NMDA receptor antagonists such as Ketamine and PCP are potent psychoactive drugs used recreationally. This class of drug induces a number of phenomena in humans similar to those associated with schizophrenia including reduced selective attention, altered working memory, thought disorders and hallucinations. These psychotomimetic drugs have thus been used as a longstanding model to study this disease in animals. Importantly, such animal models allow for recording of brain activity using invasive techniques that are inappropriate in humans. Previous electrophysiological studies have shown that MK-801, a potent non-competitive NMDA receptor antagonist, increases gamma-frequency oscillations and produces a state of disinhibition in the prefrontal cortex of rats wherein the activity of putative excitatory pyramidal neurons increases while the activity of putative inhibitory interneurons decreases. These features are relevant to schizophrenia because molecular evidence suggests dysfunction of inhibitory cortical interneurons, while electroencephalographic recordings show altered gamma-frequency oscillations in this disease. It has been hypothesized that the disinhibited cortical state produces “noisy” information processing, but this has not been directly observed in the interaction of neuronal firing in either humans or animal models. We therefore tested this hypothesis by examining the synchronization of neural activity in the NMDA receptor antagonist model of schizophrenia. We used high-density electrophysiological recordings in the medial prefrontal cortex of freely moving rats before and after systemic injection of MK-801. Analysis of these recordings revealed that drug administration: (i) increases gamma power in field potentials in a manner dissociated from increased locomotion; (ii) does not change the gamma power in multi-unit activity; (iii) decreases spike synchronization among putative pyramidal neurons in the gamma range (30ms), and despite of this it (iv) does not change the synchronization between gamma-range field potentials or between sum-of-spikes and field potentials. These effects in synchronization may be revealing of potent cognitive effects associated with NMDA receptor antagonism, and may reflect impaired communication processing hypothesized to occur in schizophrenia. / xi, 42 leaves : ill. ; 29 cm

Overexpression of the epidermal growth factor receptor (EGFR), a member of the ErbB family, and its closest homologue HER2, have been associated with aggressive tumour progression and reduced sensitivity to DNA-damaging agents. In order to block the proliferation of refractory tumors overexpressing EGFR, a novel strategy has been developed that sought to design molecules capable of not only blocking EGFR-TK, but also damaging DNA. These molecules, termed combi-molecules (CMs), have been shown to degrade under physical conditions to release another inhibitor of EGFR, and to be potent against tumor cells of various origins including breast, prostate and carcinoma of the vulva. However, despite their potency, their growth inhibitory IC50 values were still in the high micromolar range. In order to augment the potency of the CMs, here they were re-designed to contain two quinazoline moieties and a central N,N-bis(2-aminoethyl)methylamine spacer which, following degradation, could yield higher concentrations of free inhibitors and a more cytotoxic bifunctional DNA damaging species. Here, we describe the mechanism of action of the first prototype of this approach, JDE52, which we now classify as a double-arm CM, in comparison with ZRBA1, its closest single-arm counterpart. The results indicated that JDE52 was capable of inducing significant blockade of EGFR, DNA single-strand breaks and inter-strand cross-links. ZRBA1, its single-arm counterpart, was capable of only forming DNA single-strand breaks. The fluorescent property of FD105, the secondary inhibitor that both JDE52 and ZRBA1 are capable of releasing, has permitted the analysis of its levels in tumor cells by UV flowcytometry. It was found that JDE52 was indeed capable of significantly releasing higher levels of fluorescence (p<0.05) in human tumor cells, compared with levels of fluorescence released by ZRBA1. More importantly, JDE52 induced higher levels of apoptosis and cell killing than ZRBA1. Apoptosis was triggered by JDE52 at a faster rate than ZRBA1. The results in toto suggest that the superior potency of JDE52, when compared with ZRBA1, may be imputed to mechanisms associated with the generation of higher levels of FD105 intracellularly, and the induction of DNA cross-links, which are known to be more cytotoxic. These combined mechanisms (blockade of EGFR-TK and formation of cross-links) contributed to an accelerated rate of apoptosis in cells treated with JDE52. This study conclusively demonstrated that designing molecules as prodrugs of high levels of quinazoline inhibitors of EGFR and bifunctional DNA cross-linking species is a valid strategy to enhance the potency of CMs against refractory tumors.

Objectives. beta-blockers can alter bone turnover and increase bone formation in animals. It is unknown whether beta-blockers have similar bone protective effects in humans. We aimed to estimate the effects of beta-blockers on bone mineral density (BMD) and fractures using data from the Canadian Multicentre Osteoporosis Study, a large prospective cohort study. / Methods. All medications, including beta-blockers, taken at baseline and after five years of follow-up were recorded. BMD was measured at baseline. During the five years of follow-up, incident minimal trauma fractures were documented by yearly questionnaires. To compare users of beta-blockers to non-users while controlling for possible confounders, multiple linear regression was utilized to estimate between group differences in BMD and multivariate logistic regression was employed to estimate differences in fracture risk. / Results. Of the 9423 participants, 236 of 2884 males (8.2%) and 600 of 6539 females (9.2%) used beta-blockers at some point during the study. In men, beta-blocker users had differences of +1.1% (95% confidence interval [CI] -0.9%, 3.0%) and +1.2% (95% CI -0.5%, 4.0%) in baseline BMD at the total hip and at the lumbar spine, respectively, compared to non-users. In women, beta-blocker users had differences of +0.05% (95% CI -1.2%, 1.3%) and +0.2% (95% CI -1.3%, 1.7%) for the BMD of the total hip and the lumbar spine, respectively, compared to non-users. For users of beta-blockers at baseline, the adjusted odds ratio (OR) for any minimal trauma fracture was 1.23 (95% CI 0.67--2.25) in men and 1.02 (95% CI 0.76--1.35) in women. Chronic use (user at baseline and year 5) in men had an OR for any minimal trauma fracture of 2.1 (95% CI 1.0--4.3). In women who used beta-blockers at baseline but not at year 5, the OR for hip fracture was 6.3 (95% CI 2.0--19.3). The risk of fractures for other sites was inconclusive owing to wide confidence intervals. / Conclusion. Despite relatively large numbers of subjects, wide confidence intervals do not permit strong conclusions with regards to the effect of beta-blockers on BMD in men. Using a 2% limit of clinical importance for BMD, there appears to be no effect of beta-blockers on BMD in women. There is some evidence from our study that beta-blockers may be associated with an increased risk of fractures in certain subsets of users.

At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of this form remains unknown as does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Methoprene is a Juvenile Hormone (JH) analogue commonly used for the control of mosquito larvae. When applied to a mosquito breeding site, methoprene enters the haemolymph, where it mimics the function of JH and interferes with normal metamorphosis, resulting in larval mortality. Methoprene is commonly used for the control of larvae and has not been used as an adulticide, due to an absence of acute effects. This study evaluated possible chronic effects caused by the exposure of adult Aedes mosquitoes to methoprene. Methoprene was applied, in both technical grade and the commercially available Altosid®, topically to adults through droplet application on the abdomen and as a spray application. Mosquitoes were examined for treatment effects on ovary development, adult male and female mortality, and fecundity. The results demonstrate that relatively high doses are required to affect adult survivorship. In contrast, significant impacts on both fecundity and egg hatch were observed for females treated at the lower dosages. I discuss the results in relation to autocidal strategies for mosquito control in which the release of fecund females is to be avoided.

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

Background and rationale: Parkinson’s disease (PD) is a progressive, degenerative disorder of the central nervous system and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The loss of functional dopamine in the striatum is thought to be responsible for the typical symptoms of PD. Cardinal features of PD include bradykinesia, muscular rigidity, resting tremor and impairment of postural balance. This study focuses on the inhibition of monoamine oxidase B (MAO-B) and antagonism of A2A receptors as therapeutic strategies for PD. Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)-containing mitochondrial bound isoenzyme which consists of two isoforms namely MAO-A and MAO-B. The primary function of MAO is to catalyze the oxidative deamination of dietary amines, monoamine neurotransmitters and hormones. MAO-A is responsible for the oxidative deamination of serotonin (5-HT) and norepinephrine (NE), while MAO-B is responsible for the oxidative deamination of dopamine (DA). The formation of DA takes place in the presynaptic neuron where it is stored in vesicles and released into the presynaptic cleft. The released DA then either binds to D1 and D2 receptors which results in an effector response. The excess DA in the presynaptic cleft is metabolized by MAO-B which may result in the formation of free radicals and a decrease in DA concentrations. Under normal physiological conditions free radicals are removed from the body via normal physiological processes, but in PD these normal physiological processes are thought to be unable to remove the radicals and this may lead to oxidative stress. Oxidative stress is believed to be one of the leading causes of neurodegeneration in PD. The rationale for the use of MAO-B inhibitors in PD would be to increase the natural DA levels in the brain and also diminish the likelihood of free radicals to be formed. Adenosine is an endogenous purine nucleoside and yields a variety of physiological effects. Four adenosine receptor subtypes have been characterized: A1, A2A, A2B and A3. They are all part of the G-protein-coupled receptor family and have seven transmembrane domains. The A2A receptor is highly concentrated in the striatum. There are two important pathways in the basal ganglia (BG) through which striatal information reaches the globus pallidus, namely the direct pathway containing A1 and D1 receptors and the indirect pathway containing A2A and D2 receptors. The direct pathway facilitates willed movement and the indirect pathway inhibits willed movement. A balance of the two pathways is necessary for normal movement. In PD, there is a decrease in DA in the striatum, thus leading to unopposed A2A receptor signaling and ultimately resulting in overactivity of the indirect pathway. Overactivity of the indirect pathway results in the locomotor symptoms associated with PD. Treatment with an A2A antagonist will block the A2A receptor, resulting in the restoration of balance between the indirect and direct pathways, thus leading to a decrease in locomotor symptoms. Aim: In this study, caffeine served as a lead compound for the design of dual-targeted drugs that are selective, reversible MAO-B inhibitors as well as A2A antagonists. Caffeine is a very weak MAO-B inhibitor and a moderately potent A2A antagonist. Substitution on the C8 position of caffeine yields compounds with good MAO-B inhibition activities and A2A receptor affinities. An example of this behaviour is found with (E)-8-(3-chlorostyryl)caffeine (CSC), which is not only a potent A2A antagonist but also a potent MAO-B inhibitor. The goal of this study was to identify and synthesize dual-targeted xanthine compounds. Recently Swanepoel and co-workers (2012) found that 8-phenoxymethyl substituted caffeines are potent reversible inhibitors of MAO-B. Therefore, this study focused on expanding the 8-(phenoxymethyl)caffeine series and evaluating the resulting compounds as both MAO-A and -B inhibitors as well as A2A antagonists. Synthesis: Two series were synthesized namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines. The analogues were synthesized according to the literature procedure. 1,3-Dimethyl-5,6-diaminouracil or 1,3-diethyl-5,6-diaminouracil were used as starting materials and were acylated with a suitable substituted phenoxyacetic acid in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) as an activating reagent. The intermediary amide was treated with sodium hydroxide, which resulted in ring closure to yield the corresponding 1,3-dimethyl-8-phenoxymethyl-7Hxanthinyl or 1,3-diethyl-8-phenoxymethyl-7H-xanthinyl analogues. These xanthines were 7-N-methylated in the presence of an excess of potassium carbonate and iodomethane to yield the target compounds. In vitro evaluation: A radioligand binding assay was performed to determine the affinities of the synthesized compounds for the A2A receptor. The MAO-B inhibition studies were carried out via a fluorometric assay where the MAO-catalyzed formation of H2O2 was measured. Results: Both series showed good to moderate MAO-B inhibition activities, while none of the compounds had activity towards MAO-A. Results were comparable to that of a known MAOB inhibitor lazabemide. For example, lazabemide (IC50 = 0.091 μM) was twice as potent as the most potent compound identified in this study, 8-(3-chlorophenoxymethyl)caffeine (compound 3; IC50 = 0.189 μM). Two additional compounds, 8-(4-iodophenoxymethyl)caffeine and 8-(3,4-dimethylphenoxymethyl) caffeine, also exhibited submicromolar IC50 values for the inhibition of MAO-B. The structure-activity relationships (SARs) indicated that 1,3-diethyl substitution resulted in decreased inhibition potency towards MAO-B and that 1,3-dimethyl substitution was a more suitable substitution pattern, leading to better inhibition potencies towards MAO-B. The compounds were also evaluated for A2A binding affinity, and relatively weak affinities were recorded with the most potent compound, 1,3-diethyl-7-methyl-8-[4-chlorophenoxymethyl]xanthine (compound 16), exhibiting a Ki value of 0.923 μM. Compared to KW-6002 (Ki = 7.94 nM), a potent reference A2A antagonist, compound 16 was 35-fold less potent. Comparing compound 16 to CSC [Ki(A2A) = 22.6 nM; IC50(MAO-B) = 0.146 nM], it was found that compound 16 is 31-fold less potent as an A2A antagonist and 21-fold less potent as a MAO-B inhibitor. Loss of MAO-B inhibition potency may be attributed to 1,3-diethyl substitution which correlates with similar conclusions reached in earlier studies. In addition, the replacement of the styryl functional group (as found with CSC and KW-6002) with the phenoxymethyl functional group (as found with the present series) may explain the general reduction in affinity for the A2A receptor. This suggests that the styryl side chain is more appropriate for A2A antagonism than the phenoxymethyl functional group. Conclusion: In this study two series of xanthine derivatives were successfully synthesized, namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines (11 compounds in total). Three of the newly synthesized compounds were found to act as potent inhibitors of MAO-B, with IC50 values in the submicromolar range. None of the compounds were however noteworthy MAO-A inhibitors. The most potent A2A antagonist among the examined compounds, compound 16, proved to be moderately potent compared to the reference antagonists, CSC and KW-6002. It may be concluded that the styryl functional group (as found with CSC and KW-6002) is more optimal than the phenoxymethyl functional group (as found with the present series) for A2A antagonism. 1,3-Diethyl substitution of the xanthine ring was found to be less optimal for MAO-B inhibition compared to 1,3-dimethyl substitution. These results together with known SARs provide valuable insight into the design of 8-(phenoxymethyl)caffeines as selective and potent MAO-B inhibitors. Such drugs may find application in the therapy of PD. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Background and rationale - Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of dopamine in the brain. The cause of Parkinson's disease is still unknown; however several theories pertaining to the etiology exist. Current treatment mainly aims at dopamine replacement, with agents such as levodopa and dopamine agonists that provide patients with symptomatic relief. This relief is unfortunately only temporary as the progression of the disease is not halted. Furthermore, these therapies are associated with a range of side effects and novel approaches to the treatment are thus urgently required. Adenosine A2A receptor antagonists recently emerged as a promising non-dopaminergic alternative, not only as symptomatic treatment, but also as potential neuroprotective therapy. Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors (A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine depletion to give symptomatic relief. There are also several mechanisms where by adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A antagonists have already reached clinical trials and promising results were obtained, especially when combined with levodopa. Consequently, A2A antagonists are realistic prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor antagonists. Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of inhibition comes with certain disadvantages as it may take up to several weeks after termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast will have much better safety profiles seeing that they will not inactivate the enzyme permanently and allow for competition with the substrate. When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease. MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the progression of the disease by preventing the formation of these byproducts. Promising MAOB inhibitory activities have been reported for chalcones, and since the intermediates obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B. Results - Design and synthesis: A series of 2-aminopyrimidines were designed using known active structures and literature pharmacophores. A molecular modelling study (Discovery Studio 3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked in the binding site of the adenosine A2A receptor. Binding orientations and observed interactions with important residues in the active site were similar to those observed for known A2A antagonists. It was therefore concluded that these compounds may be potential A2A antagonists and the designed compounds were thus synthesised. Structures were primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry. MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay and kynuramine as substrate. The compounds were potent and selective inhibitors of the MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1- (5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of 0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A. Some preliminary structure activity relationships were derived, for example, phenyl substitution with an electron withdrawing chlorine group generally resulted in better activity than substitution with electron donating methoxy groups. Further investigation of structure activity relationships are however required as a very small series of chalcones were screened. Reversibility studies and mode of inhibition: A dilution assay was used to determine whether compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor complex. The results from the reversibility studies showed that the inhibition of the most potent compound (1c) is reversible as the catalytic activities are recovered to approximately 80% and 50% respectively, compared to the control measured in the absence of an inhibitor. For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver- Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor of the MAO-B enzyme. In vitro adenosine A2A assays: Radioligand binding assays were used to determine the affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine (CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM. The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part, be explained by the molecular modellling studies. In the docking experiments an additional hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271 indicating that this structural feature is a major contributing factor to the improved affinity observed for these derivatives. In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to determine whether the two compounds with the highest affinity for the adenosine A2A receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine A2A antagonists. The objectives of this study as set out were thus successfully realised and promising results were obtained. During this study, several novel 2-aminopyrimidines and chalcones were synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase inhibitory activities for all of the screened compounds were determined for the first time. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

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.

Focal adhesion kinase (FAK), a non-receptor kinase, is a key regulator of integrin and focal adhesion signaling required for cancer cell survival, cell migration, and cell invasion. Amplification/Overexpression of FAK occurs in a wide variety of human cancers, supporting a role in carcinogenesis. Moreover, preclinical studies using cancer models where FAK is genetically inhibited indicate that this kinase is a potential therapeutic target to interfere with cancer progression. However, very little progress has been made in the identification of chemical inhibitors for potential therapeutic applications, in contrast to other kinases. Herein, I report optimization of the high-throughput in vitro Glo kinase assay for screening inhibitors of FAK kinase activity. Screening a large library of small molecule chemicals using these assays identified at least twenty FAK inhibitors, including a new FAK inhibitor developed by Pfizer and undergoing human clinical trials, and the non-specific kinase inhibitor staurosporine. Molecular studies of selective FAK inhibitors are undergoing in my host laboratory. In addition to this in vitro assay, I established similar assays to examine FAK kinase and adapter function in intact cells. The latter consists of ErbB-transformed cells deficient in FAK, and their matched cells where wild-type or kinase-dead FAK was restored. Biological characterization of these models revealed that both FAK kinase and adaptor activities cooperate for the regulation of cell migration, cell invasion, and tumor formation.