>Magister Scientiae - MSc / Alzheimer’s Disease (AD) is a neurodegenerative disease which results from the irreversible loss of neurons in the brain. The disease is characterized by progressive cognitive impairment with recurrent short-term memory loss. AD is the leading cause of dementia and 4th leading cause of death in the elderly. Success in the treatment of AD has been limited, with drugs only treating it at a symptomatic level due to its pathology being complex and poorly understood. However, it is known that the cholinesterase and MAO-B enzymes play an important role in the disease through their association with production of amyloid plaques and oxidative stress respectively, two mechanisms associated with cell death and the symptoms seen in AD.

Magister Pharmaceuticae - MPharm / Neurodegenerative disorders (NDs) are a range of chronic brain disorders that includes amongst others motor function loss. Parkinson’s disease (PD) is one of the common NDs that has an insidious onset and diagnosed when dopaminergic neurons in the substantia nigra are already lost. The loss creates a deficiency of the dopamine (neurotransmitter) thereby causing neurochemical imbalance resulting in the signs and symptoms of PD. NDs overlap at multiple levels so some of the symptoms overlap as well. NDs currently have no cure yet and current drug therapies only improve the quality of life of the patients by targeting the symptoms mainly. Treatment of PD currently involves different classes of drugs and depending on the stages of the disease, some drugs can be only used as an adjunct therapy. Anti-oxidants and monoamine oxidase inhibitors (MAO-I) are part of the treatment options.

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Monoamine oxidase (MAO) is a mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of monoamines in a wide array of organisms. While the enzyme monoamine oxidase has been studied extensively in its role in moderating behavior in mammals, there is a paucity of research investigating this role in invertebrates, where the latter utilizes this enzyme in a major pathway to degrade monoamines. There is especially a dismal lack of information on how MAO influences activity in invertebrates, particularly in account of the circadian cycle. Previous studies revealed MAO degrades serotonin and norepinephrine in arachnids, but did not investigate other critically important compounds like octopamine. Larinioides cornutus is a species of orb-weaving spider that exhibits diel fluctuations in behavior, specifically levels of aggression. The monoamines octopamine and serotonin have been shown to influence aggressive behaviors in L. cornutus, thus this species was used to investigate if MAO is a potential site of regulation throughout the day. Not only did gene expression of MAO orthologs and MAO activity fluctuate at different times of day, but the enzymatic activity was substrate-specific producing a higher level of degradation of octopamine as compared to serotonin in vitro. This study further supports evidence that MAO has an active role in monoamine inactivation in invertebrates and provides a first look at how MAO ultimately may be regulating behavior in an invertebrate.

The parkinsonian inducing drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated in a reaction catalyzed by the flavoenzyme monoamine oxidase B (MAO-B) to form the corresponding dihydropyridinium (MPDP+) subsequently pyridinium (MPP+) metabolites. As part of our ongoing studies to characterize the structural features responsible for this unexpected biotransformation, we have synthesized and examined the MAO-B substrate properties of a variety of MPTP analogs bearing various heteroaryl groups at the 4-position of the tetrahydropyridinyl ring. The results of these SAR studies indicate that electronic features, steric features and polar interactions can contribute to the substrate activities. Additionally, isotope effects have been examined to investigate the mechanism and stereoselectivity of the MAO-B catalytic pathway. The synthesis and characterization of regio and stereoselectively deuterated MPTP analogs have been achieved. The results indicate that the catalytic step occurs exclusively at the allylic C-6 position and is rate-determining for both good and poor substrates. The two enantiomers of MPTP bearing a deuterium atom at C-6 have been prepared via chiral aminooxazolinyl derivatives and have been characterized by 2H NMR in a chiral liquid crystal matrix. These enantiomers were used to determine the selectivity of the MAO-B catalyzed a C-H bond cleavage reaction leading to the dihydropyridinium metabolite MPDP+. Some of the cyclopropyl analogs of MPTP have also been synthesized as the potential inhibitors. / Ph. D.

The neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) protects against the neurotoxicity of MPTP in a mouse model of neurodegeneration. Since 7-NI also inhibits the monoamine oxidase-B (MAO-B) catalyzed bioactivation of MPTP, the role of nNOS inhibition as a mediator of 7-NI's neuroprotective properties have been challenged. In order to examine in greater detail the neuroprotective effects of indazolyl derivatives, the synthesis of water soluble indazolyltetrahydropyridinyl derivatives as potential "prodrugs" that may undergo MAO bioactivation in the brain was undertaken. During the course of the studies on the synthesis of indazolylpyridinium derivatives, precursors to these "prodrugs", an interesting reaction involving the rearrangement of 4-(2H-indazolyl)-1-methylpyridinium iodide to the corresponding 1H-isomer was encountered. A detailed investigation of this rearrangement reaction is reported in this thesis. The syntheses and interaction of nitroindazolyltetrahydropyridinyl "prodrugs" with MAO-B have been investigated previously. Molecular docking studies that attempt to explain the MAO-B substrate and inhibitor properties of members of this series of compounds are described. Finally, the MAO-A substrate properties of nitroindazolyltetrahydropyridinyl derivatives are reported. / Ph. D.

The flavin-containing monoamine oxidases (MAO) A and B catalyze the oxidative deamination of primary and secondary amines. The overall process involves a two electron oxidation of the amine to the iminium with concomitantreduction of the flavin. Based on extensive studies with a variety of chemical probes, Silverman and colleagues have proposed a catalytic pathway for the processing of amine substrates and inactivators by MAO-B that is initiated by a single electron transfer (SET) step from the nitrogen lone pair to the oxidized flavin followed by α-proton loss from the resulting amine radical cation that leads to a carbon radical. Subsequent transfer of the second electron leads to the reduced flavin and the iminium product. In the case of N-cyclopropylamines, the initially formed amine radical cation is proposed to undergo rapid ring opening to form a highly reactive primary carbon centered radical that is thought to be responsible for inactivation of the enzyme. In this thesis we have exploited the unique substrate and inactivator properties of 1,4-disubstituted tetrahydropyridine derivatives to probe the mechanism of MAO-B catalysis. Reports of the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a structurally unique substrate of MAO-B initiated these studies. Consistent with the SET pathway, the N-cyclopropyl analog of MPTP proved to be an efficient time and concentration dependent inactivator but not a substrate of MAO-B. On the other hand, the 4-benzyl-1-cyclopropyl analog is both a substrate and inactivator of MAO-B. These properties may not be consistent with the obligatory formation of a cyclopropylaminyl radicalcation intermediate. In an attempt to gain further insight into the mechanism associated with the MAO catalyzed oxidation of 1,4-disubstituted tetrahydropyridines, deuterium isotope effects studies on both the substrate and inactivation properties of the 4-benzyl-1- cyclopropyl derivative were undertaken. A series of 1-methyl- and 1-cyclopropyltetrahydropyridine derivatives bearing various heteroaro-matic groups at C-4 also have been examined. The MAO-B substrate properties, inactivator properties and partition ratios for these compounds together with preliminary results from chemical model studies are discussed in terms of the MAO-B catalytic pathway. / Ph. D.

Smoking is one of the leading causes of death in the world. About 80% of smokers start smoking before the age of 18. In the Appalachian area and the South in the United States, smoking percentages among adults and adolescents are higher than in other regions. Female smoking shows a variety of different trends from male smoking, and smoking brings particular health problems related to production to female smokers. These findings highlighted the importance of studying female teenage smokers in southwest Virginia. The initial project aimed to identify risk factors that might prevent smoking in an early stage. Dr. Helen Crawford led the Cognitive Neuroscience Lab at Virginia Tech in discovering the psychophysiological vulnerabilities of female teenage smokers. Toward this end, event-related potential (ERP), personality, and behavioral data were collected in teenage female smokers and non-smokers. These data were analyzed to examine possible psychophysiological vulnerabilities in female teenage smokers such as deficits in brain and cognitive function, personality traits, and environment influences. The purpose of this dissertation is to further analyze these data to elaborate and clarify the relationships among these vulnerabilities toward understanding teenage smoking behavior. Participants were 49 teenage girls (smokers and non-smokers) with age from 14 to 18. The measures included sensory gating, platelet MAO-B activity, attention, memory, temperament, schizotypal personality, recognition of facial expressions, taste and smell. The initial set of analyses compared smokers and non-smokers, including those classified as high and low dependent, on all dependent measures. The results suggested some psychophysiological vulnerabilities in female teenage smokers, which have been used as support for the self-medication and the orbito-frontal dysfunction models of why teenagers smoke (Crawford et al., 2004). Further examination of these factors may help teenagers to reduce the smoking dependency and possibly improve cognitive function. Specifically, this dissertation focused on the role of the variable of monoamine oxidase-B (MAO-B) in the correlations among sensory gating, MAO and other cognitive and personality measures. All smokers were divided into high and low MAO groups first. Comparison analyses were conducted between them. The high MAO group showed better sensory gating function than the low MAO group. Correlation analyses were conducted among all of the measures. The significant linear relationships between MAO and sensory gating, MAO and CO level and MAO and temperament were demonstrated. MAO activity positively correlated with the sensory gating function and negatively correlated with CO level and temperament characteristics. Finally, to explore the mechanisms of the relationship between MAO and sensory gating, the neurotransmitter systems related to MAO and sensory gating were discussed. / Ph. D.

The unexpected monoamine oxidase (MAO) substrate properties of 1-cyclopropyl-4-substituted-1 ,2,3,6-tetrahydropyridines have been interpreted in terms of partitioning of these tertiary cyclic allylamines between substrate turnover and ring opening inactivation processes. To evaluate further this proposal, we examined the bioactivities of the related saturated analogs. Several 1,4-disubstituted piperidine derivatives were synthesized and their interactions with MAO-A and MAO-B were characterized. These compounds displayed poor substrate properties toward MAO-A and MAO-B and led to the expected α-carbon oxidized metabolites which were fully characterized. Both the N-methyl and N-cyclopropyl derivatives were good inactivators of MAO-B, suggesting that some species other than the radical resulting from cyclopropyl ring opening is responsible for the inactivation. Both the N-methyl and N-cyclopropyl derivatives also inactivated MAO-A. In this instance, the N-cyclopropyl analogs were much more potent inactivators than the N-methyl analogs. These results suggest that the radical derived from cyclopropyl ring opening may be involved in this inactivation process. The MAO substrate/inactivator properties of these piperidine analogs are discussed in terms of current proposed mechanisms for the MAO catalyzed oxidation of amines. / Master of Science

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