Deaza-analogues of Adenosine and 2-Chloroadenosine as Agonists of Adenosine Receptors

Abstract

A variety of adenosine analogues have been recently evaluated in order Lo find more potent and selective agonists on adenosine receptors. The most potent adenosine analogues acting on A₁ receptor, a high affinity receptor inhibitory to adenylate cyclase, are N⁶-substituted compounds. So ⁶-cyclohexyladenosine (CHA) and ⁶-L-phenylisopropyladenosine (L-PIA) are extremely potent agonists on A₂ receptor, whereas they are relatively weak agonists on A receptor, a lower affinity receptor which is stirnulatory to cyclase, and they have no effect on the adenosine P site.     

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract

Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A₁ rat brain adenosine receptor, follows the order N⁷ > N³ > N¹. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Molecular Modelling of the Antagonist Binding Site on the Adenosine A,Receptor

Abstract

With the aid of molecular modelling both adenosine and adenosine A, receptor antagonists belonging to various chemical classes were compared. A model for the antagonist binding site was developed. As a consequence 1H-imidazo[4, 5-c]-quinolin-4-amines were synthesized, constituting a novel class of potent non-xanthine adenosine receptor antagonists.     

Recent Progress in the Purification of Adenosine Receptors

Abstract

A₁ adenosine receptors were purified to an apparent homogeneity from rat brain and testicular membranes by a novel affinity chromatography system using xanthine amine congener (XAC) as an immobilized ligand. This affinity chromatography was also useful for the purification of human brain A1 adenosine receptor.     

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1 Receptor in the Rat Cortex

Abstract

The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [³H]XCC and [³H]XAC², and [³H]CPX³) which bind with high and selective affinity to the adenosine A₁ receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 8216⁵, represents the first reported non-xanthine structure that potently blocks adenosine receptors⁶. CGS 15943 has nanomolar affinity for both A₁ and A₂ receptor subtypes⁶. However, in contrast to many of the xanthine adenosine receptor antagonists, CGS 15943 is not a phosphodiesterase inhibitor and does not interact with adenosine transporter sites⁶. This compound is a potent and selective adenosine receptor antagonist in vivo ⁷ with a solubility/affinity ratio of greater than 1000⁷. In the present studies, the binding of [³H]CGS 15943 to the adenosine A₁ receptor was characterized.     

Cardiovascular Effects of Adenosine Derivatives in Conscious Normotensive Rats

Abstract

All adenosine receptor agonists, regardless of their A₁/A₂ selectivity ratio, dose dependently reduced blood pressure (MAP) whereas their effects on heart rate (HR) and plasma renin activity (PRA) depended on their receptor subtype selectivity. Thus an adenosine receptor agonist with an optimal A₁- and A₂- receptor selectivity (no increase in HR and PRA) and which does not penetrate the brain, might be a useful antihypertensive drug.     

Nucleoside Modification and Concerted Mutagenesis of the Human A3 Adenosine Receptor to Probe Interactions Between the 2-Position of Adenosine Analogs and Gln167 in the Second Extracellular Loop

Residues of the second extracellular loop are believed to be important for ligand recognition in adenosine receptors. Molecular modeling studies have suggested that one such residue, Gln ¹⁶⁷ of the human A ³ receptor, is in proximity to the C2 moiety of some adenosine analogs when bound. Here this putative interaction was systematically explored using a neoceptor strategy, i.e., by site-directed mutagenesis and examination of the affinities of nucleosides modified to have complementary functionality. Gln ¹⁶⁷ was mutated to Ala, Glu, and Arg, while the 2-position of several adenosine analogs was substituted with amine or carboxylic acid groups. All compounds tested lost affinity to the mutant receptors in comparison to the wild type. However, comparing affinities among the mutant receptors, several compounds bearing charge at the 2-position demonstrated preferential affinity for the mutant receptor bearing a residue of complementary charge. 13, with a positively-charged C2 moiety, displayed an 8.5-fold increase in affinity at the Q167E mutant receptor versus the Q167R mutant receptor. Preferential affinity for specific mutant receptors was also observed for 8 and 12. The data suggests that a direct contact is made between the C2 substituent of some charged ligands and the mutant receptor bearing the opposite charge at position 167.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.     

Effect of Endogenous Phospholipids on the [3H]R-Pia Binding to A1 Adenosine Receptors from PIG Cerebral Cortex

Abstract

The effect of phospholipases on the membrane-bound and solubilized A₁ adenosine receptor has been studied. The results indicate that phospholipids are essential for the functionality of the A₁ adenosine receptor and that there is a co-solubilization of receptor and phospholipids.     

Adenosine Receptors in Isolated Tissue Preparations

Abstract

Adenosine receptors in isolated tissues from guinea-pigs have been investigated using agonists and antagonists. The receptor mediating decreases in rate and force of contraction of the atria is of the A₁ sub-type. Responses of smooth muscle preparations to adenosine and its analogues are complex involving relaxation mediated by both A₂ receptors an by non-receptor mechanisms. Contractions mediated by an A₁ receptor can also be detected.     

Xanthine-7-Ribosides as Adenosine Al Receptor Antagonists: Further Evidence for Adenosine's Anti Mode of Binding

Abstract

The synthesis and A₁ adenosine receptor affinity of some xanthine-7-ribosides is described. It appears that these compounds are A, receptor antagonists. The orientation of the ribose moiety, as determined by ′H-NMR spectroscopy and theoretical chemical calculations, is compared with the orientation of the ribose in the agonist adenosine. Implications for the syn vs anti modes of binding to the receptor are discussed.     

A Computer Generated Model of Adenosine Receptors Rationalising Binding and Selectivity of Receptor Ligands

Abstract

Computer graphic analyses on a broad spectrum of adenosine receptor ligands has shown that both the A1 and A2 adenosine receptors have three binding sites. The spatial relationship of these three binding sites has been defined. Adenosine orientation at A1 and A2 is different.     

R-N6-Phenylisopropyladenosine Produces Tracheal Contraction Through A1 Adenosine Receptor and Challenges Prostanoid Mechanisms

Abstract

The type of purinergic receptor involved in tracheal contraction by R-N⁶-phenylisopropyladenosine (PIA) and the influence of this adenosine analogue on prostaglandin release were studied in normal and in actively sensitized tracheae. Results suggest a balance between adenosine and eicosanoids in the regulation of the airway system.     

Role of Adenosine Kinase in the Biological (Antiviral and Anticellular) Activities of Adenosine Analogues

Abstract

A paired adenosine kinase-positive/adenosine kinase-negative cell system is proposed to distinguish those adenosine analogues that need to be phosphorylated to exert their biological effects from those that are mainly targeted at S-adenosyl-L-homocysteine hydrolase.     

Regional Changes in Rat Brain Adenosine A1 Receptors Following Seizures

Abstract

Generalized seizures induced a widespread upregulation of adenosine A1 receptors linked to G proteins in the rat brain. Changes in receptor density were more pronounced in structures mediating seizure activity and were age-dependent.     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

Background

Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A₃ receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A₃ receptor agonists in coronary blood vessels using the isolated perfused heart preparation.

Methods

mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A₃ receptor mediated coronary vasodilation in the rat heart.

Results

Adenosine A₃ receptor agonists induced coronary vasodilation. The expression of adenosine A₃ receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A₃ receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A₃ receptor agonists.

Conclusions

This study demonstrated alterations in the expression of adenosine A₃ receptors occurred in a tissue specific mode, and reduced adenosine A₃ receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A₃ receptor in hypertensive hearts suggest that adenosine A₃ receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A₃ receptor agonists.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 2′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 2′-deoxy-2′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Survey of Nonxanthine Derivatives as Adenosine Receptor Ligands

Abstract

The binding affinities at rat A₁, A_2a, and A₃ adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [³H]PIA or [³H]CGS 21680 in rat brain membranes or [¹²⁵I]AB-MECA in CHO cells stably transfected with rat A₃ receptors. Several new classes of adenosine antagonists (e. g. 5- oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11- hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidin-one, folic acid, and cytochalasin H and J bound to A₃ receptors selectively. Moreover, cytochalasin A, which bound to A₁ adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A₁ receptor binding.