ISOLATION AND CHARACTERIZATION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETEROPHYLLUM WALL AND RELATED SPECIES BY Mr. HANIF AHMAD DOCTOR OF PHILOSPHY (PhD) IN CHEMISTRY DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2016 ISOLATION AND CHARACTERIZATION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETEROPHYLLUM WALL AND RELATED SPECIES BY Mr. HANIF AHMAD Thesis submitted to the Department of Chemistry, University of Malakand for the partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (PhD) IN CHEMISTRY DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2016 Declaration I declare that the thesis “ISOLATION AND CHARACTERIZTION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETROPHYLLUM WALL. AND RELATED SPECIES” is my original work and has never been presented for the award of any degree at any other university before and that all the information sources I have used and or quoted have been indicated and acknowledged by means of complete reference. Hanif Ahmad Certificate It is recommended that thesis submitted by Mr. HANIF AHMAD entitled “ISOLATION AND CHARACTERIZTION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETROPHYLLUM WALL. AND RELATED SPECIES” be accepted as fulfilling this part of the requirements for the degree of Doctor of Philosophy (PhD) in Chemistry. ______________________ _______________________ SUPERVISOR CO-SUPERVISOR Dr. Manzoor Ahmad Prof. Dr. Farzana Shaheen Associate Professor HEJ Research Institute of Chemistry ICCBS, University of Karachi ____________________ _______________________ EXTERNAL EXAMINER CHAIRMAN Department of Chemistry University of Malaknd Dedicated To My Beloved Mother Contents ACKNOWLEDGMENTS .................................................................................................... i Aims of the Study ........................................................................................................... iii SUMMARY .................................................................................................................... iv List of Tables .................................................................................................................. ix List of Figures ................................................................................................................. xi List of Schemes ............................................................................................................ xiv List of Abbreviations ..................................................................................................... xv INTRODUCTION ........................................................................................................... 1 1.1 Plants as a source of medicine ............................................................................... 1 1.2 Strategies in the search of new natural compounds ............................................... 3 1.3 Ranunculaceae ....................................................................................................... 5 1.3.1 The Genus Aconitum ....................................................................................... 5 1.3.2 Aconitum heterophyllum Wall. ........................................................................ 6 1.3.3 The Aconitum laeve Royle .............................................................................. 6 1.3.4 Systematic Position ......................................................................................... 7 1.3.5 Traditional importance of Aconitum Species .................................................. 7 1.3.6 Chemistry of the Genus Aconitum .................................................................. 8 1.3.7 Literature survey on A. heterophyllum Wall and A. laeve Royle. ................... 8 1.4 The Genus Delphinium .......................................................................................... 9 1.4.1 Systematic Position ....................................................................................... 10 1.4.2 Delphinium denudatum Wall. ....................................................................... 10 1.4.3 Ethnobotanical importance of the Genus Delphinium .................................. 11 1.4.4 Literature survey on Delphinium denudatum Wall. ...................................... 12 1.5 Diterpenoid alkaloids ........................................................................................... 27 1.6 Classification of diterpenoid alkaloids................................................................. 27 1.6.1 C20 diterpene alkaloids ................................................................................... 27 1.6.2 C19-diterpenoid alkaloids ............................................................................... 29 1.6.3 C18 diterpene alkaloids .................................................................................. 31 1.7 Biosynthesis of diterpenoid alkaloids .................................................................. 31 EXPERIMENTAL......................................................................................................... 36 2.1 General Experimental Conditions ........................................................................ 36 2.1.1 Physical constants ......................................................................................... 36 2.1.2 Spectroscopic techniques .............................................................................. 36 2.1.3 Column chromatography ............................................................................... 36 2.1.4 Materials for chromatography ....................................................................... 36 2.1.5 Solvents ......................................................................................................... 37 2.2 Plant materials ...................................................................................................... 37 2.2.1 Extraction ...................................................................................................... 37 2.2.2 Crude alkaloids fractionation ........................................................................ 38 2.2.3 Isolation of diterpenoids from A. heterophyllum Wall. ................................ 40 2.2.4 Isolation of diterpenoids from A. laeve Royle .............................................. 41 2.2.5 Isolation of diterpenoids from D. denudatum Wall ...................................... 43 2.3 X-Rays Diffraction Studies .................................................................................. 45 2.3.1 DFT Calculations .......................................................................................... 45 2.4 Cholinesterase inhibition assay and determination of IC50 .................................. 45 2.5 Experimental data of new diterpenoids from A. heterophyllum Wall. ................ 46 2.5.1 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1) ...................................... 46 2.5.2 1,11,13α, Trihydroxyl atisine (2) .................................................................. 47 2.5.3 2α, 6β Dihydroxy atisine (3) ......................................................................... 47 2.6 Experimental data of new diterpenoids from A. laeve Royle. ............................. 48 2.6.1 Swatinine-C (4) ............................................................................................. 48 2.6.2 Swatinine-D (5) ............................................................................................. 48 2.6.3 Methyl 2 acetamidobenzoate (6) ................................................................... 49 2.6.4 Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7) ....................... 49 2.7 Experimental data of new diterpenoids from D. denudatum Wall. ..................... 50 2.7.1 1β-hydroxy, 14β-acetyl condelphine (8) ....................................................... 50 2.7.2 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitane (9) ........ 50 2.7.3 1α,8,16,18β-Tetramethoxy, 14α-hydroxy-N-ethyl-5-aconitene (10) ............ 51 2.8 Experimental data of known diterpenoids from A. heterophyllum Wall. ............ 52 2.8.1 Isoatisine (11) ................................................................................................ 52 2.8.2 19-Epiisoatisine (12) ..................................................................................... 52 2.8.3 Atidine (13) ................................................................................................... 53 2.8.4 Heteratisine (14) ............................................................................................ 53 2.8.5 Hetisinone (15) .............................................................................................. 54 2.9 Experimental data of known diterpenoids from A. leave Royle. ......................... 55 2.9.1 Aconorine (16) .............................................................................................. 55 2.9.2 Lappaconitine (17) ........................................................................................ 55 2.10 Experimental data of known diterpenoids from D. denudatum Wall. .............. 56 2.10.1 Isotalatizidine hydrate (18) ........................................................................ 56 2.10.2 Dihydropentagynine (19) ........................................................................... 57 3.1 Diterpenoids isolated from A. heterophyllum Wall. ............................................ 58 3.2 New Diterpenoids from A. heterophyllum