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.

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.

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.