The Synthesis, Isolation and Evaluation of Novel Anticancer and Antibacterial Therapeutics Derived from Natural Products NICHOLAS FRANCIS OMONGA School of Environment and Life Sciences University of Salford Submitted in partial Fulfilment of the Requirements of the Degree of Doctor of Philosophy 2020 i Table of Contents Page Table of Contents ............................................................................................................ .... ....i Table of Figures ................................................................................................................... ...vi Table of Structures ............................................................................................................... .viii Table of Tables .................................................................................................................... ...xi Acknowledgements ............................................................................................................. .xiii Declaration................. .......................................................................................................... .xiv Abbreviations........... ............................................................................................................ ..xv Abstract............................ ........................................................................................................ xviii Chapter 1 Introduction......................................................................................................................1 1.1 Cancer; an Overview ................................................................................................ ....1 1.1.1 What is cancer? ...................................................................................................... ....1 1.1.2 Cancer Epidemiology ................................................................................................ ....9 1.1.3 Cancer Treatment ..................................................................................................... ..15 1.2 Infectious Disease. ..................................................................................................... ..42 1.2.1 Microbial Infection ..................................................................................................... ..42 1.2.2 Antibacterial Drugs ................................................................................................... ..52 1.2.3 Fungal infection......................................................................................................... ..67 1.3 Phytochemicals as Anticancer and Antimicrobial Agents .............................................. ..71 1.3.1 The Mevalonate (MVA) and 2C-methyl-d-erythritol 4-phosphate (MEP) Pathway Provides Isoprenoid, the Building Block of Most Phytochemicals ................. ..71 1.3.2 Phenylpropanoid Pathway for Synthesis of Flavonoids ............................................. ..73 1.4 Inula Species .............................................................................................................. ..76 1.4.1 Traditional Uses of Inula Species .............................................................................. ..76 1.4.2 Isolated Metabolites from Inula ................................................................................. ..78 1.4.3 Pharmacological and Biological Activities.................................................................. ..87 1.4.4 Cytotoxic / Antitumour Activity ................................................................................... ..90 1.4.5 Antimicrobial Activity of Inula ..................................................................................... ..96 1.5 Flavonoids .................................................................................................................. ..99 1.5.1 Classification ............................................................................................................. 100 1.5.2 Pharmacological Activities of Flavonoids .................................................................. 102 1.5.3 Chrysin – a Flavonoid ............................................................................................... 109 1.5.4 7-O-Chrysin Derivatives Previously Synthesised ....................................................... 117 1.5.5 Justification / Rational behind the Synthesis of 7-O-Chrysin Derivatives .................... 118 1.5.6 Flavonoids in Clinical Use ......................................................................................... 118 1.6 Project Aims ............................................................................................................... 120 ii 1.6.1 Isolation Work ........................................................................................................... 120 1.6.2 Synthetic Work ........................................................................................................... 121 Chapter 2 Methodology.............................................................................. ……………………….122 2.1 Isolation and Purification of Natural Products Experimental ........................................ 122 2.1.1 Extraction of compounds from Inula helenium ........................................................... 122 2.1.2 Determination of extraction yield ........................................................................... .... 123 2.1.3 Statistical analysis ..................................................................................................... 124 2.1.4 High-performance liquid chromatography (HPLC) ..................................................... 124 2.2 Synthetic Chemistry of Chrysin derivatives Methodology ............................................ 124 2.2.1 Synthesis of 7-O-bromoalkylchrysin derivatives (C13-C17) ....................................... 125 2.2.2 Synthesis of 7-O-alkylchrysin Derivatives (C4-C12) .................................................. 126 2.2.3 Synthesis of Methylated 7-O-alkylchrysin derivatives ................................................ 127 2.2.4 Synthesis of other Chrysin Derivatives (C1-C3) ........................................................ 128 2.2.5 Determination of Hydrophilicity of Chrysin Derivatives .............................................. 130 2.3 Antimicrobial Studies Experimental ............................................................................ 131 2.3.1 Culture Media and Microbial Strain ........................................................................... 131 2.3.2 Bacterial Preparation................................................................................................ 131 2.3.3 Bacterial positive control ........................................................................................... 131 2.3.4 Agar well diffusion assay ........................................................................................... 132 2.3.5 Colorimetric assay for Minimum Inhibitory Concentration (MIC) ................................. 132 2.3.6 Screening for antifungal activity ................................................................................ 133 2.4 Anticancer Studies Experimental ................................................................................ 133 2.4.1 Cell lines ................................................................................................................... 133 2.4.2 Maintenance of cells ................................................................................................. 135 2.4.3 Splitting Matured Confluent Cells .............................................................................. 135 2.4.4 Dosing procedure ...................................................................................................... 135 2.4.5 Cell viability assay using MTT ................................................................................... 136 2.4.6 MTS Assay ............................................................................................................... 136 2.4.7 Cell Culture and dosing of samples used for Hypoxia, Normoxia, Hyperglycaemia and Normoglycaemia Studies .......................................................... 137 Chapter 3 Extraction and Purification of Bioactive Agents from Inula helenium.........................................................................................................138 3.1 Extracts from Inula helenium ................................................................................. 138 3.1.1 Extraction of compounds from Inula helenium ...................................................... 138 3.1.2 Centrifugation of compounds extracted from Inula helenium ..................................... 140 3.1.3 High-performance liquid chromatography (HPLC) ..................................................... 143 Chapter 4 Chemical Synthesis of Modified Chrysin Derivatives .............................................158 4.1 4-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)butyl acetate (C1) or 7- O-Chrysinbutyl acetate – novel compound ............................................................ 158 4.2 7-((3,5-Dimethylbenzyl)oxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (C2) or 7-O-Chrysin-3,5-dimethylbenzene – novel compound ........................................... 159 iii 4.3 7-(2,4-Dinitrophenoxy)-5-hydroxy-3-phenyl-4H-chromen-4-one (C3) or 7-O- Chrysin-2,4-dinitrobenzene – novel compound .....................................................