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Chapter 1 of thesis is a literature review of adenosine research. The central importance of the contributions of both classical pharmacology and, more recently, molecular biology to adenosine research is demonstrated. These disciplines have enabled the classification and characterisation of adenosine receptors and as well an understanding of the physiological significance of endogenous adenosine. The significant benefits of developing therapeutics for regulation of the diverse physiological functions of adenosine, by regulation of adenosine receptors, is outlined. For this therapeutic potential to be realised both high affinity and subtype selective adenosine agonists and antagonists are required. The structure-activity relationships for agonists and xanthine antagonists are discussed. The assimilation of these structure-activity relationships have guided the development of ligand based models of the adenosine receptor pharmacophore. The 'flipped', 'N6-C8' and 'three binding domain' models were described. These models aim to direct the future design of high affinity and selective ligands for adenosine receptors. The development of receptor based models by modelling of the receptor-ligand complex is also presented. The main body of this thesis presents a study of the structure-activity relationships for pyrazolo(3,4-d) pyrimidines binding to adenosine Ai and A2a receptors. Prior to this study few non-xanthine adenosine antagonists had been well defined or optimised in terms of structure-activity relationships. However, the value of such ligands is immense, facilitating further definition of structural requirements for high affinity and selective adenosine receptor binding. These ligands should complement existing agonists and xanthine antagonists in developing an understanding of adenosine receptor binding. The experimental approach to development of the lead compound of this study, a-(6-(l'-carbamoylethylthio)- l-phenylpyrazolo(3,4-d)pyrimidin-4-ylthio)propanamide (5), is outlined in Chapter 2 of this thesis. 5 is substituted at C-4, C-6 and N-i of the pyrazolo(3,4-d)pyrimidine heterocycle. The experimental approach to optiniising 5 was approached in a rational manner, requiring an iterative approach i.e. design of generation I target compounds --synthesis -- biological evaluation -- structure-activity relationships -- design of generation II target compounds, etc. Chapters 3, 4 and 5 of this thesis describe this experimental approach as it relates to optimising the lead compound, 5, for adenosine receptor affinity and subtype selectivity. The importance of receptor interactions with multiple ligand domains, to achieve both potency and selectivity, was recognised so that optimisation of the C-4, C-6 and N-i substituents of the lead compound was targeted and achieved. Previous structure-activity studies with agonists and xanthine antagonists have concentrated on modifying a single ligand domain. Chapter 3 presents twelve generation I target compounds to examine C-4 and C-6 substituent structure-activity relationships. Chapter 4 presents twelve generation II target compounds to further examine C-4 and C-6 substituent structure-activity relationships. Chapter 5 presents sixteen generation ifi target compounds to examine N-I substituent structure-activity relationships. A major outcome from the research presented in these chapters was the development of highly potent and highly selective ligands for the adenosine A1 receptor subtype. a(4-Methylamino- I -phenylpyrazolo(3,4-d)pyrimidin-6-ylthio)hexanamide (29) was the most potent ligand at the Ai receptor identified in this study, and is one of the most potent Ai selective antagonists ever reported. 29 has an A1 K1 value of 0.745±0.045 nM and is 332-fold selective for the A1 receptor over the A2a receptor. a-(1-Phenyl-4-propylthiopyrazolo(3,4-d)pyrimidin-6-ylthio)butanainide (27) was the most selective ligand of this study. It is four orders of magnitude selective for the A1 receptor (up to 16900-fold), and one of the most selective antagonists ever reported. This high selectivity has been achieved with the maintenance of good A1 affinity (A1 K1 = 29.5±6.6 nM). These results prove the value of modifying multiple substituents of adenosine receptor ligands, generating ligands which bind with high potency and selectivity to adenosine Al receptors compared to adenosine A2a receptors.

Graduation date: 1998

Master of Science / Department of Kinesiology / Brett J. Wong / Mechanisms underlying the cutaneous vasodilation response to local skin heating and whole body heating in humans remain unresolved. Although nitric oxide (NO) is known to contribute to these responses, it remains unclear as to the source of NO. Adenosine receptors induce vasodilation in many human tissues and may work, in part, through NO. As these receptors are also known to be located in the cutaneous vasculature, the studies contained in this thesis were designed to investigate a potential contribution of adenosine receptor activation to the rise in skin blood flow elicited by local skin and whole body heating. The study presented in chapter IV was designed to determine a potential role for adenosine receptors in contributing to cutaneous thermal hyperemia. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments designed to elucidate the contribution of A[subscript]1/A[subscript]2 adenosine receptors during local skin heating. Each site was locally heated from a baseline temperature of 33°C to 42°C at a rate of 1°C/10 s and skin blood flow was monitored via laser-Doppler flowmetry (LDF). The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation directly contributes to cutaneous thermal hyperemia. These data further suggest a portion of the NO response may be explained by A[subscript]1/A[subscript]2 adenosine receptor activation; however, a substantial portion of the NO response is independent of the adenosine receptor contribution. The study presented in chapter V was designed to determine a potential role for A[subscript]1/A[subscript]2 adenosine receptors in contributing to cutaneous active vasodilation. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments, as above, and skin blood flow was monitored via LDF. Whole body heat stress, sufficient to raise oral temperature at least 0.8°C above baseline, was induced via water-perfused suits. The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation does not directly contribute to cutaneous active vasodilation; however, a role for A[subscript]1/A[subscript]2 adenosine receptor activation is unmasked when NO synthase is inhibited. The data from this study further suggest that A[subscript]1/A[subscript]2 adenosine receptor activation may be responsible for a portion of the known NO component of cutaneous active vasodilation.

Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.

Graduation date: 1990

Graduation date: 1993

Caffeine is one of the most widely used neurostimulants in the world and there is considerable debate on its effect in immune cells. One of its main targets is proposed to be adenosine receptors which mediate an anti-inflammatory switch in activated immune cells while another target is phosphodiesterase where it acts as an inhibitor. In macrophages, caffeine has been shown to cause both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. If the primary effect of caffeine on macrophages were to antagonize adenosine receptors we would expect cells exposed to caffeine to have a prolonged M1 response. However, we show that caffeine suppresses phagocytosis at physiological concentrations (an indicator of M2 phenotype). This suppression was reversed when macrophages were pretreated with protein kinase A inhibitor, suggesting that at physiological concentrations caffeine's phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an M2 fate. However, mRNA expression profiling suggests that caffeine can modulate A2A receptor expression and suppress MKP-1 expression, a hallmark of M1 macrophages. Caffeine is, therefore an immunomodulator that can suppress or prolong inflammatory responses in macrophages, which may account for the abundance of contradicting evidence in the literature. Additionally, these effects are complicated by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in macrophage activation.

2. Abstract Adaptation to chronic hypoxia is in addition to ischemic preconditioning one of the two known cardioprotective mechanisms. The precise molecular basis of these processes is still not fully explained. There are some studies that suggest the possible involvement of the adenosinergic signaling system in this adaptation. In this work, we focused on the characterization of the adenosinergic system in the myocardium of rats adapted to two regimens of chronic hypoxia - a protective continuous normobaric hypoxia (CNH) and non-protective intermittent hypoxia (INH/R, 23 h hypoxia and 1 h normoxia). Initially, we compared the total amount of adenosine receptors in samples from different groups of adapted animals. We discovered changes mainly at A2B receptor, which increased at CNH and declined in INH/R. This result suggests the possible involvement of A2B receptors in cardioprotection afforded by adaptation to chronic hypoxia. Furthermore, we investigated the distribution of various types of adenosine receptors and transporters in the plasma membrane of cardiac cells. We observed that A2A and A3 localize in membrane microdomains together with membrane enzyme CD73 that produces adenosine in the extracellular space by degrading AMP. A1 and A2B receptors similarly as nucleoside transporters ENT1, ENT2 and...

Adenosine plays a critical role in the heart signalling while affecting heart rate, contractility or coronary flow. Nowadays, four adenosine receptor subtypes are distinguished which are present in most of tissues and cells: A1, A2A, A2B and A3. All these receptors belong to the family of G protein-coupled receptors. Upon activation, their main target is an enzyme adenylyl cyclase which produces an important second messenger cAMP. The main goal of this thesis was characterization of adenosine receptors in the rat myocardium, assessment of their distribution, binding properties and signalling. We examined a possible disparity in receptors distribution between the left and right ventricles using SDS-PAGE electrophoresis and Western blotting. The same methods have been used in studies of adenosine receptor distribution in lipid rafts. Samples of lipid rafts and soluble fraction were prepared using a nonionic detergent Triton X-100. We did not find any evidence of different distribution between the left and right ventricles and our results did not confirm compartmentation of the receptors either. For determination of binding properties of the receptors we used radioligand binding assays with the A1 selective radioligand [H3 ]DPCPX. We did not observe any significant difference between the receptor...