UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Bioactive natural products from nature Brastianos, Harry Charilaos 2007

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
ubc_2007_fall_brastianos_harry_charilaos.pdf [ 4.82MB ]
[if-you-see-this-DO-NOT-CLICK]
Metadata
JSON: 1.0061735.json
JSON-LD: 1.0061735+ld.json
RDF/XML (Pretty): 1.0061735.xml
RDF/JSON: 1.0061735+rdf.json
Turtle: 1.0061735+rdf-turtle.txt
N-Triples: 1.0061735+rdf-ntriples.txt
Original Record: 1.0061735 +original-record.json
Full Text
1.0061735.txt
Citation
1.0061735.ris

Full Text

Bioactive Natural Products From Nature  By Harry Charilaos Brastianos  B.Sc., The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA October 2007 © Harry Charilaos Brastianos, 2007  II  Abstract Bioassay guided fractionation of a crude extract of the marine sponge Neopetrosia exigua resulted in the first reported isolation of exiguamines A (2.58)  and B (2.59). These pyrroloquinone alkaloids have an unprecedented hexacyclic skeleton that has not been previously encountered in natural products. Biological studies have identified exiguamine A (2.58) as a potent in vitro inhibitor of the enzyme indoleamine-2,3-dioxygenase (IDO). tumor cells to evade the immune system.  IDO is an enzyme expressed by Inhibitors against this enzyme may  allow the immune system to attack cancer cells, making this enzyme a potential drug target for anti-cancer agents.  2.58  2.59  Investigation of the crude extract of a Bacillus sp. collected in Dominica led to the isolation of the known diketopiperazine cyclo(S-Val-S-Phe) (3.9). In vitro biological studies revealed that cyclo(S-Val-S-Phe) (3.9) is able to promote neurite outgrowth, even in the presence of physiological inhibitors.  In vivo  studies have shown that cyclo(S-VaI-S-Phe) (3.9) is able promote sprouting in serotonergic and adrenergic axons. Synthesis of the other three diastereomers led to the discovery that cyclo(R-Val-R-Phe) (3.22) is also an in vitro activator of axonal outgrowth.  III  39  3.22  Inhibitors of the G 2 checkpoint are able to increase the cytotoxicity of DNA damaging chemotherapeutics. Bioassay guided fractionation of an extract of the South American plant Duguetia odorata led to the isolation of the G 2 checkpoint abrogator, oliveroline (4.32). This investigation also led to the isolation of the previously unreported alkaloid N-methylguatterine (4.33), and the known alkaloids dehydrodiscretine (4.34) and pseudopalmatine (4.35).  4.32  4.33  OH 4.34  0  4.35  Chemical investigation of the marine sponge Myrmekioderma granulatum led to the isolation of the new compounds abolenone (5.24) and myrmekioside C (5.26), as well as the known compounds curcudiol (5.23), curcuphenol (5.25), abolene (5.22) and sesquiterpenoid (5.21).  Biological studies of these  compounds revealed that curcudiol is a ligand of the sex hormone-binding globulin (SHBG).  This protein is involved in transporting and regulating the  iv  concentration of steroids such as testosterone and estradiol. Many pathological conditions have a lower plasma concentration of these steroids.  Ligands to  SHBG can release steroids into the blood, so this protein is a potential drug target to treat conditions where a hormone insufficiency is present. H  iJ 5.23  5.22  5.21  5.24  5.25  XyIose  2  5.26  V  Table of Contents Abstract  ii  .  Table of Contents  v  List of Tables  ix  List of Figures  x  List of Schemes  xx  List of Abbreviations  xxi  Acknowledgements  xxxi  Chapter 1: Introduction to the Field of Natural Products  I  1.1. Historical Overview of Natural Products as Therapeutic Drugs  I  1.2. Bioactive Metabolites from Terrestrial Plants  2  1.3 Overview of Natural Products from Microorganisms  5  1.4 Overview of Marine Natural Products from Invertebrates  9  1.5 Conclusions  12  1.6. Preview of Thesis  13  1.7 References  17  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 20 2.1. Preview of Chapter2  20  2.2 Biology of Indoleamine-2,3-dioxygenase (IDO)  20  2.3 Inhibitors of IDO as Treatments for Cancer  24  2.4 Pyrroloquinones from Marine Sources  28  2.5 Alkaloids isolated from Neopetrosia sp  33  vi  2.6 Isolation of exiguamines A and B  .  36  2.7 Structure Elucidation of exiguamine A  38  2.8 Structure Elucidation of exiguamine B  64  2.9 Proposed Biogenesis of Exiguamine A  83  2.10 Stereochemistry of the exiguamines  84  2.11 Biological activity of Exiguamine A  88  2.12. General Experimental Methods  90  2.13. Isolation of exiguamines A and B  91  2.14. Physical Data  92  2.15. References  92  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  97  3.1. Preview of Chapter 3  97  3.2. Inhibitions that Prevent Spinal Cord Repair  97  3.3. Neuroprotective Properties of Diketopiperazines  100  3.4. Isolation of Neurite Outgrowth Activator from Bacillus sp  102  3.5. Stucture Elucidation of Cyclo(S-Va)-S-Phe)  103  3.6. Synthesis of Cyclo(S-Val-S-Phe) and its Diastereomers  105  3.7. Biology of Diketopiperazines  110  3.8. Concluding Remarks  115  3.9. General Experimental Section  116  3.10. Bacterial Culture  117  3.11. Identification of bacterial culture from sediment  117  3.12. Isolation of Cyclo(S-Val-S-Phe) from Bacillus sp  118  3.13. Physical Data of Isolated Diketopiperazine from Bacillus sp  119  3.14. Synthetic Experimental Section  120  VII  3.15 References. 130 Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  132  4.1. Preview of Chapter 4  132  4.2. The Cell cycle  132  4.3. G to M Transition  134  4.3. Rationale for using G 2 Checkpoint Inhibitors  136  4.5. Known G 2 Checkpoint Inhibitors  139  4.6. Description of the G 2 Checkpoint Assay  142  4.7. Chemistry of Duguetia sp  143  4.8. Isolation of alkaloids from Duguetia odorata  146  4.9. Structure Elucidation of N-methylguatterine  147  4.10. Biology of the Alkaloids Isolated from Duguetia odorata  163  4.11. General Experimental Methods  166  4.12. Isolation procedure of the alkaloids from Duguetia odorata  167  4.13. Checkpoint inhibitor activity  168  4.14. Description of the Cell Viability Assay  169  4.15. Physical Data of Alkaloids From Duguetia odorata  169  4.16. References  171  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 175 5.1. Preview of Chapter 5  175  5.2. Biology of the Sex-Hormone Binding Globulin Protein  175  5.3. Compounds Isolated from the genus Myrmekioderma  177  5.4. Isolation of bisabolane sesquiterpenes and myrmekioside C  180  5.5. Structure Elucidation of Abolenone  181  VIII  5.6. Structure Elucidation of Myrmekioside C peracetate  198  5.7. Biology of Secondary Metabolites isolated from Myrmekioderma styx  219  5.8. Acetylation of myrmekioside C  220  5.9. General Experimental Methods  220  5.10. Isolation of bisabolane sesquiterpenes and myrmekioside C  222  5.11. Physical data of secondary metabolites from Myrmekioderma styx  223  5.12. References  225  Chapter 6: Conclusions  227  6.1. Conclusions  227  6.2. References  230  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 231 Al. Data Collection  231  A.2. Data Reduction  231  A.3 Structure Solution and Refinement  232  A.4.1. Experiemental Details, Crystal Data  234  A.4.2. Experimental Details, Intensity Measurements  235  A.4.3. Experimental Details, Structure Solution and Refinement  236  A.5. References  248  ix  List of Tables Table 2.7.1. ID and 2D NMR data for Exiguamine A  47  Table 2.8.1. ID and 2D NMR data of Exiguamine B recorded in DMSO-d 6  72  Table 3.5.1. 1 H chemical shift values for 3.9, and the literature 1 H chemical shift values for both cyclo(S-Val-S-Phe) and cyclo(S-Val-R-Phe) 105 Table 4.9.1. ID and 2D NMR data of N-methylguatterine  154  Table 5.5.1. ID and 2D NMR data of abolenone (5.25)  188  Table 5.6.1. ID and 2D NMR data of myrmekioside C peracetate in C D 6  206  Table A.4. 1. Atomic coordinates (x I O”4) and equivalent isotropic displacement parameters (AA2 x I 0A3) for exigumaine A 237 Table A.4.2. Bond lengths [A] and angles [deg] for exiguamine A  238  Table A.4.3. Anisotropic displacement parameters (A”2 x I 0”3) for exiguamine A 243 Table A.4.4. Hydrogen coordinates (x I 0A4) and isotropic displacement parameters (AA2 x I 0A3) for exiguamine A  244  Table A.4.5. Torsion angles [degi for exiguamine A  245  Table A.4.6. Hydrogen Bonds  248  x  List of Figures Figure 11.1. Structures of salicin (1.1) and aspirin (1.2)  2  Figure 1.2.2. Examples of plant derived natural products recently approved for medicinal use 5 Figure 1.3.1. Significant natural products isolated from microorganisms  8  Figure 1.3.2. Significant natural products isolated from microorganisms in 2006.9 Figure 1.4.1. Significant marine natural products  11  Figure 1.4.2. Promising marine natural products isolated in 2006  12  Figure 1.6.1. Procedure of bioassay guided fractionation  15  Figure 2.2.1. Kynurenine pathway  22  Figure 2.2.2. Mechanism of formation of adduct between cs—crystalin and kynurenine  24  Figure 2.3.1. Analogs of tryptophan as competitive inhibitors of IDO  25  Figure 2.3.3. Isolated natural product lDO inhibitors  27  Figure 2.4.1. The discorhabdins and the epinardins  29  Figure 2.4.2. Batzelline family of natural products  30  Figure 2.4.3. Makaluvamines and veiutamine  31  Figure 2.4.4. Makaluvic Acids  32  Figure 2.4.5. Bispyrroloquinones from marine sources  33  Figure 2.5.1. Alkaloids isolated from Xestospongia/Neopetrosia exigua  35  Figure 2.6.1. Secondary metabolites isolated from neopetrosia exigua  36  xi  Figure 2.6.2 Neopotrosia exigua collected in Papua New Guinea  37  Figure 2.7.1. Numbering Scheme for exiguamine A  38  Figure 2.7.2. Three substructures of exiguamine A  39  Figure 2.7.3. 1 H NMR spectrum of exiguamine A (2.58) acquired at 600 MHz in 6 DMSO-d 40 Figure 2.7.4. 13 C NMR spectrum of exiguamine A (2.58) acquired at 150 MHz in DMSO-d 6 41 Figure 2.7.5. DEPT spectrum of exiguamine A (2.58) acquired at 150 MHz in 6 DMSO-d 42 Figure 2.7.6. HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d 6 43 Figure 2.7.7. HMBC spectrum of exiguamine A (2.58) acquired at 600 MHz in 6 DMSO-d 44 Figure 2.7.8. COSY spectrum of exiguamine A (2.58) acquired at 600 MHz in 6 DMSO-d 45 Figure 2.7.9. 1 H, 15 N LR-HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d 6 46 Figure 2.7.10. (a) 1 H and (b) ‘ C and 15 3 N chemical shifts of substructure I of exiguamine A  49  Figure 2.7.11. Key HMBC and COSY correlations of substructure I exiguamine A 49 Figure 2.7.12. Expansion of the 1 H, 15 N LR-HMQC spectra of the key correlations of substructure I of exiguamine A  52  Figure 2.7.13. COSY expansion of the correlations for substructure I of exiguamineA  53  Figure 2.7.14. HMBC correlations observed for H-13 of substructure! of exiguamine A (2.58)  54  XII  Figure 2.7.15. (a) 1 H and (b) 13 C and 15 N chemical shifts of substructure II of exiguamineA 54 Figure 2.7.16. Key HMBC and COSY correlations of substructure II of exiguamine A  55  Figure 2.7.17. Expansion of the 1 H, 15 N LR-HMQC spectra of the key correlations of substructure II of exiguamine A  57  Figure 2.7.18. Key COSY correlation for substructure II of exiguamine A  58  Figure 2.7.19. HMBC correlations observed for H-I of substructure II of exiguamine A (2.58)  59  Figure 2.7.20. (a) 1 H and (b) 13 C and 15 N chemical shifts of substructure lii of exiguamineA 59 Figure 2.7.21. HMBC correlations of substructure Ill of exiguamine A (2.58)... 60 Figure 2.7.22. Expansion of the 15 N LR-HMQC spectrum of the key correlations of substructure Ill of exiguamine A 61 Figure 2.7.23. Expansion of the HMBC spectrum of the key correlations of substructure Ill of exiguamine A (2.58)  62  Figure 2.7.24. ORTEP diagram of exiguamine A  63  Figure 2.8.1. Numbering scheme of exiguamine B (2.59)  64  Figure 2.8.2. Three substructures of exiguamine B  65  Figure 2.8.3. 1 H NMR spectrum of exiguamine B (2.59) run at 600 MHz in 6 DMSO-d  66  Figure 2.8.4. ‘ C NMR spectrum of exiguamine B run at 150 MHz in 6 3 DMSO-d . 67 Figure 2.8.5. DEPT spectrum of exiguamine B run at 150 MHz in . 6 DMSO -d  ..  68  Figure 2.8.6. HMQC spectrum of exiguamine B run at 600 MHz in . 6 DMSO . -d 69 Figure 2.8.7. HMBC spectrum of exiguamine B run at 600 MHz in . 6 DMSO -d 70 .  XIII  Figure 2.8.8. COSY spectrum of exiguamine B run at 600 MHz in . 6 DMSO .. -d 71 Figure 2.8.9. (a) 1 H NMR and (b) 13 C NMR of substructure I of exiguamine B. 73 Figure 2.8.10. Key HMBC correlations of substructure I of exiguamine B  73  Figure 2.8.11. HMBC correlations observed for substructure I of exiguamine B. 74 Figure 2.8.12. (a) 1 H NMR and (b) 13 C NMR of substructure II of exiguamine B. 75 Figure 2.8.13. Key HMBC and COSY correlations of substructure II of exiguamine B  75  Figure 2.8.14. Key COSY correlation of substructure II of exiguamine B  77  Figure 2.8.15. HMBC correlations for H-I of substructure II of exiguamine B (2.59)  78  Figure 2.8.16. (a) 1 H NMR and (b) 13 C NMR of substructure III of exiguamine B. 78 Figure 2.8.17. Key HMBC and COSY correlations of substructure II of exiguamine B  79  Figure 2.8.18. COSY correlations of substructure III of exiguamine B  81  Figure 2.8.19. HMBC correlations observed for H-13 of substructure Ill of exiguamine B  82  Figure 2.9.1. Proposed biogenesis of exiguamine A  84  Figure 2.10.1. CD spectrum of exiguamine A  85  Figure 2.10.2. Possible equilibrium between the enantiomers of exiguamine A.86 Figure 2.10.3. CD-spectrum of exiguamine B  87  Figure 2.10.4. Proposed mechanism of isomerization for C-17  87  xiv  Figure 2.10.5. 1 H NMR of expansions of exiguamine B. 88 Figure 2.10.6. 1 H NMR of expansions of exiguamine B  88  Figure 2.11.1. Description of the chemical reactions present in the in vitro IDO inhibition assay 89 Figure 2.11.2. Proposed pharmacophore of the exiguamines  90  Figure 3.2.1. Nogo-A, MAG, and OMgp are inhibitory proteins found in myelin. 99 Figure 3.2.2. Inhibitors of ROCK as potential axonal outgrowth activators  100  Figure 3.3.1. TRH (3) and Cyclo(S-His-S-Pro)  100  Figure 3.3.2. Neuroprotective Diketopiperazines  102  Figure 3.4.1. Cyclo(S-Val-S-Phe) (3.9), a compound promoting axonal outgrowth  102  Figure 3.5.1. 1 H NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 600 MHz in DMSO-d 6 104 Figure 3.5.2. 13 C NMR spectrum of cyclo(S-Val-S-Phe) (3.9) acquired at 150 MHz in DMSO-d 6 104 Figure 3.6.1. Preferred conformation of enolate 3.15  107  Figure 3.6.2. Preferred conformations of 3.12, 3.16, 3.9  108  Figure 3.7.1. The procedure of the cell migration assay to isolate neurite outgrowth activators  111  Figure 3.7.2. To evaluate the ability of the extracts to promote cell migration, each well is viewed under a microscope 111 Figure 3.7.3. Addition of 32 tM of cyclo[S-VaI-S-Phe] increases the neurite.. 113 Figure 3.7.4. Addition of Cyclo(S-VaI-S-Phe) (3.9) enhances the neurite length of axons even in the presence of inhibitory substrates from the central nervous system 114  xv  Figure 3.7.5. Addition of cyclo(S-Val-S-Phe) increased the axon sprouting in both serotonergic and adrenergic sprouting in the dorsal horn 115 Figure 3.8.1. Comparison of the structures of cyclo(S-Val-S-Phe) (3.9) and cyclo(R-Val-R-Phe) (3.22)  116  Figure 4.2.1. The cell cycle  134  Figure 4.3.1. 2 G / M transition  135  Figure 4.3.2. G 2 checkpoint pathway  136  Figure 4.4.1. Rationale for using G 2 checkpoint inhibitors  138  Figure 4.5.1. ATM/ATR inhibitors of the G 2 checkpoint pathway  139  Figure 4.5.2. Indole alkaloids inhibiting the G 2 checkpoint through Chkl  140  Figure 4.5.3 Alkaloids inhibiting the G 2 checkpoint through Chkl  141  Figure 4.5.4 Polyketide derived G 2 checkpoint inhibitors  142  Figure 4.6.1. Description of the G 2 checkpoint inhibition assay  143  Figure 4.7.1. Aporphine alkaloids from Duguetia  145  Figure 4.7.2 Alkaloids from Duguetia sp  146  Figure 4.8.1. Alkaloids isolated from D. odorata  147  Figure 4.9.1. Numbering scheme of N-methylguatterine  147  Figure 4.9.2 Substructures of N-methylguatterine deduced from the COSY and HMBC spectra 148 Figure 4.9.3. 1 H NMR spectrum of N-methylguatterine at 500 MHz in 6 DMSO-d . 149 Figure 4.9.4. “ C spectrum NMR of N-methylguatterine at 100 MHz in 6 3 DMSO-d . 150  xvi  Figure 4.9.5. HMQC spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d 151 Figure 4.9.6. HMBC spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d 152 Figure 4.9.7. COSY spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d 153 Figure 4.9.8. (a) 1 H chemical shifts and coupling constants for substructure I and (b) 13 C chemical shifts for substructure I 155 Figure 4.9.9. Key COSY and HMBC correlations observed for substructure I of 33 155 Figure 4.9.10. COSY correlations for substructure I of 4.33  157  Figure 4.9.11. HMBC correlations observed for H-13 and H-14 for substructure I of 4.33 158 Figure 4.9.12. (a) 1 H chemical shifts and (b) 13 C chemical shifts for substructure II 158 Figure 4.9.13. Key HMBC and COSY correlations observed for substructure II of 33 159 Figure 4.9.14. Expansion of the aromatic region of the COSY spectrum for 4.33. 160 Figure 4.9.15. HMBC correlations linking substructures I and II for 4.33  160  Figure 4.9.16. 1 H NMR of substructures III (a) and IV (b)  161  Figure 4.9.17. 13 C NMR of substructures III (a) and IV (b)  161  Figure 4.9.18. HMBC correlations for substructure III and IV for 4.33  161  Figure 4.9.19. 13 C chemical shifts for guatterine (4.36), an aporphine alkaloid related to 4.33 162 Figure 4.9.20. CD spectrum of N-methylguatterine (dashed line) and oliveroline (solid line) 163  xvii  Figure 4.10.1 Flow cytometry analysis of A DMSO, B isogranulatimide and C oliveroline 165 Figure 4.10.2. Concentration dependence of checkpoint inhibition activity of oliveroline and the other alkaloids 165 Figure 4.10.3. Other alkaloids tested in the G 2 checkpoint assay  166  Figure 5.2.1. Several examples of ligands that bind to SHBG  177  Figure 5.3.1. Linear diterpenes from M. styx  177  Figure 5.3.2. Cyanthiwigins isolated from Myimekioderma sp  178  Figure 5.3.3. Sesquiterpenoids isolated from Myrmekioderma sp  179  Figure 5.3.4. Glycolipids isolated from Myrmekioderma sp  180  Figure 5.4.1. Compounds isolated from Myrmekioderma styx  181  Figure 5.5.1: Abolenone  181  Figure 5.5.2. Substructures of abolenone as deduced from the HMBC and the COSY data 182 Figure 5.5.3. 1 H NMR spectrum of abolenone (5.25) at 600 MHz in C D 6  183  Figure 5.5.4. 13 C NMR spectrum of abolenone (5.25) at 150 MHz in C D 6  184  Figure 5.5.5. HMQC spectrum of abolenone (5.25) at 600 MHz in C D 6  185  Figure 5.5.6. HMBC spectrum of abolenone (5.25) at 600 MHz in C D 6  186  Figure 5.5.7. COSY spectrum of abolenone (5.25) at 600 MHz in C D 6  187  Figure 5.5.8. (a) H and (b) 1 C chemical shifts of substructure I of abolenone 13 (5.24) 189 Figure 5.5.9. Key HMBC correlations of substructure I of abolenone (5.25).... 189  xviii  Figure 5.5.10. HMBC correlations for H-15 of substructure I of abolenone (5.25). 190 Figure 5.5.11. (a) H and (b)’ 1 C chemical shifts of substructure II of abolenone 3 (5.25) 190 Figure 5.5.12. Key HMBC correlations of substructure II of abolenone (5.25). 191 Figure 5.5.13. COSY expansion for substructure II of abolenone (5.25)  192  Figure 5.5.14. (a) H and (b) 1 C chemical shifts of substructure Ill of abolenone 13 (5.25) 192 Figure 5.5.15. Key HMBC correlations of substructure UI of abolenone (5.25).193 Figure 5.5.16. COSY expansion for substructure Ill of abolenone (5.25)  195  Figure 5.5.17. HMBC expansion for substructure Ill of abolenone (5.25)  196  Figure 5.5.18. Key HMBC correlations of substructure of abolenone (5.25).... 196 Figure 5.5.19. CD spectrum of curcuphenol (dashed line) and abolene (solid line) 197 Figure 5.6.1: Myrmekioside C peracetate (5.28)  198  Figure 5.6.2. Five substructures of myrmekioside C peracetate (5.28)  199  Figure 5.6.3. 1 H NMR spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C D 6 200 Figure 5.6.4. C 13 NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C D 6 201 Figure 5.6.5. DEPT NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C D 6 202 Figure 5.6.6. HMQC spectrum of myrmekioside C peracetate (5.28) at 600 MHz D 6 in C 203 Figure 5.6.7. HMBC spectrum of myrmekioside C peracetate (5.28) at 600 MHz D 6 in C 204  xix  Figure 5.6.8. COSY spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C D 6 205 Figure 5.6.9. (a) H chemical shifts and coupling constants and (b) 13 1 C chemical shifts of substructure I of myrmekioside C peracetate (5.28) 208 Figure 5.6.10. Key HMBC correlations of substructure I of myrmekioside C peracetate (5.28)  208  Figure 5.6.11. COSY expansion for substructure I of myrmekioside C peracetate (5.28) 210 Figure 5.6.12. (a) H chemical shifts and coupling constants and (b) 13 1 C chemical shifts of substructure II of myrmekioside C peracetate (5.28) 210 Figure 5.6.13. Key HMBC correlations of substructure II of myrmekioside C peracetate (5.28) 211 Figure 5.6.14. (a) 1 H chemical shifts and coupling constants and (b) 13 C chemical shifts of substructure Ill of myrmekioside C peracetate (5.28)  212  Figure 5.6.15. Key HMBC correlations of substructure Ill of myrmekioside C peracetate (5.28) 212 Figure 5.6.17. (a) 1 H chemical shifts and coupling constants and (b) 13 C chemical shifts of substructure IV of myrmekioside C peracetate (5.28)  214  Figure 5.6.18. Key HMBC correlations of substructure IV of myrmekioside C peracetate (5.28) 215 Figure 5.6.19. Key HMBC correlations of myrmekioside C peracetate (5.28). 216 Figure 5.6.20. (a) 1 H chemical shifts and coupling constants and (b) ‘ C 3 chemical shifts of substructure V of myrmekioside C peracetate (5.28)  217  Figure 5.6.21. Key HMBC correlations of substructure V of myrmekioside C peracetate (5.28) 217 Figure 5.7.1. Dose response curve of (+)-curcudiol (5.24) in the SHBG assay. 220  xx  List of Schemes Scheme 3.6.1. Synthesis of cyclo(S-Val-S-Phe) (3.9) and cyclo(S-VaI-R-Phe) (3.14) 107 Scheme 3.6.2. Synthesis of cyclo(R-VaI-S-Phe) (3.21) and cyclo(R-VaI-R-Phe) (3.22) 109  xxi  List of Abbreviations o  -degrees -degrees Celsius  ID  -one dimensional  2D  -two dimensional  (-)  -negative optical rotation  (+)  -positive optical rotation  H 1  -proton  1, 15 H N LR-HMQC  -(, 15 H 1 N) long range heteronuclear multiple quantum coherence -carbon-13  3OHKG  -3-hydroxykynurenine glucoside  3OHKyn  -3-hydroxykynurenine  ci.  -1, 2 relative position or below the plane of the ring  ABP  -androgen binding protein -specific rotation at wavelength of sodium D line at 25° C  ACN  -acetonitrile  Ara-C  -cytosine arabinoside  ArH  -aromatic proton(s)  ATM  -ataxia telangiectasia mutated kinase  ATR  -ataxia telangiectasia mutated-related kinase -1, 3 relative position or above the plane of the ring  BC  -before Christ  xxii  BLAST  -Basic Local Alignment Search Tool  BnBr  -benzyl bromide  bs  -broad singlet  Bu  -butyl  c  -concentration  C  -carbon(s)  CAN  -ceric ammonium nitrate  D 6 C  -deuterated benzene  calc’d  -calculated  CD  -circular dichroism  3 CDCI  -deuterated chloroform  cdc2  -cyclin-dependent kinase I  cdc25c  -cell division cycle 25C kinase  CH  -methine  2 CH  -methylene  3 CH  -methyl  CI CH 2  -methylene chloride  3 CHCI  -chloroform  NO 3 CH 2  -nitromethane  ChkI  -CHKI checkpoint homolog  Chk2  -CHK2 checkpoint homolog  cm  -centimeter(s)  CNS  -central nervous system  xxiii  coil no  -collection number  COSY  (, H H 1 1) homonuclear correlation spectroscopy  CSPG  -chondroitin-sulfate proteoglycans  C-X  -carbon number X  Cys  -cysteine  d  -doublet -carbon chemical shift (in parts per million from tetramethyl silane)  dd  -doublet of doublets -extinction coefficient difference -proton chemical shift (in parts per million from tetramethyl silane)  DEPT  -distortionless enhancement by polarization transfer spectroscopy  di  -two  DMF  -N,N-dimethylformamide  DMSO  -dimethyl sulfoxide  6 DMSO-d  -deuterated dimethyl sulfoxide -nitrogen chemical shift (in parts per million from nitrous methane)  DNA  -deoxyribonucleic acid  DOPA  -3,4-dihydroxy-phenylalan me  Dr.  -doctor  s  -extinction coefficient  xxiv  50 EC  -concentration required for obtaining 50% of a maximum effect in vivo  ELISA  -enzyme linked immunosorbant assay  EPOCH II  -etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin  ESI  -electrospray ionization  EtOAc  -ethyl acetate  EtOH  -ethanol  FDA  -U.S. Food and Drug Administration  g  -grams  1 G  -growth phase one of the cell cycle -growth phase two of the cell cycle  Glul  -glucose one  Glul-C-X  -glucose one, carbon numberX  Glul-H-X  -glucose one hydrogen number X  G1u2  -glucose two  Glu2-C-X  -glucose two, carbon number X  G1u2-H-X  -glucose two, hydrogen number X  Gly  -glycerol  GIy-C-X  -glycerol carbon number X  Gly-H-X  -glycerol hydrogen number X  GTPase  -guanosine triphosphatase  Gy  -gray  h  -hour(s)  xxv  H  -hydrogen(s)  HCI  -hydrochloric acid  HCT-116  -human colon carcinoma cell line  [ H 3 ]-DHT  -tritium labeled d ihydrotestoterone  His  -histidine  0 2 H  -water  HL-60  -human leukemia cell line  HMBC  -(, 13 H 1 C) heteronuclear multiple bond coherence  HMQC  -(, 13 H 1 C) heteronuclear multiple quantum coherence  HPLC  -high performance liquid chromatography  H-Ras  -V.-Ha-Ras Harvey Rat Sarcoma Viral Oncogene Homolog  HRESIMS  -high resolution electrospray ionization mass spectrometry  HRESIMS-TOF  -high resolution electrospray ionization mass spectrometry time of flight  4 S 2 H 0  -sulphuric acid  H-X  -hydrogen number X  Hz  -hertz  50 IC  -the half maximal inhibitory concentration  IDO  -indoleamine-2,3-dioxygenase  J  -coupling constant in hertz  K  -dissociation constant for inhibitor binding  Kyn  -kynurenine  L  -liter(s) or levorotatory  xxvi  LC  -liquid chromatography  50 LC  -the dose required to kill half the population  LCT  -liquid chromatrograph-time of flight  LHMDS  -lithium hexamethyl disilazide  LRESIMS  -  low resolution electrospray ionization mass spectrometry  ’2 max  -wavelength at maximum intensity in nanometers  m  -multiplet or meter  M  -mitosis  [M]  -molecular ion  3 m  -meter cubed  MAG  -myelin-associated g lycoprotein  [M+HJ  -molecule plus hydrogen ion  [M+Na]  -molecule plus sodium ion  MCF-7  -human breast adenocarcinoma cell line  Me  -methyl  Me-X  -methyl number X  MeOD  -deuterated methanol  MeOH  -methanol  mg  milligram(s) -microgram(s)  4 MgSO  -magnesium sulphate  MHz  -megahertz  MIC  -minimum inhibitory concentration  xxvii  mm  -minute(s)  mL  -milliliter(s)  p.M  -micromolar  p.m  -micrometer(s)  mm  -millimeter(s)  mmol  -millimole(s)  MS  -mass spectrometry  mp53  -mutated cellular tumor antigen p53  M-Phase  -mitotic phase  m/z  -mass to charge ratio  N 15  -nitrogen-15  NaCI  -sodium chloride  NAD  -nicotinamide adenine dinucleotide  NADP  -nicotinamide adenine dinucleotide phosphate  NaH  -sodium hydride  NAPS  -Nucleic Acids and Protein Services, U.B.C.  n-BuLi  -n-butyl lithium  n-BuOH  -1-butanol  ng  -nanogram(s)  NgR  -Nogo receptor  CI 4 NH  -ammonium chloride  NCI  -National Cancer Institute  nm  -nanometer(s)  xxviii  nM  -nanomolar  NMR  -nuclear magnetic resonance  N-X  -nitrogen number X  ODS  -octadecyl silane  OH  -hydroxide  OMe  -methoxy  OMgp  -oligodendrocyte-myelin glycoprotein  p  -para  P  -phosphorylated  p53  -cellular tumor antigen p53  PCR  -polymerase chain reaction  pMeOBnCl  -  paramethoxybenzyl chloride  PLL  -poly-L-lysine  ppm  -parts per million  Pro  -proline  R  -rectus (configuration)  ref no  -reference number  RhoA  -ras homolog gene family, member A  RNA  -ribonucleic acid  RNase  -ribonuclease  rRNA  -ribosomal ribonucleic acid  ROCK s  -  RhoA associated coiled-coil-containing protein kinase  -singlet  xxix  S  -synthesis phase of cell cycle or sinister (configuration) or south  SAB  -standardized azide buffer  SCI  -spinal cord injuries  SCUBA  -self-contained underwater breathing apparatus  sec  -seconds  Sept  -September  Ser  -serine  SHBG  -sex hormone-binding globulin  sp.  -species  2 sp  2 hybrid orbital -sp  3 sp  3 hybrid orbital -sp  S-phase  -synthesis phase  t  -triplet  t  -tertiary  TCA  -trichioroacetic acid  TDO  -trypotophan-2,3-dioxygenase  tert  -tertiary  THF  -tetrahydrofuran  Thr  -threonine  TFA  -trifluoroacetic acid  TG-3  -thyroglobulin antibody three  TLC  -thin layer chromatography  TM  -trademark  xxx  TRH  -thyrotopin-releasing hormone  Tyr  -tyrosine  Val  -valine  XH  -x number of hydrogens  Xyl  -xylose  XyI-C-X  -xylose carbon number X  XyI-H-X  -xylose hydrogen number X  U.B.C.  -University of British Columbia  U.S.  -United States  UV  -ultraviolet  wt  -weight  xxxi  Acknowledgements First and foremost, I would like to express my gratitude to my supervisor, Dr. Raymond Andersen for the opportunity to be a graduate student in his laboratory.  His mentorship throughout the years have given me the skills to  succeed in any scientific endeavor. I am indebted to Dr. David Williams. He has been helpful throughout my graduate student career, and has always been willing to answer my countless questions. The Andersen lab is a wonderful environment to work in, and all the members have helped make my time in this lab unforgettable. Special thanks go to Rob Keyzers, and Gavin Carr for their assistance in my thesis research. Dr. Eduardo Vottero, Dr. Chris Sturgeon, Jennifer Wong, and Maghid Fallahi have conducted the biological aspects of the research.  Mike LeBlanc collected the  sponge samples, as well as consistently providing assistance with the equipment in the lab. Finally, I will always leave the best for last. My parents, John and Maria Brastianos, and  my sister Dr.  Priscilla  Brastianos have supported me  tremendously throughout my life. Without their many sacrifices, I would not be the person I am today.  Chapter 1: Introduction to the Field of Natural Products  I  Chapter 1: Introduction to the Field of Natural Products 1.1. Historical Overview of Natural Products as Therapeutic Drugs  For thousands of years, humanity has used the extracts of organisms to cure ailments. These drugs were usually preparations of herbs, shrubs, or other plants. An example of a plant that was used extensively in ancient times was the bark of the willow tree. Willow tree was first used by the Assyrians (4000 BC) and Babylonians (600 BC) as an anti-inflammatory and analgesic agent. The Greek physician Hippocrates in 400 BC recognized its pain-relieving properties and used it to treat the pain of child bearing in women. 1 For over two thousand years this bark was used as a cure for pain before chemical studies were undertaken to discover the source of the biological activity.  In 1829, the French pharmacist Henri Leroux isolated the pure  crystalline bioactive material known as salicin (1.1, Figure 1.1.1).  Synthetic  modifications of salicin led to aspirin (1.2, Figure 1.1.1), which is among the highest selling drugs of all time. The development of aspirin is an early landmark in natural product chemistry.  Salicin represents one of the earliest bioactive  compounds ever purified, and aspirin is the first synthetic drug based on a natural product lead.’  Chapter 1: Introduction to the Field of Natural Products  2  OH HO  OH  HOX  Figure lii. Structures of salicin (1.1) and aspirin (1.2). The use of secondary metabolites from organisms as a resource for the treatment of diseases has had a tremendous impact in medicine. From 19812002, 28% of all drugs that entered the market were either natural products or natural-product derived compounds. 2 Furthermore, an additional 24% of the drugs introduced were synthetic derivatives of natural product lead compounds. More than half (52%) the small molecule therapeutics were developed from natural products. 2 The impact of natural products is even more pronounced in the fields of oncology and infectious diseases where 60 and 75 percent, respectively, of drugs entering the market in that 21-year period were from a natural product origin. 2 Clearly, secondary metabolites from nature will continue to play a prominent role in the development of novel pharmaceuticals. 1.2. Bioactive Metabolites from Terrestrial Plants Terrestrial plant secondary metabolites have been the main source of therapeutics since ancient times, and currently it has been estimated that approximately 80% of the world’s population uses plant-based medicines. 3 Current estimations indicate that there are approximately 350,000 different species of plants growing on earth. Out of the 350,000 plants, one-third of these plants have not been 4 discovered. Out of the remaining two-thirds, only a small  Chapter 1: Introduction to the Field of Natural Products  3  fraction (15%) of these species have been studied for biologically active secondary metabolites, so there remains potential to find novel bioactive 5 compounds. The natural products of plants have played a key role in the treatment of cancer. A significant discovery in cancer therapy was the isolation of paclitaxel (taxolTM, 1.3, Figure 1.2.1) as the cytotoxic component from the Pacific yew tree, Taxus brevifolia. 6  Elucidation of its biological activity showed that it induces  mitotic arrest by promoting the polymerization of tubulin. 7 Paclitaxel has become one of the most important drugs for the treatment of ovarian and breast 9 Vinblastine (1.4, Figure 1.2.2) and vincristine (1.5, Figure 1.2.2) are ’ 8 cancers. two other plant natural products currently used clinically that interact with tubulin. These alkaloids isolated from the periwinkle known as Catharanthus roseus are mainly used to treat leukemias and lymphomas. ° Other plant entities in clinical 1 use include derivatives of the antineoplastic agent camptothecin (1.6, Figure 1.2.2).  Camptothecin was originally isolated from the extracts of the Chinese  ornamental tree, Camptotheca acuminatea. Since camptothecin was too toxic to be used in the clinic, its analogues topotecan (1.7) and irinotecan (1.8) were developed and are currently used to treat various cancers.  Camptothecin is  cytotoxic due to its interactions with DNA-topoisomerase I, which ultimately leads to the inhibition of DNA synthesis and cell death. 11  Chapter 1: Introduction to the Field of Natural Products  9  HOO  4  OH  OH  c; o  H C 3 O  1  j 1.4  1.3  3 OCOCH 3 C 2 1’CO H HQH 3 CH ‘:  N  ONZ / 2 C 3 H C O H COjJ\ 3 H QcocH CH 2 1<,CO 3 1.5  ,I  0 HJ 1.6  CHO  HO 1.7  0  1.8 ‘  0  Figure 1.2.1. Plant derived anti-cancer compounds. Numerous plant derived natural products have been approved for clinical use in the last seven years. Galantamine hydrobromide (1.9) is an alkaloid that is used to treat Alzheimer’s disease by slowing the process of neural degeneration.  This compound was isolated from the plant Galanthus nivalis,  which is found in Turkey and Bulgaria. 12 Another neuroactive alkaloid that has been approved in the clinic is apomorphine hydrochloride (1.10), which is used for Parkinson’s disease. 13 It is clear that based on the number of compounds recently being approved for medicinal use, plant-derived secondary metabolites remain a promising field for drug discovery.  Chapter 1: Introduction to the Field of Natural Products  1.9  5  1.10  Figure 1.2.2. Examples of plant derived natural products recently approved for medicinal use. 1.3 Overview of Natura’ Products from Microorganisms  Current estimations indicate that only 5% of fungal and 1% of bacterial species have ever been cultured in the laboratory, and even smaller numbers have been examined for secondary metabolites.  Despite the low number of  species studied, over 22,000 bioactive compounds have been isolated from microorganisms. This illustrates the impressive chemical diversity of secondary metabolites  produced  by microorganisms.  As  culturing  conditions for  microorganisms improve, the potential to study an even greater number of microorganisms and isolate additional novel biologically active compounds increases tremendously 14. The explosion of the use of microorganisms as a source of medicinally relevant compounds started in the 1930’s and 1940’s with the discovery of penicillin (1.11).  After that discovery, drug companies realized that culturing  microorganisms provided access to a wide chemical diversity of bioactive secondary metabolites and an almost limitless supply of a drug. Therefore, drug companies started isolating large collections of cultivatable microorganisms which led to the discovery of antibiotics such as streptomycin (1.12) and chlorotetracycline (1.13) during the 1950’s. 15  Microorganisms have not only  Chapter 1: Introduction to the Field of Natural Products  6  been studied for potential antibiotics, but also for compounds that affect cell metabolism and signaling pathways. Other drugs produced by microorganisms that are used clinically include the immunosuppressive drug FK-506 (1.14), which is produced by Streptomyces tsukubaensis 16, the cholesterol-lowering agent lovastatin (1.15), isolated from Aspergillus terreus, 17 and the antidiabetic drug acarbose (1.16), from the Actinoplanes sp. 18 In the past year, there have been several interesting bioactive secondary metabolites that were isolated from microorganisms. The compound garnering perhaps the most attention was the novel antibiotic platensimycin (1.17)19 Platensimycin was isolated from the extracts of Streptomyces platensis, a soil bacterium collected in South Africa. This compound contains a unique tetracycle and an uncommon 3-amino-2,4-dihydroxybenzoic acid head group. Biologically, platensimycin selectively inhibits lipid biosynthesis in both Staphylococus aureas and Staphylococus pneumoniae and does not affect other metabolic processes. In vitro studies reveal that platensimycin has potent activity against Grampositive bacteria including ones resistant to antibiotics. Studies in mice infected with S. aureas show that platensimycin has promising in vivo activity as well. 19 In an era of increasing antibiotic resistance, the discovery of novel antibiotics can have a substantial effect on the course of human disease. Other promising novel antibacterial compounds isolated in the past year include marinomycins A-D.  These polyketide-derived secondary metabolites  were isolated from a previously unclassified species of marine actinomycete. Fenical and co-workers suggested the name of Marinispora for the bacterial  Chapter 1: Introduction to the Field of Natural Products  7  ° Marinomycin A (1.18) was found to be the most potent antibacterial 2 genus. agent of all the marinomycins with an in vitro minimum inhibitory concentration (MIC) of 130 nM against menthicillin-resistant S. aureus and vancomycin resistant Streptococcus faecium. The marinomycins were found to be inactive as anti-fungal agents, with only marinomycin A showing weak activity against Candida albicans. The marinomycins also demonstrated potent and selective anti-tumor activity. When the marinomycins were tested in the NCI’s panel of 60 cancer lines, marinomycin A, B and C were very active against six out of the eight melanoma cell lines. More importantly though, the marinomycins showed only very weak activity against the leukemia cell lines which suggests selective ° 2 cytotoxicity. Rhizoxin (1.19) is one of the most potent anti-mitotic agents known and it was found to be very active against human and murine tumor cells in vitro. Due to its promising biological activity, rhizoxin has undergone clinical trials as a compound to treat cancer. 21 Unfortunately, due to low in vivo activity, rhizoxin was removed from clinical trials.  This compound was first isolated from the  pathogenic plant fungus Rhizopus microsporus, which causes rice seedling blight.  In 2000, Andersen and co-workers discovered several analogues of  rhizoxin from a bacterium in the genus Pseudomona. This was the first time rhizoxin derivatives were isolated from bacteria and not from a fungus. 22 Other studies have shown that rhizoxin is biosynthesized by the bacterium Burkholderia rhizoxina, which are endosymbiotic bacteria that reside in the fungus. recently, the symbiotic bacteria were cultivated and  Very  numerous rhizoxin  Chapter 1: Introduction to the Field of Natural Products  derivatives were isolated from the bacteria.  8  Three derivatives, rhizoxin MI  (1.20), M2 (1.21), and Z2 (1.22) were 1000-10000 times more active than rhizoxin at inhibiting the proliferation of K-562 leukemia cells and were found to be among the most potent anti-mitotic agents ever found.  Perhaps these  derivatives will yield more promising in vivo activity than rhizoxin. 21 H  H  __  S.  0 COOH 1.11  ‘CHO CIHOH:  I .12 OH  0  OH  0  1.13 HOO  1.15  CH O 2 H  OH HO-7j HO HO_N7 HO HO 1.16  OH  OHOH  Figure 1.3.1. Significant natural products isolated from microorganisms.  ChaDter 1: Introduction to the Field of Natural Products  9  1.17 HO 118  3 “COOCH  1.19  OH 3 “COOCH  1.21  1.22  Figure 1.3.2. Significant natural products isolated from microorganisms in 2006. 1.4 Overview of Marine Natural Products from Invertebrates Oceans cover approximately two-thirds of the world’s surface and contain  over 100,000 species of marine invertebrates; however, only a small fraction of these species have been examined for the presence of biologically active compounds. Many of the invertebrates that live in the ocean including porifera, echinodermata, bryozoa and coelentara have soft bodies and are sessile, yet they are able to thrive in the ocean.  These organisms contain secondary  metabolites which protect them from predators, deter competitors, and assist  Chapter 1: Introduction to the Field of Natural Products  10  them in catching prey. 23 Chemists have exploited these compounds produced by marine invertebrates to yield very promising medicinally active drugs. The field of marine natural products is relatively young, being only studied extensively in the last thirty five years. Despite its youth, several compounds of marine origin that have been approved to be used clinically, as well as numerous marine invertebrate-derived drug candidates that are in clinical trials. Among the earliest bioactive compounds from marine invertebrates were the nucleosides spongouridine (1.26) and spongothymidine (1.27) from the Caribbean sponge Ciyptotheca ciypta. 24 Synthetic modifications of the two nucleosides led to the discovery of cytosine arabinoside (Ara-C; 1.28), the first drug introduced in the clinic that was based on a marine natural product lead. Cytosine arabinoside was approved by the FDA in 1969 as an anti-cancer agent, and is currently used to treat leukemia and lymphomas. 24 A promising drug from the sea to enter clinical trials is the isoquinoline alkaloid ecteinascidin 743 (1.29), which was isolated from the marine tunicate Ecteinascidia turbianta.  This alkaloid is currently in phase Ill clinical trials for  numerous cancers including ovarian and soft tissue 25 sarcoma. 2 ’ 6 Another anti tumor drug in phase II clinical trials is alpidine (1.30), which was first isolated from the Mediterranean ascidian, Aplidium albicans. The mechanism of its anti cancer action is that it arrests cells at the G 1 or G 2 phases of the cell cycle, and is an inhibitor of angiogenesis. It is presently in phase II clinical trials for various cancers including melanoma, pancreatic, and non-Hodgkin lymphoma. 27  Chanter 1 Introduction to the Field of Netural Products  0  0  HN 0  HO  1.26  N  11  2 NH  HN HO  0” ‘N  0  HO  1.27  N  1.28  H C 3 ,  /  1.29  1.30  Figure 1.4.1. Significant marine natural products. In the past year, there have been several novel bioactive compounds isolated from marine invertebrates. One anti-cancer agent recently discovered was the polyketide palmerolide A (1.31) which was isolated from the Antarctic tunicate Synoicum adareanum. This macrolide targets melanoma 50 (LC 18 nM) = with three orders of magnitude greater sensitivity relative to other cell lines that were tested. 2829 Specificity for certain cell lines is beneficial therapeutically when used in humans, because of fewer side effects. Cortistatins A-D were isolated from a MeOH extract of Corticium simplex, which was collected in Indonesia. The cortistatins contain an isoquinoline moiety and a bicyclic octene which are both rare structural elements in steroids. All four cortistatins were able to selectively inhibit the proliferation of human umbilical vein endothelial tumor cells. Cortistatin A (1.32) was also found to be a potent in  Chantar 1 introduction to tha Fiald of Natural Products  12  vivo inhibitor of 3 angiogenesis. Other anti-cancer agents isolated from sponges ° were azumamides A-E. These cyclic peptides were isolated from the Japanese sponge Mycale izuensis and were active inhibitors of histone deacetylases. Furthermore, azumamide A (1.33) was also found to inhibit angiog 31 enesis. OH  o0H  oo  0 N 2 H  OH  rfl<. 1.32  1.33  Figure 1.4.2. Promising marine natural products isolated in 2006. 1.5 Conclusions The investigation of bioactive secondary metabolites from nature plays an important role in the medical sciences. First, bioactivity-guided natural product investigation can lead to the discovery of novel chemical entities, or the discovery of new biological activity for known compounds. Once a lead drug candidate is isolated, it may be modified synthetically to make it more efficacious or less toxic. Second, bioactive secondary metabolites may also be powerful biological tools to discover new drug targets. This was especially evident with camptothecin, where it was the first time inhibitors of DNA topoisomerase I were seen as drug  Chapter 1: Introduction to the Field of Natural Products  candidates against cancer 11.  13  Finally, searching for compounds in nature may  also yield a source for a drug that may be very difficult or expensive to manufacture synthetically.  This includes natural products produced from  microorganisms where the fermentation of microbes may provide an industrial scale supply of the desired bioactive corn 32 pound. Currently, it has been estimated that only one-third of diseases can be treated effectively. 33 Furthermore, with bacteria, cancer and viruses becoming resistant to the current therapeutic regimens, the need for novel drug candidates has never been greater. Despite the need for novel drug pharmacophores, major pharmaceutical companies in the last ten years have either abandoned, or drastically reduced funding for the research and development of novel bioactive compounds from 34 nature. 3 ’ 5 Hopefully this trend will cease, as the majority of Earth’s natural biological resources remain untapped for novel drug leads. 1.6. Preview of Thesis This thesis focuses on the purification and structure elucidation of bloactive secondary metabolites from marine, terrestrial and microbial sources. The Andersen lab has access to a large library of extracts from organisms, which equates to a wide diversity of secondary metabolites.  Furthermore, the  Andersen lab also has access to a large number of novel biological assays. The combination of having access to unique biological assays, and a large library of extracts represents a unique opportunity to discover new bioactive small molecules.  Chapter 1: Introduction to the Field of Natural Products  14  The isolated bioactive natural products can serve various purposes in the biological sciences. Firstly, these compounds may be used as lead compounds to develop potential therapeutic agents. Secondly, the isolated small molecules may serve as biochemical tools to discover new drug targets, as well as to probe the molecular basis for diseases. Finally, the isolated molecules may assist in the development of novel biological assays and serve as a proof of principle that the biological screen may be used to search for bioactive compounds from biological extracts. 5 The emphasis of the Andersen group is to use bioassay guided fractionation (Figure 1.6.1) to isolate the bioactive molecules from the crude extracts of organisms, and to use spectroscopic techniques to identify the structure of the bioactive compounds. In bioassay guided fractionation, a library of crude extracts are evaluated for a particular biological 4 response. 3 ’ 3 The active crude extract is further separated using various chromatographic techniques to obtain semi-purified fractions, which are evaluated in a biological assay. Only the biologically active fractions are further separated and evaluated in the bioassay. This process is repeated until the biologically active components are purified (Figure 1.6.1).  The structures of the purified compounds are then  determined using various spectroscopic techniques.  Chanter 1: Introduction to the Field of Natural Products  15  4  Separation  No Purification  Bioassay/I  \\ Bioassay Inactive Fractions  Repeated Separatioi Repeated Bioassays  I  Pure Bioactive Compound(s)  Jr  I  No [[Purifica tion  Figure 1.6.1. Procedure for bioassay guided fractionation.  I I  Chapter 1: Introduction to the Field of Natural Products  16  The second chapter of the thesis will discuss the search for inhibitors for the enzyme indoleamine-2,3-dioxygenase (IDO).  I DO is a protein that is  expressed by many tumors in order to suppress the immune system, therefore, inhibitors of this enzyme have the potential to be used in cancer therapy. Bioassay-guided fractionation of the crude extract of the sponge Neopetrosia exigua yielded the novel alkaloids exiguamines A and B. Exiguamine A is one of the most active IDO inhibitors known to date. 36 The third chapter will deal with the isolation and structure elucidation of compounds that induce neurite outgrowth. When there is an injury to the central nervous system, inhibitors are present that prohibit the spontaneous repair of axons. Compounds that stimulate neuronal outgrowth in the presence of these inhibitors have the potential to aid in the repair of the nervous system following traumatic spinal cord injury.  Bioassay-guided fractionation of an extract from  cultures of a marine Bacillus sp. yielded the diketopiperazine cyclo(S-Val-S-Phe) as the active component. Synthesis of all four diastereomers established that cyclo(R-Val-R-Phe) was also an axonal outgrowth activator. The fourth chapter of the thesis will discuss the isolation of compounds that inhibit the G 2 checkpoint. Both the G 1 and the G 2 checkpoints are involved in repairing damaged DNA.  It has been found that most tumors lack the Ci  checkpoint, so inhibitors of the G 2 checkpoint would make tumor cells more sensitive to DNA-damaging chemotherapeutics such as cisplatin.  Bioassay  guided fractionation of the MeOH extract of the plant Duguetia odorata yielded  Chapter 1: Introduction to the Field of Natural Products  17  oliveroline as the active compound and led to the isolation of three more alkaloids, including the new aporphine alkaloid, 3 N-methylgua tterine. 7 The isolation of ligands for the sex hormone binding globulin will be the focus of the fifth chapter. Sex hormone-binding globulin (SHBG) is involved in regulating and binding steroids such as testosterone, estradiol, and 5cLdihydrotestosterone. Many conditions result in low levels of these steroids, so ligands that bind to SHBG may release bound steroids into the bloodstream. Bioassay-guided fractionation of the sponge Myrmekioderma granulatum yielded the known terpene (+)-curcudiol as the active component.  Five additional  inactive compounds were isolated, including a new glycolipid and two new terpenes.  1.7 References  (1)  Mahdi J.G.; Mahdi A.J.; Mahdi A.J.; Bowen l.D. Cell Proliferation 2006, 39, 147-155.  (2)  Newman D.J.; Cragg G.M.; Snader K.M. Journal of Natural products 2003, 66, 1022-1037.  (3)  Ramawat K.G. Biotechnology of Medicinal Plants; Science Publishers, Inc.: Enfield, NH, 2004.  (4)  Cseke L.J.; Kirakosyan A.; Kaufman RB.; Waiter S.L.; Duke J.A.; Brielmann H.L. Natural Products From Plants; 2nd ed.; Taylor & Francis: New York, 2006.  (5)  Trignali C. Bioactive Compounds from Natural Sources: Isolation, Characterisation and Biological Properties; Taylor & Francis: New York, 2001.  (6)  Wani M.C.; Taylor H.L.; Wall M.E.; Coggon P.; McPhail A.T. Journal of the American Chemical Society 1971, 93, 2325-2327.  (7)  Schiff P.B.; Fant J.; Horwitz S.B. Nature 277, 665-667.  Chapter 1: Introduction to the Field of Natural Products  18  (8)  McGuire W.P.; Rowinsky E.K.; Rosenheim N.B.; Grubine F.C.; Ettinger D.S.; Armstrong D.K.; Donnehower R.C. Annals of Internal Medicine 1989, 273-279.  (9)  Holmes F.A.; Walters R.S.; Therlault R.L.; Forman A.D.; Newton L.K.; Raber M.N.; Buzdar A.U.; Fyre D.K.; Hortobagyi G.N. Journal of the National Cancer Institute 1991, 83, 1797-1805.  (10)  Duffin J Canadian Bulletin of Medical Histoiy 2000, 17, 155-192.  (11)  Oberlies N.H.; Kroll D.J. Journal of Natural products 2004, 67, 129-135.  (12)  Heinrich M.; Teoh H.L. Journal of Ethnopharmacology 2004, 92, 147-162.  (13)  Deleu D.; Hanssens Y.; Northway M.G. Drugs and Aging 2004, 21, 687709.  (14)  Demain A.L. Journal of Industrial Microbiology & Biotechnology 2006, 33, 486-495.  (15)  Chin Y.W.; Balumas M.J.; Chai H.B.; Kinghorn A.D. The APPS Journal 2006, 8, E239-E253.  (16)  Tanaka H.; Kuroda A.; Marusawa H.; Hatanaka H.; Kino T.; Goto T.; Hash imoto M. Journal of the American Chemical Society 1987, 109, 50315033.  (17)  Endo A. Journal of Lipid Research 1992, 33, 1569-1582.  (18)  FrommerW.; Junge B.; Muller L.; Schmidt D.; Truscheit E. Planta Medica 1979, 35, 195-217.  (19)  Wang J.; Soisson S. M.; Young K.; W. Shoop; Kodali S.; Galgoci A.; Painter R.; G. Parthasarathy; Tang Y. S.; Cummings R.; Ha S.; K. Dorso; Motyl M.; Jayasuriya H.; Ondeyka J.; Herath K.; Zhang C.; Hernandez L.; Allocco J.; Basilio P.; Tormo J. R.; Genilloud 0.; Vicente F.; Pelaez F.; Coiwell L.; Lee S. H.; Michael B.; T. Felcetto; Gill C.; Silver L. L.; Hermes J. D.; Bartizal K.; Barrett J.; Schmatz 0.; Becker J. W.; Cully D.; Singh S. B. Nature 2006, 441, 358-361.  (20)  Kwon H.C.; Kauffman C.A.; Jensen P.R.; Fenical W. Journal of the American Chemical Society 2006, 128, 1622-1632.  (21)  Scherlach K.; Patida-Martinez L.P.; Dahse H.M.; Hertwick C. Journal of the American Chemical Society 2006, 128, 11529-11536.  (22)  Roberge M.; Cinel B.; Anderson H.J.; Lim L.; Jiang X.; Xu L.; Bigg C.M.; Kelly M.T.; Andersen R.J. Cancer Research 2000, 60, 5052-5058.  Chapter 1: Introduction to the Field of Natural Products  19  (23)  Haefner B. Drug Discover,’ Today 2003, 8, 536-544.  (24)  Newman D.J.; Cragg G.M. Current Medicinal Chemist,y 2004, 11, 16931713.  (25)  Simmons T.L.; Andrianasolo E.; McPhail K.; Flatt P.; Gerwick W.H. Molecular Cancer Therapeutics 2005, 4, 333-342.  (26)  Markman M. The Oncologist 2007, 12, 186-190.  (27)  Taraboletti G.; Poli M.; Dossi R.; Manenti L.; Borsotti P.; Faircoth G.T.; Broggini M.; D’lncalci M.; Ribatti D.; Giavazzi R. British Journal of Cancer 2004, 90, 2771-2784.  (28)  Diyabalanage T.; Amsier C.D.; McClintock J.B.; Baker B.J. Journal of the American Chemical Society 2006, 128, 5630-5631.  (29)  Jiang X.; Liu B.; Lebreton S.; DeBradander J.K. Journal of the American Chemical Society 2007, ASAP.  (30)  Aoki S.; Watanabe Y.; Sanagawa M.; Satiawan A.; Kotoku N.; Kobayashi M. Journal of the American Chemical Society 2006, 128, 3 148-3149.  (31)  Noako Y.; Yoshida S.; Matsunaga S.; Shindoh N.; Terada Y.; Nagai K.; Yamashita J.K.; Ganesa A.; Soest R.W.M. van; Fuesetani N. Angewandte Chemie 2006, 45, 7553-7557.  (32)  Robbers J.E.; Speedie M.K.; Tyler V.E. Phamacognosy and Pharmacobiotechnology; Williams & Wilkins: Baltimore, 1996.  (33)  Mulzer J.; Bohiman R. The Role of Natural of Natural Products in Drug Discovery; Springer: New York, 2000.  (34)  Newman D.J.; Cragg G.M. Journal of Natural products 2007, 70,461-477.  (35)  Cordell G.A. Phytochemistty Reviews 2002, 1, 26 1-273.  (36)  Brastianos H.C.; Vottero E.; Patrick B.C.; Soest R. van; Matainaho T.; Mauk A.G.; Andersen R.J. Journal of the American Chemical Society 2006, 128, 16046-16047.  (37)  Brastianos H.C.; Sturgeon C.M.; Roberge M.; Andersen R.J. Journal of Natural Products 2007, 70, 287-288.  Chapter 2: Isolation of Inhibitors of lridoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 20  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3dioxygenase (100) from the Marine Sponge Neopetrosia exiguaa 2.1. Preview of Chapter 2 Tumor cells express high levels of IDO and use this enzyme to gain protection from T-cell attack. 1  The rationale for using IDO inhibitors as anti  cancer drugs would be to prevent tumor cells from evading the immun e system, therefore, this enzyme is an attractive target for treating cancer 2. This chapter will discuss the isolation and structure elucidation of inhibitors of IDO from the marine sponge Neopetrosia exigua. 2.2 Biology of Indoleamine-2,3-dioxygenase (IDO) The vital indole amino acid L-tryptophan is necessary for the biosynthesis of proteins and several important secondary metabolites. A small part of the ingested tryptophan is converted to serotonin and melatonin. The majority of tryptophan digested from food is metabolized by the kynurenine pathway (Figur e 2.2.1) which synthesizes nicotinamide, a key component in several co-enzymes such as NAD and NADP 3. The first and rate limiting step in the kynurenine pathway is the oxidative cleavage of the indole ring. This is catalyzed by either tryptophan-2,3-dioxygenase (TDO), which is mainly found in the liver, or  a.  Reproduced in part with permission from Brastianos H.C.; Vottero E.; Patrick B.O.; Soest van; Matairiaho T.; Mauk A.G.; Andersen R.J. Journal of the American Chemical Society R. 2006, 128, 16046-16047. Copyright 2006 American Chemical Society.  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 21 indoleamine-2,3-dioxygenase (lDO), which is found in the epididymis, thymu s, gut, lung, placenta and dendritic cells. 4 It has been shown that IDO plays an immunological function. Interferon-’y activation in cells such as macrophages induces the activity of lDO. Tryptophan is an essential amino acid for protein synthesis. Induction of IDO depletes local extracellular concentrations of tryptophan causing pathogens sensitive to tryptophan concentrations to arrest in G 1 of the cell cycle. 3  Pathogens  suppressed by the lack of tryptophan include: Chiamydia psittaci, C. trachomatis, C. pneumoniae, Staphylococcus aureus and the measles virus. 4 Mann and co-workers showed that there was an increased inclination for pregnant mice to lose their fetus when they were exposed to an inhibitor of IDO (1-methyl tryptop 5 han). Loss of IDO function resulted in increased T-ceII attack on the fetus, thus, causing pregnancy failure. These results suggest that the placenta expresses IDO to protect itself from the maternal T-cell attack. 6  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 22  2 lH  Tryptophan  2 NH Kynurenine  N-formyl kynurenine  Kynurenine 3-hydroxylase H 2 CO CO H 2 _3-hydroxyanthranilic acid N H 2 CO Quinolinic acid Quinolinic-acid phosphoribosy! transferase  [cooH Kynureninase 2 NH  2  OH 3-hydroxyanthranhlic acid  OH 3-Hydroxykynurenine  NH2 Nicotinamide  Figure 2.2.1. Kynurenine pathway (Adapted from Stone et al.). 7  IDO also plays a critical role in the progression of cancer. Tumor cells expressing IDO are protected from attack by the killer T-cells of the host. Several molecular mechanisms explain the how tumors evade the immune system. One mechanism suggests that secondary metabolites in the kynurenine pathway are cytotoxic toward T-cells and are able to induce 8 apoptosis. T-cells are also sensitive to the local tryptophan concentration. IDO in cancer cells is able to deplete the concentration of tryptophan in the tumor environment, thus IDO is able to arrest T-cells in the C 1 phase of the cell cycle. 2 Tumor cells have adaptive mechanisms which are able to offset the low intracellular tryptophan concentrations and are able to continue to proliferate. 1 The importance of IDO in cancer stems from the fact that a large number of human tumors express IDO. Patients with tumors that express IDO in ovarian, 9 endometrial’° and colorectal  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 23 11 have been found to have a poor prognosis for disease progression and cancer overall survival. The presence of IDO has also been confirmed in the lens of the eye. 12 Several key secondary metabolites that result from the kynurenine pathway are UV protection agents in the eye.  The major UV filter is 3-hydroxykynurenine  glucoside (3OHKG) which is specifically found in the lens of primates. Other UV filters from  the  kynurenine  pathway include  kynurenine  (Kyn) and  3-  hydroxykynurenine (3OHKyn). These small molecules protect the lens and the cornea by absorbing the harmful UV radiation between 300 and 400 nm. 13 Unfortunately, these compounds have also been found responsible for the undesirable yellowing of the lens and have been implicated in the formation of 14 catarac ts. The mechanism that yields the coloration of the lenses begins with the spontaneous deamination of 3OHKG, Kyn or 3OHKyn to afford an c3—unsaturated ketone.  The cysteine residues in cL—crystalin (the most  abundant protein in the lens) covalently bind with the kynurenine metabolites in a Michael fashion.  3OHKyn oxidation, after forming adducts with cc—crystalin,  results in cross-linked and insoluble proteins which may play a role in the development of age-related nuclear cataracts (Figure 2.2.2).15  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 24 0  Kynurenine  3 NH  2  a,3 unsaturated ketone  2 NH C00H  Cys-Kyn adduct  2 CH  Protein  Figure 2.2.2. Mechanism of formation of adduct between a—crystalin and kynurenine (Adapted from 15 Truscott).  2.3 Inhibitors of IDO as Treatments for Cancer Immune tolerance towards tumors is one of the hallmarks of cancer 2. Cancer cells that express IDO are able to induce immune escape by inhibiting T cell attack at the tumor site.  Abrogators of IDO would enhance anti-tumor  immunity by targeting the processes cancer cells use to evade T-cells. As a target for cancer, IDO is an attractive candidate. Knockout mice that have the gene for IDO removed are found to be viable and healthy, making it unlikely that IDO inhibitors will be highly toxic drugs. 2 One strategy that was used to develop new inhibitors of IDO has involved synthesizing analogues of tryptophan or indole compounds.  This is not  surprising since the first step in the kynurenine pathway is the oxidative cleavage of the indole ring of tryptophan. The most common lDO inhibitors synthesized are compounds that have a substituent on the indole ring of tryptophan. These act as competitive inhibitors of IDO. Some of the more potent inhibitors with a  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 25 substituted indole ring include: 1-methyl tryptophan (2.1),16 7-fluoro tryptophan (2.2),17 5,7-difluoro tryptophan (2.3),17 and methyithiohy dantoin tryptophan (2.4).18  All these inhibitors have K values ranging between 11-40 tM.  00C  00C  N 3 +H -CCC  2.1  2.2 O7S NH  Figure 2.3.1. Analogs of tryptophan as competitive inhibitors of IDO.  Derivatives of f3-carboline were found to be non-competitive inhibitors of IDO. The more active analogs of f3-carboline include compounds 2.5 and 2.6, which have 1 K ’ s of 3.3 and 7.4 jiM, respec 19 tively.  Unfortunately, these  compounds have unfavorable side effects in the central nervous system making them unlikely to be used for cancer 2 treatment. °  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exqua 26 0 F.  / N  \ N H  /r\z— N  2.5  N H  2.6  Figure 2.3.2. f3-carbolines as inhibitors of IDO. Among the most potent IDO inhibitors published to date are annulins A (2.7), B (2.8), and C (2.9) which have 1 K ’ s of 124, 140, and 690 nM, respectively. The annulins were isolated from a MeOH extract of the Northeastern Pacific marine hydroid Ga,veia annulata. These marine-derived polyketides contain a quinone moiety which appears to be essential for the activit 21 y.  The natural  product brassinin (2.10) was identified as an IDO inhibitor and has a K of 97.7 i22 1 M. A structure-activity relationship study was undertaken to determine which areas of the molecule are required for the inhibition of IDO.  An unexpected  finding was that the indole ring was not necessary to cause inhibition of lDO. It can be replaced with a wide range of aromatic substrates and still be able to prevent the activity of IDO. This may be a positive finding as indole compounds may cause neurological side effects 22. Further synthetic experiments established that the dithiocarbamate moiety in brassinin was crucial for the biological activity. Finally, replacing the S-methyl group with an aromatic moiety such as naphthalene greatly increased the potency of 22 brassinin.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 27 °OMe°  OH  0  0  OH  oOxLO 0  /\  2.7  H  2.8  2.9  2.10  Figure 2.3.3. Isolated natural product IDO inhibitors. The most widely used inhibitor of IDO is 1-methyl tryptophan (2.1). In vitro data show that 1-methyl tryptophan has a K of 34 jiM.  When 1-methyl  tryptophan was used in vivo against the MMTV-Neu transgenic mouse model of breast cancer, very little inhibition of tumor growth was observed. Similarly, the use of paclitaxel did very little to slow down tumor growth in this particular mouse 18. Signficant tumor regression was observed when combining 1-methyl model tryptophan with paclitaxel. This was also observed with other chemotherapeutic agents such as doxorubin, cisplatin and cyclophosphamide.  Increased  cytotoxicity towards cancer cells was not observed with other anti-cancer drugs such as 5-fluorouracil and methotrexate.  These results indicate that IDO  inhibitors can be used as adjuvants to enhance the efficacy of only certain chemotherapeutic drugs. 18 It is evident that combining IDO immunotherapy with chemotherapy is a potentially exciting new approach to cancer treatment. Most of the studies done on 1-methyl tryptophan have used the racemic (R, S) mixture. One very recent study has compared the biological activity of the  two enantiomers in vitro and in vivo to determine which of the two isomers would  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 28 be more effective in tumor 23 regression. The S-isomer of 1-methyl tryptophan  was found to be more effective in inhibiting IDO in vitro using HeLa cells and the purified enzyme. The R-isomer was found to be significantly more effective than the S-isomer when combining IDO immunotherapy with chemotherapy in mouse models of melanoma and breast cancer. Because of the greater efficacy of the R-isomer in vivo, it is more likely that R-1-methyl tryptophan would be more appropriate for human clinical trials. 23 2.4 Pyrroloquinones from Marine Sources Marine derived alkaloids from the pyrroloiminoquinone family are characterized by their biological activity. The first example of this family from a marine source was discorhabdin C (2.11), which was isolated from a marine sponge of the genus Latrunculia collected in New Zealand. Discorhabdin C was found to be a potent cytotoxin toward P-388 murine leukemia cells with an IC 50 of 40 nglmL. 24 Furthermore, discorhabdin C was found to be an antibacterial agent with activity against both Gram-positive and Gram-negative bacteri 25 a.  The  dischorhabdin family is characterized by an iminoquinone with a spiro cyclohexanone. Later, the structures of discorhabdin A (2.12), B (2.13), and D (2.14) were elucidated and these compounds were found to be potent cytotoxins against P-388 murine leukemia cells as well. These alkaloids had an additional sulfur containing ring. 25 More recently, the first discorhabdin dimer, discorhabdin W (2.15), was discovered and its biological activity was equivalent to that of discorhabdin B. 26  Epinardins A-D are very similar to the discorhabdins.  However, the epinardins contain an allylic alcohol rather than an unsaturated  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 29 ketone. Epinardin C (2.16) displayed the most toxicity against murine leukemia cells with an IC 50 of 0.32 p,gImL 27.  Br 2.11  2.12  2.13  2.14  BrBr 2.15  2.16  Figure 2.4.1. The discorhabdins and the epinardins. Batzellines A (2.17), B (2.18) and C (2.19) were discovered in 1980 by Sakemi et at. from the sponge BatzeI!a sp. collected in the Carrib 28 ean. Initially, no biological activity was found for these compounds, but later batzelline A was found to be cytotoxic against non-small cell lung carcinoma A-549 cells. 29 More recently, isobatzellines A-D (2.20-2.23) were discovered as being cytotoxic against P388 murine leukemia cells (1C 50  =  0.42-20 j.igfmL), and having anti  fungal activity against Candida albicans (MIC = 3.1-50 ° 3. Other structures ig/mL) related to the batzellines were the secobatzellines A (2.24) and B (2.25) isolated from Batzella sp..  Secobatzelline A was found to be a potent inhibitor of  calcineurin and was one of the few known compounds to have nM potency against this target. 31  This particular group of secondary metabolites have a  bicyclic core, while the batzellines and the isobatzellines are tricyclic. Damirones  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 30 A (2.26) and B (2.27) were isolated from the Palauan sponge Damiria sp. and have similar structures to the batzell 32 ines. SMe  SMe N—a,  HN-\  N—  2.18  2.19  Th  oz 2.17 SMe  ‘  SMe  ‘  N  oç H2N-N  H2N—N’ 2 H N —N 2.22  2.21  2.20  HN—__ OH O. jj OH N j’NH 2 H CI 2.24  2.23  HN—  OH  Q OH H N 2 ’O CI 2.25  O  2.26  2.27  Figure 2.4.2. Batzelline family of natural products. Makaluvamines A-F (2.28-2.33) were first isolated from Zyzza fuliginosa. These pyrroloiminoquinones have potent in vitro activity against the human colon tumor cell line HCT-116 and can inhibit topoisomerase II in vitro. 33  The  makaluvamines were isolated along with the discorhabdins, indicating that a biosynthetic relationship may be presen 33 t. The pyrroloquinone veiutamine (2.34) along with makaluvamines A-D were isolated from Zyzza fuliginosa collected in 34 Fiji.  Veiutamine (2.34) has a unique substitution pattern compared with  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 31 makaluvamine D, however, it was shown to be seven times more potent than makaluvamine D against the human colon tumor cell line HCT-1 0  o 2 NH  2 NH  2 NH  N  2.31  2.32  0  2.33  OH Br  2 NH  2.34  Figure 2.4.3. Makaluvamines and veiutamine. Makaluvic acids A (2.35) and B (2.36) were first isolated from the sponge Zyzzya fuliginosa. 35 These compounds can be seen as the oxidation products of the batzellines, isobatzellines, and the makavulamines. Keyzers et al. isolated N-1-f-D-ribofuranosylmakaluvic acid C (2.37) from Strongylodesma aliwaliensis and it was found to have moderate activity against esophageal cancer cells (1C 50 =  61 ig/mL) 36.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 32 IMe HN—/  ‘OH MeN— )oH  NH  NMe  OH OH  2.35  2.36  2.37  Figure 2.4.4. Makaluvic Acids. Wakayin (2.38), isolated from the ascidian Clavelina sp., is an example of a bispyrroloiminoquinone.  It was reported to be cytotoxic against the human  colon tumor cell line HCT-1 16 (1C 50  =  0.5 ig/mL), an inhibitor of topoisomerase II,  and a antimicrobial agent against Bacillus subtillus (MIC  =  0.3 1 i37 gImL).  Tsitsikammamines A (2.39) and B (2.40) were isolated from a South African Latrunculid  38. sponge  These  compounds  are  also  examples  of  bispyrroloquinones, but contain a phenol ring rather than the indole ring that is present in wakayin.  Studies have shown that tsitsikammamines have  antimicrobial activity, cytotoxicity to tumor cells, and antifungal activity, however, these compounds do not inhibit topoisomerase 11.38  More recently, the  zyzzyanones A-D (2.412.44)3940 were isolated from the Australian sponge, Zyzzya fuliginosa.  These bispyrroloquinones were found to lack the imine  present in both the wakayins and the tsitsikammamines. All of the zyzzyanones were found to have moderate cytotoxicity against Ehrlich carcinoma cells 50 (1C 25 pgImL). This may indicate that the presence of the imine is vital for the cytotoxic  activity of the 3 ’ pyrrolo immin 3 5 oquinones.  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 33  <x5 OH 2.40  OH 2.39  2.38  Ji  OHC  OH 2.41  2.42  Me  OH  2.43  2.44  Figure 2.4.5. Bispyrroloquinones from marine sources. 2.5 Alkaloids isolated from Neopetrosia sp. Neopetrosia and Xestospongia are two very similar genera of sponges with Xestospongia skeletons being composed of large spicules while Neopetrosia has smaller spicule 41 s.  In 2002, it was decided that Xestospongia exigua and  Neopetrosia exigua were in fact the same species. Xestospongia/Neopetrosia exigua is a reddish brown sponge mainly found in the shallow tropical waters of the Indo-West Pacific. This species lives in colonies of up to I m 3 in 42 size. 4 ’ 3 The first alkaloids isolated from Xestospongia/Neopetrosia exigua were xestospongins A-D (2.45-2.48).  These quinolizidine alkaloids were isolated  from a sponge collected in Australia. Their structures were determined using NMR and X-ray crystallography and they were found to be in vivo vasodi 44 lators. Other similar quinolizidine alkaloids include the araguspongines A, C, K, and L (49-52) which were isolated from N. exigua collected in the Red sea.  No  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Søonae Neoyetrosia exigua 34 biological activity was reported for araguspongine K (2.51) and L (2.52), however, araguspongine C (2.50) had anti-parasitic activity against Plasmodium falciparum,  as  well  as  antituberculosis  activity  against  Mycobacterium  45 Araguspongine M (2.53) was isolated from N. exigua collected in tuberculosis. Palau. This alkaloid showed cytotoxic activity against the human leukemia cell line HL-60 with an IC 50 value of 5.5 M, but did not show any anti-bacterial 42 Xestosin A (2.54) was isolated from a N. exigua sample collected in activity. Papua New Guinea. No biological activity was reported for this compound. 46 Bioassay guided fractionation of a sample of a MeOH extract of N. exigua from  Papua  New  Guinea  yielded  neoamphimedine  (2.55)  and  5-  methoxyneoamphimedine (2.56). These bisannulated acridines were found to be cytotoxic against murine cancer cells. 47 Other compounds isolated from N. exigua include the motuporamines. These heterocyclic alkaloids were isolated from an extract of N. exigua collected in Papua New Guinea. Biological studies revealed that these alkaloids are anti-angiogenic compounds with the most potent angiogenic inhibitor being motuporamine C (2.57).48  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 35  /  &°/  :2  H1  H°t  H°t  2.45  2.46  o  H/  H°t  2.47  2.48  HO  2.49  2.50  2.51 2.52 H’H°  2.54  2.53  3 OCH  2.55  2.56  N’_  NH  2.57  Figure 2.5.1. Alkaloids isolated from XestospongialNeopetrosia exigua.  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 36 2.6 Isolation of exiguamines A and B Neopetrosia exigua (Figure 2.6.2) was collected by hand using SCUBA from Mime Bay in Papua New Guinea.  A MeOH extract of the sponge was  suspended in 2 H 0 , and then sequentially partitioned with EtOAc and with n butanol.  The active butanol extract was subjected to size exclusion  chromatography,  flash  reversed-phase  column  chromatography,  gradient  reversed-phase HPLC and isocratic reversed phase HPLC to yield exiguamine A (2.58) and exiguamine B (2.59) (Figure 2.6.1). For full experimental details, see Section 2.13.  r 2 H  2.58  2.59  Figure 2.6.1. Secondary metabolites isolated from Neopetrosia exigua.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exgua 37  Figure 2.6.2 Neopetrosia exigua collected in Papua New Guinea.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase ((DO) from the Marine Sponge Neoyetrosia exigua 38  2.7 Structure Elucidation of exiguamine A 0 25 /‘4  N 2 H  /  29  o ‘N ° 2  ‘-‘  26 2cçioH  H 1  19  0  17’  /13  15 \ —N—161G  27  28  Figure 2.7.1. Numbering Scheme for exiguamine A. Exiguamine A gave a [M] ion at m/z 492.1883 in the HRESIMS indicating a molecular formula of 5 N C 2 H 6 0 5 (calc’d 492.1883). 6  The LRESIMS  measurement in MeOH yielded a molecular ion peak at m/z 492.2, while the LRESIMS measurement in MeOD afforded a molecular ion peak at m/z 496.2, demonstrating that four exchangeable protons are present.  The 1 H NMR  spectrum (Figure 2.7.3) of exiguamine A acquired in DMSO-d 6 at 600 MHz displayed an indole proton protons  (OH  (oH  13.10), a phenolic proton  (OH  10.42), two amine  7.82), two protons connected to sp 2 hybridized carbons  (OH  7.52 and  7.30), and a series of methine and methyl protons connected to sp 3 hybridized carbons attached to either a nitrogen or an sp 2 hybridized carbon  (OH  2.44-4.17).  The “ C NMR spectrum (Figure 2.7.4) indicated the presence of 25 carbons, 3 confirming that no symmetry was present.  The DEPT and the HMQC data  (Figures 2.7.5 and 2.7.6) indicated four carbonyls (Oc 179.7, 173.0, 168.4, 154.5), 11 quaternary carbons (Oc 146.5, 142.8, 142.7, 138.8, 131.6, 130.5, 122.8, 121.3, 120.7, 114.7, 85.4), two methines (Oc 126.5, 108.7), four methylenes (Oc 67.4, 38.3, 28.5, 23.3), and four methyls (Oc 54.3, 53.2, 26.0, 25.2). The 1 H, 15 N  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 39 LR-HMQC spectrum (Figure 2.7.9), which was referenced to an external  standard of 2 NO revealed five nitrogens 3 CH ,  (oN  -349, -310, -275, -248 and -218).  After using HMQC to assign proton resonances to their respective carbon atoms (Table 2.7.1), it was possible to deduce three substructures (I, II, UI, Figure 2.7.2) from the HMBC (Figure 2.7.7), COSY (Figure 2.7.8), and H 1, 15 N LR-HMQC spectra. o  I  II  Figure 2.7.2. Three substructures of exiguamine A.  /  III  Chapter  2:  Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the  Marine Sponge Neopetrosia ex,gua  40  .  -___  -=. —  C  Q -  0  0 0 Z /0Z  C’4  z  Figure 2.7.3. 1 H NMR spectrum of exiguamine A (2.58) acquired at 600 MHz in . 6 DMSO-d  E Q..  z N  C)  C,’  00  ‘TI  D CD  C  C.  CD  -  3 o  C  a  CD  Co  .g  cI,  ar;  N H  2 N\ H  /  0  I  I I  )i.pi  OH  srar  9’N%Q  0  150  p IW  100  UHI$IN 50  _____ ____ _____ __ _____ __ _____  I  _____ _____ __  0) CO CD  CD  9.  C.)  CD  C)  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 42  -e In  I 0  Figure 2.7.5. DEPT spectrum of exiguamine A (2.58) acquired at 150 MHz in 6 DMSO-d  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 43  +  —50  —100  + + ppm ppm  8.0  7.0  6.0  5.0  4.0  3.0  2.0  1.0  Figure 2.7.6. HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in . 6 DMSO-d  Chapter 2: Isolation of Inhibitors of lndoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 44 o /  4  0I  4  100  :  I 8  150  + ppm ppm  10.0  5.0  Figure 2.7.7. HMBC spectrum of exiguamine A (2.58) acquired at 600 MHz in . 6 DMSO-d  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 45 -N  /  e  1  +  -10.0  +  + ppm ppm  10.0  5.0  Figure 2.7.8. COSY spectrum of exiguamine A (2.58) acquired at 600 MHz in . 6 DMSO-d  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 46 Q  0  liii,,  0  0  I  I  0  a)  0  0 U)  0  0 U)  0  0 U)  Figure 2.7.9. 1 H, 15 N LR-HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d . 6  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Syonqe Neopetrosia exigua 47  Table 2.7.1. 1 D and 2D NMR data for Exiguamine A. a  Position  öf  öN  H 8  (J in Hz)  1, 13 H C-HMBC  1, 15 H N-  COSY  HMQC 1  -218  13.10, brs  C-2, C-3, C-4 C-  H-2  5, C-8, C-9 2  126.5  3  120.7  4C  121.3  d 5  179.7  6  126.5  7  138.8  8”  173.4  9C  131.6  10  114.7  II  146.5  12  142.7  13  108.7  7.30, d,  C-3, C-4, C-5, C-  (2.2)  8, C-9, C-24  7.52, brs  C-ID, C-Il, C-12,  N-I  H-I  N-15  C-14, C-18 14e  142.8  15 16  -310 67.4  16b 17  28.5  3.84, q,  C-17,C-18, C-27,  H-16a, H  (10.8)  C-28  17b  4.17, t,  C-14, C-17, C-18,  H-16b, H  (8.6)  C-28  17a  3.22, bdd  C-b, C-14, C-16,  (16.9, 7.5)  C-18  N-15  H-16a, H17b  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Svonae Neopetrosia exigua 48 Position 8 H, 13 1 C-HMBC C oN OH (J in Hz) H, 15 1 N- COSY  HMQC 17b  3.73, m  C-10, C-13, C-14, C-16, C-18  18  122.8  19  85.4  20 21  -275 154.5  22  -248  23  168.6  24a  23.3  24b 25  38.3  26  -349  2.92, m  C-2, C-3, C-25  3.02, m  C-2, C-3, C-25  2.99, m  C-3, C-24  N-26  N-26  7.82, br  54.3  3.43, s  C-14, C-16, C-28  N-15  53.2  3.51, s  C-14, C-16,C-27  N-15  29  26.0  2.44, s  C-19, C-21  N-20  30  25.2  3.07,s  C-21,C-23  N-22  10.42,brs  C-11,C-12,C-13  12-OH a  H and 13 1 C chemical shifts [ppm] are referenced to 6 DMSO-d 39.51 ppm respectively)  H 6 (  2.50 and O  bThe 15 N spectrum was not calibrated with an external standard. The 6 value has an accuracy of about 1 ppm in reference to 2 NO (0 ppm). 3 CH C  C4 and C9 are interchangeable signals d C5 and C8 are interchangeable signals a  C12 and C14 are interchangeable signals H24a and H24b are interchangeable proton chemical shifts g C27 and C28 are interchangeable signals ‘  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 49  11o  42 -LOHl ,b O.  I  I  1147  J%%  /L% 3., bdd (16.9, 7.5 Hz)  ‘12.7QH  ç  122)* 7.52,  S  28.5  3.84, q (10.78 Hz) 4.17, t (8.57)  7142.8  67.4 3.51,  108.7  -310  S  53.2  a  b  Figure 2.7.10. (a) 1 H and (b) 13 C and 15 N chemical shifts of substructure I of exiguamine A (2.58).  1QH  1 0 H 17\  (Th H M BC  27  Figure 2.7.11. Key HMBC and COSY correlations of substructure I exiguamine A (2.58).  Two singlet proton resonances at  H 6  3.43 (H-27: HMQC to  54.3) and  H 8  3.51 (H-28: HMQC to c 53.2) displayed 1 H, 15 N, LR-HMQC correlations to the nitrogen resonance at  oN  -310 (N-15) (Figure 2.7.12). HMBC correlations were  observed between the proton resonance at  OH  3.42 (H-27) and the carbon  resonance at öc 53.2 (C-28). An additional HMBC correlation between the proton  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 50 resonance at  oH  3.51 (H-28) and the carbon resonance at Oc 53.2 (C-28) implied  that Me-27 and Me-28 were geminal, and their chemical shifts indicated that they were attached to nitrogen (N-15). Both the proton resonances at and  H 0  OH  3.42 (H-27)  3.51 (H-28) showed HMBC cross-peaks to the sp 2 hybridized carbon  resonance at & 142.8 (C-14), which confirmed the linkage between the C-14 (Oc 142.8) and N-15  (ON  -310) (Figures 2.7.10 and 2.7.11).  correlations between the proton resonances at  H 8  Three bond HMBC  3.42 (H-27) and OH 3.51 (H-28)  and the methylene carbon resonance at 0 c 67.4 (C-16) established the bond between C-16 (Oc 67.4) and N-15 at  OH  (ON  -310). Both methylene proton resonances  3.84 (H-16a: HMQC to Oc 67.4) and  H 0  4.17 (H-16b: HMQC to 0 c 67.4)  showed COSY correlations to the proton resonances at 8 H 3.22 (H-17a: HMQC to oc 28.5) and OH 3.73 (H-17b: HMQC to oc 28.5), which assigned C-16 (Oc 67.4) next to C-17 (Oc 28.5) (Figure 2.7.13). All four proton resonances at  H 8  3.84 (H  16a), OH 4.17 (H-16b), OH 3.22 (H-17a) and OH 3.73 (H-17b) showed HMBC correlations to the aromatic carbon resonance at Oc 122.8 (C-18).  This  determined the connectivity between C-17 (Oc 28.5) and C-18 (122.8). Three proton resonances at OH 4.17 (H-16b), OH 3.22 (H-17a) and oH 3.73 (H-17b) showed HMBC cross-peaks to the carbon resonance at Oc 142.8 (C-14). This assigned C-14 (Oc 142.8) next to C-18 (oc 122.8) and established the presence of an N,N-dimethyldihydropyrrole moiety (Figures 2.7.10 and 2.7.11). Both methylene proton resonances at  H 0  3.22 (H-17a) and OH 3.73 (H-17b)  showed HMBC correlations with the carbon resonating at Oc 114.7 (C-b), which  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sijonqe Neopetrosia exigua 51  established the bond between C-b methine proton resonance at  H  (öc 114.7) and C-18 (öc 122.8). An aromatic  7.52 (H-13: HMQC to öc 108.7) displayed HMBC  correlations with the carbon resonance at ö 142.8 (C-14) and H 1, 15 N LR-HMQC correlations with the nitrogen resonance at  N  -310 (N-15). This confirmed the  linkage between C-13 (c 108.7) and C-14 (öc 142.8). The chemical shift of the carbon resonating at & 142.7 (C-12) was consistent for an oxygenated aromatic carbon. This was confirmed by HMBC correlations between the exchangeable phenolic proton resonance at oH 10.42 (12-OH) and the carbon resonance at Oc 142.7 (C-12).  The bond between C-12 (Oc 142.7) and C-13 (Oc 108.7) was  deduced from a three bond HMBC correlation between the proton resonance at OH  10.42 (12-OH) and the carbon resonance at & 108.7 (C-13). Both proton  resonances at oH 10.42 (12-OH) and oH 7.52 (H-13) showed three bond HMBC correlations to the oxygenated carbon resonance at Oc 146.5 (C-i 1), which allowed the determination of the linkage between C-Il (Oc 146.5) and C-12 (Oc 142.7).  A four bond HMBC correlation was present between the aromatic  methine proton resonance at  H 0  7.52 (H-13) and the quaternary aromatic carbon  resonance at Oc 114.7 (C-b) (Figure 2.7.14). This assigned C-b and revealed substructure I (Figures 2.7.10 and 2.7.11).  next to C-Il  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 52  H-28 H-I 3  -315.  L  -305.  --.300.  pm ppm  7,0  6.0  5.0  4.0  3.0  Figure 2.7.12. Expansion of the 1 N LR-HMQC spectrum of the key H, 15 correlations of substructure I of exiguamine A (2.58).  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 53  H-17b H-16a  —3.00  3.50  -4.00  0  -4.50 ppm  ppm  4.50  4.00  3.50  3.00  Figure 2.7.13. COSY expansion of the correlations for substructure I of exiguamine A (258).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopotrosia exigua 54 H-I 3 -  ..-110  c-I 0 -120  c-I 8  —130  —140  C-I 2/14 c-Il—150  ppm 7.600  ppm  7.550  7.500  Figure 2.7.14. HMBC correlations observed for H-13 of substructure I of exiguamine A (2.58). 2.99, m  7.82, br  2.92, m 3.02,m  Q  112 Fl  38.3  H N 2 ’  7.30, d(2.22 Hz)  126 -218  0  13.10,br  a  N  II  H  0 b  Figure 2.7.15. (a) 1 H and (b) 13 C and 15 N chemical shifts of substructure II of exiguamine A (2.58).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 55  0 26  cosy ‘HMBC  8  Figure 2.7.16. Key HMBC and COSY correlations of substructure II of exiguamine A (2.58). The methylene proton resonance at  H 6  2.99 (H-25: HMQC to ö 38.3)  showed COSY correlations to a broad exchangeable singlet at oH 7.82 (H-26) and LR-HMQC correlations to a nitrogen resonance at  N 8  -349 (N-26).  This  confirmed C-25 ( NH moiety (N-26). The methylene c 38.3) was adjacent to an 2 0 proton resonance at OH 2.99 (H-25) displayed COSY correlations to both proton resonances at  H 8  3.02 (H-24a: HMQC to öc 23.3) and  H 0  2.92 (H-24b: HMQC to  Oc 23.3), which allowed the determination of the C-24 (oc 23.3) and C-25 (Oc 38.3) linkage. All of the above is consistent for an ethylamine moiety. All three proton resonances at  oH  3.02 (H-24a), 8 H 2.92 (H-24b) and oH  2.99 (H-25) showed HMBC correlations to the sp 2 hybridized carbon resonance at Oc 120.7 (C-3), thereby linking C-3 (Oc 120.7) to C-24 (Oc 23.3).  The  connection between C-2 (Oc 126.5) and C-3 ( c 120.7) was deduced from three 8 bond HMBC correlations between both methylene proton resonances at oH 3.02 (H-24a) and  H 8  2.92 (H-24b), and the methine carbon resonance at Oc 126.5 (C  2). COSY correlations were observed between the methine proton resonance at  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 56 H 6  7.30 (H-2: HMQC to öc 126.5) and the exchangeable proton resonance at  13.10 (H-I) (Figure 2.7.18). observed between  H 6  H 3  Additional H N, LR-HMQC correlations were 1, 15  7.30 (H-2) and  N 6  -218 (N-I), which confirmed that C-2 (öc  126.5) was linked to N-I (oN -218) (Figure 2.7.17). The proton resonances at  H 8  13.10 (H-I) and oH 7.30 (H-2) showed HMBC correlations to the quaternary sp 2 hybridized carbon  resonance at 8 c  131.6 (C-9), thereby,  allowing the  determination of the N-I (ON -218) and C-9 bond ( c 131.6). 8  Both proton  resonances at OH 13.10 (H-I) and  H 8  7.30 (H-2) displayed additional HMBC  cross-peaks to the carbon resonating at Oc 121.3 (C-4). This established that C3 (120.7) was connected to C-4 (Oc 121.3), which in turn was bonded to C-9 (Oc 131.6). All of the above is consistent for a tn-substituted pyrrole ring. Weak four bond HMBC correlations were observed between the proton resonance at OH 7.30 (H-2) and the two carbonyl carbon resonances at O 179.7 (C-5) and Oc 173.4 (C-8). Additional HMBC correlations were observed between the proton resonance at OH 13.10 (H-i) and the carbonyl resonances at O 179.7 (C-5) and Oc 173.4 (C-8) (Figure 2.7.19). This confirmed that both C-4 and C-9 were linked to carbonyls, thus completing substructure II (Figures 2.7.15 and 2.7.16).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 57  N-26  400  0 ppm  7.0  6.0  50  4.0  3.0  Figure 2.7.17. Expansion of the 1 H, 15 N LR-HMQC spectrum of the key correlations of substructure II of exiguamine A (2.58)..  -  -250  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 58  7,0  8.0  -9.0  10.0  -itO  :12,0  —  iao  ,ppm 11!1’.  13.0  ppm  12.0  11.0  10.0  9.0  8.0  7.0  Figure 2.7.18. Key COSY correlation for substructure II of exiguamine A (2.58).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 59 H-i  C-3 C-4  Figure 27.19. HMBC correlations observed for H-I of substructure II of exiguamine A (2.58). 3.07, s  0  /  2.44,s  25.2  0  ____ N-248 1 ; 3 26.0 ‘c: 154.5  168.6  N 10  a  0  8  b  Figure 2.7.20. (a) 1 H and (b) 13 C and 15 N chemical shifts of substructure Ill of exiguamine A (2.58).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Stjonge Neopetrosia exigua 60  HMBC rr Figure 2.7.21. HMBC correlations of substructure Ill of exiguamine A (2.58). The singlet methyl proton resonance at oH 3.07 (H-30: HMQC to öc 25.2) showed H 1, 15 N LR-HMQC correlations to oN -248 (N-22), which established an N-methyl moiety (Figure 2.7.22). methyl proton resonance at  H 0  Three bond HMBC couplings between the 3.07 (H-30) and the two carbonyl carbon  resonances at O 154.5 (C-21) and oc 168.6 (C-23) implies that N-22  (ON  -248), is  flanked by two carbonyls (Figures 2.7.21 and 2.7.23). This is further confirmed by observation of the chemical shift of N-22 (ON -248), which is consistent for an amide moiety. 49  Another N-methyl moiety was confirmed by a LR-HMQC  correlation between  H 0  2.44 (H-29: HMQC to 0 c 26.0) and  N 8  -275 (N-20). The  HMBC spectrum revealed cross peaks between the proton resonance at OH 2.44 (H-29) and the carbonyl resonance at Oc 154.5 (C-21), which yielded a second amide group. A three bond HMBC correlation between the proton resonance at H 8  2.44 (H-29) and the carbon resonance at Oc 85.4 (C-19) established the bond  between C-19 (Oc 85.4) and N-20  (ON  -275) (Figures 2.7.21 and 2.7.23). The  chemical shift of C-I 9 (Oc 85.4) is typical for an sp 3 hybridized carbon connected to two heteroatoms. Since all the nitrogens of exiguamine A were accounted for,  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 61 the second heteroatom on C-19 was determined to be oxygen. All of the above data are consistent with substructure III (Figures 21.20 and 2.7.21).  N-22  26O  --250  N-20  ppm  2.50  Figure 2.7.22. Expansion of the 15 N LR-HMQC spectrum of the key correlations of substructure III of exiguamine A (2.58).  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 62 H-30  H-29  J L_  -  C-19  —  —I’...  -100  —  -  C-21  -150 -iIr  —  C-23 ppm ppm  3.10  3.00  2.90  2.80  2.70  2.60  2.50  2.40  Figure 2.7.23. Expansion of the HMBC spectrum of the key correlations of substructure Ill of exiguamine A (2.58).  The NMR data of exiguamine A accounted for the fragments I-Ill. Unfortunately, due to the lack of proton resonances and the large number of quaternary carbons and hetero-atoms, the NMR data were inadequate for connecting fragments I-Ill.  Therefore, x-ray crystallography was needed to  establish the complete structure of exiguamine A. Exiguamine A was suspended in I N HCI and the solution was evaporated in vacuo. This process was repeated four times to generate the HCI salt. Deep red crystals of exiguamine A were obtained by the slow evaporation of a methanol solution of the HCI salt. The crystals were appropriate for single crystal x-ray diffraction analysis. Dr. Brian Patrick from the department of chemistry at the University of British Columbia  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 63 performed the x-ray diffraction analysis and the structure was unequivocally established as that proposed for exiguamine A (Figure 2.7.24). For the full x-ray diffraction analysis parameters, see appendix I.  Figure 2.7.24. ORTEP diagram of exiguamine A (2.58). The x-ray diffraction analysis was performed by Dr. Brian Patrick.  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 64  2.8 Structure Elucidation of exiguamine B 25  24  H N 2 26  Figure 2.8.1. Numbering scheme of exiguamine B (2.59). Exiguamine B (2.59) gave a [M] ion at m/z 508.1850 in the HRESIMS which afforded a molecular formula of 5 N C 2 H 7 0 5 (calc’d 508.1832). This differs 6 from the molecular formula of exiguamine A by the addition of one oxygen atom. The alkaloid, when subjected to LRESIMS in MeOH, was found to have a molecular ion peak at m/z 508.3.  When the LRESIMS measurement was  performed in MeOD, the molecular mass was determined to be 513.3, which is consistent with five exchangeable protons in the molecule.  The 1 H NMR  spectrum (Figure 2.8.3) of exiguamine B acquired in DMSO-d 6 at 600 MHz contained five exchangeable protons  (oH  13.12, 10.71, 7.79, 6.07), two aromatic  protons (OH 7.62 and 7.35), a deshielded oxymethine proton (OH 5.75), and a series of methines and methyl protons on carbons adjacent to either nitrogen, or an aromatic carbon (OH 2.44-4.45). The 13 C NMR spectra (Figure 2.8.4) run in 6 at 150 MHz contained 25 carbon resonances. DMSO-d  Observation of the  DEPT (Figure 2.8.5) and HMQC (Figure 2.8.6) data revealed four carbonyls (Oc 179.4, 173.4, 168.6, 154.5), 11 quaternary carbons ( c 148.5, 143.7, 142.8, 0 138.7, 131.4, 130.7, 125.3, 121.9, 121.0, 113.8, 85.5), three methines (Oc 126.9, 108.6, 69.1), three methylenes (Oc 73.5, 38.3, 23.3), and four methyls (Oc 57.3,  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Svone Neopetrosia exigua 65 55.4, 26.2, 25.3). After assignment of all the protons to their respective carbons (Table 2.8.1), three independent spin systems (I, II, III, Figure 2.8.2) were deduced from the HMBC and the COSY data (Figures 2.8.7 and 2.8.8). OH HOj  I  \N’ NN O  ii  Figure 2.8.2. Three substructures of exiguamine B (2.59).  III  z N  0) 0 0  C  1  CD  01  r%)  w  CD  D  B  C  x  CD  0 -t  B  C  —‘  g  -o CD  Cl)  2J  z z  Co  ppm  9’N  /  HO  10.0  oH  N 2 H  o  K 5.0  CD’  -  0  0•  J  I.  CD 3 ()  0  CDO  CD  0  -  DCI)  0 Ct  I. 1  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neoøetrosia exiqua 67  -o In  I  Figure 2.84. 13 C NMR spectrum of exiguamine B (2.59) run at 150 MHz in DMSO-d . 6  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 68  —  0  -o  C.  Figure 2.8.5. DEPT spectrum of exiguamine B (2.59) run at 150 MHz in DMSO d . 6  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 69 Q\ / H N 2 ”\ / N H  0 N 0 0H  o  H0’  ppm  8.0  7.0  6.0  5.0  4.0  3.0  2.0  1.0  Figure 2.8.& HMQC spectrum of exiguamine B (2.59) run at 600 MHz in DMSO  . 6 d  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 70  o  /  Hn H0’  /  [  H -50 I  p  -100 •  p.  .  4  -150  -200  ppm ppm  10.0  5.0  Figure 2.8.7. HMBC spectrum of exiguamine B (2.59) run at 600 MHz in DMSO . 6 d  Chapter  2:  Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (lDO) from the  71  Marine Sponge Neopetrosia exigua  0  /  —5.0  a  •1  if  $1 0  K  —10.0 +00  ppm ppm  10.0  5.0  Figure 2.8.8. COSY spectrum of exiguamine B (2.59) run at 600 MHz in DMSO d . 6  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 72 Position I 2 3 5C  6 7 8’ 10 11 12 13 14 15 I 6a  Table 2.8.1. 1D and 2D NMR data of Exiguamine C-HMBC H, 13 1 H (J in Hz) 8 13.10, brs C-2, C-3, C-4, C-9 126.9 7.35 ,d, (2.2) C-3, C-4, C-9 121.0 121.9 173.0 130.7 138.7 179.4 131.4 113.8 148.5 143.7 108.6 7.62, s C-b, C-Il, C-12, C-14, C-18 142.8  B 8 COSY H-2 H-2  73.5  C-14, C-18, C-27, C-28  H-16b, H-17  C-14, C-18, C-17, C-28  H-16a, H-17  I 6b 17  69.1  4.45, dd, (12.4 Hz, 5.8) 3.95, dd, (12.4, 2.5) 5.75, m  H-16a, H-16b, 17-OH  18 125.3 19 85.5 20 21 154.5 22 23 168.4 24 23.3 2.94, m C-2, C-3, C-4, C-25 H-25 25 38.3 3.04, m C-3, C-24 H-24, H-26 26 7.79, brs C-24, C-25 H-25 27” 55.4 3.58, s C-14, C-16, C-28 28” 57.3 3.53, s C-14, C-16, C-27 29 26.2 2.44,s C-19,C-21 30 25.3 3.10,s C-21,C-23 12-OH 10.71, brs C-Il, C-12, C-13 17-OH 6.07, d, (5.0) C-16, C-17, C-18 H-17 a H and 1 1 C chemical shifts [ppm] are referenced to the DMSO-d 6 (2.50 and 39.51 ppm respectively) b C4 and C9 are interchangeable signals C C5 and C8 are interchangeable signals d C27 and C28 are interchangeable signals  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 73 3.1O,s  O  25.3  0  /  /  26.2  2.44,s  168.4  N 85.5  r0  J%r  a  b  Figure 2.8.9. (a) 1 H NMR and (b) 13 C NMR assignments for substructure I of exiguamine B (2.59).  29  (ThHMBC 0  Figure 2.8.10. Key HMBC correlations observed for substructure I of exiguamine B (2.59).  The 1 H chemical shift of the methyl protons H-30  (oH  3.10, s) of  exiguamine B (2.59) is very similar to that of H-30 (OH 3.07, s) for exiguamine A (2.58), which confirmed a nitrogen bearing methyl. HMBC correlations between the methyl proton resonance at OH 3.10 (H-30: HMQC to Oc 25.3) and both the carbon resonances at Oc 154.5 (C-21) and Oc 168.4 (C-23) established an Nmethyl moiety adjacent to two carbonyls (Figures 2.9.10 and 2.9.11). The 1 H chemical shift of the proton resonance H-29 (OH 2.44, s) of exiguamine B (2.59) is identical to the H-29 (OH 2.44, s) proton resonance of exiguamine A (2.58). This reveals an additional N-methyl moiety. Cross-peaks in the HMBC were present  m  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine S,,onqe Neopetrosia exjgua  74  between the methyl proton resonance at oH 2.44 (H-29: HMQC to 8 26.2) and the carbon resonance at öc 154.5 (C-21), thus yielding an additional amide moiety. A bond between C-19 (Oc 85.5) and N-20 was deduced from an HMBC correlation between the methyl proton resonance at  2.44 (H-29) and the  H 8  carbon resonance at 0 c 85.5 (C-19) (Figures 2.9.10 and 2.9.11).  The 13 C  chemical shift of carbon C-19 (Oc 85.5) of exiguamine B is very similar to that of C-19 (öc 85.4) for exiguamine A, which allowed the determination of an aminal carbon. This confirmed substructure  ,  analogous to that found in exiguamine A  (Figures 2.8.9 and 2.8.10). H-25  I  H-24  ‘.jL_____  4  )  C-19-  -  :1o0  150  C-21—  -  C-23-— I  ppm  I  3.00  I  2.50  Figure 2.8.11. HMBC correlations observed for substructure I of exiguamine B (2.59).  Chapter 2: Isolation of Inhibitors of lndoieamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 75  7.79br  N 2 H 3.04, m  7.35, d, 2.21 Hz  13.10,br  N 2 H 2.94, m  23.3  “  126.9  N H b  [-I  a  Figure 2.8.12. (a) 1 H NMR and (b) 13 C NMR assignments of substructure II of exiguamine B (2.59).  cosy ‘HMBC  Figure 2.8.13. Key HMBC and COSY correlations observed for substructure II of exiguamine B (2.59). The 1 H chemical shift of the exchangeable proton resonance H-26  (oH  7.79, brs) of exiguamine B (2.59) is very similar to the chemical shift found for H26  (OH  7.82, brs) in exiguamine A (2.58).  Therefore, one can establish the  presence of a primary amine. The proton resonance at COSY correlations to the multiplet resonating at  oH  H 3  7.79 (H-26) showed  3.04 (H-25: HMQC to Oc  38.3), which, in turn, showed COSY correlations to the methylene proton resonance at  OH  2.94 (H-24: HMQC to & 23.3). This was consistent with an  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 76 ethylamine moiety, which was confirmed by key correlations in the HMBC data (Figure 2.8.13). Both methylene proton resonances at  oH  2.94 (H-24) and oH 3.04 (H-25)  showed HMBC correlations to the quaternary sp 2 hybridized carbon resonating at Oc 121.0 (C-3), thus assigning C-24 (oc 23.3) next to C-3 (Oc 121.0). The linkage between C-2 ( c 126.9) and C-3 (Oc 121.0) was deduced from three bond HMBC 8 correlations between the multiplet resonating at  H 0  2.94 (H-24) and the sp 2  hybridized carbon resonance at Oc 126.9 (C-2). Both proton resonances at OH 7.35 (H-2: HMQC to 0 c 126.9) and  oH  2.94 (H-24) displayed HMBC correlations  to the carbon resonance at & 121.9 (C-4), thereby placing C-3 (Oc 121.0) next to C-4 (öc 121.9). The exchangeable proton H-I (OH 13.10, brs) of exiguamine B (2.59) had an identical chemical shift to H-I  (OH  13.10, brs) of exiguamine A  (2.58), which is consistent for a proton on a pyrrole nitrogen. A proton resonance at OH 7.35 (H-2) showed a COSY correlation to the proton resonating at  H 0  13.10  (H-I), which confirms that C-2 (Oc 126.9) is adjacent to an NH moiety (Figure 2.8.14). The proton resonances at  H 8  7.35 (H-2) and  oH  13.10 (H-I) both showed  HMBC correlations to the quaternary carbon resonating at Oc 131.6 (C-9), thus allowing the determination of the N-I and C-9 (Oc 131.6) bond.  A linkage  between C-4 (Oc 121.9) and C-9 (Oc 131.6) was confirmed from a three bond HMBC correlation between the proton resonating at  H 8  13.10 (H-i), and the  carbon resonance at Oc 121.3 (C-4) (Figures 2.8.13 and 2.8.15). All of the above  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (lDO) from the Marine Sponge Neopetrosia exigua 77 data is consistent with a tn-substituted pyrrole moiety, and substructure II (Figures 2.8.12 and 2.8.13).  7.0  8.0  9.0  10.0  11.0  12.0  13.0  rPPm ppm  13.0  12.0  11.0  10.0  9.0  8.0  7.0  Figure 2.8.14. Key COSY correlation of substructure II of exiguamine B (2.59).  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 78  j —110  i.-120  -:  C-2 —130  C  H 140  r ppm  13.250  13.200  13.150  13.100  13.050  13.000  12.950  Figure 2.8.15. HMBC correlations for H-I of substructure II of exiguamine B (2.59).  OH 5.75m* S  6.07, d, 4.97 Hz  HO  4.45, dd. 12.4 and 5.81 Hz 3.95,dd, 12.4 and2.49 Hz  j485QH 143.7  125.3  11108.6  HO/N I  69.1\  \— I\i—  I  3.53, S  a  75.5  I 57.3  b  Figure 2.8.16. (a) 1 H NMR and (b) 13 C NMR of substructure III of exiguamine B (2.59).  Chapter 2: isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 79  OH COSY (ThHMBC  Figure 2.8.17. Key HMBC and COSY correlations of substructure II of exiguamine B (2.59). HMBC cross-peaks were observed between the proton resonance at  H 6  3.58 (H-27: HMQC to öc 55.4) and the carbon resonance at öc 57.3 (C-28). A methyl proton resonance at  oH  3.51 (H-28: HMQC to Oc 57.3) showed HMBC  correlations to 8 55.4 (C-27), which established that Me-27 and Me-28 were attached to the same nitrogen (N-15). Both methyl proton resonances at OH 3.58 (H-27) and 3.51 (H-28) showed HMBC correlations to the methylene carbon resonating at 8 c 73.5 (C-16), thereby placing C-16 ( c 73.5) next to the N 8 dimethyl moiety.  All four proton resonances at  73.5), 3.95 (H-16b: HMQC to 0 c 73.5),  H 8  H 8  445 (H-16a: HMQC to 0 c  3.58 (H-27) and 3.51 (H-28) showed  HMBC correlations to the quaternary sp 2 hybridized carbon resonating at Oc 142.8 (C-14). This allowed the determination of the C-14 (Oc 142.8) and N-15 bond. Observation of COSY correlations between the proton resonances at OH 4.45 (H-16a) and 3.95 (H-16b) and the methine proton resonating at  H 8  5.75 (H-  17: HMQC to O 69.1) established the connectivity between C-16 (O 73.5) and C-  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 80 17 (8 69.1) (Figure 2.8.18).  Methine C-17 (E 69.1) was linked to an alcohol  moiety (17-OH) from observation of a COSY correlation between the proton resonating at  H 6  5.75 (H-17) and the exchangeable proton resonance at  (17-OH). The proton resonances at  oH  4.45 (H-16a), 3.95 (H-16b) and  H 6  6.07  OH  6.07  (17-OH) all showed HMBC correlations to the carbon resonance at O 125.3 (C18), which allowed the assignment of the C-17 (O 69.1) and C-18 bond (O 125.3) (Figure 2.8.17). The chemical shift of the carbon at 8 143.7 (C-12) is indicative of an oxygenated aromatic carbon.  This was confirmed from a two bond HMBC  correlation between the phenolic proton resonating at aromatic carbon resonance at H 8  80  H 3  4.45 (12-OH) and the  143.7 (C-12). The phenolic proton resonance at  4.45 (12-OH) showed three bond HMBC correlations to the carbon  resonances at 8 148.5 (C-lI) and 8 108.6 (C-13), thereby placing C-12 (8 143.7) between C-Il (8 148.5) and C-13 (8 108.6). An aromatic methine proton resonating at  H 8  7.62 (H-13: HMQC to 8 108.6) had HMBC cross-peaks to the  aromatic carbon resonance at Oc 142.8 (C-14), thus linking C-13 (8 108.6) to C14 (Oc 142.8) (Figures 2.8.17 and 2.8.19). The aromatic carbon C-14 (Oc 142.8) was assigned next to C-18 (3 125.3) from observation of a three bond HMBC correlation between the proton resonance at  H 8  7.62 (H-13) and the quaternary  aromatic carbon resonance at 8 125.3 (C-18).  Finally, a four bond HMBC  correlation was present between the proton resonance at  H 8  7.62 (H-I 3) and the  aromatic carbon resonance at o 113.8 (C-b) (Figure 2.8.19). This established  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 81  that C-18 (6 125.3) was linked to C-b  (ö 113.8), which, in turn, was linked to C  11 (6 148.5). All of the above is consistent with substructure substructure Ill (Figures 2.8.16 and 2.8.17).  -3.50  —4.00  r 1-  4.50  -  5.00  -5.50  —6.00  ppm  6.00  5.50  5.00  4.50  4.00  Figure 2.8.18. COSY correlations of substructure III of exiguamine B (2.59).  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 82 H-I 3  1\  /\  -110  c-ID—  -zn.  .  L 1 20  —=  c-I 8—  -  -130  —  :  -140 :  CIl  —  c -150  -160  ppm  7.700  7.650  —rT 7.600  ppm I 7.550  I  Figure 2.8.19. HMBC correlations observed for H-13 of substructure Ill of exiguamine B (2.59).  Unfortunately, there was insufficient NMR data to assign a constitution to exiguamine B. Attempts to crystallize exiguamine B by soaking in IN HCI and crystallizing from methanol failed. However, comparison of the available NMR data between exiguamine A (2.58) and exiguamine B (2.59) showed that the only difference was in the placement of the hydroxyl group on C-17 (o 69.1), consistent with the difference in the molecular formulae of the two natural products. Therefore, the structure of exiguamine B (2.59) was established based upon the comparison of the NMR data of exiguamine A (2.58) with those of B.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 83 2.9 Proposed Biogenesis of Exiguamine A  A proposed biogenesis (Figure 2.9.1) of the skeleton of exiguamines involves tryptophan, DOPA, and a hydantoin moiety.  Methylation of the two  amides on the hydantoin (2.60) moiety occurs via S-adenosyl methionine to yield N,N-dimethylhydantoin (2.61).  DOPA (2.62) undergoes a decarboxylation  followed by an oxidation of the catechol ring to yield an ortho quinone moiety (2.63).  The primary amine on 2.63 attacks in a Michael fashion to yield a  bicyclic analog of DOPA (2.64).  This is then followed by methylation via S  adenosyl methionine to afford 2.65.  A decarboxylation occurs on tryptophan  (2.66), followed by a series of oxidations to yield tryptamine hydroquinone (2.67). The tryptamine analog (2.67) couples to the DOPA analog (2.65) in a Michael fashion followed by rearomatization and oxidation to yield 2.68. Base catalyzed attack of the N,N-dimethylhydantoin followed by reformation of the quinone yields the exiguamine precursor 2.69. Tautomerisation of 2.69 followed by a cyclization establishes the hexacyclic precursor to exiguamine A (2.71). Finally, oxidation of 2.71 affords exiguamine A (2.58) (Figure 2.9.1). The exiguamines are novel alkaloids of the pyrroloquinone family of natural products. This family of natural products is characterized by having a pyrrole ring adjacent to a quinone moiety. As is evident in the biogenesis, the exiguamines contains dopamine, hydantoin, and tryptamine fragments.  Even  though these are very common biosynthetic elements, their connectivity yields an unprecedented hexacyclic skeleton.  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (ID 0) from the Marine Sponge Neopetrosia exigua 84  2.64 2 NH  OH  2.67  Figure 2.9.1. Proposed biogenesis of exiguamine A. 2.10 Stereochemistry of the exiguamines From the x-ray diffraction analysis, it was discovered that exiguamine A was isolated as a racemic mixture. When exiguamine A crystallized, it belonged in the space group C21c.  The c-glide plane in this space group produces a  symmetrical mirror relationship which means that a racemic mixture is present in  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 85  the unit cell. The lack of optical activity, as well as the lack of any peaks in the CD-spectrum (Figure 2.10.1) confirmed the racemate.  When observing the  biogenesis of the exiguamines, one could envisage that the oxygen on C-il may attack from either face of the alkene, thus yielding a racemic mixtur e (Figure 2.10.2).  Another explanation for the presence of a racemate is perhaps the  exiguamines exist in equilibrium between the two enantiomers in an acidic solution (Figure 2.10.2). The purification of exiguamine A was performed using acidic solvent conditions.  The acidic environment may have catalyzed the  cleavage of the C-19/N-20 bond to yield a pentacyclic structure and an electrophilic imine.  Nucleophilic attack of the phenol oxygen onto the  electrophilic imine (C-il) from both faces yields the racemic mixture.  1.5  0.5 0  E  —1 -1.5 -2 -2.5 -3  Wavelength (nm)  Figure 2.10.1. CD spectrum of exiguamine A.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (ID 0) from the Marine Sponge Neopetrosia exiqua 86 O  H N 2 L  0 -N-—  1 N N’ 3 H  \ N H  jjN  ,  OH 0 —  N—  Figure 2.10.2. Possible equilibrium between the enantiomers of exiguamine A.  There are two chiral carbons in exiguamine B (2.59), C-17, and C-19. Similar to exiguamine A, 2.59 was purified in the presence of TFA, therefore, it is possible that a mixture of both configurations of C-19 are present. The optical rotation of exiguamine B was found to be zero and there were no peaks present in the CD-spectrum (Figure 2.10.3), suggesting the presence of equal quantities of four possible stereoisomers. Attempts to crystallize exiguamine B involved I N HCI. The presence of the strong acid may have induced the isomerisation of the stereocentre on C-17, yielding a mixture of all four diastereomers (Figure 2.10.4). Another explanation for the lack of optical activity or a peak in the CD spectrum may be that the light emitted by the polarimeter or the CD spectrometer could not penetrate exiguamine B. The intense colour of the alkaloid may have prevented the measurement of a meaningful optical rotation or a CD curve.  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 87 Observing the 1 H NMR spectrum of exiguamine B, one could see the presence of minor peaks adjacent to the H-i, H-2, and H-26 resonances (Figure 2.10.5). The presence of these minor peaks may confirm that exiguamine B is present as a mixture of unequal quantities of diastereomers.  There were no  minor peaks present adjacent to either H-17 or 17-OH (Figure 2.10.6). This was unexpected as the largest deviations in chemical shift for diastereomers usually occur at the epimeric centre.  £ 4 3 2 0  E E U  :i  r  510  610  -3 -4 -5  Wavelength (nm)  Figure 2.10.3. CD-spectrum of exiguamine B.  N 3 H  Q  0  /‘  H® HO\N  I  Q 2 H  ®  /) 0 H Iii  Figure 2.10.4. Proposed mechanism of isomerization for C-17.  HO  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 88  H-2  H-26  i-i  --—  3 00  7.00  750  730  7.00  7.50  7.40  a Figure 2.10.5. 1 H NMR expansions of exiguamine B. Minor peaks adjacent to (a) H-I and (b) H-2 and H-26 confirm that exiguamine B was isolated as a diastereotopic mixture.  17-OH H-i 7  fr  ppm  6.10  6.00  5.90  5.80  5.70  Figure 2.10.6. 1 H NMR of expansions of exiguamine B. No minor peaks were present adjacent to 17-OH and H-17, the proton on the epimeric carbon. 2.11 Biological activity of Exiguamine A To screen for inhibitors against IDO, a high throughput assay was run by the laboratory of Professor Grant Mauk in the department of biochemistry at U.B.C.. IDO was added to a reaction mixture that contained tryptophan (2.66) and the desired extract to be tested for inhibition. A reaction was carried out for  730  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 89 30 minutes and stopped by the addition of trichioroacetic acid (TCA) which yields kynurerine (2.73). The reaction mixture was then heated at 65°C for 15 minutes, after which p-dimethylaminobenzaldehyde (2.74) was added to convert any kynurenine  (2.73)  present  dimethylaminobenzylidene (2.75).  to  the  fluorescing  kynurenine  N-p  The concentration of this compound was  measured at 480 nm, and gave an indication of the activity of 50 IDO. 5 ’ 1 A large concentration of 2.75 afforded an intense yellow color and indicated that the extract being tested did not inhibit IDO.  Conversely, a small concentration of  2.75 yielded no color, and signified IDO inhibition. Exiguamine A was found to be a potent inhibitor of IDO in this assay, with a K of 210 nM, making it one of the most potent in vitro IDO inhibitors known to date.  0  Desired  ctextract  COCY  -  TCA  +  NH  inhibition  60°C, 10 mm  2 NH  H 2.66  o  0  coo-  2.73  2.72  H 75monitor:d:t  +  activityofiDO .  2.73  N  2.74  2.75  Figure 2.11.1. Description of the chemical reactions present in the in vitro IDO inhibition assay.  Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 90 As mentioned previously, most IDO inhibitors are analogs of tryptophan. The most potent IDO inhibitors found have been the annulins, 21 which contain a quinone moiety that may be necessary for the potent activity.  Exiguamines  combine both of these elements of inhibition in that the proposed pharmacophore contains both an analog of trytophan, and a quinone moiety (Figure 2.12.2). We suggest that the presence of a substituted pyrroloquinone in the exiguamines is the reason these alkaloids are potent IDO abrogators.  Currently, synthetic  analogs of this pharmacophore are being developed to make novel inhibitors of IDO.  Proposed ph  of exguamine A  Figure 2.11.2. Proposed pharmacophore of the exiguamines. 2.12. General Experimental Methods All solvents used (except for NMR solvents) were HPLC grade (Fisher) and no further purification was done on them unless for use on the HPLC. Those solvents were filtered through a 0.45 p.m filter (Osmonics, Inc) before use. Reversed-phase 1 C 8 silica gel Sep PaksTM (lOg) were purchased from Waters, Inc.. Separations on the HPLC was accomplished using either a Waters 2487 dual channel detector/system controller (Waters Series 515 pump; chart recorder, 0.25 cm/mm), or a Waters 1500 series HPLC pump and a Waters 2487  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 91 dual channel detector. The HPLC column used was a Whatman Partisil 10 ODS 3 Magnum column. The conditions of the HPLC separation were 2.0 mLlmin at 254 nm.  Thin-layer chromatography (TLC) plates were Whatman MKCI8F  (reversed phase) and Kieselgel 60F 2 (normal phase).  TLC was visualized  using either a dip solution of p-an isaldehyde (1% p-an isaldehyde, 2% , 4 S 2 H 0 20% acetic acid and 77% ethanol) or under ultraviolet light (254 nm). NMR spectra were recorded on a Bruker AV600 spectrometer fitted with a inverse triple resonance 1 (, 13 H C, 15 N) cryoprobe. NMR solvents were purchased from Cambridge Isotope laboratories and were referenced to solvent peaks for 6 ( DMSO-d c 39.5 ppm and 6  H  7.24 ppm). Low resolution ESI mass spectra  were recorded on a Bruker Esquire LC mass spectrometer. High resolution ESI mass spectra was obtained using a Micromass LCT mass spectrometer. Optical rotations were recorded with a JASCO J-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm micro cell.  The CD spectra were  determined using a JASCO J-710 spectropolarimeter with a 1 mm micro cell. 2.13. Isolation of exiguamines A and B Neopetrosia exigua (138.5 g wet wt) was collected on Sept 17, 2003 in Milne Bay in Papua New Guinea, 10° 32.02’ 5, 150° 39.07’ E.  This is a  red/brown smooth encrusting sheet sponge, 2 mm x 10 cm, collected from an overhang at 15 m depth.  The sponge was identified by Dr. R. van Soest  (University of Amsterdam) and a voucher sample has been kept at the Zoologisch Museum, Amsterdam (ref no ZMAPORI9II3).  The material was  frozen and stored until workup. The frozen sponge was extracted four times with  Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neoyetrosia exigua 92 MeOH (4 X I L). The combined MeOH extracts were reduced in vacuo to give a brown solid (5.6 g). The brown solid was suspended in 400 mL of 2 H 0 , and then sequentially partitioned with EtOAc (3 X 200 mL) and with n-butanol (3 X 200 mL). The active butanol fraction (1.2 g) was subjected to Sephadex  TM  LH-20  size exclusion chromatography eluting with MeOH. Six hundred milligrams was further purified using gradient elution on a reversed phase Sep PakTM (H 0 to 2 MeOH) to attain 300 mg of the active fraction. The bioactive material was then subjected to gradient reversed phase HPLC (lnertsil C , 1 8 9.4 X 250 mm, 2 H to 0 ACN in 0.1% TFA, UV detection at 254 nm) giving 98.3 mg. Finally, this material was purified by reversed phase HPLC (lnertsil C , 1 8 9.4 X 250 mm, 9:1:0.1 2 H 0 : ACN: TFA) to obtain exiguamine A (58, 20 mg), and exiguamine B (59, 4.5 mg). 2.14. Physical Data Exiguamine A (2.58): UV (MeOH) 2max (log e) 212 (3.44), 263 (3.08), 330 (2.79) nm; [a] 13  0 (c 5.3, MeOH); CD (MeOH, 0.2 mg/mL) no absorption; HRESIMS  [M]m/z 492.1882 (calc’d for 5 N C 2 H 4 6 0 92.188 5 6 3); 1 H and ‘ C NMR data see 3 Table 2.7.1. Exiguamine B (2.59): UV (MeOH) ?max (log e) 215 (3.54), 267 (2.98), 332 (2.69) nm; [a]o 0 (c 3.3, MeOH); CD (MeOH, 0.3 mg!mL) no absorption; HRESIMS 23 [M]m/z 508.1850 (calc’d for 5 N C 2 H 7 0 5 508.1832); ‘H and ‘ 6 C NMR data see 3 Table 2.8.1. 2.15. References (1)  Munn, D. H.; Mellor, A. L. Trends in Molecular Medicine 2004, 10, 15-18.  Chapter 2: Isolation of Inhibitors of lndoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 93 (2)  Muller, A.; Prendergast, G. C. Cancer Research 2005, 65, 8065-8068.  (3)  Schrocksnadel, K.; Wirleitner, B.; Winkler, C.; Fuchs, D. Clinica Chimica Acta 2006, 364, 82 90. —  (4)  Takikawa, 0. Biochemical and Biophysical Research Communications 2005, 338, 12-19.  (5)  Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S. J.; Marshall, B.; Brown, C.; Mellor, A. L. Science 1998, 281, 1191-1193.  (6)  Mellor, A. L.; Munn, D. H. Journal of Reproductive Immunology 2001, 52, 5-13.  (7)  Stone, T. W.; Darlington, L. G. Nature Reviews Drug Discovery 2002, 1, 609-620.  (8)  Mellor, A. Biochemical and Biophysical Research Communications 2005, 338, 20-24.  (9)  Okamoto, A.; Nikaido, T.; Qchiai, K.; Takakura, S.; Saito, M.; Aoki, Y.; lshii, N.; Yanaihara, N.; amada, K. Y.; Takikawa, 0.; Kawaguchi, R.; Isonishi, S.; Tanaka, T.; Urashima, M. Clinical Cancer Research 2005, 11, 6030-6039.  (10)  mo, K.; Yoshida, N.; Kajiyama, H.; Shibata, K.; Yamamoto, E.; Kidokoro, K.; Takahashi, N.; Terauchi, M.; Nawa, A.; Nomura, S.; Nagasaka, T.; Takikawa, 0.; Kikkawa, F. British Journal of Cancer2006, 95, 1555-1561.  (11)  Brandacher, G.; Perathoner, A.; Ladurner, R.; Schneeberger, S.; Obrist, P.; Winkler, C.; Werner, E. R.; G. Werner-Felmayer; Weiss, H. G.; Go, G.; Margreiter, R.; A. Konigsrainer; Fuchs, D.; Amberger, A. Clinical Cancer Research 2006, 12, 1144-1151.  (12)  Malina, H. Z.; Martin, X. D. Graefe’s Archive for Clinical and Experimental Ophthalmology 1993, 231, 482-486.  (13)  Korlimbinis, A.; Truscott, R. J. W. Biochemistry 2006, 45, 1950-1960.  (14)  Takikawa, 0.; Truscott, R. J. W.; Fukao, M.; Miwa, S. Advances in Experimental Medicine and Biology 2003, 527, 277-285.  (15)  Truscott, R. J. W. The International Journal of Biochemistry & Cell Biology 2003, 35, 1500-1504.  (16)  Cady, S. G.; Sono, M. Archives of biochemistry and biophysics 1991, 326333.  Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 94 (17)  Sono, M.; Roach, M. P.; Coulter, E. D. Chemical Reviews 1996, 96, 28412888.  (18)  Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.; Prendergast, G. C. Nature Medicine 2005, 11, 312-319.  (19)  Peterson, A. C.; Loggia, A. J. L.; Hamaker, L. K. Medicinal chemistry research 1993, 4, 473-482.  (20)  Braestup, C.; Nielson, M.; Olsen, C. E. Proceeding of the National Academy of Science 1980, 77, 2288-2292.  (21)  Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R. J. Journal of Natural Products 2006, 69, 1496-1499.  (22)  Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast, G. C.; DuHadaway, J.; Bennett, S.; Donovan, A. M. Journal of Medicinal Chemistry 2006, 49, 684-692.  (23)  Hou, D. Y.; Muller, A. J.; Sharma, M. D.; DuHadaway, J.; Baneijee, T.; Johnson, M.; Mellor, A. I.; Prendergast, G. C.; Munn, D. H. Cancer Research 2007, 67, 792-801.  (24)  Perry, N. B.; Blunt, J. W.; McCombs, J. D.; Munro, M. H. G. Journal of the American Chemical Society 1986, 51, 5476-5478.  (25)  Copp, B. R.; Fulton, K. F.; Perry, N. B.; Blunt, J. W.; Munro, M. H. G. Journal of Organic Chemistry 1994, 59, 8233-8238.  (26)  G. Lang; Pinkert, A.; Blunt, J. W.; Munro, M. H. G. Journal of Natural Products 2005, 68, 1796-1798.  (27)  D’Ambrosio, M.; Guerriero, A.; Chiasera, G.; Pietra, F.; Tato, M. 1996, 52, 8899-8906.  (28)  Sakemi, S.; Sun, H. H.; Jefford, C. W.; Berdardinelli, G. Tetrahedron Letters 1989, 30, 251 7-2520.  (29)  Longley, R. E.; McConnel, 0. J.; Essich, E.; Harmony, D. Journal of Natural Products 1993, 56, 915-920.  (30)  Sun, H. H.; Sakemi, S.; Burres, N.; McCarthy, P. Journal of Organic Chemistry 1990, 55, 4964-4966.  (31)  Gunasekera, S. P.; McCarthy, P. J.; Longley, R. E.; Pomponi, S. A.; Wright, A. E. Journal of Natural Products 1999, 62, 1208-1211.  Chapter 2: Isolation of Inhibitors of Indoieamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 95 (32)  Stierle, D. B.; Faulkner, D. J. Journal of Natural Products 1991, 54, 11311133.  (33)  Radisky, D. C.; Radisky, E. S.; Barrows, L. R.; Copp, B. R.; Kramer, R. A.; Ireland, C. M. Journal of the American Chemical Society 1993, 115.  (34)  Venables, D. A.; Barrows, L. R.; Lassota, P.; Ireland, C. M. Tetrahedron Letters 1997, 38, 721-711.  (35)  Fu, X.; Ng, P. L.; Schmitz, F. J.; Hossain, M. B.; Helm, D. V. d.; Kelly Borges, M. Journal of Natural Products 1996, 59, 1104-1106.  (36)  Keyzers, R. A.; Arendse, C. E.; Hendricks, D. T.; Samaai, T.; Davies Coleman, M. T. Journal of Natural Products 2005, 68, 506-510.  (37)  Copp, B. R.; Ireland, C. M. Journal of Organic Chemistry 1991, 56, 45964597.  (38)  Hooper, G. J.; Davies-Coleman, M. T.; Kelly-Borges, M.; Coetzee, P. S. Tetrahedron Letters 1996, 37, 7135-71 38.  (39)  Utkina, N. K.; Makrchenko, A. E.; Denisenko, V. A.; Dmitrenok, P. 5. Tetrahedron Letters 2004, 45, 7491-7494.  (40)  Utkina, N. K.; Makarchenko, A. E.; Denisenko, V. A. Journal of Natural Products 2005, 68, 1424-1427.  (41)  Hooper, J. N. A.; Soest, R. W. M. V. System Porifera A Guide to Classification of Sponges; Kiuwer Academic/Plenum Publishers: New York, 2002.  (42)  Liu, H.; Mishima, Y.; Fujiwara, T.; Nagai, H.; Kitazawa, A.; Mine, Y.; Kobayashi, H.; Yao, X.; Yamada, J.; Oda, T.; Namikoshi, M. Marine Drugs 2004, 2, 154-163.  (43)  Hildemann, W. H. Transplantation 1981, 32, 77-80.  (44)  Nakagawa, M.; Endo, M. Tetrahedron Letters 1984, 25, 3227-3230.  (45)  Orabi, K.; Sayed, K. A. E.; Hamann, M. T.; Dunbar, D. C.; Al-Said, M. S.; Higa, T.; Kelly, M. Journal of Natural Products 2002, 65, 1782-1785.  (46)  Iwagawa, T.; Kaneko, M.; Okamura, H.; Nakatani, M.; Soest, R. W. M. v.; Shiro, M. Journal of Natural Products 2000, 63, 1310-1311.  (47)  Thale, Z.; Johnson, T.; Tenney, K.; Wenzel, P. J.; Lobkovsky, E.; Clardy, J.; Media, J.; Pietraszkiewicz, H.; Valeriote, F. A.; Crews, P. Journal of Organic Chemistry 2002, 67, 9384-9391.  Chapter 2: Isolation of Inhibitors of lndoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 96 (48)  Williams, D. E.; Craig, K. S.; Patrick, B.; McHardy, L. M.; Soest, R. V.; Roberge, M.; Andersen, R. J. Journal of Organic Chemistiy 2002, 67, 245258.  (49)  Nelson, J. H. In Nuclear Magnetic Spectroscopy Nelson, J. H., Ed.; Pearson Education, Inc.: Upper Saddle River, 2003.  (50)  Sono, M.; Cady, S. G. Biochemistry 1989, 28, 5392-5399.  (51)  Takikawa, 0.; Kuroiwa, T.; Yamazaki, F.; Kido, R. J. Journal of Biological Chemistry 1988, 263,2041-2048.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  97  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 3.1. Preview of Chapter 3 After an injury to the spinal cord, the axons within the lesion will attempt to repair the damage.  Unfortunately, inhibitory components within the central nervous  system prevent the spontaneous regeneration of axons. 1 Compounds that can activate neurite outgrowth and overcome the inhibitory cues of the central nervous system have the potential to be used to treat traumatic spinal cord 1 This chapter will discuss the isolation and synthesis of compounds that injury. can induce neurite outgrowth. 3.2. Inhibitions that Prevent Spinal Cord Repair Traumatic spinal cord injuries (SCI) can result in severe disability. Patients may become either paraplegic or quadriplegic, lose their tactile sensation, lose the ability to coordinate voluntary movements and often have chronic pain issues and spasticity. 2 Unfortunately, treatment options are limited and damage to the spinal cord cannot be adequately treated by any therapy. 3 In 2004, it was estimated that 11,000 new cases of spinal cord injuries would be diagnosed per year in the United States. 4  The prevalence of SCI and the  devastating effects it has on patients has led to considerable research to yield novel interventions that can repair the spinal cord.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  98  Following a traumatic lesion in the spinal cord, the nerve fibers begin a brief attempt to repair the damage by sprouting over the area of damage. Unfortunately, the environment of the central nervous system (CNS) makes it difficult for axons to bypass the injury site. 1 One of the factors contributing to the lack of regeneration is the development of scar tissue at the lesion site. This tissue contains chondroitin-sulfate proteoglycans (CSPG) which inhibit axonal regeneration.  The mechanism by which CSPGs inhibits neurite outgrowth is  1 unclear. The lack of regeneration in the CNS is also due to the presence of inhibitory compounds within myelin, which is the electrically insulating layer that surrounds the axons of many 5 neurons. After damage to the spinal cord, myelin is disrupted, which leaves a high concentration of inhibitory molecules present in the lesion.  Three proteins from myelin have been identified as the major  inhibitors of axon regeneration.  These are Nogo-A, myelin-associated  glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp). 5 All of these proteins bind to the Nogo receptor (NgR), which then activates the RhoA GTP-ase. RhoA then serves to trigger the ROCK (RhoA associated coiled-coil containing protein kinase) serine-threonine protein kinase which leads to the inactivation of neurite outgrowth (Figure 3.2.1).6 Compounds that induce axonal regeneration might be used to treat the dysfunctions brought on by spinal cord injury. Inhibitors of the ROCK kinase are compounds that can potentially induce neurite outgrowth by overcoming the inhibitory proteins of myelin. The two most studied inhibitors of the ROCK kinase  Chanter 3: Isolation of Comnounds That Can Induce Neurite Outarowth  99  are the isoquinoline alkaloid fasudil (3.1, Figure 32.2) and Y-27632 (3.2, Figure 3.2.2). Fasudil inhibits the ROCK kinase with a K of 330 nM, 7 but unfortunately, fasudil is a non-specific kinase inhibitor and is unlikely to be used to treat spinal cord injuries. 8 Y-27632 has a K 1 of 140 nM and is more potent at inhibiting ROCK than fasudil, 7 but this amino-pyridine is not a promising drug candidate to treat spinal cord injuries because it too is a non-specific kinase inhibitor. 8  Membrane  Inhibition of Neurite Outgrowth Figure 3.2.1. Nogo-A, MAG, and OMgp are inhibitory proteins found in myelin. These bind to the Nogo receptor (Ngr) which activates the RhoA GTPase. This then triggers ROCK which inhibits neurite outgrowth.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  100  N 2 H H  3.2  3.1  Figure 3.2.2. Inhibitors of ROCK as potential axonal outgrowth activators. 3.3. Neuroprotective Properties of Diketopiperazines The thyrotropin-releasing hormone (TRH) (3.3, Figure 3.3.1) is a tn peptide hormone that is produced by the hypothalamus.  It is distributed  throughout the CNS and has many neurological functions including regulating changes in temperature, and also interacting with opioid receptors. 9 TRH is metabolized in the central nervous system and in the blood into the diketopiperazine cyclo(S-H is-S-Pro) (3.4, Figure 3.3.1). This diketopiperazine is also present throughout the CNS and has significant neurological roles. Levels of cyclo(S-His-S-Pro) increase in the presence of alcohol in the brain. Studies have revealed that 3.4 assists in diminishing the sedative effects of alcohol. Other  behavioral  effects  of  cyclo(S-His-S-Pro)  include  hypothalamus to reduce the intake of food. ° 1 çNH2 /(O N NHHNO  ° N  NH N ‘0 3.3  Figure 3.3.1. TRH (3) and Cyclo(S-His-S-Pro).  3.4  acting  on  the  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  101  In the presence of a spinal cord injury, it has also been shown that the thyrotopin-releasing hormone has neuroprotective effects. cerebral blood flow as a neuroprotective mechanism.  TRH increases  In a small clinical trial,  twenty patients with spinal cord injuries were treated with TRH. Examination of these patients after four months of treatment revealed significant increases in motor and sensory functions. 11  Unfortunately, TRH would be a poor drug  candidate to treat spinal cord injuries due to the large number of physiological processes this hormone is involved in. 12 As mentioned above, TRH is also metabolized to the bioactive diketopiperazine cyclo(S-His-S-Pro) (3.4) which has similar biological properties. Synthesis of diketopiperazines may provide compounds that can potentially provide similar neuroprotective effects to that of TRH. 9 Based on this fact, a series of cyclized dipeptides were synthesized and evaluated for their neuroprotective actions. One diketopiperazine similar to cyclo(S-His-S-Pro) was synthesized in which the histidine functionality was replaced by a 3,5-di-tertbutyltyrosine.  Evaluation of its neuroprotective properties established that  cyclo((di-tert-Bu)Tyr-Pro) (3.5, Figure 3.3.2) protected neurons from free-radical mediated death. 9 The most promising cyclized dipeptide is the compound referred to as 35b  (3.6, Figure 3.3.2).  In vitro studies have revealed that 35b provides  neuroprotection against apoptotic and mechanical cell death. 13 Administering 35b to rats and mice with brain injuries reduced their lesions and improved cognitive and motor outcomes.  This compound displayed no harmful effects  ChaDter 3: Isolation of Comoounds That Can Induce Neurite Outarowth  even at 100 times the optimal therapeutic dosage.  102  Furthermore, 35b did not  have any endocrine effects, nor did it interact with any TRH receptors.  The  specificity of 35b makes it a promising drug candidate. 12 Other diketopiperazines with neuroprotective properties include 3.7 and 3.8, however these two cyclic  dipeptides are less potent neuroprotective agents. 12  3.5  3.6  3.7  3.8  Figure 3.3.2. Neuroprotective Diketopiperazines. 3.4. Isolation of Neurite Outgrowth Activator from Bacillus sp. A MeOH extract of a Bacillus sp. collected in Dominica was suspended in a 9:1 MeOH: H 0 mixture and then partitioned with hexanes. The MeOH/H 2 0 2 partition showed axonal outgrowth activity and was subjected to size exclusion chromatography, flash reversed-phase column chromatography and reversedphase HPLC to yield pure cyclo(S-Val-S-Phe) (3.9). The structure of the known diketopiperazine was confirmed by comparing the optical rotation, NMR, and MS data to the literature values. 14 For full experimental details, see Section 3.10. 13 14r12 15  HN  16  i 3  6  Z  9  NH  10  0  3.9  Figure 3.4.1. Cyclo(S-VaI-S-Phe) (3.9), a compound promoting axonal outgrowth.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  103  3.5. Stucture Elucidation of Cyclo(S-Val-S-Phe) Cyclo(S-Val-S-Phe) (3.9) was isolated as a white powder that gave a [M  +  Na] ion at m/z 269.1269 in the HRESIMS, appropriate for a molecular formula of  Na C H 1 O 4 (calc’d for 269.1266) and requiring seven degrees of unsaturation. The 1 H NMR spectrum was acquired in DMSO-d 6 at 600 MHz. Examination of the 1 H NMR spectrum (Figure 3.5.1) revealed two exchangeable protons and 7.91), five aromatic protons  (oH  H 6 (  8.11  7.17-7.25), four protons on carbons adjacent  to either a heteroatom or an aromatic ring (OH 4.21, 3.52, 3.15, 2.86), and two methyl doublets  (OH  0.64 and 0.25). Analysis of the ‘ C NMR (Figure 3.5.2) and 3  the HMQC spectra indicated the presence of two carbonyls (Oc 166.5 and 166.3), one quaternary carbon ( c 136.2), six methine carbon resonances (Oc 130.2, 8 127.9, 126.4, 59.0, 54.9, 30.9), one methylene (Oc 37.7) and two methyls (Oc 18.2 and 16.0).  The planar structure of 3.9 was determined as cyclo(Val-Phe) by  extensive examination of the ID and 2D NMR data. Comparison of the 1 H NMR data of 3.9 to previously published data of cyclo(S-Val14 S-Phe) and cyclo(S-Val 15 established a cis-diketopiperazine (Table 3.6.1). The optical rotation of R-Phe) 3.9 ([c]i? -45.82 (c 0.3, DMSO)) was similar to that found for cyclo(S-Val-S-Phe) 22 -43.30 (c 0.3, DMSO)) in the literature, ([c]D 15 which established the absolute configuration as 3S and 6S.  _ Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  104  H”  IL I  I  I  ppm  I  8.0  I  7.0  6.0  I  I  5.0  I  I 4.0  I  I  I  3.0  I  2.0  I  1.0  Figure 3.5.1. 1 H NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 600 MHz in DMSO-d . 5  aNH  pp  100  50  Figure 3.5.2. 13 C NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 150 MHz in DMSO-d . 6  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  105  Table 3.5.1. 1 H chemical shift values for 3.9, and the literature 1 H chemical shift values for both cyclo(S-Val-S-Phe) and cyclo(SValRPhe).a Chemical shift values Literature chemical Literature chemical of 3.9 shift values of shift values of cyclo(S cyclo(S-Val-S-Phe) Val-R-Phe) 14 15 Position 8 H (J in Hz) oH (J in Hz) OH (J in Hz) 1 8.11, bs 8.14, bs 8.14, bs 2 3 3.52, m 3.52, m 3.38, m 4 5 7.91, brs 7.92, brs 7.93, brs 6 4.21, m 4.22, m 4.16, m 7 1.69,m 1.71,m 2.00,m b 8 0.64, d, (7.2) 0.66, d, (7.1) 0.79, d, (7.0) gb 0.25, d, (7.2) 0.27, d, (6.8) 0.72, d, (7.0) lOa 3.15, m 3.16, dd, (13.5, 4.5) 3.14, dd, (13.6, 3.7) lOb 2.86, m 2.88, dd, (13.5, 5.0) 2.86, dd, (13.6, 3.7) 11-16 7.17-7.25, m 7.16-7.28, m 7.05-7.30, m a H chemical shifts [ppm] were all referenced to DMSO-d 1 6 (2.50 ppm) b H-8 and H-9 are interchangeable signals  3.6. Synthesis of Cyclo(S-VaI-S-Phe) and its Diastereomers Isolation and structure elucidation of the active component from the Bacillus sp. extract had established that cyclo(S-Val-S-Phe) was inducing axonal outgrowth.  Further biological studies were necessary, so cyclo(S-Val-S-Phe)  was generated by employing the synthesis developed by Bull et. a!. (Scheme 3.6.1).1416  To analyze if the stereochemistry played a role in the biological  activity of 3.9, the three other diastereomers were also synthesized (Schemes 3.6.1 and 3.6.2). The  commercially  available  diketopiperazine  (S)-(+)-3-isopropyl-2,5-  piperazinedione (3.10, Scheme 3.6.1) was added to a solution of sodium hydride and DMF and stirred at 0°C.  p-methoxybenzyl chloride was added and the  reaction was stirred for 4 h. The reaction was quenched and purified by flash  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  silica gel chromatography to afford the protected diketopiperazine 3.11  106  : 22 (cD  -  49.8 (C 0.8, 3 CHCI ) ) with an overall yield of 75% (Scheme 3.6.2). To a stirring solution of LHMDS in THE, 3.11 was added and allowed to stir at -78°C for I h. To the solution, benzyl bromide was added and the reaction stirred for an additional 3 h at -78°C. The solution was quenched with ammonium chloride and the (3S, 6R) benzylated diketopiperazine 3.12 (65% yield) (Scheme 3.6.2) was separated from the reaction mixture via flash silica gel chromatography. Molecular modeling studies have shown that 3.15 (enolate of 3.11) prefers a conformation where the (3S)-isopropyl group is syn to the N-I protecting group, and anti to the N-4 protecting group (Eigure 3.6.1). This conformation sterically inhibits benzylation at the Si face of enolate 3.15. Benzylation occurs anti to both the N-I protecting group and the isopropyl moiety which yields the (3S, 6R) benzylated diketopiperazine 3.12.1617 Treatment of 3.12 with ceric ammonium nitrate followed by separation with a reversed phase Sep PakTM afforded the diastereomerically pure cyclo(S-Val-R-Phe) (3.14, ccD : -65.4 (c 0.25, DMSO); 2 65%) (Scheme 3.6.1). The structure and absolute stereochemistry of 3.14 was confirmed by comparing the optical rotation, MS and NMR data to the literature 16 values.  Chaoter 3: Isolation of Comoounds That Can Induce Neurite Outarowth  107  Ph  (ii)di-tert-butylphenol 3.10  3.12 3.11 CAN,  XXPh 3.14  CAN,  XxPh 3.9  Scheme 3.6.1. Synthesis of cyclo(S-Val-S-Phe) (3.9) and cyclo(S-Val-R-Phe) (3.14)  6  3.15  Figure 3.6.1. Preferred conformation of enolate 3.15. To obtain the other diastereomer, 3.12 was added to a solution of n-BuLi in THF and allowed to stir for I h at -78°C. The reaction was quenched with 2,6di-tert-butylphenol, and flash silica chromatography afforded the (3S, 6S) benzylated diketopiperazine 3.13 (Scheme 3.6.1) in an 80% yield. The preferred conformation of 3.12 has the isopropyl group syn to the N-I protecting group and anti to the N-4 protecting group.  The branched isopropyl group and the N-4  protecting group provide steric hindrance to n-BuLi.  This results in selective  Chanfar  Lcnlation of Comnniind That Can Indut”.a Naijrifa Outarnwth  108  deprotonation at C-6 to obtain the enolate 3.16. When 3.13 was treated with n BuLl and deuterated with MeOD, there was no deuterium incorporation on C-3. Therefore, only the proton on C-6 was abstracted by n-BuLi (Figure 3.6.2).17 When a bulky proton source such as 2,6-di-tert-butylphenol is used, the C-3 alkyl group and the N-I protecting group provide enough steric hindrance, which results in selective reprotonation trans to both the C-3 and N-I allyl substituents to obtain the (3S, 6S) diketopiperazine 3.13.  Deprotection of 3.13 was  accomplished by the oxidative removal of the p-methoxybenzyl groups using ceric ammonium nitrate.  Chromatographic purification of the reaction mixture  with a reversed phase Sep Pak and reversed phase HPLC obtained the diastereomerically pure cyclo(S-Val-S-Phe) (3.9) in 70% yield (cD : -45.82 (c 22 0.3, DMSO)).  The structure and absolute stereochemistry of diketopiperazine  3.9 was confirmed by comparing the optical rotation, MS and NMR data to the literature values 14.  3.12  3.16  3.13  Figure 3.6.2. Preferred conformations of 3.12, 3.16, 3.13. Cyclo(R-Val-S-Phe) (3.21) was prepared in a similar fashion to cyclo(S Val-R-Phe) (3.14) (Scheme 3.62).  Comparison of the NMR data of  Chapter 3: Isolation of Compounds That Can Induce NeuriteOutqrowth  1 09  diketopiperazine 3.21 to both diketopiperazine 3.14 and the literature established the structure and relative stereochemistry of 3.21 16 diketopiperazine 3.21  : 21 (cD  sign, to diketopiperazine 3.14 these  two  The optical rotation of  69.3, (c 0.3 DMSO)) was similar, but opposite in : 21 (cLD  -65.4, (c 0.25 DMSO)). 16 This confirmed that  molecules are enantiomers and the absolute stereochemistry of  cyclo(R-Val-S-Phe) was determined. To prepare cyclo (R-Val-R-Phe) (3.22), a similar synthesis was employed to that of its enantiomer, cyclo(S-Val-S-Phe) (3.9) (Scheme 3.6.2). The NMR data of cyclo(R-Val-R-Phe) (3.22) was compared to those of both 3.9 and the literature to obtain the structure and relative stereochemistry of 3.21 16  The  diketopiperazine 3.22 (ctD : 43.45 (c 0.29, DMSO)) had an optical rotation that 22 was similar but opposite in sign to that of 3.9 (ctD : -45.82 (c 0.3, DMSO)). This 22 established  that  both  molecules  are  enantiomers;  thus,  the  absolute  stereochemistry of cyclo(R-Val-R-Phe) was confirmed.  3.17  3.19 3.18  I  CAN, ACN-H 0 2  Ph 3.21  CAN, ACN-H 0 2  Ph  3.22  Scheme 3.6.2. Synthesis of cyclo(R-Val-S-Phe) (3.21) and cyclo(R-Val-R-Phe) (3.22).  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  110  3.7. Biology of Diketopiperazines  Actin is an abundant protein in cells that polymerizes to form actin filaments. These filaments are dispersed throughout the cell and are critical for cell motility. Cell migration and neurite outgrowth are very similar processes in that they both require the organized polymerization of actin filaments. 18 Because of the parallels that exist between cell migration and neurite outgrowth, Dr. Tim O’Connor from the Department of Anatomy at the University of British Columbia has developed a novel high-throughput assay to look for compounds that are able to promote cell migration. In this screen, HEK293 cells are cultured in 96well plates and allowed to grow to confluency.  A 96-pin Biogrid robot then  scratches the middle of each well and natural product extracts are added. The treated cells are incubated for 18 hours and then evaluated for their ability to reenter the scratch.  Positive candidates stimulate migration into the scratch,  while cells exposed to inactive compounds will not reinvade the scratch (Figures 3.7.1 and 3.7.2). Bioassay guided fractionation of the Bacillus sp. extract led to the discovery of cyclo(S-Val-S-Phe) (3.9) as a promoter of cell migration at a concentration range of 20-40 riM. The synthetic enantiomer, cyclo(R-Val-R-Phe) (3.22), was also found to be active in the cell migration assay at a similar concentration range. The other two synthetic diastereomers, cyclo(S-Val-R-Phe) (3.14) and cyclo(R-Val-S-Phe) (3.21), were found to be inactive. These results  indicate that the cyclo(cis-Val-Phe) will promote neurite outgrowth while the cyclo(trans-Val-Phe) is not active.  Chanter 3: Isolation of Comnounds That Can Induce Neurite Outarowth  111  1) Confluent plate of 293 cells (96 well plate) 2) 96 pin tool makes a scratch in the middle of each well  3) Robot adds extracts to each well  *  4) Incubate for 18 hours 5) Assess cell migration  Figure 3.7.1. The procedure of the cell migration assay to isolate neurite outgrowth activators.  Figure 3.7.2. To evaluate the ability of the extracts to promote cell migration, each well is viewed under a microscope. These images were generated by Jennifer Wong of the O’Connor lab. As is evident in b, when compounds induce cell migration, the scratch becomes inhabited. Extracts may also inhibit cell migration (c), or promote apoptosis (d). In the last two cases, cells do not enter the scratch.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  112  To further assess the ability of cyclo(S-Val-S-Phe) to act as an activator of neurite outgrowth, a secondary assay was performed. In this assay, day eight dorsal root ganglia isolated from chick embryos were cultured on glass coverslips coated with 100 tg/mL poly-L-lysine (PLL).  The dorsal root ganglia were  incubated on the coverslips for 2 h and the natural product candidates were added, Increases in the neurite length measurements establish an activator of axonal outgrowth. neurite length.  Hence, addition of 32 .tM of cyclo(S-Val-S-Phe) enhanced  To analyze the ability of cyclo(S-Val-S-Phe) to induce axonal  outgrowth in a physiological environment, the secondary assay was done in the presence of inhibitors present in the lesion site. Day 8 dorsal root ganglia from chick embryos were cultured with both 20 j.iglmL of poly-L-lysine and 4 j.ig/mL of CSPG. These neurons showed axonal outgrowth when 32 tM of cyclo(S-Val-S Phe), was added. Similar results were present when 32 jiM of cyclo(S-VaI-S Phe) was added to day 14 dorsal root ganglia from chick embryos cultured in 20 .Lg/mL of PLL and 40 jig/mL of myelin (Figures 3.7.3 and 3.7.4).  Chanter 3: Isolation of Comnounds That Can Induce Neurite Outarowth  113  •PLL LICSPG  In  2  0  E .= ‘I  -J 1  z  0  Control  Cyclo [S-Val-S-Phe]  Figure 3.7.3. Addition of 32 M of cyclo(S-Val-S-Phe) increases the neurite length even in the presence of inhibitors present in the central nervous system. This data was obtained by Jennifer Wong of the O’Connor lab.  Charter 3: Isolation of Comvounds That Can Induce Neurite Outarowth  Control  114  Cyclo (S-Val-S-Phe)  PLL  Myelin  CPSG  Figure 3.7.4. Addition of cyclo(S-Val-S-Phe) (3.9) enhances the neurite length of axons even in the presence of inhibitory substrates from the central nervous system. These images were generated by Jennifer Wong of the O’Connor lab. Further studies were done to analyze the in vivo effects of cyclo(S-Val-S Phe). Sprague Dawley rats underwent a septuptie dorsal rhizotomy. This was then followed by addition of either DMSO or 32 tM of cyclo(S-Val-S-Phe) intrathecally via a cannula attached to a subcutaneously implanted osmotic pump. The presence of cyclo(S-Val-S-Phe) produced an increase in both the  Chapter 3: Isolation of Compounds That Can Induce Neurite Outqrowth  115  serotonergic and adrenergic axons sprouting in both injured and uninjured dorsal horns (Figure 3.7.5).  • •  2  E  Adrenergic  Q.  •1-  0  4-  (‘3  C  a)  C 0  Control  Cyclo (S-Va I-S-P he)  Figure 3.7.5. Addition of cyclo(S-Val-S-Phe) increased the axon sprouting in both serotonergic and adrenergic sprouting in the dorsal horn. This data was obtained by Jennifer Wong of the O’Connor lab. 3.8. Concluding Remarks The two diketopiperazines, cyclo(S-Val-S-Phe) (3.9) and cyclo(R-Val-R Phe) (3.22) were in vitro activators of neurite outgrowth.  More importantly  though, in vitro studies showed that these compounds are able to promote axonal outgrowth even in the presence of inhibitory substrates naturally found in the nervous system. In vivo tests also revealed that following a dorsal rhizotomy, cyclo(S-Val-S-Phe) promoted sprouting of uninjured dorsal roots over the injured site. The two enantiomers, cyclo(S-Val-S-Phe) and cyclo(R-Val-R-Phe) both showed equivalent biologically activity.  This was unexpected because one  assumes that there should be a difference in biological activity between two enantiomers.  Comparison of the chemical structures of the two enantiomers  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  116  reveals that there is pseudosymmetry between the two compounds (Figure 3.8.1). Due to their similar structure, both compounds are able to induce neurite outgrowth.  3.22  Figure 3.8.1. Comparison of the structures of cyclo(S-Val-S-Phe) (3.9) and cyclo(R-Val-R-Phe) (3.22). 3.9. General Experimental Section All solvents used for extraction and chromatography were HPLC grade. When used for HPLC, solvents were filtered through a 0.45 pm filter (Osmonics, Inc). Reversed-phase 1 C 8 silica gel Sep PaksTM (10 g) and normal-phase Si gel Sep PaksTM (2 g) were purchased from Waters, Inc.. HPLC separations were carried out on a Waters 2487 dual channel detector/system controller (Waters Series 515 pump; chart recorder, 0.25 cm/mm), or a Waters 600 controller and Waters 486 Tunable Absorbance Detector. A 5 pm lnertsil column from Chromatography Sciences (Montreal, PQ) was used for reversed phase HPLC, and separations were carried out at 2.0 mL/min, monitoring with UV absorption at 220 nm. Thin-layer chromatography (TLC) plates were Whatman MKCI8F (reversed phase) and Kieselgel 60F 254 (normal phase).  TLC spots were  visualized using either a dip solution of p-anisaldehyde (1 % p-anisaldehyde, 2% H2S04, 20% acetic acid and 77% ethanol) or under ultraviolet light (254 nm). All synthetic reagents were purchased from Aldrich Canada.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  117  NMR spectra were recorded on a Bruker AV600 spectrometer fitted with a  inverse triple resonance 1 (, 13 H C, 15 N) cryoprobe. NMR solvents were purchased from Cambridge Isotope laboratories and were referenced to solvent peaks for 6 DMSO-d  H 6 (  2.49 ppm and 6 3 c 39.5 ppm) and CDCI  (oH  7.24 ppm and 0 c 77.0  ppm). Low resolution ESI mass spectra were recorded on a Bruker Esquire LC mass spectrometer. High resolution ESI mass spectra was obtained using a Micromass LCT mass spectrometer. Optical rotations were recorded with a JASCO J-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10mm micro cell. 3.10. Bacterial Culture The Bacillus sp. culture was isolated from a sediment sample collected by Mike LeBlanc in Dominica in June 2003. It was originally grown on Ml agar and subsequent pans were also made of this agar. To make Ml agar, 10 g of soluble starch, 2 g of bacto-peptone and 18 g of agar were immersed in IL of sterile seawater (30 g/L NaCI in distilled H20) and then autoclaved.  The  autoclaved agar was dispensed into large stainless steel pans at 400 mL per pan and was subsequently incubated for seven days before harvest. The cells and the agar were freeze dried before extraction with MeOH. 3.11. Identification of bacterial culture from sediment Identification of the bacterial species was performed by Helen Wright of the biological services laboratory at the UBC department of Chemistry. The pure culture of the sediment bacterial strain was grown at room temperature on Ml  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  118  plates. Extraction of genomic DNA was then performed by using the DNeasy Tissue Kit (QIAGEN, Mississauga, ON, Canada) in accordance with the manufacturer’s instructions. PCR reactions were performed in 25 pL reaction volumes that contained 12.5 pL of iQ Master mix (BioRad Laboratories), and a mixture of 0.2 pM each 1387r (reverse) and 27f forward primers, and 6.5 pL of sterile distilled 2 H 0 . Primers were synthesized by the NAPS (Nucleic Acids and Protein Services, UBC). The PCR reactions were set up as follows: 95°C for 3 mm, 30 cycles of 95°C for 15 sec (denaturation), 60°C for 15 sec (annealing) and 72°C for 15 sec (elongation). The amplification product was cut from the 0.1% agarose gel and a sequencing reaction was performed by NAPS. The results of the BLAST search of the GenBank database (National Center for Bioinformatics, website http://www.ncbi.nih.pov) confirmed that the PCR product had a sequence corresponding to the 16s rRNA of the Bacillus sp. 3.12. Isolation of Cyclo(S-VaI-S-Phe) from Bacillus sp. The bacterial species (coIl no 101516) was collected from a sediment sample off the coast of Dominica and identified by the Biological Services at UBC as Bacillus sp. Twenty pans of the freeze dried Bacillus sp. were extracted five times with MeOH (5 X I .5L). The MeOH extracts were combined and reduced in vacuo to give a golden brown solid (6 g). The crude extract was then dissolved in 500 mL of a 9:1 MeOH: H 0 mixture which was then partitioned with hexanes 2 (3 X 200mL). The active MeOH/H 0 fraction (800 mg) was then subjected to 2 Sephadex  TM  LH-20 size exclusion chromatography eluting with MeOH which  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  119  afforded an active fraction of 122.3 mg. This material was further purified using a stepped gradient reversed phase Sep PakTM (H 0 to MeOH) where the active 2 fraction eluted with 6:4 MeOH: H 0 (7.6 mg). The crude brown solid was further 2 purified using reversed phase HPLC (lnertsil C , 1 8 9.4 X 250 mm, 1:1 2 H 0 : MeOH, UV detection at 220 nm  )  to yield 1.3 mg of the cyclic dipeptide cyclo(S-Val-S  Phe) (3.9, Figure 3.3.1) as the bioactive compound. The structure of the known diketopiperazine was confirmed by comparing the optical rotation, NMR and MS data to the literature values 14. 3.13. Physical Data of Isolated Diketopiperazine from Bacillus sp. (3S,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.9): white powder. [ct]o : 22 45.82 (c 0.3, DMSO); ‘H NMR (600 MHz, 6 DMSO-d ) :  H 6  -  8.11 (1H, bs, NH), 7.91  (IH, bs, NH), 7.22-7.25 (2H, m, ArH), 7.17-7.18 (3H, m, ArH), 4.21 (IH, m, H-6), 3.52 (IH, m, H-3), 3.15 (IH, m, H-IDa), 2.86 (IH, dd, J 1.69 (IH, m, H-7), 0.64 (3H, d, J  =  =  13.4, 4.9 Hz, H-lOb),  7.2, H-8 or H-9), 0.25 (3H, d, J  =  7.2 Hz, H-9  or H-8): 13 C NMR (150 MHz, 6 DMSO-d ) : Sc 166.5 (C, C-4), 166.3 (C, C-I), 136.2 (C, C-lI), 130.2 (CH, C-12, C-16), 127.9 (CH, C-13, C-15), 126.4 (CH, C-14), 59.0 (CH, C-3), 54.9 (CH, C-6), 37.7 (CH , C-b), 30.9 (CH, C-7), 18.2 (CH 2 , C-8 3 or C-9), 16.0 (CH , C-8 or C-9); LRESIMS m/z 269.1; HRESIMS m/z 269.1269 3 (calc’d for 1 H C 42 8 N N O a 269.1266).  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  120  3.14. Synthetic Experimental Section Preparation of (S)-N,N’-bis(p-methoxybenzyl)-3-isopropylpiperazine-2,5dione (3.11)  I NO ON  o___’  —  To 30 mL of dimethyl formamide, 20 mg (0.75 mmol) of NaH was added and the  mixture was subsequently cooled to 0°C. The addition of 43 mg (0.3 mmol) of (S)-3-isopropylpiperazine-2,5-dione (3.10) was followed by the slow addition of p methoxybenzyl chloride (100 giL; 0.75 mmol) over a period of I h.  After the  reaction mixture was stirred for 4 h, the solution was quenched with H 0 (5 mL), 2 followed by the addition of excess 4 NH C I.  Extraction of the mixture was  accomplished with EtOAc (3 X 10 mL) and dried with MgSO , filtered through 4 CeliteTM and concentrated to dryness in vacuo.  Purification of the reaction  mixture was accomplished using flash chromatography (40 X 2 cm; 1:1 EtOAc: Hexanes) and removal of trace solvents (vacuum pump) provided 96 mg (75% yield) of the protected diketopiperazine (3.11) as a white solid. (S)-N,N’-bis(p-methoxybenzyl)-3-isopropylpiperazine-2,5-dione (3.11): white powder. [cL]D CHCI ) : -49.8 (c 0.8, 3 22 ;1 H NMR (600 MHz, CDCI ) 3 m), 5.26 (IH, d, J  =  14.8 Hz), 4.84 (IH, d, J  =  16.5 Hz), 3.89 (IH, d, J  =  Hz), 3.99 (IH, d, J  =  s), 3.81 (IH, d, J  16.5 Hz), 3.77 (IH, d, J  =  =  H 6  6.79-7.15 (8H,  14.2 Hz), 4.27 (IH, d, J  =  14.2  14.8 Hz), 3.85 (3H, s), 3.85 (3H,  4.8 Hz), 2.24 (IH, m), 1.05 (3H, d, J  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  =  7.2 Hz), 0.90 (3H, d, J  =  121  7.2 Hz); 13 C NMR (150 MHz, 3 CDCI ) : öc 167.2, 165.8,  158.5, 132.1, 130.7, 128.4, 115.8, 65.7, 55.1, 50.5, 47.2, 46.5, 32.3, 20.1, 18.5; LRESIMS m/z 379.2; HRESIMS m/z 379.2321 (calc’d for 2 H C 4 379.2324). 0 2 3N 8 Preparation of (3S,6R)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6benzylpiperazine-2,5-dione (3.12). 1  ON  n-BuLl (237 jiL; 0.379 mmol) was added to a cold (-78°C) stirred solution of hexamethyldisilizane (131 tL; 0.625 mmol) in dry THF (10 mL) under an argon atmosphere. The resulting solution was then warmed to 0°C before being added to a solution of 3.11 (96 mg; 0.253 mmol) in dry THF at -78°C under an argon atmosphere. The reaction mixture was stirred for I h at -78°C which was then followed by the addition of benzyl bromide (55 tL; 0.506 mmol).  After the  solution was stirred for 3 h, the reaction mixture was quenched by the addition of excess saturated 4 NH C I. The volatiles were removed in vacuo and the solution was subsequently extracted with EtOAc (10 mL), dried with 4 MgSO and , concentrated in vacuo. Purification of the reaction mixture was accomplished using flash chromatography (40 X 2 cm; 1:4 EtOAc: Hexanes) and removal of trace solvents (vacuum pump) provided 80 mg (65%) of the protected diketopiperazine (3.12) as a white solid.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  122  (3S,6R)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6-benzylpiperazine-2,5diane (3.12): [aID : 22  +  49.4 (C 0.76, 3 CHCI ) ; 1 H NMR (600 MHz, CDCI ) 3  7.60 (13H, m), 5.71 (IH, d, J  =  14.7 Hz), 4.34 (1H, t, J  14.5), 3.90 (3H, s), 3.86 (3H, s), 3.77 (IH, d, J 4.2), 3.42 (IH, dd, J (3H, d, J  =  =  =  =  =  6.63-  4.2 Hz), 3.98 (IH, d, J  14.7 Hz), 3.50 (IH, dd, J  13.9, 4.2), 3.29 (IH, d, J  7.0 Hz), 0.69 (3H, d, J  =  oH  3.0 Hz)  ,  =  =  13.9,  2.22 (IH, m), 1.05  7.0 Hz); 13 C NMR (150 MHz, 3 CDCI ) : c 0  168.5, 166.1, 161.5, 159.6, 136.2, 131.7, 130.9, 130.1, 129.3, 128.5, 127.6, 127.1, 115.7, 114.9, 61.7, 59.1, 55.3, 47.1, 45.8, 34.9, 31.3, 18.9, 16.0; LRESIMS m/z 487.2597; HRESIMS m/z 487.2597 (calc’d for 3 H C 0 N 5 4 0 2 487.2597). Preparation of 3S,6S-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6benzylpiperazine-2,5-dione (3.13):  n-BuLi (140 j.iL; 0.22 mmol) was added to a cold (-78°C) stirred solution of 3.12 (54 mg; 0.11 mmol) in dry THF (5mL) under an argon atmosphere. After the solution was stirred for 3 h at -78°C, the reaction mixture was quenched by the addition of an excess of a solution of 2,6-di-tert-butylphenol in THF at -78°C. The volatiles were removed in vacuo and the solution was subsequently extracted with EtOAc (5 mL), dried with MgSO , and concentrated in vacuo. Purification of 4 the reaction mixture was accomplished using flash chromatography (40 X 2 cm;  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  123  1:4 EtOAc: Hexanes) and removal of trace solvents (vacuum pump) provided 43 mg (80%) of the protected diketopiperazine (3.13) as a white solid. (3S,6S-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6-benzylpiperazine-2,5-dione (3.13): white powder;  : 22 [c]D  -199 (c 0.85, 3 CHCI ) ; H 1 NMR (600 MHz, CDCI ) 3  oH  7.21—7.32 (3H, m), 7.16—7.20 (2H, m), 6.89—6.95 (2H, m), 6.65—6.73 (2H, m), 5.38 (IH, d, J (IH, d, J =  =  =  14.8), 5.15 (IH, d, J  =  14.6), 4.10 (IH,dd, J  14.8), 3.69 (3H, s), 3.67 (3H, s), 3.49 (IH, d, J  14.3, 4.1), 3.00 (IH, dd, J  1.07 (3H, d, J  =  =  14.3, 4.1), 2.98 (IH, d, J  7.0), 1.01 (3H, d, J  =  =  7.9, 4.0), 3.76  =  7.9), 3.37 (IH, dd, J  =  14.6), 1.86 (IH, m),  7.0); 13 C NMR (150 MHz, 3 CDCI ) : Oc 167.9,  166.5, 160.1, 159.7, 138.2, 129.6, 129.4, 129.2, 129.0, 128.8, 127.6, 127.1, 126.9, 115.1, 114.7, 64.1, 61.2, 55.4, 55.3, 49.2, 47.2, 41.2, 33.8, 21.1, 20.0; LRESIMS m/z 509.2; HRESIMS m/z 509.2423 (calc’d for 3 H C 0 2 5 N N 4 O a 509.2416). Preparation of (3S,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.14): H  XXC To a solution of 3.12 (26 mg; 0.05 mmol) in ACN-H 0 (1:1; 5 mL), ceric 2 ammonium nitrate (54 mg; 0.1 mmol) was added and stirred for 4 h. Reversed phase silica gel was added and the solvent was removed in vacuo, and the residue was purified using a 10 g reversed-phase Sep Pak (eluent: 1:9 MeOH: H 0 2 )to afford 10mg of 3.14(75% yield). (3S,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.14): white powder. [ct]D : 21 -65.4 (c 0.25, DMSO); 1 H NMR (600 MHz, DMSO-d ) OH 8.10 (IH, br s), 8.00 6  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  (IH, br s), 7.10-7.34 (5H, rn), 4.22 (IH, m), 3.15 (IH, dd, J (IH, m), 2.90 (IH, dd, J 0.72 (3H, d, J  =  =  124  =  13.4, 3.9 Hz), 2.95  13.4, 3.9 Hz), 1.95 (IH, m), 0.81 (3H, d, J  =  6.8 Hz),  6.8 Hz); 13 C NMR (150 MHz, 3 CDCI ) : öc 167.0, 166.1, 134.9,  129.6, 126.1, 125.7, 59.1, 55.1, 38.1, 31.3, 18.1, 16.4; LRESIMS m/z 269.1; HRESIMS m/z 269.1266 (calc’d for 1 H C 42 8 N N O a 269.1266). Preparation of (3S,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.9): I  H  N” H  To a solution of 3.13 (43 mg; 0.08 mmol) in 2 ACN-H (1:1; 5 mL), ceric 0 ammonium nitrate (87.7 mg ; 0.16 mmol) was added and the solution stirred for 4 hours. Reversed-phase silica gel was added and the solvent was removed in vacuo, and the residue purified using a reversed-phase Sep PakTM (10 g)(eluent: 9:1 0 2 MeOH ) :H to afford 15 mg of 3.9 (70% yield). (3S,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.9): white powder. [aID : 23 47.32 (c 0.5, DMSO); ‘H NMR (600 MHz, ) 6 DMSO : -d  oH  -  8.11 (IH, bs, NH), 7.91  (IH, bs, NH), 7.22-7.25 (2H, m), 7.17-7.18 (3H, m), 4.21 (IH, m), 3.52 (IH, m), 3.15 (IH, m), 2.86 (IH, dd, J = 13.4, 4.9 Hz), 1.69 (IH, m), 0.64 (3H, d, J 0.25 (3H, d, J  =  =  7.2),  7.2 Hz); “ C NMR (150 MHz, ) 3 6 DMSO : -d Oc 166.5, 166.3, 136.2,  130.2, 127.9, 126.4, 59.0, 54.9, 37.7, 30.9, 18.2, 16.0; LRESIMS m/z 269.1; HRESIMS m/z 269.1269 (calc’d for 1 H C 42 8 N N O a 269.1266).  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  125  Preparation of (R)-N,N’-bis(p-methoxybenzyl)-3-isopropylpiperazine-2,5dione (3.18)  To 50 mL of dimethyl formamide, 20 mg (0.92 mmol) of NaH was added and the mixture  was  subsequently  cooled  to  0°C.  The  addition  of  (R)-3-  isopropylpiperazine-2,5-dione (57 mg; 0.37 mmol) was then followed by the slow addition of p-Methoxybenzyl chloride (124 tL; 0.92 mmol) over a period of I h. After the reaction mixture was stirred for 4 h, the solution was quenched with H 0 2 (6 mL), followed by the addition of excess 4 NH C I. Extraction of the mixture was accomplished with EtOAc (3 X 5 mL) and was dried with MgSO , filtered through 4 CeliteTM and concentrated to dryness in vacuo.  Purification of the reaction  mixture was accomplished using flash chromatography (40 X 2 cm; 1:1 EtOAc: Hexanes) and removal of trace solvents (vacuum pump) provided 100 mg (70% yield) of the protected diketopiperazine (3.18) as a white solid. (R)-N,N’-bis(p-methoxybenzyl)-3-isopropylpiperazine-2,5-dione (3.18): white powder. [c]D : 44.6 (c 0.76, 3 22 CHCI ) ; 1 H NMR (600 MHz, CDCI ) 3 (8H, m), 5.26 (IH, d, J  =  14.2 Hz), 3.99 (IH, d, J (3H, s), 3.81 (IH, d, J (3H, d, J  =  =  =  14.8 Hz), 4.84 (IH, d, J 16.5 Hz), 3.89 (IH, d, J 16.5 Hz), 3.77 (IH, d, J  7.2 Hz), 0.90 (3H, d, J  =  =  H 6  6.79-7.15  14.2 Hz), 4.27 (IH, d, J  =  =  14.8 Hz), 3.85 (3H, s), 3.85  =  4.8 Hz), 2.24 (IH, m), 1.05  7.2 Hz); 13 C NMR (150 MHz, 3 CDCI ) : öc  Chaoter 3: isolation of Comnounds That Can Induce Neurite Outarowth  126  167.2, 165.8, 158.5, 132.1, 130.7, 128.4, 115.8, 65.7, 55.1, 50.5, 47.2, 46.5, 32.3, 20.1, 18.5; LRESIMS m/z 379.2; HRESIMS m/z 379.2321 (calc’d for 2 H C 3 8 4 379.2322). 0 2 N Preparation of (3R,6S)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6benzylpiperazine-2,5-dione (3.19).  n-BuLl (62 j.L; 0.39 mmol) was added to a cold (-78°C) stirred solution of hexamethyldisilizane (135 tL; 0.52 mmol) in dry THF (10 mL) under an argon atmosphere. The resulting solution then was warmed to 0°C before being added to a solution of 3.18 (100 mg; 0.26 mmol) in dry THF at -78°C under an argon atmosphere. The reaction mixture was stirred for I h at -78°C which was then followed by the addition of benzyl bromide (62 pL; 0.52 mmol). After the solution was stirred for 3 h, the reaction mixture was quenched by the addition of excess saturated 4 NH C I.  The volatiles were removed in vacuo and the solution was  subsequently extracted with EtOAc (3 X 5 mL), dried with MgSO , and 4 concentrated in vacuo. Purification of the reaction mixture was accomplished using flash chromatography (40 X 2 cm; 1:4 EtOAc: Hexanes) and removal of trace solvents (vacuum pump) provided 79 mg (60% yield) of the protected diketopiperazine (3.19) as a white solid.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  127  (3R,6S)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6-benzylpiperazine-2,5dione (3.19): [cL]o : -46.2 (c 0.71, 3 22 CHCI ) ; 1 H NMR (600 MHz, 3 CDCI ) : 7.60 (13H, m), 5.71 (IH, d, J  =  14.7 Hz), 4.34 (IH, t, J  14.5 Hz), 3.90 (3H, s), 3.86 (3H, s), 3.77 (1H, d, J 13.9, 4.2 Hz), 3.42 (IH, dd, J m), 1.05 (3H, d, J  =  =  =  =  6.63-  4.2 Hz), 3.98 (IH, d, J  =  14.7 Hz), 3.50 (IH, dd, J  =  =  13.9, 4.2 Hz), 3.29 (IH, d, J  7.0 Hz), 0.69 (3H, d, J  oH  =  3.0 Hz) 2.22 (IH, ,  7.0 Hz); 13 C NMR (150 MHz,  CDCI ) 3 : öc 168.5, 166.1, 161.5, 159.6, 136.2, 131.7, 130.9, 130.1, 129.3, 128.5, 127.6, 127.1, 115.7, 114.9, 61.7, 59.1, 55.3, 47.1, 45.8, 34.9, 31.3, 18.9, 16.0; LRESIMS m/z 487.3; HRESIMS m/z 487.2590 (calc’d for 3 H C 02 5 N 487.2597). 4 0 Preparation of 3R,6R-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6benzylpiperazine-2,5-dione (3.20):  tJ NO O  n-BuLi (295 tL; 0.18 mmol) was added to a cold (-78°C) stirred solution of 3.19 (45 mg; 0.092 mmol) in dry THF (5 mL) under an argon atmosphere. After the solution was stirred for 3 h at -78°C, the reaction mixture was quenched by the addition of an excess of a solution of 2,6-di-tert-butylphenol in THF at -78°C. The volatiles were removed in vacuo and the solution was subsequently extracted with EtOAc (3 X 5mL), dried with MgSO , and concentrated in vacuo. Purification 4 of the reaction mixture was accomplished using silica gel flash chromatography (40 x 2 cm; 1:4 EtOAc: Hexanes) and removal of trace solvents (vacuum pump)  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  128  provided 31 mg (69% yield) of the protected diketopiperazine (3.20) as a white solid. (3R,6R-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6-benzylpiperazine-2,5-d lone (3.20): white powder;  : 22 [c]D  189 (C 0.79, 3 CHCI ) ; ‘H NMR (600 MHz, CDCI ) 3  oH  7.21—7.32 (3H, m), 7.16—7.20 (2H, m), 6.89—6.95 (2H, m), 6.65—6.73 (2H, m), 5.38 (IH, d, J  =  14.8 Hz), 5.15 (IH, d, J  =  14.6 Hz), 4.10 (IH, dd, J  =  7.9, 4.0  Hz), 3.76 (IH, d, J  =  14.8 Hz), 3.69 (3H, s), 3.67 (3H, s), 3.49 (1H, d, J  Hz), 3.37 (IH, dd, J  =  14.3, 4.1 Hz), 3.00 (1H, dd, J  J  =  14.6 Hz), 1.86 (1 H, m), 1.07 (3H, d, J  =  =  =  7.9  14.3, 4.1 Hz), 2.98 (IH, d,  7.0 Hz), 1.01 (3H, d, J  =  7.0 Hz); 13 C  NMR (150 MHz, 3 CDCI ) : 0 c 167.9, 166.5, 160.1, 159.7, 138.2, 129.6, 129.4, 129.2, 129.0, 128.8, 127.6, 127.1, 126.9, 115.1, 114.7, 64.1, 61.2, 55.4, 55.3, 49.2, 47.2, 41.2, 33.8, 21.1, 20.0; LRESIMS m/z487.3; HRESIMS m/z487.2588 (calc’d for 3 H C 0N 5 4 487.2597). 0 2 Preparation of (3R,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.21):  :  :  To a 5 mL solution of 3.19 (34 mg; 0.41 mmol) in ACN-H 0 (1:1), ceric 2 ammonium nitrate (0.45 mg; 0.82 mmol) was added and the solution was stirred for 4 h.  Reversed-phase silica was added and the solvent was removed in  vacuo, and the residue was purified using a reversed-phase Sep PakTM (9:1 H 0: 2 MeOH) to afford 13 mg (65% yield) of 3.21. (3R,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.21): white powder. [c]o : 21 69.3 (c 0.27, DMSO); 1 H NMR (600 MHz, ) 6 DMSO -d 0 H 8.10 (IH, br s), 8.00 (IH,  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  br s), 7.10-7.34 (5H, m), 4.22 (IH, m), 3.15 (dd, J 2.90 (IH, dd, J d, J  =  =  =  129  13.4, 3.9 Hz), 2.95 (IH, m),  13.4, 3.9 Hz), 1.95 (IH, m), 0.81 (3H, d, J  =  6.8 Hz), 0.72 (3H,  6.8 Hz); 13 C NMR (150 MHz, 3 CDCI ) : öc 167.0, 166.1, 134.9, 129.6, 126.1,  125.7, 59.1, 55.1, 38.1, 31.3, 18.1, 16.4; LRESIMS m/z 269.1; HRESIMS m/z 269.1268 (calc’d for 1 H C 42 8 N N O a 269.1266). Preparation of (3R,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.22):  To a 5 mL solution of 3.20 (31 mg; 0.06 mmol) in ACN-H 0 (1:1), 65mg of ceric 2 ammonium nitrate was added and stirred for 4 hours. Reversed-phase silica was added and the solvent was removed in vacuo, and the residue purified using a reversed-phase Sep PakTM (10 g)(eluent: 9:1 0 2 MeOH ) :H to afford 12 mg (70 % yield) of 3.22. (3R,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione  (3.22):  [cL]o: 43.45 (c 0.29, DMSO); 1 H NMR (600 MHz, ) 6 DMSO : -d  white oH  powder.  8.11 (IH, bs,  NH), 7.91 (IH, bs), 7.22-7.25 (2H, m), 7.17-7.18 (3H, m), 4.21 (IH, m), 3.52 (IH, m), 3.15 (IH, m), 2.86 (1H, dd, J 7.19), 0.25 (3H, d, J  =  =  13.4, 4.9 Hz), 1.69 (IH, m), 0.64 (3H, d, J  =  7.2 Hz); ‘ C NMR (150 MHz, ) 3 6 DMSO : -d O 166.5, 166.3,  136.2, 130.2, 127.9, 126.4, 59.0, 54.9, 37.7, 30.9, 18.16, 16.0; LRESIMS m/z 269.1; HRESIMS m/z 269.1267 (calc’d for 1 H C 42 8 N N O a 269.1266).  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  130  3.15 References (1)  Fawcett J.F. Journal of Neurotrama 2006, 23, 371-383.  (2)  Ditunno J.F.; Formal C.S. New England Journal of Medicine 1994, 330, 550-556.  (3)  Bradbury E.J.; McMahon S.B. Nature Reviews Neuroscience 2006, 7, 644-653.  (4)  Lim P.A.C.; Tow A.M. Annals of the Academy of Medicine, Singapore 2007, 36, 49-57.  (5)  Skaper S.D. Annals of the New York Academy of Sciences 2005, 1053, 376-385.  (6)  Lee D.H.S.; Strittmatter S.M.; Sah D.W.Y. Nature Reviews Drug Discovery 2003, 2, 1-7.  (7)  Uehata M.; Ishizaki T.; Satoh H.; Ono T.; Kawahara T.; Morishita T.; Tamakawa H.; Yamgami K.; lnui J.; Maekawa M.; Narumiya S. Nature 1997, 30, 990-994.  (8)  Mueller B.K.; Mack H.; Teusch N. Nature Reviews Drug Discovery 2005, 4, 387-398.  (9)  Prakash K. R. C.; Tang Y.; Kozikowski A.P.; Flippen-Anderson J.L.; Knoblachc S.M.; Fadenc A.l. Bloorganic and Medicinal Chemistry 2002, 10, 3043-3048.  (10)  Prasad C. Peptides 1995, 16, 151-164.  (11)  Baptiste D.C.; Fehlings M.G. Journal of Neurotrama 2006, 23, 31 8-334.  (12)  Faden A.I.; Knoblack S.M.; Movsesyan V.A.; IV P.M. Lea; Cernak I. Annals of the New York Academy of Science 2005, 1053, 472-481.  (13)  Faden A.l.; Knoblach S.M.; Movseyan V.A.; Cernak I. Journal of Alzheimer’s Disease 2004, 6, S93-S97.  (14)  Bull S.D.; Davies S.G.; GarnerA.C.; O’Shea M.D. 2001, J. Chem. Soc., Perkin Trans. 1, 3281-3287.  (15)  Lopez-Cobenas A.; Cledera P.; Sanchez J.D.; Lopez-Alvarado P.; Ramos M.T.; Avendano C.; Menedez J.C. Synthesis 2005, 19, 3412-3422.  (16)  Bull S.D.; Davies S.G.; Epstein S.W.; Leech M.A.; Ouzman J.V.A. Journal of the Chemical Society, Perkin Trans. 11998, 2321-2330.  Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth  (17)  Bull S.D.; Davies S.G.; Epstein S.W.; Ouzman J.V.A. Tetrahedron: Assymmetiy 1998, 9, 2795-2798.  (18)  Meyer G.; Feldman EL. Journal of Neurochemistiy 2002, 83, 490-503.  131  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  132  Chapter 4: Structure Elucidation of G 2 Checkpoint Odorat aa inhibitors from Duguetia 4.1. Preview of Chapter 4  High-throughput screening of plant extracts from the U.S. National Cancer Institute’s Open Repository collection identified the South American plant Duguetia odorata as having activity in the G 2 checkpoint inhibition assay. Bioassay guided fractionation of the plant extract led to the discovery of oliveroline (4.32) as an abrogator of G 2 arrest. This investigation also yielded the new aporphine alkaloid N-methylguatterine (4.33), as well as the known alkaloids dehydrodiscretine (4.34) and pseudopalmatine (4.35). 4.2. The Cell cycle  The cell cycle (Figure 4.2.1) is a process the cell undergoes until it has reproduced itself. Interphase is the first part of cell division, where the cell grows, prepares for cell division and metabolism take place. It is divided into the G , S, 1 and G 2 phases. The G 1 phase is where metabolism takes place. At the end of , centrioles replicate and prepare for cell division. Before progressing to the S 1 G phase, sensors scan to check the fidelity of the DNA.  Should there be any  anomalies present on the DNA, the G 1 checkpoint would stall the cell cycle to allow the damage to be repaired.  In the S-phase, DNA synthesis and  chromosome replication take place. Critical proteins and enzymes required for a.  Reproduced with permission from Brastianos H.C.; Sturgeon C.M.; Roberge M.; Andersen R.J. Journal of Natural Products 2007, 70, 287-288. Copyright 2007 American Chemical Society.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  133  cell division are synthesized in the G 2 phase of interphase. Similar to the G 1 checkpoint, the G 2 checkpoint halts the cell cycle to repair any damaged DNA. The M-phase is the second part of cell division which consists of both mitosis and cytokinesis.  Mitosis is where the cell divides the duplicated chromosomes to  obtain identical sets and is divided into four stages. The first stage of mitosis is prophase where the chromosomes condense and the centrioles move toward the opposite poles of the cell.  This is then followed by metaphase, where the  chromosomes line up at the equator of the cell. During anaphase, the two sets of chromosomes split and move towards the opposite poles of the cell. The final step of mitosis is telophase where the nuclear envelope forms around each chromosome. Cytokinesis is the final part of the cell cycle where division of the cytoplasm takes place and two independent cells are obtain 1 ed.  Chapter 4: Structure Elucidation of G Checjpintinhibitors from Duquetia Odorata 2  134  Interphase  1 checkpoint G  checkpoint  M-phase  Figure 4.2.1. The cell cycle consists of interphase and the M-phase (Adapted from Voet & Voet). 1 Interphase is the first part of the cell cycle and it consists of the G , S, and G 1 2 phases of the cell cycle. The M-phase consists of mitosis where the chromosomes are divided, and cytokinesis where two separate cells are obtained. 4.3. G to M Transition The formation of the cyclinB/cdc2 complex is crucial for cells to enter mitosis. This complex is known as the M-phase promoting factor. Throughout mitosis and G , the levels of cyclinB are low. At the end of the S-phase however, 1 cyclinB is synthesized, which leads to the formation of the cylcinB/cdc2 complex. During  , 2 G  the  cyclinB/cdc2  phosphorylations on cdc2.  complex  is  held  inactive  by  inhibitory  These phosphorylations are carried out by Wee 1.  Cdc25c acts as a positive regulator of the cdc2/cyclin B complex by  ChaDter 4: Structure Elucidation of G, Checkooint inhibitors from Duauetia Odorata  135  dephosphorylating cdc2. Dephosphorylation of cdc2 activates the complex and triggers mitosis (Figure 4.3.1 )•24  Active  Figure 4.3.1. 2 G / M transition (adapted from Foijer) 4. It is vital for the DNA not to be damaged before entering mitosis. This is ensured by the G 2 checkpoint pathway. This checkpoint’s purpose is to prohibit cdc25c from activating the cyclinB /cdc25c complex, therefore, the checkpoint arrests the onset of mitosis to repair any damaged DNA. Upon DNA damage, ATM/ATR induces the activation of Chkl/Chk2, which goes on to phosphorylate cdc25c. The phosphorylation on cdc25c also creates a binding site for the 14-33a proteins.  The 14-3-3a/cdc25c complex is then sequestered out of the  nucleus. Since cdc25c is not present to activate the cyclinB/cdc2 complex, the cell cycle is arrested (Figure 4.3.2).56  Chanter 4: Structure Elucidation of G Checknoint inhibitors from Ducuetia Odorata  136  P cdc25c  I 4-3-3a  Inactive  Active  Figure 4.3.2. G 2 checkpoint pathway (Adapted from Samuel et al.). 6  4.3. Rationale for using G 2 Checkpoint Inhibitors The p53 protein is critical for protecting against cellular damage. It is a tumor suppressor protein that is significant in cell-cycle control, apoptosis, and maintaining genetic stability. A critical role of p53 is to activate the G, checkpoint to allow time to repair any lesions in the DNA. It has been found, however, that at least 60% of tumors lack p53 and in the presence of DNA damage, mp53 cells fail to arrest at G 1 to repair their DNA. The mp53 cells may then either die, or continue to proliferate with a blemished genom 7 e.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  137  Due to their genetic instability, it had been thought that mp53 cells would be more sensitive to treatments that involved DNA damaging agents. It has been found, however, that there is no correlation between p53 function and radiosensitivity. An explanation may lie in the fact that cells still arrest at the 2 G checkpoint.  This allows tumor cells time to recover and repair their DNA. 9 ’ 8  Tumor cells lacking p53 may also have a growth advantage over wild type cells because the mp53 cells do not arrest at the G 1 checkp 7 oint. DNA damaging agents such as cisplatin are often used to treat tumors. Upon DNA damage, the wild type cells activate both the G 1 and the C 2 checkpoints, but mp53 cells activate only the G 2 checkpoint. If a C 2 checkpoint inhibitor is used, the wild type cell is still able to arrest at G , but mp53 cells 1 would not have any mechanism to repair their DNA. Tumor cells would enter mitosis with a large portion of their genome damaged, which is a lethal event (Figure 4.4.1).b0  Chanter 4: Structure Elucidation of G, Checknoint inhibitors from Duciuetia Odorata  A) Wild Type Cell  1 G  II I—. 2 checkpoint G  S  2 G  I-  1 checkpoint G  M  —  G Checkpoint lnhbitor 2 DNA Damaging Agent 1 G  S  2 G  1 chckpoint G  2 cheoint G  B) mp53 cells G  S  G2  1 cDkpoint G  ‘I  ii  M  G checkpoint 2 2 Checkpoint Inhbitor G DNA Damaging Agent  S 1 G  M 2 ch G  Figure 4.4.1. Rationale for using G 2 checkpoint inhibitors.  nt  138  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  139  4.5. Known G 2 Checkpoint Inhibitors There have been numerous G 2 checkpoint inhibitors that have been discovered. The first G 2 checkpoint inhibitor found was caffeine (4.1), and it was found to inhibit ATM/ATR mediated phosphorylation of Chkl.  Unfortunately,  caffeine is not practical to use in the clinic due to its numerous pharmacological activities and its cytotoxicity to cells at millimolar ° 1 concentration s.  Other  compounds that can inhibit ATM/ATR include the polyketide kinase inhibitor wortmannin (4.2), which has been found to be a strong G 2 checkpoint abrogator at 10 ” iM. 1 2  4.2  Figure 4.5.1. ATM/ATR inhibitors of the G 2 checkpoint pathway. Staurosporine (4.3) is a very potent G 2 checkpoint inhibitor (IC 50 0.2 nM). 1 ’ 10 2 Unfortunately, this indole alkaloid is a non-specific kinase inhibitor and is highly toxic. 13  In efforts to find staurosporine analogs, UCN-01 (4.4) (7-  hydroxystaurosporine) has been found to be a potent kinase inhibitor, a C 2 checkpoint inhibitor (1C 50 50-1 00 nM),’ 4 and an in vitro inhibitor of Chkl (1C 50 1025 nM). 15 UCN-01’s promising biological activities allowed it to undergo clinical trials in both Japan and the U.S.  One case report found that a patient with  lymphoma chemotherapeutically resistant to EPOCH II (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) had complete remission after one  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duguetia Odorata  140  cycle of UCN-O1 proceeded by an EPOCH II dosage. 16 As evidenced by this study, further trials with this promising compound need to be done. SB-218078 (4.5) has also been shown as a compound that can inhibit G 2 arrest. This indole alkaloid was discovered by testing a series of SerlThr kinase inhibitors for their ability to inhibit Chkl in vitro.  It is a potent Chkl inhibitor that can inhibit G 2  arrest with an IC 50 of 15 nM. Paradoxically, SB-218078 was found to induce G 2 arrest at higher concentration 17 s. N  0  HO  45  Figure 4.5.2. Indole alkaloids inhibiting the G 2 checkpoint through Chkl. Bioassay-guided fractionation of the Brazilian  ascidian  Didemnum  granulatum led to the isolation of two G 2 checkpoint inhibitors: granulatimide (4.6) and isogranulatimide (4.7). Each of these alkaloids inhibited G 2 arrest with an 50 of 6 tM. IC ° Isogranulatimide was able to inhibit Chk I with an IC 1 50 of 0.432 jtM, while granulatimide was more potent (1C 50 0.081 iM).” 8 Other marine G 2 checkpoint inhibitors discovered include the alkaloids hymenialdisine (4.8) and debromohymenialdisine (4.9) isolated from a MeOH extract of the marine sponge Stylissa flabeliformis. Both of these alkaloids were able to inhibit G 2 arrest (IC 50 6-8 jiM) and were found to be in vitro inhibitors of Chkl (1C 50 3 jiM).’ 9  Chanter 4: Structure Elucidation of G., Checknoint inhibitors from Duauetia Odorata  141  4.6 N 2 H  N 2 H  0  0 4.8  Figure 4.5.3 Alkaloids inhibiting the G 2 checkpoint through Chkl Other inhibitors of the C 2 checkpoint include 1 3-hydroxy-1 5-oxozoapatlin (4.10), which was isolated from a MeOH extract of Parinari curatellifona bark obtained from the NCI Natural Products Repository. This compound was able to inhibit the C 2 checkpoint (1C 50 5-7 pM), however, the target of this small molecule is unknown, as it was found to be neither an inhibitor of ATM or of Chkl  20  One  hypothesis for the biological activity is that the presence of the a, 13-unsaturated ketone makes it reactive to thiols in proteins.  Other polyketide derived G 2  checkpoint abrogators include okadaic acid (4.11), which has an 1C 50 of 0.5 jiM. Unfortunately, okadaic acid is a carcinogen and a food poison, so it is unlikely to be used in the clinic. 21 Fostriecin (4.12) is an anti-tumor drug that has activity against lung, breast, and colon cancer. This polyketide was shown to inhibit the 2 checkpoint pathway with an (C G 50 of 3.2 tM. 21  Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duouetia Odorata  142  OH  OH 4.12  Figure 4.5.4. G 2 checkpoint inhibitors 4.6. Description of the G 2 Checkpoint Assay Dr. Michel Roberge has developed the first assay to search for G 2 checkpoint inhibitors in crude extracts. ° In the assay, MCF-7 mp53 cells are 1 cultured and allowed to grow for 24 hours. The cells are then irradiated and after 16 hours, extracts are added to the cells along with nocodazole. Caffeine is used as a positive control in this assay.  Cells are incubated for eight hours after  adding the crude extracts, and cells that enter mitosis are measured by ELISA. The TG-3 antibody used in the ELISA assay recognizes a phosphorylated form of nucleolin present only in mitotic cells (Figure 4.6.1).b0  Chanter 4: Structure Elucidation of  Checknoint inhibitors from Duauetia Odorata  143  MCF-7 mp53 in 96 well plate  Jr  Irradiate with 6.5 Gy  Cells exposed to G 2 checkpoint inhibitors enter mitosis  Jr  Cells not exposed to G 2 checkpoint inhibitors remain arrested in G 2  I  Quantitate mitosis Quantitate mitosis by ELISA using TG-3 by ELISA using TG-3  Jr  Positive Signal  Negative Signal  Figure 4.6.1. Description of the G 2 checkpoint inhibition assay. 4.7. Chemistry of Duguetia sp. The Annonaceae family contains numerous shrubs, trees, and lianas, and is distributed in the tropics of South America, Africa, and Asia. This particular family is known for the acetogenins, a group of chemical compounds that have been discovered to have potent anti-tumor, cytotoxic, and anti-microbial  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata  144  22 Duguetia is a genus within the Annonaceae family and contains properties. approximately 80 species. Even though numerous chemical studies have been done on the Duguetia genus, biological studies have not been as detailed. 23 Plants in the genus Duguetia have medicinal potential as components of D. glabriuscula have been found to have anti-neoplastic activity, 24 and extracts of D. furfuracea and D. lanceolata have anti-parasitic activity. 25 Three aporphinoid alkaloids, R-(-) dicentrine (4.13), duguetine (4.14) and norglaucine (4.15), were the first natural products isolated from a Duguetia sp. collected in 23 Brazil. Aporphine alkaloids that were first discovered from the bark of D. spixiana include reomerolidine (4.16), nornuciferidine (4.17), rurrebanine (4.18), and rurrebanidine (4.19). None of these natural products were reported to have any biological activity. 26 More recently, an ethanol extract of D. furufuracea collected in Brazil afforded N-nitrosoanonaine (4.20) and N-nitrosoxylopine (4.21). Their structures were determined using both NMR spectroscopy and X ray crystallograp 23 hy.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  NH  —  4.13  <:  145  4.14  4.15  :O)NH  OH OH OH  4.18  4.17  <:zg  4.19  K:cIN  4.20  O  4.21  Figure 4.7.1. Aporphine alkaloids from Duguetia. Chemical studies of an extract of D. eximia led to the isolation of several known oxoaporphines including O-methylmoschatoline (4.22) and oxostephanine (4.23), and the first report of oxo-O-methylpukateine  (4.24).27  Later, biological  studies on O-methylmoschatoline revealed this oxoaporphine to have moderate antiparasitic activity against Leishmania braziliensis, and Leishmania guyanesis as well as cytotoxicity in the brine shrimp assay with an IC 50 of 3.80 28 jtg/mL. 2 ’ 9 Oxostephanine was discovered to have promising activity against the Herpes simplex virus. 30 More recently, duguevaline (4.25) was isolated from a CH CI 2 extract of D. vallicola collected in Columbia. 31  Other isoquinoline alkaloids  isolated from the bark of D. spixiana include codamine-N-oxide (4.26), spiguetine (4.27), and spiguetidine  (4.28)2632  An EtCH extract of D. hadrantha has also  yielded a series of bioactive alkaloids. Hadranthine A (4.29), sampangine (4.30)  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  146  and 1-methoxysampangine (4.31) were found to display in vitro antimalarial activity against Plasmodium falciparum (1C 50 120, 68, 95 ng/mL). Sampangine was also cytotoxic towards human malignant melanoma cells (1C 50 370 nglmL).  o  N  KQXXN  N  0 4.22  423  ZO3Ng3 oc 4.26  ‘c  4.24  4.25  Y  4.27  i  4.28  NN  x:e 4.29  4.30  4.31  Figure 4.7.2 Alkaloids from Duguetia sp.. 4.8. Isolation of alkaloids from Duguetia odorata A MeOH extract of D. odorata (MacBride 1929) (Annonaceae) was obtained from the NCI repository of natural products and found to have bioactivity in the G 2 checkpoint assay. The MeOH extract was suspended in 2 H 0 , and then sequentially partitioned with hexanes, , CI EtOAc and n-butanol. The active 2 CH n-butanol extract was subjected to size exclusion chromatography, flash reversed-phase column chromatography and reversed-phase HPLC to obtain oliveroline (4.32), a new alkaloid N-methylguatterine (4.33), dehydrodiscretine  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata  (4.34), and pseudopalmatine (4.35).  147  The structures of the known alkaloids  oliveroline (4.32),’ dehydrodiscretine (4.34),  35,36  and pseudopalmatine (4.35)  were all confirmed by comparing their NMR and MS data to the literature values. For full experimental details, see section 4.12.  j’OH 4.32  4.34  4.33  0  0H 0  4.35  I  Figure 4.8.1. Alkaloids isolated from D. odorata. 4.9. Structure Elucidation of N-methylguatterine  12<\  0  ibi G— 13 1f4 H14 11  ‘0H  1111 1O8  9 4.33  Figure 4.9.1. Numbering scheme of N-methylguatterine. N-methylguatterine (4.33) was isolated as an optically active colorless solid that gave a [M] ion at m/z 340.1534 in the HRESIMS, which is consistent with a molecular formula of 4 N0 C 2 H 0 (calc’d for 340.1549). 2  The H 1 NMR  spectrum (Figure 4.9.3) was acquired in DMSQ-d 6 at 500 MHz and was found to be similar to the 1 H NMR spectrum of oliveroline, suggesting that 4.33 was an  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  aporphine alkaloid.  Examination of the  aromatic protons  H 6 (  deshielded protons  (oH  148  H NMR spectrum revealed four 1  7.42-7.87), an exchangeable proton  H 6 (  6.95), two  6.25 and 6.07), a series of protons on carbons attached to  a heteroatom (OH 3.17-5.02) and two protons on a carbon attached to an sp 2 hybridized carbon (OH 2.93). Analysis of the 13 C NMR spectra (Figure 4.9.4) and the HMQC data (Figure 4.9.5) identified 12 sp 2 hybridized carbons (Oc 145.1, 138.9, 136.9, 135.9, 128.0, 127.9, 127.6, 125.4, 123.9, 119.2, 116.3, 109.5), an acetal carbon (c 101.7), six sp 3 hybridized carbons attached to heteroatoms (Oc 70.8, 68.2, 61.8, 59.4, 56.8, 42.1) and one shielded 3 -sp hybridized carbon ( c 0 18.6). The HMQC data allowed the assignment of the proton resonances to their respective carbons (Table 4.9.1). It was possible to deduce four substructures (Figure 4.9.2) using the HMBC and COSY data ((Figures 4.9.6 and 4.9.7).  II  Iv  Figure 4.9.2. Substructures of N-methylguatterine deduced from the COSY and HMBC spectra.  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors fmm Duquetia Odorata  149  C Cy,  q  o L  Q CD  0 1%:  /D 2 0  Figure 4.9.3. H 1 NMR spectrum of N-methylguatterine at 500 MHz in DMSO-d . 6  Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duauetia Odorata  150  0  u)  0 0  /°<D  0 LC)  ozo  E a-  Figure 4.9.4. ‘ C spectrum NMR of N-methylguatterine at 100 MHz in DMSO-d 3 . 6  Chapter 4: Structure Elucidation of G,Checkoint inhibitors from Duquetia Odorata  JUL  LJLLJJ__  ppm  7.0  6.0  5.0  4.0  3.0  Figure 4.9.5. HMQC spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d  151  Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duouetia Odorata  152  K:  50  100  ppm ppm  7.0  6.0  5.0  4.0  3.0  Figure 49.6. HMBC spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d  Chapter 4: Structure Elucidation of G, CheckpoTht inhibitors from Duquetia Odorat a  153  2.0  • 3.0  4.0  •5.0  6.0  7.0  ppir  7.0  6.0  5.0  4.0  3.0  Figure 4.9.7. COSY spectrum of N-methylguatterine at 500 MHz in . 6 DMSO -d  Chaoter 4: Structure Elucidation of G 9 Checkooint inhibitors from Duauetia Odorata  Position 1 Ia lb 2 3 3a 4 5a 5b 6 6a 7 7a 8  b 9  0” 11 ha 12  154  Table 4.9.1. ID and 2DNMR data of Nmethylguatterine.a ö (J in Hz) H, 13 1 C-HMBC C COSY 145.1 109.5 119.2 135.9 138.9 116.3 18.6 2.93, m H-5a, H-5b 61.8 3.61, m C-14 H-4, H-5b 3.75, m C-4 H-4, H-5a  70.8  4.74, d, (12.0)  68.2 136.9 123.9 127.9 128.0 125.4 127.6 101.7  5.02,dd, (12.0, 7.0)  C-Ib, C-7, C-7a, C-13, C14 C-lb, C-6a, C-7a  7-OH, H-6a  7.65, m 7.41,m 7.42,m 7.87, m  C-7, C-9, C-l0 C-Il C-8 C-lIa, C-la  H-9 H-8,H-I0 H-9,H-1I H-b  H-7  6.25 (a), s C-I, C-2 H-12b 6.07(b),s C-l,C-2 H-12a 13 42.1 3.17, s C-5, C-6a, C-I4 H-14 14 56.8 3.61, s C-5, C-6a, C-13 H-13 15 59.4 4.02, s C-3 7-OH 6.95, d, (7.0) C-6a, C-7 H-7 a: H and 1 1 C chemical shifts [ppm] are referenced to DMSO-d 6 (ö- 2.50 and öc 39.51 respectively) b: Signals may be interchanged  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duciuetia Odorata  4.74, d, (12.00 Hz)  155  68.2  OH  0H6.95,d,(6.82Hz) 5.02, cld, (12.00, 7 Hz)  a  b  Figure 4.9.8. (a) “H chemical shifts and coupling constants for substructure I and (b) 13 C chemical shifts for substructure I.  )icosy  13  Figure 4.9.9. Key COSY and HMBC correlations observed for substructure I of 33. A singlet methyl proton resonance at  H  3.17 (H-13: HMQC to c 42.1)  showed HMBC correlations to a methyl carbon resonance at ö 56.8 (C-14). HMBC cross-peaks were also observed between the methyl proton resonance at oN  3.61 (H-14: HMQC to Oc 56.8) and the methyl carbon resonance at öc 42.1 (C  13). This implied that Me-13 and Me-14 were geminal, and their chemical shifts indicated that they were attached to nitrogen (N-6) (Figure 4.9.11). methyl proton resonances at  H 6  3.17 (H-13) and  H 6  Both the  3.61 (H-14) showed HMBC  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duijuetia Odorata  correlations to the carbon resonance at & 61.8 (C-5). correlations between the proton resonance at  oH  156  Further HMBC  3.61 (H-5a: HMQC to 0 c 61.8)  and the carbon resonance at Oc 56.8 (C-14) established the bond between C-5 and N-6 (Figures 4.9.8 and 4.9.11).  The downfield chemical shift of the  methylene carbon C-5 (öc 61.8) confirmed the attachment to the N-dimethyl moiety (Figure 4.9.8). Both methylene proton resonances at OH  H 0  3.61 (I-I-5a) and  3.75 (H-5b) displayed COSY cross-peaks with the methylene proton  resonance at  oH  2.93 (H-4), which established the connectivity between C-4 and  C-5 (Figure 4.9.9 and 4.9.10). Both the proton resonances at OH 3.17 (H-13) and  H 0  3.61 (H-14)  displayed HMBC correlations to the methine carbon resonance at 0 c 70.8 (C-6a). A methine proton resonance at OH 4.74 (H-6a: HMQC to Oc 70.8) displayed HMBC correlations to the methyl resonances at Oc 56.8 (C-14) and 0 c 42.1 (C13), which established the connectivity between C-6a and N-6 (Figure 4.9.8). Observation of the carbon chemical shift of C-6a (Oc 70.8) also confirmed this linkage (Figure 4.9.8).  The proton resonance at  H 0  COSY correlation to the methine proton resonance at  4.74 (H-6a) contained a H 0  5.02 (H-7: HMQC to Oc  68.2), which indicated a linkage between C-6a (Oc 70.8) and C-7 (öc 68.2) (Figures 4.9.8 and 4.9.9).  No HMQC correlations were present for the proton  resonance at OH 7.95 (7-OH), which indicated the presence of an exchangeable alcohol proton. COSY correlations between the exchangeable proton resonance at  H 0  7.95 (7-OH), with the methine proton resonance at OH 5.02 (H-7) placed the  Chanter 4: Structure Elucidation of G, Checkøoint inhibitors from Duquetia Odorata  157  alcohol moiety on C-7 ( c 68.2) (Figures 4.9.8 and 4.9.9). The chemical shift of 6 C-7 (öc 68.2) is typical of an alcohol moiety attached to a carbon. Both methine proton resonances at correlations to  oH  oH  4.74 (H-6a) and  H 0  5.02 (H-7) showed HMBC  119.2 (C-lb), which indicated that H-6a was neighboring an  -sp 2 hybridized carbon. All of the above data was consistent with substructure I (Figures 4.9.8 and 4.9.9).  H5bjJJ  7.0  ppm  6.0  5.0  4.0  Figure 4.9.10. COSY correlations for substructure I of 4.33.  HH4  Uu  3.0  ChRntcr 4: Strutiirn EIImidRfinn nf ( Chcknnint inhthitn,s frnm  DIJnIJRt1R  flrkrnt  158  H-i 4  ppm  3.60  3.50  3.40  3.30  3.20  3.10  Figure 4.9.11. HMBC correlations observed for H-13 and H-14 for substructure I of 4.33.  7.87, m  125.4  7.42, m  128.0  7.41, m  a  127.9  b  Figure 4.9.12. (a) 1 H chemical shifts and (b) ‘ C chemical shifts for substructure 3 II.  Chaoter 4: Structure Elucidation of G 2 Checkooint inhibitors from Duauetia Odorata  159  cosy ‘HMBC  I 8  Figure 4.9.13. Key HMBC and COSY correlations observed for substructure II of 4.33.  The aromatic proton resonance at  oH  7.65 (H-8: HMQC to Oc 123.9)  showed COSY correlations to the proton resonance at  H 0  7.41 (H-9: HMQC to 0 c  127.9), which in turn had COSY correlations to the proton resonance at (H-10: HMQC to Oc 128.0).  H 8  7.42  Additional COSY cross-peaks were observed  between the proton resonance at  H 3  7.42 (H-b) and the proton resonance at  H 0  7.87 (H-lI: HMQC to O 125.4) (Figures 4.9.13 and 4.9.14). All of the above data are consistent with four contiguous aromatic methines (C-8 to CII) and the presence of a 1,2 disubstituted benzene ring.  This was also confirmed by  observation of the HMBC data (Figure 4.9.13). The proton resonance at  H 8  7.87  (H-Il) showed HMBC correlations to the quaternary aromatic carbon resonance at 0 c 109.5 (C-la), thus establishing substructure II (Figures 4.9.12 and 4.9.13).  ChaDter 4: Structure Elucidation of G 9 CheckDoint inhibitors from Duauetia Odorata  160  H-9/1O H-il _)  H-8  -  -7.50  ‘r  —  —8.00  ppm  ppm  8.00  7.90  7.80  7.70  7.60  7.50  7.40  7.30  Figure 4.9.14. Expansion of the aromatic region of the COSY spectrum for 4.33.  HMBC  Figure 4.9.15. HMBC correlations linking substructures I and II for 4.33. Both methine proton resonances at  H  4.74 (H-6a), and  H  5.02 (H-7)  showed HMBC correlations to the quaternary aromatic carbon resonance at 8 c 136.9 (C-7a). This indicated that carbon resonances C-7 (öc 68.2) and C-7a (öc  Chapter 4: Structure Elucidation of G 2 Checjcix,int inhibitors from Duquetia Odorata  136.9) were linked.  161  Finally, HMBC correlations between the aromatic methine  proton resonance at  oH  7.65 (H-8) and the oxygenated carbon methine  resonance at 8 68.2 (C-7) confirmed that substructure I was adjacent to substructure II (Figure 4.9.15). 4.02, s  II  6.25, s 6.07,s<  b  a  Figure 4.916. 1 H NMR of substructures Ill (a) and IV (b)  59.4  0 /  11135.9  II  101.7 ‘  a  1451  b  Figure 4.9.17. 13 C NMR of substructures Ill (a) and IV (b)  ‘HMBC  Figure 4.9.18. HMBC correlations for substructure Ill and IV for 4.33.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  162  The chemical shift of the resonance at & 101.7 (C-12) is consistent for that of a methylene-dioxy carbon. The methylene proton resonances at (H-12a: HMQC to 6 c 101.7) and  H 6  H  6.25  6.07 (H-12b: HMQC to öc 101.7) showed  three bond HMBC couplings to the 2 -sp hybrizided carbon resonances at c 145.1 (C-I) and ö 135.9 (C-2) (Figure 4.9.18). All of the above is consistent with substructure III (Figures 4.9.16 and 4.9.17).  The 1 H NMR spectrum of 4.33  lacked a proton resonance that belonged to C-3. HMBC correlations between the proton resonance at  H  4.02 (H-15) and the carbon resonating at öc 138.9 (C-  3) placed the methyl ether on C-3 (substructure IV) (Figures 4.9.16, 4.9.17, and 4.9.18). Closely related aporphine alkaloids with a methyl ether on C-3 display similar 13 C chemical shifts.’ 38 Finally, comparison of the 13 C chemical shifts of 4.33, to that of the related aporphine alkaloid guatterine (4.36), confirmed the constitution of N-methylguatterine (Figure 4.9.19).  I  \  I  124.11  39.0 128.7j  125.7  69.7J.  9 ’ OH . f’ 7  126.9 126.9 4.36  Figure 4.9.19. 13 C chemical shifts for guatterine (4.36), an aporphine alkaloid related to 4.33.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Ductuetia Odorata  163  Observation of the scalar coupling constant between H-6a and H-7 (J 12.0 Hz) in 4.33 revealed a trans-relationship between the two protons.  =  The  scalar coupling of 4.33 was very similar to the scalar coupling present in oliveroline between H-6a and H-7 (J  =  13.0 Hz) which indicated that the relative  configurations of C-6a and C-7 were identical. The CD-spectra (Figure 4.9.20) of both oliveroline and N-methylguatterine were similar. This established that N methylguatterine had S configurations on both stereocentres. 60 40 20 4’ —  0  c.r-  -20  E  2 0  ,‘2 0  300  i  ‘.  \  350  4  ,.  -40 -60 -80 -100• Wavelength  Figure 4.9.20. CD spectrum of N-methylguatterine (dashed line) and oliveroline (solid line). 4.10. Biology of the Alkaloids Isolated from Duguetia odorata Oliveroline was found to be active in the 2 G checkpoint assay at concentrations above 10 i1 M (Figure 4.10.2). There were insufficient amounts of N-methylguatterine to biologically test this molecule, while dehydrodiscretine and  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata  pseudopalmatine were all found to be inactive.  164  To test if a structure activity  relationship was present, the known alkaloids boldine (4.37), apomorphine (4.38), berberine (4.39) and palmatine (4.40) were also tested and no biological activity was found (Figures 4.10.2 and 4.10.3). Flow cytometry analysis indicated that 43 +1- 12% of cells entered mitosis in the presence of 10 i1 M of the known G 2 checkpoint inhibitor isogranulatimide (B, Figure 4.10.1), while 50 jiM of oliveroline was required to induce the same activity (C, Figure 4.10.1).  In the  presence of the drug carrier DMSO, 16% of cells had escaped G 2 arrest (A, Figure 4.10.1). Oliveroline, dehydrodiscretine and pseudopalmatine were found to be moderate inhibitors of cell proliferation with lC ’s of 45, 250, and 75 i1 50 M respectively, but were 2-3 times more potent when cells were irradiated with 6.5 Gy (IC 50 20, 80, 50 j.tM respectively). Oliveroline is an efficacious but moderate inhibitor of the G 2 checkpoint. It was discovered that oliveroline is not an inhibitor of Chkl, which means that this compound is a potential biological tool that can be used to discover new targets in the G 2 checkpoint pathway.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata A  B  165  C  C  I  C  ( U  C  Gi  G2  DNA  a  .io  Pt-H  1023  P1-H  P1-Il  Figure 4.10.1 Flow cytometry analysis of A DMSO, B isogranulatimide and C oliveroline. These graphs were obtained by Dr. Chris Sturgeon of the Roberge laboratory.  60  OHveroine Boldine ADomorphine Pseudopalmatine Dehydrodiscretine —a-— Berberine ‘ Palmatine —0---  50  —  40 30  20  10  0  0.01  0.1  1  10  100  Concentration (LM)  Figure 4.10.2. Concentration dependence of checkpoint inhibition activity of oliveroline and the other alkaloids. The graph was obtained by Dr. Chris Sturgeon of the Roberge laboratory.  Chanter 4: Structure Elucidation of G CheckDoint inhibitors from Duauetia Odorata  ‘166  $CH3 HOj> HO  4.38  3 OCH H C 3 O  4.40  4.39  3 OCH  Figure 4.10.3. Other alkaloids tested in the G 2 checkpoint assay 4.11. General Experimental Methods  All solvents used (except for NMR solvents) were HPLC grade (Fisher) and no further purification was done on them unless for use on the HPLC. Solvents for HPLC were filtered through a 0.45 jtm filter (Osmonics, mc) before use.  Pure alkaloids screened in the G 2 checkpoint assay (boldine (4.37),  apomorphine (4.38), berberine (4.39) and palmatine (4.40)) were purchased from Aldrich. Reversed-phase 1 C 8 silica gel Sep PaksTM (10 g) were purchased from Waters, Inc..  Separations on the HPLC were accomplished using either a  Waters 2487 dual channel detector/system controller (Waters Series 515 pump; chart recorder, 0.25 cm/mm), or a Waters 1500 series HPLC pump and a Waters 2487 dual channel detector. The HPLC column used was a Whatman Partisil 10 ODS-3 Magnum column.  The conditions of the HPLC separation were as  follows: 2.0 mL/min with UV observation at 220 nm. Thin-layer chromatography (TLC) plates were Whatman MKCI8F (reversed phase) and Kieselgel 60F 254 (normal phase). TLC was visualized using either a dip solution of p-anisaldehyde  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  167  (1% p-anisaldehyde, 2% , 4 S 2 H 0 20% acetic acid and 77% ethanol) or under ultraviolet light (254 nm). The 13 C spectra were obtained with the Bruker AM400 spectrometer. 1 H spectra and 2-D data sets were taken with Bruker AMX500, and Bruker AV400 spectrometers.  NMR solvents were purchased from Cambridge Isotope  laboratories and were referenced to solvent peaks for DMSO-d 6 ( c 39.5 ppm 6 and  H 6  2.50 ppm). Low resolution ESI mass spectra were recorded on a Bruker  Esquire LC mass spectrometer. High resolution ESI mass spectra were obtained using a Micromass LCT mass spectrometer. Optical rotations were recorded with a JASCO J-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm micro cell.  The CD spectra were determined using a JASCO J-710  spectropolarimeter with a 1 mm micro cell. 4.12. Isolation procedure of the alkaloids from Duguetia odorata A sample of D. odorata was obtained from Peru in February 1992 by the New York Botanical Gardens as part of a contract with the NCI.  A voucher  specimen is found at the National Herbarium in Washington, D.C. (OCKHOI64). A crude MeOH extract of Duguetia odorata (MacBride 1929) (Annonaceae) was obtained from the NCI repository (N075679-Z/3) of natural products and found to have bioactivity in the G 2 checkpoint assay. The crude extract (4 g) was first suspended in 100 mL of 2 H 0 , and then sequentially partitioned with hexanes (3 X 50 mL), CH CI (3 X 50 mL), EtOAc (3 X 50 mL) and butanol (3 X 50 mL). 2 Four hundred milligrams of the bioactive butanol fraction was subjected to Sephadex  TM  LH-20 size exclusion chromatography eluting with 100% MeOH.  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata  168  This was followed by further purification on a gradient reverse phase Sep PakTM (eluent: H 0 to MeOH) to yield one biologically active fraction. This fraction was 2 subjected to repeated reversed phase HPLC (lnertsil C , 1 8 9.4 X 250 mm, 6:4:0.1 H 0 2 : MeOH: TFA, UV detection at 220 nm ) to yield oliveroline (4.32, 1.7 mg), N methylguatterine  (4.33,  1.3  mg),  dehydrodiscretine  (4.34 3.3  mg),  and  pseudopalmatine (4.35, 2.6 mg).  4.13. Checkpoint inhibitor activity Cells were seeded at 2 x i0 5 cells/dish in 35 mm-diameter dishes and subsequently cultured for 24 h. Cells were then irradiated with 6.5 Gy using a Co source (1.2 Gy/min, Gammacell 220, Atomic Energy Commission of 60 Canada). Sixteen hours later, when 90% of cells were arrested in 2 G , ’° drugs were added with 100 ng/mL nocodazole, and cells were cultured for another 8 h.  Cells were then collected in SAB (phosphate buffered saline with I % fetal bovine serum and 0.1% sodium azide) and fixed in 10 volumes of 70% ethanol at 4°C overnight. Cells were washed in 0.5% Tween-20 in SAB and incubated with a mitosis-specific antibody GF-7 ° for I h, washed twice, and suspended with 2 1:500 diluted Alexa 488-conjugated goat anti-rabbit (Molecular Probes A-I 1029) antibody for 30 mm. Following two more washes, cells were suspended in RNase A (Roche Diagnostics, 500 units/mL in 4 mM sodium citrate buffer, pH 8.4) for 30 mm at 37°C. An equal volume of 50 pg/mL propidium iodide prepared in 4 mM sodium citrate pH 8.4 was added and incubated for an additional 20 mm. Cells were resuspended at a final concentration of IxIO 6 cells/mL in 25 pg/mL propidium iodide solution and stored in the dark overnight. Cells were analyzed  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duquetia Odorata  169  with a Becton-Dickson FACSCalibur, collecting 20,000 events per sample. All data was analyzed using WinMDl freeware. 4.14. Description of the Cell Viability Assay MCF-7 mp53 cells were seeded at 1000 cells/well in 96-well plates, grown overnight, and treated or not treated with compound for 24 h, immediately followed by irradiation or not. DMSO carrier did not exceed 1% final concentration. The drug was removed, and cells were allowed to grow in fresh medium until those not treated with the drug approached confluence, which was typically 4-6 days. Cell proliferation was measured as follows: 25 iL of a 5 mg/mL solution  of  3(4,5-dimethylthiazol-2-yl)-2 ,5-diphenyltetrazolium  bromide  in  phosphate-buffered saline was added to cells in the presence of 100 pL of cell culture medium. After a 2 h incubation at 37°C, 100 tL of 20% sodium dodecyl sulfate dissolved in DMF/H 0 (1:1), pH 4.7, was added, and the absorbance at 2 570 nm was measured after overnight incubation. 4.15. Physical Data of Alkaloids From Duguetia odorata (-)-Oliveroline (4.32): Brown oil. [cUD : -16.9 (c 0.3, MeOH) UV (MeOH) 23 (log  G):  233 (3.91), 271 (3.88), 315 (3.39); CD (MeOH)  314.36);  H NMR (500 MHz, ) 1 6 DMSO : -d  (IH, m, H-9 or H-b), 7.43 (IH, m, H-b  H  8.00 (IH, d, J  max (AE) =  Xmax  232 nm  (-  6.7 Hz, H-lI), 7.43  or H-9), 6.89 (IH, s, H-3), 6.82 (IH, d,  5.90 Hz, 7-OH), 6.24 (IH, s, H-12a or H-12b), 6.06 (IH, s, H-12b or H-12a), 4.92 (IH, dd, J  =  12.4, 5.9 Hz, H-7), 4.62 (IH, d, J  =  12.4 Hz, H-6a), 3.51 (2H, m, H-  5), 3.16 (IH, m, H-4a or H-4b), 2.90 (IH, m, H-4 or H-4a), 2.83 (IH, s, H-13); ‘ C 3  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata  170  NMR (100 MHz, ) 6 DMSO : -d & 148.1 (C, C-2), 143.1 (C, C-I), 136.9 (C, C-7a), 128.4 (CH, C-9 or C-b), 128.1 (C, C-lb), 127.9 (CH, C-9 or C-la), 126.2 (CH, C-lI), 124.2 (CH, C-8), 116.5 (C, C-Ia), 115.1 (C, C-ha), 107.9 (CH, C-3), 101.5 (CH , C-12), 65.6 (CH, C-7), 62.5 (CH, C-6a), 49.5 (CH, C-5), 32.1 (CH 2 , 3 C-13), 21.3 (CH , C-4); LRESIMS m/z 295; HRESIMS m/z 295.12029 [M+H] 2 (calc’d for 3 N0 C 1 H 8 295.12084). 7 (+)-N, N-methylguatterine (4.33): Brown oil. [c]D : 6.17 (C 0.13, MeOH); UV 21 (MeOH) 162.82);  ?max  (log s): 242 (3.48), 279 (3.42); CD (MeOH)  ?.max  (&) 239 nm  (-  H NMR and 13 1 C NMR see Table 4.9.1; LRESIMS m/z295; HRESIMS  m/z 295.12029 [Mt] (calc’d for 3 N0 C 1 H 8 295.12084). 7 Dehydrodiscretine (4.34): Yellow powder. UV (MeOH)  max  (log  242 (3.59), 340 (3.52), 378 (3.15); 1 H NMR (400 MHz, ) 6 DMSO : -d  6):  oH  289 (3.81), 10.05 (IH,  s, OH-3), 9.48 (IH, s, H-8), 8.79 (IH, s, H-13), 7.68 (IH, s, H-9), 7.65 (IH, s, H9), 7.58 (IH, s, H-12), 6.84 (IH, s, H-4), 4.74 (2H, t, J= 6.4 Hz, H-6), 4.07 (3H, s, OMe-lO), 4.00 (3H, s, OMe-Il), 3.93 (3H, s, OMe-2), 3.22 (2H, m, H-5); ‘ C 3 NMR (100 MHz, ) 6 DMSO : -d  0 1 D  157.3 (C, C-b), 152.0 (C, C-Il), 150.0 (C, C-3),  147.7 (C, C-2), 138.7 (C, C-14), 136.6 (C, C-8a), 128.7 (C, C-4a), 121.8 (C, C 12a), 117.6 (C, C-14a), 117.5 (CH, C-13), 114.9 (CH, C-4), 109.2 (CH, C-I), 56.5 , OMe-lO), 56.2 (CH 3 (CH , OMe-hl), 56.0 (CH 3 , OMe-2), 54.6 (CH 3 , C-6), 25.8 2 , C-5); LRESIMS m/z 338; HRESIMS m/z 338.1394 [Mt] (calc’d for 2 (CH N0 C 1 H 3 8 338.1392). 7  Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duguetia Odorata  Pseudopalmatine (4.35): Yellow powder. UV (MeOH)  2max  171  (log e): 287 (4.02),  239 (3.75), 338 (3.65), 373 (3.24); 1 H NMR (400 MHz, ) 6 DMSO : -d  oH  9.52 (1H, s,  H-8), 8.84 (IH, s, H-13), 7.71 (IH, s, H-9), 7.67 (IH, s, H-I), 7.60 (IH, s, H-12), 7.10 (IH, s, H-4), 4.78 (2H, t, J  =  6.1 Hz, H-6), 4.07 (3H, s, 3 OCH I0), 4.00 (3H,  s, OMe-Il), 3.93 (3H, s, OMe-3), 3.86 (3H, s, OMe-2), 3.21 (2H, t, J= 6.1 Hz, H5); 13 C NMR (100 MHz, ) 6 DMSO : -d oc 157.5 (C, C-b), 151.5 (C, C-Il), 151.5 (C, C-2), 151.5 (C, C-3), 145.5 (C, C-H), 138.4 (C, C-14), 136.6 (C, C-8a), 128.6 (C, C-4a), 122.0 (C, C-12a), 117.9 (CH, C-13), 111.3 (C, C-4), 108.6 (CH, C-I), 106.5 (CH, C-9), 56.6 (CH , OMe-lO), 56.3 (CH 3 , OMe-Il), 56.0 (CH 3 , OMe-3), 3 55.8 (CH , OMe-2), 54.7 (CH, C-6), 26.0 (CH 3 , C-5); LRESIMS m/z 353; 2 HRESIMS m/z 352.1541 [M] (calc’d for 4 N0 C 2 H 1 352.1549). 2 4.16. References (I)  Voet D.; Voet J.G. In Biochemisti’y; 3 ed.; Wiley: New Jersey, 2004.  (2)  Molinari M. Cell Proliferation 2000, 33, 261-274.  (3)  Hwang A.; Muschel R.J. Radiation Research 1998, 150, S52-S59.  (4)  Foijer F.; Riele H. te Cell Cycle 2006, 5, 831-836.  (5)  Pientenpol J.A.; Steward Z.A. Toxicology 2002, 181, 475-481.  (6)  Samuel T.; Weber H.O.; Funk JO. Cell Cycle 2002, 1, 162-168.  (7)  Greenblat M.S.; Bennet W.P.; Hollstein B.M.; Harris C.C. Cancer Research 1994, 54, 4855-4878.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  172  (8)  Mclfwrath A.J.; Vasey P.A.; Ross G.M.; Brown R. Cancer Research 1994, 53, 3667-3699.  (9)  SlichenmeyerW.J.; Nelson W.G.; Slebos R.J.; Kastan M.B. Cancer Research 1993, 53, 4164-4168.  (10)  Roberge M.; Berlink R.G.; Xu L.; Anderson H.J.; Lim L.Y.; Curman D.; Stringer C.M.; Friend S.H.; Davies P.; Vincent I.; Haggarty S.J.; Kelly M.T.; Britton R.; Piers E.; Andersen R.J. Cancer Research 1998, 58, 5701-5706.  (11)  Sarkaria J.N.; Tibbets R.S.; Busby E.C.; KennedyA.P.; Hill D.E.; Abrahm R.T. Cancer Research 1998, 58, 4375-4382.  (12)  Blasina A.; Weyer I. van de; Laus M.C.; McGowan C. Current Biology 1999, 9, 1-10.  (13)  Tamaoki T. Methods in Enzymology 1991, 201, 340-347.  (14)  Bunch R.T.; Eastman A. Clinical Cancer Research 1996, 2, 791-797.  (15)  Busby E.G.; Leistritz D.F.; Abraham R.T.; Karnitz L.M.; Sarkaria J.N. Cancer Research 2000, 60, 2108-2112.  (16)  Wilson W.H.; Sorbara L.; Figg W.D.; Mont E.K.; Sausville E.; Warren K.E.; Balis F.M.; Bauer K.; Raffeld M.; Senderowicz A.M.; Monks A. Clinical Cancer Research 2000, 6, 415-421.  (17)  Jackson J.R.; Gilmartin A.; lmburgia C.; Winkler J.D.; Marshall L.A.; Roshak A. Cancer Research 2000, 60, 566-572.  (18)  Hénon H.; Messaoudi S.; Anizon F.; Aboab B.; Kucharczyk N.; Léonce S.; Golsteyn R.M.; Pfeiffer B.; Prudhomme M. European Journal of Pharmacology 2007, 554, 106-112.  (19)  Curman D.; Cinel B.; Williams D.E.; Rundle N.; BlockW.D.; GoodarzA.A.; Hutchins J.R.; Clarke P.R.; Zhou B.B.; Lees-Miller S.P.; Andersen R.J.; Roberge M. Journal of Biological Chemistiy 2001, 276, 17914-17919.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Ducwetia Odorata  173  (20)  Rundle N.T.; Xu L.; Andersen R.J.; Roberge M. Journal of Biological Chemistiy 2001, 276, 48231-48326.  (21)  Anderson H.J.; Andersen R.J.; Roberge M. Progress in Cell Cycle Research 2003, 5, 423-430.  (22)  Bermejo A.; Figadere B.; Zafra-Polo M.C.; Barrachina I.; Estornell E.; Cortes D. Natural Product Reports 2005, 22, 269-303.  (23)  Carollo C.A.; Siqueira J.M. de; Garcez W.S.; Diniz R.; Fernandes N.G. Journal of Natural Products 2006, 69.  (24)  Matos M.F.C.; Leite L.I.S.P.; Brustolim D.; Siqueira J.M. de; Carollo C.A.; HeHmann A.R.; Pereira N.F.G.; Silva D.B. da Fitoterapia 2006, 77, 227— 229.  (25)  Tempone A.G.; Borborema S.E. Treiger; Jr. H.F. de Andrade; Gualda N.C. de Amorim; Yogi A.; Carvaiho C. Salerno; Bachiega D.; Lupo F.N.; Bonotto S.V.; Fischer D.C.H. Phytomedicine 2005, 12, 382-390.  (26)  Ramasamizafy S.; Hocquemiller R.; Cave A. Journal of Natural Products 1987, 50, 674-679.  (27)  Gottlieb O.R.; Magalhaes A.F.; Maia J.G.S.; Marsioli A.J. Phytochemistiy 1978, 17, 837-838.  (28)  Costa E.V.; Piheiro M.L.B.; Xavier C.M.; Silva J.F.R.; Amaral A.C.F.; Souza A.D.L; Barison A.; Campos F.R.; Ferreira A.G.; Machado G.M.C.; Leon L.L.P. Journal of Natural Products 2006, 69, 292-294.  (29)  Rahman M.M.; Lopa S.S.; Sadik G.; Rashid H.O.; Islam R.; Khondkar P.; Alam A.H.M.K.; Rashid M.A. Fitoterapia 2005, 76, 758-761.  (30)  Montanha J.A.; Amoros M.; Boustie J.; Girre L. Plant Medica 1995, 61, 419-424.  (31)  Perez E.; Saez J.; Blair S.; Franck X.; Figadere B. Letters in Organic Chemistiy 2004, 1, 102-104.  Chapter 4: Structure Elucidation of G 2 Checkpoint inhibitors from Duguetia Odorata  174  (32)  Debourges D.; Roblot F.; Hocquemiller R.; Cave A. Journal of Natural Products 1987, 50, 852-859.  (33)  Hamonniere M.; Leboeuf M.; Cave A. Phytochemistry 1977, 16, 10291034.  (34)  Shamma M.; Stephens R.L.; Wenkert E.; Leboeuf M.; Cave A.J. Journal of Natural Products 1979, 42, 437-439.  (35)  Duah F.K.; Owusu P.D.; Slatkin D.J.; Schiff P.L. Phytochemist,y 1983, 22, 321-322.  (36)  Chen C.H.; Chen T.M.; Lee. C.J. Journal of Pharmaceutical Sciences 1980, 69, 1061-1065.  (37)  Patra A.; Montgomery C.T.; Freyer A.J.; Guinaudeau H.; Shamma M.; Tantisewie B.; Pharidai K. Phytochemistry 1987, 26, 547-549.  (38)  Marsaioli A.J.; Reis F.A.M.; Maglhaes A.F.; Ruveda E.A.; Kuck A.M. Phytochemist,y 1979, 18.  (39)  Jackman L.M.; Trewella J.C.; Moniot J.L.; Shamma M.; Stephens R.L.; Wenkert E. Journal of Natural Products 1979, 42, 437-449.  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  175  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 5.1. Preview of Chapter 5 Sex hormone-binding globulin (SHBG) is a protein that is vital in the transport of unbound steroids such as testosterone, estradiol, and 5c-dihydrotestoterone. Furthermore, SHBG also plays a role in regulating the concentration of these hormones in the blood. 1 Elevated SHBG levels are present in various disorders including anorexia, osteoporosis and hypogonadal men. 2  Many of these  pathological conditions are associated with a lower plasma concentration of hormones. Ligands that bind to SHBG can release steroids into the blood, so these Hgands can be viewed as potential drug candid 3 ates. This chapter will deal with the isolation and structure elucidation of ligands for SHBG from the marine sponge Myrmekioderma granulatum. 5.2. Biology of the Sex-Hormone Binding Globulin Protein The sex-hormone binding gobulin (SHBG) is a homodimeric glycoprotein primarily synthesized in the liver. 1  SHBG found in the testes is commonly  referred to as the androgen binding protein (ABP). 2 Monomeric SHBG contains 373 amino acid residues and 3 carbohydrate side chains mainly composed of sialic acid and N-glucosami 4 ne.  SHBG strongly binds to planar steroids that  have a 17 hydroxyl group through Van der Waals forces and polar attractions.  Chapter 5: Isolation of I,gands for the Human Sex Hormone Bindincji Globulin  176  Several examples of steroids that bind to SHBG include estradiol, testosterone, and 5cz-dihydrot 5 estosterone. SHBG interacts with hormones and has a key role in regulating their distribution and biological function.  It has been estimated that less than two  percent of steroids circulate freely in the blood, with the remainder sequestered in SHBG 4. Free steroids diffuse into the cells altering cellular function, so a role of SHBG is to regulate the concentration of unbound hormone in the plasma. SHBG may also directly transport steroids to the plasma membranes of some tissues to induce intracellular signaling pathwa 2 ys. The ABP synthesized in the testes is subsequently taken to the epididymis where it is thought to aid in transporting androgens vital for sperm maturation. 5 Various pathological conditions have higher levels of SHBG which results in a lower concentration of free steroids in the blood. Hypogonadal males have higher concentrations of SHBG which leads to decreased plasma testosterone levels. This results in testicular failure and defective gonadotropin secreti 6 on. Other conditions with elevated SHBG levels include anorexia nervosa where low estradiol concentrations prevent women from ovulating. 7  Lower plasma  concentrations of estradiol due to increased SHBG levels have also been associated with an increased rate of bone loss and osteop 8 orosis. It is evident that ligands capable of binding to SHBG could release bound steroids into the bloodstream. Therefore, SHBG could represent an attractive drug target for conditions where a hormone insufficiency is present. The first potent ligand discovered was (-)-3,4-divanillyltetrahydrofuran  (5•j),3  which has an  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindino Globulin  177  50 of 2.6 jiM. Other ligands developed include compounds 5.2 and 5.3, which IC have 50 1C of 13.6 and I jiM, ’ ’s 9 respec 1 0 tively. H HO  5.1  OH  5.2  5.3  Figure 5.2.1. Several examples of ligands that bind to SHBG. 5.3. Compounds Isolated from the genus Myrmekioderma Sponges in the genus Myrmekioderma (family Heteroxyidae) are distributed in the shallow oceans of the Indo-P 11 acific.  In 1992, the first  secondary metabolites from a Myrmekioderma sp. were discovered when Faitorusso and co-workers isolated four oxygenated linear diterpenes from the sponge M. styx. 12 All four diterpenoids were active in the brine shrimp assay with styxenol (5.4) and 5.5 showing the most cytotoxicity (LC = 154 jiglmL and 3 50 .1 tg/mL respectively).  12  Figure 5.3.1. Linear diterpenes from M. styx. The cyanthiwigin family of diterpenoids play a predominant role in the chemistry of the genus Myrmekioderma. Cyanthiwigin C (5.6) was the first of this  Chaoter 5: Isolation of liaands for the Human Sex Hormone Bindinci Globulin  family  of  5,6,7  tricarbocyclic  diterpenoids  to  be  isolated  178  from  the  1 Myrm ekiode 3 rma. In 2002, Peng et a!. reported the isolation of twenty-seven previously unreported cyanthiwigins from M. styx. 14 The most biologically active cyanthiwigins isolated from this study include the cytotoxins cyanthiwigin D (5.7) and cyanthiwigin F (5.8), which had 1C ’s of 5 .igImL and 3 tgImL, respectively, 50 against human primary tumor cells. 14 In 2003, Hamann and co-workers isolated several unreported diterpenoids of the cyanthiwigin class from M. styx.’ 5 Cyanthiwigin AC (5.9) was found to contain a six-membered Spiro ring rather than a seven-membered ring, while cyanthiwigin AD (5.10) was found to have a 5,6,6 tricarbocyclic structure rather than the 5,6,7 tricarbocyclic ring formation. No biological screening was done on these compounds due to the very small quantities that were isolated from the sponge 15. H0  HQ  OH  5.7  J 5.9  5.10  Figure 5.3.2. Cyanthiwigins isolated from Myrmekioderma sp.. Several bisabolane sesquiterpenes have been isolated from sponges of the genus Myrmekioderma.  The bisabolanes (÷)-curcuphenol (5.11) and (+)-  curcudiol (5.12) were isolated from M. dendyl, and were found to have antifouling activity against the cypris larvae of the barnacle Balanus amphitrite with 50 EC of ’s  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  2.5 and 2.8 pg/mL, respectively. 16  179  Compounds (5.13), (5.14) and (5.15) are  three biologically inactive bisabolane sesquiterpenes that were first isolated from M. dendyi. 17  Styxone A (5.16) and B (5.17) are other biologically inactive  sesquiterpenes that were first discovered from M. styx.’ 8  5.9  5.10  Figure 5.3.3. Sesquiterpenoids isolated from Myrmekioderma sp.. Other biologically active natural products that have been discovered from Myremekioderma are myrmekiosides A (5.18) and B (5.19)19 These glycolipids have been found to alter the tumor cell morphology of H-Ras transformed NIH2T3 fibroblasts at 5 tgImL.  Furthermore, myrmekioside A has also been  found to prevent NIH2T3 cells from entering the S-phase of the cell cycle. Compounds 5.20 and 5.21 are similar glycolipids, and were isolated from Myrmekioderma by Letourneux et at. 17  Chapter 5: Isolation of liciands for the Human Sex Hormone Bindir&q Globulin  180  OH Xylose Glucose 1  “OH 5.18: 16 RC 3 H 3 5.19:  6 1 H 4  8  Xylose N-Acetyl glucosamine  “OH  NHAc  5.20: Q R=— C 2 CH H H 16 C(CH 2 R=H 5.21: O H 17  Figure 5.3.4. Glycolipids isolated from Myrmekioderma sp.. 5.4. Isolation of bisabolane sesquiterpenes and myrmekioside C A MeOH extract of the Myrmekioderma granulatum collected in Indonesia was subjected to flash reversed-phase column chromatography to yield two biologically active fractions. The more active fraction was subjected to reversedphase HPLC to obtain 5.22,20 abolene (5.23 mg) as a diastereotopic ° 2 mixtur e, (+)-curcudiol  (5.24),21  and abolenone (5.25 mg). Biological studies revealed (+)-  curcudiol (5.24) to be a ligand of SHBG. The less active fraction was purified using reversed-phase HPLC to yield (+)-curcuphenol (5.26),21 and myrmekioside C (5.27).  The structures of (+)-curcudiol  curcuphenol  (5.26)21  (5.24),21  abolene (5.23),20 and (+)-  were confirmed by comparing the optical rotation, NMR and  the MS data to the literature values. The optical rotation of sesquiterpenoid 5.22  Chaoter 5: Isolation of Ikiands for the Human Sex Hormone Bindinci Globulin  181  is opposite in sign to the literature values, thus a new enantiomer was ° 2 isolate d. For full experimental details, see Section 5.8.  5.23  5.22  5.24  5.25  5.26  L  OH  OH  ,-,  GlucoselL HO” “r HO  Y  0 (‘H  OH Xylose “OH  oJ  HO”  jGlucose2  ( OH  ‘OH  5.27  Figure 5.4.1. Compounds isolated from Myrmekioderma styx. 5.5. Structure Elucidation of Abolenone  15’  4  5.25  Figure 5.5.1: Abolenone. Abolenone (5.25, figure 5.5.1) was isolated as an optically active yellow oil that gave a [M+Na] ion at m/z 255.1360 in the HRESI-TOF, corresponding to a molecular formula of 2 0 1 C 2 H 5 and requiring six degrees of unsaturation. I D and 0 2D NMR experiments were run in both DMSO 6 -d and in C . D 6  The best  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  182  dispersion in the 1 H NMR spectrum was found in 6 C (Figure 5.5.3). The proton D NMR revealed three aromatic proton resonances exchangeable proton  H 8 (  (oH  7.01, 6.72,  ) 6 . 6 OH 7  an  6.63), two olefinic protons (OH 5.36 and 5.16), and three  methyl proton resonances (OH 2.14 and 1.68). Analysis of the 13 C NMR spectrum (Figure 5.5.4), and the HMQC data (Figure 5.5.5) revealed four methines (Oc 125.5, 120.6, 116.8 and 30.4), three methylenes ( c 124.5, 33.9 and 31.9), three methyls ( 8 c 20.3, 18.7, 17.5), and 8 four quaternary carbons ( c 202.4, 143.8, 136.4, 128.6). After using the HMQC 8 data to assign proton resonances to their respective carbon atoms (Table 5.5.1), it was possible to deduce three substructures (I, II, N, Figure 5.5.2) from the HMBC (Figure 5.5.6) and COSY data (Figure 5.5.7).  13  Figure 5.5.2. Substructures of abolenone as deduced from the HMBC and the COSY data.  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  I 83  0 r  0 C..’  0 C.,  0  0 U, .0  :wo 0 U,  C 1-.  Figure 5.5.3. 1 H NMR spectrum of abolenone (5.25) at 600 MHz in C . D 6  0)  C,  N  -  01 C  01  01  CD  0  D  0  0  3  -I  a  CD  C,)  ppm  200  150  I  ii  100  50  ii  M  uL  0 -  C)  0.  txl  0 CD  -‘  0  >c  0)  CD  CD  0  CD  0  J LLLJLJtIL  0  HayHb  z  OH  C)  -  C,’  1  ChaDter 5: Isolation of liciands for the Human Sex Hormone Bindina Globulin  ppm  Figure 5.5.5. HMQC spectrum of abolenone (5.25) at 600 MHz in C . D 6  185  Chapter 5: Isolation of liqands for the Human Sex Hormone Bindinci Globulin  186  HaHb  00  .  ,  p  •  ?  8 -50  -100 ‘0  .0,  I  I  I  I I  0  •0  a 0’  150  •.  ppm  7.0  6,0  [-200 ppm 5.0  4.0  3.0  2.0  1.0  Figure 5.5.6. HMBC spectrum of abolenone (5.25) at 600 MHz in C . D 6  ChaDter 5: Isolation of Iiaands for the Human Sex Hormone Bindina Globulin  HaHb  ppm  Figure 5.5.7. COSY spectrum of abolenone (5.25) at 600 MHz in C . D 6  187  Chapter 5: Isolation of I,gands for the Human Sex Hormone Binding Globulin  Position 1 2 3 4 5 6 7 8ab 8bb 9aC  9bC  10 II I2ad I2bd 13 14 15 1-OH  188  Table 5.5.1. 1 D and 2D NMR data of abolenone (5.25).8 6 HMBC H (J in Hz) 6 COSY 154.9 116.8 6.67, s C-I, C-6, C-4, C-15 H-15 136.4 120.6 6.72, d, (8.0) C-2, C-4, C-6, C-15 H-5 125.5 7.01,d,(8.0) C-1,C-3,C-7 H-4 128.6 30.4 2.95, m C-I, C-5, C-6, C-8, C-9, H-8a, H-8b, H-14 C-14 31.9 1.92, m C-6, C-7, C-9, C-10, C- H-7, H-8b, H-9a, 14 H-9b 1.43, m C-6, C-7, C-9, C-10, C- H-7, H-8a, H-9a, 14 H-9b 33.9 2.34, m C-7, C-8, C-10 H8a, H8b, H9b 2.14, m C-7, C-8, C-b H8a, H8b, H9a 202.4 143.8 124.6 5.36, s C-I0, C-13 H-12b, H-13 5.16, s C-10, C-13 H-12a, H-I3 17.5 1.68, s C-b, C-Il, C-12 H-12a, H-12b 18.7 1.07, d, (6.8) C-6, C-7, C-8 H-7 20.4 2.14, s C-2, C-3, C-4 H-2 6.63, s C-I, C-2, C-6  a.  1 and 13 H C chemical shifts (ppm) are referenced to the C D 6 öc 128 ppm) b: H-8a and H-8b are interchangeable signals C: H-9a and H-9b are interchangeable signals d: H-12a and H-12b are interchangeable signals  (H  7.15 ppm and  ChaDter 5: Isolation of Ikiands for the Human Sex Hormone Bindina Globulin  Ha  Hb  189  124.5  5.36, s  202.3  0 b  a  Figure 5.5.8. (a) H and 13 1 (b) chemical shifts of substructure I of abolenone C (5.24).  HMBC 13  Figure 5.5.9. Key HMBC correlations of substructure I of abolenone (5.25). Both proton resonances at  H 3  5.36 (H-12a) and  H  5.16 (H-12b) showed  identical HMQC correlations to the carbon resonance at 6 c 124.5 (C-12), thus establishing the presence of an olefenic methylene. The methyl singlet proton resonance at  oH  1.68 (H-13: HMQC to & 17.5) showed HMBC correlations to the  carbon resonance at Oc 143.8 (C-lI), which established that Me-13 was connected to C-Il (Figures 5.5.9 and 5.5.10). Further HMBC cross-peaks were observed between the proton resonance at  oH  1.68 (H-I 3) and the carbon  resonance at Oc 124.5 (C-12) (Figures 5.5.9 and 5.5.10). The methylene proton resonances at  oH  5.36 (H-12a) and  oH  5.16 (H-12b) showed HMBC correlations to  the methyl carbon resonance at Oc 17.5 (C-I3), which established that a methyl group was adjacent to an olefenic methylene. Finally, the proton resonances at OH 3 . 5 6 (H-12a), 0 H 5.16 (H-12b) and  H 0  1.68 (H-13) all showed HMBC  _________  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulln  190  correlations to the carbonyl resonance at c 202.4 (C-b). This established the presence of an ct, n-unsaturated ketone shown as substructure I (Figures 5.5.8 and 5.5.9). H-I 5  -  C-I2  -  Th c_Il— 1-150  C-ID  — —  iz—  ppm 1.700  1.650  Figure 5.5.10. HMBC correlations for H-15 of substructure of abolenone (5.25). 18.7  1.07, d (6.8 Hz) 2.34, m 2.14, m  2.95, m  1.92,m 1.43, m  a  304  31.9  b  Figure 5.5.11. (a) H and (b) 1 C chemical shifts of substructure II of abolenone 13 (5.25).  Chanter 5: Isolation of Iiaands for the Human Sex Hormone Bindina Globulin  191  cosy HMBC Figure 5.5.12. Key HMBC correlations of substructure II of abolenone (5.25). The methyl proton resonance at  oH  1.07 (H-14: HMQC to öc 18.7) showed  a COSY correlation to the methine proton resonance at  H 0  2.95 (H-7: HMQC to Oc  30.3) which linked C-14 (Oc 18.7) to C-7 (Oc 30.3). Additional COSY correlations were present between the proton resonance at  OH  2.95 (H-7) and the two  methylene proton resonances at 0 H 1.92 (H-8a: HMQC to 8 c 31.9) and  Oi-  1.43 (H  8b: HMQC to Oc 31.9), which indicated the connectivity between C-7 (Oc 30.3) and C-8 (Oc 31.9). Finally, the methylene C-8 (Oc 31.9) was linked to methylene C-9 (Oc 33.9) due to COSY correlations between H-8a/H-8b  (OH  1.92 and 1.43,  respectively) and H-9a/H-9b (OH 2.34 and 2.14, respectively) (Figure 5.5.13). These connections were supported by the HMBC data (Figure 5.5.12), and the above data is consistent with substructure II (Figure 5.5.11 and 5.5.12).  Chaoter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  192  H-14 H-7  H-8a  Figure 5.5.13. COSY expansion for substructure II of abolenone (5.25).  OH 154.9  6.67,s  116.8 128.6 136.4 7.01, d (8.0 Hz)  2.14,  S  6.72, d (8.0 Hz)  a  .—  20.4  125.5  120.6  b  Figure 55.14. (a) H and (b) 1 C chemical shifts of substructure III of abolenone 13 (5.25).  ciipter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  193  cosy ‘HMBC Figure 5.5.15. Key HMBC correlations of substructure Ill of abolenone (5.25).  The proton resonance at E 6.63 (1-OH) did not show any HMQC correlations and was assigned as an exchangeable alcohol proton.  HMBC  correlations were present between the exchangeable proton resonance at 6.63 (I-OH) and the aromatic carbon resonance at öc 154.9 (C-I).  H 6  This  established that C-I (ö 154.9) contained an alcohol moiety and its chemical shift was typical for an oxygenated aromatic carbon. Additional HMBC correlations were present between the proton resonance at  H 6  6.63 (1-OH) and the  quaternary aromatic carbon resonance at öc 128.6 (C-6), which established the connectivity between C-I (c 154.9) and C-6 ( c 128.6) (Figure 5.5.17). HMBC 6 cross-peaks were also observed between the proton resonance at and the aromatic methine carbon at  o  116.9 (C-2).  H  6.63 (I-OH)  The HMBC correlations  between the aromatic methine proton at öH6.67 (H-2: HMQC to ) and the 9 . 6 cIl oxygenated carbon resonance at o 154.9 (C-I) determined the linkage between C-I ( c 154.9) and C-2 (öc 116.9) (Figures 5.5.15 and 5.5.17). A methyl proton 6 resonance at  2.14 (H-15: HMQC to c 20.3) had an HMBC correlation to the  aromatic carbon resonance at 6 c 116.9 (C-2). between  oH  Cross-peaks in the HMBC  6.67 (H-2) and Oc 20.3 (C-15) established that Me-15 was ortho to an  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  aromatic methine proton (H-2).  An aromatic methine proton at  194  oH  6.72 (H-4:  HMQC to 8 120.6) contained HMBC cross-peaks to the methyl carbon resonance at Oc 20.3 (C-I 5). Additonal HMBC couplings between the proton resonance at OH 2.14 (H-15) and the carbon resonance at Oc 120.6 (C-4) established that Me-15 was ortho to an additional aromatic methine proton (H-4) (Figure 5.5.17).  The linkage between C-4 (Oc 120.6) and C-5 (Oc 125.5) was  established from observation of a COSY correlation between the proton resonance at  H 8  6.72 (H-4), and the aromatic methine proton resonance at OH  7.01 (H-5: HMQC to O 125.5) (Figure 5.5.16).  Finally, a three bond HMBC  correlation was observed between the proton resonance at  oH  7.01 (H-5) and the  carbon resonance at Oc 154.9 (C-I), which showed that C-5 (Oc 125.5) and C-6 (Oc 128.6) were connected.  The above data is consistent with a 1, 2, 4  trisubstituted aromatic ring shown as substructure Ill (Figures 5.5.14 and 5.5.15).  Chapter 5: Isolation of liqands for the Human Sex Hormone Bindina Globulin  H-5  H6  A 1  I  195  11  -6.50  cc:’  -  1  7.00  ppm I  ppm  7.10  7.00  6.90  6.80  6.70  I  6.60  Figure 5.5.16. COSY expansion for substructure Ill of abolenone (5.25).  I  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  196  H-2 1-OH  H-5  AA C-i 5—i’—-—— C-7—_ -50  C-2  -100  C-3— C-i  -150  —  ppm ppm  7.00  6.90  6.80  6.70  6.60  6.50  Figure 5.5.17. HMBC expansion for substructure Ill of abolenone (5.25).  14  12  HMBC  1  2  15  13  5  0  4  Figure 5.5.18. Key HMBC correlations of substructure of abolenone (5.25). The methylene protons H-8a/H-8b and H-9a/H-9b all showed HMBC correlations to a carbonyl resonance at öc 202.4 (C-b), which established the connectivity between C-9 ( c 33.9) and C-b 6 chemical shifts of H-9a  (oH  (ö 202.4).  Observation of the  2.32) and H-9b (OH 2.14) is consistent with this  Chaoter 5: Isolation of Iiaands for the Human Sex Hormone Bindina Globulin  197  assignment, therefore, C-9 ( c 33.9) is linked to substructure I (Figure 5.5.18). 6 The aromatic methine proton H-5  (oH  7.01) had HMBC correlations to the methine  carbon C-7 (Oc 30.4). Additional HMBC correlations were observed between the methine proton resonance H-7 (OH 2.95, m) and the aromatic carbon resonances C-I (Oc 154.9), C-5 (Oc 125.5) and C-6 (Oc 128.6).  This established the link  between the trisubstituted benzene (substructure III) to the alkyl chain at C-7, which was further supported by key HMBC correlations (Figure 5.5.18). The CD spectra of both (+)-curcuphenol (5.26) and (+)-abolenone (5.25) were similar, which established that both molecules had identical 7S configurations (Figure 5.5.19).  CD Spectrum of Abolenone and (+)-Curcuphenol 5.00 0.00 309.00  -5.00  329.00  .-1o.0o -15.00  E °-20.00 -25.00 -30.00 -35.00  Wavelength (nm) Figure 5.5.19. CD spectrum of curcuphenol (dashed line) and abolene (solid line).  (hntRr 15 LcnItinn nf Iicinds fnr th Humcin Sx Hormnnn Rindino (InhuIin  I 9A  5.6. Structure Elucidation of Myrmekioside C peracetate Myrmekioside C (5.27, figure 5.4.1) was isolated as an optically active yellow oil that gave a [M+HJ ion at m/z 815.4640 in the HRESI-TOF mass spectrum, corresponding to a molecular formula of 1 0 3 C 7 H 2 and requiring three 8 degrees of unsaturation. The LRESIMS in MeOH gave a molecular ion peak at  m/z 839.9, while the same experiment using MeOD afforded a molecular ion peak at m/z 850.8, thus establishing eleven exchangeable protons in the molecule.  The H 1 NMR signals were poorly dispersed in DMSO 6 -d (Figure  5.6.2), so acetylation of myrmekioside C (for acetylation procedure see section 5.9) was performed to yield myrmekioside C peracetate (5.28, Figure 5.6.1). The H NMR resonances of 5.28 were well dispersed in C 1 D and therefore all the 6 NMR data was obtained with this solvent. Glycerol (Gly)  OAc 10  (Xyl)  Glucose 1 (Glul)  Glucose 2 (G1u2)  5.28  Figure 5.6.1: Myrmekioside C peracetate (5.28). Myrmekioside C peracetate (5.28, figure 5.6.1) was obtained as an optically active yellow oil that gave a [M÷H] ion at m/z 1301.774 in the HRESI TOF mass spectrum, corresponding to a molecular formula of 2 0 6 C 9 H 0 and 4 9 requiring 14 degrees of unsaturation. The increase in mass was suitable for the  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  199  addition of eleven acetate groups, consistent with the eleven exchangeable proton resonances noted for myremkioside C. The 1 H NMR spectrum (Figure 5.6.3) contained signals from  H 6  0.9 and 1.8 consistent with the presence of  aliphatic methylenes. A series of acetate methyl proton resonances were found between öHl.6and 2.0. H 6  The 1 H NMR spectrum also revealed peaks from  3.3 to 5.5 which is suitable for protons attached to oxygenated sp 3 carbons.  Observation of the 13 C (Figure 5.6.4), DEPT (Figure 5.6.5) and the HMQC (Figure 5.5.6) spectra confirmed the presence of 11 carbonyls (öc169-I7O), three anomeric carbons ( c 101.4, 101.5, 100.9), twenty carbons attached to oxygen 6 atoms (öc 61.7-79.0) and a series of acetate methyls and aliphatic methylenes  (  20.1-30.5). After the assignment of the proton resonances was done using the HMQC data (Table 5.6.1), five substructures of myrmekioside C (Figure 5.6.9) were deduced using the HMBC and COSY spectra (Figures 5.6.7 and 5.6.8). AcOd0  ACOL0  AcOQAc0  iv  iii  V  Figure 5.6.2. Five substructures of myrmekioside C peracetate (5.28).  Chapter 5: Isolation of I,gands for the Human Sex Hormone Bindinci Globulin  200  C  1’-  0  Li  Li  Figure 5.6.3. H 1 NMR spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C . D 6  Chanter 5: Isolation of licands for the Human Sex Hormone Bindina Globulin  201  0  C 0  Figure 5.6.4. 13 C NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C . 6 D 5  Chanter 5: Isolation of lioands for the Human Sex Hormone Rindinci Globulin  2fl2  -  C  I E  & Figure 5.6.5. DEPT NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C . 5 D 6  Chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin  LJ  AcO”  i  .  .  203  I  —-...  50  a  —100  ppm I,,.’I,’  ppm  5.0  4.0  I  3.0  2.0  1.0  Figure 5.6.6. HMQC spectrum of myrmekioside C peracetate (5.28) at 600 MHz . D 6 in C  chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin  204  AcO”  on  U  -50 a.. 4D;,4 I  I  •  0 I  ,*  •  Iae, ••*  *  •i  ..  II  ‘Ic  .  *  I  .8  -100  -150 .0.0  5.0  4.0  3.0  2.0  1.0  Figure 5.6.7. HMBC spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C . D 6  Chapter 5: Isolation of liqands for the Human Sex Hormone Bindinci Globulin  I  ppm  7.0  I  6.0  I  IIli  5.0  4.0  I  II  3.0  I  II  2.0  205  I  II  1.0  II  I  0.0  Figure 5.6.8. COSY spectrum of myrmekioside C peracetate (5.28) at 600 MHz . D 6 in C  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  206  Table 5.6.1. ID and 2D NMR data of myrmekioside C peracetate. a Position HMBC H (J in Hz) 6 O’AIkyI Chain I 72.1 3.53, m GIy-C-1, C-2, C-3 2 30.5 1.70, m C-I, C-3, C-4 3 26.8 1.49, m 4-15 20.00.80-1.80 30.5, (24 H total) 12C total 16 26.3 1.19, m C-14, C-15 17 29.0 1.46, m C-15, C-16, C-18 64.4 4.00, t, (6.7) C-16, C-17 Glycerol (Gly) Gly- I 70.4 3.70, d, (5.4) Gly-C-2, Gly-C-3, C-I Gly2d 78.0 4.06, m Glu-C -1, Gly-C -1 Gly-3a 69.4 4.10, dd, Xyl-C -1, GIy-C -1 (10.17, 4.2) Gly3bc 3.73, m Xyl-C -1 Xylose (XyI) Xyl-1 101.5 4.42, d, (7.3) Gly-C-3, Xyl-C -5 Xyl-2 71.5 5.30, t, (8.3) XyI-C -1, Xyl-C -2 Xyl-3 72.2 5.43, t, (8.9) Xyl-C -2, Xy-C 1-4, Xy-C -5 Xyl-4 69.5 5.10, m Xyl-C-3, Xyl-C -5 Xyl-5a b, e  62.4  Xyl5be  3.10, t, (10.1) 3.97, m  Xyl-C -1, Xy-C -3, Xyl-C -4 Xyl-C -1, XyI-C -3, XyI-C 4  -  Glucosel (Glul) GluI-1  101.4  4.53, d, (8.0)  Glul-2  78.9  3.68, t, (8.9)  Gly-C -2, GIul-C -3, GIul C-5 GluI-C -1, GIul-C -3  GLu1-3  74.7  5.37, t, (9.5)  GIuI-C -2, GIul-C -4  Glul-4  68.8  5.20, t, (9.9)  71.8  3.29, d, (10.2) 4.05, m  Glul-C GIui-C GluI-C Glul-C GIul-C  4.30, m  GIul-C -4  Glul-5  61.7 h  -3, GIul-C -5, -6 -1, Glul-C -3, -4 -4  COSY H-2 H-i, H-3 H-2  H-16, H-18 H-17 Gly-H-2 Gly-H-2, GIy-H-3b Gly-H-2, Gly-H-3b GIy-H-2, Gly-H -3a Xyl-H-2 Xyl-H-1, XyI-H -3 XyI-2, Xyl-4 Xyl-H -3, XyI-H -5a, Xyl-H -5b Xyl-H -4, XyI-H -5b Xy-H 1-4, Xyl-H -5a  Glul-H -2 Glul-H -1, GIul-H 3 Glul-H -2, GIul-H 4 Glul-H -3, GIul-H 5 Glul-H -4, Glul-H 6a, Glui-H -6b Glul-H -5, GIul-H 6b GIul-H -5, GIul-H 6a  -  -  -  -  -  -  Chapter 5: Isolation of ligands for the Human Sex Hormone Bindinci Globulin  Position Glucose 2 (Glu2) G1u2-1 G1u2-2  100.9 72.6  4.80, d, (8.0) 5.25, t, (9.1)  G1u2-3  73.6  5.47, t, (9.2)  (J in Hz)  Glu2-4  68.6  G1u2-5  72.2  GIu26ah Glu2-6b’  62.0  5.37, t, (10.2) 3.59, d, (10.1) 4.29, m 4.47, dd, (5.1, 12.4)  HMBC  Glul-C G1u2-C Glu2-C G1u2-C  COSY -2, G1u2-C -2 -1, Glu2-C -3, -4 -2, G1u2-C -4  G1u2-C -3, G1u2-C -6 Glu2-C-1, G1u2-C -4 G1u2-C -4, G1u2-C -5 G1u2-C -4, G1u2-C -5  1 and 13 H C chemical shifts (ppm) are referenced to the C D 6 c 128 ppm). 0 b: H-18 and Xyl-5a are overlapping signals C: Gly-1 and Gly-3b are overlapping signals d: G1y2 and Glul-6a are overlapping signals e. Xyl-5a and Xyl-5b are interchangeable signals Glul-3 and G1u2-4 are overlapping signals Glul-6a and Glul-6b are interchangeable signals Ii: Glul-6b and G1u2-6a are overlapping signals ‘:G1u2-6a and G1u2-6b are interchangeable signals  G1u2-H -2 G1u2-H -1, G1u2-H 3 G1u2-H -2, G1u2-H 4 G1u2-3, Glu2-5 G1u2-H -4, G1u2-H 6a, G1u2-H -6b G1u2-H -5, G1u2-H 6b G1u2-H-5, G1u2-H6a  a.  ‘:  207  (oH  7.15 ppm and  -  -  -  Chanter 5: Isolation of liaands for the Human Sex Hormone Rindina Globulin  208  5.10, m  AcO  5.43, t (8.9 Hz)  a  4.42, d (6.7 Hz)  b  Figure 5.6.9. (a) H chemical shifts and coupling constants and (b) 13 1 C chemical shifts of substructure I of myrmekioside C peracetate (5.28).  ‘HMBC OLø1d.  Figure 5.6.10. Key HMBC correlations of substructure I of myrmekioside C peracetate (5.28). The chemical shift of Xyl-C-1 ( c 101.5) is typical of an sp 6 3 hybridized carbon attached to two oxygen atoms. The proton resonance at  H 6  4.42 (Xyl-H  1: HMQC to 8 c 101.5) showed COSY correlations to the methine proton at  i-  5.30 (XyI-H-2: HMQC to 6 c 71.5), which in turn had COSY correlations to the proton resonance at  H  5.43 (Xyl-H-3: HMQC to 6 c 72.2) (Figure 5.6.11). The  COSY spectrum revealed that the proton resonance at  H  to & 69.5) had correlations to the proton resonances at 3.10 (Xyl-H-5a: HMQC to Oc 62.4) and  oH  5.10 (Xyl-H-4: HMQC H 8  5.43 (XyI-H-3),  oH  3.97 (XyI-H-5b: HMQC to Oc 62.4). All  of the above is consistent for fragment of five consecutive oxygenated carbons and this was supported by numerous HMBC correlations (Figure 5.6.10). Both  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  the methylene protons at  oH  3.10 (Xyl-H-5a) and  H 8  209  3.97 (Xyl-H5b) displayed  HMBC correlations to the oxygenated methine carbon resonance at O 101.5 (XyI-C-1), which allowed the establishment of a pentose in its pyranose form. The vicinal coupling constants of the pentose from Xyl-H-1 to Xyl-H-4 showed a range from 7.3-10.1 Hz (Figure 5.6.9). This is consistent with all the protons having axial/axial coupling, therefore, the sugar was found to be a xylose residue in its -anomeric form (substructure I, Figures 5.6.9 and 5.6.10).  Chapter 5: Isolation of liqands forthe Human Sex Hormone Bindinq Globulin  210  XyI-H-1  ppm  5.50  5.00  4.50  Figure 5.6.11. COSY expansion for substructure I of myrmekioside C peracetate (5.28). 5.20,t (9.9 Hz)  H  3.68, (8.9 Hz)  QAc rn  j  ’ 0 H  Q  AcQAQ (102Hz)  3.68, (8.9 Hz)  a  F H  b  Figure 5.6.12. (a) H chemical shifts and coupling constants and (b) 13 1 C chemical shifts of substructure II of myrmekioside C peracetate (5.28).  Chaoter 5: Isolation of liaands for the Human Sex Hormone Bindinci Globulin  211  .0  HMBC  Figure 5.6.13. Key HMBC correlations of substructure II of myrmekioside C peracetate (5.28). The downfield shift of the carbon at öc 101.4 (Glul-C-1) indicated a dioxy methine. COSY cross-peaks were present between the proton resonance at 4.53 (Glul-H-1: HMQC to öc 101.4) and the methine proton at HMQC to 3 78.9).  The proton resonance at  H 0  oH  H 6  3.68 (Glul-H-2:  3.68 (Glul-H-2) had COSY  correlations to the methine proton at O- 5.37 (Glul-H-3: HMQC to Oc 74.7), which contained an additional COSY correlation to the proton resonance at  H 8  5.20  (Glul-H-4: HMQC to 0 c 68.8). Further COSY correlations were present between the proton resonance at 01-1 5.20 (Glul-H-4) and the oxygenated methine proton resonance at 0 H 3.29 (Glul-H-5: HMQC to Oc 71.8). Finally, COSY cross-peaks between the proton resonance at OH 3.29 (Glul-H-5) and both proton resonances at  H 8  4.05 (Glul-H-6a: HMQC to Oc 61.7) and  H 8  4.30 (Glul-H-6b: HMQC to O  61.7) revealed a fragment of six adjacent oxygenated carbons. This fragment was confirmed by several key HMBC correlations (Figure 5.6.13). HMBC cross peaks were present between the methine proton resonance at Oi-i 4.53 (Glul-H-5) and the dioxy methine carbon at Oc 101.4 (Glul-C-1). This established a hexose moiety in its pyranose form. Examination of the vicinal coupling constants from  ChQntr 5: lso!ition of lion nds for th Humnn Sx Hormonn Rindino Globulin  212  Glul-H-1 to Glul-H-5 revealed a coupling constant range from 7.9-10.1 Hz, which is consistent with all the protons having axial/axial coupling (Figure 5.6.13). It can be deduced that a glucose moiety is present in its -anomeric form (substructure II, Figures 5.6.12 and 5.6.13).  5.25, t  H H  a  b  Figure 5.6.14. (a) 1 H chemical shifts and coupling constants and (b) 13 C chemical shifts of substructure Ill of myrmekioside C peracetate (5.28). QAc °  HMBC  Figure 5.6.15. Key HMBC correlations of substructure Ill of myrmekioside C peracetate (5.28).  The methine carbon resonance G1u2-C-1 (c 100.9) displayed a chemical shift suitable for a carbon attached to two oxygeris. showed that the proton resonance at  oH  The COSY spectrum  4.80 (G1u2-H-2: HMQC to Oc 100.9) had  a correlation to the proton resonance at  H 8  5.25 (G1u2-H-2: HMQC to 8 c 72.6),  which in turn had a correlation to methine proton at  oH  5.47 (G1u2-H-3: HMQC to  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  c 73.6). The proton resonance at 8  oH  213  5.37 (G1u2-L-I-4: HMQC to Oc 68.6) showed  COSY correlations to both proton resonances at  OH  5.47 (G1u2-H-3) and  H 8  5.47  (Glu2-H-5: HMQC to Oc 72.2). Finally, COSY correlations were present between the proton resonance at  H 8  3.59 (G1u2-H-5) and the proton resonances at  (G1u2-H-6a: HMQC to Oc 62.0) and  H 0  oH  4.29  4.47 (Glu2-H-6b: HMQC to 0 c 62.0). All of  the above data is consistent for a fragment of six consecutive oxygenated carbons which was confirmed by observation of the HMBC data (Figure 5.6.15). A key HMBC correlation between the proton resonance at  H 8  3.29 (G1u2-H-5) and  the dioxy carbon resonance at 100.9 (G1u2-C-1) established the presence of a hexose in its pyranose form. The vicinal coupling constant range for Glul-H-1 to Glul-H-5 was found to be 8.0-10.2 Hz which corresponds to all the protons having axial/axial coupling.  This is consistent for a glucose moiety in its 3-  anomeric form and substructure Ill (Figures 5.6.14 and 5.6.15).  hpter5jsolation ofjJgnds for the Human Sex Hormone Binding Globulin  214  G1u2-H-4  I V  G1u2-H-3——-*’,  ppm  5.50  G1u2-H-1 ..,  5.40  5.30  5.20  5.10  5.00  4.90  4.80  4.70  Figure 5.6.16. COSY expansion for substructure UI of myrmekioside C peracetate (5.28).  3.7, d (5.4 Hz)  70.4  °‘ldt. 4.10, dd (10.2, 4.2 Hz) 3.73, m  a  69.4  b  Figure 5.6.17. (a) “H chemical shifts and coupling constants and (b) 13 C chemical shifts of substructure IV of myrmekioside C peracetate (5.28).  Chanter 5: Isolation of liqands for the Human Sex Hormone Bindinq Globulin  215  ‘HMBC 0  Figure 5.6.18. Key HMBC correlations of substructure IV of myrmekioside C peracetate (5.28). The oxygenated methylene protons at  H 6  3.70 (Gly-H-1: HMQC to 8 c 70.4)  displayed COSY correlations to the methine proton at  oH  4.06 (Gly-H-2: HMQC  to E 78.0), which established the linkage between GIy-C-1 (Oc 70.4) and Gly-C-2 (öc 78.0). Both methylene proton resonances at 69.4) and  H 6  oH  4.10 (Gly-H-3a: HMQC to O  3.73 (Gly-H-3b: HMQC to 8 c 69.4) showed COSY correlations to the  proton resonance at  oH  4.06 (Gly-H-2), which allowed the connectivity between  Gly-C-2 ( c 78.0) and Gly-C-3 (Oc 69.4). All of this is consistent with a linear 0 chain of three oxygenated carbons and a glycerol moiety (substructure IV, Figure 5.6.17). This was supported by numerous correlations in the HMBC spectrum (Figure 5.6.18).  Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin  216  QAc XyI-3  Glucose I XyI-5  Xylose (Xyl)  “QAc  HMBC Glucose 2 (Glu2)  G1u2-5  AcO”  ‘QAc QAc  Figure 5.6.19. Key HMBC correlations of myrmekioside C peracetate (5.28). HMBC cross-peaks were present between the anomeric proton resonance at  oH  4.80 (G1u2-H-1) and the oxygenated carbon methine at & 78.9 (Glul-C-2).  This established the linkage between Glul and Glu2 and supported a disaccharide moiety (Figure 5.6.20). The anomeric proton methine at  oH  4.53  (Glul-H-1) had HMBC correlations to the oxygenated carbon resonance at c 70.0 (Gly-C-2). Additional three bond HMBC couplings were present between 8 the methine proton resonance at  oH  4.06 (Gly-H-2) and the oxygenated carbon  resonance at 0 c 101.4 (Glul-C-1). This confirmed the link between the glycerol and the disaccharide moieties at Glul-C-1 (Figure 5.6.20). resonances at  H 0  4.10 (Gly-H-3a) and  H 0  Both proton  3.73 (Gly-H-3b) showed HMBC  correlations to the anomeric carbon resonance at 8 c 101.5 (Xyl-C-1). Additional HMBC cross-peaks were observed between the anomeric proton resonance at  Charter 5: Isolation of liqands for the Human Sex Hormone Bindinq Globulin  oH  217  4.42 (Xyl-H-1) and Oc 69.4 (Gly-C-3), which established the link between the  xylose and glycerol moieties at Gly-C-3 (Figure 5.6.20). 1.4  4.00, t  72 1  Figure 5.6.20. (a) ‘H chemical shifts and coupling constants and (b) C 13 chemical shifts of substructure V of myrmekioside C peracetate (5.28).  Gly-3  Glycerol  GIy-2  Figure 5.6.21. Key HMBC correlations of substructure V of myrmekioside C peracetate (5.28). Analysis of the NMR data to this point had revealed a glycerol and three sugar subunits which is consistent with a molecular formula of C 41 , 67 H 2 0 3 leaving 0 C 4 H 9 2 1 , unaccounted for. Analysis of the H 1, 3 ‘, and DEPT NMR spectra C revealed no methyl doublets or triplets, 16 aliphatic methylenes, and two oxymethylenes.  The proton resonance at  H 0  1.19 (H-16: HMQC to Oc 26.3)  contained COSY correlations to the proton resonance at  oH  1.46 (H-I 7: HMQC to  c 29.0), which linked methylenes C-16 (oc 26.3) and C-17 ( 8 c 29.0). Additional 0 COSY cross-peaks between  H 8  1.46 (H-17) and H 0 . 4 O° (H-18: HMQC to Oc 64.4)  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  218  linked methylenes C-17 ( c 29.0) to C-18 (öc 64.4). These connections were 6 supported by correlations in the HMBC (Figure 5.6.22). The downfield chemical shift of C-18 (öc 64.4) is typical of a carbon attached to oxygen. A methylene proton resonance at  oH  4.00 (H-18) showed an HMBC correlation to an acetate  carbonyl (Oc 170.2) (Figure 5.6.21).  This reveals that an aliphatic chain is  terminated by an oxymethylene. The methylene proton at  H 0  1.49 (H-3: HMQC to  c 26.8) showed a COSY correlation with the proton resonance at 0  oH  1.19 (H-2:  HMQC to O 30.5), which confirmed the connection between C-2 (Oc 30.5) and C3( c 26.8). The methylene C-I ( 8 c 72.1) was bonded to methylene C-2 ( 3 c 30.5) 0 from observation of a COSY correlation between HMQC to öc 72.1).  H 8  1.19 (H-2) and OH 3.53 (H-I:  These linkages were supported by numerous HMBC  correlations (Figure 5.6.21). The chemical shift of the methylene C-I (Oc 72.1) is consistent with a carbon attached to oxygen.  From the above data, one can  deduce a linear eighteen carbon aliphatic chain flanked by two terminal oxymethylenes (substructure V, Figures 5.6.20 and 5.6.21). HMBC cross-peaks were observed between the methylene proton reasonance at  oH  3.53 (H-I) and  the oxygenated methine carbon resonance at Oc 70.4 (Gly-C-1). Finally, HMBC correlations between the proton resonance at O,- 3.70 (Gly-H-1) and the carbon resonance at Oc 72.1 established that one terminal of the aliphatic chain (C-I) is attached to the glycerol moiety at Gly-C-I, thus completing the structure of myrmekioside C peracetate (5.28).  Even though the carbon skeleton of  myrmekioside C is known, it contains a rare saturated lipid moiety that is  ChaDter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  219  oxygenated on both ends of the linear chain. Only two other naturally occurring compounds, 5.20 and 5.21, contain this rare lipid moiety.  17  5.7. BioJogy of Secondary Metabolites isolated from Myrmekioderma styx To screen for active ligands against SHBG, assays were run by the Hammond laboratory in the Child and Family research institute at the University of British Columbia.  In this assay, 22 SHBG is saturated with tritium labeled  dihydrotestoterone 3 ([ H ]-DHT) and any excess steroid is removed. The desired ligand is then added to the H 3 SHB ]-DH G/[ T mixture and incubated overnight. After removal of the displaced 3 [ H 1-DHT, the quantity of [ H]-DHT bound to SHBG 3 in the presence of the ligand is compared to the amount of 3 [ H ]-DHT bound to SHBG when no ligand was added. The determination of the IC 50 concentration was achieved when the ligand released more than 50% of 3 [ H 1-DHT from 22. All of the pure natural compounds isolated were tested in the SHBG SHBG ligand binding assay. Only (+)-curcudiol (5.24; IC 50 100 jtM) was identified as a ligand able to displace 3 [ H ]-DHT from SHBG (Figure 5.7.1).  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  100  •  220  Cortisci Curcucitoi  90 80 70 60 50 40 30 20 10 0  ,.••.“,I  0.1  1  10  100  1000  10000  100000 1000000  Ligand Concentration nM  Figure 5.7.1. Dose response curve of (+)-curcudiol (5.24) in the SHBG assay. The graph was generated by Magid Fallahi of the Hammond laboratory. 5.8. Acetylation of myrmekioside C  Acetylation of 5.27 (1.1 mg, 0.001 mmol) was accomplished by stirring pyridine (1 mL; 12 mmol) and acetic anhydride (1 mL; 10 mmol) for 24 hours. The reaction mixture was dried in vacuo and purification was accomplished using a normal phase silica gel Sep PakTM (eluent: 4:1 Hexanes: EtOAc) to obtain 5.28 (1.0mg, 0.0007 mmol) in a 78% yield. 5.9. General Experimental Methods  All solvents used (except for NMR solvents) were HPLC grade (Fisher) and no further purification was performed.  Solvents for HPLC were filtered  through a 0.45 i.im filter (Osmonics, Inc) before use.  Acetic anhydride and  pyridine were acquired from Aldrich and were used without further purification.  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  221  Reversed-phase 1 C 8 silica gel Sep PaksTM (10 g) and normal-phase silica gel Sep PaksTM (2 g) were purchased from Waters, Inc.. Separations on the HPLC was accomplished using either a Waters 2487 dual channel detector/system controller (Waters Series 515 pump; chart recorder, 0.25 cm/mm), or a Waters 600 controller and Waters 486 Tunable Absorbance Detector (chart recorder, 0.25 cm/mm).  The HPLC column used was a 5 im lnertsil column from  Chromatography Sciences (Montreal, PQ).  The conditions of the HPLC  separation were as follows: 2.0 mL/min, monitoring at 220 nm.  Thin-layer  chromatography (TLC) plates were Whatman MKCI8F (reversed-phase) and Kieselgel 60F 254 (normal phase). TLC was visualized using either a dip solution of p-anisaldehyde (1% p-anisaldehyde, 2% , 4 S 2 H 0 20% acetic acid and 77% EtCH) or under ultraviolet light (254 nm). The ‘ C spectra were recorded with either a Bruker AV600, AMX500, 3 AM400, or AV400 spectrometer. ‘H spectra and 2D data sets were taken with either a Bruker AV600, AV500, or AV 400 spectrometer.  NMR solvents were  purchased from Cambridge Isotope laboratories and were referenced to solvent peaks C D 6  H 6 (  7.15 ppm and öc 128.0 ppm), DMSO 6 -d  (oH  2.49 ppm and 8 c 39.5  ppm), and CDCI 3 (OH 7.24 ppm and 0 c 77.23 ppm). Low resolution ESI mass spectra were recorded on a Bruker Esquire LC mass spectrometer.  High  resolution ESI mass spectra was obtained using a Micromass LCT mass spectrometer. Optical rotations were recorded with a JASCO J-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm micro cell.  The CD  Chapter 5: Isolation of I,gands for the Human Sex Hormone Binding Globulin  222  spectra were recorded using a JASCO J-710 spectropolarimeter with a 1 mm micro cell. 510. Isolation of bisabolane sesquiterpenes and myrmekioside C Myrmekioderma granulatum (75 g wet wt.) was collected by hand using  SCUBA from Latondo Island of Besar, Takabonerati, Indonesia.  The sponge  was identified by Dr. R. van Soest (University of Amsterdam) and a voucher sample has been kept at the Zoologisch Museum, Amsterdam (ref. No. ZMA POR 18337).  The material was frozen and stored until workup. The frozen  sponge sample was extracted four times with MeOH (4 X I L). The combined MeOH extracts were reduced in vacuo to give a brown solid (2.6 g). The solid was subjected to a gradient reversed-phase Sep PakTM to yield two biologically active fractions. The most active fraction was subjected to repeated reversedphase HPLC (Inertsil C, , 9.4 X 250 mm, 4:6 2 8 H 0 : MeOH, UV detection at 220 nm) to obtain the biologically active compound (+)-curcudiol (5.24, 16.7 mg), 21 abolene (5.23, 15.7 mg), ° abolenone (5.25, 5.2 mg) and the bisabolane 2 sesquiterpenoid 5.22 (32.8 mg). ° 2  The less active fraction was subjected to  repeated reversed phase HPLC (lnertsil C , 1 8 9.4 X 250 mm, 3:7 2 H 0 : MeOH, UV detection at 220 nm) to yield (+)-curcuphenol (5.26, myrmekioside C (5.27, 4.3 mg).  16.7 mg), 21  and  Chapter 5: Isolation of l,gands for the Human Sex Hormone Binding Globulin  223  5.11. Physical data of secondary metabolites from Myrmekioderma styx  (+)-Curcudiol (5.24): yellow oil. [c]D : +3.9 (c 0.3, MeOH); UV (MeOH) 22 (log  ) 219  2max  nm (3.17), 270 (3.10); ‘H NMR (500 MHz, ) 6 DMSO :6H -d 8.96 (IH, bs,  1-OH) 6.91 (IH, d, J  =  7.63 Hz, H-5), 6.55 (IH, s, H-2), 6.53 (IH, d, J  =  7.63 Hz,  H-4), 3.98 (IH, bs, 11-OH)) 3.00 (1H, m, H-7), 2.15 (3H, s, H-15), 1.49 (IH, m, H 8a), 1.39 (IH, m, H-8b), 1.29 (2H, m, H-la), 1.23 (2H, m, H-9), 1.08  (  3H, d, J  =  7.05 Hz, H-14), 1.00 (3H, s, H-12), 0.99 (3H, s, H-13); ‘ C NMR (100 MHz, 3  6 DMSO ) : -d ö 154.4 (C, C-I), 135.0 (C, C-3), 130.3 (C, C-6), 126.3 (CH, C-5), 119.6 (CH, C-4), 115.6 (CH, C-2), 68.6 (C, C-lI), 43.7 (CH , C-b), 37.8 (CH 2 , 2 C-8), 31.1 (CH, C-7), 29.3 (CH , C-12 or C-13), 29.1 (CH 3 , C-12 or C-13), 21.9 3 , C-I5), 21.1 (CH 3 (CH , C-14), 20.6 (CH 3 , C-9); LRESIMS m/z 259; HRESIMS 2  m/z 259.1672 (calc’d for O 14 C 2 H N 2 5 a 259.1674). Abolene (diastereotopic mixture) (5.23): yellow oil; UV (MeOH) 276 ( 3.51); 1 H NMR (500 MHz, ) 6 DMSO : -d =  oH  max  (log  E)  9.01 (IH, b, 9-OH), 6.90 (IH, d, J  7.6 Hz, H-5), 6.56 (IH, s, H-2), 6.53 (IH, d, J  =  7.6 Hz, H-4), 4.86 (I-H, s, H  12a or H-12b), 4.80 (1H, s, H-12b or H-12a), 4.59 (IH, s, 9-OH), 3.81 (IH, bm, H-b), 2.99 (IH, m, H-7), 2.16 (3H, s, H-15), 1.55 (3H, s, H-13), 1.3-1.5 (2H, m, H-8a, H-8b), 1.24 (2H, m, H-9), 1.08 (3H, bd, J  =  6.8 Hz, H-14); 13 C NMR (100  MHz, ) 6 DMSO :O -d 154.8 (C, C-I), 148.6 (C, C-Il), 148.6 (C, C-lI), 135.5 (C, C-  3), 130.4 (C, C-6), 126.8 (CH, C-5), 120.0 (CH, C-4), 116.0 (CH, C-2), 110.3 , C-l2), 110.0 (CH 2 (CH , C-12), 74.6 (C, C-b), 74.2 (C, C-b), 32.9 (CH 2 , C-9), 2 32.5 (CH , C-8), 32.5 (CH 2 , C-8), 31.1 (CH, C-7), 30.8 (CH, C-7), 21.1 (CH, C2  Chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin  224  14), 21.0 (CH , C-14), 20.6 (CH 3 , C-15), 17.4 (CH 3 , C-13), 17.2 (CH 3 , C-13); 3 LRESIMS m/z 257; HRESIMS m/z 257.1512 (calc’d for 2 O 1 C 2 H N 5 2 a 257.1517). Phenol,  2-(5-hydroxy-1 ,5-dimethyl-3-hexenyl)-5-methyl-,  yellow oil. [aID : -6.9 (c 1.8, MeOH); UV (MeOH) 22 (3.09), 241 (3.02); 1 H NMR (500 MHz, 6 C ) D :  H  max  [R-(E)]  (5.22):  (logE) 277 (3.10), 227  7.05 (IH, d, J  =  7.6, H-3), 6.71  (IH, d, J= 7.6 Hz, H-4), 6.40 (1H, bs, H-2), 5.57 (IH, m, H-9), 5.52 (IH, bd, J= 15.3 Hz, H-b), 3.33 (IH, m, H-7), 2.42 (IH, m, H-8a or H-8b), 2.28 (IH, m, H 8b, or H8a), 2.14 (IH, s, H-15), 1.28 (3H, d, J  =  7.0 Hz, H-14), 1.15 (3H, s, H-12  or H-13), 1.14 (3H, s, H-13 or H-12); 1 H NMR (500 MHz, ) 6 DMSO : -d b, 9-OH), 6.91 (IH, d, J  =  oH  9.09 (IH,  7.63 Hz, H-5), 6.58 (IH, s, H-2), 6.53 (IH, d, J  =  7.63  Hz, H-4), 5.53-5.39 (2H, m, H-9 and H-la), 4.35 (IH, b, Il-OH), 3.03 (IH, m, H7), 2.28-2.05 (2H, m, H-8a, H-8b), 2.16 (3H, s, H-15), 1.16 (6H, bs, H-l2, H-13), 1.09 (3H, bd, H-14); 13 C NMR (100 MHz, ) 6 DMSO : -d & 154.1 (C, C-I), 140.3 (CH, C-b), 135.2 (C, C-3), 129.7 (C, C-6), 126.3 (CH, C-5), 123.8 (CH, C-9), 119.4 (CH, C-4), 115.1 (CH, C-2), 68.7 (C, C-lI), 38.8 (CH , C-8), 31.4 (CH, C2 7), 29.7 3 (CH C-12, C-13), 20.6 (CH , , C-15), 19.7 (CH 3 , C-14); LRESIMS m/z 3 257; HRESIMS m/z 257.1515 (calc’d for 2 O 1 C 2 H N 5 0 a 257.1517). Abolenone (5.25): yellow oil. [aID : +8.8 (C 2.6, MeOH); UV (MeOH) 22 277 (2.89); CD  ax (AE  2max  (log  8)  (MeOH) 280 nm (-187.87); 1 H NMR and C 13 NMR see  Table 5.5.1; LRESIMS m/z 255; HRESIMS m/z 255.1360 (calc’d for 2 O 1 C 2 H N 5 0 a 255.1361).  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  (+)-Curcuphenol (5.26): yellow oil. ?max  : 21 [cL]D  (logE) 277 (3.03), 240 (2.65); CD  NMR (500 MHz, 3 CDCI ) :  H  max  225  +25.2 (c 0.84, 3 CHCI ) ; UV (MeOH) (& (MeOH) 280 nm (-141.18); 1 H  7.04 (1 H, d, J = 7.8 Hz, H-5), 6.73 (1 H, d, J  =  7.8 Hz,  H-4), 6.59 (1H, s, H-2), 5.13 (IH, m, H-b), 4.63 (IH, s, OH), 2.97 (IH, m, H-7), 2.27 (3H, s, H-15), 1.94 (2H, m, H-9), 1.69 (3H, s, H-12 or H-13), 1.69-1.56 (2H, m, H-8a, H-8b), 1.54 (3H, s, H-13 or H-12), 1.23 (3H, d, J  =  7.0 Hz, H-14); 13 C  NMR (125 MHz, 3 CDCI ) : c 3 153.0 (C, C-I), 136.5 (C, C-6), 131.9 (C, C-Il), 130.0 (C, C-3), 126.8 (CH, C-5), 124.6 (CH, C-b), 121.6 (CH, C-4), 116.2 (CH, C-2), 37.3 (CH , C-8), 31.4 (CH, C-7), 26.1 (CH 2 , C-9), 25.7 (CH 2 , C-12 or C-13), 3 21.1 (CH , C-15), 20.9 (CH 3 , C-14), 17.7 3 3 (CH C-13 or C-12); LRESIMS m/z , 217; HRESIMS m/z 217.1590 [Mt] (calc’d for H 0 1 C 2 5 217.1592). 1 Myrmekioside C peracetate (5.28): yellow oil. [cz]D : -18.2 (c 0.45, EtOAc); For 21 H NMR and 13 1 C NMR see Table 5.6.1.; LRESIMS m/z 1301; HRESIMS m/z 1301.5774 (calc’d for N 69 C 9 H 2 O 0 4 a 1301.5778). 5.12. References (1)  Hammond G.L. Trends in Endocrinology and Metabolism 1995, 6, 398304.  (2)  Hammond G.L.; Avvakumov G.V.; Muller Y.A. Journal of Steroid Biochemistty and Molecular Biology 2003, 85, 195-200.  (3)  Schottner M.; Spiteller G. Journal of Natural Products 1998, 61, 119-121.  (4)  Selby C. Annals of Clinical Biochemista’y 1990, 27, 532-541.  (5)  Joseph D.R. Vitamins and Hormones 1994, 49, 197-280.  (6)  Handelsman D.J.; Swerdloff R.S. Clinics in endocrinology and metabolism 1985, 14, 89-124.  Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin  226  (7)  Estour B.; Pugaeat M.; Lang F.; Dechaud H.; Pellet J.; Rousset H. Clinical Endocrinology 1986, 24, 571-576.  (8)  Rapuri P.B.; Gallagher J.C.; Haynatzki G. The Journal of Clinical Endocrinology and Metabolism 2004, 89, 4954-4962.  (9)  CherkasovA.; Shi Z.; Fallahi M.; Hammond G.L. Journal of Medicinal Chemistiy 2005, 48, 3203-3213.  (10)  Charkasov A.; Ban F.; Li Y.; Fallahi M.; Hammond G.L. Journal of Medicinal Chemistry 2006, 49, 7466-7478.  (11)  Hooper J.N.A.; Soest R.W.M Van System Porifera A Guide to Classification of Sponges; Kluwer Academic/Plenum Publishers: New York, 2002.  (12)  Albrizio S.; Faitorusso E.; Magno S.; Mangoni A. Journal of Natural Products 1992, 55, 1287-1293.  (13)  Sennet S.H.; Pomponi S.A.; Wright A.E. Journal of Natural Products 1992, 55, 1421-1429.  (14)  Peng J. Walsh K., Weedman V., Bergthold J.D., Lynch J., Lieu K.L., Braude l.A., Kelly M., Hamann M.T. Tetrahedron 2002, 58, 7809-7819.  (15)  Peng J. Avery M.A., Hamann M.T. Organic Letters 2003, 5, 4575-4578.  (16)  Tsukamato S.; Haryko K.; Hirota H.; Fusetani N. Biofouling 1997, 11, 283291.  (17)  Letourneux Y.; Brunel J.M.; Fernandez R.; Dherbomez M.; Debitus C. Heterocyclic Communications 2005, 11, 291-298.  (18)  Peng J.; Franzblau S.G.; Zhang F.; Hamann M.T. Tetrahedron Letters 2002, 43, 9699-9702.  (19)  Aoki S.; Higuchi K.; Kato A.; Murakami N.; Kobayashi M. Tetrahedron 1999, 55, 14865-14870.  (20)  Butler M.S.; Capon R.J.; Nadeson R.; Beveridge A.A. Journal of Natural Productrs 1991, 54, 619-623.  (21)  WrightA.E.; Pomponi S.A.; McConnell O.J.; Kohmoto S.; McCarthy P.J. Journal of Natural Productrs 1987, 50, 976-978.  (22)  Hammond G.L.; Lahteenmaki P.L. Clinica Chimica Acta 1983, 132, 101110.  Chanter 6: Conclusions  227  Chapter 6: Conclusions 6.1. Conclusions The overarching goal in the Andersen lab is to isolate bloactive small molecules that can be potential drug leads.  The research presented in the  second chapter of the dissertation describes a successful example of this goal. The MeOH extract of the sponge Neopetrosia exigua displayed potent inhibitory activity against IDO.  Bioassay guided fractionation of N. exigua led to the  isolation and identification of two novel alkaloids, exiguamines A (2.58) and B (2.59).1  The proposed pharmacophore of the exiguamines is the tryptamine  quinone moiety. Currently, synthetic analogs of the tryptamine-quinone moiety are being prepared and evaluated as novel inhibitors of IDO. Very recently, one of the synthetic tryptamine-quinones was found to be an inhibitor of IDO in the yeast based assay. 3 Clearly, based on this result, derivatives of the tryptamine ’ 2 quinone moiety represent a new drug lead to develop inhibitors of IDO as potential treatments for cancer. Biological studies found that exiguamine A had a K of 210 nM, making it among the most potent IDO inhibitors found to date. Unfortunately, exiguamine A was unable to inhibit IDO in a yeast based assay. 2  The presence of the  quaternary ammonium cation in exiguamine A most likely prohibited exiguamine A from crossing the cell membrane. Even though exiguamine B was found to be an inhibitor of IDO, a K value was not obtained for this alkaloid. Finding the K value may establish the effect on the biological activity of having a hydroxyl group on C-17.  More biological studies on the exiguamines are needed to  Chapter 6: Conclusions  228  determine if this family of alkaloids are competitive or non-competitive inhibitors. Finally, work is currently being done to crystallize exiguamine A with IDO. This may establish which parts of the compound form the pharmacophore. Future investigations are required to determine the stability of the two enantiomers of exiguamine A. If the two enantiomers of exiguamine A can be separated, then one can evaluate to see if the configuration at c-I 9 plays a role in the inhibition of IDO.  Finally, further purifications need to be performed to  separate the diastereomers of exiguamine B. Biological studies may reveal the importance of the stereochemistry at both C-I 7 and C-I 9. Another goal of the Andersen lab is to assist in the development of biological screens. Chapter three of this dissertation provides an example of this goal. Cyclo(S-Val-S-Phe) (3.9) and cyclo(R-Val-R-Phe) (3.22) were found to be neurite outgrowth activators using a novel bioassay. The study validates that this screen may be used to discover new axonal outgrowth activators from natural sources. The discovery of cyclo(S-Val-S-Phe) (3.9) and cyclo(R-Val-R-Phe) (3.22) as both in vivo and in vitro activators of neuronal outgrowth may have an impact  in the search for pharmaceuticals to promote spinal cord repair. Currently, the biological mechanism of these two compounds is unknown. Elucidation of how these two diketopiperazines overcome the inhibition of spinal cord repair may yield new protein targets and potentially a new class of drugs.  Furthermore,  because of the simple structures of both compounds, a combinatorial library of cis-diketopiperazines may yield a more potent neurite outgrowth activator.  Chapter 6: Conclusions  229  Chapter four described the purification and structure elucidation of compounds inhibiting the G 2 checkpoint pathway. The MeOH extract of Duguetia odorata showed G 2 checkpoint inhibitory activity. Fractionation of a crude extract of D. odorata led to the isolation of the known alkaloids oliveroline (4.32), dehydrodiscretine (4.34), pseudopalmatine (4.35), and the new alkaloid, N methylguatterine (4•33),4 Oliveroline was active in the G 2 checkpoint assay at concentrations above 10 tM. This alkaloid is structurally distinct from other G 2 checkpoint inhibitors and does not inhibit Chkl. Finding oliveroline’s mechanism of inhibition may yield new information about the G 2 checkpoint pathway, and may potentially lead to the discovery of a new target against cancer. Chapter five describes the isolation and identification of potential ligands for SHBG. The MeOH extract of the marine sponge Myrmekioderma granulatum displayed activity in the SHBG ligand binding assay.  Chromatographic  separation of a crude extract of M. granulatum led to the isolation and identification of 5.22, abolene (5.23), (4)-curcudiol (5.24), abolenone (5.25), (+)curcuphenol (5.26), and myrmekioside C (5.27).  Myrmekioside C (5.27)  contained a rare saturated lipid moiety that is oxygenated on both ends of the linear chain. Biological studies have revealed (+)-curcudiol to be a weak ligand of SHBG. This terpenoid may be used as a lead structure to develop stronger binding SHBG ligands. The discovery of (+)-curcudiol represents the first SHBG ligand that was discovered from a marine source. This research provides proof of principle that marine organisms can provide new ligands for SHBG.  Chapter 6: Conclusions  230  6.2. References (1)  Brastianos H.C.; Vottero E.; Patrick B.O.; Soest R. van; Matainaho T.; Mauk A.G.; Andersen R.J. Journal of the American Chemical Society 2006, 128, 16046-16047.  (2)  Vottero E.; Balgi A.; Woods K.; Tugendreich S.; Melese T.; Andersen R.J.; Mauk A.G.; Roberge M. Biotechnology Journal 2006, 1, 282-288.  (3)  Andersen R.J. Personal Communication, June 2007.  (4)  Brastianos H.C.; Sturgeon C.M.; Roberge M.; Andersen R.J. Journal of Natural Products 2007, 70, 287-288.  Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A  231  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A A.1. Data Collection An  irregular  red  crystal  of  C 2 . 6 5 N . 2 H 4 O Cl 2 .4V H 5 7 S  having  approximate dimensions of 0.05 x 0.25 x 0.30 mm was mounted on a glass fiber. All measurements were made on a Bruker X8 APEX diffractometer with graphite monochromated Mo-KcL radiation. The data were collected at a temperature of -100.0 ± 0.1°C to a maximum 28 value of 45.20. Data were collected in a series of  and co scans in 0.500  oscillations with 45.0 second exposures. The crystal-to-detector distance was 38.85 mm. A.2. Data Reduction The material crystallizes as a two-component twin with the two components related by a 1800 rotation about the (1 0 0) reciprocal axis. Data were integrated for both twin components, including both overlapped and nonoverlapped reflections. In total 41468 reflections were integrated (18959 from component one only, 18804 from component two only, 3667 overlapped). Data were collected and integrated using the Bruker SAINT 1 software packages. The linear absorption coefficient,  i,  for Mo-Kcc radiation is 4.65 cm. Data were 1  corrected for absorption effects using the multi-scan technique (TWIN 2 ABS), with minimum and  maximum  transmission coefficients of 0.682 and  respectively. The data were corrected for Lorentz and polarization effects.  0.977,  Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A  232  A.3 Structure Solution and Refinement The structure was solved by direct methods using non-overlapped data from the major twin compo 3 nent. Subsequent refinements were carried out using an HKLF 5 format data set containing complete data from both twin components. It was immediately evident that the two anions in this material  (C1 and  V(SH) are very different. The chloride anion was easily identified, however ) 2 S the vanadium anion was less evident. Residual electron density clearly showe d a disordered tetrahedron residing on a two-fold axis. The bond distances to the central atom (2.2 —2.35 A) were too long to be any common organic anion (i.e. phosphate, chlorate, etc).  Additionally, the electron density surrounding the  central atom was greater than what one would expect for oxygen atoms. The residual electron density of the central atom is consistent with an early first-ro w transition metal.  Ultimately, vanadium was chosen as the central atom, and  refinement of its site-occupation factor (sof) gave a value of 1.08 (1).  The  disordered atoms surrounding the central V are consistent with sulfur atoms (i.e. refinement of their populations as sulfur gives a value nearly equal to 1, and V-S and V=S distances are consistent with those found in ’ 4 literatu 5 re). Additionally, two disordered water molecules are found in the lattice. All non-hydrogen atoms except C(1 1) were refined anisotropically. All hydrogen atoms were included in calculated positions but not refined.  The batch scale refinement showed a  roughly 96:4 ratio between the major and minor twin components. The final cycle of full-matrix least-squares refinem 6 ent on F 2 was based on 21334 reflections from both twin components and 395 variable parameters and converged (largest  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  233  parameter shift was 0.00 times its esd) with unweighted and weighted agreem ent factors of: RI  wR2  =  =  IlFol  2 [ ( w (Fo  -  -  lFcII I  ) )I 2 Fc  IFol  =  0.247  w(F ] ) 2 l/ o  =  0.515  The standard deviation of an observation of unit weight was I .027 The weighting scheme was based on counting statistics.  The maximum and  minimum peaks on the final difference Fourier map corresponded to 1.04 and —1.07 e/A , respectively. 3 Neutral atom scattering factors were taken from Cromer and Waber 8. Anomalous dispersion effects were included in Fcalc, 9 the values for At’ and At” were those of Creagh and ° 1 McAu ley.  The values for the mass attenuation  coefficients are those of Creagh and ’ 1 Hubbe ll. All refinements were performed using the SHELXTL crystallographic software package of Bruker 2 -AXS.’  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  A.4.1. Experiemental Details, Crystal Data  Empirical Formula  H 2 C 3 5. 2 C1V 2 S 8 0 N 5  Formula Weight  655.60  Crystal Color, Habit  red, irregular  Crystal Dimensions  0.05 X 0.25 X 0.30 mm  Crystal System  monoclinic  Lattice Type  C-centered  Lattice Parameters  a = 32.833(7) A b = 8.462(2) A c=23.947(5) A = 90° = 114.891(9)0 900 .Y= V = 6035(2) A 3  Space Group  C 2/c (#15)  Zvalue  8  DIc  1.443 g/Cm 3  FOOD  2732.00  .t(MoKc)  4.65 cm 1  234  Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A  235  A.4.2. Experimental Details, Intensity Measurements  Diffractometer  Bruker X8 APEX  Radiation  MoKx ( = 0.71073 A) graphite monochromated  Data Images  1105 exposures  Detector Position  38.85 mm  max 29  45.2°  No. of Reflections Measured  Total: 21334  Corrections  Unique: 41468 (Rint = 0.108) Absorption (Tmin = 0.682, Tmax 0.977) Lorentz-polarization  45.0 seconds  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  236  A.4.3. Experimental Details, Structure Solution and Refinement  Structure Solution  Direct Methods (S1R97)  Refinement  Full-matrix least-squares on F 2  Function Minimized  w (Fo 2  -  ) 2 Fc  Least Squares Weights  w= I I(a 2 (Fo )+(O. I 479P) 2 +8I23206P) 2  Anomalous Dispersion  All non-hydrogen atoms  No. Observations (l>O.OOLI(l))  21334  No. Variables  390  Reflection/Parameter Ratio  54.70  Residuals (refined on F , all data): RI; wR2 2  0.247; 0.515  Goodness of Fit Indicator  1.02  No. Observations (l>2.OOcr(I))  12745  Residuals (refined on F): Ri; wR2  0.188; 0.481  Max Shift/Error in Final Cycle  0.00  Maximum peak in Final Duff. Map  1.04 eIA 3  Minimum peak in Final Duff. Map  -1 3 .06e/A  Appendix: Experimental Details for X-ray Diffraction Analysis of Exigua mine A Table A.4.1. Atomic coordinates (x 1OM) and equivalent isotropic displac ement parameters (AA2 x 10A3) for exigumaine A. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.  x C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(11) C(12) C(13) C(14) C(16) C(17) C(18) C(19) C(21) C(23) C(24) C(25) C(27) C(28) C(29) C(30) N(1) N(15) N(20) N(22) N(26) 0(1) 0(2A) 0(2B) 0(4A) 0(5) 0(8) 0(12) 0(21) 0(23) Cl(1) V(1) S(1) S(2) S(3A) S(3B) 0(4B)  3365(4) 3303(4) 2821(3) 2569(3) 2068(3) 1857(4) 2152(4) 2626(3) 1395(3) 1144(3) 672(4) 416(4) 646(4) 817(4) 1221(4) 1128(4) 1806(3) 1853(4) 1999(4) 3690(5) 3728(4) 92(4) 277(6) 2125(4) 1602(4) 2941(3) 445(3) 1759(3) 1988(3) 4072(4) 1360(2) 3196(6) 3850(6) 4207(5) 2704(2) 1995(2) 466(3) 1851(3) 2071(2) 882(1) 5000 4847(4) 5049(3) 4468(3) 4369(3) 4682(12)  y 4485(12) 3273(12) 3141(11) 2065(11) 2260(10) 3481(10) 4401(10) 4292(12) 3585(9) 2258(10) 2140(10) 3472(11) 4803(11) 7503(12) 6550(11) 4942(10) 1137(10) 256(11) -597(15) 231 5(14) 2450(15) 6684(11) 6163(18) -2602(1 0) 2959(13) 5060(9) 6295(9) 1501(8) -1005(9) 1234(13) 931(7) -2396(18) -1629(17) 4842(13) 1123(8) 5183(8) 880(8) 198(8) -1406(7) 6298(3) -5491(2) -5644(9) -8072(7) -4341(8) -4036(15) 4750(30)  z 5790(5) 6108(5) 5948(5) 6141(5) 5794(4) 5347(5) 5142(5) 5506(4) 5073(4) 5137(4) 4889(5) 4565(6) 4515(5) 4527(5) 4656(6) 4782(4) 5957(5) 6893(5) 6099(6) 6575(5) 7224(4) 4430(5) 3534(6) 6905(5) 6650(6) 5376(5) 4259(4) 6523(4) 6625(4) 7616(5) 5447(3) 6616(7) 7091(7) 8858(5) 6551(3) 4647(3) 4971(5) 7395(4) 5714(4) 6078(1) 7500 6461(3) 7749(3) 7709(4) 7086(7) 9096(14)  U(eq) 60(3) 58(3) 43(3) 44(3) 38(3) 44(3) 47(3) 46(3) 37(3) 35(2) 54(3) 62(3) 50(3) 64(3) 64(3) 48(3) 39(3) 45(3) 64(4) 74(4) 67(4) 57(3) 131(7) 58(3) 81(4) 63(3) 51(2) 50(2) 46(2) 104(4) 55(2) 61(6) 86(6) 61(5) 56(2) 60(2) 85(3) 70(2) 51(2) 64(1) 31(1) 105(3) 80(2) 87(2) 182(7) 125(13)  237  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  Table A.4.2. Bond lengths [A] and angles [deg] for exiguamine A. Bonds C(2)-C(3) C(2)-N(1) C(2)-H(2) C(3)-C(4) C(3)-C(24) C(4)-C(9) C(4)-C(5) C(5)-O(5) C(5)-C(6) C(6)-C(7) C(6)-C(19) C(7)-C(10) C(7)-C(8) C(8)-O(8) C(8)-C(9) C(9)-N(1) C(10)-C(18) C(10)-C(11) C(11)-O(1) C(11)-C(12) C(12)-O(12) C(12)-C(13) C(13)-C(14) C(13)-H(13) C(14)-N(15) C(14)-C(18) C(16)-C(17) C(16)-N(15) C(16)-H(16A) C(16)-H(16B) C(17)-C(18) C(17)-H(17A) C(17)-H(17B) C(19)-N(20) C(19)-O(1) C(19)-C(23) C(21)-O(21) C(21)-N(20) C(21)-N(22) C(23)-O(23) C(23)-N(22) C(24)-C(25) C(24)-H(24A) C(24)-H(24B) C(25)-N(26) C(25)-H(25A) C(25)-H(25B) C(27)-N(1 5) C(27)-H(27A) C(27)-H(27B) C(27)-H(27C) C(28)-N(15) C(28)-H(28A)  Bond Lenghts FM (angle (deg)) 1.343(14) 1.412(14) 0.9500 1.467(15) 1.527(15) 1.381(13) 1.430(14) 1.196(11) 1.508(14) 1.439(12) 1.441(13) 1.379(14) 1.476(14) 1.263(11) 1.431(14) 1.367(13) 1.435(12) 1.439(12) 1.368(10) 1.411(14) 1.320(11) 1.424(13) 1.390(14) 0.9500 1.437(11) 1.440(15) 1.471(15) 1.513(13) 0.9900 0.9900 1.454(13) 0.9900 0.9900 1.461(13) 1.472(11) 1.577(14) 1.206(12) 1.326(12) 1.410(12) 1.249(14) 1.320(14) 1.510(15) 0.9900 0.9900 1.525(13) 0.9900 0.9900 1.420(13) 0.9800 0.9800 0.9800 1.589(15) 0.9800  238  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  C(28)-H(28B) C(28)-H(28C) C(29)-N(22) C(29)-H(29A) C(29)-H(29B) C(29)-H(29C) C(30)-N(20) C(30)-H(30A) C(30)-H(30B) C(30)-H(30C) N(1)-H(1) N(26)-H(26A) N(26)-H(26B) N(26)-H(26C) O(4A)-O(4B) O(12)-H(12) V(1 )-S(3A)#1 V(1 )-S(3A) V(1 )-S(3B)#1 V(1)-S(3B) V(1)-S(2)#1 V(1 )-S(2) V(1)-S(1) V(1 )-S(1 )#1 S(1 )-S(3A)#1 S(2)-S(2)#1 S(3A)-S(3B) S(3A)-S(1 )#1 C(3)-C(2)-N(1) C(3)-C(2)-H(2) N(1 )-C(2)-H(2) C(2)-C(3)-C(4) C(2)-C(3)-C(24) C(4)-C(3)-C(24) C(9)-C(4)-C(5) C(9)-C(4)-C(3) C(5)-C(4)-C(3) O(5)-C(5)-C(4) O(5)-C(5)-C(6) C(4)-C(5)-C(6) C(7)-C(6)-C(19) C(7)-C(6)-C(5) C(19)-C(6)-C(5) C(1 0)-C(7)-C(6) C( I 0)-C(7)-C(8) C(6)-C(7)-C(8) O(8)-C(8)-C(9) O(8)-C(8)-C(7) C(9)-C(8)-C(7) N(1 )-C(9)-C(4) N(1 )-C(9)-C(8) C(4)-C(9)-C(8) C(7)-C(10)-C(1 8) C(7)-C(10)-C(1 1) C(18)-C(10)-C(1 1) O(1)-C(11)-C(12)  0.9800 0.9800 1.492(11) 0.9800 0.9800 0.9800 1.418(12) 0.9800 0.9800 0.9800 0.8800 0.9100 0.9100 0.91 00 1.42(3) 0.8400 2.233(7) 2.233(7) 2.249(10) 2.249(10) 2.252(6) 2.252(6) 2.324(7) 2.324(7) 2.543(13) 1.099(13) 1.412(14) 2.543(13) 108.8(10) 125.6 125.6 108.4(9) 122.8(12) 128.7(10) 123.5(10) 104.3(9) 132.1(9) 128.7(10) 117.8(9) 113.4(8) 121.4(9) 124.0(8) 114.6(8) 119.2(9) 123.5(8) 116.7(9) 121.0(10) 121.5(10) 117.4(8) 111.1(9) 124.4(9) 123.1(9) 127.4(9) 117.7(8) 114.5(9) 113.3(8)  239  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A  O(1)-C(1 1)-C(10) C(12)-C(1 1)-C(10) 0(12)-C(12)-C(1 1) 0(12)-C(12)-C(13) C(1 1)-C(12)-C(13) C(14)—C(13)—C(12) C(14)-C(13)-H(13) C(12)-C(13)-H(13) C(13)-C(14)-N(15) C(13)-C(14)-C(18) N(1 5)-C(14)-C(1 8) C(1 7)-C(1 6)-N(1 5) C(17)-C(16)-H(16A) N(15)-C(16)-H(16A) C(17)-C(16)-H(16B) N(15)-C(16)-H(16B) H(16A)-C(16)-H(16B) C(18)-C(17)-C(16) C(18)-C(17)-H(17A) C(16)-C(17)-H(17A) C(18)-C(17)-H(17B) C(16)-C(17)-H(17B) H(17A)-C(17)-H(17B) C(10)-C(18)-C(14) C(10)-C(18)-C(17) C(14)-C(18)-C(17) C(6)-C(19)-N(20) C(6)-C(19)-0(1) N(20)-C(19)-0(1) C(6)-C(19)-C(23) N(20)-C(1 9)-C(23) 0(1 )-C(1 9)-C(23) 0(21)-C(21)-N(20) 0(21)-C(21)-N(22) N(20)-C(21)-N(22) 0(23)-C(23)-N(22) 0(23)-C(23)-C(1 9) N(22)-C(23)-C(19) C(25)-C(24)-C(3) C(25)-C(24)-H(24A) C(3)-C(24)-H(24A) C(25)-C(24)-H(24B) C(3)-C(24)-H(24B) H(24A)-C(24)-H(24B) C(24)-C(25)-N(26) C(24)-C(25)-H(25A) N(26)-C(25)-H(25A) C(24)-C(25)-H(25B) N(26)-C(25)-H(25B) H(25A)-C(25)-H(25B) N(1 5)-C(27)-H(27A) N(15)-C(27)-H(27B) H(27A)-C(27)-H(27B) N(1 5)-C(27)-H(27C) H(27A)-C(27)-H(27C) H(27B)-C(27)-H(27C)  120.6(8) 126.0(8) 122.4(8) 119.9(10) 117.7(9) 118.1(10) 121.0 121.0 125.4(11) 124.3(9) 109.9(8) 102.4(8) 111.3 111.3 111.3 111.3 109.2 107.8(10) 110.1 110.1 110.1 110.1 108.5 118.9(8) 135.3(11) 105.8(9) 114.8(8) 110.6(8) 109.8(8) 116.2(9) 100.7(8) 103.8(8) 126.8(10) 124.0(9) 109.1(9) 130.6(11) 122.3(10) 106.2(11) 113.2(10) 108.9 108.9 108.9 108.9 107.7 107.5(10) 110.2 110.2 110.2 110.2 108.5 109.5 109.5 109.5 109.5 109.5 109.5  240  Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A  N(1 5)-C(28)-H(28A) N(15)-C(28)-H(288) H(28A)-C(28)-H(28B) N(15)-C(28)-H(28C) H(28A)-C(28)-H(28C) H(28B)-C(28)-H(28C) N(22)-C(29)-H(29A) N(22)-C(29)-H(29B) H(29A)-C(29)-H(29B) N(22)-C(29)-H(29C) H(29A)-C(29)-H(29C) H(29B)-C(29)-H(29C) N(20)-C(30)-H(30A) N(20)-C(30)-H(30B) H(30A)-C(30)-H(30B) N(20)-C(30)-H(30C) H(30A)-C(30)-H(30C) H(30B)-C(30)-H(30C) C(9)-N(1 )-C(2) C(9)-N(1)-H(1) C(2)-N(1)-H(1) C(27)-N(1 5)-C(14) C(27)-N(15)-C(16) C(14)-N(15)-C(16) C(27)-N(15)-C(28) C(14)-N(15)-C(28) C(16)-N(15)-C(28) C(21)-N(20)-C(30) C(21 )-N(20)-C(1 9) C(30)-N(20)-C(1 9) C(23)-N(22)-C(21) C(23)-N(22)-C(29) C(21 )-N(22)-C(29) C(25)-N(26)-H(26A) C(25)-N(26)-H(26B) H(26A)-N(26)-H(26B) C(25)-N(26)-H(26C) H(26A)-N(26)-H(26C) H(26B)-N(26)-H(26C) C(11)-O(1)-C(19) C(12)-O(12)-H(12) S(3A)#1 -V(1)-S(3A) S(3A)#1-V(1)-S(3B)#1 S(3A)-V(1)-S(3B)#1 S(3A)#1 -V(1 )-S(3B) S(3A)-V(1 )-S(3B) S(3B)#1-V(1 )-S(3B) S(3A)#1 -V(1 )-S(2)#1 S(3A)-V(1 )-S(2)#1 S(3B)#1-V(1)-S(2)#1 S(3B)-V(1)-S(2)#1 S(3A)#1-V(1 )-S(2) S(3A)-V(1 )-S(2) S(3B)#1 -V(1 )-S(2) S(3B)-V(1)-S(2) S(2)#1-V(1)-S(2)  109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 107.0(9) 126.5 126.5 111.4(8) 109.4(8) 105.5(8) 112.5(10) 106.9(8) 110.9(11) 123.5(10) 111.4(8) 124.9(9) 112.1(9) 122.8(10) 125.0(9) 109.5 109.5 109.5 109.5 109.5 109.5 117.4(7) 109.5 128.3(4) 36.7(4) 108.9(3) 108.9(3) 36.7(4) 113.6(6) 109.8(3) 120.5(3) 126.7(3) 117.7(4) 120.5(3) 109.8(3) 117.7(4) 126.7(3) 28.2(3)  241  Appendix: Experimental Details for X-ray Diffraction Analysis of Exipuamine A  S(3A)#1 -V(1 )-S(1) S(3A)-V(1)-S(1) S(3B)#1-V(1)-S(1) S(3B)-V(1)-S(1) S(2)#1 -V(1 )-S(1) S(2)-V(1)-S(1) S(3A)#1 -V(1 )-S(1 )#1 S(3A)-V(1 )-S(1 )#1 S(3B)#1-V(1)-S(1)#1 S(3B)-V(1 )-S(1 )#1 S(2)#1 -V(1 )-S(1 )#1 S(2)-V(1 )-S(1 )#1 S(1)-V(1)-S(1)#1 V(1)-S(1)-S(3A)#1 S(2)#1-S(2)-V(1) S(3B)-S(3A)-V(1) S(3B)-S(3A)-S(1)#1 V(1 )-S(3A)-S(1 )#1 S(3A)-S(3B)-V(1)  67.8(4) 115.2(4) 104.3(5) 79.3(5) 72.7(3) 100.9(3) 115.2(4) 67.8(4) 79.3(5) 104.3(5) 100.9(3) 72.7(3) 173.6(4) 54.4(3) 75.88(17) 72.3(6) 129.7(7) 57.8(3) 71.0(5)  Symmetry transformations used to generate equivalent atoms: #1 -x+1,y,-z+312  242  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  243  Table A.4.3. Anisotropic displacement parameters (AA2 x I 0”3) for exiguamine A. The anisotropic displacement factor exponent takes the form: -2 piA2 [hA2 a*2 Ull + + 2 h k a* b* U12] ...  C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(12) C(13) C(14) C(16) C(17) C(18) C(19) C(23) C(24) C(25) C(27) C(28) C(29) C(30) N(1) N(15) N(20) N(22) N(26) 0(1) 0(5) 0(8) 0(12) 0(21) 0(23) Cl(1) V(1) S(1) 8(2) S(3A) S(3B)  Ull 43(8) 56(9) 31(7) 39(7) 30(6) 47(8) 64(8) 28(7) 34(7) 42(7) 45(8) 64(9) 68(9) 73(9) 65(8) 21(6) 27(7) 81(10) 55(8) 75(9) 173(19) 71(9) 58(9) 58(7) 60(7) 57(6) 34(6) 66(8) 32(4) 38(5) 50(5) 40(5) 89(7) 51(5) 74(2) 37(2) 167(9) 77(5) 76(6) 42(5)  U22 56(7) 66(7) 48(6) 45(6) 33(5) 26(5) 25(5) 63(7) 26(5) 24(5) 46(7) 36(6) 51(7) 41(6) 24(5) 32(5) 76(9) 74(8) 108(10) 38(6) 155(15) 31(5) 72(8) 42(5) 36(5) 30(5) 53(5) 170(11) 50(4) 59(4) 55(4) 32(4) 57(5) 25(4) 58(2) 23(1) 79(5) 67(4) 69(4) 236(13)  U33 66(8) 44(7) 47(6) 56(7) 47(6) 47(6) 48(7) 36(6) 41(6) 65(7) 91(9) 49(7) 63(8) 77(9) 30(6) 62(7) 92(10) 70(9) 30(6) 67(8) 70(10) 58(7) 98(11) 100(8) 40(5) 59(6) 49(5) 84(8) 77(5) 63(5) 65(5) 167(9) 83(6) 80(5) 65(2) 26(1) 62(5) 86(6) 135(7) 232(14)  U23 8(6) 22(6) -4(5) 15(5) 4(4) 13(4) 21(5) 17(5) 2(4) 6(5) -29(6) 6(5) 17(6) 6(6) 5(4) 5(5) 49(8) 18(7) 5(6) -5(5) 41(10) 11(5) -16(7) 8(5) 13(4) 3(4) 20(4) 91(8) 28(4) 30(4) 3(4) 33(5) 21(4) 16(4) 6(1) 0 15(4) 23(3) -30(5) 166(11)  U13 7(6) 15(6) 13(5) 27(6) 13(5) 10(6) 19(6) 3(5) 5(5) -9(6) 24(7) 24(6) 16(7) 31(7) -4(6) 15(5) 29(7) 34(8) 12(6) 40(7) 55(12) 14(7) 18(8) 44(7) 5(5) 20(5) 15(5) 39(7) 17(4) 14(4) 16(4) 27(6) 53(5) 32(5) 33(2) 6(1) 43(5) 25(6) 62(5) 23(7)  U12 -17(6) 11(6) -10(5) 13(5) -9(4) 1(5) 6(5) -3(5) 1(4) -6(5) -25(6) -5(5) 15(7) 7(6) -6(5) -1(4) 20(6) 21(7) -11(7) 14(6) 95(13) -5(5) 16(7) -9(5) 16(5) 5(4) 4(4) 72(8) 8(3) -2(4) -16(4) -2(3) 11(4) 8(3) 17(2) 0 7(5) 19(4) -19(4) 3(6)  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A  Table A.4.4. Hydrogen coordinates ( x 10’4) and isotropic displacement parameters (AA2 x 10A3) for exiguamine A. x  H(2) H(13) H(16A) H(16B) H(17A) H(17B) H(24A) H(24B) H(25A) H(25B) H(27A) H(27B) H(27C) H(28A) H(28B) H(28C) H(29A) H(29B) H(29C) H(30A) H(30B) H(30C) H(1) H(26A) H(26B) H(26C) H(12)  3647 97 785 823 1297 1479 3649 3975 3433 3826 113 -198 117 98 538 93 2261 1861 2344 1622 1289 1786 2890 4020 4049 4353 314  y  4888 3451 8354 7976 6567 6985 1189 2679 2241 3528 6016 6506 7797 5200 6122 7086 -3195 -3172 -2490 2942 3118 3824 5778 297 1101 1580 454  z  U(eq)  5836 4389 4228 4908 4297 5016 6451 6568 7230 7386 4775 4079 4553 3386 3434 3335 6675 6889 7334 7070 6357 6610 5088 7411 7978 7693 4629  73 75 77 77 77 77 89 89 80 80 85 85 85 197 197 197 87 87 87 121 121 121 76 156 156 156 128  244  Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A  Table A.4.5. Torsion angles [deg] for exiguamine A. N(l)-C(2)-C(3)-C(4) N( I )-C(2)-C(3)-C(24) C(2)-C(3)-C(4)-C(9) C(24)-C(3)-C(4)-C(9) C(2)-C(3)-C(4)-C(5) C(24)-C(3)-C(4)-C(5) C(9)-C(4)-C(5)-O(5) C(3)-C(4)-C(5)-O(5) C(9)-C(4)-C(5)-C(6) C(3)-C(4)-C(5)-C(6) O(5)-C(5)-C(6)-C(7) C(4)-C(5)-C(6)-C(7) O(5)-C(5)-C(6)-C(1 9) C(4)-C(5)-C(6)-C(1 9) C(1 9)-C(6)-C(7)-C(1 0) C(5)-C(6)-C(7)-C(1 0) C(1 9)-C(6)-C(7)-C(8) C(5)-C(6)-C(7)-C(8) C(1 0)-C(7)-C(8)-O(8) C(6)-C(7)-C(8)-O(8) C(1 0)-C(7)-C(8)-C(9) C(6)-C(7)-C(8)-C(9) C(5)-C(4)-C(9)-N(1) C(3)-C(4)-C(9)-N(1) C(5)-C(4)-C(9)-C(8) C(3)-C(4)-C(9)-C(8) O(8)-C(8)-C(9)-N(1) C(7)-C(8)-C(9)-N(1) O(8)-C(8)-C(9)-C(4) C(7)-C(8)-C(9)-C(4) C(6)-C(7)-C(1 0)-C(1 8) C(8)-C(7)-C(10)-C(18) C(6)-C(7)-C(10)-C(1 1) C(8)-C(7)-C(10)-C(1 1) C(7)-C(1 0)-C(1 I )-O(1) C(1 8)-C(1 0)-C(1 I )-O(1) C(7)-C(10)-C(1 1)-C(12) C(18)—C(10)—C(11)—C(12) O(1)—C(11)—C(12)—O(12) C(10)-C(1 1)-C(12)-O(12) O(1)—C(11)—C(12)—C(13) C(1 0)-C(1 I )-C(1 2)-C(1 3) O(12)-C(1 2)-C(1 3)-C(14) C(1 I )-C(1 2)-C(1 3)-C(1 4) C(12)-C(1 3)-C(14)-N(1 5) C(1 2)-C(1 3)-C(14)-C(1 8) N(1 5)-C(16)-C(1 7)-C(1 8) C(7)-C(10)-C(18)-C(14) C(1 I )-C(1 0)-C(1 8)-C( 14) C(7)-C(1 0)-C(I 8)-C(1 7) C(I 1)-C(10)-C(18)-C(17) C(13)-C(14)-C(18)-C(10) N(1 5)-C(14)-C(18)-C(10) C(13)-C(14)-C(18)-C(1 7)  -6.2(13) 176.8(10) 2.8(12) 179.6(11) 179.0(11) -4(2) -173.1(10) 11(2) 2.6(15) -173.0(11) 171.6(9) -4.6(14) -6.8(14) 176.9(9) 0.7(15) -177.6(9) -170.5(9) 11.1(14) -10.1(15) 160.7(9) 174.0(10) -15.2(13) -174.9(10) 1.7(12) -8.0(17) 168.7(10) 3.5(16) 179.5(10) -161.7(10) 14.3(15) 158.2(9) -31.2(16) -14.5(14) 156.1(9) -1.5(14) -175.1(8) -1 79.7(10) 6.7(15) 2.0(16) -179.7(10) 179.1(9) -2.6(17) 176.9(11) -0.2(16) -173.6(10) -1.5(17) -29.0(12) 179.3(10) -7.8(13) 2(2) 174.7(11) 5.9(16) 179. 0(9) -175.9(11)  245  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A  N(1 5)-C(14)-C(1 8)-C(17) C(16)-C(17)-C(18)-C(1 0) C(1 6)-C(1 7)-C(1 8)-C(14) C(7)-C(6)-C(1 9)-N(20) C(5)-C(6)-C(19)-N(20) C(7)-C(6)-C(1 9)-0(1) C(5)-C(6)-C(19)-0(1) C(7)-C(6)-C(19)-C(23) C(5)-C(6)-C(19)-C(23) C(6)-C(1 9)-C(23)-0(23) N(20)-C(19)-C(23)-0(23) 0(1 )-C(1 9)-C(23)-0(23) C(6)-C( I 9)-C(23)-N(22) N(20)-C(19)-C(23)-N(22) 0(1 )-C(1 9)-C(23)-N(22) C(2)-C(3)-C(24)-C(25) C(4)-C(3)-C(24)-C(25) C(3)-C(24)-C(25)-N(26) C(4)-C(9)-N(1 )-C(2) C(8)-C(9)-N(1 )-C(2) C(3)-C(2)-N(1 )-C(9) C(1 3)-C(14)-N(1 5)-C(27) C(1 8)-C(1 4)-N(1 5)-C(27) C(1 3)—C(14)—N(1 5)—C(16) C(18)-C(14)-N(15)-C(16) C(1 3)-C(14)-N(1 5)-C(28) C(1 8)-C(14)-N(1 5)-C(28) C(1 7)-C(1 6)-N(1 5)-C(27) C(1 7)-C(1 6)-N(1 5)-C(14) C(1 7)-C(1 6)-N(1 5)-C(28) 0(21 )-C(21 )-N(20)-C(30) N(22)-C(21 )-N(20)-C(30) 0(21 )-C(21 )-N(20)-C(1 9) N(22)-C(21)-N(20)-C(19) C(6)-C(1 9)-N(20)-C(2 1) 0(1 )-C(1 9)-N(20)-C(21) C(23)-C(19)-N(20)-C(21) C(6)-C(1 9)-N(20)-C(30) 0(1 )-C(1 9)-N(20)-C(30) C(23)-C(1 9)-N(20)-C(30) 0(23)-C(23)-N(22)-C(21) C(1 9)-C(23)-N(22)-C(2 1) O(23)-C(23)-N(22)-C(29) C(1 9)-C(23)-N(22)-C(29) 0(21 )-C(21 )-N(22)-C(23) N(20)-C(21 )-N(22)-C(23) 0(21 )-C(21 )-N(22)-C(29) N(20)-C(21 )-N(22)-C(29) C(1 2)-C(1 I )-0(1 )-C(1 9) C(10)-C(11)-0(1)-C(19) C(6)-C(19)-0(1)-C(1 1) N(20)-C(19)-0(1)-C(1 1) C(23)-C(19)-0(1)-C(1 1) S(3A)-V( I )-S(1 )-S(3A)#1 S(3B)#1 -V(1 )-S(1 )-S(3A)#1 S(3B)-V(1 )-S(1 )-S(3A)#1  -2.8(12) -161.8(11) 20.5(12) -98.6(11) 79.9(11) 26.3(13) -155.2(8) 144.2(10) -37.2(13) -58.7(15) 176.6(11) 63.0(13) 131.0(10) 6.3(11) -107.3(10) 116.6(14) -59.7(16) 170.6(10) -5.4(12) -172.2(10) 7.2(13) 39.2(14) -133.8(9) 157.8(11) -15.2(11) -84.0(14) 103.0(12) 146.6(9) 26.6(12) -88.8(10) -5.3(19) 178.4(10) 178.8(11) 2.5(12) -130.9(9) 103.8(9) -5.2(11) 53.3(14) -72.1(12) 179.0(10) -174.6(12) -5.4(12) 8(2) 177.3(8) -174.3(11) 2.2(13) 3.0(17) 179.5(9) -150.5(9) 31.1(12) -41.8(12) 86.0(9) -167.1(8) 123.2(4) 3.8(4) 115.7(3)  246  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  S(2)#1 -V(1 )-S(1 )-S(3A)#1 S(2)-V( I )-S( 1 )-S(3A)#1 S(1)#1-V(1)-S(1)-S(3A)#1 S(3A)#1 -V(1 )-S(2)-S(2)#1 S(3A)-V(1)-S(2)-S(2)#1 S(3B)#1-V(1)-S(2)-S(2)#1 S(3B)-V(1)-S(2)-S(2)#1 S(1)-V(1 )-S(2)-S(2)#1 S(1)#1-V(1)-S(2)-S(2)#1 S(3A)#1 -V(1 )-S(3A)-S(3B) S(3B)#1-V(1)-S(3A)-S(3B) S(2)#1 -V(1 )-S(3A)-S(3B) S(2)-V( I )-S(3A)-S(3B) S( I )-V(1 )-S(3A)-S(38) 8(1 )#1 -V( I )-S(3A)-S(3B) S(3A)#1 -V(1 )-S(3A)-S(1 )#1 S(3B)#1 -V( I )-S(3A)-S( I )#1 S(3B)-V(1 )-S(3A)-S(1 )#1 S(2)#1 -V(1 )-S(3A)-S( I )#1 S(2)-V(1 )-S(3A)-S(1 )#I S(1 )-V(1 )-S(3A)-S(1 )#1 S(1 )#1 -S(3A)-S(3B)-V(1) S(3A)#1 -V(1 )-S(3B)-S(3A) S(3B)#1 -V( 1 )-S(3B)-S(3A) S(2)#1 -V( I )-S(3B)-S(3A) S(2)-V(1 )-S(3B)-S(3A) S(1 )-V( I )-S(3B)-S(3A) S(1)#I-V(1 )-S(3B)-S(3A)  -120.7(3) -118.6(3) -119.6(2) -74.7(10) 117.9(9) -116.8(9) 80.5(11) -4.1(10) 175.8(10) 68.4(6) 104.4(10) -96.4(6) -125.4(6) -12.4(7) 173.8(7) -105.3(3) -69.4(5) -173.8(7) 89.8(3) 60.8(3) 173.9(4) 6.9(7) -129.5(5) -90.3(7) 104.8(6) 73.0(8) 168.6(6) -6.0(6)  Symmetry transformations used to generate equivalent atoms: #1 -x+1 ,y,-z+3/2  247  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  248  Table A.4.6. Hydrogen Bonds  Donor N(1) 0(12) N(26) N(26) N(26) N(26)  H H(1) H(12) H(26A) H(26B) H(26C) H26(C)  Acceptor 0(23) *S(1) >0(2B) C1(1) *S(2) *S(2)  [ARU] [7556.01] [7546.02]  [  ]  [6546.04] [1565.02] [2666.02]  D-H 0.88 0.84 0.91 0.91 0.91 0.91  H.. .A 2.05 2.44 1.79 2.18 2.25 2.61  D. A 2.8333(13) 3.145(13) 2.685(18) 3.067(12) 3.143(17) 3.400(14) . .  D-H. 148 142 168 165 166 146  .  Translation of ARU-code to Equivalent Position Code [7556.] 1/2-x, 1/2-y, 1-z [7546.] 112-x, -lI2-y, 1-z [1565.]= x, li-y,z [2666.]= 1-x, 1+y, 312-z [6546.1= 112-x, -112+y, 312-z  A.5. References (1)  SAINT; Version 7.03A; Bruker AXS Inc.: Madison, Wisconsin, USA, 19972003.  (2)  TWINABS. Bruker Nonius scaling and absorption for twinned crystals; V1.05; BrukerAXS Inc.: Madison, Wisconsin, USA, 2003.  (3)  Altomare A.; Burla M.C.; Camalli M.; Cascarano G.L.; Giacovazzo G.L.; Guagliardi A.; Moliterni A.G.G.; Polidori G.; Spagna R. Journal of Applied Ctystallography 1999, 32, 115-119.  (4)  Lee S.C.; Li J.; Mitchell J.C.; Hoim R.H. Inorganic Chemistiy 1992, 31, 4333-4338.  (5)  Heinrich D.D.; Folting K.; Hoffman J.C.; J.G. Reynolds; Christou G. Inorganic Chemistiy 1991, 30, 300-305.  (6)  Least squares function minimized:  (7)  Standard deviation of an observation of unit weight:  Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A  249  1 [w / ) 2 (N F 1 o-N (F 2 c v)] o (8)  Cromer D.T.; Waber J.T. International Tables for X-ray Crystallography, Vol IV; The Kynoch Press: Birmingham, England, 1974, Table 2.2A.  (9)  lbers J.A.; Hamiltion W.C. Acta Ciystallographica 1964, 17, 781-782.  (10)  Creagh D.C.; McAuley W.J. In International Tables for Crystallography, Vol. C; Wilson, A. J. C., Ed.; Kiuwer Academic Publishers: Boston, 1992; 21 9-222.  (11)  Creagh D.C.; Hubbell J.H. In International Tables for Crystallography, Vol C; Wilson, A. J. C., Ed.; Kiuwer Academic Publishers: Boston, 1992, 200206.  (12)  SHELXTL; Version 5.1; BrukerAXS Inc.: Madison, Wisconsin, USA, 1997.  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 22 0
Canada 18 0
Germany 14 16
United States 10 2
Japan 3 0
Iran 2 0
Algeria 2 0
Czech Republic 2 1
India 1 0
France 1 0
Mexico 1 0
Russia 1 0
Colombia 1 1
City Views Downloads
Qingdao 16 0
Braunschweig 14 13
Vancouver 9 0
Kingston 5 0
Beijing 5 0
Unknown 4 3
Montreal 4 0
Tokyo 3 0
Ashburn 2 0
Burlington 2 0
Redmond 2 0
Cambridge 2 0
Ceska 2 1

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0061735/manifest

Comment

Related Items