Thesis (Ph. D.)--University of Virginia, 2008. / In title: [A] is subscript upper case A; [B] is subscript upper case B. Includes bibliographical references. Also available online through Digital Dissertations.

A hipertensão arterial é um problema de saúde pública no Brasil, pois aproximadamente 20 % da população adulta desenvolve hipertensão essencial, cujas causas ainda não são conhecidas. No entanto, sua gênese pode estar relacionada com disfunção nas áreas do sistema nervoso central (SNC) que regulam o sistema cardiovascular. O núcleo do trato solitário (NTS) e o bulbo ventrolateral são áreas importantes no controle neural da pressão arterial. Os receptores de adenosina A2a (rA2a) são encontrados em todo o SNC e estão relacionados com estudos terapêuticos de diversas doenças. No NTS a estimulação dos rA2a provoca ajustes pontuais em outros sistemas de neurotransmissão, além de diminuir a pressão arterial. A nicotina é uma molécula com uma vasta faixa de efeitos modulatórios em nosso organismo. Entre esses efeitos se destacam a capacidade de interagir com diversos sistemas de neurotransmissão nas áreas do bulbo relacionadas com a regulação da pressão arterial e de antecipar e/ou intensificar o desenvolvimento da hipertensão em sujeitos com pré-disposição genética. Desta forma, o objetivo do presente trabalho é avaliar o efeito modulatório da nicotina sobre o rA2a em cultura mista de neurônios e células gliais da porção dorso-medial do bulbo de ratos geneticamente hipertenso (SHR) e normotensos (WKY). Para isso, utilizaram-se técnicas como a de PCR em tempo real, Western Blotting e análise de ligação do receptor. Nossos resultados demonstraram que: 1) em condição basal células de ratos normotensos apresentam maior ligação do rA2a do que células de ratos hipertensos; 2) tratamento com nicotina resultou na diminuição da ligação do receptor em ambas as cepas, com um efeito de maior magnitude em células de ratos WKY; 3) nas duas linhagens o tratamento com nicotina alterou os níveis protéicos do rA2a, assim como o RNAm do receptor; 4) a linhagem e o tratamento separadamente, como a interação entre ambos influenciaram na expressão do RNAm , níveis protéicos e ligação do rA2a nas células dos ratos WKY e SHR. Por fim, os resultados apresentados aqui indicam que o rA2a em células de ratos hipertensos tem sua função deprimida em comparação com as células de ratos normotensos; e que a nicotina foi capaz de modular o funcionamento do rA2a, o qual pode influenciar no controle da pressão arterial. Esses dados são bastante interessantes, pois abrem novas perspectivas de análise dos mecanismos intracelulares envolvidos na modulação dos rA2a pela nicotina, assim como a importância desse sistema no desenvolvimento da hipertensão / Hypertension is one of the most common worldwide diseases afflicting humans. Because of the associated with morbidity and mortality and the cost to the society, it became an important public health challenge in Brazil. The mechanisms involved in development of hypertension still remain unclear However, hypertension can result from neuronal network imbalance in areas of the central nervous system that control blood pressure. The nucleus tractus solitarius (NTS) plays an important role in cardiovascular control. Within the NTS there are several neurotransmitters and neuromodulatory substances, such as adenosine, which acts on purinoreceptors A2a (A2ar). The A2ar modulates neurotransmission in the NTS and its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that cross the blood-brain barrier and acts in several areas of central nervous system including the NTS. In this nucleus, nicotine is able to interact with some neurotransmitter systems and contributes for the development of hypertension in subjects with genetic predisposition to this disease. The goal of this study was to analyze the modulatory effects of nicotine on A2ar in cultured neurons and glial cells from medulla oblongata of normotensive (WKY) and spontaneously hypertensive rats (SHR). By means of real time PCR, Western Blotting and binding receptor assay. We have demonstrated that in basal condition cells of WKY presents increased binding of A2ar than the cells of SHR. Nicotine treatment induced a decrease in the binding of A2ar in both strains, however, this response was more pronounced in cells of WKY than SHR. Changes in mRNA and protein levels of A2ar was also observed in response to nicotine treatment. The strains and treatment separately, as well as the interaction between them influenced mRNA expression, protein level and binding of A2ar in NTS cells of WKY and SHR rats. Finally, these results show for the first time changes in A2ar mRNA expression, protein level and binding in cells from the medulla oblongata of WKY and SHR rats, as well as, the nicotine modulation upon this system, which might influence cardiovascular control. These data open up new approaches for the study of intracellular mechanisms involved in the modulation of adenosine A2a receptor by nicotine, as well as the importance of this interaction in the development of hypertension.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A doenÃa de Parkinson (DP) à uma desordem neurodegenerativa, caracterizada pela destruiÃÃo dos neurÃnios nigroestriatais dopaminÃrgicos. O tratamento atual para esta doenÃa està restrito ao alÃvio sintomÃtico, porque atà o presente momento nÃo existem agentes capazes de inibir a degeneraÃÃo neuronal. Existem evidÃncias experimentais de que antagonistas de receptores A2A da adenosina poderiam ser Ãteis no tratamento de DP. Com a finalidade de investigar essa possibilidade, o presente trabalho demonstrou os efeitos da cafeÃna e do CSC (8-(3-chlorostyryl caffeine) no comportamento rotacional e nas alteraÃÃes neuroquÃmicas em ratos lesionados com 6-OHDA, como modelo da doenÃa de Parkinson. Os animais (ratos Wistar machos, 250-280g) foram tratados com cafeÃna (10 e 20 mg/kg, i.p.) diariamente durante 14 dias, iniciando 1h apÃs a lesÃo ou 7 dias, iniciando seis dias apÃs a lesÃo com 6-OHDA ou com CSC (1 e 5 mg/kg, i.p.) diariamente durante 7 dias, iniciando 6 dias apÃs a lesÃo com 6-OHDA, sozinho ou associado com L-DOPA (CSC 1 mg/kg, i.p. + L-DOPA 50mg/kg + Benzerazida 12,5 mg/kg, i.p.). Os resultados mostraram que houve um aumento significativo do nÃmero de rotaÃÃes induzidas por apomorfina nos animais lesionados com 6-OHDA (50 vezes) quando comparados aos animais falso operados. O tratamento com cafeÃna, principalmente durante 14 dias e o tratamento com CSC produziram uma recuperaÃÃo motora parcial com reduÃÃo do nÃmero de rotaÃÃes. A 6-OHDA provocou morte neuronal evidenciada pela reduÃÃo dos nÃveis de monoaminas (75-85%) quando comparadas ao lado contralateral. Nos grupos tratados com cafeÃna ou CSC sozinho ou associado com L-DOPA a reduÃÃo dos nÃveis de DA, 5HT e seus metabÃlitos foi menor. As concentraÃÃes dos aminoÃcidos glutamato e GABA foram significativamente aumentadas (3,8 e 3 vezes, respectivamente) no estriado de ratos lesionados. O CSC reverteu essas alteraÃÃes significativamente e foi observada uma potencializaÃÃo desses efeitos na associaÃÃo com L-DOPA. Os experimentos in vitro demonstraram que a cafeÃna e o CSC apresentaram um forte efeito neuroprotetor nas cÃlulas mesencefÃlicas de rato expostas a 6-OHDA. O tratamento com CSC ou cafeÃna aumentou significativamente o nÃmero de cÃlulas viÃveis apÃs a exposiÃÃo das cÃlulas a 6-OHDA, como foi demonstrado pelo teste do MTT. A exposiÃÃo das cÃlulas mesencefÃlicas a 6-OHDA aumentou os conteÃdos de nitrito e a peroxidaÃÃo lipÃdica, que retornaram a concentraÃÃes normais apÃs tratamento com CSC ou cafeÃna. AlÃm disso, a 6-OHDA reduziu o nÃmero de cÃlulas normais e aumentou o nÃmero de cÃlulas apoptÃticas e o tratamento com CSC ou cafeÃna reverteu esses efeitos da 6-OHDA, promovendo aumento do nÃmero de cÃlulas viÃveis e reduÃÃo do nÃmero de cÃlulas apoptÃticas. Houve uma reduÃÃo do nÃmero de microglias ativadas apÃs a exposiÃÃo das cÃlulas a cafeÃna e a 6-OHDA, o mesmo nÃo ocorreu apÃs a exposiÃÃo das cÃlulas ao CSC e a 6-OHDA. O tratamento com cafeÃna reduziu o aumento do nÃmero de astrÃcitos reativos induzidos pela 6-OHDA, enquanto o CSC nÃo apresentou esse efeito. Esses resultados mostraram que ambos, a cafeÃna e o CSC apresentaram aÃÃes neuroprotetoras em cÃlulas mesencefÃlicas de rato expostas a 6-OHDA. O presente trabalho mostrou que a cafeÃna e o CSC reverteram Ãs alteraÃÃes comportamentais e neuroquÃmicas da 6-OHDA, apresentando efeitos possivelmente benÃficos no tratamento da DP. / Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. Antagonists of the A2A subtype of adenosine receptor have emerged as a target for nondopaminergic antiparkinsonian agents. The present work showed the effects of caffeine and 8-(-3-chlorostyryl)-caffeine (CSC), A2A receptors antagonists, on behavior and biochemical alterations in 6-OHDA-lesioned rats, as a model of PD. Animals (male Wistar rats, 260-280 g) were injected daily with caffeine (10 and 20 mg/kg,i.p., 1h after 6-OHDA lesion for 14 days or six days after 6-OHDA lesion for 7 days), or CSC (1 and 5 mg/kg, i.p., 1h after 6-OHDA lesion for 7 days) alone or associated with L-DOPA (CSC 1 mg/kg, i.p. + L-DOPA 50mg/kg + Benzerazida 12,5 mg/kg, i.p., six days after 6-OHDA lesion for 7 days). Fourteen days after 6-OHDA, the animalsâ behavior was assessed by monitoring body rotations induced by apomorphine (3 mg/kg, i.p.). The results showed that the drastic increase in body rotation, induced by the 6-OHDA lesion, after the apomorphine challenge, was significantly (50 times) and dose-dependently reversed by CSC or caffeine. The decreased striatal levels of DA and metabolites, in the 6-OHDA-lesioned rats (75-85%), were blocked after caffeine or CSC alone or in association with L-DOPA treatment as well as the concentrations of NE, 5-HT and 5-HIAA. These effects were potentiated in 6-OHDA-lesioned animals treated with the association of CSC and L-DOPA. Concentrations of the amino acids glutamate and GABA were significantly increased (3.8 and 3 times, respectively) in the 6-OHDA-lesioned rat striatum. Similarly, CSC also reversed these alterations significantly. We also demonstrated protective effects against 6-OHDA-induced cytotoxicity in rat mesencephalic cells. Caffeine or CSC significantly increased the number of viable cells after their exposure to 6-OHDA, as measured by the MTT assay. While nitrite levels and lipid peroxidation in the cells were drastically increased by 6-OHDA, its concentration was brought toward normality after caffeine or CSC. 6-OHDA decreased the number of normal cells while increasing the number of apoptotic cells. Caffeine or CSC, significantly recovered the number of viable cells, and decreased the number of apoptotic cells, as compared to the group treated with 6-OHDA alone. Interestingly, while a significant lower number of activated microglia was seen after cells exposure to caffeine plus 6-OHDA, this was not the case after cells exposure to CSC plus 6-OHDA. While caffeine lowered the percentage of reactive astrocytes increased by 6-OHDA, CSC showed not effect. These results showed a strong neuroptrotection afforded by caffeine or CSC on rat mesencephalic cells exposed to 6-OHDA. In conclusion, we showed that CSC or caffeine reversed behavior and biochemical alterations, observed in the 6-OHDA-lesioned rats, pointing out to the potential benefit of A2A receptors antagonists as non-dopaminergic therapeutic targets for the treatment of PD.

A hipertensão arterial é um problema de saúde pública no Brasil, pois aproximadamente 20 % da população adulta desenvolve hipertensão essencial, cujas causas ainda não são conhecidas. No entanto, sua gênese pode estar relacionada com disfunção nas áreas do sistema nervoso central (SNC) que regulam o sistema cardiovascular. O núcleo do trato solitário (NTS) e o bulbo ventrolateral são áreas importantes no controle neural da pressão arterial. Os receptores de adenosina A2a (rA2a) são encontrados em todo o SNC e estão relacionados com estudos terapêuticos de diversas doenças. No NTS a estimulação dos rA2a provoca ajustes pontuais em outros sistemas de neurotransmissão, além de diminuir a pressão arterial. A nicotina é uma molécula com uma vasta faixa de efeitos modulatórios em nosso organismo. Entre esses efeitos se destacam a capacidade de interagir com diversos sistemas de neurotransmissão nas áreas do bulbo relacionadas com a regulação da pressão arterial e de antecipar e/ou intensificar o desenvolvimento da hipertensão em sujeitos com pré-disposição genética. Desta forma, o objetivo do presente trabalho é avaliar o efeito modulatório da nicotina sobre o rA2a em cultura mista de neurônios e células gliais da porção dorso-medial do bulbo de ratos geneticamente hipertenso (SHR) e normotensos (WKY). Para isso, utilizaram-se técnicas como a de PCR em tempo real, Western Blotting e análise de ligação do receptor. Nossos resultados demonstraram que: 1) em condição basal células de ratos normotensos apresentam maior ligação do rA2a do que células de ratos hipertensos; 2) tratamento com nicotina resultou na diminuição da ligação do receptor em ambas as cepas, com um efeito de maior magnitude em células de ratos WKY; 3) nas duas linhagens o tratamento com nicotina alterou os níveis protéicos do rA2a, assim como o RNAm do receptor; 4) a linhagem e o tratamento separadamente, como a interação entre ambos influenciaram na expressão do RNAm , níveis protéicos e ligação do rA2a nas células dos ratos WKY e SHR. Por fim, os resultados apresentados aqui indicam que o rA2a em células de ratos hipertensos tem sua função deprimida em comparação com as células de ratos normotensos; e que a nicotina foi capaz de modular o funcionamento do rA2a, o qual pode influenciar no controle da pressão arterial. Esses dados são bastante interessantes, pois abrem novas perspectivas de análise dos mecanismos intracelulares envolvidos na modulação dos rA2a pela nicotina, assim como a importância desse sistema no desenvolvimento da hipertensão / Hypertension is one of the most common worldwide diseases afflicting humans. Because of the associated with morbidity and mortality and the cost to the society, it became an important public health challenge in Brazil. The mechanisms involved in development of hypertension still remain unclear However, hypertension can result from neuronal network imbalance in areas of the central nervous system that control blood pressure. The nucleus tractus solitarius (NTS) plays an important role in cardiovascular control. Within the NTS there are several neurotransmitters and neuromodulatory substances, such as adenosine, which acts on purinoreceptors A2a (A2ar). The A2ar modulates neurotransmission in the NTS and its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that cross the blood-brain barrier and acts in several areas of central nervous system including the NTS. In this nucleus, nicotine is able to interact with some neurotransmitter systems and contributes for the development of hypertension in subjects with genetic predisposition to this disease. The goal of this study was to analyze the modulatory effects of nicotine on A2ar in cultured neurons and glial cells from medulla oblongata of normotensive (WKY) and spontaneously hypertensive rats (SHR). By means of real time PCR, Western Blotting and binding receptor assay. We have demonstrated that in basal condition cells of WKY presents increased binding of A2ar than the cells of SHR. Nicotine treatment induced a decrease in the binding of A2ar in both strains, however, this response was more pronounced in cells of WKY than SHR. Changes in mRNA and protein levels of A2ar was also observed in response to nicotine treatment. The strains and treatment separately, as well as the interaction between them influenced mRNA expression, protein level and binding of A2ar in NTS cells of WKY and SHR rats. Finally, these results show for the first time changes in A2ar mRNA expression, protein level and binding in cells from the medulla oblongata of WKY and SHR rats, as well as, the nicotine modulation upon this system, which might influence cardiovascular control. These data open up new approaches for the study of intracellular mechanisms involved in the modulation of adenosine A2a receptor by nicotine, as well as the importance of this interaction in the development of hypertension.

Background and rationale - Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of dopamine in the brain. The cause of Parkinson's disease is still unknown; however several theories pertaining to the etiology exist. Current treatment mainly aims at dopamine replacement, with agents such as levodopa and dopamine agonists that provide patients with symptomatic relief. This relief is unfortunately only temporary as the progression of the disease is not halted. Furthermore, these therapies are associated with a range of side effects and novel approaches to the treatment are thus urgently required. Adenosine A2A receptor antagonists recently emerged as a promising non-dopaminergic alternative, not only as symptomatic treatment, but also as potential neuroprotective therapy. Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors (A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine depletion to give symptomatic relief. There are also several mechanisms where by adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A antagonists have already reached clinical trials and promising results were obtained, especially when combined with levodopa. Consequently, A2A antagonists are realistic prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor antagonists. Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of inhibition comes with certain disadvantages as it may take up to several weeks after termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast will have much better safety profiles seeing that they will not inactivate the enzyme permanently and allow for competition with the substrate. When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease. MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the progression of the disease by preventing the formation of these byproducts. Promising MAOB inhibitory activities have been reported for chalcones, and since the intermediates obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B. Results - Design and synthesis: A series of 2-aminopyrimidines were designed using known active structures and literature pharmacophores. A molecular modelling study (Discovery Studio 3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked in the binding site of the adenosine A2A receptor. Binding orientations and observed interactions with important residues in the active site were similar to those observed for known A2A antagonists. It was therefore concluded that these compounds may be potential A2A antagonists and the designed compounds were thus synthesised. Structures were primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry. MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay and kynuramine as substrate. The compounds were potent and selective inhibitors of the MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1- (5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of 0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A. Some preliminary structure activity relationships were derived, for example, phenyl substitution with an electron withdrawing chlorine group generally resulted in better activity than substitution with electron donating methoxy groups. Further investigation of structure activity relationships are however required as a very small series of chalcones were screened. Reversibility studies and mode of inhibition: A dilution assay was used to determine whether compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor complex. The results from the reversibility studies showed that the inhibition of the most potent compound (1c) is reversible as the catalytic activities are recovered to approximately 80% and 50% respectively, compared to the control measured in the absence of an inhibitor. For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver- Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor of the MAO-B enzyme. In vitro adenosine A2A assays: Radioligand binding assays were used to determine the affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine (CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM. The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part, be explained by the molecular modellling studies. In the docking experiments an additional hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271 indicating that this structural feature is a major contributing factor to the improved affinity observed for these derivatives. In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to determine whether the two compounds with the highest affinity for the adenosine A2A receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine A2A antagonists. The objectives of this study as set out were thus successfully realised and promising results were obtained. During this study, several novel 2-aminopyrimidines and chalcones were synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase inhibitory activities for all of the screened compounds were determined for the first time. / Thesis (MSc (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

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.

Background and rationale - Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of dopamine in the brain. The cause of Parkinson's disease is still unknown; however several theories pertaining to the etiology exist. Current treatment mainly aims at dopamine replacement, with agents such as levodopa and dopamine agonists that provide patients with symptomatic relief. This relief is unfortunately only temporary as the progression of the disease is not halted. Furthermore, these therapies are associated with a range of side effects and novel approaches to the treatment are thus urgently required. Adenosine A2A receptor antagonists recently emerged as a promising non-dopaminergic alternative, not only as symptomatic treatment, but also as potential neuroprotective therapy. Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors (A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine depletion to give symptomatic relief. There are also several mechanisms where by adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A antagonists have already reached clinical trials and promising results were obtained, especially when combined with levodopa. Consequently, A2A antagonists are realistic prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor antagonists. Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of inhibition comes with certain disadvantages as it may take up to several weeks after termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast will have much better safety profiles seeing that they will not inactivate the enzyme permanently and allow for competition with the substrate. When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease. MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the progression of the disease by preventing the formation of these byproducts. Promising MAOB inhibitory activities have been reported for chalcones, and since the intermediates obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B. Results - Design and synthesis: A series of 2-aminopyrimidines were designed using known active structures and literature pharmacophores. A molecular modelling study (Discovery Studio 3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked in the binding site of the adenosine A2A receptor. Binding orientations and observed interactions with important residues in the active site were similar to those observed for known A2A antagonists. It was therefore concluded that these compounds may be potential A2A antagonists and the designed compounds were thus synthesised. Structures were primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry. MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay and kynuramine as substrate. The compounds were potent and selective inhibitors of the MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1- (5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of 0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A. Some preliminary structure activity relationships were derived, for example, phenyl substitution with an electron withdrawing chlorine group generally resulted in better activity than substitution with electron donating methoxy groups. Further investigation of structure activity relationships are however required as a very small series of chalcones were screened. Reversibility studies and mode of inhibition: A dilution assay was used to determine whether compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor complex. The results from the reversibility studies showed that the inhibition of the most potent compound (1c) is reversible as the catalytic activities are recovered to approximately 80% and 50% respectively, compared to the control measured in the absence of an inhibitor. For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver- Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor of the MAO-B enzyme. In vitro adenosine A2A assays: Radioligand binding assays were used to determine the affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine (CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM. The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part, be explained by the molecular modellling studies. In the docking experiments an additional hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271 indicating that this structural feature is a major contributing factor to the improved affinity observed for these derivatives. In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to determine whether the two compounds with the highest affinity for the adenosine A2A receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine A2A antagonists. The objectives of this study as set out were thus successfully realised and promising results were obtained. During this study, several novel 2-aminopyrimidines and chalcones were synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase inhibitory activities for all of the screened compounds were determined for the first time. / Thesis (MSc (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