BACKGROUND AND RATIONALE Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder affecting the central nervous system, primarily, the substantia nigra. It is characterized by loss of dopaminergic neurons in the nigro-striatal pathway, and ultimately patients with Parkinson’s disease may lose up to 80% of their dopamine-producing cells in the brain. Symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Symptomatic relief is obtained by using levodopa and various adjunct therapy including dopamine agonists, catechol-O-methyltransferase inhibitors and monoamine oxidase B inhibitors. Levodopa is used as the gold-standard for treatment of this disease. It effectively controls motor symptoms, however, motor complications that impair the quality of life develop with continued levodopa use. No treatments currently available can halt disease progression, therefore novel drugs that can slow down or stop disease progression are urgently required. The monoamine oxidase (MAO) A and B enzymes are flavoenzymes that play an important role in the oxidative degradation of amine neurotransmitters such as dopamine, serotonin and epinephrine. Early attempts to block dopamine metabolism in the brain using nonselective MAO inhibitors was effective but led to side effects such as hypertensive crisis, thus they lost favor. The MAO-B enzyme is of particular importance in Parkinson’s disease because it is more active than MAO-A in the basal ganglia, and is thus primarily responsible for the catabolism of dopamine in the brain. Selegiline and rasagiline, both irreversible, selective MAO-B inhibitors have proven efficacy in symptomatic treatment of Parkinson’s disease, but due to the irreversible nature of their binding, it can take several weeks after treatment termination for the enzyme to recover. Use of reversible inhibitors such as lazabemide and safinamide do not have this disadvantage, and have safer side effect profiles. Unfortunately, clinical trials for lazabemide use in Parkinson’s disease have been discontinued. Therefore, due to the lack of disease modifying agents for Parkinson’s disease, as well as safety concerns of current PD therapy, an urgent need exists for novel, safe and efficient MAO inhibitors. Current research is thus aimed at designing selective or non-selective reversible inhibitors that bind competitively to the enzyme. The MAO inhibitory potential of chromone derivatives has been illustrated previously. Evaluation of C6- and C7-alkyloxy substituted chromones, for example revealed that these compounds were potent, selective and reversible MAO-B inhibitors. It has further been shown that chromone 3-carboxylic acid is a potent selective, irreversible MAO-B inhibitor. Phenylcarboxamide substitution in position 3 of chromone 3-carboxylic acid also results in potent, selective MAO-B inhibitory activity. Therefore, further evaluation of the effect of substitution with flexible side chains in the 3-position to evaluate MAO-B inhibition is of importance. The chromone ring system is thus a privileged scaffold for the design of inhibitors that are selective for MAO-B and has the additional advantages of generally exhibiting low mammalian toxicity and ease of synthesis. AIM The aim of this study was to design, synthesize and evaluate novel chromone derivatives as inhibitors of monoamine oxidase. RESULTS Design and Synthesis 3-Aminomethylene-2,4-chromandiones and ester chromone derivatives were synthesized by coupling several aromatic and aliphatic amines and alcohols, to chromone 3-carboxylic acid, in the presence of CDI (carbonyldiimidazole). 15 Compounds were successfully synthesized and characterized by using NMR and IR spectroscopy, as well as mass spectrometry. X-ray crystallography was used to obtain a crystal structure for the 3-aminomethylene-2,4- chromandione derivative, 46, in a bid to verify the structures of the synthesized compounds. Melting points of all compounds were determined, and the purity determined using HPLC techniques. MAO inhibition studies A fluorometric assay was employed using kynuramine as substrate, to determine the IC50 (50% inhibition concentration) values and SI (selectivity index) of the synthesized compounds. Generally, the esters exhibited weak MAO-A and MAO-B inhibition, while the 3- aminomethylene-2,4-chromandione derivatives showed promise as selective MAO-B inhibitors, with IC50 values in the micromolar range. Compound 38, 3- [(benzylamino)methylidene]-3,4-dihydro-2H-1-benzopyran-2,4-dione, was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM and a SI of 122 for MAO-B inhibition. Interesting trends were revealed through analysis of the structure activity relationships, for example, for the 3-aminomethylene-2,4-chromandione derivatives, the presence of a chlorine moiety in the side chains of the compounds resulted in a decrease of MAO-B inhibition activity. Chain elongation further also resulted in weakening the MAO-B inhibition activity, while chain elongation in the ester derivatives led to a slight increase in MAO-B inhibition activity. Reversibility studies The reversibility of binding of the most potent compound in the 3-aminomethylene-2,4- chromandione series, 38, was evaluated. None of the synthesized inhibitors were potent MAO-A inhibitors, therefore reversibility of MAO-A inhibition was not examined. Recovery of enzyme activity was determined after dialysis of the enzyme-inhibitor complexes. Analysis of the kinetic data obtained showed that MAO-B catalytic activity was recovered to 115% of the control value. This suggests that compound 38 is a reversible inhibitor of MAO-B. Mode of inhibition A set of Lineweaver-Burk plots were constructed to determine mode of inhibition of compound 38. The results show linear lines that intersect at a single point just to the left on the y-axis. This indicates that compound 38 interacts competitively with the MAO-B enzyme. In conclusion, chromone derivatives were synthesized and evaluated as inhibitors of MAO. Compound 38 was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM. The effect of chain elongation and introduction of flexible substituents in position 3 of the chromone 3-carboxylic acid nucleus was explored and the results showed that 3- aminomethylene-2,4-chromandione substitution is preferable over ester substitution. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

BACKGROUND AND RATIONALE Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder affecting the central nervous system, primarily, the substantia nigra. It is characterized by loss of dopaminergic neurons in the nigro-striatal pathway, and ultimately patients with Parkinson’s disease may lose up to 80% of their dopamine-producing cells in the brain. Symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Symptomatic relief is obtained by using levodopa and various adjunct therapy including dopamine agonists, catechol-O-methyltransferase inhibitors and monoamine oxidase B inhibitors. Levodopa is used as the gold-standard for treatment of this disease. It effectively controls motor symptoms, however, motor complications that impair the quality of life develop with continued levodopa use. No treatments currently available can halt disease progression, therefore novel drugs that can slow down or stop disease progression are urgently required. The monoamine oxidase (MAO) A and B enzymes are flavoenzymes that play an important role in the oxidative degradation of amine neurotransmitters such as dopamine, serotonin and epinephrine. Early attempts to block dopamine metabolism in the brain using nonselective MAO inhibitors was effective but led to side effects such as hypertensive crisis, thus they lost favor. The MAO-B enzyme is of particular importance in Parkinson’s disease because it is more active than MAO-A in the basal ganglia, and is thus primarily responsible for the catabolism of dopamine in the brain. Selegiline and rasagiline, both irreversible, selective MAO-B inhibitors have proven efficacy in symptomatic treatment of Parkinson’s disease, but due to the irreversible nature of their binding, it can take several weeks after treatment termination for the enzyme to recover. Use of reversible inhibitors such as lazabemide and safinamide do not have this disadvantage, and have safer side effect profiles. Unfortunately, clinical trials for lazabemide use in Parkinson’s disease have been discontinued. Therefore, due to the lack of disease modifying agents for Parkinson’s disease, as well as safety concerns of current PD therapy, an urgent need exists for novel, safe and efficient MAO inhibitors. Current research is thus aimed at designing selective or non-selective reversible inhibitors that bind competitively to the enzyme. The MAO inhibitory potential of chromone derivatives has been illustrated previously. Evaluation of C6- and C7-alkyloxy substituted chromones, for example revealed that these compounds were potent, selective and reversible MAO-B inhibitors. It has further been shown that chromone 3-carboxylic acid is a potent selective, irreversible MAO-B inhibitor. Phenylcarboxamide substitution in position 3 of chromone 3-carboxylic acid also results in potent, selective MAO-B inhibitory activity. Therefore, further evaluation of the effect of substitution with flexible side chains in the 3-position to evaluate MAO-B inhibition is of importance. The chromone ring system is thus a privileged scaffold for the design of inhibitors that are selective for MAO-B and has the additional advantages of generally exhibiting low mammalian toxicity and ease of synthesis. AIM The aim of this study was to design, synthesize and evaluate novel chromone derivatives as inhibitors of monoamine oxidase. RESULTS Design and Synthesis 3-Aminomethylene-2,4-chromandiones and ester chromone derivatives were synthesized by coupling several aromatic and aliphatic amines and alcohols, to chromone 3-carboxylic acid, in the presence of CDI (carbonyldiimidazole). 15 Compounds were successfully synthesized and characterized by using NMR and IR spectroscopy, as well as mass spectrometry. X-ray crystallography was used to obtain a crystal structure for the 3-aminomethylene-2,4- chromandione derivative, 46, in a bid to verify the structures of the synthesized compounds. Melting points of all compounds were determined, and the purity determined using HPLC techniques. MAO inhibition studies A fluorometric assay was employed using kynuramine as substrate, to determine the IC50 (50% inhibition concentration) values and SI (selectivity index) of the synthesized compounds. Generally, the esters exhibited weak MAO-A and MAO-B inhibition, while the 3- aminomethylene-2,4-chromandione derivatives showed promise as selective MAO-B inhibitors, with IC50 values in the micromolar range. Compound 38, 3- [(benzylamino)methylidene]-3,4-dihydro-2H-1-benzopyran-2,4-dione, was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM and a SI of 122 for MAO-B inhibition. Interesting trends were revealed through analysis of the structure activity relationships, for example, for the 3-aminomethylene-2,4-chromandione derivatives, the presence of a chlorine moiety in the side chains of the compounds resulted in a decrease of MAO-B inhibition activity. Chain elongation further also resulted in weakening the MAO-B inhibition activity, while chain elongation in the ester derivatives led to a slight increase in MAO-B inhibition activity. Reversibility studies The reversibility of binding of the most potent compound in the 3-aminomethylene-2,4- chromandione series, 38, was evaluated. None of the synthesized inhibitors were potent MAO-A inhibitors, therefore reversibility of MAO-A inhibition was not examined. Recovery of enzyme activity was determined after dialysis of the enzyme-inhibitor complexes. Analysis of the kinetic data obtained showed that MAO-B catalytic activity was recovered to 115% of the control value. This suggests that compound 38 is a reversible inhibitor of MAO-B. Mode of inhibition A set of Lineweaver-Burk plots were constructed to determine mode of inhibition of compound 38. The results show linear lines that intersect at a single point just to the left on the y-axis. This indicates that compound 38 interacts competitively with the MAO-B enzyme. In conclusion, chromone derivatives were synthesized and evaluated as inhibitors of MAO. Compound 38 was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM. The effect of chain elongation and introduction of flexible substituents in position 3 of the chromone 3-carboxylic acid nucleus was explored and the results showed that 3- aminomethylene-2,4-chromandione substitution is preferable over ester substitution. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Magister Pharmaceuticae - MPharm / Alzheimer's disease is a progressive neurodegenerative disease characterised by low acetylcholine (ACh) levels in the hippocampus and cortex of the brain, causing symptoms like progressive memory loss, decline in language skills and other cognitive impairments to occur. The hallmarks of AD include the presence of extracellular insoluble amyloid beta plaques, intracellular neurofibrillary tangles, and the decrease in ACh concentration. The pathophysiology of AD is not well understood, however, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and monoamine oxidases (MAO) are conspicuous role players in AD pathogenesis. Based on the cholinergic hypothesis, the AChE inhibitor donepezil was developed and has been used effectively clinically in the management of AD, with minimal side effects. Studies regarding the binding interactions of donepezil with AChE has shown that the benzyl-piperidine moiety of this compound shows substantial binding interactions at the CAS site of AChE where it blocks AChE activity. Coumarin is a compound of natural source that has shown some MAO inhibitory activity. Further studies done to clarify the potential of coumarin as a drug against AD has shown that coumarin has the capacity to bind at the PAS site of AChE, thus giving it the potential to prevent AChE induced amyloid plaque formation. Due to the multifactorial nature of AD, the drugs in the market show limited therapeutic benefits and are mainly for symptomatic relief. In order to address this limitation in AD treatment, researchers are exploring the possibility of designing a multi-target-directed-ligand (MTDL). The aim of this study was to synthesise a series of compounds out of pharmacophoric groups of donepezil and coumarin that will be able to inhibit both cholinesterases and MAO B. Four series of 5 compounds per series were synthesised. The first series of compounds consisted of the coumarin moiety to which a 1,4-dibromo benzene moiety was attached. The second series represented the coumarin moiety to which a piperidine (donepezil moiety shown to confer cholinesterase inhibitory property) was attached. The third series represented the coumarin moiety to which bromobenzyl-piperazine was attached and in the last series were compounds similar in structure to series 1 with an unsubstituted benzyl moiety as opposed to the dibromobenzyl moiety. Prior to the synthesis, molecular modelling was conducted in order to have an idea of the binding capacity of the compounds to MAO A and B and cholinesterases. In vitro biological evaluation of the compounds was done and used to determine the IC₅₀ values of the compounds. Nineteen compounds were synthesised and purified successfully as shown by their NMR, MS and IR spectra. The compounds to which dual inhibitory activity was conferred were those in series 2 and 3, of which series 2 showed the best overall inhibitory activity with IC₅₀ values within the low μM range. The compound with the best overall activity was Cp 9. Molecular modelling of Cp 9 showed that the coumarin core was located in the PAS region of AChE while the benzyl-piperidine moiety was situated in the CAS region of the enzyme. This compound orientation demonstrates the potential of Cp 9 to inhibit AChE induced amyloid beta plaque formation. Cp 9 showed no inhibitory activity towards MAO A, but showed good inhibitory activity towards MAO B with an IC₅₀ value of 0.30 μM. Its inhibitory activity towards cholinesterases also fell within the low μM range (AChE IC50 = 9.1 μM and BuChE IC₅₀ = 5.9 μM). From the results, it can be concluded that Cp 9 was able to inhibit both cholinesterase and MAO B catalytic activities at low μM concentrations. This thus means that our novel compound will not only increase ACh levels in the brain thus improving cognitive activity, but it will also have neuroprotective effect from its MAO B inhibitory property and also potentially slow down amyloid plaque formation due to AChE activity. / National Research Foundation (NRF)

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

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

R-(-)-Phenylephrine (PE) is the most commonly used nonprescription oral nasal decongestant in the United States. It is a selective α1-adrenergic receptor agonist and has many years of safe usage. However, the efficacy of PE is controversial, due to its extensive pre-systemic metabolism, which leads to low and variable oral bioavailability (38 ± 9%, mean ± SD). Sulfation plays a very important role in pre-systemic metabolism of PE. The sulfation of PE occurs at its phenolic group, which is the preferred structural feature of many sulfotransferase (SULT) substrates. Compounds with phenolic groups have similar structures to PE, which may share the same SULT isoforms with PE and have the potential to inhibit PE sulfation. Co-administration of the phenolic compounds from the Food and Drug Administration’s (FDA) “Generally Recognized as Safe” (GRAS) list, Everything Added to Food in the United States (EAFUS), or dietary supplements along with PE could be an effective strategy to inhibit the pre-systemic sulfation of PE. The primary side effect of PE is hypertension. Since monoamine oxidase (MAO) inhibitors may increase the risk of hypertension, they should not be taken with PE. In order to increase the oral bioavailability and eventually improve the efficacy of PE, this research project aimed to investigate the feasibility of inhibiting the pre-systemic sulfation of PE with phenolic dietary compounds. Considering the safety issue, this research project also aimed to investigate whether these phenolic dietary compounds have inhibitory effects on MAO-A/B. A human colon adenocarcinoma epithelial cell line (LS180), which shows sulfation activity, was used as a model to test the effect of these phenolic compounds on the sulfation of PE. The extent of disappearance of PE was significantly (p < 0.05) decreased to the following (mean ± SEM, as % of control) when incubated with phenolic dietary compounds in LS180 cells for 14 - 19 hrs: curcumin 24.5 ± 14.0%, guaiacol 51.3 ± 8.0%, isoeugenol 73.9 ± 4.3%, pterostilbene 70.6 ± 4.2%, resveratrol 14.2 ± 28.0%, zingerone 52.4 ± 14.6%, and the combinations eugenol + propylparaben 42.6 ± 8.4%, vanillin + propylparaben 37.0 ± 11.2%, eugenol + propylparaben + vanillin + ascorbic acid 31.1 ± 10.9%, eugenol + vanillin 57.5 ± 20.6%, and pterostilbene + zingerone 36.5 ± 7.0%. The combinations of curcumin + resveratrol and curcumin + pterostilbene + resveratrol + zingerone almost completely inhibited PE disappearance. PE sulfate formation was inhibited 67.0 ± 4.2% (mean ± SEM, as % of control) by guaiacol and 71.7 ± 2.6% by pterostilbene + zingerone. The combinations of curcumin + resveratrol and curcumin + pterostilbene + resveratrol + zingerone inhibited ≥ 99% of PE sulfate formation. These results were consistent with those from analysis of the disappearance of PE in LS180 cells. These phenolic inhibitors for sulfation were also tested to see whether they have any inhibitory effects on MAO-A or B. Significant inhibition was found with curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone on both MAO-A and B. Further kinetic studies were conducted to investigate the concentration of an inhibitor at which the enzyme activity is reduced by half (IC50) (mean ± SEM) of these inhibitors. The most potent inhibitor for MAO-A was resveratrol (0.313 ± 0.008 μM) followed by isoeugenol (3.72 ± 0.20 μM), curcumin (12.9 ± 1.3 μM), pterostilbene (13.4 ± 1.5 μM), zingerone (16.3 ± 1.1 μM), and guaiacol (131 ± 6 μM). The most potent inhibitor for MAO-B was pterostilbene (0.138 ± 0.013 μM), followed by curcumin (6.30 ± 0.11 μM), resveratrol (15.8 ± 1.3 μM), isoeugenol (102 ± 5 μM), and guaiacol (322 ± 27 μM). Since these phenolic compounds all have relatively low oral bioavailability, any MAO inhibition which could occur systemically is expected to be limited. Most inhibitory effects on MAO-A and B if any would be limited to the GI tract and liver. In conclusion, several compounds and combinations showed inhibition on PE sulfation in LS180 cell model, which may have potential to inhibit the pre-systemic sulfation of PE to improve its oral bioavailability. These compounds also showed the unexpected inhibition on human MAO-A and B with different potency, which could guide the selection of phenolic dietary compounds for further studies, along with the sulfation inhibition results and their pharmacokinetic (PK) properties such as bioavailability.

The plant Garcinia gardneriana (Planch. & Triana) Zappi, popularly known in Brazil as "bacupari" has traditionally been used for various types of inflammatory diseases and the evaluation of their chemical composition, mainly of leaves, has resulted in biflavonoids as major compounds. These phenolic compounds have shown anti-inflammatory activity validating the popular use of the plant. In this work was isolated from dried branches of Garcinia gardneriana the biflavonoids: morelloflavone, that is an naringenin covalently linked to luteolin, Gb-2a which is an naringenin linked to eriodictyol and Gb-2a- 7-O-glucose. These compounds have been previously evaluated in various activities such as anti-inflammatory and anti-antioxidants but there is no report of its activity as enzymatic inhibitors. However, the monomers that form it, have been evaluated in the inhibition of aromatase and antidepressant activity with positive outcome, which commonly are used MAO-A inhibitors. In the isolation process were also founded terpenoid compounds as lupeol and friedelin The isolated and purified biflavonoids were used to evaluate enzyme inhibition "in vitro" in monoamine oxidases (MAO-A MAO-B) and aromatase. The compounds showed a positive response even of IC50 5,47 μM and 1,35 μM for MAO-A inhibition of and aromatase enzyme respectively; discovering a way for a new proposal to link both enzymes for treatment of hormone-dependent cancers and anxiety and depression disorders. / La planta Garcinia gardneriana (Planch. & Triana) Zappi, popularmente conocida en Brasil como "bacupari" ha sido tradicionalmente usada para varios tipos de enfermedades inflamatorias y la evaluación de su composición química, principalmente de las hojas, ha resultado en biflavonoides como compuestos mayoritarios. Estos compuestos fenólicos han demostrado actividad anti-inflamatória validando el uso popular de la planta. En este trabajo se asilaron a partir de tallos secos de la Garcinia gardneriana los biflavonoides: moreloflavona, que consiste en una naringenina unida covalentemente a luteolina, Gb-2a que es un compuesto que consiste en una naringenina unida a un eriodictyol y Gb-2a-7-O-glucose. Estos compuestos ya han sido previamente evaluados en diversas actividades como anti inflamatorios y anti antioxidantes pero no se tiene reporte de su actividad como inhibidores enzimáticos. Sin embargo, los monomeros que los conforman han sido evaluados en la inhibición de la aromatasa y con resultados positivos como en la actividad antidepresiva, para la cual comúnmente son usados los inibidores de MAO-A. En el proceso de aislamiento también fueron encontrados compuestos terpenoides como lupeol y friedelina. Los biflavonoides aislados y purificados se usaron para evaluar la inhibición enzimática “in vitro” en monoaminooxidasas (MAO-A, MAO-B) y aromatasa. Los compuestos presentaron una respuesta positiva calculada con IC50 de hasta 5,47 μM y 1,35 μM para la inhibición de las enzimas MAO-A y aromatasa respectivamente, abriendo el camino a una nueva propuesta de relacionar estas dos enzimas para tratamiento de cánceres hormonodependientes y transtornos de ansiedad y depresión.

No description available.

Background and rationale - Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of dopamine in the brain. The cause of Parkinson's disease is still unknown; however several theories pertaining to the etiology exist. Current treatment mainly aims at dopamine replacement, with agents such as levodopa and dopamine agonists that provide patients with symptomatic relief. This relief is unfortunately only temporary as the progression of the disease is not halted. Furthermore, these therapies are associated with a range of side effects and novel approaches to the treatment are thus urgently required. Adenosine A2A receptor antagonists recently emerged as a promising non-dopaminergic alternative, not only as symptomatic treatment, but also as potential neuroprotective therapy. Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors (A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine depletion to give symptomatic relief. There are also several mechanisms where by adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A antagonists have already reached clinical trials and promising results were obtained, especially when combined with levodopa. Consequently, A2A antagonists are realistic prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor antagonists. Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of inhibition comes with certain disadvantages as it may take up to several weeks after termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast will have much better safety profiles seeing that they will not inactivate the enzyme permanently and allow for competition with the substrate. When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease. MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the progression of the disease by preventing the formation of these byproducts. Promising MAOB inhibitory activities have been reported for chalcones, and since the intermediates obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B. Results - Design and synthesis: A series of 2-aminopyrimidines were designed using known active structures and literature pharmacophores. A molecular modelling study (Discovery Studio 3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked in the binding site of the adenosine A2A receptor. Binding orientations and observed interactions with important residues in the active site were similar to those observed for known A2A antagonists. It was therefore concluded that these compounds may be potential A2A antagonists and the designed compounds were thus synthesised. Structures were primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry. MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay and kynuramine as substrate. The compounds were potent and selective inhibitors of the MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1- (5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of 0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A. Some preliminary structure activity relationships were derived, for example, phenyl substitution with an electron withdrawing chlorine group generally resulted in better activity than substitution with electron donating methoxy groups. Further investigation of structure activity relationships are however required as a very small series of chalcones were screened. Reversibility studies and mode of inhibition: A dilution assay was used to determine whether compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor complex. The results from the reversibility studies showed that the inhibition of the most potent compound (1c) is reversible as the catalytic activities are recovered to approximately 80% and 50% respectively, compared to the control measured in the absence of an inhibitor. For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver- Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor of the MAO-B enzyme. In vitro adenosine A2A assays: Radioligand binding assays were used to determine the affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine (CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM. The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part, be explained by the molecular modellling studies. In the docking experiments an additional hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271 indicating that this structural feature is a major contributing factor to the improved affinity observed for these derivatives. In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to determine whether the two compounds with the highest affinity for the adenosine A2A receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine A2A antagonists. The objectives of this study as set out were thus successfully realised and promising results were obtained. During this study, several novel 2-aminopyrimidines and chalcones were synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase inhibitory activities for all of the screened compounds were determined for the first time. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Background and rationale: Parkinson’s disease (PD) is a progressive, degenerative disorder of the central nervous system and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The loss of functional dopamine in the striatum is thought to be responsible for the typical symptoms of PD. Cardinal features of PD include bradykinesia, muscular rigidity, resting tremor and impairment of postural balance. This study focuses on the inhibition of monoamine oxidase B (MAO-B) and antagonism of A2A receptors as therapeutic strategies for PD. Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)-containing mitochondrial bound isoenzyme which consists of two isoforms namely MAO-A and MAO-B. The primary function of MAO is to catalyze the oxidative deamination of dietary amines, monoamine neurotransmitters and hormones. MAO-A is responsible for the oxidative deamination of serotonin (5-HT) and norepinephrine (NE), while MAO-B is responsible for the oxidative deamination of dopamine (DA). The formation of DA takes place in the presynaptic neuron where it is stored in vesicles and released into the presynaptic cleft. The released DA then either binds to D1 and D2 receptors which results in an effector response. The excess DA in the presynaptic cleft is metabolized by MAO-B which may result in the formation of free radicals and a decrease in DA concentrations. Under normal physiological conditions free radicals are removed from the body via normal physiological processes, but in PD these normal physiological processes are thought to be unable to remove the radicals and this may lead to oxidative stress. Oxidative stress is believed to be one of the leading causes of neurodegeneration in PD. The rationale for the use of MAO-B inhibitors in PD would be to increase the natural DA levels in the brain and also diminish the likelihood of free radicals to be formed. Adenosine is an endogenous purine nucleoside and yields a variety of physiological effects. Four adenosine receptor subtypes have been characterized: A1, A2A, A2B and A3. They are all part of the G-protein-coupled receptor family and have seven transmembrane domains. The A2A receptor is highly concentrated in the striatum. There are two important pathways in the basal ganglia (BG) through which striatal information reaches the globus pallidus, namely the direct pathway containing A1 and D1 receptors and the indirect pathway containing A2A and D2 receptors. The direct pathway facilitates willed movement and the indirect pathway inhibits willed movement. A balance of the two pathways is necessary for normal movement. In PD, there is a decrease in DA in the striatum, thus leading to unopposed A2A receptor signaling and ultimately resulting in overactivity of the indirect pathway. Overactivity of the indirect pathway results in the locomotor symptoms associated with PD. Treatment with an A2A antagonist will block the A2A receptor, resulting in the restoration of balance between the indirect and direct pathways, thus leading to a decrease in locomotor symptoms. Aim: In this study, caffeine served as a lead compound for the design of dual-targeted drugs that are selective, reversible MAO-B inhibitors as well as A2A antagonists. Caffeine is a very weak MAO-B inhibitor and a moderately potent A2A antagonist. Substitution on the C8 position of caffeine yields compounds with good MAO-B inhibition activities and A2A receptor affinities. An example of this behaviour is found with (E)-8-(3-chlorostyryl)caffeine (CSC), which is not only a potent A2A antagonist but also a potent MAO-B inhibitor. The goal of this study was to identify and synthesize dual-targeted xanthine compounds. Recently Swanepoel and co-workers (2012) found that 8-phenoxymethyl substituted caffeines are potent reversible inhibitors of MAO-B. Therefore, this study focused on expanding the 8-(phenoxymethyl)caffeine series and evaluating the resulting compounds as both MAO-A and -B inhibitors as well as A2A antagonists. Synthesis: Two series were synthesized namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines. The analogues were synthesized according to the literature procedure. 1,3-Dimethyl-5,6-diaminouracil or 1,3-diethyl-5,6-diaminouracil were used as starting materials and were acylated with a suitable substituted phenoxyacetic acid in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) as an activating reagent. The intermediary amide was treated with sodium hydroxide, which resulted in ring closure to yield the corresponding 1,3-dimethyl-8-phenoxymethyl-7Hxanthinyl or 1,3-diethyl-8-phenoxymethyl-7H-xanthinyl analogues. These xanthines were 7-N-methylated in the presence of an excess of potassium carbonate and iodomethane to yield the target compounds. In vitro evaluation: A radioligand binding assay was performed to determine the affinities of the synthesized compounds for the A2A receptor. The MAO-B inhibition studies were carried out via a fluorometric assay where the MAO-catalyzed formation of H2O2 was measured. Results: Both series showed good to moderate MAO-B inhibition activities, while none of the compounds had activity towards MAO-A. Results were comparable to that of a known MAOB inhibitor lazabemide. For example, lazabemide (IC50 = 0.091 μM) was twice as potent as the most potent compound identified in this study, 8-(3-chlorophenoxymethyl)caffeine (compound 3; IC50 = 0.189 μM). Two additional compounds, 8-(4-iodophenoxymethyl)caffeine and 8-(3,4-dimethylphenoxymethyl) caffeine, also exhibited submicromolar IC50 values for the inhibition of MAO-B. The structure-activity relationships (SARs) indicated that 1,3-diethyl substitution resulted in decreased inhibition potency towards MAO-B and that 1,3-dimethyl substitution was a more suitable substitution pattern, leading to better inhibition potencies towards MAO-B. The compounds were also evaluated for A2A binding affinity, and relatively weak affinities were recorded with the most potent compound, 1,3-diethyl-7-methyl-8-[4-chlorophenoxymethyl]xanthine (compound 16), exhibiting a Ki value of 0.923 μM. Compared to KW-6002 (Ki = 7.94 nM), a potent reference A2A antagonist, compound 16 was 35-fold less potent. Comparing compound 16 to CSC [Ki(A2A) = 22.6 nM; IC50(MAO-B) = 0.146 nM], it was found that compound 16 is 31-fold less potent as an A2A antagonist and 21-fold less potent as a MAO-B inhibitor. Loss of MAO-B inhibition potency may be attributed to 1,3-diethyl substitution which correlates with similar conclusions reached in earlier studies. In addition, the replacement of the styryl functional group (as found with CSC and KW-6002) with the phenoxymethyl functional group (as found with the present series) may explain the general reduction in affinity for the A2A receptor. This suggests that the styryl side chain is more appropriate for A2A antagonism than the phenoxymethyl functional group. Conclusion: In this study two series of xanthine derivatives were successfully synthesized, namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines (11 compounds in total). Three of the newly synthesized compounds were found to act as potent inhibitors of MAO-B, with IC50 values in the submicromolar range. None of the compounds were however noteworthy MAO-A inhibitors. The most potent A2A antagonist among the examined compounds, compound 16, proved to be moderately potent compared to the reference antagonists, CSC and KW-6002. It may be concluded that the styryl functional group (as found with CSC and KW-6002) is more optimal than the phenoxymethyl functional group (as found with the present series) for A2A antagonism. 1,3-Diethyl substitution of the xanthine ring was found to be less optimal for MAO-B inhibition compared to 1,3-dimethyl substitution. These results together with known SARs provide valuable insight into the design of 8-(phenoxymethyl)caffeines as selective and potent MAO-B inhibitors. Such drugs may find application in the therapy of PD. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.