Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required. Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment. Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results - Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring. Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5- methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with Ki values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively. A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > pfluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin- 4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor. The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log Ki (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 μM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities. Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy. Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

La dysoxie (hypoxie/hyperoxie) résulte de l’inadéquation entre la consommation en oxygène et ses apports, provoquant altérations du métabolisme cellulaire et impact physiopathologique majeur. L’hypoxie et l’inflammation font intervenir les facteurs HIF-1a et NF-kB qui activent le système adénosinergique. L’hypoxie augmente la concentration d’adénosine, entraîne une surexpression d’A2AR et induit une immunosuppression lymphocytaire T. Nous avons fait l‘hypothèse que l’inflammation via NF-kB influençait la suppression lymphocytaire adénosinergique; qu’une hyperhomocystéinémie favoriserait l’inflammation en modifiant la viabilité lymphocytaire; qu’in vivo, la réponse adénosinergique était modulée par l’hyperoxie. Des lymphocytes T ont été soumis à une inflammation (agents mitogènes) et à une hypoxie chimique (CoCl2). Nous avons analysé l’expression de NF-kB, HIF-1α, A2AR et évalué les concentrations en adénosine, adénosine déaminase, AMPc, et homocystéine. Enfin, nous avons étudié les effets de l’hyperoxie/hyperbarie sur la réponse adénosinergique. L’hypoxie stimule la réponse adénosinergique : NF-kB induit HIF-1α qui augmente l’expression d’A2AR, favorisant l’immunosuppression. L’inhibition de NF-kB par H2S bloque l’immunosuppression via HIF-1α/A2AR. In vivo, l’hyperoxie inhibe la réponse adénosinergique via la diminution de l’expression d’A2AR. Hypoxie et hyperoxie ont un effet en miroir sur le système adénosinergique. Manipuler la concentration en O2 permet de piloter système immunitaire et inflammation via A2AR. L’utilisation d’H2S pourrait traiter des pathologies à fort impact en santé publique, tels des troubles cardiovasculaires favorisés par l’hyperhomocystéinémie. / Dysoxia (hypoxia/hyperoxia) results from an impaired balance between oxygen-supply concentration and cellular metabolism causing various disorders. Hypoxia and inflammation involve HIF-1a and NF-kB factors and are linked via the adenosinergic response. Hypoxia increase adenosine concentration and A2A receptors (A2AR) expression which induces T-lymphocyte suppression. We hypothesized that during hypoxia, inflammation influences adenosinergic immunosuppression via NF-kB. As homocysteine promotes inflammation, which is considered as a risk factor, we hypothesized that hyperomocysteinemia affects T-cell viability and adenosinergic response. Effects of hyperoxic and hyperbaric conditions on adenosinergic system remain unclear. NF-kB, HIF-1α, and A2AR expression were studied using T-cells stimulated by mitogens under hypoxic conditions (CoCl2). Adenosine, adenosine deaminase, cAMP concentration and homocysteine metabolism were analyzed. Effect of hyperoxia on the adenosinergic pathway was addressed in a rat model using pressure chambers. HIF-1α production was induced by hypoxia, A2AR expression increased following NF-kB activation that enhanced lymphocyte-suppression. Inhibition of NF-kB by H2S resulted in improved cell-viability by down-regulating A2AR-mediated-immunosuppression. Hyperhomocysteinemia increased H2S production (transsulfuration-pathway). We also found in rat that hyperoxia repressed the adenosinergic response. Manipulating blood oxygen level constitutes an effective mean to control the immune response and inflammation via the adenosinergic system. Acting on A2AR expression via H2S production may control cardiovascular-disorders with high impact on public health.

<p><!-- @page { margin: 2cm } P { margin-bottom: 0.21cm; background: transparent; page-break-before: auto } P.western { font-size: 14pt } --></p><p>Rheumatoid arthritis is an autoimmune disease, where joints are attacked by the own immune system, leading to chronic inflammation and destruction of bone and cartilage. Inflammation is a complex process, controlled by many different substances. One of them is adenosine, which has anti-inflammatory properties. In this project, three polymorphisms in different genes, involved in synthesis and signaling of adenosine, were genotyped for 188 patients with RA and 362 controls without RA. The results shows that for the polymorphism in A2a, a gene coding for an adenosine receptor, there was no significant difference in genotype distribution between the groups. There were, however, some differences in the general sensation of pain and well-being reported by the patients. For the polymorphism in NT5E, a gene coding for a nucleotidase for extracellular adenosine synthesis, there were differences both regarding genotype distribution between the groups, and in the progression of the disease. The NT5E-AA genotype seems to increase inflammation, but decrease the number of tender joints. In the case of the polymorphism in ADA, which codes for adenosine deaminase, the minor allele frequency was too low for any conclusions to be made. An attempt was made to analyze the gene polymorphisms in relation to drinking habits, but the population was too small to generate any reliable conclusions. The project shows that the polymorphism in NT5E, whose functional consequences are yet unknown, might have an effect on the extracellular adenosin synthesis and RA pathogenesis. Further studies are required to shed more light on this matter.</p><p> </p> / <p>Reumatoid artrit är en autoimmun sjukdom där leder angrips av det egna immunförsvaret, vilket leder till kronisk inflammation och nedbrytning av brosk och ben. Inflammation är en komplex mekanism som regleras av många olika substanser, varav en är adenosin, som i förhöjda koncentrationer fungerar som en broms av inflammationen. I detta arbetet har tre olika polymorfier i gener inblandade i syntes och signalering av adenosin genotypats för 188 patienter med nydiagnostiserad RA, samt hos en kontrollgrupp på 362 frivilliga utan reumatisk sjukdom. Avseende en polymorfi i A2a, som kodar för en adenosinreceptor, fanns inga signifikanta skillnader i fördelning mellan fall och kontroller. Beträffande sjukdomsförloppet sågs dock vissa skillnader i patienternas bedömning av smärta och välbefinnande. För polymorfin i NT5E, som kodar för ett nukleotidas för extracellulär adenosinsyntes, hittades skillnader både i fördelningen mellan RA-gruppen och kontrollgruppen, samt i sjukdomsförloppet, där NT5E-AA genotypen var relaterad till högre inflammation, men samtidigt till lägre smärtupplevelse. För den tredje polymorfin (ADA) var antalet individer med mutant allel för litet för att tillåta några slutsatser. Tester gjordes även för att se om de olika adenosinpolymorfierna korrelerade till alkoholkonsumtion, men underlaget var för litet för att några slutsatser skulle kunna dras. Arbetet visar att polymorfin i NT5E, vars funktionella effekt är okänd, kan ha en effekt på nukleotidasets förmåga att producera extracellulärt adenosin. Kompletterande studier på större material behövs för att bringa klarhet i dessa frågor.</p>

Rheumatoid arthritis is an autoimmune disease, where joints are attacked by the own immune system, leading to chronic inflammation and destruction of bone and cartilage. Inflammation is a complex process, controlled by many different substances. One of them is adenosine, which has anti-inflammatory properties. In this project, three polymorphisms in different genes, involved in synthesis and signaling of adenosine, were genotyped for 188 patients with RA and 362 controls without RA. The results shows that for the polymorphism in A2a, a gene coding for an adenosine receptor, there was no significant difference in genotype distribution between the groups. There were, however, some differences in the general sensation of pain and well-being reported by the patients. For the polymorphism in NT5E, a gene coding for a nucleotidase for extracellular adenosine synthesis, there were differences both regarding genotype distribution between the groups, and in the progression of the disease. The NT5E-AA genotype seems to increase inflammation, but decrease the number of tender joints. In the case of the polymorphism in ADA, which codes for adenosine deaminase, the minor allele frequency was too low for any conclusions to be made. An attempt was made to analyze the gene polymorphisms in relation to drinking habits, but the population was too small to generate any reliable conclusions. The project shows that the polymorphism in NT5E, whose functional consequences are yet unknown, might have an effect on the extracellular adenosin synthesis and RA pathogenesis. Further studies are required to shed more light on this matter. / Reumatoid artrit är en autoimmun sjukdom där leder angrips av det egna immunförsvaret, vilket leder till kronisk inflammation och nedbrytning av brosk och ben. Inflammation är en komplex mekanism som regleras av många olika substanser, varav en är adenosin, som i förhöjda koncentrationer fungerar som en broms av inflammationen. I detta arbetet har tre olika polymorfier i gener inblandade i syntes och signalering av adenosin genotypats för 188 patienter med nydiagnostiserad RA, samt hos en kontrollgrupp på 362 frivilliga utan reumatisk sjukdom. Avseende en polymorfi i A2a, som kodar för en adenosinreceptor, fanns inga signifikanta skillnader i fördelning mellan fall och kontroller. Beträffande sjukdomsförloppet sågs dock vissa skillnader i patienternas bedömning av smärta och välbefinnande. För polymorfin i NT5E, som kodar för ett nukleotidas för extracellulär adenosinsyntes, hittades skillnader både i fördelningen mellan RA-gruppen och kontrollgruppen, samt i sjukdomsförloppet, där NT5E-AA genotypen var relaterad till högre inflammation, men samtidigt till lägre smärtupplevelse. För den tredje polymorfin (ADA) var antalet individer med mutant allel för litet för att tillåta några slutsatser. Tester gjordes även för att se om de olika adenosinpolymorfierna korrelerade till alkoholkonsumtion, men underlaget var för litet för att några slutsatser skulle kunna dras. Arbetet visar att polymorfin i NT5E, vars funktionella effekt är okänd, kan ha en effekt på nukleotidasets förmåga att producera extracellulärt adenosin. Kompletterande studier på större material behövs för att bringa klarhet i dessa frågor.

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.

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.

A cafeína é o psicoestimulante mais consumido mundialmente. Sua ação farmacológica consiste em bloquear os receptores de adenosina A1 e A2A. A administração crônica de cafeína previne déficits cognitivos decorrentes da idade e em modelos experimentais da doença de Alzheimer. Esses efeitos preventivos são também observados pela administração do antagonista seletivo do receptor de adenosina A2A. Nesse trabalho investigou-se a participação dos receptores de adenosina A1 e A2A na prevenção dos déficits cognitivos induzidos por escopolamina. Também foi investigado se a manipulação dos receptores A2A teria algum impacto na memória em camundongos naive. Para isso, foram utilizadas três tarefas comportamentais que avaliaram três tipos de memória: a tarefa de reconhecimento de objetos (RO), a esquiva inibitória (EI) e o labirinto em Y. A administração intraperitoneal de escopolamina (1,0 mg/kg) prejudicou o desempenho da memória de curto prazo nas três tarefas utilizadas. O antagonista de receptores A1 (DPCPX, 1,0 mg / kg, i.p.) preveniu a amnésia induzida por escopolamina no RO e na EI, enquanto o antagonista de receptores A2A (SCH 58261, 0,5 mg / kg, i.p.) preveniu a amnésia em todos os testes. Além disso, ambos os antagonistas não apresentaram efeitos sobre a memória ou a locomoção em animais naive. Também foi observado que a ativação dos receptores A2A, a partir da administração de CGS 21680 (0,1 mg/kg, i.p.) antes da sessão de treino, foi suficiente para provocar prejuízos na memória em animais naive também nas três tarefas, e este efeito foi prevenido por meio da administração de SCH 58261 (0,5 mg/kg, i.p.). Por fim, a administração intracerebroventricular (i.c.v) de CGS 21680 (50 nmol) também prejudicou o desempenho dos animais na tarefa de RO. Em conjunto, estes resultados sugerem que a ativação dos receptores A2A é suficiente para provocar déficits de memória e ainda sugerem que os receptores A1 também participam de maneira seletiva no controle dos déficits de memória relacionados ao sistema colinérgico. / Caffeine, a non-selective adenosine receptor antagonist, prevents memory deficits, an effect mimicked by adenosine A2A receptor (A2AR), but not receptor A1 (A1R), antagonists upon aging and Alzheimer´s disease. We tested if A2AR were also necessary for the memory impairment upon direct perturbation of the cholinergic system with scopolamine and if A2AR activation was sufficient to trigger memory deficits in naive mice using 3 tests, to probe for short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. The intra-peritoneal (i.p.) administration of scopolamine (1.0 mg/kg) impaired short-term memory performance in 3 tests, namely the object recognition task, inhibitory avoidance and modified Y-maze. The scopolamine-induced amnesia was prevented by the A2AR (SCH 58261, 0.5 mg/kg, i.p.) as well as by A1R antagonist (DPCPX, 1 mg/kg, i.p.) in all tests, except for the modified Y-maze, and both antagonists were devoid of effects on memory or locomotion in naive rats. Notably, the activation of A2AR with CGS 21680 (0.1 mg/kg, i.p.) before the training session was sufficient to trigger memory impairment in the 3 tests in naive mice, and effect prevented by SCH 58261 (0.5 mg/kg, i.p.). Furthermore, the intracerebroventricular administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2AR are necessary and sufficient to trigger memory impairment and they further suggest that A1R might also be selectively engaged to control the cholinergic driven memory impairment.

A cafeína é o psicoestimulante mais consumido mundialmente. Sua ação farmacológica consiste em bloquear os receptores de adenosina A1 e A2A. A administração crônica de cafeína previne déficits cognitivos decorrentes da idade e em modelos experimentais da doença de Alzheimer. Esses efeitos preventivos são também observados pela administração do antagonista seletivo do receptor de adenosina A2A. Nesse trabalho investigou-se a participação dos receptores de adenosina A1 e A2A na prevenção dos déficits cognitivos induzidos por escopolamina. Também foi investigado se a manipulação dos receptores A2A teria algum impacto na memória em camundongos naive. Para isso, foram utilizadas três tarefas comportamentais que avaliaram três tipos de memória: a tarefa de reconhecimento de objetos (RO), a esquiva inibitória (EI) e o labirinto em Y. A administração intraperitoneal de escopolamina (1,0 mg/kg) prejudicou o desempenho da memória de curto prazo nas três tarefas utilizadas. O antagonista de receptores A1 (DPCPX, 1,0 mg / kg, i.p.) preveniu a amnésia induzida por escopolamina no RO e na EI, enquanto o antagonista de receptores A2A (SCH 58261, 0,5 mg / kg, i.p.) preveniu a amnésia em todos os testes. Além disso, ambos os antagonistas não apresentaram efeitos sobre a memória ou a locomoção em animais naive. Também foi observado que a ativação dos receptores A2A, a partir da administração de CGS 21680 (0,1 mg/kg, i.p.) antes da sessão de treino, foi suficiente para provocar prejuízos na memória em animais naive também nas três tarefas, e este efeito foi prevenido por meio da administração de SCH 58261 (0,5 mg/kg, i.p.). Por fim, a administração intracerebroventricular (i.c.v) de CGS 21680 (50 nmol) também prejudicou o desempenho dos animais na tarefa de RO. Em conjunto, estes resultados sugerem que a ativação dos receptores A2A é suficiente para provocar déficits de memória e ainda sugerem que os receptores A1 também participam de maneira seletiva no controle dos déficits de memória relacionados ao sistema colinérgico. / Caffeine, a non-selective adenosine receptor antagonist, prevents memory deficits, an effect mimicked by adenosine A2A receptor (A2AR), but not receptor A1 (A1R), antagonists upon aging and Alzheimer´s disease. We tested if A2AR were also necessary for the memory impairment upon direct perturbation of the cholinergic system with scopolamine and if A2AR activation was sufficient to trigger memory deficits in naive mice using 3 tests, to probe for short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. The intra-peritoneal (i.p.) administration of scopolamine (1.0 mg/kg) impaired short-term memory performance in 3 tests, namely the object recognition task, inhibitory avoidance and modified Y-maze. The scopolamine-induced amnesia was prevented by the A2AR (SCH 58261, 0.5 mg/kg, i.p.) as well as by A1R antagonist (DPCPX, 1 mg/kg, i.p.) in all tests, except for the modified Y-maze, and both antagonists were devoid of effects on memory or locomotion in naive rats. Notably, the activation of A2AR with CGS 21680 (0.1 mg/kg, i.p.) before the training session was sufficient to trigger memory impairment in the 3 tests in naive mice, and effect prevented by SCH 58261 (0.5 mg/kg, i.p.). Furthermore, the intracerebroventricular administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2AR are necessary and sufficient to trigger memory impairment and they further suggest that A1R might also be selectively engaged to control the cholinergic driven memory impairment.