Insights Into the Chemical Composition and Ecology Of

Insights into the Chemical Composition and Ecology of Nudibranchs of the Genus Goniobranchus Louise Catherine Forster Bachelor of Science, Honours in Chemistry Class I

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 School of Chemistry and Molecular Biosciences

Abstract The aim of this PhD was to isolate and elucidate the natural products of nudibranchs from the genus Goniobranchus and to explore the biological role of the compounds discovered. A total of fifty- six compounds, twenty-three of which were new, have been isolated from six Goniobranchus species. Structure elucidation was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). X-ray crystallographic analysis and chemical correlation studies were carried out on selected metabolites to aid in the determination of the relative and absolute configurations. Computational studies involving conformational modelling, density functional theory (DFT) calculations and DP4 probability predictions were also used as complementary tools to aid in determining the preferred diastereomers of selected metabolites. Selected purified compounds were screened for cytotoxicity, deterrency against Palaemon shrimp, antiviral activity against Dengue fever, Ross River fever and influenza viral strains, and lastly, antimicrobial activity against the Gram- positive bacteria S. aureus. The metabolite composition of the six Goniobranchus species was studied. From the nudibranch G. collingwoodi a new spongian diterpene 7α-acetoxyisoagatholactone (2.1) was isolated along with four spongian diterpenes (1.19, 1.24, 1.32, and 1.33) and a linear furanoditerpene (1.16). The investigation on the nudibranch G. aureopurpureus yielded five new highly substituted spongian diterpenes (2.2-2.6) and a new dendrillolide A analogue (4.4), along with eleven known rearranged spongian diterpenes (1.16, 1.24, 1.32, 1.144, 1.145, 1.154, 1.155, 1.158, 1.161, 1.164, and 1.165). The new spongian diterpenes showed varying levels of oxidation, in particular at positions C- 6, C-7, C-13 and C-20. Metabolites 2.5 and 2.6 were crystallized and their X-ray structures aided in establishing the relative and absolute configurations. Goniobranchus sp. 1 was found to contain spongian diterpenes, six of which were new (2.7- 2.12) and eight were known (1.19, 1.24, 1.32, and 1.38-1.43). The new monosubstituted (2.9), disubstituted (2.7, 2.8 and 2.10) and trisubstituted (2.11 and 2.12) spongian diterpenes demonstrated a high level of oxidation at positions C-11, C-12, C-13 and C-20. The new metabolites isolated were only found in mantle tissue. The chemical composition of G. leopardus was found to consist of both spongian diterpenes and their rearranged congeners. Two new metabolites, the isospongian diterpene 12α- acetoxypolyrhaphin D (2.13) and the spiroepoxide (3.4) were isolated together with eleven known metabolites (1.130-1.132, 1.142, 1.144, 1.145, 1.160, 1.163-1.165, and 1.172). From the mantle and viscera of G. coi, eight new rearranged spongian diterpenes possessing a perhydroazulene motif (3.1-3.5, 4.1 and 4.2) and a perhydronapthalene analogue (3.6) with a fused cyclopropyl group, were isolated along with thirteen known metabolites (1.130, 1.132, 1.142, 1.143- 1.146, 1.148, 1.149, 1.162, 1.163, 1.165, and 1.171). Epoxide 3.1 and ketone 3.2 showed broadened i signals in the 1H and 13C NMR, suggesting conformational averaging. As a result, the dynamic nature of the perhydroazulene motif and 2,8-dioxabicyclo[3.3.0]octane moiety was explored through variable temperature NMR experiments and computational modelling. The relative configuration of the fused epoxide 3.1 was confirmed by X-ray crystallographic analysis. The relative configuration of the spiroepoxide 3.4 was explored through chemical correlation experiments, where aldehyde 3.5 was identified as a ring opened artefact of the C-10 epimer of 3.4. Lactols 4.1 and 4.2 were isolated as an inseparable mixture of diastereomers. Molecular modelling and computational studies aided in correctly assigning their relative configurations. Chemical correlation experiments further confirmed the structural assignments of 4.1 and 4.2. The final species G. geometricus was found to contain two new metabolites with a dendrillane carbon skeleton (4.3 and 4.4), as well as sixteen known oxygenated rearranged diterpenes (1.130- 1.132, 1.142, 1.143-1.145, 1.152, 1.154, 1.158, 1.160, 1.162-1.164, 1.166, and 1.171). In secoshahamin (1.164), the configurational assignments of C-13 and C-14 had not been rigorously defined in an earlier isolation study. Chemical correlation experiments and computational studies were therefore undertaken and conclusively assigned the relative and absolute configuration of 1.164. All specimens, excluding the G. geometricus specimens, were dissected prior to extraction. The anatomical distribution of the metabolites isolated from the mantle and viscera tissue were studied. Purified samples of dendrillolide A (1.142), macfarlandin E (1.143) and aplyviolene (1.144) were screened for deterrency against Palaemon shrimp.

Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis.

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Publications included in this thesis Forster, L. C.; White, A. M.; Cheney, K. L.; Garson, M. J. Oxygenated Diterpenes from the Indo- Pacific Nudibranchs Goniobranchus splendidus and Goniobranchus collingwoodi. Nat. Prod. Commun. 2018, 13, 299-302.

Forster, L. C.; Pierens, G. K.; Garson, M. J. Elucidation of Relative and Absolute Configurations of Highly Rearranged Diterpenoids and Evidence for a Putative Biosynthetic Intermediate from the Australian Nudibranch Goniobranchus geometricus. Journal of Natural Products 2019, 82, 449-455. DOI: 10.1021/acs.jnatprod.8b00713.

Forster, L. C.; Pierens, G. K.; Clegg J. K.; Garson, M. J. Dynamic NMR and Computational Studies Inform the Conformational Description of Dendrillane Terpenes from the Nudibranch Goniobranchus coi. Journal of Natural Products 2020, in press. DOI: 10.1021/acs.jnatprod.9b01051.

Submitted manuscripts included in this thesis No manuscripts submitted for publication at the time of thesis submission.

Other publications during candidature Peer-reviewed papers Forster, L. C.; Winters, A. E.; Cheney., K. L.; Dewapriya, P.; Capon, R. J.; Garson, M. J. Spongian- 16-one Diterpenes and Their Anatomical Distribution in the Australian Nudibranch Goniobranchus collingwoodi. J. Nat. Prod. 2017, 80, 670-675. DOI: 10.1021/acs.jnatprod.6b00936

Forster, L. C.; Pierens, G. K.; White, A. M.; Cheney, K. L.; Dewapriya, P.; Capon, R. J.; Garson, M. J. Cytotoxic Spiroepoxide Lactone and Its Putative Biosynthetic Precursor from Goniobranchus Splendidus. ACS Omega 2017, 2, 2672-2677. DOI: 10.1021/acsomega.7b00641

Daygon, V. D.; Calingacion, M.; Forster, L. C.; De Voss, J. J.; Schwartz, B. D.; Ovenden, B.; Alonso, D. E.; McCouch, S. R.; Garson, M. J.; Fitzgerald, M. A. Metabolomics and genomics combine to unravel the pathway for the presence of fragrance in rice. Sci. Rep. 2017, 7, 8767. DOI: 10.1038/s41598-017-07693-9

Winters, A. E.; White, A. M.; Dewi, A. S.; Mudianta, I. W.; Wilson, N. G.; Forster, L. C.; Garson, M. J.; Cheney, K. L. Distribution of Defensive Metabolites in Nudibranch Molluscs. J. Chem. Ecol. 2018, 44, 384-396.

Conference abstracts Forster, L. C.; Pierens, G. K.; White, A. M.; Winters, A. E.; Cheney, K. L.; Garson, M. J. Chromodorididae molluscs: the isolation, characterization and stereochemical elucidation of natural products. 15th International Symposium on Marine Natural Products, Fortaleza, Brazil, 29th August – 2nd September 2016 (abstract, poster presentation and short 5 min oral presentation).

Forster, L. C.; Pierens, G. K.; White, A. M.; Cheney, K. L.; Dewapriya, P.; Capon, R. J.; Garson, M. J. Extensively rearranged cytotoxic norditerpenes from the Australian nudibranch Goniobranchus splendidus. RACI Centenary Congress, Melbourne, Australia, 23rd-28th July 2017 (abstract and poster presentation).

Forster, L. C.; Pierens, G. K.; White, A. M.; Cheney, K. L.; Dewapriya, P.; Capon, R. J.; Garson, M. J. Cytotoxic Spiroepoxide Lactone and Its Putative Biosynthetic Precursor from Goniobranchus v

Splendidus. 10th European Conference on Marine Natural Products, Kolymbari, Crete, Greece, 3rd-7th September 2017 (abstract and poster presentation).

Forster, L. C.; Cheney, K. L.; Garson, M. J. Looking into the mirror: a detailed investigation into nudibranch mimicry. 13th Annual SCMB Research Student Symposium, University of Queensland, Brisbane, Australia, 23rd November 2017 (abstract and poster presentation).

Forster, L. C.; Cheney, K. L.; Garson, M. J. Looking into the mirror: a detailed investigation into nudibranch mimicry. 2nd Queensland Annual Chemistry Symposium, Queensland University of Technology, Brisbane, Australia, 27th November 2017 (abstract and 15 min oral presentation).

Forster, L. C.; Pierens, G. K.; White, A. M.; Cheney, K. L.; Dewapriya, P.; Capon, R. J.; Garson, M. J. Molecular modelling in conjunction with high field NMR secures the structure of an extensively rearranged cytotoxic norditerpene from the Australian nudibranch Goniobranchus splendidus. 11th Australia and New Zealand Society for Magnetic Resonance Conference, Kingscliff, New South Wales, Australia, 2nd-6th December 2017 (abstract, poster presentation and short 5 min oral presentation).

Forster, L. C.; Pierens, G. K.; Cheney, K. L.; Garson, M. J. Inconspicuous carbon NMR signals and fluxional effects complicate the structural elucidation of norditerpenes from Goniobranchus coi. 16th International Symposium on Marine Natural Products and 11th European Conference on Marine Natural Products, Peniche, Portugal, 1st-5th September 2019 (abstract and 15 min oral presentation).

Forster, L. C.; Pierens, G. K.; Clegg, J. K.; Cheney, K. L.; Garson, M. J. Dynamic NMR studies and conformational analyses inform stereochemical analysis of dendrillane terpenes from the nudibranch Gonibranchus coi. 4th Queensland Annual Chemistry Symposium, University of Queensland, Brisbane, Australia, 29th November 2019 (abstract and 15 min oral presentation).

Contributions by others to the thesis Chapter 1 None.

Chapter 2 Dr Karen Cheney research group (School of Biological Sciences, UQ) collected and identified the nudibranchs. Associate Professor Jack Clegg and PhD student Kasun Sankalpa Athukorala Arachchige (School of Chemistry and Molecular Biosciences, UQ) performed the X-ray crystallographic analysis. Dr Gregory Pierens (Center of Advance Imaging, UQ) provided training and assistance in conducting NMR experiments on the 700 MHz NMR machine.

Chapter 3 The nudibranch collection was carried out by Dr A. Roberts-Thomson (QLD Salty Pets Pty. Ltd) and Gary Cobb (Permit #183990QLD General Fisheries). Associate Professor Jack Clegg and PhD student Kasun Sankalpa Athukorala Arachchige (School of Chemistry and Molecular Biosciences, UQ) performed the X-ray crystallographic analysis. Dr Tri Le (School of Chemistry and Molecular Biosciences, UQ) supervised the author during variable temperature experiments on the 500 MHz NMR machine. Dr Gregory Pierens (Center of Advance Imaging, UQ) provided training and assistance in conducting NMR experiments on the 700 MHz NMR machine as well as, carried out molecular modelling and DFT calculations.

Chapter 4 Dr Karen Cheney research group (School of Biological Sciences, UQ) collected and identified the nudibranchs. The chemical extraction of Goniobranchus geometricus was performed by Honors student Holly H. Urquhart (School of Biological Sciences, UQ). Dr Gregory Pierens (Center of Advance Imaging, UQ) provided training and assistance in conducting NMR experiments on the 700 MHz NMR machine as well as, carried out molecular modelling and DFT calculations.

Chapter 5 Dr Karen Cheney supervised the author during Palaemon shrimp palatability assays. Dr Pradeep Dewapriya from Professor Robert Capons research group (Institute of Molecular Biosciences, UQ) conducted the cytotoxicity assays. Dr Daniel Watterson and Dr Christopher McMillan (School of Chemistry and Molecular Biosciences, UQ) conducted the antiviral assays. PhD candidate Jessie Adams from Professor Bill Bakers research group (University of South Florida) carried out the antimicrobial assays.

Chapter 6 None.

Chapter 7 None.

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Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects No animals requiring ethics approval or human subjects were involved in this research. The animals used were all invertebrates: Nudibranchs (mollusks) and Palaemon serenus (rockpool shrimp).

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Acknowledgements The completion of this PhD degree could not have been achieved without the scientific assistance and moral support of many people. This has been a truly life-changing experience.

First and foremost, I would like to express my sincere gratitude and appreciation to my supervisor Professor Mary Garson, for her support, continual patience, and immense knowledge. It was a great honor to work under her guidance. Mary is an inspiring mentor, not only as a brilliant natural product chemist but as a strong, independent women. I am proud to be one of her last chemistry students. Her passion and dedication to science inspires me to do great things. I would also like to thank my first co-supervisor Dr Karen Cheney, for her moral support throughout this whole process and her valuable insights to marine ecology during both field work and laboratory experiments. You are a great dive buddy too! Finally, thank you to my second co-supervisor Professor James De Voss, for his insights into synthetic derivatisation and biosynthesis. His entertaining comments about ‘surviving’ the Northern Territory made me a little less homesick. I am very grateful to Dr Gregory Pierens for the assistance with the 700 MHz NMR experiments, molecular modelling and computational calculations. His dedication to helping me obtain high quality spectra and the invaluable time spent modelling is really appreciated. I would like to acknowledge Dr Tri Le for NMR training and assistance with variable temperature NMR experiments; Dr Amanda Nouwens and Peter Josh for mass spectrometry; Dr Jack Clegg and Kasun Sankalpa Athukorala Arachchige for the X-ray crystallographic studies; Prof Robert Capon and Dr Krisahntha Pradeep for cytotoxicity screening; Dr Daniel Watterson and Dr Christopher McMillan for antiviral screening; Jessie Adams and Professor Bill Baker for antimicrobial screening; Prof James De Voss for access to the GC/MS instruments; Dr Aston Roberts-Thomson, Gary Cobb, Cedric van den Berg, Dr Anne Winters and Dr Karen Cheney for collection of nudibranch specimens; and Debra Aston for nudibranch pictures. I would like to thank the past and present members of the Garson group for their friendship and continual help during my candidature. In particular, Andrew for his chemistry discussions over daily coffee catch ups and Weili for her spontaneous singing, laughter and moral support during our parallel PhD candidatures. My appreciation also extends to my laboratory colleagues that are a part of the Level 10 family, for their friendship, banter and for letting me ‘borrow’ small quantities of reagents for my tiny scale reactions. In particular, thank you to Bella, I am grateful to have had such a witty, supportive mate to undertake my candidature with. I would like to say a special thank you to Professor Joanne Blanchfield for her continual moral support and confidence in me. ix

A huge thank you to Nano Café and all the staff, for being my daily source of delicious coffee throughout my candidature. I would also like to express my gratitude to Michelle and her herd of horses, working with them is the best therapy. The journey of my PhD was made possible by the unconditional love, support, and encouragement from my family. I express my wholehearted gratitude to my parents for always encouraging me to push my boundaries, they are my golden standard for tenacity. My siblings Norman, Jenna, Ben and Sara, I love you all so dearly, thank you for being so understanding throughout these past four years, always encouraging me to go out of my comfort zone and to follow my passion. A special thank you to my Aunty Lynne and Uncle Robert who inspired me to follow the academic pathway, their advice and love mean the world to me. To Kate, my best mate, who was there through the worst and the best moments; thank you for your friendship.

To anyone that I have been remiss in mentioning, I appreciate you too, however through the fog of thesis writing I have failed to mention you.

Where there is a will there is a way! ~Awillaway~

Financial support This research was supported by an Australian Government Research Training Program (RTP) Scholarship. This research was further supported by the Australian Pacific Science Foundation (APSF) and the University of Queensland Graduate School. The UQ School of Chemistry and Molecular Biosciences provided funding for conference travel.

Keywords Nudibranch, goniobranchus, diterpenes, molecular modelling, nmr, marine natural product, stereochemistry.

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 030502, Natural Products Chemistry, 100%

Fields of Research (FoR) Classification FoR code: 0305, Organic Chemistry, 100%

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Table of Contents

Chapter 1: Introduction ...... 1 1.1 Natural products from an evolutionary perspective ...... 2 1.2 Marine invertebrates – sponges and nudibranchs...... 2 1.3 Biosynthesis and bioactivity of diterpenes in the marine environment...... 3 1.3.1 Spongian diterpenes ...... 5 1.3.2 Rearranged spongian diterpenes ...... 10 1.4 Computational studies complementing natural product elucidation ...... 26 1.5 Aims of the thesis ...... 32

Chapter 2: The chemistry of spongian diterpenes ...... 35 2.0 Introduction ...... 36 2.0.1 Nudibranchs and their sponge prey ...... 36 2.0.2 Reported spongian metabolites from Goniobranchus species ...... 37 2.1 Results and discussion ...... 37 2.1.1 The isolation of metabolites from Goniobranchus collingwoodi (Rudman, 1987) ...... 37 2.1.1.1 Characterization of spongian-16-one (1.24) ...... 38 2.1.1.1 Characterization of 7α-acetoxyisoagatholactone (2.1) ...... 39 2.1.2 The isolation of metabolites from Goniobranchus aureopurpureus (Collingwood, 1881) ...... 40 2.1.2.1 Structure elucidation of new oxygenated spongian diterpenes from Goniobranchus aureopurpureus ...... 42 2.1.3 The isolation of metabolites from Goniobranchus sp 1...... 51 2.1.3.1 Structure elucidation of new spongian diterpenes from Goniobranchus sp 1...... 52 2.1.4 The isolation of metabolites from Goniobranchus leopardus (Rudman, 1987) ...... 59 2.1.4.1 Structure elucidation of 12α-acetoxypolyrhaphin D (2.13) ...... 60 2.2 Conclusions ...... 63

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes ...... 66 3.0 Introduction ...... 67 3.0.1 Reported metabolites from Goniobranchus species ...... 67 3.1 Results and discussion ...... 68 3.1.1 The isolation of metabolites from Goniobranchus coi (Risbec, 1956) ...... 68 3.1.1.1 Introduction to conformational averaging in metabolites from G. coi ...... 70 3.1.1.1a Structure elucidation of a known metabolite dendrillolide A (1.142) ...... 71 3.1.1.1b Structure elucidation of 5,9-epoxydendrillolide A (3.1) ...... 71 3.1.1.1c Conformational averaging observed for 5,9-epoxydendrillolide A (3.1) ...... 75 3.1.1.1e Molecular modelling of dendrillolide A (1.142) ...... 78 3.1.1.1f Structure elucidation of 10-oxonordendrillolide A (3.2) ...... 79 3.1.1.1g Mass spectrometry experiments on 10-oxonordendrillolide A (3.2) ...... 79 3.1.1.1h Molecular modelling 10-oxonordendrillolide A (3.2) ...... 80 3.1.1.2 Structure elucidation of 5-hydroxydendrillolide A (3.3) ...... 81 3.1.1.3 Structure elucidation of 10,20-epoxydendrillolide A (3.4) ...... 83 3.1.1.3a Molecular modelling of 10,20-epoxydendrillolide A (3.4) ...... 84 3.1.1.3b Structure elucidation of aldehyde rearrangement product (3.5) ...... 87

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3.1.1.3c Chemical correlation experiments to probe the configuration at C-10 of 3.4 and 3.5 ...... 88 3.1.1.4 Characterization of a cyclopropyl functionalized metabolite (3.6) from G. coi ...... 91 3.1.1.4a Structure elucidation of cyclopropyl metabolite from G. coi ...... 92 3.2 Conclusions ...... 96

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes ...... 98 4.0 Introduction ...... 99 4.0.2 Reported metabolites from G. geometricus ...... 99 4.1 Results and discussion ...... 100 4.1.1 Structure elucidation of diastereomeric metabolites (4.1 and 4.2) from G. coi ...... 100 4.1.1.1 Molecular modelling of lactol diastereomers (4.1 and 4.2) ...... 104 4.1.1.2 Chemical correlation study on the lactol diastereomers (4.1 and 4.2) ...... 107 4.1.2 The isolation of metabolites from Goniobranchus geometricus (Risbec, 1928) ...... 107 4.1.2.1 Structure elucidation of secoshahamin (1.164) ...... 109 4.1.2.2 Molecular modelling for secoshahamin (1.164) ...... 112 4.1.2.3 Chemical correlation study of secoshahamin (1.164) ...... 113 4.1.2.4 Structure elucidation of shahamin L (4.3)...... 116 4.1.2.5 Molecular modelling of truncated structure to inform elucidation of shahamin L (4.3) ...... 117 4.1.2.6 Characterization of 15-desacetoxy-12-acetoxydendrillolide A (4.4)...... 118 4.1.2.7 Biosynthesis of metabolites from G. geometricus ...... 119 4.2 Conclusions ...... 120

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites122 5.0 Introduction ...... 123 5.1 Anatomical distribution of metabolites within Goniobranchus nudibranchs ...... 123 5.1.1 Anatomical distribution of metabolites in G. collingwoodi ...... 124 5.1.2 Anatomical distribution of metabolites in G. aureopurpureus ...... 125 5.1.3 Anatomical distribution of metabolites in Goniobranchus sp 1...... 125 5.1.4 Anatomical distribution of metabolites in G. leopardus ...... 126 5.1.5 Anatomical distribution of metabolites in G. coi ...... 127 5.2 Ecological bioactivity screening ...... 128 5.2.1 Palatability assays ...... 129 5.3 Biological screening ...... 134 5.3.1 Cytotoxicity of spongian diterpenes ...... 134 5.3.2 Antiviral screening ...... 136 5.3.3 Antimicrobial screening ...... 141 5.4 Conclusions ...... 144

Chapter 6: General conclusions ...... 146 6.1 General conclusions ...... 147 6.2 Future work ...... 149 6.3 Summary of isolated compounds ...... 151

Chapter 7: Experimental ...... 161 7.1 General experimental procedures ...... 161 xiv

7.1.1 Solvents...... 161 7.1.2 Thin layer chromatography ...... 161 7.1.3 Normal phase flash chromatography ...... 161 7.1.4 High performance liquid chromatography ...... 161 7.1.5 Mass spectrometry ...... 162 7.1.6 Nuclear magnetic resonance spectroscopy ...... 162 7.1.7 Gas chromatography/mass spectrometry ...... 162 7.1.8 Specific rotation ...... 163 7.1.9 X-ray crystallography ...... 163 7.1.10 Minimum biofilm eradication concentration (MBEC) determination assays ...... 163 7.1.11 Antiviral screening methods ...... 164 7.1.12 Palatability assays with shrimp (Palaemon serenus) methods ...... 164 7.1.13 Cytotoxicity screening methods ...... 165 7.2 Sample collection ...... 166 7.3 Isolation of spongian diterpenes from Goniobranchus collingwoodi ...... 167 7.3.1 Isolation of diterpenes from Goniobranchus collingwoodi ...... 167 7.4 Isolation of spongian diterpenes from Goniobranchus aureopurpureus ...... 167 7.4.1 Isolation of diterpenes from Goniobranchus aureopurpureus ...... 167 7.4.2 Crystallographic data for (–)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2- methyl)-butanoate (2.5) ...... 169 7.4.3 Crystallographic data for (–)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3- methyl)-butanoate (2.6) ...... 169 7.5 Isolation of oxygenated terpenes from Goniobranchus sp 1...... 169 7.5.1 Isolation of diterpenes from Goniobranchus sp 1...... 169 7.6 Isolation of spongian diterpenes from Goniobranchus leopardus ...... 171 7.6.1 Isolation of diterpenes from Goniobranchus leopardus ...... 171 7.7 Isolation of oxygenated terpenes from Goniobranchus coi ...... 172 7.7.1 Isolation of diterpenes from Goniobranchus coi ...... 172 7.7.2 Crystallographic data for 5, 9-epoxydendrillolide A (3.1) ...... 173 7.7.3 Procedure for the acetylation of lactol diastereomers (4.1 and 4.2) ...... 174 7.7.4 Procedure for epoxidation of dendrillolide A (1.142) ...... 174 7.8 Isolation of oxygenated diterpenes from Goniobranchus geometricus...... 175 7.8.1 Isolation of oxygenated diterpenes from Goniobranchus geometricus ...... 175 7.8.2 Procedure for the saponification and lactonization of rearranged diterpenes ...... 176 7.8.3 Procedure for the acetylation of 12-deoxyshahamin E (4.6) ...... 177

References ...... 178

Appendices ...... 191

List of Figures

Figure 1.1 Rearranged spongian metabolites with a perhydroindane motif. Image adapted from Keyzers et al. 2006.42 ...... 17 Figure 1.2 Rearranged spongian metabolites with a perhydroazulene motif. Image adapted from Keyzers et al. 2006.42 ...... 20 Figure 1.3 Clarification of structure and name for (A) aplyviolene (1.144), (B) macfarlandin E (1.143) and (C) dendrillolide A (1.142)...... 21 Figure 1.4 Clarification of structure and name for selected diterpenes. (A) Confirmed structure and absolute configuration of cheloviolene A (1.148); (B) revised structure of cheloviolene B (1.149); and (C) incorrect configuration originally assigned to cheloviolene B...... 22 Figure 1.5 Computational approach to calculate electron density around atomic nuclei and spectroscopic properties of candidate structures. Diagram reproduced with permission of Dr Gregory Pierens...... 27 Figure 2.1 The nudibranchs Goniobranchus collingwoodi (A), Goniobranchus aureopurpureus (B), Goniobranchus sp. 1 (C), and Goniobranchus leopardus (D)...... 36 Figure 2.2 Metabolites (1.42-1.47) previously isolated from G. collingwoodi...... 37 Figure 2.3 Isolation scheme of G. collingwoodi compounds...... 38 Figure 2.4 Spongian-16-one (1.24) with trans ring junctions highlighted in pink and the cis ring junction in blue...... 39 Figure 2.5 Isolation scheme of G. aureopurpureus compounds...... 41 Figure 2.6 Known compounds isolated from G. aureopurpureus...... 42 Figure 2.7 Molecular modelling for 2.2 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 43 Figure 2.8 Molecular modelling for 2.3 showing the proposed relative configuration (A) and with hydrogen atoms omitted from the model for clarity (B)...... 46 Figure 2.9 Molecular modelling for 2.4 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 47 Figure 2.10 Molecular modelling for 2.5 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 48 Figure 2.11 ORTEP184 representation of the crystal structure of (5S, 7R, 8R, 9R, 10R, 13S, 14R, 2’S)- 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate 2.5 shown with 30% probability ellipsoids...... 49 Figure 2.12 Molecular modelling for 2.6 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 50 Figure 2.13 ORTEP184 representation of the crystal structure of 13-acetoxy-20-hydroxy-7α- oxyspongian-16-one-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate 2.6 shown with 30% probability ellipsoids. Only one component of each region of disorder is shown...... 51 Figure 2.14 Isolation scheme of Goniobranchus sp. 1 compounds...... 52 Figure 2.15 Molecular modelling for 2.7 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 53 Figure 2.16 Molecular modelling for 2.8 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 56 Figure 2.17 Molecular modelling for 2.9 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 56 Figure 2.18 Molecular modelling for 2.10 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 57 Figure 2.19 Molecular modelling for 2.11 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 58 Figure 2.20 Molecular modelling for 2.12 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 59 Figure 2.21 Isolation scheme of G. leopardus compounds...... 60 xvi

Figure 2.22 Molecular modelling for 2.13 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B)...... 61 Figure 3.1 Isolation scheme of G. coi compounds...... 68 Figure 3.2 Known oxygenated terpenes isolated from Goniobranchus coi...... 69 Figure 3.3 New oxygenated terpenes isolated from Goniobranchus coi...... 69 Figure 3.4 Selected 2D NMR correlations for 5,9-epoxydendrillolide A (3.1)...... 72 Figure 3.5 ORTEP184 representation of the crystal structure of epoxide 3.1 shown with 50% probability ellipsoids. Only one of the two chemically identical but crystallographically unique molecules in the asymmetric unit is shown...... 74 Figure 3.6 Overlay of the 2,8-dioxabicyclo[3.3.0]octane ring system in the X-ray crystal structure of 3.1 (purple) and macfarlandin C (1.166) (yellow).111 ...... 75 Figure 3.7 Stacked of 1H NMR spectra for 3.1 in the temperature range 298 K to 193 K (500 MHz, d6-acetone)...... 76 Figure 3.8 Overlay of energy-minimized stereostructures of epoxide conformers 3.1a (green), 3.1b (blue) and 3.1c (pink), with hydrogen atoms omitted for clarity...... 77 Figure 3.9 Overlay of energy-minimized stereostructures of dendrillolide A conformers 1.142a (green) and 1.142b (blue), with hydrogen atoms omitted for clarity...... 78 Figure 3.10 Selected 2D NMR correlations for 10-oxonordendrillolide A (3.2)...... 79 Figure 3.11 Energy-minimized stereostructure of the major conformer 3.2a, with hydrogen atoms omitted for clarity...... 81 Figure 3.12 Molecular modelling for 5-hydroxydendrillolide A (3.3). (A) showing key NOESY correlations and (B) with hydrogen atoms omitted from the model for clarity...... 82 Figure 3.13 Selected 2D NMR correlations for 10,20-epoxydendrillolide A (3.4)...... 84 Figure 3.14 Candidate stereostructures (3.4a-3.4b) proposed for 10,20-epoxydendrillolide A (3.4)...... 85 Figure 3.15 (A) Lowest energy conformer of stereoisomer 3.4a of (10R)-spiroepoxide (3.4) and (B) truncated and rotated image of 3.4a showing the conformation of the cycloheptane ring; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety...... 86 Figure 3.16 (A) Lowest energy conformer of stereoisomer 3.4b of (10S)-spiroepoxide (3.7) and (B) truncated and rotated image of 3.4b showing the conformation of the cycloheptane ring; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety...... 86 Figure 3.17 Truncated structures of candidate diastereomers 3.4a and 3.4b for spiroepoxide 3.4, showing distances between key atoms for each stereoisomer; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety...... 91 Figure 3.18 Examples of oxygenated rearranged spongian diterpenes with cyclopropyl functionality.76, 85, 100, 113, 117, 120 ...... 92 Figure 4.1 The nudibranchs Goniobranchus geometricus (A) and Goniobranchus coi (B)...... 99 Figure 4.2 Comparison of selected structures for rearranged metabolites possessing a 2,8- dioxabicyclo[3.3.0]octane moiety, with the three established configurations indicated in blue, green and red...... 101 Figure 4.3 Candidate structures (4.1a, 4.1b, 4.2a and 4.2b) proposed for lactols 4.2 and 4.1...... 104 Figure 4.4 (A) Energy-minimized stereostructure of the major conformer of 4.1a, (B) with hydrogen atoms omitted for clarity...... 104 Figure 4.5 (A) Energy-minimized stereostructure of the major conformer of 4.2a, (B) with hydrogen atoms omitted for clarity...... 105 Figure 4.6 Overlay of energy-minimized stereostructures of lactol conformers 4.1a-1 (green) and 4.1a-2 (pink), with hydrogen atoms omitted for clarity...... 105 Figure 4.7 Overlay of energy-minimized stereostructures of lactol conformers 4.2a-1 (green), 4.2a-2 (pink) and 4.2a-3 (blue), with hydrogen atoms omitted for clarity...... 106 Figure 4.8 Isolation scheme of G. geometricus compounds...... 108 Figure 4.9 Known oxygenated terpenes isolated from G. geometricus...... 109

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Figure 4.10 Relative configurational assignment for the C-13/C-14 segment for the possible diastereomers 4.5a and 4.5b: (Top) three possible staggered conformers for 4.5a; (Bottom) three possible staggered conformers for 4.5b...... 111 Figure 4.11 Candidate diastereomers 4.5a and 4.5b for the truncated structure of secoshahamin (1.164), with carbon numbers matching those of 1.164...... 112 Figure 4.12 Candidate diastereomers 4.7a and 4.7b for the truncated structure of shahamin L (4.3), with carbon numbers matching those of (4.3)...... 117 Figure 4.13 Substitution patterning on the dendrillane scaffold at C-12 and C-15...... 118 Figure 5.1 Schematic depiction of pellet recipes for feeding deterrency assay (Images provided by Dr Karen Cheney)...... 131 Figure 5.2 Palaemon shrimp demonstrating (A) a red spot indicating a positive uptake of the pellet; (B) the absence of a red spot indicating rejection of the pellet (Images provided by Dr Karen Cheney)...... 131 Figure 5.3 Comparison of unpalatability of dendrillolide A (1.142), macfarlandin E (1.143) and aplyviolene (1.144) against Palaemon serenus at 10 and 20 mg/mL...... 132 Figure 5.4 The [3.3.0]- and [3.2.1]-diaxoabicyclooctane ring systems of dendrillolide A, aplyviolene and macfarlandin E ring open to a common dialdehyde intermediate...... 133 Figure 5.5 Cytotoxicity screening for spongian diterpenes 1.42-1.47. (a) NCIH-460 (human lung carcinoma cell line), (b) SW620 (human colorectal carcinoma), (c) HepG2 (hepatocellular carcinoma) (Data output provided by Dr Pradeep Dewapriya of Professor Robert Capon’s group, IMB)...... 136 Figure 5.6 Structures of metabolites selected for antiviral screening...... 138 Figure 5.7 Cytotoxicity screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142- 1.145, and 5.12-5.14 (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ)...... 139 Figure 5.8 Antiviral screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142-1.145, and 5.12-5.14 against Ross River virus strain (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ)...... 140 Figure 5.9 Antiviral screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142-1.145, and 5.12-5.14 against Dengue virus strain (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ)...... 140 Figure 5.10 Structures of metabolites selected for antimicrobial screening...... 143 Figure 5.11 Structure of 1.105, 1.106, 1.110 and 1.116 comparing the structural similarities. In blue the trimethylcyclohexane moiety and in pink is the 2,8-dioxabicyclo[3.3.0]octane moiety...... 144 Figure 5.12 Minimum biofilm eradication percent measured for mollusk compounds against multi- drug resistant S. aureus at 50 µg mL-1. Note data not compared with drug controls and represented as percent eradication. Error = ± SEM. MBEC90/50: level to which 90% or 50% of the population was eradicated compared to no treatment (Data output provided by Jessie Adams of Professor Bill Bakers research group, USF)...... 144 Figure 6.1 Representative structural motifs for oxygenated rearranged diterpenes and norditerpenes...... 147

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List of Tables

Table 2.1 1H NMR assignments for spongian-16-one analogues 1.24, 1.16 and 2.1a,b ...... 40 Table 2.2 1H NMR assignments for new spongian-16-one analogues 2.2-2.6a ...... 44 Table 2.3 13C NMR assignments for new spongian-16-one analogues 2.2 – 2.6a ...... 45 Table 2.4 1H NMR assignments for new spongian-16-one analogues 2.7 - 2.12a ...... 54 Table 2.5 13C NMR assignments for new spongian-16-one analogues 2.7 – 2.12a ...... 55 Table 2.6 1H NMR assignments for 12α-acetoxypolyrhaphin D (2.13)a,b ...... 62 Table 3.1 1H and 13C NMR assignments of 3.1, 3.2 and 1.142 a ...... 73 Table 3.2 1H and 13C NMR assignments of 5-hydroxydendrillolide A (3.3) a,b,c ...... 82 Table 3.3 1H and 13C NMR assignments of 10,20-epoxydendrillolide A (3.4) and 10S aldehyde derivative (3.5)a,b ...... 84 Table 3.4 1H and 13C NMR assignments of epoxidation products 10S-spiroepoxide (3.7) and 10R- aldehyde (3.8) a,b...... 90 Table 3.5 1H and 13C NMR assignments of cyclopropyl derivative (3.6) and omriolide B (1.176). . 94 Table 4.1 1H and 13C NMR Assignments of 15-dendrillactol (4.1), 15-epidendrillactol (4.2), cheloviolene A (1.148) and cheloviolene B (1.149).a,b,c ...... 102 Table 4.2 Comparison of selected 1H NMR data for rearranged metabolites possessing a 2,8- dioxabicyclo[3.3.0]octane moiety. a,b ...... 103 Table 4.3 Comparison of selected 13C NMR data for rearranged metabolites possessing a 2,8- dioxabicyclo[3.3.0]octane moiety.a,b ...... 103 Table 4.4 Comparison of calculated (black) and experimental (blue) key coupling constants for the 2,8-dioxabicyclo[3.3.0]octane moiety of the major component (4.1) ...... 106 Table 4.5 Comparison of calculated (black) and experimental (blue) key coupling constants for the 2,8-dioxabicyclo[3.3.0]octane moiety of the minor component (4.2) a ...... 107 Table 4.6 Comparison of 1H and 13C NMR assignments for secoshahamin (1.164), 12- desacetoxyshahamin C (1.162) and 12-desacetoxypolyrhaphin A (1.163), saponification product (4.6), shahamin K (1.161) and shahamin E (1.159).a,b ...... 115 Table 4.7 1H and 13C NMR assignments for shahamin L (4.3).a,b ...... 116 Table 4.8 1H and 13C NMR assignments for 15-desacetoxy-12-acetoxydendrillolide A (4.4)a,b .... 119 Table 5.1 Distribution of diterpenes in G. collingwoodi organs...... 124 Table 5.2 Distribution of diterpenes in G. aureopurpureus organs...... 125 Table 5.3 Distribution of diterpenes in Gonibranchus sp. 1 organs...... 126 Table 5.4 Distribution of diterpenes in G. leopardus organs...... 126 Table 5.5 Distribution of diterpenes in G. coi organs...... 127 Table 6.1 Summary of isolated compounds from Goniobranchus species investigated in this thesis...... 151

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List of Schemes

Scheme 1.1 Proposed biosynthesis of the spongian diterpenes scaffold.1 ...... 5 Scheme 1.2 Proposed biosynthesis of the gracilin, spongionellin, cadlinolide, aplysulphurin and pourewic acid classes, as well as the metabolites darwinolide, gonioline and epoxygoniolide-1.73, 86- 89 Diagram adapted from Keyzers et al. 2006 and from Forster et al. 2017.42, 86 ...... 13 Scheme 1.3 Proposed biosynthesis of the glaciolane-type scaffold.95 ...... 16 Scheme 1.4 Chemical correlation of chelonaplysin C (1.131) and norrisolide (1.130).99 ...... 18 Scheme 1.5 Proposed biosynthesis of the norrisolide class of compounds and linked chromodorane scaffold pathway.103 ...... 19 Scheme 1.6 Proposed biosynthesis of the dendrillane-scaffold class of compounds.110 ...... 21 Scheme 1.7 Proposed biosynthesis of the shahamin-type scaffold.118 ...... 24 Scheme 2.1 Putative biosynthesis for isospongian 12α-acetoxypolyrhaphin D (2.13)...... 62 Scheme 2.2 Putative metabolic transformations in nudibranchs of the genera G. collingwoodi, G. aureopurpureus and Goniobranchus sp. 1...... 64 Scheme 3.1 Proposed solvolysis of 3.1 to give a dimethoxy adduct...... 73 Scheme 3.2 Proposed solvolysis of 3.2 to give a dimethoxy product...... 80 Scheme 3.3 Mechanism of degradation/isomerization to the aldehyde from a spiroepoxide (Scheme reproduced from Li et al.).211 ...... 88 Scheme 3.4 Oxidation of alloaromadendrene (3.9) and subsequent reduction of the epoxide diastereomers (3.10 and 3.11) to afford the diastereomeric alcohols (3.14 and 3.15) (Scheme reproduced from Bombarda et al.).213 ...... 88 Scheme 3.5 Chemical correlation of spiroepoxides (3.4 and 3.7) and aldehyde products (3.5 and 3.8) by epoxidation of dendrillolide A (1.142)...... 89 Scheme 3.6 Putative biosynthesis of cyclopropyl-containing (nor)diterpenes.42, 76 ...... 95 Scheme 4.1 Chemical correlation of lactol 4.1 and 4.2 mixture with dendrillolide A (1.142) by acetylation...... 107 Scheme 4.2 Chemical correlation of secoshahamin (1.164) with 12-desacetoxyshahamin C (1.162) (of 13R, 14R configuration) and 12-desacetoxypolyrhaphin A (1.163) by saponification and lactonization, each giving the same 13R, 14R δ-lactone product (12-deoxyshahamin E (4.6))...... 113 Scheme 4.3 Putative biosynthetic pathway to the diterpene metabolites of Gonibranchus geometricus. The green, pink and orange dots highlight carbons derived from C-12, C-13 and C-14 of the precursor spongian diterpene framework...... 120

List of Abbreviations

1H NMR Proton Nuclear Magnetic Resonance 13C NMR Carbon Nuclear Magnetic Resonance

[α]D Specific optical rotation 1D One Dimensional 2D Two Dimensional br s broad singlet br d broad doublet calcd. calculated CUF Colony-forming unit d doublet DMSO Dimethyl sulfoxide DCM Dichloromethane dd doublet of doublets ddd doublet of doublets of doublets dt doublet of triplets DENV Dengue Virus DFT Density Functional Theory DIPE Diisopropyl ether DMSO Dimethyl sulfoxide ECD Electronic circular dichroism

ED50 Median effective dose ESIMS Electrospray Ionization Mass Spectrometry EtOAc Ethyl acetate

Et2O Diethyl ether EtOH Ethanol EPS Extracellular polymeric substances FC Fermi contact Flu Influenza gCOSY Gradient Correlation Spectroscopy GC/MS Gas Chromatography/Mass Spectrometry HMBC Heteronuclear Multiple Bond Correlation HSQC Heteronuclear Single Quantum Correlation HPLC High Performance Liquid Chromatography

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HRESIMS High Resolution Electrospray Ionization Mass Spectrometry HeLa Henrietta Lacks HepG2 Hepatocellular carcinoma HSV-1 Herpes simplex virus type 1

LC50 Median lethal concentration

LD50 Lethal Dose at 50 LCMS Liquid Chromatography Mass Spectrometry LRESIMS Low Resolution Electrospray Ionization Mass Spectrometry MAE Mean Absolute Error MCMM Monte Carlo Multiple Minimum MDF Mantle dermal formation Me Methyl MeOH Methanol MeCN Acetonitrile m Multiplet MIC Minimum inhibitory concentration MBEC Minimum biofilm eradication concentration MPA O-methylmandelic acid MRSA methicillin-resistant Staphylococcus aureus MS Mass spectrometry MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor NBO Natural bond orbital NOESY Nuclear Overhauser Enhancement Correlation Spectroscopy NP-HPLC Normal Phase High Performance Chromatography NCIH-460 Human lung carcinoma cell line

OD600 Optical density PBS Phosphate buffered saline ppm parts per million PRNT Plaque reduction neutralization test PUF Plaque-forming units q quartet RRV Ross River Virus SW620 Human colorectal carcinoma SSCC Spin-spin coupling constant

xxii

S singlet t triplet td triplet of doublet TDDFT Time-dependent density-functional theory TLC Thin Layer Chromatography TOCSY Total Correlation Spectroscopy UV Ultraviolet VSV Vesicular stomatitis virus

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Chapter 1: Introduction

Chapter 1: Introduction 1.1 Natural products from an evolutionary perspective Natural chemical diversity is observed in terrestrial and marine environments from a variety of sources: flora, fauna, fungi, and microorganisms. Most organisms, both terrestrial and marine, contain a complex mixture of primary and secondary metabolites (natural products). The latter appear not to be involved in growth, development, or reproduction, but instead provide the host organism with an adaptive advantage, such as anti-predator adaptations. These adaptations, both morphological and chemical, are congruent with Darwin’s theory of natural selection. It has been hypothesized that the secondary metabolic pathways evolved to exploit excess (waste) acetate, shikimate, and amino acids that were surplus to primary metabolic pathways.1-3 However, with the emergence of the field of chemical ecology in the late 1960s, it is now more commonly accepted that secondary metabolites fulfill an important role in the survival of individual organisms and species. These include defensive compounds that protect species from predation, and which have frequently been shown to be associated with conspicuous visual signals, such as the brightly colored patterns of frogs4 and butterflies.5,6 Alteration in the genetic makeup of an organism can lead to consequential changes in the production of proteins and enzymes. Genetic mutations can result from many external factors; exposure to UV radiation, radioactivity, certain chemicals (e.g. carcinogens), or viruses. However, they are most commonly the result of modifications in DNA replication. New enzymes formed as a result of changes to DNA may be detrimental or useful. Beneficial changes confer an evolutionary advantage, such as the production of a chemical arsenal against predation, the ability to consume a previously toxic food source, improve their courtship or hunting abilities. A terrestrial example of this is the survival mechanism employed by the monarch butterfly; it consumes the milkweed plant, which contains highly unpalatable cardenolides, and incorporates the plant toxins into its tissue.6

1.2 Marine invertebrates – sponges and nudibranchs Two marine invertebrates well-known for their interesting chemical profiles and chemical ecology are sponges and nudibranchs.7 Sponges (Porifera) are primitive, multicellular, sessile marine filter feeders.8 Despite being immobile, many sponges are not preyed upon by fish, possibly due to the presence of physical defenses (spicules) and/or a chemical arsenal of secondary metabolites to deter predators. Although sponges possess the enzymatic machinery for the production of these natural products, it has also been postulated that sponges contain bacteria that are the true producers of the secondary metabolites, which are then utilized by the sponge for protection against predators or for maintaining space to grow.9 A fascinating example of this is the macrocyclic alkaloid latrunculin A (1.1), first isolated in 1980 from the Red Sea sponge Latrunculia (= Negombata) magnifica.10 Field observations showed N. magnifica grows without competition for space and is 2

Chapter 1: Introduction avoided by reef fish, despite its defenseless appearance;11 latrunculin A has been shown to be significantly ichthyotoxic.11,12

Nudibranchs are small, slow moving, shell-less, brightly colored molluscs from the order Opisthobranchia (Mollusca: gastropoda).13 The literal translation of nudibranch is ‘naked gills’, describing the exposed respiratory gills on the dorsal posterior end of the body. Their lack of physical defense would imply high vulnerability; however, nudibranchs have evolved special adaptations that allow them to feed on chemically defended organisms such as sponges and sequester the secondary metabolites from their prey to use as their own chemical arsenal.13-19 In many species, their toxicity or unpalatability is advertised though highly conspicuous colorations and patterning (aposematism).5,14 Studies on butterflies, and more recently on nudibranchs, have indicated there is a positive correlation between conspicuousness and toxicity, independent of phylogenetic relatedness.5,20 The chemical profile of a nudibranch is dependent on its dietary preferences; therefore, it is significantly affected by the high diversity in sponge species observed in marine communities, which varies not only as a result of geographical location but also with depth. Studies have demonstrated the effect of geographical variation on nudibranchs, such as the differences in chemical profiles of Doriprismatica (= Glossodoris) atromarginata populations. Specimens collected from Sri Lanka21 and Australia22,23 have been found to contain predominately oxygenated diterpenes, whereas specimens collected from India24 contain sesterterpenes.

1.3 Biosynthesis and bioactivity of diterpenes in the marine environment From an ecological perspective, it is vital to understand the source of secondary metabolites, and thus the biosynthetic pathways utilized by the organisms of origin. A plethora of secondary metabolites have been isolated and elucidated from sponges and nudibranchs, many possessing novel carbon skeletons, functionality and bioactivity. The scaffolds of secondary metabolites can be derived from one of several pathways, in which key structural motifs are characteristic of the biosynthesis. Some common classes of secondary metabolites are: polyketides, alkaloids, steroids and terpenoids.25 Steroids and terpenoids are derived from acetyl-Coenzyme A.1 These natural products are assembled using isoprenoid C5 units derived from isopentenyl (3-methylbut-3-en-1-yl)

Chapter 1: Introduction

1 pyrophosphate (IPP), now named as a diphosphate rather than as a pyrophosphate. The isoprene (C5) units are joined in a head-tail fashion. Terpenes are classified into sub-groups based on the number of C5 isoprene units they contain: monoterpenoid (C10), sesquiterpenoid (C15), diterpenoid (C20), sesterterpenoid (C25), triterpenoid (C30) and carotenoid (C40), while steroids are derived from tetracyclic triterpenoids. The most commonly isolated terpenes from marine sponges and nudibranchs are sesquiterpenoids, diterpenoids and sesterterpenoids. Sesquiterpenes, both oxygenated and nitrogenous, have been shown to possess a wide range of biological activities, including antimalarial, antifungal, cytotoxic as well as anti-fouling activity. Representative examples of the different carbon skeletons of sesquiterpenes include: the cytotoxic sesquiterpene ether arenaran A (1.5),26 nitrogen- containing axisonitrile-2 (1.6) (antimalarial activity),27 axisonitrile-3 (1.7) (antimalarial activity),28 and halichonadin C (1.8);29 as well as the furanosesquiterpenes euryfuran (1.9) (cytotoxic and antibacterial activity),30 pallescensone (1.10)31 and dendrolasin (1.11).32

The sesterterpenes, while not as commonly found in the marine environment as diterpenes and sesquiterpenes, have been shown to be biologically active. Representative sesterterpenes isolated from marine invertebrates include: the cytotoxic scalaranes23,24,33,34 heteronemin (1.12)35 and scalaradial (1.13) (anti-inflammatory activity);36 and the manoalides37 manoalide (1.14) (anti- inflammatory activity)38,39 and deoxysecomanoalide (1.15) (antimicrobial).40

Chapter 1: Introduction Diterpenes and the truncated norditerpenes are quite prolific in marine sponges and nudibranchs. There are several classes that make up marine oxygenated diterpenes; these include both spongian diterpenes and their rearranged derivatives. This group of marine metabolites will be the focus of my PhD and selected compounds from the literature are discussed below.

1.3.1 Spongian diterpenes Spongian diterpenes are bioactive natural products isolated exclusively from sponges which are metabolites usually sequestered by nudibranchs or shell-less mollusks.41 These diterpenoids have previously been isolated from two chemotaxonomic orders Dictyoceratida and Dendroceratida.42 It has been speculated that the following terpenes are the linear precursors to the tetracyclic spongian diterpene skeleton.1 Luffarin-X (1.16) was first reported by Butler and Capon from the marine sponge Luffariella geometrica.43 Ambliofuran (1.17) was isolated from the sponge Dysidea amblia, and its structure solved through spectroscopic analysis.44

A plethora of biosynthetically related metabolites can all be traced back to the same spongian diterpene scaffold (1.18). Biosynthetically, the protonation of geranylgeranyl diphosphate (GGPP) can initiate a concerted cyclization sequence to form a tertiary carbocation (Scheme 1.1). Deprotonation of the cation, hydrolysis of the diphosphate followed by the addition of the resulting alcohol on to the exomethylene leads to the formation of ether ring D.1

Scheme 1.1 Proposed biosynthesis of the spongian diterpenes scaffold.1

The first example of the spongian diterpene scaffold was isoagatholactone (1.19), isolated from the Mediterranean sponge Spongia officinalis by Cimino et al., with the structure and absolute

Chapter 1: Introduction configuration established through chemical correlation with grindelic acid.45 The total synthesis of (+)-isoagatholactone was achieved in 1996 by Zaragozā and co-workers and the synthetic product was found to be identical in all spectral aspects to the natural isolate.46 Derivatives of this scaffold (1.20-1.23) were also isolated from the sponge S. officinalis collected from Tenerife, Canary Islands.47 The initial crude extract of S. officinalis showed antimicrobial activity against the Gram- positive bacteria Staphylococcus aureus and Bacillus sphaericus, as well as the Gram-negative bacteria Pseudomonas aeruginosa.47 Compounds 1.19-1.21 showed cytostatic activity against the HeLa cell line in the range of 1-10 µg mL-1.48

A common feature of the spongian diterpene skeleton is a carbonyl at C-15 and/or C-16. The metabolites spongian-16-one (1.24) and spongian-15,16-dione (1.25) were isolated from the New Zealand sponge Dictyodendrilla cavernosa.49 The structure of 1.24 was determined through exhaustive NMR studies carried out independently by two groups, those of Kernan et al.49 and Hambley et al.50 Spongian-16-one has also been isolated from several nudibranch species, including Chromodoris obsoleta.51 Metabolite 1.24 exhibited moderate anti-neoplastic activity against L1210 -1 -1 51 (IC50 = 5.0 µg mL ) and KB (IC50 = 9.2 µg mL ) cell lines.

Oxidation of the methyl group at C-19 has also been observed. For example, from the Micronesian sponge Spongia matamata, six spongian diterpenes (1.26-1.31) with C-19 oxidized were

Chapter 1: Introduction isolated. In this group of tetracyclic diterpenes, further oxidation is commonly seen at C-7, C-11, C- 12, C-13, C-15, C-17 and C-20, as shown in structures 1.32-1.47. The monosubstituted spongian metabolite 7α-acetoxyspongian-16-one (1.32) was isolated from the sponge Dendrilla rosea,52 with the name later updated to aplyroseol-7.53 Along with 1.32, 7α-hydroxyspongian-16-one (1.33) was isolated from a Japanese nudibranch Chromodoris obsoleta. The structure of 1.33 was confirmed by single-crystal X-ray analysis.51 Metabolites 1.33 and 1.32 -1 exhibited moderate anti-neoplastic activity against L1210 (IC50 = 7.5 and 2.2 µg mL , respectively) -1 51 and KB (IC50 = 10.2 and 16.0 µg mL , respectively) cell lines.

R1 R2 R3 R4 R5 R6 R7 R8 Spongian-16-one 1.24 H H H H H H H H Monosubstituted 1.32 OAc H H H H H H H 1.33 OH H H H H H H H 1.34 H OAc H H H H H H 1.35 H H H H H H OAc H 1.38 H H H OAc H H H H 1.39 H H H H H H H OAc 1.40 H H H H H H H OCOPr Disubstituted 1.36 OAc H H H H OH H H 1.41 H H H OCOPr H H H OCOPr 1.42 H H H OAc H H H OAc 1.43 H H H OAc H H H OCOPr 1.44 H H OAc H H H H OAc 1.45 H H OAc H H H H OCOPr 1.46 iVal OCOPr H H H H H H Trisubstituted 1.37 OAc H H H OH H OAc H 1.47 iVal H OH H H H H OCOPr OH = Hydroxy; OAc = Acetate; OCOPr = Propionate; iVal = Isovalerate The metabolite 11α-acetoxyspongian-16-one (1.34) was isolated from two specimens of the dorid nudibranch Chromodoris reticulata54 and the structure solved by spectroscopic comparison with the epimeric (C-11) sample derived by hydrogenation of the C-12/C-13 bond of an 11β-OAc containing metabolite 1.21 from Spongia officinalis.47 From the dendroceratid sponge Aplysilla rosea collected near Sydney, aplyroseols-14 (1.35), -15 (1.36) and -16 (1.37) were isolated, providing examples of mono-, di- and trisubstituted spongian-16-one scaffolds.55 The sponge Coscinoderma matthewsi and the nudibranch Chromodoris albopunctata, collected within the Gneerings reefs, offshore from Mooloolaba in South East Queensland, yielded the monosubstituted tetracyclic spongian diterpenes 12α-acetoxyspongian-16-one (1.38), 20-acetoxyspongian-16-one (1.39), and 20- oxyspongian-16-one-propionate (1.40), as well as the disubstituted metabolite 12α,20- diacetoxyspongian-16-one-dipropionate (1.41).18 Finally, from an individual nudibranch specimen of 7

Chapter 1: Introduction Goniobranchus collingwoodi, collected from the Great Barrier Reef, five highly oxygenated disubstituted spongian diterpenes (1.42-1.46) and a trisubstituted spongian diterpene (1.47) were isolated.56 These metabolites showed substitution at C-7, C-11, C-12 and/or C-20, with substituents including hydroxy, acetate, propionate and isopentanoate groups. It has been proposed that furanoterpenes could be derived biosynthetically from an α, β- unsaturated ketone precursor, in a process mediated by cytochrome P450 enzymes.57 However, biosynthetically it appears that the ring D of the spongian scaffold (1.18) could alternatively be converted directly into a furan via (P450-mediated) oxidation. Spongian furanoditerpenes often have a high level of substitution on ring A. The first examples of a spongian diterpene scaffold with a furan ring (1.48-1.51) were elucidated in 1979 by Kazlauskas et al., and these metabolites were isolated from an Australian Spongia sp. collected from the Great Barrier Reef.58 Later, an analogue of the furanospongian scaffold, 19-acetoxy-3α-hydroxyspongia-13(16),14-diene-2-one (1.52), was isolated from the Red Sea sponge Spongia arabica.59 A further three related furanospongian diterpenes, 3α,17- dihydroxyspongia-13(16),14-diene-2-one (1.53), 3α-hydroxyspongia-13(16),14-diene-2-one (1.54) and the C-3 epimer (1.55) of compound 1.53, were reported from a sample of Spongia sp. collected from the Exmouth Gulf in Western Australia.60

Also isolated by Kazlauskas et al. were the C-3 epimers (1.56-1.59) of compounds (1.48-1.51).58 Within this series, Cambie et al. isolated 3β-acetoxy,19- hydroxyspongia-13(16),14-diene-2-one (1.60) from the sponge Hyattella intestinalis, collected off the coast of Darwin in Northern Australia.61 Isolated along with 1.60 were two C-3 keto-functionalized furanoditerpenes, 19- hydroxyspongia-13(16),14-diene-2-one (1.61) and 2α,19-dihydroxyspongia-13(16),14-diene-2-one (1.62).61 The dictyoceratid sponge collected from the Exmouth Gulf also yielded two related metabolites, 2α,17-dihydroxyspongia-13(16),14-diene-2-one (1.63) and 2α,19-dihydroxyspongia- 13(16),14-diene-2-one (1.64), the corresponding C-2 epimer with C-3 ketone functionality.60 8

Chapter 1: Introduction

From the sponge Spongia officinalis, collected off Laing Island in Papua New Guinea, three furanoditerpenes, spongia-13(16),14-diene (1.65), spongia-13(16),14-dien-19-oic acid (1.66), and spongia-13(16),14-dien-19-al (1.67) were isolated.62 Furano-spongian diterpenes have been isolated from nudibranchs, with the first examples being from the nudibranch Casella (= Glossodoris) atromarginata, collected from Sri Lanka. Metabolites 1.68 and 1.69 demonstrated further structural variation within this furano-spongian class.63 Another Glossodoris atromarginata specimen, collected from Australia, later yielded two metabolites spongia-13(16),14-dien-3-one (1.70) and 3α- acetoxy-19-hydroxyspongia-13(16),14-dien-2-one (1.71).22,64 Somerville et al. conducted anatomical dissections of G. atromarginata, which revealed the selective compartmentalization of metabolite 1.49 in the mantle glands. The authors proposed enzymatic systems could reduce C-3 selectively, which may be the reason for the difference in stereochemistry at C-3 of the metabolites in the viscera compared with those in the mantle.22,64 Nine furano spongian diterpenes were also isolated (1.72- 1.80) from a Spongia sp. specimen, collected near Mooloolaba in Australia.23 In the same study, metabolite 1.73 was isolated from the nudibranch Dorisprismatica (= Glossodoris) atromarginata.23 Compound 1.64 was later crystallized, and the structure reported along with the crystal structures of 1.48 and 1.56, defining the absolute configurations.65

Chapter 1: Introduction Within this section, the biosynthetic origins of the spongian scaffold have been discussed, including the lactone and furan spongian diterpene groups. While a selection of metabolites have been chosen to represent these groups, these by no means summarize all of the spongian diterpenes reported in literature. Other structural variations exist within the tetracyclic spongian diterpene group, including the aplyroseol scaffold group, which have an additional ring linking C-13 and C-17. The reviews by Fontana et al., Keyzers et al., and Dean et al. provide a comprehensive catalogue up to 2006, 2000, and 2017, respectively, for sponge and nudibranch diterpene metabolites.42,66,67 Furthermore, the annual reviews of the marine natural product series by Blunt et al., or by Carroll et al., offer a thorough update on all the scaffolds isolated each year from the marine environment; the most recent review was published in 2019.68

1.3.2 Rearranged spongian diterpenes The previously discussed spongian diterpenes are proposed to biosynthetically undergo extensive rearrangements, forming new classes of scaffolds. Herein these rearranged spongian scaffold classes are discussed in regard to their unique structural features, total synthesis, biological and ecological activity and their biosynthetic origins. The following metabolites (1.81-1.126) possess a trimethylcyclohexane motif as a common feature. Norditerpenes, as well as bisnorditerpenes, are a unique group of structures found in marine organisms, with the gracilin series (A-Q) representing a significant number of these types of compounds. Gracilin A (1.81), isolated from a Mediterranean dendroceratid sponge Spongionella gracilis, was the first example of a rearranged spongian diterpene with the gracilane scaffold.69 The unusual bisnorditerpene gracilin B (1.82), was also isolated from the sponge S. gracilis by the same group.70 When Mayol et al. later isolated gracilins C (1.83) and D (1.84) it became apparent that the ring A exocyclic alkene of gracilin B had been erroneously assigned with Z-geometry and it was then revised to have an E-configuration.71 The total synthesis of 1.82 and 1.83 was first achieved by Corey and Letavic in 1995.72 Later, gracilins E (1.85) and F (1.86) were also isolated from a sample of S. gracilis, collected from the Bay of Naples.73 The analogue of 1.81, 9,11-dihydrogracilin A (1.87), was isolated from the Antarctic sponge Dendrilla membranosa and was found to inhibit the growth of Bacillus subtilis at 100 µg per disc.74 To establish the relative configuration at C-10, X-ray analysis was conducted on a C-8 ketone derivative formed following ozonolysis of 1.87.75 The metabolites 16-deacetoxy-9,11-dihydrogracilin A (1.88) and 15,16-deacetoxy-15-hydroxy-9,11-dihydrogracilin A (1.89) were separately isolated from two nudibranch specimens of Goniobranchus splendidus.76 In addition to 1.88 and 1.89, White et al. also isolated an unusual cyclopropyl functionalized metabolite, verrielactone (1.90), for which the planar structure and relative configuration were established through detailed spectroscopic analysis complemented by computational experiments. Compound 10

Chapter 1: Introduction 1.90 also has a 2,7-dioxabicyclo[3.2.1]octane moiety, which features in several rearranged spongian diterpenes.76 The relative configuration of C-9 was determined through computational studies, where the H-9α and H-9β stereoisomers of 1.90 were modelled. The calculated chemical shifts were compared with the experimental NMR data and the H-9β conformer was statistically preferred. Computational methods used in natural product structure elucidation will be discussed in detail later in this chapter.

Continuing the gracilin series, gracilins G-I (1.91-1.93), were isolated from the sponge Spongionella pulchella.77 The structure of gracilin H (1.92) was later confirmed by single-crystal X-ray analysis.78 Gracilins J-L (1.94-1.96) were also isolated from a Spongionella sp. and exhibited activity against 78 the K562 leukemia cell line, with IC50 values of 15, 8.5 and 2.7 µM, respectively. The Garson group later reported the structures and relative configuration of six additional metabolites named as gracilins M-Q (1.97-1.101).79 Within this study, the structure of 1.91 was revised, where 1.91 had initially been erroneously assigned as having a biosynthetically disfavoured 1,2,2-trimethylcyclohexyl framework and was revised to a 1,3,3-trimethylcyclohexyl moiety.79 The gracilins isolated from G. splendidus were screened for cytotoxicity against HeLa S3 cells and it was found that the most potent were 1.81 -1 and 1.99-1.101, each with IC50 <0.30 µg mL , while the 6Z isomer of gracilin B (1.82) showed an 11

Chapter 1: Introduction

-1 79 IC50 of 0.32 µg mL . Gracilin L (1.94) and synthetic derivatives of 1.94 have shown potential in treating immunosuppressive and chronic inflammatory diseases, by inhibiting inflammatory mediators and enhancing anti-oxidative mechanisms in activated microglia.80 The gracilins have shown significant potential for therapeutic application in neurodegenerative diseases, especially in Alzheimer’s disease, due to their multi-target activities.81 The neuroprotective and antioxidant abilities of gracilins has been explored for gracilins A, H, J, K and L.82,83 In another study, in vitro assays demonstrated that certain gracilins were able to inhibit β-secretase (BACE1), reduce tau hyperphosphorylation and inhibit extracellular signal-regulated kinases (ERK).84

The norditerpene spongionellin (1.102) was isolated from the Mediterranean sponge Spongionella gracilis, and possesses a 2,8-dioxabicyclo[3.3.0]octane moiety.73 The structurally related metabolite daphnelactone (1.103), was isolated from the nudibranch Goniobranchus daphne, however, even with detailed spectroscopic analysis, the configuration of the C-9 stereogenic centre remained unresolved.79 The metabolite spongionellin-2 (1.104) was isolated by the Garson group from a single specimen of Goniobranchus splendidus.85 The unusual rearranged structure epoxygoniolide-1 (1.105) was also isolated from G. splendidus, and possessed spiroepoxide lactone, enal, and masked dialdehyde functionality.86 Metabolite 1.105 is a rare example of a C-12 functionalized 2,8-dioxabicyclo[3.3.0]octane moiety. The co-isolated metabolite gonioline (1.106) was crucial to proposing a putative biosynthesis. Biosynthetically 1.105 and 1.106 were proposed to be derived from a spongian diterpene such as 1.18, where upon C-17 methyl migration, oxidative cleavage of C-5/C-6 involving the loss of C-6, and cleavage of the C-9/C-11 bond, the spongionellin framework is afforded (Scheme 1.2). Subsequent epoxidation of the C-6/C-7 bond is followed by cyclohexyl ring formation, yielding an allylic alcohol intermediate. Oxidation at C-12, then acetylation and lactonization, provides gonioline (1.106). Formation of 1.105 possibly occurs via a Grob-type mechanism, due to the C-6/C-7 bond and the 12-OAc group of 1.106 being optimally oriented from a stereoelectronic perspective and as a result generating the alkene-functionalized intermediate. Finally, epoxidation and lactone formation provide 1.105. When 1.105 was screened against human lung (NCIH-460), colorectal (SW620) and liver (HepG2) cancer cells, using

Chapter 1: Introduction vinblastine as a positive control, the metabolite showed IC50 values of 10.2, 10.18, and 15.72 µM, respectively.86

Scheme 1.2 Proposed biosynthesis of the gracilin, spongionellin, cadlinolide, aplysulphurin and pourewic acid classes, as well as the metabolites darwinolide, gonioline and epoxygoniolide-1.73,86-89 Diagram adapted from Keyzers et al. 2006 and from Forster et al. 2017.42,86

Chapter 1: Introduction

The cadlinolides and aplysulphurin class possesses a tricyclic ring system attached to the trimethylcyclohexane moiety. Cadlinolides A (1.107) and B (1.108) were the first of this class, isolated from the sponge Aplysilla glacialis, with 1.107 and tetrahydroaplysulphurin-1 (1.109) also isolated from the dorid nudibranch Cadlina luteomarginata.90 Compound 1.108 was isolated as an equilibrium mixture of two C-16 hydroxy epimers, which when treated with acetic anhydride and pyridine converged to form one acetylated product identified as tetrahydroaplysulphurin-1 (1.109). Consequently the comparison of the 1H and 13C NMR chemical shift values for the semi-synthetic product and the literature data for 1.109 revealed the 1H NMR assignments were erroneous and therefore the structure of 1.109 was revised.90 Metabolite 1.109 was previously reported from an Australian Darwinella sponge species,53 and is structurally related to aplysulphurin (1.110), which was isolated from another Australian sponge Aplysilla sulphurea.91 The structure of 1.110 was determined via chemical, spectroscopic and X-ray analysis.53,92 Unfortunately, many authors have mistakenly misspelt aplysulphurin as aplysulfurin. Isolated from the New Zealand sponge Darwinella oxeata was compound 1.109, along with the olefin isomers tetrahydroaplysulphurin-2 (1.111) and tetrahydroaplysulphurin-3 (1.112).53 From the nudibranch Goniobranchus splendidus, three cadlinolide-like metabolites were isolated: tetrahydroaplysulphurin-4 (1.113), splendidalactone-1 (1.114), and splendidalactone-2 (1.115), with the structure of each metabolite determined through spectroscopic methods.76 The structure of 1.115 is proposed to be formed from 1.109, or an equivalent structure, through ring expansion involving the C-20 methyl. The compound darwinolide (1.116) was isolated from the Antarctic sponge Dendrilla membranosa; the unique structure was determined through spectroscopic analysis, and the absolute configuration was established through X-ray analysis.93 The unusual position of the cycloheptane ring was proposed by Baker and co-workers to 14

Chapter 1: Introduction be the result of a divergent biosynthetic pathway compared to the gracilins, membranolides and aplysulphurins (Scheme 1.2). The ring expansion that forms the new seven-membered carbocyclic ring was proposed to occur from a C-8/C-14 bond migration to C-7. Subsequent oxidative cleavage of the C-5/C-6 bond and lactonization provides darwinolide (1.116). Similar to metabolite 1.105, darwinolide (1.116) is another example of the uncommon C-12 functionalized 2,8- dioxabicyclo[3.3.0]octane moiety. Metabolite 1.116 exhibited a 4-fold selectivity against the biofilm phase of methicillin-resistant Staphylococcus aureus compared to the planktonic phase.93

Metabolites cadlinolides C (1.117) and D (1.118) were isolated from a New Zealand marine sponge, Chelonaplysilla violacea. Along with 1.117 and 1.118 a series of carboxylic acid functionalized compounds were isolated: pourewic acid A (1.119), 15-methoxypourewic acid B (1.120), the methyl ester of pourewic acid B (1.121) and pourewanone (1.122).89 Metabolites 1.117, 1.119 and 1.120 showed moderate anti-inflammatory activity.89 The isolation of the pourewic acids with intact carboxylic acid functionality provided further evidence to support the proposed biosynthesis of the cadlinolides and gracilins (Scheme 1.2).

Structurally related to the cadlinolides are the membranolides; two separate collections of the Antarctic sponge Dendrilla membranosa provided membranolide74 (1.123) and membranolides B-D (1.124-1.126).94 Compound 1.123 was shown to inhibit growth of Bacillus subtilis at 100 µg per disc and displayed mild activity against Staphylococcus aureus,74 whereas 1.125 and 1.126 showed modest, broad spectrum antimicrobial activity at 200 µg per disc.94 Biosynthetically, the trimethylcyclohexane motif is formed via a Wagner-Meerwein rearrangement involving the C-17 methyl group, with concomitant oxidative cleavage of the C-5/C- 6 bond. The rearrangement of the gracilane norditerpene compounds has been proposed to occur by

Chapter 1: Introduction either decarboxylation of the C-6 carboxylic acid moiety, as a result of the oxidative cleavage of the C-5/C-6 bond; or lactonization of the acid functionality with a C-15 hydroxy group (Scheme 1.2).73,87

There are few examples in the marine diterpenoid literature of the glaciolane-scaffold.88 The first example of this carbon skeleton was a norditerpene (1.127) isolated in 1988 from the Mediterranean sponge Spongionella gracilis.95 The metabolite glaciolide (1.128), later isolated from a Canadian sponge Aplysilla glacialis and from a predatory nudibranch Cadlina luteomarginata, is a structurally related analogue of 1.127.90 Another related metabolite dendrinolide (1.129) was isolated from the Antarctic sponge Dendrilla membranosa.96 A putative biosynthetic pathway was proposed from a spongian precursor for these glaciolane-type metabolites, where the ring A contraction occurs from a C-1/C-10 bond migration to C-5 (Scheme 1.3).95

Scheme 1.3 Proposed biosynthesis of the glaciolane-type scaffold.95

Chapter 1: Introduction

Figure 1.1 Rearranged spongian metabolites with a perhydroindane motif. Image adapted from Keyzers et al. 2006.42

A significant class of rearranged spongian diterpenes are the norrisolide-type scaffolds, based on a perhydroindane motif (Figure 1.1). Norrisolide (1.130) was first of this class, isolated by Kozikowski and Goldstein from the brightly colored dorid nudibranch Chromodoris norrisi, with the structure elucidated through single crystal X-ray diffraction.97 Similar to 1.105 and 1.116, metabolite 1.130 also possessed a 2,8-dioxabicyclo[3.3.0]octane moiety. The total synthesis of (+)-norrisolide was first achieved by Brady et al. in 2005.98 Chelonaplysin C (1.131) was isolated from a Pohnpeian sponge Chelonaplysilla species, and the structure elucidated through comparison with that of 1.130, where instead of a 2,8-dioxabicyclo[3.3.0]octane moiety, 1.131 had a 2,7-dioxabicyclo[3.2.1]octane motif.99 To further confirm the structural similarity of 1.131 to norrisolide, Bobzin and Faulkner carried out chemical correlation experiments, where both metabolites were separately treated with

LiAlH4 to reduce the acetate, open the lactone ring and cleave the remaining acetal bond (Scheme 1.4). The subsequent triol was acetylated to form the triacetate. Both 1.131 and 1.130 gave the same triacetate product with identical 1H NMR, LRMS and TLC, confirming the structural and stereochemical similarity of these metabolites.99

Chapter 1: Introduction

Scheme 1.4 Chemical correlation of chelonaplysin C (1.131) and norrisolide (1.130).99

The structures of cheloviolene C (1.132) and E (1.133), isolated along with 1.131 from the New Zealand sponge Chelonaplysilla violacea, were determined through detailed spectroscopic analysis and comparison with 1.130.100 The structure of 1.133 was originally named seconorrisolide B, which was isolated from a Dysidea species, collected in the Gulf of Suez, along with metabolites seconorrisolide C (1.134) and norrlandin (1.135).101 Compounds 1.130 and 1.135 have shown cytotoxicity at 1.5 and 1.2 µg mL-1, respectively.101 Chromolactol (1.136) was isolated from the nudibranch Goniobranchus coi, collected from a reef off Mackay, Australia. Metabolite 1.136 was found to have a hydroxy group at the C-15 position, instead of an acetate as seen in norrisolide (1.130). The relative configuration of 1.136 in the 2,8- dioxabicyclo[3.3.0]octane moiety is changed, where there is a trans configuration between both H- 13/14 and H-14/H-15.102 The 2,8-dioxabicyclo[3.3.0]octane ring systems of 1.130 has a cis configuration between H-13/H-14 and a trans configuration between H-14/H-15. Through detailed spectroscopic and computational analysis, the unique change in the relative configuration of the oxygenated chiral domain was determined and the proposed configuration of 1.136 was in accordance with biosynthetic expectations.102 The cheloviolenes A and B (discussed below) are also other examples of structures with a trans configuration between H-13/14 in a 2,8-dioxabicyclo[3.3.0]octane moiety. The putative biosynthetic pathway for the norrisolide-class of metabolites proposes cleavage of the C-9/C-11 bond and subsequent contraction of ring B with a C-7/C-8 bond migration to C-9, thereby forming the hydrocarbon domain of the scaffold (Scheme 1.5).

Chapter 1: Introduction

Scheme 1.5 Proposed biosynthesis of the norrisolide class of compounds and linked chromodorane scaffold pathway.103

The chromodorolides, isolated from the nudibranch Chromodoris cavae, possess the same perhydroindane motif as the norrisolide class, however the functionality has evolved into an extensively oxygenated tricyclic moiety (Scheme 1.5). The structure of chromodorolide A (1.137) was first determined by detailed spectroscopic analysis,104 and later established by X-ray crystallographic analysis.103 Chromodorolide B (1.138) was isolated alongside 1.137 in the latter study and the structure determined by NMR analysis.103 The proposed biosynthesis for the chromodorane scaffold is shown in Scheme 1.5. The enantioselective total synthesis of (-)- chromodorolide B (1.138) was achieved in 21 steps by Overman and co-workers in 2016. The synthesis of 1.138 involved a photoredox radical cascade reaction that allowed butenolide and trans- hydroindane fragments to be combined. The subsequent late-stage fragment coupling between a tertiary carbon radical and an electron-deficient alkene united the last two ring systems, affording 1.138.105 The metabolite chromodorolide C (1.139) was isolated along with 1.137 and 1.138 from an Australian aplysillid sponge.106 When screened against a P388 cell line at a concentration of 10 µg mL-1, 1.137-1.139 displayed inhibition (66 (± 3), 70 (±2) and 42 (±4)%, respectively).106 Chromodorolides D (1.140) and E (1.141) were identified as analogues of 1.137 and 1.138, and were isolated from an encrusting Dysidea sponge from Mooloolaba, Australia.18,107 Metabolite 1.140 was isolated by two separate groups simultaneously, but named as chromodorolide E by Katavic et al. and

Chapter 1: Introduction as chromodorolide D by Uddin et al.18,108 For clarity, Katavic et al. later reversed the names of chromodorolide D and E to be consistent with the name and structure of chromodorolide D (1.140) reported by Uddin et al.107

Another significant class of rearranged spongian diterpenes are the dendrillane-type scaffolds, which are based on a perhydroazulene motif (Figure 1.2).

Figure 1.2 Rearranged spongian metabolites with a perhydroazulene motif. Image adapted from Keyzers et al. 2006.42 20

Chapter 1: Introduction

The dendrillane-type scaffold was first elucidated by Sullivan and Faulkner, where it is biosynthetically formed from a spongian diterpene scaffold involving the expansion of ring A to a seven-membered carbocycle, with the concomitant contraction of ring B to a five-membered ring (Scheme 1.6). The C-9/C-11 bond cleavage and introduction of an X group (e.g [X] = OH or OR) may occur through a P450 catalyzed process.109

Scheme 1.6 Proposed biosynthesis of the dendrillane-scaffold class of compounds.110

Figure 1.3 Clarification of structure and name for (A) aplyviolene (1.144), (B) macfarlandin E (1.143) and (C) dendrillolide A (1.142).

The first metabolite to be elucidated in this class with a perhydroazulene motif was dendrillolide A. However as shown in Figure 1.3 the structures of dendrillolide A, dendrillolide B, macfarlandin E and aplyviolene have a confusing history. The structure of dendrillolide A, isolated from a marine sponge Dendrilla sp. collected from Palau, was erroneously assigned.110 Sullivan and Faulkner (1984) initially elucidated a perhydroazulene motif in conjunction with a 2,7- dioxabicyclo[3.2.1]octane ring system (cf. 1.144) (Figure 1.3 A).110 The structures of dendrillolide A and B were re-examined when the Faulkner group (1986) isolated macfarlandin D (1.145) and E (1.144) from the nudibranch Chromodoris macfarlandi, which provided data that established the differences between the [3.3.0]- and [3.2.1]-dioxabicyclooctane ring systems.111 During 1986, Hambley and co-workers isolated aplyviolene (1.144) from the marine sponge Chelonaplysilla 21

Chapter 1: Introduction violacea and this structure was elucidated by X-ray analysis.112 The crystal structure for 1.144 exhibited the same planar structure and relative configuration of that earlier proposed for dendrillolide A (Figure 1.3 A). Consequently, Bobzin and Faulkner (1989) revised the name of the structure first identified as dendrillolide B to dendrillolide A (1.142) (Figure 1.3 C).113 In addition, the Hambley group isolated aplyviolacene which has an identical planar structure and relative configuration as macfarlandin E (1.143), published by the Faulkner group that same year (Figure 1.3 B).112 Metabolite 1.143 has been shown to induce irreversible fragmentation of the Golgi apparatus, where the fragments remain in the pericentriolar region of the cell.114 To date the correct structure of dendrillolide B remains unknown. The structure of dendrillolide C (1.146) remains unchanged; this metabolite was isolated along with dendrillolide A from the sponge Dendrilla species.110 The total synthesis of dendrillolide C was achieved by Overman and co-workers.115,116

Figure 1.4 Clarification of structure and name for selected diterpenes. (A) Confirmed structure and absolute configuration of cheloviolene A (1.148); (B) revised structure of cheloviolene B (1.149); and (C) incorrect configuration originally assigned to cheloviolene B.

A derivative of 1.143 and 1.144 was chelonaplysin A (1.147) isolated with chelonaplysin B (1.148) from the Pohnpei sponge Chelonaplysilla species.99 Metabolites 1.142 and 1.148 have been shown to inhibit growth of the Gram-positive bacterium B. subtilis. The metabolite chelonaplysin B (1.148) was later isolated, by Bergquist et al. who named the structure cheloviolene A (Figure 1.4 A).100 The cheloviolenes B (1.149), D (1.150) and F (1.151) were isolated along with 1.148 from the New Zealand sponge Chelonaplysilla violacea. The structure of 1.148 was confirmed by X-ray crystal analysis. Bergquist et al. inferred 1.149 was the C-15 epimer based on the J-value of H-15 (JH-15,H-14 = 1.0 Hz). In recent years the total syntheses of 1.148, cheloviolene B (1.149) and dendrillolide C (1.146) were achieved. Overman and co-workers first synthesized cheloviolene A, crystallizing the product and confirming the relative and absolute configuration of C-13, C-14, C-15 and C-16 to be consistent with the previously published literature data for 1.148. The pivotal step toward synthesizing 1.148 was the coupling of a tertiary radical generated from a carboxylic acid or alcohol precursor with a 5-alkoxybutenolide, providing access to the 2,8-dioxabicyclo[3.3.0]octane ring system. The relative configuration of synthetic (+)-cheloviolene B was established by X-ray crystal

Chapter 1: Introduction analysis and comparison with the reported 1H and 13C NMR data of the synthetic and natural samples of cheloviolene B (1.149) showed that the spectra were indistinguishable. The corrected structure of 1.149 has a cis configuration between H-13/H-16 and a trans configuration between H-14/H-15; this is epimeric at C-13, C-14, C-15 and C-16 to cheloviolene A (1.148) (Figure 1.4 B and C).115,116 The assumption made by Bergquist et al. that the C-13, C-14, and C-16 configurations would be identical in cheloviolenes A and B was erroneous.100 The 12-acetate analogue of dendrillolide A, 12- acetoxydendrillolide A (1.152), was isolated from the nudibranch Goniobranchus albonares.34 The polyrhaphins A (1.153) and B (1.154) were isolated from the dendroceratid sponge Aplysilla polyrhaphis.117 Polyrhaphin A (1.153) has also been isolated from the nudibranch Chromodoris norrisi collected from the same location.117 From two Dysidea sponge species, Carmely et al. reported the structures of shahamins A-E (1.155-1.159), elucidated through detailed spectroscopic and chemical correlation studies.118 For example, the two hydroxy groups in shahamin B (1.156) were readily lost during acquisition of the mass spectrum and to establish their presence, 1.156 was acetylated to give 15,16-diacetoxyshahamin B (1.160).118 Shahamin K (1.161) was extracted from the skin of a dorid nudibranch Chromodoris gleniei, collected off the coast of Sri Lanka, with the planar structure and relative configuration determined through spectroscopic analysis.21 The enantioselective total synthesis of (+)-shahamin K (1.161) was first achieved by Valentekovich and co-workers in 2001.119

The metabolites 12-desacetoxyshahamin C (1.162) and 12-desacetoxypolyrhaphin A (1.163) were isolated from the Palauan sponge Dendrilla species.113 Shahamin C (1.157) was found to inhibit feeding by rainbow wrasse (Thalossoma lucasanum) at a concentration of 100 µg per mg of food. In contrast, metabolites 1.142, 1.145 and 1.154 did not exhibit feeding deterrence when tested at the same concentration. Polyrhaphin B (1.154), however, exhibited inhibition of B. subtilis growth at 10 µg per disc.117 Finally, secoshahamin 1.164 was first isolated by Tanaka and co-workers and the planar structure established by spectroscopic analysis, however the relative configuration was inconclusively assigned.108 Tanaka and co-workers could not deduce which diastereomer was isolated based solely on 2D NMR, due to the ease of rotation about the C-8/C-14 bond. Since this metabolite is potentially involved in the formation of the polyrhaphins, shahamins, dendrillolides, and

Chapter 1: Introduction macfarlandins, it is important that this be resolved. Consequently, the relative and absolute configuration of 1.164 is a topic that will be explored later in this thesis (Chapter 4). The next class of rearranged spongian diterpenes are those that possess a hydronaphthalene motif, including the remaining reported shahamins. The first members of this class to be isolated were macfarlandin C (1.165) and macfarlandin D (1.145), from the dorid nudibranch Chromodoris macfarlandi. The structure of 1.165 was established through a single-crystal X-ray experiment.111 Next to be elucidated were the shahamins F-J (1.166-1.170), isolated from two Dysidea sponge species.118 The Kashman group also proposed a biosynthetic pathway for the shahamins (Scheme 1.7), analogous to the biosynthetic pathway proposed for the perhydroazulene scaffold (Scheme 1.6).

Scheme 1.7 Proposed biosynthesis of the shahamin-type scaffold.118

From the marine sponge A. polyrhaphis, the cyclopropyl functionalized polyrhaphin C (1.171) was isolated along with 1.153 and 1.154. The structure was determined through detailed spectroscopic analysis and comparison with the structure of 1.145.117 Metabolite 1.171 was found to inhibit the growth of S. aureus at 100 µg per disc and B. subtilis at 10µg per disc.117 The structure of dendrillolide E (1.172) was determined through spectroscopic comparison with that of 1.142, suggesting a 2,8- dioxabicyclo[3.3.0]octane moiety, instead of the 2,7-dioxabicyclo[3.2.1]octane motif seen in 1.171.113 The 12-acetoxy analogue of 1.171, 12-acetoxypolyrhaphin C (1.173) was isolated from the dorid nudibranch G. splendidus, with the structure of 1.173 established by spectroscopic methods.85 Cheloviolin (1.174) is another metabolite that was elucidated through spectroscopic comparison with 24

Chapter 1: Introduction 1.172, revealing that they have the same 2,7-dioxabicyclo[3.2.1]octane moiety, and only differ in the position of the cyclopropyl group.100

The metabolites omriolide A (1.175) and B (1.176) were isolated from the southern Kenyan sponge Dictyodendrilla aff. retiara. Metabolite 1.175 shows an unusual tricyclic motif, spiro attached at C-8. A biosynthesis for 1.175 was proposed by Rudi et al.120 The structure of omriolide B (1.176) was determined through comparison with 1.174 for the hydronaphthalene motif and with norrisolide (1.130) for the 2,8-dioxabicyclo[3.3.0]octane moiety. It was noted that 1.176 was epimeric at C-15 compared to norrisolide (1.130) and macfarlandin C (1.165), giving a cis configuration between H- 14/H-15 instead of the more commonly observed trans configuration. Rudi et al. determined the anomalous relative configuration at C-15 by NOESY correlations and J-values; however on close inspection, the J-value reported for H-15 (d, J = 5.0 Hz) of 1.176 is well within the range of reported J-values for metabolites with cis configuration between C-14/C-15 in a 2,8-dioxabicyclo[3.3.0]octane moiety (1.130: d, J = 3.5 Hz; 1.165: d, J = 6.6 Hz). In addition, Rudi et al. did not provide convincing spectroscopic evidence for the C-13 acetate configuration, although the proposed 13β-OAc may be correct as it is energetically unfavourable to have a trans configuration at the ring junction in a 2,8- dioxabicyclo[3.3.0]octane moiety. Both 1.175 and 1.176 lacked cytotoxic activity against several tumor cells, or activity on the Golgi membrane.120 Metabolites 1.171-1.174 and 1.176 are representative marine spongian diterpenes with cyclopropyl functionality; related structures will be discussed in Chapter 3 of this thesis.

Chapter 1: Introduction 1.4 Computational studies complementing natural product elucidation The foregoing Sections 1.1-1.3 have highlighted that natural products are a prolific source of new structural scaffolds. However, a significant restriction in elucidating natural products is the small quantity of sample extracted from the natural source. Often, secondary metabolites are minor chemical components in the organism, and therefore the quantity of purified sample is in the sub- milligram range. The classic route for elucidating natural products is through mass spectrometry, and 1H and 13C NMR, as well as 2D NMR experiments, including heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC), total correlation spectroscopy (TOCSY), and gradient correlation spectroscopy (gCOSY). These experiments aid in establishing the planar structure. In addition, scalar spin-spin coupling (J-coupling) is an important structural parameter in structure elucidation. The magnitude of a coupling constant is dependent on the number of bonds between coupled nuclei, as well as the configuration and spatial arrangement of the electrons.121 Since J-coupling is mediated by bonding electrons, information regarding the connectivity of coupled nuclei and individual conformers of a structure can be determined. NMR data derived from nuclear Overhauser effect spectroscopy (NOESY) and rotating frame Overhauser effect spectroscopy (ROESY) pulse sequences play an important role in solving the relative configuration of natural products, particularly when informed by J-based configurational analysis (JBCA). This approach, which utilizes the information deduced from homonuclear and heteronuclear coupling constants to elucidate relative configuration, is well understood for 1, 2-dimethine and related acyclic systems.122,123 Computational studies are a complementary approach to classic spectroscopic natural product elucidation.124 Hoye and co-workers have described in detail the computational protocol for validating the structural assignments of new chemical entities.125 A recent comparative computational study revealed that the calculated shifts depended on the operating system when using the “Willoughby−Hoye” Pythonscripts and consequently could lead to incorrect conclusions. This glitch is apparent in the LINUX (Ubuntu16) and Mac (Mojave) operating systems.126 The protocol utilized through this thesis was not affected by this glitch as the data was processed in Powershell on a Windows based operating system. The process employed by Dr Gregory Pierens for the computation studies executed in this thesis is shown in Figure 1.5.

Chapter 1: Introduction

1. Generate conformations of the stereoisomer of interest and select a suitable energy window (MacroModel, 21 kJ/mol or 5 kcal/mol)

2. Preliminary DFT optimization and remove of duplicates and high energy conformers

3. DFT optimization at a higher level of theory and calculation of free energy of individual conformers (Boltzmann population)

4. Single point calculation of NMR chemical shifts for each conformer

5. Calculate the Boltzmann averaged chemical shifts (averaging due to symmetry, e.g. CH3)

6. Metric to calculate the goodness of fit to the experiment data (MAE)

Figure 1.5 Computational approach to calculate electron density around atomic nuclei and spectroscopic properties of candidate structures. Diagram reproduced with permission of Dr Gregory Pierens.

Computational methods86,125,127 that involve predictions of chemical shift values and associated mean absolute error (MAE) values have provided a valuable complementary approach to traditional structure elucidation techniques. The MAE is obtained by calculating the absolute error in each chemical shift and determining the mean of the total. These computational methods include molecular mechanics-based modelling, such as a MacroModel128 conformational search, and/or quantum mechanics-based modelling, like that of the density functional theory (DFT) based approach. MacroModel is used to generate conformer libraries quickly, due to the predefined parameters of bond length, bond angles and bond torsions; however, the energy differences between the conformers may be less accurate.129 Quantum mechanics-based methods are generally accepted as the more reliable approach, as the parameters for bonds are not predefined, and are instead calculated based on optimized electron density calculations.125,130 DFT-based modelling can be used subsequent to MacroModel, where conformers within a defined energy window are selected to be optimized through DFT. The conformers from DFT optimization are then weighted by their Boltzmann population. The computed free energies along with an appropriate value for temperature are fed into the Boltzmann equation:131-133 27

Chapter 1: Introduction

e−i /kT i = M e−i /kT i=1 Where: pi = probability of state i,

εi = the energy of state i, k = the Boltzmann constant, T = temperature of the system, and M = the number of all states accessible to the system of interest.

The DFT-based approach has been used to distinguish between possible diastereomers of natural products. For example, Litaudon and co-workers reported the isolation of the metabolite jatrohemiketal (1.177), which had an unusual tricyclic ring system with a hemiketal substructure.134 The two-dimensional structure was determined through detailed spectroscopic analysis and the relative configuration, apart from the C-6 and C-9 stereocenters, was established. To probe the configuration of C-6 and C-9, four diastereomers of 1.177 were considered (1.177a-1.177d). The quantum chemical calculations based on DFT provided a reliable prediction of the NMR parameters for the lowest energy conformer of each diastereomer, where, on comparison with the experimental data, diastereomer 1.177a was the most plausible candidate of 1.177. To further validate these results, the vicinal dihedral angles and interproton distances of the modelled diastereomers were compared with the experimental NMR data and found to be consistent.134

In addition, probability predictions calculated using DP4135 or DP4+136 or J-DP4137 algorithms aid in the selection of preferred diastereoisomers.138 The DP4 method, developed by Smith and Goodman in 2011, is a probability based analysis used to compare theoretically calculated 1H and 13C NMR chemical shifts to experimental NMR-derived data. Smith and Goodman demonstrated the

Chapter 1: Introduction application of DP4 by modelling the structures of twenty-one different natural products where the configuration was originally erroneously assigned, and could only be correctly assigned through extensive synthesis of diastereomers.135 Their statistically based method has been shown to be significantly more successful at determining correct assignments with high confidence, compared to other methods that are based on the correlation coefficient and mean absolute error parameters. For example, the alkaloid nobilistine A (1.178), isolated from the South African plant Clivia noblis,139 was originally reported with an incorrect relative configuration. Lodewyk and Tantillo used the DP4 method to assess the relative configuration of 1.178, modelling seven diastereomers of 1.178 and ultimately determining the most probable relative configuration as 1.178a.140

The DP4 method was later improved by Sarotti and co-workers and termed DP4+. The DP4+ method includes the use of both scaled and unscaled NMR data computed at higher levels of theory compared to the original DP4 method.141,142 Sarotti and co-workers showed the DP4+ method is a reliable, alternative method to aid in the configurational assignment of spiro and terminal epoxides, where previously the DP4 method afforded poor results. In previous work undertaken as part of my Honors thesis, the structure of an unusual oxygenated diterpene epoxygoniolide-1 (1.105) was established through spectroscopic analysis, however, the C-12 spiroepoxide configuration could not be determined by NMR alone. Consequently, four candidate diastereomers (1.105a-1.105d) of 1.105 were modelled using MacroModel and the DFT-refined conformers were then used to calculate the Boltzmann-averaged 1H and 13C NMR chemical shifts for each conformer. Considering both the 1H and 13C NMR data, DP4+ was used to statistically compare the diastereomers, which resulted in a 90.39% probability that the 10S*,12R* configuration was the likely diastereomer of 1.105.

Chapter 1: Introduction These spectroscopic methods have been aided by recent advances in theoretical prediction of coupling constants. There are several methods for calculating proton−proton spin−spin coupling constants (SSCCs), including those that consider the Fermi contact (FC) interactions in nuclear spin scalar couplings.143 To clarify, FC interactions are the magnetic interaction between an electron and an atomic nucleus and constitute the largest portion of the total spin-spin coupling constant.144 Kutateladze and Mukhina have developed a fast and accurate method (rff/DU8c parametric method) that uses both empirical scaling parameters and the natural bond orbital (NBO) hybridization coefficients to calculate spin-spin coupling constants.145 A wealth of structural information can be found in SSCCs, where in some natural products the values have been instrumental in structural assignments (and misassignments).146-148 Arguably, many structural misassignments could have been avoided if the experimental SSCC values were cross checked with computed SSCC values. As a recent example, cordycepol A (1.179), isolated from a fungus Cordyceps ophioglossoides that colonizes other fungal species.149 The originally proposed structure of 1.179 was found to be erroneous. Using the rff/DU8c parametric method, Reddy and

Kutateladze calculated the JH-1,H-2b and JH-1,H-2a values, as well as chemical shifts of C-1, C-2, C-6, C- 7, C-10, and C-12, finding that the computed values significantly diverged from the experimental NMR spectroscopic data. Conversely, the calculated SSCC values and chemical shifts for the C-1/C- 11 epimer (1.179a) closely matched the experimental values. As a result, it was proposed that the structure of cordycepol A be revised to structure 1.179a.150

3 Adaptations have been made to incorporate JHH couplings into the DP4 probability-based analyses, aptly named the J-DP4 method. The three alternative DP4 strategies developed by Grimblat et al. are the dJ-DP4 (direct) approach, iJ-DP4 (indirect) approach and the combined iJ-DP4/ dJ- DP4.137 Of the three methods, the combined approach provides a 2.5-fold improvement in performance, compared to DP4, at an equal or lower computational cost. The authors note that the reliability of J-DP4 calculations is dependent on the accuracy of the data provided to the program.137 A significant limitation in computational studies to date is the accuracy of the modelling output for structures featuring high flexibility and/or intramolecular H-bonding interactions.151 Other approaches that can be applied to assign the absolute configuration of natural products include comparison of specific rotations and/or ECD measurements, X-ray crystallography, Mosher’s analysis, as well as the aforementioned computational methods. X-ray crystallography no longer 30

Chapter 1: Introduction requires a heavy atom for determination of absolute configuration.152,153 For metabolites containing secondary alcohols, analysis of Mosher ester products by NMR spectroscopy is routinely used to determine absolute configurations.154,155 For example, Yong et al. isolated the plakortolides K-S, as well as a number of seco-plakortolides and plakortones from the Australian sponge Plakinastrella clathrata. Mosher ester analyses were conducted on plakortolides K-N (1.180-1.183), where the absolute configurations were determined from 1H NMR analyses on O-methylmandelic acid (MPA) esters derived from Zn/AcOH reduction products.156

Computational methods86,125,127 comparing experimental and time-dependent density- functional theory (TDDFT) derived electronic circular dichroism (ECD) data157 can facilitate the assignment of absolute configuration for a natural product providing a chromophore is present and spatially near the stereogenic center(s).158 For example, TDDFT-ECD data were instrumental in aiding the elucidation of the metabolite pustulosaisonitrile-1 (1.184), isolated from the nudibranch Phyllidiella pustulosa. This approach determined the C-3 absolute configuration in the 3,3,6- trisubstituted cyclohexene unit of 1.184, which guided the decision to synthesize diastereomers (1.184a, 1.184b and 1.184c). Subsequently, the relative and absolute configurations were confirmed as shown in 1.184a.138

Chapter 1: Introduction Despite the plethora of NMR pulse sequences available,159 and the emergence of the above- mentioned computational approaches, such studies may fail to establish the full relative and absolute configuration of natural products in cases where there are multiple elements of chirality. In such cases, chemical correlation of newly isolated natural metabolites with a compound of known configuration can inform the structure elucidation.118,156 Within the field of marine natural products, examples of highly rearranged and oxygenated diterpenes with remote chiral domains for which chemical correlation studies have proven valuable include the shahamins,118 the polyrhaphins,117 and chelonaplysin C.99

1.5 Aims of the thesis Aim 1: The first aim was to discover new metabolites and catalogue the natural products from nudibranchs. This project would isolate and elucidate the structure of the natural products from the nudibranch family Chromodorididae, with particular interest in the Goniobranchus genus. Based on the anatomical differences, Goniobranchus has been recently separated from the genus Chromodoris in which it was previously grouped.160 The high diversity of the genus Goniobranchus, including their conspicuous color patterns amongst many species and variable diet resulting to diverging chemistries with potential bioactivity, make them a fascinating group to study. In addition, their indiscriminate sequestration of metabolites relative to geographical location renders them a promising source for discovering novel, biologically active diterpenes. Earlier studies by the Garson group has led to cataloging the chemical compositions of a range of Goniobranchus species. All studied species were found to contain oxygenated diterpenes in varying amounts.161 Chapter 2 of this thesis describes the isolation and structural elucidation of spongian diterpenes from four Goniobranchus species (G. collingwoodi, G. aureopurpureus, G. sp.1 and G. leopardus). X-ray crystallography was also used to determine the absolute configuration of selected metabolites. Chapters 3 and 4 explored the chemistry of the left and right motifs of the dendrillane scaffold (mentioned in Chapter 1), isolated from G. coi and G. geometricus. The structures of metabolites discussed in Chapter 3 were confirmed by NMR conducted under different temperatures and computational studies to better understand the dynamic nature of the perhydroazulene motif and 2,8-dioxabicyclo[3.3.0]octane moiety. Chapter 4 discusses a further two metabolites isolated from G. coi and three metabolites from G. geometricus, where the relative configuration was investigated through chemical correlation experiments, molecular modelling, and computational calculations (DFT and DP4+). X-ray crystallography was also used to establish the absolute configuration of selected metabolites. Aim 2: The second aim would be to explore the biological function of the compounds found in nudibranchs. Chapter 5 investigated the role of oxygenated diterpenes in interspecific 32

Chapter 1: Introduction (prey/predation) communication. Nudibranchs prey upon sponges, sequestering the secondary metabolites found in the sponges and using the compounds as a chemical weapon for defense; generally nudibranchs store the more toxic chemicals in their mantle.12 As a result, the chemical composition of nudibranchs relative to the anatomical distribution (mantle and viscera) of the metabolites would be considered. The anatomical distribution could be used as an indicator of the potential ecological role of individual metabolites. The possible function of these metabolites in the crude extracts and as individual purified compounds would be tested using feeding deterrency assays with generalist feeders (Palaemon serenus (rockpool shrimp)). Within this aim, the biological activities of purified oxygenated nor/diterpenes would be explored, including, and not limited to cytotoxicity, antiviral, and antimicrobial activity. Previous studies in the Garson group in collaboration with the research group of Professor Robert Capon have explored the cytotoxicity of isonitriles and rearranged oxygenated diterpenes, with some showing moderate activity against cancer cell lines (NCIH-460, SW620 and HepG2).56,76,86

Chapter 2: The chemistry of spongian diterpenes Publications included in Chapter 2 Forster, L. C.; White, A. M.; Cheney, K. L.; Garson, M. J. Oxygenated Diterpenes from the Indo- Pacific Nudibranchs Goniobranchus splendidus and Goniobranchus collingwoodi. Nat. Prod. Commun. 2018, 13, 299-302.

Candidate contributions Louise C. Forster was responsible for the following work, incorporated into Section 2.1.1: • 25% Conception and design • 70% Data interpretation • 50% Manuscript drafting

Contributions by others Mary J. Garson and Andrew M. White both contributed to the conception and design of this project, as well as, data interpretation and manuscript drafting. Karen L. Cheney collected and identified the nudibranch specimens and also contributed to the manuscript drafting.

Chapter 2: The chemistry of spongian diterpenes Chapter 2: The chemistry of spongian diterpenes

Chapter 2: The chemistry of spongian diterpenes 2.0 Introduction Nudibranchs of the family chromodorididae have been the focus of many chemical investigations and the source of an impressive catalogue of novel natural products.67 The genus Chromodoris has drawn much of the attention of natural products chemists but has recently undergone a significant taxonomic revision.162 Due to the recent advancements in genetic analysis procedures, many nudibranchs species originally classified as Chromodoris sp. have been reclassified to Felimida,97,117,163-167 Doriprismatica,168 Glossodoris,164,169 Hypselodoris,16,170 and Goniobranchus.18,19,51,54,103,104,107,171,172 This chapter details the investigation of the secondary metabolites from four Goniobranchus nudibranch species collected along the East Coast of Australia (Figure 2.1). From Goniobranchus collingwoodi (Section 2.1.1), one new oxygenated rearranged diterpene was isolated along with seven known spongian diterpenes. Inspection of the second nudibranch species Goniobranchus aureopurpureus (Section 2.1.2) uncovered a total of seventeen spongian diterpenes, including eleven known (eight of which were rearranged spongian diterpenes) and six new metabolites (one of which is a rearranged spongian diterpene). Investigation of the third nudibranch species, Goniobranchus sp. 1 (Section 2.1.3), revealed eight known and six new spongian diterpenes. The extraction of Goniobranchus leopardus (Section 2.1.4) yielded eleven known oxygenated rearranged spongian diterpenes and two new diterpenes.

(A) (B) (C) (D)

Figure 2.1 The nudibranchs Goniobranchus collingwoodi (A), Goniobranchus aureopurpureus (B), Goniobranchus sp. 1 (C), and Goniobranchus leopardus (D).

2.0.1 Nudibranchs and their sponge prey Nudibranchs of the Chromodoris genus, and those formally classified within this genus, feed exclusively on marine sponges. They are known to sequester terpenoid metabolites from their sponge prey and have been found to contain sesquiterpenes,16,163,170,173 norditerpenes,69,73,76,86,165,174 diterpenes,19,21,51,54,56,97,102-104,111,117,166,167,175 and sesterterpenes.40,164,176-178 The diverse range of sponges that have been confirmed as being consumed by these nudibranchs include species from the genus Aplysilla,111,117 Darwinella,179 and Dysidea.18,170 Studies have also shown that nudibranchs from the family Chromodorididae contain secondary metabolites commonly found in

Chapter 2: The chemistry of spongian diterpenes Chelonaplysilla, Dendrilla, and Spongionella sponges, it has been assumed that nudibranchs have preyed upon these sponges although they have not been directly observed feeding on them.66-67

2.0.2 Reported spongian metabolites from Goniobranchus species In my previous Honors work, I investigated the chemistry of G. collingwoodi.56 To date this is the only study on an individual G. collingwoodi specimen examined for its chemical profile.56 From the specimen, six highly oxygenated spongian-16-one analogues (1.42-1.47) were isolated (Figure 2.2).

Figure 2.2 Metabolites (1.42-1.47) previously isolated from G. collingwoodi.

The chemical analysis of G. aureopurpureus, Goniobranchus sp. 1, and G. leopardus has not been reported elsewhere. The current study therefore represents the first report on the secondary metabolite profile of G. aureopurpureus, Goniobranchus sp. 1, and G. leopardus.

2.1 Results and discussion 2.1.1 The isolation of metabolites from Goniobranchus collingwoodi (Rudman, 1987)

Eight specimens of G. collingwoodi were collected from Nelson Bay in March 2016. Specimens were dissected into their mantle and viscera and each body part was finely chopped, extracted with acetone and the extract concentrated under vacuum. Distilled water was then added to the residues and the aqueous suspensions were partitioned with Et2O to yield an orange oil from the mantle and a green oil from the viscera. A 1H NMR spectrum was obtained for the extracts from each body part of each individual and the mantle and viscera extracts of all eight specimens were compared 37

Chapter 2: The chemistry of spongian diterpenes but were found to be indistinguishable and so were combined, respectively. The combined mantles and combined visceras were partitioned by normal phase (NP) flash chromatography (Figure 2.3) with a stepwise solvent gradient (hexanes: hexanes/DCM: DCM/EtOAc: EtOAc: MeOH). All fractions were compared using TLC and were subject to 1H NMR analysis, resulting to pooling fractions with similar spectra. 1H NMR data identified the presence of peaks characteristic of protons in the environment of esters and hydroxy groups, suggesting the occurrence of oxygenated diterpenes. In total six compounds were isolated, with five spongian diterpenes extracted from the mantle, including spongian-16-one (1.24),49,50 7α-acetoxyspongian-16-one (1.32),52 7α-hydroxyspongian-16- one (1.33)51, isoagatholactone (1.19)45, and the new spongian diterpene 7α-acetoxyisoagatholactone (2.1). While the furanone diterpene luffarin-X (1.16)43 was found in the viscera, along with 1.19 and 1.24.

G. collingwoodi [8 specimens]

Dissected Acetone Extraction

Partition with Et2O /H2O

Mantle Viscera

Crude Extract (20.8 mg) Crude Extract (50.0 mg)

NP-flash chromatography NP-flash chromatography (hexanes: DCM: EtOAc: MeOH) (hexanes: DCM: EtOAc: MeOH)

Fractions D-H Fractions A-B

NP-HPLC NP-HPLC 10-25% EtOAc/ hexanes 10% EtOAc/hexanes

1.19, 1.24, 1.32, 1.33, 2.1 1.16, 1.19, 1.24

Figure 2.3 Isolation scheme of G. collingwoodi compounds.

2.1.1.1 Characterization of spongian-16-one (1.24) Spongian-16-one (1.24) was isolated as a white amorphous solid from NP-HPLC (10% EtOAc in hexanes). An adduct ion at m/z 327.2 [M+Na]+, which is compatible to the molecular 1 formula (C20H32O2) of spongian-16-one in the presence of a sodium adduct. The H NMR spectral

Chapter 2: The chemistry of spongian diterpenes data (Table 2.1) of 1.24 was consistent with the known compound spongian-16-one. Two research groups independently reported 1.24 from the marine sponge Dictyodendrilla cavernosa and Chelonaplysilla violacea.49,50 The relative configuration of 1.24 was initially defined through coupling constants and NOE correlations, from which the cis configuration of the C/D ring junction was established, with a 6.0 Hz coupling between H-13 and H-12ax placing H-13 in an equatorial orientation (Figure 2.4).49,50 (±)-Spongian-16-one (1.24) was synthesized in 1996 by Pattenden and Roberts.180 The relative configuration across the ring junctions has been established as trans for the A/B and B/C rings, while cis for the C/D ring. To date the absolute configuration of spongian-16-one has not been conclusively established.

Figure 2.4 Spongian-16-one (1.24) with trans ring junctions highlighted in pink and the cis ring junction in blue.

2.1.1.1 Characterization of 7α-acetoxyisoagatholactone (2.1) 7α-Acetoxyisoagatholactone (2.1) was isolated as a colorless oil from the mantle tissue of G. collingwoodi. High resolution electrospray ionisation mass spectrometry (HRESIMS) analysis of the terpene displayed a pseudomolecular ion peak at m/z 359.2247 [M-H]- corresponding to a molecular 1 formula of C22H32O4. Inspection of the H NMR spectral data (Table 2.1) revealed some features that closely matched those of spongian-16-one (1.24)49,50 and isoagatholactone (1.19).45 Notable 1 differences in the H NMR spectrum of 2.1 included an acetate methyl group at δH 2.11 (s) and an oxymethine proton at δH 4.84 (t), and the absence of the (H2-7) methylene signals found in 1.19. The 13 C NMR spectrum of 2.1 revealed a signal at δC 74.5 which was assigned to C-7 due to its HMBC correlations with Me-17 (δH 0.86) and H-5 (δH 1.39). The spectrum also contained signals consistent with an acetate ester at δC 169.7 and 21.3. HMBC correlations from the acetate methyl (δH 2.11) to

C-7 (δC 74.5) indicated that the acetate group was located at C-7. The triplet signal for H-7 with a J- value of 2.9 Hz, established the equatorial orientation of this proton. Axially-orientated oxygen- bearing substituents are commonly encountered at the C-7 position of marine diterpenes isolated from 52 21 nudibranchs. Since the specific optical rotation of 2.1 showed a value of [α] D -51 (c 0.02, CHCl3), the name of compound 2.1 was proposed as (-)-7α-acetoxyisoagatholactone.

Chapter 2: The chemistry of spongian diterpenes

Table 2.1 1H NMR assignments for spongian-16-one analogues 1.24, 1.16 and 2.1a,b Position 1.2449-50 1.16c45 2.1 1H (mult., J, Hz) 1H (mult., J, Hz) 1H (mult., J, Hz) 13C 1 eq 1.72, br d (12.8) - 1.65, m 39.6, CH2 1ax 0.77, m 0.95, m 2 eq 1.60, m - 1.62, m 18.3, CH2 2ax 1.43, m 1.46, m 3eq 1.35, m - 1.43, m 41.6, CH2 3ax 1.12, dt (4.2, 13.8) 1.19, m 4 - - - 32.5, C 5 0.77, m - 1.39, m 48.1, CH 6eq 1.55, m - 1.77, m 23.4, CH2 6ax 1.32, m 1.71, m 7eq 1.81, dt (3.3, 13.0) - - 74.5, CH 7ax 1.02, dt (3.5, 13.0) 4.84, t (2.9) 8 - - - 37.5, C 9 0.75, m - 1.66, m 49.8, CH 10 - - - 37.1, C 11eq 1.26, m 2.35, m 2.39, m 24.4, CH2 11ax 1.53, m 2.35, m 2.16, m 12eq 2.29, dd (4.7, 13.8) 6.87, dd (3.5, 7.0) 6.85, q (3.6) 135, CH 12ax 1.60, m - - 13 2.52, t (8.0) - - 126.6, C 14 2.07, dd (5.5, 8.0) 2.80, m 3.24, m 43.2, CH 15eq 4.21, d (9.9) 4.37, t (9.0) 4.20, t (9.2) 66.8, CH2 15ax 4.09, dd (5.5, 9.9) 4.05, t (9.0) 4.02, t (9.2) 16 - - - 169.4, C 17 0.85, s 0.88, s 0.86, s 14.2, CH3 18 0.88, s 0.93, s 0.80, s 33.0, CH3 19 0.84, s 0.84, s 0.82, s 21.4, CH3 20 0.81, s 0.78, s 0.95, s 15.0, CH3 7-OCOCH3 - - 169.7, C 7-OCOCH3 2.11, s 21.3, CH3 a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. Partial data

2.1.2 The isolation of metabolites from Goniobranchus aureopurpureus (Collingwood, 1881) Six specimens of G. aureopurpureus were collected by SCUBA from Nelson Bay in March 2016. The preparation of mantle and viscera extracts was carried out following the previously described procedure (Figure 2.5). The respective mantle and viscera extracts were combined based on the similarity of their 1H NMR spectra prior to fractionation by NP-flash chromatography and

Chapter 2: The chemistry of spongian diterpenes subsequent NP-HPLC. Similarly, 1H NMR data identified the presence of oxygenated diterpenes. In total seventeen compounds were isolated (Figure 2.5 and 2.6), with metabolites macfarlandin E 1.143,111 aplyviolene 1.144112 polyrhaphin B 1.154,117 shahamin C 1.157,118 secoshahamin 1.164,108 7α-acetoxy-6α-hydroxyspongian-16-one (2.2), 6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)- butanoate (2.3), 20-acetoxy-6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.4), 13- acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.5), 13-acetoxy-20- hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate (2.6), and 15-desacetoxy-12- acetoxydendrillolide A (4.4) isolated from the mantle. While from the viscera luffarin-X 1.16,43 spongian-16-one 1.24,49,50 7α-acetoxyspongian-16-one 1.32,52 polyrhaphin A 1.153,117 15,16- diacetoxyshahamin B 1.160,118 and 12-desacetoxypolyrhaphin A 1.163,113 along with 1.24, 1.32, 1.143, 1.144, .1.154 and 1.164 were isolated. The new spongian diterpenes (2.2-2.6) show varying levels of oxidation, particularly at positions C-6, C-7, C-13 and C-20. The structures and relative configuration were determined from the analysis of their 2D NMR spectra obtained from COSY, HSQC, HMBC and NOESY experiments as well as where applicable by X-ray crystallography for elucidating the absolute configuration.

G. aureopurpureus [6 specimens]

Dissected Acetone Extraction

Partition with Et2O /H2O

Mantle Viscera

Crude Extract (51.9 mg) Crude Extract (56.1 mg)

NP-flash chromatography NP-flash chromatography (hexanes: DCM: EtOAc: MeOH) (hexanes: DCM: EtOAc: MeOH)

Fractions D-G Fractions B-E

NP-HPLC NP-HPLC 25% EtOAc/ hexanes 25% EtOAc/hexanes

1.24, 1.32, 1.143, 1.144, 1.154, 1.16, 1.24, 1.32, 1.143, 1.144, 1.157, 1.164, 2.2, 2.3, 2.4, 2.5, 1.153, 1.154, 1.160, 1.163, 1.164 2.6, 4.4 Figure 2.5 Isolation scheme of G. aureopurpureus compounds.

Chapter 2: The chemistry of spongian diterpenes

Figure 2.6 Known compounds isolated from G. aureopurpureus.

2.1.2.1 Structure elucidation of new oxygenated spongian diterpenes from Goniobranchus aureopurpureus

Diterpene 2.2 was isolated as a colorless oil and displayed a sodiated molecular ion peak in + 1 the HRESIMS at m/z 401.2293 [M+Na] , corresponding to a molecular formula of C22H34O5. The H 13 and C NMR spectral data (Tables 2.2 and 2.3) presented an acetoxy methyl singlet at δH 2.09 along with its carbonyl signal at δC 178.9. HMBC correlations from the signals at δH 4.84 (H-7, d) and 2.09

(7-OCOCH3, s) to the signal at δC 169.7 confirmed the position of the acetoxy group at C-7 and were 42

Chapter 2: The chemistry of spongian diterpenes comparable to those in the 1H NMR spectrum of 7α-acetoxyspongian-16-one (1.32).52 The final 16 mass units was attributed to an additional hydroxy moiety which was consistent with the calculated molecular formula. The H-7 (J = 3.1 Hz) doublet suggested a substituent at C-6. gCOSY correlations from H-6 (δH 4.19, dd, J = 3.1, 6.6 Hz) to H-5 and H-7 confirmed a vicinal proton at C-6. The down field chemical shift of C-6 suggested an oxygen substituent, thus determining the position of the hydroxy group. The relative configuration of 2.2 was assigned by its NOESY correlations between

H-6/H-7, H-6/Me-17 and H-7/Me-17 positioning 6-OH and 7-OCOCH3 relatively in the same orientation as Me-17, which is above the plane as illustrated in Figure 2.7. NOESY correlations between H-5/H-9, H-9/H-14, and H-13/H-14, alternatively, are similarly annotated but opposite (trans-axial) to the proton H-6, established the overall relative configuration of 2.2 (Figure 2.7). The 21 specific optical rotation of 2.2 showed a value of [α] D -65 (c 0.022, CHCl3). Compound 2.2 was assigned the systematic name (-)-7α-acetoxy-6α-hydroxyspongian-16-one.

(A) (B)

Figure 2.7 Molecular modelling for 2.2 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Chapter 2: The chemistry of spongian diterpenes Table 2.2 1H NMR assignments for new spongian-16-one analogues 2.2-2.6a Position 2.2b 2.3c,e 2.4c 2.5b 2.6c 1 eq 1.74, m 1.75, m 2.34, m 2.22, br d (12.5) 2.22, m 1ax 0.86, m 0.86, m 0.78, m 0.89, m 0.89, m 2 eq 1.71, m 1.73, m 1.61, m 1.60, m 1.61, m 2ax 1.46, m 1.46, m 1.46, m 1.51, m 1.51, m 3eq 1.39, m 1.39, m 1.45, m 1.49, m 1.48, m 3ax 1.20, m 1.19, m 1.24, m 1.23, m 1.24, m 4 - - - - - 5 1.16, d (2.0) 1.16, m 1.38, m 1.45, m 1.50, m 6eq - - - 1.81, m 1.81, m 6ax 4.19, dd (3.1, 6.6) 4.18, br s 4.17, m 1.63, m 1.63, m 7eq - - - - - 7ax 4.84, d (3.1) 4.84, d (2.6) 4.87, d (3.2) 4.85, t (2.8) 4.86, t (2.6) 8 - - - - - 9 1.05, dd (2.2, 12.5) 1.05, m 1.14, m 1.49, m 1.44, m 10 - - - - - 11eq 1.57, m 1.58, m 1.80, m 1.92, m 1.88, m 11ax 1.45, m 1.47, m 1.58, m 1.89, m 1.88, m 12eq 2.32, m 2.32, m 2.31, m 2.28, dt (13.9, 5.8) 2.32, dt (14.1, 5.6) 12ax 1.60, m 1.60, m 1.55, m 2.02, m 1.99, m 13 2.60, t (8.2) 2.57, t (7.8) 2.57, t (7.9) - - 14 2.43, dd (5.6, 8.2) 2.44, dd (5.6, 7.8) 2.49, m 2.92, dd (1.5, 6.5) 2.91, dd (1.2, 6.3) 15eq 4.26, d (10.2) 4.27, d (10.1) 4.25, d (10.2) 4.22, dd (6.5, 9.9) 4.25, d (6.3, 9.9) 15ax 3.98, dd (5.6, 10.2) 3.95, dd (5.6, 10.1) 3.94, dd (5.5, 10.2) 4.20, dd (1.5, 9.9) 4.20, dd (1.2, 9.9) 16 - - - - - 17 1.21, s 1.22, s 1.27, s 1.10, s 1.09, s 18 0.90, s 0.90, s 0.90, s 0.81, s 0.81, s 19 1.19, s 1.19, s 1.18, s 0.81, s 0.80, s 20 1.19, s 1.19, s 4.79, m 4.05, d (11.8) 4.04, d (11.8) 4.73, m 3.92, d (11.8) 3.91, d (11.8) d d d 6-OH - - 7-OCOCH3 2.09, s - - - - 7-OCOCH2CH(CH3)2 - - - - 2.22, m 2.22, m 7-OCOCH2CH(CH3)2 - - - - 2.11, m 7-OCOCH2CH(CH3)2 - - - - 0.95, d (6.6) 0.96, d (6.6) 7-OCOCHCH3CH2CH3 - 2.45, m 2.41, q (6.9) 2.40, sx (7.1) - 7-OCOCHCH3CH2CH3 - 1.15, d (7.2) 1.15, d (6.9) 1.15, d (6.9) - 7-OCOCHCH3CH2CH3 - 1.68, m 1.68, dt (13.6, 7.4) 1.68, dt (13.6, 7.4) - 1.49, m 1.50, m 1.48, m 7-OCOCHCH3CH2CH3 - 0.90, t (7.2) 0.91, t (7.4) 0.89, t (7.4) - 13-OCOCH3 - - - 2.04, s 2.04, s 20-OCOCH3 - - 2.03, s - - 20-OH - - - d d a b c d e Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. At 700 MHz. Not observed. Data acquired using in a Shigemi NMR tube.

Chapter 2: The chemistry of spongian diterpenes Table 2.3 13C NMR assignments for new spongian-16-one analogues 2.2 – 2.6a Position 2.2b 2.3c,d 2.4c 2.5b 2.6c

1 42.4, CH2 42.4, CH2 36.6, CH2 34.6, CH2 34.9, CH2 2 18.7, CH2 18.7, CH2 18.6, CH2 18.7, CH2 18.8, CH2 3 43.9, CH2 44.1, CH2 43.4, CH2 41.7, CH2 41.7, CH2 4 33.2, C 33.7, C 33.5, C 32.2, C 32.3, C 5 51.2, CH 51.3, CH 51.8, CH 48.1, CH 47.9, CH 6 70.5, CH 70.6, CH 69.6, CH 23.1, CH2 23.1, CH2 7 76.1, CH 75.7, CH 75.2, CH 73.4, CH 73.6, CH 8 37.7, C 37.7, C 37.9, C 39.5, C 39.3, C 9 51.8, CH 51.9, CH 52.4, CH 50.1, CH 50.3, CH 10 36.7, C 36.8, C 40.8, C 39.5, C 41.5, C 11 17.4, CH2 17.4, CH2 19.1, CH2 18.7, CH2 18.8, CH2 12 22.0, CH2 21.8, CH2 22.5, CH2 27.3, CH2 27.5, CH2 13 37.0, CH 37.0, CH 36.9, CH 81.1, C 81.0, CH 14 41.9, CH 41.6, CH 41.9, CH 45.9, CH 45.9, CH 15 67.4, CH2 67.2, CH2 67.1, CH2 66.9, CH2 66.9, CH2 16 178.9, C 178.9, C 178.5, C 174.0, C 173.7, C 17 14.9, CH3 15.0, CH3 15.1, CH3 16.1, CH3 15.9, CH3 18eq 33.4, CH3 33.2, CH3 33.8, CH3 33.4, CH3 33.6, CH3 19ax 24.5, CH3 24.3, CH3 24.6, CH3 21.8, CH3 21.9, CH3 20 18.0, CH3 17.6, CH3 64.1, CH2 62.1, CH2 62.1, CH2 - - 7-OCOCH3 169.7, C - - - - 7-OCOCH3 21.4, CH3 - - - 7-OCOCH2CH(CH3)2 - - - - 171.9, C 7-OCOCH2CH(CH3)2 - - - - 44.0, CH2 7-OCOCH2CH(CH3)2 - - - 25.7, CH 7-OCOCH2CH(CH3)2 - - - - 22.5, CH3 22.5, CH3 7-OCOCHCH3CH2CH3 - 175.3, C 174.9, C 175.4, C - 7-OCOCHCH3CH2CH3 - 41.6, CH 41.2, CH 41.8, CH - 7-OCOCHCH3CH2CH3 - 16.9, CH3 16.9, CH3 16.9, CH3 - 7-OCOCHCH3CH2CH3 - 26.9, CH2 26.8, CH2 26.8, CH2 - 7-OCOCHCH3CH2CH3 - 11.7, CH3 11.8, CH3 11.8, CH3 - 13-OCOCH3 - - - 169.7, C 169.6, C 13-OCOCH3 - - - 21.5, CH3 21.6, CH3 20-OCOCH3 - - 170.6, C - - 20-OCOCH3 - - 21.2, CH3 - - a b c d Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. At 700 MHz. Data acquired using a Shigemi NMR tube.

Diterpene 2.3 was isolated as a colorless oil and displayed an adduct ion at m/z 443.2779 + [M+Na] in HRESIMS analysis, which established the molecular formula of C25H40O5. The HRMS data indicated an additional five carbons and three oxygens, when compared with spongian-16-one (1.24). Due to the small sample quantity (<0.06 mg), a Shigemi NMR tube was employed to increase the sensitivity of signal detection in the very diluted sample.181 The glass in Shigemi tubes is matched 1 13 to the magnetic susceptibility of the solvent used, in this case CDCl3. Examination of the H and C

NMR spectral data (Tables 2.2 and 2.3) revealed a methyl doublet at δH 1.15 and a methyl triplet at

δH 0.90 corresponding to the methyl groups in a 2-methylbutanoate moiety, along with a carboxylic ester resonance at δC 175.3. The HMBC spectrum established the presence of an ester (δC 175.3), in addition to the typical lactone carbonyl (δC 178.9) observed in spongian-16-one analogues.

Chapter 2: The chemistry of spongian diterpenes Identification of these two functional groups accounted for four of the oxygens represented in the molecular formula. The final oxygen in the molecule was inferred to be a hydroxy group. As with spectral analysis of 2.2, the doublet appearance of H-7 (J = 2.6 Hz) suggested a substituent at C-6.

HMBC correlations from H-6 (δH 4.18) to C-5 (δC 51.3), C-7 (δC 75.7) and C-9 (δC 51.9) confirmed the hydroxy group at C-6. Owing to the small quantity of the sample NOESY data could not be obtained. HMBC correlations from both δH 4.84 (H-7, d, J = 2.6 Hz) and 1.15 (7-

OCOCHCH3CH2CH3, d) to the signal at δC 175.3 confirmed the position of the 2-methylbutanoate group at C-7 (Figure 2.8). To explore the configuration of the 2’-methyl in the butanoate substituent a chiral GCMS study was carried out. The C-7 substituent should be dissociated from the main spongian structure and compared with GCMS standards of 2R- and 2S-methylbutanoate. A similar study was carried out by Rettinger et al. and Maas and co-workers.182-183 However, owing to the small sample size this was not explored further. It should be noted that in nature, the S-configuration tends to be the preferred configuration.183 The relative configuration of the tetracyclic scaffold was in parallel with 2.2, where the J-value of H-7 of 2.3 (J = 2.6 Hz) was comparable to that of 2.2 (J = 3.1 Hz), thereby suggesting a similar equatorial orientation for H-7. The specific optical rotation of 2.3 21 showed a value of [α] D -167 (c 0.006, CHCl3). The name of compound 2.3 was assigned as (-)-6α- hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate.

(A) (B)

Figure 2.8 Molecular modelling for 2.3 showing the proposed relative configuration (A) and with hydrogen atoms omitted from the model for clarity (B).

Diterpene 2.4 was isolated as a colorless oil and found to have a molecular formula of + C27H42O7 inferred from HRESIMS, which exhibited an adduct ion at m/z 501.2824 [M+Na] . This value is 58 mass units larger than that of 2.3, suggesting the addition of an acetate group. Examination of the 1H and 13C NMR spectral data (Tables 2.2 and 2.3) revealed similar signals to those of 2.3, including a methyl doublet at δH 1.15 (J = 6.9 Hz) and a methyl triplet at δH 0.91 (J = 7.4 Hz) corresponding to the methyl groups in a 2-methylbutanoate ester, in addition to an acetate methyl singlet at δH 2.03. Identical HMBC correlations for H-7 were also observed as found in 2.3.The configuration of the 2’-methyl in the ester sidechain could also not be established further owing to 46

Chapter 2: The chemistry of spongian diterpenes the small quantity (0.07 mg) of the sample. The occurrence of a doublet signal at δH 4.87 for H-7 (J = 3.2 Hz), was similar to that of 2.2 and 2.3, suggesting an identical substituent at C-6. HMBC correlations for H-6 were identical to those of 2.2 and 2.3 and confirmed the hydroxy group at C-6. NOESY correlations between H-5/H-9, H-9/H-14, H-13/H-14, H-6/H-7, H-6/Me-17 and H-7/Me-17 placed the C-6 and C-7 substituents on the opposite face to Me-17 and the same face as H-9 and H-

14 (Figure 2.9). HMBC correlations from H-20a (δH 4.79) and H-20b (δH 4.73) to the signal at δC 170.6 confirmed the position of the acetoxy group at C-20. A NOESY correlation from H-20 to Me- 17 confirmed the C-20 substituent to be on the same side of the plane as Me-17 (Figure 2.9). The 21 specific optical rotation of 2.4 showed a value of [α] D -71 (c 0.007, CHCl3). Compound 2.4 was assigned the systematic name (-)-20-acetoxy-6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)- butanoate.

(A) (B)

Figure 2.9 Molecular modelling for 2.4 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Metabolite 2.5 was isolated as a colorless oil and also displayed a sodiated ion at m/z 501.2831 + [M+Na] from HRESIMS for C27H42O7, which corresponded to the same molecular formula as 2.4. Inspection of the 1H and 13C NMR spectral data (Table 2.2 and 2.3) similarly revealed the occurrence of a 2-methylbutanoate substituent as observed in 2.3 and 2.4. The presence of three ester carbonyls

(δC 169.7, 174.0, and 175.4) identified six of the oxygen atoms present in the molecular formula.

HMBC correlations from both δH 4.85 (H-7, t, J = 2.8 Hz) and 1.15 (7-OCOCHCH3CH2CH3, d) to the signal at δC 175.4 again confirmed the position of the 2-methylbutanoate group at C-7. The Me-

20 of spongian-16-one (1.24) was replaced with an oxymethylene H2-20 (δH 4.05 and δH 3.92) in 2.5. It was inferred that the hydroxy group was located at C-20, identifying the final oxygen atom. The J- value of H-14 (δH 2.92, dd, J = 1.5, 6.5 Hz) of 2.5 had changed from that seen in 2.2 (J = 5.6, 8.1 Hz) and 2.3 (J = 5.6, 7.8 Hz), which suggested a substituent at C-13. HMBC correlations from H-12a (δH 47

Chapter 2: The chemistry of spongian diterpenes

2.28), H-14 (δH 2.92) and H-15b (δH 4.20), as well as the signals at δH 2.04 (13- OCOCH3) and 2.92

(H-14) to the signal at δC 81.1 (C-13), confirmed that the position of the acetate group at C-13. NOESY correlations observed between H-7/Me-17 and H-20/Me-17 confirmed the relative configuration was identical to 2.4, however, the configuration of the acetate at C-13 and the 2’-methyl in the butanoate substituent could not be confirmed by NMR (Figure 2.10).

(A) (B)

Figure 2.10 Molecular modelling for 2.5 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Metabolite 2.5 was crystallized from 10% EtOAc/hexanes producing small needle-shaped crystals which were suitable for diffraction (Figure 2.11). The crystal structure confirmed the relative configuration at C-13 and the absolute configuration was assigned as 5S, 7R, 8R, 9R, 10R, 13S, 14R, 2’S. The cyclohexane rings A, B and C adopted a chair conformation. As a result, adjacent molecules interact through hydrogen bonds O(7)H•••O(4) = 2.17 Å, 167 ° resulting in the formation of an undulating one-dimensional polymeric chains that extend down parallel to the crystallographic a- 21 axis. The specific optical rotation of 2.5 showed a value of [α] D -12 (c 0.12, CHCl3). The name of compound 2.5 was assigned as (-)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)- butanoate.

Chapter 2: The chemistry of spongian diterpenes

Figure 2.11 ORTEP184 representation of the crystal structure of (5S, 7R, 8R, 9R, 10R, 13S, 14R, 2’S)- 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate 2.5 shown with 30% probability ellipsoids.

The new spongian-16-one analogue 2.6, a colorless oil, exhibited an adduct ion at m/z + 501.2831 [M+Na] in the HRESIMS, corresponding to a molecular formula of C27H42O7, which was the same molecular formula observed for 2.4 and 2.5. Scrutiny of the 1H and 13C NMR spectral data

(Table 2.2), revealed an acetate methyl singlet at δH 2.04 as well as an oxymethylene signals at δH

4.04 (d, J = 11.8 Hz) and 3.91 (dd, J = 11.8 Hz) corresponding to H2-20, similar to comparable signals for 2.5. The major difference however was the presence of two methyl doublets at δH 0.95 (d, J = 6.6 Hz) and 0.96 (dd, J = 6.6 Hz), instead of the doublet and triplet signals for the methyl groups of the 2-methylbutanoate substituent in 2.3, 2.4 and 2.5, suggesting a possible isomer. HMBC correlations from the signals at δH 4.86 (H-7, t, J = 2.6 Hz), 2.11 (7-OCOCH2CH(CH3)3, m) and the methylene signals at δH 2.22 (7-OCOCH2CH(CH3)3, m, 2H) to the carbon at δC 171.9 confirmed the 3- methylbutanoate group was attached at C-7. These values were comparable to those in the 1H NMR spectrum of 7α-11α-dioxyspongian-16-one-7α-isopentanoate-11α-propionate (1.47).56 The oxymethylene signals at δH 4.04 (d, J = 11.8 Hz) and 3.91 (dd, J = 11.8 Hz) in 2.6, were comparable to those seen in 2.5, suggesting a hydroxy group was located at C-20. The similarity of the signal pattern corresponding to H-14 (δH 2.91, dd, J = 1.2, 6.3 Hz) to that in 2.5 confirmed the occurrence of the acetate substituent at C-13. Furthermore, HMBC correlations from both δH 2.04 (13-OCOCH3) and 2.91 (H-14) to δC 81.0 (C-13) established the position of the acetate group at C-13. NOESY

Chapter 2: The chemistry of spongian diterpenes correlations determined the relative configuration of C-7 and C-10 to be identical to that of 2.4 and 2.5, however the configuration at C-13 again could not be confirmed by NMR (Figure 2.12). The 21 specific optical rotation of 2.6 showed a value of [α] D -19 (c 0.08, CHCl3). Compound 2.6 was assigned the name (-)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate.

(A) (B)

Figure 2.12 Molecular modelling for 2.6 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

13-Acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate 2.6 was also crystallized from 10% EtOAc/hexanes producing small needle-shaped crystals (Figure 2.13). The cyclohexane rings A, B and C adopt a chair conformation. The crystal structure confirmed the relative configuration at C-13, however the C-7 substituent showed disorder at the terminus of the 3- methylbutanoate. On scrutiny of the 1H NMR of 2.6 prior to crystallization it became apparent that the sample contained a very minor quantity of metabolite 2.5. A 1H NMR of a single crystal of 2.6 was measured at 500 MHz and diagnostic signals for the C-7 substituent methyl signals in 2.5 and 2.6 were observed in a 1:0.8 ratio, respectively. With this evidence of co-crystallization the crystal data was re-examined and modelled with 25% occupancy of 2.6 while the remaining 75% was metabolite 2.5, which gave the appearance of disorder at C-2’, C-3’, C-4’ and C-5’(Figure 2.13). The crystal packing was similar to that observed in the crystal structure of 2.5, being dominated by hydrogen bonds O(7)H•••O(4) = 2.16 Å, 150 ° resulting in the formation of undulating one- dimensional polymeric chains that extend down parallel to the crystallographic a-axis.

Chapter 2: The chemistry of spongian diterpenes

Figure 2.13 ORTEP184 representation of the crystal structure of 13-acetoxy-20-hydroxy-7α- oxyspongian-16-one-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate 2.6 shown with 30% probability ellipsoids. Only one component of each region of disorder is shown.

2.1.3 The isolation of metabolites from Goniobranchus sp 1. Three specimens of Goniobranchus sp. 1 were collected from Mudjimba and Gneerings Reef (Mooloolaba, Queensland) in October 2016. The extraction and chemical profile of the metabolites from the mantle and viscera tissue were carried out based on the previously described procedures (Figure 2.15). 1H NMR spectral data again suggested the presence of oxygenated diterpenes. A total of fifteen spongian diterpene metabolites were isolated from G. sp 1. From the mantle isoagatholactone (1.19),45 12α-acetoxyspongian-16-one (1.38),18 20-acetoxyspongian-16-one (1.39),18 20-oxyspongian-16-one-propoionate (1.40),18 12α,20-dioxyspongian-16-one-dipropionate (1.41),18 12α,20-diacetoxyspongian-16-one (1.42),56 12α-acetoxy, 20-oxyspongian-16-one-20- propionate (1.43),56 20-acetoxy-12α-oxyspongian-16-one-12α-propionate (2.7), 20-acetoxy-13- hydroxyspongian-16-one (2.8), 12-hydroxyspongian-16-one (2.9), 12-hydroxy-20-oxyspongian-16- one-20-propionate (2.10), 12-hydroxy-11,20-dioxyspongian-16-one-11,20-dipropionate (2.11), and 11-hydroxy-12,20-dioxyspongian-16-one-12,20-dipropionate (2.12) were also isolated. While from the viscera spongian-16-one (1.24)49,50 and 7α-acetoxyspongian-16-one (1.32),52 along with 1.19, 1.38, 1.39 and 1.43 were isolated. The new compounds (2.7-2.12) demonstrate a high level of oxidation, in particular at positions C-11, C-12, C-13 and C-20. The structures and relative 51

Chapter 2: The chemistry of spongian diterpenes configuration were determined from the analysis of 2D NMR spectra; COSY, HSQC, HMBC, and NOESY experiments.

Goniobranchus sp 1 [3 specimens]

Dissected Acetone Extraction

Partition with Et2O /H2O

Mantle Viscera

Crude Extract (96.8 mg) Crude Extract (71.2 mg)

NP-flash chromatography NP-flash chromatography (hexanes: DCM: EtOAc: MeOH) (hexanes: DCM: EtOAc: MeOH)

Fractions D-H Fractions B-E

NP-HPLC NP-HPLC 30% EtOAc/ hexanes 30% EtOAc/hexanes

1.19, 1.38, 1.39, 1.40, 1.41, 1.19, 1.24, 1.32, 1.38, 1.39, 1.42, 1.43, 2.7, 2.8, 2.9, 2.10, 1.43 2.11, 2.12

Figure 2.14 Isolation scheme of Goniobranchus sp. 1 compounds.

2.1.3.1 Structure elucidation of new spongian diterpenes from Goniobranchus sp 1.

Chapter 2: The chemistry of spongian diterpenes Diterpene 2.7 was obtained as a colorless oil from NP-HPLC and exhibited a sodiated + 1 13 molecular ion peak in the HRESIMS at m/z 457.2566 [M+Na] (C25H38O6). The H and C NMR spectral data (Tables 2.4 and 2.5) indicated an acetoxy methyl singlet at δH 2.09 and a quartet (2H) at δH 2.33 and a triplet at δH 1.16 and carbonyl resonance at δC 173.0 corresponding to propionate methylene and methyl signals. Carbon to proton correlations of two ester carbonyls (δC 170.8 and

173.0) were observed in the HMBC spectrum, in addition to the typical lactone carbonyl (δC 174.9) observed in spongian-16-one analogues and accounted for all six oxygen atoms expected from the molecular formula. The NMR data was found to be very similar to 12α-acetoxy,20-oxyspongian-16- one-20-propionate (1.43), which was previously elucidated as part of my Honors thesis. When compared with 2.7, there were some obvious differences in the location of substituents.56

Oxymethylene signals at δH 4.56 (d) and 4.13 (m) corresponded to those of H2-20 in 20- 18 acetoxyspongian-16-one (1.39). These two signals, plus the acetate methyl signal at δH 2.03 (s), all correlated to the carbon signal at δC 170.3 and C-10 (δC 39.8), confirming the position of the acetate group at C-20. There was a NOE from H-20b to Me-17, placing the acetate on the same orientation as Me-17 above the plane as illustrated in Figure 2.15. HMBC correlations from the signals at δH

5.44 (H-12), methylene signals δH 2.33 (2H, q) and 2.09 (12-OCOCH2CH3, t) to the carbon of δC 173.0 confirmed the position of the propionate group at C-12 and were comparable to those in the 1H NMR spectrum of 12α,20-dioxyspongian-16-one-dipropionate (1.41), a compound isolated by a previous member of the Garson group.18 The relative configuration of 2.7 was assigned as follows: NOESY correlations between H-5/H-9, H-9/H-14, H-13-H-14, H-20/Me-17 and H-12/Me-17 placed H-12 and H-20 on the same face as Me-17 (Figure 2.15). The specific optical rotation of 2.7 showed 21 a value of [α] D -29 (c 0.021, CHCl3). Compound 2.7 was named (-)-20-acetoxy-12α-oxyspongian- 16-one-12α-propionate.

(A) (B)

Figure 2.15 Molecular modelling for 2.7 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Chapter 2: The chemistry of spongian diterpenes Table 2.4 1H NMR assignments for new spongian-16-one analogues 2.7 - 2.12a Position 2.7c 2.8c 2.9b 2.10b 2.11c 2.12c 1 eq 2.03, m 2.12, m 1.68, br d (12.8) 2.07, br d (13.2) 2.03, br d (13.4) 2.02, br d (13.6) 1ax 0.62, m 0.80, m 0.83, m 0.79, td (13.2, 2.3) 0.75, m 1.15, m 2 eq 1.54, m 1.56, m 1.61, m 1.57, m 1.61, m 1.62, m 2ax 1.43, m 1.45, m 1.42, m 1.46, m 1.47, m 1.50, m 3eq 1.45, m 1.45, m 1.38, m 1.45, m 1.45, m 1.45, br d (12.9) 3ax 1.16, m 1.17, m 1.15, td (13.2, 3.7) 1.19, m 1.16, m 1.18, m 4 ------5 1.03, m 1.01, dd (2.1, 12.4) 0.89, m 1.08, dd (1.7, 12.3) 1.05, dd (12.7, 2.4) 1.11, m 6eq 1.57, m 1.56, m 1.55, m 1.58, m 1.66, m 1.63, m 6ax 1.40, m 1.38, m 1.35, m 1.39, m 1.45, m 1.49, m 7eq 1.92, m 1.88, m 1.82, dt (12.8, 3.3) 1.91, dt (12.8, 3.3) 1.76, dt (12.6, 3.1) 1.76, dt (12.6, 3.2) 7ax 1.16, m 1.16, m 1.09, dt (12.8, 3.5) 1.17, m 1.06, m 1.06, td (12.6, 3.7) 8 ------9 1.33, m 1.04, m 1.32, dd (6.3, 9.1) 1.51, m 1.35, d (2.9) 1.34, d (2.9) 10 ------11eq 2.00, m 1.88, m 1.63, m 1.87, m - - 11ax 1.80, dd (3.4, 13.2) 1.49, m 1.63, m 1.85, m 5.95, t (3.4) 4.46, t (3.0) 12eq 5.44, br s 2.63, m 4.52, br s 4.49, br s 4.36, m 5.54, dd (9.1, 3.5) 12ax - 1.62, m - - - - 13 2.67, dt (8.0, 1.5) - 2.66, d (8.0) 2.65, d (7.9) 2.84, dd (10.9, 9.4) 3.00, dd (10.6, 9.3) 14 2.29, dd (5.2, 8.0) 1.94, dd (5.6, 7.8) 2.33, dd (5.4, 8.0) 2.37, dd (5.4, 7.9) 2.44, m 2.44, m 15eq 4.26, d (9.9) 4.42, dd (5.6, 9.4) 4.23, d (9.7) 4.26, d (9.9) 4.33, m 4.28, m 15ax 4.12, m 4.13, dd (, 7.8, 9.4) 4.11, dd (5.4, 9.7) 4.13, dd (5.4, 9.9) 4.33, m 4.28, m 16 ------17 0.90, s 0.88, s 0.82, s 0.89, s 0.95, s 0.94, s 18 0.89, s 0.89, s 0.86, s 0.90, s 0.87, s 0.87, s 19 0.83, s 0.83, s 0.81, s 0.85, s 0.81, s 0.82, s 20 4.56, d (12.4) 4.55, d (13.1) 0.82, s 4.59, d (12.1) 4.74, d (12.0) 4.61, d (12.2) 4.13, m 4.14, d (13.1) 4.17, d (12.1) 3.96, d (12.0, 1.9) 4.02, d (12.2, 1.8) 11-OH - - - - - 2.08, br s 11-OCOCH2CH3 - - - - 2.34, m - 2.34, m 11-OCOCH2CH3 - - - - 1.15, t (7.7) - d d 12-OH - - 2.79, br s - 12-OCOCH3 ------12-OCOCH2CH3 2.33, q (7.6) - - - - 2.46, m 2.33, q (7.6) 2.40, m 12-OCOCH2CH3 1.16, t (7.7) - - - - 1.18, t (7.6) 13-OH - d - - - - 20-OCOCH3 2.03, s 2.02, s - - - - 20-OCOCH2CH3 - - - 2.31, q (7.7) 2.46, m 2.50, m 2.31, q (7.7) 2.46, m 2.45, m 20-OCOCH2CH3 - - - 1.13, t (7.7) 1.18, t (7.7) 1.12, t (7.5) a b c d Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. At 700 MHz. Not observed.

Chapter 2: The chemistry of spongian diterpenes Table 2.5 13C NMR assignments for new spongian-16-one analogues 2.7 – 2.12a Position 2.7c 2.8c 2.9b 2.10b 2.11c 2.12c 1 35.1, CH2 35.4, CH2 39.9, CH2 35.1, CH2 33.8, CH2 33.8, CH2 2 18.3, CH2 18.5, CH2 18.5, CH2 18.4, CH2 18.2, CH2 18.2, CH2 3 41.5, CH2 41.6, CH2 42.1, CH2 41.7, CH2 41.3, CH2 41.5, CH2 4 32.8, C 33.0, C 33.1, C 33.1, C 33.0, C 32.9, C 5 57.1, CH 57.0, CH 56.8, CH 56.9, CH 58.0, CH 58.2, CH 6 17.8, CH2 17.9, CH2 18.1, CH2 17.9, CH2 17.6, CH2 18.1, CH2 7 42.2, CH2 42.8, CH2 42.0, CH2 42.2, CH2 42.1, CH2 41.9, CH2 8 35.6, C 36.3, C 35.5, C 36.0, C 35.1, C 33.2, C 9 50.0, CH 56.7, CH 48.8, CH 49.0, CH 62.6, CH 64.2, CH 10 39.8, C 40.4, C 36.1, C 40.2, C 41.2, C 40.7, C 11 25.6, CH2 19.4, CH2 27.1, CH2 29.2, CH2 70.4, CH 67.7, CH 12 67.9, CH 28.1, CH2 65.1, CH 64.9, CH 66.4, CH 69.5, CH 13 43.1, CH 83.7, C 45.5, CH 45.6, CH 41.6, CH 38.7, CH 14 49.1, CH 54.5, CH 48.3, CH 48.6, CH 47.6, CH 47.7, CH 15 67.7, CH2 67.1, CH2 67.9, CH2 68.0, CH2 67.7, CH2 66.9, CH2 16 174.9, C 173.6, C 176.3, C 176.4, C 180.3, C 178.2, C 17 15.3, CH3 15.5, CH3 15.4, CH3 15.2, CH3 17.7, CH3 17.6, CH3 18eq 33.9, CH3 33.9, CH3 33.5, CH3 33.9, CH3 33.6, CH3 33.6, CH3 19ax 22.1, CH3 22.0, CH3 21.6, CH3 21.9, CH3 21.9, CH3 21.7, CH3 20 64.3, CH2 64.3, CH2 16.5, CH3 64.5, CH2 64.4, CH2 64.7, CH2 11-OCOCH2CH3 - - - - 173.6, C - 11-OCOCH2CH3 - - - - 27.9, CH2 - 11-OCOCH2CH3 - - - - 9.2, CH3 - 12-OCOCH2CH3 173.0, C - - 174.5, C - 172.6, C 12-OCOCH2CH3 28.0, CH2 - - 27.9, CH2 - 27.7, CH2 12-OCOCH2CH3 9.4, CH3 - - 9.3, CH3 - 9.1, CH3 20-OCOCH3 170.8, C 170.8, C - - - - 20-OCOCH3 21.2, CH3 21.2, CH3 - - - - 20-OCOCH2CH3 - - - - 174.2, C 175.5, C 20-OCOCH2CH3 - - - - 27.6, CH2 27.4, CH2 20-OCOCH2CH3 - - - - 9.0, CH3 9.4, CH3 a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. At 700 MHz.

Metabolite 2.8, which was isolated as a colorless oil, exhibited an adduct ion at m/z 401.2291 [M+Na]+ from HRESIMS, which corresponded to the same molecular formula as 2.2, implying a spongian-16-one carbon skeleton with an acetate group and a hydroxy group. This was confirmed on scrutiny of the 1H and 13C NMR spectral data (Table 2.4), which revealed an acetate methyl singlet at δH 2.02. Oxymethylene signals at δH 4.55 (d, J = 13.1 Hz) and 4.14 (d, J = 13.1 Hz) were consistent with those of H2-20 in 2.7. The HMBC correlations from the signals at δH 4.55 and 4.14, as well as from δH 2.02 to the carbon at δC 170.8 and C-10 ( δC 40.4) confirmed the position of an acetate group at C-20. The C-20 assignment matched similar signals in 20-acetoxyspongian-16-one (1.39).18 There was a NOE between H-20a/Me-19 and H-20b/Me-17, which placed the C-20 acetate on the same orientation (Figure 2.16). The upfield change in chemical shift for H-14 (δH 1.94, dd, J = 5.6, 7.8 Hz) suggested the occurrence of an electronegative substituent at C-13. HMBC correlations from signals at δH 1.94 (H-14) and 4.13 (H-15) to the quaternary carbon at δC 81.0 (C-13), confirmed the position of the hydroxy group at C-13. The configuration of the tertiary hydroxy group at C-13 was not explored further owing to the small quantity (0.2 mg) of the sample. NOESY correlations observed between H-5/H-9 and H-9/H-14 confirmed the remaining overall stereochemistry. The specific

Chapter 2: The chemistry of spongian diterpenes

21 optical rotation of 2.8 showed a value of [α] D -22 (c 0.017, CHCl3). The name compound 2.8 was assigned the systematic name (-)-20-acetoxy-13-hydroxyspongian-16-one.

(A) (B)

Figure 2.16 Molecular modelling for 2.8 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Metabolite 2.9 was isolated as a colorless oil and displayed a sodiated ion at m/z 343.2245 + [M+Na] from HRESIMS for C20H32O3, corresponding to the addition of 16 mass units to spongian- 16-one (1.24),49-50 suggesting the addition of a hydroxy group. gCOSY correlations from the oxygenated methine proton signal at δH 4.52 (br s) to δH 1.63 (H2-11) and 2.66 (H-13) established the hydroxy group at the C-12 position. HMBC correlations further confirmed the assignment of the hydroxy group at C-12. The cis configuration of the C/D ring junction was confirmed by the 8.0 Hz coupling and NOE correlation between H-13 and H-14. The remaining NOESY correlations observed between H-5/H-9, H-9/H-14 and H-12/Me-17 confirmed the overall stereochemistry (Figure 2.17). 21 The specific optical rotation of 2.9 showed a value of [α] D -58 (c 0.017, CHCl3). Compound 2.9 was named as (-)-12α-hydroxyspongian-16-one. (A) (B)

Figure 2.17 Molecular modelling for 2.9 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Diterpene 2.10 was isolated as a colorless oil and produced an adduct ion at m/z 415.2458 + 1 [M+Na] from HRESIMS for C23H36O5. The H NMR spectrum (Table 2.4 and 2.5) indicated a quartet (2H) at δH 2.31 and a triplet at δH 1.13 corresponding to propionate methylene and methyl signals. Oxymethylene signals at δH 4.59 (d) and 4.17 (m) corresponded to those of H2-20 in 20-

Chapter 2: The chemistry of spongian diterpenes

18 oxyspogian-16-one-propionate (1.40). The HMBC correlations from the signals at δH 4.59 and 4.17, as well as from δH 2.31 (2H) and 1.13 to the carbon at δC 174.5 and C-10 ( δC 40.2) confirmed the position of a propionate group at C-20. The signals at δH 4.49 (H-12, br s) and 2.65 (H-13) were comparable to those in the 1H NMR spectrum of 2.9, positioning the hydroxy group at C-12. The cis configuration of the C/D ring junction was established by the 7.9 Hz coupling between H-13 and H- 14. An NOE between Me-17, H-12 and H-20 positioned the propionate on the similar side of the plane and the 12-OH on the opposite side (Figure 2.18). The specific optical rotation of 2.10 showed 21 a value of [α] D -7 (c 0.097, CHCl3). The name of compound 2.10 was assigned as (-)-12α-hydroxy- 20-oxyspongian-16-one-20-propionate.

(A) (B)

Figure 2.18 Molecular modelling for 2.10 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

Metabolite 2.11, a colorless oil, exhibited an adduct ion at m/z 487.2668 [M+Na]+ in the

HRESIMS, corresponding to a molecular formula of C26H40O7. The observed mass was 16 mass units larger than 12α, 20-dioxyspongian-16-one-dipropionate (1.41).18 The 1H NMR spectrum revealed two multiplets at δH 2.34 (2H) and 2.46 (2H), and two methyl triplets at δH 1.15 (J = 7.7 Hz) and 1.18

(J = 7.7 Hz). The addition of two ester carbonyls (δC 173.6 and 174.2) and the lactone carbonyl (δC 176.4) located six of the oxygen atoms, with the seventh oxygen atom inferred to be an additional hydroxy group. Oxymethylene signals at δH 4.59 (d, J = 12.0 Hz) and 3.96 (dd, J = 1.9, 12.0 Hz) 18 corresponded to those of H2-20 in 2.10 and 20-oxyspongian-16-one-propionate (1.40). HMBC correlations (Table 2.4) from the signals at δH 4.59 and 3.96, as well as from δH 2.46 (2H) and 1.18 to the carbon at δC 174.2 and C-10 (δC 41.2) confirmed the position of a propionate group at C-20. A substituent was proposed to be attached at C-11, since the multiplicity of the adjacent H-9 signal at

δH 1.35 was changed to a doublet rather than a doublet of doublets as seen in 2.9. HMBC correlations from H-11 (δH 5.95) and 11-OCOCH2CH3 (δH 2.34 and 1.15) to the propionate carbonyl at δC 173.6 confirmed the propionate group on C-11. gCOSY correlations from H-11 and H-13 (δH 2.84) to H-

Chapter 2: The chemistry of spongian diterpenes 12 confirmed the hydroxy group at C-12. NOESY correlations observed between H-5/H-9, H-9/H- 14, H-13/H-14, H-9/H-11, H-20b/Me-17, H-12/Me-17 confirmed the overall stereochemistry (Figure 2.19) which is similar to those of 2.7, 2.8, 2.9 and 2.10. The specific optical rotation of 2.11 showed 21 a value of [α] D -35 (c 0.017, CHCl3). Compound 2.11 was named systematically as (-)-12α-hydroxy- 11β,20-dioxyspongian-16-one-11β,20-dipropionate.

(A) (B)

Figure 2.19 Molecular modelling for 2.11 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

The new spongian-16-one analogue 2.12 was isolated as a colorless oil and produced an adduct ion at m/z 487.2668 [M+Na]+, giving the same molecular formula as 2.11, implying two propionate groups and a hydroxy group. This was established on scrutiny of the 1H and 13C NMR spectral data (Table 2.4), which revealed two multiplets at δH 2.46-2.40 (2H) and δH 2.50-2.45 (2H), and two triplets at δH 1.18 (J = 7.6 Hz) and 1.12 (J = 7.5 Hz). Similar to 2.11, two ester carbonyls (δC

173.6 and 174.2) and a lactone carbonyl (δC 176.4) were identified, and the remaining oxygen assigned to a hydroxy group. HMBC correlations (Table 2.5) from the signals at δH 4.61 (d, J = 12.2

Hz) and 4.02 (dd, J = 1.8, 12.2 Hz), as well as from δH 2.50, 2.46 and 1.12 to the carbon at δC 175.5 confirmed the position of a propionate group at C-20. The H2-20 oxymethylene signals were 18 consistent with those of H2-20 in 20-oxyspongian-16-one-propionate (1.40). HMBC correlations from signals at δH 5.54 (H-12, dd, J = 3.5, 9.1 Hz), 2.46 (m), 2.40 (m) and 1.18 (t, J = 7.6 Hz) to the signal at δC 172.6, confirmed the position of the second propionate group at C-12. These signals were consistent with the signals seen for H-12 in 2.7. The NOE correlation from H-12 to Me-17 placed the propionate substituent in the α-position. Lastly, the occurrence of a signal at δH 1.34 (d, J = 2.9 Hz) for H-9, suggested a substituent at the C-11 position. gCOSY correlations from H-12 and H-9 to H- 11 established the hydroxy group at C-11. The cis configuration of the C/D ring junction was deduced by the 10.6 Hz coupling and NOE correlation between H-13 and H-14. In addition, the large 9.1 Hz coupling between H-13 and H-12ax placed H-13 in an equatorial orientation. The remaining NOESY correlations observed between H-5/H-9, H-9/H-14, H-9/H-11, H-20b/Me-17, H-12/Me-17 confirmed

Chapter 2: The chemistry of spongian diterpenes the overall stereochemistry (Figure 2.20). The specific optical rotation of 2.12 showed a value of 21 [α] D - 64 (c 0.011, CHCl3). Compound 2.12 was named as (-)-11β-hydroxy-12α,20-dioxyspongian- 16-one-12α,20-dipropionate.

(A) (B)

Figure 2.20 Molecular modelling for 2.12 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

2.1.4 The isolation of metabolites from Goniobranchus leopardus (Rudman, 1987) Five specimens of G. leopardus were collected from Coolum (2017), QLD, Australia. The specimens were dissected into mantle and viscera tissue and each body part was separately chopped finely, extracted using previously described methods. Due to similarities in the 1H NMR profile for each extract the mantle extracts were combined, as were the viscera extracts. A total of thirteen diterpenes were isolated using NP-HPLC (Figure 2.21). In total thirteen oxygenated diterpenes were isolated from G. leopardus, with the new isospongian diterpene analogue 12α-acetoxypolyrhaphin D (2.13), along with the known metabolites norrisolide (1.130),97 dendrillolide A (1.142),110,111 aplyviolene (1.144),112 macfarlandin D (1.145),111 15,16-diacetoxyshahamin B (1.160),118 12- desacetoxypolyrhaphin A (1.163),113 12-desacetoxyshahamin C (1.163),113 secoshahamin (1.164),108 and macfarlandin C (1.165)111 (see Chapter 1) were isolated from both the mantle and viscera tissue. While the metabolites chelonaplysin C (1.131)100 cheloviolene C (1.132),100 and dendrillolide E (1.172)113 were only isolated from the viscera tissue and the new metabolite 10,20-epoxydendrillolide A (3.4) (Chapter 3) was isolated from the mantle tissue. The new isospongian diterpene analogue 2.13 possesses a rare lactone moiety.

Chapter 2: The chemistry of spongian diterpenes

G. leopardus [5 specimens]

Dissected Acetone Extraction

Partition with EtOAc /H2O

Mantle Viscera

Crude Extract (43.3 mg) Crude Extract (49.7 mg)

NP-flash chromatography NP-flash chromatography (hexanes: DCM: EtOAc: MeOH) (hexanes: DCM: EtOAc: MeOH)

Fractions B-D Fractions C-E

NP-HPLC NP-HPLC 25% EtOAc/hexanes 25% EtOAc/hexanes

1.130, 1.142, 1.144, 1.145, 1.162, 1.130, 1.131, 1.132, 1.142, 1.144, 1.163, 1.164, 1.165, 2.13, 3.4 1.145, 1.162, 1.163, 1.164, 1.165, 1.172, 2.13

Figure 2.21 Isolation scheme of G. leopardus compounds.

2.1.4.1 Structure elucidation of 12α-acetoxypolyrhaphin D (2.13) A new spongian-16-one analogue 2.13 was isolated from both the viscera and mantle of G. leopardus as a colorless oil. The HRESIMS analysis of the compound showed an adduct ion at m/z 383.2191 [M+Na]+, corresponding to 2 mass units less than that of 12α-acetoxyspongian-16-one (1.38),18 which suggested the presence of a double bond. This was established on scrutiny of the 1H 13 and C NMR spectral data (Table 2.6), which revealed an alkene proton at δH 5.95 and an additional acetate methyl signal at δH 2.08. HMBC correlations from the olefinic proton signal at δH 5.95 to the distinctive lactone carbonyl carbon at δC 172.8 (C-15) and δC 89.6 (C-16) strongly suggesting a Δ13,14 double bond giving an α,β-unsaturated γ-lactone. Additional HMBC correlations from the olefinic H-

14 (δH 5.95) to C-13 (δC 163.1) and C-12 (δC 66.2) allowed assignment of these signals. Assignment 1 of the C-12 signal allowed confirmation that the low field H NMR signal at δH 5.79 corresponded to

H-12. This proton signal displayed HMBC correlations to the acetate carbonyl at δC 169.6 which also exhibited a correlation to the acetate methyl at 2.08. These data confirmed the position of the acetate 60

Chapter 2: The chemistry of spongian diterpenes group at C-12 and were comparable to the signals in the 1H NMR spectrum of 12α-acetoxyspongian- 16-one (1.38).18 It was concluded therefore that the alkene in 2.13 was adjacent to the acetate substituent, thus the terpene was an α,β,γ-unsaturated ester in addition to being an α,β-unsaturated lactone. This unusual motif is an example of the less common isospongian skeleton, previously observed in polyrhaphin D (2.14).117 Polyrhaphin D (2.14) was isolated from the sponge Aplysilla polyrhaphis, collected from California (USA). It has a γ‐butenolide as the D ring instead of the typically isolated furan ring structure found in other isospongian diterpenes.117 The gCOSY spectrum for 2.13 showed a long range coupling from H-16 at δH 4.58 (d, J = 1.6 Hz) to H-14 at δH 5.95 (d, J = 1.6 Hz). NOESY correlations from H-5/H-9, H-9/H-16, Me-20/Me-17 established the relative 21 configuration (Figure 2.22). The specific optical rotation of 2.13 showed a value of [α] D + 48 (c

0.038, CHCl3). The name of compound 2.13 was assigned as (+)-12α-acetoxypolyrhaphin D.

(A) (B)

Figure 2.22 Molecular modelling for 2.13 showing key NOESY correlations (A) and with hydrogen atoms omitted from the model for clarity (B).

The formation of the unsaturated lactone ring seen in 2.13 is proposed to occur through different biosynthetic steps, compared to that of spongian-16-one (1.24) (Scheme 2.1). The key difference is the precursor, where the precursor geranylgeranyl diphosphate (GGPP) undergoes double bond isomerization prior to concerted cyclization forming ring A, B and C. Subsequent hydrolysis of the diphosphate to an alcohol, followed by oxidation, lactonization and acetylation provides 12α-acetoxypolyrhaphin D.

Chapter 2: The chemistry of spongian diterpenes

Scheme 2.1 Putative biosynthesis for isospongian 12α-acetoxypolyrhaphin D (2.13).

Table 2.6 1H NMR assignments for 12α-acetoxypolyrhaphin D (2.13)a,b Position 13C 1H (mult., J, Hz) HMBC COSY NOESY

1 eq 40.3, CH2 1.65, m Me-20 - - 1ax 0.89, m 2eq 18.5, CH2 1.61, m - - - 2ax 1.45, m 3eq 41.7, CH2 1.41, m - - - 3ax 1.19, m 4 33.2, C - H-5, Me-18, Me-19 - - 5 56.6, CH 0.96, m H2-6, H2-7, Me-18, Me-19, Me-20 H-6 - 6eq 18.2, CH2 1.66, m H-7eq, Me18 H-5, H-7 H-7ax 6ax 1.37, m 7eq 40.3, CH2 2.05, m H-16, Me-17 H-6 H2-6, Me-17 7ax 1.48, m H-5 8 42.3, C - H-16, Me-17 - - 9 48.9, CH 1.49, m H-11ax, H-12, Me-17, Me-20 H-11 - 10 37.3, C - H-5, H-9, Me-20 H-9 - 11eq 27.1, CH2 1.99, m H-9 - H2-11 11ax 1.64, m d 12eq 66.2, CH 5.79, dd (2.5, 3.2) H-9, H-11eq, H-14, 12-COOCH3 H-14, - 13 163.1, C - H-11eq, H-12, H-14, H-16 - - 14 115.5, C 5.95, d (1.6) H-12, H-16 H-12,d H-16,d Me-17 15 173.3, C - H-14, H-16 - - 16 89.9, CH 4.58, d (1.6) H-7ax/H-9, H-12, H-14, Me-17 H-14,d H-7ax 17 11.7, CH3 0.67, s H2-7, H-16 - H-6ax, H-7eq, Me-20 18 33.5, CH3 0.90, s - H-3ax, Me-19 19 21.6, CH3 0.83, s H-3ax, H-5, Me-18 - H-11ax, Me-17 20 16.2, CH3 0.85, s H-5, H-9 - - 7-OCOCH3 169.6, C - H-12, 12-COOCH3 - - 7-OCOCH3 21.2, CH3 2.08, s - - a b c d Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. HMBC correlations from C to H. W- coupling.

Chapter 2: The chemistry of spongian diterpenes 2.2 Conclusions A thorough isolation work was conducted on G. collingwoodi, G. aureopurpureus, Goniobranchus sp. 1, and G. leopardus affording a total of thirteen new spongian diterpenes. The new spongian diterpenes elucidated showed a proliferation of oxidation occurring at various positions on C-6, C-7, C-11, C-12, C-13 and/or C-20. A new metabolite 7α-acetoxyisoagatholactone (2.1), along with five known metabolites (1.16, 1.19, 1.24, 1.32, and 1.33) were isolated from G. collingwoodi. G. aureopurpureus, which was found to consist of both spongian diterpenes and their rearranged congeners, where six metabolites (2.2-2.6 and 4.4) were identified as new spongian-16- one analogues. The two X-ray structures of 2.5 and 2.6, respectively also provided insight into the absolute configuration of the parent structure spongian-16-one (1.24). From Goniobranchus sp. 1 six new spongian metabolites (2.7-2.12) were isolated, along with eight known metabolites (1.19, 1.24, 1.32, and 1.38-1.43). Finally, G. leopardus was found to contain two new metabolites (2.13 and 3.4) in conjunction with eleven known metabolites (1.130-1.132, 1.142, 1.144, 1.145, 1.160, 1.163-1.165 and 1.172). Notably all the new spongian-16-one analogues 2.1-2.12 had negative specific optical rotations with the exception of 2.13 which had a positive specific optical rotation. As many of the new highly oxygenated spongian diterpenes were only isolated in the mantle tissue, it could be argued that the nudibranch could be oxidising a precursor to increase the activity of these compounds to use a chemical defense against predation. The anatomical distribution will be further discussed for all Goniobranchus species in Chapter 5. Biosynthetically, metabolites 2.1-2.12 are all derived from the parent scaffold spongian-16-one (1.24) displayed in Scheme 2.2. As the metabolites found in these nudibranchs may have likely been sequestered from the nudibranchs sponge prey, further investigation should be undertaken into the dietary sponges of Goniobranchus to better understand the biosynthetic origins of these metabolites.

Chapter 2: The chemistry of spongian diterpenes

Scheme 2.2 Putative metabolic transformations in nudibranchs of the genera G. collingwoodi, G. aureopurpureus and Goniobranchus sp. 1.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes Publications included in Chapter 3: Forster, L. C.; Pierens, G. K.; Clegg J. K.; Garson, M. J. Dynamic NMR and Computational Studies Inform the Conformational Description of Dendrillane Terpenes from the Nudibranch Goniobranchus coi. Journal of Natural Products 2020, in press. DOI: 10.1021/acs.jnatprod.9b01051.

Candidate contributions Louise C. Forster was responsible for the following work, incorporated into Section 3.1.1.1: • 50% Conception and design • 50% Data interpretation • 50% Manuscript drafting

Contributions by others Mary J. Garson contributed to the conception and design of this project, as well as, data interpretation and manuscript drafting. Gregory K. Pierens conducted computational studies for this project. Jack K. Clegg conducted X-ray crystallographic analyses for this project.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.0 Introduction As previously discussed in Chapter 1, there are five key structural motifs that can derive from rearrangement of ring A and/or B of a spongian diterpene scaffold. This chapter will focus on the perhydroazulene and perhydronaphthalene motifs. Goniobranchus coi was found to contain thirteen known and seven new oxygenated rearranged diterpenes with a perhydroazulene motif and one with a perhydronaphthalene motif. All metabolites were elucidated using classic 1H NMR, 13C NMR, 2D NMR spectroscopy and mass spectrometry. Where applicable, variable temperature experiments, molecular modelling, X-ray crystallographic and chemical correlation studies were carried out on the new metabolites.

Figure 3.1 The nudibranch Goniobranchus coi.

3.0.1 Reported metabolites from Goniobranchus species To date only two G. coi specimens collected from Lizard Island and Mackay, Queensland, Australia have been examined for their chemical profile. Dr Ariyanti Dewi, a previous member of the Garson group, isolated norrisolide (1.130) as the major component from these specimens, as well as macfarlandin C (1.165), cheloviolene C (1.131), dendrillolide A (1.142) and chromolactol (1.136).102

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1 Results and discussion 3.1.1 The isolation of metabolites from Goniobranchus coi (Risbec, 1956) Two specimens of G. coi were collected from Percy Isles (2015) and Coolum (2017), Australia. The preparation of mantle and viscera extracts was carried out based on the previously described procedures (Figure 3.1). The various mantle extracts were combined based on similar 1H NMR spectra, as were the viscera extracts. Subsequent NP-HPLC purification provided a total of twenty-one oxygenated rearranged terpenes (Figure 3.2), of which thirteen were identified as known diterpenes. Structure elucidation of seven new perhydroazulene-containing metabolites (3.1–3.5, 4.1, and 4.2) (Figure 3.3) and a cyclopropyl-functionalized diterpene (3.6) was provided by detailed spectroscopic, computational, chemical correlation and where possible X-ray crystallographic analysis.

G. coi [2 specimens]

Dissected Acetone Extraction

Partition with EtOAc /H2O

Mantles Visceras

Crude Extract (53.4 mg) Crude Extract (94.5 mg)

NP-flash chromatography NP-flash chromatography (hexanes: DCM: EtOAc: MeOH) (hexanes: DCM: EtOAc: MeOH)

Fractions B-F Fractions D-G

NP-HPLC NP-HPLC 25% EtOAc/hexanes 25% EtOAc/hexanes

1.142, 1.143, 1.144, 1.146, 1.148, 1.130, 1.132, 1.142, 1.143, 1.144, 1.149, 1.162, 1.163, 1.165, 3.1, 3.2, 1.145, 1.162, 1.163, 1.165, 1.171, 3.3, 3.4, 3.5, 3.6, 4.1, 4.2 3.1, 3.2, 3.4, 3.5, 3.6, 4.1, 4.2

Figure 3.1 Isolation scheme of G. coi compounds.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.2 Known oxygenated terpenes isolated from Goniobranchus coi.

Figure 3.3 New oxygenated terpenes isolated from Goniobranchus coi. 69

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1.1.1 Introduction to conformational averaging in metabolites from G. coi Dynamic NMR spectroscopy is an important tool for understanding conformational equilibria in organic molecules and probes the consequences of chemical exchange processes on the NMR timescale. Kinetic information, including the magnitude of activation energy barriers, can be obtained by “bandshape” analysis of exchange-broadened spectra.185 In cyclic systems, conformational averaging can be caused by ring inversions such as ring flips; the interconversion between chair, twist chair, twist boat or boat forms. In cases such as cycloheptane or cyclohexane, the energy barrier has been calculated to be between 7-10 kcal mol-1.186-191 Conformational averaging is also commonly encountered in fused ring systems. For example, House et al. explored the flexibility of the perhydroazulene ring system through X-ray crystallographic analyses, NMR studies and molecular mechanic conformational modelling, where the activation barrier was found to range between 19-22 kcal mol-1.192-193 Hendrickson pioneered a procedure that allowed for discrimination amongst different configurational and conformational isomers based on thermodynamics.194 Other fused ring systems that show conformational averaging include 1,5-benzodiazepines195 and 2-diazepines.196 Another example is that of guaianolide sesquiterpenes,197,198 where low temperature experiments combined with semiempirical calculations determined the activation barriers to be within 13-15 kcal mol-1. A second form of conformational averaging is restricted rotation. In substituted ethanes (sp3- sp3), restricted rotation has been studied both experimentally and theoretically. The energy barrier in simple hydrocarbons is 3 kcal mol-1, but is reported to be as high as 10-25 kcal mol-1 in halogenated ethanes.199 Conversely, the energy barrier to rotation in isopropyl- and t-butyl-substituted hydrocarbons is generally within 5-10 kcal mol-1.200,201 Classic examples from the natural product literature include restricted rotation about amide bonds in the sesquiterpene formamide metabolites isolated from marine sponges and their dietary molluscs,153 as well as in peptides.202 In amides such as N-methyl acetamide and N-methyl formamide, slow exchange at room temperature enables observation of both conformers, with an activation barrier up to ~15 kcal mol-1.203-205 The dendrillane scaffold, first named by Sullivan and Faulkner,110 possesses both a perhydroazulene ring system112,113,117,118 that has the potential for conformational change and a 2,8- dioxabicyclo[3.3.0]octane ring moiety79, 111 that is linked to the hydrocarbon moiety by a highly substituted carbon-carbon bond. The following Sections 3.1.1.1a-h describes two oxygenated terpenes (3.1-3.2), derived from the dendrillane scaffold, and explores the origin of line broadening effects observed in the 1H and 13C NMR spectra of each metabolite. A computational rationale is provided for the conformational rate processes detected.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1.1.1a Structure elucidation of a known metabolite dendrillolide A (1.142) Dendrillolide A (1.142) was isolated as a colorless oil from both the mantle and viscera by NP-HPLC (25% EtOAc in hexanes). In the LRMS an adduct ion of m/z 399.2 [M+Na]+ was observed, which is comparable to the molecular formula (C22H32O5) of dendrillolide A in the presence of a 1 sodium adduct. The H NMR spectral data (Table 3.1) showed three methyl singlets at δH 0.93 (Me-

18), 0.94 (Me-19) and 0.97 (Me-17), an acetate methyl singlet at δH 2.11 (15-OAc), two acetal protons

δH 6.05 (d, J = 4.4 Hz, H-16) and 6.44 (d, J = 6.6 Hz, H-15), as well as exomethylene signals at δH

4.83 (d, J = 2.2 Hz, H-20a) and 4.57 (d, J = 2.2 Hz, H-20b). The Me-17, Me-18, Me-19 and H2-20 signals were diagnostic of a perhydroazulene motif and the H-15, H-16 and lactone carbonyl at δC 175.5 was indicative of a 2,8-dioxabicyclo[3.3.0]octane ring system. As previously described in the Chapter 1, the structure of dendrillolide A (1.142) has a confusing structure elucidation history.

3.1.1.1b Structure elucidation of 5,9-epoxydendrillolide A (3.1)

Metabolite 3.1 was isolated as a white amorphous solid from both the mantle and viscera and + exhibited an adduct ion at m/z 413.1931 [M+Na] by HRESIMS, corresponding to C22H30O6. By comparison with the molecular formula of dendrillolide A (1.142), the loss of two hydrogens and the addition of one oxygen was suggestive of an epoxide functionality. The 700 MHz 1H NMR spectrum

(Table 3.1) acquired at 298 K indicated three methyl singlets at δH 0.98, 1.05 and 1.10, an acetate methyl at δH 2.11, and two acetal protons (δH 6.04 (d) and 6.49 (d)). However, some signals were broad, particularly those eventually assigned to H2-1, H-3b, H-7b, H-14, Me-17, Me-18, and H-20a. Notably, the signal for H-20a presented as a significantly broadened hump. Heating the sample at 323

K sharpened the H2-1, H-3b, H-7b, H-14, Me-17, Me-18, and H-20a signals, confirming the presence of a conformational rate process. When an initial HSQC spectrum was run at 298 K, 10 carbon signals were exhibited instead of the 15 signals expected for the protonated carbons in this particular type of diterpene, the 2D datasets subsequently acquired for 3.1 were all run at 323 K. Inspection of the gCOSY, TOCSY, HSQC, and HMBC data established a two-dimensional structure for 3.1.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.4 Selected 2D NMR correlations for 5,9-epoxydendrillolide A (3.1).

In the 2,8-dioxabicyclo[3.3.0]octane ring system, gCOSY connectivities linked H-14 (δH -

3.10) to both H-13 (δH 3.09) and H-15 (δH 6.49), as well as H-13 to H-16 (δH 6.04) (Figure 3.4). There were HMBC correlations from H-15 to δC 48.1 (C-14) and the acetate carbonyl at δC 169.7, as well as correlations from H-16 to δC 96.6 (C-15), 41.8 (C-13), 29.2 (C-12), and the lactone at δC 175.0 (C-

11). gCOSY and TOCSY correlations established the H2-1/H2-2/H2-3 and H2-6/H2-7 spin systems of the perhydroazulene fragment, as well as the position of the exomethylene moiety. However, the H- 5 and H-9 signals characteristic of the dendrillane skeleton110 were notably missing in the 1H NMR spectrum. Accordingly, the bridgehead methine carbon signals of dendrillolide A (1.142) were replaced by quaternary signals at δC 72.1 (C-5) and 75.3 (C-9), suggestive of epoxide functionality. The perhydroazulene fragment was linked to the 2,8-dioxabicyclo[3.3.0]octane ring system through

HMBC correlations from H-14 to δC 30.2 (C-7), 45.6 (C-8) and 75.3 (C-9). During the data collection for metabolite 3.1, the mass spectrometry (MS) sample was first dissolved in MeOH and gave a molecular adduct of m/z 413.2 [M+Na]+, consistent with the molecular formula of C22H30O6. When the MS sample was re-run for HRMS, a different ion adduct of m/z + 417.2243 [M-CH2CO+Na] was observed that was consistent with the molecular formula C22H34O6. The exposure to MeOH resulted in the bridge-head epoxide undergoing ring opening and subsequent formation of a dimethoxy artefact (Scheme 3.1). To confirm, metabolite 3.1 was treated with CD3OD, + 2 and the observed adduct ion at m/z 423.2 [M-CH2CO+Na] attributed to C21H28 H6O6 was noted, indicating the incorporation of six deuterium atoms.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Scheme 3.1 Proposed solvolysis of 3.1 to give a dimethoxy adduct.

Table 3.1 1H and 13C NMR assignments of 3.1, 3.2 and 1.142 a 3.1b 3.2b Dendrillolide A (1.142)c Position 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 1 36.5, CH2 a2.29, m 45.9, CH2 a2.45, m 37.6, CH2 a2.38, dd (4.8, 12.5) b2.04, m b2.42, m b1.82, td (1.8, 12.5) 2 25.3, CH2 a1.56, m 22.0, CH2 a1.71, m 28.5, CH2 a1.74, m b1.51, m b1.55, m b1.37, qt (2.8, 13.5) 3 37.3, CH2 a1.52, m 40.4, CH2 a1.70, m 38.1, CH2 a1.58, td (3.9, 14.1) b1.09, m b1.43, m b1.28, br d (14.1) 4 34.5, C - 36.4, C - 36.2, C - 5 72.1, C - 50.7, CH 2.03, m 54.6, CH 1.76, m 6 25.6, CH2 a1.95, m 26.7, CH2 a1.87, m 27.0, CH2 a1.73, m b1.90, m b1.83, m b1.73, m 7 30.2, CH2 a1.62, q (10.2) 38.4, CH2 a1.74, m 38.2, CH2 a1.71, m b1.19, m b1.54, m b1.47, m 8 45.6, C - 45.6, C - 46.8, C - 9 75.3, C - 63.1, CH 2.71, d (9.7) 56.2, CH 2.68, m 10 144.4, C - 214.2, C - 153.6, C - 11 175.0, C - 175.6, C - 175.5, C - 12 29.2, CH2 a2.60, dd (9.1, 17.7) 21.1, CH2 a2.66, dd (9.6, 17.9) 29.1, CH2 a2.71, dd (9.9, 17.9) b2.49, dd (8.9, 17.7) b2.61, dd (9.6, 17.9) b2.52, dd (9.4, 17.9) 3.18, t dd (4.5, 6.8, 13 41.8, CH 3.09, m 42.1, CH 42.0, CH 3.14, m 9.7) 14 48.1, CH 3.10, m 54.7, CH 2.79, t (6.8) 54.7, CH 2.67, m 15 96.6, CH 6.49, d (6.3) 96.4, CH 6.40, d (6.7) 97.4, CH 6.44, d (6.6) 16 104.4, CH 6.04, d (4.2) 104.9, CH 6.04, d (4.5) 105.2, CH 6.05, d (4.4) 17 23.8, CH3 1.10, s 22.5, CH3 1.13, s 24.1, CH3 0.97, s d d 18 28.0, CH3 0.98, s 27.8, CH3 0.86, s 25.7, CH3 0.93, s d d 19 24.1, CH3 1.05, s 29.5, CH3 0.95, s 34.4, CH3 0.94, s 20 117.7, CH2 a5.53, br s - - 114.8, CH2 a4.83, d (2.2) b5.27, s - b4.57, d (2.2) 15-OAc 169.7, C - 170.0, C - 169.7, C - 15-OAc 20.9, CH3 2.11, s 21.2, CH3 2.10, s 21.2, CH3 2.11, s a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. At 500 MHz. dInterchangeable assignments.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 5,9-Epoxydendrillolide A (3.1) was crystallized from 30% EtOAc/hexanes producing small needle-shaped crystals which were analyzed using synchrotron radiation (Figure 3.5). The crystal structure confirmed the relative configurations of the bridgehead epoxide relative to the 2,8- dioxabicyclo[3.3.0]octane ring system and the absolute configuration (5R, 8S, 9R, 13R, 14R, 15R, 16R) was selected for uniformity with other terpene perhydroazulenes,115 based on a common biosynthesis.

Figure 3.5 ORTEP184 representation of the crystal structure of epoxide 3.1 shown with 50% probability ellipsoids. Only one of the two chemically identical but crystallographically unique molecules in the asymmetric unit is shown.

The cycloheptane and cyclopentane rings in 3.1 adopted chair and envelope conformations, respectively. The 2,8-dioxabicyclo[3.3.0]octane ring system, which had a concave shape with a 67° angle of intersection of the mean planes of its two fused rings, had an identical shape to that of the 2,8-dioxabicyclo[3.3.0]octane moiety in the crystal structure of macfarlandin C (1.166) (Figure 3.6).111 The compact ‘crown-like’ conformation206,207 of the perhydroazulene moiety was relatively flattened, and the crystal packing was dominated by weak intermolecular interactions including exomethylene-epoxide contacts (H•••O = 2.7 Å). The C(13)-C(14)-C(8)-C(9) dihedral angle was 172.2(4)° placing the acetate group relatively close to the epoxide (C(21)•••O(1) = 3.34 Å).

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.6 Overlay of the 2,8-dioxabicyclo[3.3.0]octane ring system in the X-ray crystal structure of 3.1 (purple) and macfarlandin C (1.166) (yellow).111

3.1.1.1c Conformational averaging observed for 5,9-epoxydendrillolide A (3.1) An explanation for the line broadening in 3.1 was sought through a dynamic NMR study conducted at 500 MHz in CDCl3 combined with molecular modelling. Potentially, two different rate processes were involved, either a conformational change in the cycloheptane ring or rotation about the 1 congested C-8/C-14 bond. At 298 K, the H NMR spectrum showed broadened signals for H2-1, H- 3b, H-7b, H-14, Me-17, Me-18, and H-20a. All signals in the 1H NMR spectrum exhibited further broadening as the temperature was lowered; the signals for H-14, H-15 and H-16 coalesced at a temperature between 253 K and 263 K (Figure 3.7). On further cooling below 253 K, the signals for H-14, H-15, and OAc-15 started to separate into pairs corresponding to different conformers.

Eventually at 223 K there was clear separation of the conformer signals for H-14, H-15, H2-20, and OAc-15, but also evident for H-16 and the three methyl signals. Integration of the signals for H-15 gave a 1:0.3 ratio of conformers. The major conformer showed signals at δH 3.12 (H-14), 6.44 (H- 15), 6.11 (H-16), 5.70 (H-20a), 5.26 (H-20b), and 2.16 (OAc-15); in the minor conformer these signals appeared at δH 3.18 (H-14), 6.41 (H-15), 6.09 (H-16), 5.21 (H-20a), 5.19 (H-20b), and 2.12 (OAc-15), respectively. Using the difference in chemical shifts of H-15 for the major and minor conformers, the activation barrier for interconversion was calculated as 15.5 kcal mol-1 with the Sandstrom185 version of the Eyring equation (below). Alternatively using the difference in chemical shift values of the acetate methyls, the activation barrier was calculated as 15.0 kcal mol-1.

‡ ∆퐺푐 = 푎 × 푇푐 × [9.972 + 푙표푔(푇푐⁄∆푣)] Where: 푎 = 4.575 x 10-3 for values expressed in kcal mol-1

Tc = coalescence temperature in K ∆푣 = difference in frequencies (Hz) for the non-equivalent protons.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes At 223 K, the chemical shifts of the acetate methyl signals were 2.1568 ppm (major conformer) and 2.1146 ppm (minor conformer) corresponding to ∆푣 of 21.2 Hz, and with a ‡ -1 coalescence temperature estimated at 263 K gave a value for ΔGc of 15.0 kcal mol . At 223 K, the chemical shifts of the acetate methyl signals were 2.1568 ppm (major conformer) and 2.1146 ppm (minor conformer) corresponding to ∆푣 of 21.2 Hz, and with a coalescence temperature estimated at ‡ -1 253 K gave a value for ΔGc of 14.4 kcal mol . Repeating the calculation for the acetal (H-15) signal ‡ at 6.4 ppm, with ∆푣 of 13.8 Hz and with a coalescence temperature of 263 K gave ΔGc of 15.5 kcal mol-1. The difference in calculated values may be attributed to a small difference in the actual temperature at which individual non-equivalent proton signals coalesce, in effect leading to partial averaging at the temperature at which the data were recorded. When the 1H NMR spectrum of 3.1 was acquired in d6-acetone at 193 K, signals corresponding to three conformers in a ratio of 1:0.2:0.3 were apparent (Figure 3.7). 298 K 278 K 268 K

263 K 253 K 243 K

233 K 223 K

213 K

203 K 193 K

Figure 3.7 Stacked of 1H NMR spectra for 3.1 in the temperature range 298 K to 193 K (500 MHz, d6-acetone).

Although the separation of the exomethylene signals of 3.1 at 223 K was fully consistent with a cycloheptane conformational change, the separation of the H-14, H-15, H-16, and OAc-15 signals was unexpected. This led us to consider whether a rate process involving rotation about the pentasubstituted C-8/C-14 bond might be also involved. Restricted rotation in substituted ethanes has been studied both experimentally and theoretically. The energy barrier in regular hydrocarbons is 3 kcal mol-1, but is reported to be as high as 10-25 kcal mol-1 in halogenated ethanes.199 The barrier to rotation in isopropyl- and t-butyl-substituted hydrocarbons is generally within 5-10 kcal mol-1.200 A

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes computational investigation, carried out in parallel with the variable temperature NMR study described above, provided further insight into the origin of the conformational averaging in 3.1.

3.1.1.1d Molecular modelling of 5,9-epoxydendrillolide A A Monte Carlo Multiple Minimum (MCMM) conformational search of 3.1 was undertaken using MacroModel (Schrodinger Inc),128 providing eight conformers, and selected conformers (< 3 kcal mol-1 of the global minimum) were next optimized by DFT calculations at the B3LYP/6- 31G(d,p) method with IEFPCM chloroform solvent (Gaussian16W (Revision B.01)) (Appendix).208 The three lowest energy conformers, which were all within 0.6 kcal mol-1 of the global minimum and thus represented >99.8% of the conformational population, all had an identical orientation of the 2,8- dioxabicyclo[3.3.0]octane ring relative to the perhydroazulene moiety (Figure 3.8). For all three conformers, the C(13)-C(14)-C(8)-C(9) dihedral angle was between 172° and 174° placing the acetate group close to the epoxide, as had been observed in the crystal structure. The difference between the major conformer (3.1a) and the conformers 3.1b and 3.1c was the shape of the cycloheptane ring. In the lowest energy conformer (3.1a) the conformation of the cycloheptane ring was a twist chair, whereas in conformers 3.1b and 3.1c, which were comparable in energy, it was a chair and an inverted chair, respectively. The Boltzmann percentages of conformer 3.1a (59.7%) compared to 3.1b (21.8%) and 3.1c (18.5%) corresponded to a ratio of 1:0.67. It was noted that the perhydroazulene moiety in the crystal structure has the same overall shape as conformer 3.1c. The interconversion between the major conformer (3.1a) and conformer 3.1b is a pseudorotation that occurs when C-2 and C-3 flip upward relative to the epoxide group. The conversion to conformer 3.1c from 3.1a, is also a pseudorotation but instead requires C-1 to flip in a downward direction relative to the epoxide group, forcing the exomethylene (C-20) to move in an upward direction.

Figure 3.8 Overlay of energy-minimized stereostructures of epoxide conformers 3.1a (green), 3.1b (blue) and 3.1c (pink), with hydrogen atoms omitted for clarity.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes A recent study on plant sesquiterpenes also describes a conformational change in the cycloheptane ring of guaianolide lactones with an activation barrier of ~13-15 kcal mol-1,197,198 similar to the value calculated for 3.1. A difference was that the conformational change was attributed to a chair to twisted chair interconversion. The plant study described pairs of well resolved resonances for many of the 1H and 13C NMR signals, from which we inferred that conformers related by pseudorotation could show distinct signal pairs for atoms that are not adjacent to the cycloheptane ring.

3.1.1.1e Molecular modelling of dendrillolide A (1.142) We questioned why the parent dendrillane scaffold did not show conformational averaging. We therefore carried out a parallel computational study on dendrillolide A (1.142), resulting in an output of four conformers (< 3 kcal mol-1 of the global minimum); the two lowest energy conformers had a 0.65 kcal mol-1 energy difference and so represented >89.4% of the conformational population. The lowest energy conformer 1.142a represented 67.2% of the conformational population. These two conformers (1.142a and 1.142b) both had a chair conformation for the cycloheptane ring and differed only in the orientation of the 2,8-dioxabicyclo[3.3.0]octane moiety relative to the perhydroazulene fragment (Figure 3.9). Conformers 1.142a and 1.142b showed a C(13)-C(14)-C(8)-C(9) dihedral angle of 174.7° and 65.7°, respectively. When variable temperature 1H NMR studies were conducted on dendrillolide A, ranging from 323 K down to 223 K, no line broadening was detected. This information reinforced our interpretation that the rate process detected in 3.1 was linked to a conformational change in the cycloheptane ring rather than rotation about the C-8/C-14 bond.

Figure 3.9 Overlay of energy-minimized stereostructures of dendrillolide A conformers 1.142a (green) and 1.142b (blue), with hydrogen atoms omitted for clarity.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1.1.1f Structure elucidation of 10-oxonordendrillolide A (3.2)

Figure 3.10 Selected 2D NMR correlations for 10-oxonordendrillolide A (3.2).

Metabolite 3.2 was isolated from both the mantle and viscera as a colorless oil and displayed + a sodiated molecular ion peak at m/z 401.1934 [M+Na] by HRESIMS, corresponding to C21H30O6. The 1H NMR spectral data (Table 3.1) displayed a striking resemblance to that of dendrillolide A and revealed three methyl singlets at δH 0.86, 0.95 and 1.13, an acetate methyl at δH 2.10, and two acetal protons at δH 6.04 (d, J = 4.5 Hz) and 6.40 (d, J = 6.7 Hz). Notably, the lack of the diagnostic exomethylene signals suggested a different functionality at C-10/C-20 compared to dendrillolide A. The HSQC spectrum run at 298 K showed correlations to 14 carbon signals instead of the 16 signals expected for the protonated carbons in this norditerpene. Inspection of the gCOSY, HSQC, HMBC and TOCSY data run at 323 K established the two-dimensional structure of this rearranged norditerpene (Figure 3.10). As with compound 3.2, heating the sample at 323 K sharpened signals, and so allowed the missing carbon to proton correlations (for C-3 and Me-19) to be revealed in the

HSQC spectrum. A C-10 ketone was established by HMBC correlations from δC 214.2 to H2-1 (δH

2.45 and 2.42), H2-2 (δH 1.71 and 1.55), H-5 (δH 2.03) and H-9 (δH 2.71). The cis relative configuration of the perhydroazulene ring system was established by NOESY correlation between H-5 and H-9. From a biosynthetic perspective, the ketone may be formed via a C-10/C-20 spiro epoxide intermediate, followed by oxidative cleavage of the epoxide carbon-carbon bond.209

3.1.1.1g Mass spectrometry experiments on 10-oxonordendrillolide A (3.2) Before the presence of ketone functionality was identified the LRMS sample was dissolved in MeOH and an ion adduct of m/z 401.2 [M+Na]+ was observed, which corresponded to molecular formula C21H30O6. When the HRMS analysis was later carried out, a different molecular adduct of + m/z 405.2241 [M-CH2CO+Na] was noted, corresponding to the molecular formula C21H34O6. We proposed that the ketone functionality had converted to a dimethoxyacetal (Scheme 3.2). This mass change was explored in a MS study, where metabolite 3.2 was treated with CD3OD and the molecular + adduct m/z 411.2 [M-CH2CO+Na] corresponding to the incorporation of six deuterium atoms was observed, consistent with the proposed conversion (Scheme 3.2).

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Scheme 3.2 Proposed solvolysis of 3.2 to give a dimethoxy product.

3.1.1.1h Molecular modelling 10-oxonordendrillolide A (3.2) To probe the conformational equilibrium in 3.2, a computational study was undertaken resulting in ten conformers (< 3 kcal mol-1 of the global minimum), with four of the lowest energy conformers (< 0.4 kcal mol-1 of the global minimum) representing >79.6% of the conformational population (Appendix). These four conformers, within a 0.32 kcal mol-1 energy difference, showed conformational change in both the cycloheptane ring and 2,8-dioxabicyclo[3.3.0]octane moiety. The major conformer (3.2a) showed a C(13)-C(14)-C(8)-C(9) dihedral angle of 175.6° and a chair conformation for the cycloheptane ring (Figure 3.11). The 1H NMR spectrum of 3.2 was sharp and well resolved at 298 K, but exhibited broadening as the temperature was lowered, suggesting dynamic conformational averaging. At 223 K the signals were too broad and unresolved to identify individual conformers, however signals corresponding to at least 3 conformers were observed (Appendix).

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.11 Energy-minimized stereostructure of the major conformer 3.2a, with hydrogen atoms omitted for clarity.

3.1.1.2 Structure elucidation of 5-hydroxydendrillolide A (3.3)

Terpene 3.3 was isolated from the mantle as a colorless oil and displayed an adduct ion at m/z + 415.2086 [M+Na] from HRESIMS, which afforded the same molecular formula as 3.1 (C22H32O6). The additional oxygen compared to the molecular formula of dendrillolide A, may suggest an epoxide or hydroxy group. Inspection of the 1H NMR spectrum (Table 3.2) revealed one acetoxy methyl singlet at δH 2.10, two acetal proton signals at δH 6.58 (d, J = 6.5 Hz) and 6.05 (d, J = 4.1 Hz), as well 1 as exomethylene signals at δH 4.64 (d, J = 2.3 Hz) and 4.86 (d, J = 2.3 Hz). Comparison with the H NMR spectrum of dendrillolide A (1.142)110,111 supported a dendrillane-derived carbon skeleton, however signals for the methine at the bridgehead of the perhydroazulene motif seen in 1.142 (δH

1.76, δC 54.6) were missing. Instead there was a tertiary hydroxy group at C-5, based on HMBC correlations from the three methyl signals at δH 0.99 (Me-18), 0.94 (Me-19) and 0.98 (Me-17) to a carbon at δC 87.6. NOESY correlations observed between H-13/H-14, H-13/H-16 and H-14/Me-17 confirmed the configuration of the 2,8-dioxabicyclo[3.3.0]octane moiety (Figure 3.12). NOESY correlations were also observed between H-9/Me-18 and so confirmed the orientation of H-9. The configuration of the tertiary hydroxy group was not explored further owing to the small quantity (0.4 21 mg) of the sample. The specific optical rotation of 3.3 showed a value of [α] D +19 (c 0.044, CHCl3). The name of compound 3.3 was assigned as (+)-5-hydroxydendrillolide A.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

(A) (B)

Figure 3.12 Molecular modelling for 5-hydroxydendrillolide A (3.3). (A) showing key NOESY correlations and (B) with hydrogen atoms omitted from the model for clarity.

Table 3.2 1H and 13C NMR assignments of 5-hydroxydendrillolide A (3.3) a,b,c Position 13C 1H (mult., J, Hz)

1 37.1, CH2 a2.37, m b1.84, m 2 27.9, CH2 a1.72, m b1.47, m 3 39.4, CH2 a1.64, m b1.41, m 4 40.3, C - 5 87.6, C - 6 34.2, CH2 a2.06, m b1.60, m 7 34.7, CH2 a2.20, m b1.38, m 8 46.8, C - 9 64.4, CH 2.76, m 10 150.9, C - 11 175.3, C - 12 29.0, CH2 a2.72, dd (17.9, 10.1) b2.50, dd (17.9, 9.3) 13 42.0, CH 3.12, m 14 54.4, CH 2.73, m 15 97.7, CH 6.58, d (6.5) 16 104.5, CH 6.05, d (4.1) 17 24.4, CH3 0.98, s 18 20.7, CH3 0.99, s 19 28.9, CH3 0.94, s 20 115.5, CH2 4.64, d (2.3) 4.86, d (2.3) 15-OCOCH3 169.5, C - 15-OCOCH3 21.1, CH3 2.10, s a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. HMBC connectivity from C to H.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1.1.3 Structure elucidation of 10,20-epoxydendrillolide A (3.4)

Metabolite 3.4 was isolated from both the mantle and viscera as a colorless oil and displayed a sodiated molecular ion peak in the HRESIMS at m/z 415.2083 [M+Na]+, corresponding to a 1 molecular formula of C22H32O6. Comparison of the H NMR spectral data (Table 3.3) of 3.4 with dendrillolide A (1.142)110,111 supported a dendrillane-derived skeleton, with the absence of exomethylene signals suggested a change in functionality at the C-10/C-20 position. The exomethylene signals at δH 4.83 (d) and 4.57 (d) were replaced with signals for an epoxide methylene at δH 2.64 (d, J = 5.2 Hz) and 2.40 (dd, J = 5.2, 1.5 Hz). HMBC correlations from the epoxide protons

(H2-20) to signals at δC 37.2 (C-1), 53.4 (C-9) and 61.2 (C-10) established the spiroepoxide at the C-

10 position. gCOSY correlations confirmed the H2-1/H2-2/H2-3 and H-9/H-5/H2-6/H2-7 spin systems of a perhydroazulene fragment (Figure 3.13). The perhydroazulene fragment was linked to the 2,8- dioxabicyclo[3.3.0]octane ring system through HMBC correlations from H-14 to C-7 (δC 38.4), C-8

(δC 47.7) and C-9 (δC 53.4). The cis relative configuration of the perhydroazulene ring system was established by NOESY correlation between H-5 and H-9. In the 2,8-dioxabicyclo[3.3.0]octane ring system, gCOSY connectivities linked H-14 (δH 2.70) to both H-13 (δH 3.05) and H-15 (δH 6.38), as well as H-13 to H-16 (δH 5.98). There were HMBC correlations from H-15 to C-14 at δC 56.2 and the acetate carbonyl at δC 169.5, as well as correlations from H-16 to C-15 (δC 96.9), C-13 (δC 42.4), C-

12 (δC 29.1), and the lactone at δC 175.0 (C-11). The configuration of the 2,8- dioxabicyclo[3.3.0]octane ring system was established through NOESY correlations between H- 14/H-16, H-13/H-16 and H-13/H-14 which revealed that these protons were all on the same side of the plane. Accordingly, 3.4 was determined to be a new analogue and named as 10,20- epoxydendrillolide A.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.13 Selected 2D NMR correlations for 10,20-epoxydendrillolide A (3.4).

Table 3.3 1H and 13C NMR assignments of 10,20-epoxydendrillolide A (3.4) and 10S aldehyde derivative (3.5)a,b 3.4 3.5 Position 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz)

1 37.2, CH2 a2.06, m 30.3, CH2 a1.72, m b1.21, br dd (13.7, 4.9) b1.53, m 2 24.1, CH2 a1.66, m 24.5, CH2 a1.66, m b1.45, m b1.66, m 3 37.2, CH2 a1.58, m 44.1, CH2 a1.62, m b1.32, m b1.35, m 4 36.3, C - 36.4, C - 5 54.1, CH 1.69, m 52.6, CH 2.10, m 6 27.2, CH2 a1.69, m 26.9, CH2 a1.81, m b1.69, m b1.62, m 7 38.4, CH2 a1.62, m 36.5, CH2 a1.72, m b1.41, m b1.44, m 8 47.7, C - 46.3, C - 9 53.4, CH 1.73, m 49.5, CH 2.39, t (9.9) 10 61.2, C - 52.8, C 2.55, m 11 175.7, C - 175.1, C - 12 29.1, CH2 a2.75, dd (18.0, 9.2) 29.5, CH2 a2.64, dd (17.1, 9.9) b2.67, dd (18.0, 10.1) b2.52, m 13 42.4, CH 3.05, tdd (9.6, 6.4, 4.4) 41.8, CH 3.05, tdd (9.7, 6.6, 4.4) 14 56.2, CH 2.70, t (6.6) 52.1, CH 2.54, m 15 96.9, CH 6.38, d (6.8) 96.7, CH 6.41, d (6.7) 16 104.8, CH 5.98, d (4.3) 104.7, CH 6.00, d (4.5) 17 24.1, CH3 1.34, s 22.1, CH3 1.00, s 18 34.8, CH3 0.97, s 25.1, CH3 0.86, s 19 26.5, CH3 0.99, s 32.8, CH3 0.99, s 20 56.7, CH3 2.64, d (5.2) 204.8, CH 9.57, d (4.4) 2.40, dd (5.2, 1.5) 15-OCOCH3 169.5, C - 169.6, C - 15-OCOCH3 21.1, CH3 2.09, s 21.4, CH3 2.14, s a b Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz.

3.1.1.3a Molecular modelling of 10,20-epoxydendrillolide A (3.4)

Although NOESY correlations were observed from the epoxide proton at δH 2.40 (H-20b) to

Me-19 (δH 0.99) and H-9 (δH 1.73), at this stage in the characterization process, the configuration at C-10 could not be conclusively assigned. To investigate the configuration at the C-10 position two candidate stereostructures (3.4a-3.4b) (Figure 3.14) were proposed for 3.4 and molecular modelling was undertaken by Dr Gregory Pierens, from the Centre for Advanced Imaging. 84

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Figure 3.14 Candidate stereostructures (3.4a-3.4b) proposed for 10,20-epoxydendrillolide A (3.4).

A Monte Carlo Multiple Minimum (MCMM) conformational search was undertaken using MacroModel (Schrodinger Inc)128 providing eight conformers for 3.4a and twelve conformers for 3.4b. The lowest energy conformers (< 3 kcal mol-1 of the global minimum) were next optimized by DFT calculations using the B3LYP/6-31G(d,p) method with IEFPCM chloroform solvent (Gaussian16W (Revision B.01)), converging to six and five conformers for 3.4a and 3.4b respectively. The two lowest energy conformers of 3.4a were within 0.07 kcalmol-1 of the global minimum and thus represented >75.6% of the conformational population. These two conformers both had a chair conformation for the cycloheptane ring and differed only in the orientation of the 2,8- dioxabicyclo[3.3.0]octane moiety relative to the perhydroazulene fragment. The lowest energy conformer for 3.4a represented 40.3% of the conformational population (Figure 3.15). The two conformers of 3.4a showed a C(13)-C(14)-C(8)-C(9) dihedral angle of 68.5° and 173.2°, respectively. The lowest energy conformer of 3.4b represented >84.6% of the conformational population and, similar to 3.4a, had a chair conformation for the cycloheptane ring (Figure 3.16). Conformer 3.4b had a C(13)-C(14)-C(8)-C(9) dihedral angle of 175.7°.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

(A) (B)

Figure 3.15 (A) Lowest energy conformer of stereoisomer 3.4a of (10R)-spiroepoxide (3.4) and (B) truncated and rotated image of 3.4a showing the conformation of the cycloheptane ring; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety.

(A) (B)

Figure 3.16 (A) Lowest energy conformer of stereoisomer 3.4b of (10S)-spiroepoxide (3.7) and (B) truncated and rotated image of 3.4b showing the conformation of the cycloheptane ring; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety.

The free energies of 3.4a and 3.4b were used to calculate the Boltzmann-weighted 1H and 13C NMR chemical shifts in chloroform solvent (IEF-PCM) (see Appendices for full details). The calculated chemical shifts were then examined using the DP4 computational approach developed by Smith and Goodman to assign the most probable diastereomer.135,210 Using both the 1H and 13C NMR data, the probability was 99.99% that the C-10 configuration was as shown in stereoisomer 3.4a (R- configuration).

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.1.1.3b Structure elucidation of aldehyde rearrangement product (3.5)

An aldehyde product (3.5) was purified as a colorless oil from both the mantle and the viscera and displayed a sodiated molecular ion peak in the HRESIMS at m/z 415.2092 [M+Na]+. The corresponding molecular formula of C22H32O6 was identical to that of spiroepoxide 3.4. Comparison of the 1H NMR spectral data (Table 3.3) of 3.5 with dendrillolide A (1.142) 110,111 supported again a dendrillane-derived skeleton, but with the absence of exomethylene signals at δH 4.83 (d) and 4.57

(d) found in 1.142 replaced by a methine signal at δH 2.55 (m) and an aldehyde methine at δH 9.57 (d,

J = 4.4 Hz, H-20). HMBC correlations from the aldehyde proton (H-20) to the signals at δH 30.3 (C- 1), 49.5 (C-9) and 52.8 (C-10) established the position of an aldehyde at the C-10 position. In a similar manner to 3.4, through HMBC and gCOSY correlations, the two-dimensional structure was established and the relative configuration of the perhydroazulene and 2,8-dioxabicyclo[3.3.0]octane moieties were found to be consistent with a dendrillolide A-like scaffold. Signals for compound 3.5 were not observed in 1H NMR spectrum of the crude extract and were only observed after the sample was purified through silica chromatography, implying that 3.5 is a rearrangement product formed during the purification process. As explored below, it is well established that epoxides are labile and isomerize into aldehyde compounds when in acid conditions.211 The isomerization of epoxides into carbonyl compounds is named as the Meinwald rearrangement or Meinwald reaction (Scheme 3.3).211,212 When considering the mechanism for formation of the aldehyde, it is understood that a hydride migration occurs across the unhindered face of the epoxide, antiperiplanar to the C-O bond (Scheme 3.3). As a result of this preference, the R- epoxide was expected to convert to the R-aldehyde, likewise, the S-epoxide would convert to the S- aldehyde. However, at this stage it was not clear whether epoxide 3.4 or its C-10 diastereomer had rearranged to form 3.5.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes Scheme 3.3 Mechanism of degradation/isomerization to the aldehyde from a spiroepoxide (Scheme reproduced from Li et al.).211 3.1.1.3c Chemical correlation experiments to probe the configuration at C-10 of 3.4 and 3.5 A recent study on plant sesquiterpenes has used chemical correlation experiments to aid in confirming the C-10 configuration of the two sesquiterpenoid alcohols viridifloral (3.14) and ledol (3.15).213 Bombarda et al. reported that oxidation of alloaromadendrene (3.9), using m- chloroperbenzoic acid (mCPBA) produced two diastereoisomeric epoxides (3.10 and 3.11) and two aldehyde isomers (3.12 and 3.13). Subsequently, the epoxides were then reduced using lithium aluminum hydride (LiAlH4) to yield the two alcohol diastereomers (Scheme 3.4). The study by Bombarda et al. found that the minor epoxide (3.11) was more labile than the major epoxide (3.10), as it rapidly isomerized to an aldehyde during column chromatography, and therefore the epoxide products (3.10 and 3.11) were characterized from a mixture. For spiroepoxide 3.10, NOESY correlations between the epoxide protons (H2-14) and the cyclopropyl protons (H-6 and H-7) established the C-10 configuration as S. The aldehyde metabolites 3.12 and 3.13 were not separated and were isolated in a ratio of 30:70, determined by 1H NMR data. However, the authors were able to distinguish the NMR signals for the major aldehyde 3.13 in the spectra of the mixture.

Scheme 3.4 Oxidation of alloaromadendrene (3.9) and subsequent reduction of the epoxide diastereomers (3.10 and 3.11) to afford the diastereomeric alcohols (3.14 and 3.15) (Scheme reproduced from Bombarda et al.).213

By analogy with the study by Bombarda et al., the expectation was that epoxidation of dendrillolide A (1.142) would provide both epoxide diastereomers and their subsequent aldehyde degradation products for comparison with the natural isolates. Therefore in our study, the relative configuration at the C-10 position was investigated for both the spiroepoxide (3.4) and aldehyde (3.5),

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes where a sample of dendrillolide A (1.142) was reacted with mCPBA in a biphasic solution with phosphate buffer and DCM to generate a mixture of the 10R and 10S spiroepoxides (Scheme 3.5).

Commercial mCPBA is known to contain H2O and m-chlorobenzoic acid, which is also formed as a by-product during the reaction. The buffer was used to counteract epoxide product degradation resulting from acid sensitivity.

Scheme 3.5 Chemical correlation of spiroepoxides (3.4 and 3.7) and aldehyde products (3.5 and 3.8) by epoxidation of dendrillolide A (1.142).

Spectroscopic analysis was carried out on the crude reaction mixture, due to the labile nature of the product epoxides. From the initial 1H NMR of the crude reaction mixture, the signals for the major component were found to be consistent with those of the natural epoxide isolate (3.4). However, when a 1H NMR spectrum was run following an overnight acquisition of 2D NMR data it was apparent that the signals for the epoxide diastereomers had disappeared, and aldehyde signals were instead present. Fortunately, the degradation process was relatively slow, so the initially collected HSQC and HMBC data provided sufficient information for characterization of the minor epoxide (3.7) from the reaction mixture (Table 3.4). Due to their labile and acid sensitive nature, the epoxides 3.4 and 3.7 could not be isolated by column chromatography. In an attempt to characterize the aldehyde products, the reaction mixture was purified directly using NP-HPLC (25% EtOAc in hexanes) to afford both aldehyde products (3.5 and 3.8), whereupon the NMR chemical shifts of aldehyde 3.5 were found to match the NMR data for the isolated aldehyde from G. coi. The two- dimensional structure of the other aldehyde reaction product (3.8) was characterized through spectroscopic analysis (Table 3.4).

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Table 3.4 1H and 13C NMR assignments of epoxidation products 10S-spiroepoxide (3.7) and 10R-aldehyde (3.8) a,b (10S)-Spiroepoxide (3.7) (10R)-Aldehyde (3.8) Position 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz)

1 35.5, CH2 a1.82, m 32.7, CH2 a2.19, dt (14.8, 40) b1.27, m b1.46, m 2 22.1, CH2 a1.77, m 20.3, CH2 a1.84, m b1.50, m b1.71, m 3 37.7, CH2 a1.61, m 39.4, CH2 a1.79, m b1.41, m b1.30, m 4 35.8, C - 35.1, C - 5 55.7, CH 1.72, m 55.0, CH 1.95, m 6 26.9, CH2 a1.73, m 27.0, CH2 a1.70, m b1.70, m b1.61, m 7 37.8, CH2 a1.73, m 38.0, CH2 a1.68, m b1.46, m b1.39, m 8 46.4, C - 46.4, C - 9 54.4, CH 1.71, m 54.7, CH 2.58, m 10 60.1, C - 54.7, C 2.58, m 11 175.1, C - 174.9, C - c 12 28.4, CH2 a2.72, m 28.7, CH2 a2.61, m b2.52, dd (18.0, 9.8) b2.57, m 13 42.0, CH 3.13, mc 41.6, CH 3.05, t dd (9.7, 6.6, 4.6) 14 55.9, CH 2.65, mc 53.3, CH 2.63, m 15 97.0, CH 6.42, d (6.6) 96.7, CH 6.39, d (6.8) 16 105.0, CH 6.04, d (4.5) 104.3, CH 6.01, d (4.2) 17 25.8, CH3 1.19, s 26.6, CH3 1.14, s 18 34.7, CH3 1.97, s 26.0, CH3 1.05, s 19 25.8, CH3 1.11, s 32.7, CH3 1.03, s 20 56.4, CH3 2.84, d (4.9) 204.4, CH 9.95, s 2.66, mc 15-OCOCH3 169.5, C - 169.2, C - 15-OCOCH3 21.1, CH3 2.09, s 21.2, CH3 2.15, s a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. . Obscured.

Inspection of a model of the 10R-stereoisomer (3.4a) revealed the epoxide protons are orientated towards the center of the cycloheptane ring, placing them in proximity (2.65 Å) to one of the geminal dimethyl protons (Me-19) (Figure 3.17). In contrast, the model of the 10S-stereoisomer (3.4b), showed the epoxide protons orientated outward from the cycloheptane ring, placing one epoxide proton in proximity to Me-17 (2.54 Å). The steric hindrance afforded by the geminal dimethyls in the 10R-stereoisomer (3.4a) resulted in shielding the epoxide from rapidly undergoing a Meinwald rearrangement to form the 10R-aldehyde. In the 10S-stereoisomer, where the epoxide protons are less hindered, the epoxide can rapidly isomerize in mild acidic conditions to the 10S- aldehyde.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

2.54 Å

4.13 Å 2.65 Å 3.4a 3.4b Epoxide protons “inside” Epoxide proton “outside”

Figure 3.17 Truncated structures of candidate diastereomers 3.4a and 3.4b for spiroepoxide 3.4, showing distances between key atoms for each stereoisomer; with the blue dot indicating the location of the 2,8-dioxabicyclo[3.3.0]octane moiety.

Since the minor epoxide (3.7) degraded before NOESY data could be collected, its experimental NMR chemical shifts were compared to the previously modelled 10S-stereoisomer 3.4b and agreed with a 10S-configuration. Consequently, it was inferred that along with the 10R-epoxide (3.4), the 10S-epoxide was originally present in the crude extracts of G. coi, however when the extract was exposed to silica chromatography the 10S-epoxide isomerized to form the 10S-aldehyde product (3.5). The specific optical rotation measurement of the aldehyde reaction product gave a value of 21 21 [α] D + 11 (c 0.042, CHCl3) that was comparable to that of the isolated aldehyde artefact 3.5 [α] D

+16 (c 0.055, CHCl3). The sample of the 10R-aldehyde reaction product (3.8) degraded on storage and its specific optical rotation could not therefore be measured.

3.1.1.4 Characterization of a cyclopropyl functionalized metabolite (3.6) from G. coi The first examples in the sponge diterpene literature with cyclopropyl functionality are the metabolites polyrhaphin C (1.171), isolated from Aplysilla polyrhaphis117 and dendrillolide E (1.172), isolated from a Dendrilla species (Figure 3.18).113 Bobzin and Faulkner discovered both metabolites in 1989 and found that the metabolites possessed the same perhydronaphthelene ring system with the cyclopropyl moiety fused across the C-9/C-10 bond. The difference between these two compounds was the dioxabicyclooctane ring system, where 1.172 had a 2,8-dioxabicyclo[3.3.0]octane moiety and 1.171 possessed a 2,7-dioxabicyclo[3.2.1]octane moiety.113,117 Later discovered were the diterpenes cheloviolin (1.174) by Bergquist et al. in 1993 and omriolide B (1.176) by Kashman co- workers in 2005. These compounds (1.174 and 1.176) possessed the same hydrocarbon fragment, with the cyclopropyl moiety fused across the C-8/C-9 bond. Cheloviolin (1.174), like 1.171, has a 2,7-dioxabicyclo[3.2.1]octane moiety, whereas 1.176 has a 2,8-dioxabicyclo[3.3.0]octane moiety.100,120 As presented in Chapter 1, the structure of omriolide B (1.176) was noted to have an 91

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes alternative configuration at C-15 in the 2,8-dioxabicyclo[3.3.0]octane moiety, which is anomalous compared to other structures described in the marine literature. The C-12 acetate analogue of polyrhaphin C, 12α-acetoxypolyrhaphin C (1.173), was later isolated from the nudibranch Goniobranchus splendidus by a member of our group, Andrew White.85 Also isolated by our group was verrielactone (1.90), which was proposed to follow an alternative biosynthetic pathway to that of the other cyclopropyls previously isolated in literature.76

Figure 3.18 Examples of oxygenated rearranged spongian diterpenes with cyclopropyl functionality.76,85,100,113,117,120

3.1.1.4a Structure elucidation of cyclopropyl metabolite from G. coi

Metabolite 3.6 was isolated from both the mantle and the viscera as a colorless oil and displayed a sodiated molecular ion peak in the HRESIMS at m/z 399.2154 [M+Na]+ which 1 corresponded to a molecular formula of C22H32O5. The H NMR spectrum (Table 3.5) presented an acetate methyl singlet at δH 2.07 and cyclopropyl signals at δH 0.28, 0.47 and 0.76, each of the latter integrating for one proton. Comparison of the 1H NMR spectral data of 3.6 with literature examples of rearranged diterpenes that possess a cyclopropyl functionality aided in establishing the structure

(Figure 3.18). HMBC correlations from the H-5 bridgehead methine (δH 0.72) to the signal for C-18

(δC 33.1), C-19 (δC 21.5) and C-20 (δC 21.1) were consistent with a perhydronaphthalene motif.

HMBC correlations from the cyclopropyl methylene protons (H2-17) to C-7 (δC 29.8), C-9 (δC 35.6),

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

C-10 (δC 32.2) and C-14 (δC 54.8) confirmed the cyclopropyl group to be fused across the C8/C9 bond, as seen in cheloviolin (1.174)100 and omriolide B (1.176).120 The right side motif was next elucidated, in particular gCOSY correlations linked H-14 (δH 1.99) to H-13 (δH 3.29) and H-15 (δH

6.20), as well as H-13 to H-16 (δH 6.11) and H2-12 (δH 2.99 and 2.67), suggesting a 2,8- dioxabicyclo[3.3.0]octane moiety, similarly seen in dendrillolide E (1.172),113 dendrillolide A (1.142),110,111,113 and macfarlandin C (1.165).111 A 2,7-dioxabicyclo[3.2.1]octane moiety as seen in polyrhaphin C (1.171)117 and cheloviolin (1.174)100 was also considered, but was later disregarded 13 since the C value observed for the lactone carbonyl (δC 174.7) did not fit within the typical range 87,111 for a δ-lactone carbonyl (δC 165-168), instead the value supported a γ-lactone. The 2,8- dioxabicyclo[3.3.0]octane ring system was further confirmed through HMBC correlations from H-15 to C-13 (δC 39.9) and the acetate carbonyl at δC 169.2, as well as correlations from H-16 to C-15 (δC

100.8), C-14 (δC 54.8), C-12 (δC 29.6) and the lactone carbonyl at δC 174.7 (C-11). The 2,8- dioxabicyclo[3.3.0]octane moiety was shown to be linked to the perhydronaphthalene motif at C-8/C-

14, though HMBC correlations from H-14 (δH 1.99) to C-9 (δC 35.6) and C-17 (δC 11.9), as well as

H-15 (δH 6.20) to C-8 (δC 19.0). NOESY correlations between H-14/H-16, H-13/H-16 and H-13/H- 14 revealed that these protons were all on the same face, establishing the configuration of the 2,8- dioxabicyclo[3.3.0]octane ring system. The configuration of the cyclopropyl was determined through NOESY correlations between H-14/H-17b and H-17a/Me-20, placing the cyclopropyl group on the same face as Me-17. In the marine literature, cyclopropyl functionalized diterpenes are proposed to originate from the spongian scaffold and can be formed through one of three pathways (Scheme 3.6). Pathway A begins with migration of the C-8 methyl and oxidative cleavage of the C-5/C-6 bond. Subsequently, the loss of CO2 leads to the gracilin A carbon skeleton, while oxidative cleavage of the C-9/C-11 bond provides the spongionellin skeleton, from which verrielactone (1.90) can be derived.76 Pathway B starts with an oxidative cleavage of the C-9/C-11 bond and C-8 methyl migration, from which the C-8/C-9 cyclopropyl can be formed.42 We also propose an alternative where following oxidative C- 9/C-11 bond cleavage, loss of HX from C-9 (where X = OH, OR etc. leaving group) and subsequent cyclopropyl formation from Me-17. Final cyclization gives cheloviolin (1.174) and the new metabolite 3.6, which can then be further oxidized to provide 1.176. Pathway C follows similar steps to pathway B, with oxidative cleavage of the C-9/C-11 bond, however instead migration of C-10 methyl occurs to provide the C-9/C-10 cyclopropyl structure, where subsequent cyclization provides 1.171, 1.172 and 1.173.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Table 3.5 1H and 13C NMR assignments of cyclopropyl derivative (3.6) and omriolide B (1.176). 3.6 Omriolide B (1.176)120 Position 13C 1H (mult., J, Hz) a,b 13C 1H (mult., J, Hz) c,d

1 43.4, CH2 a1.60, m 43.5, CH2 a1.58, m b1.41, m b1.40, m 2 20.0, CH2 a1.68, m 20.0, CH2 a1.71, m b1.50, m b1.52, m 3 41.7, CH2 a1.41, m 41.8, CH2 a1.45, br d (13.1) b1.22, m b1.19, td (5.0, 13.1) 4 33.1, C - 32.4, C - 5 54.4, CH 0.72, m 55.0, CH 0.67, d (11.9) 6 17.1, CH2 a1.44, m 17.1, CH2 a1.43, m b0.82, m b0.86, m 7 29.8, CH2 a1.77, m 32.3, CH2 a2.01, bd (12.5) b1.57, m b1.85, dt (5.6, 12.5) 8 19.0, C - 18.1, C - 9 35.6, CH 0.76, dd (5.8, 9.9) 34.2, CH 1.01, dd (5.6, 9.9) 10 32.2, C - 33.3, C - 11 174.7, C - 172.8, C - 12 29.6, CH2 a2.99, dd (4.9, 18.7) 36.3, CH2 a3.59, d (18.6) b2.67, dd (10.4, 18.7) b2.94, d (18.6) 13 39.9, CH 3.29, m 88.2, CH - 14 54.8, CH 1.99, dd (3.3, 8.9) 55.6, CH 2.68, d (5.0) 15 100.8, CH 6.20, d (3.3) 97.3, CH 6.24, d (5.0) 16 106.9, CH 6.11, d (6.0) 109.3, CH 6.02, s 17 11.9, CH2 a0.47, dd (5.3, 5.8) 11.5, CH2 a0.42, t (5.6) b0.28, ddd (0.8, 5.3, 9.9) b0.37, dd (5.6, 9.9) 18 21.5, CH3 0.73, s 21.4, CH3 0.75, s 19 33.1, CH3 0.83, s 33.0, CH3 0.83, s 20 21.1, CH3 0.93, s 21.0, CH3 0.95, s 13-OCOCH3 - - 170.2, C - 13-OCOCH3 - - 21.3, CH3 2.13, s 15-OCOCH3 169.2, C - 168.5, C - 15-OCOCH3 21.1, CH3 2.07, s 21.2, CH3 2.04, s a b c Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. Chemical shifts (ppm) referenced to d CHCl3 (δH 7.26, δC 77.0). At 400 MHz.

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes

Scheme 3.6 Putative biosynthesis of cyclopropyl-containing (nor)diterpenes.42,76

Chapter 3: Modifications to the hydrocarbon fragment of nor/diterpenes 3.2 Conclusions The analysis of Goniobranchus coi, collected from the East coast of Australia, has yielded a range of dendrillane analogues (3.1-3.4), as well as an aldehyde generated during the extraction process (3.5) and a perhydronaphthalene analogue with cyclopropyl functionality (3.6). The new dendrillane analogues 3.1 and 3.2 showed broadening of individual 1H and 13C NMR signals at 298 K, which was suggestive of an occurring conformational rate process. A variable temperature NMR study revealed the cause of the line broadening as a result of pseudorotation between a twist chair and two different chair conformations. An X-ray crystallographic study of 3.1 coupled with biosynthetic reasoning established the absolute configuration (5R, 8S, 9R, 13R, 14R, 15R, 16R). The ketone 10- oxonordendrillolide A (3.2) demonstrated similar dynamic conformational changes, however owing to the small energy barrier between conformers involved rapid interconversion that signals for the individual conformers could not be separated at 223 K. Molecular modelling was carried out by Dr Gregory Pierens to further explore the conformations of 3.1 and 3.2 relative to the parent scaffold dendrillolide A (1.142). The computational studies indicated conformational changes in the perhydroazulene moiety is likely the cause of the conformational averaging observed as line broadening in the NMR experiments. The structurally related hydroxy metabolite (3.3) was elucidated solely by NMR spectroscopic analysis, however the tertiary hydroxy configuration could not be determined through X-ray crystallographic analysis owing to the small quantity of sample isolated. For the spiroepoxide 3.4 and aldehyde 3.5, chemical correlation experiments coupled with computational studies enabled the determination of their relative configurations. Finally, the new metabolite 3.6 possessed cyclopropyl functionality on a perhydronaphthalene scaffold instead of a perhydroazulene motif present in the other new metabolites. These studies illustrated how combining experimental NMR-derived and computational data can support the stereochemical analysis of conformationally flexible molecules and aid in determining the relative configuration. The anatomical distribution of these metabolites within the tissues of G. coi is discussed in Chapter 5.

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes Publications included in Chapter 4: Forster, L. C.; Pierens, G. K.; Garson, M. J. Elucidation of Relative and Absolute Configurations of Highly Rearranged Diterpenoids and Evidence for a Putative Biosynthetic Intermediate from the Australian Nudibranch Goniobranchus geometricus. Journal of Natural Products 2019, 82, 449-455. DOI: 10.1021/acs.jnatprod.8b00713.

Candidate contributions Louise C. Forster was responsible for the following work, incorporated into Section 4.1.2: • 40% Conception and design • 80% Data interpretation • 50% Manuscript drafting

Contributions by others Mary J. Garson contributed to the conception and design, data interpretation and manuscript drafting for this project. Gregory K. Pierens also contributed to the manuscript drafting and conducted computational studies.

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes 4.0 Introduction As previously discussed in Chapter 1 the spongian diterpenes have been biosynthetically proposed to undergo significant rearrangements to form an array of compounds. The precursors can undergo structural changes to either the hydrocarbon moiety, as shown in Chapter 3, or structural changes to the highly oxygenated motif, which will be explored in this chapter. As discussed in Chapter 1, computational studies including molecular modelling, DFT and DP4/DP4+ methods can provide insight into determining the relative configuration and in some cases the absolute configuration of natural products.133 A significant limitation in natural product elucidation is the restricted quantity of an isolated metabolite, which can reduce the types of characterization techniques available for planar structure determination, as well as establishing the relative and absolute configuration.159 This chapter continues the investigation of the secondary metabolites from Goniobranchus coi, previously described in Chapter 3, and includes the description of two diastereomeric lactol metabolites (4.1, 4.2) that shows stereochemical changes to the 2,8-dioxabicyclo[3.3.0]octane ring system. The current chapter also reassesses the relative and absolute configuration of a previously characterized scaffold, proposed as a key intermediate in the putative biosynthesis of rearranged metabolites with a perhydroazulene motif. Finally, we introduce two new perhydroazulene containing metabolites isolated from G. geometricus (Figure 4.1). In this chapter, the combined application of NMR, molecular modelling and computational approaches is illustrated, which together with chemical correlation experiments enabled the determination of the relative and absolute configuration of oxygenated rearranged spongian diterpenes.

(A) (B)

Figure 4.1 The nudibranchs Goniobranchus geometricus (A) and Goniobranchus coi (B).

4.0.2 Reported metabolites from G. geometricus To date, prior to this study, the chemical analysis of G. geometricus has not been reported elsewhere. Herein, the role of the carbon skeleton of secoshahamin (1.164) in the biogenetic pathway of rearranged diterpenes was being explored. In addition, the isolation and configurational analysis of two other rearranged oxygenated terpenes (4.3 and 4.4) from Goniobranchus geometricus are also described.

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes 4.1 Results and discussion 4.1.1 Structure elucidation of diastereomeric metabolites (4.1 and 4.2) from G. coi

Metabolites 4.1 and 4.2 were isolated from both the mantle and the viscera as an inseparable mixture by NP-HPLC, but the key individual signals could be identified in the 1H NMR spectrum (Table 4.1). In the 1H NMR spectrum, integration of the two sets of signals indicated a 2:1 ratio of 4.1 and 4.2, respectively. The colorless oil displayed a sodiated molecular ion peak in the HRESIMS at m/z 357.2045, corresponding to a molecular formula of C20H30O4, consistent with the loss of an acetate from dendrillolide A and the addition of a hydroxy group. Each terpene showed exomethylene signals (4.1: δH 4.81 (d) and 4.53 (d); 4.2: δH 4.82 (d) and 4.59 (d)), three quaternary methyl signals

(4.1: δH 0.95 (s, Me-18), 0.97 (s, Me-19), 1.04 (s, Me-17); 4.2: δH 0.94 (s, Me-18), 0.96 (s, Me-17),

0.97 (s, Me-19), and the diagnostic bridging methine proton (4.1: δH 2.70 (d, J = 9.3 Hz, H-9); 4.2:

δH 2.88 (d, J = 8.8 Hz, H-9)), indicative of a perhydroazulene ring system. The cis relative configuration of the perhydroazulene ring system in both 4.1 and 4.2 was confirmed by NOESY correlation between H-5 and H-9. The perhydroazulene fragment was linked to the 2,8- dioxabicyclo[3.3.0]octane ring system through HMBC correlations from H-14 to C-7 (4.1: δC 37.6;

4.2: δC 37.8), C-8 (4.1: δC 47.0; 4.2: δC 46.6), and C-9 (4.1: δC 57.7; 4.2: δC 56.2). In the 2,8- dioxabicyclo[3.3.0]octane ring system of each terpene, gCOSY connectivities and HMBC correlations linked H-14 (4.1: δH 2.16; 4.2: δH 2.35) to both H-13 (4.1: δH 3.15; 4.2: δH 3.17) and H-

15 (4.1: δH 5.64 (d, J = 3.9 Hz); 4.2: δH 5.63 (d, J = 7.1 Hz)), as well as H-13 to H-16 (4.1: δH 6.05 (d,

J = 6.1 Hz); 4.2: δH 6.06 (d, J = 4.7 Hz)). This spectroscopic analysis established two-dimensional structure of 4.1 and 4.2 with the lactol group at C-15, that is consistent with those of the known metabolites cheloviolene A and cheloviolene B.100 The configuration of the 2,8-dioxabicyclo[3.3.0]octane motif was next considered, comparing the relative configuration with those of dendrillolide A (1.142),110,111 macfarlandin C (1.165)111 and norrisolide (1.130),97 as well as those of cheloviolene A (1.148),100 cheloviolene B (1.149)100 and chromolactol (1.136).102 The 2,8-dioxabicyclo[3.3.0]octane ring system seen in 1.142, 1.165, and 1.130 has a cis configuration between H-13/H-14 and a trans configuration between H-14/H-15. In contrast 1.136, 1.148, and 1.149, have a trans configuration between H-13/H-14 and between H- 14/H-15. The relative and absolute configuration of cheloviolene A (1.148) was recently confirmed

100

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes by single-crystal X-ray analysis, following an eleven-step enantioselective total synthesis carried out by the Overman group (Figure 4.2).115-116

Figure 4.2 Comparison of selected structures for rearranged metabolites possessing a 2,8- dioxabicyclo[3.3.0]octane moiety, with the three established configurations indicated in blue, green and red.

Cheloviolene A (13S, 14S, 15S, 16S) and cheloviolene B (13R, 14R, 15R, 16R) are the first examples in the marine diterpene literature of which there is a change in the configuration of H-14 relative to H-13 and H-15 in the 2,8-dioxabicyclo[3.3.0]octane moiety.100 When the 1H NMR spectra of lactols 4.1 and 4.2 were compared with those of cheloviolene A (1.148) and B (1.149) there were obvious differences. The coupling constants of H-12a (dd, J = 18.3, 9.0 Hz), H-12b (dd, J = 18.3, 9.7 Hz), H-15 (d, J = 7.1 Hz), and H-16 (d, J = 4.7 Hz) were in accordance with those observed for 1.142,110,111 suggesting a cis configuration between H-13/H-14 and a trans configuration between H- 14/H-15 (Table 4.2 and Table 4.3). Notably, the configuration of the 2,8-dioxabicyclo[3.3.0]octane ring system of 4.2 was comparable to that of dendrillolide A (1.142) (Table 4.2 and Table 4.3). For 4.1, the coupling constant of H-15 (d, J = 3.7 Hz) suggested a change in configuration at C-15 (Table 4.2).

101

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

Table 4.1 1H and 13C NMR Assignments of 15-dendrillactol (4.1), 15-epidendrillactol (4.2), cheloviolene A (1.148) and cheloviolene B (1.149).a,b,c

100, 116 100, 116 15-Dendrillactol (4.1) 15-Epidendrillactol (4.2) Cheloviolene A (1.148) Cheloviolene B (1.149) Position 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13Cd 1H (mult., J, Hz) d 1a 37.6, CH2 2.37, m 37.6, CH2 2.20, m 37.0, CH2 2.35, br dd (5.0, 13.0) 39.3, CH2 2.35, dd (5.1, 12.9) 1b 1.88, m 1.82, m 1.84, br t (13.0) 1.90, td (2.3, 12.9) 2a 28.5, CH2 1.73, m 28.5, CH2 1.72, m 28.8, CH2 1.80-1.70, m 31.5, CH2 1.75, m 2b 1.38, m 1.38, m 1.39, br dd (13.0, 13.5) 1.39, m 3a 38.3, CH2 1.61, m 38.4, CH2 1.72, m 37.8, CH2 1.63, m 40.2, CH2 1.69, m 3b 1.27, m 1.46, m 1.27, br d (14.0) 1.25, dt (3.6, 14.4) 4 36.2, C - 36.2, C - 36.1, C - 38.6, C - 5 54.5, CH 1.87, m 54.4, CH 1.72, m 54.3, CH 1.93, ddd (8.0, 9.0, 11.0) 56.4, CH 2.12, dt (8.2, 11.8) 6a 27.1, CH2 1.70, m 26.8, CH2 1.74, m 26.3, CH2 1.80-1.70, m 28.5, CH2 1.77, m 6b 1.70, m 1.74, m 1.80-1.70, m 1.77, m 7a 37.6, CH2 1.82, m 37.8, CH2 1.72, m 38.4, CH2 1.80-1.70, m 40.8, CH2 1.64, m 7b 1.51, m 1.38, m 1.58, m 1.55, ddd (4.0, 8.5, 12.7) 8 47.0, C - 46.6, C - 46.9, C - 49.9, C - 9 57.7, CH 2.70, d (9.3) 56.2, CH 2.88, d (8.9) 56.5, CH 2.55, d (9.0) 59.0, CH 2.75, d (8.7) 10 153.8, C - 153.5, C - 153.9, C - 157.1, C - 11 177.0, C - 176.2, C - 175.5, C - 177.7, C - 12a 31.2, CH2 3.26, dd (6.9, 18.4) 29.7, CH2 2.72, dd (9.3, 18.1) 36.8, CH2 2.94, dd (11.0, 18.4) 39.4, CH2 2.95, dd (11.0, 18.1) 12b 2.53, dd (10.9, 18.4) 2.55, dd (9.8, 18.1) 2.73, ddd (0.5, 3.9, 18.1) 2.61, ddd (0.5, 3.6, 18.1) 3.10, dddd (2.2, 3.9, 6.2, 3.10, dddd (2.6, 3.6, 6.2, 13 39.1, CH 3.17 m 41.9, CH 3.15, m 40.2, CH 43.0, CH 11.0) 11.0) 14 55.2, CH 2.16, dd (3.7, 7.0) 56.2, CH 2.35, m 66.0, CH 2.25, br d (2.2) 68.5, CH 2.25, d (2.5) 15 100.3, CH 5.64, d (3.7) 99.7, CH 5.63, d (7.0) 103.1, CH 5.52, br s 105.6, CH 5.62, d (3.3) 16 107.4, CH 6.05, d (6.1) 104.1, CH 6.06, d (4.8) 109.6, CH 6.07, d (6.2) 112.1, CH 6.06, d (6.2) 17 24.9, CH3 1.04, s 24.1, CH3 0.96, s 21.2, CH3 0.81, s 23.4, CH3 0.79, s 18 34.5, CH3 0.95, s 34.5, CH2 0.94, s 34.4, CH3 0.95, s 36.6, CH3 0.95, s 19 26.0, CH2 0.97, s 26.0, CH3 0.97, s 25.7, CH3 0.99, s 27.8, CH3 1.01, s 20a 114.2, CH2 4.81, d (2.4) 114.7, CH2 4.82, d (2.4) 114.4, CH2 4.83, ddd (0.7, 0.8, 2.3) 116.6, CH2 4.84, (2.4) 20b 4.53, d (2.4) 4.59, d (2.4) 4.62, dd (0.8, 2.3) 4.68, dd (2.4) 15-OH - 3.08 br s - c - 3.07, br s - - a b c d Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz. Not detected. Chemical shifts (ppm) referenced to (CD3)2CO.

102

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes Table 4.2 Comparison of selected 1H NMR data for rearranged metabolites possessing a 2,8-dioxabicyclo[3.3.0]octane moiety. a,b Position 15-Dendrillactol (4.1) 15-Epidendrillactol (4.2) Dendrillolide A (1.142)110,111 Chromolactol (1.136)102 Cheloviolene A (1.148)100 Cheloviolene B (1.149)100 12a 3.26, dd (6.9, 18.3) 2.72, dd (9.3, 18.1) 2.71, dd (9.9, 17.9) 2864, dd (11.4, 18.6) 2.94, dd (11.0, 18.4) 2.96, ddd (0.5, 3.6, 18.1) 12b 2.53, dd (10.9, 18.3) 2.55, dd (9.8, 18.1) 2.52, dd (9.4, 17.9) 2.75, dd (5.3, 18.6) 2.73, ddd (0.5, 3.9, 18.4) 2.61, ddd (0.5, 3.6, 18.1) 13 3.17 m 3.15, m 3.14, m 3.01, ddd (5.3, 6.2, 11.4) 3.10, dddd (2.2, 3.9, 6.2, 11.0) 3.10, dddd (2.6, 3.6, 6.2, 11.0) 14 2.16, dd (3.7, 7.0) 2.35, m 2.67, m 2.76, br s 2.25, br d (2.2) 2.25, ddd (0.5, 1.0, 2.6) 15 5.64, d (3.7) 5.63, d (7.0) 6.44, d (6.6) 5.54, s 5.52, br s 5.61, d (1.0) 16 6.05, d (6.1) 6.06, d (4.8) 6.05, d (4.4) 6.18, d (6.2) 6.07, d (6.2) 6.06, d (6.2) a b Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz.

Table 4.3 Comparison of selected 13C NMR data for rearranged metabolites possessing a 2,8-dioxabicyclo[3.3.0]octane moiety.a,b Position 15-Dendrillactol (4.1) 15-Epidendrillactol (4.2) Dendrillolide A (1.142)110,111 Chromolactol (1.136)102 Cheloviolene A (1.148)100 Cheloviolene B (1.149)100

12 31.2, CH2 29.7, CH2 29.1, CH2 35.8, CH2 29.7, CH2 28.5, CH2 13 39.1, CH 41.9, CH 42.0, CH 45.2, CH 40.2, CH 40.8, CH 14 55.2, CH 56.2, CH 54.7, CH 58.5, CH 66.1, CH 68.5, CH 15 100.3, CH 99.7, CH 97.4, CH 104.1, CH 103.2, CH 105.6, CH 16 107.4, CH 104.1, CH 105.2, CH 108.9, CH 109.5, CH 112.1, CH a b Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 500 MHz.

103

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

4.1.1.1 Molecular modelling of lactol diastereomers (4.1 and 4.2) To further explore the relative configuration of 4.1 and 4.2, four candidate stereoisomers (4.1a, 4.1b, 4.2a and 4.2b) were proposed (Figure 4.3). Molecular modelling was undertaken by Dr Gregory Pierens, from the Centre for Advanced Imaging.

Figure 4.3 Candidate structures (4.1a, 4.1b, 4.2a and 4.2b) proposed for lactols 4.2 and 4.1.

A Monte Carlo Multiple Minimum (MCMM) conformational search for four diastereomers (4.1a, 4.1b, 4.2a and 4.2b) was undertaken in gas phase with Merck Molecular Force Field (MMFF) using MacroModel v12.128 The selected conformers (< 5 kcal/mol of the global minimum, 4.1a: 5 conformers, 4.1b: 5 conformers, 4.2a: 6 conformers and 4.2b: 10 conformers) were optimized by density functional theory (DFT) using Gaussian software (G16W).208 The calculated NMR parameters were scaled relative to their Boltzmann population and the vibrational frequencies were checked for a true minimum, i.e. no negative frequencies. The magnetic field tensors were calculated using B3LYP/6-311+G(d,p) and their DP4+ probabilities calculated136 relative to the experimental chemical shifts. It was found that conformer 4.1a had a 100% probability for the major component 15-dendrillactol (4.1) and conformer 4.2a had a 99.9% probability for the minor component 15- epidendrillactol (4.2). In parallel the DP4 probabilities133,210,214 were calculated that 4.1a had a 99.7% probability of representing the major component (4.1) and 4.2a had a 98.5% probability for the minor component (4.2), and was in complete agreement with the DP4+ output (Figure 4.4 and Figure 4.5).

Figure 4.4 (A) Energy-minimized stereostructure of the major conformer of 4.1a, (B) with hydrogen atoms omitted for clarity.

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Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

Figure 4.5 (A) Energy-minimized stereostructure of the major conformer of 4.2a, (B) with hydrogen atoms omitted for clarity.

The final NMR chemical shifts were calculated with mpw1pw91/6-311+G(2d,p) for proton NMR and wB97XD/6-311+g(2d,p) for carbon NMR with chloroform solvent (IEF-PCM). The magnetic field tensors were converted to scaled chemical shifts by the methods outlined above and the chemical shifts weighted by their Boltzmann populations for diastereomers 4.1a and 4.2a only. The mean absolute error (MAE) of stereoisomer 4.1a compared to the experimental chemical shifts for 4.1 were 0.07 ppm and 1.5 ppm for proton and carbon, respectively. The maximum deviations were 0.30 ppm and 3.6 ppm. Conversely, the MAE of stereoisomer 4.2a compared to the experimental chemical shifts for 4.2 were 0.09 ppm and 1.5 ppm for proton and carbon, respectively. The maximum deviations were 0.24 ppm and 2.8 ppm. The two lowest energy conformers of 4.1 represented >87.6% of the conformational population. These two conformers (4.1a-1 and 4.1a-2) both had a chair conformation for the cycloheptane ring and differed only in the orientation of the 2,8-dioxabicyclo[3.3.0]octane moiety relative to the perhydroazulene fragment (Figure 4.6). Conformers 4.1a-1 and 4.1a-2 showed a C(13)-C(14)-C(8)-C(9) dihedral angle of 175.1° and 68.5°, respectively.

Figure 4.6 Overlay of energy-minimized stereostructures of lactol conformers 4.1a-1 (green) and 4.1a-2 (pink), with hydrogen atoms omitted for clarity. 105

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes The three lowest energy conformers of 4.2, that represented >92.8% of the conformational population, all had an identical chair conformation for the cycloheptane ring and differed in the orientation of the 2,8-dioxabicyclo[3.3.0]octane moiety relative to the perhydroazulene fragment (Figure 4.7). Conformers 4.2a-1, 4.2a-2 and 4.1a-3 exhibited a C(13)-C(14)-C(8)-C(9) dihedral angle of 174.8°, 64.7° and 43.7°, respectively.

Figure 4.7 Overlay of energy-minimized stereostructures of lactol conformers 4.2a-1 (green), 4.2a- 2 (pink) and 4.2a-3 (blue), with hydrogen atoms omitted for clarity.

The coupling constants for the 2,8-dioxabicyclo[3.3.0]octane moiety in 4.1 and 4.2 were calculated using the method of Kutateladze et al. (Table 4.4 and Table 4.5).145 For both metabolites 4.1 and 4.2, the calculated and experimental coupling constants were in agreement, which further supported the proposed configuration of 15R for 4.1 and 15S for 4.2.

Table 4.4 Comparison of calculated (black) and experimental (blue) key coupling constants for the 2,8-dioxabicyclo[3.3.0]octane moiety of the major component (4.1)

H 12b 13 14 15 16 18.5 7.5 - - - 12a 18.4 6.9 - - - - 10.8 - - - 12b - 10.9 - - - - - 6.9 - 6.0 13 - - 7.0 - 6.1 - - - 3.7 - 14 - - - 3.7 -

106

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes Table 4.5 Comparison of calculated (black) and experimental (blue) key coupling constants for the 2,8-dioxabicyclo[3.3.0]octane moiety of the minor component (4.2) a

H 12b 13 14 15 16 17.1 10.5 - - - 12a 18.1 9.8 - - - - 9.2 - - - 12b - 9.3 - - - - - 6.9 - 4.2 13 - - a - 4.8 - - - 5.9 - 14 - - - 7.0 - aExperimental value obscured in 1H NMR.

4.1.1.2 Chemical correlation study on the lactol diastereomers (4.1 and 4.2) Based on the computational studies, the minimum energy conformer of 4.1a had the lowest energy compared to that of 4.2a, with an energy difference of -0.87 kcal mol-1. Metabolite 4.1 was isolated as the major diastereomer. Since lactols can undergo ring opening, there would be an equilibrium mixture of diastereomers. As expected, following treatment of the lactol mixture (4.1 and 4.2) with acetic anhydride and pyridine, the epimers converged to form a single acetylated product identified as dendrillolide A (1.142) (Scheme 4.1). The presence of one acetylated product is likely due to the increased steric hindrance afforded by the bulky acetate group. The specific optical rotation 21 measurement of the reaction product gave a value of [α] D +26 (c 0.02, CHCl3) that was comparable 21 110,111 to that of the natural isolate 1.142 [α] D +87 (c 0.31, CHCl3).

Scheme 4.1 Chemical correlation of lactol 4.1 and 4.2 mixture with dendrillolide A (1.142) by acetylation.

4.1.2 The isolation of metabolites from Goniobranchus geometricus (Risbec, 1928) Eight specimens of Goniobranchus geometricus were collected from Mooloolaba and Gold Coast, Australia between 2012 and 2016. The specimens were finely chopped then extracted with acetone and the extract was concentrated under vacuum. The aqueous suspension was then partitioned with Et2O to yield a yellow oil. A portion of the extract was utilized for ecological assays and the remaining extract (46 mg) was partitioned by NP-flash chromatography with a stepwise solvent

107

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes gradient. Fractions were combined on the basis of TLC analysis, with the resulting 1H NMR spectra identifying the presence of oxygenated diterpenes (Figure 4.8).

G. geometricus [8 specimens] Acetone Extraction

Partition with Et2O /H2O NP-flash chromatography (Hexanes: DCM: EtOAc: MeOH)

Fraction D Fraction E Fraction F & G hexanes/DCM 1:1 hexanes/DCM 1:4 100% DCM & DCM/EtOAc 4:1

NP-HPLC NP-flash chromatography NP-HPLC 25% EtOAc/hexanes (hexanes: DCM: EtOAc: MeOH) 30% EtOAc/hexanes

1.142, 1.143, 1.154, 4.3 NP-HPLC 1.131, 1.142, 1.143, 1.145, 10% EtOAc/hexanes 1.144, 1.154, 1.157, 1.162, 1.164, 1.165, 1.166, 1.171 1.130, 1.131, 1.132, 1.142, 1.143, 1.144, 1.152, 1.154, 1.157, 1.162, 1.163, 1.165, 1.166, 1.171, 4.4

Figure 4.8 Isolation scheme of G. geometricus compounds.

A total of sixteen known rearranged terpenes were isolated using NP-HPLC: norrisolide (1.130),97 chelonaplysin C (1.131),100 cheloviolene C (1.132),100 dendrillolide A (1.142),110,111 macfarlandin E (1.143),111 aplyviolene (1.144),112 macfarlandin D (1.145),111 12α- acetoxydendrillolide A (1.152),34 polyrhaphin B (1.154),117 shahamin C (1.157),118 12- desacetoxyshahamin C (1.162),113 12-desacetoxypolyrhaphin A (1.163),113 shahamin F(1.166),118 polyrhaphin C (1.171),117 and 15,16-diacetoxyshahamin B (1.160), previously reported as a synthetic derivative of shahamin B (1.156).118 Each known compound was identified though the comparison of mass spectrometric data, literature and in-house NMR data as explained in the previous chapter (Figure 4.9). In addition, we provide rigorous characterization of a perhydroazulene-containing methyl ester, first isolated from a marine sponge,108 and herein named as secoshahamin (1.164). Two new rearranged terpenes 4.3 and 4.4 were also characterized.

108

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

Figure 4.9 Known oxygenated terpenes isolated from G. geometricus.

4.1.2.1 Structure elucidation of secoshahamin (1.164)

109

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes Diterpene 1.164 was isolated as a colorless oil from NP-HPLC (25% EtOAc in hexanes). In the LRESIMS, there was a peak at m/z 459.2 [M+Na]+. The 1H NMR spectrum showed signals indicative of a substituted perhydroazulene ring, including exomethylene signals at δH 4.82 (br s) and

4.91 (d, J = 1.9 Hz) for H-20, three quaternary methyl signals at δH 0.85 (s), 0.95 (s), and 1.01 (s), 110 and a diagnostic H-9 signal at δH 2.83 (d, J = 8.8 Hz). The cis relative configuration of the perhydroazulene ring system was established by NOESY correlation between H-5 and H-9. The signals at δH 2.05 (s), 2.07 (s) and 3.68 (s) identified two acetate methyls and a methyl ester, respectively. One of the acetoxy groups was confirmed at C-15 through HMBC correlations, as each of the H-15 signals at δH 3.98 (dd, J = 9.7, 11.6 Hz) and 4.36 (dd, J = 4.0, 11.6 Hz) and the 15-OAc methyl at δH 2.05 (s) correlated to the carbonyl at δC 170.8. HMBC correlations from the H-16 signals at δH 4.02 (dd, J = 6.8, 10.8 Hz) and 4.07 (dd, J = 8.3, 10.8 Hz) and the 16-OAc methyl at δH 2.07 (s) to the carbonyl at δC 170.9 confirmed the second acetoxy group at C-16. The methyl ester was located at C-12 through HMBC correlations, with both the H-12 signals at δH 2.21 (dd, J = 11.0, 15.8 Hz) and 2.52 (dd, J = 2.2, 15.8 Hz) and the methoxy signal at δH 3.68 (s) correlated to the carbonyl at δC 173.1. At this point, it was recognized that the 1H and 13C NMR data matched those of a metabolite first isolated from an unidentified Japanese marine sponge by Tanaka and colleagues,108 but which was not assigned a name by these authors. In this study, diterpene 1.164 was assigned the name secoshahamin. Tanaka et al. inferred that H-13 and H-14 of 1.164 were anti to each other on the basis of an nOe between H-13 and H-17, together with the absence of an nOe between H-14 and H-17. However, their analysis was incomplete as it did not consider the conformational implications of the 1, 2-acyclic system. The NOESY spectrum of the G. geometricus sample of secoshahamin (1.164) showed correlations from δH 0.85 (s, Me-17) to δH 2.94 (m, H-13) and to δH 1.84 (br d, H-14). Further investigation of the C-13/C-14 syn or anti configuration and the configuration relative to Me-17 was desirable. A J-Based Configurational Analysis (JBCA) was undertaken, with focus on the coupling 3 constants between protons separated by three bonds ( JHH), that are directly related to their dihedral angles through the Karplus equation. Further information can be obtained from dihedral angles, where empirical equations that consider substituent patterns can allow for accurate predictions of J-values from dihedral angles. We investigated the (R,R) and (R,S) configurations on the 1,2-dimethine system (4.5a and 4.5b, respectively). There are three possible lowest energy (staggered) conformers for each of the diastereomers 4.5a and 4.5b. 3 The C-13/C-14 configuration was first explored by JBCA involving determination of the JH- 3 3 13/H-14 and JCH values (Figure 4.10). H-13 presented as an apparent quartet, however J-values of 6.8 3 Hz and 8.3 Hz were assigned from inspection of the H2-16 signals, and J-values of 11.0 Hz and 2.2 110

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes

3 Hz from inspection of the H2-12 signals. H-14 presented as a doublet of doublets with J-values of 3 4.0 Hz and 9.7 Hz to the two H-15 signals. Therefore, the JH-13/H-14 value was clearly small (<1 Hz) and this conclusion was further reinforced from inspection of COSY and 1D TOCSY data, neither of which showed a correlation between H-13 and H-14. Contributions of the anti-conformers are 3 presumed minimal, since the JH-13/H-14 value was small. In the HMBC spectrum of 1.164, it was noticeable that there were no correlations from H-13 to C-8 or C-15, which suggested that the 3 heteronuclear JCH values were also small. However, it was not possible to measure the actual values of these coupling constants using HECADE,215 EXSIDE216 or IPAP-HSQMBC,217 since only 0.2 mg of material was available; these pulse sequences generally require ~ 1 mg of material for optimal 3 signal to noise, nor can small JCH values easily be measured accurately.

NOESY correlations were observed from H-13 to H-9, H2-12, H-14, H2-16 and Me-17. In addition, NOESY correlations from H-14 to H-9, H-15, H-16 and Me-17, as well as from H-15 to H- 16 and Me-19 were observed. The rotation about the substituted ethane bonds connected to C-14 and C-13 may result in conformational averaging, therefore the experimental NOESY correlations are inconclusive.

111

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes 4.1.2.2 Molecular modelling for secoshahamin (1.164)

Figure 4.11 Candidate diastereomers 4.5a and 4.5b for the truncated structure of secoshahamin (1.164), with carbon numbers matching those of 1.164.

Next, the truncated structures 4.5a and 4.5b, in which a t-butyl group replaced the conformationally flexible perhydroazulene ring of secoshahamin, were selected for computational analysis (Figure 4.11). A Monte Carlo conformational search of the (13R, 14R)-4.5a and (13S, 14R)- 4.5b diastereomers was undertaken with Merck Molecular Force Field (MMFF) using Macromodel.128 The conformers (< 5 kcal/mol of the global minimum) were further optimized by 208 density functional theory (DFT) using Gaussian software, and for CHCl3 solvent (IEF-PCM) (Appendix).218 The mean absolute error (MAE) values for 4.5a and 4.5b were then considered. For the 1H data, the MAE were 0.17 and 0.21 ppm for the 4.5a and 4.5b diastereomers, respectively, while the 13C MAE were 1.68 and 1.90 ppm for the 4.5a and 4.5b diastereomers, respectively. Although the MAE values were similar, there was a slight preference for the 4.5a diastereomer. When the Boltzmann-averaged 1H chemical shifts were examined using DP4135 and considering both 1H and 13C chemical shifts, there was a high probability that the 4.5a diastereomer was preferred. This was primarily due to the large difference in the calculated chemical shifts for H- 14 for the two diastereomers; the values for 4.5a and 4.5b were 1.82 ppm and 1.26 ppm, respectively, while the experimental value was 1.84 ppm. Using the 13C chemical shifts, the DP4 output resulted in a 4.5a:4.5b probability of 77.5:22.5%. Although the 13C DP4 calculation showed a preference for the 4.5a isomer, the result was considered inconclusive as the probability value was < 80%.136 Using both 1H and 13C chemical shifts in the DP4 probability output resulted in a 99.97% preference for the 4.5a diastereomer over the 4.5b diastereomer. To further verify the proposed relative configuration, the coupling constants for H-14 were calculated using the method of Kutateladze et al.145 The calculated coupling constants for H-14 in 4.5a were 9.1, 3.5, and 0.9 Hz, while in 4.5b the values were 3.5, 1.5, 0.9, and 0.8 Hz. Therefore, this provided further evidence to support the 13R, 14R diastereomer 4.5a as the preferred stereoisomer of secoshahamin.

112

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes 4.1.2.3 Chemical correlation study of secoshahamin (1.164) The relative configuration of secoshahamin (1.164) was next investigated by chemical correlation with 12-desacetoxyshahamin C (1.162)113 and with 12-desacetoxypolyrhaphin A (1.163);113 the relative configuration of each of these metabolites has been established by NMR analysis. The ester groups of 1.162-1.164 were hydrolyzed using potassium hydroxide followed by hydrochloric acid work up. In each case, a δ-lactone (4.6) (named as 12-deoxyshahamin E for consistency with the literature)118 was isolated rather than the alternative γ-lactone product (Scheme 4.2).

Scheme 4.2 Chemical correlation of secoshahamin (1.164) with 12-desacetoxyshahamin C (1.162) (of 13R, 14R configuration) and 12-desacetoxypolyrhaphin A (1.163) by saponification and lactonization, each giving the same 13R, 14R δ-lactone product (12-deoxyshahamin E (4.6)).

δ-Lactone carbonyls typically have a smaller carbonyl chemical shift than γ-lactones (δC 175 219 vs. 178). The lactone moiety of 4.6 showed a carbonyl chemical shift of δC 173.6, but, given that the δ-lactone-containing 12-desacetoxyshahamin C (1.162) and γ-lactone-containing 12- desacetoxypolyrhaphin A (1.163) have closely similar carbonyl chemical shifts (δC 170.8 and 169.1, respectively), additional data was desirable. Fortunately, the δC values for C-14, C-15 and C-16 of 4.6 matched closely those of 1.162 rather than the values for 1.163. Furthermore, when the 1H NMR data were compared, the chemical shift values for the H-13 and H2-15 signals and the J-values for

H2-15 of 4.6 closely matched those of 1.162 rather than those of 1.163 (Table 4.6). In 4.6, the J- values (JH-12a/H-13 = 3.4 Hz; JH-12b/H-13 = 6.0 Hz; JH-14/H-15b = 10.6 Hz) together with a strong NOE between H-12b and H-15b were in full agreement with the previous assignment of these protons in 1.162113 and in shahamins C-E (1.157-1.159).118 The lactone ring of 4.6 adopted a twisted-boat conformation with H-13 and H-14 in a trans arrangement.118 This preferred conformation places the 16-OH and the lactone carbonyl in close proximity for hydrogen bonding stabilization, which could explain why a δ-lactone product was isolated rather than the alternative γ-lactone. In this way the

113

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes overall relative configuration of the δ-lactone (4.6), and thereby of secoshahamin (1.164) was established.

As expected, treatment of lactone 4.6 with Ac2O/pyridine produced 12-desacetoxyshahamin C (1.162) with same positive sign of specific rotation as the natural isolate. 1.162 shares the same sign of optical rotation as shahamin K (1.161), whose full relative and absolute configuration has been verified by enantioselective synthesis.21,119 Therefore we infer the same absolute configuration for secoshahamin (1.164).

114

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes Table 4.6 Comparison of 1H and 13C NMR assignments for secoshahamin (1.164), 12-desacetoxyshahamin C (1.162) and 12- desacetoxypolyrhaphin A (1.163), saponification product (4.6), shahamin K (1.161) and shahamin E (1.159).a,b Secoshahamin (1.164)108 12-desacetoxyshahamin C 12-desacetoxypolyrhaphin A 12-deoxyshahamin E (4.6) Shahamin K (1.161)e119,171 Shahamin E (1.159)d118 (1.162)113 (1.163)113 Position 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) 13C 1H (mult., J, Hz) d 1 37.2, CH2 a2.34, br d (12.6) 37.2, CH2 a2.34, m 37.0, CH2 a2.35, br dd (12.8, 37.1, CH2 a2.35, m 36.7, CH2 a2.39, br dd (12.7, 37.2, CH2 a2.37, br dd 5.2) 5.1) b1.82, m b1.79, m b1.83, m b1.80, m b1.95, dd (12.7, 12.6) b1.80, br t (12.7) d 2 29.0, CH2 a1.74, m 28.8, CH2 a1.74, m 28.8, CH2 a1.75, m 28.9, CH2 a1.74, m 28.8, CH2 28.9, CH2 a1.77, m b1.37, m b1.37, m b1.38, m b1.38, m b1.38, m d 3 37.9, CH2 a1.64, m 37.8, CH2 a1.60, m 38.8, CH2 a1.59, m 37.9, CH2 a1.59, m 37.8, CH2 37.7, CH2 a1.65, dt b1.25, m b1.25, m b1.51, m b1.26, m b1.28, m 4 36.1, C - 37.6, C - 37.0, C - 36.0, C - 36.0, C - 48.4, C - 5 53.8, CH 1.95, m 54.5, CH 1.90, m 54.4, CH 1.87, m 54.6, CH 1.92, m 48.7, CH 1.82, m 54.8, CH 1.93, td (10.0, 8.8) 6 26.5, CH2 a1.70, m 26.0, CH2 a1.70, m 26.5, CH2 a1.72, m 26.0, CH2 a1.71, m 32.5, CH2 a2.17, m 26.1, CH2 a1.74, m b1.62, m b1.70, m b1.68, m b1.71, m b1.80, m b1.75, m d 7 39.3, CH2 1.67, m 37.8, CH2 1.56, m 37.8, CH2 1.60, m 37.9, CH2 1.56, m 78.5, CH2 4.98, dd (9.5) 37.9, CH2 1.75, m 1.51, m 1.49, m 1.27, m 1.49, m 1.50, m 8 49.3, C - 48.0, C - 38.8, C - 48.2, C - 49.7, C - 36.3, C - 9 56.0, CH 2.83, d (8.8) 54.9, CH 2.72, d (8.7) 57.2, CH 2.58, d (8.7) 54.9, CH 2.72, d (8.6) 54.2, CH 2.80, d (7.7) 54.7, CH 2.77, d (8.8) 10 153.5, C - 153.3, C - 153.9, C - 153.6, C - 153.0, C - 153.7, C - 11 173.1, C - 172.6, C - 177.0, C - 173.6, C - 172.6, C - 165.8, C - d 12 33.7, CH2 2.52, dd (15.8, 2.2) 32.5, CH2 2.54, m 31.0, CH2 2.45, d (10.1) 31.9, CH2 2.70, dd (15.3, 6.6) 32.2, CH2 2.56, dd 68.4, CH2 4.52, d (7.4) 2.21, dd (15.8, 11.0) 2.54, m 2.45, d (10.1) 2.51, dd (15.3, 11.7) 2.55, dd d 13 34.0, CH 2.94, m 32.1, CH 2.46, m 35.2, CH 3.09, m (9.4, 1.4) 35.0, CH 2.34, m 31.5, CH 2.49, m 45.0, CH 2.64, dddd (7.4, 6.9, 4.0, 2.7) 14 46.6, CH 1.84, dd (9.7, 4.0) 44.2, CH 1.78, br d (9.7) 48.6, CH 1.81, m 43.9, CH 1.76, m 44.6, CH 1.80, m 39.8, CH 2.01, ddd (12.3, 6.6, 4.0) 15 63.6, CH2 4.36, dd (11.6, 4.0) 67.5, CH2 4.31, dd (11.8, 6.1) 73.5, CH2 4.44, t (8.9) 68.0, CH2 4.31, dd (11.7, 5.7) 67.9, CH2 4.28, dd (12.0, 9.8) 66.4, CH2 4.36, dd (11.8, 6.6) 3.98, dd (11.6, 9.7) 4.20, dd (11.8, 10.0) 4.12, t (8.9) 4.22, dd (11.7, 10.6) 4.20, dd (12.0, 6.0) 4.19, dd (12.3, 11.8) 16 68.4, CH2 4.07, dd (10.8, 8.3) 68.1, CH2 4.17, dd (11.2, 4.3) 63.1, CH2 4.40, dd (11.7, 4.1) 67.3, CH2 3.69, dd (10.6, 4.5) 67.0, CH2 4.21, dd (11.2, 4.4) 65.3, CH2 3.81, dd (11.3, 2.7) 4.02, dd (10.8, 6.8) 3.83, dd (11.2, 7.8) 4.02, dd (11.7, 9.3) 3.49, dd (10.6, 8.4) 3.89, dd (11.2, 7.5) 3.61, dd (11.3, 6.9) 17 21.2, CH3 0.85, s 21.5, CH3 0.91, s 21.2, CH3 0.82, s 21.6, CH3 0.92, s 21.2, CH3 0.93, s 21.3, CH3 0.97, s 18 34.6, CH3 0.95, s 34.5, CH3 0.93, s 34.5, CH3 0.94, s 34.6, CH3 0.94, s 34.4, CH3 0.95, s 34.5, CH3 0.95, s 19 25.6, CH3 1.01, s 25.8, CH3 0.99, s 25.6, CH3 0.98, s 25.8, CH3 0.98, s 25.3, CH3 1.01, s 25.6, CH3 0.99, s 20 115.3, CH2 4.91, d (1.9) 115.0, CH2 4.85, d (2.0) 114.9, CH2 4.86, d (1.8) 115.2, CH2 4.86, d (1.9) 116.3, CH2 4.94, br d (1.7) 115.5, CH2 4.88, d (1.9) 4.82, br s 4.61, d (2.0) 4.63, br s 4.63, br s 4.69, br d (1.7) 4.67, d (0.9) 7- 16.1, CH3 2.07, s OCOCH3 7- 170.7, C - OCOCH3 15- 21.0, CH3 2.05, s OCOCH3 15- 170.8, C - OCOCH3 16- 21.0, CH3 2.07, s 20.9, CH3 2.07, s 21.0, CH3 2.06, s 20.8, CH3 2.06, s OCOCH3 16- 170.9, C - 170.0, C - 170.5, C - 172.6, C - OCOCH3 12- 51.7, CH3 3.68, s CO2CH3 12-OH NDc 16-OH NDc NDc a b c d e Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz. ND – Not Detected. At 360 MHz, in CDCl3. At 400 MHz, CDCl3.

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4.1.2.4 Structure elucidation of shahamin L (4.3).

Metabolite 4.3, named as shahamin L, was likewise isolated as a colorless oil and produced + 1 an adduct ion at m/z 413.2328 [M + Na] , corresponding to a molecular formula of C23H34O5. The H

NMR spectrum (Table 4.7) showed exomethylene signals at δH 4.79 (br s) and 4.55 (br s) assigned to H-20. These signals, in addition to three quaternary methyl signals at δH 0.87 (s), 0.95 (s), 0.97 (s) and the diagnostic bridging proton at δH 2.81 (d, J = 9.0 Hz, H-9), were indicative of a perhydroazulene ring system.110 The cis relative configuration of the perhydroazulene ring system was confirmed by a NOESY correlation between H-5 and H-9.

Table 4.7 1H and 13C NMR assignments for shahamin L (4.3).a,b Position 13C 1H (mult., J, Hz) 1 37.0, CH2 a2.32, br d (12.8) b1.84, m 2 28.7, CH2 a1.72, m b1.36, m 3 37.8, CH2 a1.61, m b1.25, m 4 35.8, C - 5 54.0, CH 2.06, m 6 26.6, CH2 a1.73, m b1.73, m a1.84, m 7 37.5, CH2 b1.47, m 8 49.5, C - 9 54.5, CH 2.81, d (9.0) 10 153.3, C - 11 166.2, C - 12 115.4, CH 5.98, q (2.0) 13 157.6, C - 14 56.7, CH 3.83, t (1.4) 15 101.9, CH 6.36, s a4.57, dt (14.4, 2.0) 16 73.4, CH2 b4.54, dd (14.4, 2.0) 17 22.5, CH3 0.87, s 18 34.3, CH3 0.95, s 19 25.6, CH3 0.97, s 4.79, br s 20 114.4, CH2 4.55, br s 11-CO2CH3 51.3, CH3 3.72, s 15-OCOCH3 21.5, CH3 2.04, s

15-OCOCH3 170.2, C - a b Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz.

There were signals at δH 2.04 (s), 3.72 (s), and 6.36 (s) for an acetate methyl, methyl ester, and an acetal, respectively. Other NMR signals corresponded to an oxymethylene group (δH 4.57 and

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4.54; δC 73.4), and an alkene (δH 5.98 q; δC 115.4 (CH) and 157.6 (C)). There was a methine proton at δH 3.83 (t, J = 1.4 Hz, H-14) linked directly to a carbon at δC 56.7 and connected by gCOSY to the acetal proton at δH 6.36, by HMBC to C-7, C-8 and C-17 of the perhydroazulene ring, as well as to the carbon signals at δC 157.6 and 73.4; this signal was therefore assigned to H-14. The gCOSY spectrum connected the alkene proton at δH 5.98 (H-12) to the oxymethylene protons at δH 4.57 and n 4.54 (H2-16). In the HMBC spectrum ( JCH 8 Hz), the methyl ester signal at δH 3.72 correlated to the carbonyl signal at δC 166.2, while the correlations of (H2-16) to C-12 and C-13, and of H-15 at δH

6.36 to C-13, C-14, and C-16, as well the acetate carbonyl signal at δC 170.2, confirmed the two- dimensional structure. It was noticeable that there were no visible HMBC correlations from the alkene n H-12 to adjacent carbons even when the HMBC experiment was rerun with an JCH value of 4 Hz.

4.1.2.5 Molecular modelling of truncated structure to inform elucidation of shahamin L (4.3)

Figure 4.12 Candidate diastereomers 4.7a and 4.7b for the truncated structure of shahamin L (4.3), with carbon numbers matching those of (4.3).

The relative configuration of the 2-(dihydrofuran-3(2H)-ylidene)acetate moiety was next considered. An NOE between H-12 and both H-16 protons established an E configuration for the exocyclic double bond. Although the small coupling (J = < 0.5 Hz) between the H-14 and H-15 signals suggested a trans arrangement of these protons,111 it was uncertain to what extent the conformation of the five membered ring was affected by the presence of the exocyclic substituent, therefore modelling was undertaken. The truncated structures 4.7a and 4.7b, in which a t-butyl group replaced the conformationally flexible perhydroazulene ring, were selected for computational analysis (Figure 4.12). A Monte Carlo conformational search of the (14S, 15R)-4.7a and (14S, 15S)- 4.7b diastereomers, further optimization by DFT using Gaussian software, and calculation of MAE values for 4.7a and 4.7b was undertaken. For the 1H data, the MAE were 0.16 and 0.24 ppm for 4.7a and 4.7b, respectively, while the 13C MAE were 1.61 and 2.15 ppm for 4.7a and 4.7b, respectively. Although the MAE values were similar, there was a slight preference for the 4.7a diastereomer. The DP4 probabilities135 were calculated for both diastereomers resulting in a conclusive 99.97% probability for the (14S, 15R)-4.7a diastereomer (Appendix).

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Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes 4.1.2.6 Characterization of 15-desacetoxy-12-acetoxydendrillolide A (4.4).

Diterpene 4.4, also a colorless oil, displayed an adduct ion at m/z 399.2142 [M+Na]+ by

HRESIMS. The corresponding molecular formula of C22H32O5 was identical to that of dendrillolide 110,111 1 A (1.142). Inspection of the signals for H2-20, H-9 and the methyl groups in the H NMR spectrum (Table 4.8) confirmed a perhydroazulene skeleton, and further revealed an acetate methyl group at δH 2.16 (s) and an oxymethine proton at H-12 δH 5.49 (d); these signals were comparable to those of 12-acetoxydendrillolide A (1.152).34 For 4.4, the position of the acetoxy group was confirmed at C-12 through HMBC data, as both the signals at δH 5.49 (d, J = 6.6 Hz) and 2.16 (12-

OAc, s) correlated to the carbonyl at δC 169.4. However, metabolite 4.4 differed from 1.152 in lacking a C-15 acetate substituent since the oxymethine signal of 1.142 was replaced by signals for H2-15 at

δH 4.10 (dd, J = 6.7, 8.9 Hz) and 3.92 (dd, J = 8.9, 11.8 Hz). Accordingly, 4.4 was elucidated to be 15-desacetoxy-12-acetoxydendrillolide A. In both 4.4 and 1.152, NOESY correlations from H-13 to H-14 and H-16 positioned these protons on the same side of the plane. In our earlier publication on 12-acetoxydendrillolide, the structure of 1.152 was inadvertently drawn with an incorrect configuration at C-15 (Figure 4.13).34

Figure 4.13 Substitution patterning on the dendrillane scaffold at C-12 and C-15.

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Table 4.8 1H and 13C NMR assignments for 15-desacetoxy-12-acetoxydendrillolide A (4.4)a,b Position 13C 1H (mult., J, Hz) 1 37.5, CH2 a2.36, br d (12.6) b1.81, m 2 28.6, CH2 a1.74, m b1.38, m 3 37.8, CH2 a1.60, m b1.26, m 4 37.7, C - 5 53.9, CH 1.77, m 6 27.1, CH2 a1.75, m b1.75, m 7 37.0, CH2 a1.66, m b1.50, m 8 36.9, C - 9 57.8, CH 2.61, d (7.9) 10 152.3, C - 11 171.7, C - 12 68.0, CH 5.49, d (6.6) 13 45.9, CH 3.18, br q (6.7) 14 51.2, CH 2.53, br td (11.8, 6.7) 15 68.6, CH2 a4.10, dd (8.9, 6.7) b3.92, dd (11.8, 8.9) 16 105.8, CH 6.07, d (5.6) 17 23.7, CH3 0.96, s 18 34.4, CH3 0.94, s 19 25.9, CH3 0.96, s 20 114.5, CH2 4.82, d (2.2) 4.59, d (2.2) 12-OCOCH3 20.6, CH3 2.16, s

12-OCOCH3 169.4, C - a b Chemical shifts (ppm) referenced to CHCl3 (δH 7.26, δC 77.16). At 700 MHz.

4.1.2.7 Biosynthesis of metabolites from G. geometricus Rearranged diterpenes with the perhydroazulene motif have been proposed to originate from modification of a spongian skeleton, in which cleavage of the C-9/C-11 bond of ring C, generating intermediate 4.8,110 is followed by expansion of ring A via 1, 2-alkyl migration of the C-5/C-10 bond, with concomitant contraction of ring B to give 4.9 (Scheme 4.3).118 Secoshahamin (1.164) is the diacetylated methyl ester of 4.9. Shahamin L (4.3) may be derived by cyclization of C-12 to a lactol (4.10), following oxidation at C-15. The exocyclic double bond could be formed either by dehydrogenation or perhaps through an acetylated intermediate (cf. polyrhaphin B).117 Lactonization of 4.9 followed by acetylation provides the metabolites 12-desacetoxyshahamin C (1.162) and 12- desacetoxypolyrhaphin A (1.163),113 as well as the new metabolite 4.4. Based on this biosynthetic hypothesis, we infer that 4.3 and 4.4 have the same absolute configuration as 1.164, given that the three metabolites co-occur in the nudibranch Goniobranchus geometricus.

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Scheme 4.3 Putative biosynthetic pathway to the diterpene metabolites of Gonibranchus geometricus. The green, pink and orange dots highlight carbons derived from C-12, C-13 and C-14 of the precursor spongian diterpene framework.

4.2 Conclusions The investigation into characterizing the mixture of lactols (4.1 and 4.2) isolated from G. coi, successfully assigned the relative configuration at C-15 as 15R and 15S, respectively for 4.1 and 4.2, through spectroscopic analysis, computational studies and chemical correlation. The anatomical distribution of the metabolites isolated from G. coi will be explored in Chapter 5. Furthermore, the isolation and structure elucidation of three oxygenated rearranged diterpene derivatives (1.164, 4.3 and 4.4) from Goniobranchus geometricus have been described. A combination of spectroscopic, modelling, and computational approaches, together with chemical correlation involving conversion to the δ-lactone product 4.6, enabled the relative configuration of secoshahamin (1.164) to be established as 13R,14R as well as by comparison with 12- desacetoxyshahamin C (1.162) and 12-desacetoxypolyrhaphin A (1.163). Finally, the carbon skeleton of 1.164 as a central intermediate in the biosynthesis of these highly rearranged oxygenated diterpenes

120

Chapter 4: Modifications to the oxygenated fragment of rearranged diterpenes from a spongian diterpene scaffold has also been explored in this study. The isolation of secoshahamin (1.164) from both a sponge and nudibranch suggested 1.164 was of dietary origin in the nudibranch.

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites

122

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites 5.0 Introduction This chapter covers the investigation of the anatomical distribution of the metabolites isolated from Goniobranchus sp.1, G. collingwoodi, G. aureopurpureus, G. leopardus, and G. coi. To investigate the ecological role of the metabolites isolated from these nudibranchs, the 1H NMR spectra of the extracts obtained from the mantle and gut (viscera) were compared. To explore how nudibranchs protect themselves against predators, the anti-feedant properties of purified metabolites dendrillolide A, aplyviolene and macfarlandin E were investigated through Palaemon shrimp palatability assays. The synergistic effect of these metabolites was also explored. To assess the bioactivity of the compounds found in the nudibranch and understand whether these compounds may be useful for other applications, selected purified compounds were screened for cytotoxic, antiviral and antimicrobial activity. The metabolites were screened for cytotoxic activity against human lung (NCIH-460), colorectal (SW620), and liver (HepG2) cells. Screening was carried out on metabolites for anti-infective activity against Ross River fever, Dengue fever and influenza viral strains. Lastly, antimicrobial assays against Staphylococcus aureus in both broth and biofilm forms were carried out.

5.1 Anatomical distribution of metabolites within Goniobranchus nudibranchs Many species, both terrestrial and marine, utilize toxic and/or unpalatable natural products as a form of chemical defense. These compounds protect the host organism through the employment of aversive smells, distasteful flavours, painful irritants and/or lethal toxins.220 Nudibranchs are one such group that have evolved chemical defenses, due to their lack of physical protection. Many species of nudibranchs obtain secondary metabolites from dietary sponges, so the chemical composition of a nudibranch can be dependent on its dietary preferences.67 Understanding the predator/prey relationship between nudibranchs and sponges will aid in understanding nudibranch chemical ecology.221 The anatomical distribution can be used as an indicator of the potential ecological role of individual metabolites, as some nudibranchs have been found to store the more toxic chemicals in their mantle and peripheral (mantle rim) relative to the viscera.12,20 As mentioned in Chapter 1, some nudibranch species have a broad diet and selectively accumulate one defensive metabolite in their peripheral tissues, as observed for Chromodoris species which accumulate the highly toxic latrunculin A (1.1) in the mantle rim.20 Conversely, other nudibranch species may only feed on selected sponge species and accumulate several defensive metabolites in their mantle.17,76,86 For example, a previous member of our laboratory group found Chromodoris (= Goniobranchus) reticulata to contain twenty- two metabolites, with fifteen spongian diterpenes in the mantle, one linear furan diterpene in the viscera and six metabolites in both tissues. The major component in the tissues was different, with aplysulphurin (1.110) in the viscera and aplyroseol-2 (5.1) in the mantle tissue.54 Other nudibranch 123

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites species, such as those from the genus Glossodoris, can modify the chemicals obtained from their sponge diet. It was shown by Manzo et al. that Glossodoris nudibranchs not only sequester scalarane metabolites from their sponge prey, but also have the ability to modify these metabolites into less toxic forms, such as deoxoscalarin (5.2), by a detoxification process.33 Winters et al. investigated 10 genera of sponge-eating nudibranchs (28 species), exploring the ability of nudibranchs to bioaccumulate and store toxins obtained from their sponge prey, by comparing chemical profiles of gut and mantle tissue extracts.161

5.1.1 Anatomical distribution of metabolites in G. collingwoodi In our study, spongian diterpenes were isolated from all eight specimens of G. collingwoodi. Following careful dissection of each specimen, the 1H NMR spectra of mantle and viscera extracts were compared to investigate the anatomical distribution of the metabolites. The distribution of metabolites in G. collingwoodi is summarized in Table 5.1. The mantle tissue contained the new diterpene 2.1 together with the known metabolites 1.32 and 1.33, while luffarin-X (1.16) was only found in the viscera. The remaining metabolites spongian-16-one (1.24) and isoagatholactone (1.19) were isolated from both tissue types. 7α-Acetoxyspongian-16-one (1.32) was the major component in the mantle, whereas metabolites 1.19 and 1.24 were the major component in the viscera.

Table 5.1 Distribution of diterpenes in G. collingwoodi organs. Compound Present in 7α-acetoxyisoagatholactone (2.1) Mantle 7α-acetoxyspongian-16-one (1.32) Mantle 7α-hydroxyspongian-16-one (1.33) Mantle luffarin-X (1.16) Viscera isoagatholactone (1.19) Both tissues spongian-16-one (1.24) Both tissues

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites 5.1.2 Anatomical distribution of metabolites in G. aureopurpureus The six specimens of G. aureopurpureus were dissected into their mantle and viscera. The body parts were extracted separately, and the extracts compared by 1H NMR spectroscopy (Table 5.2). The mantle was found to contain all of the new spongian diterpenes (2.2-2.6 and 4.4), along with the known metabolite shahamin C (1.157). From the viscera, metabolites 1.16, 1.153, 1.160, and 1.163 were isolated. The metabolites 1.24, 1.32, 1.143, 1.144, 1.154, and 1.164 were found in both the mantle and viscera, while the new metabolites (2.2-2.6) were only isolated from the mantle tissue.

Table 5.2 Distribution of diterpenes in G. aureopurpureus organs. Compound Present in 15-desacetoxy-12-acetoxydendrillolide A (4.4) Mantle 7α-acetoxy-6α-hydroxyspongian-16-one (2.2) Mantle 6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.3) Mantle 20-acetoxy-6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.4) Mantle 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.5) Mantle 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate (2.6) Mantle shahamin C (1.157) Mantle luffarin-X (1.16) Viscera polyrhaphin A (1.153) Viscera 15,16-diacetoxyshahamin B (1.160) Viscera 12-desacetoxypolyrhaphin A (1.163) Viscera spongian-16-one (1.24) Both tissues 7α-acetoxyspongian-16-one (1.32) Both tissues macfarlandin E (1.143) Both tissues aplyviolene (1.144) Both tissues polyrhaphin B (1.154) Both tissues secoshahamin (1.164) Both tissues

5.1.3 Anatomical distribution of metabolites in Goniobranchus sp 1. The three specimens of Goniobranchus sp. 1 were dissected into the mantle and viscera. The anatomical distribution was explored through the comparison of the mantle and viscera chemical profiles (Table 5.3). The mantle tissue contained the new diterpenes 2.7-2.12 together with the known metabolites 1.40, 1.41 and 1.43, while spongian-16-one (1.24) and 7α-acetoxyspongian-16-one (1.32) were only found in the viscera. The remaining diterpenes 1.19, 1.38, 1.39 and 1.42, were isolated from both tissue types. Like G. collingwoodi and G. aureopurpureus only the mantle was found to have the newly elucidated metabolites.

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites Table 5.3 Distribution of diterpenes in Gonibranchus sp. 1 organs. Compound Present in 20-acetoxy,12α-oxyspongian-16-one-12α-propionate (2.7) Mantle 20-acetoxy-13-hydroxy-spongian-16-one (2.8) Mantle 12-hydroxyspongian-16-one (2.9) Mantle 12-hydroxy-20-oxyspongian-16-one-20-propionate (2.10) Mantle 12-hydroxy-11,20-dioxyspongian-16-one-11,20-dipropionate (2.11) Mantle 11-hydroxy-12,20-dioxyspongian-16-one-12,20-dipropionate (2.12) Mantle 20-oxyspongian-16-one-propoionate (1.40) Mantle 12α,20-dioxyspongian-16-one-dipropionate (1.41) Mantle 12α-acetoxy,20-oxyspongian-16-one-20-propionate (1.43) Mantle spongian-16-one (1.24) Viscera 7α-acetoxyspongian-16-one (1.32) Viscera isoagatholactone (1.19) Both tissues 12α-acetoxyspongian-16-one (1.38) Both tissues 20-acetoxyspongian-16-one (1.39) Both tissues 12α,20-diacetoxyspongian-16-one (1.42) Both tissues

5.1.4 Anatomical distribution of metabolites in G. leopardus After the careful dissection and analysis of the five G. leopardus specimens, the 1H NMR spectra of the mantle and viscera extracts were compared (Table 5.4). The majority of the metabolites (1.130, 1.142, 1.144, 1.145, and 1.162-1.165) were found in both the mantle and the viscera tissue, with the exception of 10,20-epoxydendrillolide A (3.4) which was found only in the mantle. In addition, chelonaplysin C (1.131), cheloviolene C (1.132) and dendrillolide E (1.172) were only found in the viscera. In contrast to G. collingwoodi, G. aureopurpureus and G. leopardus the new metabolite 12α-acetoxypolyrhaphin D (2.13) was found in the viscera, as well as the mantle.

Table 5.4 Distribution of diterpenes in G. leopardus organs. Compound Present in 10,20-epoxydendrillolide A (3.4) Mantle chelonaplysin C (1.131) Viscera cheloviolene C (1.132) Viscera dendrillolide E (1.172) Viscera 12α-acetoxypolyrhaphin D (2.13) Both tissues norrisolide (1.130) Both tissues dendrillolide A (1.142) Both tissues aplyviolene (1.144) Both tissues macfarlandin D (1.145) Both tissues 12-desacetoxyshahamin C (1.162) Both tissues 12-desacetoxypolyrhaphin A (1.163) Both tissues secoshahamin (1.164) Both tissues macfarlandin C (1.165) Both tissues

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites 5.1.5 Anatomical distribution of metabolites in G. coi The two specimens of G. coi were dissected into the mantle and viscera, and the subsequent extracts were compared through their 1H NMR spectra. In summary, several of the metabolites were found in both the mantle and viscera tissues (3.1, 3.2, 3.4-3.6, 4.1, 4.2, 1.142-1.144, 1.148, 1.149, 1.162, 1.163, and 1.165). From the mantle tissue the new metabolite 5-hydroxydendrillolide A (3.3) and the known compound dendrillolide C (1.146) were present. From the viscera tissue the known compounds norrisolide (1.130), cheloviolene C (1.132), macfarlandin D (1.145), and polyrhaphin C (1.171) were found.

Table 5.5 Distribution of diterpenes in G. coi organs. Compound Present in dendrillolide C (1.146) Mantle 5-hydroxydendrillolide A (3.3) Mantle norrisolide (1.130) Viscera cheloviolene C (1.132) Viscera macfarlandin D (1.145) Viscera polyrhaphin C (1.171) Viscera 5,9-epoxydendrillolide A (3.1) Both tissues 10-oxonordendrillolide A (3.2) Both tissues 10,20-epoxydendrillolide A (3.4) Both tissues 10R-aldehyde artefact (3.5) Both tissues cyclopropyl derivative (3.6) Both tissues dendrillactol (4.1) Both tissues 15-epidendrillactol (4.2) Both tissues dendrillolide A (1.142) Both tissues macfarlandin E (1.143) Both tissues aplyviolene (1.144) Both tissues cheloviolene A (1.148) Both tissues cheloviolene B (1.149) Both tissues 12-desacetoxyshahamin C (1.162) Both tissues 12-desacetoxypolyrhaphin A (1.163) Both tissues macfarlandin C (1.165) Both tissues

The Goniobranchus species investigated within this study tended to sequester the newly elucidated metabolites in the mantle rather than the viscera, excluding G. coi, where majority of the new metabolites were found in both tissues. The metabolites isolated from the mantle are proposed to be utilized by the animal as an unpalatable or toxic deterrent against predation from fish and crustaceans. For examples, a previous study found 7α-acetoxyspongian-16-one (1.32) to be cytotoxic 51 against L1210 (IC50 6.1 μM) and KB (IC50 44.1 μM) cells. In our study metabolite 1.32 was found to be the major component isolated from the mantle of G. collingwoodi. All Goniobranchus specimens examined in this study were found to accumulate several metabolites in their mantle, which is consistent with what has been previously described in the literature.56,86,161 In addition, for G. aureopurpureus and G. coi the metabolites macfarlandin E 127

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites (1.143) and aplyviolene (1.144) were found in both the mantle and the viscera. These metabolites have been previously found only in the viscera of Chromodoris kuiteri and the toxic metabolite latrunculin A (1.1) was the only metabolites found in the mantle.20 Therefore, the biological activity of the mantle metabolites was explored.

5.2 Ecological bioactivity screening To assess the relative feeding deterrency of nudibranch and sponge extracts, anti-feedant assays (measured as effective dose, ED50) have previously been performed using intertidal generalist crustaceans such as hermit crabs (Pagurus longicarp),222 crabs (Leptodius spp.)223 or prawns (Palaemon serenus20 or Palaemon elegans12,224). Other studies have also conducted laboratory-based palatability assays using fish.225 It is important to test extracts against multiple types of predators as it has been observed that different species may respond differently to secondary metabolites due to the mechanisms of uptake.226 Such assays involve making food pellets using various media (dried squid, fish food, etc.), which is then mixed with varying concentrations of extract and fed to the crustacean, alongside pellets lacking extract as controls. The pellets are fed to the target species, then whether the pellet is consumed or rejected is recorded. However, palatability assays utilizing generalist crustacean scavengers may not be ecologically relevant as some of these species are not known to hunt nudibranchs, therefore some assays assessing the relative feeding deterrency of nudibranch and sponge extracts have instead been conducted on fish using the generalist predators, Tetractenos hamiltoni (toadfish) and Rhinecanthus aculeatus (Picasso triggerfish). These fish cohabit with some nudibranchs, with T. hamiltoni being shown to include molluscs in its diet.227 Avoidance learning assays, such as those that use zebra fish (Danio rerio), are also a valuable assay to assess the memory recall of post-ingestion aversive effects (vomiting) and link this to mechanisms of hunting; i.e tasting prey first or hunting based on sight.228 Previous studies have utilized natural assemblages of reef fish in the field (Guam) to assess the palatability of nudibranch extracts.117,229 Paul et al. noted that three Glossodoris nudibranch species fed on the sponge Hyrtios erecta, which have been shown to contain sesterterpenes. The organic extracts of the Hyrtios sponge exhibited feeding deterrency at half of natural concentrations in some laboratory and field feeding assays. Conversely, the organic extracts of the nudibranchs G. hikeurensis and G. cincta which also contain sesterterpenes were not deterrent at half of natural concentrations.

A brine shrimp lethal dose 50 (LD50) assay is a quick and convenient test to give an indication of the potential toxicity of nudibranch extracts; though this assay is again argued to be not ecologically relevant.5 Previous assays from the Cheney group have shown Goniobranchus crude extracts to be non-toxic but are moderately or highly unpalatable.225 Winters et al. screened the crude extracts of the mantles and guts of each species for toxicity to brine shrimp and palatability to three generalist

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites marine predators (P. serenus, T. hamiltoni and R. aculeatus) using lethal dose (LD50) and effective 161 dose (ED50) values, respectively. Winters et al. found that there was no correlation between toxicity and palatability of nudibranch chemical extracts. Instead extracts could be categorized into four groups: 1) highly toxic and unpalatable (Chromodoris and Doriprismatica); 2) highly toxic but weakly unpalatable (Ceratosoma and Mexichromis), 3) weakly toxic but highly unpalatable (Goniobranchus); and 4) extracts that did not display activity in any assays (Sebadoris fragilis and Aphelodoris varia). The findings of Winters et al. gave insight on the evolution of diverse sponge- based chemical defenses that are utilized by nudibranchs.161

5.2.1 Palatability assays The Palaemon shrimp assay used in our study was first developed by Mollo et al. to investigate the role of a selection of the purified compounds, including brominated tetrahydropyran 224 (5.3) isolated from Haminoea cyanomarginata and PEG2-1,15-lactone (5.4) from Melibe viridis. At natural concentration each metabolite caused a significant rejection rate with respect to the control.

Mollo and co-workers also used this method to screen purified metabolites sequestered into mantle dermal formations (MDFs) of six nudibranch species, including Chromodoris sinensis.12 The two metabolites isolated from C. sinensis, aplyroseol-2 (5.1) and the corresponding dialdehyde (5.5), were in a 1:3 mixture. A pure sample of aplyroseol-2 was screened, while the dialdehyde was screened as the major component in the crude extract, since it easily converted to the co-isolated cyclic- hemiacetal (aplyroseol-2), which is proposed to be the masked form of this dialdehyde. It was found that the feeding deterrent activity of the mixture, which include 5.1 and 5.5, was more deterrent relative to the purified aplyroseol-2.

Other examples of unpalatable dialdehydes include the sesquiterpenoid, polygodial (5.6),230 and the sesterterpene, scalaradial (5.7).229 When screened for feeding deterrency against two species of co-occurring fishes (the mummichog Fundulus heteroclitus and the striped blenny Chasmodes 129

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites bosquianus), 5.6 was found to be unpalatable and was believed to be responsible for the deterrency of the nudibranch extract.230 The reactive aldehyde groups of polygodial have been suggested to be masked by the formation of cyclic bis acetals.231 Indeed, this cyclization was observed in the presence of an acid.232 Rogers and Paul demonstrated the major component 5.7, found in the nudibranch G. hikeurensis, was only deterrent against Melichthys vidua (trigger fish) at high concentrations of 2.5% dry weight and when screened as a 1:1 mixture with scalarin at a combined concentration of 1.0% dry weight, suggesting there may be some synergistic effect of the mixture.229 Cimino et al. suggest that the nudibranch is able to actively convert scalaradial into deoxoscalarin (5.2).33

Our group regularly uses the palatability assay protocol of Mollo et al.224 to screen crude extracts and purified metabolites for their unpalatability. The common rock shrimp (Palaemon serenus) were collected from rockpools during low tide. This shrimp species was selected for its clear carapace, allowing the observation of a red spot in the digestive tract when the shrimp consumes the red colored pellets. The pellet preparation recipe involved combining the dry ingredients of freeze- dried squid, alginic acid and purified sea sand. The extract dissolved in DCM was then added, the ingredients mixed and the solvent evaporated. The mixture was homogenized in water, to which a drop of red food dye was added. Subsequently to produce the spaghetti-like strands, the mixture was exuded via syringe into a 0.25 M calcium chloride solution. The alginic acid reacts with the calcium chloride to form calcium alginate, a gelatinous substance, where the strands can be cut into even sized pellets (Figure 5.1). Using tweezers these pellets were then fed to the shrimp individually housed in small aquaria. A shrimp is considered to have ‘accepted’ a pellet when a red spot is observed through the clear carapace and ‘rejected’ when the pellet is not consumed, thus no red spot is present (Figure 5.2). For details of the feeding deterrency protocol see Chapter 7.

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Extract dissolved in DCM, Mixtures combined and Homogenized in water freeze dried squid, alginic solvent evaporated and red food dye acid and purified sea sand

“Spaghetti-like” strand is cut Loaded & extruded into into pellets for shrimp feeding calcium chloride solution

Figure 5.1 Schematic depiction of pellet recipes for feeding deterrency assay (Images provided by Dr Karen Cheney).

A B

Figure 5.2 Palaemon shrimp demonstrating (A) a red spot indicating a positive uptake of the pellet; (B) the absence of a red spot indicating rejection of the pellet (Images provided by Dr Karen Cheney).

A member of our group, Dr Ariyanti Dewi, previously investigated the chemistry of G. coi and conducted Palaemon shrimp palatability assays on the crude extract at concentrations ranging from 1.23 mg/mL up to the natural concentration (NC = 39.4 mg/mL). At natural concentration 50% rejection was observed, however when the crude extract was screened at 1/2, 1/4 and 1/8 of natural concentration 25.0%, 34.5% and 44.4% rejection was observed, respectively. The results were

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites deemed inconclusive, due to the inconsistent trend. The major component of the crude extract screened was dendrillolide A (1.142), with aplyviolene (1.144) and macfarlandin E (1.143) present in minor quantities. Within the current study, dendrillolide A was also isolated as the major component in the crude extract of G. coi. In the 1H NMR spectrum of the crude mantle extract of G. coi, the integration of the 1.142 signals was compared to signals for the minor components 1.144 and 1.143, showing a relative integration of 242:1:8, respectively. The preponderance of 1.142 over the minor components suggested that the nudibranch may be sequestering dendrillolide A as a noxious defensive metabolite. Therefore, purified samples of 1.142, 1.143 and 1.144 were screened at 10 mg/mL and 20 mg/mL to explore the unpalatability of each metabolite. Dendrillolide A exhibited 45% rejection against Palaemon serenus at 20 mg/mL (Figure 5.3). Further screening at higher concentration is required, as the natural concentration of the crude extract, screened by Dr Ariyanti Dewi, was 39.4 mg/mL and showed 50% rejection. However, further screening at higher concentration could not be achieved due to the limited quantity of extract. We also speculated if a minor component could be responsible for the potential unpalatability of the crude extracts. Next, 1.143 and 1.144 were screened separately at 10 and 20 mg/mL. Aplyviolene (1.144) exhibited 33% rejection at 10 mg/mL and 40% rejection at 20 mg/mL, whereas macfarlandin E (1.143) showed 0% rejection (10 mg/mL) and 17% rejection (20 mg/mL).

Figure 5.3 Comparison of unpalatability of dendrillolide A (1.142), macfarlandin E (1.143) and aplyviolene (1.144) against Palaemon serenus at 10 and 20 mg/mL.

Dendrillolide A (1.142) and aplyviolene (1.144) produced similar rejection percentages at 10 mg/mL (30% and 33% rejection, respectively) and at 20 mg/mL (45% and 40% rejection, respectively). The key structural difference between the metabolites was that 1.142 possessed a 2,8- dioxabicyclo[3.3.0]octane moiety while 1.144 has a 2,7-dioxabicyclo[3.2.1]octane moiety. 132

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites

Model studies have been conducted by Overman and co-workers on truncated analogues of 1.142 and 1.143, where the perhydrozulene motif was replaced with a t-butyl group on the [3.3.0]- and [3.2.1]-diaxoabicyclooctane ring systems. These studies demonstrated that the t-butyl analogues of the fused and bridged diaxoabicyclooctane ring systems were found to react with the primary amine end groups of lysine residues present in hen egg white lysozyme (HEWL) to form pyrrole products, presumable via the dialdehyde intermediate (Figure 5.4).114,115 A dialdehyde functionality has been shown to produce an unpalatable response, as described in the study by Mollo and co-workers on metabolites within MDFs.12 Therefore, it was hypothesized that aplyviolene and dendrillolide A should produce an unpalatable response, assuming that the bioactivity originates from the oxygenated moiety ring opening to the dialdehyde.

Figure 5.4 The [3.3.0]- and [3.2.1]-diaxoabicyclooctane ring systems of dendrillolide A, aplyviolene and macfarlandin E ring open to a common dialdehyde intermediate.

Macfarlandin E (1.143) differs to 1.144 only in the addition of an acetate group at C-12. Compared with 1.142 and 1.144, macfarlandin E (1.143) showed a significantly lower response, with

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites 0% rejection at 10 mg/mL and 17% rejection at 20 mg/mL (Figure 5.3). It was hypothesized that the additional acetate may increase the unpalatability of 1.143, as the C-12 and C-16 acetate groups on 1.143 have been found to contribute to activity as a Golgi modifier.114 However, macfarlandin E (1.143) was found to be more palatable relative to aplyviolene (1.144). Unfortunately, the limited quantity of the purified compounds again restricted further screening at higher concentrations of metabolites. With the initial screening of macfarlandin E (1.143) exhibiting 0% rejection at 10 mg/mL, the sample was considered palatable at this concentration. The bioactivity of metabolites isolated from Goniobranchus species may be the result of individual metabolites or a synergistic effect. We explored the possible synergy of these compounds using the residual sample of dendrillolide A (1.142) and macfarlandin E (1.143). It was found that 7% rejection was observed using a 1:4 ratio of macfarlandin E and dendrillolide A at a concentration of 10 mg/mL against P. serenus. In contrast, the mixture of macfarlandin E and dendillolide A (4:1 ratio) at 10 mg/mL showed 50% rejection. These preliminary results showed the addition of 1.142 increased the unpalatability of 1.143 at 10 mg/mL against P. serenus.

5.3 Biological screening Historically, natural products have been invaluable in treating human ailments, thus forming the basis of medicine. Initially natural products were utilized in crude form however with advancements in technology they are now more commonly used in purified form. Natural products have been found to result in a diverse range of pharmacological effects, making them a source for potential drug leads. When subjected to drug development studies a compound in its naturally isolated form may be utilized as a starting point to be synthetically modified to improve activity.233,234 Due to the rich species diversity, the marine environment is a prospective source for novel compounds.234,235 Spongian and rearranged diterpenes have previously been shown to exhibit a variety of bioactivities, including cytotoxicity,51,79 anti-infective,236 antimicrobial,47,93,117 anti-inflammatory89 and neuroprotective effects.84

5.3.1 Cytotoxicity of spongian diterpenes Cancer is the abnormal, uncontrolled proliferation of cells, which can potentially invade or spread to other parts of the body, leading to various deleterious side effects that can result in death. The predisposition to different types of cancer can be inherited, however, the genetic mutations that cause majority of cancers are due to several environmental and lifestyle factors.237 The marine environment is a rich source for novel cytotoxic compounds to be utilized as anti-cancer agents.234,235,238A notable example is the sponge-derived compound jorumycin (5.8) from the Pacific nudibranch Jorunna funebris.239,240 Metabolite 5.8 showed potent cytotoxicity against the P388 cell

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites line. Modifications to the marine natural product resulted in pharmaceutical PM00104, trade name Zalypsis® (5.9), which has a potent preclinical anti-myeloma effect based on the sensitivity of malignant plasma cells to DNA-damage induction. Preclinical in vivo investigations have indicated significant activities against breast, prostate and renal cancer cell lines, as well as reasonable antitumor activity against colon cancer.235 Zalypsis® is currently in Phase II trials, with studies extending to solve the side effects of reversible hematological disorder or liver enzymes imbalance.235

Previous studies have investigated the cytotoxicity of oxygenated diterpenes with the spongian-16-one skeleton.51,79,241 Spongian-16-one (1.24) has been reported to show moderate 51 activity against L1210 (IC50 16.4 μM) and KB (IC50 30.2 μM) cells, and some activity against HeLa 79 S3 cells, while 7α-acetoxyspongian-16-one (1.32) was active against L1210 (IC50 6.1 μM) and KB 51 (IC50 44.1 μM) cells.

To investigate the cytotoxicity of spongian diterpenes, metabolites 1.42-1.47 (isolated prior to PhD commencement) were screened against human lung (NCIH-460), colorectal (SW620), and liver (HepG2) cancer cells and using vinblastine as a positive control.

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites These assays were undertaken by a member of Professor Robert Capon’s research group, Dr Pradeep Dewapriya, at the Institute of Molecular Biosciences (IMB). All of the diterpenes tested had

IC50 values of >30 μM and so did not exhibit cytotoxicity toward these cell types (Figure 5.5). As a result, the newNCI-H460 spongian Human analogues lung cancer reported in this thesis wereSW620 not beHuman screened colon cancer for cytotoxic activity.

150 150 1 1 (a) 2 (b) 2 3 3 4 4

y 5

t t

i 100

l i

6 i 6

b b

a vinblastine

50 50

% %

0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 Concentration (mM) Concentration (mM) HEP-G2 Hepatocyte carcinoma 1 = 12α,20-diacetoxyspongian-16-one (1.42) 2 = 12α-acetoxy-20-oxyspongian-16-one propionate 150 (c) (1.43) 1 3 = 11β,20-diacetoxyspongian-16-one (1.44) 2 4 = 11β-acetoxy-20-oxyspongian-16-one propionate

y 3 t

i 100 l

i 4

b (1.45)

a 5

i v

l 5 = 7α,11α-dioxyspongian-16-one-7α-isopentanoate- l

e vinblastine

C 11α-propionate (1.46) 50 % 6 = 11β-hydroxy-7α,20-dioxyspongian-16-one-7α- isopentanoate-20-propionate (1.47)

0 0.001 0.01 0.1 1 10 100 Concentration (mM) Figure 5.5 Cytotoxicity screening for spongian diterpenes 1.42-1.47. (a) NCIH-460 (human lung carcinoma cell line), (b) SW620 (human colorectal carcinoma), (c) HepG2 (hepatocellular carcinoma) (Data output provided by Dr Pradeep Dewapriya of Professor Robert Capon’s group, IMB).

5.3.2 Antiviral screening To date, the available therapeutics for a number of infectious diseases are limited. Diseases caused by viral pathogens are a specific group that require new medicines, due to the increasing appearance of resistance to available treatments. In particular, viruses with RNA as their genetic material are a difficult group to treat, due to the high mutation rates in their genomes.242 An escalating issue with RNA viruses is their mode of transmission, as many of these viruses involve arthropod vectors in their life cycle, which proliferates their distribution.243 Notable human diseases caused by RNA viruses include influenza, Dengue fever and Ross River fever. Influenza is a highly contagious viral infection that has rapidly evolved to form many strains and affects humans worldwide.244 Dengue (DENV) and Ross River (RRV) viruses are both mosquito-borne single-strand RNA viruses of the genus Flavivirus and Alphavirus, respectively. Dengue fever is a global health concern and 136

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites affects more than 100 countries around the world, including in Europe and the United States.245 Conversely, Ross River fever is endemic to Australia, Papua New Guinea and other islands in the South Pacific.246,247 Marine organisms have the potential to provide an array of leads, with diverse chemical structures, that could be developed into antiviral drugs. For example, the diterpenes reiswigins A (5.10) and B (5.11) isolated from the deepwater sponge Epipolasis reiswigi, exhibited in vitro antiviral activity. It was found at 2 µg both metabolites inhibited the DNA virus herpes simplex virus type 1 (HSV-1) and the RNA virus vesicular stomatitis virus (VSV). Antiviral activity was also observed at 20 µg levels against the RNA virus coronavirus A59.248

The furanoditerpenes spongiadiol (1.76), epispongiadiol (1.48), and isospongiadiol (1.50), presented in Chapter 1, were initially isolated from an Australian Spongia species58 and later isolated from a Caribbean deepwater Spongia. These natural products exhibited antiviral activity against -1 249 HSV-I, with IC50 values of 0.25, 12.5, and 2 µg mL , respectively.

Natural products that have a similar carbon scaffold (1.24, 1.32 and 1.110) to spongiadiol (1.76) or biosynthetically related (1.105, 1.110, 1.130 and 1.142-1.145) have been selected for in vitro screening. Our group has a history of working on isonitrile metabolites,138,153,250 so the following oxygenated sesquiterpenes have been selected from the group repository: arenaran A (5.12), isonitrile sesquiterpene (5.13) and isonitrile diterpene (5.14) for comparison with the oxygenated terpenes (Figure 5.6). The samples of oxygenated diterpenes 1.24, 1.32, 1.130, 1.142-1.145, 1.152 and 4.4 described in Chapter 1-4 that were isolated from the Goniobranchus species have been investigated in this study and described. Epoxygoniolide-1 (1.105) was isolated from my previous investigation on the nudibranch G. splendidus in 2015.86 The sample of aplysulphurin91 (1.110) was isolated by a previous member of the Garson Group, Sharna Graham, from the encrusting sponge Darwinella tango.92 The

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites sample of arenaran A26 (5.12) was isolated by a previous member of the Garson Group, Patrick Narbutas, from the nudibranch Chromodoris strigata.251 The sample of axisonitrile-328 (5.13) was isolated by a member of the Garson Group, Pinus Jumaryatno, from the bright orange sponge Acanthella cavernosa.252 The sample of diisocyanoadociane253 (5.14) was isolated from the sponge Amphimedon terpenensis and was provided by Professor Mary Garson.254

Figure 5.6 Structures of metabolites selected for antiviral screening.

The following assays in our study were undertaken by members of Professor Paul Young’s research group, at the University of Queensland (UQ). Dr Daniel Watterson conducted the Dengue Virus (DENV) and Ross River Virus (RRV) assays and Dr Christopher McMillan carried out the cytotoxicity and influenza (Flu) virus assays. The antiviral ability of compounds was assayed in vitro via the microneutralization assay with visualisation of plaque formation by immunostaining. Fluorescent intensity was calculated using Image Studio software and antiviral activity quantified using a three-parameter dose response model analysis (Graphpad Prism 8 software). The screening of metabolites was performed using two replicates, with antiviral activity determined through comparison of the fluorescent intensity of treated to untreated samples. In addition, all compounds were also screened for cytotoxicity activity. For detailed experimental procedure refer to Chapter 7.

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites Metabolites 1.24, 1.32, 1.105, 1.110, 1.130, 1.142-1.145, and 5.12-5.14 were screened for cytotoxicity (Figure 5.6). Epoxygoniolide-1 (1.105) was the only metabolite that showed cytotoxic activity with an IC50 = 12.64 µM.

Figure 5.7 Cytotoxicity screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142- 1.145, and 5.12-5.14 (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ).

Unfortunately, there was no inhibition detected for any of the compounds when tested at 50 µM against the influenza viral strain, with the exception of compound 1.105 which showed inhibition. However, it was apparent that this was due to the cytotoxicity of epoxygoniolide-1 (1.105) (Figure 5.7). A sample was considered to cause no inhibition if the R2 value of the fitted inhibition curve was below 0.8.

In the RRV assay, inhibition was observed at 50 µM for spongian-16-one (1.24: IC50 = 6.8

µM), 7α-acetoxyspongian-16-one (1.32: IC50 = 8.3 µM), macfarlandin E (1.142: IC50 = 9.5 µM), aplyviolene (1.144: IC50 = 17.6 µM) and norrisolide (1.130: IC50 = 12.2 µM). Metabolite 1.105 also showed inhibition however this was also attributed to the cytotoxic activity. No inhibition was seen for 1.110, 1.142, 1.145 and 5.12-5.14 (Figure 5.8).

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Figure 5.8 Antiviral screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142-1.145, and 5.12-5.14 against Ross River virus strain (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ).

In the DENV assay inhibition was observed at 50 µM for norrisolide (1.130: IC50 = 7.4 µM). All other metabolites (1.24, 1.32, 1.105, 1.110, 1.142-1.145 and 5.12-5.14) showed no inhibition (Figure 5.9). There was more variability in the DENV microneutralization quantification due to the smaller viral foci (Appendix).

Figure 5.9 Antiviral screening for spongian diterpenes 1.24, 1.32, 1.105, 1.110, 1.130, 1.142-1.145, and 5.12-5.14 against Dengue virus strain (Data output provided by Dr Daniel Watterson of Professor Paul Young’s research group, UQ).

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites 5.3.3 Antimicrobial screening Antibiotics were considered to be ‘miracle drugs’ when they first became available in the 1920s, however their popularity rapidly led to overuse. The efficacy of antibiotics and antimicrobials has declined over the decades, due to the continuous evolution of resistance in pathogens. Antimicrobial resistance threatens the effective prevention and treatment of infections caused by bacteria. It is now urgent to discover new potent antimicrobial compounds, due to the increasing prevalence of antibiotic-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial active compounds are also of particular interest in medicine and the food industry for the eradication of biofilms, which are biologically active matrices of cells and extracellular substances in association with a solid surface.255 A review by Costerton et al. later elaborated this definition, describing biofilms as a functional consortium of microorganisms attached to a surface and embedded in the extracellular polymeric substances (EPS) produced by the microorganisms.256 In most situations biofilms are undesirable, for example they can lead to biofouling, which refers to the formation of a layer of living microorganisms and their decomposition products as deposits on surfaces in contact with liquid media, which can cause significant structural and functional deficiencies.257 Biofouling in industrial water systems and on the hull of marine vessels can considerably affect the functioning of the equipment.258 In addition, medical biofouling on prosthetic implants, biosensors, catheters, dental implants and medical equipment can have a detrimental impact leading to implant rejection, malfunction of biosensors and spread of infectious diseases.257 Lastly, biofouling can interrupt the efficiency of equipment in agriculture, such as compromising the processing of milk in the dairy industry.259 Few diterpenes from the marine literature, as crude extracts47 or purified metabolites, have been screened for activity against microbes. Commonly screened microbes include the Gram-positive bacteria Bacillus subtilis,74,99,117 and Staphylococcus aureus.74,117 Aplyviolene (1.144) has been previously screened against the Gram positive bacterium B. subtilis, where at a concentration of 5 µg/disk it exhibited inhibition of growth.99 The highly methicillin-resistant Staphylococcus aureus (MRSA) can be screened in both the planktonic state and as a biofilm. In the case of darwinolide (1.116) Baker and co-workers carried out a broth dilution assay using S. aureus. As described by Fleeman et al.260 this assay involves broth cultures of the S. aureus strain being grown overnight before being diluted 1 in 1000 in fresh media. Subsequently, sterile 96-well plates were loaded with culture, and 1.116 (in DMF) was added at decreasing concentrations to equal a total volume of 200 μL per well. Plates were then incubated at 37 °C, and minimum inhibitory concentrations (MICs) determined after 24 h by visual inspection for a lack of turbidity in wells. The MIC for darwinolide (1.116) was found to be 132.9 μM against S.

141

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites aureus. In addition, after a cell recovery experiment, 1.116 was determined to be cytotoxic, rather than cytostatic, towards S. aureus; only 1.6% of the treated bacteria were able to recover and grow.93

Darwinolide (1.116) was also screened for activity against S. aureus as a biofilm, where the minimum biofilm eradication concentration (MBEC) was determined using the method reported by 93 Baker and co-workers. The metabolite darwinolide (1.116), exhibited an IC50 value of 33.2 µM against the S. aureus biofilm.93 The following assays in our study were undertaken by a member of Professor Bill Baker’s research group, at the University of South Florida (USF). A selection of purified compounds was screened against S. aureus at 50 µg mL-1 using the Baker group’s own minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) assays (Figure 5.10). Based on the four-fold selectivity of darwinolide for MRSA biofilms over planktonic cells, we selected natural products that have similar structural features (1.105, 1.106 and 1.110) to darwinolide or were biosynthetically related (1.24, 1.130, 1.142-1.145, 1.152, and 4.4). Our group has a history of working on isonitrile metabolites.138,153,250 Isonitrile sesquiterpenes and diterpenes have been shown to have varying degrees of antimicrobial activity. As a result, we selected two isonitrile sesquiterpenes (5.13 and 5.15) and two isonitrile diterpenes (5.14 and 5.16) for comparison with the oxygenated terpenes. The samples of oxygenated diterpenes 1.24, 1.130, 1.142-1.145, 1.152 and 4.4 were isolated from the Goniobranchus species investigated in this study, discussed in Chapters 1-4. Metabolites 1.142, 1.152 and 4.4 represent the comparison of substitution patterning, with an acetate at either C- 15 or C-12, or at both positions on the dendrillane scaffold. Epoxygoniolide-1 (1.105) and gonioline (1.106) were isolated from my previous investigation on the nudibranch G. splendidus in 2015.86 Isocyanoclovene153 (5.15) and pustulosaisonitrile138 (5.16) were isolated by a member of the Garson Group, Andrew White, from two different nudibranch species, Phyllidia ocellata and Phyllidiella pustulosa, respectively. The samples of aplysulphurin (1.110), axisonitrile-3 (5.13), and diisocyanoadociane (5.14) were sourced from the same batch of isolate as submitted for the antiviral assays.28,91,253

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites

Figure 5.10 Structures of metabolites selected for antimicrobial screening.

Unfortunately, there was no inhibition in the MIC assay for any of the compounds when tested at 50 µg mL-1 against S. aureus, and owing to the limited quantity of isolated compounds, higher concentrations could not be tested. There was partial inhibition seen for diisocyanoadociane (5.14) (Figure 5.11). To be noted, axisonitrile-3 (5.13), isocyanoclovene (5.15) and pustulosaisonitrile (5.16) also showed very mild activity. From the selection of oxygenated diterpenes screened, only aplyviolene (1.144) and norrisolide (1.130) showed mild activity. Epoxygoniolide-1 (1.105), gonioline (1.106) and aplysulphurin (1.110) all share structural similarities with darwinolide (1.116); metabolites 1.105 and 1.106 also have substitution at C-12 of a 2,8-dioxabicyclo[3.3.0]octane moiety, and both 1.106 and 1.110 have oxygenated tricyclic moieties. Even though these oxygenated

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Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites diterpenes (1.105, 1.106, and 1.110) have structural similarities to darwinolide (1.116), none of these three terpenes showed activity.

Figure 5.11 Structure of 1.105, 1.106, 1.110 and 1.116 comparing the structural similarities. In blue the trimethylcyclohexane moiety and in pink is the 2,8-dioxabicyclo[3.3.0]octane moiety.

Legend: 1 = spongian-16-one (1.24) 2 = epoxygoniolide-1 (1.106) 3 = aplysulphurin (1.110) 4 = dendrillolide A (1.142) 5 = 12-acetoxydendrillolide A (1.152) 6 = 15-desacetoxy-12- acetoxydendrillolide A (4.4) 7 = aplyviolene (1.144) 8 = macfarlandin E (1.143) 9 = macfarlandin D (1.145) 10 = norrisolide (1.130) 11 = axisonitrile-3 (5.13) 12 = diisocyanoadociane (5.14) 13 = gonioline (1.105) 14 = isocyanoclovene (5.15) 15 = pustulosaisonitrile (5.16)

Figure 5.12 Minimum biofilm eradication percent measured for mollusk compounds against multi- drug resistant S. aureus at 50 µg mL-1. Note data not compared with drug controls and represented as percent eradication. Error = ± SEM. MBEC90/50: level to which 90% or 50% of the population was eradicated compared to no treatment (Data output provided by Jessie Adams of Professor Bill Bakers research group, USF).

5.4 Conclusions The anatomical distribution of metabolites in Goniobranchus species were investigated, with the exception of Goniobranchus geometricus. Species of this genus tend to sequester several metabolites into their mantle tissue. In G. collingwoodi, G. aureopurpureus and Goniobranchus sp. 1 the new metabolites, characterized in Chapter 2, were all only found in the mantle tissues. Whereas in G. leopardus and G. coi, the new metabolites were found in both the mantle and viscera tissues, with the exception of 10,20-epoxydendrillolide A (3.4) and 5-hydroxydendrillolide A (3.3) which were only found in the mantles of G. leopardus and G. coi, respectively. The anatomical distribution

144

Chapter 5: Anatomical distribution and bioactive properties of nudibranch metabolites of the five Goniobranchus species was found to be similar to literature,56,261 where Goniobranchus nudibranchs accumulate an eclectic mix of metabolites into their mantle from their sponge diet. The Goniobranchus specimens investigated within this study tended to sequester more metabolites in the mantle than the viscera. The metabolites isolated from the mantle are proposed to be utilized by the animal as an unpalatable or toxic deterrent against predation from fish and crustaceans. The mechanism of internally transporting these potentially noxious metabolites from the viscera to the mantle is unclear, but has been shown to be linked with the storage of metabolites in mantle dermal formations (MDFs).262 The major component of G. coi, dendrillolide A (1.142), was screened for unpalatability against Palaemon shrimp, where at 20 mg/mL 45% rejection was observed. The biosynthetically related metabolites 1.143 and 1.144, which were minor components isolated from the crude extract of G. coi, were also screened for feeding deterrency and at 20 mg/mL exhibited 40% and 17% rejection, respectively. Due to the complexity of the crude mantle extract, we hypothesized the unpalatability of the mantle tissue may be due synergistic effects. The deterrency of macfarlandin E (1.143) was found to improve when administered in a 4:1 ratio with dendrillolide A (1.142) at 10 mg/mL, however due to limited sample quantity further screening could not be carried out. A selection of biological screening assays were also carried out by collaborators, including cytotoxicity, antiviral and antimicrobial assays. Unfortunately, no significant cytotoxicity was observed for metabolites 1.42-1.47. Metabolite 1.105 showed cytotoxicity with an IC50 = 12.64 µM. In the antiviral assays, no inhibition was observed against influenza, except for 1.105 whose activity was attributed to its cytotoxicity. Inhibition was observed at 50 µM for spongian-16-one

(1.24: IC50 = 6.8 µM), 7α-acetoxyspongian-16-one (1.32: IC50 = 8.3 µM), macfarlandin E (1.142: IC50

= 9.5 µM), and aplyviolene (1.144: IC50 = 17.6 µM) against Ross River Virus. Norrisolide (1.130) exhibited inhibition at 50 µM against Ross River Virus (IC50 = 12.2 µM) and Dengue Virus (IC50 = 7.4 µM) strains. No other metabolites screened showed inhibition. In the MIC assay against S. aureus, no inhibition was observed for all metabolites (1.24, 1.105, 1.106, 1.110, 1.130, 1.142-1.145, 1.152, 4.4, and 5.13-5.16) when tested at 50 µg mL-1. In the MBEC assay there was partial inhibition seen for 5.14 and mild activity exhibited for 5.13, 5.15 and 5.16. From the selection of oxygenated diterpenes screened, only 1.144 and 1.130 showed mild activity. While none of these compounds are prospective leads as anti-cancer, antiviral or antimicrobial agents, it may be that their true biological targets have yet to be identified. Further studies need to be undertaken to explore the structural origins of bioactivity in these scaffolds, as there is not a clear pattern.

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Chapter 6: General conclusions

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Chapter 6: General conclusions

6.1 General conclusions The aim of this PhD was to isolate and elucidate the natural products of nudibranchs from the genus Goniobranchus and to explore the biological role of the compounds discovered. As discussed in Chapter 1, there are several key motifs that can be derived from the spongian diterpene scaffold (6.1), where rearranged diterpenes can be organised into dimethylcyclopentane (6.2), trimethylcyclohexane (6.3), perhydroindane (6.4), perhydronaphthalene (6.5) and perhydroazulene (6.6) structural motifs (Figure 6.1).

Figure 6.1 Representative structural motifs for oxygenated rearranged diterpenes and norditerpenes.

A total of fifty-six compounds, twenty-three of which are new, have been isolated from six nudibranch species from the Goniobranchus genus. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. X-ray crystallographic analysis and chemical correlation were carried out on selected metabolites to aid in the determination of the relative and absolute configurations. Computational studies involving conformational modelling, DFT calculations and DP4 probability predictions were also used as complementary tools to aid in determining the preferred diastereomers of selected metabolites. Several purified compounds were screened for cytotoxicity, deterrency against Palaemon shrimp, antiviral activity against Dengue fever, Ross River fever and influenza viral strains and lastly, antimicrobial activity against the Gram- positive bacteria S. aureus. The metabolites isolated from this study are summarized in Table 6.1. From the nudibranch G. collingwoodi a new spongian diterpene 7α-acetoxyisoagatholactone (2.1) was isolated along with four spongian diterpenes (1.19, 1.24, 1.32, and 1.33) and a linear furanoditerpene (1.16). The new metabolite 2.1 was found to only be present in the mantle tissue of the specimens. The investigation on the nudibranch G. aureopurpureus yielded five new highly substituted spongian diterpenes (2.2-2.6) and a new dendrillolide A analogue (4.4), along with eleven known rearranged spongian diterpenes (1.16, 1.24, 1.32, 1.144, 1.145, 1.154, 1.155, 1.158, 1.161, 1.164, and 1.165). The new spongian diterpenes showed varying levels of oxidation, in particular at positions C- 6, C-7, C-13 and C-20. Metabolites 2.5 and 2.6 were crystallized and their X-ray structures aided in establishing the relative and absolute configurations. The new metabolites 2.2-2.6 were all only present in the mantle tissue of the specimens.

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Chapter 6: General conclusions Goniobranchus sp. 1 was found to contain spongian diterpenes, six of which were new (2.7- 2.12) and eight were known (1.18, 1.24, 1.32, and 1.38-1.43). The new monosubstituted (2.9), disubstituted (2.7, 2.8 and 2.10) and trisubstituted (2.11 and 2.12) spongian diterpenes elucidated demonstrated a high level of oxidation at positions C-11, C-12, C-13 and C-20. The new metabolites were only found in mantle tissue. The G. leopardus specimens were found to contain of both spongian diterpenes and rearranged spongian diterpenes. Two new metabolites, the isospongian 12α-acetoxypolyrhaphin D (2.13) and the spiroepoxide (3.4) were isolated together with eleven known metabolites (1.130-1.132, 1.142, 1.144, 1.145, 1.160, 1.163-1.165, and 1.172). Metabolite 2.13 was found in both the viscera and the mantle, whereas 3.4 was found only in the mantle. Notably all the new spongian-16-one analogues 2.1-2.12 had negative specific optical rotations with the exception of 2.13 which had a positive specific optical rotation. Biosynthetically, metabolites 2.1-2.12 can all be derived from the parent scaffold spongian-16-one (1.24). Since many of the new highly oxygenated spongian diterpenes were only isolated in the mantle tissue of the specimens, it could be argued that the nudibranch may be oxidizing a precursor to increase the activity of these compounds to use as a chemical defense against predation. However, the metabolites found in these nudibranchs may have been sequestered from their sponge diet, and therefore further investigation should be undertaken into the sponges on which the Goniobranchus specimens feed, to better understand the origins of the oxidation in these metabolites.263 From the mantle and viscera of G. coi, eight new rearranged spongian diterpenes possessing a perhydroazulene motif (3.1-3.5, 4.1 and 4.2) or a perhydronapthalene analogue (3.6) with a fused cyclopropyl group, were isolated along with thirteen known metabolites (1.130, 1.132, 1.142, 1.143- 1.146, 1.148, 1.149, 1.162, 1.163, 1.165, and 1.171). Epoxide 3.1 and ketone 3.2 showed broadened signals in the 1H and 13C NMR, suggesting conformational averaging. As a result, the dynamic nature of the perhydroazulene motif and 2,8-dioxabicyclo[3.3.0]octane moiety was explored through variable temperature NMR experiments and computational modelling. The relative configuration of the fused epoxide 3.1 was confirmed by X-ray crystallographic analysis. Metabolite 3.3 was isolated as a very minor component from the mantle of G. coi. Owing to the sub-milligram quantity of 3.3, the configuration of the C-5 hydroxy group could not be established. The relative configuration of the spiroepoxide 3.4 was explored through chemical correlation experiments, where aldehyde 3.5 was identified as a ring opened artefact of the C-10 epimer of 3.4. Lactols 4.1 and 4.2 were isolated as an inseparable mixture of diastereomers. Molecular modelling and computational studies aided in correctly assigning their relative configurations. Chemical correlation experiments further confirmed the structural assignments of 4.1 and 4.2.

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Chapter 6: General conclusions The final species G. geometricus was found to contain two new metabolites with a perhydroazulene moiety (4.3 and 4.4), as well as sixteen known oxygenated rearranged diterpenes (1.130-1.132, 1.142, 1.143-1.145, 1.152, 1.154, 1.158, 1.160, 1.162-1.164, 1.166, and 1.171). In secoshahamin (1.164), the configurational assignments of C-13 and C-14 had not been rigorously defined in an earlier isolation study. Chemical correlation experiments and computational studies were therefore undertaken and conclusively assigned the relative and absolute configuration of 1.164. All specimens, excluding the G. geometricus specimens, were dissected prior to extraction. The anatomical distribution of the metabolites isolated from the mantle and viscera tissue was found to be consistent with literature, where Goniobranchus nudibranchs accumulate several metabolites into their mantle from their sponge diet. The Goniobranchus species investigated within this study tended to sequester more metabolites in the mantle than the viscera. The metabolites isolated from the mantle are proposed to be utilized by the animal as an unpalatable or toxic deterrent against predators such as fish and crustaceans. The major component of G. coi, dendrillolide A (1.142), was screened for unpalatability against Palaemon shrimp, where at 20 mg/mL 45% rejection was observed. The deterrency of macfarlandin E (1.143), which exhibited 0% rejection at 10 mg/mL, was found to improve when administered in a 4:1 ratio with dendrillolide A (1.142) at 10 mg/mL suggestive of a synergistic effect, however due to limited sample quantity further screening could not be carried out. A selection of metabolites were also screened for cytotoxicity, antiviral and antimicrobial activities. Unfortunately, no significant cytotoxicity was observed for metabolites 1.42-1.47.

Metabolite 1.105 showed cytotoxicity with an IC50 = 12.64 µM. In the antiviral assays, no inhibition was observed against influenza, except for 1.105 whose activity was attributed to its cytotoxicity. At 50 µM inhibition was observed for 1.24, 1.32, 1.142 and 1.144 against Ross River Virus. Norrisolide (1.130) exhibited inhibition at 50 µM against Ross River Virus and Dengue Virus. All other metabolites screened showed no inhibition. In the MIC assay against S. aureus no inhibition was observed for all metabolites (1.24, 1.105, 1.106, 1.110, 1.130, 1.142-1.145, 1.152, 4.4, and 5.13-5.16) when tested at 50 µg mL-1. When screened in the MBEC assay there was partial inhibition seen for 5.14 and mild activity exhibited for 5.13, 5.15 and 5.16. From the selection of oxygenated diterpenes, only 1.144 and 1.130 showed mild inhibition in the MBEC assay.

6.2 Future work The new metabolites discovered during my PhD adds to the growing corpus of research on marine natural products. The minute quantities were a significant limiting factor in establishing the absolute configuration of the metabolites described in this thesis. The complete determination of the 149

Chapter 6: General conclusions absolute configuration of several of these new natural products (2.1-2.4, 2.6-2.13, 3.2-3.6, and 4.1- 4.4) can in principle be established through TDDFT/ ECD calculations,158 or by X-ray crystallographic analysis or total enantioselective synthesis, however these studies were outside the scope of this thesis. Future research should consider the potential role these metabolites play in the survival of the nudibranchs that have sequestered these compounds, where they may have been used as a chemical weapon to deter predators by being unpalatable or toxic. As presented in Chapter 5, metabolites 1.142-1.144 were explored for their individual palatability and their potential synergy with other minor metabolites. It will be important to explore and confirm the initial results of the palatability assays in this thesis. Further studies could also probe the association between nudibranchs and their dietary sponges. Recent studies in other laboratories have investigated sponge-microbiome interactions, where the symbionts provide chemical defence for the host organism.264 The possibility that bacteria may be the source of terpenes warrants further investigation. In addition, research is required to disentangle the origin of spongian diterpenes to aid in the validation of the putative biosynthetic pathways described this thesis. For example, research utilizing genome sequencing may provide unique insights in understanding the diversity and distributions of natural product biosynthetic gene clusters.265 While none of the compounds screened during my PhD showed significant cytotoxic, antiviral or antimicrobial activity, spongian diterpenes may possess activity that has yet to be discovered. Further studies need to be undertaken to explore the potential of bioactivity in these scaffolds. As discussed in Chapter 5 the apparent protected dialdehyde scaffold of 1.142 and 1.144 may require a biological trigger to activate the potency known for dialdehydes; for example, when a nudibranch is attacked by a predator. Although not performed within the scope of this thesis, neuroprotective screening of spongian diterpenes and their congeners may be a potential avenue for future investigation. Examples of spongian diterpenes with neuroprotective activity include the gracilins, as mentioned in Chapter 1.84,266 Romo and co-workers examined the structure-activity relationship of truncated substructures of gracilin A, where simpler congeners were identified and screened for potent neuroprotective and immunosuppressive activity.83 The new metabolites isolated during my PhD possess substructures that may hold potential potency. All metabolites isolated and characterized in this thesis are summarized in Table 6.1 below.

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Chapter 6: General conclusions 6.3 Summary of isolated compounds Table 6.1 Summary of isolated compounds from Goniobranchus species investigated in this thesis. Source Structure and name Properties Mass Comment (mg) G. collingwoodi MW 308 1.9 Known G. aureopurpureus

G. collingwoodi MW 302 2.1 Known Goniobranchus sp. 1

G. collingwoodi MW 304 1.9 Known G. aureopurpureus Goniobranchus sp. 1

G. collingwoodi MW 362 3.4 Known G. aureopurpureus Goniobranchus sp. 1

G. collingwoodi MW 320 0.5 Known

Goniobranchus sp. 1 MW 362 0.4 Known

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Chapter 6: General conclusions Goniobranchus sp. 1 MW 362 2.9 Known

Goniobranchus sp. 1 MW 376 0.3 Known

Goniobranchus sp. 1 MW 448 0.4 Known

Goniobranchus sp. 1 MW 420 0.4 Known

Goniobranchus sp. 1 MW 434 0.3 Known

G. leopardus MW 376 0.9 Known G. coi G. geometricus

152

Chapter 6: General conclusions G. leopardus MW 376 0.8 Known G. geometricus

G. leopardus MW 339 0.2 Known G. coi G. geometricus

G. leopardus MW 376 2.6 Known G. coi G. geometricus

G. aureopurpureus MW 434 9.0 Known G. coi G. geometricus

G. aureopurpureus MW 376 6.1 Known G. leopardus G. coi G. geometricus

153

Chapter 6: General conclusions G. leopardus MW 376 0.8 Known G. coi G. geometricus

G. coi MW 316 0.2 Known

G. coi MW 334 0.1 Known

G. geometricus MW 434 1.8 Known

G. aureopurpureus MW 376 0.2 Known

154

Chapter 6: General conclusions G. aureopurpureus MW 450 4.7 Known G. geometricus

G. aureopurpureus MW 420 0.3 Known G. geometricus

G. aureopurpureus MW 464 0.3 Known G. geometricus

G. leopardus MW 362 0.4 Known G. coi G. geometricus

G. aureopurpureus MW 362 0.6 Known G. leopardus G. coi G. geometricus

155

Chapter 6: General conclusions G. aureopurpureus MW 436 0.2 Known G. leopardus G. geometricus

G. leopardus MW 376 0.4 Known G. coi G. geometricus

G. geometricus MW 376 0.1 Known

G. coi MW 376 0.1 Known G. geometricus

G. leopardus MW 376 0.1 Known

G. collingwoodi MW 360 0.2 New

[α]D -51

156

Chapter 6: General conclusions G. aureopurpureus MW 378 0.22 New

[α]D -65

G. aureopurpureus MW 420 0.06 New

[α]D -167

G. aureopurpureus MW 478 0.07 New

[α]D -71

G. aureopurpureus MW 478 1.2 New

[α]D -12

G. aureopurpureus MW 478 0.8 New

[α]D -19

157

Chapter 6: General conclusions Goniobranchus sp. 1 MW 434 0.21 New

[α]D -29

Goniobranchus sp. 1 MW 378 0.71 New

[α]D -22

Goniobranchus sp. 1 MW 320 0.08 New

[α]D -58

Goniobranchus sp. 1 MW 392 0.97 New

[α]D -7

Goniobranchus sp. 1 MW 464 0.17 New

[α]D -35

Goniobranchus sp. 1 MW 464 0.11 New

[α]D -64

158

Chapter 6: General conclusions G. leopardus MW 360 0.38 New

[α]D +48

G. coi MW 390 1.35 New

[α]D +14

G. coi MW 378 1.17 New

[α]D +43

G. coi MW 392 0.44 New

[α]D +19

G. leopardus MW 392 0.21 New

G. coi [α]D +44

G. coi MW 392 0.55 New

[α]D +16

159

Chapter 6: General conclusions G. coi MW 376 0.32 New

[α]D -10

G. coi MW 334 6.55 New

G. geometricus MW 390 0.1 New

[α]D -48

G. aureopurpureus MW 376 0.3 New

G. geometricus [α]D -2

160

Chapter 7: Experimental

7.1 General experimental procedures 7.1.1 Solvents Analytical reagent grade solvents were distilled prior to use for extraction, partition and flash column chromatography. HPLC grade solvents were purchased and used for HPLC purification. LCMS grade methanol was used for dilutions of samples prior to mass spectrometric measurements. Protic and deuterated chloroform were filtered through basic and neutral alumina (1:1) to remove traces of acid prior to use. Anhydrous solvents were dried according to standard procedures and distilled under vacuum or nitrogen atmosphere.

7.1.2 Thin layer chromatography Normal phase TLC was performed on Merck Art 5554 aluminium backed plates precoated with silica gel 60 F254. Plates were visualized under UV (254 and 365 nm) and by staining using vanillin stain reagent followed by heating with hot air for 10s. To prepare the vanillin stain, vanillin (15 g) was dissolved in 96% ethanol (250 mL), followed by the addition of concentrated sulfuric acid (2.5 mL). The solution was stored in a sealed container in the dark.

7.1.3 Normal phase flash chromatography Normal phase flash chromatography was carried out on Merck silica gel 60 (0.063-0.200 mm, 70-230 mesh ASTM). Gradient elution used hexanes to methanol under pressure of compressed air with combinations of the resulting fractions guided by TLC. Sep-Pak® chromatography was carried out with Waters Sep-Pak® silica cartridges (normal phase).

7.1.4 High performance liquid chromatography Normal phase (NP) high performance liquid chromatography (HPLC) was conducted with a Waters 515 pump in combination with a Gilson® 132 refractive index detector. All HPLC separations were performed using a semi preparative Waters μPorasil® column 10 μm (7.8×300 mm) or Phenomenex Luna 5μ silica column (250×10 mm); with isocratic elution conditions using premixed, filtered and degassed mobile phases. Flow rate was 2 mL/min for mobile phases up to 30% EtOAc in hexanes.

161

Chapter 7: Experimental 7.1.5 Mass spectrometry Low resolution electrospray ionisation mass spectrometry (LRESIMS) was performed on a Bruker Esquire HCT 3D ion trap spectrometer or a Thermo LCQFleet ion trap spectrometer, in positive or negative mode. High resolution electrospray ionisation mass spectroscopy (HRESIMS) was performed on a MicroTof-Q or an Orbitrap Elite instrument with a standard ESI source (sodium formate). Samples were introduced into the source using MeOH or MeCN.

7.1.6 Nuclear magnetic resonance spectroscopy Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker Avance 500 spectrometer using a 5 mm SEI probe, a Bruker Ascend 500 spectrometer using a 5 mm SEI probe, or a Bruker Avance DRX 700 spectrometer with a 5 mm TXI Zgrad probe. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were recorded on a Bruker Avance DRX 700 spectrometer with a 5 mm TXI Zgrad probe. Measurements were made in deuterated chloroform (CDCl3, referenced at: δH 7.26 ppm, δC 77.16 ppm) or deuterated benzene (C6D6, referenced at: δH 7.16 ppm,

δC 128.06 ppm) as specified. Chemical shifts (δ) were recorded in parts per million (ppm) and coupling constants (J-values) were measured in Hertz (Hz). The selective gradient enhanced 1D- NOESY NMR spectra were recorded on a Bruker Avance 500. The signal for irradiation was selected using gradient pulses of 1 millisecond, and a refocusing 180 Gaussian pulse of 50 milliseconds. Two- dimensional NMR (2D NMR) data were acquired from Bruker Avance 500 and 700 MHz instruments. Gradient enhanced HMBC (geHMBC) and HSQC (geHSQC) NMR were obtained with n 8 to 64 transients per increment with the evolution delay set at JCH of 4 Hz or 8 Hz (geHMBC) and 1 JCH of 135 Hz (geHSQC). Gradient COSY (gCOSY) was recorded with 8 to 32 transients per increment with a pulse delay of 2.0 seconds. NOESY NMR spectra were obtained with 32 to 64 transients per increment, a recycle time between scans of 3.4 seconds and mixing times of 0.6, 0.7 or 1.5 seconds. TOCSY NMR spectra were obtained with 8 to 32 transients per increment and a mixing time of 0.08 seconds. One-dimensional 13C NMR experiments were optimized for quaternary carbon signals by using a 60o pulse angle and a delay time of 5 sec.

7.1.7 Gas chromatography/mass spectrometry Gas chromatography/mass spectrometry (GC/MS) spectra were recorded on a Shimadzu GCMS-QP2010 Plus, ZB-5MS column (30 m). GC/MS programme: flow rate 1.5 mL/min; initial oven temperature 100 oC (isothermal for 3 minutes); ramped 16 oC/min to 270 oC held for 10 min; injection temperature 250 oC.

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Chapter 7: Experimental 7.1.8 Specific rotation

Specific rotation [α]D measurements were performed on a Jasco P-2000 polarimeter, with the sodium D-line of 589.5 nm. Measurements were recorded using an optical path length of 10 cm at o 22-25 C for solutions in CHCl3. Concentrations are reported in g/100 mL. Specific rotation [α]D measurements were not recorded for samples less than 0.1 mg or where the sample contained impurities (>10%).

7.1.9 X-ray crystallography Data were with collected by Associate Professor Jack Clegg and PhD student Kasun Sankalpa Athukorala Arachchige (School of Chemistry and Molecular Biosciences, UQ) using either an Oxford

Rigaku Synergy-S employing confocal mirror monochromated Mo-K radiation generated from a microfocus source (0.71073 Å) with ω and ψ scans at 100(2) K267 or at the Australian Synchrotron with Silicon Double Crystal monochromated radiation at 100(2) K (λ = 0.7108 Å).268 Data integration and reduction were undertaken with CrysAlisPro267 or XDS.269 Subsequent computations were carried out using Olex2.270 Structures were solved with ShelXT271 and refined and extended with ShelXL272 Carbon-bound hydrogen atoms were included in idealised positions and refined using a riding model. Queries regarding crystallography can be directed to A/Prof Jack K. Clegg, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane St Lucia, QLD, Australia, 4072, [email protected].

7.1.10 Minimum biofilm eradication concentration (MBEC) determination assays A clinical, multi-drug resistant strain of methicillin-resistant Staphylococcus aureus (MRSA) (CBD-635) was used in these studies for minimum inhibitory concentration (MIC) determination and assessment of anti-biofilm properties. These assays were carried out by PhD candidate Jessie Adams from Professor Bill Bakers research group (University of South Florida), using the procedure described below:260 The minimum biofilm eradication concentration (MBEC) was determined in 96-well microtiter plates as follows. Broth cultures of ESKAPE strains were grown using the conditions described above. Biofilms for each of the ESKAPE pathogens were generated from these as we have previously described for S. aureus, however, human serum was not used for non-staphylococcal organisms.273-274 For all organisms, biofilms were developed by standardizing an overnight culture into fresh media to an OD600 of 0.5 and adding 150 μL into each well of a 96-well microtiter plate. Biofilms were allowed to develop for 24 h before the media was carefully removed and 200 μL of fresh media added containing a range of front-runner agent (above and below MIC). These cultures were incubated at 37 °C overnight alongside no drug controls. After 24 h, the media was removed 163

Chapter 7: Experimental from wells and the biofilm resuspended in phosphate buffered saline (PBS). Cultures were mixed by vigorous pipetting before being serially diluted in PBS and plated in duplicate on relevant agar. Plates were incubated at 37 °C for 24 h and colony-forming unit (CFUs) determined by enumeration. Each analysis was performed using three technical replicates, and anti-biofilm activity was determined by comparing treated to untreated samples.

7.1.11 Antiviral screening methods The following procedure described was used by the research group of Professor Paul Young (School of Chemistry and Molecular Biosciences, UQ) to assay a selection of compounds against Ross River fever, Dengue fever and influenza virus strains. The antiviral ability of compounds was assayed in vitro via the plaque reduction neutralisation test (PRNT) with visualisation of plaque formation by immunostaining. The wells of 96-well plates were seeded with 50,000 cells/well of Vero cells and incubated at 37 °C in a 5% CO2 humidified incubator overnight. The next day, compounds were serially diluted in serum-free OPTI-MEM in a U-bottomed 96-well plate. Virus was made up at a concentration of ~100 plaque-forming units (PFU)/mL in OPTI-MEM before being added to compound dilutions in a 1:1 v/v ratio. For influenza antiviral assays, the virus was diluted in OPTI- MEM containing 4 µg mL-1 of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. Virus-compound solutions were incubated at 37 °C for 1 h before being added to Vero cells that had previously been washed with serum-free OPTI-MEM. Infection was allowed to proceed for 1 h at 37 °C before media was removed and 100 µL/well of overlay medium was added (1.5% carboxymethyl cellulose (CMC), 2% heat-inactivated FBS, 100 U/mL penicillin and 100 µg mL-1 streptomycin). Three days post-infection, overlay medium was removed and cells were fixed by addition of ice-cold 80% acetone in PBS followed by incubation at -20 °C for 20-60 min. Plates were air-dried, blocked with 5% milk diluent blocking concentrate (SeraCare) diluted in PBS supplemented with 0.05% Tween-20 for 30 min at 25 °C. Plaques visualised by addition of the virus-specific mAbs (2 µg mL-1 diluted in blocking buffer) for 1 h at 37 °C, followed by an IR dye 800CW goat anti-Human IgG (H+L) flurophore secondary antibody (LI-COR Biosciences) diluted 1:5000 in blocking buffer. Imaging was done using the LI-COR Biosciences Odyssey infrared imager. Fluorescent intensity was calculated using Image Studio software and antiviral activity quantified using a three-parameter dose response model analysis (Graphpad Prism 8 software).

7.1.12 Palatability assays with shrimp (Palaemon serenus) methods The purified metabolites dendrillolide A (1.142), macfarlandin E (1.143) and aplyviolene (1.144) were tested for their feeding deterrent activity against a common generalist shrimp species (Palaemon serenus). Shrimp were collected from intertidal zones in SE Queensland and housed

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Chapter 7: Experimental communally in large aquaria until needed. Assays were conducted based on previously describe methods12,220,224 using artificially dyed (red) food pellets treated with purified compounds of different concentrations. The pellets were made using mixture of ground freeze-dried squid mantle (50 mg), alginic acid (30 mg) and purified sea sand (30 mg). The nudibranch extract/purified compound was dissolved in 0.5 mL of DCM, was added to the dry mixture and left for 30 min for the solvent to evaporate. One drop of red food coloring was added and 0.5 mL of distilled water. Food coloring was added for easy detection of the food in the digestive tract of the shrimp. The pellet ingredients were mixed carefully with a spatula, and then placed in to a 1mL syringe. The contents were extruded into a 0.25 M calcium chloride solution to create spaghetti-like strands and left for 2 min to harden. The pellets were then rinsed with distilled water and cut into pellets that were approx. 5 mm in length. Control foods were made in the same manner, with the addition of 0.5 mL of DCM, but without the nudibranch extract. Randomly selected shrimps (20–35 mm, total length) were individually placed in a section of an 8 compartment polypropylene translucent partitioned container (270 × 98 × 325 mm; each partition: 135 × 98 × 90 mm). Small holes were drilled into the side of each container to allow water flow through the container to allow the shrimp to be housed for 1–2 weeks. Two partitioned boxes were placed in larger aquaria (650 × 350 × 350 mm) with 15 cm depth of seawater and air stones to create water circulation. Shrimp were left for 3 days to acclimatize and fed green fish flakes (Ocean Nutrition, Formula 2) once per day. Shrimp were then starved for 3 days before assays began. Ten randomly selected shrimp were given a colored pellet of a particular concentration with a pair of tweezers. Most shrimp readily accepted the pellet with it their clawed appendages (chelipeds). The shrimps were then monitored at 5, 10, 30, 60 min after being given the pellet, and it was recorded whether the shrimp appeared to be eating the pellet, or not eating. After 60 min, the presence of a red spot in the transparent gastric mill was considered acceptance of the pellet by the shrimp, while the absence of a red spot was considered a rejection. If a shrimp had rejected the pellet, it was then given a control pellet and again observed for 30 min. If the shrimp rejected the control pellet (< 1%), then that shrimp was removed from the final analysis, as these shrimp were often in the process of moulting or about to expire. Shrimp were not reused. The effective dose response curve and the concentration at which 50% of the pellets were rejected by the shrimp (ED50) were calculated.

7.1.13 Cytotoxicity screening methods Dr Pradeep Dewapriya from the research group of Professor Robert Capon (Institute of Molecular Biosciences, UQ) conducted the MTT assay which was modified from that previously described.275 Adherent cell lines SW620 (adherent epithelial like, human colorectal carcinoma) and NCIH-460 (adherent epithelial like, human lung carcinoma) were cultured in RPMI medium 1640,276 165

Chapter 7: Experimental and HepG2277 (adherent human hepatocellular carcinoma) were cultured in DMEM medium as adherent mono-layer in flasks supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 100 unit/mL penicillin and 100 µg mL-1 streptomycin in a humidified 37 C incubator supplied with 5%

CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 2,000 cells/well for SW620, NCIH-460, and HepG2 and then incubated for 18 h at 37 C with 5%

CO2 (to allow cells to attach). Testing compounds were dissolved in 1% DMSO in PBS (v/v) and aliquots (10 µL) tested over a series of final concentrations ranging from 10 nM to 30 µM. Control wells were treated with 1% aqueous DMSO. After 48 h incubation at 37 C with 5% CO2 an aliquot (20 µL) of MTT in PBS (5 mg/mL) was added to each well (final concentration of 0.5 mg/mL), and the microtiter plates incubated for a further 4 h at 37 C with 5% CO2. After this final incubation the medium was aspirated and precipitated formazan crystals dissolved in DMSO (100 µL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS Microplate Reader from

Bio-Tek Instruments Inc. (Vinooski, VT). IC50 values were calculated using Prism 5.0 (GraphPad Software Inc., La Jolla, CA), as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). All experiments were performed in duplicate and vinblastine was used as a positive control.

7.2 Sample collection All nudibranch specimens were collected using SCUBA at depths between ~2-16 m. Eight individuals of Goniobranchus collingwoodi (#1457, 1458, 1459, 1463, 1464, 1465, 1467 and 1468) were collected from Nelson Bay, New South Wales in March 2016. Six individuals of Goniobranchus aureopurpureus were collected from Nelson Bay (#1469-1474), New South Wales in March 2016. Three individuals of Goniobranchus (Chromodoris) sp. 1 were collected from Mudjimba (#1563 and #1368) and Gneerings Reef (#1575) (Mooloolaba, Queensland) in October 2016. Five individuals of Goniobranchus leopardus we collected from Coolum in Sunshine Coast, Queensland (#1725, 1726, 1728-1730) in August 2017. All collections were stored in individual containers at -20 oC until dissection and extraction. Three individuals of Goniobranchus coi were collected from Percy Isles (#1426), Cairns Marine (#1440) and Coolum (#1647) in Queensland between 2015-2017. Eight specimens of Goniobranchus geometricus were collected near Mudjimba, Mooloolaba (#1378, 1551, 1195); Wave Break Island Gold Coast Seaway (#312 and #311); the south west wall of the Gold Coast Seaway (#351 and #195); and Gneerings Reef (#504) Mooloolaba between 2012-2016. Animals from Queensland were collected under the following permits: QLD General Fisheries Permit #183990QLD. Animals from New South Wales were collected under the following permits: NSW Department of Primary Industries Scientific Collection Permits F86/2163-7.0 and P16/0052-1.0. 166

Chapter 7: Experimental 7.3 Isolation of spongian diterpenes from Goniobranchus collingwoodi 7.3.1 Isolation of diterpenes from Goniobranchus collingwoodi Eight frozen nudibranchs (12.3 g) (collection numbers: #1457, 1458, 1459, 1463, 1464, 1465, 1467, 1468) were dissected into their mantle and guts. Each body part of each specimen was finely chopped, extracted with acetone (3 x 10 mL) and sonicated (5 min) separately. The extracts were separately filtered through cotton wool and concentrated to an aqueous suspension before partitioning between H2O (2 mL) and Et2O (4 x 5 mL). The organic layer was dried over anhydrous Na2SO4, filtered through cotton wool and evaporated under N2 to yield an orange oil for the mantle extracts and a green-black oil for the gut extracts. After 1H NMR analysis, the mantle extracts were combined (20.8 mg) and gut extracts were combined (50 mg) to produce two extracts. The extracts were further separated by NP-flash column chromatography with a stepwise solvent gradient from 100% hexanes to 100% MeOH. Mantle fractions eluting from hexanes/DCM (1:1) were screened by 1H NMR and were separated by NP-HPLC (10% EtOAc in hexanes) to provide spongian-16-one (1.24: 0.4 mg), isoagatholactone (1.19:1.0 mg). Mantle fractions eluting from hexanes/DCM (1:4) and 100% hexanes were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to isolate 7α- acetoxyspongian-16-one (2.1: 4.0 mg), 7α-hydroxyspongian-16-one (1.33: 0.5 mg) and 7α- acetoxyisoagatholactone (1.32: 0.2 mg). Viscera fractions eluting from hexanes/DCM (1:4) hexanes were screened by 1H NMR and were separated by NP-HPLC (10% EtOAc in hexanes) isolating spongian-16-one (1.24: 1.5 mg), isoagatholactone (1.19: 1.1 mg), and luffarin-X (1.16: 1.9 mg).

21 1 (–)-7α-Acetoxyisoagatholactone (2.1): colorless oil (0.2 mg); [α] D – 51 (c 0.02, CHCl3); H 13 - NMR and C NMR (CDCl3, 700 MHz), Table 2.1; HRESIMS m/z 359.2228 [M - H] (calcd. for

C22H31O4, 359.2247).

7.4 Isolation of spongian diterpenes from Goniobranchus aureopurpureus 7.4.1 Isolation of diterpenes from Goniobranchus aureopurpureus Six frozen nudibranchs (collection numbers: #1469, 1470, 1471, 1472, 1473, 1474) were dissected into their mantles and visceras. Each body part of each specimen was finely chopped, extracted with acetone (3 x 2 mL) and sonicated (5 min) separately. The extracts were separately filtered through cotton wool and concentrated to an aqueous suspension before partitioning between

H2O (2 mL) and Et2O (4 x 5 mL). The organic layer was dried over anhydrous Na2SO4, filtered through cotton wool and evaporated under N2 to yield an orange oil for the mantles and a purple- brown oil for the visceras. The NMR profile of all six of specimens of the mantle and viscera extracts were compared and showed similar chemistry; as a result for the specimens #1469, 1470, 1471 and 1474, the mantle extracts were combined (51.9 mg) and the viscera extracts combined (56.1 mg) to

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Chapter 7: Experimental produce two extracts. The specimens # 1472 and 1473 the extracts for the mantle and viscera were put aside for assays. The extracts were further separated by NP-flash column chromatography with a stepwise solvent gradient from 100% hexanes to 100% MeOH. Mantle fractions eluting from hexanes/DCM (1:1) were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to provide spongian-16-one (1.24: 0.4 mg). Mantle fractions eluting from hexanes/DCM (1:1) and (1:4) were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to isolate 15-desacetoxy-12-acetoxydendrillolide A (4.4: 0.4 mg), spongian-16-one (1.24: 2.1 mg), macfarlandin E (1.143: 1.7 mg), aplyviolene (1.144: 1.5 mg), and 7α-acetoxyspongian-16-one (1.32: 0.5 mg). Mantle fractions eluting from DCM/EtOAc (4:1, 1:1, 1:4) and 100% EtOAc were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to isolate macfarlandin E (1.143: 0.1 mg), polyrhaphin B (1.154:0.1 mg), secoshahamin (1.164: 0.1 mg), aplyviolene (1.144: 0.1 mg), shahamin C (1.157: 0.1 mg), 7α-acetoxyspongian-16-one (1.32: 0.1 mg), 7α-acetoxy-6α- hydroxyspongian-16-one (2.2: 0.2 mg), 6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)- butanoate (2.3: 0.06 mg), 20-acetoxy-6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.4: 0.07 mg), 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.5: 1.2 mg) and 13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate (2.6: 0.8 mg). Viscera fractions eluting from hexanes/DCM (1:4) were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to isolate luffarin-X (1.16: 0.22 mg), spongian-16-one (1.24: 0.43 mg) and macfarlandin E (1.143: 0.32 mg). Viscera fractions eluting from DCM/EtOAc (4:1) and 100% DCM were screened by 1H NMR and were separated by NP-HPLC (30% EtOAc in hexanes) to isolate macfarlandin E (1.143: 0.14 mg), polyrhaphin B (1.154: 0.1 mg), secoshahamin (1.164: 0.12 mg), polyrhaphin A (1.153: 0.32 mg), 12-desacetoxypolyrhaphin A (1.163: 0.14 mg), 15,16- diacetoxyshahamin B (1.160: 0.14 mg), aplyviolene (1.144: 0.79 mg), and 7α-acetoxyspongian-16- one (1.32: 0.44 mg).

21 (–)-7α-Acetoxy-6α-hydroxyspongian-16-one (2.2): colorless oil (0.22 mg); [α] D – 65 (c 1 13 0.022, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.2 and 2.3; HRESIMS m/z + 401.2293 [M + Na] (calcd. for C22H34NaO5, 401.2298).

(–)-6α-Hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.3): colorless oil (0.06 21 1 13 mg); [α] D – 167 (c 0.006, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.2 and 2.3; + HRESIMS m/z 443.2779 [M + Na] (calcd. for C25H40NaO5, 443.2768).

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Chapter 7: Experimental (–)-20-Acetoxy-6α-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.4): colorless 21 1 13 oil (0.07 mg); [α] D – 71 (c 0.007, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.2 + and 2.3; HRESIMS m/z 501.2824 [M + Na] (calcd. for C27H42NaO7, 501.2823).

(–)-13-Acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2.5): colorless 21 1 13 oil (1.2 mg); [α] D – 12 (c 0.12, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.2 and + 2.3; HRESIMS m/z 501.2812 [M + Na] (calcd. for C27H42NaO7, 501.2823).

(–)-13-Acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate (2.6): colorless 21 1 13 oil (0.8 mg); [α] D – 19 (c 0.08, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.2 and + 2.3; HRESIMS m/z 501.2831 [M + Na] (calcd. for C27H42NaO7, 501.2823).

7.4.2 Crystallographic data for (–)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2- methyl)-butanoate (2.5)

C27H42O7 (M =478.60 g/mol): orthorhombic, space group P212121 (no. 19), a = 7.9081(2) Å, b = 11.0710(3) Å, c = 30.2192(8) Å, V = 2645.73(12) Å3, Z = 4, T = 100.01(10) K, μ(MoKα) = 0.085 -1 3 mm , Dcalc = 1.202 g/cm , 34647 reflections measured (4.562° ≤ 2Θ ≤ 56.56°), 6562 unique (Rint =

0.0495, Rsigma = 0.0347) which were used in all calculations. The final R1 was 0.0416 (I > 2σ(I)) and wR2 was 0.1191 (all data).

7.4.3 Crystallographic data for (–)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3- methyl)-butanoate (2.6)

C27H39O7 (M =478.35 g/mol): orthorhombic, space group P212121 (no. 19), a = 7.9438(3) Å, b = 11.2958(8) Å, c = 28.897(2) Å, V = 2593.0(3) Å3, Z = 4, T = 99.99(10) K, μ(MoKα) = 0.087 mm-1, 3 Dcalc = 1.225 g/cm , 24810 reflections measured (4.578° ≤ 2Θ ≤ 50.246°), 4642 unique (Rint =

0.0727, Rsigma = 0.0429) which were used in all calculations. The final R1 was 0.0821 (I > 2σ(I)) and wR2 was 0.2356 (all data).

7.5 Isolation of oxygenated terpenes from Goniobranchus sp 1. 7.5.1 Isolation of diterpenes from Goniobranchus sp 1. Three frozen nudibranchs (collection numbers: #1368, 1563, 1575) were dissected into their mantle and viscera. Each body part of each specimen was finely chopped, extracted with acetone (5 x 5 mL) and sonicated (5 min) separately. The extracts were separately filtered through cotton wool and concentrated to an aqueous suspension before partitioning between H2O (2 mL) and Et2O (4 x 5 mL). The organic layer was dried over anhydrous Na2SO4, filtered through cotton wool and

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Chapter 7: Experimental concentrated under N2 to yield an orange oil for the mantles and a green-brown/yellow oil for the visceras. The NMR profile of all the mantles and visceras were compared and showed similar chemistry; as a result, the mantles were combined (96.8 mg) and visceras (71.2 mg) to produce two extracts. The extracts were further separated by NP-flash column chromatography with a stepwise solvent gradient from 100% hexanes to 100% MeOH. Mantle fractions eluting from 100% DCM were screened by 1H NMR and were separated by NP-HPLC (30% EtOAc in hexanes) to provide isoagatholactone (1.19: 0.5 mg), 12α-acetoxyspongian-16-one (1.38: 0.23 mg), 20-acetoxyspongian- 16-one (1.39: 7.63 mg), 20-oxyspongian-16-one-propionate (1.40: 0.35 mg), 12α,20- diacetoxyspongian-16-one (1.42: 0.15 mg), 12α,20-dioxyspongian-16-one-dipropionate (1.41: 1.05 mg), 12α-acetoxy,20-oxyspongian-16-one-20-propionate (1.43: 0.19 mg), 20-acetoxy-12α- oxyspongian-16-one-12α-propionate (2.7: 0.21 mg), and 20-acetoxy-13-hydroxyspongian-16-one (2.8: 0.17 mg). Mantle fractions eluting from DCM/EtOAc 4:1 and 1:1 were screened by 1H NMR and were separated by NP-HPLC (30% EtOAc in hexanes) to isolate 20-acetoxyspongian-16-one (1.39: 0.31 mg), 12α,20-diacetoxyspongian-16-one (1.42: 0.28 mg), 12α,20-dioxyspongian-16-one- dipropionate (1.41: 0.25 mg), 12α-acetoxy-20-dioxyspongian-16-one-20-propionate (1.43: 0.19 mg), 20-acetoxy-12α-oxyspongian-16-one-12α-propionate (2.7: 0.15 mg), 12-hydroxyspongian-16-one (2.9: 0.08 mg), 12-hydroxy-20-oxyspongian-16-one-20-propionate (2.10: 0.97 mg), 12-hydroxy- 11,20-dioxyspongian-16-one-11,20-dipropionate (2.11: 0.13 mg), and 11-hydroxy-12,20- dioxyspongian-16-one-12,20-dipropionate (2.12: 0.10 mg). Viscera fractions eluting from hexanes/DCM (1:1 and 1:4), 100% DCM and DCM/EtOAc 4:1 were screened by 1H NMR and were combined to be separated by NP-HPLC (30% EtOAc in hexanes) isolating isoagatholactone (1.19: 0.16 mg), spongian-16-one (1.24: 2.59 mg), 7α-acetoxyspongian-16-one (1.32: 0.57 mg). 12α- acetoxyspongian-16-one (1.38: 1.01 mg), 20-acetoxyspongian-16-one (1.39: 0.30 mg) and 12α,20- diacetoxyspongian-16-one (1.42: 0.83 mg).

(–)-20-Acetoxy-12α-oxyspongian-16-one-12α-propionate (2.7): colorless oil (0.21 mg); 21 1 13 [α] D – 29 (c 0.021, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.4 and 2.5; + HRESIMS m/z 457.2566 [M + Na] (calcd. for C25H38NaO6, 457.2561).

21 (–)-20-Acetoxy-13-hydroxyspongian-16-one (2.8): colorless oil (0.17 mg); [α] D – 22 (c 1 13 0.017, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.4 and 2.5; HRESIMS m/z + 401.2291 [M + Na] (calcd. for C22H34NaO5, 401.2298).

21 (–)-12-Hydroxyspongian-16-one (2.9): colorless oil (0.08 mg); [α] D – 58 (c 0.01, CHCl3); 1 13 + H NMR and C NMR (CDCl3, 500 MHz), Table 2.4 and 2.5; HRESIMS m/z 343.2245 [M + Na]

(calcd. for C20H32NaO3, 343.2244). 170

Chapter 7: Experimental

21 (–)-12-Hydroxy-20-oxyspongian-16-one-20-propionate (2.10): colorless oil (0.97 mg); [α] D 1 13 – 7 (c 0.097, CHCl3); H NMR and C NMR (CDCl3, 500 MHz), Table 2.4 and 2.5; HRESIMS m/z + 415.2458 [M + Na] (calcd. for C23H36NaO5, 415.2455).

(–)-12-Hydroxy-11,20-dioxyspongian-16-one-11,20-dipropionate (2.11): colorless oil (0.17 21 1 13 mg); [α] D – 35 (c 0.011, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.4 and 2.5; + HRESIMS m/z 487.2668 [M + Na] (calcd. for C26H40NaO7, 487.2666).

(–)-11-Hydroxy-12,20-dioxyspongian-16-one-12,20-dipropionate (2.12): colorless oil (0.11 21 1 13 mg); [α] D – 64 (c 0.011, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 2.4 and 2.5; + HRESIMS m/z 487.2667 [M + Na] (calcd. for C26H40NaO7, 487.2666).

7.6 Isolation of spongian diterpenes from Goniobranchus leopardus 7.6.1 Isolation of diterpenes from Goniobranchus leopardus Five frozen nudibranchs (collection numbers: #1725, 1726, 1728, 1729, 1730) were dissected into their mantles and visceras. Each body part of each specimen was finely chopped, extracted with acetone (3 x 2 mL) and sonicated (5 min) separately. The extracts were separately filtered through cotton wool and concentrated to an aqueous suspension before partitioning between H2O (2 mL) and

EtOAc (4 x 2 mL). The organic layer was dried over anhydrous Na2SO4, filtered through cottonwool and evaporated under N2 to yield an orange oil for the mantles and a purple-brown oil for the visceras. Comparison of the NMR profiles for the specimen mantle and viscera extracts showed similar chemistry; as a result the mantle extracts were combined (43.3 mg) and viscera extracts (49.7 mg) to produce two extracts. The extracts were further separated by NP-flash column chromatography with a stepwise solvent gradient from 100% hexanes to 100% MeOH. Mantle fractions eluting from hexanes/DCM (1:4) and 100% DCM were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to provide isolate dendrillolide A (1.142: 13.87 mg), macfarlandin C (1.165: 0.68 mg), secoshahamin (1.164: 0.59 mg), 12α-acetoxypolyrhaphin D (2.13: 0.38 mg), 12- desacetoxypolyrhaphin A (1.163: 1.07 mg), aplyviolene (1.144: 1.53 mg), norrisolide (1.130: 0.43 mg), macfarlandin D (1.145: 0.22 mg), 12-desacetoxyshahamin C (1.162: 0.5 mg) and 10,20- epoxydendrillolide A (3.4: 0.37 mg). Viscera fractions eluting from hexanes/DCM (1:4) and 100% DCM were screened by 1H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to isolate dendrillolide A (1.142: 11.99 mg), macfarlandin C (1.165: 0.87 mg), secoshahamin (1.164: 2.6 mg), 12α-acetoxypolyrhaphin D (2.13: 0.3 mg), dendrillolide E (1.172: 0.1 mg), 12- desacetoxypolyrhaphin A (1.163: 0.46 mg), aplyviolene (1.144: 1.92 mg), norrisolide (1.130: 0.5 mg), macfarlandin D (1.145: 0.41 mg), cheloviolene C (1.132: 0.1 mg), chelonaplysin C (1.131: 0.1 mg), and 12-desacetoxyshahamin C (1.162: 0.46 mg). 171

Chapter 7: Experimental

21 (+)-12α-Acetoxypolyrhaphin D (2.13): colorless oil (0.38 mg); [α] D + 48 (c 0.038, CHCl3); 1 13 + H NMR and C NMR (CDCl3, 700 MHz), Table 2.6; HRESIMS m/z 383.2191 [M + Na] (calcd. for C22H32NaO4, 383.2193).

7.7 Isolation of oxygenated terpenes from Goniobranchus coi 7.7.1 Isolation of diterpenes from Goniobranchus coi Two frozen nudibranch specimens (collection numbers: #1426 and 1647) were each dissected into viscera and mantle body segments. Individually, each body segment was finely chopped, extracted with acetone (3 x 2 mL), and sonicated (5 min). The extracts were then filtered through cotton wool, reduced to an aqueous suspension before partitioning between H2O (2 mL) and EtOAc

(4 x 2 mL). The organic extracts were dried over anhydrous Na2SO4, filtered through cotton wool and concentrated under N2 to yield a yellow oil (29.3 mg and 24.1 mg) from the mantle tissues and an orange oil (51.1 mg and 43.4 mg) from the viscera tissues. The 1H NMR profile of each extract was recorded. The extracts of the mantle were then combined, and the viscera extracts likewise combined. Each extract was subjected to NP-flash column chromatography with a gradient elution of 100% hexanes to 100% MeOH via CH2Cl2 and EtOAc. Selected fractions were combined and further subject to NP-HPLC to yield the purified compounds. For the mantle, fractions eluting from 1 hexanes/CH2Cl2 (1:1) to CH2Cl2 (100%) were screened by H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to provide 15-dendrillactol (4.1: 6.55 mg), 15-epidendrillactol (4.2: 6.55 mg), dendrillolide C (1.146: 0.2 mg), dendrillolide A (1.142: 21.1 mg), macfarlandin C (1.165: 0.9 mg), macfarlandin E (1.143: 0.4 mg), 12-desacetoxypolyrhaphin A (1.163: 2 mg), aplyviolene (1.144: 0.8 mg), 12-desacetoxyshahamin C (1.162: 0.6 mg), 5,9-epoxydendrillolide A (3.1: 0.8 mg), 5- hydroxydendrillolide A (3.3: 0.44 mg), cyclopropyl derivative (3.6: 0.32 mg), aldehyde product (3.5: 0.9 mg), 10-oxonordendrillolide A (3.2: 1.2 mg), and 10,20-epoxydendrillolide A (3.4: 0.2 mg). For the viscera fractions eluting from hexanes/CH2Cl2 (1:1) to CH2Cl2/EtOAc (4:1) were separated by NP-HPLC (25% EtOAc in hexanes) yielding 15-dendrillactol (4.1: 6.55 mg), 15-epidendrillactol (4.2: 6.55 mg), dendrillolide A (1.142: 14.5 mg), macfarlandin E (1.143: 0.6 mg), macfarlandin C (1.165: 0.6 mg), 12-desacetoxypolyrhaphin A (1.163: 1.2 mg), 12-desacetoxyshahamin C (1.162: 0.5 mg), macfarlandin D (1.145: 0.4 mg), aplyviolene (1.144: 1.0 mg), polyrhaphin C (1.171: 0.2 mg), cheloviolene C (1.132: 0.1 mg), norrisolide (1.130: 0.2 mg), 5,9-epoxydendrillolide A (3.1: 0.6 mg), cyclopropyl derivative (3.6: 0.1 mg), aldehyde product (3.5: 0.2 mg), 10-oxonordendrillolide A (3.2: 0.6 mg), and 10,20-epoxydendrillolide A (3.6: 0.2 mg).

172

Chapter 7: Experimental

21 1 (+)-5,9-Epoxydendrillolide A (3.1): colorless oil (1.35 mg); [α] D + 14 (c 0.135, CHCl3); H 13 + NMR and C NMR (CDCl3, 700 MHz), Table 3.1; HRESIMS m/z 413.1931 [M + Na] (calcd. for

C22H30NaO6, 413.1935).

21 1 (+)-10-Oxonordendrillolide A (3.2): colorless oil (1.17 mg); [α] D + 43 (c 0.117, CHCl3); H 13 + NMR and C NMR (CDCl3, 700 MHz), Table 3.3; HRESIMS m/z 401.1934 [M + Na] (calcd. for

C21H30NaO6, 401.1935).

21 1 (+)-5-Hydroxydendrillolide A (3.3): colorless oil (0.44 mg); [α] D + 19 (c 0.044, CHCl3); H 13 + NMR and C NMR (CDCl3, 700 MHz), Table 3.2; HRESIMS m/z 415.2086 [M + Na] (calcd. for

C22H32NaO6, 415.2091).

21 (+)-10,20-Epoxydendrillolide A (3.4): colorless oil (0.21 mg); [α] D + 44 (c 0.021, CHCl3); 1 13 + H NMR and C NMR (CDCl3, 700 MHz), Table 3.3; HRESIMS m/z 415.2083 [M + Na] (calcd. for C22H32NaO6, 415.2091).

21 1 (+)-Aldehyde product (3.5): colorless oil (0.55 mg); [α] D + 16 (c 0.055, CHCl3); H NMR 13 + and C NMR (CDCl3, 700 MHz), Table 3.3; HRESIMS m/z 415.2092 [M + Na] (calcd. for

C22H32NaO6, 415.2091).

21 1 (–)-Cyclopropyl derivative (3.6): colorless oil (0.32 mg); [α] D – 10 (c 0.032, CHCl3); H 13 + NMR and C NMR (CDCl3, 700 MHz), Table 3.5; HRESIMS m/z 399.2154 [M + Na] (calcd. for

C22H32NaO5, 399.2142).

1 13 15-Dendrillactol (4.1): colorless oil (6.55 mg); H NMR and C NMR (CDCl3, 700 MHz), + Table 4.1; HRESIMS m/z 357.2045 [M + Na] (calcd. for C20H30NaO4, 357.2036). Isolated as an epimeric mixture with 4.2.

1 13 15-Epidendrillactol (4.2): colorless oil (6.55 mg); H NMR and C NMR (CDCl3, 700 MHz), + Table 4.1; HRESIMS m/z 357.2045 [M + Na] (calcd. for C20H30NaO4, 357.2036). Isolated as an epimeric mixture with 4.1.

7.7.2 Crystallographic data for 5, 9-epoxydendrillolide A (3.1)

C22H30O6 (M =390.46 g/mol): monoclinic, space group P21 (no. 4), a = 17.159(3) Å, b = 6.4980(13) Å, c = 19.759(4) Å, β = 113.42(3)°, V = 2021.6(8) Å3, Z = 4, T = 100(2) K, 173

Chapter 7: Experimental μ(Synchrotron) = 0.092 mm-1, Dcalc = 1.280 g/cm3, 31040 reflections measured (2.246° ≤ 2Θ ≤

56.566°), 9157 unique (Rint = 0.1021, Rsigma = 0.0956) which were used in all calculations. The final

R1 was 0.0758 (I > 2σ(I)) and wR2 was 0.1945 (all data).

7.7.3 Procedure for the acetylation of lactol diastereomers (4.1 and 4.2) Lactol diastereomers (4.1 and 4.2) (324.2 g/mol, 1.0 mg, 0.00296 mmol) was dissolved in a mixture of anhydrous pyridine (200 µL) and acetic anhydride (200 µL). After 18 h at 25 °C, the reaction mixture was quenched with Milli-Q H2O (1 mL). The aqueous layer was extracted with EtOAc (4 x 1 mL), the combined organic extracts were washed with aqueous HCl (1 mL, 0.1 M) and with Milli-Q H2O (1 mL), then dried over anhydrous Na2SO4, filtered, and concentrated under nitrogen to yield a clear oil, which was further purified by NP-flash chromatography (1:1 DCM:EtOAc) (1.09 mg), and finally by NP-HPLC (25% EtOAc in hexanes) (0.2 mg, 20% yield).

21 110 21 (+)-Dendrillolide A (1.142): colorless oil (0.2 mg); [α] D + 26 (c 0.02, CHCl3); lit. [α] D 1 13 + 87 (c 0.31, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 3.1; LRESIMS m/z 399.2 [M + Na]+.

7.7.4 Procedure for epoxidation of dendrillolide A (1.142)

A solution of m-chloroperbenzoic acid (mCPBA) (1.5 mg, 0.00638 mmol) in CH2Cl2 (1.5 mL) was washed with phosphate buffer (pH 7.6, 100 mmol, 2 x 2 mL). Fresh buffer (pH 7.6, 2 mL) was then added and the reaction mixture cooled to 0°C and added to a solution of dendrillolide A

(1.142) (2.0 mg, 376.2 g/mol, 0.00532 mmol) in DCM (0.5 mL). The biphasic solution mixture was left at room temperature (25°C) for 3 days and monitored by TLC (100% DCM; visualised UV 254 nm and vanillin dip). The phosphate buffer layer was removed and the organic layer washed with a saturated aqueous solution of sodium thiosulfate (2 x 2 mL), then with distilled H2O (1 x 2 mL). The organic layer was then dried over MgSO4, filtered through cotton wool and dried under nitrogen. 2D NMR analysis was carried out on the crude reaction mixture. The sample was then purified by NP- HPLC (25% EtOAc in hexanes) to yield aldehyde 3.5 (0.42 mg) and aldehyde 3.8 (0.90 mg). Epoxide 3.4 and epoxide 3.7 degraded before purification could be achieved.

21 1 (+)-Aldehyde product (3.4): colorless oil (0.42 mg); [α] D + 11.8 (c 0.04, CHCl3); H NMR 13 + and C NMR (CDCl3, 700 MHz), Table 3.4; HRESIMS m/z 431.2030 [M + K] (calcd. for

C22H32KO6, 431.1830).

174

Chapter 7: Experimental

1 13 Aldehyde product (3.8): colorless oil (0.90 mg); H NMR and C NMR (CDCl3, 700 MHz), Table 3.4; Sample degraded before specific optical rotation could be measured.

7.8 Isolation of oxygenated diterpenes from Goniobranchus geometricus 7.8.1 Isolation of oxygenated diterpenes from Goniobranchus geometricus Eight frozen nudibranchs (13 g) (collection numbers: #1551, 1378, 1195, 504, 351, 312, 311, 195) were finely chopped, extracted with acetone (4 x 10 mL), and sonicated (2 min). The extract was then filtered through cotton wool, reduced to an aqueous suspension before partitioning between

H2O (2 mL) and Et2O (10 x 3 mL). The organic extract was dried over anhydrous Na2SO4, filtered 1 through cotton wool and concentrated under N2 to yield an orange oil (46 mg). The H NMR profile of the extract was recorded. The extract was subjected to NP-flash column chromatography with a gradient elution of hexanes: hexanes/CH2Cl2: CH2Cl2: CH2Cl2/EtOAc: EtOAc: MeOH. Selected fractions were further subject to NP-HPLC to yield the purified compounds. Fractions eluting from 1 hexanes/ CH2Cl2 (1:1) were screened by H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) to provide dendrillolide A (1.142: 0.1 mg), macfarlandin E (1.143: 0.1 mg), shahamin L

(4.3: 0.1 mg), and polyrhaphin B (1.154: 0.1 mg). Fractions eluting from CH2Cl2 (100%) were also separated by NP-HPLC (30% EtOAc in hexanes) isolating dendrillolide A (1.142: 0.1 mg), macfarlandin E (1.143: 0.2 mg), macfarlandin D (1.145: 0.1 mg), aplyviolene (1.144: 0.3 mg), chelonaplysin C (1.146: 0.2 mg), polyrhaphin B (1.154: 0.2 mg), shahamin C (1.157: 0.2 mg), polyrhaphin C (1.171: 0.1 mg), shahamin F (1.166: 0.1 mg), secoshahamin (1.164: 0.2 mg), and 12- desacetoxyshahamin C (1.162: 0.1 mg). Fractions eluting from CH2Cl2/EtOAc (4:1) were further separated by NP-HPLC (30% EtOAc in hexanes) isolating macfarlandin E (1.143: 0.2 mg), macfarlandin D (1.145: 0.1 mg), aplyviolene (1.144: 0.2 mg), chelonaplysin C (1.131: 0.2 mg), polyrhaphin B (1.154: 0.2 mg), polyrhaphin C (1.171: 0.1 mg), shahamin F (1.166: 0.1 mg), and shahamin C (1.157: 0.2 mg). Fractions eluting from hexanes/ CH2Cl2 (1:4) were fractionated again with a stepwise solvent gradient from 100% hexanes to 100% MeOH. The fractions eluting from 1 hexanes/ CH2Cl2 (40:60) were screened by H NMR and were separated by NP-HPLC (25% EtOAc in hexanes) isolating dendrillolide A (1.142: 0.4 mg), 15-desacetoxy-12-acetoxydendrillolide A (4.4:

0.3 mg), and macfarlandin E (1.143: 1.1 mg). Fractions eluting from hexanes/ CH2Cl2 (30:70) were screened by 1H NMR, then separated by NP-HPLC (25% EtOAc in hexanes) to isolate dendrillolide A (1.142: 0.6 mg), 12α-acetoxydendrillolide A (1.152: 0.5 mg), macfarlandin E (1.143: 7.8 mg), polyrhaphin C (1.171: 0.1 mg), shahamin F (1.166: 0.1 mg), and aplyviolene (1.144: 0.5 mg). 1 Fractions eluting from hexanes/ CH2Cl2 (20:80) were screened by H NMR then separated by NP- HPLC (25% EtOAc in hexanes) to provide dendrillolide A (1.142: 1.2 mg), 12α-acetoxydendrillolide A (1.152: 1.3 mg), macfarlandin E (1.143: 3.5 mg), macfarlandin D (1.145: 0.8 mg), norrisolide 175

Chapter 7: Experimental (1.130: 0.9 mg), aplyviolene (1.144: 3.4 mg), chelonaplysin C (1.131: 0.3 mg), cheloviolene C (1.132: 0.8 mg), polyrhaphin B (1.154: 3.5 mg), polyrhaphin C (1.171: 0.1 mg), shahamin F (1.166: 0.1 mg), and 12-desacetoxypolyrhaphin A (1.163: 0.9 mg). Lastly, the fractions eluting from CH2Cl2/EtOAc 1 (9:1 to 1:1) were screened by H NMR then separated by NP-HPLC (25% EtOAc in hexanes) to provide dendrillolide A (1.142: 0.2 mg), 12α-acetoxydendrillolide A (1.152: 0.1 mg), macfarlandin E (1.143: 0.8 mg), macfarlandin D (1.145: 0.3 mg), aplyviolene (1.144: 1.9 mg), chelonaplysin C (1.131: 0.3 mg), polyrhaphin B (1.154: 0.8 mg), 12-desacetoxypolyrhaphin A (1.163: 0.6 mg), shahamin C (1.157: 0.3 mg), polyrhaphin C (1.171: 0.1 mg), shahamin F (1.166: 0.1 mg), 15,16- diacetoxyshahamin B (1.160: 0.3 mg), and 12-desacetoxyshahamin C (1.162: 0.3 mg).

28 26 1 (+)-Secoshahamin (1.164): colorless oil (0.2 mg); lit. [α] D + 10.6 (c 0.2, CHCl3); H NMR 13 + and C NMR (CDCl3, 700 MHz), Table 4.6; LRESIMS m/z 459.2 [M + Na] .

21 1 13 (–)-Shahamin L (4.3): colorless oil (0.1 mg); [α] D – 48 (c 0.01, CHCl3); H NMR and C + NMR (CDCl3, 700 MHz), Table 4.7; HRESIMS m/z 413.2328 [M + Na] (calcd. for C23H34NaO5, 413.2298).

21 (–)-15-Desacetoxy-12-acetoxydendrillolide A (4.4): colorless oil (0.3 mg); [α] D – 2 (c 0.03, 1 13 + CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 4.8; HRESIMS m/z 399.2142 [M + Na]

(calcd. for C22H32NaO5, 399.2127).

7.8.2 Procedure for the saponification and lactonization of rearranged diterpenes 12-Desacetoxyshahamin C (1.162) (362 g/mol, 0.3 mg, 0.83 µmol) was dissolved in a mixture of MeOH (100 µL) and Milli-Q H2O (100 µL) and treated with potassium hydroxide solution (1 M, 14 µL, 0.00663 mmol). The reaction was warmed to 50 °C for 3 h and monitored by TLC. The reaction was cooled to 25 °C, aqueous HCl (28 µL, 1 M) added, then the mixture was passed through ® a Sep Pak silica cartridge, which was flushed with Milli-Q H2O (0.5 mL), then with MeOH (2 x 0.5 mL), and finally with EtOAc (1.0 mL) collected separately. The fractions were dried over Na2SO4, filtered, and concentrated under nitrogen, with the EtOAc fraction providing 12-deoxyshahamin E (4.6) as a clear oil (0.26 mg, 87% yield). This procedure was repeated using 12-desacetoxypolyrhaphin A (1.163) (362 g/mol, 0.9 mg, 0.0025 mmol) providing 0.82 mg (91%) of 4.6. The hydrolysis of secoshahamin (1.164) (436 g/mol, 0.2 mg, 0.00046 mmol) required three equivalents of KOH to yield 0.12 mg (65%) of 4.6.

176

Chapter 7: Experimental

21 (+)-12-Desacetoxyshahamin C (1.162): colorless oil (0.3 mg); [α] D + 4 (c 0.03, CHCl3); 113 1 13 lit. [α]D +54.0 (c 0.23, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 4.6; LRESIMS m/z 385.2 [M + Na]+.

21 (+)-12-Desacetoxypolyrhaphin A (1.163): colorless oil (0.9 mg); [α] D + 5 (c 0.09, CHCl3); 113 1 13 lit. [α]D +14.3 (c 0.23, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 4.6; LRESIMS m/z 385.2 [M + Na]+.

21 1 (+)-12-Deoxyshahamin E (4.6): colorless oil (0.26 mg); [α] D + 4 (c 0.026, CHCl3); H NMR 13 + and C NMR (CDCl3, 700 MHz), Table 4.6; HRESIMS m/z 343.2251 [M + Na] (calcd. for

C20H32NaO3, 343.2244).

7.8.3 Procedure for the acetylation of 12-deoxyshahamin E (4.6) 12-Deoxyshahamin E (4.6) (320.2 g/mol, 0.82 mg, 0.00256 mmol) was dissolved in a mixture of anhydrous pyridine (200 µL) and acetic anhydride (200 µL). After 17 h at 25 °C, the reaction mixture was worked up by addition of EtOAc (1 mL) and Milli-Q H2O (1 mL). The aqueous layer was extracted with EtOAc (3 x 1 mL), the combined organic extracts were washed with 5% HCl and with Milli-Q H2O, then dried over anhydrous Na2SO4, filtered, and concentrated under nitrogen to yield a clear oil (0.8 mg), which was purified by NP-HPLC (30% EtOAc in hexanes) (0.2 mg, 25% yield).

21 (+)-12-Desacetoxyshahamin C (1.162): colorless oil (0.2 mg); [α] D + 23 (c 0.02, CHCl3); 113 21 1 13 lit. [α] D + 54 (c 0.44, CHCl3); H NMR and C NMR (CDCl3, 700 MHz), Table 4.6; LRESIMS m/z 385.2 [M + Na]+.

177

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Appendices Appendices

Appendix 1. Spectral data of known compounds isolated in this investigation...... 194 1 Appendix 2. H NMR spectrum of 2.1 (700 MHz, CDCl3)...... 202 Appendix 3. HSQC spectrum of 2.1 (700 MHz, CDCl3)...... 202 Appendix 4. HMBC spectrum of 2.1 (700 MHz, CDCl3)...... 203 Appendix 5. COSY spectrum of 2.1 (700 MHz, CDCl3)...... 203 1 Appendix 6. H NMR spectrum of 2.2 (700 MHz, CDCl3)...... 204 Appendix 7. HSQC spectrum of 2.2 (700 MHz, CDCl3)...... 204 Appendix 8. HMBC spectrum of 2.2 (700 MHz, CDCl3)...... 205 Appendix 9. COSY spectrum of 2.2 (700 MHz, CDCl3)...... 205 Appendix 10. NOESY spectrum of 2.2 (700 MHz, CDCl3)...... 206 1 Appendix 11. H NMR spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube. .. 206 Appendix 12. HSQC spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube...... 207 Appendix 13. HMBC spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube...... 207 Appendix 14. COSY spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube...... 208 1 Appendix 15. H NMR spectrum of 2.4 (700 MHz, CDCl3)...... 208 Appendix 16. HSQC spectrum of 2.4 (700 MHz, CDCl3)...... 209 Appendix 17. HMBC spectrum of 2.4 (700 MHz, CDCl3)...... 209 Appendix 18. COSY spectrum of 2.4 (700 MHz, CDCl3)...... 210 Appendix 19. NOESY spectrum of 2.4 (700 MHz, CDCl3)...... 210 1 Appendix 20. H NMR spectrum of 2.5 (700 MHz, CDCl3)...... 211 Appendix 21. HSQC spectrum of 2.5 (700 MHz, CDCl3)...... 211 Appendix 22. HMBC spectrum of 2.5 (700 MHz, CDCl3)...... 212 Appendix 23. COSY spectrum of 2.5 (700 MHz, CDCl3)...... 212 Appendix 24. NOESY spectrum of 2.5 (700 MHz, CDCl3)...... 213 1 Appendix 25. H NMR spectrum of 2.6 (700 MHz, CDCl3)...... 213 Appendix 26. HSQC spectrum of 2.6 (700 MHz, CDCl3)...... 214 Appendix 27. HMBC spectrum of 2.6 (700 MHz, CDCl3)...... 214 Appendix 28. COSY spectrum of 2.6 (700 MHz, CDCl3)...... 215 Appendix 29. NOESY spectrum of 2.6 (700 MHz, CDCl3)...... 215 1 Appendix 30. H NMR spectrum of 2.7 (700 MHz, CDCl3)...... 216 Appendix 31. HSQC spectrum of 2.7 (700 MHz, CDCl3)...... 216 Appendix 32. HMBC spectrum of 2.7 (700 MHz, CDCl3)...... 217 Appendix 33. COSY spectrum of 2.7 (700 MHz, CDCl3)...... 217 Appendix 34. NOESY spectrum of 2.7 (700 MHz, CDCl3)...... 218 1 Appendix 35. H NMR spectrum of 2.8 (700 MHz, CDCl3)...... 218 Appendix 36. HSQC spectrum of 2.8 (700 MHz, CDCl3)...... 219 Appendix 37. HMBC spectrum of 2.8 (700 MHz, CDCl3)...... 219 Appendix 38. COSY spectrum of 2.8 (700 MHz, CDCl3)...... 220 1 Appendix 39. H NMR spectrum of 2.9 (700 MHz, CDCl3)...... 220 Appendix 40. HSQC spectrum of 2.9 (500 MHz, CDCl3)...... 221 Appendix 41. HMBC spectrum of 2.9 (700 MHz, CDCl3)...... 221 Appendix 42. COSY spectrum of 2.9 (500 MHz, CDCl3)...... 222 Appendix 43. NOESY spectrum of 2.9 (700 MHz, CDCl3)...... 222 1 Appendix 44. H NMR spectrum of 2.10 (500 MHz, CDCl3)...... 223 Appendix 45. HSQC spectrum of 2.10 (500 MHz, CDCl3)...... 223 Appendix 46. HMBC spectrum of 2.10 (500 MHz, CDCl3)...... 224 Appendix 47. COSY spectrum of 2.10 (500 MHz, CDCl3)...... 224 Appendix 48. NOESY spectrum of 2.10 (500 MHz, CDCl3)...... 225 1 Appendix 49. H NMR spectrum of 2.11 (700 MHz, CDCl3)...... 225

191

Appendices

Appendix 50. HSQC spectrum of 2.11 (700 MHz, CDCl3)...... 226 Appendix 51. HMBC spectrum of 2.11 (700 MHz, CDCl3)...... 226 Appendix 52. COSY spectrum of 2.11 (700 MHz, CDCl3)...... 227 Appendix 53. NOESY spectrum of 2.11 (700 MHz, CDCl3)...... 227 1 Appendix 54. H NMR spectrum of 2.12 (700 MHz, CDCl3)...... 228 Appendix 55. HSQC spectrum of 2.12 (700 MHz, CDCl3)...... 228 Appendix 56. HMBC spectrum of 2.12 (700 MHz, CDCl3)...... 229 Appendix 57. COSY spectrum of 2.12 (700 MHz, CDCl3)...... 229 Appendix 58. NOESY spectrum of 2.12 (700 MHz, CDCl3)...... 230 1 Appendix 59. H NMR spectrum of 2.13 (700 MHz, CDCl3)...... 230 Appendix 60. HSQC spectrum of 2.13 (700 MHz, CDCl3)...... 231 Appendix 61. HMBC spectrum of 2.13 (700 MHz, CDCl3)...... 231 Appendix 62. COSY spectrum of 2.13 (700 MHz, CDCl3)...... 232 Appendix 63. NOESY spectrum of 2.13 (700 MHz, CDCl3)...... 232 1 Appendix 64. H NMR spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 233 Appendix 65. HSQC spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 233 Appendix 66. HMBC spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 234 Appendix 67. gCOSY spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 234 Appendix 68. TOCSY spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 235 Appendix 69. NOESY spectrum of 3.1 at 318 K (700 MHz, CDCl3)...... 235 Appendix 70. Overlay of 1H NMR spectra for 3.1 in the temperature range 323 K to 223 K (500 MHz, CDCl3)...... 236 Appendix 71. Overlay of 1H NMR spectra for 3.1 in the temperature range 298 K to 193 K (500 MHz, d6-acetone)...... 236 1 Appendix 72. H NMR spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 237 Appendix 73. HSQC spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 237 Appendix 74. HMBC spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 238 Appendix 75. gCOSY spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 238 Appendix 76. TOCSY spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 239 Appendix 77. NOESY spectrum of 3.2 at 318 K (700 MHz, CDCl3)...... 239 Appendix 78. Overlay of 1H NMR spectra for 3.2 in the temperature range 323 K to 223 K (500 MHz, CDCl3)...... 240 Appendix 79. Overlay of 1H NMR spectra for dendrillolide A (1.142) in the temperature range 323 K to 223 K (500 MHz, CDCl3)...... 240 1 Appendix 80. H NMR spectrum of 3.3 (700 MHz, CDCl3)...... 241 Appendix 81. HSQC spectrum of 3.3 (700 MHz, CDCl3)...... 241 Appendix 82. HMBC spectrum of 3.3 (700 MHz, CDCl3)...... 242 Appendix 83. COSY spectrum of 3.3 (700 MHz, CDCl3)...... 242 Appendix 84. NOESY spectrum of 3.3 (700 MHz, CDCl3)...... 243 1 Appendix 85. H NMR spectrum of 3.4 (700 MHz, CDCl3)...... 243 Appendix 86. HSQC spectrum of 3.4 (700 MHz, CDCl3)...... 244 Appendix 87. HMBC spectrum of 3.4 (700 MHz, CDCl3)...... 244 Appendix 88. COSY spectrum of 3.4 (700 MHz, CDCl3)...... 245 Appendix 89. NOESY spectrum of 3.4 (700 MHz, CDCl3)...... 245 1 Appendix 90. H NMR spectrum of 3.5 (700 MHz, CDCl3)...... 246 Appendix 91. HSQC spectrum of 3.5 (500 MHz, CDCl3)...... 246 Appendix 92. HMBC spectrum of 3.5 (700 MHz, CDCl3)...... 247 Appendix 93. COSY spectrum of 3.5 (500 MHz, CDCl3)...... 247 Appendix 94. NOESY spectrum of 3.5 (700 MHz, CDCl3)...... 248 1 Appendix 95. H NMR spectrum of 3.6 (700 MHz, CDCl3)...... 248 Appendix 96. HSQC spectrum of 3.6 (700 MHz, CDCl3)...... 249 Appendix 97. HMBC spectrum of 3.6 (700 MHz, CDCl3)...... 249 192

Appendices

Appendix 98. COSY spectrum of 3.6 (700 MHz, CDCl3)...... 250 Appendix 99. NOESY spectrum of 3.6 (700 MHz, CDCl3)...... 250 1 Appendix 100. H NMR spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3). . 251 Appendix 101. HSQC spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3)...... 251 Appendix 102. HMBC spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3). ... 252 Appendix 103. COSY spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3)...... 252 Appendix 104. NOESY spectrum of diastereomeric mixture of 4.1 and 4.2 (700 MHz, CDCl3). .. 253 1 Appendix 105. H NMR spectrum of 1.164 (700 MHz, CDCl3)...... 253 Appendix 106. HSQC spectrum of 1.164 (700 MHz, CDCl3)...... 254 Appendix 107. HMBC spectrum of 1.164 (700 MHz, CDCl3)...... 254 Appendix 108. COSY spectrum of 1.164 (700 MHz, CDCl3)...... 255 Appendix 109. NOESY spectrum of 1.164 (700 MHz, CDCl3)...... 255 1 Appendix 110. H NMR spectrum of 4.3 (700 MHz, CDCl3)...... 256 Appendix 111. HSQC spectrum of 4.3 (700 MHz, CDCl3)...... 256 Appendix 112. HMBC spectrum of 4.3 (700 MHz, CDCl3)...... 257 Appendix 113. COSY spectrum of 4.3 (700 MHz, CDCl3)...... 257 Appendix 114. NOESY spectrum of 4.3 (700 MHz, CDCl3)...... 258 1 Appendix 115. H NMR spectrum of 4.4 (700 MHz, CDCl3)...... 258 Appendix 116. HSQC spectrum of 4.4 (700 MHz, CDCl3)...... 259 Appendix 117. HMBC spectrum of 4.4 (700 MHz, CDCl3)...... 259 Appendix 118. COSY spectrum of 4.4 (700 MHz, CDCl3)...... 260 Appendix 119. NOESY spectrum of 4.4 (700 MHz, CDCl3)...... 260 Appendix 120. Computational methods for 5,9-epoxydendrillolide A (3.1), 10-oxonordendrillolide A (3.2) and dendrillolide A (1.142) ...... 261 Appendix 121. Computational methods for 10,20-epoxydendrillolide A (3.4) ...... 261 Appendix 122. Computational methods for 15-dendrillactol (4.1) and 15-epidendrillactol (4.2). .. 263 Appendix 123. Computational methods for secoshahamin (1.164) ...... 266 Appendix 124. Computational methods for shahamin L (4.3) ...... 268 Appendix 125 Microneutralization assay raw data for Ross River fever, Dengue fever and Influenza...... 271

193

Appendices Appendix 1. Spectral data of known compounds isolated in this investigation.

43 21 43 (–)-Luffarin-X (1.16): colorless oil (1.9 mg); [α] D – 2 (c 0.18, CHCl3), lit.: [α]D + 19 (c 0.0013, 1 CHCl3); H NMR (CDCl3, 700 MHz) δ 7.10 (1H, s, H-3), 4.76, (2H, m, H-1), 2.27 (2H, m, H-5), 0.86 (3H, s, Me-17), 0.86 (3H, s, Me-20), 0.84 (3H, s, Me-18), 0.81 (3H, s, Me-19); (+)-LRESIMS m/z 331.2 [M+Na]+.

45 23 45 (–)-Isoagatholactone (1.19): colorless oil (2.1 mg); [α] D – 2 (c 0.01, CHCl3), lit: [α]D + 6 1 (concentration and solvent were not reported); H NMR (CDCl3, 500 MHz) δ 6.87 (1H, dd, J = 7.0, 3.5 Hz, H-12), 4.37 (1H, t, J = 9.0 Hz, H-15a), 4.05 (1H, t, J = 9.0 Hz, H-15b), 2.80 (1H, m, H-14), 2.35 (2H, m, H-11), 0.93 (3H, s, Me-18), 0.88 (3H, s, Me-17), 0.84 (3H, s, Me-20), 0.78 (3H, s, Me- 19); (+)- LRESIMS m/z 325.1 [M+Na]+.

49-50 21 49-50 (±)-Spongian-16-one (1.24): amorphous solid (1.9 mg); [α] D – 3 (c 0.03, CHCl3), lit.: [α]D 1 + 53 (c 0.0011, CHCl3); H NMR (CDCl3, 500 MHz) δ 4.21 (1H, d, J = 9.9 Hz, H-15β), 4.09 (1H, dd, J = 5.5, 9.9 Hz, H-15α), 2.52 (1H, t, J = 8.0 Hz, H-13), 2.29 (1H, dd, J = 4.7, 13.8 Hz, H-12β), 2.07 (1H, dd, J = 5.2, 8.0 Hz, H-14), 1.81 (1H, dt, J = 3.3, 12.8 Hz, H-7β), 1.72 (1H, br d, J = 12.8 Hz, H- 1β), 1.60 (1H, m, H-12α), 1.60 (1H, m, H-2α), 1.55 (1H, m, H-6α), 1.53 (1H, m, H-11β), 1.43 (1H, m, H-2β), 1.35 (1H, m, H-3β), 1.32 (1H, m, H-6β), 1.26 (1H, m, H-11α), 1.12 (1H, ddd, J = 4.2, 13.8, 13.8 Hz, H-3α), 1.02 (1H, ddd, J = 3.5, 13.2, 13.2 Hz, H-7α), 0.88 (3H, s, Me-18α), 0.85 (3H, s, Me- 17), 0.84 (3H, s, Me-19β), 0.81 (3H, s, Me-20), 0.77 (1H, m, H-1α), 0.77 (1H, m, H-5), 0.75 (1H, m, H-9); (+)-LRESIMS m/z 327.2 [M+Na]+.

52 1 (–)-7α-Acetoxyspongian-16-one (1.32): amorphous solid (3.4 mg); [α]D – 31 (c 0.11, CHCl3); H

NMR (CDCl3, 500 MHz) δ 4.93 (1H, t, J = 2.9, H-7), 4.22 (1H, d, J = 10.3 Hz, H-15α), 3.98 (1H, dd, J = 5.4, 10.3 Hz, H-15β), 2.58 (1H, t, J = 8.0 Hz, H-13), 2.50 (1H, dd, J = 5.4, 8.0 Hz, H-14), 2.29

(1H, dd, J = 5.0, 14.0 Hz, H-12α), 2.10 (3H, s, 7- OCOCH3), 1.74 (1H, br d, J = 12.8 Hz, H-1α), 1.69 (2H, m, H-6), 1.61 (1H, m, H-2α), 1.59 (1H, m, H-12β), 1.53 (1H, m, H-11α), 1.45 (1H, m, H-2β), 1.41 (1H, m, H-3α), 1.34 (1H, m, H-5), 1.32 (1H, m, H-11β), 1.17 (1H, td, J = 4.3, 13.5 Hz, H-3α), 1.05 (1H, dd, J = 2.2, 12.3 Hz, H-9), 0.94 (3H, s, Me-17), 0.86 (3H, s, Me-1β), 0.85 (3H, s, Me-20), + 0.79 (6H, s, Me-18 and Me-19); (+)-LRESIMS m/z 385.2 [M+Na] . No [α]D reported in literature.

51 (–)-7α-Hydroxyspongian-16-one (1.33): amorphous solid (0.5 mg); [α]D – 24 (c 0.05, CHCl3); 51 28 1 lit.: [α] D + 30.3 (c 0.22, CHCl3); H NMR (CDCl3, 500 MHz) δ 4.20 (1H, d, J = 10.0 Hz, H-15β), 4.10 (1H, dd, J = 5.6, 10.0 Hz, H-15α), 3.69 (1H, br s, H-7β), 3.08 (1H, dd, J = 5.6, 8.3 Hz, H-14),

194

Appendices 2.57 (1H, t, J = 8.3 Hz, H-13), 2.26 (1H, dd, J = 3.6, 13.8 Hz, H-12), 1.80 (1H, dt, J = 2.2, 12.2 Hz, H-6β), 1.71 (1H, br d, J = 12.7 Hz, H-1β), 1.43 (1H, m, H-11β), 1.42 (1H, m, H-2α), 1.40 (1H, br d, J = 12.9 Hz, H-3β), 1.33 (1H, br d, J = 13.2 Hz, H-5), 1.30 (1H, m, H-11α), 1.17 (1H, dt, J = 4.9, 13.7 Hz, H-3α), 1.04 (1H, dd, J = 2.0, 12.2 Hz, H-9), 0.84 (3H, s, Me-18), 0.83 (3H, s, Me-20), 0.81 (3H, s, Me-17), 0.759 (3H, s, Me-19); (+)-LRESIMS m/z 343.2 [M+Na]+.

21 18 (+)-12α-Acetoxyspongian-16-one (1.38): colorless oil (0.4 mg); [α] D + 37 (c 0.04, CHCl3), lit.: 1 [α]D – 72 (c 0.007, CHCl3); H NMR (CDCl3, 500 MHz) δ 5.47 (H1, br s, H-12α), 4.24 (1H, d, J = 9.9 Hz, H-15β), 4.12 (1H, dd, J = 5.5, 9.9 Hz, H-15α), 2.69 (1H, d, J = 7.9 Hz, H-13), 2.27 (1H, dd,

J = 5.5, 7.9 Hz H-14), 2.07 (3H, s, OCOCH3), 1.85 (1H, ddd, J = 3.2, 3.2, 12.9 Hz, H-7β), 1.78 (1H, dd, J = 2.0, 15.5 Hz, H-11β), 1.65 (1H, d, J = 12.9 Hz, H-1β), 1.58 (1H, m, H-2β), 1.55 (1H, m, H- 6β), 1.55 (H1, m, H-11α), 1.42 (1H, m, H-6α), 1.39 (1H, m, H-3β), 1.34 (H1, m, H-2α), 1.19 (1H, dd, J = 2.2, 12.5 Hz, H-9), 1.14 (1H, m, H-3α), 1.08 (1H, m, H-7α), 0.89 (1H, m, H-5), 0.88 (3H, s, Me- 18), 0.86 (3H, s, Me-17), 0.84 (3H, s, Me-19), 0.81 (3H, s, Me-20), 0.71 (1H, ddd, J = 3.6, 12.9, 12.9 Hz, H-1α); (+)-LRESIMS m/z 385.2 [M+Na]+.

21 18 (–)-20-Acetoxyspongian-16-one (1.39): colorless oil (2.9 mg); [α] D – 11 (c 0.19, CHCl3), lit.: 1 [α]D – 21 (c 0.0013, CHCl3); H NMR (CDCl3, 500 MHz) δ 4.55 (1H, d, J = 12.0 Hz, H-20a), 4.23 (1H, d, J = 9.7 Hz, H-15β), 4.16 (1H, d, J = 12.0 Hz, H-20b), 4.12 (1H, dd, J = 5.4, 9.7 Hz, H-15α), 2.53 (1H, t, J = 7.3 Hz, H-13), 2.28 (1H, d, J = 9.7 Hz, H-12β), 2.13 (1H, m, H-1β), 2.12 (1H, m, H-

14), 2.03 (3H, s, OCOCH3), 1.90 (1H, ddd, J = 3.2, 3.2, 12.7 Hz, H-7β), 1.72 (1H, br d, J = 8.8 Hz, H-11α), 1.55 (1H, m, H-2α), 1.55 (1H, m, H-6α), 1.54 (1H, m, H-12α), 1.50 (1H, m, H-11β),1.46 (1H, m, H-3β), 1.46 (1H, m, H-6 β), 1.40 (1H, m, H-2β), 1.17 (1H, ddd, J = 4.2, 13.2, 13.2 Hz, H- 3α), 1.11 (1H, ddd, J = 4.2, 13.2, 13.2 Hz, H-7α), 1.00 (1H, dd, J = 1.6, 12.5 Hz, H-5), 0.91 (3H, s, Me-17), 0.89 (3H, s, Me-18), 0.84 (1H, m, H-9), 0.84 (3H, s, Me-19), 0.75 (1H, ddd, J = 3.8, 13.2, 13.2 Hz, H-1α); (+)-LRESIMS m/z 385.2 [M+Na]+.

18 18 20-Oxyspongian-16-one propionate (1.40): amorphous solid (0.3 mg); lit.: [α]D – 13 (c 0.007, 1 CHCl3); H NMR (CDCl3, 500 MHz) δ 4.56 (1H, d, J = 12.3 Hz, H-20a), 4.23 (1H, d, J = 9.8 Hz, H- 15β), 4.16 (1H, d, J = 12.3 Hz, H-20b), 4.12 (1H, dd, J = 5.4, 9.8 Hz, H-15α), 2.53 (1H, t, J = 6.9 Hz,

H-13), 2.31 (2H, q, J = 7.7 Hz, OCOCH2CH3), 2.27 (1H, m, H-12β), 2.12 (1H, m, H-1β), 2.12 (1H, m, H-14), 1.91 (1H, ddd, J = 3.0, 3.0, 12.9 Hz, H-7β), 1.73 (1H, d, J = 8.7 Hz, H-11α), 1.55 (1H, m, H-2α), 1.55 (1H, m, H-6α), 1.55 (1H, m, H-11β), 1.55 (1H, m, H-12α), 1.45 (1H, m, H-3β), 1.45 (1H, m, H-6 β), 1.39 (1H, m, H-2β), 1.18 (1H, ddd, J = 3.7, 12.8, 12.8 Hz, H-3α), 1.12 (1H, m, H-7α), 1.12

(3H, t, J = 7.7 Hz, OCOCH2CH3), 0.99 (1H, dd, J = 1.8, 12.4 Hz, H-5), 0.91 (3H, s, Me-17), 0.89 195

Appendices (3H, s, Me-18), 0.87 (1H, m, H-9), 0.84 (3H, s, Me-19), 0.74 (1H, ddd, J = 3.3, 13.2, 13.2 Hz, H-1α); (+)-LRESIMS m/z 399.2 [M+Na]+.

18 18 12α,20-Dioxyspongian-16-one-diprpionate (1.41): colorless oil (0.4 mg); lit. [α]D – 57 (c 0.007, 1 CHCl3); H NMR (CDCl3, 500 MHz) δ 5.45 (1H, br s, H-12), 4.59 (1H, d, J = 12.4 Hz, H-20a), 4.26 (1H, d, J = 9.9 Hz, H-15β), 4.14 (1H, m, H-20b), 4.13 (1H, m, H-15α), 2.68 (1H, d, J = 7.8 Hz, H-

13), 2.35 (2H, q, J = 7.6 Hz, OCOCH2CH3), 2.32 (2H, m, OCOCH2CH3), 2.32 (1H, m, H-14), 2.05 (1H, m, H-1a), 2.03 (1H, m, H-11a), 1.92 (1H, ddd, J = 3.3, 3.3, 13.0 Hz, H-7a), 1.82 (1H, ddd, J = 3.4, 13.0, 13.0 Hz, H-11b), 1.60 (1H, m, H-6a), 1.47 (1H, m, H-2a), 1.45 (1H, m, H-3a), 1.44 (1H, m, H-2b), 1.40 (1H, m, H-6b), 1.34 (1H, d, J = 12.7 Hz, H-9), 1.20 (1H, m, H-3b), 1.18 (1H, m, H-

7b), 1.17 (3H, t, J = 7.6 Hz, OCOCH2CH3), 1.13 (3H, t, J = 7.6 Hz, OCOCH2CH3), 1.04 (1H, dd, J = 1.9, 12.9 Hz, H-5), 0.91 (3H, s, H-17), 0.91 (3H, s, H-18), 0.84 (3H, s, H-19), 0.64 (1H, ddd, J = 3.2, 12.9, 12.9 Hz, H-1α); (+)-LRESIMS m/z 471.2 [M+Na]+.

56 56 21 12α,20-Diacetoxyspongian-16-one (1.42): colorless oil (0.4 mg); lit. [α] D + 14 (c 0.04, CHCl3); 1 H NMR (CDCl3, 500 MHz) δ 5.44 (1H, br s, H-12), 4.58, (1H, d, J = 12.3 Hz, H-20a), 4.27 (1H, d, J = 9.9 Hz, H-15a), 4.15 (1H, m, H-20b), 4.13 (1H, m, H-15b), 2.69 (1H, dt, J = 1.5, 7.7 Hz, H-13),

2.31 (1H, dd, J = 5.1, 7.7 Hz, H-14), 2.09 (3H, OCOCH3), 2.04 (3H, OCOCH3), 2.04 (1H, m, H-1a), 2.02 (1H, d, J = 5.9 Hz, H-11a), 1.94 (1H, dt, J = 3.0, 12.9 Hz, H-7a), 1.82 (1H, ddd, J = 3.3, 12.9, 15.5 Hz, H-11b), 1.59 (1H, m, H-6a), 1.55 (1H, m, H-2a), 1.46 (1H, m, H-2b), 1.46 (1H, m, H-3a), 1.41 (1H, m, H-6b), 1.35 (1H, m, H-9), 1.20 (1H, m, H-3b), 1.17 (1H, m, H-7b), 1.05 (1H, dd, J = 2.1, 12.6 Hz, H-5), 0.91 (3H, s, H-17), 0.90 (3H, s, H-18), 0.84 (3H, s, H-19), 0.66 (1H, ddd, J = 1.8, 4.0, 12.9 Hz, H-1α); (+)-LRESIMS m/z 443.2 [M+Na]+.

56 56 21 12α-Acetoxy-20-oxyspongian-16-one-20-propionate (1.43): colorless oil (0.3 mg); lit. [α] D – 1 49 (c 0.03, CHCl3); H NMR (CDCl3, 500 MHz) δ 5.44 (1H, br s, H-12), 4.60, (1H, d, J = 12.4 Hz, H-20a), 4.27 (1H, d, J = 9.9 Hz, H-15a), 4.14 (1H, m, H-20b), 4.13 (1H, m, H-15b), 2.69 (1H, dt, J =

1.5, 8.2 Hz, H-13), 2.31 (1H, dd, J = 5.2, 8.2 Hz, H-14), 2.31 (2H, m, OCOCH2CH3), 2.08 (3H,

OCOCH3), 2.04 (1H, m, H-1a), 2.01 (1H, d, J = 5.9 Hz, H-11a), 1.94 (1H, dt, J = 2.9, 12.8 Hz, H- 7a), 1.82 (1H, ddd, J = 3.3, 13.2, 15.7 Hz, H-11b), 1.59 (1H, m, H-6a), 1.54 (1H, m, H-2a), 1.46 (1H, m, H-3a), 1.45 (1H, m, H-2b), 1.41 (1H, m, H-6b), 1.34 (1H, m, H-9), 1.20 (1H, m, H-3b), 1.16 (1H, m, H-7b), 1.13 (3H, t, J = 7.7 Hz, OCOCH2CH3), 1.05 (1H, dd, J = 1.7, 12.5 Hz, H-5), 0.91 (3H, s, H-17), 0.91 (3H, s, H-18), 0.85 (3H, s, H-19), 0.65 (1H, ddd, J = 1.7, 4.0, 13.2 Hz, H-1α); (+)- LRESIMS m/z 457.2 [M+Na]+.

196

Appendices

97 22 97 (+)-Norrisolide (1.130): colorless oil (0.9 mg); [α] D + 57 (c 0.03, CHCl3), lit.: [α]D + 1 (c 1.0, 1 CHCl3); H NMR (CDCl3, 500 MHz) δ 6.44 (1H, d, J = 3.3 Hz, H-15), 6.14 (1H, d, J = 5.9 Hz, H- 16), 5.15 (1H, s, H-17a), 5.09 (1H, br s, H-17b), 3.36 (1H, m, H-13), 3.07 (1H, dd, J = 3.3, 9.1 Hz,

H-14), 2.56 (2H, d, J = 7.0 Hz, H-12), 2.07 (3H, s, 15-OCOCH3), 2.14 (1H, m, H-9), 1.68 (1H, m, H- 6a), 1.66 (2H, m, H-7), 1.63 (1H, m, H-1a), 1.55 (2H, m, H-2), 1.46 (1H, m, H-3a), 1.45 (1H, m, H- 6b), 1.15 (1H, dd, J = 6.8, 13.2 Hz, H-5), 1.06 (1H, m, H-3b), 1.01 (1H, m, H-1b), 0.86 (3H, s, Me- 20), 0.85 (3H, s, Me-19), 0.66 (3H, s, Me-18), ; (+)-LRESIMS m/z 399.2 [M + Na]+.

99 99 (–)-Chelonaplysin C (1.131): colorless oil (0.8 mg); [α]D – 39 (c 0.03, CHCl3), lit.: optical rotation 1 was not reported; H NMR (CDCl3, 500 MHz) δ 6.27 (1H, s, H-16), 5.88 (1H, dd, J = 1.1, 3.5 Hz, H-15), 5.25 (1H, br dd, J = 1.2, 2.4 Hz, H-12b), 5.17 (1H, br d, J = 2.2 Hz, H-17), 3.21 (1H, m, H- 14), 3.14 (1H, dd, J = 6.3, 19.4 Hz, H-12β), 2.81 (1H, m, H-13), 2.62 (1H, m, H-12a), 2.17 (1H, m,

H-9), 2.11 (1H, s, 16-OCOCH3), 1.27 (1H, dd, J = 7.0, 12.5 Hz, H-5), 0.89 (3H, s, Me-19), 0.87 (3H, s, Me-20), 0.72 (3H, s, Me-18); (+)-LRESIMS m/z 399.2 [M+Na]+.

Cheloviolene C (1.132):100 colorless oil (0.2 mg), lit.:100 optical rotation was not reported; 1H NMR

(CDCl3, 500 MHz) δ 5.04 (1H, s, H-17a), 5.03 (1H, s, H-17b), 4.46 (1H, dd, J = 9.4, 7.8 Hz, H-16a), 4.04 (1H, dd, J = 11.6, 3.7 Hz, H-15a), 3.97 (1H, dd, 11.6, 6.6 Hz, H-15b), 3.87 (1H, t, J = 9.4 Hz, H-16b), 2.91 (1H, dddd, J = 10.4, 9.4, 8.4, 7.8 Hz, H-13), 2.73 (1H, dd, J = 17.5, 8.4 Hz, H- 12a), 2.35 (1H, dd, J = 17.5, 10.3 Hz, H-12b), 2.25 (1H, ddd, J = 10.4, 6.6, 3.7 Hz, H-14), 2.04 (4H, m,

15-OCOCH3, H-8), 1.66 (2H, m, H-7), 1.61 (2H, m, H-6), 1.56 (1H, m, H-1a), 1.53 (2H, m, H-2), 1.44 (1H, dt, J = 12.5, 3.1 Hz, H-3a), 1.28 (1H, m, H-5), 1.15 (1H, td, J = 12.5, 4.6 Hz, H-1b), 1.06 (1H, td, J = 12.5, 4.6 Hz, H-3b), 0.86 (6H, s, Me-19, Me-20), 0.67 (3H, s, Me-18); (+)-LRESIMS m/z 325.2 [M+Na]+.

113 113 (+)-Dendrillolide A (1.142): colorless oil (2.6 mg); [α]D + 61 (c 0.88, CHCl3), lit.: [α]D + 87 (c 1 0.31, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.44 (1H, d, J = 6.6 Hz, H-15), 6.05 (1H, d, J = 4.4 Hz, H-16), 4.83 (1H, d, J = 2.2 Hz, H-20a), 4.57 (1H, d, J = 2.2 Hz, H-20b), 3.14 (1H, m, H-13), 2.71 (1H, dd, J = 9.4, 18.0 Hz, H-12a), 2.68 (1H, m, H-9), 2.67 (1H, m, H-14), 2.52 (1H, dd, J = 9.4, 18.0

Hz, H-12b), 2.38 (1H, dd, J = 4.8, 12.5 Hz, H-1a), 2.11 (3H, s, 15-OCOCH3), 1.82 (1H, td, J = 1.8,

12.5 Hz, H-1b), 1.76 (1H, m, H-5), 1.74 (1H, m, H-2a), 1.73 (2H, m, H2-6), 1.71 (1H, m, H-7a), 1.58 (1H, td, J = 14.1, 3.9 Hz, H-3a), 1.47 (1H, m, H-7b), 1.37 (1H, qt, J = 13.5, 2.8 Hz, H-2b), 1.28 (1H, br d, J = 14.1 Hz, H-3b), 0.97 (3H, s, Me-17), 0.94 (3H, s, Me-19), 0.93 (3H, s, Me-18); (+)-LRESIMS m/z 399.2 [M+Na]+.

197

Appendices

111 111 (–)-Macfarlandin E (1.143): colorless oil (9.0 mg); [α]D – 27 (c 0.11, CHCl3), lit.: [α]D –29 (c 1 1.0, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.49 (1H, s, H-16), 5.80 (1H, d, J = 5.0 Hz, H-12), 5.73 (1H, d, J = 1.4 Hz, H-15), 4.89 (1H, d, J = 1.4 Hz, H-20a), 4.67 (1H, d, J = 1.4 Hz, H-20b), 2.87 (1H, ddd, J = 4.0, 4.4, 5.5 Hz, H-13), 2.74 (1H, d, J = 9.2 Hz, H-9), 2.67 (1H, dd, J = 3.0, 4.4 Hz, H-14),

2.42 (1H, dd, J = 4.7, 12.4 Hz, H-1a), 2.21 (3H, s, 12- OCOCH3), 2.11 (3H, s, 16- OCOCH3), 1.42 (1H, m, H-2a), 1.31 (1H, m, H-3a), 1.08 (3H, s, Me-17), 0.99 (3H, s, Me-19), 0.95 (3H, s, Me-18); (+)-LRESIMS m/z 457.2 [M+Na]+.

112, 278 112, 278 1 Aplyviolene (1.144): colorless oil (6.1 mg); lit.: [α]D – 18 (c 0.36, CHCl3); H NMR

(CDCl3, 500 MHz) δH 6.15 (1H, s, H-16), 5.73 (1H, d, J = 1.6 Hz, H-15), 4.86 (1H, d, J = 2.1 Hz, H- 20a), 4.60 (1H, d, J = 1.9 Hz, H-20b), 2.89 (1H, dd, J = 5.5, 19.5 Hz, H-12a), 2.71 (1H, J = 8.5, Hz, H-13), 2.68 (1H, J = 19.0 Hz, H-12b), 2.63 (1H, br t, J = 4.1 Hz, H-14), 2.44 (1H, br t, J = 2.5 Hz),

2.39 (1H, dd, J = 5.0, 13.0 Hz), 2.11 (3H, s, 16-OCOCH3), 1.03 (3H, s, Me-17), 0.99 (3H, s, Me-19), 0.95 (3H, s, Me-18); (+)-LRESIMS m/z 399.2 [M+Na]+.

111 21 111 (–)-Macfarlandin D (1.145): amorphous solid (0.8 mg); [α] D – 54 (c 0.07, CHCl3), lit.: [α]D – 1 169 (c 1.2, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.12 (1H, s), 5.75 (1H, d, J = 1.9 Hz), 5.33 (1H, br t, J = 3.5 Hz), 3.06 (1H, dd, J = 6.3, 19.9 Hz), 2.64 (1H, d, J = 19.9Hz), 2.62 (1H, m), 2.45 (1H, t,

J = 2.7 Hz), 2.22 (1H, br q, J = 5.7 Hz), 2.10 (3H, s, OCOCH3), 2.04 (2H, m), 1.75, (1H, m), 1.69, (1H, m), 1.48, (1H, br d, J = 13.3 Hz), 1.36 (1H, ddd, J = 7.0, 8.5, 12.5 Hz), 1.16 (1H, dt, J = 4.9, 12.9 Hz), 1.04 (3H, d, J = 7.0 Hz), 0.88 (3H, s, Me), 0.87 (3H, s, Me), 0.83 (3H, s, Me); (+)-LRESIMS m/z 399.2 [M + Na]+.

99, 115 99, 115 1 Dendrillolide C (1.146): colorless oil (0.2 mg); lit: [α]D + 133 (c 0.3, CHCl3); H NMR

(CDCl3, 500 MHz) δ 6.36 (1H, d, J = 6.8 Hz), 6.19 (1H, d, J = 1.6 Hz), 4.85 (1H, d, J = 2.2 Hz), 4.57 (1H, d, J = 2.2 Hz), 3.68 (1H, m), 2.84 (1H, dd, J = 9.1, 18.3 Hz), 2.79 (1H, m), 2.55 (1H, m), 2.34 (1H, dd, J = 95.6, 13.0 Hz), 1.99 (1H, q, J = 9.4 Hz), 1.86 (1H, td, J = 1.8, 12.5 Hz), 1.01 (3H, s), 0.96 (3H, s), 0.89 (3H, s); (+)-LRESIMS m/z 339.2 [M+Na]+.

100, 115, 279 100, 115 1 Cheloviolene A (1.148): colorless oil (0.1 mg); lit: [α]D + 53 (c 0.11, CHCl3); H

NMR (CDCl3, 500 MHz) δ 6.07 (1H, d, J = 6.8 Hz), 6.19 (1H, d, J = 1.6 Hz), 4.85 (1H, d, J = 2.2 Hz), 4.57 (1H, d, J = 2.2 Hz), 3.68 (1H, dddd, J = 8.8,6.4, 3.9, 1.7 Hz), 2.84 (1H, dd, J = 18.3, 9.1 Hz), 2.79 (1H, dd, J = 18.3, 3.9 Hz), 2.55 (1H, d, J = 7.9 Hz), 2.34 (1H, dd, J = 13.0, 5.6 Hz), 1.99 (1H, q, J = 9.4 Hz), 1.86 (1H, ddd, J = 13.1, 8.4, 4.7 Hz), 1.81 –1.71 (4H, m), 1.70 –1.63 (1H, m),

198

Appendices 1.63 –1.56 (1H, m), 1.41 –1.35 (1H, m), 1.25 (1H, dd, J = 10.8, 3.8 Hz), 1.01 (3H, s), 0.96 (3H, s), 0.89 (3H, s); (+)-LRESIMS m/z 357.2 [M+Na]+.

100, 115 100, 115 1 Cheloviolene B (1.149): colorless oil (0.1 mg); lit: [α]D + 26 (c 0.7, CHCl3); H NMR

(CDCl3, 500 MHz) δ 6.09 (1H, d, J = 6.0 Hz), 5.64 (1H, s), 4.83 (1H, d, J = 2.0 Hz), 4.61 (1H, d, J = 2.0 Hz), 2.98 (1H, dddd, J = 11.4, 5.9, 3.1, 2.1 Hz), 2.85 (1H, br s), 2.92 (1H, dd, J = 17.6, 11.1 Hz), 2.65 (1H, d, J = 8.8 Hz), 2.61 (1H, dd, J = 17.5, 2.9 Hz), 2.35 (1H, dd, J = 13.3, 5.8 Hz), 2.24 (1H, d, J = 1.5 Hz), 1.99 (1H, dt, J = 12.0, 8.2 Hz), 1.83 (1H, dd, J = 13.1, 2.3 Hz), 1.80 –1.72 (2H, m), 1.72 –1.64 (1H, m), 1.60 (1H, dd, J = 13.9, 4.1 Hz), 1.57 –1.52 (1H, m), 1.51 –1.46 (1H, m), 1.43 –1.34 (1H, m), 1.25 (1H, dt, J = 14.3, 3.3 Hz), 0.99 (3H, s), 0.94 (3H, s), 0.76 (3H, s); (+)-LRESIMS m/z 357.2 [M+Na]+.

34 34 (+)-12-Acetoxydendrillolide A (1.152): colorless oil (1.8 mg); [α]D + 3 (c 0.13, CHCl3), lit.: [α]D 1 = +24 (c 0.07 CHCl3); H NMR (CDCl3, 500 MHz) δ 6.46 (1H, d, J = 6.3 Hz, H-15), 6.07 (1H, d, J = 4.7 Hz, H-16), 5.49 (1H, d, J = 8.6 Hz, H-12), 4.82 (1H, d, J = 2.2 Hz, H-20a), 4.58 (1H, J = 2.2 Hz, H-20b), 3.36 (1H, m, H-13), 2.77 (1H, t, J = 6.7 Hz, H-14), 2.69 (2H, br d, J = 6.7 Hz, H-9),

2.37 (1H, m, H-1a), 2.17 (3H, s, 12-OCOCH3), 2.12 (3H, s, 15-OCOCH3), 1.80 (1H, m, H-1b), 1.75 (1H, m, H-5), 1.74 (1H, m, H-6/H-7a), 1.72 (1H, m, H-2a), 1.57 (1H, m, H-3a), 1.47 (1H, m, H-7b), 1.36 (1H, m, H-2b), 1.27 (1H, m, H-3b), 0.97 (3H, s, Me-17), 0.94 (3H, s, Me-19), 0.92 (3H, s, Me- 18); (+)-LRESIMS m/z 457.2 [M+Na]+.

117 117 1 Polyrhaphin A (1.153): colorless oil (0.3 mg); lit: [α]D – 23 (c 0.80, CHCl3); H NMR (CDCl3, 500 MHz) δ 5.69 (1H, d, J = 10.5 Hz), 4.87 (1H, d, J = 1.4 Hz), 4.61 (1H, d, J = 1.4 Hz), 4.56 (1H, dd, J = 3.2, 11.8 Hz), 4.49 (1H, dd, J = 8.8, 9.1 Hz), 4.04 (1H, dd, J = 9.1, 10.0 Hz), 3.94 (1H, dd, J = 9.6, 11.8 Hz), 3.19 (1H, m), 2.54 (1H, dd, J = 8.7 Hz), 2.35 (1H, s), 2.14 (3H, s), 2.08 (3H, s), 0.98 (3H, s), 0.95 (3H, s), 0.84 (3H, s); (+)-LRESIMS m/z 443.2 [M+Na]+.

117 117 1 Polyrhaphin B (1.154): colorless oil (4.7 mg); lit.: [α]D + 54 (c 0.40, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.27 (1H, d, J = 3.7 Hz), 5.19 (1H, d, J = 6.2 Hz), 4.81 (1H, d, J = 2.0 Hz), 4.57 (1H, d, J = 2.0 Hz), 4.02 (1H, dd, J = 5.5, 8.8 Hz), 3.88 (1H, dd, J = 6.2, 8.8 Hz), 3.77 (3H, s), 2.85 (1H, m), 2.74 (1H, d, J = 8.7 Hz), 2.56 (1H, dd, J = 3.7, 6.8 Hz), 2.35 (1H, m), 2.15 (3H, s), 2.07 (3H, s), 1.02 (3H, s), 0.95 (3H, s), 0.94 (3H, s); (+)-LRESIMS m/z 473.2 [M+Na]+.

118 118 1 Shahamin C (1.157): colorless oil (0.3 mg); lit.: [α]D + 81 (c 0.1, CHCl3),; H NMR (CDCl3, 500 MHz) δ 5.44 (1H, d, J = 7.2 Hz, H-12β), 4.88 (1H, d, J = 1.9 Hz, H-20E), 4.62 (1H, d, J = 1.9 199

Appendices Hz, H-20Z), 4.39 (1H, dd, J = 6.9, 12.1 Hz, H-15α), 4.32 (1H, dd, J = 4.1, 11.3 Hz, H-16β), 4.27 (1H, dd, J = 12.1, 12.3 Hz, H-15β), 4.16 (1H, dd, J = 2.9, 11.3 Hz, H-16α), 2.78 (1H, d, J = 8.8 Hz, H- 12β), 2.68 (1H, dddd, J = 2.9, 3.9, 4.1, 7.2 Hz, H-13), 2.36 (1H, m, H-1α), 2.30 (1H, ddd, J = 3.9, 6.9,

12.3 Hz, H-14), 2.22 (3H, s, 12-OCOCH3), 2.07 (1H, s, 16-OCOCH3), 1.93 (1H, dt, J = 8.8, 10.0 Hz, H-5α), 1.82 (1H, m, H-1β), 1.78 (2H, m, H-2β, H-6β), 1.75 (1H, m, H-7α), 1.61 (1H, m, H-3β), 1.53 (1H, m, H-7β), 1.41 (1H, m, H-2α), 1.24 (1H, m, H-3α), 1.01 (3H, s, Me-17), 0.96 (3H, s, Me-18), 0.88 (3H, s, Me-19); (+)-LRESIMS m/z 443.2 [M+Na]+.

118 1 (–)-15,16-Diacetoxyshahamin B (1.160): colorless oil (0.2 mg); [α]D – 10 (c 0.03, CHCl3); H

NMR (CDCl3, 500 MHz) δ 6.37 (1H, s), 4.82 (1H, d, J = 2.7 Hz), 4.69 (1H, d, J = 2.7 Hz), 4.51 (1H, d, J = 7.1 Hz), 4.31 (1H, dd, J = 7.1, 10.8 Hz), 4.25 (1H, dd, J = 2.7, 10.8 Hz), 4.25 (1H, dd, J = 2.7, 10.8 Hz), 3.78 (3H, s), 2.74 (1H, m), 2.66 (1H, d, J = 8.8 Hz), 2.11 (3H, s), 2.07 (3H, s), 0.99 (3H, s), 0.94 (3H, s), 0.85 (3H, s); (+)-LRESIMS m/z 487.2 [M+Na]+.Note: no optical rotation recorded in literature.

113 113 (+)-12-Desacetoxyshahamin C (1.162): colorless oil (0.4 mg); [α]D + 4 (c 0.03, CHCl3), lit: 1 [α]D + 54 (c 0.44, CHCl3); H NMR (CDCl3, 500 MHz) δ 4.86 (1H, d, J = 2.0 Hz, H-20E), 4.63 (1H, d, J = 2.0 Hz, H-20Z), 4.32 (1H, dd, J = 6.1, 11.8 Hz, H-15), 4.21 (1H, dd, J = 10.0, 11.8 Hz, H-15), 4.19 (1H, dd, J = 4.3, 11.2 Hz, H-16), 3.84 (1H, dd, J = 7.8, 11.2 Hz, H-16), 2.73 (1H, d, J = 8.7 Hz,

H-9), 2.55 (2H, m, H2-12), 2.48 (1H, m, H-13), 2.36 (1H, m, H-1), 2.08 (3H, m, 16-OCOCH3), 1.92 (1H, d, J = 8.6 Hz, H-9), 1.00 (3H, s, Me-19), 0.95 (3H, s, Me-18), 0.92 (3H, s, Me-17); (+)-LRESIMS m/z 385.2 [M+Na]+.

113 113 (+)-12-Desacetoxypolyrhaphin A (1.163): colorless oil (0.6 mg); [α]D + 5 (c 0.09, CHCl3), lit.: 1 [α]D + 14 (c 0.23, CHCl3),; H NMR (CDCl3, 500 MHz) δ 4.84 (1H, d, J = 1.7 Hz), 4.61 (1H, d, J = 1.7 Hz), 4.43 (1H, t, J = 8.8 Hz), 4.38 (1H, dd, J = 4.0, 11.8 Hz), 4.10 (1H, t, J = 8.8 Hz), 3.99 (1H, dd, J = 9.1, 11.8 Hz), 3.07 (1H, m), 2.56 (1H, d, J = 8.3 Hz), 2.43 (1H, d, J = 9.7 Hz), 2.34 (1H, m), 2.04 (3H, s), 1.85 (1H, m), 0.96 (3H, s), 0.92 (3H, s), 0.80 (3H, s); (+)-LRESIMS m/z 385.2 [M+Na]+.

108 108 1 Secoshahamin (1.164): colorless oil (0.2 mg); lit: [α]D + 10.6 (c 0.2, CHCl3); H NMR (CDCl3, 500 MHz) δ 4.91 (1H, d, J = 1.9 Hz, H-20a), 4.82 (1H, br s, H-20b), 4.36 (1H, dd, J = 4.0, 11.6 Hz, H-15a), 4.07 (1H, dd, J = 8.3, 10.8 Hz, H-16a), 4.02 (1H, dd, J = 6.8, 10.8 Hz, H-16b), 3.98 (1H, dd,

J = 9.7, 11.6 Hz, H-15b), 3.68 (3H, s, 12-CO2CH3), 2.94 (1H, br q, J = 8.3 Hz, H-13), 2.83 (1H, d, J = 8.8 Hz, H-13), 2.52 (1H, dd, J = 2.2, 15.8 Hz, H-12a), 2.34 (1H, br d, J = 12.6 Hz, H-1a), 2.21 (1H, dd, J = 11.0, 15.8 Hz, H-12b), 2.07 (3H, s, 16-OCOCH3), 2.05 (3H, s, 15-OCOCH3), 1.95 (1H, m, H- 200

Appendices 5), 1.84 (1H, br d, J = 9.7 Hz, H-14), 1.82 (1H, m, H-1b), 1.74 (1H, m, H-2a), 1.70 (1H, m, H-6a), 1.67 (1H, m, H-7a), 1.64 (1H, m, H-3a), 1.62 (1H, m, H-6b), 1.51 (1H, m, H-7b), 1.37 (1H, m, H- 2b), 1.25 (1H, m, H-3b), 1.01 (3H, s, Me-17), 0.95 (3H, s, Me-18), 0.85 (3H, s, Me-19); (+)-LRESIMS m/z 459.2 [M+Na]+.

111 111 1 Macfarlandin C (1.165): colorless oil (0.4 mg); lit.: [α]D –29 (c 0.75, CHCl3); H NMR

(CDCl3, 500 MHz) δ 6.53 (1H, d, J = 7.2 Hz, H-15), 6.04 (1H, d, J = 4.0 Hz, H-16), 5.32 (1H, s, H- 1), 3.03 (1H, m, H-13), 2.81 (1H, t, J = 6.7 Hz, H-14), 2.74 (1H, dd, J = 9.9, 17.3 Hz, H-H-12a),

2.55 (1H, dd, J = 9.0, 17.3 Hz, H-12b), 2.10 (3H, s, 15-OCOCH3,), 2.02 (2H, m, H2-2), 1.90 (1H, m, H-9), 1.72 (1H, m, H-6a), 1.63 (1H, m, H-7a), 1.59 (1H, m, H-7b), 1.40 (1H, m, H-5), 1.36 (1H, m, H-3a), 1.24 (1H, m, H-6b),1.16 (1H, m, H-3b), 1.01 (3H, d, J = 7.0 Hz, Me-20), 0.88 (3H, s, Me- 18), 0.84 (3H, s, Me-19), 0.82 (3H, s, Me-17); (+)-LRESIMS m/z 399.2 [M + Na]+.

118 118 1 Shahamin F (1.166): colorless oil (0.2 mg); lit: [α]D – 49 (c 0.001, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.04 (1H, s, H-16β), 5.62 (1H, dd, J = 1.0, 2.8 Hz, H-15α), 2.97 (1H, dd, J = 5.9, 19.7 Hz, H-12β), 2.68 (1H, dd, J = 2.8, 3.7 Hz, H-14), 2.66 (1H, br d, J = 19.7 Hz, H-12α), 2.47 (1H, ddd,

J = 1.0, 3.7, 5.9 Hz, H-13), 2.16 (1H, m, H-6β), 2.04 (3H, s, 16-OCOCH3), 2.02 (1H, m, H-1β), 1.98 (1H, m, H-1α), 1.77 (1H, br q, J = 6.9 Hz H-9α), 1.67 (1H, m, H-6α), 1.62 (1H, m, H-2β), 1.62 (1H, m, H-7β), 1.57 (1H, m, H-2α), 1.57 (1H, m, H-7α), 1.46, (1H, ddd, J = 3.9, 7.5, 11.9 Hz, H-3β), 1.46, (1H, ddd, J = 3.7, 7.8, 11.9 Hz, H-3α), 0.98 (3H, s, Me-17), 0.97 (3H, s, Me-19), 0.86 (3H, s, Me- 18), 0.94 (3H, d, J = 6.9 Hz, Me-20); (+)-LRESIMS m/z 399.2 [M + Na]+.

117 117 1 Polyrhaphin C (1.171): colorless oil (0.2 mg); lit: [α]D – 25 (c 0.30, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.15 (1H, s), 5.71 (1H, dd, J = 1.1, 2.7 Hz), 3.03 (1H, dd, J = 5.8, 19.6 Hz), 2.70 (1H, m), 2.68 (1H, br d, J = 19.6 Hz), 2.35 (1H, dd, J = 2.7, 2.8 Hz), 2.10 (3H, s, 15-OCOCH3), 1.07 (3H, s), 0.87 (3H, s), 0.85 (3H, s), 0.53 (1H, br s), 0.52 (1H, m), 0.25 (1H, dd, J = 5.6, 10.1 Hz); (+)- LRESIMS m/z 399.2 [M+Na]+.

113 113 1 Dendrillolide E (1.172): colorless oil (0.1 mg); lit: [α]D + 21 (c 0.25, CHCl3); H NMR (CDCl3, 500 MHz) δ 6.46 (1H, d, J = 5.5 Hz), 6.06 (1H, d, J = 4.8 Hz), 3.20 (1H, m), 3.05 (1H, dd, J = 8.9, 17.9 Hz), 2.57 (1H, dd, J = 2.7, 5.5 Hz), 2.54 (1H, dd, J = 8.9, 17.9 Hz), 2.07 (3H, s), 0.94 (3H, s), 0.92 (3H, s), 0.82 (3H, s), 0.47 (1H, br s), 0.44 (1H, m), 0.24 (1H, m); (+)-LRESIMS m/z 399.2 [M+Na]+.

201

Appendices Assorted spectra

1 Appendix 2. H NMR spectrum of 2.1 (700 MHz, CDCl3).

Appendix 3. HSQC spectrum of 2.1 (700 MHz, CDCl3).

202

Appendices

Appendix 4. HMBC spectrum of 2.1 (700 MHz, CDCl3).

Appendix 5. COSY spectrum of 2.1 (700 MHz, CDCl3).

203

Appendices

1 Appendix 6. H NMR spectrum of 2.2 (700 MHz, CDCl3).

Appendix 7. HSQC spectrum of 2.2 (700 MHz, CDCl3).

204

Appendices

Appendix 8. HMBC spectrum of 2.2 (700 MHz, CDCl3).

Appendix 9. COSY spectrum of 2.2 (700 MHz, CDCl3).

205

Appendices

Appendix 10. NOESY spectrum of 2.2 (700 MHz, CDCl3).

1 Appendix 11. H NMR spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube.

206

Appendices

Appendix 12. HSQC spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube.

Appendix 13. HMBC spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube.

207

Appendices

Appendix 14. COSY spectrum of 2.3 (700 MHz, CDCl3), collected in a Shigemi NMR tube.

1 Appendix 15. H NMR spectrum of 2.4 (700 MHz, CDCl3).

208

Appendices

Appendix 16. HSQC spectrum of 2.4 (700 MHz, CDCl3).

Appendix 17. HMBC spectrum of 2.4 (700 MHz, CDCl3).

209

Appendices

Appendix 18. COSY spectrum of 2.4 (700 MHz, CDCl3).

Appendix 19. NOESY spectrum of 2.4 (700 MHz, CDCl3).

210

Appendices

1 Appendix 20. H NMR spectrum of 2.5 (700 MHz, CDCl3).

Appendix 21. HSQC spectrum of 2.5 (700 MHz, CDCl3).

211

Appendices

Appendix 22. HMBC spectrum of 2.5 (700 MHz, CDCl3).

Appendix 23. COSY spectrum of 2.5 (700 MHz, CDCl3).

212

Appendices

Appendix 24. NOESY spectrum of 2.5 (700 MHz, CDCl3).

1 Appendix 25. H NMR spectrum of 2.6 (700 MHz, CDCl3).

213

Appendices

Appendix 26. HSQC spectrum of 2.6 (700 MHz, CDCl3).

Appendix 27. HMBC spectrum of 2.6 (700 MHz, CDCl3).

214

Appendices

Appendix 28. COSY spectrum of 2.6 (700 MHz, CDCl3).

Appendix 29. NOESY spectrum of 2.6 (700 MHz, CDCl3).

215

Appendices

1 Appendix 30. H NMR spectrum of 2.7 (700 MHz, CDCl3).

Appendix 31. HSQC spectrum of 2.7 (700 MHz, CDCl3).

216

Appendices

Appendix 32. HMBC spectrum of 2.7 (700 MHz, CDCl3).

Appendix 33. COSY spectrum of 2.7 (700 MHz, CDCl3).

217

Appendices

Appendix 34. NOESY spectrum of 2.7 (700 MHz, CDCl3).

1 Appendix 35. H NMR spectrum of 2.8 (700 MHz, CDCl3).

218

Appendices

Appendix 36. HSQC spectrum of 2.8 (700 MHz, CDCl3).

Appendix 37. HMBC spectrum of 2.8 (700 MHz, CDCl3).

219

Appendices

Appendix 38. COSY spectrum of 2.8 (700 MHz, CDCl3).

1 Appendix 39. H NMR spectrum of 2.9 (700 MHz, CDCl3).

220

Appendices

Appendix 40. HSQC spectrum of 2.9 (500 MHz, CDCl3).

Appendix 41. HMBC spectrum of 2.9 (700 MHz, CDCl3).

221

Appendices

Appendix 42. COSY spectrum of 2.9 (500 MHz, CDCl3).

Appendix 43. NOESY spectrum of 2.9 (700 MHz, CDCl3).

222

Appendices

1 Appendix 44. H NMR spectrum of 2.10 (500 MHz, CDCl3).

Appendix 45. HSQC spectrum of 2.10 (500 MHz, CDCl3).

223

Appendices

Appendix 46. HMBC spectrum of 2.10 (500 MHz, CDCl3).

Appendix 47. COSY spectrum of 2.10 (500 MHz, CDCl3).

224

Appendices

Appendix 48. NOESY spectrum of 2.10 (500 MHz, CDCl3).

1 Appendix 49. H NMR spectrum of 2.11 (700 MHz, CDCl3).

225

Appendices

Appendix 50. HSQC spectrum of 2.11 (700 MHz, CDCl3).

Appendix 51. HMBC spectrum of 2.11 (700 MHz, CDCl3).

226

Appendices

Appendix 52. COSY spectrum of 2.11 (700 MHz, CDCl3).

Appendix 53. NOESY spectrum of 2.11 (700 MHz, CDCl3).

227

Appendices

1 Appendix 54. H NMR spectrum of 2.12 (700 MHz, CDCl3).

Appendix 55. HSQC spectrum of 2.12 (700 MHz, CDCl3).

228

Appendices

Appendix 56. HMBC spectrum of 2.12 (700 MHz, CDCl3).

Appendix 57. COSY spectrum of 2.12 (700 MHz, CDCl3).

229

Appendices

Appendix 58. NOESY spectrum of 2.12 (700 MHz, CDCl3).

1 Appendix 59. H NMR spectrum of 2.13 (700 MHz, CDCl3).

230

Appendices

Appendix 60. HSQC spectrum of 2.13 (700 MHz, CDCl3).

Appendix 61. HMBC spectrum of 2.13 (700 MHz, CDCl3).

231

Appendices

Appendix 62. COSY spectrum of 2.13 (700 MHz, CDCl3).

Appendix 63. NOESY spectrum of 2.13 (700 MHz, CDCl3).

232

Appendices

1 Appendix 64. H NMR spectrum of 3.1 at 318 K (700 MHz, CDCl3).

Appendix 65. HSQC spectrum of 3.1 at 318 K (700 MHz, CDCl3).

233

Appendices

Appendix 66. HMBC spectrum of 3.1 at 318 K (700 MHz, CDCl3).

Appendix 67. gCOSY spectrum of 3.1 at 318 K (700 MHz, CDCl3).

234

Appendices

Appendix 68. TOCSY spectrum of 3.1 at 318 K (700 MHz, CDCl3).

Appendix 69. NOESY spectrum of 3.1 at 318 K (700 MHz, CDCl3).

235

Appendices

323 K 298 K 288 K 278 K 273 K 268 K

263 K 258 K 253 K 243 K 233 K 223 K

Appendix 70. Overlay of 1H NMR spectra for 3.1 in the temperature range 323 K to 223 K (500 MHz, CDCl3).

298 K 278 K 268 K

263 K

253 K

243 K

233 K

223 K 213 K 203 K

193 K

Appendix 71. Overlay of 1H NMR spectra for 3.1 in the temperature range 298 K to 193 K (500 MHz, d6-acetone).

236

Appendices

1 Appendix 72. H NMR spectrum of 3.2 at 318 K (700 MHz, CDCl3).

Appendix 73. HSQC spectrum of 3.2 at 318 K (700 MHz, CDCl3).

237

Appendices

Appendix 74. HMBC spectrum of 3.2 at 318 K (700 MHz, CDCl3).

Appendix 75. gCOSY spectrum of 3.2 at 318 K (700 MHz, CDCl3).

238

Appendices

Appendix 76. TOCSY spectrum of 3.2 at 318 K (700 MHz, CDCl3).

Appendix 77. NOESY spectrum of 3.2 at 318 K (700 MHz, CDCl3).

239

Appendices

323 K 298 K 288 K 278 K 273 K 268 K

263 K 258 K 253 K 243 K 233 K 223 K

Appendix 78. Overlay of 1H NMR spectra for 3.2 in the temperature range 323 K to 223 K (500 MHz, CDCl3).

323 K 298 K 288 K 278 K 273 K 268 K

263 K 258 K 253 K 243 K 233 K 223 K

Appendix 79. Overlay of 1H NMR spectra for dendrillolide A (1.142) in the temperature range 323 K to 223 K (500 MHz, CDCl3).

240

Appendices

1 Appendix 80. H NMR spectrum of 3.3 (700 MHz, CDCl3).

Appendix 81. HSQC spectrum of 3.3 (700 MHz, CDCl3).

241

Appendices

Appendix 82. HMBC spectrum of 3.3 (700 MHz, CDCl3).

Appendix 83. COSY spectrum of 3.3 (700 MHz, CDCl3).

242

Appendices

Appendix 84. NOESY spectrum of 3.3 (700 MHz, CDCl3).

1 Appendix 85. H NMR spectrum of 3.4 (700 MHz, CDCl3).

243

Appendices

Appendix 86. HSQC spectrum of 3.4 (700 MHz, CDCl3).

Appendix 87. HMBC spectrum of 3.4 (700 MHz, CDCl3).

244

Appendices

Appendix 88. COSY spectrum of 3.4 (700 MHz, CDCl3).

Appendix 89. NOESY spectrum of 3.4 (700 MHz, CDCl3).

245

Appendices

1 Appendix 90. H NMR spectrum of 3.5 (700 MHz, CDCl3).

Appendix 91. HSQC spectrum of 3.5 (500 MHz, CDCl3).

246

Appendices

Appendix 92. HMBC spectrum of 3.5 (700 MHz, CDCl3).

Appendix 93. COSY spectrum of 3.5 (500 MHz, CDCl3).

247

Appendices

Appendix 94. NOESY spectrum of 3.5 (700 MHz, CDCl3).

1 Appendix 95. H NMR spectrum of 3.6 (700 MHz, CDCl3).

248

Appendices

Appendix 96. HSQC spectrum of 3.6 (700 MHz, CDCl3).

Appendix 97. HMBC spectrum of 3.6 (700 MHz, CDCl3).

249

Appendices

Appendix 98. COSY spectrum of 3.6 (700 MHz, CDCl3).

Appendix 99. NOESY spectrum of 3.6 (700 MHz, CDCl3).

250

Appendices

1 Appendix 100. H NMR spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3).

Appendix 101. HSQC spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3).

251

Appendices

Appendix 102. HMBC spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3).

Appendix 103. COSY spectrum of diastereomeric mixture of 4.1 and 4.2 (500 MHz, CDCl3).

252

Appendices

Appendix 104. NOESY spectrum of diastereomeric mixture of 4.1 and 4.2 (700 MHz, CDCl3).

1 Appendix 105. H NMR spectrum of 1.164 (700 MHz, CDCl3).

253

Appendices

Appendix 106. HSQC spectrum of 1.164 (700 MHz, CDCl3).

Appendix 107. HMBC spectrum of 1.164 (700 MHz, CDCl3).

254

Appendices

Appendix 108. COSY spectrum of 1.164 (700 MHz, CDCl3).

Appendix 109. NOESY spectrum of 1.164 (700 MHz, CDCl3).

255

Appendices

1 Appendix 110. H NMR spectrum of 4.3 (700 MHz, CDCl3).

Appendix 111. HSQC spectrum of 4.3 (700 MHz, CDCl3).

256

Appendices

Appendix 112. HMBC spectrum of 4.3 (700 MHz, CDCl3).

Appendix 113. COSY spectrum of 4.3 (700 MHz, CDCl3).

257

Appendices

Appendix 114. NOESY spectrum of 4.3 (700 MHz, CDCl3).

1 Appendix 115. H NMR spectrum of 4.4 (700 MHz, CDCl3).

258

Appendices

Appendix 116. HSQC spectrum of 4.4 (700 MHz, CDCl3).

Appendix 117. HMBC spectrum of 4.4 (700 MHz, CDCl3).

259

Appendices

Appendix 118. COSY spectrum of 4.4 (700 MHz, CDCl3).

Appendix 119. NOESY spectrum of 4.4 (700 MHz, CDCl3).

260

Appendices Appendix 120. Computational methods for 5,9-epoxydendrillolide A (3.1), 10- oxonordendrillolide A (3.2) and dendrillolide A (1.142) A conformational search was with the Monte Carlo Multiple Minimum (MCMM) using MacroModel (Schrodinger Inc). All conformers less then 3kcal/mol from the lowest energy conformer were then further optimized by DFT calculations using B3LYP/6-31G(d,p) method with IEFPCM chloroform solvent (Gaussian16W (Revision B.01)). A single point energy was calculated using M062X/6-31+g(d,p) with IEFPCM chloroform solvent. Duplicates and high energy conformers (> 3 kcalmol-1) were removed and the free energies were calculated using a single point calculations at M062X/6-31+g(d,p) with IEFPCM chloroform solvent and the vibrational frequencies where checked for a true minimum, i.e. no negative frequencies. The free energies were used to calculate the Boltzmann populations.

Appendix 121. Computational methods for 10,20-epoxydendrillolide A (3.4) A conformational search was with the Monte Carlo Multiple Minimum (MCMM) using MacroModel (Schrodinger Inc) for R-3.4a and S-3.4b isomers. All conformers less then 3kcal/mol from the lowest energy conformer were then further optimized by DFT calculations using B3LYP/6- 31G(d,p) method with IEFPCM chloroform solvent (Gaussian16W (Revision B.01)). A single point energy was calculated using M062X/6-31+g(d,p) with IEFPCM chloroform solvent. Duplicates and high energy conformers (>3kcal/mol) were removed and the free energies were calculated using a single point calculations at M062X/6-31+g(d,p) with IEFPCM chloroform solvent and the vibrational frequencies where checked for a true minimum, i.e. no negative frequencies. The free energies were used to calculate the Boltzmann populations. Unique structures after MM After DFT opt (<3 kcal/mol + removal of duplicates) R-3.4a 8 6 S-3.4b 12 5 The magnetic field tensors were calculated using mpw1pw91/6-311+g(2d,p) and wB97XD/6-

311+g(2d,p) for 1H and 13C respectively. The unscaled chemical shifts (δu) were computed using

TMS as reference standard according to δu= σ0-σx, where σx is the Boltzmann averaged magnetic field tensors (over all significantly populated conformations) and σ0 is the magnetic field tensors of

TMS computed at the same level of theory employed for σx. The scaled chemical shifts (δs) were computed as δs= δum − b, where m and b are the slope and intercept, respectively, resulting from a linear regression calculation on a plot of δu against δexp. The DP4 probabilities were calculated using the procedure of Sarotti et al. using their supplied spreadsheet using scaled shifts. The mean absolute error (MAE) of 3.4a compared to the experimental chemical shifts was 0.08 and 1.0 for proton and carbon respectively. The maximum deviations were 0.27 and 2.6 ppm.

261

Appendices 1H NMR chemical shift experimental and 13C NMR chemical shift experimental and Compound 3.4a Boltzmann populations calculated chemical shifts for R-3.4a and S- calculated chemical shifts for R-3.4a and S- Conformer delta kcal/mol Percent 3.4b. 3.4b. 3.4a-1 1.568 2.86 1 13 H Expt R-3.4a S-3.4b C Expt R-3.4a S -3.4b 3.4a-2 0.416 19.97 1a 2.06 2.07 1.76 1 37.2 37.3 35.5 3.4a-3 0.0784 35.30 1b 1.21 1.11 1.19 2 24.1 24.6 23.4 3.4a-4 2.090 1.18 2a 1.66 1.56 1.79 3 37.2 36.2 37.1 3.4a-5 0.000 40.30 2b 1.45 1.48 1.38 4 36.3 38.1 38.7 3.4a-7 2.765 0.38 3a 1.58 1.61 1.63 5 54.1 53.5 55.6 3b 1.32 1.27 1.25 6 27.2 27.5 27.7 Compound 3.4b Boltzmann populations 5 1.69 1.75 1.73 7 38.4 37.9 37.5 Conformer delta kcal/mol Percent 6a 1.69 1.70 1.75 8 47.7 49.9 49.2 3.4b-1 0.000 84.58 6b 1.69 1.62 1.72 9 53.4 53.4 55.0 3.4b-2 1.446 7.36 7a 1.62 1.65 1.67 10 61.2 61.3 60.0 3.4b-7 2.691 0.90 3.4b-8 1.705 4.76 7b 1.41 1.37 1.46 11 175.7 175.2 175.0 3.4b-12 2.110 2.40 9 1.73 1.92 1.87 12 29.1 30.3 29.9

12a 2.75 2.75 2.64 13 42.4 44.3 44.0 12b 2.67 2.63 2.41 14 56.2 57.4 57.1 Computed DP4 probabilities for the truncated compounds 3.4a and 3.4b. 13 3.05 3.04 3.08 15 96.9 98.9 99.0 3.4a 3.4b 14 2.7 2.73 2.77 16 104.8 103.0 103.2 15 6.38 6.11 6.04 17 24.1 23.3 23.8 DP4 + (Proton data only) 99.97% 0.03% 16 5.98 6.14 6.19 18 34.8 32.2 32.7 DP4 + (Carbon data only) 80.23% 19.77% 17 1.34 1.25 1.09 19 26.5 25.2 24.7 DP4+ (All data) 99.99% 0.01% 18 0.97 0.95 1.07 20 56.7 55.1 55.2

19 0.99 0.90 0.88 15-OCOCH3 169.5 169.5 169.3 20a 2.64 2.64 2.87 20b 2.4 2.52 2.51 15-OCOCH3 21.1 20.5 20.8

15-OCOCH3 2.09 2.32 2.29

262

Appendices Appendix 122. Computational methods for 15-dendrillactol (4.1) and 15-epidendrillactol (4.2). A Monte Carlo conformational search for four diastereomers (4.1a, 4.1b, 4.2a and 4.2b) was undertaken in gas phase with Merck Molecular Force Field (MMFF) using MacroModel v12. The selected conformers (< 5 kcal/mol of the global minimum, 4.1a: 5 conformers, 4.1b: 6 conformers, 4.2a: 5 conformers, and 4.2b: 10 conformers) were optimized by density functional theory (DFT) calculations at the B3LYP/6-31+G(d,p) level with chloroform solvent (IEF-PCM) using Gaussian software (G16W, ref below). A single point energy of the optimized conformers was calculated using M062X/6-31+G(d,p) with chloroform solvent (IEF-PCM) and duplicate conformers and conformers with <1% of the Boltzmann population removed. The free energies of the resulting diastereomers for 4.1a, 4.1b, 4.2a and 4.2b were calculated with M062X/6-31+G(d,p) with PCM implicit solvent model for chloroform were used to scale the calculated NMR parameters relative to their Boltzmann population and the vibrational frequencies where checked for a true minimum, i.e. no negative frequencies. The magnetic field tensors were calculated using B3LYP/6-311+G(d,p) and their DP4+probabilities calculated relative to the experimental chemical shifts using the Excel spreadsheet available for free at sarotti-nmr.weebly.com. It was found the 4.1a had a 100% probability for the major component and 4.2a had a 99.9% probability for the minor component.

The unscaled chemical shifts (δu) were computed using TMS as reference standard according to δu= σ0-σx, where σx is the Boltzmann averaged magnetic field tensors (over all significantly populated conformations) and σ0 is the magnetic field tensors of TMS computed at the same level of theory employed for σx. The scaled chemical shifts (δs) were computed as δs= δum − b, where m and b are the slope and intercept, respectively, resulting from a linear regression calculation on a plot of

δu against δexp. The DP4 probabilities were calculated and supported the DP4+ result that 4.1a had a 99.7% probability of represented the major component and 98.5% for 4.2a. The final NMR chemical shifts were calculated with mpw1pw91/6-311+G(2d,p) for proton NMR and wB97XD/6-311+g(2d,p) for carbon NMR with chloroform solvent (IEF-PCM). The magnetic field tensors were converted to scaled chemical shifts by the method outline above and the chemical shifts weighted by their Boltzmann populations for diastereomers 4.1a and 4.2a only. The mean absolute error (MAE) of 4.1a compared to the experimental chemical shifts was 0.07 and 1.5 for proton and carbon respectively. The maximum deviations were 0.30 and 3.6 ppm. While the 4.2a compared to the experimental chemical shifts was 0.09 and 1.5 for proton and carbon respectively. The maximum deviations were 0.24 and 2.8 ppm The coupling constants were also calculated using the method of Kutateladze et al. and key coupling constants (Hz) are shown in the tables below.

263

Appendices Carbon experimental and calculated chemical Proton experimental and calculated chemical shifts for the major diastereomer 4.1 compared shifts for the major diastereomer 4.1 compared with 4.1a, respectively. with 4.1a, respectively. Major compound δ Scaled Major compound δ Scaled 13C 4.1a 1H 4.1a 37.6 38.0 2.37 2.34 28.5 29.9 1.88 1.95 38.3 37.3 1.73 1.69 36.2 38.4 1.38 1.38 54.5 53.9 1.61 1.63 27.1 27.8 1.27 1.25 37.6 37.4 1.87 1.88 47.0 49.4 1.70 1.71 57.7 57.8 1.70 1.63 153.8 157.4 1.82 2.12 177.0 175.9 1.51 1.53 31.2 32.8 2.70 2.77 39.1 41.2 3.26 3.10 55.2 54.8 2.53 2.43 100.3 98.8 3.17 3.10 107.4 105.8 2.16 2.16 24.9 22.5 5.64 5.54 34.5 32.2 6.05 5.90 26.0 24.5 1.04 1.02 114.2 112.2 0.95 0.89 MAE 1.5 0.97 0.94 MAX 3.6 4.81 4.97 4.53 4.72 Computed DP4 probabilities for 4.1a, 4.1b, MAE 0.07 4.2a and 4.2b, compared with the experimental MAX 0.30 data for 4.1.

Major compound DP4+ DP4 Computed DP4 probabilities for 4.1a, 4.1b, 4.2a and 4.2b, compared with the experimental 4.1a 100% 99.7% data for 4.2. 4.2a 0.0% 0.0% Minor compound DP4+ DP4 4.1b 0.0% 0.3% 4.1a 0.0% 0.0%

4.2b 0.0% 0.0% 4.2a 99.9% 98.5%

4.1b 0.0% 0.0% 4.2b 0.1% 1.5%

264

Appendices Proton experimental and calculated chemical Carbon experimental and calculated chemical shifts for the minor diastereomer 4.2 compared shifts for the minor diastereomer 4.2 compared with 4.2a, respectively. with 4.2a, respectively. Minor compound δ Scaled Minor compound δ Scaled 1H 4.2a 13C 4.2a 2.20 2.29 37.6 37.5 1.82 1.87 28.5 30.0 1.72 1.68 38.4 37.0 1.38 1.34 36.2 38.5 1.72 1.62 54.4 53.4 1.46 1.22 26.8 27.1 1.72 1.84 37.8 37.8 1.74 1.71 46.6 49.1 1.74 1.69 56.2 56.3 1.72 1.92 153.5 156.1 1.38 1.39 176.2 174.7 2.88 2.95 29.7 30.7 2.72 2.60 41.9 44.0 2.55 2.39 56.2 55.4 3.15 3.01 99.7 97.6 2.35 2.36 104.1 102.3 5.63 5.61 24.1 22.3 6.06 5.92 34.5 32.0 0.96 0.88 26.0 24.5 0.94 0.87 114.7 111.9 0.97 0.91 MAE 1.5 4.82 4.95 MAX 2.8 4.59 4.73 MAE 0.09 Calculated coupling constants for 4.1a MAX 0.24 4.1a 12b 13 14 15 16 12a 18.5 7.5 - - - 12b - 10.8 - - - 13 - - 6.9 - 6.0 14 - - - 3.7 -

Calculated coupling constants for 4.2a

4.2a 12b 13 14 15 16 12a 17.1 10.5 - - - 12b - 9.2 - - - 13 - - 6.9 - 4.2

14 - - - 5.9 -

265

Appendices Appendix 123. Computational methods for secoshahamin (1.164) The truncated structures 4.5a and 4.5b, in which a t-butyl group replaced the conformationally flexible perhydroazulene ring, were selected for computational analysis. A Monte Carlo conformational search of the (13R, 14R)- 4.5a and (13S, 14R)- 4.5b diastereomers was undertaken with Merck Molecular Force Field (MMFF) using MacroModel. The conformers (< 5 kcal/mol of the global minimum, 4.5a: 43 conformers and 4.5b: 21 conformers) were further optimized by density functional theory (DFT) using Gaussian software. The initial DFT optimization was performed using B3LYP/6-31+G(d,p) level of theory with chloroform solvent (IEF-PCM) followed by a single point energy calculation using M062X/6-31+G(d,p) with chloroform solvent (IEF-PCM). The conformers that contributed less than 1% of the Boltzmann population, as determined from the M062X/6- 31+G(d,p) energy, were not used in subsequent calculations. The Gibbs free energies of the resulting 4.5a and 4.5b conformers, determined using B3LYP/6-31+G(d,p) with PCM implicit solvent model for chloroform, were used to calculate Boltzmann averaged chemical shifts. The vibrational frequencies were checked for a true minimum, i.e. no negative frequencies. The NMR calculations were performed using mpw1pw91/6-311+G(2d,p) with chloroform solvent (IEF-PCM) and the resulting magnetic field tensors were converted to chemical shifts using linear scaling (1H slope: - 1.0717, intercept: 31.8721 and 13C slope: -1.0417, intercept: 186.3455). The mean absolute error (MAE) values for 4.5a and 4.5b were then considered. For the 1H data, the MAE were 0.17 and 0.21 ppm for the 4.5a and 4.5b diastereomers, respectively, while the 13C MAE were 1.68 and 1.90 ppm for the 4.5a and 4.5b diastereomers, respectively. Although the MAE values were similar, there was a slight preference for the 4.5a diastereomer. When the Boltzmann-averaged 1H chemical shifts were examined using DP4 and considering both 1H and 13C chemical shifts, there was a high probability that the 4.5a diastereomer was preferred. This was primarily due to the large difference in the calculated chemical shifts for H-14 for the two diastereomers; the values for 4.5a and 4.5b were 1.82 ppm and 1.26 ppm, respectively, while the experimental value was 1.84 ppm. Using the 13C chemical shifts, the DP4 output resulted in a 4.5a: 4.5b probability of 77.5:22.5%. Although the 13C DP4 calculation showed a preference for the 4.5a isomer, the result was considered inconclusive as the probability value was < 80%. Using both 1H and 13C chemical shifts in the DP4 probability output resulted in a 99.97% preference for the 4.5a diastereomer over the 4.5b diastereomer. To further verify the proposed stereochemistry, the coupling constants for H-14 were calculated using the method of Kutateladze et al. The H-14 multiplet of 1.164 is best described as a broad doublet with a large coupling of 9.7 Hz. The calculated coupling constants for H-14 in 4.5a were 9.1, 3.5, and 0.9 Hz, while in 4.5b the values were 3.5, 1.5, 0.9, and 0.8 Hz. Therefore, this

266

Appendices provided further evidence to support the 13R, 14R diastereomer 4.5a as the preferred stereoisomer of secoshahamin (1.164).

Proton experimental and calculated chemical shifts for the truncated compounds (13R, 14R)-4.5a and (13S, 14R)-4.5b. 1H Expt. 4.5a 4.5b 12a 2.52 2.69 2.59 12 2.21 2.54 2.45 13 2.94 2.55 2.48 14 1.84 1.82 1.26 15a 4.36 4.24 4.41 15 3.98 4.18 4.20 16a 4.07 3.99 4.51 16 4.02 4.26 4.08

15-OCOCH3 2.05 2.13 2.08

16-OCOCH3 2.07 2.20 2.06

12-CO2CH3 3.68 3.56 3.57

267

Appendices Carbon experimental and calculated chemical shifts for the truncated compounds (13R, 14R)-4.5a and (13S, 14R)-4.5b. 13C Expt. 4.5a 4.5b 11 173.1 176.11 175.2 12 33.7 33.35 37.6 13 34 36.74 35.5 14 46.6 47.01 49.4 15 63.6 67.40 66.1 16 68.4 70.58 68.8

15-OCOCH3 21 19.95 19.5

15-OCOCH3 170.8 173.09 173.4

16-OCOCH3 21 20.10 19.6

16-OCOCH3 170.9 172.49 173.0

12-CO2CH3 51.7 51.82 51.7

Computed DP4 probabilities for the truncated compounds 4.5a and 4.5b. 4.5a 4.5b

DP4 + (Proton data only) 99.88% 0.12% DP4 + (Carbon data only) 77.52% 22.48% DP4+ (All data) 99.97% 0.03%

Appendix 124. Computational methods for shahamin L (4.3) The truncated structures (14S, 15R)-4.7a and (14S, 15S)-4.7b, in which a t-butyl group replaced the conformationally flexible perhydroazulene ring, were selected for computational analysis. A Monte Carlo Conformational search was performed using MacroModel (Schrodinger Inc) for diastereomers 4.7a and 4.7b. Torsional sampling (MCMM) was performed with 1000 steps per rotatable bond. Each step was minimized with the MMFF force field using TNCG method with maximum iterations of 50,000 and energy convergence threshold of 0.02. All other parameters were unchanged. This resulted in four and six conformers for the 4.7a and 4.7b diastereomers respectively for an energy cut off of < 5 kcal/mol from the lowest energy conformer. All conformations were optimized in Gaussian, using b3lyp/6-31+g(d,p) with PCM implicit solvent model for chloroform followed by a single point energy using M062X/6-31+g(d,p) with PCM implicit solvent model for chloroform. The duplicate structure were removed resulting in two and three conformers for 4.7a and 4.7b diastereomers respectively. The Boltzmann populations were calculated from the free energies calculated from a single point frequency calculation using M062X/6-31+g(d,p) with PCM implicit solvent model for chloroform and the vibrational frequencies where checked for a true minimum, i.e. no negative frequencies. 268

Appendices The NMR parameters (nmr=giao) were calculated with a single-point calculation (1H: mpw1pw91/6-311+g(2d,p) and 13C: wB97XD/6-311+g(2d,p) with PCM implicit solvent model for chloroform). The computed isotropic shielding constants were converted to chemical shifts by empirical scaling factors determined by the methods reported previously. For proton chemical shifts scaling factor were slope of -1.072 and intercept of 31.873 and carbon chemical shift were a slope of -1.0517 and intercept of 187.4476. The mean absolute error (MAE) values for 4.7a and 4.7b were then considered. For the 1H data, the MAE were 0.16 and 0.24 ppm for the 4.7a and 4.7b diastereomers, respectively, while the 13C MAE were 1.61 and 2.15 ppm for the 4.7a and 4.7b diastereomers, respectively. It was found for both 1H and 13C the 14S, 15R was found to be the favored diastereomer. The DP4 probabilities were calculated for both diastereomers resulting in a conclusive result of 99.97% probability for the 14S, 15R.

Proton experimental and calculated chemical shifts for the truncated compounds (14S, 15R)- 4.7a and (14S, 15S)- 4.7b. 1H Expt. 4.7a 4.7b 12 5.98 5.99 5.90 14 3.83 3.94 4.04 15 6.36 5.79 5.75 16a 4.54 4.54 4.26 16b 4.57 4.63 4.72

11-CH3 3.72 3.61 3.61

15-CH3 2.04 2.30 2.32 MAE 0.16 0.24

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Appendices Carbon experimental and calculated chemical shifts for the truncated compounds (14S, 15R)- 4.7a and (14S, 15S)- 4.7b. 13C Expt. 4.7a 4.7b 11 166.2 167.4 167.5 12 115.4 115.9 116.4 13 157.6 164.3 164.9 14 56.7 57.9 52.8 15 101.9 103.9 101.7 16 73.4 73.2 71.0

11-CH3 51.3 51.5 51.6 15-CO 170.2 172.2 171.8

15-CH3 21.5 21.1 20.1 MAE 1.61 2.15

Computed DP4 probabilities for the truncated compounds 4.7a and 4.7b. 4.7a 4.7b

DP4 + (Proton data only) 98.87% 1.13% DP4 + (Carbon data only) 97.22% 2.78% DP4+ (All data) 99.97% 0.03%

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Appendices Appendix 125 Microneutralization assay raw data for Ross River fever, Dengue fever and Influenza.

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