Tesis sobre el tema "Adenosine receptor antagonists"

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Starting from chemical structure of N-benzo-[1,3]dioxol-5-yl-2-[5-(2,6dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol3-yloxy]-acetamide, MRE2029F20* various structural modifications were realized to afford a new series of A2B antagonists. The bioisosteric replacement of the anilide moiety with benzimidazole or quinazoline rings, the effect of the substitution of pyrazole with isoxazole moiety were investigated. Amide bond has been also replaced with the 5phenyl-1,2,4-oxadiazole nucleus on the basis of other adenosine pharmacophores reported previously. In this context the effect of the nitrogen at the 9-position has been also studied preparing four 9-deaza direct analogs of 8pyrazol-xanthine compounds to compare affinity and selectivity at A2B adenosine receptor. The most significant result was obtained by bioisosteric replacement of the anilide moiety with benzimidazole, achieving antagonists with high affinity and selectivity toward the A2BAR. In particular compound 8-[5-(4-Chloro-6-trifluoromethyl-1H-benzoimidazol-2-ylmethoxy)-2methyl-2H-pyrazol-3-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (hA1 Ki = 2530 nM, hA2A Ki > 1000 nM, hA2B Ki = 9.4 nM, hA3 Ki > 1000 nM) and compound 8-[5-(4,6-Bis-trifluoromethyl-1H-benzoimidazol-2-ylmethoxy)-2methyl-2H-pyrazol-3-yl]-1,3-dipropyl-3,7 dihydro-purine-2,6-dione (hA1 Ki = 4462 nM, hA2A Ki > 1000 nM, hA2B Ki = 25 nM, hA3 Ki > 1000 nM), showed the best biological data. These new selective and potent A2B antagonists will aid in the elucidation of the physiological role of this receptor and possibily lead to therapeutilally useful agents for treating asthma, diabetes and other diseases.

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

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
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G protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes, ranging from the transmission of the light and odorant signals to the mediation of neurotransmission and hormonal actions. GPCRs are dysfunctional or deregulated in several human diseases and are estimated to be the target of more than 40% of drugs used in clinical medicine today. The crystal structures of rhodopsin and the recent published crystal structures of beta-adrenergic receptors and human A2A Adrenergic Receptor provide the information of the three-dimensional structure of GPCRs, which supports homology modeling studies and structure-based drug-design approaches. Rhodopsin-based homology modeling has represented for many years a widely used approach to built GPCR three-dimensional models. Structural models can be used to describe the interatomic interactions between ligand and receptor and how the binding information is transmitted through the receptor. Both agonist and antagonist like states can be described by several different conformational receptor states depending on the nature of both ligand and receptor. Considering different complementarities, we might explore different conformations of the same pharmacological state. We investigated the molecular pharmacology of adenosine receptors and, in particular, the human A3 adenosine receptor (hA3AR) by using an interdisciplinary approach to speed up the discovery and structural refinement of new potent and selective hA3AR antagonists. Human A3AR belongs to adenosine receptors family of GPCRs, which consists of four distinct subtypes: A1, A2A, A2B, A3 that are ubiquitously expressed in the human body. The hA3AR, which is the most recently identified adenosine receptor, is implicated in a variety of important physiological processes. Activation of A3ARs increases the release of inflammatory mediators, such as histamine from rodent mast cells, and it inhibits the production of tumor necrosis factor-alpha. The activation of the hA3AR seems to be involved in immunosuppression and in the response to ischemia of the brain and heart. Agonists or antagonists of A3ARs are potential therapeutic agents for the treatment of ischemic and inflammatory diseases. The first model of human A3AR has been built using a conventional rhodopsin-based homology modeling approach. The model has been used to probe atomic level specific interactions, detected using site-directed mutagenesis analysis. The rhodopsin-based model of the hA3AR in its resting state (antagonist-like state) has been revisited, taking into account a novel strategy to simulate the possible receptor reorganization induce by the antagonist-binding. We called this new strategy ligand-based homology modeling (LBHM). It is an evolution of a conventional homology modeling algorithm: any selected atoms will be included in energy tests and in minimization stages of the modeling procedure. Ligand-based option is very useful when one wishes to build a homology model in the presence of a ligand docked to the primary template. Starting from the conventional rhodopsin-based homology model and applying our ligand-based homology modeling implementation we can generate other antagonist-like conformational states of hA3AR in which the ligand recognition cavity is expanded. Using different antagonist-like conformational states, we are able to rationalize the observed activities for all the compounds analyzed. Many severe analysis concerning false-positives and false-negatives situations are usually conducted. To strictly validate this methodology as novel tool to address the multi-conformational space of GPCRs, we have analyzed different classes of known human A3 antagonists in the corresponding putative ligand binding site: for example triazoloquinoxalin-1-one derivatives, arylpyrazolo-quinoline derivatives and pyrazolo-triazolo-pyrimidines derivatives. These studies led to the identification of groups for every class of antagonists that, introduced one by one in a suitable position, afford high hA3AR affinity and good selectivity. Starting from these binding requirements, we decided to perform an in silico molecular simplification approach to identify a suitable fragmentation route of the 4-amino-triazoloquinoxalin-1-one scaffold and explore which of the structural features were essential to guarantee efficient ligand-receptor recognition. With the availability of new three dimensional templates different from rhodopsin, we built new models of hA3AR. All the models were used for a molecular dynamic simulation in a POPC bilayer to investigate the topological fluctuation of the binding pocket.
I recettori accoppiati alle proteine G (GPCR) costituiscono una grande famiglia di proteine integrali di membrana caratterizzate da sette eliche transmenmbrana, che mediano un'ampia gamma di processi fisiologici che vanno dalla trasmissione della luce e dei segnali olfattivi alla mediazione della neurotrasmissione e dell'azione degli ormoni. I GPCR mancano di una corretta regolazione in molte patologie umane ed è stato stimato che costituiscano il target del 40% dei medicinali utilizzati attualmente in clinica. La struttura cristallografica della rodopsina e le strutture più recenti del recettore beta adrenergico e del recettore adenosinico A2A forniscono l'informazione strutturale che sta alla base della costruzione di modelli per omologia e degli approcci di structure-based drug design dei GPCR. La costruzione di modelli di GPCR per omologia basati sulla struttura della rodopsina ha rappresentato per molti anni un approccio ampiamente utilizzato. Questi modelli possono essere usati per descrivere le interazioni interatomiche tra ligando e recettore e come le informazioni sono trasmesse attraverso il recettore. Diversi stati conformazionali del recettore possono essere in grado di descrivere la conformazione del recettore che lega l'agonista e quella che lega l'antagonista, a seconda della natura di ligando e recettore. Se si considerano diverse complementarietà, si possono esplorare diversi stati conformazionali di uno stesso stato farmacologico. Noi abbiamo studiato la farmacologia molecolare dei recettori adenosinici e, in particolare, del recettore adenosinico A3 umano (hA3AR), utilizzando un approccio interdisciplinare al fine di massimizzare la scoperta e l'ottimizzazione strutturale di nuovi antagonisti potenti e selettivi per il hA3AR. Il hA3AR fa parte della famiglia dei recettori adenosinici che consiste in quattro diversi sottotipi (A1, A2A, A2B, A3) che sono espressi in tutto il corpo umano. Il recettore adenosinico A3 è stato identificato più recentemente ed è implicato in importanti processi fisologici. L'attivazione del hA3AR aumenta il rilascio di mediatori dell'infiammazione, come l'istamina dalle mastcellule, e inibisce la produzione del TNF-alpha. L'attivazione del hA3AR sembra essere coinvolta nell'immunosoppressione e nella risposta ischemica di cuore e cervello. Agonisti o antagonisti del hA3AR sono potenziali agenti terapeutici nel trattamento di patologie ischemiche e infiammatorie. Il primo modello di hA3AR è stato costruito usando un approccio convenzionale di homology modeling basato sulla rodopsina ed è nel suo stato che lega l'antagonista. Dopo essere stato utilizzato per verificare le interazioni a livello molecolare che erano state evidenziate da studi di mutagenesi, il modello è stato rivisto prendendo in considerazione una nuova strategia che simula la possibile riorganizzazione del recettore indotta dal legame con l'antagonista. Abbiamo chiamato questa strategia ligand-based homology modeling. E' un'evoluzione dell'algoritmo convenzionale di homology modeling: ogni atomo selezionato viente preso in considerazione nei test energetici e nelle fasi di minimizzazione della procedura di modeling. L'opzione ligand-based è molto utile quando si vuole costruire un modello per omologia in presenza di un ligando nella sua ipotetica conformazione di legame nel templato iniziale. A partire dal modello ottenuto dalla rodopsina e applicando la tecnica del LBHM, possiamo generare altri stati conformazionali del recettore hA3AR che legano l'antagonista, nei quali la cavità di riconoscimento del ligando è espansa. Usando diversi stati conformazionali che legano l'antagonista, possiamo razionalizzare l'attività misurata sperimentalmente di tutti i composti analizzati. Sono condotte severe analisi relative a falsi positivi e falsi negativi. Per validare la metodologia come nuovo strumento per indirizzare lo spazio multiconformazionale dei GPCR, abbiamo analizzato diverse classi di antagonisti con attività nota sul hA3AR: ad esempio derivati triazolo-chinossalinonici, derivati arilpirazolo-chinolinici e derivati pirazolo-triazolo-pirimidinici. Questi studi hanno portato all'identificazione di gruppi per ogni classe di antagonisti che, se introdotti in una precisa posizione, portano ad un'alta affinità e ad una buona selettività per il hA3AR. A partire dalle caratteristiche risultate importanti per il legame, abbiamo applicato una tecnica di semplificazione molecolare in silico per identificare una possibile via di frammentazione della struttura 4-amino-triazolochinoassalin-1-onica ed esplorare quali sono le caratteristiche strutturali essenziali per garantire un'efficiente riconoscimento ligando-recettore. Con la disponibilità di nuove strutture tridimensionali da utilizzare come templati diversi dalla rodopsina, abbiamo costruito nuovi modelli del recettore hA3AR. Tutti i modelli sono stati usati per una simulazione di dinamica molecolare in un doppio strato fosfolipidico, per analizzare le fluttuazioni topologiche della tasca di legame.

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.

The adenosine A2A receptor has emerged as an attractive target for the treatment of Parkinson's disease (PD). Evidence suggests that antagonists of the A2A receptor (A2A antagonists) partially alleviate the symptoms of PD, prevent the development of motor complications and may also slow the underlying neurodegenerative process. It was recently reported that several members of the (E)-8-styrylcaffeine class of A2A antagonists also are potent inhibitors of monoamine oxidase B (MAO-B).
Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2009.

Background: Pulmonary hypertension (PH) is a rare condition characterized by endothelial dysfunction and vascular remodelling, leading to increased pulmonary vascular resistance (PVR) and right ventricular heart failure. Endothelial dysfunction is associated with an imbalance between vasoconstrictor compounds, such as endothelin and thromboxane A2, and vasodilator compounds, such as prostacyclin and nitric oxide (NO). Asymmetric dimethylarginine (ADMA), a methyl derivate of L-arginine, inhibits synthesis of NO. Vardenafil, a phosphodiesterase type 5 inhibitor (PDE5-inhibitors), causes vasodilation through the NO/cGMP pathway. Aim: This thesis investigates the pharmacological effects and diagnostic utility of vardenafil in PH patients. In addition, to evaluate the change of L-arginine and dimethylarginines before and during PAHspecific therapy in PAH patients compared to patients with left ventricular heart failure (LVHF) and healthy subjects. Methods: The pharmacokinetics and hemodynamic effects of vardenafil were examined during right heart catheterization (RHC) in 16 PH patients and plasma concentrations were measured for up to nine hours after oral administration. In 20 PH patients, acute vasoreactivity test with vardenafil was performed during RHC. Hemodynamic responses were recorded, responders were defined and followed for up to seven years. Additionally, plasma ADMA, symmetric dimethylarginine (SDMA), L-arginine, L-citrulline and L-ornithine levels before and after PAH drug treatment were monitored in 21 PAH patients and compared to values measured in 14 LVHF patients and 27 healthy subjects. Results: Vardenafil concentrations increased rapidly to maximum plasma concentration (tmax 1h) and elimination half-life was 3.4 h. Patients co-medicated with bosentan had reduced vardenafil concentration. Significant acute hemodynamic responses were observed for mean pulmonary artery pressure (mPAP) (p<0.001), pulmonary vascular resistance (PVR) (p<0.001), cardiac output (CO) (p=0.015), cardiac index (CI) (p=0.010), systemic vascular resistance (SVR) (p<0.001) and PVR/SVR (p=0.002) and were related to plasma vardenafil concentrations. PAH patients had significantly higher ADMA and SDMA levels and significantly lower L-arginine levels and L-arginine/ADMA ratio compared with healthy subjects (p<0.001). L-arginine was also lower in PAH patients compared to patients with LVHF (p<0.05). WHO functional class and six minutes walking distance (6MWD) correlated to Larginine and L-arginine/ADMA ratio in PAH at baseline (p<0.05). At follow-up, patients on mono- or combinationtherapy with endothelin receptor antagonists (ERA) had lower ADMA levels than patients without ERA (p<0.05). In contrast, patients on PDE5-inhibitors had higher ADMA levels compared to patients without PDE5-inhibitors (p<0.05). Conclusion: Vardenafil is safe in acute vasoreactivity test in PH patients. Cardiopulmonary hemodynamic response was related to plasma drug concentrations. There was a high inter-individual variability of vardenafil pharmacokinetics and co-medication with bosentan caused a pharmacokinetic drug interaction. Baseline L-arginine and dimethylarginines levels were different in PAH patients compared to LVHF patients and healthy controls. PAH-specific treatment influenced L-arginine and dimethylarginines. Our data suggest that L-arginine might be useful for differentiating PAH from LVHF, and L-arginine/ADMA ratios were related to the severity of PAH and might be useful for follow-up evaluations of PAH patients.

The neuromodulator adenosine affects a wide variety of physiopathological processes through activation of four receptors, classified as A1, A2A, A2B, and A3 subtypes. Adenosine receptors (ARs) belong to family A of G protein-coupled receptors (GPCRs) and are ubiquitously expressed in the human body. Activation or blockade of ARs is responsible for a wide range of effects in numerous organ systems; and therefore the regulation of ARs can have many potential therapeutic applications. The main objective of this project has been the investigation of the in silico molecular pharmacology of adenosine receptors and, in particular, of the human A2A and A3 adenosine receptors to guide the discovery and the structural refinement of new potent and selective AR antagonists. The recently published crystal structures of the human A2A adenosine receptor (hA2AAR) provide detailed three-dimensional information useful to support homology modeling studies and receptor-based drug design approaches. In particular, the 2.6 Å crystallographic structure of the hA2AAR in complex with the potent and selective antagonist ZM241385 was used as template to build a homology model of the hA3AR. In order to validate the molecular docking protocols for the adenosine receptors family, the hA2AAR crystal structure was used to perform in parallel molecular docking studies using different docking software. Then RMSD values between predicted and crystallographic poses of ZM241385 were calculated to select the docking protocol able to better reproduce this molecular system and to be used in the following molecular docking studies. Subsequently, molecular docking studies of different ARs antagonists were performed at the hA3AR model and at the hA2AAR crystal structure, enabling the exploration of the potential effects of chemical modifications of these compounds, and thus facilitating the lead optimization process. Different series of new compounds belonging to known adenosine antagonists classes, including triazolo-triazines and pyrazolo-triazolo-pyrimidines, have been analyzed and modified with the aim to modulate their affinity towards different adenosine receptor subtypes, to increase their solubility, or to overcome their metabolic instability. Moreover, several compounds with simplified scaffolds have been proposed as new adenosine receptor antagonists; such as pyrazolo-pyrimidinones, phthalazinones and triazolo-pyrimidines. Finally, the knowledge gained through the docking studies led to the identification of structural features of antagonist compounds important for the interaction with the hA3AR and was applied to the design of fluorescent ligands for this subtype, of particular interest as pharmacological probes. In conclusion, the integration of in silico studies with synthetic work and pharmacological tests resulted to be a good strategy for the development of new compounds as adenosine receptors antagonists and led to a better understanding at the molecular level of this class of GPCRs.
L’adenosina è un neuromodulatore che regola molti processi fisiopatologici attraverso l’attivazione di quattro diversi recettori accoppiati a proteine G (GPCRs), classificati come sottotipi A1, A2A, A2B e A3. I recettori adenosinici sono ubiquitari nell’organismo umano e la loro attivazione è responsabile di numerosi effetti in diversi organi. Proprio per questo motivo la regolazione dell’attività di questi recettori può avere interessanti applicazioni terapeutiche. Il principale obiettivo di questo progetto è stato l’analisi in silico a livello molecolare dei recettori adenosinici, ed in particolare dei recettori adenosinici umani A2A e A3, per guidare la scoperta e l’ottimizzazione strutturale di nuovi antagonisti adenosinici potenti e selettivi. Le strutture cristallografiche del recettore adenosinico umano A2A, recentemente pubblicate, forniscono dettagliate informazioni strutturali utili per supportare studi di homology modeling e approcci di drug design di tipo structure-based. In particolare, la struttura cristallografica del recettore adenosinico umano A2A, in complesso con l’antagonista potente e selettivo ZM241385, è stata utilizzata come templato per la costruzione di un modello per omologia del recettore adenosinico umano A3. Inoltre, con l’intento di selezionare il protocollo di docking molecolare più adatto per la famiglia dei recettori adenosinici, la struttura cristallografica del recettore adenosinico A2A è stata utilizzata per effettuare simulazioni di docking con diversi softwares in parallelo. Successivamente, le conformazioni ottenute dal docking sono state confrontate con la pose cristallografica di ZM241385 per selezionare il protocollo di docking che fosse in grado di riprodurre al meglio questo sistema molecolare e che potesse quindi essere usato per i successivi studi di docking. Sono stati quindi effettuati studi di docking molecolare di vari antagonisti adenosinici sul modello del recettore A3 e sulla struttura cristallografica del recettore A2A, in modo da ricavare informazioni che potessero facilitare il processo di ottimizzazione dei composti. Sono stati infatti analizzati numerosi nuovi composti appartenenti a classi note di antagonisti adenosinici, tra cui composti triazolotriazinici e tirazolotriazolopirimidinici, in modo da suggerire modifiche strutturali in grado di modularne l’affinità nei confronti dei vari sottotipi recettoriali adenosinici, di aumentarne la solubilità o di superarne i punti di instabilità metabolica. Diversi derivati con strutture semplificate, come per esempio composti pirazolopirimidinonici, ftalazinonici e triazolotriazinici, sono stati inoltre proposti come nuovi composti con attività antagonista nei confronti dei recettori adenosinici. Le informazioni ricavate grazie agli studi di docking hanno permesso l’identificazione di caratteristiche strutturali degli antagonisti adenosinici fondamentali per l’interazione con questi recettori. Queste informazioni sono state quindi applicate alla progettazione di derivati fluorescenti per il recettore adenosinico A3, che risultano particolarmente interessanti per il loro potenziale utilizzo in saggi farmacologici. In conclusione, quindi, questo studio sui recettori adenosinici dimostra come l’integrazione di metodologie computazionali con il lavoro sintetico e farmacologico risulta essere una strategia efficace per lo sviluppo di nuovi ligandi dei recettori adenosinici, a potenziale interesse terapeutico, e per il chiarimento di importanti aspetti strutturali riguardanti questa famiglia recettoriale e più in generale tutti i GPCRs.

The aim of this work was the design and synthesis of new A2A and A3 adenosine receptors antagonists. Two different studies have been performed. The first one based on structural modifications of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pirimidine nucleus, reported in literature. In order to identify a new series of A2A or A3 AR antagonists and with the aim to better investigate the role of the nitrogen at the 7- position on the interaction with ARs, it was performed a synthetic strategy for the preparation of the pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus which can be considered the 7-deaza-analogue of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine core. In order to complete the SAR studies on this class of compounds, it has been synthesised a novel series of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives which can be considered the structural isomers of the parent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. From the biological data obtained, we can assert that the concomitant presence of the N7 and N8 is fundamental for the selectivity of these A2A/A3 ligands versus the remaining ARs subtype. Our results confirmed the importance of the presence of the NH at the 5- position of the PTPs nuclus, this could be due to the formation of an essential ligand-receptor hydrogen bond. Most of these compounds, revealed to be non selective ARs antagonists. Derivative 5-{[(4-methoxy-phenyl)carbamoyl]amino}-(2-furan-2-yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine resulted to be the best compound of the series in term of both affinity (hA3Ki = 15nM) and selectivity. The aim of the second project was to obtain A3 antagonists with improve water-solubility. A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-one derivatives have been synthesised and evaluated in radioligand binding assays to determine their affinities at the human A1, A2A, and A3 adenosine receptors. Efficacy at the hA2B AR and antagonism of selected ligands at the hA3 AR were also assessed through cAMP experiments. All the synthesised molecules exhibited high affinity at the hA3 AR (Ki values ranging from 1.46 to 44.8 nM) as well as remarkable selectivity versus A1, A2A and A2B AR subtypes. In view of the chirality of the tricycles, for selected compounds both the racemic mixtures and the pure enantiomers have been prepared with the purpose of determine a possible receptor stereoselectivity. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one showed to be the most potent hA3 AR ligand of the series (hA3Ki = 1.46 nM), in addition with very high selectivity over all the other ARs (hA2AKi/ hA3Ki > 3425; hA2BIC50/ hA3Ki > 3425; hA1Ki/ hA3Ki = 1,729).

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.

Pyrazolo[3,4-d]pyrimidines were known as adenosine antagonists at the rat A1 and A2A adenosine receptors based on our previous studies. In this study, 245 pyrazolo[3,4-d]pyrimidines derivatives with various benzyl substitutents at N-1 and various hydrophobic side chains at C-4 and C-6 were synthesized and screened at the human A1, A2A and A3 adenosine receptors. 14 out of 245 compounds were resynthesized and purified to determine the Ki values of these compounds at the human A1 adenosine receptor. Chapter 1 of the thesis is a literature review of adenosine research. It describes the physiology of adenosine and the discovery and characterization of all adenosine receptors namely A1, A2A, A2B and A3. It also looks at the medical application of adenosine, adenosine analogs, adenosine agonists and adenosine antagonists. The final part of the chapter discusses the discovery and development of adenosine agonists and antagonists. Chapter 2 of the thesis describes the rational design of the pyrazolo[3,4-d]pyrimidines template using ligand-based molecular modelling technique and describes the synthesis of the template. Chapter 3 and chapter 4 describe the application of silicon chemistry and attempts to synthesise a series of pyrazolo[3,4-d]pyrimidines heterocycle by solid phase synthesis. Chapter 5 and chapter 6 describe the synthesis of a series of pyrazolo[3,4-d]pyrimidines heterocycle using the solution phase parallel synthesis and the binding studies of a library of 245 compounds and the resynthesis of 14 target compounds. Chapter 7 describes the cell culture and membrane preparation of the human A1, A2A, A2B and A3 adenosine receptors and radioligands binding assays.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
Full Text

AGUIAR, Lissiana Magna Vasconcelos. Antagonismo do receptor da adenosina A2a : nova perspectiva para o tratamento da doença de Parkinson. 2009. 215 f. Tese (Doutorado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009.
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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 animals’ 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 doença de Parkinson (DP) é uma desordem neurodegenerativa, caracterizada pela destruição dos neurônios nigroestriatais dopaminérgicos. O tratamento atual para esta doença está restrito ao alívio sintomático, porque até o presente momento não existem agentes capazes de inibir a degeneração neuronal. Existem evidências experimentais de que antagonistas de receptores A2A da adenosina poderiam ser úteis no tratamento de DP. Com a finalidade de investigar essa possibilidade, o presente trabalho demonstrou os efeitos da cafeína e do CSC (8-(3-chlorostyryl caffeine) no comportamento rotacional e nas alterações neuroquímicas em ratos lesionados com 6-OHDA, como modelo da doença de Parkinson. Os animais (ratos Wistar machos, 250-280g) foram tratados com cafeína (10 e 20 mg/kg, i.p.) diariamente durante 14 dias, iniciando 1h após a lesão ou 7 dias, iniciando seis dias após a lesão com 6-OHDA ou com CSC (1 e 5 mg/kg, i.p.) diariamente durante 7 dias, iniciando 6 dias após 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 número de rotações induzidas por apomorfina nos animais lesionados com 6-OHDA (50 vezes) quando comparados aos animais falso operados. O tratamento com cafeína, principalmente durante 14 dias e o tratamento com CSC produziram uma recuperação motora parcial com redução do número de rotações. A 6-OHDA provocou morte neuronal evidenciada pela redução dos níveis de monoaminas (75-85%) quando comparadas ao lado contralateral. Nos grupos tratados com cafeína ou CSC sozinho ou associado com L-DOPA a redução dos níveis de DA, 5HT e seus metabólitos foi menor. As concentrações dos aminoácidos glutamato e GABA foram significativamente aumentadas (3,8 e 3 vezes, respectivamente) no estriado de ratos lesionados. O CSC reverteu essas alterações significativamente e foi observada uma potencialização desses efeitos na associação com L-DOPA. Os experimentos in vitro demonstraram que a cafeína e o CSC apresentaram um forte efeito neuroprotetor nas células mesencefálicas de rato expostas a 6-OHDA. O tratamento com CSC ou cafeína aumentou significativamente o número de células viáveis após a exposição das células a 6-OHDA, como foi demonstrado pelo teste do MTT. A exposição das células mesencefálicas a 6-OHDA aumentou os conteúdos de nitrito e a peroxidação lipídica, que retornaram a concentrações normais após tratamento com CSC ou cafeína. Além disso, a 6-OHDA reduziu o número de células normais e aumentou o número de células apoptóticas e o tratamento com CSC ou cafeína reverteu esses efeitos da 6-OHDA, promovendo aumento do número de células viáveis e redução do número de células apoptóticas. Houve uma redução do número de microglias ativadas após a exposição das células a cafeína e a 6-OHDA, o mesmo não ocorreu após a exposição das células ao CSC e a 6-OHDA. O tratamento com cafeína reduziu o aumento do número de astrócitos reativos induzidos pela 6-OHDA, enquanto o CSC não apresentou esse efeito. Esses resultados mostraram que ambos, a cafeína e o CSC apresentaram ações neuroprotetoras em células mesencefálicas de rato expostas a 6-OHDA. O presente trabalho mostrou que a cafeína e o CSC reverteram às alterações comportamentais e neuroquímicas da 6-OHDA, apresentando efeitos possivelmente benéficos no tratamento da DP.

CARMO, Marta Regina Santos do. Efeito neuroprotetor do antagonismo do receptor P2X7 no Parkinsonismo experimental induzido por 6-OHDA. 2015. 110 f. Tese (Doutorado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015.
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Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and a concomitant decrease of dopamine (DA) in the striatum, which can be modeled by 6-OHDA administration. Since ATP released from damaged cells can exert noxious effects on neurons, acting through its P2X7 receptors (P2X7R), the aim of the present study was to investigate the effects of a P2X7R antagonist, Brilliant Blue G (BBG) on 6-OHDA-induced neurotoxicity. Male Wistar rats received stereotaxic injections of 6-OHDA (18 µg/3µl) into the right striatum and were treated with BBG (45 mg/kg, i.p. 48/48 h) for two weeks. In an additional experiment, animals were treated with the selective antagonist A-438079 (10 µM i.c.v.) for two weeks. BBG decreased the number of contralateral rotations in the apomorphine test, an effect mimicked by the selective P2X7R antagonist A438079. BBG has also improved the animals’ performance in the passive avoidance test (short-term memory) and in the cued version of the Morris Water maze. The antagonism of P2X7R by BBG has also prevented the reduction of dopamine content in the striatum and SN as well as the loss of dopaminergic neurons, and microgliosis and astrogliosis in the striatum. BBG treatment also decreased the IL-1β levels in striatum, despite the observed effect not being statistically significant. To grasp the mechanism of action of BBG, we used in vitro models exploring synaptotoxicity (striatal synaptosomes) and neurotoxicity (dopamine-differentiated SH-SY5Y cells). Besides showing that P2X7R are present in striatal dopaminergic terminals, we observed that BBG 100 nM prevented the 6-OHDA-induced synaptosomal dysfunction. Furthermore, we have shown the presence of P2X7 receptors in SH-SY5Y cells, their co-localization with tyrosine hydroxilase, and that BBG attenuates the cell damage, evaluated through lactate dehydrogenase release. The present results suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in the disease.
A doença de Parkinson (DP) é caracterizada por uma degeneração progressiva dos neurônios da substância negra (SN) e uma concomitante diminuição do conteúdo de dopamina no estriado, que pode ser mimetizada pela administração de 6-OHDA. Visto que o ATP liberado das células danificadas exerce efeitos deletérios diretos sobre os neurônios, agindo através de receptores P2X7, o objetivo do presente trabalho foi estudar os efeitos do Brilliant Blue G (BBG), um antagonista dos receptores P2X7, sobre a neurotoxicidade induzida por 6-OHDA. Ratos Wistar machos receberam injeções estereotáxicas de 6-OHDA (18 µg/3µl) no estriado direito e foram tratados com BBG (45 mg/kg, i.p. 48/48hs) durante 14 dias. Em uma série adicional de experimentos, os animais receberam o antagonista seletivo A-438079 (10 µM i.c.v.) por 14 dias. O tratamento com BBG diminuiu o número de rotações contralaterais no teste da apomorfina, efeito que foi mimetizado pelo antagonista seletivo A-438079. O BBG também melhorou o desempenho dos animais no teste da esquiva passiva (memória de curta duração), assim como na versão com plataforma sinalizada do labirinto aquático. O antagonismo dos receptores P2X7 pelo BBG preveniu ainda a redução do conteúdo de dopamina no estriado e SN, assim como a perda de neurônios dopaminérgicos, a microgliose e astrogliose no estriado. O tratamento com BBG também diminuiu os níveis de IL-1β no estriado, apesar das diferenças observadas entre os grupos não serem estatisticamente diferentes. Com o objetivo de entender melhor o mecanismo de ação do BBG, foram utilizados modelos in vitro explorando a sinaptotoxicidade (sinaptossomas estriatais) e neurotoxicidade (células SH-SY5Y dopaminérgicas diferenciadas). Além da demonstração da presença dos receptores P2X7 nos terminais nervosos estriatais, foi observado que o BBG 100 nM preveniu a disfunção sinaptossomal. Também demonstramos a presença dos receptores P2X7 nas células SH-SY5Y, assim como sua co-localização com tirosina hidroxilase, onde o pré-tratamento com BBG 100 nM diminuiu o dano celular, avaliado através da liberação de lactato desidrogenase. Os resultados obtidos no presente estudo sugerem que os receptores P2X7 contribuem para a patogênese da DP através de um triplo mecanismo sobre a sinaptotoxicidade, gliose e neurotoxicidade, ressaltando o potencial terapêutico dos antagonistas desses receptores na doença.

L’adénosine est un nucléoside ubiquitaire qui exerce un contrôle puissant sur les systèmes nerveux,immunitaire et cardiovasculaire par l’intermédiaire de quatre récepteurs membranaires : A1R, A2AR, A2BR etA3R. L’étude des récepteurs de l’adénosine est nécessaire à la compréhension de physio‐pathologieshumaines non encore élucidées. Pour étudier l’expression des A2AR, nous avons, dans une première étude,produit un anticorps monoclonal, appelé Adonis, d’isotype IgM, . Adonis reconnait un épitope linéaire desept acides aminés sur la partie C‐terminale de la seconde boucle extra‐cellulaire de l’A2AR humain. Adonisrévèle, par Western blotting sur lysats cellulaires, une bande de 45 KDa, correspondant à l’A2AR. Adonis secomporte comme un « agonist‐like » en augmentant la production d’AMPc et en inhibant la proliférationcellulaire via la stimulation des A2AR. Dans une deuxième étude, nous avons utilisé Adonis pour montrerque l’expression des A2AR de cellules mononucléées, qui mime celle des tissus cardiaques, permet dedifférencier certains patients souffrant de syncope neurocardiogénique. Nous avons monté dans unetroisième étude, qu’Adonis induit une « down‐régulation » de l’expression des co‐récepteurs CXCR4 etCCR5 des cellules T via la stimulation des A2AR, et qu’à ce titre il pouvait être un outil thérapeutique dans lesinfections par HIV. Dans une quatrième étude, nous avons évalué les effets anti‐nociceptifs d’Adonis qui,administré par voie intra‐cérébro‐ventriculaire, augmente de manière dose‐dépendante les latencesobtenues avec le test du Hot‐plate et du Tail‐flick chez la souris. Ces effets sont renversés par deuxantagonistes des A2AR mais aussi par un antagoniste des récepteurs aux opioïdes. Ceci suggère que leseffets anti‐nociceptifs d’Adonis sont médiés par la libération d’opioïdes endogènes. En marge de sesétudes, nous avons également testé les propriétés biologiques de nouveaux ligands des A1R dans le cadred’une collaboration entre chimistes et biologistes. Ainsi, nous montrons, dans une cinquième étude, queparmi la trentaine de molécules synthétisées, quatre sont des antagonistes et deux autres des agonistesavec un EC50 de l’ordre du micromolaire pour la production d’AMPc. De tels agonistes des A1R pourraientêtre utiles dans le traitement des douleurs neuropathiques, tandis que les antagonistes le seraient dansl’insuffisance cardiaque ou utilisés comme diurétique. Enfin dans une sixième étude, nous avons testé unemolécule originale, puisque bivalente, possédant un pôle d’activité pour les récepteurs aux opioïdes μ et unautre pour les A1R. Cette molécule est un antagoniste pour les deux récepteurs. Elle pourrait avoir desapplications cliniques dans certaines pathologies comme le choc hypovolémique ou le sevrage aux opiacés
Adenosine interacts on its cell surface receptors, namely A1R, A2AR, A2BR and A3R, to exertphysiological effects on target tissues. Modulation of these adenosine receptors appears to be a currenttopic of research which may bring more comprehensions on human pathophysiology yet to be elucidated.In order to study A2AR expression, we produced, in study 1, a monoclonal antibody anti‐human A2AR, calledAdonis being of IgM, isotype. Adonis recognized a linear epitope of seven amino acids on the C‐terminalpart of the A2AR second extra‐cellular loop. By Western blotting, Adonis reveals a 45 KDa band of A2AR incell lysates. Adonis behaves as an agonist‐like which increases the cAMP production and inhibits cellproliferation through A2AR stimulation. In study 2, we showed that using Adonis, to measure the A2ARexpression of peripheral blood mononuclear cells which mimic those of the cardiac tissue, was able todifferentiate some patients with suspected neurally mediated syncope. We showed, in study 3, that A2ARstimulation by Adonis leads to a down‐regulation of CXCR4 and CCR5 expression on T‐cells, suggesting thatAdonis would be a potential drug to treat HIV infections. In study 4, we showed that intracereboventricularinjection of Adonis increased the Hot‐plate and Tail‐flick test latencies in mice in a dose‐dependent manner.Such increases were prevented by two A2AR antagonists and by an opiate receptor antagonist, suggestingthat the anti‐nociceptive effects of Adonis were mediated, at least in part, by endogenous opioid liberation.The last section focused on biological evaluation of new A1R ligands in collaborative studies betweenchemists and biologists. Indeed we showed, in study 5, that among thirty synthesized molecules, four act asA1R antagonists and two turn out to be A1R agonists with a micromolar EC50 on cAMP production. ThoseA1R agonists would be used in neuropathic pains, whereas other antagonists could be used in cardiacfailure or as diuretic. Finally, in study 6, we tested an original hybrid molecule which was revealed to be abivalent antagonist to μ opiate receptors and A1R. This hybrid compound may have applications in somepathologies such as hypovolemic shock and opiate addiction

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

Orientador: Gentil Alves Filho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias medicas
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Resumo: A policitemia é uma complicação freqüente após o transplante renal, estando associada a um maior risco de fenômenos tromboembólicos. Apesar de controvérsias a respeito de sua patogênese, várias terapêuticas têm sido propostas. Para analisar o efeito de dois grupos de drogas sobre a policitemia pós-transplante, foram estudados 27 transplantados renais. A primeira fase do estudo compreendeu 17 pacientes tratados seqüencialmente com aminofilina e enalapril, por períodos de 12 semanas, intercalados com períodos sem droga. A segunda fase compreendeu 27 pacientes, tratados com enalapril durante 12 semanas, e avaliados a cada quatro semanas. A cada período foram analisados dados referentes ao hematócrito, hemoglobina, total de hemácias e de reticulócitos, níveis séricos de ferro, ferritina, eritropoetina e creatinina, além da pressão arterial média. Antes e durante o tratamento com enalapril, também foram avaliados os fluxos plasmático e sangüíneo renais e a resistência vascular renal. A utilização de' aminofilina não promoveu alterações nos parâmetros hematológicos nem na função renal. Entretanto, a introdução do enalapril cursou com ,redução da eritropoese, caracterizada pela diminuição do hematócrito, da hemoglobina, do total de hemácias e de reticulócitos, além do aumento dos estoques de ferro, a partir da quarta .semana de tratamento. Esta inibição da eritropoese foi independente de variações nos níveis de eritropoetina ou de alterações do fluxo plasmático renal. Após a suspensão do renalapril, houve retomada da eritropoese, novamente caracterizando policitemia. mDesta forma, para este grupo de pacientes, o enalapril provou ser eficaz para o controle da policitemia pós-transplante renal, promovendo inibição da eritropoese independentemente dos níveis circulantes de eritropoetina.ss
Abstract: Polycythemia is a frequent complication of the post-transplant period, increasing the risk of thromboembolic complications. The pathogenesis of post-transplant polycythemia is unknown, but many studies have suggested its treatment. The aim of the present study was to evaluate the effect of two different drugs in the treatment of posttransplant polycythemia. Twenty-seven renal transplant patients were studied. During the first phase of the study protocol, 17 patients received sequentially ammynophylline and enalapril during 12 weeks, altemately with no drug periods. In the second phase, 27 patients were treated with enalapril during 12 weeks, and analyzed each 4 weeks intervals. Data were collected to hematocrit, hemoglobin, red cell and retyculocyte count, iron, ferritin, erythropoietin and creatinine levels. Mean blood pressure was measured in each period. Before and during enalapril treatment, renal plasma flow, renal blood flow and renal vascular resistance were determined. During the ammynophylline period, no changes were observed neither in hematological data nor in renal function. When enalapril was started, we observed a erythropoiesis inhibition, characterized by a reduction in hematocrit, hemoglobin levels, reduction in red cell and retyculocyte count associated to a increase in iron stores. These changes were observed from the 4th week of enalapril treatment. No changes in erythropoietin levels occurred, and the renal plasma flow was unchanged. With ena:lapril withdrawal, an increase of erythropoie sis was observed, with re-establishment of polycythemia. In conciusion, enalapril proved to be useful in control of post- transplant polycythemia in this study. The drug promoted a erythropoiesis inhibition, independently of erythropoietin levels
Doutorado
Doutor em Clínica Médica

L’adénosine (ADO) est un nucléoside ubiquitaire issu de l’ATP et du cycle de la méthionine. Via les récepteurs A1 (A1R), elle favorise la fibrillation atriale (FA). Via les récepteurs A2A (A2AR), elle induit une dilatation coronaire. L’ADO est donc un intermédiaire métabolique et un neurotransmetteur du système cardiovasculaire.La 1ère étude montre que, chez les patients coronariens, l’ADO est corrélée à l’homocystéine (Hcy) et l’uricémie. De plus, l’ADO et l’Hcy sont corrélées au score évaluant l’étendue de l’athérosclérose (score SYNTAX). Enfin, sur un modèle d’hépatocyte, l’ADO induit la production d’Hcy selon un effet dose et un effet temps. L’hyperadénosinémie semble ainsi participer à l’augmentation de l’homocystéinémie et de l’uricémie. Ces données apportent un nouvel éclairage sur la physiopathologie de la maladie coronarienne.Dans le 2nd travail, l’ADO augmente significativement uniquement chez les patients coronariens à test d’effort positif. De plus, leur expression des A2AR est plus faible que les patients à test négatif. Ainsi, la faible expression A2AR chez les coronariens à test d’effort positif participe au défaut d’adaptation coronaire durant le test. Un faible niveau d’A2AR pourrait être alors un biomarqueur de coronaropathie.Dans la 3ème étude, les patients avec FA sans cardiopathie sous-jacente ont une adénosinémie normale et une surexpression des A2AR. Sachant que l’ADO peut favoriser la FA, la surexpression des A2AR pourrait donc participer au déclenchement de FA en augmentant la sensibilité à l’adénosine.En conclusion, les médicaments modulant le système adénosinergique pourraient être utiles au traitement de la coronaropathie ou de la FA
Adenosine (ADO) is an ubiquitous nucleoside that comes from ATP and from the methionine cycle. Via A1 receptors (A1R), it promotes atrial fibrillation (AF). Via A2A receptors (A2AR), it leads to coronary vasodilatation. Thus, adenosine is a metabolic intermediate and a neurotransmitter of the cardiovascular system.The first study showed that adenosine plasma level (APL) is correlated with homocystein (Hcy) and uric acid in coronary artery disease (CAD) patients. Furthermore, APL and Hcy are correlated with the SYNTAX score which evaluate CAD severity. Finally, in cellulo, ADO induced a dose and time dependant increase of HCY production by human hepatocytes. We concluded that high APL may participate into the high HCY and uric acid levels. These data bring new highlight on the physiopathology of CAD.In the second work, APL increased significantly only in patients with positive exercise stress testing (EST). Furthermore, A2AR expression was lower in positive EST patients compared with those with negative EST. Then, we concluded that the low expression of A2AR in CAD patients with positive EST, participates in the lack of adaptive response (coronary vasodilatation) to the EST. This result suggests that low A2AR expression may be a biological marker of CAD.In the third study, patients with AF and no structural heart disease have a normal APL but an increase in A2AR expression. Because adenosine promotes AF, we concluded that high A2AR expression may participate into the triggering of AF by increasing the sensitivity to adenosine.In conclusion, drugs that modulate the purinergic system should be useful tools for the treatment of CAD or AF

La maladie d’Alzheimer (MA) est la maladie neurodégénérative touchant le plus de personnes dans le monde. Jusqu’à présent, aucun traitement curatif n’existe pour soigner cette maladie, d’où la nécessité d’identifier et d’étudier de nouvelles cibles thérapeutiques.La découverte des effets bénéfiques de la caféine, antagoniste du récepteur à adénosine A2A (A2AR), conjuguée à une surexpression de ce dernier chez les patients atteints de la MA, font de ce récepteur une cible d’intérêt. En effet, des antagonistes des A2ARs ont montré leur capacité à améliorer les performances cognitives de par une diminution de la charge amyloïde associée à une diminution la phosphorylation de la protéine Tau.Bien que plusieurs antagonistes aient été développés pour le traitement de maladies neurodégénératives, ceux-ci présentent un manque d’efficacité corrélée à de faibles propriétés pharmacocinétiques. Ainsi, à partir d’études de modélisation moléculaire, deux nouvelles familles d’antagonistes présentant un noyau central benzoxazole ou quinazoline ont été conçus, synthétisés et évalués pharmacologiquement. Trois composés ont été sélectionnés et font actuellement l’objet d’études pharmacologiques complémentaires sur modèles animaux
Alzheimer’s disease (AD) is the most prevalent form of dementia in the aged population. So far, there is no way to halt or slow-down AD. Therefore, there is a constant need of developing novel therapeutic strategies.In recent years, adenosine A2A receptor (A2AR) has attracted a growing interest since it has been proved that this receptor is over-expressed during AD. Also, epidemiological studies showed that people consuming regularly caffeine-based beverages over a lifetime are substantially less likely to develop this disease. Indeed, A2AR antagonists improve memory performance as it reduces β-amyloid deposits and Tau-phosphorylation.Though several antagonists have been developed for the treatment of neurodegenerative diseases, current research efforts are focus on developing new antagonists with relevant ADME properties and a better efficacy. Based on a molecular modeling-guided design, we synthesised new A2AR antagonists with benzoxazole and quinazoline as central scaffold. Three molecules were selected and will be subject to evaluation on animal’s model

博士
國立成功大學
化學系碩博士班
101
Pyrazole is the aromatic heterocyclic compound with the five-membered ring, and it is usually considered a kind of alkaloid. Derivatives of pyrazole are important compounds in pharmaceutical industry, and they are used for their antipyretic, anti-inflammatory, antipyretic, tranquilizing, muscle relaxing and antibacterial activities. In addition, they are also applied in organic light-emitting diode(OLED). The potential therapeutical applications of antagonizing the A3 adenosine receptor(A3 AR) by derivatives of pyrazole have been investigated in recent year. The derivatives of arylpyrazoloquinolinone are A3 AR antagonists. This kind of antagonist with highly selectivity is being sought as antiasthmatic, anti-inflammatory and cerebroprotective agents. In recent years, the electroluminescence materials with blue light-emitting have attracted great attention. As well known, pyrazole is a kind of blue light-emitting material. It is suitable to be applied in OLED after introduce fluorescent group in the pyrazole core. In this investigation, a series of arylpyrazoloquinolinones were synthesized rapidly via 1,3-dipolar cycloaddition and Suzuki coupling. Compare with previous references, we used cheaper reagents and convenience method. In addition, a new category of small molecular electroluminescence materials was synthesized by the synthetic routes we designed. The properties of optics, electrochemistry and thermology were performed by UV, PL, CV and DSC. The EL devices of materials were also performed by vacuum deposition and brightness meter.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD.
MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Dissertação de mestrado em Investigação Biométrica, apresentada à Faculdade de Medicina da Universidade de Coimbra
O glaucoma é uma doença neurodegenerativa e sem cura, caracterizada pela morte das células ganglionares da retina e pela atrofia do nervo ótico. É a segunda causa de cegueira irreversível em todo o mundo, estimando-se que a sua prevalência aumente para o dobro até 2040. O aumento da pressão intraocular (PIO) é considerado o principal fator de risco para o desenvolvimento de glaucoma e, os atuais tratamentos focam-se no controlo da PIO. Contudo, alguns doentes continuam a perder a visão mesmo com a PIO dentro de valores considerados normais. Assim, torna-se extremamente necessário desenvolver novas estratégias terapêuticas sendo a proteção das células ganglionares da retina uma estratégia com potencial. A neuroinflamação tem um papel crucial no desenvolvimento de glaucoma, e tem sido dada especial atenção ao aumento da reatividade das células da microglia observada em modelos animais e pacientes com glaucoma. A adenosina é um neuromodulador do sistema nervoso central (SNC), envolvida em respostas inflammatórias, que atua através da activação de recetores acoplados a proteínas G: A1, A2A, A2B and A3. Sabe-se atualmente que o bloqueio do recetor A2A de adenosina confere neuroproteção em várias doenças neurodenegerativas através do controlo da neuroinflamação, e diversos estudos focam-se no uso de antagonistas do recetor A2A como potencial estratégia neuroprotetora. Neste estudo pretendemos avaliar o efeito do aumento da pressão hidrostática, estímulo usado para mimitezar a hipertensão ocular, no sistema adenosinérgico das células da microglia. Adicionalmente, pretendemos compreender se o bloqueio do recetor A2A previne a resposta inflamatória resultante do aumento da pressão hidrostática. Foram para isso utilizados dois modelos in vitro: as células BV-2 (linha celular de microglia), nas quais o bloqueio do recetor A2A foi efetuado farmacologicamente usando um antagonista seletivo (SCH 58261), e culturas organotípicas de retina de ratinhos knock-out (KO) para o recetor A2A. A exposição das células BV-2 a pressão hidrostática elevada levou à alteração do sistema adenosinérgico, promovendo um aumento dos níveis de adenosina. Este aumento dos níveis de adenosina pode ativar os recetores A2A, cuja expressão aumentou em condições de pressão hidrostática elevada. Adicionalmente, o aumento da pressão hidrostática diminuiu a expressão do recetor A1 e não intereferiu com a expressão do recetor A3. O viii CRoesnutemnots bloqueio do recetor A2A preveniu a expressão e libertação de iNOS e TNF, respetivamente, nas células da microglia. Nas culturas organotípicas de retina, a exposição a pressão elevada aumentou os níveis de IL-1β e TNF e a morte das células ganglionares. A inativação genética do recetor A2A não preveniu o aumento da libertação de TNF. Em síntese, os nossos resultados mostraram que a pressão elevada conduz a alterações no sistema adenosinérgico nas células da microglia e que o bloqueio do recetor A2A pode ser uma estratégia promissora na modulação da reatividade da microglia após a exposição a pressão elevada.
Glaucoma is the second leading cause of irreversible blindness worldwide and it is expected that the prevalence of this disease increases to the double until 2040. It is a neurodegenerative disease, characterized by the progressive loss of retinal ganglion cells (RGCs) and nerve optic atrophy (RGC axons), leading to vision loss. Intraocular pressure (IOP) is the one of the major risk factors for the development of glaucoma and the current therapeutic approaches are focused on lowering IOP. However, some patients continue to loose vision despite the effective control of IOP, highlighting the urgent need to develop new therapeutic strategies, and neuroprotection of RGCs is consider to have great potential. Neuroinflammation has a preponderant role in the pathogenesis of glaucoma. In particular, special attention has been given to the exacerbated microglial cell response in glaucoma experimental models and in human patients. Adenosine is a neuromodulator in CNS involved in inflammatory responses that acts by the activation of four G protein-coupled receptors, A1, A2A, A2B and A3. The blockade of A2AR has been shown to afford robust neuroprotection in several neurodegenerative diseases, probably by the control of neuroinflammation. Therefore, several studies have been focused in A2AR antagonists as a neuroprotective strategy. In this study, we aimed to investigate the potential alterations induced by elevated hydrostatic pressure (EHP), a stimulus use to mimic increased IOP, in the adenosinergic system in microglial cells. Moreover, we aimed to assess whether the blockade of A2AR could prevent the pro-inflammatory environment prompted by EHP. In order to accomplish our aims, two in vitro models were used: the BV-2 cells (microglial cell line), using the selective antagonist SCH 58261 and retinal organotypic cultures obtained from wild-type and knock-out (KO) mice for A2AR. The exposure of BV-2 cells to EHP showed an impairment of the adenosinergic system promoting increased levels of extracellular adenosine. The increase in adenosine might activate A2AR, which we found to be upregulated in BV-2 cells exposed to EHP. We also found that EHP decreased the protein levels of A1R, without interfering with A3R protein levels. The blockade of A2AR prevented some inflammatory features (iNOS and TNF levels), in microglia. In organotypic cultures, the exposure to EHP increase both IL-1β and TNF levels and RGCs loss. Nevertheless, the genetic blockade of A2AR did not prevent the increased of viii CAobnsttreanctts TNF levels. In summary, our results demonstrated that EHP impacts the adenosinergic system in microglia and the blockade of A2AR may be promising strategy to modulate microglia reactivity triggered by increased pressure