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.

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

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

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

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

Phenelzine (PLZ) is a monoamine oxidase (MAO) inhibitor that also inhibits the activity of GABA-transaminase (GABA-T), causing significant and long-lasting increases in brain GABA levels. Inhibition of MAO prior to PLZ administration has been shown to prevent the GABAergic effects of the drug, strongly suggesting that a metabolite of PLZ formed by the action of MAO is responsible for the GABAergic effects. While PLZ has been used clinically for decades for its antidepressant and antipanic effects, it has more recently been shown to be neuroprotective in an animal model of ischemia. The aim of the experiments described in this thesis was to identify the active metabolite of PLZ, and to determine the neurochemical mechanisms by which PLZ and this metabolite exert their neuroprotective effects (with a particular focus on degenerative mechanisms observed in cerebral ischemia and Alzheimers disease (AD)). The development of an analytical assay for -phenylethylidenehydrazine (PEH) was a major breakthrough in this project and permitted the positive identification of this compound as the active metabolite of PLZ. Further experiments demonstrated that PLZ and PEH could be neuroprotective in cerebral ischemia and AD not only by reducing excitotoxicity via increased GABAergic transmission, but also by (a) increasing brain ornithine, which could potentially lead to a decrease in glutamate synthesis and/or a decrease in polyamines (whose metabolism produces toxic aldehydes); (b) inhibiting the activity of human semicarbazide-sensitive amine oxidase (SSAO), an enzyme whose activity is increased in AD producing excessive amounts of the toxic aldehyde formaldehyde (FA); (c) by sequestering FA in vitro, forming a non-reactive hydrazone product. Since PEH appears to mediate or share the neurochemical effects of PLZ, two propargylated analogs of PEH were synthesized and tested for their potential as PEH prodrugs. Surprisingly these analogs were not particularly effective prodrugs in vivo, but they possessed an interesting neurochemical properties on their own (the ability to elevate brain levels of glycine), and warrant further investigation as potential antipsychotic agents. Together, these results suggest that PLZ and its active metabolite, PEH, should be further investigated for their neuroprotective potential in cerebral ischemia and in AD. / Neurochemistry

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neurochemistry, Department of Psychiatry. Title from pdf file main screen (viewed on October 23, 2009). Includes bibliographical references.

Thesis (M. SC.)--Carleton University, 2001. / Includes bibliographical references (p. 35-53). Also available in electronic format on the Internet.

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.

Selenium is an element that can modulate the dopaminergic neurotransmission. Studies show that diphenyl diselenide, an organic compound of selenium, has antioxidant activity improves depressive-like behavior and reduce the activity of the enzyme monoamine oxidase (MAO). However, there are few studies concerning about possible alterations of diphenyl diselenide in dopaminergic system. Thus, the purpose of the present study was to evaluate the effects of acute and sub-chronic treatment of diphenyl diselenide on amphetamine-induced behavioral and biochemical alterations in mice. In the acute treatment, the mice were treated with diphenyl diselenide (5 and 10 mg/kg, s.c.) or vehicle (10% Tween 80, s.c.) 30 min before administration of amphetamine (1.25 mg/kg, i.p.). After 25 min, locomotor activity was assessed with an open field and, also, the time of stereotypy and immobility was assessed in a glass cage. Sub-chronic treatment was conducted with seven administrations of diphenyl diselenide (5 and 10 mg/kg, s.c.), or its vehicle being one administration per day. On the eighth day, amphetamine (1.25 mg/kg, i.p.) was administered and the behavioral tests were conducted after 25 min. In both treatments ex vivo tests were performed: isoform activity MAO-A and MAO-B, and measurement of total protein and non-protein thiol levels, oxidation of diclorofluorescein. Amphetamine increased the number of crossing and rearing in the open field test and diphenyl diselenide prevented only the increase in the number of crossings when acutely administered to mice. Furthermore, amphetamine increased the time of immobility and stereotypy in mice. Diphenyl diselenide did not prevent these effects. By contrary, at 10 mg/kg, sub-chronic administration of diphenyl diselenide increased per se the time of immobility and stereotypy. It was also found a positive correlation between immobility and stereotypy in acute and sub-chronic treatment with diphenyl diselenide. It was also detected a decrease in brain MAO-B activity caused by sub-chronic treatment with diphenyl diselenide either alone or in combination of amphetamine. Any change was detected in oxidative stress parameters. In conclusion, sub-chronic administration of diphenyl diselenide can promote a behavioral sensitization that seems to be, at least in part, dependent of MAO-B inhibition. / O selênio é um elemento químico capaz importante para o funcionamento celular, envolvido também na modulação do sistema dopaminérgico. Estudos mostram que o disseleneto de difenila, um composto orgânico de selênio, possui atividade antioxidante, melhora o comportamento tipo-depressivo relacionado à inibição da atividade da enzima monoamino oxidase (MAO). No entanto, existem poucos estudos acerca dos efeitos do disseleneto de difenila sobre o sistema dopaminérgico. Desta forma, o objetivo do presente estudo foi avaliar os efeitos do tratamento agudo e sub-crônico do disseleneto de difenila sobre as alterações bioquímicas e comportamentais induzidas por anfetamina em camundongos. No tratamento agudo, os camundongos foram tratados com disseleneto de difenila (5 e 10 mg/kg, s.c.) ou veículo (10% de tween 80, s.c.) 30 minutos antes da administração de anfetamina (1,25 mg/kg, i.p.). Após 25 minutos, foram avaliados a atividade locomotora através do teste de campo aberto, além do tempo de estereotipia e imobilidade, avaliado em caixa espelhada. O tratamento subcrônico foi realizado com sete administrações de disseleneto de difenila ou veículo (5 e 10 mg/kg, s.c.) sendo uma administração por dia. No oitavo dia foi administrada a anfetamina (1,25 mg/kg, i.p.) e realizados os testes comportamentais 25 minutos após. Em ambos os tratamentos os testes ex-vivo realizados foram: atividade das isoformas MAO-A e MAO-B, níveis de tióis totais e não-protéico, oxidação da diclorofluoresceína. O tratamento com anfetamina aumentou o número de cruzamentos e de levantadas no teste do campo aberto e o disseleneto de difenila preveniu somente o número de cruzamentos quando administrado agudamente aos camundongos. Além disso, o tratamento com anfetamina aumentou o tempo de imobilidade e estereotipia em camundongos. O disseleneto de difenila não preveniu estes efeitos. Pelo contrário, na dose de 10 mg/kg, a administração subcrônica de disseleneto de difenila aumentou per se o tempo de imobilidade e de estereotipia. Uma correlação positiva entre o tempo de estereotipia e de imobilidade foi também encontrada tanto para o tratamento agudo como subcrônico com disseleneto de difenila. Também foi detectada uma diminuição na atividade cerebral da MAO-B causada pelo tratamento subcrônico com disseleneto de difenila tanto per se quanto em combinação com a anfetamina. Não foram encontradas alterações em parâmetros de estresse oxidativo. Em conclusão, o tratamento subcrônico com disseleneto de difenila pode promover uma sensibilização comportamental que parece ser, pelo menos em parte, dependente da inibição da MAO-B.