeded by its scarcity. In fact, the licensing agreement to advance the development of eleutherobin (61)  by Bristol-Myers Squibb was  recently terminated because "we couldn't get any of it" [Fenical].  60  Me  55 56 57 58  R  Me  R = H  R  Et  R = Ff  R  Me  R = OH  R  Me  R =OAc  59  2  2  2  2  Me i N  60  In addition to the five main structural classes listed above (taxane, epothilone, discodermolide, laulimalide and sarcodictyin/eleutherobin), there have been recent reports of other compounds that demonstrate the ability to stabilize microtubules. hexacyclic W S 9 8 8 5 B (64) from a Streptomyces species, estradiol,  62  61  These include the  some steroidal derivatives (65, 66) of  and certain polyisoprenylated benzophenones (67, 68) from a Malaysian plant.  63  It  is clear from the success of paclitaxel (4) that further investigations into microtubule-stabilizing compounds w i l l yield promising new anticancer therapeutics.  66  R = Ac  gg  A  36,37  2.2. Results and Discussion 2.2.1. Isolation of Antimitotic Compounds A s part of a general collection of marine invertebrates from the tropical waters off Dominica, specimens of an encrusting gorgonian were collected near Prince Rupert Bay using  51 SCUBA.  Samples were harvested by hand from the shallow water reefs, frozen on site, and  transported to Vancouver over dry ice.  The organism was identified as Erythropodium  caribaeorum (Duchassaing & Michelotti, 1860) by Dr. Leen van Ofwegan of the Nationaal Natuurhistorisch Museum in Leiden, The Netherlands.  Voucher samples are stored both at the  University of British Columbia in Vancouver and in Leiden. A n initial, smaller collection of E. caribaeorum in the summer of 1997 resulted in the isolation of nearly equivalent amounts of two diterpene glycosides, one new and one previously reported. What is described below is the equivalent workup on a second, larger collection later that same year, resulting in the isolation of a total of six novel compounds. Frozen samples of E. caribaeorum (5.3 kg wet weight) were thawed and extracted exhaustively with MeOFf over a period of several days. The combined methanolic extracts were concentrated in vacuo to give a dark brown gum (280 g). A sample of this concentrated gum showed potent activity in Roberge's cell-based antimitotic assay.  64  In addition, microscopic  examination of the cells arrested in mitosis exhibited evidence of tubulin bundling, similar to the characteristic  morphological effects  of paclitaxel (4).  The combined crude extract  suspended in two liters of water and partitioned against E t O A c (2 liters x 5). fraction was concentrated  was  The E t O A c  in vacuo, suspended in two liters of 9:1 M e O H / F L O , and then  partitioned against hexanes (2 liters x 5). Next, water was added to the remaining methanolic fraction in order to achieve a 6:4 M e O H / F L O ratio. This fraction was finally partitioned against two liters of C H C I 3 (2 liters x 5). Bioassay-guided fractionation showed the antimitotic activity was entirely present in the E t O A c , the 9:1 M e O H / H 0 , and the C H C I 3 fractions, respectively 2  (see Scheme 2.3).  Crude Methanol Extract  Octocoral Erythropodium caribaeorum  H 0  EtOAc  2  9:1 M e O H : H 0  Hexane  6:4 M e O H : H 0  CHCI3  2  2  Reversed-Phase chromatography  80%  70%  60%  50%  40%  30%  20 %  10%  H 0/MeOH  H 0/MeOHj  H 0/MeOH  H,0/MeOHi  H 0/MeOH|  H 0/MeOH|  H 0/MeOH|  H 0/MeOH|  2  2  2  2  2  2  2  100% MeOH  Normal Phase chromatography  2% MeOH/EtOAc  4% MeOH/EtOAc  6% MeOH/EtOAc  8 %  10%  12 %  14 %  MeOH/EtOAc  MeOH/EtOAc  MeOH/EtOAc  MeOH/EtOAc  H i g h Performance liquid chromatography  74 ( l m g )  71 (3mg) 72 (2mg)  61 (50mg) 69 (7mg)  70(6mg)  73 ( l m g )  Scheme 2.3. Isolation procedure for antimitotic diterpenoids from E. caribaeorum.  53 Fractionation of the active chloroform fraction (59 g) was accomplished with vacuum, reversed-phase, flash liquid chromatography by first adsorbing the active extract onto C-18 silica gel. The dried extract and powder were then subjected to liquid chromatography using step-gradient elution (80:20 F f 0 / M e O H to MeOFf in 10% increments). The antimitotic activity 2  was only observed in the 30:70 F E O / M e O H fraction. This active fraction was further subjected to normal phase, flash liquid chromatography using a step-gradient elution scheme (EtOAc to 20:80 M e O H / E t O A c  in 2% increments).  Instead  of using the traditional thin layer  chromatographic analysis and combination of like-fractions, a sample of each fraction was individually placed on a 96-well plate and screened for antimitotic activity. This was the first time this technique was used and was made possible by the development of the new antimitotic bioassay. The biological activity associated with each fraction is presented in Figure 2.3. A t this stage, the antimitotic activity was centered in the three fractions corresponding to 6, 8 and 10% M e O H / E t O A c .  ' H N M R spectral screening of these and surrounding fractions revealed  signals for a family of related diterpene glycosides. 0.8  r-  <  0  10  20  30  40  50  Fraction # Figure 2.3. Antimitotic activity (reported as Absorbance^nm) of each fraction collected from liquid chromatography.  54 A  final  purification  step  involving  normal  phase,  high  performance  liquid  chromatography ( H P L C ) using isocratic elution conditions (7:93 M e O H / C H C l ) yielded pure 2  2  samples of 61 (50 mg), 69 (7 mg), 70 (6 mg), 71 (3 mg), and 72 (2 mg). The disappearance o f certain signals in the *H N M R spectra appeared to suggest the presence of compounds that were partially decomposing when subjected to the H P L C conditions.  Thus, these fractions were  ultimately purified on cyano-bonded-phase H P L C using a 56:42:2 EtOAc/hexane/('Pr) NH 2  solvent mixture system as the eluent and gave small amounts of pure 73 (1 mg) and 74 (1 mg).  OH  (E)  71 R j = H ; R = A c ; R = M e ; A 2  3  72 R , = A c ; R = H ; R = M e ; A ' ' (Z) 2  2  2.2.2.  3  3  Structure Elucidation of Antimitotic Diterpenoids The structures of eleutherobin (61), desmethyleleutherobin (69), desacetyleleutherobin  (70), isoeleutherobin A (71), Z-eleutherobin (72), caribaeoside (73), and caribaeolin (74) were solved by extensive analysis of 1-dimensional and 2-dimensional N M R spectroscopic data recorded in D M S O - d g at 500 M H z . L o w and high resolution F A B mass spectrometry provided important information on the molecular formula of the compounds. Proton spin systems were identified from C O S Y experiments.  data and proton-carbon attachments  were determined by H M Q C  The H M B C data proved invaluable for determining the connectivity within the  55 compounds and also in the assignment of any quaternary carbons present.  The relative  stereochemistry and conformational information was obtained by analysis of N O E difference, R O E S Y , and X-ray diffraction experiments presented later in this chapter. A s mentioned earlier, an initial, smaller scale workup of the crude extract from E. caribaeorum resulted in the isolation of nearly equivalent amounts of the two diterpene glycosides, desmethyleleutherobin (69) present in much larger quantities  and eleutherobin (61). in the  Although compound 61 was  second, larger scale isolation, the  structural  determination of 69 preceded that of the previously reported eleutherobin (61) and thus will be presented in detail first.  2.2.2.1. Desmethyleleutherobin OH  Desmethyleleutherobin (69) was isolated as a white amorphous solid that gave an [ M + H ] ion in the H R F A B M S at m/z 643.32230 appropriate for a molecular formula of +  C34H46N2O10  ( A M -1.21 ppm) and thirteen degrees of unsaturation. A quick analysis of the I D and 2D N M R spectra indicated the presence of two esters, eight olefinic protons, one methyl singlet attached to a nitrogen heteroatom, and one acetal suggesting a glycoside moiety. A summary of all N M R assignments, C O S Y , and H M B C correlations for desmethyleleutherobin (69) can be found in Table 2.1.  56 Beginning with the signals furthest downfield in the ' H N M R spectrum, there are two singlets (5 7.68 and 5 7.57 ppm) integrating for one proton each and showing H M Q C correlations to carbon signals in the aromatic region of the  C N M R spectrum.  HMBC  correlations were observed from these two carbons into a singlet proton resonance occurring at 5 3.66 ppm, appropriate for a methyl group attached to a nitrogen atom (see Figure 2.4). The two proton resonances at 5 7.68 and 5 7.57 ppm showed H M B C correlations into a quaternary carbon at 5 136.9 ppm, thus suggesting the presence of an Af(l)-methylimidazole functional group. The H M B C experiment revealed further connectivity to this quaternary carbon from two isolated olefinic protons appearing at 5 7.51 and 8 6.34 ppm. The coupling constant for these protons was determined to be 15.5 H z , corresponding to an £-olefinic configuration. Finally, both o f these proton resonances displayed H M B C correlations to an ester carbon at 5 166.0 ppm, forming an .E-o^P-unsaturated ester. The resonance effects of this conjugated system result in the observed downfield shift o f the P-proton. Together with the attached methyl-substituted imidazole, the a,P-unsaturated ester forms an extended conjugated system displaying a strong U V absorbance at 290 nm. Substructure A of 69 was thus identified as a /V-methylurocanic acid ester functionality.  HMBC  COSY  Figure 2.4. Selected H M B C and C O S Y correlations for substructure A of compound 69.  57 Investigation of the ' H N M R spectrum indicated several proton resonances between 5 3.3 and 8 5.0 ppm, characteristic o f a sugar moiety. A detailed analysis o f the various coupling constants, C O S Y correlations, and chemical shift considerations indicated the presence of an a2"-0-acetyl-arabinoside.  The  1 3  C N M R and H M Q C spectra supported this by exhibiting a  diagnostic anomeric carbon resonance at 8 92.9 ppm linked to its anomeric proton at 8 4.71 ppm.  This proton appears as a doublet, with a coupling constant value of 3.2 H z , and C O S Y  correlations were observed between this proton and a proton further downfield at 8 4.82 ppm. The downfield proton showed additional C O S Y correlations to a third proton resonance at 8 3.73 ppm and appeared as a doublet of doublets with coupling constants of 3.2 and 10.0 Hz, respectively. The large J value locks the downfield proton into an axial position together with the third proton, and thus establishes an equatorial configuration for the anomeric proton (see Figure 2.5).  The presence of an acetate ester linked to the glycoside was suggested by the  downfield proton and carbon chemical shifts observed at the 2" position ( ' H : 8 4.82;  1 3  C : 8 70.8  ppm). This was further supported by L R F A B M S evidence of a mass spectral fragment at m/z 451 corresponding to the loss of a C H i i 0 7  COSY  6  moiety (cleavage of the - O — C I " - bond).  HMBC  COSY  Figure 2.5. Selected H M B C and C O S Y correlations for substructure B of compound 69.  A key heteronuclear correlation was observed from an up field proton at 8 3.44 ppm into the anomeric carbon resonance.  This upfield proton and its geminal partner (8 3.62 ppm)  58 established the 5" position of the glycoside and exhibited typical geminal coupling (/ = 11.9 Hz) and C O S Y  correlations.  The downfield  partner  characteristic for a deshielded equatorial substituent.  was assigned  the  equatorial position  The multiplicity of the axial H - 5 "  resonance in the ' H N M R , coupling constant value o f 2.6 H z , and C O S Y correlation into § 3.73 ppm revealed a neighbouring equatorial proton at the H - 4 " position. Both the H - 4 " and H - 3 " proton resonances lie in the 8 3.70-3.75 ppm region and their coupling constant values and C O S Y correlations are obscured by signal overlap. However, the full complement of I D and 2D N M R spectral data fully confirms substructure B of compound 69 as an a-2"-6>-acetylarabinoside. Analysis of the remaining atoms and degrees of unsaturation required by the molecular formula indicated substructures A and B of compound 69 account for all of the nitrogen atoms, eight of the ten oxygen atoms, and seven of the thirteen sites of unsaturation. What remains is a C20 unit and, based on the related chemistry of E. caribaeorum and other gorgonians, this suggested a diterpenoid structure. Inspection of the *H and  1 3  C N M R spectra in addition to the C O S Y and H M Q C  experiments revealed an isolated methylene group (8 4.24 and 5 3.77 ppm) attached to an oxygen heteroatom and a quaternary carbon center. H M B C correlations were observed between these methylene protons and the anomeric carbon of substructure B , thus establishing the glycosidic linkage (see Figure 2.6). These methylene protons also showed H M B C correlations to an olefinic carbon signal at 8 133.0 ppm whose attached proton (8 5.29 ppm) only exhibited homonuclear coupling to a neighbouring allylic methine ( ' H : 8 3.95,  1 3  C : 8 33.5 ppm). H M B C  correlations were also present from the methylene protons to a carbon at 8 111.6 ppm that perhaps suggested a tetrahedral carbon deshielded by two oxygen atoms of a hemiketal/ketal. Heteronuclear correlations o f a hydroxyl proton to the carbons at 8 111.6 and 8 135.0 ppm supported the hemiketal structure and led to the assignment of the quaternary allylic carbon.  59  /"  \  COSY  Figure 2.6. Selected H M B C and C O S Y correlations for substructure C of compound 69.  The H N M R spectrum of compound 69 showed evidence of an isolated cw-olefin from ]  the multiplicity and the characteristic value of the coupling constant (7 = 5.6 H z , typical for a five-membered ring).  These olefinic proton resonances at 5 6.07 and 8 6.11 ppm displayed  H M B C correlations into the carbon resonance at 8 111.6 ppm and an additional signal at 8 88.6 ppm.  Due to the cw-configuration of the olefin and the limit of two oxygen atoms unaccounted  for in substructures A and B , the five-membered cyclic hemiketal system was identified. A methyl singlet at 8 1.35 ppm in the ' H N M R spectrum exhibited H M B C correlations into the downfield carbon of the c/s-olefin, the deshielded carbon resonance at 8 88.6 ppm, and an additional carbon signal at 8 80.6 ppm. The proton (8 4.61 ppm) attached to this carbon atom provided the link to substructure A in the form of a heteronuclear correlation to the ester carbon of the /V-methylurocanic acid moiety.  With all of the heteroatoms accounted for and the  connectivities to substructures A and B established, all that remained was a Cio hydrocarbon portion providing three additional degrees of unsaturation. Investigation o f the C O S Y spectrum revealed that the proton at 8 4.61 ppm was correlated into a proton at 8 1.32 ppm. This proton is one of the two methylene protons attached to the carbon at 8 31.1 ppm and is also coupled into a neighbouring methine proton at 8 2.45  60 ppm (see Figure 2.7). The final methyl singlet present in the ' H N M R spectrum (8 1.47 ppm) exhibited H M B C correlations into the downfield shifted carbon (8 38.4 ppm) attached to the neighbouring methine proton, a quaternary carbon resonance at 8 133.8 ppm, and the remaining olefinic carbon signal at 8 120.8 ppm.  The proton attached to this olefinic carbon was the  starting point o f a series of C O S Y correlations that flowed to a vicinal methylene group (5 2.28 and 8 1.95 ppm), onto a methine proton (8 1.14 ppm), and ultimately to the single proton resonance at 8 3.95 ppm identified earlier in substructure C .  The remaining upfield signals  present in the ' H N M R spectrum (8 0.92 and 8 0.93 ppm) were readily identified as belonging to an isopropyl group attached to the carbon at 8 42.1 ppm. A final H M B C correlation from a methylene proton at 8 1.32 ppm into the carbon resonance at 8 33.5 ppm completed the cyclohexene ring and accounted for all the degrees of unsaturation expected from the molecular formula.  Figure 2.7. Selected H M B C and C O S Y correlations for substructure D of compound 69.  The I D and 2 D N M R data obtained for compound 69, shown in Figures 2.8 to 2.12, was in  complete  agreement  with  the  structure  of  desmethyleleutherobin.  The  relative  stereochemistry of desmethyleleutherobin (69) was found to be identical to that of eleutherobin (61), presented later in this chapter.  A summary of all N M R assignments and correlations for  desmethyleleutherobin (69) can be found in Table 2.1.  Table 2.1. N M R data for desmethyleleutherobin (69) recorded in DMSO-J6. position 1 2 3' 4 4-OH 5 6 7 8 9a b 10 11 12 13a b 14 15a b 16 17 18 19 20 r 2' 3' 4' 5' 6'-NMe 7' 1" 2" 3" • 3"-OH 4" 4"-OH 5"eq ax 1"' 2"'  5'H (7 value in H z ) 3.95 (m) 5.29 (d, 7=9.1) a  6.40 (s) 6.07 (d, 7=5.6) 6.11 (d, 7=5.6) 4.61 (d, 7=7.2) 1.46 (m) 1.32 (m) 2.45 (m) 5.26 (m) 2.28 (m) 1.95 (m) 1.14 (m) 4.24 (d, 7=12.5) 3.77 (d, 7=12.5) 1.35 (s) 1.47 (s) 1.46 (m) 0.93 (d, 7=6.7) 0.92 (d, 7=6.7)  COSY Correlations  HMBC Correlations  33.5 133.0 135.0 111.6  H2 HI  H9b H15a/15b H15a, 4-OH H2, 5,6, 15b, 4-OH  131.0 133.5 88.6 80.8 31.1  H6* H5*  H6 H5, 8, 16 H5, 6, 8, 16 H9a*, 16*  6 C 1 3  38.4 133.8 120.8 23.9 42.1 68.2  25.1 21.7 28.8 20.3 22.0 166.0 6.34 (d, 7=15.5) 113.7 7.51 (d, 7=15.5) 137.6 136.9 7.57 (s) 124.8 3.66 (s) 33.2 7.68 (s) 140.0 4.71 (d, 7=3.2) 92.9 4.82 (dd, 7=3.2,10.0) 70.8 3.73 (m) 68.6 4.78 (d, 7=3.6) 3.76 (m) 66.2 4.88 (d, 7=6.4) 3.62 (d, 7=11.9) 63.1 3.44 (dd, 7=11.9,2.6) 170.0 2.01 (s) 21.0  b  H9b H9b H9a, 8, 10 H9b H13b H13b, 14 H13a, 12, 14 H13a/b, 1 H15b H15a  H8, 17 H17 H17  H2, 19, 20 H2 H8  H19, 20 H18 H18  H19, 20 H20 H19 H8, 2', 3'  H3' H2' H 3 \ 5', 7' 6'-NMe  H2" H I " , 3" H2", 3"-OH, 4"* H3" H3"*, 4"-OH, 5"ax H4" H5"ax H5"eq, 4"  6'-NMe H15a, 5"ax H5"ax H2", 5"ax  H2'"  recorded at 500 M H z . recorded at 100 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap.  a  62  64 OH  8.0  6.0  4.0  2.0  0.0  (ppm)  Figure 2.10. C O S Y spectrum of desmethyleleutherobin (69) recorded in D M S O - d 6 at 500 M H z .  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  Figure 2.11. H M Q C spectrum of desmethyleleutherobin (69) recorded in D M S C M 6 at 500 M H z .  66  Figure 2.12.  H M B C spectrum of desmethyleleutherobin (69) recorded in DMSO-J6 at 500 M H z .  67  2.2.2.2. Eleutherobin OH  Me i N  OH  N  61 The structural elucidation of 61 was solved by extensive analysis of I D and 2 D N M R spectral data and analysis of the mass spectrometric data. The final structure was found to have been previously published months earlier by other authors.  55  Eleutherobin (61) was isolated as a white amorphous solid that gave an [ M + H ] ion in +  the H R F A B M S at m/z 657.33719 appropriate for a molecular formula of  C35H48N2O10  (AM -  2.32 ppm). This molecular formula differed from the value obtained for desmethyleleutherobin (69) by the gain of fourteen mass units, corresponding to the addition of C H . 2  The *H N M R  spectrum of 61 differed from the ' H N M R spectrum of desmethyleleutherobin (69) only by the presence of the singlet methyl resonance at 5 3.09 ppm, consistent with a methoxyl substituent. The  mass  spectral  and  'H  N M R data  also  suggested  that  61  was  identical  to  desmethyleleutherobin (69) except for the presence of a methoxyl group instead of a hydroxyl group at C-4 on the diterpene core. Detailed analysis of the H M Q C and H M B C experimental data confirmed the presence of this group, showing a three-bond correlation from the methoxyl proton into the quaternary ketal carbon at 8 115.6 ppm (see Figure 2.13). A l s o , the replacement of a hydrogen atom by a methyl group results in the observed downfield shift of the ketal carbon from 8 111.6 ppm in desmethyleleutherobin (69) to 8 115.6 ppm in eleutherobin (61).  68  x  ( ^~^\ /  X  HMBC COSY  Figure 2.13. Selected H M B C and C O S Y correlations for the cyclic ketal subfragment of compound 61.  The I D and 2 D N M R data obtained for 61 was in complete agreement with this assignment and all spectra reacquired in CDCI3 matched those previously reported in that solvent.  55  A detailed analysis of the relative stereochemistry by R O E S Y and N O E difference  experiments can be found in Section 2.2.4. correlations for eleutherobin (61)  A summary of all N M R assignments  and  in D M S O - J 6 can be found in Table 2.2 and additional 2D  spectra can be found in the Appendix.  Table 2.2. N M R data for eleutherobin (61) recorded in D M S O - J 6 . position 1 2  3 4 4-OMe 5 6 7 8 9a b 10 11 12 13a b 14 15a b 16 17 18 19 20 r 2'  3' 4' 5' 6'-NMe 7' 1" 2" 3" 3"-OH 4" 4"-OH 5"eq ax 1"'  5'H (7 value in H z ) 3.88 (m) 5.39 (d, 7=9.4) a  3.09 (s) 6.08 (d, 7=5.9) 6.28 (d, 7=5.9) 4.65 (d, 7=7.3) 1.49 (m) 1.32 (m) 2.45 (m) 5.27 (m) 2.27 (m) 1.95 (m) 1.14 (m) 4.16 (d, 7=12.6) 3.78 (d, 7=12.6) 1.37 (s) 1.47 (s) 1.45 (m) 0.93 (d, 7=6.6) 0.91 (d, 7=6.6) 6.35 (d, 7=15.6) 7.53 (d, 7=15.6)  5 C 13  b  33.5 134.9 133.3 115.6 49.1 130.6 133.6 89.2 80.7 30.9  COSY Correlations  HMBC Correlatior  H2, 14 HI  H9a H15a H15a H2, 5, 6, 15  H6 H5  H6 H5, 8 H5, 6, 8, 16 H9a, 16  H9b H9a  38.4 133.7 120.9 23.9 42.0 67.7 23.9 21.5 28.8 20.2 21.9 166.0 113.5 137.8 136.8 124.8 33.2 140.0 92.9 70.8 68.5  H8 H17 H17  HI H15b H15a  H8 H19, 20 H18 H18  b  H19, 20  H8, 2', 3' H3' H2'  7.57 (s) 3.66 (s) 7.69 (s) 4.70 (d, 7=3.0) H2" 4.82 (dd, 7=3.0,9.4) H I " , 3" 3.73 (m) H2", 3"-OH, 4"* 4.83 (d, 7=3.2) H3" 3.76 (m) H3"*, 4"-OH, 5"ax 66.1 4.92 (d, 7=6.0) H4" 3.59 (d, 7=11.8) 63.0 H5"ax 3.44 (dd, 7=11.8,2.2) H5"eq, 4" 170.1 2.01 (s) 20.9 T" recorded at 500 M H z . recorded at 100 M H z . * unambiguous assignments not possible due to signal overlap.  a  H2, 19, 20 H2  H 2 \ 3', 5', 6'-NMe 6'-NMe H15a, 5"ax  H2", 5"ax  HI" H2", 2"'  71  72 2.2.2.3. Desacetyleleutherobin OH  Me i  OH  N N  70 Desacetyleleutherobin (70) was isolated as a white amorphous solid that gave an [ M + H ] ion in the H R F A B M S at +  m/z 615.32813  appropriate for a molecular formula of  C33H46N2O9  ( A M -0.05 ppm). This molecular formula differed from the value obtained for eleutherobin (61) by the loss of 42 mass units, corresponding to the loss of C H 0 . 2  2  The ' H N M R spectrum of 70  displayed a strong resemblance to the H N M R spectrum of eleutherobin (61) except for the !  absence of a methyl singlet resonance at 5 2.01 ppm that had been assigned to the 2 " acetyl substituent and differences in the chemical shifts of the signals attributed to the arabinose protons. The mass spectral and ' H N M R data thus suggested that 70 was simply the desacetyl analogue of eleutherobin (61).  Detailed analysis of the C O S Y and H M Q C experimental data  confirmed the absence of this group by showing a homonuclear correlation from the anomeric proton at 8 4.57 ppm to the neighbouring 2 " proton at 8 3.55 ppm (see Figure 2.16). The upfield shift in both the H - 2 " and C - 2 " resonances demonstrated they no longer experienced the deshielding effects of an attached, electron-withdrawing acetyl group. Relative to the H - 2 " and H - 3 " axial proton resonances, the H N M R chemical shift assignments for H - 4 " and H-5"eq !  displayed the expected downfield shifts of equatorial substituents.  73  COSY Figure 2.16. Selected C O S Y correlations for the arabinose subunit of compound 70.  In order to confirm the identity of the arabinose sugar moiety, compound 70 was acetylated  with  acetic anhydride in pyridine.  The resulting product  was identical to  diacetyleleutherobin (75) prepared by acetylation of eleutherobin (61) using the same reaction conditions (see Section 2.2.2.8), thus confirming the proposed structure of desacetyleleutherobin (70).  The 2D N M R data obtained for 70 was in complete agreement with this assignment.  A  summary of all N M R assignments and correlations for desacetyleleutherobin (70) in DMSO-c/6 can be found in Table 2.3 and additional 2 D spectra can be found in the Appendix.  Table 2.3. N M R data for desacetyleleutherobin (70) recorded in D M S O - J 6 . position  5'H (7 value in H z ) 3.89 (m) 5.51 (d, 7=9.2) a  5 C 13  b  COSY Correlations  HMBC Correlations  1 33.3 H2, 14 2 H15a/15b 132.3 HI 3 H15a 133.9 4 115.6 H2, 5, 6, 15b, 4-OMe 4-OMe 3.09 (s) 49.0 5 6.23 (d, 7=6.0) 130.9 H6 H6 6 6.26 (d, 7=6.0) 134.0 H5 H5, 8, 16 7 89.1 H5, 8, 16 8 4.66 (d, 7=6.6) 80.7 H9a/9b H16 9a 1.51 (m) 31.1 H9b, 8 b 1.41 (m) H9a, 8 10 2.46 (m) 38.4 H8 11 133.5 H17 12 5.26 (m) 121.2 H13b H17 2.35 (m) 13a 23.9 b 1.93 (m) H12 14 1.14 (m) 42.2 HI H2, 19, 20 15a 4.16 (d, 7=12.9) 67.9 H15b H2 b 3.83 (d, 7=12.9) H15a 16 1.37 (s) 23.9 17 1.47 (s) 21.7 18 1.49 (m) 28.7 H19, 20 H19, 20 19 0.94 (d, 7=6.9) 20.2 H18 20 0.92 (d, 7=6.9) 22.0 H18 r 166.0 H8, 3' 2' 6.35 (d, 7=15.7) 113.5 H3' 3' 7.53 (d, 7=15.7) 137.8 H2' 4' 136.8 H2',3\5',7' 5' 7.56 (s) 124.8 6'-NMe 6'-NMe 3.66 (s) 33.1 7' 7.68 (s) 140.0 6'-NMe 1" 4.57 (br.d) 97.1 H2" H15a 2" 3.55 (m) 69.2* H I " , 3"* * 2"-OH 3" 3.55 (m) 68.5* H2"* * 3"-OH 4" 3.66 (m) 68.1* * 4"-OH 5"eq 3.54 (d, 7=12.3) 63.1 H5"ax ax 3.37 (dd, 7=12.3,3.3) H5"eq recorded at 500 MHz. recorded at 100 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap or poor signal to noise. a  b  77  2.2.2.4. Isoeleutherobin A  OH  Me i  OAc  NN  71 Isoeleutherobin A (71) was isolated as a white amorphous solid that gave an [ M + H] ion in the H R F A B M S at m/z 657.33834 appropriate for a molecular formula of  +  C35H48N2O10  ( A M -0.58 ppm). This molecular formula was identical to that of eleutherobin (61).  The ' H  N M R spectrum of 71 displayed a strong resemblance to the *H N M R spectrum of eleutherobin (61)  except for differences in the chemical shifts of the signals attributed to the arabinose  protons.  The mass spectral and ' H N M R data also suggested that 71 differed only in the  acetylation pattern on the arabinose fragment.  Detailed analysis of the C O S Y and H M Q C  experimental data confirmed the absence of the acetyl group from the C - 2 " position by showing a homonuclear correlation from the anomeric proton at 5 4.64 ppm to the neighbouring 2" proton at § 3.81 ppm (see Figure 2.19). The upfield shift in both the H - 2 " and C - 2 " resonances relative to those observed in eleutherobin (61) deshielding effects  showed they no longer experienced the  of an attached, electron-withdrawing acetyl group.  Likewise,  COSY  correlations observed between the axial C - 5 " methylene proton at 5 3.38 ppm and a methine resonance at 8 3.83 ppm revealed the acetate was not at C-4". This H - 4 " methine resonance also displayed a C O S Y correlation to a downfield resonance at 5 4.80 ppm, which was thus assigned to the remaining H - 3 " position. The significant deshielding of both the H - 3 " and C - 3 "  78 resonances of compound 71 relative to the corresponding chemical shifts in eleutherobin  (61)  indicated the electron-withdrawing acetate group was present at C - 3 " .  Figure 2.19. Selected C O S Y correlations for the arabinose subunit of compound 71.  The splitting pattern observed in the ' H N M R spectrum for H - 3 " was consistent with axial-axial coupling to H - 2 " (/ = 10.1 Hz) and axial-equatorial coupling to H - 4 " (7 = 2.6 Hz). However, in order to confirm the identity of the arabinose sugar moiety, compound 71 was acetylated  with  acetic  anhydride in pyridine.  The resulting product  was identical to  diacetyleleutherobin (75) prepared by acetylation of eleutherobin (61) using the same reaction conditions (see Section 2.2.2.8), thus confirming the proposed structure of isoeleutherobin A (71).  The 2 D N M R data obtained for 71 was in complete agreement with this assignment.  A  summary of all N M R assignments and correlations for isoeleutherobin A (71) in D M S O - d 6 can be found in Table 2.4 and additional 2 D spectra can be found in the Appendix.  Table 2.4. N M R data for isoeleutherobin A (71) recorded in D M S O - J 6 . position  5'H (7 value in H z ) 3.90 (m) 5.54 (d, .7=10.1) a  8 C l 3  b  COSY Correlations  HMBC Correlations  1 33.1 H2 2 H15a/15b 132.3 HI 3 H15a 133.7 4 115.7 H2, 5,6, 15b, 4-OMe 4-OMe 3.09 (s) 49.0 5 6.23 (d, 7=5.7) 130.7 H6 H6 6 6.26 (d, 7=5.7) 133.9 H5 H5, 8 7 89.2 H5, 6, 8, 16 8 4.67 (d, 7=7.1) 80.8 H9b H16 9a 1.52 (m) 31.1 H9b b 1.43 (m) H9a, 8, 10 10 2.47 (m) 38.4 H9b H8, 17 11 133.3 H17 12 5.28 (m) 121.4 H13b H17 13a 2.37 (m) 23.9 HI 3b, 14 b 1.93 (m) H13a, 12 14 1.14 (m) 42.1 H13a H2, 19, 20 15a 4.17 (d, 7=12.7) 67.6 H15b H2 b 3.89 (d, 7=12.7) H15a 16 1.37 (s) 23.9 17 1.47 (s) 21.7 18 1.48 (m) 28.8 H19, 20 H19, 20 19 0.94 (d, 7=6.6) 20.2 H18 20 0.92 (d, 7=6.6) 22.0 H18 r 166.0 H8, 3' 2' 6.35 (d, 7=15.4) 113.6 H3' 3' 7.53 (d, 7=15.4) 137.6 H2' 4' 136.8 H 2 \ 5' 5' 7.57 (s) 124.8 6'-NMe 6'-NMe 3.66 (s) 33.2 7' 7.70 (s) 140.0 6'-NMe 1" 4.64 (d, 7=3.2) 97.0 H2" H15a 2" 3.81 (m)* 65.9* H I " , 2"-OH, 3"* 2"-OH 4.90 (d, 7=5.8)* H2"* 3" 4.80 (dd, 7=2.6,10.1) 72.7 H2", 4"* 4" 3.83 (m)* 64.9* H3", 4"-OH, 5"ax 4"-OH 4.96 (br.d)* H4"* 5"eq 3.62 (d, 7=12.2) 63.1 H5"ax ax 3.38 (m) H5"eq, 4"* 1"' 170.2 H2'" 2" 2.01 (s) 21.0 recorded at 500 M H z . recorded at 125 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap or poor signal to noise. 1  b  80  81  2.2.2.5. Z-eleutherobin OH O  -V  72 Z-eleutherobin (72) was isolated as a white amorphous solid that gave an [ M + H ] ion +  in the H R F A B M S at m/z 657.33830 appropriate for a molecular formula of 0.65 ppm).  C35H48N2O10  This molecular formula was again identical to that of eleutherobin (61).  (AM The H l  N M R spectrum of 72 displayed a strong resemblance to the ' H N M R spectrum of eleutherobin (61)  except  for differences  in the chemical shifts  methylurocanic acid ester residue.  of the signals attributed  to the  N-  Detailed analysis of the ' H N M R spectrum suggested that  the only structural change was in the configuration o f the A  2 3  olefin, changing from an E-  configuration with a coupling constant of 15.6 H z in eleutherobin (61) to a Z-configuration with a coupling constant of 12.6 H z in compound 72. Further inspection of the 2 D N M R data confirmed this change in configuration and allowed the assignment of all the nuclear resonances.  In particular, the three-bond  HMBC  correlation from the olefinic proton at 8 6.94 ppm into an imidazole ring carbon (8 126.0 ppm) revealed this proton to be H - 3 ' (see Figure 2.21). The methine proton attached to the imidazole carbon was found at 8 8.25 ppm, most likely a result o f its spatial proximity to the anisotropic deshielding effects of the carbonyl functionality. Finally, H M B C correlations from the methyl singlet at 8 3.71 ppm and from the highly deshielded methine proton at 8 8.25 ppm established connectivity to the remaining imidazole carbons.  82  3.71  Figure 2.21. Selected H M B C and C O S Y correlations for the /Y-methylurocani.c acid ester subunit of compound 72.  The N M R sample of compound 72 partially isomerized over time to eleutherobin  (61),  thus confirming the assigned structure of 72 as Z-eleutherobin. The 2 D N M R data obtained for 72 was in complete agreement with this assignment.  A summary of all N M R assignments and  correlations for Z-eleutherobin (72) in DMSO-<i6 can be found in Table 2.5 and additional 2D spectra can be found in the Appendix.  Table 2,5. N M R data for Z-eleutherobin (72) recorded in D M S O - J 6 . position 1 2 3 4 4-OMe 5 6 7 8 9a b 10 11 12 13a b 14 15a b 16 17 18 19 20 1' 2' 3' 4' 5' 6'-NMe 7' 1" 2" 3" 3"-OH 4" 4"-OH 5"eq ax 1"'  T"  5'H  a  (J value in H z ) 3.92(m) 5.40 (d, 7=9.4)  3.09 (s) 6.07 (d, 7-5.6) 6.18 (d, 7=5.6) 4.66 (d, 7=7.2) 1.51 (m) 1.33 (m) 2.47 (m) 5.29 (m) 2.28 (m) 1.95 (m) 1.14 (m) 4.15 (d, 7=12.6) 3.78 (d, 7=12.6) 1.36 (s) 1.49 (s) 1.47 (m) 0.95 (d, 7=6.6) 0.93 (d, 7=6.6) 5.75 (d, 7=12.6) 6.94 (d, 7=12.6) 8.25 (s) 3.71 (s) 7.69 (s) 4.70 (d, 7=3.0) 4.82 (dd, 7=3.0,9.6) 3.76 (m) 4.81 (d, 7=2.8)* 3.78 (m) 4.90 (d, 7=6.0)* 3.59 (d, 7=12.0) 3.44 (dd, 7=12.0,2.1)  5 C I 3  b  33.6 134.9 133.3 115.6 49.1 130.7 133.4 89.2 80.8 30.8 38.4 133.8 121.1 23.9 42.4 67.8 24.0 21.3 28.8 20.4 21.8 165.4 112.3 138.9 136.2 126.0 33.2 138.6 93.2 70.9 68.5 66.3 63.2 170.0 20.8  COSY Correlations  HMBC Correlations  H2  H9a H15a/15b H15a H2, 5,6, 15b, 4-OMe  H6 H5  H6 H5, 8, 16 H5, 6, 8, 16 H16  H9b H9b H9a, 8  H13b H13b H13a, 12 H15b H15a  H19, 20 H18 H18  H8, 17 H17 H17 H18 • H2, 19, 20 H2  H19, 20 H20 H19 H8  H3' H2' H5' 6'-NMe  H2" H I " , 3" H2", 3"-OH*, 4"* H3"* H3"*, 4"-OH* H4"* H5"ax H5"eq  6'-NMe HI5a, 5"ax  H5"ax  HI" H2'"  2.00 (s) recorded at 500 M H z . recorded at 125 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap.  a  b  84  85 2.2.2.6. Caribaeoside  OH  Me i  OH  N N  13  73 Caribaeoside (73)  was isolated as a colourless glass that gave an [ M + H ] ion in the +  H R F A B M S at m/z 673.33474 appropriate for a molecular formula of ppm).  C35H48N2O11  ( A M 1.64  This molecular formula differed from the value obtained for eleutherobin (61) by the  gain of sixteen mass units, indicating the presence of an additional oxygen atom.  Detailed  analysis of the N M R data revealed that caribaeoside (73) was a diterpene glycoside possessing the same A^-(6')-methylurocanic acid ester and 2"-0-acetylarabinose subunits present on the diterpene core of eleutherobin (61).  However, it was evident that the central core of  caribaeoside (73) differed from eleutherobin (61) in the C - l 1 to C-13 region. In comparing the ' H N M R spectra of 73 and eleutherobin (61), two characteristic features were missing: the C - l 7 olefinic methyl resonance at 6 1.47 ppm and the H-12 olefinic methine signal at 8 5.27 ppm. The 2 D N M R data for caribaeoside (73) revealed these signals were replaced by a "methyl singlet resonance at 8 0.82 ppm and a pair o f overlapping olefinic methine resonances around 8 5.52 ppm.  Several key H M B C correlations from the methyl  singlet at 8 0.82 ppm defined the changes observed in caribaeoside (73) (see Figure 2.23). This Me-17  singlet showed correlations into an olefinic carbon resonance at 8 137.4 ppm, a  quaternary carbon resonance at 8 68.3 ppm, and a methine resonance at 8 45.7 ppm.  The  olefinic carbon resonance was linked to one o f the protons at 8 5.52 ppm by an H M Q C  86 correlation, the quaternary carbon resonance suggested a hydroxyl substituent (consistent with the molecular formula), and the methine carbon bore a proton resonating at 8 2.07 ppm in the ' H N M R spectrum. This methine proton showed C O S Y correlations into a neighbouring methine proton at 8 4.00 ppm, corresponding to H - l of eleutherobin (61),  and into two methylene  protons, corresponding to H-9ab. The second olefinic proton at 8 5.53 ppm led to a series of C O S Y correlations that ultimately defined an isopropyl group from two overlapping methyl doublets at 8 0.94-0.95 ppm. Thus, the resulting structure for compound 73 maintained the C - l , C-2, C I O , and C14 centers of eleutherobin (61),  shifted the cw-olefin to C-12 and C-13, and  added a hydroxyl substituent to C - l 1.  5.52  5.53  HMBC  5.52  5.53  COSY  Figure 2.23. Selected H M B C and C O S Y correlations for the cyclohexene ring subunit of compound 73.  The relative stereochemistry about the cyclohexene ring of caribaeoside was determined by analysis of R O E S Y and scalar coupling constant data. R O E S Y correlations were observed between the isopropyl methyl proton resonances (Me-19 and Me-20) and the proton resonances at 8 4.00 and 8 2.07 ppm ( H - l and H-10, respectively; see Figure 2.24). This demonstrated that the isopropyl group, H - l , and H-10 were on the same face of the caribaeoside molecule, as in eleutherobin (61).  Additional R O E S Y correlations between H-8 and both H-10 and Me-16, and  between Me-16 and OMe-21 confirmed that caribaeoside (73)  and eleutherobin (61)  had  87 identical relative stereochemistries at these centers.  The Me-17 resonance at 8 0.82 ppm  showed a strong R O E S Y correlation to the H-2 proton resonance, indicating the Me-17 and H-2 lie cis to each other.  Typically, the methyl resonance would be expected at ~ 8 1.2 ppm,  however, models of caribaeoside (73) show that the methyl group can sit in the shielding region of the A ppm). (73)  2 , 3  olefin, resulting in a significant upfield shift for the methyl proton resonance (8 0.82  Finally, the chemical shifts and vicinal coupling constant of H - l and H-2 in caribaeoside were nearly identical to those seen in eleutherobin (61),  indicating the dihedral angle  between these protons was essentially the same for both compounds. A summary of all N M R assignments and correlations for caribaeoside (73) can be found in Table 2.6.  Figure 2.24. Selected R O E S Y correlations for the diterpene core of compound 73.  Table 2.6. N M R data for caribaeoside (73) recorded in DMSO-c/6. position  5'H (7 value in H z ) 4.00 (m) 5.38 (d, 7=9.7) a  6 C 13  b  COSY Correlations  HMBC Correlations  H2, 10 33.8 1 136.3 HI 2 H15a/15b 131.3* 3 H4-OMe 4 115.4 4-OMe 3.08 (s) 49.0 6.13 (d, 7=5.5) H6 5 130.9 6.28 (d, 7=5.5) H16 6 133.8 H5 H16 7 90.0 4.85 (d, 7=7.5) 78.7 H9b H16 8 9a 1.56 (rn) H9b, 10 29.1 1.38 (m) b H9a, 8, 10 10 2.07 (m) 45.7 H9a/9b, 2 H17 11 H17 68.3 4.32 (s) 11-OH 12 5.52 (s) 137.4 H17 13 5.53 (d, 7=6.3) 125.6 H14 14 1.54 (m) 46.5 H19, 20 H13, 18 4.14 (d, 7=12.4) 15a 68.4 H15b b 3.71 (d, 7=12.4) H15a 16 1.33 (s) 24.1 17 0.82 (s) 25.0 18 1.68 (m) 32.1 H19, 20, 14 0.94 (d, 7=6.8) 19 21.1 H18 20 0.95 (d, 7=6.8) 21.8 H18 1' 165.6* H2' 2' 6.36 (d, 7=15.5) 113.5 H3' 3' 7.52 (d, 7=15.5) 137.7 H2' 4' 136.9* H5' 7.57 (s) 5' 124.6 6'-NMe 3.66 (s) 33.2 7' 7.69 (s) 140.0 6'-NMe 1" 4.76 (d, 7=3.3) 94.4 H2" 2" 4.82 (m) 71.1 H I " , 3"* 3.74 (m) 3" 68.6 H2"*, 3"-OH*, 4"* 3"-OH 4.84 (br.d)* H3"* 4" 3.76 (m) 66.3 H3"*, 4"-OH* 4"-OH 4.92 (d, 7=5.8)* H4"* 5"eq 3.59 (d, 7=12.4) 63.1 H5"ax ax 3.47 (m) H5"eq 1"' 170.1 H2'" 2"' 2.03 (s) 20.9 recorded at 500 M H z . recorded at 125 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap or poor signal to noise. a  b  90  Figure 2.26.  Expanded R O E S Y spectrum of caribaeoside (73) recorded in DMSO-J6 at 500 M H z .  91  2.2.2.7. Caribaeolin Me ,OAc  N  20  19 13  74 Caribaeolin (74) was isolated as a clear oil that gave an [ M + H ] ion in the H R F A B M S +  at  m/z  541.29111 appropriate for a molecular formula of  C30H40N2O7  ( A M -0.49 ppm). This  molecular formula differed from the value obtained for caribaeoside (73) mass units, indicating a loss of C H 0 4 . 8  5  caribaeolin (74)  by the loss of 132  Detailed analysis of the N M R data revealed that  was a diterpenoid possessing a Af-(6')-methylurocanic acid ester subunit  attached to the diterpene core of caribaeoside (73) but was missing the 2"-0-acetylarabinose. Several key H M B C correlations from the protons of a methylene group at 5 4.46 ppm defined the changes observed in caribaeolin (74)  (see Figure 2.27).  This methylene group  showed correlations into an olefinic carbon resonance at 5 135.7 ppm, a quaternary olefinic carbon resonance at 8 131.4 ppm, and a carbon at 8 115.6 ppm that suggested a tetrahedral carbon deshielded by two oxygen atoms of a ketal.  Further inspection of the  HMQC  experimental data revealed this C H group was attached to an oxygen atom, as expected by the 2  carbon chemical shift of 65.0 ppm.  Finally, H M B C correlations were observed between a  carbonyl resonance at 8 169.9 ppm and both the methylene protons at 8 4.46 ppm and a methyl singlet resonance at 8 1.97 ppm, thus establishing the presence of a C - l 5 acetyl substituent for caribaeolin (74) in place of the C-l5 arabinose sugar residue found in caribaeoside (73).  Figure 2.27. Selected H M B C correlations around C-15 of compound 74. The relative stereochemistry of caribaeolin (74) was determined by analysis of R O E S Y data and found to be identical to caribaeoside (73).  The Me-17 resonance at 5",0.77 ppm showed  a strong R O E S Y correlation to the H-2 proton resonance, indicating the Me-17 and H-2 lie cis to each other as in caribaeoside (73) (see Figure 2.28). R O E S Y correlations were also observed between the isopropyl methyl proton resonances (Me-19 and Me-20) and the proton resonances at 5 4.01 and 5 2.07 ppm ( H - l and H-10, respectively). This demonstrated that the isopropyl group, H - l , and H-10 were on the same face of the caribaeolin molecule, as in eleutherobin (61).  Additional R O E S Y correlations between H-8 and both H-10 and Me-16, and between M e -  16 and OMe-21 confirmed that caribaeolin (74) and caribaeoside (73) share the same relative stereochemistry. A l l the 2 D N M R data was in complete agreement with the structure proposed for 74 and a summary of all N M R assignments and correlations for caribaeolin (74) can be found in Table 2.7.  ROESY  Figure 2.28. Selected R O E S Y correlations for the diterpene core of compound 74.  Table 2.7. N M R data for caribaeolin (74) recorded in DMSO-r/6. position 1 2 3 4 4-OMe 5 6 7 8 9a b 10 11 11-OH 12 13 14 15a b 16 17 18 19 20 r  5H (J value in Hz) 4.01 (m) 5.38 (d, 7=10.0)  5 C  3.08 (s) 6.16 (d, 7=5.9) 6.34 (d, 7=5.9)  49.1 130.4 134.6 90.2 78.4 29.2  !  a  4.85 (d, 7=7.7) 1.55 (m) 1.41 (m) 2.07 (m) 4.31 (s) 5.53 (m) 5.53 (m) 1.54 (m) 4.46 (s) 4.46 (s) 1.34 (s) 0.77 (s) 1.69 (m) 0.95 (d, 7=6.6) 0.96 (d, 7=6.6)  13  b  33.4 135.7 131.4 115.6  45.5 68.3 137.3 125.7 46.2 65.0 23.8 24.8 32.0 20.9 21.8 165.6 113.6 137.8 136.9 124.6 33.1 140.0 169.9 20.6  COSY Correlations H2, 10 HI  H6 H5 H9b H9b, 10 H9a, 8, 10 H9a/9b, 2  HMBC Correlations H9a*, 14* H15a/15b H15a/15b H2, 5,6, 15a/15b, 4-OMe H6 H5, 8, 16 H5, 6, 8, 161 H16  H8, 17 H17 H17  H14 H13 H15b* H15a*  H12*, 13*, 19, 20 H2  H19, 20 H18 H18  11-OH H19, 20 H20 H19 H8, 3'  2' 6.35 (d,.7=15.7) H3' 3' 7.53 (d, 7=15.7) H2' 4' H5',7' 5' 7.56 (s) 6'-NMe 6'-NMe 3.66 (s) 7' 7.68 (s) 6'-NMe 1"' 2"' 1.97 (s) recorded at 500 MHz. recorded at 125 MHz; exact assignments based on HMQC and HMBC data. * unambiguous assignments not possible due to signal overlap or poor signal to noise.  a  b  94  Figure 2.30. Expanded R O E S Y spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  96  2.2.2.8. Diacetyleleutherobin OAc  Me i  OAc  N  75 Diacetyleleutherobin  (75)  was  formed  from  three separate acetylation  performed on eleutherobin (61), desacetyleleutherobin (70), and isoeleutherobin A (71).  reactions These  compounds were acetylated with acetic anhydride in pyridine in order to confirm the identity of the arabinose sugar moiety.  The discussion presented below specifically refers to data from  acetylated eleutherobin (61),  however, a comparison of the I D and 2 D N M R data revealed all  the acetylation products were identical. Diacetyleleutherobin A (75) was isolated as a white amorphous solid that gave an [ M + H ] ion in the H R F A B M S at m/z 741.33834 appropriate for a molecular formula of +  C39H52N2O12  ( A M -1.58 ppm). This molecular formula differed from the value obtained for eleutherobin (61) by the gain of 84 mass units, corresponding to the gain of two  C2H2O  spectrum of 75 differed from the spectrum of eleutherobin (61)  units.. The ' H N M R  by the presence of two  additional singlet methyl resonances at 8 2.08 and 8 1.95 ppm and the absence o f two hydroxyl signals. In addition, the signals attributed to H - 3 " and H - 4 " on eleutherobin (8 3.73-3.76 ppm) were replaced by two overlapping signals further downfield at 8 5.22 ppm. The M S and N M R data suggested that 75 was the fully acetylated analogue of eleutherobin  (61).  Detailed analysis of the N M R experimental data confirmed the presence of two additional acetate groups on the arabinose sugar at the C - 3 " and C - 4 " positions. The C O S Y  97 spectrum showed homonuclear correlations from the anomeric proton at 8 4.87 ppm to the neighbouring 2 " proton at 8 4.96 ppm (see Figure 2.31).  Likewise C O S Y correlations were  observed between this axial 2" proton and the deshielded H - 3 " resonance at 8 5.22 ppm. The splitting pattern observed in the H N M R spectrum for H - 2 " was consistent with axial-equatorial !  coupling to H - l " (7= 3.4 Hz) and axial-axial coupling to H - 3 " (J= 10.7 H z ) .  o  COSY  HMBC  Figure 2.31. Selected C O S Y and H M B C correlations for the arabinose subunit of compound 75.  The H M B C data showed correlations from the axial 5" methylene proton at 8 3.60 ppm to both the anomeric C - l " (8 92.9 ppm) and the neighbouring C - 4 " (8 66.5 ppm), whereas the equatorial H - 5 " at 5 3.85 ppm displayed correlations into C - 3 " (8 68.6 ppm).  The ' H N M R  chemical shifts of the arabinose protons (H-2", H - 3 " , and H-4") showed they all experienced the significant deshielding effects of an electron-withdrawing acetyl group. A summary of all the N M R assignments and correlations for diacetyleleutherobin (75) in DMSO-rf6 can be found in Table 2.8 and additional 2 D spectra can be found in the Appendix.  Table 2.8. N M R data for diacetyleleutherobin (75) recorded in D M S O - d 6 . Position  5'H (7 value in Hz) 3.90 (m) 5.43 (d, 7=9.5) a  5 C 1 3  b  COSY Correlations  HMBC Correlations  H2 33.4 1 H15a 135.4 HI 2 H15a 133.1 3 H5,6, 15b, 4-OMe 115.5 4 3.10 (s) 49.1 4-OMe H6 H6 6.08 (d, 7=5.9) 130.5 5 H5, 16 H5 6.30 (d, 7=5.9) 133.9 6 H5, 6, 8, 16 89.3 7 H16 H9b 4.66 (d, 7=7.6) 80.7 8 H9b 9a 1.51 (m) 30.9 H9a, 8, 10 b 1.30 (m) 2.47 (m) H9b H8, 17 10 38.3 H17 133.7 11 H17 12 5.30 (m) 121.0 H13b 13a 24.0 H13b .2.29 (m) b 1.96 (m) H13a, 12 14 H19, 20 1.15 (m) 42.0 15a 4.24 (d, 7=12.7) H15b H2 67.9 3.85 (d, 7=12.7) H15a b 16 1.38 (s) 24.0 17 1.48 (s) 21.4 18 1.49 (m) 28.8 H19, 20 H19, 20 0.93 (d, 7=7.0) H20 19 20.2 H18 20 0.92 (d, 7=7.0) 21.9 H18 H19 166.0 H3' r 2' 6.35 (d, 7=15.6) 113.5 H3' 3' 7.53 (d, 7=15.6) 137.9 H2' 4' 136.9 H2',3',5\7' 5' 7.56 (s) 6'-NMe 124.8 6'-NMe 3.66 (s) 33.2 7' 7.68 (s) 140.1 6'-NMe 4.87 (d, 7=3.4) 1" 92.9 H2" H15a/15b, 5"ax 2" 4.96 (dd, 7=3.4,10.7) 67.6 H I " , 3" 2"-OAc H2'" 2.02 (s)* 169.8*, 20.6 3" 5.22 (m) H2" 68.6 H5"eq 3"-OAc 169.8*, 20.6 H3"' 2.08 (s)* 4" 5.22 (m) H5"ax 66.5 4"-OAc 169.6*, 20.4 H5"ax H4'" 1.95 (s)* 5"eq 3.85 (d, 7=12.8) 60.2 H5"eq ax 3.60 (dd, 7=12.8,1.5) recorded at 500 M H z . recorded at 125 M H z ; exact assignments based on H M Q C and H M B C data. * unambiguous assignments not possible due to signal overlap or poor signal to noise. a  b  99  100  102  2.2.2.9. Known compounds Sarcodictyin A Me N  "4  ,OMe  N  O 2(1  19  55 Sarcodictyin A (55)  was isolated as a clear o i l that gave an [ M + H ]  +  ion in the  H R F A B M S at m/z 497.26514 appropriate for a molecular formula of C 8 H 3 6 N 0 ( A M -0.04 2  ppm).  2  6  Detailed analysis of the I D and 2 D N M R data recorded in D M S O - J 6 identified the N-  methylurocanic acid ester functionality and diterpene core common to the previously described. evidence  eleutherobins  However, instead of a sugar substituent attached to C-15, there was  of a methyl ester.  In addition, compound  functionality also present in desmethyleleutherobin (69).  55  displayed the cyclic hemiketal  The final structure of sarcodictyin A  (55) was ultimately confirmed by comparison of spectra obtained in pyridine-t/5 with previously published data.  50  A complete assignment of the ' H and  Experimental Section.  1 3  C resonances can be found in the  103  Erythrolide A and E  Erythrolide A (36) was isolated as a white, amorphous solid that gave an [ M ] ion in the +  H R E P M S at m/z 538.15613 appropriate for a molecular formula of C 6 H i O C l ( A M -0.34 2  ppm).  3  Detailed analysis of the I D and 2 D N M R data recorded in C D C 1  1 0  3  identified three  acetates, one ketone, a y-lactone, two double bonds, and a single chlorine atom.  The  characteristic signals of an exocyclic olefinic methylene in the H N M R spectra and a shielded !  quaternary carbon in the from E. caribaeorum.  C N M R spectra suggested one of the previously isolated erythrolides The final structure of erythrolide A (36)  comparison of spectra with previously published data. ' 7  1 3  41  was ultimately confirmed by  A complete assignment of the ' H and  C resonances can be found in the Experimental Section.  Erythrolide E (41)  was isolated as a white, amorphous solid that gave an [ M ] ion in the +  H R E I M S at m/z 496.15080 appropriate for a molecular formula of C24H29O9CI ( A M -0.26 ppm). Detailed analysis of the I D and 2 D N M R data recorded in C D C I 3 indicated the presence of two acetates, one ketone, a y-lactone, one double bond, and a chlorine atom.. The final structure of erythrolide E (41) previously published data.  39  in the Experimental Section.  was ultimately confirmed by comparison of spectra with  A complete assignment of the ' H and  l 3  C resonances can be found  104  2.2.3. Biological Activity In the search for biologically active secondary metabolites from marine and terrestrial sources, over 24,000 crude extracts were screened for antimitotic activity using the new cellbased assay developed in the laboratory of M i c h e l Roberge.  The majority of antimitotic  64  agents known at this time had been discovered by serendipity, by cytotoxicity screening, or by displaying a similar activity profile in a 60 cell-line panel to other known antimitotic agents. Roberge's newly developed, rational drug screen identified 119 extracts that demonstrated significant  antimitotic  activity.  These  extracts  were  then  rescreened  using  tubulin  immunofluorescence microscopy to examine their effects on microtubule structure. The crude 65  methanolic extract of a Caribbean octocoral, Erythropodium caribaeorum, was selected for further investigation based on the morphological characteristics displayed by cells treated with this extract. The E. caribaeorum extract produced paclitaxel-like bundling of microtubules that suggested a similar mechanism of action. A t this point, the only antimitotic agents known to exhibit  this  mechanism  of action  were  paclitaxel (4),  the  epothilones  (52,  53),  and  discodermolide (17). A normal cell that has not been exposed to an antimitotic agent morphologically displays a complete, fibrous-like network of microtubules that permeates throughout the cell.  Cells  treated with an agent such as paclitaxel (4) that stabilizes microtubules against depolymerization exhibit distinctive bundling and stacking of the microtubules. treated with eleutherobin (61)  66  Figure 2.35 shows A549 cells  and visualized using tubulin immunofluorescence microscopy.  The appearance of these microtubules is consistent with the morphology of microtubules treated with paclitaxel (4).  On the contrary, cells treated with an antimitotic agent that promotes  destabilization of microtubules, such as nocodazole, result in a diffuse, ubiquitous white staining of tubulin throughout the cell as a result of the impaired polymerization into microtubule fibers.  105  Figure 2.35. hnmunofluorescence photograph of A549 cells after exposure to eleutherobin (61) at a concentration of 60 ng/ml. 67  The antimitotic assay was found to be well-suited for bioassay-guided fractionation of the crude E. caribaeorum extract and for quantitative evaluation of antimitotic activity of the pure compounds isolated. This is a useful measure of a compound's pharmacological potential since it not only evaluates the interaction of a compound and its target, as an in vitro targetbased assay would do, but also its ability to permeate the cell and interact with its target inside the environment of a cell. For the bioassay, human breast cancer MCF-7 cells were treated at various concentrations of the compounds for 20 hours and the extent of mitotic arrest was determined by ELICA. The activity profiles of eleutherobin (61) and the six related diterpenes (69-74), as well as diacetyleleutherobin (75) and sarcodictyin A (55), were determined and appear in Figure 2.36.  106  100  80  #— Eleutherobin (61) *?— Desmethyleleutherobin (69) •0— Desacetyleleutherobin (70) Isoeleutherobin A (71) Z-Eleutherobin (72) -•—Caribaeoside (73) — Caribaeolin (74) •—Diacetyleleutherobin (75) 6— Sarcodictyin A (55)  60  ' 40  20  o t ^ ^ - f f . ,r 0.01  0.1  1  0.1  0.01  100  10  1  10  Concentration (uM)  Concentration ((iM)  Figure 2.36. Antimitotic activity of the compounds isolated from E. caribaeorum. Absorbance values were converted to percentage of mitotic cells using a standard curve. Bars, S D . 68  The results of the antimitotic activity of compounds 61, 69-75, and 55 revealed important information on the structural features required for biological activity. Analysis of the IC  5 0  values  obtained  from  desmethyleleutherobin (69),  the  graphs  (see  Table  2.9)  showed  eleutherobin  (61),  diacetyleleutherobin (75), and isoeleutherobin A (72) all displayed  very similar levels of biological activity, with values ranging from 20-50 n M . This suggested the hydroxyl substitution on the cyclic ketal of eleutherobin (61) and the acetyl group position on the arabinose sugar do not result in significant changes to the antimitotic activity of these compounds. A slight decrease in activity was observed, however, when the configuration of the C - 2 ' , 3 ' olefin group was changed from E to Z as in Z-eleutherobin (72), perhaps reflecting the preference for a specific configuration to access the active site on the tubulin target. Similarly, removal of the acetate group found at the 2" position on the sugar moiety of eleutherobin results in a slight loss of antimitotic activity, as shown by desacetyleleutherobin (71).  (61)  This may  107 indicate an unfavourable interaction that occurs because of the presence of an additional hydroxyl group on the arabinose subunit.  Table 2.9. Summary of antimitotic activity of the compounds isolated from E. caribaeorum. ic  Compound desmethyleleutherobin (69) eleutherobin (61) diacetyleleutherobin (75) isoeleutherobin A (71) Z-eleutherobin (72) desacetyleleutherobin (70) sarcodictyin A(55)  (in n M ) 20 30 30 50 250 400 2000  caribaeoside (73) caribaeolin (74)  20000 20000  s  s  Structural changes from eleutherobin (61)  5 0  C-4 hydroxyl instead of methoxyl group C - 3 " and C - 4 " acetyl groups on arabinose C - 3 " acetyl group instead of C-2?' Z-configuration of C - 2 ' , 3 ' olefin C - 2 " hydroxyl instead of acetyl group C-15 methyl ester instead of arabinose, C-4 hydroxyl instead of methoxyl group C - l l hydroxyl and C-12,13 olefin C - l l hydroxyl and C-12,13 olefin, C-15 acetate group instead of arabinose  synthetically acetylated  More significant decreases in antimitotic activity were seen compounds.  In sarcodictyin A (55),  with the remaining  the sugar residue is replaced with a methyl ester and a  hydroxyl group replaces the C-4 methoxyl group, resulting in a 100-fold decrease in biological activity. Since desmethyleleutherobin (69) was fully active, it is the C-15 linked 2"-0-acetylarabinopyranose side chain and not the C-4 methoxyl group that was required for activity. The most significant loss in antimitotic activity in this series of compounds was associated with the caribaeoside (73) and caribaeolin (74) structures. The introduction of a hydroxyl group at C - l 1 and migration o f the olefin group to the A ' 1 2  1 3  position resulted in nearly a 1000-fold decrease in  antimitotic potency relative to eleutherobin (61).  This change altered both the shape and  polarity of the diterpene core and revealed for the first time the critical importance of the C - l l to C - l 3 region for microtubule-stabilizing activity. The additional replacement of the arabinose  108 fragment in caribaeoside (73) with a simple acetate residue, as in caribaeolin (75), resulted in no further loss of activity. Experiments on M C F - 7 cells showed the cytotoxicity I C compounds corresponded to their antimitotic I C  values for all these  5 0  values, suggesting cell death was ultimately a  5 0  result of the overstabilization of microtubules. Indeed, cells treated with eleutherobin (61) for one hour and then washed clean of the drug would eventually all arrest in mitosis and die, suggesting irreversible damage to its microtubule targets. Results of the antimitotic activity of eleutherobin (61) and desmethyleleutherobin (69) against a multi-drug resistant ( M D R ) cell line indicated a greater than 10-fold drop in antimitotic activity, consistent with previous results reported for eleutherobin (61).  57  This M D R phenotype is associated with the overexpression of  P-glycoprotein, an energy-dependent drug efflux pump that maintains a low intracellular drug concentration.  69  Since the discovery of eleutherobin (61) by Lindel et al. in 1997, there have been many 55  synthetic analogues that have been prepared as part of structure-activity relationship ( S A R ) studies. '  70 71  However, to date they have all been based on the eleutherobin/sarcodictyin  diterpenoid core. Recently, Ojima et al. proposed a common pharmacophore for microtubulestabilizing compounds that accommodates the paclitaxel (4), eleutherobin (61), epothilone (52, 53),  and discodermolide (17)  structural classes.  72  Their model used molecular dynamics to  predict three areas of common overlap that are important domains for binding to tubulin (labeled A, B, and C in Figures 2.37 and 2.38).  Figure 2.37. Overlay of nonataxel (cyan) with a) paclitaxel b) epothilone B c) eleutherobin and d) discodermolide, all in y e l l o w . 72  110 The antimitotic compounds isolated from E. caribaeorum (61, 69-74, 55) appeared to agree with the proposed pharmacophore model. Changes in the A region identified by Ojima, such as altering the A activity.  2 , 3  ' configuration in Z-eleutherobin (72), resulted in a small decrease in  Changes in the arabinose fragment, representing alterations in the C region of the  pharmacophore, resulted in a little change (i.e., isoeleutherobin A (71)) all the way to a significant decrease in activity (i.e., sarcodictyin A (55)). Replacement of the methoxyl group by a hydroxyl group (i.e., desmethyleleutherobin (69)) had no real effect on the antimitotic potency and correspondingly, this alteration formally exists outside the proposed tubulin binding regions. The pharmacophore presented by Ojima suggested changes in the C - l l to C 13 region of eleutherobin should have an impact on the ability of analogues to act as microtubulin stabilizers. Indeed, caribaeoside (73) is the first such analogue ever tested in an antimitotic assay and its significant loss of activity associated with the introduction of a hydroxyl  group at C - l l and migration o f the olefin  to A  1 2 1 3  further  strengthens the  pharmacophore model. Although these structural analogues were found to support Ojima's proposal, the creation of such a predictive pharmacophore model depends on comprehensive knowledge about the conformation of all the known microtubule-stabilizing compounds. A t the time this model was proposed, there had been no published solid-state or solution conformational analyses of eleutherobin (61) and the molecular dynamics calculations used to define its lowest energy conformation were carried out without any N O E constraints.  2.2.4.  Solid-State and Solution Conformations During the course of characterizing the antimitotic diterpenes from E. caribaeorum,  several crystals of eleutherobin (61) were obtained by the fortuitous slow evaporation of a concentrated N M R sample dissolved in D M S O - J 6 . The crystals proved to be suitable for X-ray  Ill 73  diffraction analysis and provided the first solid-state conformation for eleutherobin (61). ' The computer-generated O R T E P for eleutherobin (61) is shown in Figure 2.39.  08  C20  Figure 2.39. O R T E P drawing of eleutherobin (61). Hydrogen atoms have been omitted for clarity.  In order to facilitate a comparison of the solid-state conformation of eleutherobin  (61)  with its solution conformation, a series of R O E S Y and difference N O E experiments were performed in both a polar and nonpolar solvent. N M R samples were prepared in both D M S O d6 and CDCI3 and then degassed, purged of oxygen, and subsequently flushed with argon and the tubes sealed.  The difference N O E results obtained in C D C 1  3  and the R O E S Y results  collected in CDCI3 and DMSO-c/6 are found in Table 2.10 along with the solid-state internuclear distances between protons measured from the X-ray diffraction analysis.  1 12  Table 2,10.  Difference N O E and R O E S Y data for eleutherobin (61) in C D C h and DMSO-J6. a  H- Position (5 in CDCI.0  b  H- Position (5 in CDCI.0  Internuclear Distance from X-ray data (A) H2 (5.54) "Hl7a'(2.29) ~ 2.12 H2 H14 (1.21) 2.61 2.44. H8 (4.80) HI (3.94) H10 (2.60) 2.30 H8 H16 (1.43) H8 2.52 2.64 H8 H19(0.96) H10(2.60) HI (3.94) 2.33 H10 2.30 H8 (4.80) H10 HI9 (0.96) 2.51 H13cc(2.29) H2 (5.54) 2.12 HI (3.94) H14 (1.21) 2.37 H19 (0.96) 2.02 HI H19 H8 (4.80) 2.64 H19 H10 (2.60) 2.51 H-20 (0.94) H13f3(1.97) 2.03 H16 (.1.43) OMe (3.20) 2.28  Difference NOE ( % enhancement) 7.49 3.51 4.28 6.16 0.91 0.52 4.79 2.16 0.77 . 9.69 6.40 0.97 0.32 0.42 0.96 1.01  0  a  d  d  ROESY correlations (CDCh)  ROESY correlations  y y y y • y y y y n  y y y y y n  y y y y n  y y  y y  y y  y y n  V  n n  Acquired at 400 M H z . Acquired at 500 M H z . ' H in first column was irradiated. p and a are defined as being above and below the plane of diterpenoid core as in the structural representations of 61. y = R O E S Y correlation clearly observed, n = R O E S Y correlation not clearly observed. a  b  c  d  Examination of the solid-state conformation represented in Figure 2.40 revealed the presence of a twist in the cyclohexene ring relative to the remaining diterpene core of eleutherobin (61).  Analysis of a number of key N O E s indicated that the solution conformation  of the diterpenoid core in both the polar and nonpolar solvent was extremely similar, if not identical, to the solid-state conformation.  In particular, irradiation of the H - l 3 a resonance  resulted in a significant enhancement of the H-2 resonance, highlighting the close proximity of these protons that was also evident in the solid-state conformation. Detailed analysis of this X ray diffraction experiment revealed a torsional angle of -61° for the C 2 - C 1 - C 1 4 - C 1 3 bonds, thus o  placing H-13a a distance o f only 2.118 A from H-2 and consistent with the strong difference N O E observed (see Figure 2.40).  Additionally, irradiation of the H-8 resonance resulted in  113  noticeable enhancements in the H-10, H - l , and Me-19 resonances, in full accord with the spatial arrangement of the isopropyl group relative to H-8 seen in the solid-state conformation. The X ray diffraction analysis gave a torsional angle of -66° for C8-C9-C10-C1 which placed H-8 a distance of 2.304 A from H-10 and 2.438 A from H - l , in good agreement with the solution difference N O E s observed. Furthermore, it was possible to explain the weak difference N O E present between the Me-19 proton resonances and the H-8 resonance by correlation to a solidstate C 9 - C 1 0 - C 1 - C 1 4 torsional angle of 177.7°.  Figure 2.40. Torsional angles around the cyclohexene ring of eleutherobin o  Distances are in A .  (61).  114  115  Figure 2.43. ROESY spectrum of eleutherobin (61) recorded in CDC1 at 500 MHz. 3  117  Figure 2.44. R O E S Y spectrum of eleutherobin (61) recorded in D M S O - J 6 at 500 M H z .  118 Taken together, the combination of the H-2 to H - l 3 a and the.H-8 to H-10, H - l , and M e 19 difference N O E s represent an extremely restrictive set of conformational restraints.  The  interproton distances necessary for the observed N O E s between these protons are extremely difficult to attain with any significant changes from the solid-state conformation. In addition, the 2 D R O E S Y experiments in C D C 1 and D M S O - d 6 revealed the corresponding correlations 3  between H-2 to H - l 3 a and the H-8 to H-10, and H - l , providing further evidence of a major contributing solution conformation for the diterpenoid core that is essentially identical to the solid-state conformation. In light of details provided by the solid-state and solution conformational analyses of eleutherobin (61),  the pharmacophore proposed by Ojima et al. for microtubule-stabilizing  agents was re-evaluated.  A s mentioned earlier, the molecular dynamics calculations used to  obtain a conformation for eleutherobin (61) were performed without using any torsional angle or 72  N O E constraints.  Analysis of the eleutherobin conformation presented in their paper indicated  a torsional angle approaching 180° for the C2-C1-C14-C13 bonds and a torsional angle greater than -90° for the C 8 - C 9 - C 1 0 - C 1 substructure (see Figure 2.45). Such a conformation would not be expected to show the difference N O E ' s or R O E S Y correlations that were described earlier. Indeed, a significant difference exists in the spatial orientation of the C11-C12-C13 tubulin binding region identified and illustrated by Ojima compared to the solid-state and solution conformation present here.  This key difference in the diterpene core conformation relative to  the urocanic acid side chain and arabinose moiety of eleutherobin (61) suggests the solid-state and solution conformation would display dramatically different overlay fits with the other microtubule-stabilizing structures used to construct the Ojima pharmacophore.  119  Figure 2.45. Comparison of the conformations of eleutherobin (61). Left: the Ojima pharmacophore conformation (yellow overlayed with nonataxel in cyan); Right: the solid-state conformation obtained by X-ray diffraction analysis.  Recently, two papers have been published proposing alternate pharmacophore models for the microtubule-stabilizing compounds. ' 74  75  Both reports are based on studies involving the  taxane and/or epothilone classes of antimitotic agents and extend their findings to the eleutherobin/sarcodictyin group. B y evaluating the biological activity of a taxane analogue and then using molecular modeling studies to investigate its binding to tubulin, He et al. observed that the C - l 3 side chain of paclitaxel (4) is not an absolute requirement for biological activity and that the C-2 side chain can play a critical role in the binding to tubulin. The authors proposed that the 14-member tricyclic ring system of eleutherobin (61), like the macrolide ring of the epothilones (i.e., 53), overlays with the taxane ring of paclitaxel (4) and that the urocanic acid side chain, like the thiazole side chain of the epothilones, mimics the C-2 side chain in paclitaxel (see Figure 2.46).  74  120  53 OH  61 Figure 2.46. Proposed common overlap (blue and red) for paclitaxel (4), epothilone B (53), and eleutherobin (61).  A n alternate pharmacophore model was presented in the work of Giannakakou et al who investigated the binding of taxane and epothilone analogues on (3-tubulin from an epothilone-resistant cell line.  75  Molecular modeling studies of different conformations and  docking studies on tubulin identified one preferred conformation for the epothilones. pharmacophore  The  model proposed was consistent' with structure-activity studies of various  analogues and predicted a single common theme for the taxanes, epothilones, and sarcodictyins (see Figure 2.47). The authors suggested the isopropyl group at C - l 4 of sarcodictyin A (55) corresponds to the gem-dimethyl groups of the epothilones (i.e., 53) and taxanes (i.e., 4) while the ether oxygen attached to C-4 and C-l correlates to the oxetane and epoxide oxygen atoms of the taxanes and epothilones, respectively.  It is important to note that the sarcodictyin A  121 conformation presented in this model is consistent with the X-ray crystal structure and solution conformation  presented  earlier  for  eleutherobin  (61).  This  model  also  shows  the  methylimidazole functionality of the sarcodictyins lies in the vicinity of the C - l 3 side chain of the taxanes and the aromatic thiazole side chain of the epothilones.  4  Me i  55 Figure 2.47.  Proposed common overlap (blue) and pharmacophore (red) for paclitaxel (4), epothilone B (53), and sarcodictyin A (55).  Since both these models were generated prior to the publication of this thesis research, it is anticipated that the availability of solid-state and solution conformation data for eleutherobin (61)  will facilitate further refinements of the microtubule-stabilizing pharmacophore models  and, consequently, aid the eventual design of new antimitotic compounds.  122 The sample of eleutherobin (61) isolated and purified from Erythropodium caribaeorum was subjected  to further  solution conformational analysis in D M S O - J 6  researchers at Emory University in Atlanta.  76  and C D C 1  3  by  Extensive I D and 2 D N M R experiments were  performed including five different R O E S Y spectra acquired with varying mixing times to determine the linearity of the cross-relaxation buildup. From these and additional experiments, a total of 40 intramolecular R O E distances and five side-chain three bond coupling constants ( 7('H, C)) were obtained. 3  13  Although these measured quantities are averages derived from a  rapidly equilibrating ensemble of conformations, the authors were able to deconvolute the average N M R spectra into constituent conformers using a combination of multiple force field conformational searching and the N A M F I S ( N M R analysis of molecular flexibility in solution) procedure. '  77 78  These  results  provided nine distinct and  preferred  conformations  for  eleutherobin (61) ranging in populations from 1-19%. The conformational diversity arose from the urocanic acid and arabinose side chains, with the nine-membered B-ring of the diterpene core existing as an invariant twist-boat-chair as seen in the solid-state (see Figure 2.48). The second most populated conformer obtained by this detailed analysis was the conformation observed by X-ray diffraction analysis (16%), again in full agreement with the solid-state and solution conformation determined in this thesis research.  123  Figure 2.48. Superposition of nine conformations of eleutherobin (61) as determined by N A M F I S analysis of R O E S Y and 7 ( ' H , C ) data. 3  I 3  76  The X-ray conformation is shown in red. a and b denote the two most populated arabinose locations.  2.2.5. Additional Results Since previous studies on octocorals have revealed significant geographic variation in the diterpenoid content,  79  evidence for the presence of eleutherobin (61)  was investigated by  H P L C and biological activity from two additional collections of Erythropodium caribaeorum. Crude methanolic extracts of E. caribaeorum from Florida and the Bahamas showed potent activity in the antimitotic assay and were subjected to bioassay-guided fractionation similar to the original isolation scheme.  The antimitotic activity corresponded to the fractions expected  for eleutherobin (61).  The final normal phase H P L C purification step resulted in a small peak  eluting at the  anticipated for standard  chromophore ( X  time m a x  eleutherobin  and exhibited the  same U V  290 nm). After collecting the entire H P L C run in a deep-well, 96-well plate,  124 the fractions were tested for antimitotic activity. The results confirmed activity associated with the fractions corresponding to eleutherobin  (61).  During the antimitotic screening of over 60,000 natural extracts obtained from the Open Repository Program of the N C I Developmental Therapeutics Program, it was found that eleven additional octocoral extracts exhibited activity in the assay. Since these extracts originated from organisms that were taxonomically related to Erythropodium caribaeorum, they might also contain related chemistry (see Figure 2.49).  Sarcodictyin A , for instance, was first described  from the Mediterranean stolonifer Sarcodictyon roseum by Pietra et al.,  50  later isolated from a 53  South African alcyonacean along with the eleuthosides A and B by Kashman et al.,  and now  reported from the Caribbean gorgonian E. caribaeorum (see Scheme 2.4 and Figure 2.50). Thus, the eleven additional active extracts may possess sarcodictyin or  eleutherobin-type  compounds.  Phylum  Class  Order  Cnidaria  Anthozoa Gorgonacea  Alcyonacea  Family  Genus  Species  Anthothelidae Gorgoniidae  Erythropodium caribaeorum Rumphella ? Rumphella sp. 8 Rumphella sp. e Isididae Mopsea whiteleggei Muricellisis sp. a Subergorgiidae Subergorgia sp. 1 cf. M o l l i s Subergorgia cf. M o l l i s Ellisellidae Junceella sp. d Verrucella sp. b Alcyoniidae Sinularia sp. c Sinularia sp. h  Figure 2.49. Taxonomy of the antimitotic extracts from the N C I Open Repository relative to E. caribaeorum.  Porifera (sponges)  PHYLUM  CLASS  Hydrozoa (hydras)  Cnidaria  Chordata (tunicates)  Mollusca (nudibranchs)  Scyphozoa Cubozoa (jellyfish)  Anthozoa (sea anemones, corals, sea fans)  Subclass Octocorallia  I ORDER  Alcyonacea (soft corals)  Stolonifera  Pennatulacea (sea pens)  Gorgonacea (sea fans) (sea whips)  Sarcodictyins Sarcodictyins Eleuthosides Eleutherobin Sarcodictyin A Eleutherobins Caribaeoside/Caribaeolin Scheme 2.4. Taxonomic scheme showing the related chemistry isolated from octocorals.  J)  IB  Figure 2.50. M a p of the geographic collection locations of sarcodictyin/eleutherobinproducing organisms: A : sarcodictyins A - F from a Mediterranean stolonifer; ' B: sarcodictyin A and eleuthosides A and B from a South African alcyonacean; C : eleutherobin from a Western Australian alcyonacean; D: sarcodictyin A , eleutherobin, and analogues from a Caribbean gorgonian. 50 51  55  80  126 In order to determine i f these extracts possessed compounds similar to eleutherobin or the sarcodictyins, a similar bioassay-guided fractionation was performed on the 20 mg samples provided by the N C I . Each sample was subjected to reversed-phase liquid chromatography using the same stepwise gradient elution scheme as with the large E. caribaeorum purification (80:20 H 0 / M e O H 2  to M e O H  in 10 % increments).  A s expected for eleutherobin or  sarcodictyin-type compounds, the antimitotic activity was found to almost exclusively reside in the 30:70 H 0 / M e O H fraction for each of the extracts. Thus, the polarity of the unknown active 2  compounds seemed to agree with the observed polarity range of eleutherobin (61).  The active  fractions were then subjected to normal phase H P L C using the solvent system developed for eleutherobin (7:93 M e O H / C H C l 2 ) and the entire run was collected in deep-well, 96-well plates 2  and submitted for bioassay. The results showed antimitotic activity co-eluting with the solvent front for each of the extracts except one.  This lack of retention on the column would be  consistent with the behavior of the sarcodictyins, however, the solvent system was optimized for eleutherobin (61) and thus did not indicate the presence of that compound. One extract, from the gorgonian Subergorgia cf. M o l l i s , did show activity in the fractions corresponding to the elution of eleutherobin (61)  under these standardized conditions, but the scarcity of material  prevented further characterization and identification of this active agent. Although subsequent requests for additional crude extract material from the N C I failed to provide sufficient material to establish the identity of the active agents, it appears as though eleutherobin (61)  is also  produced by the Subergorgia octocoral and sarcodictyin-type chemistry may be present in the other extracts. In addition to the in vitro antimitotic studies on the M C F - 7 human breast cancer cell line, there have been preliminary results from the in vivo studies of desmethyleleutherobin  (71).  This work by Dr. Peggy Olive of the B . C . Cancer Research Centre is currently investigating the effect of desmethyleleutherobin (69)  on S C C V H tumours in C 3 H mice.  These mice were  127 injected, either intraperitoneal or intravenous, with a single dose of desmethyleleutherobin (69) ranging from 10-50 mg/kg and 24 hours later, the tumour was excised and the cells were analysed.  The preliminary results show that desmethyleleutherobin (69)  kills 30% of the  tumour cells regardless of whether they were close to or distant from the blood supply. While this work is currently ongoing, it does strengthen the anticancer therapeutic potential of desmethyleleutherobin (69) by showing in vivo efficacy.  2.2.6. Future Studies Examination of the antimitotic properties of the Erythropodium caribaeorum extract resulted in the discovery of eleutherobin (61)  and a series of related antimitotic diterpenes.  Their interesting biological properties along with the conformational studies have spawned numerous avenues for continued investigation. While caribaeoside (73)  and caribaeolin (74)  were the first compounds featuring key structural changes to the diterpene core to be evaluated for microtubule-stabilizing biological activity, there are many changes to the framework of eleutherobin (61) that remain to be explored. Many synthetic studies have created analogues of the urocanic acid and arabinose side chains but modifications to the C-4 to C-l ether bridge, the three olefinic groups, and the tricyclic nature of the diterpene core have yet to be investigated. In light of the recent pharmacophore model suggesting the importance of the isopropyl group,  75  alterations to this area of the eleutherobin/sarcodictyins may provide further insights into defining a common model for microtubule-stabilization. In order to better define the solution conformation of eleutherobin (61),  additional  studies using R O E S Y and three-bond coupling constants are continuing by the researchers at Emory University.  Application of these solution and solid-state conformations to docking  studies of eleutherobin (61) to its f3-tubulin target would provide an even greater understanding of the features required for activity.  Studies are also in progress using isotopically-labeled  128 eleutherobin to investigate the nature and dynamics of the binding to its cellular target. These results will prove to be invaluable in the refinement of pharmacophore models for microtubulestabilization and the eventual design of new compounds with antimitotic activity. Adding to the discovery of a new and relatively abundant source of eleutherobin  (61)  from Erythropodium caribaeorum, obtaining sufficient crude extract material from the eleven related marine octocorals may confirm the presence of eleutherobin (61)  in at least one  additional source and may provide a series of new structural analogues that are beyond the imagination of synthetic chemistry. The isolation of eleutherobin from E. caribaeorum alone has revived the pharmaceutical interest in this exciting molecule and can provide sufficient material for clinical investigations. Successive collecting trips in the waters off Dominica have revealed the potential for the aquaculture of this organism, as was successfully implemented 81  with the bryostatin-producing Bugula neritina,  due to the rapid regrowth of harvested  82  material.  Comparisons of the amounts and types of compounds isolated from the new versus  old growth may reveal new analogues, biosynthetic precursors, or greater quantities of the active agents. Indeed, continuing studies in our lab on E. caribaeorum have led to the isolation of possible biosynthetic intermediates in the pathway to eleutherobin (61) and have shown that the C-4 methyl ketal in eleutherobin arises from the isolation procedure.  83  2.3. Conclusions In searching for new antimitotic compounds that act by stabilizing microtubules against depolymerization, a recently developed, rational cell-based screen was used to isolate the known antimitotic agent eleutherobin (61) organism.  and six novel structural analogues (69-74) from a marine  The Caribbean octocoral Erythropodium caribaeorum proved to be a new and  abundant source of eleutherobin, whose pre-clinical development had been impeded by its scarcity, and led to structure-activity studies on the various analogues that were also identified.  129 The structural variations in this group included caribaeoside (73),  the first analogue to  demonstrate the critical importance of the C - l l to C - l 3 region of the di terpenoid core, and offered  key insights  compounds.  into  proposed  pharmacophore  models  for microtubule-stabilizing  In addition, single crystal X-ray diffraction analysis and difference N O E  experiments provided the first solid-state and solution conformations for eleutherobin (61), revealing a twist-boat-chair orientation for the diterpene core that has been further supported by molecular modeling and conformational analyses. 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M . ; Bhattacharya, S.K.; Chen, X . - T . ; He, L . ; Shen, H.-J.; Gutteridge, C . E . ; Horwitz, S.B.; Danishefsky, S.J. Cancer Chemother. Pharmacol. 1999, 44, 223.  7 1  Nicolaou, K . C ; X u , J . Y . ; K i m , S.; Pfefferkorn, J.; Ohshima, T.; Vourloumis, D . ; Hosokawa, S. 7. Am. Chem. Soc. 1998, 120, 8661.  7 2  Ojima, I.; Chakravarty, S.; Inoue, T.; L i n , S.; He, L . ; Horwitz, S.B.; Kuduk, S.D.; Danishefsky, S.J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4256.  7 3  Cinel, B . ; Patrick, B . O . ; Roberge, M . ; Andersen, R . J . Tetrahedron Lett.  7 4  He, L . ; Jagtap, P . G . ; Kingston, D.G.I.; Shen, H.-J.; Orr, G . A . ; Horwitz, S.B. Biochemistry 2000, 39, 3972.  7 5  Giannakakou, P.; Gussio, R.; Nogales, E . ; Downing, K . H . ; Zaharevitz, D . ; Bollbuck, B . ; Poy, G.; Sackett, D . ; Nicolaou, K . C ; Fojo, T. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2904.  7 6  Cornett, B . ; Monteagudo, E . ; Cicero, D . O . ; Cinel, B . ; Andersen, R . J . ; Roberge, M . ; Milanesio, M . ; Liotta, D . C ; Snyder, J.P. J. Am. Chem. Soc. submitted.  7 7  Cicero, D . O . ; Barbato, G . ; Bazzo, R . J. Am. Chem. Soc. 1995, 117, 1027.  7 8  Nevins, N . ; Cicero, D . ; Snyder, J.P. J. Org. Chem. 1999, 64, 3979.  7 9  X u , L . ; Patrick, B . O . ; Roberge, M . ; Allen, T.; van Ofwegen, L . ; Andersen, R . J . Tetrahedron Lett. 2000, 56, 9031; and references cited therein.  2000, 41, 2811.  on  Cinel, B . ; Roberge, M . ; Behrisch, H . ; van Ofwegen, L . ; Andersen, R . J . Organic Lett. 257. 8 1  2000, 2,  Schaufelberger, D . E . ; Koleck, M . P . ; Beutler, J . A . ; Vatakis, A . M . ; Alvarado, A . B . ; Andrews, P.; Marzo, L . V . ; Muschik, G . M . J. Nat. Prod. 1991, 54, 1265.  " Williams, D . E . , personal communication. 8 3  Britton, R.; Roberge, M . ; Berisch, H . ; Andersen, R . J . Tetrahedron Lett. 2001, 42, 2953.  135  CHAPTER 3  3.1. G2 Cell Cycle Checkpoint Inhibitors from Stylissa flabelliformis A recently proposed strategy for cancer therapy involving compounds that inhibit the G2 cell cycle checkpoint led to the development of a new high-throughput assay to screen natural extracts for G 2 checkpoint inhibitors. flabelliformis,  The crude extract from a marine sponge, Stylissa  collected in the waters off Motupore Island in Papua N e w Guinea exhibited  potent activity in the new assay. Bioassay-guided fractionation of the active extract resulted in the isolation and identification of the natural product debromohymenialdisine and three related alkaloid inhibitors of the G 2 checkpoint.  These compounds were the first G 2 checkpoint  inhibitors to be found by a rational screen and were structurally distinct from previously known G 2 checkpoint inhibitors.  The discovery of these biologically active natural products has  contributed to the understanding of the G 2 checkpoint signaling pathway and provided essential new tools to probe the molecular basis of inhibition.  3.1.1. Introduction to Marine Sponges Sponges, which  constitute the phylum Porifera,  are the oldest living group of  multicellular organisms and, although commonly thought of as primitive, are very successful and highly evolved organisms.  1  In total, there are an estimated 5000-9000 species of marine  sponges, in addition to 150 or so freshwater species, that have adapted to all the diverse ecological habitats of the world's oceans. This ecological success may arise from the simple architecture and sedentary lifestyle characteristic of sponges.  Morphologically, sponges vary  greatly in size and shape, from thin encrusting sheets scarcely a millimeter in thickness to large barrels or vases that may grow to over a meter in height and diameter.  1  Most species are often  136 brightly coloured and while some members exhibit radial symmetry, the majority are irregular and display massive, erect, encrusting, or branching growth patterns.  2  Sponges are sessile, filter-feeding organisms that lack internal organs but possess a welldeveloped system of cells arranged to form a network of canals.  1  Water is pumped into the  body of the sponge through small pores, called oscula, by a single layer of flagellated cells (choanocytes).  The water is filtered for organic food particles and oxygen and then its  unidirectional journey ends with expulsion though exit pores called oscules.  The skeletal  support in sponges is composed of either calcareous or siliceous spicules and protein spongin fibers and often represents the primary method of species identification. Porifera  is divided into four main classes,  the largest  2  While the phylum  and most diverse class is the  Demospongiae consisting of over 90% of all sponge species. Studies on the Demosponges have revealed a great range in habitat, spectacular variability in morphology, and an exquisitely rich chemistry. A s seen previously in Figure 1.1, sponges are the most prolific source of marine natural products described in the recent literature, accounting for nearly 44% of the compounds reported.'  In addition to the thousands of unique compounds isolated from these organisms,  many of the marine sponge metabolites have biosynthetic origins without known terrestrial counterparts.  4  For example, the direct incorporation of isocyanide and isothiocyanide into  compounds from the marine sponge Acanthella cavernosa (76, 77) and the unique biosynthetic 5  pathway to the 3-alkylpiperidine alkaloids such as manzamine A (78) are exclusive to the 6  marine world.  137  Marine sponges have provided remarkably complex molecules possessing a variety of potent biological activities making them an extremely valuable resource structures or new molecular targets for drug discovery programs. potent anticancer agent A r a - C (13)  in finding lead  The development of the  from compounds originally isolated by Bergmann from a  Caribbean sponge was presented in Chapter One.  The diterpenoid manoalide (79)  originally reported by deSilva and Scheuer from the Indo-Pacific sponge Laffareilla  was  variabilis  1  and subsequently found to exhibit exciting anti-inflammatory properties by inhibiting the key enzyme phospholipase A . While manoalide (79) has not achieved therapeutic use, it is widely 2  used as a commercial reagent and biochemical tool by the pharmaceutical industry and may yet lead to new synthetic analogues for the treatment of inflammation.  4  Many marine sponge-  derived natural products are currently in pre-clinical evaluation as promising antimitotic agents, including the microtubule-stabilizing compounds discodermolide (17) discussed earlier in Chapter T w o . Halichondrin B (80),  and laulimalide  (54)  originally isolated from the Japanese  sponge Halichondria okadai, also acts as an antimitotic agent but inhibits the polymerization of 9  tubulin instead of stabilizing the microtubules against depolymerization.  10  These examples and  many others illustrate how marine sponge metabolites have played important roles in defining new molecular targets against disease and providing structurally novel lead compounds for drug discovery. In fact, the use of natural products, such as sponge-derived compounds, as essential  3.1.2. Review of the Oroidin Alkaloids from Marine Sponges  12  Marine sponges have become well known as a rich source of structurally diverse and pharmacologically interesting C 1 1 N 5 pyrrole-imidazole alkaloids.  Collectively, this large  structural class of marine natural products is known as the oroidin alkaloids, named after the first member of this fascinating group of secondary metabolites. Oroidin (81) was first isolated from the marine sponge Agelas oroides in 1971 other sponges throughout the w o r l d . " 14  21  13  and subsequently has been found in various  In the thirty years since the initial discovery of oroidin  (81), over 60 pyrrole-imidazole alkaloids have been isolated from marine sponges of the orders Agelasida, Axinellida, and Halichondria.  These compounds all appear to be biogenetically-  derived from a single common precursor. The simplest structural entity related to oroidin (81)  139 is 3-amino-l-(2-aminoimidazol-4-yl)prop-l-ene (82)  which lacks the pyrrole moiety.  This  22  derivative was isolated from the Axinellidae sponge Teichaxinella morchella  and is thought to  be a possible biosynthetic precursor to the entire oroidin family of compounds.  Recently, a  pioneering biosynthetic feeding study using a cell culture of T. morchella by Kerr et al. demonstrated the  incorporation  odiline/stevensine (83) 3.1).  23  of the  labeled amino  acids histidine  and  via 82 and 4,5-dibromopyrrole-2-carboxylic acid (84)  proline (see  into  Scheme  This research finding led to the proposal of a universal chemical pathway for the  biosynthesis of all the known oroidin alkaloids, represented as distinct structural groups in Scheme 3.2, with mechanisms outlining their formation from precursors 82 and  84.  12  H  83 proline  84  Scheme 3.1. Biogenetic precursors of odiline/stevensine from the sponge T. morchella.  12  Scheme 3.2. Representative structural groups of oroidin alkaloids. Thus, Nature's combinatorial chemistry seems to have elaborated an extensive group of diverse secondary metabolites from the economical use of these two simple precursors. Structurally, the oroidin alkaloids can also be conceived as derivatives of the Q1N5 skeleton of oroidin (81) through (i) isomerization of the double bond and/or oxidation/reduction; (ii) dimerization; and (iii) cyclization.  24  To date, there have been six modes of oroidin cyclization  discovered in nature and Fattorusso et al. have proposed these modes be classified based on the atoms involved in the linkage formation. pyrrole-imidazole  alkaloids  into  six  24  Consequently, the authors have categorized the  groups  to  form  the  hymenialdisine  (85),"  141  dibromoagelaspongine  (86),  26  dibromophakellin  (87),  27  dibromoisophakellin  (88),  28  cyclooroidin (89), and agelastatin (90) structural types (see Scheme 3.3). 24  88  29  89  90  Scheme 3.3. The six cyclization modes of the oroidin skeleton.  One of the oroidin cyclization modes, the C-4/C-10 linkage, gives rise to a unique class of tricyclic marine sponge alkaloids that includes the hymenialdisines (85, 91, 92), the axinohydantoins (93, 94), odiline/stevensine (83), and hymenin (95). These compounds share a common bicyclic pyrrolo[2,3-c]azepin-8-one ring system connected to a cyclic guanidine or hydantoin ring system.  Debromohymenialdisine (91) was the first member of this group  reported and was originally isolated in 1980 from the Caribbean sponge Phakellia flabellata.  30  It has since been isolated from marine sponges of the genera Hymeniacidon, ' Axinella, and 15  33  31  32  25  Pseudoaxinyssa" along with its related analogue hymenialdisine (85).  Recently, a number of  reports have appeared describing the modulation of protein kinase C and the proinflammatory  142  transcription factor, N F K B , by both debromohymenialdisine (91) and hymenialdisine (85).  34  The studies indicate that these compounds may well have beneficial effects in the treatment of inflammatory diseases and are promising new drug candidates  against  rheumatoid and  osteoarthritis. The X-ray crystal structure of hymenialdisine (85) has been reported numerous synthetic studies  15  along with  on both hymenialdisines and the related 3-bromohymenialdisine  (92), previously isolated from the marine sponge Axinella carteri?  6  91 R ; = R = H 85 Rj = B r , R = H 92 R[ = H , R = B r  93  2  94  83 A ' 9  95  2  A ' 1 0  1 0  1  2  The structurally related axinohydantoin (93) and debromoaxinohydantoin (94) have 32  37  been reported from various marine sponges along with the hymenialdisines. Axinohydantoin (93) exhibits moderate activity against a murine P-388 lymphocytic leukemia and its crystal structure features a reversal of configuration at the A  1 0  ' " olefinic group connecting the  pyrroloazepinone ring to the hydantoin group. This suggested axinohydantoin was not simply a hydrolysis product of the hymenialdisines, but may arise from a C-4/C-10 cyclization of an oxidized linear precursor similar to the oroidin-like midpacamide (96) isolated by Scheuer et 38  al.  Although debromoaxinohydantoin (94) was originally suggested to possess the same E-  A  olefinic configuration, a careful examination of the spectral data for axinohydantoin (93)  1 0 1 1  37  and debromoaxinohydantoin (94) reveals the compound reported in the literature should be reassigned  the Z - A ' 1 0  1 1  olefinic  configuration,  consistent  with  a later  report  of Z -  143 debromoaxinohydantoin.  39  Thus, evidence of f-debromoaxinohydantoin has yet to be reported.  Odiline/stevensine (83) ' was isolated independently by two groups in 1985 and, along with 33 40  hymenin (95), features the dibrominated pyrrole functionality found in oroidin (81). 16  In terms  of biological activity, hymenin (95) was originally described as exhibiting a-adrenoceptor blocking activity while ecologically, the family of C n N  5  pyrrole-imidazole alkaloids including  odiline/stevensine (83) have been implicated in deterring the feeding of predatory reef fishes.  H O 93  41  96  3.1.3. Review of G2 Checkpoint Inhibitors As seen earlier in Chapter One, cells can activate biochemical control pathways in response to D N A damage, thus delaying cell cycle progression and allowing time for D N A repair.  42  These signal transduction pathways are called checkpoints and they can prevent  replication of damaged D N A by causing cells to arrest in the G l phase of the cell cycle or prevent the separation of damaged chromosomes during mitosis by arresting cells in G 2 . Considering the critical role checkpoints play in the normal life cycle of every cell, a more detailed understanding of checkpoints in mammalian systems is needed, especially since most of the current understanding of checkpoints stems primarily from genetic studies in yeast and mice. Finding small molecule inhibitors of checkpoint pathways would provide valuable chemical tools to probe the cellular machinery of these control mechanisms. In addition, over half of all human cancers lack a functioning G l checkpoint due to mutations in the p53 tumour suppressor  144 gene.  4j  Thus, compounds that inhibit the G 2 checkpoint may be valuable in cancer therapy to  enhance the actions of DNA-damaging agents in tumours with a defective G l checkpoint. " 44  46  A t the time this research was beginning, there were few G2 checkpoint inhibitors known and all had been found serendipitously. Studies revealed caffeine (23) and the related purine  (97), 2-aminopurine (98), and 6-dimethylaminopurine (99) can act. 47  analogues pentoxifylline  48  as G 2 checkpoint inhibitors but are not considered drug candidates due to their multiple pharmacological effects.  Staurosporine (100) and its related analogue U C N - 0 1 (24) also  49  display this biological activity but are found to be nonspecific protein kinase i n h i b i t o r s , limiting their usefulness.  49,50  thus  In addition, the protein phosphatase inhibitors okadaic acid (101) and  fostriecin (102) can act as G 2 checkpoint inhibitors but also lead to premature mitosis in the absence of D N A damage.  51  Thus, in order to identify new compounds as biochemical tools and  potential lead compounds for cancer therapy, a rational cell-based assay was developed by M i c h e l Roberge to screen natural extracts for G 2 checkpoint inhibitors. O  23  O  O  97  M e  98  x -  M  99  e  145 3.2. Results and Discussion 3.2.1. Isolation of G2 Checkpoint Inhibitors A s part of a general collection of marine invertebrates from the tropical Pacific Ocean reefs off Motupore and Madang in Papua New Guinea, specimens of a yellow marine sponge were collected using S C U B A .  Samples were harvested by hand from the shallow water reefs,  frozen on site, and transported to Vancouver over dry ice. The marine sponge was identified as Stylissa flabelliformis  (Hentschel, 1912) by Dr. Rob van Soest of the Zoologisch Museum in  Amsterdam, The Netherlands.  Voucher samples are stored both at the University of British  Columbia in Vancouver and in Amsterdam.  Figure 3.1. Stylissa flabelliformis (photo by P . L . Colin).  1  Specimens (87 g, wet weight) of Stylissa flabelliformis  were thawed and extracted  exhaustively with M e O H over a period of several days. The combined methanolic extracts were filtered and concentrated in vacuo to give a dark brown solid (-5 g).  A sample of this  146 concentrated extract exhibited potent activity in Roberge's G 2 checkpoint inhibition assay being developed at the time. Approximately 3 g of this crude extract was fractionated on a Sephadex L H - 2 0 column using methanol as the eluent, yielding nine fractions, A through I (see Scheme 3.4).  PNG sponge Stylissaflabelliformis  Sponge (wet weight 87 g)  Crude Methanol Extract Sephadex LH-20 MeOH  Fraction A  Fraction B  basify with 5% K C 0 2  Fraction C  Fraction D  Fraction E  Fraction F  Fraction G  Fraction H  Normal Phase chromatography, stepwise gradient 100% C H C 1 to 50% C H C l : M e O H sat. with N H  3  2  2  2  2  Fraction I  3  concentrate in vacuo Fraction DD  Reversed Phase H P L C 80:20 H 0 / M e O H 2  103  Fraction AA  Fraction BB  Fraction CC  Reversed Phase high performance liquid chromatography 80:20:0.05 H 0 / M e O H / C F C O O H 2  91-TFA  3  85-TFA  aldisin Acidify with 3N HC1 concentrate in vacuo 91-HC1  85-HC1  debromohymenialdisine-HCl  hymenialdisine-HCl  94 Z-debromoaxinohydantoin  Scheme 3.4. Isolation procedure for G 2 checkpoint inhibitors from  S.flabelliformis.  147 Fractions D , E , and F were found to contain mainly debromohymenialdisine (91) and hymenialdisine (85) present as their taurine salts, whereas fractions F , G , and H also contained Z-debromoaxinohydantoin (94).  These fractions all exhibited inhibitory activity in the G2  checkpoint assay and were further subjected to silica-gel flash chromatography using stepwise gradient elution ( C H C 1 to 1:1 C H C l / M e O H saturated with N H ( g ) ) . Further purification was 2  2  2  2  3  achieved by repeated fractionation on reversed-phase H P L C using 80:20:0.05 H 0 / M e O H / T F A 2  as the eluent. The pure fractions of debromohymenialdisine (91) and hymenialdisine (85), now as their trifluoroacetic acid salts, were then converted to the hydrochloride salts by addition of 3 N HC1 followed by concentration under reduced pressure.  The cleavage product 103 was  obtained by subjecting a portion of the original fraction D , containing mainly debromohymenialdisine-taurine, to a 5 % K C 0 2  reversed-phase  HPLC  using  3  80:20  solution followed by concentration in vacuo. H 0/MeOH  as  2  the  eluent  yielded  Finally, a  pure  debromopyrrololactam, aldisin (103). Samples of the four purified marine alkaloids were tested for G 2 checkpoint inhibitory activity and their structures were confirmed by N M R and mass spectrometric analyses.  H  Q  91  H o 85  H  Q  94  103  148  3.2.2. Structure Elucidation of G2 Checkpoint Inhibitors The  structures  of  debromohymenialdisine  (91),  hymenialdisine  (85),  Z-  debromoaxinohydantoin (94) and aldisin (103) were solved by extensive analysis of I D and 2D N M R spectroscopic data recorded in D M S C M 6 at 400 and 500 M H z . L o w and high resolution E I mass spectrometry provided important additional information on the molecular formula. Proton spin systems were identified from C O S Y data and proton-carbon attachments were determined by H M Q C experiments.  The H M B C data proved invaluable for determining the  connectivity within the compounds and also in the assignment of any quaternary carbons present.  3.2.2.1. Debromohymenialdisine  91 Debromohymenialdisine (91) was isolated as a yellow amorphous solid that gave an [ M ] ion in the H R E T M S at m/z 245.09128 appropriate for a molecular formula of C u H n N 0 +  5  ( A M - 1 . 6 ppm).  2  A s the free base, compound 91 was poorly soluble in the common N M R  solvents and gave poor spectra.  Sharper N M R signals and a significant improvement in  solubility were observed when 91 was converted to the hydrochloride salt. A quick examination of the I D and 2 D N M R spectra indicated the presence of two amide carbons, four quaternary sp carbons, and two isolated proton spin systems, one of which displayed chemical shifts 2  149 consistent with the a and (3 protons on a pyrrole ring. Analysis of the molecular formula and the N M R spectra obtained for compound 91 identified it as debromohymenialdisine, originally isolated from Phakellia flabellata  by Sharma et al. in 1980.  30  The authors recognized the  possibility of geometrical isomerization at the C-10/C-11 olefin, however the large downfield shift of the C-8/C-9 methylene protons (8 3.30 ppm) due to the anisotropic effects of the neighbouring C - l 2 amide carbonyl indicated a Z-olefin configuration.  This was confirmed a  few years later by the X-ray crystallographic analysis of the related hymenialdisine (85) and 13  comparison to the spectra reported from the isolation of /^-debromohymenialdisine in 1996.  52  The N M R assignments are presented in the Experimental section and a complete set of spectral data can be found in the Appendix.  150  151  152 3.2.2.2. Hymenialdisine  HN 2  14  6  Br  'NH  O  H  85  Hymenialdisine (85) was isolated as a yellow amorphous solid that gave, an [ M ] ion in +  the H R E I M S at m/z 325.08734 appropriate for a molecular formula of C H i o N 0 2 B r ( A M 8 1  n  1.0 ppm).  5  A s with debromohymenialdisine (91), the free base of compound 85 was poorly  soluble in the common N M R solvents and gave poor spectra.  Sharper N M R signals and a  significant  85  improvement  in solubility were  observed  when  was  converted  to  the  hydrochloride salt. The absence of an H-2 proton signal along with the upfield shift in the C-2 resonance from 5 123.2 to 5 104.5 ppm suggested that 85 was the 2-bromo derivative of debromohymenialdisine (91). The pronounced downfield shift of the pyrrole N H resonance in the ' H N M R spectra (5 12.92 ppm) can be attributed to the electron-withdrawing inductive effect of the neighbouring bromine atom and possible intramolecular hydrogen bonding to the adjacent C-6 carbonyl. Analysis of the molecular formula and the N M R spectra obtained for compound 85 confirmed the structure as that of hymenialdisine, originally isolated from Axinella verrucosa and Acanthella aurantiaca by C i m i n o et al. in 1982. isomer, Tf-hymenialdisine has also been recently reported.  52  15  The A ' 1 0  1 1  geometric  The N M R assignments  are  presented in the Experimental section and a complete set of spectral data can be found in the Appendix.  153  154  155  3.2.2.3. Z-Debromoaxinohydantoin  Z-Debromoaxinohydantoin (94) was isolated as a yellow amorphous solid that gave an [ M ] ion in the H R E I M S at m/z 246.07530 appropriate for a molecular formula of C 1 1 H 1 0 N 4 O 3 +  ( A M -0.2 ppm). Examination of the I D N M R spectra indicated compound 94 possessed the same pyrroloazepinone ring system present in debromohymenialdisine (91) but featured a hydantoin group in place of the aminoimidazolidinone. A reversal of configuration at the A ' 1 0  1 1  olefinic group relative to debromohymenialdisine (91) was proposed in the original isolation of debromoaxinohydantoin (94) by Pettit et al. in 1995.  37  However, the authors had failed to  compare the ' H N M R chemical shift values of the H-8 and H-9 methylene protons (both at 5 3.25 ppm) to the values in their previous isolation of £"-axinohydantoin.  32  The large downfield  shift of the H-9 methylene protons due to the anisotropic effects of the neighbouring C - l 2 amide carbonyl, almost identical to the effects observed with the hymenialdisines (85, 91), indicated a Z-olefinic configuration.  The ' H chemical shift of H - 9 protons that do not experience the  anisotropic effects, as in E-axinohydantoin, was reported as 5 2.67 ppm. Comparison of N M R spectra with the recently reported  isolation of Z-debromoaxinohydantoin  j9  confirmed the  proposed structure for 94. A complete set of N M R assignments for Z-debromoaxinohydantoin (94) is presented in the Experimental section.  156  157  158  3.2.2.4. Aldisin O  103 Aldisin (103)  was isolated as a pale yellow amorphous solid that gave an [ M ] ion in the +  HREEVIS at m/z 164.05605 appropriate for a molecular formula of C g H s ^ C h ( A M 0.5 ppm). Examination  of  the  ID  N M R spectra  indicated compound  103  possessed  the  same  pyrroloazepinone ring system present in debromohymenialdisine (91) but signals at 8 194.5 and 13  8 162.2 ppm in the group.  C N M R spectrum suggested the presence of one ketone and one amide  Analysis of the molecular formula and the N M R spectra obtained for compound 103  confirmed the structure as that of aldisin, originally isolated in 1985 by Schmitz et al. along with debromohymenialdisine (91) from Hymeniacidon The authors described aldisin (103)  aldis and an unidentified Fijian sponge.  31  as a likely degradation product of 91 formed either by  oxidation or by conjugative addition of H 2 O at C-10 followed by a reverse aldol reaction resulting in the loss of the guanidine moiety. A complete set of N M R assignments.for aldisin (103) can be found in the Experimental section.  161  3.2.3. Biological Activity Searching for new G 2 cell cycle checkpoint inhibitors using the recently developed cell based assay led to the isolation of debromohymenialdisine  (91),  hymenialdisine (85), Z -  (94), and aldisin (103). The bioassay-guided isolation procedure and  debromoaxinohydantoin  ultimate purification of active compounds  provided the first example of G 2 checkpoint  inhibitors isolated as a result of a rational screen; all previous inhibitors had been identified by chance.  This validated the potential of the new bioassay to identify novel active compounds  that were structurally distinct from previously known G 2 checkpoint inhibitors. The G 2 checkpoint inhibitory activity of debromohymenialdisine (91) and the related analogues was determined along with its effects on cell cycle checkpoint and signal transduction protein kinases.  53  Debromohymenialdisine (91) was found to exhibit dose dependent G 2  checkpoint inhibition in human breast carcinoma cells ( M C F - 7 mp53) that were arrested in G 2 phase as a result of exposure to ionizing radiation (see Figure 3.10). A n I C  5 0  value of 8 u M was  determined, with higher concentrations leading to maximal activity at 40 u M followed by a decrease  in activity,  Hymenialdisine  a phenomenon  also  seen  with  other  checkpoint  inhibitors. ' '  54 5 1  (85) exhibited similar potent G 2 checkpoint inhibitory activity with an ICso  value o f 6 u M (see Table 3.1), indicating the presence of a bulky bromine substituent at C-2 in 85 has little effect on the activity. Z-debromoaxinohydantoin (94) and aldisin (103) both show a dramatic  decrease  in  biological  activity  indicating  that  the  presence  of  the  aminoimidazolidinone subunit is required for G 2 checkpoint inhibition. The two commercial products, 2-aminoimidazole and 2-amino-4,5-imidazole-dicarbonitrile, lack the pyrrololactam and were found to be essentially inactive in the bioassay.  162  100  10  1  DBH (pM) Figure 3.10. Inhibition of the G 2 checkpoint by debromohymenialdisine (91).  Table 3.1. G 2 checkpoint inhibition by debromohymenialdisine (91) and related compounds. Compound  G 2 checkpoint inhibition I C (uM) 8±4 6±3 »200 »200 » 1 0 000 » 1 0 000 5 0  Debromohymenialdisine (91) Hymenialdisine (85) Z-debromoaxinohydantoin (94) Aldisin (103) 2-aminoimidazole* 2-amino-4,5,-imidazoledicarbonitrile* obtained from Sigma-Aldrich.  Hymenialdisine (85) was recently reported to be a potent inhibitor of several protein kinases in vitro, including cyclin-dependent kinases, glycogen synthase kinase-3[3, and casein kinase l .  5 6  The authors also present a crystal structure of a protein kinase Cdk2-hymenialdisine  163 complex that indicates key hydrogen bonding and van der Waals interactions hymenialdisine (85)  between  and the amino acid residues in the ATP-binding region of the protein.  These interactions are consistent with the structure-activity results obtained for G2 checkpoint inhibition with compounds 91, 85, 94, and 103, suggesting the mechanism of action involves inhibition of a protein kinase. In particular, the deleterious effect of Z-debromoaxinohydantoin (94) or the negligible influence of a C-2 bromine substituent can both be rationalized in terms of respective nonfavourable or nonaltered drug-kinase interactions. Although hymenialdisine (85) was previously reported to inhibit a number of kinases at nanomolar concentrations and several more at micromolar concentrations in vitro,  56  studies  performed on the hymenialdisines (91, 85) isolated from Stylissa flabelliformis reveal a much narrower spectrum of inhibitory activity in vivo. In addition, the previous investigations did not test kinases involved in the G 2 checkpoint.  Debromohymenialdisine (91)  isolated from S.  flabelliformis was found to inhibit the checkpoint kinases C h k l and C h k 2 in vitro with I C  5 0  values o f 3 u M and 3.5 u M , concentrations close to those required for in vivo checkpoint inhibition ( I C  50  8 p M ) (see Figures 3.11 and 3.12). However, there was no significant in vitro  inhibition of the phosphoinositide-3 kinases A T M , A T R , and D N A - P K , thus indicating that debromohymenialdisine (91) acts as a checkpoint inhibitor by blocking two major branches of the checkpoint pathway downstream of A T M (see Figure 1.6).  These results suggested the  molecular targets for checkpoint inhibition are C h k l and Chk2, although the untested kinases W e e l and M y t l cannot be excluded.  164  DBH  (pm  Figure 3.11. Inhibition of protein kinase C h k l by debromohymenialdisine  o-i 0.1  •  1  i  1  (91).  I  10  D B H (uM) Figure 3.12. Inhibition of protein kinase C h k 2 by debromohymenialdisine  (91).  Additional studies performed on the cellular localization of a variety of cell cycle regulatory proteins implicated in the control of the G 2 / M transition (Cdc2, Cdc25C, cyclin A ,  165 cyclin B , Cdk2, Cdc25B, W e e l ,  and  14-3-3) ruled out the possible effect  compartmentalization in G 2 checkpoint arrest by debromohymenialdisine (91).  of cellular  Because protein  kinases play an essential role in the control of signal transduction pathways, the effects of debromohymenialdisine (91) on the activation or inhibition of 24 other kinases were determined by analysis of their mobility shifts in Western blots.  None of the protein kinases tested  ( C a M K 4 , Cdk2, C K l a , C K 2 a , E r k l , Erk2, p38 H o g M A P K , M e k l , M e k 2 , M e k 3 , Mek4, Mek5, M e k k 3 , P i m l , P K B a , PKC(3, P K C s , P K C C , Rskl, p 7 0  S6k  , p46 S A P K , p54 S A P K , and T a k l )  revealed changes in abundance or mobility upon treatment with debromohymenialdisine  (91),  again supporting a narrower in vivo inhibitory spectrum. A complementary approach to classical genetics to probe the function of a single gene product or protein involves the use of exogenous ligands such as the marine alkaloids isolated from the marine sponge Stylissa flabelliformis.  Natural products research is embracing this  "chemical genetics" approach outlined earlier in Chapter One. The power of exploiting the vast structural diversity found in nature to identify small molecules capable of either inactivating or activating biochemical pathways or individual proteins is being recognized in searching for novel G 2 cell cycle checkpoint inhibitors. Since this discovery of the hymenialdisines (91,  85)  as the first G 2 checkpoint inhibitors identified from a rational search, additional studies have reported the isolation of staurosporine (100)  and the new inhibitor isogranulatimide (25).  54  These studies validate the new bioassay by identifying a known inhibitor and providing an additional structural inhibitor chemotype.  3.3. Conclusions The crude extract from a marine sponge, Stylissa flabelliformis, collected in the waters off Motupore Island in Papua New Guinea exhibited potent activity in a new bioassay searching for G 2 cell checkpoint inhibitors. Bioassay-guided fractionation of this active extract resulted in  166 the  isolation  and identification  of the natural  products  debromohymenialdisine  (91),  hymenialdisine (85), Z-debromoaxinohydantoin (94), and aldisin (103). These compounds were the first G 2 checkpoint inhibitors to be found by a rational screen and were structurally distinct from previously reported G 2 checkpoint inhibitors. The discovery of these biologically active natural products validated the power of the new bioassay and contributed to the understanding of the G 2 checkpoint pathway by providing a new biochemical tool to probe the molecular basis of inhibition. The G 2 checkpoint inhibitory activity of debromohymenialdisine (91) and the related analogues was determined along with its effects on cell checkpoint and signal transduction protein kinases. Debromohymenialdisine (91) was found to inhibit the G 2 checkpoint pathway with an IC50 of 8 • M and specifically inhibited the protein kinases C h k l (IC50 3 • M ) and Chk2 (IC  50  3.5 • M ) while not affecting A T M , A T R , or D N A - P K .  Thus, debromohymenialdisine  (91) was seen to block two branches of the checkpoint pathway downstream of A T M and was found to inhibit a narrow range of signal transduction proteins in vivo. These studies on the marine sponge alkaloids have provided small molecule ligands for a chemical genetics approach to probe the G 2 checkpoint pathway.  REFERENCES  1  2  Colin, P.L.; Arneson, A . C . In Pacific Invertebrates; Coral Reef Press: Beverly Hills, 1995; pp. 17-21. Rupert, E.E.; Barnes, R . D . In Invertebrate Zoology; Sixth edition; Saunders College Publishing: Toronto, 1994; pp 73-93. Faulkner, D . J . Nat. Prod. Rep. 2001,18, 1; and previous reports in this series.  4  Andersen, R.J.; Williams, D . E . Pharmaceuticals from the Sea. 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Y . ; Curman, D . ; Stringer, C M . ; Friend, S . H . ; Davies, P.; Haggarty, S.J.; Kelly, M . T . ; Britton, R.; Piers, E . ; Andersen, R.J. Cancer Res. 1998, 58, 5701.  5 5  5 6  1996,  Jiang, X . ; L i m , L . Y . ; Daly, J.W.; L i . A . H . ; Jacobson, K . A . ; Roberge, M . Int. J. Oncol. 2000, 16, 971. Meijer, L . ; Thunissen, A . - M . W . H . ; White, A . W . ; Gamier, M . ; Nikolic, M . ; Tsai, L . - H . ; Walter, J.; Cleverley, K . E . ; Salinas, P.C.; W u , Y . - Z . ; Biernat, J.; Mandelkow, E . - M . ; Pettit, G.R. Chem. Biol. 2000, 7, 51.  170  CHAPTER 4  4.1. G2 Cell Cycle Checkpoint Inhibitors from Cryptocarya concinna The successful application of the G 2 cell cycle checkpoint inhibitor assay in identifying new G 2 checkpoint inhibitors led to a large screening of marine and terrestrial natural extracts obtained from the N C I Open Repository. A crude extract from the Taiwanese tree Cryptocarya concinna was found to exhibit potent activity in the G 2 checkpoint inhibition assay. Bioassayguided fractionation of this extract resulted in the isolation and identification of the natural product cryptofolione and a series of related polyketide lactones.  Synthetic modifications on  these natural products yielded four additional analogues and led to mode of action and structureactivity relationship studies. The polyketide G 2 checkpoint inhibitors isolated from C. concinna were structurally distinct from previously known inhibitors and do not appear to act on the known G 2 checkpoint molecular targets.  4.1.1. Review of Plant-Derived Compounds For thousands of years, plants have formed the basis of sophisticated medicinal systems throughout the world.  These plant-based systems continue to play an essential role in health  care, with estimates indicating approximately 80% of the world's population rely mainly on traditional medicines for their primary health care.  1  Recently, there has been a resurgence in the  use of traditional herbal medicines and the importance of plant-derived continues to grow.  pharmaceuticals  There are currently 119 chemical substances, originally derived from 90  plant species, which are in use as important drugs and three quarters of these compounds were discovered from plants used in traditional medicine.  Plant-derived compounds have especially  had a significant impact on the treatment of proliferative diseases such as cancers.  171 Perhaps the best-known anticancer drugs in clinical use are the Vinca alkaloids, vincristine (21) and vinblastine (22), originally isolated from the Madagascar periwinkle Catharanthus roseus.  3  Their discovery and success in the treatment of cancer has led to many  semi-synthetic analogues currently in clinical use and pre-clinical trials, including vinorelbine  (104) and vindesine (105). The Vinca alkaloids are plant-derived antimitotic agents like the previously described taxanes,  but act by inhibiting the polymerization o f tubulin into  microtubules as opposed to stabilizing microtubules against depolymerization.  21  R ^ C H O  R =C0 Me  R =OAc  22  R = Me  R =C0 Me  R = OAc  104 R j = M e  R =CONH  R = OH  t  2  2  2  2  2  2  3  105  3  3  In addition to the Vinca alkaloids and the taxanes, two other important classes of anticancer compounds are also based on plant-derived compounds. Currently in general clinical use, the semi-synthetic  derivatives etoposide  (106) and teniposide (107) are based on  podophyllotoxin (108), an antitumour agent from the roots of various Podophyllum plants. The 4  Chinese ornamental tree Camptotheca acuminata was the source of camptothecin (109), which led to the development of the clinical therapeutics topotecan (110) and irinotecan (111).  5  172  109  R  110  Ri = OH R = CH NMe  1 =  H  111  R =H 2  2  2  2  The natural product rohitukine (112), reported from the plant Dysoxylum binectariferum, has served as the basis for the totally synthetic flavone, flavopiridol (113). This synthetic agent 6  is currently scheduled to advance to a phase U clinical trial against a broad range of tumours and acts as an inhibitor of cyclin dependent kinases that regulate the transition from G 2 to M phase.  7  The plant natural product has also served as a lead compound for a series of compounds, such as the paullones (114). These discoveries highlight the importance of plant natural product "lead" 1  compounds that give rise to semi-synthetic and synthetic compounds with a variety of potencies and specificities.  173  4.1.2. Review of Cryptocarya Phytochemistry The genus Cryptocarya, which includes about 350 species, belongs to the Lauraceae family of plants and is wide-spread throughout the world especially in tropical regions. For years, traditional healers of Southern Africa have used the bark of Cryptocarya and the related Ocotea genus for medicinal as well as mythical purposes.  9  Recently, the use and importance of  Cryptocarya species has increased due to accessibility and supply issues concerning the Ocotea plants.  This interest has led to over four decades of investigations on the chemical  10  constituents of Cryptocarya species, with numerous reports detailing the isolation of various alkaloids and a-pyrones. The alkaloids isolated from the genus Cryptocarya have been reported to display a range of interesting biological activities. Cryptopleurine (115), originally isolated from the bark of Cryptocarya pleurosperma in 1954," demonstrates cytotoxicity towards human epidermoid 12  13  carcinoma,  antiviral activity,  synthesis.  Chemical investigations into Cryptocarya chinensis, a perennial woody plant  14  and has been shown to be a highly-active inhibitor o f protein  widely distributed in the forests of Taiwan and China, have revealed it to be a rich source of pavine alkaloids (compounds 116 to 118). "  18  In addition to reports of these alkaloids  possessing behavioural and antitumour effects,  19  (-)-caryachine (118) was recently shown to  15  display exciting antiarrhythmic activity and represents a very promising new drug for the treatment of cardiac arrhythmias.  20  Most of the remaining alkaloids are structurally related to  174 reticuline (119)  from the N e w Caledonian Cryptocarya longifolia  11  via different cyclization  modes, as evident in cryptowolline (120) or cryptodorine (121). OCH  3  119  120  121  In addition to the alkaloid constituents of the various Cryptocarya species, these laurel plants also contain a collection of 6-substituted 5,6-dihydro-a-pyrones as secondary metabolites. These metabolites are biosynthesized from either acetate or mixed acetate-shikimate pathways and are widely distributed in both plants and fungi.  22  21  While many of these compounds display a  diverse range of biological activities, such as plant growth inhibitors, insect antifeedants, antifungal and antitumour agents,  the reports from Cryptocarya species are noticeably lacking  in biological information. The 6-arylalkyl-5,6-dihydro-a-pyrones from lauraceous plants are characteristic of Aniba and Cryptocarya species, however, Aniba a-pyrones commonly possess a C-4 methoxyl group while the pyrones from Cryptocarya are not substituted on C-4 but often feature a styryl group attached to the C-6 side chain.  A summary of the a-pyrones isolated  from various Cryptocarya species are presented in Table 4.1 along with additional cyclized derivatives.  175 Table 4.1. Summary of a-pyrones isolated from various Cryptocarya species. Organisms  Compounds  •  n  C . massoia  24  122 C. latifolia  123  124  125  25,26  C. caloneura C latifolia26 C. wylei  27  26  C. bourdilloni 28  C. moschata 30 C. wylei 26  C. kurzii  31  C. myrtifolia C. latifolia  9  26  C. liebertiana  32  176 Compounds  135 R = H  136 R = A c  Organisms  137  Recently, investigations into the chemistry of Cryptofolia moschata resulted in the identification of thirteen 6-[co-arylalkenyl]-5,6-dihydro-a-pyrones (126, 130, 138-148) including the known goniothalamin (126)  and cryptofolione (130).  23  Many of the compounds reported  were found to exist as diastereomers and all contain a styryl group and  5,6-dihydro-a-pyrone  connected by either a linear and hydroxylated carbonic chain or a carbonic chain with a dihydroxy-tetrahydropyrane ring.  O  138 C . = R  130 C  139  140  2  C =5 2  C  4  4  =/? =5  177  OH  O  OH  O  143 C . , C . = /?,5  146 C . , C , C . = /?,5,/?  144 C . , C . = 5,5  147 C . , C , C . = 5,5,/?  145 C . , C . = R,R  148 C . , C , C . = /?,/? 5  4  4  4  5  5  5  6  6  6  7  T  T  8  8  8  >  4.2. Results and Discussion 4.2.1. Isolation of G2 Checkpoint Inhibitors As part of a general screening of marine and terrestrial extracts obtained from the N C I Open Repository, a methanolic crude extract of the Taiwanese tree Cryptocarya concinna (Lauraceae) exhibited potent activity in the G 2 checkpoint inhibition assay. A 500 mg sample of this crude extract was suspended in one liter of water and partitioned against E t O A c (1 liter x 5). The E t O A c fraction was concentrated in vacuo, suspended in one liter of 9:1 M e O H / I U O , and then partitioned against hexanes (1 liter x 5). Next, water was added to the remaining methanolic fraction in order to achieve a 6:4 M e O H / H ^ O ratio.  This fraction was finally  partitioned against two liters of C H 2 C I 2 (1 liter x 5). Bioassay-guided fractionation showed the G2 checkpoint inhibitory activity was entirely present in the E t O A c , the 9:1 M e O H / F L O , and the  CH2CI2  fractions, respectively (see Scheme 4.1).  178 Crude Methanol Extract (approx. 500 mg)  Taiwanese Tree Cryptocarya concinna  EtOAc  H7O  9:1 M e O H : H 0  Hexane  6:4 MeOH:H Q|  CH C1  2  2  2  2  Sephadex LH-20 80:20 M e O H / C H C l 2  Fraction A  Fraction B  Fraction C  Fraction D  Fraction E  2  Fraction F  Fraction G  Normal Phase chromatography 75:25 EtOAc/Hexanes  Fraction A  Fraction B  Fraction C  Fraction D  Fraction E  Fraction F  Fraction G  Fraction H  Fraction I  High Performance liquid chromatography  131  148  (lmg)  (15mg)  130 (2 lmg)  138 (lmg)  Scheme 4.1. Isolation procedure for G 2 checkpoint inhibitors from C. concinna.  Approximately 125 mg of this dichloromethane fraction was fractionated on a Sephadex L H - 2 0 column using 80:20 M e O H / C H C l 2  2  as the eluent, yielding seven fractions, A through G.  Only fraction D exhibited significant inhibitory activity in the G 2 checkpoint assay and was thus  179 subjected to normal phase flash chromatography using an isocratic 75:25 EtOAc/hexanes elution.  The resulting nine fractions were screened for inhibitory activity and interesting ' H  N M R signals.  A final purification of four fractions was achieved by reversed-phase  using 48:52 H a O / M e O H as the eluent yielding pure cryptofolione cryptofolione ketone  HPLC  (130), Z-cryptofolione (148),  (131), and compound 138. The four purified a-pyrones were tested for G2  checkpoint inhibitory activity and their structures confirmed by detailed N M R and mass spectrometric analyses.  131  138  4.2.2. Structure Elucidation of G2 Checkpoint Inhibitors The structures of cryptofolione (130), Z-cryptofolione (148), cryptofolione ketone (131), and compound 138 were solved by extensive analysis o f I D and 2 D N M R spectroscopic data recorded in DMSO-<r/ at 400 and 500 M H z . 6  L o w and high resolution E I and C I mass  spectrometry provided important information on the molecular formula. Proton spin systems were identified from C O S Y data and proton-carbon attachments were determined by H M Q C experiments.  The H M B C data proved invaluable for determining the connectivity within the  compounds and also in the assignment o f any quaternary carbons present.  The relative  stereochemistry was obtained by formation of acetonide derivatives, analysis of circular dichroism experiments, and comparison with previously reported spectral data.  130  f-Cryptofolione (130) was isolated as a white amorphous solid that gave an [ M ] ion in +  the HREDvIS at m/z 314.15178 appropriate for a molecular formula of C19H22O4 ( A M -0.09 ppm).  A quick analysis of the I D and 2 D N M R spectra indicated the presence of a  monosubstituted benzene ring, two rrans-disubstituted olefins, a c/j-a,P-unsaturated  ester  functionality, three oxymethine protons, and three methylene groups. The H M B C and C O S Y experimental data revealed a trans-styryl double bond (coupling constant J of 15.9 Hz) connected to a 1,3-diol system and ultimately linked to an a-pyrone through a second transolefin ( / = 15.5 H z ) . Thus, the spectral data from compound 130 matched the N M R data for the 6-[co-arylalkenyl]-5,6-dihydro-a-pyrone metabolites.  structures  The final structure of £-cryptofolione (130)  spectra with previously published data. '  9 23  N M R data  23  characteristic  of Cryptocarya  secondary  was confirmed by comparison of  It should be noted that the most recent published  misidentifies the structure of £-cryptofolione (130) and incorrectly assigns the C  resonances for C - 7 ' and C - 8 ' , which should be interchanged.  , 3  A summary of all N M R  assignments, C O S Y , and H M B C correlations for ZT-cryptofolione (130) can be found in Table 4.2 and 2 D N M R spectra can be found in the Appendix.  181  Table 4.2. N M R data for £-cryptofolione (130) recorded in CDCI3. position l(O) 2 3 4 5 6 r 2' 3' 4' 4'-OH 5' 6' 6'-OH 7' 8' 1" 2'76" 3'75" 4" a  8'H (7 value in Hz) a  5.99 (d, 7=9.8) 6.83 (dt, 7=9.7, 4.5) 2.38 (m) 4.84 (m) 5.65 (dd, 7=15.5, 6.5) 5.84 (dd, 7=15.5,7.4) 2.27 (t, 7=6.7) 4.03 (m) 3.14 (br) 1.75 (m) 4.61 (m) 3.14 (br) 6.24 (dd, 7=15.9, 6.0) 6.60 (d, 7=15.9)  5 C 1 3  b  COSY Correlations  3  HMBC Correlations  3  H3 H5 H5,H6  164.1 121.4 144.9 29.6 77.8 129.7 131.2 40.3 68.1  H4 H3, H5 H4, H 6 H5, H I ' H6, H 2 ' H1',H3' H2',H4' H3',H5'  HI', H2' H6, H 3 ' H6, H 3 ' H1',H2' H3',H5?  42.2 70.2  H 4 \ H6' H5', H7'  H3' H5',H7',H8'  H6.',H8' H7'  H5' H6',H2'76" H7',H3'75" H8',H4"  131.8 129.9 136.6 7.34 (d, 7=7.9) 126.4 7.28 (t, 7=7.6) 128.5 7.20 (t, 7=7.3) 127.6 recorded at 400 M H z . recorded at 100 M H z .  H3'V5" H2'76", H 4 " H3'75"  H2'76"  b  The relative stereochemistry of the 1,3-diol in the linear unsaturated carbonic chain was determined by examination of the acetonide derivative 149. In papers presented by Rychnovsky et a / .  33  and Evans et al.,  34  acetonides formed from syn-1,3-diols are shown to exist in well-  defined chair conformations with the two bulky substituents occupying equatorial positions (Ri and R 2 in Figure 4.1).  B y contrast, acetonides formed from ann'-l,3-diols adopt a twist-boat  conformation to minimize unfavourable 1,3-diaxial interactions. The C N M R chemical shifts 1 3  of the acetonide methyl groups then become diagnostic for the relative stereochemistry of the 1,3-diols, indicating an ann'-configuration for £-cryptofolione (130). A detailed analysis of the acetonide derivative (149) follows in Section 4.2.3.1.  anti Figure 4.1. Characteristic conformations and  1 3  C N M R chemical shift values for  syn- and anti-\,3-6\o\s.  The C-6 configuration in Zs-cryptofolione (130)  was determined from the positive Cotton  effect in the C D curve at 254-272 nm due to n—>n* transitions. Using the Snatzke rule  35  and  considering a pseudo-equatorial substituent, this result defines a 6R configuration. Analysis of the complete set of N M R spectral data along with optical rotation values and C D curves suggest is-cryptofolione (130)  isolated from Cryptocarya concinna corresponds to Cavalheiro and  Yoshida's compound 5, which has a 6R,4'S,6'R configuration.  183  184  Z-Cryptofolione (148) was isolated as a white amorphous solid that gave an [ M ] ion in +  the HRCEVIS at m/z 314.15226 appropriate for a molecular formula of C19H22O4 ( A M -1.5 ppm). Analysis of the I D and 2 D N M R spectra indicated considerable similarities to compound 130 including the presence of a monosubstituted benzene ring, two disubstituted olefins, a cisa,p-unsaturated ester functionality, three oxymethine protons, and three methylene groups. The H M B C and C O S Y experimental data revealed the only difference from 130 was the presence of a c/s-styryl double bond with a coupling constant J of 11.7 H z . This group was connected to a similar 1,3-diol system and linked to an a-pyrone through a second trans-olefm (J = 15.5 Hz). The relative stereochemistry  of the 1,3-diol of Z-cryptofolione (148)  was determined by  examination of its acetonide derivative. The C N M R chemical shifts of the acetonide methyl l 3  groups again indicated an Gtnfz'-configuration for Z-cryptofolione (148). The C-6 configuration in Z-cryptofolione (130) was determined from the positive Cotton effect in the C D curve at 254272 n m due to n—*n* transitions, defining a 6R configuration.  A summary of all N M R  assignments, C O S Y , and H M B C correlations for Z-cryptofolione (148) can be found in Table 4.3 and 2 D N M R spectra can be found in the Appendix.  Table 4.3. N M R data for Z-cryptofolione (148) recorded in CDCI3. position 1 (0) 2 3 4 5 6 r 2' 3' 4' 4'-0H 5'a 5'b 6' 6'-OH T 8' 1" 2"/6" 3"/5" 4" a  5'H (7 value in H z ) a  6.00 6.83 2.37 4.84 5.63 5.83 2.25 4.04 2.84 1.79 1.73 4.92 2.66 5.75 6.50  (d, 7=9.8) (m) (m) (m) (dd, 7=15.5, 6.5) (dt, 7=15.5, 7.4) (t, 7=6.7) (m) (br)* (ddd, 7=14.5, 7.8, 3.2) (ddd, 7=14.4, 8.5 3.6) (m) (br)* (dd, 7=11.7, 9.2) (d, 7=11.7)  5 C 1 3  b  164.0 121.5 144.7 29.7 77.8 129.8 131.2 40.2 68.2 42.4 65.6  133.8 130.5 136.4 7.24 (d, 7=7.2) 128.7 7.32 (t, 7=7.7) 128.3 7.23 (t, 7=7.3) 127.3 recorded at 500 M H z . recorded at 100 M H z . b  COSY Correlations  3  H4 H3, H5 H4, H6 H5, H I ' H6, H 2 ' H1',H3' H2',H4' H3',H5'b  HMBC Correlations  H3 H5 H5, H6 H3,H1' H5, H 1 ' , H 2 ' H6, H 3 ' H6, H 3 ' Hl',H2*,H5'ab H2',H3',H5'ab  H5'b, H 6 ' , H 4 ' H5'a, H 4 ' , H 6 ' H5'a, H 7 '  H3'  H6', H8' H7'  H5'ab H2'76" H7',H3"/5" H8',H4"  H3'75" H2'76", H 4 " H3'75"  3  H5'ab, H 8 '  H2'76", H3'75"  * assignments interchangeable.  187  188  189  Cryptofolione ketone (131)  was isolated as a white amorphous solid that gave an  [ M + H ] ion in the HRCJJVIS at m/z 313.14358 appropriate for a molecular formula of C 1 9 H 2 0 O 4 +  ( A M 1.3 ppm). Analysis of the I D and 2 D N M R spectra indicated considerable similarities to compound  130  including the presence  of a monosubstituted  benzene  ring, two trans-  disubstituted olefins, a c/5-a,(3-unsaturated ester functionality, two oxymethine protons, three methylene groups, and a ketone functionality.  The H M B C and C O S Y experimental data  revealed the only difference from 130 was the presence of a ketone with a ° C N M R chemical shift of 5 200.6 ppm in place o f the C - 6 ' alcohol group. Since cryptofolione ketone (131)  can  serve as a biosynthetic precursor to cryptofolione (130), it is most likely an authentic natural product and not an isolation artifact. The relative stereochemistry at the two chiral centers of cryptofolione ketone (131)  was not determined due to the lack of sufficient material. However,  it should be noted that while N M R data for cryptofolione ketone (131) a P C C oxidation product and later as a natural product, 9  26  has been reported first as  the stereochemistry suggested in the  1^ literature was not supported by experimental data and the  C resonances for C - 7 ' and C-8'  should be interchanged. A significant difference also exists in the reported values for the H - 5 ' methylene protons and those determined from the present isolation from C. concinna (8 2.61 ppm versus 8 2.74 and 8 2.90 ppm.) The C-6 configuration in 131 was determined from the positive Cotton effect in the C D curve at 254-272 nm due to n—>7i* transitions, defining a 6R  configuration.  A summary of all N M R assignments, C O S Y , and H M B C correlations for  cryptofolione ketone (131)  can be found in Table 4.4 and 2D N M R spectra can be found in the  Appendix.  Table 4.4. N M R data for cryptofolione ketone (131) recorded in C D C 1 . 3  position 1(0) 2 3 4 5 6 r 2' 3'a 3'b 4' 4'-OH 5'a 5'b 6' 7' 8' 1" 2"/6" 3"/5" 4" a  5'H (7 value in Hz) a  6.03 6.86 2.44 4.90 5,72 5.89 2.37 2.30 4.21 3.31 2.90 2.74  (d, 7=9.6) (dt, 7=9.9, 4.2) (m) (m) (dd, 7=15.8, 6.6) (dt, 7=15.0, 7.2) (m) (m) (m) (br) (dd, 7=17.3, 2.7) (dd, 7=17.5, 9.0)  5 C 1 3  b  163.9 121.6 144.6 29.7 77.9 130.0 130.9 39.3 67.2 46.0  200.6 7.57 (d, 7=16.2) 143.9 6.72 (d, 7=16.1) 126.1 134.1 7.55 (d, 7=7.5) 128.5 7.39 (m) 129.0 7.39 (m) 130.9 recorded at 500 M H z . recorded at 100 M H z . b  COSY Correlations  3  H4 H3, H 5 H4, H 6 H5,H1' H6, H 2 ' Hl',H3'ab H2', H3'b H2',H3'a, H4' H 3 ' b , H5'ab  HMBC Correlations  3  H5 H2' H3'ab  H3'ab, H 5 ' b  H4', H5'b H5'a H5'b, H 7 ' , H 8 ' H8' H7' H375" H2"/6", H 4 " H3"/5"  H8' H7',H375", H4" H276"  191  192  193  Compound 138 was isolated as a white amorphous solid that gave an [ M ] ion in the +  HRCEVIS at m/z 288.13514 appropriate for a molecular formula o f  C17H20O4  ( A M 3.6 ppm).  Analysis of the I D and 2 D N M R spectra indicated considerable similarities to Zi-cryptofolione (130) including the presence of a monosubstituted benzene ring, one fra/u-disubstituted olefin, a cw-a,P-unsaturated ester functionality, two oxymethine protons, and three methylene groups. The H M B C and C O S Y experimental data revealed the only difference from 130 was the absence of the second trans-olef'm linking the 1,3-diol to the 5,6-dihydro-a-pyrone. The relative stereochemistry at the three chiral centers of compound 138 was not determined due to the lack of sufficient material.  However, the final structure of compound 138 was confirmed by  comparison of spectra with previously published data.  23  Analysis of the complete set of N M R  spectral data along with optical rotation values and C D curves suggest compound 138 isolated from Cryptocarya concinna corresponds to Cavalheiro and Yoshida's compound 3, which exists in the 6R,2'S,4'R configuration. A similar misassignment of the  l 3  C resonances for C - 5 ' and C -  6' exists in this report, as with £-cryptofolione (130), and thus the values should be interchanged.  A summary of all N M R assignments, C O S Y , and H M B C correlations for  compound 138 can be found in Table 4.5 and 2 D N M R spectra can be found in the Appendix.  Table 4.5. N M R data for compound 138 recorded in C D C 1 . 3  position HO) 2 3 4 5 6 l'a l'b  T 2'-OH 3' 4' 4'-0H 5' 6' 1" 2"/6" 375" 4" 3  6'H (7 value in Hz) a  6.01 6.88 2.37 4.74 1.90 1.78 4.37 2.25 1.82 4.65 2.83 6.29 6.62  (d, 7=9.9) (dt, 7=9.7, 4.2) (m) (m) (m) (m) (m) (br)* (m) (m) (br)* (dd, 7=15.9, 6.3) (d, 7=16.1)  7.37 (d, 7=7.0) 7.31 (t, 7=7.6) 7.24 (under solvent)  recorded at 500 M H z .  5 C 1 3  b  167.2 121.4 145.2 30.0 75.0 42.4  COSY Correlations  3  HMBC Correlations  3  H5 H3, H 5  65.0  H4 H3, H 5 H4, H 6 H5,Hl'a H6 H2' Hl'b, H3'  Hl'b, H3'  43.1 70.9  H2', H4' H3', H5'  H3',H5',H6'  131.4 130.6 136.4 126.6 128.6 127.9  H4', H6' H5'  recorded at 100 M H z .  H375" H276", H4" H375"  Hl'a  H4' H276" H5',H3"/5" H6', H 4 "  H2"/6" * assignments interchangeable.  195  196  4.2.3. Synthetic Derivatives of Zs-Cryptofolione 4.2.3.1. Cryptofolione acetonide  5"  149  In order to establish the relative configuration of the 1,3-diol system in compound 130, cryptofolione acetonide (149) was prepared by adding 2 ml of 2,2-dimethoxy-propane and a catalytic amount of pyridinium p-toluene sulfonate to approximately 2 mg of /f-cryptofolione  (130). After workup, cryptofolione acetonide (149) was isolated as a white amorphous solid that gave an [ M + H ] ion i n the HRCJJVIS at m/z 355.19102 appropriate for a molecular formula +  of C22H26O4 ( A M - 0 . 2 ppm). A quick analysis of the I D and 2 D N M R spectra indicated the presence of an acetonide group bridging the two alcohol oxygen atoms.  The addition of two  methyl singlets (8 1.39 and 8 1.40 ppm) in the ' H N M R spectrum and an acetal carbon resonance at 8 100.5 ppm was diagnostic for acetonide formation.  A s mentioned earlier in  Section 4.2.2.1, acetonides formed from syn- and anti- 1,3-diols are found to exist in distinct and well-defined conformations  33,34  (see Figure 4.1).  Thus, the  1 3  C N M R chemical shifts of the  acetonide methyl groups appearing at 8 24.9 and 8 25.5 ppm were diagnostic for an anticonfiguration for the 1,3-diols in E-cryptofolione cryptofolione acetonide (149)  (130).  The spectral data obtained for  was in full agreement with previously published data.  9  A  summary of all N M R assignments, C O S Y , and H M B C correlations for cryptofolione acetonide (149) can be found in Table 4.6.  Table 4.6. N M R data for cryptofolione acetonide (149) recorded in CDCI3. position 1 (0) 2 3 4 5 6 r 2' 3'a 3'b 4' 5'a 5'b 6' 7' 8' 1" 276" 375" 4" 1"' 2"' 3"' 3  5'H (7 value in H z ) a  6.03 6.86 2.42 4.88 5.68 5.83 2.34 2.25 3.93 1.84 1.75 4.49 6.20 6.54  (d, 7=9.9) (dt, 7=9.7, 4.5) (m) (m) (dd, 7=15.5, 6.5) (dd, 7=15.6, 7.3) (m) (m) (m) (m) (m) (m) (dd, 7=16.0, 6.3) (d, 7=15.9)  7.36 (d, 7=7.5) 7.28 (t, 7=7.5) 7.22 (t, 7=7.4) 1.40 (s)* 1.39 (s)*  recorded at 400 M H z .  5  1 3  C  b  164.0 121.7 144.5 29.8 77.9 129.4 130.7 38.5 65.8 37.5 67.7 129.7 130.5 136.7 126.5 128.5 127.7 100.5 24.9* 25.5*  COSY Correlations  H4 H3.-H5 H4, H 6 H5.H1' H6, H 2 ' Hl',H3'ab H2',H4' H2' H3'a, H5'b H6' H4' H5'a, H7' H6\ H8' H7' H3"/5" H2'76", H 4 " H3"/5"  3  HMBC Correlations  3  H4 H5 H5, H 6 H3 H4, H 5 , H 1 ' , H 2 ' H6, H 2 , H3'ab H6, H l ' , H 3 ' a b Hl',H2',H5'b H3'ab, H 5 ' b H3'ab, H7 H5'a, H 7 ' , H8' . H5'a, H6' H6', H2"/6" H7',H8',H375" H4" H2"/6" H2"',H3'" H3'" H2'"  recorded at 100 M H z . * assignments interchangeable.  199  200  201  Diacetylcryptofolione (150) ml  of  pyridine  to  was prepared by adding 0.5 ml of acetic anhydride and 1.5  approximately  diacetylcryptofolione (150)  2  mg  of  (130).  £-cryptofolione  After  workup,  was isolated as a white amorphous solid that gave an [ M ] ion in the +  H R E I M S at m/z 398.17274 appropriate for a molecular formula of C 3 h 6 0 2  2  6  ( A M -0.51 ppm).  A quick analysis of the I D and 2 D N M R spectra indicated the presence of two acetate groups in place of the two alcohol functionalities in 130.  The addition o f two methyl singlets (8 1.99 and  5 2.04 ppm) in the 'ff N M R spectrum and two ester carbon resonances at 8 170.2 and 5 170.5 ppm were diagnostic for acetate subunits.  A summary of all N M R assignments, C O S Y , and  H M B C correlations for diacetylcryptofolione (150)  can be found in Table 4.7.  Table 4.7. N M R data for diacetylcryptofolione (150) recorded in CDCI3. position 1 (0) 2 3 4 5 6 r  2' 3' 4' 5'a 5'b 6'  5'H (J value in Hz) a  6.03 (dt, 7=9.9, 1.8) 6.85 (dt, 7=9.7, 4.2) 2.39 (m) 4.87 (dt, 7=8.0, 6.6) 5.65 (dd, 7=15.5, 6.3) 5.76 (dd, 7=15.3, 7.4) 2.35 (m) 5.03 (m) 1.94 (m) 1.90 (m) 5.46 (m) 6.09 (dd, 7=15.9, 7.4) 6.60 (d, 7=15.9)  5 C 1 3  b  163.8 121.7 144.5 29.6 77.6 130.5 129.3 37.6' 68.7 38.5  70.7 127.0 8' 132.9 1" 136.1 276" 7.35 (d, 7=7.1) 126.6 375" 7.29 (t, 7=7.5) 128.6 4" 7.23 (t, 7=7.1) 128.1 1"' 170.2* 2"' 2.04 (s)* 21.2* 3"' 170.5* 4"' 1.99 (s)* 21.0* recorded at 500 M H z . recorded at 100 M H z . T  3  COSY Correlations  H4, H5 H3,H5 H3, H4, H6 H5, H I ' H6, H 2 ' H1',H3' H 2 \ H4' H3',H5'b H6' H4' H5'a, H7' H6',H8' H7' H3"/5" H2"/6", H4" H3"/5"  3  HMBC Correlations  3  H3 H5 H5 H3 H5, H 1 ' , H 2 ' H6, H2, H3' H6, H 1 ' , H 3 ' H1',H2' H3',H5fb H3',H7 H5'a, H7', H8' H6',H276" H7',H8',H3"/5" H8',H4" H276" H2"/6" H2'" H3'"  * assignments interchangeable.  203  204  205 4.2.3.3. p-mercaptoethanol adduct  O  3  ^  'S  4"  2"'  5"  OH  151  The P-mercaptoethanol adduct of E-cryptofolione (151) was prepared by adding 10 ul of P-mercaptoethanol in a phosphate buffer to approximately 2 mg of £-cryptofolione (130) dissolved in THF.  After workup, the P-mercaptoethanol adduct (151) was isolated as a pale  yellow oil that gave an [ M ] ion in the HRCHVIS at m/z 392.16585 appropriate for a molecular +  formula of C i H 8 0 S (AM -0.3 ppm). 2  2  5  A quick analysis of the ID and 2D N M R spectra  indicated the absence of proton signals for the cw-olefin of the a,P-unsaturated lactone (5 5.99 and 5 6.83 ppm in £-cryptofolione (130)). These proton resonances were replaced by three new methylene signals, two of which were attributed to the new p-mercaptoethanol substituent. H M B C and COSY correlations confirm the presence of P-mercaptoethanol at C-4, consistent with a Michael-type addition. The relative stereochemistry of the P-mercaptoethanol substituent at C-4 was assigned based on the coupling constants measured between H-4 and the two H-3 methylene protons. The deshielded equatorial H-3 (5 2.91 ppm) shows a coupling constant 7 of 5.6 Hz to H-4 whereas the axial H-3 (8 2.55 ppm) shows a larger coupling to H-4 (7 = 7.7 Hz). This larger coupling constant is close to the typical 7 value of 8-10 Hz expected between two axial protons, thus supporting an equatorial P-mercaptoethanol substituent (see Figure 4.14). A summary of all N M R assignments, COSY, and H M B C correlations for the P-mercaptoethanol adduct (151) can be found in Table 4.8.  COSY  Figure 4.14. Selected C O S Y correlations in the p-mercaptoethanol adduct (151).  Table 4.8. N M R data for p-mercaptoethanol adduct (151) recorded in C D C 1 . 3  position 1 (O) 2 3a 3b 4 5a 5b 6 r 2' 3' 4' 5'a 5'b 6' 7' 8' 1" 2'76" 375" 4" 1"' 2"' 3  5'H (7 value in H z ) a  2.91 2.55 3.39 2.09 2.01 5.08 5.59 5.81 2.29 4.05 1.81 1.75 4.64 6.27 6.62 7.37 7.30 7.22 2.75 3.77  (dd, 7=17.4, 5.6) (dd, 7=17.4, 7.7) (m) (m) (m) (m) (dd, 7=15.6, 5.8) (dt, 7=15.4, 7.5) (t, 7=6.7) (m) (m) (m) (m) (dd, 7=15.9, 6.2) (d, 7=15.8) (d, 7=7.0) (t, 7=7.4) (t, 7=7.2) (t, 7=5.9) (t, 7=5.9)  recorded at 400 M H z .  5 C 1 3  b  169.2 36.8 34.3 34.6 76.8 130.6 130.3 40.4 68.2 42.2 70.6 131.6 130.3 136.5 126.5 128.6 127.8 33.8 61.4  recorded at 100 M H z .  COSY Correlations  3  HMBC Correlations  3  H3ab H3b H3a H5b, H 6 H5a H5a, H I ' H6, H 2 ' H1\H3' H2', H4' H 3 ' , H5'ab H4',H5'b, H6' H4',H5'a, H6' H8' H7' H375" H276", H4" H3'75"  H3ab, H5ab, H6 H6 H5a, H 1 ' , H 2 ' H5ab, H6, H 3 ' H3' H1',H2' H2',H3',H5'ab H3',H7' H5'ab, H 7 ' , H 8 ' H5'ab H7',H8',H375 H8',H4" H2'76"  * assignments interchangeable.  4  207  208  209 4.2.3.4. Compound 152  O  152  £-cryptofolione (130) was subjected to hydrogenation conditions using H>(g) in the presence of catalytic Pd-C to yield compound 152. After workup, the hydrogenation product 152 was isolated as a white amorphous solid that gave an [ M + H ] ion in the H R C T M S at m/z +  323.22162 appropriate for a molecular formula of  C19H30O4  ( A M 1.9 ppm). A quick analysis of  the I D and 2 D N M R spectra indicated the absence of proton and carbon resonances for the three olefins groups that were present in ZT-cryptofolione (130). The absence of a third oxymethine proton resonance in the region around 5 4 to 5 ppm and a downfield shift in the carbonyl resonance at 5 177.6 ppm supported the opening of the lactone ring as first indicated by the molecular formula.  The olefin and oxymethine resonances were replaced by seven new  methylene group signals, with H M B C and C O S Y correlations consistent with the proposed structure of compound 152.  A summary of all N M R assignments, C O S Y , and H M B C  correlations for compound 152 can be found in Table 4.9.  Table 4.9. N M R data for compound 152 recorded in C D C 1 . 3  position 1 (OH) 2 3 4 5 6 r 2' 3'a .3'b 4' ' 5' 6' 7'a 7'b 8'a 8'b 1"  S'H (7 value in Hz) a  2.33 (m) 1.62 (m) 1.30 (m) 1.39 (m) 1.30-1.39 (m)* 1.30-1.39 (m)* 1.49 (m) 1.42 (m) 3.94 (br.s) 1.62 (m) 3.96 (bs) 1.84 (m) 1.77 (m) 2.78 (m) 1.75 (m)  6 C 1 3  b  177.6 33.8 24.6 28.8 25.5 29.0* 29.2* 37.4 69.4 42.5 69.0 39.1 32.2  3  HMBC Correlations  3  H3 H4 H3,H5 H4  H3'b, H4' H3'a, H4' H3'ab, H 5 ' H4',H6' H5\H7'ab H6\H7'b, H8' H 6 ' , H 7 ' a , H8 H7'ab, H 8 ' b H7'ab, H 8 ' a  H3, H 5 H3, H I ' * H3, H I ' *  H5'  H7'ab, H8'ab H8'ab  H 7 ' a , H8'ab, H375" 276" 7.18 (d, 7=8.0) 128.4 H375" H8'ab, H3"/5' H4" 7.26 (t, 7=7.4) 375" H2"/6", H 4 " 128.5 H2"/6" 4" 7.17 (t, 7=7.6) 125.9 H3"/5" H2"/6" recorded at 500 M H z . recorded at 100 M H z . * assignments interchangeable. 3  142.0  COSY Correlations  212  213 4.2.4. Biological Activity Using Roberge's cell-based assay, a large-scale screen for G 2 cell cycle checkpoint inhibitors from crude extracts obtained from the N C I Open Repository led to the isolation of Ecryptofolione (130), Z-cryptofolione (148), cryptofolione ketone (131), and compound 138. The bioassay-guided isolation and purification identified these four 6-[co-arylalkenyl]-5,6-dihydro-apyrones from the Taiwanese tree Cryptocarya concinna, consistent with the phytochemistry previously reported for this genus.  Relatively simple synthetic transformations on the major  metabolite, f-cryptofolione (130), resulted in four additional analogues for structure-activity relationship ( S A R ) studies, namely cryptofolione acetonide (149), diacetylcryptofolione (150), a p-mercaptoethanol adduct (151), and a hydrogenated cryptofolione (152). Preliminary results show the natural products £-cryptofolione (130) and its shorter homologue, compound 138, exhibited similar G 2 checkpoint inhibitory potency as the previously described debromohymenialdisine (91) (see Figure 4.19). Altering the configuration of the A ' 7  8  olefin (i.e., Z-cryptofolione (148)) or the oxidation state of the C - 5 ' alcohol (i.e.,  cryptofolione ketone (131)) resulted in almost complete loss of inhibitory activity. Among the synthetic analogues, the cryptofolione acetonide (149) and the P-mercaptoethanol adduct (151) appear to retain biological  activity while diacetylcryptofolione (150) and  cryptofolione (152) are essentially inactive.  hydrogenated  It is clear that further studies are required to  completely determine the structural features necessary for G 2 checkpoint inhibition.  214  Concentration (ug/ml)  Figure 4.19. Inhibition of the G 2 checkpoint by natural and semi-synthetic a-pyrones.  To date, all the known G 2 checkpoint inhibitors have been shown to act either as protein phosphatase inhibitors, such as okadaic acid (101) and fostriecin (102), or as protein kinase inhibitors, such as caffeine  (23), staurosporine (100), U C N - O l (24), debromohymenialdisine  (91), and isogranulatimide (25).  However, E-cryptofolione (130)  was found to be inactive  against the known phosphatase and kinase G 2 checkpoint targets, suggesting it acts by a novel mechanism on an undetermined cellular target. Structurally, Zs-cryptofolione (130) leptomycin B  possesses an a,P~unsaturated 8-lactone similar to  (154), a compound recently reported to inhibit the nuclear export protein C r m l .  3 5  C r m l normally acts to exclude the protein kinase regulatory subunit cyclin B from the nucleus until the onset of mitosis. The inhibition of C r m l  by leptomycin B thus results in the  accumulation of cyclin B in the nucleus and premature entry into mitosis, overriding the G 2 checkpoint. However, leptomycin B (153) is inactive in yeast C r m l mutants possessing a serine substitution at cysteine-529, suggesting it binds covalently through a Michael-type addition of the sulfhydryl group on Cys-529. When E-cryptofolione (130) was tested against this yeast  215 mutant, it retains G 2 checkpoint inhibitory activity, suggesting its mode of action does not involve a Michael-type addition to the a,P-unsaturated 5-lactone.  This result is further  supported by the significant inhibitory activity o f the p-mercaptoethanol  adduct (151) of E-  cryptofolione. It is still possible that £-cryptofolione (130) inhibits the action of C r m l via an alternate mechanism.  OH  O  154 In contrast, there are several nuclear export proteins that do not possess a cysteine residue in their active site, such as N i p 1.  36  The effects of is-cryptofolione (130) on these  possible cellular targets are currently under investigation in the laboratory of Michel Roberge at the University of British Columbia.  4.3. Conclusions A s a result of a large-scale screen of natural extracts for G 2 checkpoint inhibitors, a series o f a-pyrones from the Taiwanese tree Cryptocarya concinna were found to exhibit potent inhibitory activity. Bioassay-guided purification resulted in the isolation and identification of Ecryptofolione (130), Z-cryptofolione (148), cryptofolione ketone (131), and compound 138. Synthetic modifications on the major metabolite, /f-cryptofolione (130), yielded four additional analogues: cryptofolione acetonide (149), diacetylcryptofolione (150), a P-mercaptoethanol adduct (151), and a hydrogenated cryptofolione (152). The G 2 checkpoint inhibitors isolated from C. concinna were structurally distinct from previously known inhibitors and appear to act by a novel mechanism of action.  Continuing studies are underway to determine the cellular  target and structural requirements of inhibition.  216  REFERENCES 1  Newman, D . J . ; Cragg, G . M . ; Snader, K . M . Nat. Prod. Rep. 2000,17, 215.  2  Arvigo, R.; Balick, M . In Rainforest Remedies; Lotus Press: T w i n Lakes, 1993.  3  Hamel, E . Med. Res. Rev. 1996,16, 207; and references cited therein.  4  Cragg, G . M . ; Boyd, M . R . ; Cardellina, J . H . , U ; Newman, D . J . ; Snader, K . M . ; M c C l o u d , T . G . In Ethnobotany and the Search for New Drugs; Ciba Symposium N o . 185; 1994, p. 178.  3  Potmeisel, M . ; Pinedo, H . In Camptothecins: New Anticancer Agents; C R C Press: B o c a Raton, 1990. |  6  Naik, R . G . ; Kattige, S.L.; Bhat, S.V.; Alreja, B . ; de Sousa, N . J . ; Rupp, R . H . Tetrahedron 1988,44,2081.  7  Christian, M . 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Chem. 1999, 274, 17309.  218  EXPERIMENTAL The extractions and column chromatography purifications were performed using reagent grade solvents. H P L C purifications utilized H P L C grade solvents that had been filtered and degassed prior to use. A l l other solvents, reagents, and standards were reagent or commercial grade and were used without further purification.  Biichi rotary evaporators facilitated the  removal of solvents in vacuo. Normal phase flash column chromatography was carried out using 230-400 mesh Merck silica gel G60. Reversed-phase flash column chromatography was performed using Whatman brand reversed-phase C - l 8 silica and size-exclusion chromatography was performed using Sephadex L H - 2 0 beads (25-100 u.m). Normal and reversed-phase thin layer chromatography ( T L C ) was carried out on commercial Kieselgel 60 F254 and Whatman M K C 1 8 F plates, respectively. Compounds were visualized by U V detection (k 254 nm) and by charring using a vanillin/sulphuric acid spray reagent. High performance liquid chromatography ( H P L C ) separations were performed on one of two systems: a Waters 600E H P L C pump with a Waters 486 U V tunable absorbance detector, or a Waters 600E H P L C pump with a Waters 996 U V photodiode array detector.  Each system  directed the data to a personal computer using M i l l e n i u m ™ 2010 chromatography software. Normal phase H P L C separations involve using either a Whatman Partisil 10 Magnum column or an Econosil cyano-bonded S i 5(0. column. Reversed-phase H P L C purifications were performed on either a Whatman Partisil 10 O D S - 3 Magnum column or an Econosil C18 5u column. N M R data was collected on three different spectrometers: a Bruker A M X 5 0 0 , a Bruker W H 4 0 0 , or a Bruker A M 4 0 0 . Each was fitted with a 5mm probe and the spectra were obtained in either D M S O - d 6 or CDCI3 using the solvent residual peaks as references ( D M S O - J 6 : 8 2.49  219 ( ' H ) 5 39.5 ( C ) ; C D C 1 : 8 7.24 ('H), 5 77.0 ( C ) ppm). Spectra were processed using Bruker 13  ;  13  3  W i n d o w s ™ compatible W I N N M R software and coupling constants were reported in H z . L o w and high resolution E I mass spectra were collected on a Kratos M S 5 0 / D S 5 5 S M mass spectrometer while the low and high resolution F A B mass spectra were recorded on a Kratos Concept II H Q spectrometer. A l l M S data acquisition was performed by the staff at the U B C Department of Chemistry Mass Spectrometry Center. Optical rotations were measured on a Jasco P-1010 polarimeter or a Perkin-Elmer 241 M L polarimeter (1 c m quartz cell), and the [OC]D values are given in 10"'degcm g" . U V measurements were performed on a Waters 2487 2  1  Dual Absorbance Detector with maximum absorptions recorded as ^ absorptivity coefficients £ in units o f L m o f ' c m " .  m a x  in nanometers and molar  Circular dichroism (CD) spectra were  1  obtained using a Jasco J-710 spectropolarimeter. A l l antimitotic and G 2 checkpoint inhibition assays were performed by members of Dr. M i c h e l Roberge's research group at U B C .  X-ray  diffraction analysis was performed by Brian Patrick at U B C . Eleutherobin (61) crystallized i n space group ?2{2\2 with a=\2.8291(8), X  6=13.6209(6),  and c= 19.168(1) A . A crystal with dimensions 0.30 x 0.20 x 0.15 mm, was mounted on a glass fiber. Data were collected at - 1 0 0 ° C on a R i g a k u / A D S C C C D area detector in two sets of scans (O = 0.0 to 190.0°, x = 0°; and co =-18.0 to 23.0°, x =- 90°) using 0.50° oscillations with 58.0 s exposures. 5.52°.  The crystal-to-detector distance was 40.55 mm with a detector swing angle of -  O f the 7035 unique reflections measured ( M o - K a radiation, 29max=55.8°, i? =0.071, mr  Friedels not merged), 4520 were considered observed (I>3o(I)). The final refinement residuals were R=0.046 (on F , l>3o(l)) and wR2=0.141 (on F , all data). The data was processed using 2  the d * T R E K program and corrected for both Lorentz and polarization effects. The structure was solved by direct methods and all non-hydrogen atoms were refined anisotropically, while all hydrogens involved in hydrogen-bonding were refined isotropically. A l l other hydrogens were  220 included  in calculated positions.  A l l calculations were  performed  using the  teXsan  crystallographic software package of the Molecular Structure Corporation.  Desmethyleleutherobin (69): White amorphous solid; H R F A B M S [ M + H ] m/z 643.32230 +  (C34H46N2O10,  A M -1.21 ppm); [ a ] ° = -31.3°, (c = 0.2, M e O H ) ; U V ( M e O H ) X 2  D  8 3.929); ' H N M R ( D M S O - J 6 , 500 M H z ) ,  13  m a x  2 9 1 nm (log  C N M R (DMSO-c/6, 100 M H z ) , and 2D N M R data  listed in Table 2.1.  Eleutherobin (61): White amorphous solid; H R F A B M S [ M + H ] m/z 657.33719 C 5H4 N Oio +  3  ( A M -2.32 ppm); [ a ]  2 0 D  = -35.7°, (c = 0.4, M e O H ) ; U V ( M e O H ) X  N M R ( D M S O - J 6 , 500 M H z ) ,  max  l3  8  2  292 nm (log e 4.053); *H  C N M R ( D M S O - J 6 , 100 M H z ) , and 2 D N M R data listed in  Table 2.2.  Desacetyleleutherobin (70): White amorphous solid; H R F A B M S [ M + H ] m/z 615.32813 +  C33H46N2O9 ( A M -0.05 ppm); [ a ]  2 0 D  = -40.0°, (c = 0.3, M e O H ) ; U V ( M e O H ) X  e 4.076); ' H N M R (DMSO-</6, 500 M H z ) ,  13  m a x  290 nm (log  C N M R ( D M S O - ^ 6 , 100 M H z ) , and 2D N M R data  listed in Table 2.3.  Isoeleutherobin A (71): White amorphous solid; H R F A B M S [ M + H ] m/z 657.33834 +  C35H48N2O10 ( A M -0.58 ppm); [cc] ° = -42.8°, (c = 0.1, M e O H ) ; U V ( M e O H ) A™* 290 nm (log 2  D  e 4.132); ' H N M R ( D M S O - J 6 , 500 M H z ) and 2D N M R data listed in Table 2.4.  Z-eleutherobin (72): White amorphous solid; H R F A B M S C 5H48N2O 3  10  [M +  H]  +  m/z 657.33830  ( A M -0.65 ppm); ' H N M R ( D M S O - J 6 , 500 M H z ) and 2 D N M R data listed in  Table 2.5.  Caribaeoside (73): Colourless glass; H R F A B M S [ M + H ] m/z 673.33474 +  1.64 ppm); [ a ] NMR  2 0 D  = -10.0°, (c = 0.08, M e O H ) ; U V ( M e O H ) X  m a x  (DMSO-46, 500 M H z ) and 2 D N M R data listed in Table 2.6.  C35H48N2O11  (AM  288 nm (log £ 3.792);  'H  221  Caribaeolin (74): ppm); [ a ]  2 0 D  Clear o i l ; H R F A B M S [ M + H ] m/z 541.29111 +  = -12.0°, (c = 0.08, M e O H ) ; U V ( M e O H ) X  C35H48N2O11  289 nm (log e 4.071);  max  ( A M -0.49 'H NMR  ( D M S O - ^ 6 , 500 M H z ) and 2 D N M R data listed in Table 2.7.  Diacetyleleutherobin (75): Formed from three separate acetylation reactions on eleutherobin (61), desacetyleleutherobin (70), and isoeleutherobin A (71).  Approximately 1 mg of compound  was dissolved in 1.5 ml of pyridine and then 0.5 m l of acetic anhydride was added and mixture was stirred at room temperature overnight.  Isolated ~ 1 mg of white amorphous solid;  H R F A B M S [ M + H ] m/z 741.33834 C 9 H N O i 2 ( A M -1.58 ppm); H N M R ( D M S O - J 6 , 500 +  !  3  5 2  2  M H z ) and 2 D N M R data listed in Table 2.8.  Sarcodictyin A (55): Clear oil; H R F A B M S [ M + H ] m/z 497.26514 C 8 H 3 6 N 0 ( A M -0.04 +  2  2  6  ppm); ' H N M R (pyridine-^5, 400 M H z ) 5 8.05 (d, 7=15.5, 1H), 7.68 (s, 1H), 7.35 (s), 7.14 (d, 7=15.5, 1H), 7.12 (d, 7=5.6, 1H), 6.97 (d, 7=9.5, 1H), 6.28 (d, 7=5.6, 1H), 5.26 (bs, 1H), 5.18 (d, 7=7.0, 1H), 4.58 (m, 1H), 3.65 (s, 3H), 3.39 (s, 3H), 2.95 (m, 1H), 2.38 (m, 1H), 1.98 (m, 1H), 1.94 (m, 1H), 1.58 (s, 3H), 1.56 (s, 3H), 1.53 (s, 3H), 1.43 (m, 1H), 1.17 (m, 1H), 0.91 (d, 7=6.6, 3H), 0.81 (d, 7=6.6, 3H); C N M R ( p y r i d i n e - ^ , 100 M H z ) 5 168.0, 167.2, 144.0, 140.4, 138.3, 1 3  138.0, 135.6, 134.7, 134.3, 133.0, 124.5, 121.8, 115.4, 112.3, 89.6, 81.8, 51.8, 42.1, 39.3, 34.9, 33.3, 32.2, 29.0, 25.8, 24.5, 22.2, 22.1, 20.4.  Erythrolide A (36): White, amorphous solid; HREUvIS [ M ] m/z 538.15613 C H i O i o C l ( A M +  2 6  3  -0.34 ppm); ' H N M R ( C D C 1 , 500 M H z ) 5 6.36 (dd, 7=16.1, 7.1, 1H), 5.90 (s, 1H), 5.79 (d, 3  7=16.2, 1H), 5.51 (s, 1H), 5.39 (s, 1H), 5.35 (s, 1H), 5.05 (d, 7=9.3, 1H), 4.46 (d, 7=9.3, 1H), 3.42 (s, 1H), 3.08 (m, 1H), 2.86 (m, 1H), 2.25 (dd, 7=10.5, 7.9, 1H), 2.13 (s, 3H), 2.03 (s, 3H), 1.90 (s, 3H), 1.87 (d, 7=10.1, 1H), 1.48 (s, 3H), 1.27 (s, 3H), 1.10 (s, 3H);  1 3  C N M R (CDC1 , 3  100 M H z ) 5 204.7, 175.0, 171.4, 169.5, 167.5, 138.6, 132.8, 127.2, 126.8, 87.2, 83.0, 80.6, 79.9, 75.4, 59.2, 43.6, 43.4, 39.6, 37.2, 28.6, 22.3, 21.3, 21.3, 20.8, 20.5, 9.2.  222  Erythrolide E (41): White, amorphous solid; HREUvIS [ M ] m/z 496.15080 +  C24H29O9CI  (AM -  0.26 ppm); ' H N M R (CDCI3, 500 M H z ) 8 6.63 (d, 7=10.0, 1H), 6.00 (d, 7=10.0, 1H), 5.73 (dd, 7=5.2, 2.5, 1H), 5.67 (s, 1H), 5.39 (s, 1H), 4.78 (s, 1H), 4.36 (d, 7=1.2, 1H), 4.18 (d, 7=1.6, 1H), 3.95 (m, 1H), 3.21 (d, 7=2.2, 1H), 2.73 (m, 2H), 2.66 (m, 1H), 2.24 (s, 3H), 2.11 (s, 3H), 1.62 (s, 3H), 1.45 (s, 3H), 1.03 (s, 3H);  1 3  C N M R ( C D C 1 , 100 M H z ) 8 194.1, 174.1, 169.9, 169.9, 3  152.3, 138.2, 124.5, 123.0, 86.1, 85.7, 83.1, 80.5, 69.8, 68.7, 59.3, 48.9, 41.6, 41.3, 36.3, 22.3, 21.2,21.1,21.1,6.5.  Debromohymenialdisine (91)HC1: Y e l l o w , amorphous solid; HREEVIS [ M ] m/z 245.09128 +  C i H u N g O z ( A M -1.6 ppm); ' H N M R ( D M S O - J 6 , 400 M H z ) 8 12.10 (s, 1H), 9.5-8.5 (br, 3H), 8.02 (br.s, 1H), 7.10 (s, 1H), 6.49 (s, 1H), 3.29 (m, 4H);  1 3  C N M R ( D M S O - ^ 6 , 100 M H z ) 8  163.5, 162.9, 154.5, 130.2, 126.7, 122.7, 120.2, 120.0, 109.5, 39.2,31.6.  Hymenialdisine (85)-HCl: Y e l l o w ,  amorphous  solid;  HREEVIS  [M]  m/z  +  325.08734  C n H N O 2 B r ( A M - 1 . 0 ppm); ' H N M R ( D M S O - J 6 , 500 M H z ) 8 12.88 (s, 1H), 11.02 (br, 8 1  1 0  5  1H), 9.33 (br, 1H), 8.85 (br, 1H), 8.14 (br.s, 1H), 6.63 (s, 1H), 3.25 (m, 4 H ) ;  1 3  C N M R (DMSO-  d6, 100 M H z ) 8 163.0, 162.2, 154.5, 128.5, 128.3, 121.4, 120.5, 111.3, 105.2,39.7,31.9.  Z-Debromoaxinohydantoin (94): Y e l l o w , amorphous solid; HREEVIS [ M ] m/z 246.07530 +  C11H10N4O3  ( A M - 0 . 2 ppm); *H N M R ( D M S O - J 6 , 400 M H z ) 8 11.79 (br, 1H), 11.04 (br, 1H),  9.44 (br, 1H), 7.89 (br, 1H), 6.96 (br, 1H), 6.51 (s, 1H), 3.2 (m, 4 H ) ;  1 3  C N M R ( D M S O - ^ 6 , 100  M H z ) 8 165.3, 162.9, 154.3, 125.4, 122.7, 122.4, 121.9, 121.3, 109.7, 40.4,30.6.  Aldisin (103): Pale yellow, amorphous solid; HREEVIS [ M ] m/z 164.05605 +  C H N 0 2 (AM 8  8  2  0.5 ppm); ' H N M R ( D M S O - J 6 , 400 M H z ) 8 12.20 (br, 1H), 8.34 (br, 1H), 6.97 (s, 1H), 6.53 (s, 1H), 3.36 (m, 2H), 2.68 (m, 2 H ) ; 122.4, 109.4,43.5, 36.5.  1 3  C N M R (DMSO-c/6, 100 M H z ) 8 194.3, 162.2, 128.0, 123.5,  223 E - C r y p t o f o l i o n e (130): White, amorphous solid; H R E T M S [ M ] m/z 314.15178 C 1 9 H 2 2 O 4 ( A M +  -0.09 ppm); [ a ]  = 57.5°, (c = 0.5, M e O H ) ; U V ( M e O H ) k  2 0 D  (CDCI3, 400 M H z ) ,  1 3  m a x  240 nm (log e 3.867); ' H N M R  C N M R ( C D C 1 , 100 M H z ) , and 2 D N M R data listed in Table 4.2. 3  Z - C r y p t o f o l i o n e (148): White, amorphous solid; H R C I M S [ M ] m/z 314.15226 C i H +  9  -1.5 ppm); [ a ]  2 0 D  = 120.5°, (c = 1.2, M e O H ) ; U V ( M e O H ) X  (CDCI3, 4 0 0 M H z ) ,  1 3  2 2  0  (AM  4  239 nm (log e 3.462); H N M R !  m a x  C N M R ( C D C 1 , 100 M H z ) , and 2 D N M R data listed in Table 4.3. 3  Cryptofolione ketone (131): White, amorphous solid; H R C I M S [ M + H ] m/z 313.14358 +  C H o 0 4 ( A M 1.3 ppm); [ a ] 1 9  2  2 0 D  = 105.0°, (c = 0.08, M e O H ) ; U V ( M e O H ) X  3.823); ' H N M R (CDCI3, 500 M H z ) ,  1 3  C N M R ( C D C 1 , 100 M H z ) , 3  m a x  286 nm (log e  and 2 D N M R data listed in  Table 4.4. "C o m p o u n d 138: White, amorphous solid; H R C I M S [ M ] m/z 288.13514 C H o 0  ( A M 3.6  +  1 7  ppm);  [a]  = -13.8°, (c = 0.08, M e O H ) ; U V ( M e O H ) l  2 0 D  1 3  4  277 nm (log e 2.343); U N M R l  maK  (CDCI3, 500 M H z ) ,  2  C N M R ( C D C 1 , 100 M H z ) , and 2 D N M R data listed in Table 4.5. 3  Cryptofolione acetonide (149): Prepared by adding 2 m l of 2,2-dimethoxy-propane and a catalytic amount of pyridinium p-toluene sulfonate to approximately 2 mg of Zs-cryptofolione (130), stirred at 70°C overnight. Y i e l d 1.8 mg. White, amorphous solid; H R C I M S [ M + H ] m/z +  355.19102 C H 6 0 2 2  2  4  ( A M - 0 . 2 ppm); [ a ]  2 0 D  nm (log s 4.211); H N M R ( C D C 1 , 400 M H z ) , !  3  = 49.4°, (c = 0.7, M e O H ) ; U V ( M e O H ) X  max  1 3  250  C N M R ( C D C 1 , 100 M H z ) , and 2D N M R data 3  listed in Table 4.6. Diacetylcryptofolione (150): Prepared by adding 0.5 m l of acetic anhydride and 1.5 ml of pyridine to approximately 2 mg of E-cryptofolione (130), stirred at room temperature overnight. Y i e l d 1.9 mg. White, amorphous solid; H R E P M S [ M ] m/z 398.17274 C 3 H +  2  ppm);  [a]  2 0 D  = 46.9°, (c =• 0.8, M e O H ) ; U V ( M e O H ) X  (CDCI3, 400 M H z ) ,  1 3  m a x  2 6  0  6  ( A M -0.51  250 nm (log e 4.151); *H N M R  C N M R (CDCI3, 100 M H z ) , and 2 D N M R data listed in Table 4.7.  224  P-mercaptoethanol adduct (151): Prepared by adding 10 pi o f P-mercaptoethanol in a phosphate buffer ( p H 7.4) to approximately 2 mg of is-cryptofolione (130) stirred at room temperature for 1 hour. Y i e l d 2.1 mg. 392.16585 C i H 0 S ( A M -0.3 ppm); [ a ] 2  2 8  5  2 0 D  dissolved in T H F ,  Pale yellow o i l ; H R C I M S [ M ] m/z +  = -10.2°, (c = 1.6, M e O H ) ; U V (MeOH) X  max  250  nm (log s 3.881); *H N M R ( C D C 1 , 400 M H z ) , C N M R ( C D C 1 , 100 M H z ) , and 2 D N M R data 1 3  3  3  listed in Table 4.8.  Hydrogenated ^-cryptofolione (152): Prepared by using H (g) in the presence of catalytic Pd2  C in ethanol, stirred at room temperature overnight to yield 0.8 mg of compound 152. amorphous solid; H R C I M S [ M + H ] m/z 323.22162 C i H O ( A M 1.9 ppm); C +  9  0.51 ppm); [ a ]  2 0 D  = -7.2°, (c = 1.2, M e O H ) ; U V (MeOH) X  ( C D C 1 , 400 M H z ) , 3  3 0  1 3  4  m a x  2 3  H  2 6  0  6  (AM -  259 nm (log £ 2.039); ' H N M R  C N M R ( C D C 1 , 100 M H z ) , and 2 D N M R data listed in Table 4.9. 3  White,  225  APPENDIX  JUL  JUL  JU  din  ii^^juii.  as  ^ K  a©  ©.© g  •  • to a  3  2899  ::  B-  if iS JO  -  o 0 Op  •  0  oB  o •  (ppm)  Figure A . l . C O S Y spectrum of eleutherobin (61) recorded in D M S O - ^ 6 at 500 M H z .  226  Figure A . 2 . H M Q C spectrum of eleutherobin (61) recorded in D M S O - ^ 6 at 500 M H z .  Figure A . 3 . H M B C spectrum of eleutherobin (61) recorded in D M S O - ^ 6 at 500 M H z .  228  Figure A . 4 . C O S Y spectrum of desacetyleleutherobin (70) recorded in DMSO-rf6 at 500 M H z .  229  OH  (ppm)  7 00  6.00  5.00  4.00  3.00  2.0C  1.00  Figure A . 5 . H M Q C spectrum of desacetyleleutherobin (70) recorded in D M S O - J 6 at 500 M H z .  230  Figure A . 6 . H M B C spectrum of desacetyleleutherobin (70) recorded in DMSO-c/6 at 500 M H z .  Figure A . 7 . C O S Y spectrum of isoeleutherobin A (71) recorded in D M S O - J 6 at 500 M H z .  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  Figure A . 8 . H M Q C spectrum of isoeleutherobin A (71) recorded in D M S O - ^ 6 at 500 M H z .  233  Figure A . 9 . H M B C spectrum of isoeleutherobin A (71) recorded in D M S O - J 6 at 500 M H z .  234  (ppm)  Figure A . 10. C O S Y spectrum of Z-eleutherobin (72) recorded in D M S O - d 6 at 500 M H z .  235  Figure A . 11. H M Q C spectrum of Z-eleutherobin (72) recorded in DMSO-J6 at 500 M H z .  236  OH -<>"  0-  4  OMei OAc  OH  2  ,0  72 JUL_J_JU  (ppm)  ea  (ppm)  7.00  6.00  5.00  3.00  2.00  1.00  Figure A . 12. H M B C spectrum of Z-eleutherobin (72) recorded in D M S O - J 6 at 500 M H z .  Figure A . 13. C O S Y spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  Figure A . 14. H M Q C spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  Figure A . 15. H M B C spectrum of caribaeoside (73) recorded in D M S C M 6 at 500 M H z .  Figure A . 16. R O E S Y spectrum of caribaeoside (73) recorded in D M S O - J 6 at 500 M H z .  Figure A . 17. C O S Y spectrum of caribaeolin (74) recorded in D M S O - J 6 at 500 M H z .  Figure A . 18. H M Q C spectrum of caribaeolin (74) recorded in DMSO-J6 at 500 M H z .  Figure A . 19. H M B C spectrum of caribaeolin (74) recorded in DMSO-<i6 at 500 M H z .  244  Me  7.00  6.00  5.00  4.00  3.00  2.00  1.00  (ppm)  Figure A . 2 0 . R O E S Y spectrum of caribaeolin (74) recorded in D M S O - ^ 6 at 500 M H z .  (ppm)  Figure  A.21. COSY spectrum of debromohymenialdisine (91)HC1 recorded in DMSO-d6 at 500 MHz.  Figure A.22. H M Q C spectrum of debromohymenialdisine (91)HC1 recorded in D M S O - d 6 at 500 M H z .  1  (ppm)  (ppm)  Figure A.23. H M B C spectrum of debromohymenialdisine (9l)-HCl recorded in D M S 0 - J 6 at 500 M H z .  248 2' \l4 ,  H  0  85  L-JL  Figure A.24. COSY spectrum of hymenialdisine (85)HC1 recorded in DMSO-d6 at 500 MHz.  249  85  JL_JL  (ppm)  Figure A.25. HMQC spectrum of hymenialdisine (85)-HCl recorded in DMSO-J6 at 500 MHz.  250  85  (ppm)  (ppm)  Figure A . 2 6 . H M B C spectrum of hymenialdisine (85)-HCl recorded in D M S O - J 6 at 500 M H z .  Figure A . 2 7 . C O S Y spectrum of £-cryptofolione (130) recorded in C D C 1 at 500 M H z . 3  Figure A.28. HMQC spectrum of ^-cryptofolione (130) recorded in CDC1 at 500 MHz. 3  Figure A . 2 9 . H M B C spectrum of ^-cryptofolione (130)  recorded in C D C 1 at 500 M H z . 3  5'  T  3'  r  5  148  .A.  \  4  6.4  5.6  4.0  3.2  2.4  Figure A . 3 0 . C O S Y spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z . 3  148  J  LJLJLJUJU  Figure A.31. HMQC spectrum of Z-cryptofolione (148) recorded in CDC1 at 500 MHz. 3  Figure A.32. H M B C spectrum of Z-cryptofolione (148) recorded in C D C 1 at 500 M H z . 3  257 O  (pom)  6.0  4.0  2.0  (BP")  Figure A.33. C O S Y spectrum of cryptofolione ketone (131)  recorded in C D C 1 at 500 M H z . 3  258 O  Figure A.34. H M Q C spectrum of cryptofolione ketone (131)  recorded in C D C 1 at 500 M H z . 3  259  Figure A.35. H M B C spectrum of cryptofolione ketone (131) recorded in CDC1 at 500 MHz. 3  ;ure A.36. COSY spectrum of compound 138 recorded in CDC1 at 500 MHz. 3  Figure A.37. HMQC spectrum of compound 138 recorded in CDC1 at 500 MHz. 3  262  Figure A . 3 8 . H M B C spectrum of compound 138 recorded in C D C 1 at 500 M H z . 3  

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