Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Wang, Meng
Doctor of Philosophy - PhD
University of British Columbia
Natural products are one of the most important resources for the discovery and development of new pharmaceuticals. Organisms from different terrestrial and marine environments produce secondary metabolites with unique biological activities that could be utilized by humans medicinally and in other ways. In an effort to search for new potential lead compounds, we use bioassay-guided fractionation and structure elucidation tools to identify new biologically active natural products. The work presented in Chapters 2 and 3 is in an effort to search for new small molecules that activate HIV-1 provirus expression. Novel sesterterpenoids alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24), along with the known sesterterpenoids ansellones A−C (2.86−2.88), were isolated from a Phorbas sp. sponge collected in Howe Sound, British Columbia. The isolation and structure elucidations of these novel sesterterpenoids are presented. These compounds are a family of highly functionalized sesterterpenoids that have the alotane, ansellane, anvilane and phorane carbon skeletons. The unique ansellane and anvilane carbon skeletons have so far only been found in this particular sponge Phorbas sp. Several classes of natural products produced by this genus of sponge are reviewed. These sesterterpenoids are able to activate HIV-1 provirus expression. The most active analog, alotaketal C (3.9) showed 13.8 % activation of HIV-1 provirus expression at a concentration of 30 μM. Chapter 4 describes the identification of two anti-onchocerciasis natural products isolated from the extracts of traditional medicinal plants. Lantadene A (4.4) and morindicinone (4.5) were isolated from the extracts of Lantata camara and Morinda lucida, respectively. The filaricidal properties of lantadene A (4.4) and morindicinone (4.5) were investigated using an in vitro anti-Onchocerca assay.
BIOLOGICALLY ACTIVE NATURAL PRODUCTS FROM TERRESTRIAL AND MARINE ENVIRONMENTS by Meng Wang B.Sc., Wuhan University, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2018 © Meng Wang, 2018 ii Abstract Natural products are one of the most important resources for the discovery and development of new pharmaceuticals. Organisms from different terrestrial and marine environments produce secondary metabolites with unique biological activities that could be utilized by humans medicinally and in other ways. In an effort to search for new potential lead compounds, we use bioassay-guided fractionation and structure elucidation tools to identify new biologically active natural products. The work presented in Chapters 2 and 3 is in an effort to search for new small molecules that activate HIV-1 provirus expression. Novel sesterterpenoids alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24), along with the known sesterterpenoids ansellones A−C (2.86−2.88), were isolated from a Phorbas sp. sponge collected in Howe Sound, British Columbia. The isolation and structure elucidations of these novel sesterterpenoids are presented. These compounds are a family of highly functionalized sesterterpenoids that have the alotane, ansellane, anvilane and phorane carbon skeletons. The unique ansellane and anvilane carbon skeletons have so far only been found in this particular sponge Phorbas sp. Several classes of natural products produced by this genus of sponge are reviewed. These sesterterpenoids are able to activate HIV-1 provirus expression. The most active analog, alotaketal C (3.9) showed 13.8 % activation of HIV-1 provirus expression at a concentration of 30 µM. Chapter 4 describes the identification of two anti-onchocerciasis natural products isolated from the extracts of traditional medicinal plants. Lantadene A (4.4) and morindicinone (4.5) were isolated from the extracts of Lantata camara and Morinda lucida, respectively. The filaricidal iii properties of lantadene A (4.4) and morindicinone (4.5) were investigated using an in vitro anti-Onchocerca assay. iv Lay Summary Natural products are chemical compounds produced by a living organism. Organisms from different terrestrial and marine environments produce natural products with unique biological activities that could be utilized by humans medicinally and in other ways. They are one of the most important resources for the discovery and development of new pharmaceuticals, and many of them have become current drugs such as paclitaxel for the treatment of cancer and artemisinin for the treatment of malaria. In this thesis, I studied the natural products from some crude extracts of marine invertebrates and plants in an effort to search for new potential lead compounds for drug development. A series of pure compounds were isolated from the natural sources, and their chemical structures were determined using spectroscopic methods. The unique anti-HIV and antiparasitic activities of these compounds were also investigated. v Preface Part of the work descried in Chapter 3 has been published in two separate manuscripts. Wang, M.; Tietjen, I.; Chen, M.; Williams, D. E.; Daoust, J.; Brockman, M. A.; Andersen, R. J. Sesterterpenoids Isolated from the Sponge Phorbas sp. Activate Latent HIV‐1 Provirus Expression. J. Org. Chem. (2016), 81, 11324-11334. Daoust, J.; Chen, M.; Wang, M.; Williams, D. E.; Chavez, M. A. G.; Wang, Y. A.; Merchant, C. E.; Fontana, A.; Kieffer, T. J.; Andersen, R. J. Sesterterpenoids Isolated from a Northeastern Pacific Phorbas sp. J. Org. Chem. (2013), 78, 8267-8273. The original isolation of ansellones A–C (2.86–2.88) was accomplished by Dr. Julie Daoust and Dr. David E. Williams, I re-isolated ansellones A–C (2.86–2.88) to acquire enough material for biological assays. Structure elucidation of alotaketal C (3.7), phorbadione (3.10), ansellone N (3.21) and anvilone A (3.22) were a collaborative effort with Dr. David E. Williams and Dr. Min Chen, I isolated the compounds for biological assays and Dr. David E. Williams helped me with collecting NMR data and structure elucidation. The isolation and characterization/structure elucidation of alotaketals D (3.8) and E (3.9), ansellones D–M (3.11–3.20), anvilone B (3.23) and phorone C (3.24) were carried out by myself in full. Dr. Yan Alex Wang conducted the theoretical calculation of the CD spectrum for phorbadione (3.10). Dr. Ian Tietjen and Dr. Mark A. Brockman conducted all biological assays. My supervisor Professor Raymond J. Andersen wrote the majority of the original manuscript. Chapters 4 are based on work conducted in collaboration with members of Prof. Fidelis Cho-Ngwa’s laboratory. I was responsible for the isolation and characterization of the natural products. Dr. Brian O. Patrick provided the X-ray analysis. vi Table of Contents Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables .................................................................................................................................. x List of Figures .............................................................................................................................. xii List of Symbols ......................................................................................................................... xxiii List of Abbreviations ............................................................................................................... xxiv Acknowledgements ................................................................................................................. xxvii Dedication ............................................................................................................................... xxviii Chapter 1: Introduction ................................................................................................................1 1.1 Natural products chemistry ................................................................................................ 1 1.2 Natural products in drug discovery .................................................................................... 2 1.3 Marine natural products ..................................................................................................... 7 1.4 Methods and tools used for natural products drug discovery ............................................ 9 1.4.1 Isolation methods ...................................................................................................... 10 1.4.2 Structure elucidation methods ................................................................................... 12 1.5 Biosynthesis of natural products ...................................................................................... 13 1.5.1 Fatty acids and polyketides ....................................................................................... 16 1.5.2 Terpenoids and steroids ............................................................................................ 17 1.5.3 Aromatic amino acids and phenylpropanoids ........................................................... 20 vii 1.5.4 Alkaloids ................................................................................................................... 22 1.5.5 Peptides ..................................................................................................................... 24 1.6 Research goals ................................................................................................................. 25 Chapter 2: Metabolites isolated from the sponge Phorbas sp. .................................................27 2.1 The genus Phorbas .......................................................................................................... 27 2.2 Secondary metabolites produced by the genus Phorbas .................................................. 28 2.2.1 Alkaloids and polyketides from the genus Phorbas ................................................. 28 2.2.2 Terpenoids and steroids isolated from the genus Phorbas ....................................... 30 2.2.2.1 Sesterterpenoids isolated from the genus Phorbas ............................................ 35 Chapter 3: Sesterterpenoids isolated from the British Columbia sponge Phorbas sp. activate latent HIV-1 provirus expression .................................................................................39 3.1 Sesterterpenoids ............................................................................................................... 39 3.2 Biological background ..................................................................................................... 40 3.3 Search for new sesterterpenoids from the sponge Phorbas sp. that activates latent HIV-1 provirus expression ................................................................................................................... 42 3.4 Structure elucidation of alotaketals .................................................................................. 43 3.4.1 Structure elucidation of alotaketal C (3.7) ................................................................ 43 3.4.2 Structure elucidation of alotaketal D (3.8) and E (3.9) ............................................. 49 3.5 Structure elucidation of ansellones and phorbadione ...................................................... 61 3.5.1 Ansellones A, B and C (2.86−2.88) .......................................................................... 61 3.5.2 Structure elucidation of phorbadione (3.10) ............................................................. 65 3.5.3 Structure elucidation of ansellone D (3.11) .............................................................. 76 3.5.4 Structure elucidation of ansellone E (3.12) ............................................................... 81 viii 3.5.5 Structure elucidation of ansellone F (3.13) ............................................................... 87 3.5.6 Structure elucidation of ansellone G (3.14) .............................................................. 95 3.5.7 Structure elucidation of ansellone H (3.15) ............................................................ 101 3.5.8 Structure elucidation of ansellone I (3.16) .............................................................. 108 3.5.9 Structure elucidation of ansellone J (3.17) ............................................................. 114 3.5.10 Structure elucidation of ansellone K (3.18) .......................................................... 120 3.5.11 Structure elucidation of ansellone L (3.19) ........................................................... 128 3.5.12 Structure elucidation of ansellone M (3.20) ......................................................... 133 3.5.13 Structure elucidation of ansellone N (3.21) .......................................................... 139 3.6 Structure elucidation of anvilones ................................................................................. 146 3.6.1 Structure elucidation of anvilone A (3.22) ............................................................. 146 3.6.2 Structure elucidation of anvilone B (3.23) .............................................................. 153 3.7 Structure elucidation of phorone C (3.24) ..................................................................... 158 3.8 Biogenesis of alotaketals, ansellones, phorones and anvilones ..................................... 169 3.9 Biological activities ....................................................................................................... 171 3.10 Conclusions and future work ....................................................................................... 178 3.11 Experimental section .................................................................................................... 180 3.11.1 General experimental procedures ......................................................................... 180 3.11.2 Sample collection, extraction and isolation .......................................................... 181 3.11.3 Bioassays............................................................................................................... 185 Chapter 4: Anti-onchocerciasis properties of African plant extracts ...................................186 4.1 Onchocerciasis ............................................................................................................... 186 4.2 Filaricidal properties of the extracts of Lantana camara ............................................... 188 ix 4.2.1 Lantana camara as a traditional medicinal plant .................................................... 188 4.2.2 Lantadene A (4.4) ................................................................................................... 188 4.2.3 Biological activities of lantadene A (4.4) ............................................................... 198 4.3 Filaricidal properties of the extracts of Morinda lucida ................................................ 198 4.3.1 Morinda lucida as a traditional medicinal plant ..................................................... 198 4.3.2 Morindicinone (4.5) ................................................................................................ 199 4.3.3 Biological activity of morindicinone (4.5) .............................................................. 205 4.4 Experimental section ...................................................................................................... 205 4.4.1 General experimental procedures ........................................................................... 205 4.4.2 Sample preparation, extraction and isolation .......................................................... 206 4.4.2.1 Lantana camara extracts .................................................................................. 206 4.4.2.2 Morinda lucida extracts ................................................................................... 207 4.4.3 Bioassays................................................................................................................. 207 Chapter 5: Conclusion ...............................................................................................................208 References ...................................................................................................................................212 x List of Tables Table 3.1 NMR data for alotaketal B (2.73) and C (3.7) recorded in C6D6 at 600 MHz .............. 43 Table 3.2 NMR data for alotaketal C (3.7), D (3.8) and E (3.9) recorded in C6D6 at 600 MHz .. 56 Table 3.3 NMR data for phorbadione (3.10) recorded in C6D6 and CD3OD at 600 MHz ........... 67 Table 3.4 NMR data for ansellone A (2.86) and ansellone D (3.11) recorded in C6D6 at 600 MHz....................................................................................................................................................... 76 Table 3.5 NMR data for ansellone A (2.86) and ansellone E (3.12) recorded in C6D6 at 600 MHz....................................................................................................................................................... 81 Table 3.6 NMR data for ansellone A (2.86) and ansellone F (3.13) recorded in C6D6 at 600 MHz....................................................................................................................................................... 87 Table 3.7 NMR data for phorbadione (3.10) and ansellone G (3.14) recorded in C6D6 at 600 MHz .............................................................................................................................................. 95 Table 3.8 NMR data for ansellone A (2.86) and ansellone H (3.15) recorded in C6D6 at 600 MHz..................................................................................................................................................... 101 Table 3.9 NMR data for ansellone I (3.16) and ansellone H (3.15) recorded in C6D6 at 600 MHz..................................................................................................................................................... 108 Table 3.10 NMR data for ansellone A (2.86) and ansellone J (3.17) recorded in C6D6 at 600 MHz..................................................................................................................................................... 114 Table 3.11 NMR data for ansellone A (2.86) and ansellone K (3.18) recorded in C6D6 at 600 MHz ............................................................................................................................................ 120 Table 3.12 NMR data for ansellone L (3.19) and ansellone A (2.86) recorded in C6D6 at 600 MHz ............................................................................................................................................ 128 xi Table 3.13 NMR data for ansellone M (3.20) and ansellone D (3.11) recorded in C6D6 at 600 MHz ............................................................................................................................................ 133 Table 3.14 NMR data for ansellone A (2.86) and ansellone N (3.21) recorded in C6D6 at 600 MHz ............................................................................................................................................ 139 Table 3.15 NMR data for anvilone A (?) recorded in C6D6 at 600 MHz ................................... 148 Table 3.16 NMR data for anvilone A (3.22) and anvilone B (3.23) recorded in C6D6 at 600 MHz..................................................................................................................................................... 154 Table 3.17 NMR data for phorone C (3.24) recorded in C6D6 and (CD3)2SO at 600 MHz ....... 159 Table 3.18 Effects of sesterterpenoids on HIV-1 provirus / GFP expression and viability of J-Lat 9.2 cells ....................................................................................................................................... 176 Table 3.19 Effects of sesterterpenoids in combination with control LRAs to induce HIV-1 provirus / GFP expression ........................................................................................................... 177 Table 3.20 Effects of sesterterpenoids and control LRAs in combination with PKC inhibitors 178 Table 4.1 NMR data for lantadene A (4.4) recorded in CDCl3 at 600 MHz ............................... 190 Table 4.2 NMR data recorded for morindicinone (4.5) recorded in CDCl3 at 600 MHz ............ 201 xii List of Figures Figure 1.1 From cone snail toxin to Prialt® .................................................................................... 2 Figure 1.2 Morphine (1.2), salicylic acid (1.3) and their analogues Heroin (1.4) and Aspirin (1.5)......................................................................................................................................................... 3 Figure 1.3 Structures of penicillin G (1.6) and amoxicillin (1.7) ................................................... 4 Figure 1.4 Examples of natural product drugs: structures of erythromycin A (1.8), paclitaxel (1.9), avermectin B1 (1.10), artemisinin (1.11) and lovastatin (1.12) ............................................ 6 Figure 1.5 All small molecule approved drugs 1981-2014s; n=1211.22 ......................................... 7 Figure 1.6 Structures for spongothymidine (1.13), Ara A (1.14) and Ara C (1.15) ....................... 8 Figure 1.7 Examples of marine drugs: Yondelis® and Halaven® ................................................... 8 Figure 1.8 From “hit” to “drug lead”: the typical workflow of natural products chemists .......... 10 Figure 1.9 General scheme for bioassay-guided fractionation ..................................................... 11 Figure 1.10 Commonly used 2D NMR experiments .................................................................... 13 Figure 1.11 The common building blocks in natural products biosynthesis ................................ 15 Figure 1.12 The biosynthesis of erythronolide A (1.8)44 .............................................................. 17 Figure 1.13 Terpenoid biosynthesis .............................................................................................. 18 Figure 1.14 Biosynthesis of paclitaxel (1.9)46-48 ........................................................................... 19 Figure 1.15 Biosynthesis of cholesterol (1.39) ............................................................................. 20 Figure 1.16 Biosynthesis of salicylic acid (1.3) ............................................................................ 21 Figure 1.17 Biosynthesis of morphine (1.1)50 ............................................................................... 23 Figure 1.18 Biosynthesis of vancomycin (1.55) ........................................................................... 25 Figure 2.1 Phorbas sp. collected in Howe Sound, BC (picture from Mike LeBlanc) .................. 27 xiii Figure 2.2 Structures of phorbazoles A−D (2.1−2.4) ................................................................... 28 Figure 2.3 Structures of phorboxazoles A and B (2.5 and 2.6) and Hemi-phorboxazole A (2.7) 29 Figure 2.4 Structures of phorbasides A−I (2.8−2.16) ................................................................... 29 Figure 2.5 Structures of muironolide A (2.17) and callipeltosides A−C (2.18−2.20) .................. 30 Figure 2.6 Structures of phorbasins A−K (2.21−2.31) ................................................................. 31 Figure 2.7 Structures of gagunins A−Q (2.32−2.48) .................................................................... 32 Figure 2.8 Structures of gukulenins A−F (2.49−2.54) .................................................................. 33 Figure 2.9 Structures of anthosterones A and B (2.55 and 2.56) and phorbasterones A−D (2.57−2.60) .................................................................................................................................... 33 Figure 2.10 Structures of amaroxocanes A and B (2.61 and 2.62) ............................................... 34 Figure 2.11 Structures of amaranzoles A−F (2.63−2.68) ............................................................. 34 Figure 2.12 Structures of phorbaketals A−N (2.69−2.71, 2.74−2.81, 2.83−2.85), alotaketals A and B (2.72 and 2.73) and phorbin (2.82) ..................................................................................... 36 Figure 2.13 Sturctures of ansellones A−C (2.86−2.88) ................................................................ 37 Figure 2.14 Sturcture of secoepoxyansellone A (2.89) ................................................................. 37 Figure 2.15 Structures of phorbasones A, B and phorbasone A acetate (2.90−2.92) ................... 38 Figure 2.16 Structures of phorones A and B (2.93 and 2.94) and isophorbasone A (2.95) .......... 38 Figure 3.1 Examples of sesterterpenoids isolated from terrestrial fungi, insects, plants and marine sponge ........................................................................................................................................... 40 Figure 3.2 Structures of prostratin (3.5) and bryostatin 1 (3.6) .................................................... 41 Figure 3.3 J-Lat 9.2 GFP-reporter CD4+ T cell line111 ................................................................ 42 Figure 3.4 Structures of alotaketal C (3.7), alotaketal D (3.8) and alotaketal E (3.9) .................. 43 Figure 3.5 Selected COSY and HMBC correlations observed for alotaketal C (3.7) in C6D6. .... 46 xiv Figure 3.6 1H NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 ..................... 46 Figure 3.7 13C NMR spectrum of alotaketal C (3.7) recorded at 150 MHz in C6D6 .................... 47 Figure 3.8 gradHSQC NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 ........ 47 Figure 3.9 gradHMBC NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 ....... 48 Figure 3.10 gradCOSY60 NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 .. 48 Figure 3.11 tROESY NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 ......... 49 Figure 3.12 Selected COSY and HMBC correlations observed for alotaketal D (3.8) in C6D6. .. 50 Figure 3.13 1H NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 ................... 51 Figure 3.14 13C NMR spectrum of alotaketal D (3.8) recorded at 150 MHz in C6D6 ................. 51 Figure 3.15 COSY NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 ............. 52 Figure 3.16 HSQC NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 ............. 52 Figure 3.17 HMBC NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 ............ 53 Figure 3.18 tROESY NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 ......... 53 Figure 3.19 Determination of the configuration at C-4 in alotaketal D (3.8) ............................... 55 Figure 3.20 Coupling constants at C-4 for phorbaketal B (2.70) and C (2.71) ............................ 55 Figure 3.21 Selected COSY and HMBC correlations for alotaketal E (3.9) in C6D6. .................. 58 Figure 3.22 1H NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 .................... 58 Figure 3.23 13C NMR spectrum of alotaketal E (3.9) recorded at 150 MHz in C6D6 ................... 59 Figure 3.24 COSY NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 ............. 59 Figure 3.25 HSQC NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 ............. 60 Figure 3.26 HMBC NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 ............ 60 Figure 3.27 tROESY NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 .......... 61 Figure 3.28 1H NMR spectrum of ansellone A (2.86) recorded at 600 MHz in C6D6 .................. 62 xv Figure 3.29 Structures of ansellones A−N (2.86−2.88, 3.11−3.21) and phorbadione (3.10) isolated from the sponge Phorbas sp. ........................................................................................... 63 Figure 3.30 1H NMR spectrum of ansellone B (2.87) recorded at 600 MHz in C6D6 .................. 64 Figure 3.31 1H NMR spectrum of ansellone C (2.88) recorded at 600 MHz in C6D6 .................. 65 Figure 3.32 Selected COSY and HMBC correlations observed for phorbadione (3.10) in C6D6 and CD3OD. .................................................................................................................................. 67 Figure 3.33 CD spectrum for phorbadione (3.10) (panel A, experimental CD spectrum for 3.10 in CH2Cl2 at < 0.1 mg/mL; panel B, calculated theoretical spectrum for structure I; panel C, calculated theoretical spectrum for structure II) ........................................................................... 69 Figure 3.34 1H NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 ................. 70 Figure 3.35 13C NMR spectrum of phorbadione (3.10) recorded at 150 MHz in C6D6 ................ 70 Figure 3.36 gradCOSY60 NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 71 Figure 3.37 gradHSQC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 ... 71 Figure 3.38 gradHMBC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 .. 72 Figure 3.39 tROESY NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 ....... 72 Figure 3.40 1H NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD. ........... 73 Figure 3.41 13C NMR spectrum of phorbadione (3.10) recorded at 150 MHz in CD3OD ........... 73 Figure 3.42 gradCOSY60 NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD....................................................................................................................................................... 74 Figure 3.43 gradHSQC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD74 Figure 3.44 gradHMBC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD....................................................................................................................................................... 75 Figure 3.45 tROESY NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD .. 75 xvi Figure 3.46 Selected COSY and HMBC correlations observed for ansellone D (3.11) in C6D6 . 78 Figure 3.47 1H NMR spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 .................. 78 Figure 3.48 13C NMR spectrum of ansellone D (3.11) recorded at 150 MHz in C6D6 ................. 79 Figure 3.49 COSY spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 ..................... 79 Figure 3.50 HSQC spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 ..................... 80 Figure 3.51 HMBC spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 .................... 80 Figure 3.52 tROESY spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 .................. 81 Figure 3.53 Selected COSY, HMBC and tROESY correlations observed for ansellone E (3.12) in C6D6 .............................................................................................................................................. 83 Figure 3.54 1H NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 .................. 84 Figure 3.55 13C NMR spectrum of ansellone E (3.12) recorded at 150 MHz in C6D6 ................. 84 Figure 3.56 COSY NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 ............ 85 Figure 3.57 HSQC NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 ............ 85 Figure 3.58 HMBC NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 ........... 86 Figure 3.59 tROESY NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 ........ 86 Figure 3.60 Selected COSY and HMBC correlations observed for ansellone F (3.13) in C6D6 .. 90 Figure 3.61 Selected tROESY correlations observed for ansellone F (3.13) in C6D6 .................. 91 Figure 3.62 1H NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 .................. 91 Figure 3.63 13C NMR spectrum of ansellone F (3.13) recorded at 150 MHz in C6D6 ................. 92 Figure 3.64 APT NMR spectrum of ansellone F (3.13) recorded at 150 MHz in C6D6 ............... 92 Figure 3.65 COSY NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 ............ 93 Figure 3.66 HSQC NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 ............ 93 Figure 3.67 HMBC NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 ........... 94 xvii Figure 3.68 tROESY NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 ........ 94 Figure 3.69 Selected COSY, HMBC and tROESY correlations observed for ansellone G (3.14) in C6D6 .......................................................................................................................................... 97 Figure 3.70 1H NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 .................. 98 Figure 3.71 13C NMR spectrum of ansellone G (3.14) recorded at 150 MHz in C6D6 ................. 98 Figure 3.72 COSY NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 ........... 99 Figure 3.73 HSQC NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 ........... 99 Figure 3.74 HMBC NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 ........ 100 Figure 3.75 tROESY NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 ...... 100 Figure 3.76 Selected COSY, HMBC and tROESY correlations observed for ansellone H (3.15) in C6D6 ........................................................................................................................................ 104 Figure 3.77 1H NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 ................ 105 Figure 3.78 13C NMR spectrum of ansellone H (3.15) recorded at 150 MHz in C6D6 ............... 105 Figure 3.79 COSY NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 ......... 106 Figure 3.80 HSQC NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 ......... 106 Figure 3.81 HMBC NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 ........ 107 Figure 3.82 tROESY NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 ...... 107 Figure 3.83 Selected COSY, HMBC and tROESY correlations observed for ansellone I (3.16) in C6D6 ............................................................................................................................................ 110 Figure 3.84 1H NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 ................. 111 Figure 3.85 13C NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 ................ 111 Figure 3.86 COSY NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 ........... 112 Figure 3.87 HSQC NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 ........... 112 xviii Figure 3.88 HMBC NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 .......... 113 Figure 3.89 tROESY NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 ....... 113 Figure 3.90 Selected COSY and HMBC correlations observed for ansellone J (3.17) in C6D6 . 116 Figure 3.91 1H NMR spectrum of ansellone J (3.17, in red) and decomposed ansellone J (3.17, in blue) recorded at 600 MHz in C6D6 ............................................................................................ 117 Figure 3.92 1H NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 ................. 118 Figure 3.93 COSY NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 ........... 118 Figure 3.94 HSQC NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 ........... 119 Figure 3.95 HMBC NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 ......... 119 Figure 3.96 Selected COSY, HMBC and tROESY correlations observed for ansellone K (3.18) in C6D6 ........................................................................................................................................ 122 Figure 3.97 1H NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 ................ 124 Figure 3.98 13C NMR spectrum of ansellone K (3.18) recorded at 150 MHz in C6D6 ............... 125 Figure 3.99 DEPT135 NMR spectrum of ansellone K (3.18) recorded at 150 MHz in C6D6 .... 125 Figure 3.100 COSY NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 ....... 126 Figure 3.101 HSQC NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 ....... 126 Figure 3.102 HMBC NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 ...... 127 Figure 3.103 tROESY NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 .... 127 Figure 3.104 Selected COSY and HMBC correlations observed for ansellone L (3.19) in C6D6..................................................................................................................................................... 130 Figure 3.105 1H NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 .............. 130 Figure 3.106 13C NMR spectrum of ansellone L (3.19) recorded at 150 MHz in C6D6 ............. 131 Figure 3.107 COSY NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 ........ 131 xix Figure 3.108 HSQC NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 ........ 132 Figure 3.109 HMBC NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 ....... 132 Figure 3.110 Selected COSY, HMBC and tROESY correlations observed for ansellone M (3.20) in C6D6 ........................................................................................................................................ 135 Figure 3.111 1H NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 ............. 136 Figure 3.112 13C NMR spectrum of ansellone M (3.20) recorded at 150 MHz in C6D6 ............ 136 Figure 3.113 DEPT135 NMR spectrum of ansellone M (3.20) recorded at 150 MHz in C6D6 . 137 Figure 3.114 COSY NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 ....... 137 Figure 3.115 HSQC NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 ....... 138 Figure 3.116 HMBC NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 ..... 138 Figure 3.117 tROESY NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 ... 139 Figure 3.118 Selected COSY and HMBC correlations observed for ansellone N (3.21) in C6D6..................................................................................................................................................... 141 Figure 3.119 Selected ROESY correlations observed for ansellone N (3.21) in C6D6 .............. 143 Figure 3.120 1H NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 .............. 143 Figure 3.121 COSY NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 ....... 144 Figure 3.122 HSQC NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 ....... 144 Figure 3.123 HMBC NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 ...... 145 Figure 3.124 tROESY NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 .... 145 Figure 3.125 Structures of anvilones A (3.22) and B (3.23) ....................................................... 146 Figure 3.126 Fragments of anvilone A (3.22), important COSY and HMBC correlations shown...................................................................................................................................................... 147 Figure 3.127 Selected ROESY correlations observed for anvilone A (3.22) in C6D6 ................ 150 xx Figure 3.128 1H NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 ............... 150 Figure 3.129 13C NMR spectrum of anvilone A (3.22) recorded at 150 MHz in C6D6 .............. 151 Figure 3.130 COSY NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 ......... 151 Figure 3.131 HSQC NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 ......... 152 Figure 3.132 HMBC NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 ....... 152 Figure 3.133 tROESY NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 ..... 153 Figure 3.134 1H NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 ............... 155 Figure 3.135 13C NMR spectrum of anvilone B (3.23) recorded at 150 MHz in C6D6 .............. 156 Figure 3.136 COSY NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 ......... 156 Figure 3.137 HSQC NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 ......... 157 Figure 3.138 HMBC NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 ........ 157 Figure 3.139 tROESY NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 ..... 158 Figure 3.140 Structure of phorone C (3.24) ................................................................................ 158 Figure 3.141 Selected COSY and HMBC correlations observed for phorone C (3.24) in C6D6 and (CD3)2SO .................................................................................................................................... 160 Figure 3.142 Selected ROESY correlations observed for phorone C (3.24) in (CD3)2SO ......... 162 Figure 3.143 1H NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 ................ 163 Figure 3.144 13C NMR spectrum of phorone C (3.24) recorded at 150 MHz in C6D6 ............... 163 Figure 3.145 COSY NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 ......... 164 Figure 3.146 HSQC NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 ......... 164 Figure 3.147 HMBC NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 ........ 165 Figure 3.148 tROESY NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 ...... 165 Figure 3.149 1H NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO ........ 166 xxi Figure 3.150 13C NMR spectrum of phorone C (3.24) recorded at 150 MHz in (CD3)2SO ....... 166 Figure 3.151 COSY NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO .. 167 Figure 3.152 HSQC NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO .. 167 Figure 3.153 HMBC NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO . 168 Figure 3.154 tROESY NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO..................................................................................................................................................... 168 Figure 3.155 1D NOE NMR spectrum of phorone C (3.24) irradiated at 0.69 ppm recorded at 600 MHz in C6D6 ........................................................................................................................ 169 Figure 3.156 Biogenetic proposal for the formation of the alotaketals, ansellones, phorones and anvilones ..................................................................................................................................... 171 Figure 3.157 Effects of sesterterpenoids on HIV-1 provirus/GFP expression ........................... 172 Figure 3.158 Preliminary SAR for sesterterpenoids isolated from Phorbas sp. ......................... 173 Figure 3.159 Preliminary SAR for alotaketals C−E (3.7−3.9) ................................................... 175 Figure 3.160 Preliminary SAR for ansellones A and D−G (2.86 and 3.11−3.14) ...................... 175 Figure 3.161 Effects of sesterterpenoids on viability of J-Lat 9.2 cells ..................................... 176 Figure 3.162 Sesterterpene skeletons isolated from the sponge Phorbas sp. ............................. 179 Figure 4.1 Current drugs used for the treatment of onchocerciasis ............................................ 187 Figure 4.2 Bioassay-guided fractionation of the crude extracts of Lantana camara .................. 189 Figure 4.3 Structure of lantadene A (4.4) ................................................................................... 190 Figure 4.4 Fragments of lantadene A (4.4), selected COSY and HMBC correlations shown .... 193 Figure 4.5 NMR assignment for landadene A (4.4), data collected in CDCl3 at 600 MHz ........ 194 Figure 4.6 1H NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 ................. 194 Figure 4.7 13C NMR spectrum of lantadene A (4.4) recorded at 150 MHz in CDCl3 ................ 195 xxii Figure 4.8 APT NMR spectrum of lantadene A (4.4) recorded at 100 MHz in CDCl3 .............. 195 Figure 4.9 COSY NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 ........... 196 Figure 4.10 HSQC NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 ......... 196 Figure 4.11 HMBC NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 ....... 197 Figure 4.12 ORTEP diagram generated by the X-ray diffraction analysis of lantadene A (4.4) 197 Figure 4.13 Bioassay-guided fractionation of the crude extracts of Morinda lucida ................. 200 Figure 4.14 Morindicinone (4.5) ................................................................................................. 201 Figure 4.15 NMR data for morindicinone (4.5) collected in CDCl3 at 600 MHz with selected COSY and HMBC correlations .................................................................................................. 202 Figure 4.16 1H NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 ........... 202 Figure 4.17 COSY NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 .... 203 Figure 4.18 HSQC NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 .... 203 Figure 4.19 HMBC NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 ... 204 Figure 4.20 tROESY NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 . 204 Figure 5.1 Sesterterpenoids isolated from the marine sponge Phorbas sp. collected in BC ...... 209 Figure 5.2 Anti-onchocerciasis natural products isolated from plant extracts ............................ 211 xxiii List of Symbols °C degree Celsius [α]D20 specific rotation at sodium D-line recorded at 20 °C 13C carbon 13 C18 linear octadecane groups δ chemical shift in parts per million from tetramethyl silane 1H proton J coupling constant (in hertz) λ wavelength λmax wavelength, giving a local maximum absorbance m/z mass-to-charge ratio xxiv List of Abbreviations 1D one dimensional 2D two dimensional Ac acetyl BC British Columbia br broad C6D6 deuterated benzene CD circular dichroism CDCl3 deuterated chloroform CD3OD deuterated methanol COSY correlation spectroscopy d doublet DCM dichloromethane dd doublet of doublets DEPT distortionless enhancement by polarization transfer dm doublet of multiplets DMSO dimethyl sulfoxide dt doublet of triplets EC50 half maximal effective concentration EtOAc ethyl acetate FDA U.S. Food and Drug Administration xxv g gram GFP green fluorescent protein HIV human immunodeficiency virus HMBC heteronuclear multiple bond coherence HPLC high performance liquid chromatography HRESIMS high resolution electrospray ionization mass spectrometry HSQC heteronuclear single bond coherence HTS high-throughput screening Hz hertz IC50 half maximal inhibitory concentration IR infrared spectroscopy LRA latent reversal agent LRESIMS low resolution electrospray ionization mass spectrometry µg microgram µM micromolar m multiplet Me methyl MeCN acetonitrile MeOD deuterated methanol MeOH methanol mg milligram MHz megahertz xxvi min minute mL millilitre mRNA MRSA MS messenger ribonucleic acid methicillin-resistant Staphylococcus aureus mass spectrometry NMR nuclear magnetic resonance NOESY nuclear overhauser effect spectroscopy NRPS ORTEP non-ribosomal peptide synthase oak ridge thermal ellipsoid plot ppm parts per million ROESY rotating frame nuclear overhauser effect spectroscopy s singlet SAM S-adenosyl methionine SAR structure activity relationship SCUBA self-contained underwater breathing apparatus sp. species t triplet TLC thin layer chromatogrphy TOF time of flight UPLC ultra high pressure liquid chromatrogrphy UV altraviolet xxvii Acknowledgements I would first like to thank my supervisor Dr. Raymond J. Andersen for his guidance, support, patience and encouragement. It would be impossible for me to finish any of the work presented here without his mentorship. I am very fortunate to work with him. I would like to thank Dr. David E. Williams, Mike LeBlanc and Dr. Doralyn Dalisay for their help and guidance in the lab. I would also like to thank Luping, Ryan, Tina, Ben, Jack, Rosanne, Lingzhi, Andrew, Ping, Kalindi, Pamoda and all Andersen lab members for their help and support. I really enjoyed all the fun time we have together. Many thanks also go to our collaborators Dr. Ian Tietjen, Dr. Fidelis Cho-Ngwa and Dr. Gerry Krystal. I would also thank the NMR and MS lab staff Dr. Paul Zhicheng Xia, Dr. Maria Ezhova, Dr. Yun Ling and Marshall Lapawa. At last, I would like to thank my family and friends. My deepest gratitude and love go to my parents. All the support they have provided me over the years was the greatest gift. Thanks for always being supportive, patient and understanding. xxviii Dedication For Jintao and Guanghui 1 Chapter 1: Introduction 1.1 Natural products chemistry Living organisms produce a variety of organic compounds (metabolites) for their living, growth and reproduction. These compounds are essential to the life of living organisms, and these compounds can generally be classified as primary metabolites and secondary metabolites. Primary metabolites, such as sugars and proteins, are crucial to the basic living of organisms. Unlike primary metabolites, secondary metabolites are not directly related to fundamental biological pathways like the glycolytic pathway. Those compounds that are not absolutely required for the survival of the organism are classified as secondary metabolites. They are also called natural products or specialized metabolites. The reasons why these organisms produce secondary metabolites have not been conclusively found. There are several hypotheses which have been proposed: 1) secondary metabolites have (had) a role in the function and survival of the organism; 2) secondary metabolism is merely a waste process; 3) secondary metabolism is an overflow process, and the process, rather than the products, is important; 4) secondary metabolism provides organisms with a means of adjustment to changing circumstances; 5) secondary metabolites serve as a means of chemical defense or attraction for the organisms.1,2 The fifth hypothesis is generally acceptable based on some research results. For example, the Neem tree biosynthesizes a series of alkaloids to repel insects;3 the cone snail Conus sp. biosynthesizes several hundred peptide toxins for prey capture, defense and competitor deterrence.4 One of the compounds in the Conus toxin was isolated by the Olivera group,5 and it was developed to a clinical drug ziconotide (1.1, trade name Prialt®) for relieving severe and chronic pain (Figure 1.1, cone snail picture from Olivera 2 paper, Prialt® picture from Prialt® website). Figure 1.1 From cone snail toxin to Prialt® 1.2 Natural products in drug discovery Natural products have played an important role in drug discovery since ancient times, when our ancestors started to seek cures for human diseases. For example, willow bark has been used for hundreds of years to relieve pain. As a result of the development of organic chemistry, scientists started to look into the active compounds from medicinal plants used by our ancestors. One major breakthrough of natural products chemistry in drug discovery was the isolation of morphine (1.2) from Papverum somniferum (opium) by the German pharmacist Emanuel Merck in 1826. This alkaloid was the first pure active compound to be isolated from a medicinal plant.6 Also, the active compound that is responsible for pain relieving in willow bark was identified as salicylic acid (1.3) in the mid-1800s.7 3 Figure 1.2 Morphine (1.2), salicylic acid (1.3) and their analogues Heroin (1.4) and Aspirin (1.5) At the same time, synthetic chemists were trying to synthesize natural products and their analogues. The English chemist C. R. Alder Wright synthesized diacetylmorphine (1.4), an analogue of morphine (1.1), in 1874.8 The German chemist Felix Hoffman working at Bayer Pharmaceuticals then synthesized this compound independently in 1897. This analogue was found to be more potent than morphine (1.1), and Bayer commercialized it as a cough suppressant under the trade name “Heroin” in 1898.9 Aspirin (1.5), a salicylic acid (1.3) analogue, was also synthesized and commercialized by Bayer in 1900 as a pain relieving medicine.7 The advances of microbiology in the early 1900s have made microorganisms an important source of natural products. One famous example was the discovery of penicillin (1.6) by Alexander Fleming in 1929.10 Howard Florey and Ernst Chain purified penicillin G (1.6) in 1939 and people started to use it as an antibiotic to treat wound infections in World War II.11 Penicillin G (1.6) is rarely used now, but its derivative amoxicillin (1.7) is still being used as an antibiotic today. The structure of penicillin G (1.6) and amoxicillin (1.7) are shown in Figure 1.3. ONHOHOHMorphine(1.2)OHOHOSalicylic acid(1.3)ONAcOAcOHDiacetylmorphine(1.4, Heroin)OAcOHOAcetylsalicylic acid(1.5, Aspirin)Natrual products isolated from plants Synthetic analogues of natrual products 4 Figure 1.3 Structures of penicillin G (1.6) and amoxicillin (1.7) After these early discoveries of natural products and medicinal chemistry, there was a golden era of natural products discovery from the mid to late-twentieth century. During this period, a huge number of natural products with structural diversity were isolated and identified. They showed a broad range of biological activities and some of them have been approved as drugs for a wide variety of therapeutic applications. Erythromycin (1.8, Figure 1.4), formerly called ilotycin, was isolated from a soil bacteria Streptomyces erythreus by researchers working at Eli Lilly.12 It showed good clinical results as an antibiotic drug and it was commercialized by Lilly in 1952.13 Paclitaxel (1.9, Figure 1.4) was isolated from the bark of the pacific yew tree Taxus brevifolia in 1966, as part of the screening program initiated in the 1950s by Jonathan Hartwell of the National Cancer Institute to find antitumor natural products.14,15 Paclitaxel (1.9) showed cytotoxic activities in several mouse antitumor screening assays, and after clinical trials it became one of the treatments for ovarian cancer under the trade name Taxol® in 1992. The discovery of avermectin B1 (1.10, Figure 1.4) was a result of an intensive search for natural products with antiparasitic activities. In the early 1970s, following the extensive screen for anticancer and antibacterial agents, Merck started to use this screening strategy in parasitology studies. A novel bacterial species Streptomyces avermitilis was discovered and eight avermectins with antiparasitic activities were isolated. One of them was marketed by Merck and HNO NSOHOOHPenicillin G(1.6)HNO NSOHOOHAmoxicillin(1.7)NH2HO 5 became the most revolutionary parasiticide in animal health.16 The development of this drug as human medicine was also very successful, and it is now used in part of the mass distribution program to eradicate river blindness and elephantiasis.17 Artemisinin (1.11, Figure 1.4), which shows potent inhibition against the malaria parasite Plasmodium berghei, was isolated and identified from the plant Artemisia annua in the mid-1970s. The clinical trial was finished in China in 1978, and WHO started to adopt artemisinin (1.11) as part of the combination therapies for the treatment of malaria in 2001.16 Lovastatin (1.12, Figure 1.4), previously called mevinolin, was isolated from the fungus Aspergillus terreus in 1980. Its hydroxy acid form was a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase.18 It was also found in other sources including a mushroom Pleurotus ostreatus in 1994.19 Merck started the clinical studies of lovastatin in 1980, and the FDA approved this drug as a cholesterol-lowering agent under the trade name Mevacor® in 1987.20,21 6 Figure 1.4 Examples of natural product drugs: structures of erythromycin A (1.8), paclitaxel (1.9), avermectin B1 (1.10), artemisinin (1.11) and lovastatin (1.12) Nowadays, biologically active natural products continue to be discovered. Among all 1562 of the new approved drugs from 1981 to 2014, 1211 of them were small-molecule drugs, and 32.7% of the small-molecule drugs were natural products (N), natural product botanicals (NB) or natural product derivatives (ND), not to mention those that were inspired by natural products (Figure 1.522). Among all 246 anticancer drugs from the 1940s to 2014, 93 of them were natural products, natural product botanicals or natural product derivatives.22-26 Natural products are still playing an important role in drug discovery research. Among all the drugs approved globally from 2004 to 2014, although the numbers vary by year, the percentages of approved natural product drugs by year range between 21% and 50%.27 The diversity and complexity of structures of natural products, especially their three dimensional structures which OOOHOHO OHOOOOHONOOHErythromycin A(1.8)OHOONHO OAcOOOOOHHOAcOHO O OOOOOHOOOOOOHHHOAvermectin B1(1.10)OOOHHOOArtemisinin(1.11)OOOOHOHLovastatin(1.12)Paclitaxel(1.9) 7 are compatible with biological receptors, make them favorable as lead structures for drug discovery.27,28 Figure 1.5 All small molecule approved drugs 1981-2014s; n=1211.22 (N=unaltered natural product, NB=botanical drug defined mixture, ND=natural product derivative, S=synthetic drug, S/NM=synthetic drug/mimic of natural product, S*=synthetic drug with natural product pharmacophore, S*/NM=synthetic drug with natural product pharmacophore/mimic of natural product) 1.3 Marine natural products The investigation of terrestrial natural products dates back to the early 1800s, while the investigation of marine natural products started in the 1950s thanks to the progress of techniques for marine sample collection (e.g. SCUBA). The Bergmann group discovered the first marine natural product spongothymidine (1.13),29-32 and this compound inspired the development of the synthetic cancer chemotherapeutic drugs Ara A (1.14) and Ara C (1.15), which are the first clinical drugs derived from the marine environment (Figure 1.6).33 8 Figure 1.6 Structures for spongothymidine (1.13), Ara A (1.14) and Ara C (1.15) The marine environment covers around 70% of the biosphere, with a huge diversity of plants, invertebrates and microbes, and is relatively unexplored compared to the terrestrial environment, which makes the marine environment a valuable source of novel natural products that have potential as new drug leads.34 Marine natural products exhibit a broad range of biological activities including anticancer, anti-bacterial, anti-fungal, anti-viral and pest resistance etc.35,36 Other than Ara A (1.14), Ara C (1.15) and ziconotide (1.1), some examples of marine-derived drugs are shown in Figure 1.7.34,37 Figure 1.7 Examples of marine drugs: Yondelis® and Halaven® NNHOOOOHHOHO OOHHOHONNHOOOOHHOHOSpongothymidine(1.13)Ara A(1.14)Ara C(1.15)NNNNNH2NHNNHOMeOOOAcOMeSOHHOOMeMeMeOHTrabectedin 1.16(trade name: Yondelis)OOOOOOO OMeOHOH2NCH3SO3HH HHHEribulin mesylate 1.17(trade name: Halaven) 9 1.4 Methods and tools used for natural products drug discovery In order to search for natural products as potential drug leads, natural products chemists start with the collection of samples from various natural environments and the preparation of a natural products extract library, which comprises crude extracts of different types of biomass including plants, microbes and marine invertebrates. Thanks to the development of high-throughput screening (HTS), biologists can now screen a large number of natural product crude extracts more efficiently for a broad range of biological activities,38,39 which will result in finding a “hit” for the activity of interest. Natural products chemists use the bioassay-guided fractionation (see section 1.4.1) strategy for the purification of crude extracts, and then a pure compound/pure compounds with biological activities is/are isolated. Structure elucidation tools (see section 1.4.2) are utilized to identify the structure of the active compound(s). Further biological studies are then carried out to determine whether this active compound is viable as a drug lead. This general workflow is presented in Figure 1.8. 10 Figure 1.8 From “hit” to “drug lead”: the typical workflow of natural products chemists 1.4.1 Isolation methods The general strategy for natural products chemists is bioassay-guided fractionation (Figure 1.9). After each round of fractionation for the active extracts, the sub-fractions are screened for their biological activity. Guided by these results, natural products chemists are able to track the biological activity in the complex matrix of a crude extract, and continue to fractionate the mixture until one/a series of final pure compound(s) are obtained. 11 Figure 1.9 General scheme for bioassay-guided fractionation It is very common to carry out a few rounds of fractionation to obtain the final product(s). Techniques commonly used in fractionation and purification include solvent partitioning, size exclusion chromatography, silica gel column chromatography, reversed phase column chromatography, solid phase extraction, high-performance liquid chromatography (HPLC), ultra high-performance liquid chromatography (UPLC) and liquid chromatography-mass spectrometry (LC-MS), etc. These techniques separate compounds in the crude extract by their polarity, affinity to the column material, solubility or size of the molecules. A pure compound or a series of pure compounds with activities are obtained for structure elucidation and further biological studies. 12 1.4.2 Structure elucidation methods Structure elucidation of small molecules relies on spectroscopic techniques including mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR) spectroscopy, circular dichroism (CD) spectroscopy etc. High-resolution mass spectrometry (HRMS) provides information about the molecular formula, and it also provides fragmentation information in some cases depending on the ionization methods. The molecular structures of the unknown compounds are elucidated using NMR experiments. The chemical shifts (δ) of each atom in the molecule are obtained through 1D NMR experiments (e.g. 1H NMR, 13C NMR and 14N NMR), which provide general information about the electron density (shielding effect) of the nuclei. The information about the connectivity of the atoms can be established from 2D NMR experiments including correlation spectroscopy (COSY), heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple bond correlation spectroscopy (HMBC), nuclear overhauser enhancement spectroscopy (NOESY), etc. The results of these experiments are plotted as contour maps with each peak representing an interaction between two atoms. Their applications are shown in Figure 1.10. 13 Figure 1.10 Commonly used 2D NMR experiments UV-Vis and IR spectroscopy provide information about functional groups, which gives additional support to the structure assignment. X-ray crystallography is also utilized for crystalline samples, providing additional information about the absolute configuration. CD spectroscopy involves the interaction of the molecule with circularly polarized light, which serves as another tool for the assignment of the absolute configuration of the structure. Other methods like Mosher’s or Marfey’s derivatization techniques are also used to determine the absolute configurations of stereo centers.40,41 1.5 Biosynthesis of natural products Natural products are produced by organisms through different biosynthetic pathways using a series of general building blocks. These building blocks are derived from the primary metabolites that are shown in Figure 1.11. The most common C1 building block is S-adenosyl 14 methionine (SAM), which is derived from L-methionine (1.18). The C2 building block in fatty acid and polyketide biosynthesis (see section 1.5.1) is derived from acetyl-Coenzyme A (acetyl-CoA, 1.19), which is an important intermediate in the glycolysis pathway. The isoprene C5 building block is made from the mevalonic acid (MVA, 1.20) pathway or the methylerythritol phosphate (MEP, 1.21) pathway, and this building block is responsible for the terpenoid and steroid biosynthesis (see section 1.5.2). The phenylpropyl C6C3 building block is derived from L-phenylalanine (1.22) or L-tyrosine (1.23) via the shikimate biosynthetic pathway (see section 1.5.3). The nitrogen-containing building blocks are usually supplied by L-phenylalanine (1.22), L-tyrosine (1.23), L-ornithine (1.24), L-lysine (1.25) or L-tryptophan (1.26) (see section 1.5.4 and 1.5.5). 15 Figure 1.11 The common building blocks in natural products biosynthesis C2 building blockS COOHNH2H3CC1 building blockL-methionine1.18SCoAOacetyl-CoA1.19C5 building blocks SCoAOacetyl-CoA1.193CH3HOOC OHOHmevalonic acid1.20orOPOHmethylerythritol phosphate1.21COOHOOPPOOH+OH OHCOOHNH2COOHNH2HOL-phenylalanine1.22L-tyrosine1.23NH2orC6C3 or C6C2Nbuilding blocksC4N or C5Nbuilding blocks H2NCOOHNH2COOHNH2H2NL-ornithine1.24L-lysine1.25NNindole-C2Nbuilding blocksNHNH2COOHNNL-tryptophan1.26 16 1.5.1 Fatty acids and polyketides Fatty acids and polyketides are a class of natural products derived from the coupling of acetic acid (C2) building blocks. The building blocks are assembled into a long chain through Claisen condensations, and cyclization and oxidation/reduction reactions occur to synthesize the final natural products. These reactions occur in enzymes called polyketide synthases (PKSs). There are three types of PKSs: type I PKSs are large multifunctional enzymes with multiple modules with different functions, and are found in bacteria and fungi; type II PKSs are a group of smaller mono-functional enzymes working together, which are found in bacteria only; type III PKSs are monomer proteins that have a single active site to perform multiple reactions, and are found in plants, bacteria and fungi.42,43 Macrolides, one class of polyketides, are well known for their use as antibiotics. Some common antibiotics such as erythromycin (Eryc®), clarithromycin (Biaxin®) and azithromycin (Zithromax®) share a similar 14-membered macrolide ring substructure. The biosynthesis of erythromycin A (1.8), using type I PKS, is shown in Figure 1.12. In this case, methylmalonyl-CoA (1.27) instead of malonyl-CoA (1.28) is used as the building blocks for the chain.44 17 Figure 1.12 The biosynthesis of erythronolide A (1.8)44 1.5.2 Terpenoids and steroids Terpenoids and steroids are both derived from the C5 isoprene building blocks produced in the mevalonic acid (MVA, 1.20) pathway or methylerythritol phosphate (MEP, 1.21) pathway (Figure 1.13). The starting materials mevalonic acid (1.20) or methylerythritol phosphate (1.21), which are from the glycolytic pathway, yield the isopentenyl diphosphate (IPP, 1.29) C5 building block. SCoAOSCoAOOHOOSCoAOOOOOOOOO OOOOCoASSCoAOSCoAOBiotin, CO2, ATPCOOHAcetyl-CoA Malonyl-CoA1.28SCoAOSCoAOBiotin, CO2, ATPCOOHPropionyl-CoA Methylmalonyl-CoA1.27+ 6xOOO OOOOOOOHOHO OHOHOHErythronolide A1.8 18 Figure 1.13 Terpenoid biosynthesis Terpenoids are classified by the number of building blocks they contain: hemiterpenoids derive from one C5 building block isopentenyl diphosphate (1.29) and dimethylallyl diphosphate (1.30), monoterpenoids from geranyl diphosphate (GPP, 1.31, two of C5), sesquiterpenoids from farnesyl diphosphate (FPP, 1.32, three of C5), diterpenoids from geranylgeranyl diphosphate (GGPP, 1.33, four of C5), sesterterpenoids from geranylfarnesyl diphosphate (GFPP, 1.34, five of C5), triterpenoids from squalene (1.35, two of C15) and tetraterpenoids from phytoene (1.36, HOOC OHOHmevalonic acid(MVA) 1.20OPOHmethylerythritol phosphate(MEP) 1.21OH OHOPPmevalonate(MVA) pathwaymethylerythritol phosphate (MEP) pathwayisopentenyl diphosphate(IPP) 1.29 C5OPPdimethylallyl diphosphate(DMAPP) 1.30IPP DMAPP++ IPPOPPgeranyl diphosphate(GPP) 1.31 C10OPPfarnesyl diphosphate(FPP) 1.32 C15 squalene 1.35C30x2+ IPPOPPgeranylgeranyl diphosphate(GGPP) 1.33 C20phytoene 1.36C40x2+ IPPOPPgeranylfarnesyl diphosphate(GFPP) 1.34 C25Steroids 19 two of C20). Terpenoids are the largest group of natural products,45 and many natural products contain a terpenoid moiety in their structures. For example, the anticancer drug paclitaxel (1.9) contains a diterpene moiety derived from GGPP (1.33). The biosynthesis is shown in Figure 1.14. The cyclization and rearrangement of GGPP (1.33) occur in taxadiene synthase to yield taxadiene (1.37), followed by a few steps of hydroxylation, acetylation and esterification reactions to form the diterpene moiety in paclitaxel (1.9).46-48 Figure 1.14 Biosynthesis of paclitaxel (1.9)46-48 Steroids are a class of natural products derived from triterpenoids. They usually contain a fused tetracyclic ring system that came from the cyclization of squalene (1.35) and a few steps of OPPGeranylgeranyl diphosphate(GGPP) 1.33OHOONHO OAcOOOOOHHOAcOHPaclitaxel1.9OPPOHH HHHOHHHHHHTaxadiene1.37taxadienesynthaseOAcOHHO HAcOHOOOHOO 20 demethylation. An example of steroid biosynthesis, the biosynthesis of cholesterol (1.39), is shown in Figure 1.15. Figure 1.15 Biosynthesis of cholesterol (1.39) 1.5.3 Aromatic amino acids and phenylpropanoids One of the sources for the aromatic amino acids L-phenylalanine (1.22) and L-tyrosine (1.23) is the shikimate pathway, which provides the aromatic C6C3 phenylpropane building blocks for many natural products. The pathway is named after the important intermediate: shikimic acid (1.40). This pathway only occurs in microorganisms and plants,49 and this is why humans need essential amino acids including L-phenylalanine (1.22) and L-tyrosine (1.23) in our squalene1.35OsqualeneepoxidaseHO HHHHHO HHLanosterol1.38HO HHH14sterol 14-demethylaseHO HHHsterol 4-demethylaseHO HHHsterol 24-reductase424HOHHHCholesterol1.39 21 diet for the aromatic features in the structures. The starting materials of the shikimate pathway are phosphoenolpyruvate (PEP, 1.41) from the glycolytic pathway and D-erythrose 4-phosphate (1.42) from the pentose phosphate cycle. The biosynthesis of salicylic acid (1.3) is shown in Figure 1.16 as an example. Figure 1.16 Biosynthesis of salicylic acid (1.3) HOOC OP + POOHOHHOshikimate pathwayCOOHHOOHOHPhosphoenolpyruvate(PEP) 1.41D-erythrose 4-phosphate1.42 Shikimic acid1.40COOHHOOHOHShikimic acid1.40+ATP +PEPCOOHPOOHO5-enolpyruvyshikimic acid3-phosphate 1.43COOHCOOHOHOChorismic acid1.44COOH1,4-elimination of phosphoric acidCOOHOHSalicylic acid1.3OHPrephenic acid1.45HOCOOHOOHchorismate mutaseOCOOHPhenylpyruvic acid1.46COOHL-phenylalanine1.22NH2COOHCinnamic acid1.47COOHOHCOSCoAOH+HSCoA+H2OOxidation OHSCoA OHSCoAO 22 1.5.4 Alkaloids Alkaloids are nitrogen-containing natural products. The building blocks of alkaloids are usually derived from amino acid precursors including L-tyrosine (1.23), L-ornithine (1.24), L-lysine (1.25), L-tryptophan (1.26), nicotinic acid (1.48), anthranilic acid (1.49) and histidine (1.50). Building blocks from acetate, shikimate, MEP or MVA pathways may also be involved in alkaloids. The biosynthesis of morphine (1.1) is shown in Figure 1.17 as an example of alkaloid biosynthesis.50 23 Figure 1.17 Biosynthesis of morphine (1.1)50 HOCOOHNH2L-Tyr1.23HOCOOHNH2HOCOOHOHOHO OHO NH2HOHHO NHHOHOHHO NMeMeOMeOHHOHO NMeMeOMeOHOHO NMeMeOMeOHHO(R)-reticuline1.52(S)-reticuline1.51HOMeOONMeHOMeOONMeOHOHHHOMeOONMeOHHHOMeOONMeOHHHOMeOONMeOAcHOMeOONMeHOMeOONMeHOH HOMeOONMeHOMeOONMeHneopinone1.53codeinone1.54HOMeOHONMeHHOHOHONMeHmorphine1.1H 24 1.5.5 Peptides Peptides are polyamides that utilize amino acids as building blocks to form a long chain through condensation reactions of carboxyl and amino functionalities. The biosynthesis of peptides is usually categorized by where this biosynthesis takes place, either ribosomal peptide biosynthesis or non-ribosomal peptide biosynthesis. For example, botulinum toxin A (Botox®), which is used in plastic surgery to remove wrinkles, is a polypeptide synthesized from the ribosome of the bacterium Clostridium botulinum using the gene information carried by messenger RNA (mRNA). On the other hand, non-ribosomal peptide synthesis is an enzyme-controlled process that takes place in a multifunctional enzyme called non-ribosomal peptide synthase (NRPS). For example, vancomycin (1.55), an antibiotic known to be the last resort for treating MRSA infections,51 is biosynthesized by an NRPS as shown in Figure 1.18. L-leucine (Leu, 1.56), L-tyrosine (Tyr, 1.23), L-asparagine (Asn, 1.57), as well as two unusual amino acids L-4-hydroxyphenylglycine (Hpg, 1.58) and L-3,5-dihydroxyphenylglycine (Dpg, 1.59) are used as building blocks.52 25 Figure 1.18 Biosynthesis of vancomycin (1.55) 1.6 Research goals The previous success of using natural products in drug discovery has motivated us to continue seeking potential drug leads from natural sources. We followed the general workflow for natural products chemists described in section 1.4 (Figure 1.8). Starting from a hit for the biological activities, we used the bioassay-guided fractionation strategy and structure elucidation methods in order to find natural products with potential therapeutic applications. In an effort to search for new small molecules that activate HIV-1 provirus expression, a biological assay was designed to screen for the activators. A crude extract of Phorbas sp. activated the provirus expression in this assay. The major and minor metabolites of Phorbas sp. OHOCOOHOHOHOOHOHOOOHOHOH2NOHL-Hpg1.58SCoAOCOOHMalonyl-CoA1.284xOSCoAOOOOSCoAOO OCOOHHOOCOSCoAHO OHCOOHHO OHO COOHHO OHH2NL-Dpg1.59OH HONH2HOOCHOHOOC NH2ONH2HOOCOHNH2HOOC NH2OHO ONHHNONHOHOOHN CO2HHO OHO HNONH2OOHNHONHOHNH2HOOCCOOHH2NOHNH2HOOCL-Asn1.57L-Tyr1.23L-Leu1.56HO OHO OHOHOHO OONH2HOVancomycin NRPS Vancomycin1.55L-Hpg1.58L-Hpg1.58L-Dpg1.59L-Tyr1.23 26 were investigated. In Chapter 2, the metabolites that have been isolated before from the genus Phorbas are reviewed, and their biological activities are also covered in Chapter 2. In Chapter 3, the isolation and structure elucidation of the novel natural products from the sponge Phorbas sp. are described along with their biological activities. In an effort to identify small molecules from the traditional medicinal plants that have anti-onchocerciasis properties, a biological assay was designed to screen for inhibitors. In Chapter 4, the biologically active natural products isolated from two traditional medicinal plants Morinda lucida and Lantana camara are described. Their biological activities are also investigated. 27 Chapter 2: Metabolites isolated from the sponge Phorbas sp. 2.1 The genus Phorbas Sponges, which belong to the invertebrate phylum Porifera, are filter-feeding organisms that live in fresh water and marine aquatic habitats. They do not have hard shells that can protect them from their predators. One of their defense mechanisms appears to be utilizing bioactive secondary metabolites to prevent other sessile organisms such as sea squirts from growing near them or predators such as fish and nudibranchs from eating them. From 2001 to 2010, more than 2400 new natural products (secondary metabolites) have been reported from 542 genera and 671 species of sponges, contributing about 29% of the marine natural products discovered during this decade, making sponges the largest source of new natural products among all marine organisms.53 Figure 2.1 Phorbas sp. collected in Howe Sound, BC (picture from Mike LeBlanc) Sponges in the genus Phorbas has been found in several widely separated locations around the world, and several groups have studied their secondary metabolites. The wide structural variety of Phorbas sp. metabolites and their interesting biological activities have motivated further investigations into compounds produced by this genus. 28 2.2 Secondary metabolites produced by the genus Phorbas A variety of secondary metabolites have been isolated and identified from the genus Phorbas including alkaloids, polyketides, terpenoids and steroids. Compounds isolated from the genus Phorbas are reviewed in the following section covering the literature published till July 2017. Their sources and relevant biological activities are also included. 2.2.1 Alkaloids and polyketides from the genus Phorbas Phorbazoles A−D (2.1−2.4) were isolated from the sponge Phorbas clathrata collected in Sodwana Bay, South Africa by the Kashman group. They represent a new class of marine alkaloids embodying a chlorinated pyrrole moiety.54 Figure 2.2 Structures of phorbazoles A−D (2.1−2.4) Phorboxazoles A and B (2.5 and 2.6) were isolated from a Phorbas sp. collected in the Indian Ocean by the Molinski group. They exhibited in vitro antifungal activity against Candida albicans at 0.1 µg/disk and extraordinary cytostatic activity with mean GI50 < 7.9 × 10-10 M in the National Cancer Institute's 60 tumor cell line panel.55 Hemi-phorboxazole A (2.7), a minor analogue of 2.5 (~104 times less abundant), was isolated using nanomole scale techniques.56 NOHNR1R2 R3OHR4Phorbazole A (2.1): R1=H, R2=Cl, R3=Cl, R4=ClPhorbazole B (2.2): R1=Cl, R2=Cl, R3=Cl, R4=HPhorbazole C (2.3): R1=H, R2=Cl, R3=Cl, R4=HPhorbazole D (2.4): R1=H, R2=H, R3=Cl, R4=H 29 Figure 2.3 Structures of phorboxazoles A and B (2.5 and 2.6) and Hemi-phorboxazole A (2.7) Phorbasides A−I (2.8−2.16) were also isolated from the Phorbas sp. sample collected in Western Australia by the Molinski group, using nanomole-scale techniques.57-60 Figure 2.4 Structures of phorbasides A−I (2.8−2.16) OOONOONOOOHOMeBrOHOMeOR1R2Phorboxazole A (2.5): R1=OH, R2=HPhorboxazole B (2.6): R1=H, R2=OHOOONOOOH OHNHemi-phorboxazole A (2.7)OO OOH ClOOHMeOH OHROHMeOPhorbaside A (2.8): R=HPhorbaside F (2.13): R=MeOOROH ClOH OHMeOPhorbaside B (2.9): R=XPhorbaside C (2.10): R=YPhorbaside D (2.11): R=ZPhorbaside E (2.12): R=UPhorbaside G (2.14): R=VPhorbaside H (2.15): R=WPhorbaside I (2.16): R=QOOHMeO O OOHMeOOHOOHMeO O OOHHOOHXYOOHO OHUOMeO NHOOZOHOHOOOMeO OHVOOHMeO OOOMeO OHWOOHMeO OHOMeO NHCHOOHQ 30 Muironolide A (2.17) was isolated later with the chlorocyclopropane of opposite configuration to that of phorbaside A (2.8), but the same as the configuration of callipeltosides A−C (2.18−2.20), compounds previously isolated from a cyanobacterium.61,62 This result suggests that muironolide A (2.8) and phorbasides A−I (2.8−2.16) are likely the products of a heterogeneous sponge-microbe association.63 Figure 2.5 Structures of muironolide A (2.17) and callipeltosides A−C (2.18−2.20) 2.2.2 Terpenoids and steroids isolated from the genus Phorbas The diterpenoids, phorbasin A−K (2.21−2.31),64-68 were isolated from a Phorbas sp. collected in Southern Australian and from Phorbas gukulensis collected in Gagu-Do, Korea. Preliminary SAR evaluation of phorbasin B−K (2.22−2.31) for in vitro cytotoxicity against both normal and human cancer cell lines revealed aspects of the cytotoxic pharmacophore of the phorbasins.68 Phorbasin H (2.28) inhibited filamentous growth in Candida albicans by blocking the expression of mRNAs related to the cAMP–Efg1 pathway.69 HNO CCl3OOOOHClHHMuironolide A (2.17)OORO OH OHMeOCallipeltoside A (2.18): R=XCallipeltoside B (2.19): R=YCallipeltoside C (2.20): R=ZClHOMeO NHOOXOYOZNHCHOOHHOHOMeOMeO 31 Figure 2.6 Structures of phorbasins A−K (2.21−2.31) Another diterpene family, the gagunins A−G (2.32−2.38), was isolated by the Shin group from a Phorbas sp. collected in the Korean Sea.70 They showed cytotoxicity toward a human leukemia cell line (K562) with LC50 values ranging from 0.03 to 51 µg/mL. Gagunins H−Q (2.39−2.48) were isolated later and shown to be cytotoxic against the K562 cell line with LC50 values ranging from 0.45 to 50 µg/mL. Gagunins A−Q (2.32−2.48) were inactive against both Gram-positive and Gram-negative bacteria and pathogenic fungi despite a weak inhibitory activity (LC50 55−140 µg/mL) against isocitrate lyase, an important enzyme in microbial metabolism.71 Gagunin D (2.35) was found to exhibit anti-melanogenic activity by suppressing the expression of tyrosinase and increasing the rate of tyrosinase degradation.72 HOO OHPhorbasin A (2.21)OR2 OR1OHPhorbasin B (2.22): R1=H, R2=OHPhorbasin C (2.23): R1=Ac, R2=OHPhorbasin G (2.27): R1=H, R2=HPhorbasin H (2.28): R1=Ac, R2=HPhorbasin I (2.29): R1=Ac, R2=OEtOHN OHOHPhorbasin D (2.24)HO3SOOROHNSOOOOHORPhorbasin E (2.25): R=HPhorbasin F (2.26): R=AcOEtO OAcOHHOHO OHOHOEtPhorbasin J (2.30) Phorbasin K (2.31) 32 Figure 2.7 Structures of gagunins A−Q (2.32−2.48) The Rho and Shin groups isolated several diterpenoid pseudodimers, including the gukulenins A−F (2.49−2.54), from Phorbas gukulensis collected near Gageo Island and Gagu-do, South Korea.73 Gukulenins A and B (2.49 and 2.50) showed cytotoxicity against human pharynx, stomach, colon, and renal cancer cell lines. Gukulenins A (2.49) and C−F (2.51−2.54) exhibited cytotoxicity against the K562 and A549 cell lines.74 R3R2R1HOCO(CH2)2CH3H3CO2CHO HOCO(CH2)2CH3OCO(CH2)2CH3HR3R2R1 HR3R1HOCO(CH2)2CH3HOH3CO2C HGagunin A (2.32): R1=OCO(CH2)2CH3, R2=OCO(CH2)2CH3, R3=OCOCH2CH(CH3)2Gagunin B (2.33): R1=OCO(CH2)2CH3, R2=OCO(CH2)2CH3, R3=OCO(CH2)2CH3Gagunin C (2.34): R1=OCO(CH2)2CH3, R2=OCOCH3, R3=OCO(CH2)2CH3Gagunin H (2.39): R1=OCOCH2CH3, R2=OCO(CH2)2CH3, R3=OCO(CH2)2CH3Gagunin I (2.40): R1=OCOCH3, R2=OCO(CH2)2CH3, R3=OCO(CH2)2CH3Gagunin J (2.41): R1=OCO(CH2)2CH3, R2=OCOCH2CH3, R3=OCO(CH2)2CH3Gagunin K (2.42): R1=OCO(CH2)2CH3, R2=OCO(CH2)2CH3, R3=OCO(CH2)3CH3Gagunin L (2.43): R1=OCOCH3, R2=H, R3=OCO(CH2)2CH3Gagunin M (2.44): R1=OCOCH2CH(CH3)2, R2=H, R3=OCO(CH2)2CH3Gagunin D (2.35): R1=OH, R2=OCOCH3, R3=OCO(CH2)2CH3Gagunin E (2.36): R1=OCOCH3, R2=OCO(CH2)2CH3, R3=OHGagunin F (2.37): R1=OCOCH3, R2=OH, R3=OCO(CH2)2CH3OCO(CH2)2CH3OCO(CH2)2CH3HR2R1HO HOCO(CH2)2CH3R2Gagunin G (2.38): R1=OCO(CH2)2CH3, R2=H, R3=OCOCH3Gagunin N (2.45): R1=OCOCH3, R2=H, R3=OCO(CH2)2CH3Gagunin O (2.46): R1=OCO(CH2)2CH3, R2=OCO(CH2)2CH3, R3=OCO(CH2)2CH3Gagunin P (2.47): R1=OCOCH3, R2=OCOCH3Gagunin Q (2.48): R1=OCOCH2CH3, R2=OCO(CH2)2CH3 33 Figure 2.8 Structures of gukulenins A−F (2.49−2.54) Anthosterones A and B (2.55 and 2.56) and phorbasterones A−D (2.57−2.60), which are nor-ring A steroids, were isolated from Phorbas amaranthus collected in Key Largo, Florida.75 These compounds showed moderate cytotoxicity against human colon tumor cells (HCT-116) with IC50’s of 1-3 µg/mL. Figure 2.9 Structures of anthosterones A and B (2.55 and 2.56) and phorbasterones A−D (2.57−2.60) Amaroxocanes A and B (2.61 and 2.62), two sulfated dimeric sterols, were isolated from Phorbas amaranthus collected in Key Largo, Forida. Both of the compounds were subjected to a fish feeding assay, but only amaroxocane B (2.62) was responsible for feeding deterrent activity against a common reef predator, the bluehead wrasse.76 OOHHO OROHH OHOAcHHOOOHHO OOHH OHHHOGukulenin A (2.49): R=CHOGukulenin B (2.50): R=CH2OHR1R4R3H R2Gukulenin C (2.51): R1=OH, R2=H, R3=OCH3, R4=NHCOCH3Gukulenin D (2.52):R1=OH, R2=H, R3=OH, R4=NHCOCH3Gukulenin E (2.53): R1R2=O, R3=H, R4=NH(CH2)2SO3HOHHO OHO OHHHO OCOCH3HHHOOGukulenin F (2.54)RHOMeO2COAnthosterone A (2.55)Anthosterone B (2.56)R =R =R =R =R =R =Phorbasterone A (2.57)Phorbasterone D (2.60)Phorbasterone C (2.59)Phorbasterone B (2.58) 34 Figure 2.10 Structures of amaroxocanes A and B (2.61 and 2.62) Steroidal alkaloids amaranzoles A−F (2.63−2.68) were isolated from Phorbas amaranthus collected in Key Largo, Florida. Amaranzole A (2.63) did not show cytotoxicity toward human colon tumor cells (HCT-116), while its hydrolysis analogue, with the three –OSO3Na groups replaced by –OH, exhibited significant cytotoxicity (IC50 = 4.4 µg/mL).77,78 Figure 2.11 Structures of amaranzoles A−F (2.63−2.68) HOHOOSO3NaHOOSO3NaOHOSO3NaHOHHHNaO3SOHOOSO3NaHOOOSO3NaOHOSO3NaHHHAmaroxocane A(2.61)Amaroxocane B(2.62)NaO3SONaO3SOHROSO3NaHHOHNNOHHNNOONaO3SONaO3SOOSO3NaHHX YAmaranzole A (2.63): R=XAmaranzole B (2.64): R=YAmaranzole C/D(2.65/2.66)Amaranzole E/F(2.67/2.68)NaO3SONaO3SOOSO3NaHHOHNNOHHN NO O 35 2.2.2.1 Sesterterpenoids isolated from the genus Phorbas Phorbaketals A−C (2.69−2.71)79 were isolated from a Phorbas sp. collected in Gageo Island, South Korea. The phorbaketals have the same alotane carbon skeleton as alotaketals A and B (2.72 and 2.73), which were isolated from a Hamigera sp. belonging to another sponge genus.80 Phorbaketals A−C (2.69−2.71) exhibited moderate cytotoxicity against human colorectal, hepatoma and lung cancer cell lines. The cultivation of mixed bacteria from homogenized sponge afforded phorbaketal A (2.69), suggesting that 2.69 originated from an endosymbiotic microorganism in this sponge.79 Phorbaketals A−C (2.69−2.71), together with new compounds phorbaketals D−K (2.74−2.81) and phorbin (2.82), were isolated from another sponge Monanchora sp. collected in Gageo Island, South Korea. Since Monanchora sp. and Phorbas sp. share a very similar orange color and similar surface textures, the isolation of phorbaketals from both Monanchora sp. and Phorbas sp. could simply be a result of incorrect identification of sponge species. It is also possible that phorbaketals are produced by the symbiotic bacteria in both Monanchora sp. and Phorbas sp. Phorbaketals A−K (2.69−2.71, 2.74−2.81) and phorbin (2.82) were all tested for the cytotoxicity against three human cancer cell lines. Phorbaketals A−K (2.69−2.71, 2.74−2.81) showed weak activity or no activity, while phorbin (2.82) showed potent cytotoxicity against Mia-paca and Panc-1 human pancreatic cancer cell lines.81 Phorbaketals L−N (2.83−2.85) were isolated from a Phorbas sp. sample collected in Gageo Island, Korea. Phorbaketal N (2.85) exhibited cytotoxic activities against the human pancreatic cancer cell line Panc-1 (IC50 = 11.4 µM) and two human renal cancer cell lines A498 (IC50 = 18.7 µM) and ACHN (IC50 = 24.4 µM).82 36 Figure 2.12 Structures of phorbaketals A−N (2.69−2.71, 2.74−2.81, 2.83−2.85), alotaketals A and B (2.72 and 2.73) and phorbin (2.82) Ansellone A (2.86) was isolated by our group from both the nudibranch Cadlina luteromarginata and the sponge Phorbas sp. that nudibranch was feeding on, which were both collected in Howe Sound, British Columbia.83 Ansellone A (2.86) has an “ansellane” carbon OOOHO HHOORHO HHPhorbaketal A (2.69): R= OPhorbaketal B (2.70): R= H,OH (R)Phorbaketal C (2.71): R= H,OH (S)Alotaketal A(2.72)OOOHO HHAlotaketal B(2.73)OOOOOHHRPhorbaketal D (2.74): R=OOOAcHHPhorbaketal F (2.76): (R)- OAcPhorbaketal G (2.77): (S)- OAcOOHOOROHCHHPhorbaketal H (2.78): R= H,OH (R)Phorbaketal I (2.79): R= OPhorbaketal J (2.80): R= H,OAc (R)Phorbaketal K (2.81): R= H,OAc (S)HOHOOHOPhorbaketal E (2.75): R=Alotane skeleton Phorbin (2.82)OOHOHOOHHHOOHOHPhorbaketal L (2.83)OOHHHOOHPhorbaketal M (2.84)OOHHHOOHBrPhorbaketal N (2.85)OOH O 37 skeleton. Ansellones B and C (2.87 and 2.88) were isolated from the same Phorbas sp. collected in Howe Sound, British Columbia. Ansellone A (2.86) activated cAMP signaling in HEK293 cells in the absence of hormone binding with an EC50 of 14 µM, while ansellone B (2.87) was toxic at all concentrations tested.84 Figure 2.13 Sturctures of ansellones A−C (2.86−2.88) Secoepoxyansellone A (2.89) was also isolated by our group from the Howe Sound Phorbas sp. It was the first example of the degraded “secoansellane” sesterterpenoid carbon skeleton.84 Figure 2.14 Sturcture of secoepoxyansellone A (2.89) OOAcOOHHHHHAnsellone B(2.87)OOAcOOHHHHHAnsellone C(2.88)OHOHHH HHOAcOAnsellone A(2.86)Ansellane skeletonH OOOOHHOAcSecoepoxyansellone A(2.89)secoansellane skeleton 38 Phorbasones A and B (2.90 and 2.91) were isolated from a Phorbas sp. collected in South Korea. They were examined on calcium deposition in mensenchymal C3H10T1/2 cells, and phorbasone A (2.90) showed an induction effect on osteoblast differentiation at a concentration of 0.5 µg/mL.85 Figure 2.15 Structures of phorbasones A, B and phorbasone A acetate (2.90−2.92) Phorone A and B (2.93 and 2.94) and isophorbasone A (2.95) were isolated from the same Phorbas sp. collected in South Korea. An iNOS inhibitory assay was performed for these compounds by measuring NO production in RAW 264.7 LPS-induced mouse macrophage cells. Phorone A (2.93) and isophorbasone A (2.95) were not active up to 100 µM, while phorone B (2.94) and phorbasone A acetate (2.92) were found to exhibit moderate inhibitory activity on nitric oxide production with IC50 values of 4.5 and 2.8 µM, respectively.86 Figure 2.16 Structures of phorones A and B (2.93 and 2.94) and isophorbasone A (2.95) HOHOOH HOHOOHHOHOPhorbasone A(2.90)Phorbasane skeletonHOAcOOHPhorbasone A acetate(2.92)Phorbasone B(2.91)OOHOHHHHO OOHPhorone A(2.93)Phorane skeletonIsophorbasone A (2.95)Isophorbasane skeletonOOHOAcHOAcHPhorone B(2.94) 39 Chapter 3: Sesterterpenoids isolated from the British Columbia sponge Phorbas sp. activate latent HIV-1 provirus expression 3.1 Sesterterpenoids Sesterterpenoids (C25) are a class of terpenoids derived from five isoprene building blocks (C5). Although terpenoids comprise the largest group of natural products with different members found in all kingdoms of life, sesterterpenoids are a relatively rare group of terpenoids. The first sesterterpenoid reported was ophiobolin A (3.1), isolated from the plant pathogenic fungi Ophiobolus miyabeanus and Helminthosporium turcicum.87 The first linear sesterterpenoid geranylnerolidol (3.2) was isolated from Cochliobolus heterostrophus, the phyto-pathogenic fungus responsible for causing the leaf spot disease of maize,88 and the scale insect Ceroplastes albolineatus.89 The first marine sesterterpenoid scalarin (3.3)90, a penta-cyclic sesterterpenoid isolated from the sponge Cacospongia scalaris, has a scalarane terpenoid carbon skeleton resulting from concerted carbocation initiated cyclization of a linear geranylfarnesol precursor. The sesterterpenoid salvileucolide methyl ester (3.4) was isolated from the plant Salria hypoleuca.91 Novel sesterterpenoids continue to be isolated from various sources and they are notable for their diverse biological activities such as antimicrobial,92,93 cytotoxic,94 anticancer,95,96 anti-tubercular,97,98 anti-inflammatory99,100 and anti-biofilm101 activities. The varied bioactivities of sesterterpenoids have made them attractive research objects for biological studies and as synthetic chemistry targets. 40 Figure 3.1 Examples of sesterterpenoids isolated from terrestrial fungi, insects, plants and marine sponge 3.2 Biological background Highly active antiretroviral therapy (HAART) was introduced to HIV infected patients in the 1990s and the therapy suppressed the virus, reduced viral transmission and restored the immune functions of the patients. The application of HAART improved the quality of life of the patients and they could have near-normal life expectancy.102 However, this treatment does not eliminate latent HIV reservoirs. Once the treatment is stopped, virus replication will lead to re-infection by HIV. The long-term treatment has some drawbacks including toxic effects; higher risk of cardiovascular diseases, cancer and osteoporosis; and it is not cost-effective.103-105 Therefore, new treatments that eradicate HIV-1 reservoirs are urgently needed. CD4+ T cells that contain integrated HIV-1 provirus are the major reservoir of latent infection.104,105 One of the strategies that has been proposed to achieve a sterilizing cure for HIV is called “shock and kill”, which starts from the reactivation of the latent HIV-1 provirus expression using latency reversal agents (LRAs). If both LRAs and HAART are applied to the patient at the same time, the virus-producing cells will either die from the direct cytopathic OOOHCHHOHHHOphiobolin AH OHH HOAcHOScalarinHOOHGeranylnerolidol (Geranylfarnesol)OOMeO2C H OHOHSalvileucolide methyl ester3.1 3.33.23.4 41 effects of the virus or be cleared by host mechanisms, thus achieving the sterilizing cure for HIV.102 The potential targets for reactivation of the HIV-1 proviral gene expression include histone deacetylases (HDACs), histone methyltransferases (HMTs), DNA methyltransferases (DNMTs), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NFATs) and positive transcription elongation factor b (P-TEFb).106-108 One of the regulation target sites is NF-κB, the main host transcription factor for HIV-1 replication.109 Two compounds, prostratin (3.5) and bryostatin 1 (3.6), are known to induce the PKC-NF-κB pathway. Their structures are shown in Figure 3.2. New small molecules that are safe and can potently reactivate virus transcription are still needed. Figure 3.2 Structures of prostratin (3.5) and bryostatin 1 (3.6) In collaboration with Dr. Ian Tietjen (Faculty of Health Sciences, Simon Fraser University), we screened a large library of compounds and crude extracts for their potential to be LRAs. The effects of HIV-1 latency reversal on a J-Lat 9.2 GFP-reporter CD4+ T cell line model were tested. This is a Jurkat (T cell) cell line with an integrated HIV provirus that encodes a noninfectious (Δenv/Δnef) proviral genome.110,111 The crude extracts of Phorbas sp. showed a positive response in this assay. OHOOAcHOHHHProstratin3.5OOOHOOOOOOOOOOAcHOOHOHBryostatin 13.6 42 Figure 3.3 J-Lat 9.2 GFP-reporter CD4+ T cell line111 3.3 Search for new sesterterpenoids from the sponge Phorbas sp. that activates latent HIV-1 provirus expression The isolation of sesterterpenoids with biological activities from the sponge Phorbas sp. described in section 2.2.2.1 motivated further investigations into the secondary metabolites that can be isolated from this sponge. Besides, the preliminary screening result indicated that the crude extracts of this sponge gave a positive response in the screening program designed to find new LRAs that activate HIV-1 provirus gene expression.112 A search for the minor sesterterpenoid analogues from the British Columbia sponge Phorbas sp. was carried out to further investigate the biological activities of these compounds. Fresh specimens of the British Columbia sponge Phorbas sp. were collected in Howe Sound using SCUBA. The samples were extracted with MeOH and the MeOH soluble compounds were partitioned between water and EtOAc. The EtOAc fraction was subjected to Sephadex™ LH-20 chromatography, silica gel chromatography and reversed-phase semi-preparative HPLC to obtain pure compounds. The search for novel sesterterpenoids in the Howe Sound Phorbas sp. led to the discovery of alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24). 43 3.4 Structure elucidation of alotaketals Three new alotaketals (3.7−3.9, Figure 3.4) were isolated from the British Columbia sponge Phorbas sp., and the detailed isolation is described in experimental section 3.11. Figure 3.4 Structures of alotaketal C (3.7), alotaketal D (3.8) and alotaketal E (3.9) 3.4.1 Structure elucidation of alotaketal C (3.7) In collaboration with Dr. David E. Williams, Dr. Julie Daoust and Dr. Min Chen of the Andersen Lab, alotaketal C (3.7) was isolated as a white powder. It gave an [M + Na]+ ion peak in the HRESIMS at m/z 481.2555 (calculated for C27H38O6Na, 481.2568), requiring nine sites of unsaturation. Analysis of the 1D and 2D NMR data showed its structure similarity to the “alotane” carbon skeleton. The cyclohexene ring (C-1 to C-6 and C-21), the bicyclic spiroketal fragment (C-6 to C-13, C-1 and C-22) and part of the side chain (C-14 to C-17) were the same as alotaketal B (2.73) (Table 3.1). Table 3.1 NMR data for alotaketal B (2.73) and C (3.7) recorded in C6D6 at 600 MHz Alotaketal B (2.73) Alotaketal C (3.7) C# δC δH (J in Hz) δC δH (J in Hz) 1 63.4 4.33 (br s) 63.2 4.27 (dd, 3.2, 4.9) 2 139.6 6.32 (d, 6.0) 139.1 6.27 (dd, 1.2, 5.6) 3 138.9 — 138.6 — 4 197.7 — 197.7 — 5 38.4 2.37 (br d, 7.8) 38.5 2.38 (d, 8.8) 5’ — 2.38 (d, 8.8) OOOAcHOHHAcOOOOHOHHAcOOHOOOHOHHAcOAlotaketal E3.9Alotaketal D3.8Alotaketal C3.71357911 1315 17 1921222324 2520 44 Alotaketal B (2.73) Alotaketal C (3.7) C# δC δH (J in Hz) δC δH (J in Hz) 6 34.0 2.00 (m) 34.1 1.99 (td, 3.2, 9.0) 7 142.5 — 142.4 — 8 125.3 5.43 (s) 125.2 5.44 (br s) 9 96.9 — 96.8 — 10 40.5 3.07 (d, 15.0) 39.9 3.11 (dd, 14.7, 2.2) 10’ 1.23 (d, 15.0) 1.19 (d, 14.7) 11 77.4 — 77.4 — 12 43.9 1.24 (m) 44.1 1.19 (d, 14.0) 12’ 1.95 (m) 1.81 (dt, 1.1, 14.0) 13 63.9 5.15 (t, 10.8) 64.0 5.13 (m, 1.1, 14.0) 14 126.5 5.45 (d, 8.4) 126.4 5.46 (d, 10.0) 15 139.1 — 139.4 — 16 39.7 2.01 (t, 7.2) 40.2 2.11 (dt, 8.3) 17 26.4 1.58 (q, 7.2) 27.3 2.21 (m) 18 38.0 1.96 (t, 7.2) 124.7 5.23 (br t, 6.9) 19 145.8 — 132.0 — 20 110.9 4.81(br s)/4.82(br s) 18.1 1.55 (s) 21 16.6 1.83 (s) 16.3 1.77 (s) 22 63.7 3.51 (m) 63.7 3.51 (m) 23 27.6 1.48 (s) 27.3 1.45 (s) 24 17.2 1.77 (s) 17.2 1.77 (s) 25 22.79 1.63 (s) 26.2 1.67 (s) 26 172.4 — 170.4 — 27 45.0 2.12 (m) 22.4 1.77 (s) 28 25.6 2.12 (m) — — 29 23.1 0.89 (d, 5.4) — — 30 23.1 0.89 (d, 5.4) — — OH — 0.51 (t, 5.4) — 0.58 (s) The absence of the isovalerate ester (C-28 to C-30 in 2.73) indicated a difference in the ester adjacent to C-11. HMBC (Figure 3.9) correlations observed between a deshielded 13C resonance at δ 170.4 (C-26), assigned to an ester, and a methyl singlet at δ 1.77 (H-27) suggested the presence of an acetate ester substituent at C-11. Another difference between the NMR data of alotaketals C (3.7) and B (2.73) was the absence of the terminal olefinic methylene (C-20 in 2.73) in the side chain of alotaketal C (3.7). HMBC correlations observed between a 45 methyl singlet at δ 1.55 (Me-20) and a pair of olefinic carbon resonances (δ 132.0, C-19 and δ 124.7, C-18), the correlations between δ 1.67 (Me-25) and both C-19 and C-18 resonances, and the correlations between the two methyl singlets (Me-20 and Me-25) indicated a fragment with a pair of germinal methyls (Me-20 and Me-25) attached to an alkene (C-19 and C-18). In addition, COSY (Figure 3.10) correlations observed between an olefinic methine resonance at δ 5.23 (H-18) and an aliphatic methylene resonance at δ 2.21 (H-17) and between the two aliphatic methylene resonances at δ 2.21 and 2.11 (H-17 and H-16) identified a spin system comprised of four carbons (C-16 to C-19). HMBC correlations between H-16 (δ 2.11) and C-14 (δ 126.4), C-15 (δ 139.4) and C-24 (δ 17.2) completed the assignment of the side chain. The assignment of the constitution of alotaketal C (3.7) is shown in Figure 3.5, along with important HMBC and COSY correlations. Comparing the ROESY and coupling constant data of alotaketal C (3.7) with those of alotaketal B (2.73) confirmed that it had the same relative configurations at C-1, C-6, C-9, C-11 and C-13 as alotaketal B (2.73). A biogenetic argument led to the assumption that alotaketal C (3.7) also has the same absolute configurations at these stereocenters as alotaketals A (2.72) and B (2.73). 46 Figure 3.5 Selected COSY and HMBC correlations observed for alotaketal C (3.7) in C6D6. Figure 3.6 1H NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 OOOHOHHOO1357911 13151719212325274.27; 63.26.27; 139.1138.6197.71.77; 16.32.38; 38.51.99; 34.15.44; 125.2142.4139.4132.077.4170.496.83.51; 63.73.11, 1.19 39.91.191.81 44.15.13; 64.05.46; 126.41.7717.2 2.1140.2 5.23124.72.2127.31.5518.11.4527.31.6726.21.7722.40.58 COSYHMBC 47 Figure 3.7 13C NMR spectrum of alotaketal C (3.7) recorded at 150 MHz in C6D6 Figure 3.8 gradHSQC NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 48 Figure 3.9 gradHMBC NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 Figure 3.10 gradCOSY60 NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 H-27/C-26H-24/C-15H-16/C-15H-25/C-20H-20/C-25H-25/C-18H-25/C-19 H-20/C-18H-20/C-19H-12/H-13H-13/H-14H-14/H-24H-16/H-17H-17/H-18H-18/H-20H-18/H-25 49 Figure 3.11 tROESY NMR spectrum of alotaketal C (3.7) recorded at 600 MHz in C6D6 3.4.2 Structure elucidation of alotaketal D (3.8) and E (3.9) Alotaketal D (3.8) was isolated from Phorbas sp. as a white powder, which gave a [M + Na]+ ion in the ESITOFHRMS at m/z 525.2827 consistent with a molecular formula of C29H42O7 (calculated for C29H42O7Na 525.2828), requiring nine degrees of unsaturation. Analysis of the NMR data collected for alotaketal D (3.8) revealed its structure similarity to alotaketal C (3.7) with differences only at C-2 to C-5 region (Table 3.2). A spin system comprised of an olefinic methine (δ 5.61, H-2), a deshielded methine (δ 4.27, H-1), another methine (δ 1.77, H-6) and a pair of germinal methylene (δ 1.71 and 2.00, H-5 and H-5’) protons was identified from the COSY data, which was similar to the corresponding spin system in the cyclohexene ring in alotaketal C (3.7). COSY correlations between a deshielded methine resonance at δ 5.39 (H-4) and both H-5 and H-5’ further extended the spin system to H-4. An HMBC correlation between H-6/H-1H-12/H-13 50 the H-4 resonance and an olefinic carbon resonance at δ 125.5 (C-2) completed the assignment of the cyclohexene ring. Given the upfield shift of H-2 (δ 5.61 in alotaketal D 3.8 and δ 6.27 in alotaketal C 3.7) and the absence of the ketone carbonyl resonance (δ 197.7, assigned to C-4 in alotaketal C 3.7), suggested that the carbon (C-4) was substituted with an oxymethine. HMBC correlations between an ester carbonyl resonance at δ 170.0 (C-26) and both a methyl singlet at δ 1.66 (Me-27) and the methine resonance at δ 5.39 (H-4), established the acetate ester substituent at C-4. The constitution of alotaketal D (3.8) and the important COSY and HMBC correlations for its structure assignment are shown in Figure 3.12. Figure 3.12 Selected COSY and HMBC correlations observed for alotaketal D (3.8) in C6D6. OOOHOHHOO1357911 13151719212325274.27; 63.55.61; 125.5140.21.63; 19.11.71, 2.00 29.01.77; 33.25.52; 125.7142.7139.1131.877.6170.096.33.65; 64.03.16, 1.25 40.31.231.83 44.35.19; 63.85.47; 126.61.7917.2 2.1040.35.22124.92.2027.31.5418.11.4727.51.6626.21.9322.70.60 COSYHMBCOH29170.01.6620.15.39; 72.3 51 Figure 3.13 1H NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 Figure 3.14 13C NMR spectrum of alotaketal D (3.8) recorded at 150 MHz in C6D6 52 Figure 3.15 COSY NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 Figure 3.16 HSQC NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 H-1/H-2H-2/H-21H-1/H-6H-5'/H6H-4/H-5' 53 Figure 3.17 HMBC NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 Figure 3.18 tROESY NMR spectrum of alotaketal D (3.8) recorded at 600 MHz in C6D6 H-27/C-26H-4/C-26H-4/C-2H-4/C-3H-21/C-2H-21/C-3H-21/C-4H-5'/C-4H-1/H-6H-4/H-6 54 Comparison of the coupling constant and ROESY data of alotaketal D (3.8) with the corresponding data for alotaketal B (2.73) confirmed the same relative configurations at C-1, C-6, C-9, C-11 and C-13 as alotaketal B (2.73). The assumption that alotaketal D (3.8) has the same absolute configurations as alotaketals A (2.72) and B (2.73) at these chiral centers was made based on a biogenetic argument. The acetate substituent at C-4 was assigned as pseudo-equatorial and H-4 as pseudo-axial for the following reasons: 1) the 1H resonance at δ 5.39 (H-4) was a dd (J = 5.6 Hz, 10.1 Hz), and the coupling constant indicated that H-5 and H-5’, the two coupling partners of H-4, should be one anti and one gauche with H-4, which was only possible for a pseudoaxial orientation of H-4 (Figure 3.19); 2) the 1H resonance at δ 1.71 (H-5) was partially obscured but the two coupling constants of J ≈ 10-11.3 Hz could be observed clearly and there was no coupling constant of J = 5.6 Hz, which indicated that H-5 was also pseudoaxial; 3) the 1H resonance at δ 2.00 (H-5’) was a ddd (J = 11.5, 5.8 Hz, 1.6 Hz), which was consistent with H-5’ being pseudoequatorial; 4) the coupling constant of H-6 was obscured, but a ROESY correlation observed between two resonances at δ 5.39 (H-4) and δ 1.77 (H-6) indicated that H-6 was also pseudoaxial. Therefore, the H-4 was assigned as pseudoaxial and cis to H-6, then the acetate substituent at C-4 must be pseudoequatorial. 55 Figure 3.19 Determination of the configuration at C-4 in alotaketal D (3.8) In support of this assignment, the corresponding carbinol methine proton in phorbaketal B (2.70),79 which has the same relative configuration at C-4 as that assigned to alotaketal D (3.8), is reported to be a bd with J = 10.3 Hz, and phorbaketal C (2.71)79, that is epimeric at C-4 compared with alotaketal D (3.8), has a carbinol methine signal that is a dd with J = 3.7, 2.0 Hz (Figure 3.20). Figure 3.20 Coupling constants at C-4 for phorbaketal B (2.70) and C (2.71) (R)MeOAcHHHHHOH5'645(S)MeHOAcHHHHOH55'64C-4 (R) C-4 (S)H-4/H-5: antiH-4/H-5': gaucheH-4/H-5: gaucheH-4/H-5': gauche5.39 (dd, J = 10.1, 5.6 Hz)1.71 (q, J = 11.3 Hz)2.00 (ddd, J = 11.5, 5.8, 1.6 Hz)(R)MeOAcHHHHHOH5'645tROESYOO(R)HO HHOHPhorbaketal B2.70OO(S)HO HHOHPhorbaketal C2.71H Hbd, J = 10.3 Hz dd, J = 3.7, 2.0 Hz 56 Table 3.2 NMR data for alotaketal C (3.7), D (3.8) and E (3.9) recorded in C6D6 at 600 MHz Alotaketal C (3.7) Alotaketal D (3.8) Alotaketal E (3.9) C# δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) 1 63.2 4.27 (dd, 3.2, 4.9) 63.5 4.27 (br t, 4.3) 63.3 4.24 (dd, 3.4, 4.9) 2 139.1 6.27 (dd, 1.2, 5.6) 125.5 5.61 (dt, 5.2, 1.6) 139.3 6.25 (dd, 5.7, 1.4) 3 138.6 — 140.2 — 138.9 — 4 197.7 — 72.3 5.39 (dd, 10.1, 5.6) 197.3 — 5 38.5 2.38 (d, 8.8) 29.0 1.71 (q, 11.3) 38.5 2.37 (d, 8.5) 5’ 2.38 (d, 8.8) 2.00 (ddd, 11.5, 5.8, 1.6) 2.37 (d, 8.5) 6 34.1 1.99 (td, 3.2, 9.0) 33.2 1.77 (m) 34.0 1.98 (dt, 3.3, 8.5) 7 142.4 — 142.7 — 142.6 — 8 125.2 5.44 (br s) 125.7 5.52 (br s) 125.0 5.41 (br s) 9 96.8 — 96.3 — 96.7 — 10 39.9 3.11 (dd, 14.7, 2.2) 40.3 3.16 (dd, 14.6, 2.3) 39.9 3.11 (dd, 14.6, 2.3) 10’ 1.19 (d, 14.7) 1.25 (d, 14.7)a 1.19 (d, 14.6) 11 77.4 — 77.6 — 77.4 — 12 44.1 1.19 (d, 14.0) 44.3 1.23 (dd, 14.8, 11.2)a 44.0 1.20 (m) 12’ 1.81 (dt, 1.1, 14.0) 1.83 (dt, 2.1, 13.7) 1.80 (td, 2.2, 13.9) 13 64.0 5.13 (m, 1.1, 14.0) 63.8 5.19 (ddd, 11.2, 8.2, 2.0)b 64.0 5.12 (ddd, 11.2, 8.2, 2.0) 14 126.4 5.46 (d, 10.0) 126.6 5.47 (dd, 8.2, 1.0) 127.3 5.45 (dq, 8.2, 1.1) 15 139.4 — 139.1 — 137.9 — 16 40.2 2.11 (dt, 8.3) 40.3 2.10 (br t, 7.7) 43.1 2.68 (br d, 5.9) 17 27.3 2.21 (m) 27.3 2.20 (m) 128.5 5.63 (m) 18 124.7 5.23 (br t, 6.9) 124.9 5.22 (dt, 7.0, 1.4)b 137.1 5.62 (d, 15.8) 19 132.0 — 131.8 — 81.8 — 20 18.1 1.55 (s) 18.1 1.54 (s) 24.9 1.22 (s) 21 16.3 1.77 (s) 19.1 1.63 (s) 16.3 1.77 (s) 22 63.7 3.51 (m) 64.0 3.65 (m) 63.7 3.49 (m) 23 27.3 1.45 (s) 27.5 1.47 (s) 27.3 1.43 (s) 24 17.2 1.77 (s) 17.2 1.79 (d, 0.9) 17.2 1.75 (d, 1.0) 25 26.2 1.67 (s) 26.2 1.66 (br s) 24.9 1.22 (s) 26 170.4 — 170.0 — 170.1 — 27 22.4 1.77 (s) 20.1 1.66 (br s) 22.4 1.77 (s) 28 — — 170.0 — — — 29 — — 22.7 1.93 (s) — — OH — 0.58 (s) — 0.60 (br s) — 0.52 (t, 5.7) a.b Overlapped resonances Alotaketal E (3.9) was obtained as a white powder which gave a [M + Na]+ ion peak in the ESITOFHRMS at m/z 513.2452 consistent with a molecular formula of C27H38O8 (calculated 57 for C27H38O8Na 513.2464). The LRESIMS recorded in MeOD gave a [M + Na]+ ion peak at m/z 515.3, the two unit shift of the ion peak counted for two exchangeable protons in alotaketal E (3.9). The molecular formula of alotaketal E (3.9) was different from that of alotaketal C (3.8) only by addition of one oxygen atom. Analysis of the NMR data collected for alotaketal E (3.9) revealed its structure similarity to alotaketal C (3.8) with the identical methyl cyclohexenone ring (C-1 to C-6 and C-21), the bicyclic spiroketal (C-1, C-6 to C-13) and part of the side chain (C-14 to C-16, C-24). The differences were only at the side chain region from C-17 to C-20 (see Table 3.2). The hydroxyl group at C-22 was responsible for one of the two exchangeable protons. Therefore, the other exchangeable proton was a result of a hydroxyl substituent on the side chain. HMBC correlations between a six-proton singlet at δ 1.22 (Me-20 and Me-25) and carbon resonances at δ 24.9 (Me-20, Me-25), 81.8 (C-19), and 137.1 (C-18) identified a pair of identical germinal methyls attached to a deshielded quaternary carbon (C-19), which was then attached to an olefinic carbon (C-18). The quaternary carbon (C-19) was deshielded as a result of the hydroxyl substitution. COSY correlations observed between resonances at δ 5.62 (H-18) and 5.63 (H-17), and δ 2.68 (H-16) and 5.63 (H-17) suggested that the olefin was attached to a methylene (δ 2.68 H-16; 43.1 C-16). A HMBC correlation between this methylene at δ 2.68 (H-16) and an olefinic carbon resonance at δ 137.9 (C-15) completed the assignment of the constitution of alotaketal E (3.9) as shown in Figure 3.21. The coupling constant (J = 15.8 Hz) of the olefinic proton doublet at δ 5.62 (H-18) revealed that the olefin had the E configuration.112 58 Figure 3.21 Selected COSY and HMBC correlations for alotaketal E (3.9) in C6D6. Figure 3.22 1H NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 OOOHOHHOO1357911 13151719212325274.24; 63.36.25; 139.3138.9197.31.77; 16.32.37; 38.51.98; 34.05.41; 125.0142.6137.977.4170.196.73.49; 63.73.11, 1.19 39.91.201.80 44.05.12; 64.05.45; 127.31.7517.2 2.6843.1 5.62137.15.63128.51.2224.91.4327.31.7722.40.52 COSYHMBCOH81.81.2224.9 59 Figure 3.23 13C NMR spectrum of alotaketal E (3.9) recorded at 150 MHz in C6D6 Figure 3.24 COSY NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 H-16/H-17H-17/H-18 60 Figure 3.25 HSQC NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 Figure 3.26 HMBC NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 H-24/C-15H-27/C-26H-24/C-14H-16/C-15H-16/C-17H-20/C-19H-25/C-19H-20/C-18H-25/C-18H-20/C-25H-25/C-20 61 Figure 3.27 tROESY NMR spectrum of alotaketal E (3.9) recorded at 600 MHz in C6D6 3.5 Structure elucidation of ansellones and phorbadione A series of new ansellones together with the known compounds ansellones A−C (2.86−2.88) were isolated from Phorbas sp. (Figure 3.29), and the detailed isolation is described in experimental section 3.11. 3.5.1 Ansellones A, B and C (2.86−2.88) Ansellone A (2.86) was isolated as off-white oil. The 1H NMR spectrum (Figure 3.28) matched the spectrum of ansellone A (2.86) in the literature.83 The [M + Na]+ ion peak at m/z 465.4 in the LRESIMS was consistent with the molecular formula. H-6/H-1H-2/H-1 62 Figure 3.28 1H NMR spectrum of ansellone A (2.86) recorded at 600 MHz in C6D6 63 Figure 3.29 Structures of ansellones A−N (2.86−2.88, 3.11−3.21) and phorbadione (3.10) isolated from the sponge Phorbas sp. OHOMeHH HHOAcOOOAcO H HHHOHHOOAcOOOHH HHOOAcOOH HHHOHOOAcOOHHHHHAnsellone B2.87OOAcOOHHHHHAnsellone C2.88OHOHHH HHOAcOAnsellone A2.86Ansellone D3.11Ansellone E3.12Ansellone F3.13Ansellone G3.14OOAcOOH HHPhorbadione3.10Ansellone L3.19OHOHHH HHOHOOOAcO H HHHOHOOAcO H HHHOHOHH HAnsellone H3.15Ansellone I3.16OOHOAcAcOHHHAnsellone K3.18OOHH HHOAcOOHAnsellone J3.17OHOMeHH HHOHOAnsellone M3.20OHOHHHHOAcOHOAnsellone N3.2113579111315171920212224 2325 64 Ansellone B (2.87) was isolated as a white powder. The 1H NMR (Figure 3.30) spectrum matched the spectrum of ansellone B (2.87) in the literature.84 The [M + Na]+ ion peak at m/z 467.4 in the LRESIMS was consistent with the molecular formula. Ansellone C (2.88) was isolated as a white powder. The 1H NMR (Figure 3.31) spectrum matched the spectrum of ansellone C (2.88) in the literature.84 The [M + Na]+ ion peak at m/z 467.4 in the LRESIMS was consistent with the molecular formula. Figure 3.30 1H NMR spectrum of ansellone B (2.87) recorded at 600 MHz in C6D6 65 Figure 3.31 1H NMR spectrum of ansellone C (2.88) recorded at 600 MHz in C6D6 3.5.2 Structure elucidation of phorbadione (3.10) Phorbadione (3.10) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESIHRMS at m/z 461.2297 (calculated for C27H34O5Na, 461.2304), requiring eleven sites of unsaturation. The NMR data collected for phorbadione (3.10) is listed in Table 3.3. Detailed analysis of the COSY and HMBC data collected for phorbadione (3.10) identified the existence of the same C-1 to C-9 bicyclic fragment (C13H15O4) found in ansellone A (2.86), including two carbonyls (C-4 δ 196.8, C-26 δ 169.7) and two alkenes (C-2 δ 138.2, C-3 δ 138.8 and C-7 δ 133.8, C-8 δ 129.0), which accounted for six sites of unsaturation. The rest of the molecule still had 13C resonances for one carbonyl and four alkene carbons, which accounted for three sites of unsaturation. Therefore, the rest of the molecule should be bicyclic. 66 In the HMBC spectrum collected in C6D6, a methyl proton resonance at δ 0.93 (Me-20) correlated with carbon resonances assigned to Me-25 (δ 28.5), C-18 (δ 40.4), and C-14 (δ 170.4), and another methyl proton resonance at δ 0.99 (Me-25) correlated with carbon resonances assigned to Me-20 (δ 32.4), C-18 (δ 40.4), and C-14 (δ 170.4), indicating that these two methyl groups were geminal, and the quaternary carbon (C-19) attached to these two methyl groups was connected to C-14 and C-18. A spin system was identified from COSY correlations (spectrum collected in CD3OD) between aliphatic methylene proton resonances at δ 1.49/2.36 (H-16/H-16’), δ 1.67/1.99 (H-17/H-17’) and δ 1.39/1.73 (H-18/H-18’). HMBC correlations between a methyl proton resonance at δ 1.41 (Me-24) and carbon resonances at δ 45.9 (C-15), 161.4 (C-10), 176.3 (C-14), and 35.4 (C-16) suggested that Me-24 connected directly to C-15, which was then attached to C-10, C-14 and C-16. In the HMBC spectrum collected in C6D6, the cross-conjugated ketone substructure (C-10 to C-14) was confirmed by correlations between an alkene proton resonance at δ 6.48 (H-13) and an alkene carbon resonance at δ 134.3 (C-11), a carbonyl resonance at δ 186.3 (C-12), and an alkene carbon resonance at δ 170.4 (C-14), and between the methyl resonance at δ 2.10 (H-23) and alkene carbon resonances at δ 156.4 (C-10) and δ 134.3 (C-11) and a carbonyl resonance at δ 186.3 (C-12). At last, the HMBC correlation between H-9 and C-10 established a bond between C-9 and C-10 to complete the constitutional structure assignment of phorbadione (3.10) shown in Figure 3.32 and Table 3.3. 67 Figure 3.32 Selected COSY and HMBC correlations observed for phorbadione (3.10) in C6D6 and CD3OD. Table 3.3 NMR data for phorbadione (3.10) recorded in C6D6 and CD3OD at 600 MHz Phorbadione (3.10, in C6D6) Phorbadione (3.10, in CD3OD) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.7 3.57 (t, 5.0) 71.8 4.34 (br t, 5.2) 2 138.2 6.17 (dq, 5.0, 1.3) 140.9 6.74 (dq, 5.2, 1.4) 3 138.8 — 139.6 — 4 196.8 — 200.1 — 5 37.4 2.62 (m) 38.1 2.67 (m) 5’ 2.62 (m) 2.67 (m) 6 34.1 2.19 (m) 35.0 2.72 (m) 7 133.8 — 135.3 — 8 129.0 5.49 (br s) 129.8 5.91 (br s) OOOOHHOOOOOH HHO2.6237.43.57; 70.76.17; 138.2138.8196.8 1.75; 16.02.1934.15.49; 129.0133.8186.343.7170.4156.44.35/4.21 65.11.92/1.40 34.34.73; 75.52.10; 12.26.48124.40.9928.51.26/1.04 27.30.9332.41.50/1.27 18.5169.71.63; 20.337.1134.31.04 24.62.6738.14.34; 71.86.74; 140.9139.6200.1 1.82; 15.82.7235.05.91; 129.8135.3189.545.9176.3161.44.54/4.72 66.31.49/2.36 35.45.30; 76.42.10; 12.26.28124.11.3329.01.39/1.73 41.61.2332.91.67/1.99 19.3172.42.05; 20.737.1134.61.41 24.7COSYHMBCOOOOOOOOOOdata recorded in C6D6data recorded in C6D6data recorded in CD3ODdata recorded in CD3OD13597111315171921232522242720tROESY 68 Phorbadione (3.10, in C6D6) Phorbadione (3.10, in CD3OD) C# δC δH (J in Hz) δC δH (J in Hz) 9 75.5 4.73 (m) 76.4 5.30 (br s) 10 156.4 — 161.4 — 11 134.3 — 134.6 — 12 186.3 — 189.5 — 13 124.4 6.48 (s) 124.1 6.28 (s) 14 170.4 — 176.3 — 15 43.7 — 45.9 — 16 34.3 1.40 (td, 12.7, 3.5) 35.4 1.49 (dt, 12.9, 4.4) 16’ 1.92 (m) 2.36 (br d, 12.9) 17 18.5 1.27 * 19.3 1.67 (m) 17’ 1.50 (m) 1.99 (dm, 13.8) 18 40.4 1.04 * 41.6 1.39 (td, 13.3, 4.0) 18’ 1.26 * 1.73 (dt, 13.3, 4.0) 19 37.1 — 38.7 — 20 32.4 0.93 (s) 32.9 1.23 (s) 21 16.0 1.75 (br s) 15.8 1.82 (br s) 22 65.1 4.21 (d, 13.0) 66.3 4.54 (d, 12.9) 22’ 4.35 (d, 13.0) 4.72 (br d, 12.9) 23 12.2 2.10 (s) 12.3 1.90 (s) 24 24.6 1.04 * 24.7 1.41 (s) 25 28.5 0.99 (s) 29.0 1.33(s) 26 169.7 — 172.4 — 27 20.3 1.63 (s) 20.7 2.05 (s) * Overlapped resonances The tROESY correlations observed between proton resonances H-1 (δ 3.57) and H-6 (δ 2.19), and between H-1 (δ 3.57) and H-9 (δ 4.73), showing that H-1, H-6 and H-9 were on the same side of the ring system, suggested that the configurations of phorbadione (3.10) were the same as ansellone A (2.86) at C-1, C-6, and C-9. The noise in the spectrum made it impossible to determine the configuration at C-15 through tROESY data alone, and we assumed that C-15 had the same configuration as ansellone A (2.86) on the basis of a biogenetic argument. Comparison of the experimental CD spectrum with the theoretical CD spectra confirmed the final assignment of phorbadione (3.10) at C-15. The Wang Group (Department of Chemistry, University of British Columbia) calculated the CD spectra for the two possible structures84 differing only on the configuration at C-15 as shown in Figure 3.33. Panel A was the experimental CD spectrum 69 for phorbadione (3.10), panel B and C were theoretical CD spectra for the two possible epimers at C-15. Panel B showing the calculated CD spectrum of structure I matched panel A better, hence the structure I with C-15 (S) was confirmed as the actual configuration of phorbadione (3.10). Figure 3.33 CD spectrum for phorbadione (3.10) (panel A, experimental CD spectrum for 3.10 in CH2Cl2 at < 0.1 mg/mL; panel B, calculated theoretical spectrum for structure I; panel C, calculated theoretical spectrum for structure II) 70 Figure 3.34 1H NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 Figure 3.35 13C NMR spectrum of phorbadione (3.10) recorded at 150 MHz in C6D6 71 Figure 3.36 gradCOSY60 NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 Figure 3.37 gradHSQC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 H-1/H-6H-5/H-6H-1/H-2H-1/Me-21 72 Figure 3.38 gradHMBC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 Figure 3.39 tROESY NMR spectrum of phorbadione (3.10) recorded at 600 MHz in C6D6 Me-23/C-11Me-23/C-10Me-23/C-12H-13/C-15H-13/C-19H-13/C-11H-13/C-14Me-25/C-20Me-20/C-25Me-25/C-14Me-20/C-14Me-25/C-20Me-20/C-25H-1/H-6H-1/H-9 73 Figure 3.40 1H NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD. Figure 3.41 13C NMR spectrum of phorbadione (3.10) recorded at 150 MHz in CD3OD 74 Figure 3.42 gradCOSY60 NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD Figure 3.43 gradHSQC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD H-17'/H-18H-17/H-18H-17'/H-18'H-17/H-16'H-17'/H-16'H-1/H-6H-8/H-9 75 Figure 3.44 gradHMBC NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD Figure 3.45 tROESY NMR spectrum of phorbadione (3.10) recorded at 600 MHz in CD3OD Me-24/C-10Me-24/C-14Me-24/C-16Me-24/C-15H-9/C-10H-1/H-6H-1/H-9 76 3.5.3 Structure elucidation of ansellone D (3.11) Ansellone D (3.11) was isolated as a white powder. Preliminary analysis of the NMR data showed that it was closely related to ansellone A (2.86). It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 479.2778 (calculated for C28H40O5Na, 479.2773), which differed from the molecular formula of ansellone A (2.86) by adding a methylene. NMR data of ansellone D (3.11) is listed in Table 3.4. Comparing the NMR data of ansellone D (3.11) to the data of ansellone A (2.86) in Table 3.4, the major differences were the presence of a methyl singlet at δ 3.04 (Me-28), assigned to a methyl ether, and the absence of the 11-OH present in ansellone A (2.86). There were also noticeable differences in the chemical shifts of H-10 (δ 1.46 in 2.86; 1.82 in 3.11), H-12 (δ 5.52 in 2.86; 5.45 in 3.11), H-13 (δ 5.59 in 2.86; 5.81 in 3.11) and Me-23 (δ 1.50 in 2.86; 1.61 in 3.11), which indicated that the structural difference between ansellone A (2.86) and D (3.11) was located in the C-10 to C-13 region of the molecules. Table 3.4 NMR data for ansellone A (2.86) and ansellone D (3.11) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone D (3.11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 70.5 3.44 (t, 4.2) 2 139.4 6.14 (d, 4.8) 139.1 6.14 (dd, 5.6, 1.2) 3 138.6 — 138.2 — 4 197.7 — 197.5 — 5 38.6 2.67 (d, 9.0) 38.3 2.68 (m) 5’ 2.67 (d, 9.0) 2.68 (m) 6 34.8 2.10 (tt, 9.0, 3.0) 34.5 2.09 (t, 8.2) 7 131.4 — 131.1 — 8 134.8 5.70 (br s) 134.8 5.68 (br s) 9 76.9 4.77 (br s) 76.2 4.69 (br s) 10 63.8 1.46 (br s) 55.4 1.82 (br s) 11 73.1 — 78.4 — 12 135.7 5.52 (dd, 10.0, 3.0) 132.3 5.45 (dd, 10.3, 3.1) 13 128.0 5.59 (br d, 10.0) 131.7 5.81 (dd, 10.3, 1.5) 14 57.0 1.71 (m) 56.7 1.71 (m) 15 42.1 — 42.0 — 16 38.9 1.02 (m) 38.9 1.09 (m) * 16’ 1.82 (br d, 13.0) 1.87 (dm, 12.8) 77 Ansellone A (2.86, C6D6) Ansellone D (3.11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 17 18.9 1.33 (m) 18.5 1.35 (m) 17’ 1.53 (dt, 13.0, 3.5) 1.56 (dm, 13.7) 18 41.7 1.04 (m) 41.3 1.09 (m) * 18’ 1.29 (m) 1.31 (dm, 13.2) 19 33.1 — 32.7 — 20 33.6 0.86 (s) 33.2 0.85 (s) 21 16.3 1.69 (s) 15.9 1.69 (s) 22 66.1 4.27 (br d, 12.0) 65.8 4.25 (br d, 12.4) 22’ 4.42 (br d, 12.0) 4.43 (br d, 12.4) 23 26.5 1.50 (s) 25.5 1.61 (s) 24 16.8 1.20 (s) 17.0 1.26 (s) 25 22.2 0.78 (s) 21.8 0.78 (s) 26 170.2 — 169.9 — 27 20.8 1.67 (s) 20.4 1.65 (s) 28 — — 49.2 3.04 (s) * Overlapped resonances HMBC correlations observed between C-11 (δ 78.4) and Me-28 (δ 3.04), H-10 (δ 1.82), H-13 (δ 5.81) and Me-23 (δ 1.61) confirmed that a methyl ether (in ansellone D 3.11) rather than a hydroxyl substituent (in ansellone A 2.86) was present in ansellone D (3.11) at C-11. The 13C chemical shift of Me-23 in 3.11 (δ 25.5) was almost identical to the chemical shift of Me-23 in 2.86 (δ 26.5), indicating that Me-23 was also axial in ansellone D (3.11). The axial orientation of Me-23 could also be confirmed by a ROESY correlation observed between two methyl resonances at δ 1.61 (Me-23) and 1.26 (Me-24). The complete NMR assignment of ansellone D (3.11) and important COSY and HMBC correlations are shown in Figure 3.46. 78 Figure 3.46 Selected COSY and HMBC correlations observed for ansellone D (3.11) in C6D6 Figure 3.47 1H NMR spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 OOOHHO2.6838.33.44; 70.56.14; 139.1138.2197.5 1.69; 15.92.0934.55.68; 134.8131.15.45132.342.01.7156.71.8255.44.43/4.25 65.81.90/1.87 38.94.69; 76.21.61; 25.55.81131.70.7821.81.31/1.09 41.30.8533.21.56/1.35 18.5169.91.65; 20.432.778.41.26 17.03.04; 49.2O13OHOOAcO21201917 1513119752825242322COSYHMBC 79 Figure 3.48 13C NMR spectrum of ansellone D (3.11) recorded at 150 MHz in C6D6 Figure 3.49 COSY spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 H-12/H-13H-8/H-9H-13/H-14H-9/H-10 80 Figure 3.50 HSQC spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 Figure 3.51 HMBC spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 Me-28/C-11 Me-23/C-11H-13/C-11H-10/C-9H-10/C-11 81 Figure 3.52 tROESY spectrum of ansellone D (3.11) recorded at 600 MHz in C6D6 3.5.4 Structure elucidation of ansellone E (3.12) Ansellone E (3.12) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 465.2615 (calculated for C27H38O5Na, 465.2617), which was identical to the molecular formula of ansellone A (2.86). Detailed analysis of the 1D and 2D NMR data collected for this new compound showed that it had the same constitutional structure as ansellone A (2.86). The comparison of NMR data collected for ansellone A (2.86) and E (3.12) is shown in Table 3.5. The 1H and 13C chemical shifts were only different at H-10 (δ 1.46 in 2.86; 1.33 in 3.12), H-12 (δ 5.52 in 2.86; 5.85 in 3.12), Me-23 (δ 1.50 in 2.86; 1.35 in 3.12 and δ 26.5 in 2.86; 31.4 in 3.12). Table 3.5 NMR data for ansellone A (2.86) and ansellone E (3.12) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone E (3.12, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 70.9 3.50 (t, 4.3) 2 139.4 6.14 (d, 4.8) 138.8 6.03 (dd, 5.6, 1.3) Me-23/Me-24 82 Ansellone A (2.86, C6D6) Ansellone E (3.12, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 3 138.6 — 139.0 — 4 197.7 — 197.2 — 5 38.6 2.67 (d, 9.0) 38.7 2.65 (m) 5’ 2.67 (d, 9.0) 2.65 (m) 6 34.8 2.10 (tt, 9.0, 3.0) 34.9 2.05 (dt, 7.1, 2.9) 7 131.4 — 131.7 — 8 134.8 5.70 (br s) 133.3 5.72 (br s) 9 76.9 4.77 (br s) 77.5 4.76 (br s) 10 63.8 1.46 (br s) 61.5 1.33 (m) 11 73.1 — 71.7 — 12 135.7 5.52 (dd, 10.0, 3.0) 136.2 5.85 (dd, 10.1, 3.0) 13 128.0 5.59 (br d, 10.0) 125.9 5.57 (dd, 10.1, 1.7) 14 57.0 1.71 (m) 55.9 1.50 (t, 2.4) 15 42.1 — 39.7 — 16 38.9 1.02 (m) 39.4 0.96 (dt, 12.8, 3.3) 16’ 1.82 (br d, 13.0) 1.82 (dt, 12.5, 3.3) 17 18.9 1.33 (m) 18.9 1.36 (dt, 3.5) 17’ 1.53 (dt, 13.0, 3.5) 1.54 (dt, 13.7, 3.2) 18 41.7 1.04 (m) 41.7 1.06 (dt, 13.5, 3.9) 18’ 1.29 (m) 1.33 (m) 19 33.1 — 33.3 — 20 33.6 0.86 (s) 33.6 0.86 (s) 21 16.3 1.69 (s) 16.3 1.68 (s) 22 66.1 4.27 (br d, 12.0) 66.1 4.25 (br d, 12.6) 22’ 4.42 (br d, 12.0) 4.38 (br d, 12.6) 23 26.5 1.50 (s) 31.4 1.35 (s) 24 16.8 1.20 (s) 16.6 1.31 (s) 25 22.2 0.78 (s) 22.4 0.83 (s) 26 170.2 — 169.9 — 27 20.8 1.67 (s) 20.7 1.66 (s) OH — — — 3.23 (br s) The 13C chemical shifts of Me-23 (δ 31.4) indicated that this methyl group was in the equatorial orientation compared with its isomer ansellone A (2.86), where Me-23 (δ 26.5 in 2.86) was in the axial orientation. A ROESY correlation between Me-23 (δ 1.35) and H-9 (δ 4.76) also confirmed the equatorial orientation of Me-23. Therefore, ansellone E (3.12) was assigned the structure as shown in Figure 3.53, which was epimeric at C-11 compared with ansellone A (2.86). Important COSY, HMBC and tROESY correlations are also shown in the figure. 83 Figure 3.53 Selected COSY, HMBC and tROESY correlations observed for ansellone E (3.12) in C6D6 OOOHHO2.6538.73.50; 70.96.03; 138.8139.0197.2 1.68; 16.32.0534.95.72; 133.3131.75.85136.239.71.5055.91.3361.54.38/4.25 66.11.82/0.96 39.44.76; 77.51.35; 31.45.57125.90.8322.41.33/1.06 41.70.8633.61.54/1.36 18.9169.91.66; 20.733.371.71.31 16.6OH13OHOHOAcO21201917 15131197525242322COSYHMBC3.23HHOHOHHtROESY 84 Figure 3.54 1H NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 Figure 3.55 13C NMR spectrum of ansellone E (3.12) recorded at 150 MHz in C6D6 85 Figure 3.56 COSY NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 Figure 3.57 HSQC NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 H-12/H-13 H-12/H-14H-13/H-14H-9/H-10 86 Figure 3.58 HMBC NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 Figure 3.59 tROESY NMR spectrum of ansellone E (3.12) recorded at 600 MHz in C6D6 Me-23/C-11Me-23/C-10H-13/C-11Me-23/H-9H-9/H-1Me-25/Me-24 87 3.5.5 Structure elucidation of ansellone F (3.13) Ansellone F (3.13) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 479.2403 (calculated for C27H36O6Na, 479.2410), which required ten sites of unsaturation. The molecular formula of ansellone F (3.13) differed from ansellone A (2.86) by losing two hydrogen atoms and adding one oxygen atom. Table 3.6 NMR data for ansellone A (2.86) and ansellone F (3.13) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone F (3.13, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 70.8 3.38 (br t, 4.1) 2 139.4 6.14 (d, 4.8) 138.7 6.14 (dd, 5.5, 1.2) 3 138.6 — 139.1 — 4 197.7 — 197.2 — 5 38.6 2.67 (d, 9.0) 38.5 2.63 (m) * 5’ 2.67 (d, 9.0) 2.63 (m) * 6 34.8 2.10 (tt, 9.0, 3.0) 34.7 2.04 (m) 7 131.4 — 132.2 — 8 134.8 5.70 (br s) 128.7 5.99 (br s) 9 76.9 4.77 (br s) 78.2 4.12 (br s) 10 63.8 1.46 (br s) 69.7 — 11 73.1 — 60.4 — 12 135.7 5.52 (dd, 10.0, 3.0) 53.6 2.80 (d, 4.4) 13 128.0 5.59 (br d, 10.0) 49.8 2.65 (br t, 4.4) * 14 57.0 1.71 (m) 46.7 1.80 (d, 4.6) 15 42.1 — 41.2 — 16 38.9 1.02 (m) 35.8 1.27 (dd, 12.3, 3.9) 16’ 1.82 (br d, 13.0) 1.84 (dt, 13.1, 3.5) 17 18.9 1.33 (m) 19.0 1.38 (m) 17’ 1.53 (dt, 13.0, 3.5) 1.34 (m) 18 41.7 1.04 (m) 41.1 0.98 (m) 18’ 1.29 (m) 1.20 (m) 19 33.1 — 33.3 — 20 33.6 0.86 (s) 33.4 1.00 (s) 21 16.3 1.69 (s) 16.3 1.71 (s) 22 66.1 4.27 (br d, 12.0) 65.6 4.21 (br d, 13.0) 22’ 4.42 (br d, 12.0) 4.35 (br d, 13.0) 23 26.5 1.50 (s) 19.0 1.40 (s) 24 16.8 1.20 (s) 18.9 1.21 (s) 25 22.2 0.78 (s) 22.7 0.81 (s) 26 170.2 — 170.0 — 27 20.8 1.67 (s) 20.6 1.64 (s) * Overlapped resonances 88 Preliminary examination of the NMR data collected for this new compound showed that it was closely related to ansellone A (2.86). The NMR data of ansellone A (2.86) and F (3.13) is listed in Table 3.6. Detailed analysis of the 1D and 2D NMR data obtained for ansellone F (3.13) readily revealed that it had the identical C-1 to C-9 bicyclic fragment (C13H15O4) found in ansellone A (2.86), including two carbonyl carbons (C-4 δ 197.2; C-26 δ 170.0) and four alkene carbons (C-2 δ 138.7, C-3 δ 139.1 and C-7 δ 132.2, C-8 δ 128.7), which accounted for six sites of unsaturation. There were no other sp2 or sp hybridized carbons in the 13C NMR spectrum, which suggested that four other rings were present in the rest of the structure. The COSY spectrum showed a spin system beginning at methylene resonances at δ 1.84/1.27 (H-16/H-16’), proceeded to the second methylene resonances at δ 1.38/1.34 (H-17/H-17’), ending at the third methylene resonances at δ 0.98/1.20 (H-18/H-18’). A pair of geminal methyls was confirmed by the correlations between themselves (Me-25 δ 0.81 and C-20 δ 33.4, Me-20 δ 1.00 and C-25 δ 22.7), correlations between the methyl resonance at δ 1.00 (Me-20) and carbon resonances at δ 41.1 (C-18), δ 33.3 (C-19) and δ 46.7 (C-14), and correlations between the methyl resonance at δ 0.81 (H-25) and carbon resonances at δ 41.1 (C-18), δ 33.3 (C-19) and δ 46.7 (C-14). Another methyl resonance at δ 1.21 (Me-24) correlated with carbon resonances at δ 35.8 (C-16), δ 41.2 (C-15), δ 46.7 (C-14) and δ 69.7 (C-10), indicating that this methyl group was connected to a quaternary carbon C-15, which was then attached to C-10, C-14 and C-16. This trimethylcyclohexane ring (C-14 to C-20, C-24 and C-25) was similar to the corresponding trimethylcyclohexane moieties in other ansellones. The remaining unassigned five carbons included a methyl (δ 1.40 H-23, δ 19.0 C-23), two deshielded methines (δ 2.65 H-13, δ 49.8 C-13 and δ 2.80 H-12, δ 53.6 C-12), two deshielded quaternary carbons (δ 69.7 C-10 and δ 60.4 C-11) and two oxygen atoms, which accounted for the three rings and two ethers. 89 A spin system was observed in the COSY spectrum consisting of three vicinal methines at δ 1.80 (H-14), δ 2.65 (H-13) and δ 2.80 (H-12). The 13C NMR chemical shifts at δ 49.8 (C-13) and 53.6 (C-12) indicated that they were part of an epoxide functionality, which required that the remaining two non-protonated carbons at δ 69.7 (C-10) and 60.4 (C-11) and the final oxygen atom must also be part of an epoxide functionality. The methyl resonance at δ 1.40 (Me-23) correlated with carbon resonances at δ 69.7 (C-10), δ 60.4 (C-11) and δ 53.6 (C-12) in the HMBC spectrum, which suggested that the second epoxide bridged C-10 and C-11. Additional HMBC correlations observed between the methyl resonance at δ 1.21 (Me-24) and the epoxide resonance at δ 69.7 (C-10), and between the H-14 methine resonance at δ 1.80 and both of the epoxide carbon resonances at δ 69.7 (C-10) and 49.8 (C-13), showed that the four carbon bis-epoxide fragment was fused to C-15 via a non-protonated epoxide carbon (C-10) and to C-14 via an epoxy methine carbon (C-13), which required that the methyl (Me-23) at δ 1.40 had to be attached to the internal non-protonated epoxy carbon (C-11) resonating at δ 60.4 (C-11). A COSY correlation observed between H-13 (δ 2.65) and H-14 (δ 1.80) confirmed this assignment. The complete structure assignment of ansellone F (3.13) is presented in Figure 3.60, along with important COSY and HMBC correlations. 90 Figure 3.60 Selected COSY and HMBC correlations observed for ansellone F (3.13) in C6D6 A ROESY correlation observed between the Me-24 and Me-25 resonances at δ 1.21 and 0.81 demonstrated that the decalin rings were trans fused with the two methyls in the axial orientation, which was consistent with other ansellones. Additional ROESY correlations observed between the methine resonance at δ 2.65 (H-13) and both of the Me-24 (δ 1.21) and Me-25 (δ 0.81) resonances indicated that H-13 was cis to the two axial methyls, so the epoxide ring at C-12 and C-13 must be trans to Me-24 and Me-25. A ROESY correlation observed between the resonance at δ 1.21 (Me-24) and the resonance at δ 5.99 (H-8) indicated that H-8 and Me-24 were also cis, so the epoxide ring at C-10 and C-11 must be trans to Me-24. We have assumed that the absolute configurations at C-1, C-6, C-9, C-14, and C-15 are identical in ansellone A (2.86) and ansellone F (3.13), which completed the structure assignment of ansellone F (3.13). Important tROESY correlations are shown in Figure 3.61. OOOHHO2.6338.53.38; 70.86.14; 138.7139.1197.2 1.71; 16.32.0434.75.99; 128.7132.22.8053.641.21.8046.769.74.35/4.21 65.61.84/1.27 35.84.12; 78.21.40; 19.02.6549.80.8122.71.20/0.98 41.11.0033.41.38/1.34 19.0170.01.64; 20.633.360.41.21 18.9 O13OOOAcO21201917 15131197525242322COSYHMBCtROESYOHHOHOHOOHHH 91 Figure 3.61 Selected tROESY correlations observed for ansellone F (3.13) in C6D6 Figure 3.62 1H NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 OOOHHO2.6338.53.38; 70.86.14; 138.7139.1197.2 1.71; 16.32.0434.75.99; 128.7132.22.8053.641.21.8046.769.74.35/4.21 65.61.84/1.27 35.84.12; 78.21.40; 19.02.6549.80.8122.71.20/0.98 41.11.0033.41.38/1.34 19.0170.01.64; 20.633.360.41.21 18.9 O13OOOAcO21201917 15131197525242322COSYHMBCtROESYOHHOHOHOOHHH 92 Figure 3.63 13C NMR spectrum of ansellone F (3.13) recorded at 150 MHz in C6D6 Figure 3.64 APT NMR spectrum of ansellone F (3.13) recorded at 150 MHz in C6D6 93 Figure 3.65 COSY NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 Figure 3.66 HSQC NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 H-13/H-14H-12/H-13 94 Figure 3.67 HMBC NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 Figure 3.68 tROESY NMR spectrum of ansellone F (3.13) recorded at 600 MHz in C6D6 Me-24/C-10Me-24/C-16, C-15,C-14Me-23/C-12Me-23/C-11Me-23/C-10H-9/C-10H-9/C-15Me-24/H-8Me-24/Me-25Me-25/H-13Me-24/H-13 95 3.5.6 Structure elucidation of ansellone G (3.14) Ansellone G (3.14) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 479.2409 (calculated for C27H36O6Na, 479.2410), which required ten sites of unsaturation. The molecular formula of ansellone G (3.14) differed from phorbadione (3.10) by adding two hydrogen atoms and one oxygen atom. Preliminary analysis of the NMR data collected for this new compound showed that it was closely related to phorbadione (3.10). The NMR data of phorbadione (3.10) and ansellone G (3.14) are listed in Table 3.7. Detailed analysis of the NMR data collected for ansellone G (3.14) showed that it had the identical C-1 to C-9 C13H15O4 bicyclic fragment and the same C-15 to C-20/Me-24/Me-25 functionalities found in phorbadione (3.10), which accounted for seven sites of unsaturation. Table 3.7 NMR data for phorbadione (3.10) and ansellone G (3.14) recorded in C6D6 at 600 MHz Phorbadione (3.10, C6D6) Ansellone G (3.14, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.7 3.57 (t, 5.0) 70.5 3.46 (t, 4.4) 2 138.2 6.17 (dq, 5.0, 1.3) 139.4 6.13 (dd, 5.6, 1.4) 3 138.8 — 139.0 — 4 196.8 — 197.6 — 5 37.4 2.62 (m) 39.0 2.67 (dd, 15.9, 4.3) 5’ 2.62 (m) 2.57 (dd, 15.9, 13.1) 6 34.1 2.19 (m) 34.7 1.99 (dm, 12.9) 7 133.8 — 131.3 — 8 129.0 5.49 (br s) 133.6 5.58 (br s) 9 75.5 4.73 (m) 76.7 5.15 (m) 10 156.4 — 59.3 1.92 (d, 1.4) 11 134.3 — 75.5 — 12 186.3 — 203.3 — 13 124.4 6.48 (s) 120.2 6.08 (s) 14 170.4 — 180.3 — 15 43.7 — 43.1 — 16 34.3 1.40 (td, 12.7, 3.5) 40.7 0.93 (dt, 13.4, 4.1) 16’ 1.92 (m) 1.94 (m) 17 18.5 1.27 * 17.8 1.21 (m) 17’ 1.50 (m) 1.46 (m) 18 40.4 1.04 * 40.0 1.06 (m) 18’ 1.26 * 1.15 (m) 19 37.1 — 37.8 — 20 32.4 0.93 (s) 32.7 0.85 (s) 21 16.0 1.75 (br s) 16.3 1.73 (s) 96 Phorbadione (3.10, C6D6) Ansellone G (3.14, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 22 65.1 4.21 (d, 13.0) 66.0 4.19 (br d, 12.7) 22’ 4.35 (d, 13.0) 4.32 (br d, 12.8) 23 12.2 2.10 (s) 25.5 1.63 (s) 24 24.6 1.04 * 23.4 1.47 (s) 25 28.5 0.99 (s) 30.1 0.87 (s) 26 169.7 — 170.3 — 27 20.3 1.63 (s) 20.7 1.64 (s) OH — — — 3.83 (s) * Overlapped resonances The 13C NMR spectrum recorded for ansellone G (3.14) contained other sp2 hybridized carbon resonances at δ 120.2 (C-13) and δ 180.3 (C-14), along with a shielded ketone carbonyl resonance at δ 203.3 (C-12), which could be assigned to an α,β-unsaturated ketone fragment. Hence the unassigned fragment must have one other ring. HMBC correlations observed between a methyl resonance at δ 1.63 (Me-23) and the ketone carbonyl resonance at δ 203.3 (C-12), the methine carbon resonance at δ 59.3 (C-10), and between the olefinic resonance at δ 180.3 (C-14) and the methyl resonances at δ 0.85 (Me-20), 1.47 (Me-24) and 0.87 (Me-25), and between the methyl resonance at δ 1.47 (Me-24) and the methine carbon resonance at δ 59.3 (C-10), and between the olefinic methine at δ 6.08 (H-13) and the two quaternary carbon resonances at δ 43.1 (C-15) and 37.8 (C-19), placed the ketone functionality at C-12 and the αβ-olefinic carbons at C-13 and C-14, completing the last ring in the constitution of ansellone G (3.14). 97 Figure 3.69 Selected COSY, HMBC and tROESY correlations observed for ansellone G (3.14) in C6D6 ROESY correlations were observed between the methyl resonance δ 1.47 (Me-24) and two other methyl resonances at δ 1.63 (Me-23) and δ 0.87 (Me-25), which indicated that these three methyls were all axial. The 13C chemical shift of Me-23 (δ 25.5) also confirmed that it was axial, the hydroxyl group was then equatorial. Therefore, the relative configuration of ansellone G (3.14) was assigned as shown in Figure 3.69, along with important COSY, HMBC and tROESY correlations. OOOOO2.67/2.57 39.03.46; 70.56.13; 139.4139.0197.6 1.73; 16.31.9934.75.58; 133.6131.3203.343.1180.31.9259.34.32/4.19 66.01.94/0.93 40.75.15; 76.71.63; 25.56.08120.20.8730.11.15/1.06 40.00.8532.71.46/1.21 17.8170.31.64; 20.737.875.51.47 23.4COSYHMBCOOOOO13597111315171921232522242720OH3.83 OHtROESYHOHOHO 98 Figure 3.70 1H NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 Figure 3.71 13C NMR spectrum of ansellone G (3.14) recorded at 150 MHz in C6D6 99 Figure 3.72 COSY NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 Figure 3.73 HSQC NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 H-16'/H-17H-16'/H-17'H-16/H-17H-16/H-17'H-18/H-17H-17/H-18'H-9/H-8 100 Figure 3.74 HMBC NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 Figure 3.75 tROESY NMR spectrum of ansellone G (3.14) recorded at 600 MHz in C6D6 Me-25/C-14Me-20/C-14Me-24/C-14Me-24/C-10H-13/C-19H-13/C-15H-13/C-14Me-23/C-12Me-23/C-11Me-23/C-10H-9/C-10Me-24/Me-23Me-24/H-16'Me-24/Me-25H-6/H-9H-9/H-1H-1/H-6 101 3.5.7 Structure elucidation of ansellone H (3.15) Ansellone H (3.15) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 465.2621 (calculated for C27H38O5Na, 465.2617), which is identical to the molecular formula of ansellone A (2.86) and required nine sites of unsaturation. Table 3.8 NMR data for ansellone A (2.86) and ansellone H (3.15) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone H (3.15, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 71.1 3.63 (br t, 4.2) 2 139.4 6.14 (d, 4.8) 139.3 6.24 (dd, 0.9, 5.5) 3 138.6 — 138.8 — 4 197.7 — 197.6 — 5 38.6 2.67 (d, 9.0) 38.4 2.65 (m) * 5’ 2.67 (d, 9.0) 2.65 (m) * 6 34.8 2.10 (tt, 9.0, 3.0) 34.8 2.19 (dt, 8.5, 3.3) 7 131.4 — 133.7 — 8 134.8 5.70 (br s) 130.6 5.70 (br s) 9 76.9 4.77 (br s) 76.2 4.27 (br s) 10 63.8 1.46 (br s) 59.2 2.23 (br s) 11 73.1 — 133.9 — 12 135.7 5.52 (dd, 10.0, 3.0) 131.7 5.31 (br s) 13 128.0 5.59 (br d, 10.0) 69.2 4.11 (br t, 8.6) 14 57.0 1.71 (m) 57.5 0.91 (d, 10.2) 15 42.1 — 39.5 — 16 38.9 1.02 (m) 40.6 1.76 * 16’ 1.82 (br d, 13.0) 1.05 (m) * 17 18.9 1.33 (m) 19.2 1.41 (m) 17’ 1.53 (dt, 13.0, 3.5) 1.34 (m) 18 41.7 1.04 (m) 44.3 1.15 (m) 18’ 1.29 (m) 1.34 (m) 19 33.1 — 33.8 — 20 33.6 0.86 (s) 37.4 1.26 (s) 21 16.3 1.69 (s) 16.3 1.75 (s) * 22 66.1 4.27 (br d, 12.0) 66.0 4.36 (d, 12.2) 22’ 4.42 (br d, 12.0) 4.25 (d, 12.4) 23 26.5 1.50 (s) 22.9 1.75 (s) * 24 16.8 1.20 (s) 17.1 0.80 (s) 25 22.2 0.78 (s) 23.4 1.08 (s) * 26 170.2 — 170.1 — 27 20.8 1.67 (s) 20.7 1.65 (s) OH — — — 0.42 (br s) * Overlapped resonances 102 Preliminary examination of the NMR data collected for this new compound showed that the structure was closely related to ansellone A (2.86). The NMR data of ansellone A (2.86) and ansellone H (3.15) is listed in Table 3.8. Detailed analysis of the NMR data collected for ansellone H (3.15) showed that it had the identical C-1 to C-9 C13H15O4 bicyclic fragment and the same C-15 to C-20/Me-24/Me-25 functionalities found in ansellone A (2.86), which accounted for seven sites of unsaturation. A pair of geminal methyls was confirmed by the HMBC correlations between themselves (Me-25 δ 1.08 and C-20 δ 37.4, Me-20 δ 1.26 and C-25 δ 23.4), HMBC correlations between the methyl resonance at δ 1.26 (Me-20) and carbon resonances at δ 44.3 (C-18), δ 33.8 (C-19) and δ 57.5 (C-14), and HMBC correlations between the methyl resonance at δ 1.08 (Me-25) and carbon resonances at δ 44.3 (C-18), δ 33.8 (C-19) and δ 57.5 (C-14), indicated this pair of geminal methyls was connected to a quaternary carbon C-19 (δ 33.8). Another methyl resonance at δ 0.80 (Me-24) correlated with carbon resonances at δ 40.6 (C-16), δ 39.5 (C-15), δ 57.5 (C-14) and δ 59.2 (C-10), indicating that this methyl group was attached to the quaternary carbon C-15, which was then connected to C-14, C-10 and C-16. HMBC correlations between a methine resonance at δ 0.91 (H-14) and both δ 39.5 (C-15) and δ 33.8 (C-19) confirmed the trimethyl-cyclohexane ring C-14 to C-19/Me-20/Me-24/Me-25 fragment, which accounted for another site of unsaturation. The remaining unassigned carbons and protons included a methyl (δ 1.75 H-23, δ 22.9 C-23), one olefin (δ 133.9 C-11 and δ 5.31 H-12, δ 131.7 C-12), two methines (δ 4.11 H-13, δ 69.2 C-13 and δ 2.23 H-10, δ 59.2 C-10) and one hydroxyl group (δ 0.42), which accounted for the last two sites of unsaturation. A COSY correlation observed between two methine resonances at δ 0.91 (H-14) and δ 4.11 (H-13) showed that the methines were vicinal and the chemical shift δ 69.2 (H-13) of the 103 carbon attached to the proton at δ 4.11 (H-13) indicated that it was deshielded by the hydroxyl group (δ 0.42 13-OH). Another COSY correlation observed between the olefinic proton resonance at δ 5.31 (H-12) and the deshielded methine at δ 4.11 (H-13) extended this spin system to the olefin at C-11 and C-12. HMBC correlations observed between the last unassigned methyl resonance at δ 1.75 (Me-23) and carbon resonances at δ 59.2 (C-10), δ 133.9 (C-11) and δ 131.7 (C-12) indicated that Me-23 was an olefinic methyl attached to C-11. HMBC correlations between the last unassigned methine resonance at δ 2.23 (H-10) and carbon resonances at δ 39.5 (C-15), δ 133.9 (C-11) and δ 131.7 (C-12) established the decalin moiety C-10 to C-19 bearing four methyls (Me-20, Me-23, Me-24 and Me-25). Finally, a COSY correlation between proton resonances H-9 (δ 4.27) and H-10 (δ 2.23), and HMBC correlations between a proton resonance H-10 (δ 2.23) and carbon resonances C-9 (δ 76.2) and C-8 (δ 130.6) completed the constitutional structure of ansellone H (3.15) as presented in Figure 3.76 (important COSY and HMBC correlations are shown). 104 Figure 3.76 Selected COSY, HMBC and tROESY correlations observed for ansellone H (3.15) in C6D6 ROESY correlations observed between two methyl resonances at δ 0.80 (Me-24) and δ 1.08 (Me-25), and between resonances at δ 1.26 (Me-20) and δ 0.91 (H-14) indicated that the decalin rings were trans fused with both Me-24 and Me-25 axial. ROESY correlations between the proton resonance at δ 4.11 (H-13) and both δ 0.80 (Me-24) and δ 1.08 (Me-25) indicated that H-13 was also axial. A ROESY correlation observed between proton resonances at δ 2.23 (H-10) and δ 0.91 (H-14) suggested that H-10 was axial and cis to H-14, which was the same as other ansellones. OOOHHO2.6538.43.63; 71.16.24; 139.3138.8197.6 1.75; 16.32.1934.85.70; 130.6133.75.31; 131.739.50.9157.52.2359.24.36/4.25 66.01.76/1.05 40.64.27; 76.21.75; 22.94.11; 69.21.0823.41.34/1.15 44.31.2637.41.41/1.34 19.2170.11.65; 20.733.8133.90.80 17.113OHOAcO21201917 15131197525242322COSYHMBC0.42tROESYOH OHHHOHHHHO 105 Figure 3.77 1H NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 Figure 3.78 13C NMR spectrum of ansellone H (3.15) recorded at 150 MHz in C6D6 106 Figure 3.79 COSY NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 Figure 3.80 HSQC NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 H-13/H-14H-13/H-12H-12/Me-23 Me-23/H-10H-9/H-10H-9/H-8 107 Figure 3.81 HMBC NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 Figure 3.82 tROESY NMR spectrum of ansellone H (3.15) recorded at 600 MHz in C6D6 H-10/C-15H-10/C-9H-10/C-11 and C-12Me-23/C-12Me-23/C-11Me-23/C-10Me-24/Me-25Me-20/H-14H-13/Me-25 H-13/Me-24H-10/H-9 108 3.5.8 Structure elucidation of ansellone I (3.16) Ansellone I (3.16) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 481.2563 (calculated for C27H38O6Na, 481.2566), which differed from ansellone H (3.15) by only one oxygen atom. The molecular formula of ansellone I (3.16) required nine sites of unsaturation. Preliminary examination of the NMR data collected for this new compound showed that the structure was also closely related to ansellone H (3.15). The NMR data of ansellone H (3.15) and ansellone I (3.16) is listed in Table 3.9. The 1H and 13C chemical shifts were only different at H-23/H-23’ (δ 4.39/3.94, integrated as 2H in 3.16; δ 1.75 integrated as 3H in 3.15), C-23 (δ 65.3 in 3.16; 22.9 in 3.15) and one proton doublet (δ 3.05, only present in 3.16 and this proton was not attached to any carbon in HSQC NMR spectrum). Given that the ansellone I (3.16) had one additional oxygen atom compared to ansellone H (3.15), the substituent at the deshielded methylene C-23 in ansellone I (3.16) had to be an alcohol. The COSY correlation observed between the proton doublet at δ 3.05 (23-OH) and the methylene resonance at δ 3.94 (H-23’) also confirmed this hydroxyl substituent. The constitutional structure of ansellone I (3.16) is presented in Figure 3.83 and important COSY and HMBC correlations are shown. Table 3.9 NMR data for ansellone I (3.16) and ansellone H (3.15) recorded in C6D6 at 600 MHz Ansellone I (3.16, C6D6) Ansellone H (3.15, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.2 3.59 (t, 4.3) 71.1 3.63 (br t, 4.2) 2 136.8 6.12 (dd, 1.2, 5.5) 139.3 6.24 (dd, 0.9, 5.5) 3 138.4 — 138.8 — 4 196.0 — 197.6 — 5 37.1 2.71 (m) 38.4 2.65 (m) * 5’ 2.71 (m) 2.65 (m) * 6 33.4 2.13 (dm, 12.7) 34.8 2.19 (dt, 8.5, 3.3) 7 133.5 — 133.7 — 8 128.7 5.68 (br s) 130.6 5.70 (br s) 9 75.1 4.28 (br s) 76.2 4.27 (br s) 10 54.3 2.53 (br s) 59.2 2.23 (br s) 109 Ansellone I (3.16, C6D6) Ansellone H (3.15, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 11 136.7 — 133.9 — 11 136.7 — 133.9 — 12 131.1 5.52 (br s) 131.7 5.31 (br s) 13 67.8 4.07 (br t, 8.2) 69.2 4.11 (br t, 8.6) 14 55.9 0.94 (d, 10.4) 57.5 0.91 (d, 10.2) 15 37.9 — 39.5 — 16 39.4 1.68 (m) 40.6 1.76 * 16’ 1.03 (m) 1.05 (m) * 17 18.0 1.36 (m) 19.2 1.41 (m) 17’ 1.36 (m) 1.34 (m) 18 42.8 1.13 (m) 44.3 1.15 (m) 18’ 1.31 (m) 1.34 (m) 19 32.5 — 33.8 — 20 36.2 1.23 (s) 37.4 1.26 (s) 21 15.3 1.69 (s) 16.3 1.75 (s) * 22 64.7 4.30 (d, 13.9) 66.0 4.36 (d, 12.2) 22’ 4.19 (d, 12.7) 4.25 (d, 12.4) 23 65.3 4.39 (d, 13.1) 22.9 1.75 (s) * 23’ 3.94 (br t, 11.5) — — 24 16.1 0.74 (s) 17.1 0.80 (s) 25 22.2 1.06 (s) 23.4 1.08 (s) * 26 169.1 — 170.1 — 27 19.5 1.64 (s) 20.7 1.65 (s) 13-OH — 0.42 (br s) — 0.42 (br s) 23-OH — 3.05 (d, 7.9) — — * Overlapped resonances 110 Figure 3.83 Selected COSY, HMBC and tROESY correlations observed for ansellone I (3.16) in C6D6 ROESY correlations observed between two methyl resonances at δ 0.74 (Me-24) and δ 1.06 (Me-25), and between resonances at δ 1.23 (Me-20) and δ 0.94 (H-14) indicated that the decalin rings were trans fused with H-14, Me-24 and Me-25 axial. A ROESY correlation observed between proton resonances at δ 2.53 (H-10) and δ 0.94 (H-14) suggested that H-10 was axial and cis to H-14, which was the same as other ansellones. A ROESY correlation between the proton resonances at δ 4.07 (H-13) and δ 0.94 (H-14) indicated that H-13 was equatorial and cis to H-14. Therefore, the hydroxyl substituent at C-13 should be axial. OOOHHO2.7137.13.59; 70.26.12; 136.8138.4196.0 1.69; 15.32.1333.45.68; 128.7133.55.52; 131.137.90.9455.92.5354.34.30/4.19 64.71.68/1.03 39.44.28; 75.14.39/3.94 65.34.07; 67.81.0622.21.31/1.13 42.81.2336.21.3618.0169.11.64; 19.532.5136.70.74 16.113OOAcO21201917 1513119752524 2322COSYHMBC0.42tROESYOH OHHHOHHHOHOH3.05 OHOH 111 Figure 3.84 1H NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 Figure 3.85 13C NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 112 Figure 3.86 COSY NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 Figure 3.87 HSQC NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 H-8/H-9H-9/H-10H-10/H-1223-OH/H-23'H-12/H-23H-17/H-18H-17/H-16 113 Figure 3.88 HMBC NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 Figure 3.89 tROESY NMR spectrum of ansellone I (3.16) recorded at 600 MHz in C6D6 H-8/C-10 H-12/C-10H-23/C-10H-23'/C-12H-23/C-11 H-23'/C-11H-23'/C-10Me-24/Me-25Me-20/H-14H-10/H-14H-13/H-14 114 3.5.9 Structure elucidation of ansellone J (3.17) Ansellone J (3.17) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESILRMS at m/z 481.5, which was consistent with a molecular formula of C27H38O6, requiring nine sites of unsaturation. Preliminary examination of the NMR data collected for this new compound showed that the structure was closely related to ansellone A (2.86). The NMR data of ansellone J (3.17) and ansellone A (2.86) are listed in Table 3.10. Table 3.10 NMR data for ansellone A (2.86) and ansellone J (3.17) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone J (3.17, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 70.2 3.43 (br t, 3.2) 2 139.4 6.14 (d, 4.8) 138.8 6.12 (dd, 5.6, 1.4) 3 138.6 — 137.8 — 4 197.7 — 197.2 — 5 38.6 2.67 (d, 9.0) 38.3 2.67 (dd, 15.7, 4.5) 5’ 2.67 (d, 9.0) 2.60 (dd, 15.7, 13.1) 6 34.8 2.10 (tt, 9.0, 3.0) 34.1 2.04 (dm, 12.9) 7 131.4 — 130.5 — 8 134.8 5.70 (br s) 133.8 5.44 (br s) 9 76.9 4.77 (br s) 76.1 4.89 (br s) 10 63.8 1.46 (br s) 81.7 — 11 73.1 — 58.4 1.74 (br s) 12 135.7 5.52 (dd, 10.0, 3.0) 80.5 4.30 (br d, 3.0) 13 128.0 5.59 (br d, 10.0) 118.5 5.71 (br d, 3.0) 14 57.0 1.71 (m) 156.7 — 15 42.1 — 40.0 — 16 38.9 1.02 (m) 39.1 1.82 (br d, 13.2) 16’ 1.82 (br d, 13.0) 1.09 (m) 17 18.9 1.33 (m) 22.8 1.27 (m) * 17’ 1.53 (dt, 13.0, 3.5) 0.87 (m) 18 41.7 1.04 (m) 34.3 1.06 (m) 18’ 1.29 (m) 1.53 (m) 19 33.1 — 35.2 — 20 33.6 0.86 (s) 32.0 1.08 (s) 21 16.3 1.69 (s) 15.8 1.71 (s) 22 66.1 4.27 (br d, 12.0) 65.3 4.19 (br d, 12.6) 22’ 4.42 (br d, 12.0) 4.33 (br d, 12.6) 23 26.5 1.50 (s) 26.6 1.58 (s) 24 16.8 1.20 (s) 23.5 1.28 (s) * 25 22.2 0.78 (s) 31.0 0.99 (s) 26 170.2 — 169.9 — 27 20.8 1.67 (s) 20.3 1.63 (3H, s) * Overlapped resonances 115 Detailed analysis of the NMR data collected for ansellone J (3.17) showed that it had the identical C-1 to C-9 C13H15O4 bicyclic fragment found in ansellone A (2.86), which accounted for six sites of unsaturation. The remaining unassigned signals include no other deshielded carbons except a pair of olefinic carbons at δ 156.7 (C-14) and 118.5 (C-13), which accounted for one site of unsaturation. Therefore, there must be two other rings. A pair of geminal methyls was confirmed by the HMBC correlations between themselves (Me-25 δ 0.99 and C-20 δ 32.0, Me-20 δ 1.08 and C-25 δ 31.0), HMBC correlations between the methyl resonance at δ 1.08 (Me-20) and carbon resonances at δ 31.7 (C-18), δ 35.2 (C-19) and δ 156.7 (C-14), and HMBC correlations between the methyl resonance at δ 0.99 (Me-25) and carbon resonances at δ 31.7 (C-18), δ 35.2 (C-19) and δ 156.7 (C-14). The above HMBC correlations also showed that this pair of geminal methyls was attached to a quaternary carbon C-19 (δ 35.2), which was then attached to a methylene carbon C-18 and an olefinic carbon C-14. A spin system could be identified from COSY correlations between methylene resonances at δ 1.06/1.53 (H-18/H18’), δ 1.27/0.87 (H-17/H-17’) and δ 1.82/1.09 (H-16/H-16’). Another methyl resonance at δ 1.28 (Me-24) correlated with carbon resonances at δ 39.1 (C-16), δ 40.0 (C-15), δ 156.7 (C-14), δ 81.7 (C-10) and δ 58.4 (C-11), indicating that this methyl group was connected to a quaternary carbon C-15, and C-15 was next to the olefinic carbon C-14, methylene carbon C-16 and a deshielded carbon C-10. Therefore, one trimethyl-cyclohexane ring (C-14 to C-19/Me-20/Me-24/Me-25) of the two remaining rings was confirmed. The remaining unassigned carbons and protons included a methyl (δ 1.58 H-23, δ 26.6 C-23), one olefinic methine (δ 118.5 C-13, 5.71 H-13), two methines (δ 4.30 H-12, δ 80.5 C-12 and δ 1.74 H-11, δ 58.4 C-11) and one deshielded quaternary carbon (δ 81.7 C-10), which accounted for the last ring. HMBC correlations observed between the last unassigned methyl resonance at δ 1.58 (Me-23) and 116 carbon resonances at δ 58.4 (C-11), δ 81.7 (C-10) and δ 80.5 (C-12) suggested that Me-23 was a methyl attached to C-11, and C-11 was attached to C-10 and C-12. The 13C chemical shifts δ 80.5 (C-12) indicated that it was attached to the hydroxyl group. A COSY correlation between H-12 (δ 4.30) and H-13 (δ 5.71) showed that the methine protons were vicinal, which extended this fragment to the olefin C-13 and C-14. HMBC correlations observed between the methine H-11 and the quaternary carbon at δ 40.0 (C-15) established the last unassigned ring in the decalin moiety, C-10 to C-19 bearing four methyls (Me-20, Me-23, Me-24 and Me-25). The last hydroxyl was assigned to the deshielded quaternary carbon at δ 81.7 (C-10). Finally, the HMBC correlation between the H-11 proton resonance (δ 1.74) and the carbon resonance assigned to C-9 (δ 76.1) completed the constitutional structure of ansellone J (3.17) as presented in Figure 3.90, along with important COSY and HMBC correlations. Figure 3.90 Selected COSY and HMBC correlations observed for ansellone J (3.17) in C6D6 However, this compound was very unstable due to the allylic hydroxyl functionality, which led to the compound decomposing during the time required to record its NMR data as shown in Figure 3.91. We were not able to obtain the tROESY NMR spectrum needed to assign OOOHHO2.67/2.60 38.33.43; 70.26.12; 138.8137.8197.2 1.71; 15.82.0434.15.44; 133.8130.54.3080.540.0156.74.33/4.19 65.31.82/1.09 39.14.89; 76.11.58; 26.65.71118.51.0832.01.53/1.06 34.30.9931.01.27/0.87 22.8169.91.63; 20.335.21.74; 58.41.28 23.513OOHOAcO21201917 15131197525242322COSYHMBCOHOH81.7OHH 117 the relative configuration of the structure. However, we assumed that ansellone J (3.17) had the same relative configurations at C-1, C-6, C-11, C-15 and C-19 as all other ansellones based on biogenetic arguments, but the relative configurations at C-10 and C-12 could not be determined from the data we collected. Figure 3.91 1H NMR spectrum of ansellone J (3.17, in red) and decomposed ansellone J (3.17, in blue) recorded at 600 MHz in C6D6 118 Figure 3.92 1H NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 Figure 3.93 COSY NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 H-8/H-9H-12/H-13 119 Figure 3.94 HSQC NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 Figure 3.95 HMBC NMR spectrum of ansellone J (3.17) recorded at 600 MHz in C6D6 Me-23/C-11Me-23/C-12Me-23/C-10H-11/C-9Me-24/C-14Me-24/C15,C-16Me-24/C-11H-11/C-10H-11/C-15 120 3.5.10 Structure elucidation of ansellone K (3.18) Ansellone K (3.18) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 509.2885, which was consistent with a molecular formula of C29H42O6 (calculated for C29H42O6Na, 509.2879), requiring nine degrees of unsaturation. Table 3.11 NMR data for ansellone A (2.86) and ansellone K (3.18) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone K (3.18, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 65.8 5.74 (br t, 4.2) 2 139.4 6.14 (d, 4.8) 137.6 6.19 (br d, 5.7) 3 138.6 — 139.9 — 4 197.7 — 197.9 — 5 38.6 2.67 (d, 9.0) 38.3 2.78 (dd, 12.8, 16.1) 5’ 2.67 (d, 9.0) 2.62 (d, 2.9) 6 34.8 2.10 (tt, 9.0, 3.0) 42.5 3.21 (dt, 12.8, 3.6) 7 131.4 — 145.3 — 8 134.8 5.70 (br s) 42.6 2.64 (d, 2.9) 8’ — 2.83 (dd, 12.0, 14.7) 9 76.9 4.77 (br s) 68.2 6.08 (dd, 2.3, 11.8) 10 63.8 1.46 (br s) 139.6 — 11 73.1 — 128.4 — 12 135.7 5.52 (dd, 10.0, 3.0) 45.7 2.10 (dd, 18.4, 5.1) 12’ 1.69 * 13 128.0 5.59 (br d, 10.0) 65.5 4.06 (br t, 3.8) 14 57.0 1.71 (m) 53.7 0.93 (br s) 15 42.1 — 39.6 — 16 38.9 1.02 (m) 39.9 1.88 * 16’ 1.82 (br d, 13.0) 1.24 * 17 18.9 1.33 (m) 19.9 1.70 * 17’ 1.53 (dt, 13.0, 3.5) 1.48 (dt, 13.7, 3.6) 18 41.7 1.04 (m) 43.5 1.06 (dt, 13.7, 3.9) 18’ 1.29 (m) 1.30 (br d, 13.2) 19 33.1 — 34.1 — 20 33.6 0.86 (s) 34.5 0.90 (s) 21 16.3 1.69 (s) 16.1 1.67 (s) 22 66.1 4.27 (br d, 12.0) 115.3 4.71 (br s) 22’ 4.42 (br d, 12.0) 5.02 (br s) 23 26.5 1.50 (s) 22.0 1.90 (s) * 24 16.8 1.20 (s) 20.7 1.65 (s) 25 22.2 0.78 (s) 24.2 1.23 (s) * 26 170.2 — 169.9 — 27 20.8 1.67 (s) 21.0 1.64 (s) 28 — — 169.9 — 29 — — 21.0 1.64 (s) OH — — — 0.44 (d, 4.1) * Overlapped resonances 121 The NMR data of ansellone K (3.18) and ansellone A (2.86) are listed in Table 3.11. The 13C NMR spectrum (Figure 3.129) recorded for ansellone K (3.18) in C6D6 showed 28 well-resolved peaks (3 of the 31 peaks were from EtOAc, δ 60.3, 20.8 and 14.5), one carbon atom less than the formula. In the HMBC spectrum (Figure 3.102), four proton resonances correlated with a carbon resonance at δ 128.4 (C-11), which identified the missing carbon signal that was obscured by the C6D6 solvent peak (δ 128.39, t). The 1H NMR (Figure 3.98), DEPT135 (Figure 3.99) and HSQC (Figure 3.101) data identified 41 hydrogen atoms attached to carbon (7 CH3, 7 CH2 and 6 CH), and another exchangeable hydrogen atom was confirmed by a mass shift from 509.3 to 510.3 in the LRESIMS recorded in CD3OD. Downfield resonances in the 13C NMR spectrum (Figure 3.98) could be assigned to six alkene carbons at δ 137.5 (C-2), 139.8 (C-3), 145.3 (C-7), 115.3 (C-22), 139.6 (C-10) and 128.4 (C-11), one α,β-unsaturated ketone carbonyl carbon at δ 197.9 (C-4) and two ester or carboxylic acid carbonyl carbons at δ 169.7 (C-26) and 169.9 (C-28), which accounted for six degrees of unsaturation. The absence of 13C NMR evidence for additional unsaturated functionality indicated that ansellone K (3.18) was tricyclic. Detailed analysis of the COSY (Figure 3.100) and HMBC (Figure 3.102) data collected for ansellone K (3.18) identified the three fragments A, B and C as shown in Figure 3.96. 122 Figure 3.96 Selected COSY, HMBC and tROESY correlations observed for ansellone K (3.18) in C6D6 In fragment A (Figure 3.96), COSY correlations revealed a spin system including an oxymethine proton at δ 6.08 (H-9), allylic methylene protons at δ 2.64/2.83 (H-8/H-8’) and olefinic methylene protons at δ 4.71/5.02 (H-22/H-22’). In the HMBC spectrum (Figure 3.103), the carbonyl resonance at δ 169.9 (C-28) correlated with the proton resonance at δ 6.08 (H-9) and the methyl resonance at δ 1.64 (H-29), indicating the existence of an acetate group. The MeOOOHHHHHHHMeOHHMeOHMeMeMeMeHHHHOHHHHHHH197.9139.91.6716.138.3137.66.195.7442.53.2165.868.26.08115.3145.342.64.71/5.02169.91.6421.0A B1.9022.045.7128.453.739.61.6520.739.91.24/1.881.48/1.7019.91.06/1.3043.5 34.11.2324.2 0.9034.5139.64.0665.5 1.69/2.100.93169.92.62/2.781.6421.02.64/2.83135791113151719212324DHMBCMeOMeOHMeMeOAcOMeHHOCCOSYEHMeMeHMeHHHHHHHHMeOH1.69/2.104.060.930.901.231.301.061.651.90H6.0822tROESY 123 methyl cyclohexenone fragment C (Figure 3.96), typical for this family of terpenoids, was readily identified by a comparison with ansellone A (2.86). Fragment B (Figure 3.96) showed a decalin ring, similar to ansellone A (2.86). In the COSY spectrum (Figure 3.100), a spin system starting from methylene protons at δ 1.69/2.10 (H-12/H-12’) to an oxymethine proton at δ 4.06 (H-13) terminated in an aliphatic methine proton at δ 0.93 (H-14). Another spin system included six aliphatic methylene protons at δ 1.24/1.88 (H-16/H-16’), 1.48/1.70 (H-17/H-17’) and 1.06/1.30 (H-18/H-18’). HMBC correlations observed between a methyl resonance at δ 0.90 (Me-20) and carbon resonances at δ 24.2 (C-25), 43.5 (C-18) and 53.7 (C-14), and between another methyl resonance at δ 1.23 (Me-25) and carbon resonances at δ 34.5 (C-20), 43.5 (C-18) and 53.7 (C-14), revealed that these two methyl groups (Me-20 and Me-25) were geminal and attached to C-19 (δ 34.1), which was connected to C-14 (δ 53.7) and C-18 (δ 43.5). A methyl group (H-23 δ 1.90, C-23 δ 22.0) showed HMBC correlations with an aliphatic carbon at δ 45.7 (C-12) and olefinic carbons at δ 128.4(C-11) and 139.6 (C-10). Finally, HMBC correlations between another methyl group (H-24 δ 1.65, C-24 δ 20.7) and C-15, C-14, C-10, and C-16 helped to identify the bicyclic carbon skeleton. HMBC correlations between H-5 and C-7, and between H-22 and C-6 connected fragments A and C together while the correlation between H-9 and C-10, and between H-9 and C-11 linked fragments A and B. The configuration of ansellone K (3.18) was not totally defined from NMR data. In the ROESY spectrum, the H-1 resonance at δ 5.74 correlated with the H-6 resonance at δ 3.21 showing that they pointed to the same side of the cyclohexenone ring, which was consistent with other ansellones. We have assumed that this fragment has the same absolute configuration as ansellone A (2.86) based on a biogenetic argument. It was also assumed that C-24 and H-14 were trans since all ansellones isolated to date contained the trans-decalin substructure. With this 124 assumption, the tROESY spectrum revealed the relative configuration shown in panel E (Figure 3.96). However, ansellone K (3.18) is the first example lacking an ether linkage at C-1, and the free rotation at C-8 and C-9 made it impossible to determine the relative configuration by NMR studies alone. Figure 3.97 1H NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 125 Figure 3.98 13C NMR spectrum of ansellone K (3.18) recorded at 150 MHz in C6D6 Figure 3.99 DEPT135 NMR spectrum of ansellone K (3.18) recorded at 150 MHz in C6D6 126 Figure 3.100 COSY NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 Figure 3.101 HSQC NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 H-22'/H-8'H-8'/H-9H-8/H-9H-1/H-6H-1/H-2H-14/H-13H-12/H-1313-OH/H-13 127 Figure 3.102 HMBC NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 Figure 3.103 tROESY NMR spectrum of ansellone K (3.18) recorded at 600 MHz in C6D6 H-22'/C-8H-22/C-9H-22,H-22'/C-7H-9/C-28H-1/C-26Me-24/C-14Me-24,Me-23,H-12'/C-10H-13/C-11H-9/C-10,C-11H-22/C-6H-1/H-6Me-20/H-18H-13/H-14Me-25/M-24 128 3.5.11 Structure elucidation of ansellone L (3.19) Ansellone L (3.19) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 423.2509, which was consistent with a molecular formula of C25H36O4 (calculated for C25H36O4Na, 423.2511), requiring eight sites of unsaturation. The molecular formula of ansellone L (3.19) differed from the molecular formula of ansellone A (2.86) by subtracting an acetate group. Preliminary examination of the NMR data collected for this new compound showed that the structure was also closely related to ansellone A (2.86). The NMR data of ansellone L (3.19) and ansellone A (2.86) are listed in Table 3.12. Table 3.12 NMR data for ansellone L (3.19) and ansellone A (2.86) recorded in C6D6 at 600 MHz Ansellone A (2.86, C6D6) Ansellone L (3.19, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 71.0 3.52 (br t, 4.5) 2 139.4 6.14 (d, 4.8) 139.4 6.19 (dd, 5.6, 1.3) 3 138.6 — 138.4 — 4 197.7 — 197.7 — 5 38.6 2.67 (d, 9.0) 38.8 2.66 (dd, 13.3, 9.5) 5’ 2.67 (d, 9.0) 2.58 (dd, 15.2, 4.3) 6 34.8 2.10 (tt, 9.0, 3.0) 33.9 2.15 (ddd, 13.3, 7.1, 4.0) 7 131.4 — 132.6 — 8 134.8 5.70 (br s) 130.1 5.61 (br s) 9 76.9 4.77 (br s) 76.9 4.81 (br d, 1.3) 10 63.8 1.46 (br s) 63.4 1.53 (br s) * 11 73.1 — 72.8 — 12 135.7 5.52 (dd, 10.0, 3.0) 135.5 5.54 (dd, 10.1, 3.1) 13 128.0 5.59 (br d, 10.0) 127.5 5.60 (dd, 8.9, 1.5) 14 57.0 1.71 (m) 57.1 1.72 (t, 2.2) 15 42.1 — 41.9 — 16 38.9 1.02 (m) 38.7 1.06 (dm, 16.4) 16’ 1.82 (br d, 13.0) 1.85 (dt, 13.0, 3.6) 17 18.9 1.33 (m) 18.6 1.31 * 17’ 1.53 (dt, 13.0, 3.5) 1.54 (m) * 18 41.7 1.04 (m) 41.5 1.03 (dt, 13.5, 4.0) 18’ 1.29 (m) 1.29 * 19 33.1 — 32.5 — 20 33.6 0.86 (s) 33.2 0.86 (s) 21 16.3 1.69 (s) 15.9 1.71 (s) 22 66.1 4.27 (br d, 12.0) 64.3 3.58 (ddd, 13.1, 6.1) 22’ 4.42 (br d, 12.0) 3.62 (ddd, 13.1, 5.0) 23 26.5 1.50 (s) 26.5 1.52 (s) 24 16.8 1.20 (s) 16.6 1.23 (s) 25 22.2 0.78 (s) 22.0 0.79 (s) 129 Ansellone A (2.86, C6D6) Ansellone L (3.19, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 26 170.2 — — — 27 20.8 1.67 (s) — — OH — — — 0.51 (t, 5.6) OH’ — — — 0.98 (br s) * Overlapped resonances The major differences in the NMR data collected for ansellones A (2.86) and L (3.19) were the presence of a hydroxyl triplet at δ 0.51 (22-OH), and the absence of 22-OAc present in the NMR data of ansellone A (2.86). There were also noticeable differences in the chemical shifts of H-22/H-22’ (δ 4.27/4.42 in 2.86; 3.58/3.62 in 3.19, which indicated that the structural difference between ansellones A (2.86) and L (3.19) was located on C-22 of the molecules. A COSY correlation observed between the hydroxyl resonance at δ 0.51 (22-OH) and the methylene resonances at δ 3.58/3.62 (H-22/H-22’) suggested that the functional group on C-22 was a hydroxyl group instead of an acetate, which was consistent with the molecular formula. Detailed analysis of the HSQC, HMBC and COSY NMR data confirmed the structure of ansellone L (3.19). The structure of ansellone L (3.19) is shown in Figure 3.104, along with important HMBC and COSY correlations. The 13C chemical shift of Me-23 (δ 26.5) indicated that this methyl group was in the axial orientation, which was identical as ansellone A (2.86), where Me-23 (δ 26.5 in 2.86) was also in the axial orientation. We have assumed that ansellone L has the same absolute configuration as ansellone A (2.86) based on a biogenetic argument. Therefore, ansellone L (3.19) was assigned the structure as shown in Figure 3.104, in which a hydroxyl group (in 3.19) rather than an acetate substituent (in 2.86) was present at C-22. 130 Figure 3.104 Selected COSY and HMBC correlations observed for ansellone L (3.19) in C6D6 Figure 3.105 1H NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 OOOHH2.66/2.58 38.83.52; 71.06.19; 139.4138.4197.4 1.71; 15.92.1533.95.61; 130.1132.65.54135.541.91.7257.11.5363.43.62/3.58 64.31.85/1.06 38.74.81; 76.91.52; 26.55.60127.50.7922.01.29/1.03 41.50.8633.21.54/1.31 18.632.572.81.23 16.60.51OH13OHOHOHO21201917 1513119752524 2322COSYHMBCH0.98 131 Figure 3.106 13C NMR spectrum of ansellone L (3.19) recorded at 150 MHz in C6D6 Figure 3.107 COSY NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 H-13/H-14H-12/H-1322-OH/H-22H-9/H-10H-8/H-9 132 Figure 3.108 HSQC NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 Figure 3.109 HMBC NMR spectrum of ansellone L (3.19) recorded at 600 MHz in C6D6 Me-23/C-10Me-23/C-11Me-23/C-13Me-23/C-1211-OH/C-23H-13/C-11 133 3.5.12 Structure elucidation of ansellone M (3.20) Ansellone M (3.20) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 437.2666, which was consistent with a molecular formula of C26H38O4 (calculated for C26H38O4Na, 437.2668), requiring eight sites of unsaturation. The molecular formula of ansellone M (3.20) differed from the molecular formula of ansellone D (3.11) by subtracting an acetate group. Preliminary analysis of the NMR data collected for this new compound showed that the structure was also closely related to ansellone D (3.11). The NMR data obtained for ansellone M (3.20) and ansellone D (3.11) are listed in Table 3.13. Table 3.13 NMR data for ansellone M (3.20) and ansellone D (3.11) recorded in C6D6 at 600 MHz Ansellone M (3.20, C6D6) Ansellone D (3.11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 71.1 3.52 (br t, 4.4) 70.5 3.44 (t, 4.2) 2 139.8 6.19 (d, 5.6, 1.3) 139.1 6.14 (dd, 5.6, 1.2) 3 138.4 — 138.2 — 4 198.0 — 197.5 — 5 38.8 2.68 (dd, 15.5, 4.3) 38.3 2.68 (m) 5’ 2.59 (dd, 15.5, 13.2) 2.68 (m) 6 33.9 2.13 (ddd, 13.2, 6.9, 3.0) 34.5 2.09 (t, 8.2) 7 135.9 — 131.1 — 8 130.5 5.60 (br s) 134.8 5.68 (br s) 9 76.7 4.75 (br s) 76.2 4.69 (br s) 10 55.7 1.87 (br s) 55.4 1.82 (br s) 11 78.9 — 78.4 — 12 132.8 5.48 (dd, 10.3, 3.2) 132.3 5.45 (dd, 10.3, 3.1) 13 132.1 5.83 (dd, 10.3, 3.0) 131.7 5.81 (dd, 10.3, 1.5) 14 57.2 1.71 * 56.7 1.71 (m) 15 42.5 — 42.0 — 16 39.2 1.13 (dt, 12.7, 3.4) 38.9 1.09 (m) * 16’ 1.94 (dt, 12.8, 3.4) 1.87 (dm, 12.8) 17 18.9 1.32 (m) * 18.5 1.35 (m) 17’ 1.56 (m) 1.56 (dm, 13.7) 18 41.7 1.05 (dt, 13.5, 4.0) 41.3 1.09 (m) * 18’ 1.29 (m) * 1.31 (dm, 13.2) 19 33.1 — 32.7 — 20 33.6 0.86 (s) 33.2 0.85 (s) 21 16.3 1.71 (s) * 15.9 1.69 (s) 22 64.3 3.59 (dd, 13.2, 4.3) 65.8 4.25 (br d, 12.4) 22’ 3.63 (dd, 13.1, 4.3) 4.43 (br d, 12.4) 23 25.9 1.65 (s) 25.5 1.61 (s) 24 17.4 1.30 (s) * 17.0 1.26 (s) 25 22.2 0.79 (s) 21.8 0.78 (s) 134 Ansellone M (3.20, C6D6) Ansellone D (3.11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 26 49.6 3.07 (s) 169.9 — 27 — — 20.4 1.65 (s) 28 — — 49.2 3.04 (s) OH — 0.53 (t, 5.5) — — * Overlapped resonances The major differences in the NMR data collected for ansellones M (3.20) and D (3.11) were the presence of a hydroxyl triplet at δ 0.53 (22-OH), and the absence of 22-OAc present in ansellone D (3.11). There were also noticeable differences in the chemical shifts of H-22/H-22’ (δ 4.25/4.43 in 3.11; 3.59/3.63 in 3.20), which indicated that the structural difference between ansellones M (3.20) and D (3.11) was located on C-22 of the molecules. A COSY correlation observed between the hydroxyl resonance at δ 0.53 (22-OH) and the methylene resonances at δ 3.59/3.63 (H-22/H-22’), and an HMBC correlation observed between the hydroxyl resonance at δ 0.53 (22-OH) and the carbon resonance at δ 135.9 (C-7) suggested that the functional group on C-22 was a hydroxyl instead of an acetate, which was consistent with the molecular formula. Detailed analysis of the HSQC, HMBC and COSY NMR data confirmed the constitution of ansellone M (3.20), which is shown in Figure 3.110, along with important HMBC, COSY and tROESY correlations. A ROESY correlation observed between H-1 (δ 3.52) and H-6 (δ 2.13) confirmed that the methylcyclohexenone (C-1 to C-6) and dihydropyran (C-1, C-6 to C-9) rings were cis fused in ansellone M (3.20), which was the same as in ansellone D (3.11). ROESY correlations observed between the Me-24 and Me-25 resonances at δ 1.30 and 0.79 and between the Me-20 and H-14 resonances at δ 0.86 and 1.71 demonstrated that the decalin rings were trans fused with the Me-24 and Me-25 in the axial orientations, which was consistent with other ansellones. An additional ROESY correlation observed between the methyl resonance at δ 1.87 (Me-23) and the Me-24 (δ 135 1.30) resonance indicated that Me-23 was cis to the two axial methyls. Therefore, the methoxyl group was in an equatorial orientation, which was the same as ansellone D (3.11). ROESY correlations observed between the methine resonance at δ 1.87 (H-10) and both the resonances at δ 1.71 (H-14) and 4.75 (H-9) indicated that C-9 and C-10 had the same relative configuration as ansellone D (3.11). We have assumed that the absolute configurations are identical in ansellone A (2.86) and ansellone D (3.11), which completed the structure assignment of ansellone M (3.20). Figure 3.110 Selected COSY, HMBC and tROESY correlations observed for ansellone M (3.20) in C6D6 OOHOHH2.68/2.59 38.83.52; 71.16.19; 139.8138.4198.0 1.71; 16.32.1333.95.60; 130.5135.95.48132.842.51.7157.21.8755.73.59/3.63 64.31.94/1.13 39.24.75; 76.71.65; 25.95.83132.10.7922.21.29/1.05 41.70.8633.61.56/1.32 18.933.178.91.30 17.43.07; 49.6O13OHOOO21201917 1513119752625242322COSYHMBCHHHOOHHtROESYHH0.53 136 Figure 3.111 1H NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 Figure 3.112 13C NMR spectrum of ansellone M (3.20) recorded at 150 MHz in C6D6 137 Figure 3.113 DEPT135 NMR spectrum of ansellone M (3.20) recorded at 150 MHz in C6D6 Figure 3.114 COSY NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 22-OH/H-22H-9/H-8 H-9/H-10H-13/H-14H-12/H-13 138 Figure 3.115 HSQC NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 Figure 3.116 HMBC NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 Me-26/C-11 Me-23/C-11Me-23/C-10 Me-23/C-26H-10/C-11H-12/C-14H-13/C-11 139 Figure 3.117 tROESY NMR spectrum of ansellone M (3.20) recorded at 600 MHz in C6D6 3.5.13 Structure elucidation of ansellone N (3.21) In collaboration with Dr. Min Chen, ansellone N (3.21) was isolated as a white powder. It gave an [M + Na]+ ion peak in the ESITOFHRMS at m/z 481.2559, which was consistent with a molecular formula of C27H38O6 (calculated for C27H38O6Na, 481.2566), requiring nine sites of unsaturation. Preliminary examination of the NMR data collected for this new compound showed that the structure was also closely related to ansellone A (2.86). The NMR data of ansellone N (3.21) and ansellone A (2.86) are listed in Table 3.14. Table 3.14 NMR data for ansellone A (2.86) and ansellone N (3.21) recorded in C6D6 at 600 MHz Ansellone A (3, C6D6) Ansellone N (11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 70.9 3.44 (br t, 4.8) 66.9 4.44 (dd, 6.1, 2.2) 2 139.4 6.14 (d, 4.8) 138.4 6.24 (dd, 6.1, 1.3) 3 138.6 — 139.8 — 4 197.7 — 196.5 — Me-24/Me-25Me-23/Me-24H-14/Me-20H-9/H-10H-9/H-14 140 Ansellone A (3, C6D6) Ansellone N (11, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 5 38.6 2.67 (d, 9.0) 35.6 2.48 (dd, 16.4, 4.1) 5’ 2.67 (d, 9.0) 2.35 (dd, 16.4, 13.6) 6 34.8 2.10 (tt, 9.0, 3.0) 40.7 2.15 (dm, 13.6) 7 131.4 — 68.5 — 8 134.8 5.70 (br s) 102.8 4.54 (d, 1.5) 9 76.9 4.77 (br s) 157.7 — 10 63.8 1.46 (br s) 66.6 1.97 (s) 11 73.1 — 72.0 — 12 135.7 5.52 (dd, 10.0, 3.0) 134.9 5.58 (dd, 10.2, 3.1) 13 128.0 5.59 (br d, 10.0) 128.2 5.62 (dd, 10.2, 1.4) 14 57.0 1.71 (m) 55.8 1.63 (m) * 15 42.1 — 39.8 — 16 38.9 1.02 (m) 37.4 0.99 (m) 16’ 1.82 (br d, 13.0) 1.51 (m) 17 18.9 1.33 (m) 18.3 1.24 (m) * 17’ 1.53 (dt, 13.0, 3.5) 1.44 (m) 18 41.7 1.04 (m) 41.2 1.00 (m) 18’ 1.29 (m) 1.24 (m) * 19 33.1 — 32.5 — 20 33.6 0.86 (s) 33.0 0.82 (s) 21 16.3 1.69 (s) 15.8 1.65 (s) * 22 66.1 4.27 (br d, 12.0) 68.3 4.11 (d, 11.6) 22’ 4.42 (br d, 12.0) 3.87 (d, 11.6) 23 26.5 1.50 (s) 26.7 1.40 (s) 24 16.8 1.20 (s) 15.7 1.18 (s) 25 22.2 0.78 (s) 21.7 0.75 (s) 26 170.2 — 170.6 — 27 20.8 1.67 (s) 20.3 1.64 (s) * Overlapped resonances Analysis of the NMR data collected for these two ansellones revealed that ansellone N (3.21) was different from ansellone A (2.86) only at C-7 to C-9 region. The C-1 to C-6/Me-21 methylcyclohexenone ring and the C-10 to C-20/Me-23 to Me-25 decalin fragments, which were identical as ansellone A (2.86), could be confirmed from COSY and HMBC correlations, and these structures accounted for six sites of unsaturation. The remaining unassigned signals included one acetate group (δ 170.6, C-26; δ 1.64, Me-27; δ 20.3, C-27) and one olefin group (δ 157.7, C-9; δ 4.54, H-8; δ 102.8, C-8), which accounted for two sites of unsaturation. Therefore, the structure should have one more ring. The HMBC correlations observed between the 141 methylene resonances at δ 4.11/3.87 (H-22/22’) and both the carbonyl resonances at δ 170.6 (C-26) and the quaternary carbon at δ 68.5 (C-7) indicated that the acetate was attached to a methylene that was next to a deshielded quaternary carbon. The HMBC correlations between the olefinic methine resonance at δ 4.54 (H-8) and both the quaternary carbon at δ 68.5 (C-7) and the methylene at δ 68.3 (C-22), and the HMBC correlations between H-22 and C-8 indicated that the quaternary carbon (C-7) was attached to the olefin. The HMBC correlations observed between the methine resonance at δ 1.97 (H-10) and the olefinic carbon resonances at δ 157.7 (C-9) and 102.8 (C-8) indicated that the decalin was attached to an olefin. The chemical shift of 13C resonances at δ 157.7 (C-9) and 66.9 (C-1) indicated that they were attached to an oxygen atom. At last, the HMBC correlations between H-8 and C-6, H-22 and C-6, as well as H-5 and C-7, helped to construct the last ring (C-1, C-6 to C-9) in the structure. The last unassigned atoms were a hydroxyl group, which was assigned to the deshielded C-7. Important HMBC and COSY correlations are shown in Figure 3.118. Figure 3.118 Selected COSY and HMBC correlations observed for ansellone N (3.21) in C6D6 OOOHHO2.48/2.35 35.64.44; 66.96.24; 138.4139.8196.5 1.65; 15.82.1540.74.54; 102.868.55.58134.939.81.6355.81.9766.64.11/3.87 68.31.51/0.99 37.4157.71.40; 26.75.62128.20.7521.71.24/1.00 41.20.8233.01.44/1.24 18.3170.61.64; 20.332.572.01.18 15.7OH13OHOHOO21201917 15131197525242322COSYHMBCHO HOHO 142 A ROESY correlation observed between the H-1 and H-6 resonances at δ 4.44 and 2.15 demonstrated that the methylcyclohexenone (C-1 to C-6) ring and dihydropyran (C-1, C-6 to C-9) ring were cis fused, which was consistent with other ansellones. ROESY correlations observed between the H-5/H-5’ and H-22/H-22’ resonances at δ 2.48/2.35 and 4.11/3.87 indicated that C-22 was in the axial orientation and the hydroxyl was equatorial. ROESY correlations observed between the Me-24 and Me-25 resonances at δ 1.18 and 0.75 and between the Me-20 and H-14 resonances at δ 0.82 and 1.63 demonstrated that the decalin rings were trans fused with the Me-24 and Me-25 both axial, which was consistent with other ansellones. Me-23 (δ 1.40) was also axial, confirmed by its chemical shift (δ 26.7) and the ROESY correlation observed between the Me-24 and Me-23 resonances at δ 1.18 and 1.40, and then the hydroxyl group (11-OH) was equatorial. The ROESY correlation between H-10 and H-14 resonances at δ 1.97 and 1.63 indicated that H-10 was also axial. Therefore, the relative configurations at C-1, C-6, C-7, C-10, C-11, C-14 and C-15 were determined. We have assumed that the absolute configurations at C-1, C-6, C-11, C-14 and C-15 are identical in ansellone A (2.86) and ansellone N (3.21), which completed the structure assignment of ansellone N (3.21). Important ROESY correlations are shown in Figure 3.119. 143 Figure 3.119 Selected ROESY correlations observed for ansellone N (3.21) in C6D6 Figure 3.120 1H NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 HHOHOtROESYHOHHOHOOOO HHOAcOHOOHOHH13572299111315171920252423 144 Figure 3.121 COSY NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 Figure 3.122 HSQC NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 H-1/H-6H-13/H-14H-12/H-13 145 Figure 3.123 HMBC NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 Figure 3.124 tROESY NMR spectrum of ansellone N (3.21) recorded at 600 MHz in C6D6 H-8/C-6H-8/C-7H-8/C-22H-8/C-9H-22/C-7H-22/C-6H-22/C-26H-10/C-8H-10/C-9H-5/C-7H-1/H-6H-5/H-22 Me-24/Me-25H-14/Me-20Me-23/Me-24H-10/H-14 146 3.6 Structure elucidation of anvilones Two new anvilones (3.22 and 3.23, Figure 3.125) were isolated from the British Columbia sponge Phorbas sp., and the detailed isolation is described in experimental section 3.11. Anvilones A (3.22) and B (3.23) have the novel “anvilane” carbon skeleton that has never been found before in any other organisms. Figure 3.125 Structures of anvilones A (3.22) and B (3.23) 3.6.1 Structure elucidation of anvilone A (3.22) Anvilone A (3.22) was isolated as a colorless powder that showed an [M + Na]+ ion peak in the ESITOFHRMS spectrum at m/z 451.2823 (calculated for C27H40O4Na, 451.2824), which required eight sites of unsaturation. The 13C NMR spectrum of anvilone A (3.22) showed 27 well resolved peaks, containing four olefinic resonances (δ 125.3, C-8; 138.5, C-7; 135.6, C-3; and 142.9, C-2) and two carbonyl resonances (δ 169.5, 13-OAc and 197.7, C-4), which accounted for four sites of unsaturation, indicating that anvilone A (3.22) had to be tetracyclic. The NMR data collected for anvilone A (3.22) are shown in Table 3.15. Detailed analysis of 1D and 2D NMR data recorded for anvilone A (3.22) as shown in panel A, B, C, D of Figure 3.126 identified two fragments that resembled the C-1 to C-6 OOAcOAcHOH OAcOHOHHHHHAnvilone A3.22 Anvilone B3.23Anvilane skeleton135791113151719212022232425 147 methylcyclohexenone (fragment A) and C-10 to C-20/Me-23/Me-24/Me-25 decalin rings (fragment B) present in ansellone A (2.86), accounting for three of the four required rings. Figure 3.126 Fragments of anvilone A (3.22), important COSY and HMBC correlations shown. A four-carbon fragment C in Figure 3.126 resembled the C-7 to C-9/C-22 moiety that linked the decalin and cyclohexenone fragments in ansellone A (2.86). An acetyl fragment D in Figure 3.126 was identified from the HMBC correlation between Me-27 (δ 1.78) and C-26 (δ 169.5). A series of HMBC and COSY correlations identified the bonds that link fragments ABCD together to give the complete constitution of anvilone A (3.22) as shown in Figure 3.126 (ABCD). A COSY correlation observed between the fragment B methine at δ 0.82 (H-10) and the fragment C oxymethine resonance at δ 3.93 (H-9) identified the first C-9/C-10 link between OO•O••••••••OBA1.67, 1.80 30.46.11; 142.91.85; 16.0135.6197.72.02, 2.40 42.82.11; 41.7HH HHHH1.112.1341.81.391.5518.71.271.3344.01.2237.00.9822.41.2159.2 5.44;70.41.24, 1.80 49.60.80; 22.30.82;60.40.96; 17.540.933.636.9H HHHHH HHHHHCH HH1.10. 1.51 46.7138.55.03;125.33.9367.1HD1.7821.6169.5OOOOABCDH913112257COSYHMBC3115171921232425 148 fragments B and C, and an HMBC correlation observed between the fragment B methyl resonance at δ 0.80 (Me-23) and the fragment C methylene carbon resonance at δ 46.7 (C-22) identified a second C-11/C-22 connection between fragments B and C, which generated the final ring required by the molecular formula. Evidence for the C-6/C-7 linkage shown in Figure 3.126 (ABCD) came from the observation of HMBC correlations between the H-5/H-5′ proton resonances at δ 2.02 and 2.40 and the C-7 olefinic carbon resonance at δ 138.5 and between the H-8 olefinic resonance at δ 5.03 and the C-6 methine resonance at δ 41.7. An HMBC correlation between the fragment B oxymethine proton resonance at δ 5.44 (H-13) and the acetyl carbonyl resonance at δ 169.5 (C-26) showed that the C-13 oxymethine oxygen atom was acetylated. A COSY correlation between the fragment C oxymethine proton resonance at δ 3.93 (H-9) and a proton resonance at δ 0.37 (OH-9), that was not correlated to a carbon resonance in the HSQC spectrum, revealed that the oxygen atom was part of an alcohol functionality, completing the constitution of anvilone A (3.22). Table 3.15 NMR data for anvilone A (?) recorded in C6D6 at 600 MHz Avilone A (14, C6D6) C# δC δH (J in Hz) 1 30.4 1.80 (dd, 12.1, 3.8) 1’ 1.67 (m) 2 142.9 6.11 (dm, 4.9) 3 135.6 — 4 197.7 — 5 42.8 2.40 (dd, 15.4, 2.0) 5’ 2.02 (dd, 14.4,13.4) 6 41.7 2.11 (m) * 7 138.5 — 8 125.3 5.03 (br s) 9 67.1 3.93 (br s) 10 60.4 0.82 (d, 8.9) 11 36.9 — 12 49.6 1.24 (m) 12’ 1.80 (m) 13 70.4 5.44 (dt, 11.2, 3.8) 14 59.2 1.21 (m) 15 40.9 — 149 Avilone A (14, C6D6) C# δC δH (J in Hz) 16 41.8 2.13 * 16’ 1.11 (m) * 17 18.7 1.55 (m) 17’ 1.39 (dm, 13.4) 18 44.0 1.33 (m) 18’ 1.27 (dd, 13.4, 4.4) 19 33.6 — 20 37.0 1.22 (s) 21 16.0 1.85 (s) 22 46.7 1.51 (br d, 17.0) 22’ 1.10 (br d, 17.0) * 23 22.3 0.80 (s) 24 17.5 0.96 (s) 25 22.4 0.98 (s) 26 169.5 — 27 21.6 1.78 (s) OH — 0.37 (d, 8.6) * Overlapped resonances Coupling constant and ROESY data identified the relative configuration of anvilone A (3.22). The H-13 resonance was a dt with J = 3.8 and 11.2 Hz indicating that both H-13 and H-14 were axial, and the H-10 resonance was a doublet with J = 8.9 Hz consistent with a diaxial coupling with H-9. ROESY correlations observed between H-13 (δ 5.44) and both Me-23 (δ 0.80) and Me-24 (δ 0.96), between H-9 (δ 3.93) and both of Me-23 and Me-24, and between H-10 (δ 0.82) and H-14 (δ 1.21) confirmed that H-9, H-10, H-13, H-14, Me-23, and Me-24 were all axial, which established the relative configuration of the tricyclic fragment of anvilone A (3.22) as shown in Figure 3.127. It was not possible to determine the configuration at C-6 of anvilone A (3.22) from the NMR data alone. A biogenetic argument led to the assignment of the absolute configurations 6R for anvilone A (3.22), which are the same as all the other sesterterpenoids isolated from this sponge Phorbas sp. 150 Figure 3.127 Selected ROESY correlations observed for anvilone A (3.22) in C6D6 Figure 3.128 1H NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 MeMeMeMeHHHO•HAcOH0.961.213.930.800.82d,J=8.9Hz5.44td,J=11.2,3.8HztROESY 151 Figure 3.129 13C NMR spectrum of anvilone A (3.22) recorded at 150 MHz in C6D6 Figure 3.130 COSY NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 H-9/H-10H-9/9-OHH-8/H-22H-13/H-12/H-14H-1/H-2H-1/H-6 152 Figure 3.131 HSQC NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 Figure 3.132 HMBC NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 H-13/C-26H-8/C-9H-8/C-22H-22/C-8H-22/C-7Me-27/C-26Me-23/C-22H-10/C-9H-8/C-6H-5,H-5'/C-7 153 Figure 3.133 tROESY NMR spectrum of anvilone A (3.22) recorded at 600 MHz in C6D6 3.6.2 Structure elucidation of anvilone B (3.23) Anvilone B (3.23) was isolated as a white powder that gave a [M + Na]+ ion in the ESITOFHRMS spectrum at m/z 509.2885 (calculated for C29H42O6Na, 509.2879), requiring nine sites of unsaturation and differed from the molecular formula of anvilone A (3.22) by adding the fragment C2H2O2. The NMR data of anvilones A (3.22) and B (3.23) collected in C6D6 are listed in Table 3.16. The 13C and HMBC NMR spectrum of anvilone B (3.23) contained four olefinic resonances (δ 128.3, C-8; 135.0, C-7; 138.5, C-3; 138.6, C-2) and three carbonyl resonances (δ 169.3, 1-OAc; 169.3, 13-OAc; 197.6, C-4) accounting for five sites of unsaturation, indicating that anvilone B (3.23) had to be tetracyclic. H-10/H-14Me-24/H-13H-13/Me-23Me-24/H-9H-9/Me-23 154 Table 3.16 NMR data for anvilone A (3.22) and anvilone B (3.23) recorded in C6D6 at 600 MHz Avilone A (3.22, C6D6) Anvilone B (3.23, C6D6) C# δC δH (J in Hz) δC δH (J in Hz) 1 30.4 1.80 (dd, 12.1, 3.8) 66.7 5.26 (br t, 4.4) 1’ 1.67 (m) — 2 142.9 6.11 (dm, 4.9) 138.6 6.47 (dd, 5.5, 1.4) 3 135.6 — 138.5 — 4 197.7 — 197.6 — 5 42.8 2.40 (dd, 15.4, 2.0) 38.2 2.67 (dd, 16.2, 12.7) 5’ 2.02 (dd, 14.4,13.4) 2.34 (dd, 16.2, 3.5) 6 41.7 2.11 (m) * 43.2 2.18 (m) 7 138.5 — 135.0 — 8 125.3 5.03 (br s) 128.3 5.10 (br dt, 3.0, 1.0) 9 67.1 3.93 (br s) 67.6 3.98 (br t, 8.2) 10 60.4 0.82 (d, 8.9) 60.5 0.87 (d, 8.9) 11 36.9 — 37.2 — 12 49.6 1.24 (m) 50.0 1.27 (m) * 12’ 1.80 (m) 1.79 (m) 13 70.4 5.44 (dt, 11.2, 3.8) 70.5 5.46 (dt, 11.2, 3.8) 14 59.2 1.21 (m) 59.7 1.25 (m) * 15 40.9 — 41.3 — 16 41.8 2.13 * 42.2 2.18 (m) 16’ 1.11 (m) * 1.12 (m) 17 18.7 1.55 (m) 19.1 1.56 (dm, 13.8) 17’ 1.39 (dm, 13.4) 1.40 (dm, 13.8) 18 44.0 1.33 (m) 44.3 1.33 (m) 18’ 1.27 (dd, 13.4, 4.4) 1.27 (m) * 19 33.6 — 33.9 — 20 37.0 1.22 (s) 37.4 1.23 (s) 21 16.0 1.85 (s) 16.0 1.75 (s) 22 46.7 1.51 (br d, 17.0) 47.6 1.51 (br d, 16.4) 22’ 1.10 (br d, 17.0) * 1.32 (m) 23 22.3 0.80 (s) 22.4 0.85 (s) 24 17.5 0.96 (s) 17.9 0.99 (s) 25 22.4 0.98 (s) 22.8 1.00 (s) 26 169.5 — 169.3 — 27 21.6 1.78 (s) 20.4 1.62 (s) 28 — — 169.3 — 29 — — 22.0 1.79 (s) OH — 0.37 (d, 8.6) — 0.41 * Overlapped resonances Detailed analysis of the COSY and HMBC data recorded for anvilone B (3.23) revealed that it differed from anvilone A (3.22) simply by addition of a second acetoxy functionality, which was in agreement with its molecular formula, and anvilone B (3.23) was different from 155 anvilone A (3.22) only at C-1/C-2/C-6 region. A deshielded methine at δ 5.26 (H-1 in 3.23), which was correlated to a carbon resonance at δ 66.7 (C-1 in 3.23) in the HSQC spectrum, replaced the H-1/H-1′ resonances at δ 1.67 and 1.80 in the 1H NMR spectrum of anvilone A (3.22). This deshielded methine at δ 5.26 (H-1) showed COSY correlations with the olefinic proton resonance at δ 6.57 (H-2) and the methine resonance at δ 2.18 (H-6). In the HMBC spectrum, H-1 also showed correlation to a carbon resonance at δ 169.3 (C-26). Therefore, the new acetate functionality in anvilone B (3.23) was located at C-1. A ROESY correlation observed between the methine proton resonances assigned to H-1 (δ 5.26) and H-6 (δ 2.18) in anvilone B (3.23) demonstrated that the protons were cis as in all of the ansellones and alotaketals. Figure 3.134 1H NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 156 Figure 3.135 13C NMR spectrum of anvilone B (3.23) recorded at 150 MHz in C6D6 Figure 3.136 COSY NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 H-1/H-2H-1/H-6 157 Figure 3.137 HSQC NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 Figure 3.138 HMBC NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 H-1/C-26 158 Figure 3.139 tROESY NMR spectrum of anvilone B (3.23) recorded at 600 MHz in C6D6 3.7 Structure elucidation of phorone C (3.24) A new compound phorone C (3.24, Figure 3.140) was isolated from the British Columbia sponge Phorbas sp., and the detailed isolation is described in experimental section 3.11. Figure 3.140 Structure of phorone C (3.24) Phorone C (3.24) was isolated as white powder. It gave an [M + H]+ ion peak in the ESITOFHRMS at m/z 425.2692 (calculated for C27H37O4, 425.2692), which required ten sites of H-1/H-6OOHOAcPhorone C3.24135791113151719202122232425H 159 unsaturation. The NMR data of phorone C (3.24) collected in both C6D6 and (CD3)2SO is listed in Table 3.17. The 13C and HMBC NMR spectrum of phorone C (3.24) contained eight olefinic resonances (δ 117.3, C-1; 153.1, C-2; 127.3, C-3; 131.1, C-4; 138.9, C-5; 135.5, C-6; 146.7, C-7; 133.4, C-8; data recorded in C6D6) and two carbonyl resonances (δ 199.5, C-9; 170.4, C-26; data recorded in C6D6) accounting for six sites of unsaturation, indicating that phorone C (3.24) had to be tetracyclic. Table 3.17 NMR data for phorone C (3.24) recorded in C6D6 and (CD3)2SO at 600 MHz Phorone C (3.24, C6D6) Phorone C (3.24, (CD3)2SO) C# δC δH (J in Hz) δC δH (J in Hz) 1 117.3 6.59 (s) 116.6 7.08 (s) 2 153.1 — 153.5 — 3 127.3 — 126.7 — 4 131.1 8.10 (s) 129.4 7.69 (s) 5 138.9 — 136.1 — 6 135.5 — 133.9 — 7 146.7 — 148.0 — 8 133.4 6.56 (br s) 131.2 6.26 (s) 9 199.5 — 199.3 — 10 74.5 2.51 (s) 73.0 2.37 (s) 11 39.8 — 38.6 — 12 48.6 1.28 * 47.1 1.57 (dd, 11.5, 13.2) 12’ 2.69 (dd, 3.2, 13.6) 2.62 (dd, 2.1, 12.7) 13 71.0 5.45 (dt, 11.3, 3.3) 69.7 5.17 (dt, 11.2, 2.7) 14 59.8 1.30 * 58.3 1.46 (d, 11.3) 15 41.0 — 39.6 — 16 40.1 1.66 (m) 38.5 1.28 (m) * 16’ 1.37 (dt, 3.4, 13.1) 0.94 (m) 17 18.7 1.29 (m) * 17.6 1.28 (m) * 17’ 1.36 (dm, 13.4) 1.41 (br t, 13.0) 18 43.8 1.16 (m) 44.3 1.23 (br s) 18’ 1.16 (m) 1.23 (br s) 19 34.0 — 33.1 — 20 37.1 1.04 (s) 36.2 0.98 (s) 21 16.1 2.37 (s) 15.9 2.22 (s) 22 28.0 1.89 (s) 27.4 2.27 (s) 23 30.1 1.25 (s) 29.2 1.10 (s) 24 18.7 0.63 (s) 17.5 0.39 (s) 25 22.5 0.69 (s) 21.7 0.67 (s) 26 170.4 — 170.1 — 27 21.8 1.74 (s) 21.6 2.08 (s) OH — 3.90 (s) — 9.37 (s) * Overlapped resonances 160 Detailed analysis of 1D and 2D NMR data recorded for phorone C (3.24) as shown in Figure 3.141 identified a fragment that resembled the C-10 to C-20/Me-23/Me-24/Me-25 decalin rings that was present in ansellone B (2.87), accounting for two of the four required rings. The chemical shift of C-13 (δ 69.7) suggested that C-13 was attached to an oxygen atom. Figure 3.141 Selected COSY and HMBC correlations observed for phorone C (3.24) in C6D6 and (CD3)2SO 153.16.59117.3COSYHMBCOOHO5.4571.041.01.7156.71.37/1.66 40.12.69/1.28 48.60.6922.51.0437.11.36/1.29 18.834.02.51; 74.50.6318.7127.38.10131.1135.5138.92.37; 16.11.89; 28.0146.76.56133.4199.5 135.539.81.2530.1O170.41.74; 21.8153.57.08116.6OOHO5.1769.739.61.4658.31.28/0.94 38.52.62/1.57 47.10.6721.71.2342.70.9836.21.41/1.28 17.633.12.37; 73.00.3917.5126.77.69129.4133.9136.12.22; 15.92.27; 27.4148.06.26131.2199.3 135.538.61.1029.2O170.12.08; 21.6recorded in C6D6 recorded in (CD3)2SOOOHO OOOHO Orecorded in C6D6 recorded in (CD3)2SO13571121151319179232422252726 161 Analysis of the NMR data collected in (CD3)2SO gave a methylphenyl ring fragment (C-1 to C-6 and Me-21). It was established by the carbon chemical shifts (δ 116.6, C-1; 153.5, C-2; 126.7, C-3; 129.4, C-4; 136.1, C-5; 133.9, C-6), the HMBC correlations observed between a methyl singlet at δ 2.22 (Me-21) and aromatic carbon resonances at δ 153.5 (C-2), 126.7 (C-3) and 129.4 (C-4), and the HMBC correlations observed between a deshielded proton resonance at δ 7.08 (H-1) and aromatic carbon resonances at δ 153.5 (C-2), 126.7 (C-3) and 136.1 (C-5). HMBC correlations were observed (NMR data collected in C6D6) between a deshielded proton resonance at δ 8.10 (H-4) and aromatic carbon resonances at δ 153.1 (C-2) and 135.5 (C-6) and methyl carbon resonances at δ 16.1 (Me-21), which confirmed the methylphenyl ring fragment. The chemical shift of C-2 (δ 153.5) suggested that C-2 was attached to an oxygen atom. The remaining unassigned four carbons included a methyl (δ 2.27 H-22, δ 27.4 C-22), two deshielded carbons (δ 148.0 C-7 and δ 6.26 H-8, δ 131.2 C-8) and a carbonyl carbon (δ 199.3 C-9). An HMBC correlation observer between the methine resonance assigned to H-10 and the carbonyl carbon resonance assigned to C-9 indicated that the carbonyl (C-9) was attached to C-10. A four-carbon fragment (C-7 to C-9 and Me-22), which resembled the C-7 to C-9/C-22 moiety that linked the decalin and cyclohexenone fragments in ansellone B (2.87), was established by the HMBC correlations observed between the methyl proton resonance assigned to Me-22 and the carbon resonances assigned to C-7 and C-8, and the HMBC correlations observed between the olefinic proton resonance assigned to H-8 and the carbon resonances assigned to C-10 and Me-22. The last ring was established by the HMBC correlation observed between the aromatic carbon resonance assigned to C-5 and the methyl proton resonance assigned to Me-23 and the methine resonance assigned to H-10, and the HMBC correlation 162 observed between the aromatic carbon resonance assigned to C-6 and the methyl proton resonance assigned to Me-22 and the olefinic proton resonance assigned to H-8. Detailed analysis of the COSY and HMBC data recorded for phorone C (3.24) revealed that it differed from phorone A (2.93) simply by the replacement of a hydroxyl group by an acetoxy functionality, which was in agreement with its molecular formula. A ROESY correlation observed between the methyl resonances assigned to Me-27 (δ 2.08) and Me-20 (δ 0.98) indicated that the new acetoxy group in phorone C (3.24) was located on C-13 rather than C-2. The assigned structure is shown in Figure 3.141, along with important COSY and HMBC correlations. The carbon chemical shifts of Me-24 (δ 18.7) and Me-25 (δ 22.5) and the correlation between Me-25 (δ 0.69) and Me-24 (δ 0.63) and between Me-25 (δ 0.69) and H-13 (δ 5.45) observed in the 1D NOE NMR spectrum demonstrated that the decalin rings were trans fused with Me-24, Me-25 and H-13 all axial. ROESY correlations observed in Figure 3.142 suggested that the configuration of the acetoxy group (13-OAc) in phorone C (3.24) was the same as the hydroxyl group (13-OH) in phorone A (2.93).86 Figure 3.142 Selected ROESY correlations observed for phorone C (3.24) in (CD3)2SO MeMeMeMeHH O HtROESYMeO0.6721.70.9836.20.3917.51.2530.12.3773.02.0821.65.1769.71.4658.3OOHOAcPhorone C3.24OOHOHPhorone A2.9313 1313 163 Figure 3.143 1H NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 Figure 3.144 13C NMR spectrum of phorone C (3.24) recorded at 150 MHz in C6D6 164 Figure 3.145 COSY NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 Figure 3.146 HSQC NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 165 Figure 3.147 HMBC NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 Figure 3.148 tROESY NMR spectrum of phorone C (3.24) recorded at 600 MHz in C6D6 H-4/C-6H-4/C-2 166 Figure 3.149 1H NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO Figure 3.150 13C NMR spectrum of phorone C (3.24) recorded at 150 MHz in (CD3)2SO 167 Figure 3.151 COSY NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO Figure 3.152 HSQC NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO 168 Figure 3.153 HMBC NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO Figure 3.154 tROESY NMR spectrum of phorone C (3.24) recorded at 600 MHz in (CD3)2SO Me-21/C-2Me-21/C-3Me-21/C-4H-1/C-3H-1/C-5H-1/C-2Me-22/C-7Me-22/C-8H-8/C-10H-8/Me-22H-10/C-9Me-23/C-5H-10/C-5 Me-22/C-6H-8/C-6Me-27/Me-20Me-24/Me-25H-10/H-14 169 Figure 3.155 1D NOE NMR spectrum of phorone C (3.24) irradiated at 0.69 ppm recorded at 600 MHz in C6D6 3.8 Biogenesis of alotaketals, ansellones, phorones and anvilones There are a variety of sesterterpenoids isolated from the sponge Phorbas sp.86,112 A detailed biogenetic proposal for the formation of the alotaketals, ansellones, phorones and anvilones is shown in Figure 3.156. It suggests that all these sesterterpenoids derive from a linear geranylfarnesyl diphosphate (GFPP, 1.34) starting material. An early step of isomerization of GFPP gives the (Z) isomer of GFPP, followed by the first cyclization (through “a” or “b” in Figure 3.156) initiated by the ionization of the C-1 allylic pyrophosphate group leading to a cyclohexene ring (Intermediate I or II in Figure 3.156), which is a common intermediate for all of the sesterterpenoids isolated. Intermediate I has the alotane skeleton, which is the precursor for alotaketals and phorbaketals. Oxidation of intermediate II at C-1, C-4, C-9 and C-13, followed by a second cyclization initiated by the protonation of the C-18/C-19 alkene, forms a 170 tetracyclic decalin/oxocane/cyclohexenone ring system (through “c” in Figure 3.156) to give ansellone C (2.88) or a decalin ring system (through “d” in Figure 3.156) to give ansellone K (3.18). A second cyclization of intermediated II initiated by the protonation of the C-18/C-19 alkene also gives a 6,6,6 tricyclic fragment to form conjoint ring systems (through “e” in Figure 3.156), which can be oxidized and acetylated to give anvilones A (3.22) and B (3.23). Oxidation of intermediate I at C-1, C-4, C-9, C-13 and C-22, followed by a second cyclization initiated by the protonation of the C-18/C-19 alkene, forms a decalin ring system (through “f” in Figure 3.156). Another step of condensation gives ansellone A (2.86). Oxidation of intermediate I at C-1, C-4, C-5, C-9 and C-13 (through “h” in Figure 3.156), followed by dehydroxylation, keto-enol tautomerization of its cyclohexenone and a second cyclization initiated by the protonation of the C-18/C-19 alkene, forms phorones A (2.93). 171 Figure 3.156 Biogenetic proposal for the formation of the alotaketals, ansellones, phorones and anvilones 3.9 Biological activities In collaboration with Dr. Ian Tietjen (Faculty of Health Sciences, Simon Fraser University), we investigated the potential effects of the isolated sesterterpenoids on HIV-1 latency reversal. Effects of the new Phorbas sp. sesterterpenoids on latent HIV-1 provirus gene OPPOPP OPPHHHHHHHOOHHOHHOOAcHOHHOOHHOOHHHOOAcHOOAcHab abOOHHOH OHOHOHOHOOHHH2O OHOHOHOOHOHHH HOOAcOOHHHAnsellone A2.86Anvilone B3.23Anvilone A3.22OHOHOHOOH2OOHOHOHOOHOOAcOHOOOAlotaketal C3.7OHOHOOHHAnsellone C2.88OOHOAcAcOHAnsellone K3.18OOHHOOHH2OcdOHHcdOHOHOHOHOHOHOHOOOHOAcOOHPhorone C3.24ff[Ox][Ox][Ox]ggOHOHHO OH [Ox]HO HOIntermediate IIntermediate IIIntermediate IIeehGFPP1.34DMAPP1.30Phorone A2.93(E) (Z)1191181819[Ox]14913[Ox][Ox]142291318 191491351819 172 expression are shown in Figure 3.157 and Table 3.18. Prostratin (3.5) was the positive control LRA in this assay. Figure 3.157 Effects of sesterterpenoids on HIV-1 provirus/GFP expression 02468101214160.01 0.1 1 10 100HIV reactivation (% GFP-positive cells)Concentration (µM)ProstratinAlotaketal CAnsellone AAlotaketal DAlotaketal EAnsellone DAnsellone EAnsellone FAnsellone GAnvilone A00.20.40.60.811.20.01 0.1 1 10 100Viability, relative to no-drug controlConcentration (µM)ProstratinAlotaketal CAlotaketal DAlotaketal EAnsellone AAnsellone DAnsellone EAnsellone FAnsellone GAnvilone A3.5 Prostratin 3.7 Alotaketal C 3.8 Alotaketal D 3.9 Alotaketal E 2.86 Ansellone A 3.11 Ansellone D 3.12 Ansellone E 3.13 Ansellone F 3.14 Ansellone G 3.22 Anvilone A Figure 3. ffects of selected sesterterpenoids on HIV-1 provirus / GFP expression of J-lat 9.2 cells. 173 Figure 3.158 Preliminary SAR for sesterterpenoids isolated from Phorbas sp. In this J-Lat cell based assay, the most potent sesterterpenoid alotaketal C (3.7) induced GFP expression by 13.8 ± 2.2%, which was approximately two-fold greater than prostratin (3.5, OOOHOHHAcOAlotaketal C 3.7OHOHHH HHOAcOAnsellone A 2.86OOOAcHOHHAcOAlotaketal D 3.8H OAcOHOHHAnvilone A 3.22OOOHOHHAcOOHAlotaketal E 3.9OHOMeHH HHOAcOOOAcO H HHHOHHOOAcOOOHH HHOOAcOOH HHHOHAnsellone D 3.11 Ansellone E 3.12 Ansellone F 3.13Ansellone G 3.14OHOOAcHOHHHProstratin 3.5More activethan controlControlSimilar tocontrolWeakly activeInactive 174 the positive control LRA) at the same concentration (30 µM). Ansellone A (2.86), alotaketals D (3.8) and anvilone A (3.22) were found to have LRA profiles similar to the control prostratin (3.5). Alotaketals E (3.9) and ansellone G (3.14) weakly induced GFP expression at the concentration of 30 µM, with the activation of 3.6 ± 1.9% and 1.4 ± 1.1%, respectively. The other Phorbas sp. sesterterpenoids (2.87−2.89, 3.10−3.13) were inactive at a concentration of 30 µM. These results are summarized in Figure 3.158 and Table 3.18. The most active analog alotaketal C (3.7), containing an enone functionality as a potential Michael acceptor, might be one of the pan assay interference compounds (PAINS). PAINS are compounds that have nonspecific protein reactivity, and they are frequent hitters of several different and independent biological assays.113 However, alotaketal D (3.8), which is an alotaketal C (3.7) analog without the enone functionality, still had LRA profile similar to the positive control prostratin (3.5). Some sesterterpenoid analogs ansellones D−F (3.11−3.13), which contain enone functionalities in their structures, were inactive in this assay. Therefore, the LRA activities did not arise from nonspecific covalent binding to proteins by the enone functionalities. Alotaketal E (3.9), which is an alotaketal C (3.7) analog with a hydroxyl group in the side chain, only showed very weak activity compared to alotaketal C (3.7), which indicates that the side chain is important for the activity (Figure 3.159). Ansellone A (2.86) was found to have LRA profile similar to the control prostratin (3.5), but methylation of the 11-OH (ansellone D, 3.11) or a simple change in the configuration at C-11 (ansellone E, 3.12) would kill the activity (Figure 3.160). Ansellone G (3.14), an ansellone A (2.86) analog bearing a change at the C-12/C-13 olefin, was also less active (Figure 3.160). Therefore, the SAR in this family of compounds is quite subtle. 175 Figure 3.159 Preliminary SAR for alotaketals C−E (3.7−3.9) Figure 3.160 Preliminary SAR for ansellones A and D−G (2.86 and 3.11−3.14) Furthermore, the toxicity of the sesterterpenoids to J-Lat cells was also studied, and the effects of sesterterpenoids on viability of J-Lat 9.2 cells is shown in Figure 3.161 and Table 3.18. Ansellone A (2.86), alotaketal D (3.8) and anvilone A (3.22), which were as active as prostratin (3.5), showed similar toxicity profiles to that of prostratin (3.5). Alotaketal C (3.7) was more toxic than prostratin (3.5) with a CC50 of 11.0 ± 7.8 µM, and the toxicity and efficacy profiles of these two compounds corresponded to each other. Alotaketal C (3.7) Alotaketal D (3.8) Alotaketal E (3.9) Structure % HIV-reactivation (GFP-positive cells) at 30 µM 13.8 ± 2.2 7.3 ± 3.7 3.6 ± 1.9 OOOHOHHAcOAlotaketal C3.7OOOAcHOHHAcOAlotaketal D3.8OOOHOHHAcOOHAlotaketal E3.9Ansellone A (2.86) Ansellone G (3.14) Ansellone D (3.11) Ansellone E (3.12) Ansellone F (3.13) Structure % HIV-reactivation (GFP-positive cells) at 30 µM 6.9 ± 2.4 1.4 ± 1.1 inactive inactive inactive OHOHHH HHOAcOAnsellone A2.86OOAcOOH HHHOHAnsellone G3.14OHOMeHH HHOAcOOOAcO H HHHOHHOOAcOOOHH HHAnsellone D3.11Ansellone E3.12 Ansellone F3.13111213 176 Figure 3.161 Effects of sesterterpenoids on viability of J-Lat 9.2 cells Table 3.18 Effects of sesterterpenoids on HIV-1 provirus / GFP expression and viability of J-Lat 9.2 cells Compound % HIV-reactivation (GFP-positive cells) at 30 µM CC50 (µM) Prostratin (3.5) 6.4 ± 3.2 > 30 Alotaketal C (3.7) 13.8 ± 2.2 11.0 ± 7.8 Alotaketal D (3.8) 7.3 ± 3.7 > 30 Alotaketal E (3.9) 3.6 ± 1.9 > 30 Ansellone A (2.86) 6.9 ± 2.4 > 30 Ansellone B (2.87) n/d 7.1 ± 0.4 Ansellone C (2.88) n/d 29.1 ± 15.7 Secoepoxyansellone A (2.89) n/d 9.4 ± 3.7 Phorbadione (3.10) n/d > 30 Ansellone D (3.11) n/d > 30 Ansellone E (3.12) n/d > 30 Ansellone F (3.13) n/d > 30 Ansellone G (3.14) 1.4 ± 1.1 > 30 Ansellone K (3.18) n/d 7.5 ± 0.1 Anvilone A (3.22) 6.9 ± 2.7 > 30 00.20.40.60.811.20.01 0.1 1 10 100Viability, relative to no-drug controlConcentration (µM)ProstratinAlotaketal CAnsellone AAlotaketal DAlotaketal EAnsellone DAnsellone EAnsellone FAnsellone GAnvilone A00.20.40.60.811.20.01 0.1 1 10 100Viability, relative to no-drug controlConc ntration (µM)ProstratinAlotaketal CAlotaketal DAlotaketal EAnsellone AAnsellone DAnsellone EAnsellone FAnsellone GAnvilone A3.5 Prostratin 3.7 Alotaketal C 3.8 Alotaketal D 3.9 Alotaketal E 2.86 Ansellone A 3.11 A sellone D 3.12 Ansellone E 3.13 Ansellone F 3.14 Ansellone G 3.22 Anvilone A Figure 3. Effects of selected sesterterpenoids on viability of J-lat 9.2 cells. 177 We also investigated whether Phorbas sesterterpenoids activate latent HIV-1 infection using similar mechanisms as established LRAs. The known LRAs, which have different mechanisms of activation, include prostratin (3.5) and bryostatin 1 (3.6) that induce the PKC-NF-κB pathway, TNFα as a pro-inflammatory cytokine, and panobinostat that acts as an HDAC inhibitor.112 Table 3.19 Effects of sesterterpenoids in combination with control LRAs to induce HIV-1 provirus / GFP expression Sample tested % HIV-reactivation (GFP-positive cells) Add TNFα (10 ng/mL) Add panobinostat (0.3 µM) Add prostratin (3.5) (10 µM) No Drug 0.1 ± 0.0 13.2 ± 6.1 15.2 ± 4.6 6.8 ± 2.9 Prostratin (3.5) at 10 µM 6.8 ± 2.9 49.3 ± 10.5 60.9 ± 9.9 - Alotaketal C (3.7) at 1 µM 9.2 ± 7.7 51.1 ± 9.5 61.2 ± 13.7 11.1 ± 3.6 Ansellone A (2.86) at 10 µM 8.4 ± 1.4 56.1 ± 8.7 64.6 ± 3.0 9.2 ± 1.7 Alotaketal D (3.8) at 10 µM 3.0 ± 2.1 46.3 ± 9.0 50.0 ± 8.1 11.2 ± 1.6 Alotaketal E (3.9) at 30 µM 4.5 ± 1.0 52.1 ± 12.2 52.0 ± 5.4 12.1 ± 1.4 Anvilone A (3.22) at 30 µM 8.0 ± 1.1 61.2 ± 4.7 50.1 ± 5.0 13.8 ± 1.4 In these studies of the effects of sesterterpenoids in combination with control LRAs (Table 3.19), J-Lat cells treated with alotaketal C−E (3.7−3.9), ansellone A (2.86) and anvilone A (3.22) induced GFP expression in 3.0−9.2% of cells. Similarly, treatment of J-Lat cells with 10 ng/mL TNFα, 0.3 µM panobinostat, or 10 µM prostratin (3.5) induced GFP expression in 13.2 ± 6.1, 15.2 ± 4.6, and 6.8 ± 2.9% of cells, respectively. However, cells treated with sesterterpenoids plus TNFα or panobinostat induced GFP expression in 46.3−64.6% of cells, which is substantially more than expected for additive responses and demonstrates statistically significant levels of synergism. In contrast, cells treated with sesterterpenoids plus prostratin induced GFP expression in only 9.2−13.8% of cells, which is more consistent with additive 178 effects. These results suggest that sesterterpenoids induce HIV-1 proviral gene expression by mechanisms resembling those of prostratin (3.5). We also further investigated whether sesterterpenoids, like prostratin (3.5), may act as PKC agonists. GÖ-6983 and RO-31-8220 are two known pan-PKC inhibitors. We treated cells with sesterterpenoids or control LRAs for 24 hours with or without the pan-PKC inhibitors. Table 3.20 Effects of sesterterpenoids and control LRAs in combination with PKC inhibitors Sample tested % HIV-reactivation (GFP-positive cells) Add GÖ-6983 (1 µM) Add RO-31-8220 (0.3 µM) No Drug 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 TNFα at 10 ng/mL 17.0 ± 1.5 18.0 ± 1.1 17.3 ± 1.1 panobinostat at 0.3 µM 18.5 ± 8.1 20.0 ± 8.3 18.3 ± 8.4 Prostratin (3.5) at 30 µM 7.4 ± 4.6 0.3 ± 0.1 0.9 ± 0.5 Alotaketal C (3.7) at 30 µM 17.7 ± 5.8 2.3 ± 0.9 1.9 ± 0.3 Ansellone A (2.86) at 30 µM 8.3 ± 2.8 0.3 ± 0.1 0.6 ± 0.2 Alotaketal D (3.8) at 30 µM 8.7 ± 4.9 0.8 ± 0.3 1.1 ± 0.4 Alotaketal E (3.9) at 30 µM 4.2 ± 2.4 0.2 ± 0.0 0.5 ± 0.2 Anvilone A (3.22) at 30 µM 8.3 ± 4.1 0.4 ± 0.2 0.5 ± 0.1 The results in Table 3.20 show that neither 1 µM of GÖ-6983 nor 0.3 µM RO-31-8220 inhibited GFP expression in the presence of TNFα or panobinostat, both inhibited >85% of GFP expression induced by prostratin (3.5). As with prostratin (3.5), both PKC inhibitors also inhibited >85% of GFP expression induced by all tested sesterterpenoids, suggesting that sesterterpenoids, like prostratin (3.5), induce provirus expression through PKC activation pathways. 3.10 Conclusions and future work In conclusion, the new sesterterpenoids alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24), along 179 with the known sesterterpenoids ansellones A−C (2.86−2.88), have been isolated from the Phorbas sponge specimen collected in Howe Sound, British Columbia. Their structures have been elucidated by analysis of NMR and MS data. Ansellone F (3.13) contains a rare 1,2−3,4-bisepoxydecalin substructure that has only been previously found in sesquiterpenoids isolated from leaves of the medicinal plant Laurus nobilis.114 Ansellone K (3.18) represents the first example of the ansellane carbon skeleton lacking an ether linkage at C-1 of the cyclohexenone ring. Anvilones A and B (3.22 and 3.23) have an unprecedented tetracyclic anvilane sesterterpenoid carbon skeleton. Ansellones A−N (2.86−2.88, 3.11−3.21, having the ansellane carbon skeleton), secoepoxyansellone A (2.89, having the secoansellane carbon skeleton) and anvilones A and B (3.22 and 3.23, having the anvilane carbon skeleton) are so far only found in the metabolites isolated from this particular sponge Phorbas sp. Figure 3.162 Sesterterpene skeletons isolated from the sponge Phorbas sp. Using a cell culture model of latent HIV-1 infection, the biological activities of these sesterterpenoids isolated from the sponge Phorbas sp. were investigated. Among all these sesterterpenoids, alotaketal C (3.7) showed the most potent activation of the HIV-1 provirus Secoansellane skeleton Anvilane skeleton Ansellane skeleton Phorane skeletonAlotane skeleton 180 expression (13.8 ± 2.2% activation at 30 µM), which is two-fold over the positive control LRA prostratin (3.5). Ansellone A (2.86), alotaketal D (3.8) and anvilone A (3.22) were found to induce HIV proviral gene expression similar to the control compound prostratin (3.5). The preliminary SAR revealed that the side chain and enone functionality are important for the activity, and this conclusion was reached from the comparison of the structures and activities of alotaketals C−E (3.7−3.9). The SAR for LRAs in this sesterterpenoids family is quite subtle, and a single change in the configuration at C-11 of ansellone A (2.86) led to a dramatic change in the LRA properties. Biological results also suggested that alotaketals C (3.7) and D (3.8), ansellone A (2.86) and anvilone A (3.22) responded by activating PKC signaling pathway, which acted by the same mechanism as prostratin (3.5, PKC activator). Alotaketal C (3.7), alotaketal D (3.8), ansellone A (2.86) and anvilone A (3.22) are the first sesterterpenoids known to be able to activate latent HIV-1 provirus expression, and they are a new set of scaffolds for this activity. Alotaketal C (3.7), ansellone A (2.86) and anvilone A (3.22) serve as new lead structures in the development of new cell biology tools and potential drug candidates. Two total syntheses of alotaketal A (2.72)115,116 and the total synthesis of ansellones A (2.86) and B (2.87)117 have been reported, which provide access to analogs of these scaffolds. More SAR-driven studies can be done in the future. 3.11 Experimental section 3.11.1 General experimental procedures All the solvents used in the experiments were HPLC grade from Fisher Scientific Inc. without further treatment. Milli-Q H2O was used for reversed-phase Sep-pak and HPLC. Sephadex™ LH-20 purchased from Sigma was used for gel filtration chromatography. 181 SiliaFlash® G60 silica gel was used for flash chromatography. Merck Type 5554 Si gel plates and Whatman MKC18F plates were used for analytical thin-layer chromatography. Sep-pak cartridges were purchased from Waters. HPLC was performed on a Waters 2998 system with a Photodiode Array detector. An InertSustain C18 semi-preparative column (5 µm 10 × 250 mm) was used for reversed-phase HPLC; an Alltech Apollo silica semi-preparative column (5 µm 10 × 250 mm) was used for normal-phase HPLC. 1H and 13C NMR spectra were recorded on a Bruker 600 MHz spectrometer with a 5 mm cryoprobe. 1H chemical shifts are referenced to residual C6D6 (δ 7.16 ppm) or CD3OD (δ 3.31 ppm), and 13C chemical shifts are referenced to the C6D6 (δ 128.39 ppm) or CD3OD (δ 49.15 ppm) solvent peak. All NMR solvents were purchased from Cambridge Isotope Laboratories Inc. Low- and high-resolution MS were recorded using ESI ionization and a TOF mass analyzer. UV spectra were recorded using a Waters 2998 UV spectrophotometer. The optical rotations were recorded with a Jasco P-1010 polarimeter at the sodium D line (589 nm). The CD spectrum was collected with a Jasco J-815 CD spectrophotometer. 3.11.2 Sample collection, extraction and isolation The specimens of the sponge Phorbas sp. were collected by hand using SCUBA on rocky walls at the depth of 10−20 meters along the shorelines near Ansell Point and Anvil Island (Howe Sound, British Columbia) in several batches in 2012 and 2014. Freshly collected specimens were frozen on site with dry ice for storage. The first batch of frozen sponge (220 g) was extracted with MeOH (3 × 150 mL) at room temperature for 24 hours. The combined MeOH extracts were dried in vacuo to obtain 900 mg of brown crude extract. The extract was partitioned between H2O (100 mL) and EtOAc (3 × 50 mL), and the EtOAc fraction was concentrated under reduced pressure. This fraction was 182 subjected to Sephadex™ LH-20 column chromatography with 4:1 MeOH/CH2Cl2 to give six fractions A−F. Fraction B (200.1 mg) was chromatographed using a silica gel column with a slow gradient from 100% hexanes to 100% EtOAc, and the resulting sub-fractions BA−BM was further purified by C18 reversed-phase HPLC. A second batch of Phorbas sp. (330 g) was collected and extracted with MeOH (3 × 200 mL) at room temperature. The MeOH extract was concentrated in vacuo to obtain 1.5 g of brown crude oil. Partition between H2O (200 mL) and EtOAc (3 × 100 mL) gave the organic EtOAc fraction, and this fraction was chromatographed on a Sephadex™ LH-20 column eluting with 4:1 MeOH/CH2Cl2 to afford fractions A−K. Fraction D (496 mg) was subjected to silica gel chromatography with a gradient from 100% hexanes to 100% EtOAc to give sub-fractions DA−DR. Fractions DJ, DK, DL, DM and DR were further purified by C18 reversed-phase HPLC. The following compounds were isolated through HPLC: Alotaketal C (3.7): white powder (0.5 mg); [α]D25 −8.1 (c 0.05 MeOH); UV (MeOH) λmax (log ε) 230 nm (4.58); 13C NMR and 1H NMR, see Table 3.1; positive-ion ESITOFHRMS [M + Na]+ m/z 481.2565 (calculated for C27H38O6Na, 481.2566). Alotaketal D (3.8): white powder (0.5 mg); [α]D25 −5.4 (c 0.70 CD2Cl2); UV (MeOH) λmax (log ε) 231.5 nm (5.09); 13C NMR and 1H NMR, see Table 3.2; positive-ion ESITOFHRMS [M + Na]+ m/z 525.2827 (calculated for C29H42O7Na, 525.2828). Alotaketal E (3.9): white powder (0.5 mg); [α]D25 +2.2 (c 0.005 CH2Cl2); UV (MeCN−H2O 1:1) λmax (log ε) 229 nm; 13C NMR and 1H NMR, see Table 3.2; positive-ion ESITOFHRMS [M + Na]+ m/z 513.2452 (calculated for C27H38O8Na, 513.2464). 183 Ansellone A (2.86): white powder (1.2 mg); [α]D25 −13.8 (c 0.01 MeOH); UV (MeCN−H2O 1:1) λmax (log ε) 235 nm; 1H NMR, matched the literature value; positive-ion ESITOFLRMS [M + Na]+ m/z 465.4. Ansellone B (2.87): white powder (2.2 mg); [α]D25 −12.5 (c 0.05 MeOH); UV (MeCN−H2O 1:1) λmax (log ε) 228 nm; 1H NMR matched the literature value; positive-ion ESITOFLRMS [M + Na]+ m/z 467.4. Ansellone C (2.88): white powder (0.9 mg); UV (MeCN−H2O 1:1) λmax (log ε) 227 nm; 1H NMR matched the literature value; positive-ion ESITOFLRMS [M + Na]+ m/z 467.4. Phorbadione (3.10): white powder (0.6 mg); [α]D25 +34.6 (c 1.86 CH2Cl2); UV (MeCN−H2O 1:1) λmax (log ε) 250 nm; 13C NMR and 1H NMR, see Table 3.3; positive-ion ESITOFHRMS [M + Na]+ m/z 461.2297 (calculated for C27H34O5Na, 461.2304). Ansellone D (3.11): colorless powder (0.4 mg); [α]D25 −8.8 (c 0.08 CD2Cl2); UV (MeOH) λmax (log ε) 227.2 nm (4.08); 13C NMR and 1H NMR, see Table 3.4; positive-ion ESITOFHRMS [M + Na]+ m/z 479.2778 (calculated for C28H40O5Na, 479.2773). Ansellone E (3.12): white powder (0.5 mg); [α]D25 −5.4 (c 0.005 CH2Cl2); UV (MeCN−H2O 4:1) λmax (log ε) 228 nm (4.66); 13C NMR and 1H NMR, see Table 3.5; positive-ion ESITOFHRMS [M + Na]+ m/z 465.2615 (calculated for C27H38O5Na, 465.2617). Ansellone F (3.13): white powder (1.0 mg); [α]D25 −10.4 (c 0.005 CH2Cl2); UV (MeCN−H2O 3:2) λmax (log ε) 227 nm; 13C NMR and 1H NMR, see Table 3.6; positive-ion ESITOFHRMS [M + Na]+ m/z 479.2403 (calculated for C27H36O6Na, 479.2410). Ansellone G (3.14): white powder (0.5 mg); [α]D25 −30.5 (c 0.02 CD2Cl2); UV (MeOH) λmax (log ε) 234.3 nm (4.89); 13C NMR and 1H NMR, see Table 3.7; positive-ion ESITOFHRMS [M + Na]+ m/z 479.2409 (calculated for C27H36O6Na, 479.2410). 184 Ansellone H (3.15): white powder (0.5 mg); UV (MeCN−H2O 6:4) λmax (log ε) 230 nm; 13C NMR and 1H NMR, see Table 3.8; positive-ion ESITOFHRMS [M + Na]+ m/z 465.2621 (calculated for C27H38O5Na, 465.2617). Ansellone I (3.16): white powder (0.8 mg); UV (MeCN−H2O 1:1) λmax (log ε) 230 nm; 13C NMR and 1H NMR, see Table 3.9; positive-ion ESITOFHRMS [M + Na]+ m/z 481.2563 (calculated for C27H38O6Na, 481.2566). Ansellone J (3.17): white powder (< 0.5 mg, decomposed); UV (MeCN−H2O 11:9) λmax (log ε) 227 nm; 13C NMR and 1H NMR, see Table 3.10; positive-ion ESITOFLRMS [M + Na]+ m/z 481.5. Ansellone K (3.18): white powder (0.5 mg); UV (MeCN−H2O 1:1) λmax (log ε) 224 nm; 13C NMR and 1H NMR, see Table 3.11; positive-ion ESITOFHRMS [M + Na]+ m/z 509.2885 (calculated for C29H42O6Na, 509.2879). Ansellone L (3.19): white powder (0.5 mg); UV (MeCN−H2O 11:9) λmax (log ε) 228 nm; 13C NMR and 1H NMR, see Table 3.12; positive-ion ESITOFHRMS [M + Na]+ m/z 423.2509 (calculated for C25H36O4Na, 423.2511). Ansellone M (3.20): white powder (1.0 mg); UV (MeCN−H2O 7:3) λmax (log ε) 230 nm; 13C NMR and 1H NMR, see Table 3.13; positive-ion ESITOFHRMS [M + Na]+ m/z 437.2666 (calculated for C26H38O4Na, 437.2668) Ansellone N (3.21): white powder (0.3 mg); UV (MeCN−H2O 1:1) λmax (log ε) 230 nm; 13C NMR and 1H NMR, see Table 3.14; positive-ion ESITOFHRMS [M + Na]+ m/z 481.2559 (calculated for C27H38O6Na, 481.2566). 185 Anvilone A (3.22): colorless powder (2.0 mg); [α]D25 +3.2 (c 0.16 CD2Cl2); UV (MeOH) λmax (log ε) 236.6 nm (4.66); 13C NMR and 1H NMR, see Table 3.15; positive-ion ESITOFHRMS [M + Na]+ m/z 451.2823 (calculated for C27H40O4Na, 451.2824). Anvilone B (3.23): white powder (0.8 mg); [α]D25 −7.6 (c 0.01 CH2Cl2); UV (MeCN−H2O 1:1) λmax (log ε) 232 nm; 13C NMR and 1H NMR, see Table 3.16; positive-ion ESITOFHRMS [M + Na]+ m/z 509.2885 (calculated for C29H42O6Na, 509.2879). Phorone C (3.24): white powder (0.9 mg); UV (MeCN−H2O 1:1) λmax (log ε) 254 nm, 307 nm; 13C NMR and 1H NMR, see Table 3.17; positive-ion ESITOFHRMS [M + Na]+ m/z 425.2692 (calculated for C27H37O4, 425.2692). 3.11.3 Bioassays J-Lat cells were treated with sesterterpenoids and/or control LRAs for 24 hours, and GFP protein, a marker of proviral gene expression, was measured by flow cytometry. In parallel, we also assessed cell viability by measuring the percentage of cells displaying normal forward- and side-scatter parameters in LRA-treated cells compared to cultures treated with 0.1% DMSO vehicle control. J-Lat cells were treated with selected sesterterpenoids at 1−30 µM for 24 hours in the presence or absence of control LRAs with different known mechanisms of action including TNFα (pro-inflammatory cytokine), panobinostat (HDAC inhibitor), or prostratin (3.5, PKC activator) Cells were treated with sesterterpenoids or control LRAs for 24 hours with or without the pan-PKC inhibitors GÖ-6983 and RO-31-8220, these two reagents were purchased from Sigma. 186 Chapter 4: Anti-onchocerciasis properties of African plant extracts 4.1 Onchocerciasis Onchocerciasis is also known as river blindness, which is transmitted through tropical blackflies breeding near rivers. This filarial disease is caused by a parasitic nematode called Onchocerca volvulus, and infected individuals will suffer from a variety of clinical symptoms. The life cycle of river blindness starts from an infected black fly depositing larvae into the wound while biting human skin, and then the larvae develop into worms inside human skin tissue. The adult male and female worms (macrofilariae) mate to produce immature worms (microfilariae), and these microfilariae migrate through human skin tissue, which causes symptoms including severe itching, skin diseases, and blindness. Onchocerciasis is a leading cause of morbidity and socioeconomic loss for the world’s poorest populations,118,119 and it was estimated that up to172 million people require treatment for this disease.120 The recommended treatment for onchocerciasis is a broad-spectrum anti-parasitic medicine ivermectin (4.1), which immobilizes the microfilariae of Onchocerca volvulus. However, most adult worms are not killed by ivermectin (4.1) and will reproduce microfilariae.121 Also, ivermectin (4.1) was used in the treatment of other parasitic diseases such as Loa loa filaria, and the co-infected patients do not respond to ivermectin (4.1).122,123 An alternative treatment for river blindness is doxycycline (4.2), an antibiotic medicine that kills the endosymbiotic bacteria Wolbachia, which will lead to sterilization of the adult female filariae Onchocerca volvulus. Doxycycline (4.2) has been considered the most effective macrofilaricidal medicine for the treatment of onchocerciasis, but it has been found to be 187 incompatible with treatment in community settings because of slow drug action on adult worm populations and inadequate cost-benefit ratios.124 Therefore, a short duration macrofilaricide is needed as a substitute for doxycycline (4.2). The only drug in development for the treatment of onchocerciasis is moxidectin (4.3), a drug closely related to ivermectin (4.1) but one that showed greater activity.125 Its binding sites and mechanism of action are the same as ivermectin (4.1), so its ability to overcome resistance to ivermectin (4.1) remains unknown. There is still an urgent need for a macrofilaricidal medicine as an alternative to current drugs. Figure 4.1 Current drugs used for the treatment of onchocerciasis As part of a program designed to find natural products that could serve as potential treatments for onchocerciasis, our collaborator Dr. Fidelis Cho-Ngwa from the University of Buea, Cameroon has screened a library of crude extracts from traditional medicinal plants used O O OOOOOHOOOOOOHHHOIvermectin4.1OH O OH ONH2OOHNHOHOHHDoxycycline4.2OOOOOOHHHONOMoxidectin4.3 188 in African communities. Their biological activities in the screening assay have motivated us to identify the active components from the plant extracts. 4.2 Filaricidal properties of the extracts of Lantana camara 4.2.1 Lantana camara as a traditional medicinal plant The plant Lantana camara is a tropical flowering shrub in the family of Verbenaceae. Lantana camara has been found in around 50 countries,126 and it has been used in some communities as a folk medicine to treat a variety of diseases. Lantana camara is used as a remedy against rheumatism in Guatemala, Mexico and Puerto Rico.127 In Sri Lanka, the leaves of Lantana camara are used externally for joint swelling.128 Bangladesh communities use the root and flowers of Lantana camara for the treatment of cough, fever, small pox, tuberculosis and malaria.129 Some people from Southern Indian communities add the juice of Lantana camara leaves into food to improve digestion for children,130 and the leaves are boiled for tea.131 The leaves of this plant have been used in the treatment in some African communities where people are infected with parasites. The preliminary bioassay results showed that the crude hexane and CH2Cl2 extracts of Lantana camara leaves were active against adult male and female worms. Prompted by the bioassay results, we carried out a search for the active components in the crude extracts of Lantana camara leaves. 4.2.2 Lantadene A (4.4) Bioassay-guided fractionation of the crude extracts of Lantana camara leaves using Sephadex™ LH-20 chromatography and silica gel chromatographies gave crystals of an active pure compound C+D-D2. The isolation process is shown in Figure 4.2. The detailed experiments are described in Section 4.4. 189 Figure 4.2 Bioassay-guided fractionation of the crude extracts of Lantana camara C+D-D2, identified as lantadene A (4.4, Figure 4.3), was isolated as colorless needle-shaped crystals from assay guided fractionation of the extracts of Lantana camara. It gave an [M + Na]+ ion peak in the ESITOFHRMS spectrum at m/z 575.3708 (calculated for C35H52O5Na, 575.3712), which required ten sites of unsaturation. The NMR spectra of lantadene A (4.4) were recorded in CDCl3, and the preliminary analysis of the NMR data showed that it was a terpenoid. The NMR data recorded for lantadene A (4.4) along with resonance assignments are listed in Table 4.1. 190 Figure 4.3 Structure of lantadene A (4.4) Table 4.1 NMR data for lantadene A (4.4) recorded in CDCl3 at 600 MHz Lantadene A (4.4, CDCl3) C# δC δH (J in Hz) 1 39.4 1.39 (dm) * 2 34.3 2.53 (m) 2’ 2.35 (dm, 15.6) 3 217.9 — 4 47.6 — 5 55.5 1.28 * 6 19.7 1.46 * 7 32.4 1.27 * 8 27.8 — 9 47.1 1.65 (dd, 6.5, 11.0) 10 37.0 — 11 23.7 1.99 (dd, 18.9, 11.5) 11’ 1.90 * 12 122.7 5.35 (d, 3.4) 13 143.3 — 14 42.2 — 15 39.4 1.87 * 16 24.4 1.79 * 17 50.8 — 18 38.6 3.02 (br d, 13.9) 19 46.1 1.26 * 19’ 1.69 (dd, 13.9, 5.04) 20 30.2 — 21 37.9 1.74 * 21’ 1.48 * 22 76.0 5.06 (d, 3.1) 23 26.7 1.07 (br s) 24 21.7 1.02 (br s) 25 15.3 1.03 (br s) 26 17.1 0.80 (br s) 27 26.0 1.15 (br s) 28 179.7 — 29 33.9 0.87 (br s) 30 26.3 0.98 (br s) 31 166.4 — OCOOHOOLantadene A4.4HHH 191 Lantadene A (4.4, CDCl3) C# δC δH (J in Hz) 32 127.8 — 33 139.3 5.97 (m) 34 15.9 1.95 (dd, 7.0, 1.4) 35 20.8 1.73 (dd, 6.8, 1.3) * Overlapped resonances Seven unsaturated carbons were observed in the 13C NMR spectrum including one ketone carbonyl resonance at δ 217.9 (C-3), one acid carbonyl resonance at δ 179.7 (C-28), one ester carbonyl resonance at δ 166.4 (C-31) and two olefins (δ 122.7 assigned to C-12, 143.3 assigned to C-13, 127.8 assigned to C-32 and 139.3 assigned to C-33), which accounted for five sites of unsaturation. Therefore, this unknown compound had to be penta-cyclic. A gem-dimethyl substructure was identified from HMBC correlations observed between a methyl proton resonance at δ 1.07 (Me-23) and a quaternary carbon resonance at δ 47.6 (C-4), a carbonyl carbon resonance at δ 217.9 (C-3), a methine carbon resonance at δ 55.5 (C-5) and another methyl carbon resonance at δ 21.7 (Me-24), and HMBC correlations observed between the methyl proton at δ 1.02 (Me-24) and carbon resonances assigned to C-3, C-4, C-5 and Me-23. The above-mentioned HMBC correlations also indicated that this pair of geminal methyls (Me-23 and Me-24) were connected to the quaternary carbon C-4, and C-4 was attached to C-3 and C-5. HMBC correlations observed between the carbonyl resonance assigned to C-3 and the methylene resonances at δ 2.53/2.35 (H-2/H-2’) and 1.39 (H-1), and COSY correlations between H-1 and H-2, indicated a spin system (C-1 and C-2) vicinal to the carbonyl (C-3). HMBC correlations observed between a methyl proton resonance at δ 1.03 (Me-25) and a quaternary carbon resonance at δ 37.0 (C-10) and the methylene carbon at δ 39.3 (C-1), and an HMBC correlation observed between the methine proton resonance at δ 1.28 (H-5) and the quaternary 192 carbon resonance at δ 37.0 (C-10), completed the structure of the trimethylcyclohexenone ring (Figure 4.4, Fragment A). Another pair of geminal methyls could be identified from HMBC correlations observed between a methyl proton resonance at δ 0.87 (Me-29) and another methyl carbon resonance at δ 26.3 (Me-30), a quaternary carbon resonance at δ 30.2 (C-20) and two methylene carbon resonances at δ 46.1 (C-19) and 37.9 (C-21), and HMBC correlations observed between the methyl proton resonance at δ 0.98 (Me-30) and carbon resonances assigned to C-19, C-20, C-21 and Me-29. The above-mentioned HMBC correlations also indicated that this pair of geminal methyls (Me-29 and Me-30) was connected to the quaternary carbon C-20, and C-20 was attached to C-19 and C-21. A COSY correlation observed between a methine proton resonance at δ 3.02 (H-18) and the methylene proton resonance at δ 1.69 (H-19) indicated that C-18 was attached to C-19. A COSY correlation observed between a methine proton resonance at δ 5.06 (H-22) and the methylene proton resonances at δ 1.74/1.48 (H-21/H-21’) indicated that C-22 was attached to C-21. HMBC correlations observed between a quaternary carbon resonance at δ 50.8 (C-17) and proton resonances assigned to H-18, H-19’, H-21 and H-22 completed the assignment of a dimethylcyclohexane ring (Figure 4.4, Fragment B). In the HMBC spectrum, an olefinic proton resonance at δ 5.97 (H-33) correlated with an ester carbonyl at δ 166.4 (C-31) and two methyl carbon resonances at δ 15.9 (Me-34) and 20.8 (Me-35). HMBC correlations observed between Me-35 and C-31, C-32 and C-33, as well as HMBC correlations observed between Me-34 and C-32 and C-33, revealed the fragment with a dimethyl substituted α,β-unsaturated carbonyl (Figure 4.4, Fragment C). An HMBC correlation observed between the methine proton resonance at δ 5.06 (H-22) and the ester carbonyl at δ 166.4 (C-31) connected fragment B and fragment C together (Figure 4.4, Fragment B+C). 193 Figure 4.4 Fragments of lantadene A (4.4), selected COSY and HMBC correlations shown A literature search was carried out with above fragments, and the result showed that this unknown compound was lantadene A (4.4).132 A full NMR assignment of the constitutional structure of lantadene A (4.4) is presented in Figure 4.5. An X-ray diffraction analysis confirmed the constitution and the absolute configuration of lantadene A (4.4) as shown in the ORTEP diagram in Figure 4.12. O217.92.53/2.35 34.31.3939.337.047.61.0221.7 1.0726.71.2855.51.0315.33.0238.61.69/1.26 46.10.8733.9 0.9826.31.74/1.48 37.95.0676.050.830.213 51719 212325 29 3010242224 1820OO1.7320.81.9515.95.97139.3166.4127.83133353432Fragment A Fragment BFragment C Fragment B+C3.0238.61.69/1.26 46.10.8733.9 0.9826.31.74/1.48 37.95.0676.050.830.21719 2129 30221820OO1.7320.81.9515.95.97139.3166.4127.831 33353432COSYHMBC 194 Figure 4.5 NMR assignment for landadene A (4.4), data collected in CDCl3 at 600 MHz Figure 4.6 1H NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 OOOOHO217.92.53/2.35 34.31.3939.337.047.61.0221.7 1.0726.71.2855.51.0315.31.4619.71.2732.41.6547.11.99/1.90 21.75.35122.70.8017.127.842.2143.31.1526.01.8739.41.7924.43.0238.61.69/1.26 46.10.8733.90.9826.31.74/1.48 37.95.0676.01.7320.81.9515.95.97139.350.8179.7166.4127.830.213 5 7911 13151719 2123252729313335343026102481422283224 612 181620 195 Figure 4.7 13C NMR spectrum of lantadene A (4.4) recorded at 150 MHz in CDCl3 Figure 4.8 APT NMR spectrum of lantadene A (4.4) recorded at 100 MHz in CDCl3 196 Figure 4.9 COSY NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 Figure 4.10 HSQC NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 H-1/H-2H-18/H-18'/H-19H-21/H-22 197 Figure 4.11 HMBC NMR spectrum of lantadene A (4.4) recorded at 600 MHz in CDCl3 Figure 4.12 ORTEP diagram generated by the X-ray diffraction analysis of lantadene A (4.4) 198 4.2.3 Biological activities of lantadene A (4.4) Lantadene A (4.4) has been previously isolated and shown to have other biological activities. For example, lantadene A (4.4) displayed weak to moderate in vitro cytotoxic activities against four cancer cell lines including KB, HCT-116, MCF7 and L1210.133,134 Another study showed that lantadene A (4.4) inhibited the TNF-α-induced NF-κB activation in A549 lung adenocarcinoma cell line with an IC50 of 0.79 ± 0.01 µg/mL.135 Lantadene A (4.4) also showed moderate nematicidal activities against Meloidogyne incognita, a nematode causing the root knot disease for plants.136 Leishmanicidal activities have also been reported for lantadene A (4.4), with an IC50 of 20.4 ± 0.1 µg/mL in an in vitro anti-leishmanial assay.137 The mammalian toxicity of lantadene A (4.4) has also been studied. A guinea pig model study showed that lantadene A (4.4) produced a dose-dependent toxicity and the dose level of 25 mg/kg body weight was the sub-acute toxic dose which induced significant fibrosis and necrosis in the liver of guinea pigs.138 The filaricidal properties of the pure lantadene A (4.4) isolated from Lantana camara were investigated through an in vitro anti-Onchocerca assay developed by Dr. Cho-Ngwa. As a result, lantadene A (4.4) was both active against male and female adult worms at IC50’s of 2.47 and 6.02 µg/mL, respectively. Besides, lantadene A (4.4) also showed moderate activities against both Loa loa and Onchocerca ochengi microfilariae, i.e. completely killing the parasites at 50 µg/mL. 4.3 Filaricidal properties of the extracts of Morinda lucida 4.3.1 Morinda lucida as a traditional medicinal plant The plant Morinda lucida, which is in the family of Rubiaceae, is also called the brimstone tree. It occurs in tropical areas all over the world and it has been used in traditional 199 treatments for human diseases. Morinda lucida extracts have been used as a supplementary medicine to treat diabetes in some Nigerian communities, and a rat model evaluating the anti-diabetic properties of Morinda lucida leaves showed that the extracts of this plant had a strong glucose lowering effect on streptozotocin-treated rats.139 Other applications include the treatment of infections and yellow fever. It has also been reported that the treatment of malaria in West and Central Africa also involved the use of Morinda lucida leaves. An in vitro assay revealed that the crude extracts of Morinda lucida leaves showed anti-malarial activities against the pathogen P. falciparum with an IC50 of 25 µg/mL.140 Other research showed anti-trypanosomal activities of Morinda lucida leaves with an MIC of 6.25 µg/mL.141 The leaves of this plant were used in the treatment of people infected with the parasite Onchocerca volvulus in some Cameroonian communities. Preliminary bioassay results in our collaborator’s lab showed that the crude hexane and CH2Cl2 extracts of Morinda lucida leaves were active against male macrofilariae. Prompted by the bioassay results, we carried out a search for the active components in the crude extracts of Morinda lucida leaves. 4.3.2 Morindicinone (4.5) Bioassay-guided fractionation of the crude extracts of Morinda lucida gave active compounds. The isolation process, which involved subjecting the CH2Cl2 extracts of Morinda lucida leaves to Sephadex™ LH-20 chromatography and reversed-phase HPLC separations, is shown in Figure 4.13. The detailed experiments are described in Section 4.4. 200 Figure 4.13 Bioassay-guided fractionation of the crude extracts of Morinda lucida Morindicinone (peak 42) (4.5, Figure 4.14) was isolated as a pink solution (dissolved in water/acetonitrile) from the crude extracts of Lantana camara. It gave an [M + Na]+ ion peak in the ESITOFHRMS spectrum at m/z 351.0844 (calculated for C18H16O6Na, 351.0845), which required eight sites of unsaturation. The NMR spectra of morindicinone (4.5) were recorded in CDCl3, and the preliminary analysis of the NMR data showed that it was an anthraquinone. The NMR data recorded for morindicinone (4.5) is listed in Table 4.2. 201 Figure 4.14 Morindicinone (4.5) Table 4.2 NMR data recorded for morindicinone (4.5) recorded in CDCl3 at 600 MHz Morindicinone (4.5, CDCl3) C# δC δH (J in Hz) 1 145.9 — 2 154.9 — 3 120.8 7.33 (d, 8.6) 4 123.7 8.08 (d, 7.9) * 5 125.8 8.06 (d, 8.7) * 6 133.7 7.83 (d, 7.9) 7 135.8 — 8 158.0 — 9 181.5 — 10 182.6 — 11 125.1 — 12 140.3 — 13 124.9 — 14 128.8 — 15 62.3 4.00 (s) 16 69.1 4.63 (s) 17 59.0 3.48 (s) 18 62.1 3.92 (s) OH — 6.57 (s) * Overlapped resonances The NMR assignment for morindicinone (4.5) is shown in Figure 4.15, along with important HMBC and COSY correlations. The NMR data collected matched with the literature NMR data for morindicinone (4.5).142 OOOHOOOMorindicinone4.5 202 Figure 4.15 NMR data for morindicinone (4.5) collected in CDCl3 at 600 MHz with selected COSY and HMBC correlations Figure 4.16 1H NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 OOOHOOO1234 5678910111213141516 1718145.9154.97.33120.88.08123.78.06125.87.83133.7135.8158.0181.5182.6124.9125.1128.8140.34.0062.33.9262.14.6369.13.4859.0OOOHOOO1234 5678910111213141516 1718COSYHMBC6.57 203 Figure 4.17 COSY NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 Figure 4.18 HSQC NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 H-3/H-4H-5/H-6 204 Figure 4.19 HMBC NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 Figure 4.20 tROESY NMR spectrum of morindicinone (4.5) recorded at 600 MHz in CDCl3 Me-18/C-8Me-15/C-1H-4/C-13H-5/C-12H-5/C-9H-4/C-10H-6/C-8H-6/C-7H-3/C-4H-3/C-1H-16/C-17H-16/C-6H-16/C-12H-16/C-8Me-17/C-16 205 4.3.3 Biological activity of morindicinone (4.5) Morindicinone (4.5) was isolated from the stem of Morinda citrifolia and identified by the Siddiqui group in 2006.142 There is no biological activity reported for this compound so far. The filaricidal properties of the pure morindicinone (4.5) isolated from Morinda lucida leaves were investigated through an in vitro anti-Onchocerca assay developed by Dr. Cho-Ngwa. As a result, morindicinone (4.5) was active to male macrofilariae at 10 µg/mL, but inactive to female macrofilariae. Besides, the CH2Cl2 extracts of Morinda lucida leaves were screened for the acute toxicity in vivo in mice model at a limit dose of 2000 mg/kg body weight. No death was reported after the 14-day study period. The average weights of the mouse increased from 22.22g pre-treatment to 30.37 post-treatment. No abnormality was observed in the physical appearance of the mice during the study. 4.4 Experimental section 4.4.1 General experimental procedures All solvents used in the experiments were HPLC grade from Fisher Scientific Inc. without having further treatment. Milli-Q H2O was used for reversed-phase Sep-pak and HPLC. Sephadex™ LH-20 purchased from Sigma was used for gel filtration chromatography. SiliaFlash® G60 silica gel was used for flash chromatography. Merck Type 5554 Si gel plates and Whatman MKC18F plates were used for analytical thin-layer chromatography. HPLC was performed on a Waters 2998 system with a Photodiode Array detector. An InertSustain C18 semi-preparative column (5 µm 10 × 250 mm) was used for reversed-phase HPLC; an Alltech Apollo silica semi-preparative column (5 µm 10 × 250 mm) was used for normal-phase HPLC. 1H and 13C NMR spectra were recorded on a Bruker 600 MHz spectrometer with a 5 mm cryoprobe. 1H chemical shifts are referenced to residual CDCl3 (δ 7.24 ppm), and 13C chemical 206 shifts are referenced to the CDCl3 (δ 77.23 ppm) solvent peak. All NMR solvents were purchased from Cambridge Isotope Laboratories Inc. Low- and high-resolution MS were recorded using ESI ionization and a TOF mass analyzer. UV spectra were recorded using a Waters 2998 UV spectrophotometer. 4.4.2 Sample preparation, extraction and isolation Our collaborators from Dr. Fidelis Cho-Ngwa lab at the University of Buea, Cameroon, provided the plant materials. They were collected in the west regions of Cameroon. The leaves, barks and roots were air dried and grinded, then extracted with hexane, followed by methylene chloride and methanol. The solvents were evaporated and the crude extracts were stored at −20 °C before shipping. 4.4.2.1 Lantana camara extracts The CH2Cl2 extracts of Lantana camara leaves were chromatographed on a Sephadex™ LH-20 chromatography using 4:1 MeOH/CH2Cl2 as eluent to give fractions A-E. All these fractions were sent for biological assays and the active fractions C and D were combined and further fractionated on a silica gel column with a gradient from 100% hexanes to 100% EtOAc to give sub-fractions A-E. Theses sub-fractions were sent for bioassays and the active sub-fraction D was recrystallized in MeOH/CH2Cl2 solvent system to obtain needle-shaped crystals. The NMR data of the crystals was collected in CDCl3. Lantadene A (4.4): colorless crystal (4.0 mg); 1H NMR and 13C NMR data (600 MHz, CDCl3) in Table 4.1; ESITOFHRMS [M + Na]+ m/z 575.3708 (calculated for C35H52O5Na, 575.3712). The X-ray diffraction analysis was obtained and the diagram is shown in Figure 4.12. 207 4.4.2.2 Morinda lucida extracts The CH2Cl2 extracts of Morinda lucida leaves were chromatographed on a Sephadex™ LH-20 column using 4:1 MeOH/CH2Cl2 as eluent to give fractions A-I. All these fractions were sent for biological assays and the only active fraction G was further fractionated on a small scale Sephadex™ LH-20 column chromatography with 1:1 MeOH/EtOAc to give sub-fractions A-D. These sub-fractions were sent for bioassays, and the active sub-fraction C was subjected to reversed-phase HPLC (gradient MeCN/H2O). All the peaks collected were tested for their filaricidal properties. Their NMR data was collected in CDCl3. The reversed-phase HPLC fractions collected at 82 min and 106 min were active and further purification was conducted on normal-phase HPLC (gradient EtOAc/hexane). Morindicinone (4.5): yellow powder (1.1 mg); UV (MeCN−H2O 4:6) λmax (log ε) 270 nm; 1H NMR and 13C NMR data (600 MHz, CDCl3) in Table 4.2; ESITOFHRMS [M + Na]+ m/z 351.0844 (calculated for C18H16O6Na, 351.0845). 4.4.3 Bioassays Bioassys were conducted by our collaborator Adela Ngwewondo from Dr. Fidelis Cho-Ngwa lab at the University of Buea, Cameroon. 208 Chapter 5: Conclusion The work presented in this thesis was conducted in an effort to discover novel natural products with potential therapeutic properties, or to search for new biological applications of known natural products from marine invertebrates and plants. These organisms came from the HTP screening for biological activities done by our collaborators, and we conducted the isolation and structure elucidation of secondary metabolites from the crude extracts in order to identify the biologically active natural products. This is the first step in drug discovery that may lead to the development of a pharmaceutical. Several novel natural products were isolated and their structures were elucidated. These molecules not only serve as potential lead compounds, but also expand the known chemical library. The work presented in Chapter 3 was in an effort to search for new small-molecule drug leads that present HIV-1 proviral expression activating properties. As a result, novel sesterterpenoids alotaketals C−E (3.7−3.9), phorbadione (3.10), ansellones D−N (3.11−3.21), anvilones A and B (3.22 and 3.23) and phorone C (3.24), along with the known sesterterpenoids ansellones A−C (2.86−2.88), were isolated from the Phorbas sponge specimen collected in Howe Sound, British Columbia. We elucidated their structures as shown in Figure 5.1, and their biological activities were investigated. These compounds are a family of highly functionalized sesterterpenoids that have the alotane, ansellane, anvilane and phorane carbon skeletons. The ansellane, anvilane and phorane carbon skeletons have so far only been found in the metabolites isolated from the sponge Phorbas sp. 209 Figure 5.1 Sesterterpenoids isolated from the marine sponge Phorbas sp. collected in BC OOOAcHOHHAcOOOOHOHHAcOOHOOOHOHHAcOAlotaketal E3.9Alotaketal D3.8Alotaketal C3.7OHOMeHH HHOAcOOOAcO H HHHOHHOOAcOOOHH HHOOAcOOH HHHOHOOAcOAcHOH OAcOHOHHHHHOOAcOOHHHHHAnsellone B2.87OOAcOOHHHHHAnsellone C2.88OHOHHH HHOAcOAnsellone A2.86Ansellone D3.11Ansellone E3.12 Ansellone F3.13Ansellone G3.14OOAcOOH HHPhorbadione3.10Ansellone L3.19OHOHHH HHOHOOOAcO H HHHOHOOAcO H HHHOHOHHHAnsellone H3.15Ansellone I3.16OOHOAcAcOHHHAnsellone K3.18Anvilone A3.22Anvilone B3.23OOHH HHOAcOOHAnsellone J3.17OHOMeHH HHOHOAnsellone M3.20OOHOAcPhorone C3.24OHOHHHHOAcOHOAnsellone N3.21 210 Among all the isolated sesterterpenoids, alotaketal C (3.9) showed the most potent activation of the HIV-1 provirus expression (13.8 ± 2.2% activation at 30 µM), which is two-fold over the known LRA prostratin (3.5). The preliminary SAR revealed that the side chain and enone functionality are essential for the activity, and this conclusion was reached from the comparison of the structures and activities of alotaketals C−E (3.7−3.9). We also conducted some other biological studies, which showed that alotaketal C (3.9) likely acted by the same mechanism as prostratin (3.5, PKC activator). Alotaketal C (3.7), alotaketal D (3.8), ansellone A (2.86) and anvilone A (3.22) are the first sesterterpenoids known to be able to activate latent HIV-1 provirus expression, and they are a new set of scaffolds for this activity. Alotaketal C (3.7), ansellone A (2.86) and anvilone A (3.22) serve as new lead structures in the development of new cell biology tools and potential drug candidates. Two total syntheses of alotaketal A (2.72)115,116 and the total synthesis of ansellones A (2.86) and B (2.87)117 have been reported, which provide access to analogs of these scaffolds. More SAR-driven studies can be done in the future. In Chapter 4, the work presented was in an effort to find anti-onchocerciasis natural products from the extracts of traditional medicinal plants. As a result, lantadene A (4.4) and morindicinone (4.5) were isolated from the extracts of Lantata camara and Morinda lucida, respectively. The filaricidal properties of 4.4 and 4.5 were investigated through an in vitro anti-Onchocerca assay. Lantadene A (4.4) was both active against both male and female adult worms at IC50’s of 2.47 and 6.02 µg/mL, respectively. Besides, lantadene A (4.4) also showed moderate activities against both Loa loa and Onchocerca ochengi microfilariae, i.e. completely killing the parasites at 50 µg/mL. Morindicinone (4.5) was active against male adult worms at 10 µg/mL, but inactive on female adult worms. Although these two natural products are known compounds, 211 their biological activities reported here are new. 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Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Biologically active natural products from terrestrial and marine environments
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