Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015