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UBC Theses and Dissertations

Terpenoids from marine and terrestrial sources Craig, Kyle Sheldon 2003

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TERPENOIDS F R O M M A R I N E A N D TERRESTRIAL SOURCES  by  K Y L E SHELDON CRAIG B . S c , Andrews University, 1997  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 M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES THE D E P A R T M E N T OF CHEMISTRY  W e accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A M a y 2003 © K y l e S. Craig, 2003  In presenting this thesis in partial fulfilment o f the requirements for an advanced degree at the University o f British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying o f this thesis for scholarly purposes may be granted by the head o f m y department or by his or her representatives. It is understood that copying or publication o f this thesis for financial gain shall not be allowed without m y written permission.  |7 /W^rf- 2©o>  Abstract Studies on the Indonesian sponge Hippospongia  sp. led, via bioactivity-guided  fractionation, to the isolation o f six linear norsesterterpene carboxylic acids. Rhopaloic acids B (35) and C (36) were known, but rhopaloic acids D(37)-G(40) were novel i n structure. These compounds are the first known natural product inhibitors o f R C E protease, an important new target in cancer therapy.  37  Two investigations o f weedy plants, Ambrosia artemisiifolia  and Vernonia  baldwinii, whose extracts showed activity i n a cell-based assay for G 2 checkpoint inhibitors, were undertaken. Six sesquiterpenes were isolated for each weed (41-46, 47 and related compounds, respectively). W i t h respect to A. artemisiifolia,  an additional  four sesquiterpenes were semi-synthesized i n an effort to understand the requirements necessary for biological activity. W h e n 47 was hydrogenated under standard conditions it no longer contained an a-(3 unsaturated ester side chain and biological activity was lost. Sesquiterpenes 42-44 represent some o f the first compounds known to be both G 2 checkpoint inhibitors and antimitotic agents, while 47 has the ability to activate phosphorylation o f nucleolin and tau, making it a potential tool for the study o f Alzheimer's disease pathology.  11  In our continuing investigations into the chemistry o f the Caribbean octocoral, Erythropodium  caribaeorum,  two additional briarane diterpenes 48-49 were isolated. A  novel erythrane diterpene 50 and two novel aquariane diterpenes 51-52 were also isolated. The aquariane skeleton appears to be derived v i a a vinylcyclopropane rearrangement o f an erythrane precursor.  iii  Table of Contents  Abstract  ii  Table o f Contents  iv  List o f Tables  viii  List o f Figures  x  List o f Schemes  xv  List o f Abbreviations  xvi  Dedication and Acknowledgments  xx  1.1: Introduction to Natural Products  1  1.2: Introduction to Marine Natural Products  3  1.3: Natural Products and Cancer  8  1.4: Introduction to Terpenoids 1.4.1: T w o Examples o f Terpenes as Drugs  9  1.4.2: Biosynthesis o f Terpenoids  10  1.5: Thesis Preview  13  1.6: References  15  2.1: B r i e f Introduction to Bioactive Sponge Metabolites  19  2.2: Introduction to Sesterterpenes and Related Compounds  20  2.3: B r i e f Introduction to Sponges and to Hippospongia  sp.  2.3.1: General Introduction to Sponges  27  2.3.2: The Sponge Genus Hippospongia  28  2.3.3: Overview o f K n o w n Metabolites from Hippospongia  sp.  2.4: The 2.5: RCE-protease Ras Converting Inhibitors Enzyme fromand an Cancer Indonesian Hippospongia  iv  28 sp.  31  2.5.1: Isolation Procedure  35  2.5.2: Rhopaloic Acids B (35) and C (36)  36  2.5.3: Rhopaloic Acids D / E (37/38) and F (39)  43  2.5.4: Rhopaloic A c i d G (40)  56  2.5.5: Conclusions  60  2.6: References  64  2.7: Experimental Section 2.7.1: General Information  72  2.7.2: Isolation o f Rhopaloic Acids B(35)-G(40)  73  2.8: 2 D N M R Spectra for Rhopaloic Acids D(37)-G(40)  76  3.1: Introduction to Sesquiterpene Lactones  85 .  3.2: Biological Activity o f Sesquiterpene Lactones 3.2.1: K n o w n Examples o f Biological Activity  87  3.2.2: G 2 C e l l Cycle Checkpoint/Antimitotic Activity of Ambrosia artemisiifolia  91  3.2.3: T G - 3 Antibody Activity o f Vernonia baldwinii  92  3.3: Sesquiterpene Lactones from Ambrosia artemisiifolia 3.3.1: The Cell Cycle and G 2 Checkpoint Inhibitors 3.3.2: Isolation and Structure Elucidation o f Sesquiterpenes from A. 3.3.2.1: Introduction to Metabolites from A. artemisiifolia 3.3.2.2: Isolation and Structure Elucidation  93 artemisiifolia 98  3.3.2.2.1: Isolation Procedure  101  3.3.2.2.2: Psilostachyins A (42), B (43), and C (44)  102  3.3.2.2.3: Paulitin (45) and Isopaulitin (46)  109  3.3.2.2.4: Artemisiidiendioc A c i d Monomethyl Ester (41)  112  3.3.3: Synthesis and Structure Elucidation o f Derivatives 223-226 3.3.3.1: Psilostachyin A P-Mercaptoethanol Adduct: Compound 223  119  3.3.3.2: The Dihydropaulitins: Compounds 224 and 225  123  3.3.3.3: Paulitin Dehydration Product: Compound 226  129  3.3.4: Conclusions: The Bioactivity of Ambrosia artemisiifolia  Sesquiterpenes  3.4: Sesquiterpene Lactones from Vernonia baldwinii 3.4.1: Review o f Metabolites from Vernonia baIdwinii  135  3.4.2: Isolation Procedure o f T G - 3 Active Sesquiterpenes from Vernonia 3.4.3: Structure Elucidation o f Sesquiterpenes from Vernonia 3.4.3.1: Vernonataloides: Compounds 47, 227, and 228  133  baldwinii 136  baldwinii 137  3.4.3.2: Marginatins: Compounds 229 and 230  148  3.4.3.3: A Bourbenolide Sesquiterpene: Compound 231  153  3.4.3.4: 8-Desacylglaucolide A-tiglate: Compound 232  157  3.4.4: Conclusions: Alzheimer's Disease, Mitosis, and Vernonataloide (47)  160  3.5: References  163  3.6: Experimental Section 3.6.1: General Information  169  3.6.2: Isolation  171  3.7: 2 D N M R Spectra o f Artemisiidiendioc A c i d Monomethyl Ester  174  4.1: Introduction to Gorgonians and Their Secondary Metabolites 4.1.1: B r i e f Introduction to Gorgonians  177  4.1.2: Examples o f Secondary Metabolites from Gorgonians  177  4.2: Diterpenes from Erythropodium 4.2.1: Description o f E. caribaeorum  caribaeorum and B r i e f Overview o f K n o w n Metabolites 180  4.2.2: Isolation and Structure Elucidation o f Diterpenes 48-52 4.2.2.1: Isolation Procedure  183  4.2.2.2: N o v e l Briaranes: Erythrolides T (48) and U (49)  185  4.2.2.3: A N o v e l Erythrane: Erythrolide V (50) vi  193  4.2.2.4: N o v e l Aquarianes: Aquariolide B (51) and C (52)  198  4.3: Conclusions  207  4.4: References  213  4.5: Experimental Section 4.5.1: General Information  216  4.6: 2 D N M R Spectra o f Diterpenes 48-52  221  5.0: Concluding Remarks  237  vii  List of Tables Table 2.1  Rhopaloic Acids B (35) and C (36) N M R Data i n C D  Table 2.2  Rhopaloic A c i d D (37) N M R Data i n C D  Table 2.3  Rhopaloic A c i d E (38) N M R Data i n C D  6  Page 51  Table 2.4  Rhopaloic A c i d F (39) N M R Data i n C D  6  Page 54  Table 2.5  Rhopaloic A c i d G (40) N M R Data i n C D  Table 3.1  Psilostachyin A(42), B(43), and C (44) N M R Data i n C D C 1  6  6  6  6  6  Page 39  6  Page 48  6  Page 58  6  3  Page 105 Page 112  Table 3.2  Paulitin (45) and Isopaulitin (46) N M R Data i n C D C 1  Table 3.3  Artemisiidiendioc A c i d Monomethyl Ester (41) N M R Data inDMSO. Page 115  3  d 6  Table 3.4  Psilostachyin A (3-Mercaptoethanol Adduct 223 N M R Data i n C D C 1 Page 120  Table 3.5  Epoxide 224 N M R Data in C D C 1  3  Page 125  Table 3.6  Epoxide 225 N M R Data i n C D C 1  3  Page 126  Table 3.7  N M R Data for Compound 226 i n C D C 1  Table 3.8  Comparison o f Cytotoxicity and G 2 Checkpoint Inhibition Activity for 4246 and 223-226 Page 133  Table 3.9  Vernonataloide (47) and 8-deacylvernonataloide-8-0-tiglate (227) N M R DatainCDCl Page 142  3  3  Page 130  3  Table 3.10  13-desacetoxy-8-deacylvernonataloide-8-0-isobutyrate in C D C 1 3  Table 3.11  (228) N M R Data Page 145  Marginatin (229) and Marginatin Methylacrylate (230) N M R Data i n CDCl at233K Page 150 3  Table 3.12  Acetoxy-8a-tiglinoyloxy-4p-hydroxy-bourbon-7(ll)-en-6,12-olide (231) N M R Data i n C D C 1 at 273 K Page 155 3  vm  Table 3.13  8-Desacylglaucolide A-tiglate (232) N M R Data i n C D C 1 at 23 3/243 K Compared with Literature Data for Glaucolide B (260) at 2 6 0 K i n CDCI3 Page 158  Table 4.1  Erythrolide T (48) N M R Data in CDCI3  Page 187  Table 4.2  Erythrolide U (49) N M R Data i n C D C 1  Page 190  Table 4.3  Erythrolide V (50) N M R Data i n CDCI3  Page 195  Table 4.4  Aquariolide B (51) N M R Data in C D C 1  3  Page 200  Table 4.5  Aquariolide C (52) N M R Data i n CDCI3  Page 204  3  ix  3  List of Figures Figure 2.1  RJA96-141—Hippospongia sp. (Photo by M . LeBlanc)  Figure 2.2  Probable Orientation o f the Tetrahydropyran M o i e t y and a-P Carbonyl System i n 122 and 35 Page 37  Figure 2.3  ' H N M R Spectrum o f Rhopaloic A c i d B (35) i n C D  6  Page 40  Figure 2.4  ' H N M R Spectrum o f Rhopaloic A c i d C (36) i n C D  6  Page 41  6  6  C N M R Spectra o f Rhopaloic A c i d C (36) i n C D  Page 35  Figure 2.5  1 3  Figure 2.6  Important C O S Y and H M B C Correlations for 37/38  Page 44  Figure 2.7  Important H M B C correlations for Partial Structures o f 38  Page 45  Figure 2.8  C O S Y Correlations in 39 Suggesting Placement o f the A l l y l i c A l c o h o l  6  Page 42  6  Page 47 Figure 2.9 Figure 2.10  *H N M R Spectrum o f Rhopaloic A c i d D (37) i n C D 6  1 3  Figure 2.12  C N M R Spectrum o f Rhopaloic A c i d D (37) i n C D 6  Figure 2.11  ' H N M R Spectrum o f Rhopaloic A c i d E (38) in C D 6  1 3  6  ' H N M R Spectrum o f Rhopaloic A c i d F (39) i n C D  Figure 2.14  Important C O S Y and H M B C Correlations for 40  Figure 2.15  [  6  6  6  6  Page 50 Page 52  6  C N M R Spectrum o f Rhopaloic A c i d E (38) i n C D  Figure 2.13  Page 49  6  Page 53 Page 55 Page 57  H N M R Spectrum o f Rhopaloic A c i d G (40) i n C D 6  6  Page 59  Figure 2.16  C O S Y-45 o f Rhopaloic Acids D / E (37/38) i n C D  Figure 2.17  H M Q C o f Rhopaloic Acids D / E (37/38) in C D  6  Page 77  Figure 2.18  H M B C o f Rhopaloic Acids D / E (37/38) in C D  6  Page 78  Figure 2.19  C O S Y - 4 5 o f Rhopaloic A c i d F (39) i n C D  Figure 2.20  H S Q C o f Rhopaloic A c i d F (39) i n C D  Figure 2.21  H M B C o f Rhopaloic A c i d F (39) i n C D  6  6  6  6  6  6  6  6  Page 76  Page 79 Page 80  6  6  Page 81  Figure 2.22  C O S Y - 4 5 o f Rhopaloic A c i d G (40) i n C D  Figure 2.23  H M Q C of Rhopaloic A c i d G (40) i n C D  6  Page 83  Figure 2.24  H M B C of Rhopaloic A c i d G (40) i n C D  6  Page 84  Figure 3.1  Overview o f the C e l l Cycle (Diagram by M . Roberge)  Figure 3.2  Pictorial Representation o f the Logic Behind the G 2 Checkpoint Bioassay (Diagram by M . Roberge) Page 95  Figure 3.3  Ambrosia artemisiifolia  Figure 3.4  Important N M R Chemical Shifts for Psilostachyin A (42)  Figure 3.5  ' H and  1 3  Figure 3.6  H and  1 3  Figure 3.7  *H and  1 3  Figure 3.8  ' f l spectra o f Paulitin (45) and Isopaulitin (46) i n CDCI3  Figure 3.9  1 3  6  6  6  Page 82  6  Page 95  (Photo by W . Craig)  Page 99 Page 103  C N M R Spectra o f Psilostachyin A (42) i n C D C 1 Page 106 3  C N M R Spectra o f Psilostachyin B (43) i n C D C 1  3  Page 107  C N M R Spectra o f Psilostachyin C (44) i n C D C 1  3  Page 108  C spectra o f Paulitin (45) and Isopaulitin (46) in C D C 1  Page 110 3  Page 111  Figure 3.10  N M R Chemical Shift Assignments for 41  Page 113  Figure 3.11  ' H N M R Spectrum o f Artemisiidiendioc A c i d monomethyl ester (41) i n DMSO. Page 116 d 6  Figure 3.12  C N M R Spectrum o f Artemisiidiendioc A c i d monomethyl ester (41) i n DMSO_ Page 117 d 6  Figure 3.13  ' f l N M R Spectrum o f Psilostachyin A P-mercaptoethanol Adduct 223 i n CDCI3 Page 121  Figure 3.14  C N M R Spectrum o f Psilostachyin A |3-mercaptoefhanol Adduct 223 i n CDCI3 Page 122  Figure 3.15  ' H Spectra o f Epoxides 224 and 225 in C D C 1  Figure 3.16  1 3  Figure 3.17  1  Figure 3.18  1 3  1 3  Page 127  3  C Spectra o f Epoxides 224 and 225 i n CDCI3  H N M R Spectrum o f Compound 226 i n C D C 1 C N M R Spectrum of Compound 226 CDCI3  xi  Page 128 3  Page 131 Page 132  Figure 3.19  Structure-Activity Relationship of A. artemisiifolia  Sesquiterpenes Page 134  Figure 3.20  Vernonia baldwinii (Photo by C. S. Lewallen)  Page 136  Figure 3.21  Important N M R Chemical Shifts for Vernonataloide (47)  Page 140  Figure 3.22 Figure 3.23  l  U and C N M R Spectra of Vernonataloide (47) in CDC1 Page 143 *H & C Spectra of 8-deacylvernonataloide-8-0-tiglate (227) in CDC1 Page 144  Figure 3.24  !  1 3  3  1 3  3  H N M R Spectrum of 13-desacetoxy-8-deacylvernonataloide-8-0isobutyrate (228) in CDC1 Page 146 3  Figure 3.25  C N M R Spectrum of 13-desacetoxy-8-deacylvernonataloide-8-0isobutyrate (228) in CDC1 Page 147  1 3  3  Figure 3.26  ' H and C N M R Spectra of Marginatin (229) in CDC1  Figure 3.27  *H and C N M R Spectra of Marginatin Methylacrylate (230) in CDC1 Page 152  Figure 3.28  ' H and C N M R Spectra of Acetoxy-8a-tiglinoyloxy-4P-hydroxybourbon-7(l l)-en-6,12-olide (231) in CDC1 Page 156  1 3  3  Page 151  1 3  3  1 3  3  Figure 3.29  *H and C Spectra of 8-desacylglaucolide A-tiglate (232) Page 159  Figure 3.30  Structural Differences of the Vernonataloides  Figure 3.31  C O S Y of Artemisiidiendioc Acid monomethyl ester (41) in DMSO_d6 Page 174  Figure 3.32  H M Q C of Artemisiidiendioc Acid monomethyl ester (41) in DMSO_d6 Page 175  Figure 3.33  H M B C of Artemisiidiendioc Acid monomethyl ester (41) in DMSO_d6  1 3  Page 162  Page 176 Figure 4.1  Erythropodium  Figure 4.2  Erythrolide T (48) Stereochemistry as Defined by a R O E S Y Page 186 ' H N M R Spectrum of Erythrolide T (48) in CDC1 Page 188  Figure 4.3  caribaeorum. (Photo by M . Campbell)  3  xn  Page 181  Page 191  Figure 4.4  ' H N M R Spectrum o f Erythrolide U (49) i n C D C 1  Figure 4.5  1 3  Figure 4.6  ' H N M R Spectrum o f Erythrolide V (50) i n C D C 1  Figure 4.7  1 3  Figure 4.8  Important C O S Y and H M B C Correlations i n 51  Figure 4.9  *H N M R Spectrum o f Aquariolide B (51) i n C D C 1  Figure 4.10  1 3  Figure 4.11  ' H N M R Spectrum o f Aquariolide C (52) i n C D C 1  Figure 4.12  l 3  Figure 4.13  Graph o f the Cytotoxicity o f Erythrolides and Aquariolides Isolated Page 212  Figure 4.14  C O S Y - 9 0 Spectrum o f Erythrolide T (48) i n C D C 1  Figure 4.15  H M Q C Spectrum o f Erythrolide T (48) i n C D C 1  3  Page 222  Figure 4.16  H M B C Spectrum o f Erythrolide T (48) i n C D C 1  3  Page 223  Figure 4.17  R O E S Y Spectrum o f Erythrolide T (48) i n C D C 1  Figure 4.18  C O S Y - 9 0 Spectrum o f Erythrolide U (49) i n C D C 1  Figure 4.19  H M Q C Spectrum o f Erythrolide U (49) i n C D C 1  3  Page 226  Figure 4.20  H M B C Spectrum o f Erythrolide U (49) i n CDCI3  Page 227  Figure 4.21  C O S Y - 9 0 Spectrum o f Erythrolide V (50) i n C D C 1  Figure 4.22  H M Q C Spectrum o f Erythrolide V (50) i n C D C 1  3  Page 229  Figure 4.23  H M B C Spectrum o f Erythrolide V (50) i n C D C 1  3  Page 230  Figure 4.24  C O S Y - 9 0 Spectrum o f Aquariolide B (51) in C D C 1  Figure 4.25  H M Q C Spectrum o f Aquariolide B (51) i n C D C 1  3  Page 232  Figure 4.26  H M B C Spectrum o f Aquariolide B (51) i n C D C 1  3  Page 233  3  C N M R Spectrum o f Erythrolide U (49) i n C D C 1  Page 192  3  Page 196  3  C N M R Spectrum o f Erythrolide V (50) i n C D C 1  Page 197  3  Page 199 Page 201  3  C N M R Spectrum o f Aquariolide B (51) i n C D C 1  Page 202  3  Page 205  3  C N M R Spectrum o f Aquariolide C (52) i n C D C 1  Page 206  3  Page 221  3  Page 224  3  Page 225  3  Page 228  3  3  Page 231  Figure 4.27  C O S Y - 9 0 Spectrum o f Aquariolide C (52) i n C D C 1  Figure 4.28  H M Q C Spectrum o f Aquariolide C (52) in C D C 1  3  Page 235  Figure 4.29  H M B C Spectrum o f Aquariolide C (52) in CDCI3  Page 236  xiv  3  Page 234  List of Schemes Scheme 1.1  Formation o f the Biological Isoprene Units, D M A P (19) and IPP (20) Page 11  Scheme 1.2  Formation o f Cio to C25Terpene Chains  Page 12  Scheme 1.3  T w o Pathways to IPP (20)  Page 13  Scheme 2.1  Shortening o f a Terpene Chain V i a a Tetronic A c i d Moiety Page 24  Scheme 2.2  Possible ct-Cleavage Mechanism o f 40 i n D C I M S  Scheme 2.3  Possible Biosynthetic Pathway to Barangcadoic A c i d A (130) Page 61  Scheme 2.4  Possible Biogenesis o f Rhopaloic Acids A (122), B (35), and C (36) Page 62  Scheme 2.5  Possible Biogenesis o f Rhopaloic Acids D (37), E (38), F (39), and G (40) Page 63  Scheme 2.6  Some Related Sesterterpenes and Their Possible Biosynthesis From A Common Intermediate 181c Page 64  Scheme 3.1  Examples o f Sesquiterpene Skeletons  Page 86  Scheme 3.2  Formation o f Adduct 209  Page 88  Scheme 3.3  Biogenesis o f the A. artemisiifolia  Scheme 3.4  Biogenesis o f Compound 231 from Marginatin (229)  Page 154  Scheme 4.1  Biogenetic Relationship o f Some Erythrolides  Page 210  xv  Page 58  Sesquiterpenes Isolated Page 118  List of Abbreviations 25 [<X]D  , 3  specific rotation at the wavelength (589 nm) o f the sodium D line at 25° C carbon-13  C  •H  proton  ID  one dimensional (in reference to N M R )  2D  two dimensional (in reference to N M R )  Ac  acetyl group  AD  Alzheimer's disease  ATP  adenosine triphosphate  ax  axial  b  broad (in reference to N M R signals)  Bz  benzoate group  c  concentration  C-  carbon (followed by a number)  C D 6  6  deuterated benzene  CDCI3  deuterated chloroform  cm  Reciprocal centimetres, used in reference to infrared wavenumber values  CoA  coenzyme A  COSY  Correlation Spectroscopy; with a number after, eg. C O S Y - 4 5 indicates first tip angle i n the pulse sequence  5  chemical shift (in ppm = parts per million)  d  doublet (in reference to N M R signals)  DCM  dichloromethane  DMSO_d6  deuterated dimethyl sulfoxide  xvi  DCIMS  desorption chemical ionization mass spectrometry  DNA  deoxyribonucleic acid  s  molar absorptivity coefficient  Et  ethyl group  EtOH  ethanol  EtOAc  ethyl acetate  eq  equatorial  FABMS  fast atom bombardment mass spectrometry  FT  fourier transform  GDP  guanosine diphosphate  GTP  guanosine triphosphate  H-  proton or hydrogen (followed b y a number)  H"  hydride  hRCEl  human Ras converting enzyme 1  HIV  human immunodeficiency virus  HMBC  heteronuclear multiple bond multiple quantum coherence  HMQC  heteronuclear multiple quantum coherence  HPLC  high pressure (or performance) liquid chromatography  HSQC  , heteronuclear single quantum coherence  HRDCIMS  high resolution desorption chemical ionization mass spectrometry  HRFABMS  high resolution fast atom bombardment mass spectrometry  Hz  hertz  I  impurity signal (in N M R spectra)  xvii  IC50  dose resulting i n 50% inhibition  z'-Pr  isopropyl group  z-PrOH  isopropyl alcohol  IR  infrared  J  scalar coupling constant  A,  wavelength o f maximum absorption  max  m M  multiplet (in reference to N M R signals) molecular ion  +  [M+H]  molecule plus hydrogen ion  +  [M+NH4J  +  molecule plus ammonia ion  [M-H ]~  molecule minus hydrogen ion  Me  methyl group  Meac  methylacrylate group  MeCN  acetonitrile (methyl cyanide)  MeOH  methanol  m-CPBA  meto-chloroperoxybenzoic acid  MIP  mevalonate-independent pathway  MVA  mevalonic acid pathway  m/z  mass to charge ratio  +  NADP  +  Nicotinamide adenine dinucleotide phosphate ion  NADPH  Nicotinamide adenine dinucleotide phosphate (reduced)  NCI  National Cancer Institute (United States o f America)  NMR  nuclear magnetic resonance  xviii  nOe  nuclear Overhauser effect  OPP  pyrophosphate  Ph  phenyl group (C6H5)  q  quartet (in reference to N M R signals)  RCE  Ras converting enzyme  RI  refractive index  ROESY  rotating frame Overhauser effect spectroscopy  s  singlet (in reference to N M R signals)  S  signal due to solvent (note captial letter)  SAR  structure activity relationship  SCUBA  self-contained underwater breathing apparatus  sp.  species  t  triplet (in reference to N M R signals)  t-Bu  t-butyl group  TFA  trifluoroacetic acid  THF  tetrahydrofuran  Tig  tiglate group  TLC  thin-layer chromatography  UBC  University of British Columbia  UV  ultraviolet  w  water signal  WM  Wagner-Meerwein—a rearrangement  X  signal in the N M R spectrum due to the presence of an additional conformer  xix  " H i s strength is perfect when our strength is gone"—Steven Curtis Chapman, Real Life Conversations, 1988.  To Dad, M u m , and Tess: for always believing i n me.  I wish to acknowledge with profound appreciation my research supervisor, D r . Raymond Andersen. Without his guidance and direction this thesis would not have been possible.  I also wish to thank the past and present members o f the Andersen research group at U B C , particularly Dr. David Williams, Michael LeBlanc, and D r . Orazio TaglialatelaScafati, whose help i n the completion o f this work cannot be underestimated.  Appreciation is also expressed to Dr. M i c h e l Roberge and members o f his group, both past and present, for their assistance i n the completion o f this thesis. Additionally, I wish to thank Dr. Gordon Cragg o f the United States National Cancer Institute for his help in the acquisition o f the plant extracts investigated.  xx  1.1: Introduction to Natural Products In ancient times, people experimented with various plant and animal extracts to discover those that could be utilized to fulfil certain needs or desires. Perhaps they wanted to make a poison to aid in hunting, to make colourful dyes for their clothing, or to treat or cure diseases they suffered from. While the methods of experimentation with natural extracts have changed significantly over the intervening years, modern natural products chemists continue to discover useful natural product extracts. Much of the 1  current interest in natural products centres on the need to develop new medicinals, just as it may have in ancient times. Natural products chemistry is concerned with secondary metabolites, which have restricted taxonomic distribution and can, therefore, act as markers of individuality. Secondary metabolites, such as the polyketide erythromycin A (1), the terpene paclitaxel (2), and the alkaloid vincristine (3), are often produced in lesser abundance than primary metabolites. Compounds 1-3 are currently in commercial use as drugs. Primary metabolites include molecules such as proteins and nucleic acids that are essential in the day to day processes of life. Fatty acids, sugars, and steroids could be included in either group depending on their biological activity and/or their rarity in nature. This illustrates that grey areas do exist in this classification scheme. " 2  1  4  Some natural products based drugs such as paclitaxel (2), commercially known as Taxol®, have gone on to gross over a billion dollars per year, suggesting that natural 5  products are a good source o f potential medicinals. Between 1983 and 1994, 231 o f 520 new drugs approved by the United States' Food and Drug Administration ( F D A ) or similar agencies i n other countries were o f natural origin, further supporting the continued investigation o f natural products as a source for new drug candidates. It is important to note that natural origin does not necessarily mean the drug is exactly as found i n the source organism. It also includes those compounds that are either semisynthetic derivatives or fully synthetic products based on model natural products. '  1 6  Recently, a statistical survey was conducted by scientists at Bayer A G Pharma into the structural complementarity o f natural products and synthetic compounds. This study compared almost 30,000 bioactive natural products and approximately 70,000 natural products i n total with nearly 200,000 synthetic compounds. It was concluded that natural products contain a significant amount o f structural variability that is difficult to duplicate i n combinatorial chemistry. Furthermore, the authors state "the potential for new natural products is not exhausted and natural products still represent an important source for the lead-finding process." Pharmacological  7  A recent opinion article i n Trends in  Sciences stated that there may be millions o f natural products present i n  the world, though there are probably only a fraction as many molecular targets or receptors for these compounds. If nothing else, this idea supports the notion that by 8  isolating many natural products it is then possible to determine the requirements for good drugs as there would be a built-in structure-activity study for every target or receptor.  2  Plant natural products have been the main objects o f study for many centuries.  It  is thought that there are about 500,000 species o f plants present on the earth which produce an estimated 5,000-10,000 or more different metabolites (both primary and secondary) per plant. " 9  10  W h i l e science has studied only a fraction o f these plants,  traditional medicine has catalogued the medicinal uses o f a larger number. Traditional medicine, according to the W o r l d Health Organization, accounts for the treatment o f approximately 80% o f the earth's population. In western nations such as the United States, 2 5 % o f the medicines dispensed in pharmacies from 1959 to 1980 were derived from plants. Approximately 120 compounds from 90 plants are recognized as drugs i n at least one country. ' Clearly, the importance o f plants i n both traditional and western 1 6  medicine can not be overestimated. Natural products from other sources have increased i n interest since the early part o f the last century. In what is referred to as the "Golden A g e o f Antibiotics", from the early 1940's to the late 1970's, interest i n microorganism-produced natural products skyrocketed.  1  new interest.  4  During the latter part o f the 1960's marine natural products emerged as a The idea that one should look in the ocean for natural products is probably  not a new one, but no one had seriously looked there before.  1.2: Introduction to Marine Natural Products Marine organisms live in an environment that represents more than 70% o f the earth's surface.  Due to volumetric considerations, the inhabitable space i n the ocean is  much larger than on land. This extra space allows for species to diversify. In addition, the ocean is able to maintain a fairly consistent temperature, at any given latitude, due to water's high specific heat and, therefore, thermoregulation is not as important as it is i n  3  other environments. The concentrations o f various ions i n seawater are essentially uniform throughout the ocean and are similar to those o f the organisms that inhabit it, thus making the organism's osmoregulation fairly easy. In general, these characteristics o f the ocean have enabled the inhabiting organisms to diversify to a large extent over the many habitats present in the marine environment.  4  The diversity o f marine organisms is such that many have no counterpart on land due to unique environmental adaptations. For example, the invertebrate phyla Coelenterata (Cnidaria), Porifera, Bryozoa, and Echinodermata are completely aquatic and largely marine i n habitat. There are over 100,000 species o f invertebrates present i n the ocean with the potential for large numbers o f as yet unknown species, or even families o f marine invertebrates to be discovered due to the immense size o f the marine environment. In addition, many marine taxa are unique and one would expect that they would contain unique secondary metabolites. Since secondary metabolites are restricted taxonomically there is a rough correlation between species diversity and chemical diversity  4  From this standpoint alone, it would therefore make good sense to investigate  marine organisms as a source for new chemical structures. M a n y marine invertebrates are either sessile or slow moving i n nature, yet are brightly coloured and without protective shells. These organisms somehow survive and in fact flourish, so something must make them unpalatable to predators. both soft corals and sponges produce antifeedants '  11 12  4  For example,  and many contain spicules,  (skeletal fibres made o f calcium carbonate or silica ), which also makes them hard to eat. 13  The brightly coloured nudibranchs, or sea slugs, have been shown to sequester compounds o f their prey to make them unpalatable to their predators.  4  14  One could  therefore predict that many o f these metabolites might have medicinal or other commercial uses. M a n y marine organisms, particularly sponges,  15  can filter out and  concentrate microorganisms present i n seawater. These microorganisms also produce potentially useful secondary metabolites that can then be isolated from the sponge during an investigation. In addition, sponges often have microorganisms that live inside them as  symbionts.  15  It has been shown that microbial natural products have been isolated from  sponges that possibly fulfil any o f these situations. For example, xestodecalactone A (4), has been isolated from a Penicillium fungus present i n Xestospongia exigua. Another 16  example is that o f theopalauamide (5), which has been shown to be produced by bacterial cells present in the tissue o f Theonella swinhoei. A final example is okadaic acid (6), l5h  originally isolated from Halichondria okadai and later found to be dinoflagellate in origin.  18  W h i l e most o f the drugs currently on the market or under evaluation are derived from either plant or terrestrial microbial origin, there are some that are obtained from marine organisms. A number o f these compounds come directly from the source  5  organism with little or no modification, while others are based on lead compounds originally isolated from a marine organism. One o f these compounds is the anti-cancer drug ara C (7), also known as Cytarabine®, a synthetic compound based on the lead structures spongothymidine (8) and spongouridine (9) isolated from the sponge Cryptotethya cypta. Another is the anti-inflammatory drug pseudopterosin A (10), present in Resilience®, (a skin cream marketed by Estee Lauder), and isolated from the Caribbean gorgonian, Pseudopterogorgia  elisabethae.  4  A n additional promising marine-  derived natural product is bryostatin I (11), one o f a family o f macrolides isolated from  Me  6  the bryozoan Bugula neritina, that is currently in clinical trials as an anti-cancer agent for treating melanoma. This complex molecule is produced i n such small amounts that largescale aquaculture is the only way to achieve enough material for testing.  19  Molluscs o f  the genus Conus produce very potent piscotoxins. These piscotoxins are peptides produced by the mollusc to aid i n the capture o f prey. Several o f these peptides are 1 20  currently under development as analgesics and or as biochemical probes. '  Okadaic  acid (6), one o f the causative molecules o f shellfish poisoning, is an extremely potent protein phosphatase inhibitor and can be used to study the biochemistry o f this class o f 18  enzymes. The Andersen lab has also had its share o f success i n marine natural products. 21  IPL-576-092 (12) is an anti-asthma compound developed b y Inflazyme Pharmaceuticals that has made it into preliminary clinical trials and is still under development.  Its  structure is based on the lead structure o f contignasterol (13) isolated from the Papua N e w Guinean sponge Petrosia contignata by the Andersen lab.  22  A few years later our 23  lab isolated hemiasterlin (14) from the Papua N e w Guinean sponge Cymbastela sp. which served as the lead structure for HTI-286, a potent antimitotic agent currently in preliminary clinical trials.  24  The lab has also located an additional source for  eleutherobin (15), a potent antimitotic diterpene originally isolated from the rare Australian soft coral Eleutherobia Oceanography.  25  Erythropodium  sp. by Fenical's group at Scripps Institute o f caribaeorum,  a common soft coral o f the Caribbean  region, was found to contain 15 i n reasonable amounts. cultured E. caribaeorum  26  It was further shown that  produces 15 in similar amounts as the w i l d type, an important  7  discovery as a chief problem o f marine natural products is the lack o f source material for 97  clinical testing.  1.3: Natural Products and Cancer W h i l e not every secondary metabolite isolated has known bioactivity, many are discovered through the use o f some type o f biological assay. One area our lab has chosen to pursue is the search for potential anti-cancer agents. This is because cancer is the second leading cause o f death i n North America, and there is a strong likelihood o f it becoming the leading cause before the end o f this century.  It is estimated that half o f  Canadian men and one-third o f Canadian women w i l l develop some form o f cancer i n their l i f e t i m e .  28  Cancer is characterized by rapid uncontrolled cell proliferation i n any  body tissue or i n the circulatory system. The most common cancers are those o f the lung, colo-rectal, breast, and prostate. While rapidly proliferating cancers such as leukemia are now quite easily treated, many forms o f cancer, particularly solid tumour types, are still very difficult to treat.  M a n y cancers still require quite vigorous and extensive  chemotherapy, often leaving the patient very sick, due i n part to the drug's slight selectivity (therapeutic window) between its toxicity against normal and cancerous cells. W i t h recent advances i n molecular biology have come a new understanding o f the molecular basis o f cancer, which has enabled the development o f more selective bioassay screens. These bioassays typically exploit some difference between either the genotype or phenotype o f cancerous and normal cells. A n antimitotic assay, 29  29b  for example,  targets those cells that are dividing rapidly, a characteristic o f cancer, thus the cancerous cells are affected more readily than normal cells. W h i l e this is an example o f a more  8  selective target for anti-cancer agents, scientists are still looking for the elusive 'magic bullet,' which would be an anti-cancer agent that targets only cancerous cells.  1.4: Introduction to Terpenoids 1.4.1: Two Examples of Terpenes as Drugs It has been known i n traditional Chinese medicine since 168 B . C . that Artemisia annua (quinghao or wormwood) can be used as an antimalarial agent. The best description o f its usage occurs i n the colossal Ben Cao GangMu  (Compendium  of  -in  Materia Medico) compiled i n 1596 and still i n print.  In 1972, the active agent was  purified and its structure elucidated. The structure was found to be the sesquiterpene endoperoxide, artemisinin (16). Further isolation studies and chemical studies on artemisinin itself have identified an array o f similar antimalarial metabolites, all with the 1 30  endoperoxide functionality. ' Paclitaxel (2), or T a x o l ® as it is now known in the pharmaceutical industry, was first isolated i n 1971 from the bark o f the western pacific yew, Taxus brevifolia, collected in Washington state. Taxus species have been historically utilized by First Nations people as treatment for several non-cancerous conditions. In addition, T. baccata, a related species, was known i n traditional East Indian medicine for treatments o f several diseases including one report for 'cancer.' ' 1  The initial problem associated with clinical  10  use o f 2 was that enormous numbers o f trees were required: 4,000 trees to yield 360g o f the compound initially and increasing to 38,000 trees for 25kg needed for 12,000 patients in the early 1990's. This supply problem was solved shortly thereafter when Potier and co-workers discovered that T. baccata contained large amounts o f a related taxane, 10deacetylbaccatin III (17), which can be converted easily into 2 and its more active  9  analogue taxotere (18), also known as docetaxel. It had previously been shown (in 1979), that the mode o f action o f 2 was unlike any known anti-cancer agent. W h i l e vincristine (3) and related compounds destabilize microtubule polymerization during mitosis, 2 actually stabilizes microtubules. Currently 2 and several related semi-synthetic compounds are the drugs o f choice in the treatment o f refractory breast and ovarian cancer.  5  W i t h these two examples, it can be concluded that terpenes as a class show reasonable promise as a source o f drugs. Furthermore, according to researchers at Bayer A G , terpenes represented the largest group o f natural products present i n the Dictionary of Natural Products at approximately 35% o f compounds listed at that time (June 1996). Simple statistics in conjunction with historical examples such as 2 and 16 would argue that terpenes would represent an excellent class o f compounds to study in the search for potential medicinal agents.  1.4.2: Biosynthesis of Terpenoids It has been known since the early nineteenth century that terpenoids were comprised o f a simple five-carbon repeating unit. In 1953 this was formally put forth by Ruzicka as the "isoprene rule," when he concluded that terpenoids, (also called isoprenoids), were made up o f some biological equivalent o f isoprene, (a simple fivecarbon unit), linked together in a head-to-tail fashion. It was later discovered the that 2  biological isoprene unit was actually two molecules that were enzymatically  10  interconverted, dimethyl allyl pyrophosphate ( D M A P P ; 19) and isopentenyl pyrophosphate (IPP; 20), see Scheme 1.1. In this isomerization step, thepro-R hydrogen, (marked i n bold text i n this chapter's figures) i n 20 was lost or gained depending on the reaction direction. T o form 19 and 20, a six-carbon acid, 3R-mevalonic acid (21), is formed b y the condensation o f three acetate units, (in the activated form o f acetyl-CoA 22), by a pathway shown i n Scheme 1.2. When 19 and 20 are joined together in a headto-tail fashion with the stereospecific loss o f the pro-R hydrogen, geranyl pyrophosphate (27), a Cio compound that is the precursor to mono terpenes and iridoids, is formed. Additional IPP units can then be added until farnesyl pyrophosphate (28) (C15), geranylgeranyl pyrophosphate (29) (C20), and geranylfarnesyl pyrophosphate (30) (C25) were formed.  Scheme 1.1: Formation o f the Biological Isoprene Units, D M A P (19) and IPP (20). The hydrogen marked i n bold text (in 20) is pro-R. During the addition o f each IPP unit, there is stereospecific loss o f the pro-R hydrogen as with the initial condensation to form 27. The precursors to the sesqui-, di-, and sesterterpenoids are 28, 29, and 30 respectively. Larger molecules such as triterpenoids  11  (C30) and carotenoids (C40) are formed by the tail-to-tail linking o f two C15 or C20 chains, respectively. From a cyclized triterpene skeleton steroids are formed with the loss o f three methyl groups. Once the basic terpene precursor is biosynthesized, it can then undergo further enzymatic reactions such as oxidations, reductions, and cyclizations to form the highly functionalized molecules present i n many organisms. '  Scheme 1.2: Formation o f C10 to C2sTerpene Chains. In the last fifteen years, Rohmer and others have shown that an additional biosynthetic pathway to terpenoids, which does not pass through 21 as an intermediate, is 31 36  operational i n microorganisms (including cyanobacteria), algae, and higher plants. This pathway is referred to as the mevalonate-independent pathway (MIP). Rather than form 19 and 20 from acetate as i n the mevalonic acid ( M V A ) pathway, acetate is first converted to pyruvate (32) v i a the intermediates o f either the glyoxylate or the citric acid (tricarboxylic acid) cycles. In another permutation o f the M I P pathway, glucose (31) is converted to pyruvate (32) v i a glycolysis (or the closely related Entner-Doudoroff pathway). Once 32 is formed, it is then able to proceed to 20 through acetyl-CoA (22) and the M V A pathway or by condensation with D-glyceraldehyde 3-phosphate (33) to form l-deoxy-D-xylulose-5-phosphate ( D X P , 34) in the M I P pathway.  12  D X P (34) then  -5 1  undergoes a series o f enzymatic transformations to form 20.  Unlike the M V A pathway  where each o f the steps has been thoroughly studied, only the first five steps, (only one is shown i n Scheme 1.3: 32/33 to 34), and their enzymes are known for the M I P 31 37  pathway. ' Further studies have shown that i n plants and microorganisms both pathways are often present. Microorganisms such as actinomycetes are able to use either pathway. 31  During log phase, the M I P pathway is used while during stationary phase the M V A pathways is operational.  38  Investigations o f terpenoid biosynthesis i n plants, (including  Taxus chinensis—a producer o f taxanes ), have shown that the M V A pathway is used to 36  biosynthesize sterols and presumably triterpenes, while the monoterpenes, diterpenes, and carotenoids arise from the M I P pathway. Sesquiterpenes have been shown i n several studies to be produced b y either or even both pathways. In plants, the evidence suggests that the M I P pathway is localized i n the chloroplasts, while the M V A pathway is located in the cytoplasm.  31  The pigment compounds such as carotenoids, necessary for  photosynthesis, are formed directly from the glucose produced by the process they assist. CHO  HHOHH-  „„ 32 COOH  =*>  9II  ™ 33 H^YW  \=°  OH CH OH  CHO  H-  OH  H  MIP Pathway  °  -OH -H -OH  34  CH OP0 ~ 2  2  3  2  31 22  X  MVA Pathway  >  H C^SCoA 3  ^  v  Scheme 1.3: T w o Pathways to IPP (20).  13  "OPP  20  1.5: Thesis Preview In the course o f m y research it b e c a m e clear that b i o a c t i v e terpenoids w o u l d f o r m the basis for m y thesis, b o t h f r o m m a r i n e and terrestrial sources. C h a p t e r 2 discusses the r h o p a l o i c acids B ( 3 5 ) - G ( 4 0 ) , norsesterterpenoids i s o l a t e d f r o m the I n d o n e s i a n sponge Hippospongia  sp. T h e s e c o m p o u n d s , i n a d d i t i o n to t w o related c o m p o u n d s i s o l a t e d b y  m y c o l l e a g u e f r o m the same source, are the first natural p r o d u c t i n h i b i t o r s o f the R C E protease e n z y m e .  C h a p t e r 3 details t w o projects based o n the c o m m o n r a g w e e d artemisiifolia  Ambrosia  L . , and the western i r o n w e e d , Vernonia baldwinii T o r r . D u r i n g the  14  isolation o f the bioactive metabolites from A. artemisiifolia  a novel highly fimctionalized  degraded sesquiterpene 41 was isolated. In addition, four derivatives o f the known psilostachyins " (42-44) and paulitins (45-46), and one derivative o f the known 40  42  compound vernonataloide  43  44  (47) were semi-synthesized to expand the structure-activity  studies beyond those structures which occurred naturally i n each source plant. Both o f these projects involved the study o f sesquiterpene lactones, a class o f compounds prevalent i n plants that are responsible for an array o f biological activities. Chapter 4 describes the isolation and structure determination o f five novel erythrolides and aquariolides 48-52 from the cultured and w i l d type Caribbean soft coral Erythropodium  caribaeorum Duchassaing and Michelotti 1860. The erythrolides are a  large and growing group o f diterpenoids aquariolides are a new group  27  45  believed to serve as antifeedents,  12  while the  o f related compounds with an as yet undetermined  function.  1.6: References 1)  Newman, D.J.; Cragg, G . M . ; Snader, K . M . Nat. Prod. Rep. 2000,17, 215-234, and references therein.  2)  Mann, J.; Chemical Aspects of Biosynthesis, Oxford University Press: Oxford, 1994.  3)  Dewick, P . M . ; Medicinal Natural Products: A Biosynthetic Approach; John W i l e y and Sons: West Sussex, U K , 1997, chapter 5.  4)  Andersen, R.J.; Williams, D . E . In Chemistry in the Marine Environment; Hester, R . E . ; Harrison R . M . , E d . ; Issues i n Environmental Science and Technology, no. 13; The Royal Society o f Chemistry: Cambridge, U K , 2000; pp. 55-79, and references therein.  5)  Mann, J. Nature Rev. Cancer 2002, 2, 143-148 and references therein.  6)  a) Cragg, G . M . ; Newman, D.J.; Snader, K . M . J. Nat. Prod. 1997, 60, 52-60. b) Cragg, G . M . ; Newman, D.J. Exp. Opin. Invest. Drugs 2000, 9, 2783-2797.  15  no. 20 i n Oxford Chemistry Primers;  7)  Henkel, T.; Brunne, R . M . ; Miiller, H . ; Reichel, F. Angew. Chem. Int. Ed. Engl. 1999, 38, 643-647.  8)  Tulp, M . ; Bohlin, L . TIPS 2002, 23, 225-231.  9)  K u o , Y . - H . ; K i n g , M . - L . In Bioactive Compounds from Natural Sources: Isolation, Characterisation and Biological Properties; Tringali, C , E d . ; Taylor and Francis: N e w Y o r k , 2001; p. 191.  10)  Cordell, G . A . Phytochemistry  11)  Pawlik, J.R. Chem. Rev. 1993, 93, 1911-1922.  12)  Fenical, W . ; Pawlik, J.R. Mar. Ecol. Prog. Ser. 1991, 75, 1-8.  13)  Campbell, N . A . Biology, 3 p.604.  14)  Cimino, G . ; Sodano, G . In Sponges in Time and Space; V a n Soest, R . W . M . ; V a n Kempen, T . M . G . ; Braekman, J . - C , Eds.; A A . Balkema: Rotterdam, The Netherlands, 1993; pp. 459-472.  15)  a) Bewley, C . A . ; Faulkner, D . J . Angew. Chem. Int. Ed. Engl. 1998, 37, 21632178 and references therein. b) Schmidt, E . W . ; Bewley, C . A . ; Faulkner, D . J . J. Org. Chem. 1998, 63, 12541258.  16)  Edrada, R . A . ; Heubes, M . ; Brauers, G . ; Wray, V . ; Berg, A . ; Grafe, U . ; Wohlfarth, M . ; Miihlbacher, J.; Schaumann, K . ; Sudarsono; Bringmann, G . ; Proksch, P. J. Nat. Prod. 2002, 65, 1598-1604.  17)  Tachibana, K . ; Scheuer, P.J.; Tsukitani, Y . ; K i k u c h i , H . ; V a n Engen, D . ; Clardy, J.; Gopichand, Y . ; Schmitz, F.J. J. Am. Chem. Soc. 1981,103, 2469-2471..  18)  Yasumoto, T.; Murata, M . Chem. Rev. 1993, 93, 1897-1909, and references therein.  19)  Hale, K . J . ; Hummersone, M . G . ; Manaviazar, S.; Frigerio, M . 2002,19, 413-453, and references therein.  20)  Myers, R . A . ; Cruz, L . J . ; Rivier, J.E.; Olivera, B . M . Chem. Rev. 1993, 93, 19231936, and references therein.  21)  Shen, Y . ; Burgoyne, D . L . J. Org. Chem. 2002, 67, 3908-3910.  22)  Burgoyne, D . L . ; Andersen, R.J.; A l l e n , T . M . J. Org. Chem. 1992, 57, 525-528.  rd  1995, 40, 1585-1612.  Ed.; Benjamin Cummings: Menlo Park, C A , 1987;  16  Nat. Prod. Rep.  Coleman, J.; de Silva, E . D . ; K o n g , F.; Andersen, R . J . ; A l l e n , T . M . 1995, 39, 10653-10662.  Tetrahedron  Andersen, R.J. Personal communication. Lindel, T.; Jensen, P.R.; Fenical, W . ; Long, B . H . ; Casazza, A . M . ; Carboni, J.; Fairchild, C R . J. Am. Chem. Soc. 1997,119, 8744-8745. a) Cinel, B . ; Roberge, M . ; Behrisch, FL; van Ofwegen, L . ; Castro, C . B . ; Andersen, R.J. Org. Lett. 2000, 2, 257-260. b) Britton, R. Roberge, M . ; Berisch, FL; Andersen, R . J . Tetrahedron Lett. 2001, 42, 2953-2956. Taglialatela-Scafati, O.; Deo-Jangra, U . ; Campbell, M . ; Roberge, M . ; Andersen, R.J. Org. Lett. 2002, 4, 4085-4088. National Cancer Institute o f Canada. Canadian Cancer Statistics 2002. Toronto, Canada, 2002. a) Roberge, M . ; Berlinck, R . G . S . ; X u , L . ; Anderson, FL; L i m , L . ; Curman, D . ; Stringer, C M . ; Friend,S.FL; Davies, P.; Vincent, I.; Haggarty, S.J.; K e l l y , M . T . ; Britton,R.; Piers, E . ; Andersen, R.J. Cancer Research 1998, 58, 5701-5706. b) Roberge, M . ; Cinel, B . ; Anderson, H.J.; L i m , L . ; Jiang, X . ; X u , L . ; B i g g , C M . ; K e l l y , M . T . , Andersen, R . J . Cancer Research 2000, 60, 5052-5058. c) Adeji, A . A . JNCI2001, 93, 1062-1074. Haynes, R . K . ; Vonwiller, S . C Acc. Chem. Res. 1997, 30, 73-79. Rohmer, M . Nat. Prod. Rep. 1999,16, 565-574 and references therein. Proteau, P.J. J. Nat. Prod. 1998, 61, 841-843. Fontana, A . ; Kelly, M . T . ; Prasad, J.D.; Andersen, R . J . J. Org. Chem. 2001, 66, 6202-6206. Schwender, J.; Seemann, M . ; Lichtenthaler, H . K . ; Rohmer, M . Biochem. J. 1996, 316, 73-80. a) Eisenreich, W . ; Sagner, S.; Zenk, M . H . ; Bacher, A . Tetrahedron Lett. 1997, 35, 3889-3892. b) Arigoni, D . ; Sagner, S.; Latzel, C ; Eisenreich, W . ; Bacher, A . ; Zenk, M . H . Proc. Nat. Acad. Sci. USA 1997, 94, 10600-10605. Eisenreich, W . ; Menhard, B . ; Hylands, P.J.; Zenk, M . H . ; Bacher, A . Proc. Nat. Acad. Sci. USA 1996, 93, 6431-6436. a) Dewick, P . M . Nat. Prod. Rep. 1999,16, 97-130.  17  b) Kuzuyama, T.; Takagi, M . ; Kaneda, K . ; Dairi, T.; Seto, H . Tetrahedron Lett. 2000, 41, 703-706. c) Kuzuyama, T.; Takagi, M . ; Kaneda, K . ; Watanabe, H . ; Dairi, T.; Seto, H . Tetrahedron Lett. 2000, 41, 2925-2928. d) Takagi, M . ; Kuzuyama, T.; Kaneda, K . ; Watanabe, H . ; Dairi, T.; Seto, H . Tetrahedron Lett. 2000, 41, 3395-3398. a) Seto, H . ; Watanabe, H . ; Furihata, K . Tetrahedron Lett. 1996, 37, 7979-7982. b) Seto, H . ; Orihara, N . ; Furihata, K . Tetrahedron Lett. 1998, 39, 9497-9500. Craig, K . S.; Williams, D . E . ; Hollander, I.; Frommer, E . ; M a l l o n , R.; Collins, K . ; Wojciechowicz, D . ; Tahir, A . ; van Soest, R.; Andersen, R . J. Tetrahedron Lett. 2002, 43, 4801-4804. a) M i l l e r , H . E . ; Kagan, H . B . ; Renold, W . ; Mabry, T.J. Tetrahedron Lett. 1965, 38, 3397-3403. b) Mabry, T.J.; M i l l e r , H . E . ; Kagan, H . B . ; Renold, W . Tetrahedron 1966, 22, 1139-1146. c) Silva, G . L . ; Oberti, J.C.; Herz, W . Phytochemistry 1992, 31, 859-861. Mabry, T.J.; Kagan, H . B . ; M i l l e r , H . E . Tetrahedron  1966, 22, 1943-1948.  Kagan, H . B . ; M i l l e r , H . E . ; Renold, W . ; Lakshmikantham, M . V . ; Tether, L . R . ; Herz, W . ; Mabry, T.J. J. Org. Chem. 1966, 31, 1629-1632. Borges del Castillo, J.; Manresa Ferrero, M . T . ; Martin Ramon, J.L.; Rodriguez Luis, F.; Vazquez-Bueno, P.; Joseph Nathan, P.; Org. Magn. Reson. 1981 17, 232-234, and references therein. Bohlmann, F.; Zdero, C . Phytochemistry,  1982, 21, 2263-2267.  a) Look, S.A.; Fenical, W . ; V a n Engen, D . ; Clardy, J. J. Am. Chem. Soc. 1984, 106, 5026-5027. b) Pordesimo, E . O . ; Schmitz, F.J.; Ciereszko, L . S . ; Hossain, M . B . ; van der Helm, D . J. Org. Chem. 1991, 56, 2344-2357. c) Dookran, R.; Maharaj, D . ; Mootoo, B . S . ; Ramsewak, R.; M c L e a n , S.; Reynolds, W . F . ; Tinto, W . F . Nat. Prod. 1993, 56, 1051-1056. d) Banjoo, D . ; M a x w e l l , A . R . ; Mootoo, B . S . ; Lough, A . J . ; M c L e a n , S.; Reynolds, W . F . Tetrahedron Lett. 1998, 39, 1469-1472. e) Maharaj, D . ; Pascoe, K . O . ; Tinto, W . F . J. Nat. Prod. 1999, 62, 313-314. f) Banjoo, D . ; Mootoo, B . M . ; Ramsewak, R . S . ; Shaima, R.; Lough, A . J . ; M c L e a n , S.; Reynolds, W . F . J. Nat. Prod. 2002, 65, 314-318. g) Taglialatela-Scafati, O.; Craig, K . S . ; Reberioux, D . ; Roberge, M . ; Andersen, R . J . Eur. J. Org. Chem. 2003, submitted.  18  2.1: Brief Introduction to Bioactive Sponge Metabolites Marine sponges are an excellent source o f secondary metabolites. Since the birth o f the study o f marine natural products, a significant portion o f the metabolites isolated each year are from sponges. These metabolites cover the full spectrum o f both structure and bioactivity as the following examples show. The fatty acid derived acetylenic 1  sulfonic acid taurospongin A (101), isolated from a Hippospongia  sp. collected off the  coast o f Japan, is an inhibitor o f D N A polymerase p and is also an H I V reversetranscriptase inhibitor. (+)-Discodermolide (102) is a polyhydroxy-5-lactone polyketide 2  antimitotic agent isolated in 1990 from the Caribbean sponge Discodermia  dissoluta.  Hemiasterlin (14), an antimitotic tripeptide, was independently isolated by the Andersen  43  and K a s h m a n labs at roughly the same time from a Cymbastela sp. sponge collected in 4b  Papua N e w Guinea and from the South African sponge Hemiasetrella  minor,  respectively. Motuporamine C (103), an alkaloid isolated from the Papua N e w Guinean sponge Xestospongia  exigua, stops the invasion o f cancer cells from a source tumour into  the surrounding tissues (anti-invasion agent) and it is also anti-angiogenic (stops the growth o f new blood vessels). Isoprenoids have been isolated i n great abundance from 5  sponges. M a n y o f these have been steroidal structures such as contignasterol (13), 1  19  isolated from the Papua N e w Guinean sponge Petrosia contignata,  6  while others fall into  most o f the remaining terpene classes.  2.2: Introduction to Sesterterpenes and Related Compounds Sesterterpenes, including their  C21-24  degradation and  C26-27 7  alkylation products, 8  9  have been isolated from a variety o f sources including fungi, lichens, higher plants especially the genus Salvia, '  soft coral, nudibranchs, and, as we shall see, sponges.  9c f  10  11  This class o f terpenes is fewer i n number than any o f the others and examples have only been known from the late 1 9 5 0 ' s .  The first two sesterterpenes reported were  12-14  ophiobolin A (104) and gascardic acid (105), the structures o f which were not known 15  16  until 1965. Ophiobolin A (104) was isolated from the pathogenic fungus Helminthosporium  oryzae  and represents the ophiobolane class o f tricarbocyclic  15  sesterterpenes. Gascardic acid (105), isolated from the insect, Gascardia 14  madagascariensis,  16  represents an additional tricarbocyclic skeleton o f which it appears  to be the sole representative.  14  Geranylnerolidol (106) was the first linear sesterterpene to  be discovered when it was isolated i n 1968 from the fungus heterostrophus} ' '  Cochliobolus  Geranylfarnesol (107) itself was isolated from the scale insect  2c XA xl  1  Ceroplastes albolineatus a year later.  Q  Over the next fifteen years the number o f known  sesterterpenes increased to 158 compounds comprising thirty-two skeletons o f various types. T w o o f these sesterterpene skeletons are represented by the monocarbocyclic 14  albocerol (108), from the scale insect C. albolineatus,  19  (109) from C.floridensis  and the tricarbocyclic floridenol  Retigeranic acid (110) and its C-18 epimer ( l l l ) ,  20  8a  8 b  isolated  from various Himalayan lichens such as Lobaria retigera, represent an unusual pentacarbocyclic skeleton. In 1998 when the metabolite Y W 3 6 9 9 (112) from the fungus 14  20  Codinaea simplex was discovered there were approximately 350 known sesterterpenes.  2  A n interesting recent isolation is that o f aspergilloxide (113) from the marine-derived fungus Aspergillus  sp., with its novel tetracarbocyclic skelton.  22  W h i l e various marine organisms may produce sesterterpenes, those from sponges have been studied most often. ' " 1  12  14  Our survey o f sponge sesterterpenes begins with the  C21 tetranorfuranosesterterpenes such as furospongin-1 (114) isolated from both Spongia officinalis and Hippospongia  communis  and numerous related compounds.  C22 trisnorsesterterpenes including furodendin (115), a 5-lactone from a  Several  Carteriospongia  9^  sp.,  and hippospongins E (116) and F (117), from a Hippospongia  isolated.  sp., have been  These furanoterpenes are related to C24 and C25 sesterterpenes that w i l l be 21  discussed below. It appears that the only sponge bisnorsesterterpene (C23) isolated to date is luffarin-0 (118) from Luffariella geometrica with two tautomeric forms being noted (only the major enol form is shown).  27  Butler and Capon have isolated an additional  twenty-five related terpenes (with 18, 20, and 25 carbons) including the sesterterpene 97  luffarin B (119), (closely related to 118), from the same sponge.  The norsesterterpenes  98  (C24) are well represented with numerous muqubilone  a  (aikupikoxide A , 120)  antimalarial cyclic peroxides such as  isolated from Diacarnus erythaenus  andbyC24 97  furanoterpenes such as hippospongin D (121) isolated from a Hippospongia  sp.  Other  norsesterterpenes include the norscalarolides discussed below and the rhopaloic acids A (122), B (35), and C (36) isolated from two different sponges, a Rhopaloides Hippospongia  sp.  30  and a  sp.  The regular (ie non-degraded or rearranged) sesterterpenes are also well represented among sponges. Linear sesterterpenoids include the tetronic-acid-containing furanosesterterpene antibiotic  323  isolated from Ircinia variabilis} * 1  and antiinflammatory  320  variabilin (123) originally  It has subsequently been isolated from  22  o  130  Fasciospongia  fovea  129  and /. Strobilina.  double-bond isomer strobilinin (124).  33  In the latter sponge, 123 was isolated with its A s shown by this example, tetronic acids are  often known to have different double bond geometries, with the double bonds present i n 'non-biosynthetic positions.'  120  frequently  The tetronic acid portion o f these molecules is  quite reactive and it probably allows them to be degraded easily, hence the C21, C22 and 34  C24 furanosesterterpenes discussed previously. Scheme 2.1 shows how a terpene chain could be degraded b y one carbon v i a the tetronic acid moiety. Loss o f two or three more carbons (to give C22 or C21 terpenes, respectively) would involve additional biosynthetic steps similar to those shown i n Scheme 2.1. Other furanosesterterpenes include hippospongins A (125), B (126), and C (127), isolated from an Australian Hippospongia 26  sp., and related to hippospongins D (121), E (116), and F (117) mentioned above.  (This  series o f compounds should not to be confused with hippospongin (129) a different compound with the same name that was isolated from an Okinawan Hippospongia  23  sp. in  OH Oxidation.  H +  >  O  \O H  2  128B  128A  128C  H O ^  0  HO  Ho 128D  Further Degraded Furanosesterterpenes 128F  128G  Scheme 2.1: Shortening o f a Terpene Chain V i a a Tetronic A c i d Moiety. 1986. ) Barangcadoic acid A (130) is an RCE-protease inhibitor isolated from an 35  Indonesian Hippospongia  sp., and is an example o f a regular linear sesterterpene 31  without either a tetronic acid moiety or a furan ring system.  o  "^X^^J^^-H^LJ  OH  131  o,  HO x ° 132  The monocarbocyclic sesterterpenoid manoalide (131) isolated from Luffariella variabilis in 1980, is an antiinflammatory compound, ' and one o f a series o f related 36  compounds.  37  36d  e  This regular sesterterpene has also been shown to inactivate bee venom  phospholipase A 2 by the formation o f an imine with a lysine residue present in the enzyme.  36d  Dysidiolide (132) isolated from Dysidea etheria is a bicarbocyclic  sesterterpene an inhibitor o f cdc25A protein phosphatase. This compound is the first  24  naturally occurring inhibitor o f this signalling enzyme that has been shown to activate the G 2 / M transition in the cell cycle. The tricyclic cheilanthane class o f sesterterpenes is not very common among sponges,  but some examples such as compound 133 and several analogues from an  Ircinia sp. have been isolated.  A closely related and much more common cyclic  sesterterpenes class found i n sponges is the tetracarbocyclic scalarane class. ' 12  14  Scalarin  (134), isolated from Cacospongia scalaris in 1972, was the first member o f this class to 40  be discovered. A few more examples include hyrtiolide (135) isolated from Hyrtios erectus  41  and scalarolide (136) isolated from Spongia idia.  as hyrtial (137) from H. erecta  42  43,  Norscalarolides (C24) such  have also been isolated. Scalarane sesterterpenes are  often found i n the nudibranchs that are associated with a sponge, hence these compounds may be sequestered b y the mollusc upon feeding on the sponge.  120  Suberitenones A (138)  and B (139) from an Antarctic sponge o f the genus Suberites, represent a novel and  141: RT =0H; R  25  2  = H  unusual non-scalarane rearranged tetracarbocyclic skeleton. Suberitenone B (139) was shown to be an inhibitor o f the cholesteryl ester transfer protein.  44  The homoscalaranes isolated from sponges include the C26 compounds 140-141 isolated from two species o f sponges, a Dictyoceratida  sp. and a Halichondria  sp.,  and  45  based on the skeleton o f scalaradial (142) an antifeedant and antiinflammatory sesterterpene aldehyde isolated from C. scalaris. ' ~ 36d 45  Strepsichordaia  The Indonesian sponge  46  aliena studied by Scheuer's group has yielded a large series o f  norbishomoscalarane sesterterpenes including honu'enone (143) which is a 20,2447  dimethyl-25-norscalarane compound (the methyl at C-18 is l o s t ) . A (144) through E (148) isolated from Phyllospongia o f bishomoscalarane (C27) sesterterpenes.  48  47b  The phyllolactones  lamellosa are 'true' representatives  Homo and bishomoscalaranes typically  involve extra methylations at one or two o f the following carbons, C-19/C-20 and/or C 24  49  which is evident i n 140-141 and 143-148. Compound 149 isolated from  Cacospongia scalaris is methylated at C-23 and C-24, the first reported bishomoscalarane reported to do so. It is also one o f a limited number o f scalaranes that does not contain a substituent at C - 1 2 .  50  In the course o f this brief survey o f sponge  sesterterpenes, it has hopefully become clear that while they are the smallest class o f terpenoids, sesterterpenes are still quite diverse and prevalent i n sponges. 144: R-i = CO n-Pr; R = OH;R =H '3 145: R = C0 -Et; R = OH; R = H 146: Ri = COrMe; R = OH;R =H 147: R-i = C0 -Et; R = OAc; R = H 148: R! = H;R = R3 = OH  51  r  2  1  :H0  3  0  2  2  3  2  24  'OH  3  2  2  149  3  2  26  19  20  26  2.3: Brief Introduction to Sponges and Hippospongia sp. in Particular 2.3.1: General Introduction to Sponges Sponges make up the phylum Porifera ("pore-bearers") in the animal kingdom. These invertebrate animals are sessile and appear so unlikely to do anything that the ancient Greeks actually classified them as plants. Sponges can be almost any size and shape ranging in height from one cm to the barrel sponges that are over two metres tall. N o one really knows how many species o f sponges there are, but it is known that most are marine i n habitat. Sponge bodies are essentially a sac with holes i n it, hence the name o f the phylum. Inside the sponge are unique cells called collar cells or choanocytes, which are covered with cilia and which have a central flagellum. The movement o f the choanocyte flagella creates a current that pulls water i n and out o f the sponge. Choanocytes, (up to 7,000-18,000 per cubic millimetre o f sponge), line the walls o f many tiny chambers inside the sponge. Each o f these chambers can pump around 1,200 times its own volume o f water each day. This water is pumped through the pores (in cells called porocytes) into the spongocoel, the central cavity o f the sponge, and then out through a large opening called the osculum. A s the water flows through the sponge, the choanocytes, i n addition to providing the current, collect food particles as they pass by. Other cells called amoebocytes collect the food particles, digest them, and then carry the nutrients to other cells. This is only one o f the many functions o f amoebocytes, which can also function as the reproductive cells; (the choanocytes also function as gametes). Amoebocytes also form the tough fibres o f the skeleton i n the gelatinous matrix (mesohyl) that is sandwiched between the outside o f the sponge and the central cavity. These amoebocytes secrete skeletal fibres made up o f either spicules—small mineral  27  deposits o f calcium carbonate or silica—or o f a protein called spongin, which w i l l give the sponge a more flexible "skeleton." One o f the methods used i n the characterization o f 52 53  sponges is the analysis o f the type o f skeletal fibres they contain.  2.3.2: The Sponge Genus Hippospongia The marine sponge genus Hippospongia  (Schulze 1879; de Laubenfels 1936) is  one o f many in the family Spongiidae (Gray 1867). Other genera i n this family include Coscinoderma,  Phyllospongia,  Rhopaloeides,  Spongia, and Strepsichordaia.  The  Spongiidae is one o f several families i n the order Dictyoceratida, "the keratose sponges." T w o other families included i n this order are Irciniidae and Thorectidae. Dictyoceratid sponges are characterized as having no mineral spicules, though it is possible for them to accumulate spicules from another source, presumably due to sponges being filter feeders. Members o f the family Spongiidae are often encrusting i n nature and can contain a large amount o f the protein spongin. The genus Hippospongia  has been reported from the  Mediterranean, the Caribbean, and the western Pacific, including Hawaii, Indonesia, and Okinawa. " 53  54  2.3.3: Overview of Known Metabolites from Hippospongia sp. Hippospongia  has been chemically studied since 1926 when V . J . Clancey  subjected the protein spongin from the skeletal fibres o f the common bath sponge, Hippospongia  equina, to acidic hydrolysis.  55  H e discovered that spongin contained  iodogorgonic acid (150) i n addition to various amino acids including glutamic acid (151) and glycine (152). H e also concluded that spongin appeared to be related to collagen, another common animal protein. In 1970, a rare sugar i n animals, D-arabinose (153), was isolated from a polysaccharide found i n the connective tissue o f  28  Hippospongia  gossypina.  Furospongin-1 (114) isolated by Cimino et al in 1971 from both Spongia  officinalis and Hippospongia reported from & Hippospongia  communis  21,  appears to be the first secondary metabolite  species, with additional C21, C22, C24, and C25  furanoterpenes from Hippospongia  reported s i n c e .  243  ' ' The protein transport inhibitor, 26  57  ilimaquinone (154), the first o f several related merosesquiterpene quinones, was 58  59  isolated i n 1979 from Hippospongia  metachromia.  5&a  This species has been studied  extensively over the years by various groups yielding the merosesquiterpene quinones and hydroquinones metachromins A (155) through H (162) and related compounds, 60  and the merosesterterpenes hipposulfates A (163) and B (164).  61  A s can be deduced from  the preceding list o f compounds, H. metachromia produces various meroterpenes which are terpenes o f mixed biosynthetic origin containing both a terpene portion combined with some other portion such as an aromatic fragment (in this case) or an a l k a l o i d . ' 5la  29  b  Suvanine is an aldose reductase inhibitor from a Hippospongia sp. from Palau that has been use-patented.  63a  This sesterterpene was originally isolated from Coscinoderma  matthewsii, (formerly classified as an Ircinia sp.), and was reported as an acetylcholinesterase inhibitor.  6311  ' It was originally given the structure 165a, as the 0  sulfate o f a guanidinium meroterpene,  but it was revised i n 1988 to structure 165b with  the sulfate as part o f the molecule and the guanidino group as the counter-ion.  63c  Steroids, such as the trihydroxylated 5,6-secosterol hipposterol (166) and eight related 64  compounds  65  from Hippospongia communis, have also been isolated. The triterpene  hippospongic acid A was isolated i n 1996 from a Hippospongia. sp. collected near Japan.  66  Originally this compound was assigned structure 167a with all o f the double  bonds as i f it was formed from the sequential (head-to-tail) addition o f isoprene units to a growing terpene c h a i n .  663  However, upon synthesis o f the proposed structure, the  1 3  C  N M R data did not match, so the 'correct' structure 167b was synthesized. '° This time 66b  the N M R data matched, meaning hippospongic acid is formed from the tail-to-tail condensation o f two farnesyl pyrophosphates (28). Longer terpenoid derivatives such as the hepta- and octaprenylhydroquinones, 168 and 169, respectively, have also been isolated from this genus—from H. communis.  61  Several fatty-acid derivatives, including  the previously mentioned taurospongin A (101) i n addition to a series o f /V-acyl-2mefhylene-p-alanine methyl esters, the hurghamides A(170)-G(176), from a currently undescribed member o f this genus,  have also been isolated.  30  2.4: The Ras Converting Enzyme and Cancer A s has been mentioned, advances i n our understanding o f molecular biology have made it easier to selectively target cancerous cells. One o f the methods currently being explored involves the inhibition o f cellular signalling. Signal transduction is a very important process i n the cell as it controls a multitude o f cellular processes including cell proliferation, differentiation, and survival. In the case o f cancerous cells, cell proliferation pathways are not regulated as i n normal cells, and uncontrolled growth can occur. Unregulated cellular signalling occurs through either overexpression or mutation of proto-oncogenes to oncogenes. Ras is one o f these genes.  69  The Ras gene codes for a monomeric membrane-localized G protein involved in a cellular signalling pathway that primarily controls the proliferation o f cells. W h e n this gene is mutated, the proteins it encodes for are constantly active, with no internal ' o f f  31  switch' to stop the signal. These constitutively active proteins have been implicated i n the formation o f tumours.  Ras gene mutations occur i n approximately 30% o f human  cancers, particularly those in pancreas, colon, and thyroid tissues. Identified mutations occur at amino acids 12, 13, 59, and 61 in the protein sequence and are responsible for abolishing guanine triphosphate-activating protein ( G A P ) induced guanosine triphosphate (GTP) hydrolysis o f Ras, thereby causing continuous activity o f Ras and thus constant cell proliferation, otherwise known as cancer.  69  Ras belongs to a family o f proteins, H , K , M , N , R , Rap 1, Rap 2, and R a l . A l l o f these proteins share at least 50%> sequence identity and they have 30%> sequence identity with other small monomelic G proteins. W h i l e Ras proteins are the most studied, Rap proteins have similar effectors as Ras and are also o f great interest to science. The Ras gene product is a 35 k D protein that contains 190 amino acids with highly conserved sequences i n both the N and C termini. There is a region o f 25 amino acids located near the C terminus that is hypervariable and is the "business-end" o f the protein. It is this " C A A X " terminus that is the site for post-translational processing o f the protein before it is involved in cellular signalling.  69  The Ras protein cycles between an inactive form that is guanosine diphosphate ( G D P ) bound and a G T P bound active form. W h e n an external ligand binds to its receptor, the receptor is dimerized and a cascade o f events is initiated. This cascade ends with a cytostolic protein binding to Ras that causes it to change shape and release its bound G D P . Ras can then bind to G T P and become active. However, Ras cannot be active just by being bound to G T P . It must also be associated with a membrane for activity to occur, hence the post-translational prenylation o f this protein.  32  69  During the post-translational processing o f Ras, the segment o f the protein known as the C A A X terminus is modified first by prenylation at the cysteine (the ' C i n C A A X ) followed by cleavage o f the A A X portion. Methylation o f the cysteine residue occurs next. The processed peptide is then relocated to the cell membrane where further processing is possible. The enzyme responsible for the three post-translational processing steps o f the Ras gene product is Ras Converting Enzyme, ( R C E ) . This enzyme complex performs three transformations: prenylation to make the protein more hydrophobic, cleavage o f three amino acid residues, and methylation o f the resulting peptide.  69  It is  this enzyme complex that we are most interested i n targeting. The identity o f the C A A X residues at the C terminus o f Ras is quite important and specific. Starting with the cysteine residue that is always present, ( ' C ' ) , the next two are aliphatic amino acids, ( ' A A ' ) , either leucine, isoleucine, or valine, followed by a methionine, serine, another leucine or a glutamine, ( ' X ' ) . This X residue determines i f the prenyl group that is added during post-translational processing is o f the farnesyl or geranylgeranyl variety. Prenylation is important for the interaction o f many small hydrophobic proteins that are associated with membranes and cellular signalling. Several enzymes can be utilized to accomplish this task, including a farnesyl transferase which w i l l add a farnesyl pyrophosphate (28) to the cysteine residue or geranylgeranyl transferase type I (GGT-I) which w i l l add a geranylgeranyl pyrophosphate (29) instead.  69  Most o f the currently known inhibitors, both natural and synthetic, o f the Ras posttranslational processing pathway are farnesyl transferase inhibitors. ' 69  70  Unfortunately,  there are many cellular processes that involve farnesylation, so inhibition o f this enzyme is too general.  69  In addition, it is possible that geranylgeranylation may occur i f the  33  normal farnesylation is b l o c k e d . ' 71  72  Therefore, the block is bypassed v i a another route  essentially defeating its purpose. A s mentioned in the introductory chapter o f this thesis, both 28 and 29 are biosynthesized from acetate via the mevalonate pathway. One o f the intermediates i n this pathway is 3-hydroxy-3-methylglutaryl coenzyme A ( H M G - C o A , 25). Inhibition o f the enzymatic formation o f this metabolite w i l l block protein prenylation, but again it is too general for therapeutic use. Other possible inhibition sites include stopping Ras protein from being made b y blocking the expression o f the Ras gene, inhibition o f the downstream Ras effectors, and inhibition o f one o f the other enzymes involved i n posttranslational processing o f R a s .  69  It is the latter that we w i l l focus on.  Recently, the R C E endoprotease gene has been isolated, identified, and cloned for Ti  -TI-\  mice,  yeast,  1A.  and humans.  This endoprotease ( h R C E l ) represents a new and  hopefully more specific target in the Ras post-translational processing pathway. Wyeth Research i n N e w Y o r k has developed a novel bioassay to screen for RCE-protease inhibitors. The bioassay is based on a quenched fluorescent analog o f Ras that has the following structure: 2-aminobenzoyl-Lysine-Serine-Lysine-Threonine-Lysinefarnesylated Cysteine-s-dinitrophenyl Lysine-Isoleucine-Methionine. This peptide exhibits a linear increase i n fluorescence over time after treatment with h R C E l . W i t h no h R C E l present, there is no detectable increase i n fluorescence over the same time interval.  75  Therefore, i f an inhibitor is present in an extract o f a sponge, for example, one  should see similar effect when it is tested i n the presence o f h R C E l , ie no detectable increase in fluorescence. 34  2.5: RCE-protease Inhibitors f r o m an Indonesian Hippospongia sp. 2.5.1: Isolation Procedure  Based on this knowledge, marine extracts from the Andersen lab collection were screened for inhibition of h R C E l by Wyeth Research, our industrial collaborator. One positive result in the bioassay was the ethanol extract of the sponge RJA96-141. The source sponge (see Figure 2.1) was later identified as a Hippospongia sp. by Dr. Rob van Soest of the Zoologisch Museum at the University of Amsterdam in the Netherlands where a voucher specimen is kept (ZMA16774). This sponge was harvested by hand using S C U B A on the inner reef at Barangcadi Island, near Makassar, on the island of Sulawesi in Indonesia. Sixty grams of the freshly collected sponge were repeatedly extracted with ethanol. The extracts were combined and concentrated in vacuo, followed  Figure 2.1: RJA96-141—Hippospongia sp. collected in Indonesia. (Photo by M . LeBlanc, used by permission.) by suspension in water. This suspension was partitioned between water (20 mL) and EtOAc (4x7 mL). Bioassay guided fractionation of the RCE-protease inhibitory EtOAc soluble material was undertaken. The EtOAc fraction was sequentially separated by Sephadex LH-20 chromatography (eluent: 80% MeOH/DCM) and reverse-phase flash gradient column chromatography (eluent: 100% H 0 to 100% MeOH; lOOmL for each 2  fraction). At this point it was determined that two major metabolites were present. They  35  were purified using reverse-phase H P L C (eluent: 65% M e C N / 0.05% Trifluoroacetic acid ( T F A ) i n H 0 ) on a portion o f the fractions containing them. Following H P L C , the two 2  compounds were identified as the known norsesterterpene rhopaloic acid A (122),  30a  isolated as a clear oil (105 mg), and the novel sesterterpene, barangcadoic acid A (130), also isolated as a clear oil (35 mg). Several additional peaks were present i n the H P L C trace for 122, so an additional fraction from the flash-chromatography step was subjected to reverse-phase H P L C (eluent: 75% M e C N / (0.05%TFA/ H 0 ) ) to yield two additional 2  known norsesterterpenes, rhopaloic acids B (35) and C (36).  30b  Additional peaks in the  H P L C trace o f 130 with similar U V absorbance (at 204 nm) were also observed. One o f these was pure and contained rhopaloic acid G (40). Another contained several compounds so it was purified further b y reverse-phase H P L C (eluent: 58% M e C N / ( 0 . 0 5 % T F A / H O ) to yield the inseparable mixture o f rhopaloic acids D / E (37/38) 2  and pure rhopaloic acid F (39). The structures o f the two major metabolites 122 and 130 were elucidated b y m y colleague and hence w i l l not be discussed i n this thesis, while the structure elucidations o f 35-40 are presented below.  130  122  2.5.2: Rhopaloic Acids B (35) and C (36) Rhopaloic acid A (122) originally isolated from the Japanese sponge Rhopaloeides  sp. in 1996,  30a  and subsequently synthesized, was again isolated i n the 76  course o f our investigation on the Indonesian sponge Hippospongia  sp. A s mentioned  above, it appeared that several other compounds were present in the fraction containing 122. After separation, it became apparent that one o f these compounds was isomeric with  36  122. The H R F A B M S spectrum gave a [ M - H ]" ion at m/z 373.27'46 (C24H37O3), which is essentially identical to that o f 122 (373.2743; C24H37O3). Analysis o f both the I D and 2 D N M R data obtained i n C^D(, for 35 showed that it had the same constitution as 122, thus the two molecules must be diastereomers. Furthermore, the H-3 resonance at 8 4.11 ppm in the proton N M R spectrum o f 35 was a doublet with J = 10.6 H z indicating it was axial and the C-3 vinyl substituent was equatorial. The H - 2 1 broad doublet with J = 11.3 H z and the H - 2 1  ax  eq  resonance (5 3.72 ppm) was a  resonance (8 3.35 ppm) was a doublet o f  doublets with J = 11.3 and 2.6 H z , respectively. This demonstrated that H-6 was i n the equatorial position and the C-6 prenyl chain was i n the axial position. Rather than the trans 1,4 disubstituted tetrahydropyran ring present i n 122, 35 contained a cis 1,4 disubstituted ring system, though the absolute stereochemistry was not established. Figure 2.2 shows the probable conformations o f the two different 1,4 disubstituted tetrahydropyran systems o f 122 and 35. Therefore 35 is the known compound rhopaloic acid B , isolated from the same Rhopaloeides  sp. as 122, but i n 1 9 9 8 .  30b  The N M R  20  Figure 2.2: Probable conformation o f the tetrahydropyran moiety and a-p carbonyl system i n 122 and 35. The dashed line i n each o f the chair forms refers to a hydrogen bond. Approximate coupling constants obtained from the proton spectra are also shown.  37  assignments for 35 (0.1 m g isolated) can be found in Table 2.1 and the ' H N M R spectrum in Figure 2.3. 20  The other molecule (36) present i n this fraction (0.4 mg) was similar to both 35 and 122, but it gave a [M-H ]~ ion i n the H R F A B M S spectrum oim/z 371.2593 +  (C24H35O3), indicating there were two less protons and, therefore, one more site o f unsaturation is present. A n additional olefinic methine resonance at 8 5.36 ppm i n the ' H N M R spectrum was present, as were two additional olefinic carbon resonances at 8 118.1 and 8 136.6 ppm i n the  1 3  C N M R spectrum. The rest o f the data was similar to that o f  122, thus the extra degree o f unsaturation must be an additional olefin and 36 must be the known compound rhopaloic acid C reported i n the same paper as 3 5 .  30b  Complete  analysis of both the I D and 2 D N M R data further supported this conclusion. The N M R assignments for 36 can be found in Table 2.1 and the I D N M R spectra i n Figures 2.4-2.5. 20  38  Table 2.1: Rhopaloic Acids B (35) and C (36) N M R Data i n C D 6  No. 1 2 3 4  Carbon (35) 168.5 142.0 76.6 27.7  5  27.7  6 7  30.3 33.3  8 9 10 11 12 13 14 15 16 17 18 19 20  26.2 125.1 135.3 40.3 27.1 125.1 135.3 40.0 27.1 125.1 131.3 25.8 125.9  Proton (35) — —  4.11 d(ax), J=10.6 H z 1.64 m * , 1.40 m * 1.64 m * , 1.40 m * 1.32 m 1.40 m * , 1.19m 1.95 m 5.23 m —  2.18/2.09 m * 2.18/2.09 m * 5.28 m  a  118.1 136.6 33.1 26.5 124.3 135.5 40.2 27.2 124.7 135.1 40. l 27.1° 124.9 131.1 25.8 126.3  —  4.37 bd, J = 8.6 H z 2.31 bd, 2.01 m 5.36 bd  2.01 m 5.17 t, J = 6.6 H z —  2.17-2.10 m * 2.17-2.10 m * 5.28 t, J = 7.4 H z —  2.17-2.10 m * 2.17-2.10 m * 5.24 t, J = 6.4 H z  b  —  —  1.77 t, J = 7.4 H z  C  2.18/2.09 m * 2.18/2.09 m * 5.23 m  Proton (36)  —  b  —  a  Carbon (36) 168.7 141.4 72.0 31.4  6  —  1.68 s 1.67 s 6.37 bs 6.32 bs, 5.85 bs 5.99 bs 3.72 d ( e q ) , J = 1 1 . 3 H z , 68.8 4.04 d , J = 16.5 H z 21 71.6 3.99 d, J = 16.1 H z 3.35 dd (ax), J = 11.4 H z , J = 2.6 H z 1.55 s 1.57 s 16.1 22 15.9 1.61 s 1.60 s 16.1 23 16.0 17.7 1.56 s 24 18.0 1.56 s For 35, 'H NMR 400 MHz; C NMR v a l u e sfromH S Q C or H M B C correlations at 100 MHz. For 36, 'H NMR at 500 MHz; C NMR at 100 MHz f r o m a C NMR spectrum. Values with the s a m e letter could be interchanged. * O v e r l a p p i n g signals. 13  l3  13  c b ,a ,  35  39  40  41  42  2.5.3: Rhopaloic Acids D/E (37/38) and F (39) W i t h the data in hand for the known compounds 35, 36, and 122 we turned to the structure elucidation o f the inseparable mixture 37/38 and a closely related compound, 39. Initially, it was assumed that the mixture was a single compound, but as we progressed through the elucidation process, it became clear that we were dealing with a mixture o f two very similar compounds in approximately the ratio 6:4. There was only 0.2 m g o f this mixture, so we decided that further separation (of 37/38) was not possible due to the possibility o f losing some material i n the process, thereby making it impossible to solve the structures. W e w i l l begin with the structure elucidation o f mixture 37/38 and then proceed to that o f 39.  The mixture 37/38 gave a [ M + H ] ion at m/z 391.2852 (C24H39O4) i n the positive +  H R D C I M S spectrum corresponding to the presence o f an additional oxygen atom over that o f 35 and 122. Examination o f the I D and 2 D N M R data obtained i n C D for the 6  6  mixture showed the presence o f multiple olefinic resonances, several o f which corresponded to olefinic methylenes. The broad singlets at 8 6.35 and 8 5.91 ppm, each signal with an integration o f I H corresponded to one olefinic methylene.  There were  also four additional broad singlets corresponding to 0.5H each when integrated. T w o o f these signals, 8 4.94 and 8 4.78 ppm, (both broad singlets), were correlated i n the H M B C  43  spectrum to a carbon signal at 8 17.7 ppm and to an allylic carbinol methine at 8 75.4 ppm. Analysis o f the H M Q C spectrum showed resonances at 8 3.88 ppm (multiplet, integration I H ) , attached to the carbon at 8 75.4 ppm, and at 8 1.62 ppm (singlet, integration 3H), attached to the carbon at 8 17.7 ppm.  A s this latter resonance was for a  methyl group, this olefin must be at the end o f a chain o f some length. 4.94/4.78,110.6  3.88, 75.4  Figure 2.6: Important C O S Y (arcs) and H M B C (arrows) correlations for the tail o f 37. Carbon chemical shifts are i n italics. The remaining set o f olefinic methylene signals at 8 5.07 and 8 4.88 ppm, (both broad singlets), also integrated for 0.5H each. Similar analysis o f the H M B C and H M Q C spectra showed correlations into a methylene carbon at 8 31.8 ppm and into a carbinol methine at 8 75.0 ppm. Therefore, the olefinic methylene must be located somewhere along the length o f the chain, but not at the end. This was further established by the presence o f two methyl groups, at 8 1.66 and 8 1.55 ppm, which showed H M B C correlations into the same olefinic carbons at 8 131.6 and 8 124.8 ppm, (see Figure 2.7), confirming the presence o f a standard terpene head group. A s i n 35, 36, and 122, the presence o f a carboxylic acid functionality at 8 168.2 ppm, and two additional carbinols were detected at 8 76.2 and 8 73.1 ppm i n the  C  N M R spectrum. Furthermore, the exo-methylene protons at 8 6.35 and 8 5.91 ppm  44  showed H M Q C correlations to a carbon at 5 125.9 ppm and H M B C correlations to 8 168.2 ppm and 8 76.2 ppm. The proton attached to this carbon was determined to be at 8 4.08 ppm by analysis of the H M Q C spectrum. This proton showed two important H M B C correlations, a relatively weak one to a carbon at 8 141.8 ppm (the other carbon in the olefinic methylene system), and a stronger correlation to another carbinol methine at 8 73.8 ppm. Based on the former correlation it was now possible to determine the position of the o>P carbonyl system to be in the same location as in 35, 36, and 122. The H M B C correlation between a proton at 8 4.08 ppm and a carbon at 8 73.8 ppm suggested the presence of one of the following: an adjacent diol system, a diol system separated by one carbon, or an ether linkage. Based on comparison of the N M R data with the known compounds 35, 36, and 122 it was determined that 37/38 also contained a trans 1,4 disubstituted tetrahydropyran ring. Four of the six sites of unsaturation as required by the molecular formula were now accounted for. 5.07/4.88,709.4  1.55,77.7  3.94, 75.0  5.21, 124.8  Figure 2.7: Important H M B C Correlations for Partial Structures of 38.  Continuing the comparison of N M R data of the mixture 37/38 with those of the known compounds, the presence of three additional olefinic moieties was established from both the COSY and H M B C spectra. As there could only be two additional sites of  45  unsaturation per molecule, two o f these olefinic moieties must belong to 37 and one to 38. In the H M B C spectrum, there were correlations from two methyls (5 1.66 ppm and 8 1.55 ppm) to a carbon at 8 131.6 ppm and from a methylene at approximately 8 2.20 ppm to this same carbon, (see Figure 2.7). H M B C correlations between the olefinic methylene protons at 8 5.07 and 8 4.88 ppm and a carbon at 8 31.8 ppm were also observed. A l l that now remained in the structure elucidation o f 38 was the connection o f the allylic alcohol moiety to the terminal isoprene unit. There were two possibilities: no intervening isoprene (ie direct connection), or with an intervening isoprene unit. Due to the overlap o f the signals in the methylene region, (~ 8 2.20 ppm), and the presence o f a mixture o f compounds, it was difficult to unambiguously assign the position o f the allylic alcohol. However, a careful interpretation o f the data available from the H M B C spectrum suggested the placement o f this moiety at the middle isoprene unit, though we could not be certain. Turning our attention to the related compound 39, (~ 0.1 m g isolated), we noted that it gave a [ M + H ] ion at m/z 391.2839 (C24H39O4) i n the H R D C I M S spectrum, +  making it isomeric with 37/38. Inspection o f the I D and limited 2 D N M R spectra (in C6D6) showed there was indeed an allylic alcohol (8 3.78 ppm connected to 8 75.1 ppm) present with an adjacent olefinic methylene (8 4.99 and 8 4.85 ppm attached to 8 109.2 ppm). Other resonances similar to those i n 37/38 were additional exo-methylene resonances at 8 6.33 and 8 5.86 ppm (both broad singlets), two carbinols—a methine at 8 4.03 and a methylene at 8 3.85 (multiplet) and 8 2.85 (triplet), two additional doublebond protons (8 5.29 and 8 5.23 ppm), and three olefinic methyls (8 1.68, 8 1.55, 8 1.54 ppm).  46  Even with an H M B C experiment lasting approximately 20 hours, there was not enough material to see more than a few correlations, mostly those from the three methyl groups to olefinic carbons. W i t h four o f the six units o f unsaturation accounted for and the fact that 39 was isomeric with 37/38, the remaining two units must be accounted for by a carboxylic acid and a trans-1,4 tetrahydropyran ring as i n most o f the other rhopaloic acids. Again, all that remained was the assignment o f the allylic alcohol and accompanying olefinic methylene, though the lack o f a useable H M B C meant only the C O S Y data could be used. The C O S Y data showed a coupling between a triplet at 8 5.29 ppm and a multiplet at 8 2.23 ppm. This multiplet was coupled to another at approximately 8 1.98/8 2.15 ppm, which was i n turn weakly coupled to one o f the olefinic methylene protons (8 4.85 ppm) o f the allylic alcohol moiety, (see Figure 2.8). 4.99/4.85  1.61  5.29  1.56  5.23  Figure 2.8: C O S Y Correlations in 39 Suggesting Placement o f the A l l y l i c A l c o h o l .  W i t h this data for 39 and the structure o f 37 in hand, we could now propose that the allylic alcohol moiety in 38 was located at the middle isoprene unit. The structures o f both 38 and 39 are tenuous i n nature, and the possibility exists that we may have the structures reversed, but based on the N M R information available we believe they are as shown. Additional support for placement o f this moiety came from the co-elution o f 37 and 38 while 39 eluted with a different retention time. This suggested that 37 and 38  47  should have greater similarity with each other, thus the allylic alcohol nearer to the end o f the farnesyl side chain i n both. It was not possible to determine the stereochemistry of the allylic alcohols for 37-39 due to the small amounts that were isolated. The N M R data for rhopaloic acids D(37), E (38), and F (39) are in Tables 2.2-2.4, with respective I D N M R spectra following each table. Section 2.8 contains the 2 D N M R spectra for 37-39.  Table 2.2: Rhopaloic A c i d D (37) N M R Data i n C D 6  No. 1 2 3  Carbon 168.2 141.8 76.2  4  32.4  5  32.6  6 7 8 9 10 11 12 13 14 15 16 17 18 19 20  36.1 30.4 25.2 124.8 135.1 40.0* 36.1* 124.7 135.1 36.2 33.9 75.4 148.1 17.7 125.9  21  73.8  22 23 24  16.0 16.0 110.6  Proton  6  H M B C Correlations  C O S Y Correlations  —  —  —  —  —  —  H-4, H-4'  C - 2 1 , C - 2 , C-6  4.08 d (ax) J = 11.3 H z 1.88 m, 1.20 m 1.35 m, 0.92 m 1.35 m (ax) 1.64 m 1.85 m 5.16m —  2.07 m * 2.15 m * 5.26 m —  2.20 m * 2.20 m * 3.88 m —  1.62 s 6.35 bs 5.91 bs 3.92 m(eq) 2.88 m (ax) 1.54 s 1.56 s 4.94 bs 4.78 bs  G C , H-5, H-3 (to both) G C , H-4  C-4  H-21 (to both) H-21 H-ll,H-9 H-22  C-10 C-ll*  —  —  H-12 H-ll H-23,H-12*  C-10* C-ll* C-22, C-15/C-11*  —  —  H-16 H-15 H-24 (to 4.94)  C-14* C-15* C-19, C-24, C-16*, C-15*  —  —  H-24 G C , H-3  C-24, C-18, C-17 C-3,C-l  e q  G C , H - 7 (from H - 2 1 ) , H-6 H-9 H-15, H - 1 3 , H - 1 1 G C , H-19, H-17 (from 4.94) eq  C-7 C - l l * , C-10*, C - 9 * C-14*,C-13* C-19, C-17  'H NMR at 500 MHz; "Carbon NMR at 100 or 125 MHz. "C shifts from HMBC correlations are at 125 MHz, rest are at 100 MHz. * Uncertain values or assignments due to overlapping signals and/or the presence of a mixture. Not all correlations are listed. GC = Geininal Correlations, correlations between protons on the same carbon. Correlations from exo-methylenes are marked from/to which proton unless they are from both, then just the proton(s) or carbon numbers) is/are given.  48  49  Figure 2.10: Rhopaloic Acid D (37) C Spectrum in C D at 100 MHz. Not all peaks are marked or even visible; peaks labelled " E " are for rhopaloic acid E (38). 1 3  6  50  6  Table 2.3: Rhopaloic A c i d E (38) N M R Data i n C D 6  No. 1 2 3  Carbon 168.2 141.8 76.2  4  32.4  5  32.6  6 7 8 9 10 11 12 13 14 15 16 17 18 19 20  36.1 30.4 25.2 124.8 135.1 40.0 36.1 75.0 152.2 31.8 26.2* 124.8 131.6 25.7 125.9  21  73.8  22 23  16.0 109.4  24  17.7  Proton  6  H M B C Correlations  C O S Y Correlations  —  —  —  —  —  —  H-4, H-4'  C - 2 1 , C - 2 , C-6  4.08 d (ax) J = 11.3 H z 1.88 m , 1.20 m 1.35 m , 0.92 m 1.35 m (ax) 1.64 m 1.85 m 5.13 m —  2.07 m 2.11 m 3.94 m —  2.20 m * 2.22 m * 5.21 m —  1.66 s 6.35 bs 5.91 bs 3.92 m(eq); 2.88 m (ax) 1.54 s 5.07 bs 4.88 bs 1.55 s  G C , H-5, H-3 (to both) GC, H-4  C-4  H-21 (to both) H-21 q H-ll,H-9 H-22, H-8  C-10 C-22, C - l l * , C-8  —  —  e  H-12 H-ll  C-11,C-14 C-15  —  —  H-24 H-24, H-19, H-16,  C-15* C-16/C-15*  H-15*  —  —  H-24, H-17 G C , H-3  C - 1 8 , C - 1 7 , C-24 C-3, C - l  G C , H-7 (from H - 2 1 ) , H-6 H-9, H-8* GC eq  C-7 C - l l * , C-9 C-13,C-15  #  H-17, H-16  C-19, C-18, C-17  'H NMR at 500 MHz; "Carbon NMR at 100 or 125 MHz. C shifts from HMBC correlations are at 125 MHz, rest are at 100 MHz. * Uncertain values or assignments due to overlapping signals and/or the presence of a mixture. Not all correlations are listed. GC = Geminal Correlations, correlations between protons on the same carbon. Correlations from exo-methylenes are marked from/to which proton unless they are from both, then just the proton(s) or carbon numbers) is/are given. I 3  51  52  Figure 2.12: Rhopaloic A c i d E (38) C Spectrum in C D at 100 M H z . Not all peaks are marked or even visible; peaks labelled " D " are for rhopaloic acid D (37). 1 3  6  53  6  Table 2.4: Rhopaloic A c i d F (39) N M R Data i n C D 6  Proton  No.  Carbon  1 2  ? ?  3  76.1  4  32.6  1.86 m ,  5  ?  1.32 m , 0.90 m  6  29.7#  1.32 m (ax)  7  ?  8  35.7#  1.62 m 1.32 m  H-21eq, H - 5 H-21 , H-9  9  75.1  3.78 t  H-8  10 11  162.6f 31.4  2.15 m , 1.98 m  G C , H-22 (to 4.85 from both), H-12  12  ?  2.23 m  H-13, H - l l  13  124.2  5.29 t  H-23, H-12  6  H M B C CorrelationsJ  C O S Y Correlations —  4.03 d (ax)  H-5, H-4  J = 10.6 H z G C , H-3  1.20 m H-7 H-21  a x  ax  J = 5.7 H z —  —  —  J = 6.5 H z 14  135.6  15 16  39.9 26.8  17  124.5  —  —  —  2.09 m 2.18m 5.23 t  C-17, C-13 C-18  H-17 H-24, H-19, H-16  J = 6.1 H z 18  131.1  19  25.6  1.68 s  20  125.6  6.33 bs  21  73.7  3.85 dm  109.3  (eq) J= 10.3 H z 2.85 t (ax) J = 10.3 H z 4.99 bd  —  —  —  H-24, H-17  C-24, C-18, C-17  GC  5.86 bs  22  G C , H - 7 (from H - 2 1 ) eq  H - 6 / H - 8 (from H - 2 1 ) ax  ax  G C , H - l l (from 4.85)  C-10(4.85), C - l l (4.85)  H-13  C-15, C-14, C-13  J =4Hz 4.85 bs 23  15.9  1.61 s  C-19, C-18, C-17 24 17.5 1.56 s H-19, H-17 'H NMR at 400 MHz; C shifts are from HMBC/HSQC correlations and are at 100 MHz. % Sample was extremely dilute therefore most correlations were unobservable. ? Unknown values. # These values may be interchanged, f This value seems very high for this carbon, but there is a weak correlation nonetheless in the HMBC spectrum from H-22 (5 4.85 ppm). GC = Geminal Correlations, correlations between protons on the same carbon. Correlations from exo-methylenes are marked from/to which proton unless they are from both, then just the proton(s) or carbon numbers) is/are given. l3  54  2.5.4: Rhopaloic Acid G (40)  The remaining compound we obtained in this investigation gave a [ M + N H 4 ] ion at m/z 426.3219 i n the H R D C I M S spectrum corresponding to molecular formula o f C24H40O5, which required only five sites o f unsaturation. Compound 40 contained many o f the same elements as those preceding it, including a carboxylic acid conjugated to an olefinic methylene with an adjacent trans-1,4 tetrahydropyran ring, and two trisubstituted olefins, thus accounting for all five sites o f unsaturation. The obvious difference between 40 and the other members o f this family o f compounds was the presence o f a methyl group at 5 0.98 ppm (in C^Ds). This methyl group showed correlations i n the H M B C spectrum to two oxygenated carbons at 8 74.4 and 8 78.7 ppm, and to a methylene at 8 36.4 ppm. The carbon resonance at 8 78.7 ppm was correlated to a proton resonance at 8 3.08 ppm i n the H M Q C spectrum. This data suggested that one o f the internal olefins had been converted into a diol. The placement o f the diol was assigned as the one closest to the tetrahydropyran on the basis o f a peak at m/z 214.1198 (C11H18O4) i n the H R D C I M S spectrum. Diols undergo a-cleavage, where the carbon-carbon bond connecting the two carbinols breaks (see Scheme 2.2) quite readily, thus the diol must be in the proposed position. Inspection o f the remaining I D and 2 D N M R data was consistent with the proposed structure for rhopaloic acid G 40 as shown. A s we isolated only 0.1 mg o f rhopaloic acid G 40, we were unable to determine the stereochemistry o f the diol. Table 2.5 provides a listing o f the N M R data and Figure  56  HCv  HO  ^O. H  H  .-0 +  ,0 OH  178a  178b  179  Scheme 2.2: Possible a-Cleavage Mechanism o f 40 i n the D C I Mass Spectrometer.  2.14 shows important C O S Y and H M B C correlations for 40. The I D H N M R spectrum is found i n Figure 2.15 following Table 2.5, and the 2 D N M R spectra for rhopaloic acid G 40 are found i n Section 2.8. 6.35/5.88,  125.6  1.56, 17.7  Me> C_>Me H 5.23,  124.7  Figure 2.14: Important C O S Y and H M B C Correlations for 40.  57  ,  1.68,25.9  Table 2.5: Rhopaloic A c i d G (40) N M R Data in C D 6  No. 1 2 3  Carbon 168.0 141.8 76.2  4  32.3  5  ?  6 7 8  ?  30.1 28.2  9 10 11 12 13  78.6 74.4 36.3 125.3  14 15  135.3 40.2  16 17  27.2 124.7  18 19 20  131.4 25.9 125.6  21  73.7  22 23 24  23.5 16.1 17.7  Proton -  — —  4.09 d (ax) J = 10.4 H z 1.90 m , 1.22 m 1.48 m 0.90 m 1.61 m (ax) 1.31 m 1.18 m , 1.07 m 3.09 bs —  1.35 m 2.04 m 5.29 t J = 6.0 H z  ?  —  2.10m 2.19m 5.23 t J = 7.0 H z —  1.68 s 6.35 bs, 5.88 bs 3.89 m(eq) 2.90 t (ax) J = 10.3 H z 0.98 s 1.64 s 1.56 s  6  H M B C Correlations  C O S Y Correlations —  —  —  —  H-4 H - 6 ( f r o m 1.90), H-5 (to 0.90), H - 3 H - 6 , H-4 ax  a x  ax  H-7, H-5, H-4 (to 1.90) H - 6 , H - 2 1 (weak) H-9 ax  ax  H-8 —  —  H-13, H-12 H-13, H - l l H - 2 3 , H - 1 2 , H-15  C-23  —  —  H-13,H-ll H-17 H-24, H-19, H-16  C-23, C-17, C-16, C-14, C-13 C-18, C-17, C-15 C19  —  —  H-24, H-17 GC  C-24, C-18, C-17 C-3, C-2 (from 6.35), C - l  G C , H-7 (from H - 2 1 ^  H-13 H-19, H-17  C - l l . C - 1 0 , C-9 C-15, C-14, C-13 C - l 9 , C-18, C-17  'H NMR 500 MHz;. C shifts are from HMBC/HMQC correlations and are at 125 MHz. ? Unknown values. GC = Geminal Correlations which are correlations between protons on the same carbon. Correlations from exo-methylenes are marked from/to which proton unless they are from both, then just the proton(s) or carbon numbers) is/are given. I 3  58  59  2.5.5: Conclusions The rhopaloic acids A (122), B (35), C (36), mixture D / E (37/38), and barangcadoic acid A (130) were tested in the RCE-protease bioassay, which showed that these compounds had ICso's o f approximately 10 p.g/mL. For cellular cytotoxicity assays, two colon tumour cell lines were utilized, the L o V o and the CaCo. The L o V o cell line has mutated K - R a s and an activated Ras pathway, so it is very sensitive to Ras pathway inhibitors, while the C a C o cell line has normal Ras and is thus not as sensitive. In the cytotoxicity assay, the IC50 was approximately 1 p.g/mL. Compounds 35-37/38,122, and 130 were three to four times more active against L o V o than CaCo, which is the expected trend for RCE-protease inhibitors. The difference i n IC50 values may be an indication o f the compounds hitting more than one target i n the cell, or it may be an indication o f the compounds being concentrated by the cell. In any case, these compounds are the first  60  31  known natural product inhibitors o f this enzyme,  though several groups have made  77  some synthetic inhibitors. A n interesting biosynthetic question arose during the course o f these studies. Barangcadoic acid A (130) appears to have a regular sesterterpene skeleton where carbons C - l , C-2, and C-21 form the head o f the starter isoprene unit. The rhopaloic acids (35-40 and 122) are norsesterterpenes which appear at first glance to have the same skeletal type as 130 due to the tetrahydropyran moiety present i n all. However, it is not possible to relate these two skeletons simply b y loss o f one carbon (C-20) i n 130, due to the placement o f the double bonds. Instead, 35-40 and 122 are possibly assembled i n the other direction, starting with C - l 8, C-19, and C-24 as the head o f the first unit. Starting from the precursor geranylfarnesyl pyrophosphate (30), we can envision an epoxidation at C-2/C-3 to give intermediate 180a. This compound then undergoes acidic opening o f the epoxide and allylic oxidation at C-22, (allylic oxidation is quite common i n terpene biosynthesis ), to form intermediate 180b. Cyclization can then 78  occur as shown i n Scheme 2.3, followed b y oxidation o f C - l to form barangcadoic acid A (130). Note that the carbon numbering i n Scheme 2.3 is based on the skeleton o f the desired product, barangcadoic acid A (130).  180b  Scheme 2.3: Possible Biosynthetic Pathway to Barangcadoic A c i d A (130).  61  Once again starting from 30, but travelling down a different pathway via oxidation o f C-4 and C - 22 w i l l form the intermediate 181a. Cyclization (to form 181b as shown i n Scheme 2.4), followed by oxidation o f C - l to a carboxylic acid w i l l form intermediate 181c. (Note that the numbering i n Scheme 2.4 is based on that o f 30). Intermediate 181c can decarboxylate to form the norsesterterpene 181d which can be reoxidized to form rhopaloic acid C (36). Following enantiofacial reduction o f the double bond i n the dihydropyran ring, rhopaloic acids A (122) and B (35) are formed. Rhopaloic  Scheme 2.4: Possible Biogenesis o f Rhopaloic A c i d s A (122), B (35), and C (36). acid A (122) could then be epoxidized at any o f the three double bonds i n the farnesyl side chain. Opening o f the epoxides followed by elimination o f a proton gives the allylic alcohols rhopaloic acids D (37), E (38), and F (39). The structure o f 37 is fairly certain due to C O S Y and H M B C correlations from the olefinic methylene i n the allylic alcohol moiety to the terminal methyl group (and vice versa) at the farnesyl chain terminus. Furthermore, the cc-cleavage i n the D C I M S o f rhopaloic acid G (40) is proof o f a diol at  62  C-9 and C-10. These data suggest that epoxidation o f any the double bonds in the farnesyl terminus o f rhopaloic acid A (122) is quite likely to occur, followed by acidic opening to form the allylic alcohol moiety present i n rhopaloic acids D (37), E (38), and F (39), see Scheme 2.5.  Scheme 2.5: Possible Biogenesis o f Rhopaloic Acids D (37), E (38), F (39), and G (40) from Rhopaloic A c i d A (122). Note that the numbering scheme for 37-40 is based on the structural assignments given in Sections 2.5.3-2.5.5. Similar linear sesterterpenes with a 1,4-disubstitued "pyran-type" ring system have been found i n other sponges in the order Dictyoceratida. Cacospongionolide D 79 (182) isolated from the Adriatic sponge Fasciospongia  cavernosa,  and part o f a series  o f related sesterterpenes, is one example. Other examples include thorectolide (183) 80  isolated from aHyrtios sp. from N e w Caledonia and thorectolide 25-monoacetate (184) 81  isolated from the Australian sponge Thorectandra excavatus.  31e  These compounds may  be formed from a similar biosynthetic pathway to that o f the rhopaloic acids, see Scheme 2.6. (Note that it begins with a common intermediate, 181c).  63  183: R = H  184:R = Ac  182  Scheme 2.6: Some Related Sesterterpenes and Their Possible Biosynthesis From A Common Intermediate 181c. The numbering for 183 and 184 is from references 81 and 37e, respectively. Due to the lack o f further source material i n our collection and the slim possibility o f recollection as a result o f the political instability i n Indonesia, several aspects o f this project remain unresolved. First, the stereochemistries o f the hydroxyls present i n 37-40 have not been determined. Secondly, there are other extremely minor related metabolites present i n the extract that we are currently not able to work with. These metabolites may shed further light on the biosynthesis o f the rhopaloic acids 35-40 and 122. Finally, it would be exciting to determine the actual intermediates i n the two different pathways that lead to barangcadoic acid A (130) and to the rhopaloic acids, 35-40 and 122.  2.6: References 1)  Faulkner, D.J. Nat. Prod. Rep. 2002,19, 1-48, and previous reviews i n this series.  2)  Ishiyama, H . ; Ishibashi, M . ; Ogawa, A . ; Yoshida, S.; Kobayashi, J. J. Org. Chem. 1997, 62, 3831-3836. a) Gunasekera, S.P.; Gunasekera, M . ; Longley, R . E . ; Schulte, G . K . J. Org. Chem. 1990, 55, 4912-4915; Correction: J. Org. Chem. 1991, 56, 1346. b) ter Haar, E . ; K o w a l s k i , R.J.; Hamel, E . ; L i n , C . M . ; Longley, R . E . ; Gunasekera, S.P.; Rosenkranz, H.S.; Day, B . W . Biochemistry, 1996, 35, 243250.  3)  4)  a) Coleman, J.; de Silva, E . D . ; K o n g , F.; Andersen, R.J.; A l l e n , T . M . 1995, 39, 10653-10662.  64  Tetrahedron  b) Talpir, R.; Benayahu, Y . ; Kashman, Y . ; Pannell, L . ; Schleyer, M . Lett. 1994, 35, 4453-4456.  Tetrahedron  Williams, D . E . ; Craig, K . S.; Patrick, B . ; McHardy, L . M . ; van Soest. R . ; Roberge, M . ; Andersen, R . J. J. Org. Chem. 2002, 67, 245-258. Burgoyne, D . L . ; Andersen, R.J.; A l l e n , T . M . J. Org. Chem. 1992, 57, 525-528. See for example: a) Renner, M . K . ; Jensen, P.R.; Fenical, W . Org. Chem. 2000, 65, 4843-4852. b) Renner, M . K . ; Jensen, P.R.; Fenical, W . J. Org. Chem. 1998, 63, 8346-8354. c) Takahashi, H . ; Hosoe, T.; Nozawa, K . ; Kawai, K . J. Nat. Prod. 1999, 62, 1712-1713. d) Hensens, O . D . ; Zink, D . ; Williamson, J . M . ; Lotti, V . J . ; Chang, R . S . L . ; Goetz, M . A . J. Org. Chem. 1991, 56, 3389-3403. e) Kosemura, S.; Matsunaga, K . ; Yamamura, S.; Kubota, M . ; Ohba, S. Tetrahedron Lett. 1991, 32, 3543-3546. a) Kaneda, M . ; Takahashi, R.; Iitaka, Y . ; Shibata, S. Tetrahedron Lett. 1972, 45, 4609-4611 and references therein. b) Sugawara, H . ; Kasuya, A . ; Iitaka, Y . ; Shibata, S. Chem. Pharm. Bull. 1991, 59,3051-3054. See for example: a) Calderon, A . I . ; Terreaux, C ; Schenk, K . ; Pattison, P.; Burdette, J.E.; Pezzuto, J . M . ; Gupta, M . P . ; Hostettmann, K . J. Nat. Prod. 2002, 65, 1749-1753. b) D e Tommasi, N . ; D e Simone, F.; Pizza, C ; Mahmood, N . J. Nat. Prod. 1996, 59, 267-270. c) Topcu, G . ; Ulubelen, A . ; Tarn, T. C . - M . ; Che, C.-T. J. Nat. Prod. 1996, 59, 113-116. d) Moghaddam, F . M . ; Zaynizadeh, B . ; Riiedi, P. Phytochemistry 1995, 39, 715716. e) Rustaiyan, A . ; Koussari, S. Phytochemistry 1988, 27, \161-XI69.' f) Rustaiyan, A . ; Sadjadi, A . Phytochemistry 1987, 26, 3078-3079. Fontana, A . ; Ciavatta, M . L . ; Cimino, G . J. Org. Chem. 1998, 63, 2845-2849. See for example: a) Dumdei, E.J.; Kubanek, J.; Coleman, J.E.; Pika, J.; Andersen, R.J.; Steiner, J.R., Clardy, J. Can. J. Chem. 1997, 75, 773-789. b) Hellou, J.; Andersen, R.J.; Rafii, S.; Arnold, E . ; Clardy, J. Tetrahedron Lett. 1981,22,4173-4176. a) Hansen, J.R. Nat. Prod. Rep. 1996,13, 529-535. b) Hansen, J.R. Nat. Prod. Rep. 1992, 9, 481-489. c) Hansen, J.R. Nat. Prod. Rep. 1986, 3, 123-132.  65  d) Hansen, J.R. In Terpenoids and Steroids; Overton, K . H . , Ed.; Specialist Periodical Reports, V o l . 4 . The Chemical Society: London, 1974; pp. 171-182. Cordell, G . A . ; Prog. Phytochem. 1977, 4, 209-256. Crews, P.; Naylor, S. In Progress in the Chemistry of Organic Natural Products (Fortschr. Chem. Org. Naturstoffe); Herz, W . ; Grisebach, H . ; Kirby, G . W . ; Tamm, C , Ed.; N o . 48; Springer-Verlag, N e w Y o r k , 1985; pp. 203-269. Nozoe, S.; Morisaki, M . ; Tsuda, K . ; Iitaka, Y . ; Takahashi, N . ; Tamura, S.; Ishibashi, K . ; Shirasaka, M . J. Am. Chem. Soc. 1965, 87, 4968-4970. a) References 4 and 5 o f Crews, P.; Naylor, S. In Progress in the Chemistry of Organic Natural Products (Fortschr. Chem. Org. Naturstoffe); Herz, W . ; Grisebach, H . ; Kirby, G . W . ; Tamm, C , E d . ; N o . 48; Springer-Verlag, N e w Y o r k , 1985; pp. 203-269. b) Boeckman, Jr., R . K . ; B l u m , D . M . ; Arnold, E . V . ; Clardy, J. Tetrahedron Lett. 1979, 4609-4612. Nozoe, S.; Morisaki, M . ; Fukushima, K . ; Okuda, S. Tetrahedron Lett. 1968, 4457-4458. Rios, T.; Perez, C S . Chem. Commun. 1969, 214-215. Veloz, R.; Quijano, L . ; Calderon, J.S.; Rios, T. Chem. Commun.  1975, 191-192.  Naya, Y . ; Yoshihara, K . ; Iwashita, T.; Komura, H . ; Nakanishi, K . ; Hata, Y . Am. Chem. Soc. 1981,103, 7009-7011.  J.  Wang, Y . ; Dreyfuss, M . ; Ponelle, M . ; Oberer, L . ; Riezman, H . Tetrahedron 1998,54,6415-6426. Cueto, M . ; Jensen, P.R.; Fenical, W . Org. Lett. 2002, 4, 1583-1585. Cimino, G . ; De Stefano, S.; Minale, L . Tetrahedron  1971, 27, 4673-4379.  See for example: a) Cimino, G . ; De Stefano, S.; Minale, L . ; Fattorusso, E . Tetrahedron 1972, 28, 267-273; Correction: Tetrahedron 1972, 28, 2146. b) Cimino, G . ; D e Stefano, S.; Minale, L . ; Fattorusso, E . Tetrahedron 1972, 28, 333-341; Correction: Tetrahedron 1972, 28, 2146. c) Cafieri, F.; Fattorusso, E . ; Santacroce, C ; Minale, L . Tetrahedron 1972, 28, 1576-1583. d) Cimino, G.; De Stefano, S.; Minale, L . Tetrahedron 1972, 28, 5983-5991. e) Schmitz, F. J.; Chang, J.C. J. Nat. Prod. 1988, 51, 745-748.  66  f) Fontana, A . ; Albarella, L . ; Scognamiglio, G . ; U r i z , M . ; Cimino, G . J. Nat. Prod. 1996,59,869-872. g) Garrido, L . ; Zubia, E . ; Ortega, M . J . ; Salva, J. J. Nat. Prod. 1997, 60, 794-797. h) Holler, U . ; K o n i g , G . M . ; Wright, A . D . J. Nat. Prod. 1997, 60, 832-835. i) L i u , Y . ; Bae, B . H . ; A l a m , N . ; Hong, J.; Sim, C . J . ; Lee, C.-O.; Im, K . S . ; Jung, J . H . J. Nat. Prod. 2001, 64, 1301-1304. 25)  Kazlauskas, R.; Murphy, P.T.; Wells, R . J . Experientia  1980, 36, 814-815.  26)  Rochfort, S.J.; A t k i n , D . ; Hobbs, L . ; Capon, R . J . J. Nat. Prod. 1028.  27)  Butler, M . S . ; Capon, R . J . Aust. J. Chem. 1992, 45, 1705-1743.  28)  See for example: a) Albericci, M . ; Collart-Lempereur, M . ; Braekman, J . C ; Daloze, D . ; Tursch, B . ; Declercq, J.P.; Germain, G . ; van Meerssche, M . Tetrahedron Lett. 1979, 26872690. b) Albericci, M . ; Braekman, J . C ; Daloze, D . ; Tursch, B . Tetrahedron 1982, 38, 1881-1890. c) Ovenden, S.P.B.; Capon, R . J . J. Nat. Prod. 1999, 62, 214-218.  29)  a) E l Sayed, K . A . ; Hamann, M . T . ; Hashish, N . E . ; Shier, W . T . ; K e l l y , M . ; K h a n , A . A . J.Nat. Prod. 2001,64,522-524.  1996, 59, 1024-  b) Youssef, D . T . A . ; Yoshida, W . Y . ; K e l l y , M . ; Scheuer, P.J. J. Nat. Prod. 2001, 64, 1332-1335. 30)  a) Ohta, S.; U n o , M . ; Yoshimura, M . ; Hiraga, Y . ; Ikegami, S. Tetrahedron Lett. 1996,57,2265-2266. b) Y a n i , M . ; Ohta, S.; Ohta, E . ; ikegami, S. Tetrahedron 1998, 54, 15607-15612.  31)  Craig, K . S.; Williams, D . E . ; Hollander, I.; Frommer, E . ; M a l l o n , R.; Collins, K . ; Wojciechowicz, D . ; Tahir, A . ; van Soest, R.; Andersen, R . J. Tetrahedron Lett. 2002, 43, 4801-4804.  32)  a) Faulkner, D . J . Tetrahedron Lett. 1973, 3821-3822. b) Kazlauskas, R.; Murphy, P.T.; Quinn, R.J.; Wells, R . J . Tetrahedron Lett. 1976, 2635-2636. c) Escrig, V . ; Ubeda, A . ; Ferrandiz, M . L . ; Darias, J.; Sanchez, J . M . ; Alcaraz, M . J . ; Paya, M . J. Pharmacol. Exp. Then 1997, 282, 123-131.  33)  Rothberg, I.; Shubiak, P. Tetrahedron Lett. 1975, 769-772.  34)  a) Gonzalez Gonzalez, A . ; Lopez Rodriguez, M . ; San Martin Barrientos, A . J. Nat. Prod. 1983,46,256-261.  67  b) Barrow, C.J.; Blunt, J.W.; Munro, M . H . J . J. Nat. Prod. references therein.  1989, 52, 346-359 and  Kobayashi, J.; Ohizumi, Y . ; Nakamura, H . ; Hirata, Y . Tetrahedron Lett. 1986, 27,2113-2116. a) de Silva, E . D . ; Scheuer, P.J. Tetrahedron Lett. 1980, 21, 1611-1614. b) A m o o , V . E . ; D e Bernardo, S.; Weigele, M . Tetrahedron Lett. 1988, 29, 24012404. c) Soriente, A . ; Crispino, A . ; D e Rosa, M . ; D e Rosa, S.; Scettri, A . ; Scognamiglio, G . ; Villano, R.; Sodano, G . Eur. J. Org. Chem. 2000, 947-953. d) Potts, B . C . M . ; Faulkner, D.J.; de Carvalho, M . S . ; Jacobs, R . S . J. Am. Chem. Soc. 1992,774,5093-5100. e) Glaser, K . B ; Lock, Y . W . Biochem. Pharmacol.  1995,50,913-922.  See for example: a) Tsuda, M . ; Endo, T.; M i k a m i , Y . ; Fromont, J.; Kobayashi, J. J. Nat. Prod. 2002, 65, 1507-1508. b) Kobayashi, M . ; Okamoto, T.; Hayashi, K . ; Yokoyama, N . ; Sasaki, T.; Kitagawa, N . Chem. Pharm. Bull. 1994, 42, 265-270. c) Kobayashi, J.; Zeng, C . - M . , Ishibashi, M . ; Sasaki, T. J. Nat. Prod. 1993, 56, 436-439. d) Tsuda, M . ; Shigemori, H . ; Ishibashi, M . ; Sasaki, T.; Kobayashi, J.. J. Org. Chem. 1992, 57, 3503-3507. e) Cambie, R . C . ; Craw, P A . ; Bergquist, P.R.; Karuso, P. Nat. Prod. 1988, 57, 331-334. Gunasekera, S.P.; McCarthy, P.J.; Kelly-Borges, M . ; Lobkovsky, E . ; Clardy, J. J. Am. Chem. Soc. 1996, 775, 8759-8760. Buchanan, M . S . ; Edser, A . ; K i n g , G . ; Whitmore, J.; Quinn, R.J. 2001, 64, 300-303.  Nat. Prod.  Fattorusso, E . ; Magno, S.; Santacroce, C ; Sica, D . Tetrahedron 1972, 28, 59935997. Miyaoka, H . ; Nishijima, S.; Mitome, H . ; Yamada, Y . J. Nat. Prod. 2000, 63, 1369-1372. Walker, R.P.; Thompson, J.E.; Faulkner, D . J . 4979. Crews, P.; Bescansa, P.  Nat. Prod.  J. Org. Chem. 1980, 45, 4976-  1986, 49, 1041-1052.  Shin, J.; Seo, Y . ; Rho, J.-R.; Baek, E . ; K w o n , B . - M . ; Jeong, T.-S.; B o k , S.-H. J. Org. Chem. 1995, 60, 7582-7588.  68  Nakagwa, M . ; Hamamoto, Y . ; Ishihama, M . ; Hamasaki, S.; Endo, M . Tetrahedron Lett. 1987, 28, 431-434. a) Cimino, G . ; De Stefano, S.; Minale, L . Experientia 1973, 29, 934-936. b) Cimino, G . ; De Stefano, S.; D i Luccia, A . Experientia 1979, 35, 1277-1278. a) Jimenez, J.I.; Yoshida, W . Y . ; Scheuer, P.J.; Lobkovsky, E . ; Clardy, J.; K e l l y , M . J. Org. Chem. 2000, 65, 6837-6840. b) Jimenez, J.I.; Yoshida, W . Y . ; Scheuer, P.J.; Kelly, M . J. Nat. Prod. 2000, 63, 1388-1392. Chang, L . C . ; Otero-Quintero, S.; Nicholas, G . M . ; Bewley, C A . 2001,57,5731-5738.  Tetrahedron  Roy, M . C . ; Tanaka, J.; de Voogd, N . ; Higa, T. J. Nat. Prod. 2002, 65, 1838 1842. De Rosa, S.; Crispino, A . ; De Giulio, A . ; Iodice, C ; Tommonaro, G . ; Zavodnik, N . Tetrahedron 1998,54,6185-6190. For some other more recent examples see: a) Shin, J.; Rho, J.-R.; Seo, Y . ; Lee, H.-S.; Cho, K . W . ; Sim, C J . Tetrahedron Lett. 2001, 42, 3005-3007. b) D e Marino, S.; Iorizzi, M . ; Zollo, F.; Debitus, C ; Menou, J.-L.; Ospina, L . F . ; Alcaraz, M . J . ; Paya, M . J. Nat. Prod. 2000, 63, 322-326. c) Youssef, D . T . A . ; Y a m a k i , R . K . ; K e l l y , M . ; Scheuer, P.J. J. Nat. Prod. 2002, 65, 2-6. d) Charan, R . D . ; M c K e e , T . C . ; Boyd, M . R . J. Nat. Prod. 2001, 64, 661-66; Correction: J. Nat. Prod. 2003, 66, 155. e) Charan, R . D . ; M c K e e , T . C . ; B o y d , M . R . J. Nat. Prod. 2002, 65, 492-495. f) Stessman, C C ; Ebel, R.; Corvino, A . J . ; Crews, P. J. Nat. Prod. 2002, r55, 1183-1186. Campbell, N . A . Biology, 3 pp.603-604.  r d  Ed.; Benjamin Cummings: Menlo Park, C A , 1987;  Hooper, J . N . A . Sponguide. Guide to Sponge Collection and Identification. 2000 E d . Queensland Museum: South Brisbane, Qld., Australia. Downloaded from: http://www.qmuseum.qld.gov.au/organisation/sections/SessileMarineInvertebrate s/index.asp. De Laubenfels, M . W . The Sponges of the West Central Pacific. College: Corvallis, O R , 1954; pp. 9-12. Clancey, V . J .  Biochem. J. 1926, 20, 1186-1189.  69  Oregon State  Katzman, R . L . ; Lisowska, E . ; Jeanloz, R . W . Biochem. J.  1970,119, 17-19.  Some examples: a) Umeyama, A . ; Shoji, N . ; Arihara, S.; Ohizumi, Y . ; Kobayashi, J. Aust. J. Chem. 1989,42,459-462. b) Kobayashi, J.; Shinonaga, H . ; Shigemori, H . ; Sasaki, T. Chem. Pharm. Bull. 1993, 47,381-382. c) Guo, Y . W . ; Trivellone, E . Chin. Chem. Lett. 2000, 77, 327-330. a) Luibrand, R . T . ; Erdman, T.R.; Vollmer, J.J.; Scheuer, P.J.; Finer, J.; Clardy, J. Tetrahedron, 1979, 35, 609-612. b) Capon, R.J.; M a c L e o d , J . K . J. Org. Chem. 1987, 52, 5059-5060. c) Takizawa, P . A . ; Y u c e l , J . K . ; Veit, B . ; Faulkner, D . J . ; Deerinck, T.; Soto, G . ; Ellisman, M . ; Malhotra, V . Cell 1993,73,1079-1090. Nakamura, FL; Deng, S.; Kobayashi, J.; Ohizumi, Y . ; Hirata, Y . 1986, 42,4197-4201.  Tetrahedron,  a) Ishibashi, M . ; Ohizumi, Y . ; Cheng, J.; Nakamura, FL; Hirata, Y . ; Sasaki, T.; Kobayashi, J. J. Org. Chem. 1988, 53, 2855-2858. b) Kobayashi, J.; Murayama, T.; Ohizumi, Y . ; Ohta, T.; Nozoe, S.; Sasaki, T. J. Nat. Prod. 1989, 52, 1173-1176. c) Kobayashi, J.; Naitoh, K . ; Sasaki, T.; Shigemori, H . J. Org. Chem. 1992, 57, 5773-5776. Shen, Y . - C ; Chen, C . - Y . ; K u o , Y . - H . J. Nat. Prod.  2001, 64, 801-803.  Musman, M . ; Ohtani, L L ; Nagoaka, D . ; Tanaka, J.; Higa, T. J. Nat. Prod. 2001, 64, 350-352. a) Reference #47 o f Hansen, J.R. Nat. Prod. Rep. 1992, 9, 481-489. b) Manes, L . V . ; Naylor, S.; Crews, P.; Bakus, G . J . J. Org. Chem. 1985, 50, 284-286. c) Manes, L . V . ; Crews, P.; Kernan, M . R . ; Faulkner, D.J.; Fronczek, F . R . ; Gandour, R . D . J. Org. Chem. 1988, 53, 570-575. Madaio, A . ; Piccialli, V . ; Sica, D . Tetrahedron Lett. 1988, 29, 5999-6000. Madaio, A . ; Notaro, G . ; Piccialli, V . ; Sica, D . J. Nat. Prod.  1990, 53, 565-572.  a) Ohta, S.; Uno, M . ; Tokumasu, M . ; Hiraga, Y . ; Ikegami, S. Tetrahedron Lett. 1996,37,7765-7766. b) Tokumasu, M . ; Ando, H . ; Hiraga, Y . ; Kojima, S.; Ohkata, K . J. Chem. Soc., Perkin Trans. 1 1999, 489-496. c) H i o k i , H . ; O o i , H . ; Hamano, M . ; M i m u r a , Y . ; Yoshio, S.; Kodama, M . ; Ohta, S.; Yanai, M . ; Ikegami, S. Tetrahedron 2001, 57, 1235-1246.  70  Pouchus, Y . F . ; Verbist, J.F.; Biard, J.F.; Boukef, K . J. Nat. Prod. 189.  1988, 51, 188-  a) Guo, Y . - W . ; Trivellone, E . J. Asian Nat. Prod. Res. 2000, 2, 251-256. b) Guo, Y . - W . ; Gavagnin, M . ; M o l l o , E . ; Cimino, G . ; Hamdy, N . A . ; Fakhr, I.; Pansini, M . Nat. Prod. Lett. 1997,10, 143-150. Adjei, A . A . JNCI 2001, 93, 1062-1074, and references therein. Some recent examples include: a) Rawat, D.S.; Gibbs, R A . Org. Lett. 2002, 4, 3027-3030. b) Lee, S.-FL; K i m , H . - K . ; Seo, J . - M . , Kang, H . - M . ; K i m , J.H.; Son, K . - H . ; Lee, PL; K w o n , B . - M . ; Shin, J.; Seo, Y . J. Org. Chem. 2002, 67, 7670-7675. Whyte, D . B . ; Kirschmeier, P.; Hockenberry, T . N . ; Nunez-Oliva, I.; James, L . ; Catino, J.J.; Bishop, W . R . ; Pai, J . K . J. Biol. Chem. 1997, 272, 14459-14464. K i m , E . ; Ambroziak, P.; Otto, J.C.; Taylor, B . ; Ashby, M . ; Shannon, K . ; Casey, P.J.; Young, S.G.; Casey, P.J. J. Biol. Chem. 1999, 274, 8383-8390. a) Boyartchuk, V . L ; Ashby, M . N . ; Rine, J. Science 1997, 275, 1796-1800. b) Fujimura-Kamada, K . ; Nouvet, F.J.; Michaelis, S. J. Cell Biol. 1997,136, 271-285. c) Tarn, A . ; Nouvet, F.J.; Fujimura-Kamada, K . ; Slunt, PL; Sisodia, S.S.; Michaelis, S. J. Cell Biol. 1998,142, 635-649. Otto, J.C.; K i m , E . ; Young, S.G.; Casey, P.J. J. Biol. Chem. 1999, 274, 83798382. Hollander, I.; Frommer, E . ; M a l l o n , R . Anal. Biochem. 2000, 286, 129-137. a) Snider, B . B . ; He, F . Tetrahedron Lett. 1997, 38, 5453-5454. b) Takagi, R.; Sasaoka, A . ; Kojima, S.; Ohkata, K . Chem. Comm. 1997, 18871888. c) Takagi, R.; Sasaoka, A . ; Nishitani, H . ; Kojima, S.; Hiraga, Y . ; Ohkata, K . J. Chem. Soc, Perkin Trans. 1 1998, 925-934. Some examples o f totally synthetic inhibitors: a) Schlitzer, M . ; Winer-Vann, A . ; Casey, P.J. Bioorg. Med. Chem. Lett. 2001,11, 425-427. b) Dolence, E . K . ; Dolence, J . M . ; Poulter, C D . J. Comb. Chem. 2000, 2, 522-536. Dewick, P . M . Medicinal Natural Products: A Biosynthetic Approach. John W i l e y and Sons: West Sussex, U K , 1997, chapter 5.  71  79)  D e Rosa, S.; Giulio, A . ; Crispino, A . ; Iodice, C . ; Tommonaro, G . Nat. Prod. Lett. 1997,10, 261-21A.  80)  a) De Rosa, S.; Crispino, A . ; Giulio, A . ; Iodice, C ; Amodeo, P.; Tancredi, T. J. Nat. Prod. 1999, 62, 1316-1318. b) D e Rosa, S.; Crispino, A . ; Giulio, A . ; Iodice, C ; Benrezzouk, R.; Terencio, M . C ; Ferrandiz, M . L . ; Alcaraz, M . J.; Paya, M . J. Nat. Prod. 1998, 61, 931-935. c) D e Rosa, S.; Puliti, R.; Crispino, A . ; D e Guilio, A . ; D e Sena, C ; Iodice, C ; Mattia, C A . Tetrahedron 1995,57,10731-10736. d) D e Rosa, S.; Crispino, A . ; Giulio, A . ; Iodice, C ; Pronzato, R.; Zavodnik, N . J. Nat. Prod. 1995,55,1776-1780. e) De Rosa, S.; De Stefano, S.; Zavodnik, N . J. Org. Chem. 1988, 53, 50205023.  81)  Bourger-Kondracki, M . L . ; Debitus, C ; Guyot, M . J. Chem. Res. (S) 1996, 192193.  2.7: Experimental Section 2.7.1: General Information A l l reagents and solvents (except for N M R experiments) were purchased from either Fisher Scientific or Sigma-Aldrich and were used without further purification, except H P L C solvents which were filtered through a 0.45 um filter (Osmonics, Inc.) prior to use. The N M R solvent," 100 % grade" C e D (deuterobenzene or benzene-d6), was 6  purchased from Cambridge Isotopes Laboratories. Proton chemical shifts were referenced to the residual solvent peak at 8 7.15 ppm and carbon chemical shifts to 8 128.0 ppm. N M R data were acquired with the following spectrometers:  1 3  C data was acquired with a  Bruker A M - 4 0 0 spectrometer (direct detection 5mm probe); ' H spectra and 2 D data were either acquired with a Bruker A M X - 5 0 0 or a Bruker A V - 4 0 0 spectrometer (both with inverse detection 5 m m probes). 2 D N M R experiments performed were C O S Y - 4 5 ( ' H - ' H correlations), H M Q C or H S Q C ( C - ' H single bond correlations), and H M B C ( C - ' H 1 3  1 3  multiple-bond correlations). Mass spectral data (high and low resolution F A B and D C I ) were acquired by a Kratos Concept II H Q Mass Spectrometer with the assistance o f the  72  U B C Mass Spectrometry Centre staff. For negative F A B M S , the matrix 3-nitrobenzoic acid/CHCl3 was used, and for D C I M S , the carrier gas was a mixture o f CH4 and NH3. The S e p h a d e x ™ L H - 2 0 size-exclusion chromatography gel (Sigma-Aldrich; bead size 25-100 jam) was used i n a glass column approximately 110 cm long by 40 cm wide. Reverse-phase Ci8 silica Sep-Paks were obtained from Waters, Inc., while normal phase 230-400 mesh silica gel was acquired from Silicycle (Quebec City, P Q ) . For H P L C we utilized a Waters 996 Photodiode Array detector (600E Series pump/system controller; M i l l e n n i u m ™ 2010 software), or a Waters 2487 dual channel detector/system controller (Waters Series 515 pump; chart recorder, 0.25 cm/min). W i t h either H P L C system, the wavelength monitored was 204 nm, and the flow rate was 2.0 m L / m i n through a reversephase 5 p. Ci8 Inertsil column from Chromatography Sciences (Montreal, P Q ) . When a gradient H P L C system was used, the solvents were sparged with helium (Praxair, Inc., Vancouver, B C ) as well. Thin-layer chromatography ( T L C ) plates were Whatman M K C 1 8 F (reverse-phase) and Kieselgel 6OF254 (normal phase). T L C visualization was observed by one o f two methods: at 254 nm (short-wavelength U V ) , or by spraying a solution o f 90% EtOH/10%> concentrated H2SO4 plus a small amount o f vanillin (coloured spots appear upon heating).  2.7.2: Isolation of Rhopaloic Acids B(35)-G(40) The source sponge RJA96-141, later identified as a Hippospongia sp., was harvested by hand using S C U B A on the inner reef at Barangcadi Island, near Makassar, on the island o f Sulawesi i n Indonesia. U p o n collection, the sponge was stored in ethanol and transported to Vancouver. The sponge was identified by Dr. Rob van Soest o f the Zoologisch Museum at the University o f Amsterdam in the Netherlands where a voucher  73  specimen is kept (ZMA16774). Sixty grams o f the freshly collected sponge were repeatedly extracted with ethanol. The extracts were combined and concentrated in vacuo, followed b y suspension i n water. This suspension was partitioned between water (20 m L ) and E t O A c (4x7 m L ) . The E t O A c fraction was sequentially passed through Sephadex L H - 2 0 chromatography (eluent: 80% M e O H / D C M ) and reverse-phase flash gradient column chromatography (eluent: 100%> H 0 to 100%) M e O H ; lOOmL for each 2  fraction). A portion o f each o f the appropriate fractions (in the 80%-100% M e O H range) were purified using reverse-phase H P L C (eluent: 65% M e C N / 0.05%> T F A i n H 0 ) to 2  yield the known norsesterterpene rhopaloic acid A (122),  30a  isolated as a clear oil  (105mg), and the novel sesterterpenes, barangcadoic acid A (130), isolated as a clear o i l (35 mg), and rhopaloic acid G (40), also isolated as a clear oil (0.1 mg). A l s o isolated was a fraction containing three compounds. This fraction was purified further by reversephase H P L C (eluent: 58% M e C N / 0 . 0 5 % T F A / H 0 ) to yield the inseparable mixture o f 2  rhopaloic acids D / E (37/38) (0.2 mg) and pure rhopaloic acid F (39) (0.1 mg), all isolated as clear oils. Additional peaks were present i n the H P L C trace for 122, so another portion was subjected to reverse-phase H P L C (eluent: 75% M e C N / (0.05%TFA/ H 0 ) ) , to yield 2  the known norsesterterpenes rhopaloic acids B (35) and C (36),  30b  isolated as clear oils i n  0.1 mg and 0.3 m g yields, respectively. Compounds 122 and 130 were isolated by m y colleague and w i l l not be discussed. Bioassays were performed by Wyeth Research.  Rhopaloic A c i d B (35): Clear oil; *H and  1 3  75  C N M R data, see Table 2.1; F A B M S : m/z  [ M - H ] - 373 (30), 306 (15), 168 (25), 153 (100), 122 (10); H R F A B M S : m/z [M-H ]~ +  +  373.2746, (calculated for [ M - H ] \ C24H37O3, 373.2749). +  74  Rhopaloic A c i d C (36): Clear o i l ; ' H and  C N M R data, see Table 2.1; F A B M S : m/z  1 3  [M-H+]" 371 (4), 305 (25), 199 (12), 168 (40), 153 (100), 122 (10); H R F A B M S : m/z [ M H ] " 371.2593, (calculated for [ M - H ] " , C24H35O3, 371.2600). +  +  Rhopaloic Acids D / E (37/38): Clear o i l ; *H and  l 3  C N M R data, see Tables 2.2 and 2.3;  D C I M S : m/z [ M + H ] 391 (100), [ M - H 0 ] 374 (45); H R D C I M S : m/z [ M + H ] 391.2852, +  +  +  2  (calculated for [ M + H ] , C24H39O4, 391.2855), [ M - H 0 ] 374.2814, (calculated for [ M +  +  2  H 0 ] , C H 8 0 3 , 374.2807). +  2  24  3  Rhopaloic A c i d F (39): Clear o i l ; ' H and  1 3  C N M R data, see Table 2.4; D C I M S : m/z  [ M + H ] 391 (100), 374 (65); H R D C I M S : m/z [ M + H ] 391.2839, (calculated for [ M + H ] , +  +  +  C24H39O4, 391.2829). Rhopaloic A c i d G (40): Clear o i l ; *H and  1 3  C N M R data, see Table 2.5; D C I M S : m/z  [ M + N H ] 426 (65), [ M + H ] 408 (30), [ M - H 0 ] 391 (72), [ M - 2 H 0 ] 374 (30), 232 +  +  +  4  +  2  2  (36), 230 (45), [ M - C H 0 ] 214 (100), 212 (95), 195 (60), 177 (40), 168 (36), 151 (36), +  1 3  2 2  109 (36), 95 (28). H R D C I M S : m/z [ M + N H ] 426.3220, (calculated for [ M + N H ] , +  +  4  4  C H 0 N , 426.3220), [ M + H ] 408.2878, (calculated for [ M + H ] , C H O , 408.2881), +  2 4  4 4  +  5  2 4  4 0  5  [ M - H 0 ] 391.2862, (calculated for [ M - H 0 ] , C 4 H 0 4 , 391.2875), [ M - 2 H 0 ] , +  +  2  +  2  374.2818, (calculated for [ M - 2 H 0 ] , C +  2  2 4  H  2  0 , 374.2815), [ M - C i H 0 ] , 214.1198  +  2 2  8  2  +  3 8  3  (calculated for [ M - C i H 0 ] , C n H i 0 , 214.1191). 3  39  4  75  3  2 2  2.8: 2D N M R Spectra for Rhopaloic Acids D(37)-G(40)  i  (ppm)  1  1  7.00  1  1  1  1  6.00  1  1  1  1  1  1  1  4.00  5.00  i  1  1  1  1  3.00  1  1  1  1  1  1  2.00  Figure 2.16: COSY-45 of Rhopaloic Acids D / E (37/38) in C D at 500 M H z . 6  6  76  1  r  1.00  77  (ppm)  ..  (ppm)  7  M  1  6  m  Mill]  5  M  AA/V  LA  4  M  3  M  2  _  0 0  ,_  Figure 2.18: H M B C of Rhopaloic Acids D/E (37/38) in C D at 500 M H z . 6  6  20  78  0 0  79  (ppm)  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  Figure 2.20: HSQC of Rhopaloic Acid F (39) in C D at 400 MHz. 6  6  20  80  1.00  (ppm)  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 2.21: H M B C of Rhopaloic Acid F (39) in C D at 400 M H z . 6  6  20  81  0.00  82  (ppm)  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  Figure 2.23: H M Q C of Rhopaloic Acid G (40) in C D at 500 MHz. 6  6  83  1.00  (ppm)  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 2.24: H M B C of Rhopaloic Acid G (40) in C D at 500 MHz. 6  6  84  3.1: Introduction to Sesquiterpene Lactones In the introductory chapter o f this thesis, mention was made o f the antimalarial sesquiterpene lactone endoperoxide artemisinin (16), originally isolated in 1972 from Artemisia annua, (quinghao or wormwood), but known i n traditional Chinese medicine since 168 B . C . It is now known that the activity o f this extract is due to the endoperoxide 1  functionality that is present i n 16 and a wide array o f similar metabolites.  lb  W h i l e the  mechanism o f action o f 16 is not definitely known, many theories have been proposed, most o f which involve formation o f a radical species that is responsible for the bioactivity.  10  Irrespective o f this knowledge, 16 and two closely related compounds are  now used for the treatment o f malaria with the full support o f the W o r l d Health Organization.  13  16  O  Artemisinin (16), a member o f the sesquiterpene class o f isoprenoids, represents just one o f numerous diverse structures present i n this large class. This well-studied class 2  2 3  2 3  2 3  has yielded representatives from vertebrates, sponges, ' soft coral, ' microorganisms, ' algae, ' and various kinds o f plants.  There are greater than one hundred different  sesquiterpene skeletons and many thousands o f structures presently known, with plants representing the largest source. ' One o f the most important classes o f sesquiterpenes is 2 4  that o f the sesquiterpene lactones, with at least 3000 compounds presently known.  4  Members from this class o f compounds are almost always isolated from the higher plant family, Asteraceae, formerly known as the Compositae. M a n y o f these plants are also 5  85  those that have been frequently utilized by herbal medicine, both traditional and current.  6  The sesquiterpene lactones produced b y these plants fall into a number o f skeletal classes; the carbon skeletons o f some examples (not yet lactonized) are shown in Scheme 2 1 2,4,7,8 ^  § m  -  g  ^ 1 ^ ^ depicts, the different skeletal classes o f sesquiterpenes arise  from various cyclizations o f farnesyl pyrophosphate i n any o f three forms: E , E (28); E , Z (200); and Z , E (201). The majority o f these compounds arise from cyclization o f 28 to 7  form a cyclized cation that then is quenched, oxidized, and cyclized to form the germacrane skeleton, which be further cyclized and modified to give other skeletons. '  4 7  The E,Z-farnesyl pyrophosphate (200) can undergo similar reactions to give other skeleons such as the heliangane skeleton and the cw-cadinane skeleton, (on which 16 is based), while Z,E-farnesyl pyrophosphate (201) gives rise to the melampanes. M a n y 2  47  8  other skeletal types are known, including those that are highly rearranged. ' ' '  heliangane  melapane  Scheme 3.1: Examples o f Sesquiterpene Skeletons.  86  3.2: Biological Activity of Sesquiterpene Lactones 3.2.1: Known Examples of Biological Activity The original impetus for interest in sesquiterpene lactones was based on a number o f plants from the Asteraceae family that had been used as folk medicines for many years. For example, in the mid-nineteenth century a common folk remedy was 'acide helenique'. In 1963 the active ingredient o f this remedy was determined to be the 4  pseudoguaiane antispetic helenalin (202).  9  Until the discovery in 1968 that the  elemanolide vernolepin (203) was an anti-tumour agent,  10  most research with  sesquiterpenes revolved around chemotaxonomy or the isolation o f the active components o f folk remedies. The National Cancer Institute (NCI) i n the United States 4  undertook an intensive search during the 1970's for further sesquiterpene lactone antitumour agents. M a n y were found and many more were semi-synthesized, but this class o f compounds did not have a useable therapeutic margin so none made it into clinical testing  4  Kupchan et al discovered i n 1971 that the cytotoxicity was due to the presence  o f an a-methylene-y-lactone. The presence o f a conjugated ester, a cyclopentenone, or an a-methylene-8-lactone i n addition to the a-methylene-y-lactone already present increased the cytotoxicity.  11  These cc,p-unsaturated carbonyl systems i n addition to epoxides,  which can also be present, act as potent alkylating agents via a Michael-type reaction or 1,2 addition with a nucleophile, respectively. In biological systems, it is usually a cysteine residue i n a protein that reacts with its thiol moiety in this manner. ' ' 1  87  1 2  Some o f the  'antitumour' sesquiterpene lactones researched during this era include helenalin (202), vernolepin (203), parthenolide (204), and parthenin (205), ' " each having a different 4 10  12  carbon skeleton, providing evidence o f the bioactivity o f the a-methylene-y-lactone that is present. While some later studies " 13  14  suggested that the antitumour bioactivity o f  sesquiterpene lactones was a result o f inhibition o f D N A biosynthesis, nearly every other study conducted prior and since have suggested that a-methylene-y-lactones alkylate proteins, generally through thiol moieties present i n the protein. ' 5  6,15  "  20  The apparent failure o f sesquiterpene lactones as antitumour agents led researchers to explore other biological activities o f these compounds. Most sesquiterpene lactones are active against gram-positive bacteria such as Bacillus  subtilis 22  21  and Staphylococcus aureus,  while a lesser number are antifungal in activity.  Additionally, parthenoloide (204) and costunolide (206), among others, have been shown to have anti-tuberculosis activity. Helenalin (202) has been shown to be an analgesic 23  and an antiinflamatory agent.  Another example o f a possible ecological role was the  discovery that many a-methylene-y-lactones cause contact dermatitis, a possible 4  deterrent for herbivorous animals, though a bitter taste may be more o f a deterrent. It  207  208  209  Scheme 3.2: Formation o f Adduct 209, believed to be the caustive agent o f contact dermatitis.  88  24  has, been suggested the 2+2 photoadduct 209formed from the a-mefhylene-ybutyrolactone portion (as 207 i n the scheme) o f the molecule with the D N A base thymine (208) i n the presence o f sunlight, is the actual causative agent o f this malady, see Scheme 3.2.  26  Further ecological examples include the discovery that two eremophilanolides, 27  compounds 210 and 211, isolated from Senecio miser, are insect antifeedants,  and that  1 lp,13-dihydroparthenolide (212) causes 'suicidal germination' o f the parasitic root plant Striga  hermonthica.  28  Additional non-antitumour examples o f biochemical activity o f sesquiterpene lactones have also been discovered. One example is the discovery that sesquiterpene lactones from the bay leaf Laurus nobilis, such as costunolide (206), dehydrocostus lactone (213), reynosin (214) and the eudesmanolides 215 and 216, among others, are inhibitors o f alcohol absorption in rats. The results o f a structure-activity relationship ( S A R ) study indicated that either a y-butyrolactone or y-butyrolactol functionality i n conjunction with an a-methylene or an a-methyl group was essential for the observed activity.  29  In another investigation, Merfort et al tested the effect on N F - K B inhibition o f  twenty-eight sesquiterpene lactones including 202, parthenolide (204), cumambrin A  89  202  204  217  O  (217), goiazensolide (218), the 4,5-dihydrogermacranolide 219, and the melampolide 220.  These compounds are representatives o f the most potent compounds tested and are  members o f six different classes o f sesquiterpene lactones. Furthermore, most o f these compounds contain an a-methylene-y-lactone plus at least one other a,(3 or a,p,y,8unsaturated carbonyl. W h i l e currently best-known for its role i n the inflammatory 19  30  response,  NF-KB  may also have a connection with Alzheimer's Disease ( A D ) .  31  '  Reduction o f the a-methylene-y-lactone moiety does not always negatively affect the bioactivity o f the sesquiterpene lactone. For example, 10-ep/-8-deoxycumambrin B (221) was reduced with NaBFL; to give 1 ipH-13-dihydro-10-epz'-8-deoxycumambrin B (222) . W h i l e both compounds exhibited aromatase inhibition, an important activity for breast cancer treatment, the reduction o f the exo-methylene increased inhibition by a factor o f three. While this is not a great increase i n activity, the removal o f this double bond decreased the cytotoxicity o f 222 without a concurrent removal o f the desired activity.  32  Sesquiterpene lactones w i l l continue to interest biochemists as science  continues to unravel the fascinating biochemistry behind the a-methylene-y-lactone.  90  221  °  222  °  3.2.2: G2 Cell Cycle Checkpoint/Antimitotic Activity of Ambrosia artemisiifolia The National Cancer Institute (NCI) i n the United States maintains a large repository o f natural product extracts from all over the world. There are greater than 100,000 extracts present i n this repository. Our lab, i n conjunction with Dr. M i c h e l Roberge o f the U B C Department o f Biochemistry, has received permission to investigate the extracts present i n the open portion o f the N C I ' s repository. In the process o f screening for novel G 2 cell cycle checkpoint inhibitors the methanolic extract N35791, 33  from the common ragweed Ambrosia artemisiifolia  L . , was discovered to be one o f most  potent extracts tested to date by the Roberge lab. It was decided that further investigation was necessary and through bioassay-guided fractionation the psilostachyins A (42), B (43), C (44), paulitin (45), and isopaulitin (46) were isolated. Only psilostachyin A (42) and C (44) were active, so four derivatives 223-226 were semi-synthesized in an effort to determine the necessary structural features for biological activity. W h i l e 42 and 44 were determined to be inhibitors o f the G 2 cell cycle checkpoint, they also caused the cells to arrest i n mitosis, and are antimitotic agents also.  34  The isolation, structural elucidation  and biology o f 42-46 and 223-226 are discussed, i n addition to the novel inactive sesquiterpene diacid monoester 41, in Section 3.3.  91  3.2.3: TG-3 Antibody Activity of Vernonia baldwinii In the course o f screening the N C I ' s open repository for potential antimitotic compounds the methanolic extract N61397, from the western ironweed Vernonia 35  baldwinii Torr., gave a positive result. During the course o f follow-up screening, it was discovered that a modified (shorter time o f development) G2-checkpoint bioassay was much better for monitoring this particular biological activity. After bioassay-guided fractionation was completed, it was determined that venonataloide (47) and a related compound 227, (differing i n the ester at C-8), were causing an unexpected response o f the T G - 3 antibody used as a reporter i n both assays. '  When the methyl-acrylate ester  at C-8 o f 47 is reduced to an isobutyrate ester, the resulting compound, 228, is no longer active. (The acetate on the hydroxy methyl is also lost, but it is thought this does not effect bioactivity.) Four other compounds 229-232 were also isolated but were too cytotoxic to observe any additional activity. Additional biological studies are ongoing which are examining the use o f vernonataloide (47) as a tool to study Alzheimer's  92  disease. The isolation, structural elucidation and biology o f 47 and 227-232 are discussed i n Section 3.4.  3.3: Sesquiterpene Lactones from Ambrosia artemisiifolia 3.3.1: The Cell Cycle and G2 Checkpoint Inhibitors Normal cells respond to D N A damage b y activating checkpoints that temporarily stop the cell cycle to allow time for D N A repair. The first o f these checkpoints, the G l checkpoint, allows for the repair o f D N A before it is replicated i n S phase, while the second checkpoint, the G 2 checkpoint, allows for D N A repair before chromosomes are segregated i n mitosis. D N A repair at the G 2 checkpoint prevents the propagation o f genetic abnormalities to daughter cells. In approximately 50% o f all solid tumour cells, there is a mutated p53 tumour suppressor gene that does not activate the G l checkpoint in response to D N A damage. The G 2 checkpoint, even though it is usually weaker than i n normal cells, still halts the cell cycle and allows the cells time to repair their D N A after damage and then continue into mitosis. Inhibition o f the G 2 checkpoint should have no  93  effect on normal or cancer cells unless done i n conjunction with a DNA-damaging agent. In this latter case, the normal cells still have the G l checkpoint for D N A repair, while p53- cancerous cells now have no checkpoints and are thus forced into a premature and lethal mitosis. The G 2 checkpoint i n normal cells is usually strong and thus requires a higher dose o f checkpoint inhibitor to be inactivated. It has therefore been proposed that G 2 checkpoint inhibitors should enhance the effectiveness o f DNA-damaging agents i n the treatment o f p53- tumours. ' 33  36  (See Figures 3.1 and 3.2) W h e n an array o f paired cell  lines, differing only i n the presence o f an active p53 gene, were tested, the presence o f G2 checkpoint inhibitors induced a greater sensitivity to D N A damage in those without a p53 suppressor gene. This is a reasonable approximation to the concept o f a 'magic 33  bullet'—a drug that attacks only cancerous or diseased cells. Until recently, only a few G 2 checkpoint inhibitors were known, and these, caffeine (233) and its analogues such as pentoxifylline (234), i n addition to 37  38  staurosporine (235) and a related analogue, U C N 0 1 (236), also known as 739  hydroxystaurosporine,  40  were found serendipitously. Toxicity and non-selectivity o f 33  37 38  233 and 234 have limited their ability to be administered therapeutically in vivo. ' U C N 0 1 (236), currently i n clinical trials, is a non-selective kinase inhibitor that has been shown in vitro to nullify G 2 arrest either induced chemically or b y ionizing radiation  4 0  Based on these examples, we decided to look for additional G 2 checkpoint inhibitors that may be more selective than those currently known. To this end, our collaborator Dr. M i c h e l Roberge has screened our marine invertebrate collection, a portion o f the N C I ' s open repository o f natural product extracts, and the bacterial collection o f another collaborator.  94  M phase  Interphase  Prophase  Metaphase  — •  DNA Damage Checkpoint  Anaphase  - •  Division  Antimitotic Agents  Cell Cycle Checkpoint Inhibitors  Figure 3.1: Overview o f the Cell Cycle. (Diagram by M . Roberge, used by permission.)  p53+  Gl  S  G2  A  Gl  Cell Death  A  G 2 Arrest  G l Arrest  p53-  M  S  M  G2  Cell Death  A NoGl Checkpoint G2 Arrest  Figure 3.2: Pictorial Representation o f the L o g i c Behind the G 2 Checkpoint Bioassay. Larger Font = M o r e Cells (Diagram by M . Roberge, used by permission.)  95  Dr. Roberge has developed a cell-based assay for G2 checkpoint inhibition  that  relies on the T G - 3 antibody for colorimetric detection. This monoclonal antibody recognizes a phosphoepitope (recognition site) o f the nuclear protein nucleolin present only i n mitotic cells. T G 3 immunofluorescence is greater than fifty times more intense in the presence o f mitotic cells than with non-mitotic cells. Therefore, the T G - 3 antibody can be used in an E L I S A (Enzyme-Linked Immunosorbent Assay) to recognize cells i n mitosis.  41  In Roberge's G 2 checkpoint assay, cycling p53- M C F - 7 human breast cancer cells are seeded i n 96 well plates and grown for one day. The cells are then dosed with yirradiation to induce D N A damage. After sixteen hours the majority o f cells have arrested in G 2 and nocodazole (237), an antimitotic agent, plus the natural product extracts are added, followed by incubation for an additional eight hours. After this twenty-four hour time period, the number o f mitotic cells is determined using the T G - 3 based E L I S A . If a G 2 checkpoint inhibitor is present i n an extract, the cells w i l l be released from G 2 arrest and enter mitosis where 237 w i l l arrest them and a strong T G - 3 response w i l l be observed. If no G 2 checkpoint inhibitor is present, the cells remain i n G 2 arrest and no T G - 3 response is observed. Our lab has had reasonable success i n finding G 2 checkpoint inhibitors using this bioassay. U t i l i z i n g this bioassay, members o f the Andersen lab have isolated the novel alkaloids granulatimide (238) and isogranulatimide (239) from the tunicate Didemnum granulatum '  32 42  and re-isolated staurosporine (235) from a marine bacteria.  33  Furthermore, the known compound debromohymenialdisine (240) and several related compounds from the sponge Stylissa  flabelliformis  43  96  have been isolated, as have several  ent-kaurene diterpenoids including 13-hydroxy-15-oxozoapatlin (241) from the African plant Parinari curatellifolia  44  curatellifolia  44  and the related ring-opened acid 242 from both P.  and the South American plant Coccoloba  240  w  241  acuminata.  45  242  Each o f these compounds has been shown to be useful as a biochemical probe for studying the cellular processes involved i n the G 2 checkpoint. The granulatimides (238239) and debromohymenialdisine (240) have been found to inhibit some checkpoint 34  43  kinases but not others. Terpenoids 241 and 242 release cells from G 2 arrest and then arrest the cells in mitosis. To the best o f our knowledge, these are the first compounds i n a  conjunction with the psilostachyins—see below—known to have this dual mode o f action. Examination o f the arrested mitotic cells by fluorescence microscopy suggested that these terpenes interfere with attachment o f the chromosomes to microtubules  34  rather  than by disrupting tubulin polymerization as previously known antimitotics are known to do.  Microtubules are polymeric proteins made up o f a-tubulin and p-tubulin monomers.  97  They have a variety o f functions in a cell, mostly those concerned with cellular structure. During mitosis, however, they grow from a location called the kinetochore—present at each 'end' o f the cell—towards the centre where the chromosomes are aligned. The microtubules attach to each chromosome and pull it apart thereby enabling the formation of two sets o f daughter chromosomes, one for each o f the two new cells present at the end o f mitosis. M a n y currently known and medically utilized antimitotics are known to affect the polymerization o f tubulin, by hyperstabilizing or destabilizing the formation o f this polymeric protein. If they are hyperstabilized, the ends o f the microtubules grow past each other and never meet, while i f they are destabilized, the ends never meet. Either way, mitosis is 'jammed' and the cell never finishes dividing. Additional antimitotic modes o f action are desirable as some cancers are now showing resistance to previously known antimitotic agents.  46  The carboxylic acid functionality i n compound 240 has been  biotinylated and the resulting adduct is currently being used to determine the cellular targets o f this class o f compounds  4 5  3.3.2: Isolation and Structure Elucidation of Sesquiterpenes from A artemisiifoUa 3.3.2.1: Introduction to Metabolites from A artemisiifoUa The common or short ragweed, (Figure 3.3), Ambrosia artemisiifoUa L . , o f the Asteraceae family, is a summer annual that grows from 30 cm to about 2 metres i n height. This plant has small flowers at the base o f spiky stamens with fernlike leaves that alternate on the stalk often with the lowest ones opposite each other. Additionally, the plant is quite hairy, which causes it to feel rough to the touch  4 7  In North America there  are nine species o f ragweed (including the western ragweed A. psilostachya giant ragweed A. trifida L . )  4 7 b  D C and the  that account for about 90% o f the pollen that causes hay  98  fever in humans.  A. artemisiifolia  is a weed o f cultivated areas, though in areas with  frequent mowing, such as golf courses, it does not survive and is therefore rarely found.  473  Figure 3.3: Ambrosia artemisiifolia growing i n Michigan. (Photo by W . Craig, used by permission.) Chemical studies on A. artemisifolia have been ongoing since the mid-1950's though it was not until 1961 that the first compound was reported, the previously known coronopilin (243), isolated from a collection o f this plant i n F l o r i d a . next decade, cumanin (244b),  50  49  Throughout the  1 lp,13-dihydroparthenolide (212), isabelin (245), 51  psilostachyin A (42), peruvin (246) were re-isolated from A artemisiifolia 51  in addition  52  to the novel compounds dihydrocumanin (247) and artemisiifolin (248). 50  52  52  In the 1970's  ambrosic acid (249) originally synthesized from 246 was isolated as a natural product 53  from a Japanese collection of A. artemisiifolia.  54  A l s o in the 1970's, two 3-  oxopseudoguaian-6,12-olides 250 and 251 were isolated in addition to the known compounds psilostachyin A (42) and psilostachyin C (44) from collections i n Yugoslavia.  5  In 1987, another Yugoslavian collection yielded the novel compound 4-  99  oxo-3,4-seco-ambrosan-6,12-olide-3-oic acid (252), in addition to cumanin diacetate  (253) and several other sesquiterpenes already known from A. artemisiifolia.  More  recent studies of A. artemisiifolia have yielded 246 plus two 1 lccH.,13- dihydrodamsin derivatives 254 and 255 from a Czechoslovakian collection, while a Brazilian collection 57  58  yielded the previously known compounds paulitin (45) and isopaulitin (46).  Over the  course o f this time span, taxonomists have disagreed about the difference between A. artemisiifolia  and another species o f ragweed A. elatior L . , though it seems a resolution  has been reached as they are said to be the same species. Some o f the metabolites isolated from A. elatior include the known sesquiterpenes 42-44 and 4p,10aalloaromadendrane (256), and a known flavonoid from two Argentine collections.  100  66  3.3.2.2: Isolation and Structure Elucidation 3.3.2.2.1: Isolation Procedure  Two approximately two-gram vials o f the methanolic extract N35791 were obtained from the N C I . The extract i n the first vial was suspended i n water and partitioned between hexanes/water, dichloromethane (DCM)Avater, and EtOAc/water, with the G 2 checkpoint inhibitory activity going into the D C M layer. The resulting gummy substance was fractionated sequentially v i a normal phase silica flash chromatography ( D C M to E t O A c followed by polar solvent washes o f M e O H and acetone) and reverse-phase H P L C (25% M e C N / H 0 ) to yield the G 2 active compounds 2  psilostachyins A (42) and C (44). Almost pure psilostachyin B (43) was isolated from the normal phase silica gel chromatography and purified by reverse-phase H P L C (20% M e C N / H 2 0 ) . The polar solvent washes o f the silica flash chromatography were combined and subjected to reverse-phase Sep-Pak (Water to M e O H ) followed by repeated reverse-phase H P L C (25% M e C N / H 0 ; 10% M e C N / H Q ) . This isolation 2  101  2  procedure yielded several diacids o f which only the structure o f the novel inactive diacid monomethyl ester 41 was determined. W e wanted to obtain additional 42-44 for further biological testing and 41 for stereochemical studies, thus the second two-gram vial was examined. The extract i n this vial was partitioned between D C M / w a t e r only, followed by normal phase flash chromatography as before, though unfortunately no additional 41 was isolated. Furthermore, the fractions from the flash silica gel column were larger such that upon separation b y reverse-phase H P L C (25% M e C N / H 2 0 ) , seven fractions were obtained instead o f two. The first two fractions were impure, the third, sixth, and seventh contained 42, 44, and 43, respectively, while the fourth contained paulitin (45), and the fifth contained isopaulitin (46). The structure elucidation o f each is described i n next two subsections. A discussion o f the structure elucidation o f compound 41 (which we named artemisiidiendioc acid monomethyl ester) is in the third subsection, while the synthesis and structure elucidation o f compounds 223-226 is in Section 3.3.3.  3.3.2.2.2: Psilostachyins A (42), B (43), and C (44) The H and !  1 3  C N M R spectra o f 42-44 were all quite similar, thus the structure  elucidation o f 42 w i l l be presented, with notations on how 43-44 differ. H R D C I M S determined that the molecular formulas for 42-44 were C15H20O4,  C15H20O5, C15H18O4,  and  respectively, based on m/z values for [ M + H ] of 281.1389, 263.1283, and  265.1440, respectively. In the  +  1 3  C N M R spectrum o f 42, there were signals for two  lactone carbonyls (8 177.3 and 8 169.6 ppm). There were also signals for an olefinic methylene (8 139.0 and 8 121.7 ppm), corroborated by signals in the *H spectrum at 8 6.26 and 8 5.54 ppm. Additional inspection o f the both the *H and  102  1 3  C spectra indicated  the presence o f two methyl groups (8 1.19 and 8 1.02 ppm, each integrating for 3H), and three carbinols, (8 93.6, 8 83.4, and 8 79.6 ppm). The data for 43 and 44 were quite similar, except for the presence o f one less oxygen (as shown by M S data) and the presence o f an additional double bond i n 43 (8 133.1 and 8 126.2 ppm). the rest o f the  1 3  C N M R data with literature data ' ' 8  are psilostachyins A , psilostachya.  6 1  B,  6 2  53  60  Comparison o f  suggested that compounds 42-44  and C , respectively, originally isolated from A. 6 3  A n additional related compound, altamisic acid (257), previously isolated  from A. tenuifolia,  53  was also noted to be present i n a more polar fraction, but was not  fully characterized as it was inactive. Compounds 42-44 were isolated in 32.9 mg, 108 mg, and 17.1 m g yields, respectively. 1.02,  15.0  Figure 3.4: Important N M R Chemical Shifts for Psilostachyin A (42). Carbon chemical shifts are i n italics. The  1 3  C N M R data ' ' 8  53  60  for the psilostachyins 42-44 and altamisic acid (257) has  been previously reported several times, but the complete ' H chemical shift data has only  103  been reported for 43, 257,  and the P-OH epimer o f psilostachyin A (42).  Therefore,  we decided to perform H M Q C and H M B C experiments on 42-44 to complete the chemical shift assignments for 42 and 44 and to confirm that o f 43, as there was some ambiguity present i n the data reported. Table 3.1 contains the chemical shift assignments for 42-44. It is noteworthy that the literature for each o f these compounds has several incorrectly assigned carbons, usually aliphatic carbons i n the seven-membered ring. Psilostachyin A (42) also has the  1 3  C chemical shift for the spirolactone carbon and the  free carbinol incorrectly assigned. The I D N M R spectra for 42-44 can be found i n Figures 3.5 to 3.7.  9-  104  Table 3.1: Psilostachyin A(42), B(43), and C (44) N M R Data i n C D C 1 No.  Carbon  Proton  (42)  (42)  1 2  93.6 24.2  3  27.3  4 5 6  177.3 79.6 83.4  7 8  40. l 30.2  9 10 11 12 13  26.9 41.7 139.0 169.6 121.7  14 15  21.6 15.0  OH  —  a  2.52-2.40 m# 1.82-1.68 m 2.52-2.40 m#, 1.58 m  b  —  b  Proton  (43)  (43)  133.1 25.2 29.9  C  170.8 86.9 83.4  —  a  Carbon  —  4.96 d, J = 9.5 H z 3.36 m 2.75 m , 2.52-2.40 m# 1.96-1.90 m 2.20 m  41.5 25.9  C  34.8 126.2 138.4 169.7 120.6 C  — —  6.26 d, J = 3.4 H z , 5.54 dm, J = 2.9 H z 1.19 s 1.02 d J = 7.3 H z 3.46 s  Carbon  3  Proton  (44)  (44)  2.62-2.40 m#  43.1 22.5  2.62-2.40 m#  30.9  1.94 m 2.10-2.01 m#, 1.85-1.73 m# 2.66 ddd, J = 18.0 H z , J = 5.4 H z , J = 1.7 H z 2.45 m  —  — —  4.77 d, J = 9.0 H z 3.32 m 2.09-2.05 m, 1.91-1.77 m 2.49-2.40 m# —  — —  6.21 d, J = 3.4 H z , 5.51 d, J = 3.0Hz 1.49 s 1.72 s  23.7 23.0 —  —  d  169.1 89.9 86.2 41.3 23.9  d  31.6 35.3 138.4 169.6 120.4 d  18.9 14.4 —  — —  4.64 d, J = 9.5 H z 3.41 m 2.10-2.01 m#, 1.70-1.66 m 1.85-1.73 m# 2.17 m — —  6.22 d, J = 3.4 H z , 5.48 d, J = 3.4 H z 1.28 s 0.99 d, J = 7.6 H z —  ' H N M R at 500 M H z ; C at 100 M H z . Values marked with same letter are interchanged in the literature. Our values are based on H M B C and H M Q C correlations. # Overlapping signals. 1 3  a d  105  106  107  108  3.3.2.2.3: Paulitin (45) and Isopaulitin (46) The H and l  1 3  C N M R spectra o f 45 and 46 were quite similar to each other and to  the I D N M R spectra o f the psilostachyins 42-44, suggesting these compounds were related. H R D C I M S analysis for 45-46 gave m/z for [ M + H ] o f 277.1075 and 277.1076 +  both appropriate for a molecular formula o f C 1 5 H 1 6 O 5 . A check o f the  1 3  C N M R spectrum  for both 45 and 46 showed the diagnostic chemical shifts for epoxide carbons, 8 61.3 and 8 66.7 ppm for 45 and 8 61.9 and 8 70.9 ppm for 46. In addition, there were two additional olefinic carbons present i n these compounds, (8 127.2 and 8 140.2 ppm for 45, and 8 123.4 and 8 146.8 ppm for 46). Analysis o f the rest o f the *H and  1 3  C N M R data  confirmed that 45 was paulitin (32.8 mg) and 46 was isopaulitin (44.4 mg), originally isolated from A. cumanensis, '  60 64  and recently from A. artemisiifolia collected in B r a z i l .  58  To confirm the structures o f 45 and 46, H M Q C and H M B C experiments were performed resulting in our N M R assignments, which are i n Table 3.2. The I D N M R spectra for 45 and 46 can be found i n Figures 3.8 and 3.9.  109  110  Ill  Table 3.2: Paulitin (45) and Isopaulitin (46) N M R Data i n C D C 1  1 2 3 4 5 6 7 8 9 10 11 12 13  Carbon (45) 61.3 140.2 127.2 160.3 85.7 81.1 39.1 25.4 30.9 66.7 138.7 169.1 121.9  14 15  20.9 24.0  No.  a  a  b  b  Proton (45)  Carbon (46) 61.9 146.8 123.4 160.1 85.5 81.9 41.5 22.9 31.9 70.9 138.3 169.4 121.1  —  6.55 d, J = 10.4 H z 6.35 d, J = 10.1 H z — —  4.88 d, J = 8.2 H z 3.31 m 2.33-2.20 m 1.87-1.75 m  C  C  —  — —  6.23 d, J = 5.54 d, J = 1.58 1.49  2.7 H z 2.4 H z s s  20.9 21.8  3  Proton (46) —  6.34 d, J = 10.3 H z 6.19 d, J = 10.3 H z — —  4.75 d, J =8.4 H z 3.36 m 2.10m 1.99 m, 1.75 m — — —  6.18 d, J = 5.51 d, J = 1.49 1.42  3.1 H z 2.7 H z s s  H N M R at 500 M H z ; C at 100 M H z . " Values marked with same letter are interchanged in the literature. Our values are based on H M B C and H M Q C correlations. a  c  3.3.2.2.4: Artemisiidiendioc Acid Monomethyl Ester (41) 15  14  From the polar fraction o f the D C M soluble material, 1.9 m g o f compound 41 was isolated as described i n Section 3.3.2.2.1. This compound was isolated from a crude fraction that was inactive i n the G 2 checkpoint inhibition bioassay. U p o n inspection of both the *H and  1 3  C N M R spectra it was apparent we were dealing with a highly  functionalized compound. Initial analysis suggested the presence o f a methyl ketone  112  (5 2.07 ppm, singlet, 3 H ; 8 210.6 ppm), an another ketone (8 200.2 ppm; probably a,P unsaturated), two carboxylic acids or esters (8 173.8 and 8 166.8 ppm), two olefinic methylenes (8 6.09/5.73 and 8 6.24/5.71 ppm), and a methyl ester (8 3.68 ppm, singlet, 3 H ; 8 51.8 ppm). 6.09/5.73,124.6  6.24/5.71, 126.9  166.8 3.84, 2.38,  78.6  OMe  3.68,57.5  28.6  -2.88,32.(5  ^  O  o OH  Figure 3.10: N M R Chemical Shift Assignments for 41. H R D C I M S analysis gave m/z for [ M + H ] o f 327.1442 and m/z for [ M - H 0 ] o f +  +  2  309.1340 corresponding to a molecular formula o f C16H22O7, with the latter ion ten-fold more intense. While the C O S Y , H M Q C , and H M B C 2 D N M R spectra were all utilized, the high level o f functionality, the broadness o f some o f the signals in the proton spectrum, and the overlap o f several signals around 8 2.88 ppm i n the proton spectrum meant the H M Q C and H M B C spectra were the most useful. Analysis o f the H M B C spectrum showed a correlation from 8 3.68 ppm to 8 166.8 ppm, confirming the presence of a methyl ester as shown i n Figure 3.10. H M B C correlations from 8 6.24/5.71 ppm to 8 166.8 and 8 42.6 ppm, and from 8 6.24 ppm to 8 139.1 ppm confirmed the presence o f an a,P-unsaturated ester. A n H M B C correlation from a doublet at 8 3.84 ppm (J = 5.5 H z ) to 8 139.1 ppm and to 8 210.6 ppm indicated placement o f the ketone near, but not adjacent to, the a,p-unsaturated methyl ester. The singlet at 8 2.07 ppm was correlated to  113  8 210.6 ppm i n the H M B C spectrum, thus confirming the presence o f a methyl ketone as depicted i n Figure 3.10. H M B C correlations were also observed between multiplets at 8 2.88 and 8 2.38 ppm with both 8 173.8 and 8 200.2 ppm suggesting placement o f the second ketone and the carboxylic acid. The protons at 8 6.09/5.73 ppm showed H M B C correlations to 8 200.2, 8 147.5, and 8 28.6 ppm thereby confirming the presence o f an a,|3-unsaturated ketone. Analysis o f the limited C O S Y correlations showed that H-9 (8 3.84 ppm) was only coupled to H-8 (8 -2.88 ppm), which was i n turn coupled to H-7 (8 1.56 ppm). H-7 was coupled to H-8 as mentioned and to H-6/H-6' (8 2.08/1.94 ppm). The H M B C spectrum showed a correlation from H-6/H-6' to C-15 (8 124.6 ppm), thus completing the structure o f 41. A complete analysis o f the rest o f the 2 D data confirmed the proposed structure o f 41 is as shown in Figure 3.10. Compound 41 is a novel highly functionalized sesquiterpene that we have named artemisiidiendioc acid, which was isolated as its monomethyl ester. W e believe that the methylation o f one o f the carboxylic acids may have occurred during the methanol extraction of A. artemisiifolia, as methylation under acidic conditions occurs quite readily.  65  The presence o f an unusual highly degraded  sesquiterpene diacid 41 i n this extract was quite interesting and promised great challenges for stereochemical determination, however we needed additional material. Unfortunately, a re-isolation o f 41 was unsuccessful, as we could not find evidence o f this compound in the second vial we obtained. W h i l e the stereochemistry o f 41 was not determined, we believe that it should be similar to that o f the psilostachyins as it appears to arise from psilostachyin B (43), see below. Table 3.3 contains the H and  114  C NMR  assignments for 41, the I D N M R spectra are found i n Figures 3.11 and 3.12, and 2 D N M R spectra for 41 can be found in'Section 3.7.  Table 3.3: Artemisiidiendioc A c i d Monomethyl Ester (41) N M R Data in DMSO_d6 No. 1 2 3 4 5 6  Carbon 173.8 32.6 28.6 200.2 147.5 28.1  7 8 9  26.8 42.6 78.6  10 11 12 13 14  210.6 25.9 139.1 166.8 126.9  15  124.6  OMe  51.8  Proton —  H M B C Correlations  C O S Y Correlations —  —  H-3 H-2  C-4, C-3*, C - l C-4, C-2, C - l  —  —  —  —  -—  —  G C , H-7  C-15, C-7, C-5, C-4  H-6, H-6' H-9, H-7 H-8  C-8,C-7*,C-6 C-14, C - l 2 , C-9 C-12, C-10, C-8, C-7  •—  —  2.88 br.m 2.38 b r m  2.08 br m , 1.94 b r m 1.56 br m 2.88 b r m 3.84 d J = 5.5 H z —  —  C-10  —  —  —  —  —  —  GC GC  C - l 3 , C-12 (from 6.24), C-8 C-6, C-5, C-4  —  C-13  2.07 s  6.24 5.71 6.09 5.73 3.68  s s s s s  *H N M R at 500 M H z ; C at 100 M H z . * Uncertain assignments, due to overlap and broad ' H signals. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. 1 3  15  14  If we had only isolated 41, the possible biogenesis o f this compound would be an enigma as the molecule is so highly functionalized it is difficult to know where to begin. However, as 41 was isolated along with other seco-pseudoguaianolides it is possible to propose a biosynthesis that links all the natural products isolated, (see Scheme 3.3 below).  115  116  117  Starting with E,E-farnesyl pyrophosphate (28), cyclization occurs between C - l and C - l l as shown to form the germacrene cation 258a. Following a proton loss (258a/258b), and proton-initiated cyclization the guaiane skeleton 258c is formed. Three concerted Wagner-Meerwein (marked as W , M ) shifts occur, one a methyl and the other two hydride, forms the pseudoguaiane skeleton 258d, as its cation. Quenching o f the cation by water and appropriate oxidations, (258d and 258e), leads to the formation o f intermediate 258f, which can lose water to form 258g. A Baeyer-Villiger rearrangement gives psilostachyin B (43). Reduction o f the tetrasubstituted double 66  bond at C - l / C - 1 0 gives psilostachyin C (44), while epoxidation o f this double bond gives paulitin (45) and isopaulitin (46). Opening o f the 8-lactone gives altamisic acid (257), the N M R signals o f which were noted, though the compound was not characterized as the as it was inactive i n the G 2 checkpoint bioassay.  Scheme 3.3: Biogenesis o f the A artemisiifolia  118  Sesquiterpenes Isolated.  Protonation o f the C - l / C - 1 0 double bond allows for intramolecular addition o f the carboxylic acid to form the spiro-y-lactone, thereby forming psilostachyin A (42). Protonation o f the carbonyl in the 8-lactone o f 43 allows for the loss o f a proton at C-14, double bond migration from C - l / C - 1 0 to C - l / C / 5 , and the concurrent opening o f the 8lactone all in a concerted manner. Opening o f the y-lactone then occurs to give intermediate 258h. Following oxidative cleavage o f the C - l / C - 5 double bond, compound 259 is formed, which we believe is the putative natural product—41 is an artefact caused by methylation o f one o f the carboxylic acid moieties during the isolation procedure. The proposed stereochemistry o f 41, based on that o f psilostachyin B (43), its "parent compound," is SS, 9R.  3.3.3: Synthesis and Structure Elucidation of Derivatives 223-226 3.3.3.1: Psilostachyin A P-Mercaptoethanol Adduct: Compound 223 o  223  O  A 3.6 m g portion o f psilostachyin A (42) was stirred with 45 u L |3mercaptoethanol ( H S - C H C H O H ) i n 1.5 m L T H F and 1 m L 0.1 M phosphate buffer 2  2  solution for one hour according to established procedure.  67  The resulting solution was  diluted to 10 m L with water and partitioned with E t O A c ( 3 x 1 0 m L ) .  The organic layer  was then concentrated in vacuo and purified b y revered-phase H P L C (20% M e C N / H 0 ) 2  to yield 0.7 m g o f the desired product.  H and  C N M R established the loss o f the exo-  methylene as expected. Three new resonances appeared i n the  1 3  C spectrum, two from  the P-mercaptoethanol (8 35.9 and 8 60.7 ppm) and a new methylene (8 31.4 ppm).  119  Table 3.4: Psilostachyin A P-Mercaptoethanol Adduct 223 N M R Data i n C D C 1 3  No. 1 2  Carbon 94.9 23.8  3  27.5#  4 5 6  176.5 78.0 86.2  7 8 9  44.7 30.2 27.5#  10 11  40.1 27.5#  12 13  175.9 31.4  14 15  26.0 17.2  OH SCH CH OH  35.9 60.7  Proton  2  2  —  —  GC,H-3(1.13)  C-4 (from 1.95)  H-2 (both from 1.13)  C-4  —  —  —  —  —  —  H-7  C-14, C - l  H-8, H-6 H-9*, H-6 GC*  C-12  H-15,2.55  C-8, C-5, C - l C-13, C - 7 *  —  1.95 m 1.70 m 2.75 m 2.45 m#, 1.13bd*  4.55 d J = 5.4 H z 3.00 m 2.55 m# 2.75 m 2.45 m# 1.88 m 2.45 m# 2.75 m 2.45 m# —  2.75 m 2.45 m#, 2.00 m 1.52 s 0.95 d, J = 6.9 H z 2.18 bs 2.78 m 3.77 t, J = 5.5 H z  —  H M B C Correlations  C O S Y Correlations  C-7*  —  —  SCH  C-7*  2  H-10  C-6, C-5, C - l C-10, C - 9 , C - l  —  —  C H O H , H-13 SCH 2  2  SCH  2  ' H N M R at 500 M H z ; C at 100 M H z . * Uncertain assignments. # Overlapping values. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. Due to large area of overlap not all peaks in the H spectra can be fully assigned. Adduct correlations are given as the portion it, ie O C H . l 3  !  2  120  121  122  Additionally, a triplet at 5 3.77 ppm (J = 5.5 H z ) corresponding to the oxygenated methylene o f the added |3-mercaptoethanol was observed in the H spectrum. The "new" !  methine carbon ( C - l l ) and its corresponding proton, were not easily observed, and are i n 1  13  fact only tentatively assigned due to large overlap i n the region (in both the H and spectra) where they are believed to be.  C  Analysis o f the 2 D N M R data i n conjunction  with H R D C I M S (m/z for [ M + H ] = 359.1525; molecular formula C H i 0 S ) confirmed +  1 7  6  6  that the structure was otherwise unchanged from that o f 42. Table 3.4 contains the H and [  1 3  C N M R assignments for 223 and the I D N M R spectra are i n Figures 3.13 and 3.14.  Compound 223 was no longer active i n the G 2 checkpoint bioassay, confirming the necessity o f the a-methylene-y-lactone for biological activity.  3.3.3.2: The Dihydropaulitins: Compounds 224 and 225  In an effort to determine whether the epoxide moiety i n paulitin (45) and isopaulitin (46) was important for biological activity, (the additional a,p-unsaturated-8lactone made this difficult to ascertain i n 45 and 46), a 1.9 m g portion o f psilostachyin B (43) was treated with 3.8 mg o f w - C P B A in D C M at room temperature overnight.  68  After quenching the excess peroxyacid with saturated solutions o f Na2S2C>3 and NaHCC»3, the resulting solution was partitioned between D C M ( 3 x 3 0 m L ) and water. After concentration o f the organic layer in vacuo the resulting solid was purified by reversephased H P L C (25% M e C N / H 2 0 ) to yield two diastereomeric epoxides, 224 (0.5 mg) and 225 (0.8 mg). It was hoped that as with 45 and 46, the different chemical shifts o f the  123  epoxide carbons would used to determine the stereochemistry o f 224 and 225. Unfortunately, the N M R data i n Tables 3.5 and 3.6 shows that the chemical shifts o f the epoxide carbons are too similar. nOe studies on 45 showed that while other protons such as Me-14, H-6 and H-7 gave good nOe's, Me-15 did not give any useable data, thus we did not pursue nOe studies i n the characterization o f epoxides 224 and 225. H - 2 and H - 3 , which gave good nOe's i n 45, are overlapped i n 224 and 225 suggesting they would not give useable nOe results either. A comparison o f the ' H N M R chemical shifts for M e - 1 4 and Me-15 suggested that 45 (8 1.58 and 8 1.49 ppm, respectively) and 224 (8 1.60 and 8 1.42 ppm) were similar and perhaps had the same stereochemistry. The H N M R !  chemical shifts o f 46 (8 1.49 and 8 1.42 ppm) for Me-14 and Me-15 did not compare as well to those o f 225 (8 1.35 and 8 1.42 ppm). Therefore, we cannot make any definitive conclusions about the stereochemistry o f epoxides 224 and 225. The H R D C I M S for 224 gave m/z for [ M + N H ] as 296.1495 and 225 gave m/z for +  4  [M+NH4]" " as 296.1493, both consistent with a molecular formula o f C 1 5 H 1 8 O 5 . Analysis 1  of the I D and 2D N M R data confirmed that 224 and 225 differed from psilostachyin B (43) b y the presence o f an epoxide, and from paulitin (45) and isopaulitin (46) by the absence o f an a , P double bond in the 8-lactone moiety. The I D N M R spectra are i n Figures 3.15 and 3.16.  124  Table 3.5: Epoxide 224 N M R Data i n C D C 1 No. 1 2 3  Carbon 66.4 23.9* 25.9  4 5 6  168.3 87.6 82.6  7 8  39.5 27.4  9  31.3  10 11 12 13  66.0 131.6 168.9 121.7  14 15  21.4 23.6*  Proton  H M B C Correlations  C O S Y Correlations  —  2.68 m# 2.15 m,# 1.76-1.62 m#  3  —  —  G C , H-3 G C , H-2  C - l (from 1.76)  —  —  —  —  —  —  H-7  C-5  H-13 (6.25), H-6 G C , H-9  C-9 C-9  H-8  C-l  —  —  —  —  —  —  —  —  —  From 6.25 to H-7  C-12, C-7  4.68 d, J = 7.9 H z 3.32 bm 2.68 m,# 1.76 -1.62 m# 2.30 m,# 2.15 m#  6.25 d, J = 2.7 H z 5.55 d, J = 2.4 H z 1.60 s 1.42 s  C-6, C-5, C - l C-10,C-9  Very dilute sample. ' H N M R at 500 M H z ; C at 100 M H z . # Overlapping values. * Uncertain assignments. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. 1 3  125  Table 3.6: Epoxide 225 N M R Data i n C D C 1 No. 1 2 3  Carbon 65.1 28.1 29.3  4 5 6  170.5 85.6 80.9  7 8  42.0 20.3  9  33.6  10 11 12 13  66.0 137.4 169.3 120.0  14 15  19.9 22.0  C O S Y Correlations  Proton  3  H M B C Correlations  —  —  G C , H-3 G C , H-2  C-l C-4, C - l  —  —  —  —  —  —  H-7  C-14,C-5  —  2.70-2.60 m 2.19-2.12 m#, 1.83 dt, J = 14.4 H z , J - 4.8 H z ,  4.79 d, J = 9.1 H z 3.24 b m 1.95 m 1.64 m 2.38 dd, J = 15.6 H z , J = 5.8 H z , 2.19-2.12 m#  H - 1 3 , H - 6 , H-8 G C , H-9, H-7 G C , H-8  C-10, C-9, C-7, C-6 (from 1.95) C-8, C-7, C - l (from 2.38)  —  —  —  —  —  —  —  —  —  H-7  C-12, C-7  6.20 d J = 3.6 H z 5.45 d J = 3.3Hz 1.35 s 1.42 s  C-6, C-5, C - l C-9  ' H N M R at 500 M H z ; C at 100 M H z . # Overlapping values. * Uncertain assignments. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. 1 3  14  a  224,225  126  127  128  3.3.3.3: Paulitin Dehydration Product: Compound 226  °  o>  'T>r\;'  H  226  Based on preliminary bioactivity data that suggested paulitin (45) was active i n the G 2 checkpoint assay, we decided to open its epoxide moiety to see i f the resulting diol was still active. W e later discovered that 45 was actually inactive i n the bioassay (a false positive), but as we had already made compound 226 we tested it and determined it was also inactive, presumably due to the same reason 45 and 46 were inactive, an additional a, P-unsaturated carbonyl moiety. A 3.6 m g portion o f paulitin (45) i n 1.5 m L T H F was treated with 10 drops 30% perchloric acid (HCIO4) for 4 hours. After addition o f 15 m L o f water the resulting acidic solution was neutralized with saturated NaHCC»3 and the T H F removed in vacuo. The resulting water solution was partitioned with E t O A c ( 2 x 1 5 m L ) , and the organic layer was concentrated in vacuo. The resulting mixture o f products was purified by reverse-phase H P L C (25% M e C N / H 0 ) to yield the major product 226 (0.5 mg), 2  1  unreacted 45, and several minor products that were not identified. The H and  13  C NMR  spectra contained new signals for an additional exomefhylene (8 148.0/116.3 ppm and 8 5.19/4.93 ppm), and the H R D C I M S gave m/z for [ M + H ] o f 276.1007. This was +  appropriate for a molecular formula o f  C15H15O6  and suggested that the epoxide oxygen  was still present. Additionally only one methyl group (8 1.36 ppm) was present i n the H !  N M R spectrum. 2 D N M R data confirmed the presence o f an oxygen at C - l (8 73.4 ppm) and that the structure differed from 45 b y the opening o f the epoxide at C - l / C - 1 0 , 129  presumably as shown below. The structure o f compound 226 is otherwise the same as 45 and must be as shown. Table 3.7 contains the H a n d  C N M R assignments for 226,  while the I D N M R spectra are i n Figures 3.17 and 3.18.  Table 3.7: N M R Data for Compound 226 i n C D C 1 No. 1 2  Carbon 73.4 147.0  3  121.4  4 5 6 7 8  161.3 89.8 82.6 41.9 33.0  9  32.6  10 11 12 13  148.0 150.2 169.1 122.9  14 15  20.9 116.3  OH  —  —  H-3  C-5,C-4  H-2  C-l  —  —  —  —  —  —  —  6.84 d J = 9.9 H z 6.16 d J = 10.0 H z  H-7 H-8 (1.91), H-6 G C , From 1.91 to H-9 (2.42), H-7 G C , From 2.42 to H-8 (1.91)  C-14  —  —  —  —  —  —  —  —  —  GC  C-12,C-7  GC  C-6, C - 5 , C - l C-10, C-9, C - l  —  —  5.21 # 3.58 b m 1.91 m 1.65 m# 2.83 tm, J = 12.0 H z , 2.42 ddm J = 13.4 H z J = 6.2 H z  6.29 d J = 2.5 H z 5.63 d J = 2.9 H z 1.36 s 5.19 bs 4.93 bs 2.96 bs  —  H M B C Correlations  C O S Y Correlations  Proton  3  From 2.42 to C-15, C-8, C-7,C-l  ' H N M R at 400 M H z ; C at 100 M H z . # Overlapping values. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. 3  131  132  3.3.4: Conclusions: The Bioactivity of Ambrosia artemisiifolia Sesquiterpenes A n inspection o f the data i n Table 3.8 suggests that these compounds are not clinically useful as G 2 checkpoint inhibitors. This is due to the compounds either being inactive (> 100 u M ) , such as with psilostachyin B (43), or to a useless therapeutic window. For example, paulitin (45) kills cells before any G 2 checkpoint inhibition can be observed. Psilostachyin A (42), while a G 2 checkpoint inhibitor, is also cytotoxic at approximately the same concentration, which means the therapeutic window is too small. In spite o f their shortcomings as therapeutic agents, psilostachyin A (42), psilostachyin C  Compound Name  Cytotoxicity (ICsn)  Checkpoint Inhibition (ICsn)  Psilostachyin A (42)  30 u M  25 u M  Psilostachyin B (43)  > 100 u M  > 100 u M  Psilostachyin C (44)  8 uM  25 yM  Paulitin (45)  2.5 u M  Killed Cells  Isopaulitin (46)  2.5 u M  Killed Cells  > 100 u M  > 100 u M  Not Tested  > 100 u M  Not Tested  60 u M  Not Tested  > 100 u M  Psilostachyin A P-mercaptoethanol Adduct 223  Structure  HO trx'H , O  HCK  Epoxide 224  Epoxide 225  Compound 226  Table 3.8: Comparison o f Cytotoxicity and G 2 Checkpoint Inhibition Activity for 42-46 and 223-226. Not Tested = Not enough material was available for assay.  133  241  242  (44) and epoxide 225 are useful as biochemical probes. These compounds inhibit the G2 checkpoint, but then cause the cells to arrest i n mitosis; therefore, they are also antimitotic agents. Biochemical studies have suggested that psilostachyin A (42) and psilostachyin C (44) have a mechanism o f action similar to that o f 13-hydroxy-15oxozoapatlin (241) and the related diterpene 242, but at this point, the exact protein target is still under investigation, though it is believed not to be tubulin—the usual target. ' ' 34  44  45  The common structural feature shared between psilostachyins A (42) and C (44) and 241 and 242, is the presence o f an a , p-unsaturated carbonyl moiety, which is responsible for the observed G2 checkpoint inhibition.  44  While we did not undertake an exhaustive structure-activity relationship ( S A R ) study, we did determine that the presence o f the a-mefhylene lactone is essential. When A d.b. decreases cytotoxicity and G2 checkpoint inhbition; Epoxide (if present) orientation possibly important  Figure 3.19: Structure-Activity Relationship o f A artemisiifoUa Sesquiterpenes.  134  42 was treated with (3-mercaptoethanol, to produce adduct 223, the resulting compound showed no G2 checkpoint inhibition. Figure 3.19 details the limited results o f the S A R study. A s one would expect, i n order to better understand the requirements for G2 checkpoint biological activity, a much wider array o f structures is necessary.  3.4: Sesquiterpene Lactones from Vernonia baldwinii 3.4.1: Review of Metabolites from Vernonia baldwinii The large Asteraceae genus Vernonia has been w e l l studied chemically with greater than a hundred species or varieties investigated ' though to date the species V. 69  70  70 71  baldwinii Torr. has yielded only one compound, glaucolide B (260). '  Vernonia species  produce a large array o f germacranolides including numerous glaucolides (including 232, 260, and 261) and a variety o f other sesquiterpene lactones. " 69  70  W h i l e some o f the  compounds (47, 227, 231) we isolated from V. baldwinii i n the course o f our investigation had full H assignments, none had been assigned !  we have completed.  47  'o  o  K  227  229  135  228  1 3  C chemical shifts, which  Vernonia is a worldwide genus that is found extensively throughout the Americas, 69-73  Africa and Asia. "  Vernonia baldwinii, the western ironweed, is a perennial herb found  in the Great Plains o f the United States. V. baldwinii (Figure 3.20) produces pretty purple flowers, several to a plant. This herb grows to a height of between 80 and 150 cm and can range from thinly to densely haired. The preferred habitat o f V. baldwinii is upland pastures, open woodlands, roadsides and tall or mixed-grass prairies.  Figure 3.20: Vernonia baldwinii growing i n Okalahoma. (Photo by C . S. Lewallen, used by permission.) 3.4.2: Isolation Procedure of T G - 3 Active Sesquiterpenes from Vernonia baldwinii The TG-3 antibody is utilized in the G2 checkpoint and the antimitotic 33  35  bioassays, developed by Dr. Roberge's lab at U B C , as part o f the detection and visualization process.  41  This antibody was originally raised against paried helical  filaments that are present in the brain o f those who suffer from Alzheimer's disease,  74  but  apparently responds to an event in mitosis. This event, which w i l l be discussed further in Section 3.4.5, is the formation o f a phosphoepitope (antibody recognition site, here the TG-3 antibody) o f nucleolin (an important nuclear protein) present only in mitotic cells. For the purpose o f isolation, Roberge's lab used a modified G 2 checkpoint assay to monitor the biological activity throughout the isolation process. This assay involved a  136  shorter time span that enabled a better detection o f the green colour that signifies a positive result. If the time was too long, the cytotoxicity o f the compounds killed the cells, and no colour was observed. The methanolic extract (~10 grams) o f N6397 was obtained from the N C I ' s Open Repository. One-third o f this extract was suspended i n water and partitioned with D C M (4 x 200 m L ) . The active components were i n the D C M layer, which was concentrated in vacuo and a portion was loaded onto a normal phase silica gel column. Flash chromatography utilizing a gradient from hexanes to D C M and from D C M to E t O A c was undertaken. Bioactive fractions were combined and the most active were subjected to reverse-phase H P L C to yield the known compounds 47 (7.1 mg), 227 (5.6 mg), and 229232 (13.3, 3.4, 0.2, and 3.4 mg, respectively) i n addition to several other terpenes whose structures were not determined. The vernonataloides 47 and 227 were determined to be the active compounds, while the 229-232 were too cytotoxic. This isolation procedure was undertaken twice, once on a smaller scale, and once on a larger scale, the latter to obtain more o f 47 and 227 for further biological testing. Section 3.4.3 describes the structure elucidation o f all six sesquiterpene lactones isolated from Vernonia baldwinii and the synthesis o f compound 228 from 47. 3.4.3: Structure Elucidation of Sesquiterpenes from Vernonia baldwinii  3.4.3.1: Vernonataloides: Compounds 47, 227, and 228 The naturally occurring vernonataloides 47 and 227 eluted from the H P L C column first and were responsible for the bioactivity o f the extract. Comparison o f the ' H N M R and  C spectra o f 47 and 227 suggested they were very similar with one more  methyl signal and one more carbon signal, respectively, i n the N M R spectra o f 227. This  137  was confirmed by H R D C I M S ; 47 gave m/z for [ M + H ] at 407.1704, corresponding to a +  molecular formula o f C i H 6 0 , while 227 gave m/z for [ M + H ] at 421.1858, +  2  2  g  corresponding to a molecular formula o f C H 8 0 8 . Compound 227 differed from 47 by 22  2  C H either as an extra methylene or as a branching methyl group. The structure 2  elucidation o f 47 is presented below, with that o f 227 following it.  In the  l 3  C spectrum o f 47 there were two signals corresponding to carbonyl  systems (8 170.1 and 8 166.2 ppm), with the presence o f an additional signal at 8 163.6 ppm that could be an additional carbonyl or double bond carbon. There were only three other signals that could account for double bond carbons (8 134.9, 8 127.9, and 8 127.6 ppm) therefore the signal at 8 163.6 ppm was probably a double bond carbon. Further analysis o f the  1 3  C N M R spectrum showed seven oxygenated aliphatic carbons present,  (8 82.0, 66.6, 65.1, 62.2, 59.6, and 57.8 and 55.8 ppm), four o f which are suitable for epoxide carbons (8 ~ 60-70 ppm) and one suitable for a lactone carbinol (8 82.0 ppm), leaving two not yet assigned. The ' H N M R spectrum o f 47 contained a singlet with an integration o f 3 H at 8 2.04 ppm, suitable for an acetate methyl, which accounts for one o f the carbonyl signals (8 170.1 ppm) seen in the  1 3  C N M R spectrum. Further inspection o f the *H N M R  spectrum indicated the presence o f an isolated spin system (8 4.90 and 8 4.78'ppm) with large coupling constant, (J = 13.1 H z ) , typical o f geminal protons. These protons are  138  quite deshielded and must be attached to an electronegative atom, such as an oxygen atom in a hydroxy-methyl moiety, which would also account for one o f the remaining oxygenated carbons. T w o doublets i n the proton spectrum, one at 8 4.96 ppm (J = 8.8 H z ) and one at 8 2.57 (J = 8.7 H z ) are clearly coupled to each other and due to the size o f their coupling constant and are probably anti i n orientation. The proton with a chemical shift o f 8 2.57 is i n the region where epoxide protons are found while the chemical shift o f the other (8 4.96 ppm) is suitable for a lactone. Further consultation o f the ' H N M R data indicated the presence o f four methyls, one methyl was olefinc (8 1.91 ppm), another has already be ascribed to an acetate (8 2.04 ppm), while the remaining two aliphatic methyls (8 1.51 and 8 1.47 ppm) are singlets suggesting the presence o f a branched structure, possibly in a ring system. T w o singlets at 8 6.11 and 5.68 ppm suggested the presence o f an olefinic methylene that would also account for two olefinic carbons. A s there were no additional (unassigned) proton signals i n the region corresponding to olefins (8 ~ 4.50-8.00 ppm) the other olefinic carbons are tetrasubstituted. Returning to the  1 3  C N M R spectrum and based on comparison with  1 3  C chemical  shift data charts, the signals at 8 166.2, 127.6 (or 127.9), 134.9, and 18.0 ppm correspond nicely with those o f a methyl acrylate ester (8 165.7, 135.0, 127.0, and 18.0 ppm). This 8  would also account for an oxygenated carbon at its attachment point. The two remaining olefinic signals (8 163.6 and either 8 127.9 or 127.6 ppm) must correspond a conjugated system probably that o f an a,(3-unsaturated carbonyl, whose carbonyl shift must be accidentally equivalent to that o f the acetate carbonyl (8 170.1 ppm) as there are no remaining unassigned signals i n that spectral region. A check o f the H M B C spectrum shows a carbonyl that is correlated to an acetate methyl (8 2.04 ppm) and to the  139  hydroxymethyl protons (5 4.90 and 8 4.78 ppm). W h i l e both sets o f protons are two or three bonds away from the carbonyl and could show correlations to it, the other possibility is that they are two accidentally equivalent carbonyls. This would account for a remaining degree o f unsaturation, bringing the total number o f carbonyls to three. These carbonyls plus two double bonds, two epoxides, and a lactone leaves one degree o f unsaturation remaining o f the nine required b y the molecular formula for 47, which can be accounted for with the presence o f a ring.  1.51  16. (  Figure 3.21: Important N M R Chemical Shifts for Vernonataloide (47). 1  A comparison o f the H and  1 ^  C N M R spectra o f 47 with 227 showed it differed  in three ways. There was an absence o f signals for an olefinic methylene, an additional signal for one more olefinic methyl (8 ~ 1.80 ppm), and an additional multiplet at 8 6.87 ppm. U s i n g the same  C chemical shift tables, it was determined that 227 differed from  47 by the presence o f a tiglate ester side chain instead o f methyl acrylate ester. W i t h the tentative N M R data for 47 and 227 in hand, a careful comparison with that o f known sesquiterpene structures was undertaken, whereby we discovered that 47 and 227 were vernonataloide  69  and 8-deacylvernonataloide-8-0-tiglate,  75  respectively. These  compounds have a germacrane-type skeleton with a ten-membered ring and adjacent ct,|3-  140  unsaturated-y-lactone. Vernonataloide (47) was isolated from Vernonia natalensis 1  75  8-deacylvernonataloide-8-0-tiglate (227) was isolated from V. marginata.  and  The H  1 "5  N M R data o f these compounds has been determined, but the  C chemical shifts were not  in the literature. Therefore, H M Q C and H M B C 2 D N M R experiments were run to assign the carbons and confirm the structures o f 47 and 227. Figure 3.21 shows most o f the chemical shifts o f the core portion o f 47 that were obtained as a result o f these 2 D N M R experiments. Table 3.9 contains the chemical shift data for 47 and 227 and Figures 3.22 and 3.23 are their respective I D N M R spectra. According to established procedure,  we took 1.0 m g o f 47 and dissolved it i n  E t O A c that also contained palladium on carbon, (Pd/C). A balloon was filled with hydrogen gas and attached to a vial containing the aforementioned solution. The hydrogen gas was allowed to pass through the vial for ten minutes. Afterward, the P d / C was removed by suction filtration through a fine fritted filter and the E t O A c was removed under reduced pressure to give 0.4 m g o f 90% pure 13-desacetoxy-8deacylvernonataloide-8-O-isobutyrate (228). The structure o f 228 was confirmed b y H R D C I M S (m/z for [ M + H ] was 351.1810; molecular formula C H 60 ) +  l9  2  6  and both I D  and 2 D N M R data. These data showed the loss o f an acetoxy group as well as reduction o f the olefin in the side chain to give an isobutyrate ester. Compound 228 was completely inactive as a T G - 3 activator, indicating that the presence o f an available Michael acceptor such as methyl-acrylate or tiglate (in 47 or 227, respectively) is necessary. The N M R data for 228 can be found i n Table 3.10 and the I D N M R spectra i n Figures 3.24 and 3.25.  141  Table 3.9: Vernonataloide (47) and 8-deacylvernonataloide-8-0-tiglate (227) N M R Data i n C D C 1 3  No.  Carbon  (47)  (47)  1  62.2  2  22.3  3  35.9  2.68 d J = 10.6 H z 2.10 ddm J = 13.7 H z , J = 5.3 H z 1.55 m 2.28 ddd J = 13.7 H z , J = 5.3 H z , J = 1.7 H z , 1.33 m  4 5  59.6 65.1  6  82.0  7 8  163.6 66.6  9  45.5  10 11 12 13  57.8 127.9 170.1# 55.8  14 15 OR  17.1 16.6 166.2 134.9 127.6 18.0 —  OAc  170.1# 20.7  H M B C Correlations  Carbon  Proton  (47)  (227)  (227)  C-14  62.3  C-10, C-4, C - l  22.4  C-5 (from 2.28), C-4  35.9  2.68 d J = 10.4 H z 2.10 ddm J = 13.7 H z , J = 6.7 H z 1.55 m 2.28 ddd J = 13.7 H z , J = 5.5 H z , J = 1.8Hz 1.32 m  •—  59.7 65.2  Proton  —  2.57 d J = 8.7 H z 4.96 d J = 8.8 H z —  5.23 d J = 9.1 H z 2.74 d J =14.1 H z , 1.99 m  C-7,C-5  82.1  —  164.2 66.3  C-9, C-7, C-6 C-10, C-8, C-7, C - l (C-10, C-8 from 2.74)  —  —  —  —  —  —  4.90 d  C-12,C-ll  45.6  57.9 127.9 170.5 55.9  —  2.57 d J = 8.6 H z 4.97 d J = 8.8 H z —  5.21 d J = 9.2 H z 2.73 J = 13.8 H z 1.99 m — — —  4.88 d  J = 13.1 H z ,  J = 12.8 H z  4.78 d J = 13.1 H z 1.47 s 1.51 s  4.79 d J = 12.8 H z 1.46 s 1.51 s  C-10, C-9, C - l C-5, C-4, C-3  —  —  —  —  6.11 s, 5.68 s 1.91 s  134.9 (from 6.11), 166.2, 18.0 166.2, 134.9,127.6  —  —  —  —  2.04 s  170.1  17.2 16.6 166.7 140.1 127.3 14.7 11.9 170.2 20.7  —  6.87 qm J = 5.8Hz —  1.82 s 1.79 b r s —  2.03 s  H N M R at 500 M H z ; C at 100 M H z . # Overlapping signals. Correlations are from both protons unless otherwise marked. Ester correlations are given to the carbon's 5 value (in ppm).  142  143  144  Table 3.10: 13-desacetoxy-8-deacylvemonataloide-8-0-isobutyrate N M R Data i n C D C 1  (228)  3  No. 1  Carbon 61.8  2  22.3  3  35.9  4 5  59.3 66.0  6  81.7  7 8  158.2 66.0  9  45.0  10 11 12 13  58.1 130.1 172.9 10.1  14 15 OR  17.1 16.6 176.1 33.7  Proton 2.66 bd J = 2.2 H z 2.10m 1.55 m 2.30 m, 1.39m 2.47 d J = 8.7 H z 4.85 ddm J = 8.5 H z , J = 1.8 H z —  5.14d J = 9.1 H z 2.64 d J = 5.6 H z , 1.85 m  G C , H-3  C-10, C-2, C - l (all from 2.10) C-15, C - 5 , C-4 (all from 2.30)  G C , H-2  —  H-6 H-13,H-5  C - l l , C-7, C-5  —  —  H-9 (1.85)  C-10  G C , H-8 (from 1.85)  C-15, C-10, C - 8 , C - 7 , C - l >  —  —  —  —  —  —  —  —  —  H-6  C-12, C - l l , C-8, C-7  1.98 J = 1.8 1.45 1.49  d Hz s s  —  2.55 septet, J = 7.0 H z 1.16 d # J = 7.0 H z 1.14 d J = 1.14 #  18.6  H M B C Correlations C-14, C-10, C-9, C-2  —  —  18.8  C O S Y Correlations H-2 (1.55), H - 9  C-10, C - 9 , C - l C-5, C-4, C-3 —  —  1.16, 1.14 2.55, 1.14  176.1,33.7, 18.6  2.55, 1.16  176.1,33.7, 18.8  ' H N M R at 500 M H z ; C at 100 M H z . # Interchangeable correlations. G C = Geminal Correlations. Correlations are from both protons unless otherwise marked. Ester correlations are given to the carbon's or proton's 8 value (in ppm). 1 3  145  146  147  3.4.3.2: Marginatins: Compounds 229 and 230  229  230  Initial inspection o f the ' H and  1 3  C N M R spectra o f 229 and 230 suggested, as  with 47 and 227, that the only difference was the presence o f a different side chain ester. In 229 it appeared that there was a tiglate ester while there was a methyl acrylate ester in 230. The rest o f the I D N M R spectra for these compounds seemed to be identical. The H R D C I M S o f 229 gave a m/z for [ M + H ] at 405.1905 appropriate for a molecular +  formula o f C22H28O7, while 230 gave a m/z for [ M + H ] at 391.1749 appropriate for +  C21H26O7, differing b y C H  2  either as an extra methylene or as a branching methyl group.  Inspection o f the N M R data for 229 suggested the presence o f an acetate (5 2.00 ppm, singlet, 3H), an epoxide (8 61.0 ppm and 8 66.1 ppm), an a,p-unsaturated-y-lactone (8 ~ 171.0, 8 126.6 or 8 127.0, 8 167.1, 8 70.1 ppm), and a trisubstituted olefin (8 127.0 or 8 126.6 and 8 130.8 ppm). There was also evidence for five methyl groups including one already accounted for i n an acetate moiety ( 8 - 2 . 0 0 ppm). O f the remaining four, three were olefinic methyls (8 ~ 1.80 ppm) and one was an aliphatic methyl (8 1.30 ppm). Additional analysis o f the ' H and  1 3  C N M R spectral data suggested the presence o f a  tetrasubstituted olefin conjugated to carbonyl and with a hydroxy-methyl substituent similar to that present i n vernonataloide (47) and 8-deacylvernonataloide-8-Otiglate (227). The molecular formula o f compound 229 differed from that o f 227 by an oxygen atom, C 2H280 versus C22H28O8, respectively. Compounds 229 and 230 (whose 2  7  molecular formula differed from that o f 47 also by an oxygen), were closely related to  148  227 and 47 and differed only by the "replacement" o f an epoxide with a double bond. This proposal was confirmed by the similarity o f I D N M R spectra between these compounds. When we originally acquired both the H and  C spectra o f 229 and 230  they were very broad and not all signals that should be present (according to the molecular formula) were observed. After searching the chemical literature it was discovered that recording the N M R data at -40° C , (233 K ) , could lead to more useable spectra,  72d  by slowing the rate o f conformational interconversion such that we could see  discrete signals for the major and minor conformers. A s predicted, the spectra were better and we were able to solve the structure o f these compounds b y using the data o f the major conformer. The germacrane-type ten-membered ring in the case o f 229 and 230 must therefore have conformational mobility that is not present i n 227 and 47. A careful comparison o f the ' H N M R data for 229 and 230 showed they were the known sesquiterpenes marginatin, previously isolated from V. marginata,  76  marginatin-methacrylate previously isolated from V. arkansana,  11  and 8-deacyl respectively. These  compounds had not been fully characterized so Table 3.11 was constructed as per the vernonataloides. The 'ff and  1 3  C spectra for 229 and 230 are found in Figures 3.26 and  3.27.  149  Table 3.11: Marginatin (229) and Marginatin Methylacrylate (230) N M R Data in C D C 1 at 233K 3  No.  Carbon  1 2 3 4 5  128.7 20.2 36.3 61.2 66.3  6  82.7  7 8  166.8 71.7/ 69.3$ 45.5  (229)  9 10 11 12 13 14 15 OR  OAc  Proton  H M B C Correlations  (229)  (229)  5.63 bs 1.56 bs 1.47 bs  a  —  2.37 m, 2.15 m 4.98 bd, 4.90 # b  —  130.9 127.8 171.2 55.9/ 55.0$ 16.9 16.3 166.6 139.8 126.9  C-6 C-14  —  a  —  —  —  —  —  1.81 s 1.30 s  C-9,C-l C-5, C-4, C-3  c  —  —  6.85 b r s 3  166.6  —  14.8 11.9 170.7 20.8  1.80 s 1.76 s —  2.90 brt, 2.63 bd  130.8 127.9 171.2 55.9/ 55.0$ 16.9 16.3 165.9 134.6 127.6  —  170.7  —  4.90 #  170.7 21.0  —  2.00 s / 2.02 s$  b  18.3  126.9 166.6, 139.8  c  2.37 m, 2.15 m 4.95 bd, 4.91 #  — — —  4.85 # 1.83 s 1.30 s b  a  —  5.64 bs 1.56 bs 1.47 bs —  3  C-12, C - l l  4.86#  b  166.3 71.9/ 69.6$ 45.4  —  4.88#/ 3.62 br s$ 2.88 brt, 2.62 bd  3  82.6  C-7  Proton  (230)  (230) 128.6 20.3 36.3 61.3 66.2  C-10, C - l C-5, C-4  —  Carbon  —  —  6.09 s 5.68 s 1.88 s — —  2.03 s  ' H N M R at 500 M H z ; C at 100 M H z . "° Uncertain assignments due to multiple conformers present and/or overlapping signals, those shifts with the same letters may be switched. # Overlapping signals. Not all H M B C correlations are assigned due to multiple conformers present. J Multiple shifts due to conformers present at 233 K . Ester correlations are given to the carbon's 8 value (in ppm). 1 3  a  229  230  150  N  TPFT D  ^/  /  O  <  3JAJ-0V  X-  CN  oo  00  X  o r-H  = 3 H §11  0=0 !I 'L 0=3 '31 S  151  1  152  3.4.3.3: A Bourbenolide Sesquiterpene: Compound 231  Another peak from the H P L C yielded sesquiterpenoid 231, which gave a m/z [ M + H ] i n the H R D C I M S spectrum o f 405.1912, appropriate for a molecular formula o f +  1  13  C22H28O7 and accounting for eight degrees o f unsaturation. Inspection o f the H and  C  N M R data suggested the presence o f a tiglate ester (8 166.6, 8 127.6, 8 6.90, 8 1.84 and a doublet at 8 1.82 ppm; J = 7.0 H z ) , an acetate (8 2.03 ppm, singlet, 3H), and a hydroxymethyl (two doublets—8 4.88 and 8 4.74 ppm; J = 12.2 H z ; 8 54.7 ppm). The 13  carbonyls for both the tiglate and the acetate were presumably not seen i n the ' X spectrum due to a dilute sample. There were also three carbinol carbons, 8 69.7, 8 81.4 and 8 89.3 ppm. A t least two o f these carbons could be i n an ester or a lactone. The signal at 8 116.8 ppm i n the  1 3  C spectrum is likely an olefinic carbon though its partner  was not observed, possibly again due to low sample concentration. Assuming that 231 is similar to the other sesquiterpenes we have seen thus far, there should be an additional carbonyl present i n an a,p-unsaturated-y-lactone. Presuming that this is the case, we have accounted for six o f the nine degrees o f unsaturation. Consultation o f the H N M R spectrum showed it to be fairly simple with a large {  amount o f fine structure visible. Other than multiplets at 8 1.85 and 8 2.00 ppm, there was a triplet at 8 2.37 ppm (J = 7.1 H z ) , a pair o f doublets at 8 2.58 ppm (J = 8.3 H z ) , a doublet at 8 2.68 ppm (J = 6.4 H z ) , and a triplet at 8 6.34 (J = 9.6 Hz). There were also two aliphatic methyl signals at 8 1.08 and 8 1.22 ppm, both singlets. The remaining broad 153  singlet at 8 2.93 ppm may be an alcohol, which would account for the final oxygenated carbon. From the coupling constants, we surmised that the triplet at 8 2.37 ppm was probably coupled to the doublet at 8 2.68 ppm and the triplet at 8 6.34 ppm was likely coupled to 8 2.58 ppm, which was probably coupled to an additional proton. W i t h no other degrees o f unsaturation obvious from the I D N M R spectra, the remaining three would have to be accounted for by a three-ring system. A careful comparison o f the ' H N M R shifts o f 231 with those o f known Vernonia metabolites suggested that it was the known compound acetoxy-8a-tiglinoyloxy-4|3-hydroxy-bourbon-7(l l)-en-6,12-olide previously isolated from V. arkansana.  11  This compound had not been assigned  1 3  C  chemical shifts, so we completed H M Q C and H M B C experiments, which confirmed that 231 was acetoxy-8a-tiglinoyloxy-4p-hydroxy-bourbon-7(ll)-en-6,12-olide. Table 3.12 contains the results o f these experiments and our full N M R assignments for this compound, while the I D N M R spectra can be found i n Figure 3.28. Compound 231 is o f the bourbonane skeletal-type, a highly cyclized skeleton believed to be formed from intramolecular attack o f the double bond at C - l / C - l 0 in 229 with the epoxide at C-4/C-5 as depicted in Scheme 3.4.  72d  Scheme 3.4: Biogenesis o f Compound 231 from Marginatin (229). T i g = Tiglate.  154  Table 3.12: Acetoxy-8a-tiglinoyloxy-4p-hydroxy-bourbon-7(l l)-en-6,12-olide (231) N M R Data i n C D C 1 at 273 K 3  No.  Carbon  Proton  1  42.4  2.37 t, J = 7.1 H z  2  25.5  1.85 m#  3  40.1  1.85 m#  4  81.4  5  50.3  H M B C Correlations C-10, C - 6 , C - 5 , C-3 C-10*,C-5*,C-1* C-5*, C - l *  —  —  2.68 d, J = 6.4 H z  C-15, C-10, C - 7 , C - 6 , C-4, C - 3 , C-l  6  89.3  —  —  7  173.0 69.7  —  —  8 9  51.0  6.34 t, J = 9.6 H z 2.58 pair o f d , J = 8.3 H z , 2.00 m  C-12, C - l l , C - 9 , C-7 C - l 1 (from 2.00), C-10, C - 8 , C-7 (from 2.58), C - l  10  47.2  —  —  11  116.8  —  —  12 13  170.7 54.7  —  —  4.88 d, J = 12.2 H z  •  170.2 ( A c ) , C-12, C - l l , C-7  4.74 d, J = 12.2 H z 14  15.4  15  27.7  OH OR  OAc  —  1.08 s 1.22 s  C-10, C-9, C - 6 , C - l C - 5 , C-4, C-3 —  2.93 bs  166.6  —  -—  127.6  —  —  139.6  6.90 qm, J = 5.0 H z  14.6  1.84 s  12.0  1.83 d, J = 10.9 H z  170.2  —  166.6, 14.6, 12.0 166.6, 139.6 166.6, 139.6, 127.6 —  170.2 20.7 2.03 s H N M R at 500 M H z ; C at 100 M H z . Correlations are from both protons unless otherwise marked. Ester correlations are given to the carbon's 5 value (in ppm). * Uncertain assignments due to the overlapping peaks for H-2 and H-3 in the 5 1.85 ppm region. # Overlapping peaks.  155  156  3.4.3.4: 8-Desacylglaucolide A-tiglate: Compound 232  The final sesquiterpene from V. baldwinii gave a m/z for [M+H] i n the H R D C I M S of 479.1907 corresponding to a molecular formula o f C24H30O10 and nine degrees of unsaturation. This compound was also cooled to -40° C (233 K ) to sharpen the N M R spectra.  The I D N M R spectra o f 232 were quite similar to those of marginatin  (229), a compound whose molecular formula differed from that of 232 by C2H2O3. A n inspection o f the  l 3  C N M R data (at 243 K ) showed the presence o f a ketone (5 206.8  ppm) and based on the number o f carbonyl signals at 8 ~ 170.0 ppm, there appeared to be two acetates present rather than the single one present i n 229. A n additional acetate and a ketone would account for the difference o f C2H2O3. This would mean that 232 was related to glaucolide B (260) or glaucolide A (261), differing only i n the ester adjacent to 78  the a,P-unsaturated-y-lactone. A detailed N M R anaylsis o f 260 has been published, the *H and  1 3  so  C N M R chemical shifts we obtained for 232 were compared with the  literature values for 260. The chemical shifts for the sesquiterpene core o f 260 and 232 were essentially identical, (see Table 3.13), with the only difference being the presence o f a tiglate, (compared with known  C values), ester at C-8 instead o f an acetate. In  addition, the chemical shift values o f the tiglate in 232 were compared with those obtained for marginatin (229). Compound 232 was determined to be the known compound 8-desacylglaucolide A-tiglate (or 8-desacylglaucolide B-tiglate) previously isolated from V. erdverbengii.  157  Table 3.13: 8-Desacylglaucolide A-tiglate (232) N M R Data in C D C 1 at 233/243 K Compared with Literature Data for Glaucolide B (260) at 2 6 0 K 3  No.  Carbon  Proton  (232)  (232)  1 2  206.82 32.51  3  31.21  4 5 6 7 8 9  61.54 58.31 80.57 164.51 63.88 39.68  10 11 12 13  84.24 123.95 170.02 54.66  14 15 OAc,  18.57 20.82 171.42 22.34 170.62 21.03 166.70 126.53 140.57 15.01 11.92  OAc OR  2  —  2.29 b m not obs not obs not obs 2.92 ddd J = 14.9 H z , J = 10.5 H z J = 4.6 H z 2.59 dd not obs J = 13.2 H z J = 4.9 H z 1.62 m —  2.80 d, J = 9.6 H z 4.92 d, J = 9.5 H z —  4.66 d, J = 8.5 H z 2.77 m not obs 2.25 bm — — —  4.77 d, J = 4.71 d, J = 1.50 1.63  12.0 H z 12.0 H z s s  —  No.  (260)  (260)  206.81 32.53  3  31.28  4 5 6 7 8 9  61.44 58.37 80.59 162.86 63.81 39.80  10 11 12 13  84.33 124.62 169.81 54.88  14 15 OAc,  18.66 20.31 170.95 22.25 170.32 20.99 170.16 20.99  OAc  2  2.08 s —  Proton  1 2  2.08 s —  Carbon  OAc  —  — 6.87 qm, J = 6.3 H z — 1.77 s — 1.82 d, J = 7.0 H z ' H N M R at 500 M H z , 233 K ; C at 100 M H z , 243 K . li  158  3  —  2.27 ddd J = 17.2 H z , J = 4.6 H z , J = 2.9 H z 2.96 ddd J = 17.4 H z , J = 12.7 H z , J = 4.9 H z 2.60 ddd J = 17.8 H z , J = 12.9 H z , J = 4.9 H z ; 1.67-1.62 m —  2.83 d, J = 9.6 H z 4.89 d, J = 9.6 H z —  4.69 d, J = 8.6 H z 2.76 dd, J = 16.2 H z J = 8.6 H z 2.25 d, J = 16.2 H z — — —  4.91 d, J = 4.84 d, J = 1.54 1.65  12.7 H z 12.7 H z s s  —  2.11 s —  2.12 s —  2.16 s  —  —  —  —  —  —  159  3.4.4: Conclusions: Alzheimer's Disease, Mitosis, and Vernonataloide (47) The T G - 3 antibody used i n Roberge's G 2 checkpoint and antimitotic bioassays was originally developed to study tau,  74  a low molecular weight protein associated with  Alzheimer's Disease ( A D ) , a disease which affects an estimated 20 million people worldwide. A D is characterized by a progressive loss o f memory resulting i n dementia and death.  80  It has been shown that hyperphosphorylated tau is the major component o f  paired helical filaments ( P H F ) a pathological characteristic o f A D . " 8 0  major component o f neurofibrillary tangles,  81  P H F ' s are the  8 2  which i n conjunction with P-amyloid  plaques formed from the neurotoxic (in high concentration) and proinflammatory  [}-  amyloid protein, are the two hallmarks o f A D . Tau is a protein usually associated with 83  microtubules where it functions as a stabilizing agent, i n turn stabilizing the internal structure o f neurons. Abnormal phosphorylation o f tau in Alzheimer's disease abolishes its ability to bind microtubules and stimulates the formation o f P H F ' s . ' 8 1  8 2  In normal tau  proteins there are approximately thirty serine or threonine phosphate acceptor residues 82  which can be phosphorylated, representing about half o f all possible sites.  However,  normally only three o f these sites per mole o f protein are actually phosphorylated, in contrast with eleven per mole i n the abnormal tau found i n P H F ' s .  7 4  These  phosphorylation sites are clustered i n regions adjacent to those where microtubule binding occurs, thus hyperphosphorylation o f tau inhibits microtubule binding. This has been shown, for example, for serine-262 and serine-396, which are located in the first and fourth microtubule binding domains. Since the phosphorylation state o f tau is important in A D pathology, most research has focussed on the regulation o f this state by protein kinases and phosphatases,  160  though it is important to note that most o f these studies provide only circumstantial evidence. Recently, two kinases including G S K - 3 p kinase, have be implicated in in vivo regulation o f tau phosphorylation. G S K - 3 p kinase is a serine/threonine kinase that is found i n the brain i n abundance and is known to associate with microtubules.  Curman  has shown that G S K - 3 P kinase may be involved i n a currently unidentified role in the G 2 checkpoint pathway.  84  Additionally, Vincent, et al, showed i n 1996 that several  antibodies raised to bind to hyperphosphorylated tau, especially the T G - 3 antibody, also showed pronounced immunoreactivity with human neuroblastoma cells i n mitotic phase. Furthermore, this immunoreactivity was exhibited throughout the cytoplasm with no localization on the mitotic spindle, (the structure formed when the microtubules meet the chromosomes i n the centre o f the cell), or on the chromosomes. Similar results were observed with cells arrested in mitosis with the known microtubule-destabilizing agent nocodazole (237).  74  Additionally, A P P (from which p-amyloid protein can be formed)  and tau may be linked b y p i n l , a prolyl isomerase that enhances the rate o f cis to trans isomerization o f the peptide bond on the amino side o f proline in various proteins, and also stops entry o f cells into mitosis.  85  P i n l binds specifically to phosphorylated serine  or threonine residues preceding a proline residue such as the threonine-231 site on tau 86  which is hyperphosphorylated in A D brains, but not i n normal brains.  The  concentration o f soluble p i n l is vastly reduced i n A D , apparently through binding to the hyperphosphorylated tau i n P H F ' s .  8 7  Depletion o f soluble P i n l in non-neuronal cells  leads to mitotic arrest and cell death b y apoptosis. mechanism also occurs in A D .  on  161  850  It has been suggested this  This investigation began as a search for antimitotic or G 2 checkpoint inhibitory compounds, but has progressed far beyond that point. Vernonataloide (47) and its tiglate analogue 227 have been determined not to have the originally desired biological activity, but to be possible inducers o f A D . This exciting possibility was discovered through the use o f a biological assay that utilizes an antibody which recognizes a phosphoepitope on tau and on nucleolin, a connection that has been previously documented. ' 41  74  Furthermore, we have determined that only those sesquiterpenes with two epoxides, 47 and 227, are active i n this manner. The other compounds we isolated are too cytotoxic and k i l l the cells before any immune response can be seen. Vernonataloide (47) is approximately three-fold more active that 227 (-30 p.g/mL versus - 1 0 0 |ig/mL), which is probably due to methyl acrylate being a better Michael acceptor than tiglate. The ester portion o f the molecule is important for recognition, as was shown by the reduction o f the methyl acrylate ester to isobutyrate (in 228) which rendered the molecule inactive. It is  47  227  228  Imporant  Figure 3:30: Structural Differences o f the Vernonataloides. believed that the concurrent loss o f the acetate and conversion o f the hydroxymethyl to a methyl group does not affect the biological activity. This invesitgation has led us to a point where we were interested in determining what biochemical information about tau or A D can be discovered through the use o f 47 as a probe. W e have recently begun a collaborative study with Dr. Peter Davies, a world leader in this area, to determine what  162  the actual biological targets are. In addition, we would like to see i f it is possible to induce A D in an animal model system so as to better understand its pathology.  3.5: References 1)  a) Haynes, R . K . ; Vonwiller, S.C. Acc. Chem. Res. 1997, 30, 73-79. b) Avery, M . A . ; M c L e a n , G . ; Edwards, G . ; Ager, A . In Biologically Active Natural Products: Pharmaceuticals. Cutler, S.J.; Cutler, H . G . ; Eds. C R C Press: Boca Raton, F L , 2000, pp. 121-132. c) W u , Y . Acc. Chem. Res. 2002, 35, 255-259.  2)  Fraga, B . Nat. Prod. Rep. 2002,19, 650 - 672, and previous reviews i n this series.  3)  Faulkner, D.J. Nat. Prod. Rep. 2002,19, 1-48, and previous reviews i n this series.  4)  Fischer, N . H . In Methods in Plant Biochemistry; Charlwood, B . V . and Banthorpe, D . V , Eds.; V o l . 7. Academic Press: San Diego, 1991, pp. 187-211. Dirsch, V . M . ; Shipper, FL; Ellmerer-Miiller, E.P.; Vollmar, A . M . Bioorg. Med. Chem. 2000, 8, 21'41'-2753, and references therein.  5)  6)  K w o k , B . H . B . ; K o h , B . ; Ndubuisi, M . I . ; Elofsson, M . ; Crews, C M . Chem. Biol. 2001, 8, 759-766, and references therein.  7)  Dewick, P . M . Medicinal Natural Products: A Biosynthetic Approach. John W i l e y and Sons: West Sussex, U K , 1997, pp. 172-184.  8)  Budesinsky, M . ; Saman, D . In Annual Reports on NMR Spectroscopy; Webb, G . A . , E d . ; V o l . 30. Academic Press: San Diego, 1995, pp. 231-475.  9)  Herz, W . ; Romo De Vivar, A . ; Romo, J.; Viswanathan, N . J. Am. Chem. Soc. 1963,55, 19-26.  10)  Kupchan, S . M . ; Hemingway, R.J.; Werner, D . ; K a r i m , A . J. Org. Chem. 34, 3903-3908.  11)  Kupchan, S . M . ; Eakin, M . A . ; Thomas, A . M . J. Med. Chem. 1971,14, 11471152.  12)  Hall, I.H.; Lee, K . - H . ; M a r , E . C . ; Starnes, C O . ; Waddell, T . G . J. Med. Chem. 1977, 20, 333-337.  13)  Woynarowski, J . M . ; Konopa, J. Molec. Pharmacol.  163  1981,19, 97-102.  1969,  Woynarowski, J . M . ; Beerman, T . A . ; Konopa, J. Biochem. Pharmacol. 3005-3007.  1981, 30,  Burim, R . V . ; Canalle, R.; Callegari Lopes, J.L.; Takahashi, C S . Genetics Mol. Bio. 1999,22,401-406. See for example: a) Kupchan, S . M . ; Fessler, D . C ; Eakin, M . A . ; Giacobbe, T.J. Science 1970, 168, 376-378. b) Hanson, R . L . ; Lardy, H . A . ; Kupchan, S . M . Science 1970,168, 378-380. c) Lee, K . - H . ; H a l l , I.H.; Mar, E . - C ; Starnes, C O . ; ElGebaly, S.A.; Waddell, T . G . ; Hadgraft, R.I.; Ruffner, C . G . ; Weidner, I. Science 1977,196, 533-536. Schmidt, T.J.; Lyl3, G . ; Pahl, H . L . ; Merfort, I. Bioorg. Med. Chem. 1999, 7, 2849-2855, and references therein. LyB, G . ; Knorre, A . ; Schmidt, T.J.; Pahl, H . L . ; Merfort, I. J. Biol. Chem. 273, 33508-33516, and references therein.  1998,  Rungeler, P.; Castro, V . ; Mora, G . ; Goren, Vichnewski, W . ; Pahl, H . L . ; Merfort, I.; Schmidt, T.J. Bioorg. Med. Chem. 1999, 7, 2343-2352, and references therein. Schmidt, T.J. Bioorg. Med. Chem. 1997, 5, 645-653. Pieman, A . K . ; Towers, G . H . N . Biochem. System. Ecol. Pieman, A . K . Biochem. System. Ecol.  1983,11, 321-327.  1984, 72,13-18.  Fischer, N . H . L u , T.; Cantrell, C . L . ; Castanada-Acosta, J.; Quijano, L . ; Franzblau, S.G. Phytochemistry 1998, 49, 559-564. Lucas, R . A . ; Rovinski, S.; Kiesel, R J . ; Dorfman, L . ; MacPhillamy, H . B . J. Org. Chem. 1964,29, 1549-1554. H a l l , I.H.; Starnes, C O . ; Lee, K . - H . ; Waddekk, T . G . J. Pharm. Sci. 1980, 69, 537-543. Patel, B.P.; Waddell, T . G . ; Pagni, R . M . Fitoterapia  2001, 72, 511-515.  Reina, M . ; Gonzalez-Coloma, A . ; Gutierrez, C ; Cabrera, R.; Rodriguez, M . L . ; Fajardo, V . ; Villarroel, L . J. Nat. Prod. 2 0 0 1 , ^ , 6 - 1 1 . Rugutt, J . K . ; Rugutt, K . P . J. Agri. Food. Chem. 1997, 45, 4845-4849. Yoshikawa, M . ; Shimoda, H . ; Uemura, T.; Morikawa, T.; Kawahara, Y . ; Matsuda, H . Bioorg. Med. Chem. 2000,5,2071-2077.  164  Baldwin, A . S . Annu. Rev. Immunol. 1996,74,649-681. Behl, C ; Davis, J.; Lesley, R.; Schubert, D . Cell 1994, 77, 817-827. Blanco, J.G.; G i l , R . R . ; Bocco, J . L . ; Meragelman, T . L . ; Genti-Raimondi, S.; Flury, A . Pharmacol. Exp. Ther. 2001, 297, 1099-1105. Roberge, M . ; Berlinck, R . G . S . ; X u , L . ; Anderson, FL; L i m , L . ; Curman, D . ; Stringer, C . M . ; Friend,S.H.; Davies, P.; Vincent, I.; Haggarty, S.J.; K e l l y , M . T . ; Britton,R.; Piers, E . ; Andersen, R . J . Cancer Res. 1998, 5c?, 5701-5706, and references therein. Roberge, M . Personal communication. Roberge, M . ; Cinel, B . ; Anderson, H . J . ; L i m , L . ; Jiang, X . ; X u , L . ; B i g g , C M . ; K e l l y , M . T . , Andersen, R . J . Cancer Res. 2000, 60, 5052-5058. a) Crews, C M . ; Mohan, R. Cur. Op. Chem. Biol. 2000, 4, 47-53. b) Shapiro, G.I.; Harper, J.W. Clin. Invest. 1999,104, 1645-1653. c) Stewart, Z . A , ; Pietenpol, J . A . J. Mamm. Gland Biol. Neo. 1999, 4, 389-400. d) Nurse, P. Cell 1997, 91, 865-867. e) Hung, D . T . ; Jamison, T.F.; Schreiber, S.L. Chem. Biol. 1996, 3, 623-639. f) Kaufman, W . K . ; Paules, R . S . FASEB J. 1996,10, 238-247. a) Moser, B . A . ; Brondello, J . M . ; Barber-Fornari, B . ; Russell, P. Molec. Cell. Bio. 2000, 20, 4288-4294. b) Downes, C.S.; Musk, S.R.R.; Watson, J.V., Johnson, R . T . J. Cell Biol. 1990, 110, 1855-1859. c) Buse, P . M . ; Bose, S.K.; Jones, R . W . ; Tolmach, L . J . Radiat. Res. 1978, 76, 292307. Fan, S.; Smith, M . L . ; Rivet, D.J.; Duba, D . ; Zhan, Q.; K o h n , K . W . ; Fornace, J . A . ; O'Connor, P . M . Cancer Res. 1995, 55, 1649-1654. Tarn, S.W.; Schlegel, R. Cell Growth Differ.  1992, 3, 811-817.  a) Graves, P.R.; Y u , L . ; Schwarz, J . K . ; Gales, J.; Sausville, E . A . ; O'Connor, P . M . ; Piwnica-Worms, H . J. Biol. Chem. 2000, 275, 5600-5605. b) Wang, Q.; Fan, S.; Eastman, A . ; Worland, P.J.; Sausville, E . A . ; O'Connor, P . M . JNCI1996, 88, 956-965. c) Kawakami, K . ; Futami, H . ; Takahara, J.; Yamaguchi, K . Biochem. Biophys. Res. Comm. 1996, 219, 778-783. d) M i z u n o , K . ; Noda, K . ; Ueda, Y . ; Hanaki, H . ; Saido, T . C . ; Ikuta, T.; K u r o k i , T.; Tamaoki, T.; Hirai, S.; Osada, S.; Ohno, S. FEBSLetters 1995, 359, 259-261.  165  Anderson, H.J.; de Jong, G . ; Vincent, I.; Roberge, M . Exp. Cell Res. 1998, 238, 498-502. Berlinck, R . G . S . ; Britton, R.; Piers, E . ; L i m , L . ; Roberge, M . ; da R o c h a , R . M . ; Andersen, R . J . J. Org. Chem. 1998, 63, 9850-9856. Curman, D . ; Cinel, B ; Williams, D . E . ; Rundle, N . ; Block, W . D . ; Goodarzi, A . A . ; Hutchins, J.R.; Clarke, P.R.; Zhou, B . - B . ; Lees-Miller, S.P.; Andersen, R . J . ; Roberge, M . J.Biol. Chem. 2001,276, 17914-17919. Rundle, N . T . ; X u , L . ; Andersen, R.J.; Roberge, M . J. Biol. Chem. 2001, 276, 48231-48236. Nelson, J. M S C Thesis, University o f British Columbia Department o f Chemistry, 2002. Jordan, A . ; Hadfield, J.A.; Lawrence, N . J . ; M c G o w e n , A . T . Med. Res. Rev. 1998, 18, 259-296. a) U v a , R . H . ; Neal, J.C.; DiTomaso, J . M . Weeds of the Northeast. Cornell University Press: Ithaca, N Y , 1997, pp. 108-109. b) Great Plains Flora Association. Flora of the Great Plains. University Press o f Kansas: Lawrence, K A , 1986, pp.855-858. Claus, E.P.; Tyler, V . E . ; Brady, L . R . Pharmacognosy, Philadelphia, 1970, pp.446-449.  6 E d . Lea & Febiger: th  Herz, W . ; Hogenauer, G . J. Org. Chem. 1961, 26, 5011-5013. Porter, T . H . ; Mabry, T.J. Phytochemistry  1969, 8, 793-794.  Bianchi, E . ; Culvenor, C.J.; Loder, J . W . Aust. J. Chem. 1968, 21, 1109-1 111. Porter, T . H . ; Mabry, T.J. Yoshioka, H . ; Fischer, N . H . Phytochemistry 199-204. Oberti, J.C.; Silva, G . L . ; Sosa, V . E . ; Kulanthaivel, P.; Herz, W . 1986, 25, 1355-1358, and references therein.  1970, 9,  Phytochemistry  Inayama, S.; Ohkura, T.; Kawamata, T.; Yanagita, M . Chem. Pharm. Bull. 22, 1435-1436.  1914,  Stefanovic, M . ; Jokic, A . ; Behbud, A . ; Jeremic, D . Bull. T. LIVde I'Acad. Serbe des Sci. et des Arts CI. Sci. Math. Nat. Sci. Nat. 1976, 54, 43-52.  166  Stefanovic, M . ; Aljancic-Solaja, I.; Milosavljevic, S. Phytochemistry 850-852, and references therein.  1987, 26,  Bloszyk, E . ; Rychlewska, U . ; Szczepanska, B . ; Budesinsky, M . ; Drozdz, B . ; Holub, M . Collect. Czech. Chem. Commun. 1992,57,1092-1102. David, J.P.; de O. Santos, A . J.; da S. Guedes, M . L . ; David, J . M . ; Chai, H . - B . ; Pezzuto, J . M . ; Angerhofer, C . K . ; Cordell, G . A . Pharm. Biol. 1999, 37, 165-168, and references therein. Silva, G . L . ; Oberti, J.C.; Herz, W . Phytochemistry  1992,37,859-861.  Borges del Castillo, J.; Manresa Ferrero, M . T . ; Martin Ramon, J.L.; Rodriguez Luis, F.; Vazquez-Bueno, P.; Joseph Nathan, P. Org. Magn. Reson. 1981, 7 7, 232-234, and references therein. a) M i l l e r , H . E . ; Kagan, H . B . ; Renold, W . ; Mabry, T.J. Tetrahedron Lett. 1965, 38, 3397- 3403. b) Mabry, T.J.; Miller, H . E . ; Kagan, H . B . ; Renold, W . Tetrahedron 1966, 22, 1139-1146. Mabry, T.J.; Kagan, H . B . ; M i l l e r , H . E . Tetrahedron 1966, 22, 1943-1948. Kagan, H . B . ; Miller, H . E . ; Renold, W . ; Lakshmikantham, M . V . ; Tether, L . R . ; Herz, W . ; Mabry, T.J. J. Org. Chem. 1966, 31, 1629-1632. Rodriguez, J.G.; Perales, A . J. Nat. Prod.  1995, 55, 564-569.  For example, the ethylation o f altamisic acid likely occurred i n this isolation: Borges, J.; Manresa, J, L . ; Martin, J.L.; Pascual, C ; Vaquez, P. Tetrahedron Lett. 1978,1513-1514. Dewick, P . M . Medicinal Natural Products: A Biosynthetic Approach. John W i l e y and Sons: West Sussex, U K , 1997, p.26-27. Lee, I-S.; Shamon, L . A . ; Chai, H . - B . ; Chagedera, T . E . ; Besterman, J . M . ; Farnsworth, N . R . ; Cordell, G . A . ; Pezzuto, J . M . ; Kinghorn, A . D . Chem.-Biol. Interact. 1996, 99, 193-204. Procedure based on that of: Britton, R.; de Silva, E . D . ; B i g g , C M . ; McHardy, L . M . ; Roberge, M . ; Andersen, R . J . J. Am. Chem. Soc. 2001, 723, 8632-8633. Bohlmann, F.; Zdero, C . Phytochemistry  1982, 27, 2263-2267.  Abdel-Baset, Z . H . ; Southwick, L . ; Padolina, W . G . ; Yoshioka, H . ; Mabry, T . J . Phytochemistry, 1971,10, 2201-2204, and references therein.  167  Padolina, W . G . ; Yoshioka, H . ; Nakatani, N . ; Mabry, T.J.; Monti, S.A.; Davis, R . E . ; C o x , P.J.; S i m , G . A . ; Watson, W . H . ; W u , L B . Tetrahedron 1974, 30, 11611170. See for example: a) Abegaz, B . M . ; Keige, A . W . ; Diaz, J.D.; Herz, W . Phytochemistry 1994, 37, 191-196. b) Zdero, C ; Bohlmann, F.; Wasshausen, D . C ; Mungai, M . G . Phytochemistry 1991, 30, 4025-4028. c) Catalan, C . A . N . ; De Iglesias, D . I . A . ; Javka, J.; Sosa, V . E . ; Herz, W . Phytochemistry 1988, 27, 197-202. d) Jakupovic, J.; Schmeda-Hirschmann, G . ; Schuster, A . ; Zdero, C ; Bohlmann, F.; K i n g , R . M . ; Robinson, H . ; Pickardt, J. Phytochemistry 1986, 25, 145-158. Great Plains Flora Association. Flora of the Great Plains. Kansas: Lawrence, K A , 1986, p. 1017. Vincent, I.; Rosado, M . ; Davies, P. J. Cell Biol.  University Press of  1996,132, 413-425.  Jakupovic, J.; Gage, D . A . ; Bohlmann, F.; Mabry, T.J. Phytochemistry 1179-1183.  1986, 25,  Padolina, W . G . ; Nakatani, N . ; Yoshioka, H . ; Mabry, T.J.; M o n t i , S . A . Phytochemistry 1974, 13, 2225-2229. Bohlmann, F.; Singh, P.; Borthakur, N . ; Jakupovic, J. Phytochemistry 2379-2382.  1981, 20,  V i d a l Costa, A . ; de A l m e i d a Barbosa, L . C . ; Callegari Lopes, J.L.; Pilo-Veloso, D . Magn. Reson. Chem. 2000,55,675-679. Dominguez, X . A . ; Cano, G . ; Sanchez, H . ; Velazquez, G . J. Nat. Prod. 704-705.  1986, 49,  Spillantini, M . G . ; Goedert, M . TINS 1998, 21, 428-433. a) Weaver, C . L . ; Espinoza, M . ; Kress, Y . Davies, P. Neurobiol. Aging 2000, 21, 719-727. b) Bramblett, G.T.; Goedert, M . ; Jakes, R.; Merrick, S.E.; Trojanowski, J.Q.; Lee, V . M . Y . Neuron 1993,10, 1089-1099. c) Goedert, M . ; Spillantini, M . G . ; Cairns, N . J . ; Crowther, R . A . Neuron 1992, 8, 159-168. d) Glenner, G . G . Cell 1988, 52, 307-308. Lee, V . M - Y . ; Goedert, M . ; Tronjanowski, J.Q. Ann. Rev. Neuroscience 1121-1159, and references therein.  168  2001, 24,  83)  Carlson, C D . ; C z i l l i , D . L . ; Gitter, B . D . Neurobiol. Aging 2000, 21, 747-756.  84)  Curman, D . M S C Thesis, University o f British Columbia Department o f Biochemistry and Molecular Biology, 2000.  85)  a) Yaffe, M . B . ; Schutkowski, M . ; Shen, M . ; Zhou, X . Z . ; Stukenberg, P.T.; Rahfeld, J.-U.; X u , J.; Kuang, J.; Kirschner, M . W . ; Fischer, G . ; Cantley, L . C . ; L u , K . P . Science 1997, 278, 1957-1960. b) Shen, M . ; Stukenberg, P.T.; Kirschner, M . W . ; L u P . K . Genes Dev. 1998,12, 706-720. c) Zhou, X . Z . ; L u , P.-J.; Wulf, G . ; L u , K . P . Cell. Mol. Life Sci. 1999, 56, 788-806.  86)  L u , P.-J.; Zhou, X . Z . ; Shen, M . ; L u , P . K . Science 1999, 283, 1325-1328.  87)  L u , P.-J.; Wulf, G . ; Zhou, X . Z . ; Davies, P.; L u , K . P . Nature 1999, 399, 784-88.  3.6: Experimental Section 3.6.1: General Information A l l reagents and solvents (except for N M R experiments) were purchased from either Fisher Scientific or Sigma-Aldrich and were used without further purification. The only exception was that H P L C solvents were filtered through a 0.45 um filter (Osmonics, Inc.) prior to use. When a gradient H P L C system was used the solvents were sparged with helium (Praxair, Inc., Vancouver, B C ) as well. N M R solvents were purchased from Cambridge Isotopes Laboratories, and for this chapter, "100 % grade" CDCI3 (deuterochlorform) was used for all compounds except 41 where "100 % grade" DMSO-d6 was used. Proton chemical shifts were referenced to the residual solvent peak at 5 7.24 ppm and carbon chemical shifts to 8 77.0 ppm for CDCI3 and 8 2.49 ppm and 8 39.5 ppm for DMSO_d6- N M R data were acquired with the following spectrometers each 13  equipped with a 5mm probe:  C data was acquired with a Bruker A M - 4 0 0 spectrometer  (direct detection probe); *H spectra were acquired with a Bruker A M X - 5 0 0 with an inverse detection probes and low temperature cooling system (detailed below). 2 D N M R  169  experiments were C O S Y - 4 5 ( ' H - ' H correlations), H M Q C ( C - ' H single-bond 1 3  correlations), and H M B C ( C - ' H two- and three-bond correlations) and were acquired on 1 3  a Bruker A M X - 5 0 0 spectrometer equipped with a L i q u i d N 2 cooling system for low temperature work. D C I mass spectra, both high and low resolution, were acquired with either a Kratos Concept II H Q Mass Spectrometer or a Kratos M S 80 Mass Spectometer with the assistance o f the U B C Mass Spectrometry Centre staff.  The carrier gas utilized  for D C I M S was any one o f the following: C H , C H 4 / N H 3 , isobutane, or isobutane/NH3. 4  Reverse-phase Cig silica Sep-Paks were obtained from Waters, Inc., while normal phase 230-400 mesh silica gel was acquired from Silicycle (Quebec City, P Q ) . H P L C systems utilized were one o f the following: Waters 996 Photodiode Array detector/600E Series pump and system controller/Millennium™ 2010 software; Waters 486 Tunable Absorbance Detector/600E Series pump and system controller; Waters 2487 dual channel detector and system controller/Waters Series 515 pump; Waters 440 detector/Waters 501 Series pump. The last three systems were attached to chart recorders with a paper feed at 0.25 cm/min. W i t h any H P L C system, the wavelength(s) monitored were 220 nm and/or 254 nm with a flow rate o f 2.0 m L / m i n , (except for the Rad-Pak where the flow rate was at 1.0 mL/min). The reverse-phase H P L C columns utilized were a Whatman Partosil O D S - 3 Cig column or a Waters R C M 8 x 1 0 Rad-Pak. Thin-layer chromatography ( T L C ) plates were Whatman M K C 1 8 F (reverse-phase) and Kieselgel 6OF254 (normal phase). Compound visualization on T L C plates was observed by short-wavelength U V at 254 nm, or by spraying with a solution o f 90% E t O H / 1 0 % concentrated H 2 S O 4 plus a small amount o f vanillin added, (coloured spots appear upon heating).  170  3.6.2: Isolation T w o approximately two-gram vials o f the methanolic extract N35791 were obtained from the United States National Cancer Institute (NCI). The extract i n the first vial was suspended i n water and extracted with hexanes, followed by D C M , and then E t O A c . The resulting gummy substance ( D C M fraction) was fractionated sequentially via normal phase silica flash chromatography ( D C M to E t O A c followed by washes o f M e O H and acetone) and reverse-phase H P L C (25% M e C N / H 0 ) to yield 42 and 44. 2  Almost pure 43 was isolated from the normal phase silica gel chromatography and purified b y reverse-phase H P L C (20% M e C N / H 2 0 ) . The polar solvent washes o f the silica flash chromatography were combined and subjected to reverse-phase Sep-Pak (Water to M e O H ; 41 eluted i n 25%> M e O H ) followed by repeated reverse-phase H P L C (25% M e C N / H 0 ; 10% M e C N / H 0 ) to yield pure 41. The extract i n second vial was 2  2  partitioned between D C M and water followed by flash chromatography as before. The fractions from the flash silica gel column were separated by reverse-phase H P L C (25%o M e C N / H 2 0 ) , with seven fractions obtained instead o f two. Fractions thee, six, and seven contained 42, 44, and 43, respectively, while four contained 45, and five contained 46. The methanolic extract (~10 grams) o f N6397 was obtained from the N C I ' s Open Repository. One-third o f this extract was suspended i n water and partitioned with D C M (4 x 200 m L ) . The active components were i n the D C M layer, which was concentrated in vacuo and a portion was loaded onto a normal phase silica gel column (gradient from hexanes to D C M to E t O A c ) . Bioactive fractions were combined and were subjected to reverse-phase H P L C to yield the known compounds 47 (7.1 mg), 227 (5.6 mg), and 229232 (13.3, 3.4, 0.2, and 3.4 mg, respectively).  171  Psilostachyin A (42), B (43), C (44), Paulitin (45), and Isopaultin (46): White solids; *H and  1 3  C N M R data i n Tables 3.1 and 3.2. M S data were as i n the literature. ' 53  Artemisiidiendioc A c i d Monomethyl Ester (41): Clear oil; ' H and  1 3  58,60  '  64  C N M R data i n  Table 3.3. D C I M S : m/z 327 (2), 309 (17), 277 (28), 85 (100). H R D C I M S : m/z [ M + ] , +  327.1442, (calculated for C 1 6 H 2 3 O 7 , 327.1444), [ M - H 0 ] , 309.1340, (calculated for +  2  C , H 0 , 309.1338), [ M - H 0 - M e O H ] , 277.1073, (calculated for C H i 0 , 277.1076). +  6  2 1  6  2  1 5  7  5  1  13  Psilostachyin A P-mercaptoethanol Adduct 223: Clear aromatic o i l ; H and  C NMR  data i n Table 3.4. D C I M S : m/z 376 (100), 359 (12), 332 (12), 298 (95). H R D C I M S : m/z [ M + N H ] , 376.1796, (calculated for C i H o 0 N S , 376.1798), [ M + ] , 359.1525, +  +  4  7  3  6  (calculated for C i 7 H 0 S , 359.1521). 2 7  6  A 3.6 mg portion o f 42 was stirred with 45 u L P-mercaptoethanol i n 1.5 m L T H F and 1 m L 0.1 M phosphate buffer solution for one hour. After dilution to 10 m L with water and partition with E t O A c ( 3 x 1 0 m L ) , the organic layer was concentrated in vacuo and purified by revered-phase H P L C (20% M e C N / H 0 ) to yield 0.7 m g o f 223. 2  Epoxide 224: Clear oil; *H and  1 3  C N M R data i n Table 3.5. D C I M S : m/z 296 (100).  H R D C I M S : m/z [ M + N H ] , 296.1495, (calculated for C i H 0 N , 296.1492). +  4  5  Epoxide 225: Clear oil; ' H and  1 3  2 2  5  C N M R data i n Table 3.6. D C I M S : m/z 296 (100).  H R D C I M S : m/z [ M + N H ] , 296.1493, (calculated for C i 5 H 0 N , 296.1488). +  4  2 2  5  A 1.9 mg portion o f 43 was treated with 3.8 m g o f m - C P B A in D C M at room temperature overnight. The excess peroxyacid was quenched with saturated N a S 0 and 2  2  3  N a H C 0 solutions. After extraction with D C M ( 3 x 3 0 m L ) and concentration o f the 3  organic layer in vacuo, the resulting solid was purified b y reverse-phased H P L C (25% M e C N / H 0 ) to yield two diastereomeric epoxides, 224 (0.5 mg) and 225 (0.8 mg). 2  172  Compound 226: Clear oil; *H and  1 3  C N M R data i n Table 3.7. D C I M S : m/z 294 (100),  276 (68), 259 (50) 250 (20). H R D C I M S : m/z [ M + N H ] , 294.1333, (calculated for +  4  C i H o 0 N , 294.1325), [ M + f , 276.1007, (calculated for C H , 6 0 , 276.1017), [ M 5  2  5  1 5  H 0 ] , 259.0973, (calculated for +  2  C15H.5O4,  5  259.0976).  A 3.6 m g portion o f 45 i n 1.5 m L T H F was treated with 10 drops 30% perchloric acid (HCIO4) for 4 hours. After addition o f 15 m L o f water the resulting acidic solution was neutralized with saturated NaHCO"3 and the T H F removed in vacuo. The resulting solution was extracted with E t O A c ( 2 x 1 5 m L ) , and the organic layer was concentrated in vacuo. The resulting mixture o f products was purified by reverse-phase H P L C (25% M e C N / H 0 ) to yield the major product 226 (0.5 mg), unreacted 45 and other products. 2  Vernonataloide (47), 8-deacylvernonataloide-8-0-tiglate (227), Marginatin (229) and Marginatin Methylacrylate (230), Acetoxy-8a-tiglinoyloxy-4p-hydroxy-bourbon7(ll)-en-6,12-olide (231), 8-Desacylglaucolide A-tiglate (232): White solids; ' H and l 3  C N M R data i n Tables 3.9 and 3.11-3.13. M S data were as i n the literature. ' ' ' 69  75  77  13-desacetoxy-8-deacylvernonataloide-8-0-isobutyrate (228): Clear o i l ; *H and  79  1 3  C  N M R data i n Table 3.10. D C I M S : m/z 379 (20), 351 (85), 263 (100). H R D C I M S : m/z [M+f, 351.1810, (calculated for C i H 0 , 351.1812). 9  2 7  6  A 1.0 m g portion o f 47 was dissolved i n E t O A c , and palladium on carbon, (Pd/C) was added. A balloon was filled with H and attached to a vial containing the 2  aforementioned solution. The H was allowed to flow through the vial for ten minutes. 2  Afterward, the P d / C was removed by suction filtration through a fine fritted filter and the E t O A c was removed under reduced pressure to give 0.4 m g o f 90% pure 228.  173  3.7: 2D N M R Spectra of Artemisiidiendioc A c i d Monomethyl Ester  4.0  4.8  5.6  (ppm)  6.4  5.6  4.8  4.0  3.2  2.4  1.6  6.4  Figure 3.31: C O S Y o f Artemisiidiendioc A c i d Monomethyl Ester (41) in D M S O . at 500 M H z . d 6  174  (ppm)  (ppm)  6.4  5.6  4.8  4.0  3.2  2.4  1.6  Figure 3.32: H M Q C o f Artemisiidiendioc A c i d Monomethyl Ester (41) i n D M S O . at 500 M H z . d 6  175  15  14  Figure 3.33: H M B C of Artemisiidiendioc Acid Monomethyl Ester (41) in D M S O . at 500 M H z . d6  176  4.1: Introduction to Gorgonians and Their Secondary Metabolites 4.1.1: Brief Introduction to Gorgonians Gorgonians belong to the phylum Cnidaria, an almost exclusively marine invertebrate phylum, which also includes the jellyfish, sea anemones, and corals (the "harder" forms o f which create coral reefs with their exoskeletons). Each o f the Cnidarian classes share the following characteristics: a single-opening digestive cavity, radial symmetry, two layers o f cells with a jelly-like substance called mesoglea between them, and nematocysts, which are stinging capsules used i n the capture o f prey.  1  Sea anemones  and corals are members o f the class Anthozoa with corals belonging to one o f two subclasses based on the number o f tentacles present i n the polyp. " These two classes are 1  2  Hexacorallia (with multiples o f six tentacles; "hard corals") and Octacorallia (usually with eight tentacles; alcyonaceans and gorgonians, the latter o f which we are more interested in). Gorgonians are actually colonies o f animals that often exist i n either a 2  plant-like or fan-like shape about a central skeleton. W h i l e the many varieties o f corals 1  are worldwide i n distribution, the West Indies region (also known as the Caribbean) contains many members o f the order Gorgonacea (the gorgonians) only about 20% o f which have been chemically studied. " The natural products chemistry o f gorgonians is 3  4  primarily diterpene in origin , although sesquiterpenes and steroids such as gorgosterol 3  3  (300) have been isolated. Additionally, compounds o f fatty acid or polyketide origin 5  have been isolated from gorgonians.  4.1.2: Examples of Secondary Metabolites from Gorgonians The study o f the chemistry o f gorgonians began with Burkholder and Burkholder's paper i n 1958 detailing the presence o f antibiotic substances from a Puerto  177  Rican collection. In the decades that followed, many diterpenes o f the cembrane class 6  such as 301 were isolated. M a n y members o f this class have been previously isolated 7  7 8  Q  from other natural sources, primarily plants. " In addition, a multitude o f briaranes such as briarein A (302), have been isolated as well as members o f at least twenty other 10  skeletal types.  11  Other terpenoids isolated from gorgonians include the pseudopterosins  such as pseudopterosin A (10), isolated from Pseudopterogorgia  elisabethae,  12  and  13  currently used i n a skin cream marketed by Estee Lauder (Resilience®).  In 1997, Lindel and co-workers isolated eleutherobin (15) from a Western Australian soft coral o f the genus Eleutherobia (order Alcyonacea).  14  This compound  was later shown to possess antimitotic properties similar to those o f T a x o l ® (2) and synthetic chemists set to work to synthesize it, as the amount obtained by Lindel was very small.  15  A t about the same time, sarcodictyins A (303) through F (308), previously  isolated from the Mediterranean stolonifer Sarcodictyon roseum  16  antimitotic agents,  17  were also shown to be  as presumably are the closely related eleuthosides A (309) and B  (310) isolated from the South African alcyonacean Eleutherobia aurea.  178  Despite many  Me,  309: R i = H; R = A c 310: RT =Ac; R = H 2  2  311: R T = Ac; R = R = H 312: R = R = H; R = M e 313: R T =H; R = Ac; R = Me 2  1  3  3  2  2  3  Me,  315:  R =  316: R = Ac  318: R = H  synthetic attempts to produce large quantities o f these compounds or derivatives o f them with similar activities, it was not until the Andersen lab discovered that the relatively common and culturable gorgonian Erythropodium  caribaeorum produces 15 and eight  related diterpenes 311-318 i n reasonable amounts, that the possibility o f further development could be explored i n a cost-effective manner. " 19  179  20  It was further shown that  15 was actually an artefact produced b y extraction with M e O H under the naturally 21  occurring acidic isolation conditions. Due to the presence o f 15, 303, and 311-319 i n E. caribaeorum in either or both the wild type and cultured form, we decided to isolate further quantities o f these compounds, particularly eleutherobin 15 for further biological studies. In the course o f our isolation o f this important metabolite, the presence o f five new diterpenes was noted. The isolation and structural elucidation o f these diterpenes w i l l be discussed i n the remainder o f this chapter.  4.2: Diterpenes from Erythropodium caribaeorum 4.2.1: Description of E. caribaeorum and Brief Overview of Known Metabolites Erythropodium  caribaeorum Duchassaing and Michelotti 1860, (see Figure 4.1),  is an octocoral o f the Caribbean with a range extending from the southern portion o f Florida southeast to the Lesser Antilles and west into the G u l f o f M e x i c o . The genus Erythropodium  contains only a single species that is characterized b y the encrusting  nature o f its colonies and is dorsally tan and ventrally red i n colouration. The colonies 4  o f this gorgonian can range i n area from 10 to 100 square centimetres, and are fairly flat. E. caribaeorum lives at depths that are accessible by S C U B A — f r o m shallow water to depths o f 30 m .  2 2  A recent thesis  23  has reviewed the chemistry o f E. caribaeorum and  hence it shall not be reproduced in detail here. However, it is noted that the chemistry o f this genus is dominated by reports o f diterpenes o f the erythrolide series, " 24  30  which w i l l  be discussed below. Erythrolides A (319) and B (320) were isolated by Fenical's group from a Belizean collection i n 1984. These chlorinated diterpenes belong to two structural  180  Figure 4.1: Erythropodium caribaeorum. (Photo by M . Campbell, used by permission). classes, the erythranes (ie erythrolide A (319)), and the briaranes (ie erythrolide B (320)), the former being derived from the latter by a di-7i-methane rearrangement, which w i l l 24  be discussed further in Section 4.5. Seven years later, a study o f E. caribaeorum from the U S V i r g i n Islands and Jamaica yielded 319 and 320 in addition to seven more briarane erythrolides, C (321) through I (327).  25  Further studies on collections from Tobago and  Jamaica led to the isolation o f five more briarane erythrolides, J (328), K (329), 26  the "unnamed" acetate analogues o f E (330), H (331), and I (332).  28  27  and  In 2002, Banjoo et al  reported the isolation o f a novel erythrane, erythrolide L (333) in addition to five additional novel briarane erythrolides, M (334) through Q (338), from a collection in Tobago.  29  During the course o f our isolation o f large amounts o f 15 for further biological  testing, four additional briarane erythrolides, R (339) and S (340), and T (48) and U (49) were isolated from either cultured specimens or from a Dominican collection. Additionally, a novel erythrane, erythrolide V (50) was isolated from the Dominican collection, and two novel aquarianes, aquariolide B (51) and C (52), were isolated from cultured specimens.  30  Aquariolides A (341),  20  181  B (51),  30  and C (52)  30  are related to the  319: R = R = A c 333: R ^ C O C H z O A c ; 50: R i = Ac; R = H 1  2  R = Ac 2  3 2 0 : R = Ac 339: R = H  2  2  2  323: = Ac; R = H 324: R ^ C O C h ^ O A c ; R = H 327: RT = C O C H O H ; R = H 330: RT = Ac ; R =Ac 332: R i = C O C H O A c ; R =Ac 2  325  326: R = H 331: R = Ac  338: R=Ac  51: R = Me 52: R = Ac  2  2  321:R = Ac 322: R = C O C H O A c 49: R = C O C H O H  O  2  2  2  2  briarane skeleton b y sequential di-7i;-methane and vinylcyclopropane rearrangements as w i l l be discussed below. The isolation and structure elucidation o f compounds 48-52 are discussed i n the following sections, while that o f erythrolides R (339) and S (340), which were isolated b y m y colleague, w i l l not be discussed. A l s o during the course o f this investigation, erythrolides A (319), B (320), E (323), J (328), 16-0-Acetyl H (331), M  182  (334), P (337), and Q (338) were isolated, as was fra«s-7-hydroxycalamenene (342). This sesquiterpene was previously isolated from cotton Gossypium hirsutum, Australian soft coral Lemnalia cervicornis,  32  and other sources.  48  the  33  342  4.2.2: Isolation and Structure Elucidation of Diterpenes 48-52 4.2.2.1: Isolation Procedure Freshly collected wild-type specimens o f E. caribaeorum collected in Prince Rupert Bay, Dominica were frozen and transported to Vancouver over dry ice. Cultured E. caribaeorum,  (several generations removed from the wild), was grown on artificial  rocks i n shallow running seawater tanks located i n a greenhouse under ambient sunlight illumination. Freshly harvested animals were shipped from Ocean Dreams, Inc. in Florida to Vancouver, live, i n chilled seawater. Samples o f the organism from either source (374 g and 400 g wet weight respectively) were cut into small pieces and extracted multiple times. For cultured E. caribaeorum the extraction solvent was M e O H (3 x 750 m L ) , while the wild-type organism was extracted with E t O H (3 x 750 m L ) . W e used E t O H in the latter extraction to see i f the methyl ether in 51 was an artefact from the isolation procedure, see Section 4.2.2.4 for more details. The E t O H (or M e O H ) extracts were combined and concentrated to a brown-coloured gum in vacuo that was suspended i n water (300 m L ) and extracted with E t O A c (3 x 300 m L ) . The resulting organic layer was then partitioned between n-hexane (3 x 300 m L ) and M e O H / H 2 0 9:1 (300 m L ) , yielding approximately 3.0 g (5.1 g for cultured specimens). Further fractionation o f the  183  M e O H / H 2 0 9:1 extract by sequential application o f normal-phase flash silica gel (gradient elution from n-hexane/EtOAc 6:4 to E t O A c to E t O A c / M e O H 1:1 i n 5% increments) yielded approximately seventy fractions. Fractions from the cultured organisms that eluted with «-hexane/EtOAc 6:4 were further purified by normal-phase H P L C (n-hexane/EtOAc 75:25) to yield pure 16-0-Acetyl erythrolide H (331) (2.5 mg) and aquariolides B (51) (0.4 mg) and C (52) (1.7 mg). Aquariolide B (51) contained a persistent impurity that was removed b y passing the material through normal-phase H P L C again, this time using 1% z'-PrOH i n n-hexane. Fractions from both the cultured and the wild-type organisms that eluted with EtOAc/n-hexane 6:4 were further purified by 9:1 n-hexane/EtOAc to yield pure £ra«s-7-hydroxycalamenene (342) (5.1 mg). Those fractions from the wild-type organisms that eluted with EtOAc/rc-hexane 65:35 were purified by normal-phase H P L C (n-hexane/EtOAc 7:3) to yield pure erythrolides E (323) (0.1 mg) and M (334) (1.8 mg). Other fractions from the wild-type organisms that eluted with EtOAc/«-hexane 7:3 contained the appropriate *H N M R signals for aquariolide A (341), which was not purified further. This finding showed that this compound is also present in wild-type organisms. The normal-phase H P L C (n-hexane/EtOAc 7:3) conditions were applied to a similar fraction from the cultured organisms to yield pure erythrolide T (48) (0.1 mg). The erythrolides A (319) and B (320) were found i n large abundance (ie approximately 820 m g and 1.20 g, respectively, i n the w i l d type) from fractions eluting with EtOAc/n-hexane 9:1 and 8:2, respectively, i n both the cultured and wild-type, as has been previously reported. ' 20  24  Fractions from the wild-type organisms  that eluted with EtOAc/n-hexane 95:5 were purified by normal-phase H P L C (EtOAc/nhexane 75:25) to yield pure known erythrolides J (328) (3.3 mg) and Q (338) (0.5 mg), i n  184  addition to the novel erythrolides U (49) (0.3 mg) and V (50) (0.3 mg). Finally, those fractions that eluted with E t O A c / M e O H 8:2 from the normal-phase flash silica gel column contained, i n moderate abundance (~ 17.5 mg), the known erythrolide P (337). The known molecules were identified b y comparison o f their N M R and M S data with those reported i n the literature.  4.2.2.2: Novel Briaranes: Erythrolides T (48) and U (49)  16  AcO 48  Erythrolide T (48) (0.1 mg) and U (49) (0.3 mg) are novel briarane diterpenes, both o f which show the common feature o f the 2,3-epoxide moiety. Erythrolide T (48), isolated as a colourless glass, exhibited positive ion H R C I M S data (m/z [ M + N H ] o f 572, 574 in ~ +  4  3:1 ratio with m/z = 572.1899) compatible with the molecular formula  C26H31CIO11.  This  molecular formula possesses only an oxygen atom more than the molecular formula o f erythrolide B (320). Comparison o f  and  1 3  C N M R spectra o f 48 with those reported for  320 confirmed the close structural similarity o f these two metabolites, with the resonances o f the entire structural framework going from C-4 to C-14, comprising the y-lactone system and the cyclohexenone ring, almost identical to those o f 320.  24  However i n both H and !  1 3  C  N M R spectra o f 48, the resonances attributed to the A ' double bond were absent, being 2  3  replaced by signals attributable to epoxide methines (8 3.24 and 8 3.69 ppm and 8 64.2 and 8 55.2 ppm, respectively). Detailed analysis o f the C O S Y , H M Q C , and H M B C N M R spectra (see Table 4.1) allowed us to unambiguously locate the epoxide group at C-2/C-3  185  and to fully confirm the assignment o f 48 as the 2,3-epoxide derivative o f 320. To compare the stereochemistry o f 48 with that o f 320, a 2 D R O E S Y N M R experiment was run. The R O E S Y spectrum generated for 48 was used to infer the relative stereochemistry o f this new erythrolide, (see below i n Figure 4.2; arcs represent nOes).  Figure 4.2: Erythrolide T (48) Stereochemistry as Defined b y nOes From a R O E S Y Experiment. A r c = Correlation.  A s predicted on the basis o f close similarities i n ' H and  1 3  C N M R spectra the nOe contacts  for the structural moiety from C-4 to C-14 o f 48 appeared to be consistent with assigning the relative stereochemistry to be that reported for 320.  24  Additionally, the spatial  proximity o f H-2/H-10 and H-3/Me-15 defined the relative orientation o f the epoxide ring, thus completing the stereochemistry o f erythrolide T (48). Additional spectroscopic data for 48 can be found i n the experimental section, Section 4.5. The  N M R spectrum for 48 can  be found in Figure 4.3, while the 2 D N M R spectra for 48 can be found i n Section 4.6. Table 4.1 contains a summary o f the N M R data for 48.  186  Table 4.1: Erythrolide T (48) N M R Data i n C D C 1 No. 1 2 3  Carbon 40.2 64.4 55.2  4  69.5  5 6 7 8 8-OH 9  140.4 67.3 79.5 80.5  10 11 12 13  43.2 82.1 196.1 126.1  —  80.2  Proton —  3.24 bs 3.69 bd, J = 3.2 H z 5.43 d, J = 3.2 H z —  4.91 bs 5.11 bs —  3.15s 5.66 bs 4.09 bs  H M B C Correlations  C O S Y Correlations —  3  —  H-15 (weak), H-3 H-4, H-2 H-3 —  —  H-16, H-7 H-6  C-6  —  —  H-10  C-10, C-8, C-7, C - l , 9-Ac C = 0  H-9  —  —  —  —  —  —  H-14  C-l  H-13  C-12  H-2 (weak) G C , H-6  C-14, C-10, C-2, C - l From 5.46 to C-6, C-4  H-18  C-19, C-9/C-8*  H-17  C-19, C-17, C-8  —  —-  —  —  —  —  —  —  6.04 d, J = 10.4 H z  14  153.0  15 16  17.5 118.1  17  45.5  18  9.2  19 20 4-Ac  175.2 21.0 170.2 21.5* 170.0 20.2* 171.1 21.1  9-Ac 11-Ac  6.71 d, J = 10.4 H z 1.14s 5.55 bs, 5.46 bs 3.18 q, J = 7.3 H z 1.28 d, J = 7.3 H z —  C-12, C - l l , C-10  1.36 s —  4-Ac C = 0  2.18 s* —  9-Ac C = 0  2.17 s* —  11-Ac C = 0  2.07 s  The ' H N M R chemical shift values are at 500 M H z and the " C chemical shifts are at 125 M H z as they are from H M B C correlations. * Uncertain values or unambiguous assignments due to overlapping signals. # Overlapped signals. G C = Geminal Correlations which are between protons on the same carbon. Correlations from/to the exo-methylene are given, as per the proton they are from/to. Not many H M B C signals are shown due to dilute sample.  187  188  Isolation o f 48 is particularly interesting because this is the first erythrolide to possess both the 2,3-epoxide group and an acetate functionality at C - 4 . The 2,3 epoxide molecules are believed to be the precursors o f C-2/C-8 ether bridged erythrolides such 25  as erythrolides E (323) or I (327), thus it seems reasonable that 4-acetoxy-C-2/C-8 ether erythrolides are also part of the diterpenoid pool o f E. caribaeorum.  They have not yet  been reported possibly due to their presence i n limited amounts.  322: R = Ac  Inspection o f the C I M S and N M R data obtained for erythrolide U (49), a colourless glass with molecular formula C24H29CIO10 ( H R C I M S [ M + N H ] m/z +  4  530.1788), and those reported for erythrolide D (322) revealed them to very similar. A s 25  with 48 there were two [M+NFL;]" " ions i n ~ 3:1 ratio (m/z 530, 532) observed i n the C I 1  mass spectrum. The *H and  1 3  C N M R data o f 49 (Table 4.2) showed the entire  diterpenoid core to be identical (with very slight deviations) to that o f 322. The only differences i n the two series o f data were the lack o f H and l  1 3  C signals accounting for  one acetyl group and the upfield shift o f H-2" (8 4.22 and 4.27 ppm instead o f 8 4.52 and 8 4.62 ppm) i n the *H N M R spectrum o f 49. Since standard acetylation (acetic anhydride/pyridine) o f 49 afforded erythrolide D (322) i n 75% yield, erythrolide U 49 was unambiguously assigned as the 2"-desacetyl derivative o f 322.  25  Interestingly  compound 49 could be considered as the epoxide biosynthetic precursor o f erythrolide I 25  (327), a C-2/C-8 ether bridged molecule reported by Schmitz et al.  189  Table 4.2: Erythrolide U (49) N M R Data i n C D C l t 3  No. 1 2 3 4  Carbon 40.6 62.2 54.0 37.1  5 6  137.3 67.8  7  78.6  8 8-OH 9 10  80.0 80.3 41.6  11 12 13  81.4 194.5 124.4  14  153.6  15 16 17  16.4 119.1 45.8  18  9.5  19 20 11-Ac  175.6 21.6 169.0 21.3 171.8 61.0  1" 2"  2"-OH  —  —  Proton —  3.07 m# 3.43 m 2.75 dd, J = 16.1 H z , J = 5.5 H z 2.63 dd, J = 16.1 H z , J = 1.5Hz —  4.76 d, J = 2.1 H z 5.17 d, J = 2.1 H z —  C O S Y Correlations  H M B C Correlations —  —  C-l  H-3 H-4, H-2 G C , H-3  From 2.75 to C - l 6 , C-5  —  —  H-16, H - 7 H-7  C-19  —  —  H-17 (weak)  C-8 C - l l , C-8, C-7, C - l " C-20, C-15, C - l l , C-2, C-l  —  —  —  —  —  —  H-14  C-ll,C-l  H-13  C-12  G C * , H-6 H-18, 8 - O H (weak)  C-14, C-2, C - l C-6, C-4 C-19, C-18, C-9/C-8*  H-17  C-19, C-17, C-8  —  —  3.15 s 5.76 bs 4.12 s  6.03 d, J = 10.1 H z 6.68 d, J = 10.1 H z 1.08 s 5.12 b s & 5.42 bs 3.08 q, J = 7.6 1.31 d, J = 7.6 H z —  C-12, C - l l , C-10  1.37 s —  —  —  —  —  11-Ac C = 0  2.07 s —  4.27 dd & 4.22 dd, both with J = 16.2 H z , J = 2.5 H z 2.25 t J = 2.5 H z  From 4.22 to 2"-OH  To 4.22  f For explanation of NMR conditions, and the meaning of GC, *, #, and the from/to designation see Table 4.1.  190  191  192  Additional spectroscopic data for 49 can be found i n the experimental section, Section 4.5. The I D N M R spectra for 49 can be found i n Figures 4.4 and 4.5, the 2 D N M R spectra can be found i n Section 4.6, and a summary o f the N M R data i n Table 4.2.  4.2.2.3: A Novel Erythrane: Erythrolide V (50)  Erythrolide V (50) was isolated as a clear glass (0.3 mg) from a slightly more polar fraction. Data from both low and high resolution C I M S (m/z for [M+NH.4] 514 and 516 i n +  ~ 3:1 ratio with H R C I M S giving m/z 514.1850) established the molecular formula o f 50 to be C24H29CIO9. Inspection o f H and !  1 3  C N M R spectra (Table 4.3) o f 50, aided b y C O S Y ,  H M Q C , H M B C 2 D N M R experiments revealed the presence o f a cyclopropane ring (5 1.96 and 8 2.37 ppm, and 8 29.3, 8 36.6, and 8 39.7 ppm) fused with a ketone (8 204.5 ppm) containing five-membered ring. C O S Y and H M B C correlations indicated that the remaining carbon o f the three-membered ring must be directly connected to a double bond (8 6.37 and 5.98 ppm and 8 123.4 and 8 138.0 ppm) that is part o f an eleven-membered ring. These structural features are typical o f the tricyclo-[8.3.1.0 ' ]-tetradecane system o f the 13 14  erythrane diterpenoid skeleton.  24  To date only two molecules with such a skeleton are known, erythrolides A (319) and L (333). In particular, the N M R data o f erythrolide V (50) appeared very 24  29  similar to those reported for erythrolide A (319).  24  The only difference between the  N M R data for these two erythranes is the lack o f an acetyl signal and the parallel upfield  193  shift o f the signal attributed to H-4 (8 5.05 ppm instead o f 85.99 ppm) i n the H N M R !  spectrum o f 50. W e therefore concluded that erythrolide V (50) is the 4-desacetyl derivative o f 319. ..OR  2  319: = R = Ac 333: R T = C O C H O A c ; R = Ac 50: R i = Ac; R = H 2  2  2  2  Acetylation o f the secondary alcohol (as per 49) o f erythrolide V (50) to afford a compound identical (by H N M R and [a]o) to 319 proved this relationship and completely 1  defined the stereochemistry to be the same as that reported for 319. Additional spectroscopic data for 50 can be found in the experimental section, section 4.5. The I D N M R spectra for 50 can be found i n Figures 4.6 and 4.7, while the 2 D N M R spectra can be found i n Section 4.6, and summary o f the N M R data i n Table 4.3.  194  Table 4.3: Erythrolide V (50) N M R Data i n C D C l t 3  No. 1  Carbon 36.6  2  123.4  3  138.0  4  76.9  4-OH  —  5 6  142.1 60.3  7  80.3  8 8-OH 9  83.2  10  43.2  11 12 13  88.0 204.5 39.7  14 15 16  29.3 22.6 125.3  17  43.6  18  9.4  19 20 9-Ac  175.1 22.2 172.4 21.7 168.9 21.2  11-Ac  —  80.7  Proton 2.37 dd, J = 8.5 H z , J = 7.0Hz 6.37 dd, J = 17.8 H z , J = 7.0 H z 5.98 dd, J = 17.8 H z , J = 2.5 H z 5.05 dd, J = 8.3 H z , J = 2.5 H z 2.89 d, J = 8.3 H z —  4.60 d, J = 9.7 H z 5.13 d, J = 9.7 H z —  3.44 s 5.41 d, J = 2.3 H z 2.97 d, J = 2.3 H z  C O S Y Correlations H-13, H-2  H M B C Correlations C-15, C-12, C-10, C-3 (weak)  H-3, H - l  C - l 3 (weak), C-4  H-2  C-4,C-l  4-OH  C-6  H-4 —  —  H-7  C - l 6 , C-7, C-5, C-4  H-6  C-5  —  —  H-10 H-9  C-9/C-7*, C-8 C-14, C - l l , C-10, C-8, 9-Ac C = 0 C-15, C-14, C - l l , C-9, C-l  —  —  —  —  —  —  H-l  C-15, C - l l , C - l  —  —  1.96 d, J = 8.5 H z —  1.57 s 5.51 bs, 5.39 bs 3.16 q, J = 7.3 H z 1.18 d, J = 7.3 H z —  H-18  C-14, C - 1 3 , C - 1 0 , C - l C-5 (From 5.39), C-4 (Both) C-19, C-18  H-17  C-19,C-17,C-8  —  —  GC  C-12, C - l l , C-10  1.38 s —  —  —  —  —  —  9-Ac C = 0 11-Ac C O  t For explanation of N M R conditions, and the meaning of G C , *, #, and the from/to designation see Table 4.1.  195  196  197  4.2.2.4: Novel Aquarianes: Aquariolide B (51) and C (52)  Two additional diterpenes possessing the aquariane skeleton,  named  aquariolides B (51) (0.4 mg) and C (52) (1.7 mg) were isolated as colourless glasses from cultured E. caribaeorum. Aquariolide B (51), a compound with molecular formula determined to be C25H31O9CI by H R C I M S (m/z for [ M + N H ] = 528.2000), had two low +  4  resolution C I M S [ M + N H 4 ] ions at m/z 528 and 530 i n ~ 3:1 ratio, confirming the +  1  13  presence o f a chlorine atom i n the molecule. A detailed analysis o f the H and  C NMR  spectra (Table 4.4) i n conjunction with the aid o f C O S Y , H M Q C , and H M B C 2 D N M R 20  experiments suggested that 51 was very similar to aquariolide A (341).  In particular, a  sequential series o f cross-peaks i n the C O S Y spectrum from H-13 (8 3.29 ppm attached to a carbon 8 69.8 ppm) to H-4 (8 5.70 ppm attached to a carbon at 8 68.0 ppm) was determined, which encompasses the as-double bond at C - l / C - 2 . The remaining three spin systems were those o f protons i n simple two-carbon moieties, namely H-6/H-7, H 9/H-10, and H-17/H-18, see Figure 4.8. Additionally, three uncoupled methyls (8 1.04, 8 1.59, and 8 3.10 ppm) and two acetate methyls (8 2.08 and 8 2.19 ppm) were present. Furthermore, one O H singlet (8 2.79 ppm), and two olefinic methylene protons (8 6.00 and 8 5.98 ppm with long-range couplings to H-6 and H-4) were also present. Inspection of the  1 3  C N M R spectrum showed that apart from the resonances o f the seventeen  protonated carbons, assigned through the H M Q C spectrum, there were also signals for 198  Figure 4.8: Important C O S Y (arcs) and H M B C (arrows) Correlations in 51.  eight quaternary carbons. These were attributable to two acetyl carbonyls (5 170.7 and 5 168.7 ppm), lactone (8 174.9 ppm) and ketone (8 2i0.1 ppm) carbonyls, a double bond carbon (8 140.1 ppm), and three sp carbon atoms (8 49.3, 8 82.3, and 8 83.6 ppm). The 3  connection o f all the above partial structures with the use o f the H M B C spectrum, and facilitated by comparison with the N M R data  20  o f aquariolide A (341), suggested the  presence o f the tricyclic aquariane skeleton i n 51. In accordance with the molecular formula, aquariolide B (51) differed from aquariolide A (341) only b y the presence o f a methyl group in place o f a hydrogen atom on the tertiary alcohol at C - l 1. The site o f attachment o f this group was unequivocally located at this position on the basis o f the H M B C cross peak o f the singlet at 8 3.10 ppm with the oxygenated C - l l at 8 83.6 ppm. The stereochemistry o f 51 is assumed to be similar to that for 341 as the only difference is the substitution o f a methyl ether for a free hydroxyl at C - l 1. Additional spectroscopic data for 51 can be found i n the experimental section, Section 4.5. The I D N M R spectra for 51 can be found in Figures 4.9 and 4.10, while the 2 D N M R spectra can be found in Section 4.6. A summary o f the N M R data is i n Table 4.4.  199  Table 4.4: Aquariolide B (51) N M R Data i n C D C 1 No. 1 2  Carbon 131.3 126.9  3 4  63.2 68.0  5 6  140.1 62.1  7  76.4  8 8-OH 9  82.3  10  44.7  11 12 13 14 15 16  83.6 210.1 69.8 49.3 29.5 123.4  17  43.1  18  6.9  19 20 4-Ac  174.9 14.8 170.7 21.1 168.7 21.6 51.1  9-Ac 11-OMe  —  67.1  Proton 6.08 m 5.81 ddd, J = 7.0 H z , J = 2.7 H z , J = 2.0 H z 3.01 m 5.70 bd, J = 3.0Hz —  5.29 ddd, J = 4.7 H z , J = 2.0 H z , J = 1.8 H z 5.44 d, J = 4.7 H z —  3  H M B C Correlations  C O S Y Correlations H-13, H - 2 H-3, H - l  H-13, H-4, H-2 H-16, H-3  C-2, C - l (weak)  —  —  H-16, H-7  H-6  C-6  —  —  H-9, 8-OH (weak)  C-9, C-8, C-7 0 1 1 , 0 8 , C-7, 9-Ac C = 0 C-14, C-9, C-3  —  —  —  —  —  —  2.79 s 5.66 d, J = 3.5Hz 2.69 d, J-3.5 Hz  3.29 m —  1.59 s 6.00 bd, J = 2.0 H z , 5.98 bs 2.51 q, J = 7. 3 H z 1.20 d, J = 7.3 H z —  H-10 (weak) H-10  H-3, H - l —  —  G C , H-6, H - 4  C-14, 0 1 3 , 0 1 0 , C-3 From both to C-6, C-4, From 5.98 to C-5  H-l 8  C-19, C-18, C-9, C-8  H-17  C - l 9 , C-17, C-8  —  —  —  —  —  —  C-12,  1.04 s —  4-Ac C = 0  2.08 s —  011,010  9-Ac C = 0 C-ll  2.19 s 3.10s  The ' H N M R chemical shift values are at 500 M H z and the "C chemical shifts are at 125 M H z as they are from H M B C correlations. GC  = Geminal Correlations which are between protons on the same carbon. Correlations from/to the exo-methylene are given as per  the proton they are from/to.  200  201  202  A similar analysis was undertaken for the structure elucidation o f aquariolide C  (52), a colourless glass with molecular formula  C26H31O10CI  as determined by  H R D C I M S (m/z for [ M + H ] = 539.1692). In the low resolution C I M S two [ M + H ] ions +  +  at m/z 539 and 541 i n ~ 3:1 ratio also confirmed the presence o f a chlorine atom i n the molecule. Comparison o f the N M R data (Table 4.5) obtained for 52 with those.of aquariolide A (341) revealed that the only difference between the two was the presence 20  o f an additional acetyl group i n 52. The extra acetate could be i n one o f two positions, either at C-8 or at C - l 1. C-8 was connected to a free O H (8 2.70 ppm), as there were H M B C correlations from its proton to C-7, C-8, and C-9. Aquariolide C (52) was therefore deduced to be the 11-0-acetyl derivative o f aquariolide A (341). A s with 51, it is assumed that the stereochemistry o f the two molecules (341 and 52) is the same. Additional spectroscopic data for 52 can be found i n the experimental section, Section 4.5. The I D N M R spectra for 52 can be found i n Figures 4.11 and 4.12, while the 2 D N M R spectra can be found i n Section 4.6. Table 4.5 is a summary o f the N M R data. Inspection (but not purification) o f the diterpenoid fraction o f the wild-type E. caribaeorum confirmed the presence (by *H N M R signals) o f the aquariolide series o f compounds, presumably A (341), B (51), and C (52), the first, (341), previously obtained 20  from cultured specimens. The two new members o f the aquariane family (51 and 52) differ from the previously reported  20  aquariolide A (341) only i n the substituent at C - l 1. Furthermore, 52  can be considered the direct product o f vinyl cyclopropane rearrangement o f erythrolide A (319), one o f the most abundant secondary metabolites o f E. caribaeorum. However, it should be noted that the major aquariane diterpene o f this organism is not 52, but 341 by  203  Table 4.5: Aquariolide C (52) N M R Data i n C D C 1 No. 1 2  Carbon 130.4 127.5  3 4  64.4 67.6  5 6  137.6 61.4  7 8 8-OH 9  76.2 82.7  10  38.7  11 12 13 14 15 16  84.4 208.6 68.4 46.1 28.5 125.2  17  43.2  18  6.5  19 20 4-Ac  174.6 19.9 170.9 21.2 169.4 21.6 169.0 20.9  9-Ac 11-Ac  —  67.9  Proton 6.14 m 5.91 ddd, J = 7.0 H z , J = 2.7 H z , J = 2.0 H z 3.08 m 5.70 d, J = 4.0 H z  H M B C Correlations  C O S Y Correlations H-13, H - 2 H-3, H - l  —  5.22 ddd, J = 4.7 H z , J = 2.0 H z , J - 1.8 H z 5.43 m#  H-13, H-4, H - 2 H-16 (to 6.06)  C-2, C - l , 4 - A c C = 0  —  —  H-7  —  2.70 s 5.43 m#  H-6  C-17*, C-8*, C-6  —  —  H-10  3.91 d, J = 3.4 H z  H-9  C-9, C-8, C-7 C-17*, C - l l , C-10, C-8*, C-7 C-20, C-15, C-14, C-13, C - l l , C-8, C-3  —  —  —  —  —  —  3.25 m  H-3, H - l  —  —  —  1.46 s 6.31 d, J = 1.8 H z , 6.06 d, J = 2.0 H z 2.38 q, J = 7.2 H z 1.16 d. J = 7.2 H z  GC, From both to C-6, From 6.06 to H - 4  —  C-14, C-13, C-10, C-3 C-6, C-4, From 6.06 to C-5  H-18  C - l 9 , C-18, C-9, C-8  H-17  C-19, C-17, C-8  —  —  —•  —  —  —  —  —  C-12, C - l l , C-10  1.41 s —  4-Ac C = 0  2.11 s —  9-Ac C = 0  2.25 s —  11-Ac C = 0  1.89 s  The ' H N M R chemical shift values are at 500 M H z and the  3  l 3  C chemical shifts are at 100 M H z .  * Uncertain values or assignments  due to overlapping signals. # Overlapped signals. G C = Geminal Correlations which are between protons on the same carbon. Correlations from/to the ejco-methylene are given as per the proton they are from/to.  204  205  206  a factor o f about ten, which suggests involvement o f an enzymatic system for the biotransformation o f the erythrane into the aquariane skeleton. Further evidence for this hypothesis arises from the same isolation procedure on the wild-type organism from a deep collection (70-90 foot range) whereupon we still saw the presence o f the H N M R signals for the aquariolides. This isolation (as described in Section 4.2.2.1) was also conducted using E t O H for the initial extraction step to rule out the possibility that 51 was formed during the extraction procedure. The presence o f a methoxy signal i n the crude aquariolide fraction confirmed that 51 is not an isolation artefact, which is particularly remarkable since it represents the first diterpene from E. caribaeorum to bear a methoxy group. T o our knowledge, there is also no report o f methyl ether containing briarane or briarane-related diterpenes from octocorals. A s further evidence for the enzymatic origin for the methoxy group i n 51, we tried to leave erythrolide A (319) or aquariolide A (341) in M e O H at room temperature for several weeks but this only led to the recovery o f unreacted starting material.  4.3: Conclusions Gorgonians are known to contain a wide array o f diterpenoids including the briarane (3,8-cyclized cembranoids) class, the class to which erythrolides belong. Briarane diterpenes have been recently reviewed by Sung, et al with 299 structures presented. The review begins with briarein A (302), isolated in 1977 from the Jamaican 9  gorgonian Briareum asbestinum,  10  and continues through early 2002 with the isolation o f  juncenolide A (343) from the Taiwanese gorgonian Junceella juncea  34  (333) through P (338) from E. caribaeorum.  29  and erythrolides L  Since then a further twelve milolides (such  as G (344) —adding to those already known ) have been isolated from the Micronesian  207  319: R = R = Ac 333: R-i = C O C H O A c ; R = Ac 50: R T = Ac; R = H 1  2  2  2  320: R = Ac 339: R = H  2  J4/:  348:  Ki = U A C ; R T = OAc;  K R  2  2  = n = Me  (Yap Island) octocoral Briareum stechei. Additionally six new briaranes, including cyclobutenbriarein A (345) with its novel tricyclo[8.4.0.0 ' ]tetradec-4-ene ring system, IT  have recently been isolated from the Bahamian gorgonian Briareum asbestinum.  208  A very  recent article i n the Journal of Natural Products reported an additional three novel briaranes from the Taiwanese gorgonian J. juncea, juncenolides B (346), C (347), and D (348).  38  Including aquariolide A (341), erythrolides R (339) and S (340), and the five 20  new compounds reported i n this thesis, 48-52, the total number to date o f known briarane (and the closely related aquariane and erythrane skeletons) is 328, 327 o f which have been isolated from corals, one isolated from a nudibranch (though previously known from a 39  soft coral), and one novel diterpene from a sponge.  40  A m o n g the briaranes, erythrolide B (320) must be considered quite a remarkable example. This highly oxygenated briarane diterpene is the very major secondary metabolite o f E. caribaeorum accounting for about 30% o f the organic extract and 0.5 % o f wet weight in the specimens examined i n this work. This molecule probably plays a key role i n the biogenetic origin o f many or all o f the diterpenoids o f this organism. Scheme 4.1 presents a postulated summary o f the structural diversity o f some o f the diterpenoids isolated from E. caribaeorum that can be interconverted, with particular emphasis on those novel structures (48-52) presented i n this chapter. The important points o f reactivity i n erythrolide B (320) are the two double bonds 01  01  111/1  A ' and A ' . Epoxidation at A ' produces the first series o f derivatives, which, as a result o f nucleophilic attack o f 8-OH at C-2 give rise to the C-2/C-8 ether bridged molecules. Moreover, the 1,4-diene system is the substrate for the remarkable di-jxmethane rearrangement  19  yielding the erythrane skeleton found for erythrolides A (319), L  (333), and V (50). Additionally, the erythrane system possesses another structural moiety, that o f the vinylcyclopropane, which can participate in other rearrangements. T w o o f the most important reactions reported for the vinylcyclopropane moiety are the [1,5]-H shift  209  Scheme 4.1: Biogenetic Relationship o f Some Erythrolides. V C R = Vinylcyclopropane Rearrangement and the so-called vinylcyclopropane rearrangement ( V C R ) .  4 1  The direct product (343g) o f 97  [1,5]-H shift o f 319 has never been found i n nature, but Reynolds et al  synthesized it  from 319 by refluxing i n toluene for fifteen hours. This molecule loses acetic acid to form erythrolide K (332) by refluxing in a methanol suspension o f silica gel for forty-five minutes. Therefore the isolated 332 (from E. caribaeorum)  is possibly an artefact 27  produced by the 1,4 elimination o f acetic acid during the separation process on silica gel. Our recent isolation o f aquariolides A (341), B (51), and C (52) completes the picture 20  30  30  by showing us the products o f a V C R , a rearrangement that usually requires very high temperatures or in some examples photoinduction.  210  41  It is not clear i f this amazing series o f rearrangements (including the V C R ) is entirely photochemical or enzyme mediated. Although it has been shown to be possible to reproduce i n the laboratory the di-TT-mefhane rearrangement o f 3 2 0  24  and the [1,5]-H shift  of 319 by refluxing it i n toluene, the involvement o f an enzyme system for these 27  reactions in the living organism cannot be excluded. O n the other hand, observations that the photochemical irradiation o f 319 was not able to produce the aquariane system and that the major aquariane o f E. caribaeorum, 341, is not the product o f direct rearrangement o f 319, the major erythrane, point towards enzyme involvement.  This  matter w i l l obviously require further study. The possibility o f isolating the responsible enzymes for the catalysis o f these reactions would be quite exciting, as there is no precedent currently known. However, one aspect is reasonably certain. In 1984, Fenical and Djerassi showed b y C / C isotope ratio mass spectrometry that the terpenoids 1 3  1 2  isolated from octocorals are produced by them, while the steroids are produced by the symbiotic zooxanthellae.  42  Thus, it is likely that the enzyme or enzymes would be  gorgonian i n origin. Erythrolides are believed to be the major antifeedant principles used b y E. caribaeorum to deter their predation b y reef fish  4 3  M a n y briaranes are also known to be  cytotoxic, antiviral, antiinflammatory, ichthyotoxic, and insecticidal. To determine i f any 9  o f the diterpenes we isolated were cytotoxic they were subjected to a cytotoxicity test, the results o f which are shown i n Figure 4.13. Note that the numbers i n the legend refer to a particular erythrolide, while "others" refers to the remaining erythrolides, which were inactive, see below. Aquariolide A (341) was previously known to be cytotoxic, while 44  this cytotoxicity screen showed that aquariolides B (51) and C (52) are also. Amongst the  211  pairs o f erythrolides tested (such as 322 and 49), it appeared that those with an acetate (ie erythrolide D (322) with an acetoxyacetyl group; semisynthesized from U (49)) are active, while those with a free hydroxyl (ie erythrolide U (49) with a hydroxyacetyl group) are inactive. This trend was also true for erythrolide J (328) (active, with a 3-acetoxybutanoyl substituent) and erythrolide S (340) (inactive, 3-hydroxybutanoyl substituent) and for erythrolide Q (338) (active, acetate at C-12) and erythrolide P (337) (inactive, free hydroxyl at C-12).  However, other erythrolides which differed by an acetate were A  (319) and V (50), both o f which were inactive, and B (320) and R (339), both o f which were also inactive. In addition, erythrolides E (323), 16-O-acetyl H (331), M (334), and T (48) were all shown to be inactive. A full cytotoxicity screen o f all the erythrolides known  0-1  ,  0  2  ,  , 4  6  ,  r-  8  10  Concentration (ug/ml)  Figure 4.13: Graph o f the Cytotoxicity o f Erythrolides and Aquariolides Isolated.  212  would probably shed more light on what structural features are required and what are not. This would be fairly easy to accomplish as E. caribaeorum produces large quantities o f these diterpenes, some o f which can be synthetically interconverted to complete the entire array o f compounds.  4.4: References 1)  Reseck, Jr., J. Marine Biology 2 ed. Prentice H a l l (Reston Division): Englewood Cliffs, N . J . , 1988, chapter 16.  2)  C o l l , J. C . Chem. Rev. 1992, 92, 613-631, and references therein.  3)  Rodriguez, A . D . Tetrahedron  4)  Bayer, F . M . The Shallow-Water Octocorallia of the West Indian Region. Martinus Nijhoff: The Hague, The Netherlands, 1961, pp.9-21, 64-65, and 75-77.  5)  a) Ciereszko, L . S . ; Johnson, M . A . ; Schmidt, R . W . ; Koons, C B . Comp. Biochem. Physiol. 1968, 24, 899-904. b) L i n g , N . C ; Hale, R . L . ; Djerassi, C . J. Am. Chem. Soc. 1970, 92, 5281-5282. c) Withers, N . W . ; K o k k e , W . C . M . C , Fenical, W . ; Djerassi, C . Proc. Natl. Acad. Sci. USA 1982, 79, 3764-3768.  6)  Burkholder, P.R.; Burkholder, L . M .  7)  Pham, N . B . ; Butler, M . S . ; Quinn, R . J . J. Nat. Prod. 2002, 65, 1147-1150.  8)  Tius, M . A . Chem. Rev.  9)  Sung, P.-J.; Sheu, J.-H.; X u , J.-P. Heterocycles  10)  a) Burks, J.E.; V a n Der Helm, D . ; Chang, C . Y . ; Ciereszko, L . S . Acta Cryst. 1977, B33, 704-709.  n d  1995,57,4571-4618.  Science  1958, 727, 1174-1175.  1988,55,719-732. 2002, 56, 535-579.  b) Selover, S.J.; Crews, P.; Tagle, B . ; Clardy, J. J. Org. Chem. 1981, 46, 964970. 11)  See for example: a) Kennard, O.; Watson, D . G . ; di Sanseverino, L . R . ; Tursch, B . ; Bosmans, R.; Djerassi, C . Tetrahedron Lett. 1968, 2879-2884. b) Rodriguez, A . D . ; Ramirez , C ; Shi, Y . - P . J. Org. Chem. 2000, 65, 6682-6687. c) Rodriguez, A . D . ; Shi, Y . - P . J. Org. Chem. 2000, 65, 5839-5842.  213  d) Rodriguez, A . D . ; Ramirez , C ; Rodriguez, L L ; Barnes, C L . J. Org. Chem. 2000, 65, 1390-1398. e) Rodriguez, A . D . ; Ramirez , C . Org. Lett. 2000,2,507-510. f) Rodriguez, A . D . ; Shi, J.-G.; Huang, S.D. J. Nat. Prod. 1999,62, 1228-1237. g) Iwashima, M . ; Matsumoto, Y . ; Takenaka, Y . ; Iguchi, K . ; Y a m o r i , T. J. Nat. Prod. 2002, 65, 1441-1446. h) Duh, C . - Y . ; El-Gamal, A . A . H . ; Wang, S.-K.; D a i , C.-F. J. Nat. Prod. 2002, 65, 1429-1433. a) Look, S.A.; Fenical, W . ; Matsumoto, G . K . ; Clardy, J. J. Org. Chem. 1986, 51, 5140-5145. b) Look, S.A.; Fenical, W . ; Jacobs, R . S . ; Clardy, J. Proc. Natl. Acad. Sci. USA 1986, 83, 6238-6240. Andersen, R.J.; Williams, D . E . In Chemistry in the Marine Environment; Hester, R . E . ; Harrison R . M . , Ed.; Issues in Environmental Science and Technology, no. 13; The Royal Society o f Chemistry: Cambridge, U K , 2000; pp. 55-79. Lindel, T.; Jensen, P.R.; Fenical, W . ; Long, B . H . ; Casazza, A . M . ; Carboni, J.; Fairchild, C R . J. Am. Chem. Soc. 1997,119, 8744-8745. Lindel, T. Angew. Chem. Int. Ed. 1998, 37, 774-776. a) D ' A m b r o s i o , M . ; Guerriero, A . ; Pietra, F. Helv. Chim. Acta 1987, 70, 20192027. b) D ' A m b r o s i o , M . ; Guerriero, A . ; Pietra, F . Helv. Chim. Acta 1988, 71, 964976. Battistini, C ; Ciomei, M . ; Pietra, F.; D ' A m b r o s i o , M . ; Guerriero, M . Patent Application WO 96/36335, November 21, 1996. Ketzinel, S.; Rudi, A . ; Schleyer, M . ; Benayahu, Y . ; Kashman, Y . J. Nat. Prod. 1996, 59, 873-875. Cinel, B . ; Roberge, M . ; Behrisch, H . ; van Ofwegen, L . ; Castro, C . B . ; Andersen, R . J . Org. Lett. 2000, 2, 257-260. Taglialatela-Scafati, O.; Deo-Jangra, U . ; Campbell, M . ; Roberge, M . ; Andersen, R . J . Org. Lett. 2002, 4, 4085-4088. Britton, R. Roberge, M . ; Berisch, H . ; Andersen, R . J . Tetrahedron Lett. 2001, 42, 2953-2956. Williams, D . E . Personal Communication, 2002.  214  Cinel, B . Ph.D. Thesis, University o f British Columbia Department o f Chemistry, 2001. Look, S.A.; Fenical, W . ; V a n Engen, D . ; Clardy, J. J. Am. Chem. Soc. 1984,106, 5026-5027. Pordesimo, E.O.; Schmitz, F J . ; Ciereszko, L . S . ; Hossain, M . B . ; van der H e l m , D . J. Org. Chem. 1991, 56, 2344-2357. Dookran, R.; Maharaj, D . ; Mootoo, B . S . ; Ramsewak, R.; M c L e a n , S.; Reynolds, W . F . ; Tinto, W . F . J. Nat. Prod. 1993, 56, 1051-1056. Banjoo, D . ; M a x w e l l , A . R . ; Mootoo, B . S . ; Lough, A . J . ; M c L e a n , S.; Reynolds, W . F . Tetrahedron Lett. 1998, 39, 1469-1472. Maharaj, D . ; Pascoe, K . O . ; Tinto, W . F . J. Nat. Prod. 1999, 62, 313-314. Banjoo, D . ; Mootoo, B . M . ; Ramsewak, R.S.; Sharma, R.; Lough, A . J . ; M c L e a n , S.; Reynolds, W . F . J. Nat. Prod. 2002, 65, 314-318. Taglialatela-Scafati, O.; Craig, K . S . ; Reberioux, D . ; Roberge, M . ; Andersen, R . J . Eur. J. Org. Chem. 2003, submitted. Davila-Huerta, G . ; Hamada, H . ; Davis, G . D . ; Stipanovic, R . D . ; Adams, C M . ; Essenberg, M . Phytochemistry, 1995, 39, 531-536, and references therein. Bowden, B . F . ; C o l l , J . C ; Engelhardt, L . M . ; Tapiolas, D . M . ; White, A . H . Aust. J. Chem. 1986, 39, 103-121, and references therein. M c P h a i l , K . L . ; Davies-Coleman, M.T.; Starmer, J. J. Nat. Prod. 2001, 64, 11831190, and references therein. Shen, Y . - C ; L i n , Y . - C ; Chiang, M . Y .  J. Nat. Prod. 2002, 65, 54-56.  Kwak, J.H.; Schmitz, F.J.; Williams, G . C J. Nat. Prod. 2002, 65, 704-708. Kwak, J.H.; Schmitz, F.J.; Williams, G . C .  Nat. Prod. 2001, 64, 754-760.  Gonzalez, N . ; Rodriguez, J.; Kerr, R . G . ; Jimenez, C . J. Org. Chem. 2002, 67, 5117-5123. Shen, Y . - C ; L i n , Y . - C ; K o , C - L . ; Wang, L . - T . J. Nat. Prod. 2003, 66, 302-305. Williams, D . E . ; Andersen, R.J. Can. J. Chem. 1987, 65, 2244-2247.  215  40)  Yamada, A . ; Kitamura, H . ; Yamaguchi, K . ; Fukuzawa, S.; Kamijima, C ; Yazawa, K . ; Kuramoto, M . ; Wang, G . - Y . - S . ; Fujitani, Y . ; Uemura, D . Bull. Chem. Soc. Jpn. 1997, 70, 3061-3069.  41)  Sonawane, H . R . ; Naik, V . G . ; Bellur, N . S . ; Shah, V . G . ; Purohit, P . C . ; Kumar, M . U . ; Kulkarni, D . G . ; Ahuja, J.R. Tetrahedron 1991, 47, 8259-8276.  42)  K o k k e , W . C . M . C . ; Epstein, S.; Look, S.A.; Rau, G . H . ; Fenical, W . ; Djerassi, C . J. Biol. Chem. 1984, 259, 8168-8173.  43)  Fenical, W . ; Pawlik, J. R . Mar. Ecol. Prog. Ser., 1991, 75, 1-8.  44)  Roberge, M . Personal Communication, 2002.  4.5: Experimental Section 4.5.1: General Information A l l reagents and solvents (except for N M R experiments) were purchased from either Fisher Scientific or Sigma-Aldrich and were used without further purification, except for H P L C solvents, which were filtered through a 0.45 um filter (Osmonics, Inc.) prior to use. The N M R solvent, ("100 % grade" CDCI3, deuterochloroform), was purchased from Cambridge Isotopes Laboratories. *H chemical shifts were referenced to the residual solvent peak at 8 7.24 ppm and  1 3  C chemical shifts to 8 77.0 ppm. N M R data  were acquired with the following spectrometers:  l 3  C data was acquired with a Bruker  A M - 4 0 0 spectrometer (direct detection 5mm probe); *H spectra and 2 D data were acquired with a Bruker A M X - 5 0 0 spectrometer (inverse detection 5 m m probe). Typical 2 D N M R experiments were C O S Y - 9 0 ( ' H - ' H correlations), H M Q C ( C - ' H single-bond 1 3  correlations), and H M B C ( C - H multiple-bond correlations). A R O E S Y for Erythrolide 1 3  !  T (48) was also acquired. Mass spectral data (high and low resolution D C I ) were acquired by a Kratos Concept II H Q Mass Spectrometer with the assistance o f the U B C Mass Spectrometry Centre staff. The carrier gas used i n D C I M S was a mixture o f C H  216  4  and NH3. IR ( K B r ) spectra were recorded on a Bruker model IFS-48 spectrophotometer; U V spectra were obtained i n M e O H using a Beckman D U 7 0 spectrophotometer. Polarimetry data were obtained using a Perkin Elmer 192 polarimeter, equipped with a sodium D-line lamp and a 10 cm microcell. The data from these spectrophotometers were obtained with the help o f m y colleague, Dr. Orazio Taglialatela-Scafati. Normal phase 230-400 mesh silica gel was acquired from Silicycle (Quebec City, PQ) and used i n a column measuring 430 m m by 50 mm, while the thin-layer chromatography ( T L C ) plates utilized were normal-phase Kieselgel 6OF254. Compound visualization on T L C plates was observed by one o f two methods: at 254 nm (shortwavelength U V ) , or by spraying with a solution o f 90% ethanol/10% concentrated H2SO4 plus a small amount o f vanillin added (coloured spots appear upon heating). The H P L C system utilized was either a Waters 2487 dual channel detector/system controller (monitoring at 254 nm for compound 342 only) connected to a Waters Series 515 H P L C pump or a Perkin Elmer L C - 2 5 R I detector (for all diterpenes) connected to either a Waters 501 Series H P L C pump or a Waters Series 515 H P L C pump. Either H P L C system ( U V or RI) was connected to a chart recorder set at 0.25 cm/min. Normal-phase H P L C columns used were a Whatman Partosil-10 M a g n u m operated at 2.0 m L / m i n or an Alltech Econosil 5JJ. analytical column operated at 1.0 m L / m i n . Erythrolides U (49) and V (50) (150 ug o f each) were dissolved i n 0.5 m L o f dry pyridine and a few drops (approximately 2 pX) o f acetic anhydride (AC2O) were added. The resulting mixture was stirred at room temperature overnight, and the reaction solvents were removed by evaporation under high vacuum. Purification o f the product was by either normal-phase H P L C 1:1 n-hexane:EtOAc or 3:7 «-hexane:EtOAc to yield  217  the appropriate known erythrolides D (322) (100 ixg) and A (319) (50 fig), respectively. The ' r l N M R spectra and optical rotation data for the semi-synthetic compounds were identical to that o f the natural products. " 24  25  4.5.2: Isolation Freshly collected wild-type specimens o f E. caribaeorum collected i n Prince Rupert B a y , Dominica were frozen and transported to Vancouver over dry ice. Cultured E. caribaeorum, (several generations removed from the wild), was grown on artificial rocks i n shallow running seawater tanks located in a greenhouse under ambient sunlight illumination. Freshly harvested animals (still alive) were shipped i n chilled seawater from Ocean Dreams, Inc. i n Florida to Vancouver. Samples o f the organism from either source (374 g and 400 g wet weight respectively) were cut in small pieces and extracted multiple times. For cultured E. caribaeorum the extraction solvent was M e O H (3 x 750 m L ) , while the wild-type organism was extracted with E t O H (3 x 750 m L ) . The E t O H (or M e O H ) extracts were combined and concentrated to a brown-coloured gum in vacuo that was suspended i n water (300 m L ) and extracted with E t O A c (3 x 300 m L ) . The resulting organic layer was then partitioned between «-hexane (3 x 300 m L ) and M e O H / H i O 9:1 (300 m L ) , yielding approximately 3.0 g (5.1 g for cultured specimens). Further fractionation o f the M e O H / H i O 9:1 extract by sequential application o f normal-phase flash silica gel (gradient elution from n-hexane/EtOAc 6:4 to E t O A c to E t O A c / M e O H 1:1 in 5% increments) yielded approximately seventy fractions. Fractions from the cultured organisms that eluted with n-hexane/EtOAc 6:4 were purified by normal-phase H P L C («-hexane/EtOAc 75:25) to yield pure 2.5 mg o f 331, 0.4 m g o f 51, and 1.7 m g o f 52. A persistent impurity i n 51 was removed by passing the material through normal-  218  phase H P L C again, this time using 1% z-PrOH i n n-hexane. Fractions from both the cultured and the wild-type organisms that eluted with EtOAc/«-hexane 6:4 were purified by 9:177-hexane/EtOAc to yield 5.1 m g o f 342. Those fractions from the wild-type organisms that eluted with EtOAc/n-hexane 65:35 were purified by normal-phase H P L C (n-hexane/EtOAc 7:3) to yield 0.1 m g o f 323 and 1.8 m g o f 334. Normal-phase H P L C (n-hexane/EtOAc 7:3) conditions were applied to a similar fraction from the cultured organisms to yield 0.1 m g o f 48. From fractions eluting with EtOAc/rc-hexane 9:1 and 8:2, respectively, i n both the cultured and wild-type, 319 and 320 were found i n large abundance (ie approximately 820 m g and 1.20 g, respectively, i n the w i l d type. Fractions from the wild-type organisms that eluted with EtOAc/n-hexane 95:5 were purified b y normal-phase H P L C (EtOAc/w-hexane 75:25) to yield 3.3 m g o f 328, 0.5 m g o f 338, 0.3 m g o f 49, and 0.3 mg o f 50. Finally, those fractions that eluted with E t O A c / M e O H 8:2 from the normal-phase flash silica gel column contained almost pure 337 i n moderate abundance, (~ 17.5 mg).  Erythrolides A (319), B (320), E (323), J (328), 16-0-Acetyl H (331), M (334), P (337) and Q (338): *H N M R and M S data identical with those reported i n the l i t e r a t u r e . ' ' 24  7V«ns-7-hydroxycalamenene (342): H and !  •1 1  1 3  26  28,29  C N M R data and M S data identical with  -3-5  those reported in the literature. Erythrolide T (48): Colourless glass; [ a ] 3540, 1780, 1733 cm" ; U V ( M e O H ) : X 1  max  2 5 D  = + 25.7° (c = 1.0 mg/mL); IR ( K B r ) : v  214 (e 8965) nm. ' H and  1 3  m a x  C N M R (CDC1 ): 3  Table 4.1. D C I M S : m/z 572, 574 (~ 3:1) [ M + N H ] . H R C I M S : m/z 572.1899, (calculated +  4  for C26H35 C10i,N, m/z 572.1899). 35  219  Erythrolide U (49): Colourless glass; [ a ]  D  3 5 4 2 , 1 7 7 5 , 1743 cm" ; U V ( M e O H ) : X  2 1 6 (e 6500) nm. ' H and  1  max  = - 3 5 . 1 ° (c = 1.0 mg/mL); IR ( K B r ) : v  m a x  C N M R (CDC1 ):  1 3  3  Table 4 . 2 . D C I M S : m/z 5 3 0 , 5 3 2 ( - 3 : 1 ) [ M + N H ] . H R C I M S : m/z 530.1788, (calculated +  4  for C H 3 3 C 1 0 o N , m/z 530.1793). 3 5  2 4  1  Erythrolide V (50): Colourless glass; [ a ] 3 5 4 3 , 1 7 6 9 , 1745 cm" ; U V ( M e O H ) : X  2 5 D  1  = - 3 8 . 0 ° (c = 1.0 mg/mL); IR ( K B r ) : v  2 1 5 (s 8500) nm. H and !  m a x  1 3  m a x  C N M R (CDC1 ): 3  Table 4.3. D C I M S : m/z 514, 5 1 6 (~ 3:1) [ M + N H ] . H R C I M S : m/z 514.1850, (calculated +  4  for C 2 4 H 3 C 1 0 N , m/z 514.1844). 35  3  9  Aquariolide B (51): Colourless glass; [ a ] 3 4 3 0 , 1 7 7 5 , 1737 cm" ; U V ( M e O H ) : 1  ? i  m  a  x  2 5 D  = - 6 2 . 5 ° (c = 1.0 mg/mL); IR ( K B r ) : v  213 (s 3800) nm. *H and  1 3  m a x  C N M R (CDC1 ): 3  Table 4.4. D C I M S : m/z 528, 5 3 0 ( - 3 : 1 ) [ M + N H ] . H R C I M S : m/z 528.2000, (calculated +  4  for C 2 5 H  35 35  C 1 0 o N , m/z 528.2000).  Aquariolide C (52): Colourless glass; [ a ] 3 4 3 2 , 1776, 1740 cm" ; U V ( M e O H ) : X 1  2 5 D  = - 1 6 7 . 2 ° (c = 2.0 mg/mL); IR ( K B r ) : v  m a x  213 (s 3500) nm. ' H and C N M R ( C D C 1 ) : 1 3  max  3  Table 4 . 5 . D C I M S : m/z 539, 541 (~ 3:1) [ M + H ] . H R C I M S : m/z 539.1692, (calculated for +  C 6H3 2  3 5 2  C l O , m/z 539.1684). 1 0  220  4.6: 2D N M R Spectra of Diterpenes 48-52  (ppm) <  (ppm)  7.00  mu 11  6.00  5.00  i  4.00  Ul  A!  3.00  2.00  1  Figure 4.14: C O S Y - 9 0 o f Erythrolide T (48) i n C D C 1 at 500 M H z . 3  221  (ppm) J  kXh  Li  I  <L_ ^_M  I  M  J  20  40  60  80  100  120  140  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.15: H M Q C o f Erythrolide T (48) in C D C 1 at 500 M H z . 3  222  (ppm)  40  80  120  160  200 (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.16: H M B C of Erythrolide T (48) in CDC1 at 500 M H z . 3  223  224  (ppm)  1.00  2.00  3.00  4.00  5.00  6.00  7.00 (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.18: C O S Y - 9 0 o f Erythrolide U (49) i n C D C 1 at 500 M H z . 3  225  (ppm)  (PPm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.19: H M Q C of Erythrolide U (49) in C D C 1 at 500 M H z . 3  226  120  160  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  200  Figure 4.20: H M B C of Erythrolide U (49) in CDC1 at 500 M H z . 3  227  ,-OH  (ppm)  1.00  2.00  3.00  4.00  5.00  6.00  7.00  (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.21: C O S Y - 9 0 of Erythrolide V (50) in C D C 1 at 500 M H z . 3  228  (ppm)  (PPm)  7.00  6.00  5.00  4.00  3.00  1.00  2.00  Figure 4.22: H M Q C of Erythrolide V (50) in CDC1 at 500 M H z . 3  ,-OH  (ppm)  40  80  120  160  200 (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.23: H M B C of Erythrolide V (50) in C D C 1 at 500 M H z . 3  230  (ppm) 1.00  2.00  3.00  4.00  5.00  6.00  7.00 (ppm) 7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.24: C O S Y - 9 0 of Aquariolide B (51) in C D C 1 at 500 M H z . 3  231  (ppm)  (ppm)  7.00  6.00  5.00  4.00  3.00  1.00  2.00  Figure 4.25: H M Q C of Aquariolide B (51) in CDC1 at 500 M H z . 3  232  (ppm) _JL_  40  80  120  160  200 (ppm)  7.00  6.00  5.00  4.00  3.00  2.00  1.00  Figure 4.26: H M B C of Aquariolide B (51) in CDC1 at 500 MHz. 3  233  (ppm)  2.00  3.00  4.00  5.00  6.00  7.00 (ppm)  7.00  6.00  5.00  4.00  3.00  2.00.  Figure 4.27: COSY-90 of Aquariolide C (52) in CDC1 at 500 MHz. 3  234  (ppm) _JUL_  JLALLJL_LJ_  20  40  60 6  rj>  80  1P^-X L PAc H«. 13/14 JlO OHy" v ci AcO '" H y20 AcO P  0=/  r  1  52  (ppm)  7.00  6.00  5.00  4.00  18  3.00  100  -  • 120  0 2.00  Figure 4.28: H M Q C of Aquariolide C (52) in C D C 1 at 500 M H z . 3  235  (ppm)  40  80  120  160  200 (ppm) 7.00  6.00  5.00  4.00  3.00  2.00  Figure 4.29: H M B C of Aquariolide C (52) in CDC1 at 500 M H z . 3  236  5.0: Concluding Remarks In the course o f our investigations on isoprenoids, thirty-four compounds were investigated: ten novel compounds were isolated, five novel derivatives were semisynthesized, and nineteen known compounds were isolated. Chapter 2 discussed the rhopaloic acids (two known, 35 and 36; four novel, 3740), which are norsesterterpenes, isolated from the sponge Hippospongia  sp., that are the  first known natural product inhibitors o f Ras signalling. Ras signalling represents a new target for anticancer research. This project showed that it is possible to search for molecules that are active against a cloned molecular target. Furthermore, rhopaloic acids B(35), C(36), and D / E (37/38) were three to four times more active against cells that are sensitive to Ras inhibitors than to cells that are not sensitive, which is the expected trend for RCE-protease inhibitors. These compounds are more active in vivo than in the enzyme assay, which could mean several things. One possibility is that the compounds are concentrated i n the cell, and thus a lesser amount is needed. Another is the possibility that the compounds are hitting more than one molecular target and would therefore be ineffective as drugs. W h i l e there is a good chance that latter statement is true, we would like to do further work to ascertain i f the former might be true instead. However, a lack o f material and political instability i n the source country have made any further work unlikely at this point. Chapter 3 focussed on research undertaken on two weedy plants. The first part details the investigation o f G 2 checkpoint inhibition activity o f Ambrosia  artemisiifolia,  the common ragweed. Ten sesquiterpenes were discussed i n this section, one novel  237  HO.  HO.  HO.  natural product, four n o v e l semi-synthetic d e r i v a t i v e s , and five k n o w n c o m p o u n d s . b i o l o g i c a l a c t i v i t y o f these c o m p o u n d s w a s attributed to the  The  a-methylene-y-lactone  m o i e t y present. I n the course o f the b i o c h e m i c a l i n v e s t i g a t i o n o f the t w o G 2 c h e c k p o i n t active c o m p o u n d s , p s i l o s t a c h y i n A (42) and C (44), it w a s d i s c o v e r e d that these c o m p o u n d s are also a n t i m i t o t i c agents. It has since b e e n suggested that these c o m p o u n d s d o not affect t u b u l i n p o l y m e r i z a t i o n i n m i t o s i s , ( w h i c h is what m o s t k n o w n antimitotics do), but affect s o m e other aspect o f m i t o s i s . Further investigations into the b i o c h e m i c a l targets o f these c o m p o u n d s , w h i c h are r e a d i l y a v a i l a b l e i n large quantities from the source plant, w i l l p o s s i b l y shed m o r e l i g h t o n their m o l e c u l a r targets. C u r r e n t l y , there is a great deal o f interest i n a n t i m i t o t i c c o m p o u n d s that do not target t u b u l i n .  1  I n the course  o f our i n v e s t i g a t i o n , w e also i s o l a t e d an i n a c t i v e n o v e l c o m p o u n d (41) w i t h a h i g h l y f u n c t i o n a l i z e d and rearranged c a r b o n skeleton. W e k n o w o f no precedent for this interesting sesquiterpene c a r b o n skeleton.  o  238  The second portion o f Chapter 3 detailed our investigation on Vernonia  baldwinii,  the western ironweed. Six known sesquiterpenes were isolated and one novel semisynthetic derivative was made. W h i l e we were originally looking for G 2 checkpoint inhibitors or antimitotic agents, we actually discovered that vernonataloide (47) and a related compound stimulated the formation o f a T G - 3 responsive phosphoepitope o f nucleolin that is present i n mitotic cells. N o other mitotic event was observed. It was later discovered that vernonataloide (47) could possibly be utilized to study Alzheimer's disease ( A D ) . In A D pathology, a protein called tau becomes hyperphosphorylated, which is then recognized by the T G - 3 antibody. If vernonataloide (47) can cause tau to be hyperphosphorylated, it could then be used to create an animal model for A D . Currently, no animal model exists. Chapter 4 covered the isolation o f eleven compounds from the gorgonian Erythropodium  caribaeorum.  O f these eleven compounds, five are novel diterpenes o f  the erythrolide series, five are known diterpenes also o f the erythrolide series, and one is a known sesquiterpene. T w o o f the novel diterpenes (49-50) are desacyl derivatives o f previously known erythrolides. Another, erythrolide T (48), is the first erythrolide isolated that possesses a 2,3-epoxide and an acetate moiety at C-4. Further larger scale isolations may find other erythrolides o f this type. The last two diterpene isolated are aquariolides B (51) and C (52), two additional members o f this new skeletal type. These compounds represent the products o f two very interesting chemical rearrangements, a diTi-methane rearrangement, previously seen i n other erythrolides, and a vinylcyclopropane rearrangement, which is only seen in the aquariolides. Our investigation suggests that these reactions are enzymatic i n nature, and thus represent exciting new targets for  239  enzyme isolation and study, as no examples o f enzymes that catalyze di-7i-methane and vinylcyclopropane reactions are currently known.  Reference: 1. Mayer T . U . ; Kapoor, T . M . ; Haggarty, S.J.; K i n g , R . W . ; Schreiber, S.L.; Mitchison, T.J. Science 1999,255,971-974.  240  

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