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Bioactive marine natural products : isolation, structure elucidation and synthesis of pharmacophore analogues Carr, Gavin 2010

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BIOACTIVE MARINE NATURAL PRODUCTS: ISOLATION, STRUCTURE ELUCIDATION AND SYNTHESIS OF PHARMACOPHORE ANALOGUES  by GAVIN CARR B.Sc., The University of British Columbia, 2004    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Chemistry)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2010  © Gavin Carr, 2010 ii  Abstract Bioassay-guided fractionation of the marine sponge Spongia irregularis led to the isolation of a new sulfated sesterterpenoid, irregularasulfate (2.16), along with two known sulfated sesterterpenoids, halisulfate 7 (2.14) and hipposulfate C (2.15).  All three compounds (2.14-2.16) inhibit the related phosphatases calcineurin, PP1 and PP2A.  The analogue 2.23 was synthesized and showed similar phosphatase inhibitory activity to the natural products. OS O O O O H 2.14 O O SO O O 2.15 OS O O O N O H 2.16 O O PO S O 2.23  One new bafilomycin analogue, bafilomycin F (3.2), along with three known bafilomycin analogues, bafilomycin A1 (3.1), bafilomycin B1 (3.3) and bafilomycin D (3.4), were isolated from a marine-derived bacterium identified as Streptomyces sp.  All four compounds (3.1-3.4) are extremely potent inhibitors of autophagy. iii   Indoleamine 2,3-dioxygenase (IDO) is a relatively new and promising cancer drug target.  Synthetic analogues of exiguamine A, the most potent IDO inhibitor reported to date, were prepared and evaluated for their ability to inhibit IDO in vitro and in vivo.  The most potent of these analogues (4.32, 4.38, 4.39, 4.43 and 4.52) inhibit IDO in vitro with potency comparable to exiguamine A.   A new exiguamine analogue, exiguamine C (5.2), was isolated from the crude extract of eopetrosia exigua.  Exiguamine B (5.1) was also isolated from this crude extract in order to confirm the structure, and the relative configuration was determined with the aid of synthetic exiguamine B.   Bioassay-guided fractionation of the marine fungus Plectosphaerella cucumerina led to the isolation of three new alkaloids, plectosphaeroic acids A-C (6.1-6.3).  All three compounds iv  inhibit IDO with approximately the same potency, while the related compound T988 A was completely inactive.  Cinnabarinic acid was synthesized in order to aid with the structure elucidation of plectosphaeroic acids A-C.  Cinnabarinic acid and analogues were also active against IDO and represent a new pharmacophore for IDO inhibition.   The depsipeptides turnagainolide A (7.3) and turnagainolide B (7.4) were isolated from Bacillus sp.  Both of these compounds activate the enzyme SHIP1 in vitro.  Total syntheses of turnagainolides A and B were accomplished using solid-phase peptide synthesis, and comparison of the synthetic material with the natural products confirmed their structures.   Two novel compounds, the peptide 8.1 and the carotenoid 8.7, were isolated from two unidentified marine sponges.  The structure of 8.1 was confirmed by a total synthesis using solid- v  phase peptide synthesis.  Analogues of 8.1 were also prepared and showed moderate cytotoxicty against T98G cancer cells.  vi  Table of Contents Abstract ........................................................................................................................................... ii Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... xiii List of Figures ............................................................................................................................... xv List of Schemes .......................................................................................................................... xxiii List of Abbreviations .................................................................................................................. xxv Acknowledgements ................................................................................................................... xxxv Co-Authorship Statement......................................................................................................... xxxvi 1. Introduction ................................................................................................................................ 1 1.1. Introduction to Natural Products .......................................................................................... 1 1.1.1. History of Natural Products ........................................................................................... 1 1.2. Classes of Natural Products ................................................................................................. 3 1.2.1. Terpenoids ..................................................................................................................... 4 1.2.2. Polyketides .................................................................................................................... 5 1.2.3. Alkaloids ....................................................................................................................... 7 1.2.4. Nonribosomal Peptides.................................................................................................. 7 1.3. Marine Natural Products ...................................................................................................... 9 1.4. Importance of Natural Products ......................................................................................... 10 1.4.1. Natural Products as Drug Leads .................................................................................. 10 1.4.2. Natural Products and Molecular Genetics ................................................................... 11 1.5. References .......................................................................................................................... 12 2. Isolation and Synthesis of Phosphatase Inhibitors ................................................................... 15 vii  2.1. Calcineurin ......................................................................................................................... 15 2.1.1. Calcineurin and the Immune System........................................................................... 15 2.1.2. Calcineurin and Cardiac Hypertrophy ......................................................................... 16 2.1.3. Inhibitors of Calcineurin ............................................................................................. 17 2.2. Isolation of Sulfated Sesterterpenoids ................................................................................ 21 2.2.1. Structure Elucidation of Irregularasulfate ................................................................... 23 2.2.2. Biological Activity of Sulfated Terpenoids ................................................................. 34 2.2.3. Proposed Biogenesis of Irregularasulfate .................................................................... 34 2.3. Synthesis of Analogues ...................................................................................................... 36 2.4. Conclusions ........................................................................................................................ 39 2.5. Experimental Section ......................................................................................................... 39 2.5.1. General Experimental Procedures ............................................................................... 39 2.5.2. Isolation of Sulfated Sesterterpenoids ......................................................................... 40 2.5.3. Synthesis of Analogues ............................................................................................... 41 2.5.4. Phosphatase Inhibition Assay ...................................................................................... 44 2.5.5. NMR Spectra of Irregularasulfate ............................................................................... 45 2.6. References .......................................................................................................................... 51 3. Isolation of Bafilomycins as Inhibitors of Autophagy ............................................................. 55 3.1. Autophagy .......................................................................................................................... 55 3.1.1. Autophagy and Cancer ................................................................................................ 56 3.2. Bafilomycins ...................................................................................................................... 58 3.3. Isolation of Bafilomycins ................................................................................................... 60 3.4. Structure Elucidation of Bafilomycin F ............................................................................. 61 viii  3.4.1. Relative Configuration of Bafilomycin F .................................................................... 69 3.4.2. Absolute Configuration of Bafilomycin F................................................................... 73 3.5. Biological Activity of Bafilomycins .................................................................................. 75 3.6. Conclusions ........................................................................................................................ 76 3.7. Experimental Section ......................................................................................................... 77 3.7.1. General Experimental Procedures ............................................................................... 77 3.7.2. Isolation of Bafilomycins ............................................................................................ 78 3.7.3. Synthesis of Model Compounds.................................................................................. 78 3.7.4. Marfey’s Analysis of Bafilomycin F ........................................................................... 81 3.7.5. 2D NMR Spectra of Bafilomycin F ............................................................................ 82 3.8. References .......................................................................................................................... 85 4. Synthesis of IDO Inhibitory Analogues of Exiguamine A ...................................................... 92 4.1. Indoleamine 2,3-Dioxygenase (IDO) ................................................................................. 92 4.1.1. IDO as a Cancer Drug Target ...................................................................................... 93 4.1.2. IDO as a Drug Target for Other Diseases ................................................................... 94 4.2. Inhibitors of IDO ................................................................................................................ 96 4.2.1. Tryptophan Analogues ................................................................................................ 96 4.2.2. Polyketides .................................................................................................................. 97 4.2.3. Exiguamine A .............................................................................................................. 97 4.3. Synthesis of IDO Inhibitors ............................................................................................... 98 4.3.1. Synthesis of Cbz-Protected Tryptamine Quinone ....................................................... 99 4.3.2. Modification at C-6 ..................................................................................................... 99 4.3.3. Synthesis of Indolequinone and Analogues .............................................................. 105 ix  4.3.4. Cell-Based IDO Inhibitory Activity .......................................................................... 108 4.4. Conclusions ...................................................................................................................... 109 4.5. Experimental Section ....................................................................................................... 110 4.5.1. General Experimental Procedures ............................................................................. 110 4.5.2. Synthetic Procedures ................................................................................................. 111 4.5.3. IDO Inhibition Assays ............................................................................................... 127 4.6. References ........................................................................................................................ 128 5. Isolation of Exiguamine Analogues ....................................................................................... 131 5.1. Isolation of Exiguamines B and C ................................................................................... 131 5.2. Comparison of Natural and Synthetic Exiguamine B. ..................................................... 135 5.3. Stereochemistry of Exiguamine B ................................................................................... 138 5.4. Structure Elucidation of Exiguamine C ........................................................................... 143 5.5. Biological Activity of Exiguamines B and C ................................................................... 146 5.6. Experimental Section ....................................................................................................... 147 5.6.1. General Experimental Procedures ............................................................................. 147 5.6.2. Isolation of Exiguamines A-C ................................................................................... 147 5.6.3. 2D NMR Spectra of Exiguamine B ........................................................................... 149 5.6.4. 2D NMR Spectra of Exiguamine C ........................................................................... 151 5.7. References ........................................................................................................................ 153 6. Isolation of Plectosphaeroic Acids A-C ................................................................................. 154 6.1. Isolation of Plectosphaeroic Acids A-C ........................................................................... 154 6.2. Structure Elucidation of Plectosphaeroic Acids A-C ....................................................... 155 6.2.1. Structure Elucidation of Plectosphaeroic Acid A...................................................... 155 x  6.2.2. Structure Elucidation of Plectosphaeroic Acids B and C .......................................... 164 6.2.3. Absolute Configurations of Plectosphaeroic Acids A-C ........................................... 171 6.3. Proposed Biogenesis of Plectosphaeroic Acids A-C ....................................................... 173 6.4. Biological Activity of Plectosphaeroic Acids A-C .......................................................... 175 6.5. Synthesis of Cinnabarinic Acid Analogues ..................................................................... 176 6.6. Conclusions ...................................................................................................................... 178 6.7. Experimental Section ....................................................................................................... 178 6.7.1. General Experimental Procedures ............................................................................. 178 6.7.2. Isolation of Plectosphaeroic Acids A-C .................................................................... 179 6.7.3. Synthetic Procedures ................................................................................................. 180 6.7.4. 2D NMR Spectra of Plectosphaeroic Acid A ............................................................ 183 6.7.5. 2D NMR Spectra of Plectosphaeroic Acid B. ........................................................... 186 6.7.6. 2D NMR Spectra of Plectosphaeroic Acid C ............................................................ 190 6.8. References ........................................................................................................................ 192 7. Synthesis of Turnagainolides A and B ................................................................................... 193 7.1. Phosphatidylinositol-3,4,5-Triphosphate and SHIP ......................................................... 193 7.1.1. SHIP1 and Regulation of the Cell Cycle ................................................................... 194 7.1.2. SHIP1 and the Immune System................................................................................. 195 7.1.3. The PI3K Pathway as a Drug Target ......................................................................... 197 7.2. Isolation of Turnagainolides A and B .............................................................................. 197 7.2.1. Biological Activity of Turnagainolides A and B....................................................... 201 7.3. Synthesis of Turnagainolides A and B ............................................................................. 202 7.3.1. Retrosynthetic Analysis of Turnagainolides A and B ............................................... 202 xi  7.3.2. Synthesis of Polyketide/Shikimate Fragment............................................................ 202 7.3.3. Solid-Phase Synthesis of the Linear Peptide ............................................................. 203 7.3.4. Macrolactonization .................................................................................................... 206 7.4. Comparison of the Synthetic Materials to the Natural Products ...................................... 207 7.5. Absolute Configuration of Turnagainolide B .................................................................. 209 7.6. Synthesis of the Peptide Analogue 7.24 ........................................................................... 211 7.7. Conclusions ...................................................................................................................... 213 7.8. Experimental Section ....................................................................................................... 214 7.8.1. General Experimental Procedures ............................................................................. 214 7.8.2. Isolation of Turnagainolides A and B ....................................................................... 215 7.8.3. Synthetic Procedures ................................................................................................. 215 7.9. References ........................................................................................................................ 221 8. Chemical Prospecting ............................................................................................................. 224 8.1. Chemical Prospecting....................................................................................................... 224 8.2. Novel Peptide from a Marine Sponge .............................................................................. 224 8.3. Total Synthesis of 8.1 ....................................................................................................... 232 8.4. Novel Carotenoid from a Marine Sponge ........................................................................ 238 8.5. Conclusions ...................................................................................................................... 242 8.6. Experimental Section ....................................................................................................... 242 8.6.1. General Experimental Procedures ............................................................................. 242 8.6.2. Isolation Procedures .................................................................................................. 243 8.6.3. Synthetic Procedures ................................................................................................. 244 8.6.4. 2D NMR Spectra of 8.1 ............................................................................................. 246 xii  8.6.5. 2D NMR Spectra of 8.7. ............................................................................................ 249 8.7. References ........................................................................................................................ 252 9. Concluding Chapter ............................................................................................................... 253 9.1. Conclusions ...................................................................................................................... 253 9.2. Future Directions .............................................................................................................. 254 9.3. References ........................................................................................................................ 255  xiii  List of Tables Table 1.1. Examples of terpenoid natural products. ....................................................................... 4 Table 2.1. 1H and 13C NMR chemical shift values of irregularasulfate and halisulfate 7. ........... 25 Table 2.2. NMR data for lactam substructure of irregularasulfate. .............................................. 29 Table 2.3. NMR data for isobutylammonium counterion of irregularasulfate. ............................ 32 Table 2.4. IC50 values of sulfated sesterterpenoids against phosphatases (µM). .......................... 34 Table 3.1. NMR data for bafilomycin F. ...................................................................................... 65 Table 3.2. Selected 1H and 13C NMR chemical shift values of bafilomycin F. ............................ 70 Table 3.3. Selected 1H and 13C NMR chemical shift values of 3.9 and 3.10. ............................... 71 Table 3.4. Selected 1H and 13C NMR chemical shift values of 3.14 and 3.15. ............................. 72 Table 3.5. Fold increase in number of autophagosomes in the presence of 3.1-3.4. .................... 75 Table 4.1. Ki and IC50 values of synthetic exiguamine A analogues against IDO. .................... 107 Table 4.2. EC50 and LD50 values of synthetic analogues in a cell-based IDO assay. ................. 109 Table 5.1. NMR data for exiguamine B. ..................................................................................... 135 Table 5.2. NMR data for exiguamine C. ..................................................................................... 146 Table 6.1. NMR data for plectosphaeroic acid A. ...................................................................... 159 Table 6.2. NMR data for plectosphaeroic acid B........................................................................ 167 Table 6.3. NMR data for plectosphaeroic acid C........................................................................ 170 Table 6.4. IDO inhibitory activity of plectosphaeroic acids A-C and cinnabarinic acid. ........... 176 Table 7.1. NMR data for turnagainolide A. ................................................................................ 199 Table 7.2. NMR data for turnagainolide B. ................................................................................ 200 Table 8.1. NMR data for 8.1. ...................................................................................................... 227 Table 8.2. MS/MS of 8.1 on m/z 842 [M-H]- ion. ...................................................................... 231 xiv  Table 8.3. MS/MS of 8.1 on m/z 844 [M+H]+ ion. ..................................................................... 231 Table 8.4. MS/MS of 8.1 on m/z 866 [M+Na]+ ion. ................................................................... 231 Table 8.5. NMR data for 8.7. ...................................................................................................... 241  xv  List of Figures Figure 1.1. Historically significant natural products. ..................................................................... 3 Figure 1.2. Biosynthesis of squalene. ............................................................................................. 5 Figure 1.3. Biosynthesis of erythromycin A (1.7). ......................................................................... 6 Figure 1.4. Examples of biologically active polyketides. ............................................................... 6 Figure 1.5. Examples of biologically active alkaloids. ................................................................... 7 Figure 1.6. Biosynthesis of nonribosomal peptides. ....................................................................... 8 Figure 1.7. Examples of biologically active nonribosomal peptides. ............................................. 8 Figure 1.8. Examples of biologically active marine natural products. ......................................... 10 Figure 2.1. Structures of cyclosporin A (2.1) and FK506 (2.2). ................................................... 18 Figure 2.2. Protein phosphatase inhibitors. ................................................................................... 19 Figure 2.3. Inhibitors of calcineurin. ............................................................................................ 20 Figure 2.4. Structures of terpenoids isolated from Spongia irregularis. ...................................... 23 Figure 2.5. 1H NMR spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. ................... 24 Figure 2.6. 13C NMR spectrum of irregularasulfate recorded in CDCl3 at 150 MHz. ................. 24 Figure 2.7. C-1 through C-15 substructure of halisulfate 7 and irregularasulfate (I). .................. 26 Figure 2.8. Selected 1H and 13C NMR chemical shifts and 2D correlations in irregularasulfate. 27 Figure 2.9. Expanded HMBC spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. .... 27 Figure 2.10. Expanded COSY spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. ... 28 Figure 2.11. 1H NMR signals unaccounted for in irregularasulfate (CDCl3, 600 MHz). ............. 30 Figure 2.12. 1H and 13C NMR chemical shifts and 2D correlations in isobutylammonium. ........ 31 Figure 2.13. Expanded COSY spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. ... 31 Figure 2.14. Comparison of 1H NMR spectra of irregularasulfate with different counterions. ... 33 xvi  Figure 2.15. Alkylammonium counterions of halisulfate 7 and hipposulfate C. .......................... 34 Figure 2.16. Comparison of sulfate and phosphate moieties. ....................................................... 37 Figure 2.17. COSY spectrum of irregularasulfate with isobutylammonium ion. ......................... 45 Figure 2.18. HSQC spectrum of irregularasulfate with isobutylammonium ion. ........................... 45 Figure 2.19. Expanded HSQC spectrum of irregularasulfate with isobutylammonium ion. ........ 46 Figure 2.20. HMBC spectrum of irregularasulfate with isobutylammonium ion. .......................... 46 Figure 2.21. Expanded HMBC spectrum of irregularasulfate with isobutylammonium ion. ....... 47 Figure 2.22. 1H NMR spectrum of irregularasulfate with sodium ion in CDCl3 at 600 MHz......... 47 Figure 2.23. 13C NMR spectrum of irregularasulfate with sodium ion in CDCl3 at 150 MHz. ........ 48 Figure 2.24. COSY spectrum of irregularasulfate with sodium counterion (CDCl3, 600 MHz). . 48 Figure 2.25. HSQC spectrum of irregularasulfate with sodium counterion (CDCl3, 600 MHz). . 49 Figure 2.26. HMBC spectrum of irregularasulfate with sodium counterion (CDCl3, 600 MHz). 49 Figure 2.27. NOESY spectrum of irregularasulfate with sodium counterion (CDCl3, 600 MHz). 50 Figure 2.28. Expanded NOESY spectrum of irregularasulfate with sodium counterion.............. 50 Figure 3.1. Mechanism of autophagy. .......................................................................................... 55 Figure 3.2. Structures of 3.1-3.4. .................................................................................................. 61 Figure 3.3. 1H NMR spectrum of bafilomycin F recorded in CD3OD at 600 MHz. .................... 62 Figure 3.4. 13C NMR spectrum of bafilomycin F recorded in CD3OD at 150 MHz. ................... 63 Figure 3.5. Selected 1H NMR chemical shifts of bafilomycin C1 and bafilomycin F (CD3OD). . 63 Figure 3.6. Selected 13C NMR chemical shifts of bafilomycin C1 and bafilomycin F (CD3OD). 64 Figure 3.7. Selected 1H and 13C NMR chemical shifts and 2D correlations in bafilomycin F. .... 67 Figure 3.8. Expanded HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ...... 68 Figure 3.9. Expanded COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ....... 68 xvii  Figure 3.10. Comparison of NMR chemical shifts of bafilomycin F to 3.9 and 3.10. ................. 71 Figure 3.11. Comparison of NMR chemical shifts of bafilomycin F to 3.14 and 3.15. ............... 73 Figure 3.12. Accumulation of EGFP observed in the presence of 3.1-3.4. .................................. 76 Figure 3.13. COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ...................... 82 Figure 3.14. Expanded COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ..... 82 Figure 3.15. HSQC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ...................... 83 Figure 3.16. Expanded HSQC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ..... 83 Figure 3.17. HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. ..................... 84 Figure 3.18. Expanded HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. .... 84 Figure 3.19. ROESY spectrum of bafilomycin F recorded in CD3OD at 600 MHz..................... 85 Figure 4.1. The kynurenine pathway. ........................................................................................... 92 Figure 4.2. Proposed catalytic mechanism of IDO. ...................................................................... 93 Figure 4.3. Tryptophan analogues that inhibit IDO. ..................................................................... 96 Figure 4.4. Polyketide inhibitors of IDO. ..................................................................................... 97 Figure 4.5. Structure of exiguamine A (4.16). .............................................................................. 98 Figure 4.6. Proposed pharmacophore of exiguamine A (4.17). .................................................... 98 Figure 5.1. Structures of exiguamines B (5.1) and C (5.2). ........................................................ 132 Figure 5.2. 1H NMR spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz. ............... 132 Figure 5.3. 13C NMR spectrum of exiguamine B recorded in DMSO-d6 at 150 MHz. .............. 133 Figure 5.4. Selected 1H and 13C NMR chemical shifts and 2D correlations in exiguamine B. .. 134 Figure 5.5. Comparison of 1H NMR spectra of natural and synthetic exiguamine B................. 137 Figure 5.6. Comparison of 13C NMR spectra of natural and synthetic exiguamine B. .............. 137 Figure 5.7. 1H NMR spectrum of a mixture of natural and synthetic exiguamine B.................. 138 xviii  Figure 5.8. 1H NMR spectrum of natural exiguamine B with excess chiral shift reagent. ......... 139 Figure 5.9. 1H NMR spectrum of exiguamine B with and without chiral shift reagent. ............ 140 Figure 5.10. NOESY and ROESY correlations in synthetic exiguamine B. .............................. 141 Figure 5.11. 1D ROESY spectrum of synthetic exiguamine B in DMSO-d6 at 600 MHz. ........ 141 Figure 5.12. 1D ROESY spectrum of synthetic exiguamine B in DMSO-d6 at 600 MHz. ........ 142 Figure 5.13. Expanded 2D ROESY spectrum of synthetic exiguamine B. ................................ 142 Figure 5.14. Expanded 2D NOESY spectrum of synthetic exiguamine B. ................................ 143 Figure 5.15. 1H NMR spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz. ............. 144 Figure 5.16. 13C NMR spectrum of exiguamine C recorded in DMSO-d6 at 150 MHz. ............ 144 Figure 5.17. Selected 1H and 13C NMR chemical shifts and 2D correlations in exiguamine C. 145 Figure 5.18. Expanded HMBC spectrum of exiguamine C in DMSO-d6 at 600 MHz............... 145 Figure 5.19. COSY spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz. ................. 149 Figure 5.20. HSQC spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz. ................. 149 Figure 5.21. HMBC spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz................. 150 Figure 5.22. Expanded HMBC spectrum of exiguamine B in DMSO-d6 at 600 MHz............... 150 Figure 5.23. COSY spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz. ................. 151 Figure 5.24. HSQC spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz. ................. 151 Figure 5.25. HMBC spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz................. 152 Figure 5.26. Expanded HMBC spectrum of exiguamine C in DMSO-d6 at 600 MHz............... 152 Figure 6.1. Structures of Plectosphaeroic acids A (6.1), B (6.2), C (6.3) and T988 A (6.4). ..... 155 Figure 6.2. 1H NMR spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz. ................ 156 Figure 6.3. 13C NMR spectrum of plectosphaeroic acid A in DMSO-d6 at 150 MHz................ 157 Figure 6.4. 1H NMR chemical shifts of substructure I (DMSO-d6) and T988 B (MeOD). ........ 157 xix  Figure 6.5. 13C NMR chemical shifts of substructure I (DMSO-d6) and T988 B (MeOD). ....... 158 Figure 6.6. 1H NMR chemical shifts of substructure II and cinnabarinic acid (DMSO-d6). ...... 162 Figure 6.7. 13C and 15N NMR chemical shifts of II and cinnabarinic acid (DMSO-d6). ............ 163 Figure 6.8. ROESY correlations confirming linkage between N-6 and C-9’’ in 6.1. ................ 163 Figure 6.9. Expanded ROESY spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). .... 164 Figure 6.10. Structure of plectosphaeroic acid B (6.2). .............................................................. 165 Figure 6.11. 1H NMR spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. .............. 165 Figure 6.12. 13C NMR spectrum of plectosphaeroic acid B in DMSO-d6 at 150 MHz. ............. 166 Figure 6.13. Structure of plectosphaeroic acid C (6.3). .............................................................. 168 Figure 6.14. 1H NMR spectrum of plectosphaeroic acid C in DMSO-d6 at 600 MHz. .............. 169 Figure 6.15. Structures of leptosin D (6.9) and 6.10................................................................... 171 Figure 6.16. CD spectrum of plectosphaeroic acid A. ................................................................ 172 Figure 6.17. CD spectrum of plectosphaeroic acid B. ................................................................ 172 Figure 6.18. CD spectrum of plectosphaeroic acid C. ................................................................ 172 Figure 6.19. CD spectrum of T988 A. ........................................................................................ 173 Figure 6.20. Possible biogenesis of plectosphaeroic acid A (6.1). ............................................. 174 Figure 6.21. Possible biosynthesis of cinnabarinic acid (6.7). .................................................... 175 Figure 6.22. Structure of actinomycin D (1.15). ......................................................................... 178 Figure 6.23. COSY spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz. .. 183 Figure 6.24. HSQC spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz. .. 183 Figure 6.25. HMBC spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz. . 184 Figure 6.26. Expanded HMBC spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). .... 184 Figure 6.27. ROESY spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz. ............... 185 xx  Figure 6.28. Expanded ROESY spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). .. 185 Figure 6.29. 1H-15N HSQC spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz. ..... 186 Figure 6.30. COSY spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz. ... 186 Figure 6.31. HSQC spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz. ... 187 Figure 6.32. HMBC spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz. . 187 Figure 6.33. Expanded HMBC spectrum of plectosphaeroic acid B (DMSO-d6, 600 MHz). .... 188 Figure 6.34. ROESY spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. ............... 188 Figure 6.35. Expanded ROESY spectrum of plectosphaeroic acid B (DMSO-d6, 600 MHz). .. 189 Figure 6.36. 1H-15N HSQC spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. ..... 189 Figure 6.37. COSY spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz. ... 190 Figure 6.38. HSQC spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz. ... 190 Figure 6.39. HMBC spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz. . 191 Figure 6.40. Expanded HMBC spectrum of plectosphaeroic acid C (DMSO-d6, 600 MHz). .... 191 Figure 7.1. The PI3K pathway. ................................................................................................... 194 Figure 7.2. Structures of pelorol (7.1) and AQX-MN100 (7.2). ................................................. 197 Figure 7.3. Structures of turnagainolides A (7.3) and B (7.4). ................................................... 198 Figure 7.4. Retrosynthetic analysis of polyketide/shikimate fragment (7.10). ........................... 202 Figure 7.5. Comparison of 1H NMR spectra of natural and synthetic turnagainolide A. ........... 207 Figure 7.6. Comparison of 13C NMR spectra of natural and synthetic turnagainolide A. .......... 208 Figure 7.7. Comparison of 1H NMR spectra of natural and synthetic turnagainolide B. ........... 208 Figure 7.8. Comparison of 13C NMR spectra of natural and synthetic turnagainolide B. .......... 209 Figure 7.9. Selected 1H NMR chemical shift values and Δδ values of 7.22 and 7.23. ............... 211 Figure 7.10. Mosher’s analysis of turnagainolide B. .................................................................. 211 xxi  Figure 7.11. Structure of peptide analogue (7.24). ..................................................................... 212 Figure 8.1. Structure of the novel peptide 8.1............................................................................. 225 Figure 8.2. 1H NMR spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................................ 226 Figure 8.3. 13C NMR spectrum of 8.1 recorded in DMSO-d6 at 150 MHz. ............................... 226 Figure 8.4. Key HMBC correlations in 8.1. ................................................................................ 228 Figure 8.5. Expanded HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. .................. 229 Figure 8.6. Key ROESY correlations in 8.1. .............................................................................. 230 Figure 8.7. Expanded ROESY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................ 230 Figure 8.8. Comparison of 1H NMR spectra of natural and synthetic 8.1. ................................. 235 Figure 8.9. Comparison of 13C NMR spectra of natural and synthetic 8.1. ................................ 236 Figure 8.10. Structure of 8.7. ...................................................................................................... 238 Figure 8.11. Structure of paracentrone (8.8). .............................................................................. 239 Figure 8.12. 1H NMR spectrum of 8.7 recorded in CD2Cl2 at 600 MHz.................................... 239 Figure 8.13. 13C NMR spectrum of 8.7 recorded in CD2Cl2 at 150 MHz. ................................. 240 Figure 8.14. COSY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. .................................. 246 Figure 8.15. HSQC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. .................................. 247 Figure 8.16. Expanded HSQC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................. 247 Figure 8.17. HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................................. 248 Figure 8.18. Expanded HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................ 248 Figure 8.19. ROESY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ................................ 249 Figure 8.20. COSY spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. ...................................... 249 Figure 8.21. HSQC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. ...................................... 250 Figure 8.22. HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. ..................................... 250 xxii  Figure 8.23. Expanded HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. .................... 251 Figure 8.24. Expanded HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. .................... 251 Figure 9.1. Diversity-oriented synthesis of turnagainolide analogues. ....................................... 255  xxiii  List of Schemes Scheme 2.1. Isolation of sulfated sesterterpenoids. ...................................................................... 21 Scheme 2.2. Removal of isobutylammonium counterion. ............................................................ 33 Scheme 2.3. Proposed biogenesis of halisulfate 7. ....................................................................... 35 Scheme 2.4. Proposed biogenesis of irregularasulfate.................................................................. 36 Scheme 2.5. Synthesis of 2.19. ..................................................................................................... 36 Scheme 2.6. Synthesis of 2.21. ..................................................................................................... 37 Scheme 2.7. Synthesis of 2.23. ..................................................................................................... 38 Scheme 3.1. Isolation of bafilomycins. ......................................................................................... 60 Scheme 3.2. Synthesis of model compounds 3.9 and 3.10. .......................................................... 70 Scheme 3.3. Synthesis of model compounds 3.14 and 3.15. ........................................................ 72 Scheme 3.4. Chemical degradation and Marfey's analysis of bafilomycin F. .............................. 74 Scheme 4.1. Synthesis of 4.18. ..................................................................................................... 99 Scheme 4.2. Synthesis of 4.25. ................................................................................................... 100 Scheme 4.3. Synthesis of 4.27. ................................................................................................... 100 Scheme 4.4. Deprotection of 4.27. .............................................................................................. 101 Scheme 4.5. Synthesis of 4.30. ................................................................................................... 101 Scheme 4.6. Synthesis of 4.31. ................................................................................................... 102 Scheme 4.7. Synthesis of 4.32. ................................................................................................... 102 Scheme 4.8. Deprotection of 4.32. .............................................................................................. 103 Scheme 4.9. Synthesis of 4.40. ................................................................................................... 103 Scheme 4.10. Synthesis of 4.39. ................................................................................................. 103 Scheme 4.11. Deprotection of 4.39. ............................................................................................ 104 xxiv  Scheme 4.12. Proposed mechanism for the formation of 4.43. .................................................. 104 Scheme 4.13. Synthesis of 4.45. ................................................................................................. 105 Scheme 4.14. Synthesis of 4.47. ................................................................................................. 105 Scheme 4.15. Synthesis of 4.48. ................................................................................................. 106 Scheme 4.16. Synthesis of 4.50. ................................................................................................. 106 Scheme 4.17. Synthesis of 4.51. ................................................................................................. 107 Scheme 5.1. Trauner's synthesis of exiguamines A (4.16) and B (5.1). ..................................... 136 Scheme 6.1. Synthesis of plectosphaeroic acid A dimethyl ester (6.6). ..................................... 160 Scheme 6.2. Synthesis of cinnabarinic acid (6.7). ...................................................................... 162 Scheme 6.3. Synthesis of 6.12. ................................................................................................... 177 Scheme 6.4. Synthesis of 6.14. ................................................................................................... 177 Scheme 7.1. Mosher’s analysis of turnagainolide A (7.3). ......................................................... 201 Scheme 7.2. Synthesis of 7.12. ................................................................................................... 203 Scheme 7.3. Synthesis of linear peptide 7.14. ............................................................................ 205 Scheme 7.4. Macrolactonization. ................................................................................................ 206 Scheme 7.5. Methanolysis and Mosher’s analysis of turnagainolide B (7.4). ............................ 210 Scheme 7.6. Synthesis of 7.28. ................................................................................................... 212 Scheme 7.7. Synthesis of peptide analogue 7.24. ....................................................................... 213 Scheme 8.1. Solid-phase synthesis of 8.2. .................................................................................. 233 Scheme 8.2. Synthesis of 8.3. ..................................................................................................... 234 Scheme 8.3. Synthesis of 8.4. ..................................................................................................... 234 Scheme 8.4. Synthesis of 8.1. ..................................................................................................... 235 Scheme 8.5. Synthesis of 8.5. ..................................................................................................... 237 xxv  List of Abbreviations O  -  degrees oC  -  degrees Celsius 1D  -  1-dimensional 2D  -  2-dimensional 16S rRNA -  ribosomal RNA with a sedimentation coefficient of 16 Svedberg units 26S rDNA -  ribosomal DNA with a sedimentation coefficient of 26 Svedberg units [α]D 20  -  specific rotation at sodium D-line (589 nm) recorded at 20 oC acetone-d6 -  deuterated acetone AD  -  Alzheimer’s disease Ala  -  alanine aq.  -  aqueous ARN  -  age-related nuclear cataract ATP  -  adenosine triphosphate ATPase  -  adenosine triphosphatase B  -  base BBr3  -  boron tribromide B.C.  -  before Christ BF3 .Et2O -  boron trifluoride diethyl etherate Boc  -  tert-butyloxycarbonyl br.  -  broad tBu  -  tert-butyl n-BuLi  -  n-butyllithium xxvi  n-BuOH  -  n-butanol Bz  -  benzyl c  -  concentration 13C  -  carbon-13 Ca2+  -  calcium ion CBrCl3  -  bromotrichloromethane Cbz  -  carboxybenzyl CbzCl  -  benzyl chloroformate CD  -  circular dichroism CD2Cl2  -  deuterated dichloromethane CDCl3  -  deuterated chloroform CD3OD  -  deuterated methanol CH2Cl2  -  dichloromethane CHCl3  -  chloroform CH3I  -  iodomethane ClCOOCH3 -  methyl chloroformate CoA  -  coenzyme A COSY  -  correlation spectroscopy CuCl  -  copper(I) chloride CuSO4 .5H2O -  copper(II) sulfate pentahydrate Δ  -  heat δH  - 1H NMR chemical shift in parts per million from tetramethyl silane δC  - 13C NMR chemical shift in parts per million from tetramethyl silane xxvii  ΔδH  -  difference in 1H NMR chemical shifts in parts per million ΔδC  -  difference in 13C NMR chemical shifts in parts per million d  -  doublet dd  -  doublet of doublets ddd  -  doublet of doublet of doublets DDQ  -  2,3-dichloro-5,6-dicyanobenzoquinone DIAD  -  diisopropyl azodicarboxylate DIC  -  ,’-diisopropylcarbodiimide DIPEA  -  ,-diisopropylethylamine DMAP  -  4-dimethylaminopyridine DMF  -  ,-dimethylformamide DMSO  -  dimethyl sulfoxide DMSO-d6 -  deuterated dimethyl sulfoxide DNA  -  deoxyribonucleic acid DPPA  -  diphenylphosphoryl azide dq  -  doublet of quartets dt  -  doublet of triplets ε  -  molar extinction coefficient (M-1 cm-1) EC50  -  concentration giving 50% of the maximum effectiveness EGFP  -  enhanced green fluorescent protein ER  -  endoplasmic reticulum ESI-MS  -  electrospray ionization mass spectrometry ESI-MS(-) -  negative ion ESI-MS xxviii  ESI-MS(+) -  positive ion ESI-MS EtOAc  -  ethyl acetate EtOH  -  ethanol FDA  -  U.S. Food and Drug Administration FDAA  -  1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey’s reagent) Fe2+  -  iron(II) or ferrous ion Fe3+  -  iron(III) or ferric ion FKBP12 -  12 kDa FK506-binding protein Fmoc  -  9-fluorenylmethyloxycarbonyl Fmoc-Cl -  9-fluorenylmethyloxycarbonyl chloride g  -  gram(s) G1  -  growth phase one of the cell cycle GATA  -  guanosine-adenosine-thymidine-adenosine Glu  -  glutamic acid h  -  hour(s) H2  -  hydrogen gas H2SO4  -  sulfuric acid HCl  -  hydrochloric acid 3-HKG  -  3-hydroxy-L-kynurenine glycoside HMBC  -  heteronuclear multiple bond coherence HOBt  -  hydroxybenzotriazole HPLC  -  high-performance liquid chromatography HRESI-MS -  high-resolution electrospray ionization mass spectrometry xxix  Hsp90  -  heat shock protein 90 Hz  -  hertz IC50  -  concentration giving 50% inhibition IDO  -  indoleamine 2,3-dioxygenase Ile  -  isoleucine J  -  coupling constant (in hertz) Ki  -  inhibition constant ([E][I]/[EI]) K2CO3  -  potassium carbonate kDa  -  kilodalton KOH  -  potassium hydroxide λ  -  wavelength λmax  -  wavelength giving a local maximum absorbance LC3  -  light chain 3 LD50  -  concentration causing 50% lethality LDA  -  lithium diisopropylamide µg   -  microgram(s) µm   -  micrometre(s) µM   -  micromolar m  -  multiplet M  -  molar MeCN  -  acetonitrile MeOH  -  methanol mg  -  milligram(s) xxx  MgSO4  -  magnesium sulfate min  -  minute(s) MHz  -  megahertz mL  -  millilitre(s) mm  -  millimetre(s) Mo-Kα  -  molybdenum K-alpha MS  -  mass spectrometry MS/MS  -  tandem mass spectrometry 1-MT  -  1-methyl-DL-tryptophan mTOR  -  mammalian target of rapamycin (R)-MTPA-Cl -  (R)-(-)-α-methoxy-α-(trifluromethyl)phenylacetyl chloride (S)-MTPA-Cl -  (S)-(+)-α-methoxy-α-(trifluromethyl)phenylacetyl chloride m/z  -  mass-to-charge ratio nm  -  nanometre(s) nM  -  nanomolar N2  -  nitrogen gas Na+  -  sodium ion NaBH4  -  sodium borohydride NaBH3CN - sodium cyanoborohydride NAD+  -  nicotinamide adenine dinucleotide NaH  -  sodium hydride NaHCO3 -  sodium bicarbonate (or sodium hydrogen carbonate) NaOCH3 -  sodium methoxide xxxi  NaOH  -  sodium hydroxide NCI  -  National Cancer Institute N.D.  -  not determined NFAT  -  nuclear factor of activated T-cells NH4Cl  -  ammonium chloride Ni  -  nickel NLS  -  nuclear localization signal NMR  -  nuclear magnetic resonance NOESY  -  nuclear Overhauser effect spectroscopy NO(SO3K)2 -  potassium nitrosodisulfonate (Fremy’s salt) NRPS  -  nonribosomal peptide synthetase O2  -  oxygen gas OBzl  -  benzyl ester 3-OHKyn -  3-hydroxy-L-kynurenine o/n  -  overnight p  -  para p53  -  tumour protein 53 Pd/C  -  palladium on carbon PDK1  -  phosphatidylinositol-dependent kinase 1 PDT  -  1,3-propanedithiol pH  -  -log10[H +] Phe  -  phenylalanine PI-3,4,5-P3 -  phosphatidylinositol-3,4,5-triphosphate xxxii  PI-4,5-P2 -  phosphatidylinositol-4,5-biphosphate PI3K  -  phosphoinositide 3-kinase PKB  -  protein kinase B PKS  -  polyketide synthase PP1  -  protein phosphatase 1 PP2A  -  protein phosphatase 2A PP2B  -  protein phosphatase 2B (also known as calcineurin) PPh3  -  triphenylphosphine ppm  -  parts per million PPTS  -  pyridinium p-toluenesulfonate Pro  -  proline PTEN  -  phosphate and tensin homolog PyBOP  -  benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate quant.  -  quantitative quint.  -  quintuplet R  -  rectus Rf  -  retention factor RNA  -  ribonucleic acid ROESY  -  rotating frame nuclear Overhauser effect spectroscopy r.t.  -  room temperature s  -  singlet S  -  sinister SCUBA  -  self-contained underwater breathing apparatus xxxiii  SHIP  -  Src homology 2-containing inositol 5-phosphatase siRNA  -  small interfering ribonucleic acid sp.  -  species θ  -  angle t  -  triplet T98G  -  human glioblastoma TBAF  -  tetra-n-butylammonium fluoride TBDMS-Cl -  tert-butyldimethylsilyl chloride td  -  triplet of doublets TDO  -  tryptophan 2,3-dioxygenase TFA  -  trifluoroacetic acid TH  -  T-helper THF  -  tetrahydrofuran TLC  -  thin-layer chromatography Tl(OCOCF3)3 -  thallium(III) trifluoroacetate TMS-Br  -  trimethylsilyl bromide Trp  -  tryptophan pTSA  -  p-toluenesulfonic acid Tyr  -  tyrosine u  -  unlike U.S.  -  United States of America UV  -  ultraviolet UV-Vis  -  ultraviolet-visible xxxiv  Val  -  valine V-ATPase -  vacuolar-type H+-adenosine triphosphatase xxxv  Acknowledgements  First and foremost I would like to thank my supervisor Dr. Raymond Andersen for all of his support.  His passion for natural products chemistry, combined with his optimism, has kept me motivated.  Above all, I appreciate the kindness and respect that he always shows to all of his students.  I would also like to thank all of the members of the Andersen lab, both past and present, for all of their help.  Special thanks go to Mike LeBlanc and Dr. David Williams for collecting the marine organisms that I worked on, and to Helen Bottriell for culturing these organisms.  Thanks also go to the staff at the NMR and MS facilities at UBC, in particular to Dr. Nick Burlinson (NMR) and Dr. Yun Ling (MS).  I would also like to thank all of our collaborators who ran all of the biological assays.  I would like to thank Mike Shopik and Mikolaj Raszek for running all of the phosphatase inhibition assays, as well as their supervisor Dr. Charles Holmes at the University of Alberta. I would like to thank Aruna Balgi for running all of the autophagy assays, as well as her supervisor Dr. Michel Roberge.  Aruna also ran all of the yeast cell-based IDO inhibition assays. For the IDO projects, I would like to thank Marco Chung for his kinetic analysis and calculation of Ki values for all of the inhibitors, as well as his supervisor Dr. Grant Mauk.  Thanks also go to Wendy Tay, Eduardo Vottero and Sarah Andersen, also from the Mauk lab, for their work in screening for inhibitors of IDO.  Last but not least, I would like to thank my wife Kailee for always being there for me, and for helping to proofread my thesis.  I could not have done this without all of her support. xxxvi  Co-Authorship Statement  For chapter 2, my contributions to this research include the isolation and structure elucidation of the natural products, as well as the synthesis of analogues.  The biological assays were conducted by Micheal Shopik and Mikolaj Raszek from the Holmes lab at the University of Alberta.  My contributions to the writing of the manuscript include the experimental section and supporting information, while the majority of the manuscript body was written by my supervisor.  My contributions to chapter 3 include the isolation and structure elucidation of bafilomycins A1, B1, D and F, as well as the chemical degradation experiments and synthesis of model compounds.  Bafilomycins G-J were isolated and elucidated by Dr. David Williams. Biological assays were performed by Aruna Balgi from the Roberge lab.  I contributed to the writing of the manuscript, along with help from my supervisor.  My contributions to chapter 4 include the synthesis of all synthetic analogues of exiguamine A.  Biological assays were performed by Marco Chung and Eduardo Vottero from the Mauk lab.  I contributed the writing of the experimental section and supporting information of the manuscript, while the majority of the manuscript body was written by my supervisor.  My contributions to chapter 5 include re-isolating exiguamine B, confirming the structure and solving the relative configuration of exiguamine B.  I also compared natural and synthetic exiguamine A and B by reversed-phase HPLC and NMR spectroscopy.  Finally, I isolated and solved the structure of exiguamine C.  The synthesis of exiguamines A and B was done by Matthew Volgraf from the Trauner lab at UC Berkeley.  The majority of the manuscript was written by Dr. Dirk Trauner.  For chapter 6, I did the majority of the work on the isolation of plectosphaeroic acids A- C with the help of Wendy Tay.  I also contributed all of the structure elucidation of xxxvii  plectosphaeroic acids A-C and the synthesis of analogues.  The biological assays were performed by Marco Chung, Wendy Tay and Sarah Andersen from the Mauk lab.  My contributions to the writing of the manuscript include the experimental section and supporting information, while the majority of the manuscript body was written by my supervisor.  My contributions to chapter 7 include the synthesis of turnagainolides A and B, as well as the peptide analogue.  I also determined the absolute configuration of turnagainolide B at C-3 using Mosher’s method.  Turnagainolides A and B were isolated by Dr. Dehai Li, Dr. Martha Zhang and Dr. David Williams.  The manuscript has not yet been written.  My contributions to chapter 8 include the isolation, structure elucidation and synthesis of a new peptide, as well as the synthesis of several analogues.  I also isolated and elucidated the structure of a new carotenoid from a marine sponge. 1  1. Introduction 1.1. Introduction to *atural Products Metabolites are small molecules produced by organisms, and may be classified as either primary or secondary metabolites.  Secondary metabolites are more commonly known as natural products.  While there is no precise definition that distinguishes secondary metabolites from primary metabolites, secondary metabolites are usually found in a limited number of species whereas primary metabolites are usually found in most, if not all, species.  Additionally, secondary metabolites are not involved in the normal growth and development of an organism and are usually not essential for survival, although they often confer a survival advantage to that organism.  For example, a secondary metabolite found in a sponge that is toxic to animals may deter predation.  Often times, these natural products are not produced by the sponge itself but by symbiotic bacteria growing inside the sponge.  Pigments and pheromones are also secondary metabolites that are beneficial, though not necessarily essential, to the survival of the organism. 1.1.1. History of *atural Products The practice of using natural substances as medicines is at least several thousand years old. Clay tablets written between 4000 and 2600 B.C. in Sumer, and the Ebers Papyrus written in Egypt around 1500 B.C. are among the oldest known written records of the use of herbs and natural medicines.1  These documents detail the use of hundreds of different herbs, including the use of opium to relieve pain.2  Opium has been one of the most important pain relievers throughout history and pharmaceuticals derived from opium, including morphine (1.1) and codeine, are still vital painkillers today.  In ancient Greece, Hippocrates prescribed the use of herbs in the 5th century B.C., including the use of willow to treat pain and fever.3  Willow contains salicylic acid (1.2) and aspirin, one of the most commonly used pharmaceuticals today, 2  is a derivative of salicylic acid.  In ancient China the herb ephedra, which contains ephedrine, has been used for the treatment of asthma and colds for thousands of years and is one of over two hundred herbs described in the ancient Chinese text Shen ong Ben Cao Jing written approximately 2,000 years ago.4  Even today, ephedrine is used for these purposes.  The use of medicinal herbs likely predates these written records.  A burial site at Shanidar Cave in present day Iraq, dated from around 60,000 years ago, contains at least eight different herbs that may have been used medicinally.5 A resurgence in the use of natural medicines occurred during the Age of Enlightenment. The 17th century herbalist Nicholas Culpeper wrote two famous books on herbs: The English Physician and The Complete Herbal in 1652 and 1653, respectively.6  These books describe thousands of medicinal herbs.  Around the same time period, bark of the cinchona tree was being used to treat malaria in Europe.7  The active ingredient in cinchona trees is quinine (1.3), a drug which has saved countless lives.  Quinine and related compounds are still the drugs of choice in many parts of the world to treat malaria, a disease which claims approximately one million lives per year.8  In 1785, the English physician William Withering described the use of an extract of foxglove to treat heart conditions.9  Foxglove (Digitalis purpurea) contains the glycoside digoxin, which is still widely prescribed today for the treatment of cardiac arrhythmia. In the 19th and 20th centuries not only were natural medicines being used, but for the first time the active ingredients could be purified, identified and synthesized.  The first natural product to be purified was morphine in 1817.10  Quinine was purified from cinchona trees in 1820, although its structure was not determined until nearly one hundred years later in 1908.11 The total synthesis of a precursor of quinine by Robert Burns Woodward in 1944 is still 3  considered a landmark in total synthesis, and partly because of this work Woodward was awarded the Nobel Prize in Chemistry in 1965.12 Perhaps the single most important discovery in the field of natural products was the discovery of penicillin from the fungus Penicillium notatum by Alexander Fleming in 1928.13 The structure of penicillin G (1.4) was determined by Dorothy Crowfoot Hodgkin using X-ray crystallography in 1945, and mass production began in the early 1940’s led by Howard Florey and Ernst Boris Chain.14  The 1945 Nobel Prize in Medicine was awarded to Fleming, Florey and Chain for their work on penicillin.  It is impossible to say how many lives penicillin has saved, but some estimates put it as high as 200 million.15  Not surprisingly, the discovery of penicillin is considered to be one of the most important scientific discoveries of the millennium.  Figure 1.1. Historically significant natural products. 1.2. Classes of *atural Products Most natural products can be classified as terpenoids, polyketides, alkaloids or nonribosomal peptides based on how they are biosynthesized.  Some natural products have a mixed biosynthetic origin, where different parts of the molecule are synthesized by different 4  biochemical pathways.  Common examples of this are mixed polyketide-nonribosomal peptides and meroterpenoids, which are terpenoids with a non-terpenoid portion. 1.2.1. Terpenoids The basic building blocks of terpenoids are five carbon isoprene units, which are derived from dimethylallyl pyrophosphate (1.5) or isopentenyl pyrophosphate (1.6).  Terpenoids can be categorized by the number of isoprene units that they contain (table 1.1).  These units are usually joined together head to tail, but may also be joined tail to tail, especially for terpenoids containing six or more isoprenoid units (figure 1.2).  Terpenoids often undergo cyclization(s) and Wagner-Meerwein rearrangements to give a wide variety of carbon skeletons, which may undergo further modifications such as oxidation or glycosylation.  Terpenes are a sub-class of terpenoids that consist of only carbon and hydrogen (hydrocarbons), although the names “terpene” and “terpenoid” are often used interchangeably. Table 1.1. Examples of terpenoid natural products. Number of isoprene units Type         Examples 1 Hemiterpenoid Prenol 2 Monoterpenoid Menthol, Limonene 3 Sesquiterpenoid Farnesol, Artemisinin 4 Diterpenoid Phytol, Taxol 5 Sesterterpenoid Dysidiolide 6 Triterpenoid Lanosterol, Squalene 8 Tetraterpenoid β-Carotene, Lycopene > 8 Polyterpenoid Rubber 5   Figure 1.2. Biosynthesis of squalene. 1.2.2. Polyketides Polyketides are biosynthesized by the condensation of small carboxylic acid units,  which is catalyzed by polyketide synthase (PKS) enzymes.16  The starter unit, typically acetyl-CoA or propionyl-CoA, and the extender unit, typically malonlyl-CoA or methylmalonyl-CoA, are both attached to the PKS enzyme through a thioester bond.  These units then undergo a decarboxylative Claisen-like condensation to give a β-keto thioester (figure 1.3).  The β-ketone functionality may be further processed to give an alcohol, a double bond, or a methylene group. 6  Additional extender units are added and processed in the same manner before the polyketide is either cyclized or hydrolyzed from the enzyme.  The polyketide often undergoes postsynthetic modifications such as hydroxylation and glycosylation to produce the final product.  Examples of polyketides with important biological activities are erythromycin A (1.7), doxorubicin (1.8) and lovastatin (1.9) (figures 1.3 and 1.4).  Figure 1.3. Biosynthesis of erythromycin A (1.7).  Figure 1.4. Examples of biologically active polyketides. 7  1.2.3. Alkaloids Alkaloids are natural products containing at least one basic nitrogen atom, although the term alkaloid is often used to describe natural products with any nitrogen atoms.17 Biosynthetically, alkaloids are derived from amino acids such as tryptophan, tyrosine, phenylalanine and lysine.  Heterocycles such as indole, isoquinoline, piperidine and purine are common components of alkaloids.  Due to the basicity of alkaloids, and the ease with which they can be purified from plants by extracting with water and basifying, alkaloids were some of the first biologically active compounds to be isolated from nature.  Examples of biologically active alkaloids include caffeine (1.10), vincristine (1.11), atropine (1.12) and strychnine (1.13) (figure 1.5).  Figure 1.5. Examples of biologically active alkaloids. 1.2.4. *onribosomal Peptides Nonribosomal peptides are synthesized from amino acids by nonribosomal peptide synthetase (NRPS) enzymes (figure 1.6), and they often contain non-standard amino acids such as D-amino acids and amino acids with -methyl groups, halogen atoms and various other post- synthetic modifications.18  Nonribosomal peptides are often cyclized to form a macrocycle, and the side chains of serine, threonine and cysteine residues may also cyclize to form oxazole and 8  thiazole rings.  In a depsipeptide, the peptide is cyclized through the hydroxyl group of a polyketide fragment or a hydroxylated amino acid to form a macrolactone.  Examples of biologically active nonribosomal peptides include the antibiotics vancomycin (1.14) and actinomycin D (1.15) (figure 1.7).  Figure 1.6. Biosynthesis of nonribosomal peptides.  Figure 1.7. Examples of biologically active nonribosomal peptides. 9  1.3. Marine *atural Products While humans have been utilizing terrestrial plants as a source of medicine for thousands of years, marine organisms were rarely used in medicine until relatively recently.  It was not until the 1960’s and 1970’s that chemists began to explore the chemistry of marine organisms.  This was no doubt aided by the development of SCUBA technology in the 1940’s, prior to which exploring the ocean would have been difficult at best.  The novelty of the molecules isolated during these early years, often belonging to new structural classes, validated the use of marine organisms as a source of new small molecules.19  The first marine natural product to become a drug was the pain reliever ω-conotoxin MVIIA (ziconotide), a peptide that was isolated from a cone snail in 1987 and approved by the FDA in 2004.20  Two antiviral drugs, cytosine arabinoside (Ara-C) and adenine arabinoside (Ara-A), which are synthetic analogues of sponge metabolites, were approved by the FDA in 1969 and 1980, respectively.19  In 2007, the marine natural product trabectedin, which was isolated from a tunicate in 1987,21 was approved by the European Medicines Agency for the treatment of soft tissue sarcoma and it is currently in clinical trials for breast, prostate and paediatric cancers.  Many other marine natural products have been, or currently are, in clinical trials.  Examples of biologically active marine natural products that have been in preclinical development programs include didemnin B (1.16), bryostatin 1 (1.17), discodermolide (1.18) and salinosporamide A (1.19) (figure 1.8). 10  O H N O Cl OH O O O O O O O OH O HO O O OH HOH O O O O OO OH HO OH OH O O NH2 O O O NHO O N N OOO N H NHO HO O N O O N O HO 1.16 1.17 1.18 1.19 H  Figure 1.8. Examples of biologically active marine natural products. 1.4. Importance of *atural Products 1.4.1. *atural Products as Drug Leads  One of the most important reasons for studying natural products is their potential use as drugs or drug leads.  Of all of the small-molecule drugs approved between 1981 and 2006, 34% are either natural products or derived from natural products.22  When compounds inspired by natural products are included, this number jumps to 63%.  In the field of anticancer and anti- infective drugs, the importance of natural products is even more apparent.  For anti-infective drugs approved between 1981 and 2006, 48% are natural products or derived from natural products, with another 21% being inspired by natural products.  In this same time period, 42% of anticancer drugs approved are natural products or derived from natural products; an additional 36% are inspired by natural products, leaving only 22% being purely synthetic. 11  1.4.2. *atural Products and Molecular Genetics  In addition to serving as drug leads, natural products can serve as biological tools for chemical genetics, which can in turn lead to the discovery of new drug targets.  Chemical genetics is the technique of using small molecules to explore the link between a gene and a phenotype, and can be classified as either forward or reverse chemical genetics.  In forward chemical genetics, molecules are screened for the ability to elicit a particular phenotype of interest.  Determining the target protein of this compound establishes a link between this protein and the observed phenotype and can be used to discover new drug targets.  For example, the natural product colchicine has been used in medicine for over 2000 years for the treatment of gout.  In the 1930’s it was observed that colchicine could cause mitotic arrest,23 and regression of tumours.24  In the 1960’s, radiolabeled colchicine was used to identify the target of colchicine, and the result was the discovery of a protein called tubulin.25  Tubulin is the monomeric constituent of microtubules, which are the target of many of the most successful anticancer drugs.26 In reverse chemical genetics, molecules are screened for the ability to inhibit a particular enzyme of interest.  The active molecules can then be used to look at the phenotypic effects of inhibiting that enzyme.  While forward chemical genetics can be used to find new drug targets, reverse chemical genetics can be used to validate a possible new drug target.  For example, heat shock protein 90 (Hsp90) was identified as a possible cancer drug target using forward chemical genetics.27  Natural products and other small molecules were then screened for the ability to inhibit Hsp90, and these inhibitors showed good anti-tumour activity, validating Hsp90 as a cancer drug target. 12   Chemical genetics is a complementary tool to other genetic techniques such as site- directed mutagenesis and small interfering RNA (siRNA), and can offer some distinct advantages.  In chemical genetics, it is often possible to restore the activity of the enzyme by simply washing away the chemical, allowing the study of the effects of temporarily inhibiting an enzyme.  Secondly, while a small molecule may inhibit the catalytic activity of an enzyme, that enzyme can still interact with other proteins.  Therefore, inhibiting the enzyme with a small molecule may give different results than if the protein is not expressed at all.28  Chemical genetics can also often be less difficult and time consuming than other methods. 1.5. References (1) van Wyk, B.-E.; Wink, M. Medicinal Plants of the World; Timber Press, Inc.: Portland, OR, 2004, p. 12. (2) Fowler, M. W. J. Sci. Food Agric. 2006, 86, 1797-1804. (3) Mowrey, D. B. The Scientific Validation of Herbal Medicine; Keats Publishing: Lincolnwood, IL, 1986, p. 224. (4) Yaniv, Z.; Bachrach, U. Handbook of Medicinal Plants; Food Products Press and The Haworth Medical Press: Binghamton, NY, 2005, p. 215. (5) Solecki, R. S. Science 1975, 190, 880-881. (6) Culpeper, N. The Complete Herbal and English Physician Enlarged; Logos Press: New Delhi, India, 2007, pp. 1-357. (7) Willcox, M.; Bodeker, G.; Rasoanaivo, P. Traditional Medicinal Plants and Malaria; CRC Press: Boca Raton, FL, 2004, pp. 22-30. (8) World Malaria Report 2008; World Health Organization: Geneva, Switzerland, 2008, p. 12. 13  (9) Withering, W. An Account of the Foxglove and Some of its Medical Uses; M. Swinney: London, England, 1785, pp. 1-207. (10) Huxtable, R. J.; Schwarz, S. K. Mol. Interv. 2001, 1, 189-191. (11) Rabe, P. Ber. Dtsch. Chem. Ges. 1908, 41, 62-70. (12) Woodward, R. B.; Doering, W. E. J. Am. Chem. Soc. 1944, 66, 849. (13) Fleming, A. Br. J. Exp. Pathol. 1929, 10, 226-236. (14) Abraham, E. P. at. Prod. Rep. 1987, 4, 41-46. (15) Kidder, D. S.; Oppenheim, N. D. The Intellectual Devotional Modern Culture; Rodale Inc.: New York, NY, 2008, p. 110. (16) Staunton, J.; Weissman, K. J. at. Prod. Rep. 2001, 18, 380-416. (17) Moss, G. P.; Smith, P. A. S.; Tavernier, D. Pure Appl. Chem. 1995, 67, 1307-1375. (18) Schwarzer, D.; Finking, R.; Marahiel, M. A. at. Prod. Rep. 2003, 20, 275-287. (19) Fenical, W. Oceanography 2006, 19, 110-119. (20) Olivera, B. M.; Cruz, L. J.; de Santos, V.; LeCheminant, G. W.; Griffin, D.; Zeikus, R.; McIntosh, J. M.; Galyean, R.; Varga, J.; Gray, W. R.; Rivier, J. Biochemistry 1987, 26, 2086-2090. (21) Rinehart, K. L.; Holt, T. G. Purification and characterization of ecteinascidins 729, 7433, 745, 759a, 759b, and 770 having antibacterial and antitumor properties, 1987. PCT Int. Appl., WO87-US1226 19870601. (22) Newman, D. J.; Cragg, G. M. J. at. Prod. 2007, 70, 461-477. (23) Lits, F. C. R. Soc. Biol. 1934, 115, 1421-1423. (24) Amoroso, E. C. ature 1935, 135, 266-267. (25) Weisenberg, R. C.; Borisy, G. G.; Taylor, E. W. Biochemistry 1968, 7, 4466-4479. 14  (26) Jordan, M. A.; Wilson, L. at. Rev. Cancer 2004, 4, 253-265. (27) Neckers, L. Curr. Top. Med. Chem. 2006, 6, 1163-1171. (28) Knight, Z. A.; Shokat, K. M. Cell 2007, 128, 425-430. 15  2. Isolation and Synthesis of Phosphatase Inhibitors 2.1. Calcineurin Calcineurin, also known as protein phosphatase 2B (PP2B), is a member of the serine/threonine phosphatase enzyme family.  Other members of this family include protein phosphatase 1 (PP1)1 and protein phosphatase 2A (PP2A).2  All of these phosphatases play important roles in many signal transduction pathways.  Calcineurin contains an autoinhibitory domain that inhibits the enzymatic activity of calcineurin in the absence of calcium ions (Ca2+) and calmodulin.3  In the presence of Ca2+ and calmodulin, this inhibition is removed and calcineurin is active.  In this way, calcineurin mediates many of the processes resulting from increased Ca2+ concentrations. 2.1.1. Calcineurin and the Immune System  One of the most important roles for calcineurin is its role in regulating the immune system, which is illustrated by the discovery that calcineurin is the target of the immunosuppressive drugs cyclosporin A (2.1) and FK506 (also known as tacrolimus) (2.2).4 Recognition of antigens by immunoreceptors sets off a signal transduction cascade resulting in release of Ca2+ from the endoplasmic reticulum (ER) to the cytoplasm through store-operated calcium entry.5  The decrease in ER Ca2+ concentration activates calcium-release-activated calcium channels in the plasma membrane, resulting in influx of Ca2+ from the extracellular space and an increase in the intracellular Ca2+ concentration.5  Ca2+ binds to calmodulin, allowing calmodulin to bind to and activate calcineurin.6  Ca2+ also binds directly to the regulatory subunit of calcineurin (calcineurin B),7 and this binding also increases the activity of calcineurin.8  Thus, increased levels of Ca2+ activate calcineurin by these two mechanisms. 1 A version of this chapter has been published. Carr, G.; Raszek, M.; Van Soest, R.; Matainaho, T.; Shopik, M.; Holmes, C. F. B.; Andersen, R. J. (2007) Protein Phosphatase Inhibitors Isolated from Spongia irregularis Collected in Papua New Guinea. J. Nat. Prod. 70:1812-1815. 16   Once activated by Ca2+/calmodulin, calcineurin dephosphorylates multiple sites on the nuclear factor of activated T-cells (NFAT) family of transcription factors, including NFAT1, NFAT2, NFAT3 and NFAT4 (collectively referred to as NFAT).  This dephosphorylation exposes the nuclear localization signal (NLS) on NFAT, resulting in its relocation from the cytoplasm to the nucleus.9  Sustained calcineurin activity is required to keep NFAT inside the nucleus, and in the absence of calcineurin activity NFAT is rapidly rephosphorylated by kinases and exported back to the cytoplasm.9  Dephosphorylation by calcineurin also appears to enhance the DNA-binding activity of NFAT.10  Once activated by calcineurin, NFAT cooperates with other transcription factors to regulate the expression of genes involved in the production of cytokines, chemokines, and cell surface receptors that are important for the immune system.11 While NFAT is the most widely studied target of calcineurin, other transcription factors involved in the immune response are also regulated by calcineurin.11  Calcineurin and NFAT also play crucial roles in T-cell development and differentiation.9,12-13  Experiments with cyclosporin A- treated mice and calcineurin-deficient mice have demonstrated the role of calcineurin in T-cell development,12-14 while experiments with NFAT1, NFAT2 or NFAT4 knockout mice have demonstrated the role of NFAT in T helper (TH) cell differentiation. 9 2.1.2. Calcineurin and Cardiac Hypertrophy  Cardiac hypertrophy is the enlargement of heart ventricles due to an increase in the size of heart muscle cells (cardiac myocytes).  While physiological cardiac hypertrophy can be a beneficial adaptation to exercise enabling the heart to pump more blood, pathological cardiac hypertrophy is an unhealthy enlargement of heart muscles that decreases the volume of the heart chambers.15  Pathological cardiac hypertrophy is associated with an increased risk of heart disease and death.16-19 17   Various stimuli can elicit cardiac hypertrophy, and many of these involve an increase in intracellular Ca2+ concentrations.15  The increase in Ca2+ concentration activates calcineurin, which dephosphorylates NFAT3 and causes its translocation to the nucleus.  Inside the nucleus, NFAT3 can interact with other transcription factors such as GATA4 and upregulate various genes associated with cardiac hypertrophy.20  Consistent with the role of calcineurin in cardiac hypertrophy, transgenic mice expressing constitutively active calcineurin had hearts 2-3 times heavier relative to body weight than control mice.20  Many of these transgenic mice also experienced heart failure and sudden death.20  Likewise, transgenic mice expressing constitutively active NFAT3 also exhibited cardiac hypertrophy.20  However, when the constitutively active calcineurin transgenic mice were treated with the calcineurin inhibitor cyclosporin A, hypertrophy was significantly reduced.20  Numerous other studies have confirmed that blocking calcineurin activity, either with inhibitors or by genetic means, reduces cardiac hypertrophy.21  Finally, patients with cardiac hypertrophy showed elevated levels of calcineurin activity and nuclear NFAT3.22 2.1.3. Inhibitors of Calcineurin  The most widely studied calcineurin inhibitors are the immunosuppressive drugs cyclosporin A (2.1) and FK506 (2.2) (figure 2.1).  These compounds do not inhibit calcineurin directly, but rather bind to their respective immunophilins cyclophilin A and the 12 kDa FK506- binding protein (FKBP12), and these complexes in turn inhibit calcineurin.4  Cyclosporin A and FK506 cannot effectively inhibit calcineurin in the absence of these immunophilins.23-25 Cyclosporin A (formerly known as ramihyphin A) is a cyclic undecapeptide originally isolated from a Fusarium fungus,26 while FK506 is a macrocyclic polyketide originally isolated from Streptomyces tsukubaensis.27  While these compounds serve as important immunosuppressive 18  drugs to prevent organ rejection after transplantation, they exhibit serious side effects including nephrotoxicity,28 neurotoxicity,29 cardiovascular disease30 and diabetogenesis.31  Figure 2.1. Structures of cyclosporin A (2.1) and FK506 (2.2). Since the discovery of these immunosuppressive drugs, many related compounds have been discovered in an effort to find less toxic drugs.  Compounds related to FK506 include ascomycin32 and pimecrolimus,33 among others.34,35  Analogues of cyclosporin A have also been investigated.36  Although their structures are unrelated, FK506 and cyclosporin A exhibit similar side effects.  This is possibly because not only do they both inhibit the catalytic activity of calcineurin, but their respective immunophilins bind at a similar location on calcineurin and also disrupt the interaction of calcineurin with other proteins.37  Inhibitors that act directly on calcineurin might not disrupt such interactions and may have less side effects, although this remains to be shown.  While potent and specific inhibitors of the related phosphatases PP1 and PP2A are known, there is a lack of potent and specific inhibitors that act directly on calcineurin.  For example, spirastrellolide A (2.3) is 50 times more potent against PP2A (IC50 = 1 nM) than PP1 (IC50 = 50 nM) and shows no inhibition of calcineurin. 38  Likewise, okadaic acid (2.4) is much 19  more active against PP2A (IC50 = 1 nM) than PP1 (IC50 ≈ 100-500 nM) or calcineurin (IC50 = 4 µM).39  Microcystin LR (2.5), which is a member of the microcystin family of cyclic peptides, is much more active against PP1 (IC50 = 0.1 nM) and PP2A (IC50 = 0.1 nM) than calcineurin (IC50 = 200 nM).40  While most microcystins are approximately equally active against PP1 and PP2A, analogues of microcystin LA have been synthesized that are selective for PP1 over PP2A.41 Many other potent inhibitors of both PP1 and PP2A are known.42,43 O O HO OH OO HO O O O O OH HO O O Cl O HO HO O OH O O O O O OH OH O O OH H H H O N NH HN O H N H N O O NH HN O N H NH2 HN O O O O OH HO O 2.3 2.4 2.5  Figure 2.2. Protein phosphatase inhibitors. Known inhibitors of calcineurin include dibefurin (2.6), a fungal metabolite that inhibits calcineurin with an IC50 = 16 µg/mL (44 µM). 44   Three related natural products isolated from marine sponges, secobatzellines A (2.7) and B (2.8) and discorhabdin P (2.9), inhibit calcineurin with IC50’s of 0.55 µg/mL (2.2 µM), 2.21 µg/mL (8.58 µM) and 0.55 µg/mL (1.2 µM), respectively.45,46  Recently, eremoxylarins A (2.10) and B (2.11) were found to inhibit calcineurin with IC50’s of 2.7 µM and 1.4 µM, respectively, and this inhibition was shown to occur in the absence of immunophilins.47  A synthetic compound PD 144795 (2.12) inhibited 20  calcineurin in a cell-based assay with an IC50 in the low micromolar range. 48  However, in each case no information was given about whether these compounds also inhibit the related phosphatases PP1 and PP2A.  One compound that did show selectivity for calcineurin over PP1 is the endothal derivative containing a trans-cyclopropylphenyl moiety (2.13), although no data was reported on its inhibition of PP2A.49  Peptide inhibitors of calcineurin have also been reported.37,50 O O O O O HO O OH N H HN O HO OH H2N Cl N H O O HO OH H2N Cl N N H N O O Br Br O O H O OH O O O O H O OH O O S O O O NH2 O O O O O O O 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13  Figure 2.3. Inhibitors of calcineurin. 21  2.2. Isolation of Sulfated Sesterterpenoids  In an effort to find new marine natural products that inhibit calcineurin, a library of crude marine extracts was screened for calcineurin inhibitory activity by our collaborators in the lab of Dr. Charles Holmes at the University of Alberta.51  One of the hits in this screen was identified as the marine sponge Spongia irregularis collected in Papua New Guinea, which was subjected to bioassay-guided fractionation in collaboration with the Holmes lab (scheme 2.1).  Scheme 2.1. Isolation of sulfated sesterterpenoids. The frozen sponge was extracted twice with methanol (MeOH) and the MeOH was dried to give the crude extract.  The crude extract was partitioned between H2O and hexane, H2O and dichloromethane (CH2Cl2), and finally between H2O and ethyl acetate (EtOAc).  Bioassay results 22  revealed that both the hexane and CH2Cl2 layers were active against calcineurin.  The hexane and CH2Cl2 layers were separately subjected to silica gel chromatography (eluent: gradient from hexane to EtOAc to MeOH).  The active compounds eluted from the silica gel column with 10% MeOH/EtOAc in each case.  The active fractions from the silica gel column were pooled and subjected to reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  Once again, the active compounds from both the hexane and CH2Cl2 layers eluted with the same solvent system (75% MeOH/H2O) indicating that they may contain identical or similar compounds.  The active fractions were again pooled and subjected to reversed-phase HPLC (eluent: 70% MeOH/H2O).  Isolated from the hexane layer were the known sesterterpenoids halisulfate 7 (2.14) and hipposulfate C (2.15) as the active compounds (figure 2.4).  Isolated from the CH2Cl2 layer was the novel sesterterpenoid irregularasulfate (2.16) (figure 2.4), along with the known compounds halisulfate 7 and hipposulfate C.  The known sesterterpenoid igernellin (2.17) (figure 2.4) was isolated from an inactive fraction from the silica gel column of the hexane layer, which eluted with 10% EtOAc/hexane.  Although igernellin is structurally related to hipposulfate C, it was completely inactive in the calcineurin inhibition assay.  The structures of halisulfate 7, hipposulfate C and igernellin were determined by analysis of their NMR and mass spectrometry data, and confirmed by comparison of this data with literature values.52-55 23   Figure 2.4. Structures of terpenoids isolated from Spongia irregularis. 2.2.1. Structure Elucidation of Irregularasulfate  Irregularasulfate gave an [M - H]- peak at m/z 536.3407 in the HRESI-MS(-) spectrum, consistent with a molecular formula of C30H51NO5S (calculated for C30H50NO5S: 536.3410).  A comparison of the 1D (figures 2.5 and 2.6 and table 2.1) and 2D (figures 2.17 to 2.21) NMR data for irregularasulfate with the NMR data for halisulfate 7 indicated that they were closely related. The 1H and 13C NMR chemical shifts of irregularasulfate from C-1 through C-15, as well as the attached methyl groups (C-20 through C-23), were nearly identical to the corresponding chemical shifts in halisulfate 7 (table 2.1). 24   Figure 2.5. 1H NMR spectrum of irregularasulfate recorded in CDCl3 at 600 MHz.  Figure 2.6. 13C NMR spectrum of irregularasulfate recorded in CDCl3 at 150 MHz. 25  Table 2.1. 1H and 13C NMR chemical shift values of irregularasulfate and halisulfate 7. position δC mult. δH (J  in Hz) position δC mult. δH (J  in Hz) 1 117.0 CH 5.28 br., s 1 117.1 CH 5.29 br., s 2 23.5 CH2 1.98, m; 2.02, m 2 23.4 CH2 1.97, m; 2.02, m 3 31.5 CH2 1.06, m; 1.36, m 3 31.5 CH2 1.05, m; 1.34, m 4 31.5 C 4 31.5 C 5 43.7 CH 1.51, m 5 43.8 CH 1.50, m 6 30.4 CH2 1.06, m; 1.78, m 6 31.0 CH2 1.06, m; 1.77, m 7 31.5 CH2 1.43, m; 1.50, m 7 31.5 CH2 1.43, m; 1.50, m 8 44.9 CH 1.23, m 8 44.9 CH 1.23, m 9 42.9 C 9 42.8 C 10 146.4 C 10 146.3 C 11 28.7 CH2 1.56, m 11 28.6 CH2 1.55, m 12 25.0 CH2 1.05, m 12 24.6 CH2 1.05, m 13 38.7 CH 1.53, m 13 38.7 CH 1.51, m 14 31.0 CH2 1.25, m 14 30.4 CH2 1.24, m 15 24.9 CH2 1.49, m 15 26.9 CH2 1.50, m 16 26.6 CH2 2.23, m 16 25.1 CH2 2.36, t (7.5) 17 140.6 C 17 125.2 C 18 134.5 CH 6.62, s 18 111.3 CH 6.23 br., s 19 50.9 CH2 3.80, s 19 142.8 CH 7.32 br., s 20 27.9 CH3 0.83, s 20 27.9 CH3 0.83, s 21 28.2 CH3 0.84, s 21 28.2 CH3 0.84, s 22 16.8 CH3 0.81, d (7.0) 22 16.7 CH3 0.82, d (7.2) 23 23.5 CH3 0.95, s 23 23.4 CH3 0.95, s 24 70.9 CH2 3.87, t (8.6) 24 72.3 CH2 3.86, t (8.3) 3.97, dd (9.3, 3.7) 3.95, dd (9.1, 4.7) 25 172.0 C 25 139.1 CH 7.20, s 26 40.9 CH2 3.43, t (7.5) 27 37.6 CH2 1.41, m 28 26.0 CH 1.54, m 29 22.8 CH3 0.92, d (6.6) 30 22.8 CH3 0.92, d (6.6) Irregularasulfate (2.16) a Halisulfate 7 (2.14) a a Spectra recorded in CDCl3 at 600 MHz Analysis of the 2D NMR data confirmed that irregularasulfate contained the substructure I, which is identical to that found in halisulfate 7 (figure 2.7).  The major difference in the NMR data between halisulfate 7 and irregularasulfate is that the signals for the furan ring in halisulfate 7 are not present in the spectra of irregularasulfate, but are replaced with different signals. Subtracting the atoms in I from the molecular formula of irregularasulfate leaves C10H16NO unaccounted for in the molecular formula of irregularasulfate. 26  OS O O O H I 24 1513 23 2210 20 4 1  Figure 2.7. C-1 through C-15 substructure of halisulfate 7 and irregularasulfate (I). HMBC correlations from a downfield resonance at δH 6.62 (H-18) to δC 140.6 (C-17) and δC 172.0 (C-25) suggested the presence of an α,β-unsaturated carbonyl.  The chemical shift value of the carbonyl (δC 172.0) and the fact that nitrogen was the only heteroatom unaccounted for, suggested that the carbonyl must be an amide.  A COSY correlation was observed between δH 6.62 (H-18) and a resonance at δH 3.80 (H-19), which was in turn connected to a carbon at δC 50.9 (C-19).  The δH and δC values for H-19 and C-19 suggested that C-19 was connected to a heteroatom, and the presence of an HMBC correlation from this resonance at δH 3.80 (H-19) to the carbonyl carbon at δC 172.0 (C-25) indicated that it must be connected to the amide nitrogen as a γ-lactam.  HMBC correlations from δH 2.23 (H-16) to δC 140.6 (C-17) and δC 134.5 (C-18), as well as HMBC correlations from δH 6.62 (H-18) to δC 26.6 (C-16), showed that this γ-lactam was connected to C-16.  Thus, the furan ring found in halisulfate 7 has been replaced with a γ- lactam in irregularasulfate. 27  N O 26.6 140.6 134.5 50.9 172.0 40.9 37.6 26.0 22.8 22.8 N O 2.23 6.62 3.80 3.43 1.41 1.54 0.92 0.92 H C N O N O HMBC COSY A) B) 16 19 25 26 29 30 16 19 25 26 29 30  Figure 2.8. Selected 1H and 13C NMR chemical shifts and 2D correlations in irregularasulfate.  Figure 2.9. Expanded HMBC spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. 28  The chemical shift values of a downfield resonance at δH 3.43 (H-26), which was connected to a carbon at δC 40.9 (C-26), suggested that it was also connected to the amide nitrogen.  HMBC correlations from δH 3.43 (H-26) to δC 50.9 (C-19) and δC 172.0 (C-25) confirmed this assignment.  The remaining structure could be determined by COSY correlations from δH 3.43 (H-26) to δH 1.41 (H-27), from δH 1.41 (H-27) to a methine resonance at δH 1.54 (H-28), and from δH 1.54 (H-28) to a set of methyl resonances at δH 0.92 (H-29, H-30).  This assignment was confirmed by analysis of the HMBC data (table 2.2).  Figure 2.10. Expanded COSY spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. 29  Table 2.2. NMR data for lactam substructure of irregularasulfate. position δC δH (J  in Hz) HMBC COSY NOESY 16 26.6 2.23, m 17, 18, 25 17 140.6 18 134.5 6.62, s 16, 17, 19, 25 19 19 19 50.9 3.80, s 17, 18, 25 18 18, 26, 27 25 172.0 26 40.9 3.43, t (7.5) 19, 25, 27, 28 27 19, 29, 30 27 37.6 1.41, m 26, 28, 29, 30 26, 28 19, 29, 30 28 26.0 1.54, m 26, 27, 29, 30 27, 29, 30 29, 30 29 22.8 0.92, d (6.6) 27, 28, 30 28 26, 27, 28 30 22.8 0.92, d (6.6) 27, 28, 29 28 26, 27, 28 Lactam substructure (II) a a Spectra recorded in CDCl3 at 600 MHz  Once all of the atoms in the molecular formula of irregularasulfate were accounted for, there were extra signals in the 1D and 2D NMR spectra that did not show HMBC or COSY correlations to any of the atoms in irregularasulfate.  Since integration of these signals in the 1H NMR spectrum showed that they were present at the same concentration as irregularasulfate, it was unlikely that they were due to impurities and it was assumed that these signals were due to the presence of a positively charged counterion ionically bonded to the negatively charged sulfate group in irregularasulfate. 30   Figure 2.11. 1H NMR signals unaccounted for in irregularasulfate (CDCl3, 600 MHz). A broad downfield signal in the 1H NMR spectrum (figure 2.11) at δH 7.51 (H-1’), which integrated to three protons and was not attached to a carbon atom, showed a COSY correlation to a resonance at δH 2.83 (H-2’), which was in turn connected to a carbon at δC 47.5 (C-2’).  The δH and δC values for H-2’ and C-2’ are consistent with a structure where C-2’ is connected to a positively charged nitrogen atom (N-1’).  The resonance at δH 2.83 (H-2’) showed a COSY correlation to a resonance at δH 2.02 (H-3’), which in turn showed a COSY correlation to a methyl resonance at δH 1.00 (H-4’, H-5’) that integrated to six protons.  Thus, the counterion of irregularasulfate was determined to be the isobutylammonium ion, and this was supported by HMBC correlations (figure 2.12 and table 2.3). 31   Figure 2.12. 1H and 13C NMR chemical shifts and 2D correlations in isobutylammonium.  Figure 2.13. Expanded COSY spectrum of irregularasulfate recorded in CDCl3 at 600 MHz. 32  Table 2.3. NMR data for isobutylammonium counterion of irregularasulfate. position δC δH (J  in Hz) HMBC COSY 1' 7.51 br., s 2' 2' 47.5 2.83, quint. (6.2) 3', 4', 5' 1', 3' 3' 27.0 2.02, m 2', 4', 5' 2', 4', 5' 4' 20.1 1.00, d (6.9) 2', 3', 5' 3' 5' 20.1 1.00, d (6.9) 2', 3', 4' 3' Isobutylammonium a Spectra recorded in CDCl3 at 600 MHz In order to test this hypothesis, one equivalent of sodium hydroxide (NaOH) in MeOH was added to a solution of irregularasulfate in MeOH, and the solution was then dried in vacuo to remove the isobutylamine by-product (scheme 2.2).  Comparison of the 1H NMR spectra before and after treatment with NaOH (figure 2.14) showed that these extra signals disappeared, which supported this hypothesis.  Interestingly, both halisulfate 7 and hipposulfate C were also isolated with a variety of alkylammonium counterions (figure 2.15), as well as sodium, and these were easily separated by HPLC.  These alkylammonium counterions appear to be derived from the amino acids valine, leucine and phenylalanine.  The 1D and 2D NMR spectra for the sodium salt of irregularasulfate are shown in figures 2.22 to 2.28. 33  OS O O O N O H NH3 NaOH OS O O O N O H Na 1.0 equivalents H2O NH2  Scheme 2.2. Removal of isobutylammonium counterion.  Figure 2.14. Comparison of 1H NMR spectra of irregularasulfate with different counterions. 34   Figure 2.15. Alkylammonium counterions of halisulfate 7 and hipposulfate C. 2.2.2. Biological Activity of Sulfated Terpenoids  All three sulfated sesterterpenoids (2.14-2.16) inhibit the related phosphatases calcineurin, PP1 and PP2A.  Halisulfate 7 and hipposulfate C showed similar potency against calcineurin (IC50 = 69 µM and 66 µM, respectively) as they did against PP1 (IC50 = 64 µM and 71 µM, respectively), and lower activity against PP2A (IC50 = 140 µM and 130 µM, respectively) (table 2.4).  The novel sesterterpenoid irregularasulfate showed slightly more potent activity against PP1 (IC50 = 36 µM) than calcineurin (IC50 = 59 µM), while activity against PP2A was not determined (table 2.4). Table 2.4. IC50 values of sulfated sesterterpenoids against phosphatases (µM). Compound Calcineurin PP1 PP2A Halisulfate 7 69 64 140 Hipposulfate C 66 71 130 Irregularasulfate 59 36 N.D. N.D. = Not Determined 2.2.3. Proposed Biogenesis of Irregularasulfate  Irregularasulfate is a sulfated merosesterterpenoid.  The biosynthesis of the terpenoid portion of irregularasulfate is probably similar to the biosynthesis of halisulfate 7, which likely arises from the cyclization of the linear precursor geranyl farnesol (2.18).  One possible biogenesis of halisulfate 7 is shown in scheme 2.3, although the order of reactions is not known. For example, considering the structure of hipposulfate C, it is possible that the hydroxylations, sulfation and formation of the furan ring occur before the second cyclization.  The alkaloid portion of irregularasulfate is most likely derived from the decarboxylation of leucine.  A biosynthetic pathway from halisulfate 7 to the related compounds fasciospongines A and B has 35  been proposed.56  A possible alternative mechanism, which involves formation of the lactam ring directly rather than through a reaction with the furan ring of halisulfate 7, is shown in scheme 2.4. H HH H B HO OH HO OH OH OH OH OH OH OH OH OH O HO O OHHO O H HHH H H H HH O O H S O O O hydroxylation oxidation (+ proton transfer) dehydrationsulfation 2.18 2.14 isomerization hydration  Scheme 2.3. Proposed biogenesis of halisulfate 7. 36   Scheme 2.4. Proposed biogenesis of irregularasulfate. 2.3. Synthesis of Analogues In terms of structure-activity relationships, the first question was whether or not the sulfate group found in 2.14-2.16 was required for phosphatase inhibitory activity.  Therefore, halisulfate 7 was hydrolyzed with aq. HCl in MeOH (scheme 2.5) to give halisulfate 7 alcohol (2.19).  This compound was tested and found to be completely inactive against all three phosphatases.  Likewise, the natural product igernellin (2.17), which is the alcohol analogue of hipposulfate C, was completely inactive against all three phosphatases, proving that the sulfate group is critical for phosphatase inhibitory activity.  Scheme 2.5. Synthesis of 2.19. 37  Next, we wondered whether the sulfate group might be acting as a phosphate mimic in the active site of these phosphatases.  The sulfated terpenoids can be envisioned as a mimic of a phosphorylated serine residue in the natural substrate for these serine/threonine phosphatase enzymes (figure 2.16).  Figure 2.16. Comparison of sulfate and phosphate moieties. If the sulfate group is truly acting as a phosphate mimic, we hoped that the corresponding phosphate analogue might be an even more potent phosphatase inhibitor. Therefore, the phosphate analogue of halisulfate 7 (2.21) was synthesized according to scheme 2.6.  Scheme 2.6. Synthesis of 2.21.  Surprisingly, this analogue was less active against calcineurin (IC50 = 230 µM), but more active against PP1 (IC50 = 36 µM).  One possible reason why 2.21 was less active against 38  calcineurin is that it might act as a substrate for calcineurin, which could potentially hydrolyze it to the corresponding alcohol (2.19).  The fact that 2.21 was more potent against PP1 than the natural products suggests that 2.21 is not a substrate for PP1.  If the phosphate analogue of halisulfate 7 (2.21) is truly acting as a substrate for calcineurin, then analogues that are resistant to hydrolysis might make better inhibitors. Thiophosphates, where one of the non-bridging oxygen atoms is replaced with a sulfur atom, are known to be resistant to hydrolysis by phosphatase enzymes and are good phosphatase inhibitors.57,58  The thiophosphate analogue of hipposulfate C (2.23) was synthesized according to scheme 2.7.  Scheme 2.7. Synthesis of 2.23. This analogue was active against calcineurin with an IC50 = 70 µM, making it roughly as potent as the natural products (2.14-2.16).  The thiophosphate analogue of halisulfate 7 appeared to show similar activity, although an accurate IC50 was not determined due to a lack of material. The difference in activity between the phosphate and thiophosphate analogues suggests that 2.21 may indeed be acting as a substrate for calcineurin. 39  2.4. Conclusions  In conclusion, one new sulfated sesterterpenoid, irregularasulfate, was isolated along with two known sulfated sesterterpenoids, halisulfate 7 and hipposulfate C, as phosphatase inhibitors.  The sulfated sesterterpenoids all showed similar potency against calcineurin.  They were also approximately as potent against calcineurin as they were against PP1, but were less active against PP2A.  It appears that the sulfate group is acting as a phosphate mimic in the active site of the enzyme, and if this is true it would be the first such example.  Synthetic analogues were prepared in order to further develop structure-activity relationships.  The phosphate analogue of halisulfate 7 (2.21) was less active against calcineurin, but more active against PP1.  This compound may be acting as a substrate for calcineurin, but not for PP1.  The thiophosphate analogue of hipposulfate C (2.23) was approximately as potent against calcineurin as the natural products (2.14-2.16). 2.5. Experimental Section 2.5.1. General Experimental Procedures  Silica gel (from SiliCycle® Inc., 230-400 mesh) and SephadexTM LH-20 were used for silica gel chromatography and LH-20 column chromatography, respectively.  Thin-layer chromatography (TLC) was performed using Merck Kieselgel 60 F254 (for normal-phase) and Whatman MKC18F 60 A (for reversed-phase) TLC plates.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µm filter (Osmonics Inc.) prior to use.  The absorbance was monitored at 230 nm and 254 nm with a flow rate of 2.0 mL/min.  NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively. 40  The solvent used for NMR was CDCl3, and chemical shifts are referenced to the internal solvent peaks at δH 7.24 and δC 77.23.  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high-resolution spectra, respectively. Optical rotations were recorded with a JASCO P-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm microcell.  Reagents were purchased from Sigma-Aldrich®, except for bis(2-cyanoethyl)-,-diisopropylphosphoramidite, which was purchased from Toronto Research Chemicals Inc. 2.5.2. Isolation of Sulfated Sesterterpenoids  Specimens of Spongia irregularis (Ledenfeld) (670 g) were collected by hand using SCUBA near Keviang, Papua New Guinea (2˚ 45.1’ S, 150˚ 43’ E).  A voucher sample has been deposited at the University of Amsterdam (ZMAPOR 18522).  The sponge was frozen on site and transported frozen to Vancouver.  Thawed specimens were extracted twice with MeOH and the combined MeOH extracts were concentrated in vacuo to give the crude extract.  The crude extract was partitioned between H2O and CH2Cl2, and the CH2Cl2 soluble materials were subjected to silica gel chromatography (eluent: gradient from hexane to EtOAc to MeOH).  The active fraction, eluting with 10% MeOH/EtOAc, was subjected to reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  The fraction eluting with 75% MeOH/H2O was subjected to reversed-phase HPLC (eluent: 70% MeOH/H2O) to give halisulfate 7 (2.14) (63 mg), hipposulfate C (2.15) (20 mg) and irregularasulfate (2.16) (1.6 mg).  Igernellin (2.17) was isolated from an inactive fraction from the first silica gel column by further isocratic silica gel chromatography (eluent: 10% EtOAc/hexane). 41  Irregularasulfate (2.16): Isolated as a clear glass; [α]D 20 +39.8o (c 0.4, MeOH); 1H and 13C NMR data, see table 2.1; HRESI-MS(-) m/z 536.3407 (calculated for C30H50NO5S: 536.3410). 2.5.3. Synthesis of Analogues Synthesis of 2.19: Halisulfate 7 (2.14) was dissolved in 5 mL MeOH and 1 mL of 1 M aq. HCl was added.  The reaction mixture was heated to reflux for 2.5 hours.  The reaction mixture was cooled and H2O (5 mL) was added before extraction with CH2Cl2 (3 x 5 mL).  The combined CH2Cl2 layers were dried over MgSO4, filtered, concentrated in vacuo, and subjected to silica gel chromatography (eluent: 10% EtOAc/hexane) to give 2.19.  Compound 2.19: 1H NMR (400 MHz, CDCl3) δ 7.32 (1H, br. s, H-19), 7.18 (1H, s, H- 25), 6.24 (1H, br. s, H-18), 5.29 (1H, br. s, H-1), 3.50 (2H, m, H-24), 2.39 (2H, t, J = 5.0 Hz, H- 16), 1.98-2.01 (2H, m, H-2), 1.77 (1H, m, H-6a), 1.45-1.60 (8H, m, H-5, H-7, H-11, H-13, H- 15), 1.20-1.40 (4H, m, H-3b, H-8, H-14), 1.00-1.10 (4H, m, H-3a, H-6b, H-12), 0.96 (3H, s, H- 23), 0.83 (6H, s, H-20, H-21), 0.81 (3H, d, J = 6.9 Hz, H-22); 13C NMR (100 MHz, CDCl3) δ 146.4 (C, C-10), 142.9 (CH, C-19), 139.0 (CH, C-25), 125.3 (C, C-17), 117.1 (CH, C-1), 111.1 (CH, C-18), 66.1 (CH2, C-24), 44.9 (CH, C-8), 43.8 (CH, C-5), 42.8 (C, C-9), 41.5 (CH, C-13), 31.5 (CH2, C-3), 31.5 (C, C-4), 31.5 (CH2, C-7), 31.0 (CH2, C-6), 30.4 (CH2, C-14), 28.3 (CH2, C-11), 28.2 (CH3, C-21), 27.9 (CH3, C-20), 27.6 (CH2, C-15), 25.3 (CH2, C-16), 24.7 (CH2, C- 42  12), 23.4 (CH2, C-2), 23.4 (CH3, C-23), 16.7 (CH3, C-22); HRESI-MS(+) m/z 395.2937 (calculated for C25H40O2Na: 395.2926). Synthesis of 2.21: Compound 2.19 (19.0 mg, 0.051 mmol) was dissolved in MeCN (5 mL) at -10 oC under N2.  To this solution was added CBrCl3 (0.1 mL, 1.0 mmol) and DIPEA (0.1 mL, 0.57 mmol), followed by dibenzyl phosphite (0.3 mL, 1.36 mmol).  The reaction mixture was stirred at -10 oC for 1.5 hours, at which point 0.5 M aq. NaH2PO4 (5 mL, 2.5 mmol) was added.  The reaction mixture was then extracted with CH2Cl2 (3 x 5 mL), and the combined CH2Cl2 layers were dried over MgSO4, filtered, concentrated in vacuo and subjected to silica gel chromatography (eluent: 5% EtOAc/hexane) to give dibenzyl protected haliphosphate 7 (2.20) (14.1 mg, 44%).  To a flask containing 2.20 (2.6 mg, 0.0039 mmol), under N2, was added CH2Cl2 (1 mL), and the solution was cooled to 0  oC. Bromotrimethylsilane (0.05 mL, 0.38 mmol) was added and the reaction mixture was stirred at 0 oC for 45 minutes before the solvent and reagent were removed in vacuo to give crude haliphosphate 7 (2.21).  The crude phosphate (2.21) was dissolved in MeOH and purified by LH-20 column chromatography (eluent: MeOH) to give pure 2.21 (1.4 mg, 75%). O O 2.21 H 1 5 13 17 19 20 22 23 24 25 P O HO OH 10  Compound 2.21: 1H NMR (600 MHz, CDCl3) δ 7.30 (1H, br. s, H-19), 7.18 (1H, s, H- 25), 6.22 (1H, br. s, H-18), 5.29 (1H, br. s, H-1), 3.91 (2H, m, H-24), 2.36 (2H, m, H-16), 1.96- 2.02 (2H, m, H-2), 1.76 (1H, m, H-6a), 1.45-1.55 (8H, m, H-5, H-7, H-11, H-13, H-15), 1.20- 1.40 (4H, m, H-3b, H-8, H-14), 1.00-1.10 (4H, m, H-3a, H-6b, H-12), 0.94 (3H, s, H-23), 0.81 43  (6H, s, H-20, H-21), 0.80 (3H, d, J = 6.9 Hz, H-22); 13C NMR (150 MHz, CDCl3) δ 146.2 (C, C- 10), 142.9 (CH, C-19), 139.0 (CH, C-25), 125.2 (C, C-17), 117.1 (CH, C-1), 111.2 (CH, C-18), 70.9 (CH2, C-24), 44.9 (CH, C-8), 43.8 (CH, C-5), 42.8 (C, C-9), 39.5 (CH, C-13), 31.5 (CH2, C- 3), 31.5 (C, C-4), 31.5 (CH2, C-7), 30.8 (CH2, C-6), 30.4 (CH2, C-14), 28.2 (CH2, C-11), 28.1 (CH3, C-21), 27.8 (CH3, C-20), 27.2 (CH2, C-15), 25.1 (CH2, C-16), 24.3 (CH2, C-12), 23.4 (CH2, C-2), 23.4 (CH3, C-23), 16.7 (CH3, C-22); HRESI-MS(-) m/z 451.2615 (calculated for C25H40O5P: 451.2613). Synthesis of 2.23: Igernellin (2.17) (12.0 mg, 0.032 mmol) was dissolved in CH2Cl2 (3 mL) and to this solution was added bis(2-cyanoethyl)-,-diisopropylphosphoramidite (0.1 mL) and a solution of tetrazole in MeCN (0.7 mL of a 1 M solution, 0.7 mmol).  The reaction mixture was stirred overnight at room temperature.  Sulfur (75 mg, 0.29 mmol) was added and the reaction was allowed to stir for a further 4 hours before being concentrated in vacuo and subjected to silica gel chromatography (eluent: gradient from hexane to 25% EtOAc/hexane) to give 2.22 (9.5 mg, 57%).  The protected thiophosphate (2.22) (4.9 mg, 0.0085 mmol) was dissolved in 2 mL of 1 M KOH in MeOH, and the reaction mixture was stirred for 2 hours at room temperature.  The solution was concentrated in vacuo and purified by reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  The fraction eluting with 90% MeOH/H2O gave pure 2.23 (4.6 mg, quant.). 44   Compound 2.23: 1H NMR (400 MHz, CDCl3) δ 7.30 (1H, br. s, H-19), 7.26 (1H, s, H- 25), 6.21 (1H, br. s, H-18), 5.23 (1H, br. s, H-1), 5.03 (1H, br. s, H-10), 3.74 (1H, m, H-24a), 3.65 (1H, m, H-24b), 2.34 (2H, br. s, H-16), 1.85-1.95 (6H, m, H-2, H-8, H-11), 1.62 (3H, s, H- 22), 1.55 (1H, m, H-13), 1.52 (3H, s, H-23), 1.20-1.50 (10H, m, H-3a, H-5, H-7, H-12, H-14, H- 15), 1.07 (1H, br. d, J = 12.9 Hz, H-3b), 0.87 (3H, s, H-20), 0.82 (3H, s, H-21); 13C NMR (100 MHz, CDCl3) δ 142.9 (CH, C-19), 139.3 (CH, C-25), 136.9 (C, C-6), 136.1 (C, C-9), 125.4 (C, C-17), 124.6 (CH, C-10), 120.1 (CH, C-1), 111.5 (CH, C-18), 67.9 (CH2, C-24), 49.3 (CH, C-5), 40.9 (CH2, C-8), 38.6 (CH, C-13), 32.8 (C, C-4), 31.9 (CH2, C-3), 31.0 (CH2, C-7), 30.4 (CH2, C-12), 30.1 (CH2, C-14), 27.7 (CH3, C-20), 27.7 (CH3, C-21), 26.7 (CH2, C-15), 25.4 (CH2, C- 11), 25.1 (CH2, C-16), 23.7 (CH3, C-22), 23.3 (CH2, C-2), 16.3 (CH3, C-23); HRESI-MS(-) m/z 467.2387 (calculated for C25H40O4PS: 467.2385). 2.5.4. Phosphatase Inhibition Assay  Phosphatase inhibition assays were performed by the Holmes lab at the University of Alberta as previously described.51  The assay contained 50 mM TrisHCl, pH 7.4, 0.5 mM dithiothreitol, 0.2 mM CaCl2, 0.5 mM MnCl2, 0.2 mg/mL bovine serum albumin, 0.3 µM calmodulin, and 20 mM p-nitrophenyl phosphate.  The enzyme was assayed for 20-60 minutes at 30 oC, stopped by the addition of 100 µL of 2 M Na2CO3 and 100 mM EDTA, and absorbance was measured at 405 nm. 45  2.5.5. *MR Spectra of Irregularasulfate  Figure 2.17. COSY spectrum of irregularasulfate with isobutylammonium ion.  Figure 2.18. HSQC spectrum of irregularasulfate with isobutylammonium ion. 46   Figure 2.19. Expanded HSQC spectrum of irregularasulfate with isobutylammonium ion.  Figure 2.20. HMBC spectrum of irregularasulfate with isobutylammonium ion. 47   Figure 2.21. Expanded HMBC spectrum of irregularasulfate with isobutylammonium ion.  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Prod. Lett. 1995, 7, 297-301. (56) Yao, G.; Chang, L. C. Org. Lett. 2007, 9, 3037-3040. (57) Eckstein, F.; Sternbach, H. Biochim. Biophys. Acta 1967, 146, 618-619. (58) Zhao, Z. Biochem. Biophys. Res. Commun. 1996, 218, 480-484. 55  3. Isolation of Bafilomycins as Inhibitors of Autophagy 3.1. Autophagy The word autophagy is derived from the Greek words meaning “self eating”.  Autophagy is one process by which proteins and organelles are degraded and recycled.  The process of autophagy begins with the formation of a crescent-shaped double membrane structure called a phagophore.1  The phagophore grows to engulf its target and closes around it, at which point it is known as an autophagosome (figure 3.1).  The autophagosome is then acidified by a proton pump known as vacuolar-type H+-ATPase (V-ATPase).  The acidified autophagosome then fuses with a lysosome, at which point it is known as an autolysosome.  Alternatively, the autophagosome may fuse with an endosome, formed by the sequestration of extracellular material, in which case it is known as an amphisome.2,3  The amphisome then undergoes acidification and fusion with a lysosome to form an autolysosome.  Catabolic enzymes within the lysosome, such as proteases, degrade the contents of the autolysosome to monomeric units. These units are then exported to the cytosol through permease and recycled.4  Figure 3.1. Mechanism of autophagy.  2 A version of this chapter has been published. Carr, G.; Williams, D. E.; Díaz-Marrero, A. R.; Patrick, B. O.; Bottriell, H.; Balgi, A. D.; Donohue, E.; Roberge, M.; Andersen, R. J. (2010) Bafilomycins Produced in Culture by Streptomyces spp. Isolated from Marine Habitats Are Potent Inhibitors of Autophagy. J. Nat. Prod. 73:422-427. 56  Autophagy normally occurs at a basal rate and contributes to the turnover of older proteins and organelles.5-8  Autophagy may also be induced in response to various types of stress. Under low nutrient conditions autophagy is induced, presumably as a means to salvage much- needed nutrients from older cell components in order to synthesize new and essential proteins and organelles.9,10  Autophagy may be induced by low levels of amino acids,11-13 or by glucagon (a sign of low blood glucose levels).12,14  Conversely, high levels of free amino acids,11,13 ATP15 and insulin16 (a sign of high blood glucose levels) inhibit autophagy. 3.1.1. Autophagy and Cancer Although autophagy was discovered in the 1960’s, most of our understanding of autophagy has come within the last decade, and there is still a lot that is not understood.1  For example, it is not clear whether autophagy is beneficial or detrimental to cancer cells, as it appears that it can be either depending on the circumstances.  One clue that autophagy may help to protect against cancer is that tumour suppressors such as phosphate and tensin homolog (PTEN) and p53 are positive regulators of autophagy,17,18 while oncogenic proteins such as Bcl-2 inhibit autophagy.19  Beclin 1 is a protein required for autophagy that functions as a tumour suppressor,20 and deletions in Beclin 1 are found in a large number of breast, prostate and ovarian cancers.21  Heterozygous deletion of Beclin 1 in mice results in reduced autophagy and increased cell proliferation and tumourigenesis,22,23 while mice with a homozygous deletion of Beclin 1 did not survive past embryogenesis.22  Another important clue that autophagy can help to protect against cancer is that the level of autophagy is often lower in tumour cells.24,25 One mechanism by which autophagy may protect against cancer is by removing damaged proteins and organelles.  For example, damaged mitochondria can produce reactive oxygen species that could react with DNA, causing mutations and cancer.  Autophagy may help to 57  remove these damaged mitochondria.8  In fact, autophagy has been shown to limit genome damage and function as a tumour suppressor, likely through this mechanism.26,27  Autophagy may also help to slow tumour progression by slowing the growth of tumour cells.  Tumour cells must reach a critical size before they can divide and, by breaking down cell components, autophagy limits growth.28  Autophagy may even cause cell death in tumours that lack the ability to undergo apoptosis.29,30 While it appears that autophagy is a protective mechanism against genome damage and cancer, there are certain circumstances where autophagy may be beneficial to cancer cells. Tumours must often survive in poorly vascularized and low nutrient environments.  In these environments, tumour cells may use autophagy as a means of providing energy.31,32  When autophagy is inhibited under these low nutrient conditions, apoptosis is induced.32,33  Therefore, inhibiting autophagy may be useful in treating solid tumours that are poorly vascularized. However, if apoptosis is defective in these tumours, cell death may occur by necrosis resulting in inflammation and increased tumour growth.34,35  In the case where apoptosis is defective in tumour cells, stimulating autophagy to induce autophagic cell death may be a better alternative. Autophagy may also serve as a protection mechanism for tumour cells against chemotherapeutics.  Autophagy is induced by a variety of anticancer treatments, and may help to remove organelles damaged by these treatments.36   Indeed, autophagy inhibitors have been found to increase the effectiveness of various anticancer treatments.37-44  Similar results were obtained by blocking autophagy using siRNA.41,44-48  Thus, autophagy inhibitors may find usefulness in combination with other chemotherapeutics in the treatment of cancer.  On the other hand, some autophagy inhibitors have been found to decrease the effectiveness of anticancer treatments.37,49  Similarly, stimulating autophagy by inhibiting the mammalian target of 58  rapamycin (mTOR), a protein that inhibits autophagy, can increase the effectiveness of anticancer treatments.50-53  While inhibitors of mTOR are known to induce autophagy, a clear link between augmenting chemotherapy with mTOR inhibitors and autophagy has not been established.  Intriguingly, combining anticancer treatments with different autophagy inhibitors that work by different mechanisms can have drastically different results even within the same cell type and using the same therapy.37,39 Whether autophagy should be induced or inhibited in the treatment of cancer seems to depend on the nature of the cancer involved, as well as the type of treatment.  Whether or not autophagy modulators will find usefulness as chemotherapeutics will likely depend on further understanding the role of autophagy in cancer.  At the moment, it appears that inducing autophagy may help to prevent mutagenesis and cancer, and therefore could be used to prevent cancer in individuals with a high risk of developing cancer.  However, once a tumour is established, combining autophagy inhibitors with chemotherapy may enhance the effectiveness of chemotherapy. 3.2. Bafilomycins The bafilomycins are a group of macrolides with antibiotic and antifungal activity originally isolated from Streptomyces griseus.54,55  Since then, over a dozen naturally occurring bafilomycins have been reported.56-60  The bafilomycins belong to a larger family of macrolides known as plecomacrolides, which share a similar 16- or 18-membered lactone ring.61  This family also includes the concanamycins, hygrolidin and formamicin, among others.61  Members of this family exhibit a wide variety of biological activities including antibiotic,54 antifungal,54 insecticidal,62 antiparasitic,63,64 cytotoxic65 and anthelmintic66 activities. 59  In addition to antibiotic and antifungal properties, bafilomycin A1 (3.1) is known to be a potent and specific inhibitor of vacuolar-type H+-ATPase (V-ATPase), whose function is to pump H+ ions across a cell membrane.67  Bafilomycin A1 is selective for V-ATPase over similar P-type and F-type ATPases by nearly 5 orders of magnitude.67  V-ATPase plays a crucial role in bone resorption by osteoclasts, which requires an acidic environment.  Excessive bone resorption can lead to osteoporosis, so inhibitors of V-ATPase have been investigated for the treatment of osteoporosis.68  V-ATPase also plays a role in maintaining an acidic environment in tumours, and is often overexpressed in cancers.69  This acidic environment aids in the breakdown of the extracellular matrix and contributes to metastasis.  Thus, inhibitors of V-ATPase have also attracted attention in the treatment of cancer. By inhibiting V-ATPase and the acidification of autophagosomes, bafilomycin A1 blocks the fusion of autophagosomes with lysosomes and therefore inhibits autophagy.70  Inhibition of autophagy by bafilomycin A1 has been found to have antiproliferative effects against colon cancer cells by causing cell cycle arrest and apoptosis.71  Bafilomycin A1 has also been shown to enhance the effectiveness of various anticancer treatments.37-39  Further, bafilomycin A1 has found usefulness as a biological tool to better understand the process of autophagy.71 Due to its use as a biological tool, as well as the potential to serve as a drug lead for the treatment of cancer, bafilomycin A1 has attracted the attention of the synthetic community. Several total syntheses have been reported in the literature,72-78 along with many more partial syntheses.  Additionally, semi-synthetic analogues of bafilomycin A1 have been prepared in order to develop structure-activity relationships.79-83 60  3.3. Isolation of Bafilomycins The producing organism was obtained from marine sediment off the coast of British Columbia and identified by 16S rRNA gene sequencing as Streptomyces sp.  Cultures were grown as lawns on solid agar for 14 days at room temperature.  The solid agar was extracted with EtOAc and the EtOAc was removed in vacuo to give the crude extract, which showed good autophagy inhibitory activity.  The crude extract was fractionated according to scheme 3.1 Crude extract Streptomyces sp. extracted with EtOAc 25% EtOAc/ hexane Silica gel flash chromatography 50% EtOAc/ hexane 75% EtOAc/ hexane 100% EtOAc 100% MeOH100% Acetone Bafilomycin B1 Normal-phase HPLC (50% EtOAc/Hexane) Reversed-phase flash chromatography Reversed-phase flash chromatography 70% MeCN/H2O70% MeCN/H2O Bafilomycin A1 Bafilomycin D Reversed-phase flash chromatography 75% MeOH/ H2O Reversed-phase HPLC (75% MeOH/H2O) Bafilomycin F  Scheme 3.1. Isolation of bafilomycins. The crude extract was fractionated by silica gel flash chromatography (eluent: gradient from 25% EtOAc/hexane to MeOH).  The fraction eluting with 100% MeOH appeared to contain a compound related to the bafilomycins based on the 1H NMR spectrum.  This fraction was further purified by reversed-phase flash chromatography (eluent: gradient from H2O to MeOH). The fraction eluting with 75% MeOH/H2O was purified by reversed-phase HPLC (eluent: 75% MeOH/H2O) to give the new macrolide bafilomycin F (3.2) (2.0 mg) as a white solid.  The fractions eluting from the silica gel flash chromatography step with 50% EtOAc/hexane and 75% EtOAc/hexane also appeared to contain more bafilomycin analogues.  These fractions were 61  pooled and purified by normal-phase HPLC (eluent: 50% EtOAc/hexane), and reversed-phase flash chromatography (eluent: 70% MeCN/H2O) to give the known compounds bafilomycin A1 (3.1) (1.2 mg), bafilomycin B1 (3.3) (3.8 mg) and bafilomycin D (3.4) (1.0 mg) (figure 3.2). O O HO OH O OH O O H OH O O HO OH O OH O O H O O O HO OH O OH O O H O O NH S O O O O O HO OHO O H O OH O H N O O O 3.1 3.2 3.3 3.4 1 33 32 27 31 1' 29 28 21 17 15 7 4 26 30 25 23 1910 3' 7' 5' 9' 8'  Figure 3.2. Structures of 3.1-3.4. 3.4. Structure Elucidation of Bafilomycin F  Bafilomycin F gave an [M - H]- peak at m/z 822.4096 in the HRESI-MS(-) spectrum, consistent with a molecular formula of C42H65NO13S (calculated for C42H64NO13S: 822.4098). 62  Analysis of the 1D (figures 3.3 to 3.6 and table 3.1) and 2D NMR data (figures 3.13 to 3.19 and table 3.1) obtained for bafilomycin F identified the same 16-membered macrolide found in other bafilomycin analogues.  This macrolide was linked to a hemiketal through a three carbon linker, as found in most of the bafilomycins.  Comparison of the 1H and 13C NMR chemical shift values for C-1 through C-33 of bafilomycin F with literature values reported for bafilomycin C1 (3.5) showed that they were in excellent agreement (figures 3.5 to 3.6),59 and confirmed that bafilomycin F contained the identical C-1 through C-33 fragment found in bafilomycin C1.  Figure 3.3. 1H NMR spectrum of bafilomycin F recorded in CD3OD at 600 MHz. 63   Figure 3.4. 13C NMR spectrum of bafilomycin F recorded in CD3OD at 150 MHz. O O HO OH O OH O O H O 3.2 O O HO OH O OH O O H O O 3.5 OH O 3.61 6.70 1.97 5.88 2.52 1.05 3.22 1.84 0.92 2.01 1.87 5.76 6.59 5.10 3.98 3.24 5.02 2.07 0.86 4.15 1.80 0.98 2.29 1.31 4.99 1.63 0.83 3.58 1.92 0.96 0.82 3.61 6.69 1.97 5.88 2.52 1.05 3.24 1.84 0.92 2.02 1.89 5.76 6.59 5.10 3.98 3.24 5.03 2.07 0.85 4.14 1.79 2.29 1.30 4.95 1.61 0.86 3.56 1.91 0.95 0.80 0.98  Figure 3.5. Selected 1H NMR chemical shifts of bafilomycin C1 and bafilomycin F (CD3OD). 64   Figure 3.6. Selected 13C NMR chemical shifts of bafilomycin C1 and bafilomycin F (CD3OD). 65  Table 3.1. NMR data for bafilomycin F. position δC δH (J  in Hz) HMBC COSY ROESY 1 168.1 2 142.3 2-OMe 60.6 3.61, s 2 26, 30 3 134.9 6.69, s 1, 2, 5, 26 5, 13 4 133.3 26 14.1 1.97, s 3, 4, 5 2-OMe, 27 5 145.6 5.88, d (8.9) 3, 6, 7, 26 6 3, 6, 8, 27 6 38.5 2.52, m 4, 5, 27 5, 27 5, 8, 27, 28 27 17.9 1.05, d (6.9) 5, 6, 7 6 5, 6, 7, 26 7 81.2 3.24, m 5, 27, 28 8 27, 28 8 41.7 1.84, m 6, 7, 9, 10 7, 9, 28 5, 6, 9, 11 28 22.5 0.92, d (6.9) 7, 8, 9 8 6, 7, 9 9 42.5 2.02, m 7, 8, 10, 11, 29 8 8, 11, 28 10 145.0 29 20.1 1.89, s 9, 10, 11 12 11 125.6 5.76, d (10.8) 9, 12, 13, 29 12 8, 9, 12, 13 12 134.7 6.59, dd (14.9, 10.8) 10, 11, 13, 14 11, 13 11, 13, 14, 29 13 127.0 5.10, dd (14.9, 8.9) 11, 14, 15 12, 14 3, 11, 12, 14 14 84.4 3.98, t (8.4) 12, 14-OMe, 15, 16 13, 15 12, 13, 14-OMe, 15, 16, 30 14-OMe 55.9 3.24, s 14 14 15 77.7 5.03, d (7.7) 1, 13, 14, 16, 17, 30 14 14, 16 16 39.5 2.07, m 17, 30 17, 30 14, 15, 17, 30 30 10.5 0.85, d (4.4) 15, 16, 17 16 2-OMe, 14, 16, 17 17 72.0 4.14, dd (10.5, 1.4) 15, 16, 18, 19, 30, 31 16, 18 16, 18, 30, 31 18 43.6 1.79, m 19, 31 17, 31 17, 20b, 31 31 7.2 0.98, d (7.2) 17, 18, 19 18 17, 18, 20b 19 100.4 20a 40.6 2.29, dd (12.0, 4.8) 19, 21, 22 20b, 21 20b, 21 20b 1.30, m 18, 19, 21, 22 20a, 21 18, 20a, 22, 31 21 76.4 4.95, dt (10.9, 4.8) 1', 32 20a, 20b, 22 20a, 23, 32 22 39.4 1.61, m 21, 23, 24, 32 21, 23, 32 20b, 23, 32 32 12.6 0.86, d (4.1) 21, 22, 23 22 21, 22, 23 23 77.3 3.56, dd (10.4, 1.5) 21, 22, 24, 25, 33 22, 24 21, 22, 24, 32 24 29.2 1.91, m 25, 33 23, 25, 33 23, 25, 33 33 21.8 0.95, d (7.2) 23, 24, 25 24 24 25 14.6 0.80, d (6.9) 23, 24, 33 24 24 1' 171.5 2'a 42.6 3.20, m 1', 3', 4' 2'b, 3' 3' 2'b 3.01, dd (14.8, 7.6) 3', 4' 2'a, 3' 3' 3' 39.6 3.67, dd (7.2, 2.5) 1', 2', 4', 7' 2'a, 2'b 2'a, 2'b 4' 175.1 6' 60.6 4.25, m 7' 7' 33.7 3.12, m 3', 6' 6' 9' 178.1 a Spectra collected in CD3OD at 600 MHz Bafilomycin F (3.2) a  66  Subtracting the atoms in the C-1 through C-33 substructure from the molecular formula of bafilomycin F left C7H8NO4S unaccounted for in the molecular formula of bafilomycin F.  A downfield resonance at δH 4.95 (H-21) that showed an HMBC correlation to a carbonyl carbon (δC 171.5, C-1’) indicated that bafilomycin F was esterified at C-21, much like in bafilomycin C1.  However, bafilomycin C1 is linked to a fumarate moiety at this position.  Bafilomycin F lacked the olefinic resonances found in bafilomycin C1 at δH 6.48 (H-2’) and δH 6.91 (H-3’). 59 Instead, these resonances were replaced by methylene resonances at δH 3.20 (H-2’a) and δH 3.01 (H-2’b) and a methine resonance at δH 3.67 (H-3’), respectively (figure 3.7).  The link between C-2’ and C-3’ was established by COSY correlations between δH 3.20 (H-2’a) and δH 3.67 (H- 3’), and between δH 3.01 (H-2’b) and δH 3.67 (H-3’).  This was supported by HMBC correlations from δH 3.20 (H-2’a) to δC 39.6 (C-3’), from δH 3.01 (H-2’b) to δC 39.6 (C-3’), and from δH 3.67 (H-3’) to δC 42.6 (C-2’).  HMBC correlations were observed from δH 3.20 (H-2’a), δH 3.01 (H- 2’b) and δH 3.67 (H-3’) to a carbonyl carbon at δC 175.1 (C-4’), and from δH 3.20 (H-2’a) and δH 3.67 (H-3’) to a carbonyl carbon at δC 171.5 (C-1’), suggesting that both C-2’ and C-3’ were attached to carbonyl carbons.  A series of HMBC correlations from δH 3.67 (H-3’) to δC 33.7 (C- 7’) and from δH 3.12 (H-7’) to δC 39.6 (C-3’) suggested that C-3’ and C-7’ were connected to each other through a heteroatom.  The chemical shift values of these resonances suggested that the heteroatom was a sulfur atom, which accounts for the sulfur atom in the molecular formula of bafilomycin F.  COSY correlations were observed from δH 3.12 (H-7’) to a methine resonance at δH 4.25 (H-6’), which in turn was connected to a carbon at δC 60.6 (C-6’).  The δH and δC values of H-6’ and C-6’ suggested that C-6’ was connected to a nitrogen atom, which accounts for the nitrogen atom in the molecular formula of bafilomycin F.  A COSY spectrum of bafilomycin F recorded in DMSO-d6 showed a COSY correlation between H-6’ (δH 3.76) and an amide proton 67  at δH 7.01 (H-5’), indicating that N-5’ was connected to a carbonyl carbon (C-4’).  A final carbon resonance at δC 178.1 (C-9’) suggested the presence of a carboxylic acid connected to C-6’, completing the structure of bafilomycin F.  Figure 3.7. Selected 1H and 13C NMR chemical shifts and 2D correlations in bafilomycin F. 68   Figure 3.8. Expanded HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz.  Figure 3.9. Expanded COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. The presence of a carboxylic acid was consistent with the polarity of bafilomycin F on silica, as well as the ESI-MS data.  The presence of a strong [M - H]- ion in the ESI-MS(-) 69  spectrum, and a strong [M - H + 2Na]+ ion in the ESI-MS(+) spectrum suggested that an acidic proton was present in bafilomycin F.  This proposed substructure is also supported by biosynthetic considerations, and is consistent with a molecule of cysteine adding in a Michael fashion to the α,β-unsaturated ester of bafilomycin C1, followed by amide formation from the nitrogen of cysteine to the carboxylic acid of bafilomycin C1. 3.4.1. Relative Configuration of Bafilomycin F The relative configuration of the C-1 through C-33 substructure of bafilomycin F was determined by comparing the 1H and 13C NMR chemical shift values of bafilomycin F with those of bafilomycin C1.  These chemical shift values were nearly identical (figures 3.5 to 3.6), suggesting that bafilomycin F and bafilomycin C1 have the same relative configuration at these stereocenters.59  The relative configuration of the thiomorpholinone ring in bafilomycin F could not be determined from the ROESY spectrum due to difficulties arising from the presence of heteroatoms in the ring.  A search of the literature revealed that this type of substituted ring has never before been observed in a natural product.  Compounds containing this type of ring have been synthesized,84 although no stereochemical details were given.  Therefore, in order to determine the relative configuration of the thiomorpholinone ring in bafilomycin F, model compounds were synthesized according to a literature procedure as shown in scheme 3.2,84 and the 1H and 13C NMR chemical shift values were compared to the corresponding 1H and 13C NMR chemical shift values of bafilomycin F.  A conjugate addition of L-cysteine methyl ester (3.6) onto -phenylmaleimide (3.7) gave 3.8 in excellent yield as a mixture of diastereomers (scheme 3.2).  Cyclization of 3.8 gave a mixture of diastereomers 3.9 and 3.10, which were separated by silica gel chromatography. 70   Scheme 3.2. Synthesis of model compounds 3.9 and 3.10. Compound 3.9 was crystallized from MeOH and analyzed by single-crystal X-ray diffraction, which demonstrated that 3.9 is the cis isomer.  This was supported by a NOESY correlation between δH 4.09 (H-3) and δH 4.47 (H-6).  When the chemical shift values of bafilomycin F were compared to the chemical shift values of the model compounds 3.9 and 3.10, it appeared that they matched more closely with the trans isomer 3.10 (tables 3.2 and 3.3 and figure 3.10), especially at H-3’. Table 3.2. Selected 1H and 13C NMR chemical shift values of bafilomycin F. position δH (J  in Hz) δC δH (J  in Hz) δC 1' 171.5 169.9 b 2'a 3.20, m 42.6 3.04, m 41.9 2'b 3.01, dd (14.8, 7.6) 2.78, m 3' 3.67, dd (7.2, 2.5) 39.6 3.72, dd (6.6, 3.2) 37.6 4' 175.1 170.0 b N-5' 7.01 br, s 6' 4.25, m 60.6 3.76, dd (9.7, 4.2) 59.2 7a' 3.12, m 33.7 3.11, m 32.8 7b' 3.12, m 2.81, m 9' 178.1 170.4 CD3OD DMSO-d 6 a Signals are interchangeable a Spectra recorded in CD3OD or DMSO-d 6  at 600 MHz Bafilomycin F (3.2) a,b  71  Table 3.3. Selected 1H and 13C NMR chemical shift values of 3.9 and 3.10. position δH (J  in Hz) δC δH (J  in Hz) δC position δH (J  in Hz) δC δH (J  in Hz) δC 1 170.8 168.3 1 170.8 168.3 2a 3.28, m 39.5 3.06, dd (15.2, 5.3) 38.2 2a 3.04, m 40.9 2.95, dd (15.5, 5.0) 39.4 2b 2.61, dd (15.4, 8.2) 2.45, dd (15.2, 8.6) 2b 2.85, dd (15.3, 8.6) 2.73, dd (15.5, 8.9) 3 4.09, dd (8.2, 5.7) 39.4 3.99, dd (8.6, 5.3) 38.0 3 3.87, dd (8.6, 4.8) 39.0 3.70, m 37.1 4 171.4 167.9 4 171.4 168.0 N-5 7.98, d (3.6) N-5 8.16, d (4.7) 6 4.47, dd (6.3, 4.2) 57.7 4.45, q (4.2) 56.0 6 4.50, m 58.1 4.50, q (4.4) 55.9 7a 3.28, m 28.8 3.29, dd (13.5, 4.2) 27.3 7a 3.26, m 27.0 3.20, dd (13.4, 4.6) 25.9 7b 3.07, m 2.99, dd (13.7, 5.4) 7b 3.07, m 3.03, dd (13.5, 4.4) 9 171.8 170.7 9 171.8 171.0 a Spectra recorded in CD3OD or DMSO-d 6  at 400 MHz 3.9 a 3.10 a CD3OD DMSO-d 6 CD3OD DMSO-d 6  H-2'a H-2'b H-3' H-6' H-7'a H-7'b Cis 0.08 -0.4 0.42 0.22 0.16 -0.05 Trans -0.16 -0.16 0.2 0.25 0.14 -0.05 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 ∆δH for cis (3.9) and trans (3.10) vs. bafilomycin F in CD3OD (ppm) H-2'a H-2'b H-3' H-6' H-7'a H-7'b Cis 0.02 -0.33 0.27 0.69 0.18 0.18 Trans -0.09 -0.05 -0.02 0.74 0.09 0.22 -0.4 -0.2 0 0.2 0.4 0.6 0.8 ∆δH for cis (3.9) and trans (3.10) vs. bafilomycin F in DMSO-d6 (ppm) C-2' C-3' C-6' C-7' Cis -3.1 -0.2 -2.9 -4.9 Trans -1.7 -0.6 -2.5 -6.7 -8 -6 -4 -2 0 ∆δC for cis (3.9) and trans (3.10) vs. bafilomycin F in CD3OD (ppm) C-2' C-3' C-6' C-7' Cis -3.7 0.4 -3.2 -5.5 Trans -2.5 -0.5 -3.3 -6.9 -8 -6 -4 -2 0 2 ∆δC for cis (3.9) and trans (3.10) vs. bafilomycin F in DMSO-d6 (ppm)  Figure 3.10. Comparison of NMR chemical shifts of bafilomycin F to 3.9 and 3.10. Further analogues were synthesized according to scheme 3.3 in order to provide additional evidence.  Once again, the chemical shifts of bafilomycin F appeared to match more closely with the trans isomer 3.15 (tables 3.2 and 3.4 and figure 3.11).  Therefore, the relative configuration of the thiomorpholinone ring in bafilomycin F appears to be trans.  This 72  assignment is supported by NOESY correlations between H-3 and H-6 in the cis isomers, 3.9 and 3.14, and the lack of a NOESY correlation between H-3’ and H-6’ in bafilomycin F. HO O OH O O O O O MeOH H2SO4 O O O O S NH2 O O S NH O O O O O S NH O O O O O HS NH3 O O Cl DMF 3.11 3.12 3.6 3.13 3.14 3.15 91% 15% over two steps reflux, 1 h r.t., 15 min 140 oC, 6 h 11 10 98 531 1 3 5 8 9 10 11  Scheme 3.3. Synthesis of model compounds 3.14 and 3.15. Table 3.4. Selected 1H and 13C NMR chemical shift values of 3.14 and 3.15. position δH (J  in Hz) δC δH (J  in Hz) δC position δH (J  in Hz) δC δH (J  in Hz) δC 1 170.8 170.9 1 170.6 171.0 2a 2.95, m 36.8 2.89, m 35.7 2a 2.97, m 38.4 2.85, m 37.2 2b 2.60, dd (16.5, 7.3) 2.47, m 2b 2.85, dd (16.6, 7.8) 2.75, dd (16.4, 7.7) 3 4.00, t (6.8) 38.9 3.92, t (6.7) 37.6 3 3.79 (m) 38.5 3.63, m 36.7 4 172.8 167.5 4 172.8 167.5 N-5 7.93, d (2.1) N-5 8.15, d (4.1) 6 4.49, dd (6.5, 4.1) 57.5 4.44, q (4.1) 55.9 6 4.53, t (4.8) 58.0 4.47, q (4.4) 56.1 7a 3.31, m 28.8 3.29, dd (13.6, 3.8) 27.6 7a 3.28, m 27.1 3.21, m 26.1 7b 3.08, m 3.00, m 7b 3.08, m 3.02, m 9 171.6 170.6 9 172.0 170.9 a Spectra recorded in CD3OD or DMSO-d 6  at 400 MHz 3.14 a 3.15 a CD3OD DMSO-d 6 CD3OD DMSO-d 6  73  H-2'a H-2'b H-3' H-6' H-7'a H-7'b Cis -0.25 -0.41 0.33 0.24 0.19 -0.04 Trans -0.23 -0.16 0.12 0.28 0.16 -0.04 -0.6 -0.4 -0.2 0 0.2 0.4 ∆δH for cis (3.14) and trans (3.15) vs. bafilomycin F in CD3OD (ppm) H-2'a H-2'b H-3' H-6' H-7'a H-7'b Cis -0.15 -0.31 0.2 0.68 0.18 0.19 Trans -0.19 -0.03 -0.09 0.71 0.1 0.21 -0.4 -0.2 0 0.2 0.4 0.6 0.8 ∆δH for cis (3.14) and trans (3.15) vs. bafilomycin F in DMSO-d6 (ppm) C-2' C-3' C-6' C-7' Cis -5.8 -0.7 -3.1 -4.9 Trans -4.2 -1.1 -2.6 -6.6 -8 -6 -4 -2 0 ∆δC for cis (3.14) and trans (3.15) vs. bafilomycin F in CD3OD (ppm) C-2' C-3' C-6' C-7' Cis -6.2 0.0 -3.3 -5.2 Trans -4.7 -0.9 -3.1 -6.7 -8 -6 -4 -2 0 ∆δC for cis (3.14) and trans (3.15) vs. bafilomycin F in DMSO-d6 (ppm)  Figure 3.11. Comparison of NMR chemical shifts of bafilomycin F to 3.14 and 3.15. 3.4.2. Absolute Configuration of Bafilomycin F  The absolute configuration of the thiomorpholinone ring in bafilomycin F was determined by chemical degradation to alanine, followed by Marfey’s analysis.  Bafilomycin F was reduced with Raney nickel to give 3.16 (scheme 3.4), which was hydrolyzed with 6 M aq. HCl to give 3.17.  Compound 3.17 was purified by reversed-phase flash chromatography (eluent: H2O) and then analyzed by Marfey’s method (scheme 3.4). 85  The Marfey’s derivative 3.18 was compared to standards prepared from D- or L-alanine by reversed-phase TLC and reversed-phase HPLC.  Reversed-phase TLC analysis (developing solvent: 20% MeCN/H2O + 1% TFA) gave an Rf = 0.48 for 3.18, which was identical to the Rf value for the Marfey’s derivative prepared from L-alanine (Rf = 0.48), while the Marfey’s derivative prepared from D-alanine gave an Rf = 0.32.  The Marfey’s derivatives were also compared by reversed-phase HPLC (eluent: 10% MeCN/H2O).  When the Marfey’s derivative 3.18 was co-injected with the Marfey’s derivative 74  prepared from L-alanine, the two compounds eluted as a single peak.  When the Marfey’s derivative 3.18 was co-injected with the Marfey’s derivative prepared from D-alanine, the two compounds eluted as two separate peaks.  Both the TLC and HPLC analyses showed that 3.18 was identical to the Marfey’s derivative prepared from L-alanine.  Therefore, the configuration at C-6’ in bafilomycin F is R and, based on the relative configuration of the thiomorpholinone ring, the absolute configuration at C-3’ must be S. O HO O O O OH O OH O O S NH O O OH Raney Ni EtOH reflux, 1 h 3.2 O HO O O O OH O OH O O NH O O OH 3.16 aq. HCl reflux, 6 h H2N OH O 3.17 FDAA NaHCO3 aq. acetone H N OH OH N O2N NO2 H2N O 3.18 40 oC, 1 h  Scheme 3.4. Chemical degradation and Marfey's analysis of bafilomycin F.  The absolute configurations of the remaining stereocenters were assumed to be the same as the known bafilomycins based on biosynthetic considerations.  The optical rotation of bafilomycins A1, B1 and D isolated from the same Streptomyces sp. had the same sign as reported in the literature, indicating that they must have the same absolute configuration as the 75  known compounds.  Since bafilomycin F was isolated from this same Streptomyces sp. strain, it was assumed that it also has the same absolute configuration as these compounds. 3.5. Biological Activity of Bafilomycins  All four bafilomycins (3.1-3.4) were tested for the ability to modulate autophagy in a cell-based assay.  In the presence of each of 3.1-3.4, an accumulation of autophagosomes was observed, suggesting that autophagy was being modulated by these compounds.  Bafilomycin B1 (3.3) was the most potent, showing good activity at concentrations as low as 0.1 nM, while bafilomycins A1 (3.1), F (3.2) and D (3.4) showed good activity at concentrations as low as 1 nM (table 3.5).  Although it was clear that the number of autophagosomes had increased, this effect could be due either to inhibition or stimulation of autophagy. Table 3.5. Fold increase in number of autophagosomes in the presence of 3.1-3.4. Compound 0.1 nM 0.3 nM 1 nM 3 nM 10 nM 100 nM 1000 nM 10,000 nM 3.1 2.5 1.5 3.6 3.8 4.0 4.9 4.6 5.5 3.2 1.8 1.6 3.7 3.8 3.9 4.3 4.5 3.2 3.3 4.1 4.2 4.3 4.4 4.7 4.3 4.2 2.1 3.4 N.D. 1.4 4.3 3.5 3.3 4.5 3.7 4.5 N.D. = Not Determined Fold-increase   In order to determine whether these compounds were inhibiting or stimulating autophagy, the degradation of microtubule-associated light chain 3 (LC3) protein was investigated.  LC3 is a protein involved in autophagy, which is degraded by autophagy after the fusion of autophagosomes with lysosomes.  Therefore, the degradation of LC3 is a measure of autophagic activity.  Enhanced green fluorescent protein (EGFP), which is relatively resistant to degradation by autophagy, was linked to LC3.  When autophagy is stimulated, the LC3 portion of the LC3-EGFP conjugate is degraded leading to an accumulation of free EGFP, which can be detected using gel electrophoresis followed by western blotting.  In the presence of 3.1-3.4 at a concentration of 100 nM, there was no accumulation of EGFP observed (figure 3.12), indicating 76  that 3.1-3.4 do not stimulate autophagy.  Rapamycin, which does stimulate autophagy, causes an accumulation of EGFP at a concentration of 30 nM.  Therefore, 3.1-3.4 must inhibit autophagy, and this is consistent with previous reports.70  Figure 3.12. Accumulation of EGFP observed in the presence of 3.1-3.4.  All four compounds (3.1-3.4) were also tested for cytotoxicity in a variety of cell lines. No toxicity was observed for any of the compounds in any cell lines at concentrations as high as 5 µM.  At 10 µM, 3.3 began to show toxicity while 3.1, 3.2 and 3.4 were not toxic at this concentration.  Therefore, 3.1-3.4 inhibit autophagy at concentrations much lower than that at which they exhibit toxicity, suggesting that 3.1-3.4 have potential as anticancer agents in conjunction with other chemotherapeutics. 3.6. Conclusions  In conclusion, one new bafilomycin analogue, bafilomycin F, along with three known bafilomycin analogues, bafilomycins A1, B1 and D, were isolated from Streptomyces sp.  All four compounds are potent inhibitors of autophagy, active at concentrations as low as 1 nM.  The relative configuration of the thiomorpholinone ring in bafilomycin F was determined by comparison of 1H and 13C NMR chemical shift values to those of model compounds.  The 77  absolute configuration in the thiomorpholinone ring was determined by chemical degradation and Marfey’s analysis. 3.7. Experimental Section 3.7.1. General Experimental Procedures A Waters 10 g silica Sep-Pak® and a Waters 2 g C18 Sep-Pak ® were used for normal- phase and reversed-phase flash chromatography, respectively.  TLC was performed using Merck Kieselgel 60 F254 (for normal-phase) and Whatman MKC18F 60 A (for reversed-phase) TLC plates.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and an Alltech Apollo silica semi-preparative column (10 x 250 mm, 5µm) was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µm filter (Osmonics Inc.) prior to use.  The absorbance was monitored at 230 nm and 254 nm with a flow rate of 2.0 mL/min.  NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively.  Solvents used for NMR were CD3OD or DMSO-d6, and chemical shifts are referenced to the solvent at δH 3.30 and δC 49.0 (for CD3OD) and δH 2.50 and δC 39.5 (for DMSO-d6).  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high- resolution spectra, respectively.  Optical rotations were recorded with a JASCO P-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm microcell.  Single-crystal X- ray diffraction measurements were made on a Bruker X8 APEX II diffractometer with graphite monochromated Mo-Kα radiation.  The data were collected at a temperature of -100 ± 0.1 oC to a maximum 2θ value of 55.0o.  Data were collected in a series of φ and ω scans in 0.50o oscillations with 4.0 second exposures.  The crystal to detector distance was 36.00 mm. Reagents were purchased from Sigma-Aldrich®. 78  3.7.2. Isolation of Bafilomycins The producing organism was obtained from marine sediment collected in Indian Arm, British Columbia, and identified by 16S rRNA analysis as Streptomyces sp.  Production cultures were grown as lawns on solid ISP2 media for 7 days at 25 oC.  The solid agar culture was extracted with EtOAc, and the EtOAc was removed in vacuo to give the crude extract.  The crude extract was subjected to silica gel flash chromatography (eluent: gradient from hexane to EtOAc to MeOH).  The fractions eluting with 75% EtOAc/hexane were further purified by normal-phase HPLC (eluent: 50% EtOAc/hexane) and reversed-phase flash chromatography (eluent: 70% MeCN/H2O) to give bafilomycin A1 (3.1) (1.2 mg), bafilomycin B1 (3.3) (3.8 mg) and bafilomycin D (3.4) (1.0 mg).  The fractions eluting from the silica gel flash chromatography fractionation with 100% MeOH were subjected to reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  The fractions eluting with 75% MeOH/H2O were purified by reversed-phase HPLC (eluent: 75% MeOH/H2O) to give bafilomycin F (3.2) (2.0 mg). Bafilomycin F (3.2): Isolated as a white solid; UV (MeOH) λmax (log ε) 244 nm (4.35) 281 nm (4.05); [α]D 20 +10.0o (c 0.3, MeOH); 1H and 13C NMR data, see table 3.1; HRESI-MS(-) m/z 822.4096 (calculated for C42H64NO13S: 822.4098). 3.7.3. Synthesis of Model Compounds Synthesis of 3.9 and 3.10: Compounds 3.9 and 3.10 were synthesized according to literature protocol.84  A solution of cysteine methyl ester hydrochloride (3.6) (343 mg, 2.0 mmol) in MeOH was added to a solution of -phenylmaleimide (3.7) (363 mg, 2.1 mmol) in CH2Cl2. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then concentrated in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to MeOH) to give 3.8 (652 mg, 95%) as a mixture of diastereomers.  To a solution of 3.8 (652 79  mg, 1.90 mmol) in MeOH (10 mL) was added DIPEA (0.4 mL, 2.30 mmol), and the reaction mixture was stirred at room temperature for 2 days.  The reaction mixture was dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to EtOAc) to give pure 3.9 (85 mg) and pure 3.10 (26 mg) along with a mixture of 3.9 and 3.10 (320 mg, total yield = 74%). Compound 3.9 was recrystallized from MeOH.  Compound 3.9: 1H NMR (400 MHz, CD3OD) δ 7.52 (2H, d, J = 7.8 Hz, H-12/16), 7.28 (2H, t, J = 7.8 Hz, H-13/15), 7.07 (1H, t, J = 7.8 Hz, H-14), 4.47 (1H, dd, J = 6.3, 4.2 Hz, H-6), 4.09 (1H, dd, J = 8.2, 5.7 Hz, H-3), 3.78 (3H, s, H-17), 3.28 (2H, m, H-2a, H-7a), 3.07 (1H, m, H-7b), 2.61 (1H, dd, J = 15.4, 8.2 Hz, H-2b); 13C NMR (100 MHz, CD3OD) δ 171.8 (C, C-9), 171.4 (C, C-4), 170.8 (C, C-1), 139.8 (C, C-11), 129.8 (CH, C-13/15), 125.2 (CH, C-14), 121.3 (CH, C-12/16), 57.7 (CH, C-6), 53.4 (CH3, C-17), 39.5 (CH2, C-2), 39.4 (CH, C-3), 28.8 (CH2, C-7); HRESI-MS(+) m/z 331.0734 (calculated for C14H16N2O4SNa: 331.0728). Compound 3.10: 1H NMR (400 MHz, CD3OD) δ 7.52 (2H, d, J = 7.8 Hz, H-12/16), 7.28 (2H, t, J = 7.8 Hz, H-13/15), 7.07 (1H, t, J = 7.8 Hz, H-14), 4.50 (1H, m, H-6), 3.87 (1H, dd, J = 8.6, 4.8 Hz, H-3), 3.78 (3H, s, H-17), 3.26 (1H, m, H-7a), 3.07 (1H, m, H-7b), 3.04 (1H, m, H- 2a) 2.85 (1H, dd, J = 15.3, 8.6 Hz, H-2b); 13C NMR (100 MHz, CD3OD) δ 171.8 (C, C-9), 171.4 (C, C-4), 170.8 (C, C-1), 139.8 (C, C-11), 129.8 (CH, C-13/15), 125.2 (CH, C-14), 121.3 (CH, C-12/16), 58.1 (CH, C-6), 53.4 (CH3, C-17), 40.9 (CH2, C-2), 39.0 (CH, C-3), 27.0 (CH2, C-7); HRESI-MS(+) m/z 331.0732 (calculated for C14H16N2O4SNa: 331.0728). 80  Synthesis of 3.14 and 3.15: To fumaric acid (3.11) (1.45 g, 12.5 mmol) suspended in MeOH (20 mL) was added concentrated H2SO4 (0.5 mL) and the mixture was heated to reflux for 1 hour.  The mixture was then cooled in an ice bath and neutralized with 10% aq. Na2CO3. CH2Cl2 was added, and the layers were separated.  The organic layer was dried in vacuo, redissolved in CH2Cl2 and purified by silica gel chromatography (eluent: CH2Cl2) to give dimethylfumarate (3.12) (1.63 g, 91%).  To a solution of L-cysteine methyl ester hydrochloride (3.6) (858 mg, 5.0 mmol) in MeOH (20 mL) was added DIPEA (1.8 mL, 10.3 mmol), followed by a solution of dimethylfumarate (3.12) (734 mg, 5.1 mmol) in CH2Cl2.  The reaction mixture was stirred for 15 minutes at room temperature, at which point TLC analysis indicated that the reaction was complete.  The reaction mixture was purified by silica gel chromatography (eluent: gradient from CH2Cl2 to EtOAc) to give 3.13 as a mixture of diastereomers.  Compound 3.13 was dissolved in DMF and heated to 140 oC for 6 hours.  The reaction mixture was cooled, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to EtOAc) to give a mixture of 3.14 and 3.15 that could not be separated by chromatography (191 mg, 15% over two steps).  Compound 3.14: 1H NMR (400 MHz, CD3OD) δ 4.49 (1H, dd, J = 6.5, 4.1 Hz, H-6), 4.00 (1H, t, J = 6.8 Hz, H-3), 3.79 (3H, s, H-11), 3.69 (3H, s, H-10), 3.31 (1H, m, H-7a), 3.08 (1H, m, H-7b), 2.95 (1H, m, H-2a), 2.60 (1H, dd, J = 16.5, 7.3 Hz, H-2b); 13C NMR (100 MHz, CD3OD) δ 172.8 (C, C-4), 171.6 (C, C-9), 170.8 (C, C-1), 57.5 (CH, C-6), 53.4 (CH3, C-11), 81  52.5 (CH3, C-10), 38.9 (CH, C-3), 36.8 (CH2, C-2), 28.8 (CH2, C-7); HRESI-MS(+) m/z 270.0413 (calculated for C9H13NO5SNa: 270.0412). Compound 3.15: 1H NMR (400 MHz, CD3OD) δ 4.53 (1H, t, J = 4.8 Hz, H-6), 3.79 (1H, m, H-3), 3.79 (3H, s, H-11), 3.69 (3H, s, H-10), 3.28 (1H, m, H-7a), 3.08 (1H, m, H-7b), 2.97 (1H, m, H-2a), 2.85 (1H, dd, J = 16.6, 7.8 Hz, H-2b); 13C NMR (100 MHz, CD3OD) δ 172.8 (C, C-4), 172.0 (C, C-9), 170.6 (C, C-1), 58.0 (CH, C-6), 53.4 (CH3, C-11), 52.4 (CH3, C-10), 38.5 (CH, C-3), 38.4 (CH2, C-2), 27.1 (CH2, C-7). HRESI-MS(+) m/z 270.0413 (calculated for C9H13NO5SNa: 270.0412). 3.7.4. Marfey’s Analysis of Bafilomycin F Bafilomycin F (0.5 mg) was dissolved in EtOH (5 mL) and Raney nickel was added as a slurry in water.  The reaction mixture was refluxed for 1 hour and filtered to give crude 3.16. Crude 3.16 was dissolved in 6 M aq. HCl (5 mL) and the reaction mixture was refluxed for 6 hours, then cooled to room temperature and dried in vacuo.  The product was purified by reversed-phase flash chromatography (eluent: H2O) to give crude 3.17.  Crude 3.17 was dissolved in H2O (100 µL), and a solution of FDAA (2.5 mg) in acetone (180 µL) was added, followed by 1 M aq. NaHCO3 (20 µL).  The reaction was heated to 40 oC for 1 hour, dried in vacuo, and purified by reversed-phase flash chromatography (eluent: H2O to 15% MeCN/H2O) to give the Marfey’s derivative 3.18.  Standards were prepared from D-alanine and L-alanine, as described above. 82  3.7.5. 2D *MR Spectra of Bafilomycin F  Figure 3.13. COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz.  Figure 3.14. Expanded COSY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. 83   Figure 3.15. HSQC spectrum of bafilomycin F recorded in CD3OD at 600 MHz.  Figure 3.16. Expanded HSQC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. 84   Figure 3.17. HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz.  Figure 3.18. Expanded HMBC spectrum of bafilomycin F recorded in CD3OD at 600 MHz. 85   Figure 3.19. ROESY spectrum of bafilomycin F recorded in CD3OD at 600 MHz. 3.8. References (1) Klionsky, D. J. at. Rev. Mol. Cell Biol. 2007, 8, 931-937. (2) Gordon, P. B.; Seglen, P. O. Biochem. Biophys. Res. Commun. 1988, 151, 40-47. (3) Tooze, J.; Hollinshead, M.; Ludwig, T.; Howell, K.; Hoflack, B.; Kern, H. J. Cell Biol. 1990, 111, 329-345. (4) Yang, Z.; Huang, J.; Geng, J.; Nair, U.; Klionsky, D. J. Mol. Biol. Cell 2006, 17, 5094- 5104. 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This reaction is the first, and rate-limiting, step in the catabolism of tryptophan through the kynurenine pathway (figure 4.1).1  The same reaction may also be catalyzed by a related enzyme, tryptophan 2,3-dioxygenase (TDO), which is expressed mainly in the liver.2 N H COOH NH2 L-Tryptophan (4.1) IDO O COOH NH2 NH O H N'-Formyl-L-kynurenine (4.2) O COOH NH2 NH2 L-Kynurenine O COOH NH2 NH2 OH 3-Hydroxy-L-kynurenine (4.4) COOH NH2 OH 3-Hydroxyanthranilic acid COOH HOOC NH2 OHC 2-Amino-3-carboxymuconic semialdehyde N COOH COOH Quinolinic acid (4.3) NAD+ O H HO H HO H H OHH O OH NH2 O NH2 COOH 3-HKG or TDO kynurenine formamidase kynurenine 3-hydroxylase kynureninase 3-hydroxyanthranilate 3,4-dioxygenase  Figure 4.1. The kynurenine pathway. 3 A version of this chapter has been published. Carr, G.; Chung, M. K. W.; Mauk, A. G.; Andersen, R. J. (2008) Synthesis of Indoleamine 2,3-Dioxygenase Inhibitory Analogues of the Sponge Alkaloid Exiguamine A. J. Med. Chem. 51:2634-2637. 93  In the active form of IDO the heme iron is in the ferrous (Fe2+) oxidation state, while the ferric (Fe3+) form of IDO is inactive.  A proposed catalytic mechanism for IDO involves abstraction of the indole NH proton by the proximal oxygen atom of Fe2+-bound O2, and electrophilic attack from the C-3 position of indole to the terminal oxygen atom.3  Nucleophilic attack of the proximal oxygen atom to the C-2 position of indole gives a dioxetane intermediate, which collapses to give ’-formyl-L-kynurenine.  This proposed mechanism is summarized in figure 4.2.  Figure 4.2. Proposed catalytic mechanism of IDO. 4.1.1. IDO as a Cancer Drug Target  The link between IDO and the immune system was demonstrated by Munn et al. when they showed that IDO prevents allogeneic fetal rejection in pregnant mice.4  By degrading tryptophan, IDO may cause a local tryptophan shortage.  T-cells are sensitive to a shortage of tryptophan, which causes them to undergo G1 cell cycle arrest and apoptosis leading to immunosuppression.  Additionally, downstream products in the kynurenine pathway may inhibit T-cell proliferation.5,6  It is through these mechanisms that IDO is also believed to play a role in immune escape by solid tumours.  This hypothesis is supported by the observation that IDO is overexpressed in many tumours, and that expression of IDO is correlated with poor prognosis for survival.  The role of IDO in immune escape by tumours was further demonstrated in mice by Uyttenhove et al.7  When preimmunized mice are injected with tumour cells, the tumour cells are usually rejected.  However, when the mice were injected with tumour cells expressing IDO, most 94  of the mice developed tumours and died.  When these mice were treated with the IDO inhibitor 1-methyl-DL-tryptophan (1-MT) this effect was reversed, and the tumour cells were rejected.  Recently, IDO has attracted attention as a possible target for the treatment of cancer. The IDO inhibitor 1-MT potentiates cancer chemotherapy when used in combination with various chemotherapeutic agents.8,9  When 1-MT was combined with the mitotic inhibitor paclitaxel, tumour regression was observed, while either 1-MT or paclitaxel used alone led to no regression.8  Therefore, inhibitors of IDO would most likely be useful in conjunction with other anticancer agents. 4.1.2. IDO as a Drug Target for Other Diseases  While the most promising use for IDO inhibitors is as anticancer agents, IDO inhibitors may find use in the treatment of various other diseases.  By depleting tryptophan, IDO may play a role in diseases that are caused by a lack of tryptophan.  Tryptophan is the precursor to the important neurotransmitter serotonin, which is known to affect mood.  Low levels of serotonin are associated with depression, and many antidepressant drugs act by elevating serotonin levels. By depleting the precursor of serotonin, IDO may lower serotonin levels and contribute to depression.  This is supported by the observation that low dietary tryptophan is correlated with lower levels of brain serotonin in rats.10  Additionally, serotonin itself can act as a substrate for IDO, which may result in lower levels of serotonin.  One type of antidepressant drugs, known as monoamine oxidase inhibitors, act by inhibiting an enzyme that breaks down serotonin, although these drugs have serious side effects.  IDO inhibitors could potentially find use as antidepressant drugs by inhibiting the breakdown of tryptophan and serotonin.  In addition to depleting tryptophan, IDO may have detrimental effects due to the formation of downstream products in the kynurenine pathway.  Quinolinic acid (4.3) is one such 95  downstream product in the kynurenine pathway, and is known to be a neurotoxin.  Quinolinic acid and other metabolites of the kynurenine pathway have been implicated in Alzheimer’s disease (AD) and other neurodegenerative diseases, which means that IDO may play a critical role in these diseases.11-14  Both quinolinic acid and IDO are found in higher amounts in AD brains than non-AD brains.12  The ratio of L-kynurenine to tryptophan, which is a measure of IDO activity, is also higher in the blood serum of AD patients.15  These results suggest that IDO inhibitors may be useful in preventing and/or treating AD.  The formation of downstream products in the kynurenine pathway has also implicated IDO in the formation of age-related nuclear cataracts (ARNs), which are the leading cause of world blindness.16  ARNs are characterized by oxidation, colouration, insolubilization and cross- linking of proteins in the nucleus of the lens.17  The kynurenine metabolite 3-hydroxy-L- kynurenine (3-OHKyn, 4.4) is one of several kynurenine-derived UV filters found in the human lens.  3-OHKyn undergoes spontaneous deamination to give an α,β-unsaturated ketone, which can react with cysteine, lysine and histidine residues in lens proteins.17  Once attached to lens proteins, 3-OHKyn undergoes autoxidation to various coloured iminoquinones, contributing to the colour of the lens.18  Hydrogen peroxide is produced as a by-product of this reaction, which can in turn oxidize methionine and tryptophan residues in lens proteins.19  Oxidation  of these residues in α-crystallin reduces its chaperone activity leading to denaturation and aggregation, thereby contributing to cataracts.20  In addition, the iminoquinone intermediates produced by the oxidation of lens protein-bound 3-OHKyn can further react with amino acid side chains resulting in cross-linking of these proteins.17,19  Thus, 3-OHKyn can contribute to the processes that are characteristic of ARNs.  The role of IDO in the formation of ARNs is supported by the observation that overexpression of human IDO in the lens of mice resulted in cataract formation 96  in only 3 months.21  Inhibiting IDO could lower the amount of 3-OHKyn in the lens and help to prevent this disease. 4.2. Inhibitors of IDO 4.2.1. Tryptophan Analogues Much of the early work on inhibitors of IDO focused on analogues of tryptophan or other indole derivatives (figure 4.3).22,23  In particular, 1-MT (4.5) is the most widely studied inhibitor of IDO.  However, like most other tryptophan analogues, 1-MT is a relatively weak IDO inhibitor with a Ki > 10 µM (reported values for the Ki of 1-MT vary from ~ 6 µM to ~ 60 µM). Despite its low potency, 1-MT is in pre-clinical development by the U.S. National Cancer Institute (NCI) as part of their Rapid Access to Intervention Development (RAID) program, and by NewLink Genetics Corporation.24  Figure 4.3. Tryptophan analogues that inhibit IDO. 97  4.2.2. Polyketides  In an effort to find more potent inhibitors of IDO, the natural products annulins A (4.13), B (4.14) and C (4.15) were isolated by the Andersen lab from a marine hydroid, and these compounds showed submicromolar inhibition of IDO.25  The most potent of these compounds was annulin B, which inhibits IDO with a Ki = 120 nM, making it much more potent than 1-MT. Annulins A-C all contain a napthoquinone core, and it was hypothesized that napthoquinone is the pharmacophore of these compounds.  A series of compounds has been synthesized based on this proposed pharmacophore, resulting in the discovery of even more potent analogues (Ki = 61- 70 nM).26  Figure 4.4. Polyketide inhibitors of IDO. 4.2.3. Exiguamine A  Further work by the Andersen lab led to the isolation of the sponge alkaloid exiguamine A (4.16) from eopetrosia exigua.27  Exiguamine A inhibits IDO with a Ki = 41 nM, making it the most potent IDO inhibitor reported to date.  Like 1-MT, exiguamine A contains an indole ring derived from tryptophan.  However, exiguamine A is much more potent than 1-MT and other indole derivatives making it unlikely that the indole moiety is solely responsible for the IDO inhibitory activity.  Like the annulins, exiguamine A contains a quinone, and it was hypothesized that the quinone in exiguamine A might also play a role in the inhibition of IDO. 98  N H NH3 O N N O O O O HO N 4.16 Figure 4.5. Structure of exiguamine A (4.16). 4.3. Synthesis of IDO Inhibitors  Although exiguamine A is a potent inhibitor of IDO, the natural supply of exiguamine A is extremely limited and it would need to be mass produced by synthesis in order to become a drug.  Exiguamine A is a structurally complex molecule, and although a total synthesis has been completed,28 it is not a trivial synthesis.  More importantly, however, is that exiguamine A is not active in a cell-based assay.  The most likely explanation is that the positive charge on the quaternary nitrogen of exiguamine A prevents it from crossing cell membranes.  Nonetheless, exiguamine A is an interesting lead compound for the synthesis of IDO inhibitory analogues. Thus, we set out to synthesize uncharged and structurally simplified analogues of exiguamine A based on the proposed tryptamine quinone (4.17) pharmacophore (figure 4.6). N H NH3 O O 4.17 Figure 4.6. Proposed pharmacophore of exiguamine A (4.17). 99  4.3.1. Synthesis of Cbz-Protected Tryptamine Quinone  Cbz-protected tryptamine quinone (4.18) was synthesized from tryptamine (4.19) in 5 steps (scheme 4.1) based on the strategy used in the synthesis of lymphostin.29  Scheme 4.1. Synthesis of 4.18.  Despite the lack of a primary amine, as in exiguamine A and tryptophan, 4.18 showed good IDO inhibitory activity in vitro (Ki = 1.49 μM).  While this compound is not as active as exiguamine A, it is still roughly 40 times more potent than 1-MT, and validated our approach of synthesizing analogues based on the proposed tryptamine quinone pharmacophore. 4.3.2. Modification at C-6  While 4.18 showed good IDO inhibitory activity in vitro, it was expected that 4.18 would be a good Michael acceptor, which could react with various biological nucleophiles producing off-target toxicity.  When 4.18 was reacted with dodecylamine (4.24), it underwent a Michael addition, and in the presence of air was oxidized to the expected product 4.25 (scheme 4.2).  The ability of 4.18 to act as a Michael acceptor probably explains why attempts to 100  deprotect the Cbz protecting group in 4.18 to give 4.17 failed using a variety of conditions. Presumably, the free primary amine can act as a nucleophile and undergo polymerization by adding to the quinone in a Michael fashion.  Compound 4.25 was also tested for IDO inhibitory activity, but was not active in this assay.  Scheme 4.2. Synthesis of 4.25.  In order to prevent the off-target toxicity expected for 4.18, we decided to add sterically bulky groups at C-6 in order to block further additions to the quinone.  The first nucleophile that we tried adding was 2-phenylindan-1,3-dione (4.26), based on a literature report that 4.26 adds to napthoquinone.30  Compound 4.26 reacted with 4.18 in MeOH at room temperature in the presence of air to give 4.27 in high yield (scheme 4.3).  Scheme 4.3. Synthesis of 4.27. Unfortunately, 4.27 showed only weak inhibition of IDO (Ki = 10.9 μM), though it is still more potent than 1-MT.  Likewise, when 4.27 was deprotected with BBr3 (scheme 4.4), the deprotected product 4.28 was also only weakly active (Ki not determined).  The fact that 4.28 did 101  not undergo polymerization suggested that the bulky 2-phenylindan-1,3-dione group was sufficient to block further additions to the quinone.  Scheme 4.4. Deprotection of 4.27.  Another approach to add a bulky group to 4.18 was to add 1,3-diphenylisobenzofuran (4.29) as the diene in a Diels-Alder reaction, followed by treatment with BBr3. 31  In the presence of BBr3, the Cbz protecting group was also deprotected to give 4.30 (scheme 4.5).  Compound 4.30 was inactive against IDO (Ki > 500 µM).  Scheme 4.5. Synthesis of 4.30.  Based on the structure of exiguamine A, we wondered whether a substituted hydantoin could be used to block the C-6 position while retaining activity against IDO.  Based on our previous observation that β-dicarbonyl compounds easily add to 4.18, we hoped that the synthon 4.31 would add in a similar fashion to give 4.32.  The synthon 4.31 was easily prepared in three steps according to scheme 4.6.  n-Propyl isocyanate (4.37) was used instead of methyl isocyanate due to safety concerns. 102   Scheme 4.6. Synthesis of 4.31. As expected, 4.32 was produced in good yield by reacting 4.18 with 4.31 in MeOH in the presence of air (scheme 4.7).  Compound 4.32 was found to be a potent inhibitor of IDO (Ki = 260 nM), making it nearly as potent as exiguamine A.  Scheme 4.7. Synthesis of 4.32. Compound 4.32 was deprotected and decarboxylated by treatment with BBr3 (scheme 4.8) to give the product 4.38, which was also a potent inhibitor of IDO (Ki = 200 nM). Surprisingly, the free primary amine that is also found in exiguamine A appears to play little or no role in the IDO inhibitory activity of this compound.  An IC50 value against IDO was also obtained for this compound using a slightly different assay (IC50 = 2.5 µM). 103   Scheme 4.8. Deprotection of 4.32.  Encouraged by this result, we wondered whether the dimethyl hydantoin analogue 4.39 would be even more potent.  The synthon 4.40 was easily prepared in 2 steps from commercially available hydantoin (4.41) (scheme 4.9).  The synthon 4.40 was added to 4.18 in the same manner (scheme 4.10) to give 4.39 in good yield.  Scheme 4.9. Synthesis of 4.40.  Scheme 4.10. Synthesis of 4.39. Compound 4.39 also showed potent activity against IDO (IC50 = 720 nM, Ki not determined).  When 4.39 was deprotected with BBr3 the unexpected product 4.43 was produced, which contains an extra oxygen atom than the expected product 4.44 (scheme 4.11). 104   Scheme 4.11. Deprotection of 4.39. A likely explanation for this observation is that when the reaction was quenched with water, instead of working up the reaction immediately it was left overnight.  During this time, it is possible that water could act as a nucleophile and add to 4.44 according to the proposed mechanism shown in scheme 4.12.  Compound 4.43 was active against IDO with an IC50 = 410 nM (Ki not determined).  Scheme 4.12. Proposed mechanism for the formation of 4.43.  Attempts to add in other nucleophiles by quenching with MeOH or benzyl alcohol instead of water failed to give the expected product.  Instead, a mixture of 4.43 and 4.44 was observed.  Likewise, when the reaction was worked up immediately after quenching with water a 105  mixture of 4.43 and 4.44 was still observed.  A possible explanation is that the less bulky methyl group in 4.39 allows this reaction to take place faster than with the bulky propyl group in 4.32. 4.3.3. Synthesis of Indolequinone and Analogues  Based on the observation that a free primary amine was not a prerequisite for IDO inhibitory activity, we wondered whether the ethylamine side-chain was required at all. Analogues lacking this side chain would greatly simplify the synthesis, as indolequinone (4.45) can be prepared in just one step from commercially available 4-hydroxyindole (4.46) (scheme 4.13). Indolequinone (4.45) was active against IDO with a Ki = 200 nM, demonstrating that the side-chain is not required for activity.  Scheme 4.13. Synthesis of 4.45.  The synthon 4.31 was added to 4.45 in an analogous fashion to produce 4.47 (scheme 4.14).  Compound 4.47 was also found to be a potent inhibitor of IDO (Ki = 420 nM). Surprisingly, attempts to decarboxylate 4.47 with BBr3 led only to decomposition.  Scheme 4.14. Synthesis of 4.47. 106   Likewise, the synthon 4.40 was added to 4.45 to give 4.48 (scheme 4.15).  Compound 4.48 inhibited IDO with an IC50 = 1 µM (Ki not determined).  Scheme 4.15. Synthesis of 4.48.  Thiophenol (4.49) was also added to 4.45 to give 4.50, which was active against IDO with a Ki = 200 nM.  Scheme 4.16. Synthesis of 4.50.  The analogue 4.51 was synthesized in an analogous manner to that of 4.18, except that the Cbz group was replaced with a 3-phenylpropionamide group (scheme 4.17).  The rationale behind this is that an amide functional group is expected to be more metabolically stable than a carbamate functional group, which is important for any in vivo studies.  Not surprisingly, 4.51 showed similar IDO inhibitory activity to that of 4.18, with a Ki = 260 nM.  The activity of all synthetic analogues in vitro is summarized in table 4.1. 107  N H NH2 DIPEA CH2Cl2 N H NHR DDQ aq. THF N H NHRO 2. CuSO4 . 5H2O / DMF N H NHRO OHNaBH3CN THF N H NHR OHNO(SO3K)2 pH 7.2 phosphate buffer N H NHR O O 77% 81% BF3 . Et2O acetone, H2O 17% over 3 steps 4.19 4.52 4.53 4.54 4.55 4.56 Cl O O R = N H NHR O O 4.51 N N O O O O N N O O O O 4.31 MeOH, air 48% r.t., 1 h r.t., 1 h 1. Tl(OCOCF3)3, TFA r.t., 1 h 130 oC, 10 min r.t., 1 h r.t., 1 h r.t., o/n  Scheme 4.17. Synthesis of 4.51. Table 4.1. Ki and IC50 values of synthetic exiguamine A analogues against IDO. Compound Ki (μM) IC50 (μM) 4.18 1.49 N.D. 4.25 N.A. N.D. 4.27 10.9 N.D. 4.28 N.A. N.D. 4.30 N.A. N.D. 4.32 0.26 N.D. 4.38 0.2 2.5 4.39 N.D. 0.72 4.43 N.D. 0.41 4.45 0.2 N.D. 4.47 0.42 N.D. 4.48 N.D. 1 4.50 0.2 N.D. 4.51 0.26 N.D. N.D. = Not Determined N.A. = Not Active  108  4.3.4. Cell-Based IDO Inhibitory Activity  A therapeutic index is a measure of the difference between the concentration at which a drug produces the desired effects and the concentration at which it becomes lethal.  Ideally, a drug should have a fairly large therapeutic index so that it can be used effectively without significant adverse effects.  Our ultimate goal in synthesizing IDO inhibitors was to synthesize analogues that would be active against IDO in vivo at concentrations lower than that required to produce toxicity; in other words, to produce analogues that would have a high therapeutic index making them good drug candidates. To determine whether our IDO inhibitors would act inside cells, they were tested in two different cell-based assays.  The first assay is a yeast-based growth restoration assay.32  Yeast expressing IDO that are grown in media containing high concentrations of tryptophan grow normally, while yeast expressing IDO that are grown in media containing low concentrations of tryptophan grow poorly.  However, in the presence of IDO inhibitors, the yeast grown in low concentrations of tryptophan grow normally.  Therefore, by screening for compounds that can restore the growth of IDO-expressing yeast grown in low concentrations of tryptophan, it is possible to identify inhibitors of IDO that work inside yeast cells and are non-toxic to yeast. Unfortunately, compounds 4.25, 4.27, 4.28 and 4.38 gave no growth restoration in this assay, while compounds 4.32 and 4.51 gave only marginal activity at higher concentrations (20 µg/mL). The annulins (4.13-4.15), exiguamine A (4.16) and 1-MT (4.5) were also inactive in this assay. All of these compounds were also non-toxic to yeast cells at concentrations as high as 100 µM, suggesting that some of them may have problems crossing the yeast cell membrane, especially the ones that showed good in vitro activity.  Compound 4.18 showed toxicity at 10 µM, as might be expected for a Michael acceptor.  Compound 4.45 appeared to give good activity at 0.3 109  µg/mL, but was toxic at higher concentrations (3 µg/mL), giving only a narrow therapeutic index.  Oddly, the only compound that showed good activity in this assay is 4.30, which was active at 3 µg/mL, but was completely inactive in the in vitro assay.  This suggests that the growth restoration is not actually due to IDO inhibition and that 4.30 acts on a different target, or that 4.30 is metabolized into a different compound that is active against IDO.  A similar result was previously observed for the natural product caulerpin.32 The second cell-based assay was performed by NewLink Genetics Corporation on compounds 4.32, 4.39, 4.43 and 4.48.  In this assay, IDO inhibitory activity is determined based on the amount of L-kynurenine formed.  Compounds 4.32 and 4.48 showed a loss in L- kynurenine production (compound 4.32: EC50 = 11.3 µM, compound 4.48: EC50 = 11.2 µM), however they also showed toxicity at similar concentrations (compound 4.32: LD50 = 13.9 µM, compound 4.48: LD50 = 12.1 µM) suggesting that the loss of L-kynurenine production is not due to IDO inhibition but rather due to cell death.  Compound 4.39 showed a loss in L-kynurenine production with an EC50 = 11.7 µM and an LD50 = 21 µM, suggesting that it may have a narrow therapeutic index.  Finally, 4.43 did not inhibit L-kynurenine production and did not exhibit toxicity at concentrations as high as 100 µM, suggesting that it was not able to cross the cell membrane.  The results from this cell-based IDO inhibition assay are summarized in table 4.2. Table 4.2. EC50 and LD50 values of synthetic analogues in a cell-based IDO assay. Compound EC50 (µM) LD50 (µM) 4.32 11.3 13.9 4.39 11.7 21 4.43 > 100 > 100 4.48 11.2 12.1 4.4. Conclusions In conclusion, we have synthesized analogues of exiguamine A based on the putative tryptamine quinone (4.17) pharmacophore, many of which are potent inhibitors of IDO in vitro. 110  The most potent of these analogues inhibit IDO with a Ki ≈ 200-260 nM, making them over 100 times more potent than 1-MT.  These compounds can be readily synthesized in very few steps. Unfortunately, the cell-based results are not as promising.  A few compounds (4.32, 4.39, 4.45 and 4.51) appear to show a hint of activity in the cell-based assays, however the therapeutic index appears to be too small to make them likely drug candidates.  It is possible that these analogues could serve as a starting point to create new analogues that show better activity inside cells, lower toxicity or both.  They may also serve as biological tools to study IDO in vitro.  The quinone moiety appears to be essential for the IDO inhibitory activity of these analogues.  Since quinones are known to be able to oxidize Fe2+ to Fe3+, and since the Fe3+ form of IDO is inactive, it is plausible that these compounds inhibit IDO by oxidizing the heme iron from Fe2+ to Fe3+. Interestingly, Kumar et al. found no correlation between the reduction potential and potency of IDO inhibition for a series of napthoquinone-based IDO inhibitors.26  However, it is still possible that these compounds do act, at least in part, by oxidizing the heme iron in IDO.  Perhaps these compounds need to be sufficiently good oxidizers in order to oxidize Fe2+ to Fe3+, but that it is the binding of the compound to the enzyme that determines potency.  If these compounds do act by oxidizing the heme iron in IDO, this could be advantageous as the related enzyme TDO is still active in the Fe3+ state.  Therefore, these compounds could inhibit IDO in solid tumours without affecting the regular metabolism of tryptophan by the liver. 4.5. Experimental Section 4.5.1. General Experimental Procedures Waters 10 g silica Sep-Paks® and silica gel (SiliCycle® Inc., 230-400 mesh) were used for silica gel flash chromatography and silica gel chromatography, respectively.  Waters 2g C18 Sep-Paks® were used for reversed-phase flash chromatography.  TLC was performed using 111  Merck Kieselgel 60 F254 (for normal-phase) and Whatman MKC18F 60 A (for reversed-phase) TLC plates.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µM filter (Osmonics, Inc.) prior to use. NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively.  Solvents used for NMR were CD2Cl2, CDCl3, acetone-d6 or CD3OD, and chemical shifts are referenced to the internal solvent peaks at δH 5.32 and δC 53.8 (for CD2Cl2), δH 7.24 and δC 77.23 (for CDCl3), δH 2.05 and δC 29.92 (for acetone-d6) and δH 3.30 and δC 49.0 (for CD3OD).  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high- resolution spectra, respectively.  Reagents were purchased from Sigma-Aldrich®, and 4- hydroxyindole was purified by silica gel flash chromatography (eluent: 35% EtOAc/hexane) prior to use. 4.5.2. Synthetic Procedures Synthesis of 4.18: Cbz-protected tryptamine quinone (4.18) was prepared using a modified literature protocol.29  Tryptamine (4.19) (1.99 g, 12.42 mmol) was suspended in CHCl3 (20 mL) under nitrogen, and cooled to 0 oC.  To this solution was added DIPEA (4.4 mL, 25.26 mmol), followed by benzyl chloroformate (2.0 mL, 14.0 mmol).  The solution was allowed to warm to room temperature and stirred for 1 hour.  H2O (20 mL) was added, and the organic layer was separated, concentrated in vacuo, and purified by silica gel flash chromatography (eluent: gradient from 10% EtOAc/hexane to 100% EtOAc) to give 4.20 (3.71 g, quant.). Compound 4.20 (1.96 g, 6.67 mmol) was dissolved in a mixture of THF (10 mL) and H2O (2 mL).  To this solution was added a solution of DDQ (3.35 g, 14.67 mmol) in THF (8 112  mL).  The reaction mixture was stirred for 1 hour at room temperature and dried in vacuo. MeOH (20 mL) was added, the solution was filtered, and the precipitate collected to give crude 4.21.  Crude 4.21 was dissolved in a minimum volume of DMSO, MeOH was added, and the solution was allowed to stand overnight to give crystals of pure 4.21 (1.41 g, 69%). Compound 4.21 (1.41 g, 4.58 mmol), was dissolved in TFA (10 mL) under N2, and a solution of Tl(OCOCF3)3 (2.60 g, 4.79 mmol) in TFA (10 mL) was added.  The reaction mixture was stirred for 1 hour at room temperature and dried in vacuo.  Traces of TFA were removed by repeatedly adding 1,2-dichloroethane and drying in vacuo.  The solid was dissolved in DMF (20 mL) and CuSO4 .5H2O (5.50 g, 22.04 mmol) was added.  The solution was heated to 140 oC for 10 minutes and dried in vacuo.  The solid was suspended in CH2Cl2 and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/ CH2Cl2) to give crude 4.22 (616 mg, 42%). Crude 4.22 (120 mg, 0.37 mmol) was dissolved in THF (10 mL), and to this solution was added BF3 .Et2O (0.5 mL, 5.44 mmol) and excess NaBH4 (150 mg, 3.75 mmol).  The reaction mixture was stirred for 1 hour at room temperature, and quenched by the addition of saturated aq. NH4Cl (10 mL).  This solution was then extracted with CH2Cl2 (3 x 10 mL).  The organic layer was concentrated in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/CH2Cl2) to give 4.23 (39.3 mg, 34 %). Compound 4.23 (39.3 mg, 0.13 mmol) was dissolved in acetone (2 mL), and a solution of NO(SO3K)2 (112 mg, 0.42 mmol) in H2O (3 mL) and phosphate buffer (pH = 7.2, 1 mL) was added.  The reaction mixture was allowed to stir overnight at room temperature before extraction with CH2Cl2 (3 x 5 mL).  The organic layer was concentrated in vacuo, and purified by silica gel 113  flash chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/CH2Cl2) to give 4.18 (22.5 mg, 55%).  A portion of this was purified by reversed-phase HPLC (eluent: 50% MeCN/H2O).   Compound 4.18: 1H NMR (400 MHz, acetone-d6) δ 11.4 (1H, br. s, H-1), δ 7.30-7.35 (5H, m, H-16/20, H-17/19, H-18), 7.12 (1H, br. s, H-2), 6.56 (2H, d, J = 2.6 Hz, H-6, H-7), 5.04 (2H, s, H-14), 3.43 (2H, q, J = 6.8 Hz, H-11), 2.95 (2H, t, J = 6.9 Hz, H-10); 13C NMR (100 MHz, acetone-d6) δ 185.0 (C, C-5), 177.8 (C, C-8), 157.2 (C, C-13), 139.1 (CH, C-6), 138.7 (C, C-15), 136.9 (CH, C-7), 132.2 (C, C-9), 128.0-130.0 (CH, C-16/20, C-17/19, C-18), 125.5 (CH, C-2), 124.1 (C, C-4), 114.9 (C, C-3), 66.4 (CH2, C-14), 41.7 (CH2, C-11), 26.8 (CH2, C-10); HRESI-MS(+) m/z 347.1007 (calculated for C18H16N2O4Na: 347.1008). Synthesis of 4.25: To a solution of 4.18 (5.0 mg, 0.015 mmol) in CH2Cl2 (2 mL) was added excess dodecylamine (4.24) (100 mg, 0.54 mmol) in CH2Cl2 (2 mL), and the solution was stirred for 5 hours at room temperature.  The mixture was purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/hexane) to give 4.25 (6.0 mg, 77%).  A portion of this was purified by reversed-phase HPLC (eluent: 90% MeOH/H2O). 114   Compound 4.25: 1H NMR (400 MHz, CD2Cl2) δ 9.45 (1H, br. s, H-1), δ 7.30-7.35 (5H, m, H-29/33, H-30/32, H-31), 6.69 (1H, s, H-2), 6.09 (1H, s, H-7), 5.04 (2H, s, H-27), 3.43 (2H, t, J = 6.5 Hz, H-11), 3.12 (2H, q, J = 6.8 Hz, H-14), 2.90 (2H, t, J = 6.5 Hz, H-10), 1.66 (2H, m, H- 15), 1.25-1.30 (18H, br. s, H-16, H-17, H-18, H-19, H-20, H-21, H-22, H-23, H-24), 0.88 (3H, t, J = 6.8 Hz, H-25); 13C NMR (100 MHz, CD2Cl2) δ 179.0 (C, C-5), 177.5 (C, C-8), 156.0 (C, C- 26), 150.4 (C, C-6), 137.0 (C, C-28), 135.5 (C, C-9), 128.0-129.0 (CH, C-29/33, C-30/32, C-31), 123.5 (C, C-4), 121.4 (C, C-3), 119.1 (CH, C-2), 95.4 (CH, C-7), 66.6 (CH2, C-27), 43.4 (CH2, C-14), 41.6 (CH2, C-11), 32.3 (CH2, C-15), 27.0-30.0 (CH2, C-16, C-17, C-18, C-19, C-20, C-21, C-22, C-23), 26.4 (CH2, C-10), 23.1 (CH2, C-24), 14.2 (CH3, C-25); HRESI-MS(+) m/z 530.2980 (calculated for C30H41N3O4Na: 530.2995). Synthesis of 4.27: To a solution of 4.18 (5.9 mg, 0.018 mmol) in MeOH (3 mL) was added a solution of 2-phenyl-1,3-indandione (4.26) (15.0 mg, 0.068 mmol) in MeOH (2 mL), and the reaction mixture was stirred overnight at room temperature.  The reaction mixture was dried in vacuo, dissolved in CH2Cl2, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/hexane) to give 4.27 (7.6 mg, 77%).  A portion of this was purified by reversed-phase flash chromatography (eluent: gradient from H2O to 75% MeOH/H2O). 115    Compound 4.27: 1H NMR (400 MHz, CD2Cl2) δ 9.57 (1H, br. s, H-1), 7.99 (2H, m, H- 16/19), 7.86 (2H, m, H-17/18), 7.35-7.50 (5H, m, H-23/27, H-24/26, H-25), 7.30-7.35 (5H, m, H-31/35, H-32/34, H-33), 6.79 (1H, br. s, H-2), 5.97 (1H, s, H-7), 5.02 (2H, s, H-29), 3.29 (2H, t, J = 6.4 Hz, H-11), 2.78 (2H, t, J = 6.5 Hz, H-10); 13C NMR (100 MHz, CD2Cl2) δ 197.6 (C, C- 14/21), 183.5 (C, C-5), 176.6 (C, C-8), 156.8 (C, C-28), 152.1 (C, C-6), 141.7 (C, C-15/20), 139.4 (CH, C-7), 137.7 (C, C-30), 136.2 (CH, C-17/18), 132.6 (C, C-9), 128.0-130.0 (C-22, C- 23/27, C-24/26, C-25, C-31/35, C-32/34, C-33), 124.8 (CH, C-2), 124.6 (CH, C-16/19), 124.4 (C, C-4), 122.4 (C, C-3), 67.1 (CH2, C-29), 66.9 (C, C-13), 41.4 (CH2, C-11), 26.4 (CH2, C-10); HRESI-MS(+) m/z 567.1534 (calculated for C33H24N2O6Na: 567.1532). Synthesis of 4.28: Compound 4.27 (2.5 mg) was dissolved in CH2Cl2 (5 mL) under N2, and cooled to 0 oC.  A solution of BBr3 in CH2Cl2 (0.1 mL of a 1 M solution, 0.1 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for 3 hours.  The reaction mixture was extracted with H2O (3 x 5 mL) and purified by reversed-phase flash chromatography (eluent: H2O to MeOH) to give 4.28 (1.0 mg, 53%). 116   Compound 4.28: 1H NMR (600 MHz, CD3OD) δ 8.02 (2H, dd, J = 5.7, 2.9 Hz, H-16/19), 7.94 (2H, dd, J = 5.5, 3.0 Hz, H-17/18), 7.40-7.50 (5H, m, H-23/27, H-24/26, H-25), 7.06 (1H, s, H-2), 5.85 (1H, s, H-7), 3.03 (2H, t, J = 7.3 Hz, H-11), 2.91 (2H, t, J = 7.4 Hz, H-10); 13C NMR (150 MHz, CD3OD) δ 198.5 (C, C-14/21), 184.3 (C, C-5), 177.2 (C, C-8), 152.3 (C, C-6), 142.5 (C, C-15/20), 139.9 (CH, C-7), 137.1 (CH, C-17/18), 133.4 (C, C-9), 129.6-130.7 (C-22, C- 23/27, C-24/26, C-25), 126.9 (CH, C-2), 125.0 (CH, C-16/19), 124.6 (C, C-4), 121.9 (C, C-3), 67.4 (C, C-13), 40.5 (CH2, C-11), 24.9 (CH2, C-10); HRESI-MS(+) m/z 411.1348 (calculated for C25H19N2O4: 411.1345). Synthesis of 4.30: Compound 4.18 (5.0 mg, 0.015 mmol) was dissolved in CH2Cl2 (3 mL) under N2, and to this solution was added a solution of 1,3-diphenylisobenzofuran (4.29) (20.0 mg, 0.074 mmol) in CH2Cl2 (2 mL).  The reaction mixture was stirred overnight at room temperature, cooled to -78 oC, and a solution of BBr3 in CH2Cl2 (0.2 mL of a 1.0 M solution, 0.2 mmol) was added.  The reaction mixture was allowed to warm to room temperature, and then refluxed for 3 hours.  Additional CH2Cl2 (5 mL) was added, and the solution was extracted with H2O (4 x 5 mL).  The aqueous layer was purified by reversed-phase flash chromatography (eluent: gradient from H2O to MeOH to 50% CH2Cl2/MeOH) and reversed-phase HPLC (eluent: 100% MeOH) to give 4.30 (1.5 mg, 22%). 117  32 26 20 19 13 N H NH3 O O 4.30 12 10 9 4 1   Compound 4.30: 1H NMR (600 MHz, CD3OD) δ 7.20-7.75 (14H, m, H-15/19, H-16/18, H-17, H-21/24, H-22/23, H-28/32, H-29/31, H-30), 7.06 (1H, s, H-2), 3.08 (2H, t, J = 7.3 Hz, H- 11), 2.97 (2H, t, J = 7.2 Hz, H-10); 13C NMR (150 MHz, CD3OD) δ 184.2 (C, C-5), 177.1 (C, C- 8), 127.0-146.0 (C-6, C-7, C-13, C-14, C-15/19, C-16/18, C-17, C-20, C-21/24, C-22/23, C-25, C-26, C-27, C-28/32, C-29/31, C-30), 135.9 (C, C-9), 127.2 (CH, C-2), 126.1 (C, C-4), 121.0 (C, C-3), 41.2 (CH2, C-11), 25.0 (CH2, C-10); HRESI-MS(+) m/z 443.1774 (calculated for C30H23N2O2: 443.1760). Synthesis of 4.31: Compound 4.31 was synthesizing using a modified literature protocol.33  To a solution of dimethyl 2-bromomalonate (4.33) (0.5 mL, 3.81 mmol) in MeOH (10 mL) was added -benzylmethylamine (4.34) (1.0 mL, 7.77 mmol), and the reaction mixture was refluxed for 1 hour.  The mixture was allowed to cool, concentrated in vacuo, and purified by silica gel flash chromatography (eluent: gradient from hexane to 10% EtOAc/hexane) to give 4.35.  Compound 4.35 was dissolved in MeOH (10 mL), and to this solution was added Pd/C. The reaction mixture was flushed with N2, followed by H2, and a balloon filled with H2 was added via syringe.  The reaction mixture was allowed to stir overnight at room temperature, and Pd/C was removed by filtration to give dimethyl 2-(methylamino)malonate (4.36). 118  To a solution of 4.36 in CHCl3 was added n-propyl isocyanate (4.37) (0.4 mL, 4.21 mmol) and DIPEA (0.7 mL, 4.02 mmol).  The reaction mixture was refluxed for 2 hours, and then allowed to cool to room temperature.  H2O (10 mL) was added, and the organic layer was separated, concentrated in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/CH2Cl2) to give 4.31 (158 mg, 20% over three steps). N N O O O O 4.31 11 9 8 7 6 5 3 1   1H NMR (400 MHz, CDCl3) δ 5.89 (1H, s, H-4), 3.79 (3H, s, H-7), 3.21 (2H, m, H-9), 2.98 (3H, s, H-8), 1.54 (2H, m, H-10), 0.92 (3H, t, J = 7.4 Hz, H-11); 13C NMR (100 MHz, CDCl3) δ 168.1 (C, C-6), 168.1 (C, C-5), 157.9 (C, C-2), 61.2 (CH, C-4), 53.0 (CH3, C-7), 43.2 (CH2, C-9), 32.7 (CH3, C-8), 23.5 (CH2, C-10), 11.6 (CH3, C-11). Synthesis of 4.32: To a solution of 4.18 (6.4 mg, 0.020 mmol) in MeOH (3 mL) was added excess 4.31 (23 mg, 0.107 mmol) in MeOH (3 mL), and the solution was stirred overnight at room temperature.  The reaction mixture was dried in vacuo, dissolved in CH2Cl2, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 60% EtOAc/hexane) to give 4.32 (7.1 mg, 67%).  A portion of this was purified by reversed-phase HPLC (eluent: 50% MeCN/H2O). 119    Compound 4.32: 1H NMR (600 MHz, acetone-d6) δ 7.30-7.35 (5H, m, H-27/31, H- 28/30, H-29), 7.19 (1H, s, H-2), 6.59 (1H, s, H-7), 5.03 (2H, s, H-25), 3.83 (3H, s, H-19), 3.52 (2H, t, J = 7.2 Hz, H-21), 3.42 (2H, m, H-11), 2.94 (2H, m, H-10), 2.92 (3H, s, H-20), 1.68 (2H, m, H-22), 0.91 (3H, t, J = 7.6 Hz, H-23); 13C NMR (150 MHz, acetone-d6) δ 181.8 (C, C-5), 175.7 (C, C-8), 167.2 (C, C-17), 165.4 (C, C-18), 156.6 (C, C-24), 156.2 (C, C-15), 141.8 (C, C- 6), 137.1 (C, C-26), 137.0 (CH, C-7), 131.3 (C, C-9), 128.0-129.0 (CH, C-27/31, C-28/30, C-29), 125.4 (CH, C-2), 124.8 (C, C-3), 123.0 (C, C-4), 71.0 (C, C-13), 66.7 (CH2, C-25), 54.0 (CH3, C- 19), 41.8 (CH2, C-21), 41.3 (CH2, C-11), 27.6 (CH3, C-20), 26.2 (CH2, C-10), 21.5 (CH2, C-22), 11.2 (CH3, C-23); HRESI-MS(+) m/z 559.1810 (calculated for C27H28N4O8Na: 559.1805). Synthesis of 4.38: Compound 4.32 (8.0 mg, 0.015 mmol) was dissolved in CH2Cl2 (3 mL) under N2, cooled to 0 oC, and to this solution was added a solution of BBr3 in CH2Cl2 (0.2 mL of a 1.0 M solution, 0.2 mmol).  The reaction mixture was allowed to warm to room temperature, and stirred for 4 hours.  The reaction mixture was extracted with H2O (3 x 5 mL), and the aqueous layer was purified by reversed-phase flash chromatography (eluent: gradient from H2O to 10% MeOH/H2O) to give 4.38 (2.7 mg, 53%).  A portion of this was purified by reversed-phase HPLC (eluent: 5% MeOH/H2O). 120    Compound 4.38: 1H NMR (600 MHz, CD3OD) δ 7.08 (1H, s, H-2), 7.08 (1H, s, H-7), 4.59 (1H, s, H-13), 3.51 (2H, m, H-19), 3.14 (2H, t, J = 6.9 Hz, H-11), 3.00 (2H, t, J = 7.5 Hz, H-10), 2.73 (3H, s, H-18), 1.73 (2H, m, H-20), 1.00 (3H, t, J = 7.5 Hz, H-21); 13C NMR (150 MHz, CD3OD) δ 183.7 (C, C-5), 177.4 (C, C-8), 174.1 (C, C-17), 158.7 (C, C-15), 144.1 (C, C- 6), 138.3 (CH, C-7), 133.4 (C, C-9), 126.9 (CH, C-2), 123.8 (C, C-4), 121.7 (C, C-3), 49.6 (CH, C-13), 41.8 (CH2, C-19), 40.4 (CH2, C-11), 25.0 (CH3, C-18), 24.6 (CH2, C-10), 22.4 (CH2, C- 20), 11.7 (CH3, C-21); HRESI-MS(+) m/z 345.1569 (calculated for C17H21N4O4: 345.1563). Synthesis of 4.40: Hydantoin (4.41) (206 mg, 2.06 mmol) was dissolved in DMF (10 mL) and excess NaH (200 mg, 8.33 mmol) was added, followed by excess CH3I (0.4 mL, 6.42 mmol).  The reaction mixture was stirred at room temperature for 36 hours and then quenched with saturated aq. NH4Cl (10 mL), and extracted with CH2Cl2 (3 x 10 mL).  The organic layer was purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 20% EtOAc/CH2Cl2) to give 1,3-dimethylhydantoin (4.42) (168 mg, 64%). 1,3-Dimethylhydantoin (4.42) (47.5 mg) was dissolved in THF (2 mL) under N2 at -78 oC, and excess n-BuLi was added (0.4 mL of a 1.8 M solution, 0.72 mmol), followed by excess methyl chloroformate (0.2 mL, 2.59 mmol).  The reaction mixture was stirred for 45 minutes at - 78 oC, and then quenched by the addition of EtOAc (1 mL), followed by saturated aq. NH4Cl (5 mL).  The reaction mixture was extracted with CH2Cl2 (3 x 5 mL) and purified by silica gel 121  chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/CH2Cl2) to give 4.40 (49.6 mg, 72%). N N O O O O 4.40 9 8 7 6 5 1  Compound 4.40: 1H NMR (400 MHz, CDCl3) δ 4.52 (1H, s, H-4), 3.84 (3H, s, H-7), 3.00 (3H, s, H-8), 2.97 (3H, s, H-9); 13C NMR (100 MHz, CDCl3) δ 165.8 (C, C-5), 164.8 (C, C-6), 156.5 (C, C-2), 64.5 (CH, C-4), 53.8 (CH3, C-7), 28.9 (CH3, C-9), 25.7 (CH3, C-8). Synthesis of 4.39: To a solution of 4.18 (6.0 mg, 0.018 mmol) in MeOH (2 mL) was added a solution of 4.40 (4.4 mg, 0.024 mmol) in MeOH (3 mL), and the reaction mixture was stirred for 2 days at room temperature, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from 25% EtOAc/hexane to 75% EtOAc/hexane) and reversed-phase HPLC (eluent: 60% MeOH/H2O) to give 4.39 (6.8 mg, 72%).  Compound 4.39: 1H NMR (600 MHz, acetone-d6) δ 7.30-7.35 (5H, m, H-25/29, H-26/28, H-27), 7.19 (1H, s, H-2), 6.61 (1H, s, H-7), 6.38 (1H, t, J = 5.1 Hz, H-12), 5.03 (2H, s, H-23), 3.82 (3H, s, H-19), 3.41 (2H, q, J = 6.6 Hz, H-11), 3.04 (3H, s, H-21), 2.93 (2H, m, H-10), 2.93 (3H, s, H-20); 13C NMR (150 MHz, acetone-d6) δ 182.7 (C, C-5), 175.8 (C, C-8), 167.9 (C, C- 17), 166.2 (C, C-18), 157.3 (C, C-22), 156.6 (C, C-15), 142.7 (C, C-6), 138.8 (C, C-24), 138.2 122  (CH, C-7), 131.9 (C, C-9), 128.0-129.0 (CH, C-25/29, C-26/28, C-27), 126.6 (CH, C-2), 125.0 (C, C-3), 123.6 (C, C-4), 72.2 (C, C-13), 66.4 (CH2, C-23), 54.1 (CH3, C-19), 41.4 (CH2, C-11), 27.6 (CH3, C-20), 26.8 (CH2, C-10), 26.0 (CH3, C-21); HRESI-MS(+) m/z 531.1476 (calculated for C25H24N4O8Na: 531.1492). Synthesis of 4.43: Compound 4.39 (3.1 mg, 0.0061 mmol) was dissolved in CH2Cl2 (5 mL) under N2, and cooled to 0 oC.  A solution of BBr3 in CH2Cl2 (0.2 mL of a 1 M solution, 0.2 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for 3 hours.  The mixture was extracted with H2O (3 x 5 mL) and the aqueous layer was purified by reversed-phase chromatography (eluent: gradient from H2O to 10% MeOH/H2O) followed by reversed-phase HPLC (eluent: 100% H2O) to give 4.43 (1.0 mg, 40%). N H NH3 O O N N O O HO 4.43 19 18 17 13 12 10 9 4 1  Compound 4.43: 1H NMR (600 MHz, CD3OD) δ 7.08 (1H, s, H-2), 7.07 (1H, s, H-7), 3.13 (2H, t, J = 7.5 Hz, H-11), 3.07 (3H, s, H-19), 3.00 (2H, m, H-10), 2.73 (3H, s, H-18); 13C NMR (150 MHz, CD3OD) δ 183.8 (C, C-5), 177.3 (C, C-8), 174.1 (C, C-17), 157.7 (C, C-15), 144.0 (C, C-6), 138.2 (CH, C-7), 133.5 (C, C-9), 127.0 (CH, C-2), 123.8 (C, C-4), 121.7 (C, C- 3), 84.6 (C, C-13), 40.6 (CH2, C-11), 25.2 (CH3, C-19), 24.9 (CH2, C-10), 24.6 (CH3, C-18); HRESI-MS(+) m/z 333.1202 (calculated for C15H17N4O5: 333.1199). Synthesis of 4.45: To a solution of 4-hydroxyindole (4.46) (46.4 mg, 0.35 mmol) in acetone (6 mL) was added a solution of NO(SO3K)2 (187 mg, 0.70 mmol) in H2O (9 mL) and phosphate buffer (pH = 7.2, 3 mL).  After stirring for 6 hours at room temperature, the solution 123  was extracted with CH2Cl2 (3 x 10 mL), dried in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 40% EtOAc/hexane) to give 4.45 (12.0 mg, 23%).  A portion of this was purified by reversed-phase HPLC (eluent: 40% MeCN/H2O). 1N H O O 4.45 9 4   Compound 4.45: 1H NMR (600 MHz, acetone-d6) δ 11.6 (1H, br. s, H-1), δ 7.28 (1H, d, J = 2.6 Hz, H-2), 6.61 (2H, s, H-6, H-7), 6.58 (1H, d, J = 2.7 Hz, H-3); 13C NMR (150 MHz, acetone-d6) δ 183.8 (C, C-5), 177.9 (C, C-8), 138.6 (CH, C-6), 137.2 (CH, C-7), 131.8 (C, C-9), 126.7 (CH, C-2), 126.5 (C, C-4), 108.0 (CH, C-3). Synthesis of 4.47: Indolequinone (4.45) (2.7 mg, 0.018 mmol) was dissolved in MeOH (2 mL), and to this solution was added a solution of 4.31 (5.3 mg, 0.025 mmol) in MeOH (3 mL).  The reaction mixture was allowed to stir overnight at room temperature.  The mixture was then dried in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 40% EtOAc/hexane) to give 4.47 (4.2 mg, 64%).  A portion of this was purified by reversed-phase HPLC (eluent: 50% MeOH/H2O). N H O O N N O O O O 4.47 4 9 10 15 14 16 17 18 20 1   Compound 4.47: 1H NMR (600 MHz, acetone-d6) δ 11.80 (1H, br. s, H-1), δ 7.36 (1H, dd, J = 9.7, 2.8 Hz, H-2), 6.66 (1H, s, H-7), 6.63 (1H, d, J = 2.5 Hz, H-3), 3.84 (3H, s, H-16), 3.51 (2H, m, H-18), 2.92 (3H, s, H-17), 1.68 (2H, m, H-19), 0.92 (3H, t, J = 7.5 Hz, H-20); 13C 124  NMR (150 MHz, acetone-d6) δ 181.3 (C, C-5), 175.4 (C, C-8), 167.6 (C, C-14), 165.9 (C, C-15), 156.7 (C, C-12), 141.8 (C, C-6), 138.6 (CH, C-7), 131.5 (C, C-9), 127.8 (CH, C-2), 125.9 (C, C- 4), 109.1 (CH, C-3), 72.0 (C, C-10), 54.1 (CH3, C-16), 41.7 (CH2, C-18), 27.5 (CH3, C-17), 21.8 (CH2, C-19), 11.4 (CH3, C-20); HRESI-MS(+) m/z 382.1014 (calculated for C17H17N3O6Na: 382.1015). Synthesis of 4.48: To a solution of excess indolequinone (4.45) (10.0 mg, 0.068 mmol) in MeOH (2 mL) was added a solution of 4.40 (10.0 mg, 0.054) in MeOH (3 mL), and the reaction mixture was stirred for 2 days at room temperature, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/hexane) and reversed-phase HPLC (eluent: 50% MeOH/H2O) to give 4.48 (14.1 mg, 79%).  Compound 4.48: 1H NMR (600 MHz, CD2Cl2) δ 9.62 (1H, br. s, H-1), 7.19 (1H, m, H-2), 6.66 (1H, m, H-3), 6.55 (1H, s, H-7), 3.84 (3H, s, H-16), 3.08 (3H, s, H-18), 2.93 (3H, s, H-17); 13C NMR (150 MHz, CD2Cl2) δ 180.7 (C, C-5), 175.8 (C, C-8), 167.3 (C, C-15), 165.3 (C, C- 14), 156.3 (C, C-12), 141.5 (C, C-6), 137.3 (CH, C-7), 131.0 (C, C-9), 126.5 (CH, C-2), 125.7 (C, C-4), 109.2 (CH, C-3), 71.5 (C, C-10), 54.2 (CH3, C-16), 27.6 (CH3, C-17), 26.0 (CH3, C- 18); HRESI-MS(+) m/z 354.0692 (calculated for C15H13N3O6Na: 354.0702). Synthesis of 4.50: Indolequinone (4.45) (4.0 mg, 0.027 mmol) was dissolved in CH2Cl2 (3 mL) and thiophenol (4.49) (0.1 mL, 0.98 mmol) was added.  The reaction mixture was stirred 125  for 2 hours at room temperature and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 40% EtOAc/hexane) to give 4.50 (3.5 mg, 50%).  Compound 4.50: 1H NMR (400 MHz, acetone-d6) δ 11.58 (1H, br. s, H-1), 7.58-7.64 (5H, m, H-11/15, H-12/14, H-13), 7.23 (1H, d, J = 2.7 Hz, H-2), 6.64 (1H, d, J = 2.9 Hz, H-3), 5.53 (1H, s, H-7); 13C NMR (100 MHz, acetone-d6) δ 179.9 (C, C-5), 175.4 (C, C-8), 156.8 (C, C-6), 136.6 (CH, C-11/15), 136.6 (C, C-10), 132.8 (C, C-9), 131.4 (CH, C-12/14), 129.5 (CH, C- 13), 126.3 (CH, C-2), 125.8 (CH, C-7), 125.5 (C, C-4), 108.7 (CH, C-3); HRESI-MS(+) m/z 278.0258 (calculated for C14H9NSO2Na: 278.0252). Synthesis of 4.51: Tryptamine (4.19) (250 mg, 1.56 mmol) was suspended in CH2Cl2 (10 mL) under N2, and to this solution was added DIPEA (0.6 mL, 3.2 mmol) followed by hydrocinnamoyl chloride (0.25 mL, 1.70 mmol).  The solution was stirred at room temperature for 1 hour.  H2O (10 mL) was added, and the organic layer was separated, concentrated in vacuo, and purified by silica gel flash chromatography (eluent: CH2Cl2 to 20% EtOAc/CH2Cl2) to give 4.52 (351 mg, 77%). Compound 4.52 (351 mg, 1.20 mmol) was dissolved in a mixture of THF (10 mL) and H2O (2 mL).  To this solution was added a solution of DDQ (545 mg, 2.4 mmol) in THF (8 mL). The reaction mixture was stirred for 1 hour at room temperature, and dried in vacuo.  MeOH (10 mL) was added, the solution was filtered, and the precipitate collected to give 4.53 (299 mg, 81%). 126  Compound 4.53 (299 mg, 0.98 mmol) was dissolved in TFA (8 mL) under N2, and a solution of Tl(OCOCF3)3 (568 mg, 1.08 mmol) in TFA (6 mL) was added.  The solution was stirred for 1 hour at room temperature and dried in vacuo.  Traces of TFA were removed by repeatedly adding 1,2-dichloroethane and drying in vacuo.  The solid was dissolved in DMF (10 mL) and CuSO4 .5H2O (1.25 g, 5.0 mmol) was added.  The solution was heated to 130 oC for 10 minutes and dried in vacuo.  H2O (10 mL) was added, and the solution was extracted with CH2Cl2 (3 x 10 mL).  The organic layer was dried in vacuo, and purified by silica gel flash chromatography (CH2Cl2 to 10% EtOAc/CH2Cl2) to give crude 4.54 (256.5 mg). Crude 4.54 (257 mg, 0.80 mmol) was dissolved in THF (12 mL), and to this solution was added BF3 .Et2O (0.8 mL, 8.70 mmol) and excess NaBH3CN (250 mg, 3.98 mmol).  The reaction mixture was stirred for 1 hour at room temperature, and quenched by the addition of saturated aq. NH4Cl (10 mL).  This solution was then extracted with CH2Cl2 (3 x 10 mL).  The organic layer was concentrated in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 10% EtOAc/CH2Cl2) to give crude 4.55 (328 mg). Crude 4.55 (164 mg) was dissolved in acetone (6 mL), and a solution of NO(SO3K)2 (430 mg, 1.60 mmol) in H2O (9 mL) and phosphate buffer (pH = 7.2, 3 mL) was added.  The reaction mixture was stirred at room temperature for 1 hour, and extracted with CH2Cl2 (3 x 5 mL).  The organic layer was concentrated in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/CH2Cl2) to give 4.56 (24.4 mg, 17% over three steps).  A portion of this was purified by reversed-phase HPLC (45% MeCN/H2O). Compound 4.56 (24.4 mg, 0.076 mmol) was dissolved in MeOH (2 mL) and to this solution was added a solution of 4.31 (26.4 mg, 0.12 mmol) in MeOH (3 mL).  The solution was stirred overnight at room temperature, dried in vacuo, and purified by silica gel flash 127  chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/CH2Cl2) to give 4.51 (19.5 mg, 48%).  A portion of this was purified by reversed-phase HPLC (eluent: 45% MeCN/H2O).   Compound 4.51: 1H NMR (600 MHz, acetone-d6) δ 7.15-7.30 (5H, m, H-28/32, H- 29/31, H-30), 7.09 (1H, s, H-2), 6.60 (1H, s, H-7), 3.83 (3H, s, H-19), 3.51 (2H, t, J = 7.1 Hz, H- 21), 3.41 (2H, q, J = 6.6 Hz, H-11), 2.92 (3H, s, H-20), 2.86 (2H, m, H-26), 2.85 (2H, m, H-10), 2.40 (2H, t, J = 7.6 Hz, H-25), 1.67 (2H, m, H-22), 0.91 (3H, t, J = 7.5 Hz, H-23); 13C NMR (150 MHz, acetone-d6) δ 182.3 (C, C-5), 176.3 (C, C-8), 172.1 (C, C-24), 167.8 (C, C-17), 166.1 (C, C-18), 156.7 (C, C-15), 142.7 (C, C-6), 142.2 (C, C-27), 138.2 (CH, C-7), 131.9 (C, C-9), 128.0- 129.0 (CH, C-28/32, C-29/31, C-30), 126.6 (CH, C-2), 125.4 (C, C-3), 124.2 (C, C-4), 71.8 (C, C-13), 54.1 (CH3, C-19), 41.9 (CH2, C-21), 39.6 (CH2, C-11), 38.6 (CH2, C-25), 32.4 (CH2, C- 26), 27.6 (CH3, C-20), 26.6 (CH2, C-10), 21.9 (CH2, C-22), 11.4 (CH3, C-23); HRESI-MS(+) m/z 557.2021 (calculated for C28H30N4O7Na: 557.2012). 4.5.3. IDO Inhibition Assays  IDO inhibition assays were performed by the Mauk lab at the University of British Columbia.  For IC50 determinations, the inhibitor was dissolved in MeOH and added to mictrotitre plates (96-well) and dried.  The inhibitor was redissolved in DMSO and 50 mM potassium phosphate buffer, pH 6.5 containing 400 mM tryptophan, 20 mM ascorbic acid, 10 mM methylene blue, IDO and catalase.  The reaction was stopped by the addition of trichloroacetic acid, p-dimethylaminobenzaldehyde in acetic acid was added, and the mixture 128  was heated to 65 oC for 15 minutes.  The amount of product was determined by monitoring the absorbance at 480 nm.  Ki values were determined using various concentrations of tryptophan and inhibitors, and the rate of ’-formyl-L-kynurenine formation was determined from the initial rate of absorbance increase at 321 nm. 4.6. References (1) Opitz, C. A.; Wick, W.; Steinman, L.; Platten, M. Cell. Mol. Life Sci. 2007, 64, 2542-2563. (2) Rafice, S. 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Commun. 1995, 208, 675-679. (21) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. Rev. 1996, 96, 2841-2887. (22) Mailankot, M.; Staniszewska, M. M.; Butler, H.; Caprara, M. H.; Howell, S.; Wang, B.; Doller, C.; Reneker, L. W.; Nagaraj, R. H. Lab. Invest. 2009, 89, 498-512. (23) Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast, G. C. DuHadaway, J.; Bennett, S.; Donovan, A. M. J. Med. Chem. 2006, 49, 684-692. (24) Muller, A. J.; Scherle, P. A. at. Rev. Cancer 2006, 6, 613-625. (25) Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R. J. J. at. Prod. 2006, 69, 1496-1499. 130  (26) Kumar, S.; Malachowski, W. P.; DuHadaway, J. B.; LaLonde, J. M.; Carroll, P. J.; Jaller, D.; Metz, R.; Prendergast, G. C.; Muller, A. J. J. Med. Chem. 2008, 51, 1706-1718. (27) Brastianos, H. C.; Vottero, E.; Patrick, B. O.; Van Soest, R.; Matainaho, T.; Mauk, A. G.; Andersen, R. J. J. Am. Chem. Soc. 2006, 128, 16046-16047. (28) Volgraf, M.; Lumb, J.-P.; Brastianos, H. C.; Carr, G.; Chung, M. K. W.; Münzel, M.; Mauk, A. G.; Andersen, R. J.; Trauner, D. at. Chem. Biol. 2008, 4, 535-537. (29) Tatsuta, K.; Imamura, K.; Itoh, S.; Kasai, S. Tetrahedron Lett. 2004, 45, 2847-2850. (30) Buggle, K.; Donnelly, J. A.; Maher, L. J. J. Chem. Soc., Chem. Commun. 1971, 16, 955. (31) Dodge, J. A.; Bain, J. D.; Chamberlin, A. R. J. Org. Chem. 1990, 55, 4190-4198. (32) Vottero, E.; Balgi, A.; Woods, K.; Tugendreich, S.; Melese, T.; Andersen, R. J.; Mauk, A. G.; Roberge, M. Biotechnol. J. 2006, 1, 282-288. (33) Li, J. P. J. Org. Chem. 1975, 40, 3414-3417. 131  5. Isolation of Exiguamine Analogues 5.1. Isolation of Exiguamines B and C  Exiguamine B (5.1) was originally isolated, along with exiguamine A (4.16), by a former graduate student in the Andersen lab.1  However, there were extra signals in the 1H and 13C NMR spectra that could not be explained by the proposed structure and it was thought that this may be due to the presence of two diastereomers.  Therefore, in order to confirm the structure and determine the stereochemistry of exiguamine B, more exiguamine B was isolated. The aqueous layer, from a partition of the crude extract of eopetrosia exigua between n-BuOH and H2O, was subjected to LH-20 column chromatography (eluent: MeOH) and reversed-phase HPLC (see experimental section) to give a mixture of exiguamine B (5.1) and a new analogue exiguamine C (5.2).  Some of the extra signals originally observed in the NMR spectra of exiguamine B may have been due to the presence of exiguamine C.  It is also possible that some of the extra signals were due to different protonation states of the amine (N-26).  Exiguamines B and C were separated by further reversed-phase HPLC (15% MeOH + 0.2% TFA) to give pure exiguamine B and pure exiguamine C.  This pure exiguamine B did not show any extraneous NMR signals as previously observed.  Analysis of the 1D (figures 5.2 and 5.3 and table 5.1) and 2D (figures 5.19 to 5.22 and table 5.1) NMR data for exiguamine B confirmed that exiguamine B is the 17-OH analogue of exiguamine A. 4 A version of this chapter has been published. Volgraf, M.; Lumb, J.-P.; Brastianos, H. C.; Carr, G.; Chung, M. K. W.; Münzel, M.; Mauk, A. G.; Andersen, R. J.; Trauner, D. (2008) Biomimetic synthesis of the IDO inhibitors exiguamine A and B. Nat. Chem. Biol. 4:535-537. 132   Figure 5.1. Structures of exiguamines B (5.1) and C (5.2).  Figure 5.2. 1H NMR spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz. 133   Figure 5.3. 13C NMR spectrum of exiguamine B recorded in DMSO-d6 at 150 MHz. 134  N OH H H H N OH 3.53 3.58 5.75 6.11 4.45 3.95 142.8 57.2 55.4 73.5 69.1 125.3 H C N OH H HMBC COSY A) B)  Figure 5.4. Selected 1H and 13C NMR chemical shifts and 2D correlations in exiguamine B. 135  Table 5.1. NMR data for exiguamine B. position δC δH (J  in Hz) COSY HMBC 1 13.13, br. d (1.8) H-2 2 126.8 7.34, d (2.6) H-1 C-3, C-4, C-8, C-9, C-24 3 121.0 4 121.4 5 179.4 6 130.6 7 138.7 8 172.9 9 131.3 10 113.7 11 148.4 12 143.6 13 108.5 7.62, s C-10, C-11, C-12, C-14, C-18 14 142.8 15 16a 73.5 4.45, dd (12.3, 6.1) H-16b, H-17 C-14, C-18, C-27, C-28 16b 3.95, dd (12.1, 2.9) H-16a, H-17 C-14, C-17, C-18, C-28 17 69.1 5.75, dd (5.9, 2.9) H-16a, H-16b C-14, C-16, C-18 18 125.3 19 85.3 20 21 154.4 22 23 168.4 24 23.3 2.94, m H-25 C-2, C-3, C-4, C-25 25 38.3 3.04, m H-24, H-26 C-3, C-24 26 7.82, br. s H-25 C-24, C-25 27 55.4 3.58, s C-14, C-16, C-28 28 57.2 3.53, s C-14, C-16, C-27 29 26.1 2.44, s C-19, C-21 30 25.2 3.10, s C-21, C-23 12-OH 10.74, br. s 17-OH 6.11, br. s a Spectra recorded in DMSO-d 6  at 600 MHz Exiguamine B (5.1) a  5.2. Comparison of *atural and Synthetic Exiguamine B.  Both exiguamine A and B were synthesized by the Trauner lab at UC Berkeley.1 Interestingly, exiguamine B was synthesized as a by-product of the synthesis of exiguamine A before the structure of exiguamine B was reported.  The synthetic route to exiguamines A and B is shown in scheme 5.1. 136  N BocHN OBn Br Boc N BocHN OBn Boc OMe OMe N Deprotect [O] N H BocHN O OMe OMe N O N N O O N H BocHN O OMe OMe N O N N O O N H H2N O OH OH N O N N O O N H H2N O N N O O O O OH N N H H2N O N N O O O O OH N HO Stille 5.1 4.16 Ag2O Deprotect  Scheme 5.1. Trauner's synthesis of exiguamines A (4.16) and B (5.1). Synthetic exiguamine B was compared to natural exiguamine B by 1H and 13C NMR spectroscopy, and by reversed-phase HPLC.  A mixture of synthetic exiguamine B and natural exiguamine B eluted as a single peak in the reversed-phase HPLC chromatogram.  Comparison of the chemical shifts of synthetic exiguamine B and natural exiguamine B showed that the chemical shifts were nearly identical (figures 5.5 and 5.6).  Finally, a 1H NMR spectrum of a mixture of synthetic exiguamine B and natural exiguamine B (approximately 1:1 ratio) showed only one set of signals (figure 5.7).  Therefore, synthetic exiguamine B appears to be identical to natural exiguamine B. 137   Figure 5.5. Comparison of 1H NMR spectra of natural and synthetic exiguamine B.  Figure 5.6. Comparison of 13C NMR spectra of natural and synthetic exiguamine B. 138   Figure 5.7. 1H NMR spectrum of a mixture of natural and synthetic exiguamine B. 5.3. Stereochemistry of Exiguamine B  Exiguamine A was crystallized with space group C2/c, indicating that it exists as a racemate.  This is supported by the lack of optical activity and the flat circular dichroism (CD) spectrum of exiguamine A.  Exiguamine B contains an additional stereocenter at C-17. Assuming that exiguamine B also exists as a mixture of configurations at C-19, this leaves three possibilities.  Exiguamine B could exist as a mixture of two diastereomers with the configuration at C-17 fixed, as a mixture of enantiomers, or as a mixture of all four possible stereoisomers. The presence of a single sharp peak in the reversed-phase HPLC chromatogram, and only one set of signals in the 1H and 13C NMR spectra suggested that diastereomers were not present and that exiguamine B exists as a racemic mixture.  The lack of optical activity also supported this conclusion.  In order to determine whether exiguamine B exists as a racemic mixture, a 1H NMR spectrum of natural exiguamine B was taken with varying concentrations of a chiral shift reagent 139  europium tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate].  Splitting of the H-27, H-28 and H-30 signals was observed when excess of this chiral shift reagent was used (shift reagent to exiguamine B ratio of 3.4:1) indicating that exiguamine B exists as a racemate (figures 5.8 and 5.9).  Figure 5.8. 1H NMR spectrum of natural exiguamine B with excess chiral shift reagent. 140   Figure 5.9. 1H NMR spectrum of exiguamine B with and without chiral shift reagent. In order to determine the relative configuration of exiguamine B, 1D and 2D NOESY and ROESY experiments (figures 5.11 to 5.14) were performed on Trauner’s synthetic exiguamine B.  Correlations were observed between H-29 (δH 2.44) and the OH proton attached to C-17 (δH 6.16) in both the NOESY and ROESY spectra.  Thus, the relative configuration of synthetic exiguamine B was determined to be 17R*, 19S* (or u).  This was supported by NOESY correlations between δH 6.16 (17-OH) and δH 3.55 (H-28), and between δH 3.55 (H-28) and δH 2.45 (H-29).  Since natural and synthetic exiguamine B appear to be identical, natural exiguamine B must have the same 17R*, 19S* configuration. 141   Figure 5.10. NOESY and ROESY correlations in synthetic exiguamine B.   Figure 5.11. 1D ROESY spectrum of synthetic exiguamine B in DMSO-d6 at 600 MHz. 142   Figure 5.12. 1D ROESY spectrum of synthetic exiguamine B in DMSO-d6 at 600 MHz.  Figure 5.13. Expanded 2D ROESY spectrum of synthetic exiguamine B. 143   Figure 5.14. Expanded 2D NOESY spectrum of synthetic exiguamine B. 5.4. Structure Elucidation of Exiguamine C  Exiguamine C gave an [M]+ peak at m/z 506.1666 in the HRESI-MS(+) spectrum, consistent with a molecular formula of C25H24N5O7 (calculated for C25H24N5O7: 506.1676), which differed from the molecular formula of exiguamine B by the loss of two hydrogen atoms. Comparison of the 1D (figures 5.15 and 5.16 and table 5.2) and 2D (figures 5.23 to 5.26 and table 5.2) NMR data for exiguamine C with the data for exiguamines A and B indicated that they were closely related.  The methine signal at δH 5.75 (H-17) and the exchangeable proton at δH 6.11 (17-OH) in exiguamine B were absent in the NMR data for exiguamine C.  In addition, the methylene signals at δH 4.45 (H-16a) and δH 3.95 (H-16b) were shifted upfield in exiguamine C to δH 5.02 (H-16a) and δH 4.53 (H-16b), and both H-16a and H-16b showed HMBC correlations in exiguamine C to a carbonyl carbon (δC 186.2, C-17).  Therefore, exiguamine C was determined to be the C-17 keto analogue of exiguamine B.  Like exiguamines A and B, exiguamine C showed no optical rotation and therefore was assumed to exist as a racemic mixture. 144   Figure 5.15. 1H NMR spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz.  Figure 5.16. 13C NMR spectrum of exiguamine C recorded in DMSO-d6 at 150 MHz. 145  N N3.69 3.77 5.02 4.53 155.1 57.6 56.8 71.2 125.3 186.2 O O H C N O HMBC A) B)  Figure 5.17. Selected 1H and 13C NMR chemical shifts and 2D correlations in exiguamine C.  Figure 5.18. Expanded HMBC spectrum of exiguamine C in DMSO-d6 at 600 MHz. 146  Table 5.2. NMR data for exiguamine C. position δC δH (J  in Hz) COSY HMBC 1 13.18, br. s H-2 2 127.0 7.34, br. s H-1 C-3, C-4, C-8, C-9 3 121.1 4 121.8 5 178.9 6 130.6 7 137.2 8 171.1 9 130.6 10 110.9 11 148.4 b 12 143.6 b 13 107.8 7.86, br. s 14 155.1 15 16a 71.2 5.02, d (16.1) H-16b C-17, C-28 16b 4.53, d (16.5) H-16a C-14, C-17, C-27 17 186.2 18 125.3 b 19 85.5 20 21 154.2 22 23 167.9 24 23.2 2.94, t (7.5) H-25 C-2, C-3, C-4, C-25 25 38.0 3.04, m H-24, H-26 C-3, C-24 26 7.80, br. s H-25 C-24, C-25 27 56.8 3.77, s C-14, C-16, C-28 28 57.6 3.69, s C-14, C-16, C-27 29 26.3 2.54, s C-19, C-21 30 25.2 3.11, s C-21, C-23 a Spectra recorded in DMSO-d 6  at 600 MHz Exiguamine C (5.2) b Inferred from data of exiguamine B 5.5. Biological Activity of Exiguamines B and C  Exiguamines B and C were tested for their ability to inhibit IDO.  Exiguamine B inhibited IDO with a Ki ≈ 40-80 nM, while exiguamine C inhibited IDO with a Ki = 55 nM. Therefore, both exiguamine B and C inhibit IDO with potency comparable to that of exiguamine A (Ki = 41 nM).  Synthetic exiguamine B also inhibited IDO with potency comparable to that of 147  the natural product (Ki = 80 nM).  Neither exiguamine B nor exiguamine C showed any in vivo activity in the yeast cell-based assay (see section 4.3.4). 5.6. Experimental Section 5.6.1. General Experimental Procedures  SephadexTM LH-20 was used for LH-20 column chromatography.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µm filter (Osmonics Inc.) prior to use.  The absorbance was monitored at 254 nm and 330 nm with a flow rate of 2.0 mL/min.  NMR spectra were recorded on a Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 600 MHz.  The solvent used for NMR was DMSO-d6, and chemical shifts are referenced to the internal solvent peaks at δH 2.50 and δC 39.50.  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high-resolution spectra, respectively.  Optical rotations were recorded with a JASCO P-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm microcell.  Europium tris[3-(heptafluoropropylhydroxymethylene)-(+)- camphorate] was purchased from Sigma-Aldrich®. 5.6.2. Isolation of Exiguamines A-C Specimens of eopetrosia exigua (140 g) were collected by hand at a depth of 15 metres using SCUBA in Milne Bay, Papua New Guinea (10o 32.02’ S, 150o 39.07’ E).  A voucher sample has been deposited at the Zoologisch Museum, University of Amsterdam (ZMAPOR 19113).  The sponge was frozen on site and transported frozen to Vancouver.  The sponge was thawed and extracted with MeOH to give a crude extract, which was partitioned between water and n-BuOH.  The aqueous layer was subjected to LH-20 column chromatography (eluent: 148  MeOH), and the IDO inhibitory fractions were pooled and dried in vacuo.  These active fractions were then subjected to reversed-phase HPLC (eluent: gradient from H2O + 0.2% TFA to 50% MeCN + 0.2% TFA).  The orange-red fractions were recombined and subjected to further reversed-phase HPLC (eluent: 10% MeCN + 0.2% TFA) to give pure exiguamine A and a mixture of exiguamines B and C.  The mixture of exiguamines B and C was separated by reversed-phase HPLC (eluent: 15% MeOH + 0.2% TFA) to give pure exiguamine B (3.0 mg) and pure exiguamine C (1.9 mg).  Exiguamine B (5.1): Isolated as an orange-red solid; [α]D 20 0.0o (c 0.7, MeOH); 1H and 13C NMR data, see table 5.1; HRESI-MS(+) m/z 508.1850 (calculated for C25H26N5O7: 508.1832).  Exiguamine C (5.2): Isolated as an orange-red solid; [α]D 20 0.0o (c 0.7, MeOH); 1H and 13C NMR data, see table 5.2; HRESI-MS(+) m/z 506.1666 (calculated for C25H26N5O7: 506.1676). 149  5.6.3. 2D *MR Spectra of Exiguamine B  Figure 5.19. COSY spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz.  Figure 5.20. HSQC spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz. 150   Figure 5.21. HMBC spectrum of exiguamine B recorded in DMSO-d6 at 600 MHz.  Figure 5.22. Expanded HMBC spectrum of exiguamine B in DMSO-d6 at 600 MHz. 151  5.6.4. 2D *MR Spectra of Exiguamine C  Figure 5.23. COSY spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz.  Figure 5.24. HSQC spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz. 152   Figure 5.25. HMBC spectrum of exiguamine C recorded in DMSO-d6 at 600 MHz.  Figure 5.26. Expanded HMBC spectrum of exiguamine C in DMSO-d6 at 600 MHz. 153  5.7. References (1) Volgraf, M.; Lumb, J.-P.; Brastianos, H. C.; Carr, G.; Chung, M. K. W.; Münzel, M.; Mauk, A. G.; Andersen, R. J.; Trauner, D. at. Chem. Biol. 2008, 4, 535-537. 154  6. Isolation of Plectosphaeroic Acids A-C 6.1. Isolation of Plectosphaeroic Acids A-C  An extract of the fungus Plectosphaerella cucumerina, which was collected from marine sediment at -100 m in Barkley Sound, British Columbia, showed good activity in the IDO inhibition assay.  Cultures of P. cucumerina were grown as lawns on solid agar for 14 days at room temperature.  The solid agar was extracted twice with EtOAc and the EtOAc was dried to give the crude extract.  The crude extract was dissolved in MeOH (10 mL) and fractionated by LH-20 column chromatography (eluent: MeOH).  The IDO inhibitory fractions were pooled and purified by reversed-phase HPLC (step gradient from 50% MeOH/H2O + 0.2% TFA to 70% MeOH/H2O + 0.2% TFA to 75% MeOH/H2O + 0.2% TFA) to give plectosphaeroic acids A (6.1), B (6.2) and C (6.3).  The known diketopiperazine T988 A (6.4)1 was isolated from an inactive LH-20 fraction by reversed-phase HPLC (60% MeOH/H2O). 5 A version of this chapter has been published. Carr, G.; Tay, W.; Bottriell, H.; Andersen, S. K.; Mauk, A. G.; Andersen, R. J. (2009) Plectosphaeroic Acids A, B, and C, Indoleamine 2,3- Dioxygenase Inhibitors Produced in Culture by a Marine Isolate of the Fungus Plectosphaerella cucumerina. Org. Lett. 11:2996-2999. 155  N N N HN O O OH HO S S O N NH2 O COOH COOH H 6.1 14'' 1'' 7'a 3'a 16 15 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' 12'' 11'' 10'' 6'' 5'' 13'' N N N HN O OHO S S O N NH2 O COOH COOH H 6.2 14'' 1'' 7'a 3'a 16 15 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' 12'' 11'' 10'' 6'' 5'' 13'' N N N HN O O OH HO S S O N NH2 O COOH COOH H 6.3 14'' 1'' 7'a 3'a 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' 12'' 11'' 10'' 6'' 5'' 13'' S N N N HN O O OH HO S SH 7'a 3'a 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' S H 6.4 6a 6a6a6a  Figure 6.1. Structures of Plectosphaeroic acids A (6.1), B (6.2), C (6.3) and T988 A (6.4). 6.2. Structure Elucidation of Plectosphaeroic Acids A-C 6.2.1. Structure Elucidation of Plectosphaeroic Acid A  Plectosphaeroic acid A (6.1) gave an [M - H]- peak in the HRESI-MS(-) spectrum at m/z 807.1534, consistent with a molecular formula of C39H32N6O10S2 (calculated for C39H31N6O10S2: 807.1543).  An [M - D]- peak in the ESI-MS(-) spectrum at m/z 813 and an [M - D + 2Na]+ peak in the ESI-MS(+) spectrum at m/z 859 indicated that seven exchangeable protons were present in plectosphaeroic acid A.  Analysis of the 1D (figures 6.2 and 6.3 and table 6.1) and 2D (figures 6.23 to 6.29 and table 6.1) NMR data for plectosphaeroic acid A indicated that it contains a 156  substructure I that is also found in the known diketopiperazine T988 B (6.5) (figures 6.4 and 6.5).1  ROESY correlations confirmed that plectosphaeroic acid A has the same relative configuration at each stereocenter as in T988 B.  Figure 6.2. 1H NMR spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz. 157   Figure 6.3. 13C NMR spectrum of plectosphaeroic acid A in DMSO-d6 at 150 MHz.  Figure 6.4. 1H NMR chemical shifts of substructure I (DMSO-d6) and T988 B (MeOD). 158   Figure 6.5. 13C NMR chemical shifts of substructure I (DMSO-d6) and T988 B (MeOD). 159  Table 6.1. NMR data for plectosphaeroic acid A. position δC δH (mult., J  in Hz) HMBC COSY ROESY 1 164.3 2 3 69.4 4 162.4 5 5a 86.2 6.78 (1H, s) 3', 4, 6a, 10a, 10b, 11, 12 2', 4' 6 6a 149.1 7 106.7 6.00 (1H, d, 8.0) 9, 10a 8 7'', 8, 8'' 8 127.9 6.91 (1H, t, 7.6) 6a, 10 7, 9 7, 9 9 118.1 6.57 (1H, t, 7.6) 7, 10, 10a 8, 10 8, 10 10 122.5 7.57 (1H, d, 7.5) 6a, 8, 10b 9 4', 9, 11 10a 133.3 10b 58.5 11 78.6 5.45 (1H, br. s) 5a 11-OH 4', 10, 15, 16 11-OH 5.51 (1H, br. s) 11 12 74.1 13 27.8 2.94 (3H, s) 1, 3 14 14 61.7 4.02 (1H, m) 4 14 14, 15 3.56 (1H, d, 11.6) 3, 4 14 13, 14 15 12.9 1.89 (3H, s) 3 7'', 8'', 11, 14, 16 16 15.7 2.14 (3H, s) 12 11, 15 1' 10.89 (1H, br. s) 2', 3', 3'a, 7'a 2' 2' 2' 122.4 7.34 (1H, d, 2.9) 3', 3'a, 7'a 1' 1', 5a 3' 115.8 3'a 125.3 4' 121.6 8.05 (1H, d, 7.7) 3', 6', 7'a 5' 5a, 5', 10, 11 5' 118.7 7.06 (1H, m) 3'a, 7' 4', 6' 4' 6' 121.0 7.08 (1H, m) 4', 7'a 5', 7' 7' 7' 111.3 7.35 (1H, d, 8.8) 3'a, 5', 7'a 6' 6' 7'a 137.1 1'' 92.4 2'' 152.6 2''-NH2 9.72 (1H, br. s) 3'' 2''-NH2 2''-NH2 8.91 (1H, br. s) 1'' 2''-NH2 2''-NH2 3'' 178.1 4'' 105.5 6.67 (1H, s) 2'', 5'', 12'' 5'' 150.9 6'' 141.2 7'' 117.9 7.72 (1H, d, 8.6) 6'', 9'', 11'' 8'' 7, 8'', 15 8'' 130.6 7.68 (1H, d, 8.8) 6'', 10'' 7'' 7, 7'', 15 9'' 135.1 10'' 133.2 11'' 127.4 12'' 147.6 13'' 168.9 14'' 167.2 Plectosphaeroic acid A (6.1) a a Spectra recorded in DMSO-d 6 at 600 MHz 160  Plectosphaeroic acid A did not show any signals corresponding to H-6 in the 1H NMR or 2D NMR spectra, indicating that the substructure I was linked to another fragment through N-6. Subtracting the atoms in I from the molecular formula of plectosphaeroic acid A leaves C14H7N2O6 unaccounted for, including 4 exchangeable protons.  The presence of two signals in the 13C NMR spectrum at δC 168.9 (C-13’’) and δC 167.2 (C-14’’), the presence of an [M - 2H + 3Na]+ peak in ESI-MS(+) spectrum at m/z 875, and the higher polarity of plectosphaeroic acid A relative to T988 B on silica, suggested the presence of two carboxylic acids.  This was confirmed by reacting plectosphaeroic acid A with methyl iodide (scheme 6.1) to give the dimethyl ester of plectosphaeroic acid A (6.6), which gave a peak in the HRESI-MS(+) spectrum at m/z 859.1813, consistent with a molecular formula of C41H36N6O10S2 (calculated for C41H36N6O10S2Na: 859.1832).  Scheme 6.1. Synthesis of plectosphaeroic acid A dimethyl ester (6.6). The orange-red colour of plectosphaeroic acid A, and the UV-Vis λmax at 428 nm, suggested that a quinone, or a related functionality, might be present in the molecule.  The 161  chemical shift of the carbon resonance at δC 178.1 (C-3’’) is appropriate for a quinone carbonyl carbon.  However, the lack of another carbon resonance with a similar chemical shift suggested that plectosphaeroic acid A might contain an iminoquinone moiety instead.  The known compound cinnabarinic acid (6.7), an iminoquinone with two carboxylic acids, has a similar UV- Vis λmax (429 nm) to that of plectosphaeroic acid A, suggesting that a substituted cinnabarinic acid moiety may be present in plectosphaeroic acid A.  This hypothesis was confirmed by synthesizing cinnabarinic acid (scheme 6.2) from 3-hydroxyanthranilic acid (6.8) and comparing the 1H and 13C NMR chemical shifts of cinnabarinic acid with those for the corresponding substructure of plectosphaeroic acid A (II).  The 1H, 13C, and 15N chemical shifts of cinnabarinic acid were in excellent agreement with those for the substructure II (figures 6.6 and 6.7), supporting this proposed substructure.  The H-9 resonance of cinnabarinic acid (δH 7.96), which showed an HMBC correlation to the carbonyl carbon at δC 166.3 (C-14), was missing in plectosphaeroic acid A, indicating that I must be linked to II through C-9’’ in plectosphaeroic acid A.  ROESY correlations (figures 6.8 and 6.9) between δH 7.72 (H-7’’) and δH 6.00 (H-7), between δH 7.72 (H-7’’) and δH 1.89 (H-15), between δH 7.68 (H-8’’) and δH 6.00 (H-7), and between δH 7.68 (H-8’’) and δH 1.89 (H-15) confirmed this linkage. 162   Scheme 6.2. Synthesis of cinnabarinic acid (6.7).  Figure 6.6. 1H NMR chemical shifts of substructure II and cinnabarinic acid (DMSO-d6). 163   Figure 6.7. 13C and 15N NMR chemical shifts of II and cinnabarinic acid (DMSO-d6).  Figure 6.8. ROESY correlations confirming linkage between N-6 and C-9’’ in 6.1. 164   Figure 6.9. Expanded ROESY spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). 6.2.2. Structure Elucidation of Plectosphaeroic Acids B and C  Plectosphaeroic acid B (6.2) gave an [M - H]- peak in the HRESI-MS(-) spectrum at m/z 791.1599, consistent with a molecular formula of C39H32N6O9S2 (calculated for C39H31N6O9S2: 791.1594).  Comparison of the 1D (figures 6.11 and 6.12 and table 6.2) and 2D (figures 6.30 to 6.36 and table 6.2) NMR data for plectosphaeroic acid B with the data for plectosphaeroic acid A showed that they were closely related.  The major difference between the NMR data for plectosphaeroic acids A and B is that the signals for the oxygenated methylene at C-14 (δH 4.02, H-14a; δH 3.56, H-14b; δC 61.7, C-14) in plectosphaeroic acid A were replaced with signals for a methyl group (δH 1.59, H-14; δC 22.6, C-14) in plectosphaeroic acid B.  Therefore, plectosphaeroic acid B is the C-14 deoxygenated analogue of plectosphaeroic acid A. 165  N N N HN O OHO S S O N NH2 O COOH COOH H 6.2 14'' 1'' 7'a 3'a 16 15 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' 12'' 11'' 10'' 6'' 5'' 13'' 6a  Figure 6.10. Structure of plectosphaeroic acid B (6.2).  Figure 6.11. 1H NMR spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. 166   Figure 6.12. 13C NMR spectrum of plectosphaeroic acid B in DMSO-d6 at 150 MHz. 167  Table 6.2. NMR data for plectosphaeroic acid B. position δC δH (mult., J  in Hz) HMBC COSY ROESY 1 163.5 2 3 66.2 4 163.6 5 5a 86.1 6.74 (1H, s) 3', 4, 6a, 10a, 10b, 11, 12 2' 6 6a 149.1 7 106.7 5.98 (1H, d, 7.9) 9, 10a 8 8, 8'' 8 127.9 6.90 (1H, t, 7.6) 6a, 10 7, 9 7 9 118.0 6.56 (1H, t, 7.3) 7, 10, 10a 8, 10 10 10 122.4 7.60 (1H, d, 7.6) 6a, 8, 10b 9 4', 9, 11 10a 133.3 10b 58.6 11 78.5 5.44 (1H, br. s) 5a, 10a 11-OH 4', 10, 16 11-OH 5.74 (1H, br. s) 11 12 74.1 13 28.5 2.93 (3H, s) 1, 3 14 14 22.6 1.59 (3H, s) 3, 4 13 15 13.6 1.88 (3H, s) 3 7'', 8'', 16 16 15.4 2.14 (3H, s) 12 11, 15 1' 10.86 (1H, br. s) 2', 3', 3'a, 7'a 2' 2' 2' 122.5 7.34 (1H, m) 3', 3'a, 7'a 1' 1', 5a 3' 115.9 3'a 125.3 4' 121.5 8.04 (1H, d, 7.6) 3', 6', 7'a 5' 5', 10, 11 5' 118.6 7.06 (1H, m) 3'a, 7' 4', 6' 4' 6' 120.9 7.09 (1H, m) 4', 7'a 5', 7' 7' 7' 111.4 7.36 (1H, m) 3'a, 5', 7'a 6' 6' 7'a 137.1 1'' 92.4 2'' 152.6 2''-NH2 9.72 (1H, br. s) 3'' 2''-NH2 2''-NH2 8.91 (1H, br. s) 2''-NH2 2''-NH2 3'' 178.1 4'' 105.5 6.68 (1H, s) 2'', 5'', 12'' 5'' 151.0 6'' 141.2 7'' 117.8 7.72 (1H, d, 8.9) 6'', 9'', 11'' 8'' 8'', 15 8'' 130.7 7.69 (1H, d, 8.9) 6'', 10'' 7'' 7, 7'', 15 9'' 135.0 10'' 133.3 11'' 127.3 12'' 147.6 13'' 168.9 14'' 167.2 Plectosphaeroic acid B (6.2) a a Spectra recorded in DMSO-d 6 at 600 MHz 168   Plectosphaeroic acid C (6.3) gave an [M - H]- peak in the HRESI-MS(-) spectrum at m/z 809.0786, consistent with a molecular formula of C37H26N6O10S3 (calculated for C37H25N6O10S3: 809.0794).  Comparison of the 1H (figure 6.14 and table 6.3) and 2D (figures 6.37 to 6.40 and table 6.3) NMR data for plectosphaeroic acid C with the data for plectosphaeroic acids A and B showed that it was also closely related.  The major difference in the NMR data for plectosphaeroic acids A and C is that the signals for the thiomethyl groups (δH 1.89, H-15; δC 12.9, C-15; δH 2.14, H-16; δC 15.7, C-16) in plectosphaeroic acid A were not present in plectosphaeroic acid C.  The presence of an extra sulfur atom in the molecular formula of plectosphaeroic acid C relative to plectosphaeroic acid A, suggested that plectosphaeroic acid C contains a trisulfide bridge, much like that in T988 A. N N N HN O O OH HO S S O N NH2 O COOH COOH H 6.3 14'' 1'' 7'a 3'a 14 13 1211 10b 10a 10 7 6 5a 5 3 1 7' 4' 3' 1' 12'' 11'' 10'' 6'' 5'' 13'' S 6a  Figure 6.13. Structure of plectosphaeroic acid C (6.3). 169   Figure 6.14. 1H NMR spectrum of plectosphaeroic acid C in DMSO-d6 at 600 MHz. 170  Table 6.3. NMR data for plectosphaeroic acid C. position δC δH (mult., J  in Hz) HMBC COSY 1 166.9 2 3 75.5 4 162.5 5 5a 85.4 6.68 (1H, s) 3', 6a, 10a, 10b, 12 6 6a 150.4 7 108.4 6.32 (1H, d, 8.0) 9, 10a 8 8 129.4 7.06 (1H, m) 6a, 10 7, 9 9 119.3 6.71 (1H, t, 7.5) 7, 10, 10a 8, 10 10 124.8 7.61 (1H, d, 7.7) 6a, 8, 10b 9 10a 129.8 10b 58.7 11 81.4 5.54 (1H, d, 6.4) 5a 11-OH 11-OH 6.46 (1H, d, 6.6) 10b 11 12 86.4 13 27.0 3.11 (3H, s) 1, 3 14 59.2 3.78 (1H, m) 14, 14-OH 3.61 (1H, m) 4 14, 14-OH 14-OH 5.48 (1H, t, 5.9) 14 1' 10.86 (1H, br. s) 2', 3', 3'a, 7'a 2' 2' 122.3 7.36 (1H, m) 3', 3'a, 7'a 1' 3' 113.5 3'a 125.3 4' 120.8 8.02 (1H, d, 7.7) 6', 7'a 5' 5' 118.4 7.06 (1H, m) 3'a, 7' 4', 6' 6' 121.1 7.10 (1H, m) 4', 7'a 5', 7' 7' 111.5 7.37 (1H, m) 3'a, 5', 7'a 6' 7'a 137.3 1'' 92.4 b 2'' 152.7 2''-NH2 9.72 (1H, br. s) 2''-NH2 8.93 (1H, br. s) 2''-NH2 3'' 178.1 b 4'' 105.3 6.70 (1H, s) 2'', 5'', 12'' 5'' 150.4 6'' 141.4 7'' 117.1 7.74 (1H, d, 8.9) 6'', 9'', 11'' 8'' 8'' 131.0 7.95 (1H, m) 6'', 10'' 7'' 9'' 135.8 10'' 131.8 11'' 127.3 12'' 147.8 13'' 168.9 b 14'' 167.2 b Plectosphaeroic acid C (6.3) a a Spectra recorded in DMSO-d 6 at 600 MHz b Inferred from data for plectosphaeroic acid A 171  6.2.3. Absolute Configurations of Plectosphaeroic Acids A-C  The absolute configurations of plectosphaeroic acids A-C were determined by comparing their CD spectra with the reported spectra for related compounds.2,3  The reported CD spectrum of leptosin D (6.9) shows a positive Cotton effect at 229 nm (Δε + 41.7),2 while compound 6.10, which has the opposite configurations at C-3, C-5a, C-10b and C-12, shows a negative Cotton effect at 231 nm (Δε – 23.7).3  Figure 6.15. Structures of leptosin D (6.9) and 6.10. Since plectosphaeroic acids A-C all show a positive Cotton effect in this region (figures 6.16 to 6.18), it was assumed that plectosphaeroic acids A-C have the same absolute configurations as leptosin D.2  Therefore, plectosphaeroic acids A-C have the absolute configuration 3S, 5aR, 10bR, 11S, 12S.  The CD spectrum of the related compound T988 A was also measured (figure 6.19) and found to be similar to that of plectosphaeroic acids A-C, showing that it also has the same absolute configuration as expected. 172   Figure 6.16. CD spectrum of plectosphaeroic acid A.  Figure 6.17. CD spectrum of plectosphaeroic acid B.  Figure 6.18. CD spectrum of plectosphaeroic acid C. 173   Figure 6.19. CD spectrum of T988 A. 6.3. Proposed Biogenesis of Plectosphaeroic Acids A-C  Plectosphaeroic acid A appears to be derived from four molecules of tryptophan and one molecule of serine (figure 6.20).  One equivalent of tryptophan and one equivalent of serine can form regular peptide bonds to form the diketopiperazine core of plectosphaeroic acid A. This core diketopiperazine probably undergoes further modifications and dimerization to give an intermediate similar to 6.11.  A Grob fragmentation of 6.11 would give rise to the indole ring attached to C-10b in T988 B (6.5).  The cinnabarinic acid moiety of plectosphaeroic acid A is likely derived from the oxidative coupling of 3-hydroxyanthranilic acid (6.8), which is in turn derived from tryptophan through the kynurenine pathway (figure 4.1).  A possible biosynthesis of cinnabarinic acid, based on the proposed mechanism for the formation of cinnabarinic acid from 3-hydroxyanthranilic acid,4 is shown in figure 6.21.  Plectosphaeroic acid A could be synthesized from T988 B (6.5) and 3-hydroxyanthranilic acid (6.8), followed by oxidative coupling with another molecule of 3-hydroxyanthranilic acid in an analogous fashion to the proposed biosynthesis of cinnabarinic acid.  The biogenesis of plectosphaeroic acid B is likely very similar to that of plectosphaeroic acid A except that alanine rather than serine forms peptide bonds with tryptophan to give the diketopiperazine core.  Plectosphaeroic acid C is also likely biosynthesized in a similar fashion to plectosphaeroic acid A except that a trisulfide bridge is formed between C-3 and C-12. 174  N H NH2 OH O O HO NH2 OH HN NH O O OHHN N N O O N H OH H NNN O O O H H N N O O N HO HB HN N H NH2 OH O OH NH2 COOH O NH COOH H N N O O N HO HN COOH HO NH2 O NH COOH N N O O N HO HN COOH HO N COOH NH2 O S OH S S HO S S OH S S OH S S OH S N N O O N HO HN COOH S OH S O N O COOH NH2 6.1 peptide bond formation Grob fragmentation 6.11 6.5 H HHH 6.8 proton transfer tautomerization proton transfer oxidation proton transfer oxidation  Figure 6.20. Possible biogenesis of plectosphaeroic acid A (6.1). 175   Figure 6.21. Possible biosynthesis of cinnabarinic acid (6.7). 6.4. Biological Activity of Plectosphaeroic Acids A-C  Plectosphaeroic acids A-C and cinnabarinic acid were tested for IDO inhibitory activity in vitro.  Plectosphaeroic acids A and B showed similar potency against IDO, with IC50 values of 8.1 µM and 8.2 µM, respectively.  Plectosphaeroic acid C inhibited IDO with an IC50 = 12.8 µM. Cinnabarinic acid was even more potent with an IC50 = 390 nM. An enzymatic assay, performed by NewLink Genetics Corporation, gave similar results for plectosphaeroic acid A (IC50 = 5.7 µM), plectosphaeroic acid B (IC50 = 12 µM) and cinnabarinic acid (IC50 = 150 nM).  Plectosphaeroic acids A and B and cinnabarinic acid were also tested in a cell-based IDO assay by NewLink Genetics Corporation (see section 4.3.4). Plectosphaeroic acid A showed an IC50 = 13.8 µM in this assay, and an LD50 = 8.2 µM, while plectosphaeroic acid B showed an IC50 = 51 µM and an LD50 = 47 µM.  Since the LD50 is lower than the IC50 in each case, the loss of L-kynurenine production is probably due to cell-death and therefore plectosphaeroic acids A and B appear to be too toxic to be of therapeutic value. Cinnabarinic acid was not active (IC50 > 1000 µM) or toxic (IC50 > 1000 µM) in this cell-based 176  assay, suggesting that it may have problems crossing the cell membrane.  Plectosphaeroic acid C was not tested in this assay or the enzymatic assay due to having a limited amount of sample. The enzymatic and cell-based assay results are summarized in table 6.4. Table 6.4. IDO inhibitory activity of plectosphaeroic acids A-C and cinnabarinic acid. Compound Enzymatic IC50 (µM) a Enzymatic IC50 (µM) b Cell-based EC50 (µM) Cell-based LD50 (µM) Plectosphaeroic acid A 8.1 5.7 13.8 8.2 Plectosphaeroic acid B 8.2 12 51 47 Plectosphaeroic acid C 12.8 N.D. N.D. N.D. Cinnabarinic acid 0.39 0.15 >1000 >1000 a Assays performed by the Mauk lab b Assays performed by NewLink Genetics Corporation N.D. = Not Determined 6.5. Synthesis of Cinnabarinic Acid Analogues   Since cinnabarinic acid shows potent inhibition of IDO and represents a new pharmacophore for IDO inhibition, analogues of cinnabarinic acid were synthesized in order to determine structure-activity relationships.  Since the carboxylic acid groups of cinnabarinic acid might make cinnabarinic acid too polar to cross the cell membrane, the dimethyl ester of cinnabarinic acid (6.12) was synthesized according to scheme 6.3 and tested for IDO inhibitory activity.  Compound 6.12 has the potential to act as a prodrug, since the lower polarity of 6.12 might allow it to cross the cell membrane where it can be hydrolyzed to cinnabarinic acid.  The dimethyl ester (6.12) was less active than cinnabarinic acid in vitro, showing an IC50 ≈ 14-29 µM.  In the cell-based assay, 6.12 showed an IC50 = 83 µM and an LD50 = 188 µM.  While the therapeutic index is small, 6.12 is active against IDO at concentrations lower than that at which it shows toxicity.  Therefore, if analogues of 6.12 can be made that have a higher therapeutic index, these analogues have potential as prodrugs. 177   Scheme 6.3. Synthesis of 6.12.  Another analogue, 2-amino-3-phenoxazone (6.14), which lacks the carboxylic acids found in cinnabarinic acid, was synthesized from 2-aminophenol (6.15) according to scheme 6.4. This analogue showed only weak inhibition of IDO (IC50 ≈ 490 µM), showing that the carboxylic acids of cinnabarinic acid are important for activity.  Scheme 6.4. Synthesis of 6.14.  Finally, the natural product actinomycin D (1.15), which was isolated by a graduate student in the Andersen lab, Roberto Forestieri, also contains a moiety related to cinnabarinic acid.  Therefore, actinomycin D was tested for IDO inhibitory activity in vitro and showed an IC50 = 23 µM. 178  O N O NH2 O O N N H N N O O O O O O HN N N H N N O O O O O O NH 1.15 Figure 6.22. Structure of actinomycin D (1.15). 6.6. Conclusions  In conclusion, three new complex alkaloids, plectosphaeroic acids A-C, were isolated from a marine fungus Plectosphaerella cucumerina as inhibitors of IDO with IC50 values in the low micromolar range.  The analogue T988 A, lacking the cinnabarinic acid moiety, was completely inactive against IDO, while cinnabarinic acid itself was an even more potent IDO inhibitor than plectosphaeroic acids A-C.  Therefore, some portion of cinnabarinic acid represents a new pharmacophore for IDO inhibition.  Analogues of cinnabarinic acid were also synthesized, which showed that 6.12 or similar compounds may be useful prodrugs of cinnabarinic acid. 6.7. Experimental Section 6.7.1. General Experimental Procedures  SephadexTM LH-20 was used for LH-20 column chromatography.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µm filter (Osmonics Inc.) prior to use.  The absorbance was 179  monitored at 254 nm and 330 nm with a flow rate of 2.0 mL/min.  NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively.  The solvent used for NMR was DMSO-d6, and chemical shifts are referenced to the internal solvent peaks at δH 2.50 and δC 39.50.  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high- resolution spectra, respectively.  Optical rotations were recorded with a JASCO P-1010 polarimeter equipped with a halogen lamp (589 nm) and a 10 mm microcell.  CD spectra were recorded with a JASCO J-810 spectropolarimeter, using a 2 mm cuvette with MeOH as the solvent.  Reagents were purchased from Sigma-Aldrich®. 6.7.2. Isolation of Plectosphaeroic Acids A-C The producing organism was collected from marine sediment at -100 m depth in Barkley Sound, British Columbia and identified by 26S rDNA analysis as Plectosphaerella cucumerina. Production cultures were grown as lawns on solid ISP4 media for 14 days at room temperature. The solid agar culture was extracted with EtOAc, and the EtOAc was dried in vacuo to give the crude extract.  The crude extract was dissolved in MeOH and fractionated by LH-20 column chromatography (eluent: MeOH).  The active fractions were pooled and purified by reversed- phase HPLC (step gradient from 50% MeOH/H2O + 0.2% TFA to 70% MeOH/H2O + 0.2% TFA to 75% MeOH/H2O + 0.2% TFA) to give plectosphaeroic acids A (6.1) (3.2 mg), B (6.2) (2.8 mg), and C (6.3) (1.1 mg). Plectosphaeroic acid A (6.1): Isolated as an orange-red solid; UV (MeOH) λmax (log ε) 202 nm (4.22) 428 nm (3.62); [α]D 20 +96.9o (c 0.33, MeOH); 1H and 13C NMR data, see table 6.1; HRESI-MS(-) m/z 807.1534 (calculated for C39H31N6O10S2: 807.1543). 180  Plectosphaeroic acid B (6.2): Isolated as an orange-red solid; UV (MeOH) λmax (log ε) 203 nm (4.23) 430 nm (3.73); [α]D 20 +69.8o (c 0.27, MeOH); 1H and 13C NMR data, see table 6.2; HRESI-MS(-) m/z 791.1599 (calculated for C39H31N6O9S2: 791.1594). Plectosphaeroic acid C (6.3): Isolated as an orange-red solid; UV (MeOH) λmax (log ε) 200 nm (4.21) 435 nm (3.56); [α]D 20 +135.6o (c 0.17, MeOH); 1H and 13C NMR data, see table 6.3; HRESI-MS(-) m/z 809.0786 (calculated for C37H25N6O10S3: 809.0794). 6.7.3. Synthetic Procedures Synthesis of plectosphaeroic acid A dimethyl ester (6.6): To a solution of plectosphaeroic acid A (~ 1 mg) in DMF (5 mL) was added excess Na2CO3 (100 mg) and excess CH3I (0.1 mL, 1.61 mmol).  The reaction mixture was stirred overnight at room temperature before H2O (5 mL) was added and the mixture was extracted with CH2Cl2 (3 x 5 mL).  The organic layer was dried in vacuo, and purified by reversed-phase HPLC (eluent: 70% MeOH/H2O) to give plectosphaeroic acid A dimethyl ester (6.6). Synthesis of cinnabarinic acid (6.7): Cinnabarinic acid was synthesized from 3- hydroxyanthranilic acid (6.8) using a modified literature protocol.5  3-Hydroxyanthranilic acid (118.5 mg, 0.77 mmol) was dissolved in DMF (5 mL) and to this solution was added CuCl (10 mg, 0.10 mmol).  The reaction mixture was stirred for 3 days at room temperature and then dried in vacuo to give the crude product.  A portion of the crude product was dissolved in MeOH and purified by reversed-phase HPLC (50% MeOH/H2O + 0.2% TFA) to give pure cinnabarinic acid (12.0 mg, 10%). 181   Cinnabarinic acid (6.7): UV (MeOH) λmax (log ε) 231 nm (4.17) 429 nm (3.97); 1H NMR (600 MHz, DMSO-d6) δ 9.73 (1H, br. s, NH2), 8.82 (1H, br. s, NH2), 7.96 (1H, d, J = 7.7 Hz, H- 9), 7.78 (1H, d, J = 8.3 Hz, H-7), 7.61 (1H, t, J = 8.0 Hz, H-8), 6.62 (1H, s, H-4); 13C NMR (150 MHz, DMSO-d6) δ 178.1 (C, C-3), 169.1 (C, C-13), 166.3 (C, C-14), 152.5 (C, C-2), 150.5 (C, C-5), 147.6 (C, C-12), 142.5 (C, C-6), 129.1 (C, C-11), 128.8 (CH, C-8), 127.9 (CH, C-9), 126.2 (C, C-10), 120.2 (CH, C-7), 104.9 (CH, C-4), 92.7 (C, C-1); HRESI-MS(-) m/z 299.0301 (calculated for C14H7N2O6: 299.0304). Synthesis of 6.12: 3-hydroxyanthranilic acid (6.8) (85.3 mg, 0.56 mmol) was dissolved in DMF (5 mL) and to this solution was added Na2CO3 (200 mg, 5.0 mmol) and CH3I (0.2 mL, 3.22 mmol).  The reaction mixture was allowed to stir overnight at room temperature before H2O (5 mL) was added and the mixture was extracted with CH2Cl2 (3 x 5 mL).  The organic layer was dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 25% EtOAc/hexane) to give 6.13 (73.6 mg, 79%).  Compound 6.13 (32.0 mg) was dissolved in CH2Cl2 (5 mL), and to this solution was added CuCl (10 mg, 0.10 mmol).  The reaction mixture was stirred for 3 days at room temperature and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 50% EtOAc/hexane) and reversed-phase HPLC (eluent: 60% MeOH/H2O) to give 6.12 (3.0 mg, 10%). 182   Compound 6.12: 1H NMR (400 MHz, DMSO-d6) δ 7.66 (1H, dd, J = 8.1, 1.4 Hz, H-7), 7.58 (1H, dd, J = 7.4, 1.4 Hz, H-9), 7.53 (1H, t, J = 7.8 Hz, H-8), 6.46 (1H, s, H-4), 3.86 (3H, s, H-16), 3.83 (3H, s, H-15); 13C NMR (100 MHz, DMSO-d6) δ 179.0 (C, C-3), 167.3 (C, C-13), 166.8 (C, C-14), 148.3 (C, C-2), 146.7 (C, C-5), 142.5 (C, C-12), 141.6 (C, C-6), 130.7 (C, C- 11), 130.7 (C, C-10), 128.5 (CH, C-8), 124.8 (CH, C-9), 118.4 (CH, C-7), 103.2 (CH, C-4), 102.4 (C, C-1), 52.2 (CH3, C-16), 51.9 (CH3, C-15). Synthesis of 2-amino-3-phenoxazone (6.14): 2-aminophenol (6.15) (103.0 mg, 0.94 mmol) was dissolved in DMF (5 mL) and to this solution was added CuCl (31 mg, 0.31 mmol). The reaction mixture was stirred for 2 days at room temperature, dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 40% EtOAc/hexane) to give 6.14 (72.2 mg, 72%)  2-Amino-3-phenoxazone (6.14): 1H NMR (600 MHz, DMSO-d6) δ 7.69 (1H, dd, J = 7.9, 1.2 Hz, H-10), 7.49 (1H, dd, J = 8.1, 1.0 Hz, H-7), 7.45 (1H, td, J = 7.6, 1.3 Hz, H-8), 7.38 (1H, td, J = 7.5, 1.3 Hz, H-9), 6.35 (1H, s, H-1), 6.35 (1H, s, H-4); 13C NMR (150 MHz, DMSO-d6) δ 180.2 (C, C-3), 148.9 (C, C-2), 148.2 (C, C-5), 147.4 (C, C-12), 141.9 (C, C-6), 133.7 (C, C-11), 128.8 (CH, C-8), 128.0 (CH, C-10), 125.3 (CH, C-9), 115.9 (CH, C-7), 103.4 (CH, C-4), 98.4 (CH, C-1); HRESI-MS(+) m/z 213.0670 (calculated for C12H9N2O2: 213.0664). 183  6.7.4. 2D *MR Spectra of Plectosphaeroic Acid A  Figure 6.23. COSY spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz.  Figure 6.24. HSQC spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz. 184   Figure 6.25. HMBC spectrum of plectosphaeroic acid A recorded in DMSO-d6 at 600 MHz.  Figure 6.26. Expanded HMBC spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). 185   Figure 6.27. ROESY spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz.  Figure 6.28. Expanded ROESY spectrum of plectosphaeroic acid A (DMSO-d6, 600 MHz). 186   Figure 6.29. 1H-15N HSQC spectrum of plectosphaeroic acid A in DMSO-d6 at 600 MHz. 6.7.5. 2D *MR Spectra of Plectosphaeroic Acid B.  Figure 6.30. COSY spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz. 187   Figure 6.31. HSQC spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz.  Figure 6.32. HMBC spectrum of plectosphaeroic acid B recorded in DMSO-d6 at 600 MHz. 188   Figure 6.33. Expanded HMBC spectrum of plectosphaeroic acid B (DMSO-d6, 600 MHz).  Figure 6.34. ROESY spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. 189   Figure 6.35. Expanded ROESY spectrum of plectosphaeroic acid B (DMSO-d6, 600 MHz).  Figure 6.36. 1H-15N HSQC spectrum of plectosphaeroic acid B in DMSO-d6 at 600 MHz. 190  6.7.6. 2D *MR Spectra of Plectosphaeroic Acid C  Figure 6.37. COSY spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz.  Figure 6.38. HSQC spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz. 191   Figure 6.39. HMBC spectrum of plectosphaeroic acid C recorded in DMSO-d6 at 600 MHz.  Figure 6.40. Expanded HMBC spectrum of plectosphaeroic acid C (DMSO-d6, 600 MHz). 192  6.8. References (1) Feng, Y.; Blunt, J. W.; Cole, A. L. J.; Munro, M. H. G. J. at. Prod. 2004, 67, 2090-2092. (2) Takahashi, C.; Numata, A.; Ito, Y.; Matsumura, E.; Araki, H.; Iwaki, H.; Kushida, K. J. Chem. Soc. Perkin Trans. I 1994, 1859-1864. (3) Takahashi, C.; Minoura, K.; Yamada, T.; Numata, A.; Kushida, K.; Shingu, T.; Hagishita, S.; Nakai, H.; Sato, T.; Harada, H. Tetrahedron 1995, 51, 3483-3498. (4) Christen, S.; Southwell-Keely, P. T.; Stocker, R. Biochemistry 1992, 31, 8090-8097. (5) Horváth, T.; Kaizer, J.; Speier, G. J. Mol. Catal. A: Chem. 2004, 215, 9-15. 193  7. Synthesis of Turnagainolides A and B 7.1. Phosphatidylinositol-3,4,5-Triphosphate and SHIP Phosphatidylinositol is a membrane-bound phospholipid which may be phosphorylated at the 3, 4 or 5-position.  The phosphorylated forms of phosphatidylinositol are known as phosphoinositides and play important roles in regulating many cellular processes. Phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3) is a particularly important signalling molecule that regulates cell survival, proliferation and differentiation.  PI-3,4,5-P3 is synthesized by the phosphorylation of phosphatidylinositol-4,5-biphosphate (PI-4,5-P2), which is catalyzed by phosphoinositide 3-kinase (PI3K).1   This PI3K pathway is activated in response to various stimuli.  In the reverse reaction, the phosphate at the 3-position of PI-3,4,5-P3 is removed by the tumour suppressor PTEN (phosphatase and tensin homolog) to generate PI-4,5-P2.  Alternatively, PI-3,4,5-P3 may be dephosphorylated at the 5-position by the Src homology 2-containing inositol 5-phosphatase (SHIP) family of phosphatases, consisting of SHIP1, SHIP2 and sSHIP.  Thus, the level of PI-3,4,5-P3 is tightly regulated by the activity of these enzymes, as shown in figure 7.1. SHIP2 is expressed in many different cell types, while sSHIP is found primarily in stem cells and SHIP1 is found primarily in hematopoietic and immune cells.2 6 A version of this chapter will be submitted for publication. Li, D.; Carr, G.; Zhang, M.; Williams, D. E.; Amlani, A.; Bottriell, H.; Mui, A. L.-F.; Andersen, R. J. Turnagainolides A and B, SHIP Activating Cyclic Depsipeptides: Isolation, Structure Elucidation, and Synthesis. 194   Figure 7.1. The PI3K pathway. 7.1.1. SHIP1 and Regulation of the Cell Cycle When the PI3K pathway is activated, the PI-3,4,5-P3 that is produced recruits phosphatidylinositol-dependent kinase 1 (PDK1) and protein kinase B (PKB, also known as Akt) to the cell membrane.  PDK1 then phosphorylates and activates PKB, which promotes cell survival and proliferation and protects against apoptosis.  Thus, PI3K is a positive regulator of cell growth and survival, while PTEN is a negative regulator.  Alterations that favour PI3K activity over PTEN activity are common in cancer.3  Mutations in PTEN are associated with predisposition to cancer,4 and are the second most common type of mutation in cancer after mutations in p53.5  Likewise, overactivation of PKB, which occurs via the PI3K pathway, is found in many breast and lung tumour cells.6  By dephosphorylating PI-3,4,5-P3, which results in 195  a downstream deactivation of PKB, SHIP1 is also a negative regulator of cell survival and proliferation.7  Lowered SHIP1 activity, and the resulting increase in PI-3,4,5-P3 levels, has been implicated in leukemia.8  Indeed, SHIP1 expression was found to be lower or absent in patients with leukemia,9 and a mutation in SHIP1 was found in a patient with leukemia.10  SHIP1’s role as a negative regulator of osteoclast survival was demonstrated by the observation that SHIP1 knockout (SHIP1 -/-) mice are severely osteoporotic due to a large number of hyper-resorptive osteoclasts.11  SHIP1 -/- mice also have a shortened lifespan due to enhanced survival of myeloid progenitors, leading to myeloproliferative disorder and infiltration of the lungs by these myeloid cells.12 7.1.2. SHIP1 and the Immune System In response to stimuli from the immune system, PI3K is localized to the cellular membrane and activated.  The resulting increase in PI-3,4,5-P3 levels sets off a signal transduction pathway leading to Ca2+ influx and activation of the immune system.13,14  By dephosphorylating PI-3,4,5-P3, SHIP1 is a negative regulator of the immune system and therefore important for many diseases in which the immune system is overactive, such as inflammation, arthritis, asthma, autoimmune diseases and septic shock.  Not surprisingly, mice lacking SHIP1 have an overactive immune system.12,15,16  Similarly, mice heterozygous for PTEN, which also hydrolyzes PI-3,4,5-P3, show disorders of the immune system. 17  Mice heterozygous for both SHIP1 and PTEN show even more severe immune disorders, suggesting that elevated levels of PI-3,4,5-P3 play a role in diseases where the immune system is overactive.17  SHIP1 has also been found to inhibit mast cell degranulation15 and histamine release.18 196  Rheumatoid arthritis is a disease caused by inflammation of the joints.  PI-3,4,5-P3 is believed to play a role in rheumatoid arthritis, and PI3K inhibitors have been shown to suppress the inflammation associated with rheumatoid arthritis.19  Asthma is another inflammatory disorder characterized by inflammation of the lungs.  Mice lacking SHIP1 show an increase in cytokines, chemokines and histamine in the lungs, and the majority of them showed inflammation of the lungs characteristic of asthma.20  Likewise, when asthma was induced in mice, PI3K activity increased and PTEN activity decreased.21  Treating these mice with specific PI3K inhibitors lessened the inflammation, suggesting that the PI3K pathway is responsible for this inflammation.21  The PI3K pathway has also been implicated in lupus erythematosus, and inhibitors of PI3K may be useful in treating this disease.22 The immune system must find a balance between removing legitimate threats to the organism while not overreacting and attacking non-threats.  When the immune system overreacts to external threats, septic shock can occur and is often fatal.  In a normal immune system, exposure to endotoxin from a bacterial infection results in tolerance to subsequent exposures of endotoxin.  This phenomenon is known as endotoxin tolerance, and is important for protecting the host from the immune system in the case of persistent infection.  SHIP1 is upregulated in response to endotoxin exposure resulting in endotoxin tolerance, while bone marrow lacking SHIP1 does not show such tolerance.23  When SHIP1 -/- and wild type (SHIP1 +/+) mice were exposed to endotoxin, all of the SHIP1 -/- mice died while the SHIP1 +/+ mice did not.23  While increased SHIP1 activity may result in a higher chance of infection, it can protect against the septic shock that is often more serious than the infection itself. 197  7.1.3. The PI3K Pathway as a Drug Target Since elevated levels of PI-3,4,5-P3 are implicated in many diseases, lowering the level of PI-3,4,5-P3 has become an import therapeutic strategy.  Inhibiting PI3K is one way to limit the levels of PI-3,4,5-P3, and drugs have been developed that are specific for this kinase.   However, PI3K inhibitors are expected to have serious side effects.6  An alternative method to reduce PI- 3,4,5-P3 levels is to increase the activity of the SHIP family of phosphatases.  This strategy is particularly appealing in the case of SHIP1, since its localization in hematopoietic and immune cells should minimize side effects in other cell types.  Activating SHIP1 with small molecules represents a relatively new therapeutic strategy.  The first compound identified as a SHIP1 activator is the natural product pelorol (7.1), which was isolated from the marine sponge Dactylospongia elegans by the Andersen lab.24  A total synthesis of pelorol was completed by the Andersen lab,24 and pelorol has also inspired the synthesis of new SHIP1-activating analogues.24,25  The most promising of these analogues is AQX-MN100 (7.2), which shows good anti-inflammatory activity in vivo.25  Figure 7.2. Structures of pelorol (7.1) and AQX-MN100 (7.2). 7.2. Isolation of Turnagainolides A and B Another marine extract that showed promising SHIP1-activating activity is from a marine-derived bacterium, Bacillus sp., collected near Turnagain Island, British Columbia. Bioassay-guided fractionation of an EtOAc extract of Bacillus sp. led to the isolation of two new 198  peptides, turnagainolides A (7.3) and B (7.4), by Dehai Li, a former postdoctoral researcher in the Andersen lab.  Both peptides were subsequently isolated from another extract of Bacillus sp. as part of my investigation of bacterial extracts that showed IDO inhibitory activity, however neither compound was active in the IDO inhibition assay. Turnagainolides A and B both contain one isoleucine, one alanine and two valine residues, as well as a novel fragment of polyketide/shikimate origin, and are cyclized to form a depsipeptide (figure 7.3).  The polyketide/shikimate fragment is probably derived from an equivalent of acetate, which is of polyketide origin, and trans-cinnamaldehyde.  trans- Cinnamaldehyde is known to be derived from phenylalanine, which is biosynthesized by the shikimate pathway.26  Figure 7.3. Structures of turnagainolides A (7.3) and B (7.4). The constitutions of turnagainolides A and B, determined by analysis of the 1D and 2D NMR data, were found to be identical (tables 7.1 and 7.2).  Thus, turnagainolides A and B must differ only in stereochemistry. 199  Table 7.1. NMR data for turnagainolide A. position δC δH (J  in Hz) COSY HMBC 1 168.7 2a 40.1 2.88, m 2b, 3 C-1, C-3, C-4 2b 2.41, m 2a, 3 C-1 3 73.0 5.50, m 2a, 2b, 4 C-1, C-4, C-5, Val 1 -C=O 4 126.7 6.28, dd (16.0, 7.2) 3, 5 C-2, C-3, C-6 5 132.5 6.68, d (16.0) 4 C-3, C-4, C-6, C-7/11 6 135.7 7/11 126.5 7.45, d (7.5) 8/10 C-5, C-7/11, C-9 8/10 128.7 7.35, t (7.6) 7/11, 9 C-6, C-7/11, C-8/10 9 128.4 7.28, t (7.3) 8/10 C-7/11 Val 1 C=O 168.7 α 58.3 4.25, m Val 1 -Hβ, Val 1 -NH Val 1 -C=O, Val 1 -Cβ, Val 1 -CγA, Val 1 -CγB, Ile 2 -C=O β 28.5 2.25, m Val 1 -Hα, Val 1 -HγA, Val 1 -HγB Val 1 -Cα, Val 1 -CγA, Val 1 -CγB γACH3 19.6 0.89, m Val 1 -Hβ Val 1 -Cα, Val 1 -Cβ, Val 1 -CγB γBCH3 18.9 0.89, m Val 1 -Hβ Val 1 -Cα, Val 1 -Cβ, Val 1 -CγA NH 7.59, d (9.7) Val 1 -Hα Ile 2 -C=O, Val 1 -Cα, Val 1 -Cβ Ile 2 C=O 170.3 α 57.2 4.25, m Ile 2 -Hβ, Ile 2 -NH Ala 3 -C=O, Ile 2 -C=O, Ile 2 -Cβ, Ile 2 -Cγ, Ile 2 -CβCH3 β 35.6 2.05, m Ile 2 -Hα, Ile 2 -HγA/B, Ile 2 -HβCH3 Ile 2 -Cγ, Ile 2 -δCH3 γA 23.5 1.25, m Ile 2 -Hβ, Ile 2 -HδCH3 Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -CδCH3, Ile 2 -CβCH3 γB 1.25, m Ile 2 -Hβ, Ile 2 -HδCH3 Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -CδCH3, Ile 2 -CβCH3 δCH3 11.9 0.81, m Ile 2 -HγA/B Ile 2 -Cβ, Ile 2 -Cγ βCH3 15.5 0.81, m Ile 2 -Hβ Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -Cγ NH 8.10, d (9.7) Ile 2 -Hα Ala 3 -C=O, Ile 2 -Cα, Ile 2 -Cβ Ala 3 C=O 173.0 α 48.8 4.32, quin (6.6) Ala 3 -Hβ, Ala 3 -NH Val 4 -C=O, Ala 3 -C=O, Ala 3 -Cβ β 16.3 1.18, d (6.6) Ala 3 -Hα Ala 3 -C=O, Ala 3 -Cα NH 8.56, d (5.8) Ala 3 -Hα Val 4 -C=O, Ala 3 -Cα, Ala 3 -Cβ Val 4 C=O 172.3 α 57.5 4.14, t (7.6) Val 4 -Hβ, Val 4 -NH C-1, Val 4 -C=O, Val 4 -Cβ, Val 4 -CγA, Val 4 -CγB β 29.9 1.96, dq (13.5, 6.7) Val 4 -Hα, Val 4 -HγA, Val 4 -HγB Val 4 -C=O, Val 4 -Cα, Val 4 -CγA, Val 4 -CγB γACH3 19.4 0.88, m Val 4 -Hβ Val 4 -Cα, Val 4 -Cβ, Val 4 -CγB γBCH3 18.2 0.88, m Val 4 -Hβ Val 4 -Cα, Val 4 -Cβ, Val 4 -CγA NH 7.74, d (8.6) Val 4 -Hα C-1, Val 4 -C=O, Val 4 -Cα, Val 4 -Cβ a Spectra recorded on synthetic turnagainolide A in DMSO-d 6  at 600 MHz Turnagainolide A (7.3) a  200  Table 7.2. NMR data for turnagainolide B. position δC δH (J  in Hz) COSY HMBC 1 167.7 2a 41.2 2.79, dd (13.7, 4.6) 2b, 3 C-1, C-3, C-4 2b 2.31, dd (13.7, 3.7) 2a, 3 C-1 3 71.0 5.64, q (4.4) 2a, 2b, 4 C-1, C-4, C-5, Val 1 -C=O 4 126.5 6.20, dd (16.3, 5.8) 3, 5 C-3, C-5, C-6 5 129.3 6.32, d (16.0) 4 C-3, C-4, C-6, C-7/11 6 136.4 7/11 126.6 7.48, d (7.5) 8/10 C-5, C-7/11, C-9 8/10 128.3 7.29, t (7.7) 7/11, 9 C-6, C-7/11, C-8/10 9 127.5 7.21, t (7.3) 8/10 C-7/11 Val 1 C=O 169.0 α 59.6 4.05, t (9.3) Val 1 -Hβ, Val 1 -NH Val 1 -C=O, Val 1 -Cβ, Val 1 -CγA, Val 1 -CγB, Ile 2 -C=O β 29.0 2.36, m Val 1 -Hα, Val 1 -HγA, Val 1 -HγB Val 1 -Cα, Val 1 -CγA, Val 1 -CγB γACH3 19.5 0.99, d (6.6) Val 1 -Hβ Val 1 -Cα, Val 1 -Cβ, Val 1 -CγB γBCH3 19.4 0.94, d (6.9) Val 1 -Hβ Val 1 -Cα, Val 1 -Cβ, Val 1 -CγA NH 7.85, d (9.1) Val 1 -Hα Ile 2 -C=O, Val 1 -Cα, Val 1 -Cβ Ile 2 C=O 170.8 α 57.5 4.22, dd (9.0, 3.7) Ile 2 -Hβ, Ile 2 -NH Ala 3 -C=O, Ile 2 -C=O, Ile 2 -Cβ, Ile 2 -Cγ, Ile 2 -CβCH3 β 35.4 2.04, m Ile 2 -Hα, Ile 2 -HγA/B, Ile 2 -HβCH3 γA 23.5 1.29, m Ile 2 -Hβ, Ile 2 -HδCH3 Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -CδCH3, Ile 2 -CβCH3 γB 1.29, m Ile 2 -Hβ, Ile 2 -HδCH3 Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -CδCH3, Ile 2 -CβCH3 δCH3 12.1 0.82, t (7.5) Ile 2 -HγA/B Ile 2 -Cβ, Ile 2 -Cγ βCH3 15.6 0.85, d (6.4) Ile 2 -Hβ Ile 2 -Cα, Ile 2 -Cβ, Ile 2 -Cγ NH 8.22, d (8.9) Ile 2 -Hα Ala 3 -C=O, Ile 2 -Cα Ala 3 C=O 173.7 α 49.3 4.36, m Ala 3 -Hβ, Ala 3 -NH Ala 3 -C=O, Ala 3 -Cβ β 16.1 1.23, d (7.2) Ala 3 -Hα Ala 3 -C=O, Ala 3 -Cα NH 8.86, d (5.0) Ala 3 -Hα Val 4 -C=O, Ala 3 -Cα, Ala 3 -Cβ Val 4 C=O 172.6 α 57.0 4.19, t (8.3) Val 4 -Hβ, Val 4 -NH C-1, Val 4 -C=O, Val 4 -Cβ, Val 4 -CγA, Val 4 -CγB β 30.6 1.79, sextet (6.9) Val 4 -Hα, Val 4 -HγA, Val 4 -HγB Val 4 -C=O, Val 4 -Cα, Val 4 -CγA, Val 4 -CγB γACH3 19.1 0.86, d (5.8) Val 4 -Hβ Val 4 -Cα, Val 4 -Cβ, Val 4 -CγB γBCH3 18.8 0.88, d (6.9) Val 4 -Hβ Val 4 -Cα, Val 4 -Cβ, Val 4 -CγA NH 7.08, d (9.1) Val 4 -Hα C-1, Val 4 -C=O, Val 4 -Cα a Spectra recorded on natural turnagainolide B in DMSO-d 6  at 600 MHz Turnagainolide B (7.4) a  The absolute configurations of the amino acid residues in turnagainolides A and B were determined by Dehai Li using Marfey’s method.27  The isoleucine and valine residues were found to be the L-amino acids, while the alanine residue was found to be the D-amino acid, in both turnagainolides A and B.  The absolute configuration of C-3 in turnagainolide A was 201  determined by Dehai Li using Mosher’s method on the methyl ester (7.5), which was formed by the methanolysis of turnagainolide A (7.3) (scheme 7.1).  The absolute configuration of C-3 in turnagainolide A was determined to be R, thus completing the structure of turnagainolide A.  Scheme 7.1. Mosher’s analysis of turnagainolide A (7.3). Since turnagainolide B had the same amino acid configurations as turnagainolide A, it was assumed that turnagainolide B must have the opposite S configuration at C-3.  However, a limited amount of this compound precluded a rigorous analysis of this hypothesis. 7.2.1. Biological Activity of Turnagainolides A and B  Turnagainolides A and B were both found to be activators of SHIP1 in vitro, while the methyl ester of turnagainolide A (7.5) was not active in this assay.  Turnagainolides A and B both showed good activity between ≈ 10-100 µM, and were more potent than the lead compound AQX-MN100. 202  7.3. Synthesis of Turnagainolides A and B 7.3.1. Retrosynthetic Analysis of Turnagainolides A and B In order to confirm the structures of turnagainolides A and B, as well as provide additional material for biological testing, a synthesis of turnagainolides A and B was undertaken. Looking at the structures of turnagainolides A and B, the peptide portion of the structures can be synthesized using standard solid-phase peptide synthesis.  The polyketide/shikimate fragment can be envisioned as the product of an aldol reaction between commercially available trans- cinnamaldehyde (7.8) and an equivalent of acetate.  This equivalent of acetate can come from ethyl acetate (7.9), which has an ethyl ester protecting group.  Such a reaction was expected to produce a racemic mixture of both possible stereoisomers (7.10).  However, it was known that turnagainolides A and B, which were believed to differ only at the configuration of this stereocenter (C-3), could easily be separated by silica gel chromatography.  Therefore, the synthesis proceeded using this racemic mixture, and separation was left until the end when the two products could be separated as a diastereomeric mixture of turnagainolides A and B.  This method also has the advantage of providing a route to both turnagainolides A and B.  Figure 7.4. Retrosynthetic analysis of polyketide/shikimate fragment (7.10). 7.3.2. Synthesis of Polyketide/Shikimate Fragment  The synthesis of the silyl ether-protected β-hydroxy acid (7.12) was accomplished according to scheme 7.2.  A silyl ether protecting group for the alcohol was chosen for its compatibility with the acid-sensitive solid-phase 2-chlorotrityl resin.  The β-hydroxy ester (7.10) 203  was produced by an aldol reaction between ethyl acetate (7.9) and trans-cinnamaldehyde (7.8). The hydroxyl group was then protected with TBDMS-Cl to give 7.11.  Finally, the ethyl ester in 7.11 was hydrolyzed with aq. NaOH to give 7.12. H OO O 1. LDA, THF, -78oC 2. OH O O 96 % yield 7.8 7.9 7.10 TBDMS-Cl imidazole THF 78% O O O 7.11 Si1. NaOH, H2O 2. HCl workup O OH O 7.12 Si 70% -78 oC, 45 min reflux, 12 h MeOH, THF r.t., 3 h  Scheme 7.2. Synthesis of 7.12. 7.3.3. Solid-Phase Synthesis of the Linear Peptide  The solid-phase synthesis of turnagainolides A and B utilized Fmoc protecting groups for the amino acids, and 2-chlorotrityl chloride resin (7.13) as the solid-phase resin.  This strategy has advantages over the classic Boc/Merrifield strategy.28  Firstly, the latter strategy uses acid for both the deprotection and final cleavage steps, and although the deprotection steps are milder than the cleavage step, some cleavage from the solid-phase resin can occur at each deprotection step leading to lower yields.  Secondly, cleavage from the solid-phase Merrifield resin requires concentrated hydrofluoric acid, which is corrosive and difficult to handle.  In contrast, the former strategy uses mild base for each deprotection step and mild acid for the final cleavage step.  Therefore, no solid-phase cleavage takes place at each deprotection step, and the final cleavage uses much milder acidic conditions. 204   While many different peptide coupling agents are known, one of the most popular reagents is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). This reagent was chosen as it is well established to give good results.  Hydroxybenzotriazole (HOBt) was used to prevent epimerization and side-reactions.29  Using these reagents, the solid- phase synthesis of the linear peptide (7.14) was carried out using standard techniques,28 and the final product was cleaved from the solid-phase resin using TFA, with 1,3-propanedithiol (PDT) as a cation scavenger, as shown in scheme 7.3.  Synthesis of the appropriate Fmoc-protected amino acids was accomplished according to literature protocol.30 205  Cl Cl Fmoc-L-Val-OH CH2Cl2 DIPEA O Cl O NHFmoc O Cl O NH2 20 % piperidine DMF 1. Fmoc-D-Ala-OH PyBOP HOBt DIPEA 2. 20 % piperidine DMF O Cl O N H O NH2O Cl O N H O H N O NH2 1. Fmoc-L-Ile-OH PyBOP HOBt DIPEA 2. 20 % piperidine DMF 1. Fmoc-L-Val-OH PyBOP HOBt DIPEA 2. 20 % piperidine DMF O Cl O N H O H N O N H O NH2 O Cl O N H O H N O N H O H N O OH 1. 7.12, PyBOP, HOBt, DMF 2. TBAF, THF TFA, PDT, CH2Cl2 N H O H N O N H O H N O OH 7.13 7.15 7.16 7.17 7.18 7.19 7.20 7.14 HO O  Scheme 7.3. Synthesis of linear peptide 7.14. 206  7.3.4. Macrolactonization  Many different macrolactonization methods have been employed in the synthesis of macrolactone natural products.31  One such method that has been reported to give good results is the Boden-Keck cyclization.32  Therefore, the seco-peptide (7.14) (98.5 mg) was dissolved in DMF and added slowly to a refluxing solution of DIC, DMAP and pTSA monohydrate in refluxing chloroform, according to scheme 7.4.  The reaction mixture was then dried and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 5% MeOH in CH2Cl2) and silica gel chromatography (eluent: gradient from CH2Cl2 to 50% acetone in CH2Cl2) to give turnagainolide B (7.4) as the major product (31.5 mg, 66%) and turnagainolide A (7.3) as the minor product (14.0 mg, 29%) (48% combined yield).  Turnagainolides A (7.3) and B (7.4) were further purified separately by reversed-phase HPLC (eluent: 60% MeOH/H2O). N H O H N O N H O H N O OH N H HN N H NH O O O O O O N H HN N H NH O O O O O O DMAP, pTSA monohydrate DIC, CHCl3 7.14 7.3 7.4 48% combined yield HO O  Scheme 7.4. Macrolactonization. 207  7.4. Comparison of the Synthetic Materials to the *atural Products  Comparison of synthetic and natural turnagainolides A and B was done primarily by NMR spectroscopy.  A comparison of the 1H and 13C NMR chemical shift values (figures 7.5 to 7.6), as well as the 2D NMR data, showed that the NMR data for synthetic turnagainolide A was virtually identical to that of the natural product, confirming the structure of turnagainolide A. Likewise, comparison of 1H and 13C NMR chemical shift values (figures 7.7 to 7.8), as well as the 2D NMR data, showed that the NMR data for synthetic turnagainolide B was virtually identical to that of the natural product, confirming the structure of turnagainolide B.  Since the synthesis used commercial D-alanine, L-valine and L-isoleucine, the synthesis confirmed the absolute configurations of the amino acids in turnagainolides A and B.  The synthesis also confirmed that turnagainolides A and B differ only at the configuration of C-3, since the synthesis produced a mixture of stereoisomers at this position.  Figure 7.5. Comparison of 1H NMR spectra of natural and synthetic turnagainolide A. 208   Figure 7.6. Comparison of 13C NMR spectra of natural and synthetic turnagainolide A.  Figure 7.7. Comparison of 1H NMR spectra of natural and synthetic turnagainolide B. 209   Figure 7.8. Comparison of 13C NMR spectra of natural and synthetic turnagainolide B. 7.5. Absolute Configuration of Turnagainolide B  Since turnagainolide A was found to have an R configuration at C-3, it was assumed that turnagainolide B must have the opposite S configuration at this stereocenter.  In order to confirm this hypothesis, synthetic turnagainolide B was analyzed by the same procedure as for turnagainolide A.  Methanolysis of synthetic turnagainolide B gave the methyl ester 7.21, which was subjected to Mosher’s analysis (scheme 7.5). 210   Scheme 7.5. Methanolysis and Mosher’s analysis of turnagainolide B (7.4).  Comparison of the 1H NMR chemical shifts of the Mosher’s esters 7.22 and 7.23 showed that H-2a and H-2b were shielded in 7.22 relative to 7.23, while H-4 and H-5 were shielded in 7.23 relative to 7.22 (figure 7.9).  Assuming the configuration shown in figure 7.10, the phenyl ring must be on the same side as H-2a and H-2b in 7.22, while the phenyl ring must be on the same side as H-4 and H-5 in 7.23.  Therefore, based on this analysis, turnagainolide B has an S configuration at C-3 as predicted. 211   Figure 7.9. Selected 1H NMR chemical shift values and Δδ values of 7.22 and 7.23.  Figure 7.10. Mosher’s analysis of turnagainolide B. 7.6. Synthesis of the Peptide Analogue 7.24  While turnagainolides A and B both showed good SHIP1 activating activity in vitro, neither one was active in a cell-based assay.  One possibility for this lack of activity is that the labile ester functionality could be hydrolyzed in vivo to give the corresponding linear peptide (7.14), which was found to be inactive in the in vitro assay.  In an attempt to synthesize an analogue of turnagainolides A and B that is active in vivo, the peptide analogue 7.24 was synthesized. 212  N H HN N H NH H N O O O O O 7.24 11 6 3 1 Val1 Ile2 Ala3 Val4  Figure 7.11. Structure of peptide analogue (7.24). The hydroxyl group of 7.10 was converted to an amine using a Mitsunobu reaction followed by reduction of the azide 7.25 to give the amine 7.26 (scheme 7.6).33  The ethyl ester of 7.26 was hydrolyzed with aq. NaOH to give crude 7.27, which was protected with an Fmoc group to give the protected β-amino acid 7.28 (scheme 7.6). OH O O 7.10 N3 O O 7.25 PPh3, DIAD DPPA, CH2Cl2 r.t., o/n 84% NH2 O O 7.26 PPh3 H2O, THF 70 oC, o/n 55% NH2 OH O 7.27 NH OH O 7.28 O O 1. NaOH, H2O MeOH, THF r.t., 3 h 2. HCl workup 1. Fmoc-Cl, Na2CO3 H2O, dioxane r.t., 1 h 2. HCl workup 53% over two steps  Scheme 7.6. Synthesis of 7.28. 213  Compound 7.28 was used for the solid-phase synthesis of the linear peptide analogue 7.29, in an analogous fashion to the synthesis of 7.14.  The linear peptide 7.29 was cyclized with PyBOP and DMAP in CH2Cl2 to give the peptide analogue 7.24, according to scheme 7.7. While the macrolactonization of 7.14 gave both turnagainolides A and B, macrolactamization appeared to give only one product, 7.24.  The configuration at C-3 of this compound could not be determined using ROESY, and the poor solubility and lack of biological activity observed for 7.24 suggested that it was not worth pursuing further.  N H O H N O N H O H N O NH2 HO O N H HN N H NH H N O O O O O PyBOP, DMAP CH2Cl2 7.29 7.24 r.t., 2 d  Scheme 7.7. Synthesis of peptide analogue 7.24. 7.7. Conclusions In conclusion, two new depsipeptides, turnagainolides A and B, have been isolated from Bacillus sp.  Both compounds are activators of SHIP1 in vitro, and are more potent than AQX- MN100.  The polyketide/shikimate fragments of turnagainolides A and B were synthesized by an 214  aldol reaction between EtOAc and trans-cinnamaldehyde, while the peptide fragments of turnagainolides A and B were synthesized by solid-phase peptide synthesis.  This route provides access to both turnagainolides A and B, which can easily be separated by silica gel chromatography.  Comparison of synthetic turnagainolides A and B to the natural products by NMR spectroscopy showed that they have virtually identical chemical shifts, and confirmed the structures of the natural products, including the absolute configurations of the amino acids. 7.8. Experimental Section 7.8.1. General Experimental Procedures  Waters 10 g silica Sep-Paks® and silica gel (SiliCycle® Inc., 230-400 mesh) were used for silica gel flash chromatography and silica gel chromatography, respectively.  Waters 10 g C18 Sep-Paks® were used for reversed-phase flash chromatography.  TLC was performed using Merck Kieselgel 60 F254 (for normal-phase) and Whatman MKC18F 60 A (for reversed-phase) TLC plates.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC.  All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µM filter (Osmonics, Inc.) prior to use. NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively.  Solvents used for NMR were CD2Cl2, acetone-d6 or DMSO-d6 and chemical shifts are referenced to the internal solvent peaks at δH 5.32 and δC 53.8 (for CD2Cl2), δH 2.05 and δC 29.92 (for acetone-d6) and δH 2.50 and δC 39.5 (for DMSO-d6).  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low-resolution and high-resolution spectra, respectively.  Reagents were purchased from Sigma-Aldrich®. 215  7.8.2. Isolation of Turnagainolides A and B  The crude EtOAc extract of Bacillus sp. was subjected to LH-20 column chromatography (eluent: MeOH).  The fractions that eluted early from the column were pooled and subjected to silica gel flash chromatography (eluent: gradient from CH2Cl2 to EtOAc to MeOH).  The fractions eluting with EtOAc showed a UV-active TLC spot, and these fractions were pooled and subjected to silica gel chromatography (eluent: gradient from CH2Cl2 to 50% acetone/CH2Cl2) to give crude turnagainolides A and B.  Crude turnagainolide B was subjected to reversed-phase HPLC (eluent: 60% MeOH/H2O) to give pure turnagainolide B, and crude turnagainolide A was subjected to reversed-phase HPLC (eluent: 60% MeOH/H2O) to give pure turnagainolide A. 7.8.3. Synthetic Procedures Synthesis of 7.12: A flask containing a stir bar and THF (5 mL) under N2 was cooled to - 78 oC, and to this flask was added a solution of LDA in THF (10.0 mL of a 1.8 M solution, 18.0 mmol).  Ethyl acetate (7.9) (1.0 mL, 10.2 mmol) was added dropwise, followed by trans- cinnamaldehyde (7.8) (2.0 mL, 15.9 mmol).  The reaction mixture was stirred for 45 minutes at - 78 oC before being quenched by the addition of saturated aq. NH4Cl.  The mixture was extracted with CH2Cl2, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from 50% CH2Cl2/hexane to CH2Cl2) to give 7.10 (2.17 g, 96%).  To a solution of 7.10 (2.17 g, 9.88 mmol) in THF was added TBDMS-Cl (1.65 g, 10.9 mmol) and imidazole (1.02 g, 15.0 mmol), and the reaction mixture was refluxed for 12 hours. The reaction mixture was dried in vacuo and purified by silica gel chromatography (eluent: 25% CH2Cl2/hexane to 50% CH2Cl2/hexane) to give 7.11 (2.56 g, 78%). 216   To a solution of 7.11 (2.56 g, 7.65 mmol) in THF (5 mL) and MeOH (5 mL) was added 1 M aq. NaOH (5 mL), and the reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was acidified with 1 M aq. HCl and extracted with CH2Cl2. The organic layer was dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 5% MeOH/CH2Cl2) to give 7.12 (1.64 g, 70%).   Compound 7.12: 1H NMR (400 MHz, acetone-d6) δ 7.44 (2H, d, J = 7.3 Hz, H-7/11), 7.33 (2H, t, J = 7.5 Hz, H-8/10), 7.24 (1H, m, H-9), 6.67 (1H, d, J = 15.9 Hz, H-5), 6.34 (1H, dd, J = 15.9, 6.7 Hz, H-4), 4.83 (1H, m, H-3), 2.57 (2H, m, H-2), 0.91 (9H, s, H-13/14/15), 0.12 (3H, s, H-16), 0.09 (3H, s, H-17); 13C NMR (100 MHz, acetone-d6) δ 172.2 (C, C-1), 137.8 (C, C-6), 132.8 (CH, C-5), 130.7 (CH, C-9), 129.5 (CH, C-7/11), 128.5 (CH, C-4), 127.4 (CH, C-8/10), 71.7 (CH, C-3), 44.1 (CH2, C-2), 26.3 (CH3, C-13/14/15), 18.8 (C, C-12), -4.0 (CH3, C-16), -4.7 (CH3, C-17). Synthesis of Fmoc-protected amino acids: A solution of Fmoc-Cl (258 mg, 1.0 mmol) in dioxane (3 mL) was added to a solution of the appropriate amino acid (1.5 mmol, 1.5 eq.) in 10% aqueous Na2CO3 (3 mL) and dioxane (1.5 mL).  After stirring for 1 hour at room temperature, the mixture was acidified with 1 M aq. HCl, and extracted with CH2Cl2.  The organic layer was dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 10% MeOH/CH2Cl2) to give the Fmoc-protected amino acid (yield = 94-96%). Solid-phase synthesis of linear peptide 7.14: To CH2Cl2-washed polymer bound 2- chlorotrityl chloride (572 mg) under N2 was added Fmoc-L-Val-OH (330 mg, 0.97 mmol) in 217  CH2Cl2 (10 mL), followed by DIPEA (0.3 mL, 1.72 mmol). The reaction mixture was stirred for 1 hour at room temperature before the solid-phase resin was washed with CH2Cl2.  The solid- phase resin was capped by adding a solution of CH2Cl2/MeOH/DIPEA (ratio 17:2:1, 10 mL) and stirring for 1 hour at room temperature.  The solid-phase resin was then washed with CH2Cl2, followed by DMF.  The Fmoc protecting group of L-Val was removed by treatment with 20% piperidine in DMF (5 mL) for 5 minutes at room temperature.  This reaction was repeated twice more with 20% piperidine in DMF (5 mL), followed by washing the solid-phase resin with DMF, CH2Cl2 and DMF again.  The coupling of the next amino acid (L-Ile) was performed by adding a solution of Fmoc-L-Ile-OH (330mg, 0.93 mmol), PyBOP (560 mg, 1.08 mmol) and HOBt (2.0 mL of 0.5 M solution, 1.0 mmol) in DMF (10 mL), followed by the addition of DIPEA (0.2 mL, 1.15 mmol), and stirring for 1 hour at room temperature.  The above deprotection/coupling procedure was repeated for D-Ala and L-Val using approximately 1 mmol of each reagent as described above. The final coupling step was performed by adding a solution of 7.12 (306 mg, 1.0 mmol), PyBOP (560 mg, 1.08 mmol) and HOBt (2.0 mL of 0.5 M solution, 1.0 mmol) in DMF (10 mL), followed by the addition of DIPEA (0.2 mL, 1.15 mmol), and stirring for 1 hour at room temperature.  The solid-phase resin was then washed with DMF, followed by THF.  Deprotection of the silyl protecting group was accomplished by treatment with a solution of TBAF in THF (10 mL of 0.2 M solution, 2.0 mmol) for 1 hour at room temperature.  This reaction was repeated once more for an additional hour, followed by washing the solid-phase resin with THF and then CH2Cl2.  Cleavage from the solid-phase resin was accomplished by treatment with CH2Cl2/PDT/TFA (ratio 98:1:1, 10 mL) for 1 hour at room temperature under N2.  This procedure was repeated for an additional hour at room temperature, followed by washing the 218  solid-phase resin with CH2Cl2 and MeOH.  The washings were combined and dried in vacuo to give the crude linear peptide (7.14).  The linear peptide (7.14) was washed with CH2Cl2 and purified by silica gel chromatography (eluent: gradient from 10% MeOH/EtOAc to 25% MeOH/EtOAc) to give the linear peptide (7.14) (387 mg) as a mixture of diastereomers. Synthesis of turnagainolides A and B: A solution of DMAP (208 mg, 1.70 mmol), pTSA monohydrate (162 mg, 0.85 mmol) and DIC (0.75 mL, 4.79 mmol) in CHCl3 (200 mL) was heated to reflux.  To this reaction mixture at reflux was added a solution of the linear peptide (7.14) (95.8 mg, 0.167 mmol) in DMF (20 mL) dropwise over the course of ten hours, and heating was continued at reflux overnight.  The reaction mixture was then cooled, dried in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to 5% MeOH/CH2Cl2) and silica gel chromatography (eluent: gradient from CH2Cl2 to 50% acetone/CH2Cl2) to give crude turnagainolide B (7.4) (31.5 mg) and crude turnagainolide A (7.3) (14.0 mg) (combined yield = 48 %).  A portion of crude turnagainolides A and B were further purified separately by reversed-phase HPLC (eluent: 60% MeOH/H2O) to give pure turnagainolide A and pure turnagainolide B. Turnagainolide A (7.3): 1H and 13C NMR data, see table 7.1; HRESI-MS(+) m/z 579.3170 (calculated for C30H44N4O6Na: 579.3159). Turnagainolide B (7.4):  1H and 13C NMR data, see table 7.2; HRESI-MS(+) m/z 579.3151 (calculated for C30H44N4O6Na: 579.3159). Methanolysis of turnagainolide B:  Turnagainolide B (6.1 mg, 0.011 mmol) was dissolved in a solution of 5% sodium methoxide in MeOH, and stirred overnight at room temperature.  The reaction was acidified with 1 M aq. HCl, diluted with water, and extracted 219  with CH2Cl2.  The organic layer was purified by reversed-phase HPLC (eluent: 60% MeOH/H2O) to give 7.21 (3.2 mg, 51%). Synthesis of 7.22: Compound 7.21 (0.4 mg) was dissolved in CH2Cl2 (1 mL) and to this solution was added 1 crystal of DMAP and 10 µL (0.053 mmol) of (R)-MTPA-Cl.  The reaction mixture was stirred overnight at room temperature and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 25% acetone/CH2Cl2) to give 7.22. Synthesis of 7.23: Compound 7.21 (0.4 mg) was dissolved in CH2Cl2 (1 mL) and to this solution was added 1 crystal of DMAP and 10 µL (0.053 mmol) of (S)-MTPA-Cl.  The reaction mixture was stirred overnight at room temperature and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 25% acetone/CH2Cl2) to give 7.23. Synthesis of 7.28: Compound 7.10 (436 mg, 1.98 mmol) was dissolved in CH2Cl2 under N2, and PPh3 (1.20 g, 4.58 mmol) was added.  The mixture was cooled to 0 oC, and DIAD (0.8 mL, 4.06 mmol) was added, followed by DPPA (0.9 mL, 4.18 mmol).  The reaction mixture was stirred overnight at room temperature and purified by silica gel flash chromatography (eluent: CH2Cl2) and silica gel chromatography (eluent: 25% CH2Cl2/hexane) to give 7.25 (409 mg, 84%). Compound 7.25 (409 mg, 1.67 mmol) was dissolved in THF (12 mL) and H2O (3 mL), and PPh3 (625 mg, 2.38 mmol) was added.  The reaction was heated to 70 oC and stirred overnight at this temperature.  The reaction was cooled, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 10% MeOH/CH2Cl2) to give 7.26 (201 mg, 55%). Compound 7.26 (201 mg, 0.92 mmol) was dissolved in THF (5 mL) and MeOH (5 mL), and to this solution was added 1 M aq. NaOH (5 mL).  The reaction mixture was stirred for 3 220  hours at room temperature, acidified with 1 M aq. HCl and extracted with n-BuOH to give 250 mg of crude 7.27.  Crude 7.27 (250 mg) was dissolved in 10% Na2CO3 (3 mL) and dioxane (1.5 mL), and to this solution was added a solution of Fmoc-Cl (388 mg, 1.50 mmol) in dioxane (3 mL).  The reaction mixture was stirred for 1 hour at room temperature, acidified with 1 M aq. HCl, and extracted with CH2Cl2.  The organic layer was dried in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 5% MeOH/CH2Cl2) to give 7.28 (200 mg, 53% over two steps). Synthesis of 7.24: The linear peptide 7.29 was synthesized in the same fashion as 7.14, except that 7.28 was used instead of 7.12 for the final coupling step, and after this coupling the Fmoc group that was attached to 7.28 was removed with 20% piperidine/DMF.  The linear peptide 7.29 (41.2 mg, 0.072 mmol) was suspended in CH2Cl2, and excess PyBOP (260 mg, 0.50 mmol) and DMAP (122 mg, 1.00 mmol) were added.  The reaction mixture was stirred for 2 days at room temperature, dried in vacuo, and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 5% MeOH/CH2Cl2) to give crude 7.24.  Crude 7.24 was washed with CH2Cl2, MeOH and DMSO, and then purified by reversed-phase HPLC (eluent: 75% MeOH/H2O + 0.2% TFA).  The NMR spectra of 7.24 were recorded in a mixture of 5% TFA-d1 in CD2Cl2, since 7.24 was not soluble in any other solvent or combination of solvents tested.  Compound 7.24: 1H NMR (600 MHz, 5% TFA-d1 in CD2Cl2) δ 7.37 (2H, d, J = 7.2 Hz, H-7/11), 7.33 (2H, t, J = 7.5 Hz, H-8/10), 7.27 (1H, t, J = 7.2 Hz, H-9), 6.57 (1H, d, J = 15.8 Hz, H-5), 6.46 (1H, dd, J = 15.9, 7.3 Hz, H-4), 4.58 (1H, d, J = 5.3 Hz, Val1-Hα), 4.45 (1H, m, Ala3- Hα), 4.43 (1H, m, Ile2-Hα), 4.42 (1H, m, H-3), 4.04 (1H, d, J = 9.4 Hz, Val4-Hα), 3.42 (1H, dd, J = 13.5, 12.4 Hz, H-2a), 2.69 (1H, dd, J = 14.2, 3.7 Hz, H-2b), 2.42 (1H, m, Val1-Hβ), 2.12 (1H, m, Ile2-Hβ), 1.99 (1H, m, Val4-Hβ), 1.51 (1H, m, Ile2-HγA), 1.48 (3H, d, J = 6.9 Hz, Ala 3-Hβ), 221  1.24 (1H, m, Ile2-HγB), 1.04 (3H, d, J = 6.9 Hz, Ile 2-HβCH3), 1.03 (3H, d, J = 6.6 Hz, Val 4- HγACH3), 0.96 (3H, m, Val 4-HγBCH3), 0.95 (3H, m, Ile 2-HδCH3), 0.94 (3H, m, Val 1- HγACH3), 0.94 (3H, m, Val1-HγBCH3); 13C NMR (150 MHz, 5% TFA-d1 in CD2Cl2) δ 176.4 (C, Ala 3- C=O), 175.1 (C, Val4-C=O), 174.7 (C, C-1), 174.4 (C, Ile2-C=O), 173.5 (C, Val1-C=O), 136.4 (C, C-6), 133.7 (CH, C-5), 129.2 (CH, C-8/10), 128.8 (CH, C-9), 127.1 (CH, C-7/11), 125.7 (CH, C-4), 61.0 (CH, Val4-Cα), 60.8 (CH, CH, Ile2-Cα), 60.0 (CH, Val1-Cα), 54.5 (CH, C-3), 51.3 (CH, Ala3-Cα), 39.7 (CH2, C-2), 36.6 (CH, Ile 2-Cβ), 29.8 (CH, Val4-Cβ), 28.9 (CH, Val1- Cβ), 25.2 (CH2, Ile 2-Cγ), 19.7 (CH3, Val 1-CγACH3), 19.0 (CH3, Val 4-CγBCH3), 18.8 (CH3, Val 4- CγACH3), 17.9 (CH3, Val 1-CγBCH3), 15.9 (CH3, Ala 3-Cβ), 15.8 (CH3, Ile 2-βCH3), 11.4 (CH3, Ile2-δCH3); HRESI-MS(+) m/z 556.3488 (calculated for C30H46N5O5: 556.3499). 7.9. 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(10) Luo, J.-M.; Yoshida, H.; Komura, S.; Ohishi, N.; Pan, L.; Shigeno, K.; Hanamura, I.; Miura, K.; Iida, S.; Ueda, R.; Naoe, T.; Akao, Y.; Ohno, R.; Ohnishi, K. Leukemia 2003, 17, 1-8. (11) Takeshita, S.; Namba, N.; Zhao, J. J.; Jiang, Y.; Genant, H. K.; Silva, M. J.; Brodt, M. D.; Helgason, C. D.; Kalesnikoff, J.; Rauh, M. J.; Humphries, R. K.; Krystal, G.; Teitelbaum, S. L.; Ross, F. P. at. Med. 2002, 8, 943-949. (12) Helgason, C. D.; Damen, J. E.; Rosten, P.; Grewal, R.; Sorensen, P.; Chappel, S. M.; Borowski, A.; Jirik, F.; Krystal, G.; Humphries, R. K. Genes Dev. 1998, 12, 1610-1620. (13) March, M. E.; Ravichandran, K. Semin. Immunol. 2002, 14, 37-47. (14) Leung, W.-H.; Tarasenko, T.; Bolland, S. Immunol. Res. 2009, 43, 243-251. (15) Huber, M.; Helgason, C. D.; Damen, J. E.; Liu, L.; Humphries, R. K.; Krystal, G. Proc. atl. Acad. Sci. U.S.A. 1998, 95, 11330-11335. (16) MacDonald, S. M.; Vonakis, B. M. Mol. Immunol. 2002, 38, 1323-1327. (17) Moody, J. L.; Pereira, C. G.; Magil, A.; Fritzler, M. J.; Jirik, F. R. Genes Immun. 2003, 4, 60-66. (18) Vonakis, B. M.; Gibbons Jr., S.; Sora, R.; Langdon, J. M.; MacDonald, S. M. J. Allergy. Clin. Immunol. 2001, 108, 822-831. (19) Camps, M.; Rückle, T.; Ji, H.; Ardissone, V.; Rintelen, F.; Shaw, J.; Ferrandi, C.; Chabert, C.; Gillieron, C.; Françon, B.; Martin, T.; Gretener, D.; Perrin, D.; Leroy, D.; Vitte, P.-A.; Hirsch, E.; Wymann, M. P.; Cirillo, R. Schwarz, M. K.; Rommel, C. at. Med. 2005, 11, 936-943. 223  (20) Oh, S.-Y.; Zheng, T.; Bailey, M. L.; Barber, D. L.; Schroeder, J. T.; Kim, Y.-K. J. Allergy Clin. Immunol. 2007, 119, 123-131. (21) Kwak, Y.-G.; Song, C. H.; Yi, H. K.; Hwang, P. H.; Kim, J.-S.; Lee, K. S.; Lee, Y. C. J. Clin. Invest. 2003, 111, 1083-1092. (22) Barber, D. F.; Bartolomé, A.; Hernandez, C.; Flores, J. M.; Redondo, C.; Fernandez-Arias, C.; Camps, M.; Ruckle, T.; Schwarz, M. K.; Rodríguez, S.; Martinez-A, C.; Balomenos, D.; Rommel, C.; Carrera, A. C. at. Med. 2005, 11, 933-935. (23) Sly, L. M.; Rauh, M. J.; Kalesnikoff, J.; Song, C. H.; Krystal, G. Immunity 2004, 21, 227- 239. (24) Yang, L.; Williams, D. E.; Mui, A.; Ong, C.; Krystal, G.; van Soest, R.; Andersen, R. J. Org. Lett. 2005, 7, 1073-1076. (25) Ong, C. J.; Ming-Lum, A.; Nodwell, M.; Ghanipour, A.; Yang, L.; Williams, D. E.; Kim, J.; Demirjian, L.; Qasimi, P.; Ruschmann, J.; Cao, L.-P.; Ma, K.; Chung, S. W.; Duronio, V.; Andersen, R. J.; Krystal, G.; Mui, A. L.-F. Blood 2007, 110, 1942-1949. (26) Boerjan, W.; Ralph, J.; Baucher, M. Annu. Rev. Plant Biol. 2003, 54, 519-546. (27) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596. (28) Coin, I.; Beyermann, M.; Bienert, M. at. Protoc. 2007, 2, 3247-3256. (29) Madigan, D. M.; Swenton, J. S. J. Am. Chem. Soc. 1971, 93, 6318-6319. (30) Jørgensen, M. R.; Olsen, C. A.; Mellor, I. R.; Usherwood, P. N. R.; Witt, M.; Franzyk, H.; Jaroszewski, J. W. J. Med. Chem. 2005, 48, 56-70. (31) Parenty, A.; Moreau, X.; Campagne, J.-M. Chem. Rev. 2006, 106, 911-939. (32) Keck, G. E.; Boden, E. P.; Wiley, M. R. J. Org. Chem. 1989, 54, 896-906. (33) Banwell, M. G.; Kokas, O. J.; Willis, A. C. Org. Lett. 2007, 9, 3503-3506. 224  8. Chemical Prospecting 8.1. Chemical Prospecting  While bioassay-guided fractionation is often used to facilitate the isolation of biologically active natural products, a different approach involves isolating novel natural products first and then testing them for biological activity.  This is known as chemical prospecting.  While a crude extract may only have a few compounds that are active in any given assay, chemical prospecting is not limited to these few compounds and we can look at the entire array of compounds present in the extract to look for new compounds.  These new compounds can then be tested in a variety of biological assays to look for biological activity. 8.2. *ovel Peptide from a Marine Sponge  An unidentified sponge that showed activity in an anticancer assay was extracted with MeOH, and the MeOH was dried to give the crude extract.  The crude extract was partitioned between CH2Cl2 and H2O, followed by partitioning between n-BuOH and H2O.  The n-BuOH layer was dried, and purified by reversed-phase flash chromatography (eluent: gradient from H2O to MeOH), followed by reversed-phase HPLC (eluent: gradient from 50% MeOH/H2O + 0.2% TFA to 100% MeOH + 0.2% TFA) to give a new peptide (8.1) that did not show any activity in this particular anticancer assay. 225   Figure 8.1. Structure of the novel peptide 8.1.  Analysis of the 1D (figures 8.2 and 8.3 and table 8.1) and 2D (figures 8.14 to 8.19 and table 8.1) NMR data for 8.1 identified seven amino acids: three proline residues, one tyrosine residue, one phenylalanine residue, one isoleucine residue and one glutamic acid residue. Compound 8.1 gave a peak in the HRESI-MS(-) at m/z 842.4120, consistent with a molecular formula of C44H57N7O10 (calculated for C44H56N7O10: 842.4089).  The molecular formula of 8.1 is consistent with a cyclic peptide containing the seven amino acids identified from the NMR data. 226   Figure 8.2. 1H NMR spectrum of 8.1 recorded in DMSO-d6 at 600 MHz.  Figure 8.3. 13C NMR spectrum of 8.1 recorded in DMSO-d6 at 150 MHz. 227  Table 8.1. NMR data for 8.1. position δC δH (J  in Hz) COSY HMBC Ile 1 C=O 167.7 α 55.7 4.25, dd (9.7, 8.6) Ile 1 -Hβ, Ile 1 -NH Phe 7 -C=O, Ile 1 -C=O, Ile 1 -Cβ, Ile 1 -CβCH3 β 36.0 2.04, m Ile 1 -Hα, Ile 1 -HβCH3, Ile 1 -HγB Ile 1 -Cα γA 23.7 1.47, m Ile 1 -HδCH3, Ile 1 -HγB Ile 1 -Cβ, Ile 1 -CβCH3 γB 1.01, ddd (13.8, 10.2, 7.2) Ile 1 -Hβ, Ile 1 -HδCH3, Ile 1 -HγA Ile 1 -Cβ, Ile 1 -CβCH3, Ile 1 -CδCH3 δCH3 11.9 0.76, t (7.3) Ile 1 -HγA, Ile 1 -HγB Ile 1 -Cγ, Ile 1 -Cβ βCH3 16.9 0.90, d (6.4) Ile 1 -Hβ Ile 1 -Cα, Ile 1 -Cβ, Ile 1 -Cγ NH 6.90, d (8.0) Ile 1 -Hα Phe 7 -C=O Pro 2 C=O 170.3 α 58.4 4.36, dd (8.7, 4.6) Pro 2 -HβA, Pro 2 -HβB Pro 2 -Cγ βA 28.1 2.12, m Pro 2 -Hα, Pro 2 -HβB, Pro 2 -HγA, Pro 2 -HγB Pro 2 -C=O, Pro2-Cα, Pro 2 -Cγ βB 1.74, m Pro 2 -Hα, Pro 2 -HβA, Pro 2 -HγA Pro 2 -C=O, Pro 2 -Cγ, Pro 2 -Cδ γA 24.2 1.89, m Pro 2 -HβA, Pro 2 -HβB, Pro 2 -HδA, Pro 2 -HδB Pro 2 -Cα, Pro 2 -Cβ, Pro 2 -Cδ γB 1.81, m Pro 2 -HβA, Pro 2 -HδA, Pro 2 -HδB Pro 2 -Cα, Pro 2 -Cβ, Pro 2 -Cδ δA 46.8 3.47, m Pro 2 -HγA, Pro 2 -HγB, Pro 2 -HδB Pro 2 -Cα, Pro 2 -Cβ, Pro 2 -Cγ δB 3.25, m Pro 2 -HγA, Pro 2 -HγB, Pro 2 -HδA Pro 2 -Cβ, Pro 2 -Cγ Pro 3 C=O 171.9 α 60.3 4.30, d (8.9) Pro 3 -HβA, Pro 3 -HβB Pro 3 -C=O, Pro 3 -Cβ, Pro 3 -Cγ, Pro 3 -Cδ βA 31.9 2.14, m Pro 3 -Hα, Pro 3 -HβB, Pro 3 -HγA, Pro 3 -HγB Pro 3 -C=O, Pro 3 -Cα, Pro 3 -Cγ βB 1.99, m Pro 3 -Hα, Pro 3 -HβA, Pro 3 -HγA, Pro 3 -HγB Pro 3 -Cδ γA 21.8 1.78, m Pro 3 -HβA, Pro 3 -HβB, Pro 3 -HγB, Pro 3 -HδA, Pro 3 -HδB γB 1.60, m Pro 3 -HβA, Pro 3 -HβB, Pro 3 -HγA, Pro 3 -HδA, Pro 3 -HδB δA 46.8 3.68, m Pro 3 -HγA, Pro 3 -HγB, Pro 3 -HδB Pro 3 -Cβ δB 3.31, m Pro 3 -HγA, Pro 3 -HγB, Pro 3 -HδA Pro 3 -Cγ Glu 4 C=O 170.1 α 55.4 3.85, dt (9.7, 6.2) Glu 4 -HβA, Glu 4 -HβB, Glu 4 -NH Glu 4 -C=O, Glu 4 -Cβ, Glu 4 -Cγ βA 26.8 1.79, m Glu 4 -Hα, Glu 4 -HβB, Glu 4 -HγA, Glu 4 -HγB Glu 4 -C=O, Glu 4 -Cα, Glu 4 -Cγ, Glu 4 -Cδ βB 1.60, m Glu 4 -Hα, Glu 4 -HβA, Glu 4 -HγA, Glu 4 -HγB Glu 4 -C=O, Glu 4 -Cα, Glu 4 -Cγ, Glu 4 -Cδ γA 30.5 2.09, m Glu 4 -HβA, Glu 4 -HβB, Glu 4 -HγB Glu 4 -Cδ, Glu 4 -Cα, Glu 4 -Cβ γB 2.01, m Glu 4 -HβA, Glu 4 -HβB, Glu 4 -HγA Glu 4 -Cδ, Glu 4 -Cα, Glu 4 -Cβ δ 173.8 NH 8.02, d (6.9) Glu 4 -Hα Pro 3 -C=O, Glu 4 -Cα, Glu 4 -Cβ Tyr 5 C=O 171.5 α 51.5 4.85, m Tyr 5 -HβA, Tyr 5 -HβB, Tyr 5 -NH Tyr 5 -C=O, Tyr 5 -Cβ, Tyr 5 -C1 βA 37.0 3.26, m Tyr 5 -Hα, Tyr 5 -HβB Tyr 5 -C1, Tyr 5 -C2/6 βB 2.46, m Tyr 5 -Hα, Tyr 5 -HβA Tyr 5 -Cα, Tyr 5 -C1, Tyr 5 -C2/6 1 127.1 2/6 130.1 7.08, d (8.3) Tyr 5 -H3/5 Tyr 5 -Cβ, Tyr 5 -C2/6, Tyr 5 -C3/5, Tyr 5 -C4 3/5 115.0 6.66, d (8.3) Tyr 5 -H2/6 Tyr5-C1, Tyr5-C3/5, Tyr5-C4 4 155.9 OH 9.20, br. s NH 7.49, d (9.4) Tyr 5 -Hα Glu 4 -C=O Pro 6 C=O 171.6 α 62.7 3.93, t (8.3) Pro 6 -HβA, Pro 6 -HβB Pro 6 -C=O, Pro 6 -Cβ βA 28.4 1.95, m Pro 6 -Hα, Pro6-HβB Pro 6 -Cγ βB 1.47, ddd (9.7, 7.2, 2.8) Pro 6 -Hα, Pro 6 -HβA Pro 6 -C=O, Pro 6 -Cα γA 25.0 2.05, m Pro 6 -HδA, Pro 6 -HδB, Pro 6 -HγB γB 1.90, m Pro 6 -HδA, Pro 6 -HδB, Pro 6 -HγA Pro 6 -Cβ, Pro 6 -Cδ δA 46.8 3.78, m Pro 6 -HγA, Pro 6 -HγB, Pro 6 -HδB Pro 6 -Cγ δB 3.70, m Pro 6 -HγA, Pro 6 -HγB, Pro 6 -HδA Pro 6 -Cβ Phe 7 C=O 171.1 α 53.9 4.48, ddd (11.7, 8.6, 3.2) Phe 7 -HβA, Phe 7 -HβB, Phe 7 -NH Phe 7 -C=O βA 35.9 3.28, m Phe 7 -Hα, Phe 7 -HβB Phe 7 -Cα, Phe 7 -C1, Phe 7 -C2/6 βB 2.93, t (13.3) Phe 7 -Hα, Phe 7 -HβA Phe 7 -Cα, Phe 7 -C1, Phe 7 -C2/6 1 138.5 2/6 128.7 7.22, m Phe 7 -H3/5 Phe 7 -Cβ, Phe 7 -C2/6, Phe 7 -C4 3/5 128.1 7.27, m Phe 7 -H2/6, Phe 7 -H4 Phe 7 -C1, Phe 7 -C3/5 4 126.3 7.19, m Phe 7 -H3/5 Phe 7 -C2/6 NH 7.85, d (8.6) Phe 7 -Hα Pro 6 -C=O, Pro 6 -Cα a Spectra recorded in DMSO-d 6  at 600 MHz 8.1 a  228  The HMBC and ROESY data of 8.1 were used to determine the connectivity of the amino acids (figures 8.4 to 8.7).  HMBC correlations from δH 6.90 (Ile 1-NH) to δC 171.1 (Phe 7- C=O) and from δH 4.48 (Phe 7-Hα) to δC 171.1 (Phe 7-C=O) established connectivity between isoleucine and phenylalanine.  HMBC correlations from δH 7.85 (Phe 7-NH) to δC 171.6 (Pro 6- C=O) and from δH 3.93 (Pro 6-Hα) to δC 171.6 (Pro 6-C=O) showed that the phenylalanine residue was also connected to a proline residue.  Correlations from δH 7.49 (Tyr 5-NH) to δC 170.1 (Glu 4- C=O) and from both δH 3.85 (Glu 4-Hα) and δH 1.60 (Glu 4-HβB) to δC 170.1 (Glu 4-C=O) showed that tyrosine was connected to glutamic acid.  Two final key HMBC correlations from δH 8.02 (Glu4-NH) to δC 171.9 (Pro 3-C=O) and from δH 4.30 (Pro 3-Hα) to δC 171.9 (Pro 3-C=O) showed that glutamic acid was also connected to proline.  Figure 8.4. Key HMBC correlations in 8.1. 229   Figure 8.5. Expanded HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. ROESY correlations between δH 3.78 (Pro 6-HδA) and δH 4.85 (Tyr 5-Hα), and between δH 3.70 (Pro 6-HδB) and δH 4.85 (Tyr 5-Hα) established the link between proline and tyrosine. ROESY correlations between δH 3.47 (Pro 2-HδA) and δH 4.25 (Ile 1-Hα) and between δH 3.25 (Pro2-HδB) and δH 4.25 (Ile 1-Hα) showed that isoleucine was linked to a proline residue.  Finally, ROESY correlations between δH 2.12 (Pro 2-HβA) and δH 4.30 (Pro 3-Hα) and between δH 1.74 (Pro2-HβB) and δH 4.30 (Pro 3-Hα) showed that these two proline residues were linked, completing the connectivity of 8.1. 230   Figure 8.6. Key ROESY correlations in 8.1.  Figure 8.7. Expanded ROESY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. In order to confirm this proposed structure, tandem mass spectrometry (MS/MS) was also used to determine the connectivity.  Peaks in the MS/MS spectrum corresponding to fragments of 8.1 (tables 8.2 to 8.4) confirmed the connectivity that was determined by HMBC and ROESY correlations. 231  Table 8.2. MS/MS of 8.1 on m/z 842 [M-H]- ion. Mass Assigned Fragment 842 8.1 - H 824 8.1 - H2O - H 798 8.1 - CO2 - H 718 8.1 - Pro - CO - H 674 8.1 - Pro - CO - CO2 - H 564 8.1 - Ile - Phe - H2O - H 520 8.1 - Ile - Phe - H2O - CO2 - H 517 8.1 - Pro - Pro - Ile - H2O - H 361 8.1 - Pro - Ile - Phe - Pro - CO - H  (Tyr + Glu + Pro - CO - H) Table 8.3. MS/MS of 8.1 on m/z 844 [M+H]+ ion. Mass Assigned Fragment 845 8.1 + H 826 8.1 - H2O + H 816 8.1 - CO + H 731 8.1 - Ile + H 703 8.1 - Ile - CO + H 635 8.1 - Pro - Ile + H 589 8.1 - Glu - Pro - CO + H 555 8.1 - Pro - Tyr - CO + H Or   8.1 - Ile - Phe - CO + H 522 8.1 - Glu - Pro - Pro + H 487 8.1 - Pro - Ile - Phe + H 459 8.1 - Pro - Ile - Phe - CO + H 390 8.1 - Pro - Ile - Phe - Pro + H  (Tyr + Glu + Pro + H) 324 8.1 - Ile - Phe - Pro - Tyr + H  (Glu + Pro + Pro + H) 293 8.1 - Pro - Pro - Ile - Phe - Pro + H  (Glu + Tyr + H) Table 8.4. MS/MS of 8.1 on m/z 866 [M+Na]+ ion. Mass Assigned Fragment 867 8.1 + Na 838 8.1 - CO + Na 820 8.1 - CO - H2O + Na 753 8.1 - Ile + Na 725 8.1 - Ile - CO + Na 703 8.1 - Tyr + Na 675 8.1 - Tyr - CO + Na 546 8.1 - Tyr - Glu - CO + Na 515 8.1 - Glu - Pro - Pro - CO + Na 459 8.1 - Phe - Pro - Tyr + Na 431 8.1 - Phe - Pro - Tyr - CO + Na 402 8.1 - Glu - Pro - Pro - Ile - CO + Na  (Phe + Pro + Tyr - CO + Na) 352 8.1 - Pro - Tyr - Glu - Pro - CO + Na (Pro + Ile + Phe - CO + Na) 346 8.1 - Ile - Phe - Pro - Tyr + Na (Glu + Pro + Pro + Na) 318 8.1 - Ile - Phe - Pro - Tyr - CO + Na (Glu + Pro + Pro - CO + Na) 232   The absolute configurations of the amino acid residues in 8.1 were determined by Marfey’s analysis.1  A small sample of 8.1 (0.1 mg) was hydrolyzed with 6 M aq. HCl, and derivatized with Marfey’s reagent to give a mixture of derivatized amino acids.  The derivatized amino acids from 8.1 were compared with standards prepared from L-amino acids or racemic amino acids using reversed-phase HPLC (eluent: gradient from 10% MeCN/H2O + 0.2% TFA to 50% MeCN + 0.2% TFA).  A comparison of the retention times showed that all of the amino acids in 8.1 had the L-configuration.  This was confirmed by spiking the Marfey’s derivatives prepared from racemic amino acids with those derived from 8.1.  In this case, the peaks for all of the L-amino acid derivatives increased in size relative to the peaks for the corresponding D- amino acids, confirming that all of the amino acids in 8.1 have the L-configuration. 8.3. Total Synthesis of 8.1  In order to further confirm the structure of 8.1, as well as provide more material for biological testing, a total synthesis of 8.1 was undertaken using solid-phase peptide synthesis. The side chain-protected linear peptide (8.2) was constructed using the appropriate Fmoc- protected amino acids and 2-chlorotrityl resin, with PyBOP as the coupling reagent, as shown in scheme 8.1. 233  Cl Cl 1. Fmoc-L-Phe-OH CH2Cl2 DIPEA 2. 20% piperidine/ DMF R1 O R2 NH2 1. Fmoc-L-Pro-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF R1 O R2 H N O N H R1 O R2 H N O N O NH2 R3 R1 O R2 H N O N O NH R3 1. Fmoc-L-Tyr(tBu)-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF 1. Fmoc-L-Glu(OBzl)-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF O H2N R4 R1 O R2 H N O N O NH R3 O N H R4 O HN 1. Fmoc-L-Pro-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF R1 O R2 H N O N O NH R3 O NH R4O N OH N 1. Fmoc-L-Pro-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF 1. Fmoc-L-Ile-OH PyBOP HOBt DIPEA 2. 20% piperidine/ DMF R1 O R2 H N O N O NH R3 O NH R4O N O N O H2N HO O R2 H N O N O NH R3 O NH R4O N O N O H2N TFA PDT CH2Cl2 8.2 R1 = O Cl O O O R4 =R3 =R2 =  Scheme 8.1. Solid-phase synthesis of 8.2. 234   The side chain-protected linear peptide (8.2) was then cyclized with PyBOP, HOBt and DIPEA in CH2Cl2 to give 8.3, according to scheme 8.2.  Scheme 8.2. Synthesis of 8.3. The t-butyl protecting group of tyrosine was deprotected using TFA to give 8.4, according to scheme 8.3.  Finally, the benzyl protecting group of glutamic acid was deprotected by hydrogenolysis to give synthetic 8.1, according to scheme 8.4.  Scheme 8.3. Synthesis of 8.4. 235   Scheme 8.4. Synthesis of 8.1. This synthetic 8.1 was compared to the natural product using NMR spectroscopy.  The 1H and 13C NMR chemical shifts of synthetic and natural 8.1 were virtually identical (figures 8.8 and 8.9), further supporting the structure of 8.1.  Likewise, synthetic 8.1 showed very similar 2D NMR spectra to natural 8.1.  Figure 8.8. Comparison of 1H NMR spectra of natural and synthetic 8.1. 236   Figure 8.9. Comparison of 13C NMR spectra of natural and synthetic 8.1.  Finally, the linear analogue 8.5 was prepared by deprotecting the linear peptide 8.2 with TFA and hydrogenolysis, according to scheme 8.5. 237  N NH2 OO NN H O H N O N OO N H HO O O O O TFA, CH2Cl2 r.t., 1 h N NH2 OO NN H O H N O N OO N H HO O OH O O 8.2 8.6 H2, Pd/C, MeOH r.t., o/n N NH2 OO NN H O H N O N OO N H HO O OH O OH 99% over two steps 8.5 Scheme 8.5. Synthesis of 8.5. Natural and synthetic 8.1, as well as 8.2-8.5, were tested in cytotoxic and antibiotic assays.  Only compound 8.5 showed weak activity against Bacillus subtilis in the antibiotic assay at a concentration of 400 µg/mL, while the other analogues were inactive in this assay at concentrations as high as 400 µg/mL.  In the cytotoxicity assay, the side chain-protected cyclic 238  peptide (8.3) showed toxicity towards T98G cells with an IC50 ≈ 20 µg/mL.  Surprisingly, all of the other analogues, including the structurally similar 8.4 and 8.1, did not show any activity in this assay.  It is possible that the lipophilic protecting groups in 8.3 could help this molecule to cross the cell membrane, while 8.4 and 8.1 may have difficulty crossing the cell membrane. 8.4. *ovel Carotenoid from a Marine Sponge  An unidentified sponge that showed activity in an antimitotic assay was extracted with MeOH, and the MeOH was dried to give the crude extract.  The crude extract was fractionated by silica gel chromatography (eluent: gradient from 10% EtOAc/hexane to EtOAc to 50% MeOH/EtOAc) to give an orange-red fraction that eluted from the silica gel column with 75% EtOAc/hexane.  This orange-red pigment was further purified using reversed-phase flash chromatography (eluent: gradient from H2O to MeOH), reversed-phase HPLC (eluent: gradient from 75% MeOH/H2O to 85% MeOH/H2O) and normal-phase HPLC (eluent: 67% EtOAc/hexane) to give the new carotenoid 8.7.  Figure 8.10. Structure of 8.7.  Compound 8.7 gave a peak in the ESI-MS(+) at m/z 543.3, which is consistent with a molecular formula of C33H44O5 for 8.7.  From the 1D (figures 8.12 and 8.13 and table 8.5) and 2D (figures 8.20 to 8.24 and table 8.5) NMR data for 8.7, it was possible to identify a six- membered ring with attached hydroxyl and methyl groups.  This six-membered ring was bonded to an allene group, which was in turn conjugated to 7 additional double bonds.  This polyunsaturated chain contains a series of methyl substituents (δC 13.0, C-31; δH 1.98, H-31; δC 239  12.9, C-33; δH 2.00, H-33; δC 11.7, C-34; δH 1.91, H-34), and terminates in a methyl ketone (δC 199.3, C-23; δC 25.8, C-24; δH 2.33, H-24).  Compound 8.7 has a similar structure to paracentrone (8.8),2 except that C-28 is substituted with an O-acetyl group (δC 59.7, C-28; δH 4.78, H-28a; δH 4.74, H-28b; δC 171.0, C-29; δC 21.2, C-30; δH 2.02, H-30) in 8.7.  Figure 8.11. Structure of paracentrone (8.8).  Figure 8.12. 1H NMR spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. 240   Figure 8.13. 13C NMR spectrum of 8.7 recorded in CD2Cl2 at 150 MHz. Although the relative and absolute configurations of 8.7 were not determined, most known allenic carotenoids, including paracentrone,3 have a 3S, 5R, 6R configuration with all double bonds having an E geometry.4  Therefore, 8.7 is assumed to have the same relative and absolute configuration as paracentrone, and the same double bond geometry as paracentrone. However, the presence of an O-acetyl group attached to C-28 changes the priority such that the C9-C10 double bond in 8.7 has a Z geometry. 241  Table 8.5. NMR data for 8.7. position δC δH (J  in Hz) COSY HMBC 1 36.3 2a 49.8 1.89, m H-2b, H-3 C-1, C-3, C-4, C-6 2b 1.25, m H-2a, H-3 C-1, C-3, C-4, C-25, C-26 3 64.4 4.23, m H-2a, H-2b, H-4a, H-4b 4a 49.3 2.18, ddd (12.8, 4.1, 2.2) H-3, H-4b C-2, C-3, C-5, C-6 4b 1.32, m H-3, H-4a C-1, C-2, C-3, C-6 5 73.1 6 118.8 7 202.2 8 101.0 6.04, s C-1, C-5, C-6, C-7, C-9, C-10, C-25, C-28 9 130.2 10 133.0 6.32, m H-11 C-8, C-9, C-11, C-12, C-28 11 124.8 6.69, m H-10, H-12 C-9, C-10, C-12, C-13 12 140.0 b 6.44, m H-11 C-10, C-13, C-14, C-31 13 137.9 14 133.8 6.33, m H-15, H-31 C-12, C-16, C-31 15 132.3 6.75, m H-14 C-13, C-17 16 130.6 6.71, m H-17 C-14, C-17, C-18 17 136.2 6.42, m H-16, H-32 C-15, C-16, C-19, C-32 18 136.6 19 144.5 6.69, m C-17, C-20, C-21, C-32 20 124.5 6.66, m H-21 C-18, C-19, C-21, C-22 21 139.9 b 7.15, dd (10.8, 1.1) H-20, H-33 C-19, C-22, C-33 22 136.0 23 199.3 24 25.8 2.33, s C-21, C-22, C-23 25 29.2 1.32, s C-1, C-2, C-6, C-26 26 32.0 1.07, s C-1, C-2, C-6, C-25 27 31.2 1.34, s C-4, C-5, C-6 28a 59.7 4.78, d (11.6) H-28b C-8, C-9, C-10, C-29 28b 4.74, d (11.9) H-28a C-8, C-9, C-10, C-29 29 171.0 30 21.2 2.02, s C-29 31 13.0 c 1.98, s H-14 C-12, C-13, C-14 32 12.9 c 2.00, s H-17 C-17, C-18, C-19 33 11.7 1.91, s H-21 C-21, C-22, C-23 8.7 a a Spectra recorded in CD2Cl2 at 600 MHz b Signals are interchangeable c Signals are interchangeable 242  8.5. Conclusions  In conclusion, two new compounds have been isolated and their structures were determined by NMR spectroscopy and mass spectrometry.  A new peptide (8.1) was isolated from a marine sponge, and a total synthesis of this compound was accomplished by solid-phase peptide synthesis.  The synthesis of 8.1 confirmed the proposed structure, including the configuration of the amino acids.  Several analogues of this peptide were also synthesized, and one of these analogues (8.3) showed moderate cytotoxic activity against T98G cells (IC50 ≈ 20 µg/mL).  A novel carotenoid (8.7) was also isolated from a marine sponge, and found to be the C-28 O-acetyl analogue of paracentrone. 8.6. Experimental Section 8.6.1. General Experimental Procedures Silica gel (from SiliCycle® Inc., 230-400 mesh) was used for silica gel chromatography, and Waters 10 g or 2 g C18 Sep-Paks ® were used for reversed-phase flash chromatography.   TLC was performed using Merck Kieselgel 60 F254 (for normal-phase) and Whatman MKC18F 60 A (for reversed-phase) TLC plates.  A Waters 1500 Series pump system equipped with a Waters 2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column was used for HPLC. All solvents used for HPLC were HPLC grade and were filtered through a 0.45 µm filter (Osmonics Inc.) prior to use.  The absorbance was monitored at 230 nm and 254 nm with a flow rate of 2.0 mL/min.  NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 600 (equipped with a cryoprobe) spectrometer at 400 and 600 MHz, respectively.  The solvent used for NMR was CD2Cl2 or DMSO-d6, and chemical shifts are referenced to the internal solvent peaks at δH 5.32 and δC 53.8 (for CD2Cl2) and δH 2.50 and δC 39.5 (for DMSO-d6).  ESI-MS spectra were obtained with Bruker Esquire-LC and Micromass LCT mass spectrometers for low- 243  resolution and high-resolution spectra, respectively.  Reagents were purchased from Sigma- Aldrich®. 8.6.2. Isolation Procedures Isolation of 8.1: The sponge (32.6 g, dry weight) was extracted twice with MeOH, and the MeOH was dried in vacuo to give the crude extract.  The crude extract was partitioned between CH2Cl2 and H2O, and then between n-BuOH and H2O.  The n-BuOH layer was dried in vacuo, dissolved in MeOH and subjected to reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  The fractions eluting with 60-90% MeOH/H2O were recombined and fractionated by reversed-phase HPLC (eluent: gradient from 50% MeOH/H2O to MeOH).  A final purification by reversed-phase HPLC (eluent: gradient from 50% MeOH/H2O + 0.2% TFA to 100% MeOH + 0.2% TFA) gave pure 8.1 (1.0 mg). Compound 8.1 (natural): Isolated as a white solid; 1H and 13C NMR data, see table 8.1; HRESI-MS(-) m/z 842.4120 (calculated for C44H56N7O10: 842.4094). Isolation of 8.7: The sponge was extracted twice with MeOH, and the MeOH was dried in vacuo to give the crude extract.  The crude extract was suspended in CH2Cl2, and fractionated by silica gel chromatography (eluent: gradient from 10% EtOAc/hexane to 100% EtOAc to 50% MeOH/EtOAc).  The fractions eluting with 75% EtOAc/hexane had an orange-red colour.  The orange-red coloured fractions were pooled, and purified by reversed-phase flash chromatography (eluent: gradient from H2O to MeOH).  The orange-red coloured fractions, eluting with 80-85% MeOH/H2O, were combined and purified by reversed-phase HPLC (eluent: gradient from 75% MeOH/H2O to 85% MeOH/H2O) and normal-phase HPLC (eluent: 67% EtOAc/hexane) to give pure 8.7 (1.0 mg). 244  Compound 8.7: Isolated as an orange-red solid; 1H and 13C NMR data, see table 8.5. ESI-MS(+) m/z 543.3. 8.6.3. Synthetic Procedures  Synthesis of Fmoc-protected amino acids: A solution of Fmoc-Cl (258 mg, 1.0 mmol) in dioxane (3 mL) was added to a solution of the appropriate side chain-protected amino acid (except for Fmoc-L-Tyr(tBu)-OH, which was purchased from Sigma-Aldrich®) (1.5 mmol, 1.5 eq.) in 10% aqueous Na2CO3 (3 mL) and dioxane (1.5 mL).  After stirring for 1 hour at room temperature, the mixture was acidified with 1 M aq. HCl, and extracted with CH2Cl2.  The organic layer was dried in vacuo and purified by silica gel chromatography (eluent: gradient from CH2Cl2 to 10% MeOH/CH2Cl2) to give the Fmoc-protected amino acid (yield = 94-96%).  Solid-phase synthesis of linear peptide 8.2: To CH2Cl2-washed polymer bound 2- chlorotrityl chloride (303 mg) under N2 was added a solution of Fmoc-L-Phe-OH (160 mg, 0.41 mmol) in CH2Cl2 (10 mL), followed by DIPEA (0.25 mL, 1.44 mmol).  The reaction mixture was stirred for 1 hour at room temperature before the solid-phase resin was washed with CH2Cl2. The solid-phase resin was capped by adding a solution of CH2Cl2/MeOH/DIPEA (ratio 17:2:1, 10 mL) and stirring for 5 minutes at room temperature, and this procedure was repeated twice more.  The solid-phase resin was then washed with CH2Cl2, followed by DMF.  The deprotection/coupling procedure described in section 7.8.3. was repeated for each amino acid using approximately 1 mmol of each reagent.  Cleavage from the solid phase resin was accomplished by treatment with CH2Cl2:1,3-propanedithiol:TFA (98:1:1, 10 mL) for 1 hour at room temperature, and this reaction was repeated once more.  The solid-phase resin was then washed with CH2Cl2 and MeOH, and the combined washings were combined to give crude 8.2. Crude 8.2 was purified by reversed-phase flash chromatography (eluent: gradient from H2O to 245  MeOH) and reversed-phase HPLC (gradient from 30% MeCN + 0.1% TFA to 50% MeCN + 0.1% TFA to give pure 8.2 (24.2 mg).  HRESI-MS(+) m/z 1030.5261 (calculated for C55H73N7O11Na: 1030.5266).  Synthesis of 8.3: The side chain-protected linear peptide (8.2) (12.0 mg, 0.012 mmol) was suspended in CH2Cl2 (100 mL), and PyBOP (29.0 mg, 0.050 mmol), DIPEA (0.1 mL, 0.57 mmol) and a solution of HOBt in DMF (0.1 mL of a 0.5 M solution, 0.050 mmol) were added. The reaction mixture was allowed to stir for 2 days at room temperature.  The mixture was then dried in vacuo, and purified by silica gel flash chromatography (eluent: gradient from CH2Cl2 to EtOAc to 5% MeOH/EtOAc) and reversed-phase HPLC (eluent: gradient from 40% MeCN/H2O to 80% MeCN/H2O) to give pure 8.3 (5.1 mg, 43%).  HRESI-MS(+) m/z 1012.5159 (calculated for C55H71N7O10Na: 1012.5160).  Synthesis of 8.4: Compound 8.3 (4.6 mg, 0.0046 mmol) was dissolved in 50% TFA/CH2Cl2 (5 mL) under N2, and stirred for 1 hour at room temperature.  The solvent was removed in vacuo to give 8.4 (4.5 mg, quant.).  Synthesis of 8.1.: Compound 8.4 (4.5 mg, 0.0048 mmol) was dissolved in MeOH (10 mL), and Pd/C was added.  The mixture was flushed with N2, followed by H2, and a balloon filled with H2 was added via syringe.  The reaction mixture was stirred overnight at room temperature, filtered, dried in vacuo, and purified by reversed-phase HPLC (eluent: gradient from 50% MeCN/H2O + 0.1% TFA to 60% MeCN/H2O + 0.1% TFA) to give pure 8.1 (2.5 mg, 61%).  Compound 8.1 (synthetic): 1H and 13C NMR data, see table 8.1 (1H and 13C NMR chemical shifts of natural and synthetic 8.1 are identical to 2 decimal places and 1 decimal place, respectively); HRESI-MS(-) m/z 842.4105 (calculated for C44H56N7O10: 842.4094). 246   Synthesis of 8.5.: The side chain-protected linear peptide (8.2) (6.0 mg, 0.0060 mmol) was dissolved in 50% TFA/CH2Cl2 (3 mL) under N2, and stirred for 1 hour at room temperature. The solvent was removed in vacuo to give 8.6.  Compound 8.6 was dissolved in MeOH (10 mL), and Pd/C was added.  The mixture was flushed with N2, followed by H2, and a balloon filled with H2 was added via syringe.  The reaction mixture was stirred overnight at room temperature, filtered, dried in vacuo, and purified by reversed-phase HPLC (eluent: gradient from 30% MeCN/H2O + 0.1% TFA to 50% MeCN/H2O + 0.1% TFA) to give pure 8.5 (5.1 mg, 99% over two steps).  HRESI-MS(+) m/z 862.4360 (calculated for C44H60N7O11: 862.4351). 8.6.4. 2D *MR Spectra of 8.1  Figure 8.14. COSY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. 247   Figure 8.15. HSQC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz.  Figure 8.16. Expanded HSQC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. 248   Figure 8.17. HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz.  Figure 8.18. Expanded HMBC spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. 249   Figure 8.19. ROESY spectrum of 8.1 recorded in DMSO-d6 at 600 MHz. 8.6.5. 2D *MR Spectra of 8.7.  Figure 8.20. COSY spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. 250   Figure 8.21. HSQC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz.  Figure 8.22. HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. 251   Figure 8.23. Expanded HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz.  Figure 8.24. Expanded HMBC spectrum of 8.7 recorded in CD2Cl2 at 600 MHz. 252  8.7. References (1) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596. (2) Galasko, G.; Hora, J.; Toube, T. P.; Weedon, B. C. L.; André, D.; Barbier, M.; Lederer, E.; Villanueva, V. R. J. Chem. Soc. C 1969, 1264-1265. (3) Haugan, J. A. Tetrahedron Lett. 1996, 37, 3887-3890. (4) Liaaen-Jensen, S. Pure Appl. Chem. 1997, 69, 2027-2038. 253  9. Concluding Chapter 9.1. Conclusions  This thesis describes the isolation and structure elucidation of several new biologically active natural products from marine organisms.  In addition, analogues of marine natural products have been synthesized and tested for biological activity with the goal of developing new drug leads.  Chapters 3 through 6 describe new compounds that may have applications in cancer treatment, while chapters 2 and 7 are relevant to the regulation of the immune system.  Indoleamine 2,3-dioxygenase and the process of autophagy are two relatively new cancer drug targets, and inhibitors of IDO and autophagy show promise as anticancer agents in conjunction with other anticancer treatments.  In recent decades, our understanding of cancer at the molecular level has changed the way cancer is treated from using drugs that inhibit DNA synthesis and cell division to drugs that act on specific molecular targets associated with cancer. An example of such a specific drug is imatinib, which inhibits the tyrosine kinase, Bcr-Abl, that is associated with chronic myelogenous lukemia.1  Along with this change, cancer treatment has shifted from a “one disease, one drug” mentality towards individualized treatment, with the goal of increasing effectiveness while minimizing side effects.2  An example of such personalized treatment is the drug trastuzumab, which targets overexpressed HER2 receptors.3  Therefore, side effects from anticancer treatments can be minimized by ensuring that only those patients overexpressing HER2 receive trastuzumab treatment.  In this same manner, inhibitors of IDO could be used, likely in conjunction with other chemotherapeutics, specifically to treat patients that overexpress IDO.  Inhibitors of autophagy could also potentially be used for the treatment of cancer in conjunction with other chemotherapeutics.  However, whether or not to use autophagy inhibitors in the treatment of cancer will also likely depend on the individual, as autophagy 254  inhibitors used on patients with defects in apoptosis could lead to necrosis and increased tumour growth.4  Also, because the connection between autophagy and cancer is poorly understood, a better understanding of autophagy may help to make anticancer therapies more effective, and inhibitors of autophagy could be used as chemical genetic tools to better understand this process.  Irregularasulfate and turnagainolides A and B modulate two enzymes, calcineurin and SHIP1, involved in the regulation of the immune system.  Although irregularasulfate is probably too weak of a calcineurin inhibitor to be used as a drug, it could potentially be used as a biological tool to better understand this enzyme.  This would be particularly appealing if analogues could be discovered that are selective for calcineurin over other phosphatase enzymes. Turnagainolides A and B show good SHIP1-activating activity.  Although turnagainolides A and B are not particularly potent activators of SHIP1 in vitro, the product PI-3,4-P2 also activates SHIP1, so that even a relatively small activation of SHIP1 in vitro can be amplified in vivo.5 9.2. Future Directions  The research presented in this thesis provides opportunities for further research.  In particular, turnagainolides A and B are a good starting point for the synthesis of SHIP1- activating analogues.  The turnagainolides are a new class of SHIP1 activators, and analogues of these compounds have not been thoroughly explored.  Turnagainolides A and B contain a polyketide/shikimate fragment that has not previously been observed in a peptide.  Therefore, a good starting point for the synthesis of analogues is to modify this moiety.  Since turnagainolides A and B can be synthesized on solid-phase, this makes it easier to synthesize a large number of analogues using diversity-oriented synthesis.6,7  Because compound 7.12 is added to the peptide near the end of the synthesis, simply replacing 7.12 with a variety of different compounds, and 255  reacting these with 7.19, can lead to a variety of different analogues of turnagainolides A and B without having to re-do the entire synthesis.  Figure 9.1. Diversity-oriented synthesis of turnagainolide analogues. Although analogues of exiguamine A have been explored as IDO inhibitors, only a few analogues of cinnabarinic acid have been explored.  Cinnabarinic acid appears to be too polar to cross the cell-membrane, so analogues with similar polarity to cinnabarinic acid would not likely make good IDO inhibitors in vivo.  However, the dimethyl ester of cinnabarinic acid appears to show promising activity in vivo.  Therefore, a good starting place for the synthesis of novel IDO inhibitors is to make analogues of the dimethyl ester of cinnabarinic acid that can act as prodrugs of cinnabarinic acid. 9.3. References (1) Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. at. Rev. Drug Discov. 2002, 1, 493-502. (2) Kurzrock, R.; Markman, M. Targeted Cancer Therapy; Humana Press: Totowa, NJ, 2008, pp. 411-425. (3) Hudis, C. A. ew Engl. J. Med. 2007, 357, 39-51. 256  (4) Degenhardt, K.; Mathew, R.; Beaudoin, B.; Bray, K.; Anderson, D.; Chen, G.; Mukherjee, C.; Shi, Y.; Gélinas, C.; Fan, Y.; Nelson, D. A.; Jin, S.; White, E. Cancer Cell 2006, 10, 51-64. (5) Ong, C. J.; Ming-Lum, A.; Nodwell, M.; Ghanipour, A.; Yang, L.; Williams, D. E.; Kim, J.; Demirjian, L.; Qasimi, P.; Ruschmann, J.; Cao, L.-P.; Ma, K.; Chung, S. W.; Duronio, V.; Andersen, R. J.; Krystal, G.; Mui, A. L.-F. Blood 2007, 110, 1942-1949. (6) Peuchmaur, M.; Wong, Y.-S. Comb. Chem. High Throughput Screening 2008, 11, 587- 601. (7) Tan, D. S. at. Chem. Biol. 2005, 1, 74-84.

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