The synthesis and evaluation of dual inhibitors of monoamine oxidase and catechol-O-methyltransferase I Engelbrecht orcid.org 0000-0001-8007-1594 Thesis submitted for the degree Doctor of Philosophy in Pharmaceutical Chemistry at the North-West University Promoter: Dr A Petzer Co-promoter: Prof JP Petzer Graduation May 2018 Student number: 21639159 The financial assistance of the Deutscher Akademischer Austausch Dienst (DAAD) and the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. ii Preface This thesis is submitted in article format and consists of three original research articles as separate entities. The written declaration from the co-authors of the research articles is included. All scientific research conducted for this thesis (synthesis of compounds, the biological evaluation of the synthesised, natural and library compounds, writing of the thesis as well as the articles presented) was conducted by Miss I. Engelbrecht at the North-West University, Potchefstroom campus. Guidance and assistance in the preparation of this thesis was provided by the promoter and co-promoter, Prof. Anél Petzer and Prof. Jacques Petzer. Mr. André Joubert and Dr. Johan Jordaan from the SASOL Centre for Chemistry, North-West University, recorded the NMR and MS spectra. Support with the HPLC purity analyses of the synthesised compounds was provided by Prof. Jan du Preez of the Analytical Technology Laboratory, North-West University. Support with the preparation of liver tissue for the COMT activity measurements was provided by Mrs. Sharlene Lowe of Pharmacen, North-West University. Assistance with the COMT activity measurements was provided by Miss Denise Prinsloo, fellow postgraduate student at Pharmaceutical Chemistry, North-West University. iii iv v Table of contents Preface iii Declaration iv Letter of permission v Table of contents vi List of figures and tables ix List of abbreviations xv Abstract xvii Chapter 1 - Introduction 1 1.1. General background 1 1.2. Structures known to inhibit MAO and COMT 4 1.2.1. Chalcones 4 1.2.2. Flavonoids, flavones and natural compounds 5 1.2.3. Nitrocatechol compounds 8 1.2.4. Bisubstrate inhibitors 10 1.3. Hypothesis of the study 11 1.4. Aims of the study 11 1.5. Summary 12 Bibliography 13 Chapter 2 – Literature overview 21 2.1. General background of Parkinson’s disease 21 2.2. The treatment of Parkinson’s disease 23 2.2.1. L-Dopa 24 2.2.2. Dopamine agonists 26 2.2.3. MAO inhibitors 27 2.2.4. COMT inhibitors 28 2.2.5. Adenosine A2A receptor antagonists 29 2.2.6. Anticholinergic therapy 29 2.2.7. Antidepressant drugs 30 2.2.8. Drug treatment and quality of life 32 2.3. Aetiology of Parkinson’s disease 32 2.3.1. General 32 2.3.2. Environmental factors 33 2.3.3. Endogenous toxins 34 vi 2.3.4. Genetic factors 34 2.3.5. Smoking and coffee consumption 37 2.3.6. Additional contributing factors to the development of Parkinson’s 38 disease 2.4. Mechanisms leading to neuronal death in Parkinson’s disease 38 2.4.1. Multiple mechanisms underlie Parkinson’s disease pathogenesis 38 2.4.2. Dopamine metabolism yields injurious by-products 39 2.4.3. Role of iron 40 2.4.4. Lowered antioxidant status and glutathione levels 40 2.4.5. The role of NOS 41 2.4.6. Protein deposition 42 2.4.7. Dysfunctional mitochondrial respiration 42 2.4.8. Monoamine oxidase may produce toxic metabolic by-products 43 2.4.9. 6-Hydroxydopamine toxicity as a model for cytotoxicity of 44 catecholamines 2.4.10. Potential toxicity of L-dopa 45 2.5. Metabolism of L-dopa and dopamine 45 2.6. A case for multi-target-directed inhibitors for Parkinson’s disease 48 2.7. The biology of MAO 50 2.7.1. General background 50 2.7.2. The “cheese reaction” 51 2.7.3. Tissue distribution of the MAOs 52 2.7.4. The catalytic mechanism of MAO 53 2.7.5. Expression systems of the MAOs 55 2.7.6. MAO gene polymorphisms are related to the development of 56 Parkinson’s disease 2.7.7. MAO inhibition and neuroprotection 57 2.7.8. Inhibitors of MAO in Parkinson’s disease 57 2.7.9. The structure of MAO-B 58 2.7.10. The structure of MAO-A 60 2.8. COMT 62 2.8.1. General background and tissue distribution 62 2.8.2. The reaction catalysed by COMT 63 2.8.3. The purification of the COMT enzyme 65 2.8.4. Inhibitors of COMT 66 2.8.5. The therapeutic actions of COMT 67 vii 2.8.6. COMT gene polymorphisms are implicated in the development of 68 Parkinson’s disease 2.8.7. The structure of COMT 69 2.9. Summary 71 Bibliography 72 Chapter 3 – Article 1 110 The synthesis and evaluation of nitrocatechol derivatives of chalcone as dual inhibitors of monoamine oxidase and catechol-O-methyltransferase Chapter 4 – Article 2 166 The evaluation of selected natural compounds as potential dual inhibitors of catechol-O-methyltransferase and monoamine oxidase Chapter 5 – Article 3 191 The evaluation of structurally diverse monoamine oxidase inhibitors as potential dual inhibitors of catechol-O-methyltransferase Chapter 6 – Conclusion 218 Future perspective 225 Annexure 226 viii List of figures and tables Chapter 1 - Introduction Figure 1.1. The structures of chalcones discussed in text 5 Figure 1.2. The basic structure of a flavonoid 6 Figure 1.3. The chemical structures of compounds discussed in text 6 Figure 1.4. The chemical structures of some tea catechins and flavonoids 7 known to inhibit COMT Figure 1.5. The chemical structures of flavonoids with neighboring hydroxy 8 groups exhibiting a catechol structure (quercetin and rutin) and flavonoids devoid of a 3-hydroxy group (isorhamnetin and kaempferol) Figure 1.6. The chemical structures of “classic” nitrocatechol compounds that 9 inhibit COMT Figure 1.7. The chemical structures of nitrocatechol compounds discussed in 10 text Figure 1.8. The chemical structures of tropolone, 8-hydroxyquinoline and 10 pyrogallol Figure 1.9. A summary of the structure-activity relationships of bisubstrate 11 inhibitors of COMT Chapter 2 – Literature overview Figure 2.1. The “direct” and “indirect” pathways in the brain 22 Figure 2.2. The chemical structures of L-dopa, dopamine and amantadine 25 Figure 2.3. The chemical structures of rasagiline, entacapone, selegiline and L- 26 dopa-α-lipoic acid Figure 2.4. The chemical structures of ergoline dopamine agonists 27 Figure 2.5. The chemical structures of nor-ergoline dopamine agonists 27 Figure 2.6. The chemical structures of the first generation COMT inhibitors 28 Figure 2.7. The chemical structures of second generation COMT inhibitors 29 Figure 2.8. The chemical structures of anticholinergic therapy used in 30 Parkinson’s disease Figure 2.9. The chemical structures of the antidepressants used in Parkinson’s 31 disease Figure 2.10. The chemical structures of MPTP and rotenone, environmental 33 factors causing Parkinson’s disease Figure 2.11. The metabolic route of dopamine and the formation of toxic by- 34 ix products Figure 2.12. Simplified diagram explaining the genetic factors and the 37 subsequent pathogenetic pathways leading to neurodegeneration Figure 2.13. The chemical structure of 6-OHDA 44 Figure 2.14. The chemical structures of typical AADC inhibitors used in the 46 treatment of Parkinson’s diease Figure 2.15. Diagram representing the metabolic routes for L-dopa and 48 dopamine in the periphery and brain Figure 2.16. Diagram representing the “cheese reaction” 52 Figure 2.17. The polar nucleophilic mechanism of MAO catalysis 54 Figure 2.18. The single electron transfer mechanism of MAO catalysis 55 Figure 2.19. The chemical structures of selegiline, rasagiline and safinamide 58 Figure 2.20. Diagram representing the MAO-B protein with key active site 60 residues indicated Figure 2.21. Diagram representing the MAO-A protein with key active site 61 residues indicated Figure 2.22. The simple SN2 reaction mechanism of COMT showing only the 64 SAM cofactor and catechol Figure 2.23. The chemical structures of various COMT inhibitors 66 Figure 2.24. Diagram representing the structure of human COMT showing the 71 active site architecture. SAM is shown in magenta, the inhibitor (3,5-dinitrocatechol) in orange and Lys144 in cyan Chapter 3 – First article Figure 1. The structures of the nitrocatechol derivatives of chalcone (1a–k) 115 that were investigated in this study Figure 2. The structures of chalcone derivatives known to inhibit MAO 116 Figure 3. The structures of 3-nitrocatechol derivatives known to inhibit COMT 117 Figure 4. The synthetic route for the synthesis of nitrocatechol derivatives of 117 chalcone (1a–k). Reagents and conditions: (a) 60% HNO3, acetic acid; (b) AlCl3, pyridine, ethyl acetate, 80 °C, HCl; (c) appropriately substituted benzaldehyde, ethanol, 60% KOH, 0.5 N HCl Figure 5. Sigmoidal plots for the inhibition of MAO-A and MAO-B by 118 compound 1d and 1g Figure 6. Reversibility of inhibition of MAO-B by 1d. MAO-B and 1d (at a 121 concentration of 4 × IC50) were incubated for 15 min, dialysed for 24 h and the residual enzyme activity was measured (1d dialysed). x Similar incubation and dialysis of the enzyme in the absence (NI dialysed) and presence of the irreversible inhibitor, selegiline (Depr dialysed), were also carried out. The residual activity of undialysed mixtures of the enzyme and 1d was also recorded (1d undialysed) Figure 7. Lineweaver-Burk plots of MAO-B activity in the absence (filled 122 squares) and presence of various concentrations of 1d.