Antiplasmodial Neolignans from Trema Orientalis – Identification, Synthesis and Analogue Generation

Town The copyright of this thesis rests with the University of Cape Town. No quotation from it or information derivedCape from it is to be published without full acknowledgement of theof source. The thesis is to be used for private study or non-commercial research purposes only. University Antiplasmodial Neolignans from Trema orientalis – Identification, Synthesis and Analogue Generation Town by Cape Pamishaof Pillay Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY UniversityIn the Department of Chemistry Faculty of Science UNIVERSITY OF CAPE TOWN November 2011 ABSTRACT Thesis Title: Antiplasmodial neolignans from Trema orientalis – Identification, synthesis and analogue generation Author: Pamisha Pillay Date of submission: 30 November 2011 In the continued search for new classes of antimalarial agents that are safe, affordable and effective against multi-drug resistant parasites, medicinal plants still provide a useful starting point. Trema orientalis, a widely distributed evergreen tree with various medicinal properties including the treatment of malaria, was investigated as a potential source of new antimalarial lead compounds. Organic extracts of the young growing twigs of T. orientalis were reproducibly shown to be active against the chloroquine-sensitive (D10) and chloroquine- resistant (K1) strains of Plasmodium falciparum. The 8-O-4' oxyneolignans, dadahols A and B, were identified as the major active compounds using two bioassay-guided fractionation approaches. The new accelerated “HPLC biogram” methodologyTown allowed for early recognition of the active compounds in the complex plant extract, requiring considerably less time and material compared to the classical reiterative approach. Cape Although the natural products were shown to have promising antiplasmodial activity (IC50 < 1 µg/ml) and selectivity (SI > 100) in vitro, theyof lacked antimalarial activity in vivo. This could be attributed to the poor pharmacokinetic (pK) properties of the compounds, which was confirmed by an in vivo pK study. Biomimetic oxidative coupling, ion exchange chromatography and carbonyldiimidazole (CDI) coupling were shown to be useful techniques in completing the diastereo-selective synthesis of dadahol B. TheUniversity 8-O-4´ neolignan core of dadahol B, i.e. guaiacyglycerol β-O-4-coniferyl alcohol ether (GGCE), lacked antiplasmodial activity identifying the p-coumaroyl moieties as key pharmacophores of the natural product. A by-product of the oxidative coupling step, dehydrodiconiferyl alcohol (DHCA), showed better initial activity than GGCE and was subsequently also used as a scaffold for analogue generation. Using a selection of synthetic techniques and approaches, a range of GGCE and DHCA analogues were prepared, focusing on optimization of the ester and aromatic ring substituents. None of the precursors, by-products or analogues showed improved antiplasmodial activity relative to the natural products or chloroquine, and were therefore not considered as potential antimalarial lead compounds. i ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my superviser, Prof. Kelly Chibale, for his invaluable supervision and guidance throughout the duration of this work. In addition, I wish to thank my co-supervisor Dr Vinesh Maharaj, for his all encompassing technical input and support. I gratefully acknowledge the National Research Foundation Innovation Fund Grant (Project 31313), EU AntiMal FP6 and the CSIR for funding and permission to present this work for degree purposes. Special mention must be made of my CSIR colleagues Dr Chris Parkinson, Dr Chris van der Westhuyzen, Xolani Peters, Dr Paul Steenkamp,Town Nial Harding, Dr Gerda Fouche, Tasmiyah Khan, Michele Enslin and Prenitha Sewnarain for their technical advice, input and support. I also humbly acknowledge my UCT colleagues Prof Pete Smith, Carmen DeKock, Lubbe Wiesner, andCape Yassir Adam for their contribution to biological assay and analytical data. of Heartfelt thanks to my family and friends for their continuous inspiration and encouragement. I dedicate this thesis to my parents, my brothers, and my beloved Paul, for their unwavering love and support. University ii ABBREVIATIONS 1H Proton (1H) 3H Tritium 2D Two-dimensional 13C Carbon-13 [α] Specific optical rotation Acetone-d6 Hexadeuterioacetone ACT Artemisinin based combination therapies ADME Absorption, distribution, metabolism and excretion APAD 3-Acetylpyridine adenine dinucleotide APADH Reduced APAD ATP Adenosine triphosphate BLQ Below limit of quantification BPI Base Peak Intensity Town BCE Before Common Era br Broad resonance br s Broad singlet oC Degrees Celcius Cape C Carbon c Concentration of CAD Cinnamyl alcohol dehydrogenase CAD‟s Cinnamic acid derivatives calcd. Calculated CCoA-OMT Caffeoyl-CoA 3-O-methyltransferase CDCl3 Deuterated chloroform CDI N,N'–carbonyldiimidazole CCR UniversityCinnamoyl-CoA reductase CE Common Era cf. Consult CHO Chinese Hamster Ovarian cell line cm Centimetre CNS Central Nervous System COSY Correlated spectroscopy conc. Concentrated CoA Coenzyme A COX Cyclooxygenase iii CSIR Council for Scientific and Industrial Research CQ Chloroquine CYP Cytochrome P450 d Doublet dd Doublet of doublets ddd Doublet of doublets of doublets ddt Doublet of doublets of triplets DAD Diode Array Detector DCM Dichloromethane DCC N, N'-dicyclohexylcarbodiimide DHCA Dehydrodiconiferyl alcohol DIAD Diisopropyl azodicarboxylate DIBAL Diisobutylaluminum hydride dil. Dilute DP Directing protein Town DEPT Distortionless Enhancement by Polarisation Transfer DHA dihydroartemisinin DHFR dihydrofolate reductase DHPS dihydropteroate synthetaseCape DIAD diisopropyl azodicarboxylate DMAP 4-dimethyaminopyridineof DMF Dimethylformamide DMSO Dimethyl Sulfoxide DMEM Dulbecos Modified Eagles Medium DNA Deoxyribonucleic acid DNP Dictionary of Natural Products dq Doublet of quartets dt UniversityDoublet of triplets E East equiv. Equivalents ES Electrospray ESI Electrospray ionisation FBS Fetal bovine serum FSA Flora of Southern Africa g Gram GGCE Guaiacyglycerol β-O-4-coniferyl alcohol g/L Grams per litre H Proton iv GPS Global Positioning System h Hour HEPES N-[2-hydroxyethyl]-piperazine-N‟-[2-ethansulphonic acid] HIV Human immunodeficiency virus HDMS High Definition Mass Spectrometry HMBC Heteronuclear Multiple Bond Correlation HPLC High-Performance Liquid Chromatography H3PO4 Phosphoric acid HRESI-MS High-Resolution Electrospray Ionisation – Mass Spectrometry HRMS High-Resolution Mass Spectrometry HRP Horseradish peroxidase HSQC Heteronuclear Single Quantum Coherence HR High Resolution Hz Hertz IC50 Inhibitory concentration at which 50% inhibitionTown is achieved i.e. That is J Spin-spin coupling constant kD Kilodalton kg Kilogram Cape Kb Basicity constant Km Concentration of substrateof that leads to half-maximal velocity kV Kilovolt L Litre LC Liquid chromatography LDH Lactate dehydrogenase linn. Linnaeus lit. Literature m UniversityMetres m Multiplet mm Millimetre m/z Mass-to-charge ratio Me Methyl mg Milligrams mg/L Milligrams per litre mg/kg Milligrams per kilogram MgSO4 Magnesium sulfate µCi Microcurie g/mL Micrograms per millilitre v ml Millilitre min Minute M Micro molar mmol Millimoles MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW Molecular weight NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NADPH Reduced form of nicotinamide adenine dinucleotide phosphate NBT Nitroblue tetrazolium nM Nano molar nm Nano meter NMR Nuclear Magnetic Resonance Spectroscopy NOESY Nuclear Overhauser Effect Spectroscopy OMT O-methyltransferase Town P. Plasmodium PAL Phenylalanine ammonia-lyase p Para PBS Phosphate-buffered salineCape PDA Photodiode array detector PES Phenazine ethosulphateof PI Post infection pK Pharmacokinetic PLCγl Phospholipase cystathionine γ lyase pLDH Parasite lactate dehydrogenase ppm Parts per million PPTS Pyridinium para-toluene sulfonate prep UniversityPreparative (HPLC) pH cLog of the activity of dissolved hydrogen ions pfCRT Plasmodium falciparum chloroquine-resistance transporter pLDH Parasite lactate dehydrogenase PPh3 Triphenylphosphine q Quartet (NMR) QAE Quaternary aminoethyl QTOF Quadrupole time-of-flight mass spectrometer RBC Red blood cells or erythrocytes RI Resistance Index Rf Retention factor vi rt Retention time r.t. Room temperature rpm Revolutions per minute RPMI Roswell Park Memorial Institute RSA Republic of South Africa S South s Singlet SANBI South African National Biodiversity Institute SAR Structure activity relationship SI Selectivity index SiO2 Silica gel SOMO Single-occupied molecular orbitals SP Pyrimethamine-sulfadoxine SPE Solid Phase Extraction spp. Species Town St Saint STZ Streptozotocin subsp. Subspecies Swiss TPH Swiss Tropical and Public CapeHealth Instit ute T. Trema T. Trypanosoma of t Triplet TBAF tetra-n-butylammonium fluoride TBS-Cl tert-butyldimethylsilyl chloride THF Tetrahydrofuran THP Tetrahydropyranyl TPP Triphenylphosphine TLC UniversityThin Layer Chromatography TMS Tetramethylsilane TRIS Tris(hydroxymethyl)aminomethane UCT University of Cape Town UGT UDP-glucuronosyltransferase Umg-1 Enzyme unit per milligram UPLC Ultra Performance Liquid Chromatography UVmax Maximum of the UV absorption UV Ultraviolet V Volume v. Version vii var. Variety viz. namely v/v Volume ratio VLC Vacuum Liquid Chromatography Vmax Maximum initial velocity/rate that enzyme catalyses reaction w/w Weight ratio WHO World Health Organisation 1 δH H chemical shift 13 δC C chemical shift Town Cape of University

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