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Bioactive natural products from nature 2007

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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). IDO is an enzyme expressed by tumor cells to evade the immune system. Inhibitors against this enzyme may allow the immune system to attack cancer cells, making this enzyme a potential drug target for anti-cancer agents. 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. 2.58 2.59 III 39 Inhibitors of the G2 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 G2 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 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 3.22 4.35 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 5.21 5.22 5.23 5.24 5.25 XyIose 2 5.26 VTable 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 3.15References.130 Chapter 4: Structure Elucidation of G2 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 G2 Checkpoint Inhibitors 136 4.5. Known G2 Checkpoint Inhibitors 139 4.6. Description of the G2 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-d6 72 Table 3.5.1. 1H chemical shift values for 3.9, and the literature 1H 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 C6D 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 xList 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. 1H NMR spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 40 Figure 2.7.4. 13C NMR spectrum of exiguamine A (2.58) acquired at 150 MHz in DMSO-d6 41 Figure 2.7.5. DEPT spectrum of exiguamine A (2.58) acquired at 150 MHz in DMSO-d6 42 Figure 2.7.6. HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 43 Figure 2.7.7. HMBC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 44 Figure 2.7.8. COSY spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 45 Figure 2.7.9. 1H, 15N LR-HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 46 Figure 2.7.10. (a) 1H and (b) ‘3C and 15N 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 1H, 15N 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) 1H and (b) 13C and 15N 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 1H, 15N 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) 1H and (b) 13C and 15N 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 15N 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. 1H NMR spectrum of exiguamine B (2.59) run at 600 MHz in DMSO-d6 66 Figure 2.8.4. ‘3C NMR spectrum of exiguamine B run at 150 MHz in DMSO-d6. 67 Figure 2.8.5. DEPT spectrum of exiguamine B run at 150 MHz in DMSO-d6... 68 Figure 2.8.6. HMQC spectrum of exiguamine B run at 600 MHz in DMSO-d6..69 Figure 2.8.7. HMBC spectrum of exiguamine B run at 600 MHz in DMSO-d6.. 70 XIII Figure 2.8.8. COSY spectrum of exiguamine B run at 600 MHz in DMSO-d6... 71 Figure 2.8.9. (a) 1H NMR and (b) 13C 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) 1H NMR and (b) 13C 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) 1H NMR and (b) 13C 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. 1H NMR of expansions of exiguamineB.88 Figure 2.10.6. 1H 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. 1H NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 600 MHz in DMSO-d6 104 Figure 3.5.2. 13C NMR spectrum of cyclo(S-Val-S-Phe) (3.9) acquired at 150 MHz in DMSO-d6 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. G2/M transition 135 Figure 4.3.2. G2 checkpoint pathway 136 Figure 4.4.1. Rationale for using G2 checkpoint inhibitors 138 Figure 4.5.1. ATM/ATR inhibitors of the G2 checkpoint pathway 139 Figure 4.5.2. Indole alkaloids inhibiting the G2 checkpoint through Chkl 140 Figure 4.5.3 Alkaloids inhibiting the G2 checkpoint through Chkl 141 Figure 4.5.4 Polyketide derived G2 checkpoint inhibitors 142 Figure 4.6.1. Description of the G2 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. 1H NMR spectrum of N-methylguatterine at 500 MHz in DMSO-d6. 149 Figure 4.9.4. “3C spectrum NMR of N-methylguatterine at 100 MHz in DMSO-d6. 150 xvi Figure 4.9.5. HMQC spectrum of N-methylguatterine at 500 MHz in DMSO-d6. 151 Figure 4.9.6. HMBC spectrum of N-methylguatterine at 500 MHz in DMSO-d6. 152 Figure 4.9.7. COSY spectrum of N-methylguatterine at 500 MHz in DMSO-d6. 153 Figure 4.9.8. (a) 1H chemical shifts and coupling constants for substructure I and(b) 13C 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) 1H chemical shifts and (b) 13C 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. 1H NMR of substructures III (a) and IV (b) 161 Figure 4.9.17. 13C 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. 13C 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 G2 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. 1H NMR spectrum of abolenone (5.25) at 600 MHz in C6D 183 Figure 5.5.4. 13C NMR spectrum of abolenone (5.25) at 150 MHz in C6D 184 Figure 5.5.5. HMQC spectrum of abolenone (5.25) at 600 MHz in C6D 185 Figure 5.5.6. HMBC spectrum of abolenone (5.25) at 600 MHz in C6D 186 Figure 5.5.7. COSY spectrum of abolenone (5.25) at 600 MHz in C6D 187 Figure 5.5.8. (a)1H and (b)13C chemical shifts of substructure I of abolenone(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)1H and (b)’3C chemical shifts of substructure II of abolenone(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)1H and (b)13C chemical shifts of substructure Ill of abolenone(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. 1H NMR spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D 200 Figure 5.6.4. 13C NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C6D 201 Figure 5.6.5. DEPT NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C6D 202 Figure 5.6.6. HMQC spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D 203 Figure 5.6.7. HMBC spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D 204 xix Figure 5.6.8. COSY spectrum of myrmekioside C peracetate (5.28) at 600 MHzin C6D 205 Figure 5.6.9. (a)1H chemical shifts and coupling constants and (b) 13C 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)1H chemical shifts and coupling constants and (b) 13C 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) 1H chemical shifts and coupling constants and (b) 13C 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) 1H chemical shifts and coupling constants and (b) 13C 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) 1H chemical shifts and coupling constants and (b) ‘3C 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 I D -one dimensional 2D -two dimensional (-) -negative optical rotation (+) -positive optical rotation 1H -proton 1H, 15N LR-HMQC -(1H, 15N) 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 C6D -deuterated benzene calc’d -calculated CD -circular dichroism CDCI3 -deuterated chloroform cdc2 -cyclin-dependent kinase I cdc25c -cell division cycle 25C kinase CH -methine CH2 -methylene CH3 -methyl CH2I -methylene chloride CHCI3 -chloroform CH3NO2 -nitromethane ChkI -CHKI checkpoint homolog Chk2 -CHK2 checkpoint homolog cm -centimeter(s) CNS -central nervous system xxiii coil no -collection number COSY (1H, 1H) 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 DMSO-d6 -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 EC50 -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 G1 -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 [3H]-DHT -tritium labeled d ihydrotestoterone His -histidine H20 -water HL-60 -human leukemia cell line HMBC -(1H, 13C) heteronuclear multiple bond coherence HMQC -(1H, 13C) 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 H2S04 -sulphuric acid H-X -hydrogen number X Hz -hertz IC50 -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 LC50 -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 m3 -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) MgSO4 -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 15N -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 NH4CI -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 - RhoA associated coiled-coil-containing protein kinase s -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 sp2 -sp2 hybrid orbital sp3 -sp3 hybrid orbital 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 1981- 2002, 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 discovered.4 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 compounds.5 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 cancers.8’9 Vinblastine (1.4, Figure 1.2.2) and vincristine (1.5, Figure 1.2.2) are 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.1°Other plant entities in clinical 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 4 9 HOO OH OH c; o H3CO j ‘: OCOCH3 N 1’CO2CH31 HQH 1.3 1.4 CH3 ONZ H3CO2 / 0 COjJ\ H QcocH HJ 1.5 ,I 1<,CO2CH3 1.6 CHO HO 0 1.81.7 ‘ 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 5 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. Microorganisms have not only 1.9 1.10 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 Gram- positive 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 genus.2° Marinomycin A (1.18) was found to be the most potent antibacterial 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 cytotoxicity.2° 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. Very recently, the symbiotic bacteria were cultivated and numerous rhizoxin Chapter 1: Introduction to the Field of Natural Products 8 derivatives were isolated from the bacteria. 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 CIHOH: OH 0 OH 0 1.13 CH2O OH HO-7j HO OHHO_N7 HO 1.16 HO OHOH ‘CHO I .12 HOO 1.15 Figure 1.3.1. Significant natural products isolated from microorganisms. ChaDter 1: Introduction to the Field of Natural Products 9 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 1.17 HO 118 1.19 “COOCH3 “COOCH3 OH 1.21 1.22 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 sarcoma.25’6 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 G1 or G2 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 11 0 0 NH2 HN HN HO 0 N HO 0” ‘N HO 0 N 1.26 1.27 1.28 / 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 (LC50= 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 H3C, 1.29 1.30 Chantar 1 introduction to tha Fiald of Natural Products 12 vivo inhibitor of angiogenesis.3°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 angiogenesis.31 OH o0H oo H2N0 OH rfl<. 1.32 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 1.33 Chapter 1: Introduction to the Field of Natural Products 13 candidates against cancer.11 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 cornpound.32 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 nature.34’5 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 response.4’33 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 Repeated Separatioi Repeated Bioassays I Inactive Fractions Jr No Purification Separation 4 Bioassay/I\\Bioassay I Pure Bioactive Compound(s) [ No[Purification II Figure 1.6.1. Procedure for bioassay guided fractionation. 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 G2 checkpoint. Both the G1 and the G2 checkpoints are involved in repairing damaged DNA. It has been found that most tumors lack the Ci checkpoint, so inhibitors of the G2 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, N-methylguatterine.37 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 5cL- dihydrotestosterone. 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. 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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, 1693- 1713. (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 theMarine Sponge Neopetrosia exiqua 20 Chapter 2: Isolation of Inhibitors of Indoleamine-2,3- dioxygenase (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 immune 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 (Figure 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 R. van; Matairiaho T.; Mauk A.G.; Andersen R.J. Journal of the American Chemical Society 2006,128, 16046-16047. Copyright 2006 American Chemical Society. Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from theMarine Sponge Neopetrosia exiqua 21 indoleamine-2,3-dioxygenase (lDO), which is found in the epididymis, thymus, 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 G1 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 tryptophan).5 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 Kynurenine 3-hydroxylase Kynurenine CO2H CO2H_3-hydroxyanthranilic acid [cooH Kynureninase N CO2H 2 NH2 Quinolinic acid OH OH Quinolinic-acid 3-hydroxyanthranhlic acid 3-Hydroxykynurenine phosphoribosy! transferase 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 apoptosis.8 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 C1 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 Tryptophan N-formyl kynurenine lH2 NH2 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 23 cancer11 have been found to have a poor prognosis for disease progression and 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 cataracts.14 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 a,3 unsaturated ketone NH3 2 NH2 C00H Cys-Kyn adduct CH2 Protein Figure 2.2.2. Mechanism of formation of adduct between a—crystalin and kynurenine (Adapted from Truscott).15 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 Kynurenine 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 methyithiohydantoin tryptophan (2.4).18 All these inhibitors have K values ranging between 11-40 tM. 00C 00C +H3N 2.1 -CCC 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 K1’s of 3.3 and 7.4 jiM, respectively.19 Unfortunately, these compounds have unfavorable side effects in the central nervous system making them unlikely to be used for cancer treatment.2° 2.2 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from theMarine Sponge Neopetrosia exqua 26 0 F. / \ /r\ N N z— N N H H 2.5 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 K1’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 activity.21 The natural product brassinin (2.10) was identified as an IDO inhibitor and has a K of 97.7 1iM.22 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 brassinin.22 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 /\ H 2.7 2.8 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 model.18 Signficant tumor regression was observed when combining 1-methyl 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 2.9 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 28 be more effective in tumor regression.23 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 IC50 of 40 nglmL.24 Furthermore, discorhabdin C was found to be an antibacterial agent with activity against both Gram-positive and Gram-negative bacteria.25 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 IC50 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 Carribean.28 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 (1C50 = 0.42-20 j.igfmL), and having anti fungal activity against Candida albicans (MIC = 3.1-50 ig/mL).3° Other structures 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 batzellines.32 SMe SMe N—a, HN-\ N— Th oz 2.17 2.18 2.19 ‘ SMe ‘ SMe N oç H2N—N’ H2N—N H2N-N 2.20 2.21 2.23 HN—__ OH HN—O. Q OHjj OH OH H2N j’NH H2N’O CI CI 2.24 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 present.33 The pyrroloquinone veiutamine (2.34) along with makaluvamines A-D were isolated from Zyzza fuliginosa collected in Fiji.34 Veiutamine (2.34) has a unique substitution pattern compared with 2.22 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 NH2 NH2 NH2 N OH 2.32 2.33 Br 0 NH2 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 (1C50 = 61 ig/mL).36 2.31 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 32 IMe ‘OH )oHHN—/ MeN— NMe NH 2.35 2.36 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 (1C50 = 0.5 ig/mL), an inhibitor of topoisomerase II, and a antimicrobial agent against Bacillus subtillus (MIC = 0.3 1igImL).37 Tsitsikammamines A (2.39) and B (2.40) were isolated from a South African Latrunculid sponge.38 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 (1C50 25 pgImL). This may indicate that the presence of the imine is vital for the cytotoxic activity of the pyrroloimminoquinones.33’5 OH OH 2.37 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 spicules.41 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 m3 in size.42’3 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 vasodilators.44 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 lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 33 <x5 OH OH 2.38 2.39 2.40 Ji OH OHC Me OH 2.41 2.432.42 2.44 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 tuberculosis.45 Araguspongine M (2.53) was isolated from N. exigua collected in Palau. This alkaloid showed cytotoxic activity against the human leukemia cell line HL-60 with an IC50 value of 5.5 M, but did not show any anti-bacterial activity.42 Xestosin A (2.54) was isolated from a N. exigua sample collected in 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 H/ H1 H°t H°t H°t 2.45 2.46 o 2.47 2.48 HO 2.49 2.50 2.51 H’H° 2.52 2.53 2.54 OCH32.55 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 H20, 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. H2r 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 29 /‘4 o ‘N2° ‘-‘H2N 26 2cçioH H 19 1 0 /13 17’ \ 15 —N—- 27 161G 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 C25H6N506 (calc’d 492.1883). 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 1H NMR spectrum (Figure 2.7.3) of exiguamine A acquired in DMSO-d6 at 600 MHz displayed an indole proton (oH 13.10), a phenolic proton (OH 10.42), two amine protons (OH 7.82), two protons connected to sp2 hybridized carbons (OH 7.52 and 7.30), and a series of methine and methyl protons connected to sp3 hybridized carbons attached to either a nitrogen or an sp2 hybridized carbon (OH 2.44-4.17). The “3C NMR spectrum (Figure 2.7.4) indicated the presence of 25 carbons, 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 1H, 15N 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 CH3NO2,revealed five nitrogens (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 1H, 15N LR-HMQC spectra. o / IIII II Figure 2.7.2. Three substructures of exiguamine A. 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 EC’4 z Q.. Figure 2.7.3. 1H NMR spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6. C ) 0 / c I , N \ 9 ’N % Q H2 OH . g N H I 0 Co CD a C 3 o - CD C. C D CD CD C. ) 9. ‘ TI 00 CD 0) _ _ _ _ _ _ _ _ _ _ U H I$ IN _ _ _ _ _ _ _ _ I CO CD a r ; )i .p i sr a r p IW _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I I C, ’ C) 15 0 10 0 50 z N Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 42 -e In I Figure 2.7.5. DEPT spectrum of exiguamine A (2.58) acquired at 150 MHz in DMSO-d6 0 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 43 ppm 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 —50 —100 ppm Figure 2.7.6. HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6. + + + Chapter 2: Isolation of Inhibitors of lndoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 44 150 Figure 2.7.7. HMBC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6. o / 4 0I 4 I 8 100 : ppm 10.0 + 5.0 ppm Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 45 ppm 10.0 -10.0 ppm Figure 2.7.8. COSY spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6. / -N e 1 + + + 5.0 Figure 2.7.9. 1H, 15N LR-HMQC spectrum of exiguamine A (2.58) acquired at 600 MHz in DMSO-d6 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 46 Q 0 0 0 liii,, I I 0 0 a) 0 0 U) 0 U) 0 0 U) 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 8H (J in Hz) 1H, 13C-HMBC 1H, 15N- COSY HMQC 1 -218 13.10, brs C-2, C-3, C-4 C- H-2 5, C-8, C-9 2 126.5 7.30, d, C-3, C-4, C-5, C- N-I H-I (2.2) 8, C-9, C-24 3 120.7 4C 121.3 5d 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 7.52, brs C-ID, C-Il, C-12, N-15 C-14, C-18 14e 142.8 15 -310 16 67.4 3.84, q, C-17,C-18, C-27, H-16a, H (10.8) C-28 17b 16b 4.17, t, C-14, C-17, C-18, H-16b, H (8.6) C-28 17a 17 28.5 3.22, bdd C-b, C-14, C-16, N-15 H-16a, H- (16.9, 7.5) C-18 17b Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Svonae Neopetrosia exigua 48 Position 8C oN OH (J in Hz) 1H, 13C-HMBC 1H, 15N- COSY HMQC 17b 3.73, m C-10, C-13, C-14, C-16, C-18 18 122.8 19 85.4 20 -275 21 154.5 22 -248 23 168.6 24a 23.3 2.92, m C-2, C-3, C-25 N-26 24b 3.02, m C-2, C-3, C-25 25 38.3 2.99, m C-3, C-24 N-26 26 -349 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 12-OH 10.42,brs C-11,C-12,C-13 a 1H and 13C chemical shifts [ppm] are referenced to DMSO-d6(6H 2.50 and O 39.51 ppm respectively) bThe 15N spectrum was not calibrated with an external standard. The 6 value has an accuracy of about 1 ppm in reference to CH3NO2(0 ppm). 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 -LOHlO.42,b ‘12.7QH I I 1147 /L% J%% 122)* 108.7 3., bdd (16.9, 7.5 Hz) ç 7.52, S 28.5 7142.8 3.84, q (10.78 Hz) 67.4 -310 4.17, t (8.57) 3.51, S 53.2 a b Figure 2.7.10. (a) 1H and (b) 13C and 15N chemical shifts of substructure I of exiguamine A (2.58). 1QH 10H 27 17\ (Th H M BC Figure 2.7.11. Key HMBC and COSY correlations of substructure I exiguamine A (2.58). Two singlet proton resonances at 6H 3.43 (H-27: HMQC to 54.3) and 8H 3.51 (H-28: HMQC to c 53.2) displayed 1H, 15N, 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 OH 3.42 (H-27) and 0H 3.51 (H-28) showed HMBC cross-peaks to the sp2 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). Three bond HMBC correlations between the proton resonances at 8H 3.42 (H-27) and OH 3.51 (H-28) and the methylene carbon resonance at 0c 67.4 (C-16) established the bond between C-16 (Oc 67.4) and N-15 (ON -310). Both methylene proton resonances at OH 3.84 (H-16a: HMQC to Oc 67.4) and 0H 4.17 (H-16b: HMQC to 0c 67.4) showed COSY correlations to the proton resonances at 8H 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 8H 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 0H 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 (öc 114.7) and C-18 (öc 122.8). An aromatic methine proton resonance at H 7.52 (H-13: HMQC to öc 108.7) displayed HMBC correlations with the carbon resonance at ö 142.8 (C-14) and 1H, 15N 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 0H 7.52 (H-13) and the quaternary aromatic carbon resonance at Oc 114.7 (C-b) (Figure 2.7.14). This assigned C-b next to C-Il and revealed substructure I (Figures 2.7.10 and 2.7.11). ppm 7,0 6.0 5.0 4.0 3.0 Figure 2.7.12. Expansion of the 1H, 15N LR-HMQC spectrum of the key correlations of substructure I of exiguamine A (2.58). H-I 3 H-28 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 52 -315. L -305. --.300. pm Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 53 4.50ppm 4.00 3.50 3.00 —3.00 3.50 -4.00 -4.50 ppm Figure 2.7.13. COSY expansion exiguamine A (258). of the correlations for substructure I of H-17b H-16a 0 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopotrosia exigua 54 H-I 3 - c-I 0 c-I 8 C-I 2/14 c-Il- 7.600 ppm Figure 2.7.14. HMBC exiguamine A (2.58). 7.82, br 112 Fl 7.30, d(2.22 Hz) 7.550 7.500 ..-110 -120 —130 —140 —150 ppm correlations observed for H-13 of substructure I of 38.3 H2N’ 126 -218 N H II b 0 Figure 2.7.15. (a) 1H and (b) 13C and 15N chemical shifts of substructure II of exiguamine A (2.58). 2.99, m 2.92, m 3.02,m Q 13.10,br 0 a Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 55 cosy ‘HMBC Figure 2.7.16. Key HMBC and COSY correlations of substructure II of exiguamine A (2.58). The methylene proton resonance at 6H 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 8N -349 (N-26). This confirmed C-25 (0c 38.3) was adjacent to an NH2 moiety (N-26). The methylene proton resonance at OH 2.99 (H-25) displayed COSY correlations to both proton resonances at 8H 3.02 (H-24a: HMQC to öc 23.3) and 0H 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), 8H 2.92 (H-24b) and oH 2.99 (H-25) showed HMBC correlations to the sp2 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 (8c 120.7) was deduced from three bond HMBC correlations between both methylene proton resonances at oH 3.02 (H-24a) and 8H 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 0 26 8 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 56 6H 7.30 (H-2: HMQC to öc 126.5) and the exchangeable proton resonance at 3H 13.10 (H-I) (Figure 2.7.18). Additional 1H, 15N, LR-HMQC correlations were observed between 6H 7.30 (H-2) and 6N -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 8H 13.10 (H-I) and oH 7.30 (H-2) showed HMBC correlations to the quaternary sp2 hybridized carbon resonance at 8c 131.6 (C-9), thereby, allowing the determination of the N-I (ON -218) and C-9 bond (8c 131.6). Both proton resonances at OH 13.10 (H-I) and 8H 7.30 (H-2) displayed additional HMBC cross-peaks to the carbon resonating at Oc 121.3 (C-4). This established that C- 3 (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 Figure 2.7.17. Expansion of the 1H, 15N LR-HMQC spectrum of the key correlations of substructure II of exiguamine A (2.58).. N-26 400 0 - -250 7.0ppm 6.0 50 4.0 3.0 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 Figure 2.7.18. Key COSY correlation for substructure II of exiguamine A (2.58). 11!1’. 13.0 12.0 11.0 10.0 9.0 8.0 7.0ppm Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exiqua 59 Figure 27.19. HMBC correlations observed for H-I of substructure II of exiguamine A (2.58). Figure 2.7.20. (a) 1H and (b) 13C and 15N chemical shifts of substructure Ill of exiguamine A (2.58). H-i C-3 C-4 25.20 154.526.0 ‘c:3;1____ N-248 3.07, s0 / 2.44,s 10 N 8 168.6 0 a b Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Stjonge Neopetrosia exigua 60 HMBC 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 1H, 15N LR-HMQC correlations to oN -248 (N-22), which established an N-methyl moiety (Figure 2.7.22). Three bond HMBC couplings between the methyl proton resonance at 0H 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 0H 2.44 (H-29: HMQC to 0c 26.0) and 8N -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 8H 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 sp3 hybridized carbon connected to two heteroatoms. Since all the nitrogens of exiguamine A were accounted for, rr 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 N-20 Figure 2.7.22. Expansion of the 15N LR-HMQC spectrum of the key correlations of substructure III of exiguamine A (2.58). 26O --250 ppm 2.50 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’... — - — -iIr -100 -150 ppm 3.10 3.00 2.90 2.80 2.70 2.60 2.50 2.40ppm 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 C-21 C-23 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 H2N26 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 ofC25H6N507(calc’d 508.1832). This differs 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 1H NMR spectrum (Figure 2.8.3) of exiguamine B acquired in DMSO-d6 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 13C NMR spectra (Figure 2.8.4) run in DMSO-d6 at 150 MHz contained 25 carbon resonances. 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 (0c 148.5, 143.7, 142.8, 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 \N’ HOj NN O I ii Figure 2.8.2. Three substructures of exiguamine B (2.59). III o / H2N 9 ’N o H H O Co z z 2J Cl) - o CD g —‘ C B 0 -t CD x C B D CD w r%) 01 CD 1 C 0) 0 0 z N C D ’ I. 1 D C I) 0 C t - 0• 0 0 CD - C D O 0 CD 3 () I. J K pp m 10 .0 5. 0 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neoøetrosia exiqua 67 -o In I Figure 2.84. 13C NMR spectrum of exiguamine B (2.59) run at 150 MHz in DMSO-d6. 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 d6. Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 69 Q\ / H2N”\ 0 N / 0 N 0H H o H0’ Figure 2.8.& d6. HMQC spectrum of exiguamine B (2.59) run at 600 MHz in DMSO 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0ppm Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 70 o / Hn H0’ / [ H -50 -100 -150 -200 ppm Figure 2.8.7. HMBC spectrum of exiguamine B (2.59) run at 600 MHz in DMSO d6. I p p. • . 4 ppm 10.0 5.0 Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (lDO) from the Marine Sponge Neopetrosia exigua 71 0 / —5.0 a •1 if $1 K 0 —10.0 +00 _______ _____ _________________________________ __ ______ __ ______ __ __ __ __ __ __ ppm 10.0 5.0ppm Figure 2.8.8. COSY spectrum of exiguamine B (2.59) run at 600 MHz in DMSO d6. Position I 2 3 5C 6 7 8’ 10 11 12 13 14 15 I 6a I 6b 17 18 19 20 21 22 Table 2.8.1. 1D and 8H (J in Hz) 13.10, brs 7.35 ,d, (2.2)126.9 121.0 121.9 173.0 130.7 138.7 179.4 131.4 113.8 148.5 143.7 108.6 7.62, s 142.8 73.5 4.45, dd, (12.4 Hz, 5.8) 3.95, dd, (12.4, 2.5) 69.1 5.75, m 125.3 85.5 154.5 168.4 23.3 38.3 C-18 H-16b, H-17 H-16a, H-17 H-16a, H-16b, 17-OH H-25 H-24, H-26 H-25 Chapter 2: Isolation of Inhibitors of lndoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 72 2D NMR data of Exiguamine B8 1H, 13C-HMBC COSY C-2, C-3, C-4, C-9 H-2 C-3, C-4, C-9 H-2 C-b, C-Il, C-12, C-14, C-14, C-18, C-27, C-28 C-14, C-18, C-17, C-28 C-28 C-27 23 24 2.94, m C-2, C-3, C-4, C-25 25 3.04, m C-3, C-24 26 7.79, brs C-24, C-25 27” 55.4 3.58, s C-14, C-16, 28” 57.3 3.53, s C-14, C-16, 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 1H and 1C chemical shifts [ppm] are referenced to the DMSO-d6(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 O 3.1O,s/ 2.44,s J%r a Figure 2.8.9. (a) 1H NMR and (b) 13C NMR assignments for substructure I of exiguamine B (2.59). Figure 2.8.10. Key HMBC correlations observed for substructure I of exiguamine B (2.59). 25.30 26.2 / 168.4N 85.5 r0 b 29 (ThHMBC 0 The 1H 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 N- methyl moiety adjacent to two carbonyls (Figures 2.9.10 and 2.9.11). The 1H 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 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 8H 2.44 (H-29) and the carbon resonance at 0c 85.5 (C-19) (Figures 2.9.10 and 2.9.11). The 13C 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 ________ 4 _________________________ ) __________ __ ‘.jL_____ C-19- - :1o0 150 C-21— - C-23-— ____ ppm I I I 3.00 2.50ppm 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 H2N H2N 3.04, m 2.94, m 7.35, d, 2.21 Hz “ 13.10,br [-I a Figure 2.8.12. (a) 1H NMR and (b) 13C 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 1H 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 H- 26 (OH 7.82, brs) in exiguamine A (2.58). Therefore, one can establish the presence of a primary amine. The proton resonance at 3H 7.79 (H-26) showed COSY correlations to the multiplet resonating at oH 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 23.3 126.9 N H b 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 sp2 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 (8c 126.9) and C-3 (Oc 121.0) was deduced from three bond HMBC correlations between the multiplet resonating at 0H 2.94 (H-24) and the sp2 hybridized carbon resonance at Oc 126.9 (C-2). Both proton resonances at OH 7.35 (H-2: HMQC to 0c 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 0H 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 8H 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 8H 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). ppm 7.0 8.0 9.0 10.0 11.0 12.0 13.0 rPPm Figure 2.8.14. Key COSY correlation of substructure II of exiguamine B (2.59). 13.0 12.0 11.0 10.0 9.0 8.0 7.0 Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 78 OH 5.75m* S 6.07, d, 4.97 Hz HO \— I\i— 4.45, dd. 12.4 and 5.81 Hz 3.95,dd, 12.4 and2.49 Hz I Figure 2.8.16. (a) 1H NMR and (b) 13C NMR of substructure III of exiguamine B(2.59). j C-2 C -: —110 i. -120 —130 H 140 r ppm Figure 2.8.15. HMBC correlations for (2.59). 13.250 13.200 13.150 13.100 13.050 13.000 12.950 H-I of substructure II of exiguamine B j485QH 143.7 125.3 11108.6 HO/N 69.1\ I 75.5 I 3.53, S a 57.3 b 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 6H 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 8c 73.5 (C-16), thereby placing C-16 (8c 73.5) next to the N dimethyl moiety. All four proton resonances at 8H 445 (H-16a: HMQC to 0c 73.5), 3.95 (H-16b: HMQC to 0c 73.5), 8H 3.58 (H-27) and 3.51 (H-28) showed HMBC correlations to the quaternary sp2 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 8H 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 6H 5.75 (H-17) and the exchangeable proton resonance at 6H 6.07 (17-OH). The proton resonances at oH 4.45 (H-16a), 3.95 (H-16b) and OH 6.07 (17-OH) all showed HMBC correlations to the carbon resonance at O 125.3 (C- 18), 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 3H 4.45 (12-OH) and the aromatic carbon resonance at 80 143.7 (C-12). The phenolic proton resonance at 8H 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 8H 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 C- 14 (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 8H 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 8H 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). ppm -3.50 —4.00 r 1- 4.50 - 5.00 -5.50 —6.00 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. —= L120 c-I 8— - — -130 : : ___ -140 CIl — c -150 -160 __ ppm —rT I I 7.700 7.650 7.600 7.550ppm 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 (ID0) from the Marine Sponge Neopetrosia exigua 84 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 2.64 NH2 OH 2.67 Figure 2.9.1. Proposed biogenesis of exiguamine A. Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from theMarine 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 mixture (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. E 1.5 0.5 0 —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 (ID0) from the Marine Sponge Neopetrosia exiqua 86 O H2NL N H3N’ \ jjN , N OH H 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. -N-— 0 1 Chapter 2: Isolation of Inhibitors of Indoleamine-2, 3-dioxygenase (IDO) from the Marine Sponge Neopetrosia ex,gua 87 Observing the 1H 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. -5 Wavelength (nm) Figure 2.10.3. CD-spectrum of exiguamine B. Q /‘H3N 0 H® HO\N H2Q ®I /) H0 Iii £ 4 3 2 0 E E U r :i 510 610 -3 -4 HO Figure 2.10.4. Proposed mechanism of isomerization for C-17. Chapter 2: Isolation of Inhibitors of Indoleamine-2,3-dioxygenase (IDO) from the Marine Sponge Neopetrosia exigua 88 i-i H-26 H-2 --— 3 7.00 750 730 7.00 7.50 7.40 73000 a Figure 2.10.5. 1H 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. H-i 7 Figure 2.10.6. 1H 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 17-OH fr ppm 6.10 6.00 5.90 5.80 5.70 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 to the fluorescing kynurenine N-p dimethylaminobenzylidene (2.75). The concentration of this compound was measured at 480 nm, and gave an indication of the activity of IDO.50’1 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. Desired 0 COCY - ctextract + ________ TCA inhibition NH 60°C, 10 mm NH2 H 2.73 2.72 o coo- 0 H + __ _ ____ 75monitor:d:t activityofiDO .N 2.73 2.74 Figure 2.11.1. Description of the chemical reactions present in the in vitro IDO inhibition assay. 2.66 2.75 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 C18 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 60F2 (normal phase). TLC was visualized using either a dip solution of p-an isaldehyde (1% p-an isaldehyde, 2% H2S04, 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 (1H, 13C, 15N) cryoprobe. NMR solvents were purchased from Cambridge Isotope laboratories and were referenced to solvent peaks for DMSO-d6(6c 39.5 ppm and 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 H20, 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 (H20 to MeOH) to attain 300 mg of the active fraction. The bioactive material was then subjected to gradient reversed phase HPLC (lnertsil C18, 9.4 X 250 mm, H20 to ACN in 0.1% TFA, UV detection at 254 nm) giving 98.3 mg. Finally, this material was purified by reversed phase HPLC (lnertsil C18, 9.4 X 250 mm, 9:1:0.1 H20: 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 for256N506492.1883); 1H and ‘3C NMR data see Table 2.7.1. Exiguamine B (2.59): UV (MeOH) ?max (log e) 215 (3.54), 267 (2.98), 332 (2.69) nm; [a]o230(c 3.3, MeOH); CD (MeOH, 0.3 mg!mL) no absorption; HRESIMS [M]m/z 508.1850 (calc’d forC25H6N507508.1832); ‘H and ‘3C NMR data see 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, 326- 333. 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, 2841- 2888. (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, 1131- 1133. (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, 4596- 4597. (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, 245- 258. (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 injury.1 This chapter will discuss the isolation and synthesis of compounds that 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 unclear.1 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 neurons.5 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 K1 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 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. Inhibition of Neurite Outgrowth Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 100 H2N H 3.1 3.2 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 acting on the hypothalamus to reduce the intake of food.1° çNH2 /(O ° N NNHHNO NH N ‘0 3.3 3.4 Figure 3.3.1. TRH (3) and Cyclo(S-His-S-Pro). 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. TRH increases cerebral blood flow as a neuroprotective mechanism. 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-tert- butyltyrosine. 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 102 even at 100 times the optimal therapeutic dosage. 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 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: H20 mixture and then partitioned with hexanes. The MeOH/H20 partition showed axonal outgrowth activity and was subjected to size exclusion chromatography, flash reversed-phase column chromatography and reversed- phase 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 HN 3i 9 15 Z 6 NH 16 10 0 3.9 Figure 3.4.1. Cyclo(S-VaI-S-Phe) (3.9), a compound promoting axonal outgrowth. 3.5 3.6 3.7 3.8 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 C14HONa (calc’d for 269.1266) and requiring seven degrees of unsaturation. The 1H NMR spectrum was acquired in DMSO-d6at 600 MHz. Examination of the 1H NMR spectrum (Figure 3.5.1) revealed two exchangeable protons (6H 8.11 and 7.91), five aromatic protons (oH 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 ‘3C NMR (Figure 3.5.2) and the HMQC spectra indicated the presence of two carbonyls (Oc 166.5 and 166.3), one quaternary carbon (8c 136.2), six methine carbon resonances (Oc 130.2, 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 1H NMR data of 3.9 to previously published data of cyclo(S-Val-S-Phe)14and cyclo(S-Val R-Phe)15 established a cis-diketopiperazine (Table 3.6.1). The optical rotation of 3.9 ([c]i? -45.82 (c 0.3, DMSO)) was similar to that found for cyclo(S-Val-S-Phe) ([c]D22 -43.30 (c 0.3, DMSO)) in the literature,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 I I I I I I I I I I I 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0ppm Figure 3.5.1. 1H NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 600 MHz in DMSO-d5. aNH 100 50pp Figure 3.5.2. 13C NMR spectrum of cyclo(S-VaI-S-Phe) (3.9) acquired at 150 MHz in DMSO-d6 Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 105 Table 3.5.1. 1H chemical shift values for 3.9, and the literature 1H 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)14 Val-R-Phe)15 Position 8H (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,m8b 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 1H chemical shifts [ppm] were all referenced to DMSO-d6(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 106 silica gel chromatography to afford the protected diketopiperazine 3.11 (cD22: - 49.8 (C 0.8, CHCI3)) 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, ccD2: -65.4 (c 0.25, DMSO); 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 values.16 Chaoter 3: Isolation of Comoounds That Can Induce Neurite Outarowth 107 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) 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 2,6- di-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 3.10 3.11 (ii)di-tert-butylphenol 3.12 Ph 6 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 (cD22: -45.82 (c 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 The optical rotation of diketopiperazine 3.21 (cD21: 69.3, (c 0.3 DMSO)) was similar, but opposite in sign, to diketopiperazine 3.14 (cLD21: -65.4, (c 0.25 DMSO)).16 This confirmed that these two 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 (ctD22: 43.45 (c 0.29, DMSO)) had an optical rotation that was similar but opposite in sign to that of 3.9 (ctD22: -45.82 (c 0.3, DMSO)). This established that both molecules are enantiomers; thus, the absolute stereochemistry of cyclo(R-Val-R-Phe) was confirmed. Ph 3.21 CAN, ACN-H20 Ph 3.22 Scheme 3.6.2. Synthesis of cyclo(R-Val-S-Phe) (3.21) and cyclo(R-Val-R-Phe)(3.22). 3.17 3.19 3.18 I CAN, ACN-H20 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 96- well 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. Hence, addition of 32 .tM of cyclo(S-Val-S-Phe) enhanced neurite length. 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 In 2 0 E .= ‘I -J 1 z •PLL LICSPG 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. 0 Control Charter 3: Isolation of Comvounds That Can Induce Neurite Outarowth 114 PLL Myelin CPSG Cyclo (S-Val-S-Phe) 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 Control 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 • AdrenergicE Q. •1- 0 4- (‘3 C a) C 0 Figure 3.7.5. Addition of cyclo(S-Val-S-Phe) increased the axon sprouting inboth 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 Control Cyclo (S-Va I-S-P he) 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 C18 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 60F254 (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 (1H, 13C, 15N) cryoprobe. NMR solvents were purchased from Cambridge Isotope laboratories and were referenced to solvent peaks for DMSO-d6(6H 2.49 ppm and 6c 39.5 ppm) and CDCI3 (oH 7.24 ppm and 0c 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 H20. 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: H20 mixture which was then partitioned with hexanes (3 X 200mL). The active MeOH/H20fraction (800 mg) was then subjected to 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 (H20 to MeOH) where the active fraction eluted with 6:4 MeOH: H20 (7.6 mg). The crude brown solid was further purified using reversed phase HPLC (lnertsil C18, 9.4 X 250 mm, 1:1 H20: 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]o22: - 45.82 (c 0.3, DMSO); ‘H NMR (600 MHz, DMSO-d6):6H 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 = 13.4, 4.9 Hz, H-lOb), 1.69 (IH, m, H-7), 0.64 (3H, d, J = 7.2, H-8 or H-9), 0.25 (3H, d, J = 7.2 Hz, H-9 or H-8): 13C NMR (150 MHz, DMSO-d6):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 (CH2, C-b), 30.9 (CH, C-7), 18.2 (CH3, C-8 or C-9), 16.0 (CH3, C-8 or C-9); LRESIMS m/z 269.1; HRESIMS m/z 269.1269 (calc’d for C14H8N2Oa 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-2,5- dione (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 H20 (5 mL), followed by the addition of excess NH4CI. Extraction of the mixture was accomplished with EtOAc (3 X 10 mL) and dried with MgSO4, filtered through 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]D22: -49.8 (c 0.8, CHCI3);1H NMR (600 MHz, CDCI3)6H 6.79-7.15 (8H, m), 5.26 (IH, d, J = 14.8 Hz), 4.84 (IH, d, J = 14.2 Hz), 4.27 (IH, d, J = 14.2 Hz), 3.99 (IH, d, J = 16.5 Hz), 3.89 (IH, d, J = 14.8 Hz), 3.85 (3H, s), 3.85 (3H, s), 3.81 (IH, d, J = 16.5 Hz), 3.77 (IH, d, J = 4.8 Hz), 2.24 (IH, m), 1.05 (3H, d, J Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 121 = 7.2 Hz), 0.90 (3H, d, J = 7.2 Hz); 13C NMR (150 MHz, CDCI3): 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 forC23H8 N204 379.2324). Preparation of (3S,6R)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6- benzylpiperazine-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 NH4CI. The volatiles were removed in vacuo and the solution was subsequently extracted with EtOAc (10 mL), dried with MgSO4, 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-2,5- diane (3.12): [aID22 + 49.4 (C 0.76, CHCI3); 1H NMR (600 MHz, CDCI3) oH 6.63- 7.60 (13H, m), 5.71 (IH, d, J = 14.7 Hz), 4.34 (1H, t, J = 4.2 Hz), 3.98 (IH, d, J = 14.5), 3.90 (3H, s), 3.86 (3H, s), 3.77 (IH, d, J = 14.7 Hz), 3.50 (IH, dd, J = 13.9, 4.2), 3.42 (IH, dd, J = 13.9, 4.2), 3.29 (IH, d, J = 3.0 Hz) , 2.22 (IH, m), 1.05 (3H, d, J = 7.0 Hz), 0.69 (3H, d, J = 7.0 Hz); 13C NMR (150 MHz, CDCI3): 0c 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 C30H5 N204 487.2597). Preparation of 3S,6S-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6- benzylpiperazine-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 MgSO4, and concentrated in vacuo. Purification of 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; [c]D22: -199 (c 0.85, CHCI3); 1H NMR (600 MHz, CDCI3)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), 5.15 (IH, d, J = 14.6), 4.10 (IH,dd, J = 7.9, 4.0), 3.76 (IH, d, J = 14.8), 3.69 (3H, s), 3.67 (3H, s), 3.49 (IH, d, J = 7.9), 3.37 (IH, dd, J = 14.3, 4.1), 3.00 (IH, dd, J = 14.3, 4.1), 2.98 (IH, d, J = 14.6), 1.86 (IH, m), 1.07 (3H, d, J = 7.0), 1.01 (3H, d, J = 7.0); 13C NMR (150 MHz, CDCI3): 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 C30H5 N2O4a 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-H20 (1:1; 5 mL), ceric 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: H20)to afford 10mg of 3.14(75% yield). (3S,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.14): white powder. [ct]D21: -65.4 (c 0.25, DMSO); 1H NMR (600 MHz, DMSO-d6)OH 8.10 (IH, br s), 8.00 Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 124 (IH, br s), 7.10-7.34 (5H, rn), 4.22 (IH, m), 3.15 (IH, dd, J = 13.4, 3.9 Hz), 2.95 (IH, m), 2.90 (IH, dd, J = 13.4, 3.9 Hz), 1.95 (IH, m), 0.81 (3H, d, J = 6.8 Hz), 0.72 (3H, d, J = 6.8 Hz); 13C NMR (150 MHz, CDCI3): 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 C14H8N2Oa 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 ACN-H20 (1:1; 5 mL), ceric 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 MeOH:H20)to afford 15 mg of 3.9 (70% yield). (3S,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.9): white powder. [aID23: - 47.32 (c 0.5, DMSO); ‘H NMR (600 MHz, DMSO-d6): 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 = 7.2), 0.25 (3H, d, J = 7.2 Hz); “3C NMR (150 MHz, DMSO-d6):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 forC14H8N2Oa 269.1266). Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 125 Preparation of (R)-N,N’-bis(p-methoxybenzyl)-3-isopropylpiperazine-2,5- dione (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 H20 (6 mL), followed by the addition of excess NH4CI. Extraction of the mixture was accomplished with EtOAc (3 X 5 mL) and was dried with MgSO4,filtered through 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]D22: 44.6 (c 0.76, CHCI3); 1H NMR (600 MHz, CDCI3) 6H 6.79-7.15 (8H, m), 5.26 (IH, d, J = 14.8 Hz), 4.84 (IH, d, J = 14.2 Hz), 4.27 (IH, d, J = 14.2 Hz), 3.99 (IH, d, J = 16.5 Hz), 3.89 (IH, d, J = 14.8 Hz), 3.85 (3H, s), 3.85 (3H, s), 3.81 (IH, d, J = 16.5 Hz), 3.77 (IH, d, J = 4.8 Hz), 2.24 (IH, m), 1.05 (3H, d, J = 7.2 Hz), 0.90 (3H, d, J = 7.2 Hz); 13C NMR (150 MHz, CDCI3): 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 forC23H8 N204 379.2322). Preparation of (3R,6S)-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6- benzylpiperazine-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 NH4CI. The volatiles were removed in vacuo and the solution was subsequently extracted with EtOAc (3 X 5 mL), dried with MgSO4, 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 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-2,5- dione (3.19): [cL]o22 -46.2 (c 0.71, CHCI3); 1H NMR (600 MHz, CDCI3): oH 6.63- 7.60 (13H, m), 5.71 (IH, d, J = 14.7 Hz), 4.34 (IH, t, J = 4.2 Hz), 3.98 (IH, d, J = 14.5 Hz), 3.90 (3H, s), 3.86 (3H, s), 3.77 (1H, d, J = 14.7 Hz), 3.50 (IH, dd, J = 13.9, 4.2 Hz), 3.42 (IH, dd, J = 13.9, 4.2 Hz), 3.29 (IH, d, J = 3.0 Hz) , 2.22 (IH, m), 1.05 (3H, d, J = 7.0 Hz), 0.69 (3H, d, J = 7.0 Hz); 13C NMR (150 MHz, CDCI3): 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 forC30H5 N204487.2597). Preparation of 3R,6R-N,N’-Bis(p-methoxybenzyl)-3-isopropyl-6- benzylpiperazine-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 MgSO4,and concentrated in vacuo. Purification 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; [c]D22: 189 (C 0.79, CHCI3); ‘H NMR (600 MHz, CDCI3)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 = 7.9 Hz), 3.37 (IH, dd, J = 14.3, 4.1 Hz), 3.00 (1H, dd, J = 14.3, 4.1 Hz), 2.98 (IH, d, J = 14.6 Hz), 1.86 (1 H, m), 1.07 (3H, d, J = 7.0 Hz), 1.01 (3H, d, J = 7.0 Hz); 13C NMR (150 MHz, CDCI3): 0c 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 forC30H5 N204 487.2597). 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-H20 (1:1), ceric 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 H20: MeOH) to afford 13 mg (65% yield) of 3.21. (3R,6S)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.21): white powder. [c]o21: 69.3 (c 0.27, DMSO); 1H NMR (600 MHz, DMSO-d6)0H 8.10 (IH, br s), 8.00 (IH, Chapter 3: Isolation of Compounds That Can Induce Neurite Outgrowth 129 br s), 7.10-7.34 (5H, m), 4.22 (IH, m), 3.15 (dd, J = 13.4, 3.9 Hz), 2.95 (IH, m), 2.90 (IH, dd, J = 13.4, 3.9 Hz), 1.95 (IH, m), 0.81 (3H, d, J = 6.8 Hz), 0.72 (3H, d, J = 6.8 Hz); 13C NMR (150 MHz, CDCI3): 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 forC14H8N2Oa 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-H20(1:1), 65mg of ceric 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 MeOH:H20)to afford 12 mg (70 % yield) of 3.22. (3R,6R)-3-Benzyl-6-isopropyl-2,5-piperazinedione (3.22): white powder. [cL]o: 43.45 (c 0.29, DMSO); 1H NMR (600 MHz, DMSO-d6): oH 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 = 13.4, 4.9 Hz), 1.69 (IH, m), 0.64 (3H, d, J = 7.19), 0.25 (3H, d, J = 7.2 Hz); ‘3C NMR (150 MHz, DMSO-d6):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 C14H8N2Oa 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 131 (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. Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duguetia Odorata 132 Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duguetia Odorataa 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 G2 checkpoint inhibition assay. Bioassay guided fractionation of the plant extract led to the discovery of oliveroline (4.32) as an abrogator of G2 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 G1, S, and G2 phases. The G1 phase is where metabolism takes place. At the end of G1, centrioles replicate and prepare for cell division. Before progressing to the S phase, sensors scan to check the fidelity of the DNA. Should there be any anomalies present on the DNA, the G1 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 G2 Checkpoint inhibitors from Duguetia Odorata 133 cell division are synthesized in the G2 phase of interphase. Similar to the G1 checkpoint, the G2 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 obtained.1 Chapter 4: Structure Elucidation ofG2Checjpintinhibitors from Duquetia Odorata 134 G1 checkpoint checkpoint 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 G1, S, and G2 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 G1, the levels of cyclinB are low. At the end of the S-phase however, cyclinB is synthesized, which leads to the formation of the cylcinB/cdc2 complex. During G2, the cyclinB/cdc2 complex is held inactive by inhibitory phosphorylations on cdc2. These phosphorylations are carried out by Wee 1. Cdc25c acts as a positive regulator of the cdc2/cyclin B complex by Interphase M-phase 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 Figure 4.3.1. G2/M transition (adapted from Foijer).4 It is vital for the DNA not to be damaged before entering mitosis. This is ensured by the G2 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 14-3- 3a 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 Active Chanter 4: Structure Elucidation of G Checknoint inhibitors from Ducuetia Odorata 136 P cdc25c I 4-3-3a Figure 4.3.2. G2 checkpoint pathway (Adapted from Samuel et al.).6 4.3. Rationale for using G2 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 G1 to repair their DNA. The mp53 cells may then either die, or continue to proliferate with a blemished genome.7 Inactive Active Chapter 4: Structure Elucidation of G2 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 G2 checkpoint. This allows tumor cells time to recover and repair their DNA.8’9 Tumor cells lacking p53 may also have a growth advantage over wild type cells because the mp53 cells do not arrest at the G1 checkpoint.7 DNA damaging agents such as cisplatin are often used to treat tumors. Upon DNA damage, the wild type cells activate both the G1 and the C2 checkpoints, but mp53 cells activate only the G2 checkpoint. If a C2 checkpoint inhibitor is used, the wild type cell is still able to arrest at G1, but mp53 cells 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 138 G2 checkpoint G2 Checkpoint Inhbitor DNA Damaging Agent A) Wild Type Cell G1 S G2 II MI- — I—.G1 checkpoint G2 checkpoint G2 Checkpoint lnhbitor DNA Damaging Agent G1 S G2 G1 chckpoint G2 cheoint B) mp53 cells G S G2 ii M G1 cDkpoint ‘I S G1 M G2 ch nt Figure 4.4.1. Rationale for using G2 checkpoint inhibitors. Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duquetia Odorata 139 4.5. Known G2 Checkpoint Inhibitors There have been numerous G2 checkpoint inhibitors that have been discovered. The first G2 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 concentrations.1° Other compounds that can inhibit ATM/ATR include the polyketide kinase inhibitor wortmannin (4.2), which has been found to be a strong G2 checkpoint abrogator at 101iM.”2 Figure 4.5.1. ATM/ATR inhibitors of the G2 checkpoint pathway. Staurosporine (4.3) is a very potent G2 checkpoint inhibitor (IC50 0.2 nM).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 C2 checkpoint inhibitor (1C50 50-1 00 nM),’4 and an in vitro inhibitor of Chkl (1C50 10- 25 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 4.2 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 G2 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 G2 arrest with an IC50 of 15 nM. Paradoxically, SB-218078 was found to induce G2 arrest at higher concentrations.17 N 0 HO 45 Figure 4.5.2. Indole alkaloids inhibiting the G2 checkpoint through Chkl. Bioassay-guided fractionation of the Brazilian ascidian Didemnum granulatum led to the isolation of two G2 checkpoint inhibitors: granulatimide (4.6) and isogranulatimide (4.7). Each of these alkaloids inhibited G2 arrest with an IC50 of 6 tM.1° Isogranulatimide was able to inhibit Chk I with an IC50 of 0.432 jtM, while granulatimide was more potent (1C50 0.081 iM).”8 Other marine G2 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 G2 arrest (IC50 6-8 jiM) and were found to be in vitro inhibitors of Chkl (1C50 3 jiM).’9 Chanter 4: Structure Elucidation of G., Checknoint inhibitors from Duauetia Odorata 141 H2N H2N 0 0 4.8 Figure 4.5.3 Alkaloids inhibiting the G2 checkpoint through Chkl Other inhibitors of the C2 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 C2 checkpoint (1C50 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 G2 checkpoint abrogators include okadaic acid (4.11), which has an 1C50 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 G2 checkpoint pathway with an (C50 of 3.2 tM.21 4.6 Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duouetia Odorata 142 Figure 4.5.4. G2 checkpoint inhibitors 4.6. Description of the G2 Checkpoint Assay Dr. Michel Roberge has developed the first assay to search for G2 checkpoint inhibitors in crude extracts.1° In the assay, MCF-7 mp53 cells are 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 OH OH 4.12 Chanter 4: Structure Elucidation of Checknoint inhibitors from Duauetia Odorata 143 MCF-7 mp53 in 96 well plate Jr Irradiate with 6.5 Gy Jr 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 G2 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 Cells exposed to G2 checkpoint inhibitors enter mitosis Cells not exposed to G2 checkpoint inhibitors remain arrested in G2 Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata 144 properties.22 Duguetia is a genus within the Annonaceae family and contains 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 Brazil.23 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 crystallography.23 Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duquetia Odorata 145 — NH 4.13 <: :O)NH OH OH OH 4.17 4.18 <:zg 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 IC50 of 3.80 jtg/mL.28’9 Oxostephanine was discovered to have promising activity against the Herpes simplex virus.30 More recently, duguevaline (4.25) was isolated from a CH2I 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) 4.14 4.15 4.19 Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duquetia Odorata 146 and 1-methoxysampangine (4.31) were found to display in vitro antimalarial activity against Plasmodium falciparum (1C50 120, 68, 95 ng/mL). Sampangine was also cytotoxic towards human malignant melanoma cells (1C50 370 nglmL). KQXXN o N N 0 4.22 423 4.24 4.25 ZO3Ng3 ‘c oc Y 4.26 4.27 4.28 i x:e NN 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 G2 checkpoint assay. The MeOH extract was suspended in H20, and then sequentially partitioned with hexanes, CH2I EtOAc and n-butanol. The active 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 147 (4.34), and pseudopalmatine (4.35). 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 4.33 0H 0 4.34 0 4.35 I Figure 4.8.1. Alkaloids isolated from D. odorata. 4.9. Structure Elucidation of N-methylguatterine 12<\ ibi G—13 0 1f4H14 11 1111 ‘0H 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 C20H2N4 (calc’d for 340.1549). The 1H NMR spectrum (Figure 4.9.3) was acquired in DMSQ-d6at 500 MHz and was found to be similar to the 1H NMR spectrum of oliveroline, suggesting that 4.33 was an Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duquetia Odorata 148 aporphine alkaloid. Examination of the 1H NMR spectrum revealed four aromatic protons (6H 7.42-7.87), an exchangeable proton (6H 6.95), two deshielded protons (oH 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 sp2- hybridized carbon (OH 2.93). Analysis of the 13C NMR spectra (Figure 4.9.4) and the HMQC data (Figure 4.9.5) identified 12 sp2 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 sp3 hybridized carbons attached to heteroatoms (Oc 70.8, 68.2, 61.8, 59.4, 56.8, 42.1) and one shielded sp3-hybridized carbon (0c 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. 1H NMR spectrum of N-methylguatterine at 500 MHz in DMSO-d6. Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duauetia Odorata /°<D ozo 150 Figure 4.9.4. ‘3C spectrum NMR of N-methylguatterine at 100 MHz in DMSO-d6. 0 u) 0 0 0 LC) E a- Chapter 4: Structure Elucidation of G,Checkoint inhibitors from Duquetia Odorata 151 LJLLJJ__ _ JUL 7.0 6.0 5.0 4.0 3.0ppm Figure 4.9.5. HMQC spectrum of N-methylguatterine at 500 MHz in DMSO-d6. Chanter 4: Structure Elucidation of G Checknoint inhibitors from Duouetia Odorata 152 K: 50 100 ppm 7.0 6.0 5.0 4.0 3.0ppm Figure 49.6. HMBC spectrum of N-methylguatterine at 500 MHz in DMSO-d6. Chapter 4: Structure Elucidation of G, CheckpoTht inhibitors from Duquetia Odorata 153 2.0 • 3.0 4.0 •5.0 6.0 7.0 7.0 6.0 5.0 4.0 3.0ppir Figure 4.9.7. COSY spectrum of N-methylguatterine at 500 MHz in DMSO-d6. Chaoter 4: Structure Elucidation of G9 Checkooint inhibitors from Duauetia Odorata 154 a: 1H and 1C chemical shifts [ppm] are referenced to DMSO-d6(ö- 2.50 and öc39.51 respectively) b: Signals may be interchanged Table 4.9.1. ID and Position C ö (J in Hz) 1 145.1 2DNMR data of Nmethylguatterine.a 1H, 13C-HMBC COSY Ia lb 2 3 3a 4 5a 5b 6 6a 7 7a 8 9b 0” 11 ha 12 13 14 15 7-OH 109.5 119.2 135.9 138.9 116.3 18.6 61.8 70.8 68.2 136.9 123.9 127.9 128.0 125.4 127.6 101.7 42.1 56.8 59.4 2.93, m H-5a, H-5b 3.61, m C-14 H-4, H-5b 3.75, m C-4 H-4, H-5a 4.74, d, (12.0) C-Ib, C-7, C-7a, C-13, C- H-7 14 5.02,dd, (12.0, 7.0) C-lb, C-6a, C-7a 7-OH, H-6a 7.65, m C-7, C-9, C-l0 H-9 7.41,m C-Il H-8,H-I0 7.42,m C-8 H-9,H-1I 7.87, m C-lIa, C-la H-b 6.25 (a), s C-I, C-2 H-12b 6.07(b),s C-l,C-2 H-12a 3.17, s C-5, C-6a, C-I4 H-14 3.61, s C-5, C-6a, C-13 H-13 4.02, s C-3 6.95, d, (7.0) C-6a, C-7 H-7 Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duciuetia Odorata 155 4.74, d, (12.00 Hz) 68.2 0H6.95,d,(6.82Hz) OH 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) 13C chemical shifts for substructure I. 13 )icosy 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). Both the methyl proton resonances at 6H 3.17 (H-13) and 6H 3.61 (H-14) showed HMBC Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duijuetia Odorata 156 correlations to the carbon resonance at & 61.8 (C-5). Further HMBC correlations between the proton resonance at oH 3.61 (H-5a: HMQC to 0c 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 0H 3.61 (I-I-5a) and OH 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 0H 3.61 (H-14) displayed HMBC correlations to the methine carbon resonance at 0c 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 0c 42.1 (C- 13), 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 0H 4.74 (H-6a) contained a COSY correlation to the methine proton resonance at 0H 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 0H 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 (6c 68.2) (Figures 4.9.8 and 4.9.9). The chemical shift of C-7 (öc 68.2) is typical of an alcohol moiety attached to a carbon. Both methine proton resonances at oH 4.74 (H-6a) and 0H 5.02 (H-7) showed HMBC correlations to oH 119.2 (C-lb), which indicated that H-6a was neighboring an sp2-hybridized carbon. All of the above data was consistent with substructure I (Figures 4.9.8 and 4.9.9). HH4 H5bjJJUu 7.0 6.0 5.0 4.0 3.0ppm Figure 4.9.10. COSY correlations for substructure I of 4.33. ChRntcr 4: Strutiirn EIImidRfinn nf ( Chcknnint inhthitn,s frnm DIJnIJRt1R flrkrnt 158 Figure 4.9.11. HMBC correlations observed for H-13 and H-14 for substructure I of 4.33. Figure 4.9.12. (a) 1H chemical shifts and (b) ‘3C chemical shifts for substructure II. H-i 4 ppm 3.60 3.50 3.40 3.30 3.20 3.10 7.87, m 7.42, m 125.4 128.0 7.41, m 127.9 a b Chaoter 4: Structure Elucidation of G2 Checkooint inhibitors from Duauetia Odorata 159 cosy ‘HMBC 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 0H 7.41 (H-9: HMQC to 0c 127.9), which in turn had COSY correlations to the proton resonance at 8H 7.42 (H-10: HMQC to Oc 128.0). Additional COSY cross-peaks were observed between the proton resonance at 3H 7.42 (H-b) and the proton resonance at 0H 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 8H 7.87 (H-Il) showed HMBC correlations to the quaternary aromatic carbon resonance at 0c 109.5 (C-la), thus establishing substructure II (Figures 4.9.12 and 4.9.13). I 8 ChaDter 4: Structure Elucidation of G9 CheckDoint inhibitors from Duauetia Odorata 160 H-9/1O 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 8c 136.9 (C-7a). This indicated that carbon resonances C-7 (öc 68.2) and C-7a (öc H-il _) - H-8 ‘r — -7.50 —8.00 ppm 8.00 7.90 7.80 7.70 7.60 7.50 7.40 7.30ppm Figure 4.9.14. Expansion of the aromatic region of the COSY spectrum for 4.33. HMBC Chapter 4: Structure Elucidation of G2 Checjcix,int inhibitors from Duquetia Odorata 161 136.9) were linked. 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). 6.25, s 6.07,s< II a 4.02, s Figure 4.916. 1H NMR of substructures Ill (a) and IV (b) / 11135.9 101.7 ‘ II 1451 a 59.4 0 Figure 4.9.17. 13C NMR of substructures Ill (a) and IV (b) ‘HMBC b b Figure 4.9.18. HMBC correlations for substructure Ill and IV for 4.33. Chapter 4: Structure Elucidation of G2 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 6.25 (H-12a: HMQC to 6c 101.7) and 6H 6.07 (H-12b: HMQC to öc 101.7) showed three bond HMBC couplings to thesp2-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 1H 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 13C chemical shifts.’38 Finally, comparison of the 13C 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 124.11 I 39.0 128.7j 69.7J. 125.7 f’9.’OH 126.9 126.9 4.36 Figure 4.9.19. 13C chemical shifts for guatterine (4.36), an aporphine alkaloid related to 4.33. Chapter 4: Structure Elucidation of G2 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 2 0 ,‘2 0 300 350 4 c.r- -20 ‘. i E \ ,. -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 G2 checkpoint assay at concentrations above 10 1iM (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 164 pseudopalmatine were all found to be inactive. 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 1iM of the known G2 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 G2 arrest (A, Figure 4.10.1). Oliveroline, dehydrodiscretine and pseudopalmatine were found to be moderate inhibitors of cell proliferation with lC50’s of 45, 250, and 75 1iM respectively, but were 2-3 times more potent when cells were irradiated with 6.5 Gy (IC50 20, 80, 50 j.tM respectively). Oliveroline is an efficacious but moderate inhibitor of the G2 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 G2 checkpoint pathway. ( U Gi G2 60 50 40 30 20 10 0 OHveroine —0--- Boldine ADomorphine — Pseudopalmatine 1 10 100 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. Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duguetia Odorata 165 A B C _________________________ C I .io C C a 1023 DNA Pt-H 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. Dehydrodiscretine —a-— Berberine ‘ Palmatine 0.01 0.1 Concentration (LM) Chanter 4: Structure Elucidation of G CheckDoint inhibitors from Duauetia Odorata ‘166 $CH3 HOj> HO 4.38 OCH3 H3C 4.40 OCH3 Figure 4.10.3. Other alkaloids tested in the G2 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 G2 checkpoint assay (boldine (4.37), apomorphine (4.38), berberine (4.39) and palmatine (4.40)) were purchased from Aldrich. Reversed-phase C18 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 60F254 (normal phase). TLC was visualized using either a dip solution of p-anisaldehyde 4.39 Chapter 4: Structure Elucidation of G2 Checkpoint inhibitors from Duquetia Odorata 167 (1% p-anisaldehyde, 2% H2S04, 20% acetic acid and 77% ethanol) or under ultraviolet light (254 nm). The 13C spectra were obtained with the Bruker AM400 spectrometer. 1H 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-d6(6c 39.5 ppm and 6H 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 G2 checkpoint assay. The crude extract (4 g) was first suspended in 100 mL of H20, and then sequentially partitioned with hexanes (3 X 50 mL), CH2I (3 X 50 mL), EtOAc (3 X 50 mL) and butanol (3 X 50 mL). 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: H20 to MeOH) to yield one biologically active fraction. This fraction was subjected to repeated reversed phase HPLC (lnertsil C18, 9.4 X 250 mm, 6:4:0.1 H20: 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 i05 cells/dish in 35 mm-diameter dishes and subsequently cultured for 24 h. Cells were then irradiated with 6.5 Gy using a 60Co source (1.2 Gy/min, Gammacell 220, Atomic Energy Commission of Canada). Sixteen hours later, when 90% of cells were arrested in G2,’° 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-72° for I h, washed twice, and suspended with 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 IxIO6 cells/mL in 25 pg/mL propidium iodide solution and stored in the dark overnight. Cells were analyzed Chapter 4: Structure Elucidation of G2 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/H20(1:1), pH 4.7, was added, and the absorbance at 570 nm was measured after overnight incubation. 4.15. Physical Data of Alkaloids From Duguetia odorata (-)-Oliveroline (4.32): Brown oil. [cUD23: -16.9 (c 0.3, MeOH) UV (MeOH) Xmax (log G): 233 (3.91), 271 (3.88), 315 (3.39); CD (MeOH) max (AE) 232 nm (- 314.36); 1H NMR (500 MHz, DMSO-d6):H 8.00 (IH, d, J = 6.7 Hz, H-lI), 7.43 (IH, m, H-9 or H-b), 7.43 (IH, m, H-b 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); ‘3C Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duquetia Odorata 170 NMR (100 MHz, DMSO-d6):& 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 (CH2, C-12), 65.6 (CH, C-7), 62.5 (CH, C-6a), 49.5 (CH, C-5), 32.1 (CH3, C-13), 21.3 (CH2, C-4); LRESIMS m/z 295; HRESIMS m/z 295.12029 [M+H] (calc’d forC18H7N03295.12084). (+)-N, N-methylguatterine (4.33): Brown oil. [c]D21: 6.17 (C 0.13, MeOH); UV (MeOH) ?max (log s): 242 (3.48), 279 (3.42); CD (MeOH) ?.max (&) 239 nm (- 162.82); 1H NMR and 13C NMR see Table 4.9.1; LRESIMS m/z295; HRESIMS m/z 295.12029 [Mt] (calc’d forC18H7N03295.12084). Dehydrodiscretine (4.34): Yellow powder. UV (MeOH) max (log 6): 289 (3.81), 242 (3.59), 340 (3.52), 378 (3.15); 1H NMR (400 MHz, DMSO-d6): oH 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, H- 9), 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); ‘3C NMR (100 MHz, DMSO-d6):01D 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 (CH3, OMe-lO), 56.2 (CH3, OMe-hl), 56.0 (CH3, OMe-2), 54.6 (CH2, C-6), 25.8 (CH2, C-5); LRESIMS m/z 338; HRESIMS m/z 338.1394 [Mt] (calc’d for C18H7N03338.1392). Chapter 4: Structure Elucidation of G, Checkpoint inhibitors from Duguetia Odorata 171 Pseudopalmatine (4.35): Yellow powder. UV (MeOH) 2max (log e): 287 (4.02), 239 (3.75), 338 (3.65), 373 (3.24); 1H NMR (400 MHz, DMSO-d6):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, OCH3-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, H- 5); 13C NMR (100 MHz, DMSO-d6): 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 (CH3, OMe-lO), 56.3 (CH3, OMe-Il), 56.0 (CH3, OMe-3), 55.8 (CH3, OMe-2), 54.7 (CH, C-6), 26.0 (CH2, C-5); LRESIMS m/z 353; HRESIMS m/z 352.1541 [M] (calc’d forC21H2N04352.1549). 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. 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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 G2 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 G2 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, 1029- 1034. (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 candidates.3 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-glucosamine.4 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-dihydrotestosterone. 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 pathways.2 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 secretion.6 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 osteoporosis.8 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 H HO 5.1 OH 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-Pacific.11 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 (LC50= 154 jiglmL and 3 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 IC50 of 2.6 jiM. Other ligands developed include compounds 5.2 and 5.3, which have 1C50’s of 13.6 and I jiM, respectively.9’10 5.2 5.3 Chaoter 5: Isolation of liaands for the Human Sex Hormone Bindinci Globulin 178 family of 5,6,7 tricarbocyclic diterpenoids to be isolated from the Myrmekioderma.13 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 1C50’s of 5 .igImL and 3 tgImL, respectively, 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 OHHQ H0 5.7 J 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 EC50’s of 5.9 Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin 179 2.5 and 2.8 pg/mL, respectively.16 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 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 5.9 5.10 Chapter 5: Isolation of liciands for the Human Sex Hormone Bindir&q Globulin 180 Xylose “OH 5.18: RC16H33 5.19: 8 6H14 Xylose N-Acetyl glucosamine “OH NHAc 5.20: R=—HCCQH 16 5.21: R=HC(CO 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 reversed- phase HPLC to obtain 5.22,20 abolene (5.23 mg) as a diastereotopic mixture,2° (+)-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 (5.24),21 abolene (5.23),20 and (+)- curcuphenol (5.26)21 were confirmed by comparing the optical rotation, NMR and the MS data to the literature values. The optical rotation of sesquiterpenoid 5.22 OH Glucose 1 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 isolated.2° For full experimental details, see Section 5.8. 5.22 5.23 5.24 5.25 5.26 L OH OH ,-, OH 0 Y Xylose GlucoselL (‘H “OH HO” “r oJHO jGlucose2 HO” ( ‘OH OH 5.27 Figure 5.4.1. Compounds isolated from Myrmekioderma styx. 5.5. Structure Elucidation of Abolenone 15’ 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 ofC15H200and requiring six degrees of unsaturation. I D and 2D NMR experiments were run in both DMSO-d6 and in C6D. The best 4 5.25 Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 182 dispersion in the 1H NMR spectrum was found in C6D (Figure 5.5.3). The proton NMR revealed three aromatic proton resonances (oH 7.01, 6.72, OH6.7) an exchangeable proton (8H 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 13C 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 (8c 124.5, 33.9 and 31.9), three methyls (8c 20.3, 18.7, 17.5), and four quaternary carbons (8c 202.4, 143.8, 136.4, 128.6). After using the HMQC 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 Figure 5.5.3. 1H NMR spectrum of abolenone (5.25) at 600 MHz in C6D. 0 r 0 C..’ 0 C., 0 0 U, 0 U, I 83 .0 :wo C 1-. 1CD O H H ay H b C, ’ 0 - 0 C) 0 z CD C,) CD a -I CD 3 0 0) 0 >c D 0 0 -‘ CD 0 01 CD tx l 01 0. - C) 01 C N i i i i M u L C, 0) J L L L J L J tI L _ _ _ _ _ _ I 20 0 15 0 10 0 50 0 pp m - ChaDter 5: Isolation of liciands for the Human Sex Hormone Bindina Globulin 185 ppm Figure 5.5.5. HMQC spectrum of abolenone (5.25) at 600 MHz in C6D. Chapter 5: Isolation of liqands for the Human Sex Hormone Bindinci Globulin HaHb 186 7.0 6,0ppm 5.0 4.0 3.0 2.0 1.0 -50 -100 150 [-200 ppm Figure 5.5.6. HMBC spectrum of abolenone (5.25) at 600 MHz in C6D. 00 p , . • ? 8 ‘0 I I I I .0, I •0 0 a 0’ •. ChaDter 5: Isolation of Iiaands for the Human Sex Hormone Bindina Globulin 187 HaHb ppm Figure 5.5.7. COSY spectrum of abolenone (5.25) at 600 MHz in C6D. Chapter 5: Isolation of I,gands for the Human Sex Hormone Binding Globulin 188 Table 5.5.1. 1 D and 2D NMR data of abolenone (5.25).8 Position 6 6H (J in Hz) HMBC COSY 1 154.9 2 116.8 6.67, s C-I, C-6, C-4, C-15 H-15 3 136.4 4 120.6 6.72, d, (8.0) C-2, C-4, C-6, C-15 H-5 5 125.5 7.01,d,(8.0) C-1,C-3,C-7 H-4 6 128.6 7 30.4 2.95, m C-I, C-5, C-6, C-8, C-9, H-8a, H-8b, H-14 C-148ab 31.9 1.92, m C-6, C-7, C-9, C-10, C- H-7, H-8b, H-9a, 14 H-9b8bb 1.43, m C-6, C-7, C-9, C-10, C- H-7, H-8a, H-9a, 14 H-9b 9aC 33.9 2.34, m C-7, C-8, C-10 H8a, H8b, H9b9bC 2.14, m C-7, C-8, C-b H8a, H8b, H9a 10 202.4 II 143.8 I2ad 124.6 5.36, s C-I0, C-13 H-12b, H-13I2bd 5.16, s C-10, C-13 H-12a, H-I3 13 17.5 1.68, s C-b, C-Il, C-12 H-12a, H-12b 14 18.7 1.07, d, (6.8) C-6, C-7, C-8 H-7 15 20.4 2.14, s C-2, C-3, C-4 H-2 1-OH 6.63, s C-I, C-2, C-6 a. 1H and 13C chemical shifts (ppm) are referenced to the C6D (H 7.15 ppm and 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 ChaDter 5: Isolation of Ikiands for the Human Sex Hormone Bindina Globulin 189 13 Figure 5.5.9. Key HMBC correlations of substructure I of abolenone (5.25). Both proton resonances at 3H 5.36 (H-12a) and H 5.16 (H-12b) showed identical HMQC correlations to the carbon resonance at 6c 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 OH5.36 (H-12a), 0H 5.16 (H-12b) and 0H 1.68 (H-13) all showed HMBC Ha Hb 5.36, s 124.5 202.3 a b 0 Figure 5.5.8. (a)1H and (b)13C chemical shifts of substructure I of abolenone(5.24). 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). 1.07, d (6.8 Hz) 18.7 2.34, m 2.95, m 2.14, m 304 1.92,m 31.9 1.43, m a b Figure 5.5.11. (a)1H and (b)13C chemical shifts of substructure II of abolenone(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 0H 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 0H 1.92 (H-8a: HMQC to 8c 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-7 H-14 H-8a Figure 5.5.13. COSY expansion for substructure II of abolenone (5.25). OH 154.9 116.8 128.6 136.4 .— 125.5 2.14, S 20.4 120.6 b Figure 55.14. (a)1H and (b)13C chemical shifts of substructure III of abolenone(5.25). 6.67,s 7.01, d (8.0 Hz) 6.72, d (8.0 Hz) a 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 6H 6.63 (I-OH) and the aromatic carbon resonance at öc 154.9 (C-I). 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 6H 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 (6c 128.6) (Figure 5.5.17). HMBC cross-peaks were also observed between the proton resonance at H 6.63 (I-OH) and the aromatic methine carbon at o 116.9 (C-2). The HMBC correlations between the aromatic methine proton at öH6.67 (H-2: HMQC to cIl6.9)and the oxygenated carbon resonance at o 154.9 (C-I) determined the linkage between C-I (6c 154.9) and C-2 (öc 116.9) (Figures 5.5.15 and 5.5.17). A methyl proton resonance at 2.14 (H-15: HMQC to c 20.3) had an HMBC correlation to the aromatic carbon resonance at 6c 116.9 (C-2). Cross-peaks in the HMBC between oH 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 194 aromatic methine proton (H-2). An aromatic methine proton at 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 8H 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 195 I H-5 H61A 11 I I I 7.10 7.00 6.90 6.80 6.70 6.60ppm Figure 5.5.16. COSY expansion for substructure Ill of abolenone (5.25). -6.50 - 7.00 ppm cc:’ 1 Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin 196 C-i 5—i’—-—— C-7—_ C-2 C-3— C-i — H-5 AA ppm 7.00 6.90 6.80 H-2 1-OH 6.70 6.60 6.50 -50 -100 -150 ppm Figure 5.5.17. HMBC expansion for substructure Ill of abolenone (5.25). 15 13 HMBC 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 (6c 33.9) and C-b (ö 202.4). Observation of the chemical shifts of H-9a (oH 2.32) and H-9b (OH 2.14) is consistent with this 14 1 12 2 4 5 0 Chaoter 5: Isolation of Iiaands for the Human Sex Hormone Bindina Globulin 197 assignment, therefore, C-9 (6c 33.9) is linked to substructure I (Figure 5.5.18). 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 -5.00 .-1o.0o -15.00 E °-20.00 -25.00 -30.00 -35.00 Figure 5.5.19. CD spectrum of curcuphenol (dashed line) and abolene (solid line). 309.00 329.00 Wavelength (nm) (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 ofC38H7201 and requiring three 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 1H NMR signals were poorly dispersed in DMSO-d6 (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 1H NMR resonances of 5.28 were well dispersed in C6D and therefore all the NMR data was obtained with this solvent. Glycerol (Gly) OAc 10 Glucose 1 (Xyl) (Glul) Glucose 2 (G1u2) 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 C60H94029 and requiring 14 degrees of unsaturation. The increase in mass was suitable for the 5.28 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 1H NMR spectrum (Figure 5.6.3) contained signals from 6H 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. The 1H NMR spectrum also revealed peaks from 6H 3.3 to 5.5 which is suitable for protons attached to oxygenated sp3 carbons. Observation of the 13C (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 (6c 101.4, 101.5, 100.9), twenty carbons attached to oxygen 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 iii iv 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. 1H NMR spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D. Chanter 5: Isolation of licands for the Human Sex Hormone Bindina Globulin 201 Figure 5.6.4. 13C NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C5D6. 0 C 0 Figure 5.6.5. DEPT NMR spectrum of myrmekioside C peracetate (5.28) at 150 MHz in C6D5. Chanter 5: Isolation of lioands for the Human Sex Hormone Rindinci Globulin 2fl2 - C I E & Chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin 203 ppm i . . I LJ 50 —100 ppm Figure 5.6.6. HMQC spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D. AcO” —-... a I,,.’I,’ I 5.0 4.0 3.0 2.0 1.0 chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin 204 5.0 4.0 3.0 2.0 1.0 Figure 5.6.7. HMBC spectrum in C6D. of myrmekioside C peracetate (5.28) at 600 MHz AcO” onU • . * II I ‘Ic a.. 4D;,4 • 0 Iae, ••*I I * I ,* •i .. .8 .0.0 -50 -100 -150 Figure 5.6.8. COSY spectrum of myrmekioside C peracetate (5.28) at 600 MHz in C6D. Chapter 5: Isolation of liqands for the Human Sex Hormone Bindinci Globulin 205 I I I IIli I II I II I II I II 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0ppm Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin 206 72.1 30.5 26.8 20.0- 30.5, 12C Xyl-5a b, e 62.4 Xyl5be 16 17 Glycerol (Gly) Gly3bc Xylose (XyI) Xyl-1 Xyl-2 Xyl-3 Xyl-4 Glucosel (Glul) GluI-1 3.53, m 1.70, m 1.49, m 0.80-1.80 (24 H total) 1.19, m 1.46, m 4.00, t, (6.7) 3.70, d, (5.4) 4.06, m 4.10, dd, (10.17, 4.2) 3.73, m 4.42, d, (7.3) 5.30, t, (8.3) 5.43, t, (8.9) 5.10, m 3.10, t, (10.1) 3.97, m GIy-C-1, C-2, C-3 C-I, C-3, C-4 C-14, C-15 C-15, C-16, C-18 C-16, C-17 Gly-C-2, Gly-C-3, C-I Glu-C -1, Gly-C -1 Xyl-C -1, GIy-C -1 Xyl-C -1 Gly-C-3, Xyl-C -5 XyI-C -1, Xyl-C -2 Xyl-C -2, Xy-C 1-4, Xy-C -5 Xyl-C-3, Xyl-C -5 Xyl-C -1, Xy-C -3, Xyl-C -4 Xyl-C -1, XyI-C -3, XyI-C - Gly-C -2, GIul-C -3, GIul 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-2 GLu1-3 Glul-4 Glul-5 h 78.9 74.7 68.8 71.8 61.7 3.68, t, (8.9) 5.37, t, (9.5) 5.20, t, (9.9) 3.29, d, (10.2) 4.05, m 4.30, m GluI-C -1, GIul-C -3 GIuI-C -2, GIul-C -4 Glul-C -3, GIul-C -5, GIui-C -6 GluI-C -1, Glul-C -3, Glul-C -4 GIul-C -4 GIul-C -4 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 - Table 5.6.1. ID and 2D NMR data of myrmekioside C peracetate. a O’AIkyI Chain I 2 3 4-15 Position 6H (J in Hz) HMBC COSY H-2 H-i, H-3 H-2 Gly-I Gly2d Gly-3a total 26.3 29.0 64.4 70.4 78.0 69.4 101.5 71.5 72.2 69.5 4 101.4 4.53, d, (8.0) C-5 Glul-H -2 6a Chapter 5: Isolation of ligands for the Human Sex Hormone Bindinci Globulin 207 Position (J in Hz) HMBC COSY Glucose 2 (Glu2) G1u2-1 100.9 4.80, d, (8.0) Glul-C -2, G1u2-C -2 G1u2-H -2G1u2-2 72.6 5.25, t, (9.1) G1u2-C -1, Glu2-C -3, G1u2-H -1, G1u2-H Glu2-C -4 3 G1u2-3 73.6 5.47, t, (9.2) G1u2-C -2, G1u2-C -4 G1u2-H -2, G1u2-H - 4 Glu2-4 68.6 5.37, t, G1u2-C -3, G1u2-C -6 G1u2-3, Glu2-5(10.2) G1u2-5 72.2 3.59, d, Glu2-C-1, G1u2-C -4 G1u2-H -4, G1u2-H -(10.1) 6a, G1u2-H -6bGIu26ah 62.0 4.29, m G1u2-C -4, G1u2-C -5 G1u2-H -5, G1u2-H - 6b Glu2-6b’ 4.47, dd, G1u2-C -4, G1u2-C -5 G1u2-H-5, G1u2-H-(5.1, 12.4) 6a a. 1H and 13C chemical shifts (ppm) are referenced to the C6D (oH 7.15 ppm and 0c 128 ppm). b: H-18 and Xyl-5a are overlapping signals C: Gly-1 and Gly-3b are overlapping signalsd: 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 signalsIi: Glul-6b and G1u2-6a are overlapping signals ‘:G1u2-6a and G1u2-6b are interchangeable signals Chanter 5: Isolation of liaands for the Human Sex Hormone Rindina Globulin 208 5.10, m AcO 5.43, t (8.9 Hz) 4.42, d (6.7 Hz) a b Figure 5.6.9. (a)1H chemical shifts and coupling constants and (b) 13C 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 (6c 101.5) is typical of an sp3 hybridized carbon attached to two oxygen atoms. The proton resonance at 6H 4.42 (Xyl-H 1: HMQC to 8c 101.5) showed COSY correlations to the methine proton at i- 5.30 (XyI-H-2: HMQC to 6c 71.5), which in turn had COSY correlations to the proton resonance at H 5.43 (Xyl-H-3: HMQC to 6c 72.2) (Figure 5.6.11). The COSY spectrum revealed that the proton resonance at H 5.10 (Xyl-H-4: HMQC to & 69.5) had correlations to the proton resonances at 8H 5.43 (XyI-H-3), oH 3.10 (Xyl-H-5a: HMQC to Oc 62.4) and 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 209 the methylene protons at oH 3.10 (Xyl-H-5a) and 8H 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 Figure 5.6.11. COSY expansion for substructure I of myrmekioside C peracetate(5.28). QAc 3.68,5.20,t H (8.9 Hz)(9.9 Hz) rn j Q AcQAQ (102Hz) F 3.68, (8.9 Hz) a Figure 5.6.12. (a)1H chemical shifts and coupling constants and (b) 13C chemical shifts of substructure II of myrmekioside C peracetate (5.28). XyI-H-1 5.50ppm 5.00 4.50 H0’ H b 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 Cperacetate (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 6H 4.53 (Glul-H-1: HMQC to öc 101.4) and the methine proton at oH 3.68 (Glul-H-2: HMQC to 3 78.9). The proton resonance at 0H 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 8H 5.20 (Glul-H-4: HMQC to 0c 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 0H 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 8H 4.05 (Glul-H-6a: HMQC to Oc 61.7) and 8H 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 lionnds 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). Figure 5.6.14. (a) 1H chemical shifts and coupling constants and (b) 13C chemical shifts of substructure Ill of myrmekioside C peracetate (5.28). ° HMBC Figure 5.6.15. Key HMBC correlations of substructure Ill of myrmekioside Cperacetate (5.28). The methine carbon resonance G1u2-C-1 (c 100.9) displayed a chemical shift suitable for a carbon attached to two oxygeris. The COSY spectrum showed that the proton resonance at oH 4.80 (G1u2-H-2: HMQC to Oc 100.9) had a correlation to the proton resonance at 8H 5.25 (G1u2-H-2: HMQC to 8c 72.6), which in turn had a correlation to methine proton at oH 5.47 (G1u2-H-3: HMQC to 5.25, t H H a b QAc Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 213 8c 73.6). The proton resonance at oH 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 8H 5.47 (Glu2-H-5: HMQC to Oc 72.2). Finally, COSY correlations were present between the proton resonance at 8H 3.59 (G1u2-H-5) and the proton resonances at oH 4.29 (G1u2-H-6a: HMQC to Oc 62.0) and 0H 4.47 (Glu2-H-6b: HMQC to 0c 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 8H 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-1 G1u2-H-3——-*’, .., Figure 5.6.16. COSY expansion for substructure peracetate (5.28). UI of myrmekioside C 4.10, dd (10.2, 4.2 Hz) 3.73, m °‘ldt. Figure 5.6.17. (a) “H chemical shifts and coupling constants and (b) 13C chemical shifts of substructure IV of myrmekioside C peracetate (5.28). 5.50 5.40 5.30 5.20 5.10 5.00 4.90 4.80 4.70ppm 3.7, d (5.4 Hz) 70.4 a 69.4 b Chanter 5: Isolation of liqands for the Human Sex Hormone Bindinq Globulin 215 ‘HMBC Figure 5.6.18. Key HMBC correlations of substructure IV of myrmekioside Cperacetate (5.28). The oxygenated methylene protons at 6H 3.70 (Gly-H-1: HMQC to 8c 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 oH 4.10 (Gly-H-3a: HMQC to O 69.4) and 6H 3.73 (Gly-H-3b: HMQC to 8c 69.4) showed COSY correlations to the proton resonance at oH 4.06 (Gly-H-2), which allowed the connectivity between Gly-C-2 (0c 78.0) and Gly-C-3 (Oc 69.4). All of this is consistent with a linear 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). 0 Chanter 5: Isolation of liaands for the Human Sex Hormone Bindina Globulin 216 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 8c 70.0 (Gly-C-2). Additional three bond HMBC couplings were present between the methine proton resonance at oH 4.06 (Gly-H-2) and the oxygenated carbon resonance at 0c 101.4 (Glul-C-1). This confirmed the link between the glycerol and the disaccharide moieties at Glul-C-1 (Figure 5.6.20). Both proton resonances at 0H 4.10 (Gly-H-3a) and 0H 3.73 (Gly-H-3b) showed HMBC correlations to the anomeric carbon resonance at 8c 101.5 (Xyl-C-1). Additional HMBC cross-peaks were observed between the anomeric proton resonance at QAc Glucose I XyI-3 Xylose (Xyl) “QAcXyI-5 HMBC G1u2-5Glucose 2 (Glu2) AcO” ‘QAc Charter 5: Isolation of liqands for the Human Sex Hormone Bindinq Globulin 217 oH 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) 13C chemical shifts of substructure V of myrmekioside C peracetate (5.28). Gly-3 GIy-2Glycerol Figure 5.6.21. Key HMBC correlations of substructure V of myrmekioside Cperacetate (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 C41H63027, leaving C,9H4102 unaccounted for. Analysis of the 1H, ‘3C, and DEPT NMR spectra revealed no methyl doublets or triplets, 16 aliphatic methylenes, and two oxymethylenes. The proton resonance at 0H 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 8c 29.0), which linked methylenes C-16 (oc 26.3) and C-17 (0c 29.0). Additional COSY cross-peaks between 8H 1.46 (H-17) and0H4.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 (6c 29.0) to C-18 (öc 64.4). These connections were 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 0H 1.49 (H-3: HMQC to 0c 26.8) showed a COSY correlation with the proton resonance at oH 1.19 (H-2: HMQC to O 30.5), which confirmed the connection between C-2 (Oc 30.5) and C- 3 (8c 26.8). The methylene C-I (3c 72.1) was bonded to methylene C-2 (0c 30.5) from observation of a COSY correlation between 8H 1.19 (H-2) and OH 3.53 (H-I: HMQC to öc 72.1). 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 ([3H]-DHT) and any excess steroid is removed. The desired ligand is then added to the SHBG/[3H]-DHT mixture and incubated overnight. After removal of the displaced[3H1-DHT, the quantity of[3H]-DHT bound to SHBG in the presence of the ligand is compared to the amount of[3H]-DHT bound to SHBG when no ligand was added. The determination of the IC50 concentration was achieved when the ligand released more than 50% of[3H1-DHT from SHBG.22 All of the pure natural compounds isolated were tested in the SHBG ligand binding assay. Only (+)-curcudiol (5.24; IC50 100 jtM) was identified as a ligand able to displace[3H]-DHT from SHBG (Figure 5.7.1). Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 220 100 • Cortisci Curcucitoi90 ________________ 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 C18 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 60F254 (normal phase). TLC was visualized using either a dip solution of p-anisaldehyde (1% p-anisaldehyde, 2% H2S04, 20% acetic acid and 77% EtCH) or under ultraviolet light (254 nm). The ‘3C spectra were recorded with either a Bruker AV600, AMX500, 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 C6D(6H 7.15 ppm and öc 128.0 ppm), DMSO-d6(oH 2.49 ppm and 8c 39.5 ppm), and CDCI3 (OH 7.24 ppm and 0c 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 reversed- phase HPLC (Inertsil C,8, 9.4 X 250 mm, 4:6 H20: 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),2° abolenone (5.25, 5.2 mg) and the bisabolane sesquiterpenoid 5.22 (32.8 mg).2° The less active fraction was subjected to repeated reversed phase HPLC (lnertsil C18, 9.4 X 250 mm, 3:7 H20: MeOH, UV detection at 220 nm) to yield (+)-curcuphenol (5.26, 16.7 mg),21 and myrmekioside C (5.27, 4.3 mg). 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]D22: +3.9 (c 0.3, MeOH); UV (MeOH) 2max (log ) 219 nm (3.17), 270 (3.10); ‘H NMR (500 MHz, DMSO-d6):6H 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); ‘3C NMR (100 MHz, DMSO-d6): 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 (CH2, C-b), 37.8 (CH2, C-8), 31.1 (CH, C-7), 29.3 (CH3, C-12 or C-13), 29.1 (CH3, C-12 or C-13), 21.9 (CH3, C-I5), 21.1 (CH3, C-14), 20.6 (CH2, C-9); LRESIMS m/z 259; HRESIMS m/z 259.1672 (calc’d forC15H24ONa 259.1674). Abolene (diastereotopic mixture) (5.23): yellow oil; UV (MeOH) max (log E) 276 ( 3.51); 1H NMR (500 MHz, DMSO-d6):oH 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); 13C NMR (100 MHz, DMSO-d6): 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 (CH2, C-l2), 110.0 (CH2, C-12), 74.6 (C, C-b), 74.2 (C, C-b), 32.9 (CH2, C-9), 32.5 (CH2, C-8), 32.5 (CH2, C-8), 31.1 (CH, C-7), 30.8 (CH, C-7), 21.1 (CH, C- Chapter 5: Isolation of liqands for the Human Sex Hormone Binding Globulin 224 14), 21.0 (CH3, C-14), 20.6 (CH3, C-15), 17.4 (CH3, C-13), 17.2 (CH3, C-13); LRESIMS m/z 257; HRESIMS m/z 257.1512 (calc’d forC15H22ONa 257.1517). Phenol, 2-(5-hydroxy-1 ,5-dimethyl-3-hexenyl)-5-methyl-, [R-(E)] (5.22): yellow oil. [aID22: -6.9 (c 1.8, MeOH); UV (MeOH) max (logE) 277 (3.10), 227 (3.09), 241 (3.02); 1H NMR (500 MHz, C6D): H 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); 1H NMR (500 MHz, DMSO-d6): oH 9.09 (IH, b, 9-OH), 6.91 (IH, d, J = 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, H- 7), 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); 13C NMR (100 MHz, DMSO-d6): & 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 (CH2, C-8), 31.4 (CH, C- 7), 29.7 (CH3, C-12, C-13), 20.6 (CH3, C-15), 19.7 (CH3, C-14); LRESIMS m/z 257; HRESIMS m/z 257.1515 (calc’d forC15H20ONa 257.1517). Abolenone (5.25): yellow oil. [aID22: +8.8 (C 2.6, MeOH); UV (MeOH) 2max (log 8) 277 (2.89); CD ax (AE (MeOH) 280 nm (-187.87); 1H NMR and 13C NMR see Table 5.5.1; LRESIMS m/z 255; HRESIMS m/z 255.1360 (calc’d forC15H20ONa 255.1361). Chapter 5: Isolation of ligands for the Human Sex Hormone Binding Globulin 225 (+)-Curcuphenol (5.26): yellow oil. [cL]D21: +25.2 (c 0.84, CHCI3); UV (MeOH) ?max (logE) 277 (3.03), 240 (2.65); CD max (& (MeOH) 280 nm (-141.18); 1H NMR (500 MHz, CDCI3): 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); 13C NMR (125 MHz, CDCI3): 3c 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 (CH2, C-8), 31.4 (CH, C-7), 26.1 (CH2, C-9), 25.7 (CH3, C-12 or C-13), 21.1 (CH3, C-15), 20.9 (CH3, C-14), 17.7 (CH3, C-13 or C-12); LRESIMS m/z 217; HRESIMS m/z 217.1590 [Mt] (calc’d forC15H210217.1592). Myrmekioside C peracetate (5.28): yellow oil. [cz]D21: -18.2 (c 0.45, EtOAc); For 1H NMR and 13C NMR see Table 5.6.1.; LRESIMS m/z 1301; HRESIMS m/z 1301.5774 (calc’d forC60H94O29Na 1301.5778). 5.12. References (1) Hammond G.L. Trends in Endocrinology and Metabolism 1995, 6, 398- 304. (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. ClinicalEndocrinology 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 MedicinalChemistiy 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, 283- 291. (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, 101- 110. 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.2’3 Clearly, based on this result, derivatives of the tryptamine 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 G2 checkpoint pathway. The MeOH extract of Duguetia odorata showed G2 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 G2 checkpoint assay at concentrations above 10 tM. This alkaloid is structurally distinct from other G2 checkpoint inhibitors and does not inhibit Chkl. Finding oliveroline’s mechanism of inhibition may yield new information about the G2 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 Society2006, 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 ofC25H7N5O6.Cl.4VS42H 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 non- overlapped 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 SAINT1 software packages. The linear absorption coefficient, i, for Mo-Kcc radiation is 4.65 cm-1. Data were corrected for absorption effects using the multi-scan technique (TWINABS),2with minimum and maximum transmission coefficients of 0.682 and 0.977, respectively. The data were corrected for Lorentz and polarization effects. 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 component.3 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)2S)are very different. The chloride anion was easily identified, however the vanadium anion was less evident. Residual electron density clearly showed 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-row 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 literature).4’5Additionally, 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 refinement6 on F2 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 agreement factors of: RI = IlFol - lFcII I IFol = 0.247 wR2 = [ ( w (Fo2 - Fc2) )I w(Fo2]l/ = 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/A3, respectively. 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 McAuley.1° The values for the mass attenuation coefficients are those of Creagh and Hubbell.1’All refinements were performed using the SHELXTL crystallographic software package of Bruker-AXS.’2 Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 234 A.4.1. Experiemental Details, Crystal Data Empirical Formula C25H32N08SC1V. 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 .Y=900 V = 6035(2) A3 Space Group C 2/c (#15) Zvalue 8 DIc 1.443 g/Cm3 FOOD 2732.00 .t(MoKc) 4.65 cm1 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 45.0 seconds Detector Position 38.85 mm 29max 45.2° No. of Reflections Measured Total: 21334 Unique: 41468 (Rint = 0.108) Corrections Absorption (Tmin = 0.682, Tmax 0.977) Lorentz-polarization 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 F2 Function Minimized w (Fo2 - Fc2) Least Squares Weights w= I I(a2(Fo2)+(O. I 479P) +8I23206P) Anomalous Dispersion All non-hydrogen atoms No. Observations (l>O.OOLI(l)) 21334 No. Variables 390 Reflection/Parameter Ratio 54.70 Residuals (refined on F2, all data): RI; wR2 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 eIA3 Minimum peak in Final Duff. Map -1 .06e/A3 Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 237 Table A.4.1. Atomic coordinates (x 1OM) and equivalent isotropic displacement parameters(AA2 x 10A3) for exigumaine A. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(2) 3365(4) 4485(12) 5790(5) 60(3)C(3) 3303(4) 3273(12) 6108(5) 58(3)C(4) 2821(3) 3141(11) 5948(5) 43(3)C(5) 2569(3) 2065(11) 6141(5) 44(3)C(6) 2068(3) 2260(10) 5794(4) 38(3)C(7) 1857(4) 3481(10) 5347(5) 44(3)C(8) 2152(4) 4401(10) 5142(5) 47(3)C(9) 2626(3) 4292(12) 5506(4) 46(3)C(10) 1395(3) 3585(9) 5073(4) 37(3)C(11) 1144(3) 2258(10) 5137(4) 35(2)C(12) 672(4) 2140(10) 4889(5) 54(3)C(13) 416(4) 3472(11) 4565(6) 62(3)C(14) 646(4) 4803(11) 4515(5) 50(3)C(16) 817(4) 7503(12) 4527(5) 64(3)C(17) 1221(4) 6550(11) 4656(6) 64(3)C(18) 1128(4) 4942(10) 4782(4) 48(3)C(19) 1806(3) 1137(10) 5957(5) 39(3)C(21) 1853(4) 256(11) 6893(5) 45(3)C(23) 1999(4) -597(15) 6099(6) 64(4)C(24) 3690(5) 231 5(14) 6575(5) 74(4)C(25) 3728(4) 2450(15) 7224(4) 67(4)C(27) 92(4) 6684(11) 4430(5) 57(3)C(28) 277(6) 6163(18) 3534(6) 131(7)C(29) 2125(4) -2602(1 0) 6905(5) 58(3)C(30) 1602(4) 2959(13) 6650(6) 81(4)N(1) 2941(3) 5060(9) 5376(5) 63(3)N(15) 445(3) 6295(9) 4259(4) 51(2)N(20) 1759(3) 1501(8) 6523(4) 50(2)N(22) 1988(3) -1005(9) 6625(4) 46(2)N(26) 4072(4) 1234(13) 7616(5) 104(4)0(1) 1360(2) 931(7) 5447(3) 55(2)0(2A) 3196(6) -2396(18) 6616(7) 61(6)0(2B) 3850(6) -1629(17) 7091(7) 86(6)0(4A) 4207(5) 4842(13) 8858(5) 61(5)0(5) 2704(2) 1123(8) 6551(3) 56(2)0(8) 1995(2) 5183(8) 4647(3) 60(2)0(12) 466(3) 880(8) 4971(5) 85(3)0(21) 1851(3) 198(8) 7395(4) 70(2)0(23) 2071(2) -1406(7) 5714(4) 51(2)Cl(1) 882(1) 6298(3) 6078(1) 64(1)V(1) 5000 -5491(2) 7500 31(1)S(1) 4847(4) -5644(9) 6461(3) 105(3)S(2) 5049(3) -8072(7) 7749(3) 80(2)S(3A) 4468(3) -4341(8) 7709(4) 87(2)S(3B) 4369(3) -4036(15) 7086(7) 182(7)0(4B) 4682(12) 4750(30) 9096(14) 125(13) Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 238 Table A.4.2. Bond lengths [A] and angles [deg] for exiguamine A. Bonds Bond Lenghts FM (angle (deg)) C(2)-C(3) 1.343(14) C(2)-N(1) 1.412(14) C(2)-H(2) 0.9500 C(3)-C(4) 1.467(15) C(3)-C(24) 1.527(15) C(4)-C(9) 1.381(13) C(4)-C(5) 1.430(14) C(5)-O(5) 1.196(11) C(5)-C(6) 1.508(14) C(6)-C(7) 1.439(12) C(6)-C(19) 1.441(13) C(7)-C(10) 1.379(14) C(7)-C(8) 1.476(14) C(8)-O(8) 1.263(11) C(8)-C(9) 1.431(14) C(9)-N(1) 1.367(13) C(10)-C(18) 1.435(12) C(10)-C(11) 1.439(12) C(11)-O(1) 1.368(10) C(11)-C(12) 1.411(14) C(12)-O(12) 1.320(11) C(12)-C(13) 1.424(13) C(13)-C(14) 1.390(14) C(13)-H(13) 0.9500 C(14)-N(15) 1.437(11) C(14)-C(18) 1.440(15) C(16)-C(17) 1.471(15) C(16)-N(15) 1.513(13) C(16)-H(16A) 0.9900 C(16)-H(16B) 0.9900 C(17)-C(18) 1.454(13) C(17)-H(17A) 0.9900 C(17)-H(17B) 0.9900 C(19)-N(20) 1.461(13) C(19)-O(1) 1.472(11) C(19)-C(23) 1.577(14) C(21)-O(21) 1.206(12) C(21)-N(20) 1.326(12) C(21)-N(22) 1.410(12) C(23)-O(23) 1.249(14) C(23)-N(22) 1.320(14) C(24)-C(25) 1.510(15) C(24)-H(24A) 0.9900 C(24)-H(24B) 0.9900 C(25)-N(26) 1.525(13) C(25)-H(25A) 0.9900 C(25)-H(25B) 0.9900 C(27)-N(1 5) 1.420(13) C(27)-H(27A) 0.9800 C(27)-H(27B) 0.9800 C(27)-H(27C) 0.9800 C(28)-N(15) 1.589(15) C(28)-H(28A) 0.9800 Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 239 C(28)-H(28B) 0.9800 C(28)-H(28C) 0.9800 C(29)-N(22) 1.492(11) C(29)-H(29A) 0.9800 C(29)-H(29B) 0.9800 C(29)-H(29C) 0.9800 C(30)-N(20) 1.418(12) C(30)-H(30A) 0.9800 C(30)-H(30B) 0.9800 C(30)-H(30C) 0.9800 N(1)-H(1) 0.8800 N(26)-H(26A) 0.9100 N(26)-H(26B) 0.9100 N(26)-H(26C) 0.91 00 O(4A)-O(4B) 1.42(3) O(12)-H(12) 0.8400 V(1 )-S(3A)#1 2.233(7) V(1 )-S(3A) 2.233(7) V(1 )-S(3B)#1 2.249(10) V(1)-S(3B) 2.249(10) V(1)-S(2)#1 2.252(6) V(1 )-S(2) 2.252(6) V(1)-S(1) 2.324(7) V(1 )-S(1 )#1 2.324(7) S(1 )-S(3A)#1 2.543(13) S(2)-S(2)#1 1.099(13) S(3A)-S(3B) 1.412(14) S(3A)-S(1 )#1 2.543(13) C(3)-C(2)-N(1) 108.8(10) C(3)-C(2)-H(2) 125.6 N(1 )-C(2)-H(2) 125.6 C(2)-C(3)-C(4) 108.4(9) C(2)-C(3)-C(24) 122.8(12) C(4)-C(3)-C(24) 128.7(10) C(9)-C(4)-C(5) 123.5(10) C(9)-C(4)-C(3) 104.3(9) C(5)-C(4)-C(3) 132.1(9) O(5)-C(5)-C(4) 128.7(10) O(5)-C(5)-C(6) 117.8(9) C(4)-C(5)-C(6) 113.4(8) C(7)-C(6)-C(19) 121.4(9) C(7)-C(6)-C(5) 124.0(8) C(19)-C(6)-C(5) 114.6(8) C(1 0)-C(7)-C(6) 119.2(9) C( I 0)-C(7)-C(8) 123.5(8) C(6)-C(7)-C(8) 116.7(9) O(8)-C(8)-C(9) 121.0(10) O(8)-C(8)-C(7) 121.5(10) C(9)-C(8)-C(7) 117.4(8) N(1 )-C(9)-C(4) 111.1(9) N(1 )-C(9)-C(8) 124.4(9) C(4)-C(9)-C(8) 123.1(9) C(7)-C(10)-C(1 8) 127.4(9) C(7)-C(10)-C(1 1) 117.7(8) C(18)-C(10)-C(1 1) 114.5(9) O(1)-C(11)-C(12) 113.3(8) Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A 240 O(1)-C(1 1)-C(10) 120.6(8) C(12)-C(1 1)-C(10) 126.0(8) 0(12)-C(12)-C(1 1) 122.4(8) 0(12)-C(12)-C(13) 119.9(10) C(1 1)-C(12)-C(13) 117.7(9) C(14)—C(13)—C(12) 118.1(10) C(14)-C(13)-H(13) 121.0 C(12)-C(13)-H(13) 121.0 C(13)-C(14)-N(15) 125.4(11) C(13)-C(14)-C(18) 124.3(9) N(1 5)-C(14)-C(1 8) 109.9(8) C(1 7)-C(1 6)-N(1 5) 102.4(8) C(17)-C(16)-H(16A) 111.3 N(15)-C(16)-H(16A) 111.3 C(17)-C(16)-H(16B) 111.3 N(15)-C(16)-H(16B) 111.3 H(16A)-C(16)-H(16B) 109.2 C(18)-C(17)-C(16) 107.8(10) C(18)-C(17)-H(17A) 110.1 C(16)-C(17)-H(17A) 110.1 C(18)-C(17)-H(17B) 110.1 C(16)-C(17)-H(17B) 110.1 H(17A)-C(17)-H(17B) 108.5 C(10)-C(18)-C(14) 118.9(8) C(10)-C(18)-C(17) 135.3(11) C(14)-C(18)-C(17) 105.8(9) C(6)-C(19)-N(20) 114.8(8) C(6)-C(19)-0(1) 110.6(8) N(20)-C(19)-0(1) 109.8(8) C(6)-C(19)-C(23) 116.2(9) N(20)-C(1 9)-C(23) 100.7(8) 0(1 )-C(1 9)-C(23) 103.8(8) 0(21)-C(21)-N(20) 126.8(10) 0(21)-C(21)-N(22) 124.0(9) N(20)-C(21)-N(22) 109.1(9) 0(23)-C(23)-N(22) 130.6(11) 0(23)-C(23)-C(1 9) 122.3(10) N(22)-C(23)-C(19) 106.2(11) C(25)-C(24)-C(3) 113.2(10) C(25)-C(24)-H(24A) 108.9 C(3)-C(24)-H(24A) 108.9 C(25)-C(24)-H(24B) 108.9 C(3)-C(24)-H(24B) 108.9 H(24A)-C(24)-H(24B) 107.7 C(24)-C(25)-N(26) 107.5(10) C(24)-C(25)-H(25A) 110.2 N(26)-C(25)-H(25A) 110.2 C(24)-C(25)-H(25B) 110.2 N(26)-C(25)-H(25B) 110.2 H(25A)-C(25)-H(25B) 108.5 N(1 5)-C(27)-H(27A) 109.5 N(15)-C(27)-H(27B) 109.5 H(27A)-C(27)-H(27B) 109.5 N(1 5)-C(27)-H(27C) 109.5 H(27A)-C(27)-H(27C) 109.5 H(27B)-C(27)-H(27C) 109.5 Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A 241 N(1 5)-C(28)-H(28A) 109.5 N(15)-C(28)-H(288) 109.5 H(28A)-C(28)-H(28B) 109.5 N(15)-C(28)-H(28C) 109.5 H(28A)-C(28)-H(28C) 109.5 H(28B)-C(28)-H(28C) 109.5 N(22)-C(29)-H(29A) 109.5 N(22)-C(29)-H(29B) 109.5 H(29A)-C(29)-H(29B) 109.5 N(22)-C(29)-H(29C) 109.5 H(29A)-C(29)-H(29C) 109.5 H(29B)-C(29)-H(29C) 109.5 N(20)-C(30)-H(30A) 109.5 N(20)-C(30)-H(30B) 109.5 H(30A)-C(30)-H(30B) 109.5 N(20)-C(30)-H(30C) 109.5 H(30A)-C(30)-H(30C) 109.5 H(30B)-C(30)-H(30C) 109.5 C(9)-N(1 )-C(2) 107.0(9) C(9)-N(1)-H(1) 126.5 C(2)-N(1)-H(1) 126.5 C(27)-N(1 5)-C(14) 111.4(8) C(27)-N(15)-C(16) 109.4(8) C(14)-N(15)-C(16) 105.5(8) C(27)-N(15)-C(28) 112.5(10) C(14)-N(15)-C(28) 106.9(8) C(16)-N(15)-C(28) 110.9(11) C(21)-N(20)-C(30) 123.5(10) C(21 )-N(20)-C(1 9) 111.4(8) C(30)-N(20)-C(1 9) 124.9(9) C(23)-N(22)-C(21) 112.1(9) C(23)-N(22)-C(29) 122.8(10) C(21 )-N(22)-C(29) 125.0(9) C(25)-N(26)-H(26A) 109.5 C(25)-N(26)-H(26B) 109.5 H(26A)-N(26)-H(26B) 109.5 C(25)-N(26)-H(26C) 109.5 H(26A)-N(26)-H(26C) 109.5 H(26B)-N(26)-H(26C) 109.5 C(11)-O(1)-C(19) 117.4(7) C(12)-O(12)-H(12) 109.5 S(3A)#1 -V(1)-S(3A) 128.3(4) S(3A)#1-V(1)-S(3B)#1 36.7(4) S(3A)-V(1)-S(3B)#1 108.9(3) S(3A)#1 -V(1 )-S(3B) 108.9(3) S(3A)-V(1 )-S(3B) 36.7(4) S(3B)#1-V(1 )-S(3B) 113.6(6) S(3A)#1 -V(1 )-S(2)#1 109.8(3) S(3A)-V(1 )-S(2)#1 120.5(3) S(3B)#1-V(1)-S(2)#1 126.7(3) S(3B)-V(1)-S(2)#1 117.7(4) S(3A)#1-V(1 )-S(2) 120.5(3) S(3A)-V(1 )-S(2) 109.8(3) S(3B)#1 -V(1 )-S(2) 117.7(4) S(3B)-V(1)-S(2) 126.7(3) S(2)#1-V(1)-S(2) 28.2(3) Appendix: Experimental Details for X-ray Diffraction Analysis of Exipuamine A 242 S(3A)#1 -V(1 )-S(1) 67.8(4) S(3A)-V(1)-S(1) 115.2(4) S(3B)#1-V(1)-S(1) 104.3(5) S(3B)-V(1)-S(1) 79.3(5) S(2)#1 -V(1 )-S(1) 72.7(3) S(2)-V(1)-S(1) 100.9(3) S(3A)#1 -V(1 )-S(1 )#1 115.2(4) S(3A)-V(1 )-S(1 )#1 67.8(4) S(3B)#1-V(1)-S(1)#1 79.3(5) S(3B)-V(1 )-S(1 )#1 104.3(5) S(2)#1 -V(1 )-S(1 )#1 100.9(3) S(2)-V(1 )-S(1 )#1 72.7(3) S(1)-V(1)-S(1)#1 173.6(4) V(1)-S(1)-S(3A)#1 54.4(3) S(2)#1-S(2)-V(1) 75.88(17) S(3B)-S(3A)-V(1) 72.3(6) S(3B)-S(3A)-S(1)#1 129.7(7) V(1 )-S(3A)-S(1 )#1 57.8(3) S(3A)-S(3B)-V(1) 71.0(5) Symmetry transformations used to generate equivalent atoms: #1 -x+1,y,-z+312 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] Ull U22 U33 U23 U13 U12 C(2) 43(8) 56(7) 66(8) 8(6) 7(6) -17(6) C(3) 56(9) 66(7) 44(7) 22(6) 15(6) 11(6) C(4) 31(7) 48(6) 47(6) -4(5) 13(5) -10(5) C(5) 39(7) 45(6) 56(7) 15(5) 27(6) 13(5) C(6) 30(6) 33(5) 47(6) 4(4) 13(5) -9(4) C(7) 47(8) 26(5) 47(6) 13(4) 10(6) 1(5) C(8) 64(8) 25(5) 48(7) 21(5) 19(6) 6(5) C(9) 28(7) 63(7) 36(6) 17(5) 3(5) -3(5) C(10) 34(7) 26(5) 41(6) 2(4) 5(5) 1(4) C(12) 42(7) 24(5) 65(7) 6(5) -9(6) -6(5) C(13) 45(8) 46(7) 91(9) -29(6) 24(7) -25(6) C(14) 64(9) 36(6) 49(7) 6(5) 24(6) -5(5) C(16) 68(9) 51(7) 63(8) 17(6) 16(7) 15(7) C(17) 73(9) 41(6) 77(9) 6(6) 31(7) 7(6) C(18) 65(8) 24(5) 30(6) 5(4) -4(6) -6(5) C(19) 21(6) 32(5) 62(7) 5(5) 15(5) -1(4) C(23) 27(7) 76(9) 92(10) 49(8) 29(7) 20(6) C(24) 81(10) 74(8) 70(9) 18(7) 34(8) 21(7) C(25) 55(8) 108(10) 30(6) 5(6) 12(6) -11(7) C(27) 75(9) 38(6) 67(8) -5(5) 40(7) 14(6) C(28) 173(19) 155(15) 70(10) 41(10) 55(12) 95(13) C(29) 71(9) 31(5) 58(7) 11(5) 14(7) -5(5) C(30) 58(9) 72(8) 98(11) -16(7) 18(8) 16(7) N(1) 58(7) 42(5) 100(8) 8(5) 44(7) -9(5) N(15) 60(7) 36(5) 40(5) 13(4) 5(5) 16(5) N(20) 57(6) 30(5) 59(6) 3(4) 20(5) 5(4) N(22) 34(6) 53(5) 49(5) 20(4) 15(5) 4(4) N(26) 66(8) 170(11) 84(8) 91(8) 39(7) 72(8) 0(1) 32(4) 50(4) 77(5) 28(4) 17(4) 8(3) 0(5) 38(5) 59(4) 63(5) 30(4) 14(4) -2(4) 0(8) 50(5) 55(4) 65(5) 3(4) 16(4) -16(4) 0(12) 40(5) 32(4) 167(9) 33(5) 27(6) -2(3) 0(21) 89(7) 57(5) 83(6) 21(4) 53(5) 11(4) 0(23) 51(5) 25(4) 80(5) 16(4) 32(5) 8(3) Cl(1) 74(2) 58(2) 65(2) 6(1) 33(2) 17(2) V(1) 37(2) 23(1) 26(1) 0 6(1) 0 S(1) 167(9) 79(5) 62(5) 15(4) 43(5) 7(5) 8(2) 77(5) 67(4) 86(6) 23(3) 25(6) 19(4) S(3A) 76(6) 69(4) 135(7) -30(5) 62(5) -19(4) S(3B) 42(5) 236(13) 232(14) 166(11) 23(7) 3(6) Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A 244 Table A.4.4. Hydrogen coordinates ( x 10’4) and isotropic displacement parameters (AA2 x10A3) for exiguamine A. x y z U(eq) H(2) 3647 4888 5836 73 H(13) 97 3451 4389 75 H(16A) 785 8354 4228 77 H(16B) 823 7976 4908 77 H(17A) 1297 6567 4297 77 H(17B) 1479 6985 5016 77 H(24A) 3649 1189 6451 89 H(24B) 3975 2679 6568 89 H(25A) 3433 2241 7230 80 H(25B) 3826 3528 7386 80 H(27A) 113 6016 4775 85 H(27B) -198 6506 4079 85 H(27C) 117 7797 4553 85 H(28A) 98 5200 3386 197 H(28B) 538 6122 3434 197 H(28C) 93 7086 3335 197 H(29A) 2261 -3195 6675 87 H(29B) 1861 -3172 6889 87 H(29C) 2344 -2490 7334 87 H(30A) 1622 2942 7070 121 H(30B) 1289 3118 6357 121 H(30C) 1786 3824 6610 121 H(1) 2890 5778 5088 76 H(26A) 4020 297 7411 156 H(26B) 4049 1101 7978 156 H(26C) 4353 1580 7693 156 H(12) 314 454 4629 128 Appendix: Experimental Details for X-ray Diffraction Analysis of Ex,guamine A 245 Table A.4.5. Torsion angles [deg] for exiguamine A. N(l)-C(2)-C(3)-C(4) -6.2(13) N( I )-C(2)-C(3)-C(24) 176.8(10) C(2)-C(3)-C(4)-C(9) 2.8(12) C(24)-C(3)-C(4)-C(9) 179.6(11) C(2)-C(3)-C(4)-C(5) 179.0(11) C(24)-C(3)-C(4)-C(5) -4(2) C(9)-C(4)-C(5)-O(5) -173.1(10) C(3)-C(4)-C(5)-O(5) 11(2) C(9)-C(4)-C(5)-C(6) 2.6(15) C(3)-C(4)-C(5)-C(6) -173.0(11) O(5)-C(5)-C(6)-C(7) 171.6(9) C(4)-C(5)-C(6)-C(7) -4.6(14) O(5)-C(5)-C(6)-C(1 9) -6.8(14) C(4)-C(5)-C(6)-C(1 9) 176.9(9) C(1 9)-C(6)-C(7)-C(1 0) 0.7(15) C(5)-C(6)-C(7)-C(1 0) -177.6(9) C(1 9)-C(6)-C(7)-C(8) -170.5(9) C(5)-C(6)-C(7)-C(8) 11.1(14) C(1 0)-C(7)-C(8)-O(8) -10.1(15) C(6)-C(7)-C(8)-O(8) 160.7(9) C(1 0)-C(7)-C(8)-C(9) 174.0(10) C(6)-C(7)-C(8)-C(9) -15.2(13) C(5)-C(4)-C(9)-N(1) -174.9(10) C(3)-C(4)-C(9)-N(1) 1.7(12) C(5)-C(4)-C(9)-C(8) -8.0(17) C(3)-C(4)-C(9)-C(8) 168.7(10) O(8)-C(8)-C(9)-N(1) 3.5(16) C(7)-C(8)-C(9)-N(1) 179.5(10) O(8)-C(8)-C(9)-C(4) -161.7(10) C(7)-C(8)-C(9)-C(4) 14.3(15) C(6)-C(7)-C(1 0)-C(1 8) 158.2(9) C(8)-C(7)-C(10)-C(18) -31.2(16) C(6)-C(7)-C(10)-C(1 1) -14.5(14) C(8)-C(7)-C(10)-C(1 1) 156.1(9) C(7)-C(1 0)-C(1 I )-O(1) -1.5(14) C(1 8)-C(1 0)-C(1 I )-O(1) -175.1(8) C(7)-C(10)-C(1 1)-C(12) -1 79.7(10) C(18)—C(10)—C(11)—C(12) 6.7(15) O(1)—C(11)—C(12)—O(12) 2.0(16) C(10)-C(1 1)-C(12)-O(12) -179.7(10) O(1)—C(11)—C(12)—C(13) 179.1(9) C(1 0)-C(1 I )-C(1 2)-C(1 3) -2.6(17) O(12)-C(1 2)-C(1 3)-C(14) 176.9(11) C(1 I )-C(1 2)-C(1 3)-C(1 4) -0.2(16) C(12)-C(1 3)-C(14)-N(1 5) -173.6(10) C(1 2)-C(1 3)-C(14)-C(1 8) -1.5(17) N(1 5)-C(16)-C(1 7)-C(1 8) -29.0(12) C(7)-C(10)-C(18)-C(14) 179.3(10) C(1 I )-C(1 0)-C(1 8)-C( 14) -7.8(13) C(7)-C(1 0)-C(I 8)-C(1 7) 2(2) C(I 1)-C(10)-C(18)-C(17) 174.7(11) C(13)-C(14)-C(18)-C(10) 5.9(16) N(1 5)-C(14)-C(18)-C(10) 179. 0(9) C(13)-C(14)-C(18)-C(1 7) -175.9(11) Appendix: Experimental Details for X-ray Diffraction Analysis of Exiquamine A 246 N(1 5)-C(14)-C(1 8)-C(17) -2.8(12) C(16)-C(17)-C(18)-C(1 0) -161.8(11) C(1 6)-C(1 7)-C(1 8)-C(14) 20.5(12) C(7)-C(6)-C(1 9)-N(20) -98.6(11) C(5)-C(6)-C(19)-N(20) 79.9(11) C(7)-C(6)-C(1 9)-0(1) 26.3(13) C(5)-C(6)-C(19)-0(1) -155.2(8) C(7)-C(6)-C(19)-C(23) 144.2(10) C(5)-C(6)-C(19)-C(23) -37.2(13) C(6)-C(1 9)-C(23)-0(23) -58.7(15) N(20)-C(19)-C(23)-0(23) 176.6(11) 0(1 )-C(1 9)-C(23)-0(23) 63.0(13) C(6)-C( I 9)-C(23)-N(22) 131.0(10) N(20)-C(19)-C(23)-N(22) 6.3(11) 0(1 )-C(1 9)-C(23)-N(22) -107.3(10) C(2)-C(3)-C(24)-C(25) 116.6(14) C(4)-C(3)-C(24)-C(25) -59.7(16) C(3)-C(24)-C(25)-N(26) 170.6(10) C(4)-C(9)-N(1 )-C(2) -5.4(12) C(8)-C(9)-N(1 )-C(2) -172.2(10) C(3)-C(2)-N(1 )-C(9) 7.2(13) C(1 3)-C(14)-N(1 5)-C(27) 39.2(14) C(1 8)-C(1 4)-N(1 5)-C(27) -133.8(9) C(1 3)—C(14)—N(1 5)—C(16) 157.8(11) C(18)-C(14)-N(15)-C(16) -15.2(11) C(1 3)-C(14)-N(1 5)-C(28) -84.0(14) C(1 8)-C(14)-N(1 5)-C(28) 103.0(12) C(1 7)-C(1 6)-N(1 5)-C(27) 146.6(9) C(1 7)-C(1 6)-N(1 5)-C(14) 26.6(12) C(1 7)-C(1 6)-N(1 5)-C(28) -88.8(10) 0(21 )-C(21 )-N(20)-C(30) -5.3(19) N(22)-C(21 )-N(20)-C(30) 178.4(10) 0(21 )-C(21 )-N(20)-C(1 9) 178.8(11) N(22)-C(21)-N(20)-C(19) 2.5(12) C(6)-C(1 9)-N(20)-C(2 1) -130.9(9) 0(1 )-C(1 9)-N(20)-C(21) 103.8(9) C(23)-C(19)-N(20)-C(21) -5.2(11) C(6)-C(1 9)-N(20)-C(30) 53.3(14) 0(1 )-C(1 9)-N(20)-C(30) -72.1(12) C(23)-C(1 9)-N(20)-C(30) 179.0(10) 0(23)-C(23)-N(22)-C(21) -174.6(12) C(1 9)-C(23)-N(22)-C(2 1) -5.4(12) O(23)-C(23)-N(22)-C(29) 8(2) C(1 9)-C(23)-N(22)-C(29) 177.3(8) 0(21 )-C(21 )-N(22)-C(23) -174.3(11) N(20)-C(21 )-N(22)-C(23) 2.2(13) 0(21 )-C(21 )-N(22)-C(29) 3.0(17) N(20)-C(21 )-N(22)-C(29) 179.5(9) C(1 2)-C(1 I )-0(1 )-C(1 9) -150.5(9) C(10)-C(11)-0(1)-C(19) 31.1(12) C(6)-C(19)-0(1)-C(1 1) -41.8(12) N(20)-C(19)-0(1)-C(1 1) 86.0(9) C(23)-C(19)-0(1)-C(1 1) -167.1(8) S(3A)-V( I )-S(1 )-S(3A)#1 123.2(4) S(3B)#1 -V(1 )-S(1 )-S(3A)#1 3.8(4) S(3B)-V(1 )-S(1 )-S(3A)#1 115.7(3) Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 247 S(2)#1 -V(1 )-S(1 )-S(3A)#1 -120.7(3) S(2)-V( I )-S( 1 )-S(3A)#1 -118.6(3) S(1)#1-V(1)-S(1)-S(3A)#1 -119.6(2) S(3A)#1 -V(1 )-S(2)-S(2)#1 -74.7(10) S(3A)-V(1)-S(2)-S(2)#1 117.9(9) S(3B)#1-V(1)-S(2)-S(2)#1 -116.8(9) S(3B)-V(1)-S(2)-S(2)#1 80.5(11) S(1)-V(1 )-S(2)-S(2)#1 -4.1(10) S(1)#1-V(1)-S(2)-S(2)#1 175.8(10) S(3A)#1 -V(1 )-S(3A)-S(3B) 68.4(6) S(3B)#1-V(1)-S(3A)-S(3B) 104.4(10) S(2)#1 -V(1 )-S(3A)-S(3B) -96.4(6) S(2)-V( I )-S(3A)-S(3B) -125.4(6) S( I )-V(1 )-S(3A)-S(38) -12.4(7) 8(1 )#1 -V( I )-S(3A)-S(3B) 173.8(7) S(3A)#1 -V(1 )-S(3A)-S(1 )#1 -105.3(3) S(3B)#1 -V( I )-S(3A)-S( I )#1 -69.4(5) S(3B)-V(1 )-S(3A)-S(1 )#1 -173.8(7) S(2)#1 -V(1 )-S(3A)-S( I )#1 89.8(3) S(2)-V(1 )-S(3A)-S(1 )#I 60.8(3) S(1 )-V(1 )-S(3A)-S(1 )#1 173.9(4) S(1 )#1 -S(3A)-S(3B)-V(1) 6.9(7) S(3A)#1 -V(1 )-S(3B)-S(3A) -129.5(5) S(3B)#1 -V( 1 )-S(3B)-S(3A) -90.3(7) S(2)#1 -V( I )-S(3B)-S(3A) 104.8(6) S(2)-V(1 )-S(3B)-S(3A) 73.0(8) S(1 )-V( I )-S(3B)-S(3A) 168.6(6) S(1)#I-V(1 )-S(3B)-S(3A) -6.0(6) Symmetry transformations used to generate equivalent atoms: #1 -x+1 ,y,-z+3/2 Appendix: Experimental Details for X-ray Diffraction Analysis of Exiguamine A 248 Table A.4.6. Hydrogen Bonds Donor H Acceptor [ARU] D-H H.. .A D. . . A D-H. .N(1) H(1) 0(23) [7556.01] 0.88 2.05 2.8333(13) 1480(12) H(12) *S(1) [7546.02] 0.84 2.44 3.145(13) 142N(26) H(26A) >0(2B) [ ] 0.91 1.79 2.685(18) 168N(26) H(26B) C1(1) [6546.04] 0.91 2.18 3.067(12) 165N(26) H(26C) *S(2) [1565.02] 0.91 2.25 3.143(17) 166N(26) H26(C) *S(2) [2666.02] 0.91 2.61 3.400(14) 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, 1997- 2003. (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 [w(Fo2-Fc)/(No-Nv)]112 (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, 200- 206. (12) SHELXTL; Version 5.1; BrukerAXS Inc.: Madison, Wisconsin, USA, 1997.

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