Parkinson’s disease is the second most common age-related neurodegenerative disease after Alzheimer’s disease. The characteristic pathological feature of Parkinson’s disease is the loss of neurons in the substantia nigra pars compacta (SNpc), which leads to a striatal dopamine deficiency responsible for the major symptoms of Parkinson’s disease. These symptoms include tremor at rest, postural instability, bradykinesia and in the later stages of Parkinson’s disease, even psychosis. Presently, there is still no cure for Parkinson’s disease and all treatments are only symptomatic. Current research is therefore directed towards the prevention of further dopaminergic neurodegeneration, while the ultimate aim is the reversal of neurodegeneration. Monoamine oxidase (MAO) enzymes are responsible for the regulation and metabolism of monoamine neurotransmitters, such as dopamine. There are two MAO isoforms, MAO-A and MAO-B. Since MAO-B has greater activity in the basal ganglia, it is of particular importance in movement disorders, which include Parkinson’s disease. The selective inhibition of MAO-B, increases dopamine available for binding, and thus reduces Parkinson’s disease symptoms. MAO inhibitors also have neuroprotective potential and thus may slow down, halt and even reverse neurodegeneration in Parkinson’s disease. It is still unclear exactly how MAO inhibitors protect neurons, but one theory suggests that MAO inhibition decreases oxidative stress by reducing the formation of hydrogen peroxide, a metabolic by-product of MAO oxidation of monoamines. Normally, hydrogen peroxide is inactivated by glutathione (GSH), however, in Parkinson’s disease, GSH levels are low, resulting in the accumulation of hydrogen peroxide, which then becomes available for the Fenton reaction. In the Fenton reaction, Fe2+ reacts with hydrogen peroxide and generates an active free radical, the hydroxyl radical. This radical depletes cellular anti-oxidants, damage lipids, proteins and DNA. MAO inhibitors reduce the formation of hydrogen peroxide thus decreasing the formation of hydroxyl radicals and oxidative stress. The MAO inhibitory potential of natural and synthetic chalcones have been illustrated. For example, in 1987, Tanaka and co-workers determined that natural chalcones, such as isoliquiritigenin, have MAO inhibitory activity in rat mitochondria. In 2009, Chimenti and co-workers synthesized a series of 1,3-diphenyl-2-propen-1-ones which exhibited human MAO-B (hMAO-B) selective inhibitory activity. On the other hand, Robinson and co-workers (2013), synthesized novel furanochalcones which also had hMAO-B selective inhibitory activity. A reversible, competitive mode of binding was demonstrated by these compounds. Since the effect of heterocyclic substitution, other than furan on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesize and evaluate further heterocyclic chalcone analogues as inhibitors of hMAO. RESULTS Design and synthesis: Heterocyclic chalcone analogues that incorporated pyrrole, 5- methylthiophene, 5-chlorothiophene and 2-methoxypyridine substitution were synthesized using the Claisen-Schmidt condensation reaction. All compounds were characterized with 1H-NMR, 13CNMR, IR, MS, and melting points were recorded. Purity was determined with HPLC analysis. MAO inhibition studies: The 50% inhibitory concentration (IC50) values and selectivity index (SI) of all compounds were determined using a fluorometric assay and kynuramine as substrate. Eight out of the ten synthesized compounds exhibited IC50 values < 1 μM, and can thus be considered as potent MAO-B inhibitors, while all compounds showed selectivity for the MAO-B isoform. Compound 10i was the most potent MAO-B inhibitor with an IC50 value of 0.067 μM and the highest SI of 240.7. The most potent MAO-A inhibitor, compound 10f, had an IC50 value of 3.805 μM. Some structure-activity relationships were derived, for example; it was concluded that heterocyclic substitution with 5-methyl-thiophene ring resulted in optimal hMAO-B inhibition, while pyrrole substitution was less favourable. Further investigation is however required as this is only a preliminary study. Reversibility studies: To determine the reversibility of binding, the recovery of enzymatic activity after dilution of the enzyme inhibitor complexes were determined for selected compounds. Results indicated that the most potent MAO-A inhibitor, the pyrrole derivative 10f, had a reversible mode of binding to both the hMAO-B and hMAO-A isoforms, since the enzyme activities were completely recovered by dilution of the inhibitor concentration. In contrast, enzyme activity was only partially recovered after dilution of the most potent MAO-B inhibitor 10i, indicating that this methylthiophene derivative possibly exhibited tight binding to the hMAO-B isoform, and the inhibition caused by this compound was not readily reversed by dilution. In order to determine whether the tight binding as exhibited by compound 10i was due to the thiophene or phenyl moieties, reversibility of binding was also determined for the pyrrole derivative 10e. The results showed that 10e had a reversible mode of binding to the hMAO-B isoform, and enzyme activity was completely recovered by dilution of the inhibitor. Based on these results, it was concluded that the tight binding as exhibited by compound 10i was due to the presence of the thiophene moiety. To confirm that compound 10i exhibited tight, and not irreversible binding, reversibility of binding was also determined by dialysis of enzyme-inhibitor mixtures. For this purpose hMAO-B and 10i, at a concentration of 4 × IC50, were preincubated for a period of 15 min and subsequently dialyzed for 24 h. The results of this study showed that 10i had a reversible mode of binding for MAO-B, since enzyme activity was recovered to a level of 83% after dialysis. Mode of inhibition: To determine the mode of inhibition of compound 10f, Lineweaver-Burk plots were constructed for the inhibition of hMAO-A and hMAO-B. The lines of the Lineweaver-Burk plots intersected at a single point at the y-axis, indicating that 10f had a competitive mode of binding to both hMAO-B and hMAO-A isoforms. MTT viability assay: To determine the toxicity of the chalcones for cultured cells, selected compounds were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. The cytotoxicity of the test compounds were evaluated at concentrations of 1 and 10 μM, in HeLa cells. The results indicated that compound 10i was non-toxic at 1 and 10 μM, with 100% and 96% cell viability remaining after 24 h exposure of the compound to the cultured cells. Compound 10f, however, exhibited significant toxicity at 10 μM, with only 5% viable cells remaining. In contrast, compound 10e, with the same pyrrole moiety as 10f, was non-toxic at 1 μM and 10 μM, with 99% and 98%, cell viability remaining. It was concluded that the pyrrole moiety of 10f was not responsible for its higher degree of cytotoxicity, which suggests that the CF3 substituent may play a role in the higher degree of cytotoxicity observed for 10f. Further investigation is required to determine the mode of cytotoxicity, when cultured cells are exposed to 10f. Docking Studies: To complete this study and rationalise the results of the MAO inhibition studies, molecular modelling was carried out and all compounds were docked into the crystal structure of hMAO-B, by using the CDOCKER module of Discovery Studio. Some insights were obtained regarding the binding of compound 10i. This compound bound to MAO-B with the phenyl ring facing the FAD cofactor. This orientation allowed for the formation of pi-pi interaction with Tyr 398 as well as a pi-sigma interaction between the thiophene ring and Ile 199 (which is part of the gating switch of MAO-B). It is speculated that the tight binding component of hMAO-B inhibition by 10i may, at least in part, be attributed to the interaction of this compound with the gating switch amino acid, Ile 199. The docking results also showed that most compounds interacted with Tyr 326 or Tyr 398, while interactions with Cys 172, Gln 206, Ile 199 and Tyr 435 also occurred. In conclusion, novel heterocyclic chalcone analogues with promising MAO-B inhibitory activities were successfully synthesized and evaluated. / MSc (Pharmaceutical Chemistry) North-West University, Potchefstroom Campus, 2014

Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane and is considered to be a target for the treatment of diseases such as Parkinson’s disease and depression. MAO may be classified into two isoforms, MAO-A and MAO-B. Since MAO-A and MAO-B catalyzes the metabolism of serotonin and dopamine, respectively, MAO-A inhibitors are used in the therapy of depression while MAO-B inhibitors are useful in the treatment of Parkinson’s disease. The older nonselective and irreversible MAO inhibitors, however, are not frequently used because they may ellicit potentially dangerous side effects such as the “cheese reaction”. The cheese reaction occurs when irreversible MAO-A inhibitors block the metabolism of tyramine in the gastrointestinal tract. Excessive amounts of tyramine subsequently enter the systemic circulation and cause a hypertensive reaction. This problem may be overcome by the development of selective MAO-B inhibitors and reversible MAO-A inhibitors. Selective MAO-B inhibitors do not cause the cheese reaction, because tyramine is metabolized, in the intestines, by MAO-A. Tyramine also has the ability to displace reversible MAO-A inhibitors and can subsequently be normally metabolized, thus not causing the cheese reaction. Several reseach groups are therefore involved in the discovery of reversible MAO-A and MAO-B inhibitors. As mentioned above, such drugs may be used in the treatment of depression and Parkinson’s disease. One approach is the de novo design of novel molecules with affinities for MAO-A and MAO-B active sites. In a second approach, existing drugs may be reappropriated as MAO inhibitors. With this approach, approved drugs are screened for the possibility that they, in addition to their action at the indicated target, also act as inhibitors of MAO-A and/or MAO-B. Such drugs may then be applied as MAO inhibitors in the treatment of depression and Parkinson’s disease. From a toxicological point of view, it is also of importance to identify MAO-A inhibitory activities among existing drugs as this will alert to the occurance of potential side effects such as the cheese reaction. In this study the second approach will be followed. This study will screen a virtual library of approved drugs for inhibitory activity towards MAO-A and MAO-B. Molecular modeling may be used to screen virtual libraries of drugs as potential inhibitors of the MAO enzymes. This may conveniently be achieved by employing structure-based or ligand-based pharmacophore models. In this study a virtual library of approved drugs was screened for secondary inhibitory activities towards the MAO isoforms with the use of structure-based pharmacophore models. There are several advantages to this approach. Molecular modeling aims at reducing the overall cost associated with the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. It aids in understanding how a ligand binds to the active site of an enzyme. It is relatively easier to re-register a drug for a second pharmacological activity. This approach may also lead to drugs with a multi-target mode of action. The structure-based pharmacophores were constructed using the known crystallographic structures of MAO-A and MAO-B with the inhibitors, harmine and safinamide, complexed in the active sites, respectively. Employing the MAO-A and MAO-B structure-based pharmacophore model in the virtual screening of a library of approved drugs, 45 compounds were found to map to the MAO-A and MAO-B pharmacophore models. Among the hits, 29 compounds were selected for in vitro evaluation as MAO-A and MAO-B inhibitors. The IC50 values for these compounds were determined. After in vitro evaluation, 13 compounds showed inhibitory activity towards MAO. Of the 13 compounds 3 showed interesting inhibitory activities. These compounds included caffeine (IC50 = 0.761 μM for MAO-A and 5.08 μM for MAO-B), esomeprazole (IC50 = 23.2 μM for MAO-A and 48.3 μM for MAO-B) and leflunomide (IC50 = 19.1μM for MAO-A and 13.7 μM for MAO-B). The MAO inhibitory properties of caffeine and esomeprazole were further investigated. The reversibility of MAO inhibition by caffeine and esomeprazole were determined by dialysis and dilution studies. Sets of Lineweaver-Burk plots were constructed to determine the modes of binding of these inhibitors to the MAO enzymes. Both caffeine and esomeprazole were found to be reversible and competitive inhibitors of MAO. Dialysis of mixtures of caffeine with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 97% and 96%, respectively. Dialysis of mixtures of esomeprazole with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 93% and 88%, respectively. Similarly, dilution of mixtures containing esomeprazole and MAO-A/MAO-B resulted in the recovery of enzyme activity to levels of 94% and 87%, respectively.For the inhibition of MAO-A and MAO-B by caffeine and esomeprazole, the Lineweaver-Burk plots were indicative of a competitive mode of inhibition. In an attempt to gain further insignt, caffeine, esomeprazole and leflunomide were docked into models of the active sites of MAO-A and MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation The results of the present study show that screening of a virtual database of molecules with a pharmacophore model may be useful in identifying existing drugs with potential MAO inhibitory activities. The search for new reversible MAO inhibitors for the treatment of diseases, including Parkinson’s disease and depression, may be facilitated by employing a virtual screening approach. Such an approach also may be more costeffective than de novo inhibitor design. In addition, the virtual screening approach may alert to potential side effects of existing drugs that may arise as a consequence of a secondary inhibition of MAO. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Parkinson’s disease (PD) is an age related neurodegenerative disorder that presents with both motor and non-motor symptoms. The most common pathological characteristic of PD is the loss of the pigmented dopaminergic neurons of the substantia nigra pars compacta (SNpc), with the appearance of intracellular inclusions known as Lewy bodies in the affected neurons. The loss of the SNpc neurons results in a deficiency of dopamine in the nigrostriatal pathway of the brain, and it is this deficiency that is responsible for the motor symptoms of PD. Monoamine oxidase B (MAO-B) is predominantly found in the striatum and is responsible for the oxidative metabolism of dopamine. The first-line treatment of PD is dopamine replacement therapy with levodopa, the metabolic precursor of dopamine. Rapid metabolism of levodopa at central and peripheral level, however, hampers its therapeutic potential. MAO-B inhibition enhances striatal dopamine activity by means of inhibiting dopamine metabolism, and MAO-B inhibitors are thus used in the treatment of PD, particularly in combination with levodopa. The aim of this study was to design new potent, reversible MAO inhibitors with selectivity towards MAO-B for the symptomatic treatment of PD. Recent studies have shown that C5-substituted phthalide derivatives are highly potent inhibitors of human MAO-B. Phthalide derivatives were also found to be potent inhibitors of human MAO-A. The structural similarity between phthalide and 3-coumaranone suggests that 3-coumaranone may be a useful scaffold for the design of reversible MAO-B inhibitors. In the present study, 3-coumaranone derivatives were thus synthesised and evaluated as potential MAO-A and MAO-B inhibitors. By reacting 6-hydroxy-3-coumaranone with the appropriate alkylbromide in N,N-dimethylformamide in the presence of potassium carbonate, a series of twenty 3-coumaranone derivatives were synthesised. The structures of the compounds were verified with NMR spectroscopy and mass spectrometry. The purities of the compounds were determined by HPLC analyses. To determine the inhibition potencies, the recombinant human MAO-A and MAO-B enzymes were used, and the inhibition potencies were expressed as IC50 values. The results indicated that the 3-coumaranone derivatives are highly potent MAO-B inhibitors. For example, 9 of the 3-coumaranone derivatives inhibited MAO-B with IC50 values < 0.05 μM, with the most potent inhibitor exhibiting an IC50 value of 0.004 μM. Although the 3-coumaranone derivatives are selective MAO-B inhibitors, some compounds were also potent MAO-A inhibitors with the most potent inhibitor exhibiting an IC50 value of 0.586 μM. The reversibility of MAO-B inhibition by a representative inhibitor was examined by measuring the degree to which the enzyme activity recovers after dialysis of the enzyme-inhibitor complex. Since MAO-B activity was almost completely recovered after dialysis, it may be concluded that the 3-coumaranone derivatives bind reversibly to MAO-B. Lineweaver-Burk plots were constructed to show that the representative 3-coumaranone derivative is a competitive inhibitor of MAO-B. To conclude, the 3-coumaranone derivatives are potent, selective, reversible and competitive inhibitors of MAO-B. These compounds may find application in the treatment of neurodegenerative disorders such as PD. Potent MAO-A inhibitors were also discovered, which suggests that 3-coumaranone derivatives may serve as leads for the design of drugs for the treatment of depression. In addition, 3-coumaranone derivatives which inhibited both MAO-A and MAO-B, may have potential application in the therapy of both PD and depressive illness. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Parkinson’s disease is the second most common age-related neurodegenerative disease after Alzheimer’s disease. The characteristic pathological feature of Parkinson’s disease is the loss of neurons in the substantia nigra pars compacta (SNpc), which leads to a striatal dopamine deficiency responsible for the major symptoms of Parkinson’s disease. These symptoms include tremor at rest, postural instability, bradykinesia and in the later stages of Parkinson’s disease, even psychosis. Presently, there is still no cure for Parkinson’s disease and all treatments are only symptomatic. Current research is therefore directed towards the prevention of further dopaminergic neurodegeneration, while the ultimate aim is the reversal of neurodegeneration. Monoamine oxidase (MAO) enzymes are responsible for the regulation and metabolism of monoamine neurotransmitters, such as dopamine. There are two MAO isoforms, MAO-A and MAO-B. Since MAO-B has greater activity in the basal ganglia, it is of particular importance in movement disorders, which include Parkinson’s disease. The selective inhibition of MAO-B, increases dopamine available for binding, and thus reduces Parkinson’s disease symptoms. MAO inhibitors also have neuroprotective potential and thus may slow down, halt and even reverse neurodegeneration in Parkinson’s disease. It is still unclear exactly how MAO inhibitors protect neurons, but one theory suggests that MAO inhibition decreases oxidative stress by reducing the formation of hydrogen peroxide, a metabolic by-product of MAO oxidation of monoamines. Normally, hydrogen peroxide is inactivated by glutathione (GSH), however, in Parkinson’s disease, GSH levels are low, resulting in the accumulation of hydrogen peroxide, which then becomes available for the Fenton reaction. In the Fenton reaction, Fe2+ reacts with hydrogen peroxide and generates an active free radical, the hydroxyl radical. This radical depletes cellular anti-oxidants, damage lipids, proteins and DNA. MAO inhibitors reduce the formation of hydrogen peroxide thus decreasing the formation of hydroxyl radicals and oxidative stress. The MAO inhibitory potential of natural and synthetic chalcones have been illustrated. For example, in 1987, Tanaka and co-workers determined that natural chalcones, such as isoliquiritigenin, have MAO inhibitory activity in rat mitochondria. In 2009, Chimenti and co-workers synthesized a series of 1,3-diphenyl-2-propen-1-ones which exhibited human MAO-B (hMAO-B) selective inhibitory activity. On the other hand, Robinson and co-workers (2013), synthesized novel furanochalcones which also had hMAO-B selective inhibitory activity. A reversible, competitive mode of binding was demonstrated by these compounds. Since the effect of heterocyclic substitution, other than furan on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesize and evaluate further heterocyclic chalcone analogues as inhibitors of hMAO. RESULTS Design and synthesis: Heterocyclic chalcone analogues that incorporated pyrrole, 5- methylthiophene, 5-chlorothiophene and 2-methoxypyridine substitution were synthesized using the Claisen-Schmidt condensation reaction. All compounds were characterized with 1H-NMR, 13CNMR, IR, MS, and melting points were recorded. Purity was determined with HPLC analysis. MAO inhibition studies: The 50% inhibitory concentration (IC50) values and selectivity index (SI) of all compounds were determined using a fluorometric assay and kynuramine as substrate. Eight out of the ten synthesized compounds exhibited IC50 values < 1 μM, and can thus be considered as potent MAO-B inhibitors, while all compounds showed selectivity for the MAO-B isoform. Compound 10i was the most potent MAO-B inhibitor with an IC50 value of 0.067 μM and the highest SI of 240.7. The most potent MAO-A inhibitor, compound 10f, had an IC50 value of 3.805 μM. Some structure-activity relationships were derived, for example; it was concluded that heterocyclic substitution with 5-methyl-thiophene ring resulted in optimal hMAO-B inhibition, while pyrrole substitution was less favourable. Further investigation is however required as this is only a preliminary study. Reversibility studies: To determine the reversibility of binding, the recovery of enzymatic activity after dilution of the enzyme inhibitor complexes were determined for selected compounds. Results indicated that the most potent MAO-A inhibitor, the pyrrole derivative 10f, had a reversible mode of binding to both the hMAO-B and hMAO-A isoforms, since the enzyme activities were completely recovered by dilution of the inhibitor concentration. In contrast, enzyme activity was only partially recovered after dilution of the most potent MAO-B inhibitor 10i, indicating that this methylthiophene derivative possibly exhibited tight binding to the hMAO-B isoform, and the inhibition caused by this compound was not readily reversed by dilution. In order to determine whether the tight binding as exhibited by compound 10i was due to the thiophene or phenyl moieties, reversibility of binding was also determined for the pyrrole derivative 10e. The results showed that 10e had a reversible mode of binding to the hMAO-B isoform, and enzyme activity was completely recovered by dilution of the inhibitor. Based on these results, it was concluded that the tight binding as exhibited by compound 10i was due to the presence of the thiophene moiety. To confirm that compound 10i exhibited tight, and not irreversible binding, reversibility of binding was also determined by dialysis of enzyme-inhibitor mixtures. For this purpose hMAO-B and 10i, at a concentration of 4 × IC50, were preincubated for a period of 15 min and subsequently dialyzed for 24 h. The results of this study showed that 10i had a reversible mode of binding for MAO-B, since enzyme activity was recovered to a level of 83% after dialysis. Mode of inhibition: To determine the mode of inhibition of compound 10f, Lineweaver-Burk plots were constructed for the inhibition of hMAO-A and hMAO-B. The lines of the Lineweaver-Burk plots intersected at a single point at the y-axis, indicating that 10f had a competitive mode of binding to both hMAO-B and hMAO-A isoforms. MTT viability assay: To determine the toxicity of the chalcones for cultured cells, selected compounds were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. The cytotoxicity of the test compounds were evaluated at concentrations of 1 and 10 μM, in HeLa cells. The results indicated that compound 10i was non-toxic at 1 and 10 μM, with 100% and 96% cell viability remaining after 24 h exposure of the compound to the cultured cells. Compound 10f, however, exhibited significant toxicity at 10 μM, with only 5% viable cells remaining. In contrast, compound 10e, with the same pyrrole moiety as 10f, was non-toxic at 1 μM and 10 μM, with 99% and 98%, cell viability remaining. It was concluded that the pyrrole moiety of 10f was not responsible for its higher degree of cytotoxicity, which suggests that the CF3 substituent may play a role in the higher degree of cytotoxicity observed for 10f. Further investigation is required to determine the mode of cytotoxicity, when cultured cells are exposed to 10f. Docking Studies: To complete this study and rationalise the results of the MAO inhibition studies, molecular modelling was carried out and all compounds were docked into the crystal structure of hMAO-B, by using the CDOCKER module of Discovery Studio. Some insights were obtained regarding the binding of compound 10i. This compound bound to MAO-B with the phenyl ring facing the FAD cofactor. This orientation allowed for the formation of pi-pi interaction with Tyr 398 as well as a pi-sigma interaction between the thiophene ring and Ile 199 (which is part of the gating switch of MAO-B). It is speculated that the tight binding component of hMAO-B inhibition by 10i may, at least in part, be attributed to the interaction of this compound with the gating switch amino acid, Ile 199. The docking results also showed that most compounds interacted with Tyr 326 or Tyr 398, while interactions with Cys 172, Gln 206, Ile 199 and Tyr 435 also occurred. In conclusion, novel heterocyclic chalcone analogues with promising MAO-B inhibitory activities were successfully synthesized and evaluated. / MSc (Pharmaceutical Chemistry) North-West University, Potchefstroom Campus, 2014

Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane and is considered to be a target for the treatment of diseases such as Parkinson’s disease and depression. MAO may be classified into two isoforms, MAO-A and MAO-B. Since MAO-A and MAO-B catalyzes the metabolism of serotonin and dopamine, respectively, MAO-A inhibitors are used in the therapy of depression while MAO-B inhibitors are useful in the treatment of Parkinson’s disease. The older nonselective and irreversible MAO inhibitors, however, are not frequently used because they may ellicit potentially dangerous side effects such as the “cheese reaction”. The cheese reaction occurs when irreversible MAO-A inhibitors block the metabolism of tyramine in the gastrointestinal tract. Excessive amounts of tyramine subsequently enter the systemic circulation and cause a hypertensive reaction. This problem may be overcome by the development of selective MAO-B inhibitors and reversible MAO-A inhibitors. Selective MAO-B inhibitors do not cause the cheese reaction, because tyramine is metabolized, in the intestines, by MAO-A. Tyramine also has the ability to displace reversible MAO-A inhibitors and can subsequently be normally metabolized, thus not causing the cheese reaction. Several reseach groups are therefore involved in the discovery of reversible MAO-A and MAO-B inhibitors. As mentioned above, such drugs may be used in the treatment of depression and Parkinson’s disease. One approach is the de novo design of novel molecules with affinities for MAO-A and MAO-B active sites. In a second approach, existing drugs may be reappropriated as MAO inhibitors. With this approach, approved drugs are screened for the possibility that they, in addition to their action at the indicated target, also act as inhibitors of MAO-A and/or MAO-B. Such drugs may then be applied as MAO inhibitors in the treatment of depression and Parkinson’s disease. From a toxicological point of view, it is also of importance to identify MAO-A inhibitory activities among existing drugs as this will alert to the occurance of potential side effects such as the cheese reaction. In this study the second approach will be followed. This study will screen a virtual library of approved drugs for inhibitory activity towards MAO-A and MAO-B. Molecular modeling may be used to screen virtual libraries of drugs as potential inhibitors of the MAO enzymes. This may conveniently be achieved by employing structure-based or ligand-based pharmacophore models. In this study a virtual library of approved drugs was screened for secondary inhibitory activities towards the MAO isoforms with the use of structure-based pharmacophore models. There are several advantages to this approach. Molecular modeling aims at reducing the overall cost associated with the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. It aids in understanding how a ligand binds to the active site of an enzyme. It is relatively easier to re-register a drug for a second pharmacological activity. This approach may also lead to drugs with a multi-target mode of action. The structure-based pharmacophores were constructed using the known crystallographic structures of MAO-A and MAO-B with the inhibitors, harmine and safinamide, complexed in the active sites, respectively. Employing the MAO-A and MAO-B structure-based pharmacophore model in the virtual screening of a library of approved drugs, 45 compounds were found to map to the MAO-A and MAO-B pharmacophore models. Among the hits, 29 compounds were selected for in vitro evaluation as MAO-A and MAO-B inhibitors. The IC50 values for these compounds were determined. After in vitro evaluation, 13 compounds showed inhibitory activity towards MAO. Of the 13 compounds 3 showed interesting inhibitory activities. These compounds included caffeine (IC50 = 0.761 μM for MAO-A and 5.08 μM for MAO-B), esomeprazole (IC50 = 23.2 μM for MAO-A and 48.3 μM for MAO-B) and leflunomide (IC50 = 19.1μM for MAO-A and 13.7 μM for MAO-B). The MAO inhibitory properties of caffeine and esomeprazole were further investigated. The reversibility of MAO inhibition by caffeine and esomeprazole were determined by dialysis and dilution studies. Sets of Lineweaver-Burk plots were constructed to determine the modes of binding of these inhibitors to the MAO enzymes. Both caffeine and esomeprazole were found to be reversible and competitive inhibitors of MAO. Dialysis of mixtures of caffeine with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 97% and 96%, respectively. Dialysis of mixtures of esomeprazole with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 93% and 88%, respectively. Similarly, dilution of mixtures containing esomeprazole and MAO-A/MAO-B resulted in the recovery of enzyme activity to levels of 94% and 87%, respectively.For the inhibition of MAO-A and MAO-B by caffeine and esomeprazole, the Lineweaver-Burk plots were indicative of a competitive mode of inhibition. In an attempt to gain further insignt, caffeine, esomeprazole and leflunomide were docked into models of the active sites of MAO-A and MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation The results of the present study show that screening of a virtual database of molecules with a pharmacophore model may be useful in identifying existing drugs with potential MAO inhibitory activities. The search for new reversible MAO inhibitors for the treatment of diseases, including Parkinson’s disease and depression, may be facilitated by employing a virtual screening approach. Such an approach also may be more costeffective than de novo inhibitor design. In addition, the virtual screening approach may alert to potential side effects of existing drugs that may arise as a consequence of a secondary inhibition of MAO. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Parkinson’s disease (PD) is an age related neurodegenerative disorder that presents with both motor and non-motor symptoms. The most common pathological characteristic of PD is the loss of the pigmented dopaminergic neurons of the substantia nigra pars compacta (SNpc), with the appearance of intracellular inclusions known as Lewy bodies in the affected neurons. The loss of the SNpc neurons results in a deficiency of dopamine in the nigrostriatal pathway of the brain, and it is this deficiency that is responsible for the motor symptoms of PD. Monoamine oxidase B (MAO-B) is predominantly found in the striatum and is responsible for the oxidative metabolism of dopamine. The first-line treatment of PD is dopamine replacement therapy with levodopa, the metabolic precursor of dopamine. Rapid metabolism of levodopa at central and peripheral level, however, hampers its therapeutic potential. MAO-B inhibition enhances striatal dopamine activity by means of inhibiting dopamine metabolism, and MAO-B inhibitors are thus used in the treatment of PD, particularly in combination with levodopa. The aim of this study was to design new potent, reversible MAO inhibitors with selectivity towards MAO-B for the symptomatic treatment of PD. Recent studies have shown that C5-substituted phthalide derivatives are highly potent inhibitors of human MAO-B. Phthalide derivatives were also found to be potent inhibitors of human MAO-A. The structural similarity between phthalide and 3-coumaranone suggests that 3-coumaranone may be a useful scaffold for the design of reversible MAO-B inhibitors. In the present study, 3-coumaranone derivatives were thus synthesised and evaluated as potential MAO-A and MAO-B inhibitors. By reacting 6-hydroxy-3-coumaranone with the appropriate alkylbromide in N,N-dimethylformamide in the presence of potassium carbonate, a series of twenty 3-coumaranone derivatives were synthesised. The structures of the compounds were verified with NMR spectroscopy and mass spectrometry. The purities of the compounds were determined by HPLC analyses. To determine the inhibition potencies, the recombinant human MAO-A and MAO-B enzymes were used, and the inhibition potencies were expressed as IC50 values. The results indicated that the 3-coumaranone derivatives are highly potent MAO-B inhibitors. For example, 9 of the 3-coumaranone derivatives inhibited MAO-B with IC50 values < 0.05 μM, with the most potent inhibitor exhibiting an IC50 value of 0.004 μM. Although the 3-coumaranone derivatives are selective MAO-B inhibitors, some compounds were also potent MAO-A inhibitors with the most potent inhibitor exhibiting an IC50 value of 0.586 μM. The reversibility of MAO-B inhibition by a representative inhibitor was examined by measuring the degree to which the enzyme activity recovers after dialysis of the enzyme-inhibitor complex. Since MAO-B activity was almost completely recovered after dialysis, it may be concluded that the 3-coumaranone derivatives bind reversibly to MAO-B. Lineweaver-Burk plots were constructed to show that the representative 3-coumaranone derivative is a competitive inhibitor of MAO-B. To conclude, the 3-coumaranone derivatives are potent, selective, reversible and competitive inhibitors of MAO-B. These compounds may find application in the treatment of neurodegenerative disorders such as PD. Potent MAO-A inhibitors were also discovered, which suggests that 3-coumaranone derivatives may serve as leads for the design of drugs for the treatment of depression. In addition, 3-coumaranone derivatives which inhibited both MAO-A and MAO-B, may have potential application in the therapy of both PD and depressive illness. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Monoamine oxidase (MAO) is responsible for dopamine catabolism in the brain and therefore is especially important in the treatment of Parkinson's disease (PD). MAO–B inhibition provides symptomatic relief by indirectly elevating dopamine levels in the PD brain. PD is caused by the loss of dopaminergic neurons in the substantia nigra and the formation of proteinaceous structures in the brain. The cause of idiopathic PD is unknown, but one theory states that reactive oxygen species (ROS), partly derived from the catalytic cycle of MAO, may be to blame for damaging dopaminergic neurons. Since MAO inhibitors may reduce the MAO–catalyzed production of ROS, these compounds may protect dopaminergic neurons against degeneration in PD. It is commonly accepted that by the time PD symptoms manifest, about 80% of striatal dopamine has been lost. MAO is present as two subtypes in the human brain, namely MAO–A and MAO–B. MAOs are found mainly attached to the mitochondrial membrane and is responsible for the oxidative deamination of various monoamines, including dopamine. MAO is a dimeric enzyme which operates in conjunction with a co–factor, flavin adenine dinucleotide (FAD), to which it is covalently bound. The flavin is in a bent conformation, which assists the catalytic activity of MAO. As mentioned above, the catalytic action of MAO also produces harmful substances such as hydrogen peroxide, ammonia, aldehydes and may also increase the levels of hydroxyl radicals. In the healthy brain, these substances are metabolized rapidly, but the PD brain may exhibit reduced clearance of these species. Thus the inhibition of MAOs may be beneficial to the PD sufferer as it indirectly increases dopamine levels in the brain and may also slow the formation of harmful substances. MAO inhibitors, of the MAO–A type, were first used as anti–depressants. It was these drugs that first prompted researchers to explore MAO inhibitors as novel anti–parkinsonian drugs, as MAO–A inhibition slows the degradation of dopamine. Two types of inhibition modes exist, irreversible and reversible inhibition. Irreversible inhibitors do not allow for competition with the substrate and inactivate the enzyme permanently. Selegiline, a propargyl amine derivative, is an example of an irreversible MAO–B selective inhibitor. The major disadvantage of irreversible inhibitors is that after terminating treatment, recovery of the enzyme activity may require several weeks, since the turnover rate for the biosynthesis of MAO in the human brain may be as much as 40 days. Reversible inhibitors have better safety profiles since they allow for competition with the substrate. (E)–8–(3–Chlorostyryl)caffeine (CSC) is an example of a reversible inhibitor of MAO–B and is also an antagonist of the adenosine A2A receptor. Since antagonism of A2A receptors also produces an antiparkinsonian effect, dual acting compounds such as CSC, which block both the A2A receptors and MAO–B, may have an enhanced therapeutic potential in PD therapy. Current PD therapy available only treats the symptoms of PD and do not halt or slow the progression of the neurodegenerative processes. There therefore exists the need for the development of antiparkinsonian drugs with neuroprotective effects. Since both MAO–B inhibitors and A2A receptor antagonists are reported to possess protective effects in PD and PD animal models, dual acting drugs, that antagonize A2A receptors and inhibit MAO–B, may be candidates for neuroprotection. Using the structure of CSC as lead, we investigate in the current study, the possibility that aminocaffeines may also possess potent MAO–B inhibitory properties. The structures of the aminocaffeine derivatives that were investigated bear close structural resemblance to CSC as well as to a series of alkyloxycaffeine analogues that was recently found to be potent MAO inhibitors. This study therefore further explores the structural requirements of caffeine derivatives to act as MAO inhibitors by examining the possibility that aminocaffeine derivatives may be MAO inhibitors. Such compounds may act as lead compounds for the development of improved PD therapy. In this study, a series of 8–aminocaffeine derivatives were synthesized and evaluated as inhibitors of human MAO–A and B. For this purpose, 8–chlorocaffeine was reacted with the appropriate amine at high temperatures to produce the desired 8–aminocaffeine derivatives. The inhibitory activities of the compounds were determined towards recombinant human MAO–A and B and expressed as IC50 values. The results showed that human MAO–B was most potently inhibited by 8–[methyl(4–phenylbutyl)amino]caffeine with an IC50 value of 2.97 ?M. Human MAO–A was most potently inhibited by 8–[2–(3–chlorophenyl)–ethylamino]caffeine with an IC50 value of 5.78 ?M. It was found that methylation of the amine group at C8 of the caffeine ring increases inhibition but also selectivity towards MAO–B inhibition. For example, 8–[4–(phenylbutylamino)]caffeine inhibits MAO–B with an IC50 value of 7.56 ?M whereas 8–[methyl(4–phenylbutyl)amino]–caffeine has an increased inhibition potency of 2.97 ?M. The selectivity for MAO–B inhibition also increases over MAO–A when the C8 amine is methylated. It was found that the aminocaffeine derivatives bind reversibly to both enzyme isoforms and the mode of inhibition is competitive for MAO–B. From these results it can be concluded that although the 8–aminocaffeine derivatives are only moderately potent MAO–B inhibitors, they may act as lead compounds for the design of more potent reversible MAO inhibitors. Docking studies revealed that the 8–aminocaffeine and 8–[(methyl)amino]caffeine derivatives traverse both the entrance and substrate cavities of the MAO–B enzyme, with the caffeinyl moiety oriented towards the FAD co–factor while the amino–side chain protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Neurodegenerative disorders like Parkinson’s and Alzheimer’s disease affect millions of people around the world. Oxidative stress has been implicated in the pathogenesis of a number of neurodegenerative disorders, cancer and ischemia. The brain is particularly vulnerable to oxidative damage because of its high utilisation of oxygen, high levels of polyunsaturated fatty acids, relatively high levels of redox transition metal ions and low levels of antioxidants. Oxidative stress occurs due to an imbalance in the pro–oxidant and antioxidant levels. Reactive oxygen/nitrogen species (ROS/RNS) is a collective term used for free radicals and related molecules, promoting oxidative stress within cells and ultimately leading to neurodegeneration. Antioxidants counteract the excess in ROS/RNS, and is therefore of interest in the treatment and prevention of neurodegenerative disorders. Monoamine oxidases, especially monoamine oxidase B (MAO–B), also play an important role in neurodegenerative disorders. MAO–B is the main enzyme responsible for the oxidative deamination of dopamine in the substantia nigra of the brain. By inhibiting MAO–B, dopamine is increased in the brain providing symptomatic relief in Parkinson’s disease. The focus of the current study was to synthesise multifunctional compounds that could be used in the treatment and/or prevention of neurodegenerative diseases. In this study flavonoids were selected because of their wide spectrum of biological activities, including antioxidant activity and its monoamine oxidase inhibition. Flavones and chalcones are both classified under flavonoids and both structures were included. The amantadine moiety was included because of its known ability to inhibit calcium flux through the N–methyl–D–aspartate (NMDA) receptor channel. Six amantadine–flavonoid derivatives were synthesised using standard laboratory procedures and structures were determined with standard methods such as NMR, IR and mass spectrometry. The synthesised compounds were tested in a selection of biological assays, to establish the relative antioxidant properties and MAO inhibitory activity. The biological assays employed to test antioxidant properties were the thiobarbituric acid (TBA) and nitro–blue tetrazolium (NBT) assays. The TBA assay relies on the assessment of lipid peroxidation, induced via hydroxyl anions (OH), generating a pink colour with the complex formation between malondialdehyde (MDA) and TBA, which is measured spectrophotometrically at 532 nm. The principal of the NBT assay is the reduction of NBT to nitro–blue diformazan (NBD), producing a purple colour in the presence of superoxide anions (O2 –). The synthesised compounds were also evaluated for their MAO inhibitory activity toward recombinant human MAO–A and -B and inhibition values were expressed as IC50 values. The experimental data obtained in the NBT and TBA assay indicated a weak but a significant ability to scavenge O2 – and OH. In the NBT assay N–(adamantan–1–yl)–2–{3–hydroxy–4–[(2E)– 3–(3–methoxyphenyl)pro–2–enoyl]phenoxy}acetamide (6) had the best results with a 50.47 ± 1.31 uM/mg protein reduction in NBD formation, indicating that the hydroxyl group contributed to activity. The synthesised compounds were compared to the toxin (KCN) with a reduction in NDB formation of 69.88 ± 1.59 uM/mg protein. Results obtained from the TBA assay indicated that the flavone moiety had better OH scavenging ability than that of the chalcone moiety with N–(adamantan–1–yl)–2–[(5–hydroxy–4–oxo–2–phenyl–4H–chromen–7– yl)oxy]acetamide (3) showing the best activity at 0.967 ± 0.063 nmol MDA/mg tissue. The synthesised compounds were compared to the toxin (H2O2) 1.316 ± 0.028 nmol MDA/mg tissue. None of the test compounds could be compared to the results obtained with Trolox®. The IC50 values obtained for inhibition of recombinant human MAO indicated that the chalcone moiety (N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro–1–en–1–yl]benzamide (5)) showed the best inhibition of MAO–B with an IC50 of 0.717 ± 0.009 M and of MAO–A with an IC50 of 24.987 ± 5.988 M. It was further confirmed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3– phenylpro–1–en–1–yl]benzamide (5) binds reversible to MAO–B and that the mode of inhibition is competitive. Docking studies revealed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro– 1–en–1–yl]benzamide (5) traverses both cavities of MAO–B with the chalcone moiety orientated towards the FAD co–factor while the amantadine moiety protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Monoamine oxidase (MAO) is responsible for dopamine catabolism in the brain and therefore is especially important in the treatment of Parkinson's disease (PD). MAO–B inhibition provides symptomatic relief by indirectly elevating dopamine levels in the PD brain. PD is caused by the loss of dopaminergic neurons in the substantia nigra and the formation of proteinaceous structures in the brain. The cause of idiopathic PD is unknown, but one theory states that reactive oxygen species (ROS), partly derived from the catalytic cycle of MAO, may be to blame for damaging dopaminergic neurons. Since MAO inhibitors may reduce the MAO–catalyzed production of ROS, these compounds may protect dopaminergic neurons against degeneration in PD. It is commonly accepted that by the time PD symptoms manifest, about 80% of striatal dopamine has been lost. MAO is present as two subtypes in the human brain, namely MAO–A and MAO–B. MAOs are found mainly attached to the mitochondrial membrane and is responsible for the oxidative deamination of various monoamines, including dopamine. MAO is a dimeric enzyme which operates in conjunction with a co–factor, flavin adenine dinucleotide (FAD), to which it is covalently bound. The flavin is in a bent conformation, which assists the catalytic activity of MAO. As mentioned above, the catalytic action of MAO also produces harmful substances such as hydrogen peroxide, ammonia, aldehydes and may also increase the levels of hydroxyl radicals. In the healthy brain, these substances are metabolized rapidly, but the PD brain may exhibit reduced clearance of these species. Thus the inhibition of MAOs may be beneficial to the PD sufferer as it indirectly increases dopamine levels in the brain and may also slow the formation of harmful substances. MAO inhibitors, of the MAO–A type, were first used as anti–depressants. It was these drugs that first prompted researchers to explore MAO inhibitors as novel anti–parkinsonian drugs, as MAO–A inhibition slows the degradation of dopamine. Two types of inhibition modes exist, irreversible and reversible inhibition. Irreversible inhibitors do not allow for competition with the substrate and inactivate the enzyme permanently. Selegiline, a propargyl amine derivative, is an example of an irreversible MAO–B selective inhibitor. The major disadvantage of irreversible inhibitors is that after terminating treatment, recovery of the enzyme activity may require several weeks, since the turnover rate for the biosynthesis of MAO in the human brain may be as much as 40 days. Reversible inhibitors have better safety profiles since they allow for competition with the substrate. (E)–8–(3–Chlorostyryl)caffeine (CSC) is an example of a reversible inhibitor of MAO–B and is also an antagonist of the adenosine A2A receptor. Since antagonism of A2A receptors also produces an antiparkinsonian effect, dual acting compounds such as CSC, which block both the A2A receptors and MAO–B, may have an enhanced therapeutic potential in PD therapy. Current PD therapy available only treats the symptoms of PD and do not halt or slow the progression of the neurodegenerative processes. There therefore exists the need for the development of antiparkinsonian drugs with neuroprotective effects. Since both MAO–B inhibitors and A2A receptor antagonists are reported to possess protective effects in PD and PD animal models, dual acting drugs, that antagonize A2A receptors and inhibit MAO–B, may be candidates for neuroprotection. Using the structure of CSC as lead, we investigate in the current study, the possibility that aminocaffeines may also possess potent MAO–B inhibitory properties. The structures of the aminocaffeine derivatives that were investigated bear close structural resemblance to CSC as well as to a series of alkyloxycaffeine analogues that was recently found to be potent MAO inhibitors. This study therefore further explores the structural requirements of caffeine derivatives to act as MAO inhibitors by examining the possibility that aminocaffeine derivatives may be MAO inhibitors. Such compounds may act as lead compounds for the development of improved PD therapy. In this study, a series of 8–aminocaffeine derivatives were synthesized and evaluated as inhibitors of human MAO–A and B. For this purpose, 8–chlorocaffeine was reacted with the appropriate amine at high temperatures to produce the desired 8–aminocaffeine derivatives. The inhibitory activities of the compounds were determined towards recombinant human MAO–A and B and expressed as IC50 values. The results showed that human MAO–B was most potently inhibited by 8–[methyl(4–phenylbutyl)amino]caffeine with an IC50 value of 2.97 ?M. Human MAO–A was most potently inhibited by 8–[2–(3–chlorophenyl)–ethylamino]caffeine with an IC50 value of 5.78 ?M. It was found that methylation of the amine group at C8 of the caffeine ring increases inhibition but also selectivity towards MAO–B inhibition. For example, 8–[4–(phenylbutylamino)]caffeine inhibits MAO–B with an IC50 value of 7.56 ?M whereas 8–[methyl(4–phenylbutyl)amino]–caffeine has an increased inhibition potency of 2.97 ?M. The selectivity for MAO–B inhibition also increases over MAO–A when the C8 amine is methylated. It was found that the aminocaffeine derivatives bind reversibly to both enzyme isoforms and the mode of inhibition is competitive for MAO–B. From these results it can be concluded that although the 8–aminocaffeine derivatives are only moderately potent MAO–B inhibitors, they may act as lead compounds for the design of more potent reversible MAO inhibitors. Docking studies revealed that the 8–aminocaffeine and 8–[(methyl)amino]caffeine derivatives traverse both the entrance and substrate cavities of the MAO–B enzyme, with the caffeinyl moiety oriented towards the FAD co–factor while the amino–side chain protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Neurodegenerative disorders like Parkinson’s and Alzheimer’s disease affect millions of people around the world. Oxidative stress has been implicated in the pathogenesis of a number of neurodegenerative disorders, cancer and ischemia. The brain is particularly vulnerable to oxidative damage because of its high utilisation of oxygen, high levels of polyunsaturated fatty acids, relatively high levels of redox transition metal ions and low levels of antioxidants. Oxidative stress occurs due to an imbalance in the pro–oxidant and antioxidant levels. Reactive oxygen/nitrogen species (ROS/RNS) is a collective term used for free radicals and related molecules, promoting oxidative stress within cells and ultimately leading to neurodegeneration. Antioxidants counteract the excess in ROS/RNS, and is therefore of interest in the treatment and prevention of neurodegenerative disorders. Monoamine oxidases, especially monoamine oxidase B (MAO–B), also play an important role in neurodegenerative disorders. MAO–B is the main enzyme responsible for the oxidative deamination of dopamine in the substantia nigra of the brain. By inhibiting MAO–B, dopamine is increased in the brain providing symptomatic relief in Parkinson’s disease. The focus of the current study was to synthesise multifunctional compounds that could be used in the treatment and/or prevention of neurodegenerative diseases. In this study flavonoids were selected because of their wide spectrum of biological activities, including antioxidant activity and its monoamine oxidase inhibition. Flavones and chalcones are both classified under flavonoids and both structures were included. The amantadine moiety was included because of its known ability to inhibit calcium flux through the N–methyl–D–aspartate (NMDA) receptor channel. Six amantadine–flavonoid derivatives were synthesised using standard laboratory procedures and structures were determined with standard methods such as NMR, IR and mass spectrometry. The synthesised compounds were tested in a selection of biological assays, to establish the relative antioxidant properties and MAO inhibitory activity. The biological assays employed to test antioxidant properties were the thiobarbituric acid (TBA) and nitro–blue tetrazolium (NBT) assays. The TBA assay relies on the assessment of lipid peroxidation, induced via hydroxyl anions (OH), generating a pink colour with the complex formation between malondialdehyde (MDA) and TBA, which is measured spectrophotometrically at 532 nm. The principal of the NBT assay is the reduction of NBT to nitro–blue diformazan (NBD), producing a purple colour in the presence of superoxide anions (O2 –). The synthesised compounds were also evaluated for their MAO inhibitory activity toward recombinant human MAO–A and -B and inhibition values were expressed as IC50 values. The experimental data obtained in the NBT and TBA assay indicated a weak but a significant ability to scavenge O2 – and OH. In the NBT assay N–(adamantan–1–yl)–2–{3–hydroxy–4–[(2E)– 3–(3–methoxyphenyl)pro–2–enoyl]phenoxy}acetamide (6) had the best results with a 50.47 ± 1.31 uM/mg protein reduction in NBD formation, indicating that the hydroxyl group contributed to activity. The synthesised compounds were compared to the toxin (KCN) with a reduction in NDB formation of 69.88 ± 1.59 uM/mg protein. Results obtained from the TBA assay indicated that the flavone moiety had better OH scavenging ability than that of the chalcone moiety with N–(adamantan–1–yl)–2–[(5–hydroxy–4–oxo–2–phenyl–4H–chromen–7– yl)oxy]acetamide (3) showing the best activity at 0.967 ± 0.063 nmol MDA/mg tissue. The synthesised compounds were compared to the toxin (H2O2) 1.316 ± 0.028 nmol MDA/mg tissue. None of the test compounds could be compared to the results obtained with Trolox®. The IC50 values obtained for inhibition of recombinant human MAO indicated that the chalcone moiety (N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro–1–en–1–yl]benzamide (5)) showed the best inhibition of MAO–B with an IC50 of 0.717 ± 0.009 M and of MAO–A with an IC50 of 24.987 ± 5.988 M. It was further confirmed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3– phenylpro–1–en–1–yl]benzamide (5) binds reversible to MAO–B and that the mode of inhibition is competitive. Docking studies revealed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro– 1–en–1–yl]benzamide (5) traverses both cavities of MAO–B with the chalcone moiety orientated towards the FAD co–factor while the amantadine moiety protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.