University of Bath PHD Phytochemistry of norditerpenoid alkaloids Saensuk, Pilan Award date: 2007 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 Phytochemistry of norditerpenoid alkaloids Pilan Saensuk A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Pharmacy and Pharmacology September 2007 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and they must not copy it or use material from it except as permitted by law or with the consent of the author. RESTRICTION ON USE This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. UMI Number: U491156 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U491156 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 BW——— TO muft1—nWn'^'Wr | i £ 2 L uii H C H | j : AHVbSn \ H ivado AUStBAmn | Abstract Norditerpenoid alkaloids have important biological activities. Many of them are potent ligands and therefore promising leads for novel selective antagonists and/or agonists of sub- types of nicotinic acetylcholine receptors (nAChR) and voltage-gated sodium channels. Aconitum, Consolida, and Delphinium are important sources of norditerpenoid alkaloids. This thesis is focussed on the chemistry of norditerpenoid alkaloids from these three genera, starting with a review of those recently isolated from Aconitum, Consolida, and Delphinium with brief aspects of taxonomy, biological activities, and modes of action. Selected aspects of the uses in traditional medicine of Delphinium and Aconitum are presented, including data on both toxicity and detoxification. In this thesis, chemical constituents of the seeds of Delphinium cultivar Pacific Giant and the seeds of Aconitum lycoctonum were investigated. To extract the crude alkaloidal material, Soxhlet extraction was used for Delphinium cv Pacific Giant seeds and room temperature extraction for A. lycoctonum seeds. The crude extracts were purified by repeated column chromatography (over silica and alumina gels), yielding five known norditerpenoid alkaloids from Delphinium cv Pacific Giant (delavaines A and B, delpheline, methyllycaconitine (MLA), and pacinine) and two from A. lycoctonum (lycaconitine and TV-succinylanthranoyl lycoctonine) which were analysed by detailed spectroscopic methods. X-Ray crystallographic analysis of aconitine, mesaconitine, lycoctonine, and delpheline was also studied. Three compounds were obtained from semi­ synthesis starting with MLA: lycoctonine by basic hydrolysis, inuline by acidic hydrolysis and by esterification of lycoctonine, and elatine by methylenedioxy acetal formation. These known alkaloids were structurally elucidated by a variety of spectroscopic techniques. MLA is a selective competitive antagonist at a7 sub-type nAChR. From collaborative studies, the results of biological activity for methyl esters delavaines A and B (a 3:2 mixture), delpheline, and pacinine (the B-ring C6-ketone of delpheline) are reported in this thesis. Their biological activities were determined in competitive a-bungarotoxin binding assays for a7 nAChR in rat brain membranes. Delavaines A and B were potent ligands (IC50 = 50 nM, cf MLA IC50 = ~l-2 nM), whereas delpheline and pacinine displayed only modest activity at a7 nAChR (IC50 = ~1 pM). After studying TV-ethylpiperidine as a model alkaloid, the pXa (a key physico-chemical parameter) of MLA was measured by ’H NMR as 7.15. A brief comparison with p Ka data reported in the literature is made across closely related norditerpenoid alkaloids. Acknowledgements My greatest appreciation goes to my supervisors Dr. Ian S. Blagbrough and Dr. Michael G. Rowan for their excellent supervision, good advice, and support without which this work would not have been possible. They devoted their best to helping me to learn techniques that I have never learnt before. I have had a good experience working with them. I thank Dr. Mary F. Mahon in the Department of Chemistry, University of Bath, for her help with the X-ray crystallographic analysis. I also thank Prof. Susan Wonnacott and Philip Livingstone in the Department of Biology and Biochemistry, University of Bath, for their provision of the biological activity testing data. I am grateful to Dr. Timothy J. Woodman, Department of Pharmacy and Pharmacology, University of Bath, for his help with the detailed NMR spectroscopy. I also acknowledge accurate mass spectrometry provision (B. K. Stein) from the EPSRC National Mass Spectrometry Service Centre, University of Wales Swansea, Swansea SA2 8PP and Russell Barlowe, Chris Cryer, and Alison Smith at the University of Bath. I thank the research and technical staff in the Department of Pharmacy and Pharmacology at the University of Bath: Jo Carter, Kevin Smith, and Don Perry. I also gratefully acknowledge the Royal Thai Government and the Collaborative Research Networks for financial support of this study. I would like to express my appreciation to people working in the 5W 3.14 laboratory: Dr. Ian Williams, Dr. Adrian Neal, Dr. Daniel Furkert, and Dr. Benjamin Greedy are people who have helped me when I have had problems with the chemistry. Dr. Noppadon Adjimatera and Sawanya Buranaphalin encouraged me a lot. Shi Li, Yuxi Chen, Yiqiang Li, Dr. Osama Ahmed, Moustafa Soltan, Hassan Ghonaim, and Soad Bayoumi also gave me encouragement and I am grateful. Finally, but not least, I would like to thank my family for their support and for encouraging me through these studies. Abbreviations °C degrees Celsius A angstrom Ac acetyl aq aqueous As anisoyl ‘Bu isobutyiyl Bz benzoyl Cl chemical ionisation Cn cinnamoyl COSY correlated spectroscopy cv cultivar 2D two dimensional DCM dichloromethane DEPT distortionless enhancement by polarization transfer El electron impact ESI electrospray ionisation Et ethyl FAB fast atom bombardment HMBC heteronuclear multiple-bond correlation HMQC heteronuclear multiple-quantum correlation HPLC high performance liquid chromatography HRMS high resolution mass spectrometry Hz hertz IR infrared J coupling constant LC-MS liquid chromatography-mass spectrometry LC-MS-MS liquid chromatography-tandem mass spectrometry M molar Me methyl MET protonated molecular ion mp melting point MS mass spectrometry MW relative molecular weight m/z mass over charge mAChR muscarinic acetylcholine receptors nAChR nicotinic acetylcholine receptors NMR nuclear magnetic resonance NOESY nuclear overhauser effect spectroscopy ppm parts per million 'Pr isopropyl RPC reversed phase chromatography rt room temperature TLC thin layer chromatography UV ultraviolet v/v volume by volume Vr veratroyl Contents Abstract ii Acknowledgements iii Abbreviations iv 1. Introduction 1 1.1. Definition of the norditerpenoid alkaloid class 1 1.2. Taxonomy of plants examined 3 1.2.1. Aconitum lycoctonum 3 1.2.2. Delphinium cv Pacific Giant 4 1.3. Occurrence of norditerpenoid alkaloids 5 1.3.1. Overview of the norditerpenoid alkaloid literature 5 1.3.2. Norditerpenoid alkaloids from Aconitum 6 1.3.3. Norditerpenoid alkaloids from Consolida 17 1.3.4. Norditerpenoid alkaloids from Delphinium 19 1.4. Biological activities and modes of action 25 1.4.1 Aconitum 25 1 A.2. Delphinium 29 1.4.3. Modes of action 32 1.4.4. Nicotinic pharmacophore 33 1.5. Aims 35 2. Experimental 36 2.1. General techniques and instrumentation, chemicals, reagents, and solvents 36 2.2. Extraction of the seeds of Delphinium cv Pacific Giant 38 2.2.1. Extraction of the seeds of Delphinium cv Pacific Giant (1989 harvest) 38 2.2.2. Extraction of the seeds of Delphinium cv Pacific Giant (1993 harvest) 39 2.2.3. Extraction of the seeds of Delphinium cv Pacific Giant (1993 harvest) at different pH values 42 2.3. Separation of crude extracts isolated from the seeds of Delphinium 43 2.3.1. TLC of the extracts of Delphinium seeds 43 2.3.2. Vacuum Liquid Chromatography 45 2.3.3. Flash column chromatography 46 2.3.4. Isolation of delpheline 1 47 2.3.5. Isolation of methyllyaconitine 4 48 2.3.6. Optimisation of MLA isolation 49 2.3.7. Isolation of pacinine 118 52 2.3.8. Attempted purifications of other alkaloids 53 2.3.9. Isolation of delavaines A 119 and B 120 55 2.4. Isolation of lycaconitine 121 and TV-succinylanthranoyl lycoctonine 122 56 vi 2.5. Basic hydrolysis of MLA to yield lycoctonine 6 58 2.6. Acidic hydrolysis of MLA to yield inuline 7 59 2.7.