Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The generation of oxygen free radicals by the cytosolic enzyme, xanthine oxidase (XO), has been implicated in post-ischemic or reperfusion damage in several organs. XO catalyzes the conversion of hypoxanthine to urate with the concomitant production of superoxide anion free radical (0₂̅˙) and hydrogen peroxide (H₂O₂). Oxygen free radical-mediated injury has also been demonstrated in inflammatory lung disease. The possible involvement of XO in oxidative injury in the lung has not yet been studied. Therefore, this research project was designed to determine whether XO is present in the lung and to investigate its characteristics in porcine, bovine, rat and human lung and other tissues. Immunochemical analysis of xanthine oxidase in the tissues employed on polyclonal antibody raised to bovine milk XO. Proteins were separated by SDS-polyacrylamide gel electrophoresis of tissue homogenates. Proteins were transfered from the gels to nitrocellulose filters by Western blotting. After incubating the filters with a antisera containing the antibody to the purified bovine XO. XO on the filter was detected by its reaction with an enzyme-conjugated second antibody. XO was immunologically detectable in bovine lung and milk. Rat lung, kidney and liver all showed XO reactivity. XO was detectable in porcine liver but not detectable in porcine lung or kidney. Thus, the antibody to bovine XO was cross-reactive with porcine and rat XO. XO protein was not immunologically detectable in human lung possibly because the antibody was not cross reactive with the bovine antibody. In vivo, xanthine oxidase exists predominantly as a dehydrogenase rather than an oxidase. In this form as xanthine dehydrogenase (XDH) the enxyme does not produce either 0₂̅˙ or H₂O₂. The activity of both XDH and XO was measured in several tissues using a fluorometric assay which uses an artifical substrate, pterin which is catalytically converted to the fluorescent product isoxanthopterin (IXP). XO activity in porcine liver was of 1.1 x 10⁻³ µg IXP/mg protein/min although XO activity was not detectable in porcine lung and kidney, in rat lung of 1.7 x 10⁻² µg IXP/mg protein/min, rat kidney of 1.5 x 10⁻² µg IXP/mg protein/min, and rat liver of 2.2 x 10⁻² µg IXP/mg protein/min. Seven human lung biopsy samples were obtained after lung resection and initially tested for viability by determination of NADH oxidase activity and then assayed for XO-XDH. Three of these samples showed NADH oxidase activity indicating tissue viability, but only one of these three showed measurable XO activity of 5.35 x 10⁻⁶ µg IXP/mg protein/min. Irreversible conversion of XDH to XO is thought to be the result of limited proteolysis by a Ca²⁺/calmodulin activated protease, whereas reversible conversion of the enzyme occurs by oxidation of critical thiol groups. Studies on the rate and nature of fluorescence assay to detect catalytic activities of both enzyme forms. Incubation of lung homogenates with trypsin for 60 min caused irreverisble conversion of 90% of the XDH to XO. In contrast, incubation of homogenates at 15°C for 10 hours caused conversion of 100% of the XDH to XO. This conversion was reversible to the extent of 80% by reduction of thiol groups with dithiothreitol (DTT). The effects of free Ca²⁺ on the conversion of XDH to X0 was examined by using EDTA, a chelator of Ca²⁺ and other divalent cations; and EGTA, a more specific chelator of Ca²⁺. The presence of these chelating agents during homogenization of either normoxic or ischemic rat lung tissue did not inhibit reversible enzyme conversion. Increased XO activity was reversible by DTT. In the normoxic rat lung, homogenates prepared with EDTA and EGTA showed a similar conversion of 95% of XDH to XO which was reversible to 70% with DTT. In the ischemic rat lung, samples prepared with EDTA and EGTA showed a'conversion of 80% and 95% XDH to XO which was similar to control samples. The extent of reversibility to XDH was 75% with DTT incubation. In addition, perfusion of rat lungs with EDTA and DTT via a pulmonary artery cannula prior to 60 min of ischemia and homogenization did not affect the extent of XDH to XO conversion. These results indicate that irreversible Ca²⁺-mediated proteolytic conversion of XDH to XO does not occur to a great extent in the rat lung during either normoxia or ischemia. However, reversible conversion of XDH to XO does occur, suggesting that reversible thiol dependent conversion may play a role in the lung under both physiological and pathophysiological states. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

No / The FT Raman spectrum of caffeine was analysed along with that of its demethylated analogues, theobromine and theophylline. The similar but not identical structures of these three compounds allowed a more detailed assignment of the Raman bands. Noticeable differences in the Raman spectra of these compounds were apparent and key marker bands have been identified for the spectroscopic identification of these three compounds.

No / Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.

Existe uma relação entre o uso abrangente de inibidores da bomba de prótons (IBPs), como o omeprazol, e o aumento de risco cardiovascular. Essa relação está associada ao efeito dos IBPs de interferir na síntese e biodisponibilidade do óxido nítrico (NO), um fator importante na homeostase vascular. Também foi evidenciado que o omeprazol causa disfunção endotelial junto a um desequilíbrio redox em aortas, mediado pela ativação da enzima xantina oxidoredutase (XOR), responsável pelo catabolismo das purinas e geração de espécies reativas de oxigênio (ERO). As ERO decorrentes da atividade da XOR, podem aumentar a expressão e a atividade de metaloproteinases de matriz (MMPs), principalmente a MMP-2, que são promotoras de remodelamento tecidual. Assim, nosso objetivo foi analisar se o omeprazol causa remodelamento vascular em aorta de ratos, frente ao seu efeito de aumento do estresse oxidativo via XOR promovendo ativação de MMPs. Foram utilizados ratos wistar com peso entre 180-200 g (n=40), separados em 4 grupos de tratamento onde cada animal foi tratado com 0,5 mL de solução das drogas nas seguintes especificações: o grupo Controle (C) foi tratado com solução veículo tween 2% (vol./vol.) 1 vez ao dia por gavagem, o grupo Alopurinol (A) recebeu uma solução deste inibidor de XOR por gavagem (50 mg/kg/dia), o grupo Omeprazol (O) que recebeu uma solução de omeprazol diluída em tween 2% por via intraperitoneal (10 mg/kg/dia) e por fim, o grupo Omeprazol+Alopurinol (O+A) que recebeu as duas drogas concomitantemente. O protocolo experimental durou 4 semanas, durante as quais foram realizadas aferições da pressão arterial sistólica por pletismografia de cauda. Ao fim do tratamento, os animais foram submetidos à eutanásia, onde foi aferido o pH do lavado gástrico e foi coletada a aorta torácica para a análise de reatividade vascular, análise bioquímica de ERO, a análise morfométrica, e ensaio de atividade de MMPs. Não houve variação de pressão arterial em nenhum dos grupos. O tratamento com alopurinol não alterou nenhum dos parâmetros analisados em relação ao grupo controle neste estudo. O pH gástrico aumentou nos grupos tratados com omeprazol. Na reatividade vascular, observamos que o omeprazol diminuiu o efeito máximo da resposta vasodilatadora dos anéis de aorta à acetilcolina, mas que o tratamento associado ao alopurinol (O+A) preveniu essa diminuição. Em relação ao pD2, foi constatado que o tratamento com omeprazol resulta na diminuição da potência da acetilcolina em causar relaxamento vascular, e que a associação do tratamento com alopurinol, não foi capaz de prevenir essa diminuição. O grupo O também apresentou aumento de espécies reativas de oxigênio no leito vascular, observados no ensaio DHE e o tratamento com alopurinol preveniu este efeito. No ensaio de atividade gelatinolítica in situ observamos um aumento da atividade de MMPs no grupo O, e o tratamento com alopurinol também preveniu esse efeito. Na análise morfométrica observamos que o grupo O apresentou aumento dos parâmetros de remodelamento vascular, denotando um remodelamento hipertrófico, que foi prevenido pela associação com alopurinol. Com base nos resultados, é possível concluir que o tratamento com omeprazol causou remodelamento em aortas de ratos, e que esse efeito ocorreu paralelamente a outros prejuízos, como a diminuição da função vascular avaliada pela resposta à acetilcolina, aumento de espécies reativas de oxigênio e aumento de atividade de MMPs. Como todos esses efeitos resultantes do uso do omeprazol foram prevenidos pela associação do tratamento com alopurinol, é viável inferir que a XOR participe da via pela qual o omeprazol causa efeitos deletérios sobre a vasculatura / There is a relationship between the use of proton pump inhibitors (PPIs), such as omeprazole, and the increase of cardiovascular risk. This relation is associated with the effect of PPIs on nitric oxide synthesis and bioavailability, which is an important factor to vascular homeostasis. It also clear that omeprazole causes endothelial dysfunction by mechanisms involving xanthine oxidoreductase (XOR) mediated redox imbalance in aortas, which is responsible for purines catabolism, and generates reactive oxygen species (ROS). ROS derived from XOR activity, may increase matrix metalloproteinases expression and activity, mainly MMP-2 that are promoters of tissue remodeling. Thus, our aim was to analyze if omeprazole entails vascular remodeling in rat\'s aorta, with its effect of causing oxidative stress via XOR, promoting MMPs activation. Male rats weighing between 180-200g (n=40) were assigned to 4 groups with different treatments, where each animal was treated with 0.5 mL of drug solution, following the specification per group: Control group (C) was treated with the vehicle tween 2% (vol./vol.) 1 time a day by gavage; Allopurinol group (A) that received a solution of this XOR inhibitor by gavage (50mg/kg/day), Omeprazole group (O) which was treated by intraperitoneal route with a solution of omeprazole diluted at tween 2% (10 mg/kg/day) and at last, the Omeprazole+Allopurinol group (O+A), that received both drugs concomitantly. The experimental protocol lasted 4 weeks, during which, were performed systolic blood pressure measurements by tail cuff plethysmography. By the end of treatments, the animals were submitted to euthanasia, then the pH of the gastric washing was measured, and the thoracic aorta was collected to study vascular reactivity, biochemical analysis of ROS, morphometric analysis and MMPs activity assay. There was no blood pressure variation in any of the treatment groups. Treatment with allopurinol did not alter any of the parameters that were analyzed in the present study, in comparison to control group. Gastric washing pH increased in groups treated with omeprazole. In vascular reactivity, it was noticed that omeprazole decreased the maximum effect of the aortic ring\'s vasodilator response to acetylcholine, while the omeprazole treatment associated with allopurinol (O+A) prevented this decrease. Regarding to pD2, it was observed that omeprazole treatment results in decreased acetylcholine potency to cause vascular relaxation, and the association to allopurinol treatment was not capable of preventing this decrease. The O group also presented increased reactive oxygen species levels in the vascular bed, according to DHE assay, and the treatment with allopurinol prevented this effect. With respect to in situ gelatinolytic activity assay, we noticed an increase in MMPs activity in the O group, and the treatment with allopurinol prevented that. The morphometric analysis showed the O group with increased vascular remodeling parameters, denoting a hypertrophic remodeling, which was prevented by the association with allopurinol. Based on these results, is possible to conclude that the treatment with omeprazole caused aortic remodeling in rats, and combined to this effect, some other were observed, such as the vascular function impairment evaluated by the response to acetylcholine, the increase of ROS and increase in MMPs activity. As the effects of omeprazole treatment were prevented by the association of treatment with allopurinol, it is reasonable to infer that XOR participates of the pathway by which omeprazole exerts its deleterious effects on the vasculature

Die Xanthin-Dehydrogenase aus Rhodobacter capsulatus ist ein cytoplasmatisches Enzym, welches ein (αβ)₂ Heterotetramer mit einer Größe von 275 kDa bildet. Die drei Kofaktoren (Moco, 2[2Fe2S], FAD) sind auf zwei unterschiedlichen Polypeptidketten gebunden. So sind die beiden spektroskopisch unterscheidbaren Eisen-Schwefel-Zentren und das FAD in der XdhA-Untereinheit und der Moco in der XdhB-Untereinheit gebunden. Im ersten Teil dieser Arbeit sollte untersucht werden, warum die R. capsulatus XDH ein Dimer bildet und ob ein intramolekularer Elektronentransfer existiert. Dafür wurde eine chimäre XDH-Variante [(α)₂(β₁wt/β₂E730A)] erzeugt, welche eine aktive und eine inaktive XdhB-Untereinheit trägt. Mit Hilfe von Reduktionsspektren sowie mit der Bestimmung der kinetischen Parameter für die Substrate Xanthin und NAD+ konnte gezeigt werden, dass die chimäre XDH-Variante katalytisch halb so aktiv war, wie der auf gleiche Weise gereinigte XDH-Wildtyp. Dies verdeutlicht, dass die noch aktive Untereinheit der Chimären selbstständig und unabhängig Substrat binden und hydroxylieren kann und ein intramolekularer Elektronentransfer zwischen den beiden XdhB-Untereinheiten nicht stattfindet. Ein weiteres Ziel war die funktionelle Charakterisierung der Mus musculus AOX1 sowie der humanen AOX1 hinsichtlich ihrer Substratspezifitäten und ihrer biophysikalischen Eigenschaften sowie der Charakterisierung der konservierten Aminosäuren im aktiven Zentrum der mAOX1. Da bislang noch kein heterologes Expressionssystem für ein aktives und stabiles rekombinantes AO-Protein existierte, wurde ein E. coli Expressionssystem mit der gleichzeitigen Expression der entsprechenden Mocosulfurase für mAOX1 und hAOX1 in dieser Arbeit etabliert. Mit Hilfe dieser Koexpression konnte die Aktivität der rekombinanten mAOX1 um 50 % gesteigert werden, wenn gleich auch der sulfurierte Moco-Anteil nur 20 % betrug. Um die konservierten Aminosäuren im aktiven Zentrum hinsichtlich ihrer Funktion der Substratbindung zu charakterisieren, wurden folgende Varianten erzeugt: V806E, M884R, V806/M884R sowie E1265Q. Mit Hilfe von kinetischen Substratuntersuchungen konnte gezeigt werden, dass die beiden Aminosäuren Val806 und Met884 für die Erkennung und die Stabilisierung von Aldehyden und N-Heterozyklen essentiell sind. Ein Austausch dieser beiden gegen Glutamat bzw. Arginin (wie bei R. capsulatus XDH) zeigte jedoch keine Xanthin- oder Hypoxanthinumsetzung. Für das Glu1265 wurde ebenfalls die Rolle als die Katalyse initiierende Aminosäure belegt. / The main task of this work was to analyse the function of R. capsualtus Xanthine Dehydrogenase (R.c. XDH; EC 1.17.1.4) as well as to characterize the structure and function of mouse Aldehyde Oxidase (mAOX1; EC 1.2.3.1). Both enzymes are complex metallo-flavoproteins that contain two nonidentical [2Fe2S] clusters, FAD and the molybdenum cofactor (Moco) as catalytically acting units. AO and XDH are members of the xanthine oxidase family characterized by an equatorial sulfur ligand at the Moco site essential for enzyme activity. To solve the question why R.capsualtus XDH forms a dimer a chimeric variant of bacterial XDH was produced and expressed in E.coli. By means of the (alphabeta)(2) XDH heterotetramer variant, that should include only one active Moco-center, it should be analysed if the two subunits act independent without cooperativity. AO is characterized by broad substrate specificity and this makes it an important enzyme for the metabolism of drugs and xenobiotica. The biochemical and physiological function of AO is still largely obscure and only limited information is available on the physiological substrates of AO or the role of the enzyme in mammalia. The substrate specificity of the recombinant AO should be determined by different purines and aldehydes. In order to determine the function of conserved amino acides, site directed mutagenesis of amino acides at the active site (Val806Glu, Met884Arg, Glu1265Gln) were introduced and enzyme activity was determined. Bacterial XDH is highly homologous to the homodimeric mammalian xanthine oxidoreductase - in the amino acid sequence and the secondary and tertiary protein structure as well as the reaction mechanism as described by Leimkühler et al. (2004). Therefore, in the second part of this work, bacterial XDH will be used as a benchmark for mAOX1 during determination of enzyme acitivities using different purines and aldehydes as substrates. A single monogentic deficit of AO has not been described for humans yet. To identify the biochemical function and to characterize the enzyme in detail a system for a heterologous expression of functionally active hAOX1 in E.coli should be established too.

Currently L-DOPA is the drug most commonly used for the treatment of Parkinson’s disease (PD). However, the long-term use of L-DOPA is associated with the development of motor fluctuations and dyskinesias. Treatment mainly addresses the dopaminergic features of the disease and leaves its progressive course unaffected. An optimal treatment would be a combination of both motor and non-motor symptom relief with neuroprotective properties. Two drug targets have attracted the attention for PD treatment, namely monoamine oxidase B (MAOB) and adenosine A2A receptors. MAO-B inhibitors enhance the elevation of dopamine levels after L-DOPA treatment, improve motor functions and may also possess neuroprotective properties. The antagonistic interaction between A2A and dopamine receptors in the striatopallidal pathway, which modulates motor behaviour, has also become a potential strategy for PD treatment. Blockade of the A2A receptor exerts both anti-symptomatic and neuroprotective activities and offer benefit for motor symptoms and motor complications. This thesis seeks to synthesize novel drug treatments for PD by exploring both MAO-B inhibitors and adenosine A2A receptor antagonists and to assess the prospects for drug modification to increase activity. MAO-B inhibitors - Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5- sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5- (benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427–fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. It has recently been reported that nitrile containing compounds frequently act as potent MAO-B inhibitors. In an attempt to identify additional potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC50 values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC50 = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease. Adenosine A2A receptor antagonism - Most adenosine A2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists for PD and to further explore the structure-activity relationships of A2A antagonism by the xanthine class of compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthine, 8-(phenoxymethyl)xanthine and 8-(3- phenylpropyl)xanthine derivatives. The results document that among these series, the (E)-8- styrylxanthines are the most potent antagonists with the most potent homologue, (E)-1,3-dietyl- 7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8- (phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A2A receptor. It was also concluded that (E)-8-styrylxanthines are potent A2A antagonists with particularly the 1,3-dietyl-7-methylxanthine substitution pattern being most appropriate for high affinity binding. Conclusion - The results of these studies have established that all of the sulfanylphthalimides, sulfanylphthalonitriles and sulfanylbenzonitriles examined display significant MAO-B inhibitory properties in vitro with IC50 values in the low μM to nM range. Good A2A receptor affinity was demonstrated by the xanthines containing a styryl moiety, while the phenoxymethyl and phenylpropyl xanthines exhibited poor activity. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Currently L-DOPA is the drug most commonly used for the treatment of Parkinson’s disease (PD). However, the long-term use of L-DOPA is associated with the development of motor fluctuations and dyskinesias. Treatment mainly addresses the dopaminergic features of the disease and leaves its progressive course unaffected. An optimal treatment would be a combination of both motor and non-motor symptom relief with neuroprotective properties. Two drug targets have attracted the attention for PD treatment, namely monoamine oxidase B (MAOB) and adenosine A2A receptors. MAO-B inhibitors enhance the elevation of dopamine levels after L-DOPA treatment, improve motor functions and may also possess neuroprotective properties. The antagonistic interaction between A2A and dopamine receptors in the striatopallidal pathway, which modulates motor behaviour, has also become a potential strategy for PD treatment. Blockade of the A2A receptor exerts both anti-symptomatic and neuroprotective activities and offer benefit for motor symptoms and motor complications. This thesis seeks to synthesize novel drug treatments for PD by exploring both MAO-B inhibitors and adenosine A2A receptor antagonists and to assess the prospects for drug modification to increase activity. MAO-B inhibitors - Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5- sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5- (benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427–fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. It has recently been reported that nitrile containing compounds frequently act as potent MAO-B inhibitors. In an attempt to identify additional potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC50 values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC50 = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease. Adenosine A2A receptor antagonism - Most adenosine A2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists for PD and to further explore the structure-activity relationships of A2A antagonism by the xanthine class of compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthine, 8-(phenoxymethyl)xanthine and 8-(3- phenylpropyl)xanthine derivatives. The results document that among these series, the (E)-8- styrylxanthines are the most potent antagonists with the most potent homologue, (E)-1,3-dietyl- 7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8- (phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A2A receptor. It was also concluded that (E)-8-styrylxanthines are potent A2A antagonists with particularly the 1,3-dietyl-7-methylxanthine substitution pattern being most appropriate for high affinity binding. Conclusion - The results of these studies have established that all of the sulfanylphthalimides, sulfanylphthalonitriles and sulfanylbenzonitriles examined display significant MAO-B inhibitory properties in vitro with IC50 values in the low μM to nM range. Good A2A receptor affinity was demonstrated by the xanthines containing a styryl moiety, while the phenoxymethyl and phenylpropyl xanthines exhibited poor activity. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013